氮杂穴醚衍生物类无金属磷酸酯酶模拟物的设计、合成及其切割DNA和抗肿瘤活性的研究

Currently, the DNA cleavage abilities of artificial DNA cleavage agents are not efficient enough. Besides, most of them are lack of sequence selectivity and made of transition metal complexes which will generate harmful uncontrolled free radicals. These faults extremely restrict their potential application in the field of drug development. With respect to that, we intend to design and synthesis of the following azacryptand derivatives as metal-free DNA cleavage agents, by simulating the active site structures of alkaline phosphatase: (1) compounds with bisorthoamide linked by 2,6-dimethylene-4-substituted phenol; (2) compounds selected from afore-mentioned compounds according to their DNA cleavage efficiency and further conjugated with DNA intercalators or groove-recognition agents. Moreover, we'll investigate their DNA binding ability, DNA cleavage efficiency and mechanism by CD, fluorescence, UV-vis spectra, ESI-MS, gel electrophoresis, combined with theoretical calculation; determine their DNA cleavage sites by isotope labelling and the Maxam-Gilbert methods, and research on their cytotoxic activity by MTT assays. In a word, we envisage to obtain metal-free phosphatase mimics as highly-efficient, site-selective DNA cleavage agents with strong anti-tumor activity, by taking advantage of the high-efficiency strategy of natural emzymes and the structure feature of azacryptand, coupling rigidity with flexiblity, via optimization the pattern of synergism and spatial distance between the organic functional groups, and further enhancing the DNA sequence-recognition and binding abilities of these synthetic small molecules, via being conjugated with DNA intercalators or groove-recognition agents.

目前人工DNA切割剂由于存在切割效率低，绝大多数序列选择性差，且多为易产生对人体有害自由基的过渡金属配合物等缺点，严重制约了其在药物开发领域的应用发展。针对以上问题，本课题拟模拟碱性磷酸酯酶活性位点结构，设计合成氮杂穴醚衍生物作为无金属DNA 切割剂：①用2,6-二亚甲基取代苯酚链接双三氮杂穴醚；②筛选出效率高的小分子与DNA插入子和强沟区识别基团偶联。用实验与理论计算相结合的方式研究其与DNA的相互作用和对DNA的切割效率及机理；用同位素标记和Maxam-Gilbert法研究其识别切割位点；用MTT法研究其抗肿瘤活性。通过充分运用天然酶高效性策略和氮杂穴醚"刚柔并济"的结构特点，可优化有机功能基的协同作用方式和空间距离，并通过偶联DNA插入子和沟区识别基团进一步增强小分子与DNA的识别和结合能力，预计可获定位切割DNA效率高和抑制肿瘤效果好的无金属磷酸酯酶模拟物。

本课题旨在模拟天然磷酸酯酶活性位点结构，设计合成无金属DNA裂解剂，通过充分运用天然酶高效性策略和氮杂穴醚“刚柔并济”的结构特点，进一步优化有机功能基的协同作用方式和空间距离，并通过偶联DNA插入子和沟区识别基团以增强小分子与DNA的识别和结合能力，从而获得定位切割DNA效率高和抑制肿瘤效果好的无金属磷酸酯酶模拟物。. 主要内容包括：(1) 通过分别模拟两类天然酶活性位点结构，设计合成了两类无金属DNA切割剂：N-羟乙基-N’-胍乙基哌嗪和用2,6-二亚甲基对甲基或对氯苯酚链接双三氮杂穴醚的化合物；(2) 通过系列实验，研究了它们裂解DNA的活性；(3) 利用实验和理论计算相结合的方式，初步研究了它们裂解DNA的机制；(4) 并对后一类效率相对较高的DNA切割剂初步研究了它们抑制肿瘤活性；(5) 遗憾的是，总体效率并不理想，我们在将筛选出效率相对较高的小分子与DNA插入子偶联时，遇到了困难，没有成功得到目标产物。.此外，由于偶联反应的失败，我们因此开展了系列偶联反应的方法学研究，并形成了通过偶联反应构建新C-C、C-N、C-O和C-S键的新方法。