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.