Design, synthesis and therapeutic potential of 3-(2-(1H-benzo[d]imidazol-2-ylthio)acetamido)-N-(substituted phenyl)benzamide analogues

Background The emergence of bacterial resistance is a major public health problem. It is essential to develop and synthesize new therapeutic agents with better activity. The mode of actions of certain newly developed antimicrobial agents, however, exhibited very limited effect in treating life threatening systemic infections. Therefore, the advancement of multi-potent and efficient antimicrobial agents is crucial to overcome the increased multi-drug resistance of bacteria and fungi. Cancer, which remains as one of the primary causes of deaths and is commonly treated by chemotherapeutic agents, is also in need of novel and efficacious agents to treat resistant cases. As such, a sequence of novel substituted benzamides was designed, synthesized and evaluated for their antimicrobial and anticancer activities. Methodology All synthesized compounds were characterized by IR, NMR, Mass and elemental analysis followed by in vitro antimicrobial studies against Gram-positive (Staphylococcus aureus), Gram-negative (Salmonella typhi and Klebsiella pneumoniae) bacterial and fungal (Candida albicans and Aspergillus niger) strains by the tube dilution method. The in vitro anticancer evaluation was carried out against the human colorectal carcinoma cell line (HCT116), using the Sulforhodamine B assay. Results, discussion and conclusion Compound W6 (MICsa, st, kp = 5.19 µM) emerged as a significant antibacterial agent against all tested bacterial strains i.e. Gram-positive (S. aureus), Gram-negative (S. typhi, K. pneumoniae) while compound W1 (MICca, an = 5.08 µM) was most potent against fungal strains (A. niger and C. albicans) and comparable to fluconazole (MIC = 8.16 µM). The anticancer screening demonstrated that compound W17 (IC50 = 4.12 µM) was most potent amongst the synthesized compounds and also more potent than the standard drug 5-FU (IC50 = 7.69 µM).


Background
Antibiotics with a wide spectrum of activity are considered to be most potent against Gram positive or Gram-negative microbes. Nevertheless, there is now a frightening increment of resistance against commercially accessible antimicrobials which diminishes the scope of treatment against distinctive irresistible infections. Resistance against β-lactam, macrolides, quinolones, and vancomycin is amongst the most vital medical issues. This has called for the discovery of novel and potent antimicrobials with varying chemical characteristics [1].
Colorectal tumour (CRC) is one of the most widely recognized gastrointestinal malignancies. Changes in way of life, high-fat eating regimen, physical apathy and smoking are related to CRC pathogenesis. About 25% instances of CRC were presented with metastases at early analysis and nearly 50% of CRC patients would suffer from metastasis at some stage of life. To a large extent, the outcomes of treatment for these patients are unsatisfactory because usual regimens consider the probability of homogeneous distribution of tumor mass. It is increasingly recognized that CRC is sustained by a distinctive set of neoplastic cells named "cancer stem cells (CSCs)" which have an intrinsic potential of stemness (protection from treatment) and oncogenesis (malignant growth) [2].
Owing to their diverse potential, heterocyclic scaffolds are of major interest in the pharmaceutical industry. The heterocyclic entities which have a nitrogen group occupying a central position make them highly resemblance in structure with numerous naturally occurring molecules. In terms of improvement of biologically and therapeutically important molecules, benzimidazole nucleus has been proven as the most advantageous pharmacophore [3].
Benzimidazole is also a vital pharmacophore, a privileged sub-structure in medicinal chemistry which contributes as a key part for different natural activities. The prominent organic applications as demonstrated by molecules related to these cores have incited wide examinations for their synthesis. Extensive biochemical and pharmacological investigations have affirmed that benzimidazole derivatives are effective against different microorganisms. Given the basic resemblance with purine, antibacterial capacity of these scaffolds showed that their opposition with purines would bring about a particular hindrance against the combination of nucleic acids and proteins inside the bacterial cell wall [18,19]. Figure 2 reviews the biological and therapeutic profile of benzimidazoles as motivated by the structural features. As part of our continued effort in exploring new therapeutic molecules, we hereby report the synthesis, antimicrobial and anticancer evaluation as well as SAR studies of some 3-(2-(1H-benzo[d]imidazol-2-ylthio)acetamido)-N-(substituted phenyl) benzamide derivatives.

Chemistry
The new target molecules (W1-W21) had been synthesized by multistep procedure as discussed in Scheme 1. Firstly, the 3-(2-chloroacetamido) benzoic acid (int-I) was synthesized by the reaction of chloroacetyl chloride with m-amino benzoic acid, then it was refluxed with 2-mercaptobenzimidazole which yielded 3-(2-(1H-benzo[d] imidazol-2-ylthio)acetamido)benzoic acid (int-II) which on reaction with thionyl chloride in suitable alcohol resulted in formation of 3-(2-(1H-benzo[d]imidazol-2-ylthio)acetamido)benzoyl chloride (int-III). The reaction of above synthesized benzoyl chloride (int-III) with different substituted anilines in methanolic/ethanolic solvent yielded the title scaffolds (W1-W21) with appreciable yields. The physicochemical properties with Mass spectra and elemental analysis of synthesized compounds are given in experimental section. The synthesized molecules (W1-W21) were also illustrated by FT-IR, proton and carbon-NMR data which are in concurrence with the proposed molecular structures of synthesized compounds. The IR stretching vibration at 3107-3097 cm −1 and ̴ 1600 cm −1 illustrated the occurrence of aromatic C-H and C = C groups, respectively. The IR band in the range of 653 cm −1 to 651 cm −1 corresponds to the C-Br stretching of Ar-Br compounds (W5 and W21). The presence of aromatic nitro group in compounds, W1, W2, W3, W4, W6 and W12 is indicated by the appearance of stretching in the range of 1554-1484 cm −1 . IR band appearance around 2832-2820 cm −1 has established the existence of Ar-OCH 3 (an arylalkyl ether) in compounds W15, W16 and W17. Moreover, halogen group presence in compounds, W6, W12 and W13 is specified by the presence of stretching vibration at 758-742 cm −1 of Ar-Cl and in compounds W18 and W19 by presence of Ar-F stretching at 1085-1084 cm −1 . IR stretching at 2900-2869 cm −1 in the spectral data of synthesized derivatives (W9-W11) depicted the presence of Ar-CH 3 . Presence of C-S group is indicated by the appearance IR stretching at 708-679 cm −1 in synthesized compound's spectral data. The appearance of -CONH-group is suggested by the IR stretching at 1670-1662 cm −1 spectral data of synthesized compounds. The presence of -C=N-(3 o amine) and -C-NH-(2 o amine) groups is suggested by the appearance of IR stretching 1372-1321 cm −1 and 1338-1302 cm −1 of synthesized compound's spectral data respectively. The structures of N-phenylbenzamide were further confirmed by the corresponding 1 H-NMR. The multiplet signals between 7.12 and 10.75 δ ppm in 1 H-NMR spectra indicated the aromatic proton of synthesized derivatives. The presence of -CH 2 -and -CONH-between 2-mercaptobenzimidazole and para amino benzoic acid in all the synthesized derivatives were indicated by appearance of singlet at 2.51-4.34 δ ppm and 7.82-8.15 δ ppm, respectively. Owing to presence of CH 3 of Ar-CH 3 , the compounds W9, W10 and W11 reflected singlet at 2.51-2.53 δ ppm. As a result of presence of -OCH 3 of Ar-OCH 3 , compounds W15, W16 and W17 showed singlet at range of 3.73-3.74 δ ppm. Due to presence of -NH-of benzimidazole, all synthesized compounds reflected singlet at 4.31-4.37 δ ppm. The findings of elemental analysis of synthesized 2-mercaptobenzimidazoles were recorded within theoretical results of ± 0.4%. Conclusively, the 13 C-NMR spectra of synthesized benzamides were in DMSO-d6 and their molecular structures were in accordance with the spectral signals. Mass spectra of the synthesized derivatives reflected the characteristic molecular ion peaks.

In vitro antimicrobial and anticancer screening results
The antimicrobial results of synthesized of benzamides (W1-W21) are presented in Table 1 Table 1 shows the results in vitro anticancer activity of the synthesized analogues (W1-W21). The anticancer screening results showed that 4-methoxy substituted scaffold i.e. compound W17 (IC 50 = 4.12 µM) had the highest anticancer activity amongst the synthesized ones. It was found to be more potent than the standard drug, 5-FU (IC 50 = 7.69 µM).

Experimental section
The reactants and reagents for syntheses were taken from commercial resource. Micromass Q-ToF Micro instrument was used for mass spectra. Elemental analysis was performed on Perkin-Elmer 2400 C, H and N analyzer and all synthesized compounds gave C, H and N analysis within ± 0.4% of the theoretical results.

Procedure for synthesized benzamides (W1-W21)
Step a: Synthesis of int-I A mixture of m-aminobenzoic acid (0.01 mol) and triethylamine (0.01 mol) in ethanol was stirred to get a clear solution. Then the solution was cooled in ice for 30 min followed by dropwise addition of chloroacetylchloride (0.01 mol) with stirring (1 h). The resultant precipitate was strained via filtering, desiccated and recrystallized using alcohol [20].

Step b: Synthesis of int-II
To the reaction mixture of 3-(2-chloroacetamido) benzoic acid (int-I, 0.01 mol) and 2-mercaptobenzimidazole (0.01 mol) in alcohol potassium carbonate (0.01 mol) was added and then refluxed for 5-6 h and cooled to room temperature followed by evaporation to dryness. The resultant residue was washed with water and recrystallized from ethanol [1]. Step

d: Synthesis of final (W1-W21) benzamides
The reaction mixture of 3-(2-(1H-benzo[d]imidazol-2-ylthio)acetamido)benzoyl chloride (int-III, 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 [22]. Spectral data elucidation of the synthesized N-substituted phenyl benzamide derivatives.