微管蛋白和Akt激酶双重抑制剂的分子构建、合成及抗肿瘤活性筛选

A series of new compounds containing thiocyanate or selenocyanate and indolone pharmacore were reported as dual tubulin polymerization/Akt inhibitors, which can overcome the disadvantage of resistance. Our group have done an intensive study of the derivatives of 2-amino-3-cyano-4-phenyl-6-imidazolopyridine derivatives in the inhibition of tubulin polymerization, and recently we found that they also could selectively inhibit Akt, which point out the direction for the future research. .In this project, we make 4-phenyl-2-amino-3-cyanopyridine as a skeleton structure, and introduce three linkers (thiazole, imidazole and piperazindione) with the activity of inhibiting tubulin polymerization in the pyridine 6-position in order to combine the pyridine and indolone. besides, a alkyl chain with a thiocyanate or selenocyanate will also be attached to indolone 1-position.We estimate to synthesize three novel series of 400 substituted pyridine derivatives totally, and determine their activities of inhibiting Akt and tubulin polymerization by western blot and fluorescence analysis accordingly, besides, cytotoxic activity of the compounds will be evaluated in vitro against PC-3,Hela,SW620 and H522 by MTT assay, using MX-58151 as a positive control and the activity in vivo will be evaluated in mouse xenograft models. After those, the machanism will be elucidated and the 3D-QSAR model will be built, and the invention patent will also be applied. Finally, two or three compounds with outstanding activities will be studied in a deep-going way as a dual inhibitors of tubulin polymerization and Akt kinase.

一类含硫（硒）氰基团和吲哚酮双药效团的化合物被报道为微管蛋白和Akt激酶双靶点小分子抑制剂，可克服现有的微管蛋白抑制剂易产生耐药性的缺点。课题组前期对4-苯基-6-咪唑-2-氨基-3-氰基吡啶类化合物的研究中发现，该类化合物在抑制微管蛋白活性的同时，对Akt激酶也显示出突出的选择性抑制作用，这一研究结果为后续抗肿瘤药物的研发指明了方向。本课题以4-苯基-2-氨基-3-氰基吡啶为骨架，通过3个对微管蛋白抑制活性突出的结构片段（噻唑、咪唑和哌嗪二酮）为linker，进一步引入硫（硒）氰基烷基吲哚酮结构，设计合成3类约400个多取代吡啶类衍生物。采用免疫荧光分析法和Western blot法测定对微管蛋白及Akt激酶的抑制活性；进一步进行体外及体内抗肿瘤活性筛选，初步阐明其作用机制；基于体外抗肿瘤细胞活性筛选，建立3D-QSAR模型；申请发明专利；最终期望得到2~3个具有自主知识产权成药性好的

本项目以微管蛋白抑制剂Crolibulin和CA-4为先导化合物，以4-苯基-2-氨基-3-氰基吡啶为先导结构，对其进行骨架跃迁并进一步优化，设计合成了5,7-双芳基-8-氰基咪唑并[1,2-a]吡啶类及2,4,6-三芳基-3-氰基吡啶类化合物，共制备4类全新的化合物300余个，并确证其结构。对其药理活性进行了深入的研究，采用 MTT法，考察了所有目标化合物对HT29、H460、A549、MKN45以及SMMC-7721的体外细胞毒性，优选出8个化合物（如GC7、GC9、GC10、GC11等）对多个肿瘤细胞株IC50达到nM级。对候选化合物GC9进行了深入的作用机制研究。流式细胞术检测表明，GC9在0.2-1.0 μM浓度下对细胞周期有明显的阻滞作用，且发生在G2/M期；进一步的免疫荧光试验及秋水仙碱竞争结合试验表明，该化合物可与微管蛋白的秋水仙碱位点结合，这一研究也被分子对接模型得以证实，初步阐明了该类化合物的作用机制，为潜在的靶向作用于秋水仙碱结合位点的微管聚合抑制剂；激酶活性筛选实验表明，该类化合物对Akt激酶无抑制活性。该研究为抗微管药物的研究提供了一类有潜力的分子探针，具有重大的后期研究价值。本项目通过总结以上4类化合物的构效关系，并建立了3D-QSAR 模型，可指导后续新化合物的设计；前期研究已发表SCI论文3篇（单篇最高影响因子为5.228）；申请中国发明专利1项；已培养硕士研究生2人，在读博士研究生1人。

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