An efficient new method has been established for the synthesis of new nucleoside by Biginelli reaction of 2-pyrrole-carbaldehdye, ethyl 3,5-dioxohexanoate and thiourea. The titled compound was submitted to react with ethyl chloroacetate and chloroacetyl chloride under different conditions to afford some novel nucleosides as anticancer agents. Hydrazinolysis of the pyrimidinethione was studied under different conditions. The synthesized compounds were evaluated for their anticancer activity and was found to exhibit promising activities. All new compounds were tested for possible anti-cancer activity against HepG-2 cell lines in comparison to the reference drug doxorubicin (DOX). Compound 7 was the most active against the liver carcinoma cell line (HepG-2) giving promising half-maximal inhibitory concentration (IC50) value of 25.5±1.3 μg/mL, compared with DOX with IC50 value of 0.36±0.02 μg/mL. However, it has weak cytotoxic effects against normal rat hepatocytes with 50% cytotoxic concentration (CC50) = 1877.5 µg/ml (= > 500 µg/ml). Compound 7 was selected to be tested in combination with ionizing gamma radiation. Gene expression levels of the cell cycle inhibitor p21 and caspase-3 was quantified. As well as, Oxidative stress was quantified by measuring the concentration of malondialdehyde (MDA), and antioxidant activity of reduced gluthatione (GSH). This study concluded that the new derivative of the Pyrimidin-2-Thione compound has an effective anti-cancer effect, and it was found that using the new compound with ionizing radiation at a dose of 8 Gy improves the effectiveness of the compound on liver cancer cells.