Design, synthesis, and preliminary in vitro screening of novel tolmetin hydrazones against colon cancer

The present study describes the design and synthesis of twenty novel tolmetin-hydrazones (3a-t). The structures of the synthesized compounds were elucidated using FT-IR, 1H NMR, 13C NMR, and HR-MS (ESI+) spectroscopy techniques. The cytotoxic potential of all synthesized compounds was evaluated against HCT-116 human colon cancer cells and CCD-18Co normal colon fibroblasts using the CCK-8 colorimetric assay. Based on their IC50 and selectivity index (SI) values, 2-[1-methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]-N'-(thiophen-2-ylmethylene) acetohydrazide (3h) (IC50 = 10.93 ± 0.07 μM / SI = 4.01) and N'-(2,3-dichlorobenzylidene)-2-[1-methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl] acetohydrazide (3q) (IC50 = 21.41 ± 0.13 μM / SI = 2.76) were selected as the most active compounds for further mechanistic investigations. To elucidate the underlying cytotoxic mechanisms, mitochondrial membrane potential (MMP) assessment via JC-1 staining, apoptosis detection through Annexin V/PI double staining, and quantitative evaluation of COX-2 enzyme activity and PGE2 levels were performed on HCT-116 cells. Compound 3h selectively induced mitochondrial membrane potential depolarization, triggered apoptosis, exhibited dose-dependent COX-2 inhibition, and decreased prostaglandin levels. Furthermore, detailed molecular docking simulations of all compounds were performed against human COX-1 (PDB ID: 6Y3C) and COX-2 (PDB ID: 5KIR) enzymes to calculate their binding energies and identify the structural basis of their selectivity. In silico analysis revealed favorable binding orientations for compound 3h (-10.1 kcal/mol), including hydrogen-bonding interactions with key active-site and gating residues such as HIS90. Collectively, compound 3h exhibits selective anticancer activity and represents a promising hit compound for the development of COX-2-targeted anticancer agents.