Structural Optimization and MD Simulation Study of Benzimidazole Derivatives as Potent Mutant FLT3 Kinase Inhibitors Targeting AML

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor survival rates in adults, posing a significant economic burden. FMS-like tyrosine kinase 3 (FLT3) mutations are linked to poor prognosis in AML and resistance to clinically approved FLT3 inhibitors. Previously, we reported a novel benzimidazole-based FLT3 inhibitor, 4ACP, with nanomolar activities against FLT3-ITD and FLT3-TKD mutants, showing selective cytotoxicity against FLT3-ITD⁺ AML cell lines. In this study, we synthesized 31 derivatives by modifying the 4-acetamidophenyl group and varying substituents at N1-phenyl and C2 positions. We identified compound 21l (3-acetamidophenyl) as the most potent derivative (FLT3-TKD(D835Y) IC₅₀ = 1.47 nM). Linking 21l to a solvent-accessible group yielded compound 22b, which exhibited a sub-nanomolar activity against FLT-TKD(D835Y) mutant with an IC₅₀ value of 0.48 nM. Compound 22b showed preferential antiproliferative activities against MOLM-14, MV4-11, MOLM-14-D835Y, and MOLM-14-F691L AML cell lines with IC₅₀ values of 16.1, 10.5, 26.5, and 160.3 nM, respectively. 22b induced dose-dependent inhibition of FLT3, ERK, STAT5, and S6 phosphorylation, G0/G1 cell-cycle arrest, and apoptotic cell death at low nanomolar concentrations in MOLM-14 and MOLM-14-D835Y cells. It was more selective for FLT3-dependent cell lines, showing about 80-fold selectivity toward FLT3-TKD(D835Y) over KIT, indicating relative safety and lower myelosuppression potential. The molecular dynamics study of 4ACP and 22b was conducted to explain the significant changes in activity resulting from subtle structural alterations. Altogether, these findings establish 22b as a potent mutant FLT3 inhibitor, warranting further investigation and optimization to target resistant AML.