This study was undertaken to identify novel thymidylate synthase inhibitors with improved efficacy and selectivity over existing chemotherapeutics. A series of novel 6-(4-aminopiperidin-1-yl)-substituted benzyl-3-methylpyrimidine-2,4(1 H,3 H)-dione derivatives were designed, synthesized, and evaluated as potential anticancer agents. Among these, compound 5 h exhibited the most potent cytotoxicity, with IC50 values of 15.70 ± 0.28 µM and 16.50 ± 4.90 µM against SW480 (colorectal cancer) and MCF-7 (breast cancer) cell lines, respectively, which were comparable to cytometry demonstrated that 5 h significantly induced apoptosis in a dose-dependent manner (up to 62% at 32 µM) and caused marked S-phase cell cycle arrest. Molecular docking and 100-ns molecular dynamics (MD) simulations revealed strong and stable interactions of 5 h with the active site of thymidylate synthase (TS), primarily through hydrogen bonding with Asp218 and Met311. MM/GBSA analysis further supported a favorable binding free energy profile. Density functional theory (DFT) studies indicated that 5 h possessed lower Gibbs free energy and higher electron affinity than less active analogs, suggesting enhanced binding and biological stability. binding affinity predictions showed that all derivatives met Lipinski's criteria and had favorable intestinal absorption exhibited permeability. Collectively, these results highlight 5 h as a promising lead for further preclinical evaluation as a thymidylate synthase inhibitor and anticancer agent.
Keywords: Apoptosis; Cell cycle arrest; Molecular dynamics; Piperidine; Pyrimidine; Thymidylate synthase.
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