Fibroblast growth factor receptors (FGFRs) have become promising therapeutic targets in various types of cancers. In fact, several selective irreversible inhibitors capable of covalently reacting with the conserved cysteine of FGFRs are currently being evaluated in clinical trials. In this article, we optimized and discovered a novel lead compound 36with remarkable inhibitory effects against FGFR (1–3), which is a derivative of 2H-pyrazolo[3,4-d]pyrimidine. The irreversible binding to FGFRs was characterized by LC–MS. This compound has been shown to exhibit significant anti-proliferation effects against NCI-H1581 and SNU-16 cancer cell lines both in vitroand in vivo. Compound 36has also demonstrated a low toxicity profile and adequate pharmacokinetic properties and is currently under validation as a potential drug candidate.