新型Aurora激酶和JAKs多靶点抑制剂的设计、合成及生物活性研究

Aurora kinases, which are key regulators of major mitotic events, have been a hot spot in antitumor researches. The JAK-STAT pathway, which is involved with tumor survival and proliferation, is regulated by Aurora kinases, and has effect on tumor cells proliferation. Inhibition of Aurora kinases and JAKs together is much more effective at inhibiting tumor cells growth and invasion as well as at inducing apoptosis. The pyrazole-benzimidazole derivative AM-004, which was devised and synthesized in our previous study, inhibits Aurora A/B kinases (IC50 values of 36 and 69 nM, respectively) and JAK 2/3 (IC50 of 265 and 287 nM). In addition, AM-004 demonstrated a significantly inhibition of HCT116 (high expression of Aurora kinases) proliferation (IC50=67 nM). Based on the preliminary works, using AM-004 as lead compound, novel 2H-imidazo [1, 2-c] pyrazolo [3, 4-e] pyrimidine derivatives will be designed and synthesized as efficient Aurora kinases and JAKs multiple targeted inhibitors, and the biological activity of which will be assayed. The mechanism of the new compounds will be further studied in order to find a new antitumor active molecule and provide scientific basis for rational design of the multiple targeted inhibitors.

Aurora激酶调控有丝分裂，为抗肿瘤新药研发靶点。JAK-STAT通路与肿瘤的增殖有关，Aurora激酶通过JAKs调节STAT磷酸化，进而影响肿瘤的生长，且Aurora激酶和JAKs双重抑制可更有效抑制肿瘤恶变。申请者前期设计并合成的吡唑-苯并咪唑类化合物AM-004有良好的Aurora A/B激酶(IC50分别为36和69nM)和JAK 2/3（IC50分别为265和287nM）抑制活性，并对Aurora激酶高表达的HCT116细胞有明显的抑制作用（IC50=67nM）。本项目拟在此基础上，以AM-004为先导化合物，进行新型2H-咪唑并[1,2-c]吡唑并[3,4-e]嘧啶类Aurora激酶和JAKs多靶点抑制剂的设计、合成及生物活性研究，以期发现新颖且高效的Aurora激酶和JAKs多靶点抑制剂。同时初步研究目标化合物的作用机制，为此类多靶点抑制剂的合理设计提供依据。

本课题进行新型Aurora A/B激酶和JAK2/3多靶点抑制剂的设计、合成及生物活性研究。.1、新型2H-咪唑并[1,2-c]吡唑并[3,4-e]嘧啶类衍生物的设计、合成及生物活性研究.本部分内容设计合成14个全新2H-咪唑并[1,2-c]吡唑并[3,4-e]嘧啶类目标化合物。体外对K562、HCT116、U937和HT-29细胞活性筛选显示：化合物对K562和U937细胞的活性比对HCT116和HT-29细胞的活性好，其中化合物M-2对K562及U937细胞的活性最好，IC50分别为 5.597和3.512 μM。对Aurora A 和JAK 2的酶活检测显示：化合物对所测激酶抑制效果较差，可能发生脱靶效应。流式细胞仪检测结果显示：化合物 M-2诱导HCT-116细胞凋亡，同时细胞周期被阻滞在S期。此外，化合物M-2可上调Bax及下调Bcl-2表达，激活caspase-3，诱导HCT-116细胞发生凋亡。.2、新型吡唑-咪唑-苯类衍生物的设计、合成及生物活性研究.本部分设计合成得到了12个新型的Aurora A/B 激酶和JAK 2/3小分子抑制剂。酶活筛选显示，多数化合物对Aurora A/B激酶和JAK2/3的IC50值在0.1-1.0 μM范围内，其中化合物MD-1的对Aurora A 的IC50为0.145 μM。对K562、HCT116细胞活性筛选显示：多数化合物显著抑制细胞增殖，其中化合物MZ-3D抑制HCT116的IC50为1.592 μM；化合物MZ-3E抑制K562的IC50为2.503 μM。同时目标化合物对K652和HCT116细胞周期均具有一定的阻滞作用。Western blot法结果显示，化合物MZ-1E抑制靶蛋白的胞内表达水平。分子对接结果显示化合物与Aurora A通过氢键或者疏水作用力相互结合，与分子动力学（MD）模拟结果一致。