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<i>H</i>-indol-3-yl)pyrimidin-2-amine derivatives as the first covalent FGFR3 selective inhibitors. The representative compound <b>10s</b> displayed high potency against FGFR3 (IC₅₀ = 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 IC₅₀ value of 19.2 nM. Furthermore, <b>10s</b> exhibited strong antiproliferative effects in FGFR3-driven RT112/84 cells (IC₅₀ = 9.2 nM). Structural characterization using MALDI-TOF-MS and X-ray crystallography confirmed covalent binding of <b>10s</b> to FGFR3. Compound <b>10s</b> 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.