【作者】 王飞；

【导师】 闫兵；

【作者基本信息】 山东大学 ， 药物化学， 2010， 博士

【摘要】 铜是人体组织和动物组织所必需的微量元素,是人体进行正常新陈代谢过程所不可缺少的。人体内正常铜的含量为50～100 mg,在此范围内的铜可以维持正常的人体生理功能。如果人体内的铜含量过多则会引起铜中毒。随着对癌症机理的深入研究,人们发现在癌症病人血清中铜的浓度与正常人相比普遍偏高。进一步的研究发现铜在肿瘤发生与发展过程中发挥着极其重要的作用,例如肿瘤组织中血管的形成,肿瘤细胞的转移等过程。因此越来越多的研究将铜作为一个重要的靶点来开发新的癌症治疗方法。目前针对癌症组织中铜浓度过高的治疗方法有：降低癌症病人血清中铜的浓度,减少高浓度的铜对肿瘤血管形成的影响；利用有机铜配合物作为蛋白酶体抑制剂,从而诱导肿瘤细胞的凋亡；利用肿瘤组织中铜的氧化还原反应诱导癌细胞凋亡等。研究发现N,N-二硫代氨基甲酸盐是一种很好的铜螯合剂,其与铜生成的有机配合物是一种蛋白酶体抑制剂,能够有效的诱导肿瘤细胞的凋亡。这种结构代表性的药物就是作为戒酒药上市的药物-戒酒硫(Disulfirm, DSF)。通过对DSF抗癌活性的研究发现,这种药物能够有效的抑制肿瘤细胞的增殖,同时对正常细胞的副作用比较小。但是DSF可以与体内的醛脱氢降解酶(Aldehyde dehydrogenase, ALDH)发生反应,使其在进入人体内所发挥的抗癌作用大大降低。本文基于现有的研究结论提出假设：合成一种新型的N,N-二硫代氨基甲酸盐化合物,能够有效的抑制肿瘤细胞的增值并诱导癌细胞凋亡；对正常细胞的毒副作用小；并且在体内与ALDH不发生反应。本论文的主要工作分为三个部分：第一部分首先合成了一个全新的N,N-二硫代氨基甲酸盐的化合物库,并对库中化合物进行了生物活性筛选,比较了不同化合物与铜结合能力的差别,对其构效关系进行了初步的探讨；第二部分通过对前一个库中发现的活性化合物优化,合成了另一个N,N-二硫代氨基甲酸盐的化合物库,筛选出了对癌细胞的生长抑制作用比较明显的化合物;通过计算机模拟筛选出了对人体内的醛脱氢降解酶没有明显抑制作用的化合物；第三部分是使用组合化学和高通量筛选的方法开发新型饮用水中除砷剂,分别利用液相组合合成和固相组合合成的方法合成了两个化合物库,并对库中化合物的除砷能力进行了评估。论文的主要工作及其结果如下：第一部分,合成了一个新型的N,N-二硫代氨基甲酸盐的化合物库,并对库中的化合物进行了活性筛选。筛选发现库中的化合物与铜的反应生成的配合物除了#9之外,其余的配合物都对乳腺癌细胞MDA-MB-231的增殖有明显的抑制作用,能够很好的抑制肿瘤细胞中蛋白酶体的活性并诱导细胞凋亡。化合物#1与铜的络合物在5μM作用于细胞16 h,可以抑制90%的肿瘤细胞蛋白酶体活性。利用荧光滴定的方法对化合物与铜的络合能力进行了测定,发现化合物#9可以与铜离子发生反应,但是所得到的络合物不能起到对肿瘤细胞增殖的抑制。第二部分,经过优化后合成了第二个N,N-二硫代氨基甲酸盐化合物库,利用MDA-MB-231和DCIS两种肿瘤细胞系对库中的化合物进行了活性筛选。研究发现库中化合物的生物活性具有一定的构效关系。利用紫外滴定的方法对化合物与铜的络合常数进行了测定。通过计算化学以及计算机模拟Docking的方法研究了库中的化合物对两种醛脱氢降解酶活性的抑制作用,发现了#39化合物既能抑制癌细胞的蛋白酶体活性,又不与人体内的醛脱氢降解酶进行反应,从而最大限度的发挥了其诱导癌细胞凋亡的作用。第三部分,运用组合化学和高通量筛选的方法开发新型的饮用水除砷剂。研究以二硫代氨基甲酸盐为核心结构,运用液相和固相两种合成方法构建化合物库,并利用高通量的方法对化合物库中的化合物与砷的结合能力进行了筛选。在化合物的构建上充分考虑到所合成的化合物在环境领域应用的特点。高通量筛选方法的选择上则本着建立全自动仪器分析方法的原则,充分体现库中化合物筛选快速的特点。结论：1)N,N-二硫代氨基甲酸盐不仅可以络合肿瘤细胞中高浓度的铜,同时其与铜生成的有机配合物是一种良好的蛋白酶体抑制剂,能够抑制肿瘤细胞的增殖,并且诱导细胞的凋亡;2)通过对库中化合物进行的构效关系研究发现,化合物中N,N-二取代的取代基对其活性有着一定的影响,对我们有针对性的构建化合物库,从而筛选出理想的化合物提供了一定的理论支持。3)通过优化先导化合物的结构,合成了一种有良好的抗癌活性、且与ALDH不发生反应的新型N,N-二硫代氨基甲酸盐类化合物,为进一步的研究工作提供了良好的工作基础。更多还原

【Abstract】 In human and animal tissues, copper is an important essential trace element. Copper can affect many biological pathways in human body. In healthy adults, the necessary copper concentration is 50-100 mg. Copper can be absorbed from the stomach and intestine through protein transporters. Then, copper associated with albumin and transcuprein is deposited in the liver. In blood, the major copper carrying protein is ceruloplasmin and the rest of copper is transported by albumin and histidine. If the balance of copper in cell is impaired, diseases will occur. Over the past two decades, there are a lot of evidences show that the copper plays an important role in inflammation and the growth of tumors. The copper concentration in serum and tumor tissue is significant higher than that of healthy subjects. Therefore, copper became one of the important targets in anti-cancer treatment. There are some efficiently treatments, such as Copper reduction, oxidative stress, copper complexes as the inhibitors of proteasome. Some compounds demonstrate the very potent anti-cancer activity, such as tetrathiomolybdate (TM), Captoril、Zinc and Trientine, (8-hydroxyquinoline) Ox and (5-chloro-7-iodo-8-hydroxyquinoline)CQ.Dithiocarbamates (DTCs) are a sort of metal chelating and antioxidant compounds. These compounds are usually used in treating fungal diseases and bacteria. More recently, researchers have found that various DTC compounds can induce metal uptake, especial copper, and apotosis. More evidences confirm that the DTCs-copper complexes are also an important proteasome inhibitor. Disulfiram (DSF), a disulfide derivative of N, N-diethyldithiocarbamate (deDTC), is used as the alcohol-abuse deterrent with its ability of aldehyde dehydrogenase. Evidences prove that DSF can significantly inducing apoptosis with lower side effects. But the problem is the reaction between DSF and ALDH could decrease the anti-cancer concentration of DSF.Based on the research referred we presumed that by synthesizing and screening a novel library of PDTC analogues, we could find some compounds which have the very potent anti-cancer activity and have no reaction with ALDH. The content of this paper consists with two parts. In the first part, we synthesized and screened the first library of PDTC analogues. In this part, the binding constant between copper and PDTC analogues and structure activity relationship were discussed in this part. In the second part, another library of PDTC was synthesized and screened. Binding constant and SAR were also discussed. By doing this research, some compounds that have the very potent anti-cancer activity were found.The last part is about applying combinatorial chemisy in environmental sciences, we use combinatorial synthesis and high-throughput screening methodologies to discover novel agents for arsenic removal.The major research work and results of the paper are as follows:Part 1. The anti-proliferative effects on MDA-MB-231 cell line were screened with a synthesized novel library of PDTC analogues. The complexes of PDTC analogues and copper were found to have potent anti-proliferative activity, except compound 9#. Bingding constant was determined by UV titration. Structure activity relationship was also discussed.Part 2. The second library of PDTC analogues was synthesized. The biological data obtained from screening compounds by brease cancer cell line MDA-MB-231 and DCIS. UV titration method was used to determin the binding constant between PDTC analogues and copper. Results show that there is certain relationship between binding constant and anti-cancer activity. Computational docking was used to determin the relationship between PDTC analogues and ALDH. Finally, the compounds that have the very potent anti-cancer activity and have no reaction with ALDH were found.Part 3. We plan to establish a platform for doing various combinatorial synthesis and characterization. By using solid phase and solution phase synthesis method to discover novel agents for arsenic removal.Conclusions:1) The high concentrations of copper in tumor cells can be binded with dithiocarbamates, and the copper-dithiocarbamates are effective proteasome inhibitor. The complexes can inhibit tumor cell proliferation and induce cell apoptosis.2) Through discussing the SAR of DTCs analogues, it can be found that the substitutors are very important for the anti-cancer activity. It’s helpful for us to optimize the structures of the DTCs analogues to find more potent compounds.3) By using the combinatorial synthesis and high-through screening methods will help accelerating the speed of discover the lead compounds. By optimizing the structure of lead compounds, we can found dithiocarbamates which can inhibit the proliferation of tumor without reacting with ALDH.更多还原