Synthesis, Characteriztion and Antimicrobial Activities of Novel Pyrrolyl Benzimidazole Derivatives

Introduction

Heterocyclic compounds are pharmacologically active compounds that can be accessed in the laboratory and from natural sources. Among the new heterocyclic compounds, pyrrole has garnered considerable attention due to its potential as an antimalarial agent and enzyme inhibitor.¹ Tuberculosis is a deadly disease caused by Mycobacteria of the tuberculosis complex. TB has reemerged as one of the leading causes of death worldwide (nearly 3 million deaths annually) in the lastdecade.² A new antitubercular drug should fulfill the following criteria: (a) should have a validated safety profile; (b) should result in shorter, safer, cheaper, and more effective treatment alternatives for MDR-TB; (c) should be effective on newer targets to circumvent MDR-TB and XDR-TB; (d) must be compatible with antiretroviral therapy, for the treatment of a large population of HIV-TB coinfected patients; (e) should not result in drug interactions with other anti-TB drugs or drug candidates. Besides this, accurate diagnosis and proper screening for drug resistance are also important factors in combating TB.³

Pyrroles and their fused derivatives are an important class of naturally and synthetically occurring compounds with a wide range of biological activities: antibacterial, antifungal, antiviral, anticancer, and anti-inflammatory.^4-15 Pyrrolnitin and fludioxonil are two naturally occurring pyrroles that have been reported to exhibit broad-spectrum antifungal activity.^13,14 Pyrrole is the simplest example of an azole heterocyclic compound that contains nitrogen as a single heteroatom. Pyrrole is unique among the five-membered monoheterocyclic aromatic compounds, having electron-releasing ability through resonance. A lone pair of electrons from nitrogen takes part in the ring to form the aromatic bonding in pyrrole.

Amongst heterocyclic pharmacophores, the benzimidazole ring system is quite common. These substructures are often referred to as privileged due to their widespread occurrence in bioactive compounds. Although there is great interest in benzimidazole ligands and their structural chemistry, the primary interest lies in their biological activities. These biological activities include anticancer, bactericidal, fungicidal, analgesic, and antiviral properties. Some have cardiovascular applications, while others have been synthesized and evaluated for their inhibition of HIV-1 infectivity.¹⁶ Based on the above observation, combining pyrrole and benzimidazole may offer a synergistic effect against microbial infections.

Hence, this study was designed to address the need for safe, side-effect-free drugs with shorter treatment durations.

Materials and Methods

Chemicals used in synthesizing the titled compounds were purchased from Sigma Aldrich Pvt. Ltd., Hi-Media Laboratories, S.D. Fine Chem. Pvt Ltd and Spectrochem Pvt. Ltd. They were ortho-phenylenediamine, para-aminobenzoic acid, ethanol, 2,5-dimethoxytetrahydrofuran, glacial acetic acid, etc. All solvents and chemicals were purified before use through distillation or recrystallization. The melting points of the synthesized compounds were determined using a capillary method in a digital melting point apparatus with a paraffin bath. Thin layer chromatography (TLC) was performed on precoated TLC sheets of silica gel 60F254 (Merck, Darmstadt, Germany), visualized using both long- and short-wavelength ultraviolet lamps. FT-IR spectra were recorded on a Bruker spectrophotometer using KBr pellets, and values are expressed in cm^-1. The 1H NMR was recorded on Bruker AVANCE II 400/100 MHz instruments using deuterated trichloromethane (CDCl3 ) and dimethyl sulphoxide 6 (DMSO-d6) as solvent, and tetramethylsi lane (TMS) as internal standard; chemical shifts are expressed as δ values (ppm) and the splitting of the NMR spectra are termed as singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m).

General procedure for Synthesis of substituted (1H- benzo (d) imidazole-2-yl) aniline (3a-d):

The mixture of appropriate o-phenylenediamine (0.1 mol) and p-aminobenzoic acid (13.71 g, 0.1 mol) was refluxed in an ethanol-water bath for six hours. The reaction mixture was cooled, and later, 10% of the NaOH solution was added slowly with constant stirring until the solution was just alkaline. The crude product was filtered, washed with ice-cold water, and then recrystallized from ethanol in the presence of HCl.

Synthesis of substituted 2-(4-(1H- pyrrol-1-yl) phenyl)-1H-benzo (d) imidazole (4a-d):

2,5-Dimethoxytetrahydrofuran (16 g, 0.12 mol) was added to the appropriate (1H-benzo[d]imidazole-2-yl) aniline (0.1 mol) in glacial acetic acid (100 mL), and the mixture was heated to reflux for 30 minutes. The reaction mixture was poured into ice-cold water, and the precipitated solid was filtered and dried. Solid crude products were recrystallized from ethanol and obtained as brown crystals. Melting point 286-290˚C.

Synthesis of substituted 2-(4-(2,5-dimethly-1H- pyrrol-1-yl) phenyl)-1H-benzo (d) imidazole (5a-d):

Acetonyl acetone (13.69 g, 0.12 mol) was added to the appropriate (1H-benzo (d) imidazole-2-yl) aniline (0.1 mol) in glacial acetic acid (100 mL), and the mixture was heated to reflux for 30 mins. The reaction mixture was poured into ice-cold water, and the precipitated solid was f iltered and dried. Solid crude product was recrystallized from ethanol and obtained as brown crystals. Melting point 204-208°C.

Antibacterial activity

The minimum inhibitory concentration (MIC) of the tested compounds was determined in a side-by-side comparison with ciprofloxacin and norfloxacin against Gram-positive Staphylococcus aureus (ATCC 12598) and Gram-negative Escherichia coli (ATCC 25922) using the broth microdilution method.^17,18 Serial dilutions of the test compounds and reference drugs were prepared in Mueller-Hinton agar. Drugs (1 mg) were dissolved in DMSO/CDCl₃ (1 ml). Further progressive dilutions with melted Mueller-Hinton agar were performed to obtain the required concentrations of 0.2, 0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50, and 100 µg/ml. Test organisms were subcultured to nutrient agar and incubated overnight at 35 °C. The tubes containing 2 ml of Muller-Hinton agar were inoculated with five or more colonies from the agar plate, and the turbidity was adjusted to match a 1 McFarland standard (10⁵ cfu/ml). The tubes were then incubated at 37 °C for 18 hours. The MIC of the lowest concentration of the tested compound, yielded no visible growth on the plate. To ensure that the solvent did not affect bacterial growth, a control was performed with the test medium supplemented with DMSO/CDCl³ at the same dilutions as used in the experiments. The results showed that DMSO/CDCl³ did not affect the microorganisms in the concentrations studied.

Antitubercular Activity

The antimycobacterial activity of compounds was assessed against M. tuberculosis using the microplate Alamar Blue assay (MABA) method. Briefly, 200 μl of sterile deionized water was added to all outer perimeter wells of the sterile 96-well plate to minimize medium evaporation in the test wells during incubation. Hundred μL of Middlebrook 7H9 broth, and serial dilutions of the compounds were added directly to the plate. The f inal drug concentrations tested were 100 to 0.2 μg/ml. Plates were covered, sealed with parafilm, and incubated at 37ºC for five days. After the incubation period, 25 μl of a freshly prepared 1:1 mixture of Almar Blue reagent and 10% Tween 80 was added to the plate and incubated again for 24 hrs.  Blue colour in the well was interpreted as no bacterial growth, and pink colour was scored as growth. The MIC was defined as the lowest drug con centration that prevented the colour change from blue to pink.¹⁹

Results

A novel series of Pyrrolyl benzimidazole was synthesized as described in the scheme (Figure 1). The benzimidazole ring was constructed by reacting an appropriate orthophenylene diamine with P-aminobenzoic acid in the presence of ethanol to obtain substituted (1H-benzoimidazole-2-yl)anilines (3a-d) with good yields. Following this, it was refluxed with 2,5-dimethoxy tetrahydrofuran in glacial acetic acid to get substitut ed 2-(4-(1H-pyrrol-1-yl) phenyl)-1H-benzo[d]imidazole (4a-d). Further, (1H-benzo imidazole-2-yl) aniline (3 a-d) refluxed with acetonyl acetone in glacial acetic acid to get 4- substituted 2-(4-(2,5-dimethyl-1H-pyrrol-1-yl) phenyl)-1H-benzo[d]imidazole (5 a-d). Structures of these newly synthesized compounds were confirmed based on their physicochemical and spectral data, such as IR, ¹ H-NMR, ¹³C-NMR, and Mass spectra.

Spectral data as follows

2-(4-j(1H-pyrrol-1-yl) phenyl)-1H benzo(d) imidazole(4a)

Yield (%) 60, M.P 210-212, FTIR (KBr cm^-1) 3403.32 (NH.), 2923.39 (CH), 1623.45 (C=C) 1379.74(CN),1241.17(C=N) ¹H NMR (δ, ppm) 7.6 (m, 4H, pyrrole C², C⁵), 7.8(t, 4H, benzimidazole NH C2, C3 C4, C5), 7-7.2(d, 4H, bridging phenyl- C², C³ C⁵, C⁶), 12.07(d,1H benzimidazole- C⁵).

 4-Methyl (2,5-dimethyl-1H- pyrrole-1-yl) benzo(d) imidazole (4b)

Yield(%)65,M.P135-137,FTIR (KBrcm^-1)3392.52 (NH.), 2978.52 (CH), 1625.35 (C=C) 1397.76(CN),1253.97(C=N) ¹H NMR (δ, ppm) 6.3-7.3 (d, 2H, pyrrole- C², C³ C⁵, C⁶), 7.4-7.7(m, 3H, benzimidazole C², C³ C⁴,), 8.2(t, 4H, bridging phenyl- C², C³ C⁵, C⁶), 12.36(s,1H benzimidazole NH- C⁵). 13 C NMR 29.7(methyl- C², C⁵), 77.91 (pyrrole- C³ C⁴), 119.04 (benzimidazole C², C³ C⁵, C⁶), 123.26 (s, bridging phenyl- C², C³ C⁴ C⁵),131.86(d, benzimidazole-NH-C⁵). mol.wt 303.95g.

4-Nitro (2,5-dimethyl-1H- pyrrole-1-yl) benzo(d) imidazole (4c)

Yield (%) 62, M.P 148-151, FTIR (KBrcm^-1) 3419.39 (NH.), 290422. (CH), 1603.58 (C=C) 1411.67(CN),1119.45(C=N) ¹H NMR (δ, ppm) 6.3-7.1 (d, 4H, pyrrole C², C³ C⁴ C⁵), 7.2-7.4(m, 3H, benzimidazole C2, C3 C5), 2.2(s,3H,methyl C⁵),8.1-8.2(t, 4H, bridging phenyl-C², C³ C⁵, C⁶), 10.09(s,1H benzimidazole NH-C⁵).

4-Chloro (2,5-dimethyl-1H- pyrrole-1-yl) benzo(d) imidazole (4d)

Yield(%)60,M.P120-124,FTIR (KBrcm^-1)3434.30 (NH.),2923.31(CH),1608.54(C=C) 1376.76(CN),1180.56(C=N) ¹H NMR(δ, ppm) 6.3-7.4 (d, 4H, pyrrole C², C³ C⁴ C⁵ ), 8.1(d, 3H, benzimidazole C², C³ C⁵), 2.2(s,3H,methyl-C⁵),7.6 7.7(m, 4H, bridging phenyl- C², C³ C⁴ C⁵), 10.8(s,1H benzimidazole NH- C⁵).

4-(2,5-dimethyl-1H- pyrrole-1-yl) benzoic acid(5a)

Yield(%)63,M.P118-120,FTIR(KBrcm^-1) 3414.57 (NH.), 2926.08 (CH), 1604.53 (C=C) 1377.86(CN),1020.43(C=N) ¹H NMR(δ, ppm) 1.9 (d, 6H, methyl C², C⁵ ), 5.9 (d, 2H, pyrrole- C³, C⁴ ), 6.7-6.8(t, 4H, benzimidazole C², C³ C⁴ C⁵), 7-7.2(d, 4H, bridging phenyl- C², C³ C⁵, C⁶), 11.07(d,1H benzimidazole-NH- C⁵). 13 C NMR 12.36(methyl-C², C⁵ ),105.8 (pyrrole- C³, C⁴), 115.58 (benzimidazole C², C³ C⁴ C⁵), 128.48 (d, bridging phenyl- C², C³ C⁴ C⁵),144.22(benzimidazole-NH- C⁵). Mol.wt 285.971g,

4-Methyl(2,5-dimethyl-1H-pyrrole-1-yl)benzoic acid(5b)

Yield(%)61,M.P158-160,FTIR(KBrcm^-1) 3400.47 (NH.), 2919.90 (CH), 1606.14 (C=C) 1317.05(CN),1184.31(C=N) ¹H NMR(δ, ppm) 1.8(t, 6H, methylC², C⁵), 5.4 (m, 2H, pyrrole- C³, C⁴ ),8.4(d, 4H, bridging phenyl- C², C³ C⁵, C⁶), 7.2(t, 3H, benzim idazole C², C³ C⁵), 11.3(t,1H benzimidazole-NH- C⁵). Mol.wt 301.293g.

4-Nitro (2,5-dimethyl-1H-pyrrole-1-yl) benzoic acid(5c)

Yield(%)67,M.P128-130,FTIR(KBrcm^-1) 3371.31(NH.),2923.05(CH),1700.17(C=C) 1325.82(CN),1090.89(C=N)1HNMR(δ,ppm)2.07(d, 6H, methyl C², C⁵), 7.6 (m, 2H, pyrrole- C³, C⁴ ),10.2(m, 4H, bridging phenyl- C², C³ C⁵, C⁶), 7.8(t, 3H, benzimidazole C², C³ C⁵), 12.2(d,1H benzimidazole-NH-C⁵).13CNMR18.46(methyl C², C⁵),117.98(pyrrole-C³, C⁴),77.91(benzimidazole C2, C3 C4, C5), 130.75 (bridging phenyl- C², C³, C⁴, C⁵),132.61(benzimidazole-C⁵).

4-Chloro (2,5-dimethyl-1H-pyrrole-1-yl) benzoic acid(5d)

Yield(%)60,M.P135-139,FTIR(KBrcm^-1) 3444.80 (NH.), 2855.54 (CH), 1604.39 (C=C) 1383.47(CN),1115.66(C=N) 1H NMR(δ, ppm) 2.5(d, 6H, methyl C2, C5 ), 6.2 (d, 2H, pyrrole- C³, C⁴ ),7.9(d, 4H, bridging phenyl- C2, C3 C5, C6), 7.3-7.4(t, 3H, benzimidazole C², C³, C⁵), 10.1(s,1H benzimidazole NH- C⁵).

All newly synthesized compounds were screened for antibacterial activity using the broth microdilution assay method (MIC, µg/ml), and the results are shown in Table 1. This study reveals that electron-donating substituents exhibit significant activity against both Gram-positive and Gram-negative bacteria.

All the newly synthesized compounds were screened for anti-tubercular activity using the Microplate Alamar Blue Assay (MABA) method, and the results are demonstrated in Table 2. These results show that electron-with drawing substituents (Br, Cl) at the para position of the phenyl ring enhance the antitubercular activity (MIC 3.2 µg/mL) as compared to other substitutions.

Discussion

A novel series of Pyrrolyl benzimidazole was synthesized as described in the scheme. The benzimidazole ring was constructed by reacting appropriate orthophenylene diamine with P-aminobenzoic acid in the presence of ethanol to obtain substituted (1H-benzoimidazole-2-yl) aniline (3 a-d) with good yield. Following this, it was refluxed with 2,5-dimethoxy tetrahydrofuran in glacial acetic acid to get substituted 2-(4-(1H-pyr rol-1-yl) phenyl)-1H-benzo[d]imidazole (4a-d).Further, (1H-benzoimidazole-2-yl) aniline (3a-d) was refluxed with acetonyl acetone in glacial acetic acid to get 4-substituted2-(4-(2,5-dimethyl-1H-pyrrol-1-yl) phenyl)-1H benzo[d]imidazole (5 a-d). Structures of these newly synthesized compounds were confirmed based on their physico-chemical and spectral data, such as IR, ¹H-NMR, ¹³C-NMR, and Mass spectra. Using microdilution broth, all synthesized compounds were screened for antibacterial activity against Gram + ve (S. aureus) and Gram -ve (E. coli). The result showed antibacterial activity ranging from 25-100 µg/ml against Gram +ve bacteria (S. aureus) and 6.25-25 µg/ml against Gram -ve bacteria (E. coli). Compounds 4b and 5b had moderately significant activity at 12.5 µg/ml against Gram-ve bacteria (E. coli) and antitubercular activity against M. tuberculosis H37Rv by using MABA method. Compounds showed antitubercular activity ranging from 3.12 to 25 µg/ml. Compounds 4d and 5d show highly significant activity at 3.12 µg/ml, and Compounds 4c and 5c show moderately significant activity at 6.25 µg/ml against M. tuberculosis H37Rv by using MABA method.

Conclusion

A novel series of Pyrrolyl benzimidazoles was synthesized as described in the scheme. Benzimidazole ring was constructed by reacting appropriate orthophenylene diamine with P-aminobenzoic acid in the presence of ethanol to obtain substituted (1H-benzoimidazole-2-yl) aniline (3a-d) in good yield. Following this, it was refluxed with 2,5-dimethoxy tetrahydrofuran in glacial acetic acid to get substituted 2-(4-(1H-pyrrol-1-yl) phenyl) 1H-benzo[d]imidazole (4a-d). Further, (1H-benzo imid azole-2-yl) aniline (3a-d) refluxed with acetonyl acetone in glacial acetic acid to get 4-substituted 2-(4-(2,5-di methyl-1H-pyrrol-1-yl) phenyl)-1H-benzo[d]imidazole (5a-d). The structures of these newly synthesized com pounds were confirmed based on their physicochemical and spectral data, including IR, ¹H NMR, ¹³C NMR, and Mass spectra. The compounds synthesized in the pres ent work were screened for antibacterial activity against Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria using the microdilution broth method, and for antitubercular activity against M. tuberculosis H37Rv using the MABA method. These compounds exhibit ed moderate activity against both Gram-positive and Gram-negative bacteria, as well as significant activity against M. tuberculosis H37Rv. These compounds can be considered for further modification to get more potent antitubercular and antibacterial agents.

Conflict of Interest

The authors hereby declare that there is no conflict of interest for the publication.

Acknowledgement

Authors are thankful to Dr. H. V. Dambal, President, Dr. V. J. Jamkhandi, Dean, and Dr. V. H. Kulkarni, Principal, SET College of Pharmacy, Dharwad, for providing the necessary facilities for research work. We also thank Dr. K.G. Bhat of Maratha Mandal’s Dental College, Hospital and Research Centre, Belgaum, for providing antibacterial and antitubercular activities. We thank the Director, SAIF, Panjab University, Chandigarh, Panjab, India provided the NMR and mass spectral data.