Discovery of a novel 1H-pyrazole-3-amine derivative 6h with potent antitumor activity by targeting tubulin and FLT3.

A novel series of 1H-pyrazole-3-amine derivatives was designed, synthesized, and their antitumor activities were evaluated. Among them, 6h showed potent antiproliferative activity against solid tumor cell lines and leukemia cell lines, with IC50 values ranging from 3.3 to 25.0 nM. Mechanism studies revealed that 6h could effectively inhibit microtubule polymerization (IC50 = 3.6 μM) via targeting the colchicine binding site and disrupted cellular microtubule networks. Notably, the preliminary kinase selectivity results showed 6h displayed selectivity for FLT3 kinase (IC50 = 58.6 nM) over other kinases screened, including the homologous c-KIT. Western blot analysis demonstrated that 6h inhibited the phosphorylation of FLT3 and its downstream signaling mediator STAT5 in MOLM-13 cells. 6h could induce tumor cell cycle arrest and apoptosis. Furthermore, in a MOLM-13 xenograft model, 6h at the dosage of 30 mg/kg exhibited effective antitumor activity without notable body-weight loss and the major organ damage. Pharmacokinetic evaluation of 6h in rats revealed an oral bioavailability of 5.6 %. Molecular docking and dynamics simulations supported stable binding modes of 6h with both tubulin and FLT3. Collectively, these results suggested that 6h could be regarded as a promising lead compound against tubulin and FLT3 for further investigation.