Scientific paper
New Approaches for the Synthesis of Thiophene Derivatives with Anti-tumor Activities
Rafat M. Mohareb,1* Nermeen S. Abbas2 and Rehab A. Ibrahim3
1	Department of Chemistry, Faculty of Science, Cairo University, Giza, A., R. Egypt
2	Department of Chemistry, Faculty of Science, Helwan University, Cairo, A. R. Egypt
3 Higher Institute of Engineering and Technology, El-Tagammoe El-Khames, New Cairo, Egypt * Corresponding author: E-mail: raafat_mohareb@yahoo.com Received: 25-04-2012
Abstract
The reaction of either cyclohexanone or cyclopentanone with cyanoacetylhydrazine and elemental sulfur gave the 2-aminocycloaLkeno[b]thiophene derivatives 3a and 3b, respectively. The latter compounds reacted with either aromatic benzaldehydes or active methylene reagents to give the Schiff's bases 5a-d and the pyrazole derivatives 7a-d and 9a-d, respectively. On the other hand, the reaction of 3-oxo-N-p-tolylbutanamide (10) with either of malononitrile or ethyl cyanoacetate gave the thiophene derivatives 13a and 13b, respectively. Compounds 13a,b were subjected to a series of heterocyclization reactions to give heterocyclic derivatives. Their cytotoxicity against the three human tumor cells lines, namely breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and CNS cancer (SF-268) together against the normal human cell line namely the normal fibroblast cells WI 38 were measured.
Keywards: Hydrazide-hydrazone, thiophene, pyrazole, arylhydrazone, antitumor activity.
1. Introduction
The pair cycloalkene/thiophene represents one of the most prominent examples of bioisosterism1 (bioisoste-res are isosteric2 molecules that have similar or antagonistic properties in biological systems) and therefore the synthesis of thiophene analogues has attracted considerable attention especially in pharmaceutical research. The exploratory replacement of a benzene ring in successful drugs by a thiophene moiety has become a routine strategy in modern drug design and development.
The physiological effects of thiophene are similar to those of benzene (bioisostere), with frequently superior pharmacodynamic, pharmacokinetic, or toxicological properties. For example, thiophene replacement of the an-nulated benzene ring in derivatives of piroxicam, an anti-inflammatory agent used in arthritis patients, had no effect on activity.3 Similarly, the thiophene analogue of amphetamine retains complete amphetamine-like activity.4 Nevertheless, thiophene derivatives have shown numerous biological activities, such as nematocidal,5 insecticidal,6 antibacterial,7 antifungal,8 and antiviral activity.9 Recently, substituted thiophenes have been shown to possess
good anti-inflammatory activities in rats.10 In view of reported biological activities of alkynyl substituted hete-rocycles11-17 and our effort in the synthesis of thiophene derivatives18-20 of potential pharmacological significance we became interested in the synthesis of substituted cycloalkeno thiophenes. Due to the known anticancer activities of thiophene derivatives13 in this work we would like to report the synthesis of different thiophene derivatives that have been screened for antitumor activity against breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and CNS cancer (SF-268); this is beside studying their cytotoxicity against the normal human cell line namely the normal fibroblast cells WI 38.
2. Results and Discussion
2. 1. Chemistry
In the present work, cyanoacetylhydrazine was reacted with either cyclohexanone (2a) or cyclopentanone (2b) and elemental sulfur in 1,4-dioxan and in the presence of triethylamine gave the cycloalkeno[b]thiophene derivatives 3a and 3b, respectively. Compound 3b was pre-
viously reported.21-25 The structure of compound 3a was mainly elucidated by 1H- and 13C-NMR spectra. Thus, the 1H-NMR spectrum of 3a showed two multiplets at 5 1.77-1.79 and 2.20 ppm corresponding to the presence of three CH2 groups, two multiplets at 5 4.20-4.22 ppm corresponding to the NH2 group and a singlet at 5 9.01 (D2O exchangeable) for the NH group. 13C-NMR, 5: 22.0, 23.4,
30.9 (cyclophentene CH2), 122.6, 136.3, 138.9, 144.5 (thiophene C), 166.8 (C=O).
Compounds 3a,b reacted with either benzaldehyde (4a) or salicylaldehyde (4b) to give the hydrazide-hydra-zone derivatives 5a-d, respectively. Compounds 5b,d were identical to those reported by Jagtab et al.21 The analytical and spectral data of compounds 5a,c are consistent
Sheme 1. Reaction of 1 with elemental sulfur and the cycloketones 2a,b and reaction of 3a,b with aromatic aldehydes 4a,b and synthesis of the pyrazole deravatives 7a,d and 8a,d. Reagents and conditions: (a) Et3N, absolute ethanol, heat 3h; (b) 1,4-dioxan, heat 2 h; (c) 1,4-dioxan, Et3N, heat 3 h; (d) 1,4-dioxan, Et3N, heat 3h.
with their respective structures (see experimental section). On the other hand, the hydrazido moiety present in compounds 3a,b showed high activity towards cyanomethyle-ne reagents. Thus, their reaction with either malononitrile (6a) or ethyl cyanoacetate (6b) in 1,4-dioxan gave the pyrazole derivatives 7a-d. Similarly the reaction of either 3a or 3b with either acetylacetone (8a) or ethyl acetoace-tate (8b) gave the pyrazole derivatives 9a-d (Scheme 1). The structures of compounds 7a-d and 9a-d were based on their respective 1H- and 13C-NMR data. Thus, the
1H-NMR spectrum of 9a (as an example) showed beside the expected signals, two singlets at 5 2.33, 2.82 ppm corresponding to the two CH3 groups, a singlet at 5 4.23 ppm for NH2 group and a singlet at 5 6.59 ppm for the pyrazole H-3 proton. Moreover, the 13C-NMR spectrum showed 5: 16.1, 19.6 (2 CH3), 21.5, 24.7, 26.8, 34.2 (cyclopentene CH2), 123.8, 127.8, 134.2, 143.9, 145.2, 147.3, 152.8 (thiophene C, pyrazole C), 163.5 (C=O).
Next we moved towards the synthesis of another poly-functionally substituted thiophenes using 1,3-dicar-
Sheme 2. Synthesis of the thiophene derivatives 11a,b and their reaction with benzaldehyde and malononitrile. Reagents and conditions (a) S8, EtOH, Et3N, heat 1 h; (b) 1,4-dioxan, piperidine, heat 1 h; (c) 1,4-dioxan, Et3N, heat 3 h.
bonyl compound and cyanomethylene reagents. Thus, the reaction of the 3-oxo-N-p-tolylbutanamide (10) with either malononitrile (6b) or ethyl cyanoacetate (6b) and elemental sulfur as an application of the well known Gewald's thiop-hene synthesis,26 yielded the polyfunctionally substituted thiophene derivatives 11a,b, respectively. The analytical and spectral data of the latter products are in agreement with the assigned structures (see experimental section).
The reactivity of 11a,b towards some chemical reagents was studied in the aim of synthesizing thiophene derivatives with potential anti-tumor activity. Thus, the reaction of either 11a or 11b with benzaldehyde (4a) gave the benzal derivatives 12a,b, respectively. Analogous to the known Knoevenagel condensation27,28 we found that the reaction of either 11a or 11b with malononitrile (6a) gave
the corresponding Knoevenagel condensated products 13a,b, respectively (Scheme 2) .
The excellent yields of compounds 13a,b encouraged us to explore further reactions to study the coupling reactions of 13a,b with aryl diazonium salts. Thus, with the diazonium salts namely, benzenediazonium chloride (14a), 4-methylbenzenediazonium chloride (14b) and 4-chlorobenzenediazonium chloride (14c) gave the aryl hydrazono derivatives 15a-f, respectively (Scheme 3). The analytical and spectral data are in agreement with the assigned structures (see experimental section).
Encouraged by the excellent results, we next investigated the reactivity of the acetyl group present in 11a,b using the well known Gewald's reaction.29,30 Thus, the reaction of either 11a or 11b reacted with either malono-
Sheme 3. Synthesis of the arylhydrazo derivatives 15a-f. Reagents and conditions NaOH, EtOH, 0 °C.
Sheme 4. Synthesis of the thiophene derivatives 16a-d. Reagents and conditions (a) 1,4 dioxan, Et3N, S8 heat 2 h.
nitrile (6a) or ethyl cyanoacetate (6b) and elemental sulfur in the presence of triethylamine gave the thio-3-yl thiophene derivatives 16a-d, respectively (Scheme 4).
2. 2. Antitumor and Normal Cell Line Activity Tests
2. 2. 1. Reagents
Fetal bovine serum (FBS) and L-glutamine were from Gibco Invitrogen Co. (Scotland, UK). RPMI-1640 medium was from Cambrex (New Jersey, USA). Dimethyl sulfoxide (DMSO), doxorubicin, penicillin, streptomycin and sulforhodamine B (SRB) were from Sigma Chemical Co. (Saint Louis, USA).
Cell cultures: Three human tumor cell lines, MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), and SF-268 (CNS cancer) were used. MCF-7 was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK), NCI-H460, SF-268 and normal fibroblast cells (WI 38) were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt). They grow as a monolayer and are routinely maintained in RPMI-1640 medium supplemented with 5% heat inactivated FBS, 2 mM glutamine and antibiotics (penicillin 100 U/mL, streptomy-
cin 100 mg/mL), at 37 oC in a humidified atmosphere containing 5% CO2. Exponentially growing cells were obtained by plating 1.5 x 105 cells/mL for MCF-7 and SF-268 and 0.75 x 104 cells/mL for NCI-H460, followed by 24 h of incubation. The effect of the vehicle solvent (DMSO) on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration (0.5%) of DMSO used in each assay.
2. 2. 2. Tumor Cell Growth Assay
The effects of 3a,b-16a-d on the in vitro growth of human tumor cell lines were evaluated according to the procedure adopted by the National Cancer Institute (NCI, USA) in the zIn vitro Anticancer Drug Discovery Screen' that uses the protein-binding dye sulforhodamine B to assess cell growth (12). Briefly, exponentially, cells growing in 96-well plates were then exposed for 48 h to five serial concentrations of each compound, starting from a maximum concentration of 150 mM. Following this exposure period adherent cells were fixed, washed, and stained. The bound stain was solubilized and the absorbance was measured at 492 nm in a plate reader (Bio-Tek Instruments Inc., Powerwave XS, Winooski, VT, USA). For each test compound and cell line, a dose-response curve was obtained and the growth inhibition of 50% (GI50), correspon-
Table 1. Effect of compounds 3a,b-16a-d on the growth of three human tumor cell lines
Compound	GI50 (m mol L-1)
	MCF-7	NCI-H460	SF-268	WI 38
3a	18.1 ± 1.6	6.2 ± 2.8	8.6 ±2.6	44.2 ± 10.2
3b	22.6 ± 2.6	24.3 ± 0.8	30.9 ± 3.8	60.2 ± 8.0
5a	70.6 ± 16.9	38.9 ± 10.8	52.8 ± 8.6	77.8 ± 16.8
5b	30.6 ± 10.2	32.6 ± 8.6	24.4 ± 12.8	33.4 ± 12.6
5c	6.4 ± 2.2	4.1 ± 0.8	8.8 ± 4.8	20.8 ± 8.2
5d	10.8 ± 2.6	4.5 ± 0.8	4.8 ± 1.8	28.0 ± 4.1
7a	70.7 ± 18.5	40.2 ± 12.8	52.4 ± 8.6	60.2 ±12.4
7b	55.1 ± 2.7	23.2 ± 4.8	14.4 ± 2.6	44.3 ±10.6
7c	20.4 ± 2.2	30.6 ± 1.4	20.8 ± 6.4	8.3 ± 3.8
7d	0.02 ± 0.008	0.03 ± 0.006	0.05 ± 0.001	> 100
9a	12.8 ± 2.6	22.0 ± 0.4	30.5 ± 6.0	14.8 ± 1.5
9b	10.8 ± 2.6	14.1 ± 0.6	22.3 ± 0.8	8.9 ± 4.2
9c	60.2 ± 2.4	43.6 ± 1.8	58.8 ± 0.8	66.2 ±8.2
9d	22.2 ± 0.8	22.3 ± 2.5	30.6 ± 0.8	8.7 ± 2.6
11a	12.8 ± 3.6	20.6 ± 3.4	24.2 ± 0.8	6.5 ± 1.4
11b	60.6 ± 10.4	40.8 ± 10.8	22.1 ± 2.8	12.2 ± 3.8
12a	0.02 ± 0.002	0.01 ± 0.002	0.06 ± 0.008	> 100
12b	40.6 ± 2.6	22.6 ± 2.6	35.2 ± 12.8	10.5 ±5.1
13a	20.8 ± 6.8	18.2 ± 1.6	18.6 ± 4.8	> 100
13b	44.8 ± 8.6	38.3 ± 4.5	22.6 ± 5.5	14.2 ± 2.4
15a	32.4 ± 8.8	18.7 ± 6.2	30.4 ± 2.4	22.6 ± 9.2
15b	55.7 ± 8.1	26.9 ± 4.4	44.5 ± 6.9	64.0 ±12.4
15c	4.2 ± 1.8	2.8 ± 0.5	2.3 ± 0.8	2.4 ± 0.8
15d	22.4 ± 4.6	20.3 ± 2.8	18.8 ± 4.6	0.4 ± 0.01
15e	38.4 ± 6.2	24.2 ± 0.8	20.6 ± 2.6	26.6 ± 4.6
15f	68.2 ± 8.6	22.3 ± 2.6	50.5 ± 12.5	30.8 ± 4.2
16a	0.01 ± 0.002	0.03 ± 0.8	0.05 ± 0.01	> 100
16b	0.06 ± 0.02	0.03 ± 0.01	0.04 ± 0.02	0.2 ± 0.6
16c	4.6 ± 1.8	2.3 ± 1.2	2.0 ± 0.4	> 100
16d	6.4 ± 0.8	4.3 ± 2.6	2.8 ± 0.6	0.2 ± 0.6
Doxorubicin	0.04 ± 0.008	0.09±0.008	0.09±0.007	> 100
Results are given in concentrations that were able to cause 50% of cell growth inhibition (GI50) after a continuous exposure of 48 h and show means ± SEM of three-independent experiments performed in duplicate.
ding to the concentration of the compounds that inhibited 50% of the net cell growth, was calculated as described elsewhere. Doxorubicin was used as a positive control and tested in the same manner.
2. 2. 3. Effect on the Growth of Human TUmor Cell Lines
The effect of compounds 3a,b-16a-d was evaluated on the in vitro growth of three human tumor cell lines representing different tumor types, namely, breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and CNS cancer (SF-268) together with the normal fibroblast cells (WI 38) after a continuous exposure for 48h. The results are summarized in Table 1. All of the tested compounds were able to inhibit the growth of the tested human tumor cell lines in a dose-dependant manner. The results in Table 1 reveal that compounds 7d, 12a, 16a and 16b showed the highest inhibitory effect against
all the three tumor cell lines«, such activity is higher than the refrence doxorubicin. The inhibitory effect of 7a, 12a and 16a are very low against normal fibroblast cells (WI 38). While compounds 5d and 16b showed high inhibitory effects against non-small cell lung cancer (NCI-H460) and breast adenocarcinoma (MCF-7), respectively, which are less than the reference doxorubicin. Compounds 5b, 5c, 7a, 7b, 9c, 11b, 12a, 13b, 15a, 15b, 15e and 15f showed the lowest inhibitory effect. The rest of the compounds showed a moderate growth inhibitory effect. Comparing compound 5a-d, it is obvious that the presence of the 2-OH group in compounds 5c and 5d is reposible for their reactivity over 5a and 5b. Similarly comparing of 12a and 12b, it is obvious that the introduction of the CN group in 12a showed higher inhibitory effect towards the three cell lines than that of 12b. On the other hand, comparing the inhibitory effect of compounds 15a-f, one can say that compound 15c with the X = CN and Y = Cl showed the highest inhibitory effect
among the six compounds but such reactivity is lower than that of the reference doxorubicin. Similarly, comparison of compounds 16a-d showed that when X = Y = CN, like in 16a, the maximum inhibitory effect among the four compounds was obtained. However, when X = CN and Y = COOEt as in the case of 16b, the inhibitory effect was lowered but not by a large amount as the compound is still one of the most active compounds among all test compounds. On the other hand, introduction of the ester group, like in 16c, decreases the reactivity and such observation was shifted towards lower reactivity in case of 16d where X = Y = COOEt.
3. Experimental
3. 1. General
All melting points are uncorrected. IR spectra were recorded as KBr discs on a Pye Unicam SP-1000 spectrophotometer. :H- and 13C NMR spectra were measured on a Varian EM-390-200 MHz in CD3SOCD3 as the solvent using TMS as the internal standard and chemical shifts are expressed as 5. Analytical data were obtained from the Micro analytical Data Unit at Cairo University, Giza, Egypt. Physical and spectral data of compounds 3b, 5b and 5d as reported in the literature.21-25
3. 1. 1. General Procedure for the Synthesis of Tetrahydobenzo[b]thiophene Derivatives 3a,b
To a solution of either cyclopentanone (8.50 g, 0.01 mol) or cyclohexanone (9.9 g, 0.1 mol) in abs. ethanol (50 mL) containing triethylamine (2.0 mL), cyanoacetylhy-drazine (10.0 g, 0.1 mol) was added. The reaction mixture, in each case, was heated under reflux for 3 h then left to cool. The formed solid product was collected by filtration and crystallized from the proper solvent.
2-Amino-5,6-dihydro-4_ff-cyclopenta[£]thiophene-3-carbohydrazide (3a). Yellow crystals from ethanol, yield 88% (17.42 g), m.p. 230-233 °C. Anal. Calculated for C8H11N3OS (197.26): C, 48.71; H, 5.62; N, 21.30; S, 16.26. Found: C, 49.01; H, 5.51; N; 21.17; S, 16.31. MS: mJe 197 (M+, 20%), IR, v: 3483-3205 (NH2, NH), 3054 (CH, aromatic), 1699 (C=O). 1H-NMR, 5: 1.77-1.79 (m, 4H, 2CH2), 2.20 (m, 2H, CH2), 4.20-4.22 (2m, 4H, 2NH2), 9.01 (s, 1H, NH). 13C-NMR, 5: 22.0, 23.4, 30.9, 122.(5, 136.3, 138.9, 144.5, 166.8.
3. 1. 2. General Procedure for the Synthesis of the N-Arylidenocarbohydrazide Derivatives 5a-b
To a solution of either 3a (1.97 g, 0.01 mol) or 3b (2.11 g, 0.01 mol) in 1,4-dioxan (40 mL) either benzal-
dehyde (1.0 g, 0.01 mol) or salicylaldehyde (1.22 g, 0.01 mol) was added. The reaction mixture, in each case, was heated under reflux for 2 h then poured onto ice/water. The formed solid product was collected by filtration and crystallized from the proper solvent.
2-Amino-5,6-dihydro-4_ff-cyclopenta[£]thiophene-3-(N-benzal)-carbohydrazide (5a). Yellow crystals from ethanol, yield 90% (2.56 g), m.p. 214-217 °C. Anal. Calculated for C15H15N3OS (285.26): C, 63.13; H, 5.30; N, 14.73; S, 11.24. Found: C, 63.01; H, 5.44; N; 14.82; S, 11.43. MS: m/e 285.36 (M+, 5%), IR, v: 3466-3223 (NH2, NH), 3056 (CH, aromatic), 1689 (C=O). 1H-NMR, 5: 1.71-1.74 (m, 4H, 2CH2), 2.19 (m, 2H, CH2), 4.23 (m, 2H, NH2), 6.78 (s, 1H, CH), 7.28-7.38 (m, 5H, C6H5), 9.30 (s, 1H, NH). 13C-NMR, 5: 22.8, 25.6, 27.2, 33.8, 116.9, 117.4, 120.6, 122.6, 128.9, 134.3, 135.9, 145.1, 163.6, 169.8.
2-Amino-5,6-dihydro-4_ff-cyclopenta[£]thiophene-3-(N-benzal)-carbohydrazide (5c). Yellow crystals from ethanol, yield 66% (1.98 g), m.p. 120°C. Anal. Calculated for C15H15N3O2S (301.36): C, 59.78; H, 5.02; N, 13.94; S, 10.64. Found: C, 59.94; H, 5.31; N; 14.05; S, 10.48. MS: m/e 301 (M+, 15%), IR, v: 3475-3219 (NH2, NH), 3053 (CH, aromatic), 1686 (C=O). 1H-NMR, 5: 1.75-1.78 (m, 4H, 2CH2), 2.23 (m, 2H, CH2), 4.26 (m, 2H, NH2), 6.83 (s, 1H, CH), 7.29-7.36 (m, 5H, C6H5), 9.28 (s, 1H, NH), 10.51 (s, 1H, OH). 13C-NMR, 5: 23.2, 25.5, 26.9, 34.1, 116.3, 118.0, 121.3, 122.8, 129.2, 133.8, 144.2, 148.6, 164.2, 168.4.
3. 1. 3. General Procedure for the Synthesis of the Pyrazole Derivatives 7a-d
To a solution of either 3a (1.97 g, 0.01 mol) or 3b (2.11 g, 0.01 mol) in 1,4-dioxan (40 mL) containing triethylamine (1.5 mL) either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 3 h. The solid product, formed in each case, upon evaporation under vacuum and trituration with ethanol, was collected by filtration and crystallized from the proper solvent.
(3,5-Diamino-1_ff-pyrazol-1-yl)(2-amino-5,6-dihydro-4_ff-cycopenta[£]-thiophen-3-yl)methanone (7a). Orange crystals from ethanol, yield 82% (2.15 g), m.p. 188-191 °C. Anal. Calculated for C11H13N5OS (263.32): C, 50.17; H, 4.98; N, 26.60; S, 12.18. Found: C, 50.42; H, 5.24; N; 26.38; S, 12.41. MS: m/e 263 (M+, 12%), IR, v: 3472-3313 (3NH2), 3053 (CH, aromatic), 1693 (C=O). 1H-NMR, 5: 1.70-1.75 (m, 4H, 2CH2), 2.21 (m, 2H, CH2), 4.21, 4.45, 5.42 (3s, 6H, 3NH2), 6.55 (s, 1H, pyrazole H-3). 13C-NMR, 5: 21.2, 25.2, 28.0, 34.6, 125.0, 128.3, 136.3, 144.5, 146.8, 148.6, 154.2, 162.3.
(3,5-Diamino-1_ff-pyrazol-1-yl)(2-amino-4,5,6,7-te-trahydrobenzo[£]-thiophen-3-yl)methanone (7b). Yellow crystals from ethanol, yield 86% (2.38 g), m.p. 185-187 °C. Anal. Calculated for C12H15N5OS (277.35): C, 51.97; H, 5.45; N, 25.25; S, 11.56. Found: C, 51.75; H, 5.33; N; 25.06; S, 11.43. MS: m/e 277 (M+, 8%), IR, v: 3464-3236 (3 NH2), 3059 (CH, aromatic), 1686 (C=O). 1H-NMR, 5: 1.84-1.96 (m, 4H, 2CH2), 2.23-2.35 (m, 4H, 2CH2), 4.44, 4.48, 5.22 (3s, 6H, 3NH2), 6.59 (s, 1H, pyrazole H-3). 13C-NMR, 5: 22.8, 24.8, 26.7, 32.6, 36.8,
124.7,	127.8, 134.0, 144.2, 147.3, 148.6, 153.8, 160.2
(2-Amino-5,6-dihydro-4_ff-cyclopenta[£]thiophe-3-yl)(3-amino-5-hydroxy-1_ff-pyrazol-1-yl)methanone (7c). Orange crystals from ethanol, yield 77% (2.03 g), m.p. 145-146 °C. Anal. Calculated for C11H12N4O2S (264.07): C, 49.99; H, 4.58; N, 21.20; S, 12.131 Found: C, 50.11; H, 4.66; N; 21. 32; S, 12.25. MS: m/e 264 (M+, 10%), IR, v: 3462-3333 (OH, 2NH2), 3056 (CH, aromatic), 1690 (C=O). 1H-NMR, 5: 1.72-1.78 (m, 4H, 2CH2), 2.24 (m, 2H, CH2), 4.30, 4.47 (2s, 4H, 2NH2), 6.58 (s, 1H, pyrazole H-3), 10.38 (s, 1H, OH). 13C-NMR, 5: 5: 22.0, 25.3, 27.8, 34.3, 126.2, 126.8, 135.8, 138.9, 142.4, 146.6,
153.8,	164.0.
(2-amino-4,5,6,7-tetrahydrobenzo[£]thiophen-3-yl)(3-amino-5-hydroxy-1_ff-pyrazol-1-yl)methanone (7d).
Orange crystals from ethanol, yield 59% (1.64 g), m.p. 142-144 °C. Anal. Calculated for C12H14N4O2S (278.33): C, 51.78; H, 5.07; N, 20.13.; S, 11.521. Found: C, 51.52; H, 5.34; N; 20.08; S, 11.63. MS: m/e 278 (M+, 32%), IR, v: 3477-3232 (OH, 2 NH2), 3052 (CH, aromatic), 1689 (C=O). 1H-NMR, 5: 1.84-1.99 (m, 4H, 2CH2), 2.24-2.37 (m, 4H, 2CH2), 4.43, 4.49 (2s, 4H, 2NH2), 6.56 (s, 1H, pyrazole H-3), 10.11 (s, 1H, OH). 13C-NMR, 5: 22.5, 24.5, 26.2, 32.6, 35.4, 124.0, 126.9, 132.8, 138.0, 145.3, 148.0, 154.3, 162.6.
3. 1. 4. General Procedure for the Synthesis the Pyrazole Derivatives 9a-d
To a solution of either 3a (1.97 g, 0.01 mol) or 3b (2.11 g, 0.01 mol) in 1,4-dioxan (40 mL) containing triethylamine (1.5 mL) either acetylacetone (1.0 g, 0.01 mol) or ethyl acetoacetate (1.30 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 3 h. The solid product, formed in each case, upon evaporation under vacuum and trituration with ethanol, was collected by filtration and crystallized from the proper solvent.
(5-Amino-3-methyl-1_ff-pyrazol-1-yl)(2-amino-5,6-dihydro-4_ff-cyclopenta[£]-thiophe-3-yl)methanone (9a). Orange crystals from ethanol, yield 79% (2.07 g), m.p. 135-138 °C. Anal. Calculated for C13H15N3OS (261.34): C, 59.74; H, 5.79; N; 16.08; S, 12.271 Found: C, C, 59.94; H, 5.58; N, 16.26; S, 12.29. MS: m/e 261 (M+,
40%), IR, v: 3466-3337 (2 NH2), 3050 (CH, aromatic), 1690 (C=O). *H-NMR, 5: 1.71-1.78 (m, 4H, 2CH2), 2.23 (m, 2H, CH2), 2.33, 2.82 (2s, 6H, 2CH3), 4.23 (s, 2H, NH2), 6.59 (s, 1H, pyrazole H-3). 13C-NMR, 5: 16.1, 19.6,
21.5,	24.7, 26.8, 34.2, 123.8, 127.8, 134.2, 143.9, 145.2, 147.3, 152.8, 163.5.
(5-Amino-3-methyl-1_ff-pyrazol-1-yl)(2-amino-4,5,6,7-tetrahydro-benzo[Ä]thiophen-3-yl)methanone (9b).
Yellow crystals from ethanol, yield 66% (1.82 g), m.p. 133-135 °C. Anal. Calculated for C14H17N3OS (275.37): C, 61.06; H, 6.22; N, 15.26; S, 11.64. Found: C, 61.25; H, 6.73; N; 15.33; S, 16.52. MS: m/e 276 (M+, 20%), IR, v: 3458-3232 (2 NH2), 3054 (CH, aromatic), 1689 (C=O). 1H-NMR, 5: 1.86-1.99 (m, 4H, 2CH2), 2.21-2.56 (m, 4H, 2CH2), 2.49, 2.72 (2s, 6H, 2CH3), 4.43, 4.49 (2s, 4H, 2NH2), 6.63 (s, 1H, pyrazole H-3). 13C-NMR, 5: 16.8, 19.9, 22.4, 25.1, 26.2, 30.6, 36.2, 124.9, 128.2, 134.4, 143.9, 146.6, 148.8, 154.2, 162.2.
(5-Hydroxy-3-methyl-1_ff-pyrazol-1-yl)(2-amino-5,6-dihydro-4_ff-cyclopenta[è]-thiophe-3-yl)methanone (9c). Pale brown crystals from ethanol, yield 80% (2.10 g), m.p. 188-190 °C. Anal. Calculated for C12H13N3O2S (263.32): C, 54.74; H, 4.98; N, 15.96; S, 12.18. Found: C, 54.93; H, 4.78; N; 16.09; S, 12.42. MS: m/e 263 (M+, 22%), IR, v: 3474-3382 (OH, NH2), 3052 (CH, aromatic), 1688 (C=O). 1H-NMR, 5: 1.73-1.80 (m, 4H, 2CH2), 2.26 (m, 2H, CH2), 2.66 (s, 3H, CH3), 4.38 (s, 2H, NH22), 6.56 (s, 1H, pyrazole H-3), 10.42 (s, 1H, OH). 13C-NMR, 5: 5:
22.6,	25.2, 28.2, 34.4, 125.8, 127.1, 134.5, 137.2, 143.4, 145.6, 158.2, 163.8.
(5-Hydroxy-3-methyl-1_ff-pyrazol-1-yl)(2-amino-4,5,6, 7-tetrahydro-benzo[Ä]thiophen-3-yl)methanone (9d).
Yellow crystals from ethanol, yield 83% (2.30 g), m.p. 110-112 °C. Anal. Calculated for C13H15N3O2S (277.34): C, 56.30; H, 5.45; N, 15.15.; S, 11.5(5. Found: C, 56.45; H, 5.60; N; 15.28; S, 11.58. MS: m/e 277 (M+, 14%), IR, v: 3456-3241 (OH, NH2), 3050 (CH, aromatic), 1685 (C=O). 1H-NMR, 5: 1.83-1.95 (m, 4H, 2CH2), 2.22-2.38 (m, 4H, 2CH2), 2.69 (s, 3H, CH3), 4.40 (s, 2H, NH2), 6.58 (s, 1H, pyrazole H-3), 10.22 (s, 1H, OH). 13C-NMR, 5: 22.9, 24.2, 26.5, 33.1, 35.4, 122.9, 124.2, 133.5, 137.8, 145.6, 147.3, 158.3, 163.5.
3. 1. 5. General Procedure for the Synthesis of the 2-Aminothiophene Derivatives 11a,b
To a solution of acetoacetanilide (1.78 g, 0.01 mol) in ethanol (40 mL) containing triethylamine (1.5 mL), either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added followed by elemental sulfur (0.32 g, 0.01 mol). The whole reaction mixture was heated under reflux for 1 h then poured onto ice/water containing a few drops of hydrochloric acid. The solid product, for-
med in each case, was collected by filtration and crystallized using a suitable solvent.
4-(p-Tolylamino)-5-acetyl-2-aminothiophene-3-carbo-nitrile (11a). Orange crystals from ethanol, yield 73% (2.18 g), m.p. 221-223 °C. Anal. Calculated for C14H13N3OS (271.34): C, 61.97; H, 4.83; N, 15.49; S, 10.71. Found: C, 61.85; H, 4.60; N; 15.18; S, 10.59. MS: m/e 271 (M+, 18%), IR, v: 3458-3322 (NH2, NH), 3054 (CH, aromatic), 2220 (CN), 1689 (C=O). 1H-NMR, 5: 2.68, 2.93 (2s, 6H, 2CH3), 4.42 (s, 2H, NH2), 7.28-7.33 (m, 4H, C6H4), 8.72 (s, 1H, NH). 13C-NMR, 5: 24.8, 26.9, 118.5, 124.6, 126.0, 129.5, 132.5, 119.5, 135.8, 148.2,
155.7,	163.8.
Ethyl 4-(p-tolylamino)-5-acetyl-2-aminothiophene-3-carboxylate (11b). Orange crystals from ethanol, yield 88% (3.45 g), m.p. 172-175 °C. Anal. Calculated for C16H18N2O3S (318.39): C, 60.36; H, 5.70; N, 8.80.; S, 10.07. Found: C, 60.78; H, 5.42; N; 8.93; S, 10.21. MS: m/e 318 (M+, 20%), IR, v: 3456-3320 (NH2, NH), 3055 (CH, aromatic), 1693, 1687 (2 C=O). 1H-NMR, 5: 1.16 (t, 3H, J = 7.22 Hz, CH3), 2.68, 2.91 (2s, 6H, 2CH3), 4.25 (q, 2H, J = 7.22 Hz, CH2), 4.46 (s, 2H, NH2), 7.29-7.37 (m, 4H, C6H4), 8.68 (s, 1H, NH). 13C-NMR, 5: 19.7, 24.2, 26.7, 442, 122.8, 124.7, 128.8, 134.2, 118.9, 136.3,
145.8,	154.8, 160.2, 163.8.
3. 1. 6. General Procedure for the Synthesis of
Phenylacryloylthiophene Derivatives 12a,b
To a solution of either 11a (2.99 g, 0.01 mol) or 11b (3.46 g, 0.01 mol) in 1,4-dioxan (40 mL) containing pipe-ridine (1.0 mL), benzaldehyde (1.0 g, 0.01 mol) was added. The reaction mixture, in each case, was heated under reflux for 8 h then left to cool. The solid product formed upon evaporating the solution under vacuum followed by triturating the remaining product with ethanol, was collected by filtration and crystallized from a suitable solvent.
4-(p-Tolylamino)-2-amino-5-(3-phenylacryloyl)thiop-hene-3-carbontrile (12a). Orange crystals from ethanol, yield 86% (3.32 g), m.p. 232-235 °C. Anal. Calculated for C21H17N3OS (359.44): C, 70.17; H, 4.77; N, 11.69; S, 8.92. Found: C, 70.35; H, 4.68; N; 11.98; S, 8.99. MS: m/e 359 (M+, 23%), IR, v: 3468-3382 (NH2, NH), 3055 (CH, aromatic), 2227 (CN), 1687 (C=O). 1H-NMR, 5: 2.83 (s, 3H, CH3), 4.46 (s, 2H, NH2), 6.56, 6.99 (2d, 2H, CH=CH), 7.31-7.46 (m, 9H, C6H5, C6H4), 8.62 (s, 1H, NH).13C-NMR, 5: 26.4, 117.2, 118.1, 119.0, 120.3, 123.7, 129.5, 124.6, 130.0, 131.5, 132.5, 120.5, 135.8, 148.2, 155.7, 162.8.
Ethyl 4-(^-tolylamino)-2-amino-5-(3-phenylacryloyl) thiophene-3-carboxylate (12b). Orange crystals from acetic acid, yield 69% (2.99 g), m.p. 155-157 °C. Anal.
Calculated for C23H22N2O3S (406.14): C, 67.96; H, 5.46; N, 6.89; S, 7.89. Found: C, 67.66; H, 5.25; N; 6.61; S, 7.76. MS: m/e 406 (M+, 30%), IR, v: 3456-3342 (NH2, NH), 3050 (CH, aromatic), 1689-1687 (2 C=O). 1H-NMR, 5: 1.16 (t, 3H, J = 7.06 Hz, CH3), 2.69 (s, 3H, CH3), 4.25 (q, 2H, J = 7.06 Hz, CH2), 4.64 (s, 2H, NH2), 6.69, 6.86 (2d, 2H, CH=CH), 7.29-27.37 (m, 9H, C6H5,2 C6H4), 8.88 (s, 1H, NH). 13C-NMR, 5: 22.2, 26.9, 46.0, 117.4, 118.9, 120.5, 126.4, 127.6, 136.1, 117.9, 138.0, 146.4, 154.2, 162.8, 163.2.
3. 1. 7. General Procedure for the Synthesis of the Dicyanoprop-1-en-2-yl thiophene Derivatives 13a,b
To a solution of either 11a (2.99 g, 0.01 mol) or 11b (3.46 g, 0.01 mol) in 1,4-dioxan (40 mL) containing triethylamine (1.0 mL), malononitrile (0.66 g, 0.01 mol) was added. The reaction mixture, in each case, was heated under reflux for 3 h then left to cool. The solid product formed upon pouring onto ice/water containing a few drops of hydrochloric acid was collected by filtration and crystallized from a suitable solvent.
4-(p-Tolylamino)-2-amino-5-(1,1-dicyanoprop-1-en-2-yl)thiophene-3-carbonitrile (13a). Orange crystals from ethanol, yield 73% (2.53 g), m.p. 152-154 °C. Anal. Calculated for C17H13N5S (319.38): C, 63.93; H, 4.10; N, 21.93; S, 10.047. Found: C, 63.57; H, 4.01; N; 21.78; S, 10.29. MS: m/e 319 (M+, 38%), IR, v: 3472-3342 (NH2, NH), 3050 (CH, aromatic), 2228, 2220 (2 CN). 1H-NMR, 5: 2.73, 3.02 (2s, 6H, 2CH3), 4.62 (s, 2H, NH2), 7.32-7.45 (m, 9H, C6H5, C6H4), 8.76 (s, 1H, NH). 13C-NMR, 5: 24.8, 26.4, 116.5, 117.24, 118.8, 117.8, 118.9, 123.7, 124.8, 130.5, 134.6, 122.6, 133.9, 146.8, 154.9.
Ethyl 4-(p-tolylamino)-2-amino-5-(1,1-dicyanoprop-1-en-2-yl)thiophene-3-carboxylate (13b). Yellow crystals from 1,4-dioxan, yield 77% (3.03 g), m.p. 133-135 °C. Anal. Calculated for C19H18N4O2S (366.44): C, 62.28; H, 4.95; N, 15.29; S, 8.775. Found: C, 60.76; H, 4.51; N; 13.89; S, 8.62. MS: m/e 366 (M+, 18%), IR, v: 3642-3338 (NH2, NH), 3053 (CH, aromatic), 1690 (C=O). 1H-NMR, 5: 1.13 (t, 3H, J = 7.16 Hz, CH3), 2.70, 3.01 (2s, 6H, 2CH3), 4.22 (q, 2H, J = 7.16 Hz, CH2), 4.55 (s, 2H, NH2), 7.30-7.35 (m, 5H, C6H4), 8.85 (s, 1H, NH). 13C-NMR, 5: 22.0, 26.9, 29.1, 45.5, 416.4, 117.5, 117.4, 118.9, 120.8, 125.9, 126.8, 136.9, 118.3, 136.9, 145.5, 153.0, 164.6.
3. 1. 8. General Procedure for the Synthesis of the Arylhydrazone Derivatives 15a-f
To a cold solution of 13a (3.74 g, 0.01 mol) or 13b (3.94 g, 0.01 mol) in ethanol (40 mL) containing sodium acetate (2.5 g) a cold solution of the respective diazonium salt [prepared by the addition of sodium nitrite solution
(0.70 g, 0.01 mol) to a cold solution of either aniline (0.94 g, 0.01 mol), p-toluidine (1.15 g, 0.01 mol) or p-chloroa-niline (1.29 g, 0.01 mol) in concentrated hydrochloric acid (12 mL) with continuous stirring] was added while stirring. The formed solid product, in each case, upon stirring at room temperature for 1 h was collected by filtration and crystallized using a suitable solvent.
5-(3-(2-Phenylhydrazono)-1,1-dicyanoprop-1-en-2-yl)-
4-(p-tolylamino)	-2-aminothiophene-3-carbonitrile (15a). Brown crystals from ethanol, yield 80% (3.61 g), m.p. 182-184 °C. Anal. Calculated for C23H17N7S (423.49): C, 65.23; H, 4.05; N, 23.15; S, 7.57. Found: C, 65.66; H, 4.29; N; 23.48; S, 7.49. MS: m/e 423 (M+, 12%), IR, v: 3462-3348 (NH2, 2NH), 3053 (CH, aromatic), 2226, 2222-2220 (3 CN). 1H-NMR, 5: 3.02 (s, 3H, CH3), 4.42 (s, 2H, NH2), 6.62 (s, 1H, N=CH), 7.30-7.43 (m, 9H, C6H5, C6H4), 8.38, 8.56 (2s, 2H, 2NH). 13C-NMR, 5: 22.6, 116.8, 1177.6, 118.3, 117.4, 118.9, 120.3, 122.7, 123.0, 123.9, 124.2, 131.5, 136.3, 120.3, 126.8, 144.6, 152.5, 156.0, 158.3, 167.0.
5-(3-(2-p-Tolylhydrazono)-1,1-dicyanoprop-1-en-2-yl)-4-(p-tolylamino)-2-aminothiophene-3-carbonitrile (15b). Orange crystals from ethanol, yield 85% (3.95 g), m.p. 192-194 °C. Anal. Calculated for C24H19N7S (437.52): C, 65.88; H, 4.38; N, 22.41; S, 7.33. Found: C, 65.92; H, 4.29; N; 22.38; S, 7.28. MS: m/e 437 (M+, 8%), IR, v: 3460-3343 (NH2, 2NH), 3056 (CH, aromatic), 2228, 2224-2220 (3 CN). 1H-NMR, 5: 2.99, 3.01 (2s, 6H, 2CH3), 4.46 (s, 2H, NH2), 6.59 (s, 1H, N=CH), 7.28-7.46 (m, 8H, 2 C6H4), 8.39, 8.45 (2s, 2H, 2NH). 13C-NMR, 5: 24.3, 116.7, 117.4, 118.2, 119.4, 122.9, 123.4, 124.9, 134.4, 134.5, 143.9, 123.9, 125.6, 144.2, 154.8, 156.1, 159.3, 167.2.
2-(3-(2-p-Chlorohydrazono)-1,1-dicyanoprop-1-en-2-yl)-5-amino-4-(p-tolylamino)-2-aminothiophene-3-car-bonitrile (15c). Orange crystals from ethanol, yield 67% (3.25 g), m.p. 222-225 °C. Anal. Calculated for C23H16ClN7S (457.94): C, 60.32; H, 3.52; N, 21.41; S, 7.(30. Found: C, 60.36; H, 3.36; N; 21.31; S, 7.22. MS: m/e 485 (M+, 44%), IR, v: 3473-3339 (NH2,2NH), 3059 (CH, aromatic), 2224, 2224-2222 (3 CN). 1^-NMR, 5: 3.01 (s, 3H, CH3), 4.42 (s, 2H, NH2), 6.62 (s, 1H, N=CH), 7.29-7.433 (m, 8H, 2 C6H4), 82.36, 8.29 (2s, 2H, 2NH). 13C-NMR, 5: 116.3, 117.8, 118.4, 119.2, 122.4, 123.6, 123.9, 133.5, 134.7, 144.3, 124.2, 126.0, 144.1, 154.4, 156.2, 158.0, 167.0.
Ethyl 5-(3-(2-phenylhydrazono)-1,1-dicyano-1-en-2-yl)-4-(p-tolylamino)-2-aminothiophene-3-carboxylate (15d). Orange crystals from ethanol, yield 70% (3.49 g), m.p. > 300 °C. Anal. Calculated for C25H22N6O2S (470.55): C, 63.81; H, 4.71; N, 17.86; S, 6.81. Found: C, 63.69; H, 4.54; N; 17.79; S, 6.68. MS: m/e 470 (M+,
20%), IR, v: 3658-3332 (NH2, NH), 3056 (CH, aromatic), 1690 (C=O). 1H-NMR, 5: 1.14 (t, 3H, J = 7.41 Hz, CH3), 3.05 (s, 3H, CH3), 4.24 (q, 2H, J = 7.41 Hz, CH2), 4.63 (s, 2H, NH2), 7.31-7.43 (m, 9H, C6H5, C6H4), 8.44, 8.65 (2s, 2H, 2NH). 13C-NMR, 5: 16.8, 5755, 116.8, 118.6, 119.9, 122.6, 122.9, 123.9, 133.3, 134.7, 144.0, 124.0, 126.4, 144.1, 154.8, 156.8, 158.4, 167.2.
Ethyl 5-(3-(2-p-tolylhydrazono)-1,1-dicyano-1-en-2-yl)-4-(p-tolylamino)-2-aminothiophene-3-carboxylate (15e). Reddish brown crystals from ethanol, yield 65% (3.33 g), m.p. 266-269 °C. Anal. Calculated for C26H24N6O2S (484.57): C, 64.44; H, 4.99; N, 17.34; S, 6.62. Found: C, 64.33; H, 4.67; N; 17.39; S, 6.48. MS: m/e 484 (M+, 6%), IR, v: 3663-3338 (NH2, NH), 3053 (CH, aromatic), 1688 (C=O). 1H-NMR, 5: 1.13 (t, 3H, J = 7.22 Hz, CH3), 2.94, 3.08 (2s, 6H, 2CH3), 4.25 (q, 2H, J = 7.22 Hz, CH2), 4.77 (s, 2H, NH2), 7.28-7.40 (m, 8H, 2C6H4), 8.46, 8.63 (2s, 2H, 2NH). 13C-NMR, 5: 16.5. 19.8, 58.2, 116.4, 118.2, 121.5, 122.8, 123.7, 133.3, 136.7, 144.2, 124.0, 126.4, 144.1, 154.8, 156.3, 158.6, 164.8, 167.0.
Ethyl 5-(3-(2-p -chlorophenylhydrazono)-1,1-dicyano-1-en-2-yl)-4-(p-tolylamino)-2-aminothiophene-3-car-boxylate (15f). Reddish brown crystals from ethanol, yield 77% (4.10 g), m.p. 244-246 °C. Anal. Calculated for C25H21ClN6O2S (504.99): C, 59.46; H, 4.19; N, 16.64; S, 6.35. Found: C, 59.53; H, 4.03; N; 16.44; S, 6.28. MS: m/e 533 (M+, 35%), IR, v: 3660-3336 (NH2, NH), 3058 (CH, aromatic), 1688 (C=O). 1H-NMR, 5: 1.15 (t, 3H, J = 7.22 Hz, CH3), 2.96 (s, 3H, CH3), 4.23 (q, 2H, J = 7.22 Hz, CH2), 4.68 (s, 2H, NH2), 7.26-7.43 (m, 8H, 2C6H4), 8.38, 8.53 (2s, 2H, 2NH). 13C-NMR, 5: 16.9, 19.9, 58.8, 116.7, 118.4, 121.6, 122.6, 123.9, 134.2, 136.7, 144.8, 124.3, 126.8, 144.6, 154.2, 156.0, 158.8, 164.5, 167.3.
3. 1. 9. General Procedure for the Synthesis of the Thiophen-3-yl-thiophene Derivatives 16a-d
To a solution of either 11a (2.99 g, 0.01 mol) or 11b (3.46 g, 0.01 mol) in 1,4-dioxan (40 mL) containing triethylamine (1.0 mL), either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) and elemental sulfur (0.32 g, 0.01 mol) were added. The reaction mixture, in each case, was heated under reflux for 2 h then left to cool. The solid product formed upon pouring onto ice/water containing few drops of hydrochloric acid was collected by filtration and crystallized from a suitable solvent.
4-(p-Tolylamino)-2-amino-5-(5-amino-4-cyanothiop-hen-3-yl)-thiophene-3-carbonitrile (16a). Pale yellow crystals from acetic acid, yield 74% (2.80 g), m.p. 199-202 °C. Anal. Calculated for C17H13N5S2 (351.45): C, 58.10; H, 3.73; N, 19.93; S, 18.25. Found: C, 58.29; H,
3.59; N; 19.91; S, 18.39. MS: m/e 351 (M+, 12%), IR, v: 34655-3332 (NH2, 2NH), 3058 (CH, aromatic), 2229, 2220 (2 CN). 1H-iNMR, 5: 2.99 (s, 3H, CH3), 4.95, 5.05 (2s, 4H, 2NH2), 6.70 (s, 1H, thiophene H-53), 7.28-7.39 (m, 4H, C6H4), 8.38, 8.22 (s, 1H, NH). 13C-NMR, 5: 23.4, 116.4, 118.2, 120.3, 121.9, 123.4, 123.9, 124.3, 126.2, 133.8, 134.5, 144.8, 154.6.
Ethyl 4-(3-p-tolylamino)-5-amino-4-cyanothiophen-2-yl)-2-aminothiophene-3-carboxylate (16b). Colourless crystals from acetic acid, yield 66% (2.81 g), m.p. 158-162 °C. Anal. Calculated for C20H18N4O3S2 (426.51): C, 56.32; H, 4.25; N, 13.14; S, 15.040. Found: C, 56.59; H, 4.39; N; 13.09; S, 14.87. MS: m/e 426 (M+, 31%), IR, v: 3478-3351 (2NH2, NH), 3058 (CH, aromatic), 2222 (CN), 1689 (C=O). 1H-NMR, 5: 1.25 (t, 3H, J = 5.44 Hz, CH3), 2.85, (s, 3H, CH3), 4.22 (q, 2H, J = 5.44 Hz, CH2), 4.493, 5.20 (2s, 4H, 2NH2), 6.60 (s, 1H, thiophene H-5), 7.32-7.42 (m, 4H, C6H4), 8.26 (s, H, NH). 13C-NMR, 5: 17.0, 18.9, 117.9, 120.8, 121.3, 123.8, 124.0, 124.6,
125.8,	133.2, 134.5, 144.9, 154.2, 163.2, 164.5.
Ethyl 4-(p -tolylamino)-2-amino-5-(5-amino-4-cyanot-hiophen-3-yl)thiophene-3-carboxylate (16c). Pale yellow crystals from acetic acid, yield 55% (2.34 g), m.p. 177-179 °C. Anal. Calculated for C19H18N4O2S2 (398.50): C, 57.27; H, 4.55; N, 14.06; S, 16.09. Found: C, 57.46; H, 4.41; N; 14.36; S, 16.27. MS: m/e 398 (M+, 26%), IR, v: 3488-3352 (2NH2, NH), 3056 (CH, aromatic), 2220 (CN), 1684 (C=O)2 1H-NMR, 5: 1.36 (t, 3H, J = 6.72 Hz, CH3), 2.84, (s, 3H, CH3), 4.26 (q, 2H, J = 6.72 Hz, CH2), 4.4(3, 5.15 (2s, 4H, 2NH2), 6.62 (s, 1H, thiophene H-5), 7.30-7.40 (m, 4H, C6H4), 8.31 (s, H, NH). 13C-NMR, 5: 17.3, 18.6, 118.6, 120.2, 121.5, 123.5, 124.2, 124.6,
124.9,	133.2, 134.7, 146.0, 156.8, 163.7.
Ethyl 5-(4-ethoxycarbonyl)-4-(p-tolylamino)-2-ami-nothiophen-3-yl)-2-amino-thiophene-3-carboxylate (16d). Orange crystals from ethanol, yield 64% (3.03 g), m.p. 188-190 °C. Anal. Calculated for C21H23N3O4S2 (455.56): C, 56.61; H, 5.20; N, 9.43; S, 14.39. Found: C, 56.41; H, 5.54; N; 9.39; S, 14.58. MS: m/e 473 (M+, 6%), IR, v: 3675-3342 (2NH2, NH), 3058 (CH, aromatic), 1692-1688 (2 C=O). 1H-1NMR, 5: 1.13, 1.16 (2t, 6H, J = 7.02, 6.53 Hz, 2CH3), 2.89 (s, 3H, CH3), 4.23, 4.27 (2q, 4H, J = 7.02, 6.53 Hz, 2CH2), 4.48, 4.62 (2s, 4H, 2NH2), 7.26-7.40 (m, 4H, C6H4), 8.32 (s, 1H, NH). 13C-NMR, 5: 17.0, 17.8, 18.4, 117.8, 120.4, 122.7, 123.8, 125.5, 125.6, 126.0, 133.7, 134.3, 145.6, 156.2, 163.3, 164.5.
4. Conclusions
The above results allow the conclusion that administration of the tested compounds to the cancer cell lines showed promising anticancer activity. The most potent
compounds are 7d, 12a, 16a and 16b showing the highest inhibitory effects and displaying selectivities which are much higher than the reference doxorubicin.
5. Acknowledgement
R. M. Mohareb would like to thank Alexander von Humboldt Foundation in Bonn for affording him a fellowship in München during summer 2012.
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Povzetek
Pri reakciji cikloheksanona oz. ciklopentanona s cianoacetilhidrazinom ter elementarnim žveplom sta nastala 2-ami-nocikloalkeno[ft]tiofenska derivata 3a oz. 3b. Pri nadaljnji reakciji teh dveh spojin z aromatskimi benzaldehidi oz. reagenti, ki vsebujejo aktivno metilensko skupino, nastanejo Schiffove baze 5a-d oz. pirazolski derivati 7a-d in 9a-d. Po drugi strani pa pri reakciji 3-okso-N-^-tolilbutanamida (10) z malononitrilom oz. etil cianoacetatom nastaneta tiofenska derivata 13a oz. 13b. Spojini 13a,b sta v seriji heterociklizacijskih reakcij tvorili različne heterociklične derivate. Določena je bila tudi citotoksičnost pripravljenih spojin proti trem človeškim tumorskim celičnim linijam, t.j. adenokarci-nomom dojke (MCF-7), pljučnemu raku (NCI-H460) and raku centralnega živčenga sistema (SF-268). Za primerjavo je bila še dodatno določena aktivnost proti normalnim človeškim fibroblastom WI 38.