From Lead to Drug Candidate: Optimization of 3‑(Phenylethynyl)‑1<i>H</i>‑pyrazolo[3,4‑<i>d</i>]pyrimidin-4-amine Derivatives as Agents for the Treatment of Triple Negative Breast Cancer

Herein we report the sophisticated process of structural optimization toward a previously disclosed Src inhibitor, compound 1, which showed high potency in the treatment of triple negative breast cancer (TNBC) both in vitro and in vivo but had considerable toxicity. A series of 3-(phenylethynyl)-1H-pyrazolo­[3,4-d]­pyrimidin-4-amine derivatives were synthesized. In vitro cell-based phenotypic screening together with in vivo assays and structure–activity relationship (SAR) studies finally led to the discovery of N-(3-((4-amino-1-(trans-4-hydroxycyclohexyl)-1H-pyrazolo­[3,4-d]­pyrimidin-3-yl)­ethynyl)-4-methylphenyl)-4-methyl-3-(trifluoromethyl)­benzamide (13an). 13an is a multikinase inhibitor, which potently inhibited Src (IC50 = 0.003 μM), KDR (IC50 = 0.032 μM), and several kinases involved in the MAPK signal transduction. This compound showed potent anti-TNBC activities both in vitro and in vivo, and good pharmacokinetic properties and low toxicity. Mechanisms of action of anti-TNBC were also investigated. Collectively, the data obtained in this study indicate that 13an could be a promising drug candidate for the treatment of TNBC and hence merits further studies.本文报道了针对此前已披露的Src激酶抑制剂化合物1的精细化结构优化流程。化合物1在体外与体内实验中均展现出治疗三阴性乳腺癌（triple negative breast cancer, TNBC）的强效活性，但存在显著毒性。本研究合成了一系列3-(苯乙炔基)-1H-吡唑并[3,4-d]嘧啶-4-胺类衍生物。通过基于细胞的体外表型筛选、体内实验以及构效关系（structure–activity relationship, SAR）研究，最终获得了N-(3-((4-氨基-1-(反式-4-羟基环己基)-1H-吡唑并[3,4-d]嘧啶-3-基)乙炔基)-4-甲基苯基)-4-甲基-3-(三氟甲基)苯甲酰胺（13an）。13an属于多靶点激酶抑制剂，可强效抑制Src（半数抑制浓度（half maximal inhibitory concentration, IC50）= 0.003 μM）、激酶插入域受体（kinase insert domain receptor, KDR，IC50 = 0.032 μM）以及数种参与丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号转导的激酶。该化合物在体外与体内实验中均展现出强效的抗三阴性乳腺癌活性，同时具备良好的药代动力学特性与较低的毒性。本研究同时对其抗三阴性乳腺癌的作用机制进行了探究。综上，本研究所得数据表明，13an可作为治疗三阴性乳腺癌的潜在候选药物，因此值得开展进一步的研究工作。