Skip to main content
Download PDF
Download ePub

Synthesis and evaluation of antimicrobial, antitubercular and anticancer activities of 2-(1-benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-substituted acetamides

Chemistry Central Journal volume 12, Article number: 66 (2018) Cite this article

Abstract

Background

The study describes the synthesis, characterization, in vitro antimicrobial and anticancer evaluation of a series of 2-(1-benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-substituted acetamide derivatives. The synthesized derivatives were also assessed for in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv. The compounds found active in in vitro study were assessed for their in vivo antitubercular activity in mice models and for their inhibitory action on vital mycobacterial enzymes viz, isocitrate lyase, pantothenate synthetase and chorismate mutase.

Results

Compounds 8, 9 and 11 emerged out as excellent antimicrobial agents in antimicrobial assays when compared to standard antibacterial and antifungal drugs. The results of anticancer activity displayed that majority of the derivatives were less cytotoxic than standard drugs (tamoxifen and 5-fluorouracil) towards MCF7 and HCT116 cell lines. However, compound 2 (IC50 = 0.0047 µM/ml) and compound 10 (IC50 = 0.0058 µM/ml) showed highest cytotoxicity against MCF7 and HCT116 cell lines, respectively. The results of in vivo antitubercular activity revealed that a dose of 1.34 mg/kg was found to be safe for the synthesized compounds. The toxic dose of the compounds was 5.67 mg/kg while lethal dose varied from 1.81 to 3.17 mg/kg body weight of the mice. Compound 18 inhibited all the three mycobacterial enzymes to the highest level in comparison to the other synthesized derivatives but showed lesser inhibition as compared to streptomycin sulphate.

Conclusions

A further research on most active synthesized compounds as lead molecules may result in discovery of novel anticancer and antitubercular agents.

Background

In the twentieth century, greatest advances have been made to tackle microbial infections in human beings. However, the problem of developing resistance to the existing antimicrobial agents has become a nuisance for the medical professionals as the microbes have become capable of evading from the lethal action of most these agents [1]. Tuberculosis (TB) is a contagious disease caused by omnipresent mycobacteria i.e., Mycobacterium tuberculosis [2]. According to 2015 survey of WHO, the world had an estimated 10.4 million new TB cases. TB is one of the biggest killers striking people in their most productive years and accounts for 23% of the global TB burden in India alone [3]. The synergy of this disease with HIV infection and; emergence of multidrug resistance and extensively drug resistance tuberculosis (MDRTB and XDRTB) to the first-line drugs are the threatening global challenges [4]. The researchers have left no stone unturned to discover lead molecules against the disease even then no new chemical entity has appeared for use in clinical treatment of this disease over the last four decades [5].

Cancer, the most debilitating disease, has advanced to such a level that it has become one of the universal cause of human suffering and death all over the world [6, 7]. The huge arsenal of synthetic, semi-synthetic, and naturally-occurring agents for treating neoplastic diseases suffers from two major limitations; the first one being the lack of selectivity of conventional chemotherapeutic agents to cancer tissues, causing unwanted side effects [8]. The second is the acquisition of multiple-drug resistance by cancer cells to the available agents that impedes treatment of various kinds of cancer [9]. Therefore, developing novel molecules to circumvent multidrug resistances and exhibiting selective toxicity to cancer cells rather than to normal cells is need of the hour.

Heterocycles are of considerable interest to the researchers in the field of medicinal chemistry [10]. Benzimidazole is present in several natural and synthetic medicinal compounds and hence is most comprehensively studied bioactive heterocycle [11]. The broad-spectrum biological profile of benzimidazole derivatives includes, hormone antagonist [12], anti-HIV [13, 14], anthelmintic [15], antiprotozoal [16], antihypertensive [17], antioxidant, anti-inflammatory [18], analgesic [19], anxiolytic [20], anticoagulant [21], antifungal [22], antihistaminic [23], antiulcer [24], anti-obesity, antidiabetic [25], antimicrobial [26], antimycobacterial [27] and anticancer [28, 29] activities. In the light of above facts and in continuation of efforts in developing novel molecules for the treatment of tuberculosis and cancer [30, 31], in the present study we herein report the synthesis, antimicrobial, anticancer and antitubercular activities of benzimidazole derivatives i.e., 2-(1-benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-substituted acetamides.

Results and discussion

Chemistry

2-(1-Benzoyl-1H-benzo[d]imidazole-2-ylthio)-2-ylthio)-N-substituted acetamide derivatives (1–20) were synthesized according to Scheme 1 and characterized by physicochemical and spectral means. The structures of obtained compounds (1–20) were confirmed by IR, 1HNMR, 13CNMR and mass spectroscopic data which was consistent with the proposed molecular structures. The appearance of C=O stretch in the range of 1670–1630 cm−1 and N–H stretch 3350–3100 cm−1 of secondary amide indicated the formation of secondary amide in the synthesized compounds. The presence of methyl in compound 13, 16, 19 and 20 was demonstrated by the presence of CH stretch at 3103 cm−1. The multiplet corresponding to 7.14–7.78 δ ppm confirmed the presence of protons of benzimidazole and aryl nucleus. A singlet at around δ 3.8 ppm corresponded to the protons of the methylene in the synthesized compounds.

Scheme 1
scheme 1

Synthesis of 2-(1-benzoyl-1H-benzo[d]imidazole-2-ylthio)-2-ylthio)-N-substituted acetamide derivatives (1–20)

Full size image

In vitro antimicrobial activity

The results of in vitro antimicrobial activity of the synthesized compounds are presented in Table 1. The synthesized compounds were found to be highly efficient antimicrobial agents in comparison to the standard drug cefadroxil and fluconazole. Amongst the synthesized derivatives, compounds 7, 8, 9 and 11 were found to be highly potent antibacterial agents against Gram positive as well as Gram negative bacterial species with MIC of 0.027 µM/ml for each. Compound 7 (MIC = 0.027 µM/ml) showed activity against Aspergillus niger also. Compounds 8, 9 and 11 were highly active towards Candida albicans and A. niger than the standard antifungal drug fluconazole. The results of minimum bactericidal concentration/minimum fungicidal concentration (Table 2) conveyed that none of synthesized derivatives was either bactericidal or fungicidal in action (In general, a compound is said to be bactericidal/fungicidal if its MBC/MFC is less than three times of its MIC) [32].

Table 1 Antimicrobial (MIC = µM/ml) and anticancer (IC50 = µM/ml) screening results of 2-(1-benzoyl-1H-benzo[d]imidazole-2-ylthio)-2-ylthio)-N-substituted acetamides
Full size table
Table 2 MBC/MFC (µg/ml) of 2-(1-benzoyl-1H-benzo[d]imidazole-2-ylthio)-2-ylthio)-N-substituted acetamides
Full size table

In vitro antitubercular activity

The synthesized benzimidazole derivatives were evaluated for their in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv (NCFT/TB/537). The zone of inhibition as well as MIC values of the test compounds was determined. Minimum lethal concentration (MLC) of the compounds was also determined. The results of in vitro antitubercular activity are presented in Table 3.

Table 3 Antimycobacterial activity, MIC and MLC of synthesized derivatives against M. tuberculosis H37Rv
Full size table

In vivo antitubercular activity

The LD50 and ED50 were determined for the active compounds in mice models infected with Mycobacterium H37Rv (Table 4). It was found that the toxic dose of the compounds which proved fatal and highly toxic to mice was 5.67 mg/kg while LD50 varied from 1.81 to 3.17 mg/kg body weight of the mice. LD50 is the dose that killed 50% of the mice population within the group. Thus, ED50 of 1.34 mg/kg was considered safe for each of the compounds. It was observed that this dose was effective and safe for mice in different groups before infecting the mice models with specific TB bacteria as no mortality of any single animal was recorded.

Table 4 Lethal dose (in mg/kg) and percent inhibition of enzymes in Mycobacterium H37Rv groups after treatment with effective dose of 1.34 mg/kg of potent compounds and 25 µg/kg of positive control
Full size table

Mycobacterial enzyme assays

The results of mycobacterial enzyme assays were expressed in terms of percent inhibition of mycobacterial enzymes i.e., isocitrate lyase, pantothenate synthetase and chorismate mutase, by the M. tuberculosis H37Rv. The tested compounds inhibited the enzyme activity to a lesser extent that of streptomycin sulphate used as positive control (Table 4). However, compound 18 emerged as the best inhibitor of mycobacterial isocitrate lyase, pantothenate synthetase and chorismate mutase activity showing percentage inhibition of 64.56, 60.12 and 58.23% respectively which was comparable to percent inhibition of 75.12, 77.06 and 79.56% respectively of these enzymes by streptomycin sulphate.

In vitro anticancer activity

Almost all the synthesized compounds showed less cytotoxicity towards MCF7 and HCT116 cell lines in comparison to tamoxifen and 5-fluorouracil used as drugs for comparison against MCF7 and HCt116 cell lines, respectively (Table 1). However, compound 2 (IC50 = 0.0047 µM/ml) showed almost equal cytotoxicity to tamoxifen (IC50 = 0.0043 µM/ml) against MCF7 cell line. On the other hand, compound 10 (IC50 = 0.0058 µM/ml) was twice more cytotoxic against HCT116 cell line as compared to 5-fluorouracil (IC50 = 0.0125 µM/ml).

Structure activity relationship

  1. 1.

    Electron withdrawing group fluoro at ortho and para positions (compounds 2 and 3) while nitro group at meta position (compound 10) improved anticancer activity. The presence of other electron withdrawing groups like Cl, Br at ortho, meta or para positions diminished the anticancer activity.

  2. 2.

    It is also important to note that fluoro group at position-2 and nitro group at position-3 are essential requirements for anticancer activity.

  3. 3.

    Electron donating groups methoxy and methyl at para position (compound 18 and 12, respectively) have more activating influence on antitubercular activity as compared to ortho- and meta-positions of these groups and followed the order p > o > m.

  4. 4.

    In general, substitution of electron withdrawing groups like Cl, Br, NO2 etc. on the benzene ring has an activating influence on antimicrobial activity while substitution of electron releasing groups like OCH3, CH3 etc. decreases the antimicrobial activity.

Conclusion

A series of 2-(1-benzoyl-1H-benzo[d]imidazole-2-ylthio)-2-ylthio)-N-substituted acetamides was synthesized and assessed for its in vitro antimicrobial and anticancer activity against human breast cancer (MCF7) and colorectal (HCT116) cell line. The compounds were also assessed for their in vitro and in vivo antitubercular activity in M. tuberculosis H37Rv. The in vivo antitubercular evaluation in mice models infected with M. tuberculosis revealed 5.67 mg/kg to be the toxic dose of the compounds that proved fatal and highly toxic to mice while LD50 varied from 1.81 to 3.17 mg/kg body weight of the mice. A dose 1.34 mg/kg was found to be safe for each of the compounds. The compounds found to be active in in vivo evaluation were further assessed for their capacity to inhibit the mycobacterial enzymes viz., isocitrate lyase, pantothenate synthetase and chorismate mutase. The tested compounds inhibited these enzymes to a lesser extent than streptomycin sulphate used as positive control. However, compound 18 inhibited the mycobacterial isocitrate lyase, pantothenate synthetase and chorismate mutase activity to 64.56, 60.12 and 58.23% respectively as compared to inhibition of 75.12, 77.06 and 79.56%, respectively by streptomycin sulphate. Compounds 8, 9 and 11 emerged out as excellent antimicrobial agents in antimicrobial assays when compared to standard antibacterial and antifungal drugs. The results of anticancer activity displayed that majority of the derivatives were less cytotoxic towards MCF7 and HCT116 cell lines when compared with standard drugs tamoxifen and 5-fluorouracil respectively. However, compound 2 (IC50 = 0.0047 µM/ml) and compound 10 (IC50 = 0.0058 µM/ml) showed highest inhibition against MCF7 and HCT116 cell lines respectively.

Experimental

Materials and method

The reagents and chemical used for research work were of analytical grade obtained from commercial sources and used as such without further purification. Melting points were determined by open glass capillary method and are uncorrected. Media and Microbial type cell cultures (MTCC) for antimicrobial activity were obtained on order from Hi-media Laboratories and IMTECH, Chandigarh, respectively. Infrared (IR) spectra was recorded on Bruker 12,060,280, Software: OPUS 7.2.139.1294 spectrophotometer by KBr pellet method and expressed in cm−1. The proton nuclear magnetic resonance (1H NMR) and carbon nuclear magnetic resonance (13CNMR) spectra were traced in deuterated DMSO on Bruker Avance III 600 NMR spectrometer at a frequency of 600 and 150 MHz respectively downfield to tetramethylsilane standard and recorded as chemical shifts in δ ppm (parts per million). The progress of reaction was confirmed by TLC performed on silica gel-G plates and the spots were visualized in iodine chamber. The LCMS data were recorded on Waters Q-TOF micromass (ESI–MS), at Panjab University, India. Elemental analysis for synthesized derivatives was performed on CHNN/CHNS/O analyzer (Flash EA1112N series, Thermo finnigan, Italy).

Synthesis

General procedure for synthesis of 2-chloro-N-substituted acetamide

An appropriate aniline (0.025 mol) and chloro acetyl chloride (0.037 mol) were separately dissolved in 10 ml of glacial acetic acid and poured into a round bottom flask. The mixture was heated on a water bath with an air condenser till the evolution of hydrochloride gas ceases. The mixture was then cooled to an ambient temperature and about 35 ml of 0.4 M sodium acetate solution was added to it. Thick precipitate so formed was filtered and washed with cold water.

General procedure for synthesis of 2-(1H-benzo[d]imidazol-2-ylthio)-N-substituted acetamide

Equimolar (0.01 mol) quantities of 2-mercaptobenzimidazole and potassium hydroxide were dissolved in 100 ml of methanol by stirring and simultaneously heating to 50–60 °C. 2-Chloro-N-substituted-acetamide (0.01 mol) was added in small lots to the stirred mixture maintaining the temperature of the mixture at 50–60 °C. The reaction mixture was then stirred at room temperature for 12 h and then was poured into ice cold water and stirred for 30 min maintaining the temperature at 5–10 °C. The precipitate formed was filtered, washed with cold water, dried and recrystallized with ethanol.

General procedure for synthesis of 2-(1-benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-substituted acetamide derivatives (1–20)

To a round bottom flask containing 2-(1H-benzo[d]imidazol-2-ylthio)-N-substituted acetamide (0.01 mol) in about 40 ml of chloroform, 1.4 ml of benzoyl chloride (0.012 mol) and 1.66 ml of triethylamine (0.012 mol) were added. The reaction mixture was refluxed for an appropriate time. The formation of product was confirmed by TLC. The solvent was distilled off and the residue obtained was washed with water, dried and recrystallized with hexane.

Spectral data of 2-(1-benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-substituted acetamide derivatives (1–20)

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-acetamide (1)

Light brown crystals, yield 76%, mp 97–100 °C, Rf 0.60 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3466 N–H str. for 2° amide, 3069 N–H str. of imidazole, 1702 C=O str for 2° amide, 754 C–S str. of thiol. 1H NMR: δH 3.80 (s, 2H of methylene), 7.06–7.97 (m, 14H, aromatic), 10.87 (s, NH of 2° amide). 13C NMR: δc 36.70 CH2 aliphatic, (124.95, 126.07, 127.54, 128.48, 128.50, 128.74, 129.20, 130.70, 131.34, 131.79, 132.80) C of benzene, (113.13, 119.20, 123.70, 138.56, 150.17) C of benzimidazole, 142.5 CH aliphatic, 164.79 C of ketone, 167.25 C of amide. ESI–MS (m/z) [M+1]+ 388.36; Anal. Calcd. for C22H17N3O2S: C, 68.20; H, 4.42; N, 10.85; O, 8.26; S, 8.28 Found: C, 68.22; H, 4.39; N, 10.82; O, 8.25; S, 8.23.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(2-fluorophenyl)acetamide (2)

Light brown, yield 69%, mp 115–118 °C, Rf 0.69 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3445 N–H str. for 2° amide, 3046 N–H str. for imidazole, 1695 C=O str. for 2° amide, 1024 C–F str. of monofluorinated compound, 739 C–S str. of thiol. 1H NMR: δH 4.36 (s, 2H of methylene), 7.09–7.97 (m, 13H, aromatic), 10.38 (s, NH of 2° amide. 13C NMR: δc 36.86 CH2 aliphatic, (118.17, 122.89, 124.31, 125.26, 125.97, 126.04, 128.37, 129.30, 130.73, 132.62, 133.84, 166.49) C of benzene, (115.48, 123.53, 130.73, 143.00, 152.52) C of benzimidazole, 142.5 CH aliphatic, 167.24 C of ketone, 169.28 C of amide. ESI–MS (m/z) [M+1]+ 406.23; Anal. Calcd. for C22H16FN3O2S: C, 65.17; H, 3.98; F, 4.69; N, 10.36; O, 7.89; S, 7.91 Found: C, 65.19; H, 3.92; F, 4.66; N, 10.39; O, 7.83; S, 7.87.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(4-fluorophenyl)acetamide (3)

Cream colored crystals, yield 75%, mp 182–185 °C, Rf 0.80 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3439 N–H str. for 2° amide, 3056 N–H str. for imidazole, 1652 C=O str for 2° amide, 1156 C–F str. of monofluorinated compound, 744 C–S str. of thiol. 1H NMR: δH 4.65 (s, 2H of methylene), 7.14–7.72 (m, 13H, aromatic), 10.98 (s, NH of 2° amide). 13C NMR: δc 36.60 CH2 aliphatic, (115.25, 115.40, 121.03, 124.95, 128.49, 132.78, 134.98, 157.37) C of benzene, (113.12, 120.98, 129.19, 134.99, 150.12) C of benzimidazole, 142.5 CH aliphatic, 158.96 C of ketone, 164.73 C of amide. ESI–MS (m/z) [M+1]+ 406.01; Anal. Calcd. for C22H16FN3O2S: C, 65.17; H, 3.98; F, 4.69; N, 10.36; O, 7.89; S, 7.91 Found: C, 65.14; H, 3.95; F, 4.63; N, 10.37; O, 7.81; S, 7.85.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(2-chlorophenyl)acetamide (4)

Peach colored crystals, yield 82%, mp 137–140 °C, Rf 0.73 (n-hexane:ethylacetate 6:4); IR (νmax, cm-1): 3434 N–H str. for 2° amide, 2986 N–H str. for imidazole, 1694 C=O str. for 2° amide, 841 C–S str. of thiol. 1H NMR: δH 4.34 (s, 2H of methylene), 7.08–7.97 (m, 13H, aromatic), 10.06 (s, NH of 2° amide. 13C NMR: δc 36.72 CH2 aliphatic, (124.83, 125.42, 127.66, 128.50, 129.21, 129.41, 130.73, 132.79, 133.65, 134.74) C of benzene, (118.23, 123.14, 133.89, 143.00, 154.08) C of benzimidazole, 142.5 CH aliphatic, 166.94 C of ketone, 167.67 C of amide. ESI–MS (m/z) [M+1]+ 422.76; Anal. Calcd. for C22H16ClN3O2S: C, 62.63; H, 3.82; Cl, 8.40; N, 9.96; O, 7.58; S, 7.60 Found: C, 62.66; H, 3.78; Cl, 8.38; N, 9.97; O, 7.52; S, 7.63.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(3-chlorophenyl)acetamide (5)

Cream colored crystals, yield 87%, mp 132–135 °C, Rf 0.59 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3406 N–H str. for 2° amide, 2977 N–H str. for imidazole, 1637 C=O str. for 2° amide, 781 C–Cl str. of monochlorinated compound, 740 C–S str. of thiol. 1H NMR: δH 4.49 (s, 2H of methylene), 7.12–7.83 (m, 13H, aromatic), 11.00 (s, NH of 2° amide). 13C NMR: δc 36.39 CH2 aliphatic, (117.52, 118.58, 128.50, 129.20, 133.07, 135.91, 135.99) C of benzene, (113.47, 123.27, 130.47, 140.15, 149.85) C of benzimidazole, 142.5 CH aliphatic, 165.88 C of amide. ESI–MS (m/z) [M+1]+ 422.79; Anal. Calcd. for C22H16ClN3O2S: C, 62.63; H, 3.82; Cl, 8.40; N, 9.96; O, 7.58; S, 7.60 Found: C, 62.64; H, 3.80; Cl, 8.36; N, 9.98; O, 7.54; S, 7.59.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(2,5-dichlorophenyl)acetamide (6)

Yellow crystals, yield 79%, mp 138–140 °C, Rf 0.73 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3451 N–H str. for 2° amide, 3054 N–H str. for imidazole, 1690 C=O str. for 2° amide, 786 C–S str. of thiol, 710 C–S str. of polychlorinated compound. 1H NMR: δH 4.46 (s, 2H of methylene), 6.93–8.03 (m, 12H, aromatic), 10.56 (s, NH of 2° amide). 13C NMR: δc 35.83 CH2 aliphatic, (123.32, 125.67, 128.76, 129.04, 129.63, 130.81, 131.57, 132.01, 133.66, 136.40) C of benzene, (118.20, 123.84, 130.72, 142.94, 149.85) C of benzimidazole, 166.86 C of ketone, 167.24 C of amide. ESI–MS (m/z) [M+1]+ 456.17; Anal. Calcd. for C22H15Cl2N3O2S: C, 57.90; H, 3.31; Cl, 15.54; N, 9.21; O, 7.01; S, 7.03 Found: C, 57.86; H, 3.34; Cl, 15.48; N, 9.17; O, 7.04; S, 6.99.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(2-bromophenyl)acetamide (7)

Brownish white crystals, yield 76%, mp 142–144 °C, Rf 0.61 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3463 N–H str. for 2° amide, 3052 N–H str. for imidazole, 1696 C=O str. for 2° amide, 727 C–S str. of thiol. 1H NMR: δH 4.46 (s, 2H of methylene), 6.93–8.03 (m, 13H aromatic), 10.14 (s, NH of 2° amide). 13C NMR: δc 36.69 CH2 aliphatic, (122.19, 124.31, 126.60, 128.05, 128.50, 129.21, 132.64, 133.91, 135.97) C of benzene, (118.32, 123.13, 130.72, 143.05, 153.97) C of benzimidazole, 166.78 C of ketone, 167.67 C of amide. ESI–MS (m/z) [M+1]+ 467.21; Anal. Calcd. for C22H16BrN3O2S: C, 56.66; H, 3.46; Br, 17.13; N, 9.01; O, 6.86; S, 6.88 Found: C, 56.61; H, 3.42; Br, 17.07; N, 9.06; O, 6.79; S, 6.85.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(3-bromophenyl)acetamide (8)

Yellow crystals, yield 83%, mp 146–148 °C, Rf 0.63 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3474 N–H str. for 2° amide, 3173 N–H str. for imidazole, 1667 C=O str. for 2° amide, 822 C–H out of plane bending, 720 C–S str. of thiol, 659 C–Br str. aromatic. 1H NMR: δH 4.32 (s, 2H of methylene), 7.11–8.08 (m, 13H aromatic), 8.09 (s, NH of 2° amide). 13C NMR: δc 36.02 CH2 aliphatic, (117.82, 121.18, 121.41, 125.98, 127.38, 129.09, 129.29, 139.77, 140.54) C of benzene, (113.88, 121.59, 130.73, 139.30, 150.11) C of benzimidazole, 166.86 C of ketone, 170.46 C of amide. ESI–MS (m/z) [M+1]+ 467.19; Anal. Calcd. for C22H16BrN3O2S: C, 56.66; H, 3.46; Br, 17.13; N, 9.01; O, 6.86; S, 6.88 Found: C, 56.63; H, 3.39; Br, 17.09; N, 9.03; O, 6.83; S, 6.82.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(4-bromophenyl)acetamide (9)

Light yellow crystals, yield 72%, mp 162–165 °C, Rf 0.81 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3451 N–H str. for 2° amide, 3055 N–H str. for imidazole, 1710 C=O str for 2° amide, 742 C–S str. of thiol, 623 C–Br str. aromatic. 1H NMR: δH 4.43 (s, 2H of methylene), 7.25–7.61 (m, 13H aromatic), 10.85 (s, NH of 2° amide). 13C NMR: δc 36.37 CH2 aliphatic, (115.15, 121.03, 128.49, 129.21, 136.74, 136.84, 136.791) C of benzene, (113.56, 122.82, 131.58, 138.10, 149.85) C of benzimidazole, 165.81 C of amide. ESI–MS (m/z) [M+1]+ 467.10; Anal. Calcd. for C22H16BrN3O2S: C, 56.66; H, 3.46; Br, 17.13; N, 9.01; O, 6.86; S, 6.88 Found: C, 56.59; H, 3.41; Br, 17.16; N, 8.96; O, 6.79; S, 6.78.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(3-nitrophenyl)acetamide (10)

Dull cream colored crystals, yield 78%, mp 129–131 °C, Rf 0.61 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3470 N–H str. for 2° amide, 3007 N–H str. for imidazole, 1707 C=O str. for 2° amide, 1526 asymm. str. of aromatic nitro group, 1317 symm. str. of aromatic nitro group, 716 C–S str. of thiol. 1H NMR: δH 4.46 (s, 2H of methylene), 7.21–8.31 (m, 13H aromatic), 10.83 (s, NH of 2° amide). 13C NMR: δc 43.41 CH2 aliphatic, (125.32, 127.73, 128.48, 128.52, 129.21, 130.33, 130.79) C of benzene, (113.46, 118.35, 130.31, 132.81, 147.96) C of benzimidazole, 167.25 C of amide. ESI–MS (m/z) [M+1]+ 433.09; Anal. Calcd. for C22H16N4O4S: C, 61.10; H, 3.73; N, 12.96; O, 14.80; S, 7.41 Found: C, 61.03; H, 3.78; N, 12.89; O, 14.77; S, 7.35.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(4-chloro-2-nitrophenyl)acetamide (11)

Orange crystals, yield 83%, mp 89–91 °C, Rf 0.80 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3481 N–H str. for 2° amide, 3030 N–H str. for imidazole, 1701 C=O str. for 2° amide, 1568 asymm. str. of aromatic nitro group, 1334 symm. str. of aromatic nitro group, 815 C–S str. of thiol, 704 C–Cl str. of monochlorinated aromatic compound. 1H NMR: δH 4.46 (s, 2H of methylene), 7.09–8.11 (m, 12H aromatic), 10.91 (s, NH of 2° amide). 13C NMR: δc 36.68 CH2 aliphatic, (123.96, 124.22, 129.04, 129.19, 129.94, 130.15, 130.64, 132.34, 133.96, 134.09, 135.49, 141.60) C of benzene, (114.36, 123.07, 130.73, 141.11, 153.53) C of benzimidazole, 167.61 C of amide. ESI–MS (m/z) [M+1]+ 467.76; Anal. Calcd. for C22H15ClN4O4S: C, 56.59; H, 3.24; Cl, 7.59; N, 12.00; O, 13.71; S, 6.87 Found: C, 56.51; H, 3.21; Cl, 7.53; N, 11.94; O, 13.76; S, 6.81.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-p-tolylacetamide (12)

Dull yellow crystals, yield 81%, mp 175–178 °C, Rf 0.71 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3361 N–H str. for 2° amide, 3166 N–H str. for imidazole, 2958 CH3 asymm. str. of Ar-CH3, 1695 C=O str. for 2º amide, 809 C–H out of plane bending of 1, 4- disubstituted benzene ring, 706 C–S str. of thiol. 1H NMR: δH 4.46 (s, 2H of methylene), 7.10–7.97 (m, 13H aromatic), 10.80 (s, NH of 2° amide). 13C NMR: δc 20.39 C of methyl, 36.63 CH2 aliphatic, (120.39, 128.49, 128.89, 129.20, 129.99, 130.25, 131.36, 132.65, 132.78, 136.03) C of benzene, (113.17, 123.11, 130.72, 136.09, 150.18) C of benzimidazole, 167.24 C of amide. ESI–MS (m/z) [M+1]+ 467.76; Anal. Calcd. for C23H19N3O2S: C, 68.81; H, 4.77; N, 10.47; O, 7.97; S, 7.99 Found: C, 68.86; H, 4.68; N, 10.37; O, 7.91; S, 7.94.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(2,6-dimethylphenyl)acetamide (13)

Light yellow crystals, yield 73%, mp 166–168 °C, Rf 0.47 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3446 N–H str. for 2° amide, 3062 N–H str. for imidazole, 2979 CH3 asymm. str. of Ar-CH3, 1714 C=O str. for 2° amide, 740 C–H bending of trisubstituted benzene ring, 656 C–S str. of thiol. 1H NMR: δH 4.33 (s, 2H of methylene), 2.10–2.50 (m, 6H of methyl), 7.02–7.51 (m, 12H aromatic), 9.85 (s, NH of 2° amide). 13C NMR: δc (17.96, 18.17) C of two methyl, 35.15 CH2 aliphatic, (123.29, 126.47, 128.37, 128.51, 128.90, 129.22, 132.80, 134.70, 138.18) C of benzene, (113.91, 122.03, 130.74, 138.47, 149.81) C of benzimidazole, 167.27 C of amide. ESI–MS (m/z) [M+1]+ 416.37; Anal. Calcd. for C24H21N3O2S: C, 69.37; H, 5.09; N, 10.11; O, 7.70; S, 7.72 Found: C, 69.39; H, 5.13; N, 10.03; O, 7.64; S, 7.75.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(3-methoxyphenyl)acetamide (14)

Light brown crystals, yield 74%, mp 170–172 °C, Rf 0.59 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3454 N–H str. for 2° amide, 3131 C–H str. of aralkyl ether, 3070 N–H str. for imidazole, 1526 N–H in plane bending of secondary amide, 1705 C=O str. for 2° amide, 1273 C–O–C asymm. str. of aralkyl ether, 1119 C–O–C symm. str. of aralkyl ether, 706 C–S str. of thiol. 1H NMR: δH 4.61 (s, 2H of methylene), 7.34–7.99 (m, 13H aromatic), 10.53 (s, NH of 2° amide). 13C NMR: δc 36.25 CH2 aliphatic, 63.11 C of methoxy, (113.61, 117.50, 128.76, 129.35, 130.22, 130.46, 133.58, 137.20, 140.18, 166.15) C of benzene, (113.70, 123.23, 130.69, 137.40, 149.72) C of benzimidazole, 168.67 C of amide. ESI–MS (m/z) [M+1]+ 418.19; Anal. Calcd. for C23H19N3O3S: C, 66.17; H, 4.59; N, 10.07; O, 11.50; S, 7.68 Found: C, 66.07; H, 4.53; N, 10.12; O, 11.43; S, 7.57.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(4-chlorophenyl)acetamide (15)

Creamish yellow crystals, yield 81%, mp 158–160 °C, Rf 0.75 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3405 N–H str. for 2° amide, 3105 N–H str. for imidazole, 1653 C=O str. of secondary amide, 1536 N–H in plane bending of secondary amide, 741 C–Cl str. of monochlorinated aromatic compound, 624 C–S str. of thiol. 1H NMR: δH 4.64 (s, 2H of methylene), 7.36–7.70 (m, 13H aromatic), 11.06 (s, NH of 2° amide). 13C NMR: δc 36.64 CH2 aliphatic, (113.18, 120.75, 124.67, 127.25, 128.64, 133.32, 137.55, 150.05) C aromatic, 165.07 C of amide. ESI–MS (m/z) [M+1]+ 422.01; Anal. Calcd. for C22H16ClN3O2S: C, 62.63; H, 3.82; Cl, 8.40; N, 9.96; O, 7.58; S, 7.60 Found: C, 62.53; H, 3.75; Cl, 8.44; N, 9.86; O, 7.53; S, 7.57.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N–o-tolylacetamide (16)

Dark brown crystals, yield 89%, mp 102–105 °C, Rf 0.76 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3332 N–H str. for 2° amide, 3014 N–H str. for imidazole, 2915 CH3 asymm. str. of Ar-CH3, 2363 CH3 symm. str. of Ar-CH3, 1679 C=O str. for 2° amide, 845 C–H out of plane bending of disubstituted benzene ring, 687 C–S str. of thiol. 1H NMR: δH 4.44 (s, 2H of methylene), 7.13–7.98 (m, 13H aromatic), 11.02 (s, NH of 2° amide). 13C NMR: δc 36.17 CH2 aliphatic, (11.91, 113.09, 124.37, 128.99, 129.04, 129.19, 129.58, 131.88, 133.42, 165.92) C of benzene, (113.21, 123.62, 130.71, 136.36, 149.83) C of benzimidazole, 168.67 C of amide. ESI–MS (m/z) [M+1]+ 402.16; Anal. Calcd. for C23H19N3O2S: C, 68.81; H, 4.77; N, 10.47; O, 7.97; S, 7.99 Found: C, 68.85; H, 4.70; N, 10.36; O, 7.92; S, 7.90.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(2-methoxyphenyl)acetamide (17)

Light brown semisolid, yield 75%, mp—not determined (hygroscopic), Rf 0.59 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3466 N–H str. for 2° amide, 3033 C–H str. of aralkyl ether, 3077 N–H str. for imidazole, 1595 N–H in plane bending of secondary amide, 1705 C=O str. for 2° amide, 1247 C–O–C asymm. str. of aralkyl ether, 1022 C–O–C symm. str. of aralkyl ether, 718 C–S str. of thiol. 1H NMR: δH 4.43 (s, 2H of methylene), 6.99–8.03 (m, 13H aromatic), 10.54 (s, NH of 2° amide). 13C NMR: δc 35.84 CH2 aliphatic, 55.71 C of methoxy, (114.83, 122.22, 123.13, 125.63, 126.86, 129.20, 129.64, 131.56, 134.45, 164.92) C of benzene, (113.73, 123.29, 130.73, 136.86, 151.37) C of benzimidazole, 167.25 C of amide. ESI–MS (m/z) [M+1]+ 418.23; Anal. Calcd. for C23H19N3O3S: C, 66.17; H, 4.59; N, 10.07; O, 11.50; S, 7.68 Found: C, 66.08; H, 4.51; N, 10.03; O, 11.43; S, 7.72.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(4-methoxyphenyl)acetamide (18)

Dark brown semisolid, yield 79%, mp—not determined (hygroscopic), Rf 0.67 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3326 N–H str. for 2° amide, 2972 C–H str. of aralkyl ether, 2606 N–H str. for imidazole, 1557 N–H in plane bending of secondary amide, 1702 C=O str. for 2° amide, 1230 C–O–C asymm. str. of aralkyl ether, 1022 C–O–C symm. str. of aralkyl ether, 710 C–S str. of thiol. 1H NMR: δH 4.33 (s, 2H of methylene), 7.12–8.03 (m, 13H aromatic), 10.61 (s, NH of 2° amide). 13C NMR: δc 36.00 CH2 aliphatic, 55.16 C of methoxy, (114.06, 127.53, 128.34, 129.19, 131.67, 132.25, 134.97, 155.53) C of benzene, (113.56, 122.22, 131.29, 139.39, 150.71) C of benzimidazole, 167.68 C of amide. ESI–MS (m/z) [M+1]+ 418.19; Anal. Calcd. for C23H19N3O3S: C, 66.17; H, 4.59; N, 10.07; O, 11.50; S, 7.68 Found: C, 66.11; H, 4.61; N, 10.03; O, 11.45; S, 7.74.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-m-tolylacetamide (19)

Cream colored semisolid, yield 89%, mp—not determined (hygroscopic), Rf 0.69 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3406 N–H str. for 2° amide, 2957 N–H str. for imidazole, 2957 CH3 asymm. str. of Ar-CH3, 2893 CH3 symm. str. of Ar-CH3, 1633 C=O str. for 2° amide, 701 C–S str. of thiol. 1H NMR: δH 2.32 (s, 3H of methyl), 4.47 (s, 2H of methylene), 7.18–7.98 (m, 13H aromatic), 10.72 (s, NH of 2° amide). 13C NMR: δc 36.33 CH2 aliphatic, 21.17 C of methyl, (119.60, 122.72, 124.28, 128.28, 128.72, 129.34, 131.42, 134.93, 137.94, 138.69) C of benzene, (113.43, 122.94, 130.73, 139.06, 149.93) C of benzimidazole, 142.5 CH aliphatic, 164.79 C of ketone, 167.25 C of amide. ESI–MS (m/z) [M+1]+ 402.23; Anal. Calcd. for C23H19N3O2S: C, 68.81; H, 4.77; N, 10.47; O, 7.97; S, 7.99 Found: C, 68.76; H, 4.81; N, 10.41; O, 7.93; S, 7.92.

2-(1-Benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-(2,4-dimethylphenyl)acetamide (20)

Peach colored semisolid, yield 81%, mp- not determined (hygroscopic), Rf 0.74 (n-hexane:ethylacetate 6:4); IR (νmax, cm−1): 3406 N–H str. for 2° amide, 3134 N–H str. for imidazole, 2919 CH3 asymm. str. of Ar-CH3, 2876 CH3 symm. str. of Ar-CH3, 1638 C=O str. for 2° amide, 702 C–S str. of thiol. 1H NMR: δH 4.48 (s, 2H of methylene), 2.16–2.28 (m, 6H of methyl), 6.99–7.97 (m, 12H aromatic), 10.01 (s, NH of 2° amide). 13C NMR: δc (17.74, 20.55) c of two methyl, 35.97 CH2 aliphatic, (120.32, 126.55, 129.39, 129.68, 130.73, 131.35, 134.26, 134.37, 134.45, 134.53) C of benzene, (113.27, 123.23, 130.77, 135.00, 150.04) C of benzimidazole, 167.24 C of amide. ESI–MS (m/z) [M+1]+ 416.19; Anal. Calcd. for C24H21N3O2S: C, 69.37; H, 5.09; N, 10.11; O, 7.70; S, 7.72 Found: C, 69.40; H, 5.01; N, 10.03; O, 7.62; S, 7.67.

Antimicrobial activity evaluation

Determination of MIC

The in vitro antimicrobial activity of the synthesized derivatives was evaluated against Escherichia coli, Salmonella typhi (Gram-negative bacteria); Bacillus subtilis, Staphylococcus aureus, Bacillus cereus, (Gram-positive bacteria); C. albicans and A. niger (fungal strains) using tube dilution method [33]. Cefadroxil and fluconazole were used as standard antibacterial and antifungal drugs respectively. The stock solutions of 100 µg/ml concentration were prepared in dimethyl sulfoxide for both test and standard drugs. Both the standard and test compounds were serially diluted in double strength nutrient broth I.P. for bacteria and Sabouraud dextrose broth I.P. for fungi [34]. The bacterial cultures were incubated for a period of 24 h at 37 ± 2 °C. The incubation time for C. albicans was 48 h at 37 ± 2 °C and for A. niger was 7 days at 25 ± 2 °C. The results of antimicrobial activity were stated in terms of minimum inhibitory concentration (MIC).

Determination of MBC/MFC

The minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) of the synthesized benzimidazole derivatives was determined by subculturing 100 µl of culture from each tube that remained clear in MIC determination onto sterilized petri-plates containing fresh agar medium. The petri-plates were incubated and analyzed for microbial growth visually [35].

In vitro antitubercular activity evaluation

The antimycobacterial activity of synthesized compounds was performed in three level safety laboratories at National Centre of Fungal Taxonomy (NCFT), New Delhi in association with HIHT University, Jolly Grant, Dehradun (U.K). The preserved strains of M. tuberculosis viz., Mycobacterium sensitive to streptomycin (S), isoniazid (H), rifampin (R) and pyrazinamide (PZA)-H37Rv (NCFT/TB/537) was used in order to assess the antimycobacterial activity of the compounds. Middle brook 7H10 agar (Becton–Dickinson Company (DifcoTM), 7 Loveton Circle, Sparks, Maryland, USA; Lot No. 8175150) supplemented with oleic acid-albumin catalase (OADC) (Becton–Dickinson Company Lot 8136781) for antimycobacterial activity was used to revive and culture the mycobacteria for sensitivity testing. Streptomycin (500 mg), standard antimycobacterial drug, was obtained as gift sample from Shalina Laboratories Pvt. Ltd., Navi Mumbai, Maharashtra.

Preparation of the drugs/compounds dilutions

Each of the synthesized derivatives was dissolved in DMSO to obtain a concentration of 50 µg/ml and diluted further to a concentration of 25 and 12.5 µg/ml. Similarly, stock solution of 50 µg/ml concentration was prepared for standard antitubercular drug, streptomycin and diluted further to 25 µg/ml in order to check the antitubercular activity.

Preparation of growth media

It was prepared by adding dehydrated medium (19 g) to purified water (900 ml) containing glycerol (l5 ml). The mixture was stirred well to dissolve and autoclaved at 121 °C for 10 min. Oleic acid-albumin catalase (100 ml) was aseptically added to the medium after cooling to 45 °C. No adjustment for pH was made.

Preparation of inoculum for drug sensitivity testing

Preserved strains of M. tuberculosis viz, mycobacterium sensitive to S, H, R and PZA-H37Rv (NCFT/TB/537) was revived on Middle brook 7H10 agar, prior to antituberculosis susceptibility testing. Cells were scraped from freshly grown colonies (3 weeks old) on Middle brook 7H10 plates and introduced into saline (10 ml). Bacterial suspensions with 0.5 McFarland standard turbidity equivalents to 108 CFU were prepared by dilution with saline. The mixture was vortexed for 30 s in a glass bottle containing glass beads and the particles were allowed to settle [36].

Random screening of the isolated compounds for antitubercular activity (Alamar-blue assay)

The antimycobacterial activity of compounds was assessed against mycobacterium sensitive to S, H, R and PZA-H37Rv (NCFT/TB/537); using the microplate alamar blue assay (MABA) [37]. This methodology is nontoxic, uses a thermally-stable reagent and is suitable for random screening of the antimycobacterial activity. Briefly, 200 μl of sterile deionized water was added to all outer-perimeter wells of sterile 96 well plates to minimize evaporation of the medium in the test wells during incubation. The 96 well plates received 100 μl of the Middle brook 7H9 broth (having loopful inoculum of bacteria-108 CFU) and different dilutions of the respective compounds were made directly on the plate. Plates were covered and sealed with parafilm and incubated at 37 °C for 5 days. After this time, 25 μl of a freshly prepared 1:1 mixture of alamar blue reagent and 10% tween 80 was added to the plate and incubated for 24 h. A blue color in the well was interpreted as no bacterial growth (antimycobacterial activity), and a pink color was scored as growth.

Bioassay protocol for susceptibility tests of the compounds by well diffusion method

The well diffusion method was used to determine susceptibility [36, 38]. The agar well diffusion method [39] was modified and Middle brook 7H10 agar medium was used. The culture medium was inoculated with loopful bacteria separately suspended in Middle brook 7H10 broth. Wells of 8 mm diameter were punched into agar and filled each well separately with 50 µg/ml of test compound and 25 µg/ml of standard drug. The petri-dishes were then left in the hood overnight to allow diffusion of the drug and then sealed with a carbon dioxide-permeable tape. These were then incubated at 37 °C in a carbon dioxide incubator for 4 weeks. The wells were flooded with alamar-blue dye in highly sterilized chamber and de-stained further to observe the zones of inhibition. The sensitivity of the strains to the compounds was determined by measuring the diameter of zones of inhibition (in millimeter) around the well.

Determination of the minimum inhibitory concentration (MIC) by alamar blue assay

The compounds were serially diluted to determine the minimum inhibitory concentration of the drug in Middle brook 7H9 medium using microplate alamar blue assay [36, 40, 41]. The compounds which were found satisfactory by the above two methods at a maximum concentration of 50 µg/ml were diluted further to concentrations viz., 25, 12.5, 6.25, 3.125 and 1.56 µg/ml respectively. Similarly, streptomycin was further diluted to 25 µg/ml in order to check the antitubercular activity. The plates were covered and sealed with parafilm and incubated at 37 °C for 5 days. After this time, 25 μl of a freshly prepared 1:1 mixture of alamar blue reagent and 10% tween 80 was added to the plate and incubated for 24 h. A blue color in the well was interpreted as no bacterial growth (antimycobacterial activity) and appearance of pink color was determined as growth. The MIC is defined as the lowest drug concentration which prevented a color change from blue to pink.

In vivo antitubercular activity evaluation

The LD50 (lethal dose) and ED50 (optimum/effective dose) doses were determined for the active compounds in mice models infected with Mycobacterium H37Rv via ethical permission no., NCFT/EC/16/2313 assigned to Collaborative Research Group (CRG), NCFT, New Delhi, India.

Enzyme assays for antitubercular activity

The compounds found potent in in vivo evaluation were assayed for inhibition of mycobacterial enzymes viz., isocitrate lyase, pantothenate synthetase and chorismate mutase.

Mycobacterial isocitrate lyase (ICL) assay

Isocitrate lyase activity was assayed according to the protocol reported by Dixon and Kornberg (glyoxylate phenyl hydrazone formation) [42] at 10 μM of the compounds. Isoniazid was employed as a negative control (inhibition of 0%) and streptomycin sulphate (25 μg/kg) served as a positive control [43].

Mycobacterial pantothenate synthetase (PS) assay

About 60 µl of the PS reagent, including NADH, pantoic acid, -alanine, ATP, phosphoenolpyruvate, MgCl2, myokinase, pyruvate kinase, and lactate dehydrogenase in buffer was added to each well of a 96-well plate. The compounds were then added to plates in 1 µl volumes. The reaction was initiated by the addition of 39 µl PS diluted in buffer. The final concentrations in the reaction contained 0.4 mM NADH, 5 mM pantoic acid, 10 mM MgCl2, 5 mM -alanine, 10 mM ATP, 1 mM potassium phosphoenolpyruvate, and 18 units/ml each of chicken muscle myokinase, rabbit muscle pyruvate kinase and rabbit muscle lactate dehydrogenase diluted in 100 mM HEPES buffer (pH 7.8), 1% DMSO, and 5 µg/ml PS in the final volume of 100 µl. The absorbance was measured using microplate reader at 340 nm after every 12 s for 120 s. Each plate had 16 control wells in the two outside columns, of which 12 contained the complete reaction mixture with a DMSO carrier control (full reaction) and four without PS. The per cent inhibition was calculated using the following formula: 100 × (1 − compound rate − background rate)/(full reaction rate − background rate) [44, 45].

Mycobacterial chorismate mutase (CM) assay

Reaction volumes of 0.4 ml of chorismate (typically 1 mM) in 50 mM Tris HCl (pH 7.5), 0.5 mM EDTA, 0.1 mg/ml bovine serum albumin, and 10 mM -mercaptoethanol were incubated at 37 °C for 5 min. The reaction was started with the addition of 10 µl 5 pM of MtCM (i.e., 185 ng of CM equivalent to 12.5 nM final concentration of the dimer based on the molecular mass of 36,948 Da). The reaction was allowed to proceed at 37 °C and was terminated after 1–5 min with 0.4 ml 1 M HCl. After further incubation at 37 °C for 10 min, 0.8 ml 2.5 M NaOH was added to convert prephenate formed in the enzymatic reaction to phenyl pyruvate. The absorbance of phenylpyruvate chromophore was taken at 320 nm. A blank with no enzyme for every reaction was also set to account for the non-enzymatic conversion of chorismate to prephenate and enzyme was added after the addition of NaOH. The absorbance at 320 nm for the blank varied from 0.1 to 0.3, depending upon the concentration of chorismate and the duration of the reaction [46].

In vitro anticancer screening

The in vitro cytotoxicity screening of the synthesized benzimidazole derivatives was assessed on MCF7 (human breast cancer) and HCT116 (human colorectal) cell line by Sulforhodamine-B (SRB) assay [47]. The results of anticancer activity were expressed as IC50 (amount of drug necessary to reduce the cell viability by 50%) and compared with the standard anticancer drugs, tamoxifen and 5-fluorouracil for MCF7 and HCT116 cell lines, respectively.

Abbreviations

HCT116:

human colorectal cell line

MCF7:

human breast carcinoma cell line

MIC:

minimum inhibitory concentration

MLC:

minimum lethal concentration

MBC:

minimum bactericidal concentration

MFC:

minimum fungicidal concentration

IR:

infrared spectroscopy

1HNMR:

proton nuclear magnetic resonance

13CNMR:

carbon nuclear magnetic resonance

S. aureus :

Staphylococcus aureus

B. subtilis :

Bacillus subtilis

B. cereus :

Bacillus cereus

S. typhi :

Salmonella typhi

E. coli :

Escherichia coli

C. albicans :

Candida albicans

A. niger :

Aspergillus niger

References

  1. Singh LR, Avula SR, Raj S, Srivastava A, Palnati GR, Tripathi CKM, Pasupuleti M, Sashidhara KV (2017) Coumarin–benzimidazole hybrids as a potent antimicrobial agent: synthesis and biological elevation. J Antibiot. https://doi.org/10.1038/ja.2017.70

    Article  PubMed  PubMed Central  Google Scholar 

  2. Shingalapur RV, Hosamani KM, Keri RS (2009) Synthesis and evaluation of in vitro antimicrobial and antitubercular activity of 2-styryl benzimidazoles. Eur J Med Chem 44:4244–4248

    Article  CAS  PubMed  Google Scholar 

  3. WHO annual TB report 2017. http://apps.who.int/iris/bitstream/10665/254762/1/978929022584-eng.pdf. Accessed 24 July 2017

  4. Patel RV, Patel PK, Kumari P, Rajani DP, Chikhalia KH (2012) Synthesis of benzimidazolyl-1,3,4-oxadiazol-2ylthio-N-phenyl(benzothiazolyl) acetamides as antibacterial, antifungal and antituberculosis agents. Eur J Med Chem 53:41–51

    Article  CAS  PubMed  Google Scholar 

  5. Pandey J, Tiwari VK, Verma SS, Chaturvedi V, Bhatnagar S, Sinha S, Gaikwad AN, Tripathi RP (2009) Synthesis and antitubercular screening of imidazole derivatives. Eur J Med Chem 44:3350–3355

    Article  CAS  PubMed  Google Scholar 

  6. Pogorzelska A, Sławiński J, Żołnowska B, Szafrański K, Kawiak K, Chojnacki J, Ulenberg S, Zielińska J, Bączek T (2017) Novel 2-(2-alkylthiobenzenesulfonyl)-3-(phenylprop-2-ynylidene amino)guanidine derivatives as potent anticancer agents—synthesis, molecular structure, QSAR studies and metabolic stability. Eur J Med Chem. https://doi.org/10.1016/j.ejmech.2017.06.059

    Article  PubMed  Google Scholar 

  7. Sławinski J, Szafranski K, Pogorzelska A, Zołnowska B, Kawiak A, Macur K, Belka M, Baczek T (2017) Novel 2-benzylthio-5-(1,3,4-oxadiazol-2-yl)benzenesulfonamides with anticancer activity: synthesis, QSAR study and metabolic stability. Eur J Med Chem 132:236–248

    Article  CAS  PubMed  Google Scholar 

  8. Bairi KE, Ouzir M, Agnieszka N, Khalki L (2017) Anticancer potential of Trigonella foenum graecum: cellular and molecular targets. Biomed Pharmacother 90:479–491

    Article  CAS  PubMed  Google Scholar 

  9. Ghorab MM, Ragab FA, Heiba HI, Soliman AM (2016) Design and synthesis of some novel 4-chloro-N-(4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)phenyl) benzenesulfonamide derivatives as anticancer and radiosensitizing agents. Eur J Med Chem. https://doi.org/10.1016/j.ejmech.2016.04.009

    Article  PubMed  Google Scholar 

  10. El Rashedy AA, Aboul-Enein HY (2013) Benzimidazole derivatives as potential anticancer agents. Mini Rev Med Chem 13:399–407

    PubMed  Google Scholar 

  11. Rashid M, Husain A, Mishra R, Karim S, Khan S, Ahmad M, Al-wabel N, Husain A, Ahmad A, Khan SA (2015) Design and synthesis of benzimidazoles containing substituted oxadiazole, thiadiazole and triazolothiadiazines as a source of new anticancer agents. Arab J Med Chem. https://doi.org/10.1016/j.arabjc.2015.08.019

    Article  Google Scholar 

  12. Igawa H, Takahashi M, Shirasaki M, Kakegawa K, Kina A, Ikoma M, Aida J, Yasuma T, Okuda S, Kawata Y, Noguchi T, Yamamoto S, Fujioka Y, Kundu M, Khamrai U, Nakayama M, Nagisa Y, Kasai S, Maekawa T (2016) Amine-free melanin-concentrating hormone receptor-1 antagonists: novel 1-(1H-benzimidazol-6-yl)pyridin-2(1H)-one derivatives and design to avoid CYP3A4 time-dependent inhibition. Bioorg Med Chem 24:2486–2503

    Article  CAS  PubMed  Google Scholar 

  13. Al-Masoudi NA, Jafar NNA, Abbas LJ, Baqir SJ, Pannecouque C (2011) Synthesis and anti-HIV activity of new benzimidazole, benzothiazole and carbohyrazide derivatives of the anti-inflammatory drug indomethacin. Z Naturforsch B 66:953–960

    Article  CAS  Google Scholar 

  14. Miller JF, Turner EM, Gudmundsson KS, Jenkinson S, Spaltenstein A, Thomson M, Wheelan P (2013) Novel N-substituted benzimidazole CXCR4 antagonists as potential anti-HIV agents. Bioorg Med Chem Lett 20:2125–2128

    Article  CAS  Google Scholar 

  15. Sawant R, Kawade D (2011) Synthesis and biological evaluation of some novel 2-phenyl benzimidazole-1-acetamide derivatives as potential anthelmintic agents. Acta Pharm 61:353–361

    Article  CAS  PubMed  Google Scholar 

  16. Pérez-Villanueva J, Hernández-Campos A, Yépez-Mulia L, Méndez-Cuesta C, Méndez-Lucio O, Hernández-Luis F, Castillo R (2013) Synthesis and antiprotozoal activity of novel 2-{[2-(1H-imidazol-1-yl)ethyl]sulfanyl}-1H-benzimidazole derivatives. Bioorg Med Chem Lett 23:4221–4224

    Article  CAS  PubMed  Google Scholar 

  17. Sharma MC, Smita Sharma S, Sahu NK, Kohli DV (2013) 3D QSAR kNN-MFA studies on 6-substituted benzimidazoles derivatives as nonpeptide angiotensin II receptor antagonists: a rational approach to antihypertensive agents. J Saudi Chem Soc 17:167–176

    Article  CAS  Google Scholar 

  18. Arora RK, Kaur N, Bansal Y, Bansal G (2014) Novel coumarin-benzimidazole derivatives as antioxidants and safer anti-inflammatory agents. Acta Pharm Sin B 4:368–375

    Article  PubMed  PubMed Central  Google Scholar 

  19. Dixit S, Sharma PK, Kaushik N (2013) Synthesis of novel benzimidazole derivatives: as potent analgesic agent. Med Chem Res 22:900–904

    Article  CAS  Google Scholar 

  20. Nannapaneni DT, Gupta AV, Reddy MI, Sarva RC (2010) Synthesis, characterization, and biological evaluation of benzimidazole derivatives as potential anxiolytics. J Young Pharm 2:273–279

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ren W, Ren Y, Dong M, Gao Y (2016) Design, synthesis and thrombin inhibitory activity evaluation of some novel benzimidazole derivatives. Helv Chim Acta 99:325–332

    Article  CAS  Google Scholar 

  22. Bai Y, Zhang A, Tang J, Gao J (2013) Synthesis and antifungal activity of 2-chloromethyl-1H-benzimidazole derivatives against phytopathogenic fungi in vitro. J Agric Food Chem 61:2789–2795

    Article  CAS  PubMed  Google Scholar 

  23. Wang XJ, Xi MY, Fu JH, Zhang FR, Cheng GF, Yin DL, You QD (2012) Synthesis, biological evaluation and SAR studies of benzimidazole derivatives as H1-antihistamine agents. Chin Chem Lett 23:707–710

    Article  CAS  Google Scholar 

  24. Patil A, Ganguly S, Surana S (2010) Synthesis and antiulcer activity of 2-[5-substituted-1-H-benzo[d]imidazol-2-yl sulfinyl]methyl-3-substituted quinazoline-4-(3H) ones. J Chem Sci 122:443–450

    Article  CAS  Google Scholar 

  25. Kwak HJ, Pyun YM, Kim JY, Pagire HS, Kim KY, Kim KR, Rhee SD, Jung WH, Song JS, Bae MA, Lee DH, Ahn JH (2013) Synthesis and biological evaluation of amino benzimidazole derivatives with a phenylcyclohexyl acetic acid group as anti-obesity and anti-diabetic agents. Bioorg Med Chem Lett 23:4713–4718

    Article  CAS  PubMed  Google Scholar 

  26. Yadav S, Kumar P, Clercq ED, Balzarini J, Pannecouque C, Narasimhan B (2010) 4-[1-(substituted aryl/alkyl carbonyl)-benzoimidazol-2-yl]-benzenesulphonic acids: synthesis, antimicrobial activity, QSAR studies and antiviral evaluation. Eur J Med Chem 45:5985–5997

    Article  CAS  PubMed  Google Scholar 

  27. Keng Yoon Y, Ashraf Ali M, Choon TS, Ismail R, Chee Wei A, Suresh Kumar R, Osman H, Beevi F (2013) Antituberculosis: synthesis and antimycobacterial activity of novel benzimidazole derivatives. BioMed Res Int. https://doi.org/10.1155/2013/926309

    Article  PubMed  PubMed Central  Google Scholar 

  28. Yadav S, Narasimhan B, Kaur H (2016) Perspectives of benzimidazole derivatives as anticancer agents in the new era. Anticancer Agents Med Chem 16:1403–1425

    Article  CAS  PubMed  Google Scholar 

  29. Buu Bui HT, Kim Ha QT, Keun OhW, Duc Vo D, Tram Chau YN, Kim Tu CT, Canh Pham E, Thao Tran P, Thi Tran L, Van Mai H (2016) Microwave assisted synthesis and cytotoxic activity evaluations of new benzimidazole derivatives. Tetrahedron Lett 57:887–891

    Article  CAS  Google Scholar 

  30. Lata S, Narasimhan B (2014) Biological activities of benzimidazole derivatives in the new millennium. In: Narasimhan B (ed) 21st Century: the era of heterocyclic compounds in medicinal chemistry. Lambert Academic Publishing, Saarbrücken, pp 176–259

    Google Scholar 

  31. Yadav S, Narasimhan B, Lim SM, Ramasamy K, Vasudevan M, Shah SAA, Mathur A (2018) Synthesis and evaluation of antimicrobial, antitubercular and anticancer activities of benzimidazole derivatives. EJBAS 5:100–109

    Google Scholar 

  32. Emami S, Falhati M, Banifafemi A, Shafiee A (2004) Stereoselective synthesis and antifungal activity of (Z)-trans-3-azolyl-2-methylchromanone oxime ethers. Bioorg Med Chem 12:5881–5889

    Article  CAS  PubMed  Google Scholar 

  33. Cappuccino JG, Sherman N (1999) Microbiology: a laboratory manual. Addison Wesley Longman Inc, California, p 263

    Google Scholar 

  34. Pharmacopoeia of India Ministry of Health Department (1996) Govt. of India, New Delhi, vol II, pp A–88

  35. Rodriguez-Arguelles MC, Lopez- Silva EC, Sanmartin J, Pelagatti P, Zani F (2005) Copper complexes of imidazole-2-, pyrrole-2- and indol-3-carbaldehyde thiosemicarbazones: inhibitory activity against fungi and bacteria. J Inorg Biochem 99:2231–2239

    Article  CAS  PubMed  Google Scholar 

  36. Parish T, Stroker NG (1998) Mycobacteria protocols, vol 101. Methods in molecular biology. Humana Press, Totowa, pp 395–422

    Book  Google Scholar 

  37. Franzblau SG, Collins LA (1998) Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother 41:1004–1009

    Google Scholar 

  38. World Health Organization (2006) Global tuberculosis database. Online Tuberculosis Database as of 21st March 2005

  39. Perez C, Anesini C (1993) In vitro antimicrobial activity of Argentine folk medicinal plants against Salmonella typhi. J Ethnopharmacol 44:41–46

    Article  Google Scholar 

  40. Sanjay MJ (2004) Natural products: an important source for antitubercular drugs. CRISP 5:1

    Google Scholar 

  41. Tona L, Kambu K, Ngimbi N, Cimanga K, Vlietinck AJ (1998) Antiamoebic and phytochemical screening of some Congolese medicinal plants. J Ethnopharmacol 61:57–61

    Article  CAS  PubMed  Google Scholar 

  42. Dixon GH, Kornberg HL (1959) Assay methods for key enzymes of the glyoxylate cycle. Biochem J 72:3

    Google Scholar 

  43. Krátký M, Vinšová J, Rodriguez NG, Stolaříková J (2013) Antimycobacterial activity of salicylanilide benzenesulfonates. Molecules 17:492–503

    Article  CAS  Google Scholar 

  44. Samala S, Devi PB, Nallangi R, Yogeeswari P, Sriram D (2013) Development of 3-phenyl-4,5,6,7-tetrahydro-1 pyrazolo[4,3-c]pyridine derivatives as novel Mycobacterium tuberculosis pantothenate synthetase inhibitors. Eur J Med Chem 69:356–364

    Article  CAS  PubMed  Google Scholar 

  45. Wang S, Eisenberg D (2003) Crystal structures of a pantothenate synthetase from M. tuberculosis and its complexes with substrates and a reaction intermediate. Protein Sci 12:1097–1108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Adepu R, Kumar KS, Sandra S (2012) C–N bond formation under Cu-catalysis: synthesis and in vitro evaluation of N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-ones against chorismate mutase. Bioorg Med Chem 20:5127–5138

    Article  CAS  PubMed  Google Scholar 

  47. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82:1107–1112

    Article  CAS  Google Scholar 

Download references

Authors’ contributions

Authors BN and SY have designed, synthesized and carried out the antimicrobial activity of 2-(1-benzoyl-1H-benzo[d]imidazole-2-ylthio)-2-ylthio)-N-substituted acetamide derivatives. Authors SML, KR, MV, SAAS and AM have carried out the spectral analysis, interpretation, anticancer and antitubercular evaluation of synthesized compounds. All authors read and approved the final manuscript.

Acknowledgements

The author Snehlata Yadav is grateful to Indian Council for Medical Research, New Delhi, India for providing Senior Research Fellowship (No. 45/14/2011/PHA/BMS).

Competing interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

Provided within the manuscript.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Author information

Authors and Affiliations

  1. Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak, 124001, India

    Snehlata Yadav & Balasubramanian Narasimhan

  2. Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), 42300, Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia

    Siong Meng Lim, Kalavathy Ramasamy & Syed Adnan Ali Shah

  3. Collaborative Drug Discovery Research (CDDR) Group, Pharmaceutical Life Sciences, Community of Research, Universiti Teknologi MARA (UiTM), 40450, Shah Alam, Selangor Darul Ehsan, Malaysia

    Siong Meng Lim & Kalavathy Ramasamy

  4. Department of Pharmacology and Toxicology, College of Pharmacy, Qassim University, Buraidah, 51452, Saudi Arabia

    Mani Vasudevan

  5. Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA, Puncak Alam Campus, 42300, Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia

    Syed Adnan Ali Shah

  6. Rapture Biotech, Noida, India

    Abhishek Mathur

Authors
  1. Snehlata Yadav
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Siong Meng Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kalavathy Ramasamy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mani Vasudevan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Syed Adnan Ali Shah
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Abhishek Mathur
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Balasubramanian Narasimhan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Balasubramanian Narasimhan.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yadav, S., Lim, S.M., Ramasamy, K. et al. Synthesis and evaluation of antimicrobial, antitubercular and anticancer activities of 2-(1-benzoyl-1H-benzo[d]imidazol-2-ylthio)-N-substituted acetamides. Chemistry Central Journal 12, 66 (2018). https://doi.org/10.1186/s13065-018-0432-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13065-018-0432-3

Keywords

BMC Chemistry

ISSN: 2661-801X

Contact us