Structure-based design of functionalized 2-substituted and 1,2-disubstituted benzimidazole derivatives and their in vitro antibacterial efficacy

Graphical abstract

Abstract

The aim of this present study was to synthesize 2-substituted and 1,2-disubstituted benzimidazole derivatives to investigate their antibacterial diversity for possible future drug design. The structure-based design of precursors 2-(1H-benzimidazol-2-yl)aniline 1, 2-(3,5-dinitro phenyl)-1H-benzimidazole 3 and 2-benzyl-1H-benzimidazole 5 were achieved by the condensation reaction of o-phenylenediamine with anthranilic acid, 3,5-dinitrophenylbenzoic acid, and phenylacetic acid, respectively. The precursors 1, 3 and 5, upon reaction with six different electrophile-releasing agents, furnished the corresponding 2-substituted benzimidazole, 2a-f and 1,2-disubstituted benzimidazole derivatives 4a-f and 6a-f, respectively. The structural identity of the targeted compounds was authenticated by elemental analytical data and spectral information from FT-IR, UV, ¹H, and ¹³C NMR. The outcome of the findings from the in vitro screening unveiled 2-benzyl-1-(phenylsulfonyl)-1H-benzimidazole 6b as the most active derivative with lowest MIC value of 15.63 µg/mL.

Introduction

From time to time, heterocyclic templates have continued to gain respect and much interest among the medicinal chemists, because of their numerous therapeutic applications and effective reported druggability [1]. Benzimidazole is a heterocyclic aromatic organic compound that plays important functions in the development of theory in heterocyclic chemistry and organic synthesis [2]. Benzimidazole is a strongly acidic compound with a pKa of 12.75, while its conjugated acid has a pKa of 5.68, which is less basic than imidazole. Benzimidazole is readily prepared by [4+1]-cycloaddition of o-phenylenediamine with a one carbon donor source in the presence of various heterogeneous catalysts [3], such as H₂O₂/HCl [4], H₂O₂/CAN [5], H₂O/HCl [6], H₂O₂/Fe(NO₃)₃ [7], and H₂O₂/Bu₄NI [8] as efficient oxidative couples. In healthful analysis, the synthesis of novel benzimidazole derivatives remains a focus [9]. Diverse synthetic efforts for accessing benzimidazole derivatives have been documented, however, the commonest technique involves the reaction of o-phenylenediamine with alkanoic acids. From the evaluation of the works of various researchers, benzimidazole derivatives have been reported to possess antimalarial [10], anticancer [11], antimicrobial [12], [13], antioxidant [14], and anticonvulsant [15] activities among others. Some derivatives of benzimidazole are well known in corrosion studies and their corrosion inhibition efficiencies are related to their adsorption properties [16].

The outbreak of new diseases and the increase in population of drug resistant strains of bacteria, such as methicillin-resistant Staphylococcus aureus [17], vancomycin-resistant Enterococci [18], ampicillin-resistant Enterobacter aerogenes [19], gentamicin-resistant Escherichia coli [20], and chloroquine-resistant Plasmodium falciparum [21], have posed great challenges to life and wellbeing of mankind. Based on the existence of antidrug multi-resistant bacteria strains [22], the occurrence of side effects to commercially available drugs [23], adverse drug reaction in elderly patient [24], the emergence of new diseases, and global health threat that have resulted in high mortality rate [25]; it has become highly imperative to consistently and continuously engage in the synthetic preparation of novel heterocyclic templates as highly dynamic biologically active substances for therapeutic uses. Therefore, it is beneficial to design some 2-substituted- and 1,2-disubstituted benzimidazole derivatives by ecofriendly method so as to examine their antimicrobial properties for possible future drug development.

Material and methods

Chemical compounds and reagents were purchased from Sigma-Aldrich Chemicals (St. Louis, Missouri, USA) apart from Tetrahydrofuran (THF), benzenesulfonyl chloride, and anthranilic acid which were supplied by the British Drug Houses (Poole, Dorset, England). All these compounds were then made available by Department of Chemistry, Covenant University for research use. All the chemicals are pure and they were used directly without further purification. The synthesized heterocyclic frameworks were evaluated for their melting point determination using Stuart equipment and the value obtained were recorded directly. Bruker fourier-transform (ft-ir) spectrophotometer was utilized to obtain infrared data. The UV spectra of the solution of the compounds in THF were run in UV Genesys 10 s. The ¹H and ¹³C nuclear magnetic resonance of the heterocycles were NMR Bruker DPX 400 spectrometer at 400 MHz and 100 MHz, respectively in DMSO-d₆. The reference utilized was Tetramethylsilane (TMS). The reaction progress as well as the level of purity was routinely checked and monitored with Thin Layer Chromatography (TLC) using CHCl₃/CH₃OH (9:1, v/v) eluent. After reaction was completed, solvents were evaporated under reduced pressure using IKA® RV 10 Rotary evaporator. In a situation where more than one spots were observed, column chromatography was carried out to get a pure compound.

2-(1H-Benzimizadol-2-yl)aniline as precursor 1

o-Phenylene diamine (15.00 g, 140.00 mmol) was weighed and dissolved in 150 mL of ethanol in a round- bottomed flask. It was stirred for 5 min with the aid of magnetic stirrer after which anthranilic acid (19.20 g, 140.00 mmol) was gradually tipped into the solution followed by the addition of a catalytic amount of NH₄Cl (0.75 g, 14.00 mmol). The resulting solution was then heated under reflux at 60–70 °C for 2 h. The TLC was utilized to ascertain the progress of reaction. Upon completion, the resulting solution was allowed to cool down. The flask content was evaporated to dryness and triturated with ice-cold water. The solid mass formed was separated by suction filtration to furnish 2-(1H-benzimizadol-2-yl)aniline 1 (72.68%), mp = 85–87 °C, colour = gray. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 4.50 (s-br, 2H, NH₂), 6.37–6.39 (dd, J₁ = 4.54 Hz, J₂ = 8.80 Hz, 1H, Ph-H), 6.49–6.50 (dd, J₁ = 4.21 Hz, J₂ = 8.87 Hz, 1H, Ph-H), 6.84–6.86 (d, J = 8.80 Hz, 1H, Ph-H), 7.12–7.14 (d, J = 8.00 Hz, 1H, Ph-H), 7.39–7.41 (m, 2H, Ph-H), 7.59–7.63 (d, J = 8.87 Hz, 1H, Ph-H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 109.6, 115.2 (2 × CH), 116.9, 119.4, 123.1 (2 × CH), 125.5, 129.7, 141.9 (2 × C), 145.1, 155.0 ppm. λ_max in nm (log ε_max): 218 (4.2741), 253 (4.3096), 326 (3.6434). FT-IR ν in cm⁻¹: 3424 (N—H of NH₂), 3422 (N—H of NH₂), 3405 (N—H), 1620 (C C aromatic). Anal. Calcd for C₁₃H₁₁N₃ (209.25): C, 74.62; H, 5.30; N, 20.08%. Found: C, 74.80; H, 5.47; N, 19.96%.

Overall protocol towards accessing 2-substituted benzimidazole 2a-f

Precursor 1 (4.00 g, 19.10 mmol) was dissolved in 20 mL of tetrahydrofuran (THF) in a round-bottomed flask at room temperature. The medium was basified by the addition of Na₂CO₃ (4.06 g, 38.30 mmol) and cooled to 0–5 °C in ice bath. The corresponding electrophile-releasing substrate a-f (19.10 mmol) was then added and the reacting mixture was maintained on ice bath for additional 15 min after which the medium was warmed up to room temperature and stirred there for 24 h. Monitoring of reaction progress was conducted using TLC and upon reaction completion, the solvent was evaporated at reduced pressure using rotary evaporator. Cold water was added to the resulting mass, filtered by suction, and air-dried to afford crude product which upon column purification afforded 2-substituted benzimidazole derivatives 2a-f.

N-(2-(1H-Benzimidazole-2-yl)phenyl) acetamide 2a

When a = acetyl chloride, yield 57.70%, mp = 253–255 °C, colour = gray. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 2.67 (s, 3H, CH₃—CO), 6.36–6.39 (dd, J₁ = 4.58 Hz, J₂ = 8.82 Hz, 1H, Ph—H), 6.51–6.52 (dd, J₁ = 4.18 Hz, J₂ = 8.89 Hz, 1H, Ph—H), 6.86–6.88 (d, J = 8.82 Hz, 1H, Ph—H), 7.13–7.15 (d, J = 8.00 Hz, 1H, Ph—H), 7.39–7.43 (m, 2H, Ph—H), 7.62–7.65 (d, J = 8.89 Hz, 1H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 26.4 (CH₃), 109.6, 115.2 (2 × CH), 116.8, 119.6, 123.6 (2 × CH), 125.6, 129.8, 141.7 (2 × C), 145.3, 156.1, 175.4 (C O) ppm. λ_max in nm (log ε_max): 218 (4.2988), 248 (4.5563), 323 (3.8261), 464 (2.4771). FT-IR ν in cm⁻¹: 3405 (N—H), 1699 (C O). Anal. Calcd for C₁₅H₁₃N₃O (251.11): C, 71.70; H, 5.21; N, 16.72%. Found: C, 71.88; H, 5.09; N, 16.89%.

N-(2-(1H-Benzimidazole-2-yl)phenyl)benzenesulfonamide 2b

When b = benzenesulfonyl chloride, yield 97.21%, mp = N.D. (Oily), colour = black. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 6.35–6.38 (m, 3H, Ph—H), 6.46–6.49 (m, 2H, Ph—H), 6.84–6.86 (d, J = 7.16 Hz, 2H, Ph—H), 7.11–7.14 (d, J = 11.96 Hz, 2H, Ph—H), 7.40–7.42 (m, 2H, Ph—H), 7.62–7.65 (d, J = 11.88 Hz, 2H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 109.8, 115.3 (2 × CH), 116.9, 119.5, 123.2 (2 × CH), 125.6, 127.5 (2 × CH), 128.9, 129.8 (2 × CH), 131.9, 154.8, 139.7, 141.6 (2 × C), 145.3 ppm. λ_max in nm (log ε_max): 221 (4.6609), 251 (4.7332), 317 (4.2878), 428 (3.9294). FT-IR ν in cm⁻¹: 3405 (N—H), 3266 (N—H), 1620 (C C aromatic), 1575 (C N imine), 1376 (SO₂), 1185 (SO₂). Anal. Calcd for C₁₉H₁₅N₃O₂S (349.09): C, 65.31; H, 4.33; N, 12.03%. Found: C, 65.20; H, 4.15; N, 11.83%.

N-(2-(1H-Benzimidazole-2-yl)phenyl)-4-methylbenzenesulfonamide 2c

When c = p-toluenesulfonyl chloride, yield 80.03%, mp = N.D. (Oily), colour = brown. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 2.80 (s, 3H, CH₃-Ar), 6.36–6.39 (m, 3H, Ph—H), 6.49–6.51 (m, 2H, Ph—H), 6.84–6.86 (d, J = 7.04 Hz, 1H, Ph—H), 7.11–7.14 (d, J = 11.96 Hz, 2H, Ph—H), 7.40–7.42 (m, 2H, Ph—H), 7.62–7.65 (d, J = 11.88 Hz, 2H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 21.3 (CH₃), 109.8, 115.3 (2 × CH), 116.9, 119.5, 123.2 (2 × CH), 125.6, 127.3 (2 × CH), 129.1, 129.8 (2 × CH), 131.9, 139.7, 141.8 (2 × C), 145.3, 155.0 ppm. λ_max in nm (log ε_max): 212 (4.6343), 233 (5.2124), 311 (4.6750). FT-IR ν in cm⁻¹: 3407 (N—H), 3263 (N—H), 1377 (SO₂), 1187 (SO₂). Anal. Calcd for C₂₀H₁₇N₃O₂S (363.43): C, 66.10; H, 4.71; N, 11.56%. Found: C, 65.95; H, 4.63; N, 11.75%.

2-(1H-Benzimidazol-2-yl)-N-(3-chlorobenzyl)aniline 2d

When d = 3-chlorobenzyl chloride, yield 87.90%, mp = N.D. (Oily), colour = black. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 3.73 (s, 2H, CH₂), 6.31–6.35 (m, 3H, Ph—H), 6.48–6.50 (m, 2H, Ph—H), 6.84–6.87 (d, J = 8.00 Hz, 1H, Ph—H), 7.11–7.14 (d, J = 11.96 Hz, 2H, Ph—H), 7.61–7.63 (d, J = 7.96 Hz, 2H, Ph—H), 7.92–7.94 (d, J = 7.72 Hz, 1H, Ph—H), 8.21 (s, 1H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 48.2 (CH₂), 112.3, 114.1, 115.1 (2 × CH), 117.4, 123.2 (2 × CH), 125.0, 125.4, 126.1, 126.8, 129.3, 129.9, 132.2, 134.3, 141.8 (2 × C), 145.4, 154.8 ppm. λ_max in nm (log ε_max): 224 (5.0026), 251 (4.9633), 302 (4.7279). FT-IR ν in cm⁻¹: 3460, 3354 (N—H), 3107 (C—H aromatic), 2924 (CH aliphatic), 2854 (CH aliphatic), 1624 (C C Aromatic), 1581 (C N). Anal. Calcd for C₂₀H₁₆N₃Cl (333.81): C, 71.96; H, 4.84; N, 12.59%. Found: C, 72.14; H, 5.02; N, 12.38%.

5-((2-(1H-Benzimidazole-2-yl)phenyl)amino)pyrimidine-2,4(1H,3H)-dione 2e

When e = 5-bromouracil, yield 83.29%, mp > 300 °C, colour = brown. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 6.36–6.39 (m, 1H. Ph—H), 6.50–6.52 (m, 1H. Ph—H), 6.83–6.86 (d, J = 11.88 Hz, 1H, Ph—H), 7.11–7.13 (d, J = 8.00 Hz, 2H, Ph—H), 7.39–7.43 (m, 2H, Ph—H), 7.62–7.65 (d, J = 11.88 Hz, 1H, Ph—H), 8.37–8.41 (d, J = 13.92 Hz, 1H, Ph—H), 11.02 (s, IH, NH), 11.56 (s, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 109.2, 110.6, 115.6 (2 × CH), 119.3, 123.1 (2 × CH), 125.3, 125.7, 126.2, 129.4, 141.8 (2 × C), 145.0, 152.5, 169.0 (C O), 169.8 (C O) ppm. λ_max in nm (log ε_max): 202 (4.5646), 224 (4.9854), 254 (5.1392). FT-IR (ν_max in cm⁻¹): 3460, 3354 (N—H), 3107 (C—H aromatic), 2924 (CH aliphatic), 2854 (CH aliphatic), 1624 (C C aromatic), 1581 (C N). Anal. Calcd for C₁₇H₁₃N₅O₂ (319.32): C, 63.94; H, 4.10; N, 21.93%. Found: C, 63.90; H, 3.99; N, 22.01%.

N-(2-(1H-Benzimidazole-2-yl)phenyl)benzene-1,4-diamine 2f

When f = 4-chloroaniline, yield 80.74%, mp > 300 °C, colour = gray. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 5.32 (s, 2H, NH₂), 6.33–6.38 (m, 2H, Ph—H), 6.49–6.53 (m, 2H, Ph—H), 6.83–6.86 (d, J = 9.92 Hz, 1H, Ph—H), 7.11–7.14 (d, J = 10.48 Hz, 2H, Ph—H), 7.39–7.41 (d, J = 8.50 Hz, 2H, Ph—H), 7.59–7.62 (d, J = 10.48 Hz, 2H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 109.8, 115.3 (2 × C), 117.2 (2 × CH), 118.1, 119.5, 121.1 (2 × CH), 123.0 (2 × CH), 125.6, 129.8, 132.2, 137.9, 140.9, 141.8 (2 × C), 154.8 ppm. λ_max in nm (log ε_max): 224 (5.0228), 254 (1.9881), 308 (4.5453). FT-IR (ν_max in cm⁻¹): 3436 (N—H of 1° amine), 3406 (N—H of 1° amine), 3330 (N—H of 2° amine), 3060 (C—H aromatic), 1613 (C C aromatic), 1577 (C N). Anal. Calcd for C₁₉H₁₆N₄ (300.36): C, 75.98; H, 5.37; N, 18.65%. Found: C, 76.09; H, 5.40; N, 18.45%.

2-(3,5-Dinitrophenyl)-1H-benzimidazole as precursor 3

Procedure for the synthesis of precursor 1 was repeated for the reaction of o-phenylenediamine with 3,5-dinitrophenylbenzoic acid to afford precursor 3 (90.07%), mp = 177–179 °C, colour = yellow. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 7.40–7.42 (d, J = 8.00 Hz, 1H, Ph—H), 7.44–7.46 (d, J = 8.00 Hz, 1H, Ph—H), 7.72–7.74 (t, J = 7.58 Hz, 1H, Ph—H), 7.94–7.96 (m, 1H, Ph—H), 8.64 (s, 2H, Ph—H), 8.85 (s, 1H, Ar—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 115.2 (2 × CH), 125.8, 128.7 (2 × CH), 129.4 (2 × CH), 134.8, 144.2 (2 × C), 150.7 (2 × C), 156.1 ppm. λ_max in nm (log ε_max): 218 (4.6522), 248 (4.6365). FT-IR (ν_max in cm⁻¹): 3349 (N—H), 3172 (C—H aromatic), 3101 (C—H aromatic), 1606 (C C), 1572 (C N), 1543 (NO₂ asym.), 1344 (NO₂ sym.). Anal. Calcd for C₁₃H₈N₄O₄ (284.22): C, 55.13; H, 2.49; N, 19.78%. Found: C, 54.98; H, 2.54; N, 19.92%.

Overall protocol towards accessing 1,2-disubstituted-1H-benzimidazole 4a-f

Similar procedure for 2a-f was repeated herein using 2-(3,5-dinitrophenyl)-1H-benzimidazole 3 as the precursor which reacted with substrates a-f to afford 1-substituted-2-(3,5-dinitrophenyl)-1H-benzimidazoles 4a-f.

1-(2-(3,5-Dinitrophenyl)-1H-benzimidazole-1-yl)ethanone 4a

Yield 72.58%, mp = 250–252 °C, colour = brown. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 2.59 (s, 3H, CH₃—CO), 7.40–7.42 (d, J = 8.00 Hz, 1H, Ph—H), 7.44–7.46 (d, J = 8.00 Hz, 1H, Ph—H), 7.72–7.74 (t, J = 7.56 Hz, 1H, Ph—H), 7.94–7.96 (m, 1H, Ph—H), 8.64 (s, 2H, Ph—H), 8.85 (s, 1H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 28.9 (CH₃), 116.6 (2 × CH), 126.1, 128.9 (2 × CH), 129.7 (2 × CH), 135.0, 144.3 (2 × C), 150.5 (2 × C), 156.3, 174.1 (C O) ppm. λ_max in nm (log ε_max): 218 (4.3729), 248 (4.3050). FT-IR (ν_max in cm⁻¹): 3106 (C—H aromatic), 2854 (C—H aliphatic), 1699 (C O amide), 1622 (C C), 1580 (C N), 1541 (NO₂asym.), 1346 (NO₂ sym.). Anal. Calcd for C₁₅H₁₀N₄O₅ (326.26): C, 55.22; H, 3.09; N, 17.17%. Found: C, 55.13; H, 2.89; N, 17.08%.

2-(3,5-Dinitrophenyl)-1-(phenylsulfonyl)-1H-benzimidazole 4b

Yield 62.77%, mp > 300 °C, colour = gray. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 7.15–7.19 (m, 5H, Ph—H), 7.40–7.46 (m, 2H, Ph—H), 7.72–7.74 (t, J = 7.60 Hz, 1H, Ph—H), 7.94–7.96 (m, 1H, Ph—H), 8.64 (s, 2H, Ph—H), 8.85 (s, 1H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 116.6 (2 × CH), 119.6 (2 × CH), 126.1, 128.9 (2 × CH), 129.7 (2 × CH), 132.5, 135.0 (2 × C), 137.5 (2 × CH), 144.3 (2 × C), 150.5, 152.0, 156.3 ppm. λ_max in nm (log ε_max): 215 (3.9395), 251 (4.1492). FT-IR (ν_max in cm⁻¹): 3050 (C—H aromatic), 2855 (C—H aliphatic), 1620 (C C), 1580 (C N), 1541 (NO₂asym.), 1376 (SO₂), 1346 (NO₂ sym.), 1185 (SO₂ 2nd band). Anal. Calcd for C₁₉H₁₂N₄O₆S (434.39): C, 53.77; H, 2.85; N, 13.20%. Found: C, 53.95; H, 3.03; N, 13.31%.

2-(3,5-Dinitrophenyl)-1-tosyl-H-benzimidazole 4c

Yield 98.77%, mp = 112 °C, colour = brown. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 2.60 (s, 3H, CH₃—Ar), 6.90–6.92 (d, J = 8.00 Hz, 2H, Ph—H), 7.15–7.17 (d, J = 8.00 Hz, 2H, Ph—H), 7.40–7.44 (m, 2H, Ph—H), 7.73–7.75 (t, J = 7.54 Hz, 1H, Ph—H), 7.93–7.95 (m, 1H, Ph—H), 8.60 (s, 2H, Ph—H), 8.84 (s, 1H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 21.7 (CH₃), 116.6 (2 × CH), 119.6 (2 × CH), 124.8, 156.3, 152.1, 150.5, 144.3 (2 × C), 137.5 (2 × CH), 135.0, 132.5, 129.5 (2 × CH), 126.0, 128.9 (2 × CH) ppm. λ_max in nm (log ε_max): 242 (4.6138). FT-IR (ν_max in cm⁻¹): 3105 (C—H aromatic), 2852 (C—H aliphatic), 1620 (C C), 1575 (C N), 1540 (NO₂ asym.), 1377 (SO₂), 1345 (NO₂ sym.). Anal. Calcd for C₂₀H₁₄N₄O₆S (438.41): C, 54.79; H, 3.22; N, 12.78%. Found: C, 54.62; H, 3.16; N, 12.94%.

1-(3-Chlorobenzyl)-2-(3,5-dinitrophenyl-1H-benzimidazole 4d

Yield 71.16%, mp > 300 °C, colour = black. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 3.72 (s, 2H, CH₂—Ar), 7.11–7.14 (m, 1H, Ph—H), 7.25–7.27 (d, J = 7.56 Hz, 2H, Ph—H), 7.40–7.46 (m, 2H, Ph—H), 7.72–7.74 (t, J = 7.60 Hz, 1H, Ph—H), 7.94–7.96 (d, J = 7.22 Hz, 1H, Ph—H), 8.21 (s, 1H, Ph—H), 8.64 (s, 2H, Ph—H), 8.85 (s, 1H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 48.0 (CH₂), 115.2, 116.2 (2 × CH), 119.2, 125.1, 126.1, 128.9 (2 × CH), 129.7 (2 × CH), 130.0, 135.0, 139.1, 144.0 (2 × C), 150.4 (2 × C), 156.1 ppm. λ_max in nm (log ε_max): 212 (3.7076), 248 (4.1986), 467 (2.6020), 470 (2.6020). FT-IR (ν_max in cm⁻¹): 2924, 2854 (CH aliphatic), 1612 (C C Aromatic), 1584 (C N imine), 1501 (NO₂ asym.). Anal. Calcd for C₂₀H₁₃N₄O₄Cl (408.79): C, 58.76; H, 3.21; N, 13.71%. Found: C, 58.88; H, 3.32; N, 13.89%.

5-(2-(3,5-Dinitrophenyl)-1H-benzimidazol-1-yl)pyrimidine-2,4(1H,3H)-dione 4e

Yield 61.04%, mp > 300 °C, colour = gray. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 7.40–7.42 (t, J = 7.24 Hz, 1H, Ph—H), 7.44–7.46 (m, 1H, Ph—H), 7.72–7.74 (d, J = 8.00 Hz, 2H, Ph—H), 7.94–7.96 (s, 1H, Ph—H), 8.64 (s, 2H, Ph—H), 8.85 (s, 1H, Ph—H), 11.05 (s, IH, NH), 11.55 (s, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 116.6 (2 × CH), 126.1, 128.9 (2 × CH), 129.7 (2 × CH), 135.0, 138.7, 140.1, 144.3 (2 × C), 150.5 (2 × C), 156.3, 169.0 (C O), 169.7 (C O) ppm. λ_max in nm (log ε_max): 220 (4.0234), 242 (4.8730), 311 (4.0790). FT-IR (ν_max in cm⁻¹): 3362 (N—H), 3217 (N—H), 3050 (C—H aromatic), 1685 (C O amide), 1612 (C C aromatic), 1575 (C N), 1536 (NO₂ asym), 1344 (NO₂ sym.). Anal. Calcd for C₁₇H₁₀N₆O₆ (394.30): C, 51.78; H, 2.56; N, 21.31%. Found: C, 51.97; H, 2.69; N, 21.51%.

4-(2-(3,5-Dinitrophenyl)-1H-benzimidazole-1-yl)aniline 4f

Yield 66.85%, mp > 300 °C, colour = gray. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 4.50 (s, 2H, NH₂), 6.90–6.92 (d, J = 8.00 Hz, 2H, Ph—H), 7.15–7.17 (d, J = 8.00 Hz, 2H, Ph—H), 7.40–7.44 (m, 2H, Ph—H), 7.73–7.76 (t, J = 7.84 Hz, 1H, Ph—H), 7.93–7.96 (m, 1H, Ph—H), 8.69 (s, 2H, Ph—H), 8.90 (s, 1H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 115.2, 116.6 (2 × CH), 118.3 (2 × CH), 125.3, 126.1, 128.9 (2 × CH), 129.7 (2 × CH), 135.0, 144.1 (2 × C), 147.0, 147.8, 150.5 (2 × C), 155.4 ppm. λ_max in nm (log ε_max): 235 (5.4130), 302 (4.8040). FT-IR (ν_max in cm⁻¹): 3472 (N—H), 3363 (N—H), 3214, 1620 (C C aromatic), 1536 (NO₂ asym), 1321, 1377 (NO₂sym). Anal. Calcd for C₁₉H₁₃N₅O₄ (375.34): C, 60.80; H, 3.49; N, 18.66%. Found: C, 61.00; H, 3.68; N, 18.84%.

2-Benzyl-1H-benzimidazole as precursor 5

Procedure for the synthesis of precursor 1 was repeated for the reaction of o-phenylenediamine with phenyl acetic acid to afford precursor 5 (79.12%), mp = 108–110 °C, colour = brown. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 3.56 (s, 2H, CH₂—Ar), 6.35–6.38 (dd, J₁ = 3.48 Hz, J₂ = 9.12 Hz, 1H, Ph—H), 6.48–6.50 (dd, J₁ = 3.48 Hz, J₂ = 8.00 Hz, 1H, Ph—H), 7.12 (s, 5H, Ph—H), 7.24–7.26 (d, J = 9.12 Hz, 1H, Ph—H), 7.29–7.31 (d, J = 8.00 Hz, 1H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 34.9 (CH₂), 115.2 (2 × CH), 123.4 (2 × CH), 125.8, 128.7 (2 × CH), 129.4 (2 × CH), 136.9, 138.8 (2 × C), 142.7 ppm. λ_max in nm (log ε_max): 257 (5.4181), 275 (5.4133), 314 (4.4842). FT-IR (ν_max in cm⁻¹): 3415 (N—H), 2924 (C—H aliphatic), 2854 (C—H aliphatic), 1638 (C C), 1587 (C N imine). Anal. Calcd for C₁₄H₁₂N₂ (208.26): C, 80.74; H, 5.81; N, 13.45%. Found: C, 80.80; H, 6.01; N, 13.44%.

Overall protocol towards accessing 1,2-disubstituted-1H-benzimidazole 6a-f

Similar procedure for the synthesis of 2a-f was repeated herein using 2-benzyl-1H-benzimidazole 5 as the precursor which reacted with substrates a-f to afford 1-substituted-2-benzyl-1H-benzimidazoles 6a-f.

1-(2-Benzyl-1H-benzimidazole-1-yl)ethanone 6a

Yield 90.11%, mp = N.D. (Oily), colour = black. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 2.67 (s, 3H, CH₃CO), 3.56 (s, 2H, CH₂—Ar), 6.36–6.39 (dd, J₁ = 3.48 Hz, J₂ = 9.12Hz, 1H, Ph—H), 6.48–6.50 (dd, J₁ = 3.48 Hz, J₂ = 8.00Hz, 1H, Ph—H), 7.09 (s, 5H, Ph—H), 7.24–7.26 (d, J = 9.12 Hz, 1H, Ph—H), 7.29–7.31 (d, J = 8.00 Hz, 1H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 28.9 (CH₃), 34.9 (CH₂), 115.2 (2 × CH), 123.5 (2 × CH), 125.9, 128.7 (2 × CH), 129.5 (2 × CH), 136.9, 138.9 (2 × C), 142.8 ppm. λ_max in nm (log ε_max): 227 (2.2300), 315 (2.5057). FT-IR (ν_max in cm⁻¹): 2922 (C—H aliphatic), 2855 (C—H aliphatic), 1685 (C O), 1620 (C C), 1575 (C N imine). Anal. Calcd for C₁₆H₁₄N₂O (250.30): C, 76.78; H, 5.64; N, 11.19%. Found: C, 76.94; H, 5.59; N, 11.00%.

2-Benzyl-1-(phenylsulfonyl)-1H-benzimidazole 6b

Yield 87.48%, mp = N.D. (Oily), colour = black. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 3.56 (s, 2H, CH₂—Ar), 6.35–6.38 (dd, J₁ = 3.60 Hz, J₂ = 9.26 Hz, 1H, Ph—H), 6.48–6.50 (dd, J₁ = 3.60 Hz, J₂ = 8.00 Hz, 1H, Ar—H), 6.91–6.95 (m, 3H, Ph—H), 7.12 (s, 5H, Ph—H), 7.13–7.17 (m, 3H, Ph—H), 7.22–7.24 (d, J = 9.26 Hz, 1H, Ph—H), 7.30–7.32 (d, J = 8.00 Hz, 1H, Ph—H), 7.44–7.46 (d, J = 8.66 Hz, 2H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 34.7 (CH₂), 115.2 (2 × CH), 123.4 (2 × CH), 125.8, 128.1 (2 × CH), 128.7 (2 × CH), 129.0 (2 × CH), 129.9 (2 × CH), 133.9, 136.8, 137.9, 138.7 (2 × C), 142.9 ppm. λ_max in nm (log ε_max): 215 (4.4669), 254 (4.4540). FT-IR (ν_max in cm⁻¹): 2922, 2850 (CH aliphatic), 1620 (C C aromatic), 1575 (C N imine), 1375 (SO₂), 1185 (SO₂ 2nd band). Anal. Calcd for C₂₀H₁₆N₂O₂S (348.42): C, 68.94; H, 4.63; N, 8.04%. Found: C, 69.00; H, 4.82; N, 7.89%.

2-Benzyl-1-tosyl-1H-benzimidazole 6c

Yield 93.25%, mp = N.D. (Oily), colour = brown. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 2.76 (s, 3H, CH₃—Ar), 3.56 (s, 2H, CH₂—Ar), 6.35–6.38 (dd, J₁ = 3.60 Hz, J₂ = 9.26 Hz, 1H, Ph—H), 6.48–6.49 (dd, J₁ = 3.60 Hz, J₂ = 8.00 Hz, 1H, Ph—H), 6.92–6.94 (d, J = 8.76 Hz, 2H, Ph—H), 7.12 (s, 5H, Ph—H), 7.22–7.24 (d, J = 9.26 Hz, 1H, Ph—H), 7.30–7.32 (d, J = 8.00 Hz, 1H, Ph—H), 7.44–7.46 (d, J = 8.76 Hz, 2H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 21.5(CH₃), 34.9 (CH₂), 115.2 (2 × CH), 123.4 (2 × CH), 125.8, 128.1, (2 × CH), 128.7 (2 × CH), 129.5 (2 × CH), 130.1 (2 × CH), 134.7, 136.7, 138.9 (2 × C), 139.6, 142.6 ppm. λ_max in nm (log ε_max): 233 (5.3679), 299 (4.7896). FT-IR (ν_max in cm⁻¹): 2924, 2854 (CH aliphatic), 1648 (C C aromatic), 1562 (C N imine), 1376 (SO₂), 1185 (SO₂ 2nd band). Anal. Calcd for C₂₁H₁₈N₂O₂S (362.44): C, 69.59; H, 5.01; N, 7.73%. Found: C, 69.51; H, 4.88; N, 7.82%.

2-Benzyl-1-(3-chlorobenzyl)-1H-benzimidazole 6d

Yield 91.83%, mp = N.D. (Oily), colour = brown. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 3.42 (s, 2H, CH₂—Ar), 3.58 (s, 2H, CH₂—Ar), 6.35–6.38 (dd, J₁ = 3.48 Hz, J₂ = 9.18 Hz, 1H, Ph—H), 6.48–6.50 (dd, Jm₁ = 3.48 Hz, J₂ = 8.00 Hz, 1H, Ph—H), 7.10 (s, 5H, Ph—H), 7.17–7.19 (dd, J₁ = 7.20 Hz, J₂ = 7.82 Hz, 1H, Ph—H), 7.23–7.25 (d, J = 9.18 Hz, 1H, Ph—H), 7.31–7.33 (d, J = 8.00 Hz, 1H, Ph—H), 7.37–7.38 (d, J = 7.82 Hz, 1H, Ph—H), 7.51–7.52 (d, J = 7.20 Hz, 1H, Ph—H), 8.21 (s, 1H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 33.8 (CH₂), 48.0 (CH₂), 115.2 (2 × CH), 123.4 (2 × CH), 125.1 125.6, 125.8, 128.7 (2 × CH), 128.9, 129.5 (2 × CH), 130.2, 134.4, 136.7, 137.8, 138.9 (2 × C), 142.5 ppm. λ_max in nm (log ε_max): 233 (5.3877), 251 (5.0266). FT-IR (ν_max in cm⁻¹): 2984 (C—H aliphatic), 1646 (C C). 1565 (C N), 652 (C—Cl). Anal. Calcd for C₂₀H₁₇ClN₂ (332.83): C, 75.78; H, 5.15; N, 8.42%. Found: C, 75.70; H, 4.01; N, 8.25%.

5-(2-Benzyl-1H-benzimidazole-1-yl)pyrimidine-2,4(1H,3H)-dione 6e

Yield 80.58%, mp = N.D. (Oily), colour = black. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 3.56 (s, 2H, CH₂—Ar), 6.34–6.37 (dd, J₁ = 3.44 Hz, J₂ = 9.18 Hz, 1H, Ph—H), 6.48–6.50 (dd, J₁ = 3.44 Hz, J₂ = 8.00 Hz, 1H, Ph—H), 7.11 (s, 5H, Ph—H), 7.23–7.25 (d, J = 9.18 Hz, 1H, Ph—H), 7.30–7.32 (d, J = 8.00 Hz, 1H, Ph—H), 7.95–7.97 (s, 1H, Ph—H), 11.05 (s, IH, NH), 11.55 (s, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 34.8 (CH₂), 115.2 (2 × CH), 119.9, 123.3 (2 × CH), 125.7, 128.7 (2 × CH), 129.4 (2 × CH), 136.7, 138.5 (2 × C), 142.9, 150.0, 169.2 (C O), 169.7 (C O) ppm. λ_max in nm (log ε_max): 218 (4.6532), 248 (4.5775), 293 (4.1399), 437 (3.4914). FT-IR (ν_max in cm⁻¹): 3360 (N—H), 3217 (N—H), 3050 (C—H aromatic), 1685 (C O amide), 1615 (C C aromatic), 1575 (C N). Anal. Calcd for C₁₈H₁₄N₄O₂ (318.33): C, 67.91; H, 4.43; N, 17.60%. Found: C, 68.10; H, 4.57; N, 17.77%.

4-(2-Benzyl-1H-benzimidazole-1-yl) aniline 6f

Yield 86.24%, mp = N.D. (Oily), colour = brown. ¹H NMR (400 MHz, DMSO-d₆) δ_H: 3.56 (s, 2H, CH₂—Ar), 4.50 (s-br, 2H, NH₂), 6.35–6.38 (dd, J₁ = 3.46 Hz, J₂ = 9.12 Hz, 1H, Ph—H), 6.48–6.50 (dd, J₁ = 3.46 Hz, J₂ = 8.00 Hz, 1H, Ph—H), 7.11 (s, 5H, Ph—H), 7.16–7.18 (d, J = 8.20 Hz, 2H, Ph—H), 7.24–7.26 (d, J = 9.12 Hz, 1H, Ph—H), 7.29–7.31 (d, J = 8.00 Hz, 1H, Ph—H), 7.44–7.46 (d, J = 8.20 Hz, 2H, Ph—H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 34.9 (CH₂), 115.2 (2 × CH), 118.7 (2 × CH), 123.4 (2 × CH), 125.8, 126.2 (2 × CH), 128.7 (2 × CH), 129.4 (2 × CH), 136.9, 138.8 (2 × C), 142.7, 146.8, 148.7 ppm. λ_max in nm (log ε_max): 230 (5.2753), 251 (5.1322), 299 (4.8401). FT-IR (ν_max in cm⁻¹): 3404 (N—H interfered by OH), 2985 (C—H aliphatic), 1610 (C C aromatic), 1572 (C N). Anal. Calcd for C₂₀H₁₇N₃ (299.37): C, 80.24; H, 5.72; N, 14.04%. Found: C, 80.21; H, 5.90; N, 13.85%.

Antibacterial sensitivity testing of compounds 1–6f

All the synthesized compounds (1–6f) and gentamicin were screened for antibacterial activity on four bacterial strains using agar well diffusion method [26]. The detailed was as attached in Supplementary Materials.

Minimum inhibitory/bactericidal concentration (MIC and MBC) testing

The minimum inhibitory concentration (MIC) test on the chosen organisms was carried out via serial dilution technique [27] and the concentration range was from 500.00 to 15.63 µg/mL, while the minimum bactericidal concentration (MBC) was determined by a standard method [26]. The detailed description of the procedures for the determination of MIC and MBC were as presented in the supplementary material.

Results and discussion

Based on the enthusiastic outcome of an extensive review on functionalized benzimidazole [3] and in the continuation of the research effort in the area of benzo-fused imidazole moieties [6], [28], the synthesis of functionalized 2-substituted and 1,2-disubstituted benzimidazole derivatives was herein reported to evaluate their antibacterial activities. Although, various derivatives of benzimidazole moieties have been synthesized and reported in literature; high temperature for the reflux process and the uses harsh reaction condition in the presence of strong and concentrated acids such as HCl [4], [6] have been involved. On the contrary, the synthesis of 2-(1H-benzimizadol-2-yl)aniline precursor 1, was achieved herein by the use of eco-friendly reaction of o-phenylenediamine with anthranilic acid using a catalytic amount of NH₄Cl in the presence of ethyl alcohol as a solvent at refluxing temperature of 60–70 °C (Scheme 1a). The synthetic modification of NH₂ functional side chain of the reactive intermediate 1 was conducted by reacting it with six different electrophile-releasing substrates to furnish 2a-f. Prior to this, the reaction optimization study was carried out using two main parameters. First, solvent dependent condition was investigated using the synthesis of N-(2-(1H-benzimidazole-2-yl)phenyl)acetamide 2a via the reaction of 1 with acetyl chloride in either tetrahydrofuran (THF), ethanol or acetonitrile at room temperature for 24 h; this gave yield of 87.7%, 30%, and 25% respectively. In addition, the thermodynamic dependent kinetic of the synthesis of 2a was evaluated using the comparative study of the synthesis in THF at room temperature, 60 °C, and 120 °C. It was unveiled that room temperature gave the highest yield (87.71%) followed by 60 °C (38.24%), while there was no isolated product at 120 °C. Thus, the same reaction condition was adopted for reaction of 1 with the remaining five electrophile-releasing substrates b-f to produce diverse functionalized 2-substitutedbenzimidazole derivatives 2b-f (Scheme 1b). According to another route [29], the o-phenylenediamine reacted with 3,5-dinitrobenzoic acid to achieve 3 Scheme 2a, which was subsequently treated with the earlier reported six electrophile-releasing substrates to furnish 1,2-disubstituted benzimidazole derivatives 4a-f (Scheme 2b) in varying yields. Finally, the last precursor 5 in this present work was prepared by the condensation of o-phenylenediamine with phenyl acetic acid (Scheme 3a). Precursor 5 was eventually reacted with the six electrophile-releasing substrates to access the 1,2-disubstituted benzimidazole derivatives 6a-f (Scheme 3b).

Scheme 1.

(a) Synthesis of the precursor 1 (b) Synthesis of 2-substitutedbenzimidazoles 2a-f.

Scheme 2.

(a) Synthesis of the precursor 3 (b) Synthesis of 1,2-disubstitutedbenzimidazoles 4a-f.

Scheme 3.

(a) Synthesis of the precursor 5 (b) Synthesis of 1,2-disubstitutedbenzimidazoles 6a-f.

Physicochemical parameter data were presented in experimental section alongside the elemental analysis data. The result of elemental analysis for the % calculated agreed with % found for C, H N of all the synthesized compounds with high state of accuracy (the difference was not more that ±0.20 in all cases). Furthermore, the spectroscopic characterization of the targeted templates was carried out using IR and UV, ¹H, and ¹³C NMR analysis. The ¹H NMR spectra of the compounds were run in DMSO-d₆ at 400 MHz with chemical shift values recorded in ppm. The aryl-linked CH₃ of 2c, 4c, and 6c resonated upfield at δ 2.60–2.80 ppm as 3H singlets and acetyl-linked CH₃ of 2a, 4a, and 6a were seen upfield as 3H singlets at δ 2.60–2.67 ppm. The aryl-linked CH₂ of 2d, 4d, and 5 as well as 6a-f were noticed as singlets at δ 3.42–3.73 ppm. Signals of all aryl H appeared downfield of TMS around δ 6.31–8.90 ppm, NH₂ of amine in 1, 2f, 4f, and 6f were broad singlets at δ 4.50–5.32 ppm. The most downfield signals were that of N—H of amide found as singlets in compounds 2e, 4e, and 6e at δ 11.02–11.56 ppm. The ¹³C NMR spectra were run in DMSO-d₆ at 100 MHz with chemical shift values recorded in ppm. On the overall, the ¹³C NMR spectra of the structure-based benzimidazole derivatives varied from 21.3 ppm for CH₃ of compound 2c to 175.4 ppm for C O of 2a. Specifically, the aryl-linked CH₃ of 2c, 4c, and 6c appeared at δ 21.7–21.3 ppm, whereas the CH₂ signals of 2d, 4d, and 6d resonated at δ 48.0–48.2 ppm. The formation of acetamide in 2a, 4a, and 6a was validated by presence of CH₃ intense singlet signal at δ 26.4–28.9 ppm, which was absent in the precursors 1, 3 and 5, respectively from which they were derived. The UV transition was run in tetrahydrofuran (THF) for the precursors 1, 2, and 3 and their final compounds 2a-f, 4a-f, and 6a-f. The lowest wavelengths observed at 202–224 nm, were due to electronic excitation of π→π∗ peculiar to C C, which depicted the presence of benzene ring in those structures. Bathochromic shifts observed herein led to the presence of other peaks at higher wavelengths (233 nm to 470 nm). Some of these shifts were because of π→n transition, which was attributable to the presence of auxochromic C N group; which belong to K bands [28], [30]. The FT-IR data of the benzimidazole derivatives 4a-f revealed the stretching frequencies of C—H aromatic, C C and C N at 3106–3050, 1622–1600, and 1580–1575 cm⁻¹, respectively [28]. Additional bands were noticed in 2a, 4a, and 6a at 1699–1685 cm⁻¹, which represents the C O of amide. The two bands at 1377–1375 and 1187–1185 cm⁻¹_, which were domiciled in sulfonamide 2b-c, 4b-c, and 6b-c, were peculiarly assigned to SO₂ functionality. The bands at 1543–1540 cm⁻¹ depicted the presence of NO₂ (asym.) in compounds 3 and 4a-f. Therefore, the extrapolated spectroscopic information of targeted benzimidazole motifs was in concordance with the proposed structures.

Antibacterial activity

The general sensitivity testing was evaluated using the in vitro screening of the synthesized compounds against four bacterial isolates (Staphylococcus aureus, Bacillus licheniformis, Proteus vulgaris, and Pseudomonas aeruginosa). Gentamicin was used as the positive control in this study. The justification of gentamicin as a clinical standard was due to the mode of action, which involved irreversible binding at ribosomal level, thereby signaling to obstruct and interrupt protein synthesis [31]. Agar diffusion method was used for the sensitivity testing and the diameters of zones of inhibition (Z. O. I) were documented in millimeter (Table 1). Although, large zones of inhibition were noticed for most of the targeted benzimidazole derivatives final products against the screened organisms, resistance was observed in few cases such as 6c against S. aureus; 4d-f, 6a against Bacillus licheniformis; 2a, 2c, 4a, 4d, and 6f against Proteus vulgaris; 2e, 4d-f, and 6a against Pseudomonas aeruginosa, and 6f developed resistant against the effect of gentamicin. Overall, the largest zone of inhibition (40.00 ± 0.10 mm) was recorded for 1 against S. aureus, while the lowest zone of inhibition (15.00 ± 0.08 mm) was recorded for 3 against S. aureus. In comparison with gentamicin, all synthesized compounds, except 3, had better activity with higher zones of inhibition against growth potential of S. aureus. This means that the array of compounds synthesized herein might be a possible replacement for gentamicin on infectious disease caused by the S. aureus or enhance the potency of gentamicin where resistance issues occur. Compared to gentamicin, all compounds except 3 and 6c showed larger zones of inhibition against growth of S. aureus (i.e. >16 mm); and except 4d-f and 6c against B. licheniformis (i.e. >16 mm), while 3 and 4c exhibited approximately the same Z.O.I. as gentamicin against S. aureus (15 mm) and B. licheniformis (16 mm), respectively. Interestingly, more than 75% of the targeted benzimidazole derivatives were active on P. vulgaris with large zones of inhibition, whereas this organism was resistant to gentamicin (Table 1). Similarly, more than 75% of the targeted benzimidazole derivatives (Z.O.I = 25.00 ± 0.08 to 38.00 ± 0.12 mm) were more active than gentamicin (20.00 ± 0.08 mm) upon P. aeruginosa. In the present study, the choice of S. aureus and E. coli was due to the broad array of pathogenic infections and precarious health issue that are associated with these bacterial strains [32] and wide reported occurrence of resistant strain of S. aureus [1]. S. aureus has been reported to have strong relationship with death rate increase in human population, prolong admission of patients in hospitals, and the infections caused by this bacterial strain are expensive to treat [33]. Due to heat stable toxin production, S. aureus is enlisted as one of the highly invasive organisms referred to as pyogenic cocci involved in numerous adverse infectious conditions in humans [28], [32], [34]. In view of the reported predicament aforementioned and large zones of inhibition experienced via action of the benzimidazole framework herein on S. aureus, motivation was enhanced to investigate the activity index (A.I.) of the synthesized compounds against this organism (Fig. 1). It is quite impressive to note that all the synthesized benzimidazole motifs herein, showed better activity indices than gentamicin against S. aureus except 3 and 6c. The compound 3 competed favourably with gentamicin (A.I. ≈ 1.00) while 6c developed resistance; hence could not have activity index.

Table 1.

Antibacterial sensitivity testing with zones of inhibition in millimetre.

Compound No↓	S. aureus	B. licheniformis	P. vulgaris	P. aeruginosa
1	40.00 ± 0.10	26.00 ± 0.08	35.00 ± 0.09	38.00 ± 0.12
2a	27.00 ± 0.09	26.00 ± 0.08	R	28.00 ± 0.08
2b	30.00 ± 0.09	20.00 ± 0.08	25.00 ± 0.09	30.00 ± 0.08
2c	35.00 ± 0.09	25.00 ± 0.08	R	28.00 ± 0.08
2d	30.00 ± 0.10	28.00 ± 0.09	30.00 ± 0.09	30.00 ± 0.09
2e	32.00 ± 0.12	26.00 ± 0.08	35.00 ± 0.08	R
2f	30.00 ± 0.11	30.00 ± 0.12	28.00 ± 0.08	30.00 ± 0.08
3	15.00 ± 0.08	20.00 ± 0.08	30.00 ± 0.12	28.00 ± 0.09
4a	20.00 ± 0.10	24.00 ± 0.09	R	25.00 ± 0.08
4b	30.00 ± 0.10	18.00 ± 0.08	20.00 ± 0.08	25.00 ± 0.08
4c	25.00 ± 0.10	16.00 ± 0.08	20.00 ± 0.08	25.00 ± 0.08
4d	30.00 ± 0.09	R	R	R
4e	30.00 ± 0.10	R	20.00 ± 0.08	R
4f	30.00 ± 0.12	R	23.00 ± 0.08	R
5	30.00 ± 0.09	28.00 ± 0.09	32.00 ± 0.12	38.00 ± 0.08
6a	19.00 ± 0.08	R	18.00 ± 0.08	R
6b	35.00 ± 0.09	22.00 ± 0.08	30.00 ± 0.10	28.00 ± 0.08
6c	R	18.00 ± 0.08	32.00 ± 0.08	26.00 ± 0.08
6d	32.00 ± 0.10	26.00 ± 0.08	35.00 ± 0.12	30.00 ± 0.08
6e	25.00 ± 0.08	24.00 ± 0.08	25.00 ± 0.08	30.00 ± 0.09
6f	20.00 ± 0.08	26.00 ± 0.08	R	28.00 ± 0.08
Gentamicin	16.00 ± 0.09	16.00 ± 0.08	R	20.00 ± 0.08

R = Resistance. Mean ± SD of triplicate determination.

Fig. 1.

Comparative study of the activity index of synthesized benzimidazoles and gentamicin.

Furthermore, based on high susceptibility of the organisms to the synthesized benzimidazole templates 1-6f and broad spectrum of activity observed herein, the MIC testing was carried out to determine the lowest concentration of the compound solution that conveniently inhibited the bacterial growth (Table 2). It was carried out using serial dilution method (500, 250, 125, 62.50, 31.25, and 15.63 µg/mL) via an earlier reported method [27]. The MIC of benzimidazole derivatives upon S. aureus varied from 15.63 ± 1.63 to 250 ± 2.66 µg/mL; against B. licheniformis varied from 62.50 ± 2.04 to 250 ± 2.65 µg/mL; against P. vulgaris varied from 31.25 ± 1.94 to 250 ± 2.65 µg/mL; and against P. aeruginosa ranged from 15.63 ± 1.63 to 125 ± 2.45 µg/mL. The best activity against S. aureus was observed in the precursor 1 among the series of the 2-substituted benzimidazoles 1-2f. The presence of amino functionality in 1 and the ready availability of its lone pair of electron for coordination led to improved activity. The activity decreases after the NH₂ had underwent substitution as contained in 2a-f, except for 2c and 2e, which competed favorably with 1. This means that incorporation of p-toluenesulfonamido (in 2c) and pyrimidinedione (in 2e) played significant role as essential pharmacophores in increased activity observed in 2c and 2e against S. aureus. On the contrary, the series of 1,2-disubstituted benzimidazoles 4a-f were more active than the precursor 3 from which they were derived. This showed that additional substitution on position 1 of compound 3 to afford 1,2-disubstitution in 4a-f, was a worthwhile adventure in increasing the bioactivity of precursor 3, since the series of 1,2-disubstituted benzimidazole products 4a-f resulted in drastic improvement of growth inhibition in S. aureus. In addition to MIC testing, the minimum bactericidal concentration (MBC) testing was determined to authenticate the lowest concentration of the benzimidazole solution that causes death of the bacterium targeted per time and the results are shown in Fig. 2. Apart from where the MBC was not determined (N.D.) due to occurrence of resistance, all other cases showed that the MBC values were double the MIC in each consideration, except in 6a alone where MBC (500 µg/mL) was 4 times that of MIC (125 µg/mL) against P. vulgaris. From all indications, the lowest MBC trend was observed for the bio-assay screening of benzimidazole derivatives upon S. aureus, whereas the highest MBC was reported against P. aeruginosa as shown in Fig. 2.

Table 2.

Minimum inhibitory concentration (MIC) in µg/mL of targeted benzimidazoles, 1-6f.

Compound No↓	S. aureus	B. licheniformis	P. vulgaris	P. aeruginosa
1	15.63 ± 1.64	125.00 ± 2.44	62.50 ± 2.04	31.25 ± 1.98
2a	31.25 ± 1.98	125.00 ± 2.43	N.D.	62.50 ± 2.01
2b	31.25 ± 1.97	250.00 ± 2.67	125.00 ± 2.44	31.25 ± 1.94
2c	15.63 ± 1.67	125.00 ± 2.44	N.D.	62.50 ± 2.03
2d	31.25 ± 1.96	125.00 ± 2.44	62.50 ± 2.01	62.50 ± 2.03
2e	15.63 ± 1.63	250.00 ± 2.67	62.50 ± 2.01	N.D.
2f	31.25 ± 1.94	62.50 ± 2.04	125.00 ± 2.44	31.25 ± 1.94
3	250.00 ± 2.66	250.00 ± 2.67	125.00 ± 2.44	62.50 ± 2.01
4a	125.00 ± 2.45	125.00 ± 2.44	N.D.	62.50 ± 2.02
4b	31.25 ± 1.95	250.00 ± 2.66	250.00 ± 2.67	62.50 ± 2.01
4c	62.50 ± 2.01	250.00 ± 2.66	250.00 ± 2.66	31.25 ± 1.97
4d	31.25 ± 1.98	N.D.	N.D.	N.D.
4e	15.63 ± 1.65	N.D.	250.00 ± 2.65	N.D.
4f	31.25 ± 1.94	N.D.	250.00 ± 2.67	N.D.
5	15.63 ± 1.67	62.50 ± 2.04	62.50 ± 2.03	15.63 ± 1.63
6a	250.00 ± 2.66	N.D.	125.00 ± 2.44	N.D.
6b	15.63 ± 1.63	250.00 ± 2.65	62.50 ± 2.04	62.50 ± 2.02
6c	N.D.	250.00 ± 2.67	31.25 ± 1.94	125.00 ± 2.45
6d	31.25 ± 1.94	125.00 ± 2.44	31.25 ± 1.95	31.25 ± 1.96
6e	125.00 ± 2.45	125.00 ± 2.45	125.00 ± 2.44	62.50 ± 2.02
6f	125.00 ± 2.43	125.00 ± 2.45	N.D.	62.50 ± 2.01
Gentamicin	5.00 ± 1.23	2.5 ± 1.09	N.D.	7.5 ± 1.45

N.D. = Not Determined. Mean ± SD of triplicate determination.

Fig. 2.

Graphical representation of minimum bactericidal concentration (MBC).

Structure activity relationship (SAR) study

From the overview of the structure activity relationship (SAR) study, it was found that the nature of substituent on 1-position and 2-positions of the benzimidazole nucleus had significant effect on the antibacterial activity of the entire structures. The compounds series 2a-f were structurally related in the core pharmacophoric 2-phenylbenzimidazole; the SAR study showed their activity against S. aureus to be in the order 2c ≈ 2e > 2a ≈ 2b ≈ 2d ≈ 2f. This means that the presence of electron donating CH₃ on p-toluenesulfonamide moieties in the 2-position of anilino side chain played a significant role in the improvement of activity, since its counterpart 2b without CH₃ was far less active and stayed in the categories of 2a, 2d, and 2f in its activity upon S. aureus growth inhibition. Considering 4a-f on S. aureus, the activity varied in the order of: 4e > 4b ≈ 4d ≈ 4f > 4c > 4a. Thus, electron withdrawing ability and π-π stacking character in pyrimidine-dione at 1-position of 2-(3,5-dinitrophenyl)benzimidazole core worked synergistically with the electron withdrawing NO₂ on benzene at 2-position to increase the activity of 4e, thereby causing it to exhibit outstanding activity against S. aureus among the 4a-f series. Based on the in vitro screening of 6a-f against S. aureus, the order of activity was 6b > 6d > 6e ≈ 6f > 6a > 6c. Hence, presence of benzenesulfonamido group on 1-position of 2-benzylbenzimidazole in series 6a-f played a crucial role in activity boosting, making 6b to be the most active among the group and more active as compared to its isomorphic template 6c where no activity was noticed. It was interesting to note that p-methyl group in 6c which was the only group absent in 6b provided the framework 6a-f with antagonistic effect thereby causing total activity loss in 6c as compared to 6b against S. aureus.

Conclusions

Benzimidazole is an essential heterocyclic framework in agrochemicals, pharmaceuticals, and medicinal chemistry research. NH₄Cl catalyzed strategy was found to be efficient approach for accessing the reported benzimidazole precursor in good yield. Thus, mono- and disubstituted benzimidazole derivatives with improved medicinal potential were successfully synthesized via an elegant pathway. The findings of the in vitro screening unveiled the broad spectrum of activity of the synthesized benzimidazole templates in the present study. Among the series, the highest potency was exerted and experienced in 2-(1H-benzimizadol-2-yl)aniline, 1 and 2-benzyl-1-(phenylsulfonyl)-1H-benzimidazole, 6b. It will be a worthwhile adventure to advance the work further for more guidance on the pharmacokinetic and pharmacodynamic study to ascertain probable candidature of the templates for future drug design.

Conflict of interest

The authors have declared no conflict of interest.

Compliance with Ethics Requirements

This article does not contain any studies with human or animal subjects.