AbstractEpidermal growth factor receptor (EGFR) T790M acquired drug-resistance mutation has become a major clinical challenge for the therapy of non-small cell lung cancer. Here, we applied a structure-guided approach on the basis of the previous reported EGFR inhibitor (compound 9), and designed a series of C4-alkyl-1,4-dihydro-2H-pyrimido[4,5-d][1,3]oxazin-2-one derivatives as novel mutant-selective EGFR inhibitors. Finally, the most representative compound 20a was identified, which showed high selectivity at both enzymatic and cellular levels against EGFR^L858R/T790M (H1975 cell lines) over EGFR^WT (A431 cell lines). The representative compound 20a also showed promising antitumor efficiency in the in vivo antitumor efficacy study of H1975 xenograft mouse model driven by EGFR^L858R/T790M. These results provide a new scaffold for the treatment of dual-mutant-driven non-small cell lung cancer.