Sulphur containing heterocycles paved the way for active research in pharmaceutical Chemistry. Nowadays benzothiophene derivatives in combination with other ring systems are extensively used in pharmaceuticals such as antiallergic [1], analgesic [2], antiinflammatory [3] and occular hypotensive agents [4]. Raloxifene, a drug based on benzo[b]thiophene has been approved by the U.S Food and Drug Administration for the prevention and treatment of osteoporosis associated with menopause [5]. On the other hand, compounds bearing N-containing rings are very well known to exhibit powerful antimicrobial [6], anticonvulsant [7], antidepressant [8], and analgesic [9] activities. Moreover, some nitrogen and sulfur containing compounds are associated with diverse pharmacological activities [10-14]. Furthermore, various oxadiazoles [15], pyrazolin-5-ones [16], and diaryl pyrazole derivatives [17] also exhibit wide spectrum pharmacological activities.

On the way of continuing our work [18-20] on the synthesis of new heterocyclic compounds with expected biological activities, we hereby report the synthesis of some new cyclopenta[b]thiophene derivatives and their characterization by IR, NMR & Mass spectrometry techniques. Newly synthesized compounds were also screened for their antitumor evaluation against the three cancer cells namely breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and VNS cancer (SF-268). Moreover, the anti-leishmanial activity of the newly synthesized products was tested on Leishmania amastigotes.

In this research work, the reaction of cyclopentanone 1 and elemental sulfur with cyanomethylene reagents was studied. Thus, the former two reagents have been allowed to react with either malononitrile 2a or ethyl cyanoacetate 2b and gave the cyclopenta[b] thiophenes 3a,b, respectively [21].

The reaction of 3a with malononitrile 2a in ethanolic triethylamine solution gave the fused pyridine derivative 5 [22]. Formation of the latter compound is suggested to have taken place via the intermediate formation of 4 followed by intramolecular cyclization, this is explained in terms of activation of the CH₂ between the electronegative CN group and the sp² C. And the assigned structures for the previous compounds are consistent with analytical and spectral data. However, the reaction of 3a with ethyl cyanoacetate 2b gave the amide derivative 6, whose H¹NMR spectrum showed a multiplet at δ 2.22-2.32 corresponding to the hydrogens of the three (CH₂) groups in the cyclopentene moiety, a singlet at δ 4.75 and another at δ 8.23 corresponding to the methylene group protons and the NH hydrogen, respectively (scheme 3). Compound 6 underwent ready cyclization when heated in 1,4-dioxane and triethylamine to yield the cyclopenta[4,5]thieno[2,3-b] pyridine derivative 7 (scheme 1).

scheme 1: Reagents and conditions: (i) EtOH/Et₃N, reflux 1h; (ii), 1,4-dioxan/Et₃N, reflux 1h; (iii) dry conditions, oilbath 140°C, (iv) 1,4-dioxan/ Et₃N, reflux.

The reaction of compound 6 with benzaldehyde 8 gave the benzalidine derivative 9, while its reaction with salicaldehyde 10 produced the coumarin derivative 12, supposedly through the formation of the arylidine derivative 11 followed by intramolecular cyclization and hydrolysis of the C=NH group to a C=O group. Formation of the latter coumarin derivative 12 through the reaction of salicaldehyde 10 with cyanomethylene reagents was previously reported in literature [23]. On the other hand, the reaction of compound 6 with benzenediazonium chloride 13 gave the phenylhydrazone derivative 14 whose analytical and spectral data are in agreement with the proposed structure. Compound 14, upon reacting with either hydrazine hydrate 15a or phenyl hydrazine 15b, gave the pyrazole derivatives 17a and 17b, respectively. Formation of the latter compounds took place, supposedly, through the intermediates 16a,b followed by water elimination (Scheme 2).

scheme 2: Reagents and conditions: (i) 1,4-dioxane/Et₃N, reflux; (ii) 1,4-dioxane/Et3N; (iii) EtOH, NaOH. 0°C; (iv) 1,4-dioxane, reflux.

The reaction of compound 6 with either malononitrile 2a or ethyl cyanoacetate 2b gave the pyridine derivatives 19a,b, respectively. Formation of the latter pyridines is explained in terms of the intermediate formation of 18a,b followed by intramolecular cyclization, and structure elucidation is based on analytical and spectral data. Thus, the H¹NMR spectrum of 19a, for instance, showed a multiplet at δ 2.24- 2.39 corresponding to the hydrogens of the three (CH₂) groups in the cyclopentene moiety, two singlets (D₂O-exchangeable) for hydrogens of the two amino groups at δ 4.27 and 5.31 and another D2Oexchangeable singlet at δ 6.51 corresponding to H-3 on the pyridine ring. Moreover, the ¹³C NMR spectrum showed δ values of 22.1, 26.2, 32.7 (3 CH₂), 116.0 (CN), 119.8, 121.0, 121.3, 122.2, 123.7, 129.0, 139.9, 143.8, 152.3, 155.0 (thiophene C, pyridine C). Compound 3a has also reacted with phenylisothiocyanate 20 in 1,4-dioxane and triethylamine to give the phenyl thiourea derivative 21 (Scheme 3). Compound 21 has been readily cyclized by heating in 1,4-dioxane in the presence of a catalytic amount of triethylamine, to give the thioxo-pyrimidine derivative 22. The analytical data of the latter product was consistent with the proposed structure (see experimental section).

Materials and methods

Materials, methods & 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). Samples: Stock solutions of compounds 3a-9d were prepared in DMSO and kept at -20^oC. Appropriate dilutions of the compounds were freshly done just prior to the assays. Final concentrations of DMSO did not interfere with the cell growth.

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) and NCI-H460 and SF-268 were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt). They grow as monolayer and routinely maintained in RPMI-1640 medium supplemented with 5% heat inactivated FBS, 2 mM glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/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.

Effect of the synthesized compounds on the growth of human tumor cell lines

All the synthesized compounds were 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), after a continuous exposure of 48 h. The results are summarized in Table 1. It is clear that compound 22 showed the best inhibition towards the three cell lines.

Table 1: Effect of compounds 3a-21b on the growth of three human tumor cell lines.

Anti-leishmanial activity

Anti-leishmanial activity was tested on L. donovani amastigotes growing in macrophages at concentrations, which showed less than 40% cytotoxicity for the macrophage cell line THP-1. The compounds 7, 9, 14, 17, 19a and 21 showed high activity on L. donovani amastigotes growing in macrophages similar to that seen with axenic amastigotes at 50 μM, 95 %, 68 %, 80 %, 88 %, 86 % and 98 %, respectively. It is also obvious that compounds 7 and 21, namely 5-Amino-7-oxo-3,4- dihydro-2H-cyclopenta[4,5]thieno[2,3-b]pyridine-6-carbonitrile and 1-(3-Cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-3-phenylthiourea, showed maximum average inhibition values that are higher than the positive control (Table 2). Moreover, it could be deduced that substitution by a nitro group enhances the anti-leishmanial activity. This becomes evident by comparing the activities of 3a, 5 and 56 with average inhibitions of 22%, 20% and 24%, respectively, and those of 3b, 12, 17b and 19b with average inhibitions of 32%, 42%, 56% and 55% respectively.

Table 2: Anti-leishmanial activity of compounds 3a-9d at 50 μM against L. donovani axenic amastigotes.

Chemistry

All melting points were determined in open capillaries and are uncorrected. IR spectra were measured using KBr discs on a Pye Unicam SP-1000 spectrophotometer. ¹H-NMR and ¹³CNMR spectra were measured on a Varian EM390-200 MHz instrument in CD₃SOCD₃ as solvent using TMS as internal standard, and chemical shifts are expressed as δ in units of parts per million (ppm). Analytical data were obtained from the Micro analytical data unit at Cairo University. MS spectra were determined using a Shimadzu GC–MS- 2010P. Compounds 3a and 3b were synthesized according to the reported procedure.

5,7-Diamino-3,4-dihydro-2H-cyclopenta[4,5]thieno[2,3-b]pyridine-6-carbonitrile (5)

Malononitrile (0.12 g, 1.817×10^-3 mol) and 0.5 mL of triethylamine were added to a solution of 2-amino-5,6-dihydro-4H-cyclopenta[b] thiophene-3-carbonitrile 3a (0.3 g, 1.817×10^-3 mol) in 25 mL of 1,4-dioxane and the reaction mixture was heated under reflux for 3 h. It was then poured on ice and a few HCl (18.0 mol) drops (till pH₆) stirred in. Filtration of the observed precipitate was then carried out.

Light brown crystals from ethanol, yield 52.38 %, 0.22 g, m.p. 138- 141 ^oC. IR (KBr): υ/cm^-1= 3482-3346 (2 NH₂), 2892 (CH₂), 2223 (CN), 1648 (C=C), 1632 (C=N). H¹NMR (DMSO) δ = 2.24-2.38 (m, 6H, 3CH₂), 4.80, 5.31 (2s, 4H, 2NH₂). ¹³C NMR δ = 20.4, 26.9, 32.5 (3 CH₂), 116.8 (CN), 127.2, 134.4, 136.3, 144.4, 144.8, 154.8, 156.9 (thiophene C, pyridine C). Calcd for C₁₁H₁₀N₄S (230.293): C, 57.37; H, 4.38; N, 24.33, S, 13.93 %. Found: C, 57.62; H, 4.40; N, 24.43; S, 14.12 %. MS (relative intensity) m/z: 230 (M+, 100 %), 231 (M+1, 13 %), 232 (4 %), 176 (65 %).

2-Cyano-N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2- yl)acetamide (6)

Ethyl cyanoacetate (2.07 g, 0.018 mol) was fused at a temperature of 250 ^oC with 2-amino-5,6-dihydro-4H-cyclopenta[b]thiophene- 3-carbonitrile 3a (2.96 g, 0.018 mol) in a solvent-free catalyst-free reaction which yielded the target amide in 1 h. Water was then added and the precipitate filtered out. Grey crystals from ethanol yield 86.54 %, 3.60 g, m.p. 290-293 ^oC. IR (KBr): υ/cm^-1= 3469-3322 (NH), 2988 (CH₂), 2228, 2222 (2 CN), 1689 (CO), 1641 (C=C). H¹NMR (DMSO) δ = 2.20-2.33 (m, 6H, 3 CH₂), 4.75 (s, 2H, CH₂), 8.23 (s, 1H, NH). ¹³C NMR δ = 19.9, 26.7, 32.8 (3 CH₂), 27.8 (CH₂), 116.2, 117.0 (2CN), 127.0, 134.6, 137.0, 143.9, 144.5 (thiophene C), 169.0 (C=O). Calcd for C₁₁H₉N₃OS (231.278): C, 57.13; H, 3.92; N, 18.17; S, 13.87 %. Found: C, 57.25; H, 3.98; N, 18.39; S, 13.97 %. MS (relative intensity) m/z: 231 (M⁺, 100%), 232 (M+2, 5%), 191 (70%), 163 (36%).

5-Amino-7-oxo-3,4-dihydro-2H-cyclopenta[4,5]thieno[2,3-b] pyridine-6-carbonitrile (7)

Compound 6 (0.5 g, 2.162×10^-3 mol) was allowed to cyclize by heating under reflux for 2 h in a mixture of 1,4-dioxane (15 mL) and 0.5 mL of triethylamine as a catalyst. The reaction mixture was then poured on ice to which a few drops of HCl (18.0 mol) were added (till pH₆) and the formed precipitate was then filtered out.

Grey crystals from ethanol, yield 94 %, 0.47 g, m.p. <300^oC. IR (KBr): υ/cm^-1= 3464-3332 (NH₂, NH), 2893 (CH₂), 2225 (CN), 1692 (CO), 1633 (C=C). H1NMR (DMSO) δ = 2.20-2.36 (m, 6H, 3CH₂), 4.46 (s, 2H, NH₂), 8.88 (s, 1H, NH). ¹³C NMR δ = 21.2, 25.8, 32.9 (3 CH₂), 116.9 (CN), 90.7, 126.9, 136.8, 136.0, 144.8, 145.3, 158.5, 159.7 (thiophene C, pyridine C). Calcd for C₁₁H₉N₃OS (231.278): C, 57.13; H, 3.92; N, 18.17; S, 13.86 %. Found: C, 57.15; H, 3.99; N, 18.25; S, 13.97 %. MS (relative intensity) m/z: 231 (M+, 100 %), 233 (M+2, 6%), 214 (42%), 186 (16%).

2-Cyano-N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2- yl)-3-phenylacrylamide (9)

Compound 6 (0.3 g, 1.30×10^-3 mol) was dissolved in 1,4-dioxane (10 mL) and allowed to condense with benzaldehyde (0.14 g, 1.30×10^-3 mol) by heating the two under reflux for 3 h with 0.5 mL of piperidine added as a catalyst. All contents were then poured on ice, a few HCl (18.0 mol) drops added (till pH₆), and the reaction mixture stirred for a few minutes after which the precipitate was filtered out.

Brown crystals from ethanol, yield 66.67 %, 0.28 g, m.p. 292-296^oC. IR (KBr): υ/cm^-1= 3422-3320 (NH), 2890 (CH₂), 2227, 2222 (2CN), 1688 (CO), 1643 (C=C), 3055 (CH aromatic). H¹NMR (DMSO) δ = 2.23-2.35 (m, 6H, 3 CH₂), 6.21 (s, 1H, CH=C), 8.28 (s, 1H, NH), 7.25-7.34 (m, 5H, C₆H₅). ¹³C NMR δ = 21.7, 26.3, 32.6 (3 CH₂), 98.8, 153.6 (C=CH), 116.2, 117.3 (2 CN), 122.8, 125.8, 126.4, 128.7, 129.4, 136.6, 136.0, 144.8 (thiophene C, pyridine C), 168.8 (C=O). Calcd for C₁₈H₁₃N₃OS (319.38): C, 67.69; H, 4.10; N, 13.16; S, 10.04 %. Found: C, 67.87; H, 4.13; N, 13.41; S, 10.18 %. MS (relative intensity) m/z: 319 (M+, 100%), 191 (20%), 163 (32%), 92 (70 %).

N-(3-Cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-oxo-2H-chromene-3-carboxamide (12)

Compound 6 (0.3 g, 1.30×10^-3 mol) was dissolved in 1,4-dioxane (10 mL) and allowed to condense with salicaldehyde (0.16 g, 1.30×10^-3 mol) by heating the two under reflux for 2 h with piperidine (0.5 mL) added as a catalyst. All contents were then poured on ice, a few HCl (18.0 mol) drops added (till pH₆), and the reaction mixture stirred for a few minutes after which the precipitate was filtered out.

Dark brown crystals from ethanol, yield 81.82 %, 0.36 g, m.p. 289- 292 ^oC. IR (KBr): υ/cm^-1= 3467-3341(NH), 3055 (CH aromatic), 2890 (CH₂), 2225 (CN), 1693, 1688 (2CO), 1646 (C=C). H¹NMR (DMSO) δ = 2.22-2.33 (m, 6H, 3CH₂), 6.89 (s, 1H, coumarin H-4), 7.26-7.35 (m, 4H, C₆H₄), 8.30 (s, 1H, NH). ¹³C NMR δ = 21.9, 26.7, 32.2 (3 CH₂), 116.0 (CN), 120.4, 124.5, 125.3, 126.4, 127.9, 139.0, 140.8, 152.0 (thiophene C, coumarin C), 159.4, 166.8 (2 C=O). Calcd for C₁₈H₁₂N₂O₃S (336.36): C, 64.27; H, 3.60; N, 8.33; S, 9.53 %. Found: C, 64.42; H, 3.75; N 8.45; S, 9.79 %. MS (relative intensity) m/z: 336 (M⁺, 68%), 338 (M+2, %), 173 (100%), 145 (70%).

2-(3-Cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-ylamino)-2-oxo-N’-phenylacetohydrazonoyl cyanide (14)

To a solution of compound 6 (0.3 g, 1.30×10^-3 mol) in ethanol (15 mL), containing sodium hydroxide pellets (0.5 g), benzenediazonium chloride (0.18 g, 1.30×10^-3 mol) [prepared by adding an aqueous (20 mL of water) sodium nitrite solution (0.09 g, 1.30×10^-3 mol) to a cold solution of aniline (0.14 g, 1.30×10^-3 mol) in 30 mL of conc. HCl (18.0 mol) at 0-5^oC, with continuous stirring] was added and the coupling reaction was allowed to proceed. The formed precipitate after 1 h was collected by filtration.

Light brown crystals from ethanol, yield 29.55 %, 0.13 g, m.p. 296- 299^oC. IR (KBr): υ/cm^-1= 3486-3338 (2NH), 3060 (CH aromatic), 2893 (CH₂), 2223-2220 (2CN), 1687 (CO), 1648 (C=C), 1634 (C=N). H¹NMR (DMSO) δ = 2.24-2.38 (m, 6H, 3CH₂), 7.28-7.38 (m, 5H, C₆H₅), 8.32, 8.89 (2s, 2H, 2NH). ¹³C NMR δ = 21.7, 26.7, 32.0 (3 CH₂), 115.8, 116.8 (2 CN), 119.6, 120.5, 121.7, 122.6, 126.4, 125.3, 126.4, 139.0, 143.4 (thiophene C, C6H5), 163.5 (C=O), 168.4 (C=N). Calcd for C₁₇H₁₃N₅OS (335.38): C, 60.88; H, 3.91; N, 20.88; S, 9.56 %. Found: C, 60.93; H, 3.99; N, 20.99; S, 9.64 %. MS (relative intensity) m/z: 335 (M⁺, 43%), 337 (M+2, 4%), 258 (15%0, 234 (55%), 177 (80%), 92 (100%).

2-(3-Amino-4-(phenyldiazenyl)-1H-pyrazol-5-ylamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile (17a)

To a solution of the hydrazo compound 14 (0.3 g, 8.95×10^-4 mol) in 1,4-dioxane (40 mL), hydrazine hydrate (0.06 g, 8.95×10^-4 mol) was added and the reaction mixture was subjected to heating under reflux for 3 h, after which it was poured on ice to which a few drops of HCl (18.0 mol) were added (till pH₆). The precipitated crystals were then filtered out.

Yellow crystals from ethanol yield 32.26 %, 0.1 g, m.p. >300^oC. IR (KBr): υ/cm^-1 = 3522-3332 (NH₂, 2NH), 3057 (CH aromatic), 2895 (CH₂), 2222 (CN), 1645 (C=C), 1637 (C=N). H¹NMR (DMSO) δ = 2.22-2.37 (m, 6H, 3CH₂), 4.51 (s, 2H, NH₂), 7.30-7.39 (m, 5H, C₆H₅), 8.33, 8.37 (2s, 2H, 2NH). ¹³C NMR δ = 22.0, 26.7, 32.4 (3 CH₂), 116.9 (CN), 90.6, 120.3, 120.5, 121.9, 123.8, 125.6, 125.9, 128.5, 139.7, 144.0, 150.8, 154.6 (thiophene C, pyrazole C, C₆H₅). Calcd for C₁₇H₁₅N₇S (349.41): C, 58.44; H, 4.33; N, 28.06; S, 9.18 %. Found: C, 58.64; H, 4.41; N, 28.30; S, 9.31 %. MS (relative intensity) m/z: 349 (M⁺, 68%), 350 (12%), 332 (15%), 303 (25%), 187 (100%), 84 (80%).

2-(3-Amino-1-phenyl-4-(phenyldiazenyl)-1H-pyrazol-5- ylamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile (17b)

To a solution of the hydrazo compound 14 (0.3 g, 8.95×10^-4 mol) in 1,4-dioxane (40 mL), phenyl hydrazine (0.1 g, 8.95×10^-4 mol) was added and the reaction mixture was subjected to reflux for 2.5 h, after which it was poured on ice to which a few drops of HCl (18.0 mol) were added till a solid precipitate is being formed.

Yellow crystals from ethanol yield 34.21 %, 0.13 g, m.p. 289-292 ^oC. IR (KBr): υ/cm^-1= 3530-3348 (NH₂, NH), 3059 (CH aromatic), 2893 (CH₂), 2220 (CN), 1647 (C=C), 1639 (C=N). H¹NMR (DMSO) δ = 2.21-2.36 (m, 6H, 3CH₂), 4.48 (s, 2H, NH₂), 7.29-7.43 (m, 10H, 2C₆H₅), 8.36 (s, 1H, NH). ¹³C NMR δ = 22.2, 26.9, 32.2 (3 CH₂), 116.3 (CN), 92.8, 120.4, 120.8, 121.3, 122.9, 124.2, 125.9, 128.5, 129.0, 129.6, 139.9, 144.4, 152.3, 156.2 (thiophene C, pyrazole C, 2C₆H₅). Calcd for C₂₃H₁₉N₇S (425.51): C, 64.92; H, 4.50; N, 23.04; S, 7.54 %. Found: C, 65.10; H, 4.56; N, 23.19; S, 7.65 %. MS (relative intensity) m/z: 425 (M⁺, 28%), 427 (M+2), 348 (40%), 320 (57%), 236 (100%), 163 (52%).

4,6-Diamino-1-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (19a)

To a solution of compound 6 (0.3 g, 1.30×10^-3 mol) in 1,4-dioxane (50 mL), malononitrile (0.09 g, 1.30×10^-3 mol) and 0.5 mL of triethylamine were added and the reactants were heated under reflux for 2 h, after which the mixture was poured on ice to which a few HCl (18.0 mol) drops were added (till pH₆). The precipitate was allowed to coagulate and was then filtered out.

Dark brown crystals from ethanol, yield 89.74 %, 0.35 g, m.p. 285- 289 ^oC. IR (KBr): υ/cm^-1= 3496-3343 (2NH₂), 3059 (CH aromatic), 2887 (CH₂), 2229, 2220 (2CN), 1649 (C=C), 1692 (CO). H¹NMR (DMSO) δ = 2.24-2.39 (m, 6H, 3CH₂), 4.27, 5.31 (2s, 4H, 2NH₂), 6.51 (s, 1H, pyridine H-3). ¹³C NMR δ = 22.1, 26.2, 32.7 (3 CH₂), 116.0 (CN), 119.8, 121.0, 121.3, 122.2, 123.7, 129.0, 139.9, 143.8, 152.3, 155.0 (thiophene C, pyridine C). Calcd for C₁₄H₁₁N₅OS (297.34): C, 56.55; H, 3.73; N, 23.55; S, 10.78 %. Found: C, 56.71; H, 3.63; N, 23.61; S, 10.66 %. MS (relative intensity) m/z: 297 (M⁺, 100%), 299 (M+2, 6%), 281 (28%), 231 (60%).

4-Amino-1-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2- yl)-6-hydroxy-2-oxo-1,2-dihydropyridine-3-carbonitrile (19b)

To a solution of compound 6 (0.3 g, 1.30×10^-3 mol) in 1,4-dioxane (50 mL), ethyl cyanoacetate (0.15 g, 1.30×10^-3 mol) and 0.5 mL of triethylamine were added and the reactants were heated under reflux for 3 h, after which the mixture was poured on ice to which a few HCl (18.0 mol) drops were added (till pH₆). The precipitate was allowed to coagulate and then filtered out.

Pale brown crystals from ethanol, yield 51.28 %, 0.2 g, m.p. oC. IR (KBr): υ/cm^-1= 3585-3333 (OH, NH₂), 3049 (CH aromatic), 2893 (CH₂), 2227, 2222 (2CN), 1646 (C=C), 1692 (CO). H¹NMR (DMSO) δ = 2.22-2.36 (m, 6H, 3CH₂), 4.29 (s, 2H, NH₂), 6.69 (s, 1H, pyridine H-3), 10.41 (s, 1H, OH). ¹³C NMR δ = 22.1, 26.2, 32.7 (3 CH₂), 116.0 (CN), 119.8, 121.0, 121.3, 122.2, 123.7, 129.0, 139.9, 143.8, 158.9, 164.3 (thiophene C, pyridine C). Calcd for C₁₄H₁₀N₄O₂S (298.32): C, 56.37; H, 3.38; N, 18.78; S, 10.75 %. Found: C, 56.58; H, 3.51; N, 18.87; S, 10.94 %. MS (relative intensity) m/z: 298 (M⁺, 100%), 300 (6%), 280 (18%), 232 (40%).

1-(3-Cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-3-phenylthiourea (21)

To a solution of 2-amino-5,6-dihydro-4H-cyclopenta[b] thiophene-3-carbonitrile 3a (1.5 g, 9.13×10^-3 mol) in 1,4-dioxane (80 mL), phenylisothiocyanate (1.24 g, 9.13×10^-3 mol) and 0.5 mL of triethylamine were added and the reactants were subjected to heating under reflux for 2.5 h. The contents were poured on ice and a few HCl (18.0 mol) drops (till pH₆) stirred in to enhance precipitate formation. The latter was then filtered out.

Grey crystals from ethanol, yield 69.34 %, 1.90 g, m.p. 223-226 ^oC. IR (KBr): υ/cm^-1= 3491-3338 (2 NH), 3056 (CH aromatic), 2887 (CH₂), 2225 (CN), 1641 (C=C), 1204-1190 (C=S). H¹NMR (DMSO) δ = 2.20- 2.35 (m, 6H, 3CH₂), 7.32-7.45 (m, 5H, C₆H₅), 8.26, 8.46 (2s, 2H, 2NH). ¹³C NMR δ = 22.0, 26.9, 32.6 (3 CH₂), 116.0 (CN), 120.3, 120.5, 122.9, 124.2, 125.9, 128.5, 129.0, 137.0, 139.9, (thiophene C, C₆H₅). Calcd for C₁₅H₁₃N₃S₂ (299.41): C, 60.17; H, 4.38; N, 14.03; S, 21.42 %. Found: C, 60.34; H, 4.42; N, 14.11; S, 21.68 %. MS (relative intensity) m/z: 299 (M⁺, 20%), 207 (40%), 163 (100), 92 (76%).

5-Amino-6-phenyl-7-thioxo-3,4-dihydro-2H-cyclopenta[4,5] thieno[2,3-d]pyrimidine (22)

Compound 21 (0.4 g, 1.34×10^-3 mol) was allowed to undergo cyclization by being heated under reflux for 3 h in 1,4-dioxane (40 mL) to which 0.5 mL of triethylamine were added. The contents were poured on ice and a few HCl (18.0 mol) drops (till pH₆) stirred in to enhance precipitate formation. The latter was then filtered out.

Orange crystals from ethanol, yield 12.5 %, 0.05 g, m.p. oC. IR (KBr): υ/cm^-1= 3453-3420 (NH₂), 3062 (CH aromatic), 2886 (CH₂), 1641 (C=C), 1210-1192 (C=S), 1633 (C=N). H¹NMR (DMSO) δ = 2.21-2.39 (m, 6H, 3CH₂), 3.88 (s, 2H, NH₂), 7.30-7.39 (m, 5H, C₆H₅). ¹³C NMR δ = 22.4, 26.6, 32.7 (3 CH₂), 122.3, 122.9, 124.0, 123.8, 126.9, 128.3, 134.8, 138.0, 144.7, 154.2, 165.2 (thiophene C, C₆H₅). Calcd for C₁₅H₁₃N₃S₂ (299.41): C, 60.17; H, 4.38; N, 14.03; S, 21.42 %. Found: C, 60.34; H, 4.51; N, 14.20; S, 21.66 %. MS (relative intensity) m/z: 299 (100%), 301 (M+2, 3%), 222 (50%).

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