Materials and methods
The required synthetic chemicals were purchased from Merck Chemicals (India) and utilized without further purification. The reagents for the antimicrobial evaluation and for the cytotoxicity study were procured from Hi-Media Laboratories (India). The microbial strains were acquired from Institute of Microbial Technology and Genebank (IMTECH), Chandigarh. The IR spectra was recorded on the Bruker 12060280 FTIR spectrophotometer using KBr pellet method. The Bruker Avance II 400 NMR spectrometer was used for carried out the NMR spectroscopy (1H NMR and 13C NMR), for the synthesized derivatives in deuterated DMSO solvent. The structures of the synthesized derivatives were confirmed by mass spectra, taken on the Advion expression CMS, USA mass spectrometer with APCI mode as the ion source.
Synthetic procedure for indole diazenyl Schiff bases
The p-aminoacetophenone (0.01 mol, 1.35 g) was dissolved in 10 ml solution of 0.2 N HCl followed by addition of 5 ml solution of sodium nitrite (0.01 mol) at 0–5 °C to complete the diazotization process. The indole/5-nitroindole (0.01 mol) was solubilized in 20 ml of acetic/propionic acid (8:2) mixture and cooled at 0 °C. The diazotized solution was added to the indole derivative gradually at 0 °C over a period of 10–15 min followed by stirring for 1–2 h in the cold conditions. Afterwards saturated sodium carbonate solution (20–25 ml) was added to precipitate the azo dye (1, 2) with continuous stirring for further half an hour [38, 39]. The precipitated azo dye was filtered, vacuum dried and recrystallized from ethanol and used further for the synthesis of indole diazenyl Schiff bases. In 250 ml round bottom flask, indole azo dye (0.001 M) was refluxed with aromatic/heteroaromatic amine (0.001 M) in the presence of ethanol and catalytic amount of acid on a heating mantle. The refluxing was continued for 7–8 h until the reaction completion was confirmed by TLC. The reaction volume was concentrated and kept at 7–8 °C for 24 h for the precipitation of Schiff bases. The synthesized Schiff bases were purified by recrystallization and column chromatography using ethyl acetate: methanol (70:30) on silica gel 100–200 mesh size [27, 40].
Analytical data
1-[4-[(E)-1H-Indol-3-ylazo]phenyl]ethanone (1): MF: C16H13N3O; 263.29; Maroon color; Yield: 92%; mp: 90–95 °C: IR (KBr, cm−1) νmax: 3217, 2921, 1668, 1592, 1460, 1424, 1358, 1266, 1215, 1169, 1053, 1014, 959, 837, 748, 624, 594, 446; 1H NMR (400 MHz, DMSO-d6) δ: 11.96 (s, 1H, –NH– of indole), 9.01 (s, 1H, –CH(2) of indole), 8.16 (d, J = 2.4 Hz, 1H, –CH(4) of indole), 8.01 (d, J1= 8.8 Hz, 2H, ArH), 7.51–7.76 (m, 2H, –CH(6,7) of indole), 7.32 (d, J = 8.8 Hz, 2H, ArH), 7.21–7.26 (m, 1H, –CH(5) of indole), 2.18 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 198.2, 148.8, 140.1, 138.7, 133.7, 128.5, 124.8, 122.9, 117.6, 117.4, 116.7, 115.5, 113.4, 112.3, 26.2.
1-[4-[(E)-(5-Nitro-1H-indol-3-yl)azo]phenyl]ethanone (2): MF: C16H12N4O3; 308.29; Orange color; Yield: 69%; mp: 65–70 °C; IR (KBr, cm−1) νmax: 3328, 2925, 1707, 1669, 1617, 1517, 1468, 1426, 1329, 1167, 1108, 1065, 894, 826, 780, 743, 682, 588, 540, 432; 1H NMR (400 MHz, DMSO-d6) δ: 12.01 (s, 1H, –NH– of indole), 9.97 (s, 1H, –CH(2) of indole), 8.35–8.47 (m, 1H, –CH(4) of indole), 8.22 (d, J = 2.4 Hz, 1H, –CH(7) of indole), 7.98 (dd, J1= 8.8 Hz, J2= 2.4 Hz, 1H, –CH(6) of indole), 7.56–7.63 (m, 2H, ArH), 7.11 (d, J = 8.8 Hz, 2H, ArH), 2.09 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 198.6, 162.9, 141.0, 140.8, 138.81, 133.8, 128.5, 124.8, 122.8, 117.5, 117.4, 116.7, 115.1, 113.2, 112.3, 26.3.
(E)-N-(2-Furylmethyl)-1-[4-[(E)-1H-indol-3-ylazo]phenyl]ethanimine (DS-1): Dark Maroon; Yield: 75%; Rf = 0.57 (ethyl acetate/methanol 8:2); mp: 315–320 °C; IR (KBr, cm−1) νmax: 3297, 3055, 2922, 1641, 1596, 1401, 1355, 1264, 1158, 1011, 958, 884, 837, 744, 593, 498, 431; 1H NMR (400 MHz, DMSO-d6) δ: 11.19 (s, 1H, –NH– of indole), 9.02 (s, 1H, –CH(2) of indole), 8.38 (d, J = 8.0 Hz, 1H, –CH (4) of indole), 8.09 (d, J = 8.4 Hz, 2H, ArH), 7.8–8.02 (m, 1H, –CH(7) of indole), 7.63 (d, J = 8.4 Hz, 2H, ArH), 7.36–7.59 (m, 2H, –CH(5,6) of indole), 7.02 (d, J = 7.2 Hz, 1H, –CH(5) of furan), 6.84 (d, J = 7.2 Hz, 1H, –CH(4) of furan), 6.37 (d, J = 7.2 Hz, 1H, –CH(3) of furan), 4.23 (s, 2H, CH2), 1.89 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 170.2, 155.5, 152.2, 147.6, 140.9, 137.6, 135.2, 125.7, 126.5, 122.3, 121.9, 121.8, 121.0, 120.5, 117.3, 115.2, 109.1, 56.2, 23.5; APCI-MS m/z found for C21H18N4O: 342.39 (M+); Anal. calcd for C21H18N4O: C 73.67, H 5.30, N 16.36, O 4.67 found C 73.69, H 5.35, N 16.39, O 4.65.
Ethyl 2-((Z)-(1-(4-((Z)-(1H-indol-3-yl)diazenyl)phenyl)ethylidene)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (DS-2): Dark Maroon; Yield: 79%; mp: 260–265 °C; Rf = 0.49 (ethyl acetate/methanol 8:2); IR (KBr, cm−1) νmax: 3404, 3298, 3169, 2982, 2934, 2847, 1721, 1646, 1593, 1488, 1413, 1373, 1335, 1278, 1149, 1024, 967, 894, 836, 744, 601, 510, 469, 432; 1H NMR (400 MHz, DMSO-d6) δ: 11.29 (s, 1H, –NH– of indole), 8.92 (s, 1H, –CH(2) of indole), 8.10 (d, J = 8.8 Hz, 2H, ArH), 7.95 (d, J = 8.8 Hz, 2H, ArH), 7.41 (d, J = 7.6 Hz, 1H, –CH(4) of indole), 7.25–7.35 (m, 1H, –CH(7) of indole), 7.04–7.09 (m, 1H, –CH(5) of indole), 6.84 (d, J = 4.0 Hz, 1H, –CH(6) of indole), 4.13 (q, J = 9.6 Hz, 2H, –OCH2), 2.74–2.82 (m, 4H, CH2), 1.90 (s, 3H, CH3), 1.44–1.84 (m, 4H, CH2), 1.23 (t, J = 9.6 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 175.3, 169.6, 160.6, 155.3, 142.2, 139.1, 137.5, 135.3, 132.5, 129.2, 127.5, 125.4, 123.6, 123.0, 122.2, 121.4, 120.2, 115.7, 61.8, 26.5, 26.3, 24.4, 23.6, 22.5, 14.3; APCI-MS m/z found for C27H26N4O2S: 470.18 (M+); Anal. calcd for C27H26N4O2S: C 68.91, H 5.57, N 11.91, O 6.80, S 6.81 found C 68.95, H 5.54, N 11.88, O 6.82.
(Z)-5-(2-Chloro-4-nitrophenyl)-N-(1-(4-((Z)-(5-nitro-1H-indol-3-yl)diazenyl)phenyl) ethylidene)-1,3,4-thiadiazol-2-amine (DS-3): Orange; Yield: 68%; mp: 155–160 °C; Rf = 0.61 (ethyl acetate/methanol 7:3); IR (KBr, cm−1) νmax: 3347, 3107, 2924, 1707, 1669, 1617, 1519, 1467, 1332, 1221, 1143, 1062, 963, 915, 827, 782, 740, 684, 656, 590, 535; 1H NMR (400 MHz, DMSO-d6) δ: 12.65 (s, 1H, –NH– of indole), 9.37 (s, 1H, –CH(2) of indole), 8.75 (d, J = 2.4 Hz, 1H, –CH(4) of indole), 8.62 (s, 1H, CH–C–Cl), 8.34 (dd, J1= 8.4 Hz, J2= 2.4 Hz, 1H, –CH=C–NO2), 8.24 (dd, J1= 8.4 Hz, J2= 2.8 Hz, 1H, –CH(6) of phenyl), 8.15 (dd, J1= 9.2 Hz, J2= 2.4 Hz, 1H, CH(6) of indole), 7.91 (d, J = 2.4 Hz, 1H, –CH(7) of indole), 7.55–7.75 (m, 4H, ArH), 2.07 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 180.2, 173.2, 155.3, 148.5, 139.7, 139.5, 138.5, 137.5, 135.2, 132.2, 130.6, 129.5, 125.5, 124.4, 121.8, 121.7, 121.0, 117.7, 114.3, 23.6; APCI-MS m/z found for C24H15ClN8O4S: 546.94 (M+); Anal. calcd for C24H15ClN8O4S: C 52.70, H 2.76, Cl 6.48, N 20.49, O 11.70 S 5.86 found C 52.72, H 2.78, N 20.53, O 11.73.
(Z)-N-(1-(4-((Z)-(5-Nitro-1H-indol-3-yl)diazenyl)phenyl)ethylidene)-5-(4-nitrophenyl)-1,3,4-thiadiazol-2-amine (DS-5): Orange; Yield: 57%; mp: 160–165 °C; Rf = 0.55 (ethyl acetate/methanol 7:3); IR (KBr, cm−1) νmax: 3359, 2923, 2854, 1831, 1706, 1617, 1518, 1467, 1332, 1219, 1169, 1112, 1062, 929, 850, 823, 783, 742, 686, 593, 533, 449, 430; 1H NMR (400 MHz, DMSO-d6) δ: 11.92 (s, 1H, –NH– of indole), 9.89 (s, 1H, –CH(2) of indole), 8.50 (s, 1H, –CH(4) of indole), 8.32–8.42 (m, 2H, ArH), 8.14–8.15 (m, 2H, ArH), 8.13 (d, J = 2.4 Hz, 1H, –CH(7) of indole), 7.87–7.94 (m, 2H, ArH), 7.45–7.56 (m, 2H, ArH), 7.03 (d, J = 9.2 Hz, 1H, ArH), 2.02 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 180.1, 170.1, 151.2, 147.2, 143.8, 139.9, 139.8, 136.9, 135.4, 130.5, 128.9, 128.6, 124.6, 124.4, 122.6, 121.8, 120.9, 117.9, 114.3, 49.3, 23.6; APCI-MS m/z found for C24H16N8O4S: 512.5 (M+); Anal. calcd for C24H16N8O4S: C 56.25, H 3.15, N 21.86, O 12.49, S 6.26 found C 56.28, H 3.14, N 21.89, O 12.52.
1,5-Dimethyl-4-((Z)-(1-(4-((Z)-(5-nitro-1H-indol-3-yl)diazenyl)phenyl)ethylidene) amino)-2-phenyl-1H-pyrazol-3(2H)-one (DS-6): Dark Yellow; Yield: 64%; Rf = 0.51 (ethyl acetate/methanol 8:2); mp: 170–175 °C; IR (KBr, cm−1) νmax: 3348, 3124, 2924, 1705, 1619, 1518, 1468, 1425, 1329, 1252, 1167, 1112, 1062, 927, 895, 823, 785, 741, 688, 656, 591, 538, 448; 1H NMR (400 MHz, DMSO-d6) δ: 11.99 (s, 1H, –NH of indole), 9.97 (s, 1H, –CH(2) of indole), 8.57 (d, J = 2.0 Hz, 1H, –CH(4) of indole), 8.43 (dd, J1 = 9.2 Hz, J2= 2.4 Hz, 1H, –CH(6) of indole), 8.37 (d, J = 2.4 Hz, 1H, –CH(7) of indole), 8.22 (d, J = 8.4 Hz, 2H, ArH), 7.98 (dd, J1 = 9.2 Hz, J2= 2.4 Hz, 1H, ArH), 7.58–7.63 (m, 2H, ArH), 7.11 (d, J = 9.2 Hz, 1H, ArH), 6.73–6.74 (m, 1H, ArH), 2.11 (s, 3H, CH3), 1.98 (s, 3H, CH3), 1.92 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 162.2, 161.9, 151.2, 147.5, 139.9, 139.8, 139.4, 135.5, 132.1, 130.5, 129.1, 126.8, 126.4, 124.6, 124.3, 124.2, 121.9, 120.9, 118.1, 114.3, 36.2, 22.6, 13.9; APCI-MS m/z found for C27H23N7O3: 493.52 (M+); Anal. calcd for C27H23N7O3: C 65.71, H 4.70, N 19.87, O 9.73 found C 65.75, H 4.67 N 19.89, O 9.69.
(Z)-N-(1-(4-((Z)-(1H-Indol-3-yl)diazenyl)phenyl)ethylidene)-5-ethyl-1,3,4-thiadiazol-2-amine (DS-7): Dark Maroon; Yield: 70%; mp: 145–150 °C; Rf = 0.47 (ethyl acetate/methanol 7:3); IR (KBr, cm−1) νmax: 3262, 3050, 2977, 2921, 2751, 1684, 1588, 1491, 1455, 1419, 1371, 1304, 1268, 1197, 1107, 1013, 960, 922, 834, 801, 745, 701, 597, 458, 429; 1H NMR (400 MHz, DMSO-d6) δ: 12.28 (s, 1H, –NH of indole), 7.04–7.91 (m, 8H, ArH), 2.96 (q, J = 7.6 Hz, 2H, –CH2), 1.89 (s, 3H, –CH3) 1.27 (t, J = 7.6 Hz, 3H, –CH3); 13C NMR (100 MHz, DMSO-d6) δ: 175.3, 169.2, 164.4, 154.0, 138.5, 136.2, 135.3, 132.0, 130.1, 128.8, 126.1, 125.2, 123.1, 121.6, 113.2, 23.9, 23.9, 13.1; APCI-MS m/z found for C20H18N6S: 374.46 (M+); Anal. calcd for C20H18N6S: C 64.15, H 4.85, N 22.44, S 8.56 found C 64.14, H 4.81 N 22.47, O 8.59.
(Z)-2,5-Dichloro-N-(1-(4-((Z)-(5-nitro-1H-indol-3-yl)diazenyl)phenyl)ethylidene)aniline (DS-10): Orange; Yield: 72% mp: 210–215 °C; Rf = 0.64 (ethyl acetate/methanol 7:3); IR (KBr, cm−1) νmax: 3347, 2923, 2854, 1704, 1619, 1517, 1468, 1328, 1252, 1167, 1113, 1062, 927, 895, 823, 785, 740, 687, 656, 590, 536, 449; 1H NMR (400 MHz, DMSO-d6) δ: 11.99 (s, 1H, –NH of indole), 9.97 (s, 1H, –CH(2) of indole), 8.43 (dd, J1 = 9.2 Hz, J2= 2.4 Hz, 1H, –CH(6) of indole), 8.37 (d, J = 2.4 Hz, 1H, –CH(7) of indole), 8.22 (d, J = 2.4 Hz, 1H, –CH(4) of indole), 7.98 (dd, J1 = 9.2 Hz, J2= 2.4 Hz, 1H, –CH=C–Cl), 7.58–7.63 (m, 2H, ArH), 7.18 (d, J = 8.4 Hz, 2H, ArH), 7.12 (d, J = 9.2 Hz, 1H, ArH), 6.81 (d, J = 2.4 Hz, 1H, ArH), 1.91 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 169.6, 158.7, 149.2, 140.2, 138.6, 138.0, 135.2, 132.7, 130.6, 129.7, 128.6, 128.8, 127.7, 126.6, 124.2, 123.2, 122.6, 121.6, 119.5, 115.3, 23.2; APCI-MS m/z found for C22H15Cl2N5O2: 452.3 (M+); Anal. calcd for C22H15Cl2N5O2: C 58.42, H 3.34 Cl 15.68, N 15.48, O 7.07 found C 58.45, H 3.37 N 15.51, O 7.06.
4-((Z)-(1-(4-((Z)-(1H-Indol-3-yl)diazenyl)phenyl)ethylidene)amino)-N-(pyridin-2-yl)benzenesulfonamide (DS-11): Maroon; Yield: 68% mp: 135–140 °C; Rf = 0.57 (ethyl acetate/methanol 7:3); IR (KBr, cm−1) νmax: 3221, 3055, 2924, 1677, 1623, 1593, 1530, 1495, 1459, 1386, 1331, 1245, 1130, 1083, 1007, 956, 830, 747, 676, 612, 564, 451; 1H NMR (400 MHz, DMSO-d6) δ: 11.84 (s, 1H, –NH of indole), 10.92 (s, 1H, –CH(2) of indole), 8.08 (d, J = 4.0 Hz, 2H, ArH), 7.60–7.65 (m, 3H, ArH), 7.48–7.52 (m, 3H, ArH), 7.05 (d, J = 8.4 Hz, 2H, ArH), 6.98–7.10 (m, 2H, –CH (5,6) of indole), 6.87–6.90 (m, 2H), 6.52–6.55 (m, 2H, ArH), 5.91–5.98 (m, 1H, –SO2NH), 1.89 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 169.8, 156.1, 155.2, 148.4, 137.6, 137.6, 136.5, 135.4, 133.2, 130.0, 128.3, 127.8, 123.6, 123.1, 123.1, 121.8, 120.9, 120.1, 117.8, 114.5, 112.5, 22.8; APCI-MS m/z found for C27H22N6O2S: 494.56 (M+); Anal. calcd for C27H22N6O2S: C 65.57, H 4.48, N 16.99, O 6.47 S 6.48 found C 65.59, H 4.49 N 16.96, O 6.51.
(Z)-N-(1-(4-((Z)-(1H-Indol-3-yl)diazenyl)phenyl)ethylidene)-4-chloro-2-nitroaniline (DS-13): Maroon; Yield: 65%; Rf = 0.49 (ethyl acetate/methanol 8:2); IR (KBr, cm−1) νmax: 3474, 3355, 3096, 3056, 2963, 2922, 2857, 1632, 1596, 1563, 1502, 1455, 1406, 1339, 1246, 1117, 1013, 959, 887, 817, 744, 645, 591, 521, 466, 416; 1H NMR (400 MHz, DMSO-d6) δ: 11.99 (s, 1H, –NH of indole), 9.97 (s, 1H, –CH(2) of indole), 8.43 (dd, J1 = 9.2 Hz, J2= 2.4 Hz, 1H, –CH–C–Cl), 8.37 (d, J = 2.0 Hz, 1H, –CH(4) of indole), 8.22 (d, J = 2.4 Hz, 1H, –CH(7) of indole), 7.98 (dd, J1 = 9.2 Hz, J2= 2.4 Hz, 1H, ArH), 7.58–7.63 (m, 2H, ArH), 7.18 (d, J = 8.4 Hz, 2H, ArH), 7.12 (d, J = 9.2 Hz, 1H, ArH), 6.81 (d, J = 2.4 Hz, 1H, –CH(5) of indole), 6.53 (dd, J1= 8.4 Hz, J2= 2.4 Hz, –CH(6) of indole), 1.91 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 169.3, 158.3, 149.3, 145.8, 140.7, 136.5, 135.3, 135.1, 134.2, 132.4, 131.2, 129.6, 127.5, 126.5, 125.1, 122.5, 121.7, 119.3, 115.4; APCI-MS m/z found for C22H16ClN5O2: 417.85 (M+); Anal. calcd for C22H16ClN5O2: C 63.24, H 3.86 Cl 8.48, N 16.76, O 7.66 found C 63.27, H 3.89 N 16.79, O 7.68.
4-((Z)-(1-(4-((Z)-(1H-Indol-3-yl)diazenyl)phenyl)ethylidene)amino)benzoic acid (DS-14): Maroon; Yield: 74%; mp: 205–210 °C; Rf = 0.71 (ethyl acetate/methanol 8:2); IR (KBr, cm−1) νmax: 3391, 2924, 1676, 1601, 1529, 1458, 1381, 1249, 1172, 1108, 1043, 1014, 962, 746, 604, 456, 416; 1H NMR (400 MHz, DMSO-d6) δ: 11.14 (s, 1H, –NH– of indole), 10.24 (s, 1H, –COOH), 9.12 (s, 1H, –CH(2) of indole), 7.87 (d, J = 8.8 Hz, 2H, ArH), 7.67 (d, J = 7.6 Hz, 1H, –CH(4) of indole), 7.37 (d, J = 8.0 Hz, 2H, ArH), 7.14–7.28 (m, 2H, ArH), 7.01–7.05 (m, 2H, ArH), 6.78–6.89 (m, 2H, –CH(5,6) of indole), 1.97 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 173.9, 169.5, 155.3, 152.4, 140.3, 138.2, 136.9, 135.4, 133.0, 129.2, 128.1, 126.7, 123.2, 122.3, 120.9, 119.4, 118.5, 115.2; APCI-MS m/z found for C23H18N4O2: 382.41 (M+); Anal. calcd for C23H18N4O2: C 72.24, H 4.74, N 14.65, O 8.37 found C 72.26, H 4.78 N 14.61, O 8.41.
N-((4-((Z)-(1-(4-((Z)-(5-Nitro-1H-indol-3-yl)diazenyl)phenyl)ethylidene)amino)phenyl) sulfonyl)acetamide (DS-15): Dark Maroon color; Yield: 64%; mp: 110–115 °C; Rf = 0.64 (ethyl acetate/methanol 8:2); IR (KBr, cm−1) νmax: 1685, 1620, 1509, 1512, 1458, 1419, 1373, 1319, 1242, 1188, 1130, 1067, 1033, 829, 740, 686, 624, 547; 1H NMR (400 MHz, DMSO-d6) δ: 11.96 (s, 1H, –NH– of indole), 9.93 (s, 1H, –CH(2) of indole), 8.56 (s, 1H, –NH– of sulfonamide), 8.41 (dd, J1 = 9.2 Hz, J2= 2.4 Hz, 1H, –CH(6) of indole), 8.35 (d, J = 2.8 Hz, 1H, –CH(4) of indole), 8.21 (d, J = 2.0 Hz, 1H, –CH(7) of indole), 7.96 (dd, J1 = 9.2 Hz, J2= 2.4 Hz, 2H, ArH), 7.49–7.59 (m, 2H, ArH), 7.10 (d, J = 9.2 Hz, 2H, ArH), 6.58–6.98 (m, 2H, ArH), 1.89 (s, 3H, CH3), 1.85 (s, 3H, COCH3); 13C NMR (100 MHz, DMSO-d6) δ: 173.9, 160.2, 158.3, 143.1, 140.2, 135.1, 130.3, 129.2, 126.6, 125.2, 123.6, 122.7, 120.4, 116.5, 23.2; APCI-MS m/z found for C24H20N6O5S: 504.51 (M+); Anal. calcd for C24H20N6O5S: C 57.14, H 4.00 Cl12.83, N 16.66, O 15.86, S 6.36 found C 57.17, H 4.03 N 16.68, O 15.81.
(Z)-4-Fluoro-N-(1-(4-((Z)-(5-nitro-1H-indol-3-yl)diazenyl)phenyl)ethylidene)aniline (DS-16): Dark Orange; Yield: 63%; mp: 150–152 °C; Rf = 0.61 (ethyl acetate/methanol 8:2); IR (KBr, cm−1) νmax: 3398, 3056, 2923, 1612, 1596, 1456, 1426, 1338, 1269, 1154, 1013, 962, 924, 883, 816, 743, 596, 449; 1H NMR (400 MHz, DMSO-d6) δ: 11.96 (s, 1H, –NH– of indole), 9.82 (s, 1H, –CH(2) of indole), 8.56 (s, 1H, indole), 8.41 (dd, J1= 9.2 Hz, J2= 2.4 Hz, 2H, ArH), 8.35 (d, J = 2.4 Hz, 1H), 8.20 (d, J = 2.4 Hz, 1H, indole), 8.11 (d, J = 7.6 Hz, 1H, ArH), 7.95 (dd, J1= 9.2 Hz, J2= 2.4 Hz, 2H, ArH), 7.53–7.59 (m, 2H, ArH), 7.11 (d, J = 9.2 Hz, 1H, ArH), 1.89 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 169.7 166.3, 163.2, 156.4, 148.2, 145.8, 142.2, 140.3, 137.5, 132.6, 129.4, 127.7, 126.0, 124.6, 123.6, 122.8, 121.6, 120.2, 119.2, 116.5, 115.4, 114.2, 23.7; APCI-MS m/z found for C22H16FN5O2: 401.39 (M+); Anal. calcd for C22H16FN5O2: C 65.83, H 4.02 F 4.73, N 17.45, O 7.97 found C 65.85, H 4.06 N 17.43, O 7.98.
(Z)-N-(1-(4-((Z)-(1H-Indol-3-yl)diazenyl)phenyl)ethylidene)pyrimidin-2-amine (DS-17): Maroon; Yield: 68%; mp: 275–280 °C; Rf = 0.57 (ethyl acetate/methanol 8:2); Rf = 0.54 (ethyl acetate/methanol 8:2); IR (KBr, cm−1) νmax: 3386, 1624, 1575, 1465, 1384, 1353, 1268, 1221, 1160, 801, 746, 653, 523, 421; 1H NMR (400 MHz, DMSO-d6) δ: 11.53 (s, 1H, –NH– of indole), 8.54 (s, 1H, –CH(2) of indole), 8.19 (d, J = 4.8 Hz, 2H, ArH), 6.82–7.95 (m, 7H, ArH), 6.51–6.53 (m, 2H, ArH), 1.89 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 171.9, 167.4, 160.2, 155.2, 143.2, 137.4, 136.7, 132.8, 130.6, 128.5, 126.4, 125.0, 123.2, 122.3, 120.3, 116.2, 23.1; APCI-MS m/z found for C20H16N6: 340.38 (M+); Anal. calcd for C20H16N6: C 70.57, H 4.74 N 24.69 found C 70.59, H 4.78 N 24.72.
4-((Z)-(1-(4-((Z)-(1H-Indol-3-yl)diazenyl)phenyl)ethylidene)amino)-N-(pyrimidin-2-yl) benzenesulfonamide (DS-19): Orange, Yield: 71%; Rf = 0.67 (ethyl acetate/methanol 8:2); IR (KBr, cm−1) νmax: 3422, 3358, 3106, 3038, 2935, 2870, 2811, 2736, 1706, 1585, 1494, 1440, 1410, 1384, 1328, 1260, 1152, 1091, 941, 834, 799, 741, 683, 569, 452, 416; 1H NMR (400 MHz, DMSO-d6) δ: 11.96 (s, 1H, –NH– of indole), 9.20 (s, 1H, –CH(2) of indole), 8.57 (s, 1H), 8.46 (d, J = 4.8 Hz, 2H, –CH of pyrimidine), 8.21 (d, J = 4.8 Hz, 2H, ArH), 7.94–7.97 (m, 2H, ArH), 7.58–7.61 (m, 3H, ArH), 6.98–7.1 (m, 3H, ArH), 6.54–6.56 (m, 3H, ArH), 5.98 (s, 1H, –NH), 1.89 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 169.7, 160.7, 158.3, 155.0, 153.2, 141.3, 139.6, 137.2, 135.6, 131.2, 128.3, 127.4, 126.2, 124.2, 122.2, 121.9, 121.1, 120.8, 116.1, 109.3, 23.1; APCI-MS m/z found for C26H21N7O2S: 495.55 (M+); Anal. calcd for C26H21N7O2S: C 63.02, H 4.27, N 19.79, O 6.44 S 6.47 found C 63.05, H 4.29, N 19.76, O 6.48.
(Z)-N1-(1-(4-((Z)-(1H-Indol-3-yl)diazenyl)phenyl)ethylidene)-N2-(naphthalen-1-yl)ethane-1,2-diamine (DS-20): Maroon; Yield: 72%; mp: 110–115 °C; Rf = 0.42 (ethyl acetate/methanol 8:2); IR (KBr, cm−1) νmax: 3387, 1675, 1582, 1528, 1481, 1407, 1277, 1118, 1018, 959, 753, 571, 438, 418; 1H NMR (400 MHz, DMSO-d6) δ: 11.51 (s, 1H, –NH– of indole), 8.71–8.88 (m, 1H, –CH(2) of indole), 8.24–8.37 (m, 2H, ArH), 8.04–8.10 (m, 1H, –CH of indole), 7.15–7.50 (m, 9H, ArH), 6.66 (d, J = 8.0 Hz, 1H, ArH), 6.53–6.58 (m, 1H, –NH–), 6.23–6.37 (m, 2H, ArH), 3.53–3.56 (m, 2H, CH2), 3.44–3.47 (m, 2H, CH2), 1.89 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 170.4, 159.8, 146.3, 140.0, 139.3, 135.7, 134.1, 132.0, 126.9, 125.6, 124.5, 123.5, 122.3, 122.0, 121.3, 121.1, 120.2, 118.2, 116.2, 114.3, 58.2, 47.5, 25.3; APCI-MS m/z found for C28H25N5: 431.21 (M+); Anal. calcd for C28H25N5: C 77.93, H 5.84 N 16.23, found C 77.95, H 5.81 N 16.27.
(Z)-N-(1-(4-((Z)-(5-Nitro-1H-indol-3-yl)diazenyl)phenyl)ethylidene)benzo[d]thiazol-2-amine (DS-21): Dark Orange; Yield: 69%; mp: 130–135 °C; Rf = 0.46 (ethyl acetate/methanol 8:2); IR (KBr, cm−1) νmax: 3567, 3340, 3119, 2922, 1668, 1618, 1523, 1455, 1388, 1355, 1162, 1121, 1061, 1017, 961, 899, 837, 785, 744, 655, 590, 480; 1H NMR (400 MHz, DMSO-d6) δ: 11.93 (s, 1H, –NH– of indole), 9.19 (s, 1H, CH=C–N–), 8.73 (d, J = 9.2 Hz, 1H, –CH(6) indole), 8.56 (s, 1H, –CH(4) indole), 8.41 (dd, J1 = 9.2 Hz, J2 = 2.4 Hz, 1H, –CH(7) indole), 8.16–8.20 (m, 2H, ArH), 7.41–7.97 (m, 2H, ArH), 7.31 (d, J = 8.0 Hz, 1H, –CH(4) benzothiazole), 7.19 (t, J = 7.2 Hz, 1H, –CH(5)– benzothiazole), 7.10 (d, J = 7.6 Hz, 1H, –CH(7)– benzothiazole), 6.96 (t, J = 7.6 Hz, 1H, –CH(6)– benzothiazole), 1.89 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 176.9, 155.9, 153.2, 148.5, 142.5, 140.6, 138.2, 136.8, 133.6, 130.9, 128.3, 126.9, 126.0, 123.8, 122.7, 121.9, 118.8, 118.7, 111.6, 23.2; APCI-MS m/z found for C23H16N6O2S: 440.47 (M+); Anal. calcd for C23H16N6O2S: C 62.72, H 3.66, N 19.08, O 7.26 S 7.28 found C 62.75, H 3.69 N 19.04, O 7.26.
Antimicrobial evaluation
Determination of minimum inhibitory concentration (MIC)
The synthesized indole diazenyl Schiff bases were screened for antimicrobial activity through tube dilution method as per the reported procedure [41, 42]. The cefotaxime (antibacterial) and fluconazole (antifungal) were selected, as the standard drugs. The standard drugs and the test derivatives were dissolved in DMSO to make the stock solutions of the required concentration of 1000 μg/ml and further serially diluted in nutrient broth (for bacterial strains) and sabouraud dextrose broth (for fungal strains) to get the desired concentrations of (500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.90, 1.95 μg/ml). The each concentration of the test and standard compounds have been supplemented with 100 μl of microbial inoculum to give final inoculum size of 5 * 105 colony forming units (CFU) ml−1 under sterile conditions. The all test tubes with different concentration of the test and standard compounds and microbial strains were incubated for the specified time (for bacterial cultures—24 h at 37 ± 2 °C; fungal cultures—7 days at 25 ± 2 °C).
Determination of minimum bactericidal/fungicidal concentration (MBC/MFC)
After MIC assessment, the indole derivatives (DS1–DS21), were additionally evaluated for MBC and MFC values. To the sterilized petri plates, added 100 µl of culture from each test tube which demonstrated no visible growth in MIC test tubes aseptically. The 10–15 ml of nutrient agar and Sabouraud dextrose agar was added to the petri plates for bacterial and fungal samples respectively with gentle shaking of plates in order to mix the culture throughout the media. Allowed the media to solidify. The petri plates were then incubated for the predefined time and temperature as referenced already for bacterial and fungal cultures respectively. The plates were then investigated visually for the development of microbial growth. The MBC and MFC were stated as the minimum concentration of the compounds in aliquots showing no visual growth after incubation.
Cytotoxicity study
Cell culture
The cell lines used in study were initially procured from the National Centre for Cell Sciences (NCCS), Pune, India, and maintained in DMEM. The cell line was cultured in 25 cm2 tissue culture flask with DMEM supplemented with 10% FBS, sodium bicarbonate, l-glutamine, and antibiotic solution containing: streptomycin (100 μg/ml), penicillin (100 U/ml). Cultured cell line was kept at 37 °C in a humidified 5% CO2 incubator (VWR, USA).
MTT cell proliferation assay
The compounds found to have good antimicrobial potential were then screened for their cytotoxicity using MTT (3,4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) assay [43, 44]. 1 × 104 cells/well were seeded in 100 µl DMEM/MEM, supplemented with 10% FBS in each well of 96-well microculture plates and incubated for 24 h at 37 °C in a CO2 incubator. After incubation, all the prepared/synthesized compounds were added to the cells at 10, 25, 50 and 100 µg concentrations for 48 h. After 48 h of drug treatment, 10 µl MTT (5 mg/ml) was added to each well and the plates were further incubated for 4 h. Then the supernatant from each well was carefully removed, formazan crystals were dissolved in 100 µl of DMSO and absorbance at 570 nm wavelength was recorded on an ELISA reader. The IC50 value was calculated using the linear regression equation i.e. Y = Mx + C. Here, Y = 50, M and C values were derived from the viability graph. The assay was performed in triplicate.
Molecular docking
The novel indole diazenyl Schiff bases were subjected to dock in the active site of DNA gyrase enzyme using Schrodinger Glide software. The 3D-crystal structure of the ATP binding site of E. coli GyrB in complex with pyrimido [4,5-b]indole derivative (PDB ID: 4KFG, resolution 1.6 Å) had been used for the modelling studies and was retrieved from Protein Data Bank (http://www.rcsb.org/pdb/home/home). The target derivatives were investigated for the theoretical binding mode at the ATP binding site to understand the ligand-receptor possible intermolecular interactions in detail using molecular docking modelling. The selected protein structure was prepared using the Protein Preparation Wizard executed in Schrödinger Suite 2018-1. Crystallographic water molecules with fewer than three hydrogen bonds were deleted. Hydrogen atoms were added to the protein structure corresponding to a pH value of 7. The restrained minimization was performed until the heavy atoms RMSD reached a maximum cut-off to 0.30 Å. The active site was defined with a 20 Å radius around the ligand present in the crystal structure and a grid box was generated at the centroid of the active site. Low-energy conformations of all ligands were docked into the catalytic pocket of the 4KFG protein in extra precision mode (Glide, Schrödinger 2018-1) without applying any constraints. The best docked structures were selected based on the Glide score function, Glide energy and Glide energy model [45, 46].
ADME properties
ADME properties were calculated using Qikprop v3.5 tool of Schrödinger. It predicts both physicochemically significant descriptors and pharmacokinetic relevant properties. QikProp provides ranges for comparing a particular molecule’s properties with those of 95% of known drugs. Qikprop evaluates the acceptability of analogs based on Lipinski’s rule of five, which is essential to ensure drug-like pharmacokinetic profile while using rational drug design. All the analogs were neutralized before being used by Qikprop.