Isoxazole Derivatives against Carbonic Anhydrase: Synthesis, Molecular Docking, MD Simulations, and Free Energy Calculations Coupled with In Vitro Studies

[Image: see text] Heterocyclic compounds with a five-membered ring as a core, particularly those containing more than one heteroatom, have a wide spectrum of biological functions, especially in enzyme inhibition. In this study, we present the synthesis of five-membered heterocyclic isoxazole derivatives via sonication of ethyl butyrylacetate with aromatic aldehyde in the presence of a SnII-Mont K10 catalyst. The synthesized compounds were characterized using sophisticated spectroscopic methods. In vitro testing of the compounds reveals three derivatives with significant inhibitory action against carbonic anhydrase (CA) enzyme. The compound AC2 revealed the most promising inhibitory activity against CA among the entire series, with an IC(50) = 112.3 ± 1.6 μM (%(inh) = 79.5) followed by AC3 with an IC(50) = 228.4 ± 2.3 μM (%(inh) = 68.7) compared to the standard with 18.6 ± 0.5 μM (%(inh) = 87.0). Molecular docking (MD) study coupled with extensive MD simulations (400 ns) and MMPBSA study fully supported the in vitro enzyme inhibition results, evident from the computed ΔG(bind) (AC2 = −13.53 and AC3 = −12.49 kcal/mol). The in vitro and in silico studies are also augmented by a fluorescence-based enzymatic assay in which compounds AC2 and AC3 showed significant fluorescence enhancement. Therefore, on the basis of the present study, it is inferred that AC2 and AC3 may serve as a new framework for designing effective CA inhibitors.