Design and synthesis of 2-benzyl-1H-benzimidazole derivatives as direct InhA inhibitors with potential anti-tubercular activity.

The emergence of drug-resistant Mycobacterium tuberculosis underscores the urgent need for novel chemotherapeutics with improved potency and selectivity. In this study, a total of ten benzimidazole-derived derivatives were synthesized and evaluated for their in vitro anti-tubercular activity, cytotoxicity, and potential mechanism of action. Anti-TB activity was assessed using the Alamar Blue assay against M. tuberculosis H37Rv, yielding minimum inhibitory concentrations (MICs) ranging from 1.6 to 3.12 μg/mL. Cytotoxicity against RAW 264.7 macrophage cells was determined by MTT assay, and CCvalues were calculated for each active compound. Selectivity indices (SICC/MIC) indicated that BI-1 (MIC = 3.12 μg/mL), BI-3 (MIC = 3.12 μg/mL), and BI-5 (MIC = 1.6 μg/mL) exhibited favorable therapeutic windows. To explore the hypothetical molecular mechanism, in silico studies were performed, including molecular docking using CDOCKER in BIOVIA Discovery Studio and 100 ns molecular dynamics (MD) simulations with Desmond. Docking analyses revealed strong binding affinities of the selected compounds (-CDOCKER energy: BI-1 = 28.23, BI-3 = 27.18 and BI-5 = 31.81) toward M. tuberculosis enoyl-ACP reductase through non-covalent interactions with key residues Tyr158 and Lys165, while molecular dynamics simulations confirmed the stability of the resulting protein-ligand complexes. Overall, these findings highlight benzimidazole scaffolds as promising anti-tubercular agents, warranting further optimization and in vivo validation.