Design, Synthesis, and Structure–Activity Relationship Studies of 3‑(Phenylethynyl)‑1H‑pyrazolo[3,4‑d]pyrimidin-4-amine Derivatives as a New Class of Src Inhibitors with Potent Activities in Models of Triple Negative Breast Cancer

A series of 3-(phenylethynyl)-1H-pyrazolo­[3,4-d]­pyrimidin-4-amine derivatives were designed and synthesized. Structure–activity relationship (SAR) analysis of these compounds led to the discovery of compound 1j, which showed the highest inhibitory potency against the Src kinase and the most potent antiviability activity against the typical TNBC cell line MDA-MB-231 among all the synthesized compounds. Further kinase inhibition assays showed that compound 1j was a multikinase inhibitor and potently inhibited Src (IC50 = 0.0009 μM) and MAPK signaling protein kinases B-RAF and C-RAF. In an MDA-MB-231 xenograft mouse model, a once-daily dose of compound 1j at 30 mg/kg for 18 days completely suppressed the tumor growth with a tumor inhibition rate larger than 100% without obvious toxicity. It also displayed good pharmacokinetic properties in a preliminary pharmacokinetic assay. Western blot and immunohistochemical assays revealed that compound 1j significantly inhibited Src and MAPK signaling and markedly induced apoptosis in tumor tissues.本研究设计并合成了一系列3-(苯乙炔基)-1H-吡唑并[3,4-d]嘧啶-4-胺衍生物。通过对这些化合物开展构效关系（Structure–activity relationship，SAR）分析，最终筛选得到化合物1j：其在所有合成化合物中对Src激酶展现出最高的抑制活性，同时对典型三阴性乳腺癌（triple-negative breast cancer, TNBC）细胞系MDA-MB-231具有最强的抗增殖活性。后续激酶抑制实验表明，化合物1j属于多靶点激酶抑制剂，可强效抑制Src（半最大抑制浓度IC50=0.0009 μM）以及丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号通路相关蛋白激酶B-RAF与C-RAF。在MDA-MB-231细胞异种移植小鼠模型中，以30 mg/kg的每日单次剂量连续给药18天，化合物1j可完全抑制肿瘤生长，肿瘤抑制率超过100%，且未观察到明显毒性反应。在初步药代动力学实验中，该化合物亦展现出良好的药代动力学特性。蛋白质印迹法（Western blot）与免疫组织化学检测结果显示，化合物1j可显著抑制肿瘤组织中的Src及MAPK信号通路，并显著诱导细胞凋亡。