Pyrimidine scaffolds are recognized as versatile pharmacophores in the development of anticancer agents due to their diverse biological potential and ability to interact with multiple molecular targets. In this study, a novel series of substituted pyrimidine derivatives were designed, synthesized, and biologically evaluated for their anticancer potential. The compounds were prepared through a multistep synthetic route and characterized by FT-IR, ¹H NMR, ¹³C NMR, and mass spectrometric analyses, confirming the successful synthesis of the desired structures. In vitro cytotoxicity was assessed against human cancer cell lines MCF-7 (breast), HeLa (cervical), and A549 (lung) using the MTT assay. Several derivatives exhibited significant cytotoxic effects with IC₅₀ values comparable to or lower than the reference drug doxorubicin. Structure–activity relationship (SAR) analysis indicated that amide and electron-withdrawing substituents on the pyrimidine ring enhanced anticancer activity. Molecular docking studies further revealed strong binding affinities of the most active compounds toward the ATP-binding pockets of epidermal growth factor receptor (EGFR) and cyclin-dependent kinase 2 (CDK2), supporting their potential mechanism of action. Overall, these findings suggest that the newly synthesized pyrimidine scaffolds represent promising lead molecules for further development as potent anticancer therapeutics.