Chemistry
General
Melting points were measured on an Electrothermal IA 9000 series digital melting point apparatus (Bibby Sci. Lim. Stone, Staffordshire, UK). IR spectra were measured on PyeUnicam SP 3300 and Shimadzu FTIR 8101 PC infrared spectrophotometers (Shimadzu, Tokyo, Japan) in potassium bromide discs. NMR spectra were measured on a Varian Mercury VX-300 NMR spectrometer (Varian, Inc., Karlsruhe, Germany) operating at 300 MHz (1H-NMR) and run in deuterated dimethylsulfoxide (DMSO-d
6
). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCMS-QP1000 EX mass spectrometer (Tokyo, Japan) at 70 eV. Elemental analyses were measured by using a German made Elementar vario LIII CHNS analyzer. Antitumor activity of the products was measured at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt. 2-(4-Methyl-2-phenylthiazole-5-carbonyl)-N-phenylhydrazinecarbo-thioamide (3) [37], 5-(4-methyl-2-phenylthiazol-5-yl)-1,3,4-oxadiazole-2(3H)-thione (9) [37], hydrazonoyl halides 4a–g, 10a–d and 16a, b [38], and ethyl 5-hydrazono-4-phenyl-4,5-dihydro-1,3,4-thiadiazole-2-carboxylate (15) [37] were prepared as reported in the respective literature.
Synthetic procedures
Synthesis of 1,3,4-thiadiazole derivatives (6a–g, 12a–d and 18a, b)
General procedure A mixture of compound 3 (0.368 g, 1 mmol) and the appropriate hydrazonoyl chlorides 4a–g or 10a–d or 16a, b (1 mmol) in ethanol (20 mL), triethylamine (0.1 g, 1 mmol) was added. The mixture was refluxed for 4–6 h. The formed solid product was filtered, washed with methanol, dried and recrystallized from the proper solvents to afford products 6a–g, 10a–d and 18a, b, respectively. The physical constants and spectral data of the obtained products are listed below:
N′-(5-Acetyl-3-phenyl-1,3,4-thiadiazol-2(3H)-ylidene)-4-methyl-2-phenyl thiazole-5-carbohydrazide (6a)
Yellow solid (73%); m.p. 163–165 °C (EtOH); IR (KBr) v 3317 (NH), 3038, 2951 (CH), 1701, 1647 (2C=O), 1593 (C=N) cm−1; 1H-NMR (DMSO-d
6) δ 2.44 (s, 3H, CH3CO), 2.74 (s, 3H, CH3–thiazole), 6.92–8.00 (m, 10H, ArH), 11.19 (s, br, 1H, D2O-exchangeable NH); 13C-NMR (DMSO-d
6): δ 16.9, 24.9 (CH3), 114.8, 117.1, 120.9, 121.9, 123.4, 126.2, 128.9, 129.2, 129.4, 130.9, 138.3, 141.7, 159.4 (Ar–C and C=N), 167.9, 194.0 (C=O); MS m/z (%) 435 (M+, 10), 381 (13), 274 (56), 118 (31), 92 (100), 65 (38). Anal. Calcd. for C21H17N5O2S2 (435.52): C, 57.91; H, 3.93; N, 16.08. Found C, 57.86; H, 3.84; N, 16.00%.
N′-(5-Acetyl-3-(p-tolyl)-1,3,4-thiadiazol-2(3H)-ylidene)-4-methyl-2-phenyl thiazole-5-carbohydrazide (6b)
Yellow solid (75%); m.p. 149–151 °C (EtOH); IR (KBr) v 3334 (NH), 3019, 2920 (CH), 1699, 1648 (2C=O), 1597 (C=N) cm−1; 1H-NMR (DMSO-d
6) δ 2.31 (s, 3H, CH3-Ar), 2.44 (s, 3H, CH3CO), 2.73 (s, 3H, CH3–thiazole), 6.98–7.89 (m, 9H, ArH), 11.18 (s, br, 1H, D2O-exchangeable NH); 13C-NMR (DMSO-d
6): δ 16.0, 17.7, 19.4 (CH3), 116.0, 118.0, 120.8, 125.1, 126.7, 127.3, 128.1, 129.8, 131.9, 132.7, 138.2, 152.6, 159.4 (Ar–C and C=N), 166.5, 194.7 (C=O); MS m/z (%) 449 (M+, 45), 372 (54), 200 (27), 104 (36), 80 (100), 64 (35). Anal. Calcd. for C22H19N5O2S2 (449.55): C, 58.78; H, 4.26; N, 15.58. Found C, 58.65; H, 4.17; N, 15.46%.
N′-(5-Acetyl-3-(4-chlorophenyl)-1,3,4-thiadiazol-2(3H)-ylidene)-4-methyl-2-phenyl-thiazole-5-carbohydrazide (6c)
Brown solid (75%); m.p. 171–173 °C (EtOH); IR (KBr) v 3325 (NH), 3013, 2926 (CH), 1698, 1655 (2C=O), 1594 (C=N) cm−1; 1H-NMR (DMSO-d
6) δ 2.45 (s, 3H, CH3CO), 2.76 (s, 3H, CH3–thiazole), 6.93–7.96 (m, 9H, ArH), 11.25 (s, br, 1H, D2O-exchangeable NH); 13C-NMR (DMSO-d
6): δ 16.8, 24.9 (CH3), 115.1, 119.4, 120.2, 122.9, 123.8, 127.3, 128.3, 128.7, 129.0, 133.5, 138.3, 140.2, 157.9 (Ar–C and C=N), 167.8, 194.3 (C=O); MS m/z (%) 471 (M++2, 14), 469 (M+, 45), 396 (57), 200 (17), 80 (100), 64 (89). Anal. Calcd. for C21H16ClN5O2S2 (469.97): C, 53.67; H, 3.43; N, 14.90. Found C, 53.52; H, 3.37; N, 14.82%.
N′-(5-Acetyl-3-(4-methoxyphenyl)-1,3,4-thiadiazol-2(3H)-ylidene)-4-methyl-2-phenyl-thiazole-5-carbohydrazide (6d)
Brown solid (68%); m.p. 143–145 °C (EtOH); IR (KBr) v 3328 (NH), 3031, 2923 (CH), 1697, 1653 (2C=O), 1596 (C=N) cm−1; 1H-NMR (DMSO-d
6) δ 2.45 (s, 3H, CH3CO), 2.75 (s, 3H, CH3–thiazole), 3.76 (s, 3H, OCH3), 6.99–7.99 (m, 9H, ArH), 11.29 (s, br, 1H, D2O-exchangeable NH); 13C-NMR (DMSO-d
6): δ 16.5, 17.9, 54.2 (CH3), 116.2, 117.9, 120.7, 124.8, 126.3, 127.0, 127.7, 129.3, 131.9, 132.4, 137.6, 150.2, 159.0 (Ar–C and C=N), 166.2, 194.6 (C=O); MS m/z (%) 465 (M+, 39), 334 (87), 200 (63), 122 (80), 77 (100), 64 (45). Anal. Calcd. for C22H19N5O3S2 (465.55): C, 56.76; H, 4.11; N, 15.04. Found C, 56.63; H, 4.04; N, 14.95%.
N′-(5-Acetyl-3-(3-chlorophenyl)-1,3,4-thiadiazol-2(3H)-ylidene)-4-methyl-2-phenylthiazole-5-carbohydrazide (6e)
Yellow solid (70%); m.p. 166–168 °C (EtOH); IR (KBr) v 3431(NH), 3025, 2932 (CH), 1698, 1659 (2C=O), 1593 (C=N) cm−1; 1H-NMR (DMSO-d
6) δ 2.44 (s, 3H, CH3CO), 2.66 (s, 3H, CH3–thiazole), 6.98–7.90 (m, 9H, ArH), 11.23 (s, br, 1H, D2O-exchangeable NH); MS m/z (%) 471 (M++2, 10), 469 (M+, 34), 334 (46), 200 (28), 132 (48), 80 (100), 64 (68). Anal. Calcd. for C21H16ClN5O2S2 (469.97): C, 53.67; H, 3.43; N, 14.90. Found C, 53.60; H, 3.36; N, 14.79%.
N′-(5-Acetyl-3-(4-bromophenyl)-1,3,4-thiadiazol-2(3H)-ylidene)-4-methyl-2-phenyl thiazole-5-carbohydrazide (6f)
Brown solid (73%); m.p. 160–162 °C (EtOH); IR (KBr) v 3429 (NH), 3012, 2924 (CH), 1696, 1654 (2C=O), 1594 (C=N) cm−1; 1H-NMR (DMSO-d
6) δ 2.44 (s, 3H, CH3CO), 2.65 (s, 3H, CH3–thiazole), 6.95–7.94 (m, 9H, ArH), 11.25 (s, br, 1H, D2O-exchangeable NH); 13C-NMR (DMSO-d
6): δ 16.9, 24.8 (CH3), 114.8, 120.3, 122.0, 122.6, 123.8, 127.2, 127.9, 128.3, 130.2, 132.5, 136.9, 140.0, 157.5 (Ar–C and C=N), 167.6, 194.1 (C=O); MS m/z (%) 516 (51), 514 (M+, 53), 325 (76), 172 (44), 91 (80), 80 (100), 64 (47). Anal. Calcd. for C21H16BrN5O2S2 (514.42): C, 49.03; H, 3.14; N, 13.61. Found C, 48.93; H, 3.12; N, 13.53%.
N′-(5-Acetyl-3-(2,4-dichlorophenyl)-1,3,4-thiadiazol-2(3H)-ylidene)-4-methyl-2-phenyl thiazole-5-carbohydrazide (6g)
Brown solid (77%); m.p. 181–183 °C (EtOH/dioxane); IR (KBr) v 3318 (NH), 3088, 2926 (CH), 1699, 1671 (2C=O), 1597 (C=N) cm−1; 1H-NMR (DMSO-d
6) δ 2.47 (s, 3H, CH3CO), 2.67 (s, 3H, CH3–thiazole), 6.97–8.07 (m, 8H, ArH), 11.19 (s, br, 1H, D2O-exchangeable NH); MS m/z (%) 504 (M+, 14), 407 (33), 161 (14), 80 (99), 64 (100). Anal. Calcd. for C21H15Cl2N5O2S2 (504.41): C, 50.00; H, 3.00; N, 13.88. Found C, 49.88; H, 2.92; N, 13.75%.
Ethyl 5-(2-(4-methyl-2-phenylthiazole-5-carbonyl)hydrazono)-4-phenyl-4,5-dihydro-1,3,4-thiadiazole-2-carboxylate (12a)
Yellow solid (71%); m.p. 137–139 °C (EtOH); IR (KBr) v 3432 (NH), 3035, 2923 (CH), 1749, 1659 (2C=O), 1597 (C = N) cm−1; 1H-NMR (DMSO-d
6) δ 1.20 (t, 3H, J = 7.1 Hz, CH2CH
3
), 2.74 (s, 3H, CH3–thiazole), 4.21 (q, 2H, J = 7.1 Hz, CH
2
CH3),7.00–8.01 (m, 10H, ArH), 10.72 (s, br, 1H, D2O-exchangeable NH); 13C-NMR (DMSO-d
6): δ 13.7, 16.8 (CH3), 61.2 (CH2), 115.8, 117.3, 118.4, 120.9, 122.4, 126.0, 128.5, 128.9, 130.0, 132.6, 135.6, 139.6, 159.1 (Ar–C and C=N), 163.4, 166.8 (C=O); MS m/z (%): 465 (M+, 27), 334 (50), 200 (34), 104 (40), 80 (100), 64 (37). Anal. Calcd. for C22H19N5O3S2 (465.55): C, 56.76; H, 4.11; N, 15.04. Found C, 56.69; H, 4.03; N, 15.01%.
Ethyl 5-(2-(4-methyl-2-phenylthiazole-5-carbonyl)hydrazono)-4-(p-tolyl)-4,5-dihydro-1,3,4-thiadiazole-2-carboxylate (12b)
Yellow solid (70%); m.p. 147–149 °C (EtOH); IR (KBr) v 3424 (NH), 3058, 2925 (CH), 1749, 1674 (2C=O), 1595 (C=N) cm−1; 1H-NMR (DMSO-d
6) δ 1.20 (t, 3H, J = 7.1 Hz, CH2CH
3
), 2.26 (s, 3H, CH3–Ar), 2.76 (s, 3H, CH3–thiazole), 4.19 (q, 2H, J = 7.1 Hz, CH
2
CH3), 7.00–8.02 (m, 9H, ArH), 10.73 (s, br, 1H, D2O-exchangeable NH); 13C-NMR (DMSO-d
6): δ 13.9, 16.8, 20.1 (CH3), 61.5 (CH2), 114.5, 115.8, 117.1, 120.9, 121.9, 126.2, 128.1, 129.6, 130.8, 131.8, 138.3, 140.0, 159.4 (Ar–C and C=N), 163.0, 166.5 (C=O); MS m/z (%) 479 (M+, 20), 367 (25), 251 (18), 80 (85), 64 (100). Anal. Calcd. for C23H21N5O3S2 (479.57): C, 57.60; H, 4.41; N, 14.60. Found C, 57.49; H, 4.33; N, 14.51%.
Ethyl 4-(4-chlorophenyl)-5-(2-(4-methyl-2-phenylthiazole-5-carbonyl) hydrazono)-4,5-dihydro-1,3,4-thiadiazole-2-carboxylate (12c)
Yellow solid (73%); m.p. 167–169 °C (EtOH/dioxane); IR (KBr) v 3340 (NH), 3050, 2927 (CH), 1748, 1670 (2C=O), 1599 (C=N) cm−1; 1H-NMR (DMSO-d
6) δ 1.23 (t, 3H, J = 7.1 Hz, CH2CH
3
), 2.75 (s, 3H, CH3–thiazole), 4.22 (q, 2H, J = 7.1 Hz, CH
2
CH3),7.02–7.96 (m, 9H, ArH), 10.77 (s, br, 1H, D2O-exchangeable NH); 13C-NMR (DMSO-d
6): δ 13.4, 16.9 (CH3), 61.4 (CH2), 116.2, 117.0, 119.5, 120.9, 122.3, 127.2, 128.2, 129.4, 131.4, 132.2, 137.0, 139.4, 158.6 (Ar–C and C=N), 163.8, 167.2 (C=O); MS m/z (%) 501 (M++2, 13), 499 (M+, 45), 363 (39), 334 (100), 200 (35), 104 (30), 77 (50). Anal. Calcd. for C22H18ClN5O3S2 (499.99): C, 52.85; H, 3.63; N, 14.01. Found C, 52.79; H, 3.60; N, 13.87%.
Ethyl 4-(2,4-dichlorophenyl)-5-(2-(4-methyl-2-phenylthiazole-5-carbonyl) hydrazono)-4,5-dihydro-1,3,4-thiadiazole-2-carboxylate (12d)
Brown solid (75%); m.p. 173–175 °C (EtOH/dioxane); IR (KBr) v 3221 (NH), 3079, 2926 (CH), 1749, 1671 (2C=O), 1599 (C=N) cm−1; 1H-NMR (DMSO-d
6) δ 1.24 (t, 3H, J = 7.1 Hz, CH2CH
3
), 2.77 (s, 3H, CH3-thiazole), 4.23 (q, 2H, J = 7.1 Hz, CH
2
CH3),7.08–8.13 (m, 8H, ArH), 10.77 (s, br, 1H, D2O-exchangeable NH); MS m/z (%) 534 (M+, 19), 449 (78), 223 (100), 200 (54), 104 (58), 80 (85). Anal. Calcd. for C22H17Cl2N5O3S2 (534.44): C, 49.44; H, 3.21; N, 13.10. Found C, 49.29; H, 3.16; N, 13.02%.
5-(2-(4-Methyl-2-phenylthiazole-5-carbonyl)hydrazono)-N,4-diphenyl-4,5-dihydro-1,3,4-thiadiazole-2-carboxamide (18a)
Brown solid (76%); m.p. 176–178 °C (EtOH/dioxane); IR (KBr) v 3427, 3343 (2NH), 1672, 1653 (2C=O), 1597 (C=N) cm−1; 1H-NMR (DMSO-d
6) δ 2.75 (s, 3H, CH3–thiazole), 7.02–7.78 (m, 15H, ArH), 10.18 (s, br, 1H, D2O-exchangeable NH), 11.72 (s, br, 1H, D2O-exchangeable NH); 13C-NMR (DMSO-d
6): δ 17.2 (CH3), 114.6, 117.3, 118.4, 120.9, 122.6, 122.8, 124.0, 126.5, 128.5, 129.1, 130.0, 130.6, 131.9, 132.6, 138.2, 142.1, 159.2 (Ar–C and C=N), 162.6, 166.0 (C=O); MS m/z (%) 512 (M+, 8), 401 (00), 282 (10), 150 (22), 92 (26), 65 (29). Anal. Calcd. For C26H20N6O2S2 (512.61): C, 60.92; H, 3.93; N, 16.39. Found C, 60.78; H, 3.85; N, 16.32%.
4-(2,4-Dichlorophenyl)-5-(2-(4-methyl-2-phenylthiazole-5-carbonyl) hydrazono)-N-phenyl-4,5-dihydro-1,3,4-thiadiazole-2-carboxamide (18b)
Brown solid (77%); m.p. 186–188 °C (Dioxane); IR (KBr) v 3429, 3337 (2NH), 1692, 1656 (2C=O), 1591 (C=N) cm−1; 1H-NMR (DMSO-d
6) δ 2.76 (s, 3H, CH3–thiazole), 7.13–7.83 (m, 13H, ArH), 10.19 (s, br, 1H, D2O-exchangeable NH), 11.77 (s, br, 1H, D2O-exchangeable NH); MS m/z (%) 581 (M+, 38), 473 (64), 334 (72), 200 (35), 119 (65), 64 (100). Anal. Calcd. for C26H18Cl2N6O2S2 (581.50): C, 53.70; H, 3.12; N, 14.45. Found C, 53.62; H, 3.03; N, 14.32%.
Alternate synthesis of thiadiazole derivatives 6a and 18a
To a mixture of 5-(4-methyl-2-phenylthiazol-5-yl)-1,3,4-oxadiazole-2(3H)-thione (9) (0.275 g, 1 mmol) and hydrazonoyl chloride 4a or 16a (1 mmol) in absolute EtOH (25 mL), was added triethylamine (0.1 g, 0.14 mL, 1 mmol). The reaction mixture was stirred at room temperature till methyl mercaptan ceased to evolve (3 h). The solvent was evaporated and the residue was treated with ice/HCl mixture. The solid product was collected by filtration, washed with EtOH, dried, and recrystallized to give the respective compounds 6a and 18a, that was identical in all respects (m.p., mixed m.p. and IR spectra) with that obtained from the reaction of 4a or 16a with 3.
Alternate synthesis of 12a
A mixture of ethyl 4-methyl-2-phenylthiazole-5-carboxylate (1) (0.247 g, 1 mmol) and ethyl 5-hydrazono-4-phenyl-4,5-dihydro-1,3,4-thiadiazole-2-carboxylate (15) (0.264 g, 1 mmol) was refluxed in ethanol for 4 h. The solid product that separated was filtered off, washed with water and finally recrystallized to give the corresponding product, 12a which was identical in all aspects (m.p., mixed m.p. and IR spectra) with those obtained from the reaction of 3 with 10a.
Evaluation of the antitumor activity using Viability assay
Human hepatocellular carcinoma (HepG2) cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD). The detailed procedure for the in vitro antitumor assay is presented in Additional file 1.