Exploring the potential of heterocyclic-containing tail approach in the development of novel covalent inhibitors dual-targeting EGFR and HER-2: design, synthesis and biological evaluation.

Based on the structure of the existing drug afatinib, this study employed the heterocyclic-containing tail modification strategy to design and synthesize a series of novel pyrimidine derivatives, aiming to develop highly selective and low-toxicity EGFR/HER-2 dual-target covalent inhibitors. In vitro kinase inhibition assays and MTT cell viability screenings revealed that most compounds exhibited potent and selective inhibitory activity against EGFR and HER-2, while demonstrating significant antiproliferative effects against multiple lung and breast cancer cell lines (SK-BR-3, A549, NCI-H1975, and MDA-MB-231) and relatively low toxicity toward normal cells. Among these compounds, compound 10e showed the strongest inhibition and selectivity at both the enzymatic and cellular levels. Molecular docking and washout experiments confirmed that 10e formed an irreversible covalent bond with the catalytic sites of EGFR and HER-2 through its acrylamide warhead, effectively suppressing the proliferation and migration of NCI-H1975 cells. Further mechanistic studies demonstrated that 10e exerted antitumor effects via multiple pathways, including inhibition of EGFR/HER-2 phosphorylation, induction of G/G-phase cell cycle arrest, promotion of apoptosis, and enhancement of reactive oxygen species (ROS) production. In addition, compound 10e exhibited significant antitumor activity with low toxicity in NCI-H1975 xenograft models. Collectively, these results indicate that a promising covalent dual inhibitor of EGFR and HER-2 represents a potential therapeutic candidate for NSCLC.