Discovery of FLC-8 as the First Covalent FLT3 Inhibitor Targeting Cys807 for FLT3 Mutant Acute Myeloid Leukemia.

FLT3 is a validated therapeutic target in acute myeloid leukemia (AML), yet resistance mutations frequently limit current inhibitors. Here, we report a series of 6-methylisoxazolo[5,4-b]pyridin-3-amines that covalently target Cys807, a previously unexploited nucleophilic residue within the FLT3 kinase domain. Compound 18 (FLC-8) potently inhibited FLT3-WT (IC50 = 10.2 nM) and clinically relevant mutants G697R (IC50 = 11.6 nM) and N676D (IC50 = 24.1 nM). Covalent engagement of Cys807 was confirmed by mass spectrometry, peptide mapping, and loss of activity upon C807S mutation. FLC-8 suppressed FLT3-mediated STAT5, AKT, and ERK signaling and induced apoptosis in AML cells while maintaining low-nanomolar potency over 72 h. Kinome profiling revealed a narrow inhibition spectrum. In vivo, FLC-8 inhibited MV4-11 xenograft growth (TGI: 136-178% at 10-50 mg/kg) without overt toxicity. These findings identify Cys807 as a covalent binding hotspot in FLT3 and establish FLC-8 as a promising scaffold for next-generation FLT3 inhibitor development.