EDITOR-IN-CHIEF EDITORIAL BOARD ADVISORY EDITORIAL BOARD ASSOCIATE EDITORS 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 Matjaž Kristl, University of Maribor, Slovenia Franc Perdih, University of Ljubljana, Slovenia Aleš Ručigaj, University of Ljubljana, Slovenia Helena Prosen, University of Ljubljana, Slovenia Irena Vovk, National Institute of Chemistry, Slovenia ADMINISTRATIVE ASSISTANT Marjana Gantar Albreht, National Institute of Chemistry, Slovenia Eva Mihalinec, Slovenian Chemical society, Slovenia 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. 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Articles in this journal are published under the   Creative Commons Attribution 4.0 International License – Graphical Contents Graphical Contents ActaChimicaSlovenica ActaChimicaSlovenica SlovenicaActaChimica Year 2022, Vol. 69, No. 3 507–518 Analytical chemistry Development of Green Differential Pulse Voltammetric Strategy for the Determination of Alfuzosin Hydrochloride at Pencil Graphite and Modified Carbon Paste Electrodes Youstina Mekhail Metias, Emad Mohamed Hussien, Mervat Mohamed Hosny, and Magda Mohamed Ayad 519–525 Organic chemistry Synthesis, Characterization, and Investigation of Mesomorphic Properties of a new 2,5-Bis-(4- alkanoyloxybenzylidene)cyclopentan-1-one Abdullah Hussein Kshash, Omar Jamal Mahdi Al-Asafi and Hanaa Kaen Salih Scientific pAper 526–535 Organic chemistry Synthesis, Molecular Docking Studies and ADME Prediction of Some new Albendazole Derivatives as α-Glucosidase Inhibitors Sevil Şenkardeş, Necla Kulabaş and Ş. Güniz Küçükgüzel Graphical Contents 552–563 chemical, biochemical and environmental engineering Decolorization of Direct Black 22 by Photo Fenton like Method Using UV Light and Zeolite Modified Zinc Ferrite: Kinetics and Thermodynamics Serap Findik 536–551 chemical, biochemical and environmental engineering nickel Removing by Electrocoagulation of ni(II)-nH3-Co2-So2-H2o System. Kinetics, Isothermal, Mechanism and Estimated Cost of operation Armando Rojas Vargas, Margarita Penedo Medina, Alba González Vives, Noureddine Barka and Aymara Ricardo Riverón 564–570 Biomedical applications The Predictive Value of oxidative Stress Index in Patients with Confirmed SARS-CoV-2 Infection Joško Osredkar, Sara Puck, Milica Lukić, Teja Fabjan, Elizabeta Božnar Alič, Kristina Kumer, Maria Martin Rodriguez and Matjaž Jereb 571–583 Organic chemistry Carvacrol Derivatives as Antifungal Agents: Synthesis, Antimicrobial Activity and in Silico Studies on Carvacryl Esters Jelena Lazarević, Ana Marković, Andrija Šmelcerović, Gordana Stojanović, Pierangela Ciuffreda and Enzo Santaniello 584–595 Organic chemistry N-(9,10-Dioxo-9,10-dihydroanthracen-1(2)-yl)-2- (R-thio) Acetamides: Synthesis, Antioxidant and Antiplatelet Activity Maryna Stasevych, Viktor Zvarych, Olena Yaremkevych, Mykhaylo Vovk, Alla Vaskevych, Tetiana Halenova and Olexii Savchuk Graphical Contents 604–618 inorganic chemistry Ternary Transition Metal Complexes with an Azo- Imine Ligand and 2,2’-Bipyridine: Characterization, Computational Calculations, and Acetylcholinesterase Inhibition Activities Kerim Serbest, Turan Dural, Demet Kızıl, Mustafa Emirik, Ali Zengin and Barbaros Dinçer 596–603 inorganic chemistry A new Zn(II) Two-dimensional Coordination Polymer: Synthesis, Structure, Highly Efficient Fluorescence and DFT Study Fen-Fang Li 619–628 Organic chemistry Synthesis, Antimicrobial and Molecular Docking Studies of Some new Derivatives of 2,3-Dihydroquinazolin- 4(1H)-one Karim Zahmatkesh, Karim Akbari Dilmaghani and Yasin Sarveahrabi 629–637 Organic chemistry Synthesis, Crystal Structures and Urease Inhibition of Mononuclear Copper(II) and nickel(II) Complexes with Schiff Base Ligands Jian Jiang, Peng Liang, Huiyuan Yu and Zhonglu You 638–646 Analytical chemistry Electroanalytical Determination of Ziram by Differential Pulse Voltammetry with Reduced Graphene oxide/Gold nanoparticles Modified Glassy Carbon Electrode Nazife Aslan, Sema Bilge Ocak and Uğur Gökmen Graphical Contents 657–664 chemical, biochemical and environmental engineering Industrial Wastewater as a Source of External organic Carbon for the Biological nutrient Removal Bibiána Kožárová, Ronald Zakhar, Zuzana Imreová, Hana Hanuljaková, Ines Karlovská and Miloslav Drtil 647–656 physical chemistry Electronic Structures and Reactivities of CoVID-19 Drugs: A DFT Study Seyda Aydogdu and Arzu Hatipoglu 665–673 chemical, biochemical and environmental engineering Environmentally Friendly Extraction of Bioactive Compounds from Rosa canina L. fruits Using Deep Eutectic Solvent (DES) as Green Extraction Media Hyrije Koraqi, Bujar Qazimi, Cengiz Çesko and Anka Trajkovska Petkoska 674–680 inorganic chemistry Syntheses, Characterization and Crystal Structures of Dicyanamide Bridged Polynuclear Copper(II) and Zinc(II) Complexes with Urease Inhibitory Activity Li Zhang,1 Yuqing Gu, Xinhui Feng, Ting Yang, Xiaoyan Li, Jing Wang and Zhonglu You 681–693 chemical, biochemical and environmental engineering Phosphate Ion Removal from Synthetic and Real Wastewater Using MnFe2o4 nanoparticles: ... Widodo Brontowiyono, Indrajit Patra, Shaymaa Abed Hussein, Alimuddin, Ahmed B. Mahdi, Samar Emad Izzat, Dhuha Mohsin Al-Dhalemi, Ahmed Kareem Obaid Aldulaim, Rosario Mireya Romero Parra, Luis Andres Barboza Arenas and Yasser Fakri Mustafa Graphical Contents 694–699 inorganic chemistry Syntheses, Structures and Insulin-Like Activity of Two oxidovanadium(V) Complexes with Similar nicotinohydrazone Ligands Gao-Qi Zhou, Xiao-Yang Qiu, Shu-Juan Liu, Chu-Yi Wang 700–713 Organic chemistry Antiproliferative and Antiprostate Cancer Activities of Heterocyclic Compounds Derived from Cyclohexane- 1,4-dione Nadia Y. Megally Abdo and Rafat Milad Mohareb 714–721 Analytical chemistry Superparamagnetic Tragacanth Coated Fe3o4@Sio2 nanoparticles for the Loading and Delivery of Metformin Fereshte Farajian and Payman Hashemi 722–733 Materials science Investigation of Biological and Prooxidant Activity of Zinc oxide nanoclusters and nanoparticles Iliana A. Ivanova, Elitsa L. Pavlova, Aneliya S. Kostadinova, Radostina D. Toshkovska, Lyubomira D. Yocheva, Kh El-Sayed, Mohamed A. Hassan, Heba El-Sayed El-Zorkany and Hisham A. Elshoky 507Acta Chim. Slov. 2022, 69, 507–518 Metias et al.: Development of Green Differential Pulse Voltammetric ... DOI: 10.17344/acsi.2022.7348 Scientific paper Development of Green Differential Pulse Voltammetric Strategy for the Determination of Alfuzosin Hydrochloride at Pencil Graphite and Modified Carbon Paste Electrodes Youstina Mekhail Metias,1,2 Emad Mohamed Hussien,3,* Mervat Mohamed Hosny,1 and Magda Mohamed Ayad1 1 Department of Analytical Chemistry, Faculty of Pharmacy, Zagazig University 44519, Zagazig, Egypt. 2 Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Moto-oka 744, Nishi-ku, Fukuoka-shi, Fukuoka 819-0395, Japan. 3 Department of Pharmaceutical Chemistry, National Organization for Drug Control and Research (NODCAR), Giza, Egypt. * Corresponding author: E-mail: emadhussien@yahoo.com Tel.: +2 02 3749 6077 Received: 06-14-2022 Abstract A sensitive and inexpensive differential pulse voltammetric technique was applied to investigate the electrochemical behavior of alfuzosin hydrochloride at two different working electrodes: silica gel modified carbon paste and pencil graphite electrodes (PGE). The voltammetric conditions were optimized using cyclic voltammetry, showing an irre- versible anodic peak in Britton-Robinson buffered medium (pH 6) at 0.86–0.90 V. The electrochemical responses were linearly correlated with alfuzosin concentrations (R2 > 0.999) in the ranges of 0.6–20 and 0.3–20 µM, exhibiting higher electrocatalytic activity at PGE with a low detection limit/ detectability of 0.099 µM. In addition, this study was a success- ful attempt for the drug determination in tablets and spiked urine samples with green profile evaluation, employing the National Environmental Methods Index, analytical Eco-Scale score, and Green Analytical Procedure Index. Keywords: Alfuzosin hydrochloride; carbon paste electrode; cyclic voltammetry; differential pulse voltammetry; pencil graphite electrode. 1. Introduction Alfuzosin hydrochloride (ALF), a selective al- pha1-adrenoceptor antagonist, is defined as (2RS) -N- [3- [(4-Amino- 6,7 dimethoxyquinazolin-2-yl) methylamino] propyl] tetrahydrofuran-2-carboxamide hydrochloride.1 The action of ALF as a vasodilator may be less frequent, but it acts more selectively on the smooth muscle tone within the prostate and bladder neck, causing relaxation of these muscles. Therefore, it results in symptomatic relief of the benign prostatic hyperplasia, a common progressive disease encountered in aging men, within weeks.2 It allevi- ates the symptoms of urinary obstruction after oral ad- ministration of sustained release ALF by reducing outflow resistance and enhancing bladder emptying. Several analytical methods were published for ALF determination either alone or in combinations, including micellar flow injection analysis with fluorescence detec- tion,3 kinetic colorimetric,4 and UV-spectrophotometric methods.5 Spectrofluorimetric methods adopting utili- zation of ortho-phthalaldehyde6 and micellar matrix7 for ALF assay in biological fluids and stability indicating spec- troscopic studies were also developed.8 In addition, chro- matographic methods such as HPLC with UV,9 MS/MS,10 and fluorescence11, 12 detections, chiral HPLC,13 UPLC,14 and stability indicating HPTLC15 were developed. Different electrochemical techniques were also es- tablished for potentiometric,16 conductometric,17 and voltammetric analysis of ALF by linear sweep and normal pulse polarography,18 Coulometric fast Fourier transform linear sweep voltammetry,19 and differential pulse20,21 and square-wave voltammetry.22 To our knowledge from the literature review, few analytical studies were performed to investigate the elec- 508 Acta Chim. Slov. 2022, 69, 507–518 Metias et al.: Development of Green Differential Pulse Voltammetric ... trochemical behavior of ALF, as depicted in Figure 1. Meanwhile, most of the reported researches were directed towards the chromatographic and spectroscopic analysis of the drug, which required expensive instrumentations, high amounts of solvents, hazardous reagents, prolonged analysis time, or complicated steps for sample pretreat- ment. Therefore, the development of analytical methods with simpler, greener, lower-cost and faster procedures, and achieving the validation criteria such as higher sen- sitivity and selectivity is widely demanded for drug anal- ysis. The electrochemical techniques such as voltammet- ric methods are considered a satisfactory alternative23 among the available approaches and an appealing choice for pharmaceutical analysis24. Although ALF is an elec- troactive compound, few voltammetric studies were re- ported for its electrochemical analysis, employing vari- ous working electrodes such as Hg electrode,18 bare22 and modified glassy carbon electrodes with hybrid of ionic liquid-ZrO2 in graphene oxide,19 and multiwall carbon nanotubes sensors incorporated with the ionic liquid 1-hexylpyridinium hexafluorophosphate20 and nickel ox- ide nanoparticles.21 Figure 1: Classification of the analytical techniques reported for ALF determination. However, the reported voltammetric methods showed good sensitivity and satisfactory analytical per- formance, the applied electrodes exhibited some draw- backs such as mercuric toxicity, and the use of expensive nanomaterials which also required complicated and time consuming procedures for preparation of these modified electrodes. Therefore, the continuous development and chemical modification of the electrochemical sensors with low-cost, easily-prepared, and eco-friendly modifiers have received an extensive interest to enhance their perfor- mance as chemical and biological sensors in electroanal- ysis. The carbon paste electrode (CPE), a widely appli- cable electrochemical sensor, has been employed in the areas of electrochemistry and electroanalysis due to its attractive characteristics, including simple preparation, affordable implementation, easy surface renewal, a low background current, and a wide range of potential win- dow.23,25 In addition, CPE can be easily modified and sim- ply manipulated in order to obtain a stable response with the possibility of lowering the overpotential, and to in- crease the sensitivity and selectivity of some electroactive species.26 Thus, the feasibility of CPE modification with different modifiers were utilized in previous studies, such as sephadex modifier,27 and rosaniline,28 phthalo blue,29 glycine,30 and helianthium dye31 which were electropol- ymerized on CPE for their effective functioning, exhib- iting linearity for the studied analytes over the ranges of 0.005–1, 1–3.5, 0.25–1.25, 0.06–1, and 0.06–0.15 mM, re- spectively. In the present work, chemically modified graphite electrode with silica gel was employed to investigate the electrochemical behavior of ALF. Silica gel is a granular and porous form of silicon dioxide and can be easily incor- porated into the carbon paste as an inexpensive and effec- tive modifier. It possesses some attractive electrochemical properties which is extremely useful for electroanalytical purposes, such as high surface area, strong adsorption ca- pacity, insolubility in most solvents, high thermal stability, and readily surface modification.32–34 In recent years, disposable sensors composed of convenient matrices for surface renewal, such as pencil graphite electrodes (PGE), gained a large applicability to quantitative assays.35–37 PGE stand as an excellent versa- tile tool in the electroanalysis having favorable advantag- es over the traditional electrodes of being simple, cheap, commercially available, easily modified, low technology, and good mechanical rigidity. Moreover, the preparation of PGE for each measurement is faster and easier than the procedures required for other conventional carbon elec- trodes, including tedious hand mixing and polishing, and hazardous steps.38 Differential pulse voltammetry (DPV) is a simple pulse voltammetric technique that is widely applicable for the determination of various pharmaceuticals.24 Herein, we employ DPV to study the electrochemical oxidation of ALF at two different electrochemical sensors: silica gel modified carbon paste and pencil graphite electrodes, with a comparison of their performance. Thus, our study aims to develop an easy, simple, and rapid DPV method for highly sensitive estimation of ALF in tablets and biological samples. As far as we know, it is the first report to employ a disposable pencil graphite electrode for the voltammetric assessment of ALF and to demonstrate the practical usefulness of two simple elec- trodes of low cost for the direct assay of ALF in urine samples. Moreover, the ecological impact of the proposed method was also evaluated using three metrics, namely, the National Environmental Methods Index (NEMI), an- alytical Eco-Scale score, and Green Analytical Procedure Index (GAPI). 509Acta Chim. Slov. 2022, 69, 507–518 Metias et al.: Development of Green Differential Pulse Voltammetric ... 2. Experimental 2. 1. Instrumentation The electroanalytical study was performed using a SP-150 potentiostat (Bio-Logic Science Instrument, France) connected to a Lenovo computer provided with EC-Lab for windows v11.02 software. The cell potentials were measured with respect to the Ag/AgCl/3.0 M KCl reference electrode (BAS, USA) in a glass cell, comprising a working electrode and platinum wire (BAS, USA) as an auxiliary electrode. A digital analyzer pH meter (Jenway 3510, USA) and ultrasonic bath sonicator (UCI-750, RAYPA, Spain) were also used. 2. 2. Working Electrodes Voltammetric measurements were performed using two different electrodes: 2. 2. 1. Modified Carbon Paste Electrode Silica gel modified carbon paste electrode (Si-gel/ CPE) was prepared by thoroughly hand-mixing of 0.2 g sili- ca gel and 0.8 g graphite powder with 0.6 mL paraffin oil by a ceramic pestle in a glass mortar to obtain a homogeneous paste. The preparation of the bare (plain or unmodified) CPE was likewise done by blending of 1.0 g graphite powder with 0.6 mL paraffin oil. The resulting paste was packed into the electrode body hole, and then the external surface of the electrode was polished on a soft paper with figure-eight mo- tions in order to remove the excess of the paste and obtain a shiny appearance prior to using. In addition, subsequent re- newal of the carbon paste surface for the each measurement should be performed, where a small portion of the paste at the electrode tip was scraped out and replaced by a new por- tion then repolished to generate a fresh electrode surface. 2. 2. 2. Pencil Graphite Electrode XQ pencil leads of 2B grade with 0.9 mm diameter and 60 mm length from the local bookstore were em- ployed for the voltammetric measurements of ALF. An insulating tape was used for wrapping PGE gently, where 25 mm of the pencil lead at one end was inserted into a home-designed brass holder for the electrical connection with the device. Meanwhile, the exposed surface of PGE at the other end was only 10 mm devoted as the sensitive part for the voltammetric assay and was gently polished before each recording using a cloth felt pad. 2. 3. Materials and Reagents Pure sample of ALF was generously provided by Eva Pharma, Egypt (lot no. 1422R118) with purity of 100.80% according to the comparison method.1 Bi-distilled water obtained from a Milli-Q water pu- rification system and chemical reagents of highest purity were used in this study. Graphite powder, paraffin oil, Se- phadex G-50, C18 silica gel, and chitosan were supplied from Sigma–Aldrich. Methanol, sodium hydroxide, glacial acetic acid, boric acid, phosphoric acid, and hydrochloric acid were obtained from El Nasr Pharmaceutical Chemi- cals CO. (Cairo, Egypt). Britton-Robinson buffer (B-R buffer), a widely used multi-buffer system in the voltammetric studies, was employed as a non-complexing supporting elec- trolyte for the voltammetric measurements of ALF. The multi-acid B-R buffer system is consisted of three dif- ferent buffering components of diminishing strength, so a linear pH response is obtained from pH 2.5 to pH 9.2 upon adding the alkali. Thus, it was easily prepared at the desired pH value without changing the chemi- cal composition of the buffered components. It was prepared from a mixture of 0.04 M of each acid; boric, phosphoric and acetic acids, and the desired pH was ad- justed using NaOH. 2. 4. Pharmaceutical Formulation Prostetrol® modified release tablets (10 mg of ALF/ tablet), a product of Eva Pharma with batch no. (10)190238, were purchased from the Egyptian market to be analyzed by the proposed method. 2. 5. Stock and Working Standard Solutions A stock standard solution of ALF (1.0 × 10−2 M) was prepared using methanol for dissolving 42.59 mg of ALF into a 10 mL volumetric flask. Further dilution was carried out by transferring 0.25 mL of the prepared solution into a 25 mL volumetric flask and completing the final volume with bi-distilled water to obtain a standard solution of 1.0 × 10–4M. The ALF working solutions were made by further dilution of the standard solution with B-R buffer solution to cover the concentration ranges of 6 × 10−7 to 2 × 10−5 M and 3 x 10−7 to 2 × 10−5 M at Si-gel/CPE and PGE, re- spectively. 2. 6. General Procedure 2. 6. 1. Voltammetric Procedures The surface of the working electrode was conditioned at first by performing successive anodic cyclic voltamme- try (CV) scans within the potential of 0 up to 1.6 V in an electrochemical cell containing B-R buffer solution of pH 6. After achieving stable background and response, an ap- propriate volume of the ALF standard solution was added to the cell, and then the solution was stirred for 1 min at an open circuit potential followed by a rest period for 30 s. The voltammograms were then recorded at a scan rate of 200 mVs−1 and ambient temperature. 510 Acta Chim. Slov. 2022, 69, 507–518 Metias et al.: Development of Green Differential Pulse Voltammetric ... 2. 6. 2. Construction of Calibration Curves The quantitative determination of ALF was per- formed using the DPV method within a potential range of 0 to 1.3 V, employing the optimum parameters of 50 mV pulses and step height, 50 ms pulses width, and 100 ms step time. Different aliquots of the ALF standard solution (1.0 × 10–4 M) were transferred by a micropipette into an electrochemical cell containing 10 mL buffer of pH 6, and then the peak currents were measured after stirring of the cell content for 1 min. The oxidation current peak (µA) that developed at each working electrode was plotted against its corresponding drug concentration (µM) to construct two calibration curves, covering the concentration ranges of 0.6–20 and 0.3–20 µM at Si-gel/CPE and PGE, respectively, and being fit with two linear regression equations. 2. 6. 3. Assay of Pharmaceutical Preparations Ten Prostetrol® tablets were finely pulverized, and an equivalent amount to 1.0 × 10−2 M ALF was accurately weighed, transferred into a 25 mL volumetric flask, and dissolved in 20 mL methanol by sonication for 30 min in an ultrasonic bath. The final volume was made up us- ing the same solvent, and then the flask content was well mixed and allowed to settle for 15 min before filtering the supernatant.39 Further dilution was done with bi-distilled water to obtain 1.0 × 10–4 M solution of ALF. Different al- iquots from the prepared solution were investigated for ALF quantification in its dosage form, applying the proce- dures described under construction of calibration curves section and the percentage recoveries were calculated from the corresponding regression equation. 2. 6. 4. ALF Assay in Spiked Human Urine Human urine samples, collected from a healthy donor and stored at –20 °C, were allowed to be partially thawed at room temperature to collect a clear superna- tant. In order to reduce the matrix effect, the urine super- natant underwent fifty and twenty fold dilutions with the B-R buffer of pH 6 for the voltammetric measurements at Si-gel/CPE and PGE, respectively. 10 mL of the sample solution was transferred into the voltammetric cell and its voltammogram was recorded as a blank solution for sub- sequent measurements. Appropriate aliquots of 1.0 × 10–4 M of ALF solution were then spiked for their direct analy- sis in urine matrix, using the procedures described under construction of calibration curves section for the recovery studies of the spiked urine samples. 3. Results and Discussion The electrochemical behavior of ALF was investi- gated at two different electrodes: CPE and PGE using CV technique, where different chemical and electrochemical parameters were thoroughly studied for optimization its voltammetric performance. The voltammograms of the studied drug were recorded from 0 to 1.6 V in the B-R buff- er solution as the supporting electrolyte. ALF exhibited a well-defined anodic peak current at around 0.90 V with- out a cathodic peak on the reverse scan, indicating the ir- reversible electro-oxidtion process of ALF. The reasonable oxidation mechanism, as postulated by the previous elec- trochemical studies of ALF,20,22 might occur at the electro- active nitrogen atom in the amine group of the pyrimidine moiety, followed by deprotonation and dimerization. 3. 1. Optimization of the Experimental Conditions Using Cyclic Voltammetry 3. 1. 1. Effect of the Solution pH The pH of the supporting electrolyte has an impor- tant impact on the voltammetric behavior of the drug. Therefore, the change in the pH range of B-R buffer from 3 to 9 was studied using CV method and the electro-oxida- tion peak potential and peak current of 10 μM ALF were assessed. Upon increasing the pH values, the anodic peak potential shifted in the less positive direction due to the proton dependent electro-oxidation mechanism of ALF on the working electrode and followed linear regression equation 1 (Figure 2B): EPa = –0.0561pH + 1.3287 (R2 = 0.9900) (1) where: EPa is the anodic peak potential of 10 μM ALF as a function of the applied pH of B-R buffer. In addition, the slope of 0.0561V/pH is close to the theoretical value of 0.059 V /pH of the Nernst equation, indicating that equal number of electrons and protons par- ticipated in the process of drug oxidation. Moreover, the anodic peak current of ALF enhanced in less acidic medi- um, reaching the optimum at pH 6. Meanwhile, badly-de- fined oxidation peaks and a gradual decrease in the peak height were observed with the further increase in the pH values. This might be attributed to the dissociation con- stant of the studied drug, where the pKa values of ALF are 2.26 and 5.56.40 Therefore, the pH of the solution could affect the existing form of ALF as the drug would exist in its protonated (water soluble) form in the acidic medium, while at higher pH values, it would be deprotonated to its barely soluble form. Thus, the decrease in the peak cur- rents was observed at higher pH values (> pKa values of ALF). As can be seen in Figure 2, B-R buffer of pH 6 was fit for all subsequent measurements of ALF. 3. 1. 2. Effect of Surfactants as Electrolyte Additives The impact of surfactant addition to the supporting electrolyte was investigated in the present study, as sur- 511Acta Chim. Slov. 2022, 69, 507–518 Metias et al.: Development of Green Differential Pulse Voltammetric ... factants are widely used in electroanalysis for enhancing the electrochemical performance of some analytes. There- fore, the oxidation behavior of ALF was studied in the presence of different types of surfactants, including SDS, CTAB, and Brij-35 as anionic, cationic, and nonionic sur- factants, respectively. The aqueous electrolytic solution of B-R buffer showed the best response as can be seen in Figure S1, meanwhile no distinct effect was noticed upon adding the surfactants to the analyte in the B-R buffer solution of pH6. 3. 1. 3. Effect of the Stirring Time The anodic peak current of ALF in dependence of the stirring time was studied at different time intervals from 0 to 3 min using CV, where the voltammograms were recorded after stirring the solution at 400 rpm, followed by 30 s quiescent time at an open circuit potential. As shown in Figure S2, the oxidation peak current improved after the solution stirring for 1 min, then became stable up to 3 min. 3. 1. 4. Effect of the Carbon Paste Composition The incorporation of different modifiers such as silica gel, chitosan, sephadex and iron nanoparticles into the carbon paste composition were investigated to attain highly responsive sensor. The oxidation peak current of the studied drug enhanced upon modifying CPE with silica gel which exhibited a higher electrocatalytic activi- ty and sensitivity towards ALF oxidation, Figure S3. The effect of silica gel content was also checked by recording the voltammetric responses of CPE containing various proportions of silica gel. The addition of silica gel in the ratio of 20% w/w of the paste total mass showed an effi- cient synergistic effect for improving the oxidation peak current of ALF, Figure S4. 3. 1. 5. Effect of the Pencil Graphite Types Different types and diameters of disposable PGE were examined using CV for B-R solution containing 5 µM ALF. Pencil leads are usually marked with either H (hardness), B (blackness), or HB letters which mainly differ in their composition ratio of graphite and clay. PGE of the soft B type containing more graphite and with larger diameter are appropriate for quantitative assay as this type provides an easy electronic transfer which generates higher signals.38 PGE from different commercial manufacturers such as HB, rotring, faber castle, and XQ were also investigated, where electroac- tive species may show different voltammetric behavior on PGE produced from different manufacturers with the same hardness due to different interactions of the analyte with common components of PGE.38 2B XQ pencil lead of 0.9 mm diameter was the optimum which gave the best electrochemical response with relatively low noise, Figure S5. 3. 1. 6 Optimization of the Scan Rate Studying the scan rate effect gave valuable informa- tion about the electrochemical behavior of the studied drug at the working electrode, whether the electro-oxida- tion process would be under diffusion or adsorption con- Figure 2: (A) Cyclic voltammograms of 10μM ALF recorded at different pH values (3–9) of B-R buffer at 200 mV/s scan rate; (B) Dependence of the anodic peak potential and peak current on the pH values. 512 Acta Chim. Slov. 2022, 69, 507–518 Metias et al.: Development of Green Differential Pulse Voltammetric ... trolled mechanism. The variation in the peak current (ip) and peak potential (Ep) of 10 µM ALF in B-R buffer solu- tion (pH 6) at Si-gel/CPE was examined by the CV mode at different scan rates. Upon increasing the scan rate from 25 up to 300 mV/s, the anodic peak current grew gradually and the oxidation peak potential shifted slightly towards more positive direction, indicating the irreversible oxida- tion process. The scan rate of 200 mV/s was selected for the successive voltammetric measurements, exhibiting a well-shaped peak with relatively narrow width and high sensitivity, Figure 3 A. A direct relationship was found between the oxida- tion peak current of ALF and square root of the scan rate (υ1/2) over the scan rate range of 25– 200 mVs−1, based on I(μA) = 2.7885 υ1/2 (mV/s)+ 2.0843(R2 = 0.9816). Thus, the electrochemical oxidation of ALF was controlled by a diffusion process, Figure 3 B. Moreover, plotting the logarithm of the peak current versus logarithm of the scan rate exhibited a straight line following equation 2 (Figure 3 C): log Ipa (μA) = 0.4932 log υ (mV/s) + 0.4922, R2 = 0.9799 (2) where Ipa represents the anodic peak current and υ is the scan rate. Figure 3: (A) Cyclic voltammograms of 10 μM ALF in B-R buffer of pH 6 at different scan rates; (B) Linear plot of the peak current versus square root of the scan rate; (C) Dependence of the logarithm of the peak current on logarithm of the scan rate; (D) Linear plot of the peak potential as function of logarithm of the scan rate. 513Acta Chim. Slov. 2022, 69, 507–518 Metias et al.: Development of Green Differential Pulse Voltammetric ... The obtained slope value of 0.4932 was close to the theoretical value of 0.5 for the diffusion-controlled elec- trode process.41 Furthermore, the relationship between the peak potential and logarithm of the scan rate was found to be linear according to equation 3 (Figure 3 D): Epa(V)= 0.0619 logυ (V/s)+ 1.0738, R2 = 0.9909 (3) where Epa represents the anodic peak potential and υ is the scan rate. In accordance with Laviron᾿s equation given for an irreversible electrochemical process, the number of elec- trons transferred at the surface of the electrode due to ALF oxidation was calculated as follows:42 (4) Herein, the following elements in equation 4 possess their conventional meanings: the electron transfer coeffi- cient (α), the number of electrons transferred (n), temper- ature (T = 298 K), gas constant (R = 8.314 J K mol−1), Far- aday constant (F = 96 485 C mol−1), and the voltammetric scan rate (υ). k° denotes the standard heterogeneous rate constant of the surface reaction, E° represents the formal redox potential, and Ep is the anodic peak potential. The E° value can be obtained from the intercept of Ep against υ on the ordinate by extrapolating the line to the vertical axis at υ = 0. From the linear relationship be- tween Epa versus log υ, the value of αn can be deduced from the slope and found to be 0.0619. The supposed value of α equals 0.5 for the irreversible electrochemical process,43 thus the n value was calculated to be 1.911 (ap- proximately 2.0). This value referred to the participation of two electrons and protons in the electro-oxidation re- action of ALF which coincided with previously published results.20,21 3. 2. Method Validation The developed DPV method at two different elec- trodes was validated according ICH guidelines44 with re- gard to the following parameters: 3. 2. 1. Linearity and Ranges Under the optimum conditions, the linearity of ALF was investigated in the concentration ranges of 6 × 10−7–2 x 10−5 M at Si-gel/CPE and 3 × 10−7–2 × 10−5 M at PGE us- ing DPV, Figure 4. Statistical data analysis was performed by plotting the peak current height (µA) as a function of concentration (µM), fitting the resulting calibration graphs into linear regression equations as follows: Si-gel/CPE: I = 0.2501 C + 2.7407 (R2 = 0.9995) (5) PGE: I = 0.6057 C + 3.3274 (R2 = 0.9996) (6) Figure 4: DPV voltammograms of (A) 0.6–20 µM of ALF at Si-gel/ CPE; (B) 0.3–20 µM of ALF at PGE in B-R buffer solution of pH 6. (C) Calibration plot of the peak current versus concentration. 514 Acta Chim. Slov. 2022, 69, 507–518 Metias et al.: Development of Green Differential Pulse Voltammetric ... The obtained data with high correlation coefficients were indicative of good linearity of the proposed method at the two applied electrodes, Table 1. 3. 2. 2. Sensitivity The limits of detection (LOD) and quantification (LOQ) were calculated according to the ICH guidelines:44 LOD = 3.3 σ/s and LOQ = 10 σ/s, where, σ is the standard deviation of the responses of four replicated blanks ob- tained at the same potential applied for the sample and s is the slope of the calibration graph. The obtained values of LOD and LOQ indicated the sensitivity of the proposed method at the applied electrodes, as shown in Table1. 3. 2. 3. Accuracy and Precision The accuracy of the proposed method was exam- ined using nine samples of three different concentrations of the pure drug selected to cover the low, medium and high ranges of the calibration graph. The mean percentage recoveries of these ALF concentrations were calculated, as shown in Table 1. The precision was also evaluated through tripli- cate determinations of three different concentrations of pure drug within the same day (intra-day) and on three different days (inter-day). The obtained results in Table 1 exhibited acceptable values of relative standard deviation (RSD%) and percentage relative error (Er%). 3.2.4. Specificity The specificity of the proposed method was con- firmed by ALF assay in its tablet formulation and urine samples. The selectivity of DPV at the applied electrodes was also tested in the presence of commonly used excipi- ents in tablets, such as silica, lactose, PVP, talc, and mag- nesium stearate. The excipients were added to ALF at the same concentration (10–5 M) and analyzed under opti- mum conditions. Table 2 exhibits sufficiently good recoveries with no interference, while lactose showed a slight decrease in the electrochemical response of ALF. Table 2: Voltammetric analysis of ALF in the presence of some common excipients by DPV at two different electrodes. Excipients Recovery %[b] Added[a] Si-gel/CPE PGE (1x 10–5 M) (1x 10–5 M) (1x 10–5 M) Silica 103.40 101.88 Lactose 95.00 94.23 Povidone k30 102.87 97.67 Talc 100.10 100.40 Magnesium stearate 101.18 101.71 [a] Drug: excipients in the ratio of 1: 1 M. [b] Average of three determinations. 3. 3. Statistical Analysis The results of DPV at both electrodes were statisti- cally examined using the Student’s t-test and variance ra- tio F-test at the 95% confidence level. The obtained results were compared with those of the official potentiometric method for the ALF assay1 and showed no significant dif- ferences, Table 3. Table 3. Statistical analysis of the results obtained by DPV at two different electrodes and the pharmacopeial method for ALF assay. Si-gel/CPE PGE Comparison method [1][a] Mean ± SD 100.10±1.58 99.39±1.40 100.80±1.42 Variance 2.508 1.970 2.016 n 6 7 3 Student-t-test 0.642 (2.365)[b] 1.549(2.306)[b] – F-test 1.244 (5.790)[b] 1.023 (5.140)[b] – [a] Potentiometric official method for ALF assay. [b] The corresponding theoretical values for t and F tests at p = 0.05. 3. 4. Analytical Applications The employed electrodes showed satisfactory results for the ALF assay in Prostetrol® tablets and spiked urine samples using DPV. The recovery values of the target ana- lyte and the standard deviations (SD) proved the suitabili- ty of the proposed method for fast routine analysis of ALF in its tablets and human urine samples (Table 4). Thus, the proposed DPV method exhibited simpler, time saving, greener, and good practical applicability for Table 1. Regression and analytical performance data of ALF assay by DPV method at two different electrodes. Parameters Si-gel/CPE PGE Linearity range (μM) 0.6–20 0.3–20 Intercept 2.7407 3.3274 Slope 0.2501 0.6057 Correlation Coefficient(R2) 0.9995 0.9996 LOD (µM) 0.17 0.099 LOQ (µM) 0.53 0.299 Accuracy (mean recovery% ± Er%) 100.85+0.85 98.97–1.03 97.57–2.43 99.74–0.26 98.99–1.01 100.94+0.94 Precision (RSD%) Repeatability[a] 1.756 1.399 Intermediate precision[b] 1.794 1.788 [a] The intra-day and [b] inter-day RSD (n=9) of 1, 10, and 20 μM ALF at Si-gel/CPE and 1, 10, and 15 μM ALF at PGE within the same day and in three successive days. 515Acta Chim. Slov. 2022, 69, 507–518 Metias et al.: Development of Green Differential Pulse Voltammetric ... ALF analysis in real samples with acceptable percentage recoveries. As compared to the voltammetric performance of ALF at Si-gel/CPE, PGE showed a better response with a smaller background current, good conductivity, sharp ox- idation peaks, and higher sensitivity (Figure 4) due to the presence of sp2 hybridized carbon of graphite bound with clay in the pencil lead composition. Herein, clay, an aggre- gate of minerals and colloidal substances, contributed to the good sensing performance of PGE because of its highly attractive characteristics such as chemical and mechanical stability, strong sorption properties revealed in high ionic exchange capabilities, and porosity which exhibits benefi- cial ionic conductivity and electrocatalytic activity.45 Table 1 ascertains the good electrocatalytic activity of PGE to- wards the ALF oxidation, where the sensitivity obtained at PGE was two times higher than that at Si-gel/CPE with a lower LOD of 0.099 μM. 3. 5. Greenness profile of the Proposed Method The assessment of the analytical method procedures from the green perspective has recently attracted the au- thors’  concern in the field of green analytical chemistry (GAC). Electroanalytical techniques mostly comply with the GAC principles as they are free of hazardous chemicals and organic solvents, and do not produce large volumes of analytical waste compared to the classical chromatograph- ic methods. To deepen this view, the greenness profile of the de- veloped method was established using three assessment tools: NEMI, Analytical Eco-Scale, and GAPI methods. The applied metrics introduced more easier and visible information on the environmental impact of the applied analytical procedures. NEMI labeling is considered as one of the oldest tools for qualitative greenness assessment of the analytical procedures. The NEMI pictogram is symbolized by a circle divided into four fields that reflect four different criteria of the described analytical methodology: persistence, bi- oaccumulation potential, and toxicity (PBT), hazardous chemicals, corrosiveness, and waste. Each field is filled with green when its required criterion is met by the devel- oped method.46 Adopting these criteria, the pictogram of the applied method showed green-colored quadrants due to satisfaction with their requirements as shown in Table 5, except for the hazardous quadrant due to methanol usage, regardless of the minute amount used per sample. From a glance at the NEMI symbol, general infor- mation about environmental impact of the analytical pro- cedure can be easily read with no significance to energy consumption or the quantity of chemicals. Thus, the analytical Eco-Scale approach was em- ployed for the greenness assessment in a more quantitative way, where a numerical score was given for the developed method. The analytical Eco-Scale score was calculated by subtraction of the total penalty points from the basis of 100 points (ideal green analysis). The penalty points of the analytical procedure were assigned to four main categories the amount and type of chemicals, energy consumption, occupational hazard, and amount of generated waste, and the way for its treatment. The score was ranked on a scale, where the method greenness was excellent if the score is higher than 75, acceptable if it is higher than 50, and in- adequate if it is less than 50.47 The DPV method scored 80 at Si-gel/CPE and 82 at PGE (higher than 75), so it ranked as an excellent green method. As can be seen in Table 5, the analytical Eco-Scale scores were calculated in detail for assessing its greenness profile more clearly than that ob- tained by NEMI. A recent assessment tool known as GAPI was also employed, showing a specific symbol with five pentagrams segmented into 15 zones to encompass five main catego- ries: sample handling, general method type, sample prepa- Table 4. Analytical application of the DPV method for ALF determination in tablets formulation and spiked urine samples at two different elec- trodes. Si-gel/CPE PGE Prostetrol® modified Urine samples Prostetrol® modified Urine samples release tablets release tablets Parameters Taken Found Recover Taken Found Recover Taken Found Recover Taken Found Recover μM μM y [a]% μM μM y [a]% μM μM y [a]% μM μM y [a]% 5 4.02 101.92 3 3.08 102.57 0.6 0.59 98.43 5 5.10 102.01 7 4.49 99.96 5 5.12 102.36 5 4.89 97.74 7 7.19 102.66 13 6.05 101.72 10 10.10 100.95 7 6.97 99.61 10 10.02 100.24 16 6.82 101.97 15 15.15 101.03 10 10.04 100.43 12 11.80 98.85 20 7.84 102.00 12 11.65 97.10 Mean ±SD 101.51 ± 0.88 101.73 ± 0.85 98.66 ± 1.36 100.94 ± 1.73 N 5 4 5 4 SE 0.392 0.427 0.607 0.866 V 0.767 0.729 1.842 2.997 [a] Average of three determinations. 516 Acta Chim. Slov. 2022, 69, 507–518 Metias et al.: Development of Green Differential Pulse Voltammetric ... ration, reagents/solvents used and instrumentation. Thus, the GAPI approach evaluates 15 criteria covering every step in the whole analytical procedure using a color code: green, yellow, and red signifying low, medium, and high adherence to GAC standards, respectively. Color codes given to each segment were specified according to the de- tailed information described in a report by Wasylka48. As shown in Table 5, the environmental impact of employing two different electrodes in the proposed method for ALF assay was the same in all evaluation parameters represent- ed in one GAPI pictogram. The visual inspection of the first eight segments of the pictogram refers to few simple steps required for the sample preparation in every ana- lytical procedure, including the use of green solvents and reagents without macro-extraction or derivatization steps since the red zone represents special storage conditions of urine samples. Meanwhile, the segments from 9 to 11 re- flects the use of lower amount of less hazardous solvents and reagents. In addition, the lower energy consumption of our instrumentation with lower waste production per sample were represented on the last pentagram that also exhibits one red zone due to no treatment applied for the generated waste. Therefore, the resulting GAPI pictogram of the DPV method showed (6) green (7) yellow, and (2) red shaded zones that reflects its lower impact on the en- vironment. Overall, our simple study is shed light on the merits of the applied voltammetric method as a green and safe practice for ALF quantification that can be used in rou- tine work and quality control purposes in pharmaceutical industries. 3. 6. Perspectives Development of the voltammetric techniques is a growing trend in the electroanalytical field to attain more affordable, greener, easily used, and highly sensitive elec- trochemical methods. Recently, the chemical modification of electrochemical sensors and the use of disposable and multiplexed electrodes received a great deal of attention to attain stable and rapid responses with high sensitivity, accurate selectivity, and reliability. Thus, incorporation of different modifiers and fabrication of new applicable sen- sors have been utilized to construct electrodes with high conductivity, great catalytic activity, lower toxicity, and effortless synthesis. Combination of the nanotechnology with electrochemical techniques have been applied, such as the application of multi-walled carbon nanotubes and metal nanoparticles for enhancing the electrocatalytic activity of the sensors. As a result, the continuous devel- opment of the sensor technology based on the electro- chemical technique have been applied to gas sensors, envi- ronmental monitoring sensors, biosensors, etc., which can be employed in different aspects such as forensic medicine and evidence science. In addition, further modifications have been widely proposed to be reasonably integrated Table 5. Greenness assessment of the proposed method by the Analytical Eco-Scale, NEMI, and GAPI approaches. Analytical Eco–Scale score parameters Penalty points (PPs) NEMI pictograms I–Reagents/ word sign /no of pictograms Si-gel/CPE PGE Bi-distilled water/ – / 0 0 Methanol/ danger / 2 4 1M NaOH/ danger / 1 2 Silica gel / – / 0 0 – Paraffin oil/ danger / 1 2 – Graphite/ – / 0 0 0.04 M acetic acid / danger / 1 2 0.04 M orthophosphoric acid/ danger / 1 2 0.04 M boric acid/ danger / 1 2 ∑ = 14 ∑ = 12 II–Instruments a-Energy GAPI pictogram* Potentiostat 0 pH meter 0 Vortex mixer 0 Sonicator 0 b–Occupational hazards 3 c–Waste 3 ∑ = 6 Total PPs 20 18 Analytical eco–scale score = 100- total PPs 80 82 Excellent green analysis * Red zones depict high ecological impact; yellow zones represent lower impact; and green zones represent more safe effect to the environment. 517Acta Chim. Slov. 2022, 69, 507–518 Metias et al.: Development of Green Differential Pulse Voltammetric ... with portable voltammetric analyzers for on-site analysis. Moreover, the growing interest in green analytical chem- istry also requires a fresh perspective for the appropriate modification and selection of the electrochemical sensors. Thus, development and exploration of electrochemical sensors with specific selectivity, good conductivity, and economic feasibility for an eco- and user-friendly voltam- metric assay is an urgent task. 4. Conclusion In the proposed study, a differential pulse voltam- metric method was developed for rapid determination of ALF at two different electrodes with an evaluation of their green-profile. Carbon paste electrode modified with silica gel and pencil graphite electrode proved to be high efficient and sensitive; nevertheless, they are simpler and more economic than those employed in the previously re- ported methods. 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DOI:10.1016/j.talanta.2018.01.013 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Uporabljena je bila občutljiva in poceni diferencialna pulzna voltametrična tehnika za raziskovanje elektrokemičnega obnašanja alfuzosin hidroklorida na dveh različnih delovnih elektrodah: ogljikova pasta, modificirana s silikagelom, in svinčnikova grafitna elektroda (PGE). Voltametrični pogoji so bili optimizirani s ciklično voltametrijo, ki je pokazala ireverzibilni anodni vrh v Britton-Robinsonovem puferskem mediju (pH 6) pri 0,86–0,90 V. Elektrokemični odzivi so bili linearno odvisni od koncentracije alfuzosina (R2> 0,999) v razponu od 0,6–20 in 0,3–20 µM, ki kaže večjo elektrokatal- itsko aktivnost pri PGE z nizko mejo zaznave 0,099 µM. Poleg tega je bila ta študija uspešen poskus določanja zdravila v tabletah in vzorcih urina z dodatkom učinkovine z vrednotenjem zelenega profila z uporabo Nacionalnega indeksa okoljskih metod, analitične ocene Eco-Scale in Indeksa zelenih analitičnih postopkov. 519Acta Chim. Slov. 2022, 69, 519–525 Kshash et al.: Synthesis, Characterization, and Investigation of ... DOI: 10.17344/acsi.2022.7360 Scientific paper Synthesis, Characterization, and Investigation of Mesomorphic Properties of a New 2,5-Bis-(4-alkanoyloxybenzylidene)cyclopentan-1-one Abdullah Hussein Kshash,1,* Omar Jamal Mahdi Al-Asafi2 and Hanaa Kaen Salih2 1 Department of Chemistry, Education College for Pure Science, University Of Anbar, Anbar, Iraq 2 Department of Chemistry, College of Science,University of Tikrit, Tikrit, Iraq * Corresponding author: E-mail: drabdullahkshash@gmail.com Received: 01-14-2022 Abstract A new set of cyclopentanone chalcone esters 2,5-bis-(4-alkanoyloxybenzylidene)cyclopentan-1-one (B2–B10) has been synthesized and monitored by TLC. Structures of these compounds were determined by spectroscopic techniques (FTIR, 1H NMR, and mass spectrometry). Differential scanning calorimetry (DSC) and polarized optical microscopy were used to evaluate their transition temperatures and mesophase properties (POM) throughout heating and cooling scans. The thermal data indicate that the compounds B5–B10 have mesomorphic properties with thermal stabilities; the data also reveal that the compounds B6–B10 are monotropic, whereas B5 is enantiotropic. B6, B7, and B9 only have a nematic phase, but B8 and B10 have a smectic phase followed by a nematic phase, and B5 only has a smectic phase. In addition, the study reveals that the inclusion of an acyl group as a terminal chain had the opposite effect on isotropization temper- atures for compounds B6, B8, and B10, resulting in an increase in transition temperatures and a decrease in mesophase stability. The lack of a smectic phase in B7 and B9 compounds could be attributed to the narrow phase temperature range, which makes examination difficult, or to the molecules’ lack of lateral attraction. Keywords: Cyclopentanone chalcone, nematic, smectic, enantiotropic, monotropic. 1. Introduction Chalcone is the common name for flavonoids, the name chalcone comes from the Greek word “chalcos”, which means “bronze”, because most natural chalcones have a bronze color.1 Chalcones have two aromatic rings linked by an unsaturated α,β-ketone. Chemically, they are open-chain flavonoids with two aromatic rings bonded by a three-carbon atom (unsaturated carbonyl) system (Fig- ure 1). In other words, “chalcones are structural deriva- tives of 1,3-diphenylprop-2-en-1-one”.2–4 Figure 1: General structure of a chalcone The Claisen–Schmidt condensation reaction is often used to prepare chalcones, which involves the condensa- tion of aldehydes and ketones in the presence of a base or acid catalyst, followed by a dehydration process.5 Chal- cones have recently received a lot of attention, not only from the synthetic and biosynthetic standpoint, but also because of their biological activities,6 which include anti- cancer,7–9 GSK-3 inhibition and antimicrobial activity,10 anti-HIV,11 and antimalarial properties.12 The target of this study is to synthesize cyclopentanone chalcone esters and investigate their liquid crystal properties and the influence of chain length on these properties. 2. Experimental Section 2. 1. Materials All chemicals were purchased from Sigma–Aldrich and were used without further purification. Melting points 520 Acta Chim. Slov. 2022, 69, 519–525 Kshash et al.: Synthesis, Characterization, and Investigation of ... were determined in an open capillary tube and are uncor- rected. A Tensor 27 Bruker, Germany spectrometer was used to record infrared spectra as ATR (range 4000–600 cm–1). The 1H NMR spectra were recorded on a Bruker Ultershield 400 MHz NMR spectrometer, Germany, using DMSO-d6 as the solvent. The chemical shifts are reported as δ values (in ppm). Mass spectrum analyses were per- formed by the Agilent Technology MS 5973 device. POM equipped with a hot stage and Mettler Toledo DSC 823 (DSC) at a heating rate of 20 °C min–1 was used for the investigation of phase transition temperatures. 2. 2. Synthesis of 2,5-Bis((E)-4- hydroxybenzylidene)cyclopentan-1-one (A) In a 250 mL round-bottom flask containing 100 mL of ethanol, cyclopentanone (40 mmol) and 4-hydroxyben- zaldehyde (80 mmol) were introduced and thoroughly mixed. Then, 40% sodium hydroxide solution (10 mL) was added slowly, and the reaction mixture was stirred over- night at room temperature. Thereafter, the reaction mix- ture was poured into a beaker containing crushed ice to quench the reaction and then neutralized with 10% HCl. The precipitate was filtered and recrystallized from abso- lute ethanol. Green solid, yield 76%; mp > 280 °C (lit. > 300 °C), Rf = 0.8 (acetone:hexane 5:5). IR (ATR) ν 3289 (O-H phenol), 3046 (C-H aromatic), 2973 (ν C-Hal), 1668 (ν C=O), 1561, 1509 (aromatic ring) cm–1. 1H NMR (400 MHz) δ 10.06 (lit. 10.06) (s, 2H, H1,H1´), 7.53 (d, 4H, H2,2, H2´,2´), 7.34 (lit. 7.34) (s, 2H, H3, H3´), 6.83–6.90 (d, 4H, H4,4, H4´,4´), 3.01 (lit. 3.01) (t, 4H, H5,5, H5´,5´). 2. 2. Synthesis of (1E,1’E)-(2- Oxocyclopentane-1,3-diylidene) bis(methaneylylidene)bis(4,1-phenylene) dialkanoate B2–B10 To a 50 mL round-bottomed flask immersed in an ice bath and containing 20 mL of pyridine and compound A (1.7 mmol), an appropriate acid chloride (3.4 mmol) was added. The mixture was stirred at room temperature overnight. Thereafter, the mixture was poured into a beak- er containing crushed ice, acidified with 10% HCl, and the solid product was filtered, washed with water, and recrys- tallized from absolute ethanol. TLC was performed using acetone-hexane (3:7) as the eluent solution. 2. 3. Characterization of Products B2–B10 ((1E,1’E)-(2-Oxocyclopentane-1,3-diylidene)bis(meth- aneylylidene))bis(4,1-phenylene) diacetate (B2) Yellow solid, yield 77%; mp 200–202 °C (decomp.), Rf = 0.62, IR (ATR) ν 3110 (C-H aromatic), 2981 (C-Hal.), 1763 (ν C=Oester), 1671 (ν C=Oketone), 1594, 1505 (aro- matic ring) cm–1. 1H NMR (400 MHz) δ 6.78–7.95 (d, 8H, Ar-H), 7.65 (s, 2H, CH=CH), 3.00 (t, 4H, saturated five membered ring), 2.40 (s, 6H, CH3 groups). ((1E,1’E)-(2-Oxocyclopentane-1,3-diylidene)bis(meth- aneylylidene))bis(4,1-phenylene) dipropionate (B3) Yellow solid, yield 48%; mp 280–282 °C, Rf = 0.81, IR (ATR) ν 3114 (C-H aromatic), 2975 (C-Hal.), 1759 (ν C=Oester), 1675 (ν C=Oketone), 1577, 1504 (aromatic ring), 752 (γ CH2) cm–1. 1H NMR (400 MHz) δ 7.67–6.76 (d, 8H, Ar-H), 7.47 (s, 2H, CH=CH), 2.95–3.00 (t, 4H, saturated five membered ring), 1.25–2.52 (m, 10H, alkyl groups). ((1E,1’E)-(2-Oxocyclopentane-1,3-diylidene)bis(meth- aneylylidene))bis(4,1-phenylene) dibutyrate (B4) Yellow solid, yield 71%; mp 164 °C (decomp.), Rf = 0.76, IR (ATR) ν 3123 (C-H aromatic), 2978 (C-Hal.), 1754 (ν C=O ester), 1672 (ν C=Oketone), 1564, 1505 (aromatic ring), 743 (γ CH2) cm–1. 1H NMR (400 MHz) δ 7.73–6.85 (d, 8H, Ar-H), 7.40 (s, 2H, CH=CH), 2.55–3.09 (t, 4H, saturated five membered ring), 0.89–2.50 (m, 14H, alkyl groups). MS m/z 434.3. ((1E,1’E)-(2-Oxocyclopentane-1,3-diylidene)bis(meth- aneylylidene))bis(4,1-phenylene) dipentanoate (B5) Deep yellow solid, yield 45%; mp 197–199 °C, Rf = 0.72, IR (ATR) ν 3119 (C-H aromatic), 2963 (C-Hal.), 1752 (ν C=Oester), 1687 (ν C=Oketone), 1594, 1504 (aromatic ring), 754 (γ CH2) cm–1. 1H NMR (400 MHz) δ 7.25–7.74 (d, 8H, Ar-H), 7.46 (s, 2H, CH=CH), 3.10 (t, 4H, saturat- ed five membered ring), 0.95–2.50 (m, 18H, alkyl groups). MS m/z 460.7. ((1E,1’E)-(2-Oxocyclopentane-1,3-diylidene)bis(meth- aneylylidene))bis(4,1-phenylene) dihexanoate (B6) Yellow solid, yield 53%; mp 169–171 °C, Rf = 0.74, IR (ATR) ν 3057 (C-H aromatic), 2957 (C-Hal.), 1753 (ν C=Oester), 1688 (ν C=Oketone), 1593, 1504 (aromatic ring), 736 (γ CH2) cm–1. 1H NMR (400 MHz) δ 6.32–7.00 (d, 8H, Ar-H), 6.99 (s, 2H, CH=CH), 1.94–2.05 (t, 4H, saturated five membered ring), 0.30–1.11 (m, 22H, alkyl groups). MS m/z 490.4. ((1E,1’E)-(2-Oxocyclopentane-1,3-diylidene)bis(meth- aneylylidene))bis(4,1-phenylene) diheptanoate (B7) Brown solid, yield 28%; mp 170–172 °C, Rf = 0.67, IR (ATR) ν 3116 (C-H aromatic), 2987 (C-Hal.), 1759 (ν C=Oester), 1669 (ν C=Oketone), 1597, 1509 (aromatic ring), 769 (γ CH2) cm–1. 1H NMR (400 MHz) δ 6.88–7.98 (d, 8H, Ar-H), 7.51 (s, 2H, CH=CH), 2.46–3.43 (t, 4H, saturated five membered ring), 0.86–1.66 (m, 26H, alkyl groups). ((1E,1’E)-(2-Oxocyclopentane-1,3-diylidene)bis(meth- aneylylidene))bis(4,1-phenylene) dioctanoate (B8) Brown solid, yield 46%; mp 178–180 °C, Rf = 0.71, IR (ATR) ν 3080 (C-H aromatic), 2956 (C-Hal.), 1748 (ν 521Acta Chim. Slov. 2022, 69, 519–525 Kshash et al.: Synthesis, Characterization, and Investigation of ... C=Oester), 1669 (ν C=Oketone), 1597, 1509 (aromatic ring), 748 (γ CH2) cm–1. 1H NMR (400 MHz) δ 6.90–7.98 (d, 8H, Ar-H), 7.46 (s, 2H, CH=CH), 2.78–3.30 (t, 4H, saturated five membered ring), 0.86–2.38 (m, 30H, alkyl groups). ((1E,1’E)-(2-Oxocyclopentane-1,3-diylidene)bis(meth- aneylylidene))bis(4,1-phenylene) dinonanoate (B9) Brown solid, yield 84%; mp 162–164 °C, Rf = 0.71, IR (ATR) ν 3057 (C-H aromatic), 2957 (C-Hal.), 1752 (ν C=Oester), 1690 (ν C=Oketone), 1594, 1503 (aromatic ring), 747 (γ CH2) cm–1. 1H NMR (400 MHz) δ 6.88–7.75 (d, 8H, Ar-H), 7.40 (s, 2H, CH=CH), 2.50–3.35 (t, 4H, saturated five membered ring), 0.86–1.65 (m, 34H, alkyl groups). ((1E,1’E)-(2-Oxocyclopentane-1,3-diylidene)bis(meth- aneylylidene))bis(4,1-phenylene) bis(decanoate) (B10) Brown solid, yield 81%; mp 175–177 °C, Rf = 0.61, IR (ATR) ν 3101 (C-H aromatic), 2958 (C-Hal.), 1752 (ν C=Oester), 1690 (ν C=Oketone), 1596, 1503 (aromatic ring), 741 (γ CH2) cm–1. 1H NMR (400 MHz) δ 6.89–7.73 (d, 8H, Ar-H), 7.32 (s, 2H, CH=CH), 2.52–3.07 (t, 4H, saturated five membered ring), 0.87–1.64 (m, 38H, alkyl groups). MS m/z 600.5. 3. Result and Discussion 3. 1. Chemistry Target ester compounds B2–B10 were synthesized using the method presented in scheme 1. Scheme1. Synthetic route for synthesis of compounds B2–B10 The presence of α-hydrogen atoms on both sides of the carbonyl group in the cyclopentanone molecule was used in the Claisen–Schmidt condensation reaction of cyclopentanone with 4-hydroxybenzaldehyde to produce two α,β-unsaturated groups of the chalcone compound 2,5-bis((E)-4-hydroxybenzylidene)cyclopentan-1-one (A). While esters B2–B10 were synthesized by reacting phenolic hydroxyl groups in chalcone A as a nucleophile with acid chlorides in the presence of pyridine as a solvent. This reaction was carried out in two steps. The nucleop- hile (OH) first attacks the carbonyl carbon atom of an acyl chloride, forming a tetrahedral intermediate that can elim- inate the chloride as a leaving group. 3. 2. Characterization TLC was used to monitor the synthesis of chalcone A, with changes in product color and melting point serving as a preliminary evidence.13 Thereafter, the FTIR spectrum (Figure 2) for compound A revealed a broad absorption band at 3289 cm–1 attributed to the O–H group, a medium absorption at 3046 cm–1 attributed to the aromatic C–H, a strong absorption band at 2973 cm–1 attributed to the C– Hasy(aliphatic) and the absorption at 2935 cm–1 that can be assigned to the C–Hsy(aliphatic), while the C=O group stretching vibration for ketone was observed at 1668 cm–1. Figure 2: FT IR spectrum for chalcone A. The 1H NMR (Figure 3) spectrum of chalcone A re- veals three distinct regions of chemical shifts. The signal of the hydroxyl proton appears as a singlet at δ 10.09 ppm, aromatic protons (8H) signals appear as two doublets in the regions of δ 7.57–7.53 and 6.89–6.88 ppm, β-protons signal was observed as a singlet at δ 7.34 ppm, while a tri- plet signal at δ 3.01 ppm was ascribed to the two methyl- ene groups in cyclopentane. Figure 3: 1H NMR spectrum for chalcone A. 522 Acta Chim. Slov. 2022, 69, 519–525 Kshash et al.: Synthesis, Characterization, and Investigation of ... The FTIR spectra for compounds B2–B10 (see Fig- ure 4 for a representative case of B6) show the disappear- ance of the OH band for chalcone, and the appearance of an absorption band within the range of 3123–3110 cm–1 that is attributable to the aromatic C–H, a strong absorp- tion band within the range of 1762–1748 cm–1 that is at- tributable to the C=O group of ester, and absorption band within the range of 1690–1669 cm–1 assigned to the C=O group for ketone. According to 1H NMR spectra for compounds B2– B10, the OH signal at 10.09 ppm attributable to compound A has disapeared, aromatic proton signals were observed within the range δ 6.32–7.79 ppm, while proton signals for alkyl groups were observed within the range δ 0.25–3.23 ppm. See Figure 5 for a representative case of a 1H NMR spectrum for compound B5. 3. 3. Mesomorphic Properties The DSC and POM were utilized to investigate the characteristics of the phases of the 2,5-bis-(4-alkanoy- loxybenzylidene)cyclopentan-1-one compounds B2–B10 Figure 4: FT IR spectrum for chalcone B6. Figure 5: 1H NMR spectrum for compound B5. 523Acta Chim. Slov. 2022, 69, 519–525 Kshash et al.: Synthesis, Characterization, and Investigation of ... upon heating and cooling, by tracing the thermal transi- tions in the DSC and confirming mesophases type by ob- serving the texture using POM. Figure 6 shows the DSC thermogram for the compound B5 after a heating and cooling scan. The transition temperatures, enthalpies, and entro- pies of the chalcone ester compounds B5–B10 from DSC are presented in Table 1. The phase behavior investigation of chalcone ester compounds revealed that the compounds B5–B10 exhib- it liquid crystal properties with thermal stabilities. Com- pounds B6–B10 were monotropic, while B5 was enantio- tropic. Compounds B6, B7, and B9 have only a nematic phase, while B8 and B10 have a smectic phase followed by a nematic phase, and B5 has only a smectic phase (Figure 7). According to thermal degradation studies, com- pounds having odd methylene groups in the terminal alkoxy chain are more stable than those with even num- ber of methylene groups.14 While the study of the data in Table 1 revealed that the introduction of an acyl group as a terminal chain had the opposite effect on isotropization temperatures for compounds B6, B8, and B10, increasing transition temperatures and decreasing their mesophase stability (Figure 6). The lack of a smectic phase in compounds B7 and B9 could be due to the phase’s narrow temperature range, which causes investigation difficulties, or to the molecules’ lack of lateral attraction. Table 1. Phase transitions and transition enthalpy for chalcone esters B5–B10 under heating and cooling cycle Comp. Transition Temperatures, οC (ΔH, kJ mol–1) ΔT/ΔS Heating Cooling B5 Cr 103.26 (0.0829) SA 174.25 (17.22) SC 193.48 (0.994) I I 167.38 (0.61) SC 146.77 (26.0) Cr Cr 70.99/0.0002 SA 19.23/ 0.038 SC -/ 0.002 I B6 Cr 68.62 (3.058) N 168.70 (23.01) I I 100.89 (10.26) Cr Cr 100.08/0.008 N -/ 0.052 I B7 Cr 103.91 (3.39) N 164.10 (17.59) I I 142.23 (9.55) Cr Cr 60.19/ 0.009 N - / 0.040 I B8 Cr1 87.88 (6.30) Cr2 106.95 (1.08) SA 134.28 (0.71) N 173.42 (16.94) I I 147.54 (13.51) Cr Cr1 19.07/ 0.017 Cr2 27.33/ 0.002 SA 39.14 / 0.0004 N - / 0.037 I B9 Cr1 122.92 (1.72) Cr2 132.27 (0.36) N 166.99 (23.15) I I 150.77 (17.63) Cr Cr1 9.35/0.0043 Cr2 34.72/0.0009 N -/0.0526 I B10 Cr1 21.01/0.0096 Cr2 51.25/0.0019 SA 32.78/0.018 SC 8.84/0.0273 N -/0.0055 I I 145.58 (10.51) Cr Figure 6: DSC of compound B5 during heating/cooling cycles 524 Acta Chim. Slov. 2022, 69, 519–525 Kshash et al.: Synthesis, Characterization, and Investigation of ... 4. Conclusion The chalcone esters of cyclopentanone were suc- cessfully synthesized, and FTIR and 1H NMR were used to characterize them. According to thermal data, com- pounds with 2–4 carbon atoms are non-mesogenic mate- rials, while those with 5–10 carbon atoms are mesogenic materials. For compounds with 6, 8, and 10 carbon at- oms, adding an acyl group as a terminal chain had the opposite impact on isotropization temperatures, causing an increase in transition temperatures and a decrease in mesophase stability. Acknowledgements The authors are grateful to Ms. Ala’a Adnan Rashad of Al-Nahrain University for her assistance with the study’s thermal analysis.. 5. References 1. N. K. Sahu, S. S. Balbhadra, J. Choudhary, D. V. Kohli, Curr. Med. Chem. 2012, 19, 209–225. DOI:10.2174/092986712803414132 2. G. L. Santosh, U. N. Vignesh, Res. Chem. Intermed. 2017, 43, 6043–6077. DOI:10.1007/s11164-017-2977-5 3. C. Mustafa, F. Esra, Synth. Commun. 2009, 39, 1046–1054. 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DOI:10.1016/j.bioorg.2021.104681 Figure 8: Relationship of isotropization and nematic phase range with the number of carbon atoms Figure 7: Polarized optical micrographs for: (a) compound B5 (SC at 174.25 °C); (b) compound B6 (N, at 68.62 °C); (c) compound B7 (N, mosaic at 103.91 °C), (d) compound B8 (SA, at 106.95 °C); (e) compound B9 (N, Schlieren, at 132.27 °C); (f) compound B10 (N, mosaic at 164.30 °C). 525Acta Chim. Slov. 2022, 69, 519–525 Kshash et al.: Synthesis, Characterization, and Investigation of ... 11. N. Turkovic, B. Ivkovic, J. Kotur-Stevuljevic, M. Tasic, B. Marković, Z. Vujic, Curr. Pharm. Des. 2020, 26, 802–814. DOI:10.2174/1381612826666200203125557 12. M. Xu, P. Wu, F. Shen, J. Ji, K. P. Rakesh, Bioorg. Chem. 2019, 91, 103133. DOI:10.1016/j.bioorg.2019.103133 13. F. Zhao, H.-H. Dong, Y.-H. Wang, T.-Y. Wang, Z.-H. Yan, F. Yan, D.-Z. Zhang, Y.-Y. Cao, Y.-S. Jin, Med. Chem. Commun. 2017, 8, 1093–1102. DOI:10.1039/C6MD00649C 14. G. Lisa, E.-R. Cioancă, N. Tudorachi, I. Cârlescu, D. Scutaru, Thermochim. Acta 2011, 524, 179–185. DOI:10.1016/j.tca.2011.07.013 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Sintetizirali smo novo serijo ciklopentanonskih halkonskih estrov 2,5-bis-(4-alkanoiloksibenziliden)ciklopentan-1-onov (B2–B10); reakcije smo nadzorovali s TLC. Strukture teh spojin smo določili s spektroskopskimi tehnikami (FTIR, 1H NMR in masna spektrometrija). Diferenčna dinamična kalorimetrija (DSC) in polarizirana optična mikroskopija sta bili uporabljeni za določanje temperatur prehoda in mezofaznih lastnosti (POM) v celotnem območju segrevanja in ohlajan- ja vzorcev. Podatki termičnih analiz kažejo, da imajo spojine B5–B10 mezomorfne lastnosti in da so termično stabilne; podatki tudi kažejo, da so spojine B6–B10 monotropne, medtem ko je spojina B5 enantiotropna. Spojine B6, B7 in B9 imajo samo nematsko fazo; pri spojinah B8 in B10 pa smektični fazi sledi nematska faza; spojina B5 ima samo smektično fazo. Študije so tudi razkrile, da ima vključitev acilne skupine na terminalno mesto verige nasprotni učinek na izotropno temperaturo za spojine B6, B8 in B10, kar povzroči povečanje temperatur prehoda in zmanjšanje mezofazne stabilnosti. Dejstvo, da spojini B7 in B9 nimata smektične faze, lahko pripišemu ozkim temperaturnim intervalom faznih prehodov, kar povzroča eksperimentalne težave, ali pa pomanjkanju lateralnih privlačnih sil med molekulami v teh dveh primerih. 526 Acta Chim. Slov. 2022, 69, 526–535 Şenkardeş et al.: Synthesis, Molecular Docking Studies and ADME ... DOI: 10.17344/acsi.2022.7387 Scientific paper Synthesis, Molecular Docking Studies and ADME Prediction of Some New Albendazole Derivatives as α-Glucosidase Inhibitors Sevil Şenkardeş,1,* Necla Kulabaş1 and Ş. Güniz Küçükgüzel2 1 Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Maltepe, Başıbüyük, 34854, Istanbul, Turkey 2 Fenerbahçe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ataşehir, 3 4758, Istanbul, Turkey * Corresponding author: E-mail: sevil.aydin@marmara.edu.tr Tel. +90-216-777 52 00 * This study was partly presented at the International Congress on Biological and Health Sciences on 26–28 February 2021 (Online), Turkey Received: 01-28-2022 Abstract A series of novel 2-(substituted arylidene)-N-(5-(propylthio)-2,3-dihydro-1H-benzo[d]imidazol-2-yl)hydrazine-1-car- boxamide derivatives 3a–i were synthesized via condensation of N-(5-(propylthio)-1H-benzo[d]imidazol-2-yl) hydra- zinecarboxamide (2), with the corresponding ketone or aldehydes. The chemical structures of the compounds prepared were confirmed by analytical and spectral data. The compounds were screened for their α-glucosidase inhibitory activity and all of them showed better inhibition than acarbose, except 3h. In particular, compound 3a proved to be the most ac- tive compound among all synthetic derivatives having IC50 value 12.88 ± 0.98 μM. Also, molecular docking studies were carried out for the compounds to figure out the binding interactions. Compound 3a has exhibited the highest binding energy (ΔG = –9.4 kcal/mol) and the most hydrogen bond interactions with active sites. Eventually, in silico studies were in good agreement with in vitro studies. Keywords: Benzimidazole; antidiabetic; albendazole; α-glucosidase; semicarbazone; docking study 1. Introduction Diabetes Mellitus (DM), known simply as diabetes, is a major health problem as a metabolic disease and is characterized by a failure of insulin production. It can be classified into two broad categories; type 1 and type 2 dia- betes. Type 2 diabetes ranks as the most common type of diabetes worldwide among all reported cases.1 This form of diabetes results from a combination of insulin resistance and insulin secretion defects.2 Insulin plays an important role in the regulation of blood glucose levels and energy metabolism.3 If diet and exercise fail to adequately control blood glucose levels, it is recommended to start oral drug therapy such as α-glucosidase inhibitors. α-Glucosidase is a key enzyme in carbohydrate di- gestion, released from mucosal cells and plays a signifi- cant role in carbohydrate metabolism.4 The enzyme has important functions in diabetes, viral infections, and cancer. α-Glucosidase inhibitors delay the hydrolysis of carbohydrates and this action reduces the glucose ab- sorption. Acarbose, miglitol, and voglibose are used in the clinic as α-glucosidase inhibitors. Nevertheless, side effects such as diarrhea, hepatotoxicity and flatulence are observed in the long-term treatment with these inhibi- tors.5,6 Hence, developing novel α-glucosidase inhibitors with minimum side effects is always a promising medici- nal chemistry effort. Benzimidazoles are important nitrogen-con- taining heterocyclic compounds and literature review showed that there are a great number of studies on α-glucosidase inhibitory activity of benzimidazole de- rivatives. Zawawi et al.7 and Ozil et al.8 have reported that a novel series of benzimidazole derivatives I–II (Figure 1) act as a new class of α-glucosidase inhibitors. 527Acta Chim. Slov. 2022, 69, 526–535 Şenkardeş et al.: Synthesis, Molecular Docking Studies and ADME ... Ahmad et al.9 provided an overview about the α-gluco- sidase inhibitory potential of a variety of benzimidazole derivatives III (Figure 1). On the other hand, semicarbazones, as medicinally significant scaffolds, are imine derivatives formed by con- densation reaction between aldehyde/ketone function- al groups and the -NH2 group of semicarbazides. These derivatives are known to have a broad range of biologi- cal properties including antidiabetic activity.10–14 For in- stance, pyrazole-phenyl semicarbazone derivatives were reported by Azimi et al. as potent α-glucosidase inhibitors IV (Figure 1) with IC50 values in the range of 65.1–695.0 μM comparing with acarbose (IC50 = 750.0 μM).14 More recently, a (E)-2-benzylidene-N-(3-cyano-4,5,6,7-tetrahy- drobenzo[b]thiophen-2-yl) hydrazine-1-carboxamide de- rivative (V) has been reported for its α-glucosidase inhibi- tory potential15 (Figure 1). From the literature survey, we revealed that mole- cules containing benzimidazole and semicarbazone moi- ety have gained a huge interest as potent α-glucosidase inhibitors. 2. Experimental 2. 1. General All the reagents used were analytical reagent grade. All melting points were determined on a Ther- mo Scientific 9300 melting point apparatus and are uncorrected. The IR spectra were recorded on a Shi- madzu FTIR 8400S spectrophotometer. NMR spectra were measured on a Bruker Avance 300 spectrometer in DMSO-d6 solutions using TMS as the internal stand- ard. Elemental analyses were determined on CHNS-932 (LECO) analyzer. The liquid chromatographic system consists of an Agilent Technologies 1100 series instru- ment equipped with a quaternary solvent delivery sys- tem and a model Agilent series G1315 A photodiode array detector. The chromatographic data were col- lected and processed using Agilent Chemstation Plus software. Chromatographic separation was performed at ambient temperature using a reverse phase Zorbax C8 (4.0×250 mm) column. All experiments were per- formed using acetonitrile-water gradient mobile phase (50:50 from 0 to 3 min; 75:25 to 50:50 from 3 to 6 min; 100:0 to 75:25 from 6 to 12 min; the flow rate was 1.0 mL/min). 2. 2. Chemistry 2. 1. 1. Synthesis of N-(5-(Propylthio)- 1H-benzo[d]imidazol-2-yl) Hydrazinecarboxamide (2) N-(5-(Propylthio)-1H-benzo[d]imidazol-2-yl) hy- drazinecarboxamide (2) was prepared by heating hy- drazine hydrate and Albendazole (1) in methanol. The product was purified by recrystallization from methanol yielding white solid. Figure 1. Designing of target molecules via molecular hybridization strategy 528 Acta Chim. Slov. 2022, 69, 526–535 Şenkardeş et al.: Synthesis, Molecular Docking Studies and ADME ... 2. 1. 2. General Procedure for the Synthesis of 2-(Substituted Arylidene)-N-(5- (propylthio)-2,3-dihydro-1H-benzo[d] imidazol-2-yl)hydrazine-1-carboxamides 3a–i A mixture of N-(5-(propylthio)-1H-benzo[d]imida- zol-2-yl) hydrazinecarboxamide (2) (0.001 mol) and vari- ous aromatic aldehydes or ketones (0.001 mol) in absolute ethanol (20 mL), in the presence of a catalytic amount of glacial acetic acid, was refluxed for 6–7 hours. The reaction mixture was allowed to cool to room temperature and then poured onto crushed ice. The precipitated compound was filtered and washed with water and recrystallized from ab- solute ethanol. 2-(5-Chloro-2-oxoindolin-3-ylidene)-N-(5-(propylth- io)-2,3-dihydro-1H-benzo[d]imidazol-2-yl)hydra- zine-1-carboxamide (3a) Yield 79%; m.p. 296–297 °C; HPLC tR (min): 6.32; FT-IR: ν 3327 (NH), 1681 (C=O), 1630 (C=N), 1608 (C=C) cm−1; 1H NMR (300 MHz, DMSO-d6) δ 0.96 (t, 3H, -S-CH2CH2CH3), 1.56 (m, 2H, -S-CH2CH2CH3), 2.88 (t, 2H, -S-CH2CH2CH3), 7.15–8.34 (m, 6H, Ar-H), 10.92 (s, 1H, NH), 11.24 (s, 1H, NH), 11.61 (s, 1H, NH), 11.65 (s, 1H, NH); 13C NMR (75.5 MHz, DMSO-d6) δ 13.5 (CH3), 22.5 (CH2), 36.9 (CH2), 112.5, 117.1, 124.8, 126.2, 128.1, 132.0, 135.6, 136.2, 142.6, 148.8 (C=N), 153.6 (C=O), 165.0 (C=O). Anal. calcd for C19H17ClN6O2S∙4/3H2O: C, 50.39; H, 4.38; N, 18.56; S, 7.08. Found: C, 50.51; H, 4.69; N, 18.65; S, 7.02. LC/MS (ESI) m/z 429 [M+H]+. 2-(2-Fluorobenzylidene)-N-(5-(propylthio)-2,3-dihy- dro-1H-benzo[d]imidazol-2-yl)hydrazine-1-carboxam- ide (3b) Yield 72%; m.p. 196–197 °C; HPLC tR (min): 5.27; FT-IR: ν 3321 (NH), 1678 (C=O), 1631 (C=N), 1556 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 0.96 (t, 3H, -S-CH2CH2CH3), 1.56 (m, 2H, -S-CH2CH2CH3), 2.86 (t, 2H, -S-CH2CH2CH3), 7.24–8.41 (m, 8H, Ar-H and CH=N), 10.65 (s, 1H, NH), 11.34 (s, 1H, NH), 11.93 (s, 1H, NH); 13C NMR (75.5 MHz, DMSO-d6) δ 13.5 (CH3), 22.6 (CH2), 37.2 (CH2), 116.2, 122.1, 122.3, 124.5, 125.0, 125.1, 127.1, 127.6, 131.9, 132.0, 135.5, 135.6, 148.5 (C=N), 153.8 (C=O), 159.7 and 162.2 (C-F, J = 248 Hz). Anal. calcd for C18H18FN5OS: C, 58.21; H, 4.88; N, 18.86; S, 8.63. Found: C, 58.60; H, 4.92; N, 18.74; S, 8.58. LC/MS (ESI) m/z 372 [M+H]+, 394 [M+Na]+. 2-(3-Fluorobenzylidene)-N-(5-(propylthio)-2,3-dihy- dro-1H-benzo[d]imidazol-2-yl)hydrazine-1-carboxam- ide (3c) Yield 85%; m.p. 188–190 °C; HPLC tR (min): 4.94; FT-IR: ν 3348 (NH), 1674 (C=O), 1627 (C=N), 1552 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 0.96 (t, 3H, -S-CH2CH2CH3), 1.54 (m, 2H, -S-CH2CH2CH3), 2.86 (t, 2H, -S-CH2CH2CH3), 7.11–8.01 (m, 8H, Ar-H and CH=N), 11.32 (s, 1H, NH), 11.89 (s, 1H, NH), 11.92 (s, 1H, NH); 13C NMR (75.5 MHz, DMSO-d6) δ 13.5 (CH3), 22.6 (CH2), 37.2 (CH2), 113.2, 113.4, 116.7, 116.9, 124.5, 124.6, 125.9, 127.0, 131.0, 137.3, 141.7, 148.5 (C=N), 153.7 (C=O),161.8 and 164.2 (C-F, J = 242 Hz). Anal. calcd for C18H18FN5OS: C, 58.21; H, 4.88; N, 18.86; S, 8.63. Found: C, 58.40; H, 4.72; N, 18.14; S, 8.11. LC/MS (ESI) m/z 372 [M+H]+, 394 [M+Na]+. 2-(2,5-Difluorobenzylidene)-N-(5-(propylthio)-2,3-di- hydro-1H-benzo[d]imidazol-2-yl)hydrazine-1-carbox- amide (3d) Yield 74%; m.p. 202–204 °C; HPLC tR (min): 5.20; FT-IR: ν 3335 (NH), 1674 (C=O), 1631 (C=N), 1552 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 0.96 (t, 3H, -S-CH2CH2CH3), 1.56 (m, 2H, -S-CH2CH2CH3), 2.86 (t, 2H, -S-CH2CH2CH3), 7.11–8.45 (m, 7H, Ar-H and CH=N), 10.88 (s, 1H, NH), 11.44 (s, 1H, NH), 11.98 (s, 1H, NH); 13C NMR (75.5 MHz, DMSO-d6) δ 13.5 (CH3), 22.5 (CH2), 37.2 (CH2), 113.5, 117.7, 117.9, 118.0, 118.3, 118.5, 123.9, 124.0, 124.5, 127.1, 134.4, 148.4 (C=N), 153.7 (C=O), 155.9 and 158.4 (C-F, J = 243 Hz), 157.8 and 160.2 (C-F, J = 238 Hz). Anal. calcd for C18H17F2N5OS: C, 55.52; H, 4.40; N, 17.98; S, 8.23. Found: C, 55.91; H, 4.76; N, 17.80; S, 8.22. LC/MS (ESI) m/z 390 [M+H]+, 428 [M+K]+. 2-(4-Methylbenzylidene)-N-(5-(propylthio)-2,3-dihy- dro-1H-benzo[d]imidazol-2-yl)hydrazine-1-carboxam- ide (3e) Yield 82%; m.p. 199–200 °C; HPLC tR (min): 3.19; FT-IR: ν 3335 (NH), 1681 (C=O), 1625 (C=N), 1556 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 0.96 (t, 3H, -S-CH2CH2CH3), 1.53 (m, 2H, -S-CH2CH2CH3), 2.35 (s, 3H, CH3), 2.86 (t, 2H, -S-CH2CH2CH3), 7.10–7.99 (m, 8H, Ar-H and CH=N), 10.29 (s, 1H, NH), 11.15 (s, 1H, NH), 11.91 (s, 1H, NH); 13C NMR (75.5 MHz, DMSO-d6) δ 13.6 (CH3), 21.5 (CH2), 22.6 (CH3), 37.5 (CH2), 124.5, 126.6, 127.0, 127.1, 127.8, 128.8, 129,6, 129.9, 131.9, 139.9, 143.3, 148.3 (C=N), 153.3 (C=O). Anal. cal- cd for C19H21N5OS∙1/4H2O: C, 61.35; H, 5.83; N, 18.83; S, 8.62. Found: C, 61.52; H, 6.24; N, 18.56; S, 8.69. LC/MS (ESI) m/z 368 [M+H]+, 390 [M+Na]+. 2-(2,4-Dichlorobenzylidene)-N-(5-(propylthio)-2,3-di- hydro-1H-benzo[d]imidazol-2-yl)hydrazine-1-carbox- amide (3f) Yield 78%; m.p. 177–179 °C; HPLC tR (min): 4.22; FT-IR: ν 3317 (NH), 1680 (C=O), 1631 (C=N), 1556 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 0.95 (t, 3H, -S-CH2CH2CH3), 1.53 (m, 2H, -S-CH2CH2CH3), 2.51 (t, 2H, -S-CH2CH2CH3), 7.05–8.53 (m, 7H, Ar-H and CH=N), 10.56 (s, 1H, NH), 11.17 (s, 1H, NH), 11.45 (s, 1H, NH); 13C NMR (75.5 MHz, DMSO-d6) δ 13.5 (CH3), 22.5 (CH2), 37.2 (CH2), 114.5, 115.8, 116.2, 124.5,127.2, 529Acta Chim. Slov. 2022, 69, 526–535 Şenkardeş et al.: Synthesis, Molecular Docking Studies and ADME ... 128.0, 129.5, 131.0, 133.7, 135.0, 137.8, 138.5, 148.5 (C=N), 154.6, 156.5 (C=O). Anal. calcd for C18H17Cl2N5OS: C, 51.19; H, 4.06; N, 16.79; S, 7.59. Found: C, 51.04; H, 4.04; N, 16.79; S, 7.59. LC/MS (ESI) m/z 422 [M+H]+. 2-(3,4-Dimethylbenzylidene)-N-(5-(propylthio)-2,3-di- hydro-1H-benzo[d]imidazol-2-yl)hydrazine-1-carbox- amide (3g) Yield 81%; m.p. 191–193 °C; HPLC tR (min): 6.02; FT-IR: ν 3332 (NH), 1681 (C=O), 1627 (C=N), 1556 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 0.96 (t, 3H, -S-CH2CH2CH3), 1.54 (m, 2H, -S-CH2CH2CH3), 2.25 (s, 3H, -CH3), 2.26 (s, 3H, -CH3), 2.85 (t, 2H, -S-CH2CH2CH3), 7.11–7.96 (m, 7H, Ar-H and CH=N), 10.35 (s, 1H, NH), 11.16 (s, 1H, NH), 11.93 (s, 1H, NH); 13C NMR (75.5 MHz, DMSO-d6) δ 13.5 (CH3), 19.7 (CH2), 19.9 (CH3), 22.6 (CH3), 37.2 (CH2), 124.4, 125.4, 126.9, 127.1, 127.4, 128.5, 130.2, 130.7, 132.2, 137.0, 138.7, 139.6, 143.6, 148.4 (C=N), 153.4 (C=O). Anal. calcd for C20H23N5OS: C, 62.71; H, 6.24; N, 17.92; S, 8.21. Found: C, 63.04; H, 5.96; N, 17.67; S, 7.99. LC/MS (ESI) m/z 382 [M+H]+, 404 [M+Na]+. 2-(5-Bromo-2-methoxybenzylidene)-N-(5-(propylth- io)-2,3-dihydro-1H-benzo[d]imidazol-2-yl)hydra- zine-1-carboxamide (3h) Yield 81%; m.p. 198–200 °C; HPLC tR (min): 5.86; FT-IR: ν 3348 (NH), 1681 (C=O), 1627 (C=N), 1556 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 0.96 (t, 3H, -S-CH2CH2CH3), 1.54 (m, 2H, -S-CH2CH2CH3), 2.86 (t, 2H, -S-CH2CH2CH3), 3.84 (s,3H, -OCH3), 7.04–8.50 (m, 7H, Ar-H and CH=N), 10.65 (s, 1H, NH), 11.26 (s, 1H, NH), 11.95 (s, 1H, NH); 13C NMR (75.5 MHz, DMSO-d6) δ 13.5 (CH3), 22.6 (CH2), 37.2 (CH2), 56.7 (OCH3), 113.3, 114.4, 116.6, 124.5, 124.8, 127.0, 128.8, 130.3, 130.4, 133.6, 136.9, 138.9, 148.4 (C=N), 153.4 (C-OCH3) 156.9 (C=O). Anal.calcd for C19H20BrN5O2S: C, 49.36; H, 4.36; N, 15.15; S, 6.94. Found: C, 49.56; H, 4.36; N, 14.35; S, 6.55. LC/MS (ESI) m/z 462 [M+H]+, 484 [M+Na]+. 2-(4-Trifluoromethylbenzylidene)-N-(5-(propylth- io)-2,3-dihydro-1H-benzo[d]imidazol-2-yl)hydra- zine-1-carboxamide (3i) Yield 75%; m.p. 208–210 °C; HPLC tR (min): 6.72; FT-IR: ν 3348 (NH), 1681 (C=O), 1614 (C=N), 1539 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 0.96 (t, 3H, -S-CH2CH2CH3), 1.54 (m, 2H, -S-CH2CH2CH3), 2.86 (t, 2H, -S-CH2CH2CH3), 7.11–8.15 (m, 8H, Ar-H and CH=N), 11.38 (s, 1H, NH, other NH proton not ob- served). 13C NMR (75.5 MHz, DMSO-d6) δ 13.5 (CH3), 22.6 (CH2), 37.2 (CH2), 115.6, 120.6, 123.3, 125.8, 125.9, 126.0, 126.2, 127.2 (q, CF3, J = 265 Hz), 128.3, 128.7, 129.2, 130.1, 138.7, 141.3, 148.6 (C=N), 153.9 (C=O). Anal. cal- cd for C19H18F3N5OS: C, 54.15; H, 4.30; N, 16.62; S, 7.61. Found: C, 54.12; H, 4.33; N, 16.62; S, 7.43. LC/MS (ESI) m/z 422 [M+H]+, 444 [M+Na]+. 2. 3. α-Glucosidase Assay The α-glucosidase inhibitor activity was evaluated as described by Ramakrishna et al. with slight modifications described by Sen et al.16 Each concentration had three rep- licates when preliminary screened and inhibition percent- ages were calculated using the following formula. where A is the absorbance. Finally, IC50 values were also determined for the compounds having 50% or above inhibition values. 2. 4. Molecular Docking Molecular docking simulations were performed against α-glucosidase by using AutoDock Vina software.17 The protein data of α-glucosidases (PDB ID: 4J5T) was ref- erenced from Protein Data Bank.18 The target protein was prepared in three steps using the AutoDock Tools program19 for docking studies: (1) were removed water molecules; (2) were added polar hy- drogen to α-glucosidase macromolecule; (3) the obtained structure was energy-minimized. The synthesized compounds 3a–i and reference li- gands were prepared in two steps for docking studies: (1) were drawn with the Spartan 04 software (SPARTAN 04, Wavefunction, Inc., Irvine, USA)20 and optimized for each compound by using the semi-empirical PM3 method; (2) the docking input files of the most stable conformation were generated using the AutoDock Tools program. The grid box size was determined as 78 Å × 69 Å × 104 Å within 0.375 Å grid spacing and center_x = –18.44, center_y = –20.91, center_z = 8.22 dimensions were used in α-glucosidase enzyme docking studies, appropriate to the literature.18 The Vina parameter “exhaustiveness” was set to the value of 10. The reference ligands used in validation studies in- clude sugars that form part of the biological substrate, as well as known inhibitors (acarbose, glucose, miglitol, de- oxynojirimycin, and kojibiose). During study of valida- tions as well as our docking of ligands were used flexible ligands in rigid protein. The resulting files were analyzed using Accelrys Discovery Studio Visualizer 4.0 program. 3. Results and Discussion 3. 1. Chemistry Scheme 1 shows the synthetic route for the target compounds. Albendazole (1) was reacted with hydrazine hydrate, affording 2 according to the similar method re- ported in the literature.21 The new derivatives 3a–i were obtained by the reaction of 2 with appropriate aromatic 530 Acta Chim. Slov. 2022, 69, 526–535 Şenkardeş et al.: Synthesis, Molecular Docking Studies and ADME ... aldehydes or ketone in the presence of acid according to the literature procedures.22,23 The structures of the desired target compounds were confirmed by FT-IR, 1H NMR, and elemental analysis. In the IR spectra of the studied compounds, the less intense broad bands around 3317–3348 cm−1 are as- signed to ν(N–H) vibrations of the NHCO group. The band around 1674–1681 cm–1 corresponds to the carbon- yl group of CONH. The azomethine band is observed at 1625–1631 cm–1. 1H NMR spectrum revealed three signals around 10.29–11.98 ppm assigned to three NH protons. In the 1H NMR spectrum of compound 3i, the NH proton was seen as a singlet at δ 11.38 ppm and two NH protons were not observed. 3.2. α-Glucosidase Inhibitory Activity We have synthesized benzimidazoles bearing sem- icarbazones 3a–i and evaluated them for α-glucosidase inhibitory potential. All derivatives showed excellent in- hibitory activities having IC50 values ranging between 12.88–44.35 µM as compared to the standard acarbose Scheme 1. Synthetic route to target derivatives 3a–i Table 1. α-Glucosidase inhibitory activity (IC50) of albendazole derivatives 3a–i 531Acta Chim. Slov. 2022, 69, 526–535 Şenkardeş et al.: Synthesis, Molecular Docking Studies and ADME ... (IC50 = 40.06 µM) (Table 1). Their possibly potent inhibi- tory potential may be due to the benzimidazole core bear- ing a semicarbazone entity. The isatin moiety of the most active compound 3a, was determined to be important for biological activity, suggesting intramolecular hydrogen bond formation. 3. 3. ADME Prediction The potent inhibitors 3a–i were evaluated in silico for selected ADME properties using the SwissADME online tool (http://www.swissadme.ch/). Table 2 shows the ADME prediction results of the compounds. Topological Polar Surface Area (TPSA) shows the surface belonging to polar atoms in the compound and lower TPSA values are appro- priate for drug-likeness properties. The absorption per- centage was also calculated by using the following formula: as given in the literature24,25 and showed a good absorp- tion profile. According to Lipinski’s rule of five, drug candidate should have log P less than 5, its polar surface area within 140 Ų, it should have less than 10 H bond acceptors, it should have less than 5 H bond donors and its molecu- lar weight should be below 500 Dalton.26 Also, the more negative the skin permeability (log Kp) value is, the less possible for the compound to penetrate the skin barrier. The tool predicted that derivatives have suitable skin per- meability with log Kp values of –5.24 to –5.90 cm/s. Eventually, all of the molecules were shown to com- ply best with these properties used to predict drug-like- ness (Table 2). To reveal the capability of intestinal absorption and permeability of the blood–brain barrier (BBB), the boiled- egg model of the molecules was predicted using SwissAD- ME (Figure 2). Molecules that fall in the yellow field de- Table 2. ADME results of the albendazole derivatives 3a–i Figure 2. The boiled-egg plot of compounds 3a–i. HIA: Human Intestinal Absorption; BBB: Blood–Brain Barrier; PGP: Permeability Glycoprotein. 532 Acta Chim. Slov. 2022, 69, 526–535 Şenkardeş et al.: Synthesis, Molecular Docking Studies and ADME ... pict the BBB permeation, whereas the white eclipse region symbolizes gastrointestinal absorption. According to the boiled-egg plot, all of the compounds are BBB-impermea- ble and have good absorption, except compound 3i. P-gly- coprotein (P-gp) plays a significant role in drug absorption and disposition. Compounds 3a–i are non substrates of Table 3. Types of interactions of the compounds 3a–i and small reference ligands with the binding site residues of α-glucosidase enzyme. Compoundsa Binding energy Binding site Distance (Å) Hydrogen bond ΔG (kcal/mol) Interactions (A/D)b 3a –9.3 A 2.80 Asp392 (A) 1.80 Arg428 (D) 2.59 Arg428 (D) 2.66 Arg428 (D) 3b –7.7 A 2.50 Arg428 (D) 3c –8.4 A 2.25 Arg428 (D) 3d –7.9 A 2.88 Trp391 (D) 2.49 Arg428 (D) 2.25 Arg428 (D) 3e –8.4 A 2.31 Arg428 (D) 2.97 Val446 (A) 2.78 Gln447 (D) 3f –7.3 A 2.87 Glu429 (A) 3g –7.9 A 2.87 Arg428 (D) 3h –7.3 A 2.84 Arg428 (D) 3i –8.4 A 2.71 Trp391 (D) 2.27 Asp568 (A) 3.02 Glu771(A)c ACB –8.1 A 3.09 Glu443 (A) 3.16 Val446 (A) 3.09 Asp568 (A) 1.97 Trp710 (D) 3.33 Glu771 (A) GLC –5.5 A 1.84 Trp391 (D) 2.26 Arg428 (D) 2.73 Arg428 (D) 2.46 Gly566 (A) 2.72 Asp568 (D) DNJ –5.4 A 2.54 Trp391 (D) 2.13 Gly566 (A) 2.69 Gly566 (A) 1.98 Trp710 (D) 2.37 Trp710 (D) MGL –5.5 A 2.27 Gly566 (A) KJB –6.1 A 2.41 Ile362 (A) 2.75 Glu429 (A) –6.4 B 2.66 Leu50 (A) 2.21 His51 (A) 2.44 Phe56 (A) 2.46 Asp61 (A) 1.96 Arg209 (D) 2.33 Arg209 (D) 2.62 Arg209 (D) a ACB: Acarbose; GLC: Glucose; DNJ: Deoxynojirimycin; MGL: Miglitol; KJB: Kojibiose. b A: H-bond acceptor; D: H-bond donor. c Halogen bond. 533Acta Chim. Slov. 2022, 69, 526–535 Şenkardeş et al.: Synthesis, Molecular Docking Studies and ADME ... P-gp, therefore it can be theorized that they may possibly act as inhibitors of P-gp. 3. 3. Molecular Modeling The molecular docking analysis was carried out to investigate the binding mode of novel α-glucosidase inhib- itors compounds 3a–i within the binding pocket of the tar- get enzyme, and to further understand their structure-ac- tivity relationship. Firstly, we examined the interactions of the known inhibitors (acarbose, glucose, miglitol, de- oxynojirimycin, and kojibiose) with α-glucosidase active site to compare with our synthesized compounds 3a–i. The binding energies and interactions with the active site of the reference ligands and compounds 3a–i are given in Table 3. As shown in Figure 3, one of the two binding sites of α-glucosidase has been defined as “site A” containing a glutamate (Glu771) and an aspartate (Asp568), while the other is “site B”.18 It was determined that among the known Figure 4. A surface representation of α-glucosidase shown as the DG color scheme (neutral oxygen (pink), nitrogen atoms (light blue), charged oxygen (red) and nitrogen atoms (dark blue)). Positions of compound 3a (green) and acarbose (purple) in the binding site A of α-glucosidase en- zyme and 2D interactions of the compound 3a with active site residues. Figure 3. For α-glucosidase enzyme small references ligands a) GLC is in binding site A, b) DNJ is in binding site A, c) MGL is in binding site A, and d) KJB is in both binding sites A and B. A surface representation of α-glucosidase shown as the DG color scheme (neutral oxygen (pink), nitrogen atoms (light blue), charged oxygen (red) and nitrogen atoms (dark blue)). 534 Acta Chim. Slov. 2022, 69, 526–535 Şenkardeş et al.: Synthesis, Molecular Docking Studies and ADME ... ligands ACB, GLC, DNJ and MGL are docked into “site A” of α-glucosidase, while KJB docked into both of the sites. According to the in silico molecular modeling stud- ies, it has been determined that the synthesized compounds 3a–i interact with the binding site A. Binding energies of these compounds have been determined to be between –7.3 kcal/mol and –9.3 kcal/mol as higher than the known inhibitors GLC, DNJ, MGL and KJB. Compared with the binding energy of ACB (–8.1 kcal/mol) used in in vitro in- hibition studies, compound 3a was found to be remarkable with the value of –9.3 kcal/mol, while the other synthe- sized compounds showed similar results. These findings supports the results of in vitro enzyme inhibition studies. All of the synthesized novel benzimidazole derivatives, except 3f and 3i have exhibited hydrogen bond (H-bond) interaction with Arg428, like reference ligand GLC. Also, it was detected that compound 3a exhibited π–anion inter- action with active site residue Glu771, while compound 3i exhibited halogen bond interaction. Compound 3a, which was found to be the most ef- fective compound according to both in vitro and in silico studies, exhibited three H-bond interactions with Arg428. One of the three H-bond interactions were detected with =N– atom of benzimidazole moiety and the others with CO group of semicarbazone moiety. Moreover, a H-bond interaction between the NH group of 2-oxoindolin struc- ture and Asp392 has been detected. Additionally, hy- drophobic interactions have been determined between Arg428, Glu771 residues and benzimidazole (π-cation), 2-oxoindolin (π-anion) rings, respectively. These hydro- phobic interactions were supported by the π–π stacking interactions of the benzimidazole ring with Phe444 be- sides other hydrophobic interactions between compound 3a and Ile451, Leu563, Trp709 and Trp710 (Figure 4). Fi- nally, it has been detected that the compound 3a exhibits more hydrophobic interactions than all reference ligands and synthesized compounds and these findings contribute to having the highest binding energy. 4. Conclusions The targeted 2-(substituted arylidene)-N-(5-(pro- pylthio)-2,3-dihydro-1H-benzo[d]imidazol-2-yl)hydra- zine-1-carboxamides 3a–i were synthesized in good yields. All of the molecules demonstrated encouraging inhibitory activity against α-glucosidase which was also supported by molecular docking studies. Docking studies revealed that compound 3a is the most active compound with the highest binding energy value of –9.3 kcal/mol. As a result, it has been determined that the findings obtained from in vitro and in silico α-glucosidase enzyme inhibition studies were compati- ble. These compounds revealed also reasonable in silico phys- icochemical and pharmacokinetic parameters (ADME). The present findings may invite researchers to work in the area of development of the α-glucosidase inhibitors. Conflict of Interest Authors declare no conflict of interest. 5. References 1. G. Wang, M. Chen, J. Qiu, Z. Xie, A. Cao, Bioorg. Med. Chem. Lett. 2018, 28, 113–116. DOI:10.1016/j.bmcl.2017.11.047 2. J. Zhang, H. Xie, Y. Li, K. Wang, Z. Song, K. Zhu, L. Fang, J. Zhang, C. Jiang, Bioorg. Med. Chem. Lett. 2017, 52, 1–5. DOI:10.1016/j.bmcl.2017.09.048 3. E. S. Moghadam, M. H. Tehrani, R. Abdel-Jalil, M. A. Fara- marzi, M. Amini, Polycycl. Aromat. Compd. 2021, 1–19. DOI:10.1080/10406638.2021.1962369 4. H. Bischoff, Clin. Invest. Med. 1995, 18, 303–311. DOI:10.1007/978-3-322-89194-5_2 5. A. J. Krentz, C. J. Bailey, Drugs. 2005, 65, 385–411. DOI:10.2165/00003495-200565030-00005 6. S.-H. Hsiao, L.-H. Liao, P.-N. Cheng, T.-J. Wu, Ann. Pharma- cother. 2006, 40, 151–154. DOI:10.1345/aph.1G336 7. N. K. N. A. Zawawi, M. Taha, N. Ahmat, A. Wadood, N. H. Is- mail, F. Rahim, S. S. Azam, N. 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DOI:10.1016/j.addr.2012.09.019 Povzetek S pomočjo kondenzacije N-(5-(propiltio)-1H-benzo[d]imidazol-2-il) hidrazinkarboksamidov (2) z ustreznimi ketoni ali aldehidi smo sintetizirali serijo novih 2-(substituiranih ariliden)-N-(5-(propiltio)-2,3-dihidro-1H-benzo[d]imida- zol-2-il)hidrazin-1-karboksamidnih derivatov 3a–i. Kemijske strukture pripravljenih spojin smo potrdili z analitskimi in spektroskopskimi metodami. Za pripravljene spojine smo določili njihovo inhibitorno aktivnost na α-glukozidazo; vse spojine, razen 3h, so se izkazale kot boljši inhibitorji od akarboze. Še posebej zanimiva je spojina 3a, ki je pokazala največjo aktivnost (IC50 vrednost 12.88±0.98 μM) izmed vseh sintetiziranih derivatov. Da bi raziskali vezavne interakcije, smo izvedli tudi študije molekulskega sidranja. Spojina 3a je pokazala največjo vezno energijo (ΔG = –9.4 kcal/mol) in največje število interakcij z aktivnim mestom z vodikovimi vezmi. Tudi rezultati in silico študij se dobro ujemajo z rezu- ltati in vitro raziskav. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 536 Acta Chim. Slov. 2022, 69, 536–551 Vargas et al.: Nickel Removing by Electrocoagulation of ... DOI: 10.17344/acsi.2022.7408 Scientific paper Nickel Removing by Electrocoagulation of Ni(II)-NH3-CO2-SO2-H2O System. Kinetics, Isothermal, Mechanism and Estimated Cost of Operation Armando Rojas Vargas,1,* Margarita Penedo Medina,2 Alba González Vives,3 Noureddine Barka4 and Aymara Ricardo Riverón5 1 Empresa de Servicios Técnicos de Computación, Comunicaciones y Electrónica “Rafael Fausto Orejón Forment”, Nicaro, Holguín, Cuba 2 Universidad de Oriente, Facultad de Ingeniería Química, Santiago de Cuba, Cuba 3 POLYMAT, Departamento de Ciencia y Tecnología de Polímeros, Universidad del País Vasco, España 4 Sultan Moulay Slimane University of Beni Mellal, Multidisciplinary Research andInnovation Laboratory, FP Khouribga, BP.145, 25000, Khouribga, Morocco. 5 Centro de Investigaciones del Níquel “Alberto Fernández Montes de Oca”, Nicaro, Holguín, Cuba * Corresponding author: E-mail: arojas@eros.moa.minem.cu) Tel: +53-24-51-6695 Received: 02-09-2022 Abstract This study reports nickel removing by electrocoagulation of Ni(II)-NH3-CO2-SO2-H2O system at laboratory scale. Ex- periments were done using Al/Al pair electrodes at initial nickel concentration between 293 and 1356 mg L–1 and under operation parameters of pH 8.6, current density 9.8 mA cm–2, electrolysis time 30 min, and temperature 60 °C. The obtained results show removal efficiencies between 97.7 and 99.7%. Kinetics modeling suggested combined effects of external diffusion and nucleation, and as controlling step the chemical reaction and a possible autocatalytic contribution. The process followed the Langmuir´s isotherm with a maximum adsorption capacity of 7519 mg g–1. ICP-OES, XRD and FTIR characterization of the precipitates indicated a typical Ni-Al layered double hydroxide structures with 33.4–40.7% nickel and 6.3–7.0% aluminum depending on initial nickel concentration. The operation costs of energy and electrode consumption were 320–537 $ t–1 of removed nickel. Keywords: Electrocoagulation – isotherm – kinetic – layered double hydroxides –mechanism – nickel removing 1. Introduction In the production plant located in Punta-Gorda Cuba, the Ammoniacal Carbonate Leaching Technology is used for the selective recovering of nickel and cobalt form lateritic ore. In the distillation effluents a suspension of basic nickel carbonate is obtained.1 After sedimenta- tion of this suspension, the clear liquor contained several ionic species with composition according to the following proportions: 1.8 < Ni/S < 3.2, 1.5 < NH3/CO2 < 2.0, 10.4 < CO2/S < 13.8 of the Ni(II)-NH3-CO2-SO2-H2O system. The temperature of the liquor is between 70 and 85 °C and the pH from 7.4 to 9.0.2 The dissolved nickel in the clear liquor reaches con- centrations between 0.2 and 1.0 g L–1 in the form of hy- droxide and coordination compounds.1,2 It precipitates with NH4HS in a piston flow reactor leading to nickel sulfide.3 The reagents used in this process are toxic, cor- rosive and of high hazard for the environment. For these reasons, the possibility of substituting chemical precipita- tion by electrocoagulation (EC) process was analyzed in our previous study.4 The EC consists of the destabilization of suspended, emulsified or dissolved compounds in an electrolytic cell facilitating their removal.5 In relation to the mechanisms of the process, the fundamental stages have been reported:6–12 537Acta Chim. Slov. 2022, 69, 536–551 Vargas et al.: Nickel Removing by Electrocoagulation of ... 1 – Electrolytic reactions on the surface of the electrodes. 2 – Formation of coagulants in the aqueous phase. 3 – Destabilization and adsorption of pollutants on coagu- lants (coagulation). 4 – Aggregation of destabilized particles and formation of flocs (flocculation). 5 – Removal of contaminating material by means of sec- ondary treatment. It also refers to the contribution of mechanisms functioning synergistically and benefit the removal effi- ciency such as: chemistry precipitation by the formation of the pollutant metal hydroxides, reduction of metal ions, non-metal inions and gases formation at the cathode sur- face, co-precipitation and complexation of anions and or- ganic compounds.8,9,11,13, The parameters that influence the efficiency of the EC process can be classified into two categories: design parameters and operational parameters. The most impor- tant design parameters are related with material, shape, arrangement and spacing of electrodes, as well as type of power supply; either direct current (DC), alternating current (AC) or alternating pulsed current (APC). The operational parameters are current density, electrocoagu- lation time, aqueous solution pH, temperature, agitation speed, initial ions concentration and supporting electro- lyte.4,7,8,10,13,14,15 The most favorable conditions for the nickel removal from Ni(II)-NH3-CO2-SO2-H2O system by EC using Al/ Al pair electrodes were determined through a full-factorial experimental design.4 The optimum efficiency of 95% was achieved for a current density of 9.8 mA cm–2, tempera- ture of 60 °C, solution pH of 8.65 and 660 mg L–1 of ini- tial nickel concentration. This resulted in a specific energy consumption of 5.41 kWh per kg of Ni. Many authors have identified the formation of Hy- drotalcite-like layered double hydroxides (LDHs) during EC process. Zhao (2010) proposed the formation of Mg/ Al-F-LDH as one of the mechanisms for EC defluoridation in systems containing both F– and Mg2+.16 Mendoza, et al. (2018) in-situ synthesized Mg/Al-LDH using synthetic water under laboratory-scale conditions, with aluminum and AZ31 magnesium alloys electrodes at 5 mA cm–2, the coagulants were generated through electrochemical oxi- dation of the electrodes.17 Jiang (2021) in-situ synthetized Zn/Al-LDH for the removal of strontium in a simulated liquid radioactive waste.18 Finally, Ou (2021) fabricated Ni/Fe-LDH using nickel-plating wastewater.19 LDHs are represented by the general formula [M2+1-xM3+x(OH)2]x+(An–x/n) mH2O, where M2+ is a di- valent cation (Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+), M3+, is a trivalent cation (Al3+, Cr3+, Mn3+, Fe3+, Co3+, Ni3+), An–, interlayer anion (Cl–, NO3–, ClO4–, CO32–, SO42–, S2O32– and other organic compounds), and x is the charge density for the molar ratio M3+ (M2+ + M3+)–1 which varied from 0.2 and 0.35.20–31 These compounds have been extensively investigated due to their improved microstructure, increased active electrochemical sites and their wide applications. In the case of Ni/Al-LDH, it has been reported as highly efficient in the adsorption of metals (Au, Cd, Cu, Pb, Se),28,32 anions (F–, IO3–)33,34 and organic compounds.24–26,29,35,36 Ni-based LDHs in the energy storage and conversion field are still limited by their intrinsically poor conductivity, ag- gregation, limited active sites and stability.23,31 Ni/Al-LDH exhibits a specific capacitance 2128 F g−1 at 1 A g−1 and coulombic efficiency above 80% during 1000 cycles (Ni/ Al:3).37,38 In order to improve the electrochemical perfor- mance, nanostructured Ni/Al-LDH have been synthetized using different routes.23,39,40,41 This compound, followed by controlled thermal decomposition, represents an appropri- ate material for the preparation of ceramic pigments with different properties.42 Carbonate intercalated with a c-axis preferred orientation, show excellent anticorrosive perfor- mance with polarization current density of 10–9 A cm–2.43 It is active for the photocatalytic conversion of CO2 to CO in water, under UV light irradiation,44 and promising cat- alyst precursors for fine CO2 removal from hydrogen-rich gas streams through the methanation reaction and methane dry reforming.45,46 Moreover, the combination of nickel and aluminum finds applications in the production of superal- loys (53.3 ≤ Ni ≤ 73.0%, 1.2 ≤ Al ≤ 6.0%) and permanent magnets (15 ≤ Ni ≤ 26%, 8 ≤ Al ≤ 12%). It was assumed that the thermodynamics, kinetics, equilibrium analysis through adsorption isotherms, char- acterization of the adsorbent, and the analysis of chemi- cal-physical interactions through Stern’s electrical double layer model, coordination surface and the electrode pro- cesses, provide elements to propose the removal mecha- nism by electrocoagulation.30,47,48 The purpose of this work was to determine the reac- tion kinetics, the adsorption isotherm, the mechanism and the preliminary cost of operation for the nickel removing by electrocoagulation from the Ni(II)-NH3-CO2-SO2- H2O system, at different concentrations of dissolved nickel in the initial liquor. The resulted precipitate was character- ized by ICP-OES, DXR, and FTIR in order to elucidate the removal mechanics. 2. Materials and Methods 2. 1. Materials The liquor used in the electrocoagulation experi- ments was sampled spot in the distillation columns dis- charge at the production plant in Punta-Gorda Cuba. The pH was adjusted with ammonium carbonate solution (pH 11.7) or a mixture of hydrochloric and nitric acid. The in- itial nickel concentration was adjusted by dilution of the liquor using distilled water. The resulting concentrations for each sample are shown in Table 1. The material used as electrode was aluminum with a composition of 98.98% Al, 0.5% Mg, 0.33% Fe and 0.114% Si. 538 Acta Chim. Slov. 2022, 69, 536–551 Vargas et al.: Nickel Removing by Electrocoagulation of ... Table 1. Characterization of the liquor fed to the electrocoagulation cell Ni NH3 CO2 S [SO4]2– (mg L–1) (g L–1) (g L–1) (g L–1) (g L–1) 293 0.51 0.33 2.70 3.59 379 0.92 0.50 2.14 3.52 474 1.10 0.25 2.43 3.53 505 1.08 0.29 2.31 3.46 646 1.40 0.30 2.73 5.70 775 1.20 0.35 3.00 3.46 953 1.21 0.35 3.47 6.83 1356 4.70 3.27 3.20 6.82 2. 2. Methods EC experiments were done in an electrochemical cell consisted of a discontinuous cylindrical glass reactor, with a useful capacity of 500 mL. It was equipped with a pair of flat electrodes, arranged vertically, in parallel, 10 mm spacing, submerged 57 mm in the liquor with a total area of 5.6 10–3 m2 and an effective area of 4.6 10–3 m2. The cell was alimented by Direct current source of 0.01 - 30 V, maximum amperage 10 A, power supply 220 ±10%, 50 Hz and 250 W. The current density was monitored using a multimeter. The positive terminal of the current source was connected directly to the electrode (anode) and the negative terminal to the multimeter and from this (COM) to the cathode. The source allowed to regulate the voltage to keep the electric current amperage constant (Fig.1). nickel removal from the Ni(II)-NH3-CO2-SO2-H2O sys- tem.4 The nickel removal, electrode mass and electric pow- er consumption were determined at different concentra- tion of nickel [Ni] dissolved in the initial liquor. After each experiment, samples were removed from the reactor to a volumetric flask. Then, they were covered and allowed to settle for 24 h. Finally, the aliquot required for chemical analysis was pipetted. Residual nickel con- centration was measured by atomic absorption spectros- copy (AAS) using a SP-9 Spectrophotometer. The preparation of the electrodes consisted of pol- ishing the surface with coarse and fine sandpaper, and washing with distilled water. After electrocoagulation, they were cleaned with phosphoric acid solution, sodium hexametaphosphate and distilled water until the deposited layer was removed. Later these electrodes were weighed. Each anode was used for at most two experiments. an Op- tical emission spectrometer GS 1000-II was used to char- acterize the electrodes. The resulting precipitate was characterized using Inductively Coupled Plasma Optical Emission Spectrom- etry (ICP-OES), Spectro ARCOS FHX. X-ray diffraction (XRD), Bruker D8 Advance equipment, Cu anode lamp (CuKα radiation) and wavelength 1.5405 Å, constant scan- ning at a measurement interval of 2theta (2θ) between 5 – 6 to 100º with a step of 0.05º measured every 5 min, and Fourier transform infrared spectroscopy (FTIR), Nicolet 6700 Spectrometer, range between 4000 and 400 cm–1, res- olution of 4 cm–1. The overflow liquor from the basic nickel carbonate settler tank was first adjusted to the desired pH using a Philips PW-9420 pH meter and the temperature was con- trolled ASCON KR3 controller. The it was fed to the re- actor and continually stirred at 100 rpm using a hot plate stirrer with thermal control. The nickel removal experiments by electrocoagula- tion consisted in assuming the current density of 9.8 mA cm–2, pH 8.6, temperature 60 °C and electrolysis time 30 min, according to the most favorable conditions for the 2. 3. Adsorption Kinetics Models The adsorption capacity (Qt) or amount of adsorbate adsorbed per adsorbent unit (mg g–1) was determined by Eq. (1). (1) where C0 (mg L–1) is the initial concentration of nickel, Ct (mg L–1) is the concentration of nickel in the liquid phase in each time interval, V (L) is the volume of solution, ΔMF Figure 1. Experimental installation of electrocoagulation 539Acta Chim. Slov. 2022, 69, 536–551 Vargas et al.: Nickel Removing by Electrocoagulation of ... (g) is the amount of metal in solution according to Fara- day’s Law, Eq. (2). (2) where, I (A) is the current intensity, M the molecular weight of [Al] 26.98 g mol–1, t (s) the electrocoagulation time, n number of electrons for aluminum (3), F Faraday constant (96487 c mol–1). When the duration of the process is long enough, Qt is constant and determines the charge or adsorption ca- pacity (Qe, mg L–1) corresponding to the concentration at equilibrium (Ce, mg L–1). Kinetics data were correlated to pseudo-second order, Avrami, Elovich, Bangham and Weber-Morris in- tra-particle diffusion models.48,49,50 The parameters were adjusted with StatGraphic 5.1 and Microsoft Excel and the best quality of fit was decided by the highest coefficient of determination (R2). The pseudo-second order kinetic model is presented by Eq. (3) and its linear form is given by Eq. (4): (3) (4) where k2 (g mg–1 min–1) is the adsorption rate constant while h (g–1 mg min–1) is assumed as the initial reaction rate. Avrami’s fractional kinetic model is based on the Johnson-Mehl-Avrami-Erofeev-Kolmogorov (JMAEK) theory,51 and consist of phase transformations via homog- enous and spontaneous nucleation and growth of a crystal as a function of crystallization time. Although it has been assumed as an empirical model for the analysis of adsorp- tion kinetic data.47 It is represented by Eq. (5), integrated form (6) and linearized form (7). (5) (6) (7) where, kav (min–1) is the kinetic constant or global con- stant, nav (/) fractional reaction order, which refers to the nucleation, growth and orientation of crystallites or possi- ble changes in the adsorption mechanism. The Elovich kinetic model in its nonlinear and linear form is expressed by the Eq. (8) and (9), respectively. (8) (9) where, α (mg g–1 min–1) is a constant related to adsorption rate, β (g mg–1) is a constant which depicts the extent of surface coverage. Bangham’s equation was used to evaluate whether the adsorption is pore-diffusion controlled, it is represent- ed by Eq. (10). (10) where, C0 (mg∙L–1) is initial concentration, V (mL) volume of the solution, W (g L–1) weight of the adsorbent, kB (mL g–1 L) and α(/) the constants. The Weber and Morris intraparticle diffusion mod- el can be expressed by Eq. (11) and its linear form by Eq. (12). (11) (12) where, k3 (mg g–1 min–0.5) the intra-particle diffusion rate constant, c (mg g–1) is the intercept. In addition, the goodness of fit of several integral equations for the reaction kinetics was evaluated, in order to investigate the controlling mechanism in the nickel re- moving by electrocoagulation, regarding the individual or simultaneous contribution of the resistances: external dif- fusion, internal diffusion, nucleation, chemical reaction, autocatalysis (Table 2).52,53,54 The algorithm followed con- sisted of assuming a controlling mechanism, calculating the fraction of incomplete conversion and adjusting the model with StatGraphic 5.1 and Microsoft Excel, the best quality of fit was decided by the highest coefficient of de- termination (R2) and the lowest estimated error, Eq. (13). (13) where, I (/) fraction of incomplete conversion and x frac- tional conversion (/). 2. 4. Adsorption Isotherm Models The Langmuir, Freundlich, Temkin, Toth, Koble – Carrigan and Redlich – Peterson adsorption isotherm models were evaluated. 10,25,35,48,49,50 Langmuir´s isotherm in the linear form is shown in Eq. (29), and the equilibrium parameter is defined in Eq. (30). (29) (30) where, qm (mg g–1) is the maximum monolayer adsorption capacity, KL (L mg–1) the Langmuir adsorption constant that defines the affinity of the adsorbate for the adsorbent, 540 Acta Chim. Slov. 2022, 69, 536–551 Vargas et al.: Nickel Removing by Electrocoagulation of ... and RL is the equilibrium parameter of the Langmuir´s isotherm. Freundlich´s isotherm is applicable to adsorption processes that occur on heterogonous surfaces, its linear form is expressed by Eq. (31). (31) where, Kf (mg g–1)/(mg L–1)n is related to the adsorption capacity and n (dimensionless) is related to the adsorption intensity; it also indicates the relative distribution of the energy and the heterogeneity of the adsorbate sites. Temkin isotherm model takes into account the ef- fects of indirect adsorbate/adsorbate interactions on the adsorption process, Eq. (32) and (33). (32) (33) where, bT (J mol−1) is Temkin constant which is related to the heat of sorption and KT (L mg−1) is Temkin isotherm constant, T (K) the absolute temperature, R is the gas con- stant 8.31 J mol–1 K–1. The Toth´s isotherm is an empirical modification of the Langmuir equation, Eq. (34) and (35). (34) (35) where, Kh (mg g−1) is Toth isotherm constant and n (mg g−1) is the Toth constant. Koble-Carrigan isotherm model is a three-parame- ter equation which incorporates both Langmuir and Fre- undlich isotherms for representing equilibrium adsorp- tion data, Eq. (36). (36) where, Ak (Ln mg1–n g–1), Bk, (L mg)n, n (dimensionless) are Koble - Carrigan’s isotherm constants. The Redlich-Peterson isotherm is a mix of the Lang- muir and Freundlich isotherms. Its linear form can be ex- pressed by the Eq. (37). (37) where, KR (L g−1) is Redlich-Peterson isotherm constant, β(dimensionless) is constant. The verification of the consistency of adsorption models and the theoretical assumptions of adsorption models was made by Average Relative Error (ARE) and Marquardt’s Percent Standard Deviation (MPSD) calculat- ed by Eq. (38) and (39) respectively.35 (38) (39) where, n is the number of data points and P the number of parameters. 2. 5. Operating Cost Estimate The operating cost per kg of nickel removed was cal- culated by Eq. (40). (40) where, Cop ($ kg–1) operating cost, a ($ 0.090 / kWh) cost Table 2. Models used in the kinetic analysis to investigate the con- trolling mechanism in the nickel removing by electrocoagulation Name g(I, x) Eq. External diffusion 1-D (14) 2-D (15) 3-D (16) Boundary layer (17) Internal diffusion 1-D (18) 2-D (19) 3-D (Jander) (20) 3-D (Ginstling – Brounshtein) (21) Nucleation Avrami (22) Erofeév (23) Avrami – Erofeév (24) JMAEK (25) Autocatalysis Roginskii-Shultz (26) Kolmogorov (27) Chemical reaction Power law (28) 541Acta Chim. Slov. 2022, 69, 536–551 Vargas et al.: Nickel Removing by Electrocoagulation of ... of electricity, Cen (kWh) power consumption, b (1.445 $ kg–1) cost of the aluminum electrode; ΔMexp (g) experi- mental weight loss of the electrodes, mNi (kg) mass of nick- el removed. After transforming, the operating cost can be ex- pressed as Eq. (41): (41) where, U (V) voltage, I (A) current intensity, t (min) elec- trocoagulation time, [Ni] (g L–1) initial concentration of dissolved nickel, V (0.5 L) useful volume of the cell, xNi fraction converted or nickel removing. The current efficiency (η) and the specific energy consumption per kg electrode dissolved (SEC, kW-h kg–1) were determined by Eq. (42) and (43), respectively. (42) (43) 3. Results and Discussion 3. 1 Adsorption Kinetics The study of adsorption kinetics provides informa- tion on the mechanisms involved in the process. For the experimental conditions of 9.8 mA cm–2, 60 °C, pH 8.6, 30 min of electrolysis and initial concentration 293 ≤ [Ni] ≤ 953 mg L–1, the nickel removal efficiency was between 99.0 ≤ X ≤ 99.7% (Table 3). Table 3. Efficiency of nickel removal by electrocoagulation Ni (mg L–1) 293 379 474 505 646 775 953 13561 X (%) 99.7 99.3 99.3 99.5 99.0 99.0 99.0 97.7 140 min of electrolysis A model was obtained that relates the conversion time (t) as a function of the fractional conversion (x), nick- el initial concentration (mg L–1), mass of aluminum [Al] and the coefficients o constants a, b, c, d, e, f, Eq. (44). (44) From Faraday’s law to determine the mass of dis- solved aluminum, Eq. (45) is obtained. (45) where, the constant (n) refers to the conversion time, the resistance coefficient to external diffusion (a), nucleation (b), chemical reaction (c) and its autocatalytic contribu- tion (d), the nickel exponent (e), and the coefficient (kAl) for the estimate of dissolved aluminum by Faraday’s Law. The parameters of the conversion time (CVT) model (45) are shown in Table 4 for the concentration ranges: 293 ≤ Ni ≤ 646 mg L–1; 775 ≤ Ni ≤ 1356 mg L–1; 293 ≤ Ni ≤ 1356 mg L–1. It reflects between 99.18 and 99.88% of the variability in nickel removal. The coefficient of determina- tion (R2) adjusted by the degrees of freedom (g.l.) allows compare this model with others with the same number of independent variables. CVT model expresses that the nickel removing is de- termined by the combined effect of the resistances of the mechanisms: – External diffusion (a), in the film or boundary layer to the adsorbent surface, by the two-dimensional (2-D) dif- fusion model. – Nucleation and crystallization (b), by the JMAEK equa- tion, which refers to the random formation and growth of the adsorption surface due to the hydrolysis and po- lymerization reactions of aluminum, giving rise to the species monomeric, polymeric, oligomeric aluminum and Al(OH)3, where the contaminants adsorption oc- curs (Ni2+, SxOyz–, CO32–, NH3) in the active centers by electrostatic interaction and coordination surface, and subsequent crystallization; in competition with that nu- cleation that occurs when the deposits grow on the elec- trodes. Table 4. CVT model constants by Eq. (45) and quality of fit n a b c d e kAl · 10–2 R2 R2 (g. l.) 1 0.2938 8.8582 1.1429 12.4190 10.4680 0.3054 1.4834 99.41 99.18 2 0.3029 0.9418 1.7497 16.7428 18.7548 0.2812 1.5661 99.91 99.88 3 0.2985 9.9782 2.6673 13.2262 20.1508 0.2337 1.5250 99.35 99.24 1[Ni] between 293 and 646 mg L–1; 2[Ni] 775 - 1356 mg L–1; 3[Ni] 293 - 1356 mg L–1. 542 Acta Chim. Slov. 2022, 69, 536–551 Vargas et al.: Nickel Removing by Electrocoagulation of ... – The chemical reaction (c) in the active centers of the adsorption surface, by the model of spherical particles according to the Power Law. – Contribution to the chemical reaction due to the syn- ergistic effect that promotes the nickel removing and a possible autocatalytic effect is estimated, according to the Roginskii - Shultz equation (e). When the nickel concentration increases between 775 and 1356 mg L–1, there is a greater effect on the remov- al by the chemical reaction mechanism due to the process- es that exert a synergistic effect on the process; while in the lower interval (293 ≤ Ni ≤ 646 mg L–1), the chemical reac- tion and external diffusion predominate which can be seen by the coefficient’s values of the kinetic model, (Table 4). The adsorption kinetic models were ordered by their quality of fit: (1) pseudo 2nd order ≈ (2) Avrami > (3) Elovich ≈ (4) Bangham >> (5) Weber-Morris. These were used to validate the conversion time model, Eq. (45). The pseudo-second order (PSO) model showed a high quality of fit (96.1 ≤ R2 ≤ 99.8%). As the initial nickel Figure 2. Adsorption of nickel with the electrode pair Al/Al at 9.8 mA cm–2, 60 °C, pH 8.6, a) Pseudo-second order kinetic model, b) Avrami´s model, c) Elovich`s model, d) Bangham´s model, e) We- ber and Morris´s model. 543Acta Chim. Slov. 2022, 69, 536–551 Vargas et al.: Nickel Removing by Electrocoagulation of ... concentration increased, (Fig.2 a), the rate constant k2 (g mg–1 min–1) decreased. This result can be attributed to the progressive saturation of the active sites in the adsorption surface with the cation [Ni2+] and causes an increase in the necessary electrocoagulation time. The Avrami´s model was representative of dates be- cause of its high quality of fit. The removal rate (kav) for in- itial nickel concentration between 293 and 646 mg L–1 was assumed constant and equal to 0.20 (+/- 0.01) min–1 (97.94 ≤ R2 ≤ 99.62%); but in the range of 775 to 1356 mg L–1 the kinetic behavior changed and kav decreased between 50 and 60% (98.82 ≤ R2 ≤ 99.75%) due to the increase in ad- sorbate concentration. It was regarded that in the first in- terval the contribution of the mechanism of external diffu- sion resistance influenced in the higher value of kav, while in the second interval kav was lower under the mechanism control the chemical reaction resistance (Fig.2 b) (Table 4). With respect to the lines slopes that reflect the frac- tional order (nav) of Avrami´s model, in the interval 293 ≤ [Ni2+] ≤ 646 mg L–1 decreased from 1.39 to 0.66 with the increase of the cation [Ni2+]. This is attributed to the pro- gressive saturation of the active adsorption sites because there is a greater amount of adsorbate that reaches the ad- sorbent surface and therefore a longer electrocoagulation time is required. Also, the interactions augment and the tendency to change the controlling mechanism. In the in- terval of 775 ≤ [Ni2+] ≤ 1356 mg L–1, the exponential con- stant (nav) increased to 1.21 (+/- 0.05). According to Eq. (44), it may be associated with the controlling mechanism of chemical reaction resistance. The Elovich´s model (95.5 ≤ R2 ≤ 99.62%) suggests that the adsorbent active sites are heterogeneous and therefore exhibit different activation energies. This sug- gests that more than one mechanism incises the removal process such as transport in the solution phase (bulk dif- fusion) and surface diffusion.48,49,50 The initial rate kinetic constant (α) (mg g–1 min) increased proportionally to the concentration of [Ni2+]. In addition, the constant (β) (g mg–1) related to the chemisorption activation energy and the extension of the adsorption surface, decreased with the increase of cation [Ni2+] throughout the interval (Fig.2 c). The Bangham and Weber-Morris´s models were less representatives of the data due to their lower quality of fit, in correspondence with the CVT model, Eq. (45) where internal diffusion resistance could be omitted from the process due to low statistical significance. The Bangham´s model is applied to investigate pore activation for adsorb- ate diffusion. The fit quality was obtained in the interval 95.0 ≤ R2 ≤ 99.3%, which indicates that both intra-parti- cle diffusion and pore diffusion are not controlling in the process (Fig.2 d).50 With regard to the Weber and Morris´s model, it reflects the influence of external mass transfer followed by intra-particle diffusion in pores of different sizes.48,50 The plot of Qt versus t0.5 did not result in a line- ar relationship with intercept at the origin of coordinates (86.5 ≤ R2 ≤ 99.1) (Fig.2 e). This result suggests that diffu- sion is not a limiting step in the mechanism. Furthermore, the intra-particle kinetic rate constant was not directly proportional to the adsorbate concentration, suggesting that the process is not controlled by adsorption in the pores. 3. 2 Nickel Adsorption Isotherms The equilibrium concentration (Ce, mg L–1) corre- sponding to each initial nickel concentration (C0, mg L–1), and the equilibrium adsorption capacity (Qe, mg g–1) were determined. From Fig.3 it can be seen that by increasing the initial concentration, the adsorption capacity at equi- librium increased. For [Ni2+]>953 mg L–1, the formation of a plateau was obtained, which indicates saturation of the adsorption sites and a decrease in the removal efficiency at the experimental conditions studied. Figure 3. Equilibrium concentration (Ce) and adsorption capacity (Qe) versus the initial concentration (C0) for nickel removing at 9.8 mA cm–2, 60 °C and pH 8.6. Table 5 shows the isotherm constants for the adsorp- tion of Ni(II) caclutated for rach isotherm model. That table indicates that the order of goodness-of-fit (R2) of the adsorption isotherm models was: Langmuir (99.3%) > Redlich - Peterson (97.3%) > Koble - Carrigan (96.1%) ≈ ToTh (96.1%) > Temkin (93.8%) ≈ Freundlich (93.7%). The Langmuir isotherm was more representative of the data, this presented the highest quality of fit deter- mined by the coefficient of determination (R2), the lower ARE 7.6 and MPSD 0.013. This result suggests monolay- er adsorption in a specific number and fixed of accessible sites on the adsorbent surface, all active sites have the same energy. Once an adsorbate occupies a site, no farther ad- sorption can occur on that site and there is not interaction 544 Acta Chim. Slov. 2022, 69, 536–551 Vargas et al.: Nickel Removing by Electrocoagulation of ... between adsorbate species.48,49,50 The maximum adsorp- tion capacity (qm) was 7519 mg g–1, the constant (KL) was 0.216 L mg–1 and the equilibrium parameter 0.003 ≤ RL ≤ 0.013. The Redlich-Peterson´s isotherm (KR 4.84 10–4 L g–1; beta-β 0.61) and the Koble-Carrigan´s isotherm (Ak 1429 Ln mg1–n g–1, Bk 0.14 (L mg)n, n 1.08) refers that adsorption is a mixture (Langmuir and Freundlich) and not precise- ly the ideal adsorption monolayer. While the Toth´s iso- therm (Kh 0.251 mg g–1, n 5.5 mg g–1) is a modification of the Langmuir´s equation and suggest a heterogeneous adsorption (n > 1). The Temkin´s model assumes linear rather than log- arithm decrease of heat of adsorption while ignoring ex- tremely low and very high concentration. It also assumes uniform distribution of bounding energy up to some max- imum bonding energy.35,50 The heat of adsorption, bT, is equal to 1.79 J mol–1 and KT was 2.88 L mg–1. Eventually, the lower value of the determination coefficient corresponded to the Freundlich´s isotherm, which assumes a heterogeneous distribution of active sites and energy on the surface, applicable to multilayer adsorp- tion.48,50 Kf was 2001 (mg g–1)/(mg L–1)n and n was 2.51. Thus, the kinetic and equilibrium analyses suggest the control of chemisorption on a monolayer, at a fixed and specific number of accessible sites on the adsorbent surface. Although, it does not specifically follow the ide- al adsorption monolayer at identical sites. The interaction between the molecules is not neglected, due to the action of electrostatic forces and exchange reactions in the active sites of the coordination surface. In addition, the transport of solute through the internal structure of the adsorbent pores and the diffusion in the solid are neglected. 3. 3 Analysis of the Precipitate In order to investigate the nickel removal mechanism by electrocoagulation the precipitate was analyzed. The ICP-OES, DXR and FTIR analysis showed the formation of Ni/Al layered double hydroxide [Ni/Al-LDH] interca- lated by [NH3], [SO42–] and [CO32–] as the main product, and accompanied by phases impurities. From XRD patterns (Fig. 4a), the largest diffraction peaks were obtained at 2theta (2θ) Bragg angles of 10.745°, 22.101°, 34.922° and 61.067°, which are assigned to the crystalline planes, according to the Miller indices (hkl): (003), (006), (012), (110) respectively, are also of interest at 46.43° (018) and 72.676° (119). These diffraction peaks are indexed on a hexagonal system with rhombohedral sym- metry, special group R-3m (polytype of three layers). The presence of 0kl peaks anticipates the presence of stacked layers (JCPDS file 15-0087).22,27,28,34,39,55 The XRD pattern also showed phases impurities. By comparison of the characteristic reflection pattern in Fig.4a to a reference library of samples, the low intensity peaks can be attributed to the bayerite polymorphs Al(OH)3 and aluminum hydroxide or gibbsite [γ-Al(OH)3], (JCPDS 33-0018, JCPDS 20-0011, JCPDS 24-0006).15,40,56 Also nickel hydroxide [Ni(OH)2] indexed to the hexagonal [β-Ni(OH)2], the [Ni(OH)2 0.75H2O], nickel oxy-hydrox- ides corresponding to [β-NiOOH] and [γ-NiOOH] phases can be identified (JCPDS 14-0117, JCPDS 38-0715, JCPDS 06-0141, JCPDS 06-0075)57,58,59–61 The presences of nick- el aluminate were also identified: [NiAl2O4], [NiAl26O40], [NiAl32O49] and [Ni2Al18O29] (JCPDS 10-0339, JCPDS 20- 0776, JCPDS 20-0777, JCPDS 22-0451).62,63 These phases may be a consequence of the decrease in pH during the process from 8.53 (+/-0.07) to 8.35 (+/-0.08). Table 5. Isotherm constants for the adsorp- tion of Ni(II) Parameter Value Langmuir qm (mg g–1) 7519 KL(L mg–1) 0.216 R2 99.30 ARE 7.60 MPSD 0.013 Redlich - Paterson KR (L g–1) 4.84 10–4 (/) 0.61 R2 (/) 97.30 ARE 8.50 MPSD 0.017 Koble - Carrigan Ak (Ln mg1–n g–1) 1429 Bk (L mg)n 0.14 n (/) 1.08 R2 (/) 96.10 ARE 8.52 MPSD 0.026 Toth qm (mg g–1) 6650 Kh (mg g–1) 0.251 n (mg g–1) 5.50 R2 (/) 96.10 ARE 10.00 MPSD 0.081 Temkin 𝐾T (L mg–1) 2.88 bT (J mol–1) 1.79 R2 (/) 93.80 ARE 8.30 MPSD 0.015 Freundlich n (/) 2.51 Kf (mg g–1)/(mg L–1)n 2001 R2 (/) 93.70 ARE 8.31 MPSD 0.015 545Acta Chim. Slov. 2022, 69, 536–551 Vargas et al.: Nickel Removing by Electrocoagulation of ... Figure 4. XRD of Ni/Al-LDH, Cu Kα1, λ=1.540598 Å, at 9.8 mA/ cm2, 30 min, 60 °C and pH 8.6. a) Diffraction intensity for various Bragg reflection angles. b) Interaction of the basal axis c0 with the molar ratio M(II)/M(III). c) Crystallite volume interaction V (Å)3 with M(II)/M(III). Fourier transform infrared spectroscopy (FTIR) spectra of the samples are illustrated in Fig.5. The broad bands that can be seen in the region from 3423 to 3465 cm–1 are assigned to the stretching vibrations hydroxyl group (νOH) in the Ni/Al -LDH, Ni(OH)2, Al(OH)3 and the water molecules adsorbed in the interlayer.25,26,27,36,55 The peaks observed between 2077.9 and 2084.7 cm–1 are associated with stretching vibration of the N-H bond.55 The characteristic bands between 1629 and 1641 cm–1 are attributed the deformation of (HOH) angle of water mol- ecule (δH–O–H) which confirms the presence of water in the Ni/Al-LDH interlayer.17,21,25,26,36 The spectra also shows intense bands located from 1364 to 1368 cm–1 and represent symmetric stretching vibrations carbon-oxy- gen bond (C-O) of carbonate ions n3(CO32–).20,21,55,60 The adsorption peaks from 1108 to 1115 cm–1 correspond- ed to S-O stretching vibrations of the sulfate anion n3(- SO42–).20,24 The characteristic band at 1041 cm–1 represents the vibration ν(Al-OH). Furthermore, in the region be- tween 615 and 617 cm–1 the bands can be assigned to the stretching vibration of metal (M) - oxygen (Ni-O; Al-O; Ni-O-Al), related to the oxides and aluminates determined by DXR.25,36 The peaks between 409.8 and 410.8 cm–1 are assigned to nickel oxides and nickel hydroxides [Ni-O; Ni- O-H–]; and the bands between 566 – 567 cm–1 are attribut- ed to stretching vibrations [Ni3+-O] in [γ-NiOOH].14,25,60 The elemental analysis of the precipitates is giv- en in Table 6. The precipitate had a nickel concentration between 33.40 and 40.68%, aluminum from 6.43 to 7.0% and charge density (x) from 0.256 to 0.36. When the initial concentration of nickel increased, there was a tendency to increase Ni in the precipitate. The table also shows that sul- fate anion was predominant. Figure 5. FTIR spectral of Ni/Al-LDH from different nickel concen- tration, a) 505 mg L–1, b) 646 mg L–1, c) 775 mg L–1, d) 953 mg L–1 From the DXR analysis, the spacing (dhkl) of the LDHs, the crystal lattice parameters (a, c) and the crys- tallite size (Dhkl) were determined (Table 7). Parameters “a” and “c” were calculated using the relationship between the spacing (dhkl) in the planes (hkl): (003), (012), (110) and the lattice parameters (a, b, c) for the hexagonal crystal system (b=c). The data was adjusted using the Statgraphic 5.1 software in the nonlinear regression option. 546 Acta Chim. Slov. 2022, 69, 536–551 Vargas et al.: Nickel Removing by Electrocoagulation of ... The distance between (d003 planes) of the LDH, also called d spacing, basal distance or thickness of the interlay- er gallery was calculated using Bragg’s Law. The obtained values were similar to those of the compounds synthesized by coprecipitation reported in the literature: [Ni/Al-SO42–] (8.01 ≤ d003 ≤ 8.59 Å) and [Ni/Al-NO3–] (7.82 ≤ d003 ≤ 8.76 Å). The variation in the basal distance is due to the varia- tion in the amount (intercalation degree) and type of an- ions (atom size and valence) in the LDH interlayer.24,26,64 The average values of the lattice parameters (+/- standard deviation) were: a=b= 3.01 Å (+/-0.013) and “c” equal to 23.4 Å (+/-0.76), with a fit quality greater than 99.7%, confirming that it is a hexagonal crystalline system. The parameter “a” is equivalent to the average distance be- tween the center of adjacent cations in the lattice; and “c” is the basal axis, which is related to the distance between neighboring atoms and the interlayer distance. These pa- rameters are comparable to the parameters reported for the compounds obtained by coprecipitation: [Ni/Al-SO42– ]-LDH values of “a” 3.03 Å and “c” 24.05 Å; and for [Ni/ Al-CO32–]-LDH in the following ranges: 3.02 ≤ a ≤ 3.08 Å and 22.2 ≤ c ≤ 24.05 Å.21,24,28 The basal axis cell parameter for n-layers is c=n c0. For the polytype 3R with rhombohedral symmetry n=3, and with the lowest reflection (0 0 n) c0 (Å) was calculated. An increase in the basal axis as the molar ratio [Ni2+ / (Al3+ + Fe3+)] increases was observed with a coefficient of deter- mination (R2) equal to 97.42%. This is because the nickel has a larger ionic radius than iron and aluminum (0.69 Å > 0.55 Å > 0.535 Å), (Figure 4 b).21,26 The unit cell volume (V=0.866 a2 c) was 183 Å3 (+/- 6.58), similar to other [CO32–]-LDH obtained by co-pre- cipitation such as [Zn/Al] 189 Å3, [Ni/Al] 187.6 Å3 y [Mg/ Al] 180 Å3 and by the sol-gel method [Ni/Al] 148-163 Å3, the lower the molar ratio [Ni2+ / (Al3+ + Fe3+)] the smaller the volume, (Figure 4 c).21,28 The crystallite size (Dhkl) was calculated using the Scherrer equation and the mean size (D) by the Willia- son-Hall “SSP” method. Both sizes reached lower values than other Ni/Al-LDHs synthetized by coprecipitation, but those were similar to the LDHs obtained by the sol-gel technique [Ni/Al-CO32–] (2.69 ≤ D003 ≤ 8.11 nm). Crys- tallinity increased with increasing temperature, current density and constant alkaline pH.21,34,65 In that order of ideas, the average size of the crystal- lites (D) presented an inversely proportional relationship with the reaction rate constant (kav) of the Avrami´s model, fallowing a linear function (R2 95.92%). Regarding the frac- tional reaction order (nav), it was related to the preferential orientation of the crystallites, according to the peak intensity in the I003/I012 ratio (1.43 ≤ I003/I012 ≤ 1.82) with an inverse relationship and linear trend (R2 98.0%). Liu (2015) used the ratio I003/I012 in the interval 0.2 ≤ I003/I012 ≤ 2.7 to evaluate the orientation of Ni/Al-CO3-LDH. He referred that a higher I003/I012 value indicates that the LDH has a c-axis preferred orientation, while a lower value demonstrates preferential- ly ab-oriented. Based on this criterion, it was supposed that when the fractional reaction order (nav) increases the crys- tallites have a greater tendency to ab-orientation.43 3. 4 Nickel Removal Mechanism Analysis Taking into account the results of the kinetic and equilibrium analysis, the characterization of the product, Table 7. Lattice parameters and size of Ni/Al-LDH crystallites Sample Ni Spacing (Å) Cell parameters V Crystallite (g mL–1) (Å) (Å)3 size (nm) d003 d012 d110 a = b D D D003 505 8.227 2.567 1.516 3.02 23.237 184 5.40 8.45 646 8.305 2.560 1.513 3.00 24.336 174 5.69 8.92 775 8.266 2.546 1.511 3.02 23.460 185 6.35 9.90 953 8.632 2.585 1.507 3.00 22.495 190 6.71 10.5 Table 6. Characterization of Ni/Al-LDH and estimated chemical formulas [Ni2+] Concentration Chemical formulas Ni/(Al+Fe) (%w/w) (molar) Ni Al S Fe 447 33.40 8.64 7.10 0.13 [Ni0.640Al0.360(OH)2] (SO4)0.156 (CO3)0.024 xH2O 1.76 505 37.39 6.30 5.62 0.03 [Ni0.732Al0.268(OH)2] (SO4)0.113 (CO3)0.021 xH2O 2.72 646 38.47 6.67 5.72 0.09 [Ni0.726Al0.274(OH)2] (SO4)0.123 (CO3)0.014 xH2O 2.63 775 39.21 6.41 5.12 0.05 [Ni0.738Al0.262(OH)2] (SO4)0.107 (CO3)0.024 xH2O 2.80 953 40.68 6.43 4.37 0.02 [Ni0.744Al0.256(OH)2] (SO4)0.115 (CO3)0.013 xH2O 2.90 1356* 39.20 7.00 3.75 0.05 [Ni0.720Al0.280(OH)2] (SO4)0.068 (CO3)0.071 xH2O 2.57 *Sample analyzed after electrocoagulation for 40 min 547Acta Chim. Slov. 2022, 69, 536–551 Vargas et al.: Nickel Removing by Electrocoagulation of ... as well as the information consulted in the literature, it is considered that the following reactions control the nickel removing by electrocoagulation of Ni(II)-NH3-CO2-SO2- H2O system, (Fig. 6). 6,7,10,11,15,66 a) Precipitation of nickel hydroxide. b) Co-precipitation of Ni in spinels [NixAlyOz]. c) Precipitation of layered double hydroxides. d) Cathodic electro-reduction to form metallic nickel Where, the anionic ligands [CO32–], [SxOyz–], [NO32–] on the coordination surface, depending on the dissolved Ni concentration, activate a synergism on the process that benefits the removal. (47) (48) (49) Nickel compounds and other contaminants, either colloids, suspended or dissolved material begin to desta- bilize due to:6,7,11,13 1) Compression of the diffuse double layer around the charged species because of the physical-chemical in- teractions with the generated ionic species, by the electro- chemical dissolution of the sacrificial electrode (anode). Due to the simultaneous electrolytic reactions that occur on the surface of the electrodes (step 1 Fig. 6), the electro-coagulant aluminum cation [Al3+] and the hydrox- ide anion [OH–] are produced, Eq. (46) and (47). These diffuse in the solution and spontaneously the hydrolysis of aluminum occurs to form several monomeric and pol- ymeric species, oligomeric complexes and aluminum hy- droxide, Eq. (48) and (49), Step 2.67 (46) These affect the potential difference between the surface of the particles and the solution, thus decreasing the inter- particle repulsive forces. 2) Charge neutralization of the ionic species present in the solution due to the ions of opposite charge generat- ed at the anode and the processes of adsorption, precipita- tion and co-precipitation; thus, the interparticle repulsive electrostatic forces decrease, instead the Van der Walls attraction forces predominate and as a result, coagulation occurs. While monomeric aluminum species neutralize Figure 6. Schematic representation of the nickel removal mechanism by electrocoagulation of the Ni(II)-NH3-CO2-SO2-H2O system. 548 Acta Chim. Slov. 2022, 69, 536–551 Vargas et al.: Nickel Removing by Electrocoagulation of ... the charge of contaminants by adsorbing on their surface and binding to their ionized groups, polymeric species can bind several contaminant particles (or molecules) at once, Step 3. 3) Following destabilization, flocs are formed as a re- sult of aggregation of the destabilized particles, leading to sludge formation (flocculation), Step 4. 4) The hydrogen released in the cathodic reaction (2), enables the electro-flotation of the flocculated parti- cles, which is also favored by the removal of sulfur in the form of hydrogen sulfide (H2S), Step 5. In parallel, mechanisms occur that favor the removal of nickel, as explained below: Adsorption of [Ni2+] in the active centers of the surface of the aluminum species and fundamentally, on [Al(OH)3] in interaction with other ions present in solu- tion provided by the compounds CO2 - SO2 - NH3. This process happens by two mechanisms: electrostatic attrac- tion and coordination surface, Eq. (50), Step 6. (50) Result of simultaneous reactions at the anode and cathode, hydroxide ion is released and nickel hydroxide precipitates, Eq. (51), Step 7. (51) Through sequential co-precipitation, [Ni(OH)2] is incorporated into the crystal structure of [Al(OH)3] and forms spinel: NiAl2O4], [NiAl26O40], [NiAl32O49], [Ni2Al18O29], Eq. (52) and (53), Step 8. (52) (53) Anions in solution are attracted by electrostatic forc- es to balance charges and adsorbed on the active centers of the coordination surface, where (L) represents anionic ligands such as [CO32–], [SxOyz–], [NO32–], [OH–], Eq. (54) and (55).65,68 (54) (55) Subsequently, the adsorbed ions can be displaced by other competing ions in the solution (exchange ad- sorption), due to the interactions between the ions on the charged surface and in the diffuse layer around the surface, and the nickel removal by formation of Ni-Al/LDH is pro- moted. LDH of high purity at alkaline pH and maximum temperature of 80 °C has been prepared by co-precipita- tion, Eqs. (56) - (57), Step 9. 24,26,31,69,70 (56) (57) The hydrogen evolution reaction (HER) (2) occurs at the cathode to a standard potential of –0.826 V with the release of gaseous H2; and the nickel reduction on the cathode surface to a more positive standard reduction po- tential of -0.25 V, Eq. (58), Step 10. (58) The precipitation of [Ni(OH)2], [Ni/Al-LDH] and the co-precipitation of spinels Ni-Al cause a synergistic effect in the process, achieving high efficiency of nickel removing. A greater effect is reached as the initial nickel concentration in dissolution increases, which is reflected by the kinetic model TCV Eq. (45). The possibility of an electrocatalytic effect of Ni2+/ Al3+-LDH and the pair Ni(OH)2/[β-Ni3+OOH] on the anodic reaction of water electrolysis with oxygen evolu- tion (OER) is also considered. The OER presupposes the absorption in the anode deposits of the hydroxide radicals generated by the hydrogen evolution (HER) in the cathode (0.404 V), Eq. (59).71 The OER can promote the aluminum oxidation and the formation of LDH, Eq. (60) and (61). (59) (60) (61) The intercalation of molecules (H2O, NH3) and an- ions [SxOyz–], [CO32–] in the LDH interlayer let to more electrons could being transferred to the surface of the ac- tive sites of LDH [Ni1-xAlxOOH], stabilizing their high-va- lence states and increases the activity for the OER from the reversible redox pair Ni2+/Ni3+. Zhou et al. (2018) showed that intercalated anions with strong reducing ability mod- ify the electronic structure of surface metal sites and sig- nificantly improve the performance of the corresponding LDH for the OER with a linear relationship64, in the case of ions [SxOyz–] it increases from [S2O82−] to [SO32−]. Regarding NiOOH, it is a catalyst for OER under al- kaline conditions and acts as an active center in the pair [Ni(OH)2]/[β-NiOOH] for the adsorption of [OH–]. Nick- el is capable of acquiring valences (+2, +3, +3.6) making it susceptible to various electronic transitions and phase transformations, Eq. (62). The Ni(OH)2 has a large specific surface which favors contact between the active material 549Acta Chim. Slov. 2022, 69, 536–551 Vargas et al.: Nickel Removing by Electrocoagulation of ... and the electrolytic dissolution.59,72,73,74 (62) Reactions of sulfide formation (NiS, Al2S3), the re- lease of irritating gases (H2S) and the formation of depos- its on the surface of the electrodes are considered. These deposits exert resistance to the passage of electrical cur- rent, reduce charge transfer, affect the efficiency of the pro- cess and the stability of the operation, Step 11. 3. 5 Result of the Operating Cost Estimate The operating cost was estimated for electrode and electrical energy consumption for initial nickel concentra- tion in the range 0.474 ≤ Ni ≤ 0.953 g L–1, 9.8 mA cm–2, 60 °C, pH 8.6 and 98% nickel removal, for a remainder between 6 and 19 mg L–1 (Table 8). Table 8. Estimated operating costs for nickel removal by electroco- agulation Base: 98% removing, 9.8 mA cm–2, 60 °C, pH 8.6 Ni (mg L–1) 379 447 505 646 775 953 Cost ($ t–1 Ni) 320 382 509 521 537 534 SEC (kW h kg–1Al) 5.26 6.33 6.75 5.11 3.45 2.76 The operating cost amounted to between 320 and 537 $ t–1 of nickel removed, regarding the specific ener- gy consumption (SEC), it was between 2.76 and 6.75 kW h kg–1 of aluminum. The increase in nickel concentration in the initial liquor augments the electrocoagulation time necessary to achieve high removal efficiency and therefore also increases energy and electrode consumption. The higher the concentration of ionic species in the liquor, the conductivity is favored and SEC decreases. According to the analyzed aspects of the removal mechanism, it is possible to reduce costs by designing a re- actor with favorable geometric and hydrodynamic condi- tions to achieve adequate mass transfer between the phas- es. It also suggests recycling a suspension of the product obtained at the non-saturation conditions of the adsorp- tion sites, according to the isotherm model to be followed. 4. Conclusions The nickel removing by electrocoagulation from the liquor effluent of the nickel production plant in Pun- ta-Gorda Cuba, was studied in Ni(II)-NH3-CO2-SO2-H2O system. The reaction kinetics, the adsorption isotherm, the mechanism and the preliminary cost of operation at differ- ent concentrations of dissolved nickel in the initial liquor were evaluated. In the interval defined for the operating variables, a removing efficiency between 97.7 and 99.7% was obtained. A kinetic model of conversion time was proposed, which suggests that the process is determined by the combined effect of the resistances of the mecha- nisms: external diffusion, nucleation, and as controlling step the chemical reaction and a possible autocatalytic contribution. The removal was characterized by monolay- er chemisorption at a finite number of specific adsorption sites, following the Langmuir isotherm. The precipitate had between 33.4 and 40.7% nickel and from 6.3 to 7.0% aluminum, with a typical structure of Ni/Al-LDH and phases impurities Al(OH)3, Ni(OH)2/NiOOH and nick- el-aluminum spinels. The operating costs were between 320 and 537 $ t–1 of removed nickel, considering the en- ergy and electrode consumption. The research represents the opportunity to diversify production, in-situ synthesize Ni/Al-LDH, improve its properties and evaluate its appli- cations for the projection of an industrial process. Acknowledgment Thanks to Nélida Powery Ebanks, NICAROTEC Co.; and colleagues of the Chemical Analysis Laboratory, CED- INIQ-Nicaro Cuba, for their collaboration. Conflict of Interest The authors declare no conflict of interest 5. References 1. A. R. Vargas; M. E. T. Nieves; Y. G. Díaz. Acta Chimica Sloven- ica, 2020, 67, 1239–1249. 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DOI:10.20964/2017.02.08 Povzetek Raziskava poroča o odstranjevanju niklja z elektrokoagulacijo sistema Ni(II)-NH3-CO2-SO2-H2O v laboratorijskem merilu. Poskusi so bili izvedeni z Al/Al par elektrodami pri začetni koncentraciji niklja med 293 in 1356 mg L–1 in pri obratovalnih parametrih pH 8,6, gostoti toka 9,8 mA cm–2, času elektrolize 30 min in temperaturi 60 °C. Dobljeni rezu- ltati kažejo na učinkovitost odstranjevanja med 97,7 in 99,7 %. Kinetično modeliranje je predlagalo kombinirane učinke zunanje difuzije in nukleacije ter kemično reakcijo in možen avtokatalitični prispevek kot kontrolni korak. Postopek je sledil Langmuirjevi izotermi z največjo adsorpcijsko zmogljivostjo 7519 mg g–1. ICP-OES, XRD in FTIR karakterizacija oborin je pokazala tipične plastne Ni-Al dvojne hidroksidne strukture s 33,4–40,7 % niklja in 6,3–7,0 % aluminija, od- visno od začetne koncentracije niklja. Operativni stroški porabe energije in elektrod so bili 320–537 $ t–1 odstranjenega niklja. 69. J. J. 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DOI: 10.17344/acsi.2022.7431 Scientific paper Decolorization of Direct Black 22 by Photo Fenton like Method Using UV Light and Zeolite Modified Zinc Ferrite: Kinetics and Thermodynamics Serap Findik* Hitit University, Engineering Faculty, Chemical Engineering Department, Kuzey Yerleskesi, Çevre Yolu Bulvarı, 19030, Çorum, Türkiye * Corresponding author: E-mail: serapfindik@hitit.edu.tr Received: 02-24-2022 Abstract In this study, a heterogeneous catalyst was prepared to enhance photo-Fenton like oxidation of Direct Black-22 (DB-22). Zeolite modified with zinc ferrite was used as a catalyst. The prepared catalyst was characterized using FTIR, SEM, EDS and XRD. The effect of various parameters like catalyst modification with HCl, H2O2 amount, catalyst amount, CaCl2 amount, initial pH, initial concentration and temperature on the decolorization of DB-22 was studied under UV light. Kinetic and thermodynamic properties were investigated. The highest decolorization of DB-22 was found to be 93.3% under the following conditions: initial concentration: 0.070 g/L, initial temperature: 25 °C, original pH, H2O2 amount: 2.78 g/L, m-ZZF amount: 3 g/L, CaCl2 amount: 3.75 g/L, reaction time: 60 min and UV light. The activation energy was found to be –14.76 kJ/mol under the studied conditions. The decolorization reaction was an exothermic reaction, and the calculated reaction enthalpy was –17.31 kJ/mol. The activation entropy was calculated to be –0.326 kJ/mol. The standard Gibbs free energy change of the activation had a positive value, and it increased with increasing temperature. Keywords: Direct black 22, photo-Fenton process, UV, zeolite, zinc ferrite 1. Introduction Textile dyes account for more than half of the total dye production in the world. The dyes used in the dyeing process cause the formation of textile wastewater. This wastewater causes coloration in natural water resources, as well as damaging living organisms and preventing the pas- sage of sunlight. In addition, when mixed with drinking water, it harms human life due to its carcinogenic nature. Removing these dyes from water is important to prevent environmental damage.1 Conventional treatment methods such as biologi- cal treatment, coagulation, adsorption, chemical precipi- tation, solvent extraction, filtration, and electrochemical treatment are ineffective for decolorization of dyes.2,3 They also have some disadvantages such as incomplete destruc- tion of dye, high energy consumption, high operating cost, poor selectivity, incomplete ion removal, and gen- eration of toxic sludge and waste product. Advanced ox- idation methods are preferred for dye removal because of their simplicity and effectiveness.2,4,5 Advanced oxidation methods are based on the production of the OH• radicals. The homogeneous Fenton process is a Fenton reaction in which iron salts are used as catalysts. In this process, hydrogen peroxide, which is used as an oxidant, and iron ions, which are used as a catalyst, react to produce OH• radicals.6 The use of H2O2 and Fenton reagents with UV light has been known as photo-Fenton oxidation system. UV light irradiation and Fenton reagents cause an increase in the OH• formation rate. Additional OH• radicals are formed by either photoreduction of ferric ions (Fe3+) to ferrous ions (Fe2+) or hydrogen peroxide photolysis.2,3,6 Fenton oxidation systems based on the homoge- neous Fenton reagent (Fe2+/Fe3+/H2O2) have some dis- advantages such as narrow pH range, removal of sludge containing iron ions, and requirement of large amount of chemicals.2–4,6 Therefore, heterogeneous Fenton reactions are preferred to eliminate the negative effects of homoge- neous Fenton reactions. In the heterogeneous catalyst, the iron is stabilized into the catalyst interlayer space. Hydroxyl radicals are produced by oxidation of hydrogen peroxide with neither pH control nor precipitation of iron hydroxide.6 553Acta Chim. Slov. 2022, 69, 552–563 Findik: Decolorization of Direct Black 22 by Photo Fenton like ... In heterogeneous Fenton-like processes, spinel fer- rites (MFe2O4) can be used as a heterogeneous catalyst. The term M in spinel ferrite structure refers to the divalent metal ions such as Ni2+, Mn2+, Co2+, Mg2+, Cu2+, Zn2+ etc. Spinel ferrites such as ZnFe2O4, CoFe2O4, and NiFe2O4 are used in medicines, sensors, and catalyst carriers thanks to their high mechanical strength, magnetic properties, unique structure, and catalytic performance.7,8 The use of zeolites modified with semiconductors can be an alternative catalyst for a variety of important reactions.1,2 Zeolites are crystalline aluminosilicates with cavities, unique structures and chemical compositions, uniform pores and channels, high surface area, thermal stability, and an excellent adsorption ability. Convention- al zeolites are constructed by tetrahedral SiO4 and AlO4 units.1,9,10 There are at least 41 known types of natural zeolite.1 Some of the most known and abundant types of natural zeolite are clinoptilolite, phillipsite, heulandite, mordenite, chabazite, and ferrierite.11 Azo dyes are the most important synthetic dyes with –N=N– bonds in their molecular structure. They can be classified according to number of azo groups, such as monoazo, diazo, triazo, polyazo, and azoic.12 Direct black 22 (DB-22) is one of the tetra azo dyes used in textile industry for dyeing cellulosic fibers such as cotton, wool, viscose, rayon, and paper. The high concentra- tion of DB-22 in wastewater harms the environment with its carcinogenic properties, toxicity, organic matter content, and strong color.13 For this reason, wastewater containing DB-22 should be released to the environment after being treated. There are few studies in the literature on DB-22 removal. In their study, Hien et al.13 investigated the catalytic ozonation of DB-22 using different metal slags. Carvalho et al.14 exam- ined the removal of DB-22 using microaerated upflow an- aerobic sludge blanket (UASB) reactors. Gomes et al.15 inves- tigated the degradation of DB-22 using homogeneous and heterogeneous photo-Fenton advanced oxidation process. They used LED light as a source of radiation, and ferrous sul- fate and iron residue as catalysts. In another study, Menezes et al.16 investigated the effect of the combination of anaerobic process and micro-aeration (continuous and intermittent) on DB-22 removal. Shu et al.17 examined the decoloriza- tion of DB-22 using the UV/H2O2 process, ozonation, and pre-ozonation coupled with UV/H2O2. Moreover, some oth- er studies focused on the photoelectrochemical oxidation of DB-22 under elevated oxygen pressure and the adsorption of DB-22 using polymeric adsorbent.18,19 The aim of this study is to investigate photo-Fenton like oxidation of DB-22 using zeolite modified zinc fer- rite. Kinetic and thermodynamic properties of the DB-22 decolorization and the effects of the reaction parameters such as catalyst modification with HCl, catalyst amount, H2O2 amount, salt addition, pH, initial dye concentration, temperature were also investigated. The zeolite and syn- thesized catalysts were characterized using FTIR, SEM, EDS and XRD. 2. Materials and Methods 2. 1. Materials and Equipments The zeolite with a particle size of 23 µ was procured from a company in Balıkesir, Turkey. It was a commercial product and used without purification. HCl and NaOH were procured from Tekkim, DB-22 (commercial name Direct Black 22 VSF 1600) from a company named “HNY” in Turkey, H2O2 (34.5–36.5 w/w %, Cas no: 7722-84-1) from Sigma, CaCl2 from Emir Kimya, FeSO47H2O (Cas no: 7782-63-0) from Merck and ZnSO47H2O (Art no: 8881) from Merck. A magnetic stirrer (HSD-180), pH meter (C561, Consort), digital scale (Ohaus), centrifuge (Nuve, NF 200), and incubator (Ecocell) were used in the study. UV- spec- trophotometer (Hach, DR-2400) was used to measure the absorbance of the sample. A COD reactor (Hach DRB 200) was used to heat the samples before measuring the COD value. A UV lamp (Light Tech GPH212T5L/4, 10 W) with a wavelength of 254 nm was used in the photocatalytic de- colorization of DB-22. 2. 2. Preparation of the Catalyst The catalyst, zeolite/Zn/Fe was prepared by chem- ical coprecipitation method. First, iron II sulphate heptahydrate (FeSO4.7H2O) and zinc sulphate heptahy- drate (ZnSO4.7H2O) with a molar ratio of 2:1 were dis- solved in 200 ml distilled water. Then, zeolite was added to the solution and heated to 65–70 °C while stirring with a magnetic stirrer. The mixture was stirred for 30 min using a magnetic stirrer. 3 M NaOH solution was add- ed dropwise to the solution, and pH of the solution was adjusted to 12. After addition of NaOH solution, stirring was continued for one hour at 100 °C. The prepared cat- alyst was left for one day at room conditions, and then placed in water bath for 4 h at 95 °C. After that, it was dried at 95 °C for 90 h. The dried composite was soaked in 0.1 N HCl solution at room temperature for 24 hour, and then filtered and washed using distilled water. Final- ly, it was dried at 95 °C for 60 hours. The catalysts treated with and without HCl were coded as m-ZZF and ZZF, respectively. 2. 3. Characterization The catalyts used in the study were characterized by XRD, FTIR, SEM and EDS. The crystalline structure of the samples was determined by XRD analysis using Rig- aku Smart Lab with Cu-Kα radiation at 40kV and 30mA. The samples were scanned from 5°– 90° at a rate of 2°/min, and with a step size of 0.01. FTIR (PerkinElmer, Spectrum Two) was used to identify the functional groups of the ad- sorbents before and after adsorption in the range of 400– 4000 cm–1. SEM and EDS analysis were performed using Tescan, MAIA3 XMU. 554 Acta Chim. Slov. 2022, 69, 552–563 Findik: Decolorization of Direct Black 22 by Photo Fenton like ... increase of 15 °C was observed at the end of 60 minutes. No temperature control was done to constitute a natural environment and to save on cooling water cost. 3. Results and Discussion 3.1. Catalyst Characterization The XRD pattern of the zeolite (Z) and m-ZZF are shown in Fig.1. As can be seen in the Fig.1, all diffraction peaks were apparent and strong. This indicates that the Z and m-ZZF samples were in crystalline form. According to XRD data, zeolite (Z) was identified by its characteristic X-ray diffraction peaks at 2θ: 11.15°, 16.87°, 20.84°, 22.34°, 22.73°, 26.02°, 26.6°, 29.85°. These XRD peaks of the Z coded sample are compatible with the clinoptilolite with PDF card no 00-013-0304. This result shows that the zeolite was composed of clinoptilolite. As shown in Fig.1-b, all peaks of natural zeolite accept 26.74° disappeared after the preparation of m-ZZF. According to the PDF card, peaks appear at 34.96°, 52.74° and 61.97° matchwell with PDF card 00-010-0467 (Franklinite, Zn- Fe2O4). Spinel ferrite ZnFe2O4 coats the surface of zeolite. This improves the photocatalytic activity of m-ZZF. Figure 2 shows the SEM images and EDS results of zeolite (Z) and m-ZZF. As can be seen in SEM image of Z, particles had an irregular shape and porous surface. In m-ZZF image, zinc ferrite agglomerated on the surface of the zeolite. According to EDS results, zeolite was mainly composed of O, Si, and Al, in addition to small amounts of K, Mg, Ca and Fe. The percentage of iron and zinc consid- erably increased in m-ZZF. Figure 3 shows the FTIR spectra of zeolite, ZZF, m-ZZF, and m-ZZF after being used in DB-22 decoloriza- tion. As can be seen in Fig. 3, the zeolite had bands around 800, 1030, and 1630 cm–1. The band between 450 and 1100 cm–1 was related to the active zeolite lattice bands.20 The band at 800 cm–1 corresponds to the stretching vibra- tion of Si-O-Si. The absorption bands around 1030 cm–1 2. 4. Experimental In the study, a cylindrical glass reactor with a volume of 250 ml, a height of 20 cm, and a diameter of 5 cm was used. The reactor was filled with 200 ml of DB-22 solution with a known concentration, and the catalyst was added, then magnetically stirred for 30 min in the dark to ensure a complete equilibration of adsorption/desorption of DB-22 on the catalyst surface. In order to find the amount of dye adsorbed on the catalyst surface in the dark, a sample was taken from the dye solution and its absorbance was meas- ured. Then, a UV lamp with a quartz tube was placed in the center of the reactor at a distance of 2 cm from the bottom. The surface of the reactor was covered with aluminum foil to prevent light penetration. While the DB 22 solution was stirred continuously, the UV lamp was switched on and the reaction time was started immediately. Stirring rate was kept constant at 800 rpm in all experiments. The ex- periments were carried out at the original pH of the dye solution, except for the experiments in which the pH effect was examined. The samples taken from the reactor at reg- ular intervals were centrifuged at 5000 rpm for 10 minutes. Then, the absorbance of the dye solution was recorded at 481 nm using UV-spectrophotometer to calculate decolor- ization. Decolorization of DB-22 dye was calculated using the Eq. 1 Decolorization, % = (1) where, C0 and C represent the concentration of the DB- 22 at the beginning of the reaction and at corresponding time, respectively. COD was measured using Hach DR 2400 spectro- photometer, Hach COD reactor and test kits in the range 0–1500 ppm. Instructions for the Hach higher range test followed. The DB-22 solution temperature was not controlled in the experiments. The reaction was started at a room temperature of 25 °C, except for the experiments in which the effect of temperature was examined, and a temperature Fig. 1. XRD analysis of (a) Z and (b) m-ZZF 555Acta Chim. Slov. 2022, 69, 552–563 Findik: Decolorization of Direct Black 22 by Photo Fenton like ... were due to asymmetric stretching vibrations of Al and/or Si bonding with oxygen. The peak at 1030 cm–1 is the proof that the zeolite was composed of alumina silicate. The peak at 1030 cm–1 is sensitive to the change in Si and Al content. The band at 1630 cm–1 is related to the O-H stretching vi- brations and the adsorbed H2O molecules.20–23 As seen in Fig. 3, there were a few differences between Z, ZZF, and m-ZZF spectrum. In ZZF and m-ZZF, the peaks at 800 cm–1 and 1630 cm–1 disappeared. The peak at 1000 cm–1 in m-ZZF was the result of the shift of the peak at 1030 cm–1 in Z, and the transmittance at 1000 cm–1 increased. With the addition of zinc ferrite to the structure of the ze- olite, the ratio of Al and Si changed. Maybe that is why, the transmittance increased. The FTIR spectrum of m-ZZF after it was used in DB-22 decolorization was similar to that before it was used and only the transmittance at 1000 cm–1 decreased. 3.2. The effect of HCl Modification of Catalyst on Photocatalytic Decolorization of DB-22 In order to determine the effective catalyst in the photocatalytic degradation of DB-22, the experiments were performed under the following reaction conditions: initial dye concentration: 0.04 g/L, catalyst loading: 4 g/L, original pH, and UV lamp. As seen in Fig. 4-a, in the pres- ence of ZZF and m-ZZF, the decolorization rate was high- er with using UV lamp than without UV lamp. The catalyst modified with HCl (m-ZZF) gave better results than ZZF. Mesopores formed as a result of acid treatment of zeolite Fig. 2. SEM image and EDS results of zeolit and m-ZZF Fig. 3. FTIR analysis of the Z, ZZF, m-ZZF and m-ZZF after being used in DB-22 decolorization 556 Acta Chim. Slov. 2022, 69, 552–563 Findik: Decolorization of Direct Black 22 by Photo Fenton like ... are active surfaces for the adsorption and catalysis of rel- atively large molecules.22 The decolorization of DB-22 us- ing UV light and 4 g/L m-ZZF was 10.9% and 14.6% at the end of 30 min and 60 min, respectively. On the other hand, a decolorization of 6.4% and 12.8% was observed in the experimental set up with UV light and 4 g/L ZZF at the reaction times of 30 min and 60 min respectively. So, the decolorization rate increased in the system using UV light and m-ZZF. Considering the individual m-ZZF and UV processes, it is seen that the photocatalytic activity of m-ZZF is very poor. 3.3. Effect of Hydrogen Peroxide Amount The effect of hydrogen peroxide amount on the de- colorization of DB-22 was studied with the H2O2 amounts of 2.78 g/L, 5.55 g/L, and 11.1 g/L while keeping all oth- er parameters constant (initial DB-22 concentration: 0.04 g/L, m-ZZF amount: 4 g/L, UV light, and original pH). The results are presented in Fig. 4-b. As seen in Fig. 4-b, the decolorization of DB-22 without the addition of H2O2 was less than that with the addition of H2O2. The photo- catalytic decolorization of the DB-22 enhanced when the H2O2 amount was introduced to the Fenton system due to the accelerated generation of hydroxyl radicals (OH•).24 According to our results, the decolorization rate decreased with increasing H2O2 concentration. In 60 min, the decol- orization rate increased from 69.5% to 76.4% when the H2O2 concentration decreased from 5.55 g/L to 2.78g/L. At high H2O2 concentrations, excess hydrogen per- oxide reacts with the produced OH• radicals and causes the formation of less reactive radicals like hydrogen diox- ide.21,25 The reactions between OH• radicals and excess hydrogen peroxide are given by the equations below26: OH• + H2O2→HO2• + H2O (2) HO2 • + OH• →O2 + H2O (3) OH +OH• →H2O2 (4) A similar result was reported by Abharya et al.24 They reported that photocatalytic degradation decreased after the H2O2 concentration of 0.0389 M and when the amount of H2O2 was above the critical value, the reaction between ex- cess H2O2 and the generated OH• radicals caused a decrease in photocatalytic activity over time. In another study, Badvi and Javanbakht21 reported similar results for the photocata- lytic degradation of methylene blue. They found that the dye degradation decreased when the H2O2 concentration was in- creased from 250 to 750 mg/L. Moreover, they asserted that when H2O2 concentration exceeded a certain level, hydrogen peroxide acted as a scavenger of the photo produced holes and caused a decrease in the efficiency of dye degradation. 3. 4. Effect of Catalyst Amount In order to examine the effect of the amount of catalyst and to find the optimum amount of catalyst, ex- periments were carried out by changing the amount of catalyst between 1 g/L and 6 g/L while keeping the other parameters constant (H2O2 amount: 2.78g/L, initial DB-22 concentration: 0.04 g/L, UV light, original pH and initial temperature: 25 °C). The results are presented in Fig. 5-a. As can be seen in Fig. 5-a, as the catalyst amount was increased from 1g/L to 3 g/L, the decolorization rate of DB-22 increased from 78.0% to 80.8%. After the catalyst loading of 3 g/L, the decolorization rate decreased. Under the studied conditions, the optimum catalyst amount was found to be 3 g/L. This means that, the catalyst amounts higher than the optimum value may decrease the light transmittance. Abharya et al.24 investigated the photocat- alytic treatment of methylene blue and obtained similar results. They reported that the number of reactive sites increased with increasing catalyst loading and after the optimum catalyst amount, the catalyst particles tended to agglomerate, causing a decrease in the number of re- active sites and an increase in light scattering. In anoth- er study, Gan and Li27 investigated the decolorization of Rhodamine B using catalyst by Fenton like process. They reported that the decolorization of Rhodamine B increased with increasing the catalyst amount from 0.5g/dm3 to 1g/ dm3, but after 1g/dm3, it started to decrease. They also re- ported that, after the optimum catalyst amount (1 g/dm3), the catalyst surface area decreased due to the aggregation between particles, and as a result, color removal decreased. Fig. 4. Effect of (a) HCl modification of catalyst (initial dye concentration: 0.04 g/L, initial temperature: 25 °C, catalyst amount: 4 g/L, original pH) and (b) H2O2 concentration (initial dye concentration: 0.04 g/L, initial temperature: 25 °C, m-ZZF amount: 4 g/L, original pH, UV light) on the decolorization of DB-22 557Acta Chim. Slov. 2022, 69, 552–563 Findik: Decolorization of Direct Black 22 by Photo Fenton like ... Ejhieh and Khorsandi1 investigated the photocatalytic de- colorization of Eriochrome Black T using NiS-P zeolite. They reported that the decolorization of Erichrome Black T increased with increasing the catalyst amount from 0.1 g/L to 0.8 g/L, but after 0.8 g/L, it started to decrease. They asserted that as the amount of catalyst increased, the sol- id particles blocked the photon penetration. In another study, Ji et al.28 investigated the decolorization of methyl- ene blue using catalyst and heterogeneous photo Fenton system and reported that the decolorization of methylene blue increased with increasing the catalyst from 0.25 to 1.5 g/L. As the amount of catalyst increases, the number of active sites increases. In the catalyst amounts higher than 1.5 g/L, the catalyst particles prevented the transmission of UV light into the solution. Therefore, with the increase of the amount of catalyst from 1.5 to 2 g/L, the rate of decol- orization decreased. 3. 5. Effect of Initial pH The effect of pH on the decolorization of DB-22 was studied at the initial pHs of 4, ≈7.1, and 10. ≈7.1 is the original pH of the dye solution. The pH of the DB-22 solu- tion was adjusted at the beginning of the experiment. pH control was not done during the reaction. Fig. 5-b shows the results. The decolorization of DB-22 increased when the pH was increased from 4 to 10. The decolorization of DB-22 was found to be 70.8%, 80.2%, and 83.3% at the pH values of 4, 7.1, and 10 respectively. Karimi-Shamsabadi et al.29 obtained similar results for the photocatalytic degradation of Erichrome black T using NiO-ZnO doped nanozeolite X. They reported that at the basic pH, due to higher con- centration of hydroxyl ions, the concentration of hydrox- yl radicals increased, which enhanced the photocatalytic degradation rate. In another study by Ejhieh and Khor- sandi1, it was asserted that the degradation of Erichrome Black T increased with increasing the initial pH. Under the acidic conditions, Cl- ions and hydroxyl radicals combine to form inorganic radical ions (ClO−•). They reported that the reactivity of ClO−• anions was less than that of hydrox- yl radicals, therefore, these radicals did not involve in the decolorization reactions. Under basic conditions, due to the increase in the number hydroxyl radicals, the decolor- ization rate increased. 3. 6. Effect of Salt Addition The effect of salt on the decolorization of DB-22 was investigated using CaCl2. The results obtained for different CaCl2 amounts are given in Fig. 6. As can be seen in Fig. 6, the decolorization rate of DB-22 increased with the ad- dition of CaCl2. When the CaCl2 amount was increased from 2.5 to 5 g/L, no significant change was observed in the decolorization rate at the reaction time of 60 min. In the reaction time of 30 min, while the decolorization of DB-22 was found to be 83.2% in the case of the addition of 2.5 g/L CaCl2, it was 90.1% in the case of the addition of 3.75 g/L CaCl2. 3.75 g/L CaCl2 was chosen due to the higher decolorization rate at 30 min. The results show that m-ZZF is effective in the presence of CaCl2. Fig. 6. Effect of CaCl2 amount on the decolorization of DB-22 (ini- tial dye concentration: 0.040 g/L, initial temperature: 25 °C, original pH, H2O2 amount: 2.78 g/L, m-ZZF amount: 3 g/L, UV light) Gan et al.27 reported similar results in their study in- vestigating the effect of ionic strength using different con- centrations of NaCl. According to their results, increasing NaCl from 0.05 M to 0.1M did not change the degrada- tion rate. The ionic strength for the adsorption of dye on oxide surface can influence the electrostatic interactions Fig. 5. Effect of (a) m-ZZF amount (initial dye concentration: 0.04 g/L, initial temperature: 25 °C, original pH, H2O2 amount 2.78 g/L, UV light) (b) pH (initial dye concentration: 0.04 g/L, initial temperature: 25 °C, H2O2 amount 2.78 g/L, m-ZZF amount: 3 g/L, UV light) on the decolorization of DB-22 558 Acta Chim. Slov. 2022, 69, 552–563 Findik: Decolorization of Direct Black 22 by Photo Fenton like ... between the oxide surface and the dye species. In heter- ogeneous catalytic systems, the presence of excess anions may affect the equilibria between the dye molecules and the catalyst surface.27 When low concentrations of salt are used, Cl• rad- icals with high oxidizing potential are formed and they oxidize organic materials. Cl• radicals have a high affinity for a hole and they can also prevent electron-hole recom- bination, thus enhancing the efficiency of reactive species formation. If the salt amount is more than the optimum value, the dye removal efficiency decreases. This is because the Cl– competes with the dye molecules for the limited catalyst surface and the surface is deactivated.30 3. 7. Effect of Initial Dye Concentration and Reaction Time In the study, the effects of initial dye concentration and reaction time on DB-22 decolorization were also in- vestigated. The experiments were done within the initial concentration range of 0.025–0.070 g/L with a m-ZZF amount of 3 g/L, H2O2 amount of 2.78 g/L, and CaCl2 amount of 3.75 g/L, at original pH, at an initial tempera- ture of 25 °C and under UV light. Fig. 7 shows the effect of initial dye concentration and reaction time on the decol- orization of DB-22. The experimental results showed that the decolori- zation rate of DB-22 was faster in the first 10 min. After 10min, decolorization rate slowed down and remained al- most constant after the reaction time of 30 min. As seen in Fig. 7, the decolorization of DB-22 increased with the increasing initial concentrations. The decolorization of DB-22 for the reaction time of 60 min was found to be 81.3%, 90.2%, 90.5%, 91%, and 93.3% at the initial dye concentrations of 0.025, 0.040, 0.050, 0.060, and 0.070 g/L, respectively. In the literature, Ejhieh and Khorsandi1 re- ported similar results for the photocatalytic decolorization of Eriochrome Black T using NiS-P zeolite. They found that the decolorization of Erichrome Black T increased with increasing the initial concentration from 10 mg/L to 40 mg/L. Having a lifetime as short as a few nanosec- onds, hydroxyl radicals react immediately after formation. As the number of dye molecules per unit volume increas- es, so does the probability of collisions between organic matter and oxidizing species. As a result, decolorization rate increases. In their study, Gan and Li27 found that the decolorization of Rhodamine B increased with increasing the initial concentration from 2.5 to 50 mg/L using rice- hull based silica supported catalyst by Fenton like process. The probability of collision between dye molecules and near-surface activating species increases with the increase of dye concentration per volume. 3. 8. Effect of Temperature The effect of temperature on the decolorization of DB-22 was investigated at 25 °C, 35 °C, and 45 °C while keeping other parameters constant (initial concentration of dye: 0.040 g/L, initial pH: original, H2O2 amount: 2.78 g/L, m-ZZF amount: 3 g/L, CaCl2 amount: 3.75 g/L, UV light). As mentioned in the experimental section, the ex- periments were performed without cooling, and a tem- perature increase was observed. Initial decolorization rate was calculated for the reaction time of 10 min. An increase of 2 °C from the initial temperature was observed in 10 min. Figure 8 shows the effect of temperature on the ini- tial decolorization rate. As can be seen in Fig. 8, the initial decolorization of DB-22 in 10 min decreased with increas- ing temperatures. The decrease in the decolorization rate with the increasing temperatures shows that the reaction occurred under exothermic conditions. An increase in the reaction temperature causes a decrease in the oxygen solubility in the solution. The rate of electron withdrawal from the surface of the photocatalyst decreases due to the decrease in dissolved oxygen concentration.31 Andreozzi et al.3 investigated the effect of temperature on the photo- catalytic degradation of 4-nitrophenol and reported that temperature had a negative effect on the degradation at pH 3. The presence of oxygen is important to keep high the concentration of photogenerated OH• radicals on the sur- face of the catalyst. The concentration of the photogenerat- ed holes decreases due to the decrease in oxygen solubility at high reaction temperatures. Fig. 8. Effect of temperature on the initial decolorization rate of DB- 22 (initial pH: original, H2O2 amount: 2.78 g/L, m-ZZF amount: 3 g/L, CaCl2 amount: 3.75 g/L, UV light) Fig. 7. Effect of initial dye concentration on the decolorization of DB-22 (pH: original, initial temperature: 25 °C, H2O2 amount: 2.78 g/L, m-ZZF amount: 3 g/L, CaCl2 amount: 3.75 g/L, UV light) 559Acta Chim. Slov. 2022, 69, 552–563 Findik: Decolorization of Direct Black 22 by Photo Fenton like ... 3. 9. Decolorization of DB-22 using Different Processes Decolorization of DB-22 was investigated at 0.040g/L initial dye concentration and 25 °C initial temperature us- ing different processes. The results are given in Fig. 9. De- colorization rate using 3 g/L m-ZZF and (3 g/L m-ZZF + 2.78 g/L H2O2) processes were 2.4% and 5.7% respectively. The effect of UV light was studied using processes such as, 3g/L m-ZZF, 2.5g/L CaCl2 and (3 g/L m-ZZF + 2.5 g/L CaCl2). Decolorization of (UV + 3 g/L m-ZZF + 2.5 g/L CaCl2) process was greater than that of the individual pro- cesses such as UV, (UV + 3g/L m-ZZF) and (UV + 2.5g/L CaCl2). Decolorization rate was 76.1 % at 30 min and 82.3 % at 60 min using (UV + 3 g/L m-ZZF + 2.5 g/L CaCl2) process. To identify the effect of the H2O2, experiments were also done using (UV + 2.78 g/L H2O2), (UV + 2.78 g/L H2O2 + 2.5 g/L CaCl2), (UV + 2.78 g/L H2O2 + 3 g/L m-ZZF) and (UV + 2.78 g/L H2O2 + 3 g/L m-ZZF + 2.5 g/L CaCl2). (UV + 2.78 g/L H2O2) and (UV + 2.78 g/L H2O2 + 3 g/L m-ZZF) processes provided nearly the same decolol- orization rate as 82% at 60 min. Decolorization rate at 60 min using (UV + 2.78 g/L H2O2 + 3 g/L m-ZZF + 2.5 g/L CaCl2) process was found to be 91.5%. The decolorization of DB-22 for the reaction time of 30 min was found to be 76.1% at (UV+ 3 g/L m-ZZF + 2.5 g/L CaCl2) process. Addition of H2O2 increased the color removal and decolorization of DB-22 at 30 min was found to be 83.2% at (UV + 2.78 g/L H2O2 + 3 g/L m-ZZF + 2.5 g/L CaCl2) process. With increasing CaCl2 amount from 2.5 to 3.75 g/L, decolorization rate increased from 83.2% to 90.1% in 30 min reaction time. With the synergetic effect of UV, m-ZZF, H2O2 and CaCl2, the highest color removal was achieved in 30 minutes. Ionic strength effects the elec- trostatic interaction between the catalyst surface and dye molecules. Addition of anions might allow the neutraliza- tion of the positive sites on catalyst surface. Nonelectro- static interaction between dye molecules and neutral sites could occur due to van del Walls forces or low energetic H-bonds.27 According to Sudrajat and Babel30, the Cl• radi- cals formed as a result of the surface chain transfer reaction of the chlorine ion oxidizes the organic compounds. Considering these results, the possible decoloriza- tion mechanism of DB-22 over m-ZZF may consist of the following steps: Due to the non-electrostatic interaction between the dye molecules and the neutral sites, dye mol- ecules may be adsorbed on the catalyst surface in the pres- ence of Cl– ions.27 Electron/hole pairs are generated on the m-ZZF surface under UV light. Electrons on m-ZZF react with H2O2 to produce both OH – ions and OH• rad- icals. Photogenerated holes could react with OH– ions or adsorbed water to generate OH• radicals.33,34 In the pres- ence of salt, the Cl• radicals could oxidize dye molecules.30 The generated radicals react with DB-22 and degradation products are formed. 3. 10. COD Removal The COD value represents the amount of oxygen re- quired for oxidation of organics into CO2 and water. It is Fig. 9. Comparison of different processes 560 Acta Chim. Slov. 2022, 69, 552–563 Findik: Decolorization of Direct Black 22 by Photo Fenton like ... related with total organic compounds in the wastewater. In the study, the variation of COD with time was inves- tigated under the following reaction conditions: initial dye concentration: 0.04 g/L, m-ZZF amount: 3 g/L, H2O2 amount: 2.78 g/L, CaCl2 amount 3.75 g/L, original pH, and UV lamp. As seen from Fig. 10, COD removal increases with increasing time. 26.9% COD removal was obtained at the end of 60 min reaction time. While the decolorization of DB-22 was found to be 79.5% and 90.1% at the 10 min and 30 min reaction time respectively, low COD removal was achieved under the studied conditions. COD removal showed the partial oxidation of the organic pollutants to CO2 and H2O. The colorless intermediates were formed as a result of DB-22 oxidation. These intermediates cause low COD removal.34 According to results, photo-Fenton like process is more useful for decolorization of DB-22 than COD removal. The operating cost of the photo-Fenton process in- cludes cost of chemicals and energy. Electricity is used in the UV lamp and mixing the process. According to Çalık35, operating cost of the photo-Fenton process changed be- tween 13.46–20.13 €/m3 for the used chemicals and elec- trical energy for treatment of textile wastewater. Fig. 10. Variation of COD with time (initial dye concentration: 0.04g/L, pH: original, initial temperature: 25 °C, H2O2 amount:2.78 g/L, m-ZZF amount: 3 g/L, CaCl2 amount: 3.75 g/L, UV light) 3. 11. Kinetic and Thermodynamic Studies The initial rate equation for decolorization of dye is as follows: (5) where kapp is the overall observed rate constant for the re- action, n is the order of the reaction with respect to con- centration. Eq. 5 is linearized by taking the natural loga- rithm and Eq. 6 is obtained. (6) If ln(r0) is plotted against C0, the slope of the straight line gives the degree of reaction (n) and intercept gives the lnkapp value. Fig. 11 shows the plot of against for 25 °C, 35 °C, and 45 °C. The initial decolorization rate of DB-22 was calculated for the initial 10 min. The calculated n and kapp values were listed in Table 1. A high regression coefficient indicates a good compatibility. The values of n were 1.08, 1.12, and 1.16 at 25 °C, 35 °C and 45 °C respectively. It can be said that, the reaction order of the photocatalytic decol- orization of DB-22 was 1.1 under the studied conditions. Fig. 11. ln(r0) versus lnC0 (initial pH: original, H2O2 amount: 2.78 g/L, m-ZZF amount: 3 g/L, CaCl2 amount: 3.75 g/L, UV light) Table 1. The reaction orders and rate constants T (K) n kapp (mg/L)–0.1/min R2 298 1.08 0.064 0.9946 308 1.12 0.053 0.9847 318 1.16 0.044 0.9949 The activation energy of the reaction was calculated using Arrhenius equation36. kapp = A e− Ea /RT (7) where kapp is the apparent reaction rate constant, A is the Arrhenius factor, Ea is the activation energy (J/mol), R is the ideal gas constant (8.314 J/molK), and T is the temperature (K). The logarithmic form of Eq. 7 can be written as: ln kapp = ln A − Ea /RT (8) When ln kapp is plotted against 1/T, the slope gives –Ea/R. The Arrhenius plot is presented in Fig. 12-a. Acti- vation energy, Ea was calculated to be –14.76 kJ/mol under the studied conditions. This result showed that the decol- orization rate decreased with increasing temperature, as mentioned in the section 3.4. The activation energy is the minimum energy required to break the bonds of the spe- cies participating in the reaction and to form new bonds. A low activation energy indicates that less energy is re- quired to break bonds.37 561Acta Chim. Slov. 2022, 69, 552–563 Findik: Decolorization of Direct Black 22 by Photo Fenton like ... The activation enthalpy (ΔH0) and the activation en- tropy (ΔS0) were calculated by plotting ln(kapp/T) against 1/T according to the equation below27: (9) where NA is Avogadro constant (6.022*1023 mol–1) and h is Planck constant (6.626*10–34 Js). The slopes of this line gives –ΔH0/R and the intercept gives . Fig. 12-b shows the plot of vs 1/T. The reaction enthalpy was calculated to be –17.31 kJ/ mol. The sign of the enthalpy indicates an exothermic re- action. The value of activation entropy was calculated as –0.326 kJ/molK. The negative value of ΔS0 indicated that the photocatalytic decolorization of DB-22 was less ran- dom and the transition state formed in the degradation process had a lower structural freedom compared to the reactants, and this also confirms that the process was ir- reversible.37,38 The lowest absolute values of Ea, ΔH0, and ΔS0 found for the catalyst used in the study are indicative of its high- est catalytic activity. According to the literature, the value of the activation energy determines whether the reaction is diffusion or reaction rate controlled. If the activation en- ergy is lower than 29 kJ/mol, this indicates that the reac- tion is controlled by diffusion process.39,40 In this study, a low activation energy was obtained; so it can be said that the photocatalytic decolorization of DB-22 using m-ZZF was a diffusion controlled process. The standard Gibbs free energy was calculated using Eq. 1036,38 (10) Table 2 gives the thermodynamic parameters. As can be seen in Table 2, standard Gibbs free energy change (ΔG0) had a positive value and it increased with increas- ing temperatures. This result indicated no spontaneous processes and weak adsorption of dye molecules on m-ZZF.36 Table 2. Kinetic and thermodynamic parameters of the photocata- lytic degradation of DB-22 T kapp Ea ΔH0 ΔS0 ΔG0 (K) (mg/L)–0.1/min (kJ/mol) (kJ/mol) (kJ/molK) (kJ/mol) 298 0.064 –14.76 –17.31 –0.326 84.02 308 0.053 87.28 318 0.044 90.36 4. Conclusions In this study, decolorization of Direct Black 22 was investigated using photo Fenton-like method. UV lamp was used as a source of light. Zeolite modified with zinc ferrite (m-ZZF) was used as a heterogeneous catalyst. m-ZZF was prepared by coprecipitation method. The zeo- lite and m-ZZF were characterized using XRD, SEM, EDS, and FTIR analysis. Zeolite surface was successfully coated with zinc ferrite. The results showed that under UV light, the decolor- ization of DB-22 was higher with m-ZZF than with ZZF. The effect of various parameters (initial pH, initial dye concentration, catalyst amount, hydrogen peroxide con- centration, CaCl2 amount, temperature) on the DB-22 de- colorization was analysed, kinetic and thermodynamic in- vestigations were performed as well. The decolorization of DB-22 was found to be 93.3% under the following reaction conditions: initial concentration: 0.070 g/L, initial temper- ature: 25 °C, original pH, H2O2 amount: 2.78g/L, m-ZZF amount: 3 g/L, CaCl2 amount 3.75 g/L, reaction time: 60 min, and under UV light. The activation energy was found to be –14.76 kJ/mol. The decolorization reaction was exo- thermic and the calculated reaction enthalpy was –17.31 kJ/mol. The value of activation entropy was calculated to be –0.326 kJ/mol. The standard Gibbs free energy change of activation had a positive value, and it increased with increasing temperatures. Although high decolorization of DB-22 was achieved with the photo Fenton like process, low COD removal was observed at the studied conditions. Fig. 12. (a) Arrhenius plot of lnkapp against 1/T (b) ln(Kapp/T) versus 1/T 562 Acta Chim. Slov. 2022, 69, 552–563 Findik: Decolorization of Direct Black 22 by Photo Fenton like ... Acknowledgement The author thank to Hitit University for their fi- nancial support of this project under contract of MUH19001.21.003. 5. References 1. A. S. Ejhieh, M. Khorsandi, J Haz Mat. 2010, 176, 629–637. DOI:10.1016/j.jhazmat.2009.11.077 2. M. Tekbas, H.C. Yatmaz, N. Bektas, Mic. Mes. Mat. 2008, 115, 594–602. DOI:10.1016/j.micromeso.2008.03.001 3. N. Demir, G. Gündüz, M. Dükkancı, Env. Sc. Poll. Res. 2015, 22, 3193–3201. DOI:10.1007/s11356-014-2868-x 4. L. G. Devi, K. S. A. Raju, S. G. Kumar, K. E. Rajashekhar, J Taiw. Inst. Chem. Eng. 2011, 42, 341–349. DOI:10.1016/j.jtice.2010.05.010 5. F. Alakhras, E. Alhajri, R. Haounati, H. Ouachtak, A. Ait Addi, T. A. 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Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Pripravljen je bil heterogeni katalizator za izboljšanje foto-Fentonove oksidacije barvila DB-22 (Direct Black-22). Kot katalizator je bil uporabljen zeolit, modificiran s cinkovim feritom. Pripravljeni katalizator je bil okarakteriziran z up- orabo FTIR, SEM, EDS in XRD. Pod vplivom UV svetlobe je bil preučevan vpliv različnih parametrov na razbarvanje DB-22, kot so modifikacija katalizatorja s HCl, količina H2O2, količina katalizatorja, količina CaCl2, začetni pH, začetna koncentracija in temperatura. Raziskane so bile kinetične in termodinamične lastnosti. Najvišje razbarvanje DB-22 je bilo 93,3 % pod sledečimi pogoji: začetna koncentracija: 0,070 g/L, začetna temperatura: 25 °C in prvotni pH, količina H2O2: 2,78 g/L, količina m-ZZF: 3 g/L, količina CaCl2: 3,75 g/L, reakcijski čas: 60 min in UV svetloba. Aktivacijska en- ergija določena pri preučevanih pogojih je bila –14,76 kJ/mol, reakcija razbarvanja pa je eksotermna z reakcijsko entalpi- jo –17,31 kJ/mol. Izračunana vrednost aktivacijske entropije je –0,326 kJ/mol. Standardna Gibbsova sprememba proste energije za aktivacijo ima pozitivno vrednost in se z naraščanjem temperature viša. 564 Acta Chim. Slov. 2022, 69, 564–570 Osredkar et al.: The Predictive Value of Oxidative Stress Index in Patients ... DOI: 10.17344/acsi.2022.7432 Scientific paper The Predictive Value of Oxidative Stress Index in Patients with Confirmed SARS-COV-2 Infection Joško Osredkar,1,3,* Sara Pucko,1,3 Milica Lukić,2 Teja Fabjan,1,3 Elizabeta Božnar Alič,1 Kristina Kumer,1,3 Maria Martin Rodriguez4 and Matjaž Jereb 2,5 1 University Medical Centre Ljubljana, Clinical Institute of Clinical Chemistry and Biochemistry, Zaloška cesta 2, 1000 Ljubljana, Slovenia 2 University Medical Centre Ljubljana, Infectious Diseases Department, Zaloška cesta 2, 1000 Ljubljana, Slovenia 3 University Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia 4 University of Alcala, Faculty of Pharmacy, Carretera Madrid-Barcelona, Km.33,600 28871 Alcala de Henares (Madrid), Spain 5 University Ljubljana, Medical Faculty, Vrazov trg 1, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: josko.osredkar@kclj.si Received: 05-27-2022 Abstract Disbalance balance between oxidants and antioxidants is called oxidative stress and could be presented as oxidative stress index (OSI). OSI is determined by the reactive oxygen metabolites test (d-ROM test) to assess oxidants and the plasma antioxidant capacity test (PAT test) to measure antioxidants. The aim of the study was to evaluate the predictive value of OSI in the disease COVID-19. d-ROMs results were the highest in the SARS-CoV-2 POSITIVE group (365+/-112), lower in the SARS-CoV-2 NEGATIVE group (314+/-72.4), and the lowest in an INTENSIVE CARE UNIT group (ICU) (277+/-142) U.Carr. PAT test values were the lowest in the SARS-CoV-2 POSITIVE group (2762+/-387), higher in the ICU group (2772 +/-786), and the highest in the SARS-CoV-2 NEGATIVE group (2808+/-470), and are not statistically significantly different (P > 0.05), while OSI was: healthy with average value of 49 and the critical ill with average value of 109 (P = 0.016). Cut-offs for predicting ICUs admission was at OSI 62, with 80.0% sensitivity and 68.2% specificity. Keywords: Oxidative stress; SARS-CoV-2; OSI Index 1. Introduction Oxidative stress in cells and tissues is caused by an imbalance between the formation of reactive oxygen spe- cies (ROS) and the ability to detoxify ROS with the anti- oxidant system. The balance of ROS and antioxidants may be disturbed by the increased formation of ROS and/or de- creased antioxidant activity. This imbalance leads to many spontaneous oxidations in the cell and, because ROS can be reducers of almost all cellular components, oxidation of biological macromolecules such as lipids, proteins, and nucleotides. Oxidation of macromolecules leads to their denaturation and, consequently, changes in their physio- logical functions. The chronic production of ROS causes toxic effects that lead to cell damage, long-term oxidative stress, accelerated aging, and many diseases including de- mentia, inflammation, cancer, diabetes, and cardiovascu- lar disease. In contrast to these diseases, short-term acute oxidative stress does not yield typical clinical signs to di- vulge its presence. Symptoms of oxidative stress usually come to the fore only when chronic diseases develop.1,2 Although abnormal levels of ROS are detrimental, small amounts of ROS production occur normally and have physiological roles. In a homeostatic state, slightly more oxidants are present than antioxidants because small amounts of ROS are formed as by-products of oxygen metabolism. Normal levels of ROS can assist with cellular signaling by changing gene and protein expression, syn- 565Acta Chim. Slov. 2022, 69, 564–570 Osredkar et al.: The Predictive Value of Oxidative Stress Index in Patients ... thesizing certain hormones, and defending against infec- tions.3 Inflammation is the body’s normal response to inju- ries, pathogens, irritants, and other toxins. The cells of the immune system that are involved in this process include neutrophils and monocytes during acute inflammation, and macrophages, especially in chronic inflammation. These phagocytes use very strong oxidants from ROS and reactive nitrogen species (RNS) groups when microbes in- vade.1 The sudden production of large amounts of reactive species produced by phagocytes is called an oxidative erup- tion. This process is usually limited to the acute response to a pathogen, but if chronic inflammation occurs, it can cause chronic oxidative stress. In chronic inflammation, the increasing amounts of ROS and RNS lead to the oxidation of cellular components and, thus, damage and apoptosis. The entry of a virus into the cell first triggers the acti- vation of innate immune cells (macrophages, neutrophils) that arrive at the site of infection and trigger an inflamma- tory response. Macrophages secrete cytokines and produce several oxidants that they use to defend themselves against the virus. Production of oxidants by macrophages depends on NADPH oxidase, which leads to the formation of O2, and on myeloperoxidase, which catalyzes the formation of hypochlorous acid. ROS can activate epithelial cells and alveolar macrophages to generate chemotactic molecules that further attract neutrophils and, especially, monocytes and lymphocytes into the lungs, providing an ideal envi- ronment for the development of chronic inflammation. Inflammation is key in the progression of COVID-19 pathology. Presentations of SARS-CoV-2 infection have ranged from asymptomatic or mildly symptomatic to se- vere disease and death. Common symptoms include fever, headache, cough, and shortness of breath. Other symp- toms, such as malaise and acute respiratory distress syn- drome (ARDS), have also been described.4 Particular laboratory features have been associated with a more severe course of the disease and worse out- comes. A progressive decline in the lymphocyte count and rise in the D-dimer concentration was observed in those who succumbed to the disease, compared with survivors who exhibited more stable levels of the D-dimer.5 In severe cases of COVID-19, it is common to ob- serve prolonged prothrombin time, elevated levels of lac- tate dehydrogenase, deficient cellular immune response, activation of coagulation, and damage to the heart, liver, and kidneys.6,7 The immune response plays a key role in controlling the SARS-CoV-2 infection, but excessive and uncontrolled activation of the immune response can contribute to a more severe course of the disease.8 Preclinical studies suggest that increased ROS pro- duction and decreased antioxidant responses play an important role in the pathogenesis of viral infection and also in disease progression and severity. The severe course of COVID-19 disease involves the connection of sever- al pathophysiological processes such as cytokine storm, inflammation, cellular apoptosis, and redox imbalance, which contribute to the poor outcomes of COVID-19.9 Lymphocyte infiltration into the lungs may explain the lymphopenia and elevated neutrophil to lymphocyte ratio observed in critically ill COVID-19 patients. The ele- vated neutrophil to lymphocyte ratio is also used to predict the death of critically ill COVID-19 patients. The conse- quence of increased ROS secreted by neutrophils, mac- rophages, and other immune cells has so far had two out- comes: 1) ROS damages erythrocytes, which release heme into the bloodstream, which is broken down by heme oxy- genase, which releases free iron; and 2) an oxidative erup- tion occurs, leading to the formation of a superoxide rad- ical and hydrogen peroxide. Furthermore, oxidative stress and free iron convert fibrinogen into abnormal fibrin clots, leading to the formation of micro thromboses in the vas- cular system and pulmonary microcirculation.7,10 Increased ROS production also directly or indirectly triggers the NF-κB signaling pathway, and studies suggest that its activation is responsible for the more severe course of COVID-19 disease. NF-κB is one of the major media- tors of cytokine and chemokine induction and is a central coordinator of the innate and adaptive immune respons- es.7,11,12 If over-activation of all these pathways occurs, like- ly depending on the amount of virus present, a cytokine storm can develop, leading to ARDS. The cytokine storm is triggered via these oxidative stress-signaling pathways by activated leukocytes, including B and T cells, mac- rophages, monocytes, neutrophils, dendritic cells, as well as epithelial and endothelial cells.13–15 Hydroperoxides are formed by the oxidation of various biological molecules such as amino acids, pep- tides, proteins, nucleotides, and, to the greatest extent, by the oxidation of lipids. Peroxides are only one of the groups of reactive oxygen species, but they are an ear- ly marker of lipid oxidation as they are formed in the initial stages of oxidative stress unlike other markers (malondialdehyde, isoprostane). Therefore, peroxides are early indicators of oxidative stress.16–18In this study, we wanted to investigate how the OSI index may be a good predictor of the severity of COVID-19 disease. 2. Materials and Methods 2. 1. Patients Measurements of oxidants and antioxidants were performed on 171 (M/F = 42/129) samples taken at Uni- versity Medical Centre Ljubljana (UMCL). Subjects were divided into 3 groups according to the course of the dis- ease. Group 1 (SARS-CoV-2 NEGATIVE): employees of UMCL who had a negative PCR test for SARS-CoV-2 in- fection (79). 566 Acta Chim. Slov. 2022, 69, 564–570 Osredkar et al.: The Predictive Value of Oxidative Stress Index in Patients ... Group 2 (SARS-CoV-2 POSITIVE): employees of UMCL who had a positive PCR test for SARS-CoV-2 in- fection without symptoms (51). Group 3 (INTENSIVE CARE): A group of people who were hospitalized in the intensive care unit (ICU) of UMCL due to a severe course of COVID-19 (41). 2. 2. Methods We used d-ROMs to measure oxidants and a PAT test to measure serum antioxidants. From the values of both tests, we then calculated the values of the oxidative stress index (OSI index) according to the FRAS5 analyzer algorithm, which summarizes the values of d-ROMs and PAT tests into one value to facilitate the evaluation of ox- idative stress. d-ROMs fast is a photometric test that gives us the status of oxidants in a biological sample by measuring hy- droperoxides (ROOH). The d-ROMs fast test is based on the Fenton reaction. Measurement with a FRAS5 photometer was performed at 505-546 nm. The color intensity was directly proportional to the ROS concentration in the sample. The PAT test is a method that evaluates the antioxi- dant power of a biological sample. Measuring the antiox- idant power of a sample is important as antioxidants are the first line of defense in the fight against oxidative dam- age.16,18 The PAT test is used to quantify water-soluble anti- oxidants in a biological sample by measuring its ability to reduce ferric ions (Fe3+) to Fe2+ ions. The measured an- tioxidants represent the main components of plasma in defense against oxidation: vitamin C, vitamin E, uric acid, and bilirubin. The values of the OSI index are obtained by a certain arithmetic transformation and enable easier interpretation of oxidative stress for an individual sample. The OSI index does not have to replace the results of d-ROMs and PAT test, but complements them and presents the state of oxi- dative stress in the body.19 2. 3. Statistics Statistical analyses were performed with IBM SPSS (version 22). We first established whether our data sets were normally distributed with the Shapiro-Wilk test for normality and established that the distribution of the oxidative stress index was nonparametric. The data were logarithmically transformed and a follow-up Shap- iro-Wilk test determined that the logarithmically-trans- formed data were normally distributed. We used the one-factor ANOVA parametric test and the post hoc Bonferroni test and Dunn’s Method test for further anal- ysis of statistical significance. For descriptive statistics, we used mean and standard deviation (SD) to summa- rize our data. 3. Results and Discussion Table 1: Basic statistics of d-ROMs, PAT, and OSI tests. N d-ROMs PAT OSI [U. Carr] [U. Cor] Median (IRQ) SARS-CoV-2 79 NEGATIVE Mean 314 2808 46 SD 72.4 470 (28–61) SARS-CoV-2 51 POSITIVE Mean 365 2762 56 SD 112 387 (31–84) INTENSIVE 41 CARE Mean 277 2772 109 SD 142 786 (60–134) * d-ROMs - ROS concentration assay PAT- antioxidant concentration assay OSI - index of oxidative stress SARS-CoV-2 - SARS-associated coronavirus 3. 1. Comparison of Groups in the Coordinate System We used a coordinate system to show where certain groups of patients are concentrated based on their OSI (Figure 1). The purpose of the OSI index is to integrate a single value based on d-ROMs and PAT test results despite different units of measure and different value ranges. With the OSI index, we can show exactly what the disease state of each group is, for example, the levels of d-ROMs = 500 U.Carr and PAT = 1800 U.Cor can show us the same OSI value (142) as the result d-ROMs = 95 U.Carr and PAT = 3900 U.Cor, although these are completely different con- ditions. Namely, the OSI value serves as a rough picture of oxidative stress; if the values are normal (below 40) we can ascertain that the patient’s redox ratio is balanced, other- wise, when the values are higher (above 40) it is necessary to investigate the cause and look at the values of oxidants and antioxidants. We can most reliably interpret the pa- tient’s condition based on the results of all 3 parameters, clinical laboratory tests, and when the sample was taken during the patient’s illness. We entered d-ROMs test values on the y-axis and PAT test values on the x-axis. Based on these two tests, with the help of OB Manager Online copyright © H&D S.r.l. In: 2.0.16 calculated oxidative stress index values. 3. 2. The Interpretation of the Results in Specific Quadrants The first quadrant includes individuals with normal or high values of d-ROMs test and normal or high values of PAT test: – High values of d-ROMs and normal PAT values indicate 567Acta Chim. Slov. 2022, 69, 564–570 Osredkar et al.: The Predictive Value of Oxidative Stress Index in Patients ... an initial oxidative outbreak due to an innate immune response, but the patient still maintains a good antiox- idant defense. – High values of d-ROMs and high values of PAT are the result of an increase in oxidant species and an anoma- lous increase of the antioxidant reserve that might reflect a state of cellular destruction and release in circulation. The second quadrant includes individuals with normal or high values for d-ROMs test and normal or low values for the PAT test: – High values of d-ROMs and low values of PAT indicate the increase of the antioxidant species and the decrease of the antioxidant response, signs of possible inflamma- tion onset, and hospitalization. – The interpretation of high values of d-ROMs and normal PAT values remain the same as for the first quadrant. The third quadrant includes individuals with normal or low values of d-ROMs and normal or low values of PAT: – Low values of d-ROMs and normal PAT values indicate a long-term infection and exhaustion of the body. Thus, the body is unable to form normal ROS, the efficacy of the innate immune response declines, and antioxidant defenses decline due to pre-existing damage indicating a loss of redox signaling power. – Low values of d-ROMs and low PAT values show the ex- haustion of the ROS system and the antioxidant network. The fourth quadrant is comprised of individuals with normal or low d-ROMs and normal or high PAT test values. – Low values of d-ROMs and normal PAT values in the fourth quadrant indicate the same conditions as for the third quadrant. – Low values of d-ROMs and high values of PAT indicate a long-term infection, which involves extensive inflamma- tion and tissue damage. For the statistical comparison of groups, we used the parametric test one-factor ANOVA and Bonferroni post hoc test. We first performed a test of homogeneity of variances and found that there was no statistically signif- icant difference between all three groups; variances were homogeneous (P = 0.395). This indicated that we could go forward with the one-factor ANOVA and Bonferroni test. The ANOVA result showed that there was a statistically significant difference (P = 0.016) between the individual groups, as shown in Table 2. Table 2: Calculated differences between groups. Group comparison for OSI P SARS-CoV-2 POSITIVE SARS-Cov-2 NEGATIVE 0.272 SARS-CoV-2 POSITIVE INTENSIVE CARE 0.471 SARS-Cov-2 NEGATIVE INTENSIVE CARE 0.024 *OSI - index of oxidative stress SARS-CoV-2 - SARS-associated coronavirus Figure 1: Coordinate system representing OSI and four different quadrants for interpretation. OSI values less than 40 represent normal levels. Values between 41-65 are borderline alert and normal. Values ranging from 66-120 signify a concerning imbalance between oxidants and antioxidants. Values above 121 signify a critical imbalance of oxidants and antioxidants. * OSI - index of oxidative stress 568 Acta Chim. Slov. 2022, 69, 564–570 Osredkar et al.: The Predictive Value of Oxidative Stress Index in Patients ... We did not prove a statistically significant difference between the SARS-Cov-2 positive and negative groups (P = 0.272). The average oxidative stress index of the posi- tive group was 17 units higher than the negative group. According to the reference table, a value of 17 is a con- cerning value, whereas the control group is in the range of the oxidative stress borderline state. Due to the less stress- ful course of the disease (from asymptomatic patients to patients with mild symptoms, which did not require hos- pitalization of patients), there was no critically impaired state of oxidants/antioxidants. We demonstrated a statistically significant difference between the ICU COVID-19 patients and the SARS-Cov-2 negative group (P = 0.024). This difference was expected as the redox ratio of hospitalized persons in ICU was severely disrupted. Some patients had a very high amount of oxidants present, yet others had a very low amount of oxidants, both indicative of oxidative stress. Normal amounts of oxidants are necessary for the normal functioning of the patient. We did not prove statistically significant differenc- es between the SARS-Cov-2 positive group and the ICU group (P = 0.471). The SARS-CoV-2 positive group with- out symptoms had more oxidative stress than the SARS- CoV-2 negative group, but much less than the patients hospitalized in ICU. 3. 2. Interpretation of OSI Values for SARS- Cov-2 NEGATIVE Group: The vast majority of patients had normal values of d-ROMs and PAT, and, consequently, the largest share of them (43.7%) had OSI values below 40, while only 2.3% had OSI above 121. These slight deviations were likely caused by other underlying conditions (such as obesity and differences in physical activity). 3. 3. Interpretation of OSI Values for SARS- CoV-2 POSITIVE Group: Individuals from this group were concentrated in ap- proximately the same part of the coordinate system, name- ly in quadrants I and II. Normal or high values of d-ROMs and normal or high values of PAT were measured. Most in- dividuals (41.5%) of this group had an oxidative stress in- dex below 40, i.e. they had normal levels without oxidative stress. Furthermore, 26.8% of them had values between 66-120 (alert state) and 22% of individuals had values be- tween 41-65 (borderline). The last group of 66-120, which is already considered a warning state, included the fewest individuals (9.7%). The results, which were slightly above normal but not critical, were in agreement with the symp- toms of the participants, which were mild although they tested positive for SARS-CoV-2. We hypothesize that the cause of high values of d-ROMs is an oxidative outbreak due to the innate immune response, while the antioxidant system also functions to fight high amounts of ROS. 3. 4. Interpretation of OSI Values for INTENSIVE CARE Group: We observed the most diverse patient conditions in this group. The majority of individuals had OSI values be- low 40 (33.3%) and above 121 (26.7%). Based on the results of d-ROMs, PAT tests, and OSI values, this large variation in patient condition was expected in the ICU group. We observed very diverse values in intensive care patients and most (66.7%) completely disturbed the balance of oxidants/ antioxidants. In quadrant II were individuals who had most- ly elevated values from the d-ROMs test and normal or de- creased values from the PAT test. Based on these two results, we concluded that these patients were in the initial stage of COVID-19 disease and had just been admitted to ICU. Individuals in quadrants I and II were patients with very high values of d-ROMs and normal or elevated PAT values. The first quadrant includes patients who were in the initial stage of the disease. In those who had elevated levels of d-ROMs and PAT, there was an extensive immune response that triggered an oxidative outburst and conse- quently an increased response of antioxidants. Individuals in quadrants III and IV +- were patients with very low d-ROMs scores and normal PAT scores, and patients with very low d-ROMs scores and high PAT scores. In both cases, these are samples taken during hos- pitalization in the ICU after the COVID-19 infection had been going on for some time. At this point during infec- tion, the body is already exhausted and unable to form ROS, nor is there an effective innate immune response. The antioxidant system is also active, trying to remove the damage. In the group with low d-ROMs and high PAT scores, high PAT values indicated inadequate redox signa- ling and increasingly severe tissue damage. Individuals in quadrant IV were critically ill patients with low d-ROMs and high PAT values. As in the above example, there was an increasing amount of tissue damage and slow organ failure. The frequency of the OSI index is shown in Figure 2. A receiver operating characteristics (ROC) curve was constructed and Youden Index was used to determine the optimal cut-off for predicting intensive care unit (ICU) admission. The ROC curve is presented in Figure 3. 4. Conclusions The oxidative stress index serves as a predictor for the course of COVID-19 disease. Based on our data, it is reasonable to think that OSI is a good predictive index for ICU admission where a cut-off of 62 was identified. The very low d-ROMs level observed in patients 1 and 2 can be explained by the pathological status of the subjects. In contrast, high PAT levels can be explained by hemolysis processes, through which a high amount of glutathione (GSH) is released from red blood cells. Simul- 569Acta Chim. Slov. 2022, 69, 564–570 Osredkar et al.: The Predictive Value of Oxidative Stress Index in Patients ... taneously, the antioxidants are not used by the individual due to the lack of ROS species, which could contribute to why PAT levels are quite high in some cases. Moreover, this could be the reason why statistically significant differ- ences in d-ROMs and PAT were not identified in the pa- tients analyzed since the levels strongly depend upon the time course of the disease, and pathology can occur with low or high ratios of d-ROMs/PAT. With the help of the coordinate system, we evaluated the disease status of the patients. We found that the PCR negative group was concentrated approximately in the middle of the coordinate system, signifying that most of the values of the measured parameters were within nor- mal reference limits. The PCR positive group was primar- ily found in quadrants I and II, and the values of oxida- tive stress parameters indicated a shift from the normal reference limits. A wide variety of disease conditions were present in the ICU group, some of which were in the initial stage of the disease and had just been admitted to the ICU, and the results of d-ROMs, PAT, OSI were not as severe as in individuals with long-term hospitalization. A comparison of the oxidative stress index in two ICU patients with biochemical and hematological param- eters (supplemental data) showed that the values of the OSI index correlated very well with the patients’ disease state and the inflammatory parameters. We compared CRP, lymphocyte and neutrophil count, IL-6, and oxida- tive stress index. The latter varied with a lag compared to the others, but this is consistent with studies by test man- ufacturers d-ROMs and PAT, where we found that oxidant levels rise when there is actual oxidative damage and thus reflect the current state of the body. Doğan et al. published a study in which they cal- culated the OSI index from the parameters total oxidant status (TOS) and total antioxidant status (TAS). The calcu- lated OSI levels were significantly different between severe moderate and mild groups of patients infected with SARS- CoV-2.20 The same method for calculating OSI was also used by Çakırca et al. Their results revealed that the increase in oxidative stress and decrease in antioxidant levels in COV- ID-19-infected patients were associated with worsening of disease.21 The results of our study, in which we calculated OSI based on d-ROM and PAT determinations, are com- parable to the results of both of these studies, which used TOS and TAS to calculate OSI. In our study of oxidative stress, our results suggest that OSI could be a predictor of the course of SARS-CoV-2 infection and warrants further investigation. Our studies were conducted on a small number of samples so further research with more samples is necessary. Acknowledgments Funding The study was funded by the research program of the Research Agency of the Republic of Slovenia (P3-0124). Author Contributions Conceptualization JO; Writing – Original draft Preparation JO; Clinical data of the Patients ML, MJ; Lab- oratory Analysis SP, TF, EBA; Statistics TF; Writing – Re- view & Editing MJ. Figure 2: Calculated OSI of the whole study group. * OSI - index of oxidative stress Figure 3: ROC curve for predicting ICU admission for COVID-19 positive patients. * OSI - index of oxidative stress AUC - area under the ROC curve 570 Acta Chim. Slov. 2022, 69, 564–570 Osredkar et al.: The Predictive Value of Oxidative Stress Index in Patients ... Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Nation- al Ethics Committee; protocol number – 012-60/2021/5. Conflicts of interest The authors declare no conflict of interest. 5. References 1. J. Mravljak, Farm. Vestn. 2015, 66, 127–132. DOI:10.1016/j.intermet.2015.07.002 2. G. Pizzino, N. Irrera, M. Cucinotta, G. Pallio, F. Mannino, V. Arcoraci, F. Squadrito, D. Altavilla, A. Bitto, Oxid. Med. Cell. Longev. 2017, 2017. DOI:10.1155/2017/8416763 3. J. Osredkar, Zdr. Vestn. 2012, 81, 393–406. DOI:10.3982/ECTA10449 4. M. Merad, J. C. Martin, Nat. Rev. Immunol. 2020, 20, 355–362. DOI:10.1038/s41577-020-0331-4 5. D. Wang, B. Hu, C. Hu, F. Zhu, X. Liu, J. Zhang, B. Wang, H. Xiang, Z. Cheng, Y. Xiong, et al., J. Am. Med. Assoc. 2020, 323, 1061–1069. DOI:10.1001/jama.2020.1585 6. B. Vellingiri, K. Jayaramayya, M. Iyer, A. Narayanasamy, V. Govindasamy, B. Giridharan, S. Ganesan, A. Venugopal, D. Venkatesan, H. Ganesan, et al., Sci. Total Environ. 2020, 725. DOI:10.1016/j.scitotenv.2020.138277 7. R. Cecchini, A. L. Cecchini, Med. Hypotheses 2020, 143. DOI:10.1016/j.mehy.2020.110102 8. Y. R. Guo, Q. D. Cao, Z. S. Hong, Y. Y. Tan, S. D. Chen, H. J. Jin, K. Sen Tan, D. Y. Wang, Y. Yan, Mil. Med. 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H&D srl, Colorimetric determination of reactive oxygen me- tabolites (ROMs), https://innovaticslabs.com/wp-content/ uploads/2018/04/d-ROMLab-test-specification_ENG-1.pdf, (assessed: March 31, 2019). 18. H&D srl, Colorimetric determination of biological antioxi- dant potential, https://innovaticslabs.com/wp-content/up- loads/2018/04/PATLab-test-specification_ENG-1.pdf, (as- sessed: May 10, 2019). 19. H&D srl, Oxidative stress index OSI, https://innovaticslabs. com/wp-content/uploads/2018/04/OSI_Oxidative-Stress-In- dex.pdf, (assessed: March 21, 2019). 20. S. Dogan, T. Bal, M. Çabalak, N. Dikmen, H. Yaqoobi, O. Oz- can, Turkish J. Biochem. 2021, 46, 349–357. DOI: https://doi.org/10.1515/tjb-2021-0013 21. G. Çakırca, T. D. Çakırca, M. Üstünel, A. Torun, İ. Koyuncu, Ir. J. Med. Sci. 2021, 1–6. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Neravnovesje med oksidanti in antioksidanti imenujemo oksidativni stres in ga lahko prikažemo kot indeks oksidativne- ga stresa (OSI). OSI določimo s testom reaktivnih presnovkov kisika (d-ROM) za oceno oksidantov in testom plazemske antioksidativne kapacitete (PAT) za merjenje antioksidantov. Namen študije je bil oceniti napovedno vrednost OSI pri bolezni COVID-19. Rezultati tasta d-ROM so bili najvišji v skupini SARS-CoV-2 pozitivni (365+/-112), nižji v skupini SARS-CoV-2 negativni (314+/-72,4) in najnižji v skupini kritično bolnih v enoti za intenzivno nego (ICU) (277+/-142) U.Carr. Vrednosti testa PAT so bile najnižje v skupini SARS-CoV-2 pozitivni (2762+/-387), višje v skupini kritično bol- nih (2772 +/-786) in najvišje v skupini SARS-CoV-2 negativni (2808 +/-470). Skupine se med sabo statistično značilno ne razlikujejo (P>0,05). OSI se statistično značilno razlikuje med zdravimi s povprečno vrednostjo 49 in kritično bolnimi s povprečno vrednostjo 109 (P = 0,016). Določili smo mejno vrednost za napovedovanje sprejema pacienta v enoto in- tenzivne nege na osnovi analize OSI, in sicer 62, z 80,0 % občutljivostjo in 68,2 % specifičnostjo. 571Acta Chim. Slov. 2022, 69, 571–583 Lazarević et al.: Carvacrol Derivatives as Antifungal Agents: ... DOI: 10.17344/acsi.2022.7438 Scientific paper Carvacrol Derivatives as Antifungal Agents: Synthesis, Antimicrobial Activity and in Silico Studies on Carvacryl Esters Jelena Lazarević,1,* Ana Marković,2 Andrija Šmelcerović,1 Gordana Stojanović,3 Pierangela Ciuffreda4 and Enzo Santaniello5 1 Department of Chemistry, Faculty of Medicine, University of Niš, Bulevar dr Zorana Đinđića 81, 18000, Niš, Serbia 2 Department of Pharmacy, Faculty of Medicine, University of Niš, Bulevar dr Zorana Đinđića 81, 18000, Niš, Serbia 3 Department of Chemistry, Faculty of Science and Mathematics, University of Niš, Višegradska 33, 18000, Niš, Serbia 4 Dipartimento di Scienze Biomediche e Cliniche “L. Sacco”, Università degli Studi di Milano, Via G.B. Grassi 74, 20157 Milano, Italy 5 Faculty of Medicine. University of Milan, Italy * Corresponding author: E-mail: jelena217@yahoo.com; jelena.lazarevic@medfak.ni.ac.rs Tel: +381 63 1045128; fax: +381-18-42-38-770 Received: 03-03-2022 Abstract Chemical modifications of natural monoterpenoids to various derivatives have been reported to result in enhancement of biological activities when compared to parent compounds. In this context a well-known biocide and food additive, carvacrol, served as a basic scaffold onto which a phenolic functionality transformation by introducing acyl groups was performed. By using this simple methodology, we obtained a small series of 25 esters. For each of the obtained com- pounds we have performed structural characterization, in vitro antimicrobial testing and in silico calculation of physi- co-chemical, pharmacokinetic and toxicological properties. Despite numerous data on the synthesis and bioactivity of carvacryl ester lower homologues, there are scarce data on esters with acid components higher than C9, so that among 25 compounds, 10 were reported for the first time (spectral characterization for 12 are herein the first reported). Our research is also the first comprehensive study of carvacryl esters antifungal and of medium/long chain fatty acid esters antibacterial activities. Interesting result is that all the synthesized esters, regardless the nature of the R residue, have shown activity on fungal strain Aspergilus niger and on yeast Candida albicans comparable to carvacrol. Besides present- ed experimental data, implementation of in silico calculation of physico-chemical, pharmacokinetic and toxicological properties on the prepared compounds, may be valuable information in further research. Keywords: chemical transformation; carvacrol esters; in vitro antimicrobial activity; in silico calculation 1. Introduction Natural products and their scaffolds have a long his- tory of application as valuable starting points for medici- nal chemistry and drug discovery.1 Their structural modi- fication, when compared to parent compounds, has often afforded structures with enhanced pharmacological activ- ities and outstanding therapeutic possibilities.2,3 Carvacrol (2-methyl-5-(1-methylethyl)-phenol), a monoterpenoid phenol biosynthetically related to pa- ra-cymene, frequently occurs in essential oils of many La- miaceae (Origanum, Thymbra, Thymus, Satureja) and Ver- benaceae (Lippia) plants usually used as spices and for therapy/prevention purposes in folk medicine.4 A variety of biological properties including antioxidant, antimicro- bial, antiviral, insecticidal, antiparasitic, antihypertensive, immunomodulatory and antitumor, resulted from numer- ous studies overtaken in past 20 years, recently reviewed by Rathod et al. and Sharifi-Rad et al.5,6 Moreover, the Eu- ropean Commission has included carvacrol in the list of 572 Acta Chim. Slov. 2022, 69, 571–583 Lazarević et al.: Carvacrol Derivatives as Antifungal Agents: ... chemical flavors and Food and Drug Administration (FDA) approved carvacrol, together with carvacryl ethyl ether, carvacryl acetate and carvacryl propanoate, consid- ering them safe from a toxicological point of view for their use as additives in food products.7,8 Although carvacrol is well known as effective in con- trolling infection diseases, the molecular mechanisms in- volved in antimicrobial action are not yet completely eluci- dated. The antibacterial activity of carvacrol has been attributed to its considerable effects on the structural and functional properties of cytoplasmatic membrane, involving outer- and inner membrane disruption and interaction with membrane proteins and intracellular targets.9,10 The most recent study by Niu et al.11 reported carvacrol could trigger Candida albicans apoptosis, causing membrane disruption, inducing ROS production and mitochondrial dysfunction. Carvacryl derivatives, either natural or synthetic, have also been employed in biological testing with vast range of activities, such as antibmicrobial,12–15 antiinflamatory,16 an- tioxidant,17–21 anticancer,17,19,20 larvicidal,22,23 antihelmint- ic,24,25 also acting as enzyme inhibitors (acetylcholinesterase and butyrylcholinesterase,26 mushroom tyrosinase,27 Myco- bacterium tuberculosis chorismate mutase28). There are also a large number of reports on the synthe- sized esters of carvacrol and on their biological activi- ties.15,16,22,23,27,29–36 Antimicrobial assays have evaluated activ- ity of a few carvacryl esters of straight chain lower carboxylic acid homologues and diverse heteroaromatic carboxylic ac- ids.15,29,30,34,37 Interestingly, versus plentiful data on the syn- thesis and bioactivity of lower homologues, there are sporadic or no data on carvacryl esters with acid components higher than C9 (except for C12 reported in Bassanetti et al.34). In the context of diverse biological activities of car- vacrol and rich number of promising studies on carvacrol derivatives, a one-step transformation of phenolic func- tionality by introducing an acyl group was made. We have obtained a series of 25 compounds (3a–y), which, after structural characterization, have been involved in in vitro antimicrobial testing. This research is the first comprehen- sive study of the antifungal activity of the synthesized car- vacrol derivatives and the first study on antimicrobial ac- tivity of carvacryl ester medium/higher homologues. Along with experimental data we provided in silico predic- tions of physico-chemical, pharmacokinetic and toxico- logical properties for entire group of the synthetized deriv- atives. Current paper also complements our work on modifying the phenolic function of a few most active nat- ural biocides found in essential oils.38,39 2. Experimental 2. 1. Chemicals All chemicals used were of analytical reagent grade. Unless specified otherwise, all reagents and standards were purchased from Merck (Darmstadt, Germany). 2. 1. 1. General Synthetic Procedures Acetyl, benzoyl, palmitoyl, stearoyl and oleoyl chlo- ride were purchased from Sigma-Aldrich and were used directly in the synthesis of carvacryl esters. For other acyl chlorides used in the study we have utilized two synthetic approaches depending on whether the transformed acids had less (together with 2-chloroacetyl and trichloroacetyl chloride)40 or more than 10 carbon atoms,41 both de- scribed by Lazarević et al.38 and Lazarević et al.39 The material obtained following the above protocols was used directly in the synthesis of esters that was per- formed as reported in Paolini et al.42 The synthesis of car- vacryl (5-isopropyl-2-methylphenyl) esters 3a–y is repre- sented in Scheme 1. The obtained esters 3a–y were purified by column chromatography, stationary phase Silica Gel 60 (70–230 mesh), mobile phase (hexane/diethyl ether, gradi- ent 9:1 to 7:3). Data on yields are given in Table 1. 2. 2. Identification of Synthetized Compounds 2. 2. 1. GC-MS Analysis MS spectra of samples of the synthesized compounds were recorded on a 7890/7000B GC/MS/MS triple quad- rupole system (Agilent Technologies, USA, equipped with a Combi PAL auto sampler). The fused silica capillary col- umn HP-5MS (5% phenylmethylsiloxane, 30 m × 0.25 mm, film thickness 0.25 μm, Agilent Technologies, Palo Alto, CA, USA) was used. The injector, source and inter- face operated at 250, 230 and 300 °C, respectively. The temperature program: from 60 for 5 min isothermal to 300 °C at a heating rate of 8 °C/min and on 300 °C for 5 min isothermal. The solutions in hexane were injected in split ratio 10:1. The carrier gas was helium with a flow of 1.0 mL/min. Post run: back flash for 1.89 min, at 280 °C, with helium at 50 psi. MS conditions were as follows: ionization voltage of 70 eV, acquisition mass range 50–650, scan time 0.32 s. Semi-quantitative analysis was carried out directly from peak areas in the GC profile. Linear retention indices (RI) were determined based on the retention times of C8– C40 alkanes run on HP-5MS column, using the above men- tioned temperature programme.43 2. 2. 2. NMR Analysis NMR spectra were registered on a Bruker AVANCE 500 spectrometer equipped with a 5 mm broadband re- verse probe with field z-gradient operating at 500.13 and 125.76 MHz for 1H and 13C, respectively. All NMR spectra were recorded at 298 K in CDCl3 (isotopic enrichment 99.95%) solution. Chemical shifts are reported on the δ (ppm) scale and are relative to the residual CHCl3 signals (7.24 for 1H and 77.0 ppm, central line, for 13C spectra, respectively), and scalar coupling constants are reported in Hertz. The experimental error in the measured 1H–1H coupling constants was ±0.5 Hz. The signals assignment was given by a combination of 1D and 2D COSY, HSQC 573Acta Chim. Slov. 2022, 69, 571–583 Lazarević et al.: Carvacrol Derivatives as Antifungal Agents: ... and HMBC experiments, using standard Bruker pulse programs. Acquisition parameters for 1D were as follows: 1H spectral width of 5000 Hz and 32k data points provid- ing a digital resolution of ca. 0.305 Hz per point, relaxation delay 2 s; 13C spectral width of 29412 Hz and 64k data points providing a digital resolution of ca. 0.898 Hz per point, relaxation delay 2.5 s. The experiments were per- formed through standard pulse sequences. gCOSY-45 ex- periments were acquired with 512 t1 increments; 2048 t2 points; spectral/spectrum width 10.0 ppm. The acquisition data for gHSQC and gHMBC experiments were obtained with 512 t1 increments; 2048 t2 points; spectral/spectrum width 10.0 ppm for 1H and 220 ppm for 13C. Delay values were optimized for 1JC,H 140.0 Hz and nJC,H 3.0 Hz. Zero filling in F1 to 1k, p/2 shifted sine-bell squared (for gHSQC) or sinebell (for gHMBC) apodization functions were used for processing. 2. 3. Antimicrobial Activity 2. 3. 1. Microbial Strains Antimicrobial activity of the synthesized compounds was tested in vitro against a panel of laboratory control strains belonging to the American Type Culture Collec- tion Maryland, USA: Gram-positive: Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 6538; Gram-negative: Escherichia coli ATCC 8739 and Salmone- la typhimurium ATCC 14028, fungal organisms: Aspergil- lus niger ATCC 16404 and Candida albicans ATCC 10231. The Gram-negative bacteria Salmonella abony NCTC 6017 was obtained from the National Collection of Type Cultures. All microorganisms were maintained at –20 °C under appropriate conditions and regenerated twice be- fore use in the manipulations. 2. 3. 2. Screening of Antimicrobial Activity The minimal inhibitory concentration (MIC) of es- ters was determined based on a broth microdilution meth- od performed in 96-well microtitre plates.44 The inocula of the bacterial strains were prepared from overnight broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. Dimethyl sulphoxide (10% aqueous solution) was used to dissolve and to dilute samples to the highest concentration to be tested (stock concentrations 2 mg/mL). A serial doubling dilution of the samples was prepared in a 96-well microtiter plate, using the method of Sarker et al.,45 with slight modifications. The minimal bac- tericidal/fungicidal concentration (MBC/MFC) was eval- uated as the lowest concentration of tested samples at which inoculated microorganisms were 99.9% killed. Tests were carried out in triplicate. The procedure is described in detail by Lazarević et al.46 2. 4. In Silico Physico-chemical, Pharmacokinetic, and Toxicological Properties of the Synthetized Compounds Together with experimental data we provided an in silico study on physico-chemical, pharmacokinetic and tox- icological properties of the synthesized compounds 3a–y. In silico predictions were accomplished using the Molinspi- ration,47 admetSAR,48 DataWarrior,49 and Toxtree50 tools. 3. Results and Discussion 3. 1. Chemical Synthesis A small focused library of 25 carvacryl esters was synthesized according to previously published standard methodology, given in Scheme 1, with yields ranging from 95 to 52% (Table 1). To the best of our knowledge com- pounds 3m, 3o–w are new (Table 1). For solid compounds 3t, 3v, 3x and 3y melting points were determined in a Stu- art Scientific SMP3 apparatus and are uncorrected. 3. 2. Spectral Data on Synthetized Compounds 3. 2. 1. Carvacryl Acetate (3a)22,30 Chromatographic purification gave colorless oil. C12H16O2 (M = 192.25); yield 87%; RI (HP5-MS): 1384; 1H Scheme 1. Synthesis of the assayed esters 3a–y. Reagents and conditions: 2a–y solution was added dropwise to the solution of 1 and triethylamine (Et3N), all previously dissolved in dichloromethane (DCM). During the addition, temperature was maintained at 0 °C. After reaching room temper- ature, the reaction mixture was refluxed for 3 h.42 For structures and 3a–y designations, see Table 1. 574 Acta Chim. Slov. 2022, 69, 571–583 Lazarević et al.: Carvacrol Derivatives as Antifungal Agents: ... NMR (CDCl3, 500.13 MHz) δ 7.14 (d, J = 8.0 Hz, 1H, Ar-H), 7.04 (d, J = 8.0 Hz, 1H, Ar-H), 6.89 (s, 1H, Ar-H), 2.90 (spt, J = 6.9 Hz, 1H, CH), 2.34 (s, 3H, CH3) 2.16 (s, 3H, CH3), 1.25 (d, J = 6.9 Hz, 6H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 169.3 (C=O), 149.3 (CAr), 148.1 (CAr), 130.9 (CAr), 127.2 (CAr), 124.2 (CAr), 119.8 (CAr), 33.6 (CH), 23.9 (2 × CH3), 20.8 (CH3), 15.8 (CH3-Ar); MS (EI): m/z (%): 192 (M+) (8.0), 151 (6.4), 150 (55.2), 136 (9.7), 135 (100), 105 (6.8), 105 (5.2), 91 (18.5), 79 (6.6), 77 (9.9), 43 (10.1). 3. 2. 2. Carvacryl 2-Chloroacetate (3b)22,27 Chromatographic purification gave colorless oil. C12H15ClO2 (M = 226.70); yield 83%; RI (HP5-MS): 1598; 1H NMR (CDCl3, 500.13 MHz) δ 7.19 (d, J = 7.6 Hz, 1H, Ar-H), 7.08 (dd, J = 8.0, 1.7 Hz, 1H, Ar-H), 6.93 (d, J = 1.7 Hz, 1H, Ar-H), 4.34 (s, 2H, CH2), 2.91 (m, 1H, CH), 2.19 (s, 3H, CH3), 1.26 (d, J = 6.9 Hz, 6H, 2 × CH3). 13C NMR (CDCl3, 125.76 MHz) δ 165.7 (C=O), 148.8 (CAr), 148.3 (CAr), 131.1 (CAr), 126.9 (CAr), 124.7 (CAr), 119.3 (CAr), 40.7 (CH2), 33.6 (CH), 23.9 (2 × CH3), 15.7 (CH3-Ar); MS (EI): m/z (%): 226 (M+) (14.5), 151 (8.2), 150 (80.9), 136 (8.8), 135 (100), 133 (6.5), 105 (10.0), 91 (9.9), 79 (6.8), 77 (18.4). 3. 2. 3. Carvacryl Trichloroacetate (3c)22,51 Chromatographic purification gave colorless oil. C12H13Cl3O2 (M = 295.59); yield 91%; RI (HP5-MS): 1731; 1H NMR (CDCl3, 500.13 MHz) δ 7.23 (m, 1H, Ar-H), 7.14 (dd, J = 8.0 Hz, 1.4 Hz, 1H, Ar-H), 7.03 (s, 1H, Ar-H), 2.95 (spt, J = 6.9 Hz, 1H, CH), 2.27 (s, 3H, Ar-CH3), 1.29 (d, J = 6.9 Hz, 6H, CH(CH3)2). 13C NMR (CDCl3, 125.76 MHz) δ 160.4 (C=O), 149.0 (CAr), 148.7 (CAr), 131.4 (CAr), 126.8 (CAr), 125.3 (CAr), 118.6 (CAr), 89.9 (C), 33.7 (CH), 23.9 (2 × CH3), 15.5 (CH3-Ar); MS (EI): m/z (%): 296 (23.0), 294 (24.2), 283 (29.8), 281 (92.1), 279 (M+) (100.0), 133 (63.1), 117 (35.9), 105 (32.3), 91 (43.6), 77 (21.0). 3. 2. 4. Carvacryl Propanoate (3d)22,30,52 Chromatographic purification gave colorless oil. C13H18O2 (M = 206.28); yield 86%; RI (HP5-MS): 1479; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 7.6 Hz, 1H, Ar-H), 7.03 (dd, J = 7.6, 1.7 Hz, 1H, Ar-H), 6.88 (d, J = 1.7 Hz, 1H, Ar-H), 2.93–2.88 (m, 1H, CH), 2.64 (q, 2H, J = 7.4 Hz, CH2), 2.15 (s, 3H, CH3), 1.33 (t, J = 7.4 Hz, 3H, CH3), 1.26 (d, J = 6.9 Hz, 6H, CH3). 13C NMR (CDCl3, 125.8 MHz) δ 172.7 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.1 (CAr), 124.0 (CAr), 119.8 (CAr), 33.6 (CH), 27.7 (CH2), 23.9 (2 × CH3), 15.8 (CH3-Ar), 9.3 (CH3); MS (EI): Table 1. Carvacryl esters 3a–y, mass (g), yield (%) and entry. The reference is related to the previously re- ported synthesis/antimicrobial research. Entry Structure of R in carvacryl esters 3 Mass (g) Yield (%) References 3a CH3 0.59 87 22, 30 3b CH2Cl 0.63 83 22, 27 3c CCl3 0.89 91 22, 51 3d CH2CH3 0.59 86 22, 30, 52 3e CH=CH2 0.50 74 53 3f CH2CH2CH3 0.65 89 52, 54 3g CH(CH3)2 0.62 85 30 3h CH2(CH2)2CH3 0.63 82 52, 44 3i CH2CH(CH3)2 0.63 81 30 3j CH2(CH2)3CH3 0.70 85 23 3k CH2(CH2)4CH3 0.79 91 52* 3l CH2(CH2)5CH3 0.78 85 52* 3m CH2(CH2)6CH3 0.79 82 current study 3n CH2(CH2)7CH3 0.83 83 3o CH2(CH2)8CH3 0.91 87 current study 3p CH2(CH2)9CH3 0.86 78 34 3q CH2(CH2)10CH3 0.93 81 current study 3r CH2(CH2)11CH3 0.90 76 current study 3s CH2(CH2)12CH3 0.91 74 current study 3t CH2(CH2)13CH3 1.12 92 current study 3u CH2(CH2)14CH3 0.97 73 current study 3v CH2(CH2)15CH3 1.17 85 current study 3w CH2(CH2)6CH=CHCH2(CH2)6CH3 0.71 52 current study 3x Ph 0.80 95 22, 23, 30, 54 3y CH3O-Ph 0.89 95 23 * spectral data are presented in the current paper for the first time 575Acta Chim. Slov. 2022, 69, 571–583 Lazarević et al.: Carvacrol Derivatives as Antifungal Agents: ... m/z (%): 206 (M+) (8.3), 151 (7.8), 150 (67.6), 136 (9.9), 135 (100), 133 (6.1), 105 (7.5), 91 (18.6), 79 (6.8), 77 (10.2), 57 (23.7). 3. 2. 5. Carvacryl Acrylate (3e)53 Chromatographic purification gave colorless oil. C13H18O2 (M = 204.27); yield 74%; RI (HP5-MS): 1466; 1H NMR (CDCl3, 500.13 MHz) δ 7.19 (d, J = 7.8 Hz, 1H, Ar-H), 7.06 (dd, J = 6.2, 1.6 Hz, 1H, Ar-H), 6.95 (s, 1H, Ar-H), 6.65 (dd, J = 6.9, 1.3 Hz, 1H, CH2=CH), 6.4 (dd, J = 10.4, 6.9 Hz, 1H, CH=CH2) 6.05 (dd, J = 9.2, 1.4 Hz, 1H, CH2=CH), 2.88–2.97 (m, 1H, J = 6.9 Hz, CH(CH3)2), 2.18 (s, 3H, CH3-Ar), 1.28 (d, J = 6.9 Hz, 6H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 164.3 (C=O), 149.1 (CAr), 148.1 (CAr), 132.3 (=C), 130.9 (CAr), 127.9 (=C<), 127.2 (CAr), 124.2 (CAr), 119.8 (CAr), 33.6 (CH), 23.9 (2 × CH3), 15.7 (CH3-Ar); MS (EI): m/z (%):204 (M+) (25.6), 189 (5.6), 150 (44.7), 149 (5.5), 135 (43.6), 105 (7.1), 91 (15.6), 79 (5.8), 77 (9.2), 57 (100). 3. 2. 6. Carvacryl Butanoate (3f)52,54 Chromatographic purification gave colorless oil. C14H20O2 (M = 220.31); yield 89%; RI (HP5-MS): 1570; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 7.6 Hz, 1H, Ar-H), 7.04 (d, J = 8.0 Hz, 1H, Ar-H), 6.88 (s, 1H, Ar-H), 2.93–2.88 (m, 1H, CH), 2.59 (t, J = 7.5 Hz, 2H, CH2), 2.16 (s, 3H, CH3), 1.85 (sxt, J = 7.5 Hz, 2H, CH2), 1.27–1.25 (m, 6H, CH3), 1.08 (t, J = 7.5 Hz, 3H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 171.9 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.1 (CAr), 124.0 (CAr), 119.3 (CAr), 36.2 (CH2), 33.6 (CH), 23.9 (2 × CH3), 18.6 (CH2), 15.8 (CH3- Ar), 13.8 (CH3); MS (EI): m/z (%): 220 (M+) (8.1), 151 (10.6), 150 (91.2), 136 (10.0), 135 (100), 105 (8.6), 91 (19.7), 77 (9.8), 71 (16.8), 43 (19.6). 3. 2. 7. Carvacryl 2-Methylpropanoate (3g)30 Chromatographic purification gave colorless oil, C14H20O2 (M = 220.31); yield 85%; RI (HP5-MS): 1524; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 8.0 Hz, 1H, Ar- -H), 7.04 (dd, J = 7.8, 1.9 Hz, 1H, Ar-H), 6.87 (d, J = 1.7 Hz, 1H, Ar-H), 2.93–2.84 (m, 2H, CH), 2.16 (s, 3H, CH3) 1.38 (d, J = 6.9 Hz, 6H, 2 × CH3), 1.26 (d, J = 6.9 Hz, 6H, 2 × CH3); 13C NMR (CDCl3, 125.76 MHz) δ 175.3 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.1 (CAr), 123.9 (CAr), 119.8 (CAr), 34.2 (CH), 33.6 (CH), 23.9 (2 × CH3), 19.1 (2 × CH3), 15.8 (CH3-Ar); MS (EI): m/z (%): 220 (13.6), 151 (11.1), 150 (100), 136 (8.6), 135 (91.7), 105 (8.5), 91 (18.9), 77 (9.5), 71 (22.8), 43 (32.8). 3. 2. 8. Carvacryl Pentanoate (3h)52,44 Chromatographic purification gave colorless oil. C15H22O2 (M = 234.34); yield 82%; RI (HP5-MS): 1670; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 8.0 Hz, 1H, Ar-H), 7.04 (dd, J = 8.0, 1.7 Hz, 1H, Ar-H), 6.88 (d, J = 1.7 Hz, 1H, Ar-H), 2.93–2.88 (m, 1H, CH), 2.61 (t, J = 7.5 Hz, 2H, CH2), 2.16 (s, 3H, CH3), 1.79 (quin, J = 7.5 Hz, 2H, CH2), 1.5 (dq, J = 15, 7.4 Hz, 2H, CH2), 1.26 (d, J = 6.9 Hz, 6H, 2 × CH3), 1.0 (t, J = 7.5 Hz, 3H, CH3). 13C NMR (CD- Cl3, 125.76 MHz) δ 172.1 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.1 (CAr), 124.0 (CAr), 119.8 (CAr), 34.0 (CH2), 33.6 (CH), 27.2 (CH2), 23.9 (2 × CH3), 22.4 (CH2), 15.8 (CH3-Ar), 13.8 (CH3); MS (EI): m/z (%): 234 (6.8), 151 (11.7), 150 (100), 136 (8.5), 135 (86.5), 105 (8.0), 91 (17.2), 85 (11.6), 77 (8.1), 57 (28.9). 3. 2. 9. Carvacryl 3-Methylbutanoate (3i)30 Chromatographic purification gave colorless oil. C15H22O2 (M = 234.34); yield 81%; RI (HP5-MS): 1619; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 7.6 Hz, 1H, Ar-H), 7.04 (dd, J = 7.6, 1.7 Hz, 1H, Ar-H), 6.87 (d, J = 1.7 Hz, 1H, Ar-H), 2.93–2.88 (m, 1H, CH), 2.49 (d, J = 6.9 Hz, 2H, CH2), 2.34–2.26 (m, 1H, CH), 2.17 (s, 3H, CH3), 1.26 (d, J = 6.9 Hz, 6H, CH3), 1.11 (d, J = 6.6 Hz, 6H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 171.4 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.2 (CAr), 124.0 (CAr), 119.9 (CAr), 43.3 (CH2), 33.6 (CH), 25.8 (CH), 23.9 (2 × CH3), 22.5 (2 × CH3), 15.9 (CH3-Ar); MS (EI): m/z (%): 234 (M+) (8.1), 151 (12.0), 150 (100), 136 (7.9), 135 (80.2), 105 (8.5), 91 (17.9), 85 (13.2), 77 (8.5), 57 (40.3), 41 (7.1). 3. 2. 10. Carvacryl Hexanoate (3j)23 Chromatographic purification gave colorless oil, C16H24O2 (M = 248.37); yield 85%; RI (HP5-MS): 1770; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 8.0 Hz, 1H, Ar-H), 7.05 (dd, J = 7.6, 1.7 Hz, 1H, Ar-H), 6.89 (d, J = 1.7 Hz, 1H, Ar-H), 2.94–2.88 (m, 1H, CH), 2.61 (t, J = 7.5 Hz, 2H, CH2), 2.17 (s, 3H, CH3) 1.85–1.79 (m, 2H, CH2), 1.48– 1.38 (m, 4H, 2 × CH2), 1.36–1.26 (m, 6H, 2 × CH3), 0.99– 0.91 (m, 3H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 172.1 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.2 (CAr), 124.0 (CAr), 119.8 (CAr), 34.3 (CH2), 33.6 (CH), 31.4 (CH2), 24.8 (CH2), 23.9 (2 × CH3), 22,4 (CH2) 15.8 (CH3- Ar), 13.9 (CH3); MS (EI): m/z (%) 248 (M+), 151 (11.9), 150 (100), 135 (73.1), 105 (8.4), 99 (8.8), 91 (17.9), 77 (8.4), 71 (13.1), 55 (8.9), 43 (21.3). 3. 2. 11. Carvacryl Heptanoate (3k)52 Chromatographic purification gave colorless oil. C17H26O2 (M = 262.39); yield 91%; RI (HP5-MS): 1873; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 7.6 Hz, 1H, Ar-H), 7.04 (dd, J = 7.8, 1.9 Hz, 1H, Ar-H), 6.89 (d, J = 1.7 Hz, 1H, Ar-H), 2.96–2.88 (m, 1H, CH), 2.61 (t, J = 7.5 Hz, 2H, CH2), 2.17 (s, 3H, CH3), 1.81 (quin, J = 7.5 Hz, 2H, CH2), 1.50–1.44 (m, 2H, CH2), 1.39–1.37 (m, 4H, 2 × CH2), 1.27 (d, J = 6.9 Hz, 6H, CH3), 0.96–0.91 (m, 3H, 576 Acta Chim. Slov. 2022, 69, 571–583 Lazarević et al.: Carvacrol Derivatives as Antifungal Agents: ... CH3). 13C NMR (CDCl3, 125.76 MHz) δ 172.1 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.2 (CAr), 124.0 (CAr), 119.8 (CAr), 34.3 (CH2), 33.6 (CH), 31.5 (CH2), 28.9 (CH2), 25.1 (CH2), 23.9 (2 × CH3), 22.5 (CH2), 15.8 (CH3- Ar), 14.1 (CH3); MS (EI): m/z (%) 262 (M+) 151 (14.3), 150 (100), 135 (67.2), 113 (8.7), 105 (10.4), 91 (22.1), 77 (11.4), 55 (13.6), 43 (31.8), 41 (12.1). 3. 2. 12. Carvacryl Octanoate (3l)52 Chromatographic purification gave colorless oil. C18H28O2 (M = 276.42); yield 85%; RI (HP5-MS): 1978; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 7.6 Hz, 1H, Ar-H), 7.04 (dd, J = 7.8, 1.6 Hz, 1H, Ar-H), 6.89 (d, J = 1.7 Hz, 1H, Ar-H), 2.94–2.88 (m, 1H, CH), 2.61 (t, J = 7.5 Hz, 2H, CH2), 2.17 (s, 3H, CH3), 1.82 (quin, J = 7.5 Hz, 2H, CH2), 1.48–1.31 (m, 8H, 4 × CH2), 1.27 (d, J = 6.9 Hz, 6H, CH3), 0.94 (m, 3H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 172.1 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.2 (CAr), 124.0 (CAr), 119.8 (CAr), 34.3 (CH2), 33.6 (CH), 31.7 (CH2), 29.2 (CH2), 29.0 (CH2), 25.1 (CH2), 23.9 (2 × CH3), 22.6 (CH2), 15.8 (CH3-Ar), 14.1 (CH3); MS (EI): m/z (%) 276 (M+) 151 (12.3), 150 (100), 135 (52.3), 105 (7.3), 91 (14.4), 77 (6.3), 57 (22.7), 55 (14.0), 43 (11.4), 41 (8.5). 3. 2. 13. Carvacryl Nonanoate (3m) Chromatographic purification gave colorless oil. C19H30O2 (M = 290.45); yield 82%; RI (HP5-MS): 2082; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 7.6 Hz, 1H, Ar-H), 7.04 (dd, J = 8.0, 1.7 Hz, 1H, Ar-H), 6.9 (d, J = 1.7 Hz, 1H, Ar-H), 2.94–2.88 (m, 1H, CH), 2.61(t, J = 7.6 Hz, 2H, CH2), 2.17 (s, 3H, CH3), 1.82 (quin, J = 7.5 Hz, 2H, CH2), 1.50–1.44 (m, 2H, CH2), 1.41–1.33 (m, 8H, 4 × CH2), 1.27 (d, J = 6.9 Hz, 6H, CH3), 0.93 (m, 3H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 172.1 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.2 (CAr), 124.0 (CAr), 119.8 (CAr), 34.3 (CH2), 33.6 (CH), 31.8 (CH2), 29.2 (2 × CH2), 29.2 (CH2), 25.1 (CH2), 23.9 (2 × CH3), 22. 7 (CH2), 15.8 (CH3-Ar), 14.1 (CH3); MS (EI): m/z (%): 290 (M+) 151 (12.2), 150 (100), 136 (4.3), 135 (43.9), 109 (3.9), 91 (6.8), 71 (6.4), 57 (9.0), 55 (7.3), 43 (5.7). 3. 2. 14. Carvacryl Decanoate (3n) Chromatographic purification gave colorless oil. C20H32O2 (M = 304.47); yield 83%; RI (HP5-MS): 2186; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 8.0 Hz, 1H, Ar-H), 7.03 (dd, J = 8.0 Hz, 1.4 Hz, 1H, Ar-H), 6.88 (s, 1H, Ar-H), 2.93–2.88 (m, 1H, CH), 2.61 (t, J = 7.5 Hz, 2H, CH2), 2.17 (s, 3H, CH3) 1.81 (quin, J = 7.5 Hz, 2H, CH2), 1.49–1.29 (m, 14H, 7 × CH2), 1.27 (d, J = 6.9 Hz, 6H, CH3), 0.93 (m, 3H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 172.1 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.2 (CAr), 124.0 (CAr), 119.8 (CAr), 34.3 (CH2), 33.6 (CH), 31.9 (CH2), 29.5 (CH2), 29.3 (2 × CH2), 29.3 (CH2), 25.1 (CH2), 23.9 (2 × CH3), 22.7 (CH2), 15.8 (CH3-Ar), 14.1 (CH3); MS (EI): m/z (%): 304 (M+) 151 (12.5), 150 (100), 136 (3.8), 135 (39.0), 109 (4.1), 91 (5.8), 71 (4.5), 57 (5.4), 55 (7.0), 43 (6.1). 3. 2. 15. Carvacryl Undecanoate (3o) Chromatographic purification gave colorless oil. C21H34O2 (M = 318.50); yield 87%; RI (HP5-MS): 2287; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 7.6 Hz, 1H, Ar-H), 7.04 (dd, J = 7.6, 1.7 Hz, 1H, Ar-H), 6.88 (d, J = 1.7 Hz, 1H, Ar-H), 2.9 (quin, J = 7. 5 Hz, 1H, CH), 2.6 (t, J = 7.5 Hz, 2H, CH2), 2.16 (s, 3H, CH3) 1.81 (quin, J = 7.5 Hz, 2H, CH2), 1.49–1.28 (m, 16H, 8 × CH2), 1.26 (d, J = 6.9 Hz, 6H, CH3), 0.92 (m, 3H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 172.1 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.2 (CAr), 124.0 (CAr), 119.8 (CAr), 34.3 (CH2), 33.6 (CH), 31.9 (CH2), 29.6 (CH2), 29.5 (CH2), 29.3 (CH2), 29.3 (CH2), 29.2 (CH2), 25.1 (CH2), 23.9 (2 × CH3), 22.7 (CH2), 15.8 (CH3-Ar), 14.1 (CH3); MS (EI): m/z (%) 318 (M+) 151 (13.0), 150 (100), 136 (3.4), 135 (34.9), 109 (4.7), 105 (2.5), 91 (4.6), 57 (6.0), 55 (6.3), 43 (4.9). 3. 2. 16. Carvacryl Dodecanoate (3p)34 Chromatographic purification gave colorless oil. C22H36O2 (M = 332.53); yield 78%; RI (HP5-MS): 2388; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 8.0 Hz, 1H, Ar-H), 7.04 (dd, J = 7.8, 1.6 Hz 1H, Ar-H), 6.88 (d, J = 1.4 Hz, 1H, Ar-H), 2.90 (m,1H, CH), 2.60 (t, J = 7.6 Hz, 2 H, CH2), 2.16 (s, 3H, CH3) 1.81 (quin, J = 7.5 Hz, 2H, CH2), 1.49–1.43 (m, 2 H, CH2), 1.4–1.29 (m, 14H, 7 × CH2), 1.27 (d, J = 6.9 Hz, 6H, CH3), 0.92 (t, J = 6.8 Hz, 3H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 172.1 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.2 (CAr), 124.0 (CAr), 119.8 (CAr), 34.3 (CH2), 33.6 (CH), 31.9 (CH2), 29.6 (2 × CH2), 29.5 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 25.1 (CH2), 23.9 (2 × CH3), 22.7 (CH2), 15.8 (CH3-Ar), 14.1 (CH3); MS (EI): m/z (%) 332 (M+) 151 (12.8), 150 (100), 136 (3.1), 135 (32.1), 109 (5.2), 91 (4.3), 71 (2.6), 57 (7.0), 55 (7.2), 43 (5.2). 3. 2. 17. Carvacryl Tridecanoate (3q) Chromatographic purification gave colorless oil, C23H38O2 (M = 346.55); yield 81%; RI (HP5-MS): 2491; 1H NMR (CDCl3, 500.13 MHz) δ 7.18 (m, 1H, Ar-H), 7.04 (dd, J = 7.8, 1.6 Hz, 1H, Ar-H), 6.88 (s, 1H, Ar-H), 2.91 (m, 1H, CH), 2.6 (t, J = 7.5 Hz, 2 H, CH2), 2.16 (s, 3H, CH3) 1.81 (quin, J = 7.5 Hz, 2H, CH2), 1.49–1.43 (m, 2H, CH2), 1.38–1.28 (m, 16H, 8 × CH2), 1.27 (d, J = 6.9 Hz, 6H, CH3), 0.92 (t, J = 6.8 Hz, 3H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 172.1 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.2 (CAr), 124.0 (CAr), 119.8 (CAr), 34.3 (CH2), 33.6 (CH), 31.9 (CH2), 29.7 (2 × CH2), 29.5 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 29.1 (CH2), 25.1 (CH2), 23.9 (2 x 577Acta Chim. Slov. 2022, 69, 571–583 Lazarević et al.: Carvacrol Derivatives as Antifungal Agents: ... CH3), 22.7 (CH2), 15.8 (CH3-Ar), 14.1 (CH3); MS (EI): m/z (%) 346 (M+) 151 (13.3), 150 (100), 136 (2.8), 135 (28.9), 109 (5.2), 91 (3.6), 71 (2.9), 57 (6.4), 55 (6.6), 43 (4.8). 3. 2. 18. Carvacryl Tetradecanoate (3r) Chromatographic purification gave colorless oil. C24H40O2 (M = 360.58); yield 76%; RI (HP5-MS): 2590; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 7.6 Hz, 1H, Ar-H), 7.04 (dd, J = 7.8, 1.6 Hz, 1H, Ar-H), 6.88 (s, 1H, Ar-H), 2.9 (m, 1H, CH), 2.6 (t, J = 7.6 Hz, 2H, CH2), 2.16 (s, 3H, CH3), 1.81 (quin, J = 7.5 Hz, 2H, CH2), 1.49-1.43 (m, 2H, CH2), 1.4-1.28 (m, 18H, 9 × CH2) 0.91 (t, J = 6.9 Hz, 3H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 172.1 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.2 (CAr), 124.0 (CAr), 119.8 (CAr), 34.3 (CH2), 33.6 (CH), 31.9 (CH2), 29.7 (2 × CH2), 29.6 (2 × CH2), 29.5 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 25.1 (CH2), 23.9 (2 × CH3), 22.7 (CH2), 15.9 (CH3-Ar), 14.1 (CH3); MS (EI): m/z (%) 360 (M+) 151 (12.4), 150 (100), 135 (27.7), 109 (6.0), 91 (4.5), 71 (3.8), 69 (3.7), 57 (9.0), 55 (10.0), 43 (8.3). 3. 2. 19. Carvacryl Pentadecanoate (3s) Chromatographic purification gave colorless oil. C25H42O2 (M = 374.61); yield 74%; RI (HP5-MS): 2694; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J =7.6 Hz, 1H, Ar-H), 7.04 (dd, J = 8.0, 1.7 Hz, 1H, Ar-H), 6.88 (d, J = 1.7 Hz, 1H, Ar-H), 2.9 (m, 1H, CH), 2.6 (t, J = 7.5 Hz, 2H, CH2), 2.16 (s, 3H, CH3), 1.81 (quin, J = 7.5 Hz, 2H, CH2), 1.49-1.44 (m, 2H, CH2), 1.4-1.28 (m, 20H, 10 × CH2), 1.26 (d, J = 6.9 Hz, 3H, CH3), 0.92 (t, J = 6.9 Hz, 3H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 172.1 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.2 (CAr), 124.0 (CAr), 119.8 (CAr), 34.3 (CH2), 33.6 (CH), 31.9 (CH2), 29.7 (3 × CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2), 29.3 (2 × CH2), 29.2 (CH2), 25.1 (CH2), 23.9 (2 × CH3), 22.7 (CH2), 15.8 (CH3- Ar), 14.1 (CH3); MS (EI): m/z (%) 374 (M+) 151 (12.5), 150 (100), 135 (25.2), 109 (6.1), 91 (3.9), 71 (3.6), 69 (3.6), 57 (8.6), 55 (9.1), 43 (7.4). 3. 2. 20. Carvacryl Hexadecanoate (3t) Chromatographic purification gave amorphous white solid. Mp 31–32 °C. C26H44O2 (M = 388.63); yield 92%; RI (HP5-MS): 2804; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 7.9 Hz, 1H, Ar-H), 7.04 (dd, J = 7.7, 1.7 Hz, 1H, Ar-H), 6.88 (d, J = 1.6 Hz, 1H, Ar-H), 2.90 (m, 1H, CH), 2.60 (t, J = 7.6 Hz, 2H, CH2), 2.16 (s, 3H, CH3), 1.81 (quin, J = 7.6 Hz, 2H, CH2), 1.47-1.28 (bm, 24H, 12 × CH2), 1.26 (d, J = 7.0 Hz, 6H, CH3), 0.9 (m, 3H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 172.1 (C=O), 149.3 (CAr), 148.0 (CAr), 130.8 (CAr), 127.1 (CAr), 124.0 (CAr), 119.8 (CAr), 34.3 (CH2), 33.6 (CH), 31.9 (CH2), 29.7 (4 × CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 29.1 (CH2), 25.1 (CH2), 23.9 (2 × CH3), 22.7 (CH2), 15.8 (CH3-Ar), 14.1 (CH3); MS (EI): m/z (%) 388 (M+) 151 (18.7), 150 (100), 136 (2.9), 135 (29.4), 109 (6.8), 71 (2.8), 69 (2.7), 57 (5.5), 55 (5.4), 43 (3.7). 3. 2. 21. Carvacryl Heptadecanoate (3u) Chromatographic purification gave colorless oil. C27H46O2 (M = 402.66); yield 73%; RI (HP5-MS): 2902; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 8.0 Hz, 1H, Ar- -H), 7.04 (dd, J =7.6, 1.7 Hz, 1H, Ar-H), 6.88 (bs, 1H, Ar- -H), 2.9 (m, 1H, CH), 2.60 (t, J = 7.6 Hz, 2H, CH2), 2.16 (s, 3H, CH3), 1.81 (quin, J = 7.5 Hz, 2H, CH2), 1.48-1.28 (bm, 26H, 13 × CH2), 1.26 (d, J = 6.9 Hz, 6H, CH3), 0.92 (t, J = 6.9 Hz, 3H, CH3). 13C NMR (CDCl3, 125.76 MHz) δ 172.1 (C=O), 149.3 (CAr), 148.0 (CAr), 130.9 (CAr), 127.2 (CAr), 124.0 (CAr), 119.8 (CAr), 34.3 (CH2), 33.6 (CH), 32.0 (CH2), 29.7 (6 × CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 25.1 (CH2), 23.9 (2 × CH3), 22.7 (CH2), 15.8 (CH3-Ar), 14.1 (CH3); MS (EI): m/z (%) 402 (M+) 151 (14.0), 150 (100), 135 (21.9), 109 (6.0), 91 (2.6), 71 (2.9), 69 (2.9), 57 (6.4), 55 (6.3), 43 (4.8). 3. 2. 22. Carvacryl Octadecanoate (3v) Chromatographic purification gave amorphous white solid. Mp 61 °C. C28H48O2 (M = 416.69); yield 85%; RI (HP5-MS): 2945; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 7.8 Hz, 1H, Ar-H), 7.04 (dd, J = 7.8, 1.9 Hz, 1H, Ar-H), 6.88 (d, J =1.8 Hz, 1H, Ar-H), 2.9 (hept, J = 6.9 Hz, 1H, CH), 2.6 (t, J = 7.6 Hz, 2H, CH2), 2.16 (s, 3H, CH3), 1.81 (quin, J = 7.5 Hz, 2H, CH2), 1.51-1.28 (m, 28H), 1.26 (d, J = 6.9 Hz, 6H, 2 × CH3), 0.92 (t, J = 6.9 Hz, 3H, CH3); 13C NMR (CDCl3, 125.76 MHz) δ 172.1 (C=O), 149.3 (CAr), 148.0 (CAr), 130.8 (CAr), 127.1 (CAr), 124.0 (CAr), 119.8 (CAr), 34.3 (CH2), 33.6 (CH), 32.0 (CH2), 29.7 (7 × CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 25.1 (CH2), 23.9 (2 × CH3), 22.7 (CH2), 15.8 (CH3- Ar), 14.1 (CH3); MS (EI): m/z (%) 416 (M+), 151 (12.5), 150 (100), 135 (25.6), 109 (7.0), 91 (5.1), 69 (5.5), 57 (13.5), 55 (14.0), 43 (15.9), 41 (5.2). 3. 2. 23. Carvacryl Oleate (3w) Chromatographic purification gave colorless oil. C28H46O2 (M = 414.67); yield 52%; RI (HP5-MS): 2936; 1H NMR (CDCl3, 500.13 MHz) δ 7.17 (d, J = 7.8 Hz, 1H, Ar-H), 7.03 (dd, J = 7.8, 1.8 Hz, 1H, Ar-H), 6.87 (d, J = 1.8 Hz, 1H, Ar-H), 5.43-5.34 (m, 2H, CH=CH Z-configura- tion), 2.9 (hept, J = 7.0 Hz, 1H, CH), 2.6 (t, J = 7.5 Hz, 2H, CH2), 2.16 (s, 3H, CH3), 2.10-2.01 (m, 4H, 2 × CH2), 1.81 (quin, J = 7.5 Hz, 2H, CH2), 1.61 – 1.53 (m, 2H, CH2), 1.51-1.41 (m, 2H, CH2), 1.36 (d, J = 7.5 Hz, 6H, 2 × CH3), 1.33-1.23 (m, 16H, 8 × CH2), 0.91 (t, J = 6.9 Hz, 3H, CH3); 13C NMR (CDCl3, 125.76 MHz) δ 172.0 (C=O), 149.3 (CAr), 148.0 (CAr), 130.8 (CAr), 130.0 (=C-), 129.7 (=C-), 578 Acta Chim. Slov. 2022, 69, 571–583 Lazarević et al.: Carvacrol Derivatives as Antifungal Agents: ... 127.1 (CAr), 124.0 (CAr), 119.8 (CAr), 34.3 (CH2), 33.6 (CH), 31.9 (CH2), 29.8 (CH2), 29.7 (CH2), 29.5 (CH2), 29.3 (CH2), 29.2 (2 × CH2), 29.1 (CH2), 27.7 (CH2), 27.3 (CH2), 27.2 (CH2), 25.1 (CH2), 23.9 (2 × CH3), 22.7 (CH2), 15.8 (CH3-Ar), 14.1 (CH3); MS (EI): m/z (%) 414.40 (M+) 151 (12.3), 150 (100), 135 (26.4), 109 (6.1), 83 (4.4), 69 (8.0), 67 (6.3), 57 (4.9), 55 (17.3), 43 (6.1). 3. 2. 24. Carvacryl Benzoate (3x)22,23,30,54 Chromatographic purification gave amorphous white solid. Mp 31 °C. C17H18O2 (M = 254.33); yield 95%; RI (HP5-MS): 1991; 1H NMR (CDCl3, 500.13 MHz) δ 8.26 (d, J = 7.6 Hz, 2H, Ar-H), 7.68 (m, 1H, Ar-H), 7.56 (m, 2H, Ar-H), 7.23 (d, J = 8.0 Hz, 1H, Ar-H), 7.09 (d, J = 8.0 Hz, 1H, Ar-H), 7.04 (s, 1H, Ar-H), 2.94 (m, 1H, CH), 2.23 (s, 3H, CH3), 1.29 (d, J = 6.9 Hz, 6H, 2 × CH3). 13C NMR (CDCl3, 125.76 MHz) δ 164.9 (C=O), 149.5 (CAr), 148.2 (CAr), 133.5 (CAr), 130.9 (CAr), 130.2 (2 × CAr), 129.6 (CAr), 129.1 (CAr) 128.6 (2 × CAr), 127.4 (CAr), 124.2 (CAr), 119.9 (CAr), 33.6 (CH), 24.0 (2 × CH3), 15.9 (CH3-Ar); MS (EI): m/z (%): 254 (M+) (7.3), 106 (7.7), 105 (100), 91 (5.1), 79 (2.1), 78 (3.3), 77 (34.8), 65 (1.2), 51 (6.2), 50 (1.4). 3. 2. 25. Carvacryl 4-Methoxybenzoate (3y)23 Chromatographic purification gave amorphous white solid. Mp 31–32 °C. C17H18O2 (M = 284.35); yield 95%; RI (HP5-MS): 2302; 1H NMR (CDCl3, 500.13 MHz) δ 8.21 (d, J = 8.9 Hz, 2H, Ar-H), 7.21 (d, J = 7.8 Hz, 1H, Ar-H), 7.07 (d, J = 7.8 Hz, 1H, Ar-H), 7.03 (s, 1H, Ar-H), 7.01 (d, 2H, Ar-H), 3.93 (s, 3H, CH3), 3.51 (q, J = 7.0 Hz, 2H contamination EtOAc), 2.93 (m, 1H, CH), 2.21 (s, 3H, CH3), 1.29 (d, J = 6.9 Hz, 6H, 2 × CH3,), 1.24 (t, J = 7.0 Hz, 3H contamination EtOAc); 13C NMR (CDCl3, 125.76 MHz) δ 164.6 (CAr-O),163.8 (C=O), 149.6 (CAr-OCO), 148.1 (CAr), 132.2 (2 × CAr), 130.9 (CAr), 127.4 (CAr), 124.0 (CAr), 122.0 (CAr), 120.0 (CAr), 113.9 (2 × CAr), 55.5 (C-O), 33.6 (CH), 23.9 (2 × CH3), 15.8 (CH3-Ar); MS (EI): m/z (%): 284 (M+) (2.6), 136 (8.7), 135 (100), 107 (6.2), 92 (9.0), 91 (3.8), 79 (2.0), 77 (147), 64 (3.4), 63 (2.0). 3. 3. Antimicrobial Activity The antimicrobial activity was evaluated by deter- mining the minimum inhibitory concentration (MIC) and the minimum microbicidal concentration, which includes minimum bactericidal (MBC) and minimum fungicidal concentrations (MFC), using the broth microdilution method. The obtained results are given in Table 2. The as- sayed samples were less effective than antibiotic/antimy- cotic used as reference standard, and if observed, activity was never greater than the values acquired for the parent compound 1 (MIC/MBC/MFC never exceeded 0.031 mg/ mL). The panel of bacterial strains, represented by Gram-positive (B. subtilis and S. aureus) and Gram-nega- tive (E. coli, S. abony and S. typhimurium) microorgan- isms, were completely resistant to the synthesized com- pounds at tested concentration (Table 2), except for compound 3b that inhibited growth of B. subtilis and S. aureus at 0.5 and 1 mg/mL, respectively, and inhibited the growth/had cidal effect on S. typhimurium at concentra- tions comparable to 1 (0.50 / 0.25 mg/mL). On the other hand, an interesting experimental fact is that the repre- sentatives of the synthesized homologues, regardless of the nature of the R residue, have shown activity on fungal strain Aspergillus niger and on yeast Candida albicans (for MIC/MFC, see Table 2, entries 3a–y), being antimicrobials comparable to carvacrol. Compounds 3a,d,g,i,x from our study are matching samples to those tested by Mathela and collaborators,30 who were making evaluation of antibacterial activity on Streptococcus mutans (MTCC 890), S. aureus (MTCC 96), B. subtilis (MTCC 121), S. epidermidis (MTCC 435) and E. coli (MTCC 723) and also reported attenuation of the ac- tivity in comparison to 1. Compound 3p is identical to the sample tested by Bassanetti et al.34 on E. coli (isolate and ATCC 25922), S. typhimurium (isolate and ATCC 23564), S. enteritidis (isolate and ATCC 49220) and Clostridium perfringens (isolate and ATCC 13124), with identical ob- servations (regardless of the bacterial strain involved) re- lated to attenuation of the synthetic compound activity in comparison to the parent compound 1. None of two previ- ous studies did include fungi/yeast strains in antimicrobial bioassays. Moreover, according to the authors’ best knowl- edge and based on the literature search,55 only one study assaying antifungal (Botrytis cinerea) and activity against yeast (Saccharomyces cerevisiae), involving only one of the prepared compounds (carvacryl acetate), exists.37 Veldhuizen et al.56 comparing 1 with carvacrol-relat- ed compounds, indicated structural requirements in exert- ing antimicrobial activity against pathogenic bacteria such as E. coli and S. aureus. Further investigations emphasized the correlation between the free-hydroxyl group in the phenolic ring and the antimicrobial potency on ester de- rivatives obtained by replacing hydroxyl group with acyl moieties. Ultee et al.9 suggested that the crucial role for efficacy of phenolic compounds (e.g. carvacrol) is attribut- ed to the presence of OH functional group and to a system of delocalized electrons, allowing compounds to act as proton exchanger, thus reducing the gradient across the cytoplasmic membrane (resulting collapse of the pro- ton-motive force and depletion of the ATP pool lead even- tually to cell death, as reported by Ultee et al.9). The delo- calized electron system present in carvacryl derivatives implies that they are proton acceptors, however unable to release a proton through the acyl group to act as a proton exchanger.37 So far obtained data emphasized that the in- sertion of acyl groups in the carvacrol aromatic ring re- sults in a weaker antibacterial activity,57 which was also the result confirmed by Mathela et al.30 and by our current study. However, this single structural modification of phe- 579Acta Chim. Slov. 2022, 69, 571–583 Lazarević et al.: Carvacrol Derivatives as Antifungal Agents: ... Table 2. The minimal inhibitory (MIC) and minimal bactericidal/fungicidal (MBC/MFC) concentrations of the carvacrol (1) and the synthetised 3a–y esters. The initial concentration of the derivatives applied in broth microdilution assay were 2 mg/mL. Bacterial strains Fungal strains Gram-positive Gram-negative Compound B. subtilis S. aureus E. coli S. abony S. typhimurium A. niger C. albicans 1 MIC = 0.25 MIC = 0.25 MIC = 0.25 MIC = 0.50 MIC = 0.25 MIC = 0.031 MIC = MFC = 0.125 MBC = 0.50 MBC = 0.50 MBC = 0.50 MBC = 1.0 MBC = 0.50 MFC = 0.50 3a na na na na na MIC = 0.25 MIC = MFC = 0.50 MFC = 0.50 3b MIC = 0.50 MIC = 1.0 na na MIC = 0.25 MIC = 0.25 MIC = 0.125 MBC = 0.50 MFC = 0.50 MFC = 0.50 3c na na na na na MIC = 0.25 MIC = 1.0 MFC = 0.50 3d na na na na na MIC = 0.25 MIC = 1.0 MFC = 0.50 3e na na na na na MIC = MFC = 0.50 MIC = 1.0 3f na na na na na MIC = 0.25 MIC = 1.0 MFC = 0.50 3g na na na na na MIC = 0.25 MIC = 1.0 MFC = 1.0 3h na na na na na MIC = 0.25 MIC = 1.0 MFC = 1.0 3i na na na na na MIC = 0.25 MIC = 0.25 MFC = 1.0 3j na na na na na MIC = 0.50 MIC = 0.50 MFC = 1.0 3k na na na na na MIC = MFC = 1.0 MIC = 0.50 MFC = 1.0 3l na na na na na MIC = 0.25 MIC = MFC = 1.0 MFC = 0.50 3m na na na na na MIC = 0.50 MIC = 1.0 3n na na na na na MIC = 0.25 MIC = 1.0 MFC = 0.50 3o na na na na na MIC = 0.25 MIC = 1.0 MFC = 0.50 3p na na na na na MIC = 0.25 MIC = 0.50 MFC = 0.50 3q na na na na na MIC = MFC = 0.50 MIC = 0.50 MFC = 1.0 3r na na na na na MIC = MFC = 0.25 MIC = 1.0 3s na na na na na MIC = MFC = 0.25 MIC = 0.25 MFC = 1.0 3t na na na na na MIC = 0.25 MIC = 0.25 MFC = 1.0 MFC = 1.0 3u na na na na na MIC = 0.25 MIC = 0.50 MFC = 0.50 3v na na na na na MIC = 0.5 MIC = 0.5 580 Acta Chim. Slov. 2022, 69, 571–583 Lazarević et al.: Carvacrol Derivatives as Antifungal Agents: ... nolic functionality seems to have different effect on fungi/ yeasts (A. niger and C. albicans) compared to bacteria (Ta- ble 2, antibacterial vs. antifungal/anticandidal activity). Introducing an acyl group to the carvacrol results in in- creased lipophilicity of the synthesized compound (Table S1, see octanol–water partition coefficient calculation, represented as miLogP). Except preserved A. niger anti- fungal potential, for which no remarkable oscillations in values were observed, no significant (balanced) correla- tions were detected between increased lipophilicity (the chain length) and the antifungal activity (MIC/MFC) among tested carvacryl ester derivatives. Slight (negligi- ble) loss of anti-A. niger potential could be observed in those compounds where the parent phenolic (1) is substi- tuted with butanoyl, 2-methylpropanoyl, pentanoyl, 3-methylbutanoyl, hexanoyl, octadecanoyl and oleoyl moieties (Table 2. entries 3g–k,v,w). As for C. albicans, the strongest anticandidal activity, except for 1, was observed for the introduced methanoyl and 2-chloromethanoyl moieties (Table 2, entries 3a,b). There are no striking dif- ferences in anticandidal potential of the homologues high- er than C3, and from the Table 2 we can notice that there is anticandidal activity evidenced in all of the synthesized compounds (3a–y). Interestingly, Damiens et al.36 have observed and stated the importance of (a balanced) hydro- philicity/lipophilicity ratio, though in sesamol derivatives, against a phyto-pathogen fungi Zymoseptoria tritici (mod- ulating lipophilicity proved to increase the antifungal bio- logical activity for sesamol derivatives). The phenomena noticed in our research and in Damiens et al.36 does not have to be an isolated incident, it could also be a regularity based on subtle structural changes that would, within cer- tain limits, by enhancing lipophilicity affect bioactivity. It is certain that this aspect deserves further research. It is interesting to recall and compare the results of the antimicrobial assay we have obtained for acylated thy- mol (positional isomer of carvacrol) derivatives.38 Unlike esters of thymol, which effected only growth of C. albicans, carvacrol ester derivatives are strongly affecting growth of both, A. niger and C. albicans, with a more pronounced (cidal) effect on A. niger. The stronger effect is most prob- ably related to the orientation/position of the groups in (acylated) positional isomer homologs, and this item could also be worth of further research. 3. 4. In Silico Study 3. 4. 1. Physico-chemical Properties Physico-chemical properties of the studied com- pounds predicted by the Molinspiration tool47 are shown in Table S1. It can be seen that 1 and seven synthesized compounds (3a,b,d,f,g,e,i) fulfilled all Lipinski’s and Ve- ber’s rules (miLogP ≤ 5, TPSA ≤ 140 Å2, nON ≤ 10, nOHNH ≤ 5, nrotb ≤ 10, Mr ≤ 500) indicating their good oral bioavailability in vivo. However, eight compounds (3c,h,j–m,x,y) were predicted with one deviation (mi- LogP > 5), and ten compounds (3n–w) were predicted with two deviations (miLogP > 5, nrotb > 10) from the Lipinski’s and Veber’s rules, indicating their poorer bio- avalilability. Bacterial strains Fungal strains Gram-positive Gram-negative Compound B. subtilis S. aureus E. coli S. abony S. typhimurium A. niger C. albicans MFC = 1.0 MFC = 1.0 3w na na na na na MIC = 0.5 MIC = 0.5 MFC = 1.0 MFC = 1.0 3x na na na na na MIC = 0.25 MIC = 1.0 MFC = 0.50 3y na na na na na MIC = 0.25 MIC = 1. MFC = 0.50 0 Positive control (referent standard) Doxycycline MIC C MIC = 6.25 MIC = MBC MIC = MBC MIC = MBC nt nt (μg/mL) = MB = 1.56 MBC = 0.78 = 0.78 = 12.5 = 6.25 Nystatin nt nt nt nt nt MIC = MBC MIC = MBC (μg/mL) = 6.25 = 0.78 Negative control (solvent used) DMSO 10% aqueous solution na na na na na na na “na” not active, “nt” not tested, “MIC” minimum inhibitory concentration as the lowest concentration of an antimicrobial agent (synthetic com- pound) needed to inhibit the visible in vitro growth of a challenge microorganism, “MBC” minimum bactericidal concentration and “MFC” mini- mum fungicidal concentration; concentrations were evaluated as the lowest concentration at which 99.9% of the inoculated microorganisms were killed. 581Acta Chim. Slov. 2022, 69, 571–583 Lazarević et al.: Carvacrol Derivatives as Antifungal Agents: ... 3. 4. 2. Pharmacokinetic Properties Absorption properties of the studied compounds predicted by admetSAR48 are shown in Table S2. All com- pounds tested were predicted as compounds permeable across Caco-2 cells, capable of being absorbed by intestine, as well as compounds able to pass through blood-brain barrier and penetrate into the central nervous system. None of the tested compounds was predicted as a P-glyco- protein substrate, while a small number (3r–w,y) was pre- dicted as a P-glycoprotein inhibitor. Metabolic properties of the studied compounds pre- dicted by admetSAR48 are shown in Table S3. According to the results, the tested compounds differ from each other in their metabolic properties, depending on whether they are potential substrates and/or inhibitors of certain CYP450 isoenzymes. No compound was predicted as CYP450 3A4 substrate or CYP450 2D6/3A4 inhibitor. Only the parent compound 1 was predicted as CYP450 2C9/2D6 substrate. The only compound envisaged as CYP450 2C9 inhibitor is 3x. Most compounds (19 of 25) are potential inhibitors of CYP450 2C19, and all are potential inhibitors of CYP450 1A2. 3. 4. 3. Toxicological Properties Toxicological properties of the studied compounds predicted by DataWarrior49 are shown in Table S4. It can be seen that most of the studied compounds were predict- ed as non-mutagenic, non-tumorigenic and non-repro- ductive effective (22, 21 and 22 out of 25 compounds, re- spectively). Compounds 3b,c,e were predicted as highly mutagenic, compounds 3b and 3c and highly reproductive effective, while compounds 3b,e,j were predicted as highly tumorigenic. All 25 compounds tested were predicted as highly irritant. The results obtained by predicting organ toxicity, organ system toxicity, genotoxicity and ecotoxici- ty of the studied compounds using admetSAR48 are shown in Tables S5–S8. Most of the studied compounds were predicted as potentially non-hepatotoxic, with no risk of eye corrosion or eye irritation, but with the possibility of human ether-à- go-go inhibition. According to the risk of acute oral toxic- ity, the studied compounds were predicted as category III, or slightly toxic compounds, with LD50 values of 500– 5000 mg/kg. Only one compound (3b) was predicted as category II, or moderately toxic compound, with LD50 value of 50–500 mg/kg (Table S5). The results obtained by predicting the compound ability to interact with the hormonal system showed that the studied compounds have low predispositions for estro- gen receptor, aromatase and glucocorticoid receptor bind- ing, slightly higher predispositions for thyroid receptor binding and high predispositions for peroxisome prolifer- ator-activated receptor γ binding (Table S6). Regarding genotoxicity, all of the studied compounds were predicted as non-genotoxic (Table S7), and regarding ecotoxicity, all compounds tested were predicted as non-toxic to avian, but toxic to fish, honey bee and Tet- rahymena pyriformis. More than a half were predicted as toxic to crustaceans. Finally, the majority was predicted as biodegradable compounds (Table S8). Structural alerts for DNA and protein binding for the studied compounds, predicted by Toxtree,50 are pre- sented in Table S9. All of the compounds tested showed at least one structural alert for DNA or protein binding. 4. Conclusion By chemical synthesis, we have obtained a series of 25 esters, among which 10 compounds are reported for the first time. All of the synthesized compounds were em- ployed in antimicrobial bioassay, exhibiting the greatest activity on fungal strain A. niger and on yeast C. albicans, where was found that all could be antimicrobials, compa- rable to carvacrol, and can also be considered as activity holders. While the phenolic hydroxyl group of carvacrol is essential for action against bacteria, it seems that lipo- philicity plays an important role in antifungal activity. The pronounced antimicrobial selectivity is certainly a subject deserving more thorough examination either through the mechanism of action or through a greater number of di- verse compounds involved in establishing a detailed struc- ture-activity correlation. Based on our in silico study seven compounds (1 and 3a,b,d,f,g,e,i) fulfilled all Lipinski’s and Veber’s rules and were predicted to have good oral bioavailability. All compounds were recognized as compounds able to pass through blood-brain barrier, capable of being absorbed by intestine and permeable across Caco-2 cells. Metabol- ic properties differ within the studied compounds, de- pending on whether they act as substrates and/or inhibi- tors of various CYP450 enzymes. All compounds were predicted as non-genotoxic, and most were predicted as non-mutagenic, non-tumorigenic, non-reproductive ef- fective and non-hepatotoxic. Regarding the risk of acute oral toxicity, they were predicted as slightly toxic com- pounds. However, some of the compounds showed pre- dispositions to act as potential endocrine disruptors, and all of them showed at least one structural alert for DNA or protein binding. Taking in consideration the overall results, carvacryl esters are another type of phenolics that, from the aspect of enhanced lipophilicity (improved membrane permeabili- ty), could be useful in fungal control. Acknowledgements The work was funded by the Ministry of Science and Technological Development of Serbia (Projects 451-03- 821/2012-14, 451-03-68/2022-14/200113 and 451-03- 9/2022-14/200124). 582 Acta Chim. Slov. 2022, 69, 571–583 Lazarević et al.: Carvacrol Derivatives as Antifungal Agents: ... 5. References 1. T. Rodrigues, D. Reker, P. Schneider, G. Schneider, Nat. Chem. 2016, 8, 531–541. DOI:10.1038/nchem.2479 2. S. Bernardini, A. Tiezzi, V. Laghezza Masci, E. Ovidi, Nat. Prod. Res. 2018, 32, 1926–1950. DOI:10.1080/14786419.2017.1356838 3. A. G. Atanasov, S. B. Zotchev, V. M. Dirsch, Nat. Rev. Drug. Discov. 2021, 20, 200–216. DOI:10.1038/s41573-020-00114-z 4. K. H. C. Baser, Curr. Pharm. Des. 2008, 14, 3106–3119. DOI:10.2174/138161208786404227 5. N. B. Rathod, P. Kulawik, F. Ozogul, J. M. Regenstein, Y. Ozogul, Trends Food Sci. Tech. 2021, 116, 733–748. DOI:10.1016/j.tifs.2021.08.023 6. M. Sharifi-Rad, E. M. Varoni, M. Iriti, M. Martorell, W. N. Setzer, M. Del Mar Contreras, B. 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DOI:10.1080/08927014.2018.1480756 Povzetek Iz literature so poznane kemijske modifikacije naravnih monoterpenoidov v različne derivate, kar lahko okrepi njihove biološke aktivnosti v primerjavi z matičnimi spojinami. Skladno s tem smo karvakrol, znan biocid in dodatek k hrani, uporabili kot ogrodje za uvedbo acilne skupine na prvotno fenolno skupino. S to enostavno metodologijo smo pripravili majhno serijo 25 estrov. Za vsako pripravljeno spojino smo izvedli strukturno karakterizacijo, določili in vitro anti- mikrobno učinkovitost ter in silico izračunali nekatere fizikalnokemijske, farmakokinetične ter toksikološke lastnosti. Čeprav obstajajo mnogi podatki o sintezah in bioaktivnostih nižjih karvakrolnih estrskih homologov, so podatki o estrih z daljšimi karboksilnimi kislinami (več kot C9), zelo redki; izmed 25 spojin jih je kar 10 opisanih prvič (spektroskopske karakterizacije pa so prvič opisane za 12 spojin). Naša raziskava predstavlja prvo podrobno študijo karvakrolnih estrov kot učinkovin proti glivam ter prvo, kjer so karvakrolni estri, sestavljeni iz srednjedolgih ali dolgih verig maščobnih kislin, izkazovali antibakterijske aktivnosti. Zanimivo je, da vse pripravljene spojine, ne glede na naravo ostanka R, izka- zujejo aktivnost proti glivi Aspergilus niger ter proti kvasovki Candida albicans, ki je primerljiva z aktivnostjo karvakrola. Poleg predstavljenih eksperimentalnih podatkov, je tudi uporaba in silico računskih metod za določanje fizikalnokemi- jskih, farmakokinetičnih ter toksikoloških lastnosti pripravljenih spojin, pomembna informacija za nadaljnje raziskave. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 584 Acta Chim. Slov. 2022, 69, 584–595 Stasevych et al.: N-(9,10-Dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) ... DOI: 10.17344/acsi.2022.7463 Scientific paper N-(9,10-Dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) Acetamides: Synthesis, Antioxidant and Antiplatelet Activity Maryna Stasevych,1,* Viktor Zvarych,1 Olena Yaremkevych,1 Mykhaylo Vovk,2 Alla Vaskevych,2 Tetiana Halenova3 and Olexii Savchuk3 1 Department of Technology of Biologically Active Substances, Pharmacy, and Biotechnology, Lviv Polytechnic National University, 79013 Lviv, Ukraine 2 Department of Chemistry of Functional Heterocyclic Systems, Institute of Organic Chemistry of National Academy of Sciences of Ukraine, 02660 Kyiv, Ukraine 3 Educational and Scientific Center “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv, 01601 Kyiv, Ukraine * Corresponding author: E-mail: maryna.v.stasevych@lpnu.ua Received: 03-11-2022 Abstract The synthesis of new N-(9,10-dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) acetamides was carried out using reac- tion of 2-chloro-N-(9,10-dioxo-9,10-dihydroanthracene-1(2)-yl)acetamides with functionalized thiols in the presence of potassium carbonate in N,N-dimethylformamide (DMF) at room temperature. Evaluation of the synthesized com- pounds on such indicators of radical scavenging activity as lipid peroxidation (LP) and oxidative modification of proteins (OMP) in vitro in rat liver homogenate was carried out. It was determined that the compounds with a substituent in the first position of anthracedione core showed better antioxidant properties than their isomers with a substituent in the second position. The compounds 6 and 7 with the best indicators of radical-scavenging activity were determined. Anti- oxidant effect in OMP processes was also determined for compound 10. The antiplatelet activity study in vitro revealed compound 10 with the inhibited effect of ADP-induced aggregation. Keywords: N-(9,10-dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) acetamides; free-radical oxidation markers; anti- oxidant activity; antiplatelet activity; structure-activity relationship 1. Introduction Arterial thrombosis is one of the critical factors de- termining the outcome of cardiovascular and oncological diseases,1–4 which share the first place among all diseas- es, both in Ukraine and in the world.5 They cause sudden death in myocardial infarction, vascular complications of diabetes mellitus, cancer chemotherapy.6 Also, it lowers the effectiveness of surgical treatment of coronary artery dis- ease, etc. There are several mechanisms of thrombosis for- mation:7–9 the activation of platelet and coagulation chains of homeostasis, disruption of synthesis for some blood coagulation factors II (Prothrombin), VII (Proconvertin), IX (Christmas factor), and X (Stuart–Prower factor), a de- crease of fibrinolytic activity of blood, activation of lipid peroxidation, disruption of endothelium functional ac- tivity, etc.10 Modern antiplatelet and anticoagulant drugs influence the thrombocyte aggregation and blood coagu- lation system. However, their effectiveness often does not satisfy clinicians. Numerous clinical studies show that the use of modern antiplatelet drugs is often accompanied by such side effects as resistance to their action, an increased risk of uncontrolled bleeding, as well as the development of serious systemic complications.11 The high cost and the listed side effects of these drugs indicate the need for fur- ther research in the pursue for new, more effective, and safe substances and the development of antiplatelet drugs based on them. In recent years, the important role of lipid peroxi- dation in the pathogenesis of thrombus formation has 585Acta Chim. Slov. 2022, 69, 584–595 Stasevych et al.: N-(9,10-Dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) ... been shown. The influence of free radical mechanisms on the development of various types of cancer, atherosclero- sis and its thrombonecrotic consequences (heart attack, stroke), diabetes mellitus, chronic nonspecific lung dis- eases, diseases of the reproductive system, as well as ra- diation injury, hepatitis, decreased cellular and humoral immunity, etc. has been studied.12–14 Therefore, there has been a constant search for antioxidants, both natural and synthetic.15–16 The quinoid system is the structural block of many natural biologically active molecules, such as vitamins K and E, as well as compounds directly involved in oxidative metabolism, such as coenzyme Q10. Many antioxidants contained in food products are quinones (for example, flavonoids). The derivatives from quercetin (contained in vegetables and fruits), resveratrol (red vine), catechins and epicatechin (chocolate and tea) and also compounds obtained from tyrosine and tryptophan aminoacids (hy- droxytyrosol, 5-hydroxytryptophan and pyrroloquinoline quinone) are considered to be quinone compounds.17 The main advantage of quinones is their aromatic nature, which implies the stability necessary for functioning in an oxidative environment and actively participating in redox reactions.18 Many natural anthracenediones extracted from plants demonstrate antioxidant properties.19–21 Among the synthetic derivatives of 9,10-anthracenedione were discov- ered compounds with antioxidant properties22–24 and oxi- dative stress and cytotoxicity ability.25 It was demonstrated that the amount and position of substituents in the anthra- cenedion’s ring influence antioxidant properties.26–28 The scientists29,30 discovered some compounds among anthra- cenedione derivatives with antiplatelet and anticoagulant action. Dutch scientists obtained 1,4- and 1,8-derivatives of 9,10-anthracenedione included in oligodeoxynucleo- tides to investigate the influence on coagulation time of fibrinogen in the blood. The investigation showed better anticoagulation properties for 1,8-anthracenedione prod- ucts.31 Some sulfur-containing derivatives of 9,10-anthra- cenedione demonstrated high antioxidant activity.32 There are studies dedicated to researching antithrombotic drugs in Ukraine as well.33,34 Therefore, the purpose of the present work is to carry out the synthesis of new derivatives of 2-chloro-N-(9,10- dioxo-9,10-dihydroanthracen-1(2)-yl)-acetamides using functionalization by thio fragments and in vitro study of obtained derivatives for antioxidant and antiplatelet ef- fects. 2. Experimental 2. 1. Chemistry Melting points were measured in open to air-glass capillaries using a Büchi B-540 melting point apparatus and are uncorrected. Elemental analysis was performed on Perkin–Elmer 2400 CHN-analyzer, and the results were found to be in good agreement with the calculated values. 1Н and 13С NMR spectra in DMSO-d6 were record- ed on Varian Mercury-400 spectrometer with TMS as an internal standard. Mass spectra were recorded on Agilent 1100 Series G1956B LC/MSD SL LCMS system (Zorbax SBC18 column, 4.6×15 mm, 1.8 µm (PN 82(c) 75-932); solvent dimethylsulfoxide), using electrospray ionization at atmospheric pressure (70 eV). Infrared spectra were recorded on a Perkin–Elmer Spectrum Two FT-IR Spec- trometer equipped with an UATR (HR Single-Reflection with a diamond sensor) using Perkin–Elmer Spectrum 10 Spectroscopy Software with an ATR absorbance correc- tion for Spectrum Two UATR spectra. All chemicals were of reagent grade and used without further purification. The solvents were purified according to the standard pro- cedures.35 2-Chloro-N-(9,10-dioxo-9,10-dihydroanthracen- 1(2)-yl)-acetamides 1 and 2 were obtained by published methods.36,37 General Procedure for the Preparation of N-(9,10-Di- oxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) Aceta- mides 3–20 To 0.5 g (1.668 mmol) of chloroacetamide 1 or 2 in 40 mL of DMF, 1.835 mmol of the corresponding thiol and 0.507 g (3.67 mmol) of potassium carbonate were added under stirring. The reaction mixture was kept under stirring and at room temperature for 12 hours. Then, the reaction mix- ture was poured into 200 mL of water, acidified with 10% HCl solution to pH 6, and left overnight. The mixture was filtered off. The precipitate was washed with 20 mL of cold water and dried. As a result, target sulfide derivatives 3–20 were obtained with 60–93% yields. 2-((2-((9,10-Dioxo-9,10-dihydroanthracen-1-yl)ami- no)-2-oxoethyl)thio)acetic Acid (3). Yield 0.551 g (93%), mp 217 oC dec. FT-IR (UATR diamond) νmax 3196.07, 2928.41, 1751.23, 1670.84, 1652.32, 1590.18, 1517.03, 1420.10, 1345.29, 1281.09, 1169.65, 1021.33, 801.68, 705.48 cm–1. 1H NMR (400 MHz, DMSO-d6) δ 12.70 (br. s, 1H, OH), 12.43 (s, 1H, NH), 8.88 (d, J = 7.7 Hz, 1HAr), 8.08 (dd, J = 23.9, 6.1 Hz, 2HAr), 7.84 (d, J = 10.0 Hz, 3HAr), 7.79 (d, J = 7.4 Hz, 1HAr), 3.67 (s, 2H, CH2), 3.46 (s, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 186.45, 182.35, 171.26, 169.08 (C=O), 141.28, 135.97, 135.10, 135.02, 134.01, 133.87, 132.48, 127.40, 126.79, 125.83, 122.41, 118.29 (CAr), 37.65, 34.35 (CH2). LC-MS, m/z (Irel): 356 (M+H, 100). Anal. Calcd for C18H13NO5S: C, 60.84; H, 3.69; N, 3.94; S, 9.02. Found: C, 60.77; H, 3.60; N, 3.88; S, 9.12. Methyl 2-((2-((9,10-Dioxo-9,10-dihydroanthracen-1- yl)amino)-2-oxoethyl)thio)acetate (4). Yield 0.554 g (90%), mp 120 oC dec. FT-IR (UATR diamond) νmax 3193.96, 2955.61, 1754.54, 1695.98, 1652.48, 1594.73, 1516.00, 1411.04, 1340.64, 1280.24, 709.26 cm–1. 1H NMR 586 Acta Chim. Slov. 2022, 69, 584–595 Stasevych et al.: N-(9,10-Dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) ... (400 MHz, DMSO-d6) δ 12.41 (br. s, 1H, NH), 8.87 (d, J = 7.7 Hz, 1HAr), 8.07 (dd, J = 22.8, 6.6 Hz, 2HAr), 7.82 (dd, J = 18.2, 7.6 Hz, 4HAr), 3.68 (s, 2H, CH2), 3.59 (s, 3H, CH3), 3.56 (s, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 186.48, 182.32, 170.40, 168.96 (C=O), 141.26, 136.01, 135.11, 135.04, 134.03, 133.85, 132.50, 127.37, 126.81, 125.77, 122.44, 118.24 (CAr), 52.55 (CH3), 37.78, 33.84 (CH2). LC-MS, m/z (Irel): 370 (M+H, 100). Anal. Calcd for C19H15NO5S: C, 61.78; H, 4.09; N, 3.79; S, 8.68. Found: C, 61.69; H, 4.20; N, 3.60; S, 8.77. 2-((2-((9,10-Dioxo-9,10-dihydroanthracen-1-yl)ami- no)-2-oxoethyl)thio)propanoic Acid (5). Yield 0.566 g (92%), mp 182 oC dec. FT-IR (UATR diamond) νmax 3135.98, 2985.20, 2933.81, 1749.97, 1673.73, 1652.70, 1593.52, 1520.90, 1283.63 cm–1. 1H NMR (400 MHz, DM- SO-d6) δ 12.94 (br. s, 1H, OH), 12.46 (s, 1H, NH), 8.84 (d, J = 7.3 Hz, 1HAr), 8.04 (d, J = 6.5 Hz, 1HAr), 7.98 (d, J = 6.7 Hz, 1HAr), 7.85–7.79 (m, 2HAr), 7.74 (q, J = 7.5, 7.0 Hz, 2HAr), 3.73–3.63 (m, 2H, CH2), 3.57 (q, J = 6.7 Hz, 1H, CH), 1.41 (d, J = 6.9 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 186.29, 182.20, 173.84, 169.06 (C=O), 141.16, 135.90, 135.03, 134.95, 133.88, 133.74, 132.34, 127.32, 126.72, 125.70, 122.36, 118.14 (CAr), 41.62 (CH), 36.88 (CH2), 17.38 (CH3). LC-MS, m/z (Irel): 370 (M+H, 100). Anal. Calcd for C19H15NO5S: C, 61.78; H, 4.09; N, 3.79; S, 8.68. Found: C, 61.71; H, 4.22; N, 3.63; S, 8.81. 3-((2-((9,10-Dioxo-9,10-dihydroanthracen-1-yl)ami- no)-2-oxoethyl)thio)propanoic Acid (6). Yield 0.443 g (72%), mp 227 oC dec. FT-IR (UATR diamond) νmax 3183.89, 1739.22, 1679.84, 1655.50, 1594.52, 1521.37, 1339.16, 1282.47, 709.32 cm–1. 1H NMR (400 MHz, DM- SO-d6) δ 13.18 (br. s, 1H, OH) 12.46 (s, 1H, NH), 8.87 (d, J = 7.8 Hz, 1H), 8.08 (d, J = 6.8 Hz, 1H), 8.02 (d, J = 7.4 Hz, 1HAr), 7.84 (q, J = 6.5, 4.0 Hz, 2HAr), 7.78 (q, J = 8.5 Hz, 2HAr), 3.59 (s, 2H, CH2), 2.84 (t, J = 7.1 Hz, 2H, CH2), 2.62 (t, J = 7.1 Hz, 2H, CH2). 13C NMR (100 MHz, DM- SO-d6) δ 186.39, 182.27, 173.27, 169.69 (C=O), 141.25, 135.95, 135.05, 134.97, 133.97, 133.81, 132.42, 127.37, 126.74, 125.67, 122.37, 118.17 (CAr), 37.43, 34.43, 27.86 (CH2). LC-MS, m/z (Irel): 370 (M+H, 100). Anal. Calcd for C19H15NO5S: C, 61.78; H, 4.09; N, 3.79; S, 8.68. Found: C, 61.75; H, 4.01; N, 3.72; S, 8.77. 2-((2-((9,10-Dioxo-9,10-dihydroanthracen-1-yl)ami- no)-2-oxoethyl)thio)succinic Acid (7). Yield 0.613 g (89%), mp 167 oC dec. FT-IR (UATR diamond) νmax 3193.78, 3014.84, 1992.55, 1739.80, 1677.70, 1654.32, 1593.35, 1575.24, 1281.58, 1242.10, 708,99 cm–1. 1H NMR (400 MHz, DMSO-d6) δ 12.72 (br. s, 1H, OH), 12.57 (br. s, 1H, OH), 12.45 (s, 1H, NH), 8.87 (d, J = 7.9 Hz, 1HAr), 8.06 (dd, J = 23.3, 7.8 Hz, 2HAr), 7.93 (s, 1HAr), 7.86–7.78 (m, 3HAr), 3.76 (d, J = 3.8 Hz, 2H, CH2), 3.70 (dd, J = 10.2, 5.1 Hz, 1H, CH), 2.85–2.76 (m, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 186.38, 182.30, 172.64, 172.15, 168.84 (C=O), 141.16, 135.94, 135.07, 135.00, 133.98, 133.85, 132.43, 127.37, 126.76, 125.84, 122.44, 118.32 (CAr), 42.19 (CH), 36.97, 36.23 (CH2). LC-MS, m/z (Irel): 414 (M+H, 100). Anal. Calcd for C20H15NO7S: C, 58.11; H, 3.66; N, 3.39; S, 7.76. Found: C, 58.16; H, 3.76; N, 3.48; S, 7.65. 2-((2-((9,10-Dioxo-9,10-dihydroanthracen-1-yl)ami- no)-2-oxoethyl)thio)benzoic Acid (8). Yield 0.64 g (92%), mp 250–252 oC dec. FT-IR (UATR diamond) νmax 3254.37, 1711.64, 1675.22, 1646.37, 1593.72, 1540.41, 1339.99, 1276.95 cm–1. 1H NMR (400 MHz, DMSO-d6) δ 13.18 (br. s, 1H, OH), 12.62 (s, 1H, NH), 8.92 (d, J = 8.1 Hz, 1HAr), 8.15 (d, J = 7.5 Hz, 1HAr), 8.11 (d, J = 6.5 Hz, 1HAr), 7.93–7.87 (m, 4HAr), 7.84 (t, J = 8.0 Hz, 1HAr), 7.50 (t, J = 7.6 Hz, 1HAr), 7.43 (d, J = 7.7 Hz, 1HAr), 7.20 (t, J = 7.5 Hz, 1HAr), 4.16 (m, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 186.46, 182.48, 169.09, 167.90 (C=O), 141.05, 139.90, 136.05, 135.09, 134.24, 133.99, 132.97, 132.63, 131.51, 129.03, 127.42, 126.84, 125.84, 125.04, 122.57, 118.52 (CAr), 37.59 (CH2). LC-MS, m/z (Irel): 418 (M+H, 100). Anal. Calcd for C23H15NO5S: C, 66.18; H, 3.62; N, 3.36; S, 7.68. Found: C, 66.05; H, 3.65; N, 3.44; S, 7.84. 2-((2-((9,10-Dioxo-9,10-dihydroanthracen-1-yl)ami- no)-2-oxoethyl)thio)nicotinic Acid (9). Yield 0.635 g (91%), mp 192 oC dec. FT-IR (UATR diamond) νmax 3492.52, 3217.87, 3085.59, 2926.78, 1698.96, 1672.50, 1643.98, 1581.67, 1558.28, 1519.99, 1466.38, 1411.38, 1338.67, 1313.37, 1274.00, 1246.80, 1236.69, 1157.46, 1068.96, 708.93 cm–1. 1H NMR (400 MHz, DMSO-d6) δ 13.57 (br. s, 1H, OH), 12.44 (s, 1H, NH), 8.86 (d, J = 5.4 Hz, 1HAr), 8.60 (d, J = 3.1 Hz, 1HAr), 8.24 (d, J = 5.9 Hz, 1HAr), 8.02 (t, J = 9.2 Hz, 2HAr), 7.96–7.91 (m, 1HAr), 7.83 (t, J = 7.1 Hz, 2HAr), 7.78–7.76 (m, 1HAr), 7.23 (dd, J = 7.8, 4.7 Hz, 1HAr), 4.10 (s, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 186.26, 182.32, 169.50, 166.76 (C=O), 159.59, 152.44, 141.29, 139.61, 135.88, 134.98, 134.92, 134.04, 133.86, 132.48, 127.22, 126.72, 125.76, 124.23, 122.20, 120.06, 118.13 (CAr), 35.89 (CH2). LC-MS, m/z (Irel): 419 (M+H, 100). Anal. Calcd for C22H14N2O5S: C, 63.15; H, 3.37; N, 6.70; S, 7.66. Found: C, 63.17; H, 3.37; N, 6.59; S, 7.73. N-(9,10-Dioxo-9,10-dihydroanthracen-1-yl)-2-((2-hy- droxyethyl)thio)acetamide (10). Yield 0.467 g (82%), mp 145 oC dec. FT-IR (UATR diamond) νmax 3460.15, 2945.84, 1689.32, 1652.84, 1590.36, 1521.72, 1414.92, 1342.11, 1277.93, 1171.80, 707.98 cm–1. 1H NMR (400 MHz, DM- SO-d6) δ 12.53 (s, 1H, NH), 8.92 (d, J = 7.9 Hz, 1HAr), 8.13 (d, J = 8.1 Hz, 1HAr), 8.06 (d, J = 6.5 Hz, 1HAr), 7.89–7.79 (m, 4HAr), 4.87 (t, J = 5.1 Hz, 1H, OH), 3.65–3.58 (m, 4H, 2CH2), 2.73 (t, J = 6.6 Hz, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 186.07, 182.00, 169.55 (C=O), 140.91, 135.57, 134.68, 134.59, 133.70, 133.52, 132.12, 126.98, 126.38, 125.30, 121.97, 117.91 (CAr), 60.46 (CH2-OH), 37.27, 34.80 (CH2). LC-MS, m/z (Irel): 342 (M+H, 100). 587Acta Chim. Slov. 2022, 69, 584–595 Stasevych et al.: N-(9,10-Dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) ... Anal. Calcd for C18H15NO4S: C, 63.33; H, 4.43; N, 4.10; S, 9.39. Found: C, 63.32; H, 4.29; N, 4.01; S, 9.47. 2-((2,3-Dihydroxypropyl)thio)-N-((9,10-dioxo-9,10- dihydroanthracen-1-yl) Acetamide (11). Yield 0.446 g (72%), mp 162 oC dec. FT-IR (UATR diamond) νmax 3380.05, 3190.26, 3106.35, 2988.43, 2934.29, 2845.55, 2780.01, 1674.85, 1647.56, 1591.37, 1575.92, 1515.35, 1476.80, 1408.66, 1337.05, 1280.48, 1238.14, 1172.97, 1021.33, 706.82 cm–1. 1H NMR (400 MHz, DMSO-d6) δ 12.61 (s, 1H, NH), 8.96 (dd, J = 14.4, 6.6 Hz, 1HAr), 8.21 (dd, J = 5.1, 2.4 Hz, 1HAr), 8.16–8.12 (m, 2HAr), 7.95–7.89 (m, 3HAr), 4.89 (t, J = 6.1 Hz, 1H, OH), 4.66–4.60 (m, 2H, CH2), 4.25 (dd, J = 13.6, 4.2 Hz, 1H, CH), 4.12–3.90 (m, 2H), 3.61 (d, J = 3.0 Hz, 2H, CH2), 2.78 (dd, J = 13.3, 4.6 Hz, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 186.51, 182.52, 170.04 (C=O), 141.37, 136.03, 135.18, 135.07, 134.15, 133.99, 132.55, 127.47, 126.83, 125.78, 122.40, 118.41 (CAr), 71.67 (CH-OH), 64.92 (CH2-OH), 36.51, 34.43 (CH2). LC-MS, m/z (Irel): 372 (M+H, 100). Anal. Calcd for C19H17NO5S: C, 61.44; H, 4.61; N, 3.77; S, 8.63. Found: C, 61.40; H, 4.52; N, 3.68; S, 8.72. 2-((2-((9,10-Dioxo-9,10-dihydroanthracen-2-yl)amino) -2-oxoethyl)thio)acetic Acid (12). Yield 0.432 g (73%), mp 136 oC dec. FT-IR (UATR diamond) νmax 3589.84, 3516.18, 3296.49, 3203.80, 3102.15, 3064.54, 2957.96, 1719.69, 1675.77, 1656.88, 1644.88, 1589.23, 1574.78, 1548.17, 1425.65, 1332.17, 1302.35, 1226.20, 1192.26, 1133.81, 712.54 cm–1. 1H NMR (400 MHz, DMSO-d6) δ 11.36 (br. s, 1H, OH), 10.69 (s, 1H, NH), 8.28–8.26 (m, 1HAr), 8.05 (d, J = 7.8 Hz, 2HAr), 8.01 (d, J = 8.4 Hz, 1HAr), 7.94 (d, J = 7.7 Hz, 1HAr), 7.83–7.79 (m, 2HAr), 3.48 (d, J = 11.6 Hz, 4H, 2CH2). 13C NMR (100 MHz, DMSO-d6) δ 182.66, 181.58, 171.50, 168.81 (C=O), 144.79, 134.88, 134.54, 134.40, 133.42, 133.38, 128.81, 128.35, 127.08, 127.01, 124.12, 124.04, 116.24 (CAr), 36.46, 34.27 (CH2). LC-MS, m/z (Irel): 356 (M+H, 100). Anal. Calcd for C18H13NO5S: C, 60.84; H, 3.69; N, 3.94; S, 9.02. Found: C, 60.79; H, 3.64; N, 3.88; S, 9.10. Methyl 2-((2-((9,10-Dioxo-9,10-dihydroanthracen-2- yl)amino)-2-oxoethyl)thio)acetate (13). Yield 0.486 g (79%), mp 180 oC dec. FT-IR (UATR diamond) νmax 3328.94, 3297.88, 3237.78, 3104.62, 2966.01, 2921.55, 1726.79, 1709.91, 1670.16, 1651.28, 1583.47, 1538.40, 1345.28, 1293.99, 1171.15, 1126.35, 713.52 cm–1. 1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H, NH), 8.35–8.31 (m, 1HAr), 8.13–8.05 (m, 3HAr), 7.98 (d, J = 7.5 Hz, 1HAr), 7.86–7.83 (m, 1HAr), 3.67–3.60 (m, 3H, CH3), 3.56–3.54 (m, 2H, CH2), 3.52–3.48 (m, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 182.69, 181.61, 170.60, 168.67 (C=O), 144.77, 134.91, 134.57, 134.44, 133.45, 133.41, 128.86, 128.39, 127.09, 127.02, 124.12, 116.24 (CAr), 52.59 (CH3), 36.49, 33.68 (CH2). LC-MS, m/z (Irel): 370 (M+H, 100). Anal. Calcd for C19H15NO5S: C, 61.78; H, 4.09; N, 3.79; S, 8.68. Found: C, 61.93; H, 4.00; N, 3.78; S, 8.57. 2-((2-((9,10-Dioxo-9,10-dihydroanthracen-2-yl)ami- no)-2-oxoethyl)thio)propanoic Acid (14). Yield 0.462 g (75%), mp 138 oC dec. FT-IR (UATR diamond) νmax 3563.85, 3480.16, 3175.69, 3102.14, 3056.23, 2990.48, 2942.47, 1729.97, 1676.97, 1589.49, 1576.80, 1548.81, 1423.12, 1349.47, 1333.52, 1303.10, 1180.68, 851.93, 713.74 cm–1. 1H NMR (400 MHz, DMSO-d6) δ 12.70 (s, 1H, OH), 10.74 (s, 1H, NH), 8.29–8.27 (m, 1HAr), 8.06–8.03 (m, 2HAr), 8.00 (d, J = 8.5 Hz, 1HAr), 7.94 (d, J = 9.1 Hz, 1HAr), 7.82–7.79 (m, 2HAr), 3.62 (q, J = 6.7 Hz, 1H, CH), 3.56 (d, J = 3.3 Hz, 2H, CH2), 1.38 (d, J = 7.1 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 182.16, 181.08, 173.64, 168.34 (C=O), 144.32, 134.37, 134.03, 133.90, 132.93, 132.89, 128.30, 127.85, 126.58, 126.51, 123.64, 115.76 (CAr), 41.12 (CH), 35.42 (CH2), 17.28 (CH3). LC-MS, m/z (Irel): 370 (M+H, 100). Anal. Calcd for C19H15NO5S: C, 61.78; H, 4.09; N, 3.79; S, 8.68. Found: C, 61.69; H, 4.17; N, 3.82; S, 8.80. 3-((2-((9,10-Dioxo-9,10-dihydroanthracen-2-yl)ami- no)-2-oxoethyl)thio)propanoic Acid (15). Yield 0.542 g (88%), mp 149 oC dec. FT-IR (UATR diamond) νmax 3066.41, 3014.57, 2980.94, 2934.81, 1745.47, 1724.84, 1667.40, 1651.21, 1644.45, 1589.56, 1573.68, 1511.50, 1417.25, 1385.74, 1332.89, 1279.43, 1239.20, 1162.90, 1081.52, 707.49 cm–1. 1H NMR (400 MHz, DMSO-d6) δ 12.32 (s, 1H, OH), 10.90 (s, 1H, NH), 8.42–8.40 (m, 1HAr), 8.14–8.12 (m, 2HAr), 8.10–8.09 (m, 1HAr), 8.04 (d, J = 8.4 Hz, 1HAr), 7.87 (t, J = 8.0 Hz, 2HAr), 3.43–3.42 (m, 2H, CH2), 2.84 (t, J = 7.0 Hz, 2H, CH2), 2.59 (t, J = 7.0 Hz, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 182.77, 181.70, 173.32, 169.56 (C=O), 144.95, 134.95, 134.62, 134.52, 133.50, 133.47, 128.87, 128.41, 127.13, 127.05, 124.20, 116.27 (CAr), 35.98, 34.51, 27.54 (CH2). LC-MS, m/z (Irel): 370 (M+H, 100). Anal. Calcd for C19H15NO5S: C, 61.78; H, 4.09; N, 3.79; S, 8.68. Found: C, 61.71; H, 4.10; N, 3.80; S, 8.72. 2-((2-((9,10-Dioxo-9,10-dihydroanthracen-2-yl)ami- no)-2-oxoethyl)thio)succinic Acid (16). Yield 0.386 g (56%), mp 185 oC dec. FT-IR (UATR diamond) νmax 3335.94, 3008.21, 2786.81, 1717.18, 1675.41, 1589.10, 1543.68, 1468.67, 1417.92, 1339.34, 1299.14, 1235.29, 1177.40, 711.15 cm–1. 1H NMR (400 MHz, DMSO-d6) δ 12.81 (br. s, 1H, OH), 12.51 (br. s, 1H, OH), 10.98 (s, 1H, NH), 8.47 (d, J = 8.1 Hz, 1HAr), 8.16–8.08 (m, 3HAr), 7.97– 7.94 (m, 1HAr), 7.83–7.76 (m, 2HAr), 3.91–3.89 (m, 1H, CH), 3.69–3.58 (d, J = 7.6 Hz, 2H, CH2), 2.70–2.59 (m, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 182.18, 181.22, 171.68, 171.29, 168.72 (C=O), 142.77, 133.92, 133.44, 133.35, 132.62, 127.58, 127.11, 123.53, 116.01 (CAr), 42.30 (CH), 36.17, 34.78 (CH2). LC-MS, m/z (Irel): 414 (M+H, 100). Anal. Calcd for C20H15NO7S: C, 58.11; H, 3.66; N, 3.39; S, 7.76. Found: C, 58.18; H, 3.59; N, 3.27; S, 7.70. 2-((2-((9,10-Dioxo-9,10-dihydroanthracen-2-yl)ami- no)-2-oxoethyl)thio)benzoic Acid (17). Yield 0.557 588 Acta Chim. Slov. 2022, 69, 584–595 Stasevych et al.: N-(9,10-Dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) ... g (80%), mp 237 oC dec. FT-IR (UATR diamond) νmax 3568.41, 3506.01, 3244.18, 3173.85. 3101.99, 3061.49, 2931.53, 2882.55, 1671.01, 1643.70, 1590.20, 1576.63, 1541.84, 1466.10, 1331.88, 1299.86, 1259.74, 1159.88, 1119.05, 710.97 cm–1. 1H NMR (400 MHz, DMSO-d6) δ 12.73 (br. s, 1H, OH), 10.94 (s, 1H, NH), 8.37–8.32 (m, 1HAr), 8.13–8.04 (m, 2HAr), 7.98 (d, J = 8.7 Hz, 2HAr), 7.92 (d, J = 7.7 Hz, 2HAr), 7.87–7.80 (m, 2HAr), 7.57–7.52 (m, 2HAr), 7.24 (t, J = 6.6 Hz, 1HAr), 3.96–3.93 (m, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 182.25, 181.20, 167.88, 167.45 (C=O), 144.24, 140.26, 134.47, 134.13, 134.03, 133.01, 132.97, 132.52, 131.02, 128.44, 128.05, 127.91, 126.65, 126.58, 125.74, 124.40, 123.75, 115.83 (CAr), 36.71 (CH2). LC-MS, m/z (Irel): 418 (M+H, 100). Anal. Calcd for C23H15NO5S: C, 66.18; H, 3.62; N, 3.36; S, 7.68. Found: C, 66.05; H, 3.65; N, 3.44; S, 7.84. 2-((2-((9,10-Dioxo-9,10-dihydroanthracen-2-yl)ami- no)-2-oxoethyl)thio)nicotinic Acid (18). Yield 0.523 g (75%), mp 180 oC dec. FT-IR (UATR diamond) νmax 3588.77, 3528.34, 3241.81, 3174.17, 3102.68, 3000.36, 2636.05, 1670.57, 1648.96, 1632.10, 1590.54, 1575.51, 1556.74, 1543.53, 1419.68, 1389.13, 1331.30, 1301.77, 1252.08, 1233.62, 1156.54, 1129.87, 1070.10, 710.97 cm–1. 1H NMR (400 MHz, DMSO-d6) δ 12.60 (br. s, 1H, OH), 10.90 (s, 1H, NH), 8.57 (s, 1HAr), 8.37 (s, 1HAr), 8.23 (d, J = 7.1 Hz, 1HAr), 8.10–7.99 (m, 5HAr), 7.83 (s, 2HAr), 7.26– 7.21 (m, 1HAr), 4.09 (s, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 182.71, 181.59, 168.67, 166.96 (C=O), 160.38, 152.02, 145.04, 139.45, 134.84, 134.50, 134.45, 133.41, 128.81, 128.19, 127.05, 126.98, 124.42, 124.07, 119.67, 116.17 (CAr), 35.74 (CH2). LC-MS, m/z (Irel): 419 (M+H, 100). Anal. Calcd for C22H14N2O5S: C, 63.15; H, 3.37; N, 6.70; S, 7.66. Found: C, 63.09; H, 3.23; N, 6.78; S, 7.77. N-(9,10-Dioxo-9,10-dihydroanthracen-2-yl)-2-((2-hy- droxyethyl)thio)acetamide (19). Yield 0.484 g (85%), mp 187 oC dec. FT-IR (UATR diamond) νmax 3589.84, 3516.18, 3296.49, 3203.80, 3102.15, 3064.54, 2957.96, 1719.69, 1675.77, 1656.88, 1644.88, 1589.23, 1574.78, 1548.17, 1425.65, 1352.14, 1332.17, 1302.35, 1226.20, 1192.26, 1167.14, 1151.73, 1133.81, 931.71, 712.54 cm–1. 1H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H, NH), 8.40–8.37 (m, 1HAr), 8.14–8.12 (m, 2HAr), 8.11–8.09 (m, 1HAr), 8.01 (d, J = 8.4 Hz, 1HAr), 7.87 (t, J = 7.6 Hz, 2HAr), 4.88 (t, J = 5.3 Hz, 1H, OH), 3.60 (q, J = 6.1 Hz, 2H, CH2), 3.40–3.39 (m, 2H, CH2), 2.74 (t, J = 6.6 Hz, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 182.34, 181.25, 169.21 (C=O), 144.48, 134.51, 134.17, 134.10, 133.08, 133.05, 128.47, 127.99, 126.69, 126.61, 123.73, 123.65, 115.82, 115.74 (CAr), 60.44 (CH2- OH), 35.90, 34.62 (CH2). LC-MS, m/z (Irel): 342 (M+H, 100). Anal. Calcd for C18H15NO4S: C, 63.33; H, 4.43; N, 4.10; S, 9.39. Found: C, 63.20; H, 4.30; N, 4.09; S, 9.29. 2-((2,3-Dihydroxypropyl)thio)-N-((9,10-dioxo-9,10- dihydroanthracen-2-yl) Acetamide (20). Yield 0.501 g (81%), mp 163 oC dec. FT-IR (UATR diamond) νmax 3335.95, 3104.86, 3069.88, 2927.97, 1675.70, 1589.96, 1542.74, 1419.12, 1340.62, 1299.62, 1237.15, 933.59, 711.60 cm–1. 1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H, NH), 8.45 (d, J = 2.3 Hz, 1HAr), 8.20–8.12 (m, 2HAr), 8.04 (dd, J = 8.6, 2.3 Hz, 1HAr), 7.91–7.86 (m, 3HAr), 5.21 (dd, J = 19.7, 5.4 Hz, 1H, CH) 4.83 (t, J = 6.3 Hz, 1H, OH), 4.09 (t, J = 12.6 Hz, 1H, OH), 3.43 (d, J = 5.0 Hz, 2H, CH2), 3.24–3.00 (m, 2H, CH2), 2.99–2.77 (m, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 182.65, 181.64, 165.00 (C=O), 144.34, 134.96, 134.63, 134.46, 133.41, 128.87, 128.68, 127.10, 127.03, 124.30, 116.38 (CAr), 66.76 (CH- OH), 59.23 (CH2-OH), 36.27, 34.66 (CH2). LC-MS, m/z (Irel): 372 (M+H, 100). Anal. Calcd for C19H17NO5S: C, 61.44; H, 4.61; N, 3.77; S, 8.63. Found: C, 61.32; H, 4.57; N, 3.69; S, 8.52. 2. 2. Antioxidant Activity 2. 2. 1 Method of Study of Lipid Peroxidation (LP) and Oxidative Modification of Proteins (OMP) of Thioether Acetamides The LP and OMP processes study was performed in vitro on rat’s liver homogenate according to the Lush- chak’s method.38 The amount of protein in the sample was determined due to the Lowry method. This meth- od is based on LP activation by ferrous iron ions to a level recorded spectrophotometrically by reaction with thiobarbituric acid. The degree of OMP was evaluated calculating the amount of additional CGs formed in the side chains of aminoacids under the reaction with 2,4-dinitrophenylhydrazine. The methanol solutions served as control, while the standard of measurement was 10–6 М solution of quercetin. Experimental data were analyzed considering the arithmetic mean M and standard error m in the form of (M ± m), n = 5. Differ- ences between experimental data were determined via Tukey’s test of one-way analysis (ANOVA), and the dif- ferences were considered to be statistically significant at P < 0.05.39 At the beginning of our experiment, to 0.3 g of rat liver homogenate 0.3 mL methanol solutions of sulfur-con- taining derivatives of 2-chloro-N-(9,10-dioxo-9,10-dihy- droanthracen-1(2)-yl)-acetamides (10–6 М concentration) was poured. Iron(II) sulfate solution (0.3 mL of 2.8%) was introduced to the obtained solution. The reaction of LP was induced within 10 minutes. Then 0.3 mL of 4% solu- tion of hydrogen peroxide was added, and the solution was incubated for 2 hours. The reaction was stopped after introducing 1.2 mL of 40% trichloroacetic acid, precipi- tating polypeptides. Reaction mixture was centrifuged for 10 minutes at 5·103 RPM. Bioactive compounds’ influence analyses on FRO were conducted for a single sample. Car- bonyl groups were determined in the sediment, and prod- ucts of lipids interaction with TBA were determined from the supernatant. 589Acta Chim. Slov. 2022, 69, 584–595 Stasevych et al.: N-(9,10-Dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) ... at 300 g for 10 min. The supernatant (PRP) was carefully separated and used further in the aggregation assay. Plate- let-pure plasma (PPP) was prepared by further centrifuga- tion of the remained stabilized blood at 1,500 g for 30 min. 2. 3. 2. Platelet Aggregation Assay Platelet aggregation was assessed within the first 3 h after blood sampling using photo-optical aggregometer AT-02 (Medtech, Russia). Before the assessment, the plate- let count in PRP was adjusted with PPP to about 230×103– 250×103 cells/μL. The studied compounds were dissolved in pure di- methyl sulfoxide (DMSO) and their test concentrations were prepared using distilled water. The final DMSO con- centration in all experiments was fixed at 1% to minimize the effect of DMSO on the platelet aggregation. Primary screening for anti-aggregation activity of compounds 3–20 was performed in vitro: PRP was incu- bated with studied compounds (final concentration was 50 μM) in a cuvette for 2 min at 37 oC with constant stirring (500 rpm). PRP incubated with DMSO alone was used as a control. ADP (Sigma, USA) in the final concentration of 5×10–6 M was added to the sample, and the change of light transmission was monitored for 8 minutes ADP to induce platelet aggregation. In this experiment the level of spontaneous aggregation induced by addition of the tested compounds to PRP was studied. The degree of platelet ag- gregation (the maximal level of light transmission of PRP after addition of inducer) was evaluated. The degree of in- hibition of ADP-dependent aggregation under the action of obtained derivatives 3–20 relative to control, which was taken as 100%, was calculated. 3. Results and Discussion 3. 1. Synthesis of N-(9,10-Dioxo-9,10- dihydroanthracen-1(2)-yl)-2-(R-thio) Acetamides In continuation of the previous studies of our group on the functionalization of 9,10-anthracenedione,36,37,40–46 a structural modification of 2-chloro-N-(9,10-dioxo-9,10- dihydroanthracene-1(2)-yl)acetamides 1, 2 by pharma- cophore fragments, namely, sulfur-containing substituents was carried out. In this work, the reaction of chloroaceta- mides 1, 2 with a number of alkyl(aryl/hetaroaryl)thiols, additionally functionalised with mercapto, hydroxy, car- boxy and carboxylate groups was carried out (Scheme 1). It can be a key aspect for improving biological activity, including antiplatelet and antioxidant actions, as well as bioavailability due to an increase in the polarity of the ob- tained compounds and better water solubility. Modification of chloroacetamides 1, 2 with the cor- responding thiols was carried out using their interaction at 2. 2. 2. Determination of the Content of Products of Lipids Interaction with TBA in the Supernatant 1.5 mL of 0.8% TBA was added to the supernatant separated from the sediment. The reaction between TBA and MDA proceeds during the heating of this solution to 100 oС for about 1 hour. The formation of the pinky color- ed complex allows determining the content of TBA-active products: After cooling, 3 mL of butanol was added to the reac- tion mass and was left for 2 hours. After, the formation of two phases was observed. Determination of the extinction coefficient was determined from the butanol fraction at λ = 532 nm. Calculation of TBA-active products was performed according to: μmol/mg of protein (1) where E – extinction coefficient of the test sample; ε – mil- limolar extinction coefficient (ε = 156 cm2/μmol); V1 – a volume of butanol (mL); V2 – sample volume (mL); V – su- pernatant volume (mL); C – protein concentration in the supernatant (μmol). 2. 2. 3. Determination of CGs Proteins in the Precipitate To the formed precipitate 1 mL of 1% 2,4-dinitro- phenylhydrazine solution was added. The resulting solution was incubated for an hour, then centrifuged at 5·103 RPM for 10 minutes. The precip- itate was washed three times with the addition of 1 mL of ethanol-ethyl acetate solution (1:1) and centrifuged. Then 3 mL of 50% urea solution was added to the precipitate, centrifuged, and the additional CG was determined using a spectrophotometer (λ = 370 nm). 2. 3. Anti-platelet Activity 2. 3. 1. Preparation of Rabbit Platelet-rich Plasma Platelet-rich plasma (PRP) was obtained from at least 3 different healthy adult rabbits. The Scientific Eth- ics Committee of Taras Shevchenko National University of Kyiv, Ukraine approved the study protocol. Rabbit blood was collected from the ear artery into a polyethylene tube with 3.8% sodium citrate in the ratio 9:1. PRP was obtained by centrifugation of stabilized blood 590 Acta Chim. Slov. 2022, 69, 584–595 Stasevych et al.: N-(9,10-Dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) ... room temperature in the presence of potassium carbonate in DMF (Scheme 1). As a result, the corresponding sulfide derivatives 3–20 were obtained in good and high yields of up to 93%. The structures of new thioether acetamides 3–20 were reliably confirmed by modern physicochemical anal- ysis methods, namely 1H and 13C NMR, LC-MS, IR-Fou- rier spectroscopy and elemental analysis. In particular, the secondary amino group resonates at 12.41–12.62 ppm for sulfides 3–11, and in the case of sulfides 12–20 at 10.69– 10.94 ppm in the 1H NMR spectra. In turn, the methylene group of the oxoethyl frag- ment of acetamide in 1Н NMR observed for compounds 3–7, 10, 11 at 3.59–3.76 ppm, and for compounds 12– 16, 19, 20 at 3.40–3.56 ppm. In the case of the aromatic thioether substituent of 8, 9, 17, 18, the CH2 group shifts downfield and resonates within 3.94–4.16. In 13С NMR spectra, the appearance of a signal of the carbon atom of the carboxyl group at 167.88–173.84 ppm is characteristic for sulfide derivatives 4, 6–9, 12, 14–18. For sulfide de- rivatives 9 and 18 containing a fragment of nicotinic acid in their structures, the appearance of signals of the qua- ternary carbon atom of the N-C-S group of the nicotine fragment was determined at 159.59 and 160.38 ppm, re- spectively. 3. 2. Antioxidant Properties The free-radical oxidation (FRO) research was conducted in vitro using a rat liver homogenate. LP and OMP as two markers of oxidative stress were used for the evaluation of antioxidant properties of the compounds. Scheme 1. Synthesis of N-(9,10-dioxo-9,10-dihydroanthracene-1(2)-yl)-2-(R-thio) acetamides 3–20 591Acta Chim. Slov. 2022, 69, 584–595 Stasevych et al.: N-(9,10-Dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) ... This method is based on the LP activation by ferrous ions to a level registered spectrometrically by reaction with thiobarbituric acid (TBA-active products content). The de- gree of OMP was determined by the amount of additional carbonyl groups formed (CG content) in the side chains of amino acids using the reaction with 2,4-diphenylhydra- zine. The results of this investigation, in particular the content of TBA-active products and CGs, found from a comparison of isomers (compounds where the substituent is introduced in position 1 or 2). Data are presented in Fig- ures 1 and 2. All compounds were compared with control, as well as with reference antioxidant quercetin. Quercetin in the LP processes acted as a control, and in the process- es of OMP it showed antioxidant properties, reducing the level of CG relative to the control by 41.5%. The comparison antioxidant activity of isomers 3–11 and 12–20 are as follows. Compound 8 contained the res- idue of 2-((2-amino-2-oxoethyl)thio)benzoic acid in the position 1, showed minor antioxidant properties (8.3% content of TBA-active products) compared to the control as presented in Figure 1. The derivative 17 increased the content of TBA-active products by 8.6%, i.e., it had proox- idant properties relative to control. The compounds 8 and 17 demonstrated antioxidant effect in OMP processes de- creasing the CGs level by 15.7% and 26.4%, respectively. The compound 17 contained the residue of 2-((2-ami- no-2-oxoethyl)thio)benzoic acid in the position 2, de- creased the level of CGs by 10.7% more than the derivative 8 in the free radical processes of protein oxidation. There- fore, 2-((2-((9,10-dioxo-9,10-dihydroanthracen-1-yl)ami- no)-2-oxoethyl)thio)benzoic acid 8 exhibited antioxidant properties on two FRO markers. The compounds 10 and 19 contained 2-((2-hydrox- yethyl)thio)acetamide residue in positions 1 or 2 of anthra- cenedione ring demonstrated similar results. In this case, derivative 10 reduced the content of TBA-active products relative to control slightly, namely by 8.7%. Compound 19 showed high prooxidant properties and significantly (by 41.0%) increased the content of TBA-active products compared to control (Fig. 1). In contrast, compound 19 showed better results on OMP processes than compound Figure 1. Influence of compounds 3–20 on amount of TBA-active products in rat liver homogenate (*- р ≤ 0,05; M ± m; n = 5) Figure 2. Influence of compounds 3–20 on amount of CGs of proteins in rat liver homogenate (*- р ≤ 0,05; **- р ≤ 0,01; M ± m; n = 5) 592 Acta Chim. Slov. 2022, 69, 584–595 Stasevych et al.: N-(9,10-Dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) ... 10 and reduced the CG content by 33.7% compared to the control (Fig. 2). In turn, derivative 10 also exhibited anti- oxidant properties in OMP processes, reducing the level of CG proteins relative to control by 24.4%. Moreover, compounds 6 and 15 show antioxidant activity in LP processes (Fig. 1). Derivative 6 reduces the content of TBA-active products by 28.0% relative to the control (p ≤ 0.05), whereas derivative 15 decreases the val- ue by 10.1%. The studied compound 6 statistically signif- icantly (p ≤ 0.01) decreases the formation of CGs in pro- teins in FRP of OMP processes by 37.8% relative to the control (Fig. 2). Meanwhile, the content of CGs under the action of compound 15 shows no difference to the control with a value at 100.5 ± 15.2%. Comparing compounds, which contain in anthra- cenedione nuclei the residue of succinic acid in the first (7) or second (16) position, demonstrates that compound 7 has antioxidant properties to lipids and lowers the con- tent of TBA-active products by 21.1% (Fig. 1). Compound 16, in contrast to compound 7, exhibits pro-oxidant prop- erties and increases the content of TBA-active products by 25.2% relative to control. In addition, compound 7 (Fig. 2) shows antioxidant effect in OMP processes and statistically significantly (p ≤ 0.01) decreases the level of CGs proteins by 40.3% compared to the control. In turn, 2-((2-((9,10-dioxo-9,10-dihydroanthracen-2-yl)ami- no)-2-oxoethyl)thio)succinic acid 16 increases the CGs content by 11.3%. The LP study of compound 11, which contains (di- hydroxypropyl)thio)acetamide residue in the structure, has not shown antioxidant properties due to no difference in the TBA-active products content to the control (Fig. 1). Compound 20 with the ((dihydroxypropyl)thio)acetamide residue in the structure at position 2 of anthracenedione exhibits prooxidant properties and increases the content of the TBA-active product by 27.0% relative to control (Fig. 1). Compounds 11 and 20, based on the results of the study of OMP (Fig. 2), showed that the content of protein CGs does not differ from the control. Such contents for derivatives 11 and 20 are 101.6 ± 6.6% and 101.0 ± 8.9%, respectively. Thus, these compounds are neutral to the ox- idation process. In turn, compounds 3 and 12 almost equally in- crease the content of TBA-active products compared to the control, namely, by 13.6% and 12.8%, respectively (Fig. 1). The compound 3 in the OMP processes, as well as for LP, demonstrates a prooxidant effect due to an increase of CGs for 11.4% compared to the control (Fig. 2). In con- trast, compound 12 has shown minor antioxidant proper- ties and decreased CGs by 4.2%. Compound 4, due to the results of LP investiga- tion, has the same effect as the control (Fig. 1), decreasing the level of TBA-active products only by 2.8%. Compound 13, which has in structure the aminooxoethylthioacetate residue at the second position of anthraquinone, showed better antioxidant properties compared to its isomer 4 and reduced the content of TBA-active products relative to control by 12.8%. Furthermore, compound 4, according to the results of OMP (Fig. 2), exhibits antioxidant properties and reduces the content of CGs by 24.9% in comparison with the control. Moreover, compound 13 shows the op- posite effect due to the increasing content of CGs relative to the control by 17.9%, which indicates an increase in free radical processes in the proteins (Fig. 2). Hence, summarizing the obtained results, the stud- ied compounds with a substituent in the first position of anthracenedione fragment exhibit higher antioxidant properties than their isomers with a substituent in the sec- ond position. Compounds with a substitution in the first position 6, 7, 8 and 10 demonstrate the antioxidant prop- erties concerning oxidative stress markers POL and OMP. Moreover, compound 6 reduces the content of TBA-active products by 28.0% and content of CGs by 36.8%, whereas derivative 7 decreases by 21.1% and 41.3%, respectively. Test compounds 4, 10, 17, 19, as well as quercetin, showed antioxidant properties only in OMP processes. Figure 3. Effect of derivatives 3–20 at a concentration of 50 μM on ADP-induced platelet aggregation in rabbit PRP (M ± SEM; n = 6, * p ≤ 0.05 changes are statistically significant compared to the control 1% DMSO) 593Acta Chim. Slov. 2022, 69, 584–595 Stasevych et al.: N-(9,10-Dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) ... 3. 3. Antiplatelet Activity Antiplatelet activity of derivatives 3–20 was studied in vitro using rabbit PRP. As can be seen from the results (Fig. 3), among the 20 tested compounds, only six (3, 4, 5, 9, 10, 18) showed moderate antiplatelet activity. The most active compound, namely compound 10, inhibited ADP-induced aggregation by 28%, while the inhibitory effect of others ranged from 12% to 20%. The latter is associated with the structure of the thio fragment and the inhibitory effect is characteristic for five com- pounds (3, 4, 5, 9, 10), and increases in the following order of substituents (Fig. 4): Analysis of the influence of the structure of the thio fragment on the manifestation of the antiplatelet ac- tivity of compounds 3–20 showed that anthracenedione derivatives containing this residue in the first position of the anthracenedione ring (3, 4, 5, 9, 10) can inhibit platelet aggregation. It was found that the presence of a less branched 2-((2-hydroxyethyl)thio)acetamide residue (compound 10) causes the highest percentage of the de- gree of inhibition. At the same time, derivatives 12–20 with a thio substituent in position 2 of the anthracenedi- one nucleus and compounds 6–8, 11 with branched and bulky substituents near the sulfur atom do not affect the degree of inhibition. 4. Conclusions A convenient way to obtain new sulfide derivatives with a 9,10-anthracenedione ring has been proposed. It includes the interaction of 2-chloro-N-(9,10-dioxo-9,10- dihydroanthracen-1(2)-yl)-acetamides 1, 2 with a num- ber of alkyl(aryl/hetaroaryl)thiols at room temperature in the presence of potassium carbonate in DMF. The study of antioxidant activity in terms of lipid peroxidation and oxidative modification of proteins in rat liver homogenate in vitro identified compounds 6 and 7 with the best prop- erties of radical scavenging activity in terms of the con- tent of TBA-active products and CGs in the corresponding range 21,1–28 % and 36.8–41.3%. An in vitro study of the antiplatelet activity using rabbit PRP revealed derivative 10, exhibiting the highest degree of inhibition of platelet aggregation among the synthesized compounds. Sulfide derivative 10 also demonstrated antioxidant properties in OMP processes, which manifested in lowering CGs pro- tein level compared to control for 24.4%. The structure-ac- tivity relationships for the obtained N-(9,10-dioxo-9,10- dihydroanthracene-1(2)-yl)-2-(R-thio) acetamides were determined. The data obtained are the basis for further studies on molecular design and the search for new com- pounds with antioxidant and antiplatelet activity in a se- ries of new derivatives of 9,10-anthracenedione. Figure 4. Correlation of substituent of the thio fragment and the inhibitory effect for five compounds 3, 4, 5, 9, 10 Funding This research was funded by the Ministry of Ed- ucation and Science of Ukraine, Project number: 0119U002252. Supplementary Data 1H and 13C NMR spectra of compounds 3–20 are provided in supplementary material via the “Supplemen- tary Content” section of this article’s webpage. Conflicts of Interest The authors declare no conflict of interest. 5. References 1. 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DOI:10.1002/hc.20112 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek S pomočjo reakcije 2-kloro-N-(9,10-diokso-9,10-dihidroantracen-1(2)-il)acetamidov s funkcionaliziranimi tioli v pris- otnosti kalijevega karbonata v N,N-dimetilformamidu (DMF) pri sobni temperaturi smo izvedli sintezo serije novih N-(9,10-diokso-9,10-dihidroantracen-1(2)-il)-2-(R-tio) acetamidov. Za nove spojine smo s pomočjo in vitro testov na osnovi lipidne peroksidaze (LP) in oksidativne modifikacije proteinov (OMP) v homogenizatu jeter podgan določili sposobnost delovanja v vlogi lovilcev radikalov. Ugotovili smo, da spojine, ki imajo vezane substituente na položaju 1 v antracendionskem skeletu, kažejo boljše antioksidacijske lastnosti kot njihovi izomeri s substituenti na položaju 2. Kot najbolj učinkovita lovilca radikalov sta se izkazali spojini 6 in 7. Antioksidacijske lastnosti v OMP procesu smo določili tudi za spojino 10; za to spojino smo izvedli tudi in vitro študijo delovanja proti agregaciji krvnih ploščic, kjer smo ugot- ovili inhibitorno delovanje na agregacijo, povzročeno z ADP. 596 Acta Chim. Slov. 2022, 69, 596–603 Li: A New Zn(II) Two-dimensional Coordination Polymer: ... DOI: 10.17344/acsi.2022.7500 Scientific paper A New Zn(II) Two-dimensional Coordination Polymer: Synthesis, Structure, Highly Efficient Fluorescence and DFT Study Fen-Fang Li Department of Chemistry and Chemical Engineering, Jinzhong University, Jinzhong, Shanxi 030600, China. * Corresponding author: E-mail: lffspring@126.com Received: 03-26-2022 Abstract A new two-dimensional coordinate polymer, {[Zn2(pbmpd)(H2O)4]·(H2O)}n (H4pbmpd = 1,1’-(1,4-phenylenebis(meth- ylene))bis-(1H-pyrazole-3,5-dicarboxylic acid)), has been hydrothermally synthesized and characterized by IR spec- trum, elemental analysis, TGA and X-ray single-crystal/powder diffraction. Structural analyses reveal that complex 1 ex- hibits a two-dimensional sheet structure in the crystal lattice. In complex 1, the carboxylic oxygen atoms and conjugated N atoms of pbmpd4– bridge zinc(II) ions form indefinitely zigzag shaped one-dimensional chains through π···π stacking interactions which are further connected by [ZnO6] units to form a novel two-dimensional structure. Finally, π···π stack- ing interactions and intermolecular hydrogen bonds assemble the two dimensional networks into a three-dimensional framework. Furthermore, the luminescent properties are also discussed. Interestingly, the solid state photoluminescence properties of the title polymer show the enhancement effect of spectrum. Density functional theory (DFT) calculations were used to support the experimental data. Keywords: Zn(II) complex; crystal structure; 1,1’-(1,4- phenylenebis(methylene)) bis-(1H-pyrazole-3,5-dicarboxylic ac- id); fluorescence property; DFT study 1. Introduction Luminescent metal–organic frameworks (LMOFs), as important functional crystalline materials, are gaining increasing attention in sensing applications during the past few years owing to their high sensitivity, short response time and their ability to be employed both in solution and the solid phase.1–3 Therefore, the synthesis of LMOFs is the basis of such work. LMOFs can be synthesized quick- ly and conveniently through self-assembly of π-conjugat- ed multidentate organic bridging ligands with d10 metal ions or/and lanthanide metal ions.4,5 Furthermore, such ligands also possess a better selective recognition ability, higher chemical and thermal stability and so on.6,7 Polycarboxylates based pyrazole as a kind of π-elec- tron rich ligand, have great benefits for the formation 2D or 3D metal–organic frameworks (MOFs), some of these, which contain Zn2+/Cd2+ ions and/or clusters, of- ten possessing good photoluminescence properties.8,9 Furthermore, rigid multicarboxylate ligands as bridging or building blocks play crucial roles in the construction of stable coordination frameworks, but their skeleton structures are limited. In contrast, flexible multicarbo- xylate ligands have remarkable advantages because their conformational freedom and flexibility can be fine-tuned by themselves to match with the coordination preference of metal ions and lower the energetic arrangement in the self-assembly process. The synthesis of coordination poly- mers based on flexible multicarboxylate is also influenced by the chemical and structural features of organic ligands, metal-to-ligand ratio, the coordination geometries of the metal, pH value, temperature, solvent and so on. Among these factors, the key factor is the selection of the organ- ic ligand, which determines the topology of the synthetic architecture through its excellent coordination capabilities and versatile bridging modes. Therefore, multifarious tet- racarboxylate ligands have been utilized to create desired coordination polymers with fascinating frameworks and properties, such as 5,5´-(1H-1,2,3-triazole-1,4-diyl)di- isophthalic acid,10 5,5´-(1,4-Phenylenebis(methylene) bis(oxy)di-isophthalic acid,11 1,1´-bis(3,5-dicarboxyben- zyl)-4,4´-bipyridinium dichloride,12 2,3,3´,4´-diphenyl ether tetracarboxylic acid.13 To date, the coordination 597Acta Chim. Slov. 2022, 69, 596–603 Li: A New Zn(II) Two-dimensional Coordination Polymer: ... polymers built from 1,1´-(1,4-phenyl enebis(methyl ene)) bis (1H-pyrazole-3,5-dicarboxylic acid) have rarely been explored.14, 15 Herein, we selected the ligand, 1,1´-(1,4-phenyl- enebis(methylene))bis (1H-pyrazole-3,5-dicarboxylic acid) (H4pbmpd) which the four carboxyl groups linked by two flexible ‘–CH2–’ groups and two free N atoms offer ample coordination capacities and the ability to adapt its conformation to geometrical requirements leading to pro- ducing interesting structures with amazing properties and successfully synthesized a new 2D MOF, {[Zn2(pbmpd) (H2O)4]·H2O}n. The synthesized samples were character- ized by X-ray single-crystal and powder diffractions, ther- mal gravimetric analysis and infrared spectra. In addition, we have also discussed the photoluminescence mechanism via density functional theory (DFT) calculations. 2. Experimental 2. 1. Materials and Measurements H4pbmpd was purchased from Jinan Henghua Sci- ence & Technology Co. Ltd, China (Fig.1). All solvents and other reagents were commercially available and were used without further purification. The IR spectrum for complex 1 was recorded in a KBr pellet in the range of 4000∼400 cm–1 on a Bruker TENSOR27 spectrometer. Element anal- ysis was carried out using a CHNO Rapid instrument. Powder X-ray diffraction (PXRD) data were collected on a Bruker D8 Advance with Cu Kα radiation (λ = 1.5418 Å). The thermogravimetric analysis (TGA) was carried out with a Dupont thermal analyser in the temperature range of 293∼1073 K under an N2 atmosphere with a heating rate of 10 K min–1. Fig. 1. 1,1´-(1,4- Phenylenebis(methylene))bis-(1H-pyrazole-3,5- dicarboxylic acid, H4pbmpd) 2. 2. Synthesis of {[Zn2(pbmpd) (H2O)4]·(H2O)}n (1) A mixture of H4pbmpd (33.2 mg, 0.05 mmol), Zn(NO3)2·4H2O (36.5 mg, 0.10 mmol) and a 12 mL of aceto nitrile / water (2 : 10, V/V) was placed in a 15 mL of Teflon-lined stainless steel autoclave. The mixture was heated under autogenous pressure at 433 K for 72 h and then cooled to room temperature. Colourless block crys- tals were collected by filtration, washed with H2O, and dried in air. (yield: 75%, based on H4pbmpd). Analysis calculated for C18H20Zn2N4O13: C 34.33, H 2.86, N 8.90%; found: C 34.38, H 2.81, N 8.93%. IR (KBr, ν, cm-1, s for strong, m medium, w weak): 3416 m, 1594 s, 1534 s, 1485 s, 1431 m, 1349 s. 1289 m, 1208 w, 1131 m, 1023 s, 854 m, 794 s, 555 m. 2. 3. Crystal Structure Determination Diffraction data were collected using a SuperNova (Cu) X-ray Source diffractometer utilizing Cu-Kα (λ = 1.5418 Å) radiation at 173 K. The structure was solved by direct methods employed in the program SHELXS-2014, and refined by full-matrix leastsquares methods against F2 with SHELXL-2016.16 The cell parameters were deter- mined by SMART software. Data reduction was performed with SAINT Plus. Program SADABS was used for absorp- tion corrections. All non-H atoms were refined anisotrop- ically, hydrogen atoms attached to C atoms were placed geometrically and refined using a riding model approxi- mation, with C‒H = 0.93 Å and Uiso(H) = 1.2Ueq(C). H at- oms bonded to O were located firstly in a difference Fouri- er map and were refined freely. Tentative free refinements of their positional coordinates resulted in an unsatisfacto- ry wide range of O‒H and H···H (in water) distances; bond lengths were therefore restrained to 0.82 (1) Å for O‒H. The H···H (in water) distances were restrained to 1.32 (1) Å. The O‒H distances of water molecules are in the range Table 1. Crystal data and structure refinement for Complex 1 1 Empirical formula C18H20Zn2N4O13 Formula weight 631.12 Temperature 173 K Crystal system monoclinic Space group P21/c a / Å a = 12.8594 (4) b / Å b = 14.7599 (4) c / Å c = 11.4301 (4) β / (°) β = 91.303(3) V / Å3 2168.91 (1) Z 4 Density (calculated) 1.933 mg/m3 Absorption coefficient 3.477 mm–1 F(000) 1280 Crystal size 0.20 × 0.20 × 0.10 mm3 θ range for data collection 2.7 to 26.5° Reflections collected 8113 Independent reflections 3951 [Rint = 0.029] Completeness to θ = 25.50° 96.7 % Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 3951/ 0/334 Goodness-of-fit on F2 1.040 Final R indices [I >2σ(I)] R1 = 0.0306, wR2 = 0.0819 R indices (all data) R1 = 0.0389, wR2 = 0.0769 598 Acta Chim. Slov. 2022, 69, 596–603 Li: A New Zn(II) Two-dimensional Coordination Polymer: ... 0.8199∼0.8205 Å. A summary of the crystallographic data, data collection and refinement parameters for complex 1 is provided in Table 1. Selected bond lengths, bond angles and H-bonds for complex 1 are listed in Tables 2 and 3, respectively. The molecular graphics were prepared using the SHELXL-2016 and MERCURY programs.17,18 2. 4. Hirshfeld Surface Analysis The Hirshfeld surface analysis19 and the related 2D-fingerprint plots20 were calculated using Crystal Ex- plorer.21 The CIF file of the structure 1 was imported into Crystal Explorer and high resolution Hirshfeld surfaces were mapped with the function dnorm. Then, the Hirshfeld surfaces were resolved into 2D-fingerprint plots, in order to quantitatively determine the nature and type of all in- termolecular contacts experienced by the molecules in the crystal. 3. Results and Discussion 3. 1. IR Characterization The peaks of FT-IR point out that the strong band around 1522 cm–1 resulting from stretching vibration of carboxyl (C=O) in the free ligand is disappeared and split- ted into two new bands at 1594 cm–1 and 1534 cm–1 in complex 1, which are assigned to symmetric and asym- metrical stretching vibrations of carboxyl (C=O). It sug- gests that the carboxyl of the ligand had been deprotonat- ed and coordinated to the Zn(II).22 Complex 1 also shows broad absorptions in the range between 3416 cm–1 asso- ciated with hydrogen-bonded O‒H stretching vibration.23 3. 2. Crystal Structure of Complex 1 The two-dimensional coordinate polymer 1 crys- tallizes in the P21/c space group of the monoclinic crystal system and shows a 2D structure. The asymmetric unit of 1 contains two independent Zn2+ ions, one deproto- nated pbmpd4– ligand, four coordinated water molecules Table 2. Selected Bond Lengths (Å) and Bond Angles (°) for Complex 1 1 Zn(1)–O(9) 1.9886 (1) Zn(1)–O(5) 2.0330 (1) Zn(1)–N(1) 2.124 (2) Zn(1)–N(3) 2.065 (2) Zn(2)–O(3) 2.119 (2) Zn(2)–O(4) 2.400 (2) Zn(2)–O(10) 2.0237 (1) Zn(2)–O(11) 2.028 (2) Zn(1)–O(1) 2.0185 (1) Zn(2)–O(7) 2.0815 (1) Zn(2)–O(12) 2.0888 (1) O(9)–Zn(1)–O(1) 88.35 (8) O(1)–Zn(1)–O(5) 168.77 (8) O(9)–Zn(1)–O(5) 88.63 (8) O(9)–Zn(1)–N(3) 111.19 (9) O(9)–Zn(1)–N(1) 145.25 (9) O(5)–Zn(1)–N(1) 96.65 (8) O(1)–Zn(1)–N(1) 79.85 (8) O(1)–Zn(1)–N(3) 109.27 (8) O(10)–Zn(2)–O(12) 93.91 (8) O(7)–Zn(2)–O(12) 175.82 (8) O(11)–Zn(2)–O(12) 82.05 (8) O(10)–Zn(2)–O(3) 141.17 (8) O(10)–Zn(2)–O(4) 84.48 (7) O(12)–Zn(2)–O(3) 93.05 (8) O(7)–Zn(2)–O(4) 98.53 (7) O(10)–Zn(2)–O(11) 104.55 (8) O(10)–Zn(2)–O(7) 86.70 (8) O(3)–Zn(2)–(4) 58.05 (8) O(5)–Zn(1)–N(3) 81.90 (8) N(3)–Zn(1)–N(1) 103.55 (8) O(11)–Zn(2)–O(3) 114.24 (8) O(7)–Zn(2)–O(3) 89.04 (8) O(11)–Zn(2)–O(4) 165.16 (8) O(12)–Zn(2)–O(4) 85.64 (8) O(11)–Zn(2)–O(7) 93.79 (8) Table 3. Hydrogen Bond Lengths (Å) and Bond Angles (°) for Com- plex 1 D–H···A d(D–H) d(H…A) d(D…A) ∠DHA O(13)H(13B)···O(8)v 0.82 2.04 2.848 (3) 167 O(13)–H(13A)···O(6) 0.82 1.91 2.704 (3) 162 C(13)–H(13)···O(9)vi 0.95 2.57 3.289 (3) 133 C(11)–H(11B)···O(13)vii 0.99 2.50 3.388 (3) 149 C(11)–H(11A)···O(8)v 0.99 2.32 2.974 (3) 122 C(4)–H(4B)···O(4) 0.99 2.33 2.981 (4) 123 C(2)–H(2)···O(4)i 0.95 2.30 3.246 (3) 171 O(12)–H(12B)···O(2)viii 0.82 1.96 2.746 (3) 160 O(12)–H(12A)···O(2)iii 0.82 1.91 2.715 (3) 167 O(11)–H(11D)···O(1)viii 0.82 1.94 2.735 (3) 162 O(11)–H(11C)···O(13)ix 0.82 1.90 2.690 (3) 163 O(10)–H(10B)···O(3)iii 0.82 2.15 2.949 (3) 167 O(10)–H(10A)···O(6)ix 0.82 1.85 2.654 (3) 169 O(9)–H(9B)···O(8)x 0.82 1.96 2.731 (3) 157 O(9)–H(9A)···O(7)xi 0.82 1.95 2.767 (3) 172 Cg1···Cg1vi 4.117 (1) Cg1···Cg2 3.582 (1) C(6)–H(6)···Cg3 0.95 2.95 3.665 (3) 130 C(4)–H(4A)···Cg3xi 0.99 2.86 3.479 (3) 124 Symmetry codes: (i) x, -y+1/2, z-1/2; (iii) x, -y+1/2, z+1/2; (v) x-1, -y+1/2, z+1/2; (vi) -x, -y+1, -z+1; (vii) -x, y-1/2, -z+3/2; (viii) -x+1, y-1/2, -z+1/2; (ix) -x+1, y-1/2, -z+3/2; (x) -x+1, y+1/2, -z+1/2; (xi) -x+1, -y+1, -z+1. 599Acta Chim. Slov. 2022, 69, 596–603 Li: A New Zn(II) Two-dimensional Coordination Polymer: ... and one solvent water molecule. As depicted in Fig. 2, the Zn(1) and Zn(2) centers exhibit different coordinat- ed environments. Zn(2) exhibits distorted hexa-coordi- nated environments with one monodentate oxygen atom from the pbmpd4– ligand (Zn(2)–O(7)), two chelating oxygen atoms originating from another pbmpd4– ligand (Zn(2)–O(3) and Zn(2)–O(4)) and three oxygen atoms (Zn(2)–O(10), Zn(2)–O(11) and Zn(2)–O(12)) derived from the coordinated water molecules. The Zn–O coor- dination distances range from 2.081 (18) to 2.398 (2) Å which are all within the normal ranges.24–26 Zn(1) is pen- ta-coordinated and displays a square pyramid ZnN2O3 ge- ometry which is coordinated by three oxygen atoms (O(9), O(5) and O(1)) and two nitrogen atoms (N(1) and N(3)). The O(9) atom is from a terminal coordinated water mole- cule and O(1) and O(5) oxygen atoms are from monoden- tate carboxylate groups of pyrazole-carboxylate units from two different ligands. Two monodentate pyrazole nitrogen atoms (N(1) and N(3)) are derived from the two ligands. The Zn–O coordination distances range from 1.935 (7) to 2.204 (16) Å and Zn–N coordination distances range from 2.075 (4) to 2.139 (4) Å, which are also observed in re- ported zinc compounds.27, 28 Atoms O(9), O(5), O(1) and N(1), which are nearly coplanar (the mean deviation from the common best plane is 0.147 Å), complete the square base plane, while the vertex is occupied by atom N(3) of which the distance to the square plane is 2.392 (2) Å. The selected bond lengths are listed in Table 2. In the structure of 1, the carboxylic oxygen atoms and conjugated N atoms bridge Zn(1) ions form an in- definitely zig-zag shaped 1D Zn(1) chains along the c axis through π···π stacking interactions between the ring Cg1 (N(3)/N(4)/C(12)-C(14)) and the ring Cg2 (C(5)-C(10)) with the centroid distance of 3.582 (1) Å and the Zn···Zn distance is 11.43 Å (Fig. 3). Meanwhile, the Zn(1) chains are further connected by [Zn2O6] units to form a novel 2D structure (Fig. 4). The π···π stacking interaction between the adjacent rings Cg1 (N(3), N(4), C(12), C(13), C(14)) and Cg1i with the centroid distance of 4.117 (1) Å, lead to a weak inter connection of these layers into a three-dimen- sional (3D) framework. This type of self-assembled dimers has been studied in Cd(II) complex with the similar ligand. 29 In addition, C(6)–H(6)···Cg3 (N(1)/N(2)/C(1)-C(3)) and C(4)–H(4A)···Cg3ii interactions (see Fig. 5), with dis- tances of 2.86 Å and 2.95 Å respectively, are also found to stabilize the 3D network. There are interesting strong O–H···O hydrogen bonds among the rich carboxyl molecules. Viewing along bc plane, inter-molecular hydrogen bonds among carbox- ylate from pyrazole-carboxylate and coordinated water molecules O atoms, named O(13)–H(13B)···O(8)i, O(12)– H(12B)···O(2)vii, O(13)–H(13A)···O(6), O(9)–H(9B)···O(8) viii, O(9)–H(9A)···O(7)ix, O(10)–H(10B)···O(3)vi, O(12)– H(12B)···O(2)v connect the two-dimensional coordina- tion network into a three-dimensional framework (Fig. 6). Inter atomic distances ranging from 0.2654 (3) to 2.949 (3) Å and angles within 157.4∼172.4° indicated strong hy- drogen bonds. Meanwhile the intra-molecular hydrogen bonds named O (13)–H (13A)···O(6) with the distance is 2.704 (3) Å is also been found to strengthen the network. The detailed hydrogen bonds are listed in Table 3. Fig.2. The atom labels and coordination environments of the Zn(II) ions in complex 1, with displacement ellipsoids drawn at the 30% probability level. Dashed lines represent hydrogen bonds. Fig. 3. The infinite one-dimensional zigzag shaped chain formed from Zn(2) atoms and pbmpd4– ligands through p···π stacking in- teractions (blue dashed lines) along the c axis. Fig. 4. The two-dimensional sheets extending in the ac plane formed by layers packed through with [ZnO6] and [ZnN2O3] units. 600 Acta Chim. Slov. 2022, 69, 596–603 Li: A New Zn(II) Two-dimensional Coordination Polymer: ... Fig. 5. π···π stacking interactions (open dashed lines) and C–H···π interactions (dashed lines) present in complex 1. Fig. 6. The three-dimensional network through π···π stacking inter- actions (purple lines) and inter-molecular hydrogen bonds (blue lines) viewing along ac plane. White balls represent the centroid of the rings. (Symmetry codes: (i) x - 1, -y + 1/2, z + 1/2;(v) -x + 1, y - 1/2, -z + 1/2; (vi) x, -y + 1/2, z + 1/2; (vii) -x + 1, y - 1/2, -z + 3/2; (viii) -x + 1, y + 1/2, -z + 1/2; (ix) -x + 1, -y + 1, -z + 1.) 3. 3. Hirshfeld Surface Analysis The intermolecular interactions in crystal structure 1 were quantified using Hirshfeld surface analysis and fin- gerprint plots (FP). The dominant intermolecular interac- tions are viewed as a bright red area on the dnorm surface. Fig. 7 illustrates samples of Hirshfeld surfaces for structure 1. In 1, we observe a high level of O···H interactions due to the hydrogen bonds between solvents molecules and the complex. The two-dimensional fingerprint plots for com- plex 1 are shown in Fig.8. The proportions of C–H···π, π···π and O–H···O interactions are 11.2%, 8.8% and 39.6% of the total Hirshfeld surfaces for complex 1. It appears that in this complex rich in aromatic rings, contact characteris- tics of π-stacking or C–H/π interactions are less important. Fig.7. Views of the Hirshfeld surfaces for 1 mapped with dnorm. Fig. 8. Fingerprint plots of com- plex 1: C–H···π, π···π and O–H···O, listing the percentages of contacts contributed to the to- tal Hirshfeld surface area of mole- cules. 3. 4. PXRD and TG Analyses To confirm that the phase of the bulk sample is pure and the crystal structure of complex 1 is truly represent- ative of the bulk material, a powder X-ray diffraction (PXRD) experiment was carried out on a Bruker D8 Ad- vance with Cu Kα radiation (λ = 1.5418 Å). The as-synthe- sized sample of 1 is characterized by powder X-ray diffrac- tion (PXRD). As shown in Fig. 9a, the PXRD patterns are almost consistent with the simulated spectrum, demon- strating the high phase purity of the compounds. As shown in Fig. 9(b), the TGA for complex 1 shows a weight loss of 12.1% (calculated 14.2%) between 293 K and 605 K, which is associated with the loss of one solvent 601Acta Chim. Slov. 2022, 69, 596–603 Li: A New Zn(II) Two-dimensional Coordination Polymer: ... water molecule and four coordinated water molecules. When the temperature is up to 646 K, the frameworks of complex 1 begin to break down gradually. 3. 5. Fluorescence Properties and DFT Calculation The luminescent properties of complex 1 and free ligand were examined in the solid state at room tempera- ture. As depicted in Fig. 10, the emission of free H4pbmpd is weak, whereas complex 1 reveals an obviously strong emission; and the maximum emission of 1 at ca. 448 nm has a blue shift compared with that of the free H4pbmpd ligand observed at λem = 495 nm (λex = 300 nm). In order to obtain a better insight into the nature of the photolu- minescence of complex 1, we investigated the structural, electronic and optical properties with S0 using DFT cal- culations at the B3LYP/6-31+G* level of the Gaussian 09 program and S1 using TD-DFT calculations at the B3LY- P/6-31+G* level of the Gaussian 09 program.30 The ge- ometry was taken from the crystal structure. According to Kasha’s rule, 31 the fluorescence of the compounds is only emitted from the lowest singlet excited states (S1) to the singlet ground state (S0). As depicted in Fig. 11, for complex 1 , in the S1 state, the HOMO is located more on the π orbitals of the pyrazole-carboxylate moiety of the ligand, and the LUMO is mainly located on the π* orbit- al of the pyrazole-phenyl moiety. Obviously, the LUMO and HOMO orbits of complex 1 are both distributed over the ligand, and their emission bands can thus be clearly assigned to the ILCTs. Meanwhile, the HOMO–LUMO energy gap of compound 1 is larger than that of the free ligand, which leads to the blue shift of the emission peak (47 nm) compared with the free H4pbmpd ligand, indi- cating that the HOMO–LUMO energy gap decreases to make the blue shift, as reported for other pyrazole derivat- ed polycarboxylate complexes.32 The enhanced emission intensity of complex 1 may arise from the aggregation in- duced emission, where the coordination of the ligand and the metal ions can reduce the freedom of the ligands and their non-radiative transitions. 33,34 Fig. 9. (a) Comparison of the simulated and experimental PXRD patterns of complex 1. (b) Thermogravimetric analysis (TGA) curve of complex 1 Fig. 10. Solid-state emission spectra of H4pbmpd (black) and com- plex 1 (red) at room temperature (inset: enlarged view of emission spectra of H4pbmpd). Fig. 11. The frontier MOs and the DFT calculations of H4pbmpd ligand and complex 1. 602 Acta Chim. Slov. 2022, 69, 596–603 Li: A New Zn(II) Two-dimensional Coordination Polymer: ... 4. Conclusion A new 2D Zn(II) compound based on 1,1´-(1,4-phe- nylenebis(methylene))bis- (1H-pyrazole-3,5-dicarboxyl- ic acid) has been constructed successfully. Complex 1 is consisted of two independent Zn2+ ions but have different coordinated environments, one of which exhibits distorted hexa-coordinated environments, while the other displays a square pyramid ZnN2O3 geometry. In complex 1, the in- definitely zig-zag shaped 1D chains formed by the ligand pbmpd4– bridged Zn ions and [Zn2O6] units to form a 2D structure. In addition, compared to the ligand, the solid state photoluminescence properties of complex 1 show an obviously strong emission, which is assigned to the ILCTs by Density functional theory (DFT) calculations. Supplementary Material CCDC 1923324 contains the supplementary crystal- lographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/conts/retrieving. html. Acknowledgments Authors appreciated the Startup Fund of Doctors of Jinzhong University. A portion of this work was per- formed on the Scientific Instrument Center of Shanxi Uni- versity of China. 6. References 1. M. Pan, W. M. Liao, S.Y. Yin, S.S. Sun, C.Y. Su, Chem Rev 2018, 118, 8889–8935; DOI:10.1021/acs.chemrev.8b00222 2. H. Y. Li, S. N. Zhao, S. Q. Zang, J. Li, Chem Soc Rev 2020, 49, 6364–6401; DOI:10.1039/C9CS00778D 3. X. Y. Liu, W. P. Lustig, J. Li, Chem. Rev 2020, 5, 2671–2680; DOI:10.1021/acsenergylett.0c01148 4. Y. W. Li, J. Li, X. Y. Wan, D. F. Sheng, H. Yan, S. S. Zhang, H. Y. Ma, S. N. Wang, D. C. Li, Z. Y. Gao, J. M. Dou, D. Sun, Inorg. Chem 2021, 60, 671–681; DOI:10.1021/acs.inorgchem.0c02629 5. W. B. Liu, N. N. Li, X. Zhang, Y. Zhao, Z. Zong, R. X. Wu, J. P. Tong, C. F. Bi, F. Shao, Y. H. Fan, Cryst. 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V strukturi spojine 1 karboksilatni kisikovi atomi in kojugirani N atomi iz pbmpd4- povezujejo preko p···π interakcij cinkove(II) ione v enodimenzionalne verige, ki so nadalje povezane preko [ZnO6] enot v dvodimenzionalno strukturo. Dvodimenzionalne strukture so preko p···π interakcij in intermolekularnih vodikovih vezi nadalje povezane v tridimenzionalno mrežo. Raziskali smo luminiscenčne lastnosti produkta, pri čemer je zanimivo, da fotoluminiscenca v trdnem stanju kaže povečanje učinka spektra. Za podporo eksperimentalnim podat- kom smo uporabili izračune DFT. 604 Acta Chim. Slov. 2022, 69, 604–618 Serbest et al.: Ternary Transition Metal Complexes with an ... DOI: 10.17344/acsi.2022.7511 Scientific paper Ternary Transition Metal Complexes with an Azo-Imine Ligand and 2,2’-Bipyridine: Characterization, Computational Calculations, and Acetylcholinesterase Inhibition Activities Kerim Serbest,1,* Turan Dural,1 Demet Kızıl,3 Mustafa Emirik,1 Ali Zengin2 and Barbaros Dinçer1 1 Department of Chemistry, Recep Tayyip Erdogan University, 53100 Rize, Turkey 2 Pazar Vocational School, Recep Tayyip Erdogan University, 53300 Pazar/Rize, Turkey 3 Central Research Laboratory, Bursa Technical University, 16310, Bursa, Turkey * Corresponding author: E-mail: kerimserbest@yahoo.com Tel.: +90 464 2234093 Received: 03-29-2022 Abstract New mononuclear ternary transition metal complexes: [M(HL)(bipy)2]ClO4, (M: Mn(II) for 1, Ni(II) for 2), [M(HL) (bipy) (ClO4)], (M: Ni(II) for 3, Cu(II) for 4, Zn(II) for 5) with M(II), 2-[(hydroxyimino)methyl]-4-[-phenyldiazenyl] phenol, H2L, and 2,2’-bipyridine were synthesized, and their structures were investigated by using various analytical, spectroscopic techniques such as elemental analysis, FTIR, UV-Vis, NMR, MALDI-TOF mass spectrometry, thermal analysis. The theoretical studies were performed by DFT techniques by using B3LYP function with 6-311++G (d, p)/ LanLD2Z basis set. The electronic transitions charters of the complexes were further analyzed by TD-DFT/CAM-B3LYP method. IR and thermal analysis data verify the proposed structures. The inhibition activities of the complexes against acetylcholinesterase (AChE) extracted from Ricania simulans adults and nymphs were examined and all the complexes were found to be active. Among the complexes studied, the highest inhibition activity was exhibited by complex 5 with the lowest IC50 value (3.2 ± 0.8 µM) for AChE of adults and complex 3 with the lowest IC50 value (4.6 ± 0.8 µM) for AChE of nymphs. Keywords: Metal complex; TDDFT; AChE inhibitor, R. simulans. 1. Introduction Coordination compounds with the azo-imine lig- ands have gained significant importance related to their applications in several high technology areas such as liquid crystalline displays (LCD), optical storage, laser and ink- jet printers as well as in leather, textile and plastic indus- tries.1–3 They have attracted the attention of researchers because of their biological activities such as anti-micro- bial, antitumor, anticancer, anti-fungicidal.4–9 Numerous azo compounds are used in pharmaceuticals and cosmet- ics although some of them have been reported to be toxic.1 Coordination compounds have also been investigated for the treatment of Alzheimer’s disease (AD), Parkinson’s dis- ease, aging, and those showing inhibitor activity of AChE promise in the use of therapeutic applications.10–12 Some Co(II), Cu(II), Ni(II), Zn(II) complexes were reported as acetylcholinesterase inhibitors (AChEIs) and they also have the potential of use agricultural struggle because they are associated with excitation, tremors, and death in in- sects.10,13 The agricultural areas located on the coastal part of the Eastern Black Sea Region have recently been exposed to the damage caused by a different type of insect known as Ricania simulans (Walker, 1851). It has been seen in this region for the past 9 years, despite the motherland which is known to be China.14,15 R. simulans is seen to be the type of pest where the population and the extended range of 605Acta Chim. Slov. 2022, 69, 604–618 Serbest et al.: Ternary Transition Metal Complexes with an ... which are on the increase with each passing day around this region.15,16 The Ricaniidae family, which belongs to the group of hemipteran insects, is represented by 40 stripes (types) and 400 species. Although the species of this family show general propagation in tropical regions, Rica- nia species can also be seen in Palearctic regions.15,17–19 It has a broad population in Japan, Southern China, Korea, Ukraine, Russia, and Georgia.15,20 Included in the quar- antine list due to the harm it caused in Korea where R. simulans was brought from Southern Asia to Russia in the 1900s and to Georgia in the 1950s and also to the Eastern Black Sea Region of Turkey in 2006 along with the young trees with eggs and with bush saplings.15 Nymphs and adults of this pest that feed on vegetables, bushes, and trees without making a distinction of hosts rather harmful by absorbing the juice sap in the plant stems, leaves, and fruit. Tea gardens, which are among the most important product places of the coastline of the Eastern Black Sea Region, are under the threat of this pest, as well.15 Acetylcholinesterase (AChE, EC3.1.1.7) plays an im- portant role in neurotransmission by hydrolyzing the neu- rotransmitter acetylcholine and is the target site of most insecticides. Vertebrates have both AChE and BuChE, whereas insects only have AChE.21,22. With the impor- tance of AChE in neurotransmission and insect resistance, much attention has been paid to AChE studies from both mammals and insects. Most of these studies use non-puri- fied AChE from homogenates of body parts or the whole body.23 There is no study on the inhibition of acetylcho- linesterase of R. Simulans with such complexes in the lit- erature. The density functional theory (DFT) is a useful tool for prediction of molecular structure, spectroscopic prop- erties and chemical reactivity of molecular systems. Ex- perimentally obtained spectroscopic results are supported by DFT-based theoretical calculations, which is a method frequently used recently. Since transition metal complexes exhibit a wide variety of excited states, it remains difficult to accurately define the energy of excited states with the Time Dependent Density Functional Theory (TD-DFT). Because transition metal complexes exhibit a wide variety of excited states, it remains difficult to accurately describe the energy of excited states with the Time Dependent Density Functional Theory (TD-DFT). Common global descriptors of chemical reactivity of biologically active de- rivatives can be discussed using DFT methods.24–26 The study presents the synthesis, characterization, DFT calculation for the assignment of experimental IR and UV–Vis spectra and acetylcholinesterase inhibition effects of mononuclear ternary transition metal complexes (1-5) derived from azo-imine ligand 2-[(hydroxyimino) methyl]-4-[-phenyldiazenyl]phenol, H2L and 2,2’-bipyri- dine (bipy) as co-ligand (Figure 1) on AChEs of adults and nymphs of Ricania simulans. Because cell extract provides the closest composition to the cell medium, R. simulans extracts were used in the inhibition studies. This study is expected to be a starting point and of great importance in the exploration of metal-based insecticide. 2. Results and Discussion 2. 1. NMR Spectra Zn(II) complex, 5 has no solubility in common or- ganic solvents, low solubility just in DMSO, and so the pro- ton NMR spectra were taken with difficulty (Figure 2). The proton NMR spectral data of the Zn(II) complex given in the experimental section was compared with the ligands, and the data clearly proved the formation of mixed ligand Zn(II) complex. Phenolic and oxime hydroxyl group pro- tons in the ligand were observed as a singlets at 10.98 and 11.60 ppm, respectively. The oxime hydroxyl proton signal was very broadened and shifted to 11.15 ppm, while the phenolic hydroxyl proton signal disappeared. The singlet of Figure 1: The proposed structures of the complexes (1-5) in the solid-state. 606 Acta Chim. Slov. 2022, 69, 604–618 Serbest et al.: Ternary Transition Metal Complexes with an ... imine proton was also slightly shifted to 8.44 ppm. The ab- sence of phenolic OH proton and the low-field shift of the imine proton signal shows that the coordination of primary azo-oxime type ligand to Zn(II) ion is through the nitrogen of imine and phenolic oxygen atoms. The integrated intensi- ties of the aromatic protons were also confirmed that Zn(II) complex contains a bipyridine molecule as co-ligand. 2. 2. Theoretical Calculations 2. 2. 1. Molecular Structures Geometry optimizations of the ligand and its corre- sponding complexes were performed using the Gaussian 09 program in the gas phase. The optimized geometry with numbering and some of the optimized bond lengths, over- lap populations, and bond orders around the metal center obtained from DFT calculations were given in Fig. 3–8. Complexes 1, 2, form six-coordinated octahedral while complexes 3, 4, and 5 have five-coordinated distort- ed square-pyramidal structures. Addison, Reedijk and coworkers have proposed a ge- ometry index for 5-coordinate transition metal complexes that can simply be calculated by taking the two largest an- gles around the metal center (Equation 1). The 5-coordinate index, τ5 = 1 is for a perfect trigonal bipyramidal structure and τ5 = 0 for a perfect square pyramidal structure.27 The 5-coordinate index values of complexes 3, 4 and 5 were found as 0.46, 0.34 and 0.17, respectively. These complexes Figure 3: Optimized molecular structure of complex 1 and selected bond length in Å (L), overlap population (OP) and bond order (BO). Figure 2: 1H NMR spectra of Zn(II) complex, 5 in DMSO-d6. 607Acta Chim. Slov. 2022, 69, 604–618 Serbest et al.: Ternary Transition Metal Complexes with an ... Figure 4: Optimized molecular structure of complex 2 and selected bond length in Å (L), overlap population (OP) and bond order (BO). Figure 5: Optimized molecular structure of the complex 3 and selected bond length in Å (L), overlap population (OP) and bond order (BO). Figure 6: Optimized molecular structure of the complex 4 and selected bond length in Å (L), overlap population (OP) and bond order (BO). Figure 7: Optimized molecular structure of the complex 5 and selected bond length in Å (L), overlap population (OP) and bond order (BO). 608 Acta Chim. Slov. 2022, 69, 604–618 Serbest et al.: Ternary Transition Metal Complexes with an ... have highly distorted coordination geometries intermediate between square-pyramidal and trigonalbipyramidal.28 τ5 = (β–α)/60 (1) Table 1. The two greatest valence angles around metal center for the complexes 3, 4, and 5. Complex 3 N48-Ni50-N25 177.27 τ5 = 0.46 O26-Ni50-O27 149.46 Complex 4 N28-Cu27-O26 171.13 τ5 = 0.34 N25-Cu27-N38 150.87 Complex 5 N27-Zn54-O26 164.13 τ5 = 0.17 O47-Zn54-N37 153.83 The frontier orbitals’ shape and the values of ener- gies and energy gap were shown in Table 2. The energies of FMOs are important in several pharmacological and chemical fields. The electron-donating ability of a mole- cule is related to EHOMO. The electron-accepting charac- ter of a molecule can be measured via ELUMO values. The greater the EHOMO is, the greater the electron donor capa- bility, and the smaller the ELUMO is the smaller the resist- ance to accept electrons. The conceptual density functional theory-based de- scriptors can be useful to estimate the biological proper- ties. The computed quantum chemical reactivity descrip- tors were illustrated in Table 3. The reactivity descriptors Table 2. The frontier orbitals, energies, and energy gap of complexes in eV. HOMO (eV) GAP LUMO (eV) including dipole moment, highest occupied molecular or- bital energy (EHOMO), lowest unoccupied molecular orbital energy (ELUMO), chemical potential (μ), electronegativity (χ), hardness (η), softness (S), ionization potential (I), electron affinity (A), Electro-donating power (ω–), elec- tro-accepting power (ω+), and net electrophilicity (Δω) were calculated using the following equations (2–10). (2) (3) (4) (5) (6) (7) (8) (9) (10) 609Acta Chim. Slov. 2022, 69, 604–618 Serbest et al.: Ternary Transition Metal Complexes with an ... HOMO (eV) GAP LUMO (eV) The chemical reactivity increases with increasing softness and according to the calculated softness values, complex 3 is more reactive than the other complexes. It is also expected that complex 2 has more activity due to bio- logical activity is related to increased hardness. The hard- ness value of complex 2 (5.57) is higher than that of other complexes and indicates that this complex is more stable. It is also known that stable molecules should have lower electrophilicity values. The net electrophilicity of complex 2 is lower than that of other complexes.29,30 610 Acta Chim. Slov. 2022, 69, 604–618 Serbest et al.: Ternary Transition Metal Complexes with an ... 2. 2. 2. Vibrational Assignments All the IR spectra of the compounds were obtained using the FT-ATR technique in the region of 4000–650 cm–1. The IR spectra of the mixed ligand complexes were compared with the starting ligands primary azo-oxime type ligand and bipyridine to determine the coordination sites that might be involved in chelation. All the vibrational signals of metal complexes (1-5) and primary ligand were calculated by using the DFT / B3LYP method to assign the experimental signals. Some selected vibrations and corre- sponding functional groups were summarized in Table 4. In the IR spectrum of the azo-oxime ligand, signals of oxime protons (C=N–OH), –N=N– and C–O stretch- ings were observed at 3403, 1393, and 1265 cm–1, respec- tively. The primary ligand has two OH groups which are oxime and phenolic OH. The primary ligand has two OH groups which are oxime and phenolic. But, the phenolic OH signal, which is expected to appear at a lower frequen- cy than oxime OH, could not be observed due to possi- ble intramolecular or intermolecular hydrogen bonding. In addition to the absence of phenolic OH stretching in the IR spectrum of the complexes, the broad bands ob- served at approximately 3440–3190 cm–1 were attributed to oxime OH stretchings. The intense C–O vibration of the primary ligand observed at 1265 cm–1 was shifted to the upper wave number in the complexes (1314–1287 cm–1) and the intensity was also decreased compared to the free ligand. The medium intensity imine (C=N) vibration at 1621 cm–1 in the spectrum of the azo-oxime ligand shift- ed to the upper/lower wavenumber (1603–1645 cm–1) and the intensity of this band increased/decreased usually in the complexes. These can be interpreted as the coordina- tion of the metal ion to the primary ligand via the phenolic oxygen and nitrogen of imine.31,32 Free 2,2’-bipyridine has a signal at 1577 cm–1 belong- ing to ν(C=N) imine group. That the signal is observed at 1544 cm–1 for 5 and shifted to upper frequencies in the other complexes in the range of 1595–1606 cm–1 shows that the secondary bidentate ligand is coordinated to the metal center through nitrogen atoms of imine. Several bands belonging to the C=C vibrations were observed in the range of 1575–1437 cm–1 in the complexes. In addition, the characteristic out-of-plane C–H bending observed in 761–764 cm–1 was attributed to the bipyridine unit. Briefly, the obtained spectral data of the complexes confirm the coordination of the primary ligand to the central metal ion via the imine and phenolic oxygen while 2,2’-bipyridine is coordinated through the nitrogen atoms. IR spectra present evidence of the metal-perchlorate bond in solid-state. The perchlorate anion has a tetrahe- dral geometry, its point group is Td, and it has four normal vibrational modes (ν1–ν4) of the nine vibrational degrees of freedom of perchlorate, of which only two modes, the asymmetrical stretching (ν3, 1110 cm–1) and asymmetri- cal bending (ν4, 626 cm–1) are IR active.1,33 But, the ATR technique does not allow us to see the lower frequencies from 650 cm–1. The diagnostic asymmetrical stretching band (ν3) of ionic perchlorate is very broad and strong which is occasionally split. The minor shift and weak split- ting of this band may be occurred because of the lattice effects as in 2.33 The Raman active symmetrical stretching band (ν1) is theoretically forbidden in IR and observed as a weak band at 925–940 cm–1. The diagnostic asymmet- rical stretching band (ν3) was observed at 1107 for 1 and 1082, 1071 cm–1 for 2 (See the supplementary file, Fig. S12, S13).34 The asymmetrical stretching band (ν3, 1110 cm–1) of the perchlorate group splits when a coordinate bond is formed between one of its oxygen atoms and central met- al ion, so the symmetry of the perchlorate is lowered to C3v. and number of vibrations increases. The bands are 1115,1085, 1071 for 3, 1158, 1113, 1071 for 4 and 1145, 1111, 1070 cm–1 for 5 (Fig. S14-16). The splittings confirm the monodentate coordination of the perchlorate ion in solid-state complexes (Table 3).35 Based on IR data of 3, perchlorate is coordinated monodentately. However, the conductivity data shows that complex 3, which is compati- ble with the 1:1 electrolyte type, is solvolized in DMF. 2. 2. 3. UV-Vis Spectra In order to evaluate experimental absorption bands, UV–Vis characteristics of the metal complexes were inter- preted using the TDDFT/CPCM method in the implicit solvent of DMSO. The observed and predicted electronic spectra and their characters were summarized in Table 5. The calculated electronic transitions and FMO transitions that contribute to the formation of these bands are depicted in Table S1–S5. Experimentally observed bands were char- acterized according to contributions of molecular orbital Table 3. The calculated quantum chemical descriptors (eV). Comp. HOMO LUMO μ χ η I A S ω ω- ω+ Δω 1 –6.68 –1.53 –4.10 4.10 5.15 6.68 1.53 0.10 1.64 5.65 1.54 7.19 2 –6.95 –1.38 –4.17 4.17 5.57 6.95 1.38 0.09 1.56 5.55 1.38 6.94 3 –5.91 –4.82 –5.36 5.36 1.09 5.91 4.82 0.46 13.21 29.17 23.80 52.97 4 –6.03 –4.32 –5.18 5.18 1.71 6.03 4.32 0.29 7.82 18.34 13.17 31.51 5 –7.06 –3.60 –5.33 5.33 3.46 7.06 3.60 0.14 4.11 11.10 5.77 16.88 611Acta Chim. Slov. 2022, 69, 604–618 Serbest et al.: Ternary Transition Metal Complexes with an ... transition and location of FMO on molecules. Since the magnetic susceptibility measurements provide informa- tion about the strength of the ligand field of the complexes and the number of unpaired electrons, these measurements were taken into account in the spin assignment of the com- plexes in the DFT calculations using spin-only formula. Table 4. Selected vibrational frequencies (observed and calculated, cm–1) of synthesized compounds. Comp. ν(C=N–OH) ν(C–H) ν(C=N) Bipy, δ(C–H) ν(C=C) ν(N=N) ν(C–O) ν(ClO4) H2L Exp. 3403 3150–3000 1621 _ 1569 1393 1265 _ Theo. 3291(Ph), 3666 3074–2982 1654* _ 1621–1590 1514 1289 _ 1 Exp. –b 3100–3050 1623, 1595* 762 1519,1468,1439 1397 1300 1107(ν3); 922(ν1) Theo. 3501 3113–3022 1611, 1593* 755 1545–1610 1422 1299 – 2 Exp. 3416a 3150–3000 1644,1602* 763 1575,1494,1472,1440 1409 1314 1082,1071(ν3); 945(ν1) Theo. 3503 3095–3002 1620, 1613* 761 1458–1567 1387 1283 – 3 Exp. 3408a 3100–3000 1644,1606* 763 1548,1476,1440 1409 1310 1115,1085,1071(ν1,ν4); 933(ν2) Theo. 3225 3126–3034 1632,1620* 762 1507–1620 1412,1420 1301 – 4 Exp. 3440a 3185–3050 1645,1602* 764 1541, 1476,1437 1406 1313 1158,1113, 1071(ν1,ν4); 924(ν2) Theo. 3505 3146–3040 1603,1629* 789 1508–1621 1420 1310 – 5 Exp. 3190a 3100–3000 1603, 1544 * 761 1477,1441 1405 1287 1145,1111, 1070(ν1,ν4); 920(ν2) Theo. 3499 3093–2999 1612, 1594* 761 1542–1610 1418 1326, 1339 – (a: broad, b: not observed, Exp.: observed experimentally, Theo.: Theoretically calculated, *: Bipy ν(C=N) Table 5. The electronic spectral data and calculated electronic transitions of complex 1-5 and their contributions. Comp. λexp. (nm) λexp. (nm) λtheo. Osc. Major contributions in solid in DMF (nm) Strength 1 237 291 251 0.07 H-2(A)→L+4(A) (18%), HOMO(A)→L+4(A) (19%) 265 0.33 H-8(A)→LUMO(A) (12%), HOMO(A)→L+5(A) (10%) 266 0.12 H-5(A)→L+2(A) (12%) 269 0.13 H-8(A)→LUMO(A) (14%), H-1(A)→L+4(A) (14%) 374 398 374 0.11 H-1(A)→L+2(A) (18%) 375 0.73 HOMO(A)→L+1(A) (38%) 406 0.13 H-1(A)→LUMO(A) (21%), HOMO(A)→L+3(A) (18%) 461 501 0.02 HOMO(B)→L+2(B) (23%) 570 0.02 HOMO(B)→LUMO(B) (28%), HOMO(B)→L+2(B) (24%) 2 246 290 212 0.15 H-1(B)→L+2(B) (12%) 218 0.19 H-7(B)→L+9(B) (32%) 238 0.22 H-2(B)→L+3(B) (13%), H-3(A)→L+3(A) (11%) 251 0.12 H-3(B)→LUMO(B) (17%) 264 0.59 H-4(B)→L+2(B) (18%), H-5(A)→L+2(A) (15%) 312 266 0.23 H-4(B)→L+2(B) (19%), H-8(A)→LUMO(A) (15%) 433 331 0.23 HOMO(A)→L+3(A) (40%), HOMO(B)→L+3(B) (41%) 383 377 1.05 HOMO(B)→LUMO(B) (35%), HOMO(A)→L+1(A) (24%) 436 0.03 H-8(B)→L+8(B) (10%) 447 0.02 H-9(B)→L+8(B) (27%) 612 Acta Chim. Slov. 2022, 69, 604–618 Serbest et al.: Ternary Transition Metal Complexes with an ... 3 245 362 329 0.03 H-21(A)→LUMO(A) (42%) 311 344 0.02 H-2(A)→L+3(A) (17%), H-4(B)→L+1(B) (15%) 379 366 0.02 H-16(B)→LUMO(B) (20%), H-1(B)→L+4(B) (13%) 368 0.07 HOMO(B)→L+7(B) (28%), H-16(B)→LUMO(B) (15%) 378 0.02 H-6(A)→L+1(A) (37%), HOMO(B)→L+6(B) (13%) 381 0.03 HOMO(B)→L+6(B) (18%), HOMO(A)→L+5(A) (13%) 429 418 0.37 HOMO(B)→L+4(B) (20%), HOMO(A)→L+2(A) (17%) 421 0.22 HOMO(B)→L+4(B) (15%) 480 0.16 HOMO(B)→L+2(B) (25%), HOMO(B)→L+4(B) (11%) 4 253 290 311 0.02 H-6(A)→L+5(A) (14%), H-2(A)→L+5(A) (14%) 325 0.01 H-5(A)→L+1(A) (17%) 372 343 0.02 H-2(A)→L+1(A) (21%) 346 0.01 H-1(A)→L+3(A) (18%), H-2(A)→L+1(A) (14%) 367 376 0.05 HOMO(A)→L+4(A) (63%), HOMO(A)→L+5(A) (10%) 380 0.16 HOMO(A)→L+4(A) (24%), HOMO(B)→L+6(B) (23%) 410 0.08 HOMO(A)→L+3(A) (73%) 416 0.60 HOMO(A)→L+2(A) (28%), HOMO(B)→L+3(B) (29%) 459 421 0.04 H-5(B)→L+1(B) (39%), H-4(B)→L+1(B) (15%) 462 0.05 H-3(B)→LUMO(B) (12%), HOMO(B)→L+2(B) (43%) 468 0.04 HOMO(A)→L+1(A) (23%), H-3(A)→LUMO(A) (13%) 478 0.01 HOMO(B)→L+2(B) (39%) 522 0.01 H-1(B)→L+1(B) (16%), HOMO(B)→L+1(B) (10%) 5 248 234 0.20 H-2→L+6 (24%) 288 236 0.17 H-2→L+6 (21%), HOMO→L+7 (12%) 250 0.09 H-3→L+3 (28%), H-10→LUMO (25%) 292 265 0.28 H-5→L+4 (43%), H-5→L+2 (11%) 267 0.10 H-5→L+4 (14%), H-11→LUMO (10%) 352 314 0.03 H-6→L+1 (75%), HOMO→L+6 (10%) 367 322 0.16 HOMO→L+6 (68%), H-6→L+1 (13%) 368 1.09 HOMO→L+3 (87%) 385 0.01 H-2→LUMO (71%) 413 0.02 H-18→LUMO (33%) The manganese(II) complex, 1 which has a distorted octahedral geometry shows three bands at 291, 398, and 461 nm in the electronic spectrum. Considering the os- cillator powers, the first dense band consists of π→π* and d→π* transitions, with a predominance of π→π* transi- tions. The second band observed at 398 nm is mainly com- 613Acta Chim. Slov. 2022, 69, 604–618 Serbest et al.: Ternary Transition Metal Complexes with an ... posed of d→n transitions, and attributed to metal-to-li- gand charge transfer (MLCT) transition. According to the theoretical calculations, the third absorbance observed at 461 nm is attributed to d→π* transitions. the bands ob- served experimentally at 461 nm originate mainly in the HOMO(B)→ LUMO(B)/L+2(B) transition and can be in- terpreted as intra-ligand charge transfer according to the orbital character. Magnetic moment values of Mn(II) com- plex (1) were measured as 5.80 BM on the Gouy balance. Magnetic moment values measured at room temperature are compatible with S = 5/2. The nickel(II) complex, 2 which has a distorted octa- hedral geometry shows two bands, at 290 and 433 nm. The first intense band is formed by the contribution of π→π*, n→π* and ligand-to-metal charge transfer (LMCT) transi- tions, with a predominance of π→π* transitions. Consider- ing the second broadband, the contribution of π→π* tran- sitions is dominant. LMCT and d-d transitions were also contributed to the formation of this band. The measured magnetic moments of the synthesized Ni(II) complex, 2 are 2.89 B.M. The distorted squarepyramid nickel(II) complex, 3 shows two absorbance bands at 362 and 429 nm. Con- sidering the oscillatory power of the observed transitions for both bands, the contribution of π→π* transitions is predominant, with the contribution of π→π*, n→π* and d→π*(MLCT) transitions. It can be said that the contri- bution of d→π* transitions is very small. The measured magnetic moments of the synthesized Ni(II) complex, 3 are 2.60 BM. Three bands were observed in the electronic spec- trum of the copper(II) complex, 4, which has a distorted squarepyramidal geometry, at 290, 367, and 459 nm. The first dense band is attributed to π→π* transitions, the sec- ond band is attributed to π→π* and n→π* transitions, and the third band is attributed to the weighted contribution of π→π*, n→π* and ligand-to-metal charge transfer tran- sitions (LMCTs). The magnetic moments of the Cu(II) complex were measured as 2.14 BM. This observed value is consistent with the spin value of the Cu(II) ion containing an unpaired electron. The zinc(II) complex, 5, which has a distorted squarepyramidal geometry, two bands were observed at 288 and 352 nm in the electronic spectrum of compound 5 and attributed to π→π* and n→π* transitions. The Zn(II) complex is diamagnetic because the zinc ion is in the d10 system. 2. 3. Thermal Stabilities In order to determine the metal/ligand ratio and to get information about their thermal stabilities of the ternary transition metal complexes (1-5) from ambient temperature to 1000 °C in the O2 atmosphere, their thermal decompo- sition processes were investigated by TG/DTG/DTA tech- niques. All the complexes studied are air-stable and have very high thermal stability from thermal data in Table 6. The TG curves of the complexes were given in Figure 8 (see Supplementary, Fig. S1-S5 for the DTAmax). The metal oxide residues in thermograms of the complexes are compatible with the proposed structures and their stoichiometry. From the TG curve of [Mn(HL)(bipy)2] ClO4, 1 has one step decomposition stage which was observed within the temperature range of 150–507 °C and the DTA curve shows three exothermic peaks at 173, 285 and 473 °C. All of the organic moiety was removed from the structure above 507 °C and the final residue was attributed to MnO, its percentage was 10.0% (calc.10.0%). In case of [Ni(HL)(bipy)2] ClO4, 2, TG curve has three exothermic decomposition steps within the range of 25–629 °C. A rapid first step decomposition was observed at 245–360 °C (with exothermic DTA peaks at 285 and 358 °C) assigned to removal HL and a bipyridine with a mass loss of 56.0% (calc. 55.9%). The exothermic second step at 360–458 °C (with exothermic DTA peaks at 410 °C) is as- signed to removal ClO4-O with a mass loss of 11.6% (calc. 11.7%). The second bipyridine molecule was removed at 459–630 °C (with exothermic DTA peaks at 505 °C) with a mass loss of 22.8% (calc. 22.8%) in the third step and fi- nal residue was assigned to NiO with a mass of 8.6% (calc. 9.51%). In case of [Ni(HL)(bipy)(ClO4)], 3, TG curve has three step decomposition stages within the range of 25– 700 °C. The endothermic dehydration of 0.5 mol adsorbed water with a mass loss of 1.6% (calc. 1.6%) was observed at 25–55 °C (DTAmax at 41.2 °C) in the first step. The exother- mic second step at 154–299 °C (DTAmax at 291 and 305 °C) is assigned to remove a bipyridine molecule with a mass loss of 29.5 (27.7). The primary ligand, HL, and ClO4-O was removed at 299–691 °C (DTAmax at 422 and 520 °C) with a mass loss of 57.6 (58.4) in the third step and final residue was assigned to NiO with a mass of 11.3% (calc. 13.5%). In case of [Cu(HL)(bipy) (ClO4)], 4, TG curve has three step decomposition stages within the range of 25– 647 °C. The endothermic dehydration of 0.6 mol adsorbed water with a mass loss of 1.9% (calc. 1.9%) was observed at 25–56 °C (DTAmax at 47 °C) in the first step. The exo- thermic second step at 180–342 °C (DTAmax at 231 °C) is assigned to remove a bipyridine molecule with a mass loss of 27.3 (27.3). The primary ligand, HL and ClO4-O was removed at 342–647 °C (DTAmax at 370, 383, and 448 °C) with a mass loss of 54.2 (56.8) in the third step, and the final residue was assigned to NiO with a mass of 16.6% (calc. 14.2%). From the TG curve of [Zn(HL)(bipy)]ClO4, 5 has one step decomposition stage which was observed within the temperature range of 131–599 °C and the DTA curve shows four exothermic peaks at 187, 301, 368, 456 °C. All of the organic moiety was removed from the structure above 599 °C and the final residue was assigned to ZnO, its percentage was 15.70% (calc.14.5%). 614 Acta Chim. Slov. 2022, 69, 604–618 Serbest et al.: Ternary Transition Metal Complexes with an ... 2. 4. AChE Inhibition Studies The synthesized complexes of Mn(II), Ni(II), Cu(II), and Zn(II) with mixed ligands exhibited inhi- bition activity against the AChEs of adults and nymphs of R. simulans (Table 7). On AChE of adults of R. sim- ulans, the [Zn(HL)(bipy)(OClO3)], 5 (IC50 = 3.2 ± 0.8 µM) showed an inhibition effect close to edrophonium chloride (IC50 = 2.4 ± 0.3 µM) known as a competitive inhibitor of AChE. The [Mn(HL)(bipy)2]ClO4, 1 showed inhibition close to tacrine as an inhibitor of AChE, while the other complexes showed better inhibition than ta- crine. Although the inhibitory effect of these complexes on the AChE of nymphs was not as much as edrophonium and tacrine, their effect on the AChE was quite high. Especial- ly these were [Ni(HL)(bipy)(OClO3)] (3) (IC50=4.6±0.8 µM), [Mn(HL)(bipy)2](ClO4) (1) (IC50 = 5.6 ± 1.2 µM) and [Cu(HL)(bipy)(OClO3)] (4) (IC50 = 6.4 ± 0.7 µM) complexes, and their inhibition concentrations were close to each other. It was observed that while [Zn(HL)(bipy) (OClO3)] (5) (IC50 = 3.2 ± 0.8 µM) was more effective on adults, it had less effect on nymphs (IC50 =10.1 ± 2.4 µM). The inhibition effect of the [Ni(HL)(bipy)2](ClO4) (2) complex on both stages of R. simulans was observed to be the same at higher concentrations than the others. As a result of inhibition studies, it was determined that the [Zn(HL)(bipy)(OClO3)] complex was more effective Table 6. Thermal analysis data of the complexes. Comp. Decom. Decom. Temp., DTAmax, Group lost, Residue formula, Step(s) °C µV mass loss %, exp. (calc.) Residue %, exp. (calc.) 1 1 150–507 173(+), 285(+) 473(+) HL+2 bipy+ ClO4-O MnO, 90.0 (90.0) 10.0 (10.0) 1 245–360 285(+), 358(+) HL+bipy [Ni(bipy)] ClO4, 56.0 (55.9) 43.1 (44.0) 2 2 360–458 410(+) ClO4-O [Ni(bipy)]O, 11.6 (11.7) 31.5 (32.6) 3 459–630 505(+) Bipy NiO, 22.8 (22.8) 8.6 (9.51) 1 25–55 41.2(–) 0.5H2O, [Ni(HL)(bipy)(ClO4)], 1.6, (1.6) 98.4 (98.4) 3 2 154–299 291(+), 305(+) Bipy [Ni(bipy)(ClO4)], 29.5 (27.7) 68.9 (98.4) 3 299–691 422(+), 520(+) HL+ ClO4-O NiO, 57.6 (58.4) 11.3 (13,5) 1 25–56 47(–) 0.6H2O, [Ni(HL)bipy] ClO4, 1.9, (1.9) 98.1 (98.1) 4 2 180–342 231(+) Bipy [Cu(HL)] ClO4, 27.3 (27.3) 70.8 (70.8) 3 342–647 370(+), 383(+), HL+ ClO4-O CuO, 448(+) 54.2 (56.8) 16.6 (14.2) 5 1 131–599 187(+), 301(+), HL+ bipy+ ClO4-O ZnO, 368(+), 456(+) 84.3 (85.5) 15.7 (14.5) Figure 8: TG curves of the complexes (1-5). in the inhibition of AChE of the adults, and the [Ni(HL) (bipy)(OClO3)] complex was more effective in the inhibi- tion of AChE of the nymphs. Although the molecular mechanism is not fully known, it is suggested that metal complexes inhibit ace- tylcholinesterase by binding to both catalytic active site (CAS) and peripheral anionic site (PAS) in the active site of the enzyme.36 Considering the structures of the com- plexes, complex 1, 2 may bind CAS site while complex 3-5 may bind PAS site of the enzyme. From the results it can be speculated that metal complexes with low coordination are 615Acta Chim. Slov. 2022, 69, 604–618 Serbest et al.: Ternary Transition Metal Complexes with an ... more likely to bind stronger than the others to the active site of the enzyme and a higher inhibitory effect may be expected.37,38 While nymphs continue their lives in and around their hosts, they can spread to a wider area after they be- come adults. This implies that the sensory equipment, as well as the nervous system, must modify to accommodate the sensory requirements (such as host recognition, mate location, oviposition, aggregation, and defense) to the differences between nymphal and adult habitats. There- fore, adults are likely to have a more improved nervous system than nymphs.39 In another literature, the differ- ences in the activities of ACHEs obtained from different stages of Bactrocera dorsalis (H.) were appraised from two perspectives. It was papered that one of them, some alterations to the protein structure occurred during the developmental stages to meet the continuously changing demands of the development of insects, the another fac- tor, the differences in the expression level of genes en- coding specific enzymes might be resulting in changes.40 Because of such differences, it is normal for the ACHEs from nymphs and adults of R. simulans to behave differ- ently towards the inhibitors. The inhibition effect of some copper complexes syn- thesized on acetylcholinesterase from Electrophorus elec- tricus (eeAChE) was investigated and IC50 values were de- termined between 5.45 ± 0.70 and 64.67 ± 2.20 µM.41 The effects of [Cu(naringin)2], [Cu(naringenin)2], Cu(hesperetin)2, Cu(naringin)(2,2'-bipyridine), Cu(nar- ingin)(phenanthroline), Cu(naringenin)(2,2'-bipyridine), Cu(hesperidin)(phenanthroline), Cu(hesperetin)(2,2'-bi- pyridine) and Cu(hesperetin) (phenanthroline) complex- es on the activities of acetylcholinesterases obtained from human serum and electric eel were investigated. In this re- ported study, it was determined that the synthesized com- plexes have different effects on AChEs in different organ- isms. Also in this reported study, IC50 values for huAChE were 1.73 ± 0.3, 0.66 ± 0.2, 0.33 ± 0.02, 0.012 ± 0.002, 0.87 ± 0.1, 0.25 ± 0.03, 0.32 ± 0.05, 0.33 ± 0.05, 0.36 ± 0.07 µM and IC50 values for for eeAChE were 0.16 ± 0.03, 1.41 ± 0.4, 2.55 ± 0.5, 0.17 ± 0.02, 1.4 ± 0.3, 0.46 ± 0.2, 1.77 ± 0.4, 0.94 ± 0.09, 0.33 ± 0.06 µM.13 Table 7. IC50 values of acetylcholinesterase of R. simulans in the presence of the complexes (1-5). Complex IC50 (µM) IC50 (µM) For adults For nymphs [Mn(HL)(bipy)2] (ClO4), 1 22.0 ± 1.8 5.6 ± 1.2 [Ni(HL)( bipy)2] (ClO4), 2 14.0 ± 1.2 16.5 ± 1.9 [Ni(HL)(bipy)(OClO3)], 3 16.0 ± 2.1 4.6 ± 0.8 [Cu(HL)( bipy) (OClO3)], 4 7.2 ± 1.4 6.4 ± 0.7 [Zn(HL)( bipy) (OClO3)], 5 3.2 ± 0.8 10.1 ± 2.4 Edrophonium cloride 2.4 ± 0.3 0.6 ± 0.09 Tacrine 18.0 ± 1.9 1.2 ± 0.4 3. Conclusions The agricultural areas located on the coastal part of the Eastern Black Sea Region have recently been exposed to the damage caused by a different type of insect known as Ricania simulans. The acetylcholine esterase is the target site of most insecticides. So, mixed ligand metal complex- es with azo-oxime ligand and 2,2’-bipyridine as co-ligand were prepared, theoretical calculations were performed to provide information about molecular geometry, electronic structure, molecular and spectroscopic properties. Addi- tionally, their inhibitory activities against the AChEs of adults and nymphs of R. simulans in this study was report- ed. All the complexes were found to have inhibitor activi- ties. Interestingly, complexes 4 and 5 showed better inhib- itor activity than the other complexes tested and the most active of the complex has found to be complex, 5 with IC50 value of 2.4±0.3 µM for adults, and complex 3 with the lowest IC50 value (4.6±0.8 µM) exhibited the most inhi- bition activity for nymphs. Here, it has been shown that these complexes may be used as potential metal based in- secticides against R. simulans which has posed a big chal- lenge to the field of agriculture. 4. Experimental 4. 1. Materials and Methods 2,2’-Bipyridine (Bipy) and perchlorate salts of Mn(II), Ni(II), Cu(II), and Zn(II) were purchased from Merck. All chemical and solvents were analytical grade and used without any purification. 2-[(E)-(hydroxyimino) methyl]-4-[(E)-phenyldiazenyl]phenol, H2L was prepared according to the reported literature.9 4. 2. Preparation of Crude Extract About 5 g of adults or nymphs of R. simulans col- lected in July were homogenized in 20 mL phosphate buff- er (pH 7.4, 0.05 M, containing 1 mM EDTA, 0.5% Triton X-100, 0.5 M NaCl) in the ice bath. The homogenates were centrifuged at 22,000xg for 30 min at 4 °C. After the super- natant was filtered via syringe filter units (pore size 0.45 μm). The supernatant was used as a crude extract.42 4. 3. Measurements Elemental analyses were measured with a LECO truspect analyzer and 1H NMR spectra were measured with an Agilent Technologies 400/54 spectrometer at the Central Research Laboratory of Recep Tayyip Erdogan (RTE) University. MALDI-TOF mass spectra in a DHB matrix were recorded on a Bruker Microflex LT at the Gebze Institute of Technology for the complexes. IR spec- tra were recorded on a Perkin Elmer Spectrum 100 FT IR infrared spectrophotometer equipped with an ATR appa- 616 Acta Chim. Slov. 2022, 69, 604–618 Serbest et al.: Ternary Transition Metal Complexes with an ... ratus. KBr is taransparent in UV-Vis region, so solid-state electronic spectra for complexes were recorded on a Spec- traMax M5 spectrophotometer in KBr discs. Magnetic susceptibility and thermogravimetric data were collected by using Sherwood MK-1 and SII 6300 TG/DTA, respec- tively. Molar conductivity measurements of the complexes were measured with a Hanna EC 215 conductivity meter by using 0.01 M KCl water solution as a calibrant. 4. 4. Enzyme assay The activity of AChE was determined by the spec- trophotometric method using acetylthiocholine iodide (ATC) as the substrate.41 The reaction mixture, in a final volume of 3 ml contained 60 µL of 75.0 mM ATC, 60 µL of 10.0 mM 5,5’-dithiobis(2-nitrobenzoic acid), 2.780 mL of 0.1 M potassium phosphate buffer, pH 8.0 and 100 µL of enzyme solution. The enzymatic reaction was initiat- ed by adding the enzyme solution to the sample cuvette. Absorbance was read after 10 min incubation at 37 °C at 412 nm. Each activity value was taken from an average of 3–5 measurements. The inhibition effect was investigat- ed by adding different volumes of 1 mM stock inhibitor solutions to the reaction mixtures which is here the buffer volume was reduced by the volume of inhibitor solution added. One unit of AChE (EU) was defined as the amount which catalyzed the hydrolysis of 1.0 µM of ATC per min- ute at room temperature, which was calculated based on an extinction coefficient of 13.6 mM−1 cm−1.42,43 The inhibition studies were performed with tacrine and edrophonium chloride, known specific inhibitors of acetylcholinesterase, and the synthesized complexes at pH 8.0 in the presence of ATC substrate. The inhibitory concentration (IC50), which inhibits the AChE activity by 50%, was determined by using the % inhibition graph drawn against the inhibitory concentration. Inhibitors and their concentration ranges were as follows: tacrine 0.1–20.0 μM, edrophonium chloride 0.1–4.0 μM, and the complexes 2.0–30.0 μM. Each inhibitor was used in at least six concentrations.44 4. 5. Theoretical Methodology Quantum chemical calculations were performed to provide information about molecular geometry, electron- ic structure, molecular and spectroscopic properties. Fully optimized structural parameters of the ligands (H2L and bipy) and their metal (Mn(II), Ni(II), Cu(II), Zn(II)) com- plexes were calculated at the DFT level (B3LYP) with basis sets 6–311++ G(d,p) for nonmetal atoms, LANL2DZ and the effective core potential (ECP) for metal atoms. Since the CAM-B3LYP method gives a better definition of the excited state transitions compared to the B3LYP function, the elec- tronic excitations were calculated using TD-CAM-B3LYP methods and 6-311++G(2d,2p) basis set for nonmetal at- oms and LANL2DZ with the effective core potential (ECP) for metal atoms combined with a conductor-like polariza- ble continuum model (CPCM) in the implicit solvent of DMSO.45 All calculations were done using the Gaussian 09 platform.46 The optimized geometries and frontier molecu- lar orbital (FMO) densities were visualized using the Gauss View 5 software. GAUSSSUM 3.047 to interpret the UV-Vis bands and analyze fractional contributions, and the VED- A4X48 for analysis of elementary vibration modes were used. 4. 6. Synthesis of the Complexes- General Procedure Firstly, ethanolic solution of NaOH was added to the solution of the ligand, H2L (1 mmol) in 15 mL ethyl alco- hol to neutralize. To this solution was added the solution of metal(II) perchlorate (Mn(ClO4)2 · 6H2O, Ni(ClO4)2 · 6H2O, Cu(ClO4)2 · 6H2O or Zn(ClO4)2 · 6H2O) (1 mmol) in 10 mL ethyl alcohol. 2,2’-bipyridine (2.0 mmol for 1 and 2; 1.0 mmol for 3-5) in 10 mL ethyl alcohol was added to the solution, after the solution was stirred for 30 min at room temperature. The mixture was stirred for six hours at room temperature and then allowed to stand for two days at room temperature. solid precipitate was filtered and washed with water and ethyl alcohol, respectively. Final- ly, the resulting solid powders were filtered, recrystallized from a hot DMSO-H2O mixture and dried in vacuo over CaCl2. Single crystals couldn’t be obtained for X-ray dif- fraction studies though our great efforts. [Mn(HL)(bipy)2](ClO4), (1) Yield 0.25 g (35%). mp 290–296 °C (dec.). Color: Brownish khaki. FT-IR (cm–1): 3100–3050 ν(C-H); 1623, 1595 ν(C=N); 1519, 1468, 1439 ν(-C=C-); 1397 ν(N=N); 1300 ν(C-O); 1141, 1107 ν3(ClO4)–; 922 ν1(ClO4)–; 762 (bipy). UV-Vis. λmax, nm (ε, M–1 cm–1) in DMF: 291 (13880); 398 (8900); 461 (7300). Molar conductivity (Ω−1 cm2 mol−1) 60. μeff B.M. (298 K): 5.80. MALDI-TOF MS (m/z): Calc. for C33H26ClN7O6Mn: 707.0; Found: 607.9 [M-ClO4]+. Anal. Calc.: C, 56.06; H, 3.71; N, 13.87. Found: C, 56.27; H, 3.62; N, 13.81. [Ni(HL)(bipy)2](ClO4), (2) Yield 0.39 g (55%). mp 252–259 °C (dec.). Color: Greenish yellow. FT-IR (cm–1): 3416 ν(OH); 1644, 1602 ν(C=N); 1575, 1495,1472, 1440 ν(-C=C-); 1409 ν(N=N); 1314 ν(C-O); 1082, 1071 ν3(ClO4)–; 945 ν1(ClO4)–; 763 (bipy). UV-Vis. λmax, nm (ε, M–1 cm–1) in DMF: 290 (16420); 433 (5740). Molar conductivity (Ω−1 cm2 mol−1) 61. μeff B.M. (298 K): 2.60. MALDI-TOF MS (m/z): Calc. for C33H26ClN7O6Ni: 710.7; Found: 592.7 [M-(ClO4+H2O)]+. Anal. Calc.: C, 55.77; H, 3.69; N, 13.79. Found: C, 55.54; H, 3.52; N, 13.88. [Ni(HL)(bipy)(ClO4)], (3) Yield 0.24 g (43%). mp 248–257 °C (dec.). Color: Khaki. FT-IR (cm–1): 3408 ν(OH); 1644, 1606 ν(C=N); 617Acta Chim. Slov. 2022, 69, 604–618 Serbest et al.: Ternary Transition Metal Complexes with an ... 1548, 1476, 1440 ν(-C=C-); 1409 ν(N=N); 1310 ν(C-O); 1157, 1115, 1085, 1071 ν1,ν4(OClO3)–; 933 ν2(OClO3)–; 763 (bipy). UV-Vis. λmax, nm (ε, M–1 cm–1) in DMF: 362 (15080); 429 (22350). Molar conductivity (Ω−1 cm2 mol−1) 82. μeff B.M. (298 K): 1.81. MALDI-TOF MS (m/z): Calc. for C23H18ClN5O6Ni: 554.6; Found: 453.6 [M-ClO4]+. Anal. Calc.: C, 49.81: H, 3.27; N, 12.63. Found: C, 50.02; H, 3.33; N, 12.47. [Cu(HL)(bipy)(ClO4)], (4) Yield 0.26 g (46%). mp 209–215 °C (dec.). Color: Brown. FT-IR (cm–1): 3440 ν(OH); 1645, 1602 ν(C=N); 1541, 1476, 1437 ν(-C=C-); 1406 ν(N=N); 1313 ν(C-O); 1158, 1113, 1071 ν1,ν4(OClO3)–; 924 ν2(OClO3)–; 764 (bipy). UV-Vis. λmax, nm (ε, M–1 cm–1) in DMF: 290 (19760); 367 (39130); 459 (32540). Molar conductivity (Ω−1 cm2 mol−1) 5. μeff B.M. (298 K): 2.1. MALDI-TOF MS (m/z): Calc. for C23H18ClN5O6Cu: 559.4; Found: 459.7 [M-ClO4]+. Anal. Calc.: C, 49.38; H, 3.24; N, 12.52. Found: C, 49.63.74; H, 3.39 N, 12.43. [Zn(HL)(bipy)(ClO4)], (5) Yield 0.16 g (29%). mp 281–289 °C (dec.). Color: Light orange. FT-IR (cm–1): 3190 ν(OH); 1603, 1544 ν(C=N); 1477, 1441 ν(-C=C-); 1405 ν(N=N); 1287 ν(C-O); 1145, 1111, 1070 ν1,ν4(OClO3)–; 920 ν2(OClO3)–; 763 (bipy). UV-Vis. λmax, nm (ε, M–1 cm–1) in DMF: 288 (22000); 352 (36270). 1H NMR δ (400 MHz, DMSO-d6): 11.15 bs. (1H, OH), 8.68 s. (1H, HC=N), 8.44 s. (2H, Ar), 8.07 s. (1H, Ar), 7.95 s. (2H, Ar), 7.77 d. (4H, Ar, J=8.0 Hz), 7.52 dd. (4H, Ar, J=8.0 Hz), 7.44 t. (2H, Ar, J=8.0 Hz), 6.87 s. (1H, Ar). Molar conductivity (Ω−1 cm2 mol−1) 4. μeff B.M. (298 K): dia. MALDI-TOF MS (m/z): Calc. for C23H18 ClN5O6Zn: 561.3; Found: 461.3 [M-ClO4]+. Anal. Calc.: C, 49.22; H, 3.23; N, 12.48. Found C, 49.08; H, 3.32; N, 12.44. Declaration of Competing Interest The authors declare that they have no known compet- ing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments Part of this work was financially supported by the Re- search Fund of Recep Tayyip Erdogan University (Project ID:1184). The numerical calculations reported in this pa- per were performed at TUBITAK ULAKBIM, High Perfor- mance, and Grid Computing Center (TRUBA Resources). 5. References 1. K. Serbest, T. Dural, M. Emirik, A. Zengin, Ö. Faiz, J. Mol. Struct. 2020, 1229, 129579. 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Strukture dobljenih spojin smo preučili z različnimi analitskimi in spek- troskopskimi metodami, kot so elementna analiza, FTIR, UV-Vis, NMR, MALDI-TOF masna spektrometrija in ter- mična analiza. Teoretske raziskave smo opravili z DFT metodo in uporabo B3LYP funkcije z naborom osnov 6-311++G (d, p)/LanLD2Z. Elektronske prehode v kompleksih smo nadalje karakterizirali z metodo TD-DFT/CAM-B3LYP. IR meritve in termična analiza potrjujejo predpostavljene strukture. Inhibicijsko delovanje kompleksov smo dokazali s pre- iskavo učinkov na acetilholinesterazo (AChE) ekstrahirano iz odraslih primerkov in ličink Ricania simulans. Med preisk- ovanimi kompleksi ima največjo aktivnost spojina 5 z najnižjo vrednostjo IC50 (3.2±0.8 µM) za AChE odraslih osebkov in spojina 3 z najnižjo vrednostjo IC50 (4.6±0.8 µM) za AChE ličink. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 619Acta Chim. Slov. 2022, 69, 619–628 Zahmatkesh et al.: Synthesis, Antimicrobial and Molecular Docking Studies of ... DOI: 10.17344/acsi.2022.7512 Scientific paper Synthesis, Antimicrobial and Molecular Docking Studies of Some New Derivatives of 2,3-Dihydroquinazolin-4(1H)-one Karim Zahmatkesh,1 Karim Akbari Dilmaghani1,* and Yasin Sarveahrabi2 1 Department of Organic Chemistry, Faculty of Chemistry, Urmia University, Urmia, 57159, Iran. 2 Department of Biology, Central Tehran Branch, Islamic Azad University, Tehran, Iran. * Corresponding author: E-mail: k.adilmaghani@urmia.ac.ir Tel: (+98)914-443-1392; Fax: (+98)44-357153-165 Received: 03-29-2022 Abstract In the present study a series of novel 2-(substituted phenyl)-3-(5-phenyl-1,3,4-thiadiazol-2-yl)-2,3-dihydroquinazolin- 4(1H)-one derivatives were synthesized by refluxing isatoic anhydride, 5-phenyl-1,3,4-thiadiazol-2-amine and aromatic aldehydes in the presence of p-TsOH as the catalyst and in H2O as the solvent and characterized by spectroscopic data and analytical methods. Antibacterial and antifungal activity of the title compounds were evaluated against two Gram positive and two Gram negative bacterial strains and strains of fungi and compared with ·standard drugs, using well diffusion method minimum bactericidal/fungicidal concentration were determined. The potential α-amylase and α-glucosidase inhibitory activity of compounds 4a–l were investigated in silico using molecular docking simulation method. Therefore, these 2,3-dihydroquinazolin-4(1H)-one derivatives may be considered as promising candidates for the development of new classes of antimicrobial and antidiabetic drugs. Keywords: Isatoic anhydride, 2,3-dihydroquinazolin-4(1H)-one, antibacterial activity, antifungal activity, anti-diabetic activity. 1. Introduction The 2,3-dihydroquinazolin-4(1H)-one (DHQ) is an important nitrogen-containing heterocyclic scaffold. DHQ ring system is a distinguished scaffold in drug de- sign. DHQ in medicinal chemistry acting as an important pharmacophore has drawn much attention due to its broad spectrum of pharmaceutical activities, which include anti- bacterial,1,2 antifungal,3–5 anticancer,6–8 antidiabetic,9 an- ti-tuberculin,10 anti-inflammatory,11–13 cholinesterase in- hibitory,14 antihypertensive activities15,16 and insecticidal activity17. On the other hand, 2-amino-1,3,4-thiadiazole and its derivatives have drawn attention of many organic chemists during recent years, since many of these compounds are known to possess interesting biological properties such as antibacterial,18,19 antifungal,20–22 anticancer,23–25 an- tihypertensive26,27 activities. Considering the reactivity of the amine group in the derivatization process, 2-ami- no-1,3,4-thiadiazole moiety is a good scaffold for drug synthesis. Most of the DHQ derivatives are substituted on the carbons 2 and 3. Due to their attractive properties, 2-substituted DHQs are becoming prominent synthetic intermediates for organic chemists and pharmacologist. Based on the above observations we report here the synthesis of a new series of 2,3-dihydroquinazo- lin-4(1H)-one derivatives 4a–l with structure modifica- tions involving incorporation of 5-phenyl-1,3,4-thiadi- azol-2-amine (3) at position 3 and aromatic aldehydes 2a–l at position 2 of DHQ ring system. In the present study, various aryl aldehyde groups were specifically in- corporated at position 2 of the DHQ scaffold with the aim of new antibacterial, antifungal and anti diabetic drugs. 2. Experimental Starting materials, solvents, and culture environ- ments (nutrient agar/broth, Sabouraud dextrose and agar/ broth) were obtained from Merck, Germany and used without any additional filtration. Microbiological tests were performed using a Memmert INC153T2T3 incu- bator. Melting points were determined in Philip Harris 620 Acta Chim. Slov. 2022, 69, 619–628 Zahmatkesh et al.: Synthesis, Antimicrobial and Molecular Docking Studies of ... C4954718 melting point apparatus and are uncorrected. IR spectra were recorded on a Thermo Nicolet Nexus-670 FTIR spectrophotometer, using potassium bromide pellets and the frequencies are expressed in cm–1. The 1H and 13C NMR spectra were recorded with a Bruker Avance 400 MHz spectrometer, using TMS as the internal reference, with DMSO-d6 as the solvent. Chemical shifts are reported in parts per million (ppm). Mass spectra were obtained on a 5973 Network Mass Selective Detector instrument using electron impact ionization (EI, 70 eV). Elemental analysis was performed on FlashEA 1112 series (Thermo Finnigan) CHNS analyzer. 2. 1. Synthesis of 5-Phenyl-1,3,4-thiadiazol-2- amine (3) A stirring mixture of benzoic acid (50 mmol), thi- osemicarbazide (50 mmol) and phosphorus oxychloride (POCl3) (15 mL) was heated at 75 °C for 1 h. After cooling to room temperature, water was added; the reaction mix- ture was further refluxed for 4 h. After cooling, the mixture was basified to pH 8 by the dropwise addition of 50% po- tassium hydroxide solution under stirring. Thus, obtained precipitate was filtered and recrystallized from ethanol.28 This compound was obtained as yellow solid in a yield 7.53 g (85%); m.p. 225–227 °C; IR (KBr, cm–1): νmax 3267.45, 3062.96 (NH), 1629.53 (C=N), 1510.55 (strong bending NH), 664.71 (C–S); 1H NMR (500 MHz, DM- SO-d6) δ 7.42 (s, 2H, NH), 7.44–7.46 (m, 3H, ArH), 7.74 (d, J = 7.7 Hz, 2H, ArH). 13C NMR (125 MHz, DMSO-d6) δ 126.7 (2CH), 129.6 (2CH), 130.0 , 131.4 , 156.8 (C=N), 169.0 (C=N). MS m/z 177.1 (M+ + H). Anal. Calcd for C8H7N3S: C, 54.22; H, 3.98; N, 23.71; S, 18.09. Found: C, 54.23; H, 3.99; N, 23.74; S, 18.13. 2. 2. General Procedure for the Synthesis of Compounds 2-Substituted Phenyl-3- (5-phenyl-1-3,4-thiadiazol-2-yl)-2,3- dihydroquinazolin-4(1H)-ones 4a–l To a round-bottom flask containing H2O (5 mL) was added isatoic anhydride (1 mmol, 0.1631 g), relevant aldehyde (1 mmol), 1,3,4-thiadiazol-2-amine (1.1 mmol, 0.1949 g) and p-TsOH (our inventory was its monohy- drate) (0.6 mmol, 0.1141 g). The mixture was heated under reflux for 2 hours. The precipitate was filtered and recrys- tallized from EtOH.29 See Tables 1 and 2 and Scheme 1. 2-Phenyl-3-(5-Phenyl-1,3,4-thiadiazol-2-yl)-2,3-dihy- droquinazolin-4(1H)-one (4a) This compound was obtained as light brown sol- id in a yield 0.3113 g (81%); m.p. 171–173 °C. IR (KBr, cm–1): νmax 3364.47 (NH), 3033.20 (C–H), 1633.86 (C=O), 1500.59 (C=N), 1448.65 (strong bending NH), 1386.63 (C–N), 1180.94 (N–N), 689.71 (C–S). 1H NMR (500 MHz, DMSO-d6): δ 6.81 (t, J = 7.5 Hz, 1H, NH), 6.93 (d, J = 8.2 Hz, 1H, CH), 7.32 (m, 5H, ArH), 7.41 (t, J = 3.8 Hz, 1H, ArH), 7.44 (d, J = 0.58 Hz, 1H, ArH), 7.55 (m, 3H, ArH), 7.82 (d, J = 7.9 Hz, 1H, ArH), 7.98 (d, J = 6.5 Hz, 2H, ArH), 8.34 (d, J = 0.2 Hz, 1H, ArH). 13C NMR (125 MHz, DMSO-d6) δ 69.0 (N-C-N), 113.2, 116.2, 118.9, 126.1 (2CH), 127.5 (2CH), 128.9, 128.9, 129.1 (2CH), 129.9 (2CH), 130.4, 131.3, 136.2, 139.8, 147.3, 158.2 (C=O), 160.9 (C=N), 164.3 (C=N). MS m/z 384.2 (M+ + H). Anal. Calcd for C22H16N4OS: C, 68.73; H, 4.20; N, 14.57; S, 8.34. Found: C, 68.77; H, 4.25; N, 15.02; S, 8.29. 2-(2-Chlorophenyl)-3-(5-phenyl-1,3,4-thiadiazol-2-yl)- 2,3-dihydroquinazolin-4(1H)-one (4b) This compound was obtained as brownish yellow solid in a yield 0.3518 g (84%); m.p. 210–212 °C. IR (KBr, cm–1): νmax 3324.17 (NH), 3056.76 (C–H), 1661.58 (C=O), 1615.33 (C=N), 1506.09 (strong bending NH), 1439.22 (C–N), 1245.53 (N–N), 748.42 (C–Cl), 685.60 (C–S). 1H NMR (500 MHz, DMSO-d6): δ 6.86 (t, J = 7.6 Hz, 1H, NH), 6.90 (d J = 8.2 Hz, 1H, CH), 7.07 (d, J = 7.8 Hz, 1H, ArH), 7.20 (t, J = 7.7 Hz, 1H, ArH), 7.33 (t, J = 7.6 Hz, 1H, ArH), 7.41 ( t, J = 7.8 Hz, 1H, ArH), 7.53 (t, J = 3.3 Hz, 3H, ArH), 7.57 (d, J = 7.9 Hz, 1H, ArH), 7.60 (d, J = 3.3 Hz, 1H, ArH), 7.93 (t, J = 7.4 Hz, 3H, ArH), 8.11 (d, J = 4.2 Hz, 1H, ArH). 13C NMR (125 MHz, DMSO-d6) δ 67.2 (N–C–N), 112.5, 116.5, 119.1, 125.8, 127.5 (2CH), 128.1, 128.8, 129.9 (2CH), 130.3, 130.9 (2CH), 131.4 (C–Cl), 132.1, 136.3, 136.9, 146.1, 157.4 (C=O), 160.9 (C=N), 164.5 (C=N). MS m/z 418.2 (M+ + H). Anal. Calcd for C22H15ClN4OS: C, 63.08; H, 3.61; N, 13.38; S, 7.65. Found: C, 63.05; H, 3.62; N, 13.45; S, 7.77. 2-(4-Chlorophenyl)-3-(5-phenyl-1,3,4-thiadiazol-2-yl)- 2,3-dihydroquinazolin-4(1H)-one (4c) This compound was obtained as greenish yellow sol- id in a yield 0.3560 g (85%); m.p. 227–229 °C. IR (KBr, cm–1): νmax 3377.68 (NH), 3053.97 (C–H), 1650.19 (C=O), 1613.78 (C=N), 1488.61 (strong bending NH), 1447.76 (C–N), 1253.00 (N–N), 756.40 (C–Cl), 684.14 (C–S). 1H NMR (500 MHz, DMSO-d6): δ 6.82 (t, J = 7.4 Hz, 1H, NH), 6.94 (d, J = 8.2 Hz, 1H, CH), 7.33 (d, J = 8.2 Hz, 2H, ArH), 7.39 (d, J = 8.3 Hz, 2H, ArH), 7.43 (d, J = 8.6 Hz, 2H, ArH), 7.54 (t, J = 4.0 Hz, 3H, ArH), 7.82 (d, J = 7.9 Hz, 1H, ArH), 8.0 (m, 2H, ArH), 8.31 (d, J = 4.0 Hz, 1H, ArH). 13C NMR (125 MHz, DMSO-d6) δ 68.5 (N–C–N), 113.2, 116.3, 119.1, 127.5 (2CH), 128.1 (2CH), 129.0, 129.2 (2CH), 129.9 (2CH), 130.4, 131.3 (C–Cl), 133.6, 136.3, 138.8, 147.1, 158.1 (C=O), 160.7 (C=N), 164.4 (C=N). MS m/z 418.2 (M+ + H). Anal. Calcd for C22H15ClN4OS: C, 63.08; H, 3.61; N, 13.38; S, 7.65. Found: C, 63.07; H, 3.60; N, 13.41; S, 7.76. 2-(2-Nitrophenyl)-3-(5-phenyl-1,3,4-thiadiazol-2-yl)- 2,3-dihydroquinazolin-4(1H)-one (4d) This compound was obtained as yellow solid in a yield 0.3521 g (82%); m.p. 199–201 °C. IR (KBr, cm–1): 621Acta Chim. Slov. 2022, 69, 619–628 Zahmatkesh et al.: Synthesis, Antimicrobial and Molecular Docking Studies of ... νmax 3356.94 (NH), 3063.28 (C–H), 1660.59 (C=O), 1615.85 (C=N), 1509.00 (strong bending NH), 1438.99 (C–N), 1509.00 and 1338.31 (NO2), 1248.33 (N–N), 687.14 (C–S). 1H NMR (500 MHz, DMSO-d6): δ 6.88 (t, J = 0.5 Hz, 1H, NH), 7.27 (d, J = 7.4 Hz, 1H, CH), 7.41 (t, J = 8.08 Hz, 1H), 7.47 (d, J = 6.82 Hz, 1H), 7.52 (t, J = 1.98 Hz, 3H), 7.59 (t, J = 8.7 Hz, 2H, ArH), 7.92 (m, 4H, ArH), 8.02 (d, J = 0.8 Hz, 1H, ArH), 8.12 (d, J = 7.6 Hz, 1H, ArH). 13C NMR (125 MHz, DMSO-d6) δ 65.9 (N–C–N), 112.7, 117.2, 119.6, 126.2, 126.5, 127.5 (2CH), 128.8, 129.8 (2CH), 130.2, 130.7, 131.4, 134.6, 134.8, 136.4, 146.0, 147.8 (C–NO2), 157.5 (C=O), 160.6 (C=N), 164.6 (C=N). MS m/z 429.1 (M+ + H). Anal. Calcd for C22H15N5O3S: C, 61.53; H, 3.52; N, 16.31; S, 7.47. Found: C, 61.50; H, 3.55; N, 16.38; S, 7.41. 2-(3-Nitrophenyl)-3-(5-phenyl-1,3,4-thiadiazol-2-yl)- 2,3-dihydroquinazolin-4(1H)-one (4e) This compound was obtained as brownish yellow solid in a yield 0.3649 g (85%); m.p. 226–228 °C. IR (KBr, cm–1): νmax 3353.50 (NH), 3065.72 (C–H), 1659.93 (C=O), 1613.78 (C=N), 1519.31 (strong bending NH), 1444.15 (C–N), 1519.31 and 1357.73 (NO2), 1300.09 (N–N), 686.53 (C–S). 1H NMR (500 MHz, DMSO-d6): δ 6.84 (t, J = 7.6 Hz, 1H, NH), 6.98 (d, J = 8.2 Hz, 1H, CH), 7.44 (t, J = 7.5 Hz, 1H, ArH), 7.56 (t, J = 3.5 Hz, 3H, ArH), 7.58 (d, J = 3.9 Hz, 1H, ArH), 7.62 (d, J = 8.0 Hz, 1H, ArH), 7.68 (d, J = 7.8 Hz, 1H, ArH), 7.85 (d, J = 3.3 Hz, 1H, ArH), 7.98 (m, 2H, ArH), 8.14 (d, J = 8.1 Hz, 1H, ArH), 8.30 (s, 1H, ArH), 8.43 (d, J = 4.0 Hz, 1H, ArH). 13C NMR (125 MHz, DM- SO-d6) δ 68.3 (N–C–N), 113.1, 116.4, 119.4, 121.3, 124.0, 127.5 (2CH), 129.0, 129.9 (2CH), 130.3, 130.9, 131.4, 132.4, 136.4, 142.1, 146.8, 148.5 (C–NO2), 158.0 (C=O), 160.5 (C=N), 164.6 (C=N). MS m/z 429.2 (M+ + H). Anal. Calcd for C22H15N5O3S: C, 61.53; H, 3.52; N, 16.31; S, 7.47. Found: C, 61.55; H, 3.50; N, 16.36; S, 7.59. 2-(4-Nitrophenyl)-3-(5-phenyl-1,3,4-thiadiazol-2-yl)- 2,3-dihydroquinazolin-4(1H)-one (4f) This compound was obtained as light green solid in a yield 0.3606 g (84%); m.p. 190–192 °C. IR (KBr, cm–1): νmax 3371.85 (NH), 3058.92 (C–H), 1657.13 (C=O), 1609.39 (C=N), 1520.35 (strong bending NH), 1441.98 (C–N), 1490.12 and 1347.08 (NO2), 1306.95 (N–N), 693.16 (C–S). 1H NMR (500 MHz, DMSO-d6): δ 6.85 (t, J = 7.6 Hz, 1H, NH), 6.96 (d, J = 8.3 Hz, 1H, CH), 7.44 (t, J = 8.0 Hz, 1H, ArH), 7.56 (m, 3H, ArH), 7.59 (m, 3H, ArH), 7.83 (t, J = 4.0 Hz, 1H, ArH), 7.99 (d, J = 3.6 Hz, 2H, ArH), 8.20 (d, J = 8.4 Hz, 2H, ArH), 8.41 (d, J = 3.7 Hz, 1H, ArH). 13C NMR (125 MHz, DMSO-d6) δ 68.5 (N–C–N), 113.2, 116.4, 119.4, 124.4 (2CH), 127.5 (2CH), 127.6 (2CH), 129.0, 129.9 (2CH), 130.3, 131.4, 136.4, 146.8, 147.0 (C– NO2), 148.0, 158.0 (C=O), 160.5 (C=N), 164.5 (C=N). MS m/z 429.2 (M+ + H). Anal. Calcd for C22H15N5O3S: C, 61.53; H, 3.52; N, 16.31; S, 7.47. Found: C, 61.50; H, 3.54; N, 16.30; S, 7.38. 2-(2-Hydroxyphenyl)-3-(5-phenyl-1,3,4-thiadiazol-2- yl)-2,3-dihydroquinazolin-4(1H)-one (4g) This compound was obtained as green solid in a yield 0.3123 g (78%); m.p. 218–220 °C. IR (KBr, cm–1): νmax 3380.01 (NH), 3060.60 (O–H), 2946.15 (C–H), 1665.55 (C=O), 1609.81 (C=N), 1496.30 (strong bend- ing NH), 1456.32 (C–N), 1312.71 (N–N), 1236.41(C–O), 687.01 (C–S). 1H NMR (500 MHz, DMSO-d6): δ 6.61 (t, J = 7.4 Hz, 1H, NH), 6.75 (d, J = 8.5 Hz, 1H, CH), 6.90 (t, J = 8.6 Hz, 2H, ArH), 7.12 (m, 1H, ArH), 7.35 (t, J = 7.8 Hz, 1H, ArH), 7.48 (d, J = 7.0 Hz, 2H, ArH), 7.53 (m, 2H, ArH), 7.77 (m, 2H, ArH), 7.86 (d, J = 8.1 Hz, 1H, ArH), 7.93 (d, J = 5.0 Hz, 2H, ArH), 10.23 (s, 1H, OH). 13C NMR (125 MHz, DMSO-d6) δ 66.0 (N–C–N), 112.4, 116.1, 116.2, 118.4, 119.0, 125.4, 125.6, 127.4 (2CH), 128.7, 129.8 (2CH), 130.0, 130.4, 131.2, 135.9, 147.2, 155.2 (C–OH), 157.6 (C=O), 161.4 (C=N), 164.2 (C=N). MS m/z 400.2 (M+ + H). Anal. Calcd for C22H16N4O2S: C, 65.99; H, 4.03; N, 13.99; S, 8.01. Found: C, 66.07; H, 4.01; N, 13.95; S, 8.25. 2-(3-Hydroxyphenyl)-3-(5-phenyl-1,3,4-thiadiazol-2- yl)-2,3-dihydroquinazolin-4(1H)-one (4h) This compound was obtained as brown solid in a yield 0.3203 g (80%); m.p. 207–209 °C. IR (KBr, cm–1): νmax 3275.51(NH), 3075.36 (O–H), 2910.10 (C–H), 1601.72 (C=O), 1505.26 (C=N), 1505.26 (strong bend- ing NH), 1448.04 (C–N), 1379.91 (N–N), 1216.41(C–O), 683.43 (C–S). 1H NMR (500 MHz, DMSO-d6): δ 6.68 (t, J = 1.3 Hz, 1H, NH), 6.79 (d, J = 7.0 Hz, 1H, CH), 6.93 (m, 1H, ArH), 7.13 (t, J = 4.4 Hz, 3H, ArH), 7.42 (t, J = 8.7 Hz, 4H, ArH), 7.72 (d, J = 8.1 Hz, 1H, ArH), 7.83 (m, 2H, ArH), 7.99 (d, J = 8.1 Hz, 2H, ArH), 9.93 (s, 1H, OH). 13C NMR (125 MHz, DMSO-d6) δ 68.9 (N–C–N), 115.4, 115.8, 116.2, 116.9, 118.8, 127.5 (2CH), 128.9, 129.9 (2CH), 130.4, 131.3, 131.6, 134.2, 136.2, 141.3, 145.9, 147.4 (C–OH), 157.9 (C=O), 164.3 (C=N), 170.0 (C=N). MS m/z 400.2 (M+ + H). Anal. Calcd for C22H16N4O2S: C, 65.99; H, 4.03; N, 13.99; S, 8.01. Found: C, 66.02; H, 4.04; N, 14.05; S, 8.13. 2-(4-Hydroxyphenyl)-3-(5-phenyl-1,3,4-thiadiazol-2- yl)-2,3-dihydroquinazolin-4(1H)-one (4i) This compound was obtained as red solid in a yield 0.3203 g (80%); m.p. 210–212 °C. IR (KBr, cm–1): νmax 3281.73 (NH), 3152.73 (O–H), 2929.54 (C–H), 1616.59 (C=O), 1582.00 (C=N), 1526.97 (strong bending NH), 1501.20 (C–N), 1299.01 (N–N), 1258.42 (C–O), 678.90 (C–S). 1H NMR (500 MHz, DMSO-d6): δ 6.69 (m, 2H, NH, CH), 6.95 (d, J = 8.5 Hz, 2H, ArH), 7.13 (t, J = 4.0 Hz, 3H, ArH), 7.33 (t, J = 3.0 Hz, 1H, ArH), 7.55 (m, 2H, ArH), 7.78 (m, 2H, ArH), 7.98 (d, J = 7.6 Hz, 2H, ArH), 8.23 (d, J = 1.4 Hz, 1H, ArH), 9.80 (s, 1H, OH). 13C NMR (125 MHz, DMSO-d6) δ 68.8 (N–C–N), 113.1, 116.2, 118.7, 126.9 (2CH), 127.5 (2CH), 128.6 (2CH), 128.9, 129.7 (2CH), 130.5, 136.1, 138.2, 146.0, 147.4, 158.0 (C– 622 Acta Chim. Slov. 2022, 69, 619–628 Zahmatkesh et al.: Synthesis, Antimicrobial and Molecular Docking Studies of ... OH), 163.8 (C=O), 164.2 (C=N), 169.3 (C=N). MS m/z 400.2 (M+ + H). Anal. Calcd for C22H16N4O2S: C, 65.99; H, 4.03; N, 13.99; S, 8.01. Found: C, 65.90; H, 4.06; N, 13.92; S, 7.87. 2-(3-Methoxyphenyl)-3-(5-phenyl-1,3,4-thiadiazol-2- yl)-2,3-dihydroquinazolin-4(1H)-one (4j) This compound was obtained as greenish yellow sol- id in a yield 0.3149 g (76%); m.p. 192–194 °C. IR (KBr, cm–1): νmax 3259.27 (NH), 3012.24 (C–H), 1653.97 (C=O), 1615.35 (C=N), 1510.68 (strong bending NH), 1441.67 (C–N), 1295.99 (N–N), 1260.28 (C–O), 685.30 (C–S). 1H NMR (500 MHz, DMSO-d6): δ 3.68 (s, 3H, CH3), 6.81 (m, 2H, NH, CH), 6.85 (d, J = 8.1 Hz, 1H, ArH), 6.94 (d, J = 9.4 Hz, 2H, ArH), 7.22 (t. J = 8.1 Hz, 1H, ArH), 7.42 (d, J = 6.5 Hz, 2H, ArH), 7.54 (m, 3H, ArH), 7.83 (d, J = 8.0 Hz, 1H, ArH), 7.98 (d, J = 6.2 Hz, 2H, ArH), 8.32 (d, J = 1.0 Hz, 1H, ArH). 13C NMR (125 MHz, DMSO-d6) δ 55.5 (CH3), 68.8 (N–C–N), 112.8, 113.2, 113.6, 116.2, 118.2, 118.9, 127.5 (2CH), 128.9, 129.8 (2CH), 130.3, 130.4, 131.3, 136.2, 141.4, 147.4, 158.2 (C–OCH3), 159.8 (C=O), 160.9 (C=N), 164.3 (C=N). MS m/z 414.2 (M+ + H). Anal. Calcd for C23H18N4O2S: C, 66.65; H, 4.38; N, 13.52; S, 7.73. Found: C, 66.90; H, 4.34; N, 13.83; S, 7.57. 2-(4-Methoxyphenyl)-3-(5-phenyl-1,3,4-thiadiazol-2- yl)-2,3-dihydroquinazolin-4(1H)-one (4k) This compound was obtained as brownish yellow solid in a yield 0.3232 g (78%); m.p. 220–222 °C. IR (KBr, cm–1): νmax 3397.48 (NH), 3057.61 (C–H), 1660.24 (C=O), 1609.56 (C=N), 1504.27 (strong bending NH), 1455.58 (C–N), 1300.42 (N–N), 1242.84 (C–O), 686.93 (C–S). 1H NMR (500 MHz, DMSO-d6): δ 3.67 (s, 3H, CH3), 6.81 (m, 2H, NH, CH), 6.92 (d, J = 8.1 Hz, 2H, ArH), 7.13 (d, J = 8.0 Hz, 1H, ArH), 7.42 (m, 1H, ArH), 7.55 (m, 5H, ArH), 7.82 (d, J = 8.3 Hz, 2H, ArH), 7.98 (m, 2H, ArH). 13C NMR (125 MHz, DMSO-d6) δ 55.5 (CH3), 68.7 (N–C–N), 113.2, 114.4 (2CH), 116.2, 118.76, 127.0 (2CH), 128.6 (2CH), 128.9, 129.9 (2CH), 131.2, 131.7, 132.2, 136.2, 147.4, 158.1 (C–OCH3), 159.7 (C=O), 160.9 (C=N), 164.2 (C=N). MS m/z 414.2 (M+ + H). Anal. Calcd for C23H18N4O2S: C, 66.65; H, 4.38; N, 13.52; S, 7.73. Found: C, 66.73; H, 4.47; N, 13.43; S, 7.88. 3-(5-Phenyl-1,3,4-thiadiazol-2-yl)-2-(p-tolyl)-2,3-dihy- droquinazolin-4(1H)-one (4l) This compound was obtained as brownish yellow solid in a yield 0.2868 g (72%); m.p. 208–210 °C. IR (KBr, cm–1): νmax 3373.40 (NH), 3052.07 (C–H), 1649.00 (C=O), 1613.00 (C=N), 1494.77 (strong bending NH), 1448.97 (C–N), 1290.22 (N–N), 682.73 (C–S). 1H NMR (500 MHz, DMSO-d6): δ 2.20 (s, 3H, CH3), 6.80 (t, J = 8.07 Hz, 1H, NH), 6.92 (d, J = 8.2 Hz, 1H, CH), 7.11 (m, 2H, ArH), 7.14 (d, J = 7.8 Hz, 2H, ArH), 7.19 (d, J = 7.8 Hz, 2H, ArH), 7.39 (d, J = 0.3 Hz, 1H, ArH), 7.55 (m, 3H, ArH), 7.82 (d, J = 0.6 Hz, 1H, ArH), 7.97 (d, J = 6.4 Hz, 2H, ArH). 13C NMR (125 MHz, DMSO-d6) δ 21.0 (CH3), 68.9 (N–C–N), 113.2, 116.2, 126.0 (2CH), 127.5 (2CH), 128.9, 129.6 (2CH), 129.9 (2CH), 130.4, 136.2, 136.9, 138.3, 138.3 (C–CH3), 147.4, 158.2, 160.9 (C=O), 164.2 (C=N), 169.4 (C=N). MS m/z 398.2 (M+ + H). Anal. Calcd for C23H18N4OS: C, 69.33; H, 4.55; N, 14.06; S, 8.05. Found: C, 69.63; H, 4.59; N, 14.16; S, 8.23. 2. 3. Antibacterial Activity (In vitro) The antibacterial activity of synthesized compounds 4a–l was evaluated against two Gram positive and two Gram negative bacteria by using the well diffusion meth- od, minimum inhibitory concentration (MIC), and min- imum bactericidal concentration (MBC). Ciprofloxacin was employed as the standard drug to compare the results. Strains of Staphylococcus aureus PTCC1826, Staphylococ- cus epidermidis PTCC1856, Escherichia coli PTCC1789, and Pseudomonas aeruginosa PTCC1950, were taken from the Iranian industrial microorganisms collection center (lyophilized). The bacterial cultures were developed by se- lective nutrient broth at 37 °C, 24 h. Nutrient broth was used for the preparation of inoculums of the bacteria and nutrient agar and broth were used for the screening meth- od21 and their results are shown in Table 3. 2. 4. Antifungal Activity (In vitro) Also the antifungal activity of synthesized com- pounds 4a–l was evaluated against two fungal strains by using the well diffusion method, minimum inhibitory concentration (MIC), and minimum fungicidal concen- tration (MFC). Amphotericin B was employed as the standard drug to compare the results. Strains of Candida albicans PTCC5027 and Aspergillus niger PTCC5320, were taken from the Iranian industrial microorganisms collec- tion center. The fungal cultures were developed by selec- tive Sabouraud dextrose broth at 37 °C and stored at 4 °C for further use. Sabouraud dextrose broth was used for the preparation of inoculums of the fungi and Sabouraud dex- trose agar and broth were used for the screening method21 and their results are presented in Table 4. Table 1. Groups (X) attached to benzaldehyde 4 and 2 a b c d e f g h i j k l X H 2-Cl 4-Cl 2-NO2 3-NO2 4-NO2 2-OH 3-OH 4-OH 3-OCH3 4-OCH3 4-CH3 623Acta Chim. Slov. 2022, 69, 619–628 Zahmatkesh et al.: Synthesis, Antimicrobial and Molecular Docking Studies of ... 2. 5. Anti-diabetic Propertises by Molecular Docking (In silico) AutoDock Vina v.1.2.0 was used to perform all dock- ing simulations. A set of new quinazolin derivatives were subjected to docking with α-amylase (PDB ID: 1hny) and α-glucosidase (PDB ID: 2ze0) from the protein data bank (RCSB) (http://www.rcsb.org/pdb). To carry out in silico studies, the 2D structures of the synthesized ligands 4a–l were drawn by ChemDraw 19.1.1 and converted to ener- gy minimized 3D structures in the pdb file format using Chem3D. By removing the heteroatoms, water molecule and cofactors, the target protein file was prepared by leav- ing the associated residue with protein by using Discovery Studio 4.5 Client. Preparation of target protein file Auto- DockTools-1.5.6 has been done, which involves the as- signing of Gasteiger charges for all the atoms of molecules converting into AD4 type. Grid box for 1hny was 64 × 64 × 64 and for 2ze0 was 72 × 72 × 72. Docking simulations for Table 2. Structure of compounds 4a–l 4a 4g 4b 4h 4c 4i 4d 4j 4e 4k 4f 4l 624 Acta Chim. Slov. 2022, 69, 619–628 Zahmatkesh et al.: Synthesis, Antimicrobial and Molecular Docking Studies of ... the compounds 4a–l were performed against the active site of α-amylase and α-glucosidase, finally Discovery Studio 4.5 Client was used to visualize docking results, which are shown in Table 5 and Figure 1. 3. Results and Discussion 3. 1. Chemistry Considering the importance of 2,3-dihydroquina- zolin-4(1H)-ones several methods have been revealed for the synthesis of these compounds.30–32 In this study compounds 2-(substituted phenyl)-3-(5-phenyl-1-3,4- thiadiazol-2-yl)-2,3-dihydroquinazolin-4(1H)-one 4a–l were obtained by refluxing isatoic anhydride (1), 5-phe- nyl-1,3,4-thiadiazol-2-amine (3) and aromatic aldehydes 2a–l in the presence of p-TsOH as the catalyst in H2O as the solvent (Scheme 1). Compounds 4a–l contain a chiral center at the car- bon 2 that is formed during the reaction. Hence, these compounds are in the form of racemate. Racemates con- sist of an equimolar mixture of two enantiomers. About more than half of the drugs currently in use are chiral compounds and near 90% of the last ones are marketed as racemates. Indeed, numerous studies have demonstrat- ed that drug enantiomers may interact differently with biological macromolecules. Replacing existing racemates with unichiral drugs may result in improved safety and ef- ficacy profile of various racemates.33,34 5-Phenyl-1,3,4-thiadiazol-2-amine (3) was obtained by refluxing benzoic acid and thiosemicarbazide in phos- phorous oxychloride (Scheme 2). Different spectroscopic data were used to confirm 2-(substituted phenyl)-3-(5-phenyl-1-3,4-thiadiazol-2- yl)-2,3-dihydroquinazolin-4(1H)-ones 4a–l. All of the newly synthesized products 4a–l were characterized by FT-IR spectroscopy, 1H and 13C NMR spectra, mass spec- trometry and elemental analysis. In the FT-IR spectra of compounds 4a–l the strong and sharp absorption bands due to NH and (N–CO) groups were observed at around 3379–3282 cm–1 and 1655–1608 cm–1, respectively. Also, in the 1H NMR spectra, the NH proton of the 2,3-dihydroquinazolin-4(1H)-one ring appeared as a broad signal at 8.36–7.03 ppm and C–H proton of position 2 appeared at 5.8–6.5 ppm.The 13C NMR spectra showed a signal at 171–162 ppm assigned to the N-C=O group and a signal for C–H at 66–70 ppm that confirmed the synthesis of 2,3-dihydroquinazolin-4(1H)-ones. Scheme 1. Synthesis of 2-substituted-2,3-dihydroquinazolin-4(1H)-one derivatives 4a–l Scheme 2. Synthesis of 5-phenyl-1,3,4-thiadiazol-2-amine (3) 625Acta Chim. Slov. 2022, 69, 619–628 Zahmatkesh et al.: Synthesis, Antimicrobial and Molecular Docking Studies of ... 3. 2. Antibacterial Activity (In vitro) As illustrated by the inhibition zone in Table 3, all tested compounds showed antibacterial activity against all Gram positive and Gram negative strains. The greatest ef- fects of all compounds in bacteria samples were observed against E. coli. The best effect of the compounds against Gram positive bacteria: S. aureus: 4k (IZ = 26 ± 0.52, MIC = 250, MBC = 500). S. epidermidis: 4j (IZ = 28 ± 0.31, MIC = 125, MBC = 250). In this group (Gram positive bacte- ria), compound 4k has the highest performance compared with ciprofloxacin than the other compounds, 4k probably works well in penetrating the membrane of these bacte- ria and causes more degradation of peptidoglycans than the other synthetic structures. The best effect of the com- pounds against Gram negative bacteria: E. coli: 4l (IZ = 31 ± 0.96, MIC = 125, MBC = 250). P. aeruginosa: 4i (IZ = 26 ± 0.14, MIC = 250, MBC = 500). In this group (Gram negative bacteria), 4l has shown the highest performance compared with ciprofloxacin than the other compounds. This compound is likely to penetrate and destroy these bacteria by destroying the inner and outer membranes. According to the results obtained from the antibacteri- al activity of compounds 4a–l, it can be concluded that the synthesized compounds that have substituted phenyl groups along with thiadiazol and quinazoline 4a–l com- pared to other compounds in this study, can perform well in eliminating human bacterial pathogens. 3. 3. Antifungal Activity (In vitro) It was found that the synthesized compounds ex- hibited varied antifungal effects against two fungal strains (Table 4). The highest number of compounds affecting antifungal activity was observed against C. albicans. The best effect of the compounds against fungal specimens: C. albicans: 4j (IZ = 26±0.33, MIC = 250, MBC = 500) and A. niger: 4k (IZ = 21±0.66, MIC = 500, MBC = 1000). Ac- cording to the results obtained from the antifungal activ- ity of the compounds, it can be concluded that the syn- thesized compounds that have substituted phenyl groups along with thiadiazol and quinazoline 4a–l compared with amphotericin B in this study, can perform well in eliminat- ing human fungal pathogens. 3. 4. Anti-diabetic Activity (In silico) In this section, it has been performed docking sim- ulation of the newly synthesized derivatives 4a–l binding the active site of the α-amylase (PDB ID: 1hny) and α-glu- cosidase (PDB ID: 2ze0). The docking result of the test- ed compounds 4c, 4d and 4g showed lowest ΔGbind with α-amylase by –10.0 kcal/mol, and compounds 4e, 4f and 4i showed lowest ΔGbind, respectively –10.6, –9.5 and –10.1 with α-glucosidase. The binding energies, inhibition con- stants and residues involved in H-bonding are presented in Table 5 and Figures 1 and 2. Table 3. Antibacterial activity of compounds 4a–l Concentration of compounds: 1 mg/mL Inhibition Zone (IZ): mm Minimum Inhibitory Concentration (MIC): 0–1000 µg/mL Minimum Bactericidal Concentration (MBC): 0–1000 µg/mL ±: average three times Cip: Ciprofloxacin Well diameter: 9 mm Gram positive bacteria Gram negative bacteria S. aureus S. epidermidis E. coli P. aeruginosa PTCC1826 PTCC1856 PTCC1789 PTCC1950 compounds IZ MIC MBC IZ MIC MBC IZ MIC MBC IZ MIC MBC 4a 19±0.22 500 1000 21±0.23 500 1000 17±0.33 1000 1000 21±0.02 500 1000 4b 21±0.21 250 500 22±0.26 250 500 19±0.42 1000 1000 21±0.21 500 1000 4c 17±0.96 500 1000 14±0.75 1000 1000 16±0.78 1000 1000 20±0.48 500 1000 4d 14±0.33 1000 1000 15±0.34 1000 1000 12±0.92 1000 1000 16±0.92 1000 1000 4e 16±0.28 500 1000 19±0.67 500 1000 14±0.28 1000 1000 14±0.35 1000 1000 4f 16±0.66 500 1000 17±0.33 500 1000 19±0.34 1000 1000 19±0.46 1000 1000 4g 16±0.66 500 1000 16±0.33 1000 1000 18±0.39 1000 1000 19±0.44 1000 1000 4h 19±0.56 500 1000 22±0.42 250 500 21±0.37 500 1000 25±0.23 500 1000 4i 18±0.41 500 1000 19±0.22 500 1000 22±0.82 500 1000 26±0.14 250 500 4j 24±0.76 250 500 28±0.31 125 250 24±0.47 500 1000 21±0.29 500 1000 4k 26±0.52 250 500 25±0.57 250 500 26±0.33 500 1000 24±0.68 250 500 4l 24±0.19 250 500 27±0.29 125 250 31±0.96 125 250 24±0.16 250 500 Cip 41±0.35 15.62 31.25 46±0.21 7.81 15.625 37±0.33 31.25 62.50 34±0.66 62.50 125 626 Acta Chim. Slov. 2022, 69, 619–628 Zahmatkesh et al.: Synthesis, Antimicrobial and Molecular Docking Studies of ... Table 4. Antifungal activity of compounds 4a–l Concentration of compounds: 1 mg/mL Inhibition Zone (IZ): mm Minimum Inhibitory Concentration (MIC): 0–1000 µg/mL Minimum Fungicidal Concentration (MFC): 0–1000 µg/mL ±: average three times AB: Amphotericin B NA: No Activity Well diameter: 9 mm C .albicans A .niger PTCC5027 PTCC5320 compounds IZ MIC MFC IZ MIC MFC 4a 21±0.33 500 1000 17±0.66 1000 1000 4b 23±0.33 500 1000 20±0.66 500 1000 4c 20±0.33 500 1000 14±0.66 1000 1000 4d 14±0.33 1000 1000 11±0.66 NA NA 4e 19±0.33 1000 1000 15±0.66 1000 1000 4f 19±0.33 1000 1000 16±0.66 1000 1000 4g 17±0.33 1000 1000 12±0.66 NA NA 4h 21±0.33 500 1000 19±0.66 1000 1000 4i 20±0.33 500 1000 14±0.66 1000 1000 4j 26±0.33 250 500 20±0.66 500 1000 4k 25±0.33 500 1000 21±0.66 500 1000 4l 21±0.33 500 1000 19±0.66 1000 1000 AB 36±0.33 62.50 125 34±0.66 62.50 125 Table 5. Molecular docking reports for compounds 4a–l against protein 1hny and 2ze0. compounds Total Energy α-amylase α-glucosidase (Kcal/mol) PDB ID: 1hny PDB ID: 2ze0 Affinity H-Bond Affinity H-Bond (kcal/mol) (kcal/mol) 4a 11.3828 –9.6 — –8.9 Arginine: 407 4b 13.3308 –9.1 — –9.4 Arginine: 407 4c 11.6324 –10.0 Glutamic acid: 233 –9.2 Valine: 383 4d –12.5194 –10.0 Aspartic acid: 197 –9.1 Arginine: 407 Glutamic acid: 233 Histidine: 299 4e –2.1596 –9.1 — –10.6 Arginine: 407 Glutamine: 167 4f 7.2655 –8.7 — –9.5 Arginine: 197 Asparagine: 324 4g 7.8283 –10.0 Glutamic acid: 233 Aspartic acid: 300 –9.0 Arginine: 407 4h 9.9999 –9.1 — –9.0 Arginine: 407 4i 10.1567 –9.5 — –10.1 Arginine: 407 Glutamine: 167 4j 16.9915 –9.1 — –9.1 Arginine: 407 4k 17.1349 –9.5 — –9.0 — 4l 11.1987 –9.8 — –9.3 — 627Acta Chim. Slov. 2022, 69, 619–628 Zahmatkesh et al.: Synthesis, Antimicrobial and Molecular Docking Studies of ... 4. Conclusion In summary, in the present study target molecules 4a–l were synthesized via a one-pot condensation reac- tion between isatoic anhydride (1) and 5-phenyl-1,3,4-thi- adiazol-2-amine (3) with aromatic aldehydes 2a–l using p-TsOH as the catalyst in refluxing water. The newly syn- thesized compounds were characterized by mass, FT-IR, 1H NMR, 13C NMR spectra and analytical methods. The antibacterial activities of the synthesized compounds showed that compounds 4k against S. aureus, 4j against S. epidermidis, 4l against E. coli, and 4i against P. aerug- inosa have comparable inhibitory effects with the stand- ards used. Also, the antifungal activities of the synthesized compounds showed that compounds 4j against C. albicans and 4k against A. niger have comparable inhibitory effects with the standards used. All compounds showed good re- sults especially compound 4e showed the lowest ΔGbind results (–10.6 kcal/mol) against α-glucosidase and com- pounds 4c and 4g showed the lowest ΔGbind results (–10.0 kcal/mol) against α-amylase. Acknowledgments The authors are grateful to Urmia University for pro- viding a fellowship for the present work. Supplementary Data Copies of IR, 1H NMR, 13C NMR and MS spectra of compounds 4a–l are provided in supplementary material via the “Supplementary Content” section of this article’s webpage. 6. References 1. J. Zhang, J. Zhao, L. Wang, J. Liu, D. Ren,Y. Ma, Tetrahedron, 2016, 72, 936–943. DOI:10.1016/j.tet.2015.12.055 2. K. F. Sina, A. Yahyazadeh, N. Mahmoodi, Lett. Org. Chem. 2021, 18, 176–182. DOI:10.2174/1570178617999200706010203 3. K. Hemalatha, G. Madhumitha, L. Ravi, V. Gopiesh Khanna, N. Abdullah Al-Dhabi, M. Valan Arasu, J. Photochem. Photo- biol. B: Biology 2016, 161, 71–79. DOI:10.1016/j.jphotobiol.2016.05.005 4. V. Jatav, S. Kashaw, P. 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Block, A. Spannenberg, P. Langer, Org. Biomol. Chem. 2014, 12, 1865–1870. DOI:10.1039/c3ob42434k 33. K. M. Rentsch, J. Biochem. Biophys. Meth. 2002, 54, 1–9. DOI:10.1016/S0165-022X(02)00124-0 34. J. McConalthy, M. J. Owens, J. Clin. Psychiatry – Primary Care Companion 2003, 5, 70–73. 10.4088/PCC.v05n0202 Povzetek V tej študiji predstavljamo serijo novih 2-(substituiranih fenil)-3-(5-fenil-1,3,4-tiadiazol-2-il)-2,3-dihidrokinazolin- 4(1H)-onskih derivatov, ki smo jih pripravili s pomočjo refluktiranja izatojskega anhidrida, 5-fenil-1,3,4-tiadiazol-2-am- ina in aromatskih aldehidov v prisotnosti p-TsOH kot katalizatorja in v H2O kot topilu. Spojine smo karakterizirali s pomočjo spektroskopskih in analitskih metod. Določili smo antibakterijsko aktivnost proti dvema Gram pozitivnima in dvema Gram negativnima bakterijama ter aktivnost proti glivam; v vseh primerih smo izvedli tudi primerjavo s stand- ardnimi učinkovinami. Z uporabo metode difuzije smo določili minimalno baktericidno oz. fungicidno koncentracijo. Morebitno inhibitorno aktivnost spojin 4a–l za α-amilazo in α-glukozidazo smo raziskovali in silico s pomočjo metode molekulskega sidranja. Ugotovili smo, da so pripravljeni 2,3-dihidrokinazolin-4(1H)-onski derivati obetavni kandidati za nadaljnji razvoj novih razredov učinkovin, ki bodo imele delovanje proti mikrobom in diabetesu. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 629Acta Chim. Slov. 2022, 69, 629–637 Juteršek et al.: Synthesis, Crystal Structures and Urease Inhibition ... DOI: 10.17344/acsi.2022.7513 Scientific paper Synthesis, Crystal Structures and Urease Inhibition of Mononuclear Copper(II) and Nickel(II) Complexes with Schiff Base Ligands Jian Jiang,1,* Peng Liang,2 Huiyuan Yu3 and Zhonglu You3 1 College of Chemical Engineering and Machinery, Eastern Liaoning University, Dandong 118003, P. R. China 2 School of Engineering and Technology, Eastern Liaoning University, Dandong 118003, P. R. China 3 Department of Chemistry and Chemical Engineering, Liaoning Normal University, Dalian 116029, P. R. China * Corresponding author: E-mail: jiangjiandd2012@126.com Received: 03-30-2022 Abstract Three mononuclear copper(II) and nickel(II) complexes, [Cu(L1)(NCS)(CH3OH)] (1), [Cu(L2)(NCS)] (2) and [Ni(L2) (N3)] (3), where L1 and L2 are the monoanionic forms of the Schiff bases N’-(pyridin-2-ylmethylene)picolinohydrazide (HL1) and 4-methyl-2-(((pyridin-2-ylmethyl)imino)methyl)phenol (HL2), have been prepared and characterized by ele- mental analysis, IR and UV-Vis spectroscopy, as well as single crystal X-ray diffraction studies. The Cu atom in complex 1 is in a square pyramidal coordination, with the three N atoms of the ligand L and the N atom of the thiocyanate ligand in the basal plane, and with the methanol O atom at the apical position. The Cu and Ni atoms in complexes 2 and 3 are in square planar coordination, with the three donor atoms of the Schiff base ligands and the terminal N atoms of thio- cyanate and azide ligands. Complexes 1 and 2 inhibit the Jack bean urease with IC50 value of 0.33 ± 0.12 and 0.39 ± 0.10 μmol L–1, respectively. Molecular docking study was performed to investigate the interaction between the complexes and the enzyme. Keywords: Schiff base; Copper complex; Nickel complex; Crystal structure; Urease inhibition 1. Introduction Urease is a nickel-containing enzyme, which wide- ly be found in bacteria, fungi, algae, plants, and even in soil. The enzyme catalyzes the hydrolysis of urea to pro- duce NH3 with the rate 1014 times faster than that without urease. This process leads to a significant increase in pH of soil, and damage the plants.1 In human being and ani- mals, urease plays a vital role in peptic ulceration, urinary catheter incrustation, kidney stone, pyelonephritis, uro- lithiasis, hepatic encephalopathy and arthritis.2 Thus, the control of side effects of the urease is a hot topic in science. Urease inhibitors have been proved to be the best way to control the activity of urease. A variety of urease inhibi- tors have been reported, including inorganic metal salts,3 hydroxamic acid derivatives, triazoles, semicarbazones, Schiff bases, urea derivatives, oxadiazole, etc.4 However, most of them were prevented from application because of their low inhibition efficiency.5 Inorganic urease inhibitors such as the nitrate and chloride salts of copper, have ef- fective activities. However, they are harmful to both soil and living organisms.6 Recent reports indicated that some Schiff base copper(II) complexes have good urease inhib- itory activities.7 Khan and co-workers reported that some hydrazones have potential urease inhibitory activity. The diacyl hydrazide group (–NH–N=CH–) in the compounds serves as stabilizing agent in the active site and prevent the binding of substrate. In addition to stabilize the inhibitors, the –NH group of diacyl hydrazide was involved mak- ing strong hydrogen bonds with amino acid Arg439 and Ala636 of the urease.8 Considering that copper and nickel complexes with Schiff bases have a wide range of biological applications,9 in the present work, three mononuclear cop- per(II) and nickel(II) complexes, [Cu(L1)(NCS)(CH3OH)] (1), [Cu(L2)(NCS)] (2) and [Ni(L2)(N3)] (3), where L1 and L2 are the monoanionic forms of the Schiff bases N’-(pyri- din-2-ylmethylene)picolinohydrazide (HL1, Scheme 1) and 4-methyl-2-(((pyridin-2-ylmethyl)imino)methyl) phenol (HL2, Scheme 1) are presented. 630 Acta Chim. Slov. 2022, 69, 629–637 Juteršek et al.: Synthesis, Crystal Structures and Urease Inhibition ... Scheme 1. The Schiff base ligands. 2. Experimental 2. 1. Materials and Measurements 2-Pyridinecarboxaldehyde, 2-picolinyl hydrazide, 5-methylsalicylaldehyde, 2-aminomethylpyridine, copper acetate, copper nitrate, nickel nitrate, ammonium thiocy- anate, sodium azide and solvents with AR grade were pur- chased from Xiya Chemicals Co. Ltd. (China). Elemental analyses for C, H and N were performed on a Perkin-Elm- er 240C elemental analyzer. IR spectra were recorded on a Jasco FT/IR-4000 spectrometer as KBr pellets in the 4000– 400 cm–1 region. Electronic spectra were recorded on a Lambda 35 spectrophotometer. 1H and 13C NMR were recorded on a Bruker 300 MHz instrument. Single crys- tal X-ray diffraction was carried out on a Bruker SMART 1000 CCD diffractometer. 2. 2. Synthesis of N’-(Pyridin-2-ylmethylene) picolinohydrazide (HL1) 2-Pyridinecarboxaldehyde (1.1 g, 0.010 mol) and 2-picolinyl hydrazide (1.4 g, 0.010 mol) were mixed in methanol (50 mL). The mixture was stirred at 25 ºC for 30 min to give colorless solution. Then the solvent was evaporated to give gummy product, which was re-crys- tallized from ethanol to give yellow crystalline product of HL1. Yield: 1.8 g (80%). M.p. 172–173 ºC. Anal. Calc. for C12H10N4O (%): C, 63.71; H, 4.46; N, 24.76. Found (%): C, 63.53; H, 4.60; N, 24.63. IR data (cm–1): 3290w (NH), 1698s (C=O), 1647m (C=N). 1H NMR (300 MHz, CDCl3): δ 11.18 (s, 1H, NH), 8.89 (d, 1H, PyH), 8.72 (d, 1H, PyH), 8.35 (d, 1H, PyH), 7.90 (t, 1H, PyH), 7.88 (d, 1H, PyH), 7.81 (m, 2H, PyH), 7.62 (m, 1H, PyH), 7.28 (s, 1H, CH=N). 13C NMR (75 MHz, CDCl3): δ 159.79, 152.14, 151.80, 149.56, 148.31, 147.70, 137.14, 136.56, 126.46, 125.98, 122.54, 121.02. 2. 3. Synthesis of 4-Methyl-2-(((pyridin-2- ylmethyl)imino)methyl)phenol (HL2) 2-Pyridinecarboxaldehyde (1.1 g, 0.010 mol) and 2-aminomethylpyridine (1.1 g, 0.010 mol) were mixed in methanol (50 mL). The mixture was stirred at 25 ºC for 30 min to give colorless solution. Then the solvent was evaporated to give gummy product, which was re-crys- tallized from ethanol to give yellow crystalline product of HL2. Yield: 1.9 g (84%). M.p. 155–156 ºC. Anal. Calc. for C14H14N2O (%): C, 74.31; H, 6.24; N, 12.38. Found (%): C, 74.45; H, 6.32; N, 12.23. IR data (cm–1): 3378w (OH), 1638m (C=N). 1H NMR (300 MHz, CDCl3): δ 10.32 (s, 1H, OH), 8.72 (s, 1H, CH=N), 8.43 (d, 1H, PyH), 7.71 (t, 1H, PyH), 7.52 (s, 1H, PyH), 7.27 (t, 1H, PyH), 7.15–7.10 (m, 2H, PyH), 6.83 (d, 1H, PyH), 5.21 (s, 2H, CH2), 2.32 (s, 3H, CH2). 13C NMR (75 MHz, CDCl3): δ 161.23, 159.31, 158.27, 148.46, 137.87, 133.53, 132.02, 130.55, 123.72, 121.55, 120.63, 115.91, 64.83, 21.72. 2. 4. Synthesis of Methanol-isothiocyanato- (N’-(pyridin-2-ylmethylene) picolinohydrazido)copper(II) (1) HL1 (0.023 g, 0.10 mmol), copper nitrate trihy- drate (0.024 g, 0.10 mmol) and ammonium thiocyanate (0.0076 g, 0.10 mmol) were mixed in methanol (30 mL). The mixture was stirred at ambient temperature for 30 min to give blue solution. The solvent was slowly evap- orated to give single crystals. Yield: 0.013 g (34%). Anal. Calc. for C14H13CuN5O2S (%): C, 44.38; H, 3.46; N, 18.48. Found (%): C, 44.53; H, 3.55; N, 18.37. IR data (cm–1): 3438w (OH), 2081s (NCS), 1645s (C=O), 1598s (C=N), 1560s, 1475w, 1386m, 1332w, 1289w, 1251w, 1167m, 1082m, 1040m, 760w, 688w, 579w, 511w, 472w. UV–Vis data (methanol, λ/nm (ε/M–1 cm–1)): 215 (17,565), 255 (11,450), 367 (13,270). 2. 5. Synthesis of Isothiocyanato-(4-methyl- 2-(((pyridin-2-ylmethyl)imino)methyl) phenolato)copper(II) (2) Complex 2 was prepared by following the same method as described in section 2.4 for complex 1, but with HL1 replaced by HL2 (0.023 g, 0.10 mmol). Yield: 0.016 g (46%). Anal. Calc. for C15H13CuN3OS (%): C, 51.94; H, 3.78; N, 12.11. Found (%): C, 51.77; H, 3.86; N, 11.98. IR data (cm–1): 2078s (NCS), 1629s (C=N), 1528w, 1461m, 1417w, 1385m, 1318w, 1276w, 1215m, 1160m, 1121w, 1071w, 1052m, 842m, 802w, 761m, 709w, 608w, 561w, 525w, 475w. UV–Vis data (methanol, λ/nm (ε/M–1 cm–1)): 223 (19,675), 246 (17,630), 272 (15,352), 385 (4,533). 2. 6. Synthesis of Azido-(4-methyl-2- (((pyridin-2-ylmethyl)imino)methyl) phenolato)nickel(II) (3) 631Acta Chim. Slov. 2022, 69, 629–637 Juteršek et al.: Synthesis, Crystal Structures and Urease Inhibition ... Complex 3 was prepared by the same method as de- scribed for complex 2, but with ammonium thiocyanate replaced by sodium azide (0.0065 g, 0.10 mmol), and with copper nitrate trihydrate replaced by nickel nitrate hex- ahydrate (0.029 g, 0.10 mmol). Yield: 0.019 g (58%). Anal. Calc. for C14H13N5NiO (%): C, 51.58; H, 4.02; N, 21.48. Found (%): C, 51.39; H, 3.92; N, 21.37. IR data (cm–1): 2041s (N3), 1627s (C=N), 1530w, 1471m, 1449w, 1387m, 1318w, 1279w, 1222m, 1168m, 1140w, 1118w, 1074m, 1054m, 983w, 822m, 763m, 709w, 610w, 560w, 533w, 464w. UV–Vis data (methanol, λ/nm (ε/M–1 cm–1)): 223 (18,720), 242 (17,315), 283 (8,120), 383 (6,350). 2. 7. X-ray Diffraction Diffraction intensities for the complexes were collect- ed at 298(2) K using a Bruker SMART 1000 CCD area-de- tector diffractometer with MoKα radiation (λ = 0.71073 Å). The collected data were reduced with the SAINT,10 and multi-scan absorption correction was performed using the SADABS.11 The structures were solved by direct method and refined against F2 by full-matrix least-squares meth- od using the SHELXL package.12 All of the non-hydrogen atoms were refined anisotropically. Hydrogen atoms were placed in calculated positions and constrained to ride on their parent atoms. The crystallographic data for the com- plexes are summarized in Table 1. 2. 8. Urease Inhibitory Activity Assay The measurement of urease inhibitory activity was carried out according to the literature method.13 The assay mixture containing 75 μL of Jack bean urease and 75 μL of tested compounds with various concentrations (dissolved in DMSO) was preincubated for 15 min on a 96-well assay plate. Acetohydroxamic acid was used as a reference. Then 75 μL of phosphate buffer at pH 6.8 containing phenol red (0.18 mmol L–1) and urea (400 mmol L–1) were added and incubated at 25 °C. The reaction time required for enough ammonium carbonate to form to raise the pH of the phos- phate buffer from 6.8 to 7.7 was measured by a micro-plate reader (560 nm) with the end-point being determined by the color change of phenol-red indicator. 2. 6. Molecular Docking Study Molecular docking study of the molecules of com- plexes 1 and 2 into the 3D X-ray structure of the Jack bean urease was carried out by using the AutoDock 4.0 software as implemented through the graphical user interface Au- Table 1. Crystal data for the complexes 1 2 3 Chemical Formula C14H13CuN5O2S C15H13CuN3OS C14H13N5NiO Fw 378.89 346.88 326.00 T (K) 298(2) 298(2) 298(2) λ (Mo Kα) (Å) 0.71073 0.71073 0.71073 Crystal system Triclinic Monoclinic Triclinic Space group P–1 P21/n P–1 a (Å) 7.0645(10) 6.9831(12) 7.3030(11) b (Å) 10.6425(14) 15.342(1) 9.0155(10) c (Å) 11.4005(16) 13.333(1) 10.5740(12) a (°) 69.749(2) 90 88.622(1) b (°) 75.837(2) 91.873(1) 77.044(1) g (°) 85.187(2) 90 85.605(1) V (Å3) 779.71(19) 1427.7(3) 676.5(2) Z 2 4 2 m (Mo Kα) (cm–1) 1.550 1.677 1.601 Dc (g cm–3) 1.614 1.614 1.601 Reflections 4179 8002 3662 Unique reflections 2885 2639 2307 Observed reflections [I ³ 2s(I)] 2354 1418 1939 Parameters 212 191 191 Restraints 3 0 0 Goodness of fit on F2 1.030 0.984 1.054 Rint 0.0166 0.1392 0.0195 R1 [I ³ 2s(I)] 0.0474 0.0767 0.0347 wR2 [I ³ 2s(I)] 0.1106 0.1278 0.0765 R1 (all data) 0.0625 0.1538 0.0461 wR2 (all data) 0.1194 0.1490 0.0820 ∆ρmax/∆ρmin, e Å–3 1.074/–0.320 0.437/–0.380 0.393/–0.288 632 Acta Chim. Slov. 2022, 69, 629–637 Juteršek et al.: Synthesis, Crystal Structures and Urease Inhibition ... toDockTools (ADT 1.5.2). In the docking, grid box size of 40 × 50 × 58 Å3 for the complex points in x, y, and z di- rections was built, the maps were centered on the original ligand molecule in the catalytic site of the protein. A grid spacing of 0.375 Å and a distance-dependent function of the dielectric constant were used for the calculation of the energetic map. 100 runs were generated by using Lamarck- ian genetic algorithm searches. Default settings were used with an initial population of 50 randomly placed individ- uals, a maximum number of 2.5 × 106 energy evaluations, and a maximum number of 2.7 × 104 generations. A mu- tation rate of 0.02 and a crossover rate of 0.8 were chosen. The results of the most favorable free energy of binding were selected as the resultant complex structures. 3. Results and Discussion 3. 1. Chemistry The Schiff bases HL1 and HL2 were readily prepared by the condensation reaction of equimolar quantities of 2-pyridinecarboxaldehyde with 2-picolinyl hydrazide, and 5-methylsalicylaldehyde with 2-aminomethylpyridine, respectively, in methanol. The copper complexes 1 and 2 were prepared by the reaction of equimolar quantities of the Schiff bases, copper nitrate and ammonium thiocy- anate in methanol. To study the influence of the anions of copper salts on the structures of the complexes, we tried to use copper acetate in the syntheses, yet, the same struc- tures as those prepared with copper nitrate have been ob- tained. The nickel complex 3 was prepared by the reaction of equimolar quantities of the Schiff base, nickel nitrate and sodium azide in methanol. The complexes are solu- ble in methanol, ethanol, acetonitrile, DMSO and DMF. Single crystals were obtained by slow evaporation of the methanolic solution of the complexes. The free Schiff bases and the complexes are stable in air at 25 °C. 3. 2. Structure Description of Complex 1 The molecular structure of the complex is shown in Fig. 1. Selected bond lengths and angles are given in Table 2. The Cu atom is five-coordinated in a square pyramidal geometry, with the three nitrogen atoms (N1, N3, N4) of the Schiff base ligand L and the thiocyanate nitrogen atom (N5) defining the basal plane, and with the methanol ox- ygen atom (O2) occupying the apical position. The distor- tion of the square pyramidal coordination can be observed by the bond angles around the Cu center. The cis and trans angles in the basal plane are 82.31(14)–94.32(15)° and 168.66(13)–174.99(16)°, respectively. The bond angles among the apical and basal donor atoms are 89.22(14)– 96.05(11)°. The Cu–N bond lengths related to the Schiff base ligand are 1.938(3)–2.033(3) Å, and the Cu–N bond length related to the thiocyanate ligand is 1.964(4) Å, which are comparable to the copper(II) complexes with similar ligands.14 The apical bond length of Cu1–O2 is Scheme 3. The synthetic procedure for the complexes. Fig. 1. A perspective view of the molecular structure of complex 1 with the atom labeling scheme. Thermal ellipsoids are drawn at the 30% probability level. 633Acta Chim. Slov. 2022, 69, 629–637 Juteršek et al.: Synthesis, Crystal Structures and Urease Inhibition ... longer than the basal bonds, which is not uncommon for methanol coordinated complexes.15 In the crystal structure of the complex, two adjacent molecules are linked through intermolecular O‒H···N and O‒H···O hydrogen bonds (Table 3), to form a dimer. The dimers are linked through intermolecular C‒H···O hydro- gen bonds (Table 3), to form ladder like chains along the b axis (Fig. 2). Moreover, there are π···π interactions among the molecules (Table 4). Fig. 2. Molecular packing diagram of complex 1, viewed along the c axis. Hydrogen bonds are shown as dashed lines. Table 2. Selected bond lengths (Å) and angles (°) for the complexes 1 Cu1–N1 2.033(3) Cu1–N3 1.938(3) Cu1–N4 2.033(3) Cu1–N5 1.964(4) Cu1–O2 2.365(3) N3–Cu1–N5 174.99(16) N3–Cu1–N1 90.05(14) N5–Cu1–N1 94.32(15) N3–Cu1–N4 82.31(14) N5–Cu1–N4 93.00(15) N1–Cu1–N4 168.66(13) N3–Cu1–O2 92.76(12) N5–Cu1–O2 89.22(14) N1–Cu1–O2 96.05(11) N4–Cu1–O2 92.69(12) 2 Cu1–O1 1.895(4) Cu1–N1 2.004(5) Cu1–N2 1.921(5) Cu1–N3 1.936(6) O1–Cu1–N2 93.73(19) O1–Cu1–N3 89.7(2) N2–Cu1–N3 176.4(2) O1–Cu1–N1 176.10(19) N2–Cu1–N1 82.4(2) N3–Cu1–N1 94.2(2) 3 Ni1–O1 1.831(2) Ni1–N1 1.842(2) Ni1–N2 1.908(2) Ni1–N3 1.909(3) O1–Ni1–N1 94.84(10) O1–Ni1–N2 178.74(9) N1–Ni1–N2 84.92(10) O1–Ni1–N3 88.10(11) N1–Ni1–N3 176.74(11) N2–Ni1–N3 92.18(12) 3. 3. Structure Description of Complexes 2 and 3 The molecular structures of complexes 2 and 3 are shown in Figs. 3 and 4, respectively. Selected bond lengths and angles are given in Table 2. Both the complexes are of distorted square planar geometry. The Cu and Ni atoms are coordinated by the phenolate O, imino N and pyridine N atoms of the Schiff base ligands, and the N atoms of thi- ocyanate (for 2) and azide (for 3) ligands. The distortion of the square planar coordination can be observed by the bond angles around the metal centers. The cis and trans angles are 82.4(2)–94.2(2)° and 176.1(2)–176.4(2)° for 2, and 84.9(1)–94.8(1)° and 176.7(1)–178.7(1)° for 3, respec- tively. The Cu–O and Cu–N bond lengths in complex 2 are longer than the Ni–O and Ni–N bond lengths in complex 3, which are in accordance with the bond values observed in similar copper(II) and nickel(II) complexes with Schiff base ligands.14,16 In the crystal structure of complex 2, the molecules are stacked with π···π interactions (Table 4, Fig. 5). In the crystal structure of complex 3, two adjacent molecules are linked through intermolecular C‒H···N hydrogen bonds (Table 3), to form a dimer (Fig. 6). Moreover, there are π···π interactions among the molecules (Table 4). Fig. 3. A perspective view of the molecular structure of complex 2 with the atom labeling scheme. Thermal ellipsoids are drawn at the 30% probability level. Fig. 4. A perspective view of the molecular structure of complex 3 with the atom labeling scheme. Thermal ellipsoids are drawn at the 30% probability level. 3. 4. IR Spectra The infrared spectra of the free Schiff bases and the complexes were recorded in the region of 4000–400 634 Acta Chim. Slov. 2022, 69, 629–637 Juteršek et al.: Synthesis, Crystal Structures and Urease Inhibition ... cm–1 using KBr pellets. There is a weak and sharp band at 3290 cm–1 in the spectrum of HL1, which is assigned to the NH vibration. The broad and weak bands at 3378- 3438 cm–1 of HL2 and complex 1 can be assigned to the OH vibrations. The bands at 1647 cm–1 for HL1 and 1645 cm–1 for complex 1 are due to the azomethine group, μ(C=N).17 They are almost in the same frequency, indi- cates that the imine N atom is not participate in coordi- nation. The intense band for the C=O group is observed at 1698 cm–1 for HL1. The absence of the band in the spectrum of complex 1 indicates that the C=O group turned to other form. The bands at 1638 cm–1 for HL2 and 1627–1629 cm–1 for complexes 2 and 3 are due to the azomethine group.17 The shift to lower frequencies Fig. 5. Molecular packing diagram of complex 2, viewed along the c axis. Fig. 6. Molecular packing diagram of complex 3, viewed along the b axis. Hydrogen bonds are shown as dashed lines. Table 3. Hydrogen bond distances (Å) and bond angles (°) for com- plexes 1 and 3 D–H∙∙∙A d(D–H) d(H∙∙∙A) d(D∙∙∙A) Angle (D–H∙∙∙A) 1 O2–H2∙∙∙N2i 0.86(1) 2.40(3) 3.038(5) 132(3) O2–H2∙∙∙O1i 0.86(1) 2.09(2) 2.852(5) 148(3) C2–H2A∙∙∙O1 0.93 2.48(2) 3.399(5) 170(3) 3 C2–H2∙∙∙N5ii 0.93 2.62(3) 3.510(5) 161(4) Symmetry codes: (i) 1 – x, 1 – y, 1 – z; (ii) 1 – x, 1 – y, – z. Table 4. π···π interactions of the complexes Cg···Cg distance (Å) Cg···Cg distance (Å) 1 Cg1···Cg1iii 3.448(5) Cg1···Cg2iii 3.818(5) Cg1···Cg2i 4.445(5) Cg1···Cg3iii 4.645(5) Cg2···Cg2i 4.938(5) Cg2···Cg3iii 3.636(5) Cg4···Cg4i 3.935(5) Cg4···Cg3iii 4.651(5) 2 Cg5···Cg6i 3.794(4) Cg1···Cg2iv 4.967(4) Cg5···Cg8i 3.507(4) Cg1···Cg4iv 3.569(4) Cg6···Cg6i 3.389(4) Cg2···Cg2iv 3.612(4) Cg6···Cg8i 4.610(4) Cg2···Cg4iv 3.601(4) Cg7···Cg8i 3.721(4) Cg3···Cg4iv 4.823(4) 3 Cg9···Cg9i 3.982(4) Cg1···Cg2iii 4.006(4) Cg9···Cg10i 3.558(4) Cg1···Cg4iii 3.563(4) Cg9···Cg12i 4.776(4) Cg2···Cg2iii 3.479(4) Cg10···Cg10i 4.624(4) Cg2···Cg3i 3.781(4) Cg10···Cg12iii 4.530(4) Cg3···Cg4iii 3.799(4) Cg11···Cg12i 3.638(4) Symmetry codes: iii: – x, 1 – y, 1 – z; iv: 2 – x, 1 – y, 1 – z. Cg1, Cg2, Cg3 and Cg4 are the centroids of Cu1-N3-C7-C8-N4, Cu1-N1-C5- C6-N2-N3, N4-C8-C9-C10-C11-C12 and N1-C1-C2-C3-C4-C5 in complex 1, respectively. Cg5, Cg6, Cg7 and Cg8 are the centroids of Cu1-N1-C5-C6-N2, Cu1-O1-C9-C8-C7-N2, N1-C1-C2-C3-C4-C5 and C8-C9-C10-C11-C12-C13 in complex 2, respectively. Cg9, Cg10, Cg11 and Cg12 are the centroids of Ni1-N1-C6-C5-N2, Ni1- O1-C9-C8-C7-N1, N2-C1-C2-C3-C4-C5 and C8-C9-C10-C11- C12-C13 in complex 3, respectively. 635Acta Chim. Slov. 2022, 69, 629–637 Juteršek et al.: Synthesis, Crystal Structures and Urease Inhibition ... in the spectra of the complexes indicates that the imine N atoms form coordination bonds with the metal atoms. The typical absorption for the thiocyanate ligands in complexes 1 and 2 is observed at 2078–2081 cm–1.18 The typical absorption for the azide ligand in complex 3 is observed at 2041 cm–1.19 3. 5. Urease Inhibitory Activity Complexes 1 and 2 have excellent inhibitory ac- tivity on the Jack bean urease, with IC50 values of 0.33 ± 0.27 and 0.39 ± 0.10 μmol L–1, respectively, whereas the free Schiff bases HL1 and HL2, and the nickel complex 3 have weak activity (> 50 μmol L–1). The reported copper complexes have shown better activity than the reference drug acetohydroxamic acid (IC50 = 28.5 ± 2.7 μmol L–1) and the copper nitrate (IC50 = 8.6 ± 1.5 μmol L–1). The two copper complexes have better activity against urease than the copper(II) complex with the Schiff base ligand N,N’-bis(4-fluorosalicylidene)-1,2-diaminopropane (IC50 = 2.1–3.4 μmol L–1),20 and the copper(II) complex with the reduced Schiff base ligand 2,2’-((propane-1,3-diylb- is(azanediyl))bis(methylene)diphenol (IC50 = 1.6 μmol L–1).21 3. 6. Molecular Docking Study on Complexes 1 and 2 Molecular docking study was performed to inspect the binding effects between the molecules of complexes 1 and 2 with the Jack bean urease. The binding models of the complexes with the urease are depicted in Figs. 7 and 8. The results indicate that the complex molecules fit well with the active site of the urease. The interactions of the complex molecules with the urease have been established in a variety of conformations because of the flexibility of the molecules and the amino acid residues of the urease. The binding energy is –5.78 kcal/mol for 1 and –5.78 kcal/ mol for 2. It is lower than the binding energy of the AHA inhibited model (–5.01 kcal/mol). The negative values re- veal that the complex molecules combine well with the center of the urease. Fig. 7. Binding mode of the molecule of complex 1 with Jack bean urease. Left: The enzyme is shown as surface, and the complex is shown as sticks. Right: The enzyme is shown as ribbons, and the complex is shown as a filling model. Fig. 8. Binding mode of the molecule of complex 2 with Jack bean urease. Left: The enzyme is shown as surface, and the complex is shown as sticks. Right: The enzyme is shown as ribbons, and the complex is shown as a filling model. 636 Acta Chim. Slov. 2022, 69, 629–637 Juteršek et al.: Synthesis, Crystal Structures and Urease Inhibition ... 4. Conclusion In summary, the present paper intends to report the syntheses, crystal structures and urease inhibition activity of three mononuclear copper(II) and nickel(II) complexes with the tridentate Schiff base ligands N’-(pyridin-2-yl- methylene)picolinohydrazide and 4-methyl-2-(((pyri- din-2-ylmethyl)imino)methyl)phenol. Both the copper complexes have shown effective inhibitory activity on Jack bean urease. Appendix A. Supplementary material CCDC 2160252 (1), 2163190 (2) and 2163191 (3) contain the supplementary crystallographic data for this article. 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Atom Cu je v kompleksu 1 kvadratno piramidalno koordiniran s tremi N atomi liganda L in N atomom tiocianatnega liganda v ekvatorialnem položaju ter z O atomom metanola v apikalnem položaju. Atoma Cu in Ni sta v kompleksih 2 in 3 kvadratno planarno koordinirana s tremi donorskimi atomi ligandov Schiffove baze in terminalnimi N atomi tiocianatnih in azidnih ligandov. Kompleksa 1 in 2 zavirata ureazo stročnice Canavalia ensiformis z vrednostjo IC50 0,33 ± 0,12 oziroma 0,39 ± 0,10 μmol L–1. Za preučitev interakcij med kompleksi in encimom je bila izvedena študija molekularnega dockinga. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 638 Acta Chim. Slov. 2022, 69, 638–646 Aslan et al.: Electroanalytical Determination of Ziram by Differential ... DOI: 10.17344/acsi.2022.7517 Scientific paper Electroanalytical Determination of Ziram by Differential Pulse Voltammetry with Reduced Graphene Oxide/Gold Nanoparticles Modified Glassy Carbon Electrode Nazife Aslan,1* Sema Bilge Ocak2 and Uğur Gökmen3 1 Ankara Hacı Bayram Veli University, Polatlı Science and Arts Faculty, Department of Chemistry, 06900 Ankara, Turkey 2 Gazi University, Graduate School of Natural and Applied Sciences, 06560 Ankara, Turkey 3 Gazi University, Faculty of Technology Metallurgical and Materials Engineering, 06500 Ankara, Turkey * Corresponding author: E-mail: nazife.aslan@hbv.edu.tr Received: 04-04-2022 Abstract The preparation of gold nanoparticles-reduced graphene oxide-based sensor materials for the determination of zinc(II) dimethyldithiocarbamate (ziram) is described in this paper. The graphene oxide (GO) was synthesized using a modified Hummer’s method. A composite sensor consisting of gold nanoparticles (AuNPs) and reduced graphene oxide (RGO) was electrochemically fabricated on a glassy carbon electrode. The nanocomposite was evaluated utilizing scanning elec- tron microscopy (SEM). Cyclic voltammetry was used to illuminate the modified sensor’s electrochemical properties at each stage of the modification. The suggested sensor was demonstrated good analytical performance to determine ziram pesticide in water and peach juice, including a very low detection limit, a large linear range, and a low RSD. Keywords: Reduced graphene oxide; ziram; nanomaterials; sensors 1. Introduction Materials having sizes or properties ranging from 1 to 100 nm in one or more dimensions are called nanoma- terials. Superior thermal, mechanical, electrical, and bio- logical properties not available in conventional materials are the important characteristics of these materials.1 The combination of these distinctive properties with their remarkable recognition abilities has resulted in im- proved performance. Apart from their high mechanical strength and low weight, nanomaterials’ surface features, including area, roughness, energetics, and electron distri- butions, are primarily the result of their unique proper- ties. It is obvious that nanomaterials, which has applica- tions such as providing clean drinking water, improving air quality, developing new energy sources and at the same time removing dangerous and toxic substances from our environment, will help create a sustainable environment.2 Graphene is one of the most important nanomate- rials, with a wide range of applications that are expand- ing.3,4 It is made up of sp2 bonded carbon atoms with a single atom thickness, as is well known. As a result of these characteristics, it exhibits remarkable electron transport capability and catalytic behavior for particular chemicals. Overall, due to its high spesific surface area, low cost, ease of processing and safety, and superior electrical conductiv- ity, it can play a vital role in increasing the performance of sensors.5,6 With its potential application areas, it is one of the most investigated materials nowadays.7 Graphene, on the other hand, is hydrophobic and does not form stable dispersions in polar solvents.8 This severely limits its use in sensor development. An effective method for overcoming this problem is in situ chemical or electrochemical reduc- tion of highly hydrophilic graphene oxide (GO) to pro- duce graphene.9 The electrochemical reduction method is commonly used because it is a green process that does not require a strong chemical reducing agent.10 Metal nanoparticles (NP) have qualities that are de- termined by their size and form. Chemical and biosensors, catalyst synthesis, electronic device component prepa- ration, imaging systems, medical and environmental ap- plications all use a variety of metal NPs in various sizes, forms, and morphologies.11,12 Among them gold nanopar- ticles (AuNPs) have been received great interests as sensor 639Acta Chim. Slov. 2022, 69, 638–646 Aslan et al.: Electroanalytical Determination of Ziram by Differential ... devices due to its high selectivity, sensitivity, biocompabil- ity and excellent chemical stability. Especially, the intro- duction of AuNPs into modified electrodes has obvious advantages in improving the sensor performances.13,14 It has been stated in the literature that sensors made with reduced graphen oxide (RGO)-metal nanocomposites su- perior qualities such as sensitivity, lower detection limits, and faster electron transfer kinetics.15,16 RGO has hydroxyl (-OH) and carboxylate (-COOH) groups in its structure, which allows it to interact with metal nanoparticles to cre- ate a metal nanoparticle-graphene based electrochemical sensor.17,18 Therefore, AuNPs/RGO have recently been used in electrochemical sensors for pesticide and other or- ganic and inorganic pollutant detection.19–24 Pesticides are widely employed as agrochemicals to enhance agricultural production by controlling or killing insects, pests, and fungi. Uncontrolled pesticide use, on the other hand, could endanger public health.25,26 Ziram is a dithiocarbamate (DTC) fungicide that is commonly used to control moulds, black spot, rot, and blight, as well as to maintain the quality of fruits and vegetables through- out transit and storage. Ziram residues, on the other hand, can cause major health problems, such as headaches and nausea, as well as cancer. It’s also linked to skin allergies, asthma, Parkinson’s disease risk, and inflammation of the eyes and respiratory tract.27–32 Several analytical instruments, such as high-per- formance liquid chromatography followed by atomic absorption spectrometry (HPLC-AAS),33 liquid chro- matography-mass spectrometry (LC-MS/MS),34 and gas chromatography–mass spectrometry (GC–MS),35 gas chromatography-electron capture detector (GC-ECD),36 inductively coupled plasma mass spectrometry37 are wide- ly used in monitoring environmental contaminants such as ziram in agricultural products. Electrochemical detec- tion38–43 and immunoassays44 are some of the other rapid methods for detecting trace compounds that have been proposed. Because of their numerous advantages, such as rapid response, a wide dynamic range, portability, ease of modification, and low cost, electrochemical sensors are a viable and rapid instrument for detecting pesticide resi- dues in food and environmental samples. The goal of this research was to explore if composites of reduced graphene oxide and gold nanoparticles might be employed as an electrochemical sensor material for low-concentration voltammetry-based pesticide residue monitoring. A glassy carbon electrode (GCE) modified with RGO and AuNPs was used to create and measure a new voltammetric sensor for the determination of ziram. The results indicated that the AuNPs/RGO-modified glassy carbon electrode could provide a quick and easy platform for ziram detection with high sensitivity, fast response, and wide detection range. So far, only a few electrochemical methods for the detection of ziram using nanocomposite sensors have been published, and real sample applications in foods are extremely limited.45,46 As a result, it is critical to develop new methods that will serve as an alternative to existing analysis methods. Using a scanning electron microscope, the surface specimens of the produced RGO/AuNPs/GCE were ex- amined. For AuNPs electrodeposition and ziram deter- mination, cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were utilized. Furthermore, limit of detection, limit of quantification, linearity, repeatability, reproducibility and pH of the sensor were investigated in detail. The RGO/AuNPs/GCE has been successfully used as electrochemical sensor to determining of ziram fungi- cide in peach juice and tap water samples. 2. Experimental 2. 1. Reagents Graphite (Alfa Aeser, <20 µm), HAuCl4.H2O 99.995%, zinc(II) dimethylditiyocarbamate (99.9%), K3[Fe(CN)6], K4[Fe(CN)6], hydrogen peroxide, boric acid, o-phosphoric acid and sulfuric acid were provided by Sigma Aldrich; Merck supplied sodium nitrate (NaNO3), potassium permanganate (KMnO4), sodium hydroxide, sodium acetate, hydrochloric acid, sodium dihydrogen- phosphate.2H2O, sodium monohydrogenphosphate.7H2O and potassium chloride. The Britton Robinson (BR) buffer solution and all other solutions were made with ultrapure water. All experiments were carried out at room tempera- ture. All sensor applications were performed in BR buffer with a pH of 8.0 and 100 mM KCl as a supporting elec- trolyte. 2. 2.Instrumentation The CH Instruments 660B model Ivium potentio- stat/galvanostat Electrochemical Analyzer (Ivium Tech- nologies, Netherlands) was used for all electrochemical experiments. A triple electrode system was used in the experiments, including an Ag/AgCl reference electrode, a glassy carbon working electrode, and a Pt wire counter electrode. Carl Zeiss AG’s EVO® 50 Series was used to capture scanning electron microscopic (SEM) pictures. An ORI- ON Model 720A pH/ion meter and a combined glass elec- trode were used to obtain the pH readings. The pH-meter was calibrated with commercial pH 4.0; 7.0 and 10.0 buffer solutions prior to the measurements. When not in use, the glass electrode was immersed in deionized water. 2. 3. Graphene Oxide Synthesis Graphene oxide is synthesized from graphite powder using a modified Hummer process.47 5 g graphite powder, 2.5 g sodium nitrate (NaNO3), and 115 mL 96.4% sulfuric acid (H2SO4) were mixed in the first step of the synthesis process. In an ice bath, the entire mixture was agitated for 640 Acta Chim. Slov. 2022, 69, 638–646 Aslan et al.: Electroanalytical Determination of Ziram by Differential ... 1 hour and, 15 g potassium permanganate (KMnO4) was gently added to the mixture. The temperature was kept below 5 °C for the permanganate addition. The solution was taken out of the ice bath and stired for 2 hours until it turned dark green. The temperature of the mixture was kept between 35–40 °C during these procedures. 500 mL deionized water was gently added to the mixture in the second step of the synthesis process, and stirring was continued for 1 hour. To remove excess KMnO4, 8.4 mL of hydrogen per- oxide (H2O2, 35.7%) was gently dropped and stirred for 10 minutes. The exothermic process happened, and the tem- perature was allowed to fall to room temperature. Following a 10-minute centrifugation at 5000 rpm, 10 mL hydrochloric acid and 30 mL deionized water were added. After that, the supernatant was decanted, and the remaining residue was rewashed three times with an HCl/ deionized water mixture until pH 7 was achieved. As a re- sult, the prepared GO was vacuum-dried overnight at 50 °C for 24 hours. 2. 4. Characterization of Graphene Oxide Graphene oxide nanostructures were investigated using a Zeiss Evo 60 EP model Scanning Electron Micro- scope (SEM) with magnifications of 2500 X and accelerat- ing voltages of 15 kV. 2. 5. RGO/AuNPs/GCE Nanocomposite Sensor Fabrication The non-modified GCE (nGCE) was polished man- ually with Al2O3 suspension (0.3 m, ATM GMBH, Germa- ny), rinsed with deionized water, and sonicated in ethanol and double-distilled water for 5 minutes, respectively. GO was dispersed into sodium acetate buffer by stir- ring at room temperature, and the resultant liquid was ul- trasonicated for 4 hours, providing a homogeneous black dispersion containing 1 mg mL–1 GO. The buffer solution of sodium acetate serves as both a buffer and an intercalant. The intercalation of sodium ions inhibits restacking of the electrochemically reduced graphene sheets, resulting in a larger electrochemically active surface area for the RGO modified electrode. The electrode was cleaned with deionized water after elec- trochemical reduction and placed in a 50°C oven for 15 minutes to thoroughly evaporate the solvent and increase RGO molecule adherence to the electrode surface. The GO dispersion was then dropped 5 µL onto a pre-cleaned GCE and let to dry at room temperature. The GO/GCE was placed in an electrochemical cell containing an acetate buffer solution (pH = 5) and 50 cyclic voltam- metric scans between (+0.4) V and (–0.4) V were done at a scan rate of 0.050 V/s. As a result, the GO treated GCE was electrochemically reduced to RGO and dried in the open air for 10 minutes. RGO/GCE was then immersed in a 3 mmol L–1 HAuClO4.H2O solution prepared in 0.01 mol L–1 Na2SO4 and 0.01 mol L–1 H2SO4 solution in the measurement cell. To electrodeposit Au nanoparticles (AuNPs) on the RGO/GCE, 20 consecutive cycles in the potential range of 0.2 to +1.0 V at a scan rate of 0.050 V s–1 were utilized. The modified sensor was labeled RGO/ GCE/AuNPs, dried, and used as an electrochemical sen- sor. When it wasn’t in use, the sensor was kept at room temperature. 2. 6. Electrochemical Measurements Appropriate volumes of supporting electrolyte (KCl) and pesticide standard solution were added to the electro- chemical cell with a total volume of 5.0 mL in the cyclic voltammetry and differential pulse voltammetry meth- ods used in this study. To record the background signals, voltammogram of the supporting electrolyte was obtained before adding the pesticide solution to be examined. At a scanning rate of 0.050 V s–1, cyclic voltammetric measure- ments were taken. According to the potential signaled by the pesticides, the most appropriate potential range was employed in both methods. Cyclic voltammograms of the GCE and modified electrode were acquired by scanning the potential between 0.80 V and +1.00 V vs. Ag/AgCl at a scan rate of 0.050 V s–1. All other voltammetric meas- urements were performed in a BR buffer solution at room temperature (25 ± 1 °C) (0.04 mol L–1, pH 8.0) 2. 7. Optimization of the Experimental Conditions for Ziram To create a highly sensitive method with a low detec- tion limit, it’s crucial to identify the most effective experi- mental conditions. On bare and modified GCE, the effect of scan rate, pH, and supporting electrolyte on the voltam- metric response of ziram was examined. The sensitivity of the assay was shown by putting the constructed sensor to the test with real samples. 2. 8. Real Sample Application of the Sensor Tap water and peach juice samples were analyzed us- ing the spiking approach to determine the applicability of the RGO/AuNPs/GCE sensor. Tap water samples were taken from our laboratory and spiked with a certain amount of standard ziram solu- tion. To maintain a homogeneous mixture, it was agitated for 3 hours in an ultrasonic bath. This solution was added to electrochemical cells containing 100 mM KCl in Brit- ton-Robinson (BR) buffer solution (0.04 mol L–1, pH 8.0) in quantities of 250 µL, 500 µL, and 1500 µL. The DPV method was used to analyze the samples. Peach juice was also tested to the method’s appli- cation. Peaches were picked from a farmer’s garden that practices organic farming and avoids using pesticides. The 641Acta Chim. Slov. 2022, 69, 638–646 Aslan et al.: Electroanalytical Determination of Ziram by Differential ... peach juice obtained by squeezing the fruit was filtered through the filter paper and the pulp was removed. In 25 mL of peach juice, a known amount of 1.01 mM ziram stock solution was added. To get a homogenous mixture, it was sonicated for 2–3 hours in an ultrasonic bath. 25 mL acetone was added to the mixture before it was transferred to centrifuge tubes. The organic phase was filtered via a Buchner funnel using Whatman filter paper (No.4) after centrifugation at 4000 rpm for 10 minutes. To remove the solvent, the filtrate was transferred to a 250 mL rotating vacuum evaporator vessel. After the solvent had evaporat- ed, the residue was dissolved in acetone to yield a total vol- ume of 5.0 mL. The blank sample was made by following the identical steps as the peach juice sample that did not contain ziram. 3. Results and Discussion 3. 1. Characterization of Graphene Oxide SEM image of the prepared GO is presented Figure 1. From the SEM image it is evident that GO has a multiple lamellar layer structure and it is possible to distinguish the edges of individual sheets. The layers are stacked one above the other and also show wrinkled areas, which could be at- tributed to intrinsic and extrinsic factors such as thermal fluctuation, defects, and functionalization. The wrinkled structure of the GO can increase the effective surface area and thus provide a good platform for bonding the AuNPs. Figure 1. SEM images of synthesized graphene oxide. 3. 2. Optimization of RGO/AuNPs Sensor Fabrication The electropolimerization cycles were investigated to achieve the best responses for ziram determination. Effect of GO concentration on sensor response was investigated using 0.5 mg mL–1; 1.0 mg mL–1; 1.5 mg mL–1; and 2.0 mg mL–1. The highest current response was observed with the electrode prepared with 1.0 mg mL−1 GO and this value was selected as the optimum GO concentration (Fig. 2). GO concentration higher than 1.0 mg mL−1 did not in- crease the sensor response. The results could be attributed to the thicker RGO layer, which restricted electrical con- ductivity. Figure 2. Effect of GO concentration on the response of the RGO/ GCE. Fig. 3 shows the cyclic voltammogram of electro- chemical reduction peak of graphene oxide at –1.14 V. Electrons act as a reducing agent, causing RGO to occur on the GCE surface. Wang et al. reported the electrochem- ical reduction mechanism of graphene oxide with two assumptions.48 The reduction of newly formed hydrogen atoms produced near the electrode surface during the wa- ter electrolysis process was one of the expectations. The following reaction takes place during water electrolysis. Figure 3. CV of electrochemical reduction of 1.0 mg mL−1 graphene oxide on GCE surface in acetate buffer solution (pH = 5) at the scan rate of 0.050 V s−1 in the potential range of (−1.2) – (2.2) V. 642 Acta Chim. Slov. 2022, 69, 638–646 Aslan et al.: Electroanalytical Determination of Ziram by Differential ... Hydrogen gas produced at the edges of graphene oxide can also contribute to the reduction process of graphene oxide. During the electrochemical reduction process, a ca- thodic peak was observed due to the formation of reduced graphene oxide. The continuous deposition of conducting reduced graphene oxide on the electrode surface was ev- idenced by the linear increase in peak current with con- secutive cycles. The current intensity stabilized after ap- proximately 15–20 cycles and the electrochemically active surface area reached its maximum value. Then, the RGO/ GCE was washed with ultrapure water. According to the literature, gold nanoparticles were successfully deposited on the RGO/GCE surface.49 Figure 4. Repetitive cyclic voltammogram of RGO/GCE in 0.01 mol L–1 H2SO4 solution containing 3 mmol L–1 HAuClO4 and 0.01 mol L–1 Na2SO4 at a scan rate of 0.050 V s–1 in the potential range of (−0.4) – (0.4) V. 3. 3. Electrochemical Characterization of RGO/AuNPs/GCE Nanocomposite The cyclic voltammogram (CV) of the [Fe(CN)6]3–/4– redox probe is a useful method for investigating the char- acteristics of surface-modified electrodes. For this, electro- chemical characteristics of the modified and unmodified sensors were investigated in 100 mM KCl containing 5.0 mM of [Fe(CN)6]3–/4– ions. Figure 5 shows the CVs record- ed for GCE, RGO/GCE, and AuNPs/RGO/GCE. In these three voltammograms, reversible peaks of [Fe(CN)6]3–/4– were observed. Although all voltammograms showed a pair of redox peaks corresponding to Fe3+/Fe2+, the cur- rent intensity varied. Because of the RGO’s large surface area and great conductivity, it was observed that peak currents increased slightly once the GCE surface was modified with RGO. When the surface was modified with gold nanoparticles, the peak currents were significantly increased compared to the currents obtained with GCE and RGO/GCE. These changes can be interpreted as that AuNPs assisting elec- tron transfer between the redox probe and the electrode. This finding supports the idea that combining the two na- nomaterials, RGO and AuNPs, increased the electrode’s sensitivity by raising the current intensity or enhance the current due to electro catalytical effect and large surface area.50 Figure 5. Cyclic voltammograms of GCE, RGO/GCE and AuNPs/ RGO/GCE in 5.0 mM [Fe(CN)6]3–/4– containing 100 mM KCl. 3. 4. Electrochemical Performance of the Sensors In 0.04 mol L–1 BR buffer solution (pH 8.0), the CV responses of the bare GCE, RGO/GCE, and AuNPs/RGO/ GCE sensors to 1.50 × 10–3 mol L–1 ziram were individual- ly examined. Figure 6 depicts a comparison of voltammo- grams obtained using GCE, RGO/GCE, and AuNPs/RGO/ GCE sensors under the same experimental conditions. The anodic peak current of ziram was investigated using the CV results. Because of the distribution of AuNPs on the electrode surface, the current measured at AuNPs modi- fied RGO/GCE was significantly higher than the current measured at RGO/GCE and bare GCE. As a result of their distinctive properties, the simultaneous presence of RGO and AuNPs improved the sensitivity of ziram detection. A synergic effect of their combination was demonstrated as a result of a larger surface area and increased conductivity. Figure 6. The cyclic voltammograms of 1.50 × 10–3 mol L–1 ziram in BR buffer solution with pH 8.0 at scan rate 0.050 v s–1, for the bare GCE, RGO/GCE and AuNPs/RGO/GCE. 643Acta Chim. Slov. 2022, 69, 638–646 Aslan et al.: Electroanalytical Determination of Ziram by Differential ... 3. 5. Optimization of Experimental Conditions The pH of the supporting solution has critical im- portance in obtaining good analytical performance for a developed sensor. Therefore, the effect of pH was investi- gated for ziram in 0.04 mol L–1 BR buffer solutions. This study was carried out at 1.52 × 10–3 mol L–1 constant ziram concentration over the pH range from 5.0 to 10.0. The var- iation of peak currents and peak potentials of the voltam- mograms recorded for the ziram oxidation were given in Table 1. The current response of the AuNPs/RGO/GCE sensor increased with the pH increasing from 5.0 to 8.0 and then gradually decreased from 8.0 to 10.0 (Fig. 7). As seen from the Figure, at pH 5.0 AuNPs/RGO/GCE sensor showed small anodic peak at around 0.72 V. But increase of pH value causes increase of peak currents up to pH 8.0. Only a fluctuation was observed for the peak potential at pH 7.0. The voltammetric response was pH sensitive and maximum peak current was appeared at pH 8.01. As a re- sult, BR buffer solution at pH 8.01 was chosen for the fol- lowing work. The ziram’s oxidation peak potential shifted to less negative values ranging from 8.0 to 10.0, indicating proton transfer participation in the electrode reaction. Figure 7. Effect of pH of ziram solutions on the current intensity of AuNPs/RGO/GCE Table 1. Variation of peak potential and peak current of 1.52 × 10–3 mol L–1 ziram solution at a scan rate of 0.050 V s–1 and different pH in 0.04 mol L–1 BR buffer solutions. pH Peak current (µA) Peak potential (V) 5.01 2.027 0.722 6.04 5.256 0.744 7.00 5.445 0.676 8.01 6.605 0.710 9.08 5.430 0.652 10.03 3.815 0.648 The scan rate is an important parameter to evaluate the electrochemical behaviour, adsorption and diffusion properties of ziram on the electrode surface. Therefore, the effect of scan rate on the oxidation peak current of 1.0 × 10–4 mol L–1 ziram was studied. The variation of the peak current of ziram versus the square root of the scan rate was plotted (Figure 8). It has been observed that with the scan rate increasing, the anodic peak current increased. In the 0.050–0.300 V s–1 range, there was good linearity between the square root of scan rate and peak current. The linear regression equation was (µA) = 0.2783 (µA s) 0.0785 (µA) with correlation coefficient 0.9919. The correlation coeffi- cient is very close to 1.0, indicating that the oxidation pro- cess is controlled by diffusion.51,52 In addition, it was observed that logarithm of peak current changed linearly with the logarithm of scan rate and slope value for this linear line is 0.6346. For ideal dif- fusion-controlled the slope is between 0.5 and 1.0.53 Figure 8. Variation of anodic peak current (Ipa)versus the square root of scan rate (υ1/2) 3. 6. Analytical Performance Parameters of the Sensor The correlation between ziram concentrations and anodic peak currents was examined utilizing the DPV method in BR buffer solution (pH 8.0) with the AuNPs/ RGO/GCE composite sensor under optimized experimen- tal conditions. The calibration graph was shown in Fig. 9. Over the range of 1.50 × 10–5 mol L–1 to 1.63 × 10–7 mol L–1, the peak current increases linearly with the increas- ing ziram concentration. The linearity between the anodic peak current and the ziram concentration is shown in the following equation; (1) The linear range of the calibration curve is 1.50 × 10–5 mol L–1 to 1.63 × 10–7 mol L–1 with the LOD values of 1.19 × 10‒7 mol L–1 for ziram. The formulas (3 × s/m) and (10 × s/m) were used to calculate the method’s limit of de- tection (LOD) and limit of quantification (LOQ).54 Where s denotes the measurement’s standard deviation and m de- notes the calibration curve’s slope (or the sensitivity). 644 Acta Chim. Slov. 2022, 69, 638–646 Aslan et al.: Electroanalytical Determination of Ziram by Differential ... LOD value satisfies the MRLs established by the The Codex Alimentarius Commision (CAC),  for stone fruits (7.0 mg kg–1).55 Particular pesticide limit levels have also been set at 0.1 μg L–1 by the European Union. This value was decided as the LOD for all pesticides found in drink- ing water. That means the method is sensitive enough and the developed sensor can be used with high reliability in detecting the maximum allowable residue level of ziram in fresh fruits and water. All validation and regression pa- rameters are tabulated in Table 2. Table 2. Analytical performance of the RGO/AuNPs/GCE sensor for ziram. Parameters Value Linear working range, mol L–1 1.50 × 10–5 – 1.63 × 10–7 LOD (mol L–1) 1.19×10‒7 LOQ (mol L–1) 7.80×10‒7 Calibration equation Ipa (μA)= 2 × 10 7 cziram (μM) + 5.5336 Regression coefficient (R2) 0.9996 Selectivity (μA/μM) 2 × 107 Intercept 5.5336 Reproducibility (RSD, %) 2.35 Repeatability (RSD, %) 4.12 3. 7. Reproducibility and Repeatability To demonstrate the reproducibility of the RGO/ AuNPs/GCE sensor, 3 modified electrodes were prepared under the same composition. Under optimized experi- mental conditions, repeated DPV measurements (n = 5) from a solution that contains 5.0 × 10–4 mol L–1 ziram were used to identify the peak current for each electrode. The anodic peak currents for ziram had a relative standard deviation (RSD) of 2.35% (Table 2). This implies that the electrode has a high level of repeatability. Multiple DPV measurements (n = 5) were used to assess the sensor’s re- peatability, giving RSD value of 4.12% (Table 2). 3. 8. Real Sample Analysis The analytical applicability of the prepared sensor was performed with tap water and peach juice. The recov- ery of the method was evaluated by spiking tap water and peach juice samples with ziram at low, middle and high concentration levels of the calibration graph. Calculated recovery values and added ziram concentrations are given in Table 3 and Table 4. The obtained recovery values were between 96.4 and 107.6%. These results show that the developed elec- trochemical sensor can effectively be applied with high sensitivity and selectivity for ziram determination in two different matrices. Table 3. Recovery results obtained by standard addition method in tap water sample using RGO/AuNPs/GCE sensor. No Added Found Recovery, RSD, Relative ziram, ziram, % % error, % (mg L–1) (mg L–1) 1 0.56 0.54 (±0.02) 96.4 (±2.73) 2.83 –3.6 2 1.68 1.64 (±0.02) 97.6 (±1.19) 1.22 –2.4 3 2.50 2.69 (±0.04) 107.6 (±1.67) 1.55 7.6 * The average of three measurements is used for each value. RSD, Relative Standard Deviation Table 4. Recovery values obtained by standard addition method in peach juice using RGO/AuNPs/GCE sensor. No Added Found Recovery, RSD, Relative ziram, ziram, % % error, (mg L–1) (mg L–1) % 1 0.62 0.65 (±0.03) 104.8 (±4.3) 4.07 4.84 2 1.86 1.83 (±0.03) 98.8 (±1.6) 1.64 –1.61 3 2.48 2.37 (±0.04) 95.3 (±1.6) 1.71 –4.70 * The average of three measurements is used for each value. RSD, Relative Standard Deviation 4. Conclusion A sensitive electrochemical sensor for the rapid de- tection of ziram was successfully constructed by modifying AuNPs improved RGO on GCE. Using cyclic voltammetry and differential pulse voltammetry, the electrochemical behaviour and real sample applicability of RGO/AuNPs/ GCE were examined. The proposed electrochemical meth- od was validated and the constructed sensor was proven to have good sensitivity and selectivity, as well as a low detection limit. Furthermore, the method has been used Figure 9. The calibration voltammograms at different concentra- tions of ziram in BR buffer (pH 8) at AuNPs/RGO/GCE by DPV (a)1.50 ×10–5; (b) 4.70 × 10–6; (c) 3.18 × 10–6; (d) 1.63 × 10–6; (e) 6.44 × 10–7; (f)1.63 × 10–7 mol L–1. 645Acta Chim. Slov. 2022, 69, 638–646 Aslan et al.: Electroanalytical Determination of Ziram by Differential ... to accurately determine the ziram in spiked tap water and peach juice. The created RGO/AuNPs/GCE sensor is effec- tive and promising due to its relatively simple modification method and disposable feature, as well as its potential to be used for direct measurements in water and peach juice. The findings of this study add to the analytical methodolo- gies for ziram determination that have been used thus far. Data available on request from the authors The data that support the findings of this study are available from the corresponding author, [Aslan, N.], upon reasonable request. Nazife Aslan http://orcid.org/0000-0002-2622-5908 Declaration of competing interest The authors declare that they have no known com- peting financial interests or personal relationships that could have appeared to influence the work reported in this article. The authors also declare that they have no conflict of interest with suggested reviewers. Acknowledgements The authors express their gratitude for the support from Gazi University with Grand Numbers BAP- 65/2020- 03. 5. 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Zane, F. Focanti, A. Curulli, G. Padeletti, J. Nanopart. Res. 2009, 11, 1925–1936; DOI:10.1007/s11051-008-9547-0 54. M. H. Mahnashi, A.M. Mahmoud, S.A. Alkahtani, R. Ali, M. M. El-Wekil, Anal. Bioanal. Chem. 2020, 412, 355–364; DOI:10.1007/s00216-019-02245-8 55. Food and Agriculture Organization of the United Na- tions (FAO). www.fao.org/fao-who-codexalimentarius/co- dex-texts/dbs/pestres/pesticide-detail. (accessed December 22, 2021). Povzetek V tem prispevku je opisana priprava senzorskih materialov na osnovi reduciranega grafenovega oksida z nanodelci zla- ta za določanje cinkovega(II)dimetilditiokarbamata (zirama). Grafenov oksid (GO) je bil sintetiziran po modificirani Hummerjevi metodi. Kompozitni senzor, sestavljen iz nanodelcev zlata (AuNP) in reduciranega grafenovega oksida (RGO), je bil elektrokemično izdelan na elektrodi iz steklenega ogljika. Nanokompozit je bil ovrednoten z uporabo vrstične elektronske mikroskopije (SEM). Ciklična voltametrija je bila uporabljena za prikaz elektrokemičnih lastnosti modificiranega senzorja na vsaki stopnji modifikacije. Predlagani senzor je pokazal dobro analitično zmogljivost za določanje pesticida ziram v vodi in breskovem soku, vključno z zelo nizko mejo zaznave, velikim linearnim razponom in nizkim RSD. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 647Acta Chim. Slov. 2022, 69, 647–656 Aydogdu and Hatipoglu: Electronic Structures and Reactivities of COVID-19 Drugs: ... DOI: 10.17344/acsi.2022.7522 Scientific paper Electronic Structures and Reactivities of COVID-19 Drugs: A DFT Study Seyda Aydogdu and Arzu Hatipoglu* Department of Chemistry, Yildiz Technical University, 34220, Istanbul, Turkey * Corresponding author: E-mail: hatiparzu@yahoo.com Received: 04-06-2022 Abstract These days, the world is facing the threat of pandemic Coronavirus Disease 2019 (COVID-19). Although a vaccine has been found to combat the pandemic, it is essential to find drugs for an effective treatment method against this disease as soon as possible. In this study, electronic and thermodynamic properties, molecular electrostatic potential (MEP) anal- ysis, and frontier molecular orbitals (FMOs) of nine different covid drugs were studied with Density Functional Theory (DFT). In addition, the relationship between the electronic structures of these drugs and their biological effectiveness was examined. All parameters were computed at the B3LYP/6-311++g(d,p) level. The Solvent effect was evaluated using conductor-like polarizable continuum model (CPCM) as the solvation model. It was observed that electrophilic indexes were important to understand the efficiencies of these drugs in COVID-19 disease. Paxlovid, hydroxyquinone, and nita- zoxanide were found as the most thermodynamically stable molecules. Thermodynamic parameters also demonstrated that these drugs were more stable in the aqueous media. Global descriptors and the reactivity of these drugs were found to be related. Nitazoxanide molecule exhibited the highest dipole moment. The high dipole moments of drugs can cause hydrophilic interactions that increase their effectiveness in an aqueous solution. Keywords: COVID-19; SARS-COV-2; Global descriptors; DFT; Solvent effect. 1. Introduction The Covid-19 outbreak is an important threat to pub- lic health nowadays. Many people died, and this pandemic caused a significant economic crisis and panic. During the last few decades, β class of coronaviruses led to mortality diseases like SARS and MERS.1 In December 2019, in Wu- han, China, an outbreak of the new type of Coronavirus Disease (COVID-19) caused a global health and economic crisis. This virus is coronavirus 2 (SARS-COV-2), a type of β coronavirus.2 Its common symptoms are shortness of breath, fatigue, fever, cough, and flue. In some more severe cases, COVID-19 infection leads to organ failure and even death.3 The mortality rate of COVID-19 is approximate- ly 6.8%, which is smaller than the mortality rate of SARS (10%) and MERS (36%). Despite the smaller mortality rate compared to SARS and MERS, the higher contagious property of COVID-19 and the unpredictability of disease progression worsened the situation and resulted in more deaths worldwide.1,4,5 To date (January 31.2022), WHO reported that there were 223 countries and territories that suffered from coronavirus with 364,191,494 confirmed cases and death number of 5,631,457 people.6 Although a vaccine has been found for COVID-19, it is essential to have appropriate drugs that are effective, inexpensive, and easily available for treatment. Therefore, more information is urgently needed on effective drug therapy and possible therapeutics used to combat the COVID-19 pandemic. It is very difficult to develop a new antiviral drug against COVID-19 and meet the urgent need for treat- ment. Drug discovery is expensive and time-consuming, a process that takes at least 15 years for a newly designed drug to reach patients from the laboratory.7,8 These are the limiting factors for control and prevention of this global pandemic. After analyzing the genome of SARS-COV-2, it is understood that the spike S protein of the virus effectively binds to the human angiotensin-converting enzyme 2 re- ceptors. Once it enters a human cell, it releases immediate- ly and replicates itself.9,10 Based on this information, many known possible therapeutics have been tested preclinically and clinically so far, but few of them have been proven ef- fective against this disease such as chloroquine, hydroxy- chloroquine, favipiravir and so on.11 Remdisevir and chlo- roquine can be used effectively to cure COVID-19.12 The 648 Acta Chim. Slov. 2022, 69, 647–656 Aydogdu and Hatipoglu: Electronic Structures and Reactivities of COVID-19 Drugs: ... combination of favipiravir with different antiviral agents has been studied for the treatment of COVID-19 and it has been found that the combination of antivirals is an appropriate treatment.13,14 Nowadays, COVID-19 vac- cines are used to prevent the disease. It is also known that at least 54.4% of the world’s population receives a dose of vaccine15, but drug treatment is still needed to prevent the pandemic. Computational methods can be a good alternative for studying in such emergency and difficult situations. In comparison to experimental methods, computation- al ones, are not expensive or time-consuming.16 There are some studies in the literature by using computation- al methods related to COVID-19. Molecular docking for inhibition of Mpro, 3CLpro, E proteins, and RdRp enzymes against SARS-COV-2 with drugs such as chloroquine, hy- droxychloroquine, favipiravir, umifenovir, paxlovid, gali- desivir, ribavirin, molnupiravir, and remdesivir have been investigated.17–28 Electronic and optoelectronic properties of hydroxychloroquine, chloroquine, azithromycin, and favipiravir were investigated by the DFT method to un- derstand the possible drug delivery system.29–31 Although many studies have been conducted on the pharmacologi- cal properties of COVID-19 drugs, there is still lack of in- formation about the effect of the electronic properties of these molecules on their physicochemical properties and reactivities. Therefore, it is crucial to examine the elec- tronic properties of COVID-19 drugs to better understand their biological effectiveness. As mutations occur in the SARS-COV-2 protein, the need to determine the proper- ties of COVID-19 drugs with rapid and effective methods has become more urgent than ever. The purpose of this study is to calculate the elec- tronic and thermodynamic properties of already used and newly proposed COVID-19 drugs. DFT method is applied for all calculations. In this respect, some chemical descrip- tors such as hardness (η), electrophilic index (ω), chemical potential (µ), softness (S) and frontier orbital energies, and thermodynamic parameters (such as enthalpy, Gibbs free energy, and entropy) are evaluated. Hydroxychloroquine, chloroquine, nitazoxanide, favipiravir, galidesivir, ribavi- rin, fluvoxamine, molnupiravir and paxlovid are selected as model drugs owing to the differences in their electronic structures. And, their efficiencies are investigated against COVID-19. 2. Computational Details All the calculations were carried out with Density Functional Theory (DFT) method with Gaussian 09 pro- gram.32 The drug molecules were optimized using Becke’s three parameter functional which combines Becke and HF exchange with the Lee-Yang-Parr correlation term at B3LYP/6-311++g(d,p) level.33 Frequency analysis, calcu- lated at the same level of theory, indicated that the opti- mized structures were at the stationary points correspond- ing to local minima without any imaginary frequency. The structural visualizations of the drugs were prepared by using the GaussView 5.0 software.34 Since blood itself is a water-based system, the Conductor-Like Polarizable Con- tinuum Method (CPCM) was used to compute the effect of water on the properties of drugs. The solvent was water with the dielectric constant value ε = 78.3.35 Thermody- namic parameters were obtained by frequency analysis and solvation energies were also calculated. The energies were corrected by including zero-point vibrational energy (ZPVE) at the B3LYP/6-311++G(d,p) level. Quantum chemical descriptors were calculated within the conceptual framework of the DFT to determine the reactivity of drugs. The reactivity of molecules can be predicted with global descriptors, which are determined by perturbations related to the change in the number of electrons. Some of the global descriptors studied in this paper are chemical potential (µ), hardness (η), electro- philic index (ω), and softness (S). Hardness, softness, and chemical potential were calculated by Koopman’s theorem. According to this theorem ionization potential and elec- tron affinity of a system are equal to the negative value of the energy of the highest occupied molecular orbital (EHO- MO) and the energy of the lowest unoccupied molecular orbital (ELUMO). By using the Koopman’s theorem these global reactivity descriptors are defined as36–38, (1) (2) (3) (4) 3. Results and Discussion 3. 1. Energies and Global Descriptors The structures of nine studied COVID-19 drugs, hy- droxychloroquine, chloroquine, nitazoxanide, favipiravir, galidesivir, ribavirin, fluvoxamine, molnupiravir, and pax- lovid, are shown in Figure 1 and optimized geometries of drugs are given in Figure 2. Some of these drugs are func- tionalized derivatives of the classic heteroaromatic rings, such as quinoline (hydroxychloroquine and chloroquine as molecules 1, 2), thiazolide (nitazoxanide as molecule 3), pyrazine (favipiravir as molecule 4). Others are nucle- oside-based heterocyclic molecules, similar to the aden- osine base of galidesivir (molecule 5) and the guanosine base of ribavirin (molecule 6). The newly proposed alter- 649Acta Chim. Slov. 2022, 69, 647–656 Aydogdu and Hatipoglu: Electronic Structures and Reactivities of COVID-19 Drugs: ... native drugs are fluvoxamine (molecule 7), a selective ser- otonin reuptake inhibitor (SSRI), ribonucleoside antiviral prodrug molnupiravir (molecule 8), and nitrile warhead paxlovid (molecule 9). The calculated EHOMO, ELUMO, ΔE, hardness (η), chemical potential (µ), electrophilic index (ω), softness (S) and dipole moments (D) are listed in Table 1 for gas and aqueous media. The hardness is a good descriptor for chemical stability and reactivity and it is related to the en- ergy gap. Hard molecules have large band gap energies. As seen in Table 1 the hardest molecules are 6 and 9, the least hard is 3. Molecules 5, 6, 7, 8, and 9, which contain electronegative atoms such as -OH and -F in their mo- lecular structure, are those with highest hardness. Mole- cules 1, 2 and 3, which have fewer electronegative atoms in their molecular structure, are those with low hardness compared to the others. Molecule 3, which contains one sulfur atom in the ring in its structure, has the lowest hardness. The hardness of molecules increases in order of 6>9>7>5>8>4>1>2>3. Softness is the opposite of hard- ness, and posseses a similar relation. The chemical poten- tial (µ) is the measure of escaping tendency of electrons. The chemical potential also shows almost the same trend with hardness except for molecules 4, 5, and 8. The electron accepting ability of a molecule is related to its electrophilicity index value. The electrophilic index value of molecules is in order 4>3>1>6>2>8>7>9>5. Mol- ecules with an electrophilic index value higher than 1.5 eV have an electrophilic character.37 As seen in Table 1, the electrophilic index value of all drug molecules is greater than 1.5 eV. Therefore, it can be inferred that all the studied molecules have an electrophilic character. It is known that the cysteine moieties of proteins are nucleophilic. So, it is advantageous to have an electrophilic agent for the treat- ment of COVID-19.39 In general, all drugs have in com- mon the ability to accept electrons, which may increase the interaction of drugs with the SARS-COV-2 virus. The in vitro half-maximal effective concentration (EC50) values for SARS-COV-2 virus in Vero E6 values are given in Figure 3. As can be seen in the figure, the molecules with the highest EC50 values are 6, 9, and 4, re- spectively. These drugs are less efficient than the others. Molecule 9, Paxlovid, is the new drug which Pfizer has de- veloped for COVID-19 and has just been approved for use. Molecule 6, Ribavirin is in a class of antiviral medications, and molecule 4, Favipiravir, is the more efficient drug for COVID-19 disease.40 Favipiravir, as an antiviral drug, has been authorized for treating COVID-19 in several coun- tries, under emergency provisions. There is a relationship between the EC50 values of the molecules and their global descriptors. Drugs with high EC50 values have high hard- ness and lower chemical potential values. Although there Figure 1. Structures of drug molecules 650 Acta Chim. Slov. 2022, 69, 647–656 Aydogdu and Hatipoglu: Electronic Structures and Reactivities of COVID-19 Drugs: ... Figure 2. Optimized geometries of drug molecules are not enough experimental studies on the efficacy of these drugs for COVID-19, the relationship between the global descriptors of these drugs and their EC50 values can be used to select the effective drug candidates. The dipole moment is an important factor affecting the solubility of a drug. The solubility and polarity of the drug must be balanced to optimize the drug efficacy.3 In biological systems, a high dipole moment value is a de- sirable property for drug delivery.16 Dipole moments of drug molecules increase in the aqueous medium because of the hydrogen bonds. It means that the solubility of these drugs in an aqueous medium may be enhanced with the increase of polarity. The dipole moments of the mol- ecules (1-9) are around 3.04–26.10 Debye in the aqueous Figure 3. In vitro half-maximal effective concentration (EC50) of drugs 651Acta Chim. Slov. 2022, 69, 647–656 Aydogdu and Hatipoglu: Electronic Structures and Reactivities of COVID-19 Drugs: ... medium and 2.08–14.58 Debye in the gas phase. Among the calculated drug molecules the largest dipole moment value is found for molecule 3. This large dipole moment value can allow high polarity in some regions of the drug and hydrophilic interactions in the solvent that increases its activity. Dipole moments 1,2,5 and 9 are greater than those of 4,6,7 and 8. Hence, 1,2,5 and 9 are more polar- ized and may show more hydrophilic properties. This Table 1. Global descriptors: hardness (η), chemical potential (µ), electrophilic index (ω), softness (S), frontier orbitals energies (EHOMO, ELUMO) and dipole moments (D) of drugs, (values in italic apply to the gas phase) EHOMO ELUMO ΔE η S µ ω D eV eV eV eV eV eV eV Debye 1 –6.05 –1.71 4.34 2.17 0.23 –3.88 3.46 9.29 –5.99 –1.59 4.40 2.20 0.23 –3.62 3.27 6.90 2 –5.94 –1.65 4.29 2.15 0.23 –3.80 3.36 9.26 –5.81 –1.44 4.37 2.18 0.23 –3.62 3.01 6.99 3 –3.81 –2.00 1.81 0.91 0.55 –2.91 4.66 26.10 –3.07 –0.82 2.25 1.12 0.44 –1.94 1.68 14.58 4 –7.31 –2.73 4.58 2.29 0.22 –5.02 5.50 4.31 –7.37 –2.85 4.52 2.26 0.22 –5.11 5.77 3.24 5 –6.35 –1.20 5.15 2.57 0.19 –3.77 2.77 8.63 –6.37 –1.31 5.05 2.53 0.20 –3.84 2.92 6.70 6 –7.75 –1.51 6.25 3.12 0.16 –4.63 3.43 3.04 –7.45 –1.44 6.03 3.02 0.17 –4.46 3.30 2.08 7 –6.94 –1.41 5.53 2.77 0.18 –4.18 3.15 4.19 –6.83 –1.64 5.19 2.60 0.19 –4.23 3.45 5.20 8 –6.66 –1.54 5.12 2.56 0.20 –4.10 3.29 6.41 –6.71 –1.66 5.05 2.53 0.20 –4.19 3.47 5.20 9 –7.14 –1.13 6.01 3.00 0.17 –4.13 2.85 8.71 –7.15 –0.94 6.22 3.11 0.16 –4.04 2.63 5.58 Figure 4 Frontier molecular orbitals of drug molecules 652 Acta Chim. Slov. 2022, 69, 647–656 Aydogdu and Hatipoglu: Electronic Structures and Reactivities of COVID-19 Drugs: ... feature can turn these drugs into active molecules in an aqueous media. 3. 2. Frontier Molecular Orbitals Analysis Frontier Molecular Orbitals (FMOs) are important parameters used to understand the distribution of electro- philic regions of molecules and their chemical interaction parts with other molecules.41,42 The chemical reactivity of a molecule can be determined by using the energy gap val- ue of frontier orbitals (ΔE). A small energy gap indicates a more reactive molecule. As seen in Table 1, molecule 3 has the smallest ΔE values in both phases. Thus, this molecule is the most reactive one. The electron-withdrawing -NO2 group in 3, can disrupt the distribution of the π electron system, which leads to deteriorated molecular backbone conjugation, thus decreasing the chemical stability of the molecule. Molecule 6 is the least reactive molecule with the highest energy gap value of 6.25 eV. The FMOs of all studied drug molecules are given in Figure 4. As can be seen from the Figure, the HOMO orbitals are π-bonding molecular orbitals. HOMO and LUMO orbitals are mainly distributed on the quinoline ring of the molecule for 1 and 2. While HOMO of mole- cule 3 is distributed on the functionalized part of the thi- azolidine ring, LUMO is distorded all through the mole- cule. The HOMO orbital is π bonding type and the LUMO orbital is π* antibonding type for molecule 4 and they are mainly distributed all through the molecule. For mole- cules 5 and 6, the electron distribution of the HOMO or- Figure 5. Molecular Electrostatic Potential Plots of molecules 653Acta Chim. Slov. 2022, 69, 647–656 Aydogdu and Hatipoglu: Electronic Structures and Reactivities of COVID-19 Drugs: ... bital is delocalized almost throughout the molecule, while the LUMO orbital is found in its two fused aromatic rings in molecule 5 and at the 1,2,4-triazole portion in molecule 6. HOMO is distributed on the etheric part and LUMO is on the benzene trifluoro acetamide part of molecule 7. The HOMO and LUMO orbitals of the antiviral prodrug 8 are distributed over the heterocyclic rings of the mol- ecule. The boundary orbitals electron cloud is mainly on the pyrrolidine ring and isobutyl portion of molecule 9. In the study of Macchiagodena et.al., these parts of molecule 9 were found to approach to the SARS-COV-2 6LU7 pro- tein.22 Thus, according to FMOs, these sites are predicted as the active sites for the probable chemical interactions. 3. 3 MEP Surfaces Molecular Electrostatic Potential (MEP) surfaces are the three-dimensional visualization of the charge distribu- tion of atoms on a molecule.43 Such surfaces supply infor- mation about electronic distribution of molecules, their nucleophilic and electrophilic attack parts, and formation of possible hydrogen bonds.44 In these surfaces, the red color shows the more negative potential of the molecule whereas the blue color shows the more positive potential. The green color represents the neutral part of the molecule with almost zero charge.45 MEP surfaces of the molecules 1-9 are shown in Figure 5. For all molecules, at least one hydrogen bonding region is detected. Thus, hydrogen bonding stabilizations decrease the energy values of the molecules with the aque- ous media. As seen in Figure 5, the electron distributions of 1 and 2 are almost the same, but they differ in the number of hydroxyl groups. Since molecule 1 has more hydroxyl groups, its negative regions are dominant. Thereby, it is more effective against SARS-COV-2 than that of mole- cule 2.11,19,30 Molecule 3 has more red areas than the oth- ers, and they are mainly concentrated on the -NO2 group, which removes the electron density from the molecule’s π system and makes the molecule less electrophilic. The electrophilic region of molecule 4 is on the hydroxyl group while the hydrogen atoms of the amino group are the nucleophilic part. But the fluorine atom in 4 has no effect on the electronic behavior of the pyrazine ring. In molecule 5, the ribose ring has a higher electron densi- ty due to the electron-donating hydroxyl groups, while the electron-positive areas are hydrogens bonding to the nitrogen atom. In molecule 6, the most negative region belongs to the oxygen atom of the carbonyl group, and the positive potential region belongs to the hydrogen at- oms. For molecule 7, the electrophilic regions are etheric oxygen and fluorine atoms. In molecule 8, the blue color distribution is in the hydrogen atoms of the amine group. Due to the electron withdrawal properties of fluorine at- oms, this region in molecule 9 can cause electron local- ization. 3. 4. Thermodynamic Properties The solvatation-free energies of the studied drug molecules are calculated using their Gibbs free energy change values between the solvent phase and the gas phase (Figure 6). As depicted from the figure, all the molecules’ solvation-free energies are negative, indicating their spon- taneous solubility in water. This result is in accordance with the increased dipole moment values in the aqueous medium. The solvation energies of the molecules vary be- tween (–6.83) – (–53.99) kcal mol–1. As noticed in Figure 6, the solvation free energies of all molecules, are quite similar except of molecule 3. Since molecule 3 has less electronegative atoms in its structure, it differs from the others. Molecules 1 and 2, 4, and 7 are structurally very similar. Therefore their solvation-free energies are also close to each other, for 1 and 2 they are –8.66, –6.83 kcal mol–1 and for 4 and 7 –8.15, –9.14 kcal mol–1 respectively. For molecules 5, 6, 8, 9 solvation-free energies are found as –14.48, –13.81, –16.31 and –18.72 kcal mol–1, respec- tively. Figure 6. Solvation-free energies of drugs Thermodynamic parameters of the studied drug molecules have been calculated at 298.15 K. The calculated total energies (E), enthalpy (ΔH), entropy (S), Gibbs free energy (ΔG) with ZPE correction for water and gas phases are listed in Table 2. As can be seen from the table, the molecules with the lowest energies are 9, 3, and 1, while the molecules with the highest energies are 4, 6, and 5. The most thermodynamically stable molecules are found as 9, 3, and 1 due to their enthalpy and Gibbs free energy values (Table 2). The thermal stabilities of all molecules are high- er in the aqueous medium. Since the thermal stability of drug molecules is necessary for drug durability, it can be inferred from the results that all molecules are more stable in an aqueous medium.46 3. 5. The Effect of Electronic Structure on Biological Effectiveness A drug binding efficiency to an active point of an en- zyme or a protein is related to its electronic structure.47 Therefore, the electronic properties of drugs are important 654 Acta Chim. Slov. 2022, 69, 647–656 Aydogdu and Hatipoglu: Electronic Structures and Reactivities of COVID-19 Drugs: ... for predicting their biological activity. In Figure 3, the in vitro half-maximal effective concentration (EC50) values of molecules are shown for the studied molecules except for 5, 7, and 8.7,12,48 According to the results of experimental biological studies, it was understood that quinoline derivative drugs 1 and 2 in Vero E6 were more active for the SARS-COV-2 virus.7 Because the unpaired electrons of the nitrogen atom in the quinoline ring and the availability of suitable sites for the hydrogen bond affect the activities of these types of drugs positively.49,50 The reason why the EC50 value of 1 is less than that of 2 may be due to the hydrogen bonding of the hydroxyl group in its structure. Molecule 3 may be an important molecule to treat COVID-19 due to the pres- ence of sulfur atom in its structure, which may change the amino acid residue of the target compound by disulfide bond formation. In addition, the sulfur atom may be im- portant for the formation of a hydrogen bond.51 Howev- er, the electron-withdrawing feature of the -NO2 group in the structure of 3 reduces the electron conjugation, re- sulting in a higher EC50 value than for 1 and 2. The lone pair electrons, halogen atom, and electron conjugation of the heterocyclic ring make molecule 4 more effective than molecule 6 against COVID-19 disease. It is known that halogen atoms increase the electron density of the rings for π-stacking interactions as well as halogen bonding.44 It is understood from the results of the Saul et al. study8 that 1 and 2 are more effective against COVID-19 than 4. The decreased electronegativity of the halogen atom in drugs 1 and 2 can increase the electron density of the quinoline ring, which may lead to the interaction of these molecules with the target site of SARS-COV-2. Altough molecule 6 has a lower conjugate electron cloud in its structure, its highest hydrogen bonding ability causes an easier to attach to the target site in comparison to the other molecules. The trifluoroacetamide moiety of molecule 9 is the potential site for hydrogen bond interactions with the amino acid of the spike protein. The halogen-type hydrogen bonding ability and electron-withdrawing substituents are key fac- tors governing the biological effectiveness. Based on all these results, we can say that the structural modification of drugs has a significant effect on the electronic structure of drugs. Therefore, a complete characterization of the elec- tronic properties of drugs is important to understand their biological activities. 4. Conclusions The fight against COVID-19 can be achieved with both vaccine prevention and drug treatment. Electronic behavior of drugs may point out their effectiveness against genetic variants of SARS-COV-2. In this study, electronic and thermodynamic properties, and quantum chemical descriptors of nine drugs are calculated. The results can be summarized as follows; • Drug molecules containing electronegative atoms such as -OH and halogen atoms have higher hard- ness. Molecule 6 (ribavirin) is found as the hardest molecule. • Electrophilic character of drug molecules may in- crease their interaction with SARS-COV-2. • Paxlovid (9), nitazoxanide (3), and hydroxychlo- roquine (1) are found as the most thermodynam- ically stable drug molecules. All the studied mol- ecules are thermodynamically more stable in an aqueous medium. • The trifluoroacetamide in molecule 9 may be the appropriate site for binding to the amino acid of the spike protein. • Structures of drugs have a significant effect on their electronic properties. Accordingly, their bi- ological activities may also differ. • The frontier molecular orbitals and MEP surfac- es allow the prediction of reactive and possible interaction sites of drug molecules. Nucleophilic attacks may take place to the quinoline ring, two fused heterocyclic ring of 1 and 2, 1,2,4-triazole Table 2. Calculated energies and thermodynamic parameters of drugs. ZPE, E, ΔH, ΔG (in cal.mol–1), S (in cal mol–1 K–1) 1 2 3 4 5 6 7 8 9 Aqueous medium ZPE .106 0.26 0.26 0.13 0.06 0.18 0.14 0.22 0.21 0.34 E .106 –879.21 –832.00 –879.41 –381.27 –582.10 –569.30 –717.64 –751.54 –1110.63 ΔH .106 –879.20 –831.99 –879.40 –381.26 –582.09 –569.30 –717.63 –751.52 –1110.61 ΔG .106 –879.25 –832.03 –879.44 –381.30 –582.13 –569.33 –717.68 –751.57 –1110.67 S 163.53 160.47 146.88 92.70 132.54 127.72 176.80 165.37 220.36 Gas ZPE .106 0.26 0.26 0.13 0.06 0.18 0.14 0.22 0.21 0.35 E .106 –879.21 –831.99 –879.32 –381.26 –582.09 –569.29 –717.64 –751.52 –1110.61 ΔH .106 –879.19 –831.98 –879.31 –381.26 –582.08 –569.28 –717.62 –751.50 –1110.58 ΔG .106 –879.21 –832.03 –879.35 –381.28 –582.12 –569.32 –717.68 –751.55 –1110.65 S 169.13 159.39 148.30 92.02 128.92 130.39 177.00 162.18 232.45 655Acta Chim. Slov. 2022, 69, 647–656 Aydogdu and Hatipoglu: Electronic Structures and Reactivities of COVID-19 Drugs: ... parts of 6, and pyrrolidine ring, isobutyl part of the 9. 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Alhaddad, Int J Mol Sci, 2020, 21 (3922), 1–13. DOI:10.3390/ijms21113922 51. S. Shekh, K. H. Gowd, A. K. K. Reddy, J Sulfur Chem, 2020, 42 (1), 1–12. DOI:10.1080/17415993.2020.1817457 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Te dni se svet sooča z grožnjo pandemije koronavirusne bolezni 2019 (COVID-19). Čeprav je bilo najdeno cepivo za boj proti tej pandemični bolezni, je nujno, da čim prej poiščemo tudi zdravila za učinkovito metodo njenega zdravljenja. V tej študiji smo raziskali elektronske in termodinamične lastnosti in mejne molekularne orbitale (FMO) devetih ra- zličnih covidnih zdravil s teorijo gostotnega funkcionala (DFT) in z analizo molekularnega elektrostatičnega potenciala (EMP). Poleg tega smo preučili povezavo med elektronskimi strukturami teh zdravil in njihovo biološko učinkovitost. Vse parametre smo izračunali na ravni B3LYP/6-311+g(d,p). Vpliv topila smo ovrednotili z uporabo modela polarizira- jočega kontinuuma (CPCM) kot modela solvatacije. Opazili smo, da so za razumevanje učinkovitosti teh zdravil pri bolezni COVID-19 pomembni elektrofilni indeksi. Paxlovid, hidroksikinon in nitazoksanid so se izkazali za najbolj ter- modinamično stabilne molekule. Termodinamični parametri so tudi pokazali, da so bila ta zdravila stabilnejša v vodnih medijih. Ugotovili smo, da so globalni deskriptorji in reaktivnost teh zdravil povezani. Molekula nitazoksanida je imela največji dipolni moment. Visoki dipolni momenti zdravil lahko povzročijo hidrofilne interakcije, ki povečujejo njihovo učinkovitost v vodni raztopini. 657Acta Chim. Slov. 2022, 69, 657–664 Kožárová et al.: Industrial Wastewater as a Source of External Organic ... DOI: 10.17344/acsi.2022.7527 Scientific paper Industrial Wastewater as a Source of External Organic Carbon for the Biological Nutrient Removal Bibiána Kožárová*, Ronald Zakhar, Zuzana Imreová, Hana Hanuljaková, Ines Karlovská and Miloslav Drtil Department of Environmental Engineering, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovak Republic * Corresponding author: E-mail: b.kozarova@gmail.com, +421907 478 032 Received: 04-10-2022 Abstract Addition of external organic carbon source for denitrification is generally used in wastewater treatment plants (WWTPs) to intensify nitrogen removal processes. The aim of the laboratory survey was to measure the composition of concen- trated industrial wastewater, determine the possibilities of its use as an external denitrification substrate, and assess its overall impact on WWTP. The obtained results demonstrate that the analysed industrial wastewater is biodegradable, and can be used as a denitrification substrate without special adaptation of biomass. The denitrification rates with tested wastewater were in the range of 1.6 to 1.9 mgN/g·h. Negative influence of long-term dosing of industrial wastewater on activated sludge were not confirmed. The effect of imported wastewater on WWTP must be assessed comprehensively, including the impact of heavy metals from wastewater on the sludge quality. The instructions on how to calculate this balance are provided in the article. Keywords: Biodegradability of industrial wastewater, biological wastewater treatment, heavy metals in sludge, denitrifi- cation rate, semicontinuous bioreactors 1. Introduction Over the years, considerable effort has been made to advance and optimize the technologies for effective bio- logical nitrogen removal at wastewater treatment plants (WWTPs).1,2 Heterotrophic denitrification is an efficient process in which, through microbiological activity, a re- duction of nitrates and nitrites to nitrogen gas occurs.3 It has a unique place in the biological removal of nitrogen from wastewater. This is because during the denitrifica- tion, nitrogen passes from water into the air. Anoxic zones without dissolved oxygen, in which the redox potential values are in the range of approximately –50 mV to 50 mV for calomel electrode and approximately 150 mV to 250 mV for standard hydrogen electrode, are necessary for this process.4 Denitrification is most often used in wastewater treatment, where the sources of electrons for nitrogen re- duction are organic compounds.5 The process takes place even in the absence of exogenous organic carbon (Corg) but its rate is significantly lower. In this case, bacteria use their internal organic compounds as a source of electrons (endogenous process). In many WWTPs, the absence of readily biodegrad- able organic substrate in the wastewater is a limiting factor for successful removal of higher nitrogen concentrations. This occurs mainly due to the long sewerage networks in which organic compounds are anaerobically decomposed, while nitrogen remains in the wastewater. Groundwater leakage into the sewer system6 can also be a contributor of excess nitrogen in the wastewater, although this prob- lem is not commonly reported. In the groundwater of the Slovak Republic, there is NO3− usually present at dozens mg/l.7 The average groundwater infiltration into the dam- aged pipes is 36.85% of the total wastewater volume.8 If the leakage of the groundwater into sewerage is too high, then this nitrogen source is certainly interesting. Increased nitrogen input into wastewater can be also a consequence of the changes in eating habits of a population. While in the Slovak standard9 nitrogen production is reported at the level of 11 g/d per capita, Pitter4 already stated the pro- duction at 12 g/d per capita and it is possible to find in the literature the production up to 14 g/d per capita.10 In the case of insufficient concentrations of Corg in the wastewater, or too short retention time of wastewater 658 Acta Chim. Slov. 2022, 69, 657–664 Kožárová et al.: Industrial Wastewater as a Source of External Organic ... under anoxic conditions at WWTP, a possible solution is the dosing of suitable external organic substrate into the denitrification reactor (to increase the denitrification rate).11 Composition of organic compounds has a strong ef- fect on the presence of denitrifying microorganisms and thus on denitrification efficiency. Available external sourc- es of Corg are alcohols, especially methanol (cost-effective, although it requires some adaptation of biomass and in- creased demands on operational safety because it is toxic and explosive) or ethanol (does not require such adaptation of biomass but is more expensive).4 Organic acids (mainly acetic acid) also require less problematic adaptation be- cause the biomass at the WWTP recognizes them (they are formed by acidogenesis and acetogenesis in the sewerage system), but they are also more expensive. Another option is saccharides (e.g., glucose, amyloid, sucrose).12 The use of alternative Corg sources such as concentrated wastewater from industries are an interesting option.13,14 In any case, these wastewaters must be treated, and therefore their import to WWTPs, where they increase the efficiency of denitrification, will bring double benefits. The usability of wastewater from the agro-food industry  (e.g., milk bot- tling industries, potato processing industries, wastewater from winery industries) is commonly reported15, however other industries also produce external organic substrates. The specific denitrification rates reported in mgN/g·h vary considerably – mostly from tenths up to 20 mgN/g·h (referred to g of dry solids).12,16–22 For activated sludge adapted to sewage, acetate is reported as the substrate with the highest denitrification rates. Denitrification rates are affected by test conditions. The optimal reaction temper- atures were 15–35 °C in which complete denitrification was achieved and nitrite accumulation was observed at 10 °C indicating the incomplete denitrification at low temperature.23 Temperature change from 10 °C to 20 °C exerted a more significant positive effect on both the spe- cific denitrification and carbon consumption rates than a further temperature increase from 20 °C to 30 °C.24 The denitrification rate is also positively related to the pH val- ue. At lower pH values, the nitrogen oxidoreductases were progressively inhibited in such way, that the overall rate of denitrification decreased and N2O produced increased.25 The process was stable in the neutral pH range and the highest denitrification rates were obtained at the pH values from 7.1 till 7.8.26 In Cao et al.27 maximum denitrification rates were measured at pH of 6.6–7.5 with inhibited deni- trification at pH increased to 8.5 and 9.2. The values of the rates also depend on the compo- sition of the biomass during the tests, adaptation, and sludge retention time (SRT) (the higher the SRT values, the higher the increase in volumetric rates, but the specific rates related to the unit amount of biomass may also de- crease).11 If denitrification rates are measured in batch ki- netic tests, then the test conditions are different from those in the activated sludge reactor. In the batch test, there is a substrate concentration gradient (i.e., at the beginning of the test there are high substrate concentrations, and they only gradually decrease). According to the so-called Monod kinetics, the substrate removal rate decreases with decreasing substrate concentration.28,29 When assessing denitrification rates, it is also necessary to consider wheth- er the organic substrate is single- or multicomponent. According to Henze et al.29 and Phillips et al.30, the deni- trification rates achieved in batch kinetic tests are divided into 3 parts. In the first phase of the tests, the rate is the highest because an easily degradable organic substrate en- ters the denitrification; in the second phase of the test, the rate is slower because high molecular weight and insoluble organic compounds requiring hydrolysis are denitrified; and finally in the third phase, the rate is the lowest because only endogenous denitrification takes place. The rates cal- culated according to the recommendations of the technical standards9,11 are on the level of 0.5–3 mgN/g·h. Such rates are observed also at real WWTPs31. According to Henze et al.5 the denitrification rates valid for temperatures 10–20 °C are 0.1–0.2 mgN/g·h for endogenous denitrification, 0.6–2 mgN/g·h for raw wastewater, and 1–9  mgN/g·h for methanol and acetate. In summary, denitrification rates measured with a given substrate above 1 mgN/g·h can be considered a positive result. Organic matter is an essential factor for microbial growth and development. In addition to the biodegrada- bility, price and storage options12, choice of external or- ganic substrate is influenced also by the following factors: the highest possible chemical oxygen demand (CODCr) and specific CODCr expressed in mg CODCr/mg substrate; efficiency at which the bacteria are able to use it; toxicity of intermediates or substrate itself; composition stability (with the best possible homogeneity)4,5. The next factors are the lowest possible portion of nitrogen in the substrate; the highest possible portion of compounds in the substrate entering the denitrification reaction; low portion of com- pounds entering the assimilation reaction associated with the growth of new biomass; and the lowest possible ratio of high molecular weight and undissolved compounds.4,5,11 The objectives of this study were to analytically de- termine the content of components present in industrial wastewater with a high COD concentration, monitor its impact on the biological stage of WWTPs, and to present the possibilities of using concentrated industrial waste- water as an external source for the denitrification (e.g., at municipal WWTPs with the lack of denitrification capac- ity, where the accelerating of denitrification could help to achieve the legal requirements on treated wastewater and to reduce the payment of fees for discharged nitrogen).32,33 2. Materials and Methods The analysed parameters, their abbreviations, re- spective symbols, and the method of determination are 659Acta Chim. Slov. 2022, 69, 657–664 Kožárová et al.: Industrial Wastewater as a Source of External Organic ... shown in Table 1. All analyses were performed according to standard procedures.34 Activated sludge for denitrification and respirometry tests was cultivated in three long-term semicontinuous lab- scale bioreactors35 (designated R1, R2, and R3) with a total volume of 1 litre placed on magnetic stirrers. Mixing and aeration of the activated sludge in models R1, R2, and R3 were set up in the following way: 5 hours after the addition of the substrate mixing (i.e., denitrification) followed by 17 hours aeration (i.e., nitrification and oxidation of residual organic compounds with O2), and 2 hours sedimentation and draw off effluent and dosing of the substrate. The vol- umetric load expressed in kg CODCr was maintained at 0.88  kg/m3·d. The hydraulic retention time was 1.8 days and the set SRT was 15 days. Reactors were operated at the laboratory temperature 27–30 °C (experiments per- formed during summer months). Such temperatures were higher than typical municipal wastewater temperatures in Slovakia (approx. 10 °C during winter and 20–25 oC dur- ing summer8). However, these differences were neglected, because biological heterotrophic processes (like denitrifi- cation) are not significantly influenced by temperature. In addition, the main aim of the research was not to measure absolute values of denitrification rates but to evaluate bio- degradability under anoxic conditions from the differenc- es between endogenous, exogenous and total rates. The substrate was dosed every 24 hours. Substrate for the reactor R1 (i.e., reference reactor) contained glu- cose, peptone, and starch. The substrate consisting of glu- cose, peptone, starch, and industrial wastewater was dosed into reactor R2 in a ratio of 1:1 (mg CODCr,glucose + peptone + starch  :  mg CODCr, industrial wastewater). Only the industrial wastewater was dosed into reactor R3. Total concentration of CODCr in each substrate was 1,600 mg/l. Nutrients (N and P) were dosed in the form of NH4Cl and KH2PO4. The concentrations of TKN (N-NH4 + Norg) were at the level of 55 mg/l and P-PO4 at the level of 12 mg/l. To ensure the supply of micronutrients for activated sludge, reject wa- ter from dewatering of digested sludge at real municipal WWTP was added (30 mlreject water/lof substrate). The pH was adjusted with a sodium hydro-carbonate solution to 7. Denitrification tests were performed on the 0, 7th, and 22nd day of operation of the laboratory reactors R1, R2, and R3. 22 days represent 1.5 times the value of SRT; within 22 days the original activated sludge (inoculum) with SRT of 15 days is completely replaced. For more com- plicated substrates, due to slower adaptation and slower growing biomass, it is possible to recommend higher SRTs and longer duration of experiments.35 In this research, the 3 week duration of the experiments was also set according to the requirements of the industrial wastewater producer. Before the denitrification test, activated sludge was taken from the reactors R1, R2, R3 (taken as an excess sludge), diluted to a concentration of 1 g/l and poured into biochemical oxygen demand (BOD) bottles D1, D2, and D3. After a 2-hour aeration to remove residual degrada- ble organic compounds, aeration was replaced by a slow stirring and the tested substrate was added. Initial N-NO3 concentration in all 3 bottles was 30 mg/l. Organic exoge- nous substrate wasn’t added to the bottle D1 (this denitri- fication test was comparative and only endogenous deni- trification was performed). Organic substrate was added to the BOD bottles D2 and D3 to allow comparison of en- dogenous and total denitrification rates. If total respiration rates in bottles D2 and D3 were higher than the endoge- nous rate in D1, the organic substrate was degradable and usable in denitrification. Glucose was added to bottle D2 in the ratio CODCr : N-NO3 = 15 mg/mg (CODCr = 450 mg/l). Organic substrate was added excessively with the aim to eliminate denitrification rate limitation. The tested industrial wastewater was added to bottle D3, also in the ratio of CODCr : N-NO3 = 15 mg/mg. By comparing the rates in bottles D2 and D3, denitrification with industrial wastewater and a standard biodegradable compound was assessed. At the same time, nutrients N and P were added to bottles D2 and D3 to avoid limiting the denitrification by their absence. During the tests, changes in pH were also monitored and their values were continuously adjusted to the neutral range of 6.8–7.3 (with a diluted acid or alkali). During de- nitrification in a closed reactor, the pH can rise, but also fall slightly.36 Except for the decrease in N-NO3, the de- crease in CODCr and the possible formation of N-NO2 as an intermediate product of incomplete denitrification were also monitored. Tests lasted for 24 hours and the samples for analysis were taken in 3 hour intervals (during the first 9 hours, 4 samples, including taking sample at time 0); the last sample was taken after 24 hours (the significance of Table 1. Analysed parameters, their abbreviations (symbols), and the method of determination. Gravimetric methods Spectrophotometric methods Atomic absorption spectrometry total solids (TS) total chemical oxygen demand (CODCr) cadmium (Cd) suspended solids (TSS) ammonium nitrogen (N-NH4) chromium (Cr) volatile solids (VS) activated total Kjeldahl nitrogen (TKN) copper (Cu) sludge concentration (Xc) nitrite nitrogen (N-NO2) nickel (Ni) sludge volume index (SVI) nitrate nitrogen (N-NO3) lead (Pb) phosphate phosphorus (P-PO4) zinc (Zn) Note: Spectrophotometer HACH DR5000 and atomic absorption spectrometer ContrAA 700 Analytik Jena were used 660 Acta Chim. Slov. 2022, 69, 657–664 Kožárová et al.: Industrial Wastewater as a Source of External Organic ... this sample was only a control). Denitrification rates and organic substrate consumption were evaluated according to the decline in N-NO3 and CODCr concentrations. For the first 9 hours of the test, the denitrification rate was not limited by the absence of organic substrate and the decline in concentrations was linear. The specific denitrification rates in mgN/g·h could thus be calculated from the slope of decrease of the concentrations divided by time and sludge concentration. The principle of such batch denitrification tests with further details is given in Bodík et al.35 Respirometric determination of biomass activity from individual reactors R1, R2, and R3 was also performed by measuring oxygen consumption rates34,37 and compar- ing endogenous (rX,ox,en), total (rX,ox,t), and substrate (ex- ogenous) respiration rates (rX,ox) in 300 ml closed BOD bottles. The tests of anoxic biomass activity from denitri- fication tests were thus supplemented with information about oxic activity. Respirometric measurements were performed on days 0 and 22. On day 0, only one respirometric measure- ment was performed, with the exogenous substrate glu- cose. The aim was to obtain information about the activity of the sludge before the addition of industrial wastewater. On day 22, three respirometric measurements with acti- vated sludge from reactors R1, R2, and R3 were performed to assess changes in oxic activity in all 3 reactors (especially in reactors R2 and R3, where the biomass was exposed for 22 days to industrial wastewater). Before the respirometric tests, sludge taken as excess sludge from reactors R1, R2, and R3 was aerated for 2 hours to remove residual exoge- nous organic compounds. The biomass was diluted to 1 g/l and the allylthiourea (10 mg/l) was added to suppress ox- ygen consumption by nitrification. For the first 5 minutes, rX,ox,en was measured and then, for rX,ox,t measurements, the following exogenous substrates were injected into the system: on day 0, glucose was added to the sludge; on day 22, glucose was used for the biomass from reactor R1, glu- cose and industrial wastewater in the ratio CODCr = 1:1 for biomass from reactor R2 and only industrial wastewa- ter for biomass from reactor R3. The concentration of ex- ogenous CODCr in the BOD bottles after substrate dosing in all three cases was 17 mg/l. Respirograms were created by evaluating the respirometric measurements from which the respiration rates rX,ox,en, rX,ox,t, rX,ox in mgO2/g·h, and substrate consumption rate rx in mgCHSKCr/g·h were calcu- lated according to Bodík et al.35,37 3. Results and Discussion The sample of industrial wastewater (from the auto- motive industry) was partially turbid, grey in colour, and had a faint odour in concentrated form. The tested sample had concentrations of CODCr = 40.3 g/l, BOD5 : CODCr = 0.37, BOD5 = 14,8 g/l (measured with unadapted inoc- ulum), N-NH4 = 16 mg/l, N-NO3 = 11 mg/l, P-PO4 = 21 mg/l, TS (105 °C) = 41.5 g/l, TSS (105  °C) = 2.1 g/l, VS (550 °C) = 68%, and pH 6.2. Solvents based on glycol are the main fraction in the wastewater (the detailed compo- sition of organic compounds is confidential; request of the producer). The concentration of heavy metals is in Table 2, focusing on the metals included in the Act on the applica- tion of sewage sludge to soil no. 188/200338, as there is an assumption that the metals present in the wastewater will be adsorbed into activated sludge and can thus influence its treatment and handling. According to their toxicity and bioaccumulation tendency, high concentrations of met- als in sewage sludge can be also obstacle to its reuse.39,40 The other metals listed in the Act of Slovak Republic (no. 188/2003)38 (As, Hg) were not determined; their occur- rence in industrial wastewater according to its producer can be neglected. Table 2 also shows the real concentra- tions of metals in sludge from Slovak municipal WWTPs (average values valid for Slovak WWTPs according to Kozáková et al.41). These concentrations were used in the calculations to assess the acceptable amount of industrial wastewater imported to the WWTP as an external denitri- fication substrate. Results of the denitrification tests are shown in Fig. 1. Day 0 assays inform about immediate response of non-adapted biomass to the addition of industrial waste- water (i.e., biomass that has been previously fed only with glucose, peptone, and starch). Subsequently, these tests were repeated on days 7 and 22 to see how the characteris- tics and parameters of biomass change after long-term ex- posure to industrial wastewater. The comparison of values Table 2. The concentrations of heavy metals in industrial wastewater, real concentrations of metals in sludge from Slovak WWTP41, and limit concentrations of metals in sludge from WWTP applied to soil.38 Parameter Industrial wastewater Real concentrations Limit concentrations (mg/l) of metals in sludge of metals in sludge (mg/kg sludge dry matter) (mg/kg sludge dry matter) Cd ≤ 0.1 0.8 10 Cr 0.4 41 1,000 Cu 0.2 168 1,000 Ni 1.8 25 300 Pb ≤ 0.1 38 750 Zn 38.1 979 2,500 661Acta Chim. Slov. 2022, 69, 657–664 Kožárová et al.: Industrial Wastewater as a Source of External Organic ... measured on day 0 (rx,D,endo = 0.27 mgN/g·h; rx,D,total,industrial wastewater = 1.6 mgN/g·h) shows the immediate biological degradation of organic substances and no need for spe- cial adaptation of the biomass. Denitrification tests per- formed on day 7 and 22 show that industrial wastewater remains degradable for denitrification purposes even after long-term exposure (rates rX,D,endo = 0.17 mgN/g·h vs. rx,D,- total,industrial wastewater = 1.9 mgN/g·h and rX,D,endo = 0.25 mgN/ g·h vs. rx,D,total,industrial wastewater = 1.6  mgN/g·h). Industrial wastewater did not deactivate the biomass. Denitrification rates measured with industrial wastewater were lower than the rates measured with glucose as a standard organ- ic substrate (rx,D,total,industrial wastewater = 1.6–1.9 mgN/g·h vs. rx,D,total,glucose = 3.1–4.7 mgN/g·h). Nevertheless, industrial wastewater can be used as external organic substrate for denitrification at WWTP. The intermediate N-NO2 and its undissociated form HNO2 were not accumulated in any of the denitrification tests with industrial wastewater. The CODCr : N-NO3 ratio (ratio of mg CODCr in industrial wastewater consumed in denitrification of 1 mg N-NO3) was 9. According to stoichiometry of denitrification re- action involving both dissimilation and assimilation, standard ratios are in the range of 5–7 (consumption of CODCr calculated for the reduction of N-NO3 to N2 is 2,86 mgCOD/mgN; the next CODCr is consumed for a growth of new biomass, which depends on the bacteria involved in denitrification and which can not be stoichiometrically calculated).4,11 Figure 1. Rates of endogenous denitrification rX,D,endo (test D1), to- tal denitrification rX,D,total,glucose with glucose as organic substrate (test D2), and total denitrification rX,D,total,industrial wastewater with in- dustrial wastewater as organic substrate (test D3). The long-term impact of industrial wastewater on activated sludge and biomass adaptation was also evalu- ated from the concentrations of N-NH4 and CODCr in the effluent of models R1, R2, and R3. These indicators were used to monitor the influence on the nitrification and the concentration of residual and non-biodegradable organic matter from industrial wastewater (Table 3). At the same time, the values of volatile suspended solids (VSS) (as a share of organic matter in activated sludge) and sludge volume index (SVI) were evaluated (Table 3). All these parameters are important for WWTP as they influence a possible deterioration of the effluent from the WWTP, where industrial wastewater would be considered as an external denitrification substrate. Nitrification was effi- cient throughout the whole experiment, as confirmed by N-NH4 concentrations in the effluents from all 3 models (differences of 1.9 to 3.7 mg/l can be neglected). An im- portant parameter in terms of fees for treated wastewater33 is the residual CODCr. If the industrial wastewater con- tains hardly or non-biodegradable organic compounds, it is necessary to quantify the possible increase of CODCr concentration in the effluent from the WWTP. The in- crease of CODCr concentration occurred in models R2 and R3 with dosed industrial wastewater, where the average concentration increased from 59 mg/l to 129 and 145 mg/l. If we balance the average values of CODCr from industrial wastewater in the influent to models R2 and R3 (800 mg/l in model R2 and 1,600 mg/l in model R3) and CODCr in- crease in the effluent from these models, the impact is as follows: – In model R2, every 100 mg/l of CODCr from industri- al wastewater added to the activated sludge reactor in- creased the concentration of effluent CODCr by 8.8 mg/l (calculated as (129 mg/l–59 mg/l) / 800 mg/l / 100 mg/l) – In model R3, every 100 mg/l of CODCr of liquid waste added to the activated sludge reactor increased the con- centration of effluent CODCr by 5.4 mg/l (calculated as (145 mg/l–59 mg/l) / 1,600 mg/l / 100 mg/l) – If we assume that for denitrification of 10 mg/l N-NO3 it is necessary to add industrial wastewater with CODCr of 90 mg/l (ratio CODCr : N-NO3 = 9 measured in denitrifi- cation tests D3), then reduction of 10 mg/l N-NO3 in the effluent from WWTP is connected with CODCr increase 4.9–7.9 mg/l (5.4 mg/l · 90 mg/l / 100 mg/l; 8.8 mg/l · 90 mg/l / 100 mg/l). Table 3. Average effluent concentrations and their range for refer- ence model R1, model R2 with glucose and industrial wastewater, and model R3 with only industrial wastewater dosing. Parameter R1 R2 R3 N-NH4 (mg/l) 1.9 3.5 3.7 0.2−6 0.4−6 1.6−5.3 CODCr (mg/l) 59 129 145 49−97 87−169 61−167 VSS (%) 79 75 75 81−82 71−86 70−82 SVI (ml/g) 52 48 51 45−60 43−49 45−64 662 Acta Chim. Slov. 2022, 69, 657–664 Kožárová et al.: Industrial Wastewater as a Source of External Organic ... The impact of industrial wastewater on biomass was also monitored using the parameters VSS and SVI. The differences between models R1, R2, and R3 are insignificant (negligible accumulation of inorganic compounds and activated sludge retained the formation of compact flocs with good sedimentation properties). Results from respirometric measurements obtained during testing industrial wastewater are shown in Table 4. The results confirm that oxic respiration activity (i.e., ability to remove glucose as a reference substrate and in- dustrial wastewater) has changed minimally during the operation of models R1, R2, and R3. The main conclusion is that industrial wastewater was not toxic to the biomass. The measured respiration rates in Table 4 are also com- pared with the recommended rates and even though they are at the lower end of the typical values from literature35, they are still acceptable and do not affect the previous statement. In the case of additional industrial wastewater to the denitrification reactor, it is also important to assess how many m3 can be imported to the WWTP so that the per- mitted concentration limits of heavy metals in sludge are not exceeded. Heavy metals in the wastewater are at the WWTP mostly adsorbed into the primary and activated sludge, and subsequently, remain in the digested sludge removed from the WWTP. The legislation defines these concentrations for cases where the sludge from WWTP is applied to the soil.38 Application to the soil, either direct- ly or as a compost from composting plants, is currently still the most common method of sludge management in Slovakia.41 The following calculation shows an example of how to evaluate such balance for specific heavy metals and specific WWTP. The calculation assumes Cr in industrial wastewater, import of wastewater to WWTP with a capac- ity of approximately 10,000 inhabitants, inflow of 150 l/d per capita, specific production of sludge dry matter of 40 g/d per capita9,11, and concentrations of metal accord- ing to Table 2: – WWTP inflow = 10,000 inhabitants · 150 l/d = 1,500 m3/d – Daily sludge production = 10,000 inhabitants · 40 g/d = 400 kg/d – Limit concentration of Cr in sewage sludge defined by Slovak legislation = 1,000 mg/kg – Average background concentration of Cr in sludge at Slovak WWTPs: 41 mg/kg – Capacity of sludge to adsorb Cr (the limit concentration defined by legislation is not to exceeded) = 1,000 mg/ kg–41 mg/kg = 959 mg/kg – Possibility to import Cr in industrial wastewater to the WWTP = 400 kg/d · 959 mg/kg = 383,600 mg/d = 3.84 kg/d – Concentration of Cr in industrial wastewater = 0.4 mg/l – Volume of industrial wastewater with 383,600 mg/d of Cr = 383,600 mg/d / 0.4 mg/l = 959,000 l/d = 959 m3/d – Conclusion of the example calculation for Cr: at a WWTP with a capacity of 10,000 inhabitants, 959 m3/d of industrial wastewater can be imported as an external denitrification substrate and the Cr concentration in sludge will not exceed the limit of 1,000 mg/kg. This con- sideration includes simplification that Cr from industri- al wastewater is completely absorbed to the sludge. The volume of industrial water (959 m3/d) represents 64% of the WWTP inflow (1,500 m3/d). The percentage of industrial wastewater imported to the WWTP with a capacity of 10,000 inhabitants calculat- ed for other heavy metals from Table 2 are given in Table 5. Table 5. The percentage of industrial wastewater to the WWTP (with a capacity of 10,000 inhabitants) for selected heavy metals. Heavy Volume of Percentage of liquid waste metal liquid waste according to WWTP (m3/d) inflow (%) Cr 959 64 Cd 36.8 2 Cu 1,664 111 Ni 61.1 4 Pb 2,835 189 Zn 15.9 1 According to these balances, Zn represents the worst case since it reduces the percentage of daily imported Table 4. Results of respirometric measurements. Respirometric Typical values Day 0a) Day 22b) rates according to (Model R1) (Model R2) (Model R3) Bodík et al.35 rX,ox,en (mgO2/g·h) 1−10 3 4 3 4 rX,ox (mgO2/g·h) 10−100 20 22 22 20 rX,max (mgCOD/g·h) 30−200 71 68 65 61 a Reference measurement with glucose as an exogenous substrate; measured with biomass used as a common inoculum for models R1, R2, and R3 b Exogenous substrates: glucose for activated sludge from model R1, glucose + industrial wastewater (1:1) for activated sludge from model R2, industrial wastewater for activated sludge from model R3 663Acta Chim. Slov. 2022, 69, 657–664 Kožárová et al.: Industrial Wastewater as a Source of External Organic ... volume of industrial wastewater to only 1% (15.9 m3/d). However, this amount of industrial wastewater is still in- teresting. It represents approximately 424 mg/l CODCr and this concentration has a potential to denitrify 47 mg/l N-NO3 (CODCr of industrial wastewater = 40.3 g/l; ratio CODCr : N-NO3 = 9). 4. Conclusion The main results emerging from the testing of indus- trial wastewater as a possible external denitrification sub- strate imported to the municipal WWTP to increase the rate and efficiency of denitrification are as follows: – Industrial wastewater is biodegradable, also for non-adapted biomass – Denitrification resulted in nitrogen gas production with- out accumulation of intermediate products – The denitrification rates with industrial wastewater as an external substrate were in the range of 1.6 to 1.9 mgN/ g·h. Addition of this substrate improves denitrification efficiency – Negative impact of long-term dosing of industrial waste- water on activated sludge was not confirmed – Partial increase of CODCr concentration in the efflu- ent from activated sludge reactor was measured (small amount of organic compounds in industrial wastewa- ter was non-biodegradable). The addition of industrial wastewater with CODCr concentration of 100 mg/l in- creased the CODCr concentration in the WWTP efflu- ent by 5–9 mg/l. This problem can be regulated by the amount of industrial wastewater applied into the deni- trification reactor. – The impact of the imported industrial wastewater as an external denitrification substrate for WWTP must be as- sessed comprehensively, including details such as the ac- cumulation of heavy metals from the wastewater in the activated sludge. The instructions on how to calculate this balance are provided in the article. 5. References 1. T. Kurbus, J. Vrtovšek, M. Roš, Acta Chim. Slov. 2008, 55, 474–479. 2. M. Roš, J. Vrtovšek, Acta Chim. Slov. 2004, 51, 779–785. 3. J. Vrtovšek, M. Roš, Acta Chim. Slov. 2006, 53, 396–400. 4. P. Pitter, Hydrochemistry, 5th Edition, VŠCHT, Praha, 2015. 5. M. Henze, P. Harremoës, J. La Cour Jansen, E. 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Act of the Slovak Republic no. 188/2003 Coll. on the Applica- tion of Sewage Sludge and Bottom Sediments into the Land. 39. K. Cer Kerčmar, M. Zupančič, P. Bukovec, Acta Chim. Slov. 2008, 55, 1023–1029. 40. S. M. Mousavi, S. A. Hashemi, A. Babapoor, A. Savardashtaki, H. Esmaeili, Y. Rahnema, F. Mojoudi, S. Bahrani, S. Jahandi- deh, M. Asadi, Acta Chim. Slov. 2019, 66, 865–873. DOI:10.17344/acsi.2019.4984 41. K. Kozáková, L. Sumegová, I. Balážová Pijáková, Vodo- hospodársky spravodajca. 2018, 61 (1–2), 32–34. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 29. M. Henze, M. C. M. van Loosdrecht, G. A. Ekama, D. Brd- janovic, Biological Wastewater Treatment: Principles, model- ling and design. IWA Publishing, 2008. DOI:10.2166/9781780401867 30. H. M. Phillips, J. L. Barnard, C. Debarbadillo, A. R. Shaw, M. T. Steichen, C. Wallis-Lage, Proceedings of the Water Environ- ment Federation, 2009, 252–276. DOI:10.2175/193864709793901301 31. M. Drtil, I. Bodík, S. Vlčková, D. Kolníková, R. Brezina, P. Levársky, J. Tichý, Z. Imreová, M. Švorcová, SOVAK. 2018, 3, 12–16. 32. Slovak Government Regulation no. 269/2010 Coll. Require- ments for the Achievement of Good Water Status. 33. Slovak Government Regulation no. 755/2004 Coll. which Establishes the Amount of Unregulated Payments, Fees and Details Related to Charging for Water Use. 34. R. Baird, L. Bridgewater, Standard Methods for the Examina- tion of Water and Wastewater. 23rd Edition, American Public Health Association, Washington, D.C., 2017. Povzetek Dodatek zunanjega vira organskega ogljika za denitrifikacijo se običajno uporablja v čistilnih napravah odpadnih vod za namen intenziviranja procesov odstranjevanja dušika. Cilj laboratorijske raziskave je bil izmeriti sestavo koncen- trirane industrijske odpadne vode, ugotoviti možnosti njene uporabe kot zunanjega substrata za denitrifikacijo in oceniti njen celoten vpliv na čistilno napravo. Dobljeni rezultati kažejo, da je analizirana industrijska odpadna voda biološko razgradljiva in se lahko uporablja kot denitrifikacijski substrat brez posebne prilagoditve biomase. Stopnje denitrifikacije pri testirani odpadni vodi so bile v območju od 1,6 do 1,9 mg N/g·h. Negativni vpliv dolgotrajnega doziranja industrijske odpadne vode na aktivno blato ni bil potrjen. Vpliv uvožene odpadne vode na čistilno napravo je treba oceniti celovito, vključno z vplivom težkih kovin iz odpadne vode na kakovost blata, kar je navedeno tudi v tej raziskavi. 665Acta Chim. Slov. 2022, 69, 665–673 Koraqi et al.: Environmentally Friendly Extraction of Bioactive ... DOI: 10.17344/acsi.2022.7559 Scientific paper Environmentally Friendly Extraction of Bioactive Compounds from Rosa canina L. fruits Using Deep Eutectic Solvent (DES) as Green Extraction Media Hyrije Koraqi,1,2,* Bujar Qazimi,1 Cengiz Çesko2 and Anka Trajkovska Petkoska3 1 Faculty of Pharmacy, UBT-Higher Education Institution, St. Rexhep Krasniqi No.56,10000 Pristina, Kosovo 2 Faculty of Food Science and Biotechnology, UBT-Higher Education Institution, St. Rexhep Krasniqi No.56, 10000 Pristina, Kosovo 3 Faculty of Technology and Technical Sciences, St. Clement of Ohrid University of Bitola, Dimitar Vlahov, 1400 Veles, Republic of North Macedonia * Corresponding author: E-mail: hyrie.koraqi@ubt-uni.net Phone: +383 38 541 400 Fax: +383 38 542 138 Received: 04-28-2022 Abstract In this study, the green extraction of bioactive compounds from Rosehip (Rosa canina L.) fruits and their antioxidant activity were investigated. An ultrasound-assisted extraction combined with deep eutectic solvents (DES) was used for this purpose. Deep eutectic solvents based on citric acid were specially designed. Namely, hydrogen bond donor (HBD) such as glycerol and ethylene glycol as well as hydrogen bond acceptor (HBA) like citric acid were used. After choosing the best option of DES, for extraction of the bioactive ingredients, optimal extraction conditions of the ultrasonic-assist- ed extraction have been optimized through Box-Behnken design of response surface methodology (RSM). Total phe- nolics content (TPC), total anthocyanins content (TAA), and antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) have been found as 103.37 mg GAE/g DW in DES2, 92.23 mg GAE/g DW in DES1, 3.25mg C3G/100g-DW in DES2, 1.31 mg C3G/100g-DW in DES1, and 101.85% inhibition in DES2, 94.32%. The results of this study showed that this method is a competitive sustainable, green, and effective extraction of bioactive compounds from Rosehip (Rosa canina L.) fruits. Keywords: Rosa canina L. fruits, deep eutectic solvent, Green extraction, Antioxidant activity, Experimental design 1. Introduction Rosa canina L, which is also known as Rosehip, is a member of the Rosaceae family and the genus Rosa which comprises nearly 200 species that are naturally dis- tributed almost in many countries such as Europe, Asia, the Middle East, and North America.1–3 In Kosovo, Rosa canina L. fruits are found in all areas of the country and are traditionally used for food or medical purposes. Functional foods and food supplements, such as herbal food supplements and nutraceuticals, that help protect humans against oxidative stress and a variety of diseases have piqued attention all over the world. Rosa canina L. fruits are high in phenolic compounds, which operate as natural antioxidants; flavonoids, anthocyanins, and high vitamin C content; vitamins A, B1, B2, B6, D, E, and K; organic acids, such as citric acid, malic acid, carotenoids, sugars, mineral elements, and fibers.4–6 Rosa canina L. is a remarkable fruit that is a rich source of biologically ac- tive compounds with pharmacological features. Moreo- ver, it is used for a wide variety of purposes like protec- tion of health and therapy for flu, infections, protect the kidneys from oxidative stress, possesses an antidiabetic, antimicrobial, inflammatory diseases, and chronic pains. Rosa canina L. fruits have anti-ulcer and anti-aging prop- erties. Chemoprevention, antioxidant, antimutagenic, and anticarcinogenic properties are also known.7,8 Due to the above-mentioned properties, Rosa canina L. fruits 666 Acta Chim. Slov. 2022, 69, 665–673 Koraqi et al.: Environmentally Friendly Extraction of Bioactive ... are commonly used in the food, pharmaceutical, and cos- metic industries. Namely, it could be, used as food and drink such as tea, marmalades, jellies, and jams. Howev- er, it has recently been utilized as an ingredient in probi- otic drinks, yogurts, and health supplements.4 In the sci- entific literature, there is still lack of information on phenolic compounds, flavonoids and the antioxidant ac- tivity of Rosa canina L. fruits. Novel applications are giv- en in a very limited number of studies mainly on the ex- traction of phenolic compounds and antioxidant activity based on solid-liquid extraction with traditional organic solvents (methanol, ethanol, acetone, ethyl acetate, etc.) and water/organic solvent mixtures have been used to ex- tract the bioactive components from Rosa canina L. fruits. Organic solvents, on the other hand, have several disadvantages, such as toxicity, volatility, non-degrada- bility, and flammability. They are also very expensive, but their use in the extraction process poses potential dan- gers to both human health and the environment.9,10 From the point of view of green chemistry several studies have been conducted to overcome these issues by replacing conventional organic solvents with deep eutectic solvents (DES) as a new generation of eco-friendly solvents.11–13 Therefore, recently ionic liquids have been developed and entitled as deep eutectic solvents (DES). DES are designable solvents formed by molecular interactions, es- pecially hydrogen bonds.14 DES can be formed by mixing two or three inexpensive materials such as organic acids, polyols, sugars, amines, and quaternary ammonium salts.15 Ultrasound-assisted extraction (UAE) is a novel extraction method known for being very efficient and en- vironmentally friendly. The frequency of the ultrasonic bath has a significant effect on the extraction process while the ultrasound irritation helps to reduce reaction time and increase mass transfer during this operation. In addition, the ultrasound allows greater penetration of the solvent into the food matrix, which increases the contact surface area between solid and liquid phases.16 In the current study, DES containing hydrogen bond donors (polyol) and hydrogen bond acceptor (organic acid) has been synthesized and used for determination total phe- nolic content, total flavonoid content, anthocyanin con- tent, from Rosa canina L. fruits and their antioxidant ac- tivity using UAE. After the determination of the best designed DES, the UAE experiments were designed by Box-Behnken design (BBD) along with response surface methodology (RSM). In this context, our study has over- come the issues related to conventional organic solvents and replace them with DES as a new generation of eco-friendly solvents. To the best of our knowledge, there is no any report on the green extraction of antioxidant phenolic compounds from Rosa canina L. fruits using the combination of UAE-DES. Therefore, the main objectives of this study are (i) to evaluate the most effective solvent to extract phenolic compounds from Rosa canina L. fruits, (ii) to screen significant extraction variables in UAE-DES using a Box-Behnken design (BBD) along with response surface methodology (RSM), and (iii) to quantify the phenolic compounds and antioxidant activ- ity of the Rosa canina L. extract at optimum conditions. 2. Materials and methods 2. 1. Plant Material Rosa canina L. fruits were collected during Septem- ber 2021 from the spontaneous flora of the central part of Kosovo. Rosehips were washed several times with tap wa- ter and dried at room temperature. The fruits were imme- diately transferred to the laboratory in polyethylene bags and stored at –4 °C until analysis. 2. 2. Chemical Materials All chemicals used in experiments were analytical grade. Ethanol was provided from Alkaloid (Skopje, North Macedonia). Folin–Ciocalteu reagent, citric acid, glycerol, ethylene glycol, sodium carbonate, and gallic acid were purchased from Sigma–Aldrich (Germany). 2. 2. Extract Preparation Ultrasound-assisted extraction was conducted in a digital ultrasonic bath at 25 °C. Rosehip fruits (500 mg) and solvent were sealed in an Erlenmeyer flask and placed into the digital ultrasonic bath. The extract was centri- fuged at 5000×g for 25 min. After centrifugation, the su- pernatant was filtered through a 0.45 μm syringe and stored at −4 °C until analysis. 2. 3. Preparation of Deep Eutectic Solvent- DES A hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD) were dried at 45 °C for 24 h before use. Before were mixed along with heating at 80 °C by a mag- netic stirrer. When a homogeneous liquid was observed, the acidity of the liquids was measured by a pH meter. The appropriate molar ratio of the used solvents, was weight and it is listed in table 1. The prepared DES compositions were stored in a desiccator to prevent moisture absorption until further analysis. Table 1. Components and their properties used in the design of DES for the UAE of Rosa canina L. fruits DES/No. HBA Chemical HBD Chemical pH formula formula DES 1 Citric acid C6H8O7 Glycerol C3H8O3 1.5 DES 2 Citric acid C6H8O7 Ethylene glycol C2H6O2 1.0 667Acta Chim. Slov. 2022, 69, 665–673 Koraqi et al.: Environmentally Friendly Extraction of Bioactive ... 2. 4. Experimental Designs 2. 4. 1. Screening of Solvents In the initial screening of the extraction efficiencies of the solvents, the Rosa canina L. fruits samples (500 mg) were mixed with the selected solvents (5 mL). Other pa- rameters were kept constant in accordance with the con- cept that one factor at a time approach will be changed. The extraction of phenolic compounds was conducted at 40 °C for 30 min with an ultrasound amplitude of 20%. The supernatant phase was collected after centrifugation at 10,000xg for 10 min. The extracts were stored at –4 °C under dark conditions. 2. 5. Determination of bioactive properties and antioxidant activity (TPC, TAA, DPPH) Total phenolics content (TPC) was determined by Folin Ciocalteu Reagent spectrophotometrically at 765 nm using the method of Singleton et al. (1999),17 with some modifications reported by Koraqi and Lluga-Ri- zani (2022)18. The results are presented as mg gallic acid (mg GAE/g DW) equivalent per gram Rosa canina L. fruits sample. Total anthocyanin analysis (TAA) was con- ducted by the pH differential method reported by Lee et al. (2005)19 with some modifications.20 TAA is based on the measurement of the absorbance of the anthocyanins, which depends on the pH alteration (pH = 1.0 and pH = 4.5). The wavelength was 530 and 657 nm. As for Total anthocyanin content, is presented as mg cyaniding-3-glu- coside (mg C3G/g DW) equivalent per gram Rosa canina L. fruits. Regarding antioxidant activity against a free radi- cal,the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test was ap- plied at 517 nm.21 Inhibition power of the extracts towards DPPH radical is stated as a percentage (% inhibition) and it can calculated according to equation 1: Antioxidant activity % inhibition of DPPH = [(Acontrol − Asample)/ Acontrol] × 100 (1) Acontrol represents the absorbance of the diluted DPPH solution, and Asample represents the absorbance of the sample. 2. 6. Statistical Analysis The differences among extraction solvents were de- termined using analysis of variance (ANOVA), followed by Duncan tests (SPSS 22 package program for Windows, Chicago, IL, USA). Statistical significance was defined at a 95% confidence level. Design Expert v13.0 trial software (Stat-Ease, USA) was used for the construction of exper- imental designs (BBD and RSA), regression analysis of experimental data, and plotting of 3D response surface graphs. ANOVA test was used to assess the statistical sig- nificance of the regression coefficient by F-test at 95% con- fidence level. The adequacy of the fitted polynomial model was expressed by the coefficient of determination (R2) and lack of fit test. 3. Results and Discussion 3. 1. Comparison of the Deep Eutectic Solvent (DES) Citric acid-based DES has been synthesized with polyol HBD such as glycerol and ethylene glycol. Figure 2 shows that the superior yield for all dependent variables (DPPH, TPC, and TAA) has been gained by the citric acid/ Figure 1. Experimental design for extraction bioactive compounds from Rosa canina L. fruits 668 Acta Chim. Slov. 2022, 69, 665–673 Koraqi et al.: Environmentally Friendly Extraction of Bioactive ... ethylene glycol combination. Hrnčič et al. (2019)22 extract- ed Rosa canina L. fruits with conventional solvents (meth- anol solution-MeOH and water), where TPC and DPPH changed between 8.13 mg GAE/g extract and 9.01% DPPH Inhibition, respectively. Our results for TPC were 13 times better, whilst our phenolic quantity was almost twice com- pared to those of the previous studies. Su et al. (2007)23 also reported a lower value of TPC than 5.09 mg GAE/g in 50% acetone extract and 2.57 mg GAE/g in 80% methanolic ex- tract. Furthermore, Fascella et al. (2019)24 declared a lower value of TPC (6784.5 mg GAE/100 g DW), but a similar values with our results for TAA (3.86 mg CGE/100g DW) and antioxidant activity DPPH IC50 (80.8%) against DPPH radical when they extracted Rosa canina L. fruits with tra- ditional extraction method through the water.IC50 values in the DPPH assay correspond to lower antioxidant activi- ty, and vice versa.24Lower value of TPC in ethanolic extract (40%–70% EtOH) has been reported by Ilbay et al.(2013)16 as well, 47.23 mg GAE/g DW in optimal conditions (40% EtOH, at 50 °C, time 81.23 min.). Bozhuyuk et al. (2021)25 reported similar results for extraction with conventional solvents as TPC (390–532 mg GAE/100g DW; and TAA 3.62–7.81 mg/kg) extracted from Rosa canina L. fruits. Our findings for TPC were higher in comparison with these studies. Most of these findings are reported in Ta- ble 2. Even though both of the DES mixtures surpassed the conventional solvents reported in the literature, citric acid/ethylene glycol formulation was shown as a better one mainly due to its viscosity.26 Since ethylene glycol is a less viscous liquid than glycerol, therefore its mixture with cit- ric acid has been shown better in terms diffusion into the plant matrix.27 After the success of the citric acid/ethylene glycol, a statistical experimental design study was per- formed. In order to achieve a clearer liquid, water addition into the DES system showed as a good addition for rising polarity of the system.27 Hence, the water content in the DES has been chosen as a process variable for the ultra- sonic-assisted extraction of Rosa canina L. fruits (Table 3). 3. 2. Box-Behnken Design and Modeling of Ultrasonic-Assisted Extraction After the success of the citric acid/ethylene glycol, a statistical experimental design study was performed. In order to achieve a clearer and more fluid liquid, water ad- dition into the DES system is a must in addition to rising polarity.27 Hence, the water content in the DES has been chosen as a process variable for the UAE of Rosa canina L. fruits (Table 3). Table 4 gives the content of TPC, TAA, and DPPH obtained by UAE under several process conditions. Table 5 summarizes the statistical outcome of the current system depending on the ANOVA test of BBD through RSM. The final equation in terms of coded factors for TPC (Response 1) is given as equation 2: TPC =96.23–1.32A+2.52B–0.6162C+1.04AB– 0.9666AC+5.02BC+4.69A²+1.96B²+1.32C² (2) Table 3. Operation parameters of the UAE of Rosa canina L. fruits Independent Unit Symbol Levels with the codes parameter −1 0 +1 Time Min A 30 60 90 Solvent volume mL B 35 42.5 50 Water content %, v/v C 10 30 50 Figure 2. Comparative results of the selected DES on the perfor- mance of UAE of Rosa canina L. fruits F 5.32 and p-value 0.0192 for the model are indica- tions of the significance of the model. Regarding process parameters, if the p-value is less than 0.05 (p < 0.05), it means that the terms of the model are significant. Time of UAE was the most influential parameter, followed by sol- Table 2. Extraction of bioactive compounds from Rosa canina L. fruits reported in scientific literature Plant material Solvents Extraction method Reference Rosa canina L. 40% EtOH Ultrasonic-assisted extraction 16 Rosa canina L. Methanol, MeOH-water Maceration, Soxhlet extraction 22 Rosa canina L. 80% MeOH,50% acetone Conventional 23 Rosa canina L. Water Traditional extraction 24 Rosa canina L. Acetone, water, acetic acid Conventional 25 669Acta Chim. Slov. 2022, 69, 665–673 Koraqi et al.: Environmentally Friendly Extraction of Bioactive ... Table 4. Effects of operation factors on the responses of Rosa canina L. fruits extract obtained by UAE Run Factor 1 Factor 2 Factor 3 Response 1 Response 2 Response 3 A:Time B: Solvent C: Water content TPC TAA DPPH (Min) volume (mL) (%, v/v) (mg GAE/gDW) mgC3G/100gDW) (inhibition %) 1 60 35 50 90.66±0.01 2.5±0.02 102.77±0.01 2 60 42.5 30 98.44±0.03 3.25±0.03 95.01±0.01 3 30 50 30 106.72±0.03 2.41±0.02 101.65±0.02 4 90 50 30 105.64±0.02 2.46±0.02 101.15±0.03 5 60 42.5 30 95.66±0.01 1.53±0.01 101.61±0.03 6 30 35 30 102.21±0.02 1.57±0.01 93.36±0.04 7 60 50 50 104.19±0.03 2.62±0.02 99.43±0.02 8 60 42.5 30 95.61±0.04 1.8±0.01 103.66±0.02 9 30 42.5 10 101.49±0.01 2.23±0.02 101.24±0.01 10 30 42.5 50 105.12±0.03 2.03±0.03 99.77±0.03 11 60 42.5 30 92.94±0.04 1.92±0.03 97.03±0.01 12 90 35 30 96.96±0.04 0.84±0.01 99.62±0.01 13 90 42.5 50 101.06±0.02 2.07±0.01 98.07±0.02 14 90 42.5 10 101.29±0.01 2.01±0.02 107.75±0.01 15 60 50 10 98.32±0.02 2.89±0.02 101.10±0.02 16 60 35 10 104.86±0.02 2.10±0.01 101.23±0.01 17 60 42.5 30 98.50±0.01 0.98±0.01 103.51±0.03 Data are given as the mean (n = 3) ± standard deviation. vent quantity (p < 0.05). Effects of interactions between the variables were also found statistically important (p < 0.05). According to the ANOVA test, R2 was found as 0.9702, whilst adjusted-R2 was 0.9086. That means that there is a convincing relationship between the experimen- tal and calculated data as seen in Figure 3. The quadratic polynomial model derived from the BBD of RSM for TAA (response 2) is given in Equation 3: TAA=1.90–0.107A+0.4213B– 0.0013C+0.1950AB+0.0650AC–0.1675BC– (3) 0.2593A2 +0.1833B2+0.4482C2 Similarly, the model was statistically significant to represent the experimental data based on the F and p val- ues (Table 4). The most influential parameter was solvent volume (v/v) of DES (p < 0.05). However, water addition into the DES solution was not a statistically significant process parameter depending on the ANOVA test results (p > 0.05). A satisfactory relationship was also observed between the experimental and calculated data for response 2 (Figure 3), where R2 and adjusted-R2 were 0.9649 and 0.9198, respectively. The second-order equation in terms of coded factors for response 3 is given in Equation 4: DPPH = 100.17+1.32A+0.7940B–1.41C– 1.69AB–2.05AC–0.8030BC–0.3196A²– (4) 0.8954B²+1.86C² The equation derived for DPPH was statistically im- portant for making estimations about the response for giv- en levels of each factor as seen in Table 4. Time of UAE was the most effective independent factor, followed by solvent volume (p < 0.05). Unexpectedly, the amount of water in the extractant system was not a statistically effective pro- cess parameter (p > 0.05). As already seen in Figures 3 and 4, there is a convincing relationship between the actual and estimated results. Depending on the ANOVA findings, R2 was found as 0.9780, whereas adjusted-R2 was 0.9326. 3. 3. Effect of Process Parameters on Ultrasonic-Assisted Extraction of Rosa Canina L. Fruits Figures 3 presents three-dimensional (3D) surfaces for UAE of Rosa canina L. fruits. As seen in Figure 3, the time of UAE has a predominant effect on the phenolics ex- traction of the current plant material. Increasing the time leads to enhancement in the TPC extraction, where there had been quick cell breakage based on the rise in temper- ature. Regarding the solvent amount to extract the plant, there was a slight effect such as increasing the yield. Since the current DES is not too viscous, the water addition had a mild effect on the enhancement of the TPC extraction. Actually, time did not have a profound impact on the TAA yield as seen in the Figures 3 and 4 as it is presented that water content rise in the extractant system favors the TAA extraction due to the decline in surface tension and viscosity as well as an increase in polarity. In respect of DPPH, we observed similar inclinations towards the pro- cess parameters of UAE of Rosa canina L. fruits. This find- ing is in a good agreement with the correlation (r = 0.879) between the total phenolics and antioxidant activity of Rosa canina L. fruits. In the matter of TAA, its relationship 670 Acta Chim. Slov. 2022, 69, 665–673 Koraqi et al.: Environmentally Friendly Extraction of Bioactive ... (r =0.003) with the antioxidant effect against DPPH radi- cal (% Inhibition) has been found to be extremely weak. 4. Conclusions This study revealed an efficient and sustainable ap- proach for the extraction of antioxidant phenolic com- pounds from Rosa canina L. fruits. In the extraction process, the combination of green solvents-DES and ul- trasound-assisted extraction was evaluated and optimized using experimental design approaches including one var- iable at a time, solvent volume, and water content (v/v). Two different deep eutectic solvents have been prepared using glycerol and ethylene glycol (hydrogen bond donor) and citric acid (hydrogen bond acceptor). Citric acid/eth- ylene glycol mixture has produced the most efficient Rosa canina L. fruits extract through ultrasonic-assisted extrac- tion. The correlation (r > 0.99) between the phenolics and the anthocyanin contents in Rosa canina L. fruits indicates that anthocyanins contribute to the most to the phenolic in the plant. On the other hand, the proposed second-order Table 5. Analysis of variance test for the Box-Behnken design for the UAE for TPC, TAA and %DPPH Source Sum of squares Df Mean square F-value p-value TPC Model 302.04 9 33.56 5.32 0.0192 A-Time 13.97 1 13.97 2.22 0.0102 B-Solvent volume 50.95 1 50.95 8.08 0.0249 C-Water content 3.04 1 3.04 0.4817 0.5100 AB 4.34 1 4.34 0.6886 0.4340 AC 3.74 1 3.74 0.5928 0.4665 BC 100.72 1 100.72 15.97 0.0052 A2 92.65 1 92.65 14.69 0.0064 B2 16.17 1 16.17 2.57 0.1533 C2 7.32 1 7.32 1.16 0.3169 Residual 44.13 7 6.30 Lack of fit 22.59 3 7.53 1.40 0.3658 Pure error 21.55 4 5.39 Cor total 346.17 16 TAA Model 3.03 9 0.3370 0.7472 0.0257 A-Time 0.0925 1 0.0925 0.2050 0.6644 B-Solvent volume 1.42 1 1.42 3.15 0.0493 C-Water content 0.0000 1 0.0000 0.0000 0.0259 AB 0.1521 1 0.1521 0.3373 0.5796 AC 0.0169 1 0.0169 0.0375 0.8520 BC 0.1122 1 0.1122 0.2489 0.6332 A2 0.2830 1 0.2830 0.6275 0.4543 B2 0.1414 1 0.1414 0.3135 0.5930 C2 0.8460 1 0.8460 1.88 0.2131 Residual 3.16 7 0.4510 Lack of fit 0.3407 3 0.1136 Pure error 2.82 4 0.7040 0.1613 0.9171 Cor total 6.19 16 DPPH Model 83.39 9 9.27 0.6531 0.0299 A-Time 13.98 1 13.98 0.9852 0.3540 B-Solvent volume 5.04 1 5.04 0.3555 0.0298 C-Water content 15.91 1 15.91 1.12 0.0348 AB 11.42 1 11.42 0.8051 0.3994 AC 16.86 1 16.86 1.19 0.3117 BC 2.58 1 2.58 0.1818 0.6826 A2 0.4300 1 0.4300 0.0303 0.8667 B2 3.38 1 3.38 0.2379 0.6406 C2 14.61 1 14.61 1.03 0.3439 Residual 99.30 7 14.19 Lack of fit 37.36 3 12.45 0.8044 0.5532 Pure error 61.93 4 15.48 Cor total 182.68 16 671Acta Chim. Slov. 2022, 69, 665–673 Koraqi et al.: Environmentally Friendly Extraction of Bioactive ... Figure 3. A 2D contour plots and 3D response surface of TPC, TAA, and DPPH as a function of time (min) and solvent volume (mL) 672 Acta Chim. Slov. 2022, 69, 665–673 Koraqi et al.: Environmentally Friendly Extraction of Bioactive ... Figure 4. The effect of DES extraction parameters (time, solvent volume, water content) on TPC, TAA, and DPPH models of the Box-Behnken design have been decided to be satisfactory depending on the statistical indicators such as p < 0.05, R2 > 0.96 and adjusted R2 > 0.91. We could optimize the bioactive ingredients in the Rosa canina L. fruits extract obtained by ultrasonic-assisted extraction as an efficient, economically and applicable approach. On the other hand, the results of this study can be utilized for further applications of antioxidant phenolic compounds from Rosa canina L. fruits in the food, cosmetical, and pharmaceutical industries as well as this study could help in using the same approach for extraction of the bioactive compounds from other plants. 673Acta Chim. Slov. 2022, 69, 665–673 Koraqi et al.: Environmentally Friendly Extraction of Bioactive ... 5. References 1. G. Angelov, S.S. Boyadzhieva, S.S. Georgieva, Cent. Eur. J. Chem., 2014, 12, 502–508. DOI:10.2478/s11532-013-0395-0 2. C. Moldovan, M. Babotă, A. Mocan, L. Menghini, S. Cesa, A. Gavan, C. Sisea, C.D. Vodnar, I.M. Dias,C. Pereira, I.C.F.R. Ferreira, G. Crişana, L. Barros,Food&Function, 2021, 12, 3939. DOI:10.1039/D0FO02783A 3. A. Bhave, V. Schulzova, H. Chmelarova, L. Mrnka, J. Hajslo- va, Journal of food and drug analysis, 2017, 25, 681–690. DOI:10.1016/j.jfda.2016.12.019 4. N. Demir, O. Yildiz, M. Alpaslan, A. A. 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Wrolstad, Journal of AOAC INTER- NATIONAL, 2005, 88, 5, 1269–1278. DOI:10.1093/jaoac/88.5.1269 20. M. M. Giusti and R. E. Wrolstad, Current Protocols in Food Analytical Chemistry, 2001, F1.2.1–F1.2.13. DOI:10.1002/0471142913.faf0102s00 21. S. Şahin S. and R. Şamli, Ultrasonics Sonochemistry, 2013, 20, 595–602. DOI:10.1016/j.ultsonch.2012.07.029 22. K. M. Hrnčič, D. Cör, P. Kotnik, Ž Knez, Acta Chim. Slov., 2019, 66, 751–761. DOI:10.17344/acsi.2019.5253 23. L. Su, J. Yin, D. Charles, K. Zhou, J. Moore, L. Yu, Food Chem- istry, 2007, 100, 990–997. DOI:10.1016/j.foodchem.2005.10.058 24. G. Fascella, F. D’Angiolillo, M. M. Mammano, M. Amenta, V. F. Romeo, P. Rapisarda, G. Ballistreri, Food Chemistry, 2019, 289, 56–64. DOI:10.1016/j.foodchem.2019.02.127 25. M. R. Bozhuyuk, S. Ercisli, N. Karatas, H. Ekiert, H. O. Elan- sary, A. Szopa, Sustainability, 2021, 13,14, 8060. DOI:10.3390/su13148060 26. L. Duan, L. L. Dou, L. Guo, P. Li, E. H. Liu, ACS Sustainable Chem. Eng., 2016, 4, 2405–2411. DOI:10.1021/acssuschemeng.6b00091 27. E. Kurtulbaş, G. A. Pekel, M. Bilgin, P. D. Makris, S. Şahin, Biomass Conv. Bioref., 2022, 12, 351–360. DOI:10.1007/s13399-020-00606-3 Povzetek V tej študiji je bila raziskana zelena ekstrakcija bioaktivnih spojin iz plodov šipka (Rosa canina L.) in njihovo antiok- sidativno delovanje. V ta namen je bila uporabljena ultrazvočna ekstrakcija v kombinaciji z globokimi evtektičnimi topili (DES). Posebej zasnovana so bila globoka evtektična topila na osnovi citronske kisline. Uporabljeni so bili donorji vodikove vezi (HBD), kot sta glicerol in etilen glikol, ter akceptor vodikove vezi (HBA), kot je citronska kislina. Po izboru najboljše možnosti DES za ekstrakcijo bioaktivnih sestavin so bili optimalni pogoji ultrazvočne ekstrakcije op- timizirani s pomočjo Box-Behnkenovega oblikovanja metodologije odzivne površine (RSM). Skupna vsebnost fenolov (TPC), skupna vsebnost antocianinov (TAA) in antioksidativna aktivnost proti 2,2-difenil-1-pikrilhidrazilu (DPPH) je bila ugotovljena kot 103,37 mg GAE/g DW v DES2, 92,23 mg GAE/g DW v DES1, 3,25 mg C3G/100 g-DW v DES2, 1,31 mg C3G/100 g-DW v DES1 in 101,85 % inhibicija v DES2, 94,32 %. Rezultati študije so pokazali, da je predstavljena ekstrakcija bioaktivnih spojin iz plodov šipka (Rosa canina L.) konkurenčno trajnostna, zelena in učinkovita. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 674 Acta Chim. Slov. 2022, 69, 674–680 Zhang et al.: Syntheses, Characterization and Crystal Structures ... DOI: 10.17344/acsi.2022.7578 Scientific paper Syntheses, Characterization and Crystal Structures of Dicyanamide Bridged Polynuclear Copper(II) and Zinc(II) Complexes with Urease Inhibitory Activity Li Zhang,1 Yuqing Gu,1 Xinhui Feng,1 Ting Yang,2 Xiaoyan Li,3 Jing Wang1 and Zhonglu You1,* 1 Department of Chemistry, Liaoning Normal University, Dalian 116029, P. R. China 2 Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, P.R. China 3 Zibo Vocational Institute, Zibo 255314, P.R. China * Corresponding author: E-mail: youzhonglu@126.com Tel.: +8641182156989 Received: 05-16-2022 Abstract A pair of structurally similar dicyanamide bridged copper(II) and zinc(II) complexes [CuL(dca)]n (1) and [ZnL(dca)] n (2), were prepared from the fluorine containing Schiff base 5-fluoro-2-(((2-hydroxyethyl)imino)methyl)phenol (HL). The compounds were characterized by physico-chemical methods. Structures of the complexes were confirmed by single crystal X-ray diffraction. The Cu atom in complex 1 is in square pyramidal coordination, whereas the Zn atom in com- plex 2 is in trigonal bipyramidal coordination. The copper complex has effective Jack bean urease inhibitory activity, with IC50 value of 0.14 ± 0.12 μmol L–1. Keywords: Schiff base; copper and zinc complexes; crystal structure; urease inhibition 1. Introduction Schiff bases derived from salicylaldehyde and its an- alogues with various primary amines represent indispen- sable ligands in coordination chemistry because of their diversified coordination modes with a large number of in- organic salts.1 Schiff bases as interesting chemotherapeutic agents have received considerable attention in recent years. Schiff base complexes have interesting pharmaceutical ap- plications such as antifungal, antitumor, antibacterial and bio-modeling techniques.2 Urease (amidohydrolase; EC 3.5.1.5) is a nickel-con- taining enzyme that catalyzes the hydrolysis of urea to NH3 and CO2. The catalyzed reaction rate is about 1014 times faster than the uncatalyzed. Urease enzyme is wide- ly found in fungi, bacteria and plants.3 The high efficien- cy of urease increased the hydrolysis of urea into NH3, which leads to severe toxicity in air and disgusting eco- nomic damages.4 In human, urease may produce several health concerns including hepatic coma, pyelonephritis, gastric and peptic ulcer.5 In recent years, various kinds of urease inhibitors such as dithiobisacetamides, thioureas, thiosemicarbazides, hydroxamic acids are reported in the fields of medicine.6 However, most of them are not appli- cable due to the low efficiency and side effects. Therefore, it is of great interest to explore new urease inhibitors. Some Schiff bases have been reported to have urease inhibitory activities.7 Our research group has pioneered the work on urease inhibitors with complexes derived from Schiff bas- es, and found that some copper, nickel, and zinc complex- es have effective activities.8 Schiff bases with halide groups are reported to have enhanced urease inhibitory activity.9 Xiao and coworkers reported that the introduction of fluo- rine atom in the hydroxamic acid compounds can increase their urease inhibitory activities.10 In addition, dicyana- mide anion is an interesting ligand in coordination chem- istry, which can lead to the formation of metal complexes with versatile structures.11 In order to construct new struc- tures of dicyanamide bridged complexes, and explore new urease inhibitors, two copper(II) and zinc(II) complexes, [CuL(dca)]n (1) and [ZnL(dca)]n (2), were prepared from 675Acta Chim. Slov. 2022, 69, 674–680 Zhang et al.: Syntheses, Characterization and Crystal Structures ... the fluorine containing Schiff base 5-fluoro-2-(((2-hy- droxyethyl)imino)methyl)phenol (HL). 2. Experimental 2. 1. Materials and Measurements 4-Fluorosalicylaldehyde and 2-aminoethanol were purchased from TCI Inc. (Japan). Other reagents and sol- vents were obtained from Xiya Reagent Company of Chi- na. Jack bean urease was purchased from Sigma-Aldrich. Elemental analyses were performed on a Perkin-Elmer 240C elemental analyzer. IR spectra were recorded on a Jasco FT/IR-4000 spectrometer as KBr pellets in the 4000–400 cm–1 region. UV-Vis spectra were recorded on a Perkin-Elmer Lambda 900 spectrometer. Conductivi- ty measurements were performed using a Metrohm 712 conductometer at 25 °C. The urease inhibitory activity was measured on a Bio-Tek Synergy HT microplate reader. Single crystal structures were determined by Bruker D8 Venture single crystal diffraction. 2. 2. Synthesis of HL and the Complexes 2. 2. 1. 5-Fluoro-2-(((2-hydroxyethyl)imino) methyl)phenol (HL) 4-Fluorosalicylaldehyde (0.010 mol, 1.4 g) and 2-am- inoethanol (0.010 mol, 0.61 g) were mixed in methanol (30 mL). The mixture was stirred for 30 min at reflux. The solvent was evaporated by distillation to give yellow solid, which was recrystallized from ethanol to give yellow crys- talline product. The product was washed three times with cold ethanol and dried in air. Yield: 1.5 g (82%). Charac- teristic IR data (KBr, cm–1): 3372 (OH), 1635 (C=N). UV–Vis data (methanol, λ/nm): 230, 335. Anal. Calcd for C9H10FNO2: C, 59.01; H, 5.50; N, 7.65. Found: C, 58.87; H, 5.58; N, 7.73%. 2. 2. 2. c atena-(μ2-Dicyanamide)(5-fluoro-2-(((2- hydroxyethyl)imino)methyl)phenolate) copper(II) (1) The Schiff base HL (1.0 mmol, 0.18 g) was dis- solved in methanol (20 mL), to which was added drop- wise Cu(NO3)2·3H2O (1.0 mmol, 0.24 g) and NaN(CN)2 (1.0 mmol, 0.089 g) dissolved in methanol (20 mL). The mixture was stirred for 20 min at room temperature. The filtrate was kept in air for a few days, to form deep blue crystals suitable for single crystal X-ray diffraction. The isolated crystals were washed three times with cold meth- anol and dried in air. Yield: 0.17 g (55%). Characteristic IR data (KBr, cm–1): 3438 (OH), 2304, 2243, 2175 (N(CN)2), 1648 (C=N). UV–Vis data (methanol, λ/nm): 270, 352. Anal. Calcd for C11H9CuFN4O2: C, 42.38; H, 2.91; N, 17.97. Found: C, 42.51; H, 3.02; N, 17.83%. ΛM (10–3 mol L–1 in methanol): 35 Ω–1 cm2 mol–1. 2. 2. 3. catena-(μ2-Dicyanamide)(5-fluoro-2-(((2- hydroxyethyl)imino)methyl)phenolate) zinc(II) (2) The zinc complex was prepared with the same method as described for the copper complex, but with Cu(NO3)2·3H2O replaced with Zn(NO3)2·6H2O (1.0 mmol, 0.30 g). Colorless block shaped crystals suitable for single crystal X-ray diffrac- tion were obtained after 5 days. Yield: 0.20 g (64%). Char- acteristic IR data (KBr, cm–1): 3420 (OH), 2341, 2273, 2199 (N(CN)2), 1643 (C=N). UV-Vis data (methanol, λ/nm): 272, 345. Anal. Calcd for C11H9FN4O2Zn: C, 42.13; H, 2.89; N, 17.87. Found: C, 42.02; H, 2.97; N, 17.75%. ΛM (10–3 mol L–1 in methanol): 28 Ω–1 cm2 mol–1. 2. 3. X-ray Crystallography Diffraction intensities for the complexes were col- lected at 298(2) K using a Bruker D8 Venture diffractom- eter with MoKα radiation (λ = 0.71073 Å). The collected data were reduced with SAINT,12 and multi-scan absorp- tion correction was performed using SADABS.13 Struc- tures of the complexes were solved by direct methods and refined against F2 by full-matrix least-squares method us- ing SHELXTL.14 All of the non-hydrogen atoms were re- fined anisotropically. The hydroxyl H atoms of the Schiff base ligands in the complexes were located from difference Fourier maps and refined isotropically, with O–H distanc- es restrained to 0.85(1) Å. The remaining hydrogen atoms were placed in calculated positions and constrained to ride on their parent atoms. Crystallographic data for the com- plexes are summarized in Table 1. Table 1 Crystal data for the complexes 1 2 Formula C11H9CuFN4O2 C11H9FN4O2Zn FW 311.76 313.59 Crystal system Monoclinic Monoclinic Space group P21/c P21/n a (Å) 7.6028(11) 7.5125(9) b (Å) 15.340(2) 10.8296(12) c (Å) 10.6785(15) 16.1164(19) α (º) 90 90 β (º) 91.869(2) 94.623(2) γ (º) 90 90 V (Å3) 1244.7(3) 1306.9(3) Z 4 4 T (K) 298(2) 298(2) μ (MoKα) (cm–1) 1.770 1.894 Reflections/parameters 7343/175 6740/175 Unique reflections 2321 2425 Observed reflections 2055 2016 [I >2σ(I)] Restraints 1 1 Goodness of fit on F2 1.059 1.051 R1, wR2 [I >2σ(I)] 0.0356, 0.0952 0.0309, 0.0826 R1, wR2 (all data) 0.0405, 0.0985 0.0393, 0.0870 676 Acta Chim. Slov. 2022, 69, 674–680 Zhang et al.: Syntheses, Characterization and Crystal Structures ... 2. 4. Urease Inhibitory Activity Assay The measurement of urease inhibitory activity was carried out according to the literature method.15 The com- pounds (0.100 mmol) and the reference drug acetohy- droxamic acid, as well as copper and zinc perchlorate were first dissolved by 5.0 mL DMSO, then diluted to 1.0 L by distilled water. The assay mixture containing 75 μL of Jack bean urease and 75 μL of tested compounds with various concentrations (100 μmol L–1, 50 μmol L–1, 25 μmol L–1, 12.5 μmol L–1, 6.25 μmol L–1, 3.12 μmol L–1, 1.56 μmol L–1, 0.78 μmol L–1) pre-incubated for 15 min on a 96-well assay plate. Then 75 μL of phosphate buffer at pH 6.8 containing phenol red (0.18 mmol L–1) and urea (400 mmol L–1) were added and incubated at room temperature. The reaction time required for enough ammonium carbonate to form to raise the pH phosphate buffer from 6.8 to 7.7 was meas- ured by micro-plate reader (560 nm) with end-point being determined by the color change of phenol-red indicator. 3. Results and Discussion 3. 1. Chemistry The Schiff base HL was facile synthesized from 4-fluorosalicylaldehyde with 2-aminoethanol in methanol. The complexes 1 and 2 (Scheme 1) were synthesized ac- cording to the similar method from the Schiff base, sodium dicyanamide with copper nitrate and zinc nitrate, respec- tively, in methanol. If copper or zinc nitrate was replaced with chloride or bromide salt, the same structures for the complexes can be obtained. Molar conductivities of the complexes in methanol are within the normal values 20–40 Ω–1 cm2 mol–1, indicate their non-electrolytic nature.16 3. 2. Structure Description of the Complexes Complex 1 Molecular structure of the polymeric copper com- plex 1 is shown in Figure 1. Selected bond lengths and angles are given in Table 2. The smallest repeat unit of the complex contains [CuL(N(CN)2)], which is bridged by dicyanamide ligands to form one dimensional chain structure. The Cu atom is in square pyramidal geometry, with the phenolate oxygen (O1), imino nitrogen (N1) and hydroxyl oxygen (O2) atoms of the Schiff base ligand, and the N2 atom of the dicyanamide ligand located at the basal plane, and with the N4A (symmetry code for A: ‒1 + x, y, z) atom of the symmetry related dicyanamide ligand locat- ed at the apical position. The Cu atom deviates from the least-squares plane defined by the four basal donor atoms by 0.250(1) Å. The coordination geometry can be defined as distorted square pyramid because the structural index τ value is 0.30.17 The bond lengths of the Cu–O (1.9271(19)– 2.035(2) Å) and Cu–N (1.937(2)–1.977(2) Å) in the basal plane of the complex are comparable to those observed in the copper(II) complexes with Schiff base ligands.18 In the crystal structure of complex 1, the [CuL] units are linked by dicyanamide ligands, to form one di- mensional chain structure along the a axis. The chains are Scheme 1. The diagrams of the complexes. Figure 1. Molecular structure of 1, showing the atom-numbering scheme. Displacement ellipsoids for non-hydrogen atoms are drawn at 30% probability level. Atoms labeled with the suffix A are related to the symmetry operation ‒1 + x, y, z. 677Acta Chim. Slov. 2022, 69, 674–680 Zhang et al.: Syntheses, Characterization and Crystal Structures ... further linked through intermolecular hydrogen bonds of O‒H···O hydrogen bonds (O2‒H2 = 0.85(1) Å, H2···O1i = 1.90(1) Å, O2···O1i = 2.734(3) Å, O2‒H2···O1i = 170(4)°, symmetry code for i: ½ ‒ x, ‒½ + y, ½ ‒ z), to form two-di- mensional network along the ac plane (Figures 2 and 3). Figure 2. Molecular packing diagram of 1, viewed along the a axis. Hydrogen bonds are shown as dashed lines. Table 2 Selected bond lengths (Å) and angles (º) for the complexes 1 2 M1–N1 1.937(2) 2.002(2) M1–N2 1.977(2) 2.016(2) M1–O1 1.927(2) 1.988(2) M1–O2 2.035(2) 2.246(2) M1–N4A 2.249(3) 1.993(2) O1–M1–N1 93.95(9) 92.26(8) O1–M1–N2 92.28(9) 96.06(10) N1–M1–N2 155.31(11) 120.66(10) O1–M1–O2 173.23(8) 169.53(8) N1–M1–O2 81.51(9) 77.28(9) N2–M1–O2 89.87(10) 89.21(10) O1–M1–N4A 95.48(10) 98.15(10) N1–M1–N4A 101.08(11) 128.85(9) N2–M1–N4A 102.06(10) 107.86(10) O2–M1–N4A 90.34(10) 88.78(10) M = Cu for 1, Zn for 2. Complex 2 Molecular structure of the polymeric zinc complex 2 is shown in Figure 4. The smallest repeat unit of the com- plex contains [ZnL(N(CN)2)], which is bridged by dicya- namide ligands to form one dimensional chain structure. The Zn atom is in trigonal bipyramidal geometry, with the imino nitrogen (N1) atom of the Schiff base ligand, and two nitrogen (N2 and N4A, symmetry code for A: ‒1 + x, y, z) atoms from two dicyanamide ligands located at the equatorial plane, and with the phenolate oxygen (O1) and hydroxyl oxygen (O2) atoms of the Schiff base ligand lo- cated at the axial positions. The Zn atom deviates from the least-squares plane defined by the three equatorial donor atoms by 0.187(1) Å. The coordination geometry can be defined as distorted trigonal bipyramid because the struc- tural index τ value is 0.68.17 The bond lengths of the Zn–O (1.988(2)–2.247(2) Å) and Zn–N (1.993(2)–2.016(2) Å) of the complex are comparable to those observed in the zinc(II) complexes with Schiff base ligands.19 In the crystal structure of complex 1, the [ZnL] units are linked by dicyanamide ligands, to form one di- mensional chain structure along the a axis. The chains are Figure 3. Molecular packing diagram of 1, viewed along the b axis. Hydrogen bonds are shown as dashed lines. Figure 4. Molecular structure of 2, showing the atom-numbering scheme. Displacement ellipsoids for non-hydrogen atoms are drawn at 30% probability level. Atoms labeled with the suffix A are related to the symmetry operation ‒1 + x, y, z. 678 Acta Chim. Slov. 2022, 69, 674–680 Zhang et al.: Syntheses, Characterization and Crystal Structures ... further linked through intermolecular hydrogen bonds of O‒H···O hydrogen bonds (O2‒H2 = 0.85(1) Å, H2···O1i = 1.91(1) Å, O2···O1i = 2.714(3) Å, O2‒H2···O1i = 161(4)°, symmetry code for i: ‒x, ‒1 ‒ y, ‒z) along the b axis, to form two-dimensional network along the ab plane (Fig- ures 5 and 6). Figure 5. Molecular packing diagram of 2, viewed along the a axis. Hydrogen bonds are shown as dashed lines. Figure 6. Molecular packing diagram of 2, viewed along the c axis. Hydrogen bonds are shown as dashed lines. 3. 3. IR and UV-Vis Spectra The weak bands centered at 3438 and 3420 cm–1 in complexes 1 and 2, respectively, are assigned to ν(O–H). The intense absorption bands at 2304, 2243 and 2175 cm–1 in 1, and 2341, 2273 and 2199 cm–1 in 2, are assigned to the stretching vibrations of dicyanamide ligands.20 Strong absorptions at 1648 cm–1 in 1 and 1643 cm–1 in 2 are as- signed to azomethine groups, ν(C=N).20b The phenolic ν(Ar–O) appear at 1288 cm–1 in 1 and 1301 cm–1 in 2. The weak bands in the range of 400–600 cm–1 for the complex- es can be assigned to ν(M–O).21 The electronic spectra of the complexes were deter- mined with methanol as solvent. The spectra of the com- plexes reveal intense absorption bands at 270–272 nm are assigned to n-π* transition of the azomethine groups. The bands observed at 340–360 nm in the spectra of the com- plexes are assigned to ligand to metal charge transfer tran- sitions.22 3. 4. Urease Inhibitory Activity Assay The results of the inhibition assays against the urease for the synthetic compounds are listed in Table 3. The cop- per complex has good inhibitory activity on urease with IC50 value of 0.14 ± 0.12 μmol L–1, whereas the zinc com- plex has no activity. The copper complex show better ac- tivity than the reference drug acetohydroxamic acid (IC50 = 37.2 ± 4.0 μmol L–1). Both the copper and zinc com- plexes have better activities than copper and zinc perchlo- rate. Thus, the copper complex would be a potential ure- ase inhibitor that deserves further study on the treatment of diseases like hepatic coma, pyelonephritis, gastric and peptic ulcer, as well as on the application in the nitrogen containing fertilizer. Table 3 Inhibition of urease by the tested materials Tested materials Percentage Inhibition IC50 rate# (μmol L–1) HL – > 100 1 99 ± 2.1 0.14 ± 0.12 2 33 ± 1.8 3.4 ± 1.6 Copper perchlorate 87.5 ± 2.6 8.8 ± 1.4 Zinc perchlorate – > 100 Acetohydroxamic acid 85.5 ± 3.9 28.1 ± 3.6 # The concentration of the tested material is 100 μmol L–1. – indicates no activity. 4. Conclusion This work reports the syntheses, characterization and crystal structures of two new dicyanamide bridged copper and zinc complexes with fluorine containing Schiff base 5-fluoro-2-(((2-hydroxyethyl)imino)methyl)phenol. 679Acta Chim. Slov. 2022, 69, 674–680 Zhang et al.: Syntheses, Characterization and Crystal Structures ... The copper complex has effective urease inhibitory ac- tivity. The results indicated that the copper complex can be further optimized and developed as a prospective lead urease inhibitor. Supplementary data CCDC 2170031 (1) and 2170032 (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: depos- it@ccdc.cam.ac.uk. Acknowledgments This work was financially supported by the Educa- tion Office of Liaoning Province (Project No. LJKZ0984). 5. References 1. (a) H. Kargar, P. Forootan, M. Fallah-Mehrjardi, R. Behjat- manesh-Ardakani, H. A. Rudbari, K. S. Munawar, M. Ashfaq, M. N. Tahir, Inorg. Chim. Acta 2021, 523, 120414; DOI:10.1016/j.ica.2021.120414 (b) S. H. Sumrra, W. Zafar, S. A. Malik, K. Mahmood, S. S. Shafqat, S. Arif, Acta Chim. Slov. 2022, 69, 200–216; DOI:10.17344/acsi.2022.7182 (c) Y. Yuan, X.-K. Lu, G.-Q. Zhou, X.-Y. Qiu, Acta Chim. Slov. 2021, 68, 1008–1015. DOI:10.17344/acsi.2021.7070 2. (a) S. U. Parsekar, K. Paliwal, P. 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DOI:10.1016/j.poly.2009.07.017 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Sintetizirali smo dva strukturno sorodna bakrova(II) in cinkova(II) kompleksa z dicianamidnim mostovnim ligandom, [CuL(dca)]n (1) in [ZnL(dca)]n (2), z vezavo fluor vsebujoče Schiffove baze 5-fluoro-2-(((2-hidroksietil)imino)metil) fenol (HL). Spojini smo okarakterizirali s fizikalno-kemijskimi metodami. Strukturi kompleksov smo določili z mo- nokristalno rentgensko difrakcijo. V kompleksu 1 je atom Cu koordiniran kvadratno piramidalno, medtem ko je atom Zn v kompleksu 2 koordiniran trigonalno bipiramidalno. Bakrov kompleks učinkovito zavira ureazo stročnice Canavalia ensiformis z vrednostjo IC50 0,14 ± 0,12 μmol L–1. 681Acta Chim. Slov. 2022, 69, 681–693 Brontowiyono et al.: Phosphate Ion Removal from Synthetic and Real Wastewater ... DOI: 10.17344/acsi.2022.7594 Scientific paper Phosphate Ion Removal from Synthetic and Real Wastewater Using MnFe2O4 Nanoparticles: A Reusable Adsorbent Widodo Brontowiyono,1 Indrajit Patra,2 Shaymaa Abed Hussein,3 Alimuddin,4 Ahmed B. Mahdi,5 Samar Emad Izzat,6 Dhuha Mohsin Al-Dhalemi,7,* Ahmed Kareem Obaid Aldulaim,8 Rosario Mireya Romero Parra,9 Luis Andres Barboza Arenas10 and Yasser Fakri Mustafa11 1 Department of Environmental Engineering and Centre for Environmental Studies, Islamic University of Indonesia, Yogyakarta-55589, Indonesia 2 NIT Durgapur, West Bengal, India 3 Al-Manara College for Medical Sciences, Maysan, Iraq 4 Physical Sciences Section, School of Sciences, Maulana Azad National Urdu University, Hyderabad-500032, Telangana, India 5 Anesthesia Techniques Department, Al-Mustaqbal University College, Babylon, Iraq 6 Pharmacy Department, Al-Nisour University College, Baghdad, Iraq 7 Altoosi University College, Najaf, Iraq 8 Department of Pharmacy, Al-Zahrawi University College, Karbala, Iraq 9 Universidad Continental, Lima, Perú 10 Universidad Tecnológica del Perú, Perú 11 Department of Pharmaceutical Chemistry, College of Pharmacy, University of Mosul, Mosul-41001, Iraq * Corresponding author: E-mail:_dhuha14@yahoo.com Received: 06-29-2022 Abstract The purpose of this study was to eliminate phosphate (P) from wastewater using MnFe2O4 nanoparticles. BET, TGA/ DTG, FTIR, SEM, TEM, VSM, XRD and EDX/Map analyses were used to determine the MnFe2O4 surface properties. The specific surface area of the adsorbent was 196.56 m2/g and VSM analysis showed that the adsorbent has a ferro- magnetic property. The maximum P sorption efficiency using MnFe2O4 (98.52%) was achieved at pH 6, temperature of 55 °C, P concentration of 10 mg/L, time of 60 min, and sorbent dosage of 2.5 g/L, which is a significant value. Also, the thermodynamic study indicated that the P sorption process is spontaneous and endothermic. Moreover, the utmost sorption capacity of P using MnFe2O4 was 39.48 mg/g. Besides, MnFe2O4 can be used for up to 6 reuse cycles with high sorption efficiency (>91%). Also, MnFe2O4 was able to remove phosphate, COD, and BOD5 from municipal wastewater with considerable removal efficiencies of 82.7%, 75.8%, and 77.3%, respectively. Keywords: Sorption, MnFe2O4 nanoparticles, phosphate, wastewater 1. Introduction Water pollution is a serious problem that can harm any living thing.1–2 Among different contaminants, phos- phate (P) ion pollution is one of the most important en- vironmental issues worldwide. The presence of P at high concentrations in natural water has adverse impacts on water ecology.3 By increasing the concentration of P in water, algae and other aquatic plants grow and reduce the level of dissolved oxygen and eliminate photosynthesis in water.4 Detergents and chemical fertilizers are the largest source of P ions. Domestic effluents and running water from fields where phosphate fertilizers are used release large amounts of P ions into natural waters. According to 682 Acta Chim. Slov. 2022, 69, 681–693 Brontowiyono et al.: Phosphate Ion Removal from Synthetic and Real Wastewater ... the EPA, the maximum allowable level and the limit of P ion release in the environment are reported to be 0.1 ppm and less than 0.05 ppm, respectively.5 Also, the al- lowable limit of P ions in drinking water is 0.2 ppm and the standard for discharge of phosphate ions into surface water is 6 ppm. The concentration of P in urban, rural, and agricultural wastewaters is very high (between 15– 2000 ppm).5,6 P has a critical role as an essential matter for plant growth in the soil as well as a limiting element in the algae growth and eutrophication phenomenon in water. A concentration of 0.005–0.05 mg/L is required to cause the eutrophication phenomenon.7 This phenome- non leads to the abundant growth of aquatic plants, the growth of algae, and the imbalance of organisms in water resources. P in surface waters and effluents are common- ly found chemically in the form of organic phosphates (such as detergents) and mineral phosphates (polyphos- phates and orthophosphates). Organic polyphosphates and phosphates are converted to orthophosphates after hydrolysis and biodegradation.8 There are various procedures for wastewater treat- ment, including reverse osmosis, membrane technology, chemical deposition, ion exchange, nanofiltration, coagu- lation, and sorption process. Most of the physical process- es such as reverse osmosis have high operating costs.9–11 Among these processes, the sorption process is a suitable method to eliminate P ions, because this process is eco- nomical, simple, reversible, low-cost, high selectivity, and high operating speed.12 P ions are insoluble in water and can be easily sorbed on the sorbent surface. Many adsor- bents have been recently utilized for eliminating P ions, in- cluding Fe3O4,13 magnetic/clay,14 chitosan,15 goethite na- noparticles,16 aluminum hydroxide modified palygorskite nano-composites,17 zirconium oxide,18 sludge derived biochar,19 and iron/biochar.20 Magnetic particles have re- ceived much attention for elimination of pollutants from sewage due to their simple synthesis, excellent surface area, and considerable removal efficiency of contaminants.21 MFe2O4 (M = Co, Mn, Cu, Zn, Mg) with the structure of cubic spinel or MO.Fe2O3 shows an important group of iron oxides in which Fe3+ and M2+ occupy quadrilateral or octahedral sites. MFe2O4 magnetic configurations can be engineered by controlling the chemical features of M2+ to produce a wide range of magnetic features.22 Iron-manga- nese oxide spinel with MnFe2O4 structure is an example of metal oxides, which has high thermal and mechanical sta- bility. MnF2O4 spinel nanocrystalline can be synthesized by various procedures such as microwave, hydrothermal, and chemical co-precipitation processes. One of the main advantages of MnFe2O4 is its simple synthesis, which dis- tinguishes it from other adsorbents.21 To control the size of MFe2O4 in the chemical coprecipitation method, pH and temperature adjustment is essential.22 Nanoadsorbents have properties such as high sorption capacity, excellent performance even at low concentration levels and low cost. Also, they are able to be reused in several cycles with- out major loss in performance.23 Due to these properties, MnFe2O4 nanoparticles were used in this research. The purpose of this work was to eliminate P ions us- ing MnFe2O4 nanoparticles. MnFe2O4 was utilized for the first time for removing phosphate. The nanoadsorbent was synthesized by the chemical co-precipitation method and analyzed by various devices like SEM, BET, TEM, FTIR, XRD, TGA-DTG, VSM, and EDX/Map. Also, the impact of various factors was studied on the P ions removal and the best operating conditions were identified. Moreover, kinetic, isotherm, and thermodynamic behaviors of P ion sorption were studied using MnFe2O4 nanoparticles. 2. Chemicals and Procedures 2. 1. Chemicals and Devices In this study, several chemicals were utilized to syn- thesize MnFe2O4 nanoparticles as well as P ions stock solu- tion, including KH2PO4 (purity = 99%, Sandia Co., Chi- na), MnCl2 with a purity of 97%, NaOH with a purity of 99%, acetone with a purity of 99%, (NH4)6 MO7 O24 with a purity of 99%, HCl with a purity of 37%, H4NO3V with a purity of 99% (Merck Co., Germany), and FeCl3.6H2O (purity = 99%, Sigma Aldrich Co.). Also, digital scale (FX 300 I model), magnetic stir- rer (HPMT 700 model), magnet, oven (DZF-6020 model), pH meter (RPB1000 model) and UV-vis spectrophotom- eter (Shimadzu 1700 model, Japan) were utilized to weigh materials, heating and mixing, separation of nanoparticles from the solution, drying the sorbent, regulating the sam- ple pH, determining the residual concentration of P ions in the solution, respectively. 2. 2. Preparing Phosphate Stock Solution and MnFe2O4 Synthesis KH2PO4 salt was employed to prepare the P stock solution. For this purpose, 4.39 g of KH2PO4 was added to one liter of distilled water and stirred to dissolve com- pletely in water. Different concentrations of 10, 20, 30, 50, 70, and 100 ppm were prepared by diluting the initial stock solution. MnFe2O4 magnetic nanoparticles were synthesized by the chemical co-precipitation approach at 80 °C. For this purpose, 0.1 mol of MnCl2 and 0.2 mol of FeCl3.6H2O were dissolved in 100 mL of distilled water and stirred at 80 °C for 20 min by a magnetic stirrer. Next, NaOH (3 molars) was added to the suspension dropwise to regu- late the solution pH at 11. The mixture was stirred for 3 h. After that, the solution color changed to black, indicating the synthesis of MnFe2O4 nanoparticles. The formed na- noparticles were separated from the mixture by a magnet and washed with distilled water and acetone (C3H6O) to neutralize (pH = 7). Afterwards, MnFe2O4 nanoparticles were placed in an oven at 100 °C for 1 day to dry. The 683Acta Chim. Slov. 2022, 69, 681–693 Brontowiyono et al.: Phosphate Ion Removal from Synthetic and Real Wastewater ... above-mentioned nanoparticles were ground with a mor- tar and used as a sorbent.24 Several analyses were utilized for identifying the fea- tures of MnFe2O4 before and after the process, including BET (JW-DA, China) for measuring the specific surface area, SEM (TESCAN, Czech Republic) for identifying the sorbent morphology, TEM (CM120, The Nederlands) for investigating the particle distribution, EDX/Map (TES- CAN, Czech Republic) for specifying the percentage of elements, FTIR (Alpha, BRUKER, Germany) for deter- mining functional groups, XRD (D6792, The Nederlands) for determining the crystalline phases, VSM (7404, LAKE SHORE, USA) for identifying the magnetic strength of the sorbent, and TGA/DTG (PYRIS, PERKIN ELMER, USA) for specifying the thermal stability of the sorbent. 2. 3. Quantitative Determination of P To produce a suitable reagent for measuring phos- phate ions, 12.5 g of (NH4)6 MO7 O24 was dissolved in 150 mL of distilled water (solution A). In another bal- loon, 0.625 g of ammonium metavanadate (H4NO3V) was dissolved in 150 mL of distilled water and gently heated (solution B). The solution B was added to solution A and stirred for 5 min. Then, 165 mL of HCl was added to the solution A+B and its volume was raised to 500 mL by dis- tilled water. This solution was then used as the reagent. The colorimetric method was used to measure P ions by the reagent. To measure residual P ions after the sorption process, MnFe2O4 was separated from the solution using a magnet. Then, 10 ml of the solution containing P ions was mixed with 0.5 ml of the reagent solution. Finally, the concentration of P ions was measured by a UV-vis spec- trophotometer device (Shimadzu 1700 model, Japan) at a wavelength of 361 nm. 2. 4. Sorption Tests The P ion sorption tests were performed using Mn- Fe2O4 nanoparticles as a batch process. The impact of ef- fective factors such as pH (2–11), adsorbent dosage (0.5–4 g/L), time (5–13 min), temperature (25–55 °C), and P ion concentration (10–100 ppm) was studied to remove P ions. For optimization of pH, various solutions were syn- thesized in different pHs and P ion concentration of 20 mg/L. Then, 2 g/L of MFe2O4 was added to the phosphate solutions. Then, the solutions were stirred at 25 °C with a mixing rate of 500 rpm. After 60 min, the remaining con- centration of P ion was measured. Also, to investigate the impact of P ion concentration on the sorption efficiency, several experiments were done at various concentrations of P ion (10–100 ppm), pH of 6, the adsorbent dosage of 2.5 g/L, temperature of 55 °C, mixing rate of 500 rpm, and contact time of 60 min. The sorption capacity (qe) and sorption efficiency (R) were measured using Equations 1 and 2. (1) (2) In mentioned equations, Ci, Ce, W, and V are the P initial concentration, the P remaining concentration (ppm), the adsorbent amount (g/L), and the sample vol- ume (L), respectively.25 2. 5. Kinetic, Isotherm, and Thermodynamic Behaviors There are 3 steps in the sorption of contaminants using a sorbent, which can affect the sorption kinetics, in- cluding 1) transfer of contaminants from the solution to the adsorbent surface, 2) penetration of contaminants into the pores inside the adsorbent, and 3) sorption of contam- inants on the adsorbent inner surface.26 In this research, pseudo-first order (PFO) and pseudo-second order (PSO) kinetics were employed to investigate P ions sorption. To this end, several experiments were done at different con- centrations of P ions (10–100 ppm) and different contact times (5–130 min). Other factors like pH of 6, mixing rate of 500 rpm, the adsorbent dosage of 2.5 g/L, and tempera- ture of 55 °C were considered constant. Equations 3 and 4 describe the PFO and PSO models, respectively: (3) (4) In these models, qe (mg/g), qt (mg/g), K1 (min–1) and K2 (g/mg.min) are the sorption capacity at equilibrium time, sorption capacity at time t, the PFO kinetic model constant and the PSO kinetic model constant, respectively.27,28 Also, sorption isotherms describe the distribution of contaminant molecules on the adsorbent surface. The most important models are the Langmuir, Dubinnin-Ra- dushkevich (D-R) and Freundlich. The Langmuir theory is used for monolayer sorption on a homogeneous surface with an infinite number of identical sites. In this theory, it is assumed that the adsorbent sites are saturated after monolayer sorption. This model is defined below.29 (5) In this model, Ce (mg/L), qmax (mg/g) and KL (L/ mg) are the P ion equilibrium concentration, the sorption capacity at equilibrium time, and the Langmuir constant, respectively. Another isotherm model (Freundlich) describes the sorption process on a heterogeneous surface. The follow- 684 Acta Chim. Slov. 2022, 69, 681–693 Brontowiyono et al.: Phosphate Ion Removal from Synthetic and Real Wastewater ... ing relationship defines the Freundlich model: (6) Where, Kf (mg/g(L/mg)1/n) and n are the Freundlich constants. Also, n indicates whether the sorption process is desirable or not.30,31 Moreover, the D-R model is another important iso- therm and assumes that the sorption energy on the surface is homogeneous. The D-R linear model is defined below: (7) Where, qm (mol/g), β (mol2/J2), and ε are the theo- ry saturation capacity, the mean sorption free energy, and Polanyi potential, respectively. The Polanyi potential is cal- culated from Equation 8: (8) Also, the following relationship describes the type of the sorption process: (9) For E between 8–16 KJ/mol and lower than 8 KJ/ mol, the sorption process will be chemical and physical, respectively.32 Also, thermodynamic parameters like Gibbs free en- ergy changes (∆G°), entropy changes (∆S°), and enthalpy changes (∆H°) are employed to determine the nature of the sorption process. Using these parameters, it can be de- termined whether the sorption process is endothermic or exothermic.27 (10) (11) (12) In these relationships, Kd is the equilibrium constant. Negative values of ∆G° in various temperatures in- dicate the spontaneous nature of the sorption process. For –80 kJ/mol < ∆G° < 0 and –400 kJ/mol < ∆G° < –80 kJ/ mol, the sorption process will be physical and chemical, respectively.33 Also, positive values of ∆H° indicate that the sorption process is endothermic and vice versa. More- over, positive values of ∆S° indicate an increase in the sol- id-solute surface disorder during the sorption process, and its negative values display a decrease in irregularity in the sorption process.27 For investigating the thermodynamic behavior of P ion sorption using MnFe2O4 nanoparticles, several tests were performed in various temperatures (25– 55 °C), pH of 6, mixing rate of 500 rpm, nanoparticles dos- age of 2 g/L, P ion concentration of 20 mg/L and contact time of 60 min. 2. 6. Desorption Experiments and Reusability of the Adsorbent To investigate the desorption process, the sorption of P ions was done in optimal conditions using MnFe2O4 nanoparticles. Next, MnFe2O4 was separated from the solution and dried. Then, MnFe2O4 was added to 50 ml of H2SO4 solution in various concentrations (1–5 mol/liter) and stirred for 2 h. Next, the adsorbent was separated from the solutions and the concentration of residual P ions was measured. After that, the optimal concentration of H2SO4 was obtained to have the highest efficiency. Next, to study the desorption capability and reusability of the adsorbent in eight cycles, H2SO4 solution was used at the optimal concentration (4 molar). The desorption percentage of P ions was calculated as follows: (13) Where q1 and q2 are the desorption capacity (mg/g) and the sorption capacity (mg/g) of P ions, respectively.34 3. Results and Discussion 3. 1. Characteristics of MnFe2O4 For determining the surface features of MnFe2O4 nanoparticles such as specific surface area and pore size, BET analysis was used. According to Table 1, the specific surface area, pores volume and average pore size of Mn- Fe2O4 were 196.56 m2/g, 0.366 cm3/g, and 74.49 °A, re- spectively. The adsorbent pore size shows that MnFe2O4 is mesoporous. Also, the high specific surface area of the adsorbent shows that contaminants can be adsorbed on the MnFe2O4 surface. According to previous studies, the specific surface area of CoFe2O4, ZnFe2O4,35 MgFe2O4,36 Fe2O3 and Fe3O437 were 71.56, 120.1, 35.2, 150 and 130 m2/g, respectively, which are lower than our study. Table 1. Surface features of MnFe2O4 nanoparticles by BET analysis BET specific surface area 196.56 m²/g Langmuir specific surface area m²/g 273.21 Pore volume cm³/g 0.366 BJH pore volume cm³/g 0.392 Mean pore size 74.49 °A BJH average width of absorption pores 74.44 °A BJH average width of desorption pores 67.14 °A SEM, EDAX, and Mapping analyses were used to determine the morphology of MnFe2O4 nanoparticles, distribution of elements, active sites on the adsorbent sur- 685Acta Chim. Slov. 2022, 69, 681–693 Brontowiyono et al.: Phosphate Ion Removal from Synthetic and Real Wastewater ... face, and elemental compositions before and after the P ion sorption process, as shown in Figure 1. SEM image for MnFe2O4 nanoparticles shows that there are many holes and bumps, which are effective in the phosphate ion sorp- tion (Figure 1 (a)). Also, EDAX and Mapping analyses for MnFe2O4 nanoparticles indicate several elements such as Fe (46.87%), O (28.5%), and Mn (24.63%) in its surface, which confirm the correct synthesis of MnFe2O4 nanopar- ticles (Figure 1 (b and c)). After sorption of P ions, many changes were observed on the MnFe2O4 surface, which can be due to the sorption of P ions (Figure 1 (d)). Also, EDAX analysis showed that the percentage of elements has been changed. According to Figure 1 (f), the percentag- es of Fe, O, and Mn were changed to 46.1%, 22.58%, and 30.6%, respectively. Moreover, 0.73% of P was seen after the P ion sorption. TEM analysis was also employed to determine the morphology and particle size of MnFe2O4 nanoparticles (Figure 2). The outcomes show that the particle size of Mn- Fe2O4 is smaller than 50 nm. The particles in the MnFe2O4 structure have spherical and cubic morphologies with fine size distribution. A similar morphology was observed by Cabrera et al.38 Figure 3 indicates FTIR analysis for MnFe2O4 na- noparticles. For MnFe2O4 nanoparticles before sorp- tion, a wide peak was seen at 3363 cm–1, which can be attributed to the stretching vibration of hydroxyl group (-OH). Also, another absorption peak was seen at 586 cm–1, which shows the spinel ferrite crystal structure of MnFe2O4. Also, the absorption peak at 586 cm−1 shows intrinsic stretching vibrations of metals at tetrahedral sites.39 Moreover, two peaks were observed at 1624 cm–1 and 964 cm–1, which indicate C = C and C-O vibrations, Figure 1. SEM, Mapping, and EDAX analysis for MnFe2O4 nanoparticles before sorption (a-c) and after sorption of P ions (d-f) Figure 2. TEM image for MnFe2O4 nanoparticles 686 Acta Chim. Slov. 2022, 69, 681–693 Brontowiyono et al.: Phosphate Ion Removal from Synthetic and Real Wastewater ... respectively.24 After sorption of P, the range of absorption peaks in the MnFe2O4 structure was slightly changed, which can be due to the interaction of functional groups and phosphate ions. To this end, functional groups of -OH, C = C, C-C, and Fe-O in the MnFe2O4 structure were shifted to 3366, 1632, 1016, and 583 cm–1, respec- tively.40–42 Figure 3. FTIR results for MnFe2O4 nanoparticles before and after sorption of P ions Moreover, XRD analysis for determining the crys- talline phases in the MnFe2O4 structure is demonstrated in Figure 4. Several peaks with various intensities were observed at 18.04 °, 29.6 °, and 35.02 °, which are attrib- uted to the crystalline phases of (111), (220), and (311), respectively. Also, other peaks were observed at 42.42 °, 56.62 °, and 61.74 °, which are attributed to the crystal- line phases of (400), (422), and (440), respectively. These crystalline phases correspond to the card number 0449- 075-01.24,38 The peak at 35.02o is attributed to the spinel structure of Mn ferrite, which has been confirmed by Cabrera et al.38 Figure 4. XRD results for MnFe2O4 nanoparticles Furthermore, VSM analysis was used to measure the magnetic strength of MnFe2O4 nanoparticles (Figure 5). According to the results, magnetic saturation, coercive force, and magnetic resonance of MnFe2O4 nanoparticles were 6.377 emu/g, 230 Oe, and 2.245 emu/g, respectively. The amount of magnetic saturation and the resulting fig- ure shows that MnFe2O4 nanoparticles have ferromagnetic properties and can be separated from the aqueous media by a magnet (1 Tesla).43 Figure 5. Magnetic behavior of MnFe2O4 nanoparticles Eventually, the thermal stability of MnFe2O4 nano- particles was investigated by TGA-DTG analysis (Figure 6). In the temperature range of 50–300 °C, MnFe2O4 na- noparticles lost 5% by weight, which can be due to the evaporation of moisture from its surface.44 By increasing temperature from 300 to 900 °C, MnFe2O4 nanoparticles had the highest weight loss (8 wt.%), which is due to the structural degradation and dehydroxylation of MnFe2O4 nanoparticles.45 Also, its weight loss in the temperature range of 900–1000 °C was about 2% by weight. Gener- ally, MnFe2O4 nanoparticles showed a weight loss of 15 wt.%. Figure 6. TGA-DTG analysis for thermal stability of MnFe2O4 nan- oparticles 687Acta Chim. Slov. 2022, 69, 681–693 Brontowiyono et al.: Phosphate Ion Removal from Synthetic and Real Wastewater ... 3. 2. Effective Factors on the P Ion Removal The solution pH is a key factor in the sorption pro- cess and can affect the surface properties of the adsorbent. Also, pH causes the release of various forms of ions in the solution.46 Figure 7 shows the impact of pH at different temperatures on the uptake of P ions. Depending on the solution pH, phosphate species are present in water and seawater as H3PO4, H2PO4–, HPO42–, and PO43– ions. The pH value of municipal effluent normally is in the range of 6.5–7.3, and H2PO4– species is the major species of phos- phate.47 At pH> 2, H3PO4 is the predominant species of P ion in solution, which is due to the absence of electrostat- ic forces. By increasing the pH from 2 to 6, H2PO4– and HPO42– are the main species in the solution, which have a strong attraction to the MnFe2O4 adsorbent, enhanc- ing removal efficiency. At pH>6, the sorption efficiency decreases because the solution contains large amounts of H2PO4– and PO43– species, and these ions compete fiercely with OH– ions to sit on the active sites of the adsorbent.20 Therefore, the highest removal efficiency (96.56%) was ob- tained at pH 6. Also, the impact of temperature on the P ion sorp- tion is shown in Figure 7. The tests were performed at the adsorbent dosage of 2 g/L, P ion concentration of 20 mg/L, mixing rate of 500 rpm, time of 60 min, and pH of 6. As shown, the sorption efficiency of P ions enhances from 86.83% to 96.56% with raising the temperature from 25 to 55 °C, respectively, demonstrating that the sorption of P ions using MnFe2O4 nanoparticles is endothermic.20 Therefore, the optimal temperature for removing P ions using MnFe2O4 nanoparticles was 55 °C. Figure 7. Impact of pH at different temperatures on the P ion sorp- tion using MnFe2O4 nanoparticles (contact time = 60 min, mixing rate = 500 rpm, pH = 6, P ion concentration = 20 mg/L and adsor- bent dosage = 2 g/L) The initial concentration of P ions in the solution plays an important role as the driving force overcoming the resistance due to the mass transfer between the liquid and solid phases. The impact of phosphate ion concentra- tion at different contact times on the P ion sorption using MnFe2O4 nanoparticles is indicated in Figure 8. As shown, the removal efficiency of P ions decreases from 97.43% to 87.54% with increasing P ion concentration from 10 to 100 mg/L, respectively, which is due to the greater ac- cessibility of active sites at low P ion concentrations. At a constant adsorbent dose, the ratio of active sites to P ions decreases with increasing P ion concentration, resulting in a decrease in the interaction between P ions and sorption sites.48,49 Therefore, the highest removal efficiency of P ions (97.43%) was obtained at a concentration of 10 mg/L. Also, the contact time is a key factor for under- standing the equilibrium sorption rate by the adsorbent. The time-dependent sorption provides the sorption rate in which contaminants can be adsorbed on the adsorbent surface.50 Figure 8 presents the impact of contact time on the P ion sorption efficiency. As shown, the contact time has an impressive impact on the sorption process, so that with increasing time from 5 to 60 min, the P ion sorption efficiency increases from 46.26% to 97.43%, respectively. With increasing contact time, P ions in the solution have a greater chance of being located on MnFe2O4 sorption sites. However, the removal efficiency decreases at higher con- tact times, which may be due to the saturation of MnFe2O4 sorption sites.51 It can be assumed that the sorption pro- cess of P ions using MnFe2O4 mainly follows intraparti- cle diffusion and sorption complex mechanisms. Previous researchers have also found the same trend for sorption of other ions.52 Therefore, 60 min was considered as the optimal contact time. Figure 8. Impact of contact time in various concentrations of P ion on the removal efficiency (Conditions: pH = 6, mixing rate = 500 rpm, adsorbent dosage = 2 g/L, and temperature = 55 oC) Adsorbent dosage is another critical factor in the P ion sorption efficiency because it directly affects the eco- nomics of the process. The removal efficiency and sorption capacity of P ions using MnFe2O4 nanoparticles in various concentrations of MnFe2O4 (0.5–4 g/L) are illustrated in Figure 9. It is observed that the P ion removal efficiency increases with increasing MnFe2O4 concentration from 0.5 to 2.5 g/L, which is due to an increase in sorption sites. At adsorbent dosage> 2.5 g/L, no significant change in re- moval efficiency was observed because almost all P ions 688 Acta Chim. Slov. 2022, 69, 681–693 Brontowiyono et al.: Phosphate Ion Removal from Synthetic and Real Wastewater ... are adsorbed by the adsorbent and the MnFe2O4 sorption sites are saturated. Also, the sorption capacity of P ions de- creases with increasing MnFe2O4 concentration. P ions in solution aggregate at high adsorbent dosages, which leads to saturation of the adsorbent surface and thus reduces the sorption capacity.53–55 According to the results, the utmost sorption capacity of P ions using MnFe2O4 nanoparticles was attained as 9.172 mg/g. Also, the utmost sorption ef- ficiency (98.52%) was obtained at the adsorbent dosage of 2.5 g/L. Figure 9. Impact of MnFe2O4 dose on sorption efficiency and sorp- tion capacity of P ions (Conditions: pH = 6, mixing rate = 500 rpm, P ion concentration = 10 ppm, contact time = 60 min, and temper- ature = 55 °C) 3. 3. Sorption Isotherms The Langmuir, D-R, and Freundlich models were used to study the sorption isotherms of P ions using Mn- Fe2O4 nanoparticles (Figure 10 and Table 2). To this end, several experiments were performed in various P ion concentrations (10–100 ppm). According to the results, the correlation coefficient (R2) for the Freundlich model (0.978) was higher than the Langmuir (0.973) and D-R (0.814) models, indicating that the Freundlich isotherm model can better describe the P ion sorption process. Also, sorption of P ions occurs in multilayers on the heteroge- neous surfaces of MnFe2O4 nanoparticles. Moreover, the R2 value for the D-R model was small, indicating that the D-R model is not fitted well with the experimental data. The highest sorption capacity of P ions by the Langmuir model was 39.84 mg/g, which is an acceptable amount. The Langmuir separation factor RL was also between 0 and 1, indicating that the P ions sorption process is favora- ble. Besides, the value of n in the Freundlich model was greater than 1, showing that the P ions sorption process using MnFe2O4 nanoparticles is physical. Using the D-R model, the mean free energy (E) was obtained as 2.331 KJ/mol, which is less than 8 KJ/mol, and shows that the P ion sorption using MnFe2O4 nanoparticles is physical. The maximum sorption capacity by the D-R model was 23.805 mg/g, which is less than the value obtained by the Langmuir model. Also, the Langmuir (KL) and Freundlich (Kf) model constants were 0.326 L/g and 10.142 mg/g.(L/ mg)1/n, respectively. The maximum sorption capacity of P ions using Mn- Fe2O4 nanoparticles was compared with previous works, as reported in Table 3. As reported, Silica/2-methyl-1-naph- thylamine composite with the maximum sorption capac- ity of 159.12 mg/g56 and bentonite/magnesium hydroxide with the maximum sorption capacity of 4.3 mg/g57 showed the highest and lowest sorption capacities. Also, the adsor- bent used in this work (MnFe2O4 nanoparticles) with an utmost sorption capacity of 39.84 mg/g showed a suitable sorption capacity compared to other adsorbents. Figure 10. Sorption isotherms of P ions using the Langmuir (a), Freundlich (b) and D-R (c) models (Conditions: pH = 6, MnFe2O4 dose = 2 g/L, temperature = 55 °C, time = 60 min, mixing rate = 500 rpm) 689Acta Chim. Slov. 2022, 69, 681–693 Brontowiyono et al.: Phosphate Ion Removal from Synthetic and Real Wastewater ... Table 3. Comparing the maximum sorption capacity of P ions using various adsorbents Refe- qmax Adsorbent rence (mg/g) 11 52.1 cross-linked chitosan 12 13 acicular goethite nanoparticles 13 16.86 aluminum hydroxide/ palygorskite nano- composite 56 159.12 Silica/2-methyl-1-naphthylamine composite 57 4.3 Carboxymethyl cellulose/Fe 58 57.8 Magnetite 58 66.6 Ferrihydrite 58 50.5 Goethite 59 6.722 Chitosan 60 8.21 iron oxide 61 36 Fe-Mn binary oxide Present study 39.84 MnFe2O4 3. 4. Sorption Kinetics Kinetic models determine the sorption mecha- nisms. They also determine whether the sorption process follows the PFO or PSO kinetic models. The PFO and PSO models were used to study the kinetic behavior of the P ions sorption using MnFe2O4 nanoparticles. To this end, several experiments were performed at various P ion concentrations from 10 ppm to 100 ppm and different contact times from 5 min to 130 min. The results of sorp- tion kinetics are provided in Figure 11 and Table 4. As re- ported, the amount of qe,cal in different concentrations of P ions (10, 20, 30, 50, 70, and 100 ppm) for the PFO model were calculated as 3.063, 8.864, 14.042, 20.753, 27.01, and 36.205 mg/g, respectively, while these values for the PSO model were 5.23, 10.548, 16.025, 26.455, 33.67, and 47.46 Table 4. Sorption kinetics of P ions using MnFe2O4 nanoparticles Kinetic Parameter P ion concentration model 10 ppm 20 ppm 30 ppm 50 ppm 70 ppm 100 ppm PFO R2 0.9441 0.9887 0.9878 0.985 0.9779 0.9638 K1(min–1) 0.0605 0.0654 0.0567 0.0439 0.0419 0.037 qe.cal (mg/g) 3.063 8.864 14.042 20.753 27.01 36.205 qe.exp (mg/g) 4.906 9.726 14.427 23.38 29.792 40.28 PSO R2 0.9985 0.9978 0.9978 0.9979 0.9977 0.992 K2 (g/mg.min) 0.029 0.011 0.005 0.002 0.002 0.0009 qe.cal (mg/g) 5.23 10.548 16.025 26.455 33.67 47.46 qe.exp (mg/g) 4.906 9.726 14.427 23.38 29.792 40.28 Table 2. Parameters of P ions sorption isotherms using MnFe2O4 nanoparticles Model Factor Value Langmuir qm(mg/g) 39.84 KL (L/mg) 0.326 R2 0.973 RL 0.029–0.234 Freundlich n 2.362 Kf (mg/g (L/mg)1/n) 10.142 R2 0.978 D-R E (KJ/mol) 2.331 qm (mg/g) 23.805 β × 10+6 (mol2/J2) 0.092 R2 0.814 Figure 11. Sorption kinetics of P ions using MnFe2O4 nanoparticles in different concentrations of P ion (10–100 ppm) and different contact times (5–130 min), including the PFO model (a) and PSO model (b) (Other conditions: adsorbent dosage = 2 g/L, tempera- ture = 55 °C, mixing rate = 500 rpm and pH = 6) 690 Acta Chim. Slov. 2022, 69, 681–693 Brontowiyono et al.: Phosphate Ion Removal from Synthetic and Real Wastewater ... mg/g, respectively, which indicates that the amounts of qe,cal for the PSO model are larger than that of the PFO model at all P ion concentrations. Also, the PFO kinet- ic constant (K1) in these concentrations were obtained as 0.0605, 0.0654, 0.0567, 0.0439, 0.0419, and 0.037 min–1, respectively. The kinetic study shows that the PSO model has more ability to describe the kinetic behavior of P ion sorption due to higher R2 values (R2>0.99) in different concentrations of P ions compared to the PFO model with R2 between 0.94–0.98. Moreover, the kinetic constant of the PSO model (K2) is smaller than K1 in different con- centrations of P ion.19 3. 5. Thermodynamic Study of P Ion Sorption The thermodynamic parameters are calculated through the plot of LnKd against 1/T, as shown in Figure 12. The thermodynamic constants are also reported in Table 5. As given, negative values of ∆G° in various tem- peratures (–2.954 kJ/mol at 25 °C and –7.205 kJ/mol at 55 °C) show that the P ion sorption process is spontaneous. Also, the ∆G° values are between 0 to –20 kJ/mol, indicat- ing that the P ion sorption process using MnFe2O4 nano- particles is physical. Moreover, ∆H° was a positive value (38.024 kJ/mol), indicating that the P ion sorption process is endothermic, which confirms the results of the impact of temperature on the sorption process. Furthermore, ∆S° was a positive value (136.848 J/mol K), showing that irreg- ularities between the solid (adsorbent) and liquid (solu- tion) phases increase during the P ion sorption process using MnFe2O4 nanoparticles.62 Figure 12. The thermodynamic behavior of P ion sorption using MnFe2O4 nanoparticles (Conditions: mixing rate = 500 rpm, pH = 6, MnFe2O4 dose = 2 g/L, P ion concentration = 10 mg/L, and con- tact time = 60 min) 3. 6. Reusability of MnFe2O4 The reusability of the adsorbent in different cycles is very important for its industrial applications due to the cost-effectiveness of the process.63,64 After examining the sorption efficiency of MnFe2O4 nanoparticles in the re- moval of P ions from an aqueous solution, the reusabil- ity of MnFe2O4 nanoparticles was studied in eight reuse cycles to assess its industrial utilization potential (Figure 13). The solution containing H2SO4 was used to study the reusability of MnFe2O4. Figure 13 (a) shows the impact of H2SO4 concentration on the P ion sorption using Mn- Fe2O4 nanoparticles. According to the results, the desorp- tion efficiency of P ions increases with increasing H2SO4 concentration. However, no significant change was seen in the P ion desorption efficiency at H2SO4 concentration above 4 mol/liter. Therefore, the H2SO4 concentration of 4 mol/liter was considered the optimum value to study the reusability of MnFe2O4 nanoparticles. According to Figure 13 (b), MnFe2O4 nanoparticles were able to remove P ions Table 5. Thermodynamic parameters for P ion sorption using Mn- Fe2O4 nanoparticles Temperature ΔG° ΔH° ΔS° (°C) (kJ/mol) (kJ/mol) (J/mol K) 25 –2.954 38.024 136.848 35 –4.053 45 –5.123 55 –7.205 Figure 13. Desorption efficiency (a) and reusability (b) of MnFe2O4 nanoparticles for removal of P ions from aqueous solution 691Acta Chim. Slov. 2022, 69, 681–693 Brontowiyono et al.: Phosphate Ion Removal from Synthetic and Real Wastewater ... from an aqueous solution with a sorption efficiency above 91% after six cycles. However, the sorption efficiency of P ions in the 7th and 8th cycles were 85.6 and 78.8%, respec- tively, which are not suitable sorption efficiencies. There- fore, MnFe2O4 nanoparticles can be used for up to 6 reuse cycles, which is significant reusability. 3. 7. Treatment of Wastewater Using MnFe2O4 Nanoparticles MnFe2O4 nanoparticles were used to treat urban wastewater and the physical properties of the wastewa- ter before and after treatment are reported in Table 6. As shown, the initial values of COD, BOD5, pH, and phos- phate ions before sorption were 310 ppm, 185 ppm, 9.5, and 22 ppm, respectively. After adding MnFe2O4 nanopar- ticles to the wastewater, the values of COD, BOD5, pH, and phosphate ions were changed to 75 ppm, 42 ppm, 9, and 3.8 ppm, respectively. The results show that the concentration of phosphate ions has been reduced by 82.7%, which is a proper amount. Also, the removal efficiency of COD and BOD5 using MnFe2O4 was 75.8% and 77.3%, respectively. Table 6. The concentration of contaminants in urban wastewater before and after adding MnFe2O4 nanoparticles Parameter Initial After Removal value treatment percentage (%) Phosphate (ppm) 22 3.8 82.7 COD (ppm) 310 75 75.8 BOD5 (ppm) 185 42 77.3 pH 9.5 9 – 4. Conclusion The presence of P at high concentrations in water has adverse impacts on water ecology and causes eutroph- ication. Therefore, the concentration of P in water must be reduced. In this study, the sorption capability of Mn- Fe2O4 nanoparticles was investigated in the removal of P ions from synthetic and real wastewater. The physical and structural properties of the aforementioned adsor- bent were studied by several analyses such as TEM, SEM, EDAX, Mapping, XRD, VSM, FTIR, BET, and TGA. Ac- cording to these analyses, MnFe2O4 nanoparticles have a highly porous structure with many active sites, which can be effective in the sorption process. The sorption study in- dicated that the highest sorption efficiency of P ions was obtained as 98.52%, which was achieved at pH of 6, mixing rate of 500 rpm, MnFe2O4 dosage of 2.5 g/L, P ion con- centration of 10 ppm, temperature of 55 oC and contact time of 60 min. Also, the maximum sorption capacity ob- tained by the Langmuir model was 39.84 mg/g, which is an acceptable amount compared to other adsorbents for P removal. Moreover, the isotherm and kinetic studies showed that the P ion sorption process using MnFe2O4 follows the Freundlich and PSO models. Therefore, het- erogeneous surfaces of the adsorbent are very important in the P ion sorption process. Furthermore, the D-R and Freundlich isotherm models show that the P ion sorption process using MnFe2O4 is physical. The thermodynamic factors like ∆G°, ∆S°, and ∆H° displayed that the sorption of P ions using MnFe2O4 nanoparticles is spontaneous and endothermic. Besides, MnFe2O4 nanoparticles can be re- used for up to 6 cycles with high sorption efficiency. Also, MnFe2O4 nanoparticles were able to remove COD, BOD5 and P ions from municipal wastewater with high removal efficiency (>75%). In general, MnFe2O4 nanoparticles are recommended for industrial wastewater treatment. Conflict of Interests Statement No conflict of interests is declared by the authors. 5. References 1. L. Dai, Z. Wang, T. Guo, L. Hu, Y. Chen, C. Chen, G. Yu, L. Q. Ma, J. Chen. Chemosphere 2022, 293, 133576. DOI:10.1016/j.chemosphere.2022.133576 2. W. Liu, J. Zheng, X. Ou, X. Liu, Y. Song, C. Tian, W. Rong, Z. Shi, Z. Dang, Z. Lin. Environ. Sci. Technol. 2018, 52, 13336– 13342. DOI:10.1021/acs.est.8b02213 3. Q. Guan, G. Zeng, J. Song, C. Liu, Z. Wang, S. Wu. J. Environ. Manage. 2021, 293, 112961. 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Jyo. Desalination 2011, 281, 111–117. DOI:10.1016/j.desal.2011.07.047 64. X. Tian, R. Yang, T. Chen, Y. Cao, H. Deng, M. Zhang, X. Jiang. J. Hazard. Mater. 2022, 426, 128121. DOI:10.1016/j.jhazmat.2021.128121 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Namen te raziskave je odstraniti fosfat (P) iz odpadne vode z uporabo nanodelcev MnFe2O4. Za določitev površinskih lastnosti MnFe2O4 so bile uporabljene analize BET, TGA/DTG, FTIR, SEM, TEM, VSM, XRD in EDX/Map. Specifična površina adsorbenta je bila 196,56 m2/g, analiza VSM pa je pokazala, da ima adsorbent feromagnetne lastnosti. Največja učinkovitost sorpcije P z uporabo MnFe2O4 (98,52 %) je bila dosežena pri pH 6, temperaturi 55 °C, koncentraciji P 10 mg/L, času 60 min in odmerku sorbenta 2,5 g/L, kar je pomembna vrednost. Poleg tega je termodinamična študija poka- zala, da je proces sorpcije P spontan in endotermičen. Največja sorpcijska kapaciteta P z uporabo MnFe2O4 je bila 39,48 mg/g. MnFe2O4 se lahko uporablja za do 6 ciklov ponovne uporabe z visoko sorpcijsko učinkovitostjo (>91 %). Poleg tega je MnFe2O4 odstranil fosfat, KPK in BPK5 iz komunalne odpadne vode s precejšnjo učinkovitostjo odstranjevanja, in sicer 82,7 %, 75,8 % in 77,3 %. 694 Acta Chim. Slov. 2022, 69, 694–699 Zhou et al.: Syntheses, Structures and Insulin-Like Activity ... DOI: 10.17344/acsi.2022.7605 Scientific paper Syntheses, Structures and Insulin-Like Activity of Two Oxidovanadium(V) Complexes with Similar Nicotinohydrazone Ligands Gao-Qi Zhou, Xiao-Yang Qiu*, Shu-Juan Liu, Chu-Yi Wang College of Science & Technology, Ningbo University, Ningbo 315315, P. R. China * Corresponding author: E-mail: xiaoyang_qiu@126.com Received: 06-05-2022 Abstract Two new oxidovanadium(V) complexes, [VOL1(HQ)] (1) and [VOL2(SAH)] (2), were prepared by the reaction of [VO(a- cac)2] (where acac = acetylacetonate) with N’-(3-ethoxy-2-hydroxybenzylidene)nicotinohydrazide (H2L1) and 8-hydrox- yquinoline (HHQ), and N’-(2-hydroxy-4-methoxybenzylidene)nicotinohydrazide (H2L2) and salicylhydroxamic acid (HSAH), respectively, in methanol. Crystal and molecular structures of the complexes were determined by elemental analysis, infrared spectroscopy and single crystal X-ray diffraction. The V atoms in both complexes are in octahedral coordination. Thermal stability of the complexes was studied. Both complexes can decrease the blood glucose level in alloxan-diabetic mice, but the blood glucose level in the treated normal mice was not altered. Keywords: Nicotinohydrazone ligand; oxovanadium complex; crystal structure; thermal property; insulin-like activity 1. Introduction It was reported that inorganic vanadium salts exhibited insulin-like activity at 40 years ago.1 Interestingly, a pharmacological advantage of the vanadium salts is that it can be orally administered with long-term insulin- like activity in vivo.1b,2 However, inorganic vanadium salts are considered as less active and more toxic when compared with vanadium complexes with various types of ligands.3 Thus, a number of vanadium complexes have been prepared to improve the stability and membrane permeability of the vanadyl cation or decrease the toxicity of the vanadate anion.4 Metal complexes with nicotinohydrazones have received particular attention in biological and medicinal chemistry.5 8-Hydroxyquinoline (HHQ) and salicylhydroxamic acid (HSAH) are widely known bidentate ligands in coordination chemistry.6 However, only two HQ coordinated oxovanadium complexes and one SAH coordinated oxidovanadium complex with hydrazone ligands have been reported so far.7 In the present paper, two new oxovanadium(V) complexes with hydrazone and HQ or SAH ligands, [VOL1(HQ)] (1) and [VOL2(SAH)] (2) (H2L1 = N’-(3-ethoxy-2- hydroxybenzylidene)nicotinohydrazide, H2L2 = N’-(2- hydroxy-4-methoxybenzylidene)nicotinohydrazide; Scheme 1), have been presented. Scheme 1. The hydrazone, HHQ and HSAH ligands. H2L1: X = OEt, Y = H; H2L2: X = H, Y = OMe. 2. Experimental 2. 1. Materials and Measurements Commercially available 3-ethoxysalicylaldehyde, 4-methoxysalicylaldehyde and nicotinohydrazide were purchased from Sigma-Aldrich and used without further purification. Other solvents and reagents were made in China and used as received. H2L1 and H2L2 were prepared according to the literature method.8 C, H and N elemental analyses were performed with a Perkin-Elmer elemental analyser. Infrared spectra were recorded on a Nicolet AVATAR 360 spectrometer as KBr pellets in the (4000–400) cm–1 region. Thermal stability analysis was performed on a Perkin-Elmer Pyris Diamond TG-DTA thermal analyses system. Molar conductivity data were determined with a DDS-11A conductometer. 695Acta Chim. Slov. 2022, 69, 694–699 Zhou et al.: Syntheses, Structures and Insulin-Like Activity ... 2. 2. Synthesis of [VOL1(HQ)] (1) A methanolic solution (10 mL) of [VO(acac)2] (0.1 mmol, 26.5 mg) was added to a methanolic solution (10 mL) of H2L1 (0.1 mmol, 28.5 mg) and HHQ (0.1 mmol, 14.5 mg) with stirring. The mixture was stirred for 30 min at room temperature to give a deep brown solution. The resulting solution was allowed to stand in air for a few days. Brown block-shaped crystals suitable for X-ray single crystal diffraction were formed at the bottom of the vessel. The isolated products were washed three times with cold ethanol, and dried in air. The yield was 55%. Anal. calc. for C24H19N4O5V: C, 58.31; H, 3.87; N, 11.33; found: C, 58.12; H, 3.78; N, 11.46%. 2. 3. Synthesis of [VOL2(SAH)] (2) This complex was prepared according to the same method as that described for 1, with H2L1 replaced by H2L2 (0.1 mmol, 27.1 mg), and HHQ replaced by HSAH (0.1 mmol, 15.3 mg). The yield was 63%. Anal. calc. for C21H17N4O7V: C, 51.65; H, 3.51; N, 11.47; found: C, 51.56; H, 3.62; N, 11.38%. 2. 4. X-Ray Crystallography Diffraction intensities for the complexes were collected at 298(2) K using a Bruker D8 VENTURE PHOTON diffractometer with MoKa radiation (l = 0.71073 Å). The collected data were reduced using the SAINT program,9 and multi-scan absorption corrections were performed using the SADABS program.10 The structures were solved by direct methods and refined against F2 by full-matrix least-squares methods using the SHELXTL.11 All of the non-hydrogen atoms were refined anisotropically. The amino hydrogen atom in complex 2 was located from a difference Fourier map and refined isotropically, with N–H distance restrained to 0.90(1) Å. The remaining hydrogen atoms were placed in idealized positions and constrained to ride on their parent atoms. The crystallographic data for the complexes are summarized in Table 1. 2. 5. Glucose-Lowering Assay Male Kunming mice, weighing about 25–30 g, were obtained from Experimental Animal Center, Shandong Lukang Pharmaceutical Co., Ltd of China, and maintained on a light/dark cycle. All animals were allowed free access to food and water. Temperature and relative humidity were maintained at 25 °C and 50%. Mice were acclimatized for a week prior to induction of diabetes. Diabetes was induced by a single intra-peritoneal injection of freshly prepared alloxan (200 mg kg–1 body weight) in 0.9% saline. The control mice were injected with an equal volume of vehicle. After a week, blood was collected from the tail vein and serum samples were analyzed for blood glucose. Animals showing fasting (12 h) blood glucose higher than 11.1 mmol L–1 were considered to be diabetic and used for the study. The experimental animals were randomly divided into 8 groups with 4 mice each according to the blood glucose. Group 1, normal control group: normal mice treated with 0.5% carboxymethyl cellulose (CMC). Groups 2 and 3, treated normal groups: normal mice treated with 20 mg V kg–1 complexes. Group 4, diabetic control group: alloxan diabetic mice treated with 0.5% CMC. Groups 5–8, treated diabetic groups: alloxan diabetic mice treated with the complexes at doses of 10 and 20 mg V kg–1 intragastric administration. The complexes were administered as suspensions in 0.5% CMC. The substances were administered intragastrically once a day at the volume of 10 mL kg–1 for 2 weeks. 3. Results and Discussion 3. 1. General Replacement of two acetylacetonate ligands of [VO(acac)2] by hydrazone and 8-hydroxyquinoline or salicylhydroxamate ligands in methanol resulted in the formation of two structurally similar complexes. The complexes are soluble in DMF, DMSO, methanol, ethanol, and acetonitrile. Molar conductance of complexes 1 and 2 at the concentration of 10–4 mol L–1 are 16 Ω–1 cm2 mol–1 Table 1. Crystallographic data and refinement parameters for the complexes Parameter Value 1 2 Chemical formula C24H19N4O5V C21H17N4O7V Formula weight 494.4 488.3 Crystal system Monoclinic Monoclinic Space group P21/n P21/n Unit cell parameters a / Å 9.0157(5) 11.0094(8) b / Å 11.4002(6) 17.7842(12) c / Å 21.6406(11) 12.6825(10) β / º 97.130(2) 101.446(2) V / Å3 2207.0(2) 2433.8(3) Z 4 4 Dcalc / g cm–3 1.488 1.333 T / K 298(2) 298(2) μ / mm–1 0.495 0.453 F(000) 1016 1000 Unique reflections 4095 4526 Observed reflections [I > 2σ(I)] 3380 3562 Parameters 308 303 Restraints 0 1 R1, wR2 [I >2σ(I)] 0.0377, 0.0943 0.0621, 0.2062 R1, wR2 (all data) 0.0503, 0.1021 0.0797, 0.2229 Goodness of fit on F2 1.041 1.105 696 Acta Chim. Slov. 2022, 69, 694–699 Zhou et al.: Syntheses, Structures and Insulin-Like Activity ... and 23 Ω–1 cm2 mol–1, respectively, indicating they are non-electrolytes.12 3. 2. Crystal 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. Selected bond lengths and angles are given in Table 2. The V atoms in the complexes are in octahedral coordination, with the three donor atoms of the nicotinohydrazone ligands and the hydroxy O atom of the HQ ligand (for 1) or SAH ligand (for 2) defining the equatorial plane, and with one oxo O atom and the pyridine N atom of the HQ ligand (for 1) or the carbonyl O atom of the SAH ligand (for 2) occupying the axial positions. The distances between atoms V(1) and O(5) in 1, and V(1) and O(7) in 2 are 1.58–1.59 Å, indicating they are typical V=O double bonds. The V(1)–N(4) bond in 1 and V(1)–O(5) bond in 2 are significantly longer than Table 2. Selected bond distances (Å) and angles (º) for the complexes. 1 V(1)–O(1) 1.8606(14) V(1)–O(3) 1.0619(15) V(1)–O(4) 1.8501(14) V(1)–O(5) 1.5862(16) V(1)–N(1) 2.0777(17) V(1)–N(4) 2.3416(18) O(5)–V(1)–O(4) 99.57(7) O(5)–V(1)–O(1) 99.77(8) O(4)–V(1)–O(1) 107.28(6) O(5)–V(1)–O(3) 99.01(8) O(4)–V(1)–O(3) 88.15(6) O(1)–V(1)–O(3) 153.21(7) O(5)–V(1)–N(1) 96.70(7) O(4)–V(1)–N(1) 158.03(7) O(1)–V(1)–N(1) 84.23(6) O(3)–V(1)–N(1) 74.74(6) O(5)–V(1)–N(4) 175.26(7) O(4)–V(1)–N(4) 75.87(6) O(1)–V(1)–N(4) 82.99(6) O(3)–V(1)–N(4) 79.70(6) N(1)–V(1)–N(4) 87.39(7) 2 V(1)–O(1) 1.855(3) V(1)–O(2) 1.947(3) V(1)–O(5) 2.218(3) V(1)–O(6) 1.885(3) V(1)–O(7) 1.585(3) V(1)–N(1) 2.084(3) O(7)–V(1)–O(1) 99.53(14) O(7)–V(1)–O(6) 92.80(13) O(1)–V(1)–O(6) 110.89(12) O(7)–V(1)–O(2) 102.68(14) O(1)–V(1)–O(2) 149.95(13) O(6)–V(1)–O(2) 88.08(11) O(7)–V(1)–N(1) 94.18(14) O(1)–V(1)–N(1) 84.21(12) O(6)–V(1)–N(1) 162.05(12) O(2)–V(1)–N(1) 74.26(11) O(7)–V(1)–O(5) 166.78(13) O(1)–V(1)–O(5) 81.35(11) O(6)–V(1)–O(5) 74.73(10) O(2)–V(1)–O(5) 81.60(10) N(1)–V(1)–O(5) 99.02(11) Figure 1. ORTEP plot of the molecular structure of complex 1. Dis- placement ellipsoids of non-hydrogen atoms are drawn at the 30% probability level. Figure 2. ORTEP plot of the molecular structure of complex 2. Dis- placement ellipsoids of non-hydrogen atoms are drawn at the 30% probability level. 697Acta Chim. Slov. 2022, 69, 694–699 Zhou et al.: Syntheses, Structures and Insulin-Like Activity ... the other coordinate bonds, yet, it is not uncommon for such complexes.13 The bond lengths in both complexes are comparable to each other, and also similar to those observed in the mononuclear oxidovanadium(V) complexes with octahedral coordination.13 The angular distortion in the octahedral environment around V comes from the five- and six-membered chelate rings taken by the nicotinohydrazone ligands. For the same reason, the trans angles significantly deviate from the ideal values of 180°. Distortion of the octahedral coordination can be observed from the coordinate bond angles, ranging from 74.74(6)º to 107.28(6)º for the perpendicular angles, and from 153.21(7)º to 175.26(7)º for the diagonal angles for 1, and from 74.26(11)º to 110.89(12)º for the perpendicular angles, and from 149.95(13)º to 162.05(12)º for the diagonal angles for 2. The displacement of the V atoms from the equatorial plane is 0.30 Å for 1 and 0.23 Å for 2. The dihedral angles between the benzene ring and the pyridine ring of the hydrazone ligands are 6.2(3)º in 1 and 6.3(5)º in 2. In the crystal structure of 2, the adjacent two complex molecules are linked by O–H···N hydrogen bonds [O(4)–H(4A)···N(3)i: O(4)–H(4A) = 0.82 Å, H(4A)···N(3) i = 1.92 Å, O(4)···N(3)i = 2.736(4) Å, O(4)–(H4A)···N(3) i = 172°; symmetry code: i) –½ + x, 1½ – y, ½ + z], to form a dimer. The dimers are further linked by N–H···O hydrogen bonds [N(4)–H(4)···O(6)ii: N(4)–H(4) = 0.90(1) Å, H(4)···O(6)ii = 2.14(4) Å, N(4)···O(6)ii = 2.838(4) Å; symmetry code: ii) 1 – x, 2 – y, 1 – z], to form one dimensional chain. 3. 3. IR Spectra Complexes 1 and 2 exhibit typical bands at 963 cm–1 and 975 cm–1, respectively, assigned to the V=O vibration.14 The bands due to νC=O were absent in the complexes, but new C–O stretches appeared at 1266 cm–1 for 1 and 1250 cm–1 for 2. This suggests occurrence of keto- imine tautomerization of the ligands during complexation. The intense νC=N absorptions are observed at 1602 cm–1 for 1 and 2.15 The weak peaks in the low wave numbers in the region (400–650) cm–1 may be attributed to V–O and V–N bonds in the complexes. 3. 4. Thermal Property Differential thermal (DT) and thermal gravimetric analyses (TGA) were conducted to examine the stability of the complexes (Figures 3 and 4). For 1, the complex decomposed from 170 °C to 490 °C, corresponding to the loss of the nicotinohydrazone and HQ ligands and the formation of V2O5. The total observed weight loss of 82.7% is close to the calculated value of 81.6%. For 2, the complex decomposed from 170 °C to 510 °C, corresponding to the loss of the nicotinohydrazone and SAH ligands and the formation of V2O5. The total observed weight loss of 82.3% is close to the calculated value of 81.4%. Figure 3. DT-TGA curves of complex 1. Figure 4. DT-TGA curves of complex 2. 3. 5. Insulin-Like Activity of the Complexes The results are listed in Table 3, which showed that both complexes had blood glucose-lowering effect at doses of 10.0 and 20.0 mg V kg–1. Both complexes can decrease the blood glucose level in alloxan-diabetic mice, whereas the blood glucose level in the treated normal mice (20.0 mg V kg–1 by intragastric administration for 2 weeks) was not altered as compared with the untreated normal mice. The alloxan-diabetic mice exhibited significant hyperglycemia. After 2-week administration with the complexes, the blood glucose level was decreased compared with the diabetic control group. The glucose-lowering effect of both complexes is similar to each other. VOSO4 was also assayed as comparison, and showed similar activities as compared to the complexes. 4. Conclusion Two new mononuclear oxidovanadium(V) com- plexes derived from mixed ligands N’-(3-ethoxy-2- 698 Acta Chim. Slov. 2022, 69, 694–699 Zhou et al.: Syntheses, Structures and Insulin-Like Activity ... hydroxybenzylidene)nicotinohydrazide and 8-hydroxy- quinoline, and N’-(2-hydroxy-4-methoxy benzylidene) nicotinohydrazide and salicylhydroxamic acid, respectively, were prepared and structurally characterized. The V atoms are in octahedral coordination. Thermal stability of the complexes was studied. The bioassay indicated that both complexes have effective insulin-like activity on alloxan-diabetic mice, which deserve further study. Supplementary Data CCDC 979511 (1) and 979512 (2) contain the supplementary crystallographic data for the complexes. 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.un. Acknowledgments This work was financially supported by Ningbo Pub- lic Welfare Funds (Project Nos. 202002N3056 and 2021S142). 5. References 1. (a) Y. Shechter, S. J. D. Karlish. Nature 1980, 284, 556–558; DOI:10.1038/284556a0 (b) C. E. Heyliger, A. G. Tahiliani, J. H. McNeill. Science 1985, 227, 1474–1477. DOI:10.1126/science.3156405 2. J. Meyerovitch, Z. Farfel, J. Sack, Y. Shechter. J. Biol. Chem. 1987, 262, 6658–6662. DOI:10.1016/S0021-9258(18)48292-0 3. (a) M. Haratake, M. Fukunaga, M. Ono, M. 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Slov. 2016, 63, 670–677; (b) L.-W. Xue, Y.-J. Han, X.-Q. Luo. Acta Chim. Slov. 2019, 66, 622–628; DOI:10.17344/acsi.2019.5039 (c) Y. Lei. Acta Chim. Slov. 2022, 69, 235–242. DOI:10.17344/acsi.2022.7296 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Z reakcijo [VO(acac)2] (acac = acetilacetonat) z N’-(3-etoksi-2-hidroksibenziliden)nikotinohidrazidom (H2L1) in 8-hi- droksikinolinom (HHQ) ter z N’-(2-hidroksi-4-metoksibenziliden)nikotinohidrazidom (H2L2) in salicilhidroksamsko kislino (HSAH) v metanolu smo pripravili dva nova oksidovanadijeva(V) kompleksa [VOL1(HQ)] (1) in [VOL2(SAH)] (2). Kristalno in molekulsko strukturo kompleksov smo določili z elementno analizo, infrardečo spektroskopijo in mo- nokristalno rentgensko difrakcijo. Atomi vanadija v obeh kompleksih so v oktaedrični koordinaciji. Proučevali smo termično stabilnost kompleksov. Oba kompleksa lahko zmanjšata raven glukoze v krvi pri aloksan-diabetičnih miših, vendar se raven glukoze v krvi pri zdravljenih normalnih miših ni spremenila. 700 Acta Chim. Slov. 2022, 69, 700–713 Abdo and Mohareb: Antiproliferative and Antiprostate Cancer Activities ... DOI: 10.17344/acsi.2021.6886 Scientific paper Antiproliferative and Antiprostate Cancer Activities of Heterocyclic Compounds Derived from Cyclohexane-1,4-dione Nadia Y. Megally Abdo1 and Rafat Milad Mohareb*,2 1 Department of Chemistry, Faculty of Science, Cairo University, Giza, Egypt 2 Chemistry Department, Faculty of Education, Alexandria University, 21526 Alexandria, Egypt * Corresponding author: E-mail: raafat-mohareb@cu.edu.eg Received: 04-13-2021 Abstract 2-Amino-6-oxo-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (3) was prepared from the reaction of cyclohex- ane-1,4-dione with elemental sulfur and malononitrile in 1,4-dioxane and triethylamine as catalyst. The latter compound reacted with triethyl orthoformate and either malononitrile or ethyl cyanoacetate in 1,4-dioxane in the presence of tri- ethylamine to produce 4H-thieno[2,3-f]chromene derivatives 10a,b. In addition, fused pyran and pyridine derivatives were synthesized starting from compound 3. The cytotoxicities of the synthesized compounds were studied using the six cancer cell lines together with c-Met kinase and PC-3 cell line. The most active compounds were tested against five tyrosine kinases and Pim-1 kinase, most of which showed strong inhibition, encouraging further work. Keywords: Cyclohexan-1,4-dione; thiophene; thiazole; cytotoxicity; tyrosine inhihibitions 1. Introduction Sulfur-containing heterocyclic compounds have attracted much attention in recent years because of their great medicinal and pharmaceutical importance.1,2 Benzo[b]thiophene derivatives are one type of such sul- fur-containing heterocyclic compounds and are good candidates for anticancer applications.3–7 In addition, benzo[b]thiophene derivatives exhibit numerous oth- er pharmacological effects, including antitumor agents,8 anti-inflammatory agents,9,10 antimicrobial agents,11,12 anti-leishmanial agents,13,14 antioxidants,15 anti-anxi- ety agents, serotonin antagonists, and antiarrhythmic agents.16 In addition, the combination of benzo[b]thio- phene with other heterocyclic rings such as thiazole, thi- ophene, pyran, or pyridine rings increases the biological significance of such compound series.17–20 Recently, our research group focused on benzo[b]thiophene derivatives by performing further heterocyclization reactions and then investigating their anticancer activities; in particu- lar, some compounds showed inhibition of kinase and Pim-1.21–25 In extension of this work, in this manuscript we show the synthesis of 2-amino-6-hydroxy-4,7-dihy- drobenzo[b]thiophene-3-carbonitrile (3) starting from cyclohexane-1,4-dione, followed by further heterocycli- zation to prepare compounds whose antiproliferative ac- tivities and kinase inhibitions were investigated. 2. Experimental 2. 1. Generral 13C NMR and 1H NMR spectra were recorded us- ing a Bruker DPX300 instrument in DMSO with TMS as the internal standard for protons and solvent signals as the internal standard for carbon spectra. Chemical shift val- ues are given in δ (ppm). Mass spectra were checked us- ing EIMS (Shimadzu) and ESI-esquire 3000 from Bruker Daltonics. Elemental analyzes were performed using the Microanalytical Data Unit at Cairo University. All reac- tions were monitored by TLC on 2 × 5 cm, 0.25 mm thick, precoated silica gel 60 F254 plates (Merck). 2. 1. 1. Synthesisof 2-amino-6-oxo-4,5,6,7- tetrahydrobenzo[b]thiophene-3- carbonitrile (3) To a solution of cyclohexane-1,4-dione (1) (1.2 g, 0.01 mol) in 1,4-dioxane (30 mL) with triethylamine (0.50 mL) was added malononitrile (0.66 g, 0.01 mol) and ele- 701Acta Chim. Slov. 2022, 69, 700–713 Abdo and Mohareb: Antiproliferative and Antiprostate Cancer Activities ... mental sulfur (0.32 g, 0.01 mol). The reaction mixture was heated at reflux for 1 h, and the product was filtered and dried. Light brown crystals from 1,4-dioxane, yield: 75%; m.p.: 160–163 °C; IR (KBr) υmax (cm–1): 3422–3236 (OH, NH2), 2966 (CH aliphatic), 2196 (CN), 1706 (CO), 1624 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 2.65 (d,2H, J = 6.7 Hz, CH2-CH=C), 3.39 (s, 2H, D2O exchange- able, NH2), 5.54 (s, 2H, CH2), 6.82 (t, 1H, J = 6.7 Hz, CH2-CH=C), 9.97 (s, 1H, OH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) δ 22.6 (CH2-CH=C), 50.3 (CH2), 66.3 (CH2-CH=C), 116.2 (CN), 118.4, 121.7, 128.9, 134.0 (thiophene C), 161.8 (CO); EIMS (m/z, %): 192 [M+, 20]. Anal. Calcd. for C9H8N2OS: C, 56.23; H, 4.19; N, 14.57; S, 16.68. Found: C, 55.94; H, 4.08; N, 14.39; S, 16.30. 2. 1. 2. Synthesis of 2-amino-7-benzylidene-6- hydroxy-4,7-dihydrobenzo[b]thiophene-3- carbonitrile (5) Benzaldehyde (4) (1.06 g, 0.01 mol) was added to a solution of compound 3 (1.92 g, 0.01 mol) in 1,4-dioxane (30 mL) containing piperidine (0.50 mL) and heated for 1 h at reflux. The reaction mixture was cooled and poured into cold water containing a few drops of hydrochloric acid. The precipitated solid was filtered off, washed and dried. Red crystals from 1,4-dioxane, yield: 76%; m.p.: 180–182 °C; IR (KBr) υ max (cm–1): 3428–3231 (OH,NH2), 2923 (CH aliphatic), 2201 (CN), 1625 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 2.89 (d, 2H, J = 4.6 Hz, CH2- CH=C), 3.44 (s, 2H, D2O exchangeable, NH2), 7.21 (t, 1H, J = 4.6 Hz, CH2-CH=C), 7.49–7.93 (m, 6H, C6H5 and C=CH−C6H5), 10.01 (s, 1H, OH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) δ 22.2 (CH2-CH=C), 66.3 (CH2-CH=C), 77.2, 114.5 (C=C), 116.2 (CN), 119.8, 120.4, 126.2, 128.4, 129.5, 131.2, 133.4, 134.5, 154.5 (C6H5, thio- phene C); EIMS (m/z, %): 280 [M+, 32]. Anal. Calcd. for C16H12N2OS: C, 68.55; H, 4.31; N, 9.99; S, 11.44. Found: C, 68.60; H, 4.29; N, 10.29; S, 11.09. 2. 1. 3. Synthesis of 2-amino-6-hydroxy-7-(2- hydroxybenzylidene)-4,7-dihydrobenzo-[b] thiophene-3-carbonitrile (7) A solution of compound 3 (1.92 g, 0.01 mol) in 1,4-dioxane (30 mL) containing piperidine (0.50 mL) was refluxed with salycilalhyde (6) (1.22 g, 0.01 mol) for 1 h, the precipitated solid was filtered and dried after addition of cold water containing a few drops of hydrochloric acid. Reddish brown crystals from 1,4-dioxane, yield: 77%; m.p.: 190–192 °C; IR (KBr) υ max (cm–1): 3423– 3231 (OH-NH2), 2925 (CH aliphatic), 2210 (CN), 1605 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 2.99 (d, 2H, CH2-CH=C), 3.32 (s, 2H, D2O exchangeable, NH2), 6.95 (m, 2H, 2 CH=C), 7.40–7.78 (m, 4H, C6H4), 10.25, 11.26 (2s, 2H, D2O exchangeable, 2OH); 13C NMR (75 MHz, DMSO-d6) δ 20.8 (CH2-CH=C), 66.9 (CH2-CH=C), 77.9, 118.9 (C=C), 116.5 (CN), 120.2, 125.9, 127.9, 128.8, 129.6, 131.1, 133.6, 134.2, 146.7 (C6H4, thiophene C); EIMS (m/z, %): 296 [M+, 51]. Anal. Calcd. for C16H12N2O2S: C, 64.85; H, 4.08; N, 9.45; S, 10.82. Found: C, 64.60; H, 4.29; N, 9.79; S, 10.58. 2. 1. 4. Synthesis of 4H-thieno[2,3-f]chromene derivatives 10a,b Triethyl orthoformate (8) (1.48 mL, 0.01 mol) and either molononitrile (2) (0.66 g, 0.01 mol) or ethyl cyano- acetate (9) (1.13 mL, 0.01 mol) were added to a solution of compound 3 (1.92 g, 0.01 mol) in 1,4-dioxane (30 mL) with triethylamine (0.50 mL). The reaction mixture was heated at reflux for 2 h, cooled, and neutralized with cold water containing a few drops of hydrochloric acid; the pre- cipitated product was filtered off and dried. 2,7-Diamino-4H-thieno[2,3-f]chromene-3,8-dicarbo- nitrile (10a) Light brown crystals from 1,4-dioxane, yield: 47%; m.p.: >300 °C; IR (KBr) υmax (cm–1): 3424–3228 (2NH2), 2924 (CH aliphatic), 2215, 2201 (2CN), 1626 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 3.39 (s, 2H, D2O exchange- able, NH2), 7.09–7.54 (m, 4H, pyran H-4 and Ar-H),7.91 (s, 2H, D2O exchangeable, NH2); 13C NMR (75 MHz, DMSO-d6) δ 77.1 (pyran C-4), 116.5, 117.3 (2CN), 114.7, 118.9, 120.2, 125.9, 127.9, 128.8, 131.1, 133.6, 134.2, 136.7 (Ar-C, pyran, thiophene); EIMS (m/z, %): 268 [M+, 44]. Anal. Calcd. For C13H8N4OS: C, 58.20; H, 3.01; N, 20.88; S, 11.95. Found: C, 58.50; H, 3.39; N, 20.62; S, 11.69. Ethyl 2,7-diamino-3-cyano-4H-thieno[2,3-f]chromene- 8-carboxylate (10b) Pale brown crystals from 1,4-dioxane, yield: 62%; m.p.: >300 °C; IR (KBr) υmax (cm–1): 3423–3211 (2NH2), 2923 (CH aliphatic), 2201 (CN), 1706 (CO), 1621 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 1.16 (t, 3H, J = 7.21 Hz, OCH2CH3), 3.41 (s, 2H, D2O exchangeable, NH2), 4.20 (q, 2H, J = 7.21 Hz, OCH2CH3), 7.26–7.61 (m, 4H, pyran H-4 and Ar-H), 7.83 (s, 2H, D2O exchangeable, NH2); 13C NMR (75 MHz, DMSO-d6) δ8.5 (OCH2CH3), 45.5 (OCH2CH3), 77.4 (pyran C-4), 116.2 (CN), 115.6, 118.3,120.8, 121.9, 122.3, 128.1, 130.3, 132.0, 133.4, 147.4 (Ar-C, pyran, thi- ophene), 162.6 (CO); EIMS (m/z, %): 315 [M+, 56]. Anal. Calcd. for C15H13N3O3S: C, 57.13; H, 4.16; N, 13.33; S, 10.17. Found: C, 57.40; H, 4.39; N, 13.62; S, 10.49. 2. 1. 5. Synthesis of N’-(2-amino-3-cyano- 4,7-dihydrobenzo[b]thiophen-6-yl)-2- cyanoacetohydrazide (12) To a solution of compound 3 (1.92 g, 0.01 mol) in 1,4-dioxane (30 mL) was added cyanoacetylhydrazine (11) (0.99 g, 0.01 mol) and the reaction mixture was heated un- 702 Acta Chim. Slov. 2022, 69, 700–713 Abdo and Mohareb: Antiproliferative and Antiprostate Cancer Activities ... der refulx for 3 h and the resulting precipitate was collect- ed by filtration after cooling. Pale brown crystals from 1,4-dioxane, yield: 41%; m.p.: >300 °C; IR (KBr) υmax (cm–1): 3418–3205 (NH2, 2NH), 2923 (CH aliphatic), 2210, 2197 (2CN), 1698 (CO), 1621 (C=C); 1H NMR (300 MHz,DMSO-d6) δ 2.71 (d, 2H, J = 6.8 Hz, CH2-CH=), 3.37 (s, 2H, D2O exchangeable, NH2), 3.76 (s, 2H, CO-CH2-CN), 5.54 (s, 2H, CH2), 6.83 (t, 1H, J = 6.8 Hz, CH2-CH=), 8.13, 9.93 (2s, 2H, D2O ex- changeable, 2NH); 13C NMR (75 MHz, DMSO-d6) δ 35.8 (CH2), 66.3 (CH2), 77.4, 118.5 (C=C), 98.9 (CO-CH2-CN), 115.7, 116.2 (2CN), 129.9, 133.3, 136.6, 154.7 (thiophene C), 162.6 (CO); δ EIMS (m/z, %): 273 [M+, 24]. Anal. Cal- cd. for C12H11N5OS: C, 52.73; H, 4.06; N, 25.62; S, 11.73. Found: C, 52.50; H, 4.39; N, 25.82; S, 11.69. 2. 1. 6. Synthesis of ethyl 2,7-diamino- 3,8-dicyano-9-hydroxy-4,5- dihydronaphtho[1,2-b]thiophene-6- carboxylate (13) A solution of compound 3 (1.92 g, 0.01 mol) (30 mL) and ethyl cyanoacetate (9) (1.13 mL, 0.01 mol) in 1,4-diox- ane was heated at reflux with triethylamine (0.50 mL) for 3 hours. The solid formed was filtered off and dried after neutralizing the reaction mixture with cold water contain- ing a few drops of hydrochloric acid. Pale brown crystals from 1,4-dioxane, yield: 46%; m.p.: >300 °C; IR (KBr) υmax (cm–1): 3521–3209 (OH, 2NH2), 2928 (CH aliphatic), 2208, 2199 (2CN), 1704 (CO), 1624 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 1.20 (t, 3H, J = 6.90 Hz, OCH2CH3), 2.65 (m, 4H, CH2-CH2), 3.36 (s, 2H, D2O exchangeable, NH2), 4.19 (q, 2H, J = 6.90 Hz, OCH2CH3), 7.84 (s, 2H, D2O exchangeable, NH2), 9.91 (s, 1H, D2O exchangeable, OH); 13C NMR (75 MHz, DMSO-d6) δ 18.5 (OCH2CH3), 46.9 (OCH2CH3), 56.0, 67.3 (CH2-CH2), 115.5, 116.2 (2CN), 118.1, 119.7, 120.7, 122.5, 128.4, 129.3, 132.2, 133.3, 152.4 (Ph, thiophene C), 162.6 (CO); EIMS (m/z, %): 354 [M+, 52]. Anal. Calcd. for C17H14N4O3S: C, 57.62; H, 3.98; N, 15.81; S, 9.05. Found: C, 57.50; H, 3.87; N, 15.53; S, 8.84. 2. 1. 7. Synthesis of 4,7-dihydrobenzo[b]thiophene derivatives 15a,b A cold solution (0–5 °C) of compound 3 (1.92 g, 0.01 mol) in ethanol (30 mL) containing sodium acetate (2.5 g) was added to a cold solution of either benzenediazonium chloride (14a) (0.01 mol) or 4-methylbenzenediazonium chloride (14b) (0.01 mol) [prepared by adding sodium ni- trite solution (0.7 g, 0.01 mol in 10 mL water) to a cold solution of either aniline oil (0.93 g, 0.01 mol) or 4-meth- ylaniline (1.07 g, 0.01 mol) in concentrated hydrochloric acid (8 mL, 18%) with constant stirring]. The whole mix- ture was kept at room temperature for 1 hour and the re- sulting product was collected by filtration. 2-Amino-6-hydroxy-7-(2-phenylhydrazono)-4,7-dihyd- robenzo[b]thiophene-3-carbonitrile (15a) Black crystals from ethanol, yield: 81%; m.p.: >300 °C; IR (KBr) υmax (cm–1): 3518–3214 (OH, NH2, NH), 2924 (CH aliphatic), 2199 (CN), 1625 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 2.79 (d, 2H, CH2), 3.44 (s, 2H, D2O exchangeable, NH2), 7.17–7.63 (m, 6H, C6H5 and CH=C), 7.94 (s, 1H, D2O exchangeable, NH), 9.01(s, 1H, D2O exchangeable, OH); 13C NMR (75 MHz, DMSO-d6) δ 20.8(CH2), 67.2, 115.2 (CH=C), 116.4 (CN), 119.4, 121.7, 126.1, 128.4, 128.9, 132.1, 133.4, 137.1 (C6H5 and thiophene), 182.8 (C=N); EIMS (m/z, %): 296 [M+, 61]. Anal. Calcd. for C15H12N4OS: C, 60.79; H, 4.08; N, 18.91; S, 10.82. Found: C, 60.49; H, 3.87; N, 18.53; S, 10.54. 2-Amino-6-hydroxy-7-(2-(p-tolyl)hydrazono)-4,7-di- hydrobenzo[b]thiophene-3-carbonitrile (15b) Dark brown crystals from ethanol, yield: 84%; m.p.: >300 °C; IR (KBr, υmax cm–1): 3524–3226 (OH, NH2, NH), 2922 (CH aliphatic), 2200 (CN), 1626 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 2.27 (s, 3H, CH3), δ3.05 (d, 2H, CH2), 3.40 (s, 2H, D2O exchangeable, NH2), 7.17–7.59 (m, 5H, C6H4 and CH=C), 7.92 (s, 1H, D2O exchangeable, NH), 9.21 (s, 1H, D2O exchangeable, OH); 13CNMR (75 MHz, DMSO-d6) δ 16.5 (CH3), 20.8 (CH2), 66.5, 114.6 (CH=C), 117.4 (CN), 119.4, 121.7, 125.5, 128.2, 130.8, 132.7, 133.5, 137.0 (C6H5 and thiophene), 184.1 (C=N); EIMS (m/z, %): 310 [M+, 57]. Anal. Calcd. for C16H14N4OS: C, 61.92; H, 4.55; N, 18.05; S, 10.33. Found: C, 62.20; H, 4.24; N, 18.37; S, 10.41. 2. 1. 8. Synthesis of dihydrobenzo[b]thiophene derivatives 19 and 20 A solution of compound 3 (1.92 g, 0.01 mol) in di- methylformamide (30 mL) and phenyl isothiocyanate (16) (1.35 mL, 0.01 mol) was cooled overnight in the presence of potassium hydroxide (0.5 g). To the reaction mixture either α-chloroacetone (18a) (0.92 mL, 0.01 mol) or ethyl chloroacetate (18b) (1.22 mL, 0.01 mol) was added and allowed to stand overnight. The synthesized product was obtained by neutralizing the reaction mixture with a solu- tion of cold water and a few drops of hydrochloric acid, filtered and dried. 2-Amino-6-hydroxy-7-(4-methyl-3-phenylthiazol- 2(3H)-ylidene)-4,7-dihydrobenzo[b]thiophene-3-car- bonitrile (19) Dark brown crystals from ethanol, yield: 79%; m.p.: 182 °C; IR (KBr) υmax (cm–1): 3518–3220 (OH, NH2,), 2924 (CH aliphatic), 2188 (CN), 1629 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 2.56 (s, 3H, CH3), 2.72 (d, 2H, J = 4.5 Hz, CH2), 3.30 (s, 2H, D2O exchangeable, NH2), 7.06 (t, 1H, J = 4.5 Hz, CH), 7.09–7.61 (m, 6H, C6H5 and thiazole H-5), 10.07 (s, 1H, D2O exchangeable, OH); 13C NMR (75 MHz, DMSO-d6) δ 22.5 (CH3), 34.3 (CH2), 703Acta Chim. Slov. 2022, 69, 700–713 Abdo and Mohareb: Antiproliferative and Antiprostate Cancer Activities ... 74.2, 118.1 (CH=C), 116.1 (CN), 121.7, 123.6, 124.4, 125.7, 127.8, 128.4, 128.7, 129.2, 129.4, 137.5, 139.4, 153.2 (C6H5, thiazole, thiophene); EIMS (m/z, %): 365 [M+, 24]. Anal. Calcd. for C19H15N3OS2: C, 62.44; H, 4.14; N, 11.50; S, 17.55. Found: C, 62.59; H, 4.50; N, 11.22; S, 17.31. Ethyl 2-(((2-amino-3-cyano-6-oxo-5,6-dihydrobenzo [b]thiophen-7(4H)-ylidene)(phenyl-amino)methyl) thio)acetate (20) Dark brown crystals from ethanol, yield: 78%; m.p.: 150 °C; IR (KBr) υmax (cm–1): 3518–3220 (OH, NH2, NH), 2929 (CH aliphatic), 2127 (CN), 1722 (CO), 1635 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 1.19 (t, 3H, J = 7.1 Hz, OCH2CH3), 2.72 (d, 2H, CH2), 3.06 (s, 2H, CH2), 3.30 (s, 2H, D2O exchangeable, NH2), 4.15 (q, 2H, J = 7.1 Hz, OCH2CH3), 7.06–7.72 (m, 6H, C6H5 and CH), 8.96 (s, 1H, D2O exchangeable, NH), 10.07 (s, 1H, D2O exchangeable, OH); 13C NMR (75 MHz, DMSO-d6) δ 13.9 (OCH2CH3), 28.7 (CH2), 45.5 (OCH2CH3), 50.1 (CH2), 72.4, 118.7 (CH=C), 116.1 (CN), 121.2, 121.7, 123.6, 124.4, 125.7, 127.8, 128.7, 130.4, 137.5, 139.4 (C=C, C6H5, thiophene C), 163.2 (CO); EIMS (m/z, %): 413 [M+, 24]. Anal. Cal- cd. for C20H19N3O3S2: C, 58.09; H, 4.63; N, 10.16; S, 15.51. Found: C, 58.36; H, 4.50; N, 10.22; S, 15.31. 2. 1. 9. Synthesis of ethyl 2-amino-3-cyano-8- (phenylamino)-4,5-dihydrobenzo[1,2- b:5,6-c’]dithiophene-6-carboxylate (21) Compound 20 (4.13 g, 0.01 mol) was heated in a solution of 1,4-dioxane containing triethylammine (0.50 mL) for 2 h under reflux. The resulting solution was neu- tralized with an ice water solution containing a few drops of hydrochloric acid to give the synthesized solid, which was filtered and dried. Brown crystals from ethanol, yield: 78%; m.p.: 225 °C; IR (KBr) υmax (cm–1): 3418–3220 (NH2, NH), 2924 (CH aliphatic), 2199 (CN), 1722 (CO), 1633 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 1.19 (t, 3H, J = 7.2 Hz,OCH2CH3), 3.06 (m, 4H, CH2-CH2), 3.30 (s, 2H, D2O exchangeable, NH2), 4.15 (q, 2H, J = 7.2 Hz, OCH2CH3), 7.26–7.79 (m, 5H, C6H5), 8.96 (s, 1H, D2O exchangeable, NH); 13C NMR (75 MHz, DMSO-d6) δ 14.3 (OCH2CH3), 45.5 (OCH2CH3), 61.5, 62.9 (CH2-CH2), 116.1 (CN), 118.0, 121.7, 128.8, 129.2, 129.9, 130.5, 131.6, 132.4, 133.0, 134.7, 136.4, 140.0 (C6H5, thiophene) 161.5 (CO); EIMS (m/z, %): 395 [M+, 24]. Anal. Calcd. for C20H17N3O2S2: C, 60.74; H, 4.33; N, 10.62; S, 16.22. Found: C, 60.49; H, 4.21; N, 10.82; S, 15.93. 2. 1. 10. Synthesis of 5,9-dihydro-4H-thieno[2,3-f] chromene derivatives 23a-f A mixture of compound 3 (1.92 g, 0.01 mol), either malononitrile (2) (0.66 g, 0.01 mol) or ethyl cyanoacetate (9) (1.13, 0.01 mol) and either benzaldehyde (4) (1.06 g, 0.01 mol), 4-chlorobenzaldehyde (22a) (1.4 g, 0.01 mol) or 4-methoxybenzaldehyde (22b) (1.36 g, 0.01 mol) in 1,4-dioxane (40 mL) and triethylamine (0.5 mL) was heat- ed under reflux for 3 h and the precipitated product was kept under reflux. The precipitated product was recovered by adding cold water and a few drops of hydrochloric acid to the resulting mixture, filtered and dried. 2,7-Diamino-9-phenyl-5,9-dihydro-4H-thieno[2,3-f] chromene-3,8-dicarbonitrile (23a) Reddish brown crystals from ethanol, yield: 40%; m.p.: 230 °C; IR (KBr) υmax (cm–1): 3422–3210 (2NH2), 2924 (CH aliphatic), 2227, 2198 (2CN), 1625 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 3.09 (m, 4H, CH2-CH2), 3.34 (s, 2H, D2O exchangeable, NH2), 7.24–7.96 (m, 6H, pyran H-4 and C6H5), 8.54 (s, 2H, D2O exchange- able, NH2); 13C NMR (75 MHz, DMSO-d6) δ 62.1, 65.3 (CH2-CH2), 76.5 (pyran C-4), 115.6, 116.6 (2CN), 118.4, 119.3, 122.6, 123.6, 128.9, 129.5, 129.9, 130.1, 131.9, 132.5, 133.7, 154.4 (C6H5, pyran, thiophene C); EIMS (m/z, %): 346 [M+, 34]. Anal. Calcd. for C19H14N4OS: C, 65.88; H, 4.07; N, 16.17; S, 9.26. Found: C, 65.59; H, 3.88; N, 16.32; S, 9.09. 2,7-Diamino-9-(4-chlorophenyl)-5,9-dihydro-4H -thieno[2,3-f]chromene-3,8-dicarbonitrile (23b) Red crystals from ethanol, yield: 77%; m.p.: 160 °C; IR (KBr) υmax (cm–1): 3421–3206 (2NH2), 2959 (CH ali- phatic), 2225, 2195 (2CN), 1621 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 3.09 (m, 4H, CH2-CH2), 3.40 (s, 2H, D2O exchangeable, NH2), 7.26 (s, 1H, pyran H-4), 7.40– 7.97 (m, 4H, C6H4), 8.53 (s, 2H, D2O exchangeable, NH2); 13C NMR (75 MHz, DMSO-d6) δ 63.2, 65.3 (CH2-CH2), 76.8 (pyranC-4), 115.9, 116.2 (2CN), 118.4, 119.1, 120.3, 121.9, 123.9, 129.4, 129.8, 130.7, 131.4, 132.5, 133.1, 148.1 (C6H4, pyran, thiophene C); EIMS (m/z, %): 380 [M+, 45]. Anal. Calcd. for C19H13ClN4OS: C, 59.92; H, 3.44; N, 14.71; S, 8.42. Found: C, 59.95; H, 3.24; N, 14.56; S, 8.73. 2,7-Diamino-9-(4-methoxyphenyl)-5,9-dihydro-4H- thieno[2,3-f]chromene-3,8-dicarbonitrile (23c). Reddish brown crystals from ethanol, yield: 82%; m.p.: 120 °C; IR (KBr) υmax (cm–1): 3418–3220 (2NH2), 2924 (CH aliphatic), 2214, 2199 (2CN), 1633 (C=C); 1H NMR (300 MHz,DMSO-d6) δ 3.06 (m, 4H, CH2-CH2), 3.36 (s, 2H, D2O exchangeable, NH2), 3.88 (s, 3H, OCH3), 7.11–7.20 (m, 3H, pyran H-4 and Ar-H), 7.85–7.99 (m, 2H, Ar-H), 8.38 (s, 2H, D2O exchangeable, NH2); 13C NMR (75 MHz, DMSO-d6): δ 55.0 (OCH3), 62.9, 66.3 (CH2-CH2), 77.3 (pyran C-4), 115.8, 116.8 (2CN), 114.0, 114.8, 119.4, 122.1, 124.0, 129.6, 129.7, 130.5, 131.7, 132.7, 133.3, 157.2 (C6H4, pyran, thiophene C); EIMS (m/z, %): 376 [M+, 56]. Anal. Calcd. for C20H16N4O2S: C, 63.81; H, 4.28; N, 14.88; S, 8.52. Found: C, 63.69; H, 3.90; N, 14.60; S, 8.82. 704 Acta Chim. Slov. 2022, 69, 700–713 Abdo and Mohareb: Antiproliferative and Antiprostate Cancer Activities ... Ethyl 2,7-diamino-3-cyano-9-phenyl-5,9-dihydro-4H- thieno[2,3-f]chromene-8-carboxylate (23d) Brown crystals from acetic acid, yield: 83%; m.p.: 161 °C; IR (KBr) υmax (cm–1): 3425–3211 (2NH2), 2933 (CH al- iphatic), 2198 (CN), 1733 (CO), 1612 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 1.07 (t, 3H, J = 7.2 Hz, OCH2CH3), 3.17 (m, 4H, CH2-CH2), 3.39 (s, 2H, D2O exchangeable, NH2), 4.18 (q, 2H, J = 7.2 Hz, OCH2CH3),7.08 (s, 1H, pyran H-4), 7.25–7.62 (m, 5H, C6H5), 8.23 (s, 2H, D2O exchangeable, NH2); 13C NMR (75 MHz, DMSO-d6) δ 13.5 (OCH2CH3), 44.4 (OCH2CH3), 61.9, 65.1 (CH2-CH2),97.9 (pyran C-4), 116.5 (CN), 121.7, 122.9, 123.7, 124.7, 127.9, 129.2, 130.9, 131.4, 133.8, 138.9, 147.5, 155.3 (C6H5, pyran, thiophene C), 163.1 (CO); EIMS (m/z, %): 393 [M+, 32]. Anal. Cal- cd. for C21H19N3O3S: C, 64.10; H, 4.87; N, 10.68; S, 8.15. Found: C, 64.39; H, 4.60; N, 10.90; S, 8.31. Ethyl 2,7-diamino-9-(4-chlorophenyl)-3-cyano-5,9-di- hydro-4H-thieno[2,3-f]chromene-8-carboxylate (23e). Brown crystals from ethanol, yield: 79%; m.p.: 102 °C; IR (KBr) υmax (cm–1): 3423–3221 (2NH2), 2921 (CH aliphatic), 2194 (CN), 1721 (CO), 1608 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 1.31 (t, 3H, J = 6.9 Hz, OCH2CH3), 2.95 (m, 4H, CH2-CH2), 3.38 (s, 2H, D2O exchangeable, NH2), 4.31 (q, 2H, J = 6.9 Hz, OCH2CH3), 7.15 (s, 1H, pyran H-4), 7.66–8.07 (m, 4H, C6H4), 8.40 (s, 2H, D2O ex- changeable, NH2); 13C NMR (75 MHz, DMSO-d6) δ 14.1 (OCH2CH3), 55.4 (OCH2CH3), 62.4, 66.3 (CH2-CH2), 96.5 (pyran C-4), 116.0 (CN), 117.9, 121.8, 122.9, 124.1, 128.7, 129.6, 130.5, 131.9, 133.1, 137.9, 147.4, 154.9 (C6H4, pyran, thiophene C), 163.9 (CO); EIMS (m/z, %): 427 [M+, 41]. Anal. Calcd. for C21H18ClN3O3S: C, 58.94; H, 4.24; N, 9.82; S, 7.49. Found: C, 59.09; H, 4.50; N, 10.02; S, 7.31. Ethyl 2,7-diamino-3-cyano-9-(4-methoxyphenyl)-5,9- dihydro-4H-thieno[2,3-f]chromene-8-carboxylate (23f) Reddish brown crystals from ethanol, yield: 68%; m.p.: 89 °C; IR (KBr) υmax (cm–1): 3413–3212 (2NH2), 2915 (CH aliphatic), 2205 (CN), 1714 (CO), 1621 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 1.30 (t, 3H, J = 7.2 Hz, OCH2CH3), 2.91 (m, 4H, CH2-CH2), 3.33 (s, 2H, D2O ex- changeable, NH2), 3.86 (s, 3H, OCH3), 4.29 (q, 2H, J = 7.2 Hz, OCH2CH3), 7.09–7.17 (m, 3H, pyran H-4 and Ar-H), 7.81–8.10 (m, 2H, Ar-H), 8.31 (s, 2H, D2O exchangeable, NH2); 13C NMR (75 MHz, DMSO-d6) δ 13.9 (OCH2CH3), 55.1 (OCH3), 55.9 (OCH2CH3), 62.7, 66.3 (CH2-CH2), 98.5 (pyran C-4), 116.1 (CN), 114.8, 121.2, 122.6, 123.9, 128.1, 129.4, 130.7, 131.7, 133.4, 138.7, 147.8, 154.3 (C6H4, pyran, thiophene C), 163.5 (CO); EIMS (m/z, %): 423 [M+, 54]. Anal. Calcd. for C22H21N3O4S: C, 62.40; H, 5.00; N, 9.92; S, 7.57. Found: C, 62.70; H, 4.72; N, 9.92; S, 7.81. 2. 1. 11. Synthesis of 4,5,6,9-tetrahydrothieno [2,3- f]quinoline derivatives 24a-f. A mixture of compound 3 (1.92 g, 0.01 mol), either malononitrile (2) (0.66 g, 0.01 mol) or ethyl cyanoacetate (9) (1.13, 0.01 mol) and either benzaldehyde (4) (1.06 g, 0.01 mol), 4-chlorobenzaldehyde (22a) (1.4 g, 0.01 mol), or 4-methoxybenzaldehyde (22b) (1.36 g, 0.01 mol) in 1,4-dioxane (40 mL) containing ammonuim acetate (0.5 g) was heated for 3-5 h under reflux. The obtained solution was neutralized by adding a few drops of hydrochloric acid and cold water. The product was precipitated, filtered off, washed with water and dried. 2,7-Diamino-9-phenyl-4,5,6,9-tetrahydrothieno[2,3-f] quinoline-3,8-dicarbonitrile (24a). Crimson red crystals from ethanol, yield: 72%; m.p.: 110 °C; IR (KBr) υmax (cm–1): 3424–3208 (2NH2, NH), 2919 (CH aliphatic), 2214, 2194 (2CN), 1620 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 2.81 (m, 4H, CH2-CH2), 3.42 (s, 2H, D2O exchangeable, NH2), 7.10 (s, 1H, pyridine H-4), 7.26–8.07 (m, 5H, C6H5), 8.54 (s, 2H, D2O exchangeable, NH2), 10.01 (s, 1H, D2O exchangeable, NH); 13C NMR (75 MHz, DMSO-d6): δ 60.4, 64.5 (CH2-CH2), 76.1 (pyridine C-4), 116.1, 116.6 (2CN), 114.7, 115.5, 121.4, 124.8, 129.3, 132.5, 133.8, 135.7, 138.2, 139.3, 148.3, 154.3 (C6H5, pyri- dine, thiophene C); EIMS (m/z, %): 345 [M+, 34]. Anal. Calcd. for C19H15N5S: C, 66.07; H, 4.38; N, 20.27; S, 9.28. Found: C, 66.18; H, 4.50; N, 20.23; S, 8.98. 2,7-Diamino-9-(4-chlorophenyl)-4,5,6,9-tetrahydroth- ieno[2,3-f]quinoline-3,8-dicarbo-nitrile (24b) Brick red crystals from ethanol, yield: 89%; m.p.: 140 °C; IR (KBr) υmax (cm–1): 3424–3209 (2NH2, NH), 2920 (CH aliphatic), 2221, 2197 (2CN), 1622 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 2.94 (m, 4H, CH2-CH2), 3.36 (s, 2H, D2O exchangeable, NH2), 7.22 (s, 1H, pyridine H-4), 7.63–8.05 (m, 4H, C6H4), 8.54 (s, 2H, D2O exchangeable, NH2), 10.03 (s, 1H, D2O exchangeable, NH); 13C NMR (75 MHz, DMSO-d6) δ 60.3, 65.6 (CH2-CH2), 76.3 (pyridine C-4), 116.2, 117.1 (2CN), 114.3, 115.5, 123.9, 125.6, 129.7, 131.9, 133.7, 134.9, 137.9, 139.2, 147.3, 154.9 (C6H4, pyri- dine, thiophene); EIMS (m/z, %): 379 [M+, 64]. Anal. Cal- cd. for C19H14ClN5S: C, 60.07; H, 3.71; N, 18.44; S, 8.44. Found: C, 60.12; H, 3.49; N, 18.29; S, 8.54. 2,7-Diamino-9-(4-methoxyphenyl)-4,5,6,9-tetrahydro- thieno[2,3-f]quinoline-3,8-dicarbonitrile (24c) Orange crystals from ethanol, yield: 77%; m.p.: 117 °C ; IR (KBr, υmax cm–1): 3421– 3207 (2NH2, NH), 2925 (CH aliphatic), 2217, 2193 (2CN), 1614 (C=C); 1H NMR (300 MHz,DMSO-d6): δ 2.83 (m, 4H, CH2-CH2), 3.32 (s, 2H, D2O exchangeable, NH2), 3.88 (s, 3H, OCH3), 7.12– 7.20 (m, 3H, pyridine H-4 and Ar-H), 7.96-8.10 (m, 2H, Ar-H), 8.38 (s, 2H, D2O exchangeable, NH2), 9.98 (s, 1H, D2O exchangeable, NH); 13C NMR (75 MHz, DMSO-d6): δ 55.8 (OCH3), 62.6,66.1 (CH2-CH2), 76.8 (pyridine C-4),116.4, 116.9 (2CN), 114.7, 115.1, 124.0, 128.4, 129.7, 132.1, 133.3, 135.1, 138.6, 139.7, 147.8, 157.3 (C6H4, pyri- dine, thiophene C); EIMS (m/z, %): 375 [M+, 49]. Anal. 705Acta Chim. Slov. 2022, 69, 700–713 Abdo and Mohareb: Antiproliferative and Antiprostate Cancer Activities ... Calcd. for C20H17N5OS: C, 63.98; H, 4.56; N, 18.65; S, 8.54. Found: C, 64.29; H, 4.80; N, 18.42; S, 8.31. Ethyl 2,7-diamino-3-cyano-9-phenyl-4,5,6,9-tetrahy- drothieno[2,3-f]quinoline-8-carboxylate (24d) Pale brown crystals from acetic acid, yield: 69%; m.p.: 145 °C; IR (KBr) υmax (cm–1): 3419–3207 (2NH2, NH), 2981 (CH aliphatic), 2196 (CN), 1719 (CO), 1606 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 1.30 (t, 3H, J = 6.3 Hz, OCH2CH3), 2.78 (m, 4H, CH2-CH2), 3.42 (s, 2H, D2O exchangeable, NH2), 4.33(q, 2H, J = 6.3 Hz, OCH2CH3), 7.14 (s, 1H, pyridine H-4), 7.56–8.06 (m, 5H, C6H5), 8.39 (s, 2H, D2O exchangeable, NH2), 9.83 (s, 1H, D2O ex- changeable, NH); 13C NMR (75 MHz, DMSO-d6): δ 14.1 (OCH2CH3), 55.3 (OCH2CH3), 61.3, 63.5 (CH2-CH2), 98.1 (pyridine C-4), 116.6 (CN), 115.7, 121.0, 123.4, 124.3, 127.9, 130.9, 133.7, 135.8, 137.1, 139.8, 147.3, 154.7 (C6H5, pyridine, thiophene C), 163.1 (CO); EIMS (m/z, %): 392 [M+, 34]. Anal. Calcd. for C21H20N4O2S: C, 64.27; H, 5.14; N, 14.28; S, 8.17. Found: C, 64.50; H, 4.92; N, 14.56; S, 8.44. Ethyl 2,7-diamino-9-(4-chlorophenyl)-3-cyano-4,5,6,9- tetrahydrothieno[2,3-f]quinoline-8-carboxylate (24e) Reddish brown crystals from ethanol, yield: 77%; m.p.: 98–100 °C; IR (KBr) υmax (cm–1): 3422–3209 (2NH2, NH), 2978 (CH aliphatic), 2198 (CN), 1720 (CO), 1610 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 1.28 (t, 3H, J = 6.93 Hz, OCH2CH3), 2.84 (m, 4H, CH2-CH2), 3.36 (s, 2H, D2O exchangeable, NH2), 4.31 (q, 2H, J = 6.93 Hz, OCH2CH3), 7.15 (s, 1H, pyridine H-4), 7.44–8.07 (m, 4H, C6H4), 8.40 (s, 2H, D2O exchangeable, NH2), 10.01 (s, 1H, D2O exchangeable, NH); 13C NMR (75 MHz, DMSO-d6) δ 14.5 (OCH2CH3), 55.1 (OCH2CH3), 61.7, 63.4 (CH2- CH2), 97.6 (pyridine C-4), 116.4 (CN), 115.2, 121.6, 123.5, 124.5, 128.1, 131.6, 133.2, 135.6, 137.4, 139.8, 147.5, 154.7 (C6H4, pyridine, thiophene C), 163.8 (CO); EIMS (m/z, %): 426 [M+, 66]. Anal. Calcd. for C21H19ClN4O2S: C, 59.08; H, 4.49; N, 13.12; S, 7.51. Found: C, 59.30; H, 4.41; N, 13.45; S, 7.81. Ethyl 2,7-diamino-3-cyano-9-(4-methoxyphenyl)-4,5,6, 9-tetrahydrothieno[2,3-f]quinoline-8-carboxylate (24f) Brown crystals from ethanol, yield: 89%; m.p.: 87 °C; IR (KBr) υmax (cm–1): 3417–3212 (2NH2, NH), 2984 (CH aliphatic), 2203 (CN), 1716 (CO), 1625 (C=C); 1H NMR (300 MHz, DMSO-d6) δ 1.29 (t, 3H, J = 6.82 Hz, OCH2CH3), 2.68 (m, 4H, CH2-CH2), 3.34 (s, 2H, D2O ex- changeable, NH2), 3.87 (s, 3H, OCH3), 4.31 (q, 2H, J = 6.82 Hz, OCH2CH3), 7.08–7.22 (m, 3H, pyridine H-4 and Ar- H), 8.01-8.04 (m, 2H, Ar-H), 8.30 (s, 2H, D2O exchangea- ble, NH2), 9.91 (s, 1H, D2O exchangeable, NH); 13C NMR (75 MHz, DMSO-d6) δ 13.9 (OCH2CH3), 50.1 (OCH3), 55.6 (OCH2CH3), 62.0, 66.3 (CH2-CH2), 98.4 (pyridine C-4), 116.1 (CN), 115.9, 121.3, 123.8, 124.7, 127.9, 131.2, Table 1. In vitro growth inhibitory effects IC50 ± SEM (µM) of the newly synthesized compounds against cancer cell lines. Compound IC50 ± SEM (µM) No A549 H460 HT29 MKN-45 U87MG SMMC-7721 3 6.29 ± 1.63 5.59 ± 2.35 4.29 ± 2.61 6.77 ± 2.37 7.18 ± 2.57 5.82 ± 1.31 5 6.27 ± 1.80 8.61 ± 2.29 4.36 ± 1.59 3.38 ± 1.62 5.80 ± 1.08 2.49 ± 0.68 7 3.18 ± 1.63 0.42 ± 0.30 1.52 ± 0.23 4.61 ± 2.51 2.63 ± 1.38 1.79 ± 0.83 10a 8.53 ± 2.36 8.29 ± 2.13 8.34 ± 3.70 8.39 ± 2.42 9.68 ± 3.37 8.27 ± 2.91 10b 1.22 ± 0.87 0.52 ± 0.32 0.73 ± 0.48 1.49 ± 0.41 2.46 ± 0.83 1.32 ± 0.42 12 0.24 ± 0.15 0.32 ± 0.22 0.34 ± 0.09 0.42 ± 0.33 0.24 ± 0.19 0.26 ± 0.14 13 4.26 ± 2.12 3.14 ± 1.39 8.14 ± 3.52 6.91 ± 2.42 3.62 ± 1.47 4.73 ± 2.68 15a 3.25 ± 1.08 2.18 ± 0.07 2.68 ± 1.17 2.69 ± 0.98 2.80 ± 1.32 5.54 ± 2.38 15b 4.65 ± 1.36 5.43 ± 2.25 1.39 ± 0.89 1.82 ± 0.96 2.34 ± 0.29 1.80 ± 0.28 19 1.23 ± 0.39 1.44 ± 0.83 2.31 ± 0.67 1.35 ± 0.68 0.89 ± 0.46 1.25 ± 0.59 20 3.12 ± 1.68 4.29 ± 2.39 5.27 ± 3.54 3.18 ± 1.26 4.31 ± 2.82 3.27 ± 1.57 21 1.02 ± 0.95 1.28 ± 0.79 1.08 ± 2.80 2.28 ± 1.23 1.67 ± 0.85 1.62 ± 0.63 23a 1.32 ± 0.88 1.43 ± 0.87 1.74 ± 0.69 1.52 ± 0.83 0.89 ± 0.35 1.63 ± 0.69 23b 0.27 ± 0.18 0.39 ± 0.19 0.62 ± 0.35 0.82 ± 0.63 0.72 ± 0.53 1.29 ± 0.83 23c 7.26 ± 2.58 3.18 ± 2.31 6.68 ± 2.40 5.62 ± 3.42 4.71 ± 1.26 6.80 ± 2.26 23d 8.53 ± 3.57 5.72 ± 3.86 6.48 ± 2.68 7.38 ± 1.87 4.69 ± 2.41 6.50 ± 2.81 23e 0.28 ± 0.15 0.32 ± 0.14 0.36 ± 0.15 0.19 ± 0.06 0.38 ± 0.15 0.17 ± 0.08 23f 4.53 ± 2.51 6.48 ± 2.63 6.59 ± 1.42 6.29 ± 1.38 6.75 ± 2.69 6.58 ± 2.80 24a 4.59 ± 2.26 5.53 ± 2.70 6.31 ± 2.29 6.50 ± 2.63 8.53 ± 2.72 6.32 ± 2.42 24b 0.40 ± 0.33 0.23 ± 0.18 0.52 ± 0.23 0.41 ± 0.25 0.26 ± 0.19 0.25 ± 0.08 24c 3.34 ± 1.24 4.67 ± 1.50 2.80 ± 0.77 2.53 ± 1.19 3.35 ± 1.64 4.49 ± 2.06 24d 6.40 ± 2.58 6.94 ± 2.39 6.29 ± 2.43 6.58 ± 2.30 5.68 ± 2.39 6.55 ± 1.90 24e 1.27 ± 0.53 0.82 ± 0.57 0.83 ± 0.82 1.72 ± 0.94 0.79 ± 0.26 0.59 ± 0.24 24f 0.48 ± 0.26 0.56 ± 0.32 0.42 ± 0.35 0.67 ± 0.40 0.29 ± 1.85 0.69 ± 0.42 Foretinib 0.08 ± 0.01 0.18 ± 0.03 0.15 ± 0.023 0.03±0.0055 0.90 ± 0.13 0.44 ± 0.062 706 Acta Chim. Slov. 2022, 69, 700–713 Abdo and Mohareb: Antiproliferative and Antiprostate Cancer Activities ... 133.4, 135.1, 137.6, 139.4, 147.8, 154.3 (C6H4, pyridine, thiophene C), 163.4 (CO); EIMS (m/z, %): 422 [M+, 33]. Anal. Calcd. for C22H22N4O3S: C, 62.54; H, 5.25; N, 13.26; S, 7.59. Found: C, 62.77; H, 5.52; N, 13.09; S, 7.31. 2. 2. Biology Section Materials ATP (adenosine triphosphate) is used in this biology section. DMSO (dimethyl sulfoxide), MgCl2 (magnesium chloride) were purchased from Sigma. Receptor tyrosine kinases c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR were purchased from Carna Biosciences (Kobe, Japan). 2. 2. 1. Cell proliferation test The antiproliferative activities of the newly synthe- sized compounds (Table 1) were evaluated against the five c-Met-dependent cancer cell lines (A549, HT -29, MKN- 45, U87MG, and SMMC-7721) and one c-Met-independ- ent cancer cell line (H460) with foretinib as a positive con- trol using the standard MTT assay in vitro.26 The experi- mental procedure was applied according to the previously reported work.27–29 In vitro cell experiments All compounds were tested for their cytotoxicity in the six cancer cell lines using the MTT method. The results, expressed as IC50 (average of at least three inde- pendent experiments), were summarized in Table 1. The data presented in Table 1 show that the tested compounds exhibited moderate to strong cytotoxicity against the six cancer cell lines in the single-digit lM range. Compounds 12, 19, 23a, 23b, 23e, 24b, 24e, and 24f exhibited high- er cytotoxicity against U87MG than fortinib (the positive control). 2. 2. 2. Structure Activity Relationship Table 1 shows the inhibitory effect of the new com- pounds on cancer cell lines A549, H460, HT -29, MKN-45, U87MG, and SMMC-7721. There are many compounds that showed high inhibitory values, such as 10b, 12, 23b, 23e, 24b, 24e, and 24f. In addition, some compounds showed moderate inhibition, such as 7, 20, 21, 23a, and 24c. The analysis of Table 1 shows that the substituted groups and the type of heterocyclic ring have a great in- fluence on the inhibitions. Thiophene derivatives 3 and 5 had little inhibitory effect on the cancer cell lines tested. In contrast, fused derivative 7 showed moderate inhibition. Surprisingly, 4H-thieno[2,3-f]chromene derivatives 10a and 10b showed low inhibitory values, while compound 10a (Y = COOEt) showed high inhibitory values, which was attributed to the presence of the COOEt group. Hy- drazide-hydrazone derivative 12 showed strong inhibition against the tested cancer cell lines, while compounds 13 and 15a,b showed moderate inhibition. In addition, com- pounds 19 and 21 showed moderate inhibition, with com- pound 19 exhibiting high inhibition against the U87MG cell line with an IC50 of 0.89M. For thieno[2,3-f]chromene derivatives 23a-f, the different substituents played the ma- jor role in the inhibitions of the compounds. Compound 23a (X = CN, Y = H) showed moderate inhibitions, while compounds 23b (X = CN, Y = Cl) and 23e (X = COOEt, Y = Cl) showed the strongest inhibitions against the test- ed cancer cell lines. In contrast, compounds 23c, 23d, and 23f showed lower inhibitory activity. Interestingly, compounds 24a-f, 24b, 24e, and 24f showed the highest inhibitory values among the six compounds, as the high inhibitory values of compounds 24b and 24e were due to the electronegative Cl group. Compound 24f showed high inhibition values despite the electron-donating OCH3 group, while the inhibition values of compounds 24a, 24c, and 24d decreased. 2. 2. 3. HTRF Kinase Assay The c-Met kinase activity of the newly synthesized compounds was assayed using a homogeneous time-re- solved fluorescence (HTRF) assay (Table 2), as reported previously.30 In addition, the maximally active compounds 7, 10a, 10b, 13, 15a, 21, 24a, 24b, 24c, 24d, and 24e were extra assayed using the same screening method for the five tyrosine kinases (c-Kit, Flt-3, VEGFR-2, EGFR, and PDG- FR) (Table 3). The experimental technique and chemicals used were based on reported work.31 Enzymatic in vitro tests All freshly prepared benzo[b]thiophene derivatives were evaluated for their inhibitory activity against c-Met enzyme32 in a homogeneous time-resolved fluorescence (HTRF) assay, with foretinib serving as a positive control. The antiproliferative activity of all newly synthesized com- pounds against the human prostate cancer cell line PC-3 was calculated by MTT assay33,34 using SGI-1776 as the reference drug. The results, reported as IC50 (average of at least three independent experiments) for both HTRF and antiproliferative activity, are shown in Table 2. Most of the compounds tested showed potent antiproliferative activity with IC50 values of less than 30 mM. In most cases, the het- erocycles were associated with the benzothiophene moiety, and variations in substituents had a marked effect on anti- proliferative activity. The most potent compounds against c-Met kinase were compounds 7, 10b, 13, 15a, 21, 24b, 24c, 24d, and 24e. It is very surprising that compounds 10b, 13, 15a, 24b, 24c, 24d and 24e showed stronger in- hibition than the reference drug foretinib (IC50 1.16 mM). On the other hand, screening with the prostate cancer cell line PC-3 showed that compounds 10b, 23c, 23e, 24a, 24b and 24d had the highest inhibition values. All tested com- pounds showed higher inhibition than the reference drug SGI-1776, except compounds 3, 10a, 15b and 23f. 707Acta Chim. Slov. 2022, 69, 700–713 Abdo and Mohareb: Antiproliferative and Antiprostate Cancer Activities ... Table 2. c-Met enzymatic activity of the newly synthesized com- pounds. Compound No IC50 (nM) IC50 (nM) c-Met PC-3 3 4.65 ± 1.42 6.56 ± 1.38 5 10.23 ± 3.58 2.16 ± 1.13 7 1.18 ± 0.69 2.51 ± 0.34 10a 1.64 ± 0.89 6.42 ±2.51 10b 0.33 ± 0.16 0.28 ± 0.16 12 4.38 ±1.64 3.58 ±1.24 13 0.48 ± 0.15 2.48 ± 1.20 15a 0.32 ± 0.20 4.26 ± 1.42 15b 13.62 ± 4.53 8.37 ± 2.63 19 4.116 ± 5.41 8.57 ± 2.46 20 6.34 ± 2.62 2.17 ± 1.15 21 1.27 ± 0.71 2.08 ± 0.85 23a 8.32 ± 2.74 2.36 ± 1.27 23b 18.27 ± 4.58 2.39 ± 0.83 23c 5.82 ± 1.29 0.92 ± 0.32 23d 18.29 ± 4.70 1.06 ± 0.73 23e 2.41 ± 1.04 0.8 3 ± 0.41 23f 6.24 ± 2.38 8.41 ± 2.49 24a 2.08 ± 0.87 0.96 ± 0.42 24b 0.08 ± 0.03 0.16 ± 0.04 24c 0.32 ± 0.26 1.03 ± 0.69 24d 0.22 ± 0.08 0.59 ± 0.08 24e 0.06 ± 0.004 1.15 ± 0.72 24f 5.31 ± 2.62 4.33 ± 1.36 Foretinib SGI-1776 1.16 ± 0.17 4.86 ± 0.16 2. 2. 4. Inhibition of Tyrosine Kinases (Enzyme IC50 (nM) The five tyrosine kinesis c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR were used using sorafenib as the ref- erence drug to test the inhibitions of the selected com- pounds. The selection of the compounds was based on their high inhibitory activity against the six cancer cell lines. Table 2 shows that compounds 7, 10a, 10b, 13, 15a, 21, 24a, 24b, 24c, 24d, and 24e had the highest inhibitory values. The data in Table 3 show that compounds 10a, 13, 24b, and 24a had the highest inhibitory activity among the compounds tested. Table 3 showed that compounds 10a, 10b, 24b, 24c, and 24e inhibit the investigated tyrosine kinases most strongly, whereas compounds 7, 21, 24a, and 24d show only slight inhibition. 2. 2. 5. Inhibition of Selected Anti-Pim-1 Kinase Compounds In addition, compounds 10b, 12, 19, 21, 23a, 23b, 23e, 24b, 24e, and 24f were selected to investigate their inhibitory effects on Pim-1 kinase (Table 4). Based on their IC50 values in a range of 10 concentrations, these compounds showed strong inhibition against both c-Met kinase and the cancer cell lines tested. The most active compounds were 10b, 23a, 23e, 24b, and 24f, with IC50 values of 0.29, 0.036, 0.26, 0.43, and 0.31 mM, respectively. Table 4. The inhibitions of compounds 10b, 12, 19, 21, 23a, 23b, 23e, 24b, 24e and 24f toward Pim-1 kinase. Compound Inhibition ratio at 10 µ M IC50 (µM) 10b 94 0.29 12 30 > 10 19 24 > 10 21 30 >10 23a 96 0.036 23b 26 >10 23e 95 0.26 24b 88 0.43 24e 28 >10 24f 89 0.31 SGI-1776 – 0.048 3. Results and Discussion 3. 1. Chemistry In recent years, our research group has carried out numerous heterocyclic reactions with cyclohexanedione derivatives.35–37 The aim of these reactions was the synthe- sis of thiophene derivatives by Gewald’s thiophene meth- od,38–41 and the synthesis of hydrazide-hydrazone deriv- atives.42,43 The prepared compounds showed interesting results as anticancer agents. As a continuation of our work here, we demonstrated the heterocyclization of cyclohex- ane-1,4-dione and then studied its biological evaluation. The reaction sequences for the synthesis of the final com- pounds 3 to 24a-f are shown in Schemes 1-4. The chemical structures of the new compounds were secured by spectral data (IR, 1H and 13C NMR, MS). Cyclohexane-1,4-dione Table 3. Inhibition of tyrosine kinases (Enzyme IC50 (nM) by com- pounds 7, 10a, 10b, 13, 15a, 21, 24a, 24b, 24c, 24d and 24e. Com- c-Kit Flt-3 VEGFR-2 EGFR PDGFR pound 7 4.16 2.68 3.19 2.57 0.83 10a 0.43 0.29 0.61 0.39 0.71 10b 0.24 1.29 2.42 1.29 2.06 13 1.03 0.48 1.18 0.49 0.25 15a 1.69 1.22 0.63 0.52 0.69 21 2.72 4.53 5.62 3.41 1.58 24a 3.62 2.95 2.80 2.45 3.68 24b 0.36 0.42 0.53 0.29 0.31 24c 0.48 0.61 0.58 1.22 0.72 24d 1.08 2.40 2.35 3.06 2.69 24e 0.22 0.36 0.18 0.49 0.31 Foretinib 0.19 0.17 0.20 0.13 0.26 708 Acta Chim. Slov. 2022, 69, 700–713 Abdo and Mohareb: Antiproliferative and Antiprostate Cancer Activities ... was subjected to Gewald’s thiophene synthesis by reacting it with elemental sulfur and malononitrile (2) in 1,4-di- oxane with triethylamine under reflux to give 2-ami- no-6-oxo-4,5,6,7-tetrahydrobenzo[b]thiophene-3-caboni- trile (3). The spectral data showed that compound 3 was present in both the keto and enol tautomeric structures. The presence of a broad signal at n 3422 cm–1 confirmed the presence of the OH group along with the appearance of a signal at n 1706 cm–1 due to the presence of the CO group in the IR spectrum. In addition, the 1H NMR spec- trum showed the appearance of a doublet and a triplet at δ 2.65 and 6.82 ppm for the CH2−CH=C protons besides a singlet at δ 5.54 ppm for the CH2 group between OH and the sp2 carbon and two singlet at δ 3.39 and 9.97 ppm (D2O interchangeable) corresponding to the NH2 and OH groups, respectively. In addition, the 13C NMR spectrum showed signals at d 22.6 (CH2−CH=C), 50.3 (CH2), 66.3 (CH2−CH=C), 116.2 (CN), 118.4, 121.7, 128.9, 134.0 (thi- ophene C), and 161.8 (CO). Compound 3 was the major starting compound for various heterocyclization reactions because it contains an active methylene moiety between the OH group and the Schema 1: Synthesis of compounds 3, 5,7 and 10a,b. 709Acta Chim. Slov. 2022, 69, 700–713 Abdo and Mohareb: Antiproliferative and Antiprostate Cancer Activities ... sp2 carbon. For example, compound 3 reacted with ben- zaldehyde in 1,4-dioxane containing a catalytic amount of piperidine to give the arylidene derivative 5. Similarly, the reaction of compound 3 with salicylaldehyde (6) formed the 2-hydroxybenzylidene derivative 7. The synthesis, reactions, and biological activities of 4H-pyran-containing molecules have been extensively studied. In addition, 4H-pyran derivatives are also an es- sential component of some pharmaceutical agents and nat- ural products.44–46 This inspired us to synthesize 4H-pyran derivatives via the multicomponent reaction of compound 3. Thus, compound 3 was subjected to a multicomponent reaction with ethyl orthoformate and either malononitrile (2) or ethyl cyanoacetate (9) to give 4H-pyran derivatives 10a and 10b, respectively (Scheme 1). Addition of cyanoacetylhydrazine (11) to com- pound 3 in 1,4-dioxane under reflux conditions pre- pared the 2-cyanoacetohydrazide derivative 12. Exam- ination of the analytical and spectral data confirmed the structure of compound 12, with the 1H NMR spectrum showing a doublet and a triplet at δ 2.71 and 6.83 ppm confirming the presence of CH2−CH=C protons, in ad- dition to two singlet at δ 3.76 and 5.54 ppm for the pro- tons CO−CH2−CN and CH2, respectively. In addition to the presence of three singlet (D2O exchangeable) at δ 3.37, 8.13 and 9.93 ppm for NH2 and two NH groups, respectively. The 13C NMR spectrum showed signals at δ 35.8 (CH2−CH=C), 66.3 (CH2), 77.4, 118.5 (CH2− CH=C), 98.9 (CO−CH2−CN), 115.7, 116.2 (2CN), 129.9, 133.3, 136.6, 154.7 (thiophene C), 162.6 (CO). In addition, ethyl cyanoacetate (9) reacted with compound 3 in 1,4-dioxane containing a catalytic amount of tri- ethylamine to produce the dihydronaphtho[1,2-b]thio- phene derivative 13. The study of analytical and spectral data confirmed the proposed structure of compound 13 as mentioned in the experimental section. On the other Schema 2: Synthesis of compounds 12, 13 and 15a,b. 710 Acta Chim. Slov. 2022, 69, 700–713 Abdo and Mohareb: Antiproliferative and Antiprostate Cancer Activities ... hand, compound 3 reacted with either benzenediazo- nium chloride or p-tolylbenzenediazonium chloride in ethanol solution containing sodium acetate at 0−5 °C to give the corresponding arylhydrazone derivatives 15a and 15b, respectively (Scheme 2). Continuing our previous work on the synthesis of thiophene or thiazole derivatives using phenyl isothiocy- anate in basic dimethylformamide and subsequent hetero- cyclization of the intermediate potassium salt by a-haloke- tones,47,48 in this work we have demonstrated such reac- tions with the aim of preparing heterocyclic compounds with predicted biological activity. For example, the reac- tion of compound 3 with phenyl isothiocyanate and potas- sium hydroxide in dimethylformamide gave the potassium salt intermediate 17, followed by the addition of a–chloro- acetone (18a) to the intermediate 17, giving the thiazole derivative 19. However, reaction with ethyl a-chloroace- tate (18b) surprisingly gave the thioether derivative 20. Heating of compound 20 in 1,4-dioxane with a catalytic amount of triethylamine gave the condensed dithiophene derivative 21 (Scheme 3). The structures of compounds 19, 20, and 21 were confirmed by the data reported in the ex- perimental section. Moreover, the multicomponent reaction of com- pound 3 with either malononitrile or ethyl cyanoacetate and either benzaldhyde, 4-chlorobenzaldhyde, or 4-meth- oxybenzaldhyde in 1,4-dioxane in the presence of trimeth- ylamine as catalyst gave the 4H-thieno[2,3-f]chromene derivatives 23a-f. Similarly, reaction of compound 3 with malononitrile or ethyl cyanoacetate and either benzald- Schema 3: Synthesis of compounds 19, 20 and 21. 711Acta Chim. Slov. 2022, 69, 700–713 Abdo and Mohareb: Antiproliferative and Antiprostate Cancer Activities ... hyde, 4-chlorobenzaldhyde or 4-methoxybenzaldhyde in 1,4-dioxane with ammonium acetate gave the 4,5,6,9-tet- rahydrothieno[2,3-f]quinoline derivatives 24a-f. The structure of compounds 23a-f and 24a-f (Scheme 4) was determined based on the study of their spectral data and elemental analyzes (see Experimental section). 4. Conclusion The benzo[b]thiophene derivative was the major starting compound for several heterocyclization reac- tions. All new compounds were tested on the six cancer cell lines. In addition, the c-Met kinase activity of all com- pounds was calculated, and the most active compounds were tested against five other tyrosine kinases. In addition, compounds 10b, 12, 20, 21, 23a, 23b, 23e, 24b, 24e, and 24f were selected to investigate their inhibitory effect on Pim-1 kinase, as these compounds showed a large inhibi- tory effect on c-Met kinase and the cancer cell lines stud- ied. The results obtained in this work will stimulate further work in the future. Human and Animal Rights No Animals/Humans were used for studies that are basis of this research. Consent for Publication This work is consent for publication through the Journal formats. Conflict of Interest The authors declare no conflict of interest, financial or otherwise. Schema 4: Synthesis of compounds 23a-f and 24a-f. 712 Acta Chim. Slov. 2022, 69, 700–713 Abdo and Mohareb: Antiproliferative and Antiprostate Cancer Activities ... 5. References 1. M. Garcia-Valverde, T. Torroba, Molecules, 2005, 10, 318–320. DOI:10.3390/10020318 2. S. Pathania, R. K. Narang, R. K. Rawal, Eur. J. Med. Chem., 2019, 180, 486–508. DOI:10.1016/j.ejmech.2019.07.043 3. S. 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DOI:10.1002/jhet.3966 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek V prispevku je opisana priprava 2-amino-6-okso-4,5,6,7-tetrahidrobenzo[b]tiofen-3-karbonitril (3) z reakcijo ciklo- heksan-1,4-diona z elementarnim žveplom in malononitrilom v 1,4-dioksanu in s trietilaminom kot katalizatorjem. Iz pripravljene spojine so z reakcijo s trietil ortoformatom in malononitrilom ali etil cianoacetatom v 1,4-dioksanu kot topilu, v prisotnosti trietilamina, nastali 4H-tieno[2,3-f]kromenski derivati 10a,b. Poleg teh so iz spojine 3 pripravili tudi kondenzirane piranske in piridinske derivate. Citotoksičnost sintetiziranih spojin so preučevali na šestih rakavih celičnih linijah skupaj s c-Met kinazo in PC-3 celično linijo. Najbolj aktivne spojine so bile dodatno testirane na petih tirozin kinazah in Pim-1 kinazi. Večina testiranih spojin je pokazala močno inhibicijo, kar je dobra spodbuda za nadaljnje delo. 714 Acta Chim. Slov. 2022, 69, 714–721 Farajian and Hashemi: Superparamagnetic Tragacanth Coated Fe3O4@SiO2 ... DOI: 10.17344/acsi.2021.7240 Scientific paper Superparamagnetic Tragacanth Coated Fe3O4@SiO2 Nanoparticles for the Loading and Delivery of Metformin Fereshte Farajian1 and Payman Hashemi1,* Department of Chemistry, Faculty of Science, Lorestan University, Khoramabad, I.R. Iran * Corresponding author: E-mail: Hashemi.p@lu.ac.ir Phone: +986633120611 Received: 10-27-2021 Abstract A novel superparamagnetic nano-composite of Fe3O4@SiO2 coated by tragacanth gum (TG) as a natural product has been prepared. The obtained SiO2@Fe3O4@TG nanoparticles were characterized by Fourier transform infrared spectros- copy, energy dispersive X-ray analysis, scanning electron microscopy and dynamic light scattering analyzer. The mag- netic nano-composite was applied for the loading and delivery of metformin, an oral diabetes medicine. The conditions for the loading of the drug were optimized by a central composite design optimization method. The maximum loading efficiency of the sorbent for metformin was obtained at pH 7 and its maximum in-vitro release was achieved at pH 1.6, using a phosphate-buffered saline medium. The loading capacity of the sorbent was dependent on the initial metform- in concentration and exceeded to 19.6 mg/g in a 200 mg/L solution. A study of the adsorption isotherms for the drug indicated the best fitting into the Langmuir and Freundlich isotherms at the low and high metformin concentrations, respectively. The results indicated that the prepared Fe3O4@SiO2@TG adsorbent, as a non-toxic and low-cost sorbent, was quite appropriate for drug delivery applications. Keywords: Tragacanth; Hydrogel adsorbent; Magnetic nanoparticles; Metformin 1. Introduction In recent years, the interest forthe use of nanoparti- cles (NPs), especially magnetic NPs, for analytical purpos- es and in drug delivery applications has been increased. The major properties of magnetic NPs include easy separa- tion by an external magnetic field, simple syntheses, high surface area, high firmness, reusability and biocompati- bility.1,2 The superparamagnetic iron oxide core increases the binding capacity of NPs and enables them to replace the centrifugation step with magnetic separation. It also simplifies the application of NPs in immune assay.3 The oxidation potential of iron NPs is high and it is frequent- ly necessary to coat their surfaces by mineral or organic compounds.4 SiO2 is one of the materials used for coat- ing ferrite NPs as it is non-toxic, water-dispersible, and environmentally friendly.5 Coating of magnetic NPs with biopolymers is also desirable as they can increase the ad- sorption capacity and selectivity of the NPs for numerous applications.6–9 Hydrogels are materials with swellable polymeric networks storing a great quantity of water. The potential to absorb water is due to the presence of polar and hydro- philic groups in the polymer network. The durability of hydrogels, on the other hand, is affected by crosslinking. Both artificial and natural polymers have been employed for the production of hydrogels. However, the natural pol- ymeric hydrogels have several advantages such as high capacity of water absorption, low expense, long operation life, low toxicity, and high gel stability.10 Tragacanth gum (TG) is a natural adhesive mixture of polysaccharides obtained from the plant of Astragalus sp. It is a biosorbent and porous hydrogel material that is a non-toxic, abundant, low-cost and biocompatible biopoly- mer.8,9,11 TG can be modified by various functional groups such as carboxylic acid, primary and secondary hydroxyl groups and epoxy groups which would enhance its selec- tivity and provide favourable adsorption conditions.11–13 The molecular structure of TG is shown in Figure 1. TG has been used in many applications such as wound cov- ering,14 drug delivery,15 natural antibacterial16 and dis- persing and thickening agent.17 TG is a biodegradable, biocompatible, inodorous, flavorless, osteogenic, and re- sistant biopolymer upon a wide pH range.18,19 However, the pure TG alone has some weaknesses and hence, it is 715Acta Chim. Slov. 2022, 69, 714–721 Farajian and Hashemi: Superparamagnetic Tragacanth Coated Fe3O4@SiO2 ... frequently strengthened with either organic or inorganic materials such as clays,20 carbon nanotubes21 methacrylate polymers,12,13 and metal nanoparticles.22 The goal of the current study is to describe a novel synthesis of Fe3O4@SiO2@TG nanocomposite with an easy method as a magnetic adsorbent with biocompatibility, low toxicity, and low cost. The SiO2 shell is anticipated to increase the resistance of the nanoparticles in acidic con- ditions and TG is a non-toxic drug delivery platform. The synthesized adsorbents would be studied for the loading and delivery of metformin as an oral diabetes medicine. In addition to the study of the adsorption mechanisms, mul- tivariate methods are used for optimization of the loading conditions 2. Experimental Section 2. 1. Chemical and Apparatus All applied chemicals were of analytical reagent grade and were used as received. Sodium hydroxide, am- monia solution 25%, tetraethyl orthosilicate (TEOS), ferric chloride (FeCl3 6H2O), ferrous chloride (FeCl2 4H2O) and ethanol were obtained from Merck chemical company. A stock solution (1000 mg/g) of metformin was pre- pared by dissolving required quantity of the drug in meth- anol. Further dilutions were made by deionized water and prepared daily prior to use. Saline phosphate buffer solu- tion (0.15 mol/L) was prepared by dissolving 0.8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KHPO4 in 1000 mL of distilled water. This solution was used for buffering test samples after adjustment of its pH (typically on 7.0). TG was obtained from local shops with the best quality and the pieces with transparent color were used for experi- ments. All solutions were prepared with deionized water. All the spectrophotometric measurements of met- formin were accomplished at its λmax (232 nm) by a Shi- madzu UV-1650PC UV–Vis spectrophotometer (Japan). A pair of quartz cells (Esquartz, model Q124) were used for the measurements. For the measurement of the hydrodynamic sizes and zeta potential values of Fe3O4@SiO2 and Fe3O4@SiO2@ GT particles as-prepared, a zeta potential and dynamic light scattering (DLS) analyzer, SZ-100 - HORIBA (Japan) was used. The particles were diluted with 20% ethanol in deionized water (pH 7). 2. 2. Synthesis of Fe3O4 NPs For the preparation of Fe3O4 NPs an ordinary chem- ical co-precipitation method was applied.23 In brief, 50 mL of 0.001 mol/L equimolar mixture of FeCl3 6 H2O, and FeCl2 4 H2O was prepared in deionized water. The mixture of the iron salts was sonicated for 15 min at room temper- ature. The mixture was then transferred to a round bottom two neck flask and heated to 80 °C under reflux in argon atmosphere. Then, 5 mL of concentrated ammonium hy- droxide (25% w/w) was slowly added to it during 30 min. A color change from yellowish to black was observed in the mixture that was further heated to 90 °C for 1.5 h un- der the argon atmosphere. Next, the prepared Fe3O4 nan- oparticles were washed several times with deionized water after being separated by a permanent magnet. Finally, the obtained Fe3O4 NPs were re-suspended in 100 mL of deionized water. The obtained NPs were sta- ble in this form up to two months. 2. 3. Synthesis of Fe3O4@SiO2 NPs Since hydrophobic Fe3O4 nanoparticles are unstable, they are usually coated by a silica shell.3 For silanization of the magnetic NPs a typical Stöber method was used. For this purpose, 1.5 g of Fe3O4 NPs were added to a solution of 16 mL distilled water, 80 mL ethanol and 2 mL 25% ammonia. The composition was then dispersed for 15 min in an ultrasonic bath. After that, 1.0 mL of tetraethyl Figure 1. The chemical structure of TG. 716 Acta Chim. Slov. 2022, 69, 714–721 Farajian and Hashemi: Superparamagnetic Tragacanth Coated Fe3O4@SiO2 ... orthosilicate (TEOS) was added dropwise to the solution containing the NPs. The suspension was shaken on a shak- er-bath for 24 h at room temperature. Eventually, the NPs were gathered by a permanent magnet and rinsed several times by distilled water. 2. 4. Coating of Fe3O4@SiO2 NPs with TG For the coating of the silanized magnetite NPs with TG, 0.02 g of TG powder was dissolved in 10 mL distilled water at 70 °C in a glass beaker. Subsequently, 1.5 g of Fe3O4@SiO2 NPs were added to the solution under stir- ring at 1200 rpm and the solution was shaken for 4 h in room temperature. Finally, the Fe3O4@SiO2@TG NPs were washed by distilled water several times and the magnetic NPs were separated by a magnet (Figure 2) and stored in 20% ethanol. 2. 5. Central Composite Design (CCD) Optimization A CCD method was used for the optimization of sample volume, adsorbent mass, pH and contact time. For each factor a low and high level was defined based on the results of some primary trials. For each of the four studied factors, five levels were suggested by the Minitab software as shown in Table 1. The recovery or adsorption efficiency for the drug was regarded as the response or independent function for the optimization. The initiation of the design and statisti- cal analyses of the results were performed using Minitab 16 software. A drug concentration of 20 mg/L with the pH ad- justed by a phosphate buffered saline solution (0.15 mol/L) was used during the optimization. After addition of the ad- sorbent, the mixture was shaken in a thermostated water bath for a preset time and the Fe3O4@SiO2@TG particles were collected using a permanent magnet. The quantity of adsorbed metformin was computed by the absorbance measurements at 232 nm before and after the adsorption. Standard solutions of metformin in the range of 1 to 20 mg/L were utilized for the calibration. 2. 6 Calculation of the Adsorption Capacity Adsorption capacity of the adsorbent was calculated in mg/g, by its loading with different metformin concen- trations. This was done by adding 5 mg of the adsorbent to 1 mL of the drug solutions at pH 7. The residual amount of the drug in solution was calculated by absorption meas- urements at 232 nm using the UV/Vis spectrophotometer. The adsorption capacity was then calculated from equa- tion 1: q = [( C o – Ct) · V ] ∕ m (1) In this equation, q is the amount of analyte adsorbed or adsorption capacity (mg/g) of the adsorbent, Co is the metformin concentration before addition of the sorbent and Ct represents its equilibrium concentration (mg/L), V represents the volume of the solution (L) and m is the adsorbent mass (g). 3. Results and Discussion 3. 1. Characterization of the NPs In the introductory experiments, Fe3O4, Fe3O4@SiO2 and Fe3O4@SiO2@TG magnetic NPs were prepared and characterized by Fourier transform infrared spectroscopy (FT-IR), energy dispersive X-ray analysis (EDX), scanning electron microscopy (SEM) and dynamic light scattering (DLS) analyzer. Figure 3 shows the FT-IR spectra of the NPs. In the FT-IR spectrum of Fe3O4 NPs (Figure 3a), the peak that appeared at wavenumber 584 cm−1 is related to stretching vibrations of Fe-O. The bands in the range of 1200–1000 cm−1 in (Figure 3b and 3c) belong to the Si–O covalent bond vibrations and confirms the coating of sili- ca on nanoparticles. In the FT-IR spectra associated with Fe3O4@SiO2@TG magnetic NPs (Figure 3c), other major vibrational peak in 3712 cm−1 is related to stretching vibra- tion of C–H. This band is a proof of the fixation of TG layer on Fe3O4@SiO2 magnetic NPs. Table 1. The studied parameters and suggested levels in the CCD optimization. Factor Abbreviation Factors’ levels –α Low 0 High +α pH pH 2 3.5 5 6.5 8 Contact time (min) Time 20 30 40 50 60 Temperature (°C) T 25 30 35 40 45 Sample volume (mL) Vs 1 3 5 7 9 Figure 2. Schematic illustration of the procedure used for the syn- thesis of Fe3O4@SiO2@GT NPs. 717Acta Chim. Slov. 2022, 69, 714–721 Farajian and Hashemi: Superparamagnetic Tragacanth Coated Fe3O4@SiO2 ... Figure 3. FT-IR spectra of Fe3O4 (a), Fe3O4@SiO2 (b) and Fe3O4@ SiO2@TG (c) nanoparticles. The dimension and morphology of Fe3O4, Fe3O4@ SiO2 and Fe3O4@SiO2@TG nanoparticles were evaluated by SEM. Figure 4 shows the spherical morphology and narrow size spreading of the NPs with an average size of 50–70 nm for Fe3O4@SiO2@TG. Energy dispersive X-ray (EDX) analysis was used for the elemental mappings and distribution of the prepared Fe3O4@SiO2 and Fe3O4@ SiO2@TG nanoparticles. Based on the EDX results, C, N, O, Si and Fe were recognized in the study of Fe3O4@SiO2@ TG nanoparticles and as expected, the quantity of carbon in the Fe3O4@SiO2@TG nanoparticles was more than that of Fe3O4@SiO2 due to the presence of TG in the former. The results apparently confirm covering of the nanopar- ticles by TG. Zeta potential study of Fe3O4@SiO2 and GT coated adsorbents indicated a –26.6 mV potential for the Fe3O4@ SiO2 and –23 mV for the Fe3O4@SiO2@GT adsorbent. This indicates that coating of the adsorbent with GT only slightly reduces the negative charge of it. The DLS results indicated that the hydrodynamic sizes of the adsorbents are increased to 191 and 253  nm, respectively. It also showed some swelling of the SiO2 and TG shells in solu- tion that confirms a biocompatible coating for the incor- poration of the drug.24 3. 2. Effect of pH on Adsorption One of the important parameters in analyzing ad- sorption systems is pH, that influences both the chem- istry of the sample and the adsorbent binding sites.25 In the present study, the efficacy of pH on the adsorption or loading the drug was investigated in a range of pH = 2–11 with an primary drug concentration of 20 mg/L and us- ing 50 mg of the adsorbent in a mixing time of 30 min (Figure 5). As shown in the figure, by increasing pH from 2 to 7, the adsorption efficiency increases, so that at pH 7, the highest loading is obtained. At higher pH values, a moderate decrease in the efficiency is observed. Alteration of pH can change the surface charge of magnetic NPs.26 More negative surface charges are expected at a higher pH that is more suitable for the extraction of metformin. This Figure 4. SEM images of Fe3O4 (a), Fe3O4@SiO2 (b) and Fe3O4@SiO2@GT (c) MNPs. Figure 5. Effect of pH on the adsorption of metformin on the Fe3O4@SiO2@TG NPs. Sample volume, 5 mL; adsorbent mass, 50 mg; drug concentration, 20 mg/L; mixing time, 30 min; tempera- ture, 25 °C. 718 Acta Chim. Slov. 2022, 69, 714–721 Farajian and Hashemi: Superparamagnetic Tragacanth Coated Fe3O4@SiO2 ... is supported by the increase in the adsorption efficiency up to pH 7 in Figure 5. 3. 3. Optimization of the Adsorption Conditions A central composite design (CCD) method was used for the optimization of the loading of metformin on the adsorbent. CCD is one of the most usual multifunctional optimization techniques. In this process, two low and high levels are defined for each factor with the addition of at least one center point and some axial or star points. With this pattern, the approximation of both linear and quad- ratic effects are possible.27 For a reasonable estimation of the experimental error, replicate analyses are performed on the center points.28 Four factors of pH, temperature (T), volume of sam- ple (Vs) and contact time (Time) were studied and op- timized by the CCD model. The five levels designed for each factor is shown in Table 1. The levels of the factors in 31 planned experiments by the model and the received response values for each experiment is shown in Table 2. Optimization of the studied factors were accomplished using a response surface model. Some three-dimensional response plots are shown in Figure 6 to demonstrate how the response variable changes with variation of a pair of factors while all other factors stay constant. As shown in Figure 6a, at a high pH, an increase in adsorption is observed with time, while at a lower pH, such effect is not considerable which indicates the rap- id adsorption of metformin on the adsorbent. Figure 6b demonstrates the interaction between pH and temperature (T); at a low temperature, adsorption is decreased by in- creasing the pH but at a higher temperature, it is slightly increased. In Figure 6c it is shown that at a low sample volume, adsorption efficiency is increased by time, while an opposite effect is observed at a higher sample volume. The same or even more remarkable effect is detected for the dual effect of pH and sample volume (not shown in the figure). Figure 6. Three-dimensional surface plots in CCD optimization procedure. (a) Effects of pH and contact time, (b) Effects of pH and temperature (T), (c) Effects of contact time and volume of sample (Vs). Table 3 displays the results of the data analysis by the CCD model for the discrete and combined effects and second order interactions of the studied variables. Based on the t-test results, the most considerable variable is the sample volume with a large negative effect on the adsorp- tion efficiency. The other factors are statistically negligible with the order of pH > T > Time. The squared terms of the factors are also negligible but the pH × T and Time × T interactions may be considered notable at a 90% confi- dence level. The anticipated optimized conditions suggested by the model for the whole data were as follows: pH = 7, tem- perature = 25 °C, volume of sample = 1.0 mL, and contact time = 20 min. By execution of 6 replicated analyses in the optimum conditions, an adsorption efficiency of 83.27(± 6.45) % was obtained for metformin. Table 2. Conditions of the performed tests and their related adsorp- tion efficiency attained by the experiments in the CCD optimiza- tion. Run pH Vs Time T Adsorption order (mL) (min) (°C) (%) 1 10 3 50 30 45.8 2 8 7 50 30 44.9 3 9 9 40 35 31.7 4 9 5 40 35 44.6 5 9 5 40 35 55.1 6 9 5 60 35 44.5 7 8 3 30 30 75.6 8 8 3 50 30 68.9 9 8 3 30 40 60.0 10 9 5 40 25 44.1 11 9 5 40 35 44.9 12 10 3 50 40 61.4 13 11 5 40 35 57.3 14 8 7 30 30 47.9 15 10 3 30 30 63.8 16 10 3 30 40 63.8 17 9 1 40 35 79.0 18 10 7 30 40 36.4 19 10 7 30 30 41.8 20 9 5 40 35 51.5 21 9 5 40 35 59.2 22 9 5 40 35 48.3 23 9 5 40 35 56.6 24 8 7 30 40 36.0 25 8 7 50 40 38.0 26 7 5 40 35 31.7 27 10 7 50 40 61.3 28 8 3 50 40 67.0 29 9 5 40 45 58.9 30 10 7 50 30 44.7 31 9 5 20 35 44.6 719Acta Chim. Slov. 2022, 69, 714–721 Farajian and Hashemi: Superparamagnetic Tragacanth Coated Fe3O4@SiO2 ... Table 3. Effects of the factors, coded coefficients (Coef), standard errors (SE) of the coefficients, t-values and p-values of the variables obtained by the CCD model. Term Effect Coef SE Coef T-Value P-Value Constant 51.46 2.89 17.81 0.000 pH 2.66 1.33 1.56 0.85 0.407 Time 0.54 0.27 1.56 0.17 0.864 T 1.68 0.84 1.56 0.54 0.599 Vs –20.82 –10.41 1.56 –6.67 0.000 pH×pH –1.94 –0.97 1.43 –0.68 0.507 Time×Time –1.92 –0.96 1.43 –0.67 0.512 T×T 1.56 0.78 1.43 0.54 0.594 Vs×Vs 3.48 1.74 1.43 1.22 0.241 pH×Time 1.01 0.51 1.91 0.26 0.795 pH×T 7.89 3.94 1.91 2.06 0.056 pH×Vs 6.76 3.38 1.91 1.77 0.096 Time×T 7.04 3.52 1.91 1.84 0.084 Time×Vs 5.86 2.93 1.91 1.53 0.145 T×Vs –0.71 –0.36 1.91 –0.19 0.854 3. 4. Adsorption Capacity The adsorption capacity of the Fe3O4@SiO2@TG adsorbent or its maximum metformin loading was asso- ciated with the primary concentration of the drug in the sample. The capacity was calculated by suspending variant amounts of the adsorbent in a buffered solution of the an- alyte, under the optimum conditions. The effect of primary concentration of metformin on the adsorption capacity is shown in Figure 7. The adsorption capacity of the nano- composite increased with increasing the primary concen- tration of metformin. The maximum capacity obtained for 200 mg/L of metformin was 19.6 mg/g. Figure 7. The adsorption capacity of NPs as a function of the initial concentration of metformin under the optimized conditions; pH, 7; T, 25 °C; t, 20 min; sample volume, 1.0 mL; adsorbent mass, 40 mg; number of replicates, 3. 3. 5. Study of the Adsorption Isotherms The mechanism of interaction of metformin with the adsorbent was examined by studying the adsorption isotherms in batch experiments. For the analysis of the ex- perimental data, two adsorption models of Langmuir and Freundlich were used. The Langmuir adsorption isotherm relates the monolayer adsorption of a species onto the ad- sorbent superficial with some well-defined sites. Accord- ing to this model, the plot of Ce/q, in which Ce is the equi- librium concentration of the analyte in solution and q is the balanced adsorption capacity of the sorbent, versus Ce should be linear. The Freundlich isotherm is not restricted to the formation of a monolayer and is related to the re- versible adsorption. Accordingly, a linear plot is attained for plotting logq versus logCe.29 Figure 8a and 8b are the corresponding plots of the Langmuir and Freundlich models, respectively. Regard- ing R2 as an indication of the desirable fit of experimental data, the Langmuir adsorption is more appropriate at low- er than 5.8 mg/L Ce values suggesting the monolayer ad- sorption of metformin at low metformin concentrations. At higher Ce values, however, the Freundlich adsorption, Figure 8. a The Langmuir adsorption isotherm and b The Freun- dlich adsorption isotherm for the results shown in Figure 7. (b) (a) 720 Acta Chim. Slov. 2022, 69, 714–721 Farajian and Hashemi: Superparamagnetic Tragacanth Coated Fe3O4@SiO2 ... with an R2 value of 0.9772, is more appropriate than the Langmuir adsorption indicating the possibility of multi- layer adsorption mechanism in this region. The values of b (Langmuir constant), qm (maximum one-layer adsorption capacity), kF and n (Freundlich constants) were computed to be 1.36 L/mg, 1.81 mg/g, 0.68, and 1.91, respectively.29 The Freundlich constant n is higher than one, representing the appropriate conditions for the adsorption.26 3. 6. Evaluation of Drug Release The influence of contact time on the desorption or the release rate of metformin from the prepared adsorbent was studied in a range from 5 to 50 min. Metformin was first loaded on the adsorbent at the optimized conditions (pH 7, volume of sample 1.0 mL, T 25 C, and contact time 20 min). The desorption was accomplished using phos- phate buffered saline at three different pH values of 1.6, 3.0, and 7.3. The low pH values were selected as the region with minimum adsorption of metformin (see Figure 5). The release at pH 7.3 was also studied because this is the pH of the human body. The corresponding results are shown in Figure 9. The maximum drug desorption or delivery was obtained with- in 10 to 30 min by changing the pH from 1.6 to 3.0 and a maximum delivery of 85% was obtained at pH 3.0. It is also seen that at the body pH of 7.3 some 38% delivery is observed which may be useful when the drug is used for intravenous injections. Figure 9. Metformin desorption (delivery) from the loaded Fe3O4@ SiO2@TG NPs in pH 1.6, 3.0 and 7.3. Number of replicates: 3. 4. Conclusions Coating of Fe3O4@SiO2 magnetic NPs by TG as a natural hydrogel was successfully carried out using a sim- ple modifying process. Characterization of the prepared NPs by FT-IR, EDX and SEM corroborated the structure and the relatively uniform sizes of the prepared NPs. The results demonstrated that the synthesized Fe3O4@SiO2@ TG nanocomposite is a suitable carrier for the loading and releasing of metformin, as it is non-toxic, highly porous, low cost, and biocompatible. The modified nanocomposite established a high ef- ficiency for metformin adsorption with a loading capacity up to 19.6 mg/g in a short time. The adsorption on the pre- pared NPs fitted into Langmuir isotherm at low concen- trations of metformin while it was best fitted into Freun- dlich isotherm in a higher concentration area. Maximum desorption of the medicine happened in pH of 1.6 to 3.0 within 10 to 30 min. 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Villar, L. A. Escaleira, Talanta 2008, 76, 965–977. DOI:10.1016/j.talanta.2008.05.019 28. F. N. Serenjeh, P. Hashemi, M. Safdarian, Z. Kheirollahi, Jour- nal of the Iranian Chemical Society 2014, 11, 733–739. DOI:10.1007/s13738-013-0346-x 29. F. G. Adivi, P. Hashemi, A. D. Tehrani, Polymer Bulletin 2019, 76, 1239–1256. DOI:10.1007/s00289-018-2418-7 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Pripravili smo nov superparamagnetni nanokompozitni material iz Fe3O4@SiO2, prevlečen s tragakantovim gumijem (TG) kot naravnim proizvodom. Pridobljene SiO2@Fe3O4@TG nanodelce smo okarakterizirali z infrardečo spektroskopi- jo s Fourierjevo transformacijo, energijsko-disperzno rentgensko žarkovno analizo, vrstično elektronsko mikroskopijo in dinamično analizo svetlobnega sipanja. Magnetni nanokompozitni material smo uporabili za vezavo in sproščanje metformina, oralne učinkovine proti diabetesu. Pogoje za vezavo učinkovine smo optimizirali s pomočjo metode faktor- skega načrta. Največjo vezavno učinkovitost sorbenta za metformin smo dobili pri pH 7, maksimalno in-vitro sproščanje v salinem mediju s fosfatnim pufrom pa pri pH 1,6. Vezavna kapaciteta sorbenta je bila odvisna od začetne koncentracije metformina in je dosegla 19,6 mg/g v raztopini s koncentracijo 200 mg/L. Študij adsorpcijskih izoterm za učinkovino je pokazal najboljše prileganje Langmuirjevi izotermi pri nizkih koncentracijah in Freundlichovi izotermi pri visokih kon- centracijah metformina. Rezultati dokazujejo, da je pridobljeni Fe3O4@SiO2@TG adsorbent, ki je nestrupen in cenen, povsem primeren za uporabo pri dostavi zdravilnih učinkovin. 722 Acta Chim. Slov. 2022, 69, 722–733 Ivanova et al.: Investigation of Biological and Prooxidant Activity of Zinc ... DOI: 10.17344/acsi.2021.7337 Scientific paper Investigation of Biological and Prooxidant Activity of Zinc Oxide Nanoclusters and Nanoparticles Iliana A. Ivanova,1 Elitsa L. Pavlova,2 Aneliya S. Kostadinova,3 Radostina D. Toshkovska,1,4 Lyubomira D. Yocheva,5 Kh El-Sayed,6,7 Mohamed A. Hassan,6,7 Heba El-Sayed El-Zorkany6,7 and Hisham A. Elshoky6,7,8* 1 Dept. Microbiology, Faculty of Biology, Sofia University Saint Kliment Ohridski, 8 Dragan Tsankov Blvd, 1164 Sofia, Bulgaria 2 Faculty of Physics, Sofia University “St. Kliment Ohridski”, 5 James Bourchier Blvd., 1164 Sofia, Bulgaria 3 Institute of biophysics and biomedical engineering, Bulgarian academy of science, Akad. Georgi Bonchev 21str, Sofia 1113, Bulgaria 4 Institute of Organic Chemistry with Center of Phytochemistry, 9 Acad. G. Bonchev Str, Sofia 5 Faculty of Medicine, 1 Kozyak Str, 1407, Sofia, Bulgaria 6 Nanotechnology and Advanced Materials Central Lab, Agricultural Research Center, Giza, Egypt 7 Regional Center for Food and Feed, Agricultural Research Center, Giza, Egypt. 8 Tumor Biology Research Program, Department of Research, Children’s Cancer Hospital Egypt 57357, P.O Box 11441, 1 Seket Al-Emam Street, Cairo, Egypt * Corresponding author: E-mail: heshamalshoky@sci.cu.edu.eg; heshamalshoky@gmail.com Received: 12-13-2021 Abstract Zinc oxide (ZnO) nanomaterials offer some promising antibacterial effects. In this study, a new form of ZnO is synthe- sized, named ZnO nanocluster bars (NCs). Herein, ZnO NCs, ZnO nanoparticles (NPs), ZnO coated with silica (ZnO- SiOA, ZnO-SiOB), and SiO2 NPs were prepared, characterized, and their antimicrobial and prooxidant activity were tested. The prooxidant activity of all nanomaterials was studied according to free-radical oxidation reactions (pH 7.4 and pH 8.5) in chemiluminescent model systems. Each form of new synthesized ZnO nanomaterials exhibited a unique behavior that varied from mild to strong prooxidant properties in the Fenton`s system. ZnO NPs and ZnO NCs showed strong antibacterial effects, ZnO-SiOA NPs did not show any antibacterial activity representing biocompatibility. All tested NMs also underwent oxidation by H2O2. ZnO NCs and ZnO NPs exhibited strong oxidation at pH 8.5 in the O2–. generating system. While, SiO2, ZnO-SiOA and ZnO-SiOB possessed pronounced 60–80% antioxidant effects, SiO2 NPs acted as a definitive prooxidant which was not observed in other tests. ZnO NCs are strongly oxidized, assuming that ZnO NCs provide a slower release of ZnO, which leads to having a stronger effect on bacterial strains. Thus, ZnO NCs are an important antibacterial agent that could be an emergent replacement of traditional antibiotics. Keywords: ZnO; nanoclusters; nanocomposites; antimicrobial activity; ROS; chemiluminescence. 1. Introduction Multi-drug resistant (MDR) bacteria have become an important problem because of the extensive use of anti- biotics, which are often applied without proper medical in- dications. The inappropriate selection and switch between antimicrobial alternates cause “selection pressure.” All of this causes MDR bacteria. Consequently, while many studies have focused on identifying new effective bacte- ricidal materials, new alternative strategies for combating bacterial resistance remain under investigation.1–4 Nanotechnology introduces a special solution to the MDR bacteria. Several nanomaterials (NMs) have been used in antibacterial treatments, antibacterial vaccines, 723Acta Chim. Slov. 2022, 69, 722–733 Ivanova et al.: Investigation of Biological and Prooxidant Activity of Zinc ... antibiotic delivery carriers, and antibacterial coatings for implantable devices and medicinal materials to prevent infection and promote infection wound healing to help control bacterial infections. They are applied in microbial diagnostic systems too.5 ZnO is known for its anti-inflammatory, astringent, and soothing effects.6,7 Therefore, ZnO has been used in cosmetics, including sunscreens, toothpaste, and sham- poos, after the nineteenth century.8 Furthermore, the US Food and Drug Administration (FDA) has classified ZnO as “Generally Recognized as Safe” (GRAS) because of its non-toxic properties. Zn is used as a food additive too.9,10 Recently, ZnO NMs have attracted considerable attention because of their antimicrobial activity. ZnO NMs’ superi- ority in fighting microbial resistance is attributed to their nonspecific activity, small particle size, high surface area, low cost, and efficiency against various bacteria with low toxicity to human cells.11 Unfortunately, we have limited knowledge of NMs’ mechanisms of action against bacteria. The suggested mechanisms include oxidative stress induction, metal ion release, and non-oxidative mechanisms.5,12,13 The bacterial destruction by ZnO NMs is believed to follow two path- ways: binding to cell membranes, consequently disrupting their potential and integrity and inducing generation of reactive oxygen species (ROS).5 ZnO NMs are mutagens albeit weak ones.14 Many studies attempted to investigate and use ZnO nanoparticles in different applications.15,16 Smaller ZnO nanoparticles usually show higher cellular inhibition ac- tivity. Furthermore, surface modification of ZnO NMs can affect their properties that may change or improve their antimicrobial activity.17 While the generation of ROS is important for an- tibacterial activity of ZnO NMs, it is necessary to inves- tigate the kinetics of free radical generation, affected by ZnO NMs.18,19 The chemiluminescent assay is a conven- ient method for such studies. It can be used to monitor the dynamics of free radical reactions and to determine their prooxidant/antioxidant activity. The chemiluminescent technique is advantageous because of its accuracy, sensi- tivity, high speed, and relatively low cost; moreover, it re- quires a small sample volume. Many physical and chemical probes, such as luminol and lucigenin, can be used to en- hance chemiluminescence. Тhese reactions are accompa- nied by emission in the range of 480–580 nm; hence, they can be harnessed to assess the quantum yield of generated free radicals.20–22 We synthesized a completely new form of ZnO na- noaggregates in this study called ZnO nanocluster bars (ZnO NCs). Their prooxidant and antimicrobial effects were evaluated compared with different forms of ZnO NMs as spherical ZnO NPs with/without silica coating. Furthermore, the prooxidant activity of all NMs was ex- amined as free radical oxidation reactions at pH 7.4 and pH 8.5 in chemiluminescent model systems. 2. Experimental 2. 1. Materials The materials used in this study were purchased with high purity; zinc acetate dihydrate (99.5%, Merck, Germany), 2-propanol (99.9%, Sigma-Aldrich), sodium hydroxide (99.5%, Sigma-Aldrich), tetraethyl orthosilicate (TEOS, 98%, Sigma-Aldrich), iron sulphate (P. A.) (Merck, Germany), ammonia solution (25%, Sigma-Aldrich), phenazine methosulfate (PhMS) (N-methyldibenzopyra- zine methyl sulfate salt) (P. A.) (Merck, Germany), hy- drogen peroxide (30%) (Boron, Bulgaria), disodium hy- drogen phosphate (P. A.) (Boron, Bulgaria), citric acid (P. A.) (Boron, Bulgaria), lucigenin (bis-N-methylacridinium nitrate) (P. A.) (Aldrich, USA), β-nicotinamide adenine dinucleotide, reduced form (P. A.) (NАD.Н, Boehringer, Germany) and dimethyl sulfoxide (P. A.) (DMSO, Aldrich, USA). All chemicals were used as-purchased without fur- ther purification. 2. 2. Preparation of ZnO Nanocluster Bars (ZnO NCs) A solvothermal process prepared the ZnO nanoclus- ter bars as follows; 1 g of zinc acetate was ultrasonically dispersed in 80 mL of 2-propanol in a 150 mL beaker for 30 min at room temperature. Then, 2 g of oxalic acid was added followed by another 30 min of ultrasonication. To complete the hydrothermal preparation process, the mix- ture was poured from the beaker in a Teflon-based stain- less steel autoclave and placed in the oven for 24 h at 180 °C. Subsequently, the prepared NPs were washed three times with DI-H2O and ethanol by centrifugation (4500 rpm at 10 °C for 30 min.), the same step was repeated for DI-H2O and ethanol three times until the whole quantity is washed. The precipitate was dried in an oven at 180 °C for 8 h. Then, the powder was calcined in a muffle oven at 400 °C for 2 h. 2. 3. Preparation of ZnO Nanoparticles (ZnO NPs) ZnO nanoparticles were obtained using a mod- ified method as described by G. Simonelli et. al.23–25 Briefly, a 46.5 mM of zinc acetate dihydrate was pre- pared by dissolving 2.195 g in 20 mL of 2-propanol at 50 °C and then the volume was increased to 210 mL by 2-propanol. Note that 0.8 g of sodium hydroxide in 40 mL solution (35 mL 2-propanol + 5 mL DI-H2O) was added under continuous stirring in an ice bath. Then, the solution was stirred at 60 °C for 2 h, and the tem- perature was measured and followed up to ensure that it did not rise over that because this influenced the parti- cle sizes. Subsequently, the preparation vessel was kept stable at room temperature for three days for additional aging. Then, the sample was repeatedly centrifuged at 724 Acta Chim. Slov. 2022, 69, 722–733 Ivanova et al.: Investigation of Biological and Prooxidant Activity of Zinc ... 7000 rpm/15 min till all other chemical residuals were completely removed. The precipitate was dried in the oven at 180 °C for 8 h. Then, the powder was calcined in a muffle oven at 400 °C for 2 h. Characterization meas- urements were performed using DLS, zeta potential, XRD and TEM. 2. 4. Preparation of Silica Capped ZnO NPs (ZnO-SiO NPsA, B) ZnO NPs were dispersed in water as per Bartczak’s protocol with modification.18,26 In an ultrasonic bath, 0.5 g of ZnO NPs in 100 mL of 2-propanol was sonicated for 15 min at room temperature. The pH of the ZnO NP solution was increased to 10 by the dropwise addition of 1 M ammonium hydroxide solution and monitoring the change using a pH meter. Next, 100 mL of 2% TEOS in DI-H2O was added, and the suspension was sonicated for 1 h at room temperature. NPs then reacted overnight at 60 °C with stirring before purification from excess by- products and organic solvent residues by triple centrif- ugation (13000 rpm, 15 min at room temperature) with the same approach mentioned for ZnO NC preparation. (ZnO-SiOA) NPs were then dried in a hot air oven at 80 °C overnight. For the second form of SiO2 capped ZnO NPs (ZnO-SiOB), the same procedure was performed but without pH adjustment of the ZnO solution. 2. 5. Preparation of Silica Nanoparticles (SiO2 NPs) In brief, 300 mL of DI-H2O was added to 300 mL ethanol and stirred for 10 min at room temperature. Then, 45 mL of TEOS were added and sonicated for 20 min. The dropwise additions of 1 M ammonium hydroxide solution were made until pH 10 was reached, and the reaction was stirred overnight. Next, the SiO2 NPs were washed well with DI-H2O using centrifugation at 10000 rpm for 15 min, using the same procedure as before, until ammonia odor disappears and pH becomes neutral. The precipitate was dried in the oven at 45 °C overnight; finally, the yield was ground to obtain a fine silica powder.26 2. 6. Characterization of the Prepared Nanoparticles A transmission electron microscope (TEM, Tecnai G20, FEI, Netherlands) was used for imaging the nano- materials that were prepared. The bright field imaging was employed at an accelerating voltage of 200 kV using a lanthanum hexaboride (LaB6) electron source gun, and the Eagle CCD camera was used to acquire and collect transmitted electron images with an image resolution (4K x 4K). Before imaging, the aqueous suspensions of pre- pared nanoparticles were prepared in an ultrasonicator (SB-120DTN, Taiwan) for 10 min, and then particles were deposited from a dilute aqueous suspension onto a 200 mesh-carbon coated copper grid placed on filter paper and left for drying at room temperature as a common method for preparing TEM samples. However, powder X-ray diffraction (XRD − X’Pert PRO, PANalytical, The Netherlands) was used to reveal the crystal structure of the prepared NMs. XRD operated at 45 kV and 30 mA using X-ray source “Cu Kα radiation” (λ = 1.5404 Å). The step time and step sizes were 0.5 s/step and 0.02 degree/step, respectively, in the range of 4° – 80° (2θ). Peaks matching and analysis were performed using high score plus software. Particle size distribution analysis and zeta potentials of the prepared materials were measured using Zetasizer Nano S, Malvern Instruments, UK, to evaluate hydrody- namic size and surface charge. These measurements were performed in aqueous solutions after NMs were dispersed in deionized water using an ultrasonicator for 15 minutes to obtain stable suspensions. A portion of suspension was transferred in 10 mm × 10 mm cuvette (DTS1070) to measure particle size and zeta potential. 2. 7. Microorganisms The antimicrobial activity was tested against Gram-positive Bacillus cereus NBIMCC1095, Staphylococ- cus epidermidis ATCC 12228 bacteria, and Gram-negative Escherichia coli BL21DE3 bacteria. All bacteria purchased from National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC, Bulgaria) were grown in nutrient broth (NB Conda, Spain) at 37 °C and 180 rpm (shaker Rotomax, incubator ED053, Germany) for 24 h with two sub-cultivations. Microbial density of cultures in an ex- ponential phase of 0.5–0.6 was determined according to McFarland. 2. 8. Antimicrobial Activity Antibacterial influence of each type of prepared NMs was investigated using spot-diffusion in agar. Briefly, 100 µL of each bacterial suspension was homogeneously spread on nutrient agar plates. 10 µL drops of investigated material were put on inoculated solid medium. Plates were left for 2 h at 4–6 °C to afford diffusion of dispersions and cultured for 24 h, and then 48 h at 37 °C. The diameters of sterile zones were measured in mm.27,28 2. 9. Chemiluminescent Assay Тhe chemiluminescent method was applied to study effect of NMs on the kinetics of free-radical oxidation re- actions using activated chemiluminescence and the probe lucigenin.29 The higher acidity of medium favors radical for- mation reactions and enables the achievement of reliable differences. Two different pH systems were investigated – 725Acta Chim. Slov. 2022, 69, 722–733 Ivanova et al.: Investigation of Biological and Prooxidant Activity of Zinc ... Figure 1: TEM images of (A) ZnO-NPs, (B and B´) different magnifications of ZnO nanocluster bars, (C) ZnO-SiOA, (D) ZnO-SiOB, and (E) SiO2 NPs, respectively. 726 Acta Chim. Slov. 2022, 69, 722–733 Ivanova et al.: Investigation of Biological and Prooxidant Activity of Zinc ... pH 7.4 and pH 8.5, physiological and alkaline. Three ex vivo model systems were implemented in buffers and here described briefly.22 First system, generating hydroxyl rad- icals (.OH) system, it contains 0.2 mol of sodium hydro- gen phosphate buffer, with the appropriate pH, Fenton’s reagent [FeSO4 (5.10−4 mol) –H2O2 (1.5%), lucigenin (10−4 mol)] and NMs. The second system contains the oxidant hydrogen peroxide (H2O2): 0.2 mol sodium hydrogen phosphate buffer, with appropriate pH, H2O2 (1.5%), the chemilumi- nescent probe lucigenin (10–4 mol), and NMs. The third system is for the generation of superoxide radicals (O2–.) through the reaction NAD.H-PhMS. It contains 0.2 mol of sodium hydrogen phosphate buffer with the specific pH, NAD.H (10−4 mol), phenazine-metasulfate (10−6 mol), lu- cigenin (10−4 mol) and NMs. The control samples do not contain any NMs. The reactions are monitored for 3 min- utes every 3 seconds; the maximum peak for each curve was obtained. 2. 10. Statistics All experiments were performed in triple reproduc- ible measurements; statistical analysis was implement- ed using Origin 8.5 and Microsoft Office Excel 2010. To measure the strength of the relationship between tested variables, correlation coefficients (r) between the sensitivi- ty of the selected bacterial strains toward the antimicrobial effect of ZnO NPs, ZnO NCs, ZnO-SiOA, and ZnO-SiOB NMs tested by spot diffusion and chemiluminescent assays are calculated. 3. Results and Discussion 3. 1. Characterization Transmission electron microscopy (TEM) imaging Fig. 1 shows the TEM micrographs of ZnO NMs and SiO2 NPs. TEM images (Fig. 1A, B, and B´) demonstrated that ZnO NPs and ZnO NCs agglomerated to certain ex- tent. The average diameter size, measured using TEM-TIA software, of the prepared ZnO-NPs (Fig. 1A, B) and SiO2 NPs (Fig. 1E) was between 22.9–38.1 and 19–25 nm. The ZnO NCs agglomerated in NCs with a length of 2–3 µm and a width between 200 and 350 nm. The image of ZnO NCs comprised small ZnO NPs with an average particle size of 14.3–21.5 nm. However, ZnO-SiO2 NPs demon- strated two different morphological forms. Fig. 1C shows a homogeneous sphere capped ZnO- SiOA NPs with an average diameter of ~20 ± 3 nm, and the silica layer sur- rounding the ZnO NPs with an estimated layer thickness of 4 ± 0.5 nm. However, ZnO- SiOB show in Fig. 1D with less homogeneity in particle size and additional aggrega- tion than ZnO- SiOA with an average particle size of 13.4 ± 3 nm for ZnO cores and 3.5 ± 0.7 nm for SiO2 cap. Fig. 1E shows well-dispersed and homogeneous spherical SiO2 NPs with an average 38 ± 3 nm diameter. The change in the shape depends on the method of preparation, which causes the SiO2 NPs to appear to be in a good and homo- geneous shape. X-ray diffraction (XRD) analysis Fig. 2(a–e) shows the XRD patterns for ZnO NPs-, ZnO NCs-, and SiO2-coated ZnO and SiO2. All the dif- Figure 2: XRD patterns of (a) ZnO NPs, (b) ZnO NC bars, (c) ZnO-SiO2A (d) ZnO-SiO2B, and (e) SiO2 NPs, respectively. 727Acta Chim. Slov. 2022, 69, 722–733 Ivanova et al.: Investigation of Biological and Prooxidant Activity of Zinc ... fraction peaks of ZnO-containing materials are fitted to the hexagonal (wurtzite) ZnO structure (JCPDS no. 01– 080–3002) with lattice parameters (a = b = 3.25 A, c = 5.21 A), and a space group P63 mc. The primary peaks of ZnO appeared at diffraction angles of 2θ: 31.8°, 34.4°, 36.2°, 47.5°, 56.6°, 62.8°, and 67.9° for ZnO NPs and ZnO NCs. While the SiO2-coated ZnO NPs present a combina- tion of SiO2 peaks at 2θ = 23.2° and ZnO diffraction peaks with a slight peak shifting (Fig. 2c). This may indicate a complete formation of SiO2-coated ZnO nanostructure. Furthermore, Figure 2d shows the diffraction peaks of pure SiO2 with a broad distinguished peak at 23.2°, which is well-matched with the JCPDS card (no. 01–077–9207). All XRD patterns show highly pure materials with no con- tamination. Particles size distribution analysis and zeta-potential measurements Table 1 shows the particle size distributions and zeta potential measurements for ZnO NPs, ZnO NCs, SiO2-coated ZnO (A&B), and SiO2 NPs. The average hy- drodynamic diameter of ZnO, SiO2-coated ZnO (A&B), and SiO2 NPs is 46.0 ± 4.9, 49.3 ± 8.4, 48.5 ± 6.7, and 68.7 ± 9.4 nm, respectively, which demonstrates a homogene- ous size distribution. Increasing hydrodynamic diameters for SiO2-coated ZnO (A&B) rather than ZnO NPs appear from the shell layer of SiO2 on the core particles of ZnO. However, the results shown from ZnO NCs are 1968 ± 237 nm because of intensive agglomerations of ZnO nanopar- ticles suspended in an aqueous solution. However, the zeta potential measurements show a negative charge on the prepared NMs except SiO2-coated ZnO (B), which was prepared without adjusting the pH. The pH of the prepara- tion medium plays an important role in the surface charge and zeta potential results. As the pH increased, the surface tendency of the prepared materials to carry more negative charges increased. The antimicrobial activity The antimicrobial effect of NMs was determined us- ing the spot diffusion test. Most of the nanoparticles pos- sess a contact killing effect that could not be demonstrat- Table 1: The particles size distributions and zeta potential measurements of ZnO NPs, ZnO NCs, and SiO2-coated ZnO (A&B) and SiO2 ZnO ZnO cluster ZnO-SiOA ZnO-SiOB SiO2 Particle size diameter (nm) 46.0 ± 4.9 1968 ± 237 49.3 ± 8.4 48.5 ± 6.7 68.7 ± 9.4 Zeta-Potential (mV) −16.5 ± 5.4 −11.37 ± 2.7 −21.78 ± 6.3 19.3 ± 4.2 −27.2 Table 2. Inhibition zones (mm) of the tested bacteria Nanoparticles Nanoparticles Tested microorganisms* Inhibition zones (mm) Concentration E. coli B. cereus S. epidermidis (mg/mL) (BL21DE) (NBIMCC1095) (ATCC 12228) ZnO NPs 3 10 ± 0.5 6 ± 0.5 13 ± 0.5 1.5 10 ± 0.5 0 10 ± 0.5 0.5 0 0 0 0.25 0 0 0 ZnO NCs 3 10 ± 0.5 9.5 ± 0.5 15 ± 0.5 1.5 0 8 ± 0.5 8 ± 0.5 0.5 0 5 ± 0.5 0 0.25 0 4 ± 0.5 0 ZnO-SiOA 3 0 0 0 1.5 0 0 0 0.5 0 0 0 0.25 0 0 0 ZnO-SiOB 3 10 ± 0.5 10 ± 0.5 15 ± 0.5 1.5 0 8 ± 0.5 7 ± 0.5 0.5 0 0 0 0.25 0 0 0 SiO2 3 0 0 0 1.5 0 0 0 0.5 0 0 0 0.25 0 0 0 728 Acta Chim. Slov. 2022, 69, 722–733 Ivanova et al.: Investigation of Biological and Prooxidant Activity of Zinc ... Figure 3. Chemiluminescence induced in the Fenton`s system (system I: A, B), by H2O2 (system II: C, D) and O2– radicals (system III: E, F) in sec- onds, at pH 8.5 and 7.4 in the presence/absence of NMs. 729Acta Chim. Slov. 2022, 69, 722–733 Ivanova et al.: Investigation of Biological and Prooxidant Activity of Zinc ... ed if nanomaterials are dropped on paper disks or in agar wells, because of impossible agar diffusion or diminishing of the nanoparticle –bacteria interaction.27 From the results, it is obvious that the SiO2 NPs and ZnO-SiOA were completely safe at concentrations up to 3 mg/mL; however, all other tested materials showed bacte- ricidal effects at a higher concentration than 3 mg/mL. All tested bacteria show high sensitivity against ZnO NPs at 3 mg/mL, while only E.coli and S. epidermidis were inhibited at 1.5 mg/mL. While, lower concentrations of ZnO NPs were safe on all the tested bacteria. Furthermore, ZnO- SiOA was completely safe on bacteria at all concentrations; however, ZnO-SiOB showed higher toxicity compared to the naked silica particles. ZnO NCs have shown the larg- est sterile zones at the diffusion test and demonstrated the strongest antibacterial effect if used in concentrations of 3 mg/mL or less. As shown in Table 2, the most sensitive of all three tested bacteria were Bacillus cereus compared to E. coli and S. epidermidis. ZnO NMs, and many metal oxide nanoparticles, possess bactericidal properties because of the generation of ROS. The chemiluminescent method was used to trace the concentration and kinetics of ROS generation by de- termining the quantum yields of these reactions in the 480–580 nm range. Three chemiluminescent model sys- tems were applied. • System I The interaction between Fe2+ ions and H2O2 pro- duces highly reactive, short-living ·OH radicals. Generally, the resulting chemiluminescent emission is considerably higher than that from other mixtures. Fe2+ + H2O2 → Fe3+ + ·OH + −OH (1) Fe3+ + H2O2 → Fe2+ + ·OOH + H+ (2) At pH 8.5 the control chemiluminescence signal in this system reaches 17006 reference luminescent units (RLU) in the interaction between the reagents, the so- called fast flash, and usually decreases with time (Fig.3A). The sample containing SiO2 NPs follows this kinetics but with slightly lower values, representing the same levels and is not susceptible to oxidation by ROS. All other NMs exhibit mild to strong prooxidant properties. ZnO NPs intensify the luminescent signal seven times, ZnO-SiOA almost three times, ZnO-SiOB more than two times, and most pronounced oxidation is registered with ZnO NCs 18 times. All kinetics is smooth, with no obvious peaks (Fig. 3A). At physiological pH 7.4 (Fig. 3B), almost the same ef- fects are registered but at considerably lower levels; at this pH, ZnO-SiOA and ZnO-SiOB change places but maintain a mild prooxidant activity. Strong prooxidant activities ex- hibit the ZnO NCs almost four times and ZnO NPs and ZnO-SiOB NMs two times or less intensification of the signal. • System II In this system, hydrogen peroxide serves as both an oxidizing agent and a ROS. The results show that ZnO NCs are mostly oxidized at alkaline and neutral pH, respective- ly, and 30- and 5-times stronger signal than the controls. ZnO NPs exhibit almost 18 times (Fig. 3C) and about two times (Fig. 3D) stronger prooxidant activity compared to the control. At pH 8.5, ZnO-SiOB and ZnO-SiOA NPs ex- pressed mild prooxidant effects, less than three times com- pared to the control signal (Fig. 3C). At pH 7.4, ZnO-SiOA provokes three times higher oxidation than ZnO-SiOB and the control (Fig. 3D). SiO2 NPs demonstrate an extremely light prooxidant effect at both tested pH media. • System III The O2–. generation in this system is believed to fol- low the chemical scheme of Nishikimi et al.30, 31 PhMS + NAD.H + H+ → PhMS.H2 + NAD+ (3) PhMS.H2 + PhMS → 2PhMS.H. (4) PhMS.H. + O2 → PhMS + O2–. + H+ (5) At alkaline tested conditions, ZnO NCs exhibit the strongest oxidation as their signal is 24 times higher than the control. ZnO NPs demonstrate almost two times stronger prooxidant activity than the control (Fig. 3E). The other tested synthesized NMs exhibit obvious antioxidant effects against the generated O2−. radicals in the system 60 to 80% (Fig. 3E). The registered antioxidant activity is not Table 3. Correlation coefficients between the sensitivity of chosen bacterial strains toward the antimicrobial effect of ZnO, ZnO NCs, ZnO-SiOA, and ZnO-SiOB NMs tested by spot diffusion and chemiluminescent assays. Microorganism System 1 System 1 System 2 System 2 System 3 System 3 pH 8.5 pH 7.4 pH 8.5 pH 7.4 pH 8.5 pH 7.4 E. coli 0.717 0.814 0.895 0.145 0.954 0.969 BL21DE B. cereus 0.921 0.970 0.996 0.496 0.998 0.814 NBIMCC1095 S. epidermidis 0.797 0.879 0.943 0.266 0.984 0.932 ATCC 12228 730 Acta Chim. Slov. 2022, 69, 722–733 Ivanova et al.: Investigation of Biological and Prooxidant Activity of Zinc ... observed at pH 7.4 for all types of NPs. At physiological pH and provoked by the O2−. radicals, ZnO NPs exhibit the strongest prooxidant activity compared to all tested systems and conditions (Fig. 3F). ZnO NCs show 16 times stronger signal than the control, followed by SiO2 (almost seven times), ZnO-SiOB (more than five times), and ZnO- SiOA (almost two times) (Fig. 3F). It should be noticed that in this ROS-generating system (O2−.), SiO2 presents a de- finitive prooxidant activity, which is not observed in the other tested systems. Table 3 shows the correlation coefficients between the sensitivity of the chosen bacterial strains toward the antimicrobial effect of ZnO NPs, ZnO NCs, ZnO-SiOA, and ZnO-SiOB NPs tested by spot diffusion and chemi- luminescent assays (1 mg/mL). Systems 1 and 3 show a strong correlation between the two assays. System 2 in- troduces a weak correlation in the case of E. coli and S. epidermidis and moderate correlation in the case of B. ce- reus at pH 7.4 despite the same system showing a strong correlation at pH 8.5. This confirms that the luminescent assay can be successfully applied at pH 8.5 for evaluating antimicrobial activity using System 2. Nanosized ZnO’s internalization and mechanistic activity depend on their physicochemical properties such as shape, size, charge, and surface.32 Therefore, different shapes and sizes of ZnO NMs were synthesized by con- trolling the preparation conditions. Аlthough the growth mechanism of NMs was extensively studied, the actual mechanism remains unknown. The general mechanism is believed to depend on solvent and growth conditions. Generally, alcohol group is extremely important in con- tributing the unoccupied oxygen to Zn2+ to form ZnO.33 Then, small crystalline nuclei are formed by Ostwald rip- ening in a supersaturated reaction solution, followed by particle growth, and then large nanoparticles grow at the expense of small NPs.34 Moreover, by showing ZnO NCs’ morphology in our experiment, Figure 1, these NMs look similar to agglomer- ates of small spheres. The growth mechanism of this form of ZnO NMs could be subject to the oriented attachment, a recent non-classical theory of crystal growth based on the repeated merging of adjacent particles on lattice-matched crystal facets; this is supported by TEM imaging (Figure 1B, inset).34 Many research groups reported the prepara- tion of ZnO aggregates;16 however, the newly synthesized ZnO NCs in our experiment are well-packed clusters with high stability. XRD confirms the formation of hexagonal (wurtzite) structure of all synthesized ZnO NMs deter- mined by the JCPDS card no. 01-080-3002, and no other phases were observed. Based on our antibacterial test, it is clear that the SiO2 NPs and ZnO-SiOA were fully safe at concentrations up to 3 mg/mL, while all other investigated materials showed bactericidal effects at a higher concentration than 3 mg/mL. All tested bacterial E. coli, Bacillus cereus, and S. epidermidis showed high sensitivity toward ZnO NPs at 3 mg/mL. Both E. coli and S. epidermidis were inhibited at 1.5 mg/mL. Meanwhile, lower concentrations of ZnO NPs, however, were safe on all tested bacteria. It is worthy to notice that coating this nanomaterial with silica renders it completely safe for the bacteria at all concentrations. This could be attributed to the complete isolation of ZnO from the surrounding media by silica, which prevents Zn ions leakage from the particles, in addition to the safe action of silica on bacteria.35 On the contrary, ZnO-SiOB shows higher toxicity compared to the naked silica particles. This could be attributed to the incomplete shielding of ZnO by silica in this case, which afforded a chance for ZnO leakage from these particles. Moreover, by referring to TEM imag- es, ZnO-SiOB shows some aggregation that can increase the antibacterial action.28 However, ZnO NCs demonstrat- ed the strongest antibacterial effect if used in concentra- tions of 3 mg/mL and less. The mechanism that gives the advantage to ZnO NCs over ZnO NPs when the ZnO NC attaches to the cell membrane, it breaks down under the physiological conditions to its constituent of small spher- ical particles that duplicate and magnify the effect of ZnO NCs compared to one of the ZnO NPs. The most sensitive of all three tested bacteria were Bacillus cereus compared to E. coli and S. epidermidis, as shown in Table 2. Our results are consistent with other studies that have reported the bactericidal effect of ZnO NMs. A few proposed mechanisms are the penetration of the NMs that release Zn2+. Smaller NPs possibly penetrate cells, and hence they have a greater impact. Zn2+ would react with proteins, peptides, and amino acids, probably with phos- phates and carbonates too, which will suppress many im- portant cellular activities inside bacteria (active transport, metabolism, and enzyme activity), ultimately inducing the cell death.9 Others suggested that the antibacterial activity is not attributed to generated O2.− rather than H2O2, with elec- trons and H+. H2O2 penetrates the membrane of bacteria, damaging its content such as proteins, lipids, and amino acids, causing cell death.12 Moreover, we suggest that the outstanding antibacterial effect of ZnO NCs could be re- lated to the random orientation of its cluster bars. We be- lieve it is the same observation with the random-oriented ZnO nanoarrays (ROZN) outlined by Wang et al. who at- tributed the superior bactericidal effect of ROZN to cell membrane injury.36 Chemiluminescent assay results demonstrate the superiority of ZnO NCs over the rest of the tested NMs. One explanation could be that ZnO NCs are composed of small ZnO NPs, as shown in TEM images, which provide a slow release of ZnO for long periods, leading to a stronger effect on bacterial strains. In system I, the tested NPs are oxidized by the generated ROS in Fenton’s system (Fig.3) that could explain the observed anti-inflammatory and antibacterial properties of those materials in the living sys- tems. At physiological pH 7.4 (Fig.3B), almost the same ef- fects are registered but at much lower levels because of the 731Acta Chim. Slov. 2022, 69, 722–733 Ivanova et al.: Investigation of Biological and Prooxidant Activity of Zinc ... change of pH of the media to a lower value. The achieved results from system II are confirmative on the stability of the tested newly synthesized NMs against H2O2 as a typical strong oxidant, also generated in the living systems as part of their nonspecific inflammation reaction. In system III, ZnO NCs were susceptible to oxidation, followed by SiO2, ZnO-SiOB, and ZnO-SiOA. Note that, in this ROS-gener- ating system (O2–.), SiO2 presents a definitive prooxidant activity, unobserved in the other tested systems. A detailed correlation analysis was performed of the sensitivity of the selected bacterial strains toward the an- timicrobial effect of ZnO NPs, ZnO NCs, ZnO-SiOA, and ZnO-SiOB NPs, tested by the spot diffusion and chemi- luminescent assays (1 mg/mL; Table 3). System I and III show a full positive correlation between the two assays. This is confirmative on the assumptions that .OH, .OOH, and O2–. radicals are part of the antimicrobial mechanism of the tested ZnO and its derived materials. System II in- troduces H2O2 as a ROS and a strong oxidant. The corre- lation between system II and the spot-diffusion assay was moderate at pH 7.4; however, the correlation was strong at pH 8.5. This confirms that only reactions at pH 8.5 can be tested and followed to obtain reliable results on proox- idant, antimicrobial and bactericidal effects applying the chemiluminescent assay. The strength of the correlation coefficient follows the relationship level as perfect, strong, moderate, weak, and zero to the ± values of 1.0, 0.7–0.9, 0.4–0.6, 0.1–0.3, and 0.37 All achieved results are confirm- ative of the role of these ROS in the bactericidal effect in living systems. Although there are structural differences between Gram-positive and Gram-negative bacteria cell membranes, ZnO NMs show a strong effect on both types of bacteria, which depicts the broad spectrum of ZnO NMs effect.38 4. Conclusions In this study, the different forms of newly synthesized ZnO NMs were prepared and tested against Gram-neg- ative and Gram-positive bacteria. The agar diffusion test confirmed that ZnO NCs presented the best antimicrobial activity, while SiO2 and ZnO-SiOA NPs demonstrated no antibacterial activity. All NMs, except SiO2, exhibit mild to strong prooxidant properties in the Fenton’s system to generate ROS. ZnO NCs are a powerful oxidant. This could be explained by assuming that ZnO NCs are composed of small units of ZnO NPs that provide a slow release of ZnO for long periods, which leads to a stronger effect on bac- terial strains. SiO2 is unsusceptible to oxidation by ROS. The results achieved for both media demonstrate that all tested NMs are susceptible to oxidation by H2O2, a typical strong oxidant, also generated in the living systems as part of their nonspecific inflammation reaction. ZnO NCs and ZnO NPs exhibit strong oxidation in the alkaline tested conditions in system III. All other tested NMs (SiO2, ZnO-SiOA, and ZnO-SiOB) exhibit pronounced 60%−80% antioxidant effects on the generated O2−. radicals in the system. The registered antioxidant activity is not observed at pH 7.4 for any newly synthesized materials. ZnO shows the strongest prooxidant activity com- pared to all the tested systems. The prooxidant effect is observed for all other materials too. SiO2 presents a defin- itive prooxidant activity, which is not observed in other systems. The correlation analysis on the sensitivity of the chosen bacterial strains toward the antimicrobial effect of ZnO NPs, ZnO-SiOA, and ZnO NCs tested using the spot-diffusion and chemiluminescent assays is highly con- firmative on the role of these ROS (.OH, .OOH, H2O2 and O2−.) in the bactericidal effect in living systems. Thus, ZnO NCs are an important antibacterial agent that could be an emergent replacement of traditional antibiotics. Funding The Joint Research Project between The Institute of Biophysics and Biomedical Engineering, Bulgarian Acade- my of Sciences, Bulgaria and The Academy of Scientific Re- search and Technology (ASRT) and Nanotechnology and Advanced Materials Central Lab, Agricultural Research Centre, Egypt entitled “Biological activity of Nanocom- posites materials with potential medical and microbiology application”. Project “Clean Technologies for Sustainable Environment – Waters, Waste, Energy for Circular Econ- omy”, Ministry of Education and Science, Bulgaria, Con- tract Number: BG05M2OP001-1.002-0019. 5. References 1. B. Khameneh, R. Diab, K. Ghazvini and B. S. Fazly Bazzaz, Microb. Pathog. 2016, 95, 32–42. DOI:10.1016/j.micpath.2016.02.009 2. R. Y. Pelgrift an\d A. J. Friedman, Adv. Drug Delivery Rev. 2013, 65, 1803–1815. DOI:10.1016/j.addr.2013.07.011 3. N. Y. Lee, W. C. 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Povzetek Nanomateriali na osnovi cinkovega oksida (ZnO) nudijo nekaj obetavnih protibakterijskih učinkov. V okviru te študije je bila sintetizirana nova oblika ZnO, imenovana »ZnO palčke nanogrozdov« (angl. ZnO nanocluster bars, NC). Pri- pravljeni in okarakterizirani so bili ZnO NC, nanodelci ZnO (NP), ZnO, prevlečen s silicijevim dioksidom (ZnO-SiOA, ZnO-SiOB) in SiO2 nanodelci, pri čemer je bila testirana tudi njihova protimikrobna in prooksidantna aktivnost. Pro- oksidantno aktivnost vseh nanomaterialov je bila preučevana glede na reakcije oksidacije s prostimi radikali (pH 7,4 in pH 8,5) v kemiluminiscentnih modelnih sistemih. Vsaka oblika na novo sintetiziranih nanomaterialov ZnO je pokazala edinstveno obnašanje, ki je v Fentonovem sistemu zajemalo vse od blagih do močnih prooksidativnih lastnosti. ZnO NP in ZnO NCs so pokazali močne protibakterijske učinke, ZnO-SiOA NPs pa niso pokazali nobene protibakterijske aktivnosti, ki bi predstavljala biokompatibilnost. Vse testirane NM so bile tudi podvržene oksidaciji s H2O2. Pri ZnO NC in ZnO NPs se je zgodila močna oksidacija v O2–. generatorskem sistemu pri pH 8,5. Medtem ko so SiO2, ZnO-SiOA and ZnO-SiOB izkazovali izrazite 60–80 % antioksidativne učinke, so SiO2 NP delovali kot dokončni prooksidant, česar v drugih testih niso opazili. ZnO NC so močno oksidirani, ob predpostavki, da ZnO NC zagotavljajo počasnejše sproš- čanje ZnO, kar vodi v močnejši učinek na bakterijske seve. ZnO NC so torej pomembno protibakterijsko sredstvo, ki bi lahko nadomeščalo tradicionalne antibiotike. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License S73Acta Chim. Slov. 2022, 69, (3), Supplement Društvene vesti in druge aktivnosti DRUŠTVENE VESTI IN DRUGE AKTIVNOSTI SOCIETY NEWS, ANNOUNCEMENTS, ACTIVITIES Vsebina 54. Mednarodna kemijska olimpijada .................................................................................... S75 Koledar važnejših znanstvenih srečanj s področja kemije in kemijske tehnologije ......... S81 Navodila za avtorje .................................................................................................................. S84 Contents 54. International Chemistry Olympiad ................................................................................ S75 Scientific meetings – Chemistry and chemical engineering ................................................ S81 Instructions for authors .......................................................................................................... S84 S74 Acta Chim. Slov. 2022, 69, (3), Supplement Društvene vesti in druge aktivnosti S75Acta Chim. Slov. 2022, 69, (3), Supplement Društvene vesti in druge aktivnosti Na Fakulteti za kemijo in Kemijsko tehnologijo v Ljubljani se je od 10. 7. do 18. 7. 2022 tednu odvijala 54. mednarodna kemijska olimpijada. To je tekmovanje sre- dnješolcev v znanju kemije, vsako državo pa zastopa ekipa štirih najboljših. Priprave potekajo na Fakulteti za kemijo in kemijsko tehnologijo v Ljubljani, pri organizaciji do- godka pa sodelujemo z Zvezo za tehnično kulturo Sloveni- je. Mentorja ekipe sva dr. Berta Košmrlj in dr. Andrej Go- dec. V pripravah na to olimpijado so sodelovali tudi dr. Marta Počkaj, dr. Darko Dolenc, pomagali pa so še Martin Rihtaršič, Vid Kermelj in dr. Damjan Jan Pavlica. Letošnjo olimpijado je organizirala Kitajska, zaradi epidemioloških razmer v tej državi pa je potekala na dalja- vo. Našo državo so zastopali dijaki Dane Jemc (Gimnazija Škofja Loka), Matej Nastran (Gimnazija Škofja Loka), Ni- na Cankar (Gimnazija Kranj) ter Patrik Potočnik (Gim- nazija Škofja Loka).  Letošnje olimpijade se je sicer udeležilo 326 dijakov iz 84 držav. Naši dijaki so dosegli odličen rezultat: Dane, Matej, in Patrik so dobili bronasto medaljo, Nina pa častno omembo. ČESTITKE! Na tekmovanju je bilo letos 9 teoretičnih nalog, za reševanje pa je sicer na voljo 5 ur časa. Naloge na olimpija- di so izjemno težke, tako da se dijaki udeležujejo priprav, slovensko ekipo pa izberemo na treh nacionalnih izbirnih testih. Seveda letošnje naloge niso mogle zaobiti epidemije, saj so hitre in enostavne metode za zgodnje odkrivanje COVID-19 so nujno potrebne. Ena izmed obetavnih me- tod je detekcija s pomočjo nanodelcev zlata. Nanodelci zlata se zaradi njihovega visokega molskega ekstinkcijske- ga koeficienta (molske absorptivnosti) pogosto uporablja- jo kot vidni odčitki na testnih lističih. Barvni izgled nano- delcev zlata je povezan z njihovo velikostjo in razpršenostjo. V splošnem velja, da večji kot so nanodelci zlata, bolj rdeč- kasta je njihova barva. Ko se nanodelci združijo, se barva spremeni iz rdeče v modro. Ko površino nanodelcev zlata modificiramo z dvema vrstama enoverižnih fragmentov nukleinske kisline a in b, se nanodelci zlata v prisotnosti tarčne nukleinske kisline (a’ b’ ) združijo. Pri tem se v ne- kaj minutah spremeni barva iz rdeče v modro (slika). Na osnovi tega bi lahko zaznavali prisotnost tarčnih nuklein- skih kislin, značilnih za koronavirus, v vzorcih. Tema prve naloge je bila opisana detekcija tarčnih nukleinskih kislin: dijaki so okarakterizirali spekter nave- denih spojin, izračunali molarni ekstincijski koeficient raztopine nanodelcev zlata, ter na koncu še na osnovi po- datkov izračunali koncentracijo virusne nukleinske kisli- ne v originalnem vzorcu brisa žrela. Druga naloga je bila povezana z glazuro. Črn glazi- ran porcelan je vrsta kitajskega porcelana, ki je bila še po- sebej priljubljena v času dinastij Tang in Song pred približ- no 1000 leti. Tovrstni keramični izdelki kot glavno barvilo vsebujejo železove okside, ki jih zmešajo z ostalimi preho- dnimi kovinami, da dobijo različne temne odtenke, kot so kostanjeva, temno rjava ali črna barva. Črni glaziran por- celan je še danes na Kitajskem precej priljubljen. Tipična 54. Mednarodna kemijska olimpijada (Univerza Nankai, Tianjin, Kitajska) Andrej Godec UL, FKKT Slovenska ekipa na 54. Mednarodni kemijski olimpijadi: Dane, Pa- trik, Nina in Matej. Foto: Aljoša Seljak. S76 Acta Chim. Slov. 2022, 69, (3), Supplement Društvene vesti in druge aktivnosti črna glazura je sestavljena iz Fe-vsebujočih oksidov s spi- nelno strukturo. Spinelni oksidi imajo splošno formulo AB2O4 in njihova struktura je naslednja: O 2– ioni tvorijo kubični najgostejši sklad, v katerem kationi vrste A zaseda- jo eno osmino tetraedričnih praznin, kationi B pa eno po- lovico oktaedričnih praznin (slika). Črno keramično glazuro, ki ima spinelno strukturo, lahko pripravimo s praženjem Fe2O3 in Cr2O3 v določe- nem razmerju v reduktivni atmosferi. Ko Fe2O3 in Cr2O3 reagirata v masnem razmerju 63.6 : 36.4, se popolnoma spremenita v čisto stehiometrično spojino. Dijaki so morali okarakterizirati osnovno celico v takšnih spinelnih strukturah; del naloge pa je bil posvečen še vlogi kroma. Spremenljiva valenca kroma namreč ni po- membna samo za proizvodnjo pigmentov, ampak tudi za katalizo. Tipičen Philipsov katalizator za polimerizacijo etena je sestavljen iz kromovega oksida, nanesenega na po- rozen nosilec, kot je npr. amorfen silicijev dioksid. Dijaki so morali zapisati elektronsko konfiguracijo d elektronov Cr(II) iona v takšnem katalizatorju, in izračunati energijo stabilizacije kristalnega polja CFSE) ter magnetni moment µ za Cr(II) ion. Tema tretje naloge je bilo zajemanje in pretvorba ogljikovega dioksida. Tehnologija direktnega zajema zraka (DAC, ang. direct air capture), ki naj bi omogočila odstra- nitev CO2 direktno iz zraka, je obetavna. Osnova metode DAC je mokro spiranje z alkalno raztopino hidroksida (običajno NaOH), v kateri se absorbira zračni CO2 do vrednosti pH ≈ 10. Porabljen sorbent se regenerira z do- datkom kalcijevega hidroksida. Bela oborina, ki nastane, razpade pri 700 °C, pri čemer nastaneta CO2 in še ena be- la snov. Na koncu lahko pridobimo kalcijev hidroksid z hidracijo. Ta proces je energetsko zelo zahteven. ( H2CO3 : Ka1 = 4.5 × 10–7, Ka2 = 4.7 × 10–11 ). Nedolgo nazaj pa so razvili elektrokemijski proces za regeneracijo alkalne raz- topine, uporabljane v postopku mokrega spiranja za DAC. Čist plinast CO2 bi lahko ponovno pridobili in shranili ali uporabili naprej. Proces je osnovan na elektrokemijskem sistemu za recikliranje H2 (HRES; ang. H2-recycling electrochemical system), elektrokemijska celica pa vsebuje dve ionoselektivni membrani. Dijaki so morali zapisati in okarakterizirati vse navedene kemijske procese, mehaniz- me prenosa kationov v celici, ter izračunati hitrost nastaja- nja CO2. Zeolitno-imidazolatna ogrodja ZIF so podskupina organskih ogrodij MOF in so tudi obetavni materiali za zajemanje in uporabo CO2. Strukture ZIF-ov so podobne strukturam zeolitov. Zanje so značilna 3D ogrodja s tetra- edrično koordiniranimi kovinskimi ioni (npr. Zn2+, Co2+), ki so premoščeni z imidazolatnim ionom (Im-) ali njegovi- mi derivati. ZIF-8 je predstavnik ZIF-ov s sodalitnim (SOD) ogrodjem, prikazanim na sliki. ZIF-8 je prvi pripravil kitajski znanstvenik Xiao-Ming Chen s sodelavci pri re- akciji Zn2+ z 2-metilimidazolom (CH3(C3N2H3), HmIm) (produkt so sprva poimenovali MAF-4). ZIF-8 kristalizira v kubičnem sistemu s parametrom osnovne celice a = 1.632 nm (faza brez topila). Efektivni premer pore, ki je prikazana s kroglo na sliki, znaša 1.16 nm. (slika 4) Dijaki so morali okarakterizirati osnovno calico tega materiala, izračunati notranjo površino por ter poroznost materiala. ZIF-8 lahko igra tudi vlogo katalizatorja pri pre- tvorbi CO2 v kemikalije z visoko dodano vrednostjo. Eden izmed najbolj obetavnih načinov za fiksiranje CO2 je priprava cikličnih karbonatov s cikloadicijo. Spodaj je pri- kazan primer: Slika 4: (a) Topologija SOD kletke; (b) SOD kletka v ZIF-8, sestavljenem iz Zn2+ (v središčih tetraedrov) in imidazolatnih ionov (H atomi so zaradi jasnosti prikaza izpuščeni); (c) Ogrodje SOD z označeno osnovno celico (kvadrat); (d) Nekaj por v ZIF-8 je poudarjenih z navideznimi kroglami. S77Acta Chim. Slov. 2022, 69, (3), Supplement Društvene vesti in druge aktivnosti Dijaki so morali zapisati intermediate pri takšni re- akciji, ter tudi urejeno enačbo za to reakcijo. Četrta naloga je bila na temo žvepla, najprej elemen- tarnim, in potem v obliki spojine pirit (FeS2), ki je surovi- na pri industrijski proizvodnji elementarnega žvepla. S segrevanjem nastaja žveplo, pa tudi manjše količine plina SO2 in še česa. Z merjenjem količin tega plina lahko zasle- dujemo potek reakcije; dijaki so morali izračunati izkori- stek oziroma predvideti izgube žvepla v piritu v obliki stranskih produktov. Razen tega je naloga obravnavala tudi drugačno uporabo žvepla. Litij – žveplova baterija ima visoko teoretično energijsko gostoto, ki presega kon- vencionalno Li-ion baterijo. Neto reakcijo v litij – žveplo- vi bateriji lahko poenostavljeno napišemo takole: 16 Li + S8 → 8 Li2S. Žveplo je katoda, kovinski litij pa je aktivni material anode pri praznjenju baterije. Dijaki so morali zapisati elektrodne reakcije v tej bateriji, izračunati mase aktivnih elektrodnih materialov, ter izračunati, koliko ča- sa deluje in koliko bolje je to od litij-ionske baterije. V litij – žveplovih baterijah se med praznjenjem S8 ne reducira direktno do Li2S , ampak postopoma preko reakcij, pri katerih nastajajo različni topni litijevi polisulfidi (Li2Sn, n = 3–8). Ti litijevi polisulfidi lahko difundirajo na anodo in tam povzroči korozijo, kar pomeni izgubo aktivnega elek- trodnega materiala. Ta pojav imenujemo »shuttle učinek«. Teoretične študije kažejo, da dva konformera s primerlji- vima energijama, Li2S6(I) in Li2S6(II) soobstajata v 1,2-dimetoksietanu (DME), ki je običajno topilo in elek- trolit v litij-žveplovih baterijah. Disociacija Li2S6 v DME je prikazana spodaj: Dijaki so morali okarakterizirati korozijo anode, predvideti s pomočjo termodinamskih podatkov, kje se nahaja ravnotežje, izračunati disociacijsko konstanto Li2S6 v DME (1,2-dimetoksietan (DME) je običajno topilo in elektrolit v litij-žveplovih baterijah), ter redoks potenci- al litija v tem topilu. Peta naloga je bila povezana z onesnaževanjem ozra- čja. Dušikovi oksidi (vključno N2O, NO, NO2, N2O4 in še drugi, kar skupaj napišemo kot NOx) so eni od glavnih onesnaževalcev zraka. Povzročijo lahko probleme, kot so ozonska luknja, kisel dež, fotokemijski smog, in efekt tople grede. Zato moramo kontrolirati emisije in pretvorbe NOx, da bi izboljšali kvaliteto zraka. Dijaki so raziskali oksidacijo NO v NO2 preko reakcije 2NO + O2 → 2NO2. Izračunali so hitrost te reakcije, ter s termodinamskimi podatki ovrednotili smer poteka reakcije ter položaj rav- notežja. V drugem delu naloge pa so preučevali načine za zmanjšanje emisij NOx. En način za zmanjšanje emisij NOx je oksidacija NO v NO2 in nato absorpcija nastalega NO2 z absorbenti. Vendar pa je zaradi nizkih koncentracij NO v izpustih njegova spontana oksidacija v atmosferi prepočasna, da bi zadostila zahtevam industrije. Zato se za pospešitev te reakcije uporabljajo trdni katalizatorji. Oksi- dacija NO na površini specifičnega katalizatorja (CatX) poteka po zapletenem mehanizmu; predpostavili so, da se NO, NO2 in O (iz disociacije O2) adsorbirajo v eni plasti, in da so vsa adsorpcijska mesta za adsorpcijo teh zvrsti enakovredna. Deleži pokritosti površine katalizatorja θso označeni kot θNO, θNO2 in θO. Dijaki so morali izpeljati zvezo med deležem pokritosti in konstanto reakcijskih hi- trosti, ter izpeljati izraz za hitrost reakcije na začetku. Šesta naloga je bila o fosfinih. Kiralne fosfine pogos- to uporabljajo kot kiralne ligande pri katalizi s prehodnimi kovinami. V zadnjih dvajsetih letih se je močno razvilo področje organokatalize, med drugim so odkrili sintezne poti, kjer se uporablja nukleofilen fosfinski katalizator. Med temi je najbolj znana Lu-jeva (3+2) cikloadicija, ki jo je razvil kitajski znanstvenik Xiyan Lu. Naprimer etil ale- noat 1 in metil akrilat 2 dajeta pri katalizi s trifenilfosfi- nom dva ciklopentenska derivata: 3 (večinski produkt) in 4 (manjšinski produkt). Pri tej nalogi so morali dijaki dopolniti reakcijske she- me za to reakcijo in določiti stereokemijo vmesnih spojin. Tema sedme naloge je bila sinteza kompleksnih pep- tidov in proteinov. Spajanje karboksilne in aminske skupi- ne, da nastane peptidna vez, je osnovna reakcija pri sintezi peptidov in proteinov. Alenon 2 lahko uporabimo za akti- vacijo karboksilne kisline 1 pod milimi reakcijskimi pogo- ji, pri čemer nastane intermediat 3. Ta potem reagira z aminom 4 in daje amid 5 z dobrim izkoristkom. Pri tem nastane tudi stranski produkt 6. S78 Acta Chim. Slov. 2022, 69, (3), Supplement Društvene vesti in druge aktivnosti Na podoben način kot alenon lahko aktivira karbo- ksilno kislino tudi N-etinil-N-metil-p-toluensulfonamid. Dijaki so morali najprej za oba primera narisati strukture spojin pri teh reakcijah in prikazati stereokemijo na vsa- kem od stereocentrov. S tem, ko peptidna veriga raste, po- staja tvorba amidnih vezi vedno težja. Za sintezo proteinov zato klasične kondenzacijske metode niso uporabne. Pri prvi sintezi kristaliničnega govejega insulina so razvili me- todo, temelječo na kemiji acilhidrazina. S tem so dosegli zahtevno spajanje dveh peptidov z nastankom amidne vezi med njima. Tudi za to reakcijo so morali dijaki narisati strukture spojin. Poleg sinteze iz aminokislin lahko znan- stveniki uporabijo modifikacijo obstoječih proteinov. Na verigi proteina je več reaktivnih mest, kot so aminske, kar- boksilne in tiolne skupine. Najbolj nukleofilna je tiolna skupina in je zato najbolj reaktivna do elektrofilnih rea- gentov, kot je N-fenilmaleimid, na katerega se adira z Mi- chaelovo adicijo. Tudi za ta primer so morali dijaki dopol- niti reakcijsko shemo. Osma naloga je imela naslov Čudežni kiralni spiro katalizator. Kiralne spojine so med drugim pomembne za človeško zdravje. Več kot 50 % zdravil, ki so trenutno v klinični uporabi, je čistih posamičnih enantiomerov kiral- nih spojin. Sinteza kiralnih molekul v enantiomerno obo- gateni obliki je velik izziv. Skupina profesorja Qilin Zhou- -ja na nankaiski univerzi je razvila vrsto visoko aktivnih kiralnih spiro-katalizatorjev. Ti so dvignili učinkovitost asimetrične sinteze na novo raven in se široko uporabljajo v farmacevtski proizvodnji. Ti katalizatorji dosegajo enan- tiomerni presežek do 99,9 % in se uporabljajo v zelo majhnih količinah, celo do 0,00002 mol%. Dijaki so v na- logi dopolnili reakcijsko shemo za sintezo teh spojin, in uporabo kiralnega spiro katalizatorja Ir-SpiroPAP za asi- metrično totalno sintezo diterpena mulinanskega tipa. Zadnja, deveta naloga, pa je bila totalna sinteza kapi- tulaktona. Rastlina Curculigo capitulata, ki raste na juž- nem Kitajskem, se že dolgo uporablja v tradicionalni kitaj- ski medicini za zdravljenje mnogih bolezni. Iz korenin te rastline so izolirali spojino kapitulakton. Mentorice in mentorji ter dijaki slovenske, madžarske in ukrajinske ekipe na letošnji olimpijadi. Naša ekipa: Andrej (glavni mentor, 1. z leve), Dane, Nina ter Matej (8., 9. ter 10. z leve), in Berta (mentorica, 11. z leve). S79Acta Chim. Slov. 2022, 69, (3), Supplement Društvene vesti in druge aktivnosti Njena struktura z absolutno konfiguracijo je bila do- ločena z uporabo spektroskopskih podatkov in totalne sin- teze. Sintezo 1 so začeli z jodiranjem kupilnega 4-bromo- veratrola. Reakcija poteka preko več intermediatov, dijaki pa so morali tudi tukaj dopolniti reakcijsko shemo sinteze. Letošnja olimpijada je bila posebna, saj smo v goste povabili še madžarsko in ukrajinsko ekipo. Obe sta prišli v soboto, 9.7.2022, in preživeli dneve do tekmovanja v dru- ženju z našimi dijakinjami in dijaki ter na izletih. Na sliki so vse tri ekipe z mentoricami in mentorji, slika pa je bila narejena takoj po otvoritvi olimpijade v nedeljo, 10.7. Takšno obliko dogodka so omogočile Fakulteta za kemijo in kemijsko tehnologijo, madžarsko ministrstvo za notranje zadeve, Zveza za tehnično kulturo Slovenije, Slo- vensko kemijsko društvo ter zavod Bunker. Vsem se iskre- no zahvaljujemo za pomoč pri dogodku, ki je lep primer tega, da se da z meddržavnim sodelovanjem preseči trenu- tne politične in epidemiološke razmere, ter omogočiti so- delujočim izkušnjo »normalne« olimpijade. Naslednje leto bo olimpijada v Švici, upajmo, da tak- rat v živo. Tekst in foto: Andrej Godec S80 Acta Chim. Slov. 2022, 69, (3), Supplement Društvene vesti in druge aktivnosti S81Acta Chim. Slov. 2022, 69, (3), Supplement Društvene vesti in druge aktivnosti 2022 October 2022 5 – 7 7 MS FOOD DAY Florence, Italy Information: https://www.spettrometriadimassa.it/Congressi/7MS-FoodDay/index.html 14 CHEMISTRY AND CHEMICAL TECHNOLOGY 2022 Kaunas, Lithuania Information: https://cct-conference.ktu.edu/ 16 – 21 SCHOOL AND CONFERENCE ON ANALYSIS OF DIFFRACTION DATA IN REAL SPACE Grenoble, France Information: https://workshops.ill.fr/event/306/ 12 – 14 CHEMICAL RESEARCH IN FLANDERS – CHEMISTRY CONFERENCE FOR YOUNG SCIENTISTS 2022 Blankenberge, Belgium Information: https://crf-chemcys.be/ 21 – 22 XIV CONFERENCE OF CHEMISTS, TECHNOLOGISTS AND ECOLOGISTS OF THE REPUBLIC OF SRPSKA Banja Luka, Bosnia and Herzegovina Information: https://savjetovanje.tf.unibl.org/ 23 – 26 31ST INTERNATIONAL SYMPOSIUM ON THE CHEMISTRY OF NATURAL PRODUCTS AND 11TH INTERNATIONAL CONGRESS ON BIODIVERSITY (ISCNP31 & ICOB11) Naples, Italy Information: https://www.iscnp31-icob11.org/index.php 23 – 26 ENERGY, ENVIRONMENT & DIGITAL TRANSITION Milano, Italy Information: https://www.aidic.it/e2dt/ 23 – 25 5th CryoNET SYMPOSIUM Copenhagen,Denmark Information: https://eventsignup.ku.dk/cryonet2022 November 2022 8 – 11 SOLUTIONS IN CHEMISTRY 2022 Sveti Martin na Muri, Croatia Information: https://solutionsinchemistry.hkd.hr/ KOLEDAR VAŽNEJŠIH ZNANSTVENIH SREČANJ S PODROČJA KEMIJE IN KEMIJSKE TEHNOLOGIJE SCIENTIFIC MEETINGS – CHEMISTRY AND CHEMICAL ENGINEERING S82 Acta Chim. Slov. 2022, 69, (3), Supplement Društvene vesti in druge aktivnosti 6 – 11 EMBO PRACTICAL COURSE: VOLUME ELECTRON MICROSCOPY BY AUTOMATED SERIAL SEM Lausanne, Switzerland Information: https://meetings.embo.org/event/21-serial-sem December 2022 5 4TH EUROPEAN FORUM ON NEW TECHNOLOGIES - CHEMICAL ENGINEERING AS APPLIED IN MEDICINE Paris, France https://efce.info/4th+European+Forum+on+New+Technologies.htm 5 – 8 ENVIRONMENTAL MEETING ON ENVIRONMENTAL CHEMISTRY 2022 – EMEC22 Ljubljana, Slovenia Information: https://www.emec22.com/ 2023 January 2023 1 EUROPEAN FOOD CHEMISTRY CONGRESS XXI – EuroFoodChem XXI Belgrade, Serbia Information: http://horizon2020foodentwin.rs/eurofoodchemxxi/ March 2023 20 – 23 VIII INTERNATIONAL CONGRESS “ENGINEERING, ENVIRONMENT AND MATERIALS IN PROCESS INDUSTRY Jahorina, Bosnia and Hercegovina Information: https://eem.tfzv.ues.rs.ba/ February 2023 8 – 11 EMBO WORKSHOP IN SITU STRUCTURAL BIOLOGY: FROM CRYO-EM TO MULTI- SCALE MODELLING Heidelberg, Germany Information: https://www.embl.org/about/info/course-and-conference-office/events/iss23-01/ July 2023 2 – 6 FEZA 2023 – 9TH CONFERENCE OF THE FEDERATION OF THE EUROPEAN ZEOLITE ASSOCIATIONS Portorož-Portorose, Slovenia Information: https://feza2023.org/en/ 7 – 11 9TH EUCHEMS CHEMISTRY CONGRESS (ECC9) Dublin, Ireland S83Acta Chim. Slov. 2022, 69, (3), Supplement Društvene vesti in druge aktivnosti S84 Acta Chim. Slov. 2022, 69, (3), Supplement Društvene vesti in druge aktivnosti Sub mis sions Submission to ACSi is made with the implicit under- standing 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 else- where. All the listed authors should have agreed on the content and the corresponding (submitting) au- thor is responsible for having ensured that this agree- ment 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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Slov. 2022, 69, (3), Supplement Društvene vesti in druge aktivnosti 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 European Federation of Chemical Engineering https://efce.info/ International Union of Pure and Applied Chemistry https://iupac.org/ Brussels News Updates http://www.euchems.eu/newsletters/ Novice europske zveze kemijskih društev EuChemS najdete na: Koristni naslovi Komore za testiranje baterij Vakuumski sušilniki Klimatske komore Donau Lab d.o.o., Ljubljana Tbilisijska 85 SI-1000 Ljubljana www.donaulab.si office-si@donaulab.com BINDER Acta Chimica oglas.indd 1 21. 11. 2021 20:29:54 www.helios-group.eu Znanje, kreativnost zaposlenih in inovacije so ključnega pomena v okolju, kjer nastajajo pametni premazi skupine KANSAI HELIOS. Z rešitvami, ki zadostijo široki paleti potreb, kontinuiranim razvojem ter s kakovostnimi izdelki, Helios predstavlja evropski center za inovacije in poslovni razvoj skupine Kansai Paint. Razvoj in inovacije za globalno uspešnost Prehransko dopolnilo ni nadomestilo za uravnoteženo in raznovrstno prehrano. Skrbite tudi za zdrav življenjski slog. BODITE ŠE BOLJ NEUSTAVLJIVI zmanjševanju utrujenosti in izčrpanosti, normalnemu delovanju mišic. www.magnezijkrka.si MAGNEZIJ Krka 300 MAGNEZIJ Krka 400 NOVO Magneziju Krka 300 se je pridružil Magnezij Krka 400 , ki vsebuje več magnezija in tako še bolj pripomore k: 249002-2022 MgKrka 400 DailyLife SPORT Ad A4 SI.indd 1 20. 04. 2022 15:09:15