AbstractA series of novel substituted pyrimidinones and fused pyrimidinones (compounds 3–18) were synthesized starting with oxiranylmethanone 2. The in vitro cytotoxicity against a human breast adenocarcinoma (MCF‐7) cell line was investigated and most of the tested compounds showed potent cytotoxic activity against the MCF‐7 cell line comparable to the activity of the commonly used anticancer drug cisplatin. Treatment of MCF‐7 cells with increasing doses (2, 5, 10, and 20 µg/mL) of the tested compounds revealed that the activity of superoxide dismutase and the level of hydrogen peroxide were significantly increased, while the activities of catalase and glutathione peroxidase and the levels of reduced glutathione were significantly lowered compared with control MCF‐7 cells. In general, derivatives 11 and 16 revealed the highest anticancer activity among the tested compounds.