靶向作用的一氧化氮供体型齐墩果酸衍生物的合成、抗肿瘤活性及其作用机理研究

Research and development of potent and safe antitumor drugs with significant cancer-targeting effect is a highly challenging task. Based on the fact that high levels of nitric oxide (NO) can inhibit tumor cell proliferation, induce tumor cell apoptosis, and prevent tumor metastasis, O2-chemical modification of NO donor diazeniumdiolate (NONOate) is firstly carried out by various strategies for targeting some transport proteins, receptors, and enzymes which are highly or over expressed in cancer cells. Given that oleanolic acid (OA), a liver protective drug with anticancer activity, is distributed and metabolized mainly in liver after administration, the modified NONOate is then conjugated with OA to selectively release high levels of NO in cancer cells, inducing apoptosis of human hepatocellular carcinoma (HCC) and other cancer cells while sparing the normal cells. The cancer-targeting effects of the designed compounds will be examined by measuring their in vitro and in vivo anticancer activity along with NO releasing levels, evaluating their selective effects on the corresponding targets, and analyzing the distribution of NO as well as its metabolites (including detection of 3-nitrotyrosine, a marker which indicates the interaction of NO with cancer cells) in both normal and tumor tissues. Furthermore, the mechanism underlying the anticancer activity of these derivatives will be studied at levels of molecule, cell, and tissue by techniques such as flow cytometry, western blot and gene amplification, respectively. It is expected that a promising antitumor candidate compound with novel mechanism, clear effect, low toxicity and strong targeting effect will be discovered by our effort for future preclinical research.

研究肿瘤靶向性的高效、低毒抗癌药物是一项极具挑战性的课题。本项目基于高浓度的一氧化氮（NO）具有抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡、阻止肿瘤转移等作用，利用肿瘤细胞高度或过度表达的酶、受体、转运蛋白作为靶标，采取多种策略对NO供体偶氮鎓二醇盐进行针对性的O2-化学修饰；同时结合保肝药物齐墩果酸（OA）具有抗肿瘤作用，且给药后主要在肝脏分布、代谢的特点，设计、合成相应的NO供体型OA衍生物，使其选择性地在肿瘤细胞内释放大量NO，诱导肝癌细胞等凋亡，而不伤害正常细胞；通过测定衍生物体内外抗肿瘤活性及NO释放水平，考察其对相应靶标的选择性作用，分析NO及其代谢物（包括NO与肿瘤作用的产物3-硝基酪氨酸）在正常及肿瘤组织中的分布，评价衍生物的肿瘤靶向作用；应用流式细胞术、免疫印迹、基因扩增等技术，分别从分子、细胞及组织水平研究衍生物的抗肿瘤机制，期望发现一种靶向性的抗肿瘤候选药物进入临床前研究。

鉴于目前大部分抗癌药给药后在肿瘤细胞中分布相对不多，毒副反应较大，治疗效果不理想，我们试图通过研发肿瘤靶向性的药物来解决这一难题。本项目基于高浓度的一氧化氮（NO）具有抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡、阻止肿瘤转移等作用，利用肿瘤细胞高度或过度表达的酶、受体、转运蛋白作为靶标，采取多种策略对NO供体偶氮鎓二醇盐进行针对性的O2-化学修饰；同时结合保肝药物齐墩果酸（OA）具有抗肿瘤作用，且给药后主要在肝脏分布、代谢的特点，设计、合成了五大类抗肿瘤（以肝癌为主）化合物：（I）基于GSTπ的NO供体型OA衍生物，（II）O2- (半乳糖等)偶氮鎓二醇盐修饰的OA衍生物，（III）O2-乙烯基偶氮鎓二醇盐修饰的OA衍生物，(IV) 齐墩果酸内酰胺类化合物，(V) 基于寡肽转运蛋白的OA衍生物。在上述基础上，对这些化合物的抗肿瘤及相关生物活性进行了较深入的研究，发现一些目标物可选择性地在肿瘤细胞内释放大量NO，诱导肿瘤细胞凋亡，而在正常细胞中仅产生少量的NO，发挥细胞保护作用。其中3个化合物的抗肿瘤活性确切、靶向作用强、毒副作用低，对它们的作用机制已初步从分子和细胞水平上加以阐明，为将其开发成为具有我国自主知识产权的创新药物打下了坚实的基础。通过项目的实施，培养了5名博士生和4名硕士生，发表了与本项目相关的15篇高质量科研论文，其中SCI论文7篇（含5篇IF > 3, 4篇IF > 5）。此外，还申请了3项中国发明专利，其中1项已授权。综上，本项目已圆满完成原先预定的各项指标和任务。

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