Introduction

Several types of pyrimidines and thienopyrimidines have attracted much interest due to their valuable pharmacological properties such as antiviral (Hafez et al. 2010), antioxidant (Kotaiah et al. 2012) and antimalarial (Seerat et al. 2012; Ashalatha et al. 2007). The antioxidants that scavenge reactive oxygen species may be of great value in preventing the onset and propagation of oxidative diseases such as autoimmune diseases, cardiovascular diseases, neurovascular diseases (Kamat et al. 2013; Giordano et al. 2014) and neurodegenerative changes associated with aging (Esposito et al. 2002). The homeostatic balance between the reactive oxygen species (ROS) and endogenous antioxidants is important in maintaining healthy tissues. Excessive ROS states are important in diseases such as acute respiratory distress syndrome and idiopathic pulmonary fibrosis (Vianello et al. 2014). Most living organisms possess enzymatic and nonenzymatic defense systems against excessive production of the reactive oxygen species. However, different external factors (smoke, diet, alcohol and some drugs) and aging decrease the efficiency of such protecting systems, resulting in disturbances of the redox equilibrium established under healthy conditions (Ogunro et al. 2013). In addition, the pyrimidine-based derivatives such as thieno[2,3-d]pyrimidines have extreme importance in medicinal chemistry, exhibiting pharmacological and therapeutic properties such as antidepressant (Kotaiah et al. 2012), antibacterial (Aly et al. 2011; Dewal et al. 2012; Abbas et al. 2013), antifungal (Leung et al. 2013), anti-inflammatory (Rizk et al. 2012; Ashour et al. 2013), antiplatelet (Bach et al. 2013), antihypertensive (Press et al. 1989), herbicidal (El-Sherbeny et al. 1995) and plant growth regulatory (Kotaiah et al. 2012) properties.

Overview of the literature survey showed the importance of thieno[2,3-d]pyrimidines individually in the biological systems and led to assimilate that this moiety may show synergistic effect. In the present work, we report the details of the synthesis of novel thieno[2,3-d]pyrimidine derivatives and in vitro antioxidant properties of them.

Results and discussion

Herein, in order to extend our research on anticancer heterocyclic derivatives with high inhibitory effects toward some cancer cell lines, we report the synthesis of new heterocyclic compounds derived from thieno[2,3-d]pyridazine. Moreover, some of the newly synthesized products were good candidates as anticancer drugs through their screening toward cancer and normal cell lines. Thus, 4,5,6,7-tetrahydrobenzo[b]thiophene reacted with benzoylisothiocyanate to give the thiourea derivative 3. The structure elucidation of compound 3 was based on analytical and spectral data. Thus, the 1H NMR spectrum showed the presence of the four CH2 groups at δ 1.80–1.84 and 2.24–2.26 ppm, a multiplet at δ 7.2–7.35 ppm equivalent to the phenyl protons and two singlets at δ 8.28 and 8.32 (D2O exchangeable) equivalent to the two NH groups. In addition, the 13C NMR spectrum showed δ 19.2, 22.1, 25.7, 28.6 (4 CH2), 119.2, 124.1, 124.9, 128.7, 130.4, 133.8, 138.0, 144.2 (thiophene, C6H5), 165.3 (CO), 179.5 (C=S). Compound 3 underwent cyclization when heated under reflux in 1,4-dioxane, containing a catalytic amount of triethylamine, to give the (4-amino-2-thioxo-5,6,7,-tetrahydro[4,5]thieno[2,3-dpyrimidin-3(2H)-yl)(phenyl)methanone (4). Compound 4 was previously synthesized using another reaction routes (Amr et al. 2010; Hacker et al. 2009; Leistner et al. 1989). Compound 4 underwent different heterocyclization reactions. Thus, it reacted with hydrazine hydrate to give the tetrahydrobenzo[4,5]thieno[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine derivative 5 (Scheme 1). The analytical and spectral data of compound 5 are consistent with its proposed structure.

Scheme 1
scheme 1

Synthesis of compounds 3, 4, 5 and 7a, b

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Compound 4 was reacted with either ethyl cyanoacetate (6a) or ethyl acetoacetate (6b) to give the amide derivatives 7a or 7b (Scheme 1). The high yield of compounds 7a, b encouraged us to use them for further work. In order to expand the scope of the present work, compounds 7a, b underwent ready cyclization when heated in sodium ethoxide solution using a boiling water bath to give the benzo[4,5]thieno[3,2-e]pyrimido[1,2-c]pyrimidine derivatives 8a and 8b, respectively. The mass spectrum of compound 8a showed molecular ion peak at m/e 390 equivalent to the molecular formula C20H14N4OS2. The IR spectrum showed the presence of CN group stretching at υ 2220 cm−1. The 1H NMR spectrum revealed beside the expected signals, a singlet at δ 8.41 indicating the presence of the NH group. The 13CNMR displayed δ 19.0, 22.4, 25.5, 28.8 corresponding to the four CH4 groups of the cyclohexene ring, δ 116.2 indicating the CN group, δ 120.6, 123.6, 124.8, 129.3, 134.2, 136.8, 139.0, 143.7, 148.9, 151.6 (Bz, pyrimidine, thiophene C), δ 166.4 equivalent to the CO group of the pyrimidine C-3, δ 170.4 (C=N, C-7) and δ 180.6 (C=S, C-6).

Next, we studied the reactivity of compounds 7a, b toward thiophene formation via the Gewald’s thiophene synthesis (Scrowston et al. 1981; Mohareb et al., 2012; Hala et al., 2011; Patel et al. 2003). Thus, the reaction of either compounds 7a or 7b with elemental sulfur and either malononitrile (9) or ethyl cyanoacetate (6a) gave the thiophene derivatives 10a–d, respectively. On the other hand, the reaction of either 7a or 7b with either benzaldehyde (11a), 4-chlorobenzaldehyde (11b) or 4-methoxybenzaldehyde (11c) gave the benzylidene derivatives 12a–f, respectively (Scheme 2). The analytical and spectral data of the latter products are consistent with their respective structures (see “Experimental” section).

Scheme 2
scheme 2

Synthesis of compounds 8a, b; 10a–d; and 12a–f

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Compounds 7a or 7b reacted with the aromatic diazonium salts namely benzenediazonium chloride (13a), 4-chlorobenzenediazonium chloride (13b) or 4-methylbenzenediazonium chloride (13c) to give the arylhydrazo derivatives 14a-f, respectively. Reaction of compound 7b with cyanomethylene reagents using different conditions was studied. Thus, compound 7b reacted with either malononitrile (9) or ethyl cyanoacetate (6b) in the presence of ammonium acetate at 120 °C to give the condensation products 15a and 15b, respectively. On the other hand, carrying out the reaction of compound 7b with either malononitrile (9) or ethyl cyanoacetate (6a) in sodium ethoxide solution gave the pyridine-1-yl derivatives 16a and 16b, respectively (Scheme 3). The reaction took place at first through condensation followed by the Michael addition of the NH to the nitrile group. The analytical and spectral data of 16a and 16b were in agreement with their respective structures. Further confirmation for the structures of compounds 16a and 16b was obtained through their synthesis using another reaction route. Thus, either 15a or 15b when heated in sodium ethoxide solution in a boiling water bath gave the same two products 16a and 16b, respectively (same m.p. and mixed m.p. and fingerprint IR spectra).

Scheme 3
scheme 3

Synthesis of compounds 14a–f; 15a, b; and 16a, b

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Compounds 16a, b reacted with phenyl isothiocyanate (17) to give the pyrido[2,3-d]pyrimidine derivatives 18a and 18b, respectively. Structure of the latter products was confirmed on the basis of analytical and spectral data. Finally, 7a or 7b was reacted with either benzaldehyde (11a), 4-chlorobenzaldehyde (11b) or 4-methoxybenzaldehyde (11c) and malononitrile to afford the pyran derivatives 19a–f, respectively. The analytical and spectral data of 19a–f were consistent with their respective structures (Scheme 4).

Scheme 4
scheme 4

Synthesis of compounds 18a, b and 19a–f

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In vitro cytotoxic assay

Chemicals

Fetal bovine serum (FBS) and l-glutamine were purchased from Gibco Invitrogen Co. (Scotland, UK). RPMI-1640 medium was purchased from Cambrex (New Jersey, USA). Dimethyl sulfoxide (DMSO), doxorubicin, penicillin, streptomycin and sulforhodamine B (SRB) were purchased from Sigma Chemical Co. (Saint Louis, USA).

Cell cultures

The cell cultures were obtained from the European Collection of cell Cultures (ECACC, Salisbury, UK), and human gastric cancer (NUGC and HR), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma (HONE1) and normal fibroblast cells (WI38) 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 lg/mL), at 37 °C in a humidified atmosphere containing 5 % CO2. Exponentially growing cells were obtained by plating 1.5 × 105 cells/mL for the seven human cancer cell lines including cells derived from 0.75 × 104 cells/mL 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.

The heterocyclic compounds, prepared in this study, were evaluated according to standard protocols for their in vitro cytotoxicity (Combes et al. 2012; Roemer et al. 2008; Li et al. 2008) against six human cancer cell lines including cells derived from human gastric cancer (NUGC), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma (HONE1) and a normal fibroblast cells (WI38). All of IC50 values are listed in Table 1. Twelve compounds 4, 7a, 8a, 8b, 10c, 10d, 12c, 12d, 12e, 12f, 14e, 15b, 18b, 19b and 19e displayed significant cytotoxicity against most of the cancer cell lines tested (IC50 = 10–1000 nM). Normal fibroblasts cells (WI38) were affected to a much lesser extent (IC50 > 10,000 nM). The reference compound used is the CHS-828 which is a pyridyl cyanoguanidine antitumor agent.

Table 1 Cytotoxicity of the newly synthesized products against a variety of cancer cell lines [IC b50 (nM)]
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Structure activity relationship

From Table 1, the newly synthesized compounds were tested against the six cancer cell lines the human gastric cancer (NUGC), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma (HONE1) and a normal fibroblast cells (WI38). The heterocyclic compounds 4, 7a, 8a, 8b, 10c, 10d, 12c, 12d, 12e, 12f, 14e, 15b, 18b, 19b and 19e exhibited optimal cytotoxic effect against cancer cell lines, with IC50 in the nM range. Comparing the cytotoxicity of the tetrahydrobenzo[b]thiophene derivative 3 and the tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-derivatives 4 exhibited clearly that cyclization of compound 3 to the tricyclic product 4 increases the cytotoxicity of the latter compound. On the other hand, the reaction of compound 4 with hydrazine hydrate gave the 3-phenyl-6,7,8,9-tetrahydrobenzo[4,5]thieno[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-amine5 through which the cytotoxicity decreases against the tested cancer cell lines. Such decrease in cytotoxicity is attributed to the replacement of the thione moiety by the nitrogen of the hydrazine. On the other hand, the reaction of compound 4 with either ethyl cyanoacetate or ethyl acetoacetate produced compounds 7a and 7b, respectively. The amide derivative 7a displayed remarkable cytotoxicity which is more than that of compound 4 against the tested cancer cell lines. Concerning the hexahydro-1H-benzo[4,5]thieno[3,2-e]pyrimido[1,2-c]pyrimidine derivatives 8a and 8b, it is clear that both of the two compounds showed high cytotoxicity against NUGC, DLDI, HA22T, HEPG2 and HONE1 cell lines.

Compound 8b with the COCH3 moiety is more potent against the five cell lines than compound 8a with the CN moiety. For the thiophene derivatives 10a–d, compounds 10c and 10d with the COOEt moiety are more potent than 10a and 10b. Moreover, compound 10a showed potency against NUGC, DLDI, HA22T, HEPG2 and HONE1 cell lines, but compound 10b showed potency against the six cell lines.

From Table 1, it is clear that for the benzylidene derivatives 12a–f, compounds 12a and 12b with the cyano moiety are less potent than compounds 12c–f, which might be due to the presence of either the OCH3 in 12c or the COCH3 moiety as in 12d–f. It is of great value to note that among the compounds 12a–f, compound 12f with the COCH3 and the Cl moieties is responsible for its maximum potency. Similarly concerning the arylhydrazo derivatives 14a–f, it is noticed that compound 14e with the COCH3 and Cl moieties showed the highest potency among the five compounds. On the other hand, compound 14b with the CN and Cl moieties showed high potency toward NUGC, DLDI, HA22T, HEPG2 and HONE1 cell lines, but its potency is less than that of 14e. Compounds 15a, b and 16a, b showed low potency against the six cancer cell lines. Concerning the 2,3-dihydropyrido[2,3-d]pyrimidin-7(8H)-one derivatives 18a and 18b, it is clear that compound 18b with the 4-hydroxy group showed more potency against the six cancer cell lines than compound 18a with the 4-amino moiety. Finally, regarding the pyran derivatives 19a–f, it is obvious that compound 19b with electronegative groups the CN and Cl and compound 19e with COCH3 and the Cl groups showed the highest cytotoxicity through such series of compounds.

Toxicity

Bioactive compounds are often toxic to shrimp larvae. Thus, in order to monitor these chemicals in vivo lethality to shrimp larvae (Artemiasalina), Brine-Shrimp Lethality Assay (Choudhary and Thomsen 2001) was used. Results were analyzed with LC50 program to determine LC50 values and 95 % confidence intervals (Brayn et al. 1993). Results are given in Table 2 for the compounds which exhibited optimal cytotoxic effect against cancer cell lines, respectively, the following twelve compounds: 4, 7a, 8a, 8b, 10c, 10d, 12c, 12d, 12e, 12f, 14e, 15b, 18b, 19b and 19e. The shrimp lethality assay is considered as a useful tool for preliminary assessment of toxicity, and it has been used for the detection of fungal toxins, plant extract toxicity, heavy metals, cyanobacteria toxins, pesticides, and cytotoxicity testing of dental materials (Carballo et al. 2002), natural and synthetic organic compounds (Choudhary and Thomsen 2001). It has also been shown that A. salina toxicity test results have a correlation with rodent and human acute oral toxicity data. Generally, a good correlation was obtained between A. salina toxicity test and the rodent data. Likewise, the predictive screening potential of the aquatic invertebrate tests for acute oral toxicity in man, including A. salina toxicity test, was slightly better than the rat test for test compounds (Calleja and Persoone 1992).

Table 2 Toxicity of compounds 4, 7a, 8a, 8b, 10c, 10d, 12c, 12d, 12e, 12f, 14e, 15b, 18b, 19b and 19e
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In order to prevent the toxicity results from possible false effects originated from solubility of compounds and DMSO’s possible toxicity effect, solutions of the test compounds were prepared in the suggested DMSO volume ranges. It is clear from Table 2 that the N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-2-cyano-3-(4-methoxyphenyl)acrylamide (12c) and the N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-3-oxo-2-(2-(4-chlorophenyl)hydrazono)-butanamide (14e) showed nontoxicity against the tested organisms.

Experimental

13C NMR and 1H NMR spectra were recorded on Bruker DPX400 instrument in DMSO with TMS as internal standard for protons and solvent signals as internal standard for carbon spectra. Chemical shift values are mentioned in δ (ppm). Mass spectra were recorded on EIMS (Shimadzu) and ESI-esquire 3000 Bruker Daltonics instrument. Elemental analyses were carried out by the Microanalytical Data Unit at Cairo University. The progress of all reactions was monitored by TLC on 2 × 5 cm precoated silica gel 60 F254 plates of thickness of 0.25 mm (Merck). Compound 1 was synthesized through Gewald’s thiophene synthesis as reported (Gewald et al. 1966). Compound 4 was synthesized earlier according to reported literature (Amr et al. 2010; Hacker et al. 2009; Leistner et al. 1989).

N-((3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamothioyl)benzamide (3)

To s solution of benzoyl isothiocyanate [prepared by adding benzoylchloride (0.01 mol) (1.40 g, 0.01 mol) to a solution of ammonium thiocyanate (0.76 g, 0.01 mol) in 1,4-dioxane and heating for 10 min] compound 1 (1.78 g, 0.01 mol) was added. The whole reaction mixture was heated under reflux for 2 h and then poured onto ice/water, and the formed solid product was collected by filtration.

Compound 3: Yellow crystals (EtOH), yield 80 % (2.73 g), mp 180–183 °C. IR (KBr) cm−1: 3460, 3325, 3057, 2220, 1688, 1662, 1625 cm−1. 1H NMR (DMSO-d6, 400 MHz): δ = 8.28, 8.32 (2s, 2H, D2O exchangeable, 2NH), 7.28–7.35 (m, 5H, Bz), 2.24–2.26 (m, 4H, 2H-5, 2H-6), 1.80–1.84 (m, 4H, 2H-4, 2H-7).13C NMR (DMSO-d6, 75 MHz): δ = 179.5 (C=S, C-2), 165.3 (CO–Bz), 116.7 (CN), 144.2,138.0, 133.8, 130.4, 128.7, 124.9, 124.1, 119.2, (C-2, C-3, C-4′, C-5′, Bz), 28.6, 25.7, 22.1, 19.2 (C-4, C-5, C-6, C-7), EIMS m/z 341 [M]+ (20); Analysis Calcd for C17H15N3OS2 (341.45): C, 59.80; H, 4.43; N, 12.31; S, 18.78. Found: C, 59.66; H, 4.31; N, 12.46; S, 18.66.

(4-Amino-2-thioxo-5,6,7,8-tetrahyobenzo[4,5]thieno[2,3-d]pyrimidin-3(2H)-yl)(phenyl)methanone (4)

A solution of compound 3 (3.41 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL) was heated under reflux for 6 h and then left to cool. The solution was evaporated under vacuum and the remaining product was triturated with ethanol and the formed solid product was collected by filtration.

3-Phenyl-6,7,8,9-tetrahydrobenzo[4,5]thieno[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-amine (5)

To a solution of compound 4 (3.41 g, 0.01 mol) in 1,4-dioxane (40 mL) hydrazine hydrate (0.50 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 4 h and then poured onto ice/water containing few drops of hydrochloric acid, and the formed solid product was collected by filtration.

Compound 5: Yellow crystals (1,4-dioxane), yield 75 % (2.41 g), mp 177–179 °C. IR (KBr) cm−1: 3477, 3326, 3053, 1636. 1H NMR (DMSO-d6, 400 MHz): δ = 7.31–7.38 (m, 5H, Bz), 5.01 (s, 2H, D2O exchangeable, NH2), 2.24–2.29 (m, 4H, 2H-6, 2H-9), 1.79–1.84 (m, 4H, 2H-7, 2H-8). 13C NMR (DMSO-d6, 75 MHz): δ = 170.3, 172.8, 173.6 (3 C=N),145.0,141.1, 138.8, 132.6, 129.0, 124.6, 123.8, 120.3 (thiophene, pyrimidine, Bz), 28.8, 25.7, 22.3,19.2(C-6, C-7, C-8, C-9), EIMS m/z 321 [M]+ (18); Analysis Calcd for C17H15N5S (321.41): C, 63.53; H, 4.70; N, 21.79; S, 9.98. Found: C, 63.82; H, 4.85; N, 21.84; S, 10.24.

N-(3-benzoyl-2-thioxo-2,,4,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-2-cyanoacetamide(7a) and N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-3-oxobutanamide (7b)

General procedure: To a solution of compound 4 (3.41 g, 0.01 mol) in dimethylformamide (40 mL), either ethyl cyanoacetate (1.13 g, 0.01 mol) or ethyl acetoacetate (1.30 g, 0.01 mol) was added. The reaction mixture in each case was heated under reflux for 3 h and then poured onto ice/water, and the formed solid product was collected by filtration.

Compound 7a: Yellow crystals (1,4-dioxane), yield 80 % (3.27 g), mp 233–235 °C. IR (KBr) cm−1: 3463, 3324, 3058, 2988, 2220, 1687, 1679, 1633. 1H NMR (DMSO-d6, 400 MHz): δ = 8.39 (s, 1H, D2O exchangeable NH), 7.32–7.39 (m, 5H, Bz), 5.20 (s, 2H, CH2), 2.26–2.30 (m, 4H, 2H-4, 2H-7), 1.82–1.86 (m, 4H, 2H-5, 2H-6).13C NMR (DMSO-d6, 75 MHz): δ = 180.4 (C=S, C-2), 170.2 (C=N, C-1), 163.6, 166.8 (CO–Bz, CO–NH), 144.8,140.9, 136.8, 143.2, 128.5, 124.8, 124.3, 120.6 (Bz, pyrimidine, thiophene C), 116.8 (CN), 28.6, 25.4, 22.6, 19.2(C-4, C-5, C-6, C-7), EIMS m/z 408 [M]+ (31); Analysis Calcd for C20H16N4O2S2 (408.51): C, 58.80; H, 3.95; N, 13.72; S, 15.70. Found: C, 58.72; H, 4.21; N, 13.49; S, 15.63.

Compound 7b: Yellow crystals (1,4-dioxane), yield 80 % (3.40 g), mp 170–173 °C. IR (KBr) cm−1: 3476, 3364, 3055, 2983, 1710, 1681, 1633, 1200. 1H NMR (DMSO-d6, 400 MHz): δ = 8.22 (s, 1H, D2O exchangeable NH), 7.29–7.36 (m, 5H, Bz), 4.87 (s, 2H, CH2), 2.63 (s, 3H, CH3), 2.27–2.32 (m, 4H, 2H-4, 2H-7), 1.81–1.86 (m, 4H, 2H-5, 2H-6). 13C NMR (DMSO-d6, 75 MHz): δ = 180.2 (C=S, C-2), 170.2 (C=N, C-1), 163.2, 164.8, 166.0 (CO–Bz, CO–NH, COCH3), 152.3, 146.7, 138.6, 133.2, 128.5, 124.8, 124.3, 121.9 (Bz, pyrimidine, thiophene C),116.3 (CN), 60.4 (s, 2H, CH2), 28.6, 25.4, 22.8, 19.4 (C-4, C-5, C-6, C-7), 18.3 (CH3). EIMS m/z 425 [M]+ (40); Analysis Calcd for C21H19N3O3S2 (425.52): C, 59.28; H, 4.50; N, 9.88; S, 15.07. Found: C, 59.40; H, 4.43; N, 9.75; S, 15.23.

2-Oxo-4-phenyl-6-thioxo-2,6,9,10,11,12-hexahydro-1H-benzo[4,5]thieno[3,2-e]pyrimido[1,2-c]pyrimidine-3-carbonitrile (8a) and 3-acetyl-4-phenyl-6-thioxo-9,10,11,12-tetrahydrop-1H-benzo[4,5]thieno[3,2-e]pyrimido-2(6H)-one (8b)

General procedure: A suspension of either compound 7a (4.08 g, 0.01 mol) or 7b (4.25 g, 0.01 mol) in sodium ethoxide solution [prepared by dissolving sodium metal (0.46 g, 0.02 mol) in absolute ethanol (40 mL)] was heated in a boiling water for 4 h. The reaction mixture was poured onto ice/water containing few drops of hydrochloric acid (till pH 6), and the formed solid product was collected by filtration.

Compound 8a: Pale yellow crystals (1,4-dioxane), yield 66 % (2.58 g), mp 193–196 °C. IR (KBr) cm−1: 3474, 3334, 3056, 2980, 2220, 1690, 1632. 1H NMR (DMSO-d6, 400 MHz): δ = 8.41 (s, 1H, D2O exchangeable NH), 7.29–7.38 (m, 5H, Bz), 2.24–2.29 (m, 4H, 2H-9, 2H-12), 1.81–1.87 (m, 4H, 2H-10, 2H-11), 13C NMR (DMSO-d6, 75 MHz): δ = 180.6 (C=S, C-6), 170.4 (C=N, C-7), 166.4 (CO-pyrimidine C-3), 151.6, 148.9, 143.7, 139.0, 136.8, 134.2,129.3, 124.8, 123.6, 120.6(Bz, pyrimidine, thiophene C), 116.2 (CN), 28.8, 25.5, 22.4, 19.0(C-9, C-10, C-11, C-12), EIMS m/z 390 [M]+ (26); Analysis Calcd for C20H14N4OS2 (390.48): C, 61.52; H, 3.61; N, 14.35; S, 16.42. Found: C, 61.38; H, 3.59; N, 14.49; S, 16.26.

Compound 8b: Yellow crystals (1,4-dioxane), yield 76 % (3.10 g), mp 153–155 °C. IR (KBr) cm−1: 3456, 3341, 3053, 2986, 1715, 1684, 1632, 1210. 1H NMR (DMSO-d6, 400 MHz): δ = 8.26 (s, 1H, D2O exchangeable NH), 7.26–7.39 (m, 5H, Bz), 2.71 (s, 3H, CH3), 2.24–2.36 (m, 4H, 2H-9, 2H-12), 1.80–1.85 (m, 4H, 2H-10, 2H-11).13C NMR (DMSO-d6, 75 MHz): δ = 180.6 (C=S, C-6), 170.6 (C=N, C-7),166.0,163.4 (CO-pyrimidine C-3, COCH3), 152.3, 148.0, 142.7, 136.9, 133.6, 126.8, 124.9, 124.6, 123.8, 122.3 (Bz, pyrimidine, thiophene C), 28.9, 25.4, 22.5, 19.6, (C-9, C-10, C-11, C-12), 18.4 (CH3). EIMS m/z 407 [M]+ (32); Analysis Calcd for C21H17N3O2S2 (407.51): C, 61.90; H, 4.21; N, 10.31; S, 15.74. Found: C, 62.08; H, 4.13; N, 10.29; S, 15.52.

3,5-Diamino-N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-4-cyanothiophene-2-carboxamide (10a), 5-amino-N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-4-cyano-3-methylthiophene-2-carboxamide (10b), ethyl 2,4-diamino-5-((3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)carbamoyl)thiophene-3-carboxylate (10c) and ethyl 2-amino-5-((3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)carbamoyl)-4-methylthiophene-3-carboxylate (10d)

General procedure: To a solution of either compound 7a (4.08 g, 0.01 mol) or 7b (4.25 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL), both of elemental sulfur (0.32 g, 0.01 mol) and of either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) were added. The whole reaction mixture, in each case, was heated under reflux for 1 h and then poured onto ice/water containing few drops of hydrochloric acid. The formed solid product was collected by filtration.

Compound 10a: Orange crystals (1,4-dioxane), yield 73 % (3.70 g), mp 231–233 °C. IR (KBr) cm−1: 3469, 3324, 3059, 2987, 2220, 1688, 1684, 1630, 1210. 1H NMR (DMSO-d6, 400 MHz): δ = 8.38 (s, 1H, D2O exchangeable NH), 7.33–7.42 (m, 5H, Bz), 4.21,4.48 (2s, 4H, D2O exchangeable, 2 NH2), 2.25-2.28 (m, 4H, 2H-5, 2H-8), 1.82–1.89 (m, 4H, 2H-6, 2H-7).13C NMR (DMSO-d6, 75 MHz): δ = 180.3 (C=S, C-2), 170.6 (C=N, C-1), 162.8, 166.8 (CO-Bz, CO–NH), 152.8, 149.0, 148.2, 146.5, 143.8, 139.0, 135.4, 134.2, 129.2, 124.9, 122.8, 120.8 (Bz, pyrimidine, two thiophene C), 116.4 (CN), 28.8, 25.4, 22.7, 19.3, (C-5, C-6, C-7, C-8), EIMS m/z 506 [M]+ (18); Analysis Calcd for C23H18N6O2S3 (506.62): C, 54.53; H, 3.58; N, 16.59; S, 18.99. Found: C, 54.80; H, 3.41; N, 16.33; S, 19.06.

Compound 10b: Yellow crystals (1,4-dioxane), yield 67 % (3.39 g), mp 210–212 °C. IR (KBr) cm−1: 3476, 3332, 3060, 2989, 2220, 1705, 1686, 1631, 1215. 1H NMR (DMSO-d6, 400 MHz): δ = 8.28 (s, 1H, D2O exchangeable NH), 7.28–7.37 (m, 5H, Bz),4.47 (s, 2H, D2O exchangeable, NH2), 2.69 (s, 3H, CH3), 2.22–2.35 (m, 4H, 2H-5, 2H-8), 1.82–1.86 (m, 4H, 2H-6, 2H-7).13C NMR (DMSO-d6, 75 MHz): δ = 180.4 (C=S, C-2), 170.5 (C=N, C-1), 163.6, 166.3 (CO–Bz, CO–NH), 154.0, 152.7, 146.2, 142.3, 136.9, 132.8, 126.6, 124.5, 124.3, 123.0,122.6, 121.8 (Bz, pyrimidine, two thiophene C), 117.0 (CN), 28.9, 25.5, 22.7, 19.4 (C-5, C-6, C-7, C-8),18.7 (CH3). EIMS m/z 505 [M]+ (18); Analysis Calcd for C24H19N5O2S3 (505.64): C, 57.01; H, 3.79N, 13.85; S, 19.02. Found: C, 56.87; H, 4.09; N, 13.69; S, 18.89.

Compound 10c: Orange crystals (1,4-dioxane), yield 65 % (3.70 g), mp 180–184 °C. IR (KBr) cm−1: 3478, 3320, 3062, 2984, 2887, 1690, 1686, 1630, 1212. 1H NMR (DMSO-d6, 400 MHz): δ = 8.33 (s, 1H, D2O exchangeable NH), 7.32–7.40 (m, 5H, Bz), 4.23,4.45 (2s, 4H, D2O exchangeable, 2 NH2), 4.21 (q, 2H, J = 7.02 Hz, CH2), 2.22–2.29 (m, 4H, 2H-5, 2H-8), 1.83–1.89 (m, 4H, 2H-6, 2H-7), 1.14 (t, 3H, J = 7.02 Hz, CH3).13C NMR (DMSO-d6, 75 MHz): δ = 180.6 (C=S, C-2), 170.4 (C=N, C-1), 162.6, 163.8, 166.2 (CO–Bz, CO–NH, CO–OEt), 152.9, 150.3, 148.6, 146.5, 142.6, 139.0, 134.6, 132.9, 129.0, 124.9, 122.6, 120.3 (Bz, pyrimidine, two thiophene C), 28.7, 25.2, 22.9, 19.6, (C-5, C-6, C-7, C-8), 49.2 (CH2), 16.0 (CH3), EIMS m/z 553 [M]+ (28); Analysis Calcd for C25H23N5O4S3 (553.67): C, 54.23; H, 4.19; N, 12.65; S, 17.37. Found: C, 54.59; H, 4.52; N, 12.49; S, 17.22.

Compound 10d: Yellow crystals (1,4-dioxane), yield 71 % (3.92 g), mp 166–168 °C. IR (KBr) cm−1: 3483, 3328, 3063, 2984, 2887, 1702, 1689, 1633, 1213. 1H NMR (DMSO-d6, 400 MHz): δ = 8.31 (s, 1H, D2O exchangeable NH), 7.29–7.36 (m, 5H, Bz), 4.48 (s, 2H, D2O exchangeable, NH2), 4.21 (q, 2H, J = 7.30 Hz, CH2), 2.65 (s, 3H, CH3), 2.23-2.38 (m, 4H, 2H-5, 2H-8), 1.83–1.89 (m, 4H, 2H-6, 2H-7), 1.14 (t, 3H, J = 7.30 Hz, CH3).13C NMR (DMSO-d6, 75 MHz): δ = 180.0 (C=S, C-2), 170.3 (C=N, C-1), 166.8, 164.4, 163.2 (CO–Bz, CO–NH, CO–OEt), 154.2, 152.4, 146.2, 142.3, 136.9, 133.6, 126.3, 124.7, 124.6, 123.2, 122.8, 121.4 (Bz, pyrimidine, two thiophene C), 49.2 (CH2), 28.9, 25.8, 22.7, 19.7 (C-5, C-6, C-7, C-8), 18.5 (CH3), 16.3 (CH3, OCH2 CH 3 ). EIMS m/z 552 [M]+ (32); Analysis Calcd for C26H24N4O4S3 (552.68): C, 56.50; H, 4.38, N, 10.14; S, 17.41. Found: C, 56.69; H, 4.26; N, 10.09; S, 17.66.

N-(3-Benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-2-cyano-3-phenylacrylamide (12a), N-(3-Benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-2-cyano-3-(4-chlorophenyl)acrylamide (12b), N-(3-Benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-2-cyano-3-(4-methoxyphenyl)acrylamide (12c), N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-2-benzylidene-3-oxobutanamide (12d), N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-2-(4-chlorobenzylidene)-3-oxobutanamide (12e) and N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-2-(4-methoxybenzylidene)-3-oxobutanamide (12f)

General procedure: To a solution of either compound 7a (4.08 g, 0.01 mol) or 7b (4.25 g, 0.01 mol) in 1,4-dioxane (40 mL) containing piperidine (0.50 mL), either benzaldehyde (1.06 g, 0.01 mol), 4-chlorobenzaldehyde (1.40 g, 0.01 mol) or 4-methoxybenzaldehyde (1.36 g, 0.01 mol) was added. The whole reaction mixture was heated under reflux for 4 h and then evaporated under vacuum. The remaining product was triturated with diethyl ether, and the formed solid product was collected by filtration.

Compound 12a: Yellow crystals (1,4-dioxane), yield 70 % (3.48 g), mp 170–172 °C. IR (KBr) cm−1: 3455, 3331, 3056, 2984, 2222, 1689, 1680, 1632, 1210. 1H NMR (DMSO-d6, 400 MHz): δ = 8.32 (s, 1H, D2O exchangeable NH), 7.28–7.40 (m, 10H, 2Bz),6.01 (s, 1H, CH=C), 2.26–2.29 (m, 4H, 2H-5, 2H-8), 1.80–1.88 (m, 4H, 2H-6, 2H-7).13C NMR (DMSO-d6, 75 MHz): δ = 180.2 (C=S, C-2), 170.4 (C=N, C-1),163.2, 166.4 (CO–Bz, CO–NH), 152.6, 149.2, 148.2, 146.5, 143.8, 139.0, 135.4, 128.6, 124.2, 123.9, 120.4, 119.6 (2Bz, pyrimidine, thiophene C), 116.7 (CN), 109.6, 114.3 (CH=C), 28.8, 25.6, 22.4, 19.1 (C-5, C-6, C-7, C-8). EIMS m/z 496 [M]+ (21); Analysis Calcd for C27H20N4O2S2 (496.60): C, 65.30; H, 4.06; N, 11.28; S, 12.91. Found: C, 65.29; H, 3.81; N, 11.08; S, 12.76.

Compound 12b: Yellow crystals (1,4-dioxane), yield 70 % (3.72 g), mp 188-190 °C. IR (KBr) cm−1: 3483, 3312, 3058, 2984, 2220, 1710, 1688, 1630, 1211. 1H NMR (DMSO-d6, 400 MHz): δ = 8.03 (s, 1H, D2O exchangeable NH), 7.26–7.39 (m, 9H, C6H5, C6H4), 5.99 (s, 1H, CH=C), 2.24-2.38 (m, 4H, 2H-5, 2H-8), 1.80–1.87 (m, 4H, 2H-6, 2H-7).13C NMR (DMSO-d6, 75 MHz): δ = 180.6 (C=S, C-1), 170.3 (C=N, C-2), 166.1, 164.8 (CO–Bz, CO–NH), 154.2, 153.4, 146.2, 142.6, 136.9, 133.4, 129.6, 126.6, 124.5, 123.8, 122.8, 120.9 (2Bz, thiophene, pyrimidine C), 116.7 (CN), 28.7, 25.5, 22.4, 19.6 (C-5, C-6, C-7, C-8), EIMS m/z 531 [M]+ (22); Analysis Calcd for C27H19ClN4O2S2 (531.05): C, 61.07; H, 3.61; N, 10.55; S, 12.08. Found: C, 60.91; H, 3.80; N, 10.76; S, 11.89.

Compound 12c: Orange crystals (1,4-dioxane), yield 58 % (3.05 g), mp 220–222 °C. IR (KBr) cm−1: 3445, 3328, 3055, 2989, 2884, 2222, 1695, 1688, 1632, 1218. 1H NMR (DMSO-d6, 400 MHz): δ = 8.09 (s, 1H, D2O exchangeable NH), 7.31–7.38 (m, 9H, C6H5, C6H4),6.11 (s, 1H, CH=C), 2.88 (s, 3H, CH3),2.23-2.26 (m, 4H, 2H-5, 2H-8), 1.81–1.87 (m, 4H, 2H-6, 2H-7), 13C NMR (DMSO-d6, 75 MHz): δ = 180.3 (C=S, C-2), 170.9 (C=N, C-1),164.2,162.9 (CO-Bz, CO–NH),153.3, 150.3, 148.6, 145.9, 142.8, 138.2, 133.6, 136.3, 129.0, 124.9, 122.8, 120.1(2Bz, pyrimidine, thiophene C), 116.8 (CN), 28.9, 25.5, 22.9, 19.8 (C-5, C-6, C-7, C-8),18.3 (CH3). EIMS m/z 526 [M]+ (20); Analysis Calcd for C28H22N4O3S2 (526.62): C, 63.86; H, 4.21; N, 10.64; S, 12.18. Found: C, 63.93; H, 4.30; N, 10.44; S, 12.09.

Compound 12d: Yellow crystals (1,4-dioxane), yield 68 % (3.49 g), mp 213–215 °C. IR (KBr) cm−1: 3456, 3330, 3060, 2986, 2880, 1710–1687, 1638, 1218. 1H NMR (DMSO-d6, 400 MHz): δ = 8.28 (s, 1H, D2O exchangeable NH), 7.31–7.42 (m, 10H, 2Bz),6.02 (s, 1H, CH=C),2.68 (s, 3H, CH3), 2.24–2.38 (m, 4H, 2H-5, 2H-8), 1.85–1.89 (m, 4H, 2H-6, 2H-7), 13C NMR (DMSO-d6, 75 MHz): δ = 180.3 (C=S, C-2), 170.6 (C=N, C-1), 166.4, 164.2, 163.6 (CO–Bz, CO–NH, COCH3), 154.3, 152.4, 146.2, 144.2, 136.9, 133.6, 126.3, 124.7, 124.6, 123.9, 122.8, 121.6 (2Bz, pyrimidine, thiophene C), 28.5, 25.8, 22.9, 19.4 (C-5, C-6, C-7, C-8), 18.9 (CH3), EIMS m/z 513 [M]+ (21); Analysis Calcd for C28H23N3O3S2 (513.63): C, 65.48; H, 4.51, N, 8.18; S, 12.49. Found: C, 65.72; H, 4.49; N, 8.09; S, 12.66.

Compound 12e: Pale yellow crystals (1,4-dioxane), yield 87 % (4.77 g), mp > 300 °C. IR (KBr) cm−1: 3476, 3326, 3060, 2988, 2883, 1708–1688, 1634, 1209. 1H NMR (DMSO-d6, 400 MHz): δ = 8.28 (s, 1H, D2O exchangeable NH), 7.31–7.42 (m, 9H, C6H4, C6H5), 6.04 (s, 1H, CH=C), 2.73 (s, 3H, CH3), 2.26–2.36 (m, 4H, 2H-5, 2H-8), 1.86–1.90 (m, 4H, 2H-6, 2H-7). 13C NMR (DMSO-d6, 75 MHz): δ = 180.1 (C=S, C-2), 170.7 (C=N, C-1),163.4, 164.8, 166.2 (CO–Bz, CO–NH, COCH3), 154.8, 152.4, 146.2, 144.2, 136.9, 133.6, 126.3, 124.9, 124.2, 123.9, 123.0, 121.7, (2Bz, pyrimidine, thiophene C), 28.5, 25.8, 22.9, 19.6 (C-5, C-6, C-7, C-8),19.3 (CH3).EIMS m/z 548 [M]+ (21); Analysis Calcd for C28H22ClN3O3S2 (548.07): C, 61.36; H, 4.05, N, 7.67; S, 11.70. Found: C, 61.49; H, 4.25; N, 6.83; S, 11.92.

Compound 12f: Pale yellow crystals (1,4-dioxane), yield 67 % (3.64 g), mp 277–279 °C. IR (KBr) cm−1: 3484, 3318, 3058, 2988, 2887, 1720–1684, 1636, 1202. 1H NMR (DMSO-d6, 400 MHz): δ = 8.34 (s, 1H, D2O exchangeable NH), 7.30–7.46 (m, 9H, C6H5, C6H4), 6.06 (s, 1H, CH=C), 2.78, 3.01 (2s, 6H, 2CH3), 2.22–2.34 (m, 4H, 2H-5, 2H-8), 1.88–1.93 (m, 4H, 2H-6, 2H-7).13C NMR (DMSO-d6, 75 MHz): δ = 180.5 (C=S, C-2), 170.2 (C=N, C-1), 163.2, 164.5, 166.7 (CO–Bz, CO–NH, COCH3),153.6, 151.9, 146.2, 143.6, 136.9, 133.8, 126.3, 125.3, 124.6, 123.9, 122.8, 121.3(2Bz, pyrimidine, thiophene C), 24.24 (OCH3), 28.6, 25.8, 22.9, 19.4 (C-5, C-6, C-7, C-8), 19.8 (CH3). EIMS m/z 543 [M]+ (12); Analysis Calcd for C29H25N3O4S2 (543.65): C, 64.07; H, 4.64, N, 7.73; S, 11.80. Found: C, 63.86; H, 4.43; N, 7.49; S, 11.66.

2-((3-Benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-2-oxo-N′phenylacetohydrazonoyl cyanide (14a),2-((3-Benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-2-oxo-N′(4-chlorophenyl)-acetohydrazonoyl cyanide (14b), 2-((3-Benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-2-oxo-N′(4-methylphenyl)aceto-hydrazonoyl cyanide (14c), N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-3-oxo-2-(2-phenylhydrazono)butanamide (14d), N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-3-oxo-2-(2-(4-chlorophenyl)hydrazono)butanamide (14e) and N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-3-oxo-2-(2-(4-methylphenyl)hydrazono)-butanamide (14f)

General procedure: To a solution of either compound 7a (4.08 g, 0.01 mol) or 7b (4.25 g, 0.01 mol) in ethanol (40 mL) containing sodium hydroxide (5 mL, 10 %), a cold solution of either benzenediazoniumchloride (0.01 mol), 4-chlorobenzenediazonium chloride (0.01 mol) or 4-methylbenzenediazonium chloride (0.01 mol) [prepared by the addition of sodium nitrite solution (0.70 g, 0.01 mol) to a cold solution (0–5 °C) of the aniline (0.94 g, 0.01 mol), 4-chloroaniline (1.27 g, 0.01 mol) or 4-methylaniline (1.08 g, 0.01 mol) in concentrated hydrochloric acid (10 mL, 18 M) was added with continuous stirring] was added with continuous stirring. The whole reaction mixture was kept with stirring for 2 h, and the formed solid product was collected by filtration.

Compound 14a: Yellow crystals (1,4-dioxane), yield 86 % (4.41 g), mp 144–146 °C. IR (KBr) cm−1: 3478, 3321, 3053, 2986, 2220, 1688, 1683, 1634, 1212. 1H NMR (DMSO-d6, 400 MHz): δ = 8.26, 3.11 (2s, 2H, D2O exchangeable, 2NH), 7.32–7.39 (m, 10H, 2Bz), 2.21–2.28 (m, 4H, 2H-5, 2H-8), 1.82–1.84 (m, 4H, 2H-6, 2H-7). 13C NMR (DMSO-d6, 75 MHz): δ = 180.4 (C=S, C-2), 170.3, 172.3 (2C=N), 166.6, 163.8 (CO–Bz, CONH)), 153.8, 149.2, 148.7, 146.5, 143.8, 139.0, 136.3, 134.2, 129.3, 124.2, 121.5, 120.8 (2Bz, pyrimidine, thiophene C), 116.9 (CN), 29.0, 25.3, 22.6, 19.4, (C-5, C-6, C-7, C-8), EIMS m/z 512 [M]+ (28); Analysis Calcd for C26H20N6O2S2 (512.61): C, 60.92; H, 3.93; N, 16.39; S, 12.51. Found: C, 61.28; H, 3.68; N, 16.59; S, 12.66.

Compound 14b: Yellow crystals (1,4-dioxane), yield 56 % (3.06 g), mp 193–195 °C. IR (KBr) cm−1: 3480, 3310, 3055, 2986, 2221, 1690, 1687, 1632, 1214. 1H NMR (DMSO-d6, 400 MHz): δ = 8.32, 8.12 (2s, 2H, D2O exchangeable, 2NH), 7.41–7.28 (m, 9H, C6H5, C6H4), 2.26–2.37 (m, 4H, 2H-5, 2H-8), 1.82–1.87 (m, 4H, 2H-6, 2H-7).13C NMR (DMSO-d6, 75 MHz): δ = 180.6 (C=S, C-2),170.8, 172.2 (2C=N), 164.6, 166.4 (CO–Bz, CO–NH), 154.0, 153.6, 146.2, 142.6, 136.6, 133.7, 129.6, 125.3, 124.8, 122.9, 122.5, 120.4 (2Bz, thiophene, pyrimidine C), 116.9 (CN), 28.9, 25.3, 22.6, 19.5 (C-5, C-6, C-7, C-8), EIMS m/z 547 [M]+ (18); Analysis Calcd for C26H19ClN6O2S2 (547.05): C, 57.09; H, 3.50; N, 15.36; S, 11.72. Found: C, 56.93; H, 3.70; N, 15.49; S, 11.93.

Compound 14c: Orange crystals (1,4-dioxane), yield 62 % (3.27 g), mp 132-135 °C. IR (KBr) cm−1: 3458, 3319, 3053, 2988, 2882, 2220, 1705, 1686, 1630, 1220. 1H NMR (DMSO-d6, 400 MHz): δ = 8.12, 8.34 (2s, 2H, D2O exchangeable, 2NH), 7.30–7.36 (m, 9H, C6H5, C6H4), 2.82 (s, 3H, CH3), 2.24–2.28 (m, 4H, 2H-5, 2H-8), 1.83-1.89 (m, 4H, 2H-6, 2H-7).13C NMR (DMSO-d6, 75 MHz): δ = 180.1 (C=S, C-2), 172.3,170.6 (C=N), 164.2, 162.9 (CO–Bz, CO–NH), 153.8, 150.3, 148.6, 144.2, 143.2, 138.2, 135.8, 133.6, 129.0, 124.7, 123.6, 120.3 (2Bz pyrimidine, thiophene C), 116.6 (CN), 19.5 (CH3), 28.6, 25.5, 23.4, 19.4 (C-5, C-6, C-7, C-8), EIMS m/z526 [M]+ (18); Analysis Calcd for C27H22N6O2S2 (526.63): C, 61.58; H, 4.21; N, 15.96; S, 12.18. Found: C, 61.79; H, 4.44; N, 16.27; S, 12.34.

Compound 14d: Yellow crystals (1,4-dioxane), yield 81 % (4.29 g), mp 120–123 °C. IR (KBr) cm−1: 3449, 3323, 3055, 2988, 2880, 1703–1689, 1638, 1220. 1H NMR (DMSO-d6, 400 MHz): δ = 8.24, 8.32 (2s, 2H, D2O exchangeable, 2NH), 7.26–7.40 (m, 10H, 2Bz), 2.66 (s, 3H, CH3), 2.22–2.37 (m, 4H, 2H-5, 2H-8), 1.82–1.87 (m, 4H, 2H-6, 2H-7).13C NMR (DMSO-d6, 75 MHz): δ = 180.1 (C=S, C-1), 172.4, 170.1 (2C=N), 166.3, 164.2, 162.4 (CO–Bz, CO–NH, COCH3), 154.3, 151.9, 146.8, 144.2, 136.9, 133.8, 126.6, 124.7, 124.6, 123.9, 122.8, 120.8 (2Bz, pyrimidine, thiophene C), 19.0 (CH3), 28.7, 26.0, 23.6, 19.6 (C-5, C-6, C-7, C-8). EIMS m/z 529 [M]+ (18); Analysis Calcd for C27H23N5O3S2 (529.63): C, 61.23; H, 4.38, N, 13.22; S, 12.11. Found: C, 61.63; H, 4.61; N, 13.47; S, 12.36.

Compound 14e: Yellow crystals (1,4-dioxane), yield 79 % (4.46 g), mp 294–297 °C. IR (KBr) cm−1: 3477, 3340, 3060, 2989, 2885, 1705–1686, 1632, 1210. 1H NMR (DMSO-d6, 400 MHz): δ = 8.24, 8.32 (2s, 2H, D2O exchangeable, 2NH), 7.28–7.40 (m, 9H, C6H4, C6H5), 2.68 (s, 3H, CH3), 2.23–2.38 (m, 4H, 2H-5, 2H-8),1.84–1.88 (m, 4H, 2H-6, 2H-8).13C NMR (DMSO-d6, 75 MHz): δ = 180.3 (C=S, C-2), 172.3,170.4 (2C=N), 163.2, 164.6, 166.4 (CO–Bz, CO–NH, COCH3), 153.6, 152.4, 120.9, 123.3, 124.1, 124.3, 124.9, 126.8, 132.4, 135.2, 144.2, 146.2, (2Bz, pyrimidine, thiophene C), 19.6 (CH3), 28.7, 25.9, 22.6, 19.9 (C-5, C-6, C-7, C-8), EIMS m/z 564 [M]+ (16); Analysis Calcd for C27H22ClN5O3S2 (564.07): C, 57.49; H, 3.93, N, 12.42; S, 11.37. Found: C, 57.74; H, 4.02; N, 12.42; S, 11.88.

Compound 14f: Yellow crystals (1,4-dioxane), yield 77 % (4.19 g), mp > 300 °C. IR (KBr) cm−1: 3474, 3339, 3056, 2980, 2881, 1706–1686, 1633, 1210. 1H NMR (DMSO-d6, 400 MHz): δ = 8.20,8.34 (2s, 2H, D2O exchangeable, 2NH), 7.28–7.43 (m, 9H, C6H5, C6H4), 2.64, 3.11 (2s, 6H, 2CH3), 2.25-2.38 (m, 4H, 2H-5, 2H-8), 1.84–1.91 (m, 4H, 2H-6, 2H-7).13C NMR (DMSO-d6, 75 MHz): δ = 180.6 (C=S, C-2), 172.8,170.11 (C=N), 163.0, 164.6, 166.9 (CO–Bz, CO–NH, COCH3), 153.8, 151.6, 146.4, 143.9, 136.9, 133.8, 126.8, 125.0, 123.9, 122.7, 121.6, 120.2, 28.9, 25.7, 22.8, 19.8 (C-5, C-6, C-7, C-8), (2Bz, pyrimidine, thiophene C), 19.6, 19.8 (2CH3), EIMS m/z 543 [M]+ (8); Analysis Calcd for C28H25N5O3S2 (543.66): C, 61.86; H, 4.64, N, 12.88; S, 11.80. Found: C, 61.96; H, 4.84; N, 12.70; S, 11.73.

N-(3-Benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-4,4-dicyano-3-methylbut-3-enamide (15a) and ethyl 5-((3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-2-cyano-3-methyl-5-oxopent-2-enoate (15b)

General procedure: To a dry solid of 7b (4.25 g, 0.01 mol) containing ammonium acetate (0.50 g), either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added. The whole reaction mixture was heated in an oil bath at 120 °C for 1 h and the formed solid product upon cooling was triturated with ethanol and collected by filtration.

Compound 15a: Pale orange crystals (acetic acid), yield 74 % (3.50 g), mp 210–212 °C. IR (KBr) cm−1: 3483, 3324, 3058, 2986, 2223, 2220, 1690, 1688, 1630. 1H NMR (DMSO-d6, 400 MHz): δ = 8.38 (s, 1H, D2O exchangeable NH), 7.31–7.39 (m, 5H, Bz), 5.25 (s, 2H, CH2), 3.01 (s, 3H, CH3), 2.24–2.29 (m, 4H, 2H-5, 2H-8), 1.80–1.87 (m, 4H, 2H-6, 2H-7), 13C NMR (DMSO-d6, 75 MHz): δ = 19.9 (CH3), 20.3, 22.4, 25.2, 28.9 (4 CH2), 117.2,116.4 (2CN), 152.8, 151.8, 148.9, 143.7, 139.0, 135.9, 134.8, 129.1, 125.3, 123.9, 120.8 (C6H5 pyrimidine, thiophene C), 166.4,164.2 (CO–Bz, CO–NH), 170.2 (C=N, C-1), 180.3 (C=S, C-2). EIMS m/z 473 [M]+ (28); Analysis Calcd for C24H19N5O2S2 (473.57): C, 60.88; H, 4.04; N, 14.79; S, 13.54. Found: C, 60.72; H, 3.89; N, 14.53; S, 13.66.

Compound 15b: Yellow crystals (acetic acid), yield 90 % (4.69 g), mp 254–258 °C. IR (KBr) cm−1: 3456, 3341, 3055, 2986, 2220, 1688, 1683, 1630, 1212. 1H NMR (DMSO-d6, 400 MHz): δ = 8.28 (s, 1H, D2O exchangeable NH), 7.28–7.39 (m, 5H, Bz), 5.21 (s, 2H, CH2), 4.23 (q, 2H, J = 7.22 Hz, CH2, OCH 2 CH3), 2.89 (s, 3H, CH3), 2.22-2.38 (m, 4H, 2H-5, 2H-8), 1.80–1.87 (m, 4H, 2H-6, 2H-7), 1.14 (t, 3H, J = 7.22 Hz, CH3. OCH2 CH 3 ), 13C NMR (DMSO-d6, 75 MHz): δ = 180.3 (C=S, C-2), 170.8 (C=N, C-1), 164.8, 163.4, 162.6 (CO–Bz, CO–NH, COCH3),154.1, 148.0, 142.7, 136.9, 126.8, 124.9, 123.8, 122.2 (Bz, pyrimidine, thiophene C), 116.6 (CN), 36.8 (OCH 2 CH3), 28.9, 25.4, 22.6, 19.8 (C-5, C-6, C-7, C-8), 18.8 (CH3), 16.2 (OCH2 CH 3 ), EIMS m/z 520 [M]+ (18); Analysis Calcd for C26H24N4O4S2 (520.61): C, 59.98; H, 4.65; N, 10.76; S, 12.32. Found: C, 60.09; H, 4.44; N, 10.63; S, 12.52.

2-Amino-1-(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-4-methyl-6-oxo-1,6-dihydropyridine-3-carbonitrile (16a) and ethyl 2-amino-1-(3-benzoyl-2-thioxo-2,3,5,6,7,-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-4-methyl-6-oxo-1,65-dihydropyridine-3-carboxylate (16b)

Method (A): General procedure: To a suspension of 7b (4.25 g, 0.01 mol) in sodium ethoxide [prepared by dissolving sodium metal (0.46 g, 0.02 mol) in absolute ethanol (40 ml)], either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added. The whole reaction mixture was heated in a boiling water bath for 6 h and then poured onto ice/water containing few drops of hydrochloric acid (till pH 6), and the formed solid product was collected by filtration.

Method (B): To a suspension of either compound 15a (4.73 g, 0.01 mol) or 15b (5.20 g, 0.01 mol) in sodium ethoxide [prepared by dissolving sodium metal (0.46 g, 0.02 mol) in absolute ethanol (40 mL)] was heated under reflux in a boiling water bath for 4 h. The solid product formed upon pouring onto ice/water containing few drops of hydrochloric acid (till pH 6) and the formed solid product was collected by filtration.

Compound 16a: Yellow crystals (1,4-dioxane), yield 77 % (3.65 g), mp 190–192 °C. IR (KBr) cm−1: 3473, 3331, 3056, 2983, 2873, 2220, 1688, 1684, 1630. 1H NMR (DMSO-d6, 400 MHz): δ = 7.34-7.36 (m, 5H, C6H5), 6.03 (s, 1H, pyridine H-5),4.30 (s, 2H, D2O exchangeable, NH2), 3.11 (s, 3H, CH3), 2.24–2.30 (m, 4H, 2H-5, 2H-8),1.82–1.88 (m, 4H, 2H-6, 2H-7), 13C NMR (DMSO-d6, 75 MHz): δ = 180.1 (C=S, C-2), 170.8 (C=N, C-1), 164.1, 162.3 (CO–Bz, CO, pyridine C-6), 154.2, 152.4, 148.9, 143.7, 140.0, 135.3, 134.5, 129.0, 124.3, 123.9, 122.4, 120.3 (Bz, pyridine, pyrimidine, thiophene C), 116.6 (CN), 28.9, 25.0, 22.8, 20.1 (C-5, C-6, C-7, C-8), 19.8 (CH3), EIMS m/z 473 [M]+ (20); Analysis Calcd for C24H19N5O2S2 (473.57): C, 60.87; H, 4.04; N, 14.79; S, 13.54. Found: C, 60.68; H, 3.99; N, 14.61; S, 13.72..

2-Amino-1-(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-4-methyl-6-oxo-1,6-dihydropyridine-3-carbonitrile

Compound 16b: Yellow crystals (1,4-dioxane), yield 70 % (3.64 g), mp 176–178 °C. IR (KBr) cm−1: 3444, 3337, 3054, 2986, 1688, 1683, 1630, 1212. 1H NMR (DMSO-d6, 400 MHz): δ = 7.25–7.37 (m, 5H, Bz), 6.22 (s, 1H, pyridine H-5),4.63 (s, 2H, D2O exchangeable, NH2), 4.23 (q, 2H, J = 9.93 Hz, OCH 2 CH3), 2.76 (s, 3H, CH3), 2.20–2.39 (m, 4H, 2H-5, 2H-8), 1.82–1.87 (m, 4H, 2H-6, 2H-7), 1.16 (t, 3H, J = 6.93 Hz, OCH2 CH 3 ).13C NMR (DMSO-d6, 75 MHz): δ = 180.1 (C=S, C-2), 170.5 (C=N, C-1), 162.8, 163.2, 164.8 (CO-Bz, CO-OEt, CO-pyridine C-6), 154.1, 150.4, 147.2, 142.9, 136.9, 133.6, 126.2, 124.9, 123.3, 122.1, 121.5, 120.3 (Bz, pyridine, pyrimidine, thiophene C), 36.5 (OCH 2 CH3), 28.3, 25.8, 22.4, 19.9(C-5, C-6, C-7, C-8), 18.6 (CH3), 16.3 (OCH 2 CH3). EIMS m/z 520 [M]+ (22); Analysis Calcd for C26H24N4O4S2 (520.61): C, 59.98; H, 4.65; N, 10.76; S, 12.32. Found: C, 60.21; H, 4.59; N, 10.49; S, 12.48.

4-Amino-5-methyl-3-phenyl-2-thioxo—(2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]-thieno[2,3-d]pyrimidin-4-yl)-2,3-dihydropyrido[2,3-d]pyrimidin-7(8H)-one (18a) and 4-hydroxy-5-methyl-3-phenyl-2-thioxo—(2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]-thieno[2,3-d]pyrimidin-4-yl)-2,3-dihydropyrido[2,3-d]pyrimidin-7(8H)-one (18b)

General procedure: To a solution of either compound 16a (4.73 g, 0.01 mol) or 16b (5.20 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL) phenylisothiocyanate (1.30 g, 0.01 mol), the whole reaction mixture was heated under reflux for 4 h and then poured onto ice/water containing few drops of hydrochloric acid, and the formed solid product was collected by filtration.

Compound 18a: Yellow crystals (1,4-dioxane), yield 80 % (4.04 g), mp > 300 °C. IR (KBr) cm−1: 3481, 3348, 3054, 2987, 2878, 1689, 1638. 1H NMR (DMSO-d6, 400 MHz): δ = 8.29 (s, 1H, NH), 7.26–7.38 (m, 5H, Bz),6.06 (s, 1H, pyridine H-5), 4.30 (s, 2H, D2O exchangeable, NH2), 2.69 (s, 3H, CH3), 2.22–2.33 (m, 4H, 2H-5, 2H-8), 1.80–1.87 (m, 4H, 2H-6, 2H-7), 13C NMR (DMSO-d6, 75 MHz): δ = 183.2, 180.0 (2C=S, C-2, C-2′), 172.4, 170.3 (2C=N, C-1, C-1′), 163.8 (CO, pyridine C-6), 28.7, 25.3, 22.4, 20.4 (C-5, C-6, C-7, C-8), 154.0, 153.6, 150.3, 148.9, 143.9, 140.0, 135.63, 128.4, 123.7, 122.6, 121.4, 119.4 (Bz, pyridine, two pyrimidine, thiophene C), 19.6 (CH3). EIMS m/z 504 [M]+ (20); Analysis Calcd for C24H20N6OS3 (504.66): C, 57.12; H, 3.99; N, 16.65; S, 19.06. Found: C, 57.04; H, 3.68; N, 16.82; S, 18.79.

Compound 18b: Yellow crystals (1,4-dioxane), yield 65 % (3.29 g), mp 190–192 °C. IR (KBr) cm−1: 3489, 3321, 3056, 2988, 1680, 1633, 1200. 1H NMR (DMSO-d6, 400 MHz): δ = 10.20 (s, 1H, D2O exchangeable, OH), 8.22 (s, 1H, D2O exchangeable, NH), 7.23–7.38 (m, 5H, Bz), 6.21 (s, 1H, pyridine H-5), 2.22–2.37 (m, 4H, 2H-5, 2H-8), 2.77 (s, 3H, CH3), 1.80–1.87 (m, 4H, 2H-6, 2H-7).13C NMR (DMSO-d6, 75 MHz): δ = 180.2, 182.1 (2C=S, C-2, C-2′), 170.3, 171.3 (2C=N, C-1, C-1′), 163.8 (CO, pyridine C-6), 19.8, 22.2, 25.9, 28.6 (C-5, C-6, C-7, C-8), 154.3, 150.6, 147.6, 142.9, 140.3, 138.6, 136.9, 133.6, 126.2, 125.9, 124.9, 123.8, 122.1, 121.8, 120.1 (Bz, pyridine, two pyrimidine, thiophene C), 19.4 (CH3), EIMS m/z 505 [M]+ (28); Analysis Calcd for C24H19N5O2S3 (505.64): C, 57.01; H, 3.79; N, 13.85; S, 19.02. Found: C, 56.88; H, 3.84; N, 14.06; S, 19.29

2-Amino-6-((3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-4-phenyl-4H-pyran-3,5-dicarbonitrile (19a), 2-amino-6-((3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-4-(4-chlorophenyl)-4H-pyran-3,5-dicarbonitrile (19b), 2-amino-6-((3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo-[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-4-(4-methoxyphenyl)-4H-pyran-3,5-dicarbonitrile (19c), 5-acetyl-2-amino-6-((3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-4-phenyl-4H-pyran-3-carbonitrile (19d), 5-acetyl-2-amino-6-((3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-4-(4-chlorophenyl)-4H-pyran-3-carbonitrile (19e) and 5-acetyl-2-amino-6-((3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-4-(4-methoxyphenyl)-4H-pyran-3-carbonitrile (19f)

General procedure: To a solution of either compound 7a (4.08 g, 0.01 mol) or 7b (4.25 g, 0.01 mol) in ethanol (40 mL) containing triethylamine (0.50 mL) malononitrile (0.66 g, 0.01 mol), either benzaldehyde (1.08 g, 0.01 mol), 4-chlorobenzaldehyde (1.40 g, 0.01 mol) or 4-methoxybenzaldehyde (1.36 g, 0.01 mol) was added. The whole reaction mixture, in each case, was heated under reflux for 6 h and then poured onto ice/water containing few drops of hydrochloric acid, and the formed solid product was collected by filtration.

Compound 19a: Orange crystals (ethanol), yield 93 % (5.23 g), mp 183–185 °C. IR (KBr) cm−1: 3493-3332, 3056, 2989, 2228, 2226, 2222, 1686, 1630, 1218. 1H NMR (DMSO-d6, 400 MHz): δ = 8.28 (s, 1H, D2O exchangeable, NH), 7.25–7.40 (m, 10H, 2Bz), 6.13(s, 1H, pyran H-4),4.26 (s, 2H, D2O, NH2), 2.21–2.34 (m, 4H, 2H-5, 2H-8), 1.81–1.88 (m, 4H, 2H-6, 2H-7), 13C NMR (DMSO-d6, 75 MHz): δ = 180.2 (C=S, C-2), 170.8 (C=N, C-1), 164.4 (CO-Bz), 154.0, 149.2, 148.7, 146.8, 144.3, 142.5, 140.2, 139.0, 136.3, 134.2, 129.3, 125.4, 124.8, 124.0, 120.6, 120.3 (2Bz, pyran, pyrimidine, thiophene C), 117.0, 116.8 (2CN), 42.3 (pyran C-4), 29.3, 25.5, 22.8, 19.8 (C-5, C-6, C-7, C-8), EIMS m/z 562 [M]+ (21); Analysis Calcd for C30H22N6O2S2 (562.67): C, 64.04; H, 3.94; N, 14.94; S, 11.40. Found: C, 64.29; H, 3.88; N, 14.73; S, 11.62.

Compound 19b: Orange crystals (ethanol), yield 63 % (3.76 g), mp 166–168 °C. IR (KBr) cm−1: 3483, 3318, 3054, 2986, 227, 2223, 1690, 1688, 1210. 1H NMR (DMSO-d6, 400 MHz): δ = 8.18 (s, 1H, D2O exchangeable, NH), 7.26–7.40 (m, 9H, C6H5, C6H4),6.37 (s, 1H, pyran H-4), 4.40 (s, 2H, D2O exchangeable, NH2), 2.22–2.38 (m, 4H, 2H-5, 2H-8), 1.84–1.89 (m, 4H, 2H-6, 2H-7), 13C NMR (DMSO-d6, 75 MHz): δ = 180.4 (C=S, C-2), 170.2 (C=N, C-1), 164.8 (CO–Bz), 154.2, 153.2, 150.1, 148.3, 146.2, 142.6, 136.3, 134.9, 128.4, 125.8, 124.3, 123.3, 122.9, 121.9, 120.3, 119.8, (2Bz, pyran, thiophene, pyrimidine C), 117.0, 116.6 (2CN), 42.6 (pyran C-4), 28.9, 25.6, 22.7, 19.4 (C-5, C-6, C-7, C-8), EIMS m/z 597 [M]+ (21); Analysis Calcd for C30H21ClN6O2S2(597.11): C, 60.35; H,.55; N, 14.07; S, 10.74. Found: C, 60.59; H, 3.62; N, 13.4; S, 10.88.

Compound 19c: Orange crystals (1,4-dioxane), yield 79 % (4.68 g), mp 184–186 °C. IR (KBr) cm−1: 3442, 3337, 3055, 2984, 2867, 2227, 2224, 1690, 1633, 1222. 1H NMR (DMSO-d6, 400 MHz): δ = 8.28 (s, 1H, D2O exchangeable, NH), 7.32–7.38 (m, 9H, C6H5, C6H4), 6.20 (s, 1H, pyran H-4), 4.38(s, 2H, D2O exchangeable, NH2), 2.90 (s, 3H, CH3), 2.23–2.29 (m, 4H, 2H-5, 2H-8), 1.81–1.88 (m, 4H, 2H-6, 2H-7).13C NMR (DMSO-d6, 75 MHz): δ = 180.3 (C=S, C-2), 170.9, (C=N, C-1), 163.4 (CO–Bz), 154.2, 153.2, 150.3, 148.6, 144.5, 143.8, 140.5, 138.8, 135.5, 133.9, 129.3, 128.6, 124.7, 123.8, 122.4, 120.1 (2Bz, pyran, pyrimidine, thiophene C),116.8, 117.2 (2CN), 42.4 (pyran C-4), 19.8 (CH3), 28.4, 25.8, 23.7, 19.3 (C-5, C-6, C-7, C-8), EIMS m/z 592 [M]+ (22); Analysis Calcd for C31H24N6O3S2 (592.69): C, 62.82; H, 4.08; N, 14.18; S, 10.82. Found: C, 62.68; H, 4.19; N, 14.35; S, 10.44.

Compound 19d: Pale orange crystals (1,4-dioxane), yield 74 % (4.29 g), mp 222-225 °C. IR (KBr) cm−1: 3438, 3303, 3058, 2980, 2893, 2220, 1688, 1634, 1210. 1H NMR (DMSO-d6, 400 MHz): δ = 8.26 (s, 1H, D2O exchangeable, NH), 7.27–7.43 (m, 10H, 2Bz), 6.38 (s, 1H, pyran H-4), 4.40 (s, 2H, D2O exchangeable, NH2), 2.72 (s, 3H, CH3), 2.24-2.39 (m, 4H, 2H-7, 2H-8), 1.83–1.88 (m, 4H, 2H-6, 2H-7).13C NMR (DMSO-d6, 75 MHz): δ = 180.5 (C=S, C-2)170.3 (C=N, C-1), 164.2, 166.3 (CO–Bz, COCH3), 154.3, 151.9, 146.8, 144.2, 143.2, 136.9, 133.8, 129.7, 126.8, 125.2, 124.8, 123.6, 122.5, 121.3, 120.5, 119.3 (2Bz, pyran, pyrimidine, thiophene C), 116.8 (CN), 42.5 (pyran C-4), 19.4 (CH3), 28.9, 26.6, 23.8, 19.4(C-5, C-6, C-7, C-8), EIMS m/z 579 [M]+ (36); Analysis Calcd for C31H25N5O3S2 (579.69): C, 64.23; H, 4.35, N, 12.08; S, 11.06. Found: C, 64.07; H, 4.44; N, 11.95; S, 11.30.

Compound 19e: Yellow crystals (1,4-dioxane), yield 55 % (3.38 g), mp 190–193 °C. IR (KBr) cm−1: 3488, 3319, 3058, 2980, 2880, 2220, 1720-1688, 1631, 1212. 1H NMR (DMSO-d6, 400 MHz): δ = 8.30 (s, 1H, D2O exchangeable, NH), 7.27–7.41 (m, 9H, C6H4, C6H5), 6.12 (s, 1H, pyran H-4), 4.30 (s, 2H, D2O exchangeable NH2), 3.13 (s, 3H, CH3), 2.23-2.36 (m, 4H, 2H-5, 2H-8), 1.82–1.89 (m, 4H, 2H-6, 2H-7), 13C NMR (DMSO-d6, 75 MHz): δ = 180.4 (C=S, C-2), 170.9 (C=N, C-1),164.3, 165.4 (CO–Bz, COCH3), 120.5, 121.8, 122.7, 123.9, 125.3, 126.0, 127.3, 129.0, 132.5, 134.0, 137.4, 140.1, 143.9, 146.4, 152.3, 153.4 (2Bz, pyran, pyrimidine, thiophene C),116.6 (CN), 42.4 (pyran C-4),19.9, 22.4, 25.6, 28.7 (4 CH2), 19.7 (CH3). EIMS m/z 614 [M]+ (18); Analysis Calcd for C31H24ClN5O3S2 (614.13): C, 60.63; H, 3.94, N, 11.40; S, 10.44. Found: C, 60.82; H, 3.69; N, 11.28; S, 10.68.

Compound 19f: Orange crystals (1,4-dioxane), yield 73 % (4.45 g), mp 180–183 °C. IR (KBr) cm−1: 3480, 3320, 3056, 2987, 2882, 2218, 1686, 1630, 1210. 1H NMR (DMSO-d6, 400 MHz): δ = 8.30 (s, 1H, D2O exchangeable, NH), 7.29–7.38 (m, 9H, C6H5, C6H4), 6.12 (s, 1H, pyran H-4), 4.37 (s, 2H, D2O exchangeable, NH), 2.78, 2.89 (2s, 6H, 2CH3), 2.21–2.34 (m, 4H, 2H-5, 2H-8), 1.81–1.86 (m, 4H, 2H-6, 2H-7).13C NMR (DMSO-d6, 75 MHz): δ = 180.2 (C=S, C-2), 170.6 (C=N, C-1), 164.8, 165.2 (CO–Bz, COCH3), 154.6, 153.2, 152.4, 147.7, 146.2, 144.2, 135.2, 133.6, 132.4, 130.4, 126.9, 125.0, 124.9, 124.4, 123.9, 123.3, 120.2, 119.8 (2Bz, pyran, pyrimidine, thiophene C),116.3 (CN), 42.6 (pyran C-4), 19.8, 19.9 (OCH3, COCH 3 ), 28.9, 25.8, 22.3, 19.8 (C-5, C-6, C-7, C-8), EIMS m/z 609 [M]+ (28); Analysis Calcd for C32H27N5O4S2 (609.71): C, 63.04; H, 4.46, N, 11.49; S, 10.52. Found: C, 63.32; H, 4.69; N, 11.82; S, 11.63.

Conclusions

In summary, we have shown herein that our strategy is compatible with the synthesis of a wide range of tetrahydrobenzo[b]thiophene derivatives and particularly when being incorporated into heterocyclic and fused derivatives. The cytotoxicity of the newly synthesized products was evaluated against human gastric cancer (NUGC and HR), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma (HONE1) and normal fibroblast cells (WI38). The results showed that compounds 4, 7a, 8a, 8b, 10c, 10d, 12c, 12d, 12e, 12f, 14e, 15b, 18b, 19b and 19e exhibited optimal cytotoxic effect against cancer cell lines with IC50 in the nM range. In addition, compounds 12c and 14e showed no toxicity when tested in vivo lethality to shrimp larvae (Artemiasalina).