Structure-Based Design of 4-(1-Methyl-1 <i>H</i> -indol-3-yl)pyrimidin-2-amine Derivatives as the First Covalent FGFR3 Selective Inhibitors

Aberrant fibroblast growth factor receptor 3 (FGFR3) activation drives bladder carcinogenesis in humans, but currently approved pan-FGFR inhibitors lack FGFR3 isoform selectivity and fail to counter clinically acquired resistance mutations (e.g., FGFR3 V555M/L). Herein, we report the structure-based drug design of 4-(1-methyl-1 H -indol-3-yl)pyrimidin-2-amine derivatives as the first covalent FGFR3 selective inhibitors. The representative compound 10s displayed high potency against FGFR3 (IC 50 = 6.8 nM) and 5–60-fold selectivity over FGFR1/2/4. It was also effective against the common clinically acquired FGFR3 V555M resistance mutation with an IC 50 value of 19.2 nM. Furthermore, 10s exhibited strong antiproliferative effects in FGFR3-driven RT112/84 cells (IC 50 = 9.2 nM). Structural characterization using MALDI-TOF-MS and X-ray crystallography confirmed covalent binding of 10s to FGFR3. Compound 10s also showed significant antitumor efficacy in the RT112/84 bladder cancer xenograft model, offering a promising compound to address both selectivity and resistance in FGFR3-targeted therapy.