Identification of Sulfonamide‐Based Thiazolidine‐2,4‐Dione Derivatives as VEGFR‐2 and Tumor‐Associated hCA IX Inhibitors With Hypoxia‐Targeted Anti‐Colorectal Cancer Activity

ABSTRACT Given their critical roles in tumor progression and hypoxia adaptation, concurrent inhibition of carbonic anhydrase IX (hCA IX) and vascular endothelial growth factor receptor‐2 (VEGFR‐2) is a viable therapeutic approach. This study rationally developed, synthesized, and biologically assessed a novel class of thiazolidinedione‐benzenesulfonamide hybrids as possible dual hCA IX/VEGFR‐2 inhibitors. A number of agents, including 4a , 4b , 4e , 4g , 6a , and 6b , showed strong hCA IX inhibition ( K I = 15.0–37.9 nM), outperforming the reference inhibitor acetazolamide ( K I = 25 nM) and displaying better selectivity indices. Moreover, these substances efficiently suppressed VEGFR‐2, with IC 50 values ranging from 0.055 to 1.080 µM. Under both normoxic and hypoxic conditions, biological evaluation demonstrated strong anti‐proliferative activity against colorectal cancer cell lines (HT‐29 and HCT‐116). Compound 4g stood out as the most promising candidate among the investigated derivatives, showing greater activity to 5‐fluorouracil during hypoxia and comparable potency under normoxia (IC 50 = 5.89 vs. 26.66 µM). In hypoxic conditions, 4g significantly enhanced the cytotoxicity of 5‐fluorouracil, increasing its activity by more than sevenfold. 4g caused cell‐cycle arrest at the G 1 /G 2 /M phases, raised pro‐apoptotic markers (caspase‐3, caspase‐9, and BAX), downregulated the anti‐apoptotic protein Bcl‐2, and dramatically enhanced apoptotic cell death (total apoptosis: 37.78%), according to mechanistic studies. The persistent binding of 4g inside the zinc‐containing active site of hCA IX and the ATP‐binding pocket of VEGFR‐2 was validated by molecular docking and molecular dynamics simulations. Additionally, favorable pharmacokinetic and drug‐likeness features were shown by in silico ADME research. All of these results point to compound 4g as a promising mechanism‐based dual VEGFR‐2/hCA IX inhibitor with strong anti‐colorectal cancer action, especially in hypoxic tumor microenvironment circumstances.