4-(2-(1H-Benzo[d]imidazol-2-ylthio)acetamido)-N-(substituted phenyl)benzamides: design, synthesis and biological evaluation

Background Dihydrofolate reductase (DHFR) is an important target for antimetabolite class of antimicrobials because it participates in purine synthesis. 2-mercaptobenzimidazole (2MBI) has similar structural features as purine nucleotides. Given that benzimidazole and similar heteroaromatics have been broadly examined for their anticancer potential, so, we hereby report the design, synthesis and biological studies (i.e. antimicrobial and anticancer studies) of 2MBI derivatives. Methodology The antimicrobial activity of synthesized 2MBI derivatives were evaluated against Gram positive and Gram negative bacterial species as well as fungal species by tube dilution technique whereas their anticancer activity was assessed against human colorectal carcinoma cell line (HCT116) by Sulforhodamine B (SRB) assay. They were also structurally characterized by IR, NMR, MS and elemental analyses. Results, discussion and conclusion The antimicrobial activity findings revealed that compound N1 (MICbs,st,ca = 1.27, 2.54, 1.27 µM), N8 (MICec= 1.43 µM), N22 (MICkp,an= 2.60 µM), N23 and N25 (MICsa= 2.65 µM) exhibited significant antimicrobial effects against tested strains, i.e. Gram-positive, Gram-negative (bacterial) and fungal strains. The anticancer screening results demonstrated that compounds N9, N18 (IC50 = 5.85, 4.53 µM) were the most potent compounds against cancer cell line (HCT116) even more than 5-FU, the standard drug (IC50 = 9.99 µM).


Background
The global statistics of infectious diseases have reached a worrying level following the rise of multi-drug resistant Gram-positive and Gram-negative microorganisms. Advanced treatments against infections which rely on a long multidrug schedule are often impeded by patient noncompliance and the emergence of multidrug resistant pathogens. The issue of resistance could be potentially overcome by exploring new effective agents. In this regard, rational drug design has been proven to be much beneficial as the biochemical basis of mechanisms of intrinsic and acquired resistance is largely known [1].
In terms of cancer, presently used chemotherapeutic agents restrain the growth of tumour through suppression of DNA replication and transcription. Chemotherapy is, however, often compromised by development of multidrug resistance, endurance of cancer cells in anaerobic environment, involvement of ribonucleotide reductase (RNR), topoisomerase I (Topo I) and topoisomerase II (Topo II) in neoplasm growth. Nevertheless, the attempt of discovering new curative anticancer agents in last decade has led to targets of specific molecular modifications in tumour cells. The new approach now focuses mainly on the development of small biologically active molecules containing significant activity without toxicity related to the usual chemotherapy [2].
Substituted benzimidazole compounds, with excellent antimicrobial activity against bacteria and ability to permeate mammalian cell membranes as well as maintain the activity with low to no associated toxicity have made these lead compounds attractive for further research work in the field of medicine [3]. The search for new active, antitumour drugs with lower toxicity against normal cells and tissues, however, remains as one of the most significant problems of modern antitumour chemotherapy [4].

Antimicrobial screening results
The results of synthesized 2MBI benzamides (N1-N26) are presented in Table 3

Anticancer screening results
The anticancer activity of synthesized analogues (N1-N26) was evaluated against human colorectal cancer cell line (HCT116) by SRB assay. Table 3 shows the antiproliferative outcome of the synthesized analogues and 5-FU, the standard drug against HCT116. The anticancer screening results indicated that 2,6-xylidine and para-methoxy substituted scaffolds, i.e. compounds N9 (IC 50 = 5.85 µM) and N18 (IC 50 = 4.53 µM) to exhibit the greatest anticancer activity among the synthesized scaffolds. The anticancer effect was even more potent than 5-FU (IC 50 = 9.99 µM).

Toxicity study
For the selectivity index calculation of the most active compounds (N9 and N18), these compounds were tested against normal human embryonic kidney cell line (HEK-293). Compounds were dissolved into 0.1% DMSO solution. The compounds were diluted in concentration (2 μM, 4 μM, 6 μM, 8 μM and 10 μM). The cells were incubated with these compounds for 24 h and more than almost 100% of HEK-293 cells were viable at IC 50 for growth inhibition of each studied compound. Results showed the significant viability difference between the test compounds treated and control cells (at zero concentration) after 24 h with (P < 0.01). The 50% of the cells were viable at the lethal dose (LD 50 ) 8.43 μM and 8.22 μM of the active compounds, N9 and N18, respectively. As we know that higher the LD 50 value than the IC 50 higher will be the selectivity that implied that the compounds may have better safety of the each of two compounds since the IC 50 is much lower the LD 50 (Table 4).

Electronic discussion
The substitution pattern of the 2MBI benzamide derivatives was carefully selected to confer different electronic environment to the molecules. Thus, electron donating groups to aromatic ring, such as methyl and methoxy and electron withdrawing groups from aromatic ring, such as halogens, nitro were chosen as substituents on the molecular structure of the target compounds [20]. Most of 2MBI benzamides derivatives were found to possess moderate antimicrobial activity except those compounds which has electron withdrawing groups. Suggestions are made that the negative inductive effect plays a significant role in medicinal field. This means that compounds with high electron density gave poor antimicrobial activity which makes the diffusion of compounds more difficult throw the body of the bacteria cell while presence of electron withdrawing groups will cause a decrease of electronic density in 2MBI benzamides compared with electron donating groups, thereby facilitating entry of the 2MBI benzamides into the cell. This is likely to increase the antimicrobial potency. The increase in antimicrobial activity is due to faster diffusion of the 2MBI benzamides with electron withdrawing groups because the reducing the total electron density on 2MBI benzamide compounds make the diffusion faster through the bacteria cell [21].
Because of their significant medicinal importance, the synthesis of substituted benzimidazoles has become a focus of synthetic organic chemistry. The antiproliferative effect and mechanism of induction of apoptosis by various bioactive heterocyclic compounds and the impact of electron releasing and withdrawing groups on the induction of apoptosis is supported by the studies of Gowda et al. [22]. Structure-activity relationship studies of the synthesized compounds based on electronic discussion, antimicrobial and anticancer screening results, the following SAR ( Fig. 6) can be assumed: i. Substitution of aromatic ring with 2,6-dimethyl substituent (compound N9, IC 50 = 5.85 µM) considerably improved the anticancer activity of benzamide derivatives whereas substitution with 2,4-dimethyl group (compound N10, IC 50 = 23.23 µM) had no such effect. The para-OCH 3 substituted aromatic ring (compound N18, IC 50 = 4.53 µM) significantly improved the anticancer activity of benzamide derivatives while the ortho and meta-OCH 3 substituents (compound N16-N17, IC 50 = 23.12 µM) does not improved the anticancer activity of synthesized compounds. ii. The substitution of aromatic amine with di-NO 2 group (compound N1) had significant effect on B. subtilis, S. typhi and fungal strain C. albicans, compared to compounds with single -NO 2 substitution (N2, N3 and N4). The meta-Cl and -Br substituted aromatic amines (compounds N8, N22) resulted in   Thus, it very well may be stated that nitro, chloro and bromo substituents bearing derivatives are the most suitable scaffolds for accomplishing the best antimicrobial range. The role of electron withdrawing group in improving antimicrobial activities is supported by the studies of Kumar et al. [19].

Experimental section
The reactants and reagents for the synthesis were got from Hi-media Laboratory, Loba Chemie and CDH Pvt. Ltd. Microbial type cell cultures (MTCC) for biological study were procured from Institute of Microbial Technology and Gene bank, Chandigarh. Reaction steps forward was monitored by thin layer chromatography (TLC) using ethyl acetate as mobile phase. The synthetic scheme was drawn via Chem Draw 8.03. Determination of melting point was done using labtech melting point equipment. IR spectrum was recorded on Bruker 12060280, spectrometer via KBr in the range of 4000-400 cm −1 .   Step i: Synthesis of 4-(2-chloroacetamido) benzoic acid (a) p-Aminobenzoic acid (0.01 mol) and triethylamine (0.01 mol) were stirred properly in ethanol to get a clear solution and cooled it for half an hour. Now the solution was added with chloroacetylchloride (0.01 mol) and stirred for 1 h and the precipitated compound a, was strained via filtering, desiccated and recrystallized with ethanol [1]. Step
Step iv: Synthesis of final (N1-N26) 2MBI derivatives The reaction mixture of 4-(2-(1H-benzo[d]imidazol-2-ylthio)acetamido)benzoyl chloride (c, 0.01 mol) and substituted aniline (0.01 mol) in suitable solvent was refluxed for appropriate time and thin layer chromatography was used to monitor the reaction. After completion of reaction, it was poured into ice cold water and the resultant precipitate was filtered, desiccated and recrystallized using ethanol [24].

Antimicrobial evaluation (in vitro)
The antimicrobial potential of new synthesized compounds (N1-N26) was validated against Gram-positive, Gram-negative bacteria using cefadroxil and fungal strains with fluconazole, by serial dilution method. Dilutions were set up in nutrient broth I.P. for bacterial (incubated at 37 ± 1 °C for 24 h) and Sabouraud dextrose broth I.P. for fungal species (25 ± 1 °C for 7 days for A. niger) and (37 ± 1 °C for 48 h for C. albicans) [25,26].

Anticancer evaluation (in vitro)
The antiproliferative activity (expressed as IC 50 ) was assessed against HCT116 using SRB B assay. This assay was based on the ability of SRB dye to bind electrostatically and its pH-dependence on protein basic amino acid residues of trichloroacetic acid-fixed cells [27]. Briefly, HCT116 was seeded at 2500 cell/well (96 well plates) and allowed to attach overnight before being exposed to 2MBI compounds (stock solution was suspended in DMSO) for 72 h and subjected to SRB assay. DMSO less than 1% did not kill the cells and the concentration of DMSO in each compound was 0.1%. Treated cells were  . 6 Structural requirements for the antimicrobial and anticancer activities of synthesized benzimidazole analogues fixed with 10% cold trichloroacetic acid and stained in 0.4% SRB. Unincorporated dye was rinsed off with 1% acetic acid and plates were left to air-dry at room temperature overnight. The air-dried plates were placed on a plate shaker and bound SRB was solubilised in 10 mM Tris base solution. Absorbance was measured by a computer-interfaced 96-well plate spectrophotometer at 570 nm.

Conclusion
The present work involves the synthesis of new substituted benzamides linked to p-amino benzoic acid and 2-mercaptobenzimidazole which possess antibacterial, antifungal and antiproliferative activities. The synthesized compounds were evaluated in vitro against seven representative microorganisms along with the anticancer activity against carcinoma cell line (HCT116). Antimicrobial results demonstrated that the presence of nitro and halo groups in aromatic ring enhanced the antimicrobial potential. Anticancer results revealed that 4- ( were the most potent anticancer agents even higher than the standard drug 5-FU (IC 50 = 9.99 µM). These compounds were more selective towards cancer cells rather than macrophages. Further the toxicity study revealed the better selectivity index against the HEK-293 cell lines at the respective IC 50 concentration. Study suggested that compound may be safer as anticancer after required experimental evaluation. The molecular structures of active compounds are mentioned in Fig. 7.