【作者】 肖艳；

【导师】 毕彩丰； 范玉华；

【作者基本信息】 中国海洋大学 ， 海洋化学， 2009， 博士

【摘要】 氨基酸是生物体内微量金属元素的重要配体,氨基酸希夫碱配合物在医学、催化、分析化学、农业等领域的应用受到广泛关注。近几年来,有关希夫碱配合物抗肿瘤活性的研究有一定的报道,但是关于氨基酸希夫碱铜配合物对肿瘤细胞内蛋白酶体活性及细胞凋亡诱导性的作用及作用机理方面的研究鲜有报道。设计和合成不同的氨基酸希夫碱配体及其金属配合物,研究其结构、性质及对生物体的作用机制,特别是氨基酸希夫碱铜配合物对肿瘤细胞内蛋白酶体的作用和作用机理,是一项具有挑战性的研究。顺铂是治疗癌症的常用药物,但是由于其毒副作用较大而限制了使用。研究铂配合物对肿瘤细胞内的蛋白酶体活性及细胞凋亡诱导性的作用,可以为铂配合物的抗肿瘤作用机理及其毒副作用研究提供理论依据。论文设计和合成新的氨基酸希夫碱配体及其金属配合物,对其进行表征,推断出其可能的结构,对部分配合物的抑菌活性进行了研究;以蛋白酶体为作用靶点,深入研究了氨基酸希夫碱铜配合物及苯基吡啶铂配合物对肿瘤细胞内蛋白酶体的抑制作用和凋亡诱导作用;以有机铜配合物NC-CuCl为模型化合物,研究了其抗肿瘤作用机理。具体研究内容如下:选用L-天冬酰胺和L-谷氨酰胺为母体,同含有亲水基的邻香草醛缩合,得到两种希夫碱配体,使其与过渡金属反应得到10种金属配合物,采用红外光谱、紫外光谱、摩尔电导率分析、元素分析和热重分析等分析手段对配体及金属配合物进行表征。L-天冬酰胺缩邻香草醛希夫碱配体及其五种过渡金属配合物的组成分别为:HL₁Li·2H₂O,[Zn（LiL₁）（CH₃COO）（H₂O）3], [CuL₁（H₂O）3], [Mn（LiL₁） （CH₃COO）（H₂O）3],[Co（LiL₁）（CH₃COO）（H₂O）3], [Ni（LiL₁）（CH₃COO）（H₂O）]·2H₂O。L-谷氨酰胺缩邻香草醛希夫碱配体及其五种过渡金属配合物的组成分别为:HL₂Li·H₂O, [Zn2（L₂）₂（CH₃COOH）（H₂O）₂]·（H₂O）（CH₃OH）, [Cu3（L₂）₂（CH₃COO）₂ （H₂O）]·2H₂O, [Mn3（L₂）₂（CH₃COO）₂（CH₃COOH）₂]·3H₂O,[Co2（L₂）₂（CH₃COOH） （H₂O）₂]·（H₂O）（CH₃OH）, [Ni3（L₂）₂（CH₃COO）₂]·5H₂O。对金属配合物进行了非等温热分解动力学处理,研究其热分解过程和热分解机理,结果如下:L-谷氨酰胺缩邻香草醛希夫碱锰配合物第一步热分解动力学函数为:f（α）= 1/3（1-α）[-ln（1-α）]^-2,热分解速率动力学方程为:dα/dt = A·e^-E/RT·1/3 （1-α）[-ln（1-α）]^-2。其它的L-天冬酰胺和谷氨酰胺缩邻香草醛希夫碱金属配合物的第一步或第二步热分解动力学函数均为:f（α）=1/4（1-α）[-ln（1-α）]^-3,热分解速率的动力学方程均为:dα/dt = A·e-E/RT·f（α） = A·e^-E/RT·1/4（1-α）[-ln（1-α）]^-3。热分解动力学处理同时也得到了配合物某步的热分解相应的动力学参数（E, A）及活化熵变△S≠和活化吉布斯自由能变△G≠。采用抑菌圈法对L-天冬酰胺和L-谷氨酰胺希夫碱配体及其部分金属配合物进行了抑菌活性研究。研究表明所供测试化合物的抑菌活性均与浓度正相关;配合物的抑菌效果明显高于相应的配体,其中天冬酰胺缩邻香草醛镍配合物（#2）和谷氨酰胺缩邻香草醛铜配合物（#8）的抑菌效果较好,具有广谱抗菌性;对金黄色葡萄球菌、绿脓假单胞菌、大肠杆菌和枯草杆菌抑菌效果最好的分别是谷氨酰胺缩邻香草醛铜配合物（#8）、谷氨酰胺缩邻香草醛钴配合物（#7）、天冬酰胺缩邻香草醛镍配合物（#2）和天冬酰胺缩邻香草醛镍配合物（#2）。配合物的抑菌效果明显高于相应的配体,说明配合物中的过渡金属离子对细菌的生长发挥了重要的抑制作用。用溴化3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑（MTT）法对所合成的12种天冬酰胺和谷氨酰胺希夫碱配体及其金属配合物进行抗肿瘤活性筛选,筛选出对乳腺癌MDA-MB-231细胞增殖具有较好抑制作用的谷氨酰胺缩邻香草醛希夫碱铜配合物（GVC）。对GVC的进一步抗肿瘤活性研究发现:GVC对乳腺癌MDA-MB-231及血癌Jurkat T细胞内的蛋白酶体活性有抑制作用,能诱导该肿瘤细胞发生凋亡,且这种作用与GVC的使用浓度和作用时间正相关;更重要的发现是GVC能选择性的抑制肿瘤细胞,对恶性乳腺癌细胞MCF 10DCIS.com有较强细胞毒性,对正常乳腺细胞MCF 10A毒性较小。GVC对乳腺癌MDA-MB-231和血癌Jurkat T细胞内蛋白酶体的抑制作用和细胞凋亡诱导作用,说明GVC具有广谱抗肿瘤活性;对恶性乳腺癌细胞MCF 10DCIS.com和正常乳腺细胞MCF 10A的选择性凋亡诱导作用,说明了GVC具有很好的抗肿瘤选择性。以有机铜配合物NC-CuCl为模型化合物,用Image-iT^TM活细胞ROS测试法研究了有机铜配合物的抗肿瘤作用机理,研究发现有机铜配合物对蛋白酶体的抑制作用可能主要是通过铜离子和蛋白酶体的键合作用,通过对细胞内蛋白酶体活性的抑制,进而诱导细胞发生凋亡。ROS的产生参与了诱导肿瘤细胞发生凋亡,但不是主要原因。研究了四种苯基吡啶铂配合物对纯化的20S蛋白酶体的抑制作用,研究表明它们对纯化的20S蛋白酶体的抑制作用与其使用浓度正相关,其半数抑制率IC50值均小于6μM;用MTT法考察了这四种苯基吡啶铂配合物对乳腺癌MDA-MB-231细胞增殖的抑制作用,研究表明这四种苯基吡啶铂配合物比顺铂具有使用浓度更低、抑制活性更好的特性,其半数抑制率IC50值均小于10μM。对这四种四种苯基吡啶铂配合物的深入研究发现:这四种苯基吡啶铂配合物均有抑制乳腺癌MDA-MB-231细胞内的蛋白酶体活性和诱导肿瘤细胞发生凋亡的作用。此项研究为深入研究这四种苯基吡啶铂配合物在体内的抗肿瘤活性提供了必要的理论依据。合成了4-氨基安替比林缩对苯二甲醛双希夫碱,并得到其单晶。该化合物属单斜晶系,P2（1）/C空间群,化学式为C30H28N6O2,M = 504.58。晶胞参数为:a = 6.0710（2）（?）,b = 22.2948（7） （?）,c = 9.8712（3） （?）,α= 90o,β= 95.147（2）°,γ= 90°,Z = 2,V = 1330.70（7） （?）³,T = 293（2） K,Dc = 1.259mg/m³,R1 = 0.0494,wR2 = 0.1361 for I>2σ（I）,F（000） = 532。分子晶体结构显示4-氨基安替比林缩对苯二甲醛的分子不是二维共平面的,而是向三维空间伸展。分子间氢键相互作用使分子形成了一维链状结构。运用Gaussian03量子化学程序包,采用量子化学的密度泛函理论（DFT）B3LYP方法,对4-氨基安替比林缩对苯二甲醛的晶体结构进行优化,在稳定几何构型的基础上计算得到前沿分子轨道能量、各分子片富含电子的氮氧原子对前沿轨道的贡献和NBO电荷分布。计算结果表明,此化合物部分键长、键角的计算值与实验值基本吻合。由计算可知该配体与金属或其他分子作用时,N（31）、N（64）、O（33）和O（66）这4个原子是化学活性部位,可以发生各种有机反应及与金属离子配位。对4-氨基安替比林缩对苯二甲醛双希夫碱配体进行了非等温热分解动力学处理,结果如下:其热分解动力学函数为:f（α）= 3/2（1-α）^4/3[1/（1-α）^1/3-1]-1,热分解速率的动力学方程为:dα/dt = A·e-E/RT·3/2（1-α）^4/3[1/（1-α）^1/3-1]^-1,E = 1248 kJ/mol,lnA = 235.1,r = 0.9799,ΔS≠= 1888 J/mol·K,ΔG≠= 86.14 kJ/mol。更多还原

【Abstract】 Amino acids are the essential trace metal elements’ligands in vivo. Amino acid Schiff base complexes have been applied in a wide range of fields, such as medicine, catalysis, analytical chemistry, agriculture and so on. In recent years, many researchers reported that Schiff base complexes have the anticancer activity, but the anticancer activities and mechanisms of amino acid Schiff base complexes as proteasome inhibitors and apoptosis inducers were seldom reported. It is a challenging project to design and synthesize different amino acid Schiff base ligands and their metal complexes for studying their structures, properties and bioactivities, especially the ancicancer activity of the amino acid Schiff base copper complexes on tumor cells by targeting cellular proteasome and inducing apoptosis. Cisplatinum is a widely used anticancer drug, but it is limited because its side effects. The study of platinum complexes on tumor cellular proteasome and apoptosis may supply theoretical basis on anticancer mechanism and side effects of platinum complexes.This dissertation aims to design, synthesize and characterize new amino acid Schiff base ligands and their metal complexes. It focuses on interactions of various amino acid Schiff base copper complexes and platinum complexes with the proteasome, and their abilities to inhibit tumor cellular proteasome and induce apoptosis in vitro. Moreover, organic copper complex NC-CuCl as a model compound was used to study the antitumor mechanism of organic copper complexes. The main researches are as follows:Two Schiff base ligands which are derived from L-asparagine or L-glutamine and o-vanillin and their ten transition metal complexes have been synthesized. Their structures were characterized by IR spectroscopy, UV spectroscopy, molar conductance analysis, elemental analysis and thermal gravimetric analysis and so on. The composition of Schiff base ligand derived from L-asparagine and o-vanillin （L₁） and its five metal complexes are as follows: HL₁Li·2H₂O, [Zn（LiL₁）（CH₃COO） （H₂O）3], [CuL₁（H₂O）3], [Mn（LiL₁）（CH₃COO）（H₂O）3], [Co（LiL₁）（CH₃COO）（H₂O）3], [Ni（LiL₁）（CH₃COO）（H₂O）]·2H₂O. The composition of Schiff base ligand derived from L-glutamine and o-vanillin （L₂） and its five metal complexes are as follows: HL₂Li·H₂O, [Zn2（L₂）₂（CH₃COOH）（H₂O）₂]·（H₂O）（CH₃OH）, [Cu3（L₂）₂（CH₃COO）₂ （H₂O）]·2H₂O, [Mn3（L₂）₂（CH₃COO）₂（CH₃COOH）₂]·3H₂O, [Co2（L₂）₂（CH₃COOH） （H₂O）₂]·（H₂O）（CH₃OH）, [Ni3（L₂）₂（CH₃COO）₂]·5H₂O.By thermal decomposition analysis, the thermal decomposition mechanism of metal complexes has been studied. The results are as follows: The first step of the thermal decomposition kinetic function of [Mn3（L₂）₂（CH₃COO）₂（CH₃COOH）₂]·3H₂O is expressed as f（α）= 1/3（1-α）[-ln（1-α）]-2 and its kinetic equation of thermal decomposition is expressed as dα/dt = A·e-E/RT·f（α） = A·e-E/RT·1/3（1-α）[-ln（1-α）]-2. Except for the above complex, the first or second step of the thermal decomposition kinetic functions of other metal complexes derived from L-asparagine or L-glutamine and o-vanillin and transition metal are all expressed as f（α）=1/4（1-α）[-ln（1-α）]-3. Their kinetic equations of thermal decomposition are expressed as dα/dt = A·e-E/RT·f（α） = A·e-E/RT·1/4（1-α）[-ln（1-α）]-3. The kinetics parameters E and A and activation entropy change△S≠and Gibbs free energy change△G≠have been obtained.The antibacterial activities of ligands and parts of their metal complexes were also studied against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis bacteria by inhibition zone method. The antibacterial activities of tested compounds were in a concentration-dependent manner. Comparing with the ligands, their metal complexes showed higher antibacterial activity. The complexes #2 and #8 showed the higher antibacterial activity than other tested complexes and were sensitive against four tested bacteria. The best antibacterial complexes against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Bacillus subtilis were complex #8, #7, #2 and #2, respectively. The results indicate that metal complexes have higher antibacterial activities than their ligands, disclosing transition metal ions play an important role in antibacterial activity.The antiproliferaion activities of parts of metal complexes were studied by 3-[4,5-dimethyltiazol-2-yl]-2,5-diphenyl-tetrazolium bromide （MTT method） in human breast cancer MDA-MB-231 cells. It was found that GVC showed the higher antiproliferation activity than other metal complexes. So GVC was chose for further anticancer studies and found that GVC could inhibit proteasome activity and induce apoptosis in MDA-MB-231 and Jurkat T cells in a concentration- and time-dependent manner. Importantly, GVC could specifically and selectively inhibit proteasome activity and induce apoptosis in breast cancer MCF 10DCIS.com cells, but not in normal, immortalized human breast MCF 10A cells. These data demonstrate that the GVC could widely inhibit proteasome activity and induce apoptosis in human cancer cells. Moreover it could specifically and selectively induce proteasome inhibition and apoptosis in human tumor cells.The antitumor mechanism of organic copper complexes was studied by Image-iTTM live green reactive oxygen species detection kit used organic copper complex NC-CuCl as a model compound. This study found the inhibition of proteasome activity induced by organic copper complexes might be mainly through copper binding mechanism and induced cytotoxicity and apoptosis might be primarily through proteasome inhibition. ROS generation may be partially involved in the process of the copper complexes induced cell death, but not the major mechanism.The inhibition of purified 20S proteasome by four platinum complexes were investigated and found that their inhibition potency of 20S proteasome was in a dose-dependent manner. Their IC50 values were less than 6μM. Their antiproliferaion activities were studied by MTT method in human breast cancer MDA-MB-231 cells and found these four platinum complexes had much more antiproliferation activity than that of cisplatin. Their antiproliferation IC50 values were less than 10μM. The further study demonstrates that these four platinum complexes could inhibit proteasome activity and induce apoptosis in human breast cancer MDA-MB-231 cells. This research provides necessary theoretical data for further study on their anticancer activity in vivo.The crystal of the Schiff base derived from 4-aminoantipyrine and terephthalaldehyde belongs to monoclinic, P2（1）/C space group, molecular formula is C30H28N6O2, M=504.58. The unit cell parameter are: a = 6.0710（2）?, b = 22.2948（7） ?, c = 9.8712（3） ?,α= 90o,β= 95.147（2）o,γ= 90o, Z = 2, V = 1330.70（7） ?3, T = 293（2） K, Dc = 1.259mg/m3, R1 = 0.0494, wR2 = 0.1361 for I>2σ（I）, F（000） = 532. The single crystal structure indicates that the molecule is not a two-dimensional plane, but extending to the three-dimensional space. It is the intermolecular hydrogen bond interaction to form a one-dimensional chain structure.The Geometry optimization of C30H28N6O2 was performed by the hybrid density functional theory of the B3LYP method. The energy level, electronic population in the frontier molecule orbitals of the fragments of molecules, the atoms with plentiful electron like as N, O atoms and natural atomic charges distribution were obtained. The calculation results disclose that the calculated data and the tested data of parts of the bond lengthes and bond angles are similar. While this ligand coordinates with metal ions or other molecules, N（31）, N（64）, O（33） and O（66） atoms could occur in certain reactions or in coordination with metal ions.The thermal decomposition kinetic function of the compound C30H28N6O2 is expressed as f（α）= 3/2（1-α）4/3[1/（1-α）1/3-1]-1 and its kinetic equation of thermal decomposition is expressed as dα/dt = A·e-E/RT·3/2（1-α）4/3[1/（1-α）1/3-1]-1, E = 1248 kJ/mol, lnA = 235.1, r = 0.9799,ΔS≠= 1888 J/mol·K,ΔG≠= 86.14 kJ/mol.更多还原