Synthesis and mechanistic investigation of N-Alkoxybenzamide derivatives as EGFR inhibitors

In this study, a series of novel compounds with potent inhibitory activity against EGFR^L858R/T790M and EGFR^{L858R/T790M/C797S} were designed and synthesized by employing a fragment combination strategy, integrating the aminopyrimidine fragment with the N-alkoxybenzamide fragment, and further evaluated through molecular docking and molecular dynamics simulations. Among them, compounds 6c (IC₅₀ = 23.8 nM) and 6e (IC₅₀ = 47.4 nM) exhibited the most significant inhibitory effects on NCI-H1975 cells. These compounds demonstrated robust activity against EGFR^L858R/T790M (IC₅₀ = 31.0 nM and 19.5 nM, respectively) and EGFR^{L858R/T790M/C797S} (IC₅₀ = 19.1 nM and 12.0 nM, respectively), and concentration-dependently suppressed colony formation and migration of NCI-H1975 cells, induced Apoptosis, and arrested the cell cycle at the G2/M phase. Mechanistically, they inhibited the phosphorylation of EGFR and ERK, thereby blocking EGFR signal transduction. By modulating the expression of apoptosis-related proteins (Cleaved PARP, Cleaved Caspase-3, and Bcl-xL), they disrupted the anti-apoptotic signaling pathway. At higher concentrations, they activated the pro-apoptotic signaling pathway through DNA damage induction, promoting rapid progression to late-stage Apoptosis. Additionally, compounds 6c and 6e concentration-dependently inhibited the mitochondrial translocation of EGFR while upregulating mitochondrial H₂S levels. These findings provide not only a new direction to discover new EGFR inhibitors but some clues to the mode related H₂S of action of EGFR inhibitors.

EGFR inhibitor; H(2)S; Mitochondrial translocation; Molecular mechanism; Synthesis.