【作者】 胡纯琦；

【导师】 胡永洲；

【作者基本信息】 浙江大学 ， 药物化学， 2012， 博士

【摘要】 肿瘤被认为是多基因疾病,一旦控制细胞分化的基因有缺陷从而导致生长调控的失序,研究发现,p53基因是与人类肿瘤相关性最高的肿瘤抑制基因,具有维持基因组稳定、抑制或阻止细胞转化的功能；mdm2是1987年发现的癌基因,其表达产物MDM2蛋白可与p53蛋白结合,并抑制p53的正常生物学功能,从而可导致肿瘤的发生和发展。理论上,阻断MDM2对p53的作用可恢复p53正常功能,因此,抑制MDM2蛋白和p53相互作用被认为是抗肿瘤药物研究中的重要靶点之本论文在文献调研和文献工作总结的基础上,建立了基于p53-MDM2结合抑制剂小分子的药效团模型,并利用药效团模型,结合分子骨架迁越、电子等排、分子宏观性质与微观结构的集合等理性药物设计方法,对先导化合物Nutlin-1进行了结构修饰,保留咪唑啉骨架,对2位苯基、1位N上的连接侧链及4,5位芳环上的取代基进行结构改造和优化,共合成25个分子；对所得化合物进行了p53-MDM2结合抑制活性测试,初步构效关系表明,2-苯环及对位氯原子取代的4,5-二苯对保持p53-MDM2结合抑制活性是必须,N-1侧链取代基的不同以及侧链连接方式的改变对活性有较大影响。在此基础上,进行了第二轮的结构修饰,以改变侧链的结构为主要手段,分别在N-1位引入各种烷基酯、氨基酸作为连接链,连接链末端为各种杂环及含氮片断,考察N-1侧链对活性的影响,共合成化合物26个,蛋白荧光偏振测试及抗肿瘤活性测试均显示出该轮设计合成的化合物活性较阳性对照有较大提高。为了进一步深入了解该类化合物的结合模式,本文构建了3D-QSAR模型,该模型具有较好的预测和评价能力。在上述研究的基础上,论文在寻找全新母核结构的p53-MDM2结合抑制剂方面亦进行了一些新的探索。a.鉴于咪唑啉母核易被氧化成咪唑,因而设计、合成了15个2,4,5-三芳基咪唑衍生物,其p53-MDM2结合抑制活性及肿瘤细胞增殖抑制活性筛选结果表明,该类化合物具有中等强度的p53-MDM2结合抑制活性(～20μM)及中等到较强的肿瘤细胞增殖抑制活性。b.根据骨架跃迁、电子等排等经典设计思想,用3-氨基吡唑、3-甲酰吡唑等母核结构代替咪唑,合成了一系列取代芳基杂环类化合物共38个,p53-MDM2结合抑制活性及肿瘤细胞增殖抑制活性筛选结果表明,3-氨基吡唑类化合物的肿瘤细胞增殖抑制活性并未不理想,但p53-MDM2结合抑制活性较咪唑类化合物有所提高(～10μM)；3-甲酰吡唑类化合物的p53-MDM2结合抑制活性较弱。c.以目前体外活性最高的螺环-吲哚林-2-酮类化合物为先导,设计、合成了一系列螺环-吲哚林-2-酮类化合物共26个,其p53-MDM2结合抑制及肿瘤细胞增殖抑制活性结果显示,该类化合物对p53野生型细胞株活性较弱,但对p53突变型细胞株(SW620)表现出中等到较强的抑制活性,推测该类化合物具有不同的作用模式,对治疗p53突变型肿瘤具有进一步深入研究的价值。同时,为测定目标分子p53-MDM2(?)了结合抑制活性,研究过程中成功表达、纯MDM2蛋白,并建立了基于荧光偏振的活性测试方法,为全文活性测试提供了实验基础。更多还原

【Abstract】 The p53tumor suppressor is controlled by MDM2, which binds p53and negatively regulates its transcriptional activity and stability. Many tumors overproduce MDM2to impair p53function. Therefore, restoration of p53activity by inhibiting the p53-MDM2binding represents an attractive novel approach to cancer therapy. Recently developed potent and selective small-molecule antagonists of the p53-MDM2interaction have been used to demonstrate the proof-of-concept for this approach.According to previous research and literature summary, a pharmacophore model was set up based on the information of p53-MDM2binding structure and structures of known p53-MDM2binding inhibitors. The pharmacophore model revealed that three hydrophobic groups on the core structure were necessary for a good p53-MDM2binding inhibitor, and the introduction of hydrogen acceptor groups might also improve the inhibitory activities. In our first-round design, three new series of imidazoline derivatives were designed and synthesized using Nultins as lead compounds. The imidazoline scaffold of Nutlins was retained as the core structure with a focus on modifying the phenyl groups on the imidazoline ring and the N-1side chain of Nutlins. Based on the obtained data, a preliminary SAR was disclosed that the2-phenyl group and4,5-di(4-cholo)phenyls were essential for the potency, and the N-1substituents displayed a very important role in remaining the potencies as well. Then a series of novel Nutlin analogues, utilizing carbamates, amino acids, amino-acid esters or amino-acid amides as the N-1substituents were designed and synthesized to discover new p53-MDM2binding inhibitors with enhanced p53-MDM2binding inhibitory activities and anti-proliferative potencies. Then CoMFA study which was carried out based on the obtained data helped in understanding the interaction between the ligand and the receptor.Then, some preliminary work was carried out in order to discover novel scaffold p53-MDM2binding inhibitors:a. a series of152,4,5-triphenyl imidazole derivatives was designed due to the imidazoline could get oxidized into imidazole, and their p53-MDM2binding inhibitory activities were tested as well as their anti-proliferative activities. The result exhibited that some derivatives performed medium inhibitory activities (-20μM), however, most of them showed medium to high anti-proliferative activities, which lead to a new thought in discovering novel p53-MDM2binding inhibitors. Thus, by utilizing classic drug design principle, such as bioisosteric replacement and scaffold hopping, a new series of3-amino pyrazole and3-carboxyl pyrazole was designed, synthesized and evaluated their p53-MDM2binding inhibitory activities and anti-proliferative activities. The result for38obtained compounds was concluded that3-amino pyrazole compounds had improved p53-MDM2binding inhibitory activities (～10μM) though they did not show promising anti-proliferative activities, and3-carboxyl pyrazole compounds was not good for their p53-MDM2binding inhibitory activities. As for the structural diversities, we employed spiro-indolinone compounds, which was reported the most potent compounds so far in vitro, as lead compounds, designed and synthesized a series of new spiro-indolinone derivatives, however, this series showed weak activities against cell lines expressing wtp53, but to cell lines have p53mutated (SW620), some compounds exhibited very promising activities, as a matter of concern, this series of derivatives may take part in other mechanism of p53pathway, and may be valuable for treating p53mutated cancer.Meanwhile, for the purpose of testing p53-MDM2binding inhibitory activities of obtained molecules, MDM2protein was expressed and purified, and a FP-based testing assay was set up for the first time at home.更多还原