Synthesis of a new series of 20 compounds bearing the thieno[2,3-d]pyrimidine-4-one scaffold was achieved. The inhibitory activity of these compounds was performed over 60 cell lines of human tumor at single and five dose concentrations. Compounds 20, 22, and 23 exhibited potent growth inhibitions toward the majority of the tested NCI 60 cell lines. Compounds 20 and 23 were the most active compounds with (MG-MID) TGI, GI50, and LC50 values of 16.2, 3.3, 50.1 and 67.7, 6.6, 100, respectively. Also, both compounds showed 7-and 4-fold better activity, respectively, than the standard antitumor agent 5-fluorouracil. Therefore, compounds 20 and 23 were selected to measure their ability to inhibit the dihydrofolate reductase enzyme (DHFR) in comparison to methotrexate (MTX) as a reference drug. Compound 20 was a more potent inhibitor of DHFR (IC50 = 0.20 mu M) than MTX (IC50 = 0.22 mu M). Molecular modeling studies were performed in the DHFR active site, and it showed compatibility with the results obtained from biological studies. Finally, the results showed that compound 20 is a strong antitumor agent and potent inhibitor of DHFR. In addition, this compound induced cell-cycle arrest in SNB-75 cells in the G2/M phase and the apoptosis process in the Pre-G phase. Compound 20 also increased the level of both caspases-3 and 9 by 11.8-and 50.3-fold, respectively.
