ISSN isflD-3iss Pages 683-992 Year 2020, Vol. 67, No. 3 Acta ChimicaSlc Acta Chimica Slc Slovenica ActaC 67/2020 http://acta.chem-soc.si EDITOR-IN-CHIEF KSENIJA KoGEJ University of Ljubjana, Facuty of Chemstry and Chemical Technology, Večna pot 113, SI-1000 Ljubljana, Slovenija E-mail: ACSi@fkkt.uni-lj.si, Telephone: (+386)-1-479-8538 ASSOCIATE EDITORS Matjaž Kristl, University of Maribor, Slovenia Franc Perdih, University of Ljubljana, Slovenia Aleš Podgornik, University of Ljubljana, Slovenia Helena Prosen, University of Ljubljana, Slovenia Irena Vovk, National Institute of Chemistry, Slovenia ADMINISTRATIVE ASSISTANT Alen Albreht, National Institute of Chemistry, Slovenia Aleš Berlec, Jožef Stefan Institute, Slovenia Janez Cerkovnik, University of Ljubljana, Slovenia Mirela Dragomir, Jožef Stefan Institute, Slovenia Ksenija Kogej, University of Ljubljana, Slovenia Krištof Kranjc, University of Ljubljana, Slovenia Marjana Gantar Albreht, National Institute of Chemistry, Slovenia EDITORIAL BOARD Wolfgang Buchberger, Johannes Kepler University, Austria Alojz Demšar, University of Ljubljana, Slovenia Stanislav Gobec, University of Ljubljana, Slovenia Marko Goličnik, University of Ljubljana, Slovenia Günter Grampp, Graz University of Technology, Austria Wojciech Grochala, University of Warsaw, Poland Danijel Kikelj, University of Ljubljana Janez Košmrlj, University of Ljubljana, Slovenia Blaž Likozar, National Institute of Chemistry, Slovenia Mahesh K. Lakshman, The City College and The City University of New York, USA Janez Mavri, National Institute of Chemistry, Slovenia Friedrich Srienc, University of Minnesota, USA Walter Steiner, Graz University of Technology, Austria Jurij Svete, University of Ljubljana, Slovenia Ivan Švancara, University of Pardubice, Czech Republic Jiri Pinkas, Masaryk University Brno, Czech Republic Gašper Tavčar, Jožef Stefan Institute, Slovenia Christine Wandrey, EPFL Lausanne, Switzerland Ennio Zangrando, University of Trieste, Italy Chairman Branko Stanovnik, Slovenia Members Udo A. Th. Brinkman, The Netherlands Attilio Cesaro, Italy Vida Hudnik, Slovenia Venceslav Kaucic, Slovenia Zeljko Knez, Slovenia ADVISORY EDITORIAL BOARD Radovan Komel, Slovenia Stane Pejovnik, Slovenia Anton Perdih, Slovenia Slavko Pečar, Slovenia Andrej Petrič, Slovenia Boris Pihlar, Slovenia Milan Randic, Des Moines, USA Jože Škerjanc, Slovenia Miha Tišler, Slovenia Durda Vasic-Rački, Croatia Marjan Veber, Slovenia Gorazd Vesnaver, Slovenia Jure Zupan, Slovenia Boris Žemva, Slovenia Majda Žigon, Slovenia Acta Chimica Slovenica is indexed in: Academic Search Complete, Central & Eastern European Academic Source, Chemical Abstracts Plus, Chemical Engineering Collection (India), Chemistry Citation Index Expanded, Current Contents (Physical, Chemical and Earth Sciences), Digitalna knjižnica Slovenije (dLib.si), DOAJ, ISI Alerting Services, PubMed, Science Citation Index Expanded, SciFinder (CAS), Scopus and Web of Science. Impact factor for 2019 is IF = 1.263. 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Acta Chimica Slovenica appears quarterly in electronic form on the web site http://acta.chem-soc.si. In case of dedicated issues, a limited number of printed copies are issued as well. Transakcijski račun: 02053-0013322846 Bank Account No.: SI56020530013322846-Nova Ljubljanska banka d. d., Trg republike 2, SI-1520 Ljubljana, Slovenia, SWIFT Code: LJBA SI 2X Oblikovanje ovitka - Design cover: KULT, oblikovalski studio, Simon KAJTNA, s. p. Grafična priprava za tisk: Majanafin, d. o. o. ActaChimicaSlo ActaChimicaSlo SlovenicaActaC Year 2020, Vol. 67, No. 3 FEATURE ARTICLE Review articles G-Quadruplexes: Emerging Targets for the Structure-Based Design of Potential Anti-Cancer and Antiviral Therapies Petar M. Mitrasinovic SCIENTIFIC PAPER Analytical chemistry A Novel Electrochemical CuO-Nanostructure Platform for Simultaneous Determination of 6-thioguanine and 5-fluorouracil Anticancer Drugs Masoud Fouladgar Analytical chemistry Metoprolol: New and Efficient Corrosion Inhibitor for Mild Steel in Hydrochloric and Sulfuric Acid Solutions Fatemeh Mohammadinejad, Seyyed Mohammad Ali Hosseini, Mehdi Shahidi Zandi, Mohammad Javad Bahrami and Zahra Golshani Graphical Contents Analytical chemistry Dynamics of Isomerization of Hop Alpha-Acids and Transition of Hop Essential Oil Components in Beer Miha Ocvirk and Iztok J. Košir Analytical chemistry Low-level Electrochemical Analysis of Ketoconazole by Sepiolite Nanoparticles Modified Sensor in Shampoo Sample Sevda Aydar, Dilek Eskiköy Bayraktepe, Hayati Filik and Zehra Yazan Organic chemistry The Effect of Hydrogen Bonding and Azomethine Group Orientation on Liquid Crystal Properties in Benzylidene Aniline Compounds Abdullah Hussein Kshash Analytical chemistry Ionic Liquid-Based Dispersive Liquid-Liquid Microextraction for the Simultaneous Determination of Carbamazepine and Lamotrigine in Biological Samples Salumeh Ranjbar, Ameneh Porgham Daryasari and Mojtaba Soleimani Biochemistry and molecular biology Voltammetric Determination of Sulfaclozine Sodium at Sephadex-modified Carbon Paste Electrode Emad Mohamed Hussien, Hanaa Saleh, Magda El Henawee, Afaf Abou El Khair and Neven Ahmed Organic chemistry Synthesis, Characterization and Biological Activity of Some Dithiourea Derivatives Felix Odame, Eric Hosten, Jason Krause, Michelle Isaacs, Heinrich Hoppe, Setshaba D. Khanye, Yasien Sayed, Carminita Frost, Kevin Lobb and Zenixole Tshentu Chemical, biochemical and environmental engineering Alternative to Conventional Edible Oil Sources: Cold Pressing and Supercritical CO2 Extraction of Plum (Prunus domestica L.) Kernel Seed Jelena Vladic, Aleksandra Gavaric, Stela Jokic, Nika Pavlovic, Tihomir Moslavac, Ljiljana Popovic, Ana Matias, Alexandre Agostinho, Marija Banozic and Senka Vidovic Organic chemistry Sulfonamide Derived Esters: Synthesis, Characterization, Density Functional Theory and Biological Evaluation through Experimental and Theoretical Approach Muhammad Danish, Ayesha Bibi, Muhammad Asam Raza, Nadia Noreen, Muhammad Nadeem Arshad and Abdullah Mohamed Asiri 799-81 Materials science Phase Equilibria in the Ag2Te-PbTe-Sb2Te3 System and Thermodynamic Properties of the (2PbTe)1_^(AgSbTe2)x Solid Solutions Leyla Farhad Mashadiyeva,Shabnam Hamlet Mansimova, Dunya Mahammad Babanly, Yusif Amirali Yusibov, Dilqam Babir Tagiyev and Mahammad Baba Babanly 812-821 Organic chemistry Study on the Synthesis and Biological Activities of N-Alkylated Deoxynojirimycin Derivatives with a Terminal Tertiary Amine Lin Wang and Zhijie Fang inorganic chemistry Structural Diversity in Ûxadiazole-Containing Silver Complexes Dependent on the Anions Long Zhao, Long-Yan Xie, Xiu-Li Du, Kai Zheng, Ting Xie, Rui-Rui Huang, Jie Qin, Jian-Ping Ma and Li-Hong Ding Applied chemistry Prediction of Biological Activities, Structural Investigation and Theoretical Studies of meta-cyanobenzyl Substituted Benzimidazolium Salts Duygu Barut Celepci and Aydin Aktaç 842-852 Chemical, biochemical and environmental engineering Effects of Amino Acids on the Crystallization of Calcium Tartrate Tetrahydrate Sevgi Polat, Elif Aytan-Goze and Perviz Sayan inorganic chemistry Synthesis, Crystal Structures and Catalytic Property of Dioxidomolybdenum(VI) Complexes with Tridentate Hydrazones Xiao-Qiang Luo, Yong-Jun Han and Ling-Wei Xue inorganic chemistry Synthesis and X-Ray Crystal Structures of Trinuclear Nickel(II) Complexes Derived from Schiff Bases and Acetate Ligands with Biological Activity Jin-Long Hou, Hong-Yuan Wu, Cheng-Bin Sun, Ye Bi and Wei Chen Organic chemistry Molybdic Acid-Functionalized Nano-Fe304@Ti02 as a Novel and Magnetically Separable Catalyst for the Synthesis of Coumarin-Containing Sulfonamide Derivatives Jamileh Etemad Gholtash, Mahnaz Farahi, Bahador Karami and Mahsa Abdollahi Biochemistry and molecular biology Quantum Mechanics/Molecular Mechanics Study on Caspase-2 Recognition by Peptide Inhibitors Petar M. Mitrasinovic Biochemistry and molecular biology Antimüllerian Hormone and Oxidative Stress Biomarkers as Predictors of Successful Pregnancy in Polycystic Ovary Syndrome, Endometriosis and Tubal Infertility Factor Teja Fabjan, Eda Vrtacnik-Bokal, Irma Virant-Klun, Jure Bedenk, Kristina Kumer and Josko Osredkar inorganic chemistry Syntheses, Crystal Structures and Catalytic Property of Oxidovanadium(V) Complexes Derived from Tridentate Hydrazone Ligands Ya-Jun Cai, Yuan-Yuan Wu, Fei Pan, Qi-An Peng and Yong-Ming Cui Chemical education Students' Achievements in Solving Authentic Tasks with 3D Dynamic Sub-Microscopic Animations About Specific States of Water and their Transition Miha Slapničar, Valerija Tompa, Saša A. Glažar, Iztok Devetak and Jerneja Pavlin WKi[l>n«nfvi|<1lwi Hi«» 1 loj HuavMt OS äjlf "nun ' Stalï it -JV i«rc*a to vov* □ 0 □ inorganic chemistry Cyanide-Bridged Polynuclear and one-Dimensional Fem-Mnm/n Bimetallic Complexes Based-on Pentacyanoferrite(III) Building Block: Synthesis, Crystal Structures, and Magnetic Properties Xiaoyun Hao, Yong Dou, Tong Cao, Lan Qin, Zhen Zhou, Lu Yang, Dacheng Li, Qingyun Liu, Yueyun Li and Daopeng Zhang inorganic chemistry Synthesis, Crystal Structures and Catalytic Property of ûxidovanadium(V) Complexes with N'-(4-ûxopentan-2-ylidene)nicotinohydrazide and 4-Bromo-N'-(4-oxopentan-2-ylidene)benzohydrazide Qiwen Yang, Pu Wang and Yan Lei Biomedical applications Synthesis and Evaluation on Anticonvulsant and Antidepressant Activities of Naphthoquinone Derivatives Containing Pyrazole and Pyrimidine Fragments Nataliia Polish, Mariia Nesterkina, Nataliia Marintsova, Andriy Karkhut, Iryna Kravchenko, Volodymyr Novikov and Andrei Khairulin Organic chemistry A Simple and Effective Synthesis of 3- and 4-((Phenylcarbamoyl)oxy)benzoic Acids Urban Kosak and Stanislav Gobec Biomedical applications Prediction of Single Point Mutations in Human Coronavirus and Their Effects on Binding to 9-ü-Acetylated Sialic Acid and Hidroxychloroquine Petar M. Mitrasinovic Biomedical applications Synthesis, Characterization and Biological Application of Pyrazolo[1,5-a]pyrimidine Based Organometallic Re(I) Complexes Reena R. Varma, Juhee G. Pandya, Foram U. Vaidya, Chandramani Pathak, Bhupesh S. Bhatt and Mohan N. Patel Analytical chemistry A New Reagent for Spectrophotometric Determination of Ir(IV): 5-[2-(4-Hydroxyphenyl) hydrazineylidene]-4-iminothiazolidin-2-one (HPIT) Oleksandr Tymoshuk, Lesia Oleksiv, Orest Fedyshyn, Petro Rydchuk, Vasyl Matiychuk and Taras Chaban F-; I icMilHiHt ipia'ii'iflV.1 m^iiviiwix physical chemistry The Influence of Ionic Liquids on Micellization of Sodium Dodecyl Sulfate in Aqueous Solutions Bojan Šarac and Marija Bešter-Rogač SHORT cOMMUNICATION Analytical chemistry Arsenic in Sediments, Soil and Plants in a Remediated Area of the Iron Quadrangle, Brazil, and its Accumulation and Biotransformation in Eleocharis geniculata Maria Ängela de B. C. Menezes, Ingrid Falnoga, Zdenka Slejkovec, Radojko Jacimovic, Nilton Couto, Eleonora Deschamps and Jadran Faganeli DOI: 10.17344/acsi.2020.5823 Acta Chim. Slov. 2020, 67, 683-700 /^creative ^commons Review article G-Quadruplexes: Emerging Targets for the Structure-Based Design of Potential Anti-Cancer and Antiviral Therapies Petar M. Mitrasinovic* Center for Biophysical and Chemical Research, Belgrade Institute of Science and Technology, 11060 Belgrade, Serbia * Corresponding author: E-mail: pmitrasinovic.ist-belgrade.edu.rs@tech-center.com Received: 01-10-2020 Abstract G-quadruplexes (G4s) are noncanonical secondary structures that fold within guanine (G) rich strands of regulatory genomic regions. Recent evidences suggest their intimate involvement in important biological processes such as telomere maintenance, end-capping and protection, chromosome stability, gene expression, viral integration, and recombination. Mechanistic details of how and why G4 structures influence biological function indicate a rationale for treating G4s as emerging molecular targets for future therapeutics. In other words, the structural heterogeneity with well-defined binding sites, thermal stability and abundance of G4s in telomeres, oncogene promoter regions, and viral genomes make G4s attractive targets for small molecules, aimed to selectively recognize them over all other nucleic acids structures, particularly duplex forms that are most abundant in the genome. Herein, a critical survey of well-characterized G4-in-teractive ligands as potential tools in anti-cancer and antiviral therapies is presented. Effects that these ligands selectively exert in vitro and in vivo models are summarized. Unique ligands involved in specific G4 recognition are put forward. A key question, how to design and develop new G4 specific ligands that conform to the structural and physicochemical requirements for optimal biological activity, is discussed by considering both remarkable advances over the last few years and our recent contributions. Keywords: Anti-cancer and antiviral therapies, gene expression, G-quadruplex, ligand, structure-based drug design, target 1. Introduction Even though nucleic acids structures are usually imagined as a double-helical DNA that is most abundant in the genome and plays a crucial role in genetic information storage,1 only 3% of the human genome is expressed in proteins.2 Nucleic acids are essentially dynamic structures that influence important biological functions.3-5 Besides folding into canonical duplex structures, single-stranded DNA may form various noncanonical structures, such as hairpin, triplex, G-quadruplex (or G-tetra-plex), and i-motif structures. G-tetrad structure, defined by four Hoogsteen G-G base pairs (Figure 1a), was firstly noticed in 19106 and identified about fifty years later.7 G4s fold within G-rich tracts and consist of two or more stacked G-tetrads, being selectively stabilized by centrally coordinated potassium ions to O6 of the guanines at concentrations (10-50 mM) that are substantially below the 120 mM of KCl observed in most cells types.8-11 Stabilizing preference for monovalent cations follows the order K+ > Na+ > Li+.12 The intracellular monovalent cation concentration and the localized ion concentrations determine the formation of G4s and can potentially dictate their regulatory roles.13,14 G4s can be assembled in an intramolecular (backfolded) way or from two-, three-, or four DNA strands in intermolecular structures (Figure 1b) able to adopt a large diversity of conformations and folding energies.8 Most intramolecular G4 structures that are deposited in the public domain have been determined by nuclear magnetic resonance spectroscopy in solution.1,15 G4s are more compact structures than duplex DNAs and contain well-defined binding sites for selective recognition by small molecules. The presence and function of G4s in vivo are not quite clear.8 While consensus sequence for G4 folding is not experimentally established, approximately 370,000 G-rich sequences that contain putative G4-forming motif (PQS) are present in the human genome,8,16,17 dispersed throughout regulatory genomic regions (human telo-meres, oncogene promoter regions, immunoglobulin Mitrasinovic: G-Quadruplexes: Emerging Targets for 684 Acta Chim. Slov. 2020, 67, 683-700 switch regions, ribosomal DNA)18-21 and some regions of (C)-rich motifs is present in the human genome and capa-RNA.22,23 Because of the self-complementary nature of du- ble of folding into i-motif tetraplexes under slightly acidic plex DNA, approximately the same number of cytosine conditions (pH=6).8,16,17 The biological relevance of i-mo- Duplex G-quadruplex Figure 1. (a) G-tetrad structure. (b) Various G4 folding topologies. (c) One of several ways24'26-34 to affect the structural equilibrium between duplex and G4/i-motif is by small-molecule binding.8,27-29 Mitrasinovic: G-Quadruplexes: Emerging Targets for ... 685 Acta Chim. Slov. 2020, 67, 683-700 tif DNA in vivo is mainly unknown, but the possibility of been highlighted.8,24,25 When G-C rich sequences exist as a having i-motfs formed under physiological conditions due mixture of G4/i-motif and canonical duplex DNA in vitro, to molecular binding and/or crowding interactions has the structural equilibrium (Figure 1c) can be affected in external stacking intercalating groove binding - ON Telomerase Oncogene ON Teiomeric DNA ] Ligand TWIPyP4 Telomestatin Epiberberine Figure 2. (a) Different modes of noncovalent interaction between small molecules and G4. (b) Regulation of gene expression and/or inhibition of telomerase activity by G4 stabilization upon ligand binding.8 (c) Well-characterized G4 ligand structures having fused aromatic rings that are capable of stacking with the terminal G-tetrad. Mitrasinovic: G-Quadruplexes: Emerging Targets for ... 686 Acta Chim. Slov. 2020, 67, 683-700 different ways, using DNA binding proteins,26 small-molecule binding,27-29 negative supercoiling,29-33 changes in pH and temperature,34 and molecular crowding.24 Thus, cell-permeable and selective ligands may be viewed as potential tools for exploring the biological relevance and/or controlling the function(s) of one or more of these struc-tures.8 It is widely accepted that predicting or controlling quadruplex folding is a mainly intractable problem.35 G-rich DNA sequences are often intrinsically polymorphic in vitro and sensitive to pH variations, cation concentrations, or crowding conditions. An interest in the resolution of this issue is dictated by potential implementation of targeted design of quadruplexes in material, biotechnologi-cal, and therapeutic applications.35-37 Methodological advances at a much higher resolution and throughput in the identification and characterization of G4s in vivo as well as in vitro have well expanded the knowledge of G4 structure and function.38 Recent evidences have suggested involvement of G4s in key genome functions such as transcription, replication, genome stability, and epigenetic regulation, with many links to cancer biology.39,40 As far as folding topology (Figure 1b) is concerned, intramolecular G4 structures have been suggested to implicate in the regulation of gene expression and chromosome stability, while intermolecular G4s have been primarily seen as intermediates or precursors of recombination and/or viral integration.8 The mechanistic insights into G4 biology and protein interaction partners38,39 have helped to design and develop an arsenal of molecular and chemical tools for biomedical applications,38 with highlighting new opportunities for drug discovery.41 A growing number of predicted (either intramolecular or intermolecular) DNA/RNA quadruplex structures, being deposited into the public domain, enable the structure-based design of G4-interactive ligands on a continuous basis.42-46 Ligands with specificity toward certain G4s relative to others are useful for exploring the features and functions of individual G4s in the genome.44 Knowing that some small molecules directly bind to G4 and some others interfere with the binding between G4 structure and related binding proteins, tells that the insights into interaction with nucleic acids and into nucleic acid-protein interaction are very important.47 Most G4 studies consider only intramolecular G4 folding, but the potential prevalence of intermolecular DNA-RNA G4s in humans has been found by bioinformatics searches,48 indicating an urgent need for innovative research in order to be able to detect and characterize intermolecular G4 motifs in vivo.38 In other words, great experimental effort and robust analysis platforms are needed to reveal their structural conformational exchange with intramolecular G4s or other structural motifs, and their potential functions in cells,38 such as in transcription.48 A wide variety of experimental and computational methods are used to study biomolecular interactions. Experimental techniques include isothermal titration calo- Figure 3. Gene promoters (Ps) with G4 folds.43 c-Myc has one putative G4-forming sequence (PQS) and one nuclease hypersensitive element (NHE IIIj). VEGF has one PQS that is close to the transcription start site (TSS) and one hormone response element (HRE) for regulating the transcription (SP1 - specific protein). BCL2 has two G4-forming elements that attenuate the BCL2 promoter activity. c-Kit has two PQSs that interact with transcription factors (MAZ, SP1). hTERT has a few PQSs, where the presence of two tandemly positioned G4s is proposed. KRAS has three PQSs, of which PQS1 acts as a stronger transcriptional suppressor. c-Myb has several PQSs (MZF1 - protein). Mitrasinovic: G-Quadruplexes: Emerging Targets for ... 687 Acta Chim. Slov. 2020, 67, 683-700 rimetry (ITC), electrospray ionization-mass spectrometry (ESI-MS), X-ray crystallography, nuclear magnetic resonance (NMR), circular dichroism (CD), ultraviolet (UV) and fluorescence spectroscopies.49-55 Relevant computational approaches comprise virtual screening (VS), molecular docking, molecular mechanics (MM), molecular dynamics (MD), statistical thermodynamics, and bioinfor-matics.56-59 Taking into account that various conformations in static structures may be due to differences in experimental conditions or procedures, the applications of MD simulations have become particularly attractive,60-64 enabling to correlate substantial G4 domain motions and binding site rearrangements with complex formation.65-68 The structural heterogeneity, thermal stability and abundance of G4s in telomeres, oncogene promoter regions, and viral genomes make them appealing targets for future therapeutics. In this paper, an up-to-date survey of well-characterized G4s and G4-preferred ligands as potential targets and tools in anti-cancer and antiviral therapies is given. Effects that these ligands selectively exert in vitro and in vivo models without appreciably affecting normal cells are summarized. Unique ligands involved in specific G4 recognition through different modes of noncovalent interaction (Figure 2a) are put forward. Future perspective in conjunction with a daunting challenge - how to design small molecules that can bind selectively to each of the many possible G4 structures is, to some extent, addressed too. 2. G4-Preferred Ligands: Potential Tools in Anti-Cancer Therapy The visualization of G4s by immunofluorescence has been enabled by the development of specific antibodies with extremely high affinity to G4s, and has shown the presence of G4s not only in human single-stranded telo-meres, but also in duplex regions.44 G4-forming sequences are observed in the promoter regions of cancer-related genes such as c-Myc,69-72 VEGF,73 BCL2,74 c-Kit,75 hTERT,76 KRAS,77,78 and c-Myb79 (Figure 3). The ends of linear chromosomes are protected by telomeres from unwanted DNA processing events that influence genome stability. Human telomeric DNA consists of 5-30 kb tandem repeats of (TTAGGG)n, which end up by a single-stranded, from-35-to-600 bases long 3' overhang.1,80-82 Telomere binding proteins protect G-rich telo-mere repeat sequences that are prone to fold into G4 structures. Small energy differences between telomeric G4 structures have been generally observed.83,84 An intrinsic polymorphism of telomere DNA is particularly reflected through conformations adopted by the TTA loop seg-ments.85 The highly conserved telomeric sequence in higher eukaryotes means that potential formations of multiple G4s may be implicated in specific recognition of different structures by different proteins with the aim to con- trol biology.1 In the majority of human cancers cells as highly proliferative cells, telomeres are maintained by telo-merase. Telomerase and its telomere substrates are potential targets for developing novel anti-cancer drugs (Figure 2b). The discovery of the naturally occurring macrocyclic compound Telomestatin (Figure 2c) with telomerase-in-hibiting activity due to binding to telomeric G4 structures indicated the existence of G4s in vivo.44 Telomere function, besides the inhibition of telomerase, might be influenced by targeting telomeric G4-DNA. The importance of telomere structure and its position during telomerase function, as well as its associated binding proteins has been experimentally dissected.20 Many proteins that bind to double-stranded and/or single-stranded regions of the telomeric DNA make a nucleoprotein complex that maintains the structural integrity of telomeres in vivo. The effect of telomere destabilization due to small-molecule binding to DNA and consequent displacement of proteins from the complex is known as possible genotoxicity, being in relation to many G4 ligands. How this effect might be cancer-specific is unclear.8 Contrary to the formation of telomeric G4s in the single-stranded 3' overhang of telomeres, promoter G4s fold in the regions of double-stranded DNA. G4 formation in promoter regions is related to genes that are responsible for cell growth and proliferation (Figure 3). The clustering of putative G4-forming sequences (PQSs) is within 1 kb upstream of the transcriptional start site (TSS). The onco-gene promoters are typically TATA-less with G-rich regions in vicinal promoters. Unlike telomeric DNA, the PQSs are substantially more diverse and frequently have more than four G-tracts. A sequence is capable of forming multiple G4s through a wide variety of combinations of G-tracts or different loop isomers. The presence of the G3NG3 motif is a conspicuous feature that might have been naturally selected as a basis for G4 formation. Since the determination of c-Myc G4, parallel G4 folds have been commonly detected; most of them contain three G-tetrads and three loops (the first and the third are 1 nu-cleotide long, the middle loop is of variable length). In other words, each parallel G4 structure is likely to adopt unique capping and loop structures by way of its specific variable middle loop and flanking segments. The propensity of promoter sequences to form multiple and stable G4s at equilibrium is quite intriguing. For example, in the overlapping region of BCL2, the presence of two distinct con-formationally interchangeable G4s suggests a mechanism for the regulation of gene transcription through specific recognition of different G4 structures by different pro-teins.1 Many proteins with binding affinity to G4s have been identified.86 Modulation of gene expression may also be influenced using different small molecules in order to recognize distinct G4s. Thus, the targeting of G4s by small molecules, aimed to disrupt the interactions between G4s and their binding proteins, emerges as a potential anti-cancer strategy.61 Mitrasinovic: G-Quadruplexes: Emerging Targets for ... 688 Acta Chim. Slov. 2020, 67, 683-700 The different modes of noncovalent G4-ligand interaction include external stacking, intercalation, and groove/ loop binding (Figure 2a). Experimental87,88 and computational61 reports have identified the n-n stacking of ligand at the end of G4 as the most stable mode. Grooves/loops have been suggested to be viable binding sites of particular importance for blocking the interaction between G4 and its binding proteins in aqueous solution.61 The challenge of designing specific groove/loop binders stems from the groove/loop interaction mode dependence on the particular topology of groove/loop residues. However, grooves/loops offer distinct environments to gain specificity among many types of G4s by way of subtle variations of G4 topologies, groove widths, and loop sequences without affecting binding affinity.44,61 There are two distinct mechanisms to inhibit cancer growth through the selective stabilization of G4s by ligand molecules (Figure 2b). The first refers to the inhibition of the over-expression of oncogenes by promoter deactivation,8,89 while the second refers to the inhibition of telo-merase, a ribonucleoprotein complex that catalyzes the 3' extension of telomeric DNA.8,90-95 The second mechanism has been more extensively studied.8 Well-characterized G4-interactive ligands, BRA-CO-19, CX-3543 (Quarfloxin), TMPyP4, and Telomesta-tin that have modest binding affinities to duplex DNAs44 are given in Figure 2c. The chemical structure of BRA-CO-19 is composed of fused aromatic rings that are capable of stacking with the terminal G-tetrad and of three side chains that branch out of its heteroaromatic core. Many similar ligands (like CX-3543) have one or more cationic side chains that are inclined to interact with G4 grooves/ loops. An early idea that an optimal G4-preferred ligand structure contains large, planar, symmetric and cyclic rings, such as those of TMPyP4 and Telomestatin (Figure 2c) in order to maximize stacking interactions with the external G-tetrad has been closely associated with low specificity among intramolecular G4s.1 BRACO-19 is one of the most studied G4 ligands so far. The studies of BRACO-19 have greatly contributed to the treatment of telomeric G4s as potential therapeutic targets. BRACO-19 has shown high anti-cancer activities in vivo, such as in a UXF1138L uterus carcinoma xenograft and in a DU-145 prostate cancer xenograft. Despite all these favorable functional features, the lack of membrane permeability and small therapeutic window have been identified as the major limitations of BRACO-19, which must be resolved before any attempt to develop an effective clinical agent.42 Quarfloxin was the first-in-class ligand with considerable therapeutic window that had completed Phase II trials as a drug candidate, well-tolerated in patients against neuroendocrine tumors, carcinoid tumors, and lymphoma. Quarfloxin targets a G4 from the c-Myc promoter region to disrupt the G4-nucleolin complexes, and its G4-binding was reported as inhibiting RNA biogenesis. The Phase III of human cancer clinical trials is not currently proceeding due to high albumin binding.43 G4s and G4-interactive ligands as potential targets and tools in anti-cancer therapy are given in Table 1. Besides small molecules, G4-binding metal complexes have been recognized as promising anti-cancer drugs.96 In general, ligand-mediated stabilization of the G4 structure(s) effectively inhibits telomerase activity or oncogene over-expression and, when applied to cells, most G4 ligands initiate antiproliferative effect (apoptosis) and/or replicative senescence.8 Ni-P, Quercetin, TH3, IZCZ-3, Benzofuran derivative, and Furopyridazinone derivative cause negligible cytotoxicity to normal somatic cells in vivo. By avoiding many of the problems underlying the therapeutic use of oligonucleotides, the G-rich VEGFq oli-gonucleotide has contributed to a novel approach to specific inhibition of gene expression in vivo, which can be applied to the wide array of genes whose promoters contain quadruplex-forming sequences.97 The chemical structure of each ligand, underlined with its respective target topology is displayed in Figure 4. Among these fifteen li-gands, eleven prefer parallel, three prefer hybrid, and one prefers dimeric G4 binding. It is known that the induction of a quadruplex or change of a quadruplex conformation upon binding may be one of the most powerful methods to exert a desired biological effect.51 If a ligand selectively interacts with different G4 topologies, the particular ligand is expected to easily regulate the conformational switch by surpassing the energy barriers between distinct G4 structures in Na+ or K+ solution. An NMR structural analysis has revealed that a berberine derivative, epiberberine (Figure 2c), discriminates a hybrid type 2 telomere G4 from the other adoptable topologies and promoter G4s (c-Myc, BCL2, and PDGFR).98 Also the ability of epiberberine to convert the other conformations, such as telomere G4 hybrid type 1 and antiparallel (basket type) G4s, into the type 2 hybrid topology has been reported.43,98 It has been recently concluded that specific targeting of G4s by small molecules represents a promising strategy to study the function of targets inside a living cell without influencing their intact states.44 The way in which CM03 (Figure 4) has been designed to target multiple effector pathways in pancreatic ductal adenocarcinoma (PDAC) deserves more attention.99 The co-crystal structure of MM41 (Figure 4) with an intramolecular human telomeric parallel G4 has been the starting point for CM03 design. Even though the nature and structures of target G4s are unknown, NMR and crystal structures illustrate that some features are common to all G4s, particularly a core of stacked G-tetrads with small-molecule binding at the end of the core. The chromophore of MM41 has been somewhat asymmetrically stacked to the terminal G-tetrad. Indeed, one of the four substituent chains has not been positioned as the other three side chains with respect to G4 due to its orientation away from the G4 surface. The particular side chain has not been capable of making effective contacts with a G4 groove, so that its contribution to overall bind- Mitrasinovic: G-Quadruplexes: Emerging Targets for ... 689 Acta Chim. Slov. 2020, 67, 683-700 Table 1. G4 ligands with anti-cancer activities. Updated data reported previously.43 Target G4 topology Cell Line Cancer Type LigandRef' Effect telomere hybrid MDA-MB-231 MCF-7 breast cancer (adenocarcinoma) breast cancer (adenocarcinoma) Ni-p107,108 Cancer stem cell-specific apoptosis, bulk cancer-specific apoptosis and senescence, negligible cytotoxicity to normal somatic cells telomere dimeric G4s SiHa squamous cell carcinoma IZNP1109 Apoptosis, senescence telomere parallel A549 MCF-7 MIA PaCa-2 PANC-1 lung adenocarcinoma breast cancer (adenocarcinoma) pancreatic ductal adenocarcinoma MM4199 CM0399 Antiproliferative activity (apoptosis), BCL2 and KRAS as secondary targets c-Myc parallel HeLa cervical cancer Quercetin110 Apoptosis, mild cytotoxicity to normal cell line c-Myc parallel A549 HeLa lung cancer cervical cancer TH3111 Antiproliferative effect (apoptosis), negligible cytotoxicity to normal somatic cells c-Myc parallel SiHa HeLa Huh7 A375 squamous cell carcinoma cervical cancer liver cancer malignant melaoma IZCZ-3112 Antiproliferative effect (apoptosis), negligible cytotoxicity to normal somatic cells c-Myc parallel L363, MM1S, MM1R etc. myeloma Benzofuran derivative113 Antiproliferative effect (apoptosis), negligible cytotoxicity to normal cells c-Myc parallel HCT116 colorectal carcinoma Tz 1114 Apoptosis VEGF parallel A549 lung cancer VEGFq97 Autophagic apoptosis BCL2 hybrid Jurkat human acute T cell leukemia Furopyridazinone derivative115 Antiproliferative effect (apoptosis), negligible cytotoxicity to normal cells c-Kit parallel MCF-7 HGC-27 breast adenocarcinoma gastric carcinoma AQ1116 Antiproliferative effect (apoptosis) hTERT hybrid with stem loop MCF7 breast adenocarcinoma GTC365117 Apoptosis, senescence KRAS parallel HCT16 SW620 colorectal carcinoma Indoloquinoline derivatives118 Apoptosis c-Myb parallel MCF7 breast adenocarcinoma Topotecan119 Repressed expression, uncertain specificity ing has been minimal. Relative to MM41, an optimal compound has been hypothesized to contain three sub-stituents and to bind with similar affinity, as well as to have the advantage of lower molecular weight and reduced overall cationic charge. Thus, MM41 has been a suboptimal drug candidate due to its higher molecular weight and four positive charges, while CM03 has been an improved rationally designed derivative of MM41 and a novel lead candidate compound for potential therapy against human PDAC. Particular promoter G-quadru-plexes have not been assumed as targets. Global genome transcriptome profiling has been employed to determine which genes are affected by the rationally designed G4-interactive small molecule. Consequently, potential targets at the whole genome level in two pancreatic cancer cell lines have been determined. With in vitro cell assays and in vivo models for human PDAC, CM03 has been identified as a highly selective and potent G4-bind-ing ligand.99 The dynamics of noncovalent interaction between a structurally representative set of small molecules (BRA-CO-19, TMPyP4, CX-3543, 10074-G5, Telomestatin, Tet- Mitrasinovic: G-Quadruplexes: Emerging Targets for ... 690 Acta Chim. Slov. 2020, 67, 683-700 Figure 4. Chemical structures of G4 ligands (with denoted target topologies by italic) that exhibit anti-cancer activities (Table 1). Mitrasinovic: G-Quadruplexes: Emerging Targets for ... 691 Acta Chim. Slov. 2020, 67, 683-700 Figure 5. External stacking of BRACO-19, TMPyP4 and CX-3543 to apo (ligand-free) G4 from the c-Myc promoter in the stable regime of molecular dynamics (MD) simulation. In a representative set of structurally diversified ligands, these three molecules were established to have the highest affinity for G4. Only BRACO-19 was shown to be a thermodynamically favorable binder by increasing the conformational flexibility of G4 in the asymptotic (t ■ ■*>) regime of MD simulation.68 rahydropalmatine, Sanguinarine, Hoechst 33258, Benzo-phenanthridine derivative, Nitidine Chloride, Piperine, 12459, Quercetin, Quindoline, Berberine, and Flavopiri- dol) and a G-quadruplex formed in the c-Myc oncogene promoter region was recently explored in a systematic fashion from a rigorous biophysical point of view.68 In fact, OH Tetrahydropalmatine Sanguinarine Hoechst 33258 Figure 6. Groove binding of Tetrahydropalmatine, Sanguinarine and Hoechst 33258 to apo (ligand-free) G4 from the c-Myc promoter in the stable regime of molecular dynamics (MD) simulation. In a representative set of structurally diversified ligands, these three molecules were established to have the highest affinity for G4. Only Tetrahydropalmatine was shown to be a thermodynamically favorable binder by increasing the conformational flexibility of G4 in the asymptotic (t>«>) regime of MD simulation.68 Mitrasinovic: G-Quadruplexes: Emerging Targets for ... 692 Acta Chim. Slov. 2020, 67, 683-700 the thermodynamic consequences of apo (ligand-free) G4 conformational flexibility change upon ligand binding have been investigated in the asymptotic regime (t — of MD simulation, obtained by extrapolating the stable regime to infinitely long MD simulation. BRACO-19, TMP-yP4 and CX-3543 have shown the highest affinity to the G4 by stacking to the bottom G-tetrad of G4 (Figure 5). However, only BRACO-19 has been found to be a ther-modynamically preferable binder by increasing the con-formational flexibility of G4 (Figure 5), with a somewhat larger (by about 3 kcal mol-1) contribution to the additional flexibility of G4 from the sugar-phosphate backbone than from the complete system of nucleobases. In addition, Tetrahydropalmatine, Sanguinarine and Hoechst 33258 have exhibited the highest affinity to the target by groove binding (Figure 6). However, only Tetrahydropalmatine has been found to be a thermodynami-cally favorable binder by increasing the conformational flexibility of G4 (Figure 6), mainly through the complete system of nucleobases. Therefore, two distinct mechanisms by way of which small molecules interact with G4 are associated with increased conformational flexibility and increased conformational rigidity of apo G4 upon ligand binding respectively.68 Even though pure tetrad-binding mode is more stable than groove/loop binding mode, grooves/loops are viable binding sites that are of interest for the structure-based drug design. Grooves/loops with distinct environments help in tuning ligand specificity among many types of G4s without affecting binding affinity.44 Thus, multiple binding modes, which include external stacking and/or intercalation and/or groove/loop binding of two or more ligands simultaneously, have attracted certain attention.61 This type of binding is less stable than external stacking, likely due to the ability of groove/loop-binding ligands to induce loop rearrangements and destabilize the overall binding by displacing the interaction of the side chains of G-tet-rad-binding ligands with the grooves/loops of G4. There are indications that a combined - G-tetrad and groove binding of ligands enhances G4 conformational rigidity, reflected through the decreased conformational flexibility of both G-tetrads and the backbone.61 For rationalizing this aspect in the case of G4 from the c-Myc promoter region, a relevant structural basis was proposed to include two unique - thermodynamically preferred small molecules: the external stacking of BRACO-19 and the groove binding of Tetrahydropalmatine simultaneously.68 Binding sites defined by the surface features of the groove/loop regions can be used to stimulate selective binding interactions, even between closely related G4 structures.8 Subtle variations of G4 topologies, groove widths, and loop sequences are associated with a highly dynamic nature of G4 structures, which have propensity to lose conformational entropy upon ligand binding.101 This factor in determining specificity is important in order to distinguish G4s with lower ligand affinities that exist as a dynamic mixture of conformations in the unbound state (human telomere) from G4s that adopt a single conforma-tion.8 A small molecule, with binding affinity to increase the conformational entropy of G4 by stacking at the end of Figure 7. Proposal of lead candidate structure to interact with the c-Myc promoter G4 through external stacking and groove binding simultaneously. This proposal was based on HTVS experiments employing the key pharmacophore features of BRACO-19 for the search of the KEGG databases.100 Mitrasinovic: G-Quadruplexes: Emerging Targets for ... 693 Acta Chim. Slov. 2020, 67, 683-700 Table 2. G4s that are identified in viral genomes and their active ligands. Updated data reported previously.46 Virus Name Size (nm) Genome No. of G4sRef Active LigandsRef. Human Immunodeficiency Virus 1 HIV-1 0 120 (+)ssRNA 9.75 kb 12120 BRACO-19120-122 TMPyP4122'123 PIPER123 c-exNDI124 Nitidine Chloride105 Benzophenanthridine derivative105 Herpes Simplex Virus 1 HSV-1 0 125 dsDNA 152 kb 316125'126 BRACO-19126 c-exNDI127 Epstein-Barr Virus EBV 0120-180 dsDNA 172 kb 13125 BRACO-19128 PDS129 PhenDC3130 Kaposi's Sarcoma associated Herpes Virus KSHV 0 125 dsDNA 170 kb 52125'131 PhenDC3131 Human Herpes Virus 6 HHV-6 0 200 dsDNA 162 kb 43125 BRACO-19132 Hepatitis C Virus HCV 0 60 (+)ssRNA 9.6 kb 2133 TMPyP4133 PDP133 Human Papilloma Virus HPV 0 60 circular dsDNA 8 kb 8134 Zika Virus ZIKV 0 50 (+)ssRNA 11 kb 8135 Severe Acute Respiratory Syndrome Corona Virus SARS CoV 0 200 (+)ssRNA 30 kb Hepatitis B Virus HBV 0 42 partially circular dsDNA 3.2 kb 1136 TMPyP4136 PDS136 Ebola Virus EBOV cylindrical 0 80 (-)ssRNA 18.9 kb 1137 TMPyP4137 G4 from an oncogene promoter region, can be hypothesized as a unique, specific pharmacophore for the identification of new lead candidates by high-throughput virtual screening (HTVS).68,100 A lead candidate compound (Figure 7), predicted to recognize the c-Myc promoter G4 through external stacking and groove binding at the same time, was designed by HTVS experiments in combination with analog design.100 The key pharmacophore features of BRACO-19 have been used for the search of the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases in order to generate hit-to-lead candidates. Two crucial features rationalize the visible G4-stabilizing advantages of the concomitant external stacking and groove binding of the lead candidate over the external stacking of BRA-CO-19. The first is a flexible aromatic core of the lead candidate relative to a rigid one of BRACO-19. The second is a more polar surface of the lead candidate (by about 51 A2) than that of BRACO-19. The conformational flexibility of small molecules is generally more preferable compared to their locking in a presumed bioactive G4 conformation.100 Structure-based virtual screening and cell-based screening approaches, as well as biophysical and/or biological assays define an acceptable framework for the determination of completely new types of bioactive G4-interactive ligands.44,102 3. G4-Preferred Ligands: Potential Tools in Antiviral Therapy The presence of G4s in viruses has attracted more attention during the last few years. The viruses include those involved in recent epidemics, such as the Zika and Ebola viruses. Putative G4-forming sequences are usually locat- Mitrasinovic: G-Quadruplexes: Emerging Targets for ... 694 Acta Chim. Slov. 2020, 67, 683-700 PDS PhenDC3 Figure 8. Chemical structures of ligands that prefer G4s in viral genomes. ed in regulatory regions of the viral genomes and implicated in key viral processes; in some cases, their involvement in viral latency has been reported too.46 G4 ligands are tools that have been developed and tested in order to study the complexity of G4-mediated mechanisms in the viral life cycle. They have also been viewed as potential therapeutic agents.46 G4s that are identified in viral genomes, as well as their active ligands are summarized in Table 2. The chemical structures of the ligands are shown in Figure 8. Promising antiviral effects of G4 ligands have been generally related to G4-mediated mechanisms of action both at the genome level and at transcriptional level.46 G4-form-ing oligonucleotides as potential antiviral agents have been previously reviewed in great detail,103,104 so that they are not considered in the present review article. Experimental research based on ESI-MS, CD spectrometry, and DMS footprinting has indicated the formation of a G4 within a G-rich sequence that is located between -76 and -57 bp in the HIV-1 promoter.105 The CD melting experiment has also shown that, among eight natural small molecules (Nitidine Chloride - NC, Benzo- phenanthridine derivative - BPD, Jatrorrhizine, Tetrahy-dropalmatine, Toddalolactone, Coptisine, Piperine, and Astragalin), NC and BPD have the highest and nearly equal affinities to the HIV-1 promoter G4.105 The binding modes of NC and BPD have been elaborated using sophisticated computational methods,66 demonstrating that NC is a thermodynamically unfavorable binder by increasing the conformational rigidity of apo G4 and that BPD is a thermodynamically favorable binder by increasing the conformational flexibility of apo G4 in the asymptotic (t — ra) regime of MD simulation (Figure 9). In addition to HTVS methods or structure-based design with ahead-presumed features, fragment-based drug discovery (FBDD) may be a valuable approach to the generation of new pharmacophores that specifically recognize G4 structures. This approach is based on the generation of molecular fragment small libraries screened against the receptor in order to further synthetically elaborate them into lead compounds. For example, one of the heterocyclic molecules (Figure 10) has been shown to specifically recognize G4 from the HIV-1 long terminal repeat (LTR) pro- Mitrasinovic: G-Quadruplexes: Emerging Targets for ... 695 Acta Chim. Slov. 2020, 67, 683-700 Figure 9. Among eight natural small molecules, NC and BPD were shown to have the most pronounced (and mutually comparable) affinity for G4 from the HIV-1 promoter.105 NC is a thermodynamically unfavorable binder by increasing the conformational rigidity of apo G4, while BPD is a thermodynamically favorable binder by increasing the conformational flexibility of apo G4 in the asymptotic regime of MD simulation.66 X = N, Y = CH CH N Figure 10. One of the heterocyclic molecules was shown to prefer G4 from the HIV-1 LTR promoter region and to represent a potential pharmacophore for the development of novel ligands with unexpected chemical features. These compounds were developed using a FBDD approach.106 moter region and to represent a potential pharmacophore for the development of novel ligands with unexpected chemical features.106 Size and poor pharmacokinetics are the main obstacles in the development of G4-interactive ligand. FBDD can be a relevant approach to the development of compounds that have smaller sizes and more drug-like properties. 4. Conclusions and Future Perspective G-quadruplexes are naturally forming structures under physiological conditions, stabilized by monovalent cations present in cells. Over two hundreds quadruplex structures, either intramolecular or intermolecular, are currently deposited in the public domain such as the Pro- tein Data Bank. Most G4 studies consider only intramolecular G4 folding. However, the potential prevalence of intermolecular DNA/RNA G4s in humans has been indicated by bioinformatics searches. It means that innovative research is urgently needed with the aim to detect and characterize intermolecular G4 motifs in vivo, that is, their structural conformational exchange with intramolecular G4s or other structural motifs, and their potential functions in cells.38 The structural diversity, thermal stability and abundance of G4s in telomeres, onco-gene promoter regions, and viral genomes make them attractive targets for potential anti-cancer and antiviral therapies. Although the nature and structures of target G4s are unknown, NMR and crystal structures show that some features are common to all G4s; e.g. a core of stacked Mitrasinovic: G-Quadruplexes: Emerging Targets for ... 696 Acta Chim. Slov. 2020, 67, 683-700 G-tetrads with small-molecule binding at one of the ends of the core. One of the features has been recently revealed by molecular dynamics simulations,66,68 because distinct conformations that are often observed in static, experimentally determined structures may be the consequences of the differences in experimental conditions or procedures. The thermodynamic consequences of apo (li-gand-free) G4 conformational flexibility change upon complex formation have been observed in the asymptotic regime (t — of MD simulation. Two dissected mechanisms of G4-small molecule interaction are associated with increased conformational flexibility and increased conformational rigidity of apo target upon ligand binding, thereby being thermodynamically favorable and unfavorable respectively. A small molecule with binding affinity to increase the conformational flexibility of G4 through n-n stacking at the end of G4 can be conceivable as a unique, specific pharmacophore for designing novel lead candidate compounds by high-throughput virtual screening.66,68 Virtual screening has been demonstrated to be effective in reducing the initial number of potential candidates.44 In this way a lead candidate structure has been predicted to target a G4 from the c-Myc promoter region through external stacking and groove binding simultaneously.100 This approach would have useful implications for overcoming the challenge of designing specific groove/loop binders, which stems from the groove/loop interaction mode dependences on the particular G4 topologies, groove widths, and loop sequences. Therefore, the use of grooves/loops offers distinct environments aimed to gain specificity among many types of G4s without influencing binding af- finity.44 In contrast to HTVS methods or structure-based design with pre-set features, fragment-based drug discovery, which is based on the generation of molecular fragment small libraries screened against the receptor to further synthetically convert them into lead compounds, may be a valuable approach to the generation of new pharmacoph-ores that specifically recognize G4 nucleic acid structures. The sizes and poor pharmacokinetic properties of G4-in-teractive ligands are the main glitches in their development. By adding up fragments to singly recognize the target, FBDD can be seen as a relevant approach to the development of compounds that have smaller sizes and more drug-like properties.106 An appropriate framework for identifying totally new types of bioactive G4-interactive ligands is currently defined by structure-based virtual screening methods and cell-based screening approaches.44,106 Specific targeting of G4s by small molecules is and will be a promising tool for studying the behavior of targets inside a living cell without influencing their intact states.44 Particular promoter G4s should not be assumed as prior targets, indicating that single G4 promoter targeting strategy is not quite a suitable approach. In fact, the knowledge of potential targets at the whole genome level is need- ed. Global genome transcriptome profiling can be exploited for the determination of which genes are affected by a rationally designed G4-interactive small molecule. 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Mehanistične podrobnosti, kako in zakaj strukture G4 vplivajo na biološko funkcijo, kažejo na utemeljenost obravnave G4 kot potencialnih molekulskih tarč za bodoče terapevtike. Z drugimi besedami, strukturna heterogenost z natančno določenimi vezavnimi mesti, termična stabilnost in pogostnost G4 v telomerih, onkogenskih promotorskih regijah in virusnih genomih naredijo G4 za privlačne tarče za majhne molekule, katerih cilj je selektivno prepoznavanje med vsemi drugimi strukturami nukleinskih kislin, zlasti dupleksne oblike, ki so v genomu najbolj pogoste. V članku je predstavljen kritičen pregled dobro opisanih ligandov, ki interagirajo z G4, kot potencialnih orodij za zdravljenje raka in protivirusnih terapij. Učinki, ki jih ti ligandi selektivno izvajajo v in vitro in in vivo modelih, so povzeti. Predstavljeni so edinstveni ligandi, ki sodelujejo v specifičnem prepoznavanju G4. Ključno vprašanje, kako oblikovati in razviti nove G4 specifične ligande, ki ustrezajo strukturnim in fizikalno-kemijskim zahtevam za optimalno biološko aktivnost, je obravnavano ob upoštevanju izjemnega napredka v zadnjih nekaj letih in naših nedavnih prispevkov. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Mitrasinovic: G-Quadruplexes: Emerging Targets for ... DOI: 10.17344/acsi.2019.4986 Acta Chim. Slov. 2020, 67, 701-709 /^creative ^commons Scientific paper A Novel Electrochemical CuO-Nanostructure Platform for Simultaneous Determination of 6-thioguanine and 5-fluorouracil Anticancer Drugs Masoud Fouladgar1 1 Department of Biochemistry, Falavarjan Branch, Islamic Azad University, Isfahan, Iran. * Corresponding author: E-mail: Fouladgar@iaufala.ac.ir; Tel: +98 9131295016 Received: 01-23-2019 Abstract Analysis of anticancer drugs is very important and necessary for the correct administration of them in the human body. Electrochemical behavior of 6-thioguanine (6-TG) has been studied using a carbon paste electrode modified by 1-ethyl-3-methylimidazolium tetrafluorob orate (ionic liquid) (1E3MIBF4) and CuO nanoparticles (CuO/1E3MIBF4/ CPE). Using square wave voltammetry showed the linear relation between net anodic current and concentration of 6-TG in the range of 70 nmol L-1 to 520 |imol L-1 6-TG with the detection limit of 20 nmol L-1 6-TG. The proposed modified electrode had excellent repeatability (RSD = 1.31%, n = 5) and long term stability (2.9% deviation in 25 days). The diffusion coefficient of 6-TG on the CuO/1E3MIBF4/CPE was found to be 1.54 x 10-5 cm2s-1 .The CuO/1E3MIBF4/ CPE was successfully applied for the determination of 6-TG in real samples. In addition, the anodic peaks of 6-TG and fluorouracil (5-FU) in their mixture can be well separated using CuO/1E3MIBF4/CPE and simultaneous determination of them was studied. Keywords: 6-Thioguanine; 5- Fluorouracil; CuO nanoparticles; 1-ethyl-3-methylimidazolium tetrafluoroborate; voltam-metry 1. Introduction 6-Thioguanine (6-TG) is a common anticancer and antitumor drug which is an analogue of the physiological purines, guanine and hypoxanthine. In addition, thiogua-nine has been applied for treatment of hematological malignancies, psoriasis and inflammatory bowel disease, such as Crohn's disease. It has interaction with DNA and RNA and using it may have several side effects. Its main side effects are on the liver and it can cause hemotoxicity as well.1 Oral administrated of 6-TG is poorly absorbed and about 30% of administered dose being bioavailable. In hepatic metabolism of 6-TG, a methyl group is added to the sulf-hydryl group of 6-TG.2 5-Fluorouracil (5-FU) is an antimetabolite agent same as 6-thioguanine that is being used for treatment of cancer. This group of drugs disrupts nucleic acid synthesis and is toxic to normal cells. It is a fluorinated pyrimidine and inhibits synthesis of DNA by blocking thymidylate synthetase. It is used in treatment of small tumors for which surgery is contraindicated. Particularly it is em- ployed for the treatment of metastatic carcinomas of the breast, gastrointestinal tract, head and neck, and pancreas. Administered 5-Fluorouracil undergoes hepatic metabolism and about 10% of administered dose excretes unchanged in urine.2 Both 6-TG and 5-FU are in the list of World Health Organization's List of Essential Medicines.3 Control of the adverse side effects of drugs and determination the pharmacokinetics properties are the important reasons to measurement the drugs in pharmaceutical samples and in biological samples too. In this regard many analytical methods have been reported to analysis drug samples including electrochemical methods.4,5 Many modified electrochemical sensors have been suggested for determination and studying interactions of 6-TG. Madue-no et al. have been studied electrochemical oxidation of this anticancer drug (6-TG) including adsorption and phase formation on the mercury electrode.6 Wang et al. could measure 6-TG by modified gold electrode with DNA. Potassium ferricyanate was used as an electroactive indicator to probe the interaction between 6-TG and DNA.7 Ensafi et al. used the ability of 6-TG to form com- Fouladgar: A Novel Electrochemical CuO-Nanostructure 702 Acta Chim. Slov. 2020, 67, 701-709 plex with Cu(II) and cathodic striping on the mercury electrode.8 Beitollahi et al. reported application of 2,7-bis (ferrocenyl ethyl)fluoren-9-one as a modifier of carbon paste electrode. They determined 6-TG based on electro-catalytic effect of modifier.9 Eksin et al. studied interaction between 6-TG and ss-DNA on the pencil graphite electrode and obtianed data confirmed interactions between 6-TG and ss-DNA.10 Beitollahi et al. reported determination of 6-TG and folic acid using amplified sensors with ZnO-CuO nanoplates and 2-chlorobenzoyl ferrocene.11 To improve the analytical features of the electrochemical methods, applied electrodes have been modified with different materials.12-14 In recent years, nanomaterials have been widely used in electrochemical analysis methods.15-20 Electrochemical sensors amplified with conductive materials help to improving sensitivity of electroactive materials sensors and also increase the diversity of them.21-26 In between, nanomaterials with different and unique properties showed more attention for modification of electrochemical sensors.27-31 One of the effects of application of nanomateri-als is due to the change in the active surface area of elec-trodes.32-34 This effect and some other effects that appear when the size of particles decreases to nanoscale, cause to improve performance of electrochemical methods.35 However, application of nanoparticles may have disadvantage effect on the electrochemical signals and increases background currents. Copper oxide nanoparticles are semiconductor metal that not only have special electrical and magnetic properties but also have great biological properties including effective antimicrobial action. Wide applications of CuO nanoparticles has caused significant advance in synthesis approaches of CuO nanoparticles.36 Zeta potential values of CuO nanoparticles are negative or positive depending on pH of solution and can effect adsorption of elec-troactive species and improve electrochemical signals.37 In addition, using electrically conducting liquids especially ionic liquids in the structure of paste electrodes, improves the sensitivity of the electrodes.38-46 1-eth-yl-3-methylimidazolium tetrafluoroborate is a room temperature ionic liquid that has suitable electrochemical stability for voltammetric aspects. It has wide voltage range of the electrochemical window, which allows electrochemical studies on various electroactive compounds.47,48 In this work, synthesized CuO nanoparticles and 1E3MIBF4 were used for amplification of modified sensor. Composition of CuO/1E3MIBF4/CPE was optimized and CuO/1E3MIBF4/CPE was suggested to determine 6-TG in real samples. In addition, simultaneous determination of 6-TG and 5-FU was investigated using modified electrode. 2. Experimental 2. 1. Materials and Devices All chemical compounds (6-thioguanine, 5-fluoro-uracil, phosphoric acid, 1-ethyl-3-methylimidazolium tetrafluoroborate, copper(II) acetate, paraffin oil, sodium hydroxide and graphite powder) were purchased from Sigma-Aldrich Company in analytical grade and they were used as received without any further purification. Ultrapure water (18.2 MO cm, Mili-Q) was used for preparation of solutions. Phosphate buffer solutions were prepared by mixing adequate amounts of 0.1 mol L-1 sodium dihydrogen phosphate and 0.1 mol L-1 sodium hydrogen phosphate solutions. To prepare 6-TG standard solution (1 x 10-3 mol L-1), adequate amount of 6-TG was dissolved in warm (35-40 °C) 1:1 (v/v) water-ethanol solution. Electrochemical measurements were executed by Autolab PGSTAT 101 potentiostat/galvanostat (Metrohm, Netherlands) in a conventional electrochemical cell (50 ml). An Ag/AgCl electrode and a platinum wire electrode were applied as reference electrode and counter electrode, respectively. CuO/1E3MIBF4/CPE was used as working electrode. 2. 2. Real Sample Preparation To prepare the tablets sample, five tablets were exactly weighed. Then the tablets were grinded and were completely homogenized. Then a required amount of the powder was transferred to the 100 ml beaker and about 80 ml of warm (35-40 °C) 1:1 (v/v) water-ethanol solution was added. The mixture was stirred magnetically and ul-trasonicated (15 min) till the powder was dissolved. Afterward, the solution was filtered by filter paper and transferred to a flask and diluted to 100 ml with water-ethanol solution. For electrochemical measurement, adequate amount of resultant solution was transfer to electrochemical cell containing 10 ml of phosphate buffer solution (pH = 7.0). The spiked dextrose-saline solution was prepared by mixing dextrose-saline solution with the same volume of phosphate buffer solution (pH = 7.0). Then 10 ml of resultant solution was added to the electrochemical cell and adequate amount of standard solution of 6-TG was added. 2. 3. Nanoparticle Synthesis 200 ml of 0.2 mol L-1 copper(II)acetate mixed with 2 ml acetic acid solution and mixture was heated until it came to boil. Then 30 ml of 0.8 mol L-1 NaOH were added to the mixture. The color of the solution changed from blue to black. Afterwards, the mixture was boiled for 2 h. After cooling the mixture in the air, it was centrifuged into solid and water and the obtained solid was separated and washed. 2. 4. Fabrication of CuO/1E3MIBF4/CPE The composition of CuO/1E3MIBF4/CPE was optimized and optimum composition contained 10% 1E Fouladgar: A Novel Electrochemical CuO-Nanostructure 703 Acta Chim. Slov. 2020, 67, 701-709 3MIBF4 and 6% nanoparticles. Accordingly, a mixture including 10% 1E3MIBF4 as ionic liquid, 6% CuO nanoparticles and 84% graphite powder was prepared. About 1 ml of diethyl ether was added to the mixture and the mixture was mixed until a uniform mixture obtained. After vaporization of diethylether, a suitable amount of viscose paraffin was added to the mixture and components were mixed and the obtained paste was inserted into the glass tube in the presence of copper wire. 2. 5. Recommended Procedure Prepared modified electrode was polished with a white and clean paper. To measure blank signal, ten milliliters of buffer solution (pH = 7.0) were transferred to the electrochemical cell. Then, the square wave voltammo-gram was recorded from 0.35 to 1.25 V V vs. Ag/AgCl (Frequency 10 Hz). Afterward, different amounts of standard solutions of 6-TG and/or 5-FU were added to the cell and square wave voltammograms were recorded again to get the analytical signal. The difference between the blank and the analytical signal was obtained as a net peak current. Calibration plot was constructed by plotting net currents vs. concentration of drugs. 3. Results and Discussion 3. 1. Investigation of Synthesized Nanoparticles Scanning electron microscopy (SEM) of synthesized nanoparticles confirmed synthesis of uniform spherical particles with nanoscale size (Figure 1.a). In addition, obtained Energy-dispersive X-ray (EDX) spectrum from synthesized nanoparticles confirmed the existence of only oxygen and copper in the composition of nanoparticles (Figure 1.b). 3. 2. pH effect According to the previous electrochemical reported papers for analysis of 6-TG,49 we guessed the pH dependent electro-oxidation mechanism for determination of 6-TG at a surface of electrode. Therefore, linear sweep voltammograms of 6-TG (100 ^mol L-1) were recorded in the pHs range of 5-8. As can be seen in Figure 2 (inset), increasing pH of solution causes the shift of oxidation peak potential to negative potentials. The slope of plot of potential versus pH was -61.2 mV/pH, which is close to anticipated Nernstian value (Figure 2). Consequently, this indicates that the electro-oxidation of 6-TG occurred in the presence of equal value of proton and electron. The obtained result agrees with the suggested mechanism for electro-oxidation of 6-TG (Scheme 1).49 a) RC b) c,i Cu 0 1 2 3 4 5 6 7 Cu Ul ............ 7 8 9 10 11 12 keV Figure 1. a) SEM image b) EDX spectrum of synthesized CuO nanoparticles 3. 3. Effect of Modification In this step, we investigated the synergic effect of modifiers on the 6-TG electro-oxidation signal by recording the linear sweep voltammograms 6-TG (100 ^mol L-1) at a surface of CuO/1E3MIBF4/CPE (curve a), 1E3MIBF4/ CPE (curve b), CuO/CPE (curve c) and CPE (curve d). As can be seen in Figure 3, addition of CuO nanoparticles caused increasing oxidation current and shifting peak potential toward lower potentials. Addition of ionic liquid into the carbon paste had similar effects. Synergy between effects of addition of nanoparticles and ionic liquid caused to achieve maximum peak current and lower overpotential (Figure 3. a). In addition, the current density in- Fouladgar: A Novel Electrochemical CuO-Nanostructure 704 Acta Chim. Slov. 2020, 67, 701-709 Figure 2. Plot of potential vs. pH for the electrooxidation of 100 |rmol L 1 6-TG at CuO/1E3MIBF4/CPE. Inset: Linear sweep voltammograms of 100 |rmol L-1 6-TG with different pHs (scan rate = 100 mV s-1). NH2 ? \ -SH -2e -2H+ HN^N NH2 Scheme 1. Proposed mechanism for oxidation of 6-TG on the electrode. \ / -s—s- \\ //' h2N Figure 3. Linear sweep voltammograms of (a) CuO/1E3MIBF4/CPE, (b) 1E3MIBF4/CPE, (c) CuO/CPE, and (d) CPE in the presence of 100 |rmol L-1 6-TG at pH 7.0. Inset: the current densities derived from voltammograms responses at same electrodes. Fouladgar: A Novel Electrochemical CuO-Nanostructure 705 Acta Chim. Slov. 2020, 67, 701-709 creased by moving CPE to CuO/1E3MIBF4/CPE (Figure 3. inset). These effects may be relative to the conductivity effect of CuO nanoparticles and 1E3MIBF4 at a surface CPE. In addition, the active surface area of modified and non-modified electrodes CuO/1E3MIBF4/CPE, 1E3MIBF4 /CPE, CuO/CPE and CPE was obtained based on the Ran-dles-Sevcik equation (in the presence of 1 mM K4Fe(CN)6).32 The active surface areas of CPE, CuO/CPE, 1E3MIBF4/CPE and CuO/1E3MIBF4/CPE were calculated equals; 0.28, 0.31, 0.32 and 0.33 cm2, respectively. 3. 4. Electrochemical Investigations Linear sweep voltammograms of 6-TG (300 ^mol L-1) at CuO/1E3MIBF4/CPE were recorded in the scan range between 10-100 mVs-1 (Figure 4-a insert). Linear relation between peak currents and square root of scan rates confirmed that the electrode process was controlled under the diffusion step. In addition, a kinetic limitation can be observed in this investigation due to shifted oxidation peak potential toward positive value. Also, the value of charge transfer coefficient (a) was obtained ~ 0.8 using slope of Tafel plot (Figure 4-b). Chronoamperometry was also employed for investigation of 6-TG (300 and 500 ^mol L-1) electro-oxidation at CuO/1E3MIBF4/CPE by applying single potential step 800 mV at CuO/1E3MIBF4/CPE. From the slopes of plots of I (current) versus t-112 (Figure 5), the average of diffusion coefficient of 6-TG was found to be 1.54 x 10-5 cm2s-1 (Cottrell equation). Since electrode reaction is diffusion-controlled, anodic current is controlled by diffusion and hence depends on the diffusion coefficient. Modification of electrode causes increasing diffusion coefficient which in turn leads to increasing anodic current. a) 6 - 5 * < 4- 3 - 2 - 3 b) 0.59 0.57 0.55 ^ 0.53 i 0.51 0.49 y = 0.7274X - 0.9827 R5=0.9989 0.5 1.0 El V 6 7 8 vV2 / (mV/s)1'2 10 11 y= 0.1S12X+ 1.4410 R2 = 0.9953 0.47 0.45 0.3 0.4 0.5 0.0 0.7 0.0 0.9 1.0 EI V -6.5 -6.4 -6.3 — -6.2 -6.1 -i- -6.0 —i— -5.9 —i— -5.3 —i -5.7 log I Figure 4. a) The Plot of Ipa vs. v1/2 for electro-oxidation of 300 |imol L-1 6-TG (pH = 7.0). Insert; linear sweep voltammograms of CuO/1E3MIBF4/ CPE containing 300 |imol L-1 6-TG at various scan rates; a-d correspond to 10, 20, 50 and 100 mVs-1, respectively. b) Tafel plot for 300 |imol L-1 (pH = 7.0) 6-TG at CuO/1E3MIBF4/CPE. Inset: Corresponding cyclic voltammogram Fouladgar: A Novel Electrochemical CuO-Nanostructure 34 Acta Chim. Slov. 2020, 67, 701-709 Figure 5. Chronoamperograms obtained at CuO/1E3MIBF4/CPE in the presence of a) 300;and b) 500 |imol L 1 of 6-TG in the buffer solution (pH 7.0). Inset: Cottrell's plot for the data from the chronoamperograms. 4. Analytical Features In order to obtain calibration curve, square wave vol-tammograms (SWV) of solutions with different 6-TG concentrations were recorded at CuO/1E3MIBF4/CPE. Plot of net oxidation peak current versus concentration was linear in the range of 70 nmol L-1 to 520 ^mol L-1 6-TG with the regression equation being Ip(^A) = 0.076Q_yG + 0.174 (R2 = 0.998) and detection limit of the method 20 nmol L-1 6-TG (3Sb/m). This value of linear dynamic range or limit of detection compared with previous electrochemical sensor and results showed better analytical ability for proposed sensor (Table 1). The relative standard deviation for square wave signals of 25 ^mol L-1 6-TG at the surface of CuO/1E3MIBF4/ CPE was 1.31% (n = 5), which confirmed excellent repeatability. The stability of CuO/1E3MIBF4/CPE was checked by recorded square wave voltammograms of 25 ^mol L-1 6-TG over a period of 25 days. Compared to its first oxidation current, only 2.9% deviation was recorded when CuO/1E3MIBF4/CPE was used daily and stored in the laboratory. This suggests that CuO/1E3MIBF4/CPE possesses long-term stability. Table 2. Interference study for analysis of 50 |imol L-1 6-TG Species Tolerance limits (mole ratio) Glucose 8SO Na+, Br-, Cl- , K+ , Ascorbic acid* SSO Phenyl alanine, Glycine, Methionine 4OO Starch Saturation * After addition of 1 mmol L 1 ascorbic oxidize Table 1. Characteristics of several electrochemical methods for determination of 6-TG Technique Electrochemical Method Linear dynamic range Detection limit Ref. Study self-assembled monolayer of 6-TG on mercury electrode DNA-modified gold electrode Complex formation and adsorption on mercury electrode Electrocatalyst Study interaction between 6-TG and DNA Electrocatalyst and using ZnO-CuO nanoplates Modification of carbon paste with CuO nano particles and ionic liquid cyclic and ac voltammetry differential pulse stripping voltammetry cathodic adsorptive stripping differential pulse voltammetry differential pulse voltammetry, electrochemical impedance spectroscopy square wave voltammetric square wave voltammetric 2 x 1O-8- S x 1O-7 mol L-1 O.1S-1SO nmol L-1 O.O6-1O |imol L-1 and 1O-16O |mol L- 6 x 1O-9 mol L-1 7 O.OS n mol L-1 8 22 nmol L-1 SO O.OS to 2OO |imol L-1 2S n mol L-1 11 O.O7 to S2O |imol L-1 2O n mol L-1 this wok 6 1O Fouladgar: A Novel Electrochemical CuO-Nanostructure 707 Acta Chim. Slov. 2020, 67, 701-709 Table 3. Determination of 6-TG in real samples (n = 5) Sample Added Expected Founded Recovery Published method (^mol L-1) (^mol L-1) (^mol L-1) % (^mol L-1) Tablet* - 5 4.92 ± 0.35 98.4 4.95 ± 0.28 10 15 15.63 ± 0.75 104.2 15.74 ± 0.98 Intravenous - - < Limit of detection solution** 20 20 20.75 ± 0.82 103.7 19.75 ± 1.01 ±Shows the standard deviation * Kwality Pharmaceuticals limited, India. "Dextrose (3.33 %), saline (0.3 %), I.P.P.C.(Iranian Parenteral & Pharmaceutical Co), Tehran, Iran. To study the influence of various substances which may potentially interfere with the determination of 6-TG, the oxidation current of 50 ^mol L-1 6-TG was measured in the presence of different concentrations of interfering species and was compared with current that obtained from 6-TG solution by acceptable error ±5%. The results are shown in Table 2 and confirm selectivity of CuO/1E-3MIBF4/CPE for the analysis of 6-TG. To study the application of CuO/1E3MIBF4/CPE for analysis of 6-TG in real samples, the CuO/1E3MIBF4/CPE was applied for the determination of 6-TG in tablets and intravenous dextrose-saline solutions (Table 3). In addition, changing the concentration of each one had no effect on the peak current of another one. Therefore, simultaneous determination was performed by simultaneously changing the concentrations of 6-TG and 5-FU and recording the SWVs. Figure 6 shows the calibration curves of 6-TG and 5-FU. The current sensitivities towards 6-TG in the presence and in the absence of 5-FU were found to be approximately equal which confirms that the oxidation processes of 6-TG and 5-FU at CuO/1E3MIBF4/CPE are independent and simultaneous or independent measurements of two compounds are, therefore, possible without any interference. 5. Simultaneous Determination of 6-TG and 5-FU Square wave voltammogram of a solution containing 6-TG and 5-FU showed two distinguished peak currents. 6. Conclusion As a conclusion, we fabricated a novel electrochemical modified sensor amplified with CuO nanoparticles and 1E3MIBF4 for the determination of 6-TG in the presence Figure 6. Inset; SWVs of CuO/1E3MIBF4/CPE ( PBS buffer, pH 7.0) containing different concentrations of 6-TG and 5-FU in jimol L"1. (a-e) 50 + 200; 100 + 300; 200 + 400; 285 + 500 and 500 + 600, respectively. B) plot of the current as a function of 6-TG concentration. C) plot of the current as a function of 5-FU concentration. Fouladgar: A Novel Electrochemical CuO-Nanostructure 708 Acta Chim. Slov. 2020, 67, 701-709 of 5-FU, as two important anticancer drugs. The CuO/1E-3MIBF4/CPE showed good analytical ability for nanomolar determination of 6-TG. The CuO/1E3MIBF4/CPE resolved overlapping signal of 6-TG and 5-FU at an optimum condition. The CuO/1E3MIBF4/CPE was used for the analysis of 6-TG in real samples. 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Mater. 2010, 183, 402-409. DOI:10.1016/j.jhazmat.2010.07.039 Fouladgar: A Novel Electrochemical CuO-Nanostructure 709 Acta Chim. Slov. 2020, 67, 701-709 40. M. Fouladgar, Measurement 2016, 86, 141-147. D01:10.1016/j.measurement.2016.02.057 41. Y. Zhang and J. B. Zheng, Electrochim. Acta 2007, 52, 72107216. D0I:10.1016/j.electacta.2007.05.039 42. W. Sun, Y. Li, M. Yang, S. Liu and K. Jiao, Electrochem. Commun. 2008, 10, 298-301. D0I:10.1016/j.elecom.2007.12.012 43. S. Negahban, M. Fouladgar and G. Amiri, J. Taiwan Inst. Chem. Eng. 2017, 78, 51-55. D0I:10.1016/j.jtice.2017.05.032 44. M. Ashjari, H. Karimi-Maleh, F. Ahmadpour, M. Shaba-ni-Nooshabadi, A. Sadrnia and M. A. Khalilzadeh, J. Taiwan Inst. Chem. Eng. 2017, 80, 989-996. D0I:10.1016/j.jtice.2017.08.046 45. M. Fouladgar, J. Electrochem. Soc. 2016, 163, B38-B42. D01:10.1149/2.0611603jes 46. M. Fouladgar, Sens. Actuators, B 2016, 230, 456-462. D0I:10.1016/j.snb.2016.02.094 47. M. Shamsipur, A. A. M. Beigi, M. Teymouri, S. M. Pourmor-tazavi and M. Irandoust, J. Mol. Liq. 2010, 157, 43-50. D0I:10.1016/j.molliq.2010.08.005 48. J. Fuller, R. T. Carlin and R. A. Osteryoung, J. Electrochem. Soc. 1997, 144, 3881-3886. D0I:10.1149/1.1838106 49. P. Kraske, J. Electroanal. Chem. Interfacial Electrochem. 1986, 207, 101-116. DOI: 10.1016/0022-0728(86)87065-6 50. H. Karimi-Maleh, M. R. Ganjali, P. Norouzi and A. Banan-ezhad, Mater. Sci. Eng., C 2017, 3, 472-477. D0I:10.1016/j.msec.2016.12.094 Povzetek Analiza protirakavih zdravil je zelo pomembna in potrebna za njihovo pravilno uporabo v človeškem telesu. Preučevali smo elektrokemijsko obnašanje 6-tioguanina (6-TG) z uporabo elektrode iz ogljikove paste, modificirane z 1-etil-3-metil-imidazolijevim tetrafluoroboratom (ionska tekočina) (1E3MIBF4) in nanodelci CuO (CuO/1E3MIBF4/CPE). Uporaba square wave voltametrije je pokazala linearno zvezo med celotnim anodnim tokom in koncentracijo 6-TG v območju od 70 nmol L-1 do 520 |imol L-1 6-TG z mejo zaznave 20 nmol L-1 6-TG. Predlagana modificirana elektroda je imela odlično ponovljivost (RSD = 1,31 %, n = 5) in dolgoročno stabilnost (2,9 % odstopanje v 25 dneh). Ugotovljeno je bilo, da je koeficient difuzije 6-TG na CuO / 1E3MIBF4/CPE 1,54 x 10-5 cm2s-1. CuO/1E3MIBF4/CPE je bil uspešno uporabljen za določanje 6-TG v realnih vzorcih. Poleg tega je mogoče anodne vrhove 6-TG in fluorouracila (5-FU) v njuni mešanici dobro ločiti z uporabo CuO/1E3MIBF4/CPE in ju preučevati istočasno. g)® Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Fouladgar: A Novel Electrochemical CuO-Nanostructure DOI: 10.17344/acsi.2019.5301 Acta Chim. Slov. 2020, 67, 710-719 /^creative ^commons Scientific paper Metoprolol: New and Efficient Corrosion Inhibitor for Mild Steel in Hydrochloric and Sulfuric Acid Solutions Fatemeh Mohammadinejad,1 Seyyed Mohammad Ali Hosseini,2^ Mehdi Shahidi Zandi,3 Mohammad Javad Bahrami4 and Zahra Golshani5 1Department of Chemistry, Shahid Bahonar University of Kerman, P.O. Box: 76169-14111, Kerman, Iran. 2 Department of Chemistry, Shahid Bahonar University of Kerman, P.O. Box: 76169 14111, Kerman, Iran. 3 Department of Chemistry, Kerman Branch, Islamic Azad University, P.O. Box: 7635131167, Kerman, Iran. 4 Department of Science, Farhangian University, P.O. Box: 76175173, Kerman, Iran. 5 Department of Chemistry, Shahid Bahonar University of Kerman, P.O. Box: 76169 14111, Kerman, Iran. * Corresponding author: E-mail: s.m.a.hosseini@uk.ac.ir Tel: 0913-3412012 Received: 06-08-2019 Abstract The inhibition behavior of metoprolol tablet on steel alloy (st37) in 1 M hydrochloric acid and 0.5 M sulfuric acid solutions were studied by three methods (potentiodynamic polarization, electrochemical impedance spectroscopy, and scanning electronic microscopy, SEM). The obtained parameters revealed that different amounts of metoprolol drug inhibited the corrosion of mild steel in the acid solutions of HCl and H2SO4. The corrosion resistance of the alloy increased with the increase in the concentration of metoprolol up to 300 ppm but was reduced by increasing the temperature. The derived parameters from polarization curves indicated that the drug is a mixed type inhibitor. The results obtained from the different methods are consistent with each other. The adsorption of metoprolol was found to be physical, exothermic, and spontaneous, and also fitted the Langmuir adsorption model. SEM micrographs are in accordance with the adsorption performance of the tablet. Keywords: Corrosion Inhibitor; potentiodynamic Polarization; electrochemical Impedance Spectroscopy; mild steel; metoprolol. 1. Introduction Corrosion is the dissolution of metals and alloys exposed to aggressive media and is very difficult to eliminate completely. Inhibition of the corroding material would be more achievable than complete elimination. Demolition of metal and alloys development could be rapid after destruction of the protective film which depends on the composition and character of metals and aggressive media. For example, formation of oxides and diffusion of metal cations into the coating matrix, local pH changes, and electrochemical potential.1 Mild steel is an abundant and efficient building material. However, it is difficult to protect this alloy exposed to harsh environments against corrosion.2 Acidic solutions are extensively utilized in different industries for various purposes, including acid pickling, acid descaling, and oil well acidizing.3 Due to the general aggressiveness of acid media, the use of effective inhibitors is one of the reliable and economical ways for minimizing the corrosion rates and the protection of metal surfaces against corrosive media.4-6 Thus, reliable inhibitors for the corroding materials in reducing acids (hydrochloric acid and sulfuric acid) have attracted the wide attention of researchers.5 Organic compounds with n-bonds and heteroatoms (P, S, N, and O) are highly effective and available inhibitors, but most of them are expensive and toxic with Mohammadinejad et al.: Metoprolol: New and Efficient Corrosion Inhibitor Acta Chim. Slov. 2020, 67, 710-719 711 negative effect on the environment.7-12 These properties restrict their use to reduce the corrosion of metals and alloys. Therefore, it is very important and necessary to find out the least expensive and environmentally safe corrosion inhibitors.13,14 Earlier report15 indicates that the use of drugs for the protection of metals corrosion offers some advantages in comparison to the use of some organic/inorganic inhibitors due to their eco-environmental nature. Most of the drugs are nontoxic and less expensive with limited negative effects on different media, so they are suggested to replace the traditional toxic corrosion inhibi-tors.16 Thus, in the present investigation the effect of me-toprolol tablet on the corrosion resistance of mild steel in 1.0 M hydrochloric acid and 0.5 M sulfuric acid solutions was done. The selection of this drug as corrosion inhibitor is done on the ground of its low toxicity and high solubility in acidic media. Electrochemical impedance spectroscopy (EIS), potentiodynamic polarization, and scanning electronic microscopy (SEM) methods were employed to evaluate corrosion rate of the steel and inhibition efficiency of the drug. mersed in the solutions for 30 min (to get stable OCP) before each EIS test. 2. 2. Procedures 2. 2. 1. Electrochemical Measurements: Potentiodynamic Polarization and Electrochemical Impedance Spectroscopy (EIS) The electrochemical measurements were carried out with an Autolab potentiostat/galvanostat (AUTOLAB-302N, Netherlands) by using a three-electrode cell containing Ag/ AgCl as reference (with 0.197 mV), Pt electrode as counter and mild steel as working electrode used for this investigation. The potential range for potentiodynamic polarization tests was -850 to -200 mV and the scan rate was 1 mV/s. The frequency range for EIS tests was 100 mHz to 100 kHz and the EIS amplitude was 10 mV peak to peak. Before each polarization and impedance test, potential was stabilized within 30 min at 25 ± 1 °C. Finally, all of the curves were analyzed with Nova software (Utrecht, The Netherlands). 2. Experimental 2. 1. Materials and Solutions Metoprolol tablets are commercially obtained as a trade name Lopressor, Toprol-xl by Toliddaru Company. The compound in its purest state has the molecular formula of C15H25NO3 and melting point 120 °C. No attempt was made to eliminate the effect of excipients from the tablets. The molecular structure of metoprolol used in this study is given in Fig. 1. At first, solutions of 2.0 M hydrochloric and 1.0 M sulfuric acid were prepared for each experiment in distilled water using analytical grade 37% HCl and 98% H2SO4 purchased from Sigma-Aldrich. Various amounts of the tablet (1000, 800, 600, 400, and 100 ppm) were added to the acids solutions, with adequate volume of acid and inhibitor (the tablet was dissolved in distilled water and passed through the filter paper without purification) to get the desired concentration of acids (1.0 M HCl and 0.5 M H2SO4) and 500-50 ppm of inhibitor. Mild steel samples of the following composition were used (in wt. %): C 0.17; Si 0.5; Mn 1.4; S 0.045; Fe to balance. The specimens were mechanically cut into 1 cm2 pieces. Prior to all measurements, the samples were polished using different grades of emery papers (600-3000), degreased with acetone and finally washed with distilled water and dried in air, then im- Figure 1. Molecular structure of metoprolol (RS)-i-(isopropylami-no)-3-[4-(2-methoxyethyl)-phenoxy]-propan-2-ol. 2. 2. 2. Effect of Temperature The effect of temperature on the corrosion rate of mild steel in both acids solutions with and without various concentrations of inhibitor in the temperature range of 25-55 ± 1 °C was investigated by potentiodynamic polarization technique. 2. 2. 3. Scanning Electron Microscopic (SEM) Studies The surface morphology of the working electrode was examined after immersion of the alloy for about 24 h in 1 M HCl and 0.5 M H2SO4 solutions at room temperature, in the absence and presence of the optimum concentration of metoprolol using scanning electron microscopy (SEM). Scans were taken with a EM3200 instrument (accelerating voltage 0-30 kV) from KYKY company, China. 3. Results and Discussion 3. 1. Electrochemical Impedance Spectroscopy Electrochemical impedance spectroscopy (EIS) has been employed in order to investigate the surface layer formed by the inhibitor. The effect of metoprolol concentrations on the impedance behavior of mild steel in 1.0 M hydrochloric acid and 0.5 M sulfuric acid solutions at 25 ± 1 °C is shown in Fig. 2. Inhibitor efficiency can also be estimated by charge transfer resistance according to the following equation:17 Mohammadinejad et al.: Metoprolol: New and Efficient Corrosion Inhibitor 712 Acta Chim. Slov. 2020, 67, 710-719 where Rlct and R0ct are charge transfer resistances of mild steel in the absence and presence of inhibitor, respectively. Inhibition efficiency increased with the concentration of inhibitor up to 300 ppm and further increase in the inhibitor concentration did not cause any appreciable change in the inhibition performance. Fig. 3 illustrates the electrical equivalent circuit employed to analyze the impedance plots. In this Figure, Rs is the solution resistance and Rct is the charge transfer resistance. The impedance of the constant phase element (CPE) is defined as bellow:18 ZCPE = A ^ito) (2) where A is proportionality coefficient, « is angular frequency (in rad./s) and i = -1 is the imaginary number. The correct equation to convert the CPE constant, A, into the double layer capacitance, Cdi, is:19 (3) where is the frequency at which the imaginary component of the impedance is maximum. The electrochemical pa- CPE Figure 3. Equivalent circuit to estimate impedance diagrams. rameters,obtained from fitting of the recorded EIS data are listed in Table 1. In this table, the calculated double-layer capacitance (Cdl) values derived from the CPE parameters are also given and standard deviation (S.D.) and mean (X) values are calculated. Table 1. Corrosion parameters derived from Nyquist curves for mild steel in a) hydrochloric acid and b) sulfuric acid solution in absence and presence of different concentrations of inhibitor at 25 ± 1 °C. (Results derived from at least two repeats of experiment.) a) inhibitor concentration (ppm) Cdi/ ^F/cm2 Rct/ fi cm2 ± S.D. IE% X Blank 368 15.0 ± 0.00 - 15.0 50 299 25.0 ± 1.53 40 25.3 100 196 38.0 ± 2.08 60 36.7 200 116 86.0 ± 2.00 82 88.0 300 62 121.0 ± 1.53 87 122.3 400 103 97.0 ± 2.08 84 98.7 b) inhibitor concentration (ppm) Cdi/ ^F/cm2 Rct/ fi cm2 ± S.D. IE% X- Blank 607 16.0 ± 0.00 - 16.0 50 506 26.0 ± 1.53 37 26.3 100 318 31.0 ± 1.53 50 32.3 200 190 40.0 ± 1.53 58 41.3 300 136 55.0 ± 1.53 70 56.3 400 241 41.0 ± 1.53 60 40.6 a) SO 60 40 „ 20 G -20 40 x blank • 50ppm a lOOppm + 200ppm ♦ 300ppm f> 400ppm / irC' rv> ♦ j 20 40 60 r (Q) 80 100 120 b) 80 60 40 G NJ 20 -20 40 x blank • 50ppm a lOOppm + 200ppm ♦ 300ppm f> 400ppm ro ♦ j 20 40 60 Z' (£1) 80 100 120 Figure 2. Nyquist curves for mild steel in a) 1 M hydrochloric acid solution and b) 0.5 M sulfuric acid solution in absence and presence of different concentrations of inhibitor at 25 ± 1 °C. (The experiments were repeated at least two times) Mohammadinejad et al.: Metoprolol: New and Efficient Corrosion Inhibitor Acta Chim. Slov. 2020, 67, 710-719 713 3. 2. Potentiodynamic Polarization Fig. 4 presents the potentiodynamic polarization curves of mild steel in 1 M HCl and 0.5 M H2SO4 in the blank and solution containing various concentrations of tablet. The relevant parameters are gathered in Table 2, i.e. corrosion current density (icorr), corrosion potential (Ecorr), surface coverage (0), anodic and cathodic Tafel slopes (^a, ^c). The corrosion current density decreased as the concentration of inhibitor increased up to 300 ppm, then decreased. Addition of inhibitor to acid media affected both cathodic and anodic branches of the polarization curves. But the corrosion potential did not change noticably. Therefore, metoprolol behaved as mixed-type inhibitor. In addition to the above parameters, Table 2 depicts the values of corrosion inhibition efficiencies (IE) that were calculated using the appropriate equation:20 I£o/0 = ('ty"'inh) x 100 (4) where icorr and iinh are the corrosion current densities in the experiments without and with inhibition, respectively. a) J:, il 0.01 u < 0.001 OH 0.0001 1E-5 1E-6 Figure 4. Polarization curves for mild steel in a) 1 M hydrochloric acid and b) 0.5 M sulfuric acid solution in absence and presence of different concentrations of inhibitor at 25 ± 1 °C. (The experiments were repeated at least two times) Table 2. Corrosion parameters derived from polarization curves for mild steel in a) hydrochloric acid and b) sulfuric acid solution in absence and presence of different concentrations of inhibitor at 25 ± 1 °C. (Results derived from at least two repeats of experiment.) a)_ inhibitor concentration icorr/ ^A/cm2 ± S.D. -Ecorr/ mV pc/ mV/dec pa/ mV/dec 0 IE% X (ppm) Blank 50 100 200 300 400 b) inhibitor concentration (ppm) icorr/ ^A/cm2 ± S.D. -Ecorr/ mV ßc/ mV/dec ßa/ mV/dec 0 IE% X- blank 1823.0 ± 02.12 508 141 113 - - 1821.5 50 739.0 ± 6.00 488 109 62 0.59 59 745.0 100 657.0 ± 6.12 487 118 57 0.64 64 662.0 200 569.0 ± 6.11 496 123 71 0.68 68 559.7 300 308.0 ± 6.36 495 67 102 0.83 83 312.5 400 480.0 ± 6.50 491 122 59 0.74 74 479.7 \ A/ \-ï / \ • V y • Blank ■ j • 50ppm - 100ppm - 200ppm - 300ppm * 400ppm b) t o 1E-5 - -0.6 -0.5 -0.4 Potential applied (V) -0.6 -0.5 -0.4 Potential applied {V} 1553.0 ± 1.41 503 176 142 - - 1552.0 568.0 ± 6.03 508 93 101 0.63 63 574.0 415.0 ± 2.52 506 83 113 0.73 73 420.0 161.0 ± 4.70 498 74 114 0.90 90 166.0 99.0 ± 7.02 502 95 54 0.94 94 97.0 135.0 ± 5.51 498 65 107 0.91 91 141.0 Mohammadinejad et al.: Metoprolol: New and Efficient Corrosion Inhibitor ... 714 Acta Chim. Slov. 2020, 67, 710-719 The IE values indicate that the inhibition is pronounced with increasing the inhibitor amount. Higher concentrations (above 300 ppm) did not affect the IE values (experiments at above 400 ppm (i.e. 500 ppm) of inhibitor were done but the results did not reveal appreciable changes, so due to overlapping of curves, these results are not given). The results show that the drug acts as an effective inhibitor. The calculated efficiencies obtained from polarization technique are in a close correlation with those obtained from charge transfer resistance. 3. 3. Effect of Temperature The effect of temperature on the resistance performance of metoprolol on mild steel in 1 M HCl and 0.5 M H2SO4 was probed by potentiodynamic polarization measurements at optimum concentration of metoprolol. The polarization curves in the absence and presence of 300 ppm metoprolol and in temperature range of 25-55 ± 1 °C are given in Fig. 5 and the corrosion parameters are listed in Table 3. The results obtained from polarization curves revealed an increase in current density and a decrease in IE values with rising temperature. Important information on the mechanism of the inhibitor action could be obtained by comparing apparent activation energy (Ea), derived in the presence of inhibitor and its absence. Ea values were calculated from Arrhenius equation:21 lcorr Aexpg) (5) where icorr is corrosion current, A is a constant and T is the temperature. Fig. 6 shows the Arrhenius plots for the cor- Potential applied (V) Potential applied (V)' c) -0.6 -0.5 -0.4 Potential applied (V) d) -0.6 -0.5 -0.4 Potential applied (VJ Figure 5. Effect of temperature on the polarization curves in 1 M hydrochloric acid solution a) without inhibitor b) with 300 ppm of inhibitor and 0.5 M sulfuric acid solution c) without inhibitor d) with 300 ppm of inhibitor. (The experiments were repeated at least two times.) Mohammadinejad et al.: Metoprolol: New and Efficient Corrosion Inhibitor Acta Chim. Slov. 2020, 67, 710-719 715 Table 3. Corrosion parameters calculated from polarization measurements in 1 M hydrochloric acid a) without inhibitor b) with 300 ppm of inhibitor and sulfuric acid solution c) without inhibitor d) with 300 ppm of inhibitor, at different temperatures. (Results derived from at least two repeats of experiment.) a) a) temperature icorr/ ^A/cm2 -Ecorr/ mV X (C°) ± S.D. 25 1553.0 ± 1.41 502 1552.0 35 1867.0 ± 9.90 494 1860.0 45 2993.0 ± 7.80 492 2978.5 55 6873.00 ± 9.20 486 6866.50 b) temperature icorr/ ^A/cm2 -E, orr/ mV IE% X (C°) ± S.D. 25 99.0 ± 7.02 502 94 97.0 35 830.0 ± 7.80 493 56 824.5 45 1728.0 ± 7.10 491 42 1723.0 55 4622.0 ± 7.07 498 35 4627.0 c) temperature icorr/ ^A/cm2 -Ecorr/ mV X (C°) ± S.D. 25 1823.0 ± 2.12 508 1821.5 35 5260.0 ± 6.36 502 5255.5 45 5268.0 ± 5.66 503 5264.0 55 10453.0 ± 5.66 504 10457.0 d) temperature icorr/ ^A/cm2 -E, orr/ mV IE% X (C°) ± S.D. 25 308.0 ± 6.36 495 83 312.5 35 1450.0 ± 9.90 494 72 1443.0 45 2024.0 ± 7.80 487 62 2018.5 55 4623.0 ± 8.50 493 56 4629.0 b) E u Ï 9 5 7 6 S 4 3 2 1 0 0.003 « v = -1.04X10jI +4.01*10 R! =9.90x101 c) 0.0031 0.0032 0.0033 0.0034 0.0035 in-® 0 0.003 • * y — -4.60X103! +2.31x10 RÏ=9.2I><10"1 0.0031 0.0032 0.0033 I T (K) rosion of mild steel in both solutions. The Ea values were determined from the plots (ln jcorr versus 1/T) and are given in Table 4. A decrease in inhibition yield with rise in temperature with analogous increase in corrosion activation energy in the presence of inhibitor compared to its absence is frequently interpreted as being suggestive of formation of an adsorption film of physical (electrostatic) nature. 3. 4. Adsorption Isotherm To obtain the surface coverage, 0, it was assumed that the inhibition efficiency is because of the blocking effect of the adsorbed species and hence 0 = IE (%)/100. Here, an attempt was made to test the Langmuir, Temkin, and Frum-kin isotherms. The Langmuir adsorption isotherm is found d) 5 4 £ 2 Y"---.- v = -7.20x103ï+3.02x10 K^JOxiO-1 0.003 0.0031 0.0032 0.0033 0.0034 0.0035 Figure 6. Arrhenius slopes calculated from corrosion current density for mild steel in 1 M hydrochloric acid, a) without inhibitor, b) with inhibitor and 0.5 M sulforic acid solution c) without inhibitor d) with inhibitor. Mohammadinejad et al.: Metoprolol: New and Efficient Corrosion Inhibitor ... 716 Acta Chim. Slov. 2020, 67, 710-719 a) 500 450 400 350 = 30U a B 250 u 200 150 100 50 0 y = I.OO1 + 2.88x10 r--9.90x10"1 100 200 300 C (ppm) 400 500 b) 600 500 400 3 c. s. 300 -40 and <-20) kJ/mol is commonly interpreted with comprehensive adsorption (physical and chemical adsorption).27 For these solutions, the negative values of AG°ds showing the spontaneity of the adsorption process of inhibitor molecules on the steel surface. The negative signs of AH°ds reflect the exothermic nature of metoprolol behavior on the alloy. In this research, entropy of adsorption in both solutions is low and negative. 3. 6. SEM Observations Scanning electron micrographs (SEM) of the surface of mild steel immersed for 24 h in 1 M HCl and 0.5 M Table 4. Thermodynamic and kinetic parameters for adsorption of inhibitor in sulfuric acid and hydrochloric acid solutions on the metal surface. (Results derived from two repeats of experiment.) _Ea (kJ/mol) AHads (kJ/mol) AGads (kJ/mol) ASads (kJ/mol K) Hydrochloric acid 30.9 - - - Sulfuric acid 38.2 - - - Hydrochloric acid and inhibitor 86.8 -84.5 -25.9 -0.197 Sulfuric acid and inhibitor 59.9 -31.9 -26.0 -0.020 Figure 9. SEM images of mild steel in 1.0 M hydrochloric acid solution, a) blank, b) with inhibitor. Figure 10. SEM images of mild steel in 0.5 M sulfuric acid solution, a) blank, b) with inhibitor. Mohammadinejad et al.: Metoprolol: New and Efficient Corrosion Inhibitor ... 718 Acta Chim. Slov. 2020, 67, 710-719 H2SO4 solutions without and with the optimum concentration of inhibitor are shown in Fig. 9 and 10. The alloy surfaces in both blank solutions (Fig. 9a and 10a) were drastically damaged but in the presence of 300 ppm me-toprolol (Fig. 9b and 10b) were protected. This demonstrates the potential of metoprolol to act as an efficient corrosion inhibitor for the mild steel in the acid media. 4. Conclusions The below conclusions were drawn for this study: 1. Metoprolol tablet was found to be a suitable inhibitor of mild steel corrosion in acid media, especially in HCl. 2. Inhibition efficiency increased with an increase in metoprolol concentration up to 300 ppm while it decreased with increase in temperature. 3. The inhibitor concentration (300 ppm) at 25 ± 1 °C reached to a maximum inhibition efficiency. 4. The free energy of adsorption indicates that the process was spontaneous and the adsorption enthalpy indicated that the process is exothermic with negative entropy of adsorption. 5. The EIS measurements showed that with addition of the inhibitor up to 300 ppm, the charge transfer resistance enhances and the double layer capacitance (C^l) reduces. 6. Potentiodynamic polarization measurements showed that the tablet acts as a mixed-type inhibitor in both solutions. 7. The results obtained from potentiodynamic polarization and EIS are consistent. 8. The adsorption of the inhibitor on the alloy surface in the solutions obeys Langmuir isotherm. 9. The SEM investigations revealed formation of a uniform and protective film on the alloy. 5. References 1. B. E. A. Rani, B. B. J. Basu, Int. J. Corros. 2012, 2012, 1-15. DOI: 10.1155/2012/380217 2. X. Li, S. Deng, H. Fu, G. Mu, Corros. Sci. 2009, 51, 620-634. metoprolola fizikalna, eksotermna in spontana 3. M. J. Bahrami, S. M. A. Hosseini, P. Pilvar, Corros. Sci. 2010, 52, 2793-2803. DOI:10.1016/j.corsci.2010.04.024 4. R. M. Palou, O. Olivares-Xomelt, N. V. Likhanova, InTech. 2014, 432-465. 5. S. M. A. Hosseini, M. Salari, M. Ghasemi, Mater. Corros. 2009, 6G, 963-968. DOI:10.1002/maco.200905214 6. S. Bilgic, M. Sahin, Mater. Chem. Phys. 2001, 7G, 290-295. DOI:10.1016/S0254-0584(00)00534-4 7. H. D. Lece, K. C. Emregul, O. Atakol, Corros. Sci. 2008, SG, 1460-1468. DOI:10.1016/j.corsci.2008.01.014 8. G. Mu, X. Li, Q. Qu, J. Zhou, Corros. Sci. 2006, iS, 445-459. DOI:10.1016/j.corsci.2005.01.013 9. E. Samiento-Bustos, J. G. Gonzalez Rodriguez, J. Uruchurtu, G. Dominguez- Patino, V.M. Salinas-Bravo, Corros. Sci. 2008, SG, 2296-2303. DOI:10.1016/j.corsci.2008.05.014 10. A. C. Bastos, M. G. Ferreira, A. M. Simoes, Corros. Sci. 2006, iS, 1500-1512. DOI:10.1016/j.corsci.2005.05.021 11. M. Sahin, G. Gece, F. Karci, S. Bilgic, J. Appl. Electrochem. 2008, 3S, 809-815. DOI:10.1007/S10800-008-9517-3 12. G. Gece, Corros. Sci. 2008, SO, 2981-2992. DOI:10.1016/j.corsci.2008.08.043 13. R. T. Loto, C.A. Loto, A. P. I. Popoola, J. Mater. Environ. Sci. 2012, 3, 885-894. 14. D. G. Ladha, U. J. Naik, N. K. Shah, J. Mater. Environ. Sci. 2013, i 701-708. 15. N. O. Eddy, S. A. Odoemelam, J. Mater. Sci. 2008, i 87-96. 16. A. S. Mahdi, Int. J. Adv. Res. Eng. Tech. 2014, S, 99-107. 17. N. S. Patel, S. Jauhari, G. N. Mehta, Acta Chim.Slov. 2010, S7, 297-304. 18. M. Outirite, M. Lagrenée, M. Lebrini, M. Traisnel, C. Jama, H. Vezin, F. Bentiss, Electrochim. Acta. 2010, SS, 1670-1681. DOI:10.1016/j.electacta.2009.10.048 19. M. S. Al-Otaibi, A. M. Al-Mayouf, M. Khan, A. A. Mousa, S. A. Al-Mazroa, H. Z. Alkhathlan, Arabian J. Chem. 2014, 7, 340-346. DOI:10.1016/j.arabjc.2012.01.015 20. A. Samide, I. Bibicu, M. Rogalski, M. Preda, Acta Chim.Slov. 2004, SÏ, 127-136. 21. B. El Mehdi, B. Mernari, M. Traisnel, F. Bentiss, M. Lagrenee, Mater. Chem. Phys. 2003, 77, 489-496. DOI:10.1016/S0254-0584(02)00085-8 22. L. Herrag, B. Hammouti, A. Aouniti, S. El Kadiri, R. Touzani, Acta Chim. Slov. 2007, Sé, 419-423. 23. P. Atkins, J. de. Paula, Oxford University Press, Oxford, USA, Physical Chemistry, Eighth, 2006. 24. A. Pal, S. Dey, D. Sukul, Res. Chem. Intermed. 2016, Ï2, 45314549. DOI:10.1007/s11164-015-2295-8 25. Gh. Golestani, M. Shahidi, D. Ghazanfari, Appl. Surf. Sci. 2014, 3GS, 347-362. DOI:10.1016/j.apsusc.2014.04.172 26. H. Ashassi-Sorkhabi, B. Shaabani, D. Seifzadeh, Appl. Surf. Sci. 2005, 239, 154-164. DOI:10.1016/j.apsusc.2004.05.143 27. E. A. No or, A. H. Al-Moubaraki, Mater. Chem. Phys. 2008, ÏÏG, 145-154. DOI:10.1016/j.matchemphys.2008.01.028 Mohammadinejad et al.: Metoprolol: New and Efficient Corrosion Inhibitor Acta Chim. Slov. 2020, 67, 710-719 719 Povzetek Proučevali smo inhibicijo korozije jeklene zlitine (st37) s tabletami metoprolola v 1 M klorovodikovi kislini in v 0,5 M žveplovi kislini. Uporabili smo tri metode: potenciodinamično polarizacijo, elektrokemijsko impedančno spektroskopijo in vrstično elektronsko mikroskopijo, SEM. Pridobljeni parametri so pokazali, da različne količine metoprolola inhibira-jo korozijo jekla v raztopinah kislin HCl in H2SO4. Korozijska odpornost zlitine se je povečevala glede na višanje koncentracije metoprolola do 300 ppm, a se je zmanjšala pri višji temperaturi. Parametri, pridobljeni iz polarizacijskih krivulj, so pokazali, da je učinkovina inhibitor mešanega tipa. Rezultati, pridobljeni z različnimi metodami, so se med seboj ujemali. Ugotovili smo, da je adsorpcija ter da se ujema z Langmuirjevim adsorpcijskim modelom. SEM mikrografije se skladajo z adsorpcijskim obnašanjem tablete © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Mohammadinejad et al.: Metoprolol: New and Efficient Corrosion Inhibitor ... DOI: 10.17344/acsi.2020.5394 Acta Chim. Slov. 2020, 67, 720-728 /^creative ^commons Scientific paper Dynamics of Isomerization of Hop Alpha-Acids and Transition of Hop Essential Oil Components in Beer Miha Ocvirk1 and Iztok J. Košir^* 1 Institute of Hop Research and Brewing, Cesta Žalskega Tabora 2, SI-3310 Žalec, Slovenia * Corresponding author: E-mail: iztok.kosir@ihps.si Tel: 00386 371 21 608 Received: 01-13-2020 Abstract Hops' unique composition of essential oil components and bitter resins are crucial for beer aroma, which is important to consumers' acceptance of beer. In this experiment the same wort was divided into four portions and each was hopped differently. To determine the dynamics of isomerization rates the concentrations of alpha- and iso-alpha-acids were continuously measured. Measurements of hop essential oil components were performed during each process to understand the dynamics of the transition into beer. The maximum isomerization yield of alpha-acids (18.1%) was achieved after 100 min. Longer boiling increased the reduction of iso-alpha-acids, as well as essential oil components. Dry hopping also affected not only on beer aroma but also on beer bitterness. Keywords: Beer; hop; aroma; isomerization; hop essential oil 1. Introduction In the brewing process hop is a quantitatively minor ingredient, but of paramount importance to the brewing industry. With their complex chemistry, hops have been the subject of investigation for decades.1 During the brewing process hops are added into the boiling wort to provide a bitter taste and aroma to the final product.2 One of the main reasons for boiling the wort is the isomerization reaction of the hop alpha-acids into their isomerized forms. The duration of kettle hopping depends on the time required for the isomerization to take place.3 Adding hops into the boiling wort is desired with intent to achieve the desired bitterness, however it causes a reduction in the yield of essential oils in the beer due to evaporation. Consequently, these components are not present in beer in the same ratios as they are in hops. Some components are very volatile and some have low solubility in water.4 During other stages of the brewing process, essential oil components decrease because of adsorption of yeast cells on the trub in addition to evaporation. The CO2 produced during alcoholic fermentation also affects the decrease of aroma compound yields in beer.5-8 To avoid the loss of aroma constituents and to achieve better yields dry hopping technique is in use, where hops are added at a later stages of brewing process, during fermentation or maturation. The volatile aroma compounds of the hops are crucial for brewers, since they give beer its unique aroma. The identification of hop essential oil composition lasts for decades and the study of Roberts et.al concluded on the presence of over 1000 compounds in an oil fraction.9 Dried hops contain approximately 0.5-4.0% of essential oils. According to the chemical composition, compounds can be divided into three groups depending on their chemical structure: the hydrocarbon fraction which form approximately 75% of the total oil, the oxygen containing compounds forming approximately 25% of the total oil and the sulphur-containing compounds present in much lower quantities.2,10 Hydrocarbon fraction, consists of monoter-penes, sesquiterpenes and aliphatic hydrocarbons. Due to evaporation and low solubility in water, concentrations of these group of aroma compounds are very low in finished beer. Presence of alcohol in beer can increase solubility of monoterpenes. Oxygen containing compounds consist mostly of monoterpenic, sesquiterpenic and aliphatic alcohols and these components can be formed by the oxidation or reduction during beer production. In general, these compounds are more soluble in water compared to the hydrocarbons because of the higher polarity of the mole-cule.6,7 Generally, myrcene, which is the most abundant component in hop essential oil, is undesirable in beer, while linalool and geraniol - both from an oxygen fraction Ocvirk and Košir: Dynamics of Isomerization of Hop Alpha-Acids Acta Chim. Slov. 2020, 67, 720-728 721 present in lower concentrations than myrcene, present a pleasant aroma. The odour threshold is more important than the concentration level of a component in beer.11 The analytical approach to determine the volatile flavour-active compounds in beer is based on different solvent extraction techniques and separation by gas chromatography with flame ionization detection (GC-FID) or gas chromatography-mass spectrometry (GC-MS).12 A newer approach to determine the volatile aroma compound is based on a solvent-free extraction technique, head-space solid phase micro extraction (HS-SPME),13,8 head-space trap GC-MS analysis of hop essential oils14,15 and stir bar sorptive extraction.16 Much research has been done to investigate the complexity of the final flavour compounds in beer aroma, but in our study the dynamics of the transfer of hop essential oil components as well as the dynamics of the isomeriza-tion of alpha-acids were studied directly during process. The aim of this study was to explore the levels of hop essential oils and alpha-acids during short- and long-time kettle hopping. By preparing the first two beer types (AB), we investigated the behaviour of hop essential oil components (HEOC) during short-time (A) and long-time (B) wort boiling. In preparing the second two types of beer, we investigated dry hopping techniques of beer aroma compounds originated from hops. We tried to determine the difference if hop is added at the beginning of fermentation (C), or at the beginning of maturation (D). 2. Experimental 2. 1. Experimental Beer Brewing For 90 L of wort 100 L of water and 20 kg of Pilsner Malt (Castle Malt, Belgium) were used. The mashing process started at 52 °C for 30 min, then the temperature was increased to 63 °C for 30 min, the temperature was increased once more to 72 °C for 30 min and finally the temperature was increased to 76 °C and held for 10 min. After mashing and lautering the wort was divided into four parts, 1 x 60 L and 2 x 15 L. For beers A (short time boiling) and B (long time boiling) the whole amount of hop was added at the beginning of the wort boiling and left it in for 60 min. After that, the wort was divided in 2 equal parts (30 L). The second part of the wort continued with boiling for another 60 min (120 min in total). In each 30 L 250 g of hop cones were added. For beers C and D wort was boiling without hop in the same way as in the first two trials. In one 15 L fermentation tank 125 g of hop was added and left it until the end of six days of fermentation (beer C). In the second 15 L, 125 g of hop was added after the fermentation process, at the beginning of a two week maturation (beer D). Yeast strain used in fermentation process was Saflager W34/70 (Fermentis) 1 g per L. The duration of the fermentation was 5 days at 12 °C, while maturation take place at 2 °C for 2 weeks. After adding hops into boil- ing wort, samples were collected every 10 min. During fermentation, samples were collected on each day of fermentation and during maturation, samples were collected every week of maturation. 2. 2. Beer Analyses Basic beer characteristics were measured. Determination of extract, alcohol and degree of fermentation were carried out according to the MEBAK II, method 2.10.4.17 Determination of pH value was carried out according to the Analytica EBC, method 9.35.18 Bitterness of beer was measured according to the Analytica EBC, method 9.8.197 Measurement of CO2 was carried out according to the Analytica EBC, method 9.28.320 and the determination of beer colour was carried out according to the Analytica EBC, method 9.6.21 2. 3. Analytical Determination of Hop Metabolites Determination of the content of hop essential oil was carried out according to Analytica EBC, method 7.10,22 using standard steam distillation. The determination of hop essential oil components was carried out according to the Analytica EBC, method 7.1223 using GC-FID. The determination of alpha-acids was carried out according to the Analytica EBC, method 7.4.24 2. 4. Determination of Hop Essential Oil Components (HEOC) in Beer For the hop essential oil component analysis in beer, an Agilent 6890 GC, equipped with FID detector (Agilent Technologies, USA) was used. The determination of essential oils was performed on a 60 m x 0.32 mm x 0.25 ^m DB-Wax capillary column (Agilent Technologies, USA) according to the MEBAK, method 2.23.625 with some modifications, where the aroma compounds are driven out of the sample by steam distillation. The extraction of aroma compounds was performed by shaking 80 mL of distillate in 100 mL glass centrifuge tubes with 1 mL dichloromethane (Sigma-Al-drich, USA). Helium 5.0 was used as a carrier gas with a constant flow of 2.8 mL min-1. The temperatures of the injector and detector were set to 250 °C. The temperature program started at 60 °C for 4 min, then increased by 5 °C min-1 to 220 °C and was held at 220 °C for 30 min, then increased by 20 °C min-1 to 240 °C and held for 5 min at 240 °C. The sample injection volume was 4 ^L. Methyl heptanoate (Sig-ma-Aldrich, USA) was used as an internal standard. Identifications and quantifications of all investigated compounds presented in Table 1 were performed using standards purchased through Sigma-Aldrich, USA. For all measured parameters, the repeatability, accuracy and linearity for ana-lytes were determined. RSDs for repeatability were from 1.3 to 1.9%. All measurements were done in duplicate. Ocvirk and Košir: Dynamics of Isomerization of Hop Alpha-Acids 722 Acta Chim. Slov. 2020, 67, 720-728 Table 1: Composition of Dana essential oil Essential oil component % in hop oil Methyl hexanoate 0.4 a-Pinene 0.2 Myrcene 37.5 Linalool 1.1 Borneol 0.1 a-Terpineole 0.4 Methyl nonanoate 0.2 Nerol 0.3 ß-Citronellol 0.3 Geraniol 0.5 Methyl caprate 1.0 Neryl acetate 0.2 Geranyl acetate 0.4 ß-Cariophyllene 9.4 a-Humulene 21.1 ß-Farnesene 5.1 Tridecanone 0.8 Geranyl iso-butyrate 1.0 Cariophylene oxide 0.9 Farnezol 0.2 Limonene 0.3 2. 5. Alpha-Acids and Iso-Alpha-Acids Determination To determine alpha-acids and iso-alpha-acids in wort and beer, samples were taken and frozen at -20 °C until analysis. Prior to analysis, samples were warmed to room temperature and centrifuged at 3000 rpm for 15 min in Her-aeus Biofuge Primo (Switzerland) centrifuge. The clear upper phase was filtered through 0.45 ^m PET filters into 2 mL glass vials. Injection of 2 ^L was made with an auto sampler of an Agilent 1200 HPLC chromatograph, using HPLC grade methanol (Merck, Germany), distilled water and 85% orto-phosphoric acid (Sigma Aldrich, USA) in a ratio of 77.5: 21: 0.9 as a mobile phase. Separation was performed on a 150 mm long Nucleodur C18 Column (Macherey Nagel, Germany). Iso-alpha-acids were recorded at wavelength 270 nm, while alpha-acids were recorded at 314 nm. 3. Results and Discussion In each type of beer brewed, the same amount (g L-1) of the traditional Slovenian variety, Dana, with the al- pha-acids content 9.0% and 1.9 mL of hop essential oil on 100 g of dried hop was used. Basic beer specifications of all investigated beer types are presented in Table 2. Data consists of extract, alcohol, degree of fermentation, pH, bitterness, content of CO2 and content of iso-alpha-acids. 3. 1. Isomerization of Alpha-Acids The content of iso-alpha-acids is the sum of all three peaks of isomerized analogues of alpha-acids: iso-cohu-mulone (tR = 4.19 min), iso-adhumulone (tR = 4.97 min) and iso-humulone (tR = 5.42 min). In brewing science, the iso-alpha-acids are expressed as the sum of all three components, because for brewers, the sum value is relevant. The content of alpha-acids is the sum of cohumulone (tR = 8.39 min) and n+adhumulone (tR = 10.70 min). In Figure 1 where the dynamics of isomerization of alpha-acids for beer type B are presented, one can see a high isomerization rate in the first 30 min. The highest utilization of 18.1% was achieved after 100 min of boiling. Isomerization yield was calculated as the ratio of actual mass concentration of iso-alpha-acids in beer, divided by the know content of alpha-acids in hop that was added into wort. Each sample taken later had smaller concentrations of iso-alpha-acids and smaller concentrations of alpha-acids. There was also a small part (1.6% of total alpha-acids added in wort) of alpha-acids that remained un-isomerized. This is a sign that longer boiling causes further degradation of iso-alpha acids. During fermentation we can see the concentration of iso-alpha-acids decreasing but a small increase in concentration was noticed on the last, fifth day of fermentation. Concentration during the maturation process increased to the final isom-erization yield of 14.6%. The rise of IAA is occurred during maturation, where all bioprocess are finished, all the components reacts between each other's and beer gets its flavour and aroma.6 The pattern was the same (not shown) in beer type A, with one difference. The maximum isomerization yield was achieved at the end of boiling for 60 min, but after this time there is a part (4.6% of total alpha-acids added in wort) of alpha-acids that remain un-isomerized. Again, the concentrations decreased during fermentation and slightly increased during the maturation process. In beer C, the concentration of iso-alpha-acids decreased every day until the final isomerization yield was below 1% (Figure 2). Table 2. Basic beer characteristics for each beer type. Beer Extract Alcohol Degree of pH Bitterness CO2 Iso-alpha-acids type (%) (vol. %) fermentation (%) (IBU) (g L-1) (mg L-1) A 11.8 5.1 66.2 4.7 112 3.0 98 B 12.6 6.1 67.1 4.8 120 3.1 110 C 11.3 5.2 68.8 4.7 14 4.7 6 D 11.4 5.2 69.1 4.7 8 4.8 6 Ocvirk and Košir: Dynamics of Isomerization of Hop Alpha-Acids ... Acta Chim. Slov. 2020, 67, 720-728 723 Figure 1: Dynamic of isomerization of alpha-acids in beer B. Sampling was made every 10 min during boiling, every day of fermentation and every week of maturation. Figure 2. Dynamic of isomerization of alpha-acids in beer C. Ocvirk and Košir: Dynamics of Isomerization of Hop Alpha-Acids ... 724 Acta Chim. Slov. 2020, 67, 720-728 The alpha-acid content in the beer was approximately 2 mg L-1 from the beginning of the process to the end of fifth day. The cause of low isomerization yield was the low temperature of the process. The cause is also limited solubility of alpha-acids in the wort and further losses of iso-alpha-acids post wort boiling; the absorption of trub and because of losses during clarification.26-29 Knowledge of the utilization value of iso-alpha-acids for each brewery is of crucial importance for brewers to achieve a desired level of bitterness in beer. In Table 3, where the dynamics Table 3. Dynamics of isomerization in beer D Time of Alpha-acids Iso-alpha- sampling (mg L-1) acids (mg L-1) After 1 week of maturation 7.2 5.8 After 2 weeks of maturation 8.6 6.0 of isomerization in beer D is presented, one can see that the concentrations of alpha-acids are slightly higher than a) b) Figure 3a and 3b. Transition of hop essential oil components during wort boiling. Ocvirk and Košir: Dynamics of Isomerization of Hop Alpha-Acids ... Acta Chim. Slov. 2020, 67, 720-728 725 the concentrations of iso-alpha-acids. Both concentrations are very low, as the isomerization process depends on the temperature of the medium. 3. 2. Determination of the Transition of HEOC in Beer The most abundant components of Dana essential oils were focused on. These components with their relative abundance in hop are presented in Table 1. Concentrations of investigated HEOC at the beginning of the hop- ping process and at the end of the experiment are presented in Table 3. Concentrations of HEOC during wort boiling in beer types A and B (first 60 min for beers A + B and next 60 min for beer B) decreased at different rates (Figures 3a and 3b). Some were decreased to the values < LOD. The exception is geraniol, where a small increase in concentration is noticed. It is important to know that these are not the final concentrations in beer. The literature states that later in the fermentation or maturation process concentrations of some HEOC can increase, because of thermal re- a) b) 12 3 Time of sampling (days) Figure 4a and 4b. Transition of hop essential oil components during fermentation. Ocvirk and Košir: Dynamics of Isomerization of Hop Alpha-Acids ... 726 Acta Chim. Slov. 2020, 67, 720-728 actions or by biotransformation processes such as oxidation, reductions, isomerization or hydrolysis of volatiles that occur in beer brewing.30-35 Myrcene and a-humulene, which belong to the terpene hydrocarbon fraction of essential oils, are hydrophobic and would not pass from the hops through to the finished beer during the brewing process. On the other hand, hydrophilic terpene alcohols, such as linalool, are easier to retain in the beer.30,31 Due to evaporation during wort boiling, myrcene, although it is the most common component in essential oils, does not contribute to the beer aroma because the concentrations are normally far below the odour threshold level.32 In beer C we observed the transition of HEOC during fermentation (Figure 4a and 4b). In general, concentrations of HEOC begin high, and then some fluctuation in concentrations is observed, until the last day of fermentation when the HEOC are again in the same range as at the beginning. One of the main reasons for such fluctuations in concentrations during dry hopping, which seems to be simple, but from a physico-chemical point of view dry hopping is very complex and complicated, is not just extraction time and amount of hops but also the presence of yeast cells and carbon dioxide which can wash out the essential components from beer.6,7 It is interesting, that concentrations of limonene, a-terpineole, neryl acetate and geranyl-iso-butyrate remain almost the same as they were after the first day of dry hopping. Concentration of linalool increases around 50 times. The concentrations of HOEC in beer C are generally higher than in both "kettle hopped" beers A and B. In beer D, where hop was added only at the beginning of the maturation process, two types of HEOC dynamics are observed. In Table 4, some concentrations decrease with time while some of the concentrations increase. Some concentrations are higher than in beer C. One of the reasons is a low temperature during the maturation process and live environment in the fermentation process. Beside extraction of HEOC in wort or beer, chemical reactions between some components can lead to an increase of concentrations. Linalool can come from nerol or geraniol, and P-citronellol can come from geraniol. Linalool could be also cyclized to a-terpineol.31,33 Although linalool is not abundant in hops, it's contribution to beer aroma is high because of its very low odour threshold value of 2.2 ^g L-1.34 Henke et al. also reported that concentrations of ge-raniol increase during fermentation, which is in line with our results.35 4. Conclusions From this experiment, it can be concluded that the optimal time of hopping to achieve high bitterness is no longer, than 100 min. Dry hopping also effects beer bitterness, but the isomerization rates are very low, and consequently higher concentrations of un-isomerized alpha-acids were detected. During brewing, severe losses, oh hop essential oils occur by evaporation in the hot part Table 4. Concentrations of hop essential oil components in beer types A-D (LOD = 0.1 |ig L 1) A B C D Start Final Start Final Start Final Start Final [re L-1] [re L-1] [re L-1] [re L-1] [re L-1] [re L-1] [re L-1] [re L-1] Methyl hexanoate 0.9 ± 0.1 0.4 ± 0.1 0.9 ± 0.1 0.4 ± 0.1 < LOD 9.4 ± 0.2 4.2 ± 0.1 2.4 ± 0.1 a-Pinene 0.2 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 0.1 ± 0.1 < LOD < LOD < LOD < LOD Myrcene 4.8 ± 0.1 1.9 ± 0.1 4.8 ± 0.1 0.4 ± 0.1 24.3 ± 0.3 22.5 ± 0.3 0.6 ± 0.1 1.71 ± 0.1 Linalool 14.6 ± 0.2 0.9 ± 0.1 14.6 ± 0.2 0.3 ± 0.1 1.1 ± 0.1 54.7 ± 0.8 19.7 ± 0.3 28.2 ± 0.3 Borneol 1.0 ± 0.1 0.3 ± 0.1 1.0 ± 0.1 < LOD 3.7 ± 0.1 1.7 ± 0.1 0.3 ± 0.1 0.3 ± 0.1 a-Terpineole 2.5 ± 0.1 0.9 ± 0.1 2.5 ± 0.1 < LOD 8.6 ± 0.2 1.4 ± 0.1 0.4 ± 0.1 0.6 ± 0.1 Methyl nonanoate 1.5 ± 0.1 9.0 ± 0.2 1.5 ± 0.1 < LOD 0.4 ± 0.1 6.5 ± 0.1 5.7 ± 0.1 14.9 ± 0.2 Nerol 0.7 ± 0.1 0.4 ± 0.1 0.7 ± 0.1 < LOD 3.6 ± 0.1 6.2 ± 0.1 2.9 ± 0.1 11.3 ± 0.2 ß-Citronellol 43.9 ± 0.7 17.3 ± 0.4 43.9 ± 0.7 1.3 ± 0.1 16.5 ± 0.3 70.3 ± 1.1 1.3 ± 0.1 0.7 ± 0.1 Geraniol 12.7 ± 0.2 24.3 ± 1.0 12.7 ± 0.2 18.2 ± 0.3 81.3 ± 1.3 113.2 ± 1.8 4.0 ± 0.2 26.9 ± 0.4 Methyl Caprate 1.8 ± 0.1 0.7 ± 0.1 1.8 ± 0.1 0.5 ± 0.1 1.2 ± 0.1 12.2 ± 0.2 2.8 ± 0.1 1.7 ± 0.1 Neryl acetate 0.2 ± 0.1 0.1 ± 0.1 0.2 ± 0.1 1.0 ± 0.1 1.6 ± 0.1 3.0 ± 0.1 1.4 ± 0.1 0.9 ± 0.1 Geranyl acetate 0.5 ± 0.1 0.2 ± 0.1 0.5 ± 0.1 0.3 ± 0.1 0.4 ± 0.1 1.5 ± 0.1 1.1 ± 0.1 38.2 ± 0.6 ß-Cariophyllene 24.4 ± 0.3 9.8 ± 0.2 24.4 ± 0.3 4.6 ± 0.1 7.8 ± 0.1 55.2 ± 0.7 5.0 ± 0.1 2.3 ± 0.1 a-Humulene 3.1 ± 0.1 0.4 ± 0.1 3.1 ± 0.1 0.6 ± 0.1 91.1 ± 1.4 73.7 ± 1.1 1.7 ± 0.1 36.5 ± 0.6 ß-Farnesene 11.6 ± 0.2 0.2 ± 0.01 11.6 ± 0.2 < LOD 186.1 ± 2.7 229.6 ± 3.2 10.7 ± 0.2 39.4 ± 0.6 Tridecanone 1.8 ± 0.1 0.4 ± 0.1 1.8 ± 0.1 0.1 ± 0.1 15.2 ± 0.3 57.8 ± 1.0 10.6 ± 0.2 31.5 ± 0.5 Geranyl iso-butyrate 9.5 ± 0.2 7.7 ± 0.2 9.5 ± 0.2 0.5 ± 0.1 3.1 ± 0.1 1.3 ± 0.1 5.7 ± 0.1 < LOD Cariophylene oxid 3.2 ± 0.1 1.4 ± 0.1 3.2 ± 0.1 0.5 ± 0.3 37.7 ± 0.7 99.7 ± 1.8 8.6 ± 0.1 17.6 ± 0.3 Farnezol 0.5 ± 0.1 0.6 ± 0.1 0.5 ± 0.1 18.6 ± 0.3 11.3 ± 0.2 21.4 ± 0.4 4.7 ± 0.1 3.6 ± 0.1 Limonene 0.4 ± 0.1 0.3 ± 0.1 0.4 ± 0.1 0.2 ± 0.1 0.3 ± 0.1 0.2 ± 0.. 1 0.1 ± 0.1 3.1 ± 0.1 Ocvirk and Košir: Dynamics of Isomerization of Hop Alpha-Acids ... Acta Chim. Slov. 2020, 67, 720-728 727 of the process. Losses in the cold part of beer production are mainly consequence of washing out aroma compounds by carbon dioxide and adsorption of the aroma compounds on the yeast cells. Nevertheless, oxidation and reaction between aroma compounds can lead to rise of concentration of these compounds. Dry hopping does not result in increased aroma intensity in all cases. Funding Sources This work was financially supported by the Slovenian Research Agency by grant 020-2/2011-3. Abbreviations HEOC - hop essential oil components, tR - retention time 5. References 1. C. Almaguer, C. Schönberger, M. Gastl, K. E. Arendt, T. Becker, Humulus lupulus - a story that begs to be told. A review, J. Inst. Brew. 2014, 120, 289-314. DOI:10.1002/jib.160 2. K. Sterba, P. Cejka, J. Culik , M. Jurkova , K. Krofta, M. Pav-lovic, A. Mikyska, J. Olsovska, Determination of Linalool in different hop varieties using a new method based on fluidized - bed extraction with gas chromatographic mass spectromet-ric detection, J. Am. Soc. Brew. Chem. 2015, 73, 151-158. DOI:10.1094/ASBCJ-2015-0406-01 3. M. G. Malowicki, T. H. Shellhammer, Isomerization and degradation kinetics of hop (Humulus lupulus) acids in a model wort-boiling system, J. Agric. Food Chem. 2005, 53 44344439. DOI:10.1021/jf0481296 4. T. Inui, F. Tsuchiya, M. Ishimaru, K. Oka, H. Komura, Different beers with different hops. Relevant compounds for their aroma characteristics, J. Agric. Food Chem. 2013, 61, 4758-64. DOI:10.1021/jf3053737 5. M. Schnaitter, A. Kell, H. Kollmann sberger, F. Schüll, M. Gastl, T. Becker, Scale-up of dry hopping trials: importance of scale for aroma and taste perceptions, Chem. Ing. Tech, 2016, 88, 1955-1965. DOI:10.1002/cite.201600040 6. N. Rettberg, M. Biendl, L. A. Garbe, Hop Aroma and Hoppy Beer Flavour: Chemical Background and Analytical Tools-A review, J. Am. Soc. Brew. Chem. 2018, 76, 151-158. DOI:10.1080/03610470.2017.1402574 7. L. Jelinek, M. Karabin, J. Müllerova, P. Dostalek, The secret of dry hopped beers-Review. Kvasny Prum. 2018, 64, 287-296. D0I:10.18832/kp201836 8. J. Dennenlöhr, S. Thörner, A. Manowski, N. Rettberg, Analysis of selected Hop Aroma Compounds in Commercial Lager and Craft Beers Using HS-SPME-GC-MS/MS. J. Am. Soc. Brew. Chem. 2019. DOI:10.1080/03610470.2019.1668223 9. M. T. Roberts, J. P. Dufour, A. C. Lewis, Application of comprehensive multidimensional gas chromatography combined with time-of-flight mass spectrometry (GC/GC-TOFMS) for high resolution analysis of hop essential oil, J. Sep. Sci. 2004, 27, 473-478. D0I:10.1002/jssc.200301669 10. G. A. Fix, Wort Boiling in Principles of Brewing Science - a Study of Serious Brewing Issues 2nd ed., Brewers Publications, Boulder, USA, 1999, pp. 53-78. 11. C. W. Bamforth, Science principles of malting and brewing, American Society of Brewing Chemists, St. Paul, 2006. 12. M. Kovacevic, M. Kac, Determination and verification of hop varieties by analysis of hop essential oils, Food Chem. 2002, 77, 489-494. D0I:10.1016/S0308-8146(02)00114-0 13. J. Hrivnak, D. Smogrovicova, P. Nadasky , J. Lakatosova, Determination of beer aroma compounds using headspace solid-phase microcolumn extraction, Talanta. 2010, 83, 294296. D0I:10.1016/j.talanta.2010.08.041 14. G. C. da Silva, A. A. S. da Silva, L. S. N. Da Silva, R. L. D. O. Godoy, L.C. Nogouira, S.L. Quiterio, R.S.L. Raices. Method development by GC-ECD and HS-SPME-GC-MS for beer volatile analysis, Food Chem. 2015, 167, 71-77. D0I:10.1016/j.foodchem.2014.06.033 15. C. Schmidt, M. Biendl, Headspace Trap GC-MS analysis of hop aroma compounds in beer, Brewing Sci. 2016, 69, 9-15. 16. T. Horak, J. Culik, V. Kellner, P. Cejka, D. Haskova, M. Jurkova, J. Dvorak, Determination of selected beer flavours: Comparison of a stir bar sorptive extraction and a steam distillation procedure, J. Ins. Brew. 2011, 117, 617-621. D0I:10.1002/j.2050-0416.2011.tb00512.x 17. Destillationsanalyse (Referenzmethode - EBC - Methode), Brautechnischen Aanalysenmethoden Band II, 4. Auflage, MEBAK, Freising-Weihenstephan, 2002, 76-78. 18. European Brewery Convention, Analytica EBC, section 9 -Beer, method 9.35, Analytica EBC, Fachverlag Hans Carl, Nürnberg, 2004. 19. European Brewery Convention, Analytica EBC, section 9 - Beer, method 9.8, Analytica EBC, Fachverlag Hans Carl, Nürnberg, Germany 2004. 20. European Brewery Convention, Analytica EBC, section 9 -Beer, method 9.28.3, Analytica EBC, Fachverlag Hans Carl, Nürnberg, 2007. 21. European Brewery Convention, Analytica EBC, section 9 - Beer, Method 9.6, Analytica EBC, Fachverlag Hans Carl, Nürnberg, Germany, 2000. 22. European Brewery Convention, Analytica EBC, section 7 -Hops, Method 7.10, Fachverlag Hans Carl GmbH, Nürnberg, Germany, 2002. 23. European Brewery Convention, Analytica EBC, section 7 -Hops, Method 7.12, Fachverlag Hans Carl GmbH, Nürnberg, Germany, 2006. 24. European Brewery Convention, Analytica EBC, section 7 -Hops, Method 7.4, Fachverlag Hans Carl GmbH, Nürnberg, Germany, 2000. 25. Determination of Steam-volatile Aroma Compounds in Beer, method 2.23.6, MEBAK Wort, Beer, Beer-based Beverages, MEBAK, Freising, 2013, pp. 405 - 412. 26. B. Jaskula, P. Kafarski, G. Aerts, L. De Cooman, A kinetic study of isomeriazion of Hop-acids, J. Agric. Food Chem. 2008, 56, 6408-6415. D0I:10.1021/jf8004965 Ocvirk and Košir: Dynamics of Isomerization of Hop Alpha-Acids 728 Acta Chim. Slov. 2020, 67, 720-728 27. Y. Huang, J. Tippmann, T. Becker, Kinetic modelling of hop acids during wort boiling. Int. J. Biosci. Biochem. Bioinforma. 2013, 3, 47-52. 28. S. Kappler, M. Krahl, C. Geissinger, T. Becker, M. Krotten-haler, Degradation of Iso-a-acids during wort boiling, J. Ins. Brew. 2010, 116, 332-338. DOI:10.1002/j.2050-0416.2010.tb00783.x 29. B. Jaskula Goiris, G. Aerts, L. De Cooman, Hop a-acids isomerization and utilisation: an experimental review, Cere-visia. 2010, 6, 57-70. DOI:10.1016/j.cervis.2010.09.004 30. T. Kishimoto, A. Wanikawa, K. Kono, K. Shibata, Comparison of the Odor-Active Compounds in Unhopped Beer and Beers Hopped with Different Hop Varieties, J. Agric. Food Chem. 2006, 54, 8855-8861. DOI:10.1021/jf061342c 31. K. Takoi, K. Koie, Y. Itoga, Y. Katayama, M. Shimase, Y. Na-kayama, J. Watari, Biotransformation of Hop-Derived Mono-terpene Alcohols by Lager Yeast and Their Contribution to the Flavor of Hopped Beer, J. Agric. Food Chem. 2010, 58, 5050-5058. DOI:10.1021/jf1000524 32. M. Coelhan, A. Aberl, Determination of Volatile Compounds in Different Hop Varieties by Headspace-Trap GC/MS In Comparison with Conventional Hop Essential Oil Analysis, J. Agric. Food Chem. 2012, 60, 2785-2792. DOI:10.1021/jf205002p 33. M. Riu-Aumatell, P. Miro, A. Serra-Cayuela, S. Buxaderas, F. Lopez-Tamames, Assessment of the aroma profiles of low-alcohol beers using HS-SPME-GC-MS, Food Res. Int. 2014, 57, 96-202. DOI:10.1016/j.foodres.2014.01.016 34. M. Steinhaus, P. Schieberle, Comparison of the most odor-active compounds in fresh and dried hopcones(Humulus lu-pulus L.variety Spalter Select) based on GC-olfactometry and odor dilution techniques, J. Agric. Food Chem. 2000, 48, 1776-1783. DOI:10.1021/jf990514l 35. S. Hanke, M. Herrmann, J. Rückerl, C. Schönberger, W. Back, Hop Volatile Compounds (part II): Transfer Rates of Hop Compounds from Hop Pellets to Wort and Beer, Brewing Sci.-Monatsschrift. Brauwiss. 2008, 61, 140-144. Povzetek Edinstvena sestava eteričnega olja hmelja in grenkih smol je ključnega pomena za aromo, kar je pomembno za sprejemanje piva s strani potrošnikov. V tem poskusu je bilo isto pivo razdeljeno na štiri dele, vsak je bil hmeljen drugače. Za določitev dinamike hitrosti izomerizacije so bile izvedene kontinuirane meritve koncentracij alfa- izo-alfa-kislin. Prav tako so bile med postopkom merjene tudi koncentracije določenih komponent eteričnega olja hmelja, da bi razumeli dinamiko njihovega prehoda v pivo. Največji izkoristek izomerizacije alfa-kislin (18,1 %) je bil dosežen po 100 min varjenja pivine. Daljše vretje povečuje zmanjšanje koncentracij izo-alfa-kislin, kot tudi komponent eteričnega olja. Hladno hmeljenje vpliva ne le na aromo piva, ampak tudi na grenkobo. Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Ocvirk and Košir: Dynamics of Isomerization of Hop Alpha-Acids ... DOI: 10.17344/acsi.2019.5398 Acta Chim. Slov. 2020, 67, 729-738 /^creative ^commons Scientific paper Low-level Electrochemical Analysis of Ketoconazole by Sepiolite Nanoparticles Modified Sensor in Shampoo Sample Sevda Aydar,1 Dilek Eskiköy Bayraktepe,2 Hayati Filik1 and Zehra Yazan2^ 1 Faculty of Engineering, Department of Chemistry, Istanbul University, 34320 Avcilar, Istanbul, Turkey 2 Ankara University, Faculty of Science, Chemistry Department, 06560 Ankara, Turkey * Corresponding author: E-mail: zehrayazan67@gmail.com; zdurmus@science.ankara.edu.tr Phone: +903122126720/1284fax: +903122232395 Received: 07-11-2019 Abstract In this study, the nano-sepiolite modified carbon paste electrode (CCPE) was prepared for the determination of ketoconazole (KC). The effects of pH, the proportion of the electrode modifier, deposition potential, and deposition time were investigated. Ketoconazole shows one irreversible oxidation peak at about the potential value of 0.6-0.7 V at different pH values. CV studies show that the modified electrode performed a catalytic effect on the peak signal of KC compared to the bare electrode. This catalytic behavior of CCPE was used for the development of a sensitive detection method. The impact of pH and scan rates on the anodic peak potentials and currents were examined, and the scan rate results show that the oxidation behavior of KC was controlled by the adsorption process at the CCPE surface. Therefore, adsorptive stripping differential pulse voltammetry (AdsDPV) and adsorptive stripping square wave voltammetry (AdsSWV) methods were developed for KC analysis. The two different linear ranges were obtained as (0.1-1.0) nM and (3.0-10.0) nM for AdsDPV, and (0.1-10.0) nM and (3.0-10.0) nM for AdsSWV, respectively. The detection (LOD) and quantification (LOQ) limits were found to be 0.017 nM and 0.056 nM for AdsDPV and 0.025 nM and 0.083 nM for AdsSWV, respectively. Besides, the proposed new sensor has obtained very high recovery values in the analysis of KC in the pharmaceutical shampoo. Keywords: Ketoconazole; carbon paste electrode; sepiolite clay; pharmaceutical shampoo 1. Introduction Ketoconazole is 1-acetyl-4-[4-[[2RS,4SR)-2-(2,4-di-chlorophenyl)-2-(1-H-imidazol-1-yl methyl)-1,3-dioxa-lan-4-yl] methoxy] phenyl] piperazine and an imidazole derivative.1 Ketoconazole (KC) has a strong antifungal effect against many fungal, gram-positive microorganisms and yeasts. In addition, KC is used in oral administration as an antifungal drug due to its lower toxicity than most azole an-timycotics.2 The mechanism of action is caused by damage to the cytoplasmic membrane in the fungus and leads to the disruption of mitochondrial and microsomal enzymes of fungi. KC is used as an active component of antifungal formulations in creams, tablets, and anti-dandruff shampoos.3 KC can cause side effects such as urticaria, angioedema, leu-kopenia, hemolytic anemia, nausea, and thrombocytopenia. Determination of ketoconazole due to its importance in biological fluids, pharmaceutical preparations, and also cosmetic products appears to be worthwhile. Various methods have been developed for this purpose, including spectrophotometry,4-7 high-performance liquid chromatography,8-10 and liquid chromatography-triple quadrupole tandem mass spectrometry.11 These methods are often time-consuming and require expensive equipment. Also, due to their low sensitivity, requiring pretreat-ment steps, such as extraction and separation steps are needed in an organic environment. Electrochemical methods can be considered for the detection of pharmaceutical and cosmetic drugs. Among them, adsorptive stripping voltammetry-based adsorption phenomena can be preferred for the electrochemical detection of pharmaceutical and cosmetic drugs in the terms of sensitivity, trace level analysis, and simplicity.12-15 Modification of electrode with nano-materials can be used for developing electrochemical nano-sensors.16,17 Electrode modification can Aydar et al.: Low-level Electrochemical Analysis 730 Acta Chim. Slov. 2020, 67, 729-738 catalyze the electron transfer rate between the analyte and electrode. Sepiolite clay used as electrode modifier has ease of adsorption for the polar organic species and ions, imparts the electrical conductivity, and has a catalytic contribution to the electrochemical processes. As well, the intercalation and ion exchange capacity of sepiolite clay expands the sorption capacity and conductivity properties of the working electrodes.18,19 Electrochemical sensors based on nano-sepiolite clay show trace limit of detection, higher effective surface area, conductivity, and adsorption capacity properties. In our previous studies, we have tried the sepiolite mineral alone and combined with other modifiers for inducing novel properties in carbon paste electrodes.12,19-21 In this regard, we have developed two different sensitive and selective adsorptive voltammetric stripping methods (Ads-DPV and AdsSWV) for the determination of ketoconazole at sepiolite clay modified CPE. The developed methods were successfully applied to the determination of keto-conazole in shampoo samples containing ketoconazole. The developed AdsDPV and AdsSWV methods for determination of KC in a shampoo sample, compared to the other electroanalytical methods based on KC analysis in the cosmetic products1,22-24 have the widest linear working range and the lowest limit of detection. 2. Experimental 2. 1. Reagents and Apparatus Sepiolite clay, graphite powder, mineral oil, and all solvents were supplied from Sigma. Ketoconazole was also supplied from Sigma-Aldrich, and other used chemicals were analytical grade and used without a preliminary purification step. The stock solution of KC (1.0 • 10-3 M) was prepared by dissolving of KC in a few drops of 0.1 M HCl solution and water. The prepared stock solution was stored in the refrigerator at + 4 °C. 0.04 M Britton-Robinson buffer was used as the supporting electrolyte. All electrochemical measurements (CV, SWV, DPV, and EIS) were performed by using CHI 660C (USA, Texas) and C3 cell stand (Bioanalytical Systems, Inc., USA, BASi) with a solid electrode unit. Ag/AgCl (in 3.0 M NaCl, BAS MF-2052) as reference electrode and platinum wire (BASi MW-1032) as auxiliary electrode were used for electrochemical measurements. CCPE and CPE sensors were selected as working electrodes. SEM photographs and EDX graphs were recorded by using Carl Zeiss AG, EV0®50 Series. Before all assays, pH was measured with a HANNA Instruments HI2211 pH/ORPmeter. Double-distilled water was supplied mpMINIpure system. All assays were carried out at 25 °C. AdsDPV and AdsSWV methods were used for the electrochemical determination of KC. For AdsDPV method, the device parameters were: amplitude: 0.05 V, pulse width: 0.05 s, sample width: 0.0167 s, pulse period: 0.5 s. For AdsSWV; amplitude: 0.025 V, frequency 20 Hz, potential range: 0.2-0.8 V. For EIS: amplitude: 0.005 V, frequency range: 0.05-105 Hz, and Nyquist plots were recorded under open circuit potential. 2. 2. Sensor Preparation Procedure 30 mg of graphite powder and 10 ^L of mineral oil were mixed in a petri dish with a spatula to prepare CPE. Sepiolite clay and graphite powder were mixed to prepare CCP electrode and then mineral oil (10 ^L) was added. The mass ratios of the sepiolite clay in the mixture were changed between 3.3-10%. The electrical connection was provided by copper wire. The surface of the prepared sensors was smoothened with a smooth paper. Before each experiment, the surface cleaning process of modified CPE sensors was performed by washing with a water-ethanol mixture (1:1). 2. 3. Analytical Procedure KC (1.0 • 10-3 M) stock solution was used in all analyses. In all voltammetric methods, supporting electrolyte (0.04 M BR buffer pH 9.0) and KC stock solution were added to the electrochemical cell with a total volume of 10.0 mL. The CCPE, reference, and counter electrodes were immersed in the cell. After arranging all the electrode connections, the working solutions were purged with nitrogen gas (99.99% purity) to remove the oxygen and then the voltammograms were recorded in the potential window of 0.2 V-0.9 V by using AdsDPV and AdsSWV. 2. 4. Shampoo Sample Preparation The 0.5 g of Ketoral shampoo was weighed to produce the desired final concentration of the sample. After that, a few drops of 0.1 M HCl and a small amount of doubly distilled water were added. Then, the total volume of pure water to 100 mL was completed, and the solution was prepared to contain 1.9 • 10-4 M KC. This mixture was incubated overnight at 4 °C to complete the dissolution of the KC. Appropriate volumes of the resulting solution were placed in the voltammetric cell containing 10.0 mL of BR buffer (pH 9.0), and voltammograms were recorded. 3. Results and Discussion 3. 1. Surface Characterization of CPE and CCPE The surface morphological studies of both bare CPE and sepiolite clay modified CPE (CCPE) were carried out using SEM and EDX measurements. The SEM photographs of CPE and CCPE show that the CCPE electrode Aydar et al.: Low-level Electrochemical Analysis Acta Chim. Slov. 2020, 67, 729-738 731 surface has a more porous structure than bare CPE surface (Fig. 1 a,b). Meanwhile, the EDX measurement was performed to confirm the elemental content of the bare CPE and sepiolite modified CPE (Fig.1 c, d). In Fig.1 c, only one peak is seen that belongs to the carbon (C) element in CP electrode and Fig.1 d shows four peaks that belong to the carbon (C), oxygen (O), magnesium (Mg), and silicon (Si), respectively. According to EDX plots and SEM measurements of the electrodes, it can be clearly said that sepiolite clay has remained successfully on the CPE surface. Cyclic voltammetry (CV) and Electrochemical impedance spectroscopy (EIS) measurements were per- formed using 5.0 mM Fe(CN)63-/Fe(CN)64- in 0.1 M KCl solution to compare the electrochemical properties of CPE and CCPE (Fig.2 a, b). According to the obtained CV voltammograms in Fig. 2a, the higher anodic and cathodic peak currents and the lower peak separation (AEp) values were obtained at the CCPE electrode compared to CPE. Meanwhile, the Nyquist plots of the same electrode surfaces (Fig.2b) show that the charge transfer resistance (Rct) (about 4000 ohms) of CCPE is smaller than the Rct (about 7000 ohms) of CPE. CV and EIS results confirm that the sepiolite clay on the CPE surface provides an electrocata-lytic effect on the electron transfer rate. Figure 1. SEM images of CPE (a) and CCPE (b), EDX elemental mapping of CPE (c) and CCPE (d) a) 1 SO 40 t ° — -40 M -SO -120 b) 0.4 -0.1 Potential /V 5000 Z'/ii 10000 Figure 2. a) CV voltammograms b) Nyquist plots of CPE and CCPE in 5.0 mM Fe(CN)63-/Fe(CN)64- in 0.1 M KCl solution. Aydar et al.: Low-level Electrochemical Analysis 732 Acta Chim. Slov. 2020, 67, 729-738 3. 2. Optimization of Sepiolite Content The optimum amount of sepiolite clay was determined to use in the preparation of the modified CCPE electrode. For this purpose, clay modified electrodes were prepared in such a way that the amount of sepiolite clay was 3.3%, 5.0%, 6.7%, 8.3%, and 10.0%. The signals were recorded using the CV method at a scan rate of 0.1 V/s in a BR buffer solution. The peak current of KC increased up to 6.7% and decreased sharply at the higher amount of sepiolite (Fig. 3). It indicates that 6.7% was the optimum amount of sepiolite concentration. The electrode based on na-no-sepiolite clay shows the higher peak current as compared to bare CPE because of the high conductivity and catalytic effect to the electron-transfer rate of sepiolite clay.20 Figure 3. CVs of 1.0 |rM KC in BR buffer solution on CCPE with different quantities of sepiolite clay (pH 9.0; scan rate: 0.10 V/s). 3. 3. Electrochemical Behavior of KC The cyclic voltammograms of 1.0 ^M KC demonstrated the oxidation signals of KC at 0.597 V and 0.582 V potentials at CPE and CCPE electrodes in BR buffer solution (pH 9.0), respectively (Fig. 4). The CCPE sensor produced a better current response and sharper peak shapes compared to the bare electrode. The presence of sepiolite clay in the modified electrode improved the sensitivity of the method and the electro-catalytic effect on the redox signals of KC. 0.4 0.5 0,6 Potential/V Figure 4. CVs of 1.0 |rM KC at CPE and CCPE electrodes (u: 0.1 V/s, 0.04 M BR buffer, pH 9.0). 3. 4. Cyclic Voltammetric Studies The effect of the scan rate on the redox properties of KC was investigated by using the CV method. For this purpose, cyclic voltammograms were recorded at scan rates in the range 0.005-0.4 V/s in the presence of 1.0 • 10-6 M KC on scanning from 0.2 V to 0.9 V towards positive potential region on CCPE electrode (Fig. 5). These voltammograms were used for determining whether the electrochemical ox- Aydar et al.: Low-level Electrochemical Analysis Acta Chim. Slov. 2020, 67, 729-738 733 idation behaviors are reversible, irreversible, or quasi-reversible. As shown in Fig. 5, only one anodic peak at about 0.60 V was observed. No peak was observed in the reverse scan potentials. In addition, as the scan rate increased, the oxidation signal of KC is shifted to more positive potential values, and this phenomenon shows that the oxidation peak of KC exhibits irreversible redox behavior.3 To monitor the electrochemical process (adsorption or diffusion-controlled) of KC, we used CV technique. In this context, the logip-logv graph was plotted, and the slope value of the logip-logv graph is 0.78 for the oxidation peak of KC. According to this result, it can be said that the adsorption phenomenon is dominant in the electrochemical behavior of ketoconazole.2,22 The fact that pre- or post-peaks are observed in the cyclic voltammograms of KC at high scan rates is another indication that adsorption does occur on the electrode surface. Ip vs. v1/2 graph is nonlinear, which indicates electrochemical reaction is not diffusion controlled. 3. 5. Influence of pH The pH is a crucial parameter that can affect the peak currents and redox mechanism at the electrode surface in voltammetry. Therefore, the effect of pH on the peak current of KC was investigated by using the CV method. The oxidation peak currents of 1.0 • 10-6 M KC were measured at different pH (2.0 -12.0) to determine the optimum pH. Fig. 6. shows that the highest peak current was obtained at pH 9.0. This pH value was chosen to perform the electro-analytical study. To investigate the transferred electron number (n) in the electrooxidation of KC for the irreversible process, we used the following Eq. (1).25 Here, Epa is anodic peak potential, Epa/2 is the half peak potential, a is the electron transfer coefficient. The a is taken to be 0.5 for an irreversible process. In this study, the number of electrons transferred (n) was found to be 2.28. This result is in good accordance with the previously estimated number of electrons of KC at pH 9.0.26 Therefore, the oxidation process of KC involves a two-electron transfer process, and the tentative oxidation peak of KC was attributed to the oxidation of the imidazole group with the loss of electrons to form the ketone structure.26 3. 6. Optimization of Experimental Conditions for AdsDPV and AdsSWV Methods Experimental conditions such as deposition potential and time are vital parameters affected by the electrochemical signal of organic compounds. For this purpose, deposition potentials were changed in the range 0.0-1.0 V for AdsDPV and AdsSWV methods (Fig. 7 A, C). This study has shown that 0.1 V and 0.4 V provided the highest peak current for AdsDPV and AdsSWV, respectively. Therefore, these deposition potentials were used in all subsequent experiments. Similar trials for deposition time were evaluated in deposition time ranging from 0.0 to 180 s (keeping deposition potentials) (Fig. 7 B, D). It was observed that Ip values increase rapidly until 75 s, and then they decrease rapidly for AdsDPV method (Fig. 7B). The optimum value of deposition time was chosen as 75 s. Similarly, the deposition time where the highest peak current was observed was selected as 45 s for AdsSWV (Fig. 7D). Aydar et al.: Low-level Electrochemical Analysis 734 Acta Chim. Slov. 2020, 67, 729-738 A) B) 0.1 0.3 0.5 0.7 0.9 Deposition Potential A' C) 0.1 OJ 0.5 0.7 0.9 Deposition Potential / V 0.12 - 50 100 150 Deposition Time/ s D) 50 100 150 Deposition Time/ s Figure 7. Effect of deposition potential and deposition time on peak current shown in AdsDPV method (A, B) and AdsSVW method (C, D) recorded in the presence of 1.0 • 10-7 M KC at CCPE electrode in 0.04 M BR buffer pH 9.0. 3. 7. Calibration Studies and Validation of Optimized Methods The applicability of AdsDPV and AdsSWV techniques as analytical methods for the analysis of KC was studied by measuring the anodic current as a function of the KC concentration. Calibration curves were constructed for both methods at pH 9.0. Considering the slopes of calibration graphs, the results obtained AdsDPV is more sensitive than by AdsSWV (Fig. 8A-B and Table 1). It was decided that the CCPE electrode could determine KC in the two different linear concentrations ranges of 0.1-1.0 nM and 3.0-10.0 nM for the AdsDPV and AdsSWV methods. The following equations calculated the LOD and LOQ values: Where, s is the standard deviation for the studied KC concentration (1.0 • 10-9 M), and m is the slope of the calibration chart. The LOD and LOQ values for AdsDPV were 0.017 nM and 0.056 nM; the AdsSWV was 0.025 nM and 0.083 nM respectively (Table 1). A survey of the literature reveals that LOD and LOQ values of KC are the lowest results so far. AdsDPV and AdsSWV methods developed for KC determination on the CCPE electrode were compared to the results obtained by voltammetric methods in the literature (Table 2). The linear working range, LOD, and LOQ values obtained by the CCPE electrode were found to be superior to those reported methods. The repeatability, reproducibility, and stability of the modified electrode were investigated. Reproducibility of peak current and potential values (intra-day and inter-day) were determined by using AdsSWV and AdsDPV methods. The percentage of relative standard deviation (%RSD) values is shown in Table 1. %RSD values are less than 5.0%. These results indicate excellent repeatability. However, the reproducibility of the CCPE sensor was tested using five different electrodes prepared on the same day. The %RSD values of reproducibility were calculated as 2.58% and 4.62% for AdsSWV and AdsDPV, respectively. To investigate the stability of the CCPE sensor, we recorded the KC signals on different days. After the first ten days, it was found that the sensor signal retains 98.42% and 99.02% of the initial value. When the current and potential values of C oxidation signal obtained up to 40 days were examined, it was observed that peak current and potential values of KC decreased by 5.0% compared to its Aydar et al.: Low-level Electrochemical Analysis 735 Acta Chim. Slov. 2020, 67, 729-738 A) B) Figure 8. A. AdsDPV and B. AdsSWV voltammograms recorded in 0.04 M BR buffer (pH 9.0) for increasing KC concentrations under optimized conditions. Inset: Calibration graphs for KC. Table 1. The statistical results of the regression analysis obtained with AdsSWV and AdsDPV methods at the CCPE electrode. CCPE Regression parameters AdsSWV AdsDPV Potential, V 0.58 0.52 Linear working range, nM 0.1-1.0 3.0-10.0 0.1-1.0 3.0-10.0 The slope of calibration graph, |A/|iM 27.51 4.35 49.47 6.75 The intercept of calibration graph, |A 0.011 0.036 0.018 0.060 Limit of detection (LOD), nM 0.025 0.017 Limit of quantification (LOQ), nM 0.083 0.056 Regression coefficient (R2) 0.997 0.991 0.992 0.993 Repeatability of peak potential, RSD*% (intra-day) 0.76 0.68 Repeatability of peak potential, RSD*% (inter-day) 3.22 3.72 Repeatability of peak current, RSD*% (intra-day) 4.51 4.63 Repeatability of peak current, RSD*% (inter-day) 4.44 4.04 Reproducibility of peak current, RSD*% 2.58 4.62 Reproducibility of peak potential, RSD*% 0.83 0.82 *RSD is the relative standard deviation of 5 replications. Aydar et al.: Low-level Electrochemical Analysis ... 736 Acta Chim. Slov. 2020, 67, 729-738 Table 2. The comparison of analytical data reported by some different electrochemical sensors with the CCPE sensor for KC analysis. Sensor Technique Linearity range ^M LOD ^M Application Reference BDD SWV 0.287-3.13 0.0829 Tablet 3 CDMGC DPV 10.0-80.0 0.105 Shampoo 1 CPE DPV 0.024-0.48 0.0233 Tablet, Cream 22 GCE DPV 0.0001-1.0 0.00004 Tablet 27 Au disc electrode DPV 50.0-2500 50.0 Tablet, Cream 23 AuNP/GCE 20.0-100.0 2.3 Shampoo 24 AuNPs/CPE DPV, SWV 1.0-80.0 0.1 Tablet 26 CCPE AdsDPV 0.0001-0.001 0.0168 nM Shampoo This paper 0.003-0.01 AdsSWV 0.0001- 0.001 0.0248 nM 0.003-0.01 BDD: boron-doped diamond electrode; CDMGC: beta-cyclo-dextrin modified glassy carbon electrode; CPE: Carbon paste electrode; GCE: Glassy carbon electrode; AuNPs/GCE: Au nanoparticles modified glassy carbon electrode; AuNPs/CPE: Au nanoparticles modified carbon paste electrode original values. According to this result, it can be said that the stability of the prepared clay CPE electrode is maintained for up to 40 days. The prepared sensor was kept at + 4 °C after all experiments. 3. 8. Interferences The interference effect of some electroactive species, which can be found in cream and drug substances, has been investigated in voltammetric AdsDPV and AdsSWV methods developed for the determination of KC. For this purpose, the concentration of Na+, Mg2+, K+, Co2+, Fe3+, Cu2+, Zn2+, ascorbic acid, glucose, lactose, glycerin, and sodium benzo-ate was added to 100 times the concentration of KC. The percent changes in the peak current of KC in the presence of Na+, K+, Fe3+, ascorbic acid, glucose, lactose, glycerin, and sodium benzoate was found to be less than 5% compared to its original signal. The obtained results indicate that these species did not have any interference effect. The results obtained in the presence of Mg2+, Co2+, Cu2+, and Zn2+ showed that these species had a high interference effect in the electrochemical determination of KC. When these cations were added in the presence of KC, the oxidation peak of KC was observed to disappear. This result may be attributed to the formation of a complex between these metals and KC.28, 29 3. 9. Real Sample Analysis and Recovery Studies To determine the accuracy of the two methods developed, we performed a recovery study using a shampoo (Ketoral shampoo 2.0% KC). In Table 3, for AdsSWV and AdsDPV methods, the recovery values were found in the range of 99.5% to 110.4% indicating that the accuracy of the methods is really satisfactory. To evaluate the accuracies and precisions of the two developed AdsDPV and AdsSWV methods, student f-test and F-test were applied to the data obtained from the recovery study. The results show that (Table 3) there are no meaningful differences in terms of accuracy and precision between these two methods. 4. Conclusions This study demonstrates that the sepiolite clay modified carbon paste electrode was tested as a sensor for keto-conazole analysis. The ultra-sensitive detection of KC was carried out for the first time by using anodic adsorptive stripping methods. The effect of modifying agent combination, pH, deposition time, and potential values were in- Table 3. Recovery results for KC in pharmaceutical shampoo (n=5) Method Added Amount, Found Amount, Average Recovery, RSD, nM nM % % AdsSWV 3.0 3.3; 3.1; 2.3; 3.2; 3.1 2.98 ± 0.129 99.5 4.3 5.0 5.1; 5.4; 5.3; 5.2; 5.0 5.23 ± 0.188 104.6 3.5 7.0 7.2; 7.6; 8.1; 7.3; 8.2 7.73 ± 0.438 110.4 5.6 AdsDPV 3.0 3.1; 3.2; 2.3; 3.3; 3.1 3.14 ± 0.155 104.9 4.9 5.0 5.2; 5.0; 5.3; 5.4; 4.7 5.13 ± 0.163 102.7 3.1 7.0 6.0; 7.9; 6.8; 6.9; 7.3 6.96 ± 0.591 99.42 8.4 f-test F-test fd1* = 1.80 Fd1 *= 1.50 fd2* = 0.57 Fd2* = 3.06 fd3* = 1.89 Fd3* = 2.42 fk = 2.31 (N-2 = 8 at 95% confidence level); Fk = 6.39 (N1-1 = 4 and N2-1 = 4 at 95% confidence level) *1, 2 and 3 represent the data obtained at concentrations of 3.0, 5.0 and 7.0 nm, respectively. Aydar et al.: Low-level Electrochemical Analysis ... Acta Chim. Slov. 2020, 67, 729-738 737 vestigated for the determination of KC. The electrochemical determination of KC was successfully developed on the surface-modified electrode in the shampoo sample. No significant interference was found in the analysis of KC except for some cations. The linear working ranges, LOD, and LOQ values obtained by the developed methods were found to be superior to the methods in the literature. The acceptable recovery and low relative standard deviation data demonstrated that the accuracy and precision of the developed methods were satisfactory. Acknowledgements We gratefully acknowledge the financial support provided by Ankara University, Scientific Research Fund (Project number: 17H0430009). 5. References 1. M. El Ries, M. A. Ghany, L. Hussin, F. M. El-Anwar and A. Mohamed, Bull. Fac. Pharm. Cairo Univ. 2013, 51, 49-55. DOI:10.1016/j.bfopcu.2011.09.001 2. J. Borowiec, L. Wei, L. Zhu and J. Zhang, Anal. Methods 2012, 4, 444-448. DOI:10.1039/c2ay05615a 3. K. Mielech-tukasiewicz and K. Roginska, Anal. Methods 2014, 6, 7912-7922. DOI:10.1039/C4AY01421A 4. S. S. Rane and P. Padmaja, J Pharm Anal 2012, 2, 43-47. DOI:10.1016/j.jpha.2011.10.004 5. E. R. 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DOI:10.1021/ic0103087 Aydar et al.: Low-level Electrochemical Analysis 738 Acta Chim. Slov. 2020, 67, 729-738 Povzetek V predstavljeni študiji smo pripravili elektrodo iz ogljikove paste, modificirano z nano-sepiolitom (CCPE) in jo uporabili za določitev ketokonazola (KC). Raziskali smo vpliv pH, deleža elektrodnega modifikatorja, depozicijskega potenciala in časa depozicije. Ketokonazol daje samo en ireverzibilni oksidacijski vrh pri vrednosti potenciala okrog 0,6-0,7 V pri različnih vrednostih pH. CV študija je pokazala, da modificirana elektroda katalitsko učinkuje na maksimalni signal KC v primerjavi z golo elektrodo. To katalitsko obnašanje CCPE smo uporabili za razvoj občutljive metode detekcije. Raziskali smo vpliv pH in hitrosti preleta na maksimalni anodni potencial in tok. Rezultati za hitrost preleta pokažejo, da je oksidacijsko obnašanje KC kontrolirano s procesom adsorpcije na površino CCPE. Zaradi tega smo za analizo KC razvili metodi adsorpcijske inverzne diferencialne pulzne voltametrije (AdsDPV) in adsorpcijske inverzne pravokwwot-no-pulzne voltametrije (AdsSWV). Za AdsDPV smo dobili dve različni linearni območji: (0,1-1,0) nM in (3,0-10,0) nM, za AdsSWV pa (0,1-10,0) nM in (3,0-10,0) nM. Meje zaznave (LOD) in meje določitve (LOQ) smo določili kot 0,017 nM in 0,056 nM pri AdsDPV ter 0,025 nM in 0,083 nM pri AdsSWV. Predlagani novi senzor je dosegal zelo visoke izkoristke oz. točnost pri analizi KC v farmacevtskem šamponu. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Aydar et al.: Low-level Electrochemical Analysis ... DOI: I0.i7344/acsi.20i9.5648 Acta Chim. Slov. 2020, 67, 739-747 ©cwnmons Scientific paper The Effect of Hydrogen Bonding and Azome thine Group Orientation on Liquid Crystal Properties in Benzylidene Aniline Compounds Abdullah Hussein Kshash Department of Chemistry, Education College for Pure Science, University Of Anbar, 31001, Ramadi, Anbar, Iraq * Corresponding author: E-mail: fdrabdullahkshash@gmail.com Tel: +964-7830818171 Received: 10-25-2019 739 Abstract This study examines the effects of substituents and hydrogen bonding, orientations of imine linkage on the behavior of benzylidene aniline compounds as liquid crystals (LC). Compounds 4-carboxy benzylidene-4-X-aniline (X = H, F, Cl, Br, CH3, OCH3) 1a-6a were synthesized by the reaction of aniline and its substituted derivatives with 4-formylbenzoic acid. Compounds 4-X-benzylidene-4-carboxy aniline (X = H, F, Cl, Br, CH3, OCH3) 1b-6b were synthesized by the reaction of benzaldehyde and its substituted derivatives with 4-aminobenzoic acid using absolute ethanol as the solvent. Synthesized compounds were characterized by FT IR and 'H NMR spectroscopy, liquid crystal properties were investigated using differential scanning calorimetry (DSC) and polarizing optical microscopy (POM) techniques. Based on the mesomorphic properties, it was proven that the compounds 2b-4b are dimorphic exhibiting a smectic and nematic phase, compounds 5b, 6b are monomorphic exhibiting a nematic phase, while compounds 1a-6a and 1b have not shown any mesophase. For compounds 1a-6a hydrogen bonding and reversing imine linkage (in comparison with compounds 1b-6b) caused the absence of their mesomorphic properties. Keywords: Benzylideneaniline, liquid crystals, hydrogen bonding, nematic phase, smectic phase, 4-formylbenzoic acid. 1. Introduction Molecules of liquid crystals (LCs) with low molecular masses consist of a central core, generally containing phenyl rings linked by a double bond(s) and terminal groups such as alkyl and alkoxy chains, which promote molecular crystallinity and lower melting points.' Hydrogen bonding is an intermolecular attractive interaction between the hydrogen atom of a molecule X-H, where H is less electronegative than X, and a Y atom that possesses one pair of electrons in the same or another molecule, therefore, hydrogen bond donor is X-H, and the acceptor is Y or a n-bond. Three-dots symbol (•••) is usually used to depict the hydrogen bonding, such as X- H—Y-Z, where both atoms X and Y could be F, O and N.2,3 The association of some molecules via hydrogen bonding enhances the mesogenic properties of these molecules by the formation of homodimers, heterodimers, and complex structures. The first enhancement was the homodimeriza-tion of M-alkoxybenzoic acids 1 and M-alkylthio benzoic acids 2 via hydrogen bonding and formation of a supra-molecular nucleus.4,5 Heterodimerization can occur in different ratios to form supramolecular liquid crystals. For instance, hetero-complex 3 can be obtained in ratio 1:1 via the formation of Kshash et al.: The Effect of Hydrogen Bonding and Azomethine 740 Acta Chim. Slov. 2020, 67, 739-747 a single hydrogen bond (H—N) between 4-alkoxybenzoic acid and pyridine fragment.6 being more parallel to benzylidene ring than to the aniline ring12 (Fig. 1). On the other hand, heterocomplex 4 is formed in ratio 2:1 via the formation of two hydrogen bonds between the bipyridyl fragment and two carboxylic acid molecules.7 Inverted approaches of ratio 2:1 can also be found, such as in heterocomplex 5 between a dicarboxylic acid and two pyridine fragments.8 Halogen bonding is defined as an interaction between the halogen atom X and an electronegative atom A, which is generally depicted by the dotted line: D-X—A.9 The electron density around the halogen is polarized and distributed anisotropically and can additionally be amplified when halogen atom is bonded to an electron-withdrawing group.10 Nguyen and his colleagues reported that there are no mesomorphic properties of 4-alkoxy-4'-stil-bazole 6. °CnH2ll+| n= 4,6,8,10,12 Nevertheless, the mixing of equimolar amounts of stilbazoles 6 and pentafluoroiodobenzene can cause interactions between the nitrogen atom in the pyridine ring and the electronic iodine density by forming a halogen bond, that can also induce the formation of complex 7 which is exhibiting nematic and smectic phases.11 Thus, the anisotropic complex formed by halogen bonding extends the rigid-rod motif for the molecule and induces liquid crystal properties. Non-planar N-benzylidene aniline is the simplest compound of Schiff base structure; torsion angle for N-phenyl bond is around 55°, and about 10° for the benzylidene ring, so the n orbitals of azomethine group are Figure 1. The torsion angle for benzylidene aniline compounds. This study aims to investigate the influence of hydrogen bonding and the orientation of the imine linkage on liquid crystalline properties of the benzylidene aniline compounds, using 4-carboxy-benzylidene-4-X-aniline (X-C6H4-N=CH-C6H4-COOH) 1a-6a and 4-X-benzylidene-4-car-boxy-aniline (X-C6H4-CH=N-C6H4-COOH) 1b-6b as models, where X = H, F, Cl, Br, CH3, OCH3. 2. Experimental Section 2. 1. Material and Methods All chemicals were purchased from Sigma-Aldrich. They were used without further purification. Infrared spectra were recorded as ATR using Bruker-Tensor 27 spectrometer. 1H NMR spectra were recorded using Bruker 400 MHz spectrometer and DMSO-d6 as the solvent. Measurements of phase transition temperatures were made using Mettler Toledo DSC 823 (DSC) at a heating rate of 10 °C min-1, and POM equipped with hot stage. 2. 2. Synthesis of Schiff Bases To a 50 mL round-bottomed flask, that contains 20 mL of absolute ethanol, 7 mmol of aromatic aldehyde, and 5 drops of glacial acetic acid, was added 7 mmol of aromatic amine dissolved in 10 mL of absolute ethanol; the mixture was then refluxed for 3 h, thereafter cooled down to room temperature, the solid precipitate obtained was filtered, washed with cooled EtOH and recrystallized from EtOH. The compounds of 4-carboxy benzylidene-4-X-ani-line (Fig. 2) have been characterized as follows: X - H (la), F (2a), Cl (3a), Br (4a), -CH3 (5a), -OCH3 (6a) Figure 2. Molecular structure for compounds 1a-6a. 4-Carboxybenzylideneaniline (1a).13 White solid, yield 82%; m.p. 219-221 °C, IR (ATR) cm-1 2500-3500 (O-H carboxylic acid), 3077 (C-H aromatic), 3032 (vH-CN), 1679 (C=O carboxylic acid), 1620 (v C=N). 1H NMR: 6 Kshash et al.: The Effect of Hydrogen Bonding and Azomethine ... Acta Chim. Slov. 2020, 67, 739-747 741 13.18 (s, 1H, H-15), 8.73 (s, 1H, H-8), 8.08 (d, J = 8.1 Hz, 2H, H-11, H-13), 8.06 (d, J = 8.3 Hz, 2H, H-3, H-7), 7.45 (t, J = 7.7 Hz, 2H, H4, H-6), 7.32 (d, J = 7.8 Hz, 2H, H-10, H-14), 7.29 (t, J = 7.3 Hz, 1H, H-5). 4-Carboxybenzylidene-4-fluoroaniline (2a). Pale yellow solid, yield 65%; m.p. 230-232 °C, IR (ATR) cm-1 25003500 (O-H carboxylic acid), 3057 (C-H aromatic), 3031 (vH-CN), 1675 (C=O carboxylic acid), 1620 (v C=N). 1H NMR: 5 13.18 (s, 1H, H-15), 8.74 (s, 1H, H-8), 8.08 (d, J = 8.1 Hz, 2H, H-11, H-13), 8.05 (d, J = 8.1 Hz, 2H, H-3, H-7), 7.28 (t, J = 8.6 Hz, 2H, H-4, H-6), 7.40 (m, 2H, H-10, H-14). 4-Carboxybenzylidene-4-chloroaniline (3a). Pale yellow solid, yield 69%; m.p. 235-237 °C, IR (ATr) cm-1 25003500 (O-H carboxylic acid), 3064 (C-H aromatic), 3033 (vH-CN), 1675 (C=O carboxylic acid), 1620 (v C=N). 1H NMR: 5 13.24 (s, 1H, H-15), 8.75 (s, 1H, H-8), 8.08 (d, J = 8.4 Hz, 2H, H-11, H-13), 7.50 (d, J = 8.2 Hz, 2H, H-3, H-7), 8.06 (d, J = 8.1 Hz, 2H, H-4, H-6), 7.36 (d, J = 8.4 Hz, 2H, H-10, H-14). 4-Carboxybenzylidene-4-bromoaniline (4a). Pale yellow solid, yield 74%; m.p. 266-268 °C, IR (ATR) cm-1 25003500 (O-H carboxylic acid), 3061 (C-H aromatic), 3034 (vH-CN), 1680 (C=O carboxylic acid), 1621 (v C=N). 1H NMR: 5 13.23 (s, 1H, H-15), 8.74 (s, 1H, H-8), 8.07 (d, J = 8.7 Hz, 2H, H-11, H-13), 7.63 (d, J = 8.2 Hz, 2H, H-3, H-7), 8.05 (d, J = 8.2 Hz, 2H, H-4, H-6), 7.29 (d, J = 8.2 Hz, 2H, H-10, H-14). 4-Carboxybenzylidene-4-methylaniline (5a). Pale yellow solid, yield 82%; m.p. 238-240 °C, IR (ATR) cm-1 25003500 (O-H carboxylic acid), 3078 (C-H aromatic), 3028 (vH-CN), 1679 (C=O carboxylic acid), 1621 (v C=N). 1H NMR: 5 13.23 (s, 1H, H-15), 8.73 (s, 1H, H-8), 8.14 (d, J = 8.0 Hz, 2H, H-11, H-13), 8.07 (d, J = 8.3 Hz, 2H, H-3, H-7), 8.03 (d, J = 6.0 Hz, 2H, H-4, H-6), 8.04 (d, J = 5.9 Hz, 2H, H-10, H-14), 2.34 (s, 3H, CH3). 4-Carboxybenzylidene-4-methoxyaniline (6a). Pale yellow solid, yield 82%; m.p. 212-214 °C, IR (ATR) cm-1 2500-3500 (O-H carboxylic acid), 3076 (C-H aromatic), 3032 (vH-CN), 1682 (C=O carboxylic acid), 1619 (v C=N). 1H NMR: 5 12.63 (s, 1H, H-15), 8.74 (s, 1H, H-8), 8.06 (d, J = 8.0 Hz, 2H, H-11, H-13), 7.36 (d, J = 8.3 Hz, 2H, H-3, H-7), 7.01 (d, J = 8.4 Hz, 2H, H-4, H-6), 8.02 (d, J = 7.9 Hz, 2H, H-10, H-14), 3.79 (s, 3H, OCH3). X = H (I b), F (2b), CI (3b), Br (4b), -CH3 (5b), -OCH3 (6b) Figure 3. Molecular structures for compounds 1b-6b. The compounds of 4-X-benzylidene-4-carboxyani-line (Fig. 3) were characterized as follows: Benzylidene-4-carboxyaniline (1b). Pale yellow solid, yield 71%; m.p. 194.8 °C, IR (ATR) cm-1 2500-3500 (O-H carboxylic acid), 3068 (C-H aromatic), 3031 (vH-CN), 1677 (C=O carboxylic acid), 1622 (v C=N). 1H NMR: 5 12. 83 (s, 1H, H-15), 8.65 (s, 1H, H-8), 7.97 (d, J = 8.3 Hz, 2H, H-11, H-13), 7.56 (d, J = 7.5 Hz, 2H, H-3, H-7), 7.37-730 (m, 2H, H-4, H-6), 7.58 (d, J = 8.1 Hz, 2H, H-10, H-14), 7.37-7.30 (m, 1H, H-5). 4-Fluorobenzylidene-4-carboxyaniline (2b). Pale yellow solid, yield 88%; m.p. 249 °C, IR (ATR) cm-1 2500-3500 (O-H carboxylic acid), 3071 (C-H aromatic), 3019 (vH-CN), 1678 (C=O carboxylic acid), 1625 (v C=N). 1H NMR: 5 12.35 (s, 1H, H-15), 8.64 (s, 1H, H-8), 7.99 (d, J = 8.5 Hz, 2H, H-11, H-13), 7.32 (d, J = 8.0 Hz, 2H, H-3, H-7), 7.45 (t, J = 8.6 Hz, 2H, H-4, H-6), 7.62 (d, J = 8.2 Hz, 2H, H-10, H-14). 4-Chlorobenzylidene-4-carboxyaniline (3b). Pale yellow solid, yield 79%; m.p. 270.4 °C, IR (ATR) cm-1 2500-3500 (O-H carboxylic acid), 3073 (C-H aromatic), 3013 (vH-CN), 1677 (C=O carboxylic acid), 1624 (v C=N). 1H NMR: 5 12.38 (s, 1H, H-15), 8.66 (s, 1H, H-8), 7.98 (d, J = 8.1 Hz, 2H, H-11, H-13), 7.33 (d, J = 7.6 Hz, 2H, H-3, H-7), 7.93 (d, J = 8.0 Hz, 2H, H-4, H-6), 7.61 (d, J = 7.9 Hz, 2H, H-10, H-14). 4-Bromobenzylidene-4-carboxyaniline (4b). Pale yellow solid, yield 80%; m.p. 289 °C, IR (ATR) cm-1 2500-3500 (O-H carboxylic acid), 3070 (C-H aromatic), 3018 (vH-CN), 1679 (C=O carboxylic acid), 1621 (v C=N). 1H NMR: 5 12.33 (s, 1H, H-15), 8.66 (s, 1H, H-8), 7.99 (d, J = 8.1 Hz, 2H, H-11, H-13), 7.34 (d, J = 8.0 Hz, 2H, H-3, H-7), 7.91 (d, J = 8.1 Hz, 2H, H-4, H-6), 7.62 (d, J = 8.2 Hz, 2H, H-10, H-14). 4-Methylbenzylidene-4-carboxyaniline (5b). Pale yellow solid, yield 74%; m.p. 256.2 °C, IR (ATR) cm-1 2500-3500 (O-H carboxylic acid), 3070 (C-H aromatic), 3032 (vH-CN), 1678 (C=O carboxylic acid), 1627 (v C=N). 1H NMR: 5 12.50 (s, 1H, H-15), 8.59 (s, 1H, H-8), 7.98 (d, J = 8.1 Hz, 2H, H-11, H-13), 7.35 (d, J = 7.7 Hz, 2H, H-3, H-7), 7.31 (d, J = 8.0 Hz, 2H, H-4, H-6), 7.86 (d, J = 7.7 Hz, 2H, H-10, H-14), 2.39 (s, 3H, CH3). 4-Methoxybenzylidene-4-carboxyaniline (6b). Pale yellow solid, yield 72%; m.p. 289 °C, IR (ATR) cm-1 25003500 (O-H carboxylic acid), 3063 (C-H aromatic), 3030 (vH-CN), 1678 (C=O carboxylic acid), 1626 (v C=N). 1H NMR: 5 12.61 (s, 1H, H-15), 8.55 (s, 1H, H-8), 7.97 (d, J = 8.2 Hz, 2H, H-11, H-13), 7.29 (d, J = 8.1 Hz, 2H, H-3, H-7), 7.09 (d, J = 8.3 Hz, 2H, H-4, H-6), 7.91 (d, J = 8.2 Hz, 2H, H-10, H-14), 3.85 (s, 3H, OCH3). Kshash et al.: The Effect of Hydrogen Bonding and Azomethine ... 742 Acta Chim. Slov. 2020, 67, 739-747 3. Results and Discussion 3. 1. Synthesis Target Schiff bases were synthesized by the condensation reactions of aromatic aldehydes and aromatic 3 2 Characterization stituted aniline. Compounds 1b-6b were synthesized by the reaction of 4-amino benzoic acid with 4-substituted benzaldehyde (Scheme 1). amines using absolute ethanol as the solvent and glacial acetic acid as the catalyst. Compounds 1a-6a were synthe- FT IR spectra for compounds 1a-6a and 1b-6b sized by the reaction of 4-formylbenzoic acid with 4-sub- showed the absence of NH2 group stretching vibration X = H (lb), F (2 b), CI (3b), Br (4b), -CH, (5b), -OCH-, (6b) Scheme 1. Synthetic route for compounds 1a-6a and 1b-6b. 8.2 3.1 s.o 7.9 7.i 7.7 7.6 7.5 fl (ppm) Us 7 A 7.3 7.2 I I _ —1—'—1—1—1—1—1—'—1—«—1—'—1—'—;—1—1—"—1—1—1—■—1—i—1—1—1—1—i—1—1—1—1—■—1—1—1—1—1—■—1—1—1—1—1—1—]—1—1—■—1—i—1—1—1—1—s—1—1—1—1—■ 15.0 14.5 14.0 13.5 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 B.O 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 fl (ppm) Figure 4. 1H NMR spectrum of compound 2b. Kshash et al.: The Effect of Hydrogen Bonding and Azomethine ... Acta Chim. Slov. 2020, 67, 739-747 743 Figure 5. A schematic of the hydrogen bond in compounds 1a-6a and 1b-6b. bands for aromatic amine and vC=O group absorption band for aromatic aldehyde. FT IR spectra for compounds 1a-6a revealed a broad absorption within the range 2500-3500 cm-1 which is attributable to the O-H group, medium absorption within the range 3057-3078 cm-1 attributable to the aromatic C-H, strong absorption band within the range 1675-1682 cm-1 attributable to the C=O group of carboxylic acid, and absorption within the range 1619-1621 cm-1 for the C=N group. FT IR spectra for compounds 1b-6b showed the appearance of a broad absorption band within the range 2500-3500 cm-1 attributable to the O-H group, an absorption band within a range 3063-3073 cm-1 that was attributable to aromatic C-H, and a strong absorption band within the range 1677-1679 cm-1 that was attributable to C=O group of carboxylic acid, besides absorption band for C=N group observed within the range 1621-1627 cm-1. Further identification for Schiff bases was performed using 1H NMR, spectra of compounds 1a-6a were comprised of a singlet signal within the range 12.63-13.24 ppm attributed to the proton of the hydroxyl group (-COOH); a singlet signal at the down-field region within the range 8.73-8.75 ppm which evidenced the presence of the proton of the azome-thine group (H-C=N) and several different signals within the range 7.01-8.14 ppm which are ascribed to aromatic protons. 1H NMR spectra for compounds 1b-6b showed a singlet signal within the range 12.35-12.83 ppm which is ascribed to the proton of the hydroxyl group (-COOH); a singlet signal within the range 8.55-8.66 ppm which was attributed to the proton of the azomethine group (H-C=N); and several signals within the range 7.29-7.99 ppm which were attributed to aromatic protons. The 1H NMR spectrum for compound 2b is shown in Fig. 4 as a representative illustration. Chemical shift values (12.63-13.24 ppm) for the proton of the hydroxyl group in compounds 1a-6a suggest that these compounds tend to form stabilized di-mers by hydrogen bonding between carboxyl groups, while the chemical shift values (12.63-13.24 ppm) for compounds 1b-6b suggest that these compounds prefer to form weak hydrogen bonds between the carbox-yl group and terminal substituent groups on the ben-zylidene ring (Fig. 5). 3. 3. A Study of the Mesomorphic Properties of the Synthesized Compounds Polarised optical microscopy and differential scanning calorimetry were used to study the mesomorphic properties of synthesized compounds, employing careful monitoring by POM during heating and cooling scans and subsequently verified by the DSC measurements. The results showed that there are no mesomorphic properties for compounds 1a-6a, owing to spontaneous carboxylic dimerization via intermolecular hydrogen bonding and weakness in the lateral attractive force (Fig. 5). On the other hand, it was found that the melting points of these compounds increase as the terminal halogen atom size increased (Fig. 6). Mesomorphic properties of synthesized compounds 1b-6b were investigated by DSC and POM and the measurements were very similar. Transition temper- Br F Cl / H Figure 6. The dependence of melting points on the size of substituent atoms. Kshash et al.: The Effect of Hydrogen Bonding and Azomethine ... 744 Acta Chim. Slov. 2020, 67, 739-747 Tablel. Mesomorphic data and structures for compounds 1b-6b Comp. Transition Peak temp. °C DTSm DTn AH (kJ mol-1) AH (J mol1 K 1b Cr-I 197.4 - - - - Cr-Sm 166 0.2531 0.5766 2b Sm-N 197.7 31.7 70.6 7.7838 16.5365 N-I 268.3 9.1891 16.9760 Cr-Sm 224 1.5491 3.1166 3b Sm-N 263.5 39.5 14.5 2.8153 5.2475 N-I 278 2.5721 4.6680 Cr-Sm 225.2 1.4613 2.9332 4b Sm-N 247.9 22.7 28.1 1.0373 1.9913 N-I 276 0.8338 1.5186 5b Cr-N 242.1 24.2 7.3093 14.1900 N-I 266.3 7.3193 13.5718 6b Cr-N 202.9 88.9 8.7821 18.4535 N-I 291.8 1.0423 1.8454 ) atures and associated AH, AS are listed in Table 1. The investigation revealed no mesomorphic behavior for compound 1a, whereas, compounds 2b-4b were dimorphic exhibiting smectic and nematic phase, furthermore, compounds 5b and 6b were monomorphic exhibiting nematic phase. Smectic mosaic and nematic Schlieren textures were observed during POM investigation, optical photomicrographs are shown in Fig. 7 and the DSC thermogram of 6b is shown in Fig. 8 as a representative illustration. 3. 5. Influence of Reverse Imine Linkage on Mesomorphic Properties Despite the structural similarity of compounds 1a-6a and 1b-6b, the mesomorphic properties are quite Figure 7. Polarized optical micrographs for: (a) compound 2b (smectic at 166 °C); (b) compound 2b (nematic, Schlieren at 197.7 °C); (c) compound 3b (smectic at 224 °C), (d) compound 3b (nematic, Schlieren at 263.5 °C); (e) compound 4b (smectic at 225.2 °C); (f) compound 4b (nematic, Schlieren at 247.9 °C); (g) compound 5b (nematic, Schlieren at 242.1 °C); (h) compound 6b (nematic, Schlieren at 202.9 °C). Kshash et al.: The Effect of Hydrogen Bonding and Azomethine ... Acta Chim. Slov. 2020, 67, 739-747 745 1 j iTtt »•I99.K Ito=2S9.0C ro=29l8C H> »■to;«» KMÛ829JÏ 231 Tcop(0 it: 314 355 397 Figure 8. DSC thermogram for compound 6b. different. No mesomorphic properties were found for compounds 1a-6a, while inversions of imine linkage orientation in compounds 1b-6b enhanced the mesomorphic properties, except for compound 1b due to the absence of a terminal group (in 1b) and its dimerization by intermolecular hydrogen bonding. Orientations of inverted imine linkage and carboxyl group beside terminal sub-stituents cause remarkable changes on the dipole moment and improve the polarisability of the molecule by conju- gation between azomethine and terminal substituents via the phenyl ring for compounds 2b-4b, thus, increasing the lateral attraction force to enhance the formation of smectic phase and terminal attraction force by hydrogen bonding to enhanced nematic phase formation11 (Fig. 9). The appearance of the nematic phase in compounds 5b and 6b was enhanced by the terminal hydrogen bonding between carboxyl and a n orbital created by hypercon-jugation,12 while in compound 6b hydrogen bonding was between carboxyl group and the oxygen atom of the meth-oxy group (Fig. 10). 4. Conclusion Benzylidene aniline compounds were synthesized and characterized. The study indicates that the hydrogen bonding and the orientation of inverted imine C=N linkage play a significant role in liquid crystals behavior, especially in compounds 1a-6a and 1b, which is preventing the formation of liquid crystals owing to their dimeriza-tion through hydrogen bonding, while the liquid crystal properties of compounds 2b-6b were improved by the lateral interactions and terminal hydrogen bonding occurring between carboxyl and terminal substituent group. Figure 9. Hydrogen bonds for compounds 2b-4b at the smectic and nematic phases. Kshash et al.: The Effect of Hydrogen Bonding and Azomethine ... 746 Acta Chim. Slov. 2020, 67, 739-747 compound 6h Figure 10. Hydrogen bond for compounds 5b and 6b at the nematic phase. Acknowledgments The author is grateful to Ms. Alaa Adnan Rashad, Al-Nahrain University, for her help in carrying out some of the measurements in this research. 5. References 1. B. Bai, H. Wang, H. Xin, B. Long, M. Li, Liq. Cryst. 2007, 34, 659-665. DOI: 10.1080/02678290701328118 2. T. Steiner, Angew. Chem. Int. Ed. 2002, 41, 48-76. D0I:10.1002/1521-3773(20020104)41:1<48::AID-ANIE48 >3.0.C0;2-U 3. E. Arunan, G. R. Desiraju, R. A. Klein, J. Sadlej, S. Scheiner, I. Alkorta, D. C. Clary, R. H. Crabtree, J. J. Dannenberg, P. Hob-za, H. G. Kjaergaard, A. C. Legon, B. Mennucci, D. J. Nesbitt, Pure Appl. Chem. 2011, 83, 1637-1641. D0I:10.1351/PAC-REC-10-01-02 4. G. W. Gray, B. Jones, J. Chem. Soc. 1954, 2556-2562. D0I:10.1039/jr9540002556 5. Y. Arakawa, Y. Sasaki, K. Igawa, H. Tsuji, New J. Chem. 2017, 41, 6514-6522. D0I:10.1039/C7NJ00282C 6. T. Kato, J. M. J. Frechet, J. Chem. Soc. 1989, 111, 8533-8534. DOI: 10.1021/ja00204a044 7. T. Kato, J. M. J. Frechet, P. G. Wilson, T. Saito, T. Uryu, A. Fujishima, C. Jin, F. Kaneuchi, Chem. Mater. 1993, 5, 10941100. D0I:10.1021/cm00032a012 8. T. Kato, A. Fujishima, J. M. J. Frechet, Chem. Lett. 1990, 19, 919-922. D0I:10.1246/cl.1990.919 9. M. Fourmigue, Curr. Opin. Solid State Mater. Sci. 2009, 13, 36. D0I:10.1016/j.cossms.2009.05.001 10. B. Bankiewicz, M. Palusiak, Struct. Chem. 2013, 24, 12971306. D0I:10.1007/s11224-012-0157-1 11. H. L. Nguyuen, P. N. Horton, M. B. Hursthouse, A. C. Legon, D. W. Bruce, J. Am. Chem. Soc. 2004, 126, 16-17. D0I:10.1021/ja036994l 12. H. B. Bürgi, J. D. Dunitz, Helv. Chim. Acta, 1970, 53, 17471764. D0I:10.1002/hlca.19700530724 13. L. Ovari, Y. Luo, F. Leyssner, R. Haag, M. Wolf, P. Tegeder, J. Chem. Phys. 2010, 133, 1-8. D0I:10.1063/1.3460647 Kshash et al.: The Effect of Hydrogen Bonding and Azomethine ... Acta Chim. Slov. 2020, 67, 739- 747 747 Povzetek V tej študiji smo raziskali učinke substituentov in vodikovih vezi ter orientacije iminskih povezav na obnašanje benzi-liden anilinskih spojin v vlogi tekočih kristalov (LC). 4-Karboksibenziliden-4-X-aniline (X = H, F, Cl, Br, CH3, OCH3) 1a-6a smo sintetizirali s pomočjo reakcije anilina in njegovih substituiranih derivatov s 4-formilbenzojsko kislino; 4-X-benziliden-4-karboksi aniline (X = H, F, Cl, Br, CH3, OCH3) 1b-6b pa smo pripravili z reakcijo anilina in njegovih substituiranih derivatov s 4-aminobenzojsko kislino v absolutnem etanolu kot topilu. Pripravljene spojine smo karak-terizirali s pomočjo FT IR in 'H NMR spektroskopije, lastnosti tekočih kristalov pa smo raziskali s pomočjo diferenčne dinamične kalorimetrije (DSC) in s polarizirano optično mikroskopijo (POM). Glede na mezomorfne lastnosti smo dokazali, da so spojine 2b-4b dimorfne in izkazujejo smektično in nematično fazo, spojini 5b, 6b sta monofazni in izkazujeta nematično fazo, spojine 1a-6a and 1b pa niso pokazale mezofaznega obnašanja. Pri spojinah 1a-6a se zaradi vodikovih vezi in inverzne iminske povezave (glede na spojine 1b-6b) mezofazne lastnosti niso pojavile. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Kshash et al.: The Effect of Hydrogen Bonding and Azomethine ... 748 Acta Chim. Slov. 2020, 67, 748-756 ©common DOI: I0.i7344/acsi.20i9.5685 Acta Chim. Slov. 2020, 67, 748-756 V£2commons Scientific paper Ionic Liquid-Based Dispersive Liquid-Liquid Microextraction for the Simultaneous Determination of Carbamazepine and Lamotrigine in Biological Samples Salumeh Ranjbar, Ameneh Porgham Daryasari* and Mojtaba Soleimani Department of Chemistry, Lahijan Branch, Islamic Azad University, Lahijan, Iran P.O.Box 4416939515 * Corresponding author: E-mail: porgham54@gmail.com Tel.: +981342230561; Fax: +981342224756 Received: 11-07-2019 Abstract This paper describes a new approach for the determination of carbamazepine and lamotrigine in biological samples by ionic liquid dispersive liquid-phase microextraction prior to high-performance liquid chromatography with ultraviolet detection. The effects of different ionic liquids (ILs) on the extraction efficiency of carbamazepine and lamotrigine were investigated. The highest extraction efficiencies of carbamazepine and lamotrigine were obtained using 30 |L of 1-me-thyl-3-octylimidazolium hexafluorophosphate [C8MIM][PF6]. Several factors affecting the microextraction efficiency, such as the type and volume of extracting solvent, type and volume of disperser solvent, salt concentration, and pH of the sample solution have been optimized. The calibration plots were linear in the range of 0.1-20 mg L-1 for carbamazepine and 0.3-40 mg L-1 for lamotrigine with detection limits of 0.04 mg L-1 for carbamazepine and 0.07 mg L-1 for lamotrigine in plasma samples. The results confirm the suitability of the presented method as a sensitive method for the analysis of the target analytes in urine and plasma samples. Keywords: Ionic liquids; dispersive liquid-liquid microextraction; carbamazepine; lamotrigine; human urine; human plasma 1. Introduction One of the most common serious neurological disorders is epilepsy.1 Anti-epileptic drugs (AEDs) are the main form of treatment for epilepsy. Carbamazepine and newer AEDs like lamotrigine (LTG) are among the firstline medicines for treatment of seizures.2 Lamotrigine (LTG), chemically known as [6-(2,3-dichlorophenyl) -1,2,4-triazine-3,5-diamine], is used as monotherapy and as an adjunct with other antiepileptics for treatment of Carbamazepine Lamotrigine Figure 1. The structure of carbamazepine and lamotrigine partial and generalized toxic-clonic seizures. It's used as a tranquilizer and in the treatment of neurological lesions.3,4 Carbamazepine (CBZ) (5-H-dibenzo[fr,/]azepine-5 -carboxamide), is a first line antiepileptic drug used in the treatment of partial and generalized tonic-clonic seizures.5 The chemical structures of carbamazepine and lamotrigi-ne are shown in Figure 1. Most biological samples have complex matrices and the analytes are typically present at low concentration levels, which are not detectable by the analytical instrument. Therefore, a sample preparation step is generally required to extract, isolate, and concentrate the analytes of interest. Different analytical techniques that have been used for the determination of lamotrigine include planar chromatog-raphy,6 HPLC,7 TLC and HPLC,8 GC,9 HPLC and GC,10 capillary electrophoresis,11 and immunoassay.12 High performance liquid chromatography (HPLC-UV, HPLC-DAD) and immunoassay were used for determination of carba-mazepine in biological materials. Also, gas chromatography with mass spectrometry and liquid chromatography with mass spectrometry have been reported.13-15 Ranjbar et al.: Ionic Liquid-Based Dispersive Liquid-Liquid Acta Chim. Slov. 2020, 67, 748-756 749 Liquid-liquid extraction (LLE),16 solid-phase extraction (SPE),17 and stir bar-sorptive extraction (SBSE)18 have been developed for the determination of CBZ in biological fluids. These methods are time consuming and require substantial amounts of toxic organic solvents. The sample preparation methods employed for lamotrigine involve SPME,19 SPE,20 protein precipitation (PP),21 LLE,22 and microextraction by packed sorbent (MEPS).23 A novel microextraction method called dispersive liquid-liquid microextraction (DLLME) was introduced in 2006.24 DLLME utilizes an extraction solvent and a dispersive solvent to produce a cloudy solution. DLLME has become a very popular technique for the extraction of different compounds.25-27 Generally, the extraction solvent used in DLLME is highly toxic and not environmentally friendly. In environmentally friendly sample preparation methods, it is important to use liquid solvents in reduced amounts, replaced with green solvents or even completely eliminated from the analytical procedure.28 Ionic liquids (ILs) are considered to be "environmentally friendly solvents".29 The immiscibility of ILs in water and their capability to solubilize organic species has made them suitable to extract the compounds.30 Recently, ionic liquid DLLME is very popular.31 In this paper, for the first time, DLLME method using IL as extraction solvent combined with high-performance liquid chromatography has been developed for the simultaneous determination of carbamazepine and lamo-trigine in biological samples. The parameters affecting the extraction efficiency, such as the type and volume of extracting solvent, type and volume of disperser solvent, salt concentration, and pH of the sample solution have been optimized. The proposed method was successfully applied to determine carbamazepine and lamotrigine in biological samples. 2. Experimental 2. 1. Chemicals and Reagents Carbamazepine and lamotrigine were obtained from Sobhan Darou Company (Rasht, Iran). Acetone, acetoni-trile, methanol, and sodium chloride were obtained from Merck Company (Germany). Ionic liquids (ILs) [C8MIM] [PF6] and [C6MIM][PF6] were obtained from SIG-MA-ALDRICH. IL [C4MIM][PF6] was obtained from Fluka. Buffer solution (disodium hydrogen phosphate -potassium dihydrogen phosphate, pH = 6.88) was obtained from Merck. Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium hydrogen carbonate, and disodium carbonate were obtained from Merck. De-ionized water was used in all experiments. Stock standard solutions of the analytes were prepared by dissolution of each drug in methanol, having a concentration of 1000 mg L-1. Fresh standard solutions were prepared by diluting the standard solution of the analytes with deionized water of required concentration. All these solutions were stored at 4 °C in the absence of light. 2. 2. Apparatus Chromatographic analysis was performed using a Shimadzu (LC-20AD prominence, Japan) with a photodiode array detector (SPD-M20A). Separations were carried out on a ^Boundapak C18 column of15 cm x 4.6 mm with 5 ^m particles. HPLC data were acquired and processed using a Lab solution software (LC solution version 1.25 SP5). The mobile phase was phosphate buffer (pH 6.8) -methanol - acetonitrile (70:20:10, v/v/v) at a flow rate of 1.0 mL min-1 under isocratic conditions. The detection was performed at the wavelength of 284 and 308 nm for carbamazepine and lamotrigine, respectively. In the measurement of lamotrigine (LTG) and carbamazepine (CBZ) in the optimization steps and also determination in real samples, the mixture of both drugs were used. The maximum wavelengths for measurement of LTG and CBZ were 308 nm and 284 nm, respectively. Unfortunately, in 308 nm and 284 nm, both of the drugs have peaks. However, CBZ at 284 nm and LTG at 308 nm have peaks with high intensities. The maximum intensity of the peaks of each drug at the selected wavelengths were used for the subsequent experiments. A centrifuge model ALC 4232 was used to perform the centrifuge process (USA). The pH-meter model 827 Metrohm (Herisau, Switzerland) was used for pH measurements. 2. 3. Dispersive Liquid-liquid Microextraction procedure Five milliliters of sample solution containing the an-alytes was poured into a centrifuge glass vial. The pH of the solution was adjusted to 10 by using sodium bicarbonate. A mixture containing 30 ^L of [C8MIM][PF6] (as extraction solvent) and 100 ^L of methanol (as disperser solvent) was injected into the sample solution. Cloudy solution was formed as the fine droplets of the immiscible extraction solvent dispersed in the sample. This process enlarged the contact area between the extraction solvent and sample, and the analytes were extracted into the formed fine droplets. Then it was placed in ice bath for 2 min. The cloudy solution was centrifuged at 3500 rpm for 10 min to separate the phases. Finally, 100 ^L methanol was added into the collected IL and injected into the HPLC system. 3. Results and Discussion In the present study, the applicability of ionic liquid DLLME combined with HPLC was considered for the simultaneous determination of carbamazepine and lamo- Ranjbar et al.: Ionic Liquid-Based Dispersive Liquid-Liquid 750 Acta Chim. Slov. 2020, 67, 748-756 trigine in biological samples. There are several factors which affect the extraction process including type and volume of extracting solvent, type and volume of disperser solvent, salt concentration and pH of the sample solution. Optimization of the variables was performed using one variable at a time method. All experiments were replicated three times. The spiked concentration level in the optimization study was 5 mg L-1 of carbamazepine and 20 mg L-1 of lamotrigine. Enrichment factor (EF) and extraction recovery (ER) were calculated based on the following equations: EF _ Csedimented/C0 C xV FR(1 _ -'■- dim eitted_sedimented ^ j qq _ C X V 0 A ' sample (1) (2) Where, EF, Csedimented and C0 are the enrichment factor, concentration of the analyte in the sedimented phase, and initial concentration of the analyte in the sample, respectively. ER%, Vsedimented and Vsample are the extraction recovery, volume of the sedimented phase, and volume of the sample, respectively. Csedimented is calculated from a suitable direct injection calibration curve. Blank urine and plasma was obtained from ten different healthy volunteers. Different sources of blank urine and plasma (n = 3) were used for testing the endogenous interferences. There were no interfering peaks at either the carbamazepine or lamo-trigine retention time. 3. 1. Effect of pH The sample pH is an important factor in the enrichment process and can affect the extraction efficiencies of the analytes. In this study, the pH values of the sample solutions were adjusted between 7 and 11 with buffers of sodium phosphate and sodium bicarbonate. As seen in Figure 2, the best peak areas were obtained at pH 10. The pKa value for CBZ is 13.13 and the pKa value for LTG is 5.3. At the pH 10, the analytes were extracted based on hydropho-bic interaction. Also, in acidic pH, the drugs were decomposed. Thus, pH 10 was selected as the optimum value. 3. 2. Selection of Extraction Solvent In the selection of the extraction solvent, certain properties of the IL that need to be considered are: (1) to extract carbamazepine and lamotrigine well; (2) to have higher density than water; and (3) to form a cloudy solution in the presence of dispersive solvent. In this study, three ionic liquids, including [C8MIM][PF6], [C6MIM] [PF6], and [C4MIM][PF6] were investigated. By comparing them as extraction solvents, it was observed that carba-mazepine and lamotrigine exhibited a better affinity for [C8MIM][PF6], because of higher solubility of the mentioned drugs in [C8MIM][PF6] (Figure 3). Therefore, [C8MIM][PF6] was selected as extraction solvent in the subsequent experiments. [C6M1M][PF6J Type of extraction solvent Figure 3. Effect of type of extraction solvent on the extraction efficiency, Extraction conditions: extraction solvent volume: 30 |iL; dispersive solvent: methanol; dispersive solvent volume: 100 |iL; concentration of NaCl (w/v): 1.0%, pH:10 250000 200000 g 150000 c3 £ 100000 50000 —*— Lamotrigine —■— Carbamazepine pH Figure 2. Effect of pH on the extraction efficiency, Extraction conditions: extraction solvent: [C4MIM][PF6], extraction solvent volume: 30 |iL; dispersive solvent: methanol; dispersive solvent volume: 100 |iL; concentration of NaCl (w/v): 1.0% 3. 3. Effect of Extraction Solvent Volume Optimization of the volume of the IL as an extraction solvent is a further step in the development of a IL-DLLME procedure. The volume of the extraction solvent can influence formation of dispersion and thus has to be optimized. In order to study the effect of extraction solvent volume, different volumes of [C8MIM][PF6] (20-50 ^L in 10 ^L intervals) were tested. It was observed (Figure 4) that the peak areas were increased by increasing the [C8MIM] [PF6] volume up to 30 ^L for carbamazepine and lamotrigine. The peak areas of the analytes decreased by increasing the volume of [C8MIM][PF6], which was an expected result due to dilution of the extracted analytes in the extraction solvent at higher volumes. Therefore, 30 ^L of [C8MIM][PF6] was selected as the optimum volume. Ranjbar et al.: Ionic Liquid-Based Dispersive Liquid-Liquid ... Acta Chim. Slov. 2020, 67, 748-756 751 Figure 4. Effect of volume of extraction solvent on the extraction efficiency, Extraction conditions: extraction solvent: [C8MIM] [PF6], dispersive solvent: methanol; dispersive solvent volume: 100 |iL; concentration of NaCl (w/v): 1.0%, pH:10 3. 4. Effect of Type of Disperser Solvent In the IL-DLLME, the disperser solvent should be soluble in the extraction solvent and miscible in the sample solution, thus enabling the formation of fine droplets of the extraction solvent. Therefore, acetonitrile, methanol, and acetone were tested as dispersive solvents. A series of sample solutions were examined using 100 ^L of each of the disperser solvents containing 30 ^L of [C8MIM][PF6]. By using acetone and acetonitrile, the cloudy solution was not formed well. It was clear that (Figure 5) the best peak areas were obtained when methanol was used as a disperser solvent. Hence, the subsequent experiments were performed using methanol as the disperser solvent. Figure 5. Effect of type of disperser solvent on the extraction efficiency, Extraction conditions: extraction solvent: [C8MIM][PF6], extraction solvent volume: 30 |iL; dispersive solvent volume: 100 |iL; concentration of NaCl (w/v): 1.0%, pH:10 3. 5. Effect of Volume of Disperser Solvent In order to study the effect of disperser solvent volume, different volumes of methanol (50, 100, 300, 500, and 1000 ^L) were used. It is clear from Figure 6 that 100 ^L methanol gave the highest peak areas. It seems that at the volume of 100 ^L, the amount of methanol was enough for effective forming of the cloudy solution. At lower volume of methanol, cloudy solution was not properly formed resulting in a decrease in the peak areas. At higher volume of methanol, the solubility of the analytes in the sample increased resulting in a decrease in the peak areas. Thus, 100 ^L was selected as the optimum volume of methanol. Figure 6. Effect of volume of methanol on the extraction efficiency, Extraction conditions: extraction solvent: [C8MIM][PF6], extraction solvent volume: 30 |iL; dispersive solvent: methanol; concentration of NaCl (w/v): 1%; pH: 10 3. 6. Salt Addition Generally, salt addition can cause a decrease in the solubility of the analytes in sample solution and enhance extraction efficiency. To evaluate the possibility of salting-out effect, the extraction efficiency was studied with the sodium chloride ranging from 0.5 to 1.5% (w/v) (Figure 7). Due to the salting-out effect, the peak areas increased as the amount of NaCl increased from 0.5 to 1.0% (w/v). By increasing the ionic strength (NaCl concentration from 1.0 to 1.5% (w/v)), a reduction of the peak areas for carbamazepine and lamotrigine were observed because of dilution effect. Based on the results, 1.0% (w/v) of NaCl was added in all the subsequent experiments. Figure 7. Effect of NaCl concentration on the extraction efficiency, Extraction conditions: extraction solvent: [C8MIM][PF6], extraction solvent volume: 30 |iL; dispersive solvent: methanol; dispersive solvent volume: 100 |iL; pH: 10 Ranjbar et al.: Ionic Liquid-Based Dispersive Liquid-Liquid 752 Acta Chim. Slov. 2020, 67, 748-756 3. 7. Analytical Performance and Method Validation Calibration data of carbamazepine and lamotrigine were obtained using IL-DLLME-HPLC system under optimum conditions. For urine samples, the linearity of calibration curve was observed in the range of 0.07-20 mg L-1 for carbamazepine and 0.17-40 mg L-1 for lamotrigine. The coefficients of determination (R2) were 0.991 and 0.997 for carbamazepine and lamotrigine, respectively. The limits of detection (LODs) based on signal-to-noise ratio (S/N) of 3, were 0.02 and 0.05 mg L-1 for carbamazepine and lamotrigine, respectively. The limits of quantification (LOQs), based on signal-to-noise ratio (S/N) of 10, were 0.07 and 0.17 mg L-1 for carbamazepine and lamotrigine, respectively. The relative standard deviation (RSD%, n = 5) at the concentration level of 5.0 mg L-1 of carbamazepine and lamotrigine were 1.7% and 5.6% for carbamazepine and lamotrigine, respectively. Enrichment factors were 35 and 26 for carbamazepine and lamotrigine, respectively. Extraction recoveries were 70 and 52% for carbamazepine and lamotrigine, respectively. However, for plasma samples, linearity was observed in the range of 0.1-20 mg L-1 for carbamazepine and 0.3-40 mg L-1 for lamotrigine. The R2 were 0.987 and 0.995 for carbamazepine and lamotrigine, respectively. The limits of detection (LODs) based on signal-to-noise ratio (S/N) of 3, were 0.04 and 0.07 mg L-1 for carbamazepine and lamotrigine, respectively. The limits of quantification (LOQs), based on signal-to-noise ratio (S/N) of 10, were 0.1 and 0.3 mg L-1 for carbamazepine and lamotrigine, respectively. The relative standard deviation (RSD%, n = 5) at the concentration level of 5.0 mg L-1 of carbamazepine and lamotrigine were 3.2% and 8.4% for carbamazepine and la-motrigine, respectively. Enrichment factors were 27 and 19 for carbamazepine and lamotrigine, respectively. Extraction recoveries were 54 and 38% for carbamazepine and lamotrigine, respectively. The selectivity of the method was evaluated by analysing six blank plasma and urine samples to evaluate the existence of matrix endogenous substances at retention times that could interfere with carbamazepine (CBZ) and lamotrigine (LTG) peaks. The analysis of blank human plasma and urine samples from six healthy volunteers confirmed the absence of endogenous interferences at the retention times of carbamazepine and lamotrigine. The stability of CBZ and LTG stock solutions were evaluated at room temperature for 8 h and 24 h and after storage at -20 °C for 10 days. Stability was calculated by comparing the pertinent responses obtained from the tested stock solution(s) with the responses of freshly prepared ones and the results are given in Table 1. According to the results obtained, CBZ and LTG was stable in human plasma and urine samples in the different storage conditions. Absolute recoveries of the analytes were determined in triplicates at high, medium and low concentrations in plasma and urine by extracting drug-free plasma and Table 1. Summary of stability of CBZ and LTG in stock solution and human plasma and human urine Drug(n = 5) Data on Stock Solution Stability 8 h at RT 24 h at RT 10 days at -20 °C CBZ Precision (%) 1.2 1.8 1.5 Accuracy (%) LTG 100.1 99.8 98.7 Precision (%) 3.5 3.7 3.0 Accuracy (%) 99.6 99.3 98.9 Data on Stability in Plasma Samples CBZ Precision (%) 3.0 3.3 3.6 Accuracy (%) LTG 98.1 97.9 97.2 Precision (%) 7.8 8.0 7.6 Accuracy (%) 98.3 97.8 98.1 Data on Stability in Urine Samples CBZ Precision (%) 1.5 1.4 1.6 Accuracy (%) LTG 98.8 99.1 97.8 Precision (%) 4.8 5.1 5.0 Accuracy (%) 98.2 98.5 97.8 Ranjbar et al.: Ionic Liquid-Based Dispersive Liquid-Liquid ... Acta Chim. Slov. 2020, 67, 748-756 753 urine samples spiked with CBZ and LTG. Recovery was calculated by comparison of the analyte peak-areas of the extracted samples with those of the unextracted analyte standards, representing 94 and 88% recovery of CBZ and LTG, respectively, in plasma and 99 and 94% recovery of CBZ and LTG, respectively, in urine. In order to evaluate the effect of matrix samples on the performance of the proposed method, determination of CBZ and LTG in human urine and plasma samples at the three different concentration levels were performed. For doing the IL-DLLME procedure on the plasma samples, some extra processes are needed. First the human plasmas were dissolved in a suitable amount of acetoni-trile such as 1:1 (v/v) reducing the matrix effect and then the mixtures were centrifuged. Secondly, they were filtered for getting a clear solution and removing the dirty solution at the bottom of test tubes. The samples was found to be free from the drugs. Therefore, specific amounts of CBZ and LTG at the three different concentration levels were spiked to the samples and analyzed by the proposed method. The spiking recoveries of the target compounds in the urine and plasma samples are summarized in Table 2. The relative recovery (RR) is obtained from the following equation: RR% = CfoUnd - Creal /Cadded X 100 (3) where Cfound, Creai, and Cadded are the concentrations of the analytes after the addition of a known amount of standard in a real sample, the concentration of the analytes in a real sample, and the concentration of a known amount of standard, which was spiked to the real sample, respectively. The relative recoveries were between 87-103% (Table 2) and showed that the matrix had negligible effect on the performance of the proposed method. The chromato-grams of the urine and plasma sample (without spiking and spiked) are shown in Figures 8 and 9, respectively. Table 3 compares the proposed method with the other extraction methods for the determination of the target analytes in biological samples. The comparison of extraction time of the proposed method with solid-phase microextraction (SPME),32 liquid-liquid extraction (LLE),33,34 and solid-phase extraction (SPE)35 for the extraction of the target analytes indicates that this novel method has a very short equilibrium time comparing to the mentioned methods and the extraction time needed for the proposed method is a few seconds. Quantitative results of the proposed method are better than for SPE35 and LLE33,34 methods. Relative standard deviation (RSD%) of the proposed method is better than for SPME32 and LLE33 methods. Also, SPE and LLE methods are time-consuming and laborious, and the large amounts of organic solvents used in the extraction procedures cause problems with regards to health and the Table 2. Determination of carbamazepine (CBZ) and lamotrigine (LTG) in human plasma and urine by IL-DLLME-HPLC-DAD Spiked concentration (mg L-1) CBZ LTG CBZ Relative recovery (% ± SD), n = 3a LTG Human urine Human plasma 2 4 10 5 14 2 4 10 5 14 20 20 93.0 ± 3.1 89.0 ± 5.4 103.0 ± 1.5 94.0 ± 2.8 101.0 ± 1.2 90.0 ± 5.0 96.0 ± 2.5 87.0 ± 9.5 97.0 ± 3.5 91.0 ± 4.5 99.0 ± 4.1 93.0 ± 3.1 a Standard deviation Figure 8. HPLC chromatograms, (a,b) before spiking with analytes in urine at the wavelength of 308 and 284 nm for lamotrigine and carbamazepine, respectively, (c) 14 mg L-1 (lamotrigine) and (d) 4 mg L-1 (carbamazepine) spiking of analytes in urine after extraction via proposed method at optimum conditions. (The retention time of LTG was 5.1 min and the retention time of CBZ was 12.1 min at the measurement in the maximum wavelengths). Ranjbar et al.: Ionic Liquid-Based Dispersive Liquid-Liquid 754 Acta Chim. Slov. 2020, 67, 748-756 uV 22500 12500 |(d) { C) 1 __ (b) 15.0 min Figure 9. HPLC chromatograms, (a,b) before spiking with analytes in plasma at the wavelength of 308 and 284 nm for lamotrigine and carbamazepine, respectively,, (c) 14 mg L-1 (lamotrigine) and (d) 4 mg L-1 (carbamazepine) spiking of analytes in plasma after extraction via proposed method at optimum conditions. (The retention time of LTG was 5.1 min and the retention time of CBZ was 12.1 min at the measurement in the maximum wavelengths). environment. Finally, the extraction solvent used in DLLME generally is highly toxic and not environmentally friendly. Ionic liquids (ILs) are considered to be "environmental friendly solvents". In the proposed work, in DLLME method, IL was used as extraction solvent. 4. Conclusions A rapid and simple method using the ionic liquid-based dispersive liquid-liquid microextraction procedure was presented to the extract and concentrate carbamazepine and lamotrigine from biological samples. Table 3. Comparison of the proposed method with other extraction methods for the determination of carbamazepine (CBZ) and lamotrigine (LTG) Dynamic Limit of Extraction Methods Sample R.S.D.% linear range (mg L-1) detection (mg L-1) time (min) Ref. SPME-GC-TSD Plasma <10 0.06-20 (CBZ); 0.2-10 (LTG) 0.06 (CBZ); 0.2 (LTG) (Limit of 15 [32] quantitation) Precipitation and liquid extraction- Serum <12 0.625-20 (CBZ, LTG) - 5 [33] GC-MS LLE-HPLC-UV Plasma <6 1.0-30 (LTG) 0.15 (LTG) 5 [34] SPE-HPLC-DAD Plasma <8 0.2-25 (CBZ) 0.02 (CBZ) 1 [35] Urine (1.7 Urine (0.07-20 Urine (0.02 (CBZ), 5.6 (CBZ), 0.17-40 (CBZ), 0.05 (LTG) (LTG) (LTG) IL-DLLME-HPLC-DAD Urine and plasma Plasma (3.2) (CBZ), 8.4 (LTG) Plasma (0.1-20 CBZ), 0.3-40 (LTG) Plasma (0.04) (CBZ), 0.07 (LTG) A few seconds This work Ranjbar et al.: Ionic Liquid-Based Dispersive Liquid-Liquid ... Acta Chim. Slov. 2020, 67, 748-756 755 The proposed microextraction method is environmentally friendly (highly toxic chlorinated solvents are not required), rapid, and with a simple set-up. The proposed method has satisfying LODs which were in the range of 0.02-0.07 mg L-1, and precisions were in the range of 1.7-8.4%. The proposed method was also applied for the analysis of drugs in urine and plasma samples and the recoveries from spiked samples were in the range of 87103%. All these results indicated that the proposed method had advantages such as good sensitivity, simplicity, easyness to operate, limited chance of exposure to the toxic solvents, and high enrichment factor. This study provides a new perspective regarding the replacement of chlorinated solvents with less-toxic solvents in DLLME and supports the use of green analytical chemistry methods. In the final experiment, the developed method was applied to the determination of carbamazepine and lam-otrigine in biological samples and the acceptable results can be achieved. Acknowledgements Financial support by Lahijan Branch, Islamic Azad University (Lahijan, Iran) during the period of this research is gratefully acknowledged. Compliance with Ethical Standards Funding There is no funding for this study. Conflict of Interest No conflict exists; author Ame-neh Porgham Daryasari declares that she has no conflict of interest. Author Salumeh Ranjbar declares that she has no conflict of interest. Author Mojtaba Soleimani declares that he has no conflict of interest. Ethical approval This article does not comprise of any studies with human participants or animals performed by any of the authors. Informed consent No humans are involved in this study. 5. References 1. H. M. Neels, A. C. Sierens, K. Naelaerts, S.L. Scharpe, G. M. Hatfield, W. E. Lambert, Clin. Chem. Lab. 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Chem. 2005, 24, 20-27. D0I:10.1016/j.trac.2004.09.005 DOI: 10.1016/j.trac.2004.09.005 31. R. Rahnama, N. Mansoursamaei, M. R. Jamali, Acta Chim. Slov. 2014, 61, 191-196. 32. M. E. C. Queiroz, S. M. Silva, D. Carvalho, J. Chromatogr. Sci. 2002, 40, 219-223. D0I:10.1093/chromsci/40.4.219 33. J. Hallbach, H. Vogel, W. G. Guder, Eur. J. Clin. Chem. Clin. Bio-chem. 1997, 35, 755-759. D0I:10.1515/cclm.1997.35.10.755 34. E. Greiner-Sosanko, S. Giannoutsos, D. R. Lower, M. A. Vir-ji, M. D. Krasowski, J. Chromatogr. Sci. 2007, 45, 616-622. D0I:10.1093/chromsci/45.9.616 35. P. Dzodic, L. Zivanovic, A. Protic, I. Ivanovic, R. Velick-ovic-Radovanovic, M. Spasic, S. Lukic, J. Serb. Chem. Soc. 2012, 77, 1423-1436. D0I:10.2298/JSC120106084D Povzetek Članek opisuje nov pristop za določanje karbamazepina in lamotrigina v bioloških vzorcih z disperzivno mikroekstrakcijo s topili ob uporabi ionskih tekočin ter v nadaljevanju s tekočinsko kromatografijo visoke ločljivosti z ultravijolično detekcijo. Raziskali smo učinek različnih ionskih tekočin (IL) na učinkovitost ek-strakcije karbamazepina in lamotrigina. Najvišjo učinkovitost ekstrakcije karbamazepina in lamotrigina smo dobili z uporabo 30 ^L 1-metil-3-oktilimidazolijevega heksafluorofosfata [C8MIM][PF6]. Optimizirali smo še več drugih faktorjev, ki vplivajo na učinkovitost mikroekstrakcije, kot so vrsta in volumen ekstrakcijskega topila, vrsta in volumen disperzijskega topila, koncentracija soli in pH vzorca. Kalibracijske krivulje so bile za plazemske vzorce linearne v območju 0,1-20 mg L-1 za karbamazepin in 0,3-40 mg L-1 za lamotrigin, meje zaznave pa so bile 0,04 mg L-1 za karbamazepin in 0,07 mg L-1 za lamotrigin. Rezultati potrjujejo primernost predstavljene metode kot dovolj občutljive za analizo tarčnih analitov v vzorcih urina in plazme. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Ranjbar et al.: Ionic Liquid-Based Dispersive Liquid-Liquid ... DOI: 10.17344/acsi.2019.5686 Acta Chim. Slov. 2020, 67, 757-763 /^creative ^commons Scientific paper Voltammetric Determination of Sulfaclozine Sodium at Sephadex-modified Carbon Paste Electrode Emad Mohamed Hussien,1^ Hanaa Saleh,2 Magda El Henawee,2 Afaf Abou El Khair2 and Neven Ahmed1 1 National Organization for Drug Control and Research (NODCAR), Giza, Egypt. 2 Faculty of Pharmacy, Zagazig University, Zagazig, Egypt. * Corresponding author: E-mail: emadhussien@yahoo.com Tel.: +2 02 3749 6077 Received: 11-08-2019 Abstract The electrochemical behavior of Sulfaclozine Sodium (SLC) was studied at a bare and sephadex-modified carbon paste electrodes by cyclic voltammetry and square wave voltammetry. The cyclic voltammetry (CV) showed a well-defined irreversible oxidation peak at 0.94 V in Britton- Robinson buffer pH 7.0. The strong affinity of SLC to sephadex allowed accumulation of SLC at the surface of electrode and thus higher electrochemical sensitivity to SLC. The influence of sephadex loading, the pH of the solution and the scan rate on the peak current was studied. A linear calibration curve covering the concentration range from 0.005 to 1 mM was obtained using SWV. The method was successfully applied for the determination of SLC in the veterinary pharmaceutical formulations with satisfactory accuracy and precision. Keywords: Sulfaclozine Sodium; square wave voltammetry; sephadex; carbon paste electrode. 1. Introduction Coccidiosis is a parasitic disease that attacks the intestinal tract of poultry caused by protozoan parasites of the genus Eimeria. This disease is of worldwide occurrence and costs the poultry industry many millions of dollars every year to control.1 Although live vaccines were introduced, prophylactic chemotherapy is still preferred for coccidiosis control in most countries. The last half of the twentieth century marked improvements in the performance of commercially reared poultry. These improvements would not be possible without the introduction of a succession of ever more effective anticoccidial agents to control coccidiosis.2 Sulfaclozine, N1 - (6-chloropyrazinyl) sulfanilamide (Figure 1) is a sulfonamide antibacterial that has been used Figure 1. Chemical structure of Sulfaclozine in veterinary medicine.3 it is effective in the treatment of clinical coccidiosis as well as prevention of the disease.4 The analytical methods which have been reported for the determination of sulfaclozine include chromatographic methods with different detectors.5-9 and capillary electrophoresis.10 These methods are either time-consuming or use expensive instrumentation. In contrast to the reported techniques, the voltammetric techniques are simple and rapid with high sensitivity and selectivity for drug analysis. Moreover, the carbon paste electrodes which are used for voltammetric measurements have several advantages for the electrochemical investigation of organic compounds. They are cheap, easy to prepare and use; and offer surface regeneration and modification, low background current, a large potential domain, no memory effects and adsorption-extraction ca-pabilities.11,12 Furthermore, including surfactants in the experimental protocol and modification of the carbon paste (chemically or by nanomaterials) have been reported to influence the electrochemical process occurring at the surface of the electrode.13,14 Sephadex is a cross-linked dextran which is used as a stationary phase in gel filtration chromatography.15 Carbon paste electrodes modified with Hussien et al.: Voltammetric Determination of Sulfaclozine Sodium 758 Acta Chim. Slov. 2020, 67, 757-763 sephadex have been used for the sensitive determination of nifuroxazide and glibenclaide.16,17 Indeed, Numerous pharmaceutical compounds were analyzed using carbon paste electrodes. The present work reports for the first time a SWV method for the determination of SLC in veterinary formulation. The proposed method utilizes the electrochemical oxidation of SLC at a carbon paste electrode modified with sephadex in a micellar medium. The effect of sephadex on the oxidation peak current was investigated. The method was validated According to the ICH guidelines.18 2. Experimental 2. 1. Reagents and Materials Sulfaclozine sodium was obtained from Yangzhou Tianhe Pharmaceutical Co., LTD, China with potency 99.75%. Clozicocc® W.S.P (Each 100 g contains 32 g sulfaclozine sodium) was obtained from Pharco Pharmaceuticals, Alex., Egypt. Graphite powder, paraffin oil, Sephadex G-50, C18 silica gel and chitosan were supplied from Sig-ma-Aldrich. Methanol was purchased from Loba Chemie Co., India. Sodium dodecyl sulfate (SDS), Phosphoric acid and boric acid were supplied from Adwic Co., Egypt. Acetic acid was obtained from Piochem Co., Egypt. Briton Robinson buffer (BR) buffer was prepared by adding equal volumes of phosphoric acid 0.04 M, acetic acid 0.04 M and boric acid 0.04 M, the pH of the buffer was adjusted by NaOH 0.2 M to cover the pH range from 2.0 to 10.0. SDS 10.0 mM was prepared by dissolving an appropriate amount of SDS in water. Double- distilled water was used throughout the study and referred to by "water". 2. 1. 4. Standard Solution Sulfaclozine stock solution (10.0 mM) was prepared by dissolving 30.66 mg of SLC in 1.0 mL methanol, then diluting with water to 10 mL. 2. 2. Apparatus Bio-logic SP 150 electrochemical work station with a three-electrode configured stand (model C-3) was used for the voltammetric measurements. The working electrode was a bare carbon paste electrode or a sephadex-modified carbon paste electrode (SMCPE); the reference electrode was Ag/AgCl/3 M KCl (BAS, USA) and the counter electrode was a platinum wire (BAS, USA). 2. 3. Procedures 2. 3. 1. Preparation of Modified Carbon- Paste Electrode Sephadex-modified carbon paste (SMCPE) electrode was made by hand mixing of 0.4 g Sephadex and 0.8 g of graphite powder with 0.4 mL paraffin oil. Plain (unmodified) carbon paste was made by mixing 1.0 g graphite powder with 0.6 mL paraffin oil. The paste was packed into the electrode body and smoothed on a filter paper till a shiny appearance of the electrode surface was obtained. 2. 3. 2. Analytical Procedure The CV at the carbon paste was repeated between 0 and 1.4 V several times in the buffer solution (pH 7.0) till the CV becomes stable. Then the electrode was transferred into another cell containing BR buffer solution (pH 7.0), 0.005 mM to 1.0 mM SLC and 0.03 mM SDS. The solution was stirred for 30 s at an open circuit potential, afterwards, the CV was recorded between +0.4 and +1.4, at 100 mVs-1 scan rate. 2. 3. 3. Calibration Curve of SLC The SWV was performed to determine SLC in bulk powder and pharmaceutical formulations. Different ali-quots were accurately transferred from the stock standard solution to an electrochemical cell containing 10 mL buffer (pH 7.0) and 0.03 mM SDS. The SWV was recorded at SMCPE. The peak current was plotted against drug concentration of SLC in (^M). 2. 3. 4. Application to Veterinary Pharmaceutical Formulation An accurately weighed 0.96 g Clozicocc® W.S.P. containing 306.7 mg of sulfaclozine sodium was transferred into a 100-mL volumetric flask and dissolved in 10 mL methanol. The solution was sonicated for 15 min, then, the flask was completed to the mark with water to obtain 10.0 mM SLC (solution I). Further dilution was carried out from solution 1 into 10-mL volumetric flask to obtain 1.0 mM SLC (solution II). The accuracy and precision of the method was studied using 0.005, 0.47 and 0.65 mM of the sample solution, each solution was prepared in triplicate. The accuracy and precision solutions were prepared by transferring 50 ^L from solution 1; 500 ^L and 700 ^L from solution II, each into an electrochemical cell containing BR pH 7 and 0.03 mM SDS. The concentration of the sample was determined by the standard addition method using the SWV. 3. Results and Discussion 3. 1. Sulfaclozine Electrochemical Oxidation Behavior The electrochemical behavior of SLC was studied at the carbon paste by recording the CV from 0 to 1.4 V in BR pH 7. The CV (Figure 2) shows one anodic peak current Hussien et al.: Voltammetric Determination of Sulfaclozine Sodium Acta Chim. Slov. 2020, 67, 757-759 763 Figure 2. The results are the average of five separate determinations Cyclic voltammogram of 0.1 mM SLC in BR buffer of pH 7.0 at a bare carbon paste lectrode. ((Ip) = 3.5 ^A at 0.94 V) with no cathodic peak in the reverse scan, it means that the oxidation process of SLC is irreversible. The anodic peak could be due to the oxidation of the amine group in SLC.19 3. 2. Optimization of the Experimental Conditions 3. 2. 1. Effect of pH The electrochemical oxidation of organic compounds depends, in most cases, on the pH of the solution. Herein, the effect of changing the pH of the solution on the oxidation of SLC was studied in BR buffer over the pH range from 2.0-10.0. It was observed that the peak potential of SLC is shifted towards less positive values when the pH was increased. The relationship between the EP and pH at the sephadex-modified carbon paste electrode was found to be linear and controlled by the equation EP = -51pH + 1284 (R2 = 0.995) (Figure 3a). The slope (~ 51 mV per pH) is close to the expected 59 mV per pH indicating that equal number of protons and electrons involved in the oxidation process of SLC. The highest oxidation current was obtained at pH 7 (Figure 3b), therefore, all measurements were carried out at pH 7.0. 3. 2. 2. Effect of Sephadex Different materials including C18 modified silica, chitosan and sephadex were tested for possible enhancement of the oxidation current and, hence, increasing sensitivity of the electrode. (Figure 4) shows no difference in the electrochemical behavior of sulfaclozine when 30% C18 modified silica was added to the carbon paste electrode, while the addition of 30% chitosan to the paste make a little improvement in the current response. In contrast, carbon paste modified with 30% (w/w) sepha-dex exhibited a considerable oxidation current that indicating the high affinity of the drug to sephadex. This affinity has been utilized for preconcentration of the drug onto the electrode surface to increase the sensitivity to SLC. The effect of sephadex loading on the peak current is shown in Figure 5. 3. 2. 3. Effect of Sodium Dodecyl Sulfate Sulfaclozine oxidation behavior in a micellar medium was also studied using SDS. Figure 4. Cyclic voltammograms of 1.0 mM SLC in PH 7.0 using different modified and unmodified electrodes. Figure 3. Dependence of peak potential (a) and peak current (b) on The pH of 0.1 mM SLC. The scan rate is 100 mV s-1 Figure 5. Cyclic voltammograms of SLC (1.0 mM) in PH 7.0 at carbon paste electrode containing different amounts of sephadexL; the scan rate is 100 mV s-1 SDS is a hydrophobic ionic surfactant, which can be adsorbed onto the electrode surface. As a result, the electrochemical process such as the mass and electron transfer energy at the electrode/solution interface are affected.20 It Hussien et al.: Voltammetric Determination of Sulfaclozine Sodium ... 760 Acta Chim. Slov. 2020, 67, 757-763 8» -70 ,|lA 2ft 110 SDS — JO Hi SDS 20 m1 SDS Â0 \i\ SDS 40 SDS -50 ni SDS A so -> t, MA 7C f O 002 0.04 0.06 M Ë SOS J Figure 6. Cyclic voltammograms recorded in 1.0 mM SLC containing different concentrations of SDS, the measurements were carried out using 40% sephadex carbon paste electrode in BR buffer pH 7.0. has been reported that SDS can remove the oily binder (insulator) and hence lower the uncompensated resistance at the electrode/solution interface.21, 22, 23 Herein, the effect of SDS was studied by the addition of different volume of 0.01 M SDS (10-50 ^L) to the SLC solution of pH 7 and recording the CV. Figure 6 shows the relationship between the anodic current and the SDS concentrations. It was observed that the peak current increases with increasing SDS in the measuring solution, and the highest oxidation current was observed when the SLC solution contains 30 ^L of 0.01 M SDS; no further improvement in the peak current was observed above this concentration. 3. 2. 4. Effect of Scan Rate The effect of the scan rate (u) on the peak potential (Ep) and the peak current (¿p) was studied between 10 mVs-1and 250 mVs-1 in 1.0 mM sulfaclozine solution in BR buffer (pH 7.0) containing 0.03 mM SDS (Figure 7a), The relationship between the oxidation peak current of SLC and the square root of scan rate (u1/2) was found to be linear, indicating that electrochemical oxidation of SLC is a diffusion controlled process.24 Plotting the logarithm of the peak current against the logarithm of the scan rate resulted in a straight line with a slope of 0.47 (Figure 7b), this value is close to the theoretical value of 0.5 for a purely diffusion-controlled process.24 It was also found that the Ep (oxidation peak potential) was dependent on scan rate, the peak potential was shifted to more positive values when the scan rate increased, which confirms that the oxidation process is irreversible. Furthermore, the relationship between the peak potential and the logarithm of the scan rate was found to be linear (Figure 7C) in accordance with Laviron's equation (1).25 „ _0. , .2.303RT , RTkO 2.303RT ,, , E„ = E + (-)log(-) + (-)logu (1) p v anF ' v anF ' v anF ' b Here a is the transfer coefficient, k0 is the standard heterogeneous rate constant of the reaction, u is the scan a) 120 100 »0 3. 1)0 40 20 0 —scan rateli>inV.s-l "■scan rateiOmV s-1 — scan ralelSOmV s-1 -scan ralc250mV s-1 —.scan rate25 mV a-1 —scanratelODmV s-1 —scan raTcIOOmV s-L GOO 800 E, mV b) 12 10 8 < L 6 Si 4 2 0 log Ip, (lA n.47^1ng II/Vr-1 + 3.1792 II" = 0.U895 10 log II /VSJ c) 0,!>8 0.% 0,94 0,92 0,88 ■ 0,86 0,84 0,82 0,8 ft n/V = 0.0G6 log o .'VV - 0 MS2 ttz = 0.L1336 8 10 logu /Vs-1 Figure 7. (a) The CV of 1.0 mM SLC containing 0.03 mM SDS in BR buffer of pH 7.0 at 40% SMCPE at different scan rates, (b) Dependence of the logarithm of peak current Ip/^A on logarithm of scan rate (u /Vs-1). (c) Relationship between peak potential Ep/V and logarithm of scan rate log (u/Vs-1). rate, and E0 is the formal redox potential, n is the number of electrons transferred. So, the value of an can be obtained from the slope of Ep vs log u. The slope was found to be 0.068, when T = 298K and R = 8.314 JK-1 mol-1 and F = 96485 C/mol, an was found to be 0.85. According to Bard and Faulkner.26 a can be calculated from the following equation (2). (2) k0 value can be calculated from the intercept of the above plot if the value of E0' is known. E0' in Eq. (1) can be obtained from the intercept of Ep versus u curve by extrapolating to the vertical axis at u = 027. All values of an, a, n, E0'and k0 are summarized in table 1. Hussien et al.: Voltammetric Determination of Sulfaclozine Sodium Acta Chim. Slov. 2020, 67, 757-761 763 Table 1. The calculated values of an, a, n, E0 and k0 for the electro-oxidation of SLC by cyclic voltammetry (CV) at SMCPE. Table 2. Performance data of the proposed SWV method for determination of SLC Parameters SMCPE an 0.8476 a 0.611 n 1.38 E0' 0.85 k0 3.1439 Parameters SLC 3. 2. 5. Square Wave Voltammetry (SWV) Under optimal experimental conditions, the calibration curve was constructed using the SWV over the concentration range from 0.005 to 1 mM. The parameters of SWV are 50 mV pulse height, 200 ms pulse width, 10 ms step height and 100 ms step time. The solution was stirred for 30 s at 400 rpm at an open circuit potential followed by 30 s quiescent time before any measurements. Linearity range ( mM ) Slope (|A. mM -1) Intercept (|A) Correlation coefficient (r) LOD(|M ) LOQ( |M ) Accuracy (mean ± S.D.) Precision (RSD %) Interday Intraday 0.005mM to 1.0 mM 50.67 5.86 0.9995 1.04 2.99 100.18 ± 0.01 2.08 2.12 tion range from 0.005 mM to 1.0 mM (R = 0.999) with a slop of 50.67 |AmM-1 and a limit of detection 0.001 mM. The reproducibility (%RSD, n = 3) of the peak current for 0.005 mM sulfaclozine was 2.08% as shown in Table 2. 3. 3. Calibration, Detection Limit and Reproducibility A linear relationship between SLC anodic peak current of and its concentration was found in the concentra- 3. 4. Determination of Sulfaclozine in Veterinary Formulation SLC was determined in Clozicocc® W.S.P. using standard addition method; the obtained results were sta- Figure 8. SWV of SLC over the concentration range from 0.005 to 1.0 mM in BR pH 7.0 containing 0.03 mM SDS using 40% SMCPE. Table 3. determination of SLC in pharmaceutical dosage form and statistical comparison of the proposed voltammetric and the published HPLC method 5 Pharmaceutical formulation Standard addition technique Reference method5 Taken(mg) Added (mg) Found(mg) %Recoverya Clozicocc w.s.p batch No.564 0.015 0.015 0.120 1.45 0.120 2.00 0.0149 1.43 2.01 99.96 98.87 100.31 Mean 99.59 99.33 S.D 1.01 1.51 n 5 variance 1.02 2.28 Student's t-test (2.132)b F-test (6.39)b 0.64 2.24 - a The results are the average of five separate determinations b the tabulated t and F values, respectively, at P = 0.05 Hussien et al.: Voltammetric Determination of Sulfaclozine Sodium ... 762 Acta Chim. Slov. 2020, 67, 757-763 tistically compared with those obtained by a reference method.5 The calculated t- and F-values are found to be less than the theoretical ones, confirming that accuracy and precision of the two methods are comparable at 95% confidence level (Table 3). 4. Conclusion Herein, we report for the first time a novel simple and rapid SWV method for SLC determination in veterinary formulations. The method is based on a carbon paste electrode modified with sephadex. The sephadex modified carbon paste electrode showed a dramatic increase in the oxidation peak current over the plain carbon paste. The SWV method was linear over a wide concentration range of SLC from 0.005 mM to 1.0 mM with a detection limit of 1 ^M. The method was applied successfully for the determination of SLC in the veterinary formulation with satisfactory accuracy and precision. The student's t-test and F-ratio test showed no significant difference regarding the accuracy and precision between the present method and the reported method. List of abbreviations SLC : Sulfaclozine Sodium SWV : Square wave voltammetry SDS : Sodium dodecyl sulfate BR : Briton Robinson buffer SMCPE : Sephadex-modified carbon paste electrode CV : Cyclic Voltammetry W.S.P : Water soluble powder 5. References 1. Chapman, H. D., Chapter 53 - Coccidiosis in Egg Laying Poultry, in Egg Innovations and Strategies for Improvements, P.Y. Hester, Editor. 2017, Academic Press: San Diego. 571-579. DOI:10.1016/B978-0-12-800879-9.00053-6 2. Chapman, H.D., Perspectives for the control of coccidiosis in poultry by chemotherapy and vaccinationin Proceedings of the IXth International Coccidiosis Conference. 2005. Foz de Iguassu, Parana, Brazil. 99-104. 3. S. C. Sweetman, R.P.S., Martindale: The Complete Drug Reference. thirty-eights ed. Vol. 1. 2014, London: Pharmaceutical Press. 4. Md. Harun-Or-Rashid, et al., Scholars Journal of Agriculture and Veterinary Sciences, 2016, 3(4), 284-287. 5. TANG Shu, C. J., GAO Jian-long, BAO En-dong, Nanjing Agricultural Univeristy, 2012, 105-109. 6. Yu, H., et al., Journal of Chromatography B, 2011, 879(25), 2653-2662. DOI:10.1016/j.jchromb.2011.07.032 7. Bousova, K. Senyuva, and H. Mittendorf, Journal of Chromatography A, 2013, 1274, 19-27. 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A, Fresenius' Journal of Analytical Chemistry, 1999, 364, 590-594. DOI:10.1007/s002160051391 18. ICH Q2A. validation of analytical methods. International Conference on Harmonization. 2003, IFPMA: Geneva. 19. V. Momberg, et al., Analytica Chimica Acta, 1984, 159, 119127. DOI:10.1016/S0003-2670(00)84288-9 20. Sanghavi, B. J. and A. K. Srivastava, Electrochimica Acta, 2010, 55(28), 8638-8648. DOI:10.1016/j.electacta.2010.07.093 21. Jayaprakash, G. K., et al., Journal of Molecular Liquids, 2017, 240, 395-401. DOI:10.1016/j.molliq.2017.05.093 22. Manjunatha JG, et al., Int J Electrochem Sci, 2009, 4, 662-671. 23. Shankar, S. S., B. E. K. Swamy, and B. N. Chandrashekar, Journal of Molecular Liquids, 2012, 168, 80-86. DOI:10.1016/j.molliq.2012.01.012 24. D. K. Gosser, Cyclic Voltammetry; Simulation and Analysis of Reaction Mechanisms. New York (N.Y.): VCH, 1993. 25. E. Laviron, J. Electroanal. Chem. 1979, 101(1), 19-28. DOI:10.1016/S0022-0728(79)80075-3 26. A. J. Bard and L. R. Faulkner, "Electrochemical Methods Fundamentals and Applications," 2nd Edition, Wiley, Hoboken, 2004. 27. Wu, Y., X. Ji, and S. Hu, Bioelectrochemistry. 2004, 64, 91-97. DOI:10.1016/j.bioelechem.2004.03.005 Hussien et al.: Voltammetric Determination of Sulfaclozine Sodium Acta Chim. Slov. 2020, 67, 757-763 763 Povzetek Elektrokemijsko obnašanje natrijevega sulfaklozina (SLC) je bilo proučevano na golih in sephadex-modificiranih elektrodah iz ogljikove paste s ciklično voltametrijo in kvadratno valovno voltametrijo. Ciklična voltametrija (CV) je pokazala dobro definiran nepovratni vrh oksidacije pri 0.94 V v Britton-Robinson pufru pri pH 7.0. Močna afiniteta SLC do sefadeksa je omogočila kopičenje SLC na površini elektrode in s tem večjo elektrokemično občutljivost za SLC. Proučen je bil vpliv nalaganja sefadeksa, pH raztopine in hitrost skeniranja na največji tok. Z uporabo SWV smo dobili linearno kalibracijsko krivuljo, ki pokriva območje koncentracije od 0.005 do 1 mM. Metodo smo uspešno uporabili za določanje SLC v veterinarskih farmacevtskih formulacijah z zadovoljivo točnostjo in natančnostjo. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Hussien et al.: Voltammetric Determination of Sulfaclozine Sodium ... DOI: 10.17344/acsi.2019.5689 Acta Chim. Slov. 2020, 67, 764-777 /^creative ^commons Scientific paper Synthesis, Characterization and Biological Activity of Some Dithiourea Derivatives Felix Odame,1,2* Eric Hosten,2 Jason Krause,3 Michelle Isaacs,5 Heinrich Hoppe,5 Setshaba D. Khanye,4 Yasien Sayed,6 Carminita Frost,3 Kevin Lobb4 and Zenixole Tshentu2 1 Department of Basic Sciences, University of Health and Allied Sciences, PMB 31, Ho, Ghana. 2 Department of Chemistry, Nelson Mandela University, P.O. Box 77000, Port Elizabeth 6031, South Africa. 3 Department of Biochemistry and Microbiology, Nelson Mandela University, P.O. Box 77000, Port Elizabeth 6031, South Africa. 4 Department of Chemistry, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa. 5 Department of Biochemistry and Microbiology, Rhodes University, Grahamstown 6140, South Africa. 6 Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand 2050, Johannesburg 2050, South Africa. * Corresponding author: E-mail: felixessah15@gmail.com Received: 11-08-2019 Abstract Novel dithiourea derivatives have been designed as HIV-1 protease inhibitors using Autodock 4.2, synthesized and characterized by spectroscopic methods and microanalysis. 1-(3-Bromobenzoyl)-3-[2-({[(3-bromophenyl)formami-do]methanethioyl}amino)phenyl]thiourea (10) and 3-benzoyl-1{[(phenylformamido)methanethioyl]amino}thiourea (12) gave a percentage viability of 17.9 ± 5.6% and 11.2 ± 0.9% against Trypanosoma brucei. Single crystal X-ray diffraction analysis of 1-benzoyl-3-(5-methyl-2-{[(phenylformamido)methanethioyl]amino}phenyl)thiourea (1), 3-ben-zoyl-1-(2-{ [(phenylformamido)methanethioyl] amino}ethyl)thiourea (11), 3-benzoyl-1-{ [(phenylformamido)methan-ethioyl]amino}thiourea (12) and 3-benzoyl-1-(4-{[(phenylformamido)methanethioyl]amino}butyl)thiourea (14) have been presented. 1-(3-Bromobenzoyl)-3-[2-({[(3-bromophenyl)formamido]methanethioyl}amino)phenyl]thiourea (10) gave a percentage inhibition of 97.03 ± 0.37% against HIV-1 protease enzyme at a concentration of 100 |M. Keywords: Dithiourea, cytotoxicity; HIV-1 protease inhibition; plasmodium falciparum activity; trypanosoma brucei activity 1. Introduction Thiourea derivatives have been synthesized by a variety of methods.1-8 A solvent-free three-component one-pot reaction between 2,6-diaminopyridine or 1,2-diami-nobenzene and NH4SCN with subsequent addition of an aroyl chloride gave bis-1-(aroyl)-3-(aryl)thioureas in excellent yields. The thiocyanate derivatives were first synthesized and then used to prepare the thiourea deriva-tives.1 Benzoyl chloride has been reacted with ammonium thiocyanate in CH2Cl2 solution under solid-liquid phase transfer catalysis, using polyethylene glycol-400 as the catalyst, to give the corresponding benzoyl isothiocyanate. Dropwise addition of a solution of 1,4-butylenediamine in CH2Cl2 yielded 3,3'-dibenzoyl-1,1'-(butane-1,4-diyl)dith-iourea,2 while 3,3-bis(4-nitrophenyl)-1,10-(para-phenyl-ene)dithiourea has been prepared by the reaction of (pa-ra-nitro)benzoyl isothiocyanate with para-phenylenedi-amine in CH2Cl2 using polyethylene glycol-400 as a phase transfer catalyst.3 This reaction has been carried out using 1,6-hexyldiamine as the source diamine to give N,N-(1,6-hexamethylene)-bis(benzoylthiourea).4 Thio-carbonohydrazide has been converted into 1-aminothio-carbamoyl-4-aroyl-3-thiosemicarbazides and 1,5-bis(aro-ylthiocarbamoy1)thiocarbonohydrazides by the addition Odame et al.: Synthesis, Characterization and Biological Activity Acta Chim. Slov. 2020, 67, 764-765 777 of one or two equivalents of aroyl isothiocyanate, respectively. 1-Phenyl- or 1-benzylidene-thiocarbonohydrazide and aroyl isothiocyanates gave the appropriate mono-ad-duct analogues. 1-Aminothiocarbamoyl-4-benzoyl-3-thiosemicarbazide is cyclised to 3-mercapto-5-phenyl-1,2,4-triazole in alkaline medium, and to 2-benzami-do-5-mercapto-1,3,4-thiadiazole in acid media; the action of alkyl halides on the appropriate alcohol yields 2-ben-zamido-5-alkylthio-l,3,4-thiadiazoles.5 The reaction of benzoyl isothiocyanate with or-tho-phenylenediamine has been done in acetone using potassium thiocyanate as a thiocyanate source.6 Urea attacks the benzoyl isothiocyanate on one end of the molecule. Potassium thiocyanate in acetone has been reacted with benzoyl chloride at 50 °C.7 1,2-Diaminoethane, 1,3-diami-nopropane or 1,4-diaminobutane dissolved in acetone were added and stirred at room temperature for 2 h.8 2. Results and Discussion 2. 1. Synthesis and Spectroscopic Characterization The phenyl thiourea derivatives are formed by the attack of the thione carbon of the starting benzoyl isothiocyanate by the two amino groups of the other starting molecule. Scheme 1 gives the synthetic pathway for the synthesis of the diamine derivatives. Spectroscopic characterization. The dithiourea derivatives were obtained by the reaction of ammonium thiocyanate with the respective benzoyl chloride in acetone and heating under reflux for 2 h to yield the benzoyl isothiocyanate derivatives. The addition of the diamines and further heating under reflux for 3 h gave the final products 1-14. The 1H NMR gave signals between 5 14.24 and 11.24 ppm for the NH proton of the amide. Table 1 gives the structures of all the synthesized compounds and their yields. Aromatic protons gave signals between 5 11.72 and 7.01 ppm. In the 13C NMR the thione signal was observed between 5 180.8 and 171.5 ppm whilst the carbonyl occurred between 5 168.3 and 161.0 ppm. Signals for aromatic carbons were observed between 5 159.0 and 113.3 ppm. The IR gave signals for the N-H stretching between 3440 and 3071 cm-1, whilst the aliphatic C-H stretching occurred between 2993 and 2727 cm-1. The C=S stretching was observed between 1683 and 1670 cm-1, with the carbonyl stretching occurring between 1687 and 1640 cm-1 and the C=C stretching observed between 1597 and 1506 cm-1. Crystal structures of compounds 1, 11, 12 and 14. Compounds 1, 11, 12 and 14 were recrystallized from DMSO/toluene (1:1). Compound 1 was obtained as white crystals, whilst compounds 11 and 12 were obtained as brown and light brown crystals, respectively. Compound 14 recrystallized from DMSO/toluene (1:3) as a light brown solid. The crystallographic data, selected bond Scheme 1. Synthesis of phenylthiourea compounds and other diamine derivatives. Odame et al.: Synthesis, Characterization and Biological Activity ... 766 Acta Chim. Slov. 2020, 67, 764-777 Table 1. List of synthesized compounds and their yields. Compound Structure Yield (%) Compound 78.0 74.7 70.7 73.0 > M M _j N H H N-¥ \ H 76.4 h/ b- OCHj Structure Yield (%) 78.0 7 72.2 77.1 75.3 \ K / o y—n n—v o / \ / \ // H H N-H \ H h/ ch 10 80.0 11 o s 70.9 H H S O 12 71.8 13 71.6 14 80.8 A., \| M Y 1 8 2 9 3 4 5 6 Odame et al.: Synthesis, Characterization and Biological Activity ... Acta Chim. Slov. 2020, 67, 764-767 777 lengths and bond angles for the crystal structures of compounds 1, 11, 12 and 14 are provided in Tables 2 and 3. The ORTEP diagrams for compounds 1, 11, 12 and 14 are pre- sented in Figures 1, 2, 3 and 4. Compounds 1, 11 and 14 crystallized in the monoclinic space group P21/c, while compound 12 crystallized in the monoclinic space group Table 2. Crystallographic data and structure refinement summary for compounds 1, 11, 12 and 14. Property 1 11 12 14 Formula C23H20N4O2S2 C18H18N4O2S2 C16HMN4O2S2, 2(C2H6OS) C20H22N4O2S2 CCD C Number 1448382 1919730 1919731 1919732 Formula weight 448.57 386.50 514.73 414.56 Crystal system Monoclinic Monoclinic Monoclinic Monoclinic Space group P2llc P21lc P21ln P21lc a [A] 10.8288(4) 11.2036(13) 6.3738(2) 5.9962(2) b [A] 17.8575(7) 7.1780(8) 15.3854(5) 23.2946(10) c [A] 22.6276(9) 11.0901(13) 12.6585(4) 7.1680(3) « [°] 90 90 90 90 P [°] 92.581(2) 100.783(5) 93.448(1) 103.777(2) Y [°] 90 90 90 90 V [A3] 4371.2(3) 876.11(18) 1239.09(7) 972.42(7) Z 8 2 2 2 Dcalc [g/cm3] 1.363 1.465 1.380 1.416 Mu(MoKa) [/mm ] 0.272 0.325 0.417 0.298 F(000) 1872 404 540 436 Crystal size [mm] 0.23 x 0.32 x 0.54 0.14 x 0.22 x 0.25 0.15 x 0.27 x 0.33 0.06 x 0.47 x 0.58 Temperature [K] 200 200 200 200 Tot., unique data, R(int) 40580, 10903, 0.028 2175, 2175, 0.000 11663, 3088, 0.020 13405, 2402, 0.020 Observed data [7 > 2.0 a(T)] 7835 1986 2596 2022 Nef 10903 2175 3088 2402 N -^par 616 128 155 135 R, wR2, S 0.0605, 0.1408, 1.08 0.1277, 0.4138, 1.17 0.0298, 0.0821, 1.03 0.0339, 0.0931, 1.06 vlin. and max. resd. dens. [e/A3] -0.63, 0.71 -1.59, 1.64 -0.27, 0.33 -0.20, 0.32 Table 3. Selected bond lengths (A) and bond angles (°) for compounds 1, 11, 12 and 14. Bond Distances (A) 1 11 12 14 S21-C22 1.667(1) S1-C2 1.662(1) S1-C2 1.668(1) S1-C2 1.673(1) S11-C12 1.667(1) O1-C1 1.218(1) O1-C1 1.225(2) O1-C1 1.223(2) O21-C21 1.230(1) C1-C11 1.492(1) N1-C1 1.382(2) N1-C1 1.374(2) O11-C11 1.224(1) N1-C2 1.405(1) N1-C2 1.383(2) N2-C2 1.321(2) N21-C22 1.399(1) N1-C1 1.371(1) N2-N2_a 1.373(2) N2-C3 1.461(2) N22-C22 1.332(1) C3-C3_a 1.522(1) N2-C2 1.332(2) N1-C2 1.390(2) N22-C221 1.421(3) N2-C2 1.325(2) S2-O2 1.508(1) C3-C4 1.521(2) N23-C23 1.335(4) N2-C3 1.454(2) N2-C2 1.332(2) C4-C4_a 1.522(2) Bond Angles (°) 1 11 12 14 N21-C22-N22 114.5(2) C1-N1-C2 128.6(1) O2-S2-C4 106.2(1) C1-N1-C2 129.3(1) S21-C22-N22 127.6(2) C2-N2-C3 123.3(1) O2-S2-C3 105.6(1) C2-N2-C3 122.3(1) O11-C11-N11 122.4(2) O1-C1-N1 122.4(1) N2_a-N2-C2 119.6(1) O1-C1-C11 122.1(1) N21-C21-C211 117.6(2) S1-C2-N2 126.0(1) N1-C1-C11 115.1(1) N1-C2-N2 117.8(1) N11-C12-N12 114.7(2) S1-C2-N1 118.4(1) N1-C2-N2 116.1(1) S1-C2-N2 124.7(1) S22-C23-N24 118.2(2) N1-C1-C11 115.3(1) S1-C2-N1 121.2(1) N2-C3-C4 112.4(1) S22-C23-N23 126.0(2) O1-C1-C11 122.3(1) C3-S2-C4 97.1(1) S1-C2-N1 117.5(1) O22-C24-N24 122.7(3) N1-C2-N2 115.6(1) C1-N1-C2 126.5(1) N1-C1-C11 115.4(1) O22-C24-C231 121.4(3) N2-C3-C3_a 111.1(1) O1-C1-N1 122.9(1) O1-C1-N1 122.5(1) O11-C11-C111 122.4(2) C1-C11-C12 123.5(1) O1-C1-C11 122.0(1) C1-C11-C12 117.5(1) O21-C21-C211 120.7(2) C1-C11-C16 116.6(1) S1-C2-N2 122. 8(1) C1-C11-C16 123.7(1) Odame et al.: Synthesis, Characterization and Biological Activity ... 768 Acta Chim. Slov. 2020, 67, 764-777 P21/n. In compound 1 the bond distances O21-C21 and Oll-Cll are 1.230(1) Â and 1.224(1) Â which are consistent with carbonyls,9 whilst the bond distances of S21-C22 and S11-C12 which are 1.667(1) Â and 1.667(1) Â are typical of thiones.10 The bond angles of S21-C22-N22 and O11-C11-N11 are 127.6(2)° and 122.4(2)° respectively this confirms that the carbon atoms are sp2 hybridized. The bond distances of S1-C2 and O1-C1 in compound 11 are 1.662(1) Â and 1.218(1) Â for a thione and a carbonyl, respectively. The bond distance of C3-C3_a is 1.522(1) Â which is consistent with a carbon-carbon single bond.11 The bond angles of S1-C2-N2 and S1-C2-N1 are 126.0(1)° and 118.4(1)° confirming that the carbon is sp2 hybridized, whilst the bond angle of N2-C3-C3_a which is 111.1(1)° confirms the carbon is sp3 hybridized. In compound 12 the bond distance S1-C2 which was 1.668(1) Â was consistent with a thione, whilst the car-bonyl O1-C1 bond legth was 1.225(2) Â. The N2-N2_a bond distance was 1.373(2) Â. The bond angles of O1-C1-N1 and O1-C1-C11 were 122.9(1)° and 122.0(1)°, Figure 1. An ORTEP view of 1-benzoyl-3-(5-methyl-2-{[(phenylformamido)methanethioyl]amino}phenyl)thiourea (1) showing 50% probability displacement ellipsoids and the atom labelling. Figure 2. An ORTEP view of 3-benzoyl-1-(2-{[(phenylformamido)methanethioyl]amino}ethyl)thiourea (11) showing 50% probability displacement ellipsoids and the atom labelling. Odame et al.: Synthesis, Characterization and Biological Activity ... Acta Chim. Slov. 2020, 67, 764-769 777 respectively, confirming that the carbon atom involved is sp2 hybridized. In compound 14 the carbonyl O1-C1 bond length was 1.223(2) A, whilst the thione S1-C2 was 1.673(1) A. The bond angles of O1-C1-C11, N1-C2-N2 and S1-C2-N2 in compound 14 were 122.1(1)°, 117.8(1)° and 124.7(1)° which is characteristic of sp2 hybridized carbon. The crystal structure of compound 11 was reported at 293 K,12 but this work gives the crystal structure at 200 K. Both measurements gave a monoclinic space group P21/c with two molecules in the unit cell. The cell parameters obtained at 273 K were slightly higher than the measurement at 200 K. The crystal structure of compound 12 has been reported at 273 K,13 whilst this work presents the crystal structure at 200 K. Both measurements gave a monoclinic space group P21/n with two molecules in the unit cell and each molecule bonded to two molecules of dimethylsulfox- Figure 3. An ORTEP view of 3-benzoyl-1{[(phenylformido)methanethioyl]amino}thiourea dimethyl sulfoxide (12) showing 50% probability displacement ellipsoids and the atom labelling. Figure 4. An ORTEP view of 3-benzoyl-1-(4-{[(phenylformamido)methanethioyl]amino}butyl)thiourea (14) showing 50% probability displacement ellipsoids and atom labelling. Odame et al.: Synthesis, Characterization and Biological Activity ... 770 Acta Chim. Slov. 2020, 67, 764-777 ide. The cell parameters for the determination at 273 K gave consistently higher values than the measurement at 200 K. The measurement at 273 K gave a lower density (1.347 g cm-3) than the measurement at 200 K (1.380 g cm-3) The crystal structure of compound 14 has been reported at 298 K,2 whilst this work reports the crystal structure at 200 K. Both measurements gave a monoclinic space group P21/c with two molecules in the unit cell. The cell parameters for the determination at 298 K gave consistently higher values than the measurement at 200 K. The measurement at 298 K gave a lower density (1.380 g cm-3) than the measurement at 200 K (1.416 g cm-3) 3. Biological Studies The compounds were tested for their cytotoxicity using Hela cells, and tested against HIV-1 protease with ritonavir as a positive control and Plasmodium falciparum strain 3D7 (20 ^M) with chloroquine as a positive control. The compounds were also tested for their activity against Trypanosoma brucei (20 ^M) with pentamidine as a positive control. Cytotoxicity tests. The graph (Figure 5) and table (Table 4) below give the % HeLa cell viability obtained for each tested compound (1-12). Compounds 1 and 12 were found to be cytotoxic against Hela cells whilst all the other compounds were found to be non-cytotoxic. HIV-1 protease activity. Table 5 and Figure 6 give the HIV-1 screening results for the diamine derivatives of benzoyl isothiocyanate and their in silico results. The Table 4. % HeLa cell viability obtained for the compounds 1-12. Compound % Viability 1 42.48 2 68.62 3 67.55 4 67.67 5 75.44 6 91.16 7 99.55 8 72.61 9 63.29 10 65.97 11 71.01 12 49.73 screening of the compounds 1-14 was completed at 100 ^M and 10 ^M of inhibitor and ritonavir, respectively. The predicted inhibition constant for compound 1 (4-meth-yldithiourea) was 0.19 ^M whilst the HIV-1 assay gave a % inhibition of 17.69 ± 9.61%, compound 2 (unsubstituted) gave a predicted inhibition constant of 0.13 ^M and a percentage inhibition of 10.30 ± 6.12%. For compound 3 (4-nitro derivative) a predicted inhibition constant of 0.47 ^M and a percentage inhibition of 31.03 ± 0.42% were obtained whilst compound 5 (3-nitro derivative) gave predicted inhibition constant of 0.11 ^M and a percentage inhibition of 32.68 ± 11.03%. Though the predicted inhibition constant of the 3-nitro derivative seems to be better than that of the 4-nitro, the percentage inhibition for both compounds are not too different, due to their interaction with solvent molecules which does not greatly change Figure 5. % HeLa cell viability ± SD obtained for compounds 1-12. Odame et al.: Synthesis, Characterization and Biological Activity ... Acta Chim. Slov. 2020, 67, 764-771 777 their orientations in the active site. Compounds 4 (4-chloro derivative), 6 (3-methoxy derivative) and 8 (4-methoxy derivative) showed no activity in the HIV-1 protease assay with the predicted inhibition constants of 0.21, 1.90 and 0.81 ^M, suggesting that in solution the methoxy substituent on the dithiourea makes the whole molecule inactive against HIV-1 protease at a concentration of 100 ^M whilst a chloro substitution at position 4 makes the molecule ineffective at inhibiting HIV-1 protease because the chloro group interacts with the surrounding groups that interfere with its ability to fit well into the active site for effective inhibition of the protease. When the chloro group is attached at position 3, such as in compound 9 (3-chloro derivative), it gave a predicted inhibition constant of 0.06 ^M which was the best predicted inhibition constant from the set and a percentage inhibition of 1.78 ± 11% confirming that the chloro group undergoes too much interaction with polar groups in solution hence the substantial departure from the predicted inhibition. Compound 7 (4-bromo derivative) gave predicted inhibition constant of 0.12 ^M and a percentage inhibition of 29.62 ± 4.10% whilst compound 10 (3-bromo derivative) gave predicted inhibition constant of 0.095 ^M and a percentage inhibition of 97.03 ± 0.37%. Compound 10 gave the best percentage inhibition of all the compounds. In these class of compounds, a bromo substituent at position 3 gives the best percentage inhibition among this class of compounds. The size of the bromo group allows the substituent to fit the active site for effective binding to the aspartate groups and the bridging water molecules in the active site. The other diamine derivatives gave lower predicted inhibition constants than those with the phenyl backbone. In the computation, the lack of rigidity in these molecules accounts for their lower predicted inhibition constants even though their protease Table 5. HIV-1 protease screening results of the screened 1-14 diamine derivatives of benzoyl isothio-cyanate. Compound Fluorescence % Activity relative to untreated control % Inhibition relative to untreated control In silico results K (MM) Ritonavir 36.24 9.34 90.66 ± 1.88 Unsuccessful 1 186.01 82.31 17.69 ± 9.61 0.19 2 202.70 89.70 10.30 ± 6.12 0.13 3 155.85 68.97 31.03 ± 0.42 0.47 4 402.60 103.79 0 ± 4.10 0.21 5 152.13 67.32 32.68 ± 11.03 0.11 6 243.51 107.76 0 ± 4.60 1.90 7 159.05 70.38 29.62 ± 4.10 0.12 8 446.80 115.18 0 ± 2.43 0.81 9 381.00 98.22 1.78 ± 11 0.06 10 11.522 2.97 97.03 ± 0.37 0.095 11 147.94 65.4 34.53 ± 20.69 19.98 12 145.79 64.52 35.49 ± 5.24 10.98 13 268.44 69.20 30.80 ± 10.61 0.25 14 151.00 66.82 33.18 ± 0.16 1.34 Odame et al.: Synthesis, Characterization and Biological Activity ... Figure 6. HIV-1 protease screening results illustrating percentage inhibition of selected diamine derivatives of benzoyl isothiocyanate (100 |iM) and ritonavir (10 |iM) relative to untreated control. Error bars represent standard deviation of n = 3. % inhibition is comparable to the ortfoo-phenylenediamine derivatives. Compound 11 (ethanediamine derivative) gave predicted inhibition constant of 19.98 ^M and a percentage inhibition of 34.53 ± 20.69%. Compound 12 (hydrazine derivative) gave predicted inhibition constant of 10.98 ^M and a percentage inhibition of 35.49 ± 5.24%. Compound 13 (phenylhydrazine derivative) gave predicted inhibition constant of 0.25 ^M and a percentage inhibition of 30.80 ± 10.61%. Compound 14 (butyldiamine derivative) gave predicted inhibition constant of 10.98 ^M and a percentage inhibition of 33.18 ± 0.16%. Figure 7 gives the 2D representation of compound 10 in the protease active site. The presence of a polar group on this class of compounds improves the extent of interaction at the active site both in the docking studies and the bioassays making this class of compounds active against the protease. 772 Acta Chim. Slov. 2020, 67, 764-777 Figure 7. 2D representation of 1-(3-bromobenzoyl)-3-[2-({[(3-bromophenyl)formamido]methanethioyl}amino)phenyl]thiourea (10) in the HIV-1 protease binding site. Anti-malaria test. The bar graph (Figure 8) and table (Table 6) below show the percentage parasite (Plasmodium falciparum strain 3D7) viability ± SD obtained after a 48 h incubation with 20 ^M of the individual compounds 1-12. The anti-malaria test showed varying degrees of activity, with compound 6 giving the best percentage viability of 57.2 ± 1.3, whilst compound 11 was the least active with a percentage viability of 98.0 ± 13.4. Chloroquine was used as the standard in the antimalarial test. Trypanosoma brucei activity. The graph (Figure 9) and table (Table 7) below show the residual percentage parasite (Trypanosoma brucei) viability obtained after a 48 h incubation with 20 ^M of the individual compounds 1-12. Compounds 10 and 12 gave very good activity with percentage viability of 17.9 ± 5.6% and 11.2 ± 0.9%, re- Table 6. Percentage parasite (Plasmodium falciparum strain 3D7) viability ± SD obtained for the compounds 1-12. Compound Viability % 1 65.9 ± 5.0 2 80.0 ± 6.5 3 87.8 ± 7.9 4 62.6 ± 1.8 5 80.9 ± 14.2 6 57.2 ± 1.3 7 88.1 ± 3.1 8 78.2 ± 13.2 9 89.2 ± 5.9 10 64.9 ± 2.2 11 98.0 ± 13.4 12 60.6 ± 6.3 mffWTTfll Figure 8. Percentage parasite (Plasmodium falciparum strain 3D7) viability ± SD obtained for compounds 1-12. Odame et al.: Synthesis, Characterization and Biological Activity ... Acta Chim. Slov. 2020, 67, 764-773 777 T T T "JL Figure 9. The residual percentage parasite (Trypanosoma brucei) viability ± SD obtained for compounds 1-12. spectively whilst the least active was compound 3 with a percentage viability 106.1 ± 4.5%. Pentamidine was used as the standard. Table 7. Percentage parasite (Trypanosoma brucei) viability ± SD obtained for the compounds 1-12. Compound at 20 ^M Viability % 1 46.4 ± 0.4 2 101.2 ± 0.1 3 106.1 ± 4.5 4 100.1 ± 3.0 5 96.9 ± 0.5 6 104.2 ± 0.5 7 68.4 ± 4.3 8 102.2 ± 4.7 9 62.0 ± 0.6 10 17.9 ± 5.6 11 101.3 ± 2.7 12 11.2 ± 0.9 4. Experimental Chemicals and instrumentation. Analytical grade reagents and solvents for synthesis and analysis which included 3-chlorobenzoyl chloride, 4-chlorobenzoyl chloride, 4-methoxybenzoyl chloride, 3-methoxybenzoyl chloride, 3-bromobenzoyl chloride, 4-bromobenzoyl chloride, 4-nitrobenzoyl chloride, 3-nitrobenzoyl chloride, ortho-phenylenediamine, 4-methyl-ortho-phenylenediamine, 2-(2-aminophenyl)benzimidazole ethylene diamine, hydra-zine hydrate and ammonium thiocyanate were obtained from Sigma Aldrich (USA), whilst benzoyl chloride, toluene and acetone were obtained from Merck Chemicals (SA). The chemicals were used as received (i.e. without further purification). JH NMR and 13C NMR spectra were recorded on a Bruker Avance AV 400 MHz spectrometer operating at 400 MHz for and 100 MHz for 13C, using deuterated dimethyl sulfoxide as the solvent and te-tramethylsilane as the internal standard. Chemical shifts are expressed in ppm. Structural assignments of resonances have been performed with the help of 2D NMR gradient experiments (1H-1H COSY). FT-IR spectra were recorded on a Bruker Platinum ATR Spectrophotometer Tensor 27 and the data were processed using OPUS. Elemental analyses were performed using a Vario Elementar Microcube ELIII. Melting points were obtained using a Stuart Lasec SMP30 melting point apparatus and are reported uncor-rected, whilst the mass spectra were determined using an Agilent 7890A GC System connected to a 5975C VL-MSC with electron impact as the ionization mode and detection by a triple-axis detector. General method for the synthesis of dithiourea derivatives. The dithiourea derivatives were prepared by dissolving ammonium thiocyanate (0.04 mol, 3.05 g) in 80 mL of acetone, the respective benzoyl chloride (0.04 mol) was then added and heated under reflux at 100-120 °C for 2 h. The benzoyl isothiocyanate derivative (0.04 mol) obtained was filtered, 4-methyl-ortho-phenylenediamine, or-tho-phenylenediamine, ethylenediamine or hydrazine hydrate (0.04 mol) was added to the filtrate and refluxed at 100-120 °C for 3 h. 1-Benzoyl-3-(5-methyl-2-{[(phenylformamido)meth-anethioyl]amino}phenyl)thiourea (1). The product obtained was filtered and recrystallized from DMSO/toluene (1:1) as a brown solid. M.p. 172-173 °C. Yield 78.0%. 1H NMR (400 MHz, DMSO-d6) 5 12.45 (s, 1H, NH), 12.41 (s, 1H, NH), 11.72 (d, 2H, J = 8.0 Hz, NH), 7.90 (d, 4H, J = 8.0 Hz), 7.77 (m, 1H, J = 8.4 Hz), 7.73 (s, 1H), 7.64 (t, 2H, J = 7.2 Hz), 7.49 (t, 4H, J = 7.6 Hz), 7.22 (d, 1H, J = 8.0 Hz), 2.31 (s, 3H). 13C NMR (100 MHz, DMSO-d6) 5 180.40 (C=S), 168.3 (C=O), 136.8 (C), 133.2 (C), 133.1 (C), 130.9 Odame et al.: Synthesis, Characterization and Biological Activity ... 774 Acta Chim. Slov. 2020, 67, 764-777 (CH), 128.5 (CH), 128.3 (CH), 127.7 (CH), 126.9 (CH), 126.5 (CH), 20.7 (CH3). IR vmax 3186 (N-h), 2981 (C-h), 1670 (C=S), 1593 (C=O), 1512 (C=C), 1487 (C-N) cm-1. Anal. calcd. for C23H20N4O2S2: C 61.59; H, 4.49; N, 12.49; S, 14.30. Found: C 61.65; H, 4.54; N, 12.56; S, 14.46. LRMS (m/z, M+) found for C23H20N4O2S2: 448.40, expected mass: 448.56. 1-Benzoyl-3-(2-{[(phenylformamido)methanethioyl] amino}phenyl)thiourea (2). The product obtained was filtered and recrystallized from DMSO/toluene (1:1) as a light brown solid. M.p. 174-176 °C. Yield 78.0%. 1H NMR (400 MHz, DMSO-d6) 5 12.52 (s, 1H), 8.10 (d, 2H), 7.94 (d, 2H), 7.71 (m, 2H), 7.65 (m, 4H), 7.48 (m, 2H), 7.42 (m, 2H), 7.11 (m, 1H). 13C NMR (100 MHz, DMSO-d6) 5 167.3 (C=O), 166.4 (C=O), 144.2 (C), 131.8 (C), 130.7 (CH), 129.2 (CH), 128.8 (CH), 128.5 (CH), 124.4(CH) 113.5 (CH). IR vmax 3327 (N-H), 3262 (N-H), 3134 (N-H), 1673 (C=S), 1643 (C=O), 1596 (C=C), 1514 (C=C), 1486 (C-N), 1337 cm-1. Anal. calcd. for C22H18N2O2S2: C, 60.81; H, 4.18; N, 12.89; S, 14.76. Found: C, 60.56; H, 4.28; N, 12.78; S, 14.52. LRMS (m/z, M+) found for C22H18N4O2S2: 434.45, expected mass: 434.53. 1-(4-Nitrobenzoyl)-3-[2-({[(4-nitrophenyl)formamido] methanthioyl]amino}phenyl]thiourea (3). The mother liquor was allowed to stand overnight in a fume hood. The product obtained was filtered and recrystallized from DMSO/toluene (1:1) as a yellow solid. M.p. 202-204 °C Yield 74.7%. 1H NMR (400 MHz, DMSO-d6) 5 12.30 (s, 2H, NH), 12.13 (s, 2H, NH), 833 (d, 4H, J = 8.0 Hz), 8.09 (d, 4H, J = 8.0 Hz), 7.93 (m, 2H), 7.42 (m, 2H). 13C NMR (100 MHz, DMSO-d6) 5 180.1 (C=S), 161.0 (C=O), 149.7 (C), 138.1 (C), 133.3 (C), 130.20 (CH), 127.3 (CH), 126.8 (CH), 123.2 (CH). IR vmax 3200 (N-h), 3071 (N-H), 1683 (c=S), 1662 (C=O), 1508 (C=C), 1484 (C-N) cm-1. Anal. calcd. for C22H16N6O6S2: C, 50.38; H, 3.07; N, 16.02; S, 12.23. Found: C, 50.49; H, 3.11; N, 16.17; S, 12.36. LRMS (m/z, M+) found for C22H16N6O6S2: 524.20, expected mass: 524.53. 1-(4-Chlorobenzoyl)-3-[2-({[(4-chlorophenyl)forma-mido]methanethioyl}amino)phenyl thiourea (4). The product obtained was filtered and recrystallized from DMSO/toluene (1:1) as a yellow solid. M.p. 173-175 °C. Yield 70.7%. 1H NMR (400 MHz, DMSO-d6) 5 8.52 (m, 2H), 7.93 (d, 2H, J = 7.6 Hz), 7.89 (d, 2H, J = 7.6 Hz), 7.81 (br, 2H), 7.65 (br, 2H), 7.55 (t, 2H, J = 8.0 Hz), 7.50 (t, 2H, J = 8.0 Hz). 13C NMR (100 MHz, DMSO-d6) 5 143.8 (C), 132.1 (CH), 125.9 (CH), 124.0 (CH), 119.6 (CH). IR vmax 3038 (N-H), 1640 (C=O), 1578 (C=C), 1555 (C=C), 1476 (C-N), 1447 (C-N) cm-1. Anal. calcd. for C22H16Cl-2N4O2S2: C, 52.49; H, 3.20; N, 11.13; S, 12.74. Found: C, 52.56; H, 3.26; N, 11.22; S, 12.85. LRMS (m/z, M+) found for C22H16Cl2N4O2S2: 503.20, expected mass: 503.42. 1-(3-Nitrobenzoyl)-3-[2-({[(3-nitrophenyl)formamido] methane}amino)phenyl] thourea (5). The product obtained was filtered and recrystallized from DMSO/toluene (1:1) as a yellow solid. M.p. 201-203 °C. Yield 73.0%. 1H NMR (400 MHz, DMSO-d6) 5 12.34 (s, 2H, NH), 12.18 (s, 2H, NH), 8.65 (s, 2H), 8.48 (d, 2H, J = 8.0 Hz), 8.31 (d, 2H, J = 8.0 Hz), 7.96 (m, 2H), 7.78 (dd, 2H, J = 8 Hz), 7.44 (t, 2H, J = 4.0 Hz). 13C NMR (100 MHz, DMSO-d6) 5 180.2 (C=S), 166.4 (C=O), 147.3 (C), 135.1 (C), 133.7 (C), 133.3 (CH), 130.1 (CH), 127.4 (CH), 127.2 (CH), 126.6 (CH), 123.5 (CH). IR vmax 3351 (N-H), 3204 (N-H), 1687 (C=O), 1515 (C=C) cm-1. Anal. calcd. for C22H16N6O6S: C, 50.38; H, 3.07; N, 16.02; S, 12.23. Found: C, 50.24; H, 3.20; N, 16.18; S, 12.19. LRMS (m/z, M+) found for C22H-16N6O6S: 524.60, expected mass: 524.53. 1-(3-Methoxybenzoyl)-3-[2-({[(3-methoxyphenyl)for-mamido]methanethioyl}amino)phenyl] thiourea (6). The product obtained was filtered and recrystallized from DMSO/toluene (1:1) as a white solid. M.p. 164-166 °C. Yield 76.4%. 1H NMR (400 MHz, DMSO-d6) 5 12.50 (s, 2H), 11.69 (s, 2H), 7.92 (m, 2H), 7.50 (d, 2H, J = 7.6 Hz), 7.45 (s, 2H), 7.41 (t, 4H, J = 8 Hz), 7.21 (d, 2H, J = 8.8 Hz), 3.77 (s, 6H). 13C NMR (100 MHz, DMSO-d6) 5 180.4 (C=S), 168.1 (C=O), 159.0 (C), 133.4 (C), 129.8 (C), 127.1 (CH), 126.6 (CH), 120.8 (CH), 119.3 (CH), 113.3 (CH), 55.5 (CH3). IR vmax 3326 (N-H), 3184 (N-H), 3003 (N-H), 1663 (C=O), 1597 (C=C), 1506 (C=C), 1464 (c-N) cm-1. Anal. calcd. for C24H22N4O4S2: C, 58.28; H, 4.48; N, 11.33; S, 12.97. Found: C, 58.12; H, 4.29; N, 11.42; S, 12.86. LRMS (m/z, M+) found for C24H22N4O4S2: 494.35, expected mass: 494.59. 1-(4-Bromobenzoyl) -3-[2-({[ (4-bromophenyl)forma-mido]methanethioyl}amino)phenyl] thiourea (7). The product obtained was filtered and recrystallized from DMSO/toluene (1:1) as a white solid. M.p. 205-207 °C. Yield 72.2%. 1H NMR (400 MHz, DMSO-d6) 5 12.37 (s, 2H), 11.82 (s, 2H), 7.91 (m, 2H), 7.81 (d, 4H, J = 8.0 Hz), 7.73 (d, 4H, J = 7.6 Hz), 7.40 (m, 2H). 13C NMR (100 MHz, DMSO-d6) 5 180.5 (C=S), 167.4 (C=O), 133.6 (C), 131.4 (CH) 131.2 (C), 130.6 (CH), 127.2 (CH), 127.1 (C), 126.7 (CH). IR vmax 3140 (N-H), 2993 (C-H), 1681 (C=O), 1585 (C=C), 1517 (C=C), 1429 (C-N) cm-1. Anal. calcd. for C22H16Br2N4O2S2: C, 44.61; H, 2.72; N, 9.46; S, 10.83. Found: C, 44.70; H, 2.65; N, 9.40; S, 10.76. LRMS (m/z, M+) found for C22H16Br2N4O2S2: 592.20, expected mass: 592.33. 1-(4-Methoxybenzoyl)-3-[2-({[(4-methoxylphenyl)for-mamido]methanethioyl}amino)phenyl] thiourea (8). The product obtained was filtered and recrystallized from DMSO/toluene (1:1) as a white solid. M.p. 206-208 °C. Yield 77.1%. 1H NMR (400 MHz, DMSO-d6) 5 12.56 (s, 2H, NH), 11.48 (s, 2H, NH), 7.92 (m, 6H), 7.38 (m, 2H, J = 3.6, 5.6 Hz), 7.01 (d, 4H, J = 8.8 Hz), 3.82 (s, 6H). 13C NMR Odame et al.: Synthesis, Characterization and Biological Activity ... Acta Chim. Slov. 2020, 67, 764-775 777 (100 MHz, DMSO-d6) 8 180.8 (C=S), 167.5 (C=O), 163.2 (C), 133.3 (C), 131.0 (CH), 126.9 (CH), 123.9 (CH), 113.71 (CH), 55.8 (CH3). IR vmax 3404 (N-H), 3278 (n-H), 3001 (N-H), 2961 (C-H), 2837 (C-H), 1653 (C=O), 1594 (C=C), 1525 (C=C), 1489 (C-N) cm-1. Anal. calcd. for C24H22N4O4S2: C, 58.26; H, 4.48; N, 11.33; S, 12.97. Found: C, 58.13; H, 4.37; N, 11.29; S, 13.03. LRMS (m/z, M+) found for C24H22N4O4S2: 494.20, expected mass: 494.59. 1-(3-Chlorobenzoyl)-3-[2-({[(3-chlorophenyl)forma-mido]methanethioyl}amino)phenyl] thiourea (9). The product obtained was filtered and recrystallized from DMSO/toluene (1:1) as a light brown solid. M.p. 143-145 °C. Yield 75.3%. 1H NMR (400 MHz, DMSO-d6) 8 12.40 (br, 2H), 11.88 (s, 2H, NH), 8.04 (m, 1H), 7.91 (d, 3H, J = 12.8 Hz), 7.83 (d, 2H, J = 7.6 Hz), 7.70 (d, 2H, J = 8.0 Hz), 7.52 (m, 2H, J = 8.0, 7.2 Hz), 7.41 (s, 2H). 13C NMR (100 MHz, DMSO-d6) 8 180.4 (C=S), 167.0 (C=O), 134.3 (C), 133.2 (C), 132.8 (CH), 130.5 (CH), 128.5 (CH), 127.3 (CH), 127.1 (CH), 126.7 (CH). IR vmax 3440 (N-H), 3166 (N-H), 2971 (C-H), 1668 (C=O), 1593 (C=C), 1510 (C=C), 1471 (C-N), 1459 (C-N) cm-1. Anal. calcd. for C22H16Cl2N4O2S2: C, 52.49; H, 3.20; N, 11.13; S, 12.74. Found: C, 52.31; H, 3.29; N, 11.22; S, 12.86. LRMS (m/z, M+) found for C22H16Cl2N4O2S2: 503.35, expected mass: 503.42. 1-(3-Bromobenzoyl) -3-[2-({[ (3-bromophenyl)forma-mido]methanethioyl}amino)phenyl] thiourea (10). The mother liquor was allowed to stand overnight in a fume hood. The product obtained was filtered and recrystallized from DMSO/toluene (1:1) as a light yellow solid. M.p. 191-193 °C. Yield 80.0%. 1H NMR (400 MHz, DMSO-d6) 8 12.29 (s, 2H), 11.75 (s, 2H), 7.93 (s, 2H), 7.89 (m, 2H), 7.79 (d, 4H, J = 7.6 Hz), 7.44 (d, 2H, J = 8 Hz), 7.41 (m, 2H). 13C NMR (100 MHz, DMSO-d6) 8 180.65 (C=S), 167.74 (C=O), 136.3 (C), 134.5 (CH), 133.8 (C), 131.5 (CH), 131.2 (CH), 127.9 (CH), 126.9 (CH), 122.1 (CH). IR vmax 3389 (N-H), 3176 (N-H), 3016 (N-H), 1662 (C=O), 1595 (C=C), 1563 (C=C), 1456 (C-N) cm-1. Anal. calcd. for C22H16Br2N4O2S2: C, 44.61; H, 2.72; N, 9.46; S, 10.83. Found: C, 44.75; H, 2.68; N, 9.39; S, 10.70. LRMS (m/z, M+) found for C22H16Br2N4O2S2: 592.10, expected mass: 592.33. 3-Benzoyl-1-(2-{[(phenylformamido)methanethioyl] amino}ethyl)thiourea (11). The product obtained was filtered and recrystallized from DMSO/toluene (1:1) as a light brown solid. M.p. 220-222 °C. Yield 70.85%. 1H NMR (400 MHz, DMSO-d6) 8 10.98 (s, 2H), 7.91 (d, 4H, J = 7.6 Hz), 7.61 (t, 2H, J = 7.2 Hz), 7.51 (t, 4H, J = 7.6 Hz), 3.09 (s, 4H). 13C NMR (100 MHz, DMSO-d6) 8 180.8 (C=S), 167.3 (C=O), 132.9 (C), 132.2 (CH), 128.5 (CH) 43.4 (CH2). IR vmax 3420 (N-H), 3229(N-H), 3047 (N-H), 1664 (C=O), 1579 (C=C), 1507 (C=C), 1448 (C-N) cm-1. Anal. calcd. for C18H18N4O2S2: C, 55.94; H, 4.69; N, 14.50; S, 16.59. Found: C, 56.03; H, 4.74; N, 14.42; S, 16.63. LRMS (m/z, M+) found for C18H18N4O2S2: 386.30, expected mass: 386.49. 3-Benzoyl-1{[(phenylformido)methanethioyl]amino} thiourea (12). The product obtained was filtered and recrystallized from DMSO/toluene (1:1) as a white solid. M.p. 345-346 °C. Yield 71.8%. 1H NMR (400 MHz, DM-SO-d6) 6 14.24 (s, 1H, NH), 12.12 (s, 1H, NH), 8.13 (d, 2H, J = 8.0 Hz), 8.01 (d, 2H, J = 8.0 Hz), 7. 94 (d, 1H, J = 8.0 Hz), 7.65 (m, 1H), 7.55 (t, 3H, J = 8.0 Hz), 7.50 (m, 1H). 13C NMR (100 MHz, DMSO-d6) 6 171.5 (C=O), 168.3 (C=S), 167.3 (C=O), 165.0 (C=O), 156.0 (C), 150.2 (C), 134.2 (CH), 132.8 (ch), 131.6 (CH), 131.2 (CH), 130.7 (CH), 128.8 (CH), 128.4 (CH), 128.3 (CH), 128.2 (CH), 125.4 (CH). IR vmax 2988 (C-H), 2911 (C-H), 1670 (C=O), 1658 (C=O), 1536 (c=c), 1489 (c-n), 1424 (c-n) cm-1. Anal. calcd. for C16H14N4O2S2: C, 53.61; H, 3.94; N, 15.63; S, 17.89. Found: C, 53.73; H, 4.02; N, 15.60; S, 17.78. LRMS (m/z, M+) found for C16H14N4O2S2: 358.36, expected mass: 358.44. 3-Benzoyl-1-(phenylamino)thiourea (13). The product recrystallized from DMSO/toluene (1:1) as a white solid. M.p. 242-244 °C. Yield 71.6%. 1H NMR (400 MHz, DM-SO-d6) 6 8.03 (m, 2H), 7.44 (m, 2H), 7.37 (m, 5H), 7.34 (s, 1H), 7.19 (s, 1H). 13C NMR (100 MHz, DMSO-d6) 6 162.9 (C=O), 149.6 (C), 136.8 (C), 129.9 (CH), 129.4 (CH), 129.3 (CH), 128.7 (CH), 126.6 (CH), 125.1 (CH). IR vmax 3070 (N-H), 3018 (N-H), 2727 (C-H), 1591 (C=O), 1561 (C=O), 1499 (C-N), 1475 (C-N) cm-1. Anal. calcd. for C14H13N3OS: C, 62.31; H, 5.19; N, 14.42; S, 13.86. Found: C, 62.31; H, 5.19; N, 14.42; S, 13.86. LRMS (m/z, M+) found for C14H13N3OS: 271.80, expected mass: 271.97. 3-Benzoyl-1-(4-{[(phenylformamido)methanethioyl] amino}butyl)thiourea (14). The product was filtered and recrystallized from DMSO/toluene (1:1) as a light brown solid. M.p. 159-161 °C. Yield 80.8%. 1H NMR (400 MHz, DMSO-d6) 6 11.24 (s, 1H, NH), 10.94 (br, 1H, NH), 7.90 (d, 2H, J = 8.0 Hz,), 7.62 (t, 1H, J = 7.2, 7.6 Hz), 7.48 (t, 2H, J = 7.6 Hz), 3.76 (m, 4h), 2.51, (br, 2H, NH), 2.06 (t, 2H, NH). 13C NMR (100 MHz, DMSO-d6) 6 180.2 (C=S), 167.7 (C=O), 132.9 (C), 132.2 (CH), 128.4 (CH), 128.3 (CH), 42.6 (CH2), 26.7 (CH2). IR vmax 3405 (N-H), 3217 (N-H), 2929 (C-H), 1666 (C=O), 1511 (C=C), 1432 (c-N) cm-1. Anal. calcd. for C20H22N4O2S2: C, 57.95; H, 5.35; N, 13.32; S, 15.47. Found: C, 57.87; H, 5.42; N, 13.45; S, 15.36. LRMS (m/z, M+) found for C20H22N2O2S2: 414.70. Expected mass: 414.54. X-ray crystal structure determination. X-ray diffraction analyses of 1, 11, 12 and 14 were performed at 200 K using a Bruker Kappa Apex II diffractometer with monochro-mated Mo Ka radiation (A = 0.71073 Â). APEXII14 was Odame et al.: Synthesis, Characterization and Biological Activity ... 776 Acta Chim. Slov. 2020, 67, 764-777 used for data collection and15 for cell refinement and data reduction. The structures were solved by direct methods using SHELXS-2013,14 and refined by least-squares procedures using SHELXL-2013,15 with SHELXLE,14 as a graphical interface. All non-hydrogen atoms were refined aniso-tropically. Carbon-bound H atoms were placed in calculated positions (C-H 0.95 A for aromatic carbon atoms and C-H 0.99 A for methylene groups) and were included in the refinement in the riding model approximation, with (7iso(H) set to 1.2Ueq(C). The H atoms of the methyl groups were allowed to rotate with a fixed angle around the C-C bond to best fit the experimental electron density (HFIX 137 in the SHELX program suite15) with (7iso(H) set to 1.5Ueq(C). Nitrogen-bound H atoms were located on a difference Fourier map and refined freely. Data were corrected for absorption effects using the numerical method implemented in SADABS.16 5. Conclusions The work involved the design and synthesis of dith-iourea derivatives for HIV-1 protease inhibitors using Autodock 4.2, the compounds were characterized by spectroscopic techniques and microanalysis. 1-(3-Bromobenzo-yl)-3-[2-({[(3-bromophenyl)formamido]methanethioyl} amino)phenyl]thiourea (10) and 3-benzoyl-1{[(phenylfor-mido)methanethioyl]amino}thiourea (12) gave a percentage viability of 17.9 ± 5.6% and 11.2 ± 0.9% against Trypanosoma brucei. The single crystal X-ray diffraction analysis of 1-benzoyl-3-(5-methyl-2-{[(phenylformamido)meth-anethioyl]amino}phenyl)thiourea (1), 3-benzoyl-1-(2-{[(phenylformamido)methanethioyl]amino}ethyl)thiourea (11), 3-benzoyl-1{[(phenylformido)methanethioyl]amino} thiourea (12) and 3-benzoyl-1-(4-{[(phenylformamido) methanethioyl]amino}butyl)thiourea (14) have been presented. 1-(3-Bromobenzoyl)-3-[2-({[(3-bromophenyl)for-mamido]methanethioyl}amino)phenyl]thiourea (10) gave a percentage inhibition of 97.03 ± 0.37% against HIV-1 protease enzyme at a concentration of 100 ^M. Acknowledgement We thank MRC for the research funding (MRC-SIR). F. Odame thanks the National Research Foundation of South Africa for awarding him a postdoctoral Fellowship. Supplementary Information Supplementary data associated with this article can be found in the online version. CCDC numbers 1448382, 1919730, 1919731 and 1919732 contain the crystal structures associated with this article. 6. References 1. R. Mohebat, G. Mohammadian, J. Chem. Res, 2012, 36, 626628. zoil-1{[(fenilformamido)metantioil]amino}tiosecnina 2. Y. J. Ding, X. B. Chang, X. Q. Yang, W. K. Dong, Acta Cryst. 2008, E64, o658. Trypanosoma brucei. Predstavljeni so tudi rezultati rent 3. W. K. Dong, H. B. Yan, L. Q. Chai, Z. W. Lv, C. Y. Zhao, Acta Cryst. 2008, E64, o1097. DOI:10.1107/S160053680801430X 4. W. K. Dong, X. Q. Yang, L. Xu, L. Wang, G. L. Liu, J. H. Feng, Z. Kristallogr. NCS 2007, 222, 279-280. DOI:10.1524/ncrs.2007.0118 5. F. Kurzer, J. Chem. Soc. (C), 1971, 2932-2938. DOI:10.1039/j39710002932 6. S. K. Kang, N. S. Cho, M. K. Jeon, Acta Cryst. 2012, E68, o395. DOI:10.1107/S1600536812000621 7. E. I. Thiam, M. Diop, M. Gaye, A. S. Sall, A. H. Barry, Acta Cryst. 2008, E64, o776. DOI:10.1107/S1600536808008374 8. Y. H. Lee, W. S. Han, H. J. Lee, S. M. Ahn, T. K. Hong, J. Anal. Chem. 2015, 70, 621-626. DOI:10.1134/S1061934815050172 9. F. Odame, E. Hosten, R. Betz, K. Lobb, Z. R. Tshentu, Acta Chim. Slov. 2015, 62, 986-994. DOI:10.17344/acsi.2015.1703 10. F. Odame, E. C. Hosten, Z. R. Tshentu, R. Betz, Z. Kristallogr. NCS 2014, 229, 337-338. 11. F. Odame, J. Krause, E. C. Hosten, R. Betz, K. Lobb, Z. R. Tshentu, C. L. Frost, Bull. Chem. Soc. Ethiop. 2018, 32, 271284. DOI: 10.4314/bcse.v32i2.8 12. I. Samb, N. Gaye, R. Sylla-Gueye, E. I. Thiam, M. Gaye, P. Retailleau, Acta Cryst E. 2019, 75, 642-645. DOI:10.1107/S205698901900495X 13. B. M. Yamin, M. S. M. Yusof, Acta Cryst E. 2003, 59, o358-o359. DOI:10.1107/S1600536803003635 14. APEX2, SADABS and SAINT (2010) Bruker AXS Inc: Madison, WI, USA. 15. G. M. Sheldrick, A short history of SHELX, Acta. Cryst. A, 2008, 64, 112-122. DOI:10.1107/S0108767307043930 16. C. B. Hübschle, G. M. Sheldrick, B. Dittrich, ShelXle: J. Appl. Cryst. 2011, 44, 1281-1284. DOI:10.1107/S0021889811043202 Odame et al.: Synthesis, Characterization and Biological Activity ... Acta Chim. Slov. 2020, 67, 764-777 777 Povzetek Izvedli smo načrtovanje (s pomočjo Autodock 4.2) in sintezo novih ditiosečninskih derivatov kot inhibitorjev HIV-1 proteaze. Nove spojine smo karakterizirali s spektroskopskimi metodami in z mikroanalizo. Spojini 1-(3-bromoben-zoil)-3-[2-({[(3-bromofenil)formamido]metantioil}amino)fenil]tiosečnina (10) in 3-ben(12) sta dali 17.9 ± 5.6% in 11.2 ± 0.9% sposobnost preživetja za genske difrakcijske analize monokristalov spojin 1-benzoil-3-(5-metil-2-{[(fenil-formamido)metantioil]amino}fenil)tiosečnine (1), 3-benzoil-1-(2-{[(fenilformamido)metantioil]amino}etil)tioseč-nine (11), 3-benzoil-1-{[(fenilformamido)metantioil]amino}tiosečnine (12) and 3-benzoil-1-(4-{[(fenilformamido) metantioil]amino}butil)tiosečnine (14). Za spojino 1-(3-bromobenzoil)-3-[2-({[(3-bromofenil)formamido]metantioil} amino)fenil]tiosečnina (10) smo izmerili odstotno inhibicijo 97.03 ± 0.37% proti encimu HIV-1 proteaza pri koncentraciji 100 |M. Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Odame et al.: Synthesis, Characterization and Biological Activity ... DOI: 10.17344/acsi.2019.5690 Acta Chim. Slov. 2020, 67, 778-784 /^creative ^commons Scientific paper Alternative to Conventional Edible Oil Sources: Cold Pressing and Supercritical CO2 Extraction of Plum (Prunus domestica L.) Kernel Seed Jelena Vladic,1 Aleksandra Gavaric,1 Stela Jokic,2 Nika Pavlovic,3 Tihomir Moslavac,4 Ljiljana Popovic,1 Ana Matias,4 Alexandre Agostinho,4 Marija Banozic2 and Senka Vidovic1^ 1 University of Novi Sad, Faculty of Technology, Bulevar cara Lazara 1, Novi Sad, Serbia 2 J. J. Strossmayer University of Osijek, Faculty of Food Technology, Trg S. Trojstva 3, 31000 Osijek, Croatia 3 J. J. Strossmayer University of Osijek, Faculty of Medicine, Cara Hadrijana 10E, 31000 Osijek, Croatia 4 iBET - Instituto de Biologia Experimental e Tecnológica, Avenida da República, Estagäo Agronómica, 2780-157 Oeiras, Portugal * Corresponding author: E-mail: senka.vidovic@uns.ac.rs Tel.: +381 21 485 3603 Received: 11-08-2019 Abstract Plum (Prunus domestica L.) is a fruit widely cultivated across Europe and its processing generates a considerable amount of waste in form of discharged plum kernels. This creates a new opportunity to exploit plum kernels in order to provide an alternative to conventional edible oils. The main aim of this study was to obtain high-quality oil from plum kernel seeds by applying traditional cold pressing (CP) and supercritical carbon dioxide (ScCO2) extraction as a modern technology. The obtained oils were characterized based on the chemical composition of fatty acids and tocopherols. In obtained oils, twelve fatty acids were identified. The oleic acid was the most dominant in both oils (68.66% in oil obtained by ScCO2, 65.86% in oil obtained by CP), followed by linoleic acid (22.24-25.44%). While total tocopherols content in oil obtained by ScCO2 was 4 to 5.8-fold higher than CP. The results proved that the utilization of plum kernel seeds possess high potential as an alternative oil source due a high amount of oleic acid and tocopherols and a low amount of saturated fatty acids and amygdalin. Keywords: Prunus domestica; supercritical carbon dioxide; cold pressing; tocopherol; fatty acid 1. Introduction Prunus species comprises 40 different varieties. However, only two species are predominate for industrial application: the European plum (Prunus domestica L.) which is hexaploid tree, and the Japanese plum (Prunus salicina L.) which is diploid tree.1 Plum production and processing are widely spread across Europe. Food companies producing dry and canned plum, plum juices and jam; beside, in several European countries plums are used for production of alcoholic beverages.2 Through processing all are generating large amounts of waste, mostly consist of plum kernel. Apart from being use as biodiesel feed- stock,3 cheap source of bioactive peptides,4 active carbons5 or carbonaceous adsorbents,6 plum kernel (plum kernel seeds) could be used as a valuable source of oils, which yield can reach above 50%.7 Recently, nonconventional oils have gained a lot of attention due to their useful properties.8 It is already known that some fruit seeds derived from citruses,9 grapes10 and watermelon11 can be used as sources of oils, phenolics and proteins. Citrus seeds contain 20.0-78.9% of oil depending on the species and culitvation conditions.12 Mathhaus and Ozcan13 reported that in 17 different citrus seeds the content of oils oleic acid was in the range from 12.8 to 70.1%, linoleic acid from 19.5 to 58.8%, and palmitic acid from 5.1 Vladic et al.: Alternative to Conventional Edible Oil Sources: Acta Chim. Slov. 2020, 67, 778-784 779 to 28.3%. Watermelon seeds have an excellent potential for application in food formulations due to the high amount of oil from 50 to 51% and proteins from 32 to 37%.14 Watermelon seed oil is reported to contain a high content of unsaturated fatty acids from 77 to 82%, a high content of lin-oleic acid from 59 to 67.5%, and oleic acid from 14 to 18.1%. In addition, grape seeds also contain a considerable amount of oil (14-17%) and a high content of unsaturated acids compared to sunflower and corn oils, which is the main cause for their high commercial interest. Cao and Ito15 reported linoleic acid as the most abundant fatty acid (68.1078.18%) in grape seed oils. Plum oil is located in the plum kernel seed. According to Velickovic et al.16 the content of seed in the plum kernel is 136 g/kg for, while content of oil, determined using standard Soxhlet procedure, is 409 g of oil per kg of plum.16 Same authors identified six main fatty acids in the plum oil: oleic, linoleic, palmitic, stearic and ara-chidonic acid, out of which oleic (59.5%) and linoleic (27.1%) had the highest percentage.16 Several different techniques could be applied for separation of oil fraction from the seeds. Among them extraction by organic solvents, supercritical extraction by carbon dioxide (ScCO2) and cold pressing (CP), are mostly used. Yield of separated oil, oil quality and chemical composition are dependable on applied technique. Advantage of ScCO2 and CP is the production of safe products, as well as processing in accordance to "green technologies" concept. Therefore, the main aim of this research was to investigate the possibility to obtain high-quality oil from plum kernels seed, using traditional CP and modern technology such as ScCO2. The oils were characterized based on the chemical composition of fatty acids and tocopherols. The characteristics of oil obtained by ScCO2 were compared to the oil produced by CD. The outcomes may provide a preliminary estimation of the plum kernels seed as an alternative source of edible oil, based upon their fatty acid profile since it is the main quality parameter for edible oils. 2. Materials and Methods 2. 1. Material Plum kernels were obtained from fruit producer Ple-mic komerc doo (Osecina, Serbia; year of collection 2017). Material was collected in dry condition and pulverized in mill (MRC Sample mill C-SM/450-C, Holon, Israel). Sieve sets (Erweka, Germany) was in employed for determination of the particle size of the grounded material and the average particle size was 0.310 mm. The purity of CO2 used for extraction was 99.97% (w/w) and purchased from Messer, Osijek, Croatia. For determination of fatty acid composition, Food Industry FAME mix 37 standards (Cat. No. 35077) was purchased from Restek (USA). For determination of tocopherol, a-tocopherol (Dr. Ehrenstorfer Cat No. 17924300), ^-tocopherol (Supelco Cat No. 46401-U), ^tocopherol (Supelco Cat No. 4-7785) and 5-tocopherol (Su- pelco Cat No. 4-7784) were used. Amygdalin (> 99%) standard (A6005-1G) was purchased from Sigma-Aldrich, Cas No. 29883-15-6, USA. All other used chemicals were analytical grade and purchased from J. T. Baker (PA, USA). 2. 2. Cold Pressing of Plum Kernel Oil (PKO) The cold pressed plum kernel oil was obtained by pressing 1 kg of plum seeds using the following parameters: head presses temperature of 40 °C, frequency of 20 Hz and using a nozzle of ID 6 mm. The pressing of the seeds was performed in a screw expeller SPU 20 (Senta, Serbia) with capacity 20-25 kg/h. 2. 3. Supercritical CO2 Extraction of Plum Kernel Oil (ScCO2) The experiments were carried out using ScCO2 system explained in detail elsewhere.17,18 The extractor vessel was filled with the 100 g of grounded dried plum. The obtained extracts (oil) were collected in glass tubes. Extraction time was 5h. After each 30 minutes, extraction process was paused and the amount of obtained extracts was weighed. The ScCO2 extraction parameters were as follows: pressure 300 bar, temperature 40 °C, and mass flow rate of 2 kg/h. Conditions in the separator were pressure 15 bar and temperature 25 °C. 2. 4. Analysis of Fatty Acids (FA) Composition FA methyl esters were prepared according to HRN EN ISO 12966-2:2011 standard method by saponification of glycerides with NaOH in methanol and analysed by gas chromatography carried out using Gas chromatograph 7890B (Agilent Technologies, Lake Forest, USA). Gas chromatography conditions were explained elsewhere.19 Obtained results are expressed as percentage (%) of individual fatty acids to the total fatty acids. The analyses were performed in two replicates. 2. 5. Determination of Content of Tocopherols Determination of tocopherols (a, 5) content in PKO obtained by ScCO2 was performed according to modified HRN EN 12822:2014 standard.20 Analysis was performed using reversed-phase High Performance Liquid Chromatography (HPLC) Infinity 1290 Agilent Technologies (USA) instrument equipped with fluorescence detector (FLD). The analysis was monitored at wavelengths set at 290 and 325 nm, respectively. The instrument was equipped with autosampler G4226A and 1260 FLD G1321C with quaternary pump G4204A. Used column was Zorbax Eclipse XDB, C18 with particle size 5 ^m, and 250 mm long. As a mobile phase was acetonitrile:metha-nol (50:50) used with gradient run time of 16 minutes, as follow: start flow 2 mL/min and holding for 7 minutes, de- Vladic et al.: Alternative to Conventional Edible Oil Sources: 780 Acta Chim. Slov. 2020, 67, 778-784 creasing to 1.5 mL/min. Injection volume was 20 ^L and column temperature was set to 25 °C. Oil for HPLC analysis was prepared as follows. First, certain amount of oil was dissolved in volume of isopropanol providing 88.0089.00% recovery. Prepared solutions were filtered through 0.2 filters before analysis. 2. 6. Determination of Content of Amygdalin Cold pressed PKO and ScCO2 extracts (2 g) were weighed into a round-bottom flask, with added ethanol (50 mL). Prepared mixture was boiled under reflux for 120 min. The extracts were filtered and ethanol was completely evaporated under vacuum. Diethyl ether (10 mL) was added to the dried sample and mixture was vortexed about 1 min to precipitate amygdalin. Diethyl ether was evaporated on rotary evaporator and dissolved in water (5 mL). Sample was filtered through a 0.2 ^m PTFE filter before HPLC analysis.21 The method was performed with an Agilent 1290 Infinity I HPLC system equipped with Agilent DAD detector and auto-sampler using (20 ^L). The detector was set at 210 nm and the peak areas were integrated automatically, using the Agilent HPLC Data Analysis software Chemsta-tion. Used column for separation was C18 column (4.6 x 250 mm, 5 ^m) at 20 °C. Quantitative analysis was performed with the external standardization by measuring the peak areas. RP-HPLC analysis was performed by isoc-ratic elution with a flow rate of 1.0 mL min-1. Used mobile phase was water: methanol (75:25 v/v). The analyses were conducted in two replicates. 3. Results and Discussion 3. 1. Extraction Yield of PKO The kernels recovered from the plum kernels are a valuable source of oil and significant variations in oil yield have been reported in different studies.22-24 The causes of variations might be attributed to a different geographic origin, variety, applied extraction technique, etc. Matthäus and Özcan22 determined oil content in P. domestica from two difference locations (47.1 and 47.8 g/100 g) using petroleum as extraction solvent. According to Gornas et al.,23 significant impacts on the PKO yield were related to variety and applied extraction techniques. Gornas et al.23 reported that the difference between the highest and the lowest level of oil was almost 2.5-fold, while the average content of 28 varieties of two species tested was 38.2% (w/w) where oil extraction was done using n-hexane. In a study by Kostic et al.,24 plum kernel oil yield was 35.8%, determined by the Soxhlet extraction, while the PKO yield obtained by pressing was 25.5%, which was 71% of the oil content. Authors concluded that extraction is a more efficient method than pressing since part of oil remains in cake during pressing. However, having in mind environmental and economic disadvantages of organic solvents, pressing is a more appropriate method.24 In this study, efficiency of ScCO2 as a modern extraction technique and CP technique as a traditional one was compared. Extraction yield obtained with CP was 30.85%, whereas the total extraction yield obtained by ScCO2 was higher, 38.70%. ScCO2 extraction kinetics, that is, the extraction yield in function of extraction time, is presented in Figure 1. 3. 1. Composition of Fatty Acids Seed oils from Prunus species have already been reported to possess a highly desirable fatty acid composition with a high content of oleic acid, variable contents of linole-ic acid followed with low content of saturated fatty acids. According to fatty acid composition, that may result higher consumer preferences than for olive oil.22 Twelve fatty acids in the kernel oils of plum were identified. The oleic and lin-oleic acids were predominant in PKO in both cold pressed Figure 1. Extraction yield of PKO obtained by ScCO2 Vladic et al.: Alternative to Conventional Edible Oil Sources: ... Acta Chim. Slov. 2020, 67, 778-784 781 and ScCO2 extracts. These results are consistent with the ones from a published study by Kiralan et al.25 where oleic acid was predominant, followed by linoleic and palmitic acids. Kamel and Kakuda26 also reported oleic and linoleic acids as prevailing fatty acids in peach kernel oil. The oleic acid is 18-carbon monounsaturated fatty acid, essential and highly preferable in human nutrition. It is attributed to have positive effects such as reducing triglycerides, total cholesterol, and glycemic index in human metabolism. Moreover, the increase in oxidation stability in vegetable oil is often assigned to oleic acid presence.27 The PKO is characterized with a high content of oleic acid (68.66% in PKO obtained by ScCO2, 65.86% in PKO obtained by cold pressing) and a significantly lower amount of saturated fatty acids like palmitic acid and stearic acid (5.80% and 1.92% in PKO obtained by ScCO2, respectively; 5.79% and 1.62% in PKO obtained by cold pressing, respectively). Similar results were obtained by Kiralan et al.25 where the content of oleic acid was higher (75.43%), the content of palmitic acid was slightly higher (5.83%), and the content of stearic acid was slightly lower (1.35%) in P. domestica oil. The oleic (5583%) and palmitic (7.5-20%) acids were the prevailing fatty acids in olive oils, followed by stearic acid (0.5-5.0%).28 Detailed fatty acid profile is displayed in Table 2. Moreover, the composition of the obtained fatty acids has a number of similarities with other kernel oil studies.29,30 However, notable differences were not found in fatty acid profiles of PKO obtained by ScCO2 and CP. Considering its nutritional value, genus Prunus kernels represent an attractive oil for human consumption due to high values of oleic and linoleic acids. In a study by Matthäus and Özcan,22 the mean value for the content of oleic acid was 66.9% and for linoleic acid 22.7% which is in Table 2. Fatty acid composition of plum kernels obtained by ScCO2 and agreement with our findings. Regarding fatty acid composition, the Prunus kernel seed oil could be compared to mid-high-oleic rapeseed oil which does not possess a-lin-olenic acid which is sensitive to oxidation. It is convenient that the amount of this fatty acid is low (0.09% in PKO obtained by ScCO2, 0.07% in PKO obtained by CP). This is significant for the stability of PKO, especially if food processing requires heat. Therefore, taking into account the low amount of saturated fatty acids, the high amount of monounsaturated oleic acid, and a desirable fatty acid composition, PKO is highly recommended for human consumption as edible oil.22 3. 2. Tocopherol Content Tocopherol comprises several lipophilic phenolic compounds commonly found in edible oils, oil products, fatty fishes, cereals, nuts and other fat-contain products.31 This vitamin has 4 tocopherol homologues a-, p-, y-, and S-tocopherol, and 4 tocotrienol homologues, a-, p-, y-, and S-tocotrienol.32 Primary function of antioxidants and vitamin E is terminating free radicals in vivo.32 The relative reactivity of a, p, y, and S-tocopherol forms against oxygen radicals decreases following the order of a > p = y > S.33 Therefore, a-tocopherol has the highest affinity to a-to-copherol transferprotein and as the consequence highest bioavailability32 and low rate of metabolism.35 However, the deficiency of tocopherol in diet may cause circulatory disorders and influence on the metabolism pathway in muscles. Since vegetable oils provide humans with a significant part of their daily vitamin E dietary requirements,36 it was of great importance to determine tocopherols profile for PKOs. Results of quantitative HPLC analysis of tocoph- old pressing Fatty acid Average value for Average value ScCO2-PKO [%] for CP-PKO [%] C16:0 Palmitic acid 5.80 5.79 C16:1 (cis-9) Palmitoleic acid 0.90 0.93 C17:1 (cis-10) Heptadecenoic acid 0.10 0.11 C18:0 Stearic acid 1.92 1.62 C18:1 (cis-9) Oleic acid, « - 9 68.66 65.86 C18:2 (cis-9,12) Linoleic acid, « - 6 22.24 25.44 C20:0 Arachidic acid 0.13 0.11 C20:1 (cis-11) Eicosenoic acid 0.09 0.07 C18:3 (cis-9,12,15) Linolenic acid, « - 3 0.09 0.07 C22:0 Behenic acid 0.02 <0.01 C20:3 (cis-8,11,14) Eicosatrienoic acid <0.01 <0.01 C22:1 (cis-13) Erucic acid, « - 9 0.01 <0.01 C24:0 Lignoceric acid 0.01 <0.01 ^Saturated fatty acids SFA ^Unsaturated fatty acids UFA ZMonounsaturated fatty acids MUFA ^Polyunsaturated fatty acids PUFA LOD - limit of detection; <0.01 7.90 7.52 92.10 92.48 69.76 66.97 w22.33 25.51 Vladic et al.: Alternative to Conventional Edible Oil Sources: 782 Acta Chim. Slov. 2020, 67, 778-784 Table 3. Content of tocopherols in PKO PKO a-tocopherol P+y-tocopherol ô-tocopherol (mg/g oil) (mg/g of oil) (mg/g of oil) CP-PKO 0.076 1.22 0.149 ScCO2 after 1h 0.157 2.197 0.354 after 2h 0.092 1.46 0.217 after 3h 0.033 0.921 0.131 after 4h 0.019 0.720 0.096 after 5h 0.0063 0.532 0.067 Total 0.310 5.830 0.865 erols in PKOs, cold pressed or obtained by ScCO2, are expressed as mg of compound per g of oil (mg/g) in Table 3. The tocopherol homologues (a, p, y and 5) were detected in cold pressed PKO, and also in PKO obtained by ScCO2. Whole ScCO2 process lasted for 5 hours and oil fractions were collected every hour of the extraction in order to determine at which extraction time the most significant solubility of tocopherols in oil is achieved. Previous researches37-40 showed that ScCO2 extraction can provide satisfactory tocopherol yield from kernels and other plant by-products. According to Hassanein,41 tocopherol content of PKO (0.71 mg/g) was distinctly higher than that of apricot (0.43 mg/g) and peach (0.52 mg/g) kernel oils, where oil was extracted using chloroform-methanol as a solvent. The oils derived from plum contained 85.5% of Y-tocoph-erol, apricot oil 93.5% of Y-tocopherol and peach 97.7% of tocopherol. However, a-and 5-tocopherols were detected in minor amounts. Beta-tocopherol was not detected in the three above mentioned oils. Previous mentioned three kernel oils showed to be highly resistant to autoxidation due to high content of Y-tocopherol.42 In our case, the majority of p+Y-tocopherols were obtained after two hours of ScCO2. After 1h of ScCO2, the amount of a-tocopherol is 2-fold higher than the amount obtained by cold pressing. The similar ratio is noticed after 1h of ScCO2 in P+Y-to-copherols and 5-tocopherol amounts toward the ones obtained by traditional method. It is evident that the highest solubility of tocopherols was in the first hour of the extraction. Solubility decreases with further extraction, and after 5 hours of extraction, a 25 times lower amount of a-tocopherol was extracted in comparison with the first hour. A similar pattern of results was obtained for apricot kernels seeds in a study Pavlovic et al.37 where after 1 hour of ScCO2 extraction the total content of tocopherols significantly decreased. When comparing the total tocopher-ols content in PKO obtained by ScCO2 to those obtained with a conventional technique, it must be pointed out that a higher tocopherols content (4 to 5,8-fold) was obtained by applying the modern extraction technique (ScCO2). Obtained tocopherol values correlate fairly well with Gor-nas et al.43 where the extraction of PKO was conducted with hexane and assisted with ultrasound waves. Similarly to their research, Y-tocopherol was found to be predominant. 3. 3. Amygdalin Content Stones from the Prunus genus fruits are low-cost and could represent considerable sources of proteins potential sources of peptides with biological activity. However, main restriction to the use of these oil sources is the presence of cyanogenic glycosides such as amygdalin.44 Amygdalin is cyanogenic glycoside, commonly present in kernels and seeds of different fruits.45 This glycoside is potentially toxic in the presence of enzymes (P-glucosidases and a-hy-droxynitrilelyases), resulting in the release of hydrogen cyanide.46 On the other hand, amygdalin exhibits many positive biological activities such as the anti-inflammatory and anti-cancer activity. Bolarinwa et al.46 reported that seeds from Rosaceae species, especially from subspecies Pomoideae and Prunoideae contained relatively high amounts (0.1-17.5 mg/g) of amygdalin compared to seeds from non-Rosaceae species (0.01-0.2 mg/g). Senica et al.47 also reported big differences in amounts of amygdalin in plum varieties. The lowest amount of amygdalin was in seeds of Valjevka variety and the highest amount of amygdalin was in seeds of Jojo variety. Before industrial application of Rosaceae species, it is recommended to perform a determination of amygdalin.48 Garcia et al.44 reported amygdalin content of 4.39 mg/g present in the plum seeds, which is 10-fold higher in comparison to amygdalin content (0.41 mg/g) found in our sample. A possible explanation for the significant differences in amygdalin content can be found in different environmental conditions, such as geographical origin, atmospheric conditions, and so on.47 Amygdalin content in our plum seed was very low, and in both PKOs is present in traces (Table 4). Moreover, amygdalin content in both oils was significantly lower than in fresh plum kernels. The decrease in amygdalin content could be explained by enzyme degradation of amygdalin into degradation products such as cyanide. Both CP and ScCO2 methods for obtaining plum kernel seeds oil were performed at low temperature (40 °C), avoiding inactiva-tion of enzymes above 100 °C. With respect to differences in variety and extraction method, enzymatic degradation could be responsible for low amygdalin content. Generally, amygdalin content decreased in processed products for all Rosaceae and non-Rosaceae species.46 However, future Table 4. Amygdalin content in plum kernels, their oil and extract Sample Amygdalin content Plum kernels 0.4100 mg/g of kernels CP-PKO 0.0025 mg/g of oil ScCO2-PKO 0.0022 mg/g of oil LOD (limit of detection) = = 0.00024 mg/g; LOQ (limit of quantita- tion) = 0.00074 mg/g Vladic et al.: Alternative to Conventional Edible Oil Sources: ... Acta Chim. Slov. 2020, 67, 778-784 783 work should consider the determination of enzymatic degradation products. 4. Conclusions Despite the growing number of published papers, the fruit kernels are still considered as non-conventional potential oil sources. In view of the current desire for convenience food such as seedless fruits (citrus, grapes, watermelon, cherry, etc.) there is a tendency of growing kernel waste and further disposal issues. Dealing with the issue of pre-consumer or production food waste will be a crucial action prior to improving productivity and sustainability of the food production system. The high oil content in Prunus kernel seeds is comparable to commercial oils seeds such as rapeseed or sunflower seeds. Therefore kernels from genus Prunus are highly suitable for commercial oil production. Due to this, the utilization of kernels from this genus seems to be an interesting niche to create an extra value from a by-product. Fatty acids composition of PKOs obtained by ScCO2 and by cold pressing are similar, however, ScCO2 has shown as more efficient considering notably higher yields of tocopherols, especially a-tocopherol. Additionally, obtained results justify the further processing of plum kernels as by-products of the fruit industry for the production of oil for potential food and pharmaceutical applications. 5. References 1. B. L. Topp, D. M. Russell, M. Neumüller, M. A. Dalbö, W. Liu, Springer US, 2012, 571-621. DOI:10.1007/978-1-4419-0763-9_15 2. F. V. Dulf, D. C. Vodnar, C. Socaciu, Food Chem. 2016, 209, 27-36. DOI:10.1016/j.foodchem.2016.04.016 3. M. D. Kostic, A. V. Velickovic, N. M. Jokovic, O. S. Stamenk-ovic, V. B. Veljkovic, Wast. Manag. 2016, 48, 619-629. DOI:10.1016/j.wasman.2015.11.052 4. E. Gonzalez-Garcia, M. L. Marina, M. C. Garcia, J. Funct. Foods, 2014, 11, 428-437. DOI:10.1016/j.jff.2014.10.020 5. P. Nowicki, H. Wachowska, R. Pietrzak, J. Hazard. Mater. 2010, 181(1), 1088-1094. 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Academic Press, New York, 1989, 363. DOI:10.1016/B978-0-12-718051-9.50013-5 43. P. Görnas, M. Rudzinska, M. Raczyk, I. Misina, A. Soliven, G. Lacis, D. Seglina, J. Agric. Food Chem. 2016, 64, 898-905. DOI:10.1021/acs.jafc.5b05330 44. M. C. García, E. González-García, R. Vásquez-Villanueva, M. L. Marina, Food Funct. 2016, 7(11), 4693-4701. DOI:10.1039/C6F001132B 45. G. B. Donald, Med. Toxicol. Nat. Subst. 2009, 55, 336-352. 46. I. F. Bolarinwa, C. Orfila, M. R. A. Morgan, Food Chem. 2014, 152, 133-139. DOI:10.1016/j.foodchem.2013.11.002 47. M. Senica, F. Stampar, R. Veberic, M. Mikulic-Petkovsek, J. Agric. Food Chem. 2017, 65(48), 10621-10629. DOI:10.1021/acs.jafc.7b03408 48. P. Görnas, I. Misina I. Gravite, G. Lacis, V Radenkovs, A. Olsteine, D. Seglina, E. Kaufmane, E. Rubauskis, Eur. Food Res. Technol. 2015, 241, 513-520. DOI:10.1007/s00217-015-2480-4 Povzetek Sliva (Prunus domestica L.) je pogosto gojena na območju Evrope in procesiranje plodov ustvarja znatne količine odpadnih pešk, ki vsebujejo mehko jedrce. Le-ta lahko predstavljajo alternativni vir jedilnih olj. Cilj te študije je bil pridobitev visoko kvalitetnega olja iz jedrc slivovih pešk z uporabo tradicionalne tehnike hladnega stiskanja (ang. CP) in moderne tehnologije ekstrakcije s superkritičnim ogljikovim dioksidom (ScCO2). Pridobljena olja smo okaraterizirali na osnovi kemijske sestave maščobnih kislin in tokoferolov. Oleinska kislina je bila prisotna v najvišji koncentraciji v olju pri obeh tehnikah ekstrakcije (68.66 % v olju pridobljenem s ScCO2, 65.86 % pridobljenem s CP), sledila pa je linolenska kislina (22.24-25.44 %). Celotna količina tokoferolov v olju pa je bila v primeru ekstrakcije s ScCO2 4-5.8 krat višja kot pri CP. Rezultati kažejo, da imajo jedrca slivovih pešk velik potencial kot alternativni vir olj z visokim deleže oleinske kisline in tokoferola ter nizkim deležem nasičenih maščobnih kislin in amigdalina. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Vladic et al.: Alternative to Conventional Edible Oil Sources: ... DOI: 10.17344/acsi.2019.5720 Acta Chim. Slov. 2020, 67, 785-798 /^creative ^commons Scientific paper Sulfonamide Derived Esters: Synthesis, Characterization, Density Functional Theory and Biological Evaluation through Experimental and Theoretical Approach Muhammad Danish,1^ Ayesha Bibi,1 Muhammad Asam Raza,1^ Nadia Noreen,1 Muhammad Nadeem Arshad2 and Abdullah Mohamed Asiri2 1 Department of Chemistry, University of Gujrat, Gujrat 50700 Pakistan 2 Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia * Corresponding author: E-mail: drdanish62@gmail.com and asamgcu@yahoo.com Received: 11-19-2019 Abstract A series of new solid esters was synthesized by using greener chemistry strategy involving simple reaction of an alcohol with sulfonamide ligand. Characterization study of these methyl (1), ethyl (2) isopropyl (3) and «-butyl (4) ester of 4-((4-chlo-rophenylsulfonamido)methyl)cyclohexanecarboxylic acid was done by using FTIR, NMR mass spectrometry and X-ray crystallography. The compounds were optimized with Gaussian software according to basis set B3LYP/6-31G(d,p) and their different parameters related to structure were calculated. Furthermore, all compounds of the series were screened for their in vitro biological applications involving anti-bacterial (Chromohalobactor salixgens, Halomonas halofila, Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Shiegella sonnei), anti-fungal (Aspergillus niger), anti-oxidant (DPPH scavenging activity) and enzyme inhibition (acetylcholine esterase and butyrylcholine esterase) study. Sulfonamide based esters were also docked against selected enzymes (AChE and BChE) using MOE software for their mode of binding. Results obtained from these biological evaluations showed that such compounds have potential against targeted activity. Keywords: Sulfonamide derived esters; DFT; docking studies; enzyme inhibition. 1. Introduction Infectious diseases due to bacteria or fungi are the major leading causes of morbidity all over the world.1-4 The development of resistance (antibacterial) in microbes against the present antibiotics is growing now on a daily basis.5 Consequently, there is an urgent need in this area for new and improved antimicrobial agents having a broad-spectrum activity against the resistant strains. Researchers throughout the world are engaged in synthesizing and designing new drugs having widespread activity to overcome this issue.6-9 Sulfonamides form the foundation for the first drugs mainly employed as preventive and che-motherapeutic agents against different ailments.10 Sulfa drugs, having sulfonamide functionality, revolutionized the medicinal field due to their extensive biological activi-ties.11 Folic acid, an important compound for synthesis of bacterial nucleic acids, is inhibited by sulfonamides which ultimately leads to death of bacterial cells. Green chemistry synthesis is the current requirement as it has no haz- ardous by-products. Carboxylate ester formation implemented simply by reacting alcohol with carboxylic acid is such an example. Besides their applications in artificial flavoring of food and in perfume making,12 esters of various compounds are found to show promising biological applications. Variety of carboxylate esters derived from sulfonamides are found to serve in the field of health by giving meritorious biological applications. Ester derived from para-tolylbenzene sulfonamide of benzoic acid has been found to show inhibition activity for enzyme lipoxy-genase. Carboxylate ester derived from biphenyl sulfon-amide is found to show inhibitory behavior toward carbonic anhydrase enzymes as well as its isozymes, including isozymes I, II, XIV, XII and XIV.13 Moreover, for the treatment of obesity and to control Type 2 diabetes, esters derived from arylsulfonamide served to give such applica-tions.14 As anti-microbial agents, esters of N-substituted sulfonamide are found to give anti-bacterial activity against four bacterial strains, i.e. Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli and Staphylococcus au- Danish et al.: Sulfonamide Derived Esters: Synthesis, Characterization, 786 Acta Chim. Slov. 2020, 67, 785-798 reus.15 Due to the presence of amide and sulfonamide functional groups in carboxylate esters of sulfonamide derivatives, they are found to show anti-fungal activity against Aspergillus niger and Candida albicans.16 In current research work new methyl, ethyl, isopro-pyl and n-butyl esters derived from 4-((4-chlorophenyl-sulfonamido)methyl)cyclohexanecarboxylic acid were aimed to be synthesized. In vitro biological study, i.e. enzyme inhibition, anti-oxidant study, anti-bacterial and antifungal screening were also part of this research work. 2. Experimental 2. 1. Chemical and Instruments Chemicals like dimethyl sulfoxide, methanol, etha-nol, isopropanol and n-butanol were purchased from Alfa Aesar and Merck chemical industries. These were of analytical grade. Infrared spectral study of compounds was done in mid IR region (4000-400 cm-1) by KBr disc method using Perkin-Elmer System 100. Mass spectral study was done by ESI-MS technique. 2. 2. Synthesis of Carboxylate Esters of Sulfonamide 20 mL alcohol (methanol for 1, ethanol for 2, isopropanol for 3 and n-butanol for 4) was taken and then added 1 mL conc. H2SO4 as a catalyst into it. 0.5 g of 4-((4-chlorophenylsulfonamido)methyl)cyclohexanecar-boxylic acid (published by our research group) was added in 20 mL alcohol till clear solution was obtained. Then added this alcoholic solution to alcohol-sulfuric acid mixture and refluxed it for about 4-6 hours. Reaction mixture was concentrated at room temperature by slow evaporation process and ester as a solid product was obtained (Scheme 1).17 2. 3. Crystallography of 3 The compound 3 was re-crystallized to support the synthesis of series of compounds being presented in this manuscript. Microscope was used for screening of suitable crystal for data collection. The selected single crystal was fixed over a glass fiber tip fascinated in a wax supported by a hollow copper rod with magnetic base. This holder was mounted on Agilent SuperNova (Dual source) Agilent Technologies Diffractometer, equipped with graphite-monochromatic Cu/Mo Ka radiation for data collection. The data collection was accomplished using Crys-AlisPro software18 at 296 K under the Mo Ka radiation. The structure solution was performed using SHELXS-9719 and refined by full-matrix least-squares methods on F using SHELXL-97 in-built with WinGX.20 All non-hydrogen atoms were refined anisotropically by full-matrix least-squares methods.19 Figures were drawn using PLATON Scheme 1. Synthesis of carboxylate esters of sulfonamide Danish et al.: Sulfonamide Derived Esters: Synthesis, Characterization, ... Acta Chim. Slov. 2020, 67, 785-798 787 and ORTEP-3.20 All the aromatic CH hydrogen atoms were positioned geometrically and treated as riding atoms where C-H = 0.93 A and Uiso(H) = 1.2Ueq(C) for carbon atoms. The C-H bond distances are 0.96 A, 0.97 A and 0.98 A for methyl, methylene and methine groups, respectively. Uiso(H) = 1.5Ueq(C) for methyl carbon atoms, while Uiso(H) = 1.2Ueq(C) for methylene and methine carbon atoms. The N-H = 0.68(7)-0.94(4) A, hydrogen atoms were located with difference Fourier map and refined with Uiso(H) = 1.2Ueq(N). The assigned CCDC number is 1861455. 2. 4. Biological Studies Antibacterial Activity Antibacterial activity was determined according to the disc diffusion method against four bacterial strains: Chromohalobacter salexigens, Chromohalobacter israelen-sis, Halomonas halofila and Halomonas salina.21 Bacterial medium was prepared and autoclaved for 20 min at 121 °C and 15 psi. 30 mL of the sterilized medium was poured in the petri plates and seeded with respective bacterial strains. 20 ^L of sample (5 mg/mL) was applied on disks with the help of a micropipette. Streptomycin and ampicillin were used as reference drugs while solvents were used as negative. After incubation of 24 hours at 37 °C, the zone of inhibition was measured. Antifungal Activity Antifungal activity was determined against two different fungal strains: Aspergillus flavus and Aspergillus ni-gerby using the method of Samina et al. (2009) with minor modification.21 Sterilized medium of 30 mL was poured aseptically in autoclaved petri plates and seeded with the respective fungal strain. After the solidification of the medium disks were placed on it and 20 ^L of sample (5 mg/ mL) was applied on each disc. The plates were incubated at 25 °C and the zone of inhibition was measured with Vernier caliper after 48 hours. Antioxidant Activity Antioxidant activity of synthesized compounds was checked according to the method of Shahwar et al. (2012) using 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical.22 DPPH solution was prepared as 0.0025 g/mL in methanol and 100 ^L of sample (5 mg/mL) was mixed with 2 mL DPPH solution. Test tubes were kept in dark for half an hour and measured the absorbance at 517 nm using methanol as blank and gallic acid as reference standard. The scavenging of free radicals was calculated using following formula: Enzyme Inhibition Studies The AChE and BChE inhibition activities were determined according to the method of Ellman et al. (1961)23 with slight modifications. 100 ^L test compound (5 mg/ mL) was mixed with 100 ^L enzyme (AChE and BChE) and incubated at 37 °C for 10 minutes. After incubation, 0.5 mL buffer (50 mM), 50 ^L DTNB followed by the addition of 50 ^L substrate acetylthiocholine iodide and butyr-ylthiocholine iodide for AChE and BChE, respectively. After 30 minutes of incubation at 37 °C, the absorbance was measured at 410 nm using UV/VIS spectrophotome-ter. All experiments were carried out with their respective controls in triplicate. The percentage inhibition was calculated by the following formula: A -B % age inhibition --.vl 00 (2) Where A is the optical density of blank and B is the optical density of sample. 2. 5. DFT Studies Quantum chemical calculations were performed with Gaussian 09. The results are visualized with Gauss View 5.0. The geometries of the compounds are optimized without any symmetry constraints using the hybrid functional B3LYP method with 6-31G(d,p) basis set.24,25 The basis set chosen contains polarization functions on all atoms. The B3LYP method of DFT is quite reliable for the prediction of geometric and electronic properties of neutral and charged species ranging from simple molecular to polymer structures.26-28 For optimization, the input geometries are taken from the crystal structure (where available) in order to better match with the experimentally obtained structures. Frequency calculations are also performed at the same level in order to confirm these structures as true minima (absence of an imaginary frequency). 2. 6. Docking Studies Docking experiments were performed via Molecular Operating Environment (MOE). Crystal structures of AChE and BChE with PDB codes 1EVE and 1POI, respectively, were selected for these studies. All the water molecules were removed from the protein structure, then hydrogen atoms were added and energy optimization was carried out using default force field. The three-dimensional (3D) structures of compounds were modeled through the builder program implemented in MOE. The geometrical parameters for 3D structures of the compounds were optimized, and partial charges were calculated before docking. The 3D protonation of the downloaded enzymes was done and energy minimization of the retrieved protein molecule was carried out using default parameters of Danish et al.: Sulfonamide Derived Esters: Synthesis, Characterization, ... 788 Acta Chim. Slov. 2020, 67, 785-798 MOE energy minimization algorithm (gradient: 0.05, Force Field: MMFF94X). The resulting model was subjected to systematic conformational search at default parameters with RMS gradient of 0.01 kcal/mol using Site Finder. For 1EVE (AChE), the active site of the prepared enzyme was defined as the residues within 10 A of the reference ligand (donepezil). However, for 1POI (BChE), the enzyme was searched for its active site and dummy atoms were created using alpha spheres as centroids. A key tryp-tophan residue in AChE, Trp84 (TcAChE numbering), is conserved in BChE (Trp82). The backbone and residues were kept fixed and the energy minimization was performed. The lowest energy minimized pose was used for further analysis. Ligand-interaction module of MOE was used to calculate the 2D ligand-enzyme interactions. The view of the docking results and analysis of their surface with graphical representations were done using MOE and discovery studio visualizer.29 3. Results and Discussion The reaction was carried out in acid catalyzed media by simply reacting alcohol with sulfonamide ligand. The reaction gives water as by-product, hence the strategy is a type of green synthesis which is advantageous and environmentally friendly having no hazardous or harmful effects on the environment. The structure and activity of the starting material (acid) has been already published by our group.30 The reaction was monitored with TLC and after completion of the reaction, structure elucidation was done with FTIR, mass spectrometry and NMR. The exact crystal structure of 3 was confirmed with XRD analysis. 3. 1. Methyl 4-((4-Chlorophenylsulfonamido) methyl)cyclohexanecarboxylate (1) White amorphous solid; yield: 1.74 g (76%); mp: 96-97 oC; molecular formula: C15H20ClNO4S; molecular mass: 345.84 g mol-1; IR (KBr, cm-1): vmax 3270 (NH), 2922 (CH), 1320-1158 (SO2), 1699-1432 (C=O); 1H NMR (DMSO-d6, 300 MHz): 5 7.65-7.80 (dd, 4H, aromatic), 3.56 (s, 3H, -CH3), 2.59 (t, 2H, -CH2), 0.79-2.24 (m, 10H, cyclohexyl); 13C NMR (DMSO-d6, 75 MHz): 5 175.8 (C-2), 140.0 (C-4'), 137.5 (C-1'), 129.8 (C-3' and C-5'), 128.8 (C-2' and C-6'), 51.7 (C-1), 48.8 (C-3), 42.6 (C-1''), 37.1 (C-4''), 29.4 (C-2'' and C-6''), 28.5 (C-3'' and C-5''); EI-MS: m/z 344.25 [M - 1]. 3. 2. Ethyl 4-((4-Chlorophenylsulfonamido) methyl)cyclohexanecarboxylate (2) Lustrous white amorphous; yield: 0.81 g (47%); mp: 120-122 °C, molecular formula: C16H22ClNO4S; molecular mass: 359.87 g mol-1; IR (KBr, cm-1): vmax 3298 (NH), 2934 (CH), 1321-1159 (SO2), 1727 (C=O). 1H NMR (CDCl3): 5 7.5-7.8 (dd, 4H, aromatic), 4.8 (t, 3H, -CH3), 4.1 (q, 2H, -CH2), 1.0-2.2 (m, 10H, cyclo-H); 13C NMR (CDCl3): 5 175.7 (C-3), 139.1 (C-4'), 138.5 (C-1'), 129.4 (C-3' and C-5'), 128.5 (C-2' and C-6'), 60.3 (C-1), 49.1 (C-3), 43.1 (C-1''), 37.2 (C-4''), 29.5 (C-2'' and C-6''), 28.2 (C-3'' and C-5''), 14.2 (C-2); ESI-MS: m/z 358.25 [M - 1]. 3. 3. Isopropyl 4-((4-Chlorophenylsulfona-mido)methyl)cyclohexanecarboxylate (3) Lustrous white crystalline; yield: 1.97 g (84%); mp: 111-113 °C, molecular formula: C17H24ClNO4S; molecular mass: 373.89 g mol-1; IR (KBr, cm-1): vmax 3263 (NH), 2924 (CH), 1320-1158 (SO2), 1730-1432 (c=o), ESI-MS: m/z 372.25 [M - 1]. 3. 4. Butyl 4-((4-chlorophenylsulfonamido) methyl)cyclohexanecarboxylate (4) Off-white amorphous solid; yield: 1.21g (59%); mp: 106-108 °C; molecular formula: C18H26ClNO4S; molecular mass: 387.92 g mol-1; IR (KBr, cm-1): vmax 3267 (NH), 2931 (CH), 1323-1159 (SO2), 1725-1431 (C=O); 1H NMR (DMSO-d6, 300 MHz): 5 7.65-7.80 (dd, 4H, aromatic), 4.00 (t, 2H, H-4), 2.58 (t, 2H, H-6), 1.54-2.21 (m, 10H, cyclohexyl), 0.87 (t, 3H, H-1), 1.20 (m, 2H, H-2), 1.47 (m, 2H, H-3); 13C NMR (DMSO-d6, 75 MHz): 5 175.4 (C-5), 140.0 (C-4'), 137.5 (C-1'), 129.8 (C-3' and C-5'), 128.8 (C-2' and C-6'), 63.8 (C-4), 48.9 (C-5), 42.8 (C-1''), 37.1 (C-3), 30.6 (C-4''), 29.4 (C-2'' and C-6''), 28.5 (C-3'' and C-5''), 19.0 (C-2), 14.0 (C-1); ESI-MS: m/z 386.33 [M - 1]. 3. 5. Crystallography of 3 Molecule 3 is ornamented with the methyl, methylene, methine and aromatic hydrogen atoms along with the NH group. We have observed two independent molecules [(C1-C17) and (C18-C34)] per asymmetric unit cell (Figure 1). The crystallographic parameters are given in Table 1, while the selected bond lengths and bond angles are provided in Supplementary Data (Tables S1 and S2). The cyclohexane ring adopted the chair conformation in each independent molecule and the root mean square (r.m.s) deviations for the fitted atoms of this ring are 0.2342(4) A and 0.2298(4) A in molecule 1 and molecule 2, respectively. The puckering parameters were determined for the cyclohexane rings in each independent molecule and the parameters in black and white are Q = 0.5738, 6 = 0.69 and p = 22.3039 for the ring II (C8-C13), while Q = 0.5628, 6 = 1.55 and p = 40.732 for the ring IV (C25-C30). The geometry around the S atom is distorted tetrahedral which is typical behavior of sulfonamide functional group.31-34 The dihedral angle between the aromatic ring I (C1-C6) and cyclohexane ring II (C8-C13) Danish et al.: Sulfonamide Derived Esters: Synthesis, Characterization, ... Acta Chim. Slov. 2020, 67, 785-798 789 is 44.649(3)° but the same angle between the ring III (C18-C23) and ring IV (C25-C30) is 45.663(3)°. The car-boxylate and the isopropyl groups are almost perpendicular in each molecule as the dihedral angles are 78.772(7)° and 78.906(72)° in molecule 1 and molecule 2, respective- ly. The carboxylate groups are twisted at dihedral angles of 68.738(3)° and 60.883(4)° with respect to the plane produced from the fitted atoms of cyclohexane rings in molecule 1 and molecule 2, respectively. Classical hydrogen bonding of N-H—O type connects the molecule along b-axis to generate the infinite long chains. The N1 acts as donor via H1N to the O1 following the symmetry operation 1 - x, V + y, -z. The N2 acts as donor in the molecule 2 via H2N to O5 oxygen atom, where the symmetry operation is 1 - x, V + y, 1 - z as shown in Figure 2, Table 2. Table 1. Crystal data and structure refinement for 3 Figure 2. A unit cell view for 3 showing the intermolecular hydrogen bonding and formation of long chains along b-axis. CCDC number l86l455 Empirical formula Cl7H24ClNO4S Formula weight 373.88 Temperature/K 296(2) Crystal system monoclinic Space group P2l a/A l6.9426(l3) b/A 5.8454(3) c/A 2G.59lG(l8) a/° 9G ¿3/° ll2.829(9) 9G Volume/A3 l879.5(3) Z 4 Pcalcmg/mm3 l.32l ,w/mm-1 G.334 F(000) 792.G Reflections collected llGGG Independent reflections 7495[R(int) = G.G379] Data/restraints/parameters 7495/l/442 Goodness-of-fit on F2 l.G26 Final R indexes [I > 2a (I)] Rl = G.G729, wR2 = G.l776 Final R indexes [all data] Rl = G.G986, wR2 = G.2G39 Largest diff. peak/hole / e A-3 l.ll/-G.3l Flack Parameters G.G2(l2) Danish et al.: Sulfonamide Derived Esters: Synthesis, Characterization, ... 790 Acta Chim. Slov. 2020, 67, 785-798 Table 2. Hydrogen Bonds for 3 D H A d(D-H)/Â d(H-A)/Â d(D-A)/Â D-H-A/° N1 H1N oi1 1.01(6) 2.02(6) 2.992(6) 161(5) N2 H2N O5u 0.87(6) 2.12(6) 2.977(6) 169(6) 1 1 - x, -/ + y, -z 11 1 - x, / + y, 1 - z 3. 6. Antimicrobial Studies Bacteria are necessary for life function but pathogenic bacterial species are major cause of various infections in human bodies. For the treatment of infections, different antibiotics are being sold in the market and one among them is sulfa drug. Our group is also synthesizing different sulfonamide compounds and evaluating their antibacterial potential using in vitro model. Here we aimed to check the anti-bacterial potential of 1, 2, 3 and 4 against six bacterial strains, i.e. C. salixgens, H. halofila, S. aureus, B. subtilis, S. sonnei, E. coli. Results obtained showed that except C. sa-lixgens, all bacteria were inhibited by synthesized compounds. Aspergillus niger (A. niger) is the fungus that causes aspergillosis; a lung disease, for the person having extremely weak immune system.35 It is also a common cause of fungal ear infection known as otomycosis in tropical areas.36 For the treatment of these diseases, synthesis of anti-fungal agents is obvious. In current research work compounds 1, 2, 3 and 4 were screened for their anti-fungal potential against A. niger. It was found from results that 2 and 3 are more active against fungal strain (see Table 3). 3. 7. Antioxidant Potential Reactive oxygen species (ROS) are free radicals involving hydrogen peroxide, hydroxyl ion, superoxide, hydrogen peroxide and hydroxyl free radical (•OH, OH-, •O2-, -O2-2, H2O2). Diseases caused by the overproduction of ROS in body involve chronic inflammation and autoimmune diseases, infectious diseases, cancer, sensory impairment, neurological disorders, fibrotic and cardiovascular diseases.37 In order to treat the mentioned health disorders, use of antioxidants is imperative. The antioxidant po- tential of all synthesized esters has been evaluated using standard protocols and results are presented in Table 4. All compounds exhibited moderate to good activity except 1 (24.1±0.4), furthermore maximum radical scavenging activity was shown by 3 comparable to standard. 3. 8. Acetylcholine/Butyrylcholine Esterase Study Acetylcholine esterase (AChE) is mainly involved in the transmission of neurotransmitter acetylcholine in brain. AChE hydrolyzes the acetylcholine into choline and acetate group. Over-activity of AChE causes deficiency of acetylcholine, hence leads to Alzheimer's disease (AD). In order to treat Alzheimer's disease, AChE activity must be inhibited. Butyrylcholine esterase also belongs to the same class of enzyme and is actively involved in Alzheimer's disease.38 Our research group members are working on the synthesis of different compounds and evaluating their enzyme inhibition potential. This work is a continuation of our previous research, in which we aimed to check the inhibitory potential of 1, 2, 3 and 4 against both enzymes Table 4. Enzyme inhibition and antioxidant potential of synthesized esters Compound Enzyme Inhibition (%) AChE BChE Antiradical Scavenging (%) 1 47.3 ± 0.5 55.4 ± 0.7 24.1 ± 0.4 2 41.7 ± 0.9 32.2 ± 0.4 61.4 ± 1.2 3 60.4 ± 1.4 47.7 ± 1.1 77.9 ± 1.3 4 58.1 ± 1.1 54.5 ± 0.9 63.3 ± 0.9 Gallic acid - - 91.1 ± 0.9 Table 3. Antimicrobial potential of synthesized esters 1-4 Compound C. salixgens H. halofila Zone of inhibition (mm) Bacterial strains E. coli S. aureus B. subtilis S. sonnei Fungal strain A. niger 1 NIL 12.4 ± 1.1 11.1 ± 0.5 10.0 ± 0.7 NIL 25.4 ± 0.7 NIL 2 NIL 7.3 ± 0.7 15.4 ± 0.4 12.1 ± 0.9 17.5 ± 1.1 30.1 ± 1.1 4.5 ± 0.1 3 NIL 13.7 ± 0.9 11.3 ± 0.8 12.4 ± 1.3 15.1 ± 0.8 12.5 ± 0.6 6.5 ± 0.3 4 NIL 14.1 ± 1.2 NIL 8.5 ± 0.8 NIL 15.3 ± 1.0 NIL Ampicillin NIL 15.6 ± 0.7 33.5 ± 1.1 39.2 ± 1.0 41.3 ± 1.3 31.5 ± 1.1 - Fungone - - - - - - 30.2 ± 0.8 Chromohalobactor salixgens (C. salixgens), Halomonas halofila (H. halofila), Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Shiegella sonnei (S. sonnei). Danish et al.: Sulfonamide Derived Esters: Synthesis, Characterization, ... Acta Chim. Slov. 2020, 67, 785-798 791 Table 5. Docking results of synthesized esters Docking Score Binding Affinity (kcal/mol) Compound AChE BChE AChE BChE 1EVE 1POI 4BDS 1EVE 1POI 4BDS 1 -10.2116 -9.8558 - 11.6188 -6.9708 -5.2680 - 5.4057 2 -10.9667 -10.5598 - 11.2390 -6.5568 -5.4028 - 4.7353 3 -12.6362 -10.8013 - 12.4252 -6.5128 -5.0774 - 4.7111 4 -11.7883 -10.4357 - 12.1566 -7.3311 -5.6446 - 5.5252 (AChE and BChE). As shown in Table 4, all the synthesized compounds gave moderate to good enzyme inhibition activity. The order of enzyme inhibition against AChE was found to be: 3 > 4 > 1 > 2, while 1 > 4 > 3 > 2 against BChE. All four synthesized esters 1-4 were docked with AChE (1EVE) and BChE (1POI and 4BDS) by downloading their respective PDB files from the internet source using MOE software. The solvent molecules were eliminated and structures of enzyme and compounds were minimized before docking. The interaction of molecules with different amino acid residues at the active site are shown in Figure 3. Esters showed different types of interactions with residues such as hydrogen bonding, van der Waals, n-n interaction; among these some are weak while others bind the inhibitor to the active site rigidly. In AChE, inhibitors (esters 1-4) showed interactions with Phe288, Trp84, Tyr121, Ser122, Trp279, Phe290, Arg289, Phe331, Tyr334, His440, Asp72, Ser31, Tyr130, Gly123 and Gly118. Maximum interaction was demonstrated by 3 with docking score and binding affinity -12.6362, -6.5128, respectively, while others have close results (Table 5). Ester 1 interacts with Trp84 at anion-ic site, His440 at catalytic triad and Phe288 located at acyl pocket. Phen290, Trp84, Tyr334 and Phe288 are the amino acid residues exhibiting different interactions with 2. In case of 3, the amino acid residues at peripheral anionic site (PAS) Tyr70, Asp72, Tyr334, Tyr130 interacted with inhibitor, while Trp84 and Tyr130 located at anionic site also stabilized the molecule. The 4 ester also interacted with Trp84, Tyr121, Tyr 334 and His440 located at anionic, PAS and catalytic triad of the AChE (IEVE), as shown in Figure 4. The synthesized inhibitors were also docked with BChE using PDB files; 1POI and 4BDS. It is evident from the results that 3 exhibited the highest binding score -10.8013 and -12.4252 against 1POI and 4BDS, respectively. The order (1POI) of remaining esters with respect to the binding score was 2 > 4 > 1, while in 4BDS it was 4 > 1 > 2 as shown in Table 7. The inhibitors showed the interactions with Trp82, Thr120, Ser198, Gly116, Try332, His438, Gly117, Trp231, Val288, Glu197, Leu286 and Pro285 in the case of 1POI, while in 4BDS Trp82, Thr120, His438, Danish et al.: Sulfonamide Derived Esters: Synthesis, Characterization, ... 792 Acta Chim. Slov. 2020, 67, 785-798 Figure 4. Best docking-poses of the synthesized compounds in the binding site of 1EVE © CD © y^y©^ Ï © 1 2 Figure 5. Interactions of synthesized esters with BChE (1POI) ©(g) ® ® " © f A® ® S © © © ® Danish et al.: Sulfonamide Derived Esters: Synthesis, Characterization, ... Acta Chim. Slov. 2020, 67, 785-798 793 Figure 7. Interactions of synthesized esters with BChE (4BDS) Danish et al.: Sulfonamide Derived Esters: Synthesis, Characterization, ... 794 Acta Chim. Slov. 2020, 67, 785-798 Figure 8. Best docking-poses of the synthesized compounds in the binding site of 4BDS Gly117, Tyr440, Tyr332 and Ala328 are major residues which bind to the inhibitors (Figures 5-8). Ester 1 showed hydrogen binding with Gly117 and Trp231, while hydrophobic interaction with His438 and Trp82. Ester 2 demonstrated n-n interaction with Trp82 at anioic site, while hy- drogen bonding with Val288 located at acyl pocket of 1POI. Similarly, Val288 and Trp82 provide the major interactions of ester 3 with the enzyme. It was observed that different residues such as Trp82, Glu197, His438, Gly117 etc. interact with hydrogen bonds with the inhibitor 4. On Danish et al.: Sulfonamide Derived Esters: Synthesis, Characterization, ... Acta Chim. Slov. 2020, 67, 785-798 795 Figure 9. Optimized structures of synthesized compounds Table 6. Global chemical reactivity indices of synthesized esters Esters 1 2 3 4 ^ (chemical potential) -0.1402 -0.1405 -0.1540 -0.1398 q (chemical hardness) 0.2138 0.2139 0.2312 0.2137 X (electronegativity) 0.1402 0.1405 0.1540 0.1398 HOMO -0.3540 -0.3544 -0.3852 -0.3535 LUMO 0.0736 0.0734 0.0772 0.0739 (LUMO-HOMO) 0.4276 0.4278 0.4625 0.4274 Energy (Hartree) -1816.3955 -1777.5706 -1858.7156 -1894.0337 O -0.1402 0.1405 0.154 -0.0193 IP (ionization potential) 0.3540 0.3544 0.3852 0.3535 EA (electron affinity) -0.0736 -0.0734 -0.0772 -0.0739 Dipole moment 5.8484 5.8946 2.9289 6.2044 Nuclear repulsion energy 2111.4097 1983.1852 2359.5095 2341.0539 Gibbs free energy -1816.1263 -1777.3305 -1858.3458 -1893.7086 Enthalpy -1816.0521 -1777.2589 -1858.2654 -1893.6267 the active site of 4BDS, all four esters mainly interact with Trp82, Thr120, Tyr440 and 1, 2 and 3 are further stabilized by interaction with Ala328. 3. 9. Computational Studies DFT study of the targeted compounds was carried out using Gaussian software while optimized structures were visualized in Gauss view 5. The structures of all compounds were optimized using basis set B3LYP and bond lengths and bond angels of 3 were compared with experi- mental data (XRD results). It was clear from the results that there is a close resemblance between experimental and theoretical results. HOMO and LUMO were also drawn and energy gap between these was calculated and it was found that 1, 2 and 4 have very small difference in the energy gap ranging from 0.4274 to 0.4278, while 3 has 0.4625 as shown in Table 6 and Figure 10. Others parameters such as chemical potential, chemical hardness, electronegativity, Hartree energy, ionization potential, electron affinity, dipole moment, nuclear repulsion energy, Gibbs free energy were also calculated and are presented in Table 6. It was clear from Danish et al.: Sulfonamide Derived Esters: Synthesis, Characterization, ... 796 Acta Chim. Slov. 2020, 67, 785-798 ae= O i: hmj ^¿r JE = 0.4(05 _iE = 0.42~-l Figure 10. HOMO-LUMO energy diagram of synthesized esters the results that there is a slight variation among the values of calculated parameters which suggested the presence of the same functionality and having similar physical and chemical properties of the molecules. 4. Conclusion In current research work, a series of methyl (1), ethyl (2), isopropyl (3) and butyl (4) esters of 4-((4-chlorophe-nylsulfonamido)methyl)cyclohexanecarboxylic acid has been synthesized. Characterization of these compounds was done by FT-IR and mass spectrometry and NMR techniques while 3 was confirmed with X-ray crystallography. All compounds were screened for their biological applications involving anti-bacterial, anti-fungal, enzyme inhibi- tion and anti-oxidant studies. Results showed that synthesized molecules have biological potential against tested activities. HOMO and LUMO was drawn after optimizing the structures with Gaussian and computational analysis was done to check binding mode of compound 3. Conflict of Interest All authors declared that they have no conflict of interest. Acknowledgment The help of Higher Education Commission is acknowledged for funding this study under the Project No. 20-2549/NRPU/R&D/HEC/12. Danish et al.: Sulfonamide Derived Esters: Synthesis, Characterization, ... Acta Chim. Slov. 2020, 67, 785-798 797 Supplementary Data Selected bond lengths and bond angles for ester 3 are provided (Tables S1 and S2). 5. References 1. M. Pfaller, D. Diekema, D. Gibbs, V. Newell, D. Ellis, V. Tul-lio, A. Rodloff, W. Fu, T. Ling, J. Clin. Microbiol. 2010, 48, 1366-1377. DOI: 10.1128/JCM.02117-09 2. H. S. Lee, Y. Kim, J. Microbiol. Biotechnol. 2016, 26, 610-617. 3. N. Robbins, P. Uppuluri, J. Nett, R. Rajendran, G. Ramage, J. L. 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Karakterizacijo tako pripravljenega metilnega (1), etilnega (2), izopropilnega (3) in n-butil-nega (4) estra 4-((4-klorofenilsulfonamido)metil)cikloheksankarboksilne kisline smo izvedli s pomočjo FTIR, NMR masne spektrometrije in rentgenske kristalografije. Spojine smo optimizirali s pomočjo Gaussian programskega paketa z baznim setom B3LYP/6-31G(d,p) in izračunali nekaj parametrov, ki so povezani s strukturo. Za vse spojine smo in vitro izvedli nekatere biološke študije, vključno z antibakterijskim delovanjem (Chromohalobactor salixgens, Halomonas halofila, Escherichia coli, Staphylococcus aureus, Bacillus subtilis in Shiegella sonnei), delovanjem proti glivam (Aspergillus niger), antioksidacijskim delovanjem (aktivnost uničevanja DPPH) in encimsko inhibicijo (acetilholin esteraza in butirilholin esteraza). Da bi ugotovili način vezave, smo sulfonamidne estre sidrali v izbrana encima (AChE in BChE) s pomočjo MOE programske opreme. Rezultati bioloških študij kažejo, da pripravljene spojine izkazujejo potencialno aktivnost. O—® Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Danish et al.: Sulfonamide Derived Esters: Synthesis, Characterization, ... Acta Chim. Slov. 2020, 67, 799-811 ©common! DOI: I0.i7344/acsi.20i9.5748 Acta Chim. Slov. 2020, 67, 799-811 ^commons Scientific paper Phase Equilibria in the Ag2Te-PbTe-Sb2Te3 System and Thermodynamic Properties of the (2PbTe)1.^(AgSbTe2)x Solid Solutions Leyla Farhad Mashadiyeva,1 Shabnam Hamlet Mansimova,2 Dunya Mahammad Babanly,1,3 Yusif Amirali Yusibov,4 Dilqam Babir Tagiyev1 and Mahammad Baba Babanly1,2,* 1 Institute of Catalysis and Inorganic Chemistry, Azerbaijan National Academy of Science, Baku, Azerbaijan 2 Baku State University, Chemistry Department, Baku, Azerbaijan 3 Azerbaijan State Oil and Industry University, Baku, Azerbaijan 4 Ganja State University, Chemistry Department, Ganja, Azerbaijan * Corresponding author: E-mail: babanlymb@gmail.com Received: 13-29-2019 799 Abstract Phase equilibria in the Ag2Te-PbTe-Sb2Te3 system were experimentally investigated by means of differential thermal analysis, powder X-ray diffraction techniques and electromotive force (EMF) measurement method. A liquidus surface projection of the system, 750 K and 300 K isothermal sections, as well as five vertical sections of the phase diagram, were constructed. The primary crystallization fields of phases and homogeneity range of phases were also determined. The character and temperature of the various nonvariant and monovariant equilibria were identified. The studied system is characterized by the formation of a wide continuous band of a high-temperature cubic structured solid solution (P-phase) between PbTe and Ag1-xSbj + xTe2 + x intermediate phase. The partial molar thermodynamic functions of lead telluride in alloys and standard integral thermodynamic functions of the P-solid solutions along the 2PbTe-"AgSbTe2" section were calculated based on the EMF measurements results. Keywords: Ag-Sb-Pb-Te system; silver telluride; lead telluride; antimony telluride; phase equilibria; solid solution; thermodynamic functions. 1. Introduction Complex silver-containing chalcogenides are among the advanced functional materials.1-3 Thanks to the mobility of silver ions, these phases have mixed ion-electron conductivity and can be widely used in semiconductors, electrochemical energy storage materials, electrodes of fuel cells and batteries, etc.4,5 Alloys formed in Ag2X-AIVX-BV2X3 systems (where AIV = Ge, Sn, Pb; BV = Sb, Bi; X = S, Se, Te) are good thermoelectric materials with a high thermoelectric figure of merit ZT.6-10 Besides, phases AIVBV2X4, AIVBV4X7, etc. which are formed on the boundary quasi-binary systems of the Ag2X-AIVX-BV2X3 systems possess topological surface states and can be used in spintronics and quantum computing.11-14 Great interest is complex telluride phases formed in these systems with excellent thermoelectric properties, such as Ge-Sb-Ag-Te (named as TAGS) and Pb-Sb-Ag-Te (denoted as LAST).15-19 It is known that the development of new multicomponent materials is based on data on the phase equilibria of the corresponding systems and thermodynamic properties of phases formed in them.20-24 Herein, we present the phase relationships in the Ag2Te-PbTe-Sb2Te3 system over the entire concentration range. Also, we report the thermodynamic properties of the (2PbTe)1-x(AgSbTe2)x solid solution formed in the system. Earlier we report the self-consistent phase equilibria description and thermodynamic study result in such silver-based multi-component telluride systems Mashadiyeva et al.: Phase Equilibria in the Ag2Te-PbTe-Sb2Te3 System and 800 Acta Chim. Slov. 2020, 67, 799-811 Ag2Te-PbTe-Bi2Te3,25 Ag2Te-SnTe-Bi2Te3,26 and Ag2Te-SnTe-Sb2Te3.27,28 In all these studied systems, high-temperature solid solutions with a cubic structure along the AIVTe-AgBV2Te3 section are formed. 2. Literature Review 2. 1. Starting Compounds The starting binary compounds of the Ag2Te-PbTe-Sb2Te3 system are well known. Ag2Te melts congruently at 1233 K and has three polymorph modifications.29 Its room-temperature modification (RT-Ag2Te) crystallizes in the monoclinic system (P21/c space group; lattice parameters: a = 0.809 nm; b = 0.448 nm; c = 0.896; p = 112.55°) and remains stable up to 378 K with tellurium excess and up to 418 K with silver excess.30 Intermediate-temperature modification (IT-Ag2Te) crystallizes in the face-centered cubic system (Fm3m space group; a = 0.6648 nm,31) transforms into high temperature body-centered cubic form (HT-Ag2Te; Im3m space group; a = 0.529 nm,32) at 1075 K. PbTe melts congruently at 1197 K,29 and crystallizes in in the Fm3m face-centered cubic crystal structure with cell parameter; a = 6.6461(3) nm.33 Sb2Te3 melts congruently at 893 K,29 and crystallizes in the rhom-bohedral tetradymite type of structure (R3m space group) with parameters: a = 0.4264 nm; c = 3.0458 nm.34 2. 2. Boundary Quasi-Binary Systems The boundary quasi-binary systems of the Ag2Te-PbTe-Sb2Te3 system were studied well. The Ag2Te-PbTe system has a T-x diagram of eutec-tic type with limited mutual solubility of the compo-nents.35-37 The eutectic has the composition 38 mol % PbTe and crystallizes at 967 K,36 (35 mol % PbTe and 973 K,35). The solubility of the starting components in each other is 12-15% at the eutectic temperature.37 Phase diagrams of the Ag2Te-Sb2Te3 pseudobinary system were elaborated separately in.38,39 A previously reported in,40 phase with the nominal composition of Ag2SbTe2 does not exist. In fact, the only ternary intermediate phase Ag1_xSb1 + xTe2 + x with variable composition (0.08 < x < 0.41,38), which crystallizes in the NaCl structure type (Fm3m space group; a = 0.6078 nm,38) is thermo-dynamically stable. However, earlier reports differ about the temperature and compositional region of this phase.38,39 Further investigations of the Ag-Sb-Te ternary system confirmed that this phase is only stable in a limited temperature range (633 K < T < 847 K) and it decomposes into solid solutions based on starting Sb2Te3 and IT-Ag2Te compounds below 633 K.41-43 The decomposition process was additionally confirmed by temperature-dependent X-ray diffraction analysis,44 and electrochemical measurements.45 The homogeneity range of the Ag1-xSb1 + xTe2 + x phase varies with temperature from 35 to 45 mol% Sb2Te3. Recent works are devoted to the search for optimal compositions of this nonstoichiometric phase, which exhibits a high thermoelectric figure of merit.46,47 The eutectic of the Ag2Te-Sb2Te3 system crystallizes at 70 mol% Ag2Te and 817 K.38 The phase diagram of the PbTe-Sb2Te3 boundary system reported in,48 was characterized by formation only ternary compound Pb2Sb6Te11, at approximately the eutec-tic composition on peritectic reaction at 860 K. Shelimova et al.,49 showed formation in this system also PbSb2Te4 and PbSb4Te7 compounds with a layered structure. However, further studies on this system,50,51 have not confirmed the last compounds. These studies indicated the formation of only the Pb2Sb6Te11 metastable ternary compound with a 7-layer rhombohedral structure. This metastable phase is stable at high temperatures and decomposes on cooling into PbTe and Sb2Te3. However, solidification processing always yields the Pb2Sb6Te11 phase as a constituent phase observable at room temperature.50,51 There is an eutectic reaction between Pb2Sb6Te11 and Sb2Te3 at 855 K.48 3. Experimental Part 3. 1. Synthesis All starting compounds Ag2Te, PbTe, and Sb2Te3 of the title system were prepared by melting of elements in evacuated (~10-3 Pa) silica ampoules at temperatures ~50 K higher than their melting points.29 High purity simple substances from the Evochem Advanced Materials GMBH Company (Germany) of were used for synthesis: silver in granules (Ag-00047; 99.999%), antimony in granules (Sb-00002; 99.999%), lead in granules (Pb-00005; 99.9995%), tellurium pieces (Te-00005; 99.9999%). Silver telluride Ag2Te was additionally annealed at 1200 K for 3 hours and Fig. 1. Studied isopleth sections (lines) and alloys (points) of the Ag2Te-PbTe-Sb2Te3 system Mashadiyeva et al.: Phase Equilibria in the Ag2Te-PbTe-Sb2Te3 System and Acta Chim. Slov. 2020, 67, 799-811 801 then was quenched with cold water in order to obtain a homogeneous stoichiometric composition. All starting compounds have been identified using differential thermal analysis and powder X-ray diffraction techniques. More than sixty alloys of the Ag2Te-PbTe-Sb2Te3 system (Fig. 1) were prepared from the pre-synthesized initial compounds also by vacuum alloying. Considering that the non-stoichiometric phase Ag1_xSb1 + xTe2 + x decomposes upon cooling, 2 series of PbTe-"AgSbTe2" and PbTe-"Ag0.84Sb116Te216" alloys with the same compositions were prepared. Alloys from the first series were slowly cooled to 750 K after alloying and then annealed at this temperature for about 700 hours. Alloys from the second series were quenched in cold water after alloying and then also annealed at 750 K (700 h). All alloys after annealing were cooled to room temperature in the off-furnace mode. 3. 2. Analysis Differential thermal analysis (DTA) and powder X-ray diffraction (PXRD) techniques were employed to analyze both starting compounds and alloys. Thermal analysis of the equilibrated alloys was carried out using a NETZSCH 404 F1 Pegasus system. The DTA measurement was performed between room temperature and ~1300 K with a heating and cooling rate of 10 K min-1 under the inert gas (Ar) flow. Temperatures of thermal effects were taken mainly from the heating curves. NETZSCH Proteus Software was used for the evaluation of the DTA data. The PXRD analysis was performed on a Bruker D8 ADVANCE diffractometer, with CuKa1 radiation. PXRD patterns were indexed by using TopasV3.0 software by Bruker. The electromotive force (EMF) method with glycerol electrolyte,52 was used for the thermodynamic study of the (2PbTe)x(AgSbTe2)1-x solid solutions. Concentration chains of the following type were constructed and their EMF was measured in the temperature range of 300-450 K: (-) PbTe (s) | liquid electrolyte, Pb2 + | (2PbTe)x(AgSbTe2)1-x (s) ( + ) (1) Similar electrochemical cells were previously successfully used for thermodynamic studies of several chal-cogenide and other inorganic systems.53-56 Equilibrium alloys (2PbTe)x(AgSbTe2)1-x with compositions x = 0.1; 0.15; 0.2; 0.4; 0.6; 0.8 were synthesized by fusing the elementary components in the required ratios into evacuated to ~10-2 Pa and sealed quartz ampoules. To maximally approximate the alloys to the equilibrium state, the cast non-homogenized samples obtained by quenching the melts from 1100 K were ground into powder, thoroughly mixed, pressed into tablets weighing 0.3-0.5 g and annealed first at 750 K (500 hours), and then at 450 K (200 h.). Synthesized alloys were identified by PXRD. To prepare electrodes the PbTe (left electrode) and annealed alloys (2PbTe)x(AgSbTe2)1-x (right electrodes) were powdered and pressed onto molybdenum current leads in the form of tablets with a diameter of ~0.6 cm and a thickness of ~0.3 cm. A solution of KCl in glycerol with the addition of PbCl2 was used as the electrolyte. In order to prevent the presence of moisture and oxygen in the electrolyte anhydrous, chemically pure salts were used, as well as glycerin was previously dehydrated and outgassed by pumping at ~ 400 K. The electrochemical cell described in,53 was assembled. EMF measurements were carried out in an inert atmosphere using a high-voltage digital voltmeter V7-91. Before starting the measurements, the electrochemical cell was kept at ~350 K for 40-60 h, after which the first equilibrium EMF values were obtained. Subsequent measurements were carried out every 3-4 hours after the establishment of a certain temperature. The EMF values, which, regardless of the direction of the temperature change did not differ from each other at a given temperature by more than 0.2 mV, were considered to be equilibrium. Results and Discussion We have constructed the self-consistent phase diagram of the quasi-ternary Ag2Te-PbTe-Sb2Te3 system as well as determined thermodynamic functions of the PbTe-"AgSbTe2" solid solution by the combined analysis of all our experimental results and the data found in the literature on the phase equilibria for the boundary quasibinary systems. 4. 1. The Sections PbTe-"AgSbTe2" and PbTe-"Ago.84Sb1.16Te2.16" As mentioned above in Experimental Part, two series of alloys were prepared along these sections. Fig. 2 shows heating curves for PbTe-"AgSbTe2" alloys of both series with compositions 60, 80 and 90 mol% PbTe. As can be seen, the melting onset temperatures of the two series of alloys are very different. For samples of the 1st series (red curves) obtained by slow cooling, the melting onset temperatures are significantly (up to 100°) lower than the samples of the 2nd series (blue curves). Increasing the annealing time up to 1000 h did not change the DTA curves of alloys of the 2nd series, whereas for alloys of the 1st series some (~10-20°) rise in the temperatures of the onset of melting was observed. These results show that the 2nd series samples can be considered practically in equilibrium. Therefore, data from DTA curves of the 2nd series alloys were used to construct the phase diagram of the PbTe-"AgSbTe2" system (Fig. 3). The PbTe-"AgSbTe2" system is characterized by the formation of wide (up to 70 mol%) solid solutions based on PbTe (^-phase), but the system is generally Mashadiyeva et al.: Phase Equilibria in the Ag2Te-PbTe-Sb2Te3 System and ... 802 Acta Chim. Slov. 2020, 67, 799-811 Fig. 2. Fragments of the DTA curves for the PbTe-"AgSbTe2" system alloys with the compositions 60 (a), 80 (b) and 90 (c) mol% PbTe. DTA curves for the samples from the 1st series are red, and from the 2nd series are blue non-quasi-binary. The reason is the non-individuality of the starting component "AgSbTe2" which is a two-phase alloy Ag2Te + Agi-xSbi + xTe2 + x.3 38,39 The powder X-ray analysis results confirmed the formation of a wide area (30-100 mol% PbTe) of a solid solution with a cubic structure in the studied section. PbTe-poor alloys are three-phase. For example, Fig. 4 represents a powder X-ray pattern of alloy with composition 20 mol% PbTe and 80 mol% "AgSbTe2". As can be seen from Fig. 4 the PXRD pattern of this alloy consists of a set of reflection lines of the RT-Ag2Te, ^-phase, and y-phase based on Sb2Te3. Fig. 3. PbTe-"AgSbTe2" vertical section of the phase diagram of the Ag2Te-PbTe-Sb2Te3 system Fig. 4. PXRD patterns and phase composition for alloy with compo-sion 20 mol% PbTe-80 mol% "AgSbTe2" A characteristic feature of the PbTe-"AgSbTe2" system is a very large temperature range of crystallization (melting) of the ^-phase (up to 150°). For this reason, slow cooling of melts leads to strong segregation and in-homogeneity of solid solutions in composition, which makes it difficult to achieve an equilibrium state of the samples. Inhomogeneity of solid solutions in composition in the 1st series alloys is demonstrated by a powder X-ray patterns of an alloy with a composition of 70 mol% PbTe (Fig. 5 a, b). As can be seen, X-ray patterns of samples of this alloy, obtained in two various ways, differ sharply. The sample from the 1st series has very diffuse reflection peaks, while the alloy from the 2nd series has a very high-quality X-ray pattern showing much smoother peaks with minimum noise. The phase equilibria along the 2PbTe-"Ag084 Sbi.i6Te2. 16 section (Fig. 6) is qualitatively similar to the PbTe-"AgSbTe2" section. The system is non-quasibinary due to the peritectic melting of the 'Ag084Sb116Te216" Mashadiyeva et al.: Phase Equilibria in the Ag2Te-PbTe-Sb2Te3 System and Acta Chim. Slov. 2020, 67, 799-811 803 Fig. 5. Powder X-ray patterns of alloy with composition of 70 mol% PbTe: a) a sample from the 1st series; b) a sample from the 2nd series phase and its decay into Ag2Te + Sb2Te3 below 635 K. This, in turn, leads to the decomposition of p-solid solutions formed in this section in the 0-40 mol% PbTe composition range and, as a result, the a + p + y, p + y, a + y, etc. fields are formed in the system (a-phase is a solid solution based on an IT-Ag2Te). Using TopasV3.0 software the lattice parameters of the P-phase were calculated (Table 1) and the concentration dependence of these parameters was plotted (Fig. 7). As can be seen from Fig. 7, the lattice parameters are a linear function of the composition. An insignificant positive deviation of this dependence on the Vegard law is probably caused by elastic deformation of the P-phase' Table 1. Crystallographic parameters of the b-solid solutions for the 2PbSe-"Ago.84SbU6Te2 .16 system Composition, mol% Ag0.84Sb1.16Te2.16 Cubic lattice parameter; a, nm Fig. 6. 2PbTe-"Ag0.84Sb116Te216" vertical section of the phase diagram of the Ag2Te-PbTe-Sb2Te3 system 0 (PbTe) 20 40 60 80 100 (Ag0.84Sb1.i6Te2.i6) 0.6463(7) 0.6401(7) 0.6326(6) 0.6252(7) 0.6173(3) 0.6077(8) Mashadiyeva et al.: Phase Equilibria in the Ag2Te-PbTe-Sb2Te3 System and ... 804 Acta Chim. Slov. 2020, 67, 799-811 crystal lattice, due to the large difference in the crystal radius of antimony (0,09 nm) compared to silver (0.129 nm) and lead (0.133 nm). Crystal radii data were taken from.57 a, mil Fig. 7. Concentration dependence of cubic lattice parameter for p-phase along the 2PbTe-"Ag0.84Sb116Te2.16" section 4. 2. Solid-Phase Equilibria The isothermal sections at 750 K and 300 K of the phase diagram of the Ag2Te-PbTe-Sb2Te3 system have been constructed. The isothermal sections at 750 K. A homogeneity range of the intermediate phase Ag1-xSb1 + xTe2 + x along PbTe-Sb2Te3 section is 40-44 mol% Sb2Te3 at 750 K.38 A wide (up to 8 mol%) continuous band of ^-solid solution is formed in the Ag2Te-PbTe-Sb2Te3 system at 750 K (Fig. 8a). The width of a p-solid solutions is 3-4 mol% in the Ag2Te-Sb2Te3 section and expands up to 7-8 mol% with a change in composition towards PbTe. The PbTe-"Ag084Sb116Te216" section is completely in the homogene- Ag;Te PbTe 20 40 60 80 Sbje; mol % SbjTej ity area of the p-phase. The PbTe-"AgSbTe2" section in the composition range of 25-100 mol% PbTe passes through the p-phase homogeneity area, and at <25 mol% PbTe passes into the two-phase region a + p. The p-phase forms connode lines with a-phase and y-phase. The isothermal sections at 300 K (Fig. 8b). The decomposition of the Ag1-xSb1 + xTe2 + x intermediate phase below 635 K leads to partial decomposition of the p-phase in the composition range of <25 mol% PbTe and the following heterogeneous regions are formed: RT-Ag2Te + p + Y and RT-Ag2Te + y. The location and borders of phase areas on the solid-phase equilibrium diagrams (Fig. 8a, b) were established by using the PXRD technique (for example, powder X-ray pattern of the alloy #1 from Fig. 8b is presented in Fig. 4) and confirmed by the DTA, as well as by the EMF technique (see sections 4.5). 4. 3. The Liquidus Surface Projection The liquidus surface of the Ag2Te-PbTe-Sb2Te3 system consists of 4 fields corresponding to primary crystallization of the a-, p- and y- phases, as well as ternary compound Pb2Sb6Te11 (Fig. 9). The a-phase primary crystallization area is separated from the a'-phase based on HT-Ag2Te by the dashed line. Largest crystallization field in the system belongs to p-phase (field 2 in Fig. 9). This region is divided into 2 parts by the curve M1M2 connecting the minimum points of M1 and M2. The primary crystallization area of the ternary Pb2Sb6Te11 compound is very small (field 4 in Fig. 9). Transitional equilibrium U limits the extent of this area inside the concentration triangle. This is in good agreement with data,50,51 on a narrow temperature range for the existence of compound Pb2Sb6Te11. Ag,Te PbTe 20 40 60 80 Sb,Te3 mol % SbjTo, Fig. 8. (a) 750 K and (b) 300 K isothermal sections of the phase diagram of the Ag2Te-PbTe-Sb2Te3 system Mashadiyeva et al.: Phase Equilibria in the Ag2Te-PbTe-Sb2Te3 System and Acta Chim. Slov. 2020, 67, 799-811 805 Table 2. Nonvariant and monovariant equilibria in the Ag2Te-PbTe-Sb2Te3 system Point or Equilibrium Composition, mol% T, K curve in Fig. 9 Ag2Te Sb2Te3 Pi L + p ^ Pb2Sb6Te11 - 62 860 P2 L + y ^ Pb2Sb6Te11 48 52 847 ei L ^ Pb2Sb6Te11 + y - 66 855 e2 L ^ a + p 62 - 967 e3 L ^ a + Ag1-xSb1 + xTe2 + x 70 30 813 U L + Pb2Sb6Te11 ^ p + y 11 59 847 M1 L + Y ^ P 42 46 825 M2 L ^ a + p 68 27 805 PiU L + p ^ Pb2Sb6Te11 860-847 e1U L ^ Pb2Sb6Te11 + y 855-847 UK L ^ p + Y 847-830 KM1 L + Y ^ p 830-825 P2 Mi L + Y ^ p 847-825 m1m2 L ^ y 825-805 e2M2 L ^ a + p 967-805 e3M2 L ^ a + p 813-805 The primary crystallization fields of the phases are bordered by peritectic (piU, p2M1) and eutectic (e2M2, M2e3, e:U, UM1) curves (Fig. 9). Types and temperatures for all nonvariant and monovariant equilibria in the Ag2Te-SnTe-Sb2Te3 system are listed in Table 2. Fig. 9. The liquidus surface projection of the Ag2Te-PbTe-Sb2Te3 system. Primary crystallization fields of phases: 1- a (a'); 2 - p; 3 - y and 4 - Pb2Sb6Te11 4. 4. Isopleth Sections In the context of a liquidus surface projection (Fig.9, Table 2) we consider three isopleth sections which almost completely cover a studied quasi-ternary system. The section "AgPb0.5Te"-"AgSbTe2" (Fig. 10) passes through the region of primary crystallization of the P-phase and intersects the curve M1M2. Therefore, a minimum point is observed on the liquidus curve of this section. Below liquidus, crystallization proceeds according to a monovariant eutectic reaction L ^ a + p (Fig. 10, Table 2, curves e2M2, e3M2) and, as a result, a two-phase region a + p is formed in the subsolidus. The processes occurring in the system below 635 K and associated with the decomposition of the "Ag1-xSb1+xTe2+x" phase were described above. The section Ag2Te-«PbSb2Te4» (Fig. 11) passes through the areas of primary crystallization of a', a and p phases. Below the liquidus in the composition range of 0-25 mol% Ag2Te the univariant processes L + p ^ T, K. 900 800 600 500 400 \\\ L+P L L+ a + ß L+ a + ß «+ß - / 1 / a + ß +7 1 / i 412 ! 1 r 1 RT - Ag,Te + P 1 1 ' RT - Ag,Te + p+y" il 1 1 K40 813 635 "AgPb.Jc" 20 40 60 80 "AgSbTe,' mol % Figure 10. Isopleth section "AgPb0.5Te"-"AgSbTe2" Mashadiyeva et al.: Phase Equilibria in the Ag2Te-PbTe-Sb2Te3 System and ... 806 Acta Chim. Slov. 2020, 67, 799-811 Pb2Sb6Ten and L ^ p + y occur and the two-phase region P + y is formed. In the composition range 25-35 mol% Ag2Te, crystallization continues according to the L ^ p scheme. In the composition range 25-35 mol% Ag2Te, as a result of the crystallization process L ^ a + p a two-phase region RT-Ag2Te + p is formed in the system due to the a ^ RT-Ag2Te phase transition. Note that due to the formation of a' and a solid solutions, the phase transition temperature a' ^ a (~1080 K) increases slightly compared to pure Ag2Te, which leads to the establishment of the L + a' ^ a (p') peritonic equilibrium in the system. Figure 12. Isopleth section "AgPb05Te"- Sb2Te3 Figure 11. Isopleth section Ag2Te-«PbSb2Te4» Figure 13. Concentration dependence of the EMF of chains of type (1) at 298 K for the 2PbTe-"AgSbTe2" alloys The section "AgPb0.5Te"-Sb2Te3 (Fig. 12) passes through the regions of primary crystallization of p and y phases. Further, the crystallization process continues according to the monovariant reactions L ^ a + p (0-30 mol% Sb2Te3), L ~ p + y (40-90 mol% Sb2Te3) and L + y ^ P (90-98 mol% Sb2Te3). In the range of compositions ~30-38 mol% Sb2Te3, crystallization proceeds according to the L ^ p reaction and ends with the formation of the p-phase. 4. 5. Thermodynamic Properties of the (2 PbTe)1-x(AgSbTe2)x Solid Solution Obtained with EMF Measurements Measurements of the EMF of the chains of type (1) showed that the EMF values for samples (2PbTe)x(AgS-bTe2)1-x with compositions x = 0.1, 0.15 and 0.2 are the same, and with further increase of the concentration of PbTe the values of EMF continuously decrease (Fig. 13). This indicates that in this system up to 80 mol.% solid solutions are produced based on PbTe. Analysis of the temperature dependences of the EMF showed that for all samples they are linear (Fig.14). Therefore, the experimental data were processed by the least-squares method in the approximation of the linear temperature dependence of the EMF. For this purpose, the "Microsoft Office Excel 2010" software was used. The obtained linear equations are presented in Table 3 in the following form recommended in,52: Here n is the number of pairs of values of E and T; SE and Sb are the dispersions of individual measurements of EMF and coefficient b, respectively; T is average absolute temperature, t is Student's test. With a confidence level of Mashadiyeva et al.: Phase Equilibria in the Ag2Te-PbTe-Sb2Te3 System and Acta Chim. Slov. 2020, 67, 799-811 807 Figure14. Temperature dependences of the EMF of chains of type (1) for the 2PbTe-"AgSbTe2" alloys: 1 - (2PbTe)o2(AgSbTe2)o.8; 2 - (2PbTe)o4(AgS-bTe2)o.6; 3 - (2PbTe)o.6(AgSbTe2)o.4; 4 - (2PbTe)o.8(AgSbTe2)o.2 95% and the number of experimental points n > 20 the Student's test is t > 2. The partial molar functions of PbTe (AZPbTe) in alloys at 298.15 K were calculated from the data of Table 3 according to the following relations,58: (3) (4) (5) Table 3. Temperature dependences of the EMF of cells of type (1) for the (2PbTe)x(AgSbTe2)1-x alloys in the 3oo, 45o K temperature range Composition (2PbTe)o.4(AgSbTe2)o.6 E, mV = a + bT ± t x SE(T) (2PbTe)o.6(AgSbTe2)o.4 (2PbTe)o.8(AgSbTe2)o.2 and listed in Table 4. As can be seen from Fig. 15, all these functions are continuous functions of the composition in the field x > 0.2. The partial molar functions of PbTe are the difference between the partial molar values of lead in (2PbTe) x(AgSbTe2)1-x solid solutions (AZPb) and in pure PbTe: (6) rge Z=G (MHM H). PbTe is the only compound of the Pb-Te system and Figure 15. Concentration dependences of partial thermodynamic functions of PbTe in the 2PbTe-"AgSbTe2" solid solutions at 298 K. Table 4. Relative partial thermodynamic functions of PbTe in the 2PbTe-AgSbTe2 alloys at 298 K Composition -AGPbTe kJ x mole1 -AtPbTe kJ x mole1 -ASPbTe J x K-1 x mole-1 (2PbTe)o.2(AgSbTe2)o.8 io,13±o.2o 7,37±o.97 9,26±2,67 (2PbTe)o.4(AgSbTe2)o.6 6,11±o.21 4,56±1,o3 5,21±2,84 (2PbTe)o.6(AgSbTe2)o.4 3,27±o.17 2,35±o.83 3,o9±2,28 (2PbTe)o.8(AgSbTe2)o.2 1,64±o.18 1.12±o.85 1,74±2,34 Mashadiyeva et al.: Phase Equilibria in the Ag2Te-PbTe-Sb2Te3 System and ... 808 Acta Chim. Slov. 2020, 67, 799-811 has an almost constant stoichiometric composition.34 According to,58 in such cases: (7) Considering relations (6) and (7), the partial molar functions of lead in solid solutions (2PbTe)x(AgSbTe2)1-x can be calculated from the relation: AZFh=AZFbTe+AfZ'J(PbTe) (8) The values obtained by the relation (8) are presented in Table 5. The calculation of standard thermodynamic functions of the formation of a solid solution of the limiting composition (2PbTe)02(AgSbTe2)08, which is in equilibrium with Ag2Te and Sb2Te3, was performed using the following potential-forming reaction: PbTe + Ag2Te + Sb2Te3 = 2.5 (2PbTe)o.2(AgSbTe2)o.8. and entropy can be calculated from the relation: + OAS" (PbTe) + OASa(Ag2Te)+OAS°(Sb2Te3) (10). Standard integral thermodynamic functions of the formation of solid solutions with compositions x = 0.4; 0.6 and 0.8 were calculated by integrating the Gibbs-Duhem equation: (11) Errors were found by the method of accumulation of errors. The first term on the right side of equation (11) was determined by integrating by means of the trapezoid method using the "Microsoft Office Excel 2010" software. Literature data on corresponding standard integral thermodynamic functions of the Ag2Te, PbTe and Sb2Te3 Table 5. Relative partial thermodynamic functions of lead in the 2PbTe-AgSbTe2 alloys at 298 K Composition -AGpb -^H>b -A§>b kJ mole1 kJ mole1 J x K-1 mole1 (2PbTe)02(AgSbTe2)08 77,43±1,70 75,97±1,57 4,90±4,77 (2PbTe)04(AgSbTe2)06 73,41±1,71 73,16±1,63 0,84±4,94 (2PbTe)06(AgSbTe2)04 70,57±1,67 70,95±1,43 -1,74±4,38 (2PbTe)08(AgSbTe2)02 68,94±1,68 69,72±1,45 -2,62±4,44 Table 6. Standard integral thermodynamic functions of the (2PbTe)1-x(AgSbTe2)x solid solutions Phase -AfG° (298 K) -AfH° (298 K) S° (298 K), kJ mole1 kJ mole1 J x K-1 mole-1 PbTe,58 67.3±1.5 68.6±o.6 1io.o±2.1 Sb2Te3,58 56.9±1.o 56.5±o.4 246.4±2.1 Ag2Te,59 4o.2±o.3 35.o±o.5 152.o±2.o (2PbTe)o,9(AgSbTe2)o,i 128.5±2.8 13o.o±1.2 217.6±4.1 (2PbTe)o,8(AgSbTe2)o,2 119.o±2.5 121.9±1.1 218.4±3.9 122.2±2.9* 122.3±2.5* 220.0±3.5* (2PbTe)o,6(AgSbTe2)o,4 io4.3±2.o io4.2±o.9 213.7±3.4 106.4±2.3* 105.2±2.0* 216.1±3.5* (2PbTe)o,4(AgSbTe2)o,6 86.3±1.6 85.4±o.9 2o8.2±3.5 89.1±1.8* 86.8±1.5* 211.5±3.8* (2PbTe)o,2(AgSbTe2)o,8 67.3±1.1 66.o±o.7 2o2.2±3.o 69.8±1.2* 67.0±0.9* 204.9±3.7* * these values are obtained by the EMF method with solid electrolyte,61 According to this reaction, the standard Gibbs free energy of formation and the enthalpy of formation (2PbTe)0.2(AgSbTe2)0.8 can be calculated by the relation: A/Z°[(2PbTe)b.2(AgSbTe2)(1,] = 0.4AZ^ + (y) + QAhfZ\PbTe) + 0AAfZa (Ag2Te) + 0AAfZa (Sb2Te} ) compounds in addition to own experimental results (Table 4), were used at calculations of equations (9) and (10) (Table 6). The values of standard enthalpies of formation and entropies for PbTe and Sb2Te3 were taken from Ref.58 For the Ag2Te compound, the data of,59 obtained by the EMF Mashadiyeva et al.: Phase Equilibria in the Ag2Te-PbTe-Sb2Te3 System and Acta Chim. Slov. 2020, 67, 799-811 809 method were used. Using the EMF method with solid Ag + conducting electrolyte,60 thermodynamic data for investigated solid solutions previously were obtained in,61 and also are listed in Table 6. As can be seen, the results obtained by two modifications of the EMF method, agree within the margin of inaccuracies. 5. Conclusion A complete description of the phase equilibria in the quasi-ternary Ag2Te-PbTe-Sb2Te3 system, including 750 and 300 K isothermal sections and five isopleth sections of the phase diagram, as well as liquidus surface projection, were obtained. This system characterized by the formation of a wide continuous high-temperature solid solution (P-phase) with a cubic structure between PbTe and "Ag^ xSbj + xTe2 + x" intermediate phase. Below 635 K, a solid-state decomposition of the P-phase occurs and subsequently the formation of the a- and y-phases based correspondingly on IT-Ag2Te and Sb2Te3 were observed. The formation of a wide (up to 80 mol%) region of solid solutions based on PbTe along the 2PbTe-"AgSbTe2" section was confirmed by measuring EMF of the concentration chains concerning to the PbTe electrode. A new mutually agreed complex of data on standard partial thermodynamic functions of PbTe and lead, as well as integral thermodynamic functions of P-solid solutions along the above section was obtained. This data is in agreement with the results obtained earlier by the EMF method with solid Ag + conductive electrolyte.61 The presented results can be used for the design of new LAST alloys, which are of great interest as thermoelectric materials. 6. References 1. Applications of Chalcogenides: S, Se, and Te / Ed. Ahluwalia G. K, 2016, Cham.: Springer, 461p. 2. A. V. Kolobov, J. Tominaga, Chalcogenides, 2012, Springer. 3. M. -R. Gao, Y. -F. Xu, J. Jiang, S. -H. Yu, Chemical Society Reviews, 2013, 42(7), 2986. DOI:10.1007/978-3-642-28705-3 4. I. Riess, Solid State Ion., 2003, 157, 1-17. D0I:10.1039/c2cs35310e 5. H. Wada, M. Ishii, M. Onoda, M. Tansho, A. Sato, Solid State Ionics, 1996, 86-88, 159-163. D0I:10.1016/S0167-2738(02)00182-0 6. C. Gayner, K. K. Kar, Progress in Materials Science, 2016, 83, 330-382. DOI: 10.1016/j.pmatsci. 2016.07.002 7. A. V. Shevelkov, Russ. Chem. Rev., 2008, 77 (1), 1-19. DOI: 10.1070/RC2008v077n01ABEH003746 8. F. Tesfaye, M. 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Babanly, The EMF method with solid-state electrolyte in the thermodynamic investigation of ternary Copper and Silver Chalcogenides. Electromotive force and measurement in several systems. / Ed. Kara S. InTech. Org. 2011. P. 57. D0I:10.5772/28934 Mashadiyeva et al.: Phase Equilibria in the Ag2Te-PbTe-Sb2Te3 System and Acta Chim. Slov. 2020, 67, 799- 811 811 Povzetek Raziskovali smo fazna ravnotežja v sistemu Ag2Te-PbTe-Sb2Te3 z metodami diferencialne termične analize, praškovne rentgenske difrakcije in meritvami elektromotorne sile (EMF). Konstruirali smo površinsko projekcijo sistema, izoter-malne odseke pri 750 K in 300 K, ter pet vertikalnih odsekov faznega diagram. Določili smo primarna kristalizacijska področja in območja homogenosti faz. Identificirali smo vrsto in temperature različnih nevariantnih in monovariant-nih ravnotežij. Značilnost preučevanega sistema je nastanek širokega zveznega pasu visokotemperaturne kubične trdne raztopine (^-faza) med PbTe in Ag1-xSb1 + xTe2 + x. Na osnovi rezultatov meritev EMF smo izračunali parcialne molarne termodinamske funkcije svinčevega telurida v zlitinah in standardne integralne termodinamske funkcije ^-trdnih raztopin vzdolž odseka 2PbTe-«AgSbTe2«. Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Mashadiyeva et al.: Phase Equilibria in the Ag2Te-PbTe-Sb2Te3 System and ... DOI: 10.17344/acsi.2019.5778 Acta Chim. Slov. 2020, 67, 812-821 /^creative ^commons Scientific paper Study on the Synthesis and Biological Activities of N-Alkylated Deoxynojirimycin Derivatives with a Terminal Tertiary Amine Lin Wang and Zhijie Fang* School of Chemical Engineering, Nanjing University of Science & Technology, Nanjing 210094, Jiangsu, P.R. China * Corresponding author: E-mail: zjfang@njust.edu.cn Tel: +86-25-84303232; fax: +86-25-84315520 Received: 12-14-2019 Abstract A series of N-alkylated deoxynojirimycin (DNJ) derivatives connected to a terminal tertiary amine at the alkyl chains of various lengths were prepared. These novel synthetic compounds were assessed for preliminary glucosidase inhibition and anticancer activities in vitro. Potent and selective inhibition was observed among them. Compound 7d (IC50 = 0.0 52 mM) showed improved and selective inhibitory activity against p-glucosidase compared to DNJ (IC50 = 0.65 mM). In addition, analysis of the kinetics of enzyme inhibition by using Lineweaver-Burk plots indicated that 7d inhibited p-glu-cosidase in a competitive manner, suggesting that 7d was expected to bind to the active site of p-glucosidase. Compounds 8b and 8c were found to be moderate and selective inhibitors of a-glucosidase. Nevertheless, none of compounds inhibited the growth of B16F10 melanoma cells. Keywords: biological activities; glucosidase; 1-deoxynojirimycin; selective inhibition 1. Introduction Glucosidases are enzymes which catalyze the hydrolysis of glycosidic bonds in oligosaccharides or glycocon-jugates, playing a vital role in the digestion of carbohydrates and in the processing of glycoproteins and glycolipids.1 Glucosidases are also involved in carbohydrate-mediated diseases such as diabetes,2 tumor metasta-sis,3 viral infections,4 and lysosomal storage diseases.5 Inhibitors of a-glucosidase can significantly decrease postprandial blood glucose levels6 and promote glycoprotein misfolding in the endoplasmic reticulum (ER).7 In mammals, p-glucosidase enables hydrolysis of glucosylce-ramide into ceramide and glucose, which is in part performed by p-glucocerebrosidases (GBA1 or GCase)8 and GBA2.9 Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the p-glucocere-brosidase (GBA1) gene. Inhibitors of p-glucosidase could reduce the biosynthesis of glycolipids to balance the deficient activity of p-Gcase.10 In tumor cells, oligosaccharides on the surface of tumor cells play an important role in expression of the malignant phenotype and the metastatic spread of tumor cells. The synthesis of these oligosaccha-rides in endoplasmic reticulum and Golgi is dependent on carbohydrate processing enzymes such as glycosidases. Therefore, specific glycosidase inhibitors may be candidates for cancer chemotherapy.11,12 Among the families of glycosidase inhibitors reported so far, iminosugars are particularly notable. They are carbohydrate mimetics where the endocyclic oxygen has been replaced by a nitrogen atom.13-15 Their structures can mimic transition-state analogues of glycosidases, which interact with two carboxylic acid units to form strong ions and catalyze the cleavage of the glycoside bonds.1 Their most famous representative is the naturally occurring 1-deoxynojirimycin 1.2 Some N-alkylated DNJ derivatives, like N-hydroxyethyl-DNJ16 2 (miglitol, an intestinal a-glu-cosidase inhibitor), and N-butyl-DNJ17 3 (miglustat, a glu-cosylceramide synthase inhibitor) have been approved for the treatment of diabetes-type 2 and Gaucher disease, respectively. Compound 63 not only inhibited a-glucosidase (Bacillus stearothermophilus), BAEC growth and migration, but also suppressed the growth of A549 cells (Figure 1). Nevertheless, despite extensive synthesis and investigations of highly bioactive iminosugars, a remaining drawback is their limited selectivity on glucosidases, and this leads to some side effects when applied therapeutically. For example, N-butyl-DNJ 3 (Figure 1) can inhibit some other Wang and Fang: Study on the Synthesis and Biological Activities Acta Chim. Slov. 2020, 67, 812-813 821 1 R= H AMP-DNJ, 5 6 2 R= N-hydroxyethyl 3 R= W-butyl 4 R= N-nonyl Figure 1. Known potent glycosidase inhibitors derived from 1-deoxynojirimycin. enzymes nonrelated to lysosomal storage disease, such as sucrase, maltase, a-glucosidase I and II.18 Obviously, improving the selectivity of iminosugars as glycosidase inhibitors is a challenging goal. Modification or variation of a known iminosugar inhibitor, especially a natural product, is a feasible strategy to obtain more selective and stronger inhibitors. Generally, there are two main strategies for modification of imino-sugars: introduction of different alkyl groups on the amino group and alterations of the ring hydroxyl residues.19 It has been demonstrated that the potency of DNJ derivatives could be increased by introducing a hydrophobic group on the nitrogen atom of DNJ using a heteroatom linker and a carbon chain spacer. Moreover, lengthening of the alkyl chain and an increase in the size of the hydrophobic group would be also beneficial for the glucosidase inhibition. These types of modifications can be seen in the design of compounds 4,20 521 and 6 (Figure 1).3 Our group had done some work on the modification of DNJ, such as the synthesis of C-6 deutero DNJ, a potent a-glucosidase and the optimization of DNJ synthetic route.22,23 And as a part of our ongoing program devoted to the development of new glucosidase inhibitors, we embarked on a strategy starting from DNJ as the lead compound. The key DNJ scaffold was connected to a terminal tertiary amine through introduction of alkyl chains of various length. And the introduction of a nitrogen atom may lead to a polarization different from that of oxygen atom.24,25 The work reported herein describes the synthesis and biological evaluation of a small library of DNJ derivatives in which the length of the alkyl chain and the size and nature of the terminal tertiary amine substituents have been studied. 2. Experimental 2. 1. Materials and Methods All reagents and solvents were purchased from commercial suppliers and used without further purification. Reactions progression was monitored by Thin Layer Chromatography (TLC) using silica gel GF254 plates (0.2 mm thickness), spots were detected under UV-light (À = 254 nm). Visualization of the deprotected iminosugar was accomplished by exposure to iodine vapour. Flash column chromatography was carried out by silica gel (200-300 mesh). NMR spectra were recorded on Bruker Avance III 500 MHz spectrometer using CDCl3 or D2O as solvents. Chemical shifts are reported in ppm. High resolution mass spectra (HRMS) were recorded by direct injection on a mass spectrometer (Thermo Scientific LTQ Orbitrap XL) equipped with an electrospray ion source in positive mode. The following abbreviations have been used to describe the signal multiplicity: br (broad), s (singlet), d (doublet), t (triplet), q (quartet), h (hextet), m (multiplet), dd (doublet of doublets), dt (doublet of triplets). General Procedure A Nucleophilic substitution on a nitrogen atom. The starting material (1 mM) was mixed with N-bromoph-thalimide (2 mM) and K2CO3 (3 mM) in DMF (10 mL). The mixture was heated at 100 °C for 24 h. After cooling, the mixture was poured into water and extracted into ethyl acetate. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by flash column chromatography (10:1^3:1; PE:ethyl acetate). General Procedure B Hydrazinolysis. The starting material (1 mM) was mixed with 80% hydrazine hydrate (0.13 mL, 2 mM) in EtOH (10 mL). The mixture was heated under reflux for 3 h. After cooling, the solid was removed by filtration. The filtrate was concentrated and the residue was purified by flash column chromatography (20:1:0.2^20:2:0.2; ethyl acetate: MeOH:NH4OH). General Procedure C Reductive amination. The starting material (1 mM) was mixed with formaldehyde (178.38 mg, 37% aqueous solution, 0.22 mL, 2.2 mmol) and formic acid (0.19 mL, 5.0 mmol). The mixture was heated at 105 °C for 3 h. After cooling, the mixture was poured into water and extracted into ethyl acetate. The organic layer was washed with saturated NaHCO3 solution, dried and concentrated. The residue was purified by flash column chromatography (20:1:0.2^20:2:0.2; ethyl acetate: MeOH:NH4OH). Wang and Fang: Study on the Synthesis and Biological Activities 814 Acta Chim. Slov. 2020, 67, 812-821 General Procedure D Double nucleophilic substitution. The starting material (1 mM) was mixed with alkyl dibromide (2 mM) and K2CO3 (3 mM) in CH3CN (10 mL). The mixture was heated at 80 °C for 12 h. After cooling, the solution was concentrated. The residue was purified by flash column chromatography (20:1:0.2^20:2:0.2; ethyl acetate: MeOH:NH4OH). General Procedure E Catalytic hydrogenolysis. To a solution of the ben-zylated intermediate (1 mmol) in EtOH was added Pd (10%)/C (100 mg) and the mixture stirred under an atmosphere of hydrogen at room temperature for 24 h. The catalyst was filtered off, the solvents removed under reduced pressure and the residue purified by flash column chroma-tography (8:2:0.1^6:4:0.1; n-propanol:H2O:NH4OH). 2-(2-((2.R,3.R,4.R,5S)-3,4,5-Tris(benzyloxy)-2-((benzy-loxy)methyl)piperidin-1-yl)ethyl)isoindoline-1,3-dione (10a) Prepared according to procedure A. Compound 9 (2 g, 3.8 mmol), N-(4-bromoethyl)phthalimide (2.1 g, 7.6 mmol), K2CO3 (1.6 g, 11.4 mmol), DMF (40 mL). Yield: 87% (2.4 g), colourless syrup, Rf = 0.45 (3:1, PE:ethyl acetate). 1H NMR (500 MHz, CDCl3): 5 7.76 (dd, J = 5.4, 3.0 Hz, 2H, ArH), 7.65 (dd, J = 5.4, 3.0 Hz, 2H, ArH), 7.467.00 (m, 20H, ArH), 4.95 (d, J = 10.9 Hz, 1H, PhCH2), 4.77 (ddd, J = 22.9, 15.6, 11.3 Hz, 4H, PhCH2), 4.46 (d, J = 12.1 Hz, 1H, PhCH2), 4.32 (dd, J = 26.1, 11.4 Hz, 2H, PhCH2), 3.94 (dt, J = 13.9, 8.0 Hz, 1H, H-8a), 3.78-3.56 (m, 4H, H-6, H-8b, H-2), 3.51-3.43 (m, 2H, H-3, H-4), 3.41 (dd, J = 11.0, 4.8 Hz, 1H, H-1a), 3.23 (dt, J = 13.7, 8.2 Hz, 1H, H-7a), 2.65 (ddd, J = 13.3, 7.2, 3.4 Hz, 1H, H-7b), 2.42 (d, J = 8.8 Hz, 1H, H-5), 2.26 (t, J = 10.7 Hz, 1H, H-1b). 13C NMR (126 MHz, CDCl3): 5 168.35, 138.97, 138.71, 138.52, 138.07, 133.81, 132.17, 128.43, 128.37, 128.33, 128.01, 127.97, 127.94, 127.84, 127.64, 127.53, 123.16, 87.20, 78.69, 78.57, 77.33, 77.08, 76.83, 75.49, 75.20, 73.10, 72.66, 66.07, 64.50, 54.25, 49.24, 34.56. 2-(4-((2.R,3.R,4.R,5S)-3,4,5-Tris(benzyloxy)-2-((benzy-loxy)methyl)piperidin-1-yl)butyl)isoindoline-1,3-dione (10b) Prepared according to procedure A. Compound 9 (2 g, 3.8 mmol), N-(4-bromobutyl)phthalimide (1.9 g, 7.6 mmol), K2CO3 (1.6 g, 11.4 mmol), DMF (40 mL). Yield: 85% (2.3 g), colourless syrup, Rf = 0.33 (3:1, PE: ethyl acetate). 1H NMR (500 MHz, CDCl3): 5 7.83 (dt, J = 7.4, 3.7 Hz, 2H, ArH), 7.75-7.63 (m, 2H, ArH), 7.42-7.16 (m, 18H, ArH), 7.11 (d, J = 6.4 Hz, 2H, ArH), 4.95 (d, J = 11.1 Hz, 1H, PhCH2), 4.86 (d, J = 10.8 Hz, 1H, PhCH2), 4.80 (d, J = 11.1 Hz, 1H, PhCH2), 4.72-4.60 (m, 2H, PhCH2), 4.46 (s, 2H, PhCH2), 4.39 (d, J = 10.8 Hz, 1H, PhCH2), 3.63 (dt, J = 11.0, 5.9 Hz, 4H, H-6, H-2, H-10a), 3.56 (t, J = 9.4 Hz, 2H, H-10b, H-3), 3.45 (t, J = 9.1 Hz, 1H, H-4), 3.08 (dd, J = 11.1, 4.8 Hz, 1H, H-1a), 2.72 (m, 1H, H-7a), 2.63-2.50 (m, 1H, H-7b), 2.29 (d, J = 9.5 Hz, 1H, H-5), 2.18 (t, J = 10.8 Hz, 1H, H-1b), 1.65-1.33 (m, 4H, H-8, H-9). 13C NMR (126 MHz, CDCl3): 5 168.44, 139.07, 138.57, 137.80, 133.98, 132.17, 128.53, 128.44, 128.37, 127.92, 127.70, 127.58, 127.48, 123.27, 87.35, 78.61, 78.49, 77.38, 77.12, 76.87, 75.36, 75.23, 73.43, 72.79, 65.43, 63.88, 54.40, 51.76, 37.81, 26.56, 21.43. 2-((2.R,3.R,4.R,5S)-3,4,5-Tris(benzyloxy)-2-((benzyloxy) methyl)piperidin-1-yl)ethan-1-amine (11a) Prepared according to procedure B. Compound 10a (1.3 g, 1.86 mmol), 80% hydrazine hydrate (0.23 mL, 3.72 mM), EtOH (10 mL). Yield: 82% (0.87 g), colourless syrup, Rf = 0.43 (20:2:0.2; ethyl acetate:MeOH:NH4OH). 1H NMR (500 MHz, CDCl3): 5 7.46-7.18 (m, 18H, ArH), 7.18-7.04 (m, 2H, ArH), 4.95 (d, J = 11.0 Hz, 1H, PhCH2), 4.87 (d, J = 10.8 Hz, 1H, PhCH2), 4.81 (d, J = 11.0 Hz, 1H, PhCH2), 4.67 (q, J = 11.6 Hz, 2H, PhCH2), 4.50 (d, J = 12.0 Hz, 1H, PhCH2), 4.42 (dd, J = 11.4, 4.6 Hz, 2H, PhCH2), 3.77-3.44 (m, 5H, H-6, H-2, H-3, H-4), 3.11 (dd, J = 11.4, 4.8 Hz, 1H, H-1a), 2.90-2.79 (m, 1H, H-7a), 2.79-2.64 (m, 2H, H-7b, H-8a), 2.55-2.42 (m, 1H, H-8b), 2.42-2.24 (m, 3H, H-5, NH2), 2.20 (t, J = 10.9 Hz, 1H, H-1b). 13C NMR (126 MHz, CDCl3): 5 138.99, 138.53, 137.80, 128.52, 128.50, 128.43, 128.39, 127.98, 127.93, 127.79, 127.67, 127.57, 87.19, 78.55, 78.39, 77.46, 77.20, 76.95, 75.40, 75.26, 73.34, 72.92, 66.20, 64.83, 54.89, 54.27, 38.43. 4-((2.R,3.R,4.R,5S)-3,4,5-Tris(benzyloxy)-2-((benzyloxy) methyl)piperidin-1-yl)butan-1-amine (11b) Prepared according to procedure B. Compound 10b (1.2 g, 1.66 mmol), 80% hydrazine hydrate (0.21 mL, 3.31 mM), EtOH (10 mL). Yield: 85% (0.84 g), colourless syrup, Rf = 0.35 (20:2:0.2; ethyl acetate:MeOH:NH4OH). 1H NMR (500 MHz, CDCl3): 5 7.39-7.18 (m, 18H, ArH), 7.18-7.06 (m, 2H, ArH), 4.95 (d, J = 11.1 Hz, 1H, PhCH2), 4.87 (d, J = 10.9 Hz, 1H, PhCH2), 4.80 (d, J = 11.1 Hz, 1H, PhCH2), 4.67 (q, J = 11.6 Hz, 2H, PhCH2), 4.53-4.37 (m, 3H, PhCH2), 3.66 (dt, J = 8.7, 5.5 Hz, 2H, H-6), 3.62-3.51 (m, 2H, H-3, H-2), 3.47 (t, J = 9.0 Hz, 1H, H-4), 3.08 (dd, J = 11.2, 4.8 Hz, 1H, H-1a), 2.77-2.43 (m, 6H, H-7, H-10, NH2), 2.32 (d, J = 9.4 Hz, 1H, H-5), 2.21 (t, J = 10.8 Hz, 1H, H-1b), 1.37 (m, 4H, H-8, H-9). 13C NMR (126 MHz, CDCl3): 5 138.10, 137.67, 136.86, 127.48, 127.42, 126.94, 126.73, 126.63, 126.54, 86.23, 77.61, 77.54, 76.60, 76.35, 76.09, 74.35, 74.21, 72.52, 71.83, 64.59, 62.99, 53.44, 51.27, 40.79, 30.07, 20.50. AT,AT-Dimethyl-2-((2ß,3ß,4ß,5S)-3,4,5-tris(benzyloxy)-2-((benzyloxy)methyl)piperidin-1-yl)ethan-1-amine (12a) Prepared according to procedure C. Compound 11a (0.3 g, 0.53 mmol), formaldehyde (37% aqueous solution, 0.12 mL, 1.17 mmol) and formic acid (0.1 mL, 2.65 mmol). Yield: 83% (0.26 g), yellow syrup, Rf = 0.83(20:2:0.2; ethyl acetate:MeOH:NH4OH). 1H NMR (500 MHz, CDCl3): 5 7.28 (m, 18H, ArH), 7.12 (d, J = 6.4 Hz, 2H, ArH), 4.95 (d, Wang and Fang: Study on the Synthesis and Biological Activities ... Acta Chim. Slov. 2020, 67, 812-815 821 J = 11.1 Hz, 1H, PhCH2), 4.87 (d, J = 10.8 Hz, 1H, PhCH2), 4.81 (d, J = 11.1 Hz, 1H, PhCH2), 4.67 (q, J = 11.6 Hz, 2H, PhCH2), 4.47 (s, 2H, PhCH2), 4.40 (d, J = 10.8 Hz, 1H, PhCH2), 3.74-3.54 (m, 4H, H-6, H-2, H-3), 3.46 (t, J = 9.1 Hz, 1H, H-4), 3.12 (dd, J = 11.2, 4.8 Hz, 1H, H-1a), 2.932.80 (m, 1H, H-7a), 2.80-2.66 (m, 1H, H-7b), 2.47-2.33 (m, 2H, H-5, H-8a), 2.29 (td, J = 11.2, 7.2 Hz, 2H, H-1b, H-8b), 2.16 (s, 6H, 2xCH3). 13C NMR (126 MHz, CDCl3): 5 139.05, 138.58, 137.78, 128.56, 128.48, 128.46, 128.39, 127.95, 127.93, 127.91, 127.73, 127.61, 127.52, 87.36, 78.51, 78.42, 77.39, 77.14, 76.89, 75.38, 75.24, 73.56, 72.86, 65.66, 64.07, 55.05, 54.68, 50.21, 45.91. N,N-Dimethyl-4-((2R,3R,4R,5S)-3,4,5-tris(benzyloxy)-2-((benzyloxy)methyl)piperidin-1-yl)butan-1-amine (13a) Prepared according to procedure C. Compound 11b (0.17 g, 0.29 mmol), formaldehyde (37% aqueous solution, 64 ^L, 0.64 mmol) and formic acid (55 ^L, 1.45 mmol). Yield: 90% (0.16 g), yellow syrup, Rf = 0.75 (20:2:0.2; ethyl acetate:MeOH:NH4OH). 1H NMR (500 MHz, CDCl3): 5 7.45-7.19 (m, 18H, ArH), 7.19-7.05 (m, 2H, ArH), 4.95 (d, J = 11.1 Hz, 1H, PhCH2), 4.88 (d, J = 10.9 Hz, 1H, PhCH2), 4.81 (d, J = 11.1 Hz, 1H, PhCH2), 4.67 (q, J = 11.6 Hz, 2H, PhCH2), 4.45 (dt, J = 10.9, 9.9 Hz, 3H, PhCH2), 3.72-3.61 (m, 2H, H-6), 3.57 (dt, J = 9.8, 5.6 Hz, 2H, H-2, H-3), 3.45 (t, J = 9.1 Hz, 1H, H-4), 3.08 (dd, J = 11.2, 4.9 Hz, 1H, H-1a), 2.68 (m, 1H, H-7a), 2.63-2.52 (m, 1H, H-7b), 2.37-2.09 (m, 10H, H-5, H-1a, H-10, 2 x CH3), 1.54-1.20 (m, 4H, H-8, H-9). 13C NMR (126 MHz, CDCl3): 5 138.09, 137.64, 136.89, 127.43, 126.89, 126.67, 126.56, 126.47, 86.41, 77.70, 77.63, 76.36, 76.10, 75.85, 74.36, 74.21, 72.48, 71.78, 64.61, 62.83, 58.56, 53.47, 51.23, 44.35, 24.49, 20.72. (2R,3R,4R,5S)-3,4,5-Tris(benzyloxy)-2-((benzyloxy) methyl)-1-(2-(pyrrolidin-1-yl)ethyl)pip eridine (12b) Prepared according to procedure D. Compound 11a (0.3 g, 0.53 mmol), 1,4-dibromobutane (127 ^L, 1.06 mmol), K2CO3 (0.22 g, 1.59 mmol), CH3CN (5 mL). Yield: 84% (276 mg), yellow syrup, Rf = 0.55 (20:2:0.2; ethyl ace-tate:MeOH:NH4OH). 1H NMR (500 MHz, CDCl3): 5 7.45-7.21 (m, 18H, ArH), 7.18 (d, J = 6.7 Hz, 2H, ArH), 4.95 (d, J = 10.9 Hz, 1H, PhCH2), 4.90 (d, J = 10.8 Hz, 1H, PhCH2), 4.81 (d, J = 11.0 Hz, 1H, PhCH2), 4.69 (d, J = 1.5 Hz, 2H, PhCH2), 4.48 (dt, J = 18.5, 11.5 Hz, 3H, PhCH2), 3.74 (qd, J = 10.9, 2.4 Hz, 2H, H-6), 3.59 (m, 2H, H-2, H-3), 3.50 (t, J = 8.8 Hz, 1H, H-4), 3.27-3.10 (m, 2H, H-1a, H-8a), 2.86 (m, 7H, H-7, H-8b, H-9, H-12), 2.39 (d, J = 9.1 Hz, 1H, H-5), 2.26 (t, J = 10.8 Hz, 1H, H-1b), 1.83-1.60 (m, 4H, H-10, H-11). 13C NMR (126 MHz, CDCl3): 5 138.84, 138.46, 138.40, 137.76, 128.58, 128.52, 128.48, 128.45, 128.11, 128.02, 127.96, 127.93, 127.79, 127.76, 127.64, 86.59, 78.35, 78.02, 77.55, 77.30, 77.04, 75.40, 75.30, 73.40, 72.79, 66.28, 64.64, 55.05, 54.36, 51.99, 49.58, 23.15. (2R,3R,4R,5S)-3,4,5-Tris(benzyloxy)-2-((benzyloxy) methyl)-1-(2-(piperidin-1-yl)ethyl)piperidine (12c) Prepared according to procedure D. Compound 11a (0.3 g, 0.53 mmol), 1,4-dibromopentane (143 ^L, 1.06 mmol), K2CO3 (0.22 g, 1.59 mmol), CH3CN (5 mL). Yield: 80% (269 mg), yellow syrup, Rf = 0.64(20:2:0.2; ethyl ace-tate:MeOH:NH4OH). 1H NMR (500 MHz, CDCl3): 5 7.42-7.19 (m, 18H, ArH), 7.14 (d, J = 6.4 Hz, 2H, ArH), 4.95 (d, J = 11.1 Hz, 1H, PhCH2), 4.88 (d, J = 10.8 Hz, 1H, PhCH2), 4.81 (d, J = 11.1 Hz, 1H, PhCH2), 4.66 (q, J = 11.6 Hz, 2H, PhCH2), 4.50 (d, J = 12.1 Hz, 1H, PhCH2), 4.43 (d, J = 11.4 Hz, 2H, PhCH2), 3.75-3.52 (m, 4H, H-6, H-2, H-3), 3.46 (t, J = 9.1 Hz, 1H, H-4), 3.12 (dd, J = 11.2, 4.8 Hz, 1H, H-1a), 2.96-2.84 (m, 1H, H-7a), 2.84-2.70 (m, 1H, H-7b), 2.53-2.21 (m, 8H, H-8, H-9, H-13, H-5, H-1b), 1.58-1.46 (m, 4H, H-10, H-12), 1.39 (m, 2H, H-11). 13C NMR (126 MHz, CDCl3): 5 139.07, 138.59, 137.81, 128.59, 128.45, 128.40, 128.37, 127.92, 127.72, 127.61, 127.50, 87.31, 78.55, 78.42, 77.38, 77.13, 76.88, 75.36, 75.26, 73.54, 72.82, 65.52, 64.02, 55.02, 54.96, 54.19, 49.10, 25.75, 24.17. 4-(2-((2R,3R,4R,5S)-3,4,5-Tris(benzyloxy)-2-((benzy-loxy)methyl)piperidin-1-yl)ethyl)morpholine (12d) Prepared according to procedure D. Compound 11a (0.3 g, 0.53 mmol), 2-bromoethyl ether (137 ^L, 1.06 mmol), K2CO3 (0.22 g, 1.59 mmol), CH3CN (5 mL). Yield: 85% (287 mg), colourless syrup, Rf = 0.73 (20:2:0.2; ethyl acetate:MeOH:NH4OH). 1H NMR (500 MHz, CDCl3): 5 7.40-7.21 (m, 18H, ArH), 7.21-7.10 (m, 2H, ArH), 4.96 (d, J = 11.1 Hz, 1H, PhCH2), 4.89 (d, J = 10.9 Hz, 1H, PhCH2), 4.82 (d, J = 11.1 Hz, 1H, PhCH2), 4.66 (dd, J = 27.2, 11.6 Hz, 2H, PhCH2), 4.52 (d, J = 12.1 Hz, 1H, PhCH2), 4.45 (d, J = 10.9 Hz, 1H, PhCH2), 4.38 (d, J = 12.1 Hz, 1H, PhCH2), 3.72-3.52 (m, 8H, H-6, H-2, H-3, H-10, H-11), 3.46 (t, J = 9.1 Hz, 1H, H-4), 3.10 (dd, J = 11.2, 4.8 Hz, 1H, H-1a), 2.84 (m, 1H, H-7a), 2.78-2.67 (m, 1H, H-7b), 2.49-2.20 (m, 8H, H-5, H-1b, H-8, H-9, H-12). 13C NMR (126 MHz, CDCl3): 5 139.03, 138.56, 137.74, 128.65, 128.47, 128.43, 128.39, 127.97, 127.93, 127.75, 127.67, 127.54, 87.33, 78.54, 78.42, 77.43, 77.17, 76.92, 75.40, 75.30, 73.53, 72.88, 66.93, 65.46, 63.89, 55.04, 54.03, 48.86. (2R,3R,4R,5S)-3,4,5-Tris(benzyloxy)-2-((benzyloxy) methyl)-1-(4-(pyrrolidin-1-yl)butyl)piperidine (13b) Prepared according to procedure D. Compound 11b (0.3 g, 0.5 mmol), 1,4-dibromobutane (120 ^L, 1 mmol), K2CO3 (0.21 g, 1.5 mmol), CH3CN (5 mL). Yield: 81% (265 mg), pale yellow syrup, Rf = 0.49 (20:2:0.2; ethyl ace-tate:MeOH:NH4OH). 1H NMR (500 MHz, CDCl3): 5 7.39-7.19 (m, 18H, ArH), 7.15 (d, J = 6.6 Hz, 2H, ArH), 4.96 (d, J = 11.0 Hz, 1H, PhCH2), 4.89 (d, J = 10.8 Hz, 1H, PhCH2), 4.81 (d, J = 11.0 Hz, 1H, PhCH2), 4.67 (q, J = 11.6 Hz, 2H, PhCH2), 4.52-4.39 (m, 3H, PhCH2), 3.73-3.52 (m, 4H, H-6, H-2, H-3), 3.47 (t, J = 9.0 Hz, 1H, H-4), 3.07 (dd, J = 11.2, 4.8 Hz, 1H, H-1a), 2.78-2.43 (m, 8H, H-7, H-10, H-11, H-14), 2.30 (d, J = 9.5 Hz, 1H, H-5), 2.18 (t, J = 10.8 Wang and Fang: Study on the Synthesis and Biological Activities 816 Acta Chim. Slov. 2020, 67, 812-821 Hz, 1H, H-1b), 1.83 (m, 4H, H-12, H-13), 1.60-1.34 (m, 4H, H-8, H-9). 13C NMR (126 MHz, CDCl3): 5 138.99, 138.55, 137.91, 128.45, 128.38, 127.95, 127.92, 127.89, 127.73, 127.61, 127.53, 87.35, 78.67, 78.53, 77.34, 77.08, 76.83, 75.42, 75.24, 73.47, 72.80, 65.71, 64.01, 55.98, 54.33, 53.96, 51.75, 29.36, 25.92, 23.37, 21.91. (2R,3R,4.R,5S)-3,4,5-Tris(benzyloxy)-2-((benzyloxy) methyl)-1-(4-(piperidin-1-yl)butyl)piperidine (13c) Prepared according to procedure D. Compound 11b (0.3 g, 0.5 mmol), 1,4-dibromopentane (135 pL, 1 mmol), K2CO3 (0.21 g, 1.5 mmol), CH3CN (5 mL). Yield: 82% (274 mg), colourless syrup, Rf = 0.57 (20:2:0.2; ethyl ace-tate:MeOH:NH4OH). 1H NMR (500 MHz, CDCl3): 5 7.43-7.20 (m, 18H, ArH), 7.20-7.09 (m, 2H, ArH), 4.97 (d, J = 11.0 Hz, 1H, PhCH2), 4.90 (d, J = 10.8 Hz, 1H, PhCH2), 4.82 (d, J = 11.0 Hz, 1H, PhCH2), 4.68 (q, J = 11.7 Hz, 2H, PhCH2), 4.45 (t, J = 5.3 Hz, 3H, PhCH2), 3.74-3.53 (m, 4H, H-6, H-2, H-3), 3.48 (t, J = 9.0 Hz, 1H, H-4), 3.06 (dd, J = 11.2, 4.8 Hz, 1H, H-1a), 2.80-2.36 (m, 8H, H-7, H-10, H-11, H-15), 2.30 (d, J = 9.4 Hz, 1H, H-5), 2.15 (t, J = 10.8 Hz, 1H, H-1b), 1.86-1.31 (m, 10H, H-8, H-9, H-12, H-13, H-14). 13C NMR (126 MHz, CDCl3): 5 138.96, 138.53, 137.99, 128.51, 128.48, 128.43, 128.37, 128.00, 127.93, 127.88, 127.78, 127.66, 127.59, 87.33, 78.68, 78.44, 77.55, 77.30, 77.04, 75.47, 75.27, 73.43, 72.77, 65.88, 64.21, 58.12, 54.24, 53.73, 51.32, 24.21, 23.17, 22.94, 22.00. 4-(4-((2R,3R,4.R,5S)-3,4,5-Tris(benzyloxy)-2-((benzy-loxy)methyl)piperidin-1-yl)butyl)morpholine (13d) Prepared according to procedure D. Compound 11b (0.3 g, 0.5 mmol), 2-bromoethyl ether (129pL, 1 mmol), K2CO3 (0.21 g, 1.5 mmol), CH3CN (5 mL). Yield: 87% (292 mg), colourless syrup, Rf = 0.7 (20:2:0.2; ethyl ace-tate:MeOH:NH4OH). 1H NMR (500 MHz, CDCl3): 5 7.44-7.18 (m, 18H, ArH), 7.18-7.04 (m, 2H, ArH), 4.95 (d, J = 11.1 Hz, 1H, PhCH2), 4.88 (d, J = 10.8 Hz, 1H, PhCH2), 4.81 (d, J = 11.1 Hz, 1H, PhCH2), 4.67 (q, J = 11.6 Hz, 2H, PhCH2), 4.45 (dt, J = 13.5, 11.5 Hz, 3H, PhCH2), 3.63 (m, 8H, H-6, H-2, H-3, H-12, H-13), 3.46 (t, J = 9.1 Hz, 1H, H-4), 3.08 (dd, J = 11.1, 4.9 Hz, 1H, H-1a), 2.76-2.64 (m, 1H, H-7a), 2.60 (m, 1H, H-7b), 2.48-2.16 (m, 8H, H-5, H-1b, H-10, H-11, H-14), 1.51-1.28 (m, 4H, H-8, H-9). 13C NMR (126 MHz, CDCl3): 5 139.04, 138.59, 137.86, 128.44, 128.39, 128.37, 127.92, 127.86, 127.70, 127.60, 127.50, 87.41, 78.64, 78.60, 77.36, 77.10, 76.85, 75.38, 75.24, 73.50, 72.83, 67.01, 65.52, 63.72, 58.77, 54.49, 53.72, 52.21, 24.45, 21.58. (2R,3R ,4R,5S)-1-(2-(Dimethylamino)ethyl)-2-(hy-droxymethyl)piperidine-3,4,5-triol (7a) Prepared according to procedure E. Compound 12a (260 mg, 0.44 mmol), 10% Pd/C (100 mg), EtOH (10 mL), pH~1 with 1 M aq HCl. Yield: 91% (93 mg), pale yellow syrup, Rf = 0.73 (1:1:0.5; ethyl acetate:MeOH:N-H4OH). 1H NMR (500 MHz, D2O): 5 3.84 (dd, J = 12.8, 2.1 Hz, 1H, H-6a), 3.76 (dd, J = 12.8, 2.9 Hz, 1H, H-6b), 3.47 (td, J = 10.2, 4.9 Hz, 1H, H-2), 3.29 (t, J = 9.5 Hz, 1H, H-3), 3.18 (t, J = 9.2 Hz, 1H, H-4), 2.94 (dd, J = 11.4, 4.9 Hz, 1H, H-1a), 2.89-2.77 (m, 1H, H-7a), 2.73-2.63 (m, IH, H-7b), 2.56-2.45 (m, 2H, H-8), 2.33-2.14 (m, 8H, H-5, H-1b, 2xCH3). 13C NMR (126 MHz, D2O): 5 78.30, 69.95, 68.76, 65.22, 57.54, 55.92, 53.30, 48.79, 44.18. HRMS (ESI) m/z calcd for C10H23N2O4+ (M+H)+ 235.16523, found 235.16492. (2R,3R,4R,5S)-2-(Hydroxymethyl)-1-(2-(pyrrolidm-1-yl)ethyl)piperidine-3,4,5-triol (7b) Prepared according to procedure E. Compound 12b (160 mg, 0.44 mmol), 10% Pd/C (80 mg), EtOH (5 mL), pH~1 with 1 M aq HCl. Yield: 95% (64 mg), yellow syrup, Rf = 0.44 (1:1:0.5; ethyl acetate:MeOH:NH4OH). 1H NMR (500 MHz, D2O): 5 3.86 (dd, J = 12.7, 1.9 Hz, 1H, H-6a), 3.79 (dd, J = 12.8, 2.8 Hz, 1H, H-6b), 3.49 (td, J = 10.3, 4.9 Hz, 1H, H-2), 3.31 (t, J = 9.5 Hz, 1H, H-3), 3.21 (t, J = 9.2 Hz, 1H, H-4), 2.98 (dd, J = 11.4, 4.9 Hz, 1H, H-1a), 2.89 (m, 1H, H-7a), 2.81-2.68 (m, 3H, H-7b, H-8), 2.62 (m, 4H, H-9, H-12), 2.30 (t, J = 11.1 Hz, 1H, H-1b), 2.24 (d, J = 9.7 Hz, 1H, H-5), 1.74 (m, 4H, H-10, H-11). 13C NMR (126 MHz, D2O): 5 78.31, 69.97, 68.79, 65.17, 57.55, 55.92, 53.62, 50.34, 49.49, 22.73. HRMS (ESI) m/z calcd for C12H-25N2O4+ (M+H)+ 261.18088, found 261.18057. (2R,3R,4R,5S)-2-(Hydroxymethyl)-1-(2-(piperidin-1-yl)ethyl)piperidine-3,4,5-triol (7c) Prepared according to procedure E. Compound 12c (260 mg, 0.41 mmol), 10% Pd/C (100 mg), EtOH (10 mL), pH~1 with 1 M aq HCl. Yield: 92% (123 mg), yellow syrup, Rf = 0.52 (1:1:0.5; ethyl acetate:MeOH:NH4OH). 1H NMR (500 MHz, D2O): 5 3.82 (dd, J = 12.7, 2.2 Hz, 1H, H-6a), 3.75 (dd, J = 12.8, 2.9 Hz, 1H, H-6b), 3.45 (td, J = 10.3, 4.9 Hz, 1H, H-2), 3.26 (t, J = 9.4 Hz, 1H, H-3), 3.17 (t, J = 9.2 Hz, 1H, H-4), 2.94 (dd, J = 11.2, 5.3 Hz, 2H, H-1a, H-7a), 2.71 (m, 7H, H-7b, H-8, H-9, H-13), 2.24 (dd, J = 21.8, 10.5 Hz, 2H, H-5, H-1a), 1.65-1.48 (m, 4H, H-10, H-12), 1.43 (m, 2H, H-11). 13C NMR (126 MHz, D2O): 5 78.25, 69.92, 68.71, 65.08, 57.45, 55.81, 54.06, 53.38, 46.94, 24.00, 22.41. HRMS (ESI) m/z calcd for C13H27N2O4+ (M+H)+ 275.19653, found 275.19635. (2R,3R,4R,5S)-2-(Hydroxymethyl)-1-(2-morpholino-ethyl)piperidine-3,4,5-triol (7d) Prepared according to procedure E. Compound 12d (270 mg, 0.42 mmol), 10% Pd/C (100 mg), EtOH (10 mL), pH~1 with 1 M aq HCl. Yield: 94% (110 mg), yellow solid, Rf = 0.68 (1:1:0.5; ethyl acetate:MeOH:NH4OH). 1H NMR (500 MHz, D2O): 5 3.81 (dd, J = 12.9, 1.8 Hz, 1H, H-6a), 3.74 (dd, J = 12.9, 2.7 Hz, 1H, H-6b), 3.65 (m, 4H, H-10, H-11), 3.45 (td, J = 10.1, 4.9 Hz, 1H, H-2), 3.26 (t, J = 9.5 Hz, 1H, H-3), 3.16 (t, J = 9.2 Hz, 1H, H-4), 2.93 (dd, J = II.4, 4.9 Hz, 1H, H-1a), 2.88-2.80 (m, 1H, H-7a), 2.762.64 (m, 1H, H-7b), 2.59-2.41 (m, 6H, H-8, H-9, H-12), Wang and Fang: Study on the Synthesis and Biological Activities ... Acta Chim. Slov. 2020, 67, 812-817 821 2.27 (t, J = 11.1 Hz, 1H, H-1b), 2.20 (d, J = 9.7 Hz, 1H, H-5). 13C NMR (126 MHz, D2O): 5 78.25, 69.89, 68.70, 66.06, 65.11, 57.47, 55.87, 52.88, 47.71. HRMS (ESI) m/z calcd for C12H25N2O5+ (M+H)+ 277.17580, found 277.17614. (2.R,3.R,4.R,5S)-1-(4-(Dimethylamino)butyl)-2-(hy-droxymethyl)piperidine-3,4,5-triol (8a) Prepared according to procedure E. Compound 13a (200 mg, 0.32 mmol), 10% Pd/C (100 mg), EtOH (10 mL), pH~1 with 1 M aq HCl. Yield: 93% (78 mg), white solid, Rf = 0.67 (1:1:0.5; ethyl acetate:MeOH:NH4OH). 1H NMR (500 MHz, D2O): 5 3.79 (d, J = 12.6 Hz, 1H, H-6a), 3.71 (d, J = 12.4 Hz, 1H, H-6b), 3.49-3.38 (m, 1H, H-2), 3.25 (t, J = 9.4 Hz, 1H, H-3), 3.14 (t, J = 9.2 Hz, 1H, H-4), 2.90 (dd, J = 11.1, 4.2 Hz, 1H, H-1a), 2.72-2.59 (m, 1H, H-7a), 2.53 (m, 1H, H-7b), 2.30 (m, 2H, H-10), 2.25-2.04 (m, 8H, H-5, H-1a, 2xCH3), 1.36 (m, 4H, H-8, H-9). 13C NMR (126 MHz, D2O): 5 78.34, 70.07, 68.88, 64.96, 58.20, 57.52, 55.27, 51.81, 43.63, 24.18, 20.82. HRMS (ESI) m/z calcd for C12H27N2O4+ (M+H)+ 263.19653, found 263.19635. (2.R,3.R,4.R,5S)-2-(Hydroxymethyl)-1-(4-(pyrrolidin-1-yl)butyl)piperidine-3,4,5-triol (8b) Prepared according to procedure E. Compound 13b (220 mg, 0.34 mmol), 10% Pd/C (100 mg), EtOH (10 mL), pH~1 with 1 M aq HCl. Yield: 90% (88 mg), colorless syrup, Rf = 0.37 (1:1:0.5; ethyl acetate:MeOH:NH4OH). 1H NMR (500 MHz, D2O): 5 3.80 (dd, J = 12.7, 1.9 Hz, 1H, H-6a), 3.72 (dd, J = 12.8, 2.6 Hz, 1H, H-6b), 3.48-3.37 (m, 1H, H-2), 3.26 (t, J = 9.5 Hz, 1H, H-3), 3.15 (t, J = 9.2 Hz, 1H, H-4), 2.91 (dd, J = 11.4, 4.9 Hz, 1H, H-1a), 2.65 (m, 1H, H-H-7a), 2.48 (m, 7H, H-7b, H-10, H-11, H-14), 2.25-2.08 (m, 2H, H-5, H-1b), 1.66 (m, 4H, H-12, H-13), 1.39 (m, 4H, H-8, H-9). 13C NMR (126 MHz, D2O): 5 78.37, 70.10, 68.90, 65.00, 57.59, 55.31, 55.25, 53.19, 51.86, 25.67, 22.70, 21.09. HRMS (ESI) m/z calcd for C14H-29N2O4+ (M+H)+ 289.21218, found 289.21194. (2.R,3.R,4.R,5S)-2-(Hydroxymethyl)-1-(4-(piperidin-1-yl)butyl)piperidine-3,4,5-triol (8c) Prepared according to procedure E. Compound 13c (260 mg, 0.39 mmol), 10% Pd/C (100 mg), EtOH (10 mL), pH~1 with 1 M aq HCl. Yield: 95% (113 mg), yellow syrup, Rf = 0.44 (1:1:0.5; ethyl acetate:MeOH:NH4OH). 1H NMR (500 MHz, D2O): 5 3.78 (d, J = 11.9 Hz, 1H, H-6a), 3.71 (dd, J = 12.7, 2.4 Hz, 1H H-6b), 3.43 (td, J = 10.0, 5.0 Hz, 1H, H-2), 3.25 (t, J = 9.5 Hz, 1H, H-3), 3.14 (t, J = 9.2 Hz, 1H, H-4), 2.89 (dd, J = 11.4, 4.9 Hz, 1H, H-1a), 2.84-2.46 (m, 8H, H-7, H-10, H-11, H-15), 2.26-2.09 (m, 2H, H-5, H-1b), 1.58 (m, 4H, H-12, H-14), 1.53-1.31 (m, 6H, H-8, H-9, H-13). 13C NMR (126 MHz, D2O): 5 78.33, 70.04, 68.86, 65.01, 57.58, 57.31, 55.29, 53.29, 51.54, 23.74, 22.45, 22.30, 20.75. HRMS (ESI) m/z calcd for C15H31N2O4+ (M+H)+ 303.22783, found 303.22754. (2fl,3fl,4fl,5S)-2-(Hydroxymethyl)-1-(4-morpholi-nobutyl)piperidine-3,4,5-triol (8d) Prepared according to procedure E. Compound 13d (270 mg, 0.34 mmol), 10% Pd/C (100 mg), EtOH (10 mL), pH~1 with 1 M aq HCl. Yield: 96% (119 mg), pale yellow solid, Rf = 0.59 (1:1:0.5; ethyl acetate:MeOH:NH4OH). 1H NMR (500 MHz, D2O): 5 3.73 (m, 6H, H-6, H-12, H-13), 3.41 (td, J = 9.7, 4.6 Hz, 1H, H-2), 3.24 (t, J = 9.5 Hz, 1H, H-3), 3.12 (t, J = 9.2 Hz, 1H, H-4), 2.89 (dd, J = 11.4, 4.8 Hz, 1H, H-1a), 2.73-2.40 (m, 8H, H-7, H-10, H-11, H-14), 2.25-2.08 (m, 1H, H-5, H-1b), 1.40 (m, 4H, H-8, H-9). 13C NMR (126 MHz, D2O): 5 78.25, 69.93, 68.77, 65.50, 64.97, 57.58, 57.41, 55.20, 52.21, 51.64, 22.60, 20.75. HRMS (ESI) m/z calcd for C14H29N2O5+ (M+H)+ 305.20710, found 305.20676. 2. 2. Glucosidase Inhibitory Assays a-Glucosidase (yeast), ^-glucosidase (sweet almonds), and a-mannosidase (jack bean) was purchased from Sigma. 1-Deoxynojirimycin, para-nitrophenyl a-D-glucopyranoside, para-nitrophenyl ^-D-glucopyrano-side and para-nitrophenyl a-D-mannosidase were also purchased from Sigma. Inhibitory potencies were carried out by spectrophotometrically measuring the residual hy-drolytic activities of the glycosidases on the corresponding para-nitrophenyl glycoside substrates. The a-glucosidase,26 ^-glucosidase assays27 were performed in 50 mM phosphate buffer, pH 6.8 at 37 °C. The a-mannosidase assay28 was performed in 50 mM citrate buffer, pH 5.5 at 37 °C. The test compounds were pre-incubated with the enzyme solutions and buffered in a disposable 96-well microtiter plate at 37 °C for 15 min. Next, the reactions were initiated by the addition of 20 y.L of a solution of the corresponding para-nitrophenyl glycoside substrates. After the reaction mixture was incubated at 37 °C for 15 min. Thereupon, it was quenched by adding 80 y.L Na2CO3 (0.2 mol/L). Enzymatic activity was quantified by measuring the absorbance at 405 nm using a BioTek ^Quant Microplate Spectrophotometer. Each experiment was performed in triplicate. IC50 values were determined graphically with GraphPad Prism (version 8.0). 2. 3. Kinetics of Enzyme Inhibition Inhibition constant (K) measurement was performed in 50 mM phosphate buffer (pH 6.8) at 37 °C, using para-nitrophenyl ^-D-glucopyranoside as the substrate. The assay was initiated by adding ^-glucosidase (Km = 3.5 mM) to a solution of the substrate (concentrations used: 0.875 mM, 1.75 mM, 3.5 mM, 7 mM, 10.5 mM) in the presence of inhibitors (concentrations used: 0 mM, 0.1 mM, 0.2 mM). After the reaction mixture was incubated at 37 °C for 15 min, it was quenched by adding 80 y.L Na2CO3 (0.2 mol/L). The absorbance of 4-nitrophenol released from the substrate was read at 405 nm. Wang and Fang: Study on the Synthesis and Biological Activities 818 Acta Chim. Slov. 2020, 67, 812-821 2. 4. Cell Culture and Inhibition of Proliferation B16F10 Cells29 The mouse B16F10 melanoma cell line, which is derived from C57BL/6 mice was purchased from KeyGen Biotech (Nanjing, China). The cell line was cultured in DMEM supplemented with fetal bovine serum (10%), penicillin (100 U/mL) and streptomycin (100 ^g/mL) at 37 °C in humidified 5% CO2 atmosphere. Media was replenished every third day. B16F10 cells were seeded on 96-well microtiter plates in DMEM supplemented with 10% FBS and incubated overnight. The compounds (1 mM, 0.05 mM) were then added to the cells and cultured for another 48 h. Each treatment was performed in six well replicates. MTT reagent (Sigma Aldrich) was added to each well incubated for 4 h at 37 °C. After the cell culture medium was removed, formazan crystals in adherent cells were dissolved in 200 ^L DMSO and the absorbance of the formazan solution was measured at 570 nm. ra-0-benzyl-1-deoxynojirimycin 9 which was prepared according to previously published procedures in four steps.28 Treatment of O-benzyl protected DNJ 9 with N-(4-bromobutyl)phthalimide or N-(4-bromoethyl) phthalimide in the presence of K2CO3 in DMF afforded N-phthalyl protected DNJ 10 (Scheme 1). The intermediate 10 was then converted into primary amide 11 by a hy-drazinolysis reaction using N2H4 in EtOH. A generalized synthetic approach to the derivatives 7 and 8 was shown in Scheme 2. The reductive amination of 11 with HCHO-HCOOH gave compounds 12a and 13a. For compounds 12 and 13 which beared 5- and 6-membered rings, double nucleophilic substitution reaction was performed on primary amine 11 in basic conditions. All the intermediates 12 and 13 were obtained in good (80%) to excellent (90%) yields, independently of the chain length. Precursors 12 and 13 were then depro-tected by hydrogenolysis (10% Pd/C, EtOH, 1 M HCl) to afford the target derivatives 7 and 8 in almost quantitative yield. 3. Results and Discussion 3. 1. Chemistry The target compounds were prepared from the key intermediate 11 through reductive amination or double nucleophilic substitution, respectively (Figure 2). The synthesis of compound 11 commenced from 2,3,4,6-tet- 3. 2. Biological Evaluation The small library of DNJ derivatives were submitted to a panel of biological evaluations, which included inhibition of glycosidases, inhibition kinetics of ^-glucosidase, as well as inhibition of B10F16 cells growth. These experiments are summarized below. ,-OBn re BnO BnO1 n^b; n = 1,3 Figure 2. The key intermediate 11 and the general structures of the target compounds. Scheme 1. Synthesis of the primary amide 11. Reagents and conditions: (a) N-(4-bromobutyl) phthalimide or N-(4-bromoethyl) phthalimide, K2CO3, DM , 100 °C, 24 h, 87% (10a), 85% (10b); (b) N2H4 (80%), EtOH, reflux, 3 h, 82% (11a), 85% (11b). Wang and Fang: Study on the Synthesis and Biological Activities ... Acta Chim. Slov. 2020, 67, 812-819 821 OH — HO, X ho^y ÖBn ÖBn OH 11a, n = 1 12a, n = 1 7a n = 1 11b, n=3 13a,n = 3 8a!n = 3 A 12b, n = 1 OBn 13b, n = 3 0Bn „ „OH OBn 11a, n = 1 11b, n = 3 12c, n = 1 ÖBri 13c, n = 3 <=>H 7c, n = 1 8c, n = 3 N^HÎ OBn \\6dT-\ 6H 7d^=1 13d, n-3 u 8d, n = 3 Scheme 2. Synthesis of N-alkylated derivatives of 1-deoxynojirimycin. Reagents and conditions: (a) HCHO, HCOOH, 105 °C, 3 h, 83% (12a), 90% (13a); (b) alkyl dibromide, K2CO3, CH3CN, 80 °C, 12 h, 84% (12b), 81% (13b), 80% (12c), 82% (13c), 85% (12d), 87% (13d); (c) H2, 10% Pd/C, EtOH, 1 M HCl, rt, 24 h, 91% (7a), 93% (8a), 95% (7b), 90% (8b), 92% (7c), 95% (8c), 94% (7d), 96% (8d). 3. 2. 1. Inhibition of Glucosidases Glycosidase inhibitory activities of compounds 7 and 8 was evaluated against a-glucosidase (yeast), ^-glu-cosidase (almonds), a-mannosidase (jack bean), with reference to the known standard DNJ. The results were expressed as the inhibition of glucosidase activity (IC50) and are summarized in Table 1. Compounds 7a, 7b and 7c had weak inhibitory activities against a- and ^-glucosidase at 1 mM. It was, however, interesting to note that compound 7d bearing a morpholine ring was the only derivative in our library exhibiting higher and selective activity of ^-glucosidase with an IC50 of 0.052 ± 0.004 mM compared to DNJ (IC50 = 0.65 ± 0.04 mM), while none of the other glycosidases were inhibited by this compound (Table 1). This indicated that a much more favorable interaction with the ^-glucosi-dase active site. Compound 8a also had weak inhibitory activity against a- and ^-glucosidase. Derivatives 8b and 8c which possessed a longer alkyl chain were found to be more selective inhibitors of a-glucosidase than 7b and 7c, with IC50 values of 0.364 ± 0.011 mM and 0.358 ± 0.04 mM, respectively. And they had similar potencies to a-glucosi-dase. Compound 8d, which beared a morpholine ring, showed decreased inhibitory activity against a-glucosidase with an IC50 of 1.385 ± 0.137 mM compared to 8b and 8c. However, 8d showed better inhibitory effect on a-glucosi-dase than 7d which possessed a shorter alkyl chain (Table 1). Moreover, compounds 8a, 8b, 8c and 8d have reduced Table 1. Glycosidase inhibitory activity values IC50 (mM) Enzyme 7a 7b 7c 7d 8a 8b 8c 8d DNJ a-glucosidase (yeast) 10%a 24% 29% 33% 49% 0.364 ± 0.011 0.358 ± 0.04b 1.385 ± 0.137 0.155 ± 0.015 ß-glucosidase (almonds) 22% 34% 33% 0.052 ± 0.004 17% 40% 18% 29% 0.648 ± 0.036 a-mannosidase (jack bean) NIc NI NI NI NI NI NI NI NI a The inhibition rate (%) was obtained from the 1 mM of compounds. b IC50 is defined as the compound concentration at which 50% activities of glucosidases. The values are mean±SD from three independent experiments. c NI indicated no inhibition at 1 mM of compounds. Wang and Fang: Study on the Synthesis and Biological Activities 820 Acta Chim. Slov. 2020, 67, 812-821 inhibitory activity compared to DNJ. These results suggested that 8b and 8c which beared 5- and 6-membered rings would be beneficial for the interaction with a-gluco-sidase through the hydrophobic effect more than N-di-methyl DNJ derivative 8a.30 Compounds having n = 3 displayed better inhibition towards a-glucosidase than compounds having n = 1, with a trend correlating higher inhibition associated with increased chain length. In addition, introduction of a nitrogen atom seemed to display no or negligible inhibition against all the enzymes. Finally, none of these derivatives showed inactivation of jack bean a-mannosidase. 3. 2. 2. Inhibition Kinetics of 0-Glucosidase In order to explore further insight into how 7d interacted with ^-glucosidase (almonds), the mode of inhibition and inhibition constant of 7d was determined by the Lineweaver-Burk plots (Figure 3). The double reciprocal plots of 7d showed straight lines with the same vmax. This indicated that 7d (Ki = 7 ^M) inhibited ^-glu-cosidase in a competitive manner, a nearly 7-fold increase compared to DNJ26 (Ki = 47 ^M). Hence, this competitive inhibition indicated that 7d was expected to bind to the active site of ^-glucosidase and compete with their primary substrates. Moreover, a probable hydrogen bond acceptor was the carbonyl hydrogen atom of the catalytic acid.27 o.a-i- Ë 0.40.2- 0.0~ i i i i i i i i i | i i i i i i i i i | i i 0.0 0.5 1.0 1/[S]{mM"1> Figure 3. Double-reciprocal plot of the inhibition kinetics of |-glu-cosidase (almonds) by compound 7d. Substrate concentration: 0.875, 1.75, 3.5, 7, 10.5 mM, inhibitor concentration: 0 mM (control, ▼), 0.1 mM (♦), 0.2 mM (▲). 3. 3. Inhibition of B16F10 Cells Growth The inhibition of B16F10 cells growth by compounds was determined using the MTT assay and the results are summarized in Figure 4. All compounds were inactive with no significant inhibition being observed at 0.05 mM and 1 mM. This indicated that compounds by the modification of changing length of the tether, the size and nature of the terminal tertiary amine substituents had no influence on the anticancer activity. 1.5 c=1 mM Figure 4. Effect of glycosidase inhibitors on B16F10 cells growth. Each bar represents the mean (±SD, n = 6). P > 0.05 comparing with control. 4. Conclusion In summary, a series of DNJ derivatives were designed and synthesized, and the structures of synthesized compounds were confirmed by 1H NMR, 13C NMR and HRMS. Moreover, the preliminary glucosidase inhibition and anticancer activities were evaluated in vitro. Compound 7d proved to be the most potent and selective |-glucosidase inhibitor in a competitive manner, and none of the other glycosidases were inhibited by this compound at micromolar level. Compounds 8b and 8c were moderate and selective a-glucosidase inhibitors. Nevertheless, all compounds could not inhibit the growth of B16F10 melanoma cells. The collective results indicated that a lengthening of the alkyl chain linking DNJ provide better selectivity towards a-glucosidase. The size of the hydrophobic group at the alkyl chain, especially its nature, differs greatly for the selective inhibition aganist a-and |-glucosidases. Compounds 7d, 8b and 8c would be a lead for designing novel compounds, and further derivatives would be prepared by altering these specific molecules. In addition, our results provides useful clues for the design of selective glu-cosidase inhibitors. Wang and Fang: Study on the Synthesis and Biological Activities ... Acta Chim. Slov. 2020, 67, 812-821 821 5. References 1. C. S. Rye, S. G. Withers, Curr. Opin. Chem. Biol. 2000, 4, 573-580. DOI:10.1016/S1367-5931(00)00135-6 2. T. Kimura, K. Nakagawa, H. Kubota, Y. Kojima, Y. Goto, K. Yamagishi, S. Oita, S. Oikawa, T. Miyazawa, J. Agric. Food. Chem. 2007, 55, 5869-5874. DOI:10.1021/jf062680g 3. Y. X. Zhao, W. J. Liu, Y. Zhou, X. M. Zhang, P. V. 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Fischl, L. Resnick, R. Coombs, A. B. Kremer, J. C. Pottage, R. J. Fass, K. H. Fife, W. G. Powderly, A. C. Collier, R. L. Aspinall, J. Acquir. Immune Defic. Syndr. 1994, 7, 139-147. 19. R.-W. Wang, X.-L. Qiu, M. Bols, F. Ortega-Caballero, F.-L. Qing, J. Med. Chem. 2006, 49, 2989-2997. DOI:10.1021/jm060066q 20. A. Mehta, N. Zitzmann, P. M. Rudd, T. M. Block, R. A. Dwek, FEBS Lett. 1998, 430, 17-22. DOI:10.1016/S0014-5793(98)00525-0 21. T. Wennekes, B. Lang, M. Leeman, G. A. v. d. Marel, E. Smits, M. Weber, J. v. Wiltenburg, M. Wolberg, J. M. F. G. Aerts, H. S. Overkleeft, Org. Process. Res. Dev. 2008, 12, 414-423. DOI:10.1021/op700295x 22. W. F. Wang, F. Wang; T. T. Liang, Z. Fang, Chem.Res.Appl. 2017, 29, 793-798. 23. M. Iftikhar, L. Wang, Z. Fang, J. Chem. Res. 2017, 41, 460-464. DOI:10.1016/S0928-1258(17)30358-3 24. W. Yu, T. Gill, L. Wang, Y. Du, H. Ye, X. Qu, J. T. Guo, A. Cu-conati, K. Zhao, T. M. Block, X. Xu, J. Chang, J. Med. Chem. 2012, 55, 6061-6075. DOI:10.1021/jm300171v 25. C. G. F. Cooper, E. R. Lee, R. A. Silva, A. J. Bourque, S. Clark, S. Katti and V. Nivorozhkin, Org. Process. Res. Dev. 2012, 16, 1090-1097. DOI:10.1021/op2001222 26. W. L. Dong, T. Jespersen, M. Bols, T. Skrydstrup, M. R. Sierks, J. Biochem. 2006, 35, 2788. 27. N. Panday, Y. Canac, A. Vasella, Helv. Chim. Acta 2000, 83, 58. DOI:10.1002/(SICI)1522-2675(20000119)83:1<58::AID-HLCA58>3.0.C0;2-K 28. P. Compain, C. Decroocq, J. Iehl, M. Holler, D. Hazelard, T. Mena Barragán, C. Ortiz Mellet, J.-F. Nierengarten, Angew. Chem. Int. Ed. 2010, 49, 5753-5756. DOI:10.1002/anie.201002802 29. P. Dua, R. P. Gude, Cancer Chemother. Pharmacol. 2006, 58, 195-202. DOI:10.1007/s00280-005-0155-8 30. S. M. Ding, T. Lan, G. J. Ye, J. J. Huang, Y. Hu, Y. R. Zhu, B. Wang, Bioorg. Med. Chem. 2018, 26, 3370-3378. DOI:10.1016/j.bmc.2018.05.008 Povzetek Pripravili smo serijo N-alkiliranih deoksinojirimicinskih (DNJ) derivatov, povezanih s terminalno terciarno aminsko skupino na alkilni verigi različnih dolžin. Te nove sintezne spojine smo preliminarno in vitro analizirali za glukozidazno inhibicijo in antirakavo aktivnost. V nekaterih primerih smo opazili močno in selektivno inhibicijo. Spojina 7d (IC50 = 0.052 mM) je, v primerjavi z DNJ (IC50 = 0.65 mM), pokazala izboljšano in selektivno inhibitorno aktivnost proti p-glukozidazi. Dodatne analize kinetike encimske inhibicije s pomočjo Lineweaver-Burkovih diagramov so pokazale, da 7d inhibira p-glukozidazo na kompetitiven način, kar nakazuje, da se 7d verjetno veže v aktivno mesto p-glukozidaze. Spojini 8b and 8c sta pokazali zmerno, a vendar selektivno, inhibicijo a-glukozidaze. Ne glede na to, pa nobena od spojin ni inhibirala rasti B16F10 melanomskih celic. Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Wang and Fang: Study on the Synthesis and Biological Activities DOI: 10.17344/acsi.2019.5784 Acta Chim. Slov. 2020, 67, 822-829 /^.creative o'commons Scientific paper Structural Diversity in Oxadiazole-Containing Silver Complexes Dependent on the Anions Long Zhao,f1 Long-Yan Xie,f1 Xiu-Li Du,f1 Kai Zheng,1 Ting Xie,1 Rui-Rui Huang,1 Jie Qin,*1 Jian-Ping Ma*2 and Li-Hong Ding3 1 School of Life Sciences, Shandong University of Technology, Zibo 255049, P. R. China 2 College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, P. R. China 3 Lanzhou University of Arts and Sciences, Lanzhou, 730000, P. R. China * Corresponding author: E-mail: qinjietutu@163.com xxgk123@163.com; Tel.: 0086-533-2780271; Fax: 0086-533-2781329 Received: 12-17-2019 t These authors contributed equally to this work. Two coordination polymers, namely [Ag2(L)(SO3CF3)(H2O)](SO3CF3^CH2Cl2 (1) and [Ag5(L)4(H2O)2](SbF6)5^5THF (2), were obtained by reacting oxadiazole-containing tri-armed ligand 1,3,5-tri(2-methylthio-1,3,4-oxadiazole-5yl) benzene (L) and silver salts in CH2Cl2/THF medium. The two complexes crystallized in the tetragonal space group 14^a and orthorhombic space group Fdd2, respectively. The Single-crystal X-ray diffraction revealed that the two complexes exhibit strikingly different 3D polymeric structures, which can be ascribed to the different counter anions. L in compound 1 acted as a hexa-dentate ligand, binding to two types of Ag+ atoms to form a 3D polymeric structure. L in compound 2 acted as a hexa- and penta-dentate ligand, binding to three types of Ag+ atoms to form the 3D polymeric structure. The antibacterial activity of the complexes was also investigated. Keywords: Coordination polymers; oxadiazole-containing spacer; counter anions; antibacterial activity Abstract 1. Introduction medium, and inorganic counter anions can affect the self-assembly process of coordination compounds.11 Our research group has initiated a synthetic program for the construction of coordination polymers generated from ox-adiazole-containing organic ligands.11,12-14 As an extension of previous studies, we expand oxadiazole-bridging double-armed ligands to the oxadiazole-containing tri-armed ligand L, namely 1,3,5-tri(2-methylthio-1,3,4-oxa-diazole-5-yl)benzene (Scheme 1). L has three arms, each The design and synthesis of coordination polymers exhibiting intriguing structures and properties have attracted much attention because of their potential application in catalysis,1-3 magnetic properties,4-6 gas adsorption and separation,7,8 molecular sensing and luminescent materials.9,10 Many factors, including the nature of the metal ion, design of the organic ligand, auxiliary ligand, solvent hs, / S, Scheme 1. Synthesis of L. Zhao et al.: Structural Diversity in Oxadiazole-Containing Silver Acta Chim. Slov. 2020, 67, 822-823 829 comprising 2-methylthio-1,3,4-oxadiazole. As is known, N atoms on the 1,3,4-oxadiazole ring can bind to transition metals, and the S atom also has strong binding ability to soft metals (e.g., Ag+). Therefore, L can act as a multi-connector in the assembly of complexes with six oxadi-azole N-donors and three S-donors, which may result in polynuclear or high-dimensional intricate structures. The reactions of L with AgSO3CF3 and AgSbF6 in CH2Cl2/THF allow for two 3D coordination polymers, [Ag2(L)(SO3CF3) (H2O)](SO3CF3)-CH2Cl2 (1) and [^(LM^O^KSb-F6)5^5THF (2), respectively. Here, we report the synthesis and crystal structures of these compounds. Moreover, the antibacterial activities of 1 and 2 were investigated. 2. Experimental 2. 1. Physical Measurements and Materials Reagents and solvents were purchased commercially from Xiya Reagent. The reagents used in the experiment were all analytical pure, and no further purification was carried out without explanation. The intermediate A was synthesized according to the literature method.11 Infrared spectra in the range of (400-4000 cm-1) were determined by Vector22 Bruker spectrophotometer using potassium bromide tablets. The IR spectra of the synthesized compounds are given in Figure S1 of the supporting information. 1H NMR spectra were measured on a Bruker AM 500 spectrometer. 2. 2. Synthesis of L KOH (0.2 g, 3.57 mmol) was added to a solution of A (0.4 g, 1.00 mmol) in water (30 mL), the mixture was stirred for 20 minutes at ambient temperature. Then CH3I (0.8 mL) was added, the mixture was stirred for 6 hours at 0 °C, then filtered. The product was collected and purified on silica gel by column using CH2Cl2/THF (5 : 1, v/v) as the eluent to afford L as the white crystalline solid (0.34 g). Yield 80 %. M.p. = 134~136 °C, IR (KBr pellet cm-1): 3047(w), 2936(m), 1543(m), 1465(vs), 1432(s), 1323(m), 1181(vs), 1105(m), 947(s), 898(m), 781(s), 731(m), 705(s), 678(m). 1H NMR (300MHz, CDCl3, 25°C, TMS, ppm): 8.77(s, 3H, -C6H3), 2.87(s, 9H, -CH3). Single crystals suitable for X-ray crystallographic analysis were grown by slow diffusion of ethanol into dicholmethane solution of L. 2. 3. Synthesis of 1 A solution of AgSO3CF3 (19.6 mg, 0.076 mmol) in THF (10 mL) was layered carefully onto a solution of L (10 mg, 0.024 mmol) in CH2Cl2. The solutions were left at room temperature for about 1 week, and blocky colorless crystals were obtained. Yield: 55% (based on L). IR (KBr pellet cm-1): 2939(w), 1624(m), 1554(m), 1464(vs), 1432(m), 1257(vs), 1181(vs), 1033(s), 974(m), 782(m), 730(m), 678(s), 517(m). 2. 4. Synthesis of 2 A solution of AgSbF6 (26.6 mg, 0.077 mmol) in THF (10 mL) was layered carefully onto a solution of L (10 mg, 0.024 mmol) in CH2Cl2. The solutions were left at room temperature for about 2 weeks, and claviform colorless crystals were obtained. Yield: 40% (based on L). IR (KBr pellet cm-1): 2936(w), 1621(m), 1552(m), 1464(vs), 1431(m), 1182(s), 1105(w), 976(w), 784(m), 663(vs). Table 1. Crystallographic data for L, 1, and 2. L 1 2 Empirical formula C16H14Cl2N6O3S3 C18H16Ag2Cl2F6N6O10S5 C80H92Ag5F30N24O19S12Sb5 Mr 505.41 1037.31 3796.60 Crystal System triclinic tetragonal orthorhombic Space group P-1 I4Ja Fdd2 a (A) 12.3957(15) 29.635(2) 23.442(3) b (A) 13.1933(16) 29.635(2) 49.277(8) c (A) 14.4155(18) 15.200(2) 22.284(3) a (°) 111.293(2) 90.00 90.00 P (°) 106.666(2) 90.00 90.00 Y(°) 94.493(2) 90.00 90.00 V (A3) 2059.9(4) 13350(2) 25741(6) Z 4 4 8 Pc (g cm-3) 1.630 2.064 1.959 F(000) 1032 8228 14752.0 T / K 173(2) 173(2) 173(2) |(Mo-Ka)/ mm-1 0.652 1.738 2.079 GOF (F2) 1.021 1.098 1.029 Data / restraints / parameters 7495 / 0 / 547 5907 / 16 / 474 11022 / 19 / 740 Ria, wR2h (I>2o(I)) 0.0421, 0.1046 0.0493, 0.1046 0.0452, 0.1114 a R1 = £||F0| - |FC||/S|F0|. b wR2 = [£w(F02 - Fc2)2/Sw(F02)]1/2 Zhao et al.: Structural Diversity in Oxadiazole-Containing Silver ... 824 Acta Chim. Slov. 2020, 67, 822-829 2. 5. Determination of Crystal Structures The single crystal of the synthesized compounds were measured using Bruker Smart Apex CCD diffrac-tometer. The collected data were reduced using SAINT,15 and multi-scan absorption corrections were performed using SADABS.16 The structures were solved by direct methods and refined against F2 by full-matrix least-squares.17 All of the non-hydrogen atoms were refined aniso-tropically. All the hydrogen atoms were generated geometrically and refined isotropically using the riding model except the hydrogen atoms of water molecules, which were located directly from the Fourier map. Details of crystallographic parameters, data collection, and refinements are summarized in Table 1. The bond length and bond angle of the crystals are shown in Tables 2 and 3. Table 2. Selected bond distances (Â) and angles (°) for complex 1. Ag1-N2#' 2.274(4) Ag1-N4#2 2.293(4) Ag1-N1 2.316(4) Ag1-N3#3 2.311(4) Ag2-N5 2.229(4) Ag2-N6#4 2.282(4) Ag2-O4 2.566(4) Ag2-O7 2.322(4) N2#'-Ag1-N4#2 114.42(14) O7-Ag2-O4 93.19(16) N2#'-Ag1-N3#3 106.15(14) N5-Ag2-N6#4 138.06(14) N4#2-Ag1-N3#3 106.39(15) N5-Ag2-O7 119.72(16) N2#1-Ag1-N1 113.64(14) N6#4-Ag2-O7 94.29(15) N4#2-Ag1-N1 101.77(15) N5-Ag2-O4 87.70(14) N3#3-Ag1-N1 114.46(14) N6#4-Ag2-O4 116.05(14) Ag1-N2#1 2.274(4) Ag1-N4#2 2.293(4) Ag1-N1 2.316(4) Ag1-N3#3 2.311(4) Ag2-N5 2.229(4) Ag2-N6#4 2.282(4) Symmetry code: #1: -y+7/4, x+1/4, z+1/4; #2: y-1/4, -x+7/4, z+3/4 ; #3: -x+3/2, -y+2, z+1/2; #4: -y+7/4, :x+1/4, z-3/4 Table 3 Selected bond distances (Â) and angles (°) for complex 2. Ag1-N2 2.257(5) Ag1-N2#1 2.257(5) Ag1-N9 2.328(6) Ag1-N9#1 2.328(6) Ag2-N7 2.294(6) Ag2-N4#2 2.374(6) Ag2-N11#2 2.325(6) Ag2-O7 2.466(6) Ag2-S1#3 2.9549(19) Ag3-N1 2.362(6) Ag3-N10 2.325(7) Ag3-N12#' 2.384(6) Ag3-N6#4 2.413(7) Ag3-N3#1 2.548(6) N2#1-Ag1-N2 108.5(3) N2#1-Ag1-N9#1 123.1(2) N2-Ag(1)-N9#1 109.6(2) N2#1-Ag1-N9 109.6(2) N2-Ag1-N9 123.1(2) N9#1-Ag1-N9 82.1(3) N7-Ag2-N4#2 92.6(2) N1#2-Ag2-N4#2 134.8(2) N7-Ag2-N11#2 129.3(2) O7-Ag2-S1#3 178.13(17) N10-Ag3-N1 110.7(2) N10-Ag3-N6#4 82.7(2) N10-Ag3-N12#1 94.8(2) N10-Ag3-N3#1 132.2(2) N6#4-Ag3-N12#1 114.5(2) N12#'-Ag3-N3#' 132.76(19) N3#1-Ag3-N1 79.2(2) N1-Ag3-N6#4 152.4(2) Symmetry code: #1: -x+3/2, -y+3/2, z; #2: -x+1, -y+3/2, z+1/2; #3: x-1/2, y, z+1/2; #4:-x+7/4, y-1/4, z+1/4. 2. 6. Antibacterial Activity Test Four referenced bacterial strains, B. subtilis, E. coli, P. aeruginosa and S. aureus were selected. Streptomycin was used as a positive control. The IC50 (half minimum inhibitory concentrations) of the test compounds were determined by a colorimetric method using the dye MTT (3-(4,5-dimethylth-iazol-2-yl)-2,5-diphenyl tetrazolium-bromide). Stock solutions of the synthesized compounds (100 ^g/mL) were prepared in DMSO, and sequentially diluted with Mueller-Hinton medium. The antibacterial activities were evaluated by the method reported before.18 The procedure of antibacterial activity was given in detail in Supporting Information. 3. Results and Discussion 3. 1. Crystal Structure Analysis -> Figure 1. Molecular structure of L (50% probability displacement ellipsoids), front and side views are presented. Colorless crystals of L suitable for X-ray structure analysis were obtained via the slow diffusion of ethanol into a dicholmethane solution of L. L crystallized in the triclinic space group P1; there are two L and two CH2Cl2 Zhao et al.: Structural Diversity in Oxadiazole-Containing Silver Acta Chim. Slov. 2020, 67, 822-825 829 molecules in the asymmetric unit. As shown in Figure 1, L has an approximately planar structure except for the methyl H atoms stretching out of the plane. The dihedral angles between the central benzene ring and surrounding oxadiazole rings are 4.371(7)°, 6.136(2)°, and 7.257(2)°, respectively. Figure 2. The coordination environment of Ag+ in 1 at 50% probability displacement. (Solvent molecules are omitted for clarity) Symmetry codes: (i) -y+7/4, x+1/4, z+1/4; (ii) -x+3/2, -y+2, z+1/2; (iii) y-1/4, -x+7/4, z+3/4; (iv) -y+7/4, x+1/4, z-3/4. Crystallization of L with AgSO3CF3 in a CH2Cl2/ THF mixed-solvent system at room temperature produced complex 1 with a 55% yield. 1 crystallized in the tetragonal space group I41/a. Single crystal analysis revealed that two crystallographically independent Ag+ ions, one ligand L, one coordinated water molecule, one coordinated CF-3SO3-anion, one free CF3SO3-anion and one solvent CH2Cl2 molecule compose the asymmetric unit of 1. After complexation, L was no longer planar in complex 1, which was evident from the dihedral angles between the central benzene ring and surrounding oxadiazole rings being 11.004(7)°, 16.914(8)°, and 21.110(5)°, respectively. As illustrated in Figure 2, the Ag1 and Ag2 coordination environments are distinct from each other. The Ag1 center adopts a distorted tetrahedral coordination sphere, which consists of four Noxadiazole donors (N1, N2i, N3ii, and N4iii) from four separate ligands. Ag2 is also four-coordinated by two Noxadiazole atoms (N5 and N6iv) along with O atoms from a water molecule (O7) and CF3SO3-anion (O4). The Ag1-N bond lengths are in the range of 2.274(4)-2.316(4) A, which are longer than the bond lengths of Ag2-N (2.229(4)-2.282(4)) The Ag-N and Ag-O bond lengths are in agreement with those values in a previous report.14 In the extended structure, neighboring Ag1 centers are bridged by four Noxadiazole atoms into a {Ag2N4} dinu-clear core (Ag1—Ag1 distance of 3.8158(7) A), which is similar to that found in compounds generated from oxadi-azole-bridging double-armed ligands.14 Notably, the two adjacent {Ag2N4} dinuclear units are almost perpendicular to each other (dihedral angle of 89.923°) and are arranged Figure 4. 3D framework of 1 (anions and CH2Cl2 molecules are omitted for clarity), and Ag2 centers are highlighted as fuchsia polyhedrons. Figure 3. 1D chain structure consists of Ag1 in 1. Zhao et al.: Structural Diversity in Oxadiazole-Containing Silver ... 826 Acta Chim. Slov. 2020, 67, 822-829 alternately to form a 1D polymeric chain extending along the crystallographic c axis (Figure 3). The remaining oxadiazole groups on these {Ag2N4}n chains adopt a bridging mode, connecting four Ag2 centers to form a twelve-membered {Ag4N8} saddle-like ring (Figure S2). In the solid state, these {Ag4N8} clusters are located between the {Ag2N4}n chains, and further link the 1D chains into a 3D microporous supramo-lecular network with rectangular channels along the c axis (Figure 4). The CF3SO3- anions and solvent CH2Cl2 molecules are present in the channels. In summary, each L in compound 1 acts as a hexa-dentate ligand, binding to six Ag+ atoms, in which all the Noxadiazole donors are utilized to bind Ag+ ions into a 3D polymeric structure, whereas Smethylthio donors are not involved in coordination. Figure 5. The coordination environment of Ag+ in 2 at 50% probability displacement. (Anions and solvent molecules are omitted for clarity) Symmetry codes: (i) -x+3/2, -y+3/2, z; (ii) -x+1, -y+3/2, z+1/2; (iii) x-1/2, y, z+1/2; (iv) -x+7/4, y-1/4, z+1/4. To investigate the effect of the counter anions on the structural motif of the{Ag+-L} coordination system, the weakly coordinated SbF6- anion was used instead of the strongly coordinated CF3SO3- anion for the self-assembly reaction in the same CH2Cl2/THF solvent system; this produced compound 2 with a 40% yield. Single crystal analysis revealed that five crystallographically independent Ag+ ions, four ligands, two coordinated water molecules, five free SbF6-anions, and five tetrahydrofuran molecules constitute the asymmetric unit of 2. In complex 2, the dihedral angles between the central benzene ring and surrounding oxadiazole rings exhibit a large difference. For the central benzene ring involving C4-C9, the dihedral angles are 0.771(2) )°, 8.022(4) )°, and 23.962(6) )°, respectively. For the central benzene ring involving C19-C24, the dihedral angles are 4.827(6) )°, 18.136(8) )°, and 31.515(8) )°, respectively. There are three independent Ag+ centers in 2 (Figure 5). The first center, Ag1, is located in a distorted tetrahe-dral coordination sphere with four Noxadiazole donors (N2, N9, N2i, and N9i). The Ag1-N bond lengths are 2.257(5)-2.328(6) A. Ag2 is five-coordinated by three Noxadiazole donors (N7, N4ii, and N11ii), one S atom and one O atom from a water molecule. The Addison distortion index, t, (t = (^ - a)/60, where a and ^ are the two largest coordinated angles in the complex; perfect square pyramidal, t = 0; perfect trigonal bipyramidal, t = 1),19 for Ag2 is 0.72, indicating a distorted trigonal bipyramidal coordination sphere. The axial O7-Ag2-S1iii angle of 178.13(17)° is slightly smaller than the ideal value of 180°. The equatorial Ag2-N bond lengths are 2.325(6)-2.374(6) A, while the axial Ag2-O7 and Ag2-S1iii distances are 2.466(6) and 2.9549(19) A, respectively; thus, it is an elongated trigonal bipyramidal structure. Ag3 is also five-coordinated, with a Figure 6. View of 2D network in 2, front and side views are presented. Zhao et al.: Structural Diversity in Oxadiazole-Containing Silver Acta Chim. Slov. 2020, 67, 822-827 829 t of 0.33. Therefore, Ag3 is best described as distorted square pyramidal with N10 in axial positions and the other N donors (N1, N3i, N6iv, and N12i) forming the equatorial plane. The bond angles around the Ag+ center range from 79.2(2) to 152.4(2)° in the equatorial positions and from82.7(2) to 132.2(2)° for the apical positions. Ag3-N bond lengths are 2.325(7)-2.548(6) A, which is the longest among these Ag-N bonds. have been used as antimicrobials since the successful use of silver-containing creams for burn treatment.22 We have reported the antibacterial activities of silver complexes based on quinoline or hydrazone scaffolds.18,23,24 As a continuation of our efforts in exploring new antibacterial reagents to supplement structure-activity information, the in vitro antibacterial activities of the synthesized compounds were assessed. The IC50 (half minimum inhibitory concen- Figure 7. View of 3D network of 2 along a axis (anions and THF molecules are omitted for clarity). The {Ag2N4} dinuclear moieties were also found in compound 2. The shortest Ag2—Ag3 distance is 3.2013(8) A (while the Ag3—Ag1 separation is 3.713(1) A), indicating weak Ag-'-Ag interactions. In the solid state, four {Ag2N4} units are interlocked together into a butterfly-like penta-nuclear sub-building block (as shown in Figure S3). These sub-building blocks are strung together by the Ag2-N7 bond into a two-dimensional sheet extending in the crystallographic ac plane, as shown in Figure 6. These 2D layers are further connected by Ag3-N6 linkages into a three-dimensional network with triangular channels along the crystallographic a axis, in which uncoordinated SbF6-counteranions and THF guest molecules are located in place of CH2Cl2 (Figure 7). Principally, one of the L ligand in compound 2 acts as a hexa-dentate ligand, binding to six Ag+ via N1, N2, N3, N4, N6, and S1, while the other ligand acts as a penta-dentate ligand, binding to five Ag+ via N7, N9, N10, N11, and N12. 3. 2. Antibacterial Activity Among the diverse transition metals, silver has fundamental importance in bioinorganic chemistry, and the use of silver metal complexes for medicinal applications has been well documented.20,21 Silver-based complexes trations) values of the test compounds are presented in Table 4. Known antibiotic, such as streptomycin, was used as control drug. Table 4 Antimicrobial activity of the tested compounds. Compounds Half maximal inhibitory concentrations (^^/mL) Gram-negative Gram-positive E.coli P. aeruginosa B.subtilis S.aureus L >50 >50 >50 >50 1 8.62 9.35 13.41 10.41 2 9.43 7.56 15.23 6.74 AgSOaCFa 3.53 3.27 8.21 4.67 AgSbF6 2.03 0.19 7.02 3.87 Streptomycin 3.94 >50 4.12 5.24 As shown in Table 4, against all the tested bacteria, the free ligand L was inactive under the tested conditions. However, the introduction of Ag+ on the ligand resulted in improved antibacterial activity, especially against the tested gram-negative strains, with IC50 values of 7.56 to 9.43 ^g mL-1. For the tested gram-negative strains, the IC50 values ranged from6.74 to 15.23 ^g mL-1. It is evident that the Zhao et al.: Structural Diversity in Oxadiazole-Containing Silver ... 828 Acta Chim. Slov. 2020, 67, 822-829 activity of the two complexes is dependent primarily on the presence of the Ag+ ion. It has been found that the nature of ligands, chelate effect of ligands, nuclearity, and total charge are the main factors contributing to the biological activity of the Ag-compounds.25 Compared with our previously reported quinoline-silver(I) complexes and hy-drazone-silver(I) complexes, the bioactivities of 1 and 2 are slightly lower. This can be attributed to the lack of a chelate effect because the Noxadiazole donors of L in 1 and 2 are all in the bridging mode. 4. Conclusions In conclusion, we synthesized and characterized two novel 3D Ag(I) coordination polymers generated from the oxadiazole-containing tri-armed ligand L. Complexes 1 and 2 are both three-dimensional frameworks, but L features different coordination modes in them. L in complex 1 acts as a hexa-dentate ligand (binding to six Ag+ atoms via all the N donors), while in 2, one of the ligands acts as a hexa-dentate ligand (binding to six Ag+ atoms via five N donors and one S donor), and the other L acts as a pe-ta-dentate ligand (binding to five Ag+ via five N donors). The structural diversity reveals that inorganic counter anions play an important role in building up the coordination framework. In addition, complexes 1 and 2 exhibited potent antibacterial activity. 5. References 1. E. G. R. de Arruda, M. A. de Farias, S. A. V. Jannuzzi, S. D. A. Gonsales, R. A. Timm, S. Sharma, G. Zoppellaro, L. T. Kubo-ta, M. Knobel, A. L. B. Formiga, Inorg. Chim. Acta 2017, 466, 456-463. DOI:10.1016/j.ica.2017.06.073 2. A. Rühling, H. J. Galla, F. Glorius, Chem. Eur. J. 2015, 21, 12291-12294. DOI:10.1002/chem.201502542 3. J. Rodriguez, D. Bourissou, Angew. Chem. Int. Ed. 2018, 57, 386-388. DOI:10.1002/anie.201710105 4. T. P. Latendresse, V. Vieru, B. O. Wilkins, N. S. Bhuvanesh, L. F. Chibotaru, M. Nippe, Angew. Chem. Int. Ed. 2018, 57,81648169. DOI: 10.1002/anie.201804075 5. M. B. Coban, E. Gungor, H. Kara, U. Baisch, Y. Acar, J. Mol. Struct. 2018, 1154, 579-586. DOI: 10.1016/j.molstruc.2017.10.049 6. A. T. Baryshnikova, B. F. Minaev, G. V. Baryshnikov, H. Agren, Inorg. Chim. Acta 2019, 485, 73-79. DOI:10.1016/j.ica.2018.09.086 7. I. Ahmed, S. H. Jhung, Chem. Eng. J. 2017, 310, 197-215. DOI:10.1016/j.cej.2016.10.115 8. Y. Y. Jia, X. T. Liu, R. Feng, S. Y. Zhang, P. Zhang, Y. B. He, Y. H. Zha-ng, X. H. Bu, Inorg. Chem. Front 2016, 3, 1510-1515. DOI:10.1039/C6QI00191B 9. W. P. Lustig, S. Mukherjee, N. D. Rudd, A. V Desai, J. Li, S. K. Ghosh, Chem. Soc. Rev. 2017, 46, 3242-3285. DOI: 10.1039/C6CS00930A 10. M. Gon, K. Tanaka, Y. Chujo, Bull. Chem. Soc. Jpn. 2019, 92, 7-18. DOI:10.1246/bcsj.20180245 11. L. N. Wang, L. Fu, J. W. Zhu, Y. Xu, M. Zhang, Q. You, P. Wang, J. Qin, Acta Chim. Slov. 2017, 64, 202-207. DOI: 10.17344/acsi.2016.3109 12. C. W. Zhao, J. P. Ma, Q. K. Liu, X. R. Wang, Y. Liu, J. Yang, J. S. Yang, Y. B. Dong, Chem. Commun. 2016, 52, 5238-5241. DOI:10.1039/C6CC00189K 13. Y. B. Dong, Q. Zhang, L. L. Liu, J. P. Ma, B. Tang, R. Q. Huang, J. Am. Chem. Soc. 2007, 129, 1514-1515. DOI:10.1021/ja067384z 14. Y. B. Dong, H. X. Xu, J. P. Ma, R. Q. Huang, Inorg. Chem. 2006, 45, 3325-3343. DOI:10.1021/ic052158w 15. Bruker, SMART and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA, 2002. 16. G. M. Sheldrick, SADABS. Program for Empirical Ab-sorp-tion Correction of Area Detector, University of Göttingen, Germany, 1996. 17. G. M. Sheldrick, Acta Crystallogr. 2008, A64, 112-122. DOI:10.1107/S0108767307043930 18. L. Zhang, Z. W. Man, Y. Zhang, J. Hong, M. R. Guo, J. Qin, Acta Chim. Slov. 2016, 63, 891-898. DOI:10.17344/acsi.2016.2895 19. A. W. Addison, T. N. Rao. J. Chem. Soc., Dalton Trans. 1984, 1349-1356. DOI:10.1039/DT9840001349 20. K. Gholivand, F. Molaei, N. Oroujzadeh, R. Mobasseri, H. Na-deri-Manesh, Inorg. Chim. Acta 2014, 423, 107-116. DOI:10.1016/j.ica.2014.07.029 21. J. H. B. Nunes, R. E. F. de Paiva, A. Cuin, W. R. Lustri, P. P. Corbi, Polyhedron 2015, 85, 437-444. DOI:10.1016/j.poly.2014.09.010 22. S. Silver, L. T. Phung, G. Silver, J. Ind. Microbiol. Biotechnol., 2006, 33, 627-634. DOI:10.1007/s10295-006-0139-7 23. S. S. Zhao, P. Wang, L. N. Wang, L. Fu, X. M. Han, J. Qin, S. S. Qian, J. Coord. Chem. 2017, 70, 885-897. DOI: 10.1080/00958972.2017.1281915 24. Q. L. Ren, S. S. Zhao, L. X. Song, S. S. Qian, J. Qin, J. Coord. Chem. 2016, 69, 227-237. DOI: 10.1080/00958972.2015.1110240 25. M. Zampakou, S. Balala, F. Perdih, S. Kalogiannis, I. Turel, G. Psomas. RSC Adv. 2015, 5, 11861-11872. DOI: 10.1039/C4RA11682H Zhao et al.: Structural Diversity in Oxadiazole-Containing Silver Acta Chim. Slov. 2020, 67, 822-829 829 Povzetek Z reakcijo med triveznim ligandom z oksadiazolom, 1,3,5-tri(2-metiltio-1,3,4-oxadiazol-5il) benzen (L), in srebrovimi solmi v mediju CH2Cl2/THF smo sintetizirali dva koordinacijska polimera, [Ag2(L)(SO3CF3)(H2O)](SO3CF3)^CH2Cl2 (1) in [Ag5(L)4(H2O)2](SbF6)5^5THF (2). Spojini kristalizirata v tetragonalni prostorski skupini I4l/a oziroma ortoromb-ski prostorski skupini Fdd2. Rentgenska difrakcija na monokristalu je pokazala bistvene razlike med 3D polimernima strukturama obeh spojin, kar lahko pripišemo različnim protiionom. V spojini 1 je ligand L šestvezen in koordiniran na dve vrsti Ag+ ionov s katerimi tvori 3D polimerno strukturo. V spojini 2 je ligand L pet- in šestvezen in koordiniran na tri vrste Ag+ ionov s katerimi prav tako tvori 3D polimerno strukturo. Raziskovali smo tudi protibakterijsko učinkovitost novih koordinacijskih spojin. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Zhao et al.: Structural Diversity in Oxadiazole-Containing Silver ... DOI: 10.17344/acsi.2019.5790 Acta Chim. Slov. 2020, 67, 830-841 /^creative ^commons Scientific paper Prediction of Biological Activities, Structural Investigation and Theoretical Studies of meta-cyanobenzyl Substituted Benzimidazolium Salts Duygu Barut Ce^pc^* and Aydin Aktaç2,3 1 Dokuz Eyltil University, Faculty of Science, Department of Physics, Izmir, Turkey 2 inonu University, Vocational School of Health Service, 44280, Malatya, Turkey 3 Inonu University, Faculty of Science, Department of Chemistry 44280, Malatya, Turkey * Corresponding author: E-mail: duygu.barut@deu.edu.tr Received: 12-18-2019 Abstract The structural properties of meta-cyanobenzyl substituted N-heterocyclic carbene (NHC) precursors were investigated theoretically. The molecular and crystal structure of one of the compounds was determined by using the single-crystal X-ray diffraction method. Global reactivity descriptors were analyzed to understand the biological activity behaviors of the compounds with Density Functional Theory (DFT) B3LYP method with 6-31G* basis set. Vibrational frequencies, chemical shifts and absorption wavelengths were computed and compared to experimental data. A predictive study for the biological activities was done using PASS (prediction of activity spectra for biologically active structures) online software. Biological activity predictions showed the analgesic, substance P antagonist, non-opoid and antiinflammatory activities of the compounds. Keywords: N-heterocyclic precursors; crystal structure; DFT; PASS online 1. Introduction N-heterocyclic carbenes (NHC) are cyclic carbenes containing at least one amino substituent.1 NHCs were first pioneered by Öfele, Wanzlick, and Schönherr in 1968 and the isolation of the first stable crystalline carbene was performed by Arduengo in 1991.2-4 After the stability of the NHC ligands was registered, they have been attracted great interest in the field of organic and organometallic chemistry. Especially, in medical applications, there have been various studies of metal-NHCs.5-7 The chelating effect of NHC precursors with unique sigma donor properties could be effective in biological activity.8,9 In our previous study, the investigations on the biological activity of NHC compounds containing cyanobenzyl substituents show that these compounds have exhibited biological activity.10 Biological experiments are often limited in terms of sample, time and cost. In this context, DFT-based reactivity descriptors are advantageous and generally be consistent with the experimental observations.11 In recent years, the prediction of the reactivity of chemical systems is one of the main purposes of theoretical chemistry. Density functional theory (DFT) has been quite successful in providing the theoretical groundwork of this purpose. For analyzing and understanding the biological reactivity of the chemical systems, several reactivity descriptors have been proposed. In this work, biological reactivity studies of three compounds 2b, 2f and 2g were carried out through these global reactivity descriptors. Geometries of the compounds were optimized and bonding parameters were compared to the experimental data. Frontier molecule orbitals (HOMO and LUMO) and the energy values were computed. Natural bond orbital (NBO) analysis was used to analyze the stability of the molecules arising from hy-perconjugative interactions and charge delocalization. The vibrational frequencies, chemical shifts and absorption wavelengths were calculated and compared to the experimental ones. Also, PASS (prediction of activity spectra for biologically active structures) online software was used to predict the putative biological activity spectra of the compounds. DFT studies and PASS online predictions point out the similar activity results for the compounds. Celepci and Aktaç: Prediction of Biological Activities, Structural Acta Chim. Slov. 2020, 67, 830-841 831 It is believed that this kind of study will contribute to getting a better understanding of the chemical behavior of meta-cyanobenzyl substituted benzimidazolium salts. As proved by enzyme inhibition studies,10 these compounds can be a candidate as new drugs for therapy of some diseases such as glaucoma, epilepsy, gastric and duodenal ulcers, osteoporosis, mountain sickness, or neurological disturbances. 2. Experimental The synthesis, some spectroscopic results and enzyme inhibition studies of the compounds 2b, 2f, 2g and single-crystal X-ray diffraction studies of the 2f and 2g were reported previously.10 In this work, firstly, we determined the crystal structure of 2b by single-crystal X-ray diffraction method. Then, the theoretical studies were performed for all structures. 2. 1. X-ray Crystallography The single-crystal X-ray diffraction study of the compound 2b was performed by «-scan technique, using a Rigaku-Oxford Xcalibur diffractometer with an EOSCCD area detector operated at 50 kV and 40 mA using graphite-monochromated MoKa radiation (X = 0.71073 A) from an enhance X-ray source with CrysAlisPro software.12 Data reduction and analytical absorption corrections were carried out by CrysAlisPro program.13 The structure was solved by the Intrinsic Phasing method with SHELXT and refined utilizing the SHELXL program14,15 incorporated in the OLEX2 program package.16 The crys-tallographic data and some parameters of refinement are placed in Table 1. Anisotropic thermal parameters were applied to all non-hydrogen atoms. All the hydrogen atoms were placed using standard geometric models and with their thermal parameters riding on those of their parent atoms. 2. 2. Computational Details The compounds 2b, 2f and 2g were optimized, chemical shifts and frontier molecular orbital energies were carried out by using DFT/B3LYP with the basis set 6-31G* by Gaussian 09W and GaussView 6.0 molecular visualization programs.17,18 Natural bond orbital (NBO) analysis was performed using NBO 3.1 program as implemented in the Gaussian 09 package at the same level of the theory.19 The normal mode assignments of the compounds were employed by VEDA20 program and verified by GaussView 6.0. The NMR chemical shifts were computed in the gaseous state within GIAO (Gauge-Independent Atomic Orbital) approach by subtracting the shielding constants of TMS.21 The biological activity spectra of studied compounds 2b, 2f and 2g were obtained by the PASS Online Program (http:// www.way2drug.com/PASSOnline/). 3. Results & Discussions 3. 1. Crystal Structure of Compound 2b Single-crystal X-ray diffraction analyses reveal that the compound 2b crystallizes in the orthorhombic space Table 1. Crystal data and experimental details for the compound 2b. Empirical Formula C23H20BrN3 Formula Weight 418.33 Temperature (K) 293(2) Crystal System, space group Orthorhombic, Pca2l a, b, c (À) 14.6406(7), 7.9998(4), 17.0884(9) «, fi, Y (°) 90, 90, 90 y (À3) 2001.43(17) Z 4 Density (calculated) (g/cm3) 1.388 Absorbtion coefficient (|i, mm-1) 2.066 F(000) 856 Crystal size (mm3) 0.360 x 0.259 x 0.253 Radiation MoKa (X = 0.71073) 29 range for data collection (°) 6.054 to 51.364 Index ranges -10< h <17, -9< k <9, -20< l <20 Reflections collected 8295 Independent reflections 3441 [Rint = 0.0322, Rsigma = 0.0441] Restraints/Parameters 4/245 Goodness-of-fit on F2 1.015 Final R indices [I>2a(I)] R[ = 0.0335, WR2 = 0.0599 R indices R[ = 0.0529, wR2 = 0.0661 Largest diff. peak/hole (eÀ-3) 0.20/-0.20 Celepci and Akta§: Prediction of Biological Activities, Structural ... 832 Acta Chim. Slov. 2020, 67, 830-841 group Pca21. The asymmetric unit of the compound contains a meta-cyanobenzyl-substituted benzimidazolium cation and a bromide anion (Fig. 1). The single-crystal X-ray diffraction studies reveal that the benzimidazolium ring system is almost coplanar with the r.m.s. deviation of 0.007(6) A. Cyanobenzyl and methylbenzyl fragments lie to the different sides of the benzimidazole ring system, giving the cation a Z-shape. The dihedral angles between the benzimidazolium ring and the mean plane of these fragments are 67.83(9)° and 69.27(2)°, respectively. Two C-H—Br type intermolecular interactions are observed in the crystal structure; one is between the most acidic proton of the imidazolium cation and the bromide anion [C1-H1—Br1, H1-Br1 = 2.61 A, C1-Br1 = 3.545(5) A, C1-H1—Br1=155°], the other interaction is C16-H16B-Br1i [H16B-Br1i = 2.85 A, C16-Br1i = 3.813(6) A, C1-H1-Br1i = 170°]. Fig.2 displays the infinite chain occurs via these hydrogen bonds along the b-axis. The molecules stacked in the crystal structure to form a pincers-like packing motif, as shown in Fig. 3. Fig. 1. Structure of 2b with ellipsoids plotted at 30% probability. Selected bond parameters (A,°): N1-C1 1.327(6), N1-C2 1.389(5), N1-C8 1.472(5), N2-C1 1.326(5), N2-C7 1.389(6), N2-C16 1.459(6), N3-C15 1.138(7); N1-C1-N2 111.0(4), C1-N1-C8 125.6(4), N1-C8-C9 113.2(4), C2-N1-C8 126.3(4), C1-N2-C16 125.4(4), C7-N2-C16 127.0(4), N2-C16-C17 113.0(4), C11-C15-N3 177.2(8). Fig. 2. Packing of the cation molecules of 2b through the intermolecular hydrogen bonds bridged by the bromide anions, which lead to the infinite chain along the b axes. All hydrogen atoms except those participating in the hydrogen bonds were omitted for clarity. Cation molecules are shown in the stick drawing style. Celepci and Akta§: Prediction of Biological Activities, Structural ... Acta Chim. Slov. 2020, 67, 830-841 833 Fig. 3. Graphical representation of pincers-like packing motif the molecules within the unit cell for 2b. 3. 2. Geometry Optimization, Frontier Molecular Orbitals and Global Reactivity Descriptors The optimized ground state geometry of the compounds at DFT/B3LYP/6-31G* level of the theory is shown in Fig. 4. The correlations between the theoretical and experimental bonding parameters were displayed in Fig. S1 (see supplementary information file). It is clearly understood from the figure that there are some discrepancies between the experimental and computed bond parameters. While the theoretical calculations of the isolated structure were carried out in the gas phase, the fact that the experimental molecular structures were in a solid-state form likely caused these differences. Also, the experimental structures have intermolecular interactions, which may cause discrepancies in the bonding parameters. Fig. 4. Optimized geometries of the compounds 2b, 2f and 2g, respectively. Celepci and Akta§: Prediction of Biological Activities, Structural ... 834 Acta Chim. Slov. 2020, 67, 830-841 The FMOs theory involving HOMO and LUMO is one of the best theories to get an insight into the chemical stability of a molecule.22 The highest occupied molecular orbital (HOMO) represents the distribution and energy of the least tightly held electrons in the molecule and the lowest unoccupied molecular orbital (LUMO) describes the easiest route to the addition of more electrons to the system. The high value of EHOMO indicates the ease of donating an electron to the unoccupied orbital of the receptor molecule, and the small value of ELUMO means that it has small resistance to accept electrons so it will be more able to accept electrons. The difference between HOMO and LUMO energy values gives the HOMO-LUMO energy gap (Egap) and it is an important stability index.23 A molecule with large Egap is described as a hard molecule, much less polarizable, and implies high molecular stability and aromaticity low reactivity in chemical reactions.24,25 The soft systems have small Egap, they are highly polarizable and exhibit a significant degree of intramolecular charge transfer from the electron donor to the electron acceptor and conjugation that may influence the biological activity of the molecule.26 To evaluate the energetic behavior of the compounds, the HOMO-LUMO analysis was carried out by using B3LYP/6-31G* method, and the plots are depicted in Table 2. As can be seen, compounds show different localization of the HOMOs and LUMOs. The HOMO of the 2b and 2g is located on the bromide anions, while in 2f, it is distributed to the bromide anion and the benzimidazolium fragment of the cation molecule. Similarly, LUMO electron density of 2f is spread over the cyanobenzyl moiety, but for 2b and 2g, the LUMO electrons are mainly located on the benzimidazolium ring. The values of energy separation between the HOMO-LUMO were found as 2.930, 3.845 and 3.011 eV, respectively. The global reactivity descriptors calculated using the DFT method play an essential and reliable role to understand the biological activities in many studies. Some of these descriptors are; the global hardness (n), which measures the resistance to change in electron density; chemical potential (^), measures the escaping tendency of an electron; electronegativity (x), describes the ability of a molecule to attract electrons towards itself; electrophilicity index (w), measures the susceptibility of chemical species to accept electrons; softness (S), is the inverse of hardness; the maximum charge transfer (ANmax), describes the propensity of the system to acquire additional electronic charge from the environment.27,28 These global reactivity parameters can be defined as: (1) (2) where I is the ionization potential (I = -EHOMO) and A is the electron affinity (A = -ELUMO). The calculated values of reactivity descriptors of the compounds are listed in Table 3. The hardness (q) values of the compounds are following the order 2f > 2g > 2b. It is expected that the high value of softness (S), defined as the inverse of hardness, is the representative of high reactivity. According to these parameters, 2b seems the most reactive compound. Also, the Egap of 2b is the smallest one, this indicates that 2b is softer than other compounds, its electronic chemical potential and electrophilicity index (w) values are the greatest and the maximum charge transfer capability (ANmax) is the highest. The dipole moments of the compounds are 15.136 Debye for 2b, 7.264 Debye for 2f and 13.600 Debye for 2g, proved that the most stable compound is 2f, while the 2b is the most reactive. 3. 3. Mulliken Population Analysis, Natural Population Analysis (NPA) and Molecular Electrostatic Potential (MEP) Mulliken atomic charges and natural population analysis (NPA) play an important role in quantum chemistry. The atomic charge distribution of acceptor and donor atoms in molecules is directly affected by parameters such as polarizability, refractivity, dipole moment, and other electronic structural parameters.29 Mulliken population charge analysis and natural population analysis of structures were performed using B3LYP/6-31G* level of calculation and a list of all calculated atomic charges are given in Table S1 (see supplementary file). The analyses reveal the presence of electrophilic and nucleophilic atomic charges. According to the results, the bromide anions of the compounds display high nucleophilic behavior with their negative donor atomic charges, while the 2-C-H protons have the highest positive charge value. So, bromide anions attack the hydrogen atom of 2-C carbons, which is the most reactive site of the molecules. The molecular electrostatic potential (MEP) is related to the electron density and is a useful descriptor in understanding the reactive behavior in both electrophilic and nu-cleophilic reactions and hydrogen bonding reactions.30 In the MEP profile, the areas with major positive potential are specified by blue color, which demonstrates the strongest attraction, whereas the maximum negative potential sections have been presented red color, indicates the repulsion. Comparing with the X-ray data it was concluded that the MEP plots proved the intermolecular hydrogen bonds between the bromide anion and cation molecules for all structures (Table 2). The negative potential regions are over the electronegative bromide anions, which are responsible for the intermolecular C-H—Br hydrogen bonds. Also, the N=C of cyanobenzyl groups of all structures have red colors which means these regions are the electron-rich nucleop-hilic regions. The net charges of the nitrogen atoms of cya-nobenzyl groups confirmed the MEP output. Celepci and Akta§: Prediction of Biological Activities, Structural ... Acta Chim. Slov. 2020, 67, 830-841 835 Table 2. The molecular orbitals for HOMO-LUMO and MEP diagrams of the compounds. 2b 2f 2g Celepci and Akta§: Prediction of Biological Activities, Structural ... 836 Acta Chim. Slov. 2020, 67, 830-841 Table 3. Global descriptors of chemical reactivity of meia-cyanobenzyl substituted benzimidazolium salts. (eV) 2b 2f 2g EHOMO (-I) -4.799 -5.335 -4.861 elumo (-A) -1.869 -1.490 -1.850 Egap 2.930 3.845 3.011 Electronegativity x 3.334 3.413 3.356 Chemical hardness q 1.465 1.923 1.506 Electronic chemical potential p -3.334 -3.413 -3.356 Electrophilicity index a> 3.794 3.029 3.739 Softness S 0.341 0.260 0.332 Maximum charge transfer capability ANmax 2.276 1.775 2.228 3. 4. Natural Bond Orbital (NBO) Analysis The natural bond orbital analysis (NBO) is an effective method for predicting the stereoelectronic interactions on the reactivity and dynamic behaviors of chemical compounds.31 It provides a convenient basis for investigating charge transfer or conjugative interaction in molecular systems. The interactions depend on the energy difference between interacting orbitals, and the strong interactions arise between predominant donor and acceptor. The second-order perturbation analysis of the Fock matrix is used to calculate stabilization energy for each donor (i) and acceptor (j) within i^j delocalization. The estimated energy Ff can be determined as E(2) = AFj.- = q;——; where qi is donor orbital occupancy, Et and Ej are diagonal elements and Fj is the off-diagonal NBO Fock matrix element. The larger the E(2) value means the more intensive interaction between electron donors and acceptors.32 The natural bond orbitals' (NBO) calculations for the structures were performed at the DFT/B3LYP/6-31G* method. The stabilization energies of the most important interactions between donor and acceptor along with occupancy are given in Table 4. According to the table, the strongest interactions (n^-n*) occur in the NHC ligand for all structures. In the 2b and 2g, the electron donation from a lone-pair orbital on the carbon atom of benzimidazole, LP(1) C16 and LP(1) C6, to the antibonding acceptors n* N3-C7 (233.49 kJ/mol), n* C12-C14 (58.10 kJ/mol) and n* N3-C4 (219.31 kJ/mol), n* C17-C46 (58.79) orbitals have also high stabilization energies. In the compound 2f, from the lone-pair orbital of LP(1) N3 to the n* N2-C4 has the energy of 82.11 kJ/mol. 3. 5. UV-Vis Analysis of the Compounds 2b, 2fand 2g The UV-Vis spectra of the meta-cyanobenzyl substituted benzimidazolium salts 2b, 2f and 2g were recorded Table 4. The Second Order Perturbation Theory Analysis Results of the Fock Matrix in NBO Basis for 2b, 2f and 2g at B3LYP/6-31G* level of the theory. Donor(i) Acceptor(j) EDi(e) EDj(e) E(2) kJ/mol Ej-Ei (a.u) Fij (a.u) 2b n* N3-C7 n* N2-C5 0.8166 0.4510 245.94 0.01 0.063 LP(1) C16 n* N3-C7 1.0440 0.8166 233.49 0.08 0.123 LP(1) C16 n* C12-C14 1.0440 0.3062 58.10 0.16 0.104 2f n* C6-C9 n* C25-C36 0.4719 0.3343 217.58 0.02 0.082 n* C10-C11 n* C20-C47 0.3349 0.2821 190.65 0.01 0.081 n* C6-C9 n* C17-C23 0.4719 0.3274 188.55 0.02 0.082 n* C19-C45 n* C20-C47 0.3848 0.2821 179.27 0.02 0.082 LP(1) N3 n* N2-C4 1.5551 0.5650 82.11 0.21 0.122 2g n* N3-C4 n* N2-C9 0.8011 0.4644 321.23 0.01 0.064 n* C12-C26 n* C8-C33 0.3536 0.3265 261.24 0.01 0.081 LP(1) C6 n* N3-C4 1.0410 0.8011 219.31 0.08 0.122 n* C11-C19 n* C8-C33 0.3938 0.3265 177.46 0.02 0.084 LP(1) C6 n* C17-C46 1.0410 0.3086 58.79 0.16 0.104 Celepci and Akta§: Prediction of Biological Activities, Structural ... Acta Chim. Slov. 2020, 67, 830-841 837 with Shimadzu UV-1601 instrument in ethyl alcohol solutions at a concentration of 15 or 20 ^M at 25 °C and they were depicted with a range of 200-400 nm. The calculations of the title molecules were performed in ethyl alcohol solvent by using TD-DFT/B3LYP/6-31G* method. Table 5 shows the experimental and calculated UV-Vis spectroscopic parameters (absorption wavelengths (À), excitation energies and oscillator strengths f). Also, Fig. S2 displays the theoretical UV-Vis spectra of the compounds. The UV-Vis spectra of meta-cyanobenzyl substituted benzim-idazolium salts 2b, 2f and 2g display four absorption peaks at 220, 230, 250 and 270 nm (Fig. 5). As can be seen in 250 300 350 Wavelength / nm Fig. 5. UV-Vis spectra of the 2b, 2f and 2g Table 5, the calculated absorption peaks are found to be in close agreement with the experimental ones. The absorption bands between 202 and 288 nm in ethyl alcohol are practically identical and can be attributed to n-n* transitions in the benzene and benzimidazole ring or azome-thine (C=N) groups.33,34 The UV absorbance at an observed excitation wavelength (at 230 and 270 nm) indicates the lowest value for 2b. These data show that 2b possesses the lowest molar absorptivity and hence produces the highest quantum yield when compared with 2f and 2g. 3. 6. Vibrational Analysis The experimental and calculated FT-IR spectra of the compounds are illustrated in Fig. 6. The unscaled theoretical frequencies using the B3LYP level of theory with 6-31G* basis set along with their IR intensities, probable assignments and potential energy distribution (PED) performed employing VEDA program for all structures are presented in Table S2. As seen in Fig. 6, the experimental fundamentals are nearly consistent with the calculated ones. The probable discrepancies can arise as a result of anharmonic and finite temperature effects.35 As it is expected that C-H stretching modes belonging to the aromatic ring of the NHC salts were observed and calculated above 3000 cm-1.36 The 2-C-H stretching modes occur at 2960 cm-1 for 2b and 2f and 2956 cm-1 for 2g. The calculated assignments are 2772, 3256 and 3025 cm-1, respectively. The benzonitrile N-C stretching vibrations have the Table 5. The experimental and calculated UV-Vis parameters of the compounds 2b, 2f and 2g. 2b Experimental The calculated with B3LYP/6-311G(d,p) level in ethyl alcohol X (nm) X (nm) Excitation energy (eV) f (oscillator strength) 220.00 199.98 6.1999 0.0001 230.00 200.45 6.1853 0.0001 250.00 223.58 5.5454 0.0028 270.00 245.19 5.0566 0.1320 2f Experimental The calculated with B3LYP/6-311G(d,p) level in gas phase X (nm) X (nm) Excitation energy (eV) f (oscillator strength) 220.00 201.77 6.1450 0.0208 230.00 203.81 6.0836 0.1032 250.00 221.07 5.6087 0.0046 270.00 232.95 5.3223 0.0015 2g Experimental The calculated with B3LYP/6-311G(d,p) level in gas phase X (nm) X (nm) Excitation energy (eV) f (oscillator strength) 220.00 200.45 6.1853 0.0001 230.00 201.06 6.1667 0.0000 250.00 234.01 5.2983 0.0062 270.00 245.19 5.0566 0.1320 Celepci and Akta§: Prediction of Biological Activities, Structural ... 838 Acta Chim. Slov. 2020, 67, 830-841 PED contributions of 89% for all structures; the modes assigned at 2227, 2228 and 2229 cm-1, while they were calculated at 2352, 2348 and 2351 cm-1, respectively. 3. 7. Nuclear Magnetic Resonance (NMR) Studies The compounds were characterized by 1H, 13C NMR spectroscopy (Figs. S3-8). The theoretical GIAO 1H and 13C chemical shift calculations (with respect to TMS in ppm) were carried out using the DFT/B3LYP method with 6-31G* basis set and compared with experimental chemical shift values (Tables S3, S4). The chemical shifts are converted to the TMS scale by subtracting the calculated absolute chemical shielding of TMS (5 = Z0-Z) where 5 is the chemical shift, Z is the absolute shielding and Z0 is the absolute shielding of TMS, whose values are 32.18 and 189.73 ppm for B3LYP/6-31G*, respectively. There are some deviations of the chemical shift values, which may be due to the chemical environment of the C and H atoms in the molecules. According to the tables, the signals of the 2-C carbons appeared at 143.55, 142.2 and 134.4 ppm, while they were calculated at 139.27, 127.10 and 124.52 ppm, respec- tively. Hydrogen bonding has been recognized as the main interaction between the cations and anions of azolium salts leading to close arrangement between the counter anions and the most acidic 2-C-H proton.37-40 A strong hydrogen bond acceptor is expected to polarize the 2-C-H bond and slightly increase the acidity of the salt. As anticipated, bromide anions with four lone pairs of electrons are the hydrogen bonding acceptors that lead to the most downfield shifts of the 1H NMR for 2-C-H signal. These hydrogen bonding interactions were also determined by single-crystal X-ray diffraction studies.10 The most acidic protons (2-C-H) were observed at 10.13, 9.42 and 9.34 ppm, and calculated at 16.14, 9.81 and 12.79 ppm, respectively. The data also showed that the methylene carbon atoms had the least chemical shift values. 3. 8. Computer-Aided Prediction of Biological Activities of the Meta-cyanobenzyl-substituted Benzimidazolium Salts The PASS (Prediction of Activity Spectra for Substances) computer program is an estimation tool, which allows predicting the probable profile of biological activity of a Celepci and Akta§: Prediction of Biological Activities, Structural ... Acta Chim. Slov. 2020, 67, 830-841 839 Table 6. Biological activity assessment using PASS online software. Activity 2b 2f Pa Pi Pa Pi 2g Pa Pi Analgesic Substance P antagonist Analgesic, non-opioid Antiinflammatory CYP2H substrate CYP2C19 inducer Acetylcholine neuromuscular blocking agent 0.885 0.880 0.843 0.769 0.653 0.557 0.557 0.004 0.002 0.004 0.009 0.049 0.008 0.041 0.854 0.859 0.805 0.787 0.633 0.546 0.529 0.005 0.002 0.005 0.008 0.055 0.008 0.057 0.874 0.880 0.827 0.751 0.653 0.557 0.557 0.004 0.002 0.005 0.010 0.049 0.008 0.041 drug-like organic compound based on its structural formula. The average accuracy of prediction is about 95% according to leave-one-out-cross validation (LOOCV) estimation.41,42 The biological activity spectra of the meta-cyanoben-zyl substituted benzimidazolium salts were theoretically obtained by the PASS Online program and the analysis results were enlisted in Table 6. According to the data, all compounds are very likely to be analgesic, Substance P antagonist, non-opoid analgesic and anti-inflammatory with corresponding Pa values, which are higher than 0.7. Compounds also exhibit activity to the CYP2H substrate and CYP2C19 inducer. In our previous study, potential AChE inhibition properties of the compounds were investigated.10 PASS online studies proved that these compounds can be used as acetylcholine neuromuscular blocking agents, as well. 4. Conclusion As a result, this study contains theoretical aspects of three meta-cyanobenzyl substituted NHC precursors. The biological reactivity of the compounds was predicted by global reactivity descriptors and using PASS (prediction of activity spectra for biologically active structures) online. DFT and PASS online studies pointed out the activation of the compounds. The compound 2b was found to be the most reactive structure by both computational methods. These results were found to be compatible with the experimental biological activity studies. The molecular and crystal structure of 2b was also determined by using the single-crystal X-ray diffraction method. Natural bond orbital (NBO) analysis was used to analyze the stability of the molecules arising from hyperconjugative interactions and charge delocalization. The vibrational frequencies, chemical shifts and absorption wavelengths were calculated and compared to the experimental ones. Biological activity predictions showed that all structures have a high analgesic, substance P antagonist and non-opoid analgesic activities. Supplementary Crystallographic data as .cif files for the structure reported in this paper have been deposited at the Cambridge Crystallographic Data Center with CCDC 1971777 for 2b. Copies of the data can be obtained free of charge at http:// www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Center, 12, Union Road, Cambridge CB2 1EZ, UK. Fax: (+44) 1223-336-033, email: deposit@ccdc.cam.ac.uk. Acknowledgments The authors acknowledge Inonu University Scientific and Technology Center for the spectroscopic data and Do-kuz Eylul University for the use of the Oxford Rigaku Xcal-ibur Eos Diffractometer (purchased under University Research Grant No: 2010.KB.FEN.13). The authors also thank Assoc. Prof. Dr. Muhittin Aygun for the use of the Gaussian 09W/Gauss View package program. 5. References 1. S. Diez-Gonzalez, N. Marion, S. P. Nolan, Chem. Rev. 2009, 109, 3612-3676. DOI:10.1021/cr900074m 2. H. W. Wanzlick, H. J. Schönherr, Angew. Chem. Int. Ed. Engl. 1968, 7, 141-142. DOI:10.1002/anie.196801412 3. K. Öfele, J. Organomet. Chem. 1968, 12, 42-43. DOI:10.1016/S0022-328X(00)88691-X 4. A. J. III Arduengo, R. L. Harlow, M. Kline, J. Am. 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Deskriptorje globalne reaktivnosti smo analizirali z metodo gostotnega funkcionala (DFT) B3LYP z baznim setom 6-31G *, da bi razumeli biološko aktivnost spojin. Izračunali smo vibracijske frekvence, kemične premike in absorpcijske valovne dolžine in jih primerjali z eksperimentalnimi podatki. Napovedovalna študija biološke aktivnosti je bila narejena s pomočjo spletne programske opreme PASS (= prediction of activity spectra for biologically active structures) in je pokazala, da imajo spojine analgetično, antagonistično (na snov P), neopioidno in protivnetno aktivnost. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Celepci and Akta§: Prediction of Biological Activities, Structural ... 842 DOI: I0.i7344/acsi.2020.58i4 Acta Chim. Slov. 2020, 67, 842-852 ©comrnons Scientific paper Effects of Amino Acids on the Crystallization of Calcium Tartrate Tetrahydrate Sevgi Polat, Elif Aytan-Goze and Perviz Sayan* Department of Chemical Engineering, Faculty of Engineering, Marmara University, 34722, Istanbul, Turkey. * Corresponding author: E-mail: perviz.sayan@marmara.edu.tr, Phone: +90-2167773703, Fax: +90-2167773501 Received: 01-05-2020 Abstract This work assesses the effects of various amino acids, including serine, alanine, methionine, and proline, on calcium tartrate tetrahydrate (CTT) crystals. The crystallization experiments were performed in batch mode at 25 °C, pH 9 with three amino acid concentrations. The CTT crystals were characterized by XRD, FTIR, SEM, particle size and zeta potential analysis. All of the amino acids used in this study were found to significantly affect the surface electrical charge, size, and morphology of the obtained crystals. In addition, the thermal decomposition of the produced crystals obtained in pure media was examined and the obtained data were used to investigate the decomposition kinetics of the crystals with the help of three different model-free kinetic methods, namely Flynn-Wall-Ozawa (FWO), Kissinger-Akahira-Sunose (KAS), and Starink. The average activation energy of the crystals for the first, second, third, and fourth stages using the FWO, KAS, and Starink methods was calculated to be 91.0, 158.0, 249.1, and 224.8 kJ/mol; 89.6, 155.9, 250.7, and 221.1 kJ/mol; 88.6, 156.8, 250.5, and 220.4 kJ/mol, respectively. Thus, the results of this work are useful for selecting CTT morphology modifiers and explaining the decomposition kinetics of CTT crystals. Keywords: Calcium tartrate; crystallization; amino acid; kinetics, model-free 1. Introduction The physical properties of tartrate crystals hold significant research interest. Ferroelectric and piezoelectric tartrate crystals have been reported, and tartrate crystals are often used for controlling laser emissions.1,2 Among tartrate crystals, calcium tartrate has drawn the attention of many researchers because of its various applications in science and technology as well as in the field of pharmaceutical science, in addition to industrial uses.3 Besides these practical applications, calcium tartrate tetrahydrate (CTT) crystals are also reported as novel kidney stones in animals.4 CTT crystals are orthorhombic with a tetra molecular unit cell of dimensions a = 9.24, b = 10.63, and c = 9.66 Â with space group P212121. There are two asymmetric carbons in calcium tartrate, so it forms three isomers: two chiral and one non-chiral (meso-form). The chiral levoro-tatory (-) isomer is the most common form in nature. CTT crystals have been shown to have ferroelectric and non-linear optical properties.5 In recent years, researchers in the field of solid-state science have shown significant interest in the growth and characterization of CTT crystals, either pure crystallization media or with added dopants, such as barium, strontium, cobalt, nickel, manganese, zinc, and cadmium.6-9 To date, most studies on pure CTT crystals have aimed at understanding the basic principles and the nature of the crystal growth phenomenon.2 In the present study, we investigated the crystallization of CTT in the presence of the amino acid additives serine, alanine, methionine, and proline. These additives were selected because so far only limited studies have been reported on CTT crystallization in the presence of various amino acids; our work should clarify their roles as crystallization-modifying additives. Thus, in this work, we assessed the crystalline structure, morphology, and particle size of crystals prepared using solutions with and without an amino acid additive. In addition, we hope to address the lack of literature on the thermal decomposition kinetics of CTT crystals using the model-free kinetic models of Flynn-Wall-Ozawa (FWO), Kissinger-Akahira-Sunose (KAS), and Starink models. Therefore, the thermal decomposition behavior of the prepared crystals was investigated in detail and used to explain the kinetic characteristics and calculate the activation energies for the decomposition of CTT crystals. Polat et al.: Effects of Amino Acids on the Crystallization Acta Chim. Slov. 2020, 67, 842-852 843 2. Experimental 2. 1. Materials Calcium chloride dihydrate (CCD, CaCl22H2O) and tartaric acid (TA, C4H6O6) of analytical reagent grade were supplied by Merck, Darmstadt, Germany and used for the precipitation of CTT crystals. The amino acid additives alanine (C3H7NO2), methionine (C5HnNO2S), serine (C3H7NO3), and proline (C5H9NO2) of analytical reagent grade were also provided by Merck. Distilled water was used for the experiments. 2. 2. Preparation of Calcium Tartrate Tetrahydrate Crystals The CTT crystallization was carried out in batch mode at 25 °C in a double-jacketed crystallizer with an active volume of 500 ml. CCD and TA were used as the reac-tants for CTT precipitation. The experimental setup is illustrated in Figure 1. suspension in the crystallizer was stirred at a rate of 300 rpm. During the crystallization process, the pH of the solution was continuously monitored and maintained at pH 9 by adding NaOH solution using an automatic pH control system. The influence of different types and concentrations of amino acid on the crystallization of CTT was investigated in this study. At the beginning of the experiments, alanine, methionine, serine, and proline were added to the TA solution to provide the desired concentrations of 50, 100, and 200 ppm in the crystallizer. After all of the reactants had been added to the crystallizer, the suspension was left to stir for half an hour. At the end of this time, the crystallizer contents were filtered and washed thoroughly with distilled water. The washing repeated was continued until no chloride ions remained. The presence of chloride ions was checked using 0.1 M silver nitrate solution. The washed CTT crystals were then dried at room temperature and subjected to various characterization processes. Figure 1. Experimental setup. Firstly, 0.25 M CCD and 0.25 M TA solutions were prepared. To start the crystallization, 200 ml of TA solution was placed into the crystallizer, followed by the addition of 0.1 M NaOH solution to adjust the pH to 9. The solution was maintained at a constant temperature of 25 ± 0.5 °C using a thermostat. After thermal equilibrium was reached, 200 ml of CCD solution was fed into the crystal-lizer via an infusion pump at a flow rate of 1 ml/min. The 2. 3. Characterization of the Calcium Tetrahydrate Crystals The structures of the prepared crystal samples were determined using a Bruker D2 Phaser benchtop X-ray diffractometer (30 kV, 10 mA) with CuKa radiation (X = 1.5418 Â) in the 20 range of 10-60°. Fourier transform infrared spectrometry (FTIR; Shimadzu) was used to determine the functional groups of the crystals and to clarify their structures. The spectra were record- Polat et al.: Effects of Amino Acids on the Crystallization ... 844 Acta Chim. Slov. 2020, 67, 842-852 ed at room temperature in the wavenumber range of 600-4000 cm-1. The morphology of the CTT crystals was observed using a scanning electronic microscope (SEM, Zeiss EVO LS 10). The length and width of the samples were determined using Data Translation Image-Pro Plus image analysis software; at least 50 particles were counted to calculate the average aspect ratio of CTT. Zeta potential analysis was performed using a Malvern Zetasizer Nano ZS instrument to determine the surface charge of the CTT crystals obtained in pure and additive media. Measurements were repeated at least 10 times and the mean value was taken. The thermal behavior of the CTT crystals obtained in pure and amino acid-supplemented media was determined using a Setaram LABSYS Evo thermogravimetric analyzer in a nitrogen atmosphere between 30 °C and 850 °C with a heating rate of 5, 10, 20 °C/min. Using the obtained data, the thermal decomposition kinetics for the CTT crystals obtained in pure media were investigated and activation energy was calculated. 2. 4. Kinetic Model Equations Thermogravimetric analysis is a common method used to investigate the thermal decomposition behavior of solids and to determine activation energy: The decomposition rate of a solid can be defined as: dx dt - k(T) f(x) (1) where/(x), k(T), and t represent the reaction model, the reaction rate constant, and time, respectively. The rate constant, k(T), can be defined by the Arrhenius equation, as shown in Eq. (2). k(T) = A exp--— L RT, (2) where A is the pre-exponential or frequency factor (min-1), E is the activation energy (kJ/mol), T is the absolute temperature (K), and R is the ideal gas constant (8.314 J/mol K). The conversion x is shown in Eq. (3): (3) where W0 and Wf are the initial and final sample mass, respectively, and Wt is the sample weight at time t. For a constant heating rate, ft (K/min) can be defined as: (4) For non-isothermal analysis, by substituting Eq. (4) into Eq. (1), the equation is given by Eq. (5). (h A F — - k{T) fix) = - exp(—-)f{x) dT p RT Integrating Eq. (5) gives Eq. (6): m ß\ E RT )dT : AE Jr (5) P(«) (6) where p(u) and g(x) show the temperature integral and the integrated reaction model, respectively. The solution of this equation can be obtained by some approximations depending on the applied kinetic method. In this study, three common isoconversional kinetic methods, namely the Flynn-Wall-Ozawa (FWO),10,11 Kissinger-Akahira-Sunose (KAS),12 and Starink13 methods, were used to determine the activation energies of the CTT crystals. Their linear equations are presented in Eqs. (7)-(9), respectively. (7) (8) (9) For any given value, it is possible to estimate E based on the gradients of the lines obtained from the plot of ln(^) versus 1/T, ln(^/T2) versus 1/T, ln(p/TL92) versus 1/T for the FWO, KAS, and Starink, models, respectively. 3. Results and Discussion 3. 1. XRD Analysis The XRD patterns of the CTT crystals obtained with and without different amino acids are presented in Figure 2. The experimentally observed XRD patterns for the crystals closely matched the patterns simulated using Rietveld refinement. The XRD results showed that the crystals obtained in pure media were in the form of CTT. Consistent with the literature,14 the main peaks detected at 13.274, 15.700, 16.740, 18.397, 29.0250, 29.418, 33.269, 40.165, and 48.721° corresponded to the (101), (111), (020), (200), (310), (301), (132), (104), and (342) planes of the orthor-hombic structure, respectively, with space group P212121. Unit cell dimensions of a = 9.637 Â, b = 10.583 Â, c = 9.216 Â, a = fi = y = 90° were calculated for the CTT crystals using Materials Analysis Using Diffraction (MAUD) software, which was in agreement with the results of a previous study.15 Similar to the crystals prepared in pure media, all of the XRD peaks, regardless of the amino acids used, were attributed to CTT crystals and no other phase formations were observed. In other words, the addition of amino acids Polat et al.: Effects of Amino Acids on the Crystallization ... Acta Chim. Slov. 2020, 67, 842-852 845 Figure 2. XRD patterns of the CTT crystals obtained in pure media and media supplemented with various amino acids. to the crystallization media did not lead to any change in the crystal structure. Figure 2 shows that the diffraction peaks were slightly shifted to the right and the intensity of these peaks also changed with the addition of amino acids. This could be due to the adsorption of the additives on the crystal faces, which could lead to the formation of imperfections and internal strain in the resulting crystals. The unit cell dimensions for crystals prepared in media supplemented with serine, alanine, methionine, and proline were calculated and the results are shown in Table 1. Table 1. The unit cell dimensions for crystals obtained in pure media and media supplemented with various amino acids. Media « (A) Cell Units b (A) c (A) Pure 9.637 10.583 9.216 Serine 9.638 10.581 9.226 Alanine 9.647 10.593 9.233 Methionine 9.645 10.594 9.233 Proline 9.645 10.585 9.230 3. 2. FTIR Analysis FTIR analysis was performed to identify and quantify certain functional groups on the surface of the CTT crystals. Figure 3 shows the FTIR spectra of CTT crystals obtained without and with the different amino acid additives. The FTIR spectrum for the crystals prepared in pure media was in accordance with the literature.15 O-H, C=O, C-O, and metal-oxygen bonds were the main functional groups in the CTT crystals. The three peaks positioned at 3556, 3414, and 3257 cm-1 were assigned to the O-H stretching vibrations. The peak at 2986 cm-1 belongs to the C-H stretching vibration. The peaks observed at 1575 and 1381 cm-1 were attributed to the C=O stretching vibration and A(C=O) + S(O-C=O) vibrations, respectively. The peak located at 1281 cm-1 indicated the O-H plane bending vibration and the peak at 1146 cm-1 was ascribed to the S(C-H) + n(C-H). In addition, the two peaks at 1061 cm-1 and 1012 cm-1 were assigned to the O-H deformation and C-O stretching vibrations, respectively. Finally, the peaks located at 964 and 707 cm-1 were assigned to Ca-O bending vibrations. In agreement with the XRD results, FTIR analysis showed that the crystals were in the CTT form. In order to determine whether the addition of amino acids to the crystallization media affects the functional groups of the CTT and also to analyze the adsorption of these amino acids on the surface of the CTT crystals, FTIR analysis was also performed for the crystals obtained in the presence of amino acids. The FTIR spectra of the crystals prepared in media supplemented with amino acids closely matched the spectra for the crystals prepared in pure media, with all the characteristic peaks of CTT crystals present. Polat et al.: Effects of Amino Acids on the Crystallization ... 846 Acta Chim. Slov. 2020, 67, 842-852 Wavenumber (1/cm) Figure 3. FTIR spectra of the CTT crystals obtained in pure media and media supplemented with various amino acids. We did not detect any additional peaks relating to the amino acids, which could be because the small amounts of amino acids adsorbed on the surface of the crystals were below the instrument's limit of detection. This may be also attributed to the amino acids being physically adsorbed on the surface of the CTT crystals at the studied concentrations through weak Van der Waals forces. 3. 3. Morphology Analysis The effects of the different amino acids on the crystal morphology of CTT are shown in Figure 4 and the corre- sponding length, width, and aspect ratio are shown in Figure 5. As shown in Figure 4a, the crystals acquired in pure media consisted of prismatic shaped crystals with a smooth surface. There was a high tendency for twin and triplet crystal formation. These crystals had a tendency to break under hydrodynamic conditions and then to form twins and triplets again. The tips of the crystals lost their sharp corners as a result of collisions with each other, the crystallizer wall, and the stirrer. As shown in Figure 5, the aspect ratio of the CTT crystals acquired using pure media was calculated to be 0.255 by dividing the width of the crystal by its length. Polat et al.: Effects of Amino Acids on the Crystallization ... Acta Chim. Slov. 2020, 67, 842-852 847 Figure 4. SEM images of the CTT crystals obtained in pure media (a) and in the presence of 200 ppm serine (b), alanine (c), methionine (d), and proline (e). The morphology of the CTT crystals was significantly affected by the amino acids used. Figures 4b-d clearly show the morphological transformation of CTT crystals from long prismatic to short pyramidal and the surface morphology changed from smooth to rough, indicating that the addition of amino acids affected the crystal structure and led to the formation of defective crystals. The amino acids used as the additive molecules may have greater affinity with certain faces of a particular surfaces. They preferentially interact with kinks on the crystal nu- Polat et al.: Effects of Amino Acids on the Crystallization ... 848 Acta Chim. Slov. 2020, 67, 842-852 Figure 5. The width, length, and aspect ratio of CTT crystals obtained in pure media and in the presence of 200 ppm serine, alanine, methionine, and proline. cleus surface and adsorb onto the sites of active growth. These additives interact with Ca2+ ions of the crystal surface with their side terminal carboxyl groups or through hydrogen bonding. This interaction influences the crystallization rate and change the morphology of CTT crystals. In the presence of serine at a concentration of 200 ppm, the CTT crystals were shortened in length and enlarged in width, resulting in an increased aspect ratio of 0.655. In addition, rhomboid crystals were formed, compared to the prismatic crystals obtained in pure media. The formed rhomboid crystals tended to grow on top of each other and they had a different appearance. Although the surfaces of the crystals were generally smooth, the one surface in particular was affected differently from the other surfaces. Serine can selectively adsorb on the surface of CTT crystals and inhibit the crystal growth. When the SEM image for the crystals prepared with alanine added to the media was examined, it could be clearly seen that this additive had a greater effect than serine. The crystals acquired in the presence of 200 ppm alanine were defective as well as being much smaller and more agglomerated than those obtained in pure media, with an aspect ratio of 0.901. As shown in Figure 4d, the presence of methionine in the crystallization media resulted in the formation of a larger amount of irregular, rhomboid-shaped CTT crystals of non-uniform size with a smooth surface. The aspect ratio of the crystals was 0.512. When proline was used as the additive, nearly all of the crystals converted from prismatic form to short pyramidal form and some of the crystals were fractured. There were less deformations on the crystal surface than with the other amino acid additives used. In the presence of proline, there was a tendency for surface nucleation. Compared to the other additives, the tendency for surface nucleation was highest in the presence of proline. When the concentration of proline was 200 ppm, the aspect ratio was 0.793. Accordingly, it can be concluded that the addition of the amino acids serine, alanine, methionine, and proline as effective crystal modifiers results in CTT crystals with different crystal size and morphology. The most effective additive for varying the crystal shape was alanine. 3. 4. Zeta Potential Analysis To further reveal the adsorption characteristics of amino acids on CTT crystals zeta potential measurements were performed. The zeta potential analysis results for the crystals prepared with the addition of various amino acids at concentrations from 0 to 200 ppm are presented in Figure 6. The CTT crystals obtained in pure media exhibited a zeta potential of -21 ± 1.5 mV. The results showed that the addition of amino acids significantly changed the electrical surface charge of the crystals and their zeta potentials were less negative compared to the crystals obtained in pure media. Figure 6. Variation of zeta potential with amino acid concentration. When the concentration of serine was 50 ppm, the zeta potential value was -15.3 ± 0.9 mV, reaching -4.3 ± 1.8 mV at a serine concentration of 200 ppm. The zeta potentials of the CTT crystals obtained in 200 ppm alanine, methionine, and proline media were -2.5 ± 1.3 mV, -14.1 ± 1.2 mV, and -8.4 ± 0.8 mV, respectively. The zeta potential results show that alanine has the greatest effect on the surface charge of the CTT crystals and that amino acids are adsorbed onto the crystal surface. Moreover, the zeta potentials for the crystals prepared in media supplemented with amino acids increased with the agglomeration tendency of the crystals, in accordance with the SEM results. 3. 5. Thermal Analysis We applied thermogravimetric analysis to study the structural evolution and thermal decomposition of the CTT crystals. The thermogravimetric (TG) and differential thermogravimetric (DTG) analysis curves for the crystals obtained in the absence and the presence of the various amino acids at a heating rate of 10 °C/min are shown in Figure 7. In accordance with the literature,1,5 the thermal decomposition of CTT crystals obtained in pure media proceeded through four different main stages, which can be expressed as follows: Polat et al.: Effects of Amino Acids on the Crystallization ... Acta Chim. Slov. 2020, 67, 842-852 849 CaC„H4Os4HjO->CaC4H406+4H20 CaC4H406-> CaC204 + 2C + 2H,0 CaC,04->CaC03 + CO (10) The first decomposition stage included two different peaks at 137 and 191 °C, corresponding to the main weight (11) loss, which were attributed to the loss of four molecules of water of crystallization and the formation of calcium tar- (12) trate anhydride (CaC4H4O6), respectively. In the second (260-356 °C) and third (400-451 °C) decomposition stag- (13) es, the crystals decomposed into calcium oxalate (CaC2O4) and were further converted into calcium carbonate a) 80 60 O 40 20 ---N -TG V ----DTG \ rAf \ * ' \ ! 1 • ' i ',! " i; -»1 V 1 1 1 \ / -' X 1 1 1 0.6 0.3 -i 0.0 -0.3 CJj U E-Q -0.6 b) 150 300 450 600 750 Temperature (°C) c) 150 300 450 600 750 Temperature (°C) 0 150 300 450 600 750 Temperature (°C) d) e) 150 300 450 600 750 Temperature (°C) 150 300 450 600 750 Temperature (°C) Figure 7. TG/DTG curves of the CTT crystals obtained in pure media (a) and media supplemented with serine (b), alanine (c), methionine (d), and proline (e). Polat et al.: Effects of Amino Acids on the Crystallization ... 850 Acta Chim. Slov. 2020, 67, 842-852 (CaCO3) and carbon monoxide (CO) gas, respectively. The final stage was attributed to the transformation of calcium carbonate (CaCO3) to calcium oxide (CaO) and carbon dioxide (CO2) gas in the temperature range of 623776 °C. Above this temperature, the weight loss remained stable until the end of the decomposition process. The TG/ DTG results showed that the obtained crystals were in tetrahydrate form. As can be seen in Figure 7, similar to the crystals obtained in pure media, the four main decomposition stages were detected for all the amino acids used. Unlike the crystals from the pure media, a low-intensity shoulder was detected at ~297 °C owing to the decomposition of the ami-no acid additives. This may be attributed to the loss of amino group from the amino acid molecules. The weight loss from the CTT crystals obtained in pure media was 77.9%, which was in agreement with the literature.16 Increments of 1.5%, 0.8%, 0.3%, and 0.6% were observed for CTT crystals obtained in serine, alanine, methionine, and proline, respectively. The increased weight loss indicated that the amino acids had been adsorbed onto and interacted with the surface of the CTT crystals. The amino acids used had a significant effect on the temperatures of the decomposition peaks during the thermal decomposition of CTT. The peaks shifted towards the higher temperature region in the presence of the additives. 3. 6. Kinetic Analysis The FWO, KAS and Starink methods were applied to determine the activation energy of CTT was calculated in the four decomposition zones. Table 2 shows the specific decomposition temperatures, namely initial temperature, Ti, maximum peak temperature, Tmax, and final temperature, Tf, for the CTT crystals obtained in pure media. Table 2. Specific temperatures of the CTT crystals decomposition process. Media Heating Rate Ti T Amax Tf (°C/min) (°C) (°C) (°C) Stage I 5 82 183 202 10 88 191 222 20 124 198 235 Stage II 5 255 306 328 10 260 315 356 20 278 324 366 Stage III 5 390 412 429 10 400 419 451 20 409 427 470 Stage IV 5 611 722 757 10 623 737 774 20 659 773 814 Table 2 clearly shows that as the heating rate was increased, the characteristic temperatures for each decom- position stage were shifted to higher values, which was attributed to the effect of the different heat-transfer rates on the thermal decomposition kinetics. Figure 8 presents the plots of the three models of the CTT for conversion degrees from 0.1 to 0.9 at different heating rates for the first decomposition stage. The experimental results show high correlation with the three models, with R2 values of 0.9324-0.9999, which suggests that the results are highly reliable (Figure 8). The parallel lines indicate that there is similar kinetic behavior in the range of 0.10-0.40, which suggests that all of the tested models follow the same reaction mechanism for CTT decomposition. However, the reaction mechanism is not similar for all conversion values, particularly at higher conversions, which suggests that there may be a complex multi-step mechanism involving parallel, competitive, and consecutive reactions with different energies. a) 0 0019 0,0020 0.0021 0.0022 0.0023 0,0024 0.0025 0.0026 1/T b) -9.0 -9.5 C -10.0 -10.5 -11.0 - 0.1 • 0.2 ■ 0.3 ♦ 0.4 i 0.5 * 0.6 o 0.7 + 0.8 * 0.9 0.0019 0.002 0.0021 0.0022 0.0023 0.0024 0.0025 0.0026 1/T c) 0.0019 0.002 0.0021 0.0022 0.0023 0.0024 0.0025 0.0026 1/T Figure 8. Plots of (a) FWO, (b) KAS, and (c) Starink methods for the first decomposition stage of the CTT crystals obtained in pure media. Polat et al.: Effects of Amino Acids on the Crystallization ... Acta Chim. Slov. 2020, 67, 842-852 851 The activation energy (i.e., the minimum amount of energy required to initiate a reaction) is plotted as a function of the degree of CTT conversion for four different stages in Figure 9. a) 400 300 Ä200 100 0 b) 500 400 □ FWO «KAS S Starink ail BIB M BIB Bit u 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 X 300 c £ w 200 Ed 100 0 □ FWO «KAS m Starink Him 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 c) 500 400 o E 300 200 Ed 100 0 a FWO «KAS m Starink 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 X d) 5(") 400 □ FWO «KAS m Starink 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 X Figure 9. Activation energy versus conversion degree for the CTT crystals for a) Stage I b) Stage II c) Stage III d) Stage IV. The results show that the minimum mean activation energy values from the FWO, KAS, and Starink methods for the first decomposition stage were 91.0, 89.6, and 88.6 kJ/mol, respectively, which are similar to the activation energy reported in the literature for the dehydration of CTT crystals.16,17 There is good agreement among the calculated values with little deviation, demonstrating the consistency and reliability of the activation energies calculated using the three different kinetic models and various heating rates. The minor differences in the activation energy values arose from the approximations, assumptions, and mathematical formulations used in the different models. The mean activation energies for the second, third and fourth decomposition stages were calculated to be 158.0, 155.9, and 156.8 kJ/mol; 249.1, 250.7, and 250.5 kJ/mol; 224.8, 221.1, and 220.4 kJ/mol, respectively, using the FWO, KAS, and Starink models. According to the calculated activation energy values, less energy was needed for the first decomposition stage but the third decomposition stage related to the decomposition of carbon monoxide required a higher amount of energy to proceed. 4. Conclusions Different concentrations of various amino acids were used to regulate the crystal size and morphology of CTT crystals, which were characterized in detail. Moreover, the thermal decomposition kinetics of the crystals obtained in pure media were examined. The general conclusions obtained can be summarized as follows: • The XRD results showed that CTT was the only detectable crystalline phase for the crystals obtained in the absence and the presence of the amino acids. • SEM analysis results indicated that the amino acids used have the ability to change the morphology of CTT crystals. The SEM images showed that the crystal surface varied from smooth to rough, which suggested that the amino acids used affected the crystal morphology, causing the formation of defective and irregular crystals. • The morphology analysis results revealed that the CTT crystals obtained in the presence of all of the tested ami-no acids were shortened in length and enlarged in width, resulting in an increased aspect ratio. • With increasing amino acid concentration, the surface charge of the crystals became more positive. For instance, the zeta potential value changed from -21.2 ± 1.5 mV for crystals prepared in pure media to -2.5 ± 1.3 mV for the crystals prepared in media supplemented with 200 ppm alanine. • Thermal decomposition kinetics for pure media were examined using different iso-conversional methods. Increasing the conversion from 0.1 to 0.9 changed the calculated activation energy values, which led to the different decomposition characteristics of the CTT crystals. Polat et al.: Effects of Amino Acids on the Crystallization ... 852 Acta Chim. Slov. 2020, 67, 842-852 Using the different kinetic methods, the average activation energies for the first, second, third, and fourth stages were calculated to be 89.7, 156.9, 250.1, and 222.1 kJ/mol, respectively. Acknowledgements This work was supported by Marmara University Scientific Research Projects Commission under the funding FEN-C-YLP-230119-0009. 5. References 1. X. Sahaya Shajan, C. Mahadevan, Cryst. Res. Technol. 2005, 40(6), 598-602. DOI:10.1002/crat.200410389 2. S. S. Sonawane, S. J. Nandre, R. R. Ahire, S. J. Shitole, Der Pharma Chem. 2014, 6(3), 33-38. 3. M. Tailor, V. Joshi, Adv. Appl. Sci. Res. 2014, 5(6), 115-119. 4. C. Kleinguetl, J. C. Williams Jr, J. C. Lieske, M. Daudon, M. E. Rivera, P. J. Jannetto, J. Bornhorst, D. Rokke, E. T. Bird, J. E. Lingeman, M. M. El Tayeb, Urology. 2019, 126, 49-53. DOI:10.1016/j.urology.2019.01.005 5. X. Sahaya Shajan, C. Mahdevan, Bull. Mater. Sci. 2004, 27(4), 327-331. DOI:10.1007/BF02704767 6. M. E. Torres, T. Lopez, J. Stockel, X. Solans, M. Garcia-Valles, E. Rodríguez-Castellón, C. González-Silgo, J. Solid State Chem. 2002, 163, 491-497. DOI:10.1006/jssc.2001.9435 7. K. Suryanarayana, S.M. Dharmaprakash, Mater. Lett. 2000, 42, 92-96. DOI:10.1016/S0167-577X(99)00165-2 8. E.V. Shlyakhova, N. F. Yudanov, Y. V. Shubin, L. I. Yudanova, L. G. Bulusheva, A. V. Okotrub, Carbon. 2009, 47(7), 17011707. DOI:10.1016/j.carbon.2009.02.018 9. C. Gonzalez-Silgo, M. E. Torres, T. Lopez, J. Gonzalez-Platas, A. C. Yanes, J. D. Castillo, J. F. Peraza, X. Solan, Mater. Lett. 2006, 60(12), 1509-1514. DOI:10.1016/j.matlet.2005.11.068 10. T. Ozawa, Bulletin Chem. Soc. Japan. 1965, 38, 1881-1886. DOI:10.1246/bcsj.38.1881 11. J. H. Flynn, L. A. Wall, J. Res. Nat. Bur. Stand. 1966, 70, 487523. DOI:10.6028/jres.070A.043 12. T. Akahira, T. Sunose, Res. Rep. Chiba Inst. Technol (Sci Tech-nol). 1971, 16, 22-31. 13. M. Starink, Thermochim. Acta. 1966, 288, 97-104. DOI:10.1016/S0040-6031(96)03053-5 14. P. P. Pradyumnan, C. Shini, Indian J. Pure Ap. Phy. 2009, 47, 199-205. 15. B. B. Parekh, V. S. Joshi, V. Pawar, V. S. Thaker, M. J. Joshi, Cryst. Res. Technol. 2009, 44(1), 31-35. DOI: 10.1002/crat.200800405 16. V. S. Joshi, Int. J. Innov. Res. Sci. Eng. Technol. 2016, 5(5), 8191-8197. 17. V. S. Joshi, M. Joshi, lndian J. Phys. 2001, 75A(2), 159-163. Povzetek S to raziskavo smo preučili vpliv nekaterih aminokislin, konkretno serina, alanina, metionina in prolina na kristale kalcijevega tartrata tetrahidrata (CTT). Kristalizacijo smo izvajali šaržno, pri temperaturi 25 °C, pH vrednosti 9, dodali pa aminokisline v treh različnih koncentracijah. Kristale CTT smo okarakterizirali z XRD, FTIR, SEM, njihovo velikostjo in zeta potencialom. Za vse uporabljene aminokisline se je izkazalo, da znatno vplivajo na površinski naboj, velikost in morfologijo kristalov. Kristale dobljene brez dodanih aminokislin smo termično razkrojili, pri čemer smo spremljali kinetiko razkroja. Kinetiko smo preučili z uporabo treh različnih kinetičnih modelov, Flynn-Wall-Ozawa (FWO), Kissinger-Akahira-Sunose (KAS) in Starink. Povprečne aktivacijske energije kristalov za prvo, drugo, tretjo in četrto stopnjo razklopa so bile za FWO model 91.0, 158.0, 249.1, in 224.8 kJ/mol, za KAS model 89.6, 155.9, 250.7 in 221.1 kJ/ mol ter za Starink model 88.6, 156.8, 250.5 in 220.4 kJ/mol. Rezultati študije nam omogočijo, da lahko izberemo aminokislin, s katero dobimo željeno morfologijo CTT kristalov, preučena pa je tudi kinetika razklopa. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Polat et al.: Effects of Amino Acids on the Crystallization ... DOI: 10.17344/acsi.2020.5819 Acta Chim. Slov. 2020, 67, 853-859 /^creative ^commons Scientific paper Synthesis, Crystal Structures and Catalytic Property of Dioxidomolybdenum(VI) Complexes with Tridentate Hydrazones Xiao-Qiang Luo,1,2 Yong-Jun Han1 and Ling-Wei Xue1,2,* 1 School of Chemical and Environmental Engineering, Pingdingshan University, Pingdingshan Henan 467000, P.R. China 2 Henan Key Laboratory of Research for Central Plains Ancient Ceramics, Pingdingshan University, Pingdingshan Henan 467000, P.R. China * Corresponding author: E-mail: pdsuchemistry@163.com Received: 01-07-2020 Abstract New dioxidomolybdenum(VI) complexes with the formula [MoO2L(MeOH)], derived from N'-(5-chloro-2-hydroxy-benzylidene)-2-methylbenzohydrazide (H2L') and N'-(3,5-dichloro-2-hydroxybenzylidene)-2-methylbenzohydrazide (H2L2) were prepared. Crystal and molecular structures of the complexes were determined by single crystal X-ray diffraction method. Both complexes were further characterized by elemental analysis and FT-IR spectra. Single crystal X-ray structural studies indicate that the hydrazones L1 and L2 coordinate to the MoO2 cores through the enolate oxygen, phenolate oxygen and azomethine nitrogen. The Mo atoms in both complexes are in octahedral coordination. Catalytic properties for epoxidation of styrene by the complexes using PhIO and NaOCl as oxidant have been studied. Keywords: Molybdenum; hydrazine; crystal structure; hydrogen bonding; catalytic property 1. Introduction The oxidation of organic compounds is an important chemical process in the chemical industry to synthesize a large variety of organic materials. Due to the slow rate of most oxidation reactions in the absence of catalysts, the catalytic oxidation of organic substrates by metal complexes has received much attention in organic synthesis. Many transition metals have been used as homogenous or heterogeneous catalysts in various oxidation systems.1 The epoxidation products of olefins have widely applications in various fields. They are intermediates or precursors for the synthesis of pharmaceuticals, agro chemicals, and many other compounds.2 Among various metal complexes, those with Mo centers have been attracted considerable attention due H2L1 H2L2 Luo et al.: Synthesis, Crystal Structures and Catalytic Property ... to their recently discovered biochemical significance3 as well as their efficient catalytic properties in several organic synthesis procedures.4 Schiff bases are widely used as li-gands in the construction of metal complexes.5 In recent years, a large number of molybdenum complexes with Schiff bases derived from salicylaldehyde and primary amines have been reported.6 Hydrazones, bearing -C(O)-NH-N=CH- groups, are a kind of special Schiff bases, which are of particular interest in coordination chemistry and biological applications.7 However, due to the search of the Crystallographic Structural Database, the number of molybdenum(VI) complexes with hydrazone ligands are much less than other metal complexes with such type of li-gands. In this paper, two new dioxidomolybdenum(VI) complexes with the formula [MoO2L (MeOH)], derived 854 Acta Chim. Slov. 2020, 67, 853-859 from W-(5-chloro-2-hydroxybenzylidene)-2-methylben-zohydrazide (H2LJ) and N'-(3,5-dichloro-2-hydroxyben-zylidene)-2-methylbenzohydrazide (H2L2) are reported. 2. Experimental 2. 1. Materials and Measurements 5-Chlorosalicylaldehyde, 3,5-dichlorosalicylalde-hyde and 2-methylbenzohydrazide were purchased from Aldrich and used without further purification. [MoO2(a-cac)2] and other solvents and reagents were made in China and used as received. C, H and N elemental analyses were performed with a Perkin-Elmer elemental analyser. GC experiments were performed with Agilent 5977A Network GC systems. Infrared spectra were recorded on a Nicolet AVATAR 360 spectrometer as KBr pellets in the 4000-400 cm-1 region. 2. 2. Synthesis of H^1 5-Chlorosalicylaldehyde (1.0 mmol, 0.156 g) and 2-methylbenzohydrazide (1.0 mmol, 0.150 g) were dissolved in methanol (30 mL) with stirring. The mixture was stirred for about 30 min at room temperature to give a colorless solution. The solvent was evaporated to give colorless crystalline product of H2L1. Yield, 91%. For C15H13ClN2O2: anal. calcd., %: C, 62.4; H, 4.5; N, 9.7. Found, %: C, 62.2; H, 4.6; N, 9.8. 2. 3. Synthesis of H2L2 3,5-Dichlorosalicylaldehyde (1.0 mmol, 0.190 g) and 2-methylbenzohydrazide (1.0 mmol, 0.150 g) were dissolved in methanol (30 mL) with stirring. The mixture was stirred for about 30 min at room temperature to give a colorless solution. The solvent was evaporated to give colorless crystalline product of H2L2. Yield, 95%. For C15H12Cl2N2O2: anal. calcd., %: C, 55.8; H, 3.7; N, 8.7. Found, %: C, 55.7; H, 3.8; N, 8.6. 2. 4. Synthesis of [MoO2L1(MeOH)] (1) A methanolic solution (10 mL) of [MoO2(acac)2] (0.1 mmol, 32.6 mg) was added to a methanolic solution (10 mL) of H2L1 (0.1 mmol, 28.9 mg) with stirring. The mixture was stirred for 20 min to give a yellow solution. The resulting solution was allowed to stand in air for a few days. Yellow block-shaped crystals suitable for X-ray single crystal analysis were formed at the bottom of the vessel. The isolated product was washed three times with cold methanol, and dried in a vacuum over anhydrous CaCl2. Yield, 63%. For C16H15ClMoN2O5: anal. calcd., %: C, 43.0; H, 3.4; N, 6.3. Found, %: C, 43.2; H, 3.4; N, 6.2. 2. 5. Synthesis of [MoO2L2(MeOH)] (2) A methanolic solution (10 mL) of [MoO2(acac)2] (0.1 mmol, 32.6 mg) was added to a methanolic solution Table 1. Crystallographic data and refinement parameters for the complexes 1 2 Chemical formula C16H15ClMoN2O5 C16HMCl2MoN2O5 Mr 446.7 481.1 Crystal color, habit Yellow, block Yellow, block Crystal size (mm3) 0.32 x 0.30 x 0.27 0.20 x 0.20 x 0.17 Crystal system Monoclinic Monoclinic Space group P2i/c P2Jc Unit cell parameters a (A) 7.957(1) 7.961(2) b (A) 14.073(1) 14.123(2) c (A) 15.172(1) 15.981(2) P (°) 92.767(2) 92.277(2) V (A3) 1697.0(3) 1795.4(6) Z 4 4 Dcalc (g cm-3) 1.748 1.780 Temperature (K) 298(2) 298(2) p (mm-1) 0.960 1.058 F(000) 896 960 Number of unique data 3702 3919 Number of observed data [7 > 2ff(7)] 2963 3151 Number of parameters 231 240 Number of restraints 1 1 R1, wR2 [I > 2o(I)] 0.0261, 0.0595 0.0313, 0.0688 R1, wR2 (all data) 0.0388, 0.0668 0.0466, 0.0764 Goodness of fit on F2 1.080 1.043 Max and min electron density (e A-3) 0.699, -0.436 0.881, -0.710 Luo et al.: Synthesis, Crystal Structures and Catalytic Property Acta Chim. Slov. 2020, 67, 853-859 855 (10 mL) of H2L2 (0.1 mmol, 32.2 mg) with stirring. The mixture was stirred for 20 min to give a yellow solution. The resulting solution was allowed to stand in air for a few days. Yellow block-shaped crystals suitable for X-ray single crystal analysis were formed at the bottom of the vessel. The isolated product was washed three times with cold methanol, and dried in a vacuum over anhydrous CaCl2. Yield, 45%. For C16H14Cl2MoN2O5: anal. calcd., %: C, 39.9; H, 2.9; N, 5.8. Found, %: C, 40.0; H, 3.0; N, 5.6. 2. 6. Data Collection, Structural Determination and Refinement Diffraction intensities for the complexes were collected at 298(2) K using a Bruker Smart 1000 CCD area diffrac-tometer with MoKa radiation (l = 0.71073 Â). The collected data were reduced using SAINT,8 and multi-scan absorption corrections were performed using SADABS.9 Structures of the complexes were solved by direct methods and refined against F2 by full-matrix least-squares methods using SHELXTL.10 All of the non-hydrogen atoms were refined anisotropically. The methanol H atoms in the complexes were located in difference Fourier maps and refined isotropically, with O-H distances restrained to 0.85(1) Â. All other H atoms were placed in idealized positions and constrained to ride on their parent atoms. The crystallo-graphic data for the complexes are summarized in Table 1. Selected bond lengths and angles are given in Table 2. Table 2. Selected bond distances (Â) and angles (°) for the complexes 1 Mo1-O1 1.918(2) Mo1-O2 2.012(2) Mo1-O3 2.360(2) Mo1-O4 1.695(2) Mo1-O5 1.692(2) Mo1-N1 2.234(2) O1-Mo1-O2 149.57(7) O1-Mo1-N1 81.23(7) O4-Mo1-O1 103.03(8) O4-Mo1-O2 97.65(7) O4-Mo1-N1 156.30(8) O2-Mo1-N1 71.12(6) O5-Mo1-O1 99.90(9) O5-Mo1-O2 95.61(9) O5-Mo1-O4 105.77(9) O5-Mo1-N1 96.26(8) O5-Mo1-O3 170.96(8) O4-Mo1-O3 81.89(7) O1-Mo1-O3 82.69(8) O2-Mo1-O3 78.37(7) N1-Mo1-O3 75.49(6) 2 Mo1-O1 1.941(2) Mo1-O2 2.022(2) Mo1-O3 2.326(2) Mo1-O4 1.686(2) Mo1-O5 1.697(2) Mo1-N1 2.246(2) O1-Mo1-O2 149.21(8) O1-Mo1-N1 80.87(8) O2-Mo1-N1 70.86(8) O1-Mo1-O3 82.57(8) O4-Mo1-O1 98.35(10) O4-Mo1-O2 96.19(10) O4-Mo1-O5 105.94(11) O4-Mo1-N1 95.25(9) O4-Mo1-O3 170.55(9) O2-Mo1-O3 78.86(8) O5-Mo1-O1 103.64(9) O5-Mo1-O2 98.19(9) O5-Mo1-N1 157.24(9) O5-Mo1-O3 82.86(9) N1-Mo1-O3 75.56(7) 2. 7. General Method for Styrene Oxidation The oxidation reactions were carried out according to the literature method.11 The composition of the reaction mixture was 2.00 mmol of styrene, 2.00 mmol of chloro-benzene (internal standard), 0.10 mmol of the complex (catalyst) and 2.00 mmol iodosylbenzene (PhIO) or sodium hypochlorite (NaClO) as the oxidant in 5.00 mL freshly distilled acetonitrile. When the oxidant was sodium hypochlorite, the solution was buffered to pH 11.2. The composition of reaction medium was determined by GC with styrene and styrene epoxide quantified by the internal standard method (chlorobenzene). 3. Results and Discussion 3. 1. Chemistry The complexes were prepared by mixing [MoO2(a-cac)2] with the hydrazones H2L1 and H2L2 in methanol. Single crystal structures were obtained by slow evaporation of the complexes in methanol. The difference of the molecular packing modes of the complexes may be caused by the hindrance effects of the chloro-substituent groups. There is only one chloro-substituent group in complex 1, while two in complex 2. The complexes are soluble in methanol, ethanol, and acetonitrile. The molar conductance of the complexes 1 and 2 at the concentrations of 10-4 M are 25 and 20 O-1 cm2 mol-1, respectively, indicating they are non-electrolytes.12 3. 2. Structure Description of the Complexes The molecular structures and atom numbering schemes of complexes 1 and 2 are shown in Figures 1 and 2, respectively. The coordination geometry around the Mo atoms in both complexes is highly distorted octahedral. The dianionic hydrazones L1 and L2 adopt planar triden-tate manner, forming five- and one six-membered chelate rings involving the MoO2 cores. The hydrazones L1 and L2 in the complexes are bonded to the MoO2 cores in planar fashion, coordinating through the phenolate O, imino N, and enolate O, and an oxo group lying trans to the nitrogen donor. In each complex, a methanol molecule completes the distorted octahedral coordination sphere which lying trans to the other oxo group. The Mo-O(methanol) bonds are significantly longer than the other Mo-O bonds, indicating that the methanol molecules are weakly bonded to the MoO2 cores and this position holds the possibility of functioning as a substrate binding site. The atoms O1, O2, 04, and N1 show high degree of planarity from the equatorial plane, the Mo atoms displaced by 0.332(1) A for complex 1 and 0.320(1) A for complex 2 toward the axial oxo groups. The Mo=O bonds in the complexes are almost equal within the standard deviations, and are within previously reported ranges,41,13 The angular distortion in the Luo et al.: Synthesis, Crystal Structures and Catalytic Property ... 856 Acta Chim. Slov. 2020, 67, 853-859 octahedral environment around Mo atoms come from the five- and six-membered chelate rings taken by the hydra-zones L1 and L2. For the same reason, the trans angles are significantly deviate from the ideal values of 180°. The hy-drazones L1 and L2 in the complexes are distorted, with the two benzene rings make dihedral angles of 12.6(3)° for complex 1 and 9.2(3)° for complex 2. The bond lengths of C6-C7, C7-N1, N1-N2, N2-C8 and C8-O2 are comparable to those observed in similar hydrazone complexes.14 In the crystal structure of complex 1 (Figure 3), two symmetry related adjacent molecules are linked by the methanol molecules of each other through two intermolecular O3-H3A—N2 hydrogen bonds (Table 3), to form a dimeric moiety. The dimeric moieties are further linked via C-H—O interactions (Table 3), to form 3D network. In the crystal structure of complex 2 (Figure 4), two symme- Figure 1. ORTEP plot of the crystal structure of 1. Displacement ellipsoids of non-hydrogen atoms are drawn at the 30% probability level. Figure 4. Molecular packing arrangement of 2 displayed in the unit cell. Hydrogen bonds are shown as dashed lines. Figure 2. ORTEP plot of the crystal structure of 2. Displacement ellipsoids of non-hydrogen atoms are drawn at the 30% probability level. Table 3. Geometrical Parameters for Hydrogen Bonds Hydrogen bonds D-H (A) H-A (A) D-A (A) D-H-A (o) 1 O3- -H3A—N2#' 0.85(1) 1.97(1) 2.811(2) 173(4) C3- H3—O4#2 0.93 2.59(1) 3.419(2) 148(4) C7- H7—O5#3 0.93 2.55(1) 3.243(2) 131(4) C13 -H13—O4#4 0.93 2.53(1) 3.408(2) 157(4) 2 O3- -H3A—N2#5 0.90(1) 1.89(1) 2.788(3) 175(5) #1 1 - x, 1 - y, - z; #2 2 - x, -1 + y, 1/2 - z; #3 2 - x, 1 - y, - z; #4 1 - x, 1/2 + y, 1/2 - z; #5 - x, - y, 1 - z. try related adjacent molecules are linked by the methanol molecules of each other through two intermolecular O3-H3A—N2 hydrogen bonds (Table 3), to form a dimeric moiety. In addition, there are n—n interactions among the rings Mo1-O2-C8-N2-N1, C1-C2-C3-C4-C5-C6 and C9-C10-C11-C12-C13-C14, with centroid to centroid distances of 3.67-4.58 Á for complex 1 and 3.69-4.41 Á for Luo et al.: Synthesis, Crystal Structures and Catalytic Property Acta Chim. Slov. 2020, 67, 853-859 857 complex 2. The C4-Cl1—n interactions between Cl1 atom with the ring C9-C10-C11-C12-C13-C14 also contribute to the crystal packing of both complexes, with distances of 3.705(3) A for complex 1 and 3.944(3) A for complex 2. 3. 3. Infrared and Electronic Spectra The hydrazones showed stretching bands attributed to C=O, C=N, C-OH and NH at about 1654, 1629, 1150 and 1225, and 3253 cm-1. In addition, strong bands observed at 1612 cm-1 for H2L1 and H2L2 are attributed to CH=N groups.15 Both complexes exhibit intense bands at ca. 920 cm-1, assigned to the vibrations of the MoO2 cores.16 The bands due to vC=O and vNH are absent in the complexes, but new C-O stretches appeared at 1261 cm-1 for both complexes. Keto-imine tautomerism is present in molecules H2L1 and H2L2. Upon coordination to Mo atom, enol-imine tautomerism is present in ligands L1 and L2. The vC=N absorption observed at 1629 cm-1 in the free hydrazones shifted to 1603 cm-1 for the complexes upon coordination to Mo atoms.16 The weak peaks in the low wave numbers in the region 450-800 cm-1 may be attributed to Mo-O and Mo-N bonds of the complexes.17 In the electronic spectra of the two complexes, the bands ranging from 250 to 340 nm are assigned to n — n* transitions, and those at 210-230 nm assigned to n — n* transitions.18 The bands with the maximum absorption at 400-410 nm are due to the ligand to metal charge transfer transition.18 3. 4. Catalytic Property Oxidation of styrene was carried out at room temperature with the complexes as the catalysts and PhIO and NaOCl as oxidants. The orange color of the solutions containing the complexes and the substrate was intensified after the addition of oxidant indicating the formation of oxo-metallic intermediates of the catalysts. After completion of oxidation reaction of the alkene, the solution regains its initial color which suggests that the regeneration of the catalysts takes place. The two complexes as catalysts convert styrene most efficiently in the presence of PhIO or NaOCl. There is no obvious difference for the catalytic properties between the two complexes, as a result of similar structures. The complexes are selective towards the formation of styrene ep-oxide. When the reactions were carried out with PhIO, styrene conversions are 83% and 85% for 1 and 2, respectively. When the reactions were carried out with NaOCl, styrene conversions are 71% and 74% for 1 and 2, respectively. It is evident that between the oxidants PhIO and NaOCl, the former acts as a better oxidant with respect to the styrene conversion. The two complexes have similar catalytic properties on the oxidation of styrene when compared to the manganese(III) complexes with the ligand N,N'-o -phenylenebis(3-ethoxysalicylaldimine).11 Reaction of the oxidants with the complexes would likely generate a Mo-Cl or Mo-I entity, which further exchange Cl or I for ClO or IO, and homolytic cleavage of Mo-OCl or Mo-OI bond, generate effective epoxidising agent ClO-or IO-. Then, ClO- or IO- reacts with styrene to give the styrene epoxide. 4. Conclusion New dioxidomolybdenum(VI) complexes with similar hydrazones have been prepared and structurally characterized by single crystal X-ray diffraction method, as well as elemental analysis and FT-IR spectra. The hydrazones coordinate to the MoO2 cores through the enolate oxygen, phenolate oxygen and azomethine nitrogen. Methanol is a suitable solvent for the preparation of such complexes, which readily coordinates to the Mo atom as a co-ligand. 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Slov. 2020, 67, 853- 859 859 Povzetek Sintetizirali smo nove dioksidomolibdenove(VI) komplekse s formulo [MoO2L(MeOH)] z uporabo ligandov N'-(5-kloro-2-hidroksibenziiden)-2-metilbenzohidrazid (H2L') in N'-(3,5-dikloro-2-hidroksibenziliden)-2-metilben-zohidrazid (H2L2). Z rentgensko analizo na monokristalu smo določili kristalno in molekulsko strukturo obeh kompleksov. Obe spojini smo karakterizirali tudi z elementno analizo in FT-IR spektroskopijo. Strukturna analiza je pokazala, da se hidrazona L1 in L2 koordinirata na MoO2 orko enolatnega kisika, fenolatnega kisika in azometinskega dušika. Atomi Mo so v obeh spojinah oktaedrično koordinirani. Preučevali smo katalitske lastnosti obeh spojin pri epoksidaciji stirena z uporabo oksidantov PhIO in NaOCl. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Luo et al.: Synthesis, Crystal Structures and Catalytic Property ... 860 DOI: 10.17344/acsi.2020.5824 Acta Chim. Slov. 2020, 67, 860-865 ©commons Scientific paper Synthesis and X-Ray Crystal Structures of Trinuclear Nickel(II) Complexes Derived from Schiff Bases and Acetate Ligands with Biological Activity Jin-Long Hou,* Hong-Yuan Wu, Cheng-Bin Sun, Ye Bi and Wei Chen College of Chemistry and Chemical Engineering, Qiqihar University, Qiqihar 161006, P. R. China * Corresponding author: E-mail: houjinlong09@163.com Received: 01-11-2020 Abstract The reactions of Ni(OAc)2 2H2O with Schiff base ligands 5-bromo-2-((cyclopentylimino)methyl)phenol (HL1) and 5-bromo-2-(((2-(isopropylamino)ethyl)imino)methyl)phenol (HL2) in methanol afforded two discrete trinuclear complexes [Ni3(L1)2(|r2-r|1:r|1-OAc)2(DMF)2(BrSal)2] (1) and [Ni3(L2)2(^2-n1:n1-OAc)2(^2-n2:n1-OAc)2] (2), where BrSal is the monoanionic form of 4-bromosalicylaldehyde. The complexes were characterized by elemental analysis, IR and UV-Vis spectroscopy. The crystal structures of the complexes have been determined by X-ray crystallography. In both complexes, the nickel atoms are in octahedral coordination geometries. The L1 ligand coordinates to the nickel atoms through the phenolate O and imino N atoms, and the L2 ligand coordinates to the nickel atoms through the phenolate O, imino N and amino N atoms. The antimicrobial activities of the complexes were assayed. Keywords: Nickel complexes; schiff bases; crystal structures; trinuclear complexes; antimicrobial activity 1. Introduction Studies on supramolecular interaction and the self-assembly resulting from them have attracted remarkable attention in recent years. Schiff bases and their metal complexes exhibit biological activity as antibiotics, antiviral, antibacterial and antitumour agents because of their specific structures.1 Among Schiff bases, those derived from the mono-condensation of organic amines with car-bonyl compounds, are a group of classical NO or NNO donor ligands.2 These ligands react readily with nickel salts to occupy its equatorial coordination sites whereas several anionic or neutral ligands can be coordinated to the fourth coordination site of the square plane to yield different type of complexes.3 Among the co-ligands, acetate has received remarkable attention because it can coordinate to the metal centers through versatile modes, such as monodentate, chelating, bidentate bridging, monoatomic bridging, che-lating bridging, etc (Scheme 1).4 To combine Schiff bases and acetate ligands together in the complexes, a number of nickel complexes have been prepared. The complexes show interesting structures, biological, magnetic, and catalytic properties.5 However, it is still a great challenge to prepare acetate bridged multi-nuclear complexes with aiming structures. Herein, we report the synthesis and crystal structures of two new nickel(II) complexes, [Ni3(L1)2(|2- Scheme 1. Chelating modes of acetate ligand. Hou et al.: Synthesis and X-Ray Crystal Structures 861 Acta Chim. Slov. 2020, 67, 860-865 n1:n1-OAc)2(DMF)2(BrSal)2] (1) and [^(L^^-n1^1-OAc)2(^2-n2:n1-OAc)2] (2), where L1 = 5-bromo-2-((cy-clopentylimino)methyl)phenolate, L2 = 5-bromo-2-(((2 -(isopropylamino)ethyl)imino)methyl)phenolate (Scheme 2), BrSal = 4-bromosalicylaldehyde. Scheme 2. HL1 and HL2. 2. Experimental Section 2. 1. Materials and Physical Measurements 4-Bromosalicylaldehyde, cyclopentylamine, N-iso-propylethane-1,2-diamine and nickel acetate were purchased from Sigma-Aldrich. All other chemicals were commercially available and used as received. Elemental analysis was carried out using model 2400 Perkin-Elmer CHN analyzer. Infrared spectra were collected by using KBr pellets on a Jasco-5300 FT-IR spectrophotometer. Electronic spectra were carried out with a Lambda 35 spectrometer. 2. 2. Preparation of Complex 1 4-Bromosalicylaldehyde (0.20 g, 10 mmol) and cyclopentylamine (0.085 g, 10 mmol) were dissolved and stirred at ambient temperature in methanol (30 mL). Thirty minutes later, nickel acetate tetrahydrate (0.50 g, 20 mmol) was added to the solution. The color turned from yellow to deep green, and some insoluble residue was produced. Then, a few drops of DMF was added until the insoluble residue has disappeared. The reaction mixture was stirred for about 1 h and filtered. The filtrate was allowed to slow evaporate for a few days, generating green block shaped single crystals. Yield: 0.13 g (38%). Anal. Calcd for C48H54Br4N4Ni3O12: C, 41.94; H, 3.96; N, 4.08. Found: C, 41.72; H, 4.11; N, 3.97. IR data (KBr; vmax, cm-1): 3097, 3052, 2936, 2868, 1643, 1581, 1517, 1456, 1429, 1408, 1285, 1183, 1101, 1064, 914, 863, 785, 672, 601, 553, 451. UV-Vis data in methanol (X, nm (e, L mol-1 cm-1)]: 220 (1.71 x 104), 245 (1.34 x 104), 273 (9.17 x 103), 380 (3.70x103). 2. 3. Preparation of Complex 2 4-Bromosalicylaldehyde (0.20 g, 10 mmol) and N-isopropylethane-1,2-diamine (0.102 g, 10 mmol) were dissolved and stirred at ambient temperature in methanol (30 mL). Thirty minutes later, nickel acetate tetrahydrate (0.50 g, 20 mmol) was added to the solution. The color turned from yellow to deep green. The reaction mixture was stirred for about 1 h and filtered. The filtrate was al- lowed to slow evaporate for a few days, generating green block shaped single crystals. Yield: 0.21 g (43%). Anal. Calcd for C32H44Br2N4Ni3O10: C, 39.19; H, 4.52; N, 5.71. Found: C, 38.96; H, 4.61; N, 5.82. IR data (KBr; vmax, cm-1): 3107, 3075, 2935, 2858, 1627, 1582, 1550, 1473, 1430, 1409, 1396, 1295, 1188, 1157, 1102, 987, 862, 785, 667, 610, 565, 463. UV-Vis data in methanol (X, nm (e, L mol-1 cm-1)]: 222 (1.56 x 104), 240 (1.70 x 104), 265 ( 5.32 x 03), 3 3 5 (4.81 x 103). 2. 4. X-ray Crystallography X-ray data for the complexes were collected on a Bruker SMART 1000 CCD single crystal diffractometer at 298(2) K, equipped with a graphite monochromator and a Mo Ka fine-focus sealed tube (A = 0.71073 A) by the u> scan method. Reflections were corrected for Lorentz and polarization effects, and for absorption by semi-empirical methods based on symmetry-equivalent and repeated reflections. The SMART software was used for data acquisition and the SAINT-PLUS software was used for data extraction.6 The absorption corrections were performed with the help of SADABS program.7 The structures were solved by direct methods and refined on F2 by full-matrix least-squares procedures. All non-hydrogen atoms were refined using anisotropic thermal parameters. Hydrogen atoms were included at idealized positions by using a riding model. The SHELX-97 programs were used for structure solution and refinement.8 Selected crystallographic data for the complexes are listed in Table 1. Table 1. Crystal data and structure refinement parameters for complexes 1 and 2 1 2 Formula C 12H14BrCuN3OS C32H44Br2N4Ni3O10 M 391.8 980.7 Temperature (K) 298(2) 298(2) Crystal system Orthorhombic Monoclinic Space group Pca21 P2Jc a (A) 12.875(l) 11.4402(13) b (A) 6.788(1) 19.8179(17) c (A) 16.531(1) 8.6384(15) P (°) 100.8150(10) V (A3) 1444.8(2) 1923.7(4) Z 4 2 Dc( g/cm3) 1.801 1.693 p (mm-1) 4.416 3.588 F(000) 780 996 Reflections collected 12900 10436 Independent reflections 2978 3581 Rint 0.0448 0.0998 Reflections with 2644 1552 F2 > 2ff(F2) R1 [F2 > 2ff(F2)] 0.0405 0.0491 wR2 (all data) 0.1020 0.0907 Goodness-of-fit on F2 1.082 0.931 Hou et al.: Synthesis and X-Ray Crystal Structures 862 Acta Chim. Slov. 2020, 67, 860-865 3. Results and Discussion 3. 1. Chemistry Both complexes were readily prepared by reacting nickel acetate tetrahydrate with Schiff base ligands. The elemental analysis data are consistent with the general molecular formulae of the complexes. In complex 1, the acetate ligands act as bidentate bridging groups. In complex 2, two acetate ligands act as bidentate bridging groups, and the other two act as chelating bridging groups. The complexes are soluble in common organic solvents like dimethyl sulphoxide, dimethylformamide, methanol, and ethanol. 3. 2. Infrared and Electronic Spectra of the Complexes In the spectra of the free Schiff bases HL1 and HL2, the bands at 1650 and 1645 cm-1, respectively, are assigned to the azomethine groups, vC=N,9 and the broad and weak absorptions at about 3430 cm-1 are assigned to the hydrox-yl groups, vO-H. In the complexes, the absence of the O-H stretching and bonding vibrations indicates coordination through the phenolate groups. The C=N stretches of the complexes are observed at lower frequencies (1643 and 1627 cm-1) when compared to the free Schiff bases.10 The phenolic v(Ar-O) in the free ligands exhibit bands at about 1270 cm-1. However, in the complexes it appears at about 1283 cm-1. The shift to higher wave number is an indication of C-O-M bond formation. The bands at about 1582 and 1408 cm-1 are assigned to the acetate ligands.4a,5b The occurrence of new bands in the 450-670 cm-1 region confirms the presence of metal-nitrogen and metal-oxygen bonds, respectively.11 In the electronic spectra of the free Schiff bases and the complexes, the absorption frequencies ascribed to the aromatic n-n* and n-n* transitions are located in the region 240-290 nm.12 In the electronic spectra of the complexes, the absorption centered at 380 nm for complex 1 and 335 nm for complex 2 are assigned to ligand-to-metal charge transfer.13 3. 3. Structure Description of Complex 1 The molecular structure of complex 1 is depicted in Fig. 1. Bond parameters associated with the metal atom are listed in Table 2. The molecular structure is centrosym-metric with Ni2 atom on an inversion center. The structure shows a trinuclear complex consisting of three Ni atoms in a linear array, which is bridged by two acetate ligands and four phenolate groups. The Ni—Ni distance is 3.053(2) Â. The Schiff base ligand coordinates to the Ni atoms through phenolate O and imino N atoms. The ligand 4-bromosalic-ylaldehyde coordinates to the Ni atoms through carbonyl O and phenolate O atoms. The acetate ligand acts as a bidentate bridging group. The Ni2 atom is located at the inversion centre of the complex in an octahedral geometry. This atom is coordinated by four phenolate O atoms from the Schiff base and 4-bromosalicylaldehyde ligands that form a plane and, near-perpendicularly to it, by two O atoms from two acetate bridging ligands that connect the central Ni atom to the outer Ni atoms. The greatest deviation of the bond angles from those expected for an ideal octahedral geometry is found for O1-Ni2-O3 with 79.3(3)° and O1-Ni2-O3A with 100.7(3)°. The remaining bond angles are close to the ideal values for the octahedral coordination. The cis and trans coordinate bond angles for Ni2 are in the region 79.3(3)-100.7(3)° and 180°. The terminal Nil atom is in an octahedral environment and is coordinated by the four donor atoms of the Schiff base and 4-bromosa-licylaldehyde ligands in the equatorial plane, and by two O atoms from one bridging acetate group and one coordinated DMF ligand in the axial positions. The greatest deviations from an ideal octahedral geometry are found in the O2-Ni1-O6 (84.9(3)°) and in N1-Ni1-O2 (100.8(4)°) angles. The remaining bond angles are close to the ideal values for the octahedral coordination. The cis and trans coordinate bond angles for Ni1 are in the region 84.9(3)-100.8(4)° and 168.8(3)-176.6(3)°. The Ni-O and Ni-N bond lengths are similar to those reported previously.14 Figure 1. ORTEP diagram of complex 1 with thermal ellipsoids at 30% probability level. Selected bond lengths and angles are given in Table 2. Unlabeled atoms are related to the symmetry operation 1 - x, 1 - y, 1 - z. 3. 4. Structure Description of Complex 2 The molecular structure of complex 2 is depicted in Fig. 2. Bond parameters associated with the metal atom are listed in Table 2. The molecular structure is centrosymmet-ric with Ni2 atom on an inversion center. The structure shows a trinuclear complex consisting of three Ni atoms in a linear array, which are bridged by four acetate ligands and two phenolate groups. The Ni—Ni distance is 3.043(2) A. The Schiff base ligand coordinates to the Ni atoms through phenolate O, imino N and amino N atoms. Two of the acetate ligands act as bidentate bridging groups, and the other Hou et al.: Synthesis and X-Ray Crystal Structures Acta Chim. Slov. 2020, 67, 860-865 863 two act as chelating bridging groups. The Ni2 atom is located at the inversion centre of the complex in an octahedral geometry. This atom is coordinated by two phenolate O atoms from the Schiff base ligands and two O atoms from two bidentate bridging acetate groups that form a plane and, near-perpendicularly to it, by two O atoms from two chelating bridging acetate groups that connect the central Ni atom to the outer Ni atoms. The greatest deviation of the bond angles from those expected for an ideal octahedral geometry is found for O1-Ni2-O2 with 79.97(16)° and O1-Ni2-O2A with 100.03(16)°. The remaining bond angles are close to the ideal values for the octahedral coordination. The cis and trans coordinate bond angles for Ni2 are in the region 79.97(16)-100.03(16)° and 180°. The terminal Ni1 atom is in an octahedral environment and is coordinated by the three donor atoms of the Schiff base ligand and the O2 atom of the chelating bridging acetate group in the equatorial plane, and by O3 atom of the chelating bridging acetate group and O4 atom of the bidentate bridging acetate group in the axial positions. The greatest deviations from an ideal octahedral geometry are found in O2-Ni1-O3 (60.69(19)°) and in O2-Ni1-N2 (103.6(3)°) angles. The remaining bond angles are close to the ideal values for the octahedral coordination. The cis and trans coordinate bond angles for Ni1 are in the region 60.69(19)-103.6(3)° and 157.1(2)-174.1(2)°. The Ni-O and Ni-N bond lengths are comparable to those of complex 1. Figure 2. ORTEP diagram of complex 2 with thermal ellipsoids at 30% probability level. Selected bond lengths and angles are given in Table 2. Unlabeled atoms are related to the symmetry operation 1 - x, 1 - y, 1 - z. 3. 5. Antimicrobial Activity The complexes were assayed against the bacteria Escherichia coli and Salmonella typhi, and the fungi Aspergillus niger and Candida albicans by MIC (Minimum In- hibitory Concentration) method with three different concentrations of 100, 50 and 25 ^g.15 The activity was also assayed for the free Schiff bases, the pure solvent DMF and the standard gentamycine for each of antibacterial and flu-canazole for antifungal cultures. Final adjustments were made using optical density measurement for bacteria (ab-sorbance 0.05 at 580 nm). The zones of inhibition in millimeter for the compounds are presented in Table 3. The two complexes have similar activities against the bacteria and fungi, and they are more susceptible toward bacterial cells than fungicidal cells. In general, both complexes have better activities than the free Schiff bases. The trends are in Table 2. Selected bond distances (Â) and angles (°) for complexes 1 and 2 Bond distances Ni1-N1 2.035(10) Ni1 -O1 2.014(7) Ni1-O2 2.062(9) Ni1 -O3 2.023(7) Ni1-O4 2.023(7) Ni1 -O6 2.125(8) Ni2-O5 2.022(7) Ni2 -O1 2.048(6) Ni2-O3 2.071(6) Bond angles O1-Ni1-O4 91.2(3) O1- -Ni1- O3 81.3(3) O4-Ni1-O3 90.9(3) O1- -Ni1- N1 89.7(4) O4-Ni1-N1 93.2(4) O3- -Ni1- N1 170.2(4) O1-Ni1-O2 168.8(3) O4- -Ni1- O2 92.0(3) O3-Ni1-O2 88.0(3) N1- -Ni1- O2 100.8(4) O1-Ni1-O6 92.1(3) O4- -Ni1- O6 176.6(3) O3-Ni1-O6 90.5(3) N1- -Ni1- O6 85.8(4) O2-Ni1-O6 84.9(3) O5- -Ni2- O5A 180 O5-Ni2-O1A 92.3(3) O5- -Ni2- O1 87.7(3) O1-Ni2-O1A 180 O5- -Ni2- O3A 90.7(3) O1-Ni2-O3A 100.7(3) O5- -Ni2- O3 89.3(3) O1-Ni2-O3 79.3(3) O3- -Ni2- O3A 180 2 Bond distances Ni1-N1 1.976(6) Ni1 -N2 2.152(8) Ni1-O1 2.010(4) Ni1 -O2 2.100(4) Ni1-O3 2.201(6) Ni1 -O4 2.015(4) Ni2-O5 2.009(4) Ni2 -O1 2.057(4) Ni2-O2 2.117(4) Bond angles N1-Ni1-O1 90.5(3) N1- -Ni1- O4 100.9(2) O1-Ni1-O4 93.11(17) N1- -Ni1- O2 159.8(2) O1-Ni1-O2 81.45(16) O4- -Ni1- O2 98.00(18) N1-Ni1-N2 83.7(3) O1- -Ni1- N2 174.1(2) O4-Ni1-N2 89.3(2) O2- -Ni1- N2 103.6(3)-. N1-Ni1-O3 101.4(2) O1- -Ni1- O3 91.91(19) O4-Ni1-O3 157.1(2) O2- -Ni1- O3 60.69(19) N2-Ni1-O3 88.0(3) O5- -Ni2- O5A 180 O5-Ni2-O1 90.09(16) O5- -Ni2- O1A 89.91(16) O1-Ni2-O1A 180 O5- -Ni2- O2A 90.16(17) O1-Ni2-O2A 100.03(16) O5- -Ni2- O2 89.84(17) O1-Ni2-O2 79.97(16) O2- -Ni2- O2A 180 Symmetry operation for A: 1 - x, 1 - y , 1 - - z. 1 Hou et al.: Synthesis and X-Ray Crystal Structures 864 Acta Chim. Slov. 2020, 67, 860-865 Table 3. Antimicrobial activities of the compounds (zone of inhibition in mm) Compound Bacteria Escherichia coli Fungi Salmonella typhi Aspergillus niger Candida albicans 1 29 31 15 18 2 30 30 16 18 HL1 15 11 - - HL2 13 12 - - Gentamycine 28 28 - - Flucanazole - - 23 23 DMF 12 14 12 12 The concentration is 100 |ig L accordance with the literature that metal complexes are more active in the biological potential than the ligands used in the synthesis.16 When comparing the antimicrobial activity of the studied complexes to those of reference antibiotics, the inhibitory ability is found to be good. For example, the two complexes have similar or even higher activity on the bacteria Escherichia coli and Salmonella typhi than gentamycine, and have effective activity on Asper-gillus niger and Candida albicans, which is seldom seen in the literature. The results indicate that the two complexes can possibly be used in the treatment of diseases caused by the bacteria that were tested. 4. Conclusion A pair of novel acetato-bridged nickel(II) complexes derived from the Schiff base ligands 5-bromo-2-((cyclo-pentylimino)methyl)phenol and 5-bromo-2-(((2-(isopro-pylamino)ethyl)imino)methyl)phenol were prepared. Single crystal structures of the complexes indicates that both complexes are trinuclear nickel(II) compounds. All of the nickel atoms in the complexes are in octahedral coordination geometries. The complexes have good antimicrobial activities against Escherichia coli, Salmonella typhi, Asper-gillus niger and Candida albicans. Supplementary Material CCDC Nos. 1974759 (1) and 1974760 (2) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam. ac.uk/data request/cif. Crystal data and details of the data collection and refinement for the complexes are collected in Table 1. 5. References 1. (a) H. Keypour, F. Forouzandeh, S. Salehzadeh, F. Hajibabaei, S. 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Sun, Transition Met. Chem. 2019, 44, 501506; D0I:10.1007/s11243-018-00296-x (b) H. Y. Qian, Russ. J. Coord. Chem. 2017, 43, 780-786. D0I:10.1134/S1070328417110070 11. H.-Y. Qian, Inorg. Nano-Met. Chem. 2018, 48, 461-466. DOI: 10.1080/24701556.2019.1569689 12. H. Liu, D. Niu, Z. Lu, J. Coord. Chem. 2009, 62, 3763-3771. DOI: 10.1080/00958970903159131 13. A. Ray, D. Sadhukhan, G. M. Rosair, C. J. Gomez-Garcia, S. Mitra, Polyhedron 2009, 28, 3542-3550. D01:10.1016/j.poly.2009.07.017 14. (a) M. K. Taylor, J. Reglinski, D. Wallace, Polyhedron 2004, 23, 3201-3209; D0I:10.1016/j.poly.2004.10.002 (b) R. Sanyal, S. K. Dash, P. Kundu, D. Mandal, S. Roy, D. Das, Inorg. Chim. Acta 2016, 453, 394-401; D0I:10.1016/j.ica.2016.08.047 (c) L. Rigamonti, A. Forni, Inorg. Chim. Acta 2018, 473, 216222. D0I:10.1016/j.ica.2018.01.007 15. M. A. Phaniband, S. D. Dhumwad, J. Coord. Chem. 2009, 62, 2399-2410. DOI: 10.1080/00958970902803341 16. (a) L.-W. Xue, H.-J. Zhang, P.-P. Wang, Acta Chim. Slov. 2019, 66, 190-195; D0I:10.17344/acsi.2018.4868 (b) G.-X. He, L.-W. Xue, Q.-L. Peng, P.-P. Wang, H.-J. Zhang, Acta Chim. Slov. 2019, 66, 570-575; (c) S. Daravath, A. Rambabu, N. Vamsikrishna, N. Ganji, S. Raj, J. Coord. Chem. 201 9, 72, 1973-1993; D01:10.1080/00958972.2019.1634263 (d) M. A. Shaheen, W. Xiao, M. Aziz, A. Karim, M. Saleem, M. Mustaqeem, T. Mehmood, M. N. Tahir, A. Sltan, A. Si-mair, C. Lu, Russ. J. Gen. Chem. 2019, 89, 1691-1695. D0I:10.1134/S1070363219080231 Povzetek Z reakcijo Ni(OAc)2-2H2O s Schiffovo bazo 5-bromo-2-((ciklopentilimino)metil)fenolom (HL1) in 5-bromo-2-(((2-(iz-opropilamino)etil)imino)metil)fenolom (HL2) v metanolu smo izolirali dva diskretna trijedrna kompleksa [Ni3(L1)2(|i2-r|1:r|1-OAc)2(DMF)2(BrSal)2] (1) in [Ni3(L2)2(^2-n1:n1-OAc)2(^2-n2:n1-OAc)2] (2), kjer je BrSal monoanion-ska oblika 4-bromosalicilaldehida. Kompleksa sta bila okarakterizirana z elementno analizo, IR in UV-Vis spektroskopijo. Strukture kompleksov so bile določene z rentgensko monokristalno difrakcijo. V obeh kompleksih je nikljev atom v oktaedrični koordinaciji. Ligand L1 se koordinira na nikljev atom preko fenolatnega O in imino N atoma, ligand L2 se koordinira na nikljev atom preko fenolatnega O, imino N in amino N atomov. Določena je bila tudi antimikrobna aktivnost kompleksov. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Hou et al.: Synthesis andX-Ray Crystal Structures ... DOI: 10.17344/acsi.2020.5825 Acta Chim. Slov. 2020, 67, 866-875 /^creative ^commons Scientific paper Molybdic Acid-Functionalized Nano-Fe3O4@TiO2 as a Novel and Magnetically Separable Catalyst for the Synthesis of Coumarin-Containing Sulfonamide Derivatives Jamileh Etemad Gholtash, Mahnaz Farahi,* Bahador Karami and Mahsa Abdollahi Department of Chemistry, Yasouj University, P. O. Box 353, Yasouj 75918-74831, Iran * Corresponding author: E-mail: farahimb@yu.ac.ir Received: 01-11-2020 Abstract Supported molybdic acid on nano-Fe3O4@TiO2 (Fe3O4@TiO2@(CH2)3OMoO3H) has been successfully prepared, characterized and applied as a catalyst for the synthesis of sulfonamide containing coumarin moieties. The prepared Fe3O4 nanoparticles by coprecipitation of Fe2+ and Fe3+ ions were treated with tetraethyl orthotitanate to obtain Fe3O4@TiO2. By anchoring 3-chloropropyltriethoxysilan on Fe3O4@TiO2 followed by reacting with molybdic acid, the desired catalyst was produced. The synthesized catalyst was characterized using XRD, SEM, EDS, FT-IR and VSM analysis. Fe3O4@TiO2@ (CH2)3OMoO3H was used as a catalyst for the synthesis of sulfonamide containing coumarin moieties via a three-component reaction of aryl aldehydes, para-toluenesulfonamide and 4-hydroxycoumarin or 5,7-dihydroxy-4-methylcou-marin. The catalyst recovery test showed the catalyst is highly reusable without losing its activity. Keywords: Nanocatalyst; _para-toluenesulfonamide; aromatic aldehydes; 4-hydroxycoumarin; 5,7-dihydroxy-4-methyl-coumarin 1. Introduction Nanochemistry is becoming increasingly significant which involves the synthesis and application of nanoparticles of different sizes and shapes.1-9 Nanoparticles are different from their bulk counterparts and show special properties. A considerable amount of attention to nanoparticles is due to their unique properties such as ease of availability, chemical inertness, high surface area to volume ratio, high activity and selectivity, thermal stability and low toxicity.10, 11 Nanomaterials have been widely utilized as solid support material for the design of environmentally benign heterogeneous catalysts to address various economic and green chemistry issues.12-15 Among them, magnetic nanoparticles (MNPs), especially nano-Fe3O4, have been more desirable because of their remarkable advantages. Nano-Fe3O4 has been intensively used as catalytic supports owing to the capability of separation from the reaction mixture via an external magnet which achieves a simple separation without filtration.16,17 Supported catalysts on nano-Fe3O4 show not only high catalytic activity but also a high degree of chemical stability and they do not swell in organic solvents. Nano-TiO2 owing to its nontoxicity, long-term pho-tostability, and high effectiveness has broadly been applied as catalyst in many organic transformations.18,19 It has good mechanical resistance and stability in acidic and oxidative environments. Nano-TiO2 is broadly utilized in fuel processing because of its tunable porous surface and distribution, high thermal stability and mechanical strength.20 Despite the several mentioned advantages of TiO2 nanoparticles, they are difficult to separate from the reaction media beacause of their small size. To simplify the separation and in order to increase the surface area, TiO2 can be immobilized on magnetic nanoparticles to produce magnetically recoverable heterogeneous catalysts.21-23 In recent years, there has been an increased interest on the utilization of sulfonamide derivatives as basic constituents of numerous drugs include anticancer, antiinflammatory, antiviral agents and HIV-protease inhibitors.24-26 Antibacterial agents with sulfonamide structure, e.g. sulfadiazine have been therapeutically used for many decades.27 They Gholtash et al.: Molybdic Acid-Functionalized Acta Chim. Slov. 2020, 67, 866-867 875 have also been applied as azo dyes for achieving improved light stability, water solubility, and fixation of fibers. Sulfonamides are also known as inhibitors of the activity of the enzymes, such as dihydropteroate synthase (DHPS), matrix metalloproteinase, and carbonic anhydrase (CA).28 Coumarins as a class of lactones are important hetero-cycles that not only have many biological activities, but are also found in the structure of a large number of natural com-pounds.29 With an entirely looking at medicinally important heterocycles, we find numerous drugs containing coumarin moiety.30-32 Their easy synthetic modifications cause the design and synthesis of various coumarin-based derivatives with diverse activities against cancer.33 Also, coumarins are widely employed as cosmetics, pigments and agrochemicals. They can be fused with different classes of heterocycles to obtain novel useful compounds. The unique properties and wide applications of coumarins have promoted extensive studies for the synthesis of these heterocycles. Due to these benefits and as a continuation of our studies on the preparation of new heterogeneous cata-lysts,34-36 herein we have developed synthesis and characterization of Fe3O4@TiO2@(CH2)3OMoO3H as a novel recoverable nanocatalyst. Then, its application was investigated for the synthesis of sulfonamide containing coumarin moieties. 2. Results and Discussion Because of reasonable needs for clean and green heterogeneous catalysts, Fe3O4@TiO2@(CH2)3OMoO3H was synthesized following the procedure shown in Scheme 1. In the first step, the magnetic Fe3O4 nanoparticles were synthesized by coprecipitation of iron(II) and iron(III) ions.17 Consequently,the TiO2 shell was prepared by the hydrolysis of tetraethyl orthotitanate (TEOT) in an absolute ethanol/ acetonitrile mixture in the presence of the well-dispersed Fe3O4 nanocrystals.37 In continuation, the OH groups on the titanium coating magnetic nanoparticles (Fe3O4@ TiO2) can be functionalized with 3-chloropropyltriethox-ysilan molecule.38 Next, the new catalyst 1 was obtained by replacing chloride group using molybdic acid. The structure of the nanocatalyst 1 was studied and fully characterized using SEM, EDX, VSM, XRD, and FT-IR. Fig. 1 shows the wide-angle XRD patterns of na-no-Fe3O4, Fe3O4@TiO2 and Fe3O4@TiO2@(CH2)3OMoO3H. As shown in Figure 1a, all the peaks agreed on face-centered cubic (fcc) Fe3O4. The data showed diffraction peaks at 26 = 37.158, 43.173, 66.98, 74.188, and 79.171 which can be indexed to (222), (400), (442), (533), and (444) with the JCPD 01-88-0315. Additionally, the peak of the highest in- = 3^4 Ti02 Scheme 1. Synthesis of Fe3O4@TiO2@(CH2)3OMoO3H (1). Position [°2 Thetal Figure 1. The XRD patterns of (a) nano-Fe3O4, (b) Fe3O4@TiO2 and (c) Fe3O4@TiO2@(CH2)3OMoO3H. Gholtash et al.: Molybdic Acid-Functionalized 868 Acta Chim. Slov. 2020, 67, 866-875 tensity (311, 29 = 35.522) was picked out to evaluate the particle diameter of the nanoparticles. Furthermore, in Figure 1b a broad diffraction peak appeared at 29 = 20-30 nm that can be indexed to amorphous TiO2.39 The main peaks for the Fe3O4@TiO2 are similar to the standard Fe3O4 particles, which reveals that the crystal structure of Fe3O4@TiO2 is well coated. No obvious diffraction peak for the TiO2 is observed, suggesting amorphous TiO2 coating is formed by the hydrolysis of tetraethyl titanate (TEOT) in the presence of the well-dispersed Fe3O4 nano-crystals by a sol-gel process.40 In Figure 1c, the confirming peak showing the presence of molybdate group has appeared in the range of 29 = 20-30° which is covered by the broad peak of TiO2.39, 41 The FT-IR spectra of Fe3O4, Fe3O4@TiO2, Fe3O4@ TiO2@(CH2)3Cl and Fe3O4@TiO2@(CH2)3OMoO3H were compared to analyze the progress of catalyst synthesis (Fig. 2). The observed characteristic peaks due to Fe-O stretching vibration at 583 cm-1 in all compared spectra is a confirmation of which nanostructure of Fe3O4 is preserved during the process. The appeared peaks at 1118 and 1400 cm-1 in Fig. 2b could be associated with stretching vibration modes of Ti-O and Fe-O-Ti bonds, respectively.35 In Fig. 2c, the CH2 bending as broadband and symmetric CH2 and asymmetric CH2 of the alkyl chains appear at 1480 cm-1 and 2860-2923 cm-1, respectively. Fig. 2d shows new bands at 1623 and 3297-3436 cm-1 attributable to stretching vibration of Mo-O and OH.43,44 The vibrating sample magnetometer (VSM) was applied to evaluate the magnetic measurement of the prepared catalyst (Fig. 3). Based on the results, the saturation magnetization for Fe3O4@TiO2@(CH2)3OMoO3H and Fe3O4@TiO2 are 22.98 and 77.85 emu g-1, respectively. The decrease of the saturation magnetization of Fe3O4@TiO2 o wo moo /too txo iwo iooo *oe Wavenumb«r iCm-ll Figure 2. The FT-IR spectra of (a) Fe3O4, (b) Fe3O4@TiO2, (c) Fe3O4@TiO2@(CH2)3Cl, and (d) Fe3O4@TiO2@(CH2)3OMo3H. Gholtash et al.: Molybdic Acid-Functionalized ... Figure 3. Room-temperature magnetization curves of (a) Fe3O4@ TiO2@(CH2)3OMoO3H and (b) Fe3O4@TiO2. after surface coating with molybdic acid was ascribed to the increase of particle size of Fe3O4@TiO2. This evidence indicates that molybdic acid immobilized on modified TiO2-coated Fe3O4 has been successfully obtained. The surface morphology of Fe3O4@TiO2@(CH2)3O-MoO3H was observed via a scanning electron microscopy (Fig. 4). The result demonstrates that nearly spherical nanoparticles with a narrow distribution were obtained with an average diameter of about 43.25 nm. Energy-dispersive X-ray spectroscopy (EDS) analysis of Fe3O4@ TiO2@(CH2)3OMoO3H (Fig. 5) contains all expected elemental cases including Fe, Ti, Mo, C, Si and O. The EDS spectra of the catalyst confirmed the existence of molybdate and hence indicated that molybdic acid grafted successfully on the catalyst surface. Acta Chim. Slov. 2020, 67, 866-869 875 Çft > Figure 5. EDS analysis of Fe3O4@TiO2@(CH2)3OMoO3H. SEM HV: 20.0 kV WO: l.M mm M» (¡eld: 5.03 |im D*t: SE SEM MAO: ».0 kK Dflt#(m/d/yJ: 02/17/1» .........i vmaïtmcah 1 pm Figure 4. The SEM imag of Fe3O4@TiO2@(CH2)3OMoO3H. The catalytic activity of Fe3O4@TiO2@(CH2)3O-MoO3H was investigated in the synthesis of sulfonamide containing coumarins 6 via the reaction of aryl aldehydes (2), para-toluenesulfonamide (3) and compound 5 (4-hy-droxycoumarin or 5,7-dihydroxy-4-methylcoumarin) (Scheme 2). Scheme 2. Synthesis of sulfonamide containing coumarins by nanocalyst 1. Table 1. Optimization of the model reaction.1 Entry Catalyst 1 (g) Solvent Temp. (°C) Time (h) Yieldb (%) 1 - - 25 24 - 2 - - 80 24 5 3 - - 100 24 9 4 - Toluene reflux 24 3 5 - EtOH reflux 24 5 6 0.001 - 60 3 50 7 0.003 - 60 3 65 8 0.005 - 60 3 60 9 0.007 - 60 3 57 10 0.003 EtOH reflux 3 60 11 0.003 Toluene reflux 3 55 12 0.003 h2o reflux 3 48 13 0.003 CHCl3 reflux 3 53 14 0.003 - 80 1.5 75 15 0.003 - 90 1.5 85 16 0.003 - 110 1.5 80 a Reaction conditions: benzaldehyde (1 mmol), _para-toluenesulfonamide (1 mmol) and 4-hydroxycou-marin (1 mmol). b Isolated yields. Gholtash et al.: Molybdic Acid-Functionalized 870 Acta Chim. Slov. 2020, 67, 866-875 Table 2. Synthesis of 6 in the presence of Fe3O4@TiO2@(CH2)3OMoO3H. Entry Compound 5 Ar Product Mp (°C) Time (min) 6a CfiHc 231-233 90 6b 4-ClC6H4 251-253 85 6c 4-BrC6H4 176-177 85 6d 263-264 120 6e C6H5 260-26246 240 6f 4-CH3C6H4 255-25646 260 6g 4-NO2C6H4 219-22146 200 6h 4-BrC6H4 268-26946 210 4-CH3C6H4 Gholtash et al.: Molybdic Acid-Functionalized ... Acta Chim. Slov. 2020, 67, 866-871 875 Entry Compound 5 Ar Product Mp CC) Time (min) 6i 270-27146 210 6j 230-23246 200 6k 2-ClC6H4 2 1 5-2 1 646 2 30 6l 4-ClC6H4 208-21046 220 1 Isolated yields. 3-BrC6H4 3-NO2C6H4 111111 Figure 6. Reusability study of nanocatalyst 1 in the synthesis of 6a at 90 °C under solvent-free conditions. In a preliminary study, the reaction of benzaldehyde, para-toluenesulfonamide and 4-hydroxycoumarin was selected as a model reaction to determine suitable conditions. To illustrate the need of Fe3O4@TiO2@(CH2)3O-MoO3H for this condensation, we examined the model reaction in the absence of this catalyst at various conditions. The results show clearly that Fe3O4@TiO2@ (CH2)3OMoO3H is an effective catalyst for this reaction and in the absence of this catalyst the reaction did not take place, even after 24 h. Next, the model reaction was conducted in the presence of several amounts of catalyst 1 at different conditions (Table 1). Through screening we found that this reaction was efficiently completed in the presence of 0.003 g of Fe3O4@TiO2@(CH2)3OMoO3H at 90 oC under solvent-free conditions. To investigate the generality of this protocol, the reaction of different aryl aldehydes, containing both electron-donating and electron-withdrawing groups were employed in the reaction. In general, the reaction proceeded smoothly to afford the desired products 6 in good to excellent yields. Furthermore, under similar conditions, aryl aldehydes and para-toluenesulfonamide were reacted with 5,7-dihydroxy-4-methylcoumarin in the presence of the catalyst 1 (Table 2). According to the reported procedure in the literature,45 5,7-dihydroxy-4-methylcoumarin was synthesized via the ZrOCl2/SiO2-catalyzed condensation of phloroglucinol and ethyl acetoacetate. Recovery and reuse of the catalysts after catalytic reactions are important factors for sustainable process management. The recovered nanomagnetic catalyst 1 from the model reaction for the synthesis of 6a was separated by an Gholtash et al.: Molybdic Acid-Functionalized 872 Acta Chim. Slov. 2020, 67, 866-875 external magnet, washed with MeOH and reused in the next run. As outlined in Fig. 6, Fe3O4@TiO2@(CH2)3O-MoO3H exhibited no considerable decrease in activity after six cycles. This advantage besides the recoverability of the biodegradable and green catalyst makes this research highly favorable for large-scale synthesis. A plausible mechanism for the synthesis of product 6 is outlined in Scheme 3. It is reasonable to assume that pa-ra-toluenesulfonamide attacks the activated arylaldehyde by the acid catalyst to give sulfonyl aldimines 4. Then, intermediate 7 or 8 is generated from the condensation of 5 (5,7-dihydroxy-4-methylcoumarin or 4-hydroxycouma-rin) with 4 which rearranges into the desired product. The filtration test was performed to show the heterogeneity of the catalyst. For this purpose, after the development of about 40% of the model reaction (synthesis of 6a), the catalyst was removed and the progress of the residual mixture was monitored. Interestingly, no further conversion was observed in this case. Therefore, we found that the catalyst operates in a heterogeneous manner and no leaching of molybdic acid as active species occurs under the applied conditions. 3. Experimental All chemicals used in this research were purchased from Fluka and Merck chemical companies. The monitoring of the reaction progress and the purity of the compounds were accomplished using TLC performed with silica gel SIL G/UV254 plates. Melting points were determined by an electrothermal KSB1N apparatus and are uncorrected. :H NMR spectra were recorded in DMSO-d6 on a Bruker Avance Ultra Shield 400 MHz instrument spectrometers and 13C NMR spectra were recorded at 100 MHz. X-ray powder diffraction (XRD) patterns were recorded using a Bruker AXS (D8 Advance) X-ray diffrac-tometer with Cu Ka radiation (X = 0.15418 nm). The measurement was made in 20 ranging from 10° to 80° at the speed of 0.05° min-1. For the preparation of the sample for XRD, after synthesis, the sample was powder, which was clumpy, and then XRD was measured. Analysis conditions includes: voltage: 40 kV and current: 30 mA. Energy dispersive spectroscopy (EDS) was performed using a TES-CAN Vega model instrument. The morphology of the particles was observed by scanning electron microscopy (SEM) under an acceleration voltage of 26 kV. Before SEM characterization, the sample was powdered and then coated with gold. The magnetic measurement was carried out in a vibrating sample magnetometer (VSM) at Kashan University (Kashan, Iran) at room temperature. Synthesis of Fe3O4 MNPs A solution of FeCl3-6H2O (1.35 g, 5 mmol) and FeCl2 • 4H2O (0.5 g, 2.5 mmol) (dissolved in 22 mL double distilled water) stirred under argon atmosphere at 80 °C for 30 min. Next the sodium hydroxide solution (2.5 mL, 10 M) was added dropwise to the reaction mixture with stirring for 1 h under argon atmosphere. Finally, the formed nanoparticles were collected using an external magnet. The nano particles were washed with distilled water repeatedly and then dried at 60 °C.47 Synthesis of nano-Fe3O4@TiO2 Fe3O4 nanoparticles were added to a mixture of eth-anol and acetonitrile (250:90 mL) and sonicated for 25 Gholtash et al.: Molybdic Acid-Functionalized ... Acta Chim. Slov. 2020, 67, 866-873 875 min. Next an aqueous ammonia solution (1.5 mL, 25 W%) was added and the resulting mixture was stirred at 25 °C for 30 min. Then, tetraethyl orthotitanate (3 mL) dissolved in absolute ethanol (20 mL) was added. After stirring for 2 h the obtained mixture was washed completely with absolute ethanol and was collected with an external magnet.37 Synthesis of Fe3O4@TiO2@(CH2)3Cl Nano-Fe3O4@TiO2 (0.6 g) was added in 3-chloro-propyltrimethoxysilane (6 mL) and stirred for 12 h under argon atmosphere and reflux conditions. This product was then separated via a magnet and washed repeatedly using toluene, ethanol-water and distilled water. Finally, Fe3O4@ TiO2@(CH2)3Cl was dried in an oven at 60 °C.48 Preparation of Fe3O4@TiO2@(CH2)3OMoO3H A mixture of Fe3O4@TiO2@(CH2)3Cl (1 g) and Na-2MoO4-2H2O (0.5 g) in DMSO (10 mL) was stirred at reflux under argon atmosphere for 12 h. The resulting product was decanted and washed twice with DMSO, once with distilled water and was dried at 50 °C for 18 h. Then, it was added to the flask containing HCl (60 mL, 0.1 N) and stirred for 2 h at room temperature. The resulting catalyst was decanted, washed with DMSO and water, afterwards dried at room temperature. General Procedure for the Synthesis of 6 Catalyst 1 (0.003 g) was added to the mixture of pa-ra-toluenesulfonamide (1 mmol), aldehyde (1 mmol) and 4-hydroxycoumarin (1 mmol) or 5,7-dihydroxy-4-methyl-coumarin (1 mmol). The resulting mixture was stirred in an oil bath (90 °C) under solvent-free conditions. The reaction progress was monitored by TLC («-hexane/EtOAc, 2:1). After completion of the reaction, boiling methanol (10 mL) was added, and the catalyst was separated by an external magnet. The obtained powder was recrystallized from hot EtOH. Compound 6a: Yield: 0.357 g (85%), FT-IR (KBr) (umax, cm-1): 3438, 1660, 1616, 1569, 1496, 1348, 1303, 1093, 759, 816. 1H NMR (400 MHz, DMSO-d6) 5 (ppm): 2.51 (s, 3H), 6.39 (s, 1H), 7.15-7.19 (t, 3H), 7.23-7.27 (t, 2H), 7.33-7.40 (m, 4H), 7.60-7.64 (m, 2H), 7.92 (dd, J1 = 9.2 Hz, J2 = 1.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) 5 (ppm): 208.8, 166.0, 153.6, 152.1, 140.5, 139, 133.5, 132.6, 131.7, 128.6, 127.1, 126.0, 124.4, 124.3, 118.0, 116.5, 104.5, 57.0, 36.4. Compound 6b: Yield: 0.409 g (90%), FT-IR (KBr) (umax, cm-1): 3438, 1668, 1616, 1562, 1494, 1438, 1351, 1311, 887, 757. 1H NMR (400 MHz, DMSO-d6) 5 (ppm): 2.07 (s, 3H), 6.28 (s, 1H), 7.13-7.15 (d, 2H), 7.25-7.27 (d, 2H), 7.307.36 (m, 4H), 7.57-7.61 (m, 2H), 7.88 (dd, J1 = 8 Hz, J2 = 7.2 Hz, 2H), 7.92-8.5 (m, 2H). 13C NMR (100 MHz, DM-SO-d6) 5 (ppm): 208.3, 165.7, 165.3, 153.6, 152.9, 139.4, 138.7, 132.5, 131.3, 130.3, 127.4, 124.4, 123.3, 119.0, 118.2, 116.4, 115.7, 104, 31.3. Compound 6c: Yield: 0.773 g (84%), FT-IR (KBr) (umax, cm-1): 3438, 3255, 1664, 1631, 1604, 1567, 1492, 1330, 1164, 736, 673. 1H NMR (400 MHz, DMSO-d6) 8 (ppm): 2.31 (s, 3H), 6.32 (s, 1H), 7.16-7.37 (t, 8H), 7.57-7.61 (m, 2H), 7.89-8.10 (m, 4H). 13C NMR (100 MHz, DMSO-d6) 8 (ppm): 208.10, 173.16, 172.32, 162.62, 143.15, 142.42, 138.37, 129.05, 128.62, 125.37, 124.32, 122.42, 120.42, 108.15, 31.10. Compound 6d: Yield: 0.326 g (75%), FT-IR (KBr) (umax, cm-1): 3438, 1668, 1616, 1604, 1563, 1351, 1311, 1095, 736, 673. 1H NMR (400 MHz, DMSO-d6) 8 (ppm): 2.24 (s, 3H), 2.34 (s, 3H), 6.39 (s, 1H), 7.17-7.27 (m, 3H), 7.34-7.41 (m, 8H), 7.61-7.95 (m, 3H). 13C NMR (100 MHz, DMSO-d6) 8 (ppm): 208.10, 173.16, 172.32, 162.62, 143.15, 142.42, 138.37, 129.05, 128.62, 125.37, 124.32, 122.42, 120.42, 108.15, 36.11, 31.10. Compound 6k: Yield: 0.392 g (81%), FT-IR (KBr) (umax, cm-1): 3471, 3338, 3170, 1673, 1610, 1575, 1413, 1157, 1091. 1H NMR (400 MHz, DMSO-d6) 8 (ppm): 10.45 (s, 1H), 10.43 (s, 1H), 7.95 (d, 1H, J = 10.9 Hz), 7.87 (d, 1H, J = 10.0 Hz), 7.52 (d, 2H, J = 10.8 Hz), 7.28-7.08 (m, 5H), 6.20 (d, 1H, J = 10.8 Hz), 6.15 (s, 1H), 5.75 (s, 1H), 2.39 (s, 3H), 2.25 (s, 3H). 13C NMR (100 MHz, DMSO-d6) 8 (ppm): 159.2, 158.8, 157.2, 154.8, 153.6, 142.0, 138.8, 138.4, 131.4, 130.9, 128.9, 128.7, 128.2, 126.3, 125.9, 108.6, 103.9, 101.8, 98.5, 49.19, 23.6, 20.9. Compound 6l: Yield: 0.412 g (85%), FT-IR (KBr) (umax, cm-1): 3477, 3423, 3315, 1702, 1619, 1524, 1432, 1292, 1182. 1H NMR (400 MHz, DMSO-d6) 8 (ppm): 10.45 (s, 1H, OH), 7.88 (d, 1H, J = 8 Hz), 7.53 (d, 2H, J = 8 Hz), 7.11-7.28 (m, 6H), 6.20 (s, 1H), 6.16 (s, 1H), 5.77 (s, 1h), 2.41 (s, 3H), 2.28 (s, 3H). 13C NMR (100 MHz, DMSO-d6) 8 (ppm): 159.14, 157.17, 154.73, 141.98, 138.91, 138.31, 131.33, 130.90, 129.14, 128.80, 128.66, 128.12, 126.24, 125.90, 108.64, 104.12, 98.57, 49.12, 23.54, 20.88. MS (EI): m/z 485 (M+), 301 (C16H9ClO4+), 279 (C17H12O4+), 258 (C15HnClO2+). 4. Conclusion In summary, we have introduced Fe3O4@TiO2@ (CH2)3OMoO3H as a novel modified Fe3O4 MNPs. It was fully characterized by XRD, SEM, EDS, FT-IR and VSM analysis. Furthermore, the first application of this reusable nanocatalyst for the synthesis of sulfonamide containing coumarin derivatives was successfully examined. The use of Fe3O4@TiO2@(CH2)3OMoO3H as a green and safe catalyst under solvent-free conditions, high yield of pure products, short reaction times and a simple recovery procedure are the main promising points of this work. Gholtash et al.: Molybdic Acid-Functionalized 874 Acta Chim. Slov. 2020, 67, 866-875 Acknowledgements The authors gratefully acknowledge the partial support of this work by Yasouj University, Iran. 5. References 1. C. Sanchez, P. Belleville, M. Popalld, L. Nicole, Chem. Soc. Rev. 2011, 40, 696-753. DOI:10.1039/c0cs00136h 2. D. Zhang, X. Du, L. Shi, R. Gao, Dalton Trans. 2012, 41, 14455-14475. DOI:10.1039/c2dt31759a 3. M. B. Gawande, S. N. Shelke, R. Zboril, R. S. Varma, Acc. Chem. Res. 2014, 47, 1338-1348. DOI:10.1021/ar400309b 4. S. R. Bankar, S. Shelke, Res. Chem. Intermed. 2018, 44, 35073521. DOI:10.1007/s11164-018-3321-4 5. S. R. Bankar, Curr. Organocataly. 2018, 5, 42-50. DOI: 10.2174/2213337205666180611112941 6. M. B. Gawande, P. S.Branco, R. S. Varma, Chem. 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D0I:10.1039/C4CY00970C Povzetek Uspešno smo pripravili molibdensko kislino imobilizirano na nano-Fe3O4@TiO2 (Fe3O4@TiO2@(CH2)3OMoO3H), jo karakterizirali in uporabili kot katalizator za sintezo sulfonamidov, vsebujočih kumarinski fragment. Pripravi Fe3O4 nanodelcev s so-obarjanjem Fe2+ in Fe3+ ionov je sledila obdelava s tetraetil ortotitanatom, ki je vodila do nastanka Fe3O4@TiO2. Tako dobljene delce smo nato obdelali s 3-kloropropiltrietoksisilanom, nato pa reagirali še z molibdensko kislino ter dobili željeni katalizator, ki smo ga karakterizirali z XRD, SEM, EDS, FT-IR in VSM analizami. Fe3O4@ TiO2@(CH2)3OMoO3H smo uporabili kot katalizator za sintezo sulfonamidov, vsebujočih kumarinski fragment, ki smo jo izvedli v obliki trokomponentne reakcije med aril aldehidi, para-toluensulfonamidom in 4-hidroksikumarinom (ali 5,7-dihidroksi-4-metilkumarinom). Izolacija katalizatorja po končani reakciji in njegova ponovna uporaba ni pokazala nobene izgube njegove aktivnosti. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Gholtash et al.: Molybdic Acid-Functionalized DOI: 10.17344/acsi.2020.5847 Acta Chim. Slov. 2020, 67, 876-884 /^creative ^commons Scientific paper Quantum Mechanics/Molecular Mechanics Study on Caspase-2 Recognition by Peptide Inhibitors Petar M. Mitrasinovic* Center for Biophysical and Chemical Research, Belgrade Institute of Science and Technology, 11060 Belgrade, Serbia * Corresponding author: E-mail: pmitrasinovic.ist-belgrade.edu.rs@tech-center.com Received: 01-17-2020 Abstract For a variety of biological and medical reasons, the ongoing development of humane caspase-2 inhibitors is of vital importance. Herein, a hybrid (Quantum Mechanics/Molecular Mechanics - QM/MM), two-layered molecular model is derived in order to understand better the affinity and specificity of peptide inhibitor interaction with caspase-2. By taking care of both the unique structural features and the catalytic activity of human caspase-2, the critical enzyme residues (E217, R378, N379, T380, and Y420) with the peptide inhibitor are treated at QM level (the Self-Consistent-Charge Density-Functional Tight-Binding method with the Dispersion correction (SCC-DFTB-D)), while the remaining part of the complex is treated at MM level (AMBER force field). The QM/MM binding free energies (BFEs) are well-correlated with the experimental observations and indicate that caspase-2 uniquely prefers a penta-peptide such as VDVAD. The sequence of VDVAD is varied in a systematic fashion by considering the physicochemical properties of every constitutive amino acid and its substituent, and the corresponding BFE with the inhibition constant (K) is evaluated. The values of Ki for several caspase-2:peptide complexes are found to be within the experimental range (between 0.01 nM and 1 |M). The affinity order is: VELAD (Ki = 0.081 nM) > VDVAD (Ki = 0.23 nM) > VEIAD (Ki = 0.61 nM) > VEVAD (Ki = 3.7 nM) > VDIAD (Ki = 4.5 nM) etc. An approximate condition needed to be satisfied by the kinetic parameters (the Michaelis constant - KM and the specificity constant - kcat/KM) for competitive inhibition is reported. The estimated values of kcat/KM, being within the experimentally established range (between 10-4 and 10-1 |M-1 s-1), indicate that VELAD and VDVAD are most specific to caspase-2. These two particular peptides are nearly 1.5, 3 and 4 times more specific to the receptor than VEIAD, VEVAD and VDIAD respectively. Additional kinetic threshold, aimed to discriminate tightly bound inhibitors, is given. Keywords: Enzyme; human caspase-2; inhibition; peptide inhibitor; QM/MM; SCC-DFTB-D 1. Introduction Homologues that make up the caspase (casp) family of cysteine proteases are essential mediators of cellular processes, such as apoptosis, proliferation, and differentiation.1,2 They are synthesized and stored as inactive zymogens, as well as divided into inflammatory (caspase-1, -4, -5, -12 in humans and caspase-1, -11, and -12 in mice) and apoptotic (caspase-3, -6, -7, -8, and -9 in mammals) caspases according to their function and pro-domain structure. The functions of caspase-2, -10, and -14 can not be easily categorized. Apoptotic caspases are further sub-classified by their mechanism of action as initiators (caspase-8 and -9) and executioners (caspase-3, -6, and -7). The first identified mammalian member is caspase-2 and its physiological role is not quite clear. Caspase-2, one of the most evolutionarily conserved caspases, is inclined to behave as either executioner or initiator. In terms of substrate specificity, caspase-2 is similar to caspase-3 and -7 (executioner caspases). However, the long N-terminal caspase recruitment domain (CARD) of caspase-2 indicates its potential role as an initiator caspase.3 While the function of caspase-2 in the embryonic development of mice is questionable,4 its important role in stress-induced cell death pathways and tumor suppression is more cer-tain.5 The potential roles of caspase-2 in mediating nonapoptotic pathways (cell-cycle regulation and DNA repair) have been reported in terms of whether caspase-2 is mandatory for apoptosis under specific circumstanc-es,6,7 or whether it primarily functions in cell-cycle regula-tion.5 An elevated expression level of caspase-2 has been observed in the brain of patients with some neurodegener-ative disorders.8 In addition, the critical role of caspase-2 in mediating nonalcoholic steatohepatitis (NASH) patho-genesis, a chronic and aggressive liver condition not only in mice but probably in humans, has been highlighted.9 Mitrasinovic: Quantum Mechanics/Molecular Mechanics Study Acta Chim. Slov. 2020, 67, 876-884 877 Whereas many questions on caspase-2 physiology remain enigmatic, one of the key aspects for developing caspase-2 specific probes is related to the way in which caspase-2 gets activated. Peptide bonds are hydrolyzed using caspases (endo-proteases) in a reaction that depends on catalytic cysteine residues in the caspase active site and occurs only after certain aspartic acid residues in the substrate. Besides resulting in substrate inactivation, caspase-mediated processing may generate active signaling molecules that participate in apoptosis and inflammation. Caspase activities are strictly regulated by protein-protein interactions and by proteolysis.1 The crystal structures of caspase-2 in complex with several peptide inhibitors and comparison of the apo (substrate-free) and inhibited caspase-2 structures have revealed a recognition via several discrete catalytic steps: (i) activation of caspase-2 by breaking a nonconserved salt bridge between Glu217 (caspase-2 is the only human caspase with glutamate at position 217) and the invariant Arg378, (ii) formation of a catalytically competent conformation upon binding to a single substrate, and (iii) formation of the enzyme-substrate complex after having both active sites occupied by the substrate.10 Caspase-2 has been suggested to uniquely prefer a penta-peptide rather than a tetra-peptide, as required for efficient cleavage by other caspases.10 To gain more complete insights into the caspase-2/peptide recognition and further facilitate the design of caspase-2 inhibitors, a hybrid QM/MM approach is employed in this work. 2. Methods To obtain the initial atomic coordinates of the apo and inhibited caspase-2 structures, the experimental structures were retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB): 3R7S.PDB (apo caspase-2), 3R6G.PDB (caspase-2/ VDVAD), 3R5J.PDB (caspase-2/ADVAD), 3R7B.PDB (caspase-2/DVAD), and 3R6L.PDB (T380A/VDVAD).10 The sequence of the penta-peptide inhibitor was varied using single point mutations generated by applying the Mutagenesis engine of PyMol-v0.99 to the experimental structure 3R6G.PDB in a backbone-dependent fashion.11 Before running QM/MM calculations, the systems were prepared using the Amber 11 suite of programs.12,13 The solute was prepared using the Amber11 utility program tLeap in association with the ff99sb force field.14 Every inhibitor was initially prepared by parameteryzing its atom types, charges, and connectivity in order to be treated as part of the solute. The molecular geometry was optimized by Gaussian 98 at the MP2/6-31G* level of theory.15 The molecular electrostatic potential was calculated by Gaussian 98 at the HF/6-31G* level of theory,15 while the atomic charges were derived by means of the RESP fitting technique,16 which is part of AmberTools 1.5.12,13 Remaining parameters were assigned from the General Amber Force Field (GAFF),17 being entirely compatible with the ff99sb macromolecular force field.14 Every solute was solvated using a 10 A (1 A = 10-10 m) pad of TIP3P water molecules (~ 11500) and the counter ions Na+ were added to neutralize each system. To remove clashes and bad contacts, two-stage geometric minimization was performed using the Sander module of Amber11.12,13 At the outset, the positions of the solute atoms were kept fixed, while the positions of the water atoms were minimized by gradually reducing an initial harmonic restraint of 2 kcal mol-1 A-2 on all non-hydrogen non-water atoms via 5000 combined steepest descent (2500 steps) and conjugate gradient (2500 steps) minimization steps. Afterwards, the entire system was minimized without restrains by means of 10000 combined steepest descent (5000 steps) and conjugate gradient (5000 steps) minimization steps. A two-layered hybrid approach was employed to assess the binding affinities within the caspase-2:peptide complexes. The outer layer of the complex (Figure 1) was Figure 1. The two-layered QM/MM (SCC-DFTB-D/AMBER) model was used to evaluate the efficacy of peptide inhibitors towards caspase-2 (PDB ID: 3R6G). Mitrasinovic: Quantum Mechanics/Molecular Mechanics Study ... 878 Acta Chim. Slov. 2020, 67, 876-884 kept at the low level of theory (MM) with an Amber force field. The central layer of the complex (bold sticks; Figure 1) was treated at the high level of theory (QM), employing SCC-DFTB-D, the Self-Consistent-Charge Density-Functional Tight-Binding method18,19 with Dispersion energy,20 as implemented in Amberll.21,22 The inclusion of the empirical correction for dispersion energy into SCC-DFTB provided a balanced and reliable description of the interactions inside the systems.23 Pure Density Functional Theory (DFT) methods are known for their modest computational costs, but they are not able to adequately describe dispersive forces, especially within unconventional systems,24,25 as many density functionals are empirical.20 DFT was extended to include dispersion correction (DFT-D),26,27 and as such DFT-D became suitable for performing energy minimizations and vibrational analyses of extended molecular complexes containing hundreds of atoms. By being a few orders of magnitude faster than DFT-D,23,28-33 SCC-DFTB-D was suggested to be applicable to both quantumchemical simulations and calculations pertaining to a large number of extended molecular com- plexes.23 The total interaction energies were defined as: A F = A F int eraction "* mod el,high -A F -A F real, low mod el,low (1) where A£mojel denote the energies of the model system defined at high and low level of theory and AEreal denotes the whole (real) system. Therefore, the equivalent binding free energies of the complex systems were determined as: (2) The thermodynamic quantities (enthalpies, entropies, and different entropie contributions) were obtained from frequency calculations done by the Nmode module of Amberll.12,13 The different entropic contributions (translation, rotation, and vibration) for caspase-2:peptide complexes were calculated as: A S casp-2:peptide casp-2 ^peptide 1 (3) 3. Results and Discussion Kinetic measurements of competitive inhibition associated with the initial experimental structures10 are summarized in Table 1. The specificity constant (fccat/KM) identified the penta-peptide VDVAD as a preferred inhibitor, while two residues, Thr380 and Tyr420, were identified as critical for recognizing a residue at the P5 position - the first position at the left end in the peptide sequence (Figures 2b & 2c). The salt bridge between Glu217 and Arg378, which is present in the apo caspase-2 (3.37 Â, Figure 2a), is broken in the caspase-2:VDVAD complex (8.05 Â, Figure 2b), because Thr380 and Tyr420 in P5 rec- ognition move 2.1 and 3.6 A, respectively (Figure 2d). Furthermore, the specificity constant revealed that mutation of Thr380 to Ala reduces the catalytic efficiency of caspase-2 by about 40 fold (Table 1), as Thr380Ala (Figure 2c) causes the loss of the hydrogen bond between Thr380 and the P5 side chain (3.51 A, Figure 2b) due to a 2.3 A movement in the main chain in residue 380. Structurally speaking in a similar manner, mutation of Tyr420 to Ala reduces the catalytic efficiency of caspase-2 by about 4 fold (Table 1), as Tyr420Ala causes a 0.5 A movement of the side chain of residue 420 and the loss of the hydrophobic interaction between Tyr420 and the P5 side chain.10 Table 1. Kinetic data10 for experimental caspase-2:peptide inhibitor complexes: KM - Michaelis constant, kcat - catalytic constant, kcat/ Km - specificity constant, and IC50 - inhibitory concentration Complex(a) PDB ID *M(b) fccat/^M IC5o(b),(c) (^M) (s-1) (^M-1 s-1) (nM) wt: VDVAD 25 0.60 0.024 25 3R6G wt:ADVAD 150 0.81 0.0055 110 3R5J wt:DVAD 92 0.12 0.0013 710 3R7B Y420A:VDVAD 84 0.52 0.0062 314 Y420A of 3R6G T380A: VDVAD 220 0.13 0.00060 347 3R6L T380A/Y420A:VDVAD > 400 < 0.000014 N/A(d) 574 Y420A of 3R6L « wild-type (wt) casp-2, Ala (A), Asp (D), Thr (T), Tyr (Y), Val (V) (b) 1 |rM = 10-6 M, 1 nM = 10-9 M (c) IC50 represents the concentration at which a substance exerts half of its maximal inhibitory effect. (d) It is unreliable to measure the catalytic efficiency values for the slowest (kcat/KM < 10 ' reactions.36 ^M-1 s-1) and fastest (kcat/KM > 10-1 ^M-1 s-1) To perform physically realistic QM/MM calculations, the first important aspect is how to define a QM region, or what caspase-2 residues need to be included in the QM region. There are no good universal rules here. Binding site residues of caspase-2 that are involved in non-covalent interactions with a peptide inhibitor are: Arg219, His277, Gly278, Gln318, Cys320, Ala376, Arg378, Asn379, Thr380, Trp385, Arg417, Glu418, and Tyr420.10 Caspase-2 is the only human caspase with glutamate at position 217 forming a salt bridge with Arg378 in the apo caspase-2 (Figure 2a). The inhibition of caspase-2 was related to breaking the Glu217-Arg378 salt bridge, while residues Thr380 and Tyr420 were pointed out as the key elements for recognizing a preferred penta-peptide along a catalytic pathway (Figure 2b).10 An intention to define a QM region to mimic the active site has to take into account all these experimental and structural arguments. Knowing that inclusion of a different number of caspase-2 residues in the QM region is associated with different thermodynamic Mitrasinovic: Quantum Mechanics/Molecular Mechanics Study ... Acta Chim. Slov. 2020, 67, 876-884 879 Figure 2. QM region is based upon some important experimental facts10 associated with caspase-2/peptide recognition: (a) apo (ligand-free) caspase-2 with Glu217-Arg378 salt bridge (3.37 A), (b) caspase-2:VDVAD without Glu217-Arg378 (8.05 A) salt bridge, (c) Thr380Ala:VDVAD without Glu217-Arg378 (7.70 A) salt bridge, and (d) the overlay of the enzyme residues of apo caspase-2 (black), caspase-2:VDVAD (blue), and Thr380Ala:VDVAD (yellow). VDVAD is denoted by bold sticks in (b) and (c), P5 - the first position at the left end in the peptide sequence. Table 2. Binding free energies that are evaluated using QM/MM (SCC-DFTB-D/AMBER) method for experimental caspase-2:peptide structures Complex(a) PDB ID AGbind(b) AH (kcal mol-1) (kcal mol-1) TAStotal TAStrans (kcal mol-1) (kcal mol-1) TASrot TASvib (kcal mol-1 ) (kcal mol-1 ) wt:VDVAD -13.22 3R6G wt:ADVAD -9.83 3R5J Y420A: VDVAD -9.17 Y420A of 3R6G T380A:VDVAD -8.71 3R6L T380A/Y420A:VDVAD -5.53 Y420A of 3R6L wt:DVAD -2.75 3R7B -31.85 -30.35 -30.29 -29.74 -25.69 -24.28 -18.63 -20.52 -21.12 -21.03 -20.16 -21.53 -12.90 -12.87 -12.87 -12.90 -12.81 -12.81 -10.61 -10.56 -10.38 -10.80 -10.53 -10.58 4.88 2.91 2.13 2.67 3.18 1.86 (a) wild-type (wt) casp-2, Ala (A), Asp (D), Thr (T), Tyr (Y), Val (V) (b) Gibb's free energy (AG), enthalpy (AH), entropy (TAS) and entropie contri- bution, translational (TAStrans), rotational (TASrot), vibrational (TASvib) are derived from Eqs. 2 and 3. Mitrasinovic: Quantum Mechanics/Molecular Mechanics Study ... 880 Acta Chim. Slov. 2020, 67, 876-884 properties such as the binding free energies means that an appropriate QM region is supposed to generate results in agreement with experimental data. Even though one might want to have as large a QM region as possible, having more than 80-100 atoms in a QM region lead to simulations that are computationally very expensive.12,13 To reconcile all the structural, functional, and computational standpoints as much as possible, the present choice of including Glu217, Arg378, Asn379, Thr380, Tyr420, and the peptide inhibitor in the QM region (Figure 1) is carefully made in order to 0 100 200 300 400 500 600 700 800 0 .......I.In I III....... ................ I.......I .......... -1 -2 -! ■14 - IC501 nM Figure 3. Correlation of the calculated binding free energy (AG^) with the measured inhibitory concentration (IC50) for the experimental caspase-2:peptide structures. AGbind = 0.013 IC50 -12.85, R = 0.97. generate the inhibition constants that are within an experimental range - from 1 ^M (1 ^M = 10-6 M) to 0.01 nM (1 nM = 10-9 M) for inhibited caspase-2 structures.34 QM/MM binding free energies for the experimental structures are given in Table 2. Figure 3 shows quite a satisfactory linear correlation between the calculated AGbind (Table 2) and the experimental inhibitory concentration IC50 (Table 1): AGbind = 0.013 IC50 -12.85, R = 0.97. The most negative BFE for the caspase-2: VDVAD complex (-13.22 kcal mol-1) signifies that VDVAD is a favorable inhibitor. The enthalpy contribution (AH) for the complexes ranges from -31.85 to -24.28 kcal mol-1, indicating that the noncovalent complexation process is exothermic. In case of the entropy contribution (TAS), the less negative entropy change is, the more reduced degrees of freedom of an inhibitor in the protein active pocket are. The least negative entropy (-18.63 kcal mol-1) is associated with caspase-2:VDVAD, of which vibrational contribution (4.88 kcal mol-1) makes a most conspicuous difference with respect to the other complexes (Table 2). The increased and thermodynamically favorable vibrational entropy change upon binding of VDVAD to caspase-2 is the signature of preferred noncovalent complexation. To search for more effective penta-peptides, the sequence of VDVwAD is systematically varied by means of single point mutations of its constitutive residues. To make such a procedure consistent, each amino acid is mutated to its counterpart observed from a physicochemical standpoint. Val (V), an aliphatic and hydrophobic amino acid, is mutated to either Ile (I) or Leu (L). Asp (D), a polar and Table 3. QM/MM binding free energies that are within experimental range (between -8.23 and -15.09 kcal mol-1) for caspase-2:peptide complexes Complex(a) AGbind VDVAD (Ki = 0.23 nM) > VEIAD (Ki = 0.61 nM) > VEVAD (Ki = 3.7 nM) > VDIAD (Ki = 4.5 nM) etc. In order to evaluate the specificity constant for the complexes (Table 3), the correlation of the Michaelis constant with the specificity constant for the experimental caspase-2:peptide structures (Table 1) is observed. Even though two linear correlations are established (Figure 4), the first one (KM = -0.58 kcat/KM + 157.51, R = 0.74; Figure 4, top) is slightly more suitable because it reproduces the experimental value of the specificity constant for the caspase-2:VDVAD complex more accurately than the second one (Figure 4, bottom). Due to the negative slope (-0.58) of the linear regression line, KM < 157.51 ^M rep- Figure 5. Reaction of competitive inhibition.35 resents an approximate condition for the physically meaningful estimate of kcat/KM. To evaluate the functional efficiency of the complexes (Table 3) in terms of the specificity constant (kcat/KM), a competitive inhibition mechanism is considered (Figure 5). For such a reaction, the inhibition constant is defined as: K,= (if [.VI = K, = tcji I {'/[.vi» (4) where IC50 is the inhibitory concentration, KM is the Michaelis constant, and [S] is the substrate concentration.35 Solving Eq. 4 for the Michaelis constant gives: (5) The kinetic data are analyzed as follows. IC50 is evaluated using its linear correlation with AGbind (Figure 3). Km is evaluated using Eq. 5 with [S] ~ 2.7 mM (1 mM = 10-3 M) - a typical experimental value.10 Of these complexes (Table 3), those having KM < 157.51 ^M are selected (Table 4) and may be considered as competitively inhibited structures. The comparison of the values of KM (Table 4) with respect to [S] shows that [S] >> KM, what is in line with Ki << IC50 according to Eq. 4. The estimate of kcat/ Km (Table 4) is made by way of the linear correlation KM = -0.58 kcat/KM + 157.51 (Figure 4, top). The specificity constants (kcat/KM) are within the experimental range (between 10-4 and 10-1 ^M-1 s-1),36 indicating that VELAD and VDVAD are most specific to caspase-2. These two particular peptides are approximately 1.5, 3 and 4 times more specific to the enzyme than VEIAD, VEVAD and VDIAD respectively. In case of simple enzyme reaction with one substrate, if kcat << k-1, Km is conceivable as the dissociation constant that quantifies the strength of the ES complex formation. If the KM value gets smaller then the ES complex gets stronger (or more stable). In other words, the more pronounced enzyme affinity to the substrate is lined up with the more specific inhibition of the enzyme. The values of AGbind (Table 3), KM (Table 4) and kcat/KM (Table 4) conform to the trend. Mitrasinovic: Quantum Mechanics/Molecular Mechanics Study ... 882 Acta Chim. Slov. 2020, 67, 876-884 Table 4. Evaluated competitive inhibition data for caspase-2:peptide complexes: Ki - inhibition constant, IC50 - inhibitory concentration, KM -Michaelis constant, fccat - catalytic constant, and fccat/KM - specificity constant Complex(a) AGbind (kcal mol ') K (^M)(b) IC50 (nM)(b) Km (^M)(b)> (c) kcat (s 1) kcat/KM (^M-1 S-1) casp-2:VELAD -13.84 0.000081 23.88 9.19 0.24 0.026 casp-2:VDVAD -13.22 0.00023 25.00 25.00 0.58 0.023 casp-2:VEIAD -12.64 0.00061 26.15 64.48 1.03 0.016 casp-2:VEVAD -11.57 0.0037 98.46 105.43 0.94 0.0089 casp-2:VDIAD -11.45 0.0045 107.69 117.75 0.80 0.0068 (a) Ala (A), Asp (D), Glu (E), Ile (I), Leu (L), Val (V) (b) 1 |iM = 10-6 M, 1 nM = 10-9 M (c) Of the complexes given in Table 3, those having KM < 157.51 |iM are reported here. Km < 157.51 |iM is an approximate condition for the physically meaningful estimate of kcat/KM (Figure 4, top). For tightly bound inhibitors, the inhibition constant is: (6) where [£] is the enzyme concentration.35 For tightly bound inhibitors, the equation 6 takes into account the larger amounts of inhibitor bound species, thus making that the Michaelis-Menten assumption of the total enzyme concentration being equal does not hold.35,37 The Michaelis constant for tightly bound inhibitors is: (7) The condition KM < 157.51 ^M for tightly bound peptides means: (8) Therefore, Solving Eq. 9 for IC50 gives: (9) (10) For [S] ~ 2.7 mM and [£] ~ 50 nM - typical experimental values,10 Eq. 10 gives an approximate condition, IC50 (nM) > 17.14 Ki (nM) + 25, which should be satisfied by the tight binding of penta-peptides to caspase-2. The values of IC50 and Ki (Table 4) indicate that VEVAD and VDIAD satisfy this condition. By reducing [£] from 50 to 44.8 nM, the condition becomes IC50 (nM) > 17.14 Ki (nM) + 22.4 and is satisfied by VELAD, VEVAD and VDIAD. If [£] is additionally lowered to 42 nM then the condition gets IC50 (nM) > 17.14 Ki (nM) + 21, indicating that VELAD, VDVAD, VEVAD and VDIAD may be considered as tightly bound inhibitors. In most experimental investigations of enzyme kinetics, the total concentration of substrate is in excess of the enzyme concentration, thus making the free and total substrate concentrations essentially equal.35 For a set of inhibitor candidates, comparison of or IC50 values is only assumed to be valid when these values are evaluated under identical experimental conditions.35,38 Only few data exist on the catalytic efficiencies of caspase substrates, so that a more complete understanding of their true substrate preferences is impossible.36 The present results are imagined to facilitate additional experiments, which are needed to understand better the kinetics of caspase-2/peptide recognition for further research or therapeutic product development. The fact that caspase inhibition-based drug has not been approved on the market so far means that the development of therapeutic approaches that specifically target caspases is a substantial challenge of particular biological and clinical interest.39 4. Conclusions QM/MM model derived and exploited in this work has been shown to correlate with the existing experimental observations to an appreciable extent, indicating that caspase-2 uniquely prefers a penta-peptide such as VDVAD. This approach has enabled the extensive and systematic investigations of some of the important aspects both of the thermodynamics and of the kinetics of caspase-2 recognition by a large number of penta-peptides. The sequence of VDVAD has been consistently varied and the corresponding binding free energies with the inhibition constants have been evaluated. The values of the inhibition constants, being within the experimental range for several caspase-2:peptide complexes, have indicated that the affinity order is: VELAD > VDVAD > VEIAD > VEVAD > VDIAD etc. The specificity constants for competitive inhibition have been estimated to fit the experimentally predicted range, thereby suggesting that VELAD and VDVAD are most specific to caspase-2; also both are about 1.5, 3 and 4 times more specific to the receptor than VEIAD, VEVAD and VDIAD respectively. An approximate kinetic Mitrasinovic: Quantum Mechanics/Molecular Mechanics Study ... Acta Chim. Slov. 2020, 67, 876-884 883 threshold, supposed to discriminate tightly bound peptide inhibitors, has been reported. This study has demonstrated that a well-calibrated computational work may yield information inaccessible by other methods or suggest new experimental procedures. Acknowledgment Prof. David A. 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D0I:10.1073/pnas.1524900113 37. P. J. Henderson, Biochem. J. 1972, 127, 321-333. D0I:10.1042/bj1270321 38. P. J. Munson, D. Rodbard, J. Recept. Res. 1988, 8, 533-546. D01:10.3109/10799898809049010 39. J. Kudelova, J. Fleischmannova, E. Adamova, E. Matalova, J. Physiol. Pharmacol. 2015, 66(4), 473-482. Povzetek Zaradi različnih bioloških in zdravstvenih razlogov je razvoj humanih zaviralcev kaspaze-2 ključnega pomena. V članku je izpeljan hibridni (kvantno mehanski / molekularno mehanski - QM / MM), dvoplastni molekulski model s ciljem boljšega razumevanja afinitete in specifičnosti interakcije peptidnih zaviralcev s kaspazo-2. Z upoštevanjem edinstvenih strukturnih značilnosti in katalitične aktivnosti človeške kaspaze-2 se kritični aminokislinski preostanki encima (E217, R378, N379, T380 in Y420) s peptidnim zaviralcem obravnavajo na ravni QM (z uporabo t.i. Self-Consistent-Charge Density-Functional Tight-Binding method with the Dispersion correction (SCC-DFTB-D)), preostali del kompleksa pa se obravnava na ravni MM (t.i. AMBER force field). QM/MM vezavne proste energije (VPE) dobro korelirajo z eksperimentalnimi opazovanji in kažejo, da kaspaza-2 daje prednost penta-peptidu, kot je VDVAD. Zaporedje VDVAD smo sistematično spreminjali tako, da smo upoštevali fizikalno-kemijske lastnosti vsake aminokisline in njenega substituenta, pri čemer smo ovrednotili ustrezne VPE z inhibicijsko konstanto (Ki). Vrednosti Ki za več kompleksov kaspaza-2:peptid-ni inhibitor se nahajajo v eksperimentalnem območju (med 0,01 nM in 1 |M). Zaporedje afinitet je: VELAD (Ki = 0,081 nM)> VDVAD (Ki = 0,23 nM)> VEIAD (Ki = 0,61 nM)> VEVAD (Ki = 3,7 nM)> VDIAD (Ki = 4,5 nM) itd. Navajamo pogoj aproksimacije, ki mu je potrebno zadostiti s kinetičnimi parametri (Michaelisova konstanta - KM in konstanta specifičnosti - kcat / KM) za kompetitivno inhibicijo. Ocenjene vrednosti kcat / KM v eksperimentalno določenem območju (med 10-4 in 10-1 |M-1 s-1) kažejo, da sta VELAD in VDVAD najbolj specifična za kaspazo-2. Ta dva peptida sta skoraj 1,5, 3 in 4-krat bolj specifična za receptor od peptidov VEIAD, VEVAD in VDIAD. Naveden je tudi dodatni kinetični prag, ki je namenjen razločevanju med tesno vezanimi zaviralci. © ® Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Mitrasinovic: Quantum Mechanics/Molecular Mechanics Study ... DOI: 10.17344/acsi.2020.5864 Acta Chim. Slov. 2020, 67, 885-895 /^.creative o'commons Scientific paper Antimüllerian Hormone and Oxidative Stress Biomarkers as Predictors of Successful Pregnancy in Polycystic Ovary Syndrome, Endometriosis and Tubal Infertility Factor Teja Fabjan,1,3 Eda Vrtačnik-Bokal,2 Irma Virant-Klun,2 Jure Bedenk,2 Kristina Kumer1,3 and Joško Osredkar1,3^ 1 University Medical Centre Ljubljana, Institute of Clinical Chemistry and Biochemistry, Njegoševa 4, 1000 Ljubljana, Slovenia 2 University Medical Centre Ljubljana, Division of Gynaecology, Department of Human Reproduction, Šlajmerjeva 3, 1000 Ljubljana, Slovenia 3 University of Ljubljana, Faculty of Pharmacy, Aškerčeva cesta 7, 1000 Ljubljana * Corresponding author: E-mail: josko.osredkar@kclj.si Received: 01-29-2020 Abstract Oxidative stress in the follicular fluid (FF) is thought to be responsible for the abnormal development of oocytes. In our study patients with polycystic ovarian syndrome (PCOS), endometriosis, and tubal infertility factor (TIF), and healthy women with a male factor of infertility, were prospectively enrolled. From each patient, a sample of individual FF was collected from a dominant follicle. Concentration levels of TAS, 8-IP, 8-OHdG, and AMH were determined. In women with PCOS, we found significantly lower values of oxidative stress markers in the FF. 8-IP and TAS levels were lower in the FF of women with endometriosis. In women with TIF, we also found significantly lower values of all tested markers in the FF, except for 8-OHdG and AMH. We wanted to see whether the biomarker measured in the FF in an individual diagnosis could predict a successfully obtained embryo from this particular follicle. The FF 8-OHdG result in PCOS patients stood out and proved to be a good predictive marker of matured and fertilized oocytes in these patients. Further research is needed to be able to apply the acquired knowledge in improving the outcome of IVF procedures. Keywords: Oxidative stress; Antimullerian hormone; Polycystic ovary syndrome; Endometriosis; Tubal infertility factor; Infertility 1. Introduction The overall prevalence of infertility is 12.5% among women and 10.1% among men, and this rate is rising. The causes vary; among female diagnoses the most common are ovulation disorders, including PCOS, as well as endometriosis and various fallopian tubes defects. The prevalence of those seeking help has been reported even above 50%.1 Environmental and lifestyle factors affect the couple's fertility status through a series of known and unknown mechanisms. The reproductive organs have the highest number of mitochondria in the human body.2 This is needed because of the high requirement of energy production via ATP. On the other hand, this makes these organs highly susceptible to elevated levels of reactive oxygen or nitrogen species (ROS/RNS). Oxidative stress (OS) has received extensive attention in the past two decades due to the discovery that abnormal oxidation status is related to patients with chronic diseases, such as diabetes, cardiovascular, polycystic ovary syndrome (PCOS), endometriosis, cancer, and neurological diseases.3-6 Oxidative stress occurs when oxidants outnumber antioxidants, then products of peroxidation develop, and then pathological effects are caused by these phenomena. ROS are produced mainly within the mitochondrial electron transport chain and must be constantly deactivated to avoid excess formation to maintain normal cell function.7 Fabjan et al.: Antimüllerian Hormone and Oxidative Stress Biomarkers 886 Acta Chim. Slov. 2020, 67, 885-895 In vitro fertilization (IVF) is a widely accepted infertility treatment and is often the only option for infertile couples to have a baby. Unfortunately, the success of this technique, measured as an average pregnancy rate per cycle, is only 30-40% for women under age 40.8-10 Several studies have reported signs of oxidative stress in the FF of infertile women.7,11-14 It has been suggested that OS is responsible for normal oocyte development, due to DNA and cell membrane damage, which would then result in reduced oocyte quality, altered fertilization, and different embryo quality, implantation, and embryonic development. Elevated OS is also associated with ovarian ageing. Low-quality oocytes contain increased amounts of damaged DNA and chromosomal aneuploidy, secondary to age-related dysfunctions.15 It has been predicted that the concentration of AMH influences the number of oocytes retrieved during the IVF process. However, to date, the relationship between FF AMH and oocyte quality is unclear. The AMH level in the individual follicle was found to inversely correlate with the oocyte's maturity and developmental potential.16 In contrast, it was observed that oocytes capable of producing high levels of AMH were much easier to fertilize in nor-mo-ovulatory females.17 In PCOS patients, however, it has even been shown that the proportion of mature oocytes, as well as fertilization success, does not correlate with FF AMH.18 In their study, Fanchin et al. showed that FF AMH is a better predictor of fertilization and implantation of embryo than serum AMH in normo-ovulatory women.19 In Korea, these results have recently been confirmed on a smaller sample.20 However, there have been very few studies on the relationship between FF AMH levels and the quality of oocyte and embryo. The tubal factor of infertility, PCOS, and endometri-osis are the main indications in patients undergoing IVF procedures. PCOS is a disease with high heterogeneity, and its clinical features mainly include menstrual disorder, secondary amenorrhea, serum hormone abnormalities, hirsutism, acne, obesity, and infertility.22 It is estimated that it affects 3-15% of all women.23 The primary cause of the disorder is an abnormality in the ovaries, but additional agents, such as obesity and environmental factors, affect the development of individual symptoms.24 Endometriosis is also one of the most common gynecologic diseases in women of reproductive age. It is characterized by implantation and growth of endometrial tissue (glands and stroma) outside the uterine cavity. Endometriosis is an estrogen-dependent pelvic inflammatory disease. The prevalence in women with pelvic pain ranges from 30-40% of the infertile population. Endometriosis can be also asymptomatic or accompanied by symptoms such as dysmenorrhea and dyspareunia.25,26 Many studies widely accepted that oxidative stress might be implicated in the pathophysiology of endometriosis causing a general inflammatory response in the peritoneal cavity.27-31 It is not known exactly how endometriosis causes infertility, but it is probably related to the inflammatory response resulting from the overproduction of prostaglan-dins, cytokines and macrophages, and natural killer cells. The inflammatory process thus impairs the function of the ovaries, peritoneal system, fallopian tubes, and endome-trium and leads to impaired folliculogenesis, fertilization, and other conditions. Tubal infertility factor (TIF) accounts for about 35% of all infertility cases.32 Pregnancy does not occur due to mechanical obstruction in the fallopian tube. There are several causes for tubal blockage: infection, inflammation, surgery due to ectopic pregnancy, adhesions due to abnormal immunochemical environment, or rarely a congenital anomaly.33 Many studies use TIF patients as a control group because the obstacle is considered purely mechanical. We decided to include it as a pathological group because the causes of tubal infertility may also be hormonal (e.g. endometriosis) and inflammatory and this could have a significant impact on oxidative stress measurements. The aim of this study was to evaluate OS in patients undergoing IVF procedure according to various indications, capabilities of fertilization, and embryo quality. We determined three different OS biomarkers and AMH in the FF of the dominant follicle containing oocyte. We have examined how their combination affects success rates in obtaining mature and fertilized oocytes in patients with PCOS, endometriosis, and TIF during IVF procedure. 2. Selected Biomarkers Antimullerian hormone (AMH) AMH is produced in the granulosa cells and is a member of the transforming growth factor ^ family. AMH is an excellent marker of ovarian reserves.34 The hormone levels in both the peripheral blood and intrafollicular fluid correspond with the rate of follicular maturation. AMH affects oocyte development during folliculogenesis, and the levels of AMH in the follicular fluid may affect the oocyte and embryo quality.20,35-37 8-Isoprostane (8-IP) Free radical attack induces lipid peroxidation. Lipid peroxidation is a self-propagating phenomenon terminated by antioxidants and the measurement of products of lipid peroxidation has commonly been used to assess OS. Isoprostanes are a series of prostaglandin F2-like compounds, in vitro and in vivo formed by free radical-catalyzed peroxidation of phospholipid-bound arachidonic acid, a pathway that is independent of the cyclooxygen-ase pathway.38-40 F2-Isoprostanes are considered the best available biomarkers of oxidative stress status and lipid peroxidation. Measurement of the level of lipid peroxi-dation as reflected by F2-isoprostane concentrations in biological fluids may help to identify those patients most likely to benefit from antioxidant treatment.41,42 Fabjan et al.: Antimüllerian Hormone and Oxidative Stress Biomarkers ... Acta Chim. Slov. 2020, 67, 885-887 895 8-hydroxy-2'-deoxyguanosine (8-OHdG) An oxidized derivative of deoxyguanosine is one of the most common oxidative modification in mutagenic damage and is used as a biomarker of OS. Oxidation of DNA occurs normally in vivo but also increases with exposure to oxidizing agents. Guanosines are very susceptible to oxidation, and this reaction can lead to G:C^T:A mutations. These mutations could have serious consequences. Oxidized bases are usually recognized and excised by special DNA repair machinery.43,44 Total antioxidant status (TAS) The antioxidant defense system has many components. The total antioxidant status (TAS) of follicular fluid samples was determined using a special metric. The Randox TAS kit measures the total antioxidant capacity of a sample, i.e. anything that has an antioxidant effect, including both enzymatic and non-enzymatic antioxidants. The reaction rate is calibrated with Trolox, which is widely used as a traditional standard for TAS measurement assays, and the assay results are expressed in mmol Trolox equivalent/L. 45,46 3. Materials and Methods 3. 1. Participants A total of 197 women with an indication for IVF/ ICSI treatment were prospectively enrolled in this study from March 2013 to April 2014 at University Medical Centre Ljubljana, Reproductive Medicine Unit. The research was approved by the ethics committee from the Slovenian National committee on medical ethics. Written informed consent was obtained from all participants. The study included four different groups: 36 patients with polycystic ovarian syndrome (PCOS), 72 with endometriosis, 41 with TIF, and 48 healthy controls. Healthy women whose in- fertility issues were caused by male partners were enrolled as controls. The demographic characteristics of the patient groups and control group are presented in Table 1. Figure 1 shows the number and share of all eggs collected and further the embryos during observation in this study. In our study, 197 dominant follicles were aspirated. Oocytes were obtained in 54% of these follicles. 81% of the oocytes were mature and 74% of these were fertilized. In this study group, 64 embryos were obtained and of these, 54 were successfully transferred at the end. 3. 2. Samples Collection All women underwent ovarian stimulation using a combination of GnRH analogues and gonadotrophins. On the day of oocyte retrieval, the FF from the dominant follicle was aspirated. FF aspiration was performed transvagi-nally using a transvaginal ultrasound probe as a guide, and a specific oocyte aspiration needle connected to a closed vacuum system. Only FF samples without blood clots were used for the measurements, so as to minimize any possible interference with the photometric assay. Blood contamination was evaluated by visual inspection, and samples that appeared cloudy or bloodstained were discarded. The FF samples collected were centrifuged at 3500 rpm for 10 min (to precipitate blood cells and to remove cellular components). All samples were stored at -80 °C until assayed. 3. 3. Sample Analysis The effect of oxidative stress was measured by 8- iso-prostane and 8-hydroxy-2'-deoxyguanosine and enzymatic antioxidant activity by TAS (the combined effect of all antioxidants). Expression levels of AMH, 8-IP, 8-OHdG and TAS levels were determined by using commercially available enzyme-linked immunosorbent assay (ELISA) Table 1: Demographic characteristics of the participants (mean or median of individual biomarkers are statistically analyzed and the p values indicating the significance of differences between different infertility groups individually with control group obtained by the t test or Mann-Whitney test as appropriate) Endometriosis PCOS Tubal factor of infertility Control group N 72 36 41 48 Age [years]; 33.8 P = 0.0013 30.8 P = 0.3621 32.3 P = 0.1597 31.62 (95% CI (33.1-34.5) (29.4-32.2) (31-33.5) (30.5-32.7) for the mean) Height [cm]; 165.9 P = 0.1746 164.8 P = 0.0310 167.5 P = 0.9985 167.4 (95% CI (164.3-167.5) (162.8-166.7) (165.8-169.2) (165.9-169.1) for the mean) Weight [kg] 60.3 P = 0.0292 70.7 P = 0.0582 65.3 P = 0.6207 63.4 (95% CI (58.5-62.0) (65.1-76.3) (61.6-69) (61.1-65.8) for the mean) BMI 21.65 P = 0.3831 24.5 P = 0.0111 22.45 P = 0.7096 22.45 (95% CI (21.2-22.5) (23-27) (21.2-24) (21.4-23.3) for the median) Fabjan et al.: Antimüllerian Hormone and Oxidative Stress Biomarkers ... 888 Acta Chim. Slov. 2020, 67, 885-895 Figure 1: Outcome of IVF procedure in patients enrolled in the study by stages kits according to the manufacturer's instructions. For 8-IP (Cayman Chemical Company, USA),47 8-OHdG (Ja-ICA - Japan Institute for the Control of Aging, Japan)48 and AMH (Anshlab)49 the lower and upper detection limits were estimated as 0.8-500 ng/L; 0.5-200 ng/mL and 3.8-1091 ng/L, respectively. Total antioxidant status (TAS) was evaluated by colourimetric method with Randox assay (Randox Laboratories Limited, UK).50 The results of the tests used are presented in Table 2. Table 2: Characteristic of the tests 3. 4. Statistical Analysis Statistical significance was calculated by two different tests: the Mann-Whitney U test. This test is non-parametric and does not require the groups to be normally distributed; it is more stable to outliers. The predictive value of biomarkers was determined using the "Receiver Operating Characteristic" analysis (ROC). P-values <0.05 were considered as significant. All analyses were made with statistical program Medcalc. Measuring Intra-assay Inter-assay Range variation (%CV) variation (%CV) AMH [pg/mL] Low 14.2-15.5 4.7 6.9 Medium 80.0-80.8 2.9 4.3 High 609.6-942.8 3.0 4.5 8-IP [pg/mL] Low 0.80-5.10 20.0 11.1 Medium 12.80-32.00 7.7 17.4 High 80.00-500.00 12.2 13.5 8-OHdG [ng/mL] Low 8.6-10.2 2.9 6.1 Medium 28.5-32.2 1.8 4.0 High 107.3-129.7 5.5 5.4 TAS Low 0.9-1.23 5.1 4.1 Medium 1.59-1.75 1.8 3.0 High 2.10-2.40 1.3 3.9 Fabjan et al.: Antimüllerian Hormone and Oxidative Stress Biomarkers ... Acta Chim. Slov. 2020, 67, 885-889 895 4. Results Follicular fluid from the dominant follicle of 197 women undergoing IVF was analyzed in this study. The groups were generally comparable with each other; only patients with endometriosis were slightly older on average. The BMI index is higher in patients with PCOS as Table 3: Hormonal status of the participants expected. The basal levels of serum hormones that affect the characteristics of infertility indications are presented in Table 3. The different diagnosis groups show variations in the levels of different hormones where it is typically expected, e.g. LH is elevated in the PCOS group. The analyzed data are summarized in Table 4 and presented graphically in Figure 2. Endometriosis PCOS TIF Control N = 72 N = 36 N = 41 group N = 48 S-FSH 7.4 P = 0.5454 6.0 P = 0.0085 6.6 P = 0.2103 7.1 S- LH 4.1 P = 0.9681 11.2 P < 0.0001 3.8 P = 0.4426 4.1 S-PRL 10.2 P = 0.7682 10.5 P = 0.9602 10.4 P = 0.9093 10.6 Table 4: Medians of individual biomarkers and interquartile ranges analyzed and the p values indicating the significance of differences between different groups of patients obtained by the Mann-Whitney U test PCOS (N = 36) Endometriosis Endometriosis TIF (N = 72) (N = 72) (N = 41) 8-OHdG 6.82 P = 0.0001 15.11 8-OHdG 15.11 P = 0.7539 16.32 [ng/mL] (4.66-11.45) (8.76-23.45) [ng/mL] (8.76-23.45) (9.77-22.41) 8-IP 85.97 P = 0.9682 91.07 8-IP 91.07 P = 0.5985 91.78 [pg/mL] (58.81-313.12) (60.15-170.09) [pg/mL] (60.15-170.09) (47.14-213.51) TAS 0.965 P = 0.0001 1.08 TAS 1.08 P = 0.0002 0.92 [mmol/L] (0.880-1.010) (0.945-1.160) [mmol/L] (0.945-1.160) (0.858-1.008) AMH 6.85 P = 0.0093 3.52 AMH 3.52 P = 0.0340 5.54 [U/mL] (3.49-11.26) (2.06-6.56) [U/mL] (2.06-6.56) (3.63-8.15) PCOS (N = 36) TIF Endometriosis Healthy (N = 41) (N = 72) (N = 48) 8-OHdG 6.82 P = 0.0001 16.32 8-OHdG 15.11 P = 0.8262 14.81 [ng/mL] (4.66-11.45) (9.77-22.41) [ng/mL] (8.76-23.45) (9.12-25.59) 8-IP 85.97 P = 0.6357 91.78 8-IP 91.07 P < 0.0001 253.36 [pg/mL] (58.81-313.12) (47.14-213.51) [pg/mL] (60.15-170.09) (125.47-556.10) TAS 0.965 P = 0.3712 0.92 TAS 1.08 P < 0.0001 1.275 [mmol/L] (0.880-1.010) (0.858-1.008) [mmol/L] (0.945-1.160) (1.150-1.355) AMH 6.85 P = 0.3814 5.54 AMH 3.52 P = 0.0895 4.64 [U/mL] (3.49-11.26) (3.63-8.15) [U/mL] (2.06-6.56) (2.69-8.18) PCOS (N = 36) Healthy TIF Healthy (N = 48) (N = 41) (N = 48) 8-OHdG 6.82 P = 0.0001 14.81) 8-OHdG 16.32 P = 0.9672 14.81 [ng/mL] (4.66-11.45) (9.12-25.59 [ng/mL] (9.77-22.41) (9.12-25.59) 8-IP 85.97 P = 0.0005 253.35 8-IP 91.78 P < 0.0001 253.35 [pg/mL] (58.81-313.12) (125.47-556.10) [pg/mL] (47.14-213.51) (125.47-556.10) TAS 0.965 P < 0.0001 1.275 TAS 0.92 P < 0.0001 1.275 [mmol/L] (0.880-1.010) (1.150-1.355) [mmol/L] (0.858-1.008) (1.150-1.355) AMH 6.85 P = 0.2306 4.64 AMH 5.54 P = 0.6537 4.64 [U/mL] (3.49-11.26) (2.69-8.18) [U/mL] (3.63-8.15) (2.69-8.18) Fabjan et al.: Antimüllerian Hormone and Oxidative Stress Biomarkers ... 890 Acta Chim. Slov. 2020, 67, 885-895 In women with PCOS, we found significantly lower values of oxidative stress markers in the FF (8-IP: 73.21 vs. 253.36 pg/mL, P = 0.0001; 8-hydroxy-2-deoxyguanosine: 6.82 vs. 14.81 ng/mL, P = 0.0001 and total antioxidant status: 0.97 vs. 1.28 mmol/L, P < 0.0001) and no difference in AMH concentration (6.9 vs. 4.6 U/mL, P = 0.2306) compared with the control group. 8-IP levels were also significantly lower in the FF of women with endometriosis (90.11 vs. 253.36 pg/mL, P < 0.0001) compared to control group. TAS levels were also lower in FF of endometriosis patients (1.08 vs. 1.28 mmol/L, P < 0.0001). No significant differences were found in FF-8-OHdG (15.11 vs. 14.81 ng/mL, P = 0.8262) and in FF-AMH (3.5 vs. 4.6 U/mL, P = 0.0895) between endo-metriosis and control group. In women with TIF, we also found significantly lower values of oxidative stress markers in the FF (8-IP: 57.18 vs. 253.36 pg/mL, P = 0.0001; and TAS: 0.97 vs. 1.28 mmol/L, P < 0.0001) and no difference in 8-OHdG concentration: 16.32 vs. 14.81 ng/mL, P = 0.0001 and AMH concentration (5.5 vs. 4.6 U/mL, P = 0.6537) compared with the control group. In the second part, we aimed to relate our results to the outcome of the IVF procedure and determine whether a single biomarker measured in the follicular fluid in an individual diagnosis could predict a successfully obtained matured and fertilized cell from that particular follicle. Figure 2 shows the accuracy measured by the area under the ROC curve (AUC). The area measures discrimination, i.e. the test's ability to correctly classify those with and without high-quality embryos ready for transfer. An area of 1 represents a perfect test; an area of 0.5 represents a worthless test. Of all the analyses shown, the FF 8-OHdG result in PCOS patients stood out and proved to be a very good predictive marker of obtaining a mature oocyte and of successful fertilization in these patients. At the limit of 6.18 ng/mL, with a sensitivity of 85.7% and a specificity of 86.4%, 8-OHdG separated those with a mature and those with immature oocyte (p <0.0001). 8-OHdG also separated those PCOS patients with a fertilized and those with unfertilized oocyte (p <0.0001), also at the limit 6.18 ng/ mL, with sensitivity of 84.62% and specificity of 82.61%. Figure 2 graphically shows both ROC curves for this bi-omarker. All other markers of OS and also AMH showed poor predictive value both in predicting obtaining a mature cell from a particular follicle and in obtaining fertilization. 5. Discussion In this study we confirmed for the first time that FF 8-OHdG is a good predictive biomarker for oocyte maturity and fertilization in PCOS patients. The evaluation of the pathophysiology of a couple's infertility has shown that oxidative stress (OS) may be one of the causative factors of female infertility, as recent studies shown.11,28,51-53 But so far there is still a big gap in our knowledge and understanding of individual mechanisms, and further research is needed to be able to use the acquired knowledge to improve the outcome of IVF procedures. Many degenerative changes to the oocytes during ageing are due to oxidative stress. We evaluate OS in patients attended to IVF procedure according to different indicators and we have come up with some very interesting results. We therefore decided in the present study also to include AMH as one of the investigated markers in the FF. AMH levels did not differ significantly between subjects with PCOS, endometriosis, TIF, and the control. However, an interesting trend suggesting lower concentrations A) AUC = 0,88« P0.0001 -I_I_I_I_I_I_I_I_I_I_I_I_I_L. 40 60 Specificity (%) B) Figure 2: ROC curve for 8-OHdG in PCOS patient group classify on two different outcomes (A-mature oocytes; B-fertilized oocytes) Fabjan et al.: Antimüllerian Hormone and Oxidative Stress Biomarkers ... Acta Chim. Slov. 2020, 67, 885-891 895 of AMH is in the group of patients with endometriosis. Lower concentrations of AMH in FF of the leading follicle in patients with endometriosis were also detected in the Spanish research group.54 They also observed that the presence of the endometrioma itself reduce even further AMH concentration in the surrounding follicles and suggest that these results could be useful when counselling patients regarding their reproductive outcome. In the PCOS group, we detected slightly higher concentrations of AMH in FF, which is comparable to the study conducted by Liu et al.55 AMH production starts in the very small follicles. The peak of production is reached and then the production rapidly declines. AMH production within the follicles is the part of the mechanism responsible for selection of the pre-ovulatory follicle.56 All follicles in our study were leading follicles of similar size, so a similar concentration of AMH is expected. In the TIF group, we did not detect a significant difference in the concentration of AMH in the follicular fluid as expected. Similarly, others have noted this, although they regarded this group as a control group.57,58 Another objective of our study was to determine the degree of oxidative stress in vivo in follicular fluid. Our results show some interesting differences between patients with PCOS, endometriosis, and TIF compared with healthy controls. The measured 8-IP concentration was found to be significantly higher in the control group than in all three patient groups. It is unclear why we obtained such results. One would expect to see less harmful OS products in healthy patients. A possible reason would be that patients are more concerned about the process and are taking more antioxidant supplements. There are known examples in the literature where vitamin supplements affect the concentration of lipid peroxidation products. Obesity and smoking can also affect the concentration of 8-IP.59 A recent meta-analysis showed that the intake of various antioxidant supplements can alter plasma F2-iso-prostane concentrations.60 However, we do not know what this means for concentrations in follicular fluid. There are only a few studies in which the concentration of FF 8-IP is measured. Malhotra et al. found that the 8-IP concentration is associated with abortion rates and is higher in patients with PCOS. But, unlike us, they took the whole pool of follicle fluid and not just the leading follicle. However, TIF patients were taken as the control group. In these patients, we also have a slightly lower 8-IP concentration, but the difference is not significant.61 In their pilot study, Lin and colleagues found a lack of correlation between 8-IP levels and age, and further found that similar 8-IP levels between the right and left follicles suggests that oxidative stress affects both ovaries equally.62 Pier analyzed the concentration of 8-IP in the follicular fluid using mass spectrometry and reached a similar conclusion, namely, the 8-IP concentration did not significantly increase with the age of the patients. Additionally, he also did not detect an increase in 8-IP concentration associated with PCOS or endometriosis. They concluded that these findings are at odds with the conventional assumption that 8-IP is a marker for oxidative stress. Instead, they suggested that F2-isoprostanes in FF may have functions unrelated to stress or inflammation.63 To date, we have not found any other researches that would measure 8-IP in follicular fluid. Some studies measured peritoneal fluid and plasma levels of 8-IP in vivo in patients with endometriosis. They found that concentrations in both the urine and peritoneal fluid of patients with endometriosis were significantly elevated compared to those of controls.64,65 Calzada et al. measured plasma 8-IP concentrations in patients with PCOS. They found that the level of 8-IP in patients was significantly increased. Our results in follicular fluid did not confirm this, as in our case the concentration of 8-IP was significantly increased in the control group. Based on all this information, it is difficult to conclude exactly what our results mean. Perhaps the 8-IP concentration in the follicular fluid from the leading follicle is not similar to that in other body fluids. Our study also shows that the concentration of 8-IP in the leading follicle has a weak effect on the effectiveness of the IVF procedure. It should be emphasized that our results of 8-IP measurements were very scattered in all groups, and some cross-reactivity might have occurred. According to the manufacturer's instructions, some types of sample may contain contaminants that interfere with the analysis. It is also known that several different prostaglandin derivatives are present in the follicular fluid.47,66 We estimate that this assay is not good for testing in follicle fluid and therefore no significant conclusions can be drawn from concentrations in this analyze. Due to the lack of clarity and poor research, further studies are needed. Our measurements of 8-OHdG in the leading follicle show similar concentration in controls and patients with endometriosis. This runs contrary to a study done in Bra-zil,67 where higher follicular concentrations of 8-OHdG were found in the endometriosis group compared to controls. A more recent study of OS in endometriosis patients led to results similar to ours.68 The concentration of 8-OHdG was similar in the control group to that of patients with endometriosis. But in both studies, all of the follicular fluid was used, not only from the leading follicle. In discussing the reasons for such a result it is worth mentioning the very interesting information that 8-OHdG also exhibited ROS-suppressing properties in several in vitro models, suggesting its possible involvement in the fine tuning of the response to OS.69 In fact, there are already several studies that investigate the mechanism where 8-OHdG sometimes show antioxidant or anti-inflammatory-like activity that can be attributed to the Racl-GTP pathway.70-72 We further failed to find a difference in the concentration of 8-OHdG between the TIF and control groups, which is consistent with extant findings.57 In patients with PCOS, the concentration of 8-OHdG in leading follicular fluid is significantly lower. Our result is consistent with other Fabjan et al.: Antimüllerian Hormone and Oxidative Stress Biomarkers ... 892 Acta Chim. Slov. 2020, 67, 885-895 studies, where authors have claimed that decreased serum levels of 8-OHdG can reflect an enfeebled repair of oxidative DNA damage or enhanced antioxidant defense rather than low ROS production in PCOS tissue. High ROS levels are well known to promote the expression of antioxidant enzymes.73 Therefore, overexpression of these antioxidants may lead to suppression of the extent of oxidative stress and consequently to the prevention of ROS interactions with DNA, thereby diminishing 8-OHdG formation. Several studies have reported that major antioxidant enzymes are significantly induced in subjects with PCOS compared to healthy subjects.74,75 Metformin therapies have also been shown to have the effect of lowering 8-OHdG levels in the serum of patients with PCOS, which also might be a reason for our results in follicular fluid. Metformin is a drug commonly used in the treatment of insulin resistance, which is very common in obese patients with PCOS, so a correlation is possible but as yet unverified.76 It would certainly be necessary to investigate further and determine in more detail the causes of such results. To begin with, the activity of the DNA glycosylase-repairing enzymes in FF should be checked. The results of our antioxidant status measurements show statistically significant higher TAS concentrations in the FF of healthy women compared to individual patients group (PCOS, endometriosis and also TIF). Our results are in perfect agreement with the rest of the literature. Some also found a positive association between FF TAS and clinical pregnancy rates.77,78 A very interesting and maybe most important finding that our research showed was that the concentration of 8-OHdG in PCOS group in the particular follicle showed a strong association with a mature and with a fertilized oocyte. As far as we know, to date, no one has tried to relate the concentration of 8-OHdG to the outcome of the IVF procedure in patients with PCOS. We have found that 8-OHdG, measured in a particular follicular fluid, can very well predict the acquisition of a mature egg and the successful fertilization of that egg. Anyway, our study alone is not enough and this link must be checked further on a larger sample. But if these results hold, the FF 8-OHdG could be a useful predictive marker for the individual oocyte in the artificial insemination procedure in PCOS patients. 6. Conclusion OS plays a role in several physiological processes, from oocyte maturation to fertilization and embryo development. There is burgeoning literature on the involvement of OS in the pathophysiology of infertility, assisted fertility, and female reproduction. What we do know is that the role of OS in female reproduction cannot be underestimated. Our study revealed a few significant differences in the concentrations of individual markers of oxidative stress and AMH between groups with different diagnoses. But the most interesting finding, one that is definitely worth exploring further, is the strong relationship between the concentration of 8-OHdG in follicular fluid and the obtaining of a useful mature cell from this follicle in PCOS patients, as well as the successful fertilization in the end of IVF procedure. Acknowledgments The authors would like to thank all the women in IVF programs who agreed to participate in the study, and to Vera Troha who processed samples in lab. Research funding: This study was financed by the Ministry of Science and Education through the Young Researchers program. The funding organizations played no role in the study design, in the collection, analysis, and interpretation of data, in the writing of the report, or in the decision to submit the report for publication. 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DOI: 10.3109/01443615.2016.1148683 Fabjan et al.: Antimüllerian Hormone and Oxidative Stress Biomarkers ... Acta Chim. Slov. 2020, 67, 885-895 895 Povzetek Oksidativni stres v folikularni tekočini (FF) naj bi bil odgovoren za nenormalen razvoj oocitov. V našo raziskavo so bile prospektivno vključene pacientke s sindromom policističnih jajčnikov (PCOS), endometriozo in tubarnim dejavnikom neplodnosti (TIF) ter zdrave ženske z dejavnikom moške neplodnosti. Od vsake bolnice je bil odvzet vzorec FF iz dominantnega folikla. Določene so bile koncentracije TAS, 8-IP, 8-OHdG in AMH. Pri ženskah s PCOS smo ugotovili bistveno nižje vrednosti označevalcev oksidativnega stresa v FF. Stopnje 8-IP in TAS so bile v FF žensk z endometriozo nižje. Pri ženskah s TIF smo ugotovili tudi bistveno nižje vrednosti vseh testiranih označevalcev v FF, razen za 8-OHdG in AMH. Želeli smo videti, ali lahko označevalec, izmerjen v FF pri posamezni diagnozi, napoveduje uspešnost pridobitve zarodka iz tega folikla. Rezultat 8-OHdG v FF pri pacientkah s PCOS je izstopal in se je izkazal za dober napovedni označevalec dozorelih in oplojenih oocitov pri teh pacientkah. Potrebne so nadaljnje raziskave, da bi lahko pridobljeno znanje uporabili za izboljšanje rezultatov postopkov IVF. Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Fabjan et al.: Antimüllerian Hormone and Oxidative Stress Biomarkers ... DOI: 10.17344/acsi.2020.5895 Acta Chim. Slov. 2020, 67, 896-903 /^creative ^commons Scientific paper Syntheses, Crystal Structures and Catalytic Property of Oxidovanadium(V) Complexes Derived from Tridentate Hydrazone Ligands Ya-Jun Cai,1,2 Yuan-Yuan Wu,1,2 Fei Pan,1,2 Qi-An Peng1,2 and Yong-Ming Cui3,* 1 School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, P. R. China 2 Engineering Research Center for Clean Production of Textile Printing, Ministry of Education, Wuhan 430073, P. R. China 3 National Local Joint Engineering Laboratory for Advanced Textile Processing and Clean Production, Wuhan Textile University, Wuhan 430073, P. R. China * Corresponding author: E-mail: fcym981248@sohu.com Received: 02-08-2020 Abstract Two new ethyl maltolato coordinated mononuclear oxidovanadium(V) complexes [VOLa(emt)]-DMF (1) and [VOL-b(emt)] (2), where H2La = N'-(4-bromo-2-hydroxybenzylidene)-3-hydroxybenzohydrazide, H2Lb = N'-(4-bromo-2-hy-droxybenzylidene)benzohydrazide, Hemt = ethyl maltol, have been synthesized and characterized on the basis of CHN elemental analysis, FT-IR and UV-Vis spectroscopy and powder XRD analysis. Structures of the complexes were further characterized by single crystal X-ray diffraction, which indicated that the V atoms in the complexes adopt octahedral coordination. The hydrazones behave as NOO tridentate ligands. The catalytic epoxidation properties on cyclooctene of the complexes were investigated. Keywords: Hydrazone; oxidovanadium complex; crystal structure; catalytic epoxidation 1. Introduction Schiff bases are interesting ligands in the formation of versatile complexes with various metal ions.1 The complexes with Schiff base ligands have received particular attention for their facile synthesis and remarkable biological, catalytic and magnetic applications.2 Catalytic epoxidation of olefins is an important type of reactions in industrial chemistry. A number of complexes with transition metal ions are active catalysts for this process.3 In particular, among the complexes, vanadium and molybdenum complexes seem more interesting because of their excellent catalytic ability in the oxidation of olefins and sulfides.4 Vanadium complexes with hydrazones are reported to possess catalytic properties. In order to study the influence of the substituent groups of the hydrazones on the catalytic property of the vanadium complexes, in this paper, two new oxidovanadium(V) complexes, [VOLa(emt)]-DMF H2L Scheme 1. The hydrazones and Hemt. Cai et al.: Syntheses, Crystal Structures and Catalytic Property ... Acta Chim. Slov. 2020, 67, 896-897 903 (1) and [VOLb(emt)] (2), where H2La = N'-(4-bromo-2-hy-droxybenzylidene)-3-hydroxybenzohydrazide, H2Lb = N'-(4-bromo-2-hydroxybenzylidene)benzohydrazide, Hemt = ethyl maltol (Scheme 1), are presented. 2. Experimental 2. 1. Materials and Methods 3-Hydroxybenzohydrazide, benzohydrazide, 4-bro-mosalicylaldehyde, ethyl maltol and VO(acac)2 were purchased from Alfa Aesar and used as received. Reagent grade solvents were used as received. Microanalyses of the complexes were performed with a Vario EL III CHNOS elemental analyzer. Infrared spectra were recorded as KBr pellets with an FTS-40 spectrophotometer. Electronic spectra were recorded on a Lambda 900 spectrometer. The powder XRD spectra were recorded in a 20 range of 2-50° using a Bruker D8 Advance detector under ambient conditions. The catalytic reactions were followed by gas chromatography on an Agilent 6890A chromatograph equipped with an FID detector and a DB5-MS capillary column (30 m x 0.32 mm, 0.25 ^m). Molar conductance measurements were made by means of a Metrohm 712 conductom-eter in acetonitrile. 2. 2. Synthesis of the Complex [VOLa(emt)]-DMF 3-Hydroxybenzohydrazide (10 mmol, 1.52 g) and 4-bromosalicylaldehyde (10 mmol, 2.01 g) were refluxed in methanol (50 mL). Then, VO(acac)2 (10 mmol, 2.63 g) and ethyl maltol (10 mmol, 1.40 g) dissolved in methanol (30 mL) were added to the mixture and refluxed for 1 h in oil bath to give a deep brown solution with some insoluble substance. Then, a few drops of DMF were added until the insoluble substance dissolved. Single crystals of the complex were formed during slow evaporation of the reaction mixture in air. The crystals were isolated by filtration, washed with cold methanol and dried over anhydrous CaCl2. Yield: 0.38 g (61%). IR data (KBr pellet, cm-1): 1667 v(C=O), 1592 v(C=N), 1524, 1455, 1408, 1353, 1250 v(c-Ophenolate), 1188 v(N-N), 1107, 1066, 1030, 973 v(v=O), 926, 850, 796, 727, 630, 522, 468. UV-Vis data in methanol (nm): 210, 269, 320, 398. Molar conductance (10-3 mol L-1, methanol): 25 O-1 cm2 mol-1. Analysis: Found: C 46.89, H 3.85, N 6.77%. Calculated for C24H23BrN3O8V: C 47.08, H 3.79, N 6.86%. 2. 3. Synthesis of the Complex [VOLb(emt)] Benzohydrazide (10 mmol, 1.36 g) and 4-bromosa-licylaldehyde (10 mmol, 2.01 g) were refluxed in methanol (50 mL). Then, VO(acac)2 (10 mmol, 2.63 g) and ethyl maltol (10 mmol, 1.40 g) dissolved in methanol (30 mL) were added to the mixture and refluxed for 1 h in oil bath to give a deep brown solution. Single crystals of the complex were formed during slow evaporation of the reaction mixture in air. The crystals were isolated by filtration, washed with cold methanol and dried over anhydrous CaCl2. Yield: 0.21 g (40%). IR data (KBr pellet, cm-1): 1595 v(C=N), 1529, 1458, 1407, 1338, 1252 v(C-Ophenolate), 1196 v(N-N), 1131, 1070, 973 v(V=O), 919, 835, 786, 695, 639, 598, 515, 466. UV-Vis data in methanol (nm): 210, 267, 322, 396. Molar conductance (10-3 mol L-1, methanol): 33 O-1 cm2 mol-1. Analysis: Found: C 48.37, H 3.16, N 5.22%. Calculated for C21H16BrN2O6V: C 48.21, H 3.08, N 5.35%. 2. 4. Crystal Structure Determination Data were collected on a Bruker SMART 1000 CCD area diffractometer using a graphite monochro-mator Mo Ka radiation (X = 0.71073 A) at 298(2) K. The data were corrected with SADABS programs and refined on F2 with Siemens SHELXL software.5 The structures of the complexes were solved by direct methods and difference Fourier syntheses. All non-hydrogen atoms were refined anisotropically. The hydrogen atoms were placed in calculated positions and included in the last cycles of refinement. Crystal data and details of the data collection and refinement are listed in Table 1. Selected coordinate bond lengths and angles are listed in Table 2. Table 1. Crystallographic data for the complexes Parameters 1 2 Empirical formula C24H23BrN3O8V C2!H!6BrN2O6V Formula weight 612.30 523.21 Crystal system Triclinic Monoclinic Space group P-1 P2Jn a [A] 10.0508(11) 12.2708(11) b [A] 11.6192(12) 7.4066(7) c [A] 12.8769(12) 22.6657(12) a [°] 63.7350(10) 90 P [°] 68.8460(10) 94.5300(10) Y [°] 86.6150(10) 90 V [A3] 1248.5(2) 2053.5(3) Z 2 4 Pcalcd. [g cm-3] 1.629 1.692 p [mm-1] 2.052 2.471 F(000) 620 1048 Measured reflections 27647 11744 Independent reflections 4646 3809 Observed reflections 3541 2765 (I > 2a(I)) Parameters 338 281 Restraints 0 0 Final R indices [I > 2ff(I)] 0.0491, 0.1387 0.0385, 0.0914 R indices (all data) 0.0674, 0.1518 0.0632, 0.1020 Goodness-of-fit on F2 1.037 1.030 Largest difference in 0.941, -0.696 0.542, -0.482 peak and hole (e A 3) Cai et al.: Syntheses, Crystal Structures and Catalytic Property ... 898 Acta Chim. Slov. 2020, 67, 896-903 Table 2. Selected bond lengths (A) and angles (°) for the complexes V1- -O1 1.843(3) V1- O2 1.918(3) V1- -O4 1.585(3) V1- O5 2.263(3) V1- O6 1.862(2) V1- N1 2.094(3) O4- -V1- -O1 100.49(16) O4 V1 O6 100.18(12) O1- -V1- O6 95.62(11) O4 V1 O2 94.89(14) O1- -V1- -O2 155.78(12) O6 V1 O2 99.96(11) O4- -V1- -N1 101.11(12) O1 V1 N1 83.47(12) O6- -V1- -N1 158.50(11) O2 V1 N1 75.24(11) O4- -V1- O5 174.17(14) O1 V1 O5 85.16(13) O6- -V1- O5 77.68(10) O2 V1 O5 80.23(11) N1- -V1- O5 80.84(10) 2 V1- -O1 1.849(2) V1- O2 1.922(2) V1- -O3 1.588(2) V1- O4 1.872(2) V1- -O5 2.255(2) V1- N1 2.083(2) O3- -V1- -O1 99.32(12) O3 V1 O4 98.75(11) O1- -V1- -O4 102.31(9) O3 V1 O2 99.79(11) O1- -V1- -O2 153.12(10) O4 V1 O2 93.26(9) O3- -V1- -N1 98.47(11) O1 V1 N1 83.71(9) O4- -V1- -N1 160.56(10) O2 V1 N1 74.93(9) O3- -V1- O5 176.08(11) O1 V1 O5 83.49(10) O4- -V1- O5 77.91(9) O2 V1 O5 78.47(9) N1- -V1- O5 84.52(9) 2. 5. Catalytic Epoxidation Process A mixture of cyclooctene (2.76 mL, 20 mmol), ace-tophenone (internal reference) and the complex as the catalyst (0.05 mmol) was stirred and heated up to 80 °C before addition of aqueous ferf-butyl hydroperoxide (TBHP; 70% w/w, 5.48 mL, 40 mmol). The mixture is initially an emulsion, but two phases become clearly visible as the reaction progresses, a colorless aqueous one and a yellowish organic one. The reaction was monitored for 5 h with withdrawal and analysis of organic phase aliquots (0.1 mL) at required times. Each withdrawn sample was mixed with 2 mL of diethylether, treated with a small quantity of MnO2 and then filtered through silica and analyzed by GC. 3. Results and Discussion 3. 1. Synthesis The hydrazone compounds and the complexes were synthesized in a facile and analogous way (Scheme 2). The hydrazones H2La and H2Lb act as tridentate dian-ionic NOO donor ligands toward the VO3+ cores. The two complexes were obtained from refluxing mixtures of the hydrazones with VO(acac)2 in 1:1 molar proportion in the presence of ethyl maltol in methanol. Complex 1 is not soluble well in methanol, so DMF was added to improve the solubility. The complexes were isolated as single crystals from Br CHO ^ J OH H2N VO(acac)2 MeOH DMF X = H Scheme 2. The syntheses of the hydrazones and the complexes MeOH X = OH HO. VO(acac)2 MeOH 1 Cai et al.: Syntheses, Crystal Structures and Catalytic Property Acta Chim. Slov. 2020, 67, 896-899 903 the reaction mixtures by slow evaporation at room temperature. Crystals of the complexes are stable at room temperature. The low molar solution conductance of the complexes in methanol indicates their non-electrolyte behavior. Theoretical diffractograms were calculated using the PowderCell program.6 The experimental X-ray powder diffraction patterns of the bulk samples of the complexes agree well with the simulated patterns calculated from single crystal X-ray diffraction (Figures 1 and 2). The results prove the purity of the bulk samples. Figure 1. Experimental and simulated powder XRD patterns of complex 1. Figure 2. Experimental and simulated powder XRD patterns of complex 2. 3. 2. IR and Electronic Spectra The IR spectra of the hydrazones show bands centered at about 3215 cm-1 for v(N-H), 3537 cm-1 for v(O-H), and 1655 cm-1 for v(C=O).7 The peaks attributed to v(N-H) and v(C=O) are absent in the spectra of the complexes as the ligands bind in dianionic form resulting in losing proton from carbohydrazide group. An intense band at 1667 cm-1 for complex 1 ascribes to the C=O band of DMF molecule. Strong bands observed at 1592 and 1595 cm-1 for the vanadium complexes are attributed to v(C=N), which are located at lower frequencies as compared to the free hydrazones.8 The complexes exhibit characteristic bands at 973 cm-1 for the stretching of V=O bonds.9 Based on the IR absorption, it is obvious that the hydrazone ligands exist in the uncoordinated form in ke-to-amino tautomer form and in the complexes in imi-no-enol tautomeric form. This is not uncommon in the coordination of the hydrazone compounds.10 The absorptions at about 1525 and 1460 cm-1 are assigned to the v(C=O) and v(C=C) of the maltolate groups.11 The UV-Vis spectra of the hydrazones and the complexes recorded in methanol are shown in Figures 3 and 4, respectively. The absorptions at about 300 nm for the hydrazones are assigned to the n-n* transitions. The strong absorption bands centered at about 400 nm for the complexes are assigned as charge transfer transitions of N(pn)-V(dn) LMCT. The medium absorption bands centered at about 320 nm for the complexes are assigned as charge transfer transitions of O(pn)-V(dn) LMCT.12 (D O 1= ro -O n < 200 250 300 350 400 450 500 Wavelength (nm) Figure 3. The UV-Vis spectra of H2La and H2Lb. 500 600 700 Wavelength (nm) Figure 4. The UV-Vis spectra of complexes 1 and 2. Cai et al.: Syntheses, Crystal Structures and Catalytic Property ... 900 Acta Chim. Slov. 2020, 67, 896-903 3. 3. Description of the Structures of the Complexes The molecular structures of complexes 1 and 2 are depicted in Figures 5 and 6, respectively. There is a sol-vated DMF molecule in complex 1. The V atoms in both complexes are in distorted octahedral coordination with NO5 chromophore. The dianionic tridentate hydrazones coordinate to the V atoms with the phenolate oxygen (O1), the enolate oxygen (O2) and the imine nitrogen (N1). The emt ligand coordinates to the V atom with the carbonyl oxygen (O5) and phenolate oxygen (O6). The octahedral coordination is defined by the three donor atoms of the hydrazone and the O6 atom at the equatorial plane, and by the oxido oxygen (O4 for 1 and O3 for 2) and the O5 atom at the axial positions. The V atoms in complex 1 and 2 deviated from the least-squares planes defined by the equatorial donor atoms by 0.306(1) Â and 0.302(1) Â, respectively. The V1-O5 bond lengths (2.26 Â) in both complexes are longer than the remaining V-O bonds (1.58-2.10 Â), which is caused by the trans effects generated by the oxido groups. The bond lengths of V-O and V-N are within the values observed in other vanadium(V) complexes.21'4'5,13 The bond lengths of C8-O2 (1.31 Â) indicate they are more close to single bonds, which is due to the conjugation effects of the ligands.14 In addition, the bond lengths of C8-N2 (1.30 Â) and N1-N2 (1.40 Â) are intermediate between single and double bonds, which also supports the electron cloud delocalization in the hydrazone ligands. The five-mem-bered chelate rings (V1-N1-N2-C8-O2) are nearly planar, while the six-membered chelate rings (V1-O1-C2-C1-C7-N1) are obviously distorted from planarity. The benzene rings of the hydrazone ligands form dihedral angles of 1.8(3)° (1) and 8.7(4)° (2). The distortion of the octahedral coordination can be observed from the trans angles (157.8(1)-174.2(1)° for 1 and 153.8(1)-176.1(1)° for 2). In the crystal structures of complexes 1 and 2 (Figures 7 and 8), the complexes molecules are linked together by hydrogen bonds (Table 3). Figure 5. ORTEP plots (30% probability level) and numbering scheme for complex 1. Figure 6. ORTEP plots (30% probability level) and numbering scheme for complex 2. 3. 4. Catalytic Epoxidation Property The complexes showed good properties on the cy-clooctene epoxidation reaction by using aqueous TBHP as oxidant with no extra addition of organic solvents. Kinetic profiles of complexes 1 and 2 as the catalysts are presented Table 3. Hydrogen bond distances (Â) and bond angles (°) for the complexes D-H-A d(D-H), Â d(H-A), Â d(D-A), Â Angle (D-H-A), ° 1 O3-H3—O8i 0.82 1.81 2.628(5) 178(3) C19-H19—O5" 0.93 2.51 3.357(5) 151(3) C2Q-H2QA-O4m 0.97 2.53 3.264(5) 132(3) 2 C17-H17—O3iv 0.93 2.48 3.330(5) 153(3) C21-H21B—O5v 0.96 2.52 3.440(5) 160(3) Symmetry codes: i) -1 + x, y, z; ii) 1 - x, -y, 1 - z; iii) 1 - x, 1 - y, -z; iv) x, 1 + y, z; v) 2 - x, 2 - y, -z. Cai et al.: Syntheses, Crystal Structures and Catalytic Property Acta Chim. Slov. 2020, 67, 896-901 903 Figure 7. The molecular packing diagram of complex 1, viewed down the b axis. Hydrogen bonds are shown as dashed lines. Figure 8. The molecular packing diagram of complex 2, viewed down the b axis. Hydrogen bonds are shown as dashed lines. in Figure 9. The cyclooctene conversions for complexes 1 and 2 are 91% and 90% after 5 h, and the selectivities for cyclooctene oxide are 67 and 69%. The proposed mechanism is shown in Scheme 3. The TBHP molecule was considered to be coordinated to the V atom with the formation of an O-H—O hydrogen bond. Interestingly, the vanadium complexes in this work have higher conversions and selec-tivities for the epoxidation reaction of cyclooctene than the molybdenum complexes with hydrazone ligands.15 From the results, it is not obvious difference between the conversions of the complexes, but the selectivity of complex 2 with benzene as the terminal group of the hydrazone ligand (Lb) is a litter higher than complex 1 with p-hydroxylbenzene as the terminal groups of the hydrazone ligand (La). 100 Time (min) Figure 9. Kinetic monitoring of ds-cyclooctene epoxidation with TBHP-H2O in the presence of the complexes 1 and 2. 4. Conclusion In summary, two new structurally similar oxidova-nadium(V) complexes derived from the tridentate hydra- Scheme 3. Proposed mechanism for the catalytic reaction. X = OH for 1, and X = H for 2. Cai et al.: Syntheses, Crystal Structures and Catalytic Property ... 902 Acta Chim. Slov. 2020, 67, 896-903 zones N'-(4-bromo-2-hydroxybenzylidene)-3-hydroxy-benzohydrazide and N'-(4-bromo-2-hydroxybenzylidene) benzohydrazide, and the bidentate ligand ethyl maltol were prepared and structurally characterized. The hydrazone ligands coordinate to the vanadium atoms through the NOO donor set. The V atoms of the complexes are in octahedral coordination. The ethyl maltol ligand can be substituted by TBHP during the catalytic processes. The complexes have effective catalytic epoxidation properties on cyclooctene with high conversions. 5. Supplementary data CCDC numbers 1979493 for 1 and 1979494 for 2 contain the supplementary crystallographic data. These data can be obtained free of charge via http://www.ccdc. cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Center, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336 033; or e-mail: deposit@ ccdc.cam.ac.uk. 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Povzetek Sintetizirali smo dva nova etil maltolato koordinacijska enojedrna oksidovanadijeva(V) kompleksa [VOLa(emt)]-DMF (1) in [VOLb(emt)] (2), kjer je H2La = N'-(4-bromo-2-hidroksibenziliden)-3-hidroksibenzohidrazid, H2Lb = N'-(4-bro-mo-2-hidroksibenziliden)benzohidrazid, Hemt = etil maltol, ter ju okarakterizirali s CHN elementno analizo, FT-IR in UV-Vis spektroskopijo ter praškovno XRD analizo. Strukture smo določili z monokristalno rentgensko difrakcijo, ki razkrije, da imajo V atomi v kompleksu oktaedrično koordinacijo. Hidrazona sta NOO trivezna liganda. Katalitične lastnostni obeh kompleksov smo raziskali z reakcijo epoksidacije ciklooktena. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Cai et al.: Syntheses, Crystal Structures and Catalytic Property ... DOI: 10.17344/acsi.2020.5908 Acta Chim. Slov. 2020, 67, 904-915 /^.creative o'commons Scientific paper Students' Achievements in Solving Authentic Tasks with 3D Dynamic Sub-Microscopic Animations About Specific States of Water and their Transition Miha Slapničar*, Valerija Tompa, Saša A. Glažar, Iztok Devetak and Jerneja Pavlin* University of Ljubljana, Faculty of Education, Kardeljeva pl. 16, 1000 Ljubljana * Corresponding authors: E-mails: miha.slapnicar@pef.uni-lj.si and jerneja.pavlin@pef.uni-lj.si Received: 02-13-2020 Abstract This paper aims to identify differences in the justification of the selection of 3D dynamic submicroscopic-representation (SMR) of the solid and liquid states of water, as well as the freezing of water presented in selected authentic tasks. According to students' achievements in solving these tasks at different levels of education, their explanations were identified. To explain in greater detail how students attempted to solve the authentic tasks, an eye-tracking method was used to identify the differences in the total fixation durations on specific areas of interest at the specific SMRs between successful and unsuccessful students in three age groups. A total of 79 students participated in this research. The data were collected with a structured interview conducted with students when solving three authentic tasks displayed on the computer screen. The tasks comprise text (as problem and questions), macro-images (photos of the phenomena) and SMRs of the phenomena. The eye-tracker was also used to measure the students' gaze fixations at the particular area of interest. The results show that successful students' justifications for a correct SMR include macroscopic and sub-microscopic representations of the chosen concepts. Along different stages of education, the selection success increases and sufficient justifications comprise the sub-microscopic level. It could be concluded that there are mostly no significant differences between successful and unsuccessful students within the same age group in the total fixation duration at the correct SMR. Further studies are needed to investigate the information-processing strategies between high and low achievers in solving various authentic tasks comprising SMRs and those that integrate all three levels of the representation of chemical concepts. Keywords: States of water; freezing of water; authentic tasks; 3D dynamic SMR; eye tracking. 1. Introduction Most chemical concepts are comprehended as abstract for teaching and learning because they can be represented on three different levels of representation: macroscopic, sub-microscopic, and symbolic. The teaching and learning of chemical concepts can be facilitated by context-based chemistry approaches that usually start from contexts (topics, questions) that are close to students' everyday life (authentic context). These approaches increase students' interest, activate their pre-knowledge on certain topics and offer situations in which newly developed knowledge can be applied and linked to basic concepts.1 Several authors have taken this into account when designing activities and tasks for students.2-5 However, facilitating the understanding of the specific level of representation of chemical concepts is related to the use of different visualisation tools.6 Therefore, teachers should pay more attention to the representation of structures of different substances at the sub-microscopic level so that students can develop an adequate understanding of specific chemical concepts.7 For visualisation at the sub-microscopic level, teachers could use static and dynamic 2D or 3D submicro-representations.8,9 However, some researchers10 have reported that students using 3D dynamic representations constructed a better understanding of the chemical concepts than students using static 3D representations did, while other researchers11 have reported that 3D representations help students improve incomplete understandings of the concepts and influence the construction of more complete concepts. For an adequate understanding of the chemical concept, students must integrate all three levels of chemical concept simultaneously: the macroscopic, the sub-micro- Slapničar et al.: Students' Achievements in Solving Authentic Tasks Acta Chim. Slov. 2020, 67, 904-915 905 scopic, and the symbolic.12 Many studies have shown that students at all levels of education have problems in interpreting and applying SMRs.9,13,14 These difficulties indicate a lack of connection between all three levels of the representation of the chemical concept. Researchers15-17 have found that primary school, secondary school, and university students have problems in explaining the process (represented at the macroscopic level) at the sub-microscopic level. Nevertheless, knowledge about the particulate nature of matter in different stages of education is improving.18 Based on research,19,20 primary school students have problems in understanding SMRs for states of matter and transitions between them. Sixteen-year-old students achieved higher scores in tasks about states of water when concepts regarding the gaseous state of water at the sub-microscopic level were included.21,22 It is reported that most students can explain particle motion in the liquid and solid states of matter.23,24 Students aged 10 to 12 years have problems in applying particle theory to justify everyday events. Even if the students had previous theoretical knowledge about the particulate nature of matter, problems with explaining everyday events or using it to explain observed phenomena were common.25 Other researchers26 have stated that the students were unable to transfer the obtained knowledge about the particulate nature of matter to situations in everyday life. Students have problems explaining events (based on particle theory) that are related to physical changes, even if they have formed adequate particle conceptions. Teachers should use the particulate nature of matter to explain events in everyday life, which enables learning and facilitates the conceptual understanding of particle theory.24 Difficulties in the conceptual understanding of state changes have been reported in recent decades.27 However, the representation of chemical concepts using dynamic SMRs has an impact on improving students' understanding of the particulate nature of matter,28 e.g., motion29-32 and particle arrangement.33-35 The process of an individual's solving a task can be identified with eye-tracking because cognitive information processing is related to eye movements, which are used as an observable measure of visual attention.36-38 Eye-tracking studies have shown that unsuccessful task solvers have had difficulty distinguishing between relevant and irrelevant factors and in focusing on relevant factors to solve the task. Success in selecting information is crucial for successful task solving,39 and it is similar to the observation of 3D-SMRs.40 Which information is processed by the cognitive system is indicated by fixations,41 which are periods of eye stability. The eyes can only be in a stable condition for a limited time (100-500 ms).36,41,42 The most commonly used measure of eye movement is fixation duration, including a variable total fixation duration (TFD).42,43 Fixation duration measures 'the duration of each fixation within an area of interest (AOI)'.37,44 Longer fixation durations indicate the greater complexity of visual material.45 The duration of the fixation on the individual components of a display can be used to identify the AOIs. Fixation duration also indicates the time in which the information is processed.37 A longer fixation time indicates a deeper and more complex processing of the information.46 2. Research Problem and Research Questions The research results47 showed an improvement in the knowledge of the states of matter at the sub-microscopic level through years of schooling. Based on research findings, primary school students have problems understanding SMRs for states of matter and transitions between them.19,20 Unsuccessful problem-solvers had difficulty in distinguishing between relevant and irrelevant factors and in focusing on the relevant factors to solve the scientific task. Success in selecting information is crucial for successful task solving.39 From the literature presented in the introduction, it is evident that difficulties in explaining the particular nature of different states of water are found among students on different stages of education. The objectives of the research were to determine whether successful and unsuccessful students' justifications at different stages of education and aged 12 years (primary school), 16 years (upper secondary school) and 23 years (university education) differ and to identify whether successful students fix their gaze for longer times on the correct 3D-SMR than non-successful students do when solving the tasks. Two research questions were set in the research: RQ 1: How do successful and unsuccessful students of different age groups (12, 16, and 23 years) differ in the justifying of the selection of 3D dynamic SMRs (for the solid and liquid states of water and the freezing of water)? RQ 2: How do successful and unsuccessful students of different age groups (12, 16, and 23 years) differ in TFD on AOI with 3D dynamic SMRs in authentic tasks, including the solid and liquid states of water and the freezing of water? 3. Methods A quantitative non-experimental research approach with descriptive methods was used. 3. 1. Participants A non-random, convenience sample of participants was formed from a mixed urban population, including seventy-nine Slovenian students from three different age groups. The students came from the Ljubljana region and voluntarily participated in the research. The first group included thirty students who attended the seventh grade of primary school (Mdn = 12.0 years, IQR = 0.43 years). The Slapničar et al.: Students' Achievements in Solving Authentic Tasks ... 906 Acta Chim. Slov. 2020, 67, 904-915 participants of the second group (N = 29) attended the first year of upper secondary school (Mdn = 16.0 years, IQR = 1.0 years). The third group consisted of 20 students (future teachers) of the double-majors study programme of chemistry and biology/physics from the Faculty of Education of the University of Ljubljana (Mdn = 23.0 years, IQR = 2.0 years). The approval for primary and upper secondary students was obtained from school authorities, teachers, and parents/caregivers, according to the Ethics Committee for Pedagogy Research of the Faculty of Education of the University of Ljubljana. All participants had normal or cor-rected-to-normal vision, and all were competent readers. To ensure anonymity, each student was assigned a code consisting of the letter 'S' with the number of the age group and a student number (e.g., S1_7). The group of successful students included students who selected the correct 3D dynamic SMR for a particular state of water or freezing of water and gave the correct justifications for their decisions, while the group of unsuccessful students included students who were unsuccessful in selecting and/or justifying the selection of a correct 3D dynamic SMR. 3. 2. Instruments The problem set consisted of three authentic tasks. These specific tasks are three of eleven authentic science tasks that were studied from different aspects in the Slovenian Research Agency project entitled 'Explaining effec- tive and efficient problem solving of the triplet relationship in science concepts representations'. The starting point for the selection of ideas for curriculum content for the preparation of tasks was made by the review of TIMMS (Trends in International Mathematics and Science Study), the PISA Programme for International Student Assessment, and the tasks of the Slovenian national external assessment for chemistry and physics. The group of the project designed authentic context-based tasks, including 3D dynamic SMRs of chemistry concepts. The 3D dynamic SMRs were designed by science educators, as well as the authors of this paper, and, according to their developed ideas, the computer specialist completed them. The 3D dynamic SMRs were developed only for this research. The time in which the participants looked at them was not limited. When the participants needed more time to solve the tasks, the animations started again from the beginning. However, the participants did not have the possibility of controlling the animations. The text of the tasks was in the Slovenian language. For the purpose of this paper, the task texts were translated into English (see Figures 1-3). Task 1 (Figure 1) includes macroscopic and sub-microscopic levels of representation for the solid state of water, Task 2 for the liquid state of water, and Task 3 for the freezing of water. Each task was presented by displaying a screen image (slide) in the PowerPoint presentation. Task 1 and Task 2 each consisted of two slides. Task 1 included a photo of an iceberg, three 3D dynamic SMRs, and two questions related to the selection □ 00 Figure 1. Screen images of the first authentic task, part 1 (left) and part 2 (right). (Image of an iceberg from hdwpics.com). What does the photo show? Which substance constitutes what you see in the photo? What does the substance in the photo consist of? In which state of matter is the substance in the photo? Which representation from 1 to 3 illustrates this state matter? State at least two reasons to justify your selection. *m 'M □ 00 Figure 2. Screen images of the second authentic task, Part 1 (left) and Part 2 (right). (Image of flowing water from www.goingmobo.com). Slapnicar et al.: Students' Achievements in Solving Authentic Tasks ... Acta Chim. Slov. 2020, 67, 904-915 907 Figure 3. Screen image of the third authentic task. (Image of lake Bled from www.wikipedia.org). All SMRs represented the movement of water molecules in three different states of matter. All SMRs represented the correct movement and arrangement of particles (water molecules) in the gaseous state of water (on the top of SMR). The correct one is SMR 1 representing the liquid state in the lower part, the solid state in the middle part of the SMR, and the gaseous state in the upper part. SMR 2 did not represent the arrangement of the particles in the correct order (solid state in the lower part, liquid state in the middle part of the SMR, and gaseous state in the upper). SMR 3 is incorrect because particles do not move in the solid state of water. and justification of the selected sub-micro-representation. Task 2 (Figure 2) consists of a photo of liquid water, three 3D dynamic SMRs, and two questions related to the selection and justification of the selected SMR. The 3D dynamic SMRs in Tasks 1 and 2 represented the correct arrangement and movement of particles in all three states of water (solid, liquid and gaseous state) in a different order. Task 3 (Figure 3) included a photo of partially frozen Lake Bled, two questions (to select and explain selected 3D sub-micro-representation) and three animations that could represent a process of water freezing. 3. 3 Research Design To determine the time required by successful and unsuccessful students for a certain AOI (3D dynamic SMRs for the solid, liquid state of water and freezing of water), TFDs were measured with eye-tracking. 'Fixations' refer to maintaining one's gaze on a specific AOI, while 'saccades' refer to rapid eye movements from one AOI to another.48 The identification of saccades/fixations is based on the motion of gaze during each collected sample. If both the velocity and acceleration threshold (in our case: 30 degrees per second and 8000 degrees per second squared) or are exceeded, a saccade begins; otherwise, the sample was labelled as a fixation. The screen-based Eye-Link 1000 (35 mm lens, horizontal orientation) eye tracker apparatus and associated software (Experiment Builder for preparation of the experiment and a connection to Eye-Link; Data Viewer for data acquisition and basic analysis) for recordings and analyses of students' eye movements when solving authentic tasks were used. Data were collected from the right eye (monocular data collection following corneal reflection and student responses) at 500 Hz.49 The data were collected using the eye-tracking method in the laboratory of the Department of Psychology, of the Faculty of Arts, of the University of Ljubljana. The data collection was performed between November 2017 and January 2018. Before the individual testing with the eye tracker, each student was informed about the eye-tracking method, the purpose of the research, and their role in it. During testing with the eye tracker, a student sat in the front of the computer screen with chin and forehead held on a special head-supporting stand, which enabled the optimal measurement, recording and stability of the head and recordings. The distance between the participants' eyes and the computer screen was approximately 60 centimetres. After calibrating and validating the eye tracker using a nine-point algorithm, the student solved the tasks and gave the answers to the tester (structured interview), who transcribed them. The tasks were represented in the form of slides in a PowerPoint presentation. When the student solved the task presented on a slide, the tester switched to another slide (task).50 A basic analysis of the collected eye movement data was performed in the Data Viewer software. Further data analysis was conducted in the Statistical Package for Social Sciences (SPSS), version 22. The participants of all three age groups (12-, 16-, and 23-year-old students) were divided into successful and unsuccessful groups based on their successful justification together with the selection of the correct 3D dynamic SMR for the liquid and solid states of water and the freezing of water, as well as the reasons for their selection. The students' justifications (written down by the tester) were read several times by two authors independently, identifying and coding the most important meanings concerning the level of the chemical concept representations and correctness of the justification. The authors then met to compare and confirm the results. Any disagreements were resolved by discussion between the authors. The planned recourse to the third author for arbitration did not prove necessary. Slapničar et al.: Students' Achievements in Solving Authentic Tasks ... 908 Acta Chim. Slov. 2020, 67, 904-915 The mean values of the TFDs were described by a median (Mdn) and an interquartile range (IQR) for the specific 3D SMR. The distribution of data was non-normal, and the sample was small. Therefore, the Mann-Whitney U and Kruskal-Wallis non-parametric tests were used to explain the relationship between the (un)successfully solved authentic task, including SMRs, and TFD on AOIs with SMRs. Statistical hypotheses were tested at a 5% alpha error rate. To describe whether the effects have a relevant magnitude, the effect size measure eta squared r|2 was used to describe the strength of a phenomenon. Benchmarks for effects size are small (0.01), medium (0.06), and large (0.14).51,52 4. Results and Discussion The results are presented according to the research questions. 4. 1. Students' Achievements in Justifications Altogether with the Selection of SMR Research Question 1 focused on the differences between successful and unsuccessful students in three age groups in the justification of selected 3D dynamic SMRs for the solid and liquid state of water or the freezing of water. The results showed that all students in Groups 2 and 3 chose the correct SMR for the solid and liquid state of water, while one student in Group 1 chose the incorrect SMR for the solid state of water and two of them chose the incorrect SMR for the liquid state of water; 23.33% of the students in Group 1, 58.62% in Group 2, and 75.00% in Group 3 chose the correct 3D dynamic SMR for the freezing of water. Table 1 presents the relative frequencies for students' achievements in justifications of the selected SMS in three tasks related to the solid and liquid state of water or the freezing of water. It is evident that the percentage of successful students' justification for the correct SMR for the solid state of water is increasing according to the stage of education from 10.00% in Group 1 to 20.00% in Group 3. In Task 2, on the liquid state of water, the ability to correctly justify the choice rose from 17.24% to 55.00% according to the years of schooling. The relative frequencies of suc- cessful students related to the task in water freezing increased among the years of schooling from 13.33% in Group 1 to 40.00% in Group 3. The results are coherent with researchers18 who noted that knowledge about the particulate nature of matter improves according to the stages of education. Other researchers19,20,48 have also argued about improved knowledge among the stages of education. The level at which the justification of selected SMR (sub-microscopic, macroscopic, a combination of both levels) was argued is shown in Table 2 by absolute frequencies of justifications at the specific level of representation of chemical concepts in Tasks 1 (solid state of water), 2 (liquid state of water) and 3 (freezing of water). The majority (70.00%) of successful and unsuccessful students in Group 1 justified their selection for the SMR of the solid state of water (Task 1) at the macroscopic level and a combination of macroscopic and sub-microscopic levels, while most (70.00%) successful and unsuccessful students in Group 3 justified their selection at the sub-microscopic level. It is evident that the majority of successful and unsuccessful students in all three groups listed the justifications for selecting the SMR for the liquid state of water (Task 2) at the macroscopic and sub-microscopic levels, except for the unsuccessful students in Group 3, who gave the same number of justifications at the sub-microscopic or macroscopic and sub-microscopic levels. Successful and unsuccessful students in Group 1 explained the majority of the justifications in Task 3 (freezing of water) at the macroscopic level. Most of the successful and unsuccessful students of Group 2 discussed a selection of a correct SMR at the macroscopic and sub-microscopic levels. Successful students in Group 3 argued about the selection of an SMR for freezing of water at the macroscopic and sub-microscopic levels or at the macroscopic level, while unsuccessful students in Group 3 argued at the macroscopic and sub-microscopic levels. This is shown by examples of justifications for Task 3 (freezing of water) that were identified as incorrect. They are listed below by age group. Examples of incorrect justification of students of Group 1. S1_3: The ice surface is at rest, and the water underneath moves normally. S1_8: The ice does not freeze everywhere. It only freezes on the surface. Water vapour still evaporates. Table 1. Relative frequencies of successful and unsuccessful students at solving tasks on solid (Task 1), liquid state of water (Task 2) and freezing of water (Task 3). Task 1: Solid state of water Task 2: Liquid state of water Task 3: Freezing of water Group of students 123 123 123 Successful f%) 10.00 13.79 20.00 17.24 50.00 55.00 13.33 31.03 40.00 Unsuccessful f%) 90.00 86.21 80.00 82.76 50.00 45.00 86.67 68.97 60.00 Group 1: Students aged 12. Group 2: Students aged 16. Group 3: Students aged 23. Slapnicar et al.: Students' Achievements in Solving Authentic Tasks ... Acta Chim. Slov. 2020, 67, 904-915 909 S117: The layer is freezing, and not all the particles are moving. Some particles are at rest. S123: Because the lake above is icy and solid and the particles are not moving. Below is liquid. Examples of incorrect justification of students of Group 2. S2_2: Because the top layer freezes. In the lower layer, the particles are still in motion (flowing water). S2_6: The upper layer is still moving a little bit. Especially when we slide from the pressure, this part melts. The water freezes. A layer of water remains on top. S211: Because there is ice on the surface, solid state. The particles do not move. Underneath, the water is in a liquid state, and the particles move. 52 18: Because there is ice in the upper part, particles stand still; they do not move, and the lower part is liquid, particles move. This layer does not freeze. Some examples of incorrect justification from students of Group 3. S3_3: I chose animation number three because the ice structure on it is firmer because the molecules don't wobble. The molecules are connected to stronger forces during their movement than in the first animation. The ice is firmer in the third presentation and, therefore, does not break as fast as in the first presentation. S3_5: What is the difference between 1 and 3? I do not see any difference. I will say 3. The top layer represents ice, the particles do not move, they are arranged. 53 12: We have gaseous molecules at the top. In the middle, the lake is frozen, below it is running water. S3 13: The bottom layer is liquid, and the top layer is ice. Here the particles are arranged and do not move. In the liquid state, the particles move in a disorderly fashion. An example per age group of the correct justification is given as well. Example of correct justification of students of Group 1. S1_2: In this animation, water exists in three states of matter. I see this because the molecules move differently, even if they are the same. They move mainly in the gaseous state of water, then in liquid and then only around them in the solid state. Example of correct justification of students of Group 2. S2 19: In the first animation, the water in the lower part of the box is in a liquid state. In the middle of the box is ice, and on top is water vapour. The molecules in the water vapour move freely, in the solid state the water molecules in the liquid state of the water vibrate, there is something between them. Example of correct justification of students of Group 3. S3_7: 3D Animation 1 is correct. In Animation 1, we see that the water at the bottom of the window is in a liquid state, which illustrates that flowing water has a higher density than the ice above it. Above the ice or the solid state of water is the gaseous state of water or water vapour. Solid-state water molecules also oscillate in this representation of particle motion, which correctly illustrates the solid state. As can be seen from the justifications for the selections of the SMRs, students of all age groups, including Group 3 (pre-service chemistry teachers), have problems describing the sub-microscopic level with the macroscopic level and misunderstandings within the sub-microscopic level, which is in line with research findings;19,20 primary school students have problems in understanding SMRs for states of matter and transitions between them, and most students can explain the motion of particles in the liquid and solid states of matter.23,24 It was found that the proportion of tested students who used macroscopic levels to represent the state of water decreased with age.48 Other studies15-17 have also shown that primary school, secondary school, and university students have problems explaining the process (represented at the macroscopic level) at the sub-microscopic level, as the present study shows. Stu- Table 2. Relative frequencies of successful and unsuccessful students' arguments at a specific level of representation, m - macroscopic level; m & s - macroscopic and sub-microscopic level; s - sub-microscopic level. Group 1 Group 2 Group 3 m f m & s f s f m (f) m & s f s (f) m f m & s f s (f) -n Task 1: Solid state of water - 6.67 3.33 - 6.89 6.89 - 15.00 40.00 o a S Task 2: Liquid state of water - 20.00 - - 10.34 6.89 - 40.00 10.00 « Task 3: Freezing of water 13.33 - - 13.79 17.24 - 20.00 20.00 - « Task 1: Solid state of water 13.33 50.00 26.67 3.45 48.28 34.48 - 15.00 30.00 O 3 a S Task 2: Liquid state of water 6.67 40.00 33.33 - 55.17 27.59 - 25.00 25.00 5 « Task 3: Freezing of water 66.67 16.67 3.33 27.59 41.38 - 25.00 35.00 - Group 1: Students aged 12. Group 2: Students aged 16. Group 3: Students aged 23. Slapničar et al.: Students' Achievements in Solving Authentic Tasks ... 910 Acta Chim. Slov. 2020, 67, 904-915 dents often assign macroscopic properties to particulate matter, revealing their misunderstandings about the macroscopic and sub-microscopic levels of particulate matter representations.21,53,54 In terms of teacher awareness, it is important to note that switching between the macroscopic and sub-microscopic levels is generally difficult for students. It is the teachers' task to accustom the students to sub-microscopic representations gradually and to present them with different examples so that they do not only recognise what they already know. Pre-service chemistry teachers should pay particular attention to the level of chemical concept representation so that they will be able to teach their students confidently. 4. 2. TFDs of Successful and Unsuccessful Students at AOIs with SMRs of Authentic Tasks, Including the Solid, Liquid State of Water and the Freezing of Water. The second research question related to the identification of differences between successful and unsuccessful students of different age groups in the TFD on AOIs, including 3D dynamic SMRs in tasks dealing with the solid, the liquid state of water and the freezing of water. Table 3 presents Mdns and IQRs for TFD at different AOIs - 3D dynamic SMRs for successful and unsuccessful students of three age groups for Task 1 about the solid state of water. Differences in TFDs of successful and unsuccessful students of Group 1, Group 2, and Group 3 on the correct SMR 1 are not statistically significant (U = 49.000, p = 0.600; U = 20.000, p = 0.060; U = 50.000, p = 1.000, respectively). However, differences in TFDs on the incorrect SMRs of successful and unsuccessful students for students from certain age groups are statistically significant only for Group 3 on AOI with the SMR 3 (U = 22.000, p = 0.038, n2 = 0.218). The results show that Task 1 of a solid aggregate state is well known for both successful and unsuccessful students of each age group, which is reflected in the fact that there are no statistically significant differences in the processing time of the information provided by TFD. Differences in TFDs of successful and unsuccessful including all 79 students together on AOI with the correct SMR in Task 1 (SMR 1) were not statistically significant (successful students: Mdn = 11.015, IQR = 9.055; unsuccessful students: Mdn = 13.788, IQR = 9.858; U = 435.500, p = 0.185) as well as on the incorrect SMR 3 (successful students: Mdn = 0.812, IQR = 0.969; unsuccessful students: Mdn = 1.430, IQR = 2.178; U = 384.500, p = 0.054). However, statistically significant differences in TFDs between all successful and unsuccessful students appear on AOI with SMR 2 (successful students: Mdn = 4.530, IQR = 7.462; unsuccessful students: Mdn = 7.322, IQR = 8.470; U = 373.000, p = 0.040, q2 = 0.054). The size effect is small. Differences in TFDs of successful students regarding the age group on the AOI with the correct SMR in Task 1 were not statistically significant (Kruskal-Wallis x2(2) = 5.720, p = 0.075, n2 = 0.248), whereas they are statistically significant for incorrect SMRs, concretely on the AOI with SMR 2 (Kruskal-Wallis x2(2) = 7.126, p = 0.028, n2 = 0.342), and AOI with the SMR 3 (Kruskal-Wallis x2(2) = 10.724, p = .005, n2 = 0.582) (Table 3). This reflects the fact that successful students of all age groups observe the correct AOI for the same amount of time. It can be anticipated that this SMR is well known to the students. Differences in the TFDs of unsuccessful students regarding the age group on the AOI with the correct SMR in Task 1 were not statistically significant (Kruskal-Wallis X2(2) = 4.451, p < 0.000, n2 = 0.042), whereas they are statistically significant for incorrect SMRs, concretely on the AOI with SMR 2 (Kruskal-Wallis x2(2) = 20.090, p < 0.001, Table 3. Median (Mdn) and interquartile range (IQR) of TFDs on areas of interest for successful and unsuccessful students of 3 age groups for Task 1 (solid state of water). The correct SMR is SMR 1. Variable SMR 1 Mdnj IQRi AOI SMR 2 Mdn2 IQR2 SMR 3 Mdn3 IQR3 Successful (n = 3) o u Unsuccessful o (n = 27) 2 Successful Sp (n = 4) o u Unsuccessful o (n = 25) 3 Successful Sp (n = 11) o Unsuccessful u (n = 9) TFD (s) 16.40 16.18 6.60 12.49 10.82 12.86 11.62 5.52 9.83 8.92 7.57 15.79 13.15 14.03 7.15 5.46 16.44 4.23 2.53 5.64 3.83 4.06 7.55 - 2.67 2.72 1.36 1.55 0.93 0.89 0.38 0.93 1.22 0.90 Group 1: Students aged 12. Group 2: Students aged 16. Group 3: Students aged 23. Slapnicar et al.: Students' Achievements in Solving Authentic Tasks ... Acta Chim. Slov. 2020, 67, 904-915 911 n2 = 0.312), and AOI with the SMR 3 (Kruskal-Wallis x2(2) = 17.884, p < 0.001, n2 = 0.274) (Table 3). It is evident that the correct AOI is similarly interesting for unsuccessful students of different age groups. Table 4 shows medians and IQR for TFDs on different AOIs with 3D dynamic SMRs for successful and unsuccessful students of three age groups for Task 2 on the liquid state of water. Similar to Task 1 (solid state of water), Task 2 (including the liquid state of water) shows that there are no statistically significant differences in the TFD between successful and unsuccessful students of each age group on the correct SMR 3 (U = 73.000, p = 1.000; U = 68.000, p = 0.674; U = 40.000, p = 0.481). This might be justified by the fact that tasks containing three typical SMRs of water with only one aggregate state and that are well known to the students reflect the similar value of the TFD for successful and unsuccessful students. From this, it can be concluded that for easier and better known authentic tasks, successful and unsuccessful students within the age group have a similar processing time. Differences in the TFDs of successful and unsuccessful 79 students on AOI with the correct SMR in Task 2 (SMR 3) were not statistically significant (successful students: Mdn = 12.014, IQR = 9.601; unsuccessful students: Mdn = 13.186, IQR = 13.988; U = 594.000, p = 0.860) as well as on the AOI with SMR 1 (successful students: Mdn = 1.988, IQR = 1.622; unsuccessful students: Mdn = 2.475, IQR = 2.859; U = 568.000, p = 0.649) and on the AOI with SMR 2 (successful students: Mdn = 4.504, IQR = 3.530; unsuccessful students: Mdn = 4.441, IQR = 4.454; U = 582.000, p = 0.764). Differences in TFDs of only the successful students regarding the age group on the AOI with the correct SMR in Task 1 (SMR 3) were not statistically significant (Krus-kal-Wallis x2(2) = 2.202, p = 0.333); it is similar on the AOI with the SMR 1 (Kruskal-Wallis x2(2) = 1.856, p = 0.395). However, statistically significant differences in TFDs of successful students regarding the age group are determined on the AOI with SMR 2 (Kruskal-Wallis x2(2) = 7.425, p = 0.024, n2 = 0.301) (Table 4). Differences in TFDs of unsuccessful students regarding the age group on the AOI with the correct SMR (SMR 3) in task 2 were not statistically significant (Kruskal-Wal-lis x2(2) = 3.325, p = 0.190), whereas they are statistically significant for incorrect SMRs, concretely on the AOI with SMR 1 (Kruskal-Wallis x2(2) = 10.326, p = 0.006, n2 = 0.151), and AOI with the SMR 2 (Kruskal-Wallis x2(2) = 14.518, p = 0.001, n2 = 0.228) (Table 4). Table 5 shows medians and interquartile range for TFDs on AOIs (3D dynamic SMRs) for successful and unsuccessful students of three age groups for Task 3 on the freezing of water. Differences in TFDs of successful and unsuccessful students for students from the certain age group on the correct (SMR 1) and other two incorrect SMRs were not statistically significant (SMR 1: Group 1: U = 83.000, p = 0.061; Group 3: U = 141.000, p = 0.015; SMR 2: Group 1: U = 53.000, p = 0.734; Group 2: U = 78.000, p = 0.594; Group 3: U = 36.000, p = 0.384; SMR 3: Group 1: U = 38.000, p = 0.425; Group 2: U = 83.000, p = 0.764; Group 3: U = 32.000, p = 0.238). An exception appears in Group 2 on the AOI with the correct SMR (U = 141.00, p = 0.015, n2 = 0.199). The results obtained show that the interpretation of the results for the liquid aggregate state of water is highly similar to the interpretation of the results for the solid aggregate state of water, which is confirmed by the fact that the successful and unsuccessful students of each age group, individual and known SMR, are similarly interested in solving the problem. Differences in TFDs of the successful and unsuccessful 79 students on AOI with the correct SMR in Task 3 (SMR 1) were statistically significant with large effect (successful students: Mdn = 22.648, IQR = 18.018; unsuccessful students: Table 4. Median (Mdn) and interquartile range (IQR) of interest for successful and unsuccessful students of 3 age groups in Task 2 (liquid state of water). The correct SMR is SMR 3. SMR 1 AOI SMR 2 SMR 3 Variable MdnI IQRI Mdn2 IQR2 Mdn3 IQR3 Successful (n = 6) 2.30 3.30 6.17 5.28 16.85 13.32 o u o Unsuccessful (n =24) 4.21 6.56 6.20 4.98 17.05 16.24 3.0.C0;2-J 54. G. Nicoll, Int. J. Sci. Educ. 2001, 23, 707-730. D01:10.1080/09500690010025012 Slapnicar et al.: Students' Achievements in Solving Authentic Tasks ... Acta Chim. Slov. 2020, 67, 904- 915 915 Povzetek V članku predstavljena raziskava se ukvarja z identifikacijo razlik med udeleženci raziskave, ki so uspešno oz. neuspešno utemeljili izbiro 3 D dinamične submikroskopske predstavitve (SMR) trdnega in tekočega agregatnega stanja vode ter zmrzovanja vode. Preučevane so bile tudi razlike v času trajanja fiksacij na izbranih interesnih področjih med njimi. V raziskavi je sodelovalo 79 udeležencev treh starostnih skupin. Podatki so bili zbrani s strukturiranim intervjujem, ki je vključeval računalniške zaslonske slike treh avtentičnih nalog. Naloga je vsebovala besedilo (problem ali vprašanje), fotografijo pojava na makroskopski ravni in SMR pojava. Metoda očesnega sledilca je bila uporabljena za merjenje fiksacij med reševanjem avtentičnih nalog na določenem interesnem področju. Rezultati kažejo, da so uspešni posamezniki pri utemeljitvah vključevali predvsem makroskopske in submikroskopske predstavitve izbranega pojma. Po vertikali izobraževanja narašča uspešnost izbire in pravilnost utemeljitve prevladujoče na submikroskopski ravni. Med uspešnimi in neuspešnimi učečimi se iste starostne skupine, se po večini ne pojavijo razlike v času trajanja fiksacij na izbranem interesnem področju (pravilni SMR). Potrebne so nadaljnje raziskave, s katerimi bo preučeno procesiranje informacij uspešnih in neuspešnih učečih se, pri reševanju različnih avtentičnih nalog s SMR. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Slapničar et al.: Students' Achievements in Solving Authentic Tasks ... DOI: 10.17344/acsi.2020.5909 Acta Chim. Slov. 2020, 67, 916-926 /^creative ^commons Scientific paper Cyanide-Bridged Polynuclear and One-Dimensional FeIII-MnIII/I1 Bimetallic Complexes Based-on Pentacyanoferrite(III) Building Block: Synthesis, Crystal Structures, and Magnetic Properties Xiaoyun Hao,1 Yong Dou,1 Tong Cao,1 Lan Qin,1 Zhen Zhou,1^ Lu Yang,1 Dacheng Li,2 Qingyun Liu,3 Yueyun Li1 and Daopeng Zhang1^ 1 College of Chemistry and Chemical Engineering, Shandong University of Technology, Zibo 255049, China. 2 College of Chemical Engineering, Liaocheng University, Liaocheng 252059, China. 3 College of Chemical and Environmental Engineering, Shandong University of Science and Technology, Qingdao 266510, PR China. * Corresponding author: E-mail: dpzhang73@126.com Received: 02-14-2020 Abstract In this contribution, based-on the structurally confirmed pentacyanometallate (PPh4)2[Fe(CN)5(imidazole)]-(imida-zole)-H2O (1) and the manganese compounds [Mn(L)(H2O)2]ClO4 (L = N,N-ethylenebis(3-methoxysalicylideneiminate) or [Mn(MAC)(H2O)Cl]ClO4 (MAC = 2,13-dimethyl-3,6,9,12,18-pentaazabicyclo-[12.3.1]octadeca-l(l8),2,12,14,16-pentaene), two new cyanide-bridged bimetallic Fem-Mnm/n complexes {[Mn(L)(H2O)]3[Fe(CN)5(imidazole)]}(ClO4) (2) and {[Mn(MAC)][Fe(CN)5(imidazole)]-CH3OH}n (3) were successfully synthesized and characterized by elemental analysis, IR spectroscopy and X-ray structure determination. Single X-ray diffraction analysis reveals the cationic FeMn3 tetranuclear entity for complex 2, which can be further assembled into supramolecular 1D ladder-like double chain by the strong intermolecular hydrogen bond interactions. For complex 3, it can be structurally characterized as neutral one-dimensional linear single infinite chain. The magnetic investigations discover the ferromagnetic coupling between the Fem-Mnm units in complex 2 and the antiferromagnetic coupling in complex 3 between the FeIII-MnI1 units through the bridging cyanide group, respectively. Keywords: Pentacyanoferrite; cyanide-bridged; crystal structure; magnetic property 1. Introduction In the past several decades, due to their great potential in high-tech fields including quantum compute, information storage, etc., more and more attention have been paid to the research of the molecular-based magnetism.1-8 As one of the most important building block for the rational construction of molecular magnetic systems, cyanide-bridged magnetic complexes have all along received intense attention since their readily controlled molecular topological structures and theoretically predicted magnetic properties.9-13 Up to now, a great deal of cyanide-bridged molecular materials structural ranging from 0-dimension-al cluster to three-dimensional beautiful networks have been rationally designed and structurally characterized. Among those, some systems with interesting magnetic properties, such as single chain magnets (SCMs) and single molecule magnets (SMMs),14-20 spin crossover materials,21-23 high-Tc magnets,24-25 photomagnetic materials,26-29 ferromagnetic materials,30 chirality magnets31-33 et al, have been detailed magnetically studied. One of the most successful strategies used to prepare cyanide-bridged magnetic complexes34-35 is based on the assemble reactions of the decorated polycyanometallates [M(L)(CN)x]n- ((M = Fe, Cr, W, Mo, Mn, Ru; x = 1-8, L = mono- or multi-dentate organic ligand) with other counterpart paramagnetic metal compounds. Because the blocking organic ligand(s) in either the cyano precursors or the counterpart assemble segments can contribute obvious steric effect and therefore efficiently lower the dimen- Hao et al.: Cyanide-Bridged Polynuclear and One-Dimensional Acta Chim. Slov. 2020, 67, 916-926 917 [Mn(L)]+ Scheme 1. The starting materials used in this paper. sionality of the target product, many low-dimensional [M(L)(CN)x]n--based complexes have been expectedly obtained.36-38 Among all the polycyanoferrite(III) building blocks, the pentacyanoferrite(III) [Fe(L)(CN)5]2- ( L' = monodentate ligand) precursors have been comparatively limited employed in cyanide-bridged molecule magnetism field. 39-43 In this paper, we are focusing on the designed preparation of new cyanide-bridged magnetic complexes with the exploitation of pentacyanidefer-rite(III) compound [Fe(CN)5(imidazole)]2- as building block and manganese compounds as assemble segments (Scheme 1). The synthesis, structural wcharacterization and magnetic study for the two new obtained MnII/m-Fem bimetallic magnetic complexes {[Mn(L) (H2O)]3[Fe(CN)5(imidazole)]}(ClO4) (2) and {[Mn(-MAC)][Fe(CN)5(imidazole)] ■ CH3OH}n (3), as well as the structure of the cyano precursor (PPh4)2[Fe(CN)5(im-idazole)] ■ (imidazole) ■ H2O(1), will be presented in the current contribution. 2. Experimental Section 2. 1. General Procedures and Materials All the reactions were carried out at room temperature under air atmosphere with the solvents and chemicals used reagent grade without additional purification. [Ca(imidazole)(H2O)][Fe(CN)5(imidazole)] was prepared as black crystals according to the literature method for [Ca(1-CH3im)(H2O)][Fe(CN)5(1-CH3im)].41 [Mn(L) (H2O)2]ClO442 (L = N,N-ethylenebis(3-methoxysalicyli-deneiminate) and [Mn(MAC)(H2O)Cl]ClO444 (MAC = 2,13-dimethyl-3,6,9,12,18-pentaazabicyclo-[12. 3.1]octa-deca-1(18),2,12,14,16-pentaene) were available from the previous works. Caution! KCN is hypertoxic and hazardous. Perchlo-rate salts with organic ligands are potentially explosive. They should be handled in small quantities with care. Synthesis of (PPh4)2[Fe(CN)5(imidazole)] • (imidazole) • H2O (1): [Ca(imidazole)(H2O)][Fe(CN)5(imidazole)] (1.9 g, 5 mmol) was dissolved in water (20 ml), and PPh4Br (4.0 g, 10 mmol) was added to the solution. Then, the mixture was stirred in the dark overnight before the yellow powder formed was filtered out. Yield: 3.57g (70%). Recrystalliza-tion of the powder in MeOH afforded yellow single crystals. Main IR bands(cm-1): 2110, 2121(s, vCsN). The elemental analysis (experimental and theoretical) and some physical properties are given in Table 1. Synthesis of {[Mn(L)(H2O)]3[Fe(CN)5(imidazole)]} (ClO4) (2): The complex was prepared by using three layers diffusion procedure. A solution containing (PPh4)2 [Fe(CN)5(im-idazole)] ■ (imidazole) ■ H2O (102 mg, 0.10 mmol) dissolved in 5mL H2O was laid in the bottom of a tube, and then carefully upon addition of a mixture solvent of water and methanol (5 mL) with a ratio of 1:1. Finally, a solution of [Mn(L)(H2O)2]ClO4 (112 mg, 0.2 mmol) in 5 mL of CH3OH was carefully added to the top of the mixture solvent layer above formed. The dark-brown crystal appeared in the middle of the tube was filtered out 2-3 weeks later, washed by methanol and dried in air. {[Mn(L) (H2O)]3[Fe(CN)5(imidazole)]}(ClO4) (2): Yield: 49.6 mg (44.6%). Main IR bands(cm-1): 2150 (s, nC„N), 2122 (s, nC°N), 1620 (m, nC=N), 1100 (s, nCl=O). The elemental analysis (experimental and theoretical) and some physical properties are given in Table 1. Synthesis of {[Mn(MAC)][Fe(CN)5(imidazole)] • CH3OH}n (3): A solution of (PPh4)2[Fe(CN)5(imidazole)]-(imidaz-ole) ■ H2O (102 mg, 0.10 mmol) dissolved in 5mL H2O was laid in the bottom of a tube, and then carefully upon addition of a mixture solvent of water and methanol (5 mL) with a ratio of 1:1. Finally, a solution of [Mn(MAC)(H2O) Cl]ClO4 (48 mg, 0.1 mmol) in 5 mL of CH3OH was carefully added to the top of the mixture solvent layer above formed. The yellow crystal appeared in the middle of the tube 2-3 weeks later, which was filtered out, washed by methanol and dried in air. {[Mn(MAC)][Fe(CN)5(imidaz-ole)]-CH3OH}n (3): Yield: 43.6 mg, 70.9%. Main IR bands (cm-1): 2150, 2120(s, vCsN), 1630 (m, nC=N). The elemental analysis (experimental and theoretical) and some physical properties are given in Table 1. Hao et al.: Cyanide-Bridged Polynuclear and One-Dimensional ... 918 Acta Chim. Slov. 2020, 67, 916-926 Table 1. The elemental analysis (experimental and theoretical) and some physical properties for complexes 1-3. Formula Elemental Analysis(Cal.) Elemental Analysis(Found) Color Solubility Melting Point C59H50N9O P2Fe C, 69.55; H, 4.95; N, 12.37 C, 69.62; H, 5.01; N, 12.31 Yellow MeOH > 573K C69H80N13 O20ClFeMn3 C, 49.70; H, 4.84; N, 10.92. C, 49.77; H, 4.91; N, 11.00 Dark-Brown MeOH DMF > 573K C24H31N12 OFeMn C, 46.92; H, 5.09; N, 27.36 C, 46.86; H, 5.16; N, 27.47 Yellow MeOH DMF > 573K 2. 2. X-ray Data Collection and Structure Refinement Single crystals with suitable dimensions for complexes 1-3 for X-ray diffraction analysis were mounted on the glass rod and the crystal data were collected on a Bruk-er APEX II CCD area-detector with a Mo Ka sealed tube (X = 0.71073 A) at room temperature using a « scan mode. The structures were solved by direct method and expanded using Fourier difference techniques with the SHELX-TL-2018/3 program package.45 While all the hydrogen atoms were introduced as fixed contributors, the non-hydrogen atoms were refined anisotropically with anisotropic displacement coefficients. Hydrogen atoms except some ones from the solvent molecules were assigned isotropic displacement coefficients U(H) = 1.2U(C) or 1.5U(C) and their coordinates were allowed to ride on their respective carbon/nitrogen atoms using SHELXL 2018/3. For the solvent H atoms, they were refined isotrop-ically with fixed U values, during which the DFIX command was used to rationalize the bond parameter. The CCD C 1978692-1978694 for complexes 1-3 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/ data_request/cif. Details of the crystallographic parameter, data collection, and refinement are summarized in Table 2. 3. Results and Discussion 3. 1. Synthesis With comparison to the widely used hexacy-anoiron(III) in cyanide-bridged molecular magnetism field, the pentacyanoiron(III) has been relatively less used to assemble magnetic complex, and only several pentacy-anoiron(III)-based heterometallic magnetic systems have been reported.39-42, 45 By using the three-layers diffusion method, which has been shown to be a powerful way for growing single crystals,43-44 one cationic tetranuclear FeIIIMnIII3 entity and one neutral one-dimensional FeIIIM-nII complex have been successfully prepared from the reactions of [Fe(imidazole)(CN)5]2- and the manganese Table 2. Details of the crystal parameters, data collection, and refinement for compounds 1-3. 1 2 3 Formula C59H50N9OP2Fe C69H80N13O20ClFeMn3 C24H31N12OFeMn Fw 1018.87 1667.58 614.4 Crystal system monoclinic monoclinic Triclinic Space group P21/c C 2/c P-1 a/A 12.8623(7) 45.062(5) 10.883(3) b/A 23.5860(13) 13.5410(14) 11.521(3) c/A 18.4068(9) 29.308(4) 13.284(3) a/deg 90 90 90.578(4) p/deg 106.7580(10) 116.647(4) 105.693(4) y/deg 90 90 114.939(4) Z 4 8 2 V/A3 5346.9(5) 15984(3) 1439.1(6) F(000) 2124 6904 636 9/deg 1.65-25.01 1.59-25.01 1.61-25.01 Goodness-of-fit 1.016 1.019 1.046 Ri [I >2a(I)] 0.0459 0.0659 0.0692 wR2 (all data) 0.1166 0.2125 0.2086 Hao et al.: Cyanide-Bridged Polynuclear and One-Dimensional Acta Chim. Slov. 2020, 67, 916-926 919 Scheme 2. The preparation diagram for the complex 2 and 3. Schiff-base [Mn(L)]+ or the macrocyclic [Mn(MAC)]2+ compound, respectively (Scheme 2). The cyanide-bridged heterometallic complexes have been characterized by IR spectroscopy. Compared to the cyanide precursor with only one peak at about 2125 cm-1, two sharp peaks in the IR spectra of complexes 2 and 3 due to the cyanide-stretching vibration [42, 43] were observed at about 2120 and 2160 cm-1, respectively, indicating the presence of bridging and nonbridging cyanide ligands in these complexes. For complex 2, the strong peak centered at 1100 cm-1 is attributed to the free ClO4- anions. 3. 2. Crystal Structure Descriptions The crystal tructure of the cyano precursor: The selected bond lengths and angles for complex 1 are given in Table 3. The molecular structure and the H-bond resulted 1D supramolecular structure are shown in Figures 1 and 2, respectively. The asymmetric unit of (PPh4)2[Fe(CN)5(imidazole)]-(imidazole) ■ H2O (1), which crystallizes in monoclinic space group P2/c, is comprised Table 3. Selected bond lengths (A) and angles (o) for complex 1. Complex 1 Fe(1 -N(1) 2.002(2) P(1)-C(12) 1.793(3) Fe(1 -C(4) 1.951(3) P(1)-C(18) 1.786(3) Fe(1 -C(5) 1.949(3) P(1)-C(24) 1.789(3) Fe(1 -C(6) 1.925(3) P(1)-C(30) 1.797(3) Fe(1 -C(7) 1.951(3) P(2)-C(36) 1.789(3) Fe(1 -C(8) 1.958(3) P(2)-C(42) 1.797(3) Fe(1 -C(4)-N(3) 179.2(3) P(2)-C(48) 1.793(3) Fe(1 -C(5)-N(4) 175.4(3) P(2)-C(54) 1.787(3) Fe(1 -C(6)-N(5) 176.3(3) Fe(1)-C(8)-N(7) 178.4(2) Fe(1 -C(7)-N(6) 177.9(3) Figure 1. The molecular structure of complex 1. The solvent molecule, the co-crystallized imidazole and all the H atoms have been omitted for clarity. Hao et al.: Cyanide-Bridged Polynuclear and One-Dimensional ... 920 Acta Chim. Slov. 2020, 67, 916-926 Figure 2. The H-bond resulted 1D anionic supramolecular structure of the complex 1. The balanced [PPh4]+ cations have been omitted for clarity. Table 4. The selected hydrogen bond parameters (Â, °) for complexes 1-3. Complex 1 D-H...A d(H...A) d(D...A) <(DHA) O1-H1A...N3 2.116 2.941 175.22 O1-H1B...N5#1 2.317 3.042 138.02 N2-H2...N9#2 1.988 2.820 162.61 N8-H8...N5#3 2.419 3.174 146.94 Complex 2 O1-H1A...O8#1 O1-H1B...O7#1 O10-H10A...O5#1 O10-H10B...O3#1 O15-H15A...O12#1 O10-H15B...O14#1 Complex 3 O1-H1A...N8 2.390 2.804 110.57 Symmetry transformations used to generate equivalent atoms: #1 x, -y+1/2, z+1/2; #2 x, -y+1/2, z+1/2; #3 x+1, y, z (1); #1 x, -y, z-1/2 (2). by five moieties: an anionic Fe complex, one water molecule, one imidazole molecule and two tetraphenylphos-phonium cations. The [Fe(CN)5(imidazole)]2- anion contains a low-spin Fe111 center coordinated by one imidazole unit and five CN units, in which four CN groups occupy the four equatorial positions with the fifth CN group col-linearly axial with the imidazole group. The Fe-C bond lengths in a narrow range 1.925(3)-1.958(3) A are slightly shorter than the Fe-Nimidazole bond with the value 2.002(2) A. The Fe-C=N angles are within the range of 175.4(3)-179.3(3)°, indicating the almost linear conformation of these atoms. Under the O-H...N H-bond interactions from the host anion and the co-crystallized water molecules (Table 4), the [Fe(CN)5(imidazole)]2- moieties are linked into 1D supramolecular single chain structure (Figure 2). 3. 3. The Crystal Structure of the Complex 2 The cationic tetranuclear structure and the one-dimensional ladder-like double-chain structure formed by the intermolecular hydrogen bonds for complex 2 are shown in Figures 3 and 4, respectively. The selected important bond parameters for complex 2 are listed in Table 5. The complex 2 is a neutral tetranuclear cluster comprised by the cationic FeMn3 unit and an additional disordered ClO4- as counter anion. In the FeMn3 core, [Fe(im-idazole)(CN)5]2- acting as a mer-m3-coordinating-donor building block is coordinated by three [Mn(L)(H2O)]+ through three cyanide groups and forming a T-like arrangement. Each [Mn(L)(H2O)]+ moiety has an elongated octahedral geometry with Jahn-Teller distortion along the (H2O)-Mn-Ncyanide axis, in which the four equatorial positions are coordinated by N2O2 unit from the Schiff-base ligand and the two axial ones occupied by one N atom of the bridge cyanide group and one O atom from the H2O molecule. The average Mn-NschiffWOschiff-base bond lengths are 1.988(5) A and 1.874(3) A, which are obviously shorter than the Mn-Ncyanide 2.245(5) A and Mn-Owater 2.298(4) A. The Mn(1)-N(1)-C(1) angle (156.5(4)°) is slightly bigger than the other two Mn-N=C angles with values of 146.(4^ and 149.2(4)o The intramolecular Mnm-FeIII distances through bridge-cyanides are very close to each other with values 5.100, 5.100 and 5.154 A, respectively, which are slightly longer than the shortest intermo- Figure 3. The cationic crystal structure of complex 2. All H atoms and the counter ClO4- anion have been omitted for clarity. 2.257 3.029 153.15 2.435 3.119 142.07 2.279 3.033 151.02 2.155 2.984 161.87 2.461 3.022 125.88 2.416 3.022 129.69 Hao et al.: Cyanide-Bridged Polynuclear and One-Dimensional 921 Acta Chim. Slov. 2020, 67, 916-926 Table 5. Selected bond lengths (Á) and angles (°) for complexes 2-3. Complex 2 Fe(1)-C(1) 1.941(5) Mn(1)-O(2) 1.889(3) Mn(2)-O(8) 1.871(3) Fe(1)-C(2) 1.928(5) Mn(1)-O(4) 1.863(4) Mn(2)-N(2) 2.247(4) Fe(1)-C(3) 1.914(5) Mn(2)-O(6) 1.873(3) Mn(2)-N(10) 1.986(4) Fe(1)-C(4) 1.958(6) Mn(1)-N(1) 2.253(5) Mn(2)-N(11) 1.982(4) Fe(1)-C(5) 1.922(6) Mn(1)-N(8) 1.996(5) Mn(2)-O(10) 2.273(3) Fe(1)-N(6) 1.986(4) Mn(1)-N(9) 1.980(5) Mn(1)-O(1) 2.336(4) Mn(1)-N(1)-C(1) 156.1(4) Mn(2)-N(2)-C(2) 146.4(4) Mn(3)-N(3)-C(3) 149.1(4) Fe(1)-C(1)-N(1) 171.1(5) Fe(1)-C(2)-N(2) 175.3(5) Fe(1)-C(3)-N(3) 175.9(5) Fe(1)-C(4)-N(4) 175.3(5) Fe(1)-C(5)-N(5) 176.5(5) C(1)-Fe(1)-C(2) 172.0(2) N(3)-Mn(3)-O(15) 175.7(2) N(2)-Mn(2)-O(10) 170.6(1) N(1)-Mn(1)-O(1) 174.3(1) Complex 3 Fe(1)-C(1) 1.936(5) Fe(2)-C(7) 1.946(6) Mn(1)-N(1) 2.199(5) Fe(1)-C(2) 1.941(7) Fe(2)-C(8) 1.939(6) Mn(1)-N(6) 2.220(5) Fe(1)-C(3) 1.958(6) Fe(2)-C(9) 1.940(6) Mn(1)-N(11) 2.251(5) Fe(1)-N(4) 1.958(6) Fe(2)-N(9) 1.940(6) Mn(1)-N(12) 2.294(4) Mn(1)-N(13) 2.299(4) Mn(1)-N(14) 2.242(5) Fe(1)-C(1)-N(1) 179.3(5) Fe(2)-C(7)-N(6) 177.1(5) Fe(1)-C(2)-N(2) 178.9(6) Fe(2)-C(8)-N(7) 179.8(6) Fe(1)-C(3)-N(3) 173.2(9) Fe(2)-C(9)-N(8) 176.5(11) Mn(1)-N(1)-C(1) 161.7(5) Mn(1)-N(12)-C(16) 112.0(4) Mn(1)-N(6)-C(7) 152.6(4) Mn(1)-N(12)-C(17) 110.8(4) Mn(1)-N(11)-C(13) 123.9(5) Mn(1)-N(13)-C(18) 115.1(5) Mn(1)-N(11)-C(15) 116.6(4) Mn(1)-N(13)-C(19) 109.6(3) Figure 4. One-dimensional double-chain structure of complex 2 formed by intermolecular hydrogen-bond interactions. All the H atoms except those used to form hydrogen bonds and the balance ClO4- have been omitted for clarity. Hao et al.: Cyanide-Bridged Polynuclear and One-Dimensional ... 922 Acta Chim. Slov. 2020, 67, 916-926 lecular metal-metal separation. Because of the excellent encapsulation ability of the O4 compartment for the water molecule, one-dimensional ladder-like double chain structure is formed under the help of the intermolecular H-bond interactions (Table 3) between the OH2O atoms and the O atoms of the Schiff-base ligand. 3. 4. The Crystal Structure of the Complex 3 The asymmetry binuclear unit and 1D neutral chain structure of complex 3 is presented in Figure 5. The important bond parameters are given in Table 5. As can be found, complex 3 possesses one-dimensional neutral single chain structure comprising of the repeating [-NC-Fe(imidazole)(CN)3-CN-Mn(MAC)-] units. In this complex, each [Fe(imidazole)(CN)5]2- unit, functioning as bidentate ligand through its two cyanide groups in trans position, connects the Mn(II) ions of two independent macrocyclic manganese units. Similar to that in compounds 1 and 2, the coordination geometry of the Fe atom is also a slightly distorted octahedral. As listed in Table 5, the bond angle of Fe-C=N in the range of 171.1(5)— 176.5(5)° clearly indicates that the three atoms are in a good linear configuration. The Mn11 ion displays the hep- ta-coordinates mode and involved in a pentagonal-bipyra-midal geometry with the macrocyclic ligand forming the equatorial plane. The five equatorial positions are occupied by N5 unit from the macrocyclic ligand, while the axial positions are occupied by two N atoms of the bridging cyanide groups. The Mn-NMAC distances within the range 2.196-2.299 A are with no conspicuous with the Mn-Ncya-nide bond lengths (2.199(5) and 2.220(5)A), indicating the only slightly distorted pentagonal-bipyramidal geometry of the Mn(II) ion. With comparison to the Fe-C=N angle distributed in a very narrow range of 179.3(5)°-177.1(5)°, the bridging cyanide ligands coordinated to the Mn(II) ions in a bent fashion with the Mn-N=C angles are 152.6(4) and 161.7(5)CI, respectively. 3. 5. The Magnetic Properties of Complexes 2 and 3 The temperature dependences of the cmT product per FeIIIMnIII3 and FeIIIMnI1 unit for 2 and 3 measured from 2 to 300 K under an applied magnetic field of 2000 Oe are shown in Figures 6 and 7. The cmT value at room temperature is 9.11 emu K mol-1 = 8.57 BM) for 2 and 4.33 emu K mol-1 (^eff = 5.91 BM) for 3, which are slightly Figure 5. Asymmetry neutral unit and 1D neutral chain structure of complex 3. All the hydrogen atoms and solvent molecules have been omitted for clarity. Hao et al.: Cyanide-Bridged Polynuclear and One-Dimensional ... Acta Chim. Slov. 2020, 67, 916-926 923 lower than the spin only value of 9.375/4.75 emu K mol 1 for uncoupled three high spin Mn(III) (S = 2)/one high spin Mn(II) ( S = 5/2) and one low spin Fe(III) (S = 1/2) based on g = 2.00, respectively. For these two complexes, the cmT values maintains nearly constant until the temperature lowering to 20 K for 2 and 50 K for 3. After that, the cmT value for complex 2 starts to increase smoothly and reaches its highest value 10.12 emu K mol-1, and then decreases rapidly to the lowest value about 0.87 emu K mol-1 at 2 K. The suddenly decrease for the cmT value in the low temperature range can maybe attributed to the comparable strong intermolecular H-bond interaction and/or the zero field spitting of the Mn(III) ion. Different from that for complex 2, the cmT value of complex 3 presents the obvious decreasing tendency from 50 K, and attains its minimum value 2.19 emu K mol-1 at 2 K, implying the different coupling nature in these two complexes. The magnetic susceptibilities of 2 and 3 conform well to Curie-Weiss law and give the positive Weiss constant q = 1.87 K, Curie constant C = 8.90 emu K mol-1 for 2 and negative q = -1.90 K, C = 4.54 emu K mol-1 for 3. On the basis of the Weiss constant and with the combination of change tendency of cmT-T curves, the overall ferromagnetic magnetic coupling between Fe(III) and Mn(III) ions in complex 2 and antiferromagnetic coupling between Fe(III) and Mn(II) ion in 3 can be concluded. In view of the situation that the three Mn-N=C-Fe bridges are structurally independent and the coordination environment of the three Mn(III) ions are not completely same to each other with the Mn-N=C angles 146.4(4)-156.1(4)° and the Mn-NCN bond lengths 1.980(5)-2.253(5) Á, the simulation of the magnetic susceptibility for this compound should be based on three different J values J # J2 # J3), where the three J values represent the Mn(1)... Fe(1), Mn(2)...Fe(1) and Mn(3)...Fe(1) interactions through cyanide bridges, respectively. On the other hand, according to the method successfully employed to simu- T/K Figure 6. Left: The cmT-T (the solid line represents the best fit based on The field-dependent magnetization for complex 2. late the magnetic susceptibilities of 1D chain compound with alternating spins 1/2 and 2,30b the one-dimensional chain structure of the complex 3 can be considered as isotropic Heisenberg chain containing alternating spins 1/2 and 5/2 with two antiferromagnetic exchange interactions J1 and J2. In this case, the magnetic susceptibility of this complex can be calculated rationally based on a closed ring cluster model consisting of five 1/2-5/2 spin pairs, 0 50 100 150 200 250 300 T/K Figure 7. Temperature dependence of cmT of complex 3 (the solid line represents the best fit based on the parameters discussed in the text). Inset: Field dependence of magnetization at 2 K (the dotted line is the Brillouin curve for antiferromagnetic coupled Fe(III) and Mn(II) ions with g = 2.0). Scheme 4. Evaluation of the exchange coupling between the iron(III) ion and manganese(III/II) ions bridged by cyanide group in complexes 2 and 3 are carried out by MAGPACK program.47 The best-fit parameters obtained are J1 = 1.12, J2 = 1.65, J3 = 0.91 cm-1, D = -1.81 cm-1, g = 2.02, R H lOkOf the parameters discussed in the text) and cm-T curve for complex 2. Right: Hao et al.: Cyanide-Bridged Polynuclear and One-Dimensional 924 Acta Chim. Slov. 2020, 67, 916-926 Scheme 4. The magnetic simulation model for the complex 3. = Z(cobsdT-ccaldT)2/Z(cobsdT)2 = 1.61 x 10-5 for complex 2 and J¡ = -1.34, J2 = -0.54(1) cm-1, g = 1.99, R = 3.01 x 10-5 for complex 3, respectively. All the theoretical fitting results are comparable to those found in the previously reported cyanide-bridged FeIII-MnIII/I1 complexes.42-44, 48 The field-dependent magnetizations measured up to 50 kOe at 2 K for complexes 2 and 3 are shown in Figure 6 and the inset of Figure 7, respectively. The field-dependent magnetization curve for complex 2 has a sigmoid shape, implying maybe the metamagnetic behavior: The magnetization first increases slowly with increasing magnetic field until 20 kOe because of the relatively strong intermolecular hydrogen bond interaction, then increases abruptly for a phase transition at about 20 kOe, and finally attains the highest value about 7.45 Nb , which is slightly higher than the saturated value for three Mn(III) ion (S = 2) and one low spin Fe(III) ion (S = 1/2). For complex 3, the magnetization quickly increases with the field increasing until about 15 kOe, then increases smoothly up to about 3.9 Nb until 50 kOe. This data is very close to the saturated value of 4.0 Nb but obviously lower than the value of uncoupled low spin Fe(III) and Mn(II) based on g = 2.0, confirming again the overall antiferromagnetic coupling interaction between Fe(III) and Mn(II) ions bridged by cyanide group. 4. Conclusion In summary, two new heterobimetallic cyanide-bridged complexes have been prepared with pentacy-anoiron(III) as building block and manganese(III/II) compounds as the counterpart assemble segment. The single crystal X-ray analysis revealed the cationic tetranuclear FeMn3 entity or one-dimensional infinite chain structure, respectively. For the polynuclear cluster, it can be self-complementary through coordinated aqua ligand from one complex and the free O4 compartment from the neighboring complex, therefore giving interesting supramolecular one-dimensional ladders. The experimental and theoretical investigation on their magnetic properties disclose the ferro- or antiferromagnetic coupling in cyanide-bridged Fem-Mnm or Fem-Mnn units, respectively. The present results can further enrich the pentacyanometallate-based molecule magnetic system, which is helpful for fully discover the magneto-structural relation from the molecule magnetism. 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DOI:10.3390/polym11101585 Povzetek V tem prispevku je na osnovi strukturno potrjenega pentacianidometalata (PPh4)2[Fe(CN)5(imidazol)](imidazol)H2O (1) in manganovih spojin [Mn(L)(H2O)2]ClO4 (L = N,N-etilenbis(3-metoksisalicilideniminat) in [Mn(MAC)(H2O) Cl]ClO4 (MAC = 2,13-dimetil-3,6,9,12,18-pentaazabiciklo-[ 12.3.1]oktadeka-1(18),2,12,14,16-pentaen) opisana sinteza dveh novih Fenl-MnIII/n kompleksov s cianidnim mostom, {[Mn(L)(H2O)]3[Fe(CN)5(imidazol)]}(ClO4) (2) in {[Mn(MAC)][Fe(CN)5(imidazol)]CH3OH}n (3), njihova karakterizacija z elementno analizo, IR spektroskopijo in rentgensko strukturno analizo. Rentgenska analiza na monokristalu je pokazala, da je spojina 2 zgrajena iz tetranuk-learnih enot FeMn3, ki se z močnimi vodikovimi vezmi povezujejo v supramolekularne 1D dvojne verige v obliki lestve, medtem ko spojino 3 sestavljajo nevtralne enodimenzionalne enojne verige. Magnetne meritve so pokazale feromagnet-no sklopitev med Fera-Mnm enotami in antiferomagnetno sklopitev med FeIII-MnI1 enotami preko mostovnih cianidnih skupin v spojinah 2 in 3. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Hao et al.: Cyanide-Bridged Polynuclear and One-Dimensional ... DOI: 10.17344/acsi.2020.5932 Acta Chim. Slov. 2020, 67, 927-933 /^creative o'commons Scientific paper Synthesis, Crystal Structures and Catalytic Property of Oxidovanadium(V) Complexes with N'-(4-Oxopentan-2-ylidene)nicotinohydrazide and 4-Bromo-N'-(4-oxopentan-2-ylidene) benzohydrazide Qiwen Yang,1^ Pu Wang1 and Yan Lei1,2 1 College of Environment and Ecology, Chongqing University, Chongqing 400030, P. R. China 2 College of Environmental and Chemistry Engineering, Chongqing Three Gorges University, Chongqing404000, P. R. China * Corresponding author: E-mail: yangqiwen222@126.com Received: 02-21-2020 Abstract A pair of structurally similar oxidovanadium(V) complexes with the general formula [VOLL'], with the hydrazone compounds N'-(4-oxopentan-2-ylidene)mcotinohydrazide (H2L') and 4-bromo-N'-(4-oxopentan-2-ylidene)benzohy-drazide (H2L2), and the acetohydroxamic acid (HL') as ligands, have been synthesized and structurally characterized by physico-chemical methods and single crystal X-ray determination. Single crystal X-ray analysis indicates that the V atoms in the complexes are in octahedral coordination, with the ONO donor atoms of the hydrazone ligands, and the OO donor atoms of the acetohydroxamate ligands, as well as an oxido O atom. The complexes showed good property for the catalytic epoxidation of styrene. Keywords: Oxidovanadium(V) complex; hydrazone ligand; crystal structure; catalytic property 1. Introduction In recent years, due to the environmental and economic issues, green chemistry became the principle for chemical syntheses. One of the major goals in recent research is to find new and efficient catalysts for the industrially important reactions. Hydrazone compounds, the aldehyde- or ketone analogs in which the carbonyl group is replaced by an imine or azomethine group, are considered privileged ligands, because of their simple preparation in an one-pot condensation of aldehydes (or ketones) and primary amines in an alcohol solvent.1 The metal complexes of hydrazones have been widely studied for their structures, biological activities and catalytic properties.2 Among the complexes, those with V centers are of particular interest for their biological and catalytic properties.3 The epoxi-dation of alkenes is one of the most widely studied reactions in organic chemistry since epoxides are key starting materials for a wide variety of products. The catalytic epox-idation of olefins by various complexes is a hot research Scheme 1. The hydrazone compounds H2L1 and H2L2. topic.4 A number of vanadium complexes with Schiff base ligands are reported for the oxidation of various organic substrates.5 However, the vanadium complexes with hydra-zones derived from hydrazides with acetylacetone have seldom been reported. Herein we report the syntheses, crystal structures and catalytic epoxidation properties of a pair of structurally similar oxidovanadium(V) complexes, [VOL1L'] (1) and [VOL2L'] (2), where L1 and L2 are the eno-late form of N'-(4-oxopentan-2-ylidene)nicotinohydrazide (H2L1) and 4-bromo-N'-(4-oxopentan-2-ylidene)benzohy-drazide (H2L2), respectively (Scheme 1), and L' is the anionic form of acetohydroxamic acid (HL'). Yang et al.: Synthesis, Crystal Structures and Catalytic 928 Acta Chim. Slov. 2020, 67, 927-933 2. Experimental 2. 1. Materials [VO(acac)2], nicotinohydrazide and 4-bromoben-zohydrazide were purchased from Aldrich. All other reagents with AR grade were used as received without further purification. 2. 2. Physical Measurements Infrared spectra (4000-400 cm-1) were recorded as KBr discs with a FTS-40 BioRad FT-IR spectrophotometer. The electronic spectra were recorded on a Lambdar 35 spectrometer. Microanalyses (C, H, N) of the complex were carried out on a Carlo-Erba 1106 elemental analyzer. Solution electrical conductivity was measured at 298K using a DDS-11 conductivity meter. GC analyses were performed on a Shimadzu GC-2010 gas chromatograph. 2. 3. X-ray Crystallography Crystallographic data of the complexes were collected on a Bruker SMART CCD area diffractometer with graphite monochromated Mo-Ka radiation (À = 0.71073 Â) at 298(2) K. Absorption corrections were applied by using the multi-scan program.6 The structures of the complexes were solved by direct methods and successive Fourier difference syntheses, and anisotropic thermal parameters for all nonhydrogen atoms were refined by full-matrix least-squares procedure against F2.7 All non-hydrogen atoms were refined anisotropically. The amino H atoms were located from difference Fourier maps and refined isotrop-ically. The N-H distances were restrained to 0.86(1) Â. The crystallographic data and experimental details for the Table 1. Crystallographic data for the single crystal of the complexes 1 2 Empirical formula C13H15N4O5V C14H15BrN3O5V Formula weight 358.23 436.14 Temperature (K) 298(2) 298(2) Crystal system Monoclinic Triclinic Space group P2i/n P"1 a (A) 7.658(1) 7.952(1) b (A) 23.873(1) 10.371(1) c (A) 8.556(1) 11.613(1) « (°) 90 110.733(1) P (°) 96.883(1) 96.007(1) Y (°) 90 102.030(1) V (A3) 1552.9(3) 859.2(2) Z 4 2 F(000) 736 436 D ata/restraints/p arameters 2891/1/214 3187/1/222 Goodness-of-fit on F2 1.132 1.023 Rh wR2 [I > 2o(I)] 0.0491, 0.1072 0.0488, 0.1047 Ru wR2 (all data) 0.0618, 0.1126 0.0762, 0.1189 structural analysis are summarized in Table 1, and the selected bond lengths and angles are listed in Table 2. Table 2. Selected bond distances (Â) and bond angles (°) for the complexes 1 2 V1-O1 1.896(2) 1.886(3) V1-O2 1.938(2) 1.975(3) V1-O3 2.228(2) 2.201(3) V1-O4 1.854(2) 1.869(3) V1-O5 1.588(2) 1.588(3) V1-N1 2.059(2) 2.046(3) C2-O1 1.310(4) 1.315(5) C2-C3 1.351(4) 1.357(6) C4-N1 1.314(4) 1.329(5) N1-N2 1.398(3) 1.393(5) C6-N2 1.291(4) 1.302(5) C6-O2 1.303(3) 1.321(5) O5-V1-O4 96.20(11) 95.96(14) O5-V1-O1 98.02(12) 98.50(14) O4-V1-O1 104.00(9) 103.84(12) O5-V1-O2 101.39(12) 100.24(13) O4-V1-O2 90.23(9) 100.24(13) O1-V1-O2 154.51(10) 154.79(12) O5-V1-N1 100.96(11) 101.32(14) O4-V1-N1 159.58(10) 159.63(12) O1-V1-N1 84.58(9) 84.29(12) O2-V1-N1 75.66(9) 75.62(12) O5-V1-O3 171.30(10) 171.74(13) O4-V1-O3 75.52(8) 75.87(11) O1-V1-O3 81.69(9) 82.57(12) O2-V1-O3 81.58(9) 81.31(11) N1-V1-O3 87.68(9) 86.92(12) 2. 4. Synthesis of [VOL1L'] (1) Nicotinohydrazide (1.00 mmol, 0.135 g) and [VO (acac)2] (1.00 mmol, 0.265 g) were mixed and stirred in methanol (50 mL) for 30 min at 25 °C. Then, acetohy-droxamic acid (1.00 mmol, 0.0750 g) was added. The final mixture was further stirred for 30 min. The brown solution was evaporated to remove three quarters of the solvents under reduced pressure, yielding deep brown solid of the complex. Yield: 0.26 g (72%). Well-shaped single crystals suitable for X-ray diffraction were obtained by recrystalli-zation of the solid from methanol. Elemental analysis found: C, 43.75; H, 4.31; N, 15.56%. C13H15N4O5V calcd: C, 43.59; H, 4.22; N, 15.64%. IR data (KBr, cm-1): 3287, 3119, 2917, 2850, 1628, 1556, 1490, 1400, 1332, 1280, 1168, 1116, 1033, 961, 806, 712, 585. UV-Vis data in methanol [Amax (nm), e (L mol-1 cm-1)]: 235, 1.62 x 104; 273, 1.58 x 104; 345, 9.67 x 103; 460, 7.72 x 103. 2. 5. Synthesis of [VOL2L'] (2) 4-Bromobenzohydrazide (1.00 mmol, 0.214 g) and [VO(acac)2] (1.00 mmol, 0.265 g) were mixed and stirred Yang et al.: Synthesis, Crystal Structures and Catalytic Acta Chim. Slov. 2020, 67, 927-929 933 in methanol (50 mL) for 30 min at 25 °C. Then, acetohy-droxamic acid (1.00 mmol, 0.0750 g) was added. The final mixture was further stirred for 30 min. The brown solution was evaporated to remove three quarters of the solvents under reduced pressure, yielding deep brown solid of the complex. Yield: 0.34 g (77%). Well-shaped single crystals suitable for X-ray diffraction were obtained by recrystalli-zation of the solid from methanol. Elemental analysis found: C, 38.37; H, 3.53; N, 9.72%. C14H15BrN3O5V calcd: C, 38.55; H, 3.47; N, 9.63%. IR data (KBr, cm-1): 3272, 3105, 2913, 2880, 1636, 1422, 1145, 1085, 958, 846, 532. UV-Vis data in methanol [Amax (nm), e (L mol-1 cm-1)]: 275, 1.73 x 104; 343, 1.05 x 104; 435, 5.62 x 103. 2. 6. Styrene Epoxidation The epoxidation reaction was carried out at room temperature in acetonitrile under N2 atmosphere with constant stirring. The composition of the reaction mixture was 2.00 mmol of styrene, 2.00 mmol of chlorobenzene (internal standard), 0.10 mmol of the complexes (catalyst) and 2.00 mmol iodosylbenzene or sodium hypochlorite (oxidant) in 5.00 mL freshly distilled acetonitrile. When the oxidant was sodium hypochlorite, the solution was buffered to pH 11.2 with NaH2PO4 and NaOH. The composition of reaction medium was determined by GC with styrene and styrene epoxide quantified by the internal standard method (chlorobenzene). All other products detected by GC were mentioned as others. For each complex the reaction time for maximum epoxide yield was determined by withdrawing periodically 0.1 mL aliquots from the reaction mixture and this time was used to monitor the efficiency of the catalyst on performing at least two independent experiments. Blank experiments with each oxi-dant and using the same experimental conditions except catalyst were also performed. 3. Results and Discussion 3. 1. Chemistry Complexes 1 and 2 were readily prepared by reaction of VO(acac)2, acetohydroxamic acid with nicotinohydra-zide and 4-bromobenzohydrazide, respectively, in methanol (Scheme 2). The hydrazone ligands were formed by the condensation reactions of the hydrazides with the acetyl-acetone ligand of VO(acac)2. The reaction progresses are accompanied by an immediate color change of the solution from colorless to brown. The molar conductivities (AM = 37 O-1 cm2 mol-1 for 1 and 43 O-1 cm2 mol-1 for 2) measured in methanol are consistent with the values expected for non-electrolyte.8 The structure of complex 2 has been reported but with different crystal system and space group (orthorhombic Pbca).8 3. 2. Crystal Structure Description of the Complexes Single-crystal X-ray analysis shows that both complexes are structurally similar mononuclear oxidovanadi-um(V) compounds. The differences between the two complexes are the terminal groups, viz. pyridinyl for 1 and bromophenyl for 2. The ORTEP plots of the complexes 1 and 2 are shown in Figs. 1 and 2, respectively. The V atom is in distorted octahedral geometry, which is coordinated by the NO2 donor atoms of the hydrazone ligand and the hydroxyl O atom of the acetylhydroxamate ligand in the equatorial plane, and by the carbonyl O atom of the acetyl-hydroxamate ligand and the oxido O atom at the two axial positions. The metal atoms are displaced toward the axial oxido O atoms (O5) by 0.29-0.30 Â from the equatorial planes of both complexes. The distortion of the octahedral coordination of the complexes can be observed from the bond angles (Table 2) related to the V atoms. The cis- and Yang et al.: Synthesis, Crystal Structures and Catalytic ... 930 Acta Chim. Slov. 2020, 67, 927-933 Table 3. Hydrogen bond distances (Á) and bond angles (°) for the complexes D-H-A d(D-H) d(H-A) d(D"A) Angle (D-H-A) 1 N3-H3A-N4" C8-H8—O4*2 0.86(1) 0.93 1.96(1) 2.55 2.813(4) 3.053(3) 171(4) 114(5) N3-H3A-O2« C1-H1A—O1*4 C12-H12—O3#5 0.86(1) 0.96 0.93 1.99(1) 2.58 2.57 2.838(4) 3.492(5) 3.253(5) 170(5) 159(6) 131(6) Symmetry codes: #1: 2 - - y, 1 - z. - x, -y, 1 - z; #2: - -1 + x, y, z; #3: 2 - x, -y, 1 - z; #4: 2 - x, -y, -z; #5: 2 - x, 1 trans- angles related to the V atoms at the equatorial planes are in the range of 75.52(8)-104.00(9)° and 154.51(10)-171.30(10)° for 1 and 75.62(12)-103.84(12)° and 154.79(12)-171.74(13)° for 2. The deviations from the ideal octahedral geometry are mainly origin from the strain created by the five-membered chelate rings N1-V1-O2 and O3-V1-O4. The bond lengths of V-O and V-N (Table 2) of both complexes are similar to each other, and comparable to those in other V complexes in literature.9,10 The terminal V1-O5 [1.588(2) Á] bond distances of both complexes agree well with the corresponding values reported for related systems.11 Because of the trans influence of the oxido groups, the distances to the O3 atoms (2.20-2.23 Á) are considerably elongated, making the O3 atoms weakly coordinated to the V atoms. Such elongation has previously been observed in other complexes with similar struc-tures.12 The hydrazone ligands coordinate to the V atoms through dianionic form, which can be seen from the bond lengths of C6-O2, N1-N2, C2-C3 and C2-O1. The bonds C6-O2 and C2-O1 are obviously longer than typical double bonds, and the bonds C6-N2 and C2-C3 are obviously shorter than typical single bonds. This phenomenon is not uncommon for hydrazone complexes.13 In the crystal structures of complex 1, the molecules are linked through N-H—N hydrogen bonds between the amino group of the acetohydroxamate ligand and the pyridine N atom, as well as the C-H—O hydrogen bonds be- Fig. 1. ORTEP diagram of complex 1 with 30% thermal ellipsoid. tween the pyridine C-H group and the hydroxyl O atom of the acetohydroxamate ligand (Table 3), to form one dimensional zigzag chains running along the a axis (Fig. 3). In the crystal structures of complex 2, the molecules are Fig. 2. ORTEP diagram of complex 2 with 30% thermal ellipsoid. Fig. 3. Molecular packing structure of complex 1 linked by hydrogen bonds. Yang et al.: Synthesis, Crystal Structures and Catalytic Acta Chim. Slov. 2020, 67, 927-931 933 linked through N-H—O hydrogen bonds between the amino group of the acetohydroxamate ligand and the carbonyl O atom of the hydrazone ligand, as well as the C-H—O hydrogen bonds between the benzene C-H group and the carbonyl O atom of the acetohydroxamate ligand (Table 3), to generate layers parallel to the bc direction (Fig. 4). 3. 3. Infrared and Electronic Spectra The sharp absorptions at about 3280 cm-1 for the spectra of both complexes are attributed to the N-H bonds of the amino groups. The bands in the region 3120-2850 cm-1 are assigned to the C-H bonds. The intense bands at about 1630 cm-1 are assigned to the vibration of the C=N group.1,12b The characteristic of the spectra of both complexes is the exhibition of sharp bands at about 960 cm-1, corresponding to the V=O stretching vibration.1,13b The appearance of a single band in this region indicates the existence of monomeric six-coordinated V=O units instead of the polymeric units.14 This is approved by the single crystal structure determination. In the UV-Vis spectra of the complexes, the bands at about 345 nm and 275 nm are attributed to the n-n* and n-n* transitions.1313,15 The weak bands at 430-470 nm are attributed to intramolecular charge transfer transitions from the pn orbital on the nitrogen and oxygen to the empty d orbitals of the V atoms.12b,13b 3. 4. Catalytic Property The percentage of conversion of styrene, selectivity for styrene oxide, yield of styrene oxide and reaction time to obtain maximum yield using both the oxidants are shown in Fig. 5. The data reveals that the complexes as catalysts convert styrene most efficiently in the presence of both oxidants. Nevertheless, the catalysts are selective (over 90%) towards the formation of styrene epoxides de- spite of the formation of by-products like benzaldehyde, phenylacetaldehyde, styrene epoxides derivative, alcohols etc. From the data it is also clear that the complexes exhibit high efficiency for styrene epoxide yields. When the reactions were carried out with PhIO at 2 h, styrene conversions of complexes 1 and 2 are 95% and 87%, respectively. When the reactions were carried out with NaOCl at 3 h, styrene conversions of complexes 1 and 2 are 93% and 84%, respectively. It is evident that between PhIO and Na-OCl, the former acts as a better oxidant with respect to both styrene conversion and styrene epoxide selectivity. Moreover, complex 2 has better conversion values than complex 1, which is in accordance with that the presence of electronegative groups in the ligands increases the catalytic efficiency of the complexes.16 The epoxide yields for the complexes 1 and 2 using PhIO and NaOCl as oxidants are about 80% and 75%, respectively. Thus, both complexes have good and similar catalytic properties on the oxidation of styrene. 12 12 12 12 12 12 Fig. 5. Catalytic epoxidation results. The red and blue columns represent the results catalyzed by PhIO (2 h) and NaO Cl (3 h), respectively. Yang et al.: Synthesis, Crystal Structures and Catalytic ... 932 Acta Chim. Slov. 2020, 67, 927-933 4. Conclusion In summary, two oxidovanadium(V) complexes derived from hydrazone and acetylhydroxamate ligands were prepared and characterized. The V atoms in the complexes are in octahedral coordination. Both complexes have good catalytic property for the epoxidation of styrene with the good selectivity (over 90%) and high styrene epoxide and epoxide yields. The presence of electronegative groups in the ligands can increase the catalytic efficiency of the complexes. Supplementary Material CCDC 1985429 (1) and 1985432 (2) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam. ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: deposit@ ccdc.cam.ac.uk. Acknowledgements This project was supported by the Scientific and Technological Research Program of Chongqing Municipal Education Commission (Grant No. KJQN201801222), the Chunhui Project from Education Ministry of China (Grant No. Z2015140), the Key Laboratory of Water Environment Evolution and Pollution Control in Three Gorges Reservoir (Chongqing Three Georges University) (Grant No. 0969809) and the Key Cultivation Project of Chongqing Three Gorges University (Grant No. 17ZD12). 5. References 1. Grivani, G. Bruno, H. A. Rudbari, A. D. Khalaji, P. Pourtei-mouri, Inorg. Chem. Commun. 2012, 18, 15-20. DOI:10.1016/j.inoche.2011.12.044 2. (a) S. Dasgupta, S. Karim, S. Banerjee, M. Saha, K. Das Saha, D. Das, Dalton Trans. 2020, 49, 1232-1240; DOI:10.1039/C9DT04636D (b) M. Cuccioloni, L. Bonfili, V. Cecarini, M. Nabissi, R. Pet-tinari, F. Marchetti, R. Petrelli, L. Cappellacci, M. Angeletti, A. M. Eleuteri, ChemMedChem 2020, 15, 105-113; DOI:10.1002/cmdc.201900551 (c) A. A. Khandar, Z. M. 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Gray, Inorg. Chem. 1997, 36, 5927-5930; D0I:10.1021/ic970824q (c) R. Ando, H. Ono, T. Yagyu, M. Maeda, Inorg. Chim. Acta 2004, 357, 2237-2244. D0I:10.1016/j.ica.2003.12.031 15. S. Mondal, M. Mukherjee, K. Dhara, S. Ghosh, J. Ratha, P. Banerjee, A. K. Mukherjee, Cryst. Growth Des. 2007, 7, 17161721. D0I:10.1021/cg060753i 16. (a) J. Rahchamani, M. Behzad, A. Bezaatpour, V. Jahed, G. Dutkiewicz, M. Kubicki, M. Salehi, Polyhedron 2011, 30, 2611-2618; D0I:10.1016/j.poly.2011.07.011 (b) D. M. Boghaei, A. Bezaatpour, M. Behzad, J. Mol. Catal. A: Chem. 2006, 245, 12-16. D0I:10.1016/j.molcata.2005.09.022 Povzetek Sintetizirali smo dva strukturno sorodna oksidovanadijeva(V) kompleksa s splošno formulo [VOLL'], s hidrazonoma N'-(4-oksopentan-2-iliden)nikotinohidrazid (H2L') in 4-bromo-N'-(4-oksopentan-2-iliden)benzohidrazid (H2L2) in z acetohidroksamsko kislino (HL') kot ligandi ter ju okarakterizirali z fiziko-kemijskimi metodami in monokristalno rentgensko difrakcijo. Kristalna analiza razkriva, da je V atom v oktaedričnem okolju z ONO donorskimi atomi hidrazons-kega liganda, z OO donorskima atomoma acetohidroksamatnega liganda ter oksido O atomom. Kompleksa izkazujeta dobre lastnosti pri katalitični epoksidaciji stirena. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Yang et al.: Synthesis, Crystal Structures and Catalytic ... DOI: 10.17344/acsi.2020.5938 Acta Chim. Slov. 2020, 67, 934-939 /^creative ^commons Scientific paper Synthesis and Evaluation on Anticonvulsant and Antidepressant Activities of Naphthoquinone Derivatives Containing Pyrazole and Pyrimidine Fragments Nataliia Polish,1 Mariia Nesterkina,2^ Nataliia Marintsova,1 Andriy Karkhut,1 Iryna Kravchenko,2 Volodymyr Novikov1 and Andrei Khairulin3 1 Department of Technology Biologically Compounds, Pharmacy and Biotechnology, Lviv Polytechnic National University, Lviv 79013, Ukraine 2 Department of Organic and Pharmaceutical Technology, Odessa National Polytechnic University, Odessa 65044, Ukraine 3 Laboratory of Condensed Heterocyclic Compounds, Department of Sulfur Chemistry, Institute of Organic Chemistry NAS of Ukraine, Kyiv 02660, Ukraine * Corresponding author: E-mail: mashaneutron@gmail.com; ORCID 0000-0002-3201-7961 Received: 02-22-2020 Abstract Novel heterocyclic dichloronaphthoquinone derivatives have been synthesized by chlorine atom substitution in 2,3-di-chloro-1,4-naphthoquinone to pyrazole or pyrimidine fragments. The structures of these compounds have been confirmed by FT-IR, ESI-MS, 'H-NMR, 13C-NMR and elementary analysis. Synthesized compounds were evaluated for their anticonvulsant action in a pentylenetetrazole (PTZ)-convulsion model and antidepressant activity in the forced swimming test (FST). All naphthoquinone derivatives at a dose 100 mg/kg indicated anticonvulsant effect in PTZ-induced test at 3 h and 24 h after oral administration. In addition, these compounds possessed prolonged antidepressant properties significantly reducing the duration of immobility time when compared to the reference drug amitriptyline. Keywords: 2,3-dichloro-1,4-naphthoquinone; pyrazole and pyrimidine fragments; anticonvulsant activity; antidepres-sant action 1. Introduction The development of novel compounds possessing combined action on central nervous system (CNS) and, thus, capable of being used simultaneously in treatment of various CNS disorders, still remains an active field in drug discovery. Such CNS disease state as depression is concomitant pathology in patients with epilepsy while some antidepressants were found to increase the risk of seizures (bupropion) or exhibit both the anticonvulsant and pro-convulsant effect in experimental study (venlafaxine).1 In this context, synthesis of compounds contemporaneously demonstrating antidepressant and anticonvulsant activity is feasible approach to reduce aforementioned side effects. In this context, significant interest is attracted by naphtho-quinones and their derivatives as building blocks for rational drug design. A considerable amount of these com- pounds have already been reported as antifungal, anti-inflammatory, anticancer and antibacterial agents.2-6 Surprisingly, only a limited number of publications are devoted to investigation of naphthoquinones influence on CNS. For example, amide derivatives of 4-amino-1,2-naph-thoquinone were examined for anticonvulsant activity by the maximal electroshock (MES) and subcutaneous pentylenetetrazole (sc. PTZ) tests.7 The antidepressant potential of plumbagin, a medicinal plant-derived naphthoqui-none, was explored in unstressed and stressed mice and explained by inhibition of brain monoamine oxidase A (MAO-A) activity.8 Naphthoquinones derived from Lith-ospermum erythrorhizon (acetylshikonin and shikonin) were isolated and proven as inhibitors of MAO-A and MAO-B in a competitive manner that might be further used in the treatment of depression.9 Obviously, the nature and position of substituents in naphthoquinone core are Khairulin et al.: Synthesis and Evaluation on Anticonvulsant and Acta Chim. Slov. 2020, 67, 934-935 939 the crucial factors affecting the pharmacological evaluation of the structures. Bearing in mind that five- and six-membered heterocycles, pyrazole and pyrimidine, are important scaffold for CNS-active compounds,10-13 our attention was paid to the naphthoquinones containing these moieties. Thus, here we report the synthesis of ami-nopyrazole- and aminopyrimidine derivatives of 2,3-di-chloro-1,4-naphthoquinone and their anticonvulsant and antidepressant activity determined by pentylenetetrazole (PTZ) and forced swim test (FST), accordingly. 2. Experimental 2. 1. Chemistry IR spectrum was measured with a Thermo Scientific Nicolet iS10 FT-IR Spectrometer using Nicolet iZ10 module (Thermo Fisher Scientific, Madison, WI, USA) equipped with a diamond window in a range of 4000-525 cm-1. 1H NMR and 13C NMR spectra were recorded on Varian Mercury-400 (Varian Inc., Palo Alto, CA) 300 MHz/75 MHz spectrometer with DMSO-d6 or CDCl3 as solvents and TMS as an internal standard; the coupling constants are given in Hz. The elemental analysis was performed on a Euro Vector EA-3000 (Eurovector SPA, Re-davalle, Italy) microanalyzer. Elemental analyses were within ±0.4% of the theoretical values. Electrospray ionization mass spectrometry (ESI-MS) was measured by Agilent 1100 Series (LC/MSD Trap) Spectrometer applying isocrat-ic elution of acetonitrile : 0.01% formic acid aqueous solution (70:30). Separation column: Rapid Resolutionn HT Cartige 4.6x30 mm, 1.8-Micron, Zorbax SB-C18. Melting points (uncorrected) were measured in an open capillary tube using a Stuart SMP30 melting point apparatus. 2. 1. 1. General Procedure for the Synthesis of Aminopyrazole Derivatives of Naphthoquinone (3a-d) To a magnetically stirred solution of 2,3-di-chloro-1,4-naphthoquinone (1) (0.68 g, 0.3 mmol) in eth-anol (50 mL) was added a solution of aminopyrazole 2a-d (0.3 mmol) in ethanol (20 mL). The reaction was carried out at 78 °C in the presence of an equivalent amount of Na2CO3 with constant stirring for 3 h. Reaction progress was monitored by TLC analysis. After reaction completion the obtained precipitate was filtered, washed several times with water and dried. The precipitate was suspended in 20 ml of ethanol, heated to boiling, filtered from impurities, the filtrate was cooled with ice to 0 °C, the precipitated crystals were filtered, dried in vacuum over CaCl2 to afford compounds 3a-c as red and 3d as orange colored crystals. 2-Chloro-3-((1-(difluoromethyl)-1-H-pyrazol-3-yl)-amino)-naphthalene-1,4-dione (3a) Yield 42%, red crystals, m.p. = 123-125 °C. IR (KBr, cm-1): 3642 (N-H), 2968, 2895 (CHaliphatic), 1681 (C=O), 1059, 1083 (C-F). 1H NMR (300 MHz, DMSO-d6): 5 9.01 (s, 1H, NH), 7.93-8.05 (m, 4H, Ar-H), 7.12 (d, J = 4.4 Hz, 1H, CH-pyraz.), 5.95 (d, J = 4.4 Hz, 1H, CH-pyraz.), 6.63 (s, 1H, CH). 13C NMR (75 Hz, DMSO-d6) 5, ppm: 180.03 (C), 176.9 (C), 149.1 (C), 147.9 (C), 133.1 (CH), 133.2 (ch), 131.0 (CH), 130.6 (C), 129.1 (C), 123.9 (CH), 123.8 (ch), 114.7 (CH), 106.1 (C), 94.8 (CH). MS (ESI), m/z (%): calculated for C14H8ClF2N3O2 [M]+ 323, found 323 (100). Calcd: C 58.45; H 3.50; Cl 12.32; N 14.61; O 11.12. Found: C 58.32; H 3.40; Cl 12.24; N 14.50. HPLC: tr = 0.879 min. 2-Chloro-3-((1-methyl-1H-pyrazol-3-yl)amino)naph-thalene-1,4-dione (3b) Yield 78%, red crystals, m.p. = 215-217 °C. IR (KBr, cm-1): 3200 (N-H); 1680, 1652 (C=O), 2967, 2894 (CHaliphatic), 717 (C-Cl). 1H NMR (300 MHz, CDCl3): 5 9.03 (s, 1H, NH), 7.96-8.07 (m, 2H, Ar-H), 7.71-7.91 (m, 2H, Ar-H), 7.60 (d, J = 2.2 Hz, 1H, CH-pyraz.), 6.04 (d, J = 2.2 Hz, 1H, CH-pyraz.), 3.76 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) 5, ppm: 180.1 (C), 177.1 (C), 149.2 (C), 147.5 (C), 133.3 (CH), 133.2 (CH), 132.2 (CH), 131.2 (C), 130.8 (C), 125.0 (ch), 124.8 (CH), 106.7 (C), 92.7 (CH), 37.7 (CH3). MS (ESI), m/z (%): calculated for C14H10ClN3O2 [M]+ 287, found 287 (100). Calcd: C 58.45; H 3.50; Cl 12.32; N 14.61; O 11.12. Found: C 58.30; H 3.42; Cl 12.21; N 14.50. HPLC: tr = 0.961 min. 2-Chloro-3-((3-(p-tolyl)-1H-pyrazol-5-yl)amino)naph-thalene-1,4-dione (3c) Yield 56%, red crystals, m.p. = 238-240 °C. IR (KBr, cm-1): 3500 (N-H), 1672 (C=O), 1600-1572 (C=C), 720 (C-Cl). 1H NMR (300 MHz, DMSO-d6): 5 12.95 (s, 1H, NH), 9.07 (s, 1H, NH), 7.97-8.06 (m, 2H, Ar-H), 7.86 (t, 1H, Ar-H), 7.79 (t, 1H, Ar-H), 7.63 (d, J = 7.8 Hz, 2H, Ar-H), 7.26 (d, J = 7.9 Hz, 2H, Ar-H), 6.49 (s, 1H, CH-pyraz.), 2.33 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) 5, ppm: 179.9 (C), 176.8 (C), 148.2 (C), 144.3 (C), 142.1 (c), 138.1 (C), 132.9 (CH), 132.8 (CH), 131.2 (C), 131.1 (C), 129.8 (2CH), 125.9 (2CH), 125.6 (CH), 125.2 (CH), 124.9 (C), 105.3 (C), 94.2 (CH), 22.0 (CH3). MS (ESI), m/z (%): calculated for C20H-14ClN3O2 [M]+ 363, found 363 (100). Calcd: C 66.03; H 3.88; Cl 9.74; N 11.55; O 8.80. Found: C 65.62; H 3.77; Cl 9.63; N 11.25. HPLC: tr = 1.389 min. Ethyl-4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-1-phenyl-1H-pyrazol-3-carboxylate (3d) Yield 76%, orange crystals, m.p. = 162-165 °C. IR (KBr, cm-1): 3300 (N-H), 1712 (C=O), 1676, 1652 (C=O), 1604-1576 (C=C), 720 (C-Cl). 1H NMR (300 MHz, DMSO-d6): 5 9.38 (br.s, 1H, NH), 8.12 (s, 1H, CH-pyraz.), 8.01 (t, 2H, Ar-H), 7.89 (d, J = 7.4x2 Hz, 1H, Ar-H), 7.82 (d, J = 7.4x2 Hz, 1H, Ar-H), 7.66 (d, J = 7.9 Hz, 2H, Ar-H), 7.51 (t, 2H, Ar-H), 7.42 (t, 1H, Ar-H), 4.04 (q, CH2CH3, 2H), 1.02 (t, CH2CH3, 3H). 13C NMR (75 MHz, DMSO-d6) 5, ppm: 180.2 (C), 176.9 (C), 161.9 (C), 149.3 (C), 147.5 (C), 140.9 (C), 133.2 (CH), 133.1 (CH), 131.1 (C), 131.0 (C), 130.8 (CH), 130.0 (2ch), 127.1 Khairulin et al.: Synthesis and Evaluation on Anticonvulsant and ... 936 Acta Chim. Slov. 2020, 67, 934-939 (CH), 125.7 (CH), 125.3 (CH), 118.2 (2CH), 106.1 (C), 96.8 (C), 59.5 (CH2), 14.3 (CH3). MS (ESI), m/z (%): calculated for C22H16ClN3O4 [M]+ 422, found 422 (100). Calcd: C 62.64; H 3.83; Cl 8.40; N 9.96; O 15.17. Found: C 62.35; H 3.71; Cl 8.29; N 9.85. HPLC: tr = 1.059 min. 2. 1. 2. General Procedure for the Synthesis Aminopy-rimidine Derivatives of Naphthoquinone (3e-f) To a magnetically stirred solution of2,3-dichloro-1,4-naph-thoquinone (1) (0.27 g, 0.1 mmol) in DMF (15 mL) was added a solution of aminopyrimidine 2e-f (0.1 mmol) in DMF (10 mL). The reaction was carried out at 65 °C in the presence of an equivalent amount of K2CO3 with constant stirring for 4 h. Reaction progress was monitored by TLC analysis. The mixture was left to cool to room temperature (25 °C), then the reaction mixture diluted with water (30 ml) and acidified with 5% HCl to pH 6-7; the formed precipitate was filtered off. The precipitate was suspended in 20 ml of ethanol, heated to boiling, filtered from impurities, the filtrate was cooled with ice to 0 °C, the precipitated crystals were filtered and dried in vacuum over CaCl2 to afford compounds 3a-f as orange colored crystals. 2-Chloro-3-((2-(4-methyl-6-(trifluoromethyl)pyrimi-din-2-yl)ethyl)amino)naphthalene-1,4-dione (3e) Yield 73%, orange crystals, m.p. = 121-123 °C. IR (KBr, cm-1): 3656 (N-H), 2981, 2889 (CHaliphatic), 1677 (C=O), 1138 (C-F). 1H NMR (300 MHz, DMSO-d6): 5 7.91-7.99 (m, 2H, Ar-H), 7.86-7.71 (m, 2H, Ar-H), 7.54 (br.s, NH, 1H), 4.21 (t, 2H, CH2), 3.28 (t, 2H, CH2), 2.53 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) 5, ppm: 180.2 (C), 177.2 (C), 168.1 (C), 166.7 (C), 149.6 (C), 139.5 (C), 133.4 (CH), 133.3 (CH), 131.8 (c), 130.4 (c), 123.1 (ch), 122.7 (ch), 122.0 (C), 112.5 (CH), 107.8 (C), 42.9 (CH2), 31.8 (CH2), 21.3 (CH3). MS (ESI), m/z (%): calculated for C18H13ClF-3N3O2 [M]+ 395, found 395 (100). Calcd: C 54.63; H 3.30; Cl 8.96; F 14.40; N 10.62; O 8.09. Found: C 54.36; H 3.19; Cl 8.87; F 14.29 N 10.50. HPLC: tr = 1.090 min. 2-Chloro-3-((2-(4-(trifluoromethyl)-5,6,7,8-tetrahy-droquinazolin-2-yl)ethyl) amino) naphthalene-1,4-di-one (3f) Yield 53%, orange crystals, m.p. = 118-120 °C. IR (KBr, cm-1): 3659 (N-H), 2980, 2889 (CHaliphatic), 1679 (C=O), 1144, 1123 (C-F). 1H NMR (300 MHz, DMSO-d6): 5 8.04-7.67 (m, 4H, Ar-H), 7.48 (br.s, 1H, NH), 4.19 (t, 2H, CH2), 3.20 (t, 2H, CH2), 2.62-2.93 (m, 4H, 2CH2), 1.90-1.65 (m, 4H, 2CH2). 13C NMR (75 MHz, DMSO-d6) 5, ppm: 180.6 (C), 177.1 (C), 168.1 (C), 165.9 (C), 153.0 (C), 139.8 (C), 133.2 (CH), 133.1 (CH), 131.3 (C), 130.7 (C), 122.8 (CH), 122.3 (CH), 121.7 (C), 119.8 (C), 106.9 (C), 42.8 (CH2), 32.1 (CH2), 30.8 (CH2), 23.7 (CH2), 22.5 (CH2), 22.8 (CH2). MS (ESI), m/z (%): calculated for C21H17ClF-3N3O2 [M]+ 435, found 435 (100). Calcd: C 57.87; H 3.93; Cl 8.13; F 13.08; N 9.65; O 7.34. Found: C 56.89; H 3.89; Cl 8.07 F 12.21; N 9.58. HPLC: tr = 1.199 min. 2. 2. Pharmacological Evaluation 2. 2. 1. Animals Pharmacological investigations of compound 3a-f were studied using outbreed male white mice (18-22 g) as experimental animals purchased from Odessa National Medical University, Ukraine. All animals were kept under 12 h light regime and in a standard animal facility with free access to water and food, in compliance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Specific Purposes (Strasbourg, 1986). 2. 2. 2. Drug Administration Anticonvulsant and antidepressant activities of compounds 3a-f were evaluated at 3 h and 24 h after administration. The compounds were administered orally to mice in Tween 80/water emulsion at a dose of 100 mg/kg and Tween 80/water emulsion has been used as a vehicle control. Valproic acid (VPA, 400 mg/kg, p.o.) and amitriptyline (20 mg/kg, p.o.) served as reference drugs, respectively. 2. 2. 3. Anticonvulsant Activity The anticonvulsant activity of 1,4-naphtoquinone derivatives was evaluated by pentylenetetrazole model (PTZ) as described in.14,15 Doses of PTZ for inducing clonic-ton-ic convulsions (DCTC) and tonic extension (DTE) were calculated relative to control. The anticonvulsant effect of compounds was estimated at 3 h and 24 h after their administration from the increase of pentylenetetrazole MED compared with a control group. MED in percent was calculated using the formula: MED = V/m x 104 where MED - minimum effective dose of PTZ inducing DCTC or DTE; V - volume of PTZ solution, ml; m - animal weight, g. 2. 2. 4. Antidepressant Effect Forced swim test (FST) was used to determine antidepressant action of 1,4-naphtoquinone derivatives 3a-f according to procedure.16 Briefly, mice were placed individually into glass cylinder filled with water (24 ± 3 °C) and total duration of their immobility during 5 minutes has been recorded. 2. 2. 5. Statistical Analysis All results are expressed as mean ± standard error mean (SEM). One-way analysis of variance (ANOVA) was used to determine the statistical significance of the results followed by Tukey's post hoc comparison. ** p < 0.01 and * p < 0.05 was considered as significant. Khairulin et al.: Synthesis and Evaluation on Anticonvulsant and Acta Chim. Slov. 2020, 67, 934-937 939 3. Results and Discussion 3. 1. Chemistry Naphthoquinones are highly reactive compounds due to the activation of unsaturated bond by two conjugated electron-withdrawing carbonyl groups. 2,3-Di-chloro-1,4-naphthoquinone readily reacts with nucleop-hiles with substitution of one chlorine atom by one-step mechanism. A nucleophilic attack results in the formation of a-complex and then the chlorine anion is eliminated with the regeneration of quinoid structure.17,18 The activity of the second chlorine atom depends on the electronic effect of the first substituent. It reduces greatly when an electron-donating group such as an amine is bonded to a C2 atom. However, the second substitution can occur if electron-withdrawing substituent is introduced.19,20 Currently, minor information is available on the reaction of 2,3-dichloro-1,4-naphthoquinone with ami-nopyrazole derivatives. Hassan et al.21 investigated methods for the synthesis of aminopyrazole derivatives of 2,3-dichloro-1,4-naphthoquinone, 2,3-dicyano-1,4-naph-thoquinone and its isomer 2-(dicyanomethylene)in-dan-1,3-dione. According to the described method, reaction of 2 eq. of 2,3-dichloro-1,4-naphthoquinone with 1 eq. of aminopyrazole derivatives without using a base proceeds with the formation of cyclization product involving both nucleophilic centers of 2,3-dichloro-1,4-naphthoqui-none. However, in our case the aforementioned method was ineffective - only the products of monosubstitution instead of cyclization derivatives were formed and isolated with low yield after heating with triethylamine. Given the above, synthesis of novel heterocyclic di-chloronaphthoquinone derivatives (3a-f) was carried out by chlorine atom substitution of 2,3-dichloro-1,4-naph-thoquinone (1) to pyrazole (3a-d) or pyrimidine (3e-f) fragments. As illustrated in Scheme 1, target compounds (3a-f) were obtained by mixing equimolar ratios of 2,3-di-chloro-1,4-naphthoquinone with heterocyclic amines (2a-f) in ethanol with further mixture refluxing for 2 h in the presence of Na2CO3 as a base or using DMF as solvent and K2CO3 as a base with constant stirring of reaction mixture at 65 °C during 4 h. Novel heterocyclic N-derivatives naphthoquinone (3a-f) were obtained in the range of 42-78% yield as red or orange solids. The structures of products 3a-f were reliably confirmed and elucidated on the basis of spectral and analytical data. The FTIR spectra exhibited absorption peaks at 3200-3659 cm-1 (NH), 1652-1712 cm-1 (C=O), 28952980 cm-1 (CHaliphatic), 717-720 cm-1 (C-Cl) and 10591144 cm-1 (C-F). The 1H-NMR spectral data of naphtho-quinone 3a-f contain resonance signals described by their chemical shift, integration and multiplicity that are in full agreement with the presented molecular formulas. 3. 2. Pharmacological Studies In the present study, a non-competitive GABA antagonist pentylenetetrazole (PTZ) has been used to investigate anticonvulsant activity of 1,4-naphthoquinone derivatives (3a-f). PTZ-induced seizure model is positioned as a model of generalized convulsions and extensively used for evaluating the excitability of central nervous system (CNS) and, consequently, activity of gamma-aminobutyric acid (GABA).22 Valproic acid (VPA), an established antie-pileptic drug possessing anticonvulsive effect on PTZ-in-duced model, served as reference drug.23 Anticonvulsant activity of heterocyclic compounds 3a-f was estimated after single oral administration (100 mg/kg, p.o.) at short (3 h) and long (24 h) time periods. As shown in Figure 1, all synthesized compounds and VPA were found to protect animals from clonic-tonic convulsions and tonic extension at 3 h after their oral administration as evidenced by in- Scheme 1. Synthesis of aminopyrazole- (3a-d) and aminopyrimidine (3e-f) derivatives of dichloronaphthoquinone. Reagents and conditions: : EtOH/Na2CO3, reflux, 2 h; ii DMF/K2CO3, 65 °C, 4 h Khairulin et al.: Synthesis and Evaluation on Anticonvulsant and ... 938 Acta Chim. Slov. 2020, 67, 934-939 Figure 1. Anticonvulsant activity of compounds 3a-f at 3 h after oral administration. Values are given as mean ± SEM, n = 5 mice; for all groups p < 0.01 compared with control; * p < 0.05 and ** p < 0.01 compared with VPA creasing of DCTC and DTE values (p < 0.01 vs control). At this time point, DCTC and DTE values of 1,4-naphthoqui-nones 3a (p < 0.05 vs VPA) and 3b-e (p < 0.01 vs VPA) are statistically different from those defined for reference drug (VPA) indicating a decrease of seizure threshold. Control 3a 3b 3c 3d 3e 3f VPA Figure 2. Anticonvulsant activity of compounds 3a-f at 24 h after oral administration. Values are given as mean ± SEM, n = 5 mice; for all groups p < 0.01 compared with control; * p < 0.05 and ** p < 0.01 compared with VPA Evaluation of pentylenetetrazole-induced seizure susceptibility of compounds 3a-f was also carried out at long time period (24 h), as depicted in Figure 2. In this case, statistically significant difference was observed only in tonic phase of clonic-tonic seizures between naphthoquinones 3b (p < 0.01 vs VPA), 3e (p < 0.05 vs VPA), 3f (p < 0.01 vs VPA) and reference drug. It is noteworthy that antiseizure effect of compounds 3a, 3c, 3d were also demonstrated prolonged anticonvulsant action at 24 h after administration that is indicated as DCTC and DTE increase in 2 times when compared to control. As demonstrated in Table 1, mean immobility period was reduced in animals treated both with naphthoquinone derivatives 3a-f and reference drug amitriptyline compared to control at 3 h after oral administration. When compared with amitriptyline, the antidepressant activity of compounds 3a-f did not exceed that of reference drug. Table 1. Antidepressant activity of compounds 3a-f in forced swim test (FST). Compound Immobility time, s 3 h after 24 h after administration administration Control 95.0 ± 8.7 95.0 ± 8.7 ^H 3a 51.7 ± 7.8 34.7 ± 4.9** 3b 16.0 ± 6.7 39.3 ± 8.7** 3c 46.3 ± 7.8 53.7 ± 3.2** 3d 70.7 ± 5.2 20.0 ± 4.5** 3e 39.0 ± 4.4 45.7 ± 4.7** 3f 27.3 ±3.7 47.7 ± 3.0** Amitriptyline 25.7 ± 3.5 93.7 ± 4.4 All values are expressed as mean ± SEM; n = 5 mice; for all groups p < 0.01 compared with control; * p < 0.05 and ** p < 0.01 compared with amitriptyline. However, there was no statistically significant difference in immobility time between control groups of animals and that treated with amitriptyline at long time period (24 h). At this time point, all synthesized naphtho-quinone derivatives were found to possess significant anti-depressant-like effect (p < 0.01 vs. amitriptyline) indicating a prolonged action of compounds 3a-f. 4. Conclusion Heterocyclic N-derivatives naphthoquinone containing pyrazole and pyrimidine moieties have been synthesized in good yield and characterized by a series of analytical and spectroscopic methods (1H NMR, 13C NMR, FT-IR, ESI-MS, LC and elementary analysis). The activity of synthesized compounds as potential anticonvulsive and antidepressive agents was investigated on the models of PTZ-induced seizures and forced swim test (FST), accordingly. Pharmacological analyses showed that compounds 3a-f exhibit anticonvulsant and antidepressant properties at a dose 100 mg/kg both at short and long time period (3 h and 24 h after oral administration). 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Ansar, Molecules 2018, 23, 1-86. DOI:10.3390/molecules23010134 Povzetek Novi heterociklični derivati dikloronaftokinona so bili sintetizirani s substitucijo klorovega atoma v 2,3-dikloro-1,4-naf-tokinonu s fragmenti pirazola ali pirimidina. Strukture teh spojin so bile potrjene s FT-IR, ESI-MS, 1H-NMR, 13C-NMR in elementno analizo. Antikonvulzivno delovanje sintetiziranih spojin je bilo ocenjeno v pentilentetrazol (PTZ) kon-vulzijskem modelu in z antidepresivnim delovanjem v testu prisilnega plavanja (FST). Vsi derivati naftokinona so v odmerku 100 mg/kg izkazovali antikonvulzivni učinek v PTZ-induciranem testu 3 ure in 24 ur po peroralni uporabi. Poleg tega so te spojine izkazovale dolgotrajne antidepresivne lastnosti in znatno zmanjšale čas nepremičnosti v primerjavi z referenčno učinkovino amitriptilin. 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Slov. 2020, 67, 940-948 /^.creative o'commons Scientific paper A Simple and Effective Synthesis of 3- and 4-((Phenylcarbamoyl)oxy)benzoic Acids Urban Košak1 and Stanislav Gobec1'* 1 Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: stanislav.gobec@ffa.uni-lj.si Tel.: + 386 (1)4769585; fax: + 386 (1)4258031 Received: 03-23-2020 Abstract Phenserine, posiphen, tolserine and cymserine and its derivatives are experimental Alzheimer's disease drugs that contain a phenyl phenylcarbamate moiety that is responsible for their anti-Alzheimer activities. We have developed a simple (3 steps) and effective (overall yields 76-90%) method for preparing 3- and 4-((phenylcarbamoyl)oxy)benzoic acids which can be reacted with amines to produce phenyl phenylcarbamate moiety containing amides as new potential anti-Alzheimer disease drugs. The synthesized carboxylic acids are thus important building blocks with potential use in medicinal chemistry and drug discovery. Keywords: ((Phenylcarbamoyl)oxy)benzoic acids; phenyl isocyanates; carbamates; building blocks; Alzheimer's disease. 1. Introduction Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder.1 The synaptic dysfunction and neurodegeneration in AD most severely affects the cholinergic system.2 This decreases the levels of the neurotransmitter acetylcholine (ACh),3 which then produces cognitive impairment and memory loss,4 characteristic for patients with AD. Several compounds are currently being evaluated in preclinical and clinical trials for efficacy in AD, including cholinesterase (ChE) inhibitors which increase the levels of ACh in the brain: phenserine, posi-phene, tolserine and cymserine and its derivatives (Figure 1).5 These experimental Alzheimer's disease drugs all contain the phenyl phenylcarbamate moiety or its derivative. Phenserine6 and posiphen7 contain a phenyl phenyl-carbamate moiety, tolserine8 contains a phenyl or-tho-tolylcarbamate moiety and cymserine and its derivatives9,10 contain a phenyl (4-isopropylphenyl)car-bamate moiety (Figure 1). Phenserine, posiphen, tolserine and cymserine and its derivatives are pseudo-irreversible carbamate inhibitors of ChEs where the phenyl phenylcarbamate moiety is responsible for their biological activity. Their mechanism of inhibition involves a rapid initial covalent reaction between their carbamate carbonyl group and the catalytic serine in the active site of ChEs (carbamoylation). The inhibited (carbamoylated) ChE is then reactivated by a slow hydrolysis (decarbamylation) of the active enzyme serine (Scheme 1).11,12 As part of our development of new ChE inhibitors as potential anti-Alzheimer disease drugs, we designed compounds with the general formula 1 that contain the phenyl phenylcarbamate moiety (Scheme 2A). These compounds were designed based on the structures of our previously reported ChE inhibitors.13-15 We planned to synthesize compounds with the general formula 1 by utilizing one of several methods for the synthesis of carbamates,16,17 i.e. reacting phenols with the general formula 2 with various phenyl isocyanates (3) in the presence of a catalytic amount of 4-dimethylamino pyridine (4-DMAP) in CH2Cl2 or DMF (Scheme 2A).18,19 However, this reaction did not produce the desired carbamates as no reaction was observed. Therefore, we had to plan an alternative synthetic route. We decided to use 3- and 4-((phenylcarbamoyl)oxy) benzoic acids (4) and react them with various amines (5) which we have previously used to synthesize amide13,14 and sulfonamide14,15 ChE inhibitors, in the presence of coupling reagent TBTU and N,N-diisopropylethylamine (DIPEA) in CH2Cl220 to produce the designed amides (Scheme 2B). The problem was that 3- and 4-((phenylcarbamoyl) oxy)benzoic acids (4; Scheme 2B) are not commercially available and procedures for their preparation have also not been reported yet. Herein we describe how we solved Košak and Gobec: A Simple and Effective Synthesis Acta Chim. Slov. 2020, 67, 940-948 941 Dihydrobenzodioxepine cymserine Figure 1. Structures of phenyl phenylcarbamate containing experimental Alzheimer's disease drugs. ChE ChE H ChE inhibited by carbamoylatiort Reactivated ChE Scheme 1. Mechanism of ChE inhibition by phenserine. Košak and Gobec: A Simple and Effective Synthesis 942 Acta Chim. Slov. 2020, 67, 940-948 a) b) Scheme 2. Synthesis of new ChE inhibitors as potential anti-Alzheimer disease drugs with the general formula 1. this problem by developing a simple procedure to produce these building blocks in high overall yields. 2. Experimental 2. 1. General Chemistry Methods NMR and 13C NMR were recorded at 400.130 MHz and 100.613 MHz, respectively, on an NMR spectrophotometer (Bruker Avance III). The chemical shifts (5) are reported in parts per million (ppm) and are referenced to the deuterated solvent used. The coupling constants (J) are reported in Hz, and the splitting patterns are indicated as: s, singlet; br. s, broad singlet; d, doublet; dd, doublet of doublets; td, triplet of doublets; h, hextet; m, multiplet; t, triplet; br. t, broad triplet; dt, doublet of triplets; tt, triplet of triplets; q, quartet; qd, quartet of doublets. Infrared (IR) spectra were recorded on a FT-IR spectrometer (System Spectrum BX; Perkin-Elmer). ATR IR spectra were recorded on a FT-IR spectrometer (Thermo Nicolet Nexus 470 ESP). Micro-analyses were performed on a Perkin-El-mer C, H, N Analyzer 240 C. The analyses are indicated by the symbols of the elements and they were within ±0.4% of the theoretical values. Mass spectra were recorded on a LC-MS/MS system (Q Executive Plus; Thermo Scientific, MA, USA). Melting points were determined on a Leica hot-stage microscope and are uncorrected. Evaporation of the solvents was performed under reduced pressure. Reagents and solvents were purchased from Acros Organics, Alfa Aesar, Euriso-Top, Fluka, Merck, Sigma-Aldrich, and TCI Europe, and were used without further purification, unless otherwise stated. Flash column chromatography was performed on silica gel 60 for column chromatography (particle size, 230-400 mesh). Analytical thin-layer chromatography was performed on silica gel aluminum sheets (0.20 mm; 60 F254; Merck), with visualization using ultraviolet light and/or visualization reagents. Analytical reversed-phase UPLC method A was performed on an LC system (Dionex Ultimate 3000 Binary Rapid Separation; Thermo Scientific) equipped with an autosampler, a binary pump system, a photodiode array detector, a thermostated column compartment, and the Chromeleon Chromatography Data System. The detector on UPLC system was set to 210 nm and 254 nm. The column used for method A was a C18 analytical column (50 x 2.1 mm, 1.8 ^m; Acquity UPLC HSS C18SB). The column was thermostated at 40 °C. Method A: The sample solution (1 ^L; 0.2 mg/mL in MeCN) was injected and eluted at a flow rate of 0.4 mL/ min, using a linear gradient of mobile phase A (MeCN) and mobile phase B (0.1% [v/v] aqueous TFA). The gradient for method A (for mobile phase A) was: 0-2 min, 20%; 2-5 min, 20-90%; 5-8 min, 90%. 2. 2. General Synthetic Procedures 2. 2. 1. General Procedure for Synthesis of Benzyl Esters 6 and 8 (General Procedure 1) To a 100-mL round-bottom flask equipped with a stirring bar, hydroxybenzoic acid (5.000 g, 36.177 mmol, 1.0 mol. equiv.) and DMF (50 mL) were added. The resulting solution was stirred and Na2CO3 (3.837 g, 36.177 Košak and Gobec: A Simple and Effective Synthesis ... Acta Chim. Slov. 2020, 67, 940-948 943 mmol, 1.0 mol. equiv.) was added. Benzyl bromide (4.297 mL, 36.177 mmol, 1.0 mol. equiv) was added dropwise to the suspension and the reaction mixture was stirred for 24 hours at room temperature, then poured into a 500-mL separating funnel. Water (100 mL) was added and the mixture was extracted with Et2O (3 x 150 mL). The combined organic phases where transferred into a 1-L separating funnel, washed with water (3 x 450 mL) followed by sat. brine solution (450 mL), dried over anhyd. Na2SO4, and evaporated to produce the benzyl hydroxybenzoate as a colourless oil which solidified into a white solid after cooling. This product was used in the next step without further purification. 2. 2. 2. General Procedure for Synthesis of Carbamates 10-15 (General Procedure 2) To a round-bottom flask equipped with a stirring bar, benzyl hydroxybenzoate (1.0 mol. equiv.) and CH2Cl2 (c = 0.3 M) were added. The resulting solution was stirred and 4-DMAP (0.01 mol. equiv.) was added. Phenyl isocy-anate, 2-methylphenyl isocyanate or 4-isopropylphenyl isocyanate (1.0 mol. equiv.) was added dropwise and the reaction mixture was stirred for 24 hours at room temperature, then evaporated to produce the carbamates. These products were used in the next step without further purification. 2. 2. 3. General Procedure for Debenzylation of Benzyl Esters Yielding 16-21 (General Procedure 3) To a round-bottom flask equipped with a stirring bar, benzyl ester (1.0 mol. equiv.) and inhibitor-free THF (c = 0.02 g/mL) were added. The resulting solution was stirred and agitated with a stream of argon for 30 min. 10% Pd/C (5% mass of benzyl ester) was added and the resulting suspension was agitated with a stream of hydrogen for 30 min. The reaction mixture was stirred under an atmosphere of hydrogen for 24 hours then agitated with a stream of argon for 30 min, filtered with suction through a pad of Celite and evaporated to produce the carboxylic acid. 2. 3. Synthesis and Characterization of Compounds 2. 3. 1. Synthesis of Benzyl 3-Hydroxybenzoate (6) Synthesized from 3-hydroxybenzoic acid (7) (5.000 g, 36.177 mmol, 1.0 mol. equiv.), Na2CO3 (3.837 g, 36.177 mmol, 1.0 mol. equiv.) and benzyl bromide (4.297 mL, 36.177 mmol, 0.01 mol. equiv) in DMF (50 mL) via general procedure 1 to produce 7.750 g of 6 as a white solid (94% yield). Rf = 0.52 (CH2Cl2/MeOH, 20:1, v/v). 1H NMR (400.130 MHz, CDCl3): 5 5.17 (s, 1H), 5.36 (s, 2H), 7.05 (dd, J1 = 8.0 Hz, J2 = 2.4 Hz, 1H), 7.30-7.45 (m, 6H), 7.56 (s, 1H), 7.66 (d, J = 7.8 Hz, 1H). 2. 3. 2. Synthesis of Benzyl 4-Hydroxybenzoate (8) Synthesized from 4-hydroxybenzoic acid (9) (5.000 g, 36.177 mmol, 1.0 mol. equiv.), Na2CO3 (3.837 g, 36.177 mmol, 1.0 mol. equiv.) and benzyl bromide (4.297 mL, 36.177 mmol, 1.0 mol. equiv) in DMF (50 mL) via general procedure 1 to produce 7.073 g of 8 as a white solid (86% yield). Rf = 0.46 (CH2Cl2/MeOH, 20:1, v/v). 1H NMR (400.130 MHz, CDCl3): 5 5.34 (s, 2H), 5.58 (s, 1H), 6.86 (d, J = 8.7 Hz, 2H), 7.32-7.45 (m, 5H), 8.00 (d, J = 8.7 Hz, 2H). 2. 3. 3. Synthesis of Benzyl 3-((phenylcarbamoyl) oxy)benzoate (10) Synthesized from 6 (3.249 g, 14.235 mmol, 1.0 mol. equiv.), phenyl isocyanate (1.547 mL, 14.235 mmol, 1.0 mol. equiv.) and 4-DMAP (0.017 g, 0.142 mmol, 0.01 mol. equiv.) in CH2Cl2 (47 mL) via general procedure 2 to produce 4.750 g of 10 as a white solid (96% yield). Rf = 0.44 (CH2Cl2). mp 121-123 °C. IR (ATR): 3319, 1707, 1544, 1440, 1278, 1202, 1107, 732, 692 cm-1. 1H NMR (400.130 MHz, CDCl3): 5 5.37 (s, 2H), 6.96 (br. s, 1H), 7.13 (t, J = 7.3 Hz, 1H), 7.33-7.49 (m, 11H), 7.89 (s, 1h), 7.97 (d, J = 7.6 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): 5 66.41, 118.51, 122.52, 123.06, 126.23, 127.03, 127.97, 128.10, 128.46, 128.81, 129.98, 130.93, 135.90, 138.39, 150.60, 151.35, 164.82. HRMS (ESI+): m/z calcd for C21H18NO4: 348.12303; found: 348.12410. Anal. Calcd for C21H17NO4: C, 72.61; H, 4.93; N, 4.03. Found: C, 72.65; H, 4.96; N, 4.00. 2. 3. 4. Synthesis of Benzyl 3-((ortho- Tolylcarbamoyl)oxy)benzoate (11) Synthesized from 6 (3.463 g, 15.172 mmol, 1.0 mol. equiv.), 2-methylphenyl isocyanate (1.881 mL, 15.172 mmol, 1.0 mol. equiv.) and 4-DMAP (0.019 g, 0.152 mmol, 0.01 mol. equiv.) in CH2Cl2 (50 mL) via general procedure 2 to produce 5.373 g of 11 as a white solid (98% yield). Rf = 0.32 (CH2Cl2). mp 78-80 °C. IR (ATR): 3273, 1712, 1531, 1289, 1270, 1232, 1189, 1069, 1022, 747 cm-1. 1H NMR (400.130 MHz, CDCl3): 5 2.34 (s, 3H), 5.37 (s, 2H), 6.75 (br. s, 1H), 7.08 (t, J = 7.3 Hz, 1H), 7.22 (t, J = 7.6 Hz, 2H), 7.33-7.49 (m, 7H), 7.83 (br. s, 1H), 7.89 (s, 1H), 7.96 (d, J = 7.5 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): 5 17.72, 66.41, 115.66, 119.88, 120.41, 122.42, 126.08, 126.12, 126.93, 127.91, 127.97, 128.02, 128.09, 128.45, 129.92, 130.37, 130.90, 135.65, 135.91, 150.92, 152.33, 164.85. HRMS (ESI+): m/z calcd for C22H20NO4: 362.13868; found: 362.13802. Anal. Calcd for C22H19NO4: C, 73.12; H, 5.30; N, 3.88. Found: C, 73.11; H, 5.26; N, 3.92. Košak and Gobec: A Simple and Effective Synthesis 944 Acta Chim. Slov. 2020, 67, 940-948 2. 3. 5. Synthesis of Benzyl 3-(((4-Isopropylphenyl) carbamoyl)oxy)benzoate (12) Synthesized from 6 (3.242 g, 14.204 mmol, 1.0 mol. equiv.), 4-isopropylphenyl isocyanate (2.267 mL, 14.204 mmol, 1.0 mol. equiv.) and 4-DMAP (0.017 g, 0.142 mmol, 0.01 mol. equiv.) in CH2Cl2 (47 mL) via general procedure 2 to produce 5.278 g of 12 as a white solid (95% yield). Rf = 0.45 (CH2Cl2). mp 99-101 °C. IR (ATR): 3322, 2963, 1710, 1529, 1445, 1275, 1231, 1100, 741 cm-1. 1H NMR (400.130 MHz, CDCl3): 5 1.24 (d, J = 6.8 Hz, 6H), 2.84-2.94 (m, 1H), 5.37 (s, 2H), 6.92 (br. s, 1H), 7.20 (d, J = 8.3 Hz, 2H), 7.33-7.49 (m, 9H), 7.88 (s, 1H), 7.96 (d, J = 7.5 Hz, 1h). 13C NMR (100 MHz, DMSO-d6): 5 23.82, 32.74, 66.39, 118.63, 122.46, 126.13, 126.50, 126.98, 127.95, 128.07, 128.43, 129.94, 130.88, 135.88, 136.06, 143.13, 150.65, 151.35, 164.80. HRMS (ESI+): m/z calcd for C24H24NO4: 390.16998; found: 390.16931. Anal. Calcd for C24H23NO4: C, 74.02; H, 5.95; N, 3.60. Found: C, 74.05; H, 5.92; N, 3.58. 2. 3. 6. Synthesis of Benzyl 4-((Phenylcarbamoyl) oxy)benzoate (13) Synthesized from 8 (3.010 g, 13.187 mmol, 1.0 mol. equiv.), phenyl isocyanate (1.433 mL, 13.187 mmol, 1.0 mol. equiv.) and 4-DMAP (0.016 g, 0.132 mmol, 0.01 mol. equiv.) in CH2Cl2 (44 mL) via general procedure 2 to produce 4.415 g of 13 as a white solid (96% yield). Rf = 0.33 (CH2Cl2). mp 103-105 °C. IR (ATR): 3331, 1706, 1543, 1264, 1216, 1102, 1007, 752, 690 cm-1. 1H NMR (400.130 MHz, CDCl3): 5 5.34 (s, 2H), 6.95 (br. s, 1H), 7.10 (t, J = 7.3 Hz, 1H), 7.24 (d, J = 3.4 Hz, 2H), 7.30-7.43 (m, 9H), 8.10 (d, J = 8.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): 5 66.16, 115.35, 118.15, 118.55, 122.08, 123.13, 126.58, 127.86, 128.02, 128.43, 128.82, 130.82, 131.50, 136.06, 138.32, 150.93, 154.43, 164.89. HRMS (ESI+): m/z calcd for C21H18NO4: 348.12303; found: 348.12249. Anal. Calcd for C21H17NO4: C, 72.61; H, 4.93; N, 4.03. Found: C, 72.64; H, 4.96; N, 4.05. 2. 3. 7. Synthesis of Benzyl 4-((ortho- Tolylcarbamoyl)oxy)benzoate (14) Synthesized from 8 (3.453 g, 15.128 mmol, 1.0 mol. equiv.), 2-methylphenyl isocyanate (1.876 mL, 15.128 mmol, 1.0 mol. equiv.) and 4-DMAP (0.018 g, 0.131 mmol, 0.01 mol. equiv.) in CH2Cl2 (50 mL) via general procedure 2 to produce 4.975 g of 14 as a white solid (91% yield). Rf = 0.27 (CH2Cl2). mp 87-89 °C. IR (ATR): 3264, 1705, 1531, 1454, 1272, 1207, 1232, 1016, 753, 696 cm-1. 1H NMR (400.130 MHz, CDCl3): 5 2.34 (s, 3H), 5.37 (s, 2H), 6.76 (br. s, 1H), 7.09 (t, J = 7.2 Hz, 1H), 7.22 (t, J = 8.1 Hz, 2H), 7.29 (d, J = 8.6 Hz, 2H), 7.33-7.45 (m, 5H), 7.83 (br. s, 1h), 8.12 (d, J = 8.7 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): 5 17.70, 66.14, 115.34, 121.98, 124.91, 126.14, 126.40, 126.98, 127.77, 127.87, 128.03, 128.39, 128.44, 130.39, 130.79, 131.48, 135.54, 136.06, 136.42, 151.89, 154.74, 164.90. HRMS (ESI+): m/z calcd for C22H20NO4: 362.13868; found: 362.13803. Anal. Calcd for C22H19NO4: C, 73.12; H, 5.30; N, 3.88. Found: C, 73.16; H, 5.33; N, 3.91. 2. 3. 8. Synthesis of Benzyl 4-(((4-Isopropylphenyl) carbamoyl)oxy)benzoate (15) Synthesized from 8 (2.988 g, 13.091 mmol, 1.0 mol. equiv.), 4-isopropylphenyl isocyanate (2.089 mL, 13.091 mmol, 1.0 mol. equiv.) and 4-DMAP (0.016 g, 0.131 mmol, 0.01 mol. equiv.) in CH2Cl2 (44 mL) via general procedure 2 to produce 4.960 g of 15 as a white solid (97% yield). Rf = 0.37 (CH2Cl2). mp 112-114 °C. IR (ATR): 3329, 2962, 1717, 1537, 1415, 1202, 1113, 1006, 831, 689 cm-1. 1H NMR (400.130 MHz, CDCl3): 5 1.24 (d, J = 7.0 Hz, 6H), 2.84-2.95 (m, 1H), 5.37 (s, 2H), 6.91 (br. s, 1H), 7.21 (d, J = 8.3 Hz, 2H), 7.27 (d, J = 8.3 Hz, 2H), 7.33-7.45 (m, 7H), 8.12 (d, J = 8.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): 5 23.81, 32.75, 66.13, 115.32, 118.24, 118.67, 122.02, 126.52, 127.84, 128.01, 128.42, 131.47, 136.05, 143.24, 150.92, 154.50, 164.88. HRMS (ESI+): m/z calcd for C24H-24NO4: 390.16998; found: 390.17214. Anal. Calcd for C24H23NO4: C, 74.02; H, 5.95; N, 3.60. Found: C, 73.99; H, 5.99; N, 3.57. 2. 3. 9. Synthesis of 3-((Phenylcarbamoyl)oxy) benzoic Acid (16) Synthesized from 10 (5.163 g, 14.863 mmol, 1.0 mol. equiv.) and 10% Pd/C (0.258 g, 5% mass of 10) in inhibitor-free THF (258 mL) via general procedure 3 to produce 3.720 g of 16 as a white solid (96% yield). Rf = 0.00 (CH2Cl2). mp 151-153 °C. IR (ATR): 3337, 2564, 1685, 1523, 1439, 1302, 1208, 1016, 754 cm-1. 1H NMR (400.130 MHz, acetone-d6): 5 7.09 (t, J = 7.3 Hz, 1H), 7.35 (t, J = 7.9 Hz, 2H), 7.50 (d, J = 8.3 Hz, 1H), 7.57 (t, J = 7.9 Hz, 1H), 7.63 (d, J = 8.1 Hz, 2H), 7.86 (s, 1H), 7.93 (d, J = 7.6 Hz, 1H), 9.25 (s, 1H), 11.43 (br. s, 1H). 13C NMR (100 MHz, acetone-d6): 5 120.50, 124.77, 125.13, 128.32, 128.34, 130.74, 131.35, 133.91, 140.54, 152.98, 153.37, 167.96. HRMS (ESI+): m/z calcd for C14H12NO4: 258.07608; found: 258.07740. UPLC purity, 99% at 254 nm (method A, fR = 4.130 min). 2. 3. 10. Synthesis of 3-((ortho-Tolylcarbamoyl) oxy)benzoic Acid (17) Synthesized from 11 (5.292 g, 14.643 mmol, 1.0 mol. equiv.) and 10% Pd/C (0.265 g, 5% mass of 11) in inhibitor-free THF (265 mL) via general prodecure 3 to produce 3.902 g of 17 as a white solid (98% yield). Rf = 0.00 (CH2Cl2). mp 176-178 °C. IR (ATR): 3298, 2565, 1686, 1530, 1449, 1306, 1221, 1023, 942, 750 cm-1. 1H NMR (400.130 MHz, acetone-d6): 5 2.39 (s, 3H), 7.11 (t, J = 7.5 Hz, 1H), 7.19-7.26 (m, 2H), 7.49 (d, J = 8.3 Hz, 1H), 7.56 (t, J = 7.9 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.85 (s, Košak and Gobec: A Simple and Effective Synthesis ... Acta Chim. Slov. 2020, 67, 940-948 945 1H), 7.92 (d, J = 7.6 Hz, 1H), 8.50 (br. s, 1H), 11.32 (br. s, 1H). 13C NMR (100 MHz, DMSO-d6): 5 17.76, 122.54, 124.87, 125.48, 126.18, 126.34, 129.72, 130.43, 132.07, 132.28, 135.71, 150.85, 152.45, 166.64. HRMS (ESI+): m/z calcd for C15H14NO4: 272.09173; found: 272.09150. UPLC purity, 96% at 254 nm (method A, tR = 4.193 min). 2. 3. 11. Synthesis of 3-(((4-Isopropylphenyl) carbamoyl)oxy)benzoic Acid (18) Synthesized from 12 (5.193 g, 13.334 mmol, 1.0 mol. equiv.) and 10% Pd/C (0.260 g, 5% mass of 12) in inhibitor-free THF (260 mL) via general procedure 3 to produce 3.795 g of 18 as a white solid (95% yield). Rf = 0.00 (CH2 Cl2). mp 174-176 °C. IR (ATR): 3320, 2961, 2541, 1715, 1682, 1538, 1450, 1274, 1225, 1017, 840 cm-1. 1H NMR (400.130 MHz, acetone-d6): 5 1.23 (d, J = 7.0 Hz, 6H), 2.842.94 (m, 1H), 7.23 (d, J = 8.4 Hz, 2H), 7.48 (m, 4H), 7.85 (s, 1H), 7.92 (d, J = 7.7 Hz, 1H), 9.18 (s, 1H), 11.41 (br. s, 1H). 13C NMR (100 MHz, acetone-d6): 5 25.35, 35.19, 120.65, 124.76, 128.28, 128.31, 128.53, 131.32, 133.82, 138.18, 145.68, 153.03, 153.39, 167.98. HRMS (ESI+): m/z calcd for C17H18NO4: 300.12303; found: 300.12463. UPLC purity, 99% at 254 nm (method A, tR = 4.723 min). 2. 3. 12. Synthesis of 4-((Phenylcarbamoyl)oxy) benzoic Acid (19) Synthesized from 13 (4.334 g, 12.477 mmol, 1.0 mol. equiv.) and 10% Pd/C (0.217 g, 5% mass of 13) in inhibitor-free THF (217 mL) via general procedure 3 to produce 3.194 g of 19 as a white solid (99% yield). Rf = 0.00 (CH2 Cl2). mp 195-197 °C. IR (ATR): 3305, 2557, 1682, 1527, 1502, 1427, 1292, 1198, 1012, 752 cm-1. 1H NMR (400.130 MHz, acetone-d6): 5 7.10 (t, J = 7.3 Hz, 1H), 7.34-7.38 (m, 4H), 7.63 (d, J = 7.9 Hz, 2H), 8.10 (d, J = 8.1 Hz, 2H), 9.28 (s, 1H), 11.10 (br. s, 1H). 13C NMR (100 MHz, DMSO-d6): 5 115.07, 118.53, 121.89, 123.14, 127.90, 128.87, 130.82, 131.49, 138.33, 151.04, 153.98, 166.67. HRMS (ESI+): m/z calcd for C14H12NO4: 258.07608; found: 258.07647. UPLC purity, 98% at 254 nm (method A, tR = 4.153 min). 2. 3. 13. Synthesis of 4-((ortho-Tolylcarbamoyl) oxy)benzoic Acid (20) Synthesized from 14 (4.640 g, 12.839 mmol, 1.0 mol. equiv.) and 10% Pd/C (0.232 g, 5% mass of 14) in inhibitor-free THF (232 mL) via general procedure 3 to produce 3.384 g of 20 as a white solid (97% yield). Rf = 0.00 (CH2Cl2). mp 183-185 °C. IR (ATR): 3280, 2555, 1685, 1529, 1426, 1291, 1234, 1208, 1161, 748 cm-1. 1H NMR (400.130 MHz, acetone-d6): 5 2.39 (s, 3H), 7.11 (t, J = 7.5 Hz, 1H), 7.20-7.27 (m, 2H), 7.36 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 7.7 Hz, 1H), 8.09 (d, J = 8.6 Hz, 2H), 8.53 (br. s, 1H), 11.14 (br. s, 1H). 13C NMR (100 MHz, DMSO-d6): 5 17.77, 115.14, 121.81, 124.99, 125.56, 126.61, 127.80, 130.46, 130.86, 131.55, 135.63, 152.08, 154.37, 166.75. HRMS (ESI+): m/z calcd for C15H14NO4: 272.09173; found: 272.09436. UPLC purity, 97% at 254 nm (method A, tR = 4.213 min). 2. 3. 14. Synthesis of 4-(((4-Isopropylphenyl) carbamoyl)oxy)benzoic Acid (21) Synthesized from 15 (4.864 g, 12.489 mmol, 1.0 mol. equiv.) and 10% Pd/C (0.243 g, 5% mass of 15) in inhibitor-free THF (243 mL) via general procedure 3 to produce 3.720 g of 21 as a white solid (99% yield). Rf = 0.00 (CH2Cl2). mp 196-198 °C. IR (ATR): 3362, 2964, 2547, 1720, 1676, 1501, 1198, 1011, 828, 758 cm-1. 1H NMR (400.130 MHz, acetone-d6): 5 1.23 (d, J = 6.8 Hz, 6H), 2.84-2.94 (m, 1H), 7.24 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.2 Hz, 2H), 8.09 (d, J = 8.6 Hz, 2H), 9.19 (s, 1H), 11.19 (br. s, 1H). 13C NMR (100 MHz, DMSO-d6): 5 23.90, 32.83, 118.75, 121.85, 126.60, 127.87, 130.87, 136.08, 143.31, 151.12, 154.14, 166.75. HRMS (ESI+): m/z calcd for C17H18NO4: 300.12303; found: 300.12476. UPLC purity, 99% at 254 nm (method A, tR = 4.743 min). 2. 3. 15. Synthesis of 3-((1-(2,3-Dihydro-1H-inden-2-yl)piperidin-3-yl)carbamoyl) phenyl Phenylcarbamate (22) To a 50-mL round-bottom flask equipped with a stirring bar, compound 16 (0.100 g, 0.389 mmol, 1.0 equiv) was added followed by CH2Cl2 (10 mL). The resulting suspension was stirred and cooled to 0 °C. N,N-Diisopropyl-ethylamine (0.135 mL, 0.778 mmol, 2.0 equiv) was added dropwise and the suspension transformed into a solution. TBTU was added and 30 min later solution A (see below) was added dropwise. The reaction mixture was allowed to warm to room temperature and then stirred for 24 hours. During this time a white precipitate formed. The suspension was filtered with suction to produce 0.133 g of compound 22 as a white solid (75% yield). Preparation of solution A: To 25-mL round-bottom flask equipped with a stirring bar, compound 23 (0.112 g, 0.389 mmol, 1.0 equiv) was added followed by CH2Cl2 (11 mL). The resulting suspension was stirred and cooled to 0 °C. N,N-Diisopropylethylamine (0.135 mL, 0.778 mmol, 2.0 equiv) was added dropwise and the suspension transformed into a solution. Characterization of compound 22: Rf = 0.46 (CH-2Cl2/MeOH, 10:1, v/v). mp 137-139 °C. IR (ATR): 3293, 2953, 1716, 1629, 1538, 1226, 1211, 1021, 694 cm-1. 1H NMR (400.130 MHz, DMSO-d6): 5 1.33-1.43 (m, 1H), 1.48-1.60 (m, 1H), 1.73 (d, J = 13.3 Hz, 1H), 1.83 (d, J = 10.5 Hz, 1H), 1.96-2.01 (m, 2H), 2.75-2.86 (m, 3H), 2.983.05 (m, 3h), 3.20 (t, J = 7.7 Hz, 1H), 3.97 (br. s, 1h), 7.047.12 (m, 3H), 7.17-7.18 (m, 2H), 7.33 (t, J = 7.7 Hz, 2H), 7.39 (d, J = 8.4 Hz, 1H), 7.50-7.53 (m, 3H), 7.70 (s, 1H), Košak and Gobec: A Simple and Effective Synthesis 946 Acta Chim. Slov. 2020, 67, 940-948 7.76 (d, J = 7.7 Hz, 1H), 8.27 (d, J = 7.7 Hz, 1H), 10.29 (s, 1H). 13C NMR (100 MHz, DMSO-d6): 5 23.75, 29.80, 36.19, 36.28, 46.47, 50.70, 55.99, 66.07, 118.42, 120.79, 123.00, 124.17, 124.44, 124.72, 126.19, 128.81, 129.27, 135.81, 138.42, 141.27, 150.28, 151.54, 164.55. HRMS (ESI+): m/z calcd for C28H30N3O3: 456.22817; found: 456.22717. UPLC purity, 96% at 254 nm (method A, tR = 4.420 min). 3. Results and Discussion For the synthesis of 3-((phenylcarbamoyl)oxy)ben-zoic acid (16), commercially available 3-hydroxybenzoic acid (7) was treated with benzyl bromide in the presence of Na2CO3 in DMF,21 to provide benzyl 3-hydroxybenzoate (6) in 86% yield. No further purification of compound 6 was required and the diethyl ether used for the extraction of compound 6 was reused for the extraction in the synthesis of benzyl 4-hydroxybenzoate (8) (Scheme 3). In the second step, compound 6 was converted into carbamate 10 with one equivalent of phenyl isocyanate in the presence of a catalytic amount (0.01 equivalent) of 4-DMAP in CH2Cl218,19 in 96% yield. Again, no further purification of carbamate 10 was required. Using one equivalent of phenyl isocyanate, rather than 1.1019 or 1.20 equivalent,18 was found to be an advantage as no over-reaction occurred. As reported previously, excess phenyl iso-cyanate can undergo an SEAr substitution in the phenyl moiety of the carbamate to produce an amide, which can be difficult to separate from the desired carbamate.19 Additionally, 1.0 mol% rather than 5 mol%18,19 of 4-DMAP was enough to produce the desired carbamate in excellent yield (Scheme 3). In the third and final step, the benzyl ester 10 was debenzylated using classic catalytic hydrogenation with gaseous hydrogen and a catalytic amount of 10% Pd/C22 (5% mass of benzyl ester 10) in inhibitor-free THF to produce carboxylic acid 16 in 99% yield (Scheme 3). The hydrogenation was a very clean reaction: no further purification of acid 16 was required and the inhibitor-free THF was reused for the debenzylation of benzyl esters 11-15. The overall yield for the preparation of compound 16 from 3-hydroxybenzoic acid (7) using this procedure was 87% (Table 1). The same procedure was then used to prepare compounds 17-21 from the corresponding hydroxy-benzoic acids 7 or 9 via 11-15 (Scheme 3). Overall yields ranged from 76-90% and are reported in Table 1. Scheme 3. Reagents and conditions: (i) PhCH2Br, Na2CO3, DMF, rt, 24 h, 94% (for 8) and 86% (for 9); (ii) aryl isocyanate, 4-DMAP, CH2Cl2, rt, 24 h, 91-98%; (iii) H2(g), 10% Pd/C, THF, rt, 24 h, 95-99%. Scheme 4. Reagents and conditions: (i) TBTU, N,N-DIPEA, CH2Cl2, 0 °C to rt, 24 h, 75%. Košak and Gobec: A Simple and Effective Synthesis ... Acta Chim. Slov. 2020, 67, 940-948 947 Table 1. The synthesized 3- and 4-((phenylcarbamoyl)oxy)benzoic acids. Starting Final Overall hydroxybenzoic ((phenylcarbamoyl)oxy) yield % acid benzoic acid As a proof of concept that the synthesized 3- and 4-((phenylcarbamoyl)oxy)benzoic acids 16-21 can be used in the next reaction to prepare amides, carboxylic acid 16 was reacted with amine 23 (which we have previously used to synthesize amide13,14 and sulfonamide14,15 ChE inhibitors), in the presence of coupling reagent TBTU and N,N-diisopropylethylamine (N,N-DIPEA) in CH2Cl220 to produce amide 22 in 75% yield (Scheme 4). 4. Conclusions In summary, we have developed method for the synthesis of previously unreported 3- and 4-((phenylcarbam- oyl)oxy)benzoic acids from commercially available 3- and 4-hydroxybenzoic acids, respectively. The main advantages of our method are the simplicity, as no purification of intermediates or final acids is required, and effectiveness, as the overall yields are very good to excellent (76-90%). As we have shown, the synthesized carboxylic acids can be converted further, e.g. reacted with amines to produce amides with potential application in drug discovery. Acknowledgements The authors declare that there is no conflict of interest. This work was supported by the Slovenian Research Agency ARRS (grant No. Z1-9195 and core funding P1-0208). 5. References 1. C. L. Masters, R. Bateman, K. Blennow, C. C. Rowe, R. A. Sperling, J. L. Cummings, Nat. Rev. Dis. Primers 2015, 1, 15056. DOI:10.1038/nrdp.2015.56 2. E. Scherder, in: Aging and dementia: neuropsychology, motor Skills, and pain, 1st ed; VU University Press, Amsterdam, Netherlands, 2011, pp. 9-32. 3. E. Scarpini, P. Schelterns, H. Feldman, Lancet Neurol. 2003, 2, 539-547. DOI:10.1016/S1474-4422(03)00502-7 4. E. K. Perry, B. E. Tomlinson, G. Blessed, K. Bergmann, P. H. Gibson, R.H. Perry, Br. Med. J. 1978, 2, 1457-1459. DOI:10.1136/bmj.2.6150.1457 5. J. L. Cummings, G. Lee, A. Ritter, M. Sabbagh, K. 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DOI: 10.1007/s00702-008-0022-y 11. C. Bartolucci, J. Stojan, Q. Yu, N. H. Greig, D. Lamba, Biochem. J. 2012, 444, 269-277. DOI:10.1042/BJ20111675 12. G. L. Patrick, in: An Introduction to Medicinal Chemistry, Oxford Univeristy Press Inc., New York, United States, 2009, pp. 601-603. 13. U. Kosak, B. Brus, D. Knez, S. Zakelj, J. Trontelj, A. Pislar, R. Košak and Gobec: A Simple and Effective Synthesis 948 Acta Chim. Slov. 2020, 67, 940-948 Šink, M. Jukic, M. Živin, A. Podkowa, F. Nachon, X. Braz-zolotto, J. Stojan, J. Kos, N. Coquelle, K. Salat, J. P. Colletier, S. Gobec, J. Med. Chem. 2018, 61, 119-139. D01:10.1021/acs. jmedchem.7b01086 14. U. Košak, D. Knez, N. Coquelle, B. Brusa, A. Pišlar, F. Nachon, X. Brazzolotto, J. Kos, J. P. Colletier, S. Gobec, Bioorg. Med. Chem. 2017, 25, 633-645. D0I:10.1016/j.bmc.2016.11.032 15. U. Košak, B. Brus, D. Knez, R. Šink, S. Žakelj, J. Trontelj, A. Pišlar, J. Šlenc, M. Gobec, M. Živin, L. Tratnjek, M. Perše, K. Salat, A. Podkowa, B. Filipek, F. Nachon, X. Brazzolotto, A. Wi^ckowska, B. Malawska, J. Stojan, I. Mlinaric Raščan, J. Kos, N. Coquelle, J.-P. Colletier, S. Gobec, Sci. Rep. 2016, 6, 39495. D0I:10.1038/srep39495 16. A. K. Ghosh, M. Brindisi, J. Med. Chem. 2015, 58, 2895-2940. D0I:10.1021/jm501371s 17. M. T. Leffler, E. J. Matson, J. Am. Chem. Soc. 1948, 70, 34393442. D01:10.1021/ja01190a065 18. C. Stock, R. Brückner, Synlett 2010, 16, 2429-2434. D0I:10.1055/s-0030-1258552 19. C. Stock, R. Brückner, Adv. Synth. Catal. 2012, 354, 23092330. D0I:10.1002/adsc.201200303 20. S. Balalaie, M. Mahdidoust, R. Eshaghi-Najafabadi, J. Iran. Chem. Soc. 2007, 4, 364-369. D0I:10.1007/BF03245987 21. B. Laursen, M. P. Denieul, T. Skrydstrup, Tetrahedron 2002, 58, 2231-2238. D0I:10.1016/S0040-4020(02)00096-0 22. T. W. Greene, P. G. M. Wuts, in: Protective Groups in Organic Synthesis, John Wiley and Sons, Inc., New York, United States 1999, pp. 416-418. D0I:10.1002/0471220574 Povzetek Fenserin, posifen, tolserin in cimserin ter njegovi derivati so eksperimentalne učinkovine za zdravljenje Alzheimerjeve bolezni. Te učinkovine vsebujejo fenil fenilkarbamatno skupino, ki je odgovorna za njihovo delovanje proti Alzheimer-jevi bolezni. Razvili smo preprost (trije koraki) in učinkovit (skupni izkoristek 76-90%) postopek za pripravo 3- in 4-((fenilkarbamoil)oksi)benzojske kisline, ki ju lahko pri reakciji z amini pretvorimo v amide s fenil fenilkarbamatno skupino. Ti amidi so nove potencialne učinkovine za zdravljenje Alzheimerjevi bolezni. Sintetizirane karboksilne kisline so tako pomembni gradniki, ki se lahko uporabljajo v farmacevtski kemiji in pri odkrivanju zdravilnih učinkovin. © ® Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Košak and Gobec: A Simple and Effective Synthesis ... DOI: 10.17344/acsi.2020.6009 Acta Chim. Slov. 2020, 67, 949-956 /^creative ^commons Scientific paper Prediction of Single Point Mutations in Human Coronavirus and Their Effects on Binding to 9-O-Acetylated Sialic Acid and Hidroxychloroquine Petar M. Mitrasinovic* Center for Biophysical and Chemical Research, Belgrade Institute of Science and Technology, 11060 Belgrade, Serbia * Corresponding author: E-mail: pmitrasinovic.ist-belgrade.edu.rs@tech-center.com Received: 05-19-2020 Abstract Due to the current spreading of the new disease CoViD-19, the World Health Organization formally declared a world pandemic on March 11, 2020. The present trends indicate that the pandemic will have an enormous clinical and economic impact on population health. Infections are initiated by the transmembrane spike (S) glycoproteins of human coronavirus (hCoV) binding to host receptors. Ongoing research and therapeutic product development are of vital importance for the successful treatment of CoViD-19. To contribute somewhat to the overall effort, herein, single point mutations (SPMs) of the binding site residues in hCoV-OC43 S that recognizes cellular surface components containing 9-O-acetylated sialic acid (9-O-Ac-Sia) are explored using an in silico protein engineering approach, while their effects on the binding of 9-O-Ac-Sia and Hidroxychloroquine (Hcq) are evaluated using molecular docking simulations. Thr31Met and Val84Arg are predicted to be the critical - most likely SPMs in hCoV-OC43 S for the binding of 9-O-Ac-Sia and Hcq, respectively, even though Thr31Met is a very likely SPM in the case of Hcq too. The corresponding modes of interaction indicate a comparable strength of the Thr31Met/9-O-Ac-Sia and Val84Arg/Hcq (or Thr31Met/Hcq) complexes. Given that the binding site is conserved in all CoV S glycoproteins that associate with 9-O-acetyl-sialoglycans, the high hydrophobic affinity of Hcq to hCoV-OC43 S speaks in favor of its ability to competitively inhibit rapid S-mediated virion attachment in high-density receptor environments, but its considerably low specificity to hCoV-OC43 S may be one of the key obstacles in considering the potential of Hcq to become a drug candidate. Keywords: CoViD-19; coronavirus, hidroxychloroquine; pandemic; single point mutation 1. Introduction A number of enveloped, positive single-stranded RNA viruses, denoted by CoV, are involved in respiratory, enteric, hepatic and neuronal infectious diseases both in animals and in humans. Among four CoV genera (alpha, beta, gamma and delta), the beta genus comprises bCoV, hCoV-OC43, MHV, SARS-CoV and MERS-CoV.1 A new infectious disease CoViD-19, caused by a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; also referred to as hCoV-19), was first identified in 2019 in Wuhan, China,2 and has since spread through interpersonal contacts globally. The World Health Organization officially declared a world 2019-20 coronavirus pandemic on March 11, 2020.3 The rate of deaths per number of diagnosed cases varies on a daily basis and currently ranges from 0.2% to 15%, depending on age and other health problems.4 If SARS-CoV-2 continues to adapt genetically, initiating person-to-person transmission, it will presum- ably spread like wildfire throughout the globe. Such pandemic might well arise by a still-undiscovered mechanism, making coronavirus fundamental research and therapeutic product development urgent. The US FDA - United States Food and Drug Administration is testing coronavirus treatments, including hidroxychloroquine and chloro-quine, by looking at widespread clinical trials of the drugs.5 As initial therapy for patients infected with SARS-CoV-2, the use of Hcq is, presumably, more preferential than the use of Cq.6 According to the sources from the pharmaceutical industry, a vaccine will be available on the market in the next 12 to 18 months.7 SARS-CoV-2 binds the angiotensin-converting enzyme 2 (ACE2) at the surface of respiratory cells. There are biophysical and structural evidences that the SARS-CoV-2 S protein binds ACE2 with higher affinity than does SARS-CoV S.8 The shape of SARS-CoV-2 S and the size of the protein (SARS-CoV-2 S)/protein (ACE2) interaction interface make the bi-protein complex unsuitable as a mo- Mitrasinovic: Prediction of Single Point Mutations in Human 950 Acta Chim. Slov. 2020, 67, 949-956 lecular model for exploring competitive inhibition mechanism, by which a small ligand (such as Hcq) is supposed to interfere with the binding of beta CoV S glycoproteins to host receptors, particularly in terms of affinity and specificity. Structural and molecular modeling studies have suggested that the SARS-CoV-2 S protein displays two distinct domains, the receptor-binding domain (RBD) that interacts with ACE2 and the N-terminal domain (NTD) that interacts with the ganglioside-rich domain of the plasma membrane.6 Amino acid residues (111-158) of the NTD of SARS-CoV-2 S define a functional ganglio-side-binding domain (GBD) being completely conserved in clinical isolates worldwide. Hcq has been found to bind sialic acids (linked to gangliosides) with pronounced affinity, indicating its potential to block the interaction of GBD with lipid rafts.6 Therefore, specificity issue underlying the dual recognition of gangliosides and ACE2 by SARS-CoV-2 S has not been raised. One of the most representative CoV prototypes of this genus that causes common cold and pneumonia in elderly population, as well as severe lower respiratory tract infection in patients with compromised immune system is hCoV-OC43.1,9 Based on some structural knowledge of hCoV-OC43 S,10 in the present communication, the recognition modes of the hCoV-OC43 S protein by 9-O-Ac-Sia and Hcq, which share the same binding site (Figure 1), are investigated using molecular docking simulations. A computer-based protein engineering approach11,12 is employed to predict single point mutations to which the virus is prone during its genetic adaptation. A question, how the most likely SPMs affect the affinity and specificity of the hCoV-OC43/9-O-Ac-Sia and hCoV-OC43/Hcq interactions, is consequently analyzed. 2. Methods To obtain the initial coordinates of atoms, the experimental structure of the trimeric hCoV-OC43 S in complex with 9-O-Ac-Sia (PDB ID: 6NZK) was retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB).10 The FoldX molecular design toolkit version 5.013-21 was employed to systematically perform single point mutations of each residue being in the binding site of hCoV-OC43 S. FoldX provides a fast quantitative estimation of the importance of the interactions contributing to the stability of proteins and protein complexes. The algorithm uses a full atomic description of the structure of the proteins, while different energy terms taken into account are weighted using empirical data obtained from protein engineering experiments. The code mutates one amino acid to the other 24, including phosphorylated Tyr, Ser and Thr, as well as hydroxyl Proline in addition to 20 standard amino acids, and repairs the neighbor residues. The way it functions is: it mutates the selected position to Ala and anno- tates all neighbor residues; it mutates the wild-type (wt) residue to itself, and then the neighbors to themselves followed each time by the wt residue to itself. In this way it ensures that, when mutating, any residue that has not been moved in the wt reference will not move. Once this is done, the new wt reference is mutated at the selected position to the target amino acids. To prevent problems, neighbor side chains are only optimized when creating the wt reference after self mutation, but not when making the individual mutants unless a new rotamer for the neighbor is selected.11-21 The predictive power was tested on a very large set of point mutants (1088) that comprise most of the structural environments found in proteins.22 A training database of 339 mutants in nine different proteins was initially considered and the set of parameters and weighting factors that best accounted for the changes in stability of the mutants was optimized. The predictive power was then tested using a blind test mutant database of 667 mutants, as well as a database of 82 protein-protein complex mutants. The global correlation for 95% of the entire mutant database (1030 mutants) was 0.83, with a standard deviation of 0.81 kcal mol-1 and a slope of 0.76.22 In the present work, single point mutations of the binding site residues in the hCoV-OC43 S protein were predicted at pH 8, which was used for the experimental determination of the starting structure (PDB ID: 6NZK).10 It was shown that pH acidification of the medium, such as the one occurring in the endosomal compartment, does not trigger hCoV-OC43 S fusogenic conformational changes.10 Docking calculations were performed using the AScore/ShapeDock protocol from the ArgusLab 4.0.1 suite of programs.23 AScore is based on the decomposition Figure 1. The binding site of hCoV-OC43 S (PDB ID: 6NZK) consists of two loops, L1 (27-Asn-Asp-Lys-Asp-Thr-Gly-32) and L2 (80-Leu-Lys-Gly-Ser-Val-Leu-Leu-86), and two hydrophobic pockets, P1 (Leu85, Leu86 and Trp90) and P2 (Leu80, Trp90 and Phe95) given by dots. Mitrasinovic: Prediction of Single Point Mutations in Human ... Acta Chim. Slov. 2020, 67, 949-956 951 of the total protein-ligand binding free energy into the following contributions: the van der Waals interaction between the ligand and the protein, the hydrophobic effect, the hydrogen bonding between the ligand and the protein, the hydrogen bonding that involves charged donor and/or acceptor groups, the deformation effect, and the effects of the translational and rotational entropy loss in binding process, respectively. Flexible ligand docking was done by describing the ligand as a torsion tree. Groups of bonded atoms that do not have rotatable bonds are nodes, while torsions are connections between the nodes. Topology of a torsion tree is a determinative factor influencing efficient docking. The AScore/ShapeDock protocol is fast, reproducible and formally explores all energy minima.23 This particular protocol was shown to be very consistent for docking ligands into the crystal structures of viral pro-teins,24-28 while the calculated binding free energies were well-correlated with the experimental inhibitory concentrations.26 3. Results and Discussion Infections are mediated by the transmembrane S glycoproteins, binding to host receptors and fusing the viral and cellular membranes.10 Cell surface components that contain 9-O-Ac-Sia are recognized by hCoV-OC43 S.29,30 The interacting site of hCoV-OC43 S (PDB ID: 6NZK) contains the following amino acids: Asn27, Asp28, Lys29, Asp30, Thr31, Gly32, Leu80, Lys81, Gly82, Ser83, Val84, Leu85, Leu86, Trp90 and Phe95 (Figure 1). These residues are mainly located in two loops, L1 (27-Asn-Asp-Lys-Asp-Thr-Gly-32) and L2 (80-Leu-Lys-Gly-Ser-Val-Leu-Leu-86). The P1 hydrophobic pocket is defined by Leu85, Leu86 and Trp90 (Figure 1), accommodating the 9-O-Ac-Sia methyl (Figure 2, center). The P2 hydrophobic pocket is defined by Leu80, Trp90 and Phe95 (Figure 1), suiting the 5-N-acetyl methyl (Figure 2, center). The 9-O-acetyl carbonyl makes a hydrogen bond with Asn27 (2.85 A, Figure 2, right), while the 5-N atom makes a hydrogen bond with the Lys81 backbone carbonyl (2.79 A, Figure 2, right). The 9-O-Ac-Sia Cl-carboxylate makes both a salt bridge with the Lys81 side chain amine (3.24 A, Figure 2, right) and a hydrogen bond with the Ser83 side chain hy-droxyl (2.71 A, Figure 2, right). Thus, the specificity of the hCoV-OC43/9-O-Ac-Sia complex formation is Asn27Lys81Lys81Ser83. Evaluated strength of the hCoV-OC43/9-O-Ac-Sia interaction, seen through a dissociation constant Kd = 48.7 ^M (Table 1), is in agreement with an experimental observation of 49.7 ± 10.7 ^M,10 indicating rapid S-mediated virion attachment, especially in high-density receptor environments.31 The binding site is conserved in all CoV S glycoproteins that associate with 9-O-acetyl-sialoglycans, including hCoV-OC43 S, hCoV-HKU1 S, bCoV S and PHEV S.10 The particular topology of the residues that is similar to those of the ligand-binding pockets of CoV hemagglutinin esterases (HEs) and influenza virus C/D hemagglutinin-esterase fusion (HEF) glycoproteins means that the CoV S glycoproteins share the ligand specificity of influenza C/D HEF but are functionally more compatible to influenza A/B hemagglutinin.10 Each of the binding site residues is mutated to the other 24 (20 standard amino acids, phosphorylated Tyr, Ser and Thr, as well as hydroxyl Proline). All the single point mutants are energetically evaluated with reference to the original receptor (PDB ID: 6NZK). SPMs that stabilize the wt receptor structure for more than 2 kcal mol-1 are extracted as likely ones (Table 1). An average level of ther-mochemical accuracy of 2 kcal mol-1 is acceptable for the structure-based drug (or ligand) design purposes.32-44 A careful inspection of the values in Table 1 shows that the SPM Thr31Met stabilizes the wt/9-O-Ac-Sia complex to a largest extent. The introduction of Met31 (Figure 3), instead of Thr31 (Figure 2), in the binding site is asso- 9-O-Ac-Sia Figure 2. The chemical structure of 9-O-acetylated sialic acid (9-O-Ac-Sia) (left). 9-O-Ac-Sia (bold sticks) in interaction with the binding site residues of hCoV-OC43 S (PDB ID: 6NZK). The P1 (Leu85, Leu86 and Trp90) hydrophobic pocket (dots) accommodates the 9-O-Ac-Sia methyl, while the P2 (Leu80, Trp90 and Phe95) hydrophobic pocket (dots) accommodates the 5-N-acetyl methyl (center). 4 electrostatic contacts of 9-O-Ac-Sia with Asn27, Lys81, Lys81 and Ser83, respectively (right). Mitrasinovic: Prediction of Single Point Mutations in Human 952 Acta Chim. Slov. 2020, 67, 949-956 Table 1. The Binding Free Energies Obtained by Docking 9-O-Ac-Sia and Hcq in the Single Point Mutants of hCoV-OC43 S (PDB ID: 6NZK) Ligand: 9-O-Acetylated Sialic Acid Receptor(a) AGbjnd (kcal mol ') Kd (^M)(b) wt hCoV-OC43 S -5.92 48.68 Asp28Phe -6.79 11.31 Ser83Gly -6.79 11.31 Ser83Val -6.80 11.12 Gly82Ser -6.85 10.23 Asp28Tyr -6.93 8.94 Val84Leu -6.94 8.80 Ser83Arg -6.98 8.22 Leu80Ile -7.01 7.82 Asn27Leu -7.06 7.19 Leu86Met -7.07 7.07 Ser83Tpo -7.09 6.84 Lys81Met -7.12 6.50 Ser83Glu -7.12 6.50 Asp28Leu -7.13 6.40 Asp28Met -7.13 6.40 Val84Arg -7.24 5.32 Ser83Lys -7.28 4.97 Ser83Leu -7.32 4.65 Thr31Leu -7.35 4.42 Asp28Arg -7.50 3.44 Asp28Ptr -7.50 3.44 Asn27Ile -7.60 2.91 Asp28Lys -7.77 2.19 Asp28Glu -7.77 2.19 Asp28Gln -7.77 2.19 Ser83Thr -7.89 1.79 Thr31Met -7.95 1.62 Ligand: Hidroxychloroquine Receptor(a) AGbind (kcal mol-1) Kd (^M) wt hCoV-OC43 S -6.21 29.93 Ser83Gly -7.93 1.67 Asn27Ile -7.94 1.64 Asp28Arg -7.94 1.64 Asp28Gln -7.94 1.64 Asp28Glu -7.94 1.64 Asp28Leu -7.94 1.64 Asp28Lys -7.94 1.64 Asp28Met -7.94 1.64 Asp28Phe -7.94 1.64 Asp28Ptr -7.94 1.64 Asp28Tyr -7.94 1.64 Leu80Ile -7.94 1.64 Lys81Met -7.94 1.64 Leu86Met -7.94 1.64 Asn27Leu -7.95 1.62 Ser83Thr -7.95 1.62 Ser83Arg -7.96 1.59 Ser83Tpo -7.96 1.59 Thr31Leu -7.98 1.54 Ser83Glu -7.98 1.54 Ser83Leu -8.02 1.44 Gly82Ser -8.06 1.34 Val84Leu -8.07 1.32 Ser83Lys -8.08 1.30 Thr31Met -8.11 1.24 Ser83Val -8.11 1.24 Val84Arg -8.25 0.98 (a) wild-type (wt), Ptr - phosphorylated Thr, Tpo - phosphorylated Tyr; (b) AGbind - the binding free energy, Kd - the dissociation constant, AGbind = RT ln(Kd), R - the gas constant (1.9872 x 10-3 kcal K-1 mol-1), T - the absolute temperature (300 K), 1 |rM = 10-6 M Figure 3. 9-O-Ac-Sia (bold sticks) that is docked in the binding site of the Thr31Met mutant of hCoV-OC43 S. The P2 (Leu80, Trp90 and Phe95) hydrophobic pocket (dots) accommodates the 5-N-acetyl methyl (left). 4 electrostatic contacts of 9-O-Ac-Sia with Lys29, Lys81, Ser83 and Ser83, respectively (right). Mitrasinovic: Prediction of Single Point Mutations in Human ... Acta Chim. Slov. 2020, 67, 949-956 953 :,V.Phe95 s Vc.' ' Figure 4. The chemical structure of Hidroxychloroquine (Hcq) (left). Hcq (bold sticks) that is docked in the binding site of hCoV-OC43 S (PDB ID: 6NZK). Hydrophobic interaction of the P2 (Leu80, Trp90 and Phe95) pocket (dots) with the Hcq side chain (right). No electrostatic contacts are detected. ciated with the horizontal conformational flip of 9-O-Ac-Sia, making the 9-O hydrophobic side chain face outside of the active cavity (Figure 3, left). The P2 (Leu80, Trp90 and Phe95) hydrophobic pocket (dots) accommodates the 5-N-acetyl methyl (Figure 3, left). The 9-O-Ac-Sia C1-carboxylate makes a hydrogen bond with the Lys29 side chain (3.00 A, Figure 3, right); the 9-O-acetyl carbonyl makes two hydrogen bonds with the Lys81 and Ser83 side chains (3.00 A and 2.38 A, Figure 3, right), while the 9-O makes a hydrogen bond with the Ser83 side chain (2.87 A, Figure 3, right). Thus, Thr31Met changes the specificity, Asn27Lys81Lys81Ser83, of the original hCoV-OC43/9-O-Ac-Sia complex to Lys29Lys81Ser83Ser83. The specificity difference is in the substitution of an Asn by a Ser, that is, in the substitution of a small amino acid by a tiny one. Met31 is a larger and more hydrophobic amino acid than Thr31 being almost indifferent in terms of its hydropho-bicity. Among aliphatic amino acids, Met is unique by having a sulphur atom in its side chain. A water molecule in bulk water can rotate in many directions. The presence of a nonpolar residue such as Met in the binding site restricts the movement of the water, causing an entropy loss that can be regained through the hydrophobic effect and the release of protein-bound water molecules. The chemical structure of Hcq (Figure 4, left) contains an aromatic core scaffold to which both a Cl atom and a large side chain are bound, indicating a pronounced hydrophobic character of the ligand. The binding mode obtained by docking Hcq in the binding site of hCoV-OC43 S (PDB ID: 6NZK) is illustrated in Figure 4 (right). The P2 (Leu80, Trp90 and Phe95) hydrophobic pocket is in interaction with the Hcq side chain, while the Hcq aromatic core is oriented outside of the active Figure 5. Hcq (bold sticks) that is docked in the binding site of the Val84Arg mutant of hCoV-OC43 S. The P1 (Leu85, Leu86 and Trp90) hydrophobic pocket (dots) accommodates the aromatic core scaffold of Hcq. No electrostatic contacts are detected. cavity. Noteworthy is that electrostatic interactions are not involved in formation of the hCoV-OC43/Hcq complex. The values in Table 1 illustrate that the SPM Val84Arg is the most stabilizing one in the wt/Hcq complex. The introduction of Arg84 (Figure 5), instead of Val84 (Figure 4), in the binding site causes the vertical conformational Mitrasinovic: Prediction of Single Point Mutations in Human 954 Acta Chim. Slov. 2020, 67, 949-956 Figure 6. Hcq (bold sticks) that is docked in the binding site of the Thr31Met mutant of hCoV-OC43 S. The P1 (Leu85, Leu86 and Trp90) hydrophobic pocket (dots) accommodates the aromatic core scaffold of Hcq. No electrostatic contacts are detected. switch (rotation by around 90° clockwise) of Hcq, making the aromatic core scaffold of Hcq interact with the P1 hydrophobic pocket containing Leu85, Leu86 and Trp90 (Figure 5). No electrostatic interactions are detected at the Val84Arg/Hcq interface, indicating the very low (or almost negligible) specificity of Hcq binding. The values in Table 1 indicate that Thr31Met, which is the most likely SPM for the binding of 9-O-Ac-Sia, is a very likely one for the binding of Hcq as well. The hydro-phobic nature of the Thr31Met/Hcq interaction mode (Figure 6) is similar to that of the Val84Arg/Hcq interaction mode (Figure 5) qualitatively. Comparing the binding free energies (Table 1) of the complexes, formed by docking 9-O-Ac-Sia and Hcq to the same receptor such as the Thr31Met mutant of hCoV-OC43 S, shows that Hcq has slightly higher affinity to hCoV-OC43 S than does 9-O-Ac-Sia. This standpoint can be accounted for by the almost-pure hydrophobic recognition of hCoV-OC43 S by Hcq with very low specificity. However, rapid kinetics underlying S-mediated virion attachment to 9-O-Ac-Sia is associated with recognition being hydrophobic and very specific at the same time. In view of this, it is useful to contrast the chemical structures of 9-O-Ac-Sia and Hcq and to consider entropy loss upon ligand binding. Flexible docking of ligand is based on active torsions in ligand structure, which can be conceivable as particular sp3 bonds that are directly involved in finding the lowest energy receptor/ligand conformations. Entropy loss due to ligand binding is related to the loss of its degrees of freedom. The torsional potential indirectly takes care of the particular entropy amount by being proportional to the number of active torsions in ligand structure. An active torsion has been estimated to cost circa 0.3 kcal mol-1 energetically.45,46 It means that the structure of 9-O-Ac-Sia having eleven active torsions experiences a negative entropy change that roughly costs -3.3 kcal mol-1. By having only one active torsion in its structure, the binding of Hcq is associated with a tiny entropic decrease of about -0.3 kcal mol-1. The dissociation constants of the hCoV-OC43/Hcq complexes are in the micromolar (10-6 M) range (Table 1), which is acceptable for hit ligand molecule but not for drug candidate. Knowing that the binding site is conserved in all CoV S glycoproteins that associate with 9-O-acetyl-sialoglycans,10 the very low specificity of Hcq to hCoV-OC43 S does not speak in favor of the potential of Hcq to be a drug candidate. 4. References 1. J. Reguera, G. Mudgal, C. Santiago, J. M. Casasnovas, Virus Res. 2014, 194, 3-15. DOI:10.1016/j.virusres.2014.10.005 2. D. S. Hui, E. I. Azhar, T. A. Madani, C. Drosten, A. Zumla, E. Petersen, int. J. Infect. Dis. 2020, 91, 264-266. 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Org. Chem. 2010, 14, 198-211. DOI: 10.2174/138527210790069857 44. P. M. Mitrasinovic, Curr. Drug Targets 2013, 14, 817-829. DOI: 10.2174/1389450111314070009 45. G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart, R. K. Belew, A. J. Olson, J. Comput. Chem. 1998, 19,1639-1662. DOI:10.1002/(SICI)1096-987X(19981115)19:14<1639::AID-JCC10>3.0.C0;2-B 46. G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S. Goodsell, A. J. Olson, J. Comput. Chem. 2009, 30, 2785-2791. DOI:10.1002/jcc.21256 Mitrasinovic: Prediction of Single Point Mutations in Human 956 Acta Chim. Slov. 2020, 67, 949-956 Povzetek Zaradi trenutnega širjenja nove bolezni CoViD-19 je Svetovna zdravstvena organizacija 11. marca 2020 uradno razglasila svetovno pandemijo. Sedanji trendi kažejo, da bo imela pandemija izjemen klinični in gospodarski vpliv na zdravje prebivalstva. Okužbo sproži konica (S) transmembranskega glikoproteina humanega koronavirusa (hCoV), ki se veže na gostiteljski receptor. Raziskave v teku in razvoj terapevtskih izdelkov so bistvenega pomena za uspešno zdravljenje CoViD-19. Da bi prispevali k skupnemu naporu, smo v tej študiji z uporabo in silico proteinskega inženiringa proučili točkovne mutacije preostankov vezavnega mesta pri hCoV-OC43 S, ki prepoznajo celične površinske komponente, ki vsebujejo 9-O-acetilirano sialično kislino (9-O-Ac-Sia), medtem ko smo njihove učinke na vezavo 9-O-Ac-Sia in hidrok-siklorokina (Hcq) ocenili z uporabo simulacij molekulskega sidranja. Thr31Met in Val84Arg naj bi bili kritični - najverjetnejši točkovni mutaciji v hCoV-OC43 S za vezavo 9-O-Ac-Sia oz. Hcq, pri čemer je Thr31Met zelo verjetna mutacija tudi v primeru Hcq. Načini interakcije kažejo na primerljivo moč kompleksov Thr31Met / 9-O-Ac-Sia in Val84Arg / Hcq (ali Thr31Met / Hcq). Glede na to, da je pri vseh CoV ohranjeno mesto vezave S glikoproteinov, ki se povezujejo z 9-O-acetil-sialoglikani, visoka hidrofobna afiniteta Hcq do hCoV-OC43 S govori v prid njegovi sposobnosti kompet-itivne inhibicije hitre S-posredovane pritrditve viriona v okoljih z visoko gostoto receptorja, vendar je lahko njegova občutno nizka specifičnost za hCoV-OC43 S ena ključnih ovir za upoštevanje potenciala Hcq, da postane kandidat za zdravilo. Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Mitrasinovic: Prediction of Single Point Mutations in Human ... DOI: 10.17344/acsi.2020.6017 Acta Chim. Slov. 2020, 67, 957-969 /^creative ^commons Scientific paper Synthesis, Characterization and Biological Application of Pyrazolo[1,5-a]pyrimidine Based Organometallic Re(I) Complexes Reena R. Varma,1 Juhee G. Pandya,2 Foram U. Vaidya,3 Chandramani Pathak,3 Bhupesh S. Bhatt1 and Mohan N. Patel1* 1 Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar-388 120, Gujarat (INDIA) 2 Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Gujarat, (INDIA) 3 Department of Cell Biology, School of Biological Sciences and Biotechnology,Indian Institute of Advanced Research, Koba Institutional Area, Gandhinagar-382007, Gujarat (INDIA) * Corresponding author: E-mail: jeenen@gmail.com Phone number: (+912692) 226856*218 Received: 03-30-2020 Abstract The neutral rhenium(I) complexes (I-VI) of type [ReCl(CO)3Ln] {where L1 = 7-phenyl-5-(pyridin-2-yl)pyrazolo[l,5-a] pyrimidine, L2 = 7-(4-bromophenyl)-5-(pyridin-2-yl)pyrazolo[1,5-a]pyrimi- dine, L3 = 7-(4-chlorophenyl)-5-(pyri-din-2-yl)pyrazolo[1,5-a]pyrimidine, L4 = 7-(2-chlorophenyl) -5-(pyridin-2-yl)pyrazolo[1,5-a]pyrimidine, L5 = 7-(4-methoxyphenyl)-5-(pyridin-2-yl)pyrazolo [1,5-a]pyrimidine, L6 = 5-(pyridin-2-yl)-7-(p-tolyl)pyrazolo[1,5-a]py-rimidine} were synthesized and characterized by 13C-APT, 1H-NMR, IR, electronic spectra, magnetic moment and conductance measurement. The anti-proliferative activity on HCT116 cells by MTT assay suggests potent cytotoxic nature of complexes, some complexes even have better activity than standard drug cisplatin, oxaliplatin, and carboplatin. The complexes were found to have better antimicrobial activity compare to pyrazolo pyrimidine ligands. The theoretical study of compounds-DNA interactions was examined by molecular docking as a supportive tool to the experimental data, which suggests the groove mode of binding. The values of docking energy for compounds-DNA interaction were found in the range of -230.31 to -288.34 kJ/mol. The intrinsic binding constant values of complexes (1.1-3.5 x 105 M-1) were found higher than the ligands (0.32-1.8 x 105 M-1). Keywords: In vitro cytotoxicity; Molecular modelling; Anti-proliferative activity; Groove binding 1. Introduction Metal carbonyl moieties, such as {M(CO)3} (M= Cr, Mn, Re, Fe), can attach to the biomolecules capable of molecular recognition, to label and assay, specific biological receptors. When M = Tc or Re, the same idea is used to introduce radioactive 99mTc, 186Re, or 188Re at a receptor for radiopharmaceutical applications.1,2 There has been considerable interest in testing metal carbonyls for anticancer activity.3 For example, [Co2(CO)6(HC2C-CH2O2CC6H4-2-OH)] is more active than cisplatin on the human mammary tumor cell lines MCF-7 and MDA-MB-231.4 Also [{if-(4-Me2N{CH2}4OC6H4)-(4-HOC6H4)CHCHEtC5H4} Re(CO)3] has been shown to behave similarly to tamoxifen, and it appears that the observed antiproliferative effect is dependent on the oestradiol receptor a.5 pyrazolo[ 1,5-a]pyrimidine Pyrazole and pyrimidine derivatives attracted organic chemists very much due to their biological and chemo-therapeutic importance. Pyrazolo pyrimidines and related fused heterocycles are of interest as potential bioactive molecules. They are known to exhibit pharmacological ac- Varma et al.: Synthesis, Characterization and Biological Application 958 Acta Chim. Slov. 2020, 67, 957-969 tivities such as CNS depressant,6 neuroleptic,7 and tuberculostatic.8 Recently, the chemistry of pyrazolo[1,5-a]pyri-midines attracted great attention as a synthetically important class of compounds.9 They represent biologically important compounds of purine analogues and this class has attracted wide pharmaceutical interest as inhibitors of lymphocyte-specific kinase (Lck) with enzymatic, cellular, and in vivo potency.10 In 2003, a research group from NRC synthesized some pyrazolo[1,5-a]pyrimidines and studied their biological effects as an anti-inflammatory, analgesic, and antipyretic drugs in comparison to no-valgin.11 The choice of the ligand is very important for the development of new radiopharmaceuticals reagents; thus, studies on rhenium(I) complexes with ligands as aromatic N-heterocycles have shown a great effectiveness.12 In continuation of our earlier work,13 the present study illustrates the synthesis of new heterocyclic ligands and their organometallic rhenium complexes. Heterocy-clic compounds have significant biological importance upon chelation with pentacarbonyl chloro rhenium(I) and presence of carbonyls group attached with metal which further enhanced the biological activity. 2. Experimental Materials and methods: All the chemicals and solvents were of reagent grade, 2-acetyl thiophene, substituted aldehyde were purchased from Merck Limited (India), different substituted phenyl hydrazine were purchased from Thirumalai Chemicals Ltd. (TCL), potassium-tert-butox-ide, potassium hydroxide purchased from Sisco Research Laboratories Pvt. Ltd. (SRL), pentacarbonyl chloro rhenium® purchased from Sigma Aldrich (USA). Luria broth and nutrient broth were purchased from Himedia (India). Agarose and Luria Broth (LB) were purchased from Hi-media Laboratories Pvt. Ltd., India. Culture of two Gram(+ve), i.e. Staphylococcus aureus (S. aureus) (MTCC-3160) and Bacillus subtilis (MTCC-7193), and three Gram(-ve), i.e. Serratia marcescens (MTCC-7103), Pseudomonas aeruginosa (MTCC-1688) and Escherichia coli (MTCC-433), were purchased from Institute of Microbial Technology (Chandigarh, India). S. cerevices Var. Paul Linder 3360 was obtained from IMTECH, Chandigarh, India. HS DNA was purchased from Sigma Aldrich Chemical Co. (India). Human colorectal carcinoma (HCT 116) cells were obtained from the cell repository, National Center for Cell Science (NCCS), Pune, Maharashtra, India. Physical measurements: The 1H and 13C NMR spectra were recorded on a Bruker Avance (400 MHz). Infrared spectra were recorded on an FT-IR ABB Bomen MB 3000 spectrophotometer in the range 4000-400 cm-1. C, H, and N elemental analyses were performed with a Heraeus, Germany CHNO RAPID. Molar conductance was meas- ured using a conductivity meter model no. EQ-660A, Mumbai (India). Melting points (°C, uncorrected) were determined in open capillaries on the ThermoCal10 melting point apparatus (Analab Scientific Pvt. Ltd, India). The electronic spectra were recorded on a UV-160A UV-Vis spectrophotometer, Shimadzu (Japan). The minimum inhibitory concentration (MIC) study was carried out using laminar airflow cabinet (Toshiba, Delhi, India). Hydrody-namic chain length study was carried out by a viscometric measurement bath. Photo quantization of the gel after electrophoresis was carried out on AlphaDigiDocTM RT. Version V.4.0.0 PC-Image software. General method for synthesis of pyrazolo[1,5-a]pyrimi-dines ligands (L1-L6): The a,ß unsaturated carbonyl compounds (3a-3f) were synthesized using literature procedure.14 Syntheses of the pyrazolo[1,5-a]pyrimidines based ligands (L1-L6) were carried out using Lipson and coworkers method.15 To a solution of the a,ß-unsaturated carbonyl compounds (3a-3f) (~2.391 mmol) in 10 mL of DMF, 1H-pyrazol-3-amine (4a) (~198.7 mg, ~2.391 mmol) and KOH (~15 mg, ~2.391 mmol) solution were added. The reaction mixture was refluxed for 30 min. Completion of the reaction was checked by TLC plates, the excess of solvent was removed under reduced pressure and the reaction mixture was cooled on an ice bath. The reaction mixture was extracted with ethyl acetate (20 mL x 2) and washed thoroughly with water (25 mL x 2). The brine solution of sodium chloride was added to it and dried over sodium sulphate. The resulting mixture was concentrated under vacuum to obtain pyrazolo[1,5-a]pyrimidine based ligands as products. The 1H and 13C NMR spectra are shown in supplementary material 1 and 2 respectively. Synthesis of 7-phenyl-5-(pyridin-2-yl)pyrazolo[1,5-a] pyrimidine (L1): The ligand (L1) was prepared by using enone (3a) (500 mg, 2.391 mmol) and 1H-pyrazole-3-amine (4a) (198.7 mg, 2.391 mmol). Yield: 84.2%; Color: yellowish amorphous solid; mp 170 °C; Mol. wt.: 272.31g/ mol; Empirical formula: C17H12N4, Elemental analysis: Calc. (%): C, 74.98; H, 4.44; N, 20.58; found. C, 74.88; H, 4.40; N, 20.58; Mass spectra (m/z %): 272.20 (100) [M+]; 1H NMR (400 MHz, CDCl3) S/ppm: 8.75 (1H, d, J = 4.0 Hz, H6"), 8.59 (1H, d, J = 8.0 Hz, H4»), 8.22 (1H, s, H7), 8.16 (2H, dd, J = 4.4 Hz, J = 3.2 Hz, Hr, 5»), 7.89 (2H, d, J = 1.6 Hz, H2>,6>), 7.60 (1H, d, J = 3.6 Hz, H3), 7.41 (3H, m, H3, 4, 5>), 6.86 (1H, d, J = 2.4 Hz, H4). 13C NMR (100 mHz, CDCl3) S/ppm: 155.1 (C8, Cquat.), 154.5 (Cr, Cquat), 149.8 (C6>, Cquat), 149.2 (C6, CH), 146.9 (C5a, Cquat), 145.2 (C4>, -CH), 136.4 (C3, -CH), 131.6 (Cr, Cquat.) 130.9 (C3>,5, -CH), 129.4 (C4, -CH), 128.6 (C2>, 6, -CH), 124.8 (C5", -CH), 121.6 (C3>, -CH), 105.2 (C7, -CH), 97.5 (C4, -CH). [Total signal observed = 15: signal of C = 5 (phenyl ring-C = 1, pyrazolo[1,5-a]pyrimidine-C = 3, pyridine ring-C = 1), signal of CH = 10 (pyrazolo[1,5-a]pyrimi-dine-CH = 3, phenylring-CH = 3, pyridine ring-CH = 4)]; IR (KBr, 4000-400 cm- 1): 2930 v(=C-H)ar., 1551 v(C=n), Varma et al.: Synthesis, Characterization and Biological Application Acta Chim. Slov. 2020, 67, 957-959 969 1504 (C-H) bending, 1251 v(C-N), 1597 v(C=C) conjugated alkenes, 763 v(Ar-H) adjacent hydrogen. 7-(4-Bromophenyl)-5-(pyridin-2-yl)pyrazolo[1,5-a] pyrimidine (L2): The ligand (L2) was prepared by using enone (3b) (500 mg, 1.730 mmol) and 1H-pyrazole-3-amine (4a) (143.8 mg, 1.730 mmol). Yield: 84.2%; Color: yellowish amorphous solid; mp 182 °C; Mol. wt.: 351.21 g/ mol; Empirical formula: C17H11BrN4, Elemental analysis: Calc. (found) (%): C, 58.14; H, 3.16; N, 15.95; found. C, 58.08; H, 3.11; N, 15.90; Mass spectra (m/z %): 350.4 (100) [M+], 352.4 [M+2]; NMR (400 MHz, CDCl3) 8/ ppm: 8.75 (1H, d, J = 4.4 Hz, H6»), 8.59 (1H, d, J = 8.0, H4»), 8.21 (1H, s, H7), 8.14 (2H, dd, J = 3.2 Hz, 2 Hz, Hr, 5»), 7.94 (1H, d, J = 6.4 Hz, H6>), 7.91 (1H, d, J = 7.6 Hz, H2>), 7.76 (2H, d, J = 2.0 Hz, H3;h5>), 7.43 (1H, dd, J = 8.0 Hz, 1.6 Hz, H3), 6.87 (1H, d, J = 1.2 Hz, H4). 13C NMR (100 MHz, CDCl3) 8/ppm: 160.6 (C6, Cquat.),153.9 (Cr, Cquat.), 153.1 (C5a, Cquat.), 148.8 (C8, Cquat.), 148.9 (C6>, -CH),145.7 (Cr, Cquat.), 145.2 (C4>, -CH), 137.9 (C3, -CH), 130.9 (C3>,5, -CH), 125.5 (C2>,6>, -CH), 122.3 (C4, Cquat.), 121.1 (C5>, -CH), 117.6 (c3", -CH), 103.3 (C7, -CH), 97.9 (C4, -CH). [Total signal observed = 15: signal of C = 6 (p-Br-phenyl ring-C = 2, pyrazolo[1,5-a]pyrimi-dine-C = 3, pyridine ring-C = 1), signal of CH = 9 (pyra-zolo[1,5-a]pyrimidine-CH = 3, p-Br phenyl ring-CH = 2, pyridine ring-CH = 4)]; IR (KBr, 4000-400 cm- 1): 2925 v(=C-H)ar., 1558 v(C=N), 1490 (C-H) bending, 1204 v(c-N), 1597 v(C=C) conjugated alkenes, 764 v(Ar-H) adjacent hydrogen. 7-(4-Chlorophenyl)-5-(pyridin-2-yl)pyrazolo[1,5-a] pyrimidine (L3): The ligand (L3) was prepared by using enone (3c) (500 mg, 2.044 mmol) and 1H-pyrazole-3-amine (4a) (169.8 mg, 2.044 mmol). Yield: 85.4%; Color: yellowish amorphous solid; mp 178 °C; Mol. wt.: 306.75 g/ mol; Empirical formula: C17H11ClN4, Calc. (%): C, 66.56; H, 3.61; N, 18.26; found. C, 66.55; H, 3.58; N, 18.24; Mass spectra (m/z %): 306.20 (100) [M+], 308.20 [M+2]; 1H NMR (400 MHz, CDCl3) 8/ppm: 8.75 (1H, d, J = 4.8 Hz, H6»), 8.59 (1H, d, J = 8.0 Hz, H4»), 8.22 (1H, s, H7), 8.17 (2H, dd, J = 8.4, 4.0 Hz, H3»,5»), 7.93 (2H, d, J = 2.0 Hz, H2>,6>), 7.58 (1H, d, J = 8.4 Hz, H3), 7.44 (2H, d, J = 4.0 Hz, H3>,5>), 6.87(1H, d, J = 2.4 Hz, H4). 13C NMR (100 MHz, CDCl3) 8/ppm: 155.1 (C8, Cquat.), 154.3 (Cr, Cquat.), 149.6 (C6, Cquat.), 149.2 (C6>, -CH), 145.7 (C4, -CH), 145.2 (C4>, Cquat.), 137.2 (C3, -CH), 130.8 (C5a, Cquat.), 129.9 (C3>,5>, -CH), 129.4 (Cr, Cquat.), 129.0 (C2; 6, -CH), 124.9 (C5>, ' -CH), 121.7 (C3>, -CH), 104.9 (C7, -CH), 97.68 (C4, -CH). [Total signal observed = 15: signal of C = 6 (p-Cl-phenyl ring-C = 2, pyrazolo[1,5-a]pyrimidine-C = 3, pyridine ring-C = 1), signal of CH = 9 (pyrazolo[1,5-a]pyrimidine-CH = 3, p-Cl-phenyl ring-CH = 2, pyridine ring-CH = 4)]; IR (KBr, 4000-400 cm- 1): 2922 v(=C-H)ar., 1551 v(C=n), 1504 (C-H) bending, 1190 v(C-N), 1605 v(C=C) conjugated alkenes, 756 v(Ar-H) adjacent hydrogen. 7-(2-Chlorophenyl)-5-(pyridin-2-yl)pyrazolo[1,5-a] pyrimidine (L4): This ligand (L1) was prepared by using enone (3d) (500 mg, 2.044 mmol) and 1H-pyrazole-3-amine (4a) (169.8 mg, 2.044 mmol). Yield: 79.5%; Color: yellowish amorphous solid; mp 180 °C; Mol. wt.: 306.75 g/ mol; Empirical formula: C17H11ClN4, Calc. (found) (%): C, 66.56; H, 3.61; N, 18.26; found. C, 66.50; H, 3.60; N, 18.23; Mass spectra (m/z %): 306.82 (100) [M+], 308.82 [M+2]; 1H NMR (400 MHz, CDCl3) 8/ppm: 8.73 (1H, d, J = 3.6 Hz, H6»), 8.61 (1H, d, J = 8.0 Hz, H4»), 8.19 (1H, d, J = 2.4 Hz, H5"), 8.09 (1H, s, H7), 7.93 (1H, d, J = 1.6 Hz, H3»), 7.62 (2H, m, H4>,5>), 7.51 (2H, m, H3>,6>), 7.41 (1H, d, J = 5.2 Hz, H3), 6.88 (1H, d, J = 2.4 Hz, H4).13C NMR (100 MHz, CDCl3) 8/ppm: 154.9 (C8, Cquat.),153.01 (Cr, Cquat.), 149.28 (C6>, -CH), 148.9 (C6, Cquat.), 145.5 (c4>, -CH), 145.1 (C5a, Cquat.), 137.10 (C3, -Ch), 133.7 (C2, Cquat.), 131.57 (C3, -Ch), 131.1 (C5, -CH), 130.2 (C4, -CH), 128.6 (Cr, Cquat.), 127.1 (C6, -CH), 124.9 (C5>, -CH), 121.8 (C3>, -CH), 105.2 (C7, -CH), 97.72 (C4, -CH). [Total signal observed = 17: signal of C = 6 (o-Cl-phenyl ring-C = 2, pyrazolo[1,5-a]pyrimidine-C = 3, phenyl ring-C = 1), signal of CH = 11 (pyrazolo[1,5-a]pyrimi-dine-CH = 3, o-Cl phenylring-CH = 4, pyridine ring-CH = 4)]; IR (KBr, 4000-400 cm- 1): 2922 v(=C-H)ar., 1551 v(C=N), 1504 v(C-H) bending, 1190 v(C-N), 1605 v(C=C) conjugated alkenes, 758 v(Ar-H) adjacent hydrogen. 7-(4-Methoxyphenyl)-5-(pyridin-2-yl)pyra-zolo[1,5-a]pyrimidine (L5): The ligand (L5) was prepared by using enone (3e) (500 mg, 2.082 mmol) and 1H-pyra-zole-3-amine (4a) (173 mg, 2.082 mmol). Yield: 87.6%; Color: yellowish amorphous solid; mp 178 °C; Mol. wt.: 302.34 g/mol; Empirical formula: C18H14N4O, Calc. (found) (%): C, 71.51; H, 4.67; N, 18.53; found. C, 71.48; H, 4.62; N, 18.56; Mass spectra (m/z %): 302.20 (100) [M+]; 1H NMR (400 MHz, CDCl3) 8/ppm: 8.78 (1H, d, J = 4.4 Hz, H6»), 8.51 (1H, d, J = 8.0 Hz, H4»), 8.33 (1H, s, Hz, H7), 8.25 (2H, d, J = 8.8 Hz, H3>;5»), 8.10 (1H, d, J = 10.4 Hz, H6>), 8.03 (1H, d, J = 7.6 Hz, H2>), 7.58 (1H, d, J = 5.2 Hz, H3), 7.19 (2H, d, J = 8.8 Hz, H3;5>), 6.92 (1H, d, J = 2.0 Hz, H4), 3.09 (3H, s, -OCH3). 13C NmR (100 MHz, CDCl3) 8/ ppm: 161.9 (C4, Cquat.),154.7 (C8, Cquat.), 154.01 (Cr, Cquat.), 149.9 (C6>, -CH),149.7 (c6, Cquat.), 146.7 (C4>, -CH), 146.3 (C5a, Cquat.), 138.1 (c3, -CH), 131.7 (C2>,6>, -CH), 125.8 (C5>, -CH), 123.4 (C1, Cquat.), 121.5 (c3>, -CH), 114.5 (C7, -CH), 103.8 (C3>,5, -CH), 97.6 (C4, -CH), 55.9 (-OCH3). [Total signal observed = 16: signal of C = 6 (p-OCH3-phenyl ring-C = 2, pyrazolo[1,5-a]pyrimi-dine-C = 3, pyridine ring-C = 1), signal of CH = 9 (pyra-zolo[1,5-a]pyrimidine-CH = 3, p-OCH3 phenylring-CH = 2, pyridine ring-CH = 4), -OCH3 = 1]; IR (KBr, 4000-400 cm- 1): 2922 v(=C-H)ar., 1551 v(C=N), 1514 (C-H) bending, 1188 v(C-N), 1597 v(C=C) conjugated alkenes, 764 v(Ar-H) adjacent hydrogen. 5-(Pyridin-2-yl)-7-(p-tolyl)pyrazolo[1,5-a]pyrimi-dine (L6): The ligand (L6) was prepared by using enone (3f) (500 mg, 2.231 mmol) and 1H-pyrazole-3-amine (4a) (185.4 mg, 2.231 mmol). Yield: 82.5%; Color: yellowish amorphous solid; mp 175 °C; Mol. wt.: 286.34 g/mol; Em- Varma et al.: Synthesis, Characterization and Biological Application ... 960 Acta Chim. Slov. 2020, 67, 957-969 pirical formula: C18H14N4, Calc. (found) (%): C, 75.50; H, 4.93; N, 19.57; found. C, 75.46; H, 4.90; N, 19.55; Mass spectra (m/z %): 286.60 (100) [M+]; NMR (400 MHz, CDCl3) S/ppm: 8.75 (1H, d, J = 4.4 Hz, H6»), 8.59 (1H, d, J = 8.0 Hz, H4"), 8.23 (1H, s, H7), 8.22 (2H, dd, J = 2.4 Hz, 1.6 Hz, H3" 5"), 8.08 (2H, dd, J = 10.0 Hz, 8.0 Hz, H2>6>), 7.41 (3H, d, J = 7.6 Hz, H3, 3> 5>), 6.85 (1H, d, J = 2.0 Hz, H4), 2.49 (3H, s, -CH3). 13C nMR (100 MHz, CDCl3) S/ppm: 155.0 (c8, Cquat.),154.6 (Cr, Cquat.), 149.7 (C6, Cquat.), 149.2 (c6>, -CH), 147.07 (C5a, Cquat.), 145.1 (C4, -Ch), 141.3 (c4, Cquat.), 137.0 (c3, -CH), 129.3 (C3, 5, -Ch), 129.2 (C1, Cquat.), 128.6 (C2>, 6, -CH), 124.7 (C5>, -CH), 121.6 (C3>, -CH), 104.73 (C7, -CH), 97.4 (C4, -CH), 21.5 (-CH3). [Total signal observed = 16: signal of C = 6 (p-CH3 phenyl ring-C = 2, pyrazolo[1,5-a]pyrimidine-C = 3, pyridine ring-C = 1), signal of CH = 9 (pyrazolo[1,5-a]pyrim-idine-CH = 3, p-CH3-phenylring-CH = 2, pyridine ring-CH = 4), -CH3 = 1]; IR (KBr, 4000-400 cm- 1): 2923 v(=C-H)ar., 1551 v(C=N), 1512 (C-H) bending, 1196 v(C-N), 1597 v(C=C) conjugated alkenes, 764 v(Ar-H) adjacent hydrogen. General synthesis of complexes: The metal carbonyl complexes (I-VI) were synthesized using pentacarbonyl chloro rhenium(I) and ligands (L1-L6) in ethanol in a 1:1 proportion.16 Synthesis of [Re(CO)3(L')Cl] (I): Ethanolic solution of the precursor of [Re(CO)5Cl] (100 mg, 0.276 mmol) was refluxed for 10 minutes. Then a solution of ligand (L1) (75 mg, 0.276 mmol in 10mL ethanol), was added and the reaction was stirred yielding a solution. The resulting mixture was stirred at 60 °C for 5-6 hr. Progress of reaction was monitored by TLC after completion of reaction the solution was filtered through celite in order to remove solid particles and the solvent was removed under reduced pressure the orange red product was obtained. The proposed reaction for the synthesis of complexes (I-VI) is shown in scheme 1. Yield: 62.9%; Color: yellowish amorphous solid; mp 380 °C; Mol. wt.: 578.00 g/mol; Empirical formula: C20H12ClN4O3Re, Elemental analysis: Calc. (%): C, 41.56; H, 2.00; N, 9.69; Re, 32.22; Found. (%): C, 41.52; H, 1.98; N, 9.67; Re, 32.20; Conductance: 2.83 S cm2 mol-1. *H NMR (400 MHz, DMSO-d6) S/ppm: 9.18 Ri r2 L1 = -h -h L2 = -H -Br L3 = -H -c! L4 = -ci -H Ls = -H -och L6 = -h -ch3 (¡¿-¡¿J (in) if Reagent and Conditions: (i) Methanol, KOH (ii) DMF, 3-amino pyrazole (4a), K'OBu (iii) Ethanol, Re(CO)5CI, reflux 60-70 °C f l-vi I Scheme 1. Reaction scheme for the synthesis of ligands and rhenium complexes. Varma et al.: Synthesis, Characterization and Biological Application Acta Chim. Slov. 2020, 67, 957-961 969 (2H, dd, J = 8.4 Hz, 6.4 Hz, H3»>6»), 8.60 (1H, s, H7), 8.45 (2H, dd, J = 11.2 Hz, 8.0 Hz, Hc 5»), 8.3 (2H, d, J = 7.6 Hz, H2 6>), 7.91 (1H, d, J = 6.8 Hz, H3), 7.72 (3H, m, H3>,4>, 5>), 7.25 (1H, d, J = 2.4 Hz, H4). 13C NMR (100 MHz, dMSo-d6) 8/ppm: 203.1 (M-CO, Cquat.), 197.5 (2M-CO, Cquat.), 157.5 (C8, Cquat.), 154.7 (Cr, Cquat.), 153.9 (C6>, -CH), 149.8 (C6, Cquat.), 149.1 (C5a, Cquat.), 147.1 (c4>, -CH), 140.9 (c3, -CH), 132.8 (C3>,5>, -CH), 131.0 (C4, -CH), 130.0 (Cr, Cquat.), 129.4 (C26, -CH), 129.0 (C5>, -CH), 127.6 (C3», -CH), 106.1 (C7, -CH), 99.7 (C4, -CH). [Total signal observed = 17: signal of C = 7 (M-CO = 2, phenyl ring-C = 1, pyrazolo[1,5-a]pyrimidine-C = 3, pyridine ring-C = 1), signal of CH = 10 (pyrazolo[1,5-a] pyrimidine-CH = 3, phenylring-CH = 3, pyridine ring-CH = 4)]; IR (KBr, 4000-400 cm- 1): 2014, 1898 v(Re(-CO), 1550 v(C=N), 1504 (C-H) bending, 1250 v(C-N), 1604 v(C=C) conjugated alkenes, 763 v(Ar-H) adjacent hydrogen. Synthesis of [Re(CO)3(L2)Cl] (II): It was synthesized using ligand (L2) (97 mg, 0.276 mmol). Yield: 77.2%; Color: yellowish amorphous solid; mp 385 °C; Mol. wt.: 656.89 g/mol; Empirical formula: C20H11BrClN4O3Re, Elemental analysis: Calc. (%): C, 36.57; H, 1.69; N, 8.83; Re, 28.35; Found. (%): C, 36.55; H, 1.67; N, 8.80; Re, 8.33; Conductance: 5.12 S cm2 mol-1. 1H NMR (400 MHz, DMSO-d6) 8/ppm: 9.16 (2H, dd, J = 8.4 Hz, 7.6 Hz, HC6»), 8.60 (1H, s, H7), 8.46 (2H, dd, J = 6.4 Hz, 4.4 Hz, H3»,5»), 8.28 (2H, d, J = 8.4 Hz, H2>,6>), 7.95 (2H, d, J = 8.4 Hz, H3, 5>), 7.73 (1H, d, J = 7.6 Hz, H3), 7.25 (1H, d, J = 1.2 Hz, H4).' 13C NMR (100 MHz, DMSO-d6) 8/ppm: 198.9 (M-CO, Cquat.), 197.6 (2M-CO, Cquat.), 157.5 (C6, Cquat.), 154.6 (Cr, Cquat.), 153.9 (C6>, -CH), 149.8 (C5a, Cquat.), 149.2 (c8, Cquat.), 147.1 (C4>, -CH), 140.9 (C3, -CH), 132.7 (c3>,5, -CH), 132.1 (C2>,6>, -CH), 130.9 (C5>, -CH), 129.5 (c1, Cquat.), 129.06 (Cr, -CH), 126.6 (C4, -Cquat.), 106.1 (c7, -CH), 99.82 (C4, -CH). [Total signal observed = 17: signal of C = 8 (M-CO = 2, p-Br-phenyl ring-C = 2, pyra-zolo[1,5-a]pyrimidine-C = 3, pyridin ring-C = 1), signal of CH = 9 (pyrazolo[1,5-a]pyrimidine-CH = 3, p-Br phenyl-ring-CH = 2, pyridine ring-CH = 4)]; IR (KBr, 4000-400 cm- 1): 2021, 1898 v(Re(CO), 1558 v(C=N), 1481 (C-H) bending, 1196 v(C-N), 1597 v(C=C) conjugated alkenes, 764 v(Ar-H) adjacent hydrogen. Synthesis of [Re(CO)3(L3)Cl] (III): It was synthesized using ligand (L3) (84 mg, 0.276 mmol). Yield: 140 mg, 76.1%; Color: yellowish amorphous solid; mp 378 °C; Mol. wt.: 612.44 g/mol; Empirical formula: C20H11Cl2N4O3Re, Elemental analysis: Calc. (%): C, 39.22; H, 1.81; N, 9.15; Re, 30.40; Found. (%): C, 39.20; H, 1.78; N, 9.12; Re, 30.36 Conductance: 11.16 S cm2 mol-1. 1H NMR (400 MHz, DMSO-d6) 8/ppm: 9.17 (2H, dd, J = 8.4 Hz, 6.4 Hz, HC6»), 8.61 (1H, s, H7), 8.48 (2H, dd, J = 8.4 Hz, 8.0 Hz, H3»,5»), 8.36 (2H, d, J = 8.8 Hz, H2>,6>), 7.91 (1H, d, J = 6.4 Hz, H3), 7.81 (2H, d, J = 8.4 Hz, H3>,5>), 7.26 (1H, d, J = 2.0 Hz, H4). 13C NMR (100 MHz, DMSO-d6) 8/ppm: 195.5 (M-CO, Cquat.), 189.2 (M-2CO, Cquat.), 157.5 (C8, Cquat.), 154.7 (Cr, Cquat.), 153.9 (C6>, -CH), 149.3 (C6, Cquat.), 148.6 (C4, Cquat.), 147.2 (C4>, -CH), 140.9 (C3, -CH), 137.6 (C5a, Cquat.), 132.8 (C3,5, -CH), 129.5 (C2>,6, -CH), 129.2 (c5>, -CH), 128.7 (Cr, ' Cquat.), 127.5 (Cr, -CH), 106.2 (c7, -CH), 99.8 (C4, -CH). [Total signal observed = 17: signal of C = 8 (M-CO = 2, p-Cl-phenyl ring-C = 2, pyra-zolo[1,5-a]pyrimidine-C = 3, pyridin ring-C = 1), signal of CH = 9 (pyrazolo[1,5-a]pyrimidine-CH = 3, p-Cl phenyl ring-CH = 2, pyridine ring-CH = 4)]; IR (KBr, 4000-400 cm- 2021, 1898 v(Re(CO), 1551 v(C=N), 1504 (C-H) bending, 1165 v(C-N), 1597 v(C=C) conjugated alkenes, 764 v(Ar-H) adjacent hydrogen. Synthesis of [Re(CO)3(L4)Cl] (IV): It was synthesized using ligand (L4) (84 mg, 0.276 mmol). Yield: 76.1%; Color: yellowish amorphous solid; mp 368 °C; Mol. wt.: 612.44 g/mol; Empirical formula: C20H11Cl2N4O3Re, Elemental analysis: Calc. (%): C, 39.22; H, 1.81; N, 9.15; Re, 30.40, Found. (%): C, 39.20; H, 1.78; N, 9.12; Re, 30.36; Conductance: 11.30 S cm2 mol-1. NMR (400 MHz, DMSO-d6) 8/ppm: 9.20 (1H, d, J = 3.6 Hz, H6»), 9.01 (1H, d, J = 12.8 Hz, H4"), 8.54 (2H, d, J = 2.0 Hz, H3»5»), 8.44 (1H, s, H7), 7.89 (2H, m, H4>,5>), 7.77 (2H, m, H3>6>), 7.68 (1H, d, J = 7.6 Hz, H3), 7.27 ' (1H, d, J = 2.4 Hz, H4). 13C NMR (100 MHz, DMSO-d6) 8/ppm: 198.8 (M-CO, Cquat.), 197.6 (2M-CO, Cquat.), 157.6 (C8, Cquat.), 154.5 (Cr, Cquat.), 154.02 (C6>, -CH), 148.40 (C6, Cquat.), 147.9 (c5a, Cquat.), 147.3 (C4>, -CH), 141.1 (C3, -CH), 133.3 (c3, -CH), 133.1 (C2, Cquat.), 132.3 (C5, -CH), 130.2 (C4, -CH), 130.1 (Cr, Cquat.), 129.6 (C6, -CH), 128.1 (C5>, -CH), 127.5 (C3>, -CH), 107.8 (C7, -CH), 99.9 (C4, -CH). [Total signal observed = 19: signal of C = 8 (M-CO = 2, o-Cl-phenyl ring-C = 2, pyrazolo[1,5-a]pyrimidine-C = 3, pyridin ring-C = 1), signal of CH = 11 (pyrazolo[1,5-a] pyrimidine-CH = 3, o-Cl phenyl ring-CH = 4, pyridine ring-CH = 4)]; IR (KBr, 4000-400 cm- 1): 2021, 1898 v(Re(CO), 1551 v(C=N), 1504 (C-H) bending, 1165 v(c-N), 1605 v(C=C) conjugated alkenes, 756 v(Ar-H) adjacent hydrogen. Synthesis of [Re(CO)3(L5)Cl] (V): It was synthesized using ligand (L5) (84 mg, 0.276 mmol). Yield: 89.7%; Color: yellowish amorphous solid; mp 370 °C; Mol. wt.: 608.02g/mol; Empirical formula: C21H14ClN4O4Re, Elemental analysis: Calc. (%): C, 41.48; H, 2.32; N, 9.21; Re, 30.62, Found. (%): C, 41.45; H, 2.30; N, 9.18; Re, 30.60; Conductance: 15.18 S cm2 mol-1. 1H NMR (400 MHz, DMSO-d6) 8/ppm: 9.19 (2H, dd, J = 8.0 Hz, 6.0 Hz, HC6»), 8.60 (1H, s, H7), 8.46 (4H, dd, J = 7.6 Hz, 4.8 Hz, H2>,6>,3»,5»), 7.89 (1H, d, J = 6.4 Hz, H3), 7.27 (2H, d, J = 8.8 Hz, ' H3',5>), 7.22 (1H, d, J = 2.4 Hz, H4), 3.94 (3H, s, -OCH3). 13C nMr (100 MHz, DMSO-d6) 8/ppm: 199.0 (M-CO, Cquat.), 198.2 (2M-CO, Cquat.), 163.1(C4, Cquat.), 157.2 (C8, Cquat.), 154.8 (Cr, Cquat.), 153.8 (C6>, -CH), 149.4 (c6, Cquat.), 147.0 (C4>, CH), 140.9 (C3, -CH), 133.2 (C2>,6, -CH), 129.4 (C5>, -CH), 127.5 (C3>, -CH), 123.2(C5a, -Cquat.), 121.9 (C1, Cquat.), 114.7 (C7, -CH), 105.0 (C3>5, -CH ), 99.4 (C4, -CH), 56.2 (-OCH3). [Total signal ob- Varma et al.: Synthesis, Characterization and Biological Application ... 962 Acta Chim. Slov. 2020, 67, 957-969 served = 18: signal of C = 8 (M-CO = 2, p-OCH3-phenyl ring-C = 2, pyrazolo[1,5-a]pyrimidine-C = 3, pyridin ring-C = 1), signal of CH = 9 (pyrazolo[1,5-a]pyrimidine-CH = 3, p-OCH3 phenylring-CH = 2, pyridine ring-CH = 4), -OCH3 = 1]; IR (KBr, 4000-400 cm- 2021, 1921, 1898 v(Re(CO), 1551 v(C=N), 1512 (C-H) bending, 1180 v(C-N), 1597 v(C=C) conjugated alkenes, 764 v(Ar-H) adjacent hydrogen. Synthesis of [Re(CO)3(L6)Cl] (VI): It was synthesized using ligand (L6) (79 mg, 0.276 mmol). Yield: 84.9%; Color: yellowish amorphous solid; mp 374 °C; Mol. wt.: 592.03 g/mol; Empirical formula: C21H14ClN4O3Re, Elemental analysis: C, 42.60; H, 2.38; N, 9.46; Re, 31.45; Found. (%):C, 42.40; H, 2.20; N, 9.35; Re, 31.42; Conductance: 13.25 S cm2 mol1. NMR (400 MHz, DMSO-d6) S/ppm: 9.17 (2H, dd, J = 8.0 Hz, 7.6 Hz, H4»6»), 8.59 (1H, s, H7), 8.44 (2H, dd, J = 8.4 Hz, 6.4 Hz, Hr, 5»), 8.27 (2H, d, J = 8.0 Hz, H2>,6>), 7.90 (1H, d, J = 6.4 Hz, H3), 7.53 (2H, d, J = 8.4 Hz, H3,5>), 7.23 (1H, d, J = 2.4 Hz, H4), 2.49 (3H, s, -CH3 ). 13C nMr (100 MHz, DMSO-d6) S/ppm: 198.9 (M-CO, Cquat.), 197.7 (2M-CO, Cquat.), 157.3 (C8, Cquat.), 154.7 (Cr, Cquat.), 153.8 (C6>, -Ch), 149.7(C6, -Cquat.), 149.2 (C5a, Cquat.), 147.1 (C4, -CH),143.3 (C4, Cquat.), 140.9 (C3, -CH), 130.9 (C3>,5, -CH), 129.6 (C2>,6>, -CH), 129.4 (c5>, -CH), 127.5 (c3>, -CH), 126.9 (C1,> Cquat.), 105.6 (C7, -CH), 99.54 (C4, -CH), 21.7 (-CH3). [Total signal observed = 18: signal of C = 8 (M-CO = 2, p-CH3-phenyl ring-C = 2, pyrazolo[1,5-a]pyrimidine-C = 3, pyridine ring-C = 1), signal of CH = 9 (pyrazolo[1,5-a]pyrim-idine-CH = 3, p-CH3-phenylring-CH = 2, pyridine ring-CH = 4, CH3 = 1)]; IR (KBr, 4000-400 cm- 1): 2021, 1913 v(Re(CO), 1551 v(C=N), 1512 (C-H) bending, 1196 v(C-N), 1597 v(C=C) conjugated alkenes, 764 v(Ar-H) adjacent hydrogen. Biological activities: In vitro antimicrobial assay: The synthesized ligands and complexes were evaluated for their antimicrobial properties according to literature.17 In vivo brine shrimp lethality bioassay (BSLB): The brine shrimp (Artemia cysts) lethality bioassay for the synthesized compounds were carried out according to litera- ture.17 18 Cellular level bioassay using S. cerevisiae: The in vitro cytotoxicity assay was performed in the eukaryotic system where a yeast cell, S. cerevisiae was taken as a model test organism. The cytotoxic effect of compounds was determined by viability staining and represented as % viability. Lower % viability indicates high toxicity of compound on that particular biological system. Antiproliferative study: The Re(I) tricarbonyl complexes I-VI were tested for in vitro cytotoxicity against colon carcinoma (HCT116) cancerous cell lines. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine the cytotoxicity of the compounds.19 The extent of inhibition is displayed as an IC50 value, which is defined as the concentration required to inhibit cell growth to half.20,21 Stock solutions of 10-100 mg/mL of test complexes (I-VI) were prepared in dimethyl sulfoxide (DMSO). Twenty-four hours after cell plating, media was removed and replaced with fresh media containing 10, 25, 50,100,500 ^g/mL of test compounds DMSO vehicle control, for the indicated exposure times. DNA binding activity: Binding of metal complexes with DNA can be understood by absorption spectral analysis of DNA. The binding mode and binding constant (Kb) of a complex toward DNA give an idea about the strength of interaction, which can be obtained by studying UV-Vis absorbance titration.22 The binding constant values were estimated by the following equation, [DAMJ [DN4J + ■ (ea-ef) (eh-ef) Kh(eh-erJ (1) Where, [DNA] = concentration of DNA in base pairs, sa = extinction coefficient observed for the MLCT absorption band at the given DNA concentration, sf = the extinction coefficient of the complex in solution and sb = the extinction coefficient of the complex when fully bound to DNA. Viscometric experiments were performed using Ub-belohde viscometer, maintained at 25.0 (±0.5) °C in a thermostatic water bath. The total system was 3 mL, containing 100 ^M of DNA, and metal complexes were varied from 5 to 50 ^M. The flow time of solutions in phosphate buffer (pH 7.0) was recorded, and an average flow time was calculated. Data were presented as (r|/r|0)1/3 versus [Compound]/ [DNA], where n is the viscosity of DNA in the presence of complex and n0 is the viscosity of DNA alone. All the experiment was done in triplicate. The hydrodynamic length of DNA generally increases upon partial intercalation while it does not lengthen upon groove binding.23,24 Molecular docking: Docking study was measured for Re(I) complexes with deoxyribonucleic acid (DNA) sequence d(ACCGACGTCGGT)2. The main purpose of molecular docking is to identify the binding mode of metal complexes using Hex 8.0 software. The detailed process of this study is described in literature.25 Integrity of compounds on the DNA: For DNA integrity of compounds, the treated test organism's DNA subjected to Agarose gel electrophoresis. The DNA of S. cere-visiae was extracted according to the protocol described by Michael R. Green and Joseph Sambrook.26 The detailed process is described in literature.27 3. Results and Discussion 13C-APT, !H-NMR, IR, magnetic moments, conductance measurements, and electronic spectra: The 1H Varma et al.: Synthesis, Characterization and Biological Application Acta Chim. Slov. 2020, 67, 957-963 969 NMR spectra of ligands L1-L6 and complexes I-VI demonstrate peak at 6.0 - 8.0 8 ppm confirms protons of pyra-zolo[1,5-a]pyrimidine aromatic ring. 13C-APT data of ligands L1-L6 and complexes I-VI show signals at 97-160 8 ppm confirm the presence of aromatic environment.16 The crystal structure of Re(CO)5Cl show four CO at equatorial position, and one CO along with Cl atom at axial position.28 The heterocyclic bidentate ligand approach from equatorial position and replace two CO molecules to form Re(I) complexes. In keeping with the facial arrangement of the CO ligands, the 13C (APT) NMR spectra show two low-field signals in the range of 189.2-198.2 ppm and 195.5-203.1 ppm for axial and equatorial carbonyl groups of Re(I) complexes, respectively.29 Results of the FT-IR spectra of free ligands (L1-L6) show the bands at ~2922 cm-1 v(=C-H)ar, and ~1196 cm-1 for -CN stretching of pyrazolo[1,5-a]pyridine ring. The band ~590-620 cm-1 is observed due to carbon-halogen bond and band at ~977-1062 cm-1 is observed due to the para-substituted benzene ring. The bands at ~1551, and ~1597 cm-1 are assigned to v(C=N) and v(C=C) conjugated alkene.27 In complexes, the v(Re-N) band are appeared at around 570 - 578 cm-1.30 The IR spectra of Re(I) complexes exhibit three strong v(CO) bands in the range of 2020-1898 cm-1.31 The strong v(CO) bands centered at 2000 cm-1 suggests expected fac-geometry around the Re metal.31,32 The observed magnetic moment values of rheni-um(I) complexes are zero due to absence of unpaired electron i.e. low spin t2g6 eg0 configuration makes rhenium(I) complexes diamagnetic, and the oxidation state of rhenium is +1 in complexes. Molar conductance values of all the low spin Re(I) complexes are found in the range of 2.83-19.25 S cm2 mol-1. It suggests that the Re(I) complexes are non-ionic and non-electrolytic with absence of any counter ions surrounding the coordination sphere. The electronic spectra of compounds were recorded in DMSO solution (Figure 1). The ground state for t2g6 electronic configuration of rhenium(I) metal ion is 1A1g. Three bands are observed in the electronic spectrum: one band ranging in 436.0-442.50 nm region assign to MLCT, second band ranging from 332.5-354.5 nm region attribute to n-n*, and third band ranging from 286-296 nm assign to ultra-ligand charge transfer (n-n*). It suggests that Re(I) metal complexes possess octahedral geometry.33 Biological applications of synthesized ligands and complexes: In vitro antimicrobial screening: The data reveals that all the complexes have higher antimicrobial activity than neutral bidentate ligands and a metal salt (Figure 2). The antimicrobial activity of all complexes against different microorganisms is found better than that of the respective ligands are shown in supplementary material 3. The MIC values of the complexes, ligands, and metal salt are observed in the range of 60-90 ^M, 280-320 ^M, and 2500 ^M, respectively. A comparative of antimicrobial activity (MIC) values among all synthesized metal complexes and their ligands in decreasing order are as V > II > IV > VI > III > I > L5 > L4 > L3 > L1 > L2 > L6 > Re(CO)-5Cl for gram positive bacteria, and V > IV > III > I > VI > II > L4 > L5 > L3 > L6 > L2 > L1 > Re(CO)5Cl for gram negative bacteria. The complex V is the most active amongst all the complexes, due to the presence of the methoxy group to the pyridine ring in pyrazolo[1,5-a]pyrimidine ligand. The presence of a more electronegative environment in complex V and VI improves their biological properties. Two factors are applicable, that are, the ligands bound to metal ions in a multidentate fashion, and the nature of the ligand, for improving MIC values of the synthesized compounds. These may be the main reasons for the diverse antibacterial activity shown by the complexes. The pharmaco- 105 205 305 405 505 605 Wavelength (rim) Figure 1. Electronic transition spectra of the ligands (L'-L6) and complexes (I-VI). Varma et al.: Synthesis, Characterization and Biological Application ... 964 Acta Chim. Slov. 2020, 67, 957-969 logical activities of these metal compounds depend on the metal ion, its ligands, and the structure of the compounds. These factors are responsible for reaching them at the proper target site in the body. It is known that certain metal ions penetrate into bacteria and inactivate their enzymes, or some metal ions can generate hydrogen peroxide, thus killing bacteria. According to overtone's concept of cell permeability, the lipid membrane that surrounds a cell favours the passage of only lipid soluble materials so that lipo-solubility is an important factor which contributes to bactericidal ac-tivity.34 of % viability of ligands and complexes is L5 < L3 < L6 = L4 < L1 < L2 < V < VI < IV < III < II < I, respectively. Figure 2. Antibacterial study of ligands and complexes by broth dilution method in terms of MIC in |rM. Figure 3. Cellular level cytotoxicity of synthesized compounds using S. cerevisiae, dead cells are seen dark whereas live cells are seen transparent. Cellular level bioassay using S. cerevisiae: The in vitro cellular level cytotoxicity of ligands L1-L6 and complexes I-VI was found to vary with the type of substituent present in the synthesized complexes. From the results, it was found that, as the concentration of compound increases from 20 ^g/mL to 100 ^g/mL, cytotoxicity also increases which can be exhibited by decreasing % viability shown in supplementary material 4. The complexes I and II show the maximum cytotoxic effect on cells, while complexes III and IV exhibit moderate cytotoxicity, and complexes V and VI exhibit less cytotoxicity (Figure 3 and 4). The increasing order In vitro brine shrimp lethality bioassay (BSLB): This method is reliable, rapid, and economical. A plot of the log of the sample's concentration versus percentage (%) mortality of brine shrimp larvae showed a linear correlation. These results suggest that the mortality rate of brine shrimp larvae increases with increasing the concentration of the compounds. The synthesized ligands have less mortality rate as compared to the synthesized complexes. The increasing mortality rate of ligands (LC50) and complexes (LC50) is L1 (19.95) < L3 (17.96) < L5 (17.83) < L4 (16.00) < L2 (11.95) < L6 (9.84) < II (9.78) < III = V (8.03) < I (7.96) L3 IV L4 V L5 V! L6 Compounds Figure 4. Effect of compounds on S. cerevisiae cells as increasing concentration. Varma et al.: Synthesis, Characterization and Biological Application Acta Chim. Slov. 2020, 67, 957-965 969 < VI (4.01) < IV (3.98). The LC50 values of the compounds are shown in brackets in ^g/mL. Complex IV is the most potent amongst all the compounds. DNA binding activities: Binding of metal complexes with DNA via intercalation generally results in hypochro-mism and a redshift (bathochromism) in the absorption band.35 Complex bind to DNA through major or minor groove results in hypochromism and redshift. The charged rhenium complex shows intercalation due to a strong stacking interaction between an aromatic moiety of the li-gand and the base pair of the DNA,36 while neutral Re(I) complex shows groove binding.37 The increasing order of Kb is L2 < L5 < L6 < II < V < L3 < VI < L1 = L4 < I < IV < III. The observed result shows that upon successive addition of DNA (100 pL) at every 10 minutes time interval, a decrease in absorption intensity (hypochromism) and small redshift (1-6 nm) was observed (Figure 5). It suggests that all synthesized complexes show groove binding, which 0.5 - [DNA] = 0|iM [DNA] = 10 MM [DNA) = 20 pM [DNA] = 30 mM [DNA] = 40 |iM [DNA] = 50 nM 0.1 ^^V 0.05 0 - 250 350 450 550 650 750 Wavelength nm Figure 5. UV- Vis absorption spectral changes on the addition of HS DNA to the solution of complex (ligand L1 and complex I). was also confirmed by viscosity measurement and molecular docking. The organic antitumor drug netropsin has to bind within the DNA minor groove. The drug is held in place by amide hydrogen bonds to adenine N-3 and thymine O-2 atoms.38 The binding constant (Kb) values estimated from the ratio of the slop to the intercept ratio. The absorption spectral changes were monitored at around 273-296 nm for the investigation of the DNA binding mode and strength. As the DNA concentration was increased, the transition bands of the complexes I-VI exhibited hypochromicity [hypochromicity, H% = [(Afree - Abound)/Afree] x 100%] of about 11.0-40.5%, and bathochromicity of 1-6 nm. The complex IV and the ligand L4 have the highest percentage hypochromicity (IV-28.5%, L4-40.5%). The Gibb's free energies of the synthesized compounds are found negative values in the range of -34.30 to -42.20 kJ mol-1 (Table 1). The negative value of Gibbs free energy change (AG°) reveals that the binding process is spontaneous. Viscosity measurement was carried out on DNA by varying the concentration of the added Re(I) complex to get an idea of the binding mode. Groove binding typically causes less pronounced or only a minor change in the vis-cosity.39 The values of relative specific viscosity (n/n0)1/3 {(n and n0) are the specific viscosities of DNA in the presence and absence of the Re(I)complex are plotted against [Re(I)complex]/[DNA] in Figure 6. The decreasing order of the (n/n0)1/3 to the DNA is III > VI > II > IV > V > I > L6 > L5 > L4 > L1 > L2 > L3, which parallels the DNA binding affinity. The increase in viscosity, observed in the presence of I-VI is small compared to the classical DNA intercalator EtBr.40 Similar enhancement in viscosity has been observed for DNA groove binding simple and mixed ligand Fe(II) and Ru(II) complexes containing 5,6-dmp (5,6-di-methyl-1,10-phenanthroline) as a co-ligand.41,42 The enhancement in viscosity observed in the present study is Table 1 Binding constant (Kb), percentage hypochromicity (%H), bathochromicity (AX), and Gibbs free energy (AG°) values of free ligands and synthesized complexes Compounds ^mas (nm) aA\(nm) bKb cH% dAGo Free Bound (M-1)x 105 (Jmol-1) L1 277 278 1 1.8 27.8 -40,040.91 L2 279 280 1 0.3 39.2 -34,325.59 L3 281 282 1 1.3 30.1 -38,964.10 L4 277 279 2 1.8 40.5 -40,040.91 L5 276 277 1 0.5 14.9 -35,802.34 L6 272 273 1 0.7 35.4 -36,915.72 I 292 294 2 2.0 16.8 -40,389.55 II 289 291 3 1.1 15.2 -38,411.32 III 290 296 6 3.5 16.7 -42,241.30 IV 291 295 4 3.1 28.5 -41,839.72 V 286 291 5 1.2 11.2 -38,699.24 VI 286 287 1 1.7 15.1 -39,851.78 a AX = Difference between bound wavelength and free wavelength.; b Kb = Intrinsic DNA binding constant determined from the UV-visible absorption spectral titration; c H% = [(Afree - A(,olmd)/Afree] x 100%; d AG° = Change in Gibb's free energy Varma et al.: Synthesis, Characterization and Biological Application ... 966 Acta Chim. Slov. 2020, 67, 957-969 a) b) 3 2.5 2 - 1.5 0.1 0.15 [Ligand]/[DNA] 0,25 Complex-I Complex Jl Complex-Ill —Comp!ex-IV —Complex-V —Complex-VI 0.05 0.1 0.15 [Complex]/[DNA] 0.2 0.25 Figure 6. Effect of increasing concentration of (a) ligands and (b) complexes on the relative viscosity of HS DNA at 27 (±0.1) °C in phosphate buffer at pH = 7.2: also similar to minor groove binder netropsin.43 These show that complexes I-VI is more likely to have a DNA groove binding propensity.33,43 Molecular Docking with DNA sequence d(ACCGA CGTCGGT)2: Molecular docking study is attempted to have an idea on the binding sites and favoured orientation of the ligand inside the DNA groove.44, 45 The complexes and lig-ands are shown by the ball and stick model and DNA base pair shown by the VDW sphere using Hex 8.0 software shown in supplementary material 5. Structure of ligands and complexes were drawn in .CDX format using ChemBioDraw Ultra 14.0 then converted to PDB format using Chem3D (Cambridge Soft). For docking studies, the structural coordinates of DNA were obtained from the protein data bank (pdb id: 423D).46 Figure 7 shows that Re(I) complexes bind with the base pair A-T, C-G, G-C, A-T (B-DNA) minor grooves of the DNA. The energy of the docked structure (I-VI and L1-L6) is -279.72, -280.28, -283.51, -288.34, -278.84, -281.34, and -233.32, -254.18, -253.77, -252.77, -251.48, -230.31 kJ/mol. The increasing order of energy is L6 < L1 < L5 < L4 < L3 < L2 < V < I < II < VI < III < IV Effect of compounds on the integrity of DNA of S. cer-evisiae cells: To determine the DNA damaging potential of the compounds a characteristic picture of comets was observed when yeast cells were exposed to increasing concentrations of compounds, increasing in smearing was observed. Agarose gel electrophoresis is a convenient method to assess the cleavage of DNA by metal-based drugs,47 to determine the factors affecting the nucleolytic efficiency of a compound, and to compare the nucleolytic properties of different compounds. Figure 8 shows the electrophoretic separation of S. cerevisiae DNA when reacted with compounds under aerobic conditions. These clearly show that the relative binding efficacy of the complexes to DNA is much higher than the binding efficacy of pyrazolo[1,5-a]pyrimidine ligands. The difference in the DNA-cleavage efficiency of the complexes and ligands is due to the difference in binding affinity of the ligands and complexes to the DNA. In Figure 8 ligands show lesser smearing as compared to the complexes. It suggests that the cleavage efficiency of DNA is higher in the presence of complexes than the ligands. Complexes III, IV and VI show better cleavage effect of DNA, complex II shows Figure 7. Molecular docking of complex I (ball and stick) with the DNA duplex (VDW spheres) of sequence d(CGCGAATTCGCG)2. The complex is docked inside the DNA groove. Varma et al.: Synthesis, Characterization and Biological Application Acta Chim. Slov. 2020, 67, 957-967 969 î Î L1 1 L2 II L3 III |< Lt Si k^ su M i 4 W M u .'WW 4 _f L4 IV L5 V L6 VI Figure 8. Photogenic view of the cleavage of S. cerevisiae DNA with a series of compounds using % agarose gel containing 0.5 |rg/L EtBr for 24 h at 37 °C. moderate cleavage effect of DNA, and complexes I and IV show lesser cleavage effect of DNA. Antiproliferative study: Metal carbonyls as anticancer drugs in clinical and pharmaceutical trials has wide scope because of its good solubility, and carbonyl releasing ability in the biological system. The synthesized complexes tested as MTT assay using HCT 116 cell line (Supplementary material 6). As the concentration increases the % cell proliferation is deceases means inhibit the tumor cells. The increasing order of IC50 values is III > carboplatin > I > oxaliplatin > II > cisplatin > IV = V = VI. Above 500 ^g/ mL concentration solution becomes turbid, coloration, and visibility not seen properly, from these, we can conclude that below 500 ^g/mL concentration, all synthesized complexes gives good anticancer activity. The IC50 value of synthesised complex (I-VI) and standard drugs like cisplatin, carboplatin, oxaliplatin is 44.66 ^g/mL, 20.50 ^g/mL, >500 ^g/mL, <10 ^g/mL, <10 ^g/mL, <10 ^g/mL, 15.49 ^g/mL, >111.37 ^g/mL, and 22.66 ^g/mL, respectively. The complexes IV, V, and VI are most cytotoxic than other complexes and standard drugs. The approach of metal complexes having carbon monoxide (CO) and heterocy-clic compound with three to four bond distance presence of hetero atom chelated with rhenium metal is promising in terms of enhancing anticancer activity. synthesized and characterized, in search of new organo-metallic complexes with better antibacterial, cytotoxicity, genotoxicity, DNA binding, and DNA cleavage study. The synthesis was carried out by pentacarbonyl chloro rheni-um(I) as a starting material. The spectral and analytical data are in good agreement with the proposed structure and revealed the octahedral geometry, and non-electrolytic nature of complexes. Re(I) compounds treatment to Saccharomyces cerevisiae yeast cells induced genotoxicity and changes in the conformation of cell DNA. DNA binding study was carried out by absorption titration, viscosity measurement, and molecular modelling. Binding constant (Kb) values of complexes were higher than the ligands, and the studies showed groove mode of DNA binding. There was a minor change in the relative specific viscosity (n/ n0)1/3 (n and n0 are the specific viscosities) of DNA in presence and absence of the Re(I)complex, which supports absorption spectroscopy titration data of groove mode of DNA binding. In molecular modelling, docking energies of complexes were observed higher than the ligands. The presence of a more electronegative environment improves the antibacterial activity of complexes than ligands. The increasing order of LC50 values evaluated by brine shrimp lethality bioassay is L1 < L3 < L5 < L4 < L2 < L6 < II < III = V < I < VI < IV. All the complexes show potent in vitro cytotoxicity in cellular level bioassay compared to free ligands. 4. Conclusion A series of substituted pyrazolo[1,5-a]pyrimidine nucleus based organometallic rhenium(I) complexes were Acknowledgement The authors are thankful to the Head, Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar, Varma et al.: Synthesis, Characterization and Biological Application ... 968 Acta Chim. Slov. 2020, 67, 957-969 Gujarat, India, for providing necessary research facilities, Sardar Patel University, Vallabh Vidyanagar, CPEPA, UGC, New Delhi for providing chemicals facility, DST-PURSE Sardar Patel University, Vallabh Vidyanagar for LC-MS analysis. 6. References 1. G. Jaouen, S. Top, A. Vessieres and R. Alberto, Journal of Organometallic Chemistry 2000, 600, 23-36. DOI:10.1016/S0022-328X(00)00036-X 2. J. Wald, R. Alberto, K. Ortner and L. Candreia, Angewandte Chemie International Edition 2001, 40, 3062-3066. DOI:10.1002/1521-3773(20010817)40:16<3062::AID-ANIE 3062>3.0.C0;2-0 3. W. H. Mahmoud, N. F. Mahmoud and G. G. 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D0I:10.1021/ja00190a046 Povzetek Sintetizirali smo nevtralne komplekse renija(I) tipa [ReCl(CO)3Ln] {L1 = 7-fenil-5-(piridin-2-il)pirazolo[l,5-a]pirimidin, L2 = 7-(4-bromofenil)-5-(piridin-2-il)pirazolo[1,5-a]pirimidine, L3 = 7-(4-klorofenil)-5-(piridin-2-il)pirazolo[1,5-a] pirimidin, L4 = 7-(2-klorofenil)-5-(piridin-2-il)pirazolo[1,5-a]pirimidin, L5 = 7-(4-metoksifenil)-5-(piridin-2-il)pira-zolo[1,5-a]pirimidin, L6 = 5-(piridin-2-il)-7-(p-tolil)pirazolo[1,5-a]pirimidin| in jih karakterizirali s 13C-APT, 1H-NMR, IR, meritvami elektronskih spektrov, magnetnimi meritvami in meritvijo predvodnosti. Anti-proliferativna aktivnost merjena na celicah HCT116 z metodo MTT nakazuje na močno citotoksično delovanje kompleksov, ki pri nekaterih presega celo aktivnost standardnih učinkovin kot so cisplatina, oksaliplatina in karboplatina. Antimikrobno delovanje kompleksov je večje kot pri pirazolo pirimidinskih ligandih. Teoretične študije interakcij med novimi spojinami in DNK smo preučevali z metodo molekularnega priklapljanja. Vrednost interakcij DNK-kompleks je med -230.31 in -288.34 kJ/mol. Vrednosti veznih konstant za komplekse (1.1-3.5 x 105 M-1) so višje od vrednosti za ustrezne ligande (0.32-1.8 x 105 M-1). Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Varma et al.: Synthesis, Characterization and Biological Application ... DOI: 10.17344/acsi.2020.6046 Acta Chim. Slov. 2020, 67, 970-976 /^creative ^commons Scientific paper A New Reagent for Spectrophotometric Determination of Ir(IV): 5-[2-(4-Hydroxyphenyl) hydrazineylidene]-4-iminothiazolidin-2-one (HPIT) Oleksandr Tymoshuk,1 Lesia Oleksiv,1,* Orest Fedyshyn,1 Petro Rydchuk,1 Vasyl Matiychuk1 and Taras Chaban2 1 Department of Chemistry, Ivan Franko National University of Lviv, Kyryla and Mefodiya Str., 6, 79005 Lviv, Ukraine; 2 Faculty of Pharmacy, Danylo Halytsky Lviv National Medical University, Pekarska Str., 69, 79010 Lviv, Ukraine * Corresponding author: E-mail: l_lozynska@ukr.net Received: 06-15-2020 Abstract The paper presents a new azolidone derivative - 5-[2-(4-hydroxyphenyl)hydrazineylidene]-4-iminothiazolidin-2-one (HPIT) studies and its interaction results with iridium(IV) ions. The Ir(IV) with this reagent in the pH = 5.0 without heating forms a stable complex (Xmax = 328 nm). The stoichiometric ratio of Ir(IV) to the reagent in complex is 1:1. The molar absorptivity and Sandell's sensitivity are 5.57 x 103 L mol-1 cm-1 and 0.034 |g cm-2 respectively. The calibration curve is linear in the range of 1.0-11.5 |g mL-1 of Ir(IV) (R = 0.9996). The limit of detection is 0.4 |g mL-1. Based on the conducted investigation a rapid and simple, spectrophotometric method for the determination of Ir(IV) using 5-[2-(4-hydroxyphenyl)hydrazineylidene]-4-iminothiazolidin-2-one as a chromophoric reagent was developed. The iridium(IV) was determined in various synthetic mixtures and alloys. Keywords: Iridium(IV); spectrophotometry; 5-[2-(4-hydroxyphenyl)hydrazineylidene]-4-iminothiazolidin-2-one; azo-lidones. 1. Introduction The relevance of platinum metals and their compounds usage encourages the development of simple, rapid, inexpensive, selective, and sensitive methods for the determination of trace amounts of these metals in complex samples. Spectrophotometric methods of analysis are successfully used to solve this problem in analytical chemistry with the use of organic reagents containing functional-analytical groups.1,2 Spectrophotometric methods are one of the most widely used physical-chemical analysis methods in industrial and research laboratories. The main advantages of spectrophotometry are versatility, sufficient sensitivity to solve specific analytical problems, simplicity, the possibility of analysis automation, which provides it a leading place in the modern analytical chemistry. Spectrophotometric methods of platinoids determination are characterized by different sensitivity (sx~103-105 L mol-1 cm-1) depending on the choice of reagent. Their sensitivity is significantly increased using organic reagents, namely heterocyclic azo derivatives and sulfur-containing compounds.1-3 Among a large number of azo dyes, azolidones and their derivatives are of particular interest. The color reactions of noble metals with this group of reagents are highly sensitive (s = (0.4-1.5) x 104 L mol-1 cm-1) and contrast (AX = 7080 nm). Azorodanines contain several functional-analytical groups in their molecules, which makes it possible to use them as group reagents for the precious metals deter- mination.3-6 Our scientific group first investigated the analytical properties of several azolidone derivatives and successfully used them to determine the number of ions: Cu(II), Ni(II), Cd(II), Zn(II), Hg(II), Pd(II), Pt(IV), Rh(III), Ir(IV), Ru(IV).7-21 Due to the wide usage of iridium in various industries (such as jewelry, electrical equipment, dental alloys, automobile, chemical, and electronics industries, the field of photography and aviation), it is important to develop efficient analytical methods for this metal determination in various samples.22,23 Most of spectrophotometric methods for the Iridium determination are not enough sensi- Tymoshuk et al.: A New Reagent for Spectrophotometric Determination Acta Chim. Slov. 2020, 67, 970-976 971 tive, selective and need heating or extraction.1,2,4,11,24-27 For this reasons the goal of our research was to find the effective analytical reagents for spectrophotometry Ir(IV) determination using azolidones derivatives. One of them was a new reagent - 5-[2-(4-hydroxyphenyl)hydraziney-lidene]-4-iminothiazolidin-2-one (HPIT). 2. Experimental 2. 1. Equipment Spectrophotometry measurements were performed with a computerized spectrophotometer, model ULAB 108-UV, fitted with 1.0 cm quartz cells. Computerized device MTech OVA-410 with a linear potential sweep was used for voltammetric measure-ments.28 A three-electrode system including dropping mercury electrode (working electrode), a saturated calomel electrode (reference electrode), and platinum (counter electrode) were used. The pH-150 M pH-meter equipped with a combined glass electrode was used to measure the pH values of solutions. 1H NMR spectra were registered on the spectrometer Varian Mercury UX-400, DMSO-d6 was used as a solvent, tetramethylsilane as a standard. 2. 2. Reagents The stock solution of iridium(IV) chloride was prepared by melting the exact mass of pure iridium (99.99%) with the oxidizing mixture of NaNO3 + NaOH (1:3, v/v), and BaO2 at 950 K for 45-60 min. Then the fusion was dissolved in 3.0 mol L-1 hydrochloric acid. The existence form of Ir(IV) ([IrCl6]2-) in the obtained solution was confirmed by comparing its absorption spectra with the transferred data.29 The obtained solution of Ir(IV) was additionally standardized using the titration method - iodometry, due to possible losses during sintering.1,2 Standard transferred solutions of Ir(IV) were prepared by diluting an aliquot of Ir(IV) initial stock solution in 1.0 mol L-1 HCl. The solution of 5-[2-(4-hydroxyphenyl)hydraziney-lidene]-4-iminothiazolidin-2-one was prepared by dissolving the exact mass of the pre-purified reagent in dimethyl sulfoxide. Working solutions of HPIT were prepared by diluting an aliquot of the stock solution in dimethyl sulfoxide. The 5-[2-(4-hydroxyphenyl)hydraziney-lidene]-4-iminothiazolidin-2-one was synthesized by the following procedure: 0.01 mol of 4-aminophenol was dissolved in 3 ml of concentrated hydrochloric acid, after which 5 ml of water was added. The solution obtained at this stage, with cooling, was diazotized with 0.72 g of transferred nitrite dissolved in 3 mL of water. The resulting di-azonium salt was added over 30 minutes to a solution of 0.01 mol of 4-iminothiazolidin-2-one previously dissolved in 80 ml of glacial acetate acid containing 4 g of anhydrous sodium acetate (pH = 4.5-5.0) with stirring and was cooled. The mixture was left at 12 h, after which it was poured into 200 mL of water. The precipitate was filtered, washed on the filter with water, dried, and recrystallized. 1H NMR (400 MHz, DMSO-d6; 5, ppm): 6.70 (d, J = 9.0 Hz, 2H, C6H4), 7.28 (d, J = 9.0 Hz, 2H, C6H4), 8.71 (s, 1H, NH), 8.97 (s, 1H, NH), 10.17 (s, 1H, NH). The purity of HPIT was determined by chromatography-mass spectrometry. The solution of HCl was prepared by dilution of concentrated HCl. The solutions of sodium salts (to study the effect of anions), NaCl and NaOH were prepared by dissolving an appropriate amount of respective salts and NaOH in distilled water. The Britton-Robinson buffer (BRB) was prepared by mixing solutions of boric, phosphoric and acetic acids.30 The solutions of various metals (to study the effect of cations) were prepared by dissolving the exact mass of the corresponding metal in HCl or HNO3 acids either its mixture, or their salts in distilled water or dilute hydrochloric or nitric acids. The solutions of Rh(III) and Ru(IV) were prepared by sintering corresponding metal with the oxidizing mixture NaNO3 + NaOH (1:3, v/v) with further dissolving the fusion in 3.0 mol ■ L-1 HCl. All chemicals used were of analytical grade and distilled water was used for the preparation of the aqueous solution. 2. 3. Procedure Research of the HPIT spectral characteristics Aliquots of 2.0 mL HPIT working solution (2.5 x 10-4 mol L-1) were transferred into a series of 25.0 mL transferred flasks, then 2.0 mL BRB (1.5 mol L-1), 1.25 mL NaCl (2.0 mol L-1) and water (~15-20 mL) were added to each flask. The pH values (2.0-12.0) were adjusted using NaOH (4.0 mol L-1) and then diluted to volume with distilled water. The solution with pH = 1.0 was prepared as described above but without BRB addition and pH value was adjusted using HCl (6.0 mol L-1). The absorption spectra were measured against distilled water as blank. General procedure for the determination ofIr(IV) with HPIT An aliquot of Ir(IV) solution (in the range of 1.011.5 ^g mL-1 in the final volume), 2.5 mL (1.0 x 10-3 mol L-1) HPIT, 1.0 mL (1.5 mol L-1) BRB and 1.25 mL (2.0 mol L-1) NaCl were placed into 25.0 mL calibrated flasks and then distilled water was added (~15 mL). The pH was adjusted to ~5.0 with NaOH solution and diluted to the mark with distilled water. The absorbance was measured at 328 nm against a reagent blank. Determination the stoichiometric ratio of the Ir(IV)-HPIT complex The equimolar solutions of Ir(IV) and HPIT (1.0 x 10-3 mol L-1) were used to determine the metal to ligand ratio by Job's method of continuous variation. The total concentration CIr(IV) + Chpit was equal 1.0 x 10-4 mol L-1 Tymoshuk et al.: A New Reagent for Spectrophotometric Determination 972 Acta Chim. Slov. 2020, 67, 970-976 in a 25.0 mL volumetric flask. Then 1.0 mL (1.5 mol L BRB, 1.25 mL (2.0 mol L-1) NaCl, and distilled water (~15 mL) were added. The pH was adjusted to 5.0 by NaOH and diluted with distilled water to the calibration mark. The absorbance values were recorded at 328 nm. The mole-ratio method was performed in the transferred way: into a series of 25.0 mL volumetric flasks the Ir(IV) solution with fixed concentration (0.5 mL of1.0 x 10-3 mol L-1), an aliquot 0.10-10.0 mL (5.0 x 10-4 mol L-1) HPIT, 1.0 mL (1.5 mol L-1) BRB, 1.25 mL (2.0 mol L-1) NaCl, and distilled water ~ 20 mL were added. The pH was adjusted to pH = 5.0 by adding NaOH and diluted up to the mark with water. Then, the absorbance at 328 nm was measured. Alloys samples preparation The alloys (Gd2Ir3Al9, Tb2Ir3Al9) were synthesized by arc melting of pure metals (Gd > 99.86%, Tb > 99.83%, Ir > 99.9%, Al > 99.998%) under an argon atmosphere and heated to 873 K, held at that temperature for 720 h and then cooled to room temperature.31 The solutions of alloys samples were prepared by dissolving of 0.05-0.1 g of sample in 10-20 mL of HCl and HNO3 (10:1, v/v) mixture and heating for ~ 2 h. Then the black residue was filtered and filtrate transferred to a 200.0 mL volumetric flask. The residue was sintered with a NaNO3 and NaOH (1:3, v/v) mixture at 950 K (60 min). The melt was dissolved in 3.0 mol L-1 HCl. The obtained solution was transferred to the previous filtrate and distilled water was added to the mark. The 0.4-1.5 mL of alloys aliquots were taken for Ir(IV) determination with HPIT as described above.11 3. Results and Discussion 3. 1. Research Spectral Characteristics of the HPIT We researched a new reagent - 5-[2-(4-hydroxyphe-nyl)hydrazineylidene]-4-iminothiazolidin-2-one, which is a derivative of azolidone (Fig. 1). HPIT is the crystalline yellow powder poorly soluble in water and ethanol but well soluble in dimethylformamide and dimethyl sulfox-ide. The melting point is 515 K. In our previous work,32 we investigated the effect of the medium acidity on the absorption spectra of HPIT over the pH range 1.0-12.0. As shown by the results of the experiment, the HPIT absorption maximum depends on the pH of the solution, which is associated with different existence forms depending on the medium acidity (Fig. 2). At pH = 1.0, the absorption spectrum is characterized by a maximum at a wavelength of 418 nm, which corresponds to the protonated form of the reagent; at pH 2.0-10.0, the maximum of absorbance shifts slightly to the region of smaller wavelengths (400 nm), and in the alkaline medium (pH>10.0) (Fig. 3), there is a course of hydrolysis reaction with the release of ammonia (Scheme 1). The molar absorptivity at X = 418 nm is 2.04 x 104 L mol-1 cm-1 (pH = 1.0); at X = 400 nm is (1.14-1.85) x 104 L mol-1 cm-1 (pH 2.0-9.0) and 1.02 x 103 L mol-1 cm-1 (pH = 10.0). 350 400 450 Wavelengtfi/nm Fig. 2. Absorbance spectra of HPT at different pH Fig. 3. Effect pH on the absorbance of HPIT at 400 nm Fig. 1. Structual formula of the HPIT Tymoshuk et al.: A New Reagent for Spectrophotometric Determination ... Acta Chim. Slov. 2020, 67, 970-976 973 Scheme 1. Hydrolysis of 5-[2-(4-hydroxyphenyl)hydrazineylidene]-4-iminothiazolidin-2-one HPIT does not polymerize and does not form tautomeric forms at pH = 1.0 in the concentration range of 5.0 x 10-6-8.0 x 10-5 mol L-1, since in this range the Beer's law is applicable and only one maximum is observed. 3. 2. Investigation of the Interaction of Ir(IV) with HPIT It was found that the Ir(IV) ions form a complex with HPIT (Fig. 4). The absorption spectra of HPIT, Ir(IV), and Ir(IV)-HPIT were recorded over the range 200 to 550 nm. As shown on Fig. 4 the maximum of the reagent at 400 nm (pH = 5.0) is reduced in the presence of iridi-um(IV) ions. Instead, there was an increase in the absorption of Ir(IV)-HPIT in the wavelength range from 250 to 360 nm compared to the absorption of the reagent, which indicates the interaction. The largest difference in absorption of the reagent and compound is at À = 328 nm. Fig. 5. Effect of pH on the maximum yield of Ir(IV) with HPIT complex (X = 328 nm, Cir(IV) = 2 x 10-5 mol L-1, CHPIT = 4 x 10-5 mol L-1) Fig. 4. Absorbance spectra of Ir(IV), reagent and complex Ir(IV) with HPIT (pH = 5.0, Clr(IV) = 8.0 x 10-6 mol L-1, CHPIT = 2.0 x 10-5 mol L-1) The effect of various parameters on the formed products absorption intensity was studied and the reaction conditions were optimized. Effect of pH The acidity of the medium is one of the important parameters that affect the complexation. Within the pH range from 2.0 to 10.0 Ir(IV) ions form a complex with HPIT (Fig. 5). The maximum yield of complex is at pH = 5.0. Hence this value of pH was selected for further studies. Effect of time and temperature The process of iridium(IV) ions complexation with HPIT occured at room temperature (~ 291-296 K) immediately after the acidity of the medium was established. The effect of heating time on the maximum yield of the colored compound was investigated. The solutions heating in a boiling water bath (~ 371 K) caused a decrease in the absorption of the solutions, but the complex compound Ir(IV)-HPIT was not destroyed even when heated for 60 min. Therefore it is recommended that the reaction should be carried out at room temperature. The absor-bance of obtained Ir(IV)-HPIT complex was stable up to 72 h. The stoichiometric ratio of complex The Job's method of continuous variations (Fig. 6) and the mole-ratio method were used to determine the stoichiometric ratio of the complex. These methods indicate that complex with stoichiometry 1:1 was formed. A 1.5 fold excess of reagent is required for full complexation. The formal stability constant of the Ir(IV)-HPIT complex was calculated and it is equal to 8.9 x 105. Tymoshuk et al.: A New Reagent for Spectrophotometric Determination 974 Acta Chim. Slov. 2020, 67, 970-976 0 0.2 0.4 0.6 0.S 1 Fig. 6. The method of continuous variations Calibration curve The calibration graph for Ir(IV) determination with HPIT was constructed using the optimal conditions (pH = 5.0, X = 328 nm, CHPIT = 1.0 x 10-4 mol L-1, CNaCi = 0.1 mol L-1, CBRB = 0.06 mol L-1) and showed that the system obeys Beer's law in the concentration range of 1.0-11.5 ^g mL-1 of Ir(IV). The linear equation is AA = (0 ,011±0.004) + (0,0292±0.0007) x CIr(IV) (AA - absorbance, C - concentration of Ir(IV) in ^g mL-1) with correlation coefficient equal 0.9996, N = 6, SD = 2.37 x 10-3. The molar absorptivities and Sandell sensitivity are 5.57 x 103 L mol-1 cm-1 and 0.034 ^g cm-2 respectively. The limit of detection is 0.4 ^g mL-1. 3. 3. Selectivity of the Ir(IV) Determination The various cations and anions influence on the iridi-um(IV) determination were studied under the conditions of the standard procedure. The tolerance limits of tested interfering ions were calculated as the maximum concentrations that do not cause an error of more than ± 5% in an absorbance value. The tolerance limits for foreign ions are shown in Table 1. It has been found that the majority of cations do not interfere significantly. The iridium(IV) ions can be easily determined in the presence of Tb(III), Gd(II), Cd(II), Zn(II), Mn(II), Ni(II), Cu(II), Ca(II), Mg(II), Ba(II) and the studied anions. However, several ions such as Pd(II), Rh(III), Pt(IV), Ru(IV) interfere seriously. Their effect can be eliminated by using some of the studied anions as masking agents. For example, Pd(II) and Ru(IV) were masked using EDTA (the tolerance limits reach 1 for Pd(II) and 2 for Ru(IV)). This method has higher selectivity than most of the spectrophotometry methods of Iridium determination.1, 2 4 11, 24-27 3. 4. Analytical Application The developed spectrophotometric method was applied to the determination of Ir(IV) in synthetic mixtures and alloys to validate it. Analysis of Ir(IV) in synthetic mixtures Different synthetic mixtures were prepared and analyzed using the proposed developed method in order to research the precision and accuracy (Table 2). As can be seen in Table 2, the obtained results are consistent with the Table 1. Selectivity of Ir(IV) spectrophotometric determination (C^iv) = 2.5 x 10 5 mol L 1; Chpit = 1.0 x 10 4 mol L 1; CNaCl = 0.1 mol L-1; CBRB = 0.06 mol L-1; pH = 5.0; X = 328 nm; l = 1.0 cm) Foreign Tolerance limit Foreign Tolerance limit Foreign Tolerance limit ion Cion : CIr(IV) ion Cion : CIr(IV) ion Cion : CIr(IV) Pd(II) 0.1 Ni(II) 150 Ca(II), Mg(II) >200 Pt(IV) 0.25 Fe(III) 15 C2O42-* 100 Rh(III) 0.1 Cd(II)* 75 F-* 100 Ru(IV) 0.25 Pb(II)* 75 EDTA* 100 Tb(III) 100 Mn(II)* 100 Sal-* 100 Gd(III) 75 Zn(II) 75 Citr3-* 100 Cu(II) 50 Al(III) 8 Tart2-* 100 Co(II) 30 Ba(II) 75 PO43-* 100 "These ions decrease the absorbance value by ±5%, and the others all increase. Table 2. Determination of Iridium(IV) in synthetic mixtures, n = 3; P = 0.95 Composition of synthetic mixture Added Ir(IV), ^g Found Ir(IV), RSD, % 12.6 |g Ru(IV), 0.7 mg Co(II) 96 98±5 2.0 3.3 |g Pd(II), 2.8 mg Ni(II), 9.7 mg Pb(II) 96 101±7 2.8 24.4 |g Pt(IV), 3.4 mg Mn(II) 96 100±8 3.2 Tymoshuk et al.: A New Reagent for Spectrophotometric Determination ... Acta Chim. Slov. 2020, 67, 970-976 975 Table 3. Results of the determination of Ir(IV) in alloys, n = 3, P = 0,95 Alloy Spectrophotometry — pr SXl CDlr ± -p^ RSD, % Voltammetry VK RSD, % Gd2Ir3Al9 Tb2Ir3Al9 50.9 50.7 51.5 ± 1.8 51.0 ± 1.5 1.4 1.2 51.2 ± 1.4 50.8 ± 1.6 1.1 1.3 added amounts of Ir(IV). The calculated values of the relative standard deviation and the absence of a significant systematic error indicate a good reproducibility and accuracy of this spectrophotometry method. Analysis of Ir(IV) in the alloys The results of Iridium determination in the alloys are given in Table 3. The data obtained by the spectrophotometry method were compared with Ir(IV) contents determined by the voltammetric method. As seen, the results of both methods are agreemented. The results in Table 3 show that the relative error and relative standard deviation do not exceed 1.5%. 4. Conclusions The results of this research indicate that the developed spectrophotometric method based on the complex-ation of Ir(IV) with the new azolidone derivative (5-[2-(4-hydroxyphenyl)-hydrazineylidene]-4-iminothi-azolidin-2-one) can be successfully used for the determination of iridium(IV) in different samples. This method is simple, sensitive, selective towards many ions, reproducible, and rapid because it does not require heating or separation from a large number of foreign ions (such as REE, Cu(II), Ni(II), Mn(II), Zn(II), Cd(II), et al.), which are associated with Ir(IV) in its objects. The time needed for analysis about 30 min, which makes this method much faster than other spectrophotometric methods for determining Iridium, which are described in the literature. 6. References 1. Ya. A. Zolotov, G. M. Varshal, V. M. Ivanov, Analiticheskaya Khimiya Metallov Platinovoi Gruppy, Editorial URSS, Moscow, Russia, 2003, p. 592. 2. S. I. Ginzburg, N. A. Yezerskaya, I. V. Prokof'eva, N. V. Fe-dorenko, V. I. Shlenskaya, N. K. Belsky, Analiticheskaya Khimiya Platinovykh Metallov, Nauka, Moscow, Russia, 1972, p. 613. 3. R. F. Gur'eva, S.B. Savvin, Zh. Anal. Khim. 2002, 57, 980 -996. DOI:10.1023/A:1020917221896 4. R. F. Gur'eva, S. B. Savvin, Usp. Khim. 1998, 67, 236-251. DOI:10.1070/RC1998v067n03ABEH000375 5. S. B. Savvin, R. F. Gur'eva, Talanta 1987, 34, 87-101. DOI: 10.1016/0039-9140(87)80012-7 6. E. Tang, G. Yang, J. Yin, Spectrochim. Acta, Part A. 2003, 59, 651-656. DOI:10.1016/S1386-1425(02)00209-3 7. L. Lozynska, O. Tymoshuk, Chem. Chem. Technol. 2013, 7, 391-395. DOI:10.23939/chcht07.04.391 8. L. Lozynska, O. Tymoshuk, In: O. L. Berezko, The Interaction of 5-hydroxyimino-4-imino-1,3-thiazolidin-2-one with Plati-num(IV) Ions,3rd International Conference of Young Scientists CCT-13, Lviv, Ukraine, 2013, 166. 9. L. V. Lozynska, O. S. Tymoshuk, T. I. Chaban, Methods Objects Chem. Anal. 2014, 9, 50-54. DOI:10.17721/moca.2014.50-54 10. L. V. Lozynska, O. S. Tymoshuk, Issues Chem. Chem. Technol., 2014, 1, 80-85. 11. L. V. Lozyns'ka, O. S. Tymoshuk, T. Ya. Vrublevs'ka, Materials Science 2015, 6, 870-876. DOI:10.1007/s11003-015-9795-y 12. L. Lozynska, O. Tymoshuk, T. Chaban, Acta Chim Slov. 2015, 62, 159-167. DOI:10.17344/acsi.2014.866 13. A. Tupys, O. Tymoshuk, P. Rydchuk, Chem. Chem. Technol. 2016, 10, 19-25. DOI:10.23939/chcht10.01.019 14. A. Tupys, J. Kalembkiewicz, Y. Bazel, L. Zapala, M. Dranka, Y. Ostapiuk, O. Tymoshuk, E. Woznicka, J. Mol. Struct. 2017, 1127, 722-733. DOI:10.1016/j.molstruc.2016.07.119 15. A. Tupys, J. Kalembkiewicz, Y. Ostapiuk, V. Matiichuk, O. Ty-moshuk, E. Woznicka, L. Byczynski, J. Therm. Anal. Calorim. 2017, 127, 2233-2242. DOI:10.1007/s10973-016-5784-0 16. M. Fizer, V. Sidey, A. Tupys, Y. Ostapiuk, O. Tymoshuk, Y. Bazel, J. Mol. Struct. 2017, 1149, 669-682. DOI:10.1016/j.molstruc.2017.08.037 17. Y. Bazel, A. Tupys, Y. Ostapiuk, O. Tymoshuk, V. Matiychuk, J. Mol. Liq., 2017, 242, 471-477. DOI:10.1016/j.molliq.2017.07.047 18. Y. Bazel, A. Tupys, Y. Ostapiuk, O. Tymoshuk, J. Imricha, J. Sandrejova, RSC Adv., 2018, 8, 15940-15950. DOI:10.1039/C8RA02039F 19. O. Tymoshuk, L. Oleksiv, L. Khvalbota, T. Chaban, I. Patsay, Acta Chim Slov. 2019, 66, 62-69. DOI:10.17344/acsi.2018.4448 20. P. V. Rydchuk, O. S. Tymoshuk, L. V. Oleksiv, T. I. Chaban, V. S. Matiychuk, Methods Objects Chem. Anal. 2019, 14, 130139. DOI:10.17721/moca.2019.130-139 21. O. S. Tymoshuk, O. S. Fedyshyn, L. V. Oleksiv, P. V. Rydchuk, I. O. Patsai, Materials Science 2019, 55, 455-459. DOI:10.1007/s11003-019-00325-9 22. V. Draskovic, V. Vojkovic, S. Miko, Talanta 2004, 62, 489-495. DOI:10.1016/j.talanta.2003.08.031 23. L. Zhang, N. Li, P. Fan, X. Chu, S. An, J. Zhang, X.Wang, Hy- Tymoshuk et al.: A New Reagent for Spectrophotometric Determination 976 Acta Chim. Slov. 2020, 67, 970-976 drometallurgy 2012, 127-128, 8-15. D01:10.1016/j.hydromet.2012.06.012 24. V. Druškovic, V. Vojkovic, Croat. Chem. Acta 2003, 76, 49-54. 25. A. S. Amin, I. A. Zaafarany, Anal. Chem. Res. 2015, 3, 77-81. DOI: 10.1016/j.ancr.2014.10.001 26. M. A. Taher, S. Puri, R. K. Bansal, B. K. Puri, Talanta 1997, 45, 411-416. D0I:10.1016/S0039-9140(97)00149-5 27. S. Kuchekar, S. Bhumkar, H. Aher, S. H. Han, J. Mater. Environ. Sci. 2019, 10, 1200-1213. D0I:10.20959/wjpps20174-8960 28. I. Patsay, P. Rydchuk, O. Tymoshuk, Visnyk of the Lviv Univer- sity. Series Chemistry 2017, 58, 219-224. 29. N. E. Ezerskaya, I. N. Kiseleva, Zh. Anal. Khim. 2001, 967970, 855-858. D01:10.1023/A:1016768614942 30. Yu.Yu. Lure, Spravochnik po analiticheskoi khimii, Khimiya, Moscow, Russia, 1971, p. 456. 31. Yu. Lutsyshyn, Ya. Tokaychuk, P. Demchenko, R. Glady-shevskii, Visnyk of the Lviv University. Series Chemistry 2010, 51, 52-59. 32. L. Khvalbota, O. Tymoshuk, V. Matiychuk, Visnyk of the Lviv University. Series Chemistry 2016, 57, 219-225. Povzetek V prispevku je predstavljen nov derivat azolidona - 5-[2-(4-hidroksifenil)hidrazineiliden]-4-iminotiazolidin-2-on (HPIT) in rezultati njegove interakcije z ioni iridija(IV). Ir(IV) ion s tem reagentom pri pH = 5,0 brez segrevanja tvori stabilen kompleks (Xmax = 328 nm). Stehiometrično razmerje Ir(IV) in reagenta v kompleksu je 1:1. Molarna absorp-tivnost in Sandellova občutljivost sta 5,57 x 103 L mol-1 cm-1 in 0,034 |ig cm-2. Kalibracijska krivulja je linearna v območju 1,0-11,5 |g mL-1 Ir(IV) (R = 0,9996). Meja zaznave je 0,4 |g mL-1. Na podlagi izvedene raziskave je bila razvita hitra in enostavna spektrofotometrična metoda za določanje Ir(IV) z uporabo 5-[2-(4-hidroksifenil) hidrazineilidena]-4-imi-notiazolidin-2-ona kot kromofornega reagenta. Ir(IV) je bil določen v različnih sintetičnih mešanicah in zlitinah. © ® Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Tymoshuk et al.: A New Reagent for Spectrophotometric Determination ... DOI: 10.17344/acsi.2020.6052 Acta Chim. Slov. 2020, 67, 977-984 /^.creative ^commons Scientific paper The Influence of Ionic Liquids on Micellization of Sodium Dodecyl Sulfate in Aqueous Solutions Bojan Šarac* and Marija Bešter-Rogač University of Ljubljana, Faculty of Chemistry and Chemical Technology, Večna pot 113, SI-1000 Ljubljana * Corresponding author: E-mail: bojan.samc@fk.kt.uni-lj.si Received: 04-22-2020 Abstract The micellization of sodium dodecyl sulfate (SDS) in water and in aqueous solutions of three imidazolium based ionic liquids with different side-chain length, i.e. 1,3-dimethylimidazolium chloride ([CjmimjCl), 1-ethyl-3-methylimidazoli-um chloride ([C2mim]Cl), and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) was investigated by isothermal titration calorimetry (ITC) in the temperature range from 288.15 to 328.15 K. For comparison, the micellization of SDS in the presence of NaCl was studied also. ITC experimental data were analysed by the two-state mass-action model, yielding the values of critical micelle concentration (cmc), aggregation number (n), standard heat capacity (AMCp°), enthalpy (AMH°), entropy (AMS°), and Gibbs free energy (AMG°) of the micellization process. It was found that the micellization of SDS in all the studied systems is an entropy-driven at lower temperatures and an enthalpy-driven at higher temperatures. In addition, it was assumed that with the increasing nonpolar character of IL, the interactions between the SDS are stronger, leading to more negative values of AMH° and AMG°. To obtain more information about the micellar charge, the conductivity and zeta-potential measurements were performed at 298.15 K. Presumably the micellar charge is more positive in the presence of ILs due to their stronger interaction and possible incorporation into the micellar structure. This reflects in less negative zeta-potential comparing to SDS in water and consequently higher degrees of micelle ionization due to the larger portion of sodium ions in solution. Keywords: Sodium dodecyl sulfate; ionic liquids; isothermal titration calorimetry; thermodynamics; micelle ionization; zeta-potential 1. Introduction Sodium dodecyl sulfate (SDS), also known as sodium lauryl sulfate (SLS), is a well-known anionic surfactant, widely used in cleaning and hygiene products,1 as a food additive2 and also in research, as a cell disruptor, denaturat-ing agent etc.3-5 It belongs to one of the most studied surfactants and consequently several characteristics of its micellization processes in aqueous solutions as, for example, the influence of inorganic electrolytes on critical micelle concentration (cmc), the shape of micelles and thermodynamic parameters of micellization of SDS are well-known.6-12 However, the presence of organic electrolytes usually affects these parameters in much more dramatic way if their hy-drophobic chains can penetrate the micelles,13 as was already observed for many other surfactant systems.14-20 For an investigation of these effects, ionic liquids (ILs) as the most studied organic electrolytes in the last decades21,22 seem to be the most appropriate. Because of their bulky cation and anion structure over which the charge is distributed by the resonance, they tend to be liquids at temperatures below 100 °C. Due to the amphiphilic character, some of the ILs can also be classified as catanionic hydrotropes, and they can enhance the solubility of hydrophobic compounds in water.23 Their behaviour in a pure state, mixtures or solutions is unlike conventional molecular solvents by forming amphiphilic nanostructures which offer great potential as designer solvents.24 Properties of many pure protic and aprotic ILs are already well-investigated,25-27 but the knowledge of their influence on aggregation process of SDS or any other surfactant is rather scarce.28-30 Beyaz et al., for example, showed that hydrophobic ILs (e.g. 30 mM solution of 1-hexyl-3-methylimidazolium chloride ([C6mim]Cl)) decreased cmc of SDS, whereas hydrophilic ones (e.g. 30 mM [C4mim]Cl) increased it.28 Such a trend was also obtained for SDS in solutions of 1-pentyl-3-methylimidazolium hexafluorophosphate ([C5mim][PF6]), where cmc increased with increasing concentration of IL due to the solvophobic interactions around the surfactant hydrocarbon chains.29 On the contrary, Javadian et al. observed a Šarac and Bešter-Rogač: The Influence of Ionic Liquids on Micellization 978 Acta Chim. Slov. 2020, 67, 977-984 decrease of cmc of SDS in up to 5.72 mM of [C4mim]Cl. They also demonstrated that longer-chained ILs modify the structural properties of aggregates inducing the formation of wormlike micelles. It appears that the cmc values and morphology of the surfactant systems are strongly dependent upon the concentration and amphiphilicity of ILs which is still the area of extensive investigation.31,32 In the present work, the systematic study of the influence of the increasing hydrophobicity of ILs on the micel-lization process of SDS in aqueous solutions was carried out by using isothermal titration calorimetry (ITC), conductivity and zeta-potential measurements. Although SDS is one of the most studied surfactants, there is limited temperature-dependent data in the literature. Thus, the micel-lization of SDS in water was studied first, followed by the investigation of the micellization of SDS in the presence of three ILs, i.e. 1,3-dimethylimidazolium ([C1mim]Cl), 1-ethyl-3-methylimidazolium ([C2mim]Cl) and, 1-bu-tyl-3-methylimidazolium chloride ([C4mim]Cl), where the concentration of ILs was kept constant at 0.01 M. Because the comparison between ILs and "classical" electrolytes is always interesting and needed, we decided to include also the investigations of micellization of SDS in NaCl solutions. But it turned out, that NaCl affects the process in considerably less extend as ILs. Almost no difference between the thermodynamic parameters for mi-cellization of SDS in water and in 0.01 M NaCl solution was found namely, so the experiments were performed in 0.1 M NaCl solutions. On the contrary, the effect of ILs on micellization of SDS is much stronger and already at the concentration of 0.01 M the difference (in comparison to that in water) was considerable and compared to those in 0.1 M NaCl solutions. Experimental ITC data were analysed by the two-state mass-action model yielding the corresponding standard thermodynamic parameters: Gibbs free energy (AmG°), enthalpy (AMH°), and entropy (AMS°) of micellization together with cmc and aggregation number (n). From conductivity measurements, we estimated a degree of micelle ionization (a) which will be discussed in the light of the determined zeta-potentials (Z). 2. Experimental 2. 1. Chemicals Sodium dodecyl sulfate (purity > 98.5 %, M = 288.37 g mol-1) and sodium chloride (> 99.5 %, M = 58.44 g mol-1) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received. 1,3-dimethyl- (>98 %, M = 132.59 g mol-1), 1-ethyl- (>98 %, M = 146.62 g mol-1) and 1-bu-tyl-3-methylimidazolium chloride (>99 %, M = 174.67 g mol-1) were obtained from IoLiTec (Ionic Liquids Technologies GmbH, Heilbronn, Germany) and used as received. The chemicals were stored in a desiccator over P2O5. For preparation of solutions, MiliQ water was used. 2. 2. Isothermal Titration Calorimetry The heat changes associated with (de)micellization of SDS were measured using a VP-ITC microcalorimeter (MicroCal Inc., Malvern, UK). The sample cell was filled with corresponding "solvent" (water, a solution of 0.1 M NaCl or 0.01 M of IL) and successive aliquots of 6 ^L of the surfactant solution, prepared in the same "solvent", were injected at 10-15 minutes intervals by a motor-driven syringe into the sample cell while stirring at 300 rpm. For each system, experiments at five temperatures between 288.15 and 328.15 K in step of 10 K were carried out. Each added aliquot produced a heat effect (raw signal) mainly due to the demicellization of surfactant micelles, dilution of monomers, and corresponding counterions. When the cmc of surfactant was exceeded in the sample cell, the heat effects evolved only due to dilution of micelles and ions. From the integration of the raw signal (an example in Figure S1a in Supporting information) the enthalpies of dilution (AH) of the surfactant expressed per mole of added SDS were obtained by using software Origin 7.0. According to our experience, the ITC gives highly reproducible results, thus, the experiments were not repeated. 2. 3. Conductivity Measurements Electrical conductivity of solutions was recorded with a PC-interfaced LCR Meter Agilent 4284 A connected to a three-electrode measuring cell described elsewhere.33 The cell constant was determined with dilute potassium chloride solutions.34 The cell was immersed in the high precision thermostat bath (containing polydimethylsiloxane) set to 298.15 K. The temperature was additionally checked with a calibrated Pt100 resistance thermometer (MPMI 1004/300 Merz) connected to an HP 3458 A multimeter.33 After measuring the resistance of appropriate "solvent" (water, 0.1 M NaCl, 0.01 M IL) at a set temperature, successive aliquots of a stock solution of the surfactant in the same "solvent" were added by a programmable syringe pump (Model 1250, J-KEM Scientific, MO, USA) and the resistance of the solution was measured. Afterwards, the specific conductivities were calculated using previously determined cell constant. The specific conductivities of solutions were corrected by the specific conductivities of "solvent" and plotted as a function of the molar concentration of SDS in the cell. From the slopes before and after the cmc, the values of degree of micelle ionization (a) were estimated.35 The applied method for determination of electrical conductivity of solutions supplies highly reproducible data, thus, the experiments were not repeated. 2. 4. Zeta-Potential Determination Electrophoretic measurements were performed by Litesizer 500 (Anton Paar GmbH, Graz, Austria) in cuvette using Univette accessory. All the measurements were per- Šarac and Bešter-Rogač: The Influence of Ionic Liquids on Micellization ... Acta Chim. Slov. 2020, 67, 977-984 979 formed at 298.15 K, after a 1-minute temperature equilibration period. For each solution, which was prepared directly in cuvette by diluting a stock solution of surfactant (20 mM) with solvent, we performed 3 series (triplicates) of measurements, each containing 120 runs. All the ze-ta-potentials (0 were calculated using Smoluchowski approximation corrected by Henry:36, 37 m eeQ f(tca) (1) where fi is measured electrophoretic mobility, t] the viscosity of the medium, e the relative dielectric constant of the medium, eo the vacuum permittivity, k the inverse of the Debye distance, and a the radius of the micelles. Function f(ka) is for spherical micelles given by: (2) The viscosities and relative permittivities of all the solvents were taken the same as for the water (0.890 mPa s, 78.4). The radius of the micelles a = 1.81 nm for all investigated systems was taken.38 3. Thermodynamics of Micellization According to the two-state mass-action model, the process of micellization of SDS can be described as the equilibrium between negatively charged surfactant monomers (S-), corresponding positive counterions (C+), and micelles (m""-): (3) where n represents the aggregation number and a the degree of micelle ionization. The apparent constant of micellization (KM) expressed by the molalities of corresponding species, can be connected to the Gibbs free energy of micellization (AMG°) by: AH = (HS + HC)+AUH°\^\ 1 A.p.r (5) where the last term represents the change of the amount of surfactant in the micellar form at every addition of surfactant and is connected to Km or AMG°. Hs and Hc were determined from the extrapolation of the lines through the plateaus of the enthalpograms before the cmc, as it is shown on Figure S1b. AMHo and AMGo were the fitting parameters primarily determined at 298.15 K (reference temperature, To). At other temperatures (T), their values were obtained from the Kirchhoff and integrated Gibbs-Helm-holtz equations where AMCp° was treated as a temperature-independent fitting parameter. The model function (right-hand side of equation (5)) was fitted to the ITC experimental data simultaneously at all temperatures (global fitting), using the Levenberg-Marquardt nonlinear regression algorithm.40 A detailed derivation of equation (5) is given in our previous work.39 From the global analysis of ITC data, the best-fit thermodynamic parameters were extracted, i.e., enthalpy, AMHo, Gibbs free energy, AMGo, and heat capacity, AMCp°, of the micellization. Aggregation number (n) was set as the temperature-independent fitting parameter during the global analysis. The values of a were estimated from conductivity measurements at 298.15 K. Since the values of fitting parameters in the ITC data analysis are not correlated strongly to a, it was taken as a temperature-independent parameter. The entropy of micellization was calculated from the Gibbs-Helmholtz equation (7) amC° = -— In Km = -— In n " .2004.02.014 29. A. Pal, S. Chaudhary, Colloids Surf., A 2013, 430, 58-64. DOI: 10.1016/j.colsurfa.2013.04.001 30. S. Javadian, F. Nasiri, A. Heydari, A. Yousefi, A. A. Shahir, J. Phys. Chem. B 2014, 118, 4140-4150. DOI:10.1021/jp5010049 31. M. T. Lam, W. D. Adamson, S. Miao, R. Atkin, G. G. Warr, J. Colloid Interface Sci. 2019, 552, 597-603. DOI:10.1016/j.jcis.2019.05.082 32. S. J. Bryant, C. J. Jafta, R. Atkin, M. Gradzielski, G. G. Warr, J. Colloid Interface Sci. 2019, 540, 515-523. DOI:10.1016/j.jcis.2019.01.048 33. M. Bešter-Rogač, D. Habe, Acta Chim. Slov. 2006, 53, 391395. 34. J. Barthel, F. Feuerlein, R. Neueder, R. Wachter, J. Solution Chem. 1980, 9, 209-219. DOI:10.1007/BF00648327 35. J. P. Marcolongo, M. Mirenda, J. Chem. Educ. 2011, 88, 629633. DOI:10.1007/BF00648327 36. M. A. Morini, P. C. Schulz, Colloid Polym. Sci. 1997, 275, 802-805. DOI:10.1007/s003960050151 37. D. C. Henry, Proc. R. Soc. Lond. A 1931, 133, 106-129. DOI:10.1098/rspa.1931.0133 38. M. Almgren, S. Swarup, J. Phys. Chem. 1982, 86, 4212-4216. DOI:10.1021/j100218a024 39. A. Kroflič, B. Šarac, M. Bešter-Rogač, Langmuir 2012, 28, 10363-10371. DOI:10.1021/la302133q 40. W. H. Press, S. A. Teukolsky, W. T. Vetterling, B. P. Flannery, Numerical Recipes in Fortran 77: The Art of Scientific Computing, Cambridge University Press, New York, 2001, pp. 678 41. P. R. Majhi, A. Blume, Langmuir 2001, 17, 3844-3851. DOI:10.1021/la001660k 42. A. Coello, F. Meijide, M. A. Mougán, E. Núñez Rodríguez, J. Tato Vázquez, J. Chem. Educ. 1995, 72, 73-75. DOI:10.1021/ed072p73 43. S. Hayashi, S. Ikeda, J. Phys. Chem. 1980, 84, 744-751. DOI:10.1021/j100444a011 44. T. M. Perger, M. Bešter-Rogač, J. Colloid Interface Sci. 2007, 313, 288-295. DOI:10.1016/j.jcis.2007.04.043 45. B. Šarac, M. Bešter-Rogač, J. Colloid Interface Sci. 2009, 338, 216-221. DOI:10.1016/j.jcis.2009.06.027 46. K. Beyer, D. Leine, A. Blume, Colloids Surf., B 2006, 49, 31-39. DOI:10.1016/j.colsurft>.2006.02.003 47. R. Zielinski, J. Colloid Interface Sci. 2001, 235, 201-209. DOI:10.1006/jcis.2000.7364 Šarac and Bešter-Rogač: The Influence of Ionic Liquids on Micellization 984 Acta Chim. Slov. 2020, 67, 977-984 48. A. Kroflič, B. Šarac, M. Bešter-Rogač, J. Chem. Thermodyn. 2011, 43, 1557-1563. D01:10.1016/j.jct.2011.05.015 49. Ž. Medoš, M. Bešter-Rogač, Langmuir 2017, 33, 7722-7731. DOI: 10.1021/acs.langmuir.7b01700 50. A. Kroflič, B. Šarac, M. Bešter-Rogač, Acta Chim. Slov. 2012, 59, 564-570. Povzetek Z izotermno titracijsko kalorimetrijo (ITC) smo v temperaturnem območju med 15 in 55 °C proučevali micelizacijo natrijevega dodecil sulfata (SDS) v vodi in vodnih raztopinah treh imidazolijevih ionskih tekočin (IL) z različnimi dolžinami stranskih verig, in sicer 1,3-dimetil imidazolijevega klorida ([CjmimjCl), 1-etil-3-metil imidazolijevega klorida ([C2mim]Cl) ter 1-butil-3-metil imidazolijevega klorida ([C4mim]Cl). Za primerjavo smo proučili tudi micelizacijo SDS v prisotnosti NaCl. Eksperimentalne ITC podatke smo analizirali s pomočjo dvostopenjskega ravnotežnega modela, iz česar smo dobili vrednosti kritične micelne koncentracije (cmc), agregacijskega števila (n) ter vrednosti sprememb standardne toplotne kapacitete (AMCp°), standardne entalpije (AMH°), standardne entropije (AMS°) in Gibbsove proste entalpije (AMG°) za proces micelizacije. Ugotovili smo, da je micelizacija SDS v vseh proučevanih sistemih entropijsko voden proces pri nizkih temperaturah in entalpijsko voden proces pri visokih temperaturah. Predpostavimo lahko, da so z naraščajočim nepolarnim značajem ionskih tekočin interakcije z SDS bolj izražene, s čimer vrednosti AMH° in AMG° postanejo bolj negativne. Da bi pridobili več informacij o naboju micel, smo izvedli meritve električne prevodnosti in zeta-potenciala pri 25 °C. Zaradi močnejše interakcije in možnega vgrajevanja IL v micelno strukturo SDS je naboj micel bolj pozitiven. To se odraža v manj negativnih vrednostih zeta-potenciala v primerjavi s SDS v vodi, kar vodi do višjih vrednostih stopnje ionizacije micel zaradi večjega deleža natrijevih ionov v raztopini. Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Šarac and Bešter-Rogač: The Influence of Ionic Liquids on Micellization ... 51. K. D. Danov, P. A. Kralchevsky, K. P. Ananthapadmanabhan, Adv. Colloid Interface Sci. 2014, 206, 17-45. D0I:10.1016/j.cis.2013.02.001 52. D. Stigter, K. J. Mysels, J. Phys. Chem. 1955, 59, 45-51. D0I:10.1021/j150523a014 DOI: 10.17344/acsi.2019.5760 Acta Chim. Slov. 2020, 67, 985-991 /^.creative v^commons Short communication Arsenic in Sediments, Soil and Plants in a Remediated Area of the Iron Quadrangle, Brazil, and its Accumulation and Biotransformation in Eleocharis geniculata Maria Ángela de B. C. Menezes,1 Ingrid Falnoga,2 Zdenka Šlejkovec,2 Radojko Jacimovic,2 Nilton Couto,3 Eleonora Deschamps4 and Jadran Faganeli5^ 1 Nuclear Technology Development Centre/Brazilian Commission for Nuclear Energy (CDTN/CNEN), Division for Analytical Techniques, Caixa Postal 941, CEP 30161-970, Belo Horizonte, Minas Gerais, Brazil 2 Jožef Stefan Institute, Department of Environmental Sciences, Jamova cesta 39, SI-1000 Ljubljana, Slovenia 3 Fundagao Ezequiel Dias (FUNED), Rua Conde Pereira Carneiro, 80, Gameleira, CEP 30510-010, Belo Horizonte, Minas Gerais, Brazil 4 FUMEC (Universidade Fundagao Mineira de Educagao e Cultura), Rua Cobre, 200, CEP: 30.310-190 Belo Horizonte, Minas Gerais, Brazil 5 National Institute of Biology, Marine Biology Station, Fornace 41. SI-6330 Piran, Slovenia * Corresponding author: E-mail: jadran.faganeli@nib.si, Tel.: +386 5 9232911 Received: 12-04-2019 Abstract Since arsenic (As) exposure is largely due to geochemical contamination, this study focused on the remediated area of Santana do Morro, a district of Santa Bárbara, Minas Gerais, Brazil, which was previously contaminated with As due to gold mining. Total As concentrations in sediment, soil and plants were determined, next to As species, anionic arsenic compounds As(III), As(V), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), in plants samples. Total As concentrations in soil and sediments were slightly elevated (16-18 |ig g-1) and most of the plants contained low levels of As (< 1 |g g-1). The exception was a native plant Eleocharis geniculata (L.) which contained elevated levels of As (4 |g g-1). The exposure of this plant to As under controlled conditions (hydroponics) indicated its possible tolerance to elevated As levels and suggesting its potential use in phytomonitoring of As-contaminated sites. This plant is able to metabolize arsenate to arsenite and contained MMA and DMA, both in its natural habitat and under controlled conditions. Keywords: Arsenic species; soil; sediments; plants; Cyperacea; Iron Quadrangle 1. Introduction The presence of arsenic (As) in soil, water, plants and food presents a potential risk to human health1,2 due to its toxicity. Due to its potential risk, As is considered as one of the most toxic elements to human health according to the ATSDR Substance Priority List3 and has received special attention worldwide.4 The toxicity and bioavailability of As in the environment depend not only on total concentration, but also on chemical species. In soil, As is relatively immobilized due to insoluble complexes with iron and aluminum oxides.5 Generally, the toxicity decreases in the order As3+ inorganic compounds > As5+ inorganic compounds > As3+ organic compounds > As5+ organic compounds. The order of their phytotoxicity is less clear and may be plant species dependent. Finnegan and Chen6 reported that "no As form appears to be consistently most phytotoxic". In Brazil, the most significant presence of As is restricted to the state of Minas Gerais, specifically in the Iron Menezes et al.: Arsenic in Sediments, Soil and Plants 986 Acta Chim. Slov. 2020, 67, 985-991 Quadrangle region and in the city of Paracatu. The Iron Quadrangle is a known gold-producing area, where As commonly occurs as arsenopyrite (FeAsS) and as a trace element in pyrite associated with gold mineralization.7 In this region, there are many active and abandoned gold mines where the As:Au ratios range from 300 to 3000. In this region, As is also present due to past anthropogenic activities, when it was discharged into drainage systems and stored in tailing piles on the banks of several local rivers.1,8 Large dams have recently been built to store the tailings and the effluents are treated in compliance with environmental regulations since most of the mining and smelting operations, apart from some small-scale mining operations (garimpo), are carried out by large companies with modern infrastructures and facilities.8 The spring water, previously used by local inhabitants, was contaminated with As prevalently bonded onto soil particulates. In 2007, water treatment plants were constructed and water became potable.9 Various studies related to the Iron Quadrangle have described the environmental impact of As pollution from several old gold mines on stream sediments, surface water and run-off water. The impact of As pollution on the population of the municipalities of Santa Bárbara and Nova Lima1,8,10,11 has also been detected in the frame of ARSEN-EX project.12 Results showed that As in soil ranges from between 13-467 mg kg-1. In this study, levels of As and its species were determined in soil, sediments and some plants from Santana do Morro, district of Santa Bárbara, to assess the level of As contamination in this remediated area. The main objective of the study was to evaluate if certain plants are able to methylate As5+ and if they can be potential accumulators of As and used in phytomonitoring of As-contaminated sites. 2. Materials and Methods 2. 1. Study Area Samples were collected in the surroundings of the small village of Santana do Morro (Water spring coordinates: 20°1'15"S 43°28'79"W), located in the district of Santa Bárbara, Minas Gerais, Brazil (Fig. 1). This village has approximately 200 inhabitants and lacks basic services. Until 2006, the water supply originated from the local spring contaminated with As (3.3-21.5 ^g L-1).8 From 2006 onwards, the water has been treated in the water treatment plant constructed during the ARSENEX project and from there it is distributed to several houses and the As concentration varies between <0.08 and 0.64 ^g L-1.13 The climatological, hydrological and geological features of the study area were described by Almeida et al.7 Figure 1. Sampling locations in Santana do Morro (Santa Bárbara district, Minas Gerais, Brazil) (https://earth.google.com/web/@-20.02021163, 43.47656815,739.64086466a,578.07324732d,35y,0h,0t,0r) Menezes et al.: Arsenic in Sediments, Soil and Plants ... Acta Chim. Slov. 2020, 67, 985-991 987 2. 2. Collection and Preparation of Soil and Sediment Soil samples were taken from surface to the depth of 20 cm at 14 locations (Fig. 1) in the vicinity where plants were collected. From each location approximately 100 g of soil was sampled and stored in plastic bags for further analysis. Sediment samples were collected from 14 locations at the edge of the spring pool (Fig. 1) and were also stored in plastic bags. Soil and sediment samples were air-dried, homogenized and sieved to five granulometric fractions. The finest-grained fraction (<0.06 mm) was analyzed after rehomogenization. 2. 3. Collection and Preparation of Plants A total of 14 species (Table 1) were collected in the vicinity of the spring pool in Santana do Morro (Fig. 1). One part of the plants sampled was set aside for taxonom-ical identification and the other part was used for As determination and speciation. The samples for plant identification were stored in paper and pressed. For chemical analysis, all plants collected were first vigorously washed with tap water, immersed in deionized water for some minutes, air dried and weighed. Plant samples were successively frozen, freeze-dried and weighed again. Dried plants were ground to a fine powder in a laboratory mill (Knife Mill GRINDOMIX GM 200, Retsch) and analyzed for As as described below. 2. 4. As Exposure Study of Eleocharis Geniculata After identifying the plant which contained As in the environmental survey, a Cyperacea Eleocharis geniculata (L.) was exposed to As in a hydroponic system according to a procedure described by Moreno-Jimenez at al.14 Seedlings from the field were grown in a vessel with nutrient solution with commercial fertilizers prepared according to instructions (SUPERthrive®, a highly concentrated non-toxic vitamin solution suited for hydro-seeding, hydroponics and foliar spraying) without As. After producing several seedlings in As-free conditions, these seedlings were transferred to a container with fresh commercial nutrient solution and 50 ^g of As(V) per container. Ten plastic containers (capacity of 2 L each) were prepared, each one containing 3 seedlings. After one month of growth, the plants were cleaned by washing them with tap water, followed by immersion in deionized water for several minutes. The roots were separated from the shoots (leaves and stems), compounding two samples, one of shoots (22.97 g dry weight) and one of roots (3.43 g dry weight). This material was frozen, freeze-dried, homogenized and analyzed for total As and its species as described below. 2. 5. Determination of As and its Species Determination of total As in soil, sediment and plants The fc0-INAA method (Instrumental Neutron Activation Analysis) was applied to determine total As concentrations. After being weighed in polyethylene vials suitable for neutron irradiation, the samples were co-irradiated in a TRIGA Mark II reactor together with flux monitor disks of Al - 0.1 % Au from the Central Bureau for Nuclear Measurements, Geel, Belgium. After a suitable cooling time the gamma spectra were obtained in a HPGe gamma counting system with 40% efficiency15,16 and evaluated by HyperLab software.17 Concentrations were calculated using the Kayzero software.18 Standard Reference Materials from the National Research Centre for CRM (China) (GBW 07604, Poplar Leaves, 0.4 ± 0.1 mg kg-1, certified 0.37 ± 0.06 mg kg-1) and the International Atomic Energy Agency (IAEA-SOIL-7, 13 ± 1 mg kg-1, certified 13.4 ± 0.85 mg kg-1) were analyzed together with our samples to verify the accuracy of the fc0-method. Detection limits were in a range of few ng g-1. Determination of As species in plant samples Powdered plant samples were extracted with 25 mL of a Mil-li-Q water/methanol mixture (1:1) at 25 °C for 3 h. After extraction the samples were centrifuged, decanted and re-extracted using the same conditions. Both extracts were joined and evaporated to dryness using a rotary evaporator (45 °C, ~ 30 min). Dry residue was taken up into 2.00 mL of Milli-Q water, filtered through a 0.45 ^m membrane PVDF syringe filter and kept frozen until analysis. An HPLC-HG-AFS (high performance liquid chromatogra-phy-hydride generation - atomic fluorescence spectroscopy) system19 was used for determination of the anionic arsenic compounds As(III), As(V), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in extracts. An anion exchange column (Hamilton PRP X-100, with 15 mmol L-1 KH2PO4, pH 6.1) was used to separate compounds prior to hydride generation (4.4 mol L-1 HCl, 3.0 mL min-1 and 1.5% NaBH4 in 0.1% NaOH, 3 mL min-1). An Excalibur (PS Analytical, Kent, UK) AFS detector was used for the detection of volatile arsenic hydrides. Detection limits were 1 ^g kg-1 for MMA and As(III) and 2 ^g kg-1 for DMA and As(V). 3. Results and Discussion 3. 1. Total As in Soil and Sediments The mean As concentrations in soil (16 mg kg-1) and sediment (18 mg kg-1) in the vicinity of the spring water pool showed slight contamination. According to Brazilian legislation regarding soil20 they were close to prevention values (15 mg kg-1) but below the levels for residential use (55 mg kg-1). However, the concentrations were higher than the average values for world soils (5 mg kg-1) but comparable to average values for world sediments (2-22 Menezes et al.: Arsenic in Sediments, Soil and Plants ... 988 Acta Chim. Slov. 2020, 67, 985-991 mg kg-1).21 According to Adriano22, As concentrations typically vary from below 10 mg kg-1 in uncontaminated to as high as 30000 mg kg-1 in contaminated world soils. It appears therefore that mining activities did not greatly contribute to As levels in local soil and sediments.23 3. 2. Total As in Plants The taxonomic determination of the dominant species (Table 1) in the vicinity of the spring water was performed to characterize the vegetation and identify species that might be able to accumulate or exclude As. Only E. geniculata,24,25 a wetland annual or short-lived perennial plant commonly known as spike rush, was identified as a native species. Most of the plants investigated contained no detectable As concentrations, as may be expected for unexposed plants.22,26 Only E. geniculata contained higher levels of As, i.e. 4 mg kg-1, suggesting that this species is likely tolerant or able to accumulate As. The rather low As concentrations in plant can be associated with rather low As levels in the corresponding soil (16 ± 1 mg kg-1) and sediment (18 ± 1 mg kg-1). E. geniculata is known to be metal-tolerant and its metal accumulation ability was previously confirmed for lead27 but there are no data for As. Cyperaceae family is widely distributed around the world and some of the species of Eleocharis genus were suggested to be of potential use in aquatic weed management and pollution abatement28. For example, Eleocharis sp., naturally colonizing abandoned copper - tailings ponds at the Rakha mines in India, was found to accumulate copper up to 1493 mg kg-1 dry weight, with greater amounts in roots than in shoots.29 The potential for accumulation of various metals, i.e. Cu, Zn, As, Pb, Cd, and phytoremediation was reported for Eleocharis acicularis in laboratories and field experi-ments30,31 and for Eleocharis equisetina in an abandoned acid mine tailing pond in NE Australia.32 Experiments with As performed with Eleocharis macrostachya revealed that a part of As can be removed from water by rhizofiltra-tion.33 3. 3. As Species in Native and Cultivated Eleocharis Geniculata Once it was verified that E. geniculata can accumulate more As than other plants, a systematic study of As uptake was performed. Table 2 shows the results of total As and its species determined in roots and shoots of E. geniculata collected in its natural habitat at the spring water pool and in samples after 30 days of cultivation in As enriched nutrient solution in a hydroponic system. Extracta-bility of As from E. geniculata grown in the field or after hydroponic exposure to As(V) was rather low, 30% from roots and approximately 60% from shoots, as is often in plant material.4 Samples of E.geniculata from the field contained more total As in the roots than in the shoots. The root:-shoot ratio was 16.4, which is comparable to data from the literature regarding other plants from the same family. For example, E. equisetina contained from 10-146 ^g kg-1 As in roots and from 3.3-17.3 ^g kg-1 As in shoots32 and E. macrostachya contained 17-47 times more As in roots compared to shoots.33 This similarity indicates that all three plant species are able to exclude As from above-ground tissues. Poor translocation might be the result of sequestration of As into vacuoles of the root cells, helping the plant to alleviate the potentially toxic effects of As.34 However, such comparisons could be misleading due to differences in exposure conditions. Sequestration into vacuoles of the root cells during exposure to low levels is likely more efficient than at high levels of As exposure. As speciation in the E.geniculata samples highlighted the presence of mainly As(III) and small amounts of As(V) and MMA in roots while shoots from the pilot parallel ex- Table 1. Total arsenic in freeze dried plants collected around the spring water pool in Santana do Morro Species Family As (mg kg-1) Baccharis retusa DC. Asteraceae < 1 Baccharis calvescens DC. Asteraceae < 1 Cyrtocymura scorpioides (Lam.) H. Rob. Asteraceae < 1 Jacquemontia rufa (Choisy) Hallier f. Convolvulaceae < 1 Eleocharis geniculata (L.) Roem. & Schult. Cyperaceae 4 Rhynchospora marisculus Lindl. & Nees Cyperaceae < 1 Hyptis lanceolata Poir. Lamiaceae < 1 Hyptis suaveolens Poit. Lamiaceae < 1 Triunfetta semitriloba Jacg. Malvaceae < 1 Rhynchanthera grandiflora (Aubl.) DC. Melastomataceae < 1 Trembleya parviflora (D. Don) Cogn. Melastomataceae < 1 Ludwigia lagunae (Morong) H. Hara Onagraceae < 1 Melinis minutiflora P. Beauv. Poaceae < 1 Borreria capitata (Ruiz & Pav.) DC. Rubiaceae < 1 Menezes et al.: Arsenic in Sediments, Soil and Plants ... Acta Chim. Slov. 2020, 67, 985-991 989 Table 2. Arsenic and its species in Eleocharis geniculata from its natural habitat and cultivated in a hydroponic system with the addition of As(V) Sample Part of the Total As As(III) DMA MMA As(V) plants (mg kg-1) (mg kg-1) (mg kg-1) (mg kg-1) (mg kg-1) From natural Roots 4.6 0.79 < 0.001 0.005 0.010 ± 0.2 ± 0.03 ± 0.001 ± 0.001 habitat (n=5) Shoots 0.28 Na Na Na Na ± 0.05 From hydroponic Roots 11.5 2.67 0.09 0.05 1.64 system ± 0.4 ± 0.08 ± 0.01 ± 0.01 ± 0.11 (n=5) Shoots 25 4.55 0.39 0.06 9.62 ± 1 ± 0.16 ± 0.06 ± 0.01 ± 0.87 Na - not analyzed due to insufficient mass periment also contained low but detectable amounts of DMA (data not shown). It is known that As(V) is readily reduced to As(III) by plants, while biomethylation issues are less clear35 and attributed to microorganisms, in particular bacteria living in soil.35 In the hydroponic system, the seedling was subjected to a total of 500 ^g of As(V) for one month and the growing plants took up all the available As. In the hydroponic system (absence of soil), the roots contained less As than shoots, which is contradictory to natural conditions and possibly related to different exposure conditions35 including additional exposure to higher concentrations in a shorter time. In such conditions the plant was able to translocate As from roots to shoots very efficiently. It is believed that in the tolerant plants a much higher amount of assimilated As is transported to the shoots than in nontolerant plants.36 Concerning the species studied, the results also showed the presence of As(III), pointing out the ability of the plant to metabolize As(V) to As(III) as previously observed by Finnegan and Chen6 and to produce MMA and DMA. All four arsenic species were identified in roots and shoots. In the same plants growing in the field we identified three arsenic species, without DMA, in roots only. Involvement of bacterial methylation of As could not be excluded since we did not use axenic conditions. Arse-nosugars and other arsenic species were not detected in any of the samples. 4. Conclusions Total As concentrations in soil and sediments in a remediated area in Santana do Morro, a district of Santa Bárbara, Minas Gerais, Brazil, were slightly elevated (1618 ^g g-1) but most plants contained low levels of As (< 1 ^g g-1). The analysis of vegetation, mostly consisting of alien species, showed that only E. geniculata, a native plant, contained elevated levels of As. E.geniculata exposed to As in its habitat and to inorganic As (As5+) under controlled conditions (hydroponic system) was able to accumulate As. As from the treatment solution was removed completely suggesting its potential use in phytomonitoring and removing As from contaminated soils37 and further exploration is suggested. This plant was able to metabolize As(V) to As(III) and produced minor amounts of MMA and DMA in both situations. Acknowledgements The authors wish to thank Prof. Alexandre Salino and colleagues, the Botanic Department, Institute for Biological Sciences, Federal University of Minas Gerais (UFMG) for floristic analysis. This study was financially supported by CNPq (National Counsel of Technological and Scientific Development), Process n. 490059/2009-0 (Edital CNPq N 015/2009) and by the Slovenian Research Agency (ARRS) through Slovenian-Brazilian scientific cooperation (BI-BR/10-12-002). 5. References 1. E. Smith, R. Naidu, A. M. Alston, Adv. Agron. 1998, 64, 149195. D01:10.1016/S0065-2113(08)60504-0 2. A. L. Juhasz, E. Smith, J. Weber, M. Rees, A. Rofe, T. Kuchel, L. Sansom, R. Naidu, Chemosphere 2008, 71, 1963-1969. DOI: 10.1016/j.chemosphere.2007.12.021 3. ATSDR, Agency for Toxic Substances & Disease Registry, http://www.atsdr.cdc.gov/spl/, 2013. 4. A. Sarkar, B. Paul, Chemosphere 2016, 158, 37-49. D0I:10.1016/j.chemosphere.2016.05.043 5. I. Arcon, H. J. Van Elteren, A. Glass, A. Kodre, Z. Slejkovec, X-Ray Spectrom. 2005, 34, 435-438. D0I:10.1002/xrs.857 6. P. M. Finnegan, W. Chen, Front. Physiol. 2012, 3, 182, doi. org./10.3389/fphys.2012.00182. 7. K. Almeida, J. Matschullat, J. Mello, I. Meneses, Z. Viola, in: E. Deschamps, J. Matschullat (Ed.): Arsenic: Natural and Anthropogenic, CRC Press, Boca Raton, 2011, pp. 81-90. 8. O. Vasconcelos, S. Oberda, E. Deschamps, J. Matschullat, in: Menezes et al.: Arsenic in Sediments, Soil and Plants ... 990 Acta Chim. Slov. 2020, 67, 985-991 E. Deschamps, J. Matschullat (Ed.): Arsenic: Natural and Anthropogenic, CRC Press, Boca Raton, 2011, pp. 119-126. DOI:10.1201/b10772-15 9. E. Deschamps, N. Assuncao, in: E. Deschamps, J. Matschullat (Ed.): Arsenic: Natural and Anthropogenic, CRC Press, Boca Raton, 2011, pp. 183-192. DOI:10.1201/b10772-21 10. L. V. Cruz, Avaliaçâo Geoquímica ambiental da Estaçâo Ecológica do Tripuí e adjacências, sudeste do Quadrilátero Ferrífero, MG. Universidade Federal de Ouro Preto, M.Sc. Thesis, 2002. 11. J. Matschullat, R. P. Borba, E. Deschamps, B. R. Figueiredo, T. Gabrio, M. Schwenk, Appl. Geochem. 2000, 15, 181-190. DOI:10.1016/S0883-2927(99)00039-6 12. N. Couto, S. Mattos, J. Matschullat, in: E. Deschamps, J. Matschullat (Ed.): Arsenic: Natural and Anthropogenic, CRC Press, Boca Raton, 2011, pp. 153-167. 13. C. A. Ferreira, H. E. L. Palmieri, M. A. B. C. Menezes, L. M. L. A. Auler, Amer. Jour. Water Res. 2017, 5, 29-40. 14. E. Moreno-Jimenez, E. Esteban, T. Fresno, C. Lopez de Egea, J. M. Penalosa, Chemosphere 2010, 79, 513-517. DOI:10.1016/S0883-2927(99)00039-6 15. R. Jacimovic, V. Stibilj, L. Benedik, B. Smodiš, Jour. Radioanal. Nucl. Chem. 2003, 257, 545 - 549. DOI:10.1023/A:1025484313986 16. M. A. B. C. Menezes, R. Jacimovic, Jour. Radioanal. Nucl. Chem. 2008, 278, 607-611. DOI:10.1007/s10967-008-1201-x 17. HyperLab. System. Installation and Quick Start Guide, HyperLabs Software, Budapest, Hungary, 2009. 18. Kayzero for Windows® User's Manual, for reactor neutron activation analysis (NAA) using the k0 standardisation method, Ver. 2.42, 2008. 19. Z. Šlejkovec, J. T van Elteren, H. J. Glass, Z. Jeran, R. Jaci-movic, Int. Jour. Environ. Anal. Chem. 2010, 90, 784-796. DOI: 10.1080/03067310902977542 20. Brasil. Conselho Nacional de Meio Ambiente - CONAMA, Resoluçâo 420/2009, de 28 de dezembro de 2009. "Dispôe sobre critérios e valores orientadores de qualidade do solo quanto à presença de substâncias químicas e estabelece dire-trizes para o gerenciamento ambiental de áreas contaminadas por essas substâncias em decorrência de atividades antrópi-cas", Brasília. 2009. 21. C. Reimann, P. de Caritat, Chemical elements in the envi- ronment Factsheets for the geochemists and environmental scientists, Springer, New York, 1998. 22. D. C. Adriano, Trace Elements in the Terrestrial Environment. Springer, New York, 2001. DOI: 10.1007/978-0-387-21510-5 23. E. Deschamps, J. Meillo, J. Matschullat, in: E. Deschamps, J. Matschullat (Ed.): Arsenic: Natural and Anthropogenic, CRC Press, Boca Raton, 2011, pp. 127-139. D0I:10.1201/b10772 24. M. L. L. Martins, R. M. Carvalho-Okano, M. Luceño, Acta Bot. Bras. 1999, 13, 187-222. D0I:10.1590/S0102-33061999000200008 25. A. S. B. Gil, C. P. O. Bove, Arq. Mus. Nac. Rio de Janeiro 2004, 62, 131-150. 26. R. Eisler, Eisler's Encyclopedia of Environmentally Hazardous Priority Chemicals, Elsevier, 9999Amsterdam, 2007. 27. J. Yang, Z. Ye, Environ. Sci. Pollut. Res. 2015, 22, 1931-1939. D0I:10.1007/s11356-014-3610-4 28. M. S. Gonzales-Elizondo, P. M. Peterson, Taxon 1997, 46, 433-449. DOI: 10.2307/1224386 29. M. Das, S. K. Maiti, Land Contam. Reclam. 2008, 16, 135-153. DOI: 10.2462/09670513.691 30. M, Sakahibara, Y. Ohmori, N. T. H. Sano, K. Sera, Clean - Soil, Water 2011, 39, 735-741. D0I:10.1002/clen.201000488 31. A. G. Nurfitiri, S. Masayuki, S. Koichiro, Environ. Sci. Ind. Jour. 2017, 13, 131. 32. B. A. Lottermoser, P. A. Ashley, Environ. Pollut. 2011, 159, 3028-3035. D0I:10.1016/j.envpol.2011.04.014 33. M. A. Olmos-Márquez, M. T. Alarcón-Herrera, I. R. Martín-Domínguez, Environ. Sci. Pollut. Res. 2012, 19, 763-771. D0I:10.1007/s11356-011-0598-x 34. A. K. Shankar, C. Cervantes, H. Loza-Tavera, S. Avudainayag-am, Sci. Total Environ. 2005, 31, 739-753. D0I:10.1016/j.envint.2005.02.003 35. P. Wang, G. Sun, Y. S. Jia, A. A. Meharg, Y. Zhu, Jour. Environ. Sci. 2014, 26, 371-381. D0I:10.1016/S1001-0742(13)60432-5 36. N. Singh, L. Q. Ma, in: N. Willey (ed.): Phytoremediation: Methods and Reviews, Biotechnology, Humana Press, Toto-wa, 2007. 37. L. Q. Ma, K. M. Komar, C. Tu, W. Zhang, Y. Cai, E. D. Kennel-ley, Nature, 2001, 409, 579. D01:10.1038/35054664 Menezes et al.: Arsenic in Sediments, Soil and Plants ... Acta Chim. Slov. 2020, 67, 985- 991 991 Povzetek V področju kontaminiranem z arzenom (As) Santana do Morro (Santa Bárbara, Minas Gerais, Brazilija), kot posledica rudarjenja zlata, smo določili koncentracije celotnega As v sedimentu, prsti in rastlinah. V rastlinah smo analizirali tudi zvrsti As: As(III), As(V), metilarzonsko (MMA) in dimetilarzinsko kislino (DMA). Koncentracije As v prsti in sedimentu so bile le rahlo povečane (16-18 |ig g-1) in večina rastlin je vsebovala nizke koncentracije As (<1 |ig g-1). Izjema je bila avtohtona rastlina Eleocharisgeniculata (L.) s povečanimi koncentracijami As (4 |g g-1). Poskusi v kontroliranih pogojih (hidroponika) kažejo na njeno odpornost na povečane koncentracije As in možnost uporabe v fitomonitoringu in biore-mediaciji kontaminiranih področij z As. Rastlina metabolizira arzenat v arzenit in vsebuje MMA in DMA v naravnih in laboratorijskih pogojih. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Menezes et al.: Arsenic in Sediments, Soil and Plants ... Acta Chim. Slov. 2020, 67, (3), Supplement S79 DRUŠTVENE VESTI IN DRUGE AKTIVNOSTI SOCIETY NEWS, ANNOUNCEMENTS, ACTIVITIES Vsebina Navodila za avtorje.................................................................................................................. S80 Contents Instructions for authors.......................................................................................................... S80 Društvene vesti in druge aktivnosti S80 Acta Chim. Slov. 2020, 67, (3), Supplement S79 Acta Chimica Slovenica Author Guidelines Submissions Submission to ACSi is made with the implicit understanding that neither the manuscript nor the essence of its content has been published in whole or in part and that it is not being considered for publication elsewhere. All the listed authors should have agreed on the content and the corresponding (submitting) author is responsible for having ensured that this agreement has been reached. The acceptance of an article is based entirely on its scientific merit, as judged by peer review. There are no page charges for publishing articles in ACSi. The authors are asked to read the Author Guidelines carefully to gain an overview and assess if their manuscript is suitable for ACSi. 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When cited in the text, reference numbers should be superscripted, following punctuation marks. It is the sole responsibility of authors to cite articles that have been submitted to a journal or were in print at the time of submission to ACSi. Formatting of references to published work should follow the journal style; please also consult a recent issue: 1. J. W. Smith, A. G. White, Acta Chim. Slov. 2008, 55, 1055-1059. 2. M. F. Kemmere, T. F. Keurentjes, in: S. P. Nunes, K. V. Peinemann (Ed.): Membrane Technology in the Chemical Industry, Wiley-VCH, Weinheim, Germany, 2008, pp. 229-255. 3. J. Levec, Arrangement and process for oxidizing an aqueous medium, US Patent Number 5,928,521, date of patent July 27, 1999. 4. L. A. Bursill, J. M. Thomas, in: R. Sersale, C. Coll e I a, R. Aiell o (Eds.), Recent Progress Report and Discussions: 5th International Zeo I ite Conference, Naples, Italy, 1980, Gianini, Naples, 1981, pp. 25-30. 5. J. Szegezdi, F. 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When reporting experiments on animals, authors should indicate whether the institutional and national guide for the care and use of laboratory animals was followed. • To avoid conflict of interest between authors and referees we expect that not more than one referee is from the same country as the corresponding au-thor(s), however, not from the same institution. • Contributions authored by Slovenian scientists are evaluated by non-Slovenian referees. • Papers describing microwave-assisted reactions performed in domestic microwave ovens are not considered for publication in Acta Chimica Slovenica. • Manuscripts that are not prepared and submitted in accord with the instructions for authors are not considered for publication. Appendices Authors are encouraged to make use of supporting information for publication, which is supplementary material (appendices) that is submitted at the same time as the manuscript. 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Graphical abstract pictures are printed in size 6.5 x 4 cm (hence minimal resolution of 770 x 470 pixels). Cover picture is printed in size 11 x 9.5 cm (hence minimal resolution of 1300 x 1130 pixels) Authors are encouraged to submit illustrations as candidates for the journal Cover Picture*. The illustration must be related to the subject matter of the paper. Usually both proposed cover picture and graphical abstract are the same, but authors may provide different pictures as well. * The authors will be asked to contribute to the costs of the cover picture production. Statement of novelty Statement of novelty is provided in a Word file and submitted as a supplementary file in step 4 of submission process. Authors should in no more than 100 words emphasize the scientific novelty of the presented research. Do not repeat for this purpose the content of your abstract. List of suggested reviewers List of suggested reviewers is a Word file submitted as a supplementary file in step 4 of submission process. Authors should propose the names, full affiliation (department, institution, city and country) and e-mail addresses of three potential referees. Field of expertise and at least two references relevant to the scientific field of the submitted manuscript must be provided for each of the suggested reviewers. The referees should be knowledgeable about the subject but have no close connection with any of the authors. In addition, referees should be from institutions other than (and preferably countries other than) those of any of the authors. How to Submit Users registered in the role of author can start submission by choosing USER HOME link on the top of the page, then choosing the role of the Author and follow the relevant link for starting the submission process. Prior to submission we strongly recommend that you familiarize yourself with the ACSi style by browsing the journal, particularly if you have not submitted to the ACSi before or recently. Društvene vesti in druge aktivnosti Acta Chim. Slov. 2020, 67, (3), Supplement S79 Correspondence All correspondence with the ACSi editor regarding the paper goes through this web site and emails. Emails are sent and recorded in the web site database. In the correspondence with the editorial office please provide ID number of your manuscript. All emails you receive from the system contain relevant links. Please do not answer the emails directly but use the embedded links in the emails for carrying out relevant actions. Alternatively, you can carry out all the actions and correspondence through the online system by logging in and selecting relevant options. 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The submission files are in the correct format: manuscript is created in MS Word but will be submitted in PDF (for reviewers) as well as in original MS Word format (as a supplementary file for technical editing); diagrams and graphs are created in Excel and saved in one of the file formats: TIFF, EPS or JPG; illustrations are also saved in one of these formats. The preferred position of graphic files in a document is to embed them close to the place where they are mentioned in the text (See Author guidelines for details). 4. The manuscript has been examined for spelling and grammar (spell checked). 5. The title (maximum 150 characters) briefly explains the contents of the manuscript. 6. Full names (first and last) of all authors together with the affiliation address are provided. Name of author(s) denoted as the corresponding author(s), together with their e-mail address, full postal address and telephone/fax numbers are given. 7. 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Slov. 2020, 67, (3), Supplement S79 Koristni naslovi Slovensko kemijsko druStva stovwifan chwnicaf society Slovensko kemijsko društvo www.chem-soc.si e-mail: chem.soc@ki.si Wessex Institute of Technology www.wessex .ac.uk SETAC www.setac.org European Water Association http://www.ewa-online.eu/ European Science Foundation www. esf .org O EFCE European Federation of Chemical Engineering https://efce.info/ International Union of Pure and Applied Chemistry https://iupac.org/ Novice europske zveze kemijskih društev EuChemS naj'dete na: t&r EuChemS Brussels News Updates i i.irupr'.sn, ChoiniLdl Snarly http://www.euchems.eu/newsletters/ Društvene vesti in druge aktivnosti Sistemi za čisto in ultračisto vodo Kvaliteta vode 1 do 3* *v skladu s standardom ISO 3696 in ustreznimi ASTM ter CLSI Donau Lab d.o.o., Ljubljana Tbilisijska 85 SI-1000 Ljubljana www.donaulab.si office-si@donaulab.com ActaChimica Slovenica Acta ChimicaSlovenica The micellization process of sodium dodecyl sulfate in the presence of three imidazolium based ionic liquids (ILs) with different side-chain length is investigated by calorimetric, conductivity and zeta-potential measurements. The impact of ILs on critical micelle concentration, thermodynamics of micellization and charge of the micelles is discussed in terms of hydrophobic and electrostatic interactions between the species in the solution. (see page 977) ActaChimicaSlc ActaQiumcaSk Slovenica Year 2020, Vol. 67, No. 3 Acta ChimicaSlc ActaC himica Slovenica j 64/2017 Rota Hwcsiial Poqs faor Dcicnniuiior, a i Coml Siraiis fof Ftaul Muussf fmpuiiiiei iaihe 5)Hhe& ofDmi.SutaiAtti Thi lot«ik Kef. DntkpMMtfNMd IW « SkAd^iid^Su^^bilVriKbdKDeiMiMdlfg'ki Acta ChimicaSlc ActaChimic&Slc Slovenica 1 eta Roct: flnlBBiahial [klniiin*i)ii ill Ea*ik|wn Otfelc liw NitW >mnpji&dev MM Cartw har buiw I ?ie» IJkwwc HaeM^arCunntm Swiwb. rnnmiK ad DXVItirJm Slufc I A NkltI BkJ k1:th. ¡('yaetaKiul BkJ «1 IkKr^raj (flit :rLffirftN\ari IKRcjBcsiGccpTiiiai Onpi CKiwKriubM of Sloicti a Mc UiojSliMc Iwoft ad Uoiuul fnrpiMitti A Imp :Mkm rh