We describe the structural optimization of a hit compound, 1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)phenyl)-3-(3-methoxyphenyl)urea (1), which exhibits inhibitory activity but low potency against FLT3 and VEGFR2. A series of pyrazolo[3,4-d]pyrimidine derivatives were synthesized, and structure–activity relationship analysis using cell- and transgenic-zebrafish-based assays led to the discovery of a number of compounds that exhibited both high potency against FLT3-driven human acute myeloid leukemia (AML) MV4-11 cells and a considerable antiangiogenic effect in transgenic-zebrafish-based assays. The compound 1-(4-(1H-pyrazolo[3,4-d]pyrimidin −4-yloxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (33), which exhibited the highest activity in preliminary in vivo anti-AML assays, was chosen for further anti-AML studies. The results demonstrated that compound 33 is a multikinase inhibitor that potently inhibits FLT3 and VEGFR2. In an MV4-11 xenograft mouse model, a once-daily dose of compound 33 at 10 mg/kg for 18 days led to complete tumor regression without obvious toxicity. Western blot and immunohistochemical analyses were performed to determine the mechanism of action of compound 33.

中文翻译：

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吡唑并[3,4- d ]嘧啶衍生物的结构-活性关系研究导致发现一种新型的多激酶抑制剂，可有效抑制FLT3和VEGFR2的活性，并评估其在体内和体外对急性髓样白血病的活性。

我们描述了命中化合物1-（4-（1 H-吡唑并[3,4- d ]嘧啶-4-基氨基）苯基）-3-（3-甲氧基苯基）尿素（1）的结构优化抑制活性，但对FLT3和VEGFR2的效力低。合成了一系列吡唑并[3,4- d ]嘧啶衍生物，并使用基于细胞和转基因-斑马鱼的分析方法进行了结构-活性关系分析，从而发现了许多对FLT3驱动的化合物均显示出高效能的化合物。人急性髓性白血病（AML）MV4-11细胞，并在基于转基因斑马鱼的检测中具有相当大的抗血管生成作用。化合物1-（4-（1 H-吡唑并[3,4- d选择在初步的体内抗AML试验中表现出最高活性的]嘧啶−4-酰氧基）苯基）-3-（4-氯-3-（三氟甲基）苯基）脲（33）作为进一步的抗AML药物学习。结果表明，化合物33是有效抑制FLT3和VEGFR2的多激酶抑制剂。在MV4-11异种移植小鼠模型中，每天一次剂量为10 mg / kg的化合物33持续18天导致肿瘤完全消退而没有明显的毒性。进行了Western blot和免疫组化分析，确定了化合物33的作用机理。