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. • Novel benzamide–hydrazone hybrids show potent antimycobacterial activity. • INH-derived amides reach MIC ≤ 0.03 μM for M. tuberculosis via InhA inhibition. • 3,5-Dinitrofenyl-based amides act via a cell wall biosynthesis-unrelated mechanism. • Halogenation markedly enhances potency across both series. • Active derivatives show low cytotoxicity in rat precision-cut liver slices.