Design and synthesis of isoxazole-functionalized benzene sulphonamides as novel inhibitors of <i>Mycobacterium tuberculosis</i> β-carbonic anhydrases

The escalating prevalence of multidrug-resistant tuberculosis (MDR-TB) underscores the urgent need for new classes of antitubercular agents targeting novel pathways. Carbonic anhydrase, a ubiquitous metalloenzyme, catalyses the reversible hydration of carbon dioxide in the CO₂ + H₂O ⇋ HCO₃ ^- + H⁺ reaction. Suppressing this enzymatic activity has recently been identified as a new pathway for the treatment of <i>Mycobacterium tuberculosis</i>. To address this, a series of isoxazole-sulphonamides was rationally designed, incorporating an isoxazole pharmacophore as the aromatic tail, amide as a linker, and sulphonamide as the zinc-binding group. These compounds were evaluated against <i>Mycobacterium tuberculosis</i> carbonic anhydrases (MtCA 1 and 3) and two human carbonic anhydrases (hCA I and II) to identify selective inhibitors of the bacterial enzymes. The findings indicated that molecules containing an isoxazole pharmacophore with amide-linked benzene-3-sulphonamide were significantly more selective for MtCA 3 than hCA I and II. Among these compounds, 12c, 12e, and 19b had the highest inhibition against the MtCA 3 with <i>K</i> _i values between 0.08-0.09 μM compared to the standard acetazolamide with a <i>K</i> _i value of 0.10 μM. Some of the best compounds exhibited potent and selective inhibition of MtCA 3 over hCA I and II, with the <i>meta</i>- and <i>para</i>-substituted derivatives demonstrating higher selectivity and stronger inhibition. Specifically, compound 19b proved to be 199 and 38 times more selective for MtCA 3 than hCA I and hCA II respectively, compared to the standard drug acetazolamide, which is a non-selective CA inhibitor. The potential of compound 19b as a promising antitubercular agent with a MIC value of 8 μg mL^-1 against mc² 6230 was further strengthened by <i>in silico</i> ligand-target interaction studies. Thus, compound 19b is emphasised as a promising lead in the pursuit of new, selective agents targeting MtCA 3.