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.

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 (KI = 15.0-37.9 nM), outperforming the reference inhibitor acetazolamide (KI = 25 nM) and displaying better selectivity indices. Moreover, these substances efficiently suppressed VEGFR-2, with IC50 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 (IC50 = 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 G1/G2/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.