Synthesis, Cytotoxicity, and Molecular Modeling of Novel Benzamide-Derived Hydrazone Derivatives as ALDOA Inhibitors in Colorectal Cancer

Colorectal cancer remains a major global health challenge, underscoring the need for new therapeutic agents targeting cancer metabolism. In this study, a novel series of benzamide-derived hydrazone derivatives (3a–i) was designed and synthesized as the potential Aldolase A (ALDOA) inhibitors. Their antiproliferative activity was first evaluated against HT-29 human colorectal cancer cells and L929 mouse fibroblast cells. Among the series, compounds 3a (5-bromo-2-methoxyphenyl) and 3d (5-nitrofuran-2-yl) displayed particularly potent and selective cytotoxicity, with IC50 values of 0.382 ± 0.115 µM and 0.101 ± 0.066 µM, respectively. Both compounds also effectively inhibited ALDOA (72.06 ± 4.12% and 82.09 ± 7.14%, respectively, at 10 µM), reduced lactate production, downregulated hypoxia-inducible factor-1α (HIF-1α), and induced apoptosis, demonstrating a strong metabolic inhibition profile. Furthermore, 300 ns molecular dynamics simulations of the compound 3d–ALDOA complex revealed a stable and specific binding mode with the persistent intermolecular interactions, supporting its experimentally observed inhibitory potential. Collectively, these findings introduce a new hydrazone-based scaffold with enhanced potency and selectivity, representing a promising lead for further development in colorectal cancer therapy.