All chemicals were purchased from Merck (Germany) and were used without further purification. Melting points were measured on an Electrothermal 9100 apparatus and were not corrected. Mass spectra were recorded on an Agilent Technologies (HP) 5973 mass spectrometer operating at an ionization potential of 20 eV. IR spectra were recorded on a Shimadzu IR-460 spectrometer. 1H and 13C NMR spectra were measured (DMSO‑d6 solution) with Bruker DRX-300 AVANCE (at 300.1 and 75.1 MHz) spectrometer with TMS as an internal standard. α-Azido chalcones 2 were obtained from the corresponding benzylidene acetophenones in two steps following the literature procedure15.
General procedure for the preparation of 3-amino-2,4-diarylbenzo[4,5]imidazo[1,2-a]pyrimidines 3a-ag:
A solution of 2-aminobenzimidazoles 1 (1.2 mmol), α-azidochalcones 2 (1.0 mmol), Et3N (1.2 mmol) in EtOH (5.0 mL) was magnetically stirred for 2h under reflux conditions. After completion of the reaction according to the TLC analysis, the mixture was cooled to ambient temperature, the precipitated product was filtered and washed with Et2O (5.0 mL) to afford pure products as yellow powder.
General procedure for the preparation of 3-chloro-2,4-diphenylbenzo[4,5]imidazo[1,2-a]pyrimidine 4a:
To a stirring solution of concentrated sulfuric acid (1.6 mmol), sodium nitrite (2.2 mmol) was added gradually over 10-15 min. After addition was completed, the temperature was raised to 70 °C, and the mixture was stirred until sodium nitrite dissolved thoroughly. Then, the mixture is cooled to 25 °C with an ice bath, and a solution of 3-amino-2,4-diphenylbenzo[4,5]imidazo[1,2-a]pyrimidin 3a (2.0 mmol) in glacial acetic acid (4.0 ml) was added slowly with stirring, at such a rate that temperature remains below 40 °C. After 30 min, TLC monitoring confirmed compound 3a was completely converted to corresponding diazonium salt. The obtained mixture was added at 10 °C in portions to a solution of CuCl (4.4 mmol) in concentrated hydrochloric acid (4.0 mmol) over a period of about 5 minutes. Afterward, temperature was raised to 80 °C and the reaction mixture was heated for almost 5h. After completion of the reaction which was monitored by TLC, mixture was quenched by iced water. The precipitate was filtered and recrystallized in EtOH to afford the pure product 4a.
General procedure for the preparation of 2,4-diphenylbenzo[4,5]imidazo[1,2-a]pyrimidine 6a:
A mixture of 2-aminobenzimidazole 1a (1.2 mmol), chalcone 5a (1.0 mmol), and Et3N (1.2 mmol) in EtOH (5.0 mL) was heated under reflux conditions for 10h. After completion of the reaction confirmed by the TLC analysis, the solvent was removed under the reduced pressure. The residue was purified by column chromatography using n-hexane/EtOAc (3:1) as eluent to afford pure product 6a.
2,4-Diphenyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3a):
Yellow solid; yield: 89%, mp 208–210 °C. IR (KBr) (νmax/cm–1): 3459 and 3372 (NH2), 1594, 1426, 1378, 1302, 1228, 1194, 1148, 1083, 1016, 984, 906, 801, 746, 668, 624. 1H NMR (300.1 MHz, DMSO): δ 8.15 (d, J = 8.4 Hz, 2H, 2CH), 8.10–7.20 (m, 9H, 9CH), 7.11 (t, J = 7.5 Hz, 1H, CH), 6.87 (t, J = 7.4 Hz, 1H, CH), 6.18 (d, J = 8.2 Hz, 1H, CH), 4.13 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 155.2, 148.2, 144.9, 136.8, 136.4, and 130.8 (6C), 130.4 and 130.3 (2CH), 129.9 (2CH), 129.7 (2CH), 129.2 (2CH), 128.6 (CH), 127.9 (2CH), 127.4 and 126.4 (2C), 124.9, 120.0 and 119.2 (3CH). EI-MS, m/z (%): 336 (M+, 27), 133 (100), 105 (80), 79 (43), 52 (35).
2-Phenyl-4-p-tolyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3b):
Yellow solid; yield: 86%, mp 272–274 °C. IR (KBr) (νmax/cm–1): 3428 and 3362 (NH2), 1595, 1434, 1399, 1356, 1256, 1181, 1085, 1014, 991, 829, 754, 685, 650. 1H NMR (300.1 MHz, DMSO): δ 8.17 (d, J = 8.3 Hz, 2H, 2CH), 7.76 (d, J = 7.8 Hz, 1H, CH), 7.60–7.24 (m, 7H, 7CH), 7.10 (t, J = 7.5 Hz, 1H, CH), 6.91 (t, J = 7.4 Hz, 1H, CH), 6.28 (d, J = 8.2 Hz, 1H, CH), 4.04 (s, 2H, NH2), 2.36 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO-d6): δ 155.3, 148.2, 144.9, 137.5, 136.9 and 136.4 (6C), 130.9 (2CH), 130.6 (CH), 129.7 (2 × 2CH), 129.2 (CH), 128.6 (C), 127.9 (2CH), 127.4 and 126.8 (2C), 124.8, 119.9 and 119.1 (3CH), 21.1 (CH3).
4-(4-Methoxy-phenyl)-2-phenyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3c):
Yellow solid; yield: 74%, mp 267–271 °C. IR (KBr) (νmax/cm–1): 3438 and 3389 (NH2), 1592, 1414, 1367, 1283, 1182, 1144, 1068, 991, 947, 843, 805, 753, 675, 623. 1H NMR (300.1 MHz, DMSO): δ 7.91 (d, J = 8.4 Hz, 2H, 2CH), 7.74 (d, J = 8.5 Hz, 1H, CH), 7.63–7.52 (m, 5H, 5CH), 7.37–7.27 (m, 3H, 3CH), 6.94 (t, J = 7.4 Hz, 1H, CH), 6.29 (d, J = 8.4 Hz, 1H, CH), 4.03 (s, 2H, NH2), 3.91 (s, 3H, OCH3). 13C NMR (75.5 MHz, DMSO-d6): δ 160.9, 156.8, 148.1, 144.5 and 137.1 (5C), 131.2 (2CH), 130.2 and 129.9 (2CH), 129.7(C), 128.6 (2 × 2CH), 127.7, 127.2 and 126.6 (3C), 124.6, 119.9 and 119.1 (3CH), 115.7 (2CH), 55.4 (OCH3).
4-(4-Chloro-phenyl)-2-phenyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3d):
Yellow solid; yield: 90%, mp 252–253 °C. IR (KBr) (νmax/cm–1): 3448 and 3363 (NH2), 1598, 1458, 1397, 1368, 1287, 1158, 1079, 1012, 994, 935, 897, 752, 689, 623. 1H NMR (300.1 MHz, DMSO): δ 7.95–7.63 (m, 7H, 7CH), 7.60–7.47 (m, 3H, 3CH), 7.33 (t, J = 7.5 Hz, 1H, CH), 6.96 (t, J = 7.4 Hz, 1H, CH), 6.25 (d, J = 8.4 Hz, 1H, CH), 4.20 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 155.4, 148.0, 144.5, 138.8, 136.9 and 135.6 (6C), 132.0 (2CH), 131.5 (CH), 130.5 (2CH), 129.9 (CH), 128.6 (2 × 2CH), 128.1, 127.0 and 126.6 (3C), 124.6, 120.1 and 119.2 (3CH).
4-(2-Chloro-phenyl)-2-phenyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3e):
Yellow solid; yield: 68%, mp 276–277 °C. IR (KBr) (νmax/cm–1): 3436 and 3385 (NH2), 1593, 1484, 1389, 1345, 1278, 1169, 1149, 1079, 991, 936, 899, 842, 799, 753, 685, 655. 1H NMR (300.1 MHz, DMSO): δ 8.04–7.71 (m, 6H, 6CH), 7.67 (d, J = 7.6 Hz, 1H, CH), 7.63–7.47 (m, 3H, 3CH), 7.32 (t, J = 7.5 Hz, 1H, CH), 6.95 (t, J = 7.8 Hz, 1H, CH), 6.19 (d, J = 7.8 Hz, 1H, CH), 4.21 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 157.1, 147.9, 144.5, 136.9, 134.8 and 132.2 (6C), 131.8, 130.9, 129.9, 129.8 and 128.72 (5CH), 128.66 (2CH), 128.60 (2CH), 127.8, 127.0 and 126.6 (3C), 124.6, 120.1, 119.2 and 113.5 (4CH).
4-(3-Chloro-phenyl)-2-phenyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3f):
Yellow solid; yield: 73%, mp 268–269 °C. IR (KBr) (νmax/cm–1): 3443 and 3359 (NH2), 1596, 1501, 1346, 1277, 1182, 1149, 1084, 993, 825, 785, 685, 635. 1H NMR (300.1 MHz, DMSO): δ 7.96–7.85 (m, 3H, 3CH), 7.83–7.67 (m, 4H, 4CH), 7.63–7.50 (m, 3H, 3CH), 7.35 (t, J = 7.7 Hz, 1H, CH), 6.96 (t, J = 7.6 Hz, 1H, CH), 6.13 (d, J = 8.4 Hz, 1H, CH), 4.29 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 157.0, 147.7, 144.4, 136.8 and 133.8 (5C), 133.0, 132.4, 130.7, 130.0 and 129.2 (5CH), 128.7 (2CH), 128.6 (2CH), 128.5, 126.92, 126.85 and 126.1 (4C), 124.7, 120.6, 119.3 and 112.6 (4CH).
2-Phenyl-4-thiophen-2-yl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3g):
Yellow solid; yield: 69%, mp 246–248 °C. IR (KBr) (νmax/cm–1): 3458 and 3376 (NH2), 1595, 1512, 1397, 1282, 1178, 1132, 1075, 989, 923, 824, 732, 684, 652. 1H NMR (300.1 MHz, DMSO-d6): δ 8.19 (d, J = 3.5 Hz, 1H, CH), 7.89 (d, J = 5.0 Hz, 1H, CH), 7.83–7.48 (m, 6H, 6CH), 7.36–7.21 (m, 2H, 2CH), 6.95 (t, J = 7.4 Hz, 1H, CH), 6.26 (d, J = 8.3 Hz, 1H, CH), 4.25 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 156.1, 150.4, 147.9, 144.6, 141.4 and 131.6 (6C), 131.3 and 130.4 (2CH), 129.9 (2CH), 128.7 (2CH), 128.0 (CH), 127.3 and 126.6 (2C), 125.7, 124.7, 120.2, 119.4 and 113.3 (5CH). EI-MS, m/z (%): 342 (M+, 100), 266 (25), 248 (78), 168 (43), 135 (36), 105 (28), 77 (48), 51 (25).
2-(4-Chloro-phenyl)-4-phenyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3h):
Yellow solid; yield: 84%, mp 265–266 °C. IR (KBr) (νmax/cm–1): 3468 and 3373 (NH2), 1607, 1498, 1424, 1358, 1284, 1203, 1163, 1041, 991, 953, 885, 743, 696, 641. 1H NMR (300.1 MHz, DMSO-d6): δ 7.96 (d, J = 8.4 Hz, 2H, 2CH), 7.85–7.52 (m, 8H, 8CH), 7.31 (t, J = 7.7 Hz, 1H, CH), 6.89 (t, J = 7.9 Hz, 1H, CH), 6.11 (d, J = 8.4 Hz, 1H, CH), 4.14 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 155.8, 148.0, 144.6, 135.9, 134.7 and 134.1 (6C), 130.9 (CH), 130.7 (2CH), 130.4 (2CH), 130.1 (C), 129.71 (2CH), 129.66 (CH), 128.6 (2CH), 127.1 and 126.3 (2C), 124.7, 120.0 and 119.2 (3CH). EI-MS, m/z (%): 370 (M+, 100), 333 (18), 232 (25), 206 (36), 167 (29), 102 (45), 77 (51), 51 (25).
2-(4-Chloro-phenyl)-4-p-tolyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3i):
Yellow solid; yield: 78%, mp 277–278 °C. IR (KBr) (νmax/cm–1): 3468 and 3348 (NH2), 1602, 1487, 1412, 1368, 1294, 1235, 1132, 1098, 1032, 991, 928, 848, 776, 729, 687, 635. 1H NMR (300.1 MHz, DMSO-d6): δ 7.93 (d, J = 8.3 Hz, 2H, 2CH), 7.66–7.46 (m, 7H, 7CH), 7.31 (t, J = 7.9 Hz, 1H, CH), 6.90 (t, J = 7.6 Hz, 1H, CH), 6.22 (d, J = 8.5 Hz, 1H, CH), 4.09 (s, 2H, NH2), 2.43 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO-d6): δ 155.6, 148.0, 144.5, 135.9 and 134.6 (5C), 131.4 (CH), 130.9 (2CH), 130.6 (2CH), 130.2 and 129.8 (2C), 129.5 (2CH), 128.6 (2CH), 127.1, 126.6 and 126.4 (3C), 124.7, 120.0 and 119.1(3CH), 21.2 (CH3).
2-(4-Chloro-phenyl)-4-(4-methoxy-phenyl)-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3j):
Yellow solid; yield: 83%, mp 257–258 °C. IR (KBr) (νmax/cm–1): 3446 and 3372 (NH2), 1595, 1493, 1427, 1359, 1295, 1236, 1149, 1085, 1035, 972, 939, 858, 784, 740, 655, 637. 1H NMR (300.1 MHz, DMSO-d6): δ 8.18 (d, J = 8.6 Hz, 2H, 2CH), 7.95 (d, J = 8.4 Hz, 2H, 2CH), 7.74 (d, J = 8.0 Hz, 1H, CH), 7.30 (d, J = 8.4 Hz, 2H, 2CH), 7.09–7.05 (m, 1H, CH), 7.03 (d, J = 8.5 Hz, 2H, 2CH), 6.94 (t, J = 7.8 Hz, 1H, CH), 6.28 (d, J = 8.6 Hz, 1H, CH), 4.12 (s, 2H, NH2), 3.74 (s, 3H, OCH3). 13C NMR (75.5 MHz, DMSO-d6): δ 157.4, 155.6, 147.8, 145.0, 135.9 and 134.6 (6C), 131.3 (2CH), 131.2 (CH), 130.1 (C), 128.6 (2CH), 127.7 (2CH), 127.3, 127.1 and 126.7 (3C), 124.7, 120.0 and 119.1 (3CH), 115.8 (2CH), 55.4 (OCH3).
2,4-Bis-(4-chloro-phenyl)-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3k):
Yellow solid; yield: 92%, mp 271–272 °C. IR (KBr) (νmax/cm–1): 3447 and 3356 (NH2), 1599, 1506, 1436, 1348, 1294, 1233, 1172, 1061, 990, 898, 831, 786, 686, 635. 1H NMR (300.1 MHz, DMSO-d6): δ 7.93 (d, J = 8.5 Hz, 2H, 2CH), 7.82 (d, J = 8.4 Hz, 2H, 2CH), 7.75 (d, J = 8.3 Hz, 1H, CH), 7.72 (d, J = 8.5 Hz, 2H, 2CH), 7.62 (d, J = 8.4 Hz, 2H, 2CH), 7.33 (t, J = 7.8 Hz, 1H, CH), 6.96 (t, J = 7.8 Hz, 1H, CH), 6.24 (d, J = 8.4 Hz, 1H, CH), 4.25 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 155.8, 147.9, 144.5, 135.8, 135.6 and 134.7 (6C), 131.9 (2CH), 131.4 (CH), 130.6 (2CH), 130.5 (2CH), 128.6 (2CH), 128.51, 128.48, 127.0 and 126.7 (4C), 124.7, 120.2 and 119.2 (3CH). EI-MS, m/z (%): 404 (M+, 100), 333 (45), 293 (65), 270 (15), 258 (34), 166 (18), 103 (28), 77 (38), 52 (26).
3-(4-Chloro-phenyl)-1-thiophen-2-yl-benzo[4,5]imidazo[1,2-a]pyridin-2-ylamine (3l):
Yellow solid; yield: 71%, mp 246–245 °C. IR (KBr) (νmax/cm–1): 3459 and 3374 (NH2), 1586, 1502, 1435, 1370, 1283, 1256, 1082, 1046, 932, 845, 760, 638. 1H NMR (300.1 MHz, DMSO-d6): δ 8.23 (d, J = 3.6 Hz, 1H, CH), 8.02 (d, J = 8.4 Hz, 2H, 2CH), 7.90 (d, J = 4.8 Hz, 1H, CH), 7.75 (d, J = 8.3 Hz, 1H, CH), 7.40 (d, J = 8.4 Hz, 2H, 2CH), 7.32–7.21 (m, 2H, 2CH), 6.98 (t, J = 7.5 Hz, 1H, CH), 6.31 (d, J = 8.4 Hz, 1H, 1CH), 4.15 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 155.7, 151.0, 148.2, 144.8, 142.4 and 135.4 (6C), 131.1 (CH), 130.9 (2CH), 130.8 and 128.3 (2C), 128.2 (2CH), 127.4 (CH), 126.7 (C) 125.6, 124.7, 119.2, 118.9 and 114.0 (5CH).
2-(4-Bromo-phenyl)-4-phenyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3m):
Yellow solid; yield: 83%, mp 268–271 °C. IR (KBr) (νmax/cm–1): 3429 and 3384 (NH2), 1583, 1498, 1445, 1358, 1267, 1242, 1145, 1061, 995, 846, 789, 748, 674, 625. 1H NMR (300.1 MHz, DMSO-d6): δ 8.10 (d, J = 8.3 Hz, 2H, 2CH), 7.75 (d, J = 7.8 Hz, 1H, CH), 7.64–7.48 (m, 7H, 7CH), 7.33 (t, J = 7.7 Hz, 1H, CH), 6.85 (t, J = 7.4 Hz, 1H, CH), 6.17 (d, J = 8.4 Hz, 1H, CH), 4.15 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 155.1, 147.9, 144.9, 136.3 and 136.0 (5C), 131.6 (2CH), 130.92 (C), 130.86 (2CH), 130.4 and 130.1 (2CH), 129.8 (2CH), 129.1 (2CH), 127.3 and 126.4 (2C), 124.8 (CH), 123.6 (C), 119.9 and 119.2 (2CH). EI-MS, m/z (%): 416 (M+, 100), 333 (17), 232 (15), 206 (22), 167 (35), 133 (16), 102 (33), 77 (38), 51 (13).
2-(4-Bromo-phenyl)-4-p-tolyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3n):
Yellow solid; yield: 90%, mp 278–279 °C. IR (KBr) (νmax/cm–1): 3469 and 3342 (NH2), 1605, 1492, 1428, 1371, 1295, 1245, 1180, 1045, 1015, 923, 879, 753, 695, 634. 1H NMR (300.1 MHz, DMSO-d6): δ 7.87 (d, J = 8.3 Hz, 2H, 2CH), 7.80–7.65 (m, 5H, 5CH), 7.64–7.48 (m, 4H, 4CH), 7.31 (t, J = 7.5 Hz, 1H, CH), 6.91 (t, J = 7.5 Hz, 1H, CH), 6.23 (d, J = 8.7 Hz, 1H, CH), 4.10 (s, 2H, NH2), 2.29 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO-d6): δ 155.7, 148.0, 144.6, 136.2, 135.5 (5C), 131.5 (2 × 2CH), 130.89 (2CH), 130.85 (2CH), 130.7 (CH), 129.5 (2CH), 129.2, 127.1, 126.6 and 126.4 (4C), 124.7 (CH), 123.5 (C), 120.0 and 119.1 (2CH), 21.2 (CH3).
2-(4-Methoxy-phenyl)-4-phenyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3o):
Yellow solid; yield: 65%, mp 248–250 °C. IR (KBr) (νmax/cm–1): 3473 and 3359 (NH2), 1604, 1458, 1388, 1292, 1239, 1198, 1132, 1043, 994, 926, 831, 756, 698, 624. 1H NMR (300.1 MHz, DMSO-d6): δ 7.82 (d, J = 8.5 Hz, 2H, 2CH), 7.70 (d, J = 8.0 Hz, 1H, CH), 7.60–7.42 (m, 5H, 5CH), 7.38 (t, J = 7.9 Hz, 1H, CH), 7.07 (d, J = 8.5 Hz, 2H, 2CH), 6.96 (t, J = 7.6 Hz, 1H, CH), 6.30 (d, J = 8.3 Hz, 2H, 2CH), 4.13 (s, 2H, NH2), 3.82 (s, 3H, OCH3). 13C NMR (75.5 MHz, DMSO-d6): δ 160.0, 158.7, 148.2, 145.3 and 136.3 (5C), 130.5 (2CH), 130.2 and 129.9 (2CH), 129.4 (C), 129.3 (2CH), 128.6 (2CH), 127.7, 127.3 and 126.5 (3C), 124.6, 120.6 and 119.2 (3CH), 114.2 (2CH), 55.0 (OCH3).
2-(4-Methoxy-phenyl)-4-p-tolyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3p):
Yellow solid; yield: 84%, mp 274–275 °C. IR (KBr) (νmax/cm–1): 3438 and 3329 (NH2), 1605, 1458, 1401, 1376, 1273, 1178, 1075, 1023, 983, 878, 768, 659, 621. 1H NMR (300.1 MHz, DMSO-d6): δ 7.92 (d, J = 8.4 Hz, 2H, 2CH), 7.72 (d, J = 7.8 Hz, 1H, CH), 7.56 (d, J = 8.3 Hz, 2H, 2CH), 7.53 (d, J = 8.3 Hz, 2H, 2CH), 7.30 (t, J = 7.5 Hz, 1H, CH), 7.09 (d, J = 8.6 Hz, 2H, 2CH), 7.01 (t, J = 7.8 Hz, 1H, CH), 6.90 (d, J = 7.7 Hz, 1H, CH), 4.03 (s, 2H, NH2), 3.83 (s, 3H, OCH3), 2.39 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO-d6): δ 156.5, 155.2, 147.5, 144.5 and 137.7 (5C), 131.5 (CH), 130.9 (2CH), 129.9 (2CH), 129.5 (2CH), 129.3, 128.8, 127.1, 126.8 and 126.4 (5C), 124.5, 119.8 and 119.1 (3CH), 111.5 (2CH), 55.3 (OCH3), 21.2 (CH3). EI-MS, m/z (%): 380 (M+, 43), 278 (66), 167 (100), 135 (39), 77 (28), 51 (16).
2,4-Bis-(4-methoxy-phenyl)-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3q):
Yellow solid; yield: 70%, mp 291–292 °C. IR (KBr) (νmax/cm–1): 3452 and 3369 (NH2), 1587, 1498, 1354, 1298, 1134, 1065, 1022, 983, 933, 886, 798, 702, 649. 1H NMR (300.1 MHz, DMSO-d6): δ 7.96 (d, J = 8.5 Hz, 2H, 2CH), 7.74 (d, J = 8.0 Hz, 2H, 2CH), 7.72 (d, J = 7.9 Hz, 1H, CH), 7.35–7.18 (m, 5H, 5CH), 6.90 (t, J = 7.8 Hz, 1H, CH), 6.11 (d, J = 8.4 Hz, 1H, CH), 4.14 (s, 2H, NH2), 3.76 and 3.75 (2s, 6H, 2OCH3). 13C NMR (75.5 MHz, DMSO-d6): δ 160.9, 160.2, 156.5, 148.4, 144.7 and 136.2 (6C), 130.5 (CH), 129.7 (C), 129.2 (2CH), 128.6 (2CH), 128.2, 127.1 and 126.2 (3C), 124.8, 120.3 and 119.2 (3CH), 116.0 (2CH), 115.1 (2CH), 55.2 and 53.6 (2OCH3).
2-(4-Methoxy-phenyl)-4-thiophen-2-yl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3r):
Yellow solid; yield: 82%, mp 274–277 °C. IR (KBr) (νmax/cm–1): 3488 and 3362 (NH2), 1596, 1503, 1487, 1363, 1278, 1243, 1137, 1041, 985, 962, 876, 795, 687, 632. 1H NMR (300.1 MHz, DMSO-d6): δ 8.24 (d, J = 3.8 Hz, 1H, CH), 7.90–7.68 (m, 4H, 4CH), 7.28–7.08 (m, 4H, 4CH), 6.94 (t, J = 7.8 Hz, 1H, CH), 6.17 (d, J = 8.4 Hz, 1H, CH), 4.24 (s, 2H, NH2), 3.91 (s, 3H, OCH3). 13C NMR (75.5 MHz, DMSO-d6): δ 159.2, 155.9, 150.4, 148.2, 144.8 and 141.8 (6C), 131.9 (CH), 131.8 (2CH), 131.6 (CH), 128.7, 127.4 and 126.5 (3C), 125.4, 124.9, 119.8 and 119.6 (4CH), 114.2 (2CH), 112.0 (CH), 54.3 (OCH3).
4-Phenyl-2-thiophen-2-yl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3s):
Yellow solid; yield: 78%, mp 265–267 °C. IR (KBr) (νmax/cm–1): 3473 and 3345 (NH2), 1589, 1495, 1432, 1386, 1241, 1174, 1098, 1028, 934, 859, 743, 659. 1H NMR (300.1 MHz, DMSO-d6): δ 8.19 (d, J = 3.5 Hz, 1H, CH), 7.87 (d, J = 5.2 Hz, 1H, CH), 7.72 (d, J = 8.0 Hz, 1H, CH), 7.62–7.45 (m, 4H, 4CH), 7.30 (t, J = 7.4 Hz, 1H, CH), 7.24 (d, J = 4.6 Hz, 1H, CH), 6.95–6.75 (m, 2H, 2CH), 6.16 (d, J = 8.1 Hz, 1H, CH), 4.26 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 155.3, 150.2, 147.7, 144.9, 141.7, 132.3 (6C), 131.6 (CH), 130.9 (2CH), 130.8 (CH), 129.5 (2CH), 128.6 (CH), 127.2 and 126.5 (2C), 125.3, 124.7, 119.9, 119.0 and 113.6 (5C).
2-Thiophen-2-yl-4-p-tolyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3t):
Yellow solid; yield: 69%, mp 276–277 °C. IR (KBr) (νmax/cm–1): 3474 and 3352 (NH2), 1585, 1522, 1486, 1448, 1346, 1220, 1188, 1072, 952, 899, 848, 774, 646, 623. 1H NMR (300.1 MHz, DMSO-d6): δ 8.19 (d, J = 3.5 Hz, 1H, CH), 7.89 (d, J = 5.0 Hz, 1H, CH), 7.70 (d, J = 8.2 Hz, 1H, CH), 7.58 (d, J = 8.4 Hz, 2H, 2CH), 7.55 (d, J = 8.3 Hz, 2H, 2CH), 7.38–7.22 (m, 2H, 2CH), 7.13–7.04 (m, 1H, CH), 6.90 (d, J = 8.4 Hz, 1H, CH), 4.26 (s, 2H, NH2), 2.29 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO-d6): δ 155.2, 150.2, 147.7, 144.9, 141.7, 140.7 and 132.4 (7C), 131.7 (CH), 130.9 (2CH), 129.5 (2CH), 128.7 (CH), 127.2 and 126.5 (2C), 125.3, 124.8, 119.9, 119.0 and 113.6 (5CH).
4-(4-Methoxy-phenyl)-2-thiophen-2-yl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3u):
Yellow solid; yield: 72%, mp 271–273 °C. IR (KBr) (νmax/cm–1): 3468 and 3343 (NH2), 1568, 1483, 1353, 1279, 1234, 1149, 1098, 983, 886, 785, 683, 642. 1H NMR (300.1 MHz, DMSO-d6): δ 8.13 (d, J = 3.4 Hz, 1H, CH), 7.91 (d, J = 4.8 Hz, 1H, CH), 7.84 (d, J = 8.6 Hz, 2H, 2CH), 7.71 (d, J = 8.0 Hz, 1H, CH), 7.35–7.20 (m, 2H, 2CH), 7.11 (d, J = 8.6 Hz, 2H, 2CH), 6.90 (t, J = 7.6 Hz, 1H, CH), 6.31 (d, J = 8.4 Hz, 1H, CH), 4.24 (s, 2H, NH2), 3.82 (s, 3H, OCH3). 13C NMR (75.5 MHz, DMSO-d6): δ 159.9, 155.8, 150.4, 147.6, 144.9, 141.8 and 132.8 (7C), 131.5 and 128.7 (2CH), 128.6 (2CH), 127.6 and 126.9 (2C), 125.5, 124.8, 120.5 and 119.2 (4CH), 115.3 (2CH), 113.5 (CH), 55.8 (OCH3).
4-(4-Chloro-phenyl)-2-thiophen-2-yl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3w):
Yellow solid; yield: 79%, mp 283–284 °C. IR (KBr) (νmax/cm–1): 3479 and 3326 (NH2), 1598, 1543, 1478, 1398, 1306, 1211, 1189, 1090, 973, 879, 837, 768, 723, 678, 641. 1H NMR (300.1 MHz, DMSO-d6): δ 8.17 (d, J = 2.9 Hz, 1H, CH), 7.87 (d, J = 5.4 Hz, 1H, CH), 7.83 (d, J = 8.3 Hz, 2H, 2CH), 7.74 (d, J = 8.3 Hz, 2H, 2CH), 7.70 (d, J = 7.8 Hz, 1H, CH), 7.31 (t, J = 7.8 Hz, 1H, CH), 7.25 (t, J = 3.3 Hz, 1H, CH), 6.93 (t, J = 7.6 Hz, 1H, CH), 6.18 (d, J = 8.3 Hz, 1H, CH), 4.36 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 155.4, 150.3, 147.6, 144.9, 141.6 and 135.8 (6C), 131.9 (2CH), 131.6 (CH), 130.7 (C), 130.5 (2CH), 128.6 (CH), 128.4 and 127.1 (2C), 125.5, 124.7, 120.0, 118.9 and 113.3 (5CH). EI-MS, m/z (%): 376 (M+, 100), 341 (10), 266 (12), 240 (22), 205 (48), 170 (32), 138 (15), 102 (30), 75 (20), 51 (14).
2,4-Di-thiophen-2-yl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3x):
Yellow solid, yield: 72%, mp 280–282 °C. IR (KBr) (νmax/cm–1): 3458 and 3306 (NH2), 1567, 1499, 1427, 1356, 1307, 1242, 1199, 1115, 1066, 960, 876, 824, 782, 716, 683, 642. 1H NMR (300.1 MHz, DMSO-d6): δ 8.21 (d, J = 3.2 Hz, 1H, CH), 8.07 (d, J = 4.3 Hz, 1H, CH), 7.89 (d, J = 4.4 Hz, 1H, CH), 7.87–7.67 (m, 3H, 3CH), 7.45–7.29 (m, 2H, 2CH), 7.06 (t, J = 7.6 Hz, 1H, CH), 6.62 (d, J = 8.3 Hz, 1H, CH), 4.43 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 155.7, 155.4, 151.0, 144.8, 142.7 and 142.4 (6C), 132.8 (CH), 131.1 (C), 130.9 130.4, 129.1 and 128.2 (4CH), 127.4 (C), 125.6, 121.0, 119.2, 118.9 and 114.0 (5CH).
7,8-Dichloro-2,4-diphenyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3y):
Yellow solid, yield: 70%, mp 267–270 °C. IR (KBr) (νmax/cm–1): 3464 and 3325 (NH2), 1597, 1539, 1501, 1419, 1360, 1295, 1190, 1067, 966, 906, 879, 753, 683, 642. 1H NMR (300.1 MHz, DMSO-d6): δ 8.13 (dd, J = 2.1, 7.3 Hz, 2H, 2CH), 8.01 (s, 1H, CH), 7.89 (dd, J = 2.4, 7.5 Hz, 1H, CH), 7.79 (t, J = 7.4 Hz, 2H, 2CH), 7.62–7.45 (m, 5H, 5CH), 6.12 (s, 1H, CH), 4.23 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 157.3, 149.6, 144.4, 136.6, 136.4 (5C), 129.8 (2CH), 129.72 (2CH), 129.69 (CH), 129.6 (2CH), 128.71 (C), 128.67 and 128.6 (2CH), 128.1 (2CH), 127.5, 127.2, 126.6 and 126.2 (4C), 119.9 (CH).
7,8-Dichloro-4-(4-chloro-phenyl)-2-phenyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3z):
Yellow solid, yield: 74%, mp 291–293 °C. IR (KBr) (νmax/cm–1): 3461 and 3384 (NH2), 1585, 1532, 1486, 1425, 1386, 1220, 1188, 1082, 1020, 958, 848, 784, 646, 621. 1H NMR (300.1 MHz, DMSO-d6): δ 8.00 (s, 1H, CH), 7.89–7.82 (m, 4H, 4CH), 7.74 (d, J = 8.5 Hz, 2H, 2CH), 7.63–7.46 (m, 3H, 3CH), 6.22 (s, 1H, CH), 4.34 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 157.3, 149.2, 143.8, 136.6, 135.9 (5C), 132.0 (2CH), 130.6 (2CH), 130.2 (CH), 129.7 (C), 128.73 (2CH), 128.66 (2CH), 128.2, 127.9 and 127.7 (3C), 127.6 (CH), 127.2 and 126.2 (2C), 120.0 (CH).
7,8-Dichloro-2-(4-chloro-phenyl)-4-phenyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3aa):
Yellow solid, yield: 75%, mp 302–304 °C. IR (KBr) (νmax/cm–1): 3419 and 3329 (NH2), 1578, 1483, 1426, 1353, 1279, 1204, 1149, 1092, 963, 876, 832, 775, 683. 1H NMR (300.1 MHz, DMSO-d6): δ 8.15 (d, J = 8.6 Hz, 2H, 2CH), 7.99 (s, 1H, CH), 7.74–7.58 (m, 5H, 5CH), 7.45 (d, J = 8.6 Hz, 2H, 2CH), 6.15 (s, 1H, CH), 4.28 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 157.3, 149.1, 144.4, 135.2 and 134.9 (5C), 131.3 (2CH), 130.5 (CH), 129.8 (2CH), 129.3 (2CH), 129.2 and 128.7 (2C), 128.6 (CH), 127.9 (2CH), 127.3, 127.2, 126.4 and 126.2 (4C), 119.8 (CH).
7,8-Dichloro-2-(4-chloro-phenyl)-4-p-tolyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3ab):
Yellow solid, yield: 81%, mp 289–290 °C. IR (KBr) (νmax/cm–1): 3486 and 3362 (NH2), 1588, 1487, 1359, 1291, 1140, 1088, 959, 929, 846, 797, 683, 642. 1H NMR (300.1 MHz, DMSO-d6): δ 8.15 (d, J = 8.6 Hz, 2H, 2CH), 8.02 (s, 1H, CH), 7.58 (d, J = 8.2 Hz, 2H, 2CH), 7.57 (d, J = 8.4 Hz, 2H, 2CH), 7.50 (d, J = 8.2 Hz, 2H, 2CH), 6.24 (s, 1H, CH), 4.27 (s, 2H, NH2), 2.47 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO-d6): δ 157.3, 149.4, 144.4, 137.0, 136.5 and 135.2 (6C), 131.50 (2CH), 131.46 (CH), 130.7 (C), 130.0 (2CH), 129.6 (2CH), 129.2 and 128.6 (2C), 128.2 (2CH), 127.6, 126.6 and 125.6 (3C), 119.9 (CH), 21.1 (CH3).
7,8-Dichloro-2-(4-chloro-phenyl)-4-(4-methoxy-phenyl)-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3ac):
Yellow solid, yield: 75%, mp 294–295 °C. IR (KBr) (νmax/cm–1): 3466 and 3394 (NH2), 1579, 1498, 1362, 1294, 1247, 1183, 1096, 1043, 954, 879, 812, 772, 739, 690, 627. 1H NMR (300.1 MHz, DMSO-d6): δ 7.98 (s, 1H, CH), 7.86 (d, J = 8.9 Hz, 2H, 2CH), 7.77 (d, J = 8.5 Hz, 2H, 2CH), 7.61 (d, J = 8.5 Hz, 2H, 2CH), 6.98 (d, J = 8.9 Hz, 2H, 2CH), 6.50 (s, 1H, CH), 4.22 (s, 2H, NH2), 3.74 (s, 3H, OCH3). 13C NMR (75.5 MHz, DMSO-d6): δ 160.6, 157.3, 149.5, 144.8, 137.3 and 135.3 (6C), 132.7 (2CH), 131.3 (2CH), 131.0 (C), 130.1 (CH), 129.3 and 128.8 (2C), 128.6 (2CH), 127.4, 126.3 and 125.5 (3C), 119.9 (CH), 114.1 (2CH), 55.3 (OCH3).
7,8-Dichloro-2,4-bis-(4-chloro-phenyl)-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3ad):
Yellow solid, yield: 82%, mp 308–310 °C. IR (KBr) (νmax/cm–1): 3435 and 3384 (NH2), 1505, 1461, 1370, 1287, 1123, 1090, 965, 835, 791, 725, 646, 634. 1H NMR (300.1 MHz, DMSO-d6): δ 8.25 (s, 1H, CH), 8.03 (d, J = 8.5 Hz, 2H, 2CH), 7.93 (d, J = 8.4 Hz, 2H, 2CH), 7.82 (d, J = 8.5 Hz, 2H, 2CH), 7.72 (d, J = 8.4 Hz, 2H, 2CH), 6.24 (s, 1H, CH), 4.30 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 157.6, 149.5, 144.7, 137.3 and 135.7 (5C), 132.1 (2CH), 131.6 (2CH), 131.3 (C), 130.6 (CH), 129.9 (2CH), 129.5, 128.8 and 128.6 (3C), 128.4 (2CH), 127.2, 126.3 and 125.6 (3C), 119.9 (CH). EI-MS, m/z (%): 480 (M+, 100), 370 (37), 250 (45), 112 (67), 78 (19), 51 (25).
7,8-Dichloro-2-(4-chloro-phenyl)-4-thiophen-2-yl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3ae):
Yellow solid, yield: 73%, mp 275–278 °C. IR (KBr) (νmax/cm–1): 3489 and 3347 (NH2), 1585, 1522, 1486, 1448, 1346, 1340, 1288, 1173, 1062, 952, 949, 858, 774, 701, 646. 1H NMR (300.1 MHz, DMSO-d6): δ 8.15 (d, J = 3.1 Hz, 1H, CH), 7.95–7.82 (m, 4H, 4CH), 7.79 (s, 1H, CH), 7.76 (d, J = 2.0 Hz, 1H, CH), 6.11 (s, 1H, CH), 4.47 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 157.3, 150.8, 148.8, 144.1, 141.1 and 136.1 (6C), 132.4 (CH), 132.0 (2CH), 131.3 (CH), 130.6 (2CH), 130.3, 128.7, 127.7 and 127.2 (4C), 126.33 (CH), 126.25 (C), 120.6 and 114.5 (2CH).
2-(4-Bromo-phenyl)-7,8-dichloro-4-phenyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3af):
Yellow solid, yield: 84%, mp 293–296 °C. IR (KBr) (νmax/cm–1): 3487 and 3328 (NH2), 1588, 1521, 1487, 1341, 1310, 1221, 1168, 1072, 978, 924, 851, 767, 697, 642. 1H NMR (300.1 MHz, DMSO-d6): δ 8.09 (d, J = 8.6 Hz, 2H, 2CH), 8.01 (s, 1H, CH), 7.74–7.64 (m, 7H, 7CH), 6.13 (s, 1H, CH), 4.29 (s, 2H, NH2). 13C NMR (75.5 MHz, DMSO-d6): δ 157.4, 149.4, 144.5, 136.6 and 135.4 (5C), 131.7 (2CH), 131.6 (C), 131.1 (2CH), 130.7 (CH), 129.8 (2CH), 129.7 (C), 129.5 (2CH), 128.9 (CH), 127.5, 127.3, 126.5 and 123.8 (4C), 119.9 (CH).
2-(4-Bromo-phenyl)-7,8-dichloro-4-p-tolyl-benzo[4,5]imidazo[1,2-a]pyrimidin-3-ylamine (3ag):
Yellow solid, yield: 76%, mp 306–308 °C. IR (KBr) (νmax/cm–1): 3467 and 3343 (NH2), 1578, 1483, 1353, 1279, 1204, 1149, 1092, 963, 876, 832, 775, 743, 663. 1H NMR (300.1 MHz, DMSO): δ 8.05 (d, J = 8.6 Hz, 2H, 2CH), 7.95 (s, 1H, CH), 7.68 (d, J = 8.6 Hz, 2H, 2CH), 7.58 (d, J = 8.0 Hz, 2H, 2CH), 7.50 (d, J = 8.0 Hz, 2H, 2CH), 6.22 (s, 1H, CH), 4.26 (s, 2H, NH2), 2.48 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO-d6): δ 157.3, 149.4, 144.4, 136.84, 136.78 and 135.3 (6C), 131.7 (2CH), 131.6 (CH), 131.1 (2CH), 130.8 (C), 130.0 (2CH), 129.7 (2CH), 129.5, 127.6, 126.5, 125.9 and 123.8 (5C), 119.9 (CH), 21.2 (CH3). EI-MS, m/z (%): 496 (M+, 100), 342 (25), 326 (74), 263 (14), 167 (48), 133 (25), 79 (33), 51 (18).
3-Chloro-2,4-diphenyl-benzo[4,5]imidazo[1,2-a]pyrimidine (4a):
Yellow solid, yield: 56%, mp 228–229 °C. IR (KBr) (νmax/cm–1): 1578, 1529, 1474, 1437, 1376, 1297, 1251, 1214, 1175, 1091, 1026, 954, 908, 834, 805, 728, 637. 1H NMR (300.1 MHz, DMSO): δ 7.64 (dd, J = 1.6, 7.2 Hz, 2H, 2CH), 7.45–7.20 (m, 9H, 9CH), 7.00 (t, J = 7.8 Hz, 1H, CH), 6.86 (t, J = 7.6 Hz, 1H, CH), 6.32 (d, J = 8.1 Hz, 1H, CH). 13C NMR (75.5 MHz, DMSO-d6): δ 156.7, 143.1, 136.8, 135.3 and 131.2 (5C), 130.6 (CH), 130.3 (2CH), 129.5 (CH), 129.4 (2×2CH), 128.7 (2CH), 128.5 (C), 127.8 (CH), 126.2 and 126.1 (2C), 124.9, 120.2 and 119.2 (3CH). EI-MS, m/z (%): 355 (M+, 100), 204 (48), 145 (52), 167 (75), 77 (32), 51 (25).
2,4-Diphenyl-benzo[4,5]imidazo[1,2-a]pyrimidine (6a):
Yellow solid, yield: 65%, mp 206–208 °C. IR (KBr) (νmax/cm–1): 1603, 1505, 1461, 1370, 1302, 1250, 1187, 1090, 1048, 1006, 965, 897, 834, 785, 732, 634. 1H NMR (300.1 MHz, DMSO): δ 8.40 (d, J = 8.0 Hz, 2H, 2CH), 7.90–7.35 (m, 9H, 9CH), 7.29 (s, 1H, CH), 7.05 (t, J = 7.8 Hz, 1H, CH), 6.93 (t, J = 7.5 Hz, 1H, CH), 6.53 (d, J = 7.9 Hz, 1H, CH). 13C NMR (75.5 MHz, DMSO-d6): δ 159.9, 152.4, 144.5, 138.6, 136.4 and 131.8 (6C), 130.8 and 130.4 (2CH), 130.2 (2CH), 130.0 (2CH), 129.7 (2CH), 128.6 (CH), 128.2 (2CH), 127.8 and 126.9 (2C), 125.4, 120.4, 118.7 and 108.5 (4CH). EI-MS, m/z (%): 321 (M+, 100), 244 (18), 167 (25), 79 (65), 52 (45).
α-glucosidase inhibition assay
α-Glucosidase enzyme ((EC3.2.1.20, Saccharomyces cerevisiae, 20 U/mg) and substrate (p-nitrophenyl glucopyranoside) were purchased from Sigma-Aldrich. Enzyme was prepared in potassium phosphate buffer (pH 6.8, 50 mM), and ploy-substituted-2,4-diarylbenzo[4,5]imidazo[1,2-a]pyrimidines 3a-ag, 4a, and 6a was dissolved in DMSO (10% final concentration). The various concentrations of these compounds (20 mL), enzyme solution (20 mL) and potassium phosphate buffer (135 mL), were added in the 96-well plate and incubated at 37 °C for 10 min. Then, the substrate (25 mL, 4 mM) was added to the mentioned mixture and allowed to incubate at 37 °C for 20 min. Finally, the change in absorbance was measured at 405 nm by using spectrophotometer (Gen5, Power wave xs2, BioTek, America). DMSO (10% final concentration) and acarbose were used respectively as control and standard drug. The percentage of enzyme inhibition was calculated and IC50 values were obtained from non-linear regression curve using the Logit method67.
Kinetic studies
The kinetic analysis was carried out to determine inhibition mode of most potent compound 3k. The 20 mL of enzyme solution (1 U/mL) was incubated with different concentrations (0, 45, 65, and 80 mM) of compound 3k for 15 min at 30 °C. The reaction was then started by adding different concentrations of substrate (p-nitrophenyl glucopyranoside,1-4 mM), and change in absorbance was measured for 20 min at 405 nm by using spectrophotometer (Gen5, Power wave xs2, BioTek, America).
Molecular docking study
Since the x-ray crystallographic structure S. cerevisiae α-glucosidase isn’t accessible, the 3D structure of S.cerevisiae isomaltase with PDB ID: 3A4A was downloaded from RCSB web site with 84% similarity to S. cerevisiae α-glucosidase15.
Docking studies were performed using Auto Dock Tools (version1.5.6), and the pdb structure of 3A4A was taken from theBrookhaven protein database (http://www.rcsb.org) as a complex bound with α-D-glucose. The 3D structures of the selected compounds were created by MarvineSketch 5.8.3, 2012, ChemAxon (http://www.chemaxon.com) and converted to pdbqt coordinate using Auto dock Tools.. Before preparation of auto dock format of protein, the water molecules and the inhibitors were removed from it. Then, using Auto Dock Tools, polar hydrogen atoms were added, Kollman charges were assigned, and the obtained enzyme structure was used as an input file for the AUTOGRID program. In AUTOGRID for each atom type in the ligand, maps were calculated with 0.375 A spacing between grid points, and the center of the grid box was placed at x = 22.625, y = -8.069, and z = 24.158. The dimensions of the active site box were set at 50 × 50 × 50 A. Each docked system was carried out by 50 runs of the AUTODOCK search by the Lamarckian genetic algorithm. The best pose of each ligand was selected for analyzing the interactions between α-glucosidase and the inhibitor. The results were visualized using Discovery Studio 4.0 Client and LigPlot (Figure 3–6).