Synthesis of Novel Benzimidazole Derivatives as a Carbonic Anhydrase <scp>IX</scp> (Ca <scp>IX</scp> ) Inhibitor With Anticancer Potential

ABSTRACT In the present study, a series of benzimidazole‐thiazole derivatives ( 4a – 4k ) were rationally designed and synthesized to evaluate their potential inhibitory effects on carbonic anhydrase IX (CA IX) and anticancer properties. The structural elucidation of the compounds was performed using spectroscopic techniques including 1 H NMR, 13 C NMR, and HRMS. Their cytotoxic activities were assessed on L929, HT29, MCF7, and C6 cell lines through MTT assays. Following the MTT assay, compound 4h was identified as exhibiting notable cytotoxicity against HT29, MCF7, and C6 cell lines, with IC 50 values measured at 36.6 ± 11.98, 22.8 ± 5.18, and 42.06 ± 6.58 μM, respectively. Moreover, compound 4h exhibited lower toxicity toward the L929 healthy mouse fibroblast cell line compared to the reference drug cisplatin. Flow cytometry analysis demonstrated that in cancer cell lines treated with compound 4h , the proportion of apoptotic cells was markedly higher than that of necrotic (non‐programmed dead) cells. Immunofluorescence microscopy analysis also confirmed that compound 4h had cytotoxic effects on cancer cells. According to the carbonic anhydrase IX activity results, compound 4h was found to have the highest activity.