Lung cancer is one of the most common malignant tumors, with high morbidity and mortality worldwide, accounting for almost a quarter of all cancer-related deaths [1], [2]. Non-small cell lung cancer (NSCLC) comprises approximately 85 % of these cases, with the epidermal growth factor receptor (EGFR) identified as a critical driver oncogene in the majority of NSCLC instances [3]. Targeting EGFR mutations to obstruct the EGFR signaling pathway can significantly curtail tumor cell proliferation and metastasis while encouraging apoptosis [4]. Consequently, EGFR tyrosine kinase inhibitors (EGFR-TKIs) have proven effective for NSCLC patients with activating EGFR mutations [5]. To date, the development of EGFR-TKIs has marked substantial progress, leading to the approval of three generations of these inhibitors. Nonetheless, the extensive application of these drugs has led to emerging drug resistance.

Osimertinib serves as the first-line treatment drug for EGFR-mutated NSCLC [6], [7]. The emergence of the C797S mutation impedes Osimertinib's effectiveness by preventing its covalent attachment to Cys797, leading to resistance [8], [9]. Consequently, developing fourth-generation EGFR-TKIs targeting the C797S mutation has become critical. Current research focuses on fourth-generation drugs that address multiple resistant mutations, including C797S, T790M, L858R, and Del19 [10]. Among the contenders are several ATP competitive inhibitors with a 2,4-diaminopyrimidine core (such as Brigatinib, IN-7, and BLU-945) alongside allosteric inhibitors (EAI-045, JBJ-04-125-02) that have been reported, as illustrated in Fig. 1 [11], [12]. Nevertheless, as none have yet approved for treating Osimertinib-resistant NSCLC, there remains a significant need for potent and structurally novel small molecules to target EGFR with mutations.

Previously research indicated that the ALK inhibitor Brigatinib exhibits inhibitory activity against EGFRL858R/T790M/C797S mutations, with IC50 values of 5.57 nM for EGFRL858R/T790M/C797S and 53.1 nM against EGFRWT [13], [14]. Given the distant location of the L858R in the EGFR triple mutation, which was far from the molecular pocket, it showed less impact on the molecular activity. Analysis of the crystal structure of the EGFRT790M/C797S protein in conjunction with Brigatinib (PDB: 8H7U) reveals (Fig. 2A/B) that the 2,4-diaminopyrimidine scaffold forms bidentate hydrogen bonds with Met793 within the hinge region of the EGFR protein. Additionally, the N-methylpiperazine is accessible to the solvent region of the protein pocket, which possesses a spacious cavity suitable for further modifications. The solvent region pockets, enriched with polar amino acids such as Asp800 and Glu804, are anticipated to form new interactions. We also contemplated substituting the benzene ring adjacent to the solvent region with various aromatic fused heterocycles to enhance conformational stability via a cyclization strategy (Fig. 2C). Based on the rational design, we synthesized three series of 2-aminopyrimidine derivatives and assessed their biological activities. Herein, we report a structurally novel compound 52 that potently inhibits EGFRL858R/T790M/C797S enzymatic activity with an IC50 of 0.55 nM and significantly suppressed the Ba/F3 cell line harboring EGFRL858R/T790M/C797S proliferation with an IC50 of 43.28 nM. More importantly, compound 52 demonstrated good pharmacokinetic properties and exhibited potent tumor growth inhibition in the Ba/F3-EGFRL858R/T790M/C797S xenografts tumor model, making it a promising drug candidate for the next generation of EGFR inhibitors.