Synthesis, antimicrobial and antioxidant activities of 2-oxo-6-phenyl-2-yl-4-(2′-phenyl-5′-substituted 1H-indol-3′-yl)-1,2-dihydro pyridin-3-carbonitriles and their derivatives

2.1 2-Oxo-4-(2′-phenyl-5′-substituted 1H-indol-3′-yl)-6-phenylpyridin-3-carbonitriles (2a–c)

A mixture of (1a–c) (0.001 mol) and ethylcyanoacetate (0.001 mol) in glacial acetic acid (30 ml) containing anhydrous ammonium acetate (0.002 mol) was refluxed for 8 h. The reaction mixture was then cooled and poured into ice cold water. The formed product was filtered off, washed with water, dried and recrystallized from ethanol.

2.2 2-Oxo-4-(2′-phenyl-5′-chloro-1H-indol-3′-yl)-6-phenylpyridin-3-carbonitrile 2a

74% yield Yellow crystals. m.p. 245–246 °C. IR (KBr): υ 3300 (indole NH), 3264 (NH), 2206 (CN), 1677 (CO). ¹H NMR (DMSO-d₆): δ = 12.1 (s, 1H, indole NH), 9.5 (s, 1H, NH), 7.2–8.2 (m, 14H, ArH) ppm; MS: (m/z) = 421, 423 (M⁺, M⁺+2). Anal. for C₂₆H₁₆N₃OCl: calcd. C, 74.11, H 3.80, N 9.98; Found: C 74.22, H 3.99, N 10.13.

2.3 2-Oxo-4-(2′-phenyl-5′-methyl-1H-indol-3′-yl)-6-phenylpyridin-3-carbonitrile 2b

72% yield Yellow powder. m.p. 239–240 °C. IR (KBr): υ 3303 (indole NH), 3268 (NH), 2201 (CN), 1687 (CO). ¹H NMR (DMSO-d₆): δ = 12.0 (s, 1H, indole NH), 9.6 (s, 1H, NH), 7.2–8.3 (m, 14H, ArH), 2.1 (s, 3H, CH₃) ppm; MS: (m/z) = 401. Anal. for C₂₇H₁₉N₃O: calcd. C, 80.80, H 4.74, N 10.47; Found: C 80.65, H 4.85, N 10.13.

2.4 2-Oxo-4-(2′-phenyl-1H-indol-3′-yl)-6-phenylpyridin-3-carbonitrile 2c

71% yield Yellow powder. m.p. 242–240 °C. IR (KBr): υ 3307 (indole NH), 3259 (NH), 2209 (CN), 1682 (CO). ¹H NMR (DMSO-d₆): δ = 12.1 (s, 1H, indole NH), 9.5 (s, 1H, NH), 7.1–8.2 (m, 15H, ArH) ppm; MS: (m/z) = 387. Anal. for C₂₆H₁₇N₃O: calcd. C, 80.62, H 4.39, N, 10.85; Found: C 80.82, H 4.48, N 11.02.

2.5 [3-Cyano-6-phenyl-4-(2′-phenyl-5′-substituted 1H-indol-3′-yl)pyridin-2-yloxy]-acetic acid ethyl ester 3a–c

A mixture of compound (2a–c) (0.001 mol) and ethyl chloroacetate (0.002 mol) in dry acetone (40 ml) containing sodium acetate (0.001 mol) was refluxed for 7 h. The solvent was removed under vaccum, the residue was poured in cold water to remove inorganic solids, filtered to obtain compound (3a–c) and recrystallized from 1,4-dioxane.

2.6 [3-Cyano-6-phenyl-4-(2′-phenyl-5′-chloro-1H-indol-3′-yl)pyridin-2-yloxy]-acetic acid ethyl ester 3a

69% yield Pale yellow powder. m.p. 198–199 °C. IR (KBr): υ 3265 (indole NH), 2210 (CN), 1674 (CO). ¹H NMR (DMSO-d₆): δ = 11.5 (s, 1H, indole NH), 7.1–8.1 (m, 14H, ArH), 4.9 (s, 2H, CH₂), 4.1 (q, 2H, CH₂), 1.6 (t, 3H, CH₃) ppm; MS: (m/z) = 507, 509 (M⁺, M⁺+2). Anal. for C₃₀H₂₂N₃O₃Cl: calcd. C, 71.00, H 4.34, N 8.28; Found: C 71.21, H 4.25, N 8.41.

2.7 [3-Cyano-6-phenyl-4-(2′-phenyl-5′-methyl-1H-indol-3′-yl)pyridin-2-yloxy]-acetic acid ethyl ester 3b

68% yield Pale yellow powder. m.p. 185–186 °C. IR (KBr): υ 3269 (indole NH), 2216 (CN), 1680 (CO). ¹H NMR (DMSO-d₆): δ = 11.6 (s, 1H, indole NH), 7.1–8.0 (m, 14H, ArH), 4.89 (s, 2H, CH₂), 4.2 (q, 2H, CH₂), 2.2 (s, 3H, CH₃), 1.7 (t, 3H, CH₃) ppm; MS: (m/z) = 487. Anal. for C₃₁H₂₅N₃O₃: calcd. C, 76.39, H 5.13, N, 8.62; Found: C 76.52, H, 5.26, N 8.75.

2.8 [3-Cyano-6-phenyl-4-(2′-phenyl-1H-indol-3′-yl)pyridin-2-yloxy]-acetic acid ethyl ester 3c

69% yield Pale yellow powder. m.p. 180–181 °C. IR (KBr): υ 3262 (indole NH), 2214 (CN), 1678 (CO). ¹H NMR (DMSO-d₆): δ = 11.4 (s, 1H, indole NH), 7.1–8.0 (m, 15H, ArH), 4.89 (s, 2H, CH₂), 4.0 (q, 2H, CH₂), 1.6 (t, 3H, CH₃) ppm; MS: (m/z) = 473 Anal. for C₃₀H₂₃N₃O₃: calcd. C, 76.10, H 4.86, N, 8.88; Found: C 76.21, H 4.59, N 8.98.

2.9 5-{[3-Cyano-4-(2′-phenyl-5′-substituted 1H-indol-3′-yl)-6-phenylpyridin-2-yloxy]methyl}-2H-1,2,4-triazol-3(4H)-thiones 4a–c

A mixture of compound (3a–c) (0.0001 mol) and thiosemicarbazide (0.0002 mol) in pyridine (20 ml) was refluxed for 5 h. The mixture was then cooled to room temperature and poured into ice cold water and acidified with few drops of acetic acid. The formed solid was filtered, washed with water dried and recrystallized from ethanol.

2.10 5-{[3-Cyano-4-(2′-phenyl-5′-chloro-1H-indol-3-yl)-6-phenylpyridin-2′-yloxy]methyl}-2H-1,2,4-triazol-3(4H)-thione 4a

65% yield Brown solid. m.p. 285–286 °C. IR (KBr): υ 3180 (NH), 3132 (indole NH), 3105 (NH), 2206 (CN). ¹H NMR (DMSO-d₆): δ = 11.6 (s, 1H, indole NH), 10.0 (s, 1H, triazole NH), 9.6 (s, 1H, triazole NH), 7.1–7.9 (m, 14H, ArH), 4.0 (s, 2H, CH₂) ppm; MS: (m/z) = 534, 536 (M⁺, M⁺+2). Anal. for C₂₉H₁₉N₆OSCl: calcd. C, 65.17, H 3.56, N 15.73; Found: C 65.31, H 3.71, N 15.92.

2.11 5-{[3-Cyano-4-(2′-phenyl-5′-methyl-1H-indol-3′-yl)-6-phenylpyridin-2-yloxy]methyl}-2H-1,2,4-triazol-3(4H)-thione 4b

68% yield Brown solid. m.p. 276–278 °C. IR (KBr): υ 3182 (NH), 3135 (indole NH), 3110 (NH), 2214 (CN). ¹H NMR (DMSO-d₆): δ = 11.5 (s, 1H, indole NH), 10.1 (s, 1H, triazole NH), 9.5 (s, 1H, triazole NH), 7.1–7.8 (m, 14H, ArH), 4.1 (s, 2H, CH₂), 2.2 (s, 3H, CH₃) ppm; MS: (m/z) = 514. Anal. for C₃₀H₂₂N₆OS: calcd. C, 70.04, H 4.28, N 16.34; Found: C 70.25, H 4.53, N 16.13.

2.12 5-{[3-Cyano-4-(2′-phenyl-1H-indol-3′-yl)-6-phenylpyridin-2-yloxy]methyl}-2H-1,2,4-triazol-3(4H)-thione 4c

64% yield Brown solid. m.p. 281–282 °C. IR (KBr): υ 3187 (NH), 3125 (indole NH), 3103 (NH), 2209 (CN). ¹H NMR (DMSO-d₆): δ = 11.5 (s, 1H, indole NH), 10.0 (s, 1H, triazole NH), 9.5 (s, 1H, triazole NH), 7.2–7.9 (m, 15H, ArH), 4.1 (s, 2H, CH₂) ppm; MS: (m/z) = 534. Anal. for C₂₉H₂₀N₆OS: calcd. C, 69.60, H 4.00, N 16.80; Found: C 69.31, H 4.11, N 16.72.

2.13 2-(6-Oxo-5,6-dihydro-thiazolo[3,2,-b][1,2,4]triazol-2-yl methoxy)-4-(2′-phenyl-5′-substituted 1H-indol-3′-yl)-6-phenylpyridine-3-carbonitriles 5a–c

Compound (4a–c) (0.0001 mol) and chloroacetic acid (0.0001 mol) were refluxed in glacial acetic acid (9 ml) and acetic anhydride (3 ml) mixture for 4 h. Then the reaction mixture was cooled to room temperature and poured on to crushed ice. The formed solid was filtered, washed with water, dried and recrystallized from ethanol.

2.14 2-(6-Oxo-5,6-dihydro-thiazolo[3,2,-b][1,2,4]triazol-2-yl methoxy)-4-(2′-phenyl-5′-chloro-1H-indol-3′-yl)-6-phenylpyridin-3-carbonitrile 5a

69% yield Pale yellow crystals. m.p. 165–166 °C. IR (KBr): υ 3265 (indole NH), 2216 (CN), 1713 (CO). ¹H NMR (DMSO-d₆): δ = 11.6 (s, 1H, indole NH), 7.1–8.1 (m, 14H, ArH), 5.0 (s, 2H, CH₂), 4.0 (s, 2H, thiazole CH₂) ppm; MS: (m/z) = 574, 576 (M⁺, M⁺+2). Anal. for C₃₁H₁₉N₆O₂SCl: calcd. C, 64.80, H 3.31, N 14.63; Found: C 64.92, H 3.43, N 14.82.

2.15 2-(6-Oxo-5,6-dihydro-thiazolo[3,2,-b][1,2,4]triazol-2-yl methoxy)-4-(2′-phenyl-5′-methyl-1H-indol-3′-yl)-6-phenylpyridin-3-carbonitrile 5b

68% yield. Pale yellow crystals m.p. 169–170 °C. IR (KBr): υ 3260 (indole NH), 2212 (CN), 1715 (CO). ¹H NMR (DMSO-d₆): δ = 11.5 (s, 1H, indole NH), 7.1–8.0 (m, 14H, ArH), 5.1 (s, 2H, CH₂), 4.1 (s, 2H, thiazole CH₂), 2.3 (s, 3H, CH₃) ppm; MS: (m/z) = 554. Anal. for C₃₂H₂₂N₆O₂S: calcd. C, 69.31, H 3.97, N 15.16; Found: C 69.56, H 3.53, N 15.32.

2.16 2-(6-Oxo-5,6-dihydro-thiazolo[3,2,-b][1,2,4]triazol-2-yl methoxy)-4-(2′-phenyl-1H-indol-3′-yl)-6-phenylpyridin-3-carbonitrile 5c

65% yield. Pale yellow powder m.p. 162–163 °C. IR (KBr): υ 3263 (indole NH), 2215 (CN), 1717 (CO). ¹H NMR (DMSO-d₆): δ = 11.6 (s, 1H, indole NH), 7.2–8.0 (m, 15H, ArH), 5.0 (s, 2H, CH₂), 4.2 (s, 2H, thiazole CH₂) ppm; MS: (m/z) = 540. Anal. for C₃₁H₂₀N₆O₂S: calcd. C, 68.89, H 3.70, N 15.56; Found: C 68.76, H 3.63, N 15.39.

2.17 [3-Cyano-6-phenyl-4-(2′-phenyl-5′-substituted 1H-indol-3′-yl)pyridin-2-yloxy]-acetic acid hydrazides 6a–c

A mixture of compound (3a–c) (0.0001 mol) and hydrazine hydrate (99%) (0.0004 mol) was refluxed in 1,4-dioxane (30 ml) for 5 h. The excess of solvent was removed under reduced pressure. After cooling, the formed product was filtered off, washed with cold 1,4-dioxane, dried and recrystallized from methanol.

2.18 [3-Cyano-6-phenyl-4-(2′-phenyl-5′-chloro-1H-indol-3′-yl)pyridin-2-yloxy]-acetic acid hydrazide 6a

71% yield. Yellow crystals m.p. 210–211 °C. IR (KBr): υ 3418 (NH₂), 3260 (indole NH), 3141 (NH), 2211 (CN), 1680 (CO). ¹H NMR (DMSO-d₆): δ = 11.5 (s, 1H, indole NH), 8.3 (s, 1H, CONH), 7.2–8.0 (m, 14H, ArH), 4.8 (s, 2H, CH₂) ppm; MS: (m/z) = 493, 495 (M⁺, M⁺+2) Anal. for C₂₈H₂₀N₅O₂Cl: calcd. C, 68.15, H 4.06, N 14.20; Found: C 68.29, H 4.19, N 14.35.

2.19 [3-Cyano-6-phenyl-4-(2′-phenyl-5′-methyl-1H-indol-3′-yl)pyridin-2-yloxy]-acetic acid hydrazide 6b

72% yield. Yellow crystals m.p. 215–216 °C. IR (KBr): υ 3419 (NH₂), 3263 (indole NH), 3148 (NH), 2217 (CN), 1685 (CO). ¹H NMR (DMSO-d₆): δ = 11.6 (s, 1H, indole NH), 8.4 (CONH), 7.2–8.1 (m, 14H, ArH), 4.7 (s, 2H, CH₂), 2.3 (s, 3H, CH₃) ppm; MS (m/z) = 473. Anal. for C₂₉H₂₃N₅O₂: calcd. C, 73.57, H 4.86, N 14.80; Found: C 73.35, H 4.59, N 14.65.

2.20 [3-Cyano-6-phenyl-4-(2′-phenyl-1H-indol-3′-yl)pyridin-2-yloxy]-acetic acid hydrazide 6c

71% yield. Yellow crystals m.p. 205–207 °C. IR (KBr): υ 3412 (NH₂), 3262 (indole NH), 3144 (NH), 2218 (CN), 1686 (CO). ¹H NMR (DMSO-d₆): δ = 11.4 (s, 1H, indole NH), 8.4 (CONH), 7.2–7.9 (m, 15H, ArH), 4.9 (s, 2H, CH₂) ppm; MS: (m/z) = 459. Anal. for C₂₈H₂₁N₅O₂: calcd. C, 73.20, H 4.58, N 15.25; Found: C 73.36, H 4.69, N 15.35.

2.21 1-{2-[4-(2′-Phenyl-5′-substituted 1H-indol-3′-yl)-3-cyano-6-phenylpyridin-2-yloxy]acetyl}-1,2-dihydro-3-methylpyrazol-5-ones 7a–c

A mixture of compound (6a–c) (0.0001 mol), ethylacetoacetate (0.0001 mol), anhydrous NaHCO₃ (0.8 g), and anhydrous Na₂SO₄ (0.8 g) in 1,4-dioxane (35 ml) was refluxed for 10 h. Then excess of solvent was removed under reduced pressure, cooled to room temperature and the residue was poured into ice cold water. The product thus separated was filtered, washed with water and recrystallized from 1,4-dioxane.

2.22 1-{2-[4-(2′-Phenyl-5′-chloro-1H-indol-3′-yl)-3-cyano-6-phenylpyridin-2-yloxy]acetyl}-1,2-dihydro-3-methylpyrazol-5-one 7a

65% yield. Yellow needles m.p. 256–257 °C. IR (KBr): υ 3264 (indole NH), 3100 (NH), 2216 (CN), 1713 (CO), 1702 (CO). ¹H NMR (DMSO-d₆): δ = 11.4 (s, 1H, indole NH), 10.0 (s, 1H, NH), 7.1–8.2 (m, 15H, 14ArH, pyrazolone-H), 4.9 (s, 2H, CH₂), 1.6 (s, 3H, CH₃) ppm; MS: (m/z) = 559, 561 (M⁺, M⁺+2) Anal. for C₃₂H₂₂N₅O₃Cl: calcd. C, 68.69, H 3.94, N 12.52; Found: C 68.85, H 4.02, N 12.68.

2.23 1-{2-[4-(2′-Phenyl-5′-methyl-1H-indol-3′-yl)-3-cyano-6-phenylpyridin-2-yloxy]acetyl}-1,2-dihydro-3-methylpyrazol-5-one 7b

64% yield. Yellow needles. m.p. 261–262 °C. IR (KBr) υ: 3268 (indole NH), 3110 (NH), 2220 (CN), 1715 (CO), 1695 (CO). ¹H NMR (DMSO-d₆): δ = 11.5 (s, 1H, indole NH), 10.1 (s, 1H, NH), 7.0–8.2 (m, 15H, 14ArH, pyrazolone-H), 4.8 (s, 2H, CH₂), 1.6 (s, 3H, CH₃), 2.2 (s, 3H, CH₃) ppm; MS: (m/z) = 539. Anal. for C₃₃H₂₅N₅O₃: calcd. C, 73.47, H 4.64, N 12.99; Found: C 73.63, H 4.82, N 12.78.

2.24 1-{2-[4-(2′-Phenyl-1H-indol-3′-yl)-3-cyano-6-phenylpyridin-2-yloxy]acetyl}-1,2-dihydro-3-methylpyrazol-5-one 7c

66% yield. Yellow needles. m.p. 269–270 °C. IR (KBr): υ 3258 (indole NH), 3105 (NH), 2214 (CN), 1712 (CO), 1698 (CO). ¹H NMR (DMSO-d₆): δ = 11.4 (s, 1H, indole NH), 10.1 (s, 1H, NH), 7.1–8.0 (m, 16H, 15ArH, pyrazolone-H), 4.9 (s, 2H, CH₂), 1.6 (s, 3H, CH₃) ppm; MS: (m/z) = 525. Anal. for C₃₂H₂₃N₅O₃: calcd. C, 73.14, H 4.38, N 13.33; Found: C 73.33, H 4.52, N 13.57.

2.25 1-{2-[3-Cyano-6-phenyl-4-(2′-phenyl-5′-substituted 1H-indol-3′-yl)pyridin-2-yloxy]acetyl}-3-amino-1H-pyrazol-5(4H)-ones 8a-c

A mixture of compound (6a–c) (0.0001 mol) ethylcyanoacetate (0.0001 mol), anhydrous NaHCO₃ (0.8 g) and anhydrous Na₂SO₄ (0.8 g) in 1,4-dioxane (30 ml) was refluxed for 9 h. The reaction mixture was cooled to room temperature and poured onto ice cold water, the solid thus separated was filtered off, washed with cold water, dried and recrystallized from ethanol.

2.26 1-{2-[3-Cyano-6-phenyl-4-(2′-phenyl-5′-chloro-1H-indol-3′-yl)pyridin-2-yloxy]acetyl}-3-amino-1H-pyrazol-5(4H)-one 8a

61% yield. Yellow needles. m.p. 221–222 °C. IR (KBr): υ 3400 (NH₂), 3263 (indole NH), 2216 (CN), 1713 (CO), 1690 (CO). ¹H NMR (DMSO-d₆): δ = 11.5 (s, 1H, indole NH), 7.0–8.2 (m, 16H, 14ArH and NH₂), 4.9 (s, 2H, OCH₂CO), 4.2 (s, 2H, pyrazole CH₂CO) ppm; MS: (m/z) = 560, 562 (M⁺, M⁺+2). Anal. for C₃₁H₂₁N₆O₃Cl: calcd. C, 66.43, H 3.73, N 15.00; Found: C 64.57, H 3.87, N 15.15.

2.27 1-{2-[3-Cyano-6-phenyl-4-(2′-phenyl-5′-methyl-1H-indol-3′-yl)pyridin-2-yloxy]acetyl}-3-amino-1H-pyrazol-5(4H)-one 8b

62% yield. Yellow needles. m.p. 225–226 °C. IR (KBr): υ 3405 (NH₂), 3263 (indole NH), 2216 (CN), 1718 (CO), 1695 (CO). ¹H NMR (DMSO-d₆): δ = 11.4 (s, 1H, indole NH), 7.1–8.2 (m, 16H, 14ArH and NH₂), 4.8 (s, 2H, OCH₂CO), 4.1 (s, 2H, pyrazole CH₂CO), 2.3 (s, 3H, CH₃) ppm; MS: (m/z) = 540. Anal. for C₃₂H₂₄N₆O₃: calcd. C, 71.11, H 4.44, N 15.56; Found: C 71.29, H 4.67, N 15.73.

2.28 1-{2-[3-Cyano-6-phenyl-4-(2′-phenyl-1H-indol-3′-yl)pyridin-2-yloxy]acetyl}-3-amino-1H-pyrazol-5(4H)-one 8c

58% yield. Yellow powder. m.p. 216–217 °C. IR (KBr): υ 3409 (NH₂), 3263 (indole NH), 2218 (CN), 1712 (CO), 1696 (CO). ¹H NMR (DMSO-d₆): δ = 11.4 (s, 1H, indole NH), 7.0–8.1 (m, 17H, 15ArH and NH₂), 4.8 (s, 2H, OCH₂CO), 4.3 (s, 2H, pyrazole CH₂CO) ppm; MS:(m/z) = 526. Anal. for C₃₁H₂₂N₆O₃: calcd. C, 70.72, H 4.18, N 15.97; Found: C 70.51, H 4.37, N 16.11.

4.1 Ferric ions (Fe³⁺) reducing antioxidant power (FRAP)

Figs. 1–3 show the reducing power of synthesized compounds (2–8) examined as a function of their concentration. In this assay, the yellow color of the test solution changes to various shades of green and blue depending upon the reducing power of each compound. The presence of reducer (i.e. antioxidant) causes the reduction of the Fe⁺³/Ferricyanide complex to the ferrous form giving after the addition of trichloroacetic acid and ferric chloride, the perls Prussian blue that can be monitored at 700 nm. The reducing power of test compounds increases with increase in concentration. Compounds 2–6 and 8 exhibited very low absorption, whereas compounds 7a–c were found to have higher absorption but lower than the standards.

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Figure 1

Reducing power of compounds 2–8.

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Figure 2

Reducing power of compounds 2–8.

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Figure 3

Reducing power of compounds 2–8.

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The presence of enamine and α,β-unsaturated ketonic functions in the pyrazolone moiety in addition to the alkyl group may play an important role to act as a better electron donor which may enhance reducing power ability of 7. Where as such systems are not available in the rest of the test compounds. Compound 7a exhibited the maximum absorption value compare to 7b and 7c. This may be due to the chloro-substitution at 5-position of indole, which may be helping for stabilization of the free radical form after donating electron and thus to leading maximum reducing ability compared to 7b and 7c.

4.2 Radical scavenging activity (RSA)

Free radical scavenging is one of the best known mechanisms by which antioxidants inhibit lipid oxidation. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity evaluation is standard assay in antioxidant activity studies and offers a rapid technique for screening the radical scavenging activity (RSA) of specific compounds. The RSA of synthesized compounds (2–8) were tested using methanolic solution of the stable free radical DPPH. The results of the tested compounds were shown in Figs. 4–6. The freshly prepared DPPH solution exhibits a deep purple color with absorption maxima at 517 nm. The purple color generally fades/disappears when an antioxidant is present in the medium. Thus, antioxidant molecule can quench DPPH free radical (i.e. by providing hydrogen atoms or by electron donation, conceivably via a free radical attack on DPPH molecule) and convert them to a colorless/bleached product, resulting in absorbance at 517 nm. Therefore, the more rapidly the absorbance decreases, the more potent the antioxidant activity of the compounds. Hence this method is based on the reduction of alcoholic DPPH solution in the presence of a hydrogen-donating antioxidant due to the formation of the non-radical form DPPH-H by the reaction.

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Figure 4

DPPH radical scavenging activity of compounds 2–8.

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Figure 5

DPPH radical scavenging activity of compounds 2–8.

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Figure 6

DPPH radical scavenging activity of compounds 2–8.

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In the case of thiazolo[3,2,-b][1,2,4]triazoles derivative 5a, this showed highest DPPH scavenging activity with percent inhibition of 78.85 at concentration of 75 μg/ml, when compared with the other compounds. This increased activity may be due to existence of the CH₂CO group. The presence of cyano group was inadequate for antioxidant activity. All the compounds containing pyridine ring showed low RSA activity. Hence presence of cyano and pyridine substituents may drastically decrease the RSA. None of the compounds showed good RSA activity than standards (see Scheme 1).

4.3 Antimicrobial activities

The results of antimicrobial activity evaluations (Table 1) reveals that substances 4a, 5a and 7b exhibited maximum inhibitory activity against Pseudomonas aeruginosa. Compound 4a, 5a and 8a exhibited maximum zone of inhibition against gram negative bacteria. In addition, compound 4a, 5b and 8a showed maximum inhibitory growth against Aspergillus oryzae and compound 5a showed maximum inhibitory growth against Aspergillus niger. In addition, the results indicate that, rest of the compounds exhibited moderate growth inhibitory activities against all the four tested microbes.

The majority of the compounds containing indole, phenyl, 1,2,4-triazole, thiozolo[3,2,-b][1,2,4]triazole and pyrazolone in the ring system that incorporate the pyridine moiety had growth inhibitory activities. It is worth mentioning that compound (4a–c) and (5a–c) possessing 1,2,4-triazole ring appended to the pyridine ring system via methyloxy-linker showed a high antibacterial activity. In contrast, attachment of pyrazole nucleus to the pyridine nucleus via acetyloxy-linker exhibited potent antifungal activity against the two organisms used. Compound 5a exhibited consistent antimicrobial activity towards all the organisms tested. Hence, several synthesized compounds exhibited comparable activity to the gentamycin and fluconazole. However, none of the compounds exhibited zone of inhibition more than the standards.

5.1 Reducing power assay

The reducing power of the synthesized compounds was determined according to the method of Oyaizu (1986). Different concentrations of the samples (25–100 μg/ml) in DMSO (1 ml) were mixed with phosphate buffer (2.5 ml, 0.2 mol, pH = 6.6) and potassium ferricyanide (2.5 ml, 1%). The mixture was incubated at 50 °C for 20 min. after which a portion (2.5 ml) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged for 10 min. at 1000g. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃ (0.5 ml, 0.1%), and then the absorbance at 700 nm was measured in a spectrophotometer. BHA, TBHQ and ascorbic acid (A. A) were used as standards.

5.2 Radical scavenging activity (RSA) assay

The free radical scavenging activity of (2a–c)–(8a–c) was carried out in the presence of the stable free radical DPPH following Hatano’s method (Hatano et al., 1988) using 2-tert-butyl-4-methoxyphenol (butylated hydroxyl anisole, BHA), 2-(1,1-dimethylethyl)-1,4-benzenediol (2-tert. butyl hydroquinone, TBHQ) and ascorbic acid as standards. The radical scavenging activity (RSA) for methanolic solutions of compounds (2a–c)–(8a–c) at concentrations 25, 50, 75 and 100 μg/ml containing freshly prepared DPPH solution (0.004% w/v) was carried out and compared with those of standards BHA and TBHQ. All the test analyses were performed on three replicates and results are averaged. The results in percentage are expressed as the ratio of absorption decrease of DPPH in the presence of test compounds and absorption of DPPH in the absence of test compounds at 517 nm using ELICO SL 171 Mini Spec spectrophotometer. The percentage scavenging activity of the DPPH free radical was measured using the following equation $$\%\text{DPPH radical scavenging}=\frac{\text{Absorbance of control}-\text{Absorbence of test sample}}{\text{Absorbance of control}}\times 100$$

5.3 Antimicrobial assay

The antimicrobial activities (Indian Pharmacopoeia, 1985) of the compounds prepared in this effort were evaluated using P. aeruginosa (Gram negative bacteria), Klebsiella pneumoniae (gram negative bacteria), A. oryzae and A. niger (fungi). An aliquot 0.1 ml of each bacterial strain was on nutrient agar while 0.1 ml of the fungal spore suspension was spread on potato dextrose agar (PDA). An agar-well diffusion test was performed in each case. In these tests, 6 mm wells were produced by using a sterile cork borer and each well was then inoculated with 100 μl of each key substance in DMF, resulting in a final concentration of 250 μg/ml, 125 250 μg/ml and 62.5 250 μg/ml. Nutrient agar plates were incubated at 37 °C for 24 h while the PDA plates incubated at 25 °C for 72 h. The zone of inhibition around the well was determined. Gentamycin and flucanozole were used as the reference antibacterial and antifungal agents, respectively.