A series of xanthene derivatives (3a–n) was synthesized using microwave-assisted catalysis with trifluoroacetic acid (TFA) in an aqueous medium, achieving high yields within 10 min. The structural features of these compounds were comprehensively characterized by FT-IR, 1H-NMR, 13C-NMR, and mass spectrometry (MS). The inhibitory effects of the synthesized xanthene derivatives on human carbonic anhydrase isoenzymes (hCA I and hCA II) were assessed in vitro. Notably, compounds 3c and 3n demonstrated significant inhibition of these isoenzymes, with compound 3c exhibiting a Ki value of 3.619 µM for hCA I and compound 3n showing a Ki value of 0.178 µM for hCA II. In silico analyses further supported the strong inhibitory activity of compounds 3c and 3n against hCA I and hCA II. Drug-likeness and ADMET properties were evaluated according to Lipinski’s rules and computational prediction tools. Molecular docking studies indicated that both compounds formed strong interactions with their target receptors (2CAB and 1CA2) via active-site residues, including His94, His96, and His119. The binding affinities of 3c and 3n exceeded that of the reference inhibitor, acetazolamide (AAZ). Additionally, 100 ns molecular dynamics simulations confirmed the structural stability of the resulting complexes, as assessed by RMSD and RMSF analyses.
