Design, synthesis and biological evaluation of novel aminopyrimidine derivatives as potent EGFRC797S inhibitors for overcoming osimertinib resistance.

EGFRC797S mutation and bypass pathway activation are major drivers of acquired resistance to osimertinib; however, no approved therapeutic agents are currently available to effectively overcome this resistance. In this study, a series of aminopyrimidine-based small-molecule inhibitors were rationally designed and synthesized. The lead compound 6a exhibits potent single-digit nanomolar activity against EGFRL858R/T790M/C797S (IC50 = 1.76 nM), along with equipotent ALK inhibitory activity (IC50 = 1.95 nM) that may address ALK-mediated bypass resistance. The co-crystal structure of EGFRC797S in complex with 6a (PDB: 9XU9) reveals a unique hydrogen bond between the inhibitor and Ser797-the residue that confers osimertinib resistance-providing a structural basis for resistance overcoming. In cellular assays, 6a potently inhibits proliferation of osimertinib-resistant H1975-M3 cells (IC50 = 0.035 μM), suppresses downstream oncogenic signaling, induces G0/G1 cell cycle arrest and apoptosis, and impairs cell migration and invasion. In vivo, 6a demonstrates robust antitumor efficacy in an H1975-M3 xenograft model with a tumor growth inhibition rate of 86.47% at 30 mg/kg, significantly outperforming brigatinib (55.53%), while exhibiting favorable safety and pharmacokinetic profiles. Collectively, these findings establish 6a as a promising therapeutic candidate for overcoming osimertinib resistance.