【作者】 王芳；

【导师】 杨永华；

【作者基本信息】 南京大学 ， 制药工程（专业学位）， 2016， 硕士

【摘要】 目前,癌症仍是世界上死亡率最高的疾病之一。据研究报道,癌症的发病机制是极其多样的,其中涉及到了多种的分子机制。而细胞有丝分裂的失调是癌症病发的最重要原因之一。在细胞有丝分裂的整个过程中,微管扮演着极为重要的角色。微管蛋白是微管的主要组成部分,作为有丝分裂药物开发的靶点越来越受关注。紫草宁是一种天然高效的抗肿瘤药物,研究已证明其可通过有效调控微管蛋白达到抑制肿瘤细胞生长的目的;此外,由于查尔酮类化合物其结构的特殊性作为比较热门的抗癌药物活性中间体也备受关注。基于这些研究,我们将查尔酮结构和紫草宁的一部分活性结构进行结合,设计了一系列新的查尔酮类紫草宁衍生物,并推测此类化合物具有良好的抗癌活性。基于运用Discovery Studio分子模拟对接软件对所设计的紫草宁衍生物在靶点蛋白活性位点进行虚拟对接筛选,以对接的结合能以及抑制常数为判据筛选出18个新型查尔酮类紫草宁衍生物(PMMB-248-PMMB-265)进行合成。合成化合物均采用1H-NMR、元素分析、MS等分析手段进行表征以及其结构的确定。以秋水仙碱为阳性对照,所获分子均经过多次重复肿瘤细胞抑制增殖实验,抑制检测肿瘤细胞分别为Hela,MCF-7和A549。实验结果显示化合物PMMB-259显示了卓越的体外肿瘤细胞抑制活性,对A549,Hela和MCF-7三株细胞的半数抑制率(IC50)分别为4.53μM、2.3 6μM、5.84μM。可以作为以微管为靶点的新型抗肿瘤先导化合物,具有很好的研究前景。此外,用DS模拟化合物与微管蛋白分子的对接结果表明,化合物PMMB-259和微管蛋白通过3个氢键结合,表现出很好的亲和性。这也在分子结构机制上为生物活性结果提供了一定的支持。细胞凋亡实验表明,当化合物PMMB-259药物浓度达到8μM时早调达到39.8%,26.7%的细胞已经处于晚期凋亡(晚凋)状态了。随着药物浓度加大,PMMB-259对MCF-7细胞的抑制作用逐渐增强,并且有一定的时间依赖性,凋亡细胞的数目具有显著差异,证明化合物PMMB-259可以诱导细胞凋亡;在细胞周期实验中,药物浓度5μM的时候G2/M期的细胞比率为43.76%,相比于对照组细胞含量增加了29.44%,并且有时间依赖性,说明化合物PMMB-259可以阻滞肿瘤细胞有丝分裂停止在G2/M期。线粒体膜电位实验中化合物PMMB259作用于MCF-7细胞后,随着药物浓度的增加,红色荧光由86.6%降低到46.7%,绿色荧光由12.6%增加到46.7%,使用4 μM、8 μM处理的细胞相较对照组有显著变化,并且具有时间依赖性。结果说明,线粒体膜电位发生了变化,证实了化合物可通过线粒体途径诱导细胞早调。由此,我们认为PMMB-259是一种高效的微管蛋白抑制剂,具有很好的前景。最后,基于活性数据来分析构效关系的QSAR模型,也为将来在设计出新的具有更好抗微管蛋白聚合活性的药物分子的应用中提供了更多极具价值的信息。更多还原

【Abstract】 Cancer is still one of the highest mortality rate disease in the world.According to research,causes of cancer are very complicated,which involves many molecular mechanisms.And cell mitosis disorder is one of the most important reason of cancer disease.In the whole process of cell mitosis,microtubules play an extremely important role.Mitotic spindle is formed by microtubule polymerization and fasten to the kinetochore of chromosomes when microtubules extend from the cell centrosome during cell mitosis.Therefore,the inhibition of microtubule polymerization has become to be the mode of action for several clinically successful anticancer drugs.Tubulin is the major component of microtubules,and is becoming more and more important as the target of antimitotic drugs.Shikonin is a highly effective natural antitumor drugs,research has shown it can regulate tubulin to achieve the purpose of inhibiting tumor cell growth,I pharmaceutical industry pay great attention to it.Class chalcone derivatives,moreover,the value of the biological activity have been reported for a long time,chalcone ketones compounds due to the particularity of the structure as a popular anti-cancer drug active intermediates,cause the attention of many researchers.Based on these studies,we attempt to use the chalcone as the basic scaffold for the design of a series of novel tubulin polymerization inhibitors,and speculated that these compounds have good antitumor activity.18 novel shikonin derivatives(PMMB-248-PMMB-265)were designed and synthesized and characterized by 1H NMR,MS and elemental analysis.Due to there is no detail activity reported about these 18 compounds,all those compounds then were tested on three tumor cell lines Hela,MCF-7 and A549 in vitro and most of the compounds exhibited excellent antitumor activity.Among all the compounds,compound PMMB-259 showed the most potent in vitro growth inhibitory activity against Hela,MCF-7 and A549 cancer cell lines,with IC50 values of 4.53μM、2.36μM、5.84μM,respectively.The docking study based on tubulin crystal structure(PDB code:1SA0)showed compound PMMB-259 exhibits more affinity for tubulin than colchicine,which rationally proved the reason why these compounds also possess effective inhibitory activity profile against tubulin and helped us to explore the binding modes of these compounds.Furthermore,we also showed that PMMB-259 was a potent inducer of apoptosis in MCF-7 cells and it had cellular effects typical for microtubule interacting agents,causing accumulation of cells in the G2/M phase of the cell cycle.The mitochondrial membrane potential experiment confirmed that the compound could induce cell early apoptosis by mitochondria approach.Additionally,3D-QSAR(3D quantitative structure-activity relationship)model provided more information that could be applied to design new molecules with more potent tubulin inhibitory activity.更多还原