Exploration of novel indolo[2,3-b]quinoxaline hybrids as InhA inhibitors: A multi-disciplinary approach involving synthesis, antimycobacterial evaluation and computational studies.

Multidrug-resistant tuberculosis remains a formidable global health threat, necessitating novel antitubercular agents with improved potency, safety, and target engagement. Herein, a library of 52 indolo[2,3-b]quinoxaline derivatives was designed and synthesized using a pharmacophore-hybridization strategy and subjected to comprehensive biological and computational evaluation. The synthesized compounds were characterized by NMR and mass spectrometry, supplemented by representative FTIR analysis for each series, while HPLC confirmed the analytical purity of two selected lead compounds. In vitro screening against Mtb identified several potent derivatives with MIC values of 0.25-2 μg/mL. Cytotoxicity evaluation of the potent derivatives revealed favourable mammalian-cell safety profiles. From these compounds, 14b (MIC = 0.5 μg/mL; SI > 200) and 17 (MIC = 0.25 μg/mL; SI > 400) were selected for advanced evaluation to investigate the influence of linker flexibility versus rigidity on resistance profiles. Both compounds demonstrated concentration and time-dependent bactericidal activity in MBC and time-kill studies. Activity was reduced against the resistant strain, while both lead compounds demonstrated significant reductions in intracellular mycobacterial burden in infected macrophages. Mechanistic studies using resistant-mutant selection and InhA sequencing revealed a >64-fold MIC shift and mutations in key InhA residues, supporting the involvement of InhA in the antimycobacterial activity of 17. Molecular docking supported favourable binding of 14b and 17 within the InhA active site, with redocking validation yielding an RMSD of 0.52 Å. Molecular dynamic simulations indicated stable protein-ligand complexes, while physicochemical and ADMET profiling revealed favourable drug-like characteristics. Collectively, these findings highlight indolo[2,3-b]quinoxaline as promising antitubercular scaffold for further development.