Through structure-guided design approach based on our previous work on mutant-selective EGFR inhibitor (compound 9), we have designed and generated a novel 1,4-dihydro-2H-pyrimido[4,5-d][1,3]oxazin-2-one scaffold, followed by introduction of alkyl groups contacting the Met790 gatekeeper residue into the C4-position to keep the selectivity for the EGFRL858R/T790M over EGFRWT, leading to discovery of one of the most potent compound 20a. 20a not only showed remarkable selectivity of enzyme inhibition for EGFRL858R/T790M over EGFRWT, but also strongly inhibited the proliferation of H1975 cells, while significantly less effective on A431 cell lines. A further preliminary in vivo antitumor efficacy evaluation on 20a suggested that it significantly inhibited tumor growth in H1975 NSCLC xenograft mouse model via po dosing at 50 mg/kg/day for 14 days. Considering the encouraging enzymatic and cellular selectivity and in vivo antitumor efficiency of compound 20a, this agent might be a promising lead compound for further optimization as a selective EGFRL858R/T790M inhibitor to overcome T790M resistance mutation. The results also provide more insights for designing new classes of mutant-selective EGFR inhibitors. Further pre-clinical evaluations for the candidate compound are in progress and will be performed in due course.