Discovery of a highly selective FLT3-ITD inhibitor with potent antileukemic activity based on 4-(2-fluorophenoxy)pyridine scaffold

Acute Myeloid Leukemia (AML) driven by FLT3-ITD mutations remains a therapeutic challenge with limited treatment options. Herein, we report the design, synthesis, and biological evaluation of a series of 4-(2-fluorophenoxy)pyridine derivatives featuring an imidazolidinone carboxamide linker as potent FLT3-ITD inhibitors. Systematic structure-activity relationship exploration identified compound 18p as the optimal candidate, which displayed exceptional enzymatic potency against FLT3-ITD (IC₅₀ = 1.1 nM) and single-digit nanomolar antiproliferative activity against FLT3-ITD driven AML cell lines (BaF₃-FLT3-ITD IC₅₀ = 5.7 nM; MOLM-13 IC₅₀ = 1.6 nM). Compound 18p demonstrated a marked selectivity profile, discriminating FLT3-ITD from FLT3-WT and sparing normal PBMCs and a broad panel of solid tumor cells. Kinome profiling revealed that 18p maintained high selectivity over c-Kit, CDKs, FGFRs, and the majority of 60 tested Kinases, with PDGFRβ identified as the principal off-target liability. Mechanistically, 18p suppressed FLT3 autophosphorylation and downstream STAT5, Akt, and ERK signaling more effectively than gilteritinib at equimolar concentrations, and induced potent G₁-phase arrest and Apoptosis in MOLM-13 cells. In the MV4-11 xenograft model, oral administration of 18p at 10 mg/kg achieved 80% tumor growth inhibition, comparable to gilteritinib at 2.5 mg/kg, with concomitant suppression of Ki67 and p-STAT5 and induction of cleaved Caspase-3 and γH2AX in tumor tissues. Histopathological examination of major organs confirmed an absence of overt toxicity. These findings position 18p as a promising FLT3-ITD inhibitor and provide valuable insights for further development of mutation-selective AML therapeutics.