新型稠杂氮杂糖类糖苷酶抑制剂的设计、合成及活性研究

Glycosidases are the specific glycosidic bond hydrolases directly involved in the glycosylation on cell surface, and are responsible for the glycation of the proteins or lipids with important biological activities. The occurrence and development of many major diseases, such as diabetes, cancers, viral infections, autoimmune diseases, are closely related to the abnormal expression of the glycosidases. Therefore, the study of glycosidase inhibitor has become one of the hot topics in drug development. Based on the features of the transition state in glycosidase reaction, and based on the detail structure-activity relationship analysis of the azasugars as glycosidase inhibitors, in this project, we first offer a new strategy, that is, “Stabilizing the substrate intermediate at transition state” for the design of glycosidase inhibitors with high efficiency. According to the new strategy, and on the basis of the computer-aided molecular docking, we plan to design and synthesize a series of novel azasugar fused heterocycle as glycosidases inhibitors. The designed azasugars are bearing with half chair conformation which is easily recognized by glycosidase, and with the structural features which can delocalize the protonated nitrogen cation, stabilize the intermediate, and prolong the interaction time with enzyme. Such fused heterocycles have multimode synergetic interactions with the glycosidases, such as hydrogen bonding, hydrophobic and π-π interactions, which will result the improvement of the specific recognition and binding affinity between the molecule and the enzyme. As a result, the compounds would be expected to be of higher glycosidase inhibitory activity. These new compounds will also be evaluated for their anti-glycosidase activity in vitro to develop the efficient and nontoxic glycosidase inhibitors drug. The relationships between the structure and the anti-glycosidase activity will be studied. This research has important scientific and practical significance in discovering novel carbohydrate-based drug. In addition, the project will provide novel lead compounds with high glycosidase inhibitory activity, enrich chemical glycobiology and the diversity of azasugars, and offer a new idea for the design of enzyme inhibitor.

糖苷酶作为特异性苷键水解酶，直接参与细胞表面及重要生物活性蛋白、类脂的糖基化过程，许多重大疾病的发生发展，如糖尿病、肿瘤、病毒性感染、免疫性疾病等，与糖苷酶的非正常表达密切相关。因此，糖苷酶抑制剂的研究成为新药研发的热门领域之一。本课题针对糖苷酶反应过渡态，结合已有氮杂糖类糖苷酶抑制剂构效关系分析，提出基于稳定过渡态底物中间体的高效酶抑制剂设计思路。以此为依据，利用计算机模拟分子对接，拟设计合成结构新颖的稠杂氮杂糖类糖苷酶抑制剂，使其具有易于糖苷酶识别的半椅式平面、以及质子化氮正中间体离域稳定、与酶作用时间延长的结构特点。通过与酶活性中心域氢键、疏水、π-π等多方式协同作用，提高分子对酶的选择性识别和结合，发现高活性糖苷酶抑制剂，建立构效关系。为进一步研发新型高效低毒的糖类药物提供先导化合物，丰富糖化学生物学研究内容及氮杂糖的结构多样性，并为高效酶抑制剂的设计提供新的思路和重要理论依据。

糖苷酶作为特异性苷键水解酶，直接参与细胞表面及重要生物活性蛋白、类脂的糖基化过程，许多重大疾病的发生发展，如糖尿病、肿瘤、病毒性感染、免疫性疾病等，与糖苷酶的非正常表达密切相关。因此，糖苷酶抑制剂的研究成为新药研发的热门领域之一。本课题针对糖苷酶反应过渡态，结合已有氮杂糖类糖苷酶抑制剂构效关系分析，通过增强化合物与酶的识别与结合，设计合成了系列稠杂氮杂糖类糖苷酶抑制剂。本课题以天然五、六碳单糖为起始原料，经全苄基保护的糖醛基中间体，碘催化下分别与邻氨基苯甲酰胺、邻苯二胺缩合，制备了含糖基侧链的喹唑啉酮和苯并咪唑衍生物。以不保护的糖为原料，碘催化下亦可一步反应高效制备相应的糖基喹唑啉酮衍生物。继而利用Mitsunobu反应分子内关环、脱苄基二步反应，合成了系列新型喹唑啉酮并氮杂糖和苯并咪唑并氮杂糖两类稠杂三环化合物。对苯并咪唑并氮杂糖进行结构修饰，合成了糖环上含氨基或烷氧链的衍生物；以邻萘二胺为原料得到了四环稠杂氮杂糖；利用Suzuki偶联反应制备了联苯基苯并咪唑并氮杂糖。所有化合物结构及构型经NMR、HR-MS及X-Ray等波谱分析确证。测试了所合成稠杂氮杂糖的糖苷酶抑制活性。结果表明D-核糖来源的苯并咪唑并氮杂糖具优异的β-葡萄糖糖苷酶抑制活性（IC50 < 1 μM），是一类结构新颖的稠杂氮杂糖类糖苷酶抑制剂。构效分析表明糖环上羟基均处于beta-构型有利于化合物的酶抑制活性，而在苯并咪唑苯环4、5位有给电子基团，如甲基、甲氧基和苯基等，将显著提高化合物的beta-葡萄糖糖苷酶抑制活性。动力学分析揭示联苯基苯并咪唑并氮杂糖主要为竞争型抑制剂，而含甲氧基的苯并咪唑并氮杂糖为混合型抑制剂。此外，合成的新型“类蝴蝶”型噻嗪烷酮并四环、五环氮杂糖稠杂衍生物具有良好的HIV逆转录酶抑制活性。本项目研究为进一步设计合成高效稠杂氮杂糖类药物先导奠定了基础，丰富了糖化学药物的研究内容并拓展了氮杂糖的结构和生物活性的多样性。