Enhancement of antitumor activity through structural optimization of...

We used our previously developed one-pot green trans-annulation approach involving thermally induced 1 H-tetrazole ring cleavage and in situ pyrimidine formation for synthesis of combinatorial library of substituted 2-(benzyl/phenethylamino)-thieno[2,3-/3,2-d]pyrimidin-4(3H)-ones. Scaffold selection was guided by identification of 2-(benzylamino)-5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-one as a highly cytotoxic lead. Biological evaluation revealed that 4c and 4b compounds exhibited the most potent and broad-spectrum cytotoxicity. 4c compound demonstrated sub-micromolar IC₅₀ values against Jurkat (0.84 µM), KB3-1 (0.77 µM), and HCT116 (2.43 µM) cells, whereas 4b compound showed moderate cytotoxic activity (7.92-17.33 µM). Other derivatives displayed moderate cytotoxicity (8-70 µM) and generally higher IC₅₀ values in non-cancerous HaCaT, BEAS-2B, and Balb/3T3 cells. Mechanistic studies showed that 4b, 4c, and 7b compounds induced pro-apoptotic DNA damage in Jurkat T-leukemia and HCT116 wt carcinoma cells. They also significantly increased intracellular ROS levels, thus suggesting that their cytotoxicity is mediated by oxidative stress-related pathways, rather than direct DNA binding or intercalation. Compounds 4b, 4c trigger tumor cell apoptosis, characterised by activation of caspase 3 and subsequent cleavage of PARP1, and Bcl-2 suppression. Notably, the 4c compound did not exhibit acute in vivo toxicity upon extended study. Thus, the obtained findings demonstrated potential of 2-benzylaminothienopyrimidin-4(3H)-ones as promising scaffolds for further development of anticancer agents.