Design, synthesis, anticancer evaluation, and In silico studies of 2-thiopyrimidine-5-carbonitrile derivatives as potent thymidylate synthase inhibitors

Thymidylate Synthase (TS) is a validated therapeutic target against Cancer. The research examined whether the Anticancer effects of lead compound 4d surpassed the action of 5-fluorouracil (5-FU) while monitoring its impact on MCF-7 breast Cancer cells under both 2D and 3D experimental conditions. The treatment of MCF-7 cells with 4d at 1 μM and 5 μM concentrations and 5-FU at 1 μM level allowed researchers to measure cytotoxicity, Apoptosis, and cell cycle arrest activities. Furthermore, ROS production and TS expression levels, including migration dynamics and 3D spheroid structure integrity, were also examined. The cell viability declined dramatically when cells were exposed to compound 4d, which causes cell cycle blockage at the G₂/M phase and elevated ROS levels, while reducing TS expression. Severe, destructive effects on cell movement and spheroid organization resulted in a dose-dependent death of cells. The compound 4d demonstrated effects that matched the results obtained from using 5-FU. The potential of TS-targeted therapeutic candidate status for breast Cancer treatment appears promising, because compound 4d demonstrates strong Anticancer effects through multiple pathways.

Anticancer; Cyanopyrimidine; Inhibitors; MD simulation; Molecular docking; Thiopyrimidine; Thymidylate synthase.