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

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 Open Access *Correspondence: naru2000us@yahoo.com 1 Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak 124001, India Full list of author information is available at the end of the article 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.

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].

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.

In vivo antitubercular activity
The LD 50 and ED 50 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 LD 50 varied from 1.81 to 3.17 mg/ kg body weight of the mice. LD 50 is the dose that killed 50% of the mice population within the group. Thus, ED 50 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.

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 (IC 50 = 0.0047 µM/ml) showed almost equal cytotoxicity to tamoxifen (IC 50 = 0.0043 µM/ml) against MCF7 cell line. On the other hand, compound 10 (IC 50 = 0.0058 µM/ml) was twice more cytotoxic against HCT116 cell line as compared to 5-fluorouracil (IC 50 = 0.0125 µM/ml).   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. 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. In general, substitution of electron withdrawing groups like Cl, Br, NO 2 etc. on the benzene ring has an activating influence on antimicrobial activity while substitution of electron releasing groups like OCH 3 , CH 3 etc. decreases the antimicrobial activity.

Conclusion
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 LD 50 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

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.

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.

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 10 8 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-10 8 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 LD 50 (lethal dose) and ED 50 (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.