Cell-active, irreversible covalent inhibitors that selectively target the catalytic lysine of EGFR by using fluorosulfate-based SuFEx chemistry

EGFR signaling is involved in multiple cellular processes including cell proliferation, differentiation and development, making this protein kinase one of the most valuable drug targets for the treatment of non-small cell lung carcinomas (NSCLC). Herein, we describe the design and synthesis of a series of potential covalent inhibitors targeting the catalytically conserved lysine (K745) of EGFR on the basis of Erlotinib, an FDA-approved first-generation EGFR drug. Different amine-reactive electrophiles were introduced at positions on the Erlotinib scaffold proximal to K745 in EGFR. The optimized compound 26 (as well as its close analog 30), possessing a novel arylfluorosulfate group (ArOSO2F), showed excellent in vitro potency (as low as 0.19 nM in independent IC50 determination) and selectivity against EGFR and many of its drug-resistant mutants. Both intact protein mass spectrometry (MS) and site-mapping analysis revealed that compound 26 covalently bound to EGFR at K745 through the formation of a sulfamate. In addition, compound 26 displayed good anti-proliferative potency against EGFR-overexpressing HCC827 cells by inhibiting endogenous EGFR autophosphorylation. The pharma-cokinetic studies of compound 26 demonstrated the druggable potential of other ArOSO2F-containing com-pounds. Finally, competitive activity-based protein profiling (ABPP), cellular thermal shift assay (CETSA), as well as cellular wash-out experiments, all showed compound 26 to be the first cell-active, fluorosulfate-based targeted covalent inhibitor (TCI) of protein kinases capable of covalently engaging the catalytically conserved lysine of its target in live mammalian cells.