【作者】 张霞；

【导师】 毕彩丰； 范玉华；

【作者基本信息】 中国海洋大学 ， 海洋化学工程与技术， 2009， 博士

【摘要】 希夫碱及其金属配合物被证明具有抗癌、抑菌、与DNA相互作用等生物活性,引起了科学界的广泛关注。近几年来,有关希夫碱金属配合物抗肿瘤活性的研究有一定的报道,但是关于希夫碱金属配合物抑制蛋白酶体活性及诱导肿瘤细胞凋亡的机理方面的研究鲜有报道。泛素-蛋白酶体通路（Ubiquitin-proteasome pathway,UPP）在肿瘤治疗中的作用引起广泛关注。泛素-蛋白酶体通路是真核细胞蛋白质降解的重要途径。特异性阻断UPP通路,可以显著的影响许多负责细胞周期和肿瘤生长的蛋白,如细胞周期蛋白、P53、Bax、IκB等的降解,继而影响到多个细胞内过程,从而导致细胞凋亡。因此,蛋白酶体抑制剂则是一类很有希望的抗肿瘤活性化合物,成为肿瘤治疗的新靶点。本论文正是在蛋白酶体抑制剂研究的基础,合成了一系列希夫碱及其金属配合物,对其进行了表征,用MTT法及台盼兰（Trypan Blue）染色法筛选出几种抗肿瘤性能良好的铜、锌配合物,对这些高活性化合物的抗肿瘤机制进行了深入研究,确定了该类希夫碱配合物的生物靶标为蛋白酶体。具体内容如下:（1）合成了11种牛磺酸氨基酸希夫碱金属配合物,探讨其对26S蛋白酶体的抑制作用,结果表明大部分铜配合物均可抑制26S蛋白酶体的活性,MTT法筛选出对肿瘤细胞增殖具有较好抑制能力的配合物-牛磺酸缩水杨醛铜邻菲咯啉的三元配合物（Y6）,考察了Y6对纯化的20S蛋白酶体的抑制作用,结果表明其对纯化的20S蛋白酶体的抑制作用呈浓度依赖方式,半抑制率IC50约为12μM;Y6作用于人乳腺癌细胞MDA-MB-231的实验表明,此配合物以浓度及时间依赖方式抑制人乳腺癌细胞MDA-MB-231内蛋白酶体的活性进而诱导细胞凋亡;Y6作用于白血病细胞Jurkat T,台盼兰染色法表明此配合物对白血病细胞的致死率呈浓度依赖方式,且以浓度及时间依赖方式抑制白血病细胞Jurkat T内蛋白酶体的活性进而诱导细胞凋亡。Y6对人乳腺癌细胞MDA-MB-231及白血病细胞Jurkat T的蛋白酶体诱导能力及凋亡诱导方式相似,证明了其抗肿瘤广谱性。（2）合成了3,5-二溴酪氨酸希夫碱配体（KL）及其它的5种配合物,运用元素分析、红外光谱、紫外光谱、摩尔电导率及热分析等手段,对合成的配体和配合物进行了表征。各配合物的组成分别为[CuL （CH₃COO）（CH₃OH）]·2H₂O、[ZnL （CH₃COO）（CH₃OH）]·H₂O、[CdL （CH₃COO） （CH₃OH）]·H₂O、[La LNO₃] NO₃·H₂O、[SmLNO₃] NO₃·H₂O。对部分配合物进行了非等温热分解动力学研究,得出了配合物某步热分解的反应机理、热分解动力学方程、相应的动力学参数及活化熵△S≠和活化吉布斯函数△G≠,结果如下:[CuL （CH₃COO）（CH₃OH）]·2H₂O第3步热分解动力学函数为:f（α）=1/3（1-α）[-ln（1-α）]-2,热分解动力学方程为:dα/dt = A·e-E/RT·f（α） = 1/3A·e-E/RT （1-α）[-ln（1-α）]-2,E = 327.03 kJ/mol,lnA =55.24,r = 0.9952,△S≠=-209.48J/mol·K,△G≠=112.58 kJ/mol。[ZnL （CH₃COO）（CH₃OH）]·H₂O的第2步热分解动力学函数为:f（α）=1/3（1-α）[-ln（1-α）]-2 ,热分解动力学方程为:dα/dt = A·e-E/RT·f（α） = 1/3A·e-E/RT （1-α）[-ln（1-α）]-2,E = 471.70 kJ/mol,lnA =82.58,r = 0.968,△S≠=-436.19 J/mol·K,△G≠=-220.45 kJ/mol。[SmLNO₃] NO₃·H₂O的第2步热分解动力学函数为: f （α）= 1/4（1?α）[?ln（1?α）]?3,热分解动力学方程为:dα/dt = A·e-E/RT·f（α） = 1/4（1-α）[-ln（1-α）]-3A·e-E/RT,E = 295.98 kJ/mol,lnA =88.68,r = 0.9899,△S≠=-486.68 J/mol·K,△G≠=-21.95 kJ/mol。对配体KL和部分配合物的荧光特性进行了研究。配体KL的激发和发射峰λex/λem为286nm/505 nm ,配合物[CuL （CH₃COO）（CH₃OH）]·2H₂O、[ZnL（CH₃COO）]·H₂O、[LaLNO₃] NO₃·H₂O的激发和发射峰λex/λem分别为373nm/444 nm、285nm/500 nm、375nm/478nm,与配体相比较,[CuL（CH₃COO） （CH₃ OH）]·2H₂O配合物形成后,荧光强度、激发光谱和发射光谱的位置均产生了变化,尤其是荧光强度发生了明显的变化;[ZnL（CH₃COO）]·H₂O、[La LNO₃] NO₃·H₂O配合物的荧光强度、激发光谱和发射光谱的位置均未产生大的变化,荧光性质与配体较相似。（3）利用合成的3,5-二溴酪氨酸希夫碱配体KL及其铜配合物[CuL （CH₃COO）（CH₃OH）]·2H₂O（Z1）为研究对象,考察了其对人乳腺癌细胞MDA-MB-231内蛋白酶体活性抑制及凋亡诱导作用。结果表明,此铜配合物可以通过抑制蛋白酶体的活性诱导肿瘤细胞凋亡,凋亡机理与牛磺酸希夫碱铜配合物Y6相似。（4）合成了邻苯二胺缩邻香草醛希夫碱配体B2及邻苯二胺缩水杨醛希夫碱配体B7,分别将B2、B7溶液与铜盐或锌盐溶液混合得到几种配合物混合溶液。分别用各混合物溶液处理人乳腺癌细胞MDA-MB-231及白血病细胞Jurkat T,详细考察了其对两种肿瘤细胞的蛋白酶体抑制作用及细胞凋亡的诱导作用。结果表明:Cu（Ⅱ）、Zn（Ⅱ）混合液均能通过抑制蛋白酶体的活性进而诱导肿瘤细胞凋亡,Cu（Ⅱ）混合液的作用能力较Zn（Ⅱ）混合液稍强,但是二者对两种肿瘤细胞的凋亡诱导机理相似,都是钙蛋白酶参与的结果,这与Y6及Z1配合物诱导细胞凋亡机理不同。同时比较了不同结构的配体B2、B7形成的铜混合液对白血病细胞Jurkat T的蛋白酶体活性抑制能力及细胞凋亡的诱导能力,探讨了配体结构对配位能力的影响。（5）比较了Cu（I）和Cu（II）对纯化的20S蛋白酶体的抑制作用。结果表明:两种价态的铜均可抑制蛋白酶体的活性,但Cu（I）较Cu（II）抑制作用稍强;纯化的20S蛋白酶体将Cu（II）还原为Cu（I）;DMSO和乙醇等羟基自由基抑制剂部分的保护纯化的20S蛋白酶体活性被CuCl2抑制;NC-铜混合物处理MDA-MB-231细胞后,在细胞内有活性氧簇（ROS）产生,铜氧化还原产生ROS可能是铜配合物抑制蛋白酶体活性进而诱导细胞凋亡的可能原因。（6）合成了2-（2-羟基-3-甲氧基-苯基）醛缩-氨基苯并噻唑希夫碱配体,并得到了其单晶。采用量子化学的密度泛函理论（DFT）B3LYP方法,运用Gaussian03量子化学程序包,在B3LYP/6-31G+基组水平上对获得的单晶结构进行优化,计算了分子稳定构型的总能量、前沿分子轨道能量、原子自然电荷布居、自然键轨道（NBO）及稳定化能等。计算结果表明,实验值与计算值基本吻合,说明此模型是稳定的。并用此模型预测出该标题化合物的化学活性位点可能为N（28）, O（29）和O（31）这3个原子,这些活性位点易于发生某些有机反应或与金属离子配位,量子化学计算为设计合成配合物提供了理论基础。更多还原

【Abstract】 Schiff base and its metal complexes have been proved to have biological activities, such as anti-cancer, antibacterial, and interactions with DNA, which causes widespread concern in the scientific community. In recent years, several reports on anti-tumor activity of the Schiff base metal complexes have been published. However, there are rarely reports about the mechanism of Schiff base metal complexes as the proteasome inhibitor to induce apoptosis in cancer cells. As an important way for degradation of eukaryotic cells, Ubiquitin-proteasome pathway （UPP） causes wide concern in the treatment of tumor. UPP is the major proteolytic mechanism which plays a critical role in the degradation of the proteins involve in the cell cycle control and tumor growth, such as cyclin-dependent kinases, P53 , Bax, IκB and so on. Specified block the UPP, then inhibiting the degradation of such protein should have profound effects on tumor cells proliferation and cause cells to undergo apoptosis. So the proteasome inhibitor is useful compound to targeting this pathway in cancer therapy.In this dissertation, we synthesis several Schiff base metal complexes, then use proteasome activity and MTT assay to got several copper and zinc Schiff base complexes which can inhibit 26S proteasome activity and proliferation ability in cancer cell lines. The mechanism of these complexes to inhibit proteasome and induce apoptosis in MDA-MB-231 and Jurkat T cancer cells were studied, and make sure the antitumor target of those complexes is proteasome. The details of the contents are as follows,（1） 11 taurine Schiff base complexes were synthesized, and theirs proteasome inhibit activity were studied. The results showed that all of the copper complexes could inhibit the 26S proteasome activity, MTT assay showed that only taurine Schiff base copper phen complex （Y6） have anti-proliferation activity in cancer cells. Y6 potently inhibits chymotrypsin-like activity of purified 20S, the IC50 is 12μM. The experiment also showed that Y6 potently inhibits chymotrypsin-like activity of 26S proteasome and induce apoptosis in human breast cancer MDA-MB-231 in dose and time dependent manner; Trypan blue assay showed that Y6 could induce cell death in dose dependent manner, and Y6 potently inhibits activity of 26S proteasome and induce apoptosis in leukemia Jurkat T cells in dose and time dependent manner. The mechanism of Y6 induce apoptosis in these two cancer cells are same. Our results strongly suggest that Y6 are potent proteasome inhibitors.（2）The novel Schiff base ligand （KL） which is derived from 3,5-dibromotyrosine and o-vanillin has been prepared, and it’s 5 complexes have been synthesized. Those ligand and complexes were characterized by elemental analysis, IR spectroscopy, UV spectroscopy, TG-DTG analysis and molar conductance analysis. The suggested structure of the complexes are confirmed to be[CuL （CH₃COO）（CH₃OH）]·2H₂O、[ZnL （CH₃COO）（CH₃OH）]·H₂O、[CdL （CH₃COO） （CH₃OH）]·H₂O、[La LNO₃] NO₃·H₂O、[SmLNO₃] NO₃·H₂O.The kinetic equations of thermal decomposition for complexes and the corresponding kinetic parameters were gained.The detail are as follows, The thermal decomposition kinetic function of [CuL（CH₃COO）（CH₃O H）]·2H₂O in step 3 may be expressed as f（α）=1/3（1-α）[-ln（1-α）]-2, and the kinetic equation of thermal decomposition may be expressed as dα/dt = A·e-E/RT·f（α） = 1/3A·e-E/RT （1-α）[-ln （1-α）]-2,E = 327.03 kJ/mol,lnA =55.24,r = 0.9952,△S≠=-209.48J/mol·K,△G≠=112.58 kJ/mol.The thermal decomposition kinetic function of [ZnL （CH₃COO）（CH₃OH）]·H₂O in step 2 may be expressed as f（α）=1/3（1-α）[-ln（1-α）]-2, and the kinetic equation of thermal decomposition may be expressed as dα/dt = A·e-E/RT·f（α） = 1/3A·e-E/RT （1-α）[-ln（1-α）]-2,E = 471.70 kJ/mol,lnA =82.58,r = 0.968,△S≠=-436.19J/mol·K,△G≠=-220.45 kJ/mol.The thermal decomposition kinetic function of [SmLNO₃] NO₃·H₂O in step 2 may be expressed as f（α）=1/4（1-α） [-ln（1-α）]-3, and the kinetic equation of thermal decomposition may be expressed as dα/dt = A·e-E/RT·f（α） = 1/4 A·e-E/RT （1-α） [-ln（1-α）]-3,E = 295.98 kJ/mol,lnA =88.68,r = 0.9899,△S≠=-486.68 J/mol·K,△G≠=-21.95 kJ/mol.The fluorescence property of ligand（KL） and part of it’s complexes were studied. The excitation and emission peak wavelengths（λex/λem） of the schiff base ligand KL is 286nm/505 nm, and the complexes of [CuL （CH3COO）（CH3OH）]·2H2O, [ZnL（CH3COO）]·H2O, [La LNO3] NO3·H2O are 373nm/444 nm, 285nm/500 nm, 375nm/478nm. Compared with the ligand, the excitation peaks and the emission peak of [CuL （CH3COO）（CH3OH）]·2H2O have changed, and the fluorescence quenched; the excitation peaks and the emission peak of [ZnL（CH3COO）]·H2O and [La LNO3] NO3·H2O almost same as the ligand, and still have strong fluorescence.（3） The anticancer activity of ligand （KL） and copper complex [CuL （CH3COO）（CH3OH）]·2H2O （Z1） were studied. The results showed that this complex could inhibit the chymotrypsin- like activity of proteasome and induce apoptosis in highly metastatic MDA-MB-231 breast cancer cells. The mechanism of how the complex induce apoptosis are same as taurine copper Schiff base complex （Y6）.（4） The Schiff base ligands （B2, B7） which are derived from o-vanillin, salicylaldehyde with o-phenylenediamine and the mixture of the copper（or zinc） with the ligands have been prepared. The activity of proteasome inhibition and the apoptosis induction in human breast cancer MDA-MB-231 and leukemia Jurkat T cells by the mixture were studied. All the results showed that both copper and zinc mixture could inhibit proteasome and induce apoptosis in human breast cancer MDA-MB-231 and leukemia Jurkat T cells, the copper mixture a little more potent than the zinc mixture; and the calpain protein which plays a critical role in apoptosis involve in both metal mixture induce apoptosis. Compared the ability of proteasome inhibition and apoptosis induction in Jurkat T cells by the mixtures - copper with two different ligands（ B2 and B7） .The results showed that the structure of the ligands could effect theirs ability to carry metal ions.（5）The mechanism of copper inhibit proteasome were studied. The results showed that both Cu（I）and Cu（II） can inhibit purified 20S proteasome activity, the Cu（I） a little more potent than Cu（II）, the purified 20S proteasome can reduce Cu（II） to Cu（I）, the hydrogen radical scavenger can partially protect the Cu（II） inhibit the purified 20S proteasome activity, hydrogen radical scavenger is one of reactive oxygen species （ROS）, furthermore, treatment of MDA-MB-231 breast cancer cells with copper complex resulted in the production of reactive oxygen species （ROS）, which make sure the ROS is one of the reason about copper inhibit proteasome activity.（6）The crystal of Schiff base 2-（2-hydroxy-3-methoxy benzaldehyden）- aminobenzothiazole （C15H12N2O2S） was obtained. The molecular structure of 2-（2-hydroxy-3- methoxy benzaldehyden）- aminobenzothiazole is calculated using the density functional theory （DFT） with the gradient corrected B3LYP method. All calculations are performed using the Gaussian 03 program package. The crystal structure of the compound is totally optimized. The energies and components of molecular orbital （HOMO and LUMO）, natural population, NBO and stabilization energy were calculated. All data obtained from the calculations are consistent with those gained from the determination, which means the calculation model is stabilized. The possible three chemistry activities atom （N（28）, O（29）, O（31）） were predicted, that means those three atoms are easy to react with metal. The results of the calculation give a theory direction on the rational design and synthesis of metal–directed complexes in coordination chemistry.更多还原