Discovery of Potent and Selective Pyrrolo[2,3-<i>d</i>]pyrimidine Derivatives as Fourth-Generation EGFR Inhibitors Targeting Triple Mutations

Three generations of EGFR tyrosine kinase inhibitors (EGFR-TKIs) have shown clinical efficacy in nonsmall cell lung cancer (NSCLC), but acquired resistance mutations─especially the <i>cis</i>-EGFR^T790M/C797S─remain a major challenge. Here, we report the identification of a series of pyrrolo[2,3-<i>d</i>]pyrimidine derivatives that inhibit C797S-mediated EGFR triple mutants. Among them, compound <b>31r</b> shows subnanomolar IC₅₀ values against Ba/F3 EGFR^{19del/T790M/C797S} and Ba/F3 EGFR^{L858R/T790M/C797S}, while sparing wild-type EGFR. Its binding mode with EGFR^{19del/T790M/C797S} was revealed by a cocrystal structure, providing structural insights into its potency and selectivity. Compound <b>31r</b> also displays excellent kinome selectivity and drug-like properties, including good metabolic stability (<i>T</i>_1/2 = 5.9 h) and oral bioavailability (<i>F</i> = 24%). Most importantly, <b>31r</b> significantly suppressed tumor growth in PC-9 EGFR^{19del/T790M/C797S} xenograft models, achieving regression at 80 mg/kg once daily. These results highlight <b>31r</b> as a promising lead compound for overcoming resistance associated with third-generation EGFR-TKIs and support its further development for drug-resistant NSCLC.