Structure-Based Design of a Novel Covalent 4-(1-Methylindol-3-yl)pyrimidin-2-amine Series Targeting FGFR2 Resistance Mutations

Genetic alterations in FGFR2 drive multiple malignancies, most notably intrahepatic cholangiocarcinoma, where they occur in ∼10–15% of patients. While approved pan-FGFR inhibitors provide clinical benefit, their durability is limited by acquired, often polyclonal, on-target resistance mutations affecting key regions of the FGFR2 kinase domain, including the gatekeeper residue (V565), molecular brake residues (N550, E566, K642), and other key variants. These liabilities motivate the development of next-generation inhibitors. Given FGFR2-associated toxicities and the need for subtype selectivity, FGFR4 inhibition was prioritized as a selectivity determinant, while sparing FGFR1 was considered less critical. Guided by structure-based drug design, a reversible aminopyrimidine screening hit was optimized into a novel covalent inhibitor series active against FGFR2 wild-type and clinically relevant resistance mutations. An advanced lead 13 showed favorable potency, ADME properties, and demonstrated proof-of-concept in vivo efficacy in an FGFR2-amplified xenograft model comparable with the standard of care.