Synthesis, antimicrobial, anticancer and QSAR studies of 1-[4-(substituted phenyl)-2-(substituted phenyl azomethyl)-benzo[b]-[1,4]diazepin-1-yl]-2-substituted phenylaminoethanones

2.1 General procedure for the synthesis of 1-[4-(substituted phenyl)-2-(substituted phenyl azomethyl)-benzo[b]-[1,4]diazepin-1-yl]-2-substituted phenylaminoethanones (1–17)

To a cold solution of substituted aniline (0.01 mol) in water (5 mL) and concentrated hydrochloric acid (5 mL), added solution of sodium nitrite (0.01 mol) in water (8 mL) by maintaining the temperature at 0–5 °C. The diazonium salt thus obtained was filtered into cold solution of ethyl acetoacetate (0.01 mol) and sodium acetate (8 g) in ethanol (25 mL). The resulting product was filtered, washed with cold water, dried and recrystallized from ethanol.

A solution of substituted benzaldehyde (0.01 mol) in ethanol (10 mL) was added to synthesized ester (0.01 mol) in ethanol (5 mL) and sodium hydroxide (4 mL, 40%). The reaction mixture was stirred for 24 h at room temperature and then the contents were poured on to crushed ice and neutralized with 5% hydrochloric acid. The product thus separated was filtered, dried and recrystallized from ethanol.

A mixture of substituted phenyl azo-butenone (synthesized as above) (0.01 mol) and ortho-phenylenediamine (0.01 mol) in absolute ethanol (20 mL) was refluxed for 10 h. The contents were cooled and poured into ice cold water. The separated product was filtered, washed with water, dried and recrystallized from ethanol.

A mixture of the above synthesized substituted benzodiazepine (0.01 mol) and chloro acetyl chloride (0.01 mol) in absolute ethanol (20 mL) was refluxed for 8–10 h. The contents were cooled and poured into ice cold water. The separated product was filtered, washed with water, dried and recrystallized from ethanol.

A mixture of substituted benzodiazepine ethanone (synthesized as above) (0.01 mol) and substituted aniline (0.01 mol) in absolute ethanol (20 mL) was refluxed for 10–15 h. The contents were cooled and poured into ice cold water. The separated product was filtered, washed with water, dried and recrystallized from ethanol.

1-[4-(4-Dimethylamino-phenyl)-2-(p-tolylazo-methyl)-benzo[b][1,4]diazepin-1-yl]-2-phenyl aminoethanone (1): Mp (°C) 58–60; Yield – 74.34%; (KBr pellets, cm⁻¹): 2931 (C—H stretching, aromatic CH₃), 1338 (C—N stretching, aryl 3⁰ amine), 1604 (C⚌C/C⚌N stretching, diazepine ring), 1650 (C⚌O stretching, carbonyl group), 1312 (C—N stretching, aryl 2⁰ amine), 1546 (C⚌C skeletal stretching, aryl), 693 (C—C out of plane bending, mono substituted benzene), 813 (C—H out of plane bending, 1,4-disubstituted benzene), 752 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): 2.36 (s, 6H, CH₃ of ArN(CH₃)₂), 4.62 (s, 1H, diazepine ring), 6.47–7.80 (m, 17H, ArH of ArNH, ArCH₃ adjacent to N⚌N, ArN(CH₃)₂ and benzodiazepine); Anal. Calculated for C₃₃H₃₂N₆O: C, 74.98; H, 6.10; N, 15.90; Found: C, 75.00; H, 6.06; N, 15.88; MS ES+(ToF): m/z 530 [M⁺ + 1].

1-{4-(4-Methoxy-phenyl)-2-[(2-nitro-phenylazo)-methyl]-benzo[b][1,4]diazepin-1-yl}-2-phenyl amino-ethanone (2): Mp (°C) 96–98; Yield – 81.46%; (KBr pellets, cm⁻¹): 1487 (NO₂ stretching, aromatic nitro group), 1278 (C—O—C stretching, aryl conjugated), 1683 (C⚌C/C⚌N stretching, diazepine ring), 1711 (C⚌O stretching, carbonyl group), 1308 (C—N stretching, aryl 2⁰ amine), 1608 (C⚌C skeletal stretching, aryl), 690 (C—C out of plane bending, mono substituted benzene), 826 (C—H out of plane bending, 1,4-disubstituted benzene), 738 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): δ 3.33 (s, 3H, OCH₃ of ArOCH₃), 4.17 (t, 1H, NH of NHArNO₂, d, 2H, CH₂ attached to NHArNO₂), 4.38 (s, 1H, of diazepine ring), 6.60–7.70 (m, 17H, ArH of ArNH, ArCH₃ adjacent to N⚌N, ArN(CH₃)₂ and benzodiazepine); Anal. Calculated for C₃₁H₂₆N₆O₄: C, 68.12; H, 4.79; N, 15.38; Found: C, 68.14; H, 4.74; N, 15.37; MS ES+(ToF): m/z 548 [M⁺ + 1].

2-(4-chloro-phenylamino)-1-[2-[4-chloro-phenylazo)-methyl]-4-(4-dimethylamino-phenyl)-benzo[b][1,4]diazepin-1-yl]-ethanone (3): Mp (°C) 80–82; Yield – 76.55%; (KBr pellets, cm⁻¹): 1321 (C—N stretching, aryl 3⁰ amine), 1684 (C⚌C/C⚌N stretching, diazepine ring), 1719 (C⚌O stretching, carbonyl group), 1347 (C—N stretching, aryl 2⁰ amine), 1605 (C⚌C skeletal stretching, aryl), 729 (C—Cl stretching, monochlorinated aromatic), 692 (C—C out of plane bending, mono substituted benzene), 816 (C—H out of plane bending, 1,4-disubstituted benzene), 742 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): δ 3.9 (t, 1H, NH of NHArNO₂, d, 2H, CH₂ attached to NHArNO₂), 4.2 (s, 1H, of diazepine ring), 6.60–7.70 (m, 16H, ArH of ArCl, ArN(CH₃)₂ and benzodiazepine); Anal. Calculated for C₃₂H₂₈Cl₂N₆O: C, 65.87; H, 4.84; N, 14.40; Found: C, 65.82; H, 4.88; N, 14.44; MS ES+(ToF): m/z 585 [M⁺ + 1].

1-[2-[3-chloro-phenylazo)-methyl]-4-(3-methoxy-phenyl)-benzo[b][1,4]diazepin-1-yl]-2-(2-nitro-phenylamino)-ethanone (4): Mp (°C) 64–66; Yield – 48.00%; (KBr pellets, cm⁻¹): 1567 (NO₂ stretching, aromatic nitro group), 1623 (C⚌C/C⚌N stretching, diazepine ring), 1720 (C⚌O stretching carbonyl group), 1343 (C—N stretching, aryl 2⁰ amine), 1282 (C—O—C stretching, aryl conjugated), 1597 (C⚌C skeletal stretching, phenyl), 777 (C—Cl stretching, monochlorinated aromatic), 695 (C—C out of plane bending, mono substituted benzene), 822 (C—H out of plane bending, 1,4-disubstituted benzene), 739 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): 3.68 (t, 3H, H of methoxy group), 6.63–8.05 (m, 16H, ArH of ArCl, ArOCH₃, ArN(CH₃)₂, ArNO₂ and benzodiazepine); Anal. Calculated for C₃₁H₂₅ClN₆O₄: C, 64.08; H, 4.34; N, 14.46; Found: C, 64.05; H, 4.39; N, 14.51; MS ES+ (ToF): m/z 582 [M⁺ + 1].

1-[2-[2-Methyl-5-nitro-phenylazo)-methyl]-4-(3-nitro-phenyl)-benzo[b][1,4]diazepin-1-yl]-2-(3-nitro-phenylamino)-ethanone (5): Mp (°C) 92–94; Yield – 62.30%; (KBr pellets, cm⁻¹): 1532 (NO₂ stretching, aromatic nitro group), 1621 (C⚌C/C⚌N stretching diazepine ring), 1720 (C⚌O stretching carbonyl group), 1342 (C—N stretching, aryl 2⁰ amine), 1596 (C⚌C skeletal stretching, phenyl), 696 (C—C out of plane bending, mono substituted benzene), 824 (C—H out of plane bending, 1,4-disubstituted benzene), 737 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): δ 4.0 (t, 1H, NH of NHArNO₂, d, 2H, CH₂ attached to NHArNO₂), 4.25 (s, 1H, of diazepine ring), 6.98–7.50 (m, 15H, ArH of ArNO₂, ArCH₃NO₂ and benzodiazepine); Anal. Calculated for C₃₁H₂₄N₈O₇: C, 60.00; H, 3.90; N, 18.06; Found: C, 59.95; H, 3.94; N, 18.03; MS ES+ (ToF): m/z 622 [M⁺ + 1].

1-[2-[2-Chlorophenylazo)-methyl]-4-(3-methoxy-phenyl)-benzo[b][1,4]diazepin-1-yl]-2-(3-nitro-phenylamino)-ethanone (6): Mp (°C) 110–112; Yield – 54.24%; (KBr pellets, cm⁻¹): 1512 (NO₂ stretching, aromatic nitro group), 1264 (C—O—C stretching, aryl conjugated), 1621 (C⚌C/C⚌N stretching diazepine ring), 1872 (C⚌O stretching carbonyl group), 1324 (C—N stretching, aryl 2⁰ amine), 1580 (C⚌C skeletal stretching, phenyl), 761 (C—Cl stretching, monochlorinated aromatic), 667 (C—C out of plane bending, mono substituted benzene), 815 (C—H out of plane bending, 1,4-disubstituted benzene), 734 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): δ 7.00–7.49 (m, 16H, ArH of ArCl, ArOCH₃, ArNO₂ and benzodiazepine); Anal. Calculated for C₃₁H₂₅ClN₆O₄: C, 64.08; H, 4.34; N, 14.46; Found: C, 64.14; H, 4.37; N, 14.44; MS ES+ (ToF): m/z 682 [M⁺ + 1].

1-[2-[2-Chloro-4-nitrophenylazo)-methyl]-4-(4-chloro-phenyl)-benzo[b][1,4]diazepin-1-yl]-2-(4-nitro-phenylamino)-ethanone (7): Mp (°C) 142–144; Yield – 70.02%; (KBr pellets, cm⁻¹): 1504 (NO₂ stretching, aromatic nitro group), 1629 (C⚌C/C⚌N stretching diazepine ring), 1723 (C⚌O stretching carbonyl group), 1299 (C—N stretching, aryl 2⁰ amine), 1586 (C⚌C skeletal stretching, phenyl), 752 (C—Cl stretching, monochlorinated aromatic), 697 (C—C out of plane bending, mono substituted benzene), 838 (C—H out of plane bending, 1,4-disubstituted benzene), 730 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): δ 4.05 (t, 1H, NH of NHArNO₂, d, 2H, CH₂ attached to NHArNO₂), 4.22 (s, 1H, diazepine ring), 6.63–8.00 (m, 15H, ArH of ArCl, ArNO₂, ArClNO₂ and benzodiazepine); Anal. Calculated for C₃₀H₂₁Cl₂N₇O₅: C, 57.15; H, 3.36; N, 15.55; Found: C, 57.21; H, 3.39; N, 15.50; MS ES+ (ToF): m/z 631 [M⁺ + 1].

1-[2-[2-Chloro-4-nitrophenylazo)-methyl]-4-(3-chloro-phenyl)-benzo[b][1,4]diazepin-1-yl]-2-(2-nitro-phenylamino)-ethanone (8): Mp (°C) 106–108; Yield – 65.35%; (KBr pellets, cm⁻¹): 1492 (NO₂ stretching, aromatic nitro group), 1619 (C⚌C/C⚌N stretching diazepine ring), 1725 (C⚌O stretching carbonyl group), 1336 (C—N stretching, aryl 2⁰ amine), 1597 (C⚌C skeletal stretching, phenyl), 750 (C—Cl stretching, monochlorinated aromatic), 696 (C—C out of plane bending, mono substituted benzene), 819 (C—H out of plane bending, 1,4-disubstituted benzene); ¹H NMR (MeOD): δ 4.65 (s, 1H, H of azepine ring of benzodiazepine), 6.67–7.05 (m, 15H, ArH of ArCl, ArNO₂, ArClNO₂ and benzodiazepine); Anal. Calculated for C₃₀H₂₁Cl₂N₇O₅: C, 57.15; H, 3.36; N, 15.55; Found: C, 57.18; H, 3.33; N, 15.52; MS ES+ (ToF): m/z 631 [M⁺ + 1].

1-[4-[4-Hydroxy-phenyl)-2-[(4-nitro-phenylazo)-methyl]-benzo[b][1,4]diazepin-1-yl]-2-(2-methyl-5-nitro-phenylamino)-ethanone (9): Mp (°C) 85–87; Yield – 74.46%; (KBr pellets, cm⁻¹): 2898 (C—H stretching, aromatic CH₃), 1557 (NO₂ stretching, aromatic nitro group), 1627 (C⚌C/C⚌N stretching, diazepine ring), 1672 (C⚌O stretching, carbonyl group), 1337 (C—N stretching, aryl 2⁰ amine), 1612 (C⚌C skeletal stretching, phenyl), 704 (C—C out of plane bending, mono substituted benzene), 811 (C—H out of plane bending, 1,4-disubstituted benzene), 733 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): δ 7.15–7.53 (m, 15H, ArH of ArOH, ArNO₂, ArCH₃NO₂ and benzodiazepine), 2.23 (s, 3H, CH₃ of ArCH₃NO₂); Anal. Calculated for C₃₁H₂₅N₇O₆: C, 62.94; H, 4.26; N, 16.57; Found: C, 62.96; H, 4.24; N, 16.52; MS ES+ (ToF): m/z 593 [M⁺ + 1].

2-(2-Chloro-4-nitro-phenylamino)-1-{4-(4-Chloro-phenyl)-2-[(3-nitro-phenylazo)-methyl]-benzo[b][1,4]diazepin-1-yl]-ethanone (10): Mp (°C) 101–103; Yield – 64.88%; (KBr pellets, cm⁻¹): 1511 (NO₂ stretching, aromatic nitro group), 1679 (C⚌C/C⚌N stretching, diazepine ring), 1726 (C⚌O stretching, carbonyl group), 1252 (C—N stretching, aryl 2⁰ amine), 1624 (C⚌C skeletal stretching, phenyl), 748 (C—Cl stretching, monochlorinated aromatic), 702 (C—C out of plane bending, mono substituted benzene), 816 (C—H out of plane bending, 1,4-disubstituted benzene), 738 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): δ 6.98–8.04 (m, 15H, ArH of ArCl, ArNO₂, ArClNO₂ and benzodiazepine); Anal. Calculated for C₃₀H₂₁Cl₂N₇O₅: C, 57.15; H, 3.36; N, 15.55; Found: C, 57.10; H, 3.40; N, 15.58; MS ES+ (ToF): m/z 631 [M⁺ + 1].

1-{4-(3-Ethoxy-4-hydroxyphenyl)-2-[(3-nitro-phenylazo)-methyl]-benzo[b][1,4]diazepin-1-yl}-2-(2-methyl-5-nitro-phenylamino)-ethanone (11): Mp (°C) 60–62; Yield – 45.44%; (KBr pellets, cm⁻¹): 1566 (NO₂ stretching, aromatic nitro group), 1678 (C⚌C/C⚌N stretching, diazepine ring), 1731 (C⚌O stretching carbonyl group), 1283 (C—N stretching, aryl 2⁰ amine), 1623 (C⚌C skeletal stretching, phenyl), 694 (C—C out of plane bending, mono substituted benzene), 813 (C—H out of plane bending, 1,4-disubstituted benzene), 744 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): δ 6.65–8.06 (m, 14H, ArH of ArNO₂, ArCH₃NO₂, ArOH(OC₂H₅) and benzodiazepine); Anal. Calculated for C₃₃H₂₉N₇O₇: C, 62.36; H, 4.60; N, 15.43; Found: C, 62.39; H, 4.64; N, 15.47; MS ES+ (ToF): m/z 637 [M⁺ + 1].

2-(2-Chloro-4-nitrophenylamino)-1-[2[(4-chloro-phenylazo)-methyl]-4-(3-hydroxy-phenyl)-benzo [b][1,4]diazepin-1-yl]-ethanone (12): Mp (°C) 102–104; Yield – 75.36%; (KBr pellets, cm⁻¹): 1537 (NO₂ stretching, aromatic nitro group), 1639 (C⚌C/C⚌N stretching, diazepine ring), 1689 (C⚌O stretching, carbonyl group), 1294 (C—N stretching, aryl 2⁰ amine), 1603 (C⚌C skeletal stretching, phenyl), 763 (C—Cl stretching, monochlorinated aromatic), 691 (C—C out of plane bending, mono substituted benzene), 816 (C—H out of plane bending, 1,4-disubstituted benzene), 723 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): δ 6.98–8.04 (m, 15H, ArH of ArClNO₂, ArCl, ArOH and benzodiazepine); Anal. Calculated for C₃₀H₂₂Cl₂N₆O₄: C, 59.91; H, 3.69; N, 13.97; Found: C, 59.97; H, 3.74; N, 13.94; MS ES+(ToF): m/z 602 [M⁺ + 1].

1-{4-(3,4-Dimethoxy-phenyl)-2-[(2-nitro-phenylazo)-methyl]-benzo[b][1,4]diazepin-1-yl]-2-(3-nitro-phenylamino)-ethanone (13): Mp (°C) 79–81; Yield – 80.28%; (KBr pellets, cm⁻¹): 1531 (NO₂ stretching, aromatic nitro group), 1624 (C⚌C/C⚌N stretching diazepine ring), 1719 (C⚌O stretching carbonyl group), 1309 (C—N stretching, aryl 2⁰ amine), 1581 (C⚌C skeletal stretching, phenyl), 1279 (C—O—C stretching, aryl conjugated), 673 (C—C out of plane bending, mono substituted benzene), 825 (C—H out of plane bending, 1,4-disubstituted benzene), 736 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): 4.02 (t, 1H, NH of NHArNO₂, d, 2H, CH₂ attached to NHArNO₂), 4.3 (s, 1H, of diazepine), 7.35–7.40 (m, 15H, ArH of ArNH, ArNO₂ adjacent to N⚌N, Ar (OCH₃)₂ and benzodiazepine); Anal. Calculated for C₃₂H₂₇N₇O₇: C, 61.83; H, 4.38; N, 15.77; Found: C, 61.86; H, 4.44; N, 15.84; MS ES+(ToF): m/z 623 [M⁺ + 1].

2-(4-Chloro-phenylamino)-1-{4-(4-hydroxy-3-methoxy-phenyl)-2-[(2-nitro-phenylazo)-methyl]-benzo[b][1,4]diazepin-1-yl]-2)-ethanone (14): Mp (°C) 105–107; Yield – 62.87%; (KBr pellets, cm⁻¹): 1489 (NO₂ stretching, aromatic nitro group), 1608 (C⚌C/C⚌N stretching, diazepine ring), 1693 (C⚌O stretching, carbonyl group), 1314 (C—N stretching, aryl 2⁰ amine), 1570 (C⚌C skeletal stretching, phenyl), 1257 (C—O—C stretching, aryl conjugated), 712 (C—Cl stretching, monochlorinated aromatic), 691 (C—C out of plane bending, mono substituted benzene), 814 (C—H out of plane bending, 1,4-disubstituted benzene), 738 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): 6.73–8.33 (m, 15H, ArH of ArNH, ArNO₂ adjacent to N⚌N, ArOHOCH₃ and benzodiazepine); Anal. Calculated for C₃₁H₂₅ClN₆O₅: C, 62.36; H, 4.22; N, 14.08; Found: C, 62.40; H, 4.24; N, 11.12; MS ES+ (ToF): m/z 598 [M⁺ + 1].

2-(4-chloro-phenylamino)-1-[2-[4-chloro-phenylazo)-methyl]-4-(4-dimethylamino-phenyl)-benzo[b][1,4]diazepin-1-yl]-ethanone (15): Mp (°C) 140–142; Yield – 56.80%; (KBr pellets, cm⁻¹): 1525 (NO₂ stretching, aromatic nitro group), 1327 (C—N stretching, aryl 3⁰ amine), 1683 (C⚌C/C⚌N stretching, diazepine ring), 1709 (C⚌O stretching, carbonyl group), 1305 (C—N stretching, aryl 2⁰ amine), 1607 (C⚌C skeletal stretching, phenyl), 1525 (NO₂ asym. Stretching, aromatic nitro group), 692 (C—C out of plane bending, mono substituted benzene) 825 (C—H out of plane bending, 1,4-disubstituted benzene), 739 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): δ 2.6 (s, 6H, CH₃ of ArN(CH₃)₂), 3.9 (t, 1H, NH of NHArNO₂, d, 2H, CH₂ attached to NHArNO₂), 4.2 (d, 1H of diazepine ring), 6.78–8.29 (m, 17H, ArH of ArNH, ArCH₃ adjacent to N⚌N, ArN(CH₃)₂, and benzodiazepine); Anal. Calculated for C₃₃H₂₉N₇O₃: C, 68.68; H, 5.22; N, 17.52; Found: C, 68.70; H, 5.18; N, 17.47; MS ES+ (ToF): m/z 561 [M⁺ + 1].

1-[4-[4-Chloro-phenyl)-2-[(3-nitro-phenylazo)-methyl]-benzo[b][1,4]diazepin-1-yl]-2-(2-methyl-5-nitro-phenylamino)-ethanone (16): Mp (°C) 74–76; Yield – 58.68%; (KBr pellets, cm⁻¹): 1523 (NO₂ stretching, aromatic nitro group), 1627 (C⚌C/C⚌N stretching, diazepine ring), 1676 (C⚌O stretching, carbonyl group), 1313 (C—N stretching, aryl 2⁰ amine), 1601 (C⚌C skeletal stretching, phenyl), 723 (C—Cl stretching, monochlorinated aromatic), 689 (C—C out of plane bending, mono substituted benzene), 807 (C—H out of plane bending, 1,4-disubstituted benzene), 735(C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): δ 4.4 (s, 1H, of diazepine ring), 6.98–8.04 (m, 16H, ArH of ArCl, ArNO₂ and benzodiazepine); Anal. Calculated for C₃₀H₂₂ClN₇O₅: C, 60.46; H, 3.72; N, 16.45; Found: C, 60.38; H, 3.68; N, 16.46; MS ES+ (ToF): m/z 597 [M⁺ + 1].

1-{4-(3,4-Dimethoxy-phenyl)-2-[(4-nitro-phenylazo)-methyl]-benzo[b][1,4]diazepin-1-yl]-2-(3-nitrophenylamino)-ethanone (17): Mp (°C) 103–105; Yield – 70.66%; (KBr pellets, cm⁻¹): 1557 (NO₂ stretching, aromatic nitro group), 1624 (C⚌C/C⚌N stretching, diazepine ring), 1739 (C⚌O stretching, carbonyl group), 1292 (C—N stretching, aryl 2⁰ amine), 1585 (C⚌C skeletal stretching, phenyl), 1283 (C—O—C stretching, aryl conjugated), 693 (C—C out of plane bending, mono substituted benzene), 813 (C—H out of plane bending, 1,4-disubstituted benzene), 734 (C—H out of plane bending, 1,2-disubstituted benzene); ¹H NMR (MeOD): δ 7.17–7.54 (m, 15H, ArH of ArNO₂, Ar(OCH₃)₂ and benzodiazepine); Anal. Calculated for C₃₂H₂₇N₇O₇: C, 61.83; H, 4.38; N, 15.77; Found: C, 61.78; H, 4.41; N, 15.81; MS ES+ (ToF): m/z 623 [M⁺ + 1].

2.2 Evaluation of antimicrobial activity

The antimicrobial activity of synthesized 1-[4-(substituted phenyl)-2-(substituted phenyl azomethyl)-benzo[b]-[1,4]diazepin-1-yl]-2-substituted phenylaminoethanones (1–17) against Gram-positive bacteria: Staphylococcus aureus, Bacillus subtilis, Gram-negative bacterium: Escherichia coli and fungal strains: Candida albicans and Aspergillus niger was determined using the tube dilution method (Cappucino and Sherman, 1999). Dilutions of test and standard compounds were prepared in double strength nutrient broth – I.P. (bacteria) or Sabouraud dextrose broth I.P. (fungi) (Pharmacopoeia of India, 2007). The samples were incubated at 37 ± 1 °C for 24 h (bacteria), at 25 ± 1 °C for 7d (A. niger) and at 37 ± 1 °C for 48 h (C. albicans) respectively and the results were recorded in terms of MIC (the lowest concentration of test substance which inhibited the growth of microorganisms).

2.3 Determination of MBC/MFC

The minimum bactericidal concentration (MBC) and fungicidal concentration (MFC) were determined by subculturing 100 μL of culture from each tube (which remained clear in the MIC determination) on fresh medium. MBC and MFC values represent the lowest concentration of compound that produces a 99.9% end point reduction (Rodriguez-Arguelles et al., 2005).

2.4 Anticancer evaluation

The anticancer activity of synthesized 1-[4-(substituted phenyl)-2-(substituted phenyl azomethyl)-benzo[b]-[1,4]diazepin-1-yl]-2-substituted phenylaminoethanones was determined against breast cancer (MCF 7) and colon cancer (HCT116) cell lines. Cancer cell lines were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA. All cell lines were cultured in RPMI 1640 (Sigma) supplemented with 10% heat inactivated fetal bovine serum (FBS) (PAA Laboratories) and 1% penicillin/streptomycin (PAA Laboratories). Cultures were maintained in a humidified incubator at 37 °C in an atmosphere of 5% CO₂. Cytotoxicity of synthesized compounds at various concentrations was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma) assay as described by Mosmann, 1983 but with minor modification, following 72 h of incubation. Assay plates were read using a spectrophotometer at 520 nm. Data generated were used to plot a dose–response curve of which the concentration of test compounds required to kill 50% of cell population (IC₅₀) was determined. Anticancer activity was expressed as the mean IC₅₀ of three independent experiments (Mosmann, 1983).

2.5 QSAR studies

The structures of synthesized compounds were first pre-optimized with the Molecular Mechanics Force Field (MM⁺) procedure included in Hyperchem 6.03 (Hyperchem 6.0, 1993) and the resulting geometries were further refined by means of the semiempirical method PM3 (Parametric Method-3). We chose a gradient norm limit of 0.01 kcal/A° for the geometry optimization. The lowest energy structure was used for each molecule to calculate physicochemical properties like log of octanol–water partition coefficient (log P), molar refractivity (MR), Kier’s molecular connectivity (⁰χ, ⁰χ^v, ¹χ, ¹χ^v, ²χ, ²χ^v) and shape (κ₁, κα₁, κα₂, κα₃) topological indices, Randic topological index (R), Balaban topological index (J), Wiener topological index (W), Total energy (Te), energies of highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), dipole moment (μ) and electronic energy (Ele. E) by using TSAR 3.3 software for Windows (TSAR 3D Version 3.3, 2000). Further, the regression analysis was performed using the SPSS software package (SPSS for Windows, 1999).

3.1 Chemistry

A series of 1-[4-(substituted phenyl)-2-(substituted phenyl azomethyl)-benzo[b]-[1,4]diazepin-1-yl]-2-substituted phenylaminoethanones (1–17) was synthesized by the series of reactions as outlined in Scheme 1. All the compounds were obtained in appreciable yield and their physicochemical characteristics are presented in Table 1.

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Scheme 1 Synthetic route followed for the synthesis of 1-[4-(substituted phenyl)-2-(substituted phenyl azomethyl)-benzo[b]-[1,4]diazepin-1-yl]-2-substituted phenylaminoethanones.

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The formation of target compounds was ascertained on the basis of their consistent NMR and IR spectral characteristics. The formation of diazepine ring was confirmed by IR bands ranging from 1684 to 1604 cm⁻¹. Further appearance of IR bands at 1739–1650 cm⁻¹ (C⚌O str., carbonyl group) confirmed the formation of target compounds.

Presence of phenyl group in the synthesized compounds was demonstrated by C⚌C skeletal stretching of phenyl nucleus at wave number 1624–1546 cm⁻¹. Presence of IR bands in the region of 1567–1487 cm⁻¹ indicated the presence of aromatic nitro group in synthesized compounds. Presence of chloro group in compounds 3, 4, 6, 7, 8, 10, 12 and 14 was evidenced by IR stretching bands ranging from 777 to 712 cm⁻¹.

IR bands in the range of 1283–1257 cm⁻¹ indicated the presence of methoxy group in compounds 2, 4, 6, 13, 14 and 17. Presence of methyl group in compounds 1 and 9 was confirmed by IR bands at 2931 and 2898 cm⁻¹ respectively.

The formation of diazepine ring in the synthesized compounds was confirmed by the appearance of δ at 4.25–4.65 ppm and the presence of aromatic moieties was indicated by NMR signal at δ 6.47–8.29. Appearance of singlet signal in the range of δ 2.36–2.55 elicited the presence of ArN(CH₃)₂ groups in compounds 1 and 15. Presence of methoxy group in compound 2 was indicated by the appearance of singlet signal at δ 3.33 and singlet signal at δ 2.23 confirmed the presence of aromatic methyl group in compound 9. Presence of triplet signal at δ 4.02–4.20 indicated the link between acetyl group and the NH of substituted anilines, which confirmed the formation of the target compounds. Further, Mass spectra and elemental (C, H, N) analysis results of synthesized compounds were in full agreement with their assigned molecular structures.

3.2 Antimicrobial activity

The synthesized compounds (1–17) were screened in vitro for their antimicrobial potential against Gram-positive bacteria S. aureus, B. subtilis, Gram negative bacterium E. coli and fungal strains C. albicans and A. niger by tube dilution method (Cappucino and Sherman, 1999) using norfloxacin and fluconazole as reference drugs for antibacterial and antifungal activities, respectively. Double strength nutrient broth I.P. and Sabouraud dextrose broth I.P. (Pharmacopoeia of India, 2007) have been employed as media for growth of bacterial and fungal strains, respectively. The results of antimicrobial activity are presented in Table 2.

Results of antimicrobial study indicated that compound 17 was the most active antibacterial agent against S. aureus and B. subtilis having pMIC_sa and pMIC_bs values 1.70 and 2.30 μM/mL respectively. In case of Gram-negative bacterium E. coli, compound 16 (pMIC_ec = 1.98 μM/mL) emerged as the most active antibacterial candidate among the synthesized benzodiazepine derivatives. Besides having appreciable antibacterial activity, the synthesized compounds also displayed good antifungal activity and compound 11 emerged as the most active antifungal agent against both C. albicans and A. niger (pMIC_ca and pMIC_an = 1.71 μM/mL).

In general, the results of minimum bactericidal/fungicidal concentration (MBC/MFC) (Table 3) revealed that the synthesized compounds were bacteriostatic and fungistatic in action (except in case of A. niger) as their MFC and MBC values were 3-fold higher than their MIC values (a drug is considered to be bacteriostatic/fungistatic when its MFC and MBC values are 3-fold higher than its MIC values) (Emami et al., 2004). The MFC results against the A. niger demonstrated that the synthesized benzodiazepine derivatives are fungicidal against it.

3.3 The anticancer activity

The anticancer activity of the synthesized benzodiazepine derivatives was determined against breast cancer (MCF 7) and colon cancer (HCT116) cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Mosmann, 1983) and the results are presented in Table 4.

In general, all the synthesized compounds were less active anticancer agents as compared to the standard drug 5-fluorouracil (5-FU) but 1-[4-(4-dimethylamino-phenyl)-2-(p-tolylazo-methyl)-benzo[b][1,4]diazepin-1-yl]-2-phenyl amino ethanone (1) was the most effective compound against HCT116 cancer cell lines (IC₅₀ = 1.42 M/mL) and was more than standard drug, 5-FU (IC₅₀ = 1.34 uM/mL).

3.4 Structure activity relationship

From the anticancer and antimicrobial screening results of synthesized, 4-thiazolidinone derivatives the following structure activity relationship (SAR) can be derived:

1. The high antibacterial activity of compound 17 indicated the fact that the presence of electron withdrawing nitro group on diazobenzene portion increases the antimicrobial activity of synthesized benzodiazepine derivatives. The role of electron withdrawing group in improving antibacterial activity of benzodiazepine derivatives is supported by the studies of Konda et al. (2011).

2. The presence of electron releasing group on phenyl aminoethanone improves the antifungal activity of synthesized benzodiazepine derivatives as evidenced by the high antifungal activity of compound 11. It is important to note a fact here that the substitution on phenyl nucleus of diazobenzene nucleus does not affect the antimicrobial activity of synthesized benzodiazepines.

3. Presence of electron releasing groups on 4-substituted phenyl nucleus of benzodiazepine increases antimicrobial activity against Gram positive bacterial strains as well as both of the fungal strains whereas electron withdrawing groups increase antibacterial activity against Gram negative bacterium E. coli. From this result we may conclude that there are different structural requirements for a compound to be effective against different targets. This is similar to the results of Sortino et al. (2007).

4. From the anticancer potential of compound 1, we can say that the presence of electron releasing groups on diazobenzene nucleus as well as on 4-substituted phenyl nucleus of benzodiazepine increases anticancer activity of synthesized benzodiazepine derivatives. The role of electron releasing group in improving the anticancer potential of benzodiazepine derivatives is similar to the observation of Kamal et al. (2010a).

5. It is interesting to note a fact here that the substitution on the phenyl ring of phenyl amino ethanone portion of the synthesized benzodiazepine derivatives decreased their anticancer potential in comparison to the unsubstituted phenyl amino ethanone portion. This result gave an important clue that the hydrogen of unsubstituted phenyl ring may be necessary for binding of synthesized benzodiazepine derivatives with anticancer target through hydrogen bonding.

The aforementioned findings are summarized in Fig. 1.

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Figure 1 Structural requirements for the antimicrobial and anticancer activities of 1-[4-(substituted phenyl)-2-(substituted phenyl azomethyl)-benzo[b]-[1,4]diazepin-1-yl]-2-substituted phenylaminoethanones.

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3.5 QSAR studies

3.5.1

3.5.1 QSAR studies for antimicrobial activity

In the present study, we have performed the quantitative structure activity relationship study by conventional Hansch’s analysis using the linear free energy relationship model (LFER) described by Hansch and Fujita (1964). In this approach, structural features of drug molecules were quantified in terms of different parameters and these structural features were correlated with quantified biological activity through equation using regression analysis. Biological activity data determined as MIC values was first transformed into pMIC values (i.e. −log MIC) and used as a dependent variable in QSAR study.

The different molecular descriptors (independent variables) like log of octanol–water partition coefficient (log P), molar refractivity (MR), Kier’s molecular connectivity (⁰χ, ⁰χ^v, ¹χ, ¹χ^v, ²χ, ²χ^v) and shape (κ₁, κα₁, κα₂, κα₃) topological indices, Randic topological index (R), Balaban topological index (J), Wiener topological index (W), Total energy (Te), energies of highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), dipole moment (μ) and electronic energy (Ele. E) (Hansch et al., 1973; Kier and Hall, 1976; Randic, 1975, 1993; Balaban, 1982; Wiener, 1947) were calculated for benzodiazepine derivatives and the values of selected descriptors are presented in Table 5.

Table 5 Values of selected parameters used in QSAR studies of 1-[4-(substituted phenyl)-2-(substituted phenyl azomethyl)-benzo[b]-[1,4]diazepin-1-yl]-2-substituted phenylaminoethanones.

Comp.	log P	0χ	0χv	κα1	κα2	Ele. E	LUMO	HOMO	μ
Training set
1	5.92	27.90	22.95	28.18	13.33	−59505.80	−0.65	−8.30	2.72
2	5.37	28.61	22.18	28.72	13.71	−63069.80	−1.06	−8.75	4.46
3	6.49	28.77	24.26	29.70	13.92	−63164.80	−0.72	−8.37	4.23
4	5.88	29.48	23.29	29.96	14.11	−64991.60	−0.98	−8.91	7.29
5	5.99	32.79	24.14	32.74	14.64	−78238.70	−1.48	−9.48	8.51
6	5.88	29.48	23.29	29.96	14.11	−66340.10	−0.92	−9.03	8.13
7	6.61	31.22	24.27	31.79	14.44	−69865.10	−1.60	−9.32	10.19
8	6.61	31.22	24.27	31.79	14.44	−71007.60	−1.54	−9.08	7.16
9	5.76	31.22	23.32	31.19	14.03	−71388.30	−1.44	−9.10	10.07
10	6.61	31.22	24.27	31.79	14.44	−70391.00	−1.50	−9.25	10.84
11	5.84	33.50	25.36	34.07	15.54	−80672.50	−1.33	−8.90	8.41
12	6.37	29.64	23.45	30.24	13.83	−64165.30	−1.18	−9.21	8.34
13	5.07	32.63	24.69	33.09	15.34	−77009.40	−1.12	−8.71	3.88
14	5.60	30.35	23.66	30.89	14.28	−69010.60	−1.08	−8.78	4.03
Test set
15	5.41	29.48	23.21	29.68	13.91	−65460.50	−1.01	−8.30	2.73
16	6.09	30.35	23.15	30.54	14.04	−68308.60	−1.42	−8.99	5.43
17	5.07	32.63	24.69	33.09	15.34	−75244.00	−1.52	−8.86	7.94

A training set consisting of 14 benzodiazepine derivatives (1–14) was used for linear regression model generation and a test set consisting of 3 benzodiazepine derivatives (15–17) was used for cross validation of the generated models. The standard drugs norfloxacin and fluconazole were not included in model generation because of dissimilarity in structure with synthesized compounds.

From the results of our previous QSAR studies (Narang et al., 2012; Judge et al., 2012; Kumar et al., 2010, 2009), we observed that multi-target QSAR (mt-QSAR) models gave better results than one-target QSAR (ot-QSAR) models in describing the antimicrobial activity. So, in light of our past experiences, we decided to develop muti-target QSAR models directly to describe the antimicrobial activity of synthesized 1-[4-(substituted phenyl)-2-(substituted phenyl azomethyl)-benzo[b]-[1,4]diazepin-1-yl]-2-substituted phenylaminoethanones in the present study.

According to ot-QSAR models one should use five different equations to predict the activity of a new compound against five microbial species. The ot-QSAR models, which are almost in the whole literature, become unpractical to use when we have to predict each compound results for more than one target. In these cases we have to develop one ot-QSAR for each target. In opposition to ot-QSAR, the mt-QSAR model is a single equation that considers the nature of molecular descriptors which are common and essential for describing the antibacterial and antifungal activities (Prado-Prado et al., 2008; Gonzalez-Diaz and Prado-Prado, 2008; Cruz-Monteagudo et al., 2007; Gonzalez-Diaz et al., 2007).

In order to develop mt-QSAR models, initially we have calculated the average antibacterial activity, antifungal activity and antimicrobial activity values of substituted benzodiazepine derivatives which are presented in Table 2.

Preliminary analysis was carried out in terms of correlation analysis. A correlation matrix constructed for antibacterial, antifungal and antimicrobial activities is presented in Table 6. In general, high colinearity (r > 0.5) was observed between different parameters. The high interrelationship was observed between Ele. E and κα₁ (r = −0.999), and low interrelationship was observed between ⁰χ^v and log P (r = −0.002).

Table 6 Correlation matrix for the antimicrobial activity of 1-[4-(substituted phenyl)-2-(substituted phenyl azomethyl)-benzo[b]-[1,4]diazepin-1-yl]-2-substituted phenylaminoethanones.

	pMICab	pMICaf	pMICam	log P	0χ	0χv	κα1	Ele. E	Nu. E	HOMO
pMICab	1.000	0.769	0.914	0.280	0.868	0.927	−0.826	0.806	−0.730	0.546
pMICaf		1.000	0.962	0.384	0.784	0.682	−0.562	0.549	−0.410	0.448
pMICam			1.000	0.368	0.870	0.822	−0.701	0.685	−0.559	0.505
log P				1.000	0.248	−0.002	0.173	−0.195	−0.321	0.500
0χ					1.000	0.861	−0.757	0.752	−0.422	0.268
0χv						1.000	−0.968	0.960	−0.698	0.448
κα1							1.000	−0.999	0.651	−0.381
Ele. E								1.000	−0.626	0.357
Nu. E									1.000	−0.759
HOMO										1.000

The structural effects on variations in antibacterial activity of the benzodiazepine derivatives in terms of pMIC_ab were examined by regression analysis with molecular parameters shown in Table 5. For 14 benzodiazepine derivatives, Eq. (1) was derived as that of the best quality using the topological parameter, Kier’s alpha first order shape index (κα₁).

3.5.1.1

3.5.1.1 LR mt-QSAR model for antibacterial activity

(1)

$${\text{pMIC}}_{\text{ab}}=0.0132\kappa {\alpha }_1+1.169$$ $$n=14r=0.926q^2=0.790s=0.0093F=72.92$$ Here and thereafter, n – number of data points, r – correlation coefficient, q² – cross validated r² obtained by leave one out method, s – standard error of the estimate and F – Fischer statistics.

The antibacterial activity of synthesized compounds is positively correlated with the Kier’s alpha first order shape index (κα₁). The coefficient of κα₁ in Eq. (1) is positive which signifies that the activity of synthesized compounds will increase with increase in value of κα₁. This is evidenced by the antibacterial activity data of benzodiazepine derivatives (Table 2) and their κα₁ values (Table 5). Compounds 16 and 17 having high κα₁ values 30.54 and 33.09 (Table 5) respectively have maximum antibacterial activity with pMIC_ab value of 1.88 and 1.90 (Table 2) respectively. Compounds 1 and 2 having minimum κα₁ values 28.18 and 28.72 (Table 5) respectively have minimum antibacterial activity with pMIC_ab values 1.53 and 1.54 (Table 2) respectively.

According to Kier, the shape of a molecule may be partitioned into attributes, each describable by the count of bonds of various path lengths. The basis for devising a relative index of shape is given by the relationship of the number of path of length l in the molecule i, ^lP_i, to some reference values based on molecules with a given number of atoms, n, in which the values of ^lP are maximum and minimum, ^lP_max and ^lP_min (Kier and Hall, 1999).

The modified kappa shape indices are given by:

• κα₁ = (n + α)(n + α − 1)²/(¹P_i + α)²

• κα₂ = (n + α − 1)(n + α − 2)²/(²P_i + α)²

• κα₃ = (n + α − 1)(n + α − 3)²/(³P_i + α)² n is odd

• κα₃ = (n + α − 3)(n + α − 2)2/(³P_i + α)² n is even.

In order to improve the value of regression coefficient (Eq. (1)), we coupled κα₁ with lipophilic parameter, log P which improved regression coefficient from 0.926 to 0.968 (Eq. (2)).

3.5.1.2

3.5.1.2 MLR mt-QSAR model for antibacterial activity

(2)

$${\text{pMIC}}_{\text{ab}}=0.0138\log P+0.0132\kappa {\alpha }_1+1.086$$ $$n=14r=0.968q^2=0.889s=0.0064F=83.20$$

The developed QSAR model (Eq. (2)) was cross validated by its high q² value (q² = 0.889) obtained by leave one out (LOO) method. The value of q² more than 0.5 indicated that the developed model is a valid one (Golbraikh and Tropsha, 2002). Further the observed and predicted values are close to each other (Table 7), the mt-QSAR model for antibacterial activity (Eq. (2)) is a valid one. The plot of predicted pMIC_ab against observed pMIC_ab (Fig. 2) also favors the developed model expressed by Eq. (2). Further, the plot of observed pMIC_ab vs residual pMIC_ab (Fig. 3) indicated that there was no systemic error in the model development as the propagation of error was observed on both sides of zero (Kumar et al., 2007).

Table 7 Comparison of observed and predicted antimicrobial activity obtained by mt-QSAR models.

Comp.	pMICab			pMICaf			pMICam
	Obs.	Pre.	Res.	Obs.	Pre.	Res.	Obs.	Pre.	Res.
Training set
1	1.53	1.54	−0.01	1.63	1.60	0.03	1.57	1.57	0.00
2	1.54	1.54	0.00	1.49	1.58	−0.09	1.52	1.55	−0.03
3	1.57	1.57	0.00	1.67	1.68	−0.01	1.61	1.62	−0.01
4	1.57	1.56	0.01	1.67	1.65	0.02	1.61	1.60	0.01
5	1.60	1.60	0.00	1.70	1.69	0.01	1.64	1.63	0.01
6	1.57	1.56	0.01	1.67	1.65	0.02	1.61	1.60	0.01
7	1.60	1.60	0.00	1.70	1.71	−0.01	1.64	1.64	0.00
8	1.60	1.60	0.00	1.70	1.69	0.01	1.64	1.63	0.01
9	1.57	1.58	−0.01	1.68	1.66	0.02	1.61	1.61	0.00
10	1.60	1.60	0.00	1.70	1.71	−0.01	1.64	1.65	−0.01
11	1.61	1.62	−0.01	1.71	1.75	−0.04	1.65	1.67	−0.02
12	1.58	1.58	0.00	1.68	1.66	0.02	1.62	1.61	0.01
13	1.60	1.59	0.01	1.70	1.70	0.00	1.64	1.63	0.01
14	1.58	1.57	0.01	1.68	1.65	0.03	1.62	1.60	0.02
Test set
15	1.65	1.55	0.10	1.65	1.62	0.03	1.65	1.58	0.07
16	1.88	1.57	0.31	1.68	1.63	0.05	1.80	1.59	0.21
17	1.90	1.59	0.31	1.70	1.71	−0.01	1.82	1.65	0.17

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Figure 2 Plot of predicted pMIC_ab values against observed pMIC_ab values for the multiple linear regression developed model by Eq. (2).

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Figure 3 Plot of residual pMIC_ab values against observed pMIC_ab values for the multiple linear regression developed model by Eq. (2).

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The antifungal activity of benzodiazepine derivatives was best described by the topological parameter, valence zero order molecular connectivity index (⁰χ^v), (Table 6, Eq. (3)).

3.5.1.3

3.5.1.3 LR mt-QSAR model for antifungal activity

(3)

$${\text{pMIC}}_{\text{af}}={0.0541}^0{\chi }^{\text{v}}+0.3798$$ $$n=14r=0.783q^2=0.170s=0.036F=19.12$$

Antifungal activity of synthesized benzodiazepine derivatives is positively correlated with valence zero order molecular connectivity index (⁰χ^v) which means that antifungal potential of synthesized compounds will increase with an increase in ⁰χ^v values.

The molecular connectivity index, an adjacency based topological index proposed by Randic is denoted by χ and is defined as sum over all the edges (ij) as per following $$\chi ={\displaystyle \sum _{i=1}^n}{(V_i-V_j)}^{-1/2}$$ where V_i and V_j are the degrees of adjacent vertices i and j and n is the number of vertices in a hydrogen suppressed molecular structure (Lather and Madan, 2005). The topological index, χ signifies the degree of branching, connectivity of atoms and unsaturation in the molecule which accounts for variation in activity (Gupta et al., 2003).

In search of better QSAR model, we coupled valence zero order molecular connectivity index (⁰χ^v) with dipole moment (μ) which increased r value from 0.783 to 0.821.

3.5.1.4

3.5.1.4 MLR mt-QSAR model for antifungal activity

(4)

$${\text{pMIC}}_{\text{af}}={0.0494}^0{\chi }^{\text{v}}+0.005\mu +0.455$$ $$n=14r=0.821q^2=0.231s=0.034F=11.42$$

The validity and predictability of the QSAR model for antifungal activity i.e. Eq. (4) was cross validated by q² value (q² = 0.231) obtained by leave one out (LOO) method. The value of q² less than 0.5 indicated that the developed model is an invalid one. But one should not forget the recommendations of Golbraikh and Tropsha (2002) who reported that the only way to estimate the true predictive power of a model is to test their ability to predict accurately the biological activities of compounds. As the observed and predicted values are close to each other (Table 7), the mt-QSAR model for antifungal activity (Eq. (4)) is therefore a valid one (Golbraikh and Tropsha, 2002).

In case of antimicrobial activity also valence zero order molecular connectivity index (⁰χ^v) was proved as the most determinant parameter (Eq. (5)).

3.5.1.5

3.5.1.5 LR mt-QSAR model for antimicrobial activity

(5)

$${\text{pMIC}}_{\text{am}}={0.037}^0{\chi }^{\text{v}}+0.727$$ $$n=14r=0.870q^2=0.547s=0.0177F=37.48$$

The addition of dipole moment (μ) to valence zero order molecular connectivity index (⁰χ^v) resulted in better QSAR model (Eq. (6)) with regression coefficient value 0.914.

3.5.1.6

3.5.1.6 MLR mt-QSAR model for antimicrobial activity

(6)

$${\text{pMIC}}_{\text{am}}={0.0339}^0{\chi }^{\text{v}}+0.004\mu +0.780$$ $$n=14r=0.914q^2=0.650s=0.0151F=28.17$$

The validity and predictability of the QSAR model (Eq. (6)) is evidenced by its high q² value (0.650) as well as the low residual values (Table 7).

It was observed from mt-QSAR models (Eqs. (1)–(6)) that the antibacterial, antifungal and overall antimicrobial activities of synthesized benzodiazepine derivatives is governed by the topological parameter Kier’s alpha first order shape index (κα₁) and valence zero order molecular connectivity index (⁰χ^v).