Rational design and synthesis of pyrazole-based sulfonamides as dual carbonic anhydrase and PRAK-targeting anticancer agents.

Herein, a novel series of pyrazole-based derivatives 5a-f, 6a-f, 7a,b, and 8a-f was rationally designed, synthesized, and evaluated as potential anticancer agents targeting tumor-associated carbonic anhydrase (hCA) isoforms IX and XII. Sulfonamide derivatives 5d,e, and 8d-f displayed potent inhibitory activity against hCA IX (Ki = 2.2-34.9 nM) and XII (Ki = 9.9-51.9 nM). The structure of representative compound 8d was confirmed by single crystal X-ray crystallography. In vitro anticancer screening against the NCI-60 human tumor cell lines revealed that compounds 8d-f exhibited broad-spectrum cytotoxicity, with 8d inducing G0/G1 cell cycle arrest and suppressing DNA synthesis in MDA-MB-231 breast cancer cells. Kinase profiling of compound 8d against 140 kinases at 10 μM uncovered additional inhibitory activity against key cancer-related kinases, particularly YES1, BTK, MAP4K3, and most intriguingly, PRAK, suggesting a potential multitarget mechanism. To the best of our knowledge, this study represents the first dual carbonic anhydrase inhibitor capable of engaging PRAK. The concurrent inhibition of pH regulator-hCA IX and PRAK signaling by compound 8d disrupted hypoxia-driven survival pathways and promoted cell death in therapy-resistant tumor cells. Molecular docking studies supported these findings, demonstrating stable binding within the active sites of hCA IX and XII. Additionally, docking studies demonstrated that compound 8d adopts a stable binding pose within the ATP-binding cleft of PRAK, establishing favorable interactions with key active-site residues.