降血糖活性成分J306衍生物合成、构效关系及作用机制研究

Diabetes mellitus was a chronic metabolic disease characterized by hyperglycemia. The incidence of diabetes increased year by year, becoming one of the major risks to human health. Traditional anti-diabetics frequently led to various side effects, such as hypoglycemia, gastrointestinal tract reaction. Traditional Chinese medicines play an important role in the treatment of type 2 diabetes. Cyclocarya paliurus is an endemic species growing in China. Due to its function on the treatment of anti-diabetes and anti-hypertention, C. paliurus leaves have been used to make health products for a long time. In our previous study, bioactivity-guided fractionation on the active part of C. paliurus led to the isolation and identification of seventeen compounds. Seven compounds noticeably inhibited the activity of α-glucosidase with IC50 values ranged from 0.53-1.87 mg/mL. Particularly, oral administration of J306 to C57BL/6 and BKS-DB mice to significantly decreased the serum glucose level. In this project, J306 derivatives were designed and synthesized to obtain 80-100 compounds with strong activity against the α-glucosidase and to study structure-activity relationship. Then, to further evaluated the most potent compounds in diabetic BKS-DB mice and identify their major metabolites in liver microsomes and in rats by LC/MS-IT-TOF. Western blot analyses and Q-PCR technique will be used to study their potential mechanism. At the end, we hope to discover 1-2 compounds promising to be developed into a drug candidate in the treatment of type 2 diabetes.

糖尿病是一种持续高血糖慢性代谢性疾病，发病率逐年增高，成为危害人类健康的重大疾病，现有糖尿病药物易产生低血糖和胃肠道反应等副作用，中药在治疗糖尿病具有重要作用。青钱柳是我国特有的单种属植物，因其叶具有降血糖，降血压作用而用于保健品。我们前期从青钱柳中分离得到17个化合物，其中7个化合物对α-葡萄糖苷酶有较好的抑制作用，其IC50值范围为0.53-1.87 mg/mL。通过对J306进行体内活性评价，发现其对Ⅱ型糖尿病模型BKS-db小鼠的餐后血糖具有明显的降糖作用，对正常小鼠的餐后血糖也有调节作用。本研究拟在此基础上，对J306进行结构修饰合成80-100个衍生物并研究其构效关系，对高活性化合物进行BKS-db小鼠和正常小鼠模型体内药效评价，药代动力学研究和初步安全性评价，并利用Western blot分析和Q-PCR技术研究其作用机制，为发现治疗2型糖尿病的候选化合物提供化学和药学依据。

青钱柳叶具有降压、降血糖和清热解毒的功效，以青钱柳叶为主要原料的保健品已经上市。我们研究组发现来自青钱柳的达玛烷型三萜J306在200µM时，对α-葡萄糖苷酶具有一定的抑制活性，抑制率为32.2%，且能够降低正常小鼠和BKS-db小鼠的餐后血糖。为了揭示其结构-活性关系，得到高活性化合物，项目以J306为先导，对其主要功能团C-3和C-12位羟基、A和E环进行修饰，合成了51个衍生物，测试了这些化合物对α-葡萄糖苷酶和PTP1B的抑制活性。发现2个化合物 (7和36) 对α-葡萄糖苷酶具有较好的抑制活性，IC50分别为489.8和497.7 µM，比J306提高了约2倍 (J306 IC50˃ 800 µM)；3个化合物 (7、23和36) 对PTP1B的抑制活性和阳性相当，IC50分别为319.7、341.7和269.1 µM (J306 IC50˃ 800 µM；苏拉明钠 IC50 339.0 µM)，而化合物46对PTP1B的抑制活性比阳性强2.5倍。化合物7和36对α-葡萄糖苷酶和PTP1B均具有抑制作用，是一个双靶点抑制剂。酶动力学研究表明，化合物36是α-葡萄糖苷酶的混合型抑制剂，是PTP1B的非竞争性抑制剂， Ki值分别为414.4 和 110.7 μM。化合物46为PTP1B混合型抑制剂，Ki值为139.2 μM。初步构效关系是：C-3位连接含羧基酰基时对两种酶都具有较好的抑制活性，C-12位连接含羧基的酰基时对PTP1B具有较好的抑制活性，A环需要保持，E环开环后羟基脱水所得的衍生物46对PTP1B具有好的活性。研究丰富了PTP1B和α-葡萄糖苷酶抑制剂的结构类型，为双靶点抑制剂的发现提供了参考。本项目发表相关论文1篇（第一标注），申请发明专利1项。培养出站博士后1名，培养博士研究生3名，硕士研究生3名。