Based on biological results of previous synthesized pyrazolyl ureas able to interfere with angiogenesis process, we planned and synthesized the new benzyl-urea derivatives 2–4; some of them showed an interesting anti-proliferative profile and particularly 4e potently inhibited HUVEC proliferation. To shed light on the mechanism of action of 4e, its interactome has been deeply inspected to identify the most prominent protein partners, mainly taking into account kinome and phosphatome, through drug affinity responsive target stability experiments, followed by targeted limited proteolysis analysis. From these studies, PP1γ emerged as the most reliable 4e potential target in HUVEC. Molecular docking simulations on PP1γ were carried out to predict 4e binding mode. To assess its potential anti-angiogenic effect, 4e was tested in vitro to verify interference on kinase and phosphate activities. Overall, our results evidenced for 4e an interesting anti-angiogenic action, probably due to its action at intracellular level on PP1γ signalling pathways.

基于以往合成的能够干扰血管生成的吡唑脲的生物学结果，我们规划并合成了新的苄基脲衍生物2-4；其中一些显示出有趣的抗增殖特性，特别是4e有效抑制 HUVEC 增殖。为了阐明4e的作用机制，对其相互作用组进行了深入检查，以确定最突出的蛋白质伙伴，主要考虑激酶组和磷酸组，通过药物亲和力响应靶稳定性实验，然后进行靶向有限蛋白水解分析。从这些研究中，PP1γ 成为最可靠的4HUVEC 中的潜在目标。对 PP1γ 进行分子对接模拟以预测4e结合模式。为了评估其潜在的抗血管生成作用，在体外测试了4e以验证对激酶和磷酸盐活性的干扰。总体而言，我们的结果证明4e具有有趣的抗血管生成作用，可能是由于其在细胞内水平对 PP1γ 信号通路的作用。