Development of isonicotinoyl-azole hybrids as potential InhA inhibitors for anti-tubercular activity

The development of innovative treatment medicines that can overcome drug resistance and intracellular persistence is necessary since tuberculosis (TB) continues to be a significant worldwide health burden. In this work, series of hybrid compounds containing azole moieties, imine/semicarbazide linkers, and isonicotinoyl groups were synthesized, and their anti-mycobacterial activity was assessed. The microplate Alamar blue test (MABA) was used to evaluate the synthesized compounds (10a-i, 17a, b, 20a-d, and 25) against Mycobacterium tuberculosis H37Rv and M. abscessus. Compounds 17a and 17b were the most active, with sub-μg mL^-1 potency (MICs of 0.5 and 0.25 μg mL^-1, respectively). However, numerous analogues demonstrated strong inhibitory activity against M. tuberculosis (MIC = 1.0 μg mL^-1). L929 fibroblasts were used to evaluate cytotoxicity, and the results showed good selectivity, especially for compounds 17a and 17b, which had high selectivity indices (SI = 160 and 240, respectively). Compounds 17a and 17b showed high bactericidal activity at low doses (BC = 5 μg mL^-1) in the bactericidal assay, confirming that multiple compounds exhibited concentration-dependent death. Additionally, in the human macrophage model, both compounds showed notable intracellular efficacy with low host-cell toxicity, demonstrating their capacity to target intracellular M. tuberculosis. Additionally, moderate antibiofilm activity was noted, suggesting that developed mycobacterial biofilms were partially disrupted. The suggested method of action was supported by mechanistic investigations that demonstrated strong inhibition of the InhA enzyme, with compound 17b exhibiting activity similar to triclosan (IC₅₀ = 0.75 μM). The stability and favorable binding interactions of the lead compounds within the InhA active site were further validated by in silico investigations, including molecular docking and molecular dynamics simulations. All things considered, the incorporation of several pharmacophores into a single scaffold yielded promising multitarget anti-tubercular agents, with compounds 17a and 17b as potential avenues for further research and optimization.