Glycosyl Coumarins as Selective Inhibitors of Tumor-Associated Carbonic Anhydrase IX and XII: Synthesis, Structure-Activity Relationships, and Molecular Modeling.

Coumarins represent a distinctive class of non-classical carbonic anhydrase inhibitors that interact with the entrance region of the catalytic pocket rather than directly coordinating the catalytic Zn2+ ion. In this study, a series of glycosylated coumarins was synthesized through a copper-catalyzed multicomponent reaction involving propargyl glycosides, salicylaldehyde, and tosyl azide, providing efficient access to iminocoumarin-based glycosides derived from natural carbohydrates. The inhibitory activity of the synthesized compounds was evaluated against human carbonic anhydrase isoforms I, II, IX, and XII using a stopped-flow CO2 hydrase assay. The compounds showed negligible inhibition of the cytosolic isoforms hCA I and hCA II, while displaying moderate activity toward the tumor-associated isoforms hCA IX and hCA XII, with Ki values ranging from 12.9 to 41.8 μM. Among the series, 6-O-(2H-chromene-2-one-3-yl-methyl)-D-galactopyranose (10a) emerged as the most potent inhibitor of hCA IX and XII. Structure-activity relationship analysis indicated that deprotected glycosyl derivatives exhibit improved inhibitory activity compared to protected analogues. To rationalize these observations, molecular docking followed by molecular dynamics simulations and MM-GBSA binding free energy calculations were performed for both anomeric forms of compound 10a. The computational results revealed a clear preference for the β-anomer, particularly in hCA IX and XII, where favorable interactions with catalytic threonine residues and isoform-specific aromatic residues stabilize the ligand within the active-site entrance. These findings provide a molecular explanation for the experimentally observed selectivity and highlight glycosyl coumarins as potential starting points for further optimization toward selective inhibitors of tumor-associated carbonic anhydrases.