Structure-guided design of 7-azaindole FLT3 inhibitors for acute myeloid leukemia

Abstract Background Treatment options for acute myeloid leukemia (AML) remain limited by incomplete and insufficiently durable responses to currently available therapies, highlighting the need for novel chemotypes. This study aimed to optimize a bis-7-azaindole hit and identify derivatives with FMS-like tyrosine kinase 3 (FLT3) inhibitory and antileukemic activity with favorable drug-like properties. Methods A structure-guided medicinal chemistry approach combined with structure-activity relationship (SAR) studies was employed to optimize the hit. The synthesized derivatives were evaluated for inhibition of FLT3 harboring internal tandem duplication mutations, antiproliferative activity in AML cell lines, selectivity, and lipophilic ligand efficiency. Mechanistic studies examined effects on FLT3 signaling, cell-cycle progression, and apoptosis. Biochemical kinase profiling and cellular target-engagement assays were performed for the lead compound. Selected absorption, distribution, metabolism, and excretion (ADME) properties and pharmacokinetic behavior were also investigated, and ex vivo activity was assessed in FLT3-ITD or FLT3 wild-type AML samples. Results The SAR campaign identified several key pharmacophoric requirements, including the essential role of the peripheral 7-azaindole moiety, a preference for substitution at position 2, and the importance of optimizing the basic side chain. Compound 50 emerged as the lead candidate, displaying potent inhibition of FLT3 with internal tandem duplication mutations (IC 50 = 0.163 μM), strong antiproliferative activity in FLT3-mutated AML cells (GI 50 = 0.164 μM), lipophilic ligand efficiency of 8.9, differential antiproliferative response between THP-1 and MV4-11 cells (THP-1/MV4-11 anti-proliferative ratio = 108) and biochemical kinase selectivity ratio (FLT3-WT/FLT3-ITD) of 2.0. Mechanistic studies showed reduced FLT3 and downstream signaling phosphorylation, G1-phase arrest and apoptosis. However, cellular profiling did not demonstrate clear dose-dependent FLT3 engagement and identified additional kinase targets, indicating a polypharmacological profile. Compound 50 exhibited high plasma and microsomal stability and a pharmacokinetic profile characterized by rapid distribution and persistence in bone marrow. Furthermore, it reduced leukemic blast counts in FLT3-ITD patient samples at 10 μM in stromal co-culture. Conclusions Structure-guided optimization of a bis-7-azaindole hit led to the identification of compound 50 as a FLT3-pathway-modulating chemotype with a polypharmacological profile and favorable early ADME properties. These findings support further preclinical investigation of this scaffold as a starting point for antileukemic drug discovery.