Design, synthesis, and biological evaluation of novel 4-aminoquinoline benzoyl hydrazine derivatives as noncovalent BTK inhibitors for hematologic malignancies.

Bruton's tyrosine kinase (BTK) is a clinically validated target for B-cell malignancies, yet covalent inhibitors are limited by acquired resistance and cumulative off-target toxicities. To address these challenges, we employed a scaffold-hopping strategy from larotinib, by integrating a benzoyl hydrazine pharmacophore into the 4-aminoquinoline scaffold to explore a potential non-covalent binding mode. Systematic optimization yielded compound 10m, which exhibited moderate but promising BTK inhibitory activity (IC50 = 126.70 ± 16.69 nM), acceptable kinase selectivity with reduced activity against key off-targets of ibrutinib (EGFR, ITK, BMX, TEC), and potent antiproliferative activity in U-937 cells (IC50 = 0.89 ± 0.01 μM). Western blot analysis confirmed dose-dependent suppression of BTK phosphorylation (Tyr223). Flow cytometry revealed G0/G1 cell cycle arrest and concentration-dependent apoptosis induction. In the U-937 xenograft model, 10m achieved 95.5% tumor growth inhibition at 100 mg/kg i.p. without significant toxicity. Molecular docking studies showed that 10m retains the binding mode of larotinib while forming additional hydrogen bonds, consistent with a reversible binding mode supported by docking and positioned away from Cys481. These results validate the design strategy and position 10m as a promising lead for next-generation non-covalent BTK inhibitors for the treatment of hematologic malignancies.