【作者】 张燕；

【导师】 蔡苹； 程功臻；

【作者基本信息】 武汉大学 ， 无机化学， 2015， 博士

【摘要】 癌症是危害人类健康最主要的疾病之一。化疗是治疗癌症的主要方法之一,但是临床上使用的化疗药物存在较大的毒副作用和耐药性等问题,于是寻求高效、低毒的抗肿瘤药物成为研究者们长期奋斗的目标。顺铂的发现促进了金属抗肿瘤药物的研究,金属配合物的研究越来越广泛,钌配合物便是其中之一。与铂类配合物相比,钌配合物具有毒性低药效高等特点。本文设计合成了11个金属钌配合物,通过元素分析、红外光谱、核磁和电喷雾质谱对这些配合物进行表征；并研究了它们的光谱学性质和电化学性质；此外,通过体外细胞实验研究了配合物的细胞毒性,荧光光谱和凝胶电泳阻滞实验研究配合物与DNA的相互作用方式,用流式细胞仪检测配合物诱导肿瘤细胞凋亡和细胞周期的分布。具体内容如下：1.绪论介绍了钌配合物作为抗肿瘤药物的发展史及研究现状,简单介绍了钌配合物与肿瘤细胞作用的机制和研究方法,并提出本课题的选题意义和研究内容。2.第二章合成两个单齿配体和两个双齿配体,并将它们与多种过渡金属盐及cis-Ru(bpy)2Cl2·2H2O反应,通过挥发法、扩散法、水热法尝试长晶体,最后得到一个铜配合物单晶Cu(L2)2和一个钌配合物单晶Ru1。通过元素分析、核磁氢谱,红外光谱、电喷雾质谱等方法对两个配合物进行表征,用X-射线单晶衍射仪测得晶体结构。另外,通过体外细胞毒性方法检测Rul对AGS(人胃癌细胞)、BGC823(人胃癌细胞)和SGC-7901(人胃癌细胞)的细胞毒性,结果为Ru1对三种肿瘤细胞均有一定的细胞毒性,但是对AGS细胞毒性最明显。Hoechst 33258和AO/EB荧光染色实验表明Rul可以诱导AGS细胞发生凋亡,流式细胞仪凋亡实验显示随着Rul浓度的增大和作用时间的增长,凋亡细胞所占比例越来越大。细胞周期实验表明Rul可以抑制AGS细胞增殖停滞在S阶段。凝胶电泳阻滞实验得到的结论为Rul与DNA以插入方式相互作用。3.第三章设计合成六个含有-O,O或-O,N配体的单核钌配合物Ru2-Ru7,通过核磁,红外,元素分析,电喷雾质谱等表征手段证实了配合物结构的正确性。此外,对Ru2-Ru7做了紫外可见吸收光谱和荧光光谱的测试,通过循环伏安法和微分脉冲极谱法对配合物做了电化学测试,发现Ru2-Ru7在0.6-1.0 V范围内均有一个氧化还原峰,说明钌中心发生了氧化还原反应。最后,从细胞水平上研究Ru2-Ru7的生物学意义。当辅助配体相同时,端配体含有-O,O时细胞毒性比端配体是-O,N时细胞毒性明显。Ru2对SGC-7901最具有选择性；Ru3、Ru4口Ru7对Hela都有较强的选择性；Ru5对所测得的三种肿瘤细胞没有明显的活性。4.第四章设计合成三个长度不同并含有邻菲啰啉环的柔性桥配体及相应的双核金属钌配合物Ru8-Ru10,通过核磁,红外,元素分析,电喷雾质谱等表征手段对它们进行表征,并做了紫外可见吸收光谱和荧光光谱及电化学测试。通过体外细胞毒性方法检测Ru8-Ru10对Hela(宫颈癌细胞)、SGC-7901(胃癌细胞)和BGC823(胃癌细胞)的细胞毒性,发现Ru8-Ru10对三种肿瘤细胞均有一定的细胞毒性。并且三个配合物均表现出随着桥配体链中亚甲基数的增加,细胞毒性增大。可能的原因是随着桥配体链的增长,配合物亲油性越来越大,使得配合物容易累积到线粒体内。细胞周期实验表明Ru10能够将BGC823细胞增殖阻滞在G0/G1期。荧光光谱滴定方法表明Ru8-Ru10与DNA通过沟槽结合相互作用。5.第五章合成一个含有刚性桥配体的双核钌配合物Rull,通过元素分析和电喷雾质谱对它进行了表征,并做了紫外可见吸收光谱、电化学测试及氧化还原滴定。通过体外细胞毒性方法研究了Rull对Hela(宫颈癌细胞)、A549(肺癌细胞)、SGC-7901(胃癌细胞)和MCF-7(乳腺癌细胞)四种肿瘤细胞的细胞毒性,实验结果表明Rull对MCF-7最有选择性。细胞周期实验显示Rull将MCF-7细胞周期阻滞在G0/G1期。荧光光谱测试表明Rull与DNA通过插入方式相互作用。更多还原

【Abstract】 Cancer is one of the most serious disease harm to human health. Chemotherapy is a primary method for cancer treatment, but chemotherapy drug have side effects and drug resistance. So seeking for effective, low toxic anti-cancer drug become the goal for researchers. Metal complexes, especially ruthenium complex attracted extensive attention since the finding of cisplatin. Compared to cisplatin, metal ruthenium complex have the advantages of low toxicity and effective anti-cancer. In this paper, some mononuclear and dinuclear ruthenium complexes were synthesized and characterized by element analysis,1H NMR, the infrared and mass spectrometry methods. The UV-vis absorption, fluorescence spectroscopy and electrochemical behavior were studied. The cytotoxicity in vitro was assayed by MTT array, the DNA-binding behaviors were investigated by fluorescence spectroscopy and agarose gel retardation assay. The apoptosis and cell cycle arrest were performed by flow cytometry. Specific works as follows:1. The first chapter introduced the development history of ruthenium complexes as anticancer drugs and research status. The basic theory and relative research methods of ruthenium complexes as anti-cancer drug were introduced. Finally, research significance and research contents were mentioned.2. In chapter 2, two monodentate ligands and two bidentate ligands were synthesized, and let them react with a variety of transition metal salts and cis-Ru(bpy)2Cl2·2H2O. Volatilization method, diffusion method and hydrothermal method were performed in order to obtain crystal, and finally got a ruthenium complex Ru1 and a copper complex Cu(L2)2 single crystals. The complexes were characterized by element analysis,1H NMR, the infrared and mass spectrometry methods, and confirmed by X-ray diffraction studies. The cytotoxic potential of the Ru1 was tested on AGS、 BGC823 and SGC7901 gastric cancer cells, Ru1 was cytotoxic to the three kinds of tumor cells. The mode of cell apoptosis was assessed by different morphological study (Hoechst staining, Acridine orange & ethidium bromide (AO & EB) staining) and flow cytometry assay. The Ru1 induced cell apoptosis in a dose- and time-dependent manner, and the mode of cell death was essentially apoptosis though necrosis was also noticed. Cell cycle analysis by flow cytometry indicated that the Ru1 caused cell cycle arrest and accumulated cells in S phase. The DNA binding properties with Rul was studied by gel electrophoresis.3. In chapter 3, six new mononuclear ruthenium complexes Ru2-Ru7 contained -N,N or -N,O ligands were synthesized and characterized by elemental analysis, IR,1H NMR and electrospray ionization mass spectrometry. UV-vis spectroscopy, fluorescence spectroscopy and electrochemistry were also studied. All the six complexes exhibited one oxidation in the range of 0.6～1.0 V, this peak of the complexes was all assigned to the redox of the RuⅡ moieties. The cytotoxicity in vitro was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Experiments indicated that the Ru2-Ru7 showed significant dose-dependent cytotoxicity to Hela, SGC-7901 and BGC823 tumour cell lines. If auxiliary ligand was the same, the terminal ligand containing -O,O than the ligand-O,N shown obvious cytotoxicity. Ru2 had the selectivity to SGC-7901; Ru3, Ru4 and Ru7 had selectivity to Hela. Ru5 had no cytotoxicity to three test cell lines.4. In chapter 4, a series of 1,10-phenanthroline-based dinucleating bridging ligands and their dinuclear Ru(II) complexes Ru8-Ru10 were synthesized and characterized, their characterization, photophysical and electrochemical properties have been studied. The cytotoxicity in vitro was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. The study indicated that the Ru8-Ru10 showed significant dose-dependent cytotoxicity to Hela, SGC-7901 and BGC823 tumour cell lines. With the increase of the methylene of bridging ligands, the cytotoxicity of the complexes increased. It is possibly that as the growth of the bridging ligands, complexes were more and more lipophilic, so it’s easy to accumulate in the mitochondria. Cell cycle analysis by flow cytometry indicated that the Ru10 caused cell cycle arrest and accumulated cells in G0/G1 phase. The interaction between complexes and the CT-DNA may be a groove binding mode.5. In chapter 5, a dinuclear ruthenium complex Rull which contained the rigid ligand was synthesized and characterized by elemental analysis, IR, 1H NMR and electrospray ionization mass spectrometry. From the cellular level to study the biological significance of complex. The cytotoxicity in vitro was evaluated by MTT assay, the results showed that Rull was selective to MCF-7. Cell cycle analysis by flow cytometry indicated that the Rull caused cell cycle arrest and accumulated cells in G0/G1 phase. Fluorescence spectrum showed that Rull interact with DNA by inserting way.更多还原