New Approaches for the Synthesis of Thiophenes, Thiazole, Pyran Derivatives, and their Antitumor Evaluation

Research article

New Approaches for the Synthesis of Thiophenes, Thiazole, Pyran Derivatives, and their Antitumor Evaluation

Corresponding author: Dr. Rafat M. Mohareb, Department of Chemistry, Faculty of Science, Cairo University, Giza, A. R. Egypt; Email: raafat_mohareb@yahoo.com

Abstract

This research aims to a novel synthesis of thiophene, pyrans, azoles and azines derivatives in which we started with cyclopenta- none as starting material which reacted with malononitrile and ethyl cyanoacetate. The synthesized compounds were confirmed by spectral and analytical data. The biological evaluation of compounds are tested on three different tumor cell lines such as breast adenocarcinoma, CNS cancer, and non-small lung cancer and was compared to the inhibitory effect of Doxorubicin. And the results revealed that compounds 7, 13b and 17b have the highest inhibitory effect.

Keywords: Thiophene; Pyrazole; Pyrane; Pyridine; Cytotoxicity

Introduction

Multi-component reactions have been proved as an important tool for the syntheses of many multi-substituted heterocyclic ring-containing compounds [1]. Synthesis of different sub- stituted 4H-pyrans has been studied in the literature [2-5]. Pyrans and their derivatives are of considerable interest be- cause of their wide range of biological property [6], such as spasmolytic, diuretic, anti-coagulant, anti-cancer, anti anaphy- lactic activity [7–11]. In addition, they can be used as cognitive enhancers, for the treatment of neurodegenerative disease, including Alzheimer’s disease, amyotrophic lateral sclerosis, Huntington’s disease, Parkinson’s disease, AIDS-associated dementia and Down’s syndrome as well as for the treatment of schizophrenia and myoclonus [12]. 4H-Pyrans also consti- tute the structural unit of a series of natural products [13,14]. On the other hand, several synthetic routes for polyfunction- al fused or pendant pyridine systems have been reported in the literature. They mainly involve intramolecular cyclization [15,16] multi-component intermolecular cyclization [17] metal-assisted coupling [18] microwave-assisted coupling [19,20] or cycloaddition [21], azo electronic coupling [22], regioselective hetero Diels-Alder [23] and internal Mannich reaction [24]. They have been used in a wide variety of bio- logical applications [25]. For the mentioned biological values of both pyran and pyridine derivatives we demonstrate here the synthesis of a series of such compounds through the one pot multi-component reaction of cyclopentanone with aromat- ic aldehydes and either of malononitrile or ethyl cyanoacetate together with studying of the antitumor activity of these com- pounds.

Results

In the present work, we started with cyclopentanone (1) which reacted with either of malononitrile (2a) or ethyl cyanoacetate (2b) and either of benzaldehyde (3a) or 4-methoxybenzalde- hyde (3b) in absolute ethanol containing a catalytic amount of triethylamine to give the pyran derivatives 4a-d. The structure of the latter products was based on the obtained analytical and spectral data. Thus, the 1H NMR spectrum of 4a, as an example, showed multiplet signal at δ 1.78-1.84 ppm correspond- ing to the 2CH2 group, and another multiplet at δ 2.21-2.22 corresponding to CH2 group, a singlet at δ 4.84 of the 2H for the

NH2 group (D2O exchangeable), another singlet corresponding to the H-4 pyran at δ 6.12, and multiplet one at δ 7.26-7.39 for the 5H of the benzene ring. Moreover, the multi-component reaction of cyclopentanone (1) reacted with either of malono- nitrile (2a) or ethyl cyanoacetate (2b) and either of benzalde- hyde (3a) or 4-methoxybenzaldehyde (3b) in absolute etha- nol containing a catalytic amount of ammonium acetate gave the cyclopentene[b]pyridine derivatives 5a-d, respectively. Similarly, the multi-component reaction of compound 1 with cyanoacetate (6) and 4-methoxybenzaldehyde (3b) in abso- lute ethanol containing a catalytic amount of either of triethyl- amine or ammonium acetate gave the pyran 7 and the pyridine 8 derivatives, respectively (Scheme 1).

derivatives 10a and 10b, respectively. The analytical and spec- tral data of the latter products were consistent with their re- spective structures. Thus, the 1H NMR spectrum of compound 10a showed a multiplet signal at δ1.68-1.76 ppm correspond- ing to the 2CH2 group, and another multiplet at δ 2.22-2.27 cor- responding to CH2 group, a singlet at δ 4.77 for the 2H of the NH2 group (D2O exchangeable), another singlet corresponding to the H-4 pyran at δ 6.11, and multiplet one at δ 7.32-7.46 for the 4H of the benzene ring. On the other hand, carrying the same reaction but using a catalytic amount of ammonium ac- etate gave the 2,3,4,11b-tetrahydrochromeno[4,3-d]cyclopen- ta[b]pyridin-6(1H)-one derivatives 11a and 11b, respectively.

In a similar way, the reaction of compound 1 with either malo- nonitrile (2a) or ethyl cyanoacetate (2b) and furfural (12) in

absolute ethanol containing either triethylamine or ammo-

nium acetate gave either the pyran 13a,b or pyridine 14a,b

derivatives, respectively (Scheme 2).

The multi-component reaction of compound 1 with either of malononitrile (2a) or ethyl cyanoacetate (2b) and salic- ylaldehyde (9) in ethanolic triethylamine gave the 2,3-dihy- dro-1H-cyclopenta[5,6]pyrano[3,4-c]chromen-6(11bH)-one

The reactivity of compound 4a was studied to produce potentially biologically active pyranopyridine derivatives. Thus, compound 4a reacted with malononitrile (2a) in abso- lute ethanol containing a catalytic amount of triethylamine to give the 2,4-diamino-5-phenyl-5,6,7,8-tetrahydrocyclopen- ta[5,6]pyrano[2,3-b]pyridine-3-carbonitrile (15). The Analyt- ical and spectral data of compound 15 were consistent with their respective structures (Scheme 3).

The pyran derivatives 4a, 4c and 4d were converted to the corresponding pyridine derivatives 5a, 5c and 5d, respective- ly (m.p., mixed m.p. and finger print IR) upon their treatment with ammonium acetate and ethanol under reflux. Similarly, compounds 10a,b were converted to the corresponding pyri- dine derivatives 11a and 11b upon treatment with ammonium acetate in refluxing ethanol (Scheme 3).

Cyclopentanone (1) reacted with either 4-methoxybenzalde- hyde (3b) or 4-chlorobenzaldehyde (16) in ethanol containing a catalytic amount of piperidine to give the a-benzalidine deriv- atives 17a and 17b, respectively. The latter compounds were previously reported [26,27]. On the other hand, compound 1 reacted with phenylisothiocyanate (20) and elemental sulfur to give the thiazole derivative 21. The structure of the latter product was based on the analytical and spectral data. Thus, the 1H NMR spectrum revealed multiplet signal at δ 1.64-1.75 ppm corresponding to the 2CH2 group, and another multiplet at δ 2.21-2.36 ppm corresponding to CH2 group, and multiplet one at δ 7.25-7.39 for the 5H of the benzene ring.

The reactivity of compounds 17a and 17b towards thiophene synthesis using the well-known Gewald’s thiophene synthe- sis was studied. Thus, the reaction of either 17a or 17b with either malononitrile (2a) or ethyl cyanoacetate (2b) and elemental sulfur gave the thiophene derivatives 22a-d, re- spectively. Their structures were established on the basis of their respective analytical and spectral data (see experimental section).

Compound 17a reacted with 2-aminoprop-1-ene-1,1,3-tricar- bonitrile (23) in ethanolic triethylamine solution to give the 5,7-diamino-3-(4-methoxybenzylidene)-2,3-dihydro-1H-in- dene-4,6-dicarbonitrile (25). Formation of the latter product took place through the first condensation of compound 17a with 23 to give the intermediate condensation compound 24 followed by the intra-molecular Michael addition (Scheme 4).

The reaction of cyclopentanone (1) with cyanoacetyl hydra- zine (26) gave the hydrazide-hydrazone derivative 27. Com- pound 27 reacted with elemental sulfur and phenylisothiocya- nate (20) to give the thiazole derivative 28. On the other hand, compound 27 reacted with malononitrile (2a) and elemental sulfur to give the thiophene derivative 29. Structures of com- pounds 28 and 29 were confirmed on the basis of their respec- tive analytical and spectral data (see experimental section).

The multi-component reaction of compound 27 with malono- nitrile (2a) and benzaldehyde (3a) in ethanolic triethylamine gave the pyran derivative 30. Alternatively carrying the same reaction, but using ammonium acetate instead of triethylamine gave the pyridine derivative 31 (Scheme 5).

The newly synthesized products were screened towards four cancer cell lines where some of them showed high cytotoxicity.

Antitumor Evaluations

Antitumor and normal cell line activity tests

Reagents: Fetal bovine serum (FBS) and L-glutamine, were from Gibco Invitrogen Co. (Scotland, UK). RPMI-1640 medium was from Cambrex (New Jersey, USA). Dimethyl sulfoxide

(DMSO), doxorubicin, penicillin, streptomycin and sulfor- hodamine B (SRB) were from Sigma Chemical Co. (Saint Louis, USA).

Cell cultures: Three human tumor cell lines, MCF-7 (breasta- deno-carcinoma), NCI-H460 (non-small cell lung cancer), and SF-268 (CNS cancer) were used. MCF-7 was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK), NCI-H460, SF-268 and normal fibroblast cells (WI 38) were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt). They grow as monolayer and routinely maintained in RPMI-1640 medium supplemented with 5 % heat-inactivated FBS, 2 mM glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 µg/mL), at 37 oC in a humidified atmosphere containing 5 % CO2. Exponentially growing cells were obtained by plating 1.5 x 105cells/mL for MCF-7 and SF-268 and 0.75 x 104 cells/mL for NCI-H460, followed by 24 h of incubation. The effect of the vehicle solvent (DMSO) on the growth of these

cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration (0.5 %) of DMSO used in each assay.

Tumor cell growth assay

The effects of the newly synthesized compounds on the in vitro growth of human tumor cell lines were evaluated according to the procedure adopted by the National Cancer Institute (NCI, USA) in the ‘In vitro Anticancer Drug Discovery Screen’ that uses the protein-binding dye sulforhodamine B to assess cell growth. Briefly, exponentially, cells growing in 96-well plates were then exposed for 48 hr to five serial concentrations of each compound, starting from a maximum concentration of 150 µM. Following this exposure period, adherent cells were fixed, washed, and stained. The bound stain was solubilized and the absorbance was measured at 492 nm in a plate reader (Bio-Tek Instruments Inc., Power wave XS, Wincoski, USA). For

each test compound and cell line, a dose response curve was obtained and the minimum inhibitory concentration of 50 % (IC50), corresponding to the concentration of the compounds

that inhibited 50 % of the net cell growth was calculated as

described elsewhere. Doxorubicin was used as a positive con- trol and tested in the same manner.

Structure-Activity Relationship

From Table 1 it is clear that:

For human breast cancer cell line (MCF) compounds 13b, 7

and 17b showed higher effect than the reference doxorubicin.

Compound IC50 ( mol L-1)
MCF-7 NCI-H460 SF-268 WI-38
4a 16.20 ± 2.63 18.67 ± 2.2 16.81 ± 1.34 >100
4b 10.82 ± 0.04 3.09 ± 1.84 12.34 ± 2.06 >100
4c 33.69 ± 4.06 27.53 ± 10.06 11.81 ± 4.26 >100
4d 38.01 ± 7.31 29.72 ± 2.87 36.42 ± 2.93 30.6 ±1.4
5a 33.25 ± 2.44 28.64 ± 2.94 11.89 ± 4.55 >100
5b 30.23 ± 2.46 22.6 ± 2.82 13.84 ± 8.53 >100
5c 0.06 ± 0.006 0.06 ± 0.006 0.02 ± 0.008 >100
5d 11.58 ± 2.06 12.58 ± 2.50 10.22 ± 1.80 >100
7 0.01 ± 0.001 0.02 ± 0.004 0.08 ± 0.002 >100
8 30.24 ± 3.64 29.32 ± 10.5 18.63 ± 2.42 >100
10a 14.91 ± 2.68 12.60 ± 2.62 20.82 ± 4.63 >100
10b 20.75 ± 2.56 10.47 ± 8.56 22.84 ± 2.44 >100
11a 30.45 ± 5.84 25.71 ± 8.20 36.41 ± 4.45 12.6 ±5.0
11b 8.67 ± 2.01 5.65 ± 6.06 22.42 ± 2.36 >100
13a 2.62 ± 0.23 3.03 ± 0.63 0.85 ± 0.04 >100
13b 0.01 ± 0.002 0.02 ± 0.003 0.04 ± 0.002 >100
14a 1.72 ± 0.26 0.57 ± 0.04 1.53 ± 0.17 3.0 ±1.4
14b 12.61 ± 8.52 18.33 ± 2.31 14.42 ± 1.83 >100
15 20.83 ± 10.4 32.8 ± 8.24 20.11 ± 2.42 20.2 ± 0.1
17a 0.84 ± 0.04 0.62 ± 0.04 0.08 ± 0.006 40.0 ± 1.3
17b 0.02 ± 0.004 0.02 ± 0.004 0.06 ± 0.006 >100
21 0.09 ± 0.04 0.70 ± 0.04 0.39 ± 0.02 >100
22a 20.75 ± 2.06 25.58 ± 1.06 15.81 ± 2.39 >100
22b 10.62 ± 2.83 6.6 ± 2.2 10.0 ± 1.85 >100
22c 44.75 ± 4.86 39.50 ± 10.26 17.69 ± 2.09 >100
22d 22.01 ± 1.45 52.80 ± 4.38 41.30 ± 2.83 >100
25 3.71 ± 0.18 1.03 ± 0.01 0.59 ± 0.01 >100
27 0.05 ± 0.002 0.06 ± 0.003 0.02 ± 0.002 >100
28 0.22±0.12 0.99 ± 0.70 0.39 ± 0.80 >100
29 0.08 ± 0.002 0.08 ± 0.003 0.02 ± 0.002 >100
30 31.75 ± 2.06 10.55 ± 2.06 12.81 ± 2.16 >100
Doxorubicin

(Reference compound)

0.04 ± 0.008 0.09 ± 0.008 0.09 ± 0.007 >100

Results are given in concentrations that were able to cause 50 % of cell growth inhibition (IC50) after a continuous exposure of 48 h and show means ± SEM of three-independent experiments performed in duplicate.

Table 1. Effect of some new derivatives of compounds 4a-30 on the growth of three human tumor cell lines.

Moreover, compounds 5c, 21, 27 and 29 exhibited higher ef- fect but not than the reference. For non-small cell lung cancer (NCI-H460) compounds 5c, 7, 13b, 17b, 27 and 29 indicat- ed optimal anticancer effect than the reference used. For SF- 268 (CNS cancer) compounds 5c, 7, 13b, 17b, 17a, 27 and 29 showed higher effect than the doxorubicin. On the other hand compounds 15, 4d, 10a, 10b, 11a, and 11b indicated low po- tent effect. For normal fibroblast cells (WI38) all compounds indicated no cytotoxic effect.

In general, compounds 7, 13b and 17b showed significant ac- tivity on the three tumor cell lines tested. Also, compounds 5c and 27 indicated optimal activity for two cell lines, non-small cell lung cancer (NCI-H460) and SF-268 (CNS cancer). The in- hibitory effect of the other systems on tumor cell growth var- ied, according to the tested tumor cell, from high to medium or marginal effects.

It is obvious that compounds 13b, 7 and 17b exhibited opti- mal anticancer effect against cancer cell line, with IC50’s in the μM range. Comparing the cytotoxicity of the derivatives 14a and 14b, it is clear that the cytotoxicity of 14a is higher than those of 14b. The presence of the amino group is responsible for the high potent of 14a.

On the other hand, for the other series of 4,5,6,7-tetrahy- dro-1H-cyclopenta[b]pyridine-3-carbonitrilederivatives 5a-d, the presence of the amino group in compound 5c is respon- sible for the higher cytotoxicity than the doxorubicin used. Moreover, for the compound 5a and 5b didn’t reveal high cy- totoxicity.

In a similar way, the presence of the chloro group in the benza- lidine derivative 17b is responsible for its higher cytotoxicity than the derivative 17a with the methoxy group. It is clear that the cytotoxicity of thiophene derivative 29 was higher than that of the thiazole derivative 28. It is clear that the cytotox- icity of 13b is higher than that of 13a against cancer cell lines NCI-H460, MCF-7 and SF-268. Considering the low cytotoxicity of 13a to the amino group. Among the pyridine derivatives 14a and 14b, the compound 14a with the high nitrogen con- tent showed higher cytotoxicity effect.

With respect to thiophene derivatives 22a-d it is clear that compound 22b was the most effective, and this might be attributed to the cyano and para chlorophenyl groups. It is remarkable that compound 27 showed high cytotoxicity towards the three cell lines NCI-H460, MCF-7 and SF-268. Comparing the cytotoxic effect of both 7 and 8, it is obvious that the pyran derivative 7 showed much higher cytotoxic ef- fect than the pyridine derivative 8.

Experimental General

All melting points were determined on an Electrothermal dig- ital melting point apparatus and are uncorrected. IR spectra (KBr discs) were recorded on an FTIR plus 460 or PyeUnicam SP-1000 spectrophotometer. 1H NMR spectra were recorded with Varian Gemini-200 (200 MHz) and Jeol AS 500 MHz in- strument spectra were performed in DMSO-d6 as solvent using TMS as internal standard and chemical shifts are expressed as δ ppm. MS (EI) spectra were recorded with Hewlett Packard 5988 A GC/MS system and GCMS-QP 1000 Ex Shimadzu in- struments. Analytical data were obtained from the Micro-An- alytical Data Unit at Cairo University and were performed on Vario EL III Elemental Analyzer. Compounds 17a and 17b were synthesized and identified according to the reported work [26,27].

2-Amino-4-phenyl-4,5,6,7-tetrahydrocyclopenta[b]pyran-3-car- bonitrile (4a), 2-hydroxy-4-phenyl-4,5,6,7-tetrahydrocyclo- penta[b]pyran-3-carbonitrile (4b), 2-amino-4-(4-methoxy- phenyl)-4,5,6,7-tetrahydrocyclopenta[b]pyran-3-carbonitrile (4c) and 2-hydroxy-4-(4-methoxyphenyl)-4,5,6,7-tetrahydrocy- clopenta[b]pyran-3-carbonitrile (4d)

General procedure: To a solution of compound 1 (0.84 g, 0.01 mol) in ethanol (50 mL) containing triethylamine (0.50 mL) either malononitrile (0.66 g, 0.01 mol or ethyl cyanoacetate

1.13 g, 0.01 mol) and either benzaldehyde (1.06 g, 0.01 mol) or 4-methoxybenzaldehyde (1.36 g, 0.01 mol) were added. The reaction mixture in each case was heated under reflux for 4 h then poured onto ice/water containing few drops of hydro- chloric acid and the formed solid product was collected by fil- tration.

Compound 4a: Yellow crystals from ethanol, yield 78 % (1.85 g), m.p. 110–112 °C. Anal. Calculated for C15H14N2O (238.28): C, 75.61; H, 5.92; N, 11.76. Found: C, 75.44; H, 5.81; N; 11.93. MS:

m/e 238 (M+, 20 %). IR, υ: 3492, 3330 (NH2), 3056 (CH, aro- matic), 2970 (CH2), 2220 (CN), 1636 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.78-1.84 (m, 4H, 2CH2), 2.21-2.22 (m, 2H, CH2),

4.84 (s, 2H, D2O exchangeable, NH2), 6.12 (s, 1H, pyran H-4),

7.26-7.39 (m, 5H, C6H5). 13C NMR (DMSO-d6, 75 MHz): δ = 22.4,

26.3, 28.8 (3CH2), 116.8 (CN), 86.8 (pyran C-4), 120.0, 120.9,

123.7, 125.4, 126.8, 127.3, 131.8, 132.8, 133.6, 135.3 (C6H5,

pyran C).

Compound 4b: Yellow crystals from ethanol, yield 75 % (1.79 g), m.p. 177 °C. Anal. Calculated for C15H13NO2 (239.27): C, 75.30; H, 5.48; N, 5.85. Found: C, 75.39; H, 5.37; N; 5.63. MS:

m/e 239 (M+, 40 %). IR, υ: 3531-3321 (OH), 3054 (CH, aro- matic), 2983 (CH2), 2222(CN), 1636 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.76-1.84 (m, 4H, 2CH2), 2.22-2.24 (m, 2H, CH2),

6.09 (s, 1H, pyran H-4), 7.28-7.39 (m, 5H, C6H5), 10.22 (s, 1H,

OH). 13C NMR (DMSO-d6, 75 MHz): δ = 22.3, 26.5, 28.6 (3CH2),

86.6 (pyran C-4), 116.7 (CN), 122.0, 123.6, 125.3, 127.6, 128.5,

129.6, 130.2, 132.8, 133.6, 134.9 (C6H5, pyran C).

Compound 4c: Red crystals from ethanol, yield 71 % (1.90 g),

m.p. 87 °C. Anal. Calculated for C16H16N2O2 (268.12): C, 71.62; H, 6.01; N, 10.44. Found: C, 71.59; H, 5.93; N; 10.38. MS: m/e 268 (M+, 28 %). IR, υ: 3488, 3320 (NH2), 3056 (CH, aromatic), 2977 (CH2), 2220 (CN), 1636 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ

= 1.76-1.82 (m, 4H, 2CH2), 2.20-2.26 (m, 2H, CH2), 3.12 (s, 3H,

OCH3), 4.82 (s, 2H, D2O exchangeable, NH2), 6.18 (s, 1H, pyran H-4), 7.28-7.39 (2d, 4H, C6H4). 13C NMR (DMSO-d6, 75 MHz): δ

= 22.2, 26.7, 28.6 (3CH2), 24.3 (OCH3), 86.5 (pyran C-4), 116.9

(CN), 122.3, 122.9, 124.6, 126.8, 128.6, 129.5, 130.6, 131.6,

132.9, 133.8 (C6H4, pyran C).

Compound 4d: Yellow crystals from ethanol, yield 79 % (2.12 g), m.p. 156 °C. Anal. Calculated for C16H15NO3 (269.30): C, 71.36; H, 5.61; N, 5.20. Found: C, 71.55; H, 5.33; N; 5.27. MS:

m/e 269 (M+, 18 %). IR, υ: 3539-3328 (OH), 3053 (CH, aro- matic), 2977 (CH2), 2220 (CN), 1630 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.74-1.82 (m, 4H, 2CH2), 2.21-2.26 (m, 2H, CH2),

3.09 (s, 3H, OCH3), 6.18 (s, 1H, pyran H-4), 7.25-7.38 (2d, 4H,

C6H4), 10.29 (s, 1H, D2O exchangeable, OH). 13C NMR (DMSO-d6,

75 MHz): δ = 22.4, 26.8, 28.6 (3CH2), 24.2 (OCH3), 86.8 (pyran

C-4), 116.8 (CN), 122.8, 122.6, 124.4, 126.8, 128.6, 129.5,

130.8, 131.8, 133.6, 134.3 (C6H4, pyran C).

2-Amino-4-phenyl-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyr- idine-3-carbonitrile (5a), 2-hydroxy-4-phenyl-4,5,6,7-tetra- hydro-1H-cyclopenta[b]pyridine-3-carbonitrile (5b), 2-ami- no-4-(4-methoxyphenyl)-4,5,6,7-tetrahydro-1H-cyclopenta[b] pyridine-3-carbonitrile (5c) and 2-hydroxy-4-(4-methoxy- phenyl)-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carbo- nitrile (5d)

General procedure: To a solution of compound 1 (0.84 g, 0.01 mol) in ethanol (50 mL) containing ammonium acetate (0.50 g) either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) and either benzaldehyde (1.06 g, 0.01 mol) or 4-methoxybenzaldehyde (1.36 g, 0.01 mol) were added. The reaction mixture in each case was heated under reflux for 4 h then poured onto ice/water containing few drops of hydro- chloric acid and the formed solid product was collected by fil- tration.

Compound 5a: Yellow crystals from ethanol, yield 72 % (1.71 g), m.p. 76 °C. Anal. Calculated for C15H15N3 (237.30): C, 75.92; H, 6.37; N, 17.71. Found: C, 75.68; H, 6.42; N; 17.90. MS: m/e

237 (M+, 32 %). IR, υ: 3488, 3326 (NH2), 3054 (CH, aromatic),

2981 (CH2), 2220 (CN), 1632 (C=C). 1H NMR (DMSO-d6, 200

MHz): δ = 1.75-1.86 (m, 4H, 2CH2), 2.21-2.25 (m, 2H, CH2), 4.82

(s, 2H, D2O exchangeable, NH2), 6.11 (s, 1H, pyridine H-4), 7.29-

7.41 (m, 5H, C6H5), 8.18 (s, 1H, D2O exchangeable, NH). 13C NMR

(DMSO-d6, 75 MHz): δ = 22.4, 26.6, 28.5 (3CH2), 116.8 (CN),

84.5 (pyridine C-4), 120.6, 121.7, 123.7, 125.4, 128.3, 129.6,

131.5, 132.3, 134.2, 135.6 (C6H5, pyridine C).

Compound 5b:Yellow crystals from ethanol, yield 78 % (1.85 g), m.p. 170–173 °C. Anal. Calculated for C15H14N2O (238.28): C, 75.61; H, 5.92; N, 11.76. Found: C, 75.42; H, 5.77; N; 11.80. MS:

m/e 238 (M+, 20 %). IR, υ: 3560-3320 (OH), 3056 (CH, aromat-

ic), 2983 (CH2), 2222 (CN), 1634 (C=C). 1H NMR (DMSO-d6, 200

MHz): δ = 1.75-1.86 (m, 4H, 2CH2), 2.20-2.24 (m, 2H, CH2), 6.21

(s, 1H, pyridine H-4), 7.29-7.42 (m, 5H, C6H5), 8.22 (s, 1H, NH),

10.30 (s, 1H, OH). 13C NMR (DMSO-d6, 75 MHz): δ = 22.4, 26.5,

28.6 (3CH2), 84.4 (pyridine C-4), 116.9 (CN), 122.3, 122.8,

124.9, 127.6, 128.5, 129.8, 130.5, 132.5, 133.8, 134.3 (C6H5,

pyridine C).

Compound 5c: Orange crystals from ethanol, yield 70 % (1.87 g), m.p. 60–63 °C. Anal. Calculated for C16H17N3O (267.33): C, 71.89; H, 6.41; N, 15.72. Found: C, 71.59; H, 6.09; N; 15.38. MS:

m/e 267 (M+, 28 %). IR, υ: 3488, 3320 (NH2), 3056 (CH, aro- matic), 2977 (CH2), 2220 (CN), 1636 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.75-1.82 (m, 4H, 2CH2), 2.20-2.29 (m, 2H, CH2),

3.13 (s, 3H, OCH3), 4.83 (s, 2H, D2O exchangeable, NH2), 6.12

(s, 1H, pyridine H-4), 7.23-7.40 (2d, 4H, C6H4), 8.32 (s, 1H, D2O

exchangeable, NH). 13C NMR (DMSO-d6, 75 MHz): δ = 22.4,

26.5, 28.8 (3CH2), 24.6 (OCH3), 84.5 (pyridine C-4), 116.6 (CN),

122.4, 122.8, 124.8, 126.4, 128.5, 129.5, 130.8, 131.8, 132.7,

133.2 (C6H4, pyridine C).

Compound 5d: Orange crystals from ethanol, yield 74 % (1.98 g), m.p. 255 °C. Anal. Calculated for C16H16N2O2 (268.31): C, 71.62; H, 6.01; N, 10.44. Found: C, 71.80; H, 5.93; N; 10.28. MS:

m/e 268 (M+, 21 %). IR, υ: 3529-3348 (OH), 3053 (CH, aro- matic), 2979 (CH2), 2220 (CN), 1633 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.73-1.80 (m, 4H, 2CH2), 2.22-2.27 (m, 2H, CH2),

3.07 (s, 3H, OCH3), 6.16 (s, 1H, pyridine H-4), 7.27-7.39 (2d,

4H, C6H4), 8.12 (s, 1H, D2O exchangeable, NH), 10.31 (s, 1H,

D2O exchangeable, OH). 13C NMR (DMSO-d6, 75 MHz): δ = 22.4,

26.6, 28.4 (3CH2), 24.9 (OCH3), 84.6 (pyridine C-4), 116.4 (CN),

122.6, 122.6, 124.9, 126.6, 128.6, 129.5, 130.6, 131.3, 132.9,

133.1 (C6H4, pyridine C).

2-Amino-4-(4-methoxyphenyl)-4,5,6,7-tetrahydrocyclopen- ta[b]pyran-3-carboxamide (7) and 2-amino-4-(4-methoxy- phenyl)-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carbox- amide (8)

General procedure: To a solution of compound 1 (0.84 g, 0.01 mol) in ethanol (50 mL) containing either triethylamine (0.50 mL) or ammonium acetate (0.5 g) cyanoacetamide (0.84 g,

0.01 mol) and 4-methoxybenzaldehyde (1.36 g, 0.01 mol) were added. The reaction mixture in each case was heated under re- flux for 3 h then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collect- ed by filtration.

Compound 7: White crystals from ethanol, yield 68 % (1.94 g),

m.p. 198 °C. Anal. Calculated for C16H18N2O3 (286.33): C, 67.12; H, 6.34; N, 9.78. Found: C, 67.33; H, 6.51; N; 10.04. MS: m/e 286 (M+, 30 %). IR, υ: 3478, 3330 (NH2), 3058 (CH, aromatic), 2982 (CH2), 2222 (CN), 1632 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.74-1.80 (m, 4H, 2CH2), 2.23-2.29 (m, 2H, CH2), 3.09 (s, 3H, OCH3), 4.90, 5.22 (2s, 4H, D2O exchangeable, 2NH2), 6.23 (s, 1H, pyran H-4), 7.30-7.41 (2d, 4H, C6H4). 13C NMR (DMSO-d6, 75 MHz): δ = 22.3, 26.4, 28.4 (3CH2), 24.5 (OCH3), 86.5 (pyran C-4), 116.8 (CN), 120.8, 122.6, 125.3, 126.7, 128.6, 129.5, 130.8, 131.6, 132.6, 133.3 (C6H4, pyran C), 163.8 (CO).

Compound 8: Orange crystals from ethanol, yield 72 % (2.05 g), m.p. 90 °C. Anal. Calculated for C16H19N3O2 (285.34): C, 67.35; H, 6.71; N, 14.73. Found: C, 67.44; H, 6.92; N, 14.80. MS:

m/e 285 (M+, 33 %). IR, υ: 3471, 3336 (NH2), 3053 (CH, aro- matic), 2981 (CH2), 2222 (CN), 1636 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.70-1.77 (m, 4H, 2CH2), 2.20-2.27 (m, 2H, CH2),

3.10 (s, 3H, OCH3), 4.82, 5.23 (2s, 4H, D2O exchangeable, 2NH2),

6.14 (s, 1H, pyridine H-4), 7.29-7.42 (2d, 4H, C6H4), 8.22 (s, 1H,

D2O exchangeable, NH). 13C NMR (DMSO-d6, 75 MHz): δ = 22.6,

26.3, 28.6 (3CH2), 24.3 (OCH3), 84.8 (pyridine C-4), 116.5 (CN),

120.6, 122.4, 125.6, 126.9, 128.8, 129.3, 130.6, 131.8, 132.9,

133.8 (C6H4, pyran C), 164.5 (CO).

5-Amino-2,3-dihydro-1H-cyclopenta[5,6]pyrano[3,4-c] chromen-6(11bH)-one (10a) and 5-hydroxy-2,3-dihydro-1H-cy- clopenta[5,6]pyrano[3,4-c]chromen-6(11bH)-one (10b)

General procedure: To a solution of compound 1 (0.84 g, 0.01 mol) in ethanol (50 mL) containing triethylamine (0.50 mL) either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) and salicylaldehyde (1.22 g, 0.01 mol) were added. The reaction mixture in each case was heated under re- flux for 4 h then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collect- ed by filtration.

Compound 10a: Brown crystals from ethanol, yield 67 % (1.70 g), m.p. 300 °C. Anal. Calculated for C15H13NO3 (255.27): C, 70.58; H, 5.13; N, 5.49. Found: C, 70.66; H, 5.08; N, 5.62. MS:

m/e 255 (M+, 18 %). IR, υ: 3483, 3348 (NH2), 3055 (CH, aro- matic), 2988 (CH2), 1632 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.68-1.76 (m, 4H, 2CH2), 2.22-2.27 (m, 2H, CH2), 4.77 (s, 2H,

D2O exchangeable, NH2), 6.11 (s, 1H, pyran H-4), 7.32-7.46 (m,

4H, C6H4). 13C NMR (DMSO-d6, 75 MHz): δ = 22.5, 26.5, 28.3

(3CH2), 85.8 (pyran C-4), 120.8, 122.9, 125.3, 127.2, 128.6,

129.3, 130.6, 131.6, 132.6, 133.8 (C6H4, pyran C), 164.5 (CO).

Compound 10b: Reddish Browncrystals from ethanol, yield 69

% (1.76 g), m.p. 200 °C. Anal. Calculated for C15H12O4 (256.07): C, 70.31; H, 4.72. Found: C, 70.42; H, 4.68. MS: m/e 256 (M+, 41

%). IR, υ: 3540, 3331 (OH), 3055 (CH, aromatic), 2989 (CH2), 1632 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.66-1.78 (m,

4H, 2CH2), 2.19-2.27 (m, 2H, CH2), 6.13 (s, 1H, pyran H-4),

7.34-7.42 (m, 4H, C6H4), 10.28 (s, 1H, D2O exchangeable, OH).

13C NMR (DMSO-d6, 75 MHz): δ = 22.3, 26.8, 28.6 (3CH2), 85.6

(pyran C-4), 120.4, 121.4, 123.8, 125.9, 127.6, 128.2, 130.9,

131.5, 133.1, 133.3 (C6H4, pyran C), 164.8 (CO).

5-Amino-2,3,4,11b-tetrahydrochromeno[4,3-d]cyclopenta[b] pyridin-6(1H)-one (11a) and 5-hydroxy-2,3,4,11b-tetrahydro- chromeno[4,3-d]cyclopenta[b]pyridin-6(1H)-one (11b)

General procedure: To a solution of compound 1 (0.84 g, 0.01 mol) in ethanol (50 mL) containing ammonium acetate (0.50

g) either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) and salicylaldehyde (1.22 g, 0.01 mol) were added. The reaction mixture in each case was heated under re- flux for 2 h then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collect- ed by filtration.

Compound 11a: Yellowcrystals from ethanol, yield 71 % (1.81 g), m.p. 140–142 °C. Anal. Calculated for C15H14N2O2 (256.11): C, 70.85; H, 5.55; N, 11.02. Found: C, 70.66; H, 5.28; N; 10.92.

MS: m/e 256 (M+, 18 %). IR, υ: 3483, 3348 (NH2), 3055 (CH,

aromatic), 2988 (CH2), 1632 (C=C). 1H NMR (DMSO-d6, 200

MHz): δ = 1.68-1.76 (m, 4H, 2CH2), 2.22-2.27 (m, 2H, CH2), 4.77

(s, 2H, D2O exchangeable, NH2), 6.11 (s, 1H, pyridine H-4), 7.32-

7.46 (m, 4H, C6H4), 8.12 (s, 1H, D2O exchangeable, NH). 13C NMR (DMSO-d6, 75 MHz): δ = 22.3, 26.7, 28.5 (3CH2), 85.5 (pyridine C-4), 120.4, 121.7, 123.8, 125.7, 127.2, 129.6, 130.9, 131.4,

132.2, 133.5 (C6H4, pyridine C), 164.5 (CO).

Compound 11b: Brown crystals from ethanol, yield 75 % (1.91 g), m.p. 208 °C. Anal. Calculated for C15H13NO3 (255.27): C, 70.58; H, 5.13; N, 5.49. Found: C, 70.42; H, 4.88; N, 5.34. MS:

m/e 255 (M+, 41 %). IR, υ: 3540, 3331 (OH), 3055 (CH, aro- matic), 2989 (CH2), 1632 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.66-1.78 (m, 4H, 2CH2), 2.19-2.27 (m, 2H, CH2), 6.13 (s,

1H, pyridine H-4), 7.34-7.42 (m, 4H, C6H4), 8.22 (s, 1H, D2O ex-

changeable, NH), 10.28 (s, 1H, D2O exchangeable, OH). 13C NMR (DMSO-d6, 75 MHz): δ = 22.5, 26.3, 28.5 (3CH2), 85.8 (pyridine

C-4), 120.6, 121.8, 123.3, 125.5, 127.3, 127.9, 131.0, 131.5,

133.5, 133.9 (C6H4, pyridine C), 164.5 (CO).

2-Amino-4-(furan-2-yl)-4,5,6,7-tetrahydrocyclopenta[b] pyran-3-carbonitrile (13a) and 2-hydroxy-4- (furan-2-yl)

-4,5,6,7-tetrahydrocyclopenta[b]pyran-3-carbonitrile (13b)

General procedure: To a solution of compound 1 (0.84 g, 0.01 mol) in ethanol (50 mL) containing triethylamine (0.50 mL) either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) and furfural (0.96 g, 0.01 mol) were added. The reaction mixture in each case was heated under reflux for 2 h then poured onto ice/water containing few drops of hy- drochloric acid and the formed solid product was collected by

filtration.

Compound 13a: Brown crystals from 1,4-dioxane, yield 65

% (1.48 g), m.p. 114–116 °C. Anal. Calculated for C13H12N2O2 (228.25): C, 68.41; H, 5.30; N, 12.27. Found: C, 68.55; H, 5.41; N,

12.39. MS: m/e 228 (M+, 30 %). IR, υ: 3488, 3320 (NH2), 3054 (CH, aromatic), 2984 (CH2), 2223 (CN), 1632 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.63-1.76 (m, 4H, 2CH2), 2.19-2.29

(m, 2H, CH2), 4.80 (s, 2H, D2O exchangeable, NH2), 6.11 (s, 1H,

pyran H-4), 7.24-7.53 (m, 3H, furyl H). 13C NMR (DMSO-d6, 75

MHz): δ = 22.4, 26.5, 28.5 (3CH2), 116.7 (CN), 84.6 (pyran C-4),

120.4, 120.3, 122.4, 124.6, 132.4, 136.8, 142.3, 143.6 (furan,

pyran C).

Compound 13b: Reddish browncrystals from 1,4-dioxane, yield 63 % (1.44 g), m.p. 75 °C. Anal. Calculated for C13H11NO3 (229.23): C, 68.11; H, 4.84; N, 6.11. Found: C, 68.27; H, 4.63;

N, 5.92. MS: m/e 229 (M+, 22 %). IR, υ: 3532-3327 (OH), 3056 (CH, aromatic), 2989 (CH2), 2222 (CN), 1632 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.64-1.79 (m, 4H, 2CH2), 2.24-2.28

(m, 2H, CH2), 6.12 (s, 1H, pyran H-4), 7.20-7.49 (m, 3H, furyl

H), 10.20 (s, 1H, D2O exchangeable, OH). 13C NMR (DMSO-d6, 75

MHz): δ = 22.3, 26.8, 28.2 (3CH2), 116.6 (CN), 84.6 (pyran C-4),

120.2, 121.6, 123.1, 124.6, 133.5, 136.4, 142.6, 143.4 (furan,

pyran C).

  1. Amino-4-(furan-2-yl)-4,5,6,7-tetrahydro-1H-cyclopen- ta[b]pyridine-3-carbonitrile (14a) and 2-Hydroxy-4-(fu- ran-2-yl)-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carbo- nitrile (14b)

General procedure: To a solution of compound 1 (0.84 g, 0.01 mol) in ethanol (50 mL) containing ammonium acetate (0.50

g) either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) and furfural (0.96 g, 0.01 mol) were added. The reaction mixture in each case was heated under reflux for 3 h then poured onto ice/water containing few drops of hy- drochloric acid and the formed solid product was collected by filtration.

Compound 14a: Brown crystals from 1,4-dioxane, yield 68 % (1.54 g), m.p. 170 °C. Anal. Calculated for C13H13N3O (227.26): C, 68.70; H, 5.77; N, 18.49. Found: C, 68.59; H, 5.63; N, 18.28.

MS: m/e 227 (M+, 30 %). IR, υ: 3492, 3328 (NH2, NH), 3057 (CH, aromatic), 2984 (CH2), 2222 (CN), 1636 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.65-1.76 (m, 4H, 2CH2), 2.21-2.29

(m, 2H, CH2), 4.83 (s, 2H, D2O exchangeable, NH2), 6.14 (s, 1H,

pyridine H-4), 7.29-7.58 (m, 3H, furyl H), 8.49 (s, 1H, D2O ex- changeable, NH). 13C NMR (DMSO-d6, 75 MHz): δ = 22.4, 26.5, 28.5 (3CH2), 84.4 (pyran C-4), 116.5 (CN), 120.4, 121.3, 123.5,

124.3, 139.8, 140.3, 142.2, 143.7 (furan, pyridine C).

Compound 14b: Orange crystals from 1,4-dioxane, yield 67 % (1.52 g), m.p. 276 °C. Anal. Calculated for C13H12N2O2 (228.25):

C, 68.41; 5.30; N, 12.27. Found: C, 68.30; H, 5.44; N, 12.46. MS:

m/e 229 (M+, 22 %). IR, υ: 3532, 3327 (OH, NH), 3056 (CH, aro- matic), 2989 (CH2), 2220 (CN), 1632 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.66-1.79 (m, 4H, 2CH2), 2.24-2.25 (m, 2H, CH2),

6.14 (s, 1H, pyridine H-4), 7.23-7.40 (m, 3H, furyl H), 8.22 (s,

1H, D2O exchangeable, NH), 10.22 (s, 1H, D2O exchangeable, OH). 13C NMR (DMSO-d6, 75 MHz): δ = 22.5, 26.7, 28.5 (3CH2),

84.7 (pyran C-4), 116.6 (CN), 120.8, 121.9, 122.6, 126.8, 136.3,

140.2, 141.9, 142.9 (furan, pyridine C).

2,4-diamino-5-phenyl-5,6,7,8-tetrahydrocyclopenta[5,6]pyra- no[2,3-b]pyridine-3-carbonitrile (15)

To a solution of compound 4a (2.38 g, 0.01 mol) in absolute ethanol (30 mL) containing triethylamine (0.50 mL) malononi- trile (0.66 g, 0.01 mol) was added. The whole reaction mixture was heated under reflux for 6 h then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration.

Compound 15: Dark yellowcrystals from ethanol, yield 89 % (2.72 g), m.p. 95 °C. Anal. Calculated for C18H16N4O (304.35): C, 71.04; H, 5.30; N, 18.41. Found: C, 71.22; H, 5.19; N, 18.20.

MS: m/e 304 (M+, 33 %). IR, υ: 3488-3319 (2 NH2), 3052 (CH,

aromatic), 2989 (CH2), 2220 (CN), 1650 (C=N), 1632 (C=C).

1H NMR (DMSO-d6, 200 MHz): δ = 1.63-1.74 (m, 4H, 2CH2),

2.15-2.29 (m, 2H, CH2), 4.80, 5.21 (2s, 4H, D2O exchangeable, 2 NH2), 6.30 (s, 1H, pyran H-4), 7.28-7.40 (m, 5H, C6H5). 13C NMR

(DMSO-d6, 75 MHz): δ = 22.4, 26.8, 28.6 (3CH2), 84.5 (pyran

C-4), 116.8 (CN), 120.6, 121.8, 122.3, 124.9, 138.8, 140.6,

141.5, 142.3 (furan, pyridine C).

Conversion of compounds 4a, 4c and 4d into 5a, 5c and 5d:

A solution of either compound 4a (2.38 g, 0.01 mol) 4c (2.68 g, 0.01 mol) or 4d (2.69 g, 0.01 mol) in absolute ethanol (30 mL) containing ammonium acetate (0.50 g) was heated under reflux for 6 h then poured onto ice/water and the formed solid product was collected by filtration and identified as 5a, 5c and 5d, respectively (m.p., mixed m.p. and fingerprint IR).

Conversion of compounds 10a and 10b into 11a and 11b:

A solution of either compound 10a (2.55 g, 0.01 mol) or 10b (2.56 g, 0.01 mol) in absolute ethanol (30 mL) containing am- monium acetate (0.50 g) was heated under reflux for 6 h then poured onto ice/water and the formed solid product was col- lected by filtration and identified as 11a and 11b, respectively (m.p., mixed m.p. and fingerprint IR).

  1. Phenyl-3,4,5,6-tetrahydro-2H-cyclopenta[d]thiazole-2-thione (21)

To a solution of compound 1 (0.84 g, 0.01 mol) in ethanol (50

mL) containing triethylamine (0.50 mL) each of elemental sul- fur (0.32 g, 0.01 mol) and phenylisothiocyanate (1.35 g, 0.01 mol) were added. The reaction mixture in each case was heat- ed under reflux for 3 h then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration.

Compound 21: Black crystals from ethanol, yield 81 % (1.88 g), m.p. 45–48 °C. Anal. Calculated for C12H11NS2 (233.35): C, 61.76; H, 4.75; N, 6.00; S, 27.48. Found: C, 61.33; H, 4.51; N,

6.08; S, 27.33. MS: m/e 233 (M+, 32 %). IR, υ: 3056 (CH, ar- omatic), 2984 (CH2), 1632 (C=C), 1205 (C=S). 1H NMR (DM- SO-d6, 200 MHz): δ = 1.64-1.75 (m, 4H, 2CH2), 2.21-2.36 (m, 2H, CH2), 7.25-7.39 (m, 5H, C6H5).

2-Amino-4-(4-methoxybenzylidene)-5,6-dihydro-4H-cy- clopenta[b]thiophene-3-carbonitrile (22a), 2-ami- no-4-(4-chlorobenzylidene)-5,6-dihydro-4H-cyclopenta[b] thiophene-3-carbonitrile (22b), ethyl 2-amino-4-(4-methoxy- benzylidene)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbox- ylate (22c) and ethyl 2-amino-4-(4-chlorobenzylidene)-5,6-di- hydro-4H-cyclopenta[b]thiophene-3-carboxylate (22d)

General procedure: To a solution of either of compound 17a (2.02 g, 0.01 mol) or 17b (2.06 g, 0.01 mol) in absolute ethanol (40 mL) containing triethylamine (1.0 mL) each of elemental sulfur (0.32 g, 0.01 mol) and either of 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 then poured onto ice/water containing few drops of hydrochloric acid. The formed solid product, in each case, was collected by filtration.

Compound 22a: Browncrystals from ethanol, yield 87 % (2.45 g), m.p. 76 °C. Anal. Calculated for C16H14N2OS (282.36): C, 68.06; H, 5.00; N, 9.92; S, 11.36. Found: C, 68.21; H, 5.29;

N, 10.05; S, 11.47. MS: m/e 282 (M+, 12 %). IR, υ: 3486, 3327

(NH2), 3056 (CH, aromatic), 2986, 2877 (CH3, CH2), 2220 (CN), 1631 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.80-1.87 (m,

2H, CH2), 2.28-2.32 (m, 2H, CH2), 3.14 (s, 3H, OCH3), 4.92 (s, 2H,

D2O exchangeable, NH2), 6.20 (s, 1H, CH), 7.28-7.41 (2d, 4H, C6H4). 13C NMR (DMSO-d6, 75 MHz): δ = 23.6, 25.8 (2CH2), 23.6

(OCH3), 89.3, 90.8 (CH=C), 117.3 (CN), 120.6, 124.8, 127.7,

129.3, 130.5, 133.8, 138.4, 140.1 (thiophene, C6H4).

Compound 22b: Yellowish Browncrystals from ethanol, yield 84 % (2.41 g), m.p. 135 °C. Anal. Calculated for C15H- 11ClN2S(286.78): C, 62.82; H, 3.87; N, 9.77; S, 11.18. Found: C,

62.90; H, 4.01; N, 10.05; S, 11.37. MS: m/e 288 (M+, 16 %). IR,

υ: 3493, 3320 (NH2), 3058 (CH, aromatic), 2879 (CH2), 2223 (CN), 1634 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.82-1.89 (m, 2H, CH2), 2.24-2.30 (m, 2H, CH2), 4.89 (s, 2H, D2O exchange-

able, NH2), 6.18 (s, 1H, CH), 7.25-7.45 (2d, 4H, C6H4). 13C NMR

(DMSO-d6, 75 MHz): δ = 23.4, 25.6 (2CH2), 89.5, 90.3 (CH=C),

116.4 (CN), 120.6, 122.3, 124.3, 127.3, 130.3, 132.7, 136.9,

140.2 (thiophene, C6H4).

Compound 22c: Dark yellowcrystals from acetic acid, yield 81

%(2.66 g), m.p. 200 °C. Anal. Calculatedfor C18H19NO3S(329.41): C, 65.63; H, 5.81; N, 4.25; S, 9.73. Found: C, 65.88; H, 5.72; N,

4.69; S, 10.01. MS: m/e 329 (M+, 22 %). IR, υ: 3459, 3313 (NH2),

3054 (CH, aromatic), 2987, 2883 (CH3, CH2), 1632 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.13 (t, 3H, J = 7.22 Hz, CH3), 1.80-1.84 (m, 2H, CH2), 2.21-2.27 (m, 2H, CH2), 3.09 (s, 3H, OCH3), 4.22 (q, 2H, J = 7.22 Hz, CH2), 4.77 (s, 2H, D2O exchange- able, NH2), 6.16 (s, 1H, CH), 7.26-7.47 (2d, 4H, C6H4). 13C NMR

(DMSO-d6, 75 MHz): δ = 16.2 (ester CH3), 23.7, 25.8 (2CH2),

24.4 (OCH3), 52.6 (ester CH2), 89.3, 90.4 (CH=C), 120.6, 121.3,

126.8, 129.1, 130.6, 133.8, 139.2, 140.4 (thiophene, C6H4).

Compound 22d: Yellowcrystals from acetic acid, yield 89 % (2.96 g), m.p. 95 °C. Anal. Calculated for C17H16ClNO2S (333.83): C, 61.16; H, 4.83; N, 4.20; S, 9.61; Cl, 10.62. Found: C, 60.83.;

H, 5.05; N, 4.53; S, 9.58; Cl, 10.43. MS: m/e 333 (M+, 18 %). IR,

υ: 3472, 3338 (NH2), 3056 (CH, aromatic), 2989, 2880 (CH3, CH2), 1630 (C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.14 (t, 3H, J = 7.31 Hz, CH3), 1.82-1.86 (m, 2H, CH2), 2.23-2.27 (m, 2H, CH2), 4.24 (q, 2H, J = 7.31 Hz, CH2), 4.80 (s, 2H, D2O exchange- able, NH2), 6.18 (s, 1H, CH), 7.24-7.43 (2d, 4H, C6H4). 13C NMR

(DMSO-d6, 75 MHz): δ = 16.3 (ester CH3), 23.5, 25.5 (2CH2),

52.8 (ester CH2), 89.4, 90.3 (CH=C), 120.8, 121.6, 124.5, 129.3,

130.8, 134.6, 136.8, 140.8 (thiophene, C6H4).

5,7-Diamino-3-(4-methoxybenzylidene)-2,3-dihydro-1H-in- dene-4,6-dicarbonitrile (25)

To a solution of compound 17a (2.02 g, 0.01 mol) in absolute ethanol (40 mL) containing triethylamine (1.0 mL) malononi- trile dimer (1.32 g, 0.01 mol) was added. The whole reaction mixture was heated under reflux for 3 h then poured onto ice/ water containing few drops of hydrochloric acid. The formed solid product was collected by filtration.

Compound 25: Green crystals from 1,4-dioxan, yield 73 % (2.30 g), m.p. 176–179 °C. Anal. Calculated for C19H16N4O (316.13): C, 72.13; H, 5.10; N, 17.71. Found: C, 72.33 H, 5.29; N, 17.93. MS:

m/e 316 (M+, 32 %). IR, υ: 3492-3341 (2NH2), 3054 (CH, aro-

matic), 2983, 2886 (CH3, CH2), 2223, 2220 (2CN), 1636 (C=C).

1H NMR (DMSO-d6, 200 MHz): δ = 1.80-1.86 (m, 2H, CH2), 2.22-

2.26 (m, 2H, CH2), 3.16 (s, 3H, OCH3), 4.48, 5.24 (2s, 4H, D2O

exchangeable, 2NH2), 6.20 (s, 1H, CH), 7.28-7.39 (2d, 4H, C6H4).

2-Cyano-N’-cyclopentylideneacetohydrazide (27)

To a solution of compound 1 (0.84 g, 0.01 mol) in 1,4-dioxan (50 mL), cyanoacetylhydrazine (0.99 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 4 h then left to cool and the formed solid product was collected by filtration.

Compound 27: White crystals from 1,4-dioxan, yield 89 % (1.46 g), m.p. 150 °C. Anal. Calculated for C8H11N3O (165.19): C, 58.17; H, 6.71; N, 25.44. Found: C, 58.28 H, 6.39; N, 25.29. MS:

m/e 165 (M+, 30 %). IR, υ: 3488-3318 (NH), 2884 (CH2), 2256 (CN), 1660 (excocyclic C=N). ). 1H NMR (DMSO-d6, 200 MHz): δ

= 1.82-1.87 (m, 4H, 2CH2), 2.20-2.28 (m, 4H, 2CH2), 3.88 (s, 2H,

CH2), 8.30 (s, 1H, D2O exchangeable, NH). 13C NMR (DMSO-d6,

75 MHz): δ = 21.8, 22.,4, 23.9, 24.6 (4CH2), 48.6 (CH2), 116.8

(CN), 163.8 (CO), 169.4 (C=N).

4-Amino-N’-cyclopentylidene-3-phenyl-2-thioxo-2,3-dihydrothi- azole-5-carbohydrazide (28)

To a solution of compound 27 (1.65 g, 0.01 mol) in 1,4-dioxan (50 mL), containing triethylamine (0.50 g) each of elemental sulfur (0.32 g, 0.01 mol) and phenylisothiocyanate (1.35 g,

0.01 mol) were added. The whole reaction mixture was heated under reflux for 3 h then left to cool and the formed solid prod- uct was collected by filtration.

Compound 28: White crystals from 1,4-dioxan, yield 74 % (2.45 g), m.p. 239 °C. Anal. Calculated for C15H16N4OS2(332.44): C, 54.19; H, 4.85; N, 16.85; S, 19.29. Found: C, 54.05; H, 5.11;

N, 16.92; S, 19.40. MS: m/e 332 (M+, 23 %). IR, υ: 3475-3309

(NH2, NH), 2887 (CH2), 1654 (excocyclic C=N), 1630 (C=C), 1205 (C=S). 1H NMR (DMSO-d6, 200 MHz): δ = 1.81-1.89 (m,

4H, 2CH2), 2.22-2.31 (m, 4H, 2CH2), 4.87 (s, 2H, D2O exchange-

able, NH2), 7.28-7.39 (m, 5H, C6H5), 8.28 (s, 1H, D2O exchange-

able, NH).

3,5-Diamino-4-cyano-N’-cyclopentylidenethiophene-2-carbohy- drazide (29)

To a solution of compound 27 (1.65 g, 0.01 mol) in 1,4-dioxan (50 mL), containing triethylamine (0.50 g) each of elemental sulfur (0.32 g, 0.01 mol) and malononitrile (0.66 g, 0.01 mol) were added. The whole reaction mixture was heated under re- flux for 2 h then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collect- ed by filtration.

Compound 29: Brown crystals from acetic acid, yield 68 % (1.78 g), m.p. 210–213 °C. Anal. Calculated for C11H13N5OS(263.32): C, 50.17; H, 4.98; N, 26.60; S, 12.18. Found: C, 50.22; H, 5.08;

N, 26.48; S, 12.30 MS: m/e 263 (M+, 18 %). IR, υ: 3448-3327

(2NH2, NH), 2883 (CH2), 1658 (excocyclic C=N), 1632 (C=C).

11H NMR (DMSO-d6, 200 MHz): δ = 1.80-1.85 (m, 4H, 2CH2),

2.24-2.36 (m, 4H, 2CH2), 4.80, 5.28 (2s, 4H, D2O exchangeable,

2NH2), 8.31 (s, 1H, D2O exchangeable, NH).

2-Amino-6-(2-cyclopentylidenehydrazinyl)-4-phenyl-4H-pyran-3,5-di- carbonitrile(30)

To a solution of compound 27 (1.65 g, 0.01 mol) in 1,4-diox- an (40 mL) containing triethylamine (0.50 mL) malononitrile (0.66 g, 0.01 mol) and benzaldehyde (1.06 g, 0.01 mol) were added. The reaction mixture was heated under reflux for 4 h then poured onto ice/water containing few drops of hydro- chloric acid and the formed solid product was collected by fil- tration.

Compound 30: Yellow crystals from acetic acid, yield 75 % (2.39 g), m.p. 175 °C. Anal. Calculated for C18H17N5O (319.36): C, 67.70; H, 5.37; N, 21.93. Found: C, 67.32; H, 4.93; N; 22.05.

MS: m/e 319 (M+, 28 %). IR, υ: 3488-3342 (NH2, NH), 3056

(CH, aromatic), 2223, 2220 (2CN), 1650 (exocyclic C=N), 1630

(C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.60-1.84 (m, 4H, 2CH2), 2.23-2.41 (m, 4H, 2CH2), 4.87 (s, 2H, D2O exchangeable, NH2), 6.21 (s, 1H, pyran H-4), 7.26-7.42 (m, 5H, C6H5), 8.24 (s,

1H, D2O exchangeable, NH).

2-Amino-6-(2-cyclopentylidenehydrazinyl)-4-phenyl-1,4-dihy- dropyridine-3,5-dicarbonitrile (31)

To a solution of compound 27 (1.65 g, 0.01 mol) in 1,4-dioxan (40 mL) containing ammonium acetate (0.50 mL) malononi- trile (0.66 g, 0.01 mol) and benzaldehyde (1.06 g, 0.01 mol) were added. The reaction mixture was heated under reflux for 4 h then poured onto ice/water containing few drops of hy- drochloric acid and the formed solid product was collected by filtration.

Compound 31: yellow crystals from 1,4-dioxane, yield 80 % (2.54 g), m.p. 230 °C. Anal. Calculated for C18H18N6 (318.38): C, 67.90; H, 5.70; N, 26.40. Found: C, 67.88; H, 5.47; N; 26.23.

MS: m/e 319 (M+, 21 %). IR, υ: 3489-3327 (NH2, 2NH), 3053

(CH, aromatic), 2225, 2220 (2CN), 1653 (exocyclic C=N), 1630

(C=C). 1H NMR (DMSO-d6, 200 MHz): δ = 1.62-1.84 (m, 4H, 2CH2), 2.21-2.40 (m, 4H, 2CH2), 4.89 (s, 2H, D2O exchangeable, NH2), 6.11 (s, 1H, pyridine H-4), 7.28-7.36 (m, 5H, C6H5), 8.22,

8.41 (2s, 2H, D2O exchangeable, 2NH).

Conclusion

New pyran and pyridine derivatives were synthesized using efficient methods of multi-component reactions. Some com- pounds were used to produce annulated products. The antitu- mor activities of the newly synthesized product were evaluated against different tumor namely (MCF-7), (NCI-H460), (SF-268) which showed that compounds 5c and 13b are the most po- tent compounds. In most case compounds with electron neg- ative substituent showed the highest cytotoxicity among the tested compounds.

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