INTRODUCTION:

Benzimidazole nucleus a constituent of Vitamin-B₁₂¹, exhibit a wide range of biological activities². Benzimidazole derivatives are structural isosters of naturally existing nucleotides, which allow them to interact easily with the biopolymers of the living system. Some benzimidazole derivatives were show broad spectrum antimicrobial activity against various strains of microorganisms such as Staphylococcus aureus, Salmonella typhimurium, Enterococcus, Candida albicans and Pseudomonas aeruginosa^3-7. 2-Mercaptobenzimidazole and its derivatives were possessing diverse biological activities such as antibacterial⁸, antifungal⁹, antiviral^{10, 11}, antiulcerative¹², antitumor¹³, antioxidant ¹⁴, absorbents¹⁵ and important industrial inhibitors^{16, 17}. Benzimidazole-2-sulphides exhibit antimicrobial, analgesic and anti-inflammatory activities^18-23. Introducing nitro substituted phenyl ring increases antibacterial and antifungal activity of heterocyclic systems²⁴. Larry K. Keefer et al. found that the presence of 2,4-dinitrophenyl group enhances the antitumor activity both in vitro and in vivo²⁵.

Insertion of dialkylaminomethyl side chain on benzimidazole nucleus was found to enhance the antibacterial, antifungal and anti-inflammatory activities^26-29. It is therefore, planned to introduce both the 2,4-dinitrophenyl and dialkylaminomethyl side chain into mercaptobenzimidazole skeleton and screen the compounds for the antimicrobial activities. We now report for the first time the synthesis and antimicrobial activities of 1-(N,N-disubstituted)aminomethyl-2-(2,4-dinitrophenyl)sulphanyl-6-substituted-1H-benzimidazoles in continuation of our work on Mannich reactions of substituted benzimidazoles.

MATERIAL AND METHODS:

Instrumentation:

Melting points of the synthesized Mannich bases were determined with an electro thermal melting point apparatus (Seatal Scientific Ltd.) and are uncorrected. Reactions under microwave assisted conditions were carried out using Q Pro-M microwave sample preparation system. Microwaves are generated by magnetron at a frequency of 2450 MHz having an output energy range of 100-500 W and a fiber optic sensor for temperature control. The reactions were monitored by thin layer chromatography (TLC) using silica gel-G coated Al-plates (0.5mm thickness, Merck) and spots were visualized by exposing dry plates to UV light or iodine vapours. Mannich bases were purified by column chromatography using suitable solvents mixture as eluant. IR spectra (υ in cm^-1) were recorded on FT-IR spectrometer using KBr pallets, ¹H NMR (δ in ppm) spectra were recorded on 200 MHz / 400 MHz instrument using CDCl₃ or DMSO-d₆ as the solvent, TMS as the internal reference and mass spectra were acquired on a Jeol TMS D-300 spectrometer operating at 75 eV. The C; H; N and S elemental analysis of compounds were determined. The analytical data obtained was found to be in good agreement with the calculated values.

General procedure for the synthesis of compounds:

Synthesis of 5(or 6)-(un)substituted-1H-benzimidazole-2-thiols (5-8)³⁰.

4-(Un)substituted o-phenylene diamine (0.019 mol) and water (3 mL) were added to solution of sodium hydroxide (0.022 mol) in ethanol (20 mL) and carbon disulfide (0.022 mol). The mixture was heated under reflux for 3 h. Charcoal was then added cautiously and the mixture was further refluxed for 10 min. The charcoal was removed by filtration. The filtrate was heated to 60-70 °C and quenched with warm water (70 °C, 20 mL) and then 50% acetic acid (9 mL) was added. The product separated on cooling in the refrigerator for 3 h and re-crystallized using appropriate solvent.

Synthesis of 2-[(2,4-dinitrophenyl)sulphanyl]-5(or 6)-(un)substituted-1H-benzimidazoles (10-13). Conventional method:

2,4-Dinitrochlorobenzene (9, 0.04 mol) was added to a solution of 2-mercapto-5-(un)substituted benzimidazoles (5-8; 0.04 mol) and potassium hydroxide (0.04 mol) dissolved in ethanol and water. The reaction mixture was stirred using magnetic stirrer for about 30 min and then refluxed on water bath for 1-2 h. The solvent was removed and the residue was poured in ice cold water. The product that resulted was filtered, washed thoroughly with small portions of ice-cold water and purified by re-crystallization.

Microwave assisted method:

All the reactions under microwave irradiation (MWI) conditions were completed within 2.0-5.0 min. whereas similar reactions under conventional heating on steam bath (80-100 °C) gave poor yields with comparatively longer reaction time period (2 h) demonstrating that the effect of microwave irradiation facilitates the polarization of molecules under irradiation causing rapid reaction to occur. This is in consistent with reaction mechanism, which involves polar transition state. Under microwave irradiation conditions the yields of 10-13 were high (89-94 %), where as using conventional methods the yields are comparatively low (68-85 %).

2-((2,4-dinitrophenyl)sulphanyl)-1H-benzimidazole (10): Mp 180-182 °C; Anal. Calcd for C₁₃H₈N₄O₄S: C, 49.37; H, 2.55; N, 17.71. Found: C, 49.56; H, 2.72; N, 17.08%; IR (KBr): υ (cm^-1) 3320-3449 (NH), 1610 (C=N), 1525 and 1330 (Ar-NO₂), 734 (C-S-C); ¹HNMR: d 7.25-7.53 (m, 4H); 7.79 (d, 1H, J =7.5 Hz); 8.39 (d, 1H, J = 7.5 Hz); 8.79 (s, 1H); 13.02 (br s, 1H, NH); MS: m/z: 317.0 [M+H]⁺; Mol. Wt.: 316.29.

2-((2,4-Dinitrophenyl)sulphanyl)-5(or 6)-methylbenzimidazole (11):

Mp 200-202 °C; Anal. Calcd for C₁₄H₁₀N₄O₄S: C, 50.91; H, 3.05; N, 16.96. Found: C, 51.06; H, 3.18; N, 16.68%; IR (KBr): υ (cm^-1) 3330-3459 (NH), 1620 (C=N), 1535 and 1340 (Ar-NO₂), 724 (C-S-C); ¹HNMR: d 2.5 (s, 3H) 7.13 (d, 1H); 7.50(m, 2H); 7.81(d, 1H, J =7.5 Hz); 8.39 (d, 1H, J = 7.5 Hz); 8.79 (s, 1H); 13.12 (br s, 1H, NH); MS: m/z: 331.3 [M+H]⁺; Mol. Wt.: 330.32.

2-((2,4-dinitrophenyl)sulphanyl)-5(or 6)-methoxybenzimidazole (12): Mp 210-212 °C; Anal. Calcd for C₁₄H₁₀N₄O₅S: C, 48.55; H, 2.91; N, 16.18. Found: C, 48.76; H, 2.78; N, 16.38%; IR (KBr): υ (cm^-1) 3325-3545 (NH), 1615 (C=N), 1525 and 1330 (Ar-NO₂), 730 (C-S-C); ¹HNMR: d 3.85 (s, 3H) [7.24 (d, 1H, J = 7.3Hz); 7.45 (s, 1H ); 7.68 (d, 1H, J = 7.3 Hz); 7.82 (d, 1H, J = 7.5 Hz); 8.48 (d, 1H, J = 7.5 Hz); 8.94 (s, 1H ); Ar-H]; 13.20 (br s, 1H, NH); MS: m/e: 347.0 [M+H]⁺; Mol. Wt.: 346.32.

2-((2,4-Dinitrophenyl)sulphanyl)-5(or 6)-nitrobenzimidazole (13): Mp 145-147 °C; Anal. Calcd for C₁₃H₇N₅O₆S: C, 43.22; H, 1.95; N, 19.38. Found: C, 43.56; H, 1.72; N, 19.08%; IR (KBr): υ (cm^-1) 3310-3550 (NH), 1610 (C=N), 1525 and 1330 (Ar-NO₂), 724 (C-S-C); MS: m/z: 362.0 [M+H]⁺; Mol. Wt.: 361.29.

Synthesis of Mannich bases (16a-19f). 2-[(2,4-dinitrophenyl)sulphanyl]-5(or6)-substituted-1H-benzimidazole (0.005moles) in methanol (10 mL) was stirred with paraformaldehyde (500 mg), in presence of trace of concentrated hydrochloric acid for one hour at room temperature, and appropriate secondary amine (0.006 mol) was slowly added to the reaction mixture. It was refluxed on water bath for 2 h and was filtered hot and the filtrate was concentrated. The reaction mixture was poured in ice-cold water. The product that separated was filtered, dried and purified by column chromatography.

[2-(2,4-Dinitrophenylsulphanyl)benzimidazol-1-ylmethyl] dimethylamine (16a): A mixture of 2-[(2,4-dinitrophenyl) sulphanyl]-1H-benzimidazole(0.005mol) paraformaldehyde (0.5 gm), trace of concentrated hydrochloric acid and N,N-dimethyl amine (0.006 mol) was allowed to react as per the general procedure to yield the compound. The product thus formed on purification using a column of alumina (neutral) as an adsorbent and chloroform-petroleum ether (3:1) as eluant gave yellow coloured crystals 1.45 gm, (77.9 %); mp 142-144 °C; Anal. calcd for C₁₆H₁₅N₅O₄S: C, 51.47; H, 4.05; N, 18.76. Found: C, 52.08; H, 4.24; N, 18.56%; IR (KBr): υ (cm^-1) 1595.91 (C=N), 1524.52 and 1340.19 (Ar-NO₂), 1380.19 [–CH₂-N(CH₃)₂], 744.02 (C-S-C); ¹H NMR: (CDCl₃) d 2.80 (s, 6H, -NCH₃); 4.90 (s, 2H, -N-CH₂-N-); [6.9-7.19 (m, 4H,); 7.25 (d, 1H, J = 7.4 Hz); 8.30 (d, 1H, J = 7.4 Hz); 8.75 (s, 1H); Ar-H]; MS: m/z: 374.0 [M+H]⁺; Mol. Wt.: 373.39.

[2-(2,4-Dinitrophenylsulphanyl)benzimidazol-1-ylmethyl]diethylamine (16b): 1.52 gm (75.8%); mp 178-180 °C; Anal. Calcd for C₁₈H₁₉N₅O₄S: C, 53.85; H, 4.77; N, 17.45. Found: C, 54.17; H, 5.04; N, 17.35%; IR (KBr): υ (cm^-1) 1615 (C=N), 1535 and 1340 (Ar-NO₂), 744 (C-S-C); ¹H NMR: (CDCl₃) d 1.12 (t, 6H, C-CH₃,); 2.52 (q, 4H, -NCH₂,); 4.86 (s, 2H, -N-CH₂-N-); 7.22-7.64 (m, 4H); 7.80 (d, 1H, J = 7.4 Hz); 8.45 (d, 1H J = 7.4 Hz); 8.90 (s, 1H) Ar-H]; MS: m/z: 402.1 [M+H]⁺; Mol. Wt.: 401.44.

2-[[2-(2,4-Dinitrophenylsulphanyl)benzimidazol-1-ylmethyl]-(2-hydroxyethyl)amino]ethanol (16c): 1.80 gm (83.1%); mp 200-202 °C; Anal. Calcd for C₁₈H₁₉N₅O₆S: C, 49.88; H, 4.42; N, 16.16. Found: C, 49.77; H, 4.46; N, 15.98%; IR (KBr): υ (cm^-1) 3400 (OH), 1620 (C=N), 1530 and 1345 (Ar-NO₂), 744 (C-S-C); ¹H NMR: d 2.62 (t, 4H, -NCH₂,); 3.75 (t, 4H, C-CH₂-O,); 4.96 (s, 2H, -N-CH₂-N-); 5.34 (s, 2H, OH); [7.22-7.64 (m, 4H,); 7.80 (d, 1H, J = 7.5 Hz); 8.40 (d, 1H J = 7.5 Hz); 8.85 (s, 1H) Ar-H]; MS: m/z: 434.0 [M+H]⁺; Mol. Wt.: 433.44.

2-(2,4-Dinitrophenylsulphanyl)-1-morpholin-4-ylmethyl-1H-benzimidazole (16d): 1.57 gm (75.6%), mp 143-145 °C; Anal. Calcd for C₁₈H₁₇N₅O₅S: C, 52.04; H, 4.12; N, 16.86. Found: C, 52.26; H, 4.47; N, 16.23%; IR (KBr): υ (cm^-1) 1620 (C=N), 1530 and 1335 (Ar-NO₂), 740 (C-S-C); ¹H NMR: d 2.52 (t, 4H, -NCH₂,); 3.78 (t, 4H, C-CH₂-O)) 4.96 (s, 2H, -N-CH₂-N-); [7.22-7.64 (m, 4H,); 7.80 (d, 1H, J = 7.5 Hz); 8.40 (d, 1H J = 7.5 Hz); 8.85 (s, 1H) Ar-H]; MS: m/z: 416.1 [M+H]⁺; Mol. Wt.: 415.42.

2-(2,4-Dinitrophenylsulphanyl)-1-pyrrolidin-1-ylmethyl-1H-benzimidazole (16e): 1.64 gm (82.2%); mp 193-195°C; Anal. Calcd for C₁₈H₁₇N₅O₄S: C, 65.16; H, 5.72; N, 17.27. Found: C, 65.23; H, 5.74; N, 17.04%; IR (KBr): υ (cm^-1) 1610 (C=N), 1525 and 1330 (Ar-NO₂), 724 (C-S-C); ¹HNMR: d 1.54 (m, 4H, C-CH₂-C); 2.78 (t, 4H, N-CH₂-C); 4.92 (s, 2H, -N-CH₂-N-); [7.22-7.64 (m, 4H,); 7.80 (d, 1H, J = 7.5 Hz); 8.40 (d, 1H J = 7.5 Hz); 8.85 (s, 1H) Ar-H]; MS: m/z: 400.1 [M+H]⁺; Mol. Wt.: 399.42.

2-(2,4-Dinitrophenylsulphanyl)-1-piperidin-1-ylmethyl-1H-benzimidazole (16f): 1.72 gm (83.3%); mp 199-201°C; Anal. Calcd for C₁₉H₁₉N₅O₄S: C, 59.19; H, 4.63; N, 16.94. Found: C, 59.30; H, 4.53; N, 17.23%; IR (KBr): υ (cm^-1) 1620 (C=N), 1535 and 1340 (Ar-NO₂), 734 (C-S-C); ¹HNMR: d 1.34 (m, 6H, C-CH₂-C); 2.68(t, 4H, N-CH₂-C); 4.92 (s, 2H, -N-CH₂-N-); [7.22-7.64 (m, 4H); 7.82 (d, 1H, J = 7.5 Hz); 8.45 (d, 1H J = 7.5 Hz); 8.94 (s, 1H) Ar-H]; MS: m/z: 414.1 [M+H]⁺; Mol. Wt.: 413.45.

2-[(2,4-Dinitrophenylsulphanyl)-6-methylbenzimidazol-1-ylmethyl]dimethylamine (17a): 1.45 gm, 74.9%) mp 122-125 °C; Anal. Calcd for C₁₇H₁₇N₅O₄S: C, 52.70; H, 4.42; N, 18.08. Found: C, 53.20; H, 4.20; N, 18.29%; IR (KBr): υ (cm^-1) 1615.19 (C=N), 1524.5 and 1340.01 (Ar-NO₂), 1385 [–CH₂-N(CH₃)₂], 744.5 (C-S-C); ¹HNMR: d 2.29 (s, 3H, -CH₃); 2.81 (s, 6H, -NCH₃); 4.83 (s, 2H, -N-CH₂-N-); [6.84-7.15 (m, 3H); 7.25 (d, 1H, J = 7.6 Hz); 8.45 (d, 1H, J = 7.6 Hz); 8.75 (s, 1H), Ar-H]. MS: m/z: 388.1 [M+H]⁺; Mol. Wt.: 387.29.

[2-(2,4-Dinitrophenylsulphanyl)-6-methylbenzoimidazol-1-ylmethyl]diethylamine (17b): 1.70 gm (81.9%); mp 130-133 °C; Anal. Calcd for C₁₉H₂₁N₅O₄S: C, 54.93; H, 5.09; N, 16.86. Found: C, 55.10; H, 5.28; N, 16.47%; IR (KBr): υ (cm^-1) 1625 (C=N), 1535 and 1330 (Ar-NO₂), 740 (C-S-C); ¹HNMR: d1.06 (t, 6H, C-CH₃); 2.54 (q, 4H, -NCH₂); 2.68 (s, 3H, -CH₃); 4.92 (s, 2H, -N-CH₂-N-); [7.24 (d, 1H, J = 7.5 Hz); 7.45 (s, 1H, ); 7.68 (d, 1H, J = 7.5 Hz); 7.82 (d, 1H, J=7.5 Hz); 8.48 (d, 1H, J = 7.5 Hz); 8.94 (s, 1H); Ar-H]; MS: m/z: 416.0 [M+H]⁺; Mol. Wt.: 415.47.

2-[[2-(2,4-Dinitrophenylsulphanyl)-6-methylbenzimidazol-1-ylmethyl]-(2-hydroxyethyl)amino] ethanol (17c): 1.80 gm (80.5%); mp 237-239 °C; Anal. calcd for C₁₉H₂₁N₅O₆S: C, 51.00; H, 4.73; N, 15.65. Found: C, 50.89; H, 4.86; N, 15.23%; IR (KBr): υ (cm^-1) 1620 (C=N), 1535 and 1340 (Ar-NO₂), 744 (C-S-C); ¹HNMR: d 2.62 (t, 4H, -NCH₂,); 2.68 (s, 3H, -CH₃); 3.75 (t, 4H, C-CH₂-O); 4.92 (s, 2H, -N-CH₂-N-); 5.34 (s, 2H, OH); [7.24 (d, 1H, J = 7.5 Hz) 7.45 (s, 1H, ); 7.68 (d, 1H, J = 7.5 Hz); 7.82 (d, 1H, J = 7.5 Hz); 8.48 (d, 1H, J = 7.5 Hz); 8.94 (s, 1H ); Ar-H]; MS: m/z: 448.1 [M+H]⁺; Mol. Wt.: 447.46.

2-(2,4-Dinitrophenylsulphanyl)-6-methyl-1-morpholin-4-ylmethyl-1H-benzimidazole (17d): 1.60 gm (74.6%); mp 180-183 °C; Anal. Calcd for C₁₉H₁₉N₅O₅S: C, 53.14; H, 4.46; N, 16.31. Found: C, 53.10; H, 4.80; N, 16.23%; IR (KBr): υ (cm^-1) 3300 (OH), 1620 (C=N), 1535 and 1340 (Ar-NO₂), 744 (C-S-C); ¹HNMR: d 2.52 (t, 4H, -NCH₂,); 2.68 (s, 3H, -CH₃); 3.78 (t, 4H, C-CH₂-O,); 4.92 (s, 2H, -N-CH₂-N-); [7.24(d, 1H, J=7.5Hz)7.45 (s, 1H, ); 7.68 (d, 1H, J=7.5Hz); 7.82 (d, 1H, J=7.5Hz); 8.45 (d, 1H, J=7.5Hz); 8.90 (s, 1H ); Ar-H]; MS: m/z: 430.0 [M+H]⁺; Mol. Wt.: 429.45.

2-(2,4-Dinitrophenylsulphanyl)-6-methyl-1-pyrrolidin-1-ylmethyl-1H-benzimidazole (17e): 1.65 gm (80.0%); mp 130-133 °C; Anal. Calcd for C₁₉H₁₉N₅O₄S: C, 55.19; H, 4.63; N, 16.94. Found: C, 55.00; H, 4085; N, 17.08%; IR (KBr): υ (cm^-1) 1620 (C=N), 1535 and 1340 (Ar-NO₂), 744 (C-S-C); ¹HNMR: d 1.54 (m, 4H, C-CH₂-C); 2.68 (s, 3H, -CH₃); 2.78(t, 4H, N-CH₂-C); 4.92 (s, 2H, -N-CH₂-N-); [7.25-7.68 (m, 3H); 7.82 (d, 1H, J = 7.5 Hz); 8.48 (d, 1H, J = 7.5 Hz); 8.94 (s, 1H ); Ar-H]; MS: m/z: 414.0 [M+H]⁺; Mol. Wt.: 413.45.

2-(2,4-Dinitrophenylsulphanyl)-6-methyl-1-piperidin-1-ylmethyl-1H-benzimidazole (17f): 1.70 gm (79.6%); mp 135-138 °C; Anal. Calcd for C₂₀H₂₁N₅O₄S: C, 56.19; H, 4.95; N, 16.38. Found: C, 56.33; H, 5.10; N, 16.13%; IR (KBr): υ (cm^-1) 1620 (C=N), 1535 and 1330 (Ar-NO₂), 744 (C-S-C); MS: m/z: 428.1 [M+H]⁺; Mol. Wt.: 427.48.

[2-(2,4-Dinitrophenylsulphanyl)-6-methoxybenzimidazol-1-ylmethyl]dimethylamine (18a): 1.80 gm (89.3%) mp 121-123 °C; Anal. Calcd for C₁₇H₁₇N₅O₅S: C, 50.61; H, 4.25; N, 17.36. Found: C, 50.47; H, 4.39; N, 17.23%; IR (KBr): υ (cm^-1) 1620 (C=N), 1535 and 1340 (Ar-NO₂), 1385.5 [–CH₂-N(CH₃)₂], 744 (C-S-C); ¹HNMR: d 2.80 (s, 6H, -NCH₃); 3.84 (s, 3H, -OCH₃); 4.92 (s, 2H, -N-CH₂-N-); [6.82-7.2 (m, 3H,); 7.25 (d, 1H, J = 7.5 Hz); 8.45 (d, 1H, J = 7.5 Hz); 8.75 (s, 1H); Ar-H]; MS: m/z: 404.1 [M+H]⁺; Mol. Wt.: 403.41.

[2-(2,4-Dinitrophenylsulphanyl)-6-methoxy-benzimidazol-1-ylmethyl]diethylamine (18b): 1.79 gm (83.0%); mp 130-132 °C; Anal. Calcd for C₁₉H₂₁N₅O₅S: C, 52.89; H, 4.91; N, 16.23. Found: C, 52.78; H, 4.30; N, 15.99%; IR (KBr): υ (cm^-1) 1630 (C=N), 1535 and 1330 (Ar-NO₂), 1070 (C-O), 735 (C-S-C); ¹HNMR: d1.16 (t, 6H, C-CH₃,); 2.54 (q, 4H, -NCH₂, ); 3.84 (s, 3H, -OCH₃); 4.92 (s, 2H, -N-CH₂-N-); [7.24 (d, 1H, J = 7.3 Hz); 7.45 (s, 1H, ); 7.68 (d, 1H, J = 7.3 Hz); 7.82 (d, 1H, J = 7.5 Hz); 8.48 (d, 1H, J = 7.5 Hz); 8.94 (s, 1H ); Ar-H]; MS: m/z: 432.1 [M+H]⁺; Mol. Wt.: 431.47.

2-[[2-(2,4-Dinitrophenylsulphanyl)-6-methoxybenzimidazol -1-ylmethyl]-(2-hydroxyethyl)amino] ethanol (18c): 1.96 gm (84.6%); mp 148-151 °C; Anal. Calcd for C₁₉H₂₁N₅O₇S: C, 49.24; H, 4.57; N, 15.11. Found: C, 49.12; H, 4.54; N, 15.10%; IR (KBr): υ (cm^-1) 3370 (OH), 1630 (C=N), 1535 and 1330 (Ar-NO₂), 1070 (C-O), 734 (C-S-C); ¹HNMR: d 2.62 (t, 4H, -NCH₂,); 3.75 (t, 4H, C-CH₂-O); 3.86 (s, 3H, -OCH₃); 4.92 (s, 2H, -N-CH₂-N-); 5.34 (s, 2H, OH); [7.14-8.38 (m, 5H); 8.94 (s, 1H); Ar-H]; MS: m/z: 464.0 [M+H]⁺; Mol. Wt.: 463.46.

2-(2,4-Dinitrophenylsulphanyl)-6-methoxy-1-morpholin-4-ylmethyl-1H-benzimidazole (18d): 1.85 gm (83.14%); mp 110-115 °C; Anal. Calcd for C₁₉H₁₉N₅O₆S: C, 51.23; H, 4.30; N, 15.72. Found: C, 51.47; H, 4.19; N, 15.23%; IR (KBr): υ (cm^-1) 1625 (C=N), 1525 and 1330 (Ar-NO₂), 1085 (C-O), 735 (C-S-C); ¹HNMR: d 2.52 (t, 4H, -NCH₂,); 3.78 (t, 4H, C-CH₂-O); 3.85 (s, 3H, -OCH₃); 4.92 (s, 2H, -N-CH₂-N-); [7.24 - 8.28 (m, 5H), 8.98 (s, 1H) Ar-H]; MS: m/z: 446.0 [M+H]⁺; Mol. Wt.: 445.45.

2-(2,4-Dinitrophenylsulphanyl)-6-methoxy-1-pyrrolidin-4-ylmethyl-1H-benzimidazole (18e): 1.65 gm (81.9%); mp 130-132 °C; Anal. Calcd for C₁₉H₁₉N₅O₅S: C, 53.14; H, 4.46; N, 16.31. Found: C, 53.40; H, 4.76; N, 16.23%; IR (KBr): υ (cm^-1) 1610 (C=N), 1525 and 1330 (Ar-NO₂), 1075 (C-O), 744 (C-S-C); ¹HNMR: d1.54 (m, 4H, C-CH₂-C); 2.78(t, 4H, N-CH₂-C); 3.86 (s, 3H, -OCH₃); 4.92 (s, 2H, -N-CH₂-N-); [7.24-8.48 (m, 5H,); 8.92 (s, 1H); Ar-H]; MS: m/z: 430.0 [M+H]⁺; Mol. Wt.: 429.45.

2-(2,4-Dinitrophenyl-sulphanyl)-6-methoxy-1-piperidin-4-ylmethyl-1H-benzimidazole (18f): 1.80 gm (83.0%); mp 138-140 °C; Anal. Calcd for C₂₀H₂₁N₅O₅S: C, 54.17; H, 4.77; N, 15.79. Found: C, 54.37; H, 4.59; N, 15.24%; IR (KBr): υ (cm^-1) 1620 (C=N), 1523 and 1338 (Ar-NO₂), 1070 (C-O), 745 (C-S-C); MS: m/z: 444.1 [M+H]⁺; Mol. Wt.: 443.48.

[2-(2,4-Dinitrophenylsulphanyl)-6-nitrobenz-imidazol-1-ylmethyl]dimethylamine (19a): 1.50 gm, 71.7 %) mp 209-211 °C; Anal. Calcd for C₁₆H₁₄N₆O₆S: C, 45.93; H, 3.37; N, 20.09. Found: C, 46.08; H, 3.17; N, 19.80%; IR (KBr): υ (cm^-1) 1630 (C=N), 1525 and 1340 (Ar-NO₂), 1390 [–CH₂-N(CH₃)₂], 754 (C-S-C); ¹HNMR: d 2.75 (s, 6H, -NCH₃); 4.85 (s, 2H, -N-CH₂-N-); [7.45 (s, 1H,); 7.68-8.48 (m, 4H,); 8.94 (s, 1H); Ar-H]; MS: m/z: 419.0 [M+H]⁺; Mol. Wt.: 418.38.

[2-(2,4-Dinitrophenylsulphanyl)-6-nitrobenzimida- zol-1-ylmethyl]diethylamine (19b): 1.75 gm (78.4%); mp 160-163 °C; Anal. Calcd for C₁₈H₁₈N₆O₆S: C, 48.43; H, 4.06; N, 18.82. Found: C, 48.47; H, 4.10; N, 18.33%; IR (KBr): υ (cm^-1) 1630 (C=N), 1525 and 1340 (Ar-NO₂), 734 (C-S-C); ¹HNMR: d 1.16 (t, 6H, C-CH₃,); 2.54 (q, 4H, -NCH₂,); 4.92 (s, 2H, -N-CH₂-N-); [7.22 - 8.28 (m, 5H,); 8.94 (s, 1H); Ar-H]; MS: m/z: 447.0 [M+H]⁺; Mol. Wt.: 446.44.

2-[[2-(2,4-Dinitrophenylsulphanyl)-6-nitrobenzimidazol-1-ylmethyl]-(2-hydroxyethyl)amino]ethanol (19c): 1.80 gm (75.3%); mp 139-142 °C; Anal. Calcd for C₁₈H₁₈N₆O₈S: C, 45.19; H, 3.79; N, 17.57. Found: C, 45.12; H, 3.87; N, 17.33%; IR (KBr): υ (cm^-1) 3340 (OH), 1630 (C=N), 1535 and 1330 (Ar-NO₂), 1040 (C-O), 730 (C-S-C); ¹HNMR: d 2.62 (t, 4H, -NCH₂,); 3.75 (t, 4H, C-CH₂-O); 4.92 (s, 2H, -N-CH₂-N-); 5.34 (s, 2H, OH); [7.24(d, 1H, J = 7.0 Hz), 7.45 (s, 1H, ); 7.68 (d, 1H, J = 7.0 Hz); 7.82 (d, 1H, J = 7.3 Hz); 8.48 (d, 1H, J = 7.3 Hz); 8.94 (s, 1H ); Ar-H]; MS: m/z: 479.0 [M+H]⁺; Mol. Wt.: 478.44.

2-(2,4-Dinitrophenysulphanyl)-6-nitro-1-morpholin-4-ylmethyl-1H-benzimidazole (19d): 1.75 gm (76.0%); mp 180-185 °C; Anal. Calcd for C₁₈H₁₆N₆O₇S: C, 46.96; H, 3.50; N, 18.25. Found: C, 46.86; H, 3.30; N, 18.45%; IR (KBr): υ (cm^-1) 1620 (C=N), 2930 (-CH₃), 1525 and 1330 (Ar-NO₂), 730 (C-S-C); ¹HNMR: d 2.52 (t, 4H, -N-CH₂,); 3.78 (t, 4H, C-CH₂-O,); 4.92 (s, 2H, -N-CH₂-N-); [7.28 - 7.68 (m, 3H, J=7.5Hz); 7.82 (d, 1H, J=7.3Hz); 8.48 (d, 1H, J=7.3Hz); 8.94 (s, 1H); Ar-H]; MS: m/z: 461.0 [M+H]⁺; Mol. Wt.: 460.42.

2-(2,4-Dinitrophenyl-sulphanyl)-6-nitro-1-pyrrolidin-1-ylmethyl-1H-benzimidazole (19e): 1.60 gm (72.1%); mp 150-154 °C; Anal. Calcd for C₁₈H₁₆N₆O₆S: C, 48.65; H, 3.63; N, 18.91. Found: C, 48.47; H, 3.26; N, 18.64%; IR (KBr): υ (cm^-1) 1630 (C=N), 1525 and 1340 (Ar-NO₂), 744 (C-S-C); ¹HNMR: d 1.54 (m, 4H, C-CH₂-C); 2.78 (t, 4H, N-CH₂-C); 4.92 (s, 2H, -N-CH₂-N-); [7.34 (d, 1H, J = 7.0 Hz); 7.45 (s, 1H, ); 7.65 (d, 1H, J = 7.0 Hz); 7.82 (d, 1H, J = 7.3 Hz); 8.48 (d, 1H, J = 7.3 Hz); 8.94 (s, 1H ); Ar-H]; MS: m/z: 445.0 [M+H]⁺; Mol. Wt.: 444.42.

2-(2,4-Dinitrophenylsulphanyl)-6-nitro-1-piperidin-1-yl-methyl-1H-benzimidazole (19f): 1.70 gm (74.2%); mp 162-165 °C; Anal. Calcd for C₁₉H₁₈N₆O₆S: C, 49.78; H, 3.96; N, 18.33. Found: C, 49.29; H, 3.35; N, 18.12%; IR (KBr): υ (cm^-1) 1620 (C=N), 1515 and 1335 (Ar-NO₂), 742 (C-S-C); MS: m/z: 459.1 [M+H]⁺; Mol. Wt.: 458.45.

ANTIBACTERIAL ACTIVITY:

The in vitro antibacterial activity of the Mannich bases 16a-19f was tested at different concentrations following Vincent and Vincent filter paper disc method³¹. The paper discs (30-40; 4mm diameter) of Whatman filter paper No.1 were kept in contact with the test compound for 4 h by which time it was completely absorbed by the discs. Subsequently they were transferred to freshly seeded nutrient agar-plates aseptically and the plates were incubated at 37 ^°C for 48 h. The diameter of zone of inhibition of growth of bacterium was determined excluding the diameter of paper discs. A minimum of ten discs were observed and the experiment was repeated at least thrice. Simultaneously, the controls were maintained employing paper discs dipped in equal amount of acetone.

ANTIFUNGAL ACTIVITY:

The in vitro antifungal activity of compounds 16a-19f was tested at different concentrations adopting glass slide humid chamber technique³² at pH 6.8. The spore concentration was so adjusted as to appear 30-40 spores for microscopic field. The experiment was conducted in quadruplicates and repeated at least three times. Simultaneously, controls were maintained by using distilled water with appropriated quantity of acetone. The controls and treatments were incubated at room temperature (27 ± 2⁰) for 24 h. At the end of incubation period, the number of spores germinated was counted in ten different randomly selected microscopic fields.

RESULTS AND DISCUSION:

Chemistry:

1H-Benzimidazole-2-thiols were prepared by refluxing respective o-phenylenediamine (1-4) with carbon disulphide in ethanol-water solution of sodium hydroxide. The reaction between 2,4-dinitrochlorobenzene (9) and 5(or 6)-(un)substituted-1H-benzimidazole-2-thiols (5-8) in presence of a base by conventional and microwave induced methods afforded 2-[(2,4-dinitrophenyl)sulphanyl]-5(or6)-(un)substituted-1H-benzimidazoles (10-13, Scheme-1). Formation of the compounds 10-13 was evidenced by the absence of C=S absorption band at 1200 cm^-1, NHabsorption band at ~ 3300 cm^-1and appearance of new absorption bands at 1520 and 1350 cm^-1 for NO₂ group in IR spectra. ¹H NMR spectra of compound 10 showed signals at δ (ppm) 7.25-7.53 (m, 4H, Ar-H), 7.79 (d, 1H, J = 7.5 Hz), 8.39 (d, 1H, J = 7.5 Hz); 8.39 (s, 1H); 13.5 (b, 1H, benzimidazole NH) confirming the formation of substituted 2-mercapto-1H-benzimidazoles.

However, two isomeric Mannich bases i.e., 1,5-isomer and 1,6-isomer were expected (Scheme-2) owing to tautomerism in substituted benzimidazoles. Experimentally only one product was identified by TLC as per early observations³³. In order to establish whether the product is 1,6-isomer or 1,5- isomer, the product 17a was subjected to hydrogenolysis using Pd-C and hydrogen (Scheme-3). Theoretically hydrogenolysis of 1,6-isomer forms 1,6-dimethylbenzimidazole and 1,5-isomer yields 1,5-dimethylbenzimidazole. Both these compounds are known in the literature^34,
35 and have been synthesized unambiguously. In this reaction the Mannich product obtained was identical with 1,6-dimethylbenzimidazole confirming that product is 1,6-isomer. By analogy, all the Mannich bases were therefore assigned as 1,6-isomers.¹H NMR spectra of Mannich bases showed a peak at δ (ppm) ~ 4.90 (s, 2H) for -N-CH₂-N-. Absence of the benzimidazole NH signal at δ ~13.5, disappearance of the NH absorption band at ~ 3300 cm^-1 and presence of molecular ion peak in the mass spectra confirmed the formation of products. All the synthesized compounds were characterized by elemental analysis, IR, NMR and Mass spectral data.

Scheme 3. Hydrogenation of Mannich bases

ANTIBACTERIAL ACTIVITY:

The synthesized N-Mannich bases 16a-19f were evaluated for antibacterial activity with Gram negative (viz., Proteus vulgaris, Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Klebsiella pneumonia) and Gram positive bacteria (viz., Staphylococcus citreus and Staphylococcus aureus). The antibacterial screening results are presented in the Table-1.

The results reveal that all newly synthesized N-Mannich bases exhibit potent antibacterial activity. Among all the compounds tested, 16a, 16b, 16c exhibited remarkable antibacterial activity against the Klebsiella pneumonia in comparison with standard drug chloramphenicol. Compound 16d is more active towards P.aeruginosa and compound 16b, 16e towards the P.vulgaris. Compounds 17a, 18a and 19b were more active against E.coli. Remaining Mannich bases were moderately active against P. aeruginosa, E.coli and S.citreus.

N-Mannich bases with 2,4-dinitrophenyl substituent at 2^nd position with no substituents on benzimidazole moiety at 5(or 6) position have shown more activity than with substituent at 5(or 6) position. Among the 5(or 6) unsubstituted benzimidazoles, the Mannich bases with acyclic secondary amines have shown more activity than the Mannich bases with cyclic secondary amines.

ANTIFUNGAL ACTIVITY:

All those compounds screened for the evaluation of antibacterial activity was also screened for antifungal activity and tested against unicellular and multicellular organisms of both human and phyto pathogenic fungi. The human pathogenic fungus that was screened for antifungal activity was Candida albicans, where as the compounds under investigation were tested against six phyto pathogenic fungi viz., Aspergillus niger, Penicillium notatum, Alternaria alternata, Drechslera avenae, Curvularia lunata and Fusarium graminearum. The antifungal screening results are presented in Table-2 and revealed that all newly synthesized Mannich bases exhibited good antifungal activity in comparison with standard anti fungal drug flucanazole. Compounds 16d and 18a were highly active against both Penicillium notatum and Curvularia sp. Compound 16e with pyrrolidino substituent was equal in its activity against Curvularia sp., but did not show much activity against remaining fungi. [2-(2,4-Dinitrophenylsulphanyl)-6-methoxybenzimidazol-1-ylmethyl]dimethyl amine (18a) was very active against both Pencillium notatum and Curvularia sp., but moderate against remaining fungi. Compounds 17d and 19a were less active and all other compounds were moderately active.

Table 1

Compound	Bacteria species
Compound	P. vulgaris	P. aeruginosa	E. coli	S. typhi	K. pneumoniae	S. citreus	S. aureus
16a	−	−	−	++	+++	+	−
16b	+++	−	−	+	+++	−	−
16c	−	−	+	+	+++	−	−
16d	++	+++	+	−	−	++	+
16e	+++	++	+	−	−	++	+
16f	+++	++	+	−	+	−	+
17a	++	++	+++	+	+	++	+
17b	+	++	+	+	++	+	+
17c	+	++	++	++	++	++	+
17d	+	++	++	−	++	++	+
17e	+	++	++	+	++	++	+
17f	−	+	+	−	++	+	−
18a	+	++	+++	−	+	++	−
18b	+	++	+	+	++	+	+
18c	+	++	++	++	++	++	+
18d	+	++	++	+	++	++	+
18e	++	++	++	+	+	++	+
18f	+	++	+	−	++	+	−
19a	+	−	++	+	++	++	+
19b	++	++	+++	+	+	++	+
19c	+	++	+	+	++	+	+
19d	+	++	++	++	++	++	+
19e	−	++	++	−	++	++	−
19f	+	++	+	+	++	+	+
Chloramphenicol	+	++	+	+	++	+++	+

Inhibition zone is shown as + or − ; [+++ = 1.5-2.0 cm, ++ = 1.0-1.5 cm, + = 0.5 -1.0 cm, − = < 0.5 cm]

Table 2

Compound	Human Pathogen	Phyto pathogen
Compound	C.albicans	A.niger	P.notatum	Alternaria	Drechslera	Curvularia	Fusarium
16a	++	++	+	−	−	+	++
16b	++	+	−	++	−	−	−
16c	++	+	+	+	−	++	+
16d	+	+	+++	+	+	+++	+
16e	++	++	+	+	+	+++	+
16f	+	++	+	++	+	+++	−
17a	++	−	−	++	+	++	+
17b	++	+	++	++	++	++	++
17c	+	+	++	++	++	++	+
17d	+	+	−	−	−	+	−
17e	−	+	−	−	++	+	++
17f	++	++	+	+	+	++	+
18a	+	+	+++	+	++	+++	+
18b	++	++	+	++	+	++	+
18c	++	++	+	+	+	+++	+
18d	+	++	+	+	+	++	+
18e	++	++	++	+	+	++	+
18f	++	+	+	+	+	+	−
19a	+	−	−	−	+	+	++
19b	++	+	++	+	++	++	+
19c	++	−	+	++	+	++	++
19d	++	+	−	++	−	+	−
19e	+	+	−	+	++	+	+
19f	+	−	+	+	−	++	−
Fluconazole	++	+	++	+	++	++	+

Inhibition zone is shown as + or − ; [+++ = 1.5-2.0 cm, ++ = 1.0-1.5 cm, + = 0.5 -1.0 cm, − = < 0.5 cm]

CONCLUSION:

2-[(2,4-Dinitrophenyl)sulphanyl]-1H-benzimidazoles were synthesized for the first time using conventional and microwave induced methods. The microwave induced method offered significant improvement in yield with shorter reaction times. A series of N-Mannich bases were synthesized and tested for antibacterial activity against seven strains of bacteria (both gram positive and gram negative) and antifungal activity against unicellular and multicellular organisms of both human and phyto pathogenic fungi. Unsubstituted Mannich bases showed more antibacterial activity than the substituted irrespective whether it is electron releasing or electron withdrawing. Among them 16a, 16b and 16c exhibited remarkable activity against the Klebsiella pneumonia. Remaining Mannich bases were moderately active against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus citreus. In general the compounds tested were shown moderate to good activity against gram negative than the gram positive. Introduction of substituent at 5or 6 position in benzimidazole molecule was found to inhibit the bacterial activity. Majority of Mannich bases have shown moderate to good activity against plant pathogen Curvularia lunata. It is in generally found that the Mannich bases of benzimidazoles under investigation were more effective on plant pathogenic fungi rather than human pathogenic fungi. Compounds 16d and 18a were very active against Penicillium notatum and Curvularia lunata. 17d and 19f are less active and all other Mannich bases have shown prominent activity on par with fluconazole.

ACKNOWLEDGMENTS:

The authors are thankful to the Principal and Management of C.K.M. Arts and Science College for providing amenities.

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Received on 03.09.2012 Modified on 23.09.2012

Asian J. Research Chem. 5(10): October, 2012; Page 1216-1224

Mohan Rao Gangula and Vijaya Kumar Baru*