Compound 5a(Bahauddin Zakariya University)

In this study, a series of 6-ethoxyphenyl-4-fluorobenzenesulphonate-based thiosemicarbazones (5a-w) were synthesized via a two-step process and structurally characterized by ¹H NMR and ¹³C NMR spectroscopy. Their inhibitory activities against human carbonic anhydrase isoforms I and II (hCA I and hCA II) were evaluated, revealing potent inhibition at low nanomolar concentrations with IC₅₀ values ranging from 56.36 to 230.17 nM for hCA I and 30.66 to 175.45 nM for hCA II. Compounds 5a, 5g, and 5n exhibited the highest enzyme inhibition, with 5a identified as the most potent in vitro inhibitor for both isoforms. Molecular docking studies and MM-GBSA binding free energy calculations demonstrated that compound 5n displayed the strongest binding affinity toward hCA I, stabilized by key interactions including π-π stacking, hydrogen bonds, and coordination to the catalytic zinc ion. Molecular dynamics simulations over 100 ns confirmed the stability and dynamic adaptability of the 5n-hCA I and 5g-hCA II complexes, preserving critical interactions essential for binding. Validation of the docking protocol yielded RMSD values below 2.0 Å, supporting the reliability of the computational approach. Overall, these findings highlight compounds 5n and 5g as promising lead molecules for selective inhibition of hCA I and hCA II, with potential applications in the treatment of carbonic anhydrase-related disorders.