4-[(Aryloylhydrazineylidene)methyl]-N-phenylbenzamides as a new class of antimycobacterial agents: design, synthesis and biological evaluation

A novel series of 4-[(aryloylhydrazineylidene)methyl]-N-phenylbenzamides was designed, synthesized, and evaluated as potential antitubercular agents. The compounds were obtained through a two-step synthesis from pyridine-4-carbohydrazide or 3,5-dinitrobenzohydrazide precursors, using 4-formylbenzoic acid as a molecular linker and various 4-substituted anilines to fine-tune lipophilicity and electronic properties. All derivatives were tested in vitro against Mycobacterium tuberculosis (Mtb), M. avium, and M. kansasii, including multidrug-resistant clinical isolates. Several amides displayed potent and selective activity against Mtb, with minimum inhibitory concentrations ≤0.03 μM, surpassing isoniazid (INH). Structure-activity relationship analysis revealed that INH-based derivatives generally outperformed their 3,5-dinitrobenzohydrazide counterparts and that halogen or electron-donating substituents enhanced potency. Mode-of-action studies using metabolic labeling and enzyme overexpression experiments indicated that the most active compounds interfere with mycolic acid biosynthesis, consistent with an InhA-related mechanism. Cytotoxicity evaluation in rat precision-cut liver slices confirmed good tolerance. Overall, these benzamide-hydrazone hybrids represent a promising chemotype, offering a valuable platform for further optimization toward next-generation antitubercular drug candidates.