基于生漆中不饱和漆酚的HDAC靶向抑制类似物的合成机理及构效研究

Raw lacquer is a kind of important forest products in China, its main active constituents of unsaturated urushiol possess prominent antitumor biological activity. However, as the structure of urushiol is not stable, it is sensitive to oxidation and polymerization, the separation of unsaturated urushiol monomers are difficult, and study on structure modification of urushiol and its antitumor structure-activity relationship is lack at home and abroad, so the application of urushiol in antitumor drug development will be limited. The structure of unsaturated urushiol is similar to hydroxamic acid HDAC inhibitor which have been approved for use by FDA, but the key structural unit of zinc ions combined area is lacking in unsaturated urushiol. In this study the refined urushiol in raw lacquer will be used as raw material, we will prevent the oxidative polymerization of urushiol through the aldol condensation reaction. We will synthesize series of HDAC inhibitor analogs based on unsaturated urushiol, through introduction of key effect group of hydroxamic acid to the side chain tail of unsaturated urushiol by Diels-Alder, hydrolysis and hydroxyl ammonification reaction, and through introduction of pharmacophores (amides, nitrogen heterocyclic, halogen, fluoride group) of different resistance and electrical effects respectively to the benzene ring by substitution reaction. In addition, we will evaluate the inhibition ratio and IC50 value towards HDAC enzyme and tumor cell proliferation of unsaturated urushiol analogues with different structures by in vitro method, and their structure-activity relationship of HDAC inhibition will be clarified from molecular and cellular level. This project will provide well theoretical and practical foundation for the application in development of targeted anti-tumor drugs of unsaturated urushiol from raw lacquer.

生漆是我国重要的林产品，生漆中不饱和漆酚具有很好的抗肿瘤活性。但是漆酚结构不稳定，容易氧化聚合，漆酚单体分离困难，国内外缺乏漆酚结构修饰及抗肿瘤构效研究，限制了不饱和漆酚抗肿瘤药物的开发应用。不饱和漆酚结构与FDA批准使用的异羟肟酸类HDAC抑制剂结构相似，但是还缺少关键结构单元锌离子结合区。本研究以精制漆酚为原料，采用羟醛缩合反应阻断漆酚氧化聚合，分别通过Diels-Alder反应、水解和羟氨化反应，在不饱和漆酚侧链尾部引入HDAC抑制关键药效基团异羟肟酸，并通过取代反应在苯环上分别引入位阻和电性效应不同的药效基团（酰胺类、含氮杂环、卤素、含氟基团），合成系列不饱和漆酚基HDAC抑制剂类似物；采用体外法测定不同结构的不饱和漆酚类似物对HDAC酶与肿瘤细胞增殖的抑制率和IC50值，从分子和细胞水平阐明其HDAC抑制构效关系。为实现不饱和漆酚用于抗肿瘤靶向药物的研制提供良好的理论与应用基础。

生漆是我国特色经济林树种漆树的分泌物，生漆中主要成分漆酚具有很好的抗肿瘤活性，由于漆酚单体分离困难、漆酚结构不稳定、容易氧化聚合、具有致敏性，限制了不饱和漆酚作为抗肿瘤药物的开发与应用。HDAC是国内外公认的治疗癌症的重要靶点，HDAC 抑制剂发现及合成已成为国内外抗肿瘤靶向药物研究的热点。本项目以中国毛坝生漆为原料，采用丙酮提取漆酚，精制得到总漆酚，纯度为96.1%；采用Ag+配位树脂分离纯化总漆酚中三烯烷基漆酚单体，对硝酸银溶液浓度、上样量和浓度、洗脱剂、洗脱流速等条件进行优化，制备的三烯烷基漆酚单体纯度可达90.58%；通过羟醛缩合反应阻断了漆酚氧化聚合，制备得到亚甲基醚漆酚。合成了11种漆酚基异羟肟酸型HDAC 靶向抑制类似物，通过Diels-Alder、水解和缩合等反应，在三烯烷基漆酚侧链尾部引入异羟肟酸基团，通过Friedel-Crafts酰基化、Schiemann（席曼）、氧化、还原等反应在漆酚烷基链引入羟基或羰基，在苯环中引入F、Cl、氨基、磺胺基、三唑、苯甲酰胺基、羟基或硝基等功能基团，合成的目标化合物结构经1H NMR、13C NMR、 ESI-MS 、IR进行了确证。采用试剂盒检测了11种漆酚基衍生物对HDAC2和HDAC8的抑制活性，其中 7种化合物显示比阳性药SAHA更好的HDAC2/ HDAC8抑制活性。采用MTT法评价化合物对6种肿瘤细胞（Hela宫颈癌，A549肺癌，AGS胃癌，MCF-7乳腺癌，HepG2肝癌和BxPC3胰腺癌）的增殖抑制活性，9种化合物显示比阳性药SAHA更好的抗肿瘤活性。对化合物的抗肿瘤构效关系进行了评价，当苯环中引入Cl、F、硝基、羟基或甲磺酰胺基，烷基链引入羟基或羰基都可显著增加抗肿瘤广谱性、HDAC抑制和抗肿瘤生物活性。采用west-blotting和流式细胞术等手段检测化合物对肿瘤细胞中组蛋白乙酰化的表达及肿瘤细胞凋亡和周期的影响，阐明了漆酚衍生物抗肿瘤作用机制。本研究将为不饱和漆酚作为抗肿瘤靶向药物的开发提供可能，将拓展生漆在医药领域的用途，对提高我国漆树的经济开发价值具有重要意义。