The 3H-pyrazolo[4,3-f]quinoline moiety, synthesized via a Doebner or Povarov multi-component reaction, has been described as an innovative scaffold capable of interacting with the hinge region of several protein kinases, including FLT3. The receptor tyrosine kinase FLT3 and its oncogenic variants, such as ITD or D835Y, has been validated as a therapeutic target for the therapy of acute myelogenous leukemia. In this study, we demonstrate that the substitution pattern of the phenyl group at the 7-posititon of 3H-pyrazolo[4,3-f]quinoline has an impact on FLT3 kinase inhibition. Out of 48 synthesized compounds, we present here HSB401, which showed nanomolar inhibitory activities against recombinant FLT3-ITD, FLT-ITD-F691L and D835Y mutants and FLT3-driven cancer cell lines MOLM-13 and MV4-11, as well as Ba/F3 cells associated with the FLT3-ITD mutation. Approved FLT3 inhibitor quizartinib strongly inhibits both FLT3 and c-KIT, resulting in significant myelosuppression. Notably, the lead compound HSB401 inhibits c-KIT with approximately 100-fold lower potency compared to FLT3-D835Y. This suggests that HSB401 may spare normal hematopoiesis and mitigate the risk of myelosuppression. Mechanistic studies in MV4-11 cells revealed downregulated FLT3 signaling after 2 h of treatment with HSB401 and cell cycle arrest and apoptosis in the prolonged experiment. In addition, oral administration of HSB401 significantly suppressed tumor growth in the MV4-11 xenograft mouse model.