Novel staurosporine-type indolocarbazole glycoalkaloids as potent and selective FLT3-ITD inhibitors for acute myeloid leukemia.

Aberrant activation of the FMS-like tyrosine kinase 3 (FLT3) caused by internal tandem duplication (ITD) mutations is a major driver of acute myeloid leukemia (AML). To identify novel FLT3 inhibitors, two structurally distinct series of indolocarbazole glycoalkaloids, rebeccamycin-type and staurosporine-type, were designed, synthesized, and evaluated for their antiproliferative activities. Structure-activity relationship analysis revealed that the bisindolylmaleimide core and sugar modifications, particularly C2 deoxygenation and C4 methylation, critically influence both potency and selectivity toward FLT3-ITD mutant cells. Staurosporine-type glycosides bearing dual N-glycosidic linkages exhibited markedly superior activity compared with rebeccamycin-type analogs. Among them, compound 35 showed the most potent inhibition against FLT3-ITD (IC₅₀ = 3.16 ± 0.49 nM) and FLT3-WT (IC₅₀ = 294.7 ± 14.5 nM), comparable to the clinical reference midostaurin, with a ∼93-fold selectivity index. Molecular docking revealed a conserved hinge-binding mode within the ATP pocket and additional hydrogen bonds unique to compound 35, correlating with its enhanced affinity. Mechanistic studies demonstrated that compound 35 effectively suppressed FLT3 phosphorylation and downstream STAT5, Akt, and Erk signaling, induced G2/M cell-cycle arrest, and triggered apoptosis in FLT3-ITD-positive AML cells. Taken together, these findings identify compound 35 as a potent and selective FLT3 inhibitor and establish a promising scaffold for the development of next-generation therapeutics against FLT3-driven leukemias.