Design, synthesis and identification of novel substituted isothiochromene analogs as potential antiviral and cytotoxic agents

Abstract

In this study, we present the synthesis of novel isothiochromenes, thiazolidonone, thiazinone, arylidines, triazoles, and pyrimidinone compounds based on the starting material 3-amino- isothiochromene-4-carbonitrile 3. The chemical structures were confirmed using spectroscopic methods and elemental analyses. These compounds were screened for their in vitro antiviral and antitumor activities. Compounds 10a-c and 22a-b showed activity against herpes simplex virus-1 (HSV-1) and human immunodeficiency virus-1 (HIV-1). Compounds 15 and 21a-b exhibited activity against various types of cancer cell lines.

Introduction

Many biological activities in the human body are modulated by heterocyclic compounds such as nucleic acids, hormones, and neurotransmitters, etc. Heterocycles also play a crucial role in drug discovery where they are involved in many therapeutic areas. One of those heterocyclic compounds, the pyrimidine moiety, is an essential component of many classes of drugs, such as antineoplastic (Uramustine), antibacterial (Trimethoprim), antifungal (Flucytosine), and antivirals (Broxuridine) (Selvam et al. 2012; Abu-Hashem and Hussein 2015).

Substituted thiopyrans are heterocycles that exhibit an extensive range of biological activities such as anti-inflammatory (Rogier et al. 2001), antibacterial (Brown et al. 2002), effective antagonists at α1- adrenoreceptor (Quaglia et al. 2002), and dopamine D3 receptor-selective agonists (Van Vliet et al. 2000). Furthermore, isothiochromene-based compounds are reported to exhibit antitubercular, anti-HIV-1 (Bennani et al. 2007), anticancer and antitrypanosomal activities (Kaminskyy et al. 2014)

Also, thiazolopyrimidine compounds are shown to have activities as acetylcholinesterase inhibitors (Zhi et al. 2008). Meanwhile, thiazine-based compounds are known to have antimicrobial (Ram et al. 2013), antiviral (Galal et al. 2011), anti-inflammatory, analgesic, and ulcerogenic activities (Vijay 2011). Likewise, 1, 2, 4-triazolopyrimidine derivatives displayed numerous pharmacological activities as antimicrobial (Abu-Hashem et al. 2017), anti-inflammatory, analgesic (Sung and Lee 1992), and antimalarial (Havaldar and Patil 2008) agents. Also, 1, 2, 3-triazine is an interesting class of heterocyclic compounds. They are known to have pharmacological activity and may be considered as lead molecules for the development of future drugs (Kumar et al. 2014) for examples: Tubercidin (a) is used to preventing the growth of bacteria. Toyocamycin (b) is recognized as antibiotic and antineoplastic. Sangivamycin (c) is active against leukemia, lung carcinoma, against colon, and gallbladder cancer in humans. 2-Aza-adenosine (d) exhibits the highest cytotoxicity against epidermoid carcinoma cells (Migawa et al. 2005), (Fig. 1).

Fig. 1

Biologically active 1, 2, 3-triazine derivatives

Pyrrolopyrimidines are a valuable group of bioactive compounds showing antivirals, antiprotozoal, antibacterial and anticancer (De Coen et al. 2016), anti-hyperglycemic activities (Mohamed et al. 2014) and anti-cancer agents (Dholakia et al. 2015). Based on the potential biological activities of these heterocycles we synthesized new pyrimidine-, thiazolopyrimidine-, pyrimidothiazine-, triazine-, triazolo- pyrimidine-, pyrrolopyrimidine, and pyridine-based compounds incorporating isothiochromene in order to investigate their antiviral and cytotoxic activities. Thus, it is a real challenge to combine the aforementioned rings together in a molecular framework to make use of the additive effect of these rings toward the antiviral and cytotoxic activities.

Results and discussion

Chemistry

The synthetic strategies, adopted to obtain the poly-functional substituted isothiochromenes, are shown in Schemes 1–4. Compound 3 was prepared via Gewald reaction (Gewald et al. 1966) of 2, 6-bis (pyridin-4-ylmethylene) cyclohexan-1-one 1 (Somers-Edgar et al. 2009) with malononitrile and elemental sulfur in ethanol containing diethylamine. The IR spectrum of compound 3 showed absorption bands at ν 3420 (NH₂), 2212 (CN) cm⁻¹. Furthermore, the ¹H NMR spectrum of 3 exhibited two singlet signals at δ 4.62, 6.28, and 8.23 ppm for one proton (C₁-H) of the thiopyran ring and the amino group (as D₂O exchangeable) and the methine proton respectively. Moreover, its mass spectrum revealed the ion peak at m/z 358 (M⁺, 100 %), which agrees with the molecular formula (C₂₁H₁₈N₄S). Compound 3 reacted with carbon disulfide in ethanol and in the presence of aqueous potassium hydroxide according to the reported condition by (Rashad et al. 2005) to afford the pyrimidine-1, 3(2H)-dithione (5) through the intermediate 4. The IR spectrum of compound 5 revealed the absorption bands at ν 3395 (NH), 1620 (C=N) and 1355, 1370 cm⁻¹ (2 C=S). Moreover, its ¹H NMR spectrum exposed two singlet signals at δ 9.19 and 9.21 ppm due to (2NH). Subsequently, compound 5 reacted with chloroacetic acid in the presence of Ac₂O/AcOH to give the isothiochromeno[3,4-d]thiazolo[3,2-a]pyrimidin-9(10H)-one 8, the formation of 8 can be explained via nucleophilic addition of the thiol group of 1, 3(2H)-dithione 5 to in-situ formed chloroacetic anhydride 6, followed by cyclization of the formed intermediate 7. Knovenagel condensation of compounds 8 with the appropriate aromatic aldehydes derivatives namely, benzaldehyde, or 4-chlorobenzaldehyde or 4-methoxybenzaldehyde afforded the isothiochromeno[3,4-d]thiazolo[3,2-a]pyrimidin-9(10H)-one 10a-c. The IR spectra of compounds 8 displayed a characteristic absorption band at ν 1710 due to the carbonyl group of thiazolone ring respectively. The ¹H NMR spectrum of 8 exhibited multiplet signals at δ 1.19–1.69 ppm corresponding to six protons of cyclohexene (3CH₂) and two singlet signals 4.18 and 8.07 ppm due to two protons of the thiazolidinone moiety (CH₂) and the methine proton of thiopyran ring, respectively. Furthermore, the ¹H-NMR spectra of 10a-c displayed the disappearances of the singlet signal for two protons of the thiazolidinone moiety (CH₂) (Scheme 1).

Scheme 1

Synthesis of thiazolopyrimidines 8 and 10a–c. Reagents and conditions: (i) Et₂NH/EtOH, reflux, 80–90 °C, 7–9 h. (ii) KOH, stirring, 13–15 h. (iii) Ac₂O/AcOH, reflux, 7–10 h. (iv) AcOH, reflux, 12–14 h

Scheme 2

Synthesis of pyrimido[2,1-b][1,3] thiazin-9-ones 13 and, 14a–c. Reagents and conditions: (i) Ac₂O/AcOH, reflux, 7–10 h. (ii) AcOH, reflux, 12–14 h

Scheme 3

Synthesis of 1-chloro-6H-isothiochromeno[3,4-d][1,2,3]triazine 15, 3H-isothio- chromeno[3,4-d]pyrimidine 18a,b and 8H-isothiochromeno[4,3-e][1,2,4]triazolo[1,5-c] pyrimidine 22a, and 22b. Reagents and conditions: (i) NaNO₂, HCl/ AcOH, stirring, 4 h. (ii) pyridine, reflux, 10–12 h. (iii) Ac₂O, reflux, 10–12 h. (iv) dioxane, reflux, 6–8 h. (v) reflux, 4 h

Scheme 4

Synthesis of pyrrolo[3,2-d]pyrimidine 26 and 32, and isothiochromeno[3,4-b]pyrrole 30 and 31. Reagents and conditions: (i) K₂CO₃, Acetone, reflux, 10 h. (ii) K₂CO₃, DMF, reflux, 8 h. (iii) KSCN, HCl, reflux, 7 h. (iv) EtOH/EtONa, reflux, 10 h. (v) EtOH/ EtONa, reflux, 10–12 h

Similarly, 10-(sub.-benzylidene)-5-(pyridin-4-yl)-1-(pyridin-4-ylmethylene)-13-thioxo-1, 3, 4, 5, 10, 11-hexahydro-2H,9H,13H-isothiochromeno[3’,4’:4,5]pyrimido[2,1-b][1,3]thiazin-9-one 14a-c, were prepared via the reaction of 5 with 3-bromopropanoic acid, followed by the condensation of the formed pyrimido[2,1-b][1,3] thiazin-9-one derivative 13 with the same aldehydes 9a–c. The IR spectrum of compound 13 revealed a characteristic absorption bands at ν 1718 cm⁻¹ due to the carbonyl group of the thiazinone ring. Furthermore, the mass spectra of 14a–c showed the molecular ion peaks at m/z 576 (M⁺, 98 %), 611 (M⁺, 100 %), 606 (M⁺, 90 %), respectively (Scheme 2).

The treatment of 2-aminoisothiochromene 3 with sodium nitrite solution in the presence of the concentrated hydrochloric acid generated the 1-chloro-6H-isothiochromeno[3,4-d][1,2,3]triazine 15, ¹H NMR spectrum of 15 displayed two signals at δ 4.42 (s, 1H, thiopyran), and δ 8.23 (s, 1H, C=CHAr), respectively. Its mass spectrum showed the molecular ion peak at m/z 405 (M⁺, 100 %), which is in agreement with the molecular formula (C₂₁H₁₆ClN₅S). Furthermore, the 3H-isothio- chromeno[3,4-d]pyrimidine-3-thione 18a and the 3H-isothiochromeno[3,4-d]pyrimidin-3-one 18b were prepared via refluxing of compound 3 with phenylisothiocyanate 16a or phenyl isocyanate 16b in of pyridine, respectively through the formation of the intermediate 17a,b. IR spectra of 18a and 18b indicated the presence of three absorption bands at ν 3250, 3220, and 1275 cm⁻¹ corresponding to 2NH and (CS) groups. The ¹H NMR spectrum of compound 18a exhibited two characteristic broad singlet signals at δ 10.58 and 10.62 corresponding to the two protons of 2NH groups. Moreover, condensation of 3 with triethyl orthoformate 19a or triethyl orthoacetate 19b in refluxing acetic anhydride gave the ethylformimidate derivative (20a) and the ethylacetimidate (20b), respectively. Compounds 20a or 20b undergo cyclic condensation upon treating with phenylhydrazine in dioxane affording the 1H-isothiochromeno[3,4-d]pyrimidin-2(6H)-amines (21a) and (21b) respectively. The ¹H NMR spectrum of compound (21a) displayed two broad singlet signals at δ 9.35 and 10.58 ppm due to (2NH) protons. The mass spectra of 20a, 20b, 21a, and 21b revealed the molecular ion peaks at m/z 414 (M⁺, 100 %), 428 (M⁺, 90 %), 476 (M⁺, 100 %), 490 (M⁺, 88 %). Subsequently, compounds 21a, and 21b underwent cyclization upon heating under reflux in excess of triethyl orthoformate 19a to achieve the 8H-isothiochromeno[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine 22a, and 22b, respectively. ¹H NMR spectrum of 22a showed triplet and quartet signals at δ 1.31 and δ 4.22 ppm due to the ethyl group, and four singlet signals at δ 4.72 (s, 1H, thiopyran), 6.34 (s, 1H, CHOEt, triazole ring), 8.13 (s, 1H, C=CHAr), and 8.84 (s, 1H, N=CH), (Scheme 3)

The cyclocondensation of compound (3) with ethyl bromoacetate in dry acetone and anhydrous potassium carbonate gave the ethyl glycinate 23, which underwent cyclization to the corresponding ethylisothiochromeno[3,4-b]pyrrole-2-carboxylate 24 upon refluxing in dimethylformamide. The structure of 23 and 24 was recognized through the spectral and elemental analysis. The IR spectrum of 24 exhibited three characteristic absorption bands at ν 3405, 3280 and 1735 cm⁻¹ due to the presence of NH₂, NH and carbonyl group. Hence, compound 24 reacted with potassium thiocyanate to give the ethylisothiochromeno [3,4-b] pyrrole-2-carboxylate 25, which cyclized via refluxing in ethanol in the presence of sodium ethoxide to achieve the corresponding isothiochromeno[4′,3′:4,5] pyrrolo[3,2-d] pyrimidin-8(2H)-one 26. The ¹H-NMR spectra of (26) revealed three broad singlet signals at δ 10.35, 12.32, 13.13 ppm due to the presence of 3NH groups. The mass spectra of compounds 25 and 26 indicated the molecular ion peaks at m/z 503 (M⁺, 90 %), 457 (M⁺, 99 %), Finally, the treatment of compound 3 with 2-chloroacetamide or phenacyl bromide or ethyl cyanoacetate was refluxed in sodium ethoxide solution afforded the corresponding isothiochromeno [3,4-b]pyrrole-2-carboxamide 30, isothiochromeno[3,4-b]pyrrol-2-yl) phenyl methanone 31 and isothiochromeno[3,4-b]pyridine-2-carbonitrile 32, respectively. IR spectrum of compound 30 exposed the absorption band at ν 3410-3250 (br) and 1675 cm⁻¹ due to the 2NH₂, NH, and the carbonyl groups. Meanwhile, the ¹H-NMR spectrum of 31 showed two characteristic singlet signals at δ 6.43 and δ 12.67 ppm corresponding to the two protons of NH₂ and the one proton of NH groups. Furthermore, the ¹³C-NMR of 32 showed signals at δ 15.7, 21.6, 31.2 ppm for three carbon atoms of (3CH_2, cyclohexene), 65.6, 88.9 ppm for two carbon atoms of thiopyran ring, 94.1 ppm for one carbon atom of pyridine ring, 102.4 ppm for one carbon atom of cyano group, 111.9 ppm for one carbon atom of (CH), 165.7 ppm for one carbonyl group, and 172.1 ppm for one carbon atom of (C-NH₂). The MS of 30, 31, and 32 showed the molecular ion peaks at m/z 415 (M⁺, 90 %), 476 (M⁺, 95 %), 425 (M⁺, 92 %), respectively (Scheme 4).

Biological screening

In vitro anti-Herpes Simplex-1 Virus activity

The in vitro Anti-Herpes Simplex-1 Virus (HSV-1) for the isothiochromene derivatives were evaluated with the method reported by (El-Subbagh et al. 2000; Hufford et al. 1991) where Aphidicolin (0.001 µg/mL) was used as a positive control. The results in (Table 1) revealed that compounds 10a, 10b, 10c, 14b, 22a, and 22b displayed the highest activity among the tested compounds where they decrease the number of viral plaques by 82, 88, 85, 80, 90, and 95 % respectively. The remarkable anti-HSV-1 activity of these compounds may be attributable to the presence of the 3-(4-substitutedbenzylidene)-5-thioxo-3H-thiazolo[3,2-a]pyrimidin-2(5H)-one, 3-(4-chlorobenzylidene)-6-thioxo-pyrimido[2,1-b][1,3]thiazin-2(6H)-one, and 2-ethoxy-5-substituted-3-phenyl-[1,2,4]triazolo[1,5-f]pyrimidine moieties, respectively (El-Subbagh et al. 2000).

Table 1 In vitro Anti-Herpes Simplex-1 Virus (HSV-1) activity of the new compounds

In vitro anti-Human Immunodeficiency-1 Virus activity

The effect of the newly synthesized isothiochromene and the azidothymidine (AZT) drug on acutely and persistently infected (T4) cell lines in vitro were evaluated using the method reported by (Chen et al. 1992; Weislow et al. 1989). The method used to estimate the anti-Human Immunodeficiency-1 Virus (HIV-1) effectiveness is intended to discover agents acting at any phase of the virus procreative cycle. The result in (Table 2) displayed that the compounds 10a-c, 22a, and 22b have moderate activity against (HIV-1) cytopathic effect. Whereas; compounds 3, 5, 8, 13, 14a, 14b, 14c, 15, 18a, 18b, 20a, 20b, 21a, 21b, 23, 24, 25, 26, 30, 31, and 32 are inactive.

Table 2 In vitro Anti-Human Immunodeficiency-1 Virus (HIV-1) activity of the new compounds

Antitumor screening (in vitro cytotoxicity)

The isothiochromene derivatives were tested for their in vitro cytotoxicity using the standard MTT method (Mosmann 1983; Denizot and Lang 1986; Thabrew et al.1997) against the human oral carcinoma cells (KB), nasopharyngeal carcinoma cells (CNE2), breast adenocarcinoma cells (MCF-7), and gastric carcinoma cells (MGC-803). The MTT method is based on the reduction of soluble 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide. The result in (Table 3) revealed that the isothiochromenes 15, 21a, 21b, 22a, and 22b exhibited high cytotoxicity against all carcinoma cell lines as follows; the KB (IC₅₀ 11.9, 11.7, 11.5, 12.5, and 12.2 µM), the CNE2 (IC₅₀ 13.3, 12.9, 12.6, 13.8, and13.5 µM), the MCF-7 (IC₅₀ 13.1, 12.8, 12.4, 13.9, and 13.4 µM), the MGC-803 (IC₅₀ 12.4, 12.2, 11.9, 13.2, and 12.9 µM), respectively. Also, some compounds as; 5, 18a, 18b, 26, and 32 showed moderate cytotoxicity against the carcinoma cell lines and the rest of the compounds showed weak cytotoxicity activities.

Table 3 Cytotoxic activity of the new compounds against different human cancer cell lines

Structural activity relationship

By comparing the observed antiviral and cytotoxic activities of the isothiochromenes obtained in this study to their structures, the (SAR’s) were postulated. (I) the presence of the isothiochromen moiety may be necessary for the broad spectrum of the antiviral and cytotoxicity activity. (II) 8H-isothiochromeno[4,3-e][1,2,4]triazolo[1,5-c] pyrimidines 22a-b exhibited high in vitro anti-herpes simplex -1 virus activity and moderate anti-human immunodeficiency-1 virus activity. The 1, 2, 4 triazole template, either fused with a pyrimidine ring (Abu-Hashem et al. 2017) or not (Sung and Lee 1992; Havaldar and Patil 2008) display remarkable biological activity, thus the noticeable behavior of 22a-b may be attributed to the presence of the triazolo[1,5-c]pyrimidine moiety. (III) Compound 10a–c and 14a–c are more potent than compound 8 and 13: a comparison of the corresponding structures leads to the conclusion that this fact may be attributed to the presence of the arylidene moiety in the more active molecules. (IV) Compounds 15, 21a-b, and 22a-b exhibited high cytotoxicity against all carcinoma cell lines. The 1, 2, 3-triazine scaffold from 15 has a potent biological activity (Kumar et al. 2014). The iminopyrimido moiety from compounds 21 was already identified as presenting in vitro antitumor activity against human breast cancer cells MCF-7 (El-Ashmawy et al. 2013). Thus, the remarkable antineoplastic activity of the compounds may be attributable to the presence of the iminopyrimidine, chlorotriazine, and triazolo-pyrimidine moieties, respectively.

Conclusions

The objective of the present study is to synthesize and evaluate the antiviral and cytotoxic activities of some new isochromene-based compounds with the hope of discovering new structure leads serving as antiviral or antitumor agents. The data revealed that isothiochromenotriazolopyrimidines 22a,b possess promising in vitro antiviral activity against HSV-1 and HIV-1. While isothiochromenotriazine 15 and isothiochromenopyrimidin-2(6H)-amine21a, 21b showed wonderful cytotoxic activity against carcinoma cell lines when compared to the 5-fluorouracil drug.

Experimental

Materials, equipment’s, and methods

All materials used were obtained from Sigma Aldrich (Saint Lewis, USA). The melting points are in degree centigrade (uncorrected) and were determined on Gallenkamp electric melting point apparatus. TLC analysis was carried out on silica gel 60 F₂₅₄ precoated aluminum sheets. The IR spectra were recorded (KBr) on a Perkin–Elmer 1430 spectrometer (λ, cm⁻¹) in National Research Center, Egypt. ¹H and ¹³C-NMR spectra were measured on JEOL-ECA 500 and JEOL JNM-LA-400 FT NMR Spectrometers at 500/400, 125/100 MHz, respectively, using tetramethylsilane (TMS) as an internal reference and DMSO-d₆ as the solvent at the Microanalytical Center in National Research Center, Egypt. The mass spectra (EI) were recorded on GCMS-QP 1000 EX (Shimadzu) at National Research Center, Egypt. Elemental analyses (C, H, and N) were carried out at the Microanalytical Center in National Research Center, Egypt. The elemental analyses were found to agree favorably with the calculated values. The synthesized compounds were tested in vitro for their antiviral activity using HSV-1, HIV-1 and cytotoxicity activity at the Pharmacology Unit, Department of Pharmacognosy, College of Pharmacy, University of Mansoura, Mansoura, Egypt and National Cancer Institute, Cairo University, Cairo, Egypt.

Synthesis of 3-amino-1-(pyridin-4-yl)-5-(pyridin-4-ylmethylene)-5, 6, 7, 8-tetrahydro-1H-isothiochromene-4- carbonitrile (3)

A mixture of 2, 6-bis(pyridin-4-ylmethylene) cyclohexan-1-one (2.76 g, 0.01 mol), malononitrile (0.66 g, 0.01 mol), sulfur (0.32 g, 0.01 mol), and diethylamine (0.01 mol) in ethanol (30 mL) was heated at 80–90 °C for 7–9 h, then the mixture was left for 36 h at 0 °C. The formed product was collected by filtration, washed with ethanol (25 mL), dried and crystallized from absolute methanol, as yellow crystals (90%), mp 290–292 °C. IR (ν, cm⁻¹) KBr: 3420 (br. NH₂), 3050 (CH-aryl), 2952 (CH-aliph), 2212 (CN), 1624 (C=N). ¹H NMR (DMSO-d₆, ppm) δ 1.57–2.23 (m, 6H, 3CH₂, cyclohexene), 4.62(s, 1H, thiopyran), 6.28 (brs, NH_2, D₂O exchangeable),7.56–7.96 (m, 8H, Ar-H), 8.23 (s, 1H, C=CHAr), ¹³C NMR (DMSO-d₆) δ 15.6, 21.5, 31.2 (3 C, CH₂), 65.5, 88.9 (2 C, thiopyran),111.9 (CN), 116.2 (1 C, C=CHAr), 121.4, 124.8, 130.2, 130.8, 130.9, 131.5, 136.1, 147.7, 158.3 (13 C, Ar-C), 165.6 (C-NH₂); MS (70 eV, %) m/z 358 (M⁺, 100 %); Anal. Calc. (Found) for C₂₁H₁₈N₄S (358.46): C, 70.36 (70.45); H, 5.06 (5.14); N, 15.63(15.72); S, 8.94(8.51).

Synthesis of 6-(pyridin-4-yl)-10-(pyridin-4-ylmethylene)-4, 6, 7, 8, 9, 10-hexahydro-1H-isothio chromeno[3, 4-d]pyrimidine-1,3 (2H)-dithione (5)

A mixture of compound 3 (3.58 g, 0.01 mol), and carbon disulfide (excess 15 mL) was heated under reflux in ethanolic potassium hydroxide solution for 15 h. The solution was allowed to cool to 0 °C for 5 h; the solid precipitate was filtered off, washed with water (30 mL), dried and crystallized from dimethylformamide to give (5) as a yellow crystals (85%); mp 315–317 °C. IR (ν, cm⁻¹) KBr: 3395 (brs, NH), 3055 (CH-aryl), 2950 (CH-aliph), 1620 (C=N), 1355, 1370 (2 C=S).¹H NMR (DMSO-d6, ppm) δ 1.26–2.35 (m, 6H, 3CH₂, cyclohexene), 4.29 (s,1H, thiopyran),7.45–8.02 (m, 8H, Ar-H), 8.27 (s, 1H, C=CHAr), 9.19, 9.21 (brs, 2NH_, D₂O exchangeable), ¹³C NMR (DMSO-d₆) δ 16.8, 22.5, 32.5 (3 C, CH₂), 60.2, 90.8 (2 C, thiopyran), 114.7 (1 C, C=CHAr), 122.9, 123.8, 132.5, 133.9, 142.4, 145.5, 146.8, 148.7, 149.1,158.6 (14 C, Ar–C), 168.5, 185.4 (2 C, 2 C=S); MS (70 eV, %) m/z 434 (M⁺, 100 %); Anal. Calc. (Found) for C₂₂H₁₈N₄S₃ (434.59): C, 60.80 (60.75); H, 4.17 (4.25); N, 12.89 (12.94); S, 22.13 (22.06).

Synthesis of 5-(pyridin-4-yl)-1-(pyridin-4-ylmethylene)-12-thioxo-1,3,4,5,7,7a-hexa- hydro-2H,12H-isothiochromeno[3,4-d]thiazolo[3,2-a]pyrimidin-9(10H)-one (8) and 5-(pyridin-4-yl)-1-(pyridin-4-ylmethylene)-13-thioxo-1,3,4,5,7,7a,10,11-octahydro-2H,9H,13H-isothiochromeno [3′,4′:4,5] pyrimido[2,1-b][1,3]thiazin-9-one (13)

General method: A mixture of compound 5 (4.34 g, 0.01 mol), and chloroacetic acid (0.93 g, 0.01 mol) or β-bromopropionic acid (1.52 g, 0.01 mol) was heated under reflux in a solution of glacial acetic acid and acetic anhydride (8 ml, 3:1 V) in the presence of sodium acetate anhydrous (0.82 g, 0.01 mol) for 7–10 h. The reaction mixture was into crashed ice water. The formed precipitate was washed with water and filtered off, dried, and recrystallized from the suitable solvent to give 8 and 13 respectively.

Compound 8: Yellowish crystals, yield (75%), mp 328–330 °C, crystallized from EtOH; IR (ν, cm⁻¹) KBr: 3050 (CH-aryl), 2954 (CH-aliph), 1710 (C=O), 1624 (C=N), 1325 (C=S), ¹H NMR (DMSO-d6, ppm) δ 1.19–1.69 (m, 6H, 3CH₂, cyclohexene), 4.18 (s, 2H, CH_2, thiazolidinone), 4.62 (s, 1H, thiopyran), 7.10–7.74 (m, 8H, Ar-H), 8.07 (s, 1H, C=CHAr), ¹³C-NMR (DMSO-d₆) δ 15.7, 21.6, 31.2 (3 C, CH₂), 47.7 (1 C, thiopyran), 89.1 (1 C, CH_2, thiazole),111.8 (1 C, C=CHAr), 116.2, 121.5, 124.8, 130.3, 131.5, 135.8, 136.1, 136.9, 140.1, 140.2, 140.3, 140.5 (16 C,Ar-C), 165.6 (1 C, C=O), 172.5 (1 C, C=S); MS (70 eV, %) m/z 474 (M⁺, 100 %); Anal. Calc. (Found) for C₂₄H₁₈N₄OS₃ (474.62): C, 60.74 (60.78); H, 3.82 (3.75); N, 11.80 (11.90); S, 20.26 (20.20).

Compound 13: Yellow crystals, yield (70%), mp 338–340 °C, crystallized from MeOH; IR (ν, cm⁻¹) KBr: 3058 (CH-aryl), 2960 (CH-aliph), 1718 (C=O), 1621 (C=N), 1322 (C=S). ¹H NMR (DMSO-d6, ppm) δ 1.01 (t, 2H, J = 3.70 Hz, CH₂CH₂S_, thiazine), 1.75–2.41 (m, 6H, 3CH₂, cyclohexene), 3.13 (t, 2H, J = 3.74 Hz, CH₂CH₂S_, thiazine), 4.51 (s, 1H, thiopyran), 7.05–8.07 (m, 8H, Ar-H), 8.09 (s, 1H, C=CHAr),¹³C NMR (DMSO-d₆) δ 17.9, 23.6, 30.5 (3 C, CH₂), 55.8 (1 C, thiopyran), 48.5 CH₂CH₂S_, thiazine, 50.5 (CH₂CH₂S_, thiazine), 113.9 (1 C, C=CHAr), 123.8, 124.5, 128.4, 132.7, 133.9, 143.5, 145.6, 147.8, 149.4, 149.9, 155.7, 158.7 (16 C,Ar-C),169.2 (1 C, C=O), 180.1(1 C, C=S); MS (70 eV, %) m/z 488 (M⁺, 100 %); Anal. Calc. (Found) for C₂₅H₂₀N₄OS₃ (488.64): C, 61.45 (61.54); H, 4.13 (4.18); N, 11.47 (11.40); S, 19.68 (19.61).

Synthesis of 10-(4-substitute-benzylidene)-5-(pyridin-4-yl)-1-(pyridin-4-ylmethylene)-12-thioxo-1,3,4,5-tetrahydro -2H,12H-isothiochromeno[3,4-d]thiazolo[3,2-a]pyrimidin-9(10H)-one(10a-c) and 10-(4-substituted-benzylidene)-5-(pyridin-4-yl)-1-(pyridin-4-ylmethylene)-13-thioxo-1,3,4,5,10, 11-hexahydro-2H,9H,13H-isothiochromeno[3’,4’:4,5]pyrimido[2,1-b][1,3] thiazin-9-one (14a-c)

General method: A mixture of compound 8 (4.74 g, 0.01 mol) or compound 13 (4.88 g, 0.01 mol), and aromatic aldehyde derivatives, namely: benzaldehyde (1.06 g, 0.01 mol) or 4-chloro- benzaldehyde (1.40 g, 0.01 mol) or 4-methoxybenzaldehyde (1.36 g, 0.01 mol), respectively was refluxed in a solution of glacial acetic acid (10 mL) and anhydrous sodium acetate anhydrous (0.82 g, 0.01 mol) for 12–14 h. The reaction mixture was into crashed ice water and the formed precipitate product was collected by filtration and crystallized from the appropriate solvent to afford 10a–c and 14a–c, respectively.

10-(benzylidene)-5-(pyridin-4-yl)-1-(pyridin-4-ylmethylene)-12-thioxo-1, 3, 4, 5-tetrahydro-2H, 12H-isothiochromeno[3,4-d]thiazolo[3,2-a]pyrimidin-9(10H)-one(10a)

Yellowish crystals, yield (60%), mp > 340 °C, crystallized from EtOH; IR (ν, cm⁻¹) KBr: 3060 (CH-aryl), 2958 (CH-aliph), 1720 (C=O),1625(C=N), 1328 (C=S), ¹H NMR (DMSO-d6, ppm) δ 1.19–1.69 (m, 6H, 3CH₂, cyclohexene), 4.72 (s, 1H, thiopyran),7.10–8.06 (m, 13H, Ar-H), 8.13 (s, 1H, C=CHAr), 8.23 (s, 1H, C=CHAr),¹³C NMR (DMSO-d₆) δ 18.7, 23.6, 33.2 (3 C, CH₂), 44.9 (1 C, thiopyran), 110.5, 112.8 (2 C, 2 C=CHAr), 123.4, 124.6, 127.2, 127.9, 128.2, 128.5, 133.2, 133.9, 134.9, 144.1, 146.3, 147.5, 149.2, 149.9, 151.5, 155.4, 158.9 (23 C, Ar–C), 179.8 (1 C, C=O), 186.2(1 C, C=S); MS (70 eV, %) m/z 562 (M⁺, 100 %); Anal. Calc. (Found) for C₃₁H₂₂N₄OS₃ (562.72): C, 66.17 (66.24); H, 3.94 (3.90); N, 9.96 (9.92); S, 17.09 (17.15).

10-(4-chlorobenzylidene)-5-(pyridin-4-yl)-1-(pyridin-4-ylmethylene)-12-thioxo-1,3,4,5-tetrahydro-2H,12H-isothiochromeno[3,4-d]thiazolo[3,2-a]pyrimidin-9(10H)-one(10b)

Yellow crystals, yield (65%), mp > 340 °C, crystallized from hexane; IR (ν, cm⁻¹) KBr: 3064 (CH-aryl), 2960 (CH-aliph), 1628(C=N), 1330 (C=S), 1724 (C=O).¹H NMR (DMSO-d6, ppm) δ 1.21–1.73 (m, 6H, 3CH₂, cyclohexene), 4.62 (s, 1H, thiopyran),7.10–7.95(m, 12H, Ar-H), 8.09 (s, 1H, C=CHAr), 8.18 (s, 1H, C=CHAr),¹³C NMR (DMSO-d₆) δ 15.6, 21.6, 31.2 (3 C, CH₂), 65.6 (1 C, thiopyran), 107.8, 111.9 (2 C, 2 C=CHAr), 116.8, 118.2, 121.5, 124.8, 130.2, 130.8, 130.9, 131.5, 136.1, 141.7, 143.6, 147.7, 149.5, 153.6, 158.4, 165.7, 168.2 (23 C, Ar–C), 173.8 (1 C, C=O), 177.9 (1 C, C=S); MS (70 eV, %) m/z 597 (M⁺, 90 %); Anal. Calc. (Found) for C₃₁H₂₁ClN₄OS₃ (597.17): C, 62.35 (62.40); H, 3.54 (3.59); N, 9.38 (9.33); S, 16.11 (16.18).

10-(4-methoxybenzylidene)-5-(pyridin-4-yl)-1-(pyridin-4-ylmethylene)-12-thioxo-1,3,4,5-tetra hydro-2H,12H-isothiochromeno[3,4-d]thiazolo[3,2-a]pyrimidin-9(10H)-one(10c)

Brownish crystals, yield (62%), mp > 340 °C, crystallized from benzen; IR (ν, cm⁻¹) KBr: 3059 (CH-aryl), 2957 (CH-aliph), 1722 (C=O), 1626 (C=N), 1328 (C=S), ¹H NMR (DMSO-d6, ppm) δ 1.22–1.75 (m, 6H, 3CH₂, cyclohexene), 3.85 (s,3H, CH₃), 4.67 (s, 1H, thiopyran),7.12–7.94 (m, 12H, Ar-H), 8.08 (s, 1H, methine proton), 8.15 (s, 1H, C=CHAr),¹³C NMR (DMSO-d₆) δ 19.1, 23.4, 31.3 (3 C, CH₂), 44.9 (1 C, thiopyran), 56.7(1 C,OCH₃), 120.8, 121.9 (2 C, 2CH C=CHAr), 122.8, 123.1, 124.2, 127.4, 129.1, 130.4, 133.5, 134.6, 144.2, 146.7, 147.5, 149.5, 149.8, 150.3, 156.7, 158.1, 159.5 (23 C, Ar–C), 169.2 (1 C, C=O), 175.5 (1 C, C=S); MS (70 eV, %) m/z 592 (M⁺, 95 %); Anal. Calc. (Found) for C₃₂H₂₄N₄O₂S₃ (592.75): C, 64.84 (64.90); H, 4.08 (4.12); N, 9.45 (9.49); S, 16.23 (16.28).

10-(benzylidene)-5-(pyridin-4-yl)-1-(pyridin-4-ylmethylene)-13-thioxo-1,3,4,5,10,11-hexahydro-2H,9H,13H-isothiochromeno[3′,4′:4,5]pyrimido[2,1-b] [1,3]thiazin-9-one (14a)

Yellow crystals, yield (70%), mp > 340 °C, crystallized from dioxane; IR (ν, cm⁻¹) KBr: 3065 (CH-aryl), 2960 (CH-aliph), 1730 (C=O), 1629(C=N), 1335 (C=S), ¹H NMR (DMSO-d6, ppm) δ 1.25–2.31 (m, 6H, 3CH₂, cyclohexene), 4.29 (s, 2H, CH₂N, thiazine), 4.41 (s, 1H, thiopyran), 7.06–7.95 (m, 13H, Ar–H), 8.07 (s, 1H, C=CHAr), 8.15 (s, 1H, C=CHAr),¹³C NMR (DMSO-d₆) δ 20.2, 24.6, 28.8 (3 C, CH₂), 45.2 (1 C, thiopyran), 58.2 (1 C, CH₂N, thiazine), 121.2, 123.1 (2 C, 2 C=CHAr), 123.1, 124.4, 127.1, 127.9, 128.6, 128.8, 133.3, 133.8, 135.4, 141.2, 144.7, 146.1, 147.3, 149.5, 149.8, 155.6, 159.1 (23 C, Ar–C), 168.5 (1 C, C=O), 177.4 (1 C, C=S); MS (70 eV, %) m/z 576 (M⁺, 98 %); Anal. Calc. (Found) for C₃₂H₂₄N₄OS₃ (576.75): C, 66.64 (66.60); H, 4.19 (4.12); N, 9.71 (9.78); S, 16.68 (16.62).

10-(4-chlorobenzylidene)-5-(pyridin-4-yl)-1-(pyridin-4-ylmethylene)-13-thioxo-1,3,4,5,10,11-hexa hydro-2H,9H,13H-isothiochromeno[3′,4′:4,5]pyrimido[2,1-b][1,3]thiazin-9-one (14b)

Yellowish crystals, yield (75%), mp > 340 °C, crystallized from methanol; IR (ν, cm⁻¹) KBr: 3062 (CH-aryl), 2958 (CH-aliph), 1735 (C=O), 1625 (C=N), 1338 (C=S). ¹H NMR (DMSO-d6, ppm) δ 1.18–2.25 (m, 6H, 3CH₂, cyclohexene), 4.30 (s, 2H, CH₂N, thiazine), 4.33 (s, 1H, thiopyran), 7.21–7.99 (m, 12H, Ar–H), 8.01 (s, 1H, C=CHAr), 8.26 (s, 1H, C=CHAr),¹³C NMR (DMSO-d₆) δ 19.8, 24.7, 28.6 (3 C, CH₂), 45.5 (1 C, thiopyran), 57.8 (1 C, CH₂N, thiazine), 120.6, 123.3 (2 C, C=CHAr), 123.2, 124.5, 127.6, 128.9, 129.1, 133.1, 133.4, 133.8, 135.2, 140.7, 144.4, 146.6, 147.5, 149.6, 149.9, 156.2, 159.5 (23 C, Ar–C),168.2 (1 C, C=O), 178.2 (1 C, C=S); MS (70 eV, %) m/z 611 (M⁺, 100 %); Anal. Calc. (Found) for C₃₂H₂₃ClN₄OS₃ (611.19): C, 62.89 (62.80); H, 3.79 (3.72); N, 9.17 (9.11); S, 15.74 (15.78).

10-(4-methoxybenzylidene)-5-(pyridin-4-yl)-1-(pyridin-4-ylmethylene)-13-thioxo-1,3,4,5,10,11-hexahydro-2H,9H,13H-isothiochromeno[3′,4′:4,5]pyrimido[2,1-b][1,3]thiazin-9-one (14c)

Yellow crystals, yield (71%), mp > 350 °C, crystallized from DMF; IR (ν, cm⁻¹) KBr: 3060 (CH-aryl), 2955 (CH-aliph), 1738 (C=O), 1628(C=N), 1340 (C=S).¹H NMR (DMSO-d6, ppm) δ 1.20–2.28 (m, 6H, 3CH₂, cyclohexene), 3.80 (s,3H, CH₃), 4.29 (s, 2H, CH₂N, thiazine), 4.41 (s, 1H, thiopyran), 7.22–7.97 (m, 12H, Ar-H), 8.05 (s, 1H, C=CHAr), 8.22 (s, 1H, C=CHAr),¹³C NMR (DMSO-d₆) δ 24.8, 30.8, 30.9 (3 C, CH₂), 58.3 (1 C, thiopyran), 58.5 (1 C, OCH₃), 58.9 (1 C, CH₂N, thiazine), 115.8, 115.9 (2 C, C=CHAr), 116.1, 121.5, 124.8, 130.3, 130.8, 130.9, 131.5, 136.1, 140.1, 140.2, 147.7, 152.6, 152.8, 158.3, 165.5, 165.6 (23 C, Ar–C), 168.5 (1 C, C=O), 180.1 (1 C, C=S); MS (70 eV, %) m/z 606 (M⁺, 90 %); Anal. Calc. (Found) for C₃₃H₂₆N₄O₂S₃ (606.78): C, 65.32 (65.38); H, 4.32 (4.40); N, 9.23 (9.29); S, 15.85 (15.91).

Synthesis of 1-chloro-6-(pyridin-4-yl)-10-(pyridin-4-ylmethylene)-7, 8, 9, 10-tetrahydro-6H-isothiochromeno [3, 4- d] [1, 2, 3] triazine (15)

To a cool solution of compound 3 (3.58 g, 0.01 mol) in a mixture of acetic acid (15 mL) and conc. hydrochloric acid (10 mL), a sodium nitrite solution 10% (2 mL) was added with stirring for 15 min. The stirring was continued at (1–5 °C) for 4 h. The formed precipitate was filtered off, dried and crystallized from DMF to give compound 15. Yellow crystals, yield (78%), mp 325–327 °C (dec.); IR (ν, cm⁻¹) KBr: 3065 (CH-aryl), 2952 (CH-aliph), 1625(C=N). ¹H NMR (DMSO-d₆, ppm) δ 1.57–2.23 (m, 6H, 3CH₂, cyclohexene), 4.42 (s,1H, thiopyran), 7.56–7.96 (m, 8H, Ar–H), 8.23 (s, 1H, C=CHAr), ¹³C NMR (DMSO-d₆) δ 15.6, 21.6, 31.2 (3 C, CH₂), 65.6 (1 C, thiopyran), 1119 (1 C, C=CHAr), 120.7, 121.5, 124.9, 130.2, 130.8, 130.9, 131.5, 136.1, 143.2, 147.7, 154.1, 158.4 (16 C, Ar–C); MS (70 eV, %) m/z 405 (M⁺, 100 %); Anal. Calc. (Found) for C₂₁H₁₆ClN₅S (405.90): C, 62.14 (62.20); H, 3.97 (3.90); N, 17.25 (17.20); S, 7.90 (7.85).

Synthesis of 1-imino-2-phenyl-6-(pyridin-4-yl)-10-(pyridin-4-ylmethylene)-1, 2, 4, 6, 7, 8, 9, 10-octahydro-3H-isothiochromeno[3,4-d]pyrimidine-3-thione (18a) and 1-imino-2-phenyl-6-(pyridin-4-yl)-10-(pyridin-4-ylmethylene)-1,2,4,6,7,8,9,10-octahydro-3H-isothiochromeno[3,4-d]pyrimidin-3-one (18b)

General method: A mixture of compound 3 (3.58 g, 0.01 mol) and phenylisothiocyanate (1.2 mL, 0.01 mol) or phenylisocyanate (1.1 mL, 0.01 mol) in pyridine (30 mL) was refluxed for 10–12 h. The reaction mixture was left to cool, acidified with dilute hydrochloric acid; the formed product precipitate was filtered off, dried, and recrystallized from the suitable solvent to give 18a and 18b, respectively.

Compound 18a: Recrystallized from THF as yellow crystals in 68% yield, mp > 340 °C (dec.); IR (ν, cm⁻¹) KBr: 3250, 3220 (2NH), 3050 (CH-aryl), 2958 (CH-aliph), 1628 (C=N), 1275 (C=S). ¹H NMR (DMSO-d₆, ppm) δ 1.19–2.28 (m, 6H, 3CH₂, cyclohexene), 4.72 (s,1H, thiopyran), 7.01–8.01 (m, 13H, Ar–H), 8.05 (s, 1H, C=CHAr), 10.58 (br. s, 1H, NH_, D₂O exchangeable), 10.62 (br. s, 1H, NH_, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 15.9, 21.8, 31.5 (3 C, 3CH₂), 67.4, 91.6 (2 C, thiopyran), 112.8 (1 C, C=CHAr),119.4, 122.2, 128.5, 129.3, 132.7, 133.6, 138.4, 144.6, 146.9, 147.4, 149.1, 149.9, 151.5 (20 C, Ar–C), 159.5 (1 C, C=NH), 181.2 (1 C, C=S); MS (70 eV, %) m/z 493 (M⁺, 99%); Anal. Calc. (Found) for C₂₈H₂₃N₅S₂ (493.65): C, 68.13 (68.20); H, 4.70 (4.78); N, 14.19 (14.25); S, 12.99 (12.92).

Compound 18b: Recrystallized from Toluene as yellowish crystals in 66% yield, mp > 350 °C (dec.); IR (ν, cm⁻¹) KBr: 3255, 3222 (2NH), 3051 (CH-aryl), 2962 (CH-aliph), 1670 (C=O), 1629 (C=N).¹H NMR (DMSO-d₆, ppm) δ 1.20–2.30 (m, 6H, 3CH₂, cyclohexene), 4.74 (s,1H, thiopyran), 7.02–7.99 (m, 13H, Ar–H), 8.06 (s, 1H, C=CHAr), 10.60 (br. s, 1H, NH_, D₂O exchangeable), 10.65 (br. s, 1H,NH_, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 15.6, 21.5, 31.2(3 C, 3CH₂), 65.5, 88.9 (2 C, thiopyran),111.9 (1 C, C=CHAr), 116.3, 118.5, 121.6, 124.8, 130.3, 130.8, 130.9, 131.6, 136.2, 139.9, 144.3, 147.8, 152.5, 153.6 (20 C, Ar–C), 158.2 (1 C, C=NH), 160.2 (1 C, C=O); MS (70 eV, %) m/z 477 (M⁺, 100 %); Anal. Calc. (Found) for C₂₈H₂₃N₅OS (477.59): C, 70.42 (70.50); H, 4.85 (4.80); N, 14.66 (14.61); S, 6.71 (6.78).

Synthesis of Ethyl-N-(4-cyano-1-(pyridin-4-yl)-5-(pyridin-4-ylmethylene)-5,6,7,8-tetrahydro-1H-isothio- chromen-3-yl) formimidate (20a) and ethyl-N-(4-cyano-1-(pyridin-4-yl)-5-(pyridin-4-yl methylene)-5, 6, 7, 8-tetrahydro-1H-isothiochromen-3-yl) acetimidate (20b)

General method: A mixture of compound 3 (3.58 g, 0.01 mol) and triethylorthoformate (1.66 mL, 0.01 mol) or triethylorthoacetate (1.83 mL, 0.01 mol) in acetic anhydride (30 mL) was heated under reflux for 10–12 h (TLC control). The solution was cooled overnight to room temperature and then concentrated. The precipitated solid was filtered off and recrystallized to yields 20a and 20b respectively.

Compound 20a: Crystallized from dioxane, yellow crystals in 80% yield, mp 260–262 °C (dec.); IR (ν, cm⁻¹) KBr: 3055 (CH-aryl), 2960 (CH-aliph), 2210(CN), 1622(C=N). ¹H NMR (DMSO-d₆, ppm) δ 1.29 (t, J = 5.55 Hz, 3H, CH₃), 1.32–2.43 (m, 6H, 3CH₂, cyclohexene), 4.23 (q, J = 5.60 Hz, 2H, CH₂), 4.72 (s,1H, thiopyran), 7.01–7.98 (m, 8H, Ar-H), 8.03 (s, 1H, C=CHAr), 8.05 (s, 1H, N=CHO); ¹³C NMR (DMSO-d₆) δ 20.2 (1 C, CH₃), 24.4, 25.9, 28.1 (3 C, CH₂), 46.5 (1 C, thiopyran), 66.2 (1 C, OCH₂), 98.8 (1 C, thiopyran), 118.7(1 C, CN),121.1 (1 C, C=CHAr), 123.6, 124.8, 133.7, 134.6, 145.1, 146.7, 147.5, 149.4, 149.9, 153.4 (14 C, Ar–C), 155.8(1 C, N=CHOEt); MS (70 eV, %) m/z 414 (M⁺, 100 %); Anal. Calc. (Found) for C₂₄H₂₂N₄OS (414.53): C, 69.54 (69.62); H, 5.35 (5.40); N, 13.52 (13.58); S, 7.73 (7.81).

Compound 20b: Crystallized from benzene, yellowish crystals in 82% yield, mp 273–275 °C (dec.); IR (ν, cm⁻¹) KBr: 3058 (CH-aryl), 2964 (CH-aliph), 2212 (CN), 1620 (C=N). 1H NMR (DMSO-d6, ppm) δ 1.21 (s,1H,CH₃),1.33 (t, J = 5.58 Hz, 3H, _CH3), 1.36–2.45 (m, 6H, 3CH₂, cyclohexene), 4.10 (q, J = 5.62 Hz,2H, OCH₂CH₃), 4.70 (s,1H, thiopyran), 7.04–7.99 (m, 8H, Ar-H), 8.06 (s, 1H, C=CHAr); ¹³C NMR (DMSO-d6) δ 19.5 (1 C, CH₂CH₃), 21.9 (1 C, N=CCH₃), 23.6, 25.2, 27.8 (3 C, CH₂), 47.1 (1 C, thiopyran), 68.4 (1 C,OCH₂), 99.2 (1 C, thiopyran), 118.2 (1 C, CN), 121.5 (1 C, C=CHAr), 123.3, 124.5, 133.2, 134.1, 145.3, 146.6, 147.4, 149.2, 149.6, 152.1 (14 C, Ar–C), 164.5 (1 C, N=COEt); MS (70 eV, %) m/z 428 (M⁺, 90 %); Anal. Calc. (Found) for C₂₅H₂₄N₄OS (428.55): C, 70.07 (70.10); H, 5.65 (5.71); N, 13.07 (13.12); S, 7.48 (7.55).

Synthesis of 1-imino-N-phenyl-6-(pyridin-4-yl)-10-(pyridin-4-ylmethylene)-7,8,9,10-tetrahydro-1H-isothiochromeno[3,4-d]pyrimidin-2(6H)-amine (21a) and 1-imino-3-methyl-N-phenyl-6-(pyridin-4-yl)-10-(pyridin-4-ylmethylene)-7,8,9,10-tetrahydro-1H-isothiochromeno [3,4-d]pyrimidin-2(6H)-amine (21b)

General method: A suspension of compound 20a (4.14 g, 0.01 mol) or 20b (4.28 g, 0.01 mol) and phenylhydrazine (1.08 g, 0.01 mol) in dioxane (30 mL) was refluxed for 6–8 h. The solid product which formed was filtered off, washed with water, dried in air and recrystallized from the suitable solvent to afford 21a and 21b respectively.

Compound 21a: Crystallized from methanol, yellowish crystals in 78% yield, mp > 340 °C (dec.); IR (ν, cm⁻¹) KBr: 3210, 3190 (2NH), 3054 (CH-aryl), 2956 (CH-aliph), 1626(C=N). ¹H NMR (DMSO-d₆, ppm) δ 1.30–2.21 (m, 6H, 3CH₂, cyclohexene), 4.75 (s, 1H, thiopyran), 7.15–7.95 (m, 13H, Ar-H), 8.05 (s, 1H, C=CHAr), 8.30 (s, 1H, N=CH), 9.35 (br. s, 1H, NH_, D₂O exchangeable), 10.58 (br. s, 1H, NH_, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 23.8, 26.2, 28.5 (3 C, 3CH₂), 48.1, 108.2 (2 C, thiopyran),120.9 (1 C, C=CHAr), 121.8, 122.5, 123.4, 124.6, 130.5, 133.1, 134.8, 144.1, 145.3, 146.8, 147.5, 147.9, 149.4, 149.7, 154.2 (21 C, Ar-C),159.3 (1 C, C=NH); MS (70 eV, %) m/z 476 (M⁺, 100 %); Anal. Calc. (Found) for C₂₈H₂₄N₆S (476.60): C, 70.56 (70.50); H, 5.08 (5.15); N, 17.63 (17.68); S, 6.73(6.80).

Compound 21b: Crystallized from DMF, yellow crystals in 74 % yield, mp > 350 °C (dec.); IR (ν, cm⁻¹) KBr: 3215, 3195 (2NH), 3052 (CH-aryl), 2954 (CH-aliph), 1628(C=N). ¹H NMR (DMSO-d₆, ppm) δ 1.29–2.10 (m, 6H, 3CH₂, cyclohexene), 2.92(s, 3H, CH₃), 4.72 (s,1H, thiopyran), 7.21–8.01 (m, 13H, Ar-H), 8.03 (s, 1H, C=CHAr), 9.28 (br. s, 1H, NH_, D₂O exchangeable), 10.52 (br. s, 1H, NH_, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 20.4 (1 C,CH₃), 23.5, 26.6, 28.2 (3 C, 3CH₂), 46.3,105.5 (2 C,thiopyran), 120.6 (1 C, C=CHAr), 121.5, 122.7, 123.2, 124.4, 129.8, 133.5, 134.7, 144.4, 145.8, 146.5, 147.4, 149.3, 149.6, 153.9, 156.2 (21 C, Ar–C),157.9 (1 C, C=NH); MS (70 eV, %) m/z 490 (M⁺, 88 %); Anal. Calc. (Found) for C₂₉H₂₆N₆S (490.63): C, 70.99 (70.90); H, 5.34 (5.40); N, 17.13 (17.20); S, 6.53(6.61).

Synthesis of 2-ethoxy-3-phenyl-8-(pyridin-4-yl)-12-(pyridin-4-ylmethylene)-2,3,9,10,11,12-hexahydro-8H-isothiochromeno[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (22a) and 2-ethoxy-5-methyl-3-phenyl-8-(pyridin-4-yl)-12-(pyridin-4-ylmethylene)-2,3,9,10,11,12-hexahydro-8H-isothiochromeno[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (22b)

General method: A solution of compounds 21a (4.76 g, 0. 01 mol) or 21b (4.90 g, 0. 01 mol) in triethylorthoformate (10 mL) was refluxed for 2–4 h. The reaction mixture was poured into crashed ice water; the formed precipitated was collected via filtration and recrystallized to afford 22a and 22b respectively.

Compound 22a: Crystallized from chloroform, yellow crystals in 75 % yield, mp >340 °C (dec.); IR (ν, cm⁻¹) KBr: 3050 (CH-aryl), 2952 (CH-aliph), 1622(C=N). ¹H NMR (DMSO-d₆, ppm) δ 1.31 (t, J = 5.60 Hz, 3H, CH₂CH₃), 1.33–2.43 (m, 6H, 3CH₂, cyclohexene), 4.22 (q, J = 5.65 Hz, 2H, OCH₂), 4.72 (s,1H, thiopyran), 6.34 (s,1H,CHOEt, triazole ring), 7.01–7.55 (m, 13H, Ar–H), 8.13 (s, 1H, C=CHAr), 8.84 (s, 1H, N=CH); ¹³C NMR (DMSO-d₆) δ 18.5(1 C, CH₃), 22.6, 25.8, 27.2 (3 C, 3CH₂), 66.5 (1 C, OCH₂CH₃), 69.7, 190.4 (2C, thiopyran), 112.2 (1 C, C=CHAr), 121.5, 122.4, 123.5, 124.4, 127.2, 130.3, 133.6, 134.5, 142.8, 144.3, 145.9, 146.7, 149.4, 149.7, 150.1, 152.4, 155.2, (23 C, Ar–C); MS (70 eV, %) m/z 532 (M⁺, 100 %); Anal. Calc. (Found) for C₃₁H₂₈N₆OS (532.67): C, 69.90 (69.98); H, 5.30 (5.25); N, 15.78 (15.72); S, 6.02 (6.10).

Compound 22b: Crystallized from ethanol, yellowish crystals in 73% yield, mp >340 °C (dec.); IR (ν, cm⁻¹) KBr: 3053 (CH-aryl), 2954 (CH-aliph), 1621(C=N).¹H NMR (DMSO-d₆, ppm) δ 1.30 (t, J = 5.64 Hz, 3H, CH₃), 1.35–2.44 (m, 6H, 3CH₂, cyclohexene), 3.92 (s,3H,CH₃), 4.20 (q, J = 5.69 Hz,2H, CH₂), 4.70 (s,1H, thiopyran), 6.32 (s,1H, triazole ring), 7.04–7.59 (m, 13H, Ar–H), 8.11 (s, 1H, C=CHAr),¹³C NMR (DMSO-d₆) δ 15.7, 21.6(2 C,2CH₃), 31.2, 31.3, 31.9, 38.6 (4 C, 4CH₂), 65.6, 88.9 (2 C, thiopyran), 111.9 (1 C, C=CHAr), 116.2, 121.4, 124.8, 130.2, 130.8, 130.9, 131.5, 136.1, 137.2, 138.1, 140.4, 142.5, 144.2, 147.7, 150.1, 152.5, 159.3 (23 C, Ar–C); MS (70 eV, %) m/z 546 (M⁺, 90 %); Anal. Calc. (Found) for C₃₂H₃₀N₆OS (546.69): C, 70.30 (70.38); H, 5.53 (5.59); N, 15.37 (15.32); S, 5.86 (5.80).

Synthesis of Ethyl-(4-cyano-1-(pyridin-4-yl)-5-(pyridin-4-ylmethylene)-5, 6, 7, 8-tetrahydro-1H-isothiochromen-3-yl) glycinate (23)

A mixture of compound 3 (3.58 g, 0.01 mol), ethyl bromoacetate (1.11 mL, 0.01 mol) and anhydrous potassium carbonate (1.38 g, 0.01 mol) in dry acetone (35 mL) was refluxed for 8–10 h. The reaction mixture was allowed to cool to room temperature and water (30 mL) was added, then the solid that separated was filtered off, dried and crystallized from diethyl ether to give 23, yellow crystals in 88 % yield, mp 240–242 °C (dec.); IR (ν, cm⁻¹) KBr: 3250 (NH), 3055 (CH-aryl), 2960 (CH-aliph), 2215 (CN), 1730 (C=O), 1630 (C=N).¹H NMR (DMSO-d₆, ppm) δ 1.23 (t, 3H, J = 4.81 Hz, CH₃), 1.70–2.43 (m, 6H, 3CH₂, cyclohexene), 4.03 (s, 2H, NHCOCH₂), 4.21 (q, 2H, J = 4.88 Hz, OCH₂), 4.72 (s,1H, thiopyran), 7.11–7.96 (m, 8H, Ar–H), 8.24 (s, 1H, C=CHAr), 9.12 (br. s, 1H, NH_, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 15.2 (1 C, CH₃), 23.5, 26.8, 27.9 (3 C, 3CH₂), 44.1 (1 C, NHCOCH₂), 47.5 (1 C, thiopyran), 64.3 (1 C, OCH₂), 78.6 (1 C, thiopyran), 112.2 (1 C, C=CHAr), 115.5 (1 C, CN), 122.4, 124.8, 133.6, 134.8, 144.1, 146.3, 147.7, 149.1, 149.5, 165.2 (14 C, Ar–C), 168.9 (1 C, C=O); MS (70 eV, %) m/z 444 (M⁺, 100 %); Anal. Calc. (Found) for C₂₅H₂₄N₄O₂S (444.55): C, 67.55 (67.50); H, 5.44 (5.49); N, 12.60 (12.68); S, 7.21 (7.27).

Synthesis of Ethyl-1-amino-5-(pyridin-4-yl)-9-(pyridin-4-ylmethylene)-3, 5, 6, 7, 8, 9-hexahydroisothiochromeno [3, 4-b] pyrrole-2-carboxylate (24)

A solution of compound 23 (4.44 g, 0.01 mol), and anhydrous potassium carbonate (1.38 g, 0.01 mol) in dimethylformamide (30 mL) was refluxed for 5–8 h and then left to cool to room temperature and water (25 mL) was added. The formed precipitate was filtered off, dried and crystallized from ethyl acetate to give 24, yellow crystals, yield (85%), mp 250–252 °C (dec.); IR (ν, cm⁻¹) KBr: 3405(NH₂), 3280 (NH), 3058 (CH-aryl), 2962 (CH-aliph), 1735 (C=O), 1632 (C=N).¹H NMR (DMSO-d₆, ppm) δ 1.23 (t, 3H, J = 4.80 Hz, CH₃),1.68–2.42 (m, 6H, 3CH₂, cyclohexene), 4.22 (q, 2H, J = 4.85 Hz, OCH₂), 5.24 (s,1H, thiopyran), 6.27 (br. s, 2H, NH_2, D₂O exchangeable), 7.10–7.95 (m, 8H, Ar–H), 8.23 (s, 1H, C=CHAr), 10.10 (br. s, 1H, NH_, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 15.5 (1 C, CH₃), 21.8, 25.6, 30.7 (3 C, 3CH₂), 52.8 (1 C, OCH₂), 65.1, 92.6 (2 C, thiopyran), 111.8 (1 C, C=CHAr), 118.9, 122.6, 124.5, 130.8, 133.7, 135.9,143.5, 144.2, 146.8, 147.6, 149.3,149.9 (16 C, Ar–C), 162.4 (1 C, C=O); MS (70 eV, %) m/z 444 (M⁺, 92 %); Anal. Calc. (Found) for C₂₅H₂₄N₄O₂S (444.55): C, 67.55 (67.60); H, 5.44 (5.40); N, 12.60 (12.55); S, 7.21 (7.16).

Synthesis of Ethyl-5-(pyridin-4-yl)-9-(pyridin-4-ylmethylene)-1-thioureido-3, 5, 6, 7, 8, 9-hexahydroisothio- chromeno[3, 4-b] pyrrole-2-carboxylate (25)

A mixture of 24 (4.44 g, 0.01 mol) and 10 % HCl (10 mL) was refluxed with potassium thiocyanate (0.97 g, 0.01 mol) for 5–7 h. The reaction mixture was allowed to cool to room temperature. The formed solid was collected by filtration, washed with water, dried and crystallized from xylene to give 25, Yellow crystals, yield (80%), mp 302–304 °C (dec.); IR (ν, cm⁻¹) KBr: 3412 (NH₂), 3300 (NH), 3056 (CH-aryl), 2968 (CH-aliph), 1740 (C=O), 1631(C=N). ¹H NMR (DMSO-d₆, ppm) δ 1.23 (t, 3H, J = 4.79 Hz, CH₃), 1.68–2.42 (m, 6H, 3CH₂, cyclohexene), 4.22 (q, 2H, J = 4.87 Hz, CH₂), 5.82 (s,1H, thiopyran), 6.28 (br. s, 2H, NH_2, D₂O exchangeable), 7.10–7.95 (m, 8H, Ar-H), 8.23(s, 1H, C=CHAr), 9.17 (br. s, 1H, NH_, D₂O exchangeable), 9.32 (br. s, 1H, NH_, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 15.8 (1 C, CH₃), 21.6, 27.5, 31.2 (3 C, 3CH₂), 53.9 (1 C,OCH₂), 66.3, 90.5 (2 C, thiopyran), 112.5 (1 C, C=CHAr), 119.6, 122.4, 124.7, 131.1, 133.5, 135.7, 143.2, 144.8, 146.7, 147.5, 149.2, 149.6 (16 C, Ar–C), 163.5 (1 C, C=O), 181.2 (1 C, C=S); MS (70 eV, %) m/z 503 (M⁺, 90 %); Anal. Calc. (Found) for C₂₆H₂₅N₅O₂S₂ (503.64): C, 62.01 (62.10); H, 5.00 (5.09); N, 13.91 (13.97); S, 12.73 (12.80).

Synthesis of 5-(pyridin-4-yl)-1-(pyridin-4-ylmethylene)-10-thioxo-1,3,4,5,7,9,10,11-octahydro isothiochromeno [4′,3′:4,5]pyrrolo[3,2-d]pyrimidin-8 (2H)-one (26)

A solution of 25 (5.03 g, 0.01 mol) in ethanolic sodium ethoxide (0.23 g of sodium metal in 30 mL ethanol) was stirred under reflux for 8–10 h. After cooling, the reaction mixture was neutralized with 10% HCl and the solid formed was collected by filtration, washed with water, dried and then crystallized from dioxane to give 26. Yellow crystals, yield 78%, mp > 340 °C (dec.); IR (ν, cm⁻¹) KBr: 3355 (3NH), 3062 (CH-aryl), 2954 (CH-aliph), 1678 (C=O), 1628 (C=N). ¹H NMR (DMSO-d₆, ppm) δ 1.75–2.25 (m, 6H, 3CH₂, cyclohexene), 4.30 (s,1H, thiopyran), 7.11–7.93 (m, 8H, Ar-H), 8.12(s, 1H, C=CHAr), 10.35 (br. s, 1H, NH_, D₂O exchangeable), 12.32 (br. s, 1H, NH_, D₂O exchangeable), 13.13 (br. s, 1H, NH_, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 15.7, 21.6, 31.2 (3 C, 3CH₂), 89.1, 111.8 (2 C, thiopyran), 116.1 (1 C, C=CHAr), 118.1, 121.9,124.8, 128.1, 131.5, 135.8, 136.1, 142.1, 148.2, 152.1, 154.2, 159.8 (16 C, Ar-C), 165.6 (1 C, C=O),172.5 (1 C, C=S); MS (70 eV, %) m/z 457 (M⁺, 99 %); Anal. Calc. (Found) for C₂₄H₁₉N₅OS₂ (457.57): C, 63.00 (63.10); H, 4.19 (4.12); N, 15.31 (15.38); S, 14.01 (14.10).

Synthesis of 1-amino-5-(pyridin-4-yl)-9-(pyridin-4-ylmethylene)-3,5,6,7,8,9-hexa- hydroisothio- chromeno[3,4-b]pyrrole-2-carboxamide(30); (1-amino-5-(pyridin-4-yl)-9-(pyridin-4-ylmethylene)-3,5,6,7,8,9-hexahydroisothiochromeno[3,4-b]pyrrol-2-yl)(phenyl)methanone (31) and 1-amino-3-oxo-6-(pyridin-4-yl)-10-(pyridin-4-ylmethylene)-4,6,7,8,9,10-hexahydro-3H -isothiochromeno[3,4-b]pyridine-2-carbonitrile (32)

General method: A mixture of 3 (3.58 g, 0.01 mol), and the appropriate active methylene compounds namely, 2-chloroacetamide (0.93 g, 0.01 mol) or phenacyl bromides (1.99 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol), was stirred in (40 mL) of anhydrous ethanol containing sodium ethoxide (3%) for 3–4 h, and then was refluxed with stirring for 10–12 h. The reaction mixture was cooled and poured into ice water. The solid formed was filtered off, dried, and recrystallized from suitable solvent to give 30, 31 and 32 respectively.

Compound 30: The compound was obtained from the reaction of 3 (3.58 g, 0.01 mol) and 2-chloroacetamide (0.93 g, 0.01 mol), as a white crystals, crystallized from dioxane (70%), mp 348–350 °C. IR (ν, cm⁻¹) KBr: 3410–3250 broad (2NH₂ and NH), 3055 (CH-aryl), 2970 (CH-aliph), 1675 (C=O), 1625 (C=N).¹H NMR (DMSO-d₆, ppm) δ 1.85–2.27 (m, 6H, 3CH₂, cyclohexene), 4.63 (s,1H, thiopyran), 6.42, 6.45 (2 s, 2NH₂, D₂O exchangeable), 7.25–7.98 (m, 8H, Ar–H), 8.02 (s, 1H, C=CHAr), 12.68 (br., 1H, NH_, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 18.5, 22.4, 30.8 (3 C, 3 CH₂), 62.1, 94.3,110.5 (3C, thiopyran), 112.7 (1 C, C=CHAr), 122.7, 124.2, 132.6, 133.7, 135.9, 143.4, 145.6, 147.1, 148.4, 149.1, 149.5 (15 C, Ar–C), 164.6 (1 C, C=O); MS (70 eV, %) m/z 415 (M⁺, 90 %); Anal. Calc. (Found) for C₂₃H₂₁N₅OS (415.52): C, 66.48 (66.40); H, 5.09 (5.15); N, 16.85 (16.77); S, 7.72 (7.65).

Compound 31: The compound was obtained from the reaction of 3 (3.58 g, 0.01 mol) and phenacyl bromides (1.99 g, 0.01 mol), as a yellowish crystals, crystallized from DMF (73%), mp > 340 °C. IR (ν, cm⁻¹) KBr: 3400–3300 broad (NH₂ and NH), 3058 (CH-aryl), 2965 (CH-aliph), 1690 (C=O), 1627(C=N).¹H NMR (DMSO-d₆, ppm) δ 1.80–2.26 (m, 6H, 3CH₂, cyclohexene), 4.10 (s,1H, thiopyran), 6.43 (s, NH₂, D₂O exchangeable), 7.10–7.99 (m, 13H, Ar–H), 8.03 (s, 1H, C=CHAr), 12.67(br., 1H, NH_, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 17.1, 21.4, 30.9 (3 C, 3 CH₂), 59.5, 90.4, 98.8 (3C, thiopyran),111.1 (1 C, C=CHAr),122.4, 124.5,128.6, 129.5, 130.8, 132.9, 133.1, 135.2, 136.7, 144.1, 146.2, 147.5, 149.1, 149.5, 149.9 (21 C, Ar–C), 167.1 (1 C, C=O); MS (70 eV, %) m/z 476 (M⁺, 95 %); Anal. Calc. (Found) for C₂₉H₂₄N₄OS (476.60): C, 73.08 (73.14); H, 5.08 (5.14); N, 11.76 (11.70); S, 6.73 (6.78).

Compound 32: The compound was obtained from the reaction of 3 (3.58 g, 0.01 mol) and ethyl cyanoacetate (1.13 g, 0.01 mol), as a yellow crystals, crystallized from methanol (75%), mp > 340 °C. IR (ν, cm⁻¹) KBr: 3405–3280 broad (NH₂ and NH), 3054 (CH-aryl), 2960 (CH-aliph), 2210 (CN), 1676 (C=O), 1624(C=N).¹H NMR (DMSO-d₆, ppm) δ 1.75–2.25 (m, 6H, 3CH₂, cyclohexene), 4.25 (s,1H, thiopyran), 6.60 (s, NH₂, D₂O exchangeable), 7.57–7.98 (m, 8H, Ar-H), 8.12 (s, 1H, C=CHAr),12.32 (br., 1H, NH_, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 15.7, 21.6, 31.2 (3 C, 3CH₂), 65.6, 88.9 (2 C, thiopyran), 94.1 (1 C, pyridine),102.4 (1 C, CN), 111.9 (1 C, C=CHAr), 116.3, 121.5, 124.8,130.3,130.8, 130.9,131.5, 136.2, 147.7, 158.4 (14 C, Ar–C), 165.7 (1 C, C=O), 172.1(1 C, C–NH₂); MS (70 eV, %) m/z 425 (M⁺, 92 %); Anal. Calc. (Found) for C₂₄H₁₉N₅OS (425.51): C, 67.75 (67.79); H, 4.50 (4.58); N, 16.46 (16.40); S, 7.53 (7.48).