酸叶胶藤抗白血病活性化合物C21甾体结构修饰及其构效关系研究

Leukemia is a malignant tumor of the hematopoietic system，which harms mankind health and life. It is one of very important idea that an active compound which can kill some tumor cells, as a precursor from some plant, will be modified further, to increase bioactivity of these new compounds. We have separated some compounds which have antileukemia activity from a kind of plant called Ecdysanthera rosea，using the way following up biology activity . The compounds are named Ecdysantheroside B and 3β,14β,20 trihydroxy-18 oic (18→20) lactone pregnen-5, which are C21 pregnane derivative. Based on the result, we will develop a research project. We will synthetize some compounds using 3β,14β,20 trihydroxy-18 oic (18→20) lactone pregnen-5 as material, which is going to be modified at C-3, A ring and lactone loop. These compounds will be synthesized, which will be substituted at C-3 of 3β,14β,20 trihydroxy-18 oic (18→20) lactone pregnen-5 by sugar and amimo acid, and C-2 of it by cyanogen group. Lactone loop will also be opened up and substituded by piperazinedithiocarbamates derivatives. We also probe into these basic issue including catalyse system, harvest ratio and solid selectivity and the modify methods of compounds. Adopting MTT reduction assay, we value compounds about the activity of antileukemia against the human acute lymphoblastic T-cell leukaemia cells CCRF-CEM and their muhidrug resistance cells CEM-ADR5000, HL-60 and K-562. The advanced 3 D-QSAR (quantitative structure-activity reltionship) method CoMFA (comparative molecular field analysis) was used to study the QSAR relationship of compounds deriving from C21 pregnane with toxicity in vitro and led to availability CoMFA model. This CoMFA model reveal the 3D-relationship between bioactivities and structures of these compounds, and will be helpful to further design and synthetize new drugs for higher antileukemia activities. We would be able to succeed to develop a new antileukemia medicine we have our indepentence knowledge property right.

从植物中寻找有效的抗白血病恶性肿瘤药物先导化合物，再进行结构修饰，提高化合物活性是目前创新药物的重要思路之一。申请人从酸叶胶藤提取物中分离到有很强抗白血病活性新化合物Ecdysantheroside B及其皂元14β,20-二羟基-孕甾烷-5-烯-(18→20)内酯，在此基础上，本项目以14β,20-二羟基-孕甾烷-5-烯-(18→20)内酯为原料，拟对C-3、A环和内酯环进行结构修饰，得到一系列C21甾体糖基化、氨基酸衍生物、2-氰基取代、氨基二硫代甲酸酯衍生物，对反应的催化体系、收率、立体选择性等基本问题深入的研究，建立该类活性化合物结构修饰方法。采用MTT方法检测该类化合物对白血病细胞CCRF-CEM及其多药耐药细胞CEM/ADR5000、HL-60和K-562的抑制作用，进行抗白血病活性评价。用CoMFA建立3D-QSAR模型，为研制我国具有自主知识产权的抗白血病新药提供科学依据。

本项目是开展从植物中寻找有效的抗白血病恶性肿瘤药物先导化合物，再进行结构修饰，发现出更高活性化合物。通过对C21甾体化合物进行化学合成的修饰，以及通过筛选的微生物菌种对C21甾体化合物进行生物转化和从菌体提取物中分离，得到一系列化合物，通过MTT法，选用Neuro-2a细胞（小鼠神经瘤细胞）作为供试细胞，对所合成化合物和生物转化的化合物进行体外抗肿瘤活性筛选，确定活性化合物，以细胞分裂周期蛋白25B（CDC25B）和含SH2结构域蛋白酪氨酸磷酸酶2（SHP2）为靶标，进行抑制活性实验。共合成和分离了23个化合物，其中活性化合物7个，新化合物6个。发现20 -羟基- 16α，17α环氧孕甾-1,4-二烯-3-酮和灵菌红素有体外抗肿瘤活性，并能抑制细胞分裂周期蛋白25B（CDC25B）和含SH2结构域蛋白酪氨酸磷酸酶2（SHP2）的活性。文献报道，7-酮基胆固醇（7-KC）具有抑制肿瘤细胞生长活性，7β-羟基胆固醇（7-β-hydroxycholesterol）对反应性星形胶质细胞增生有明显抑制作用，并且可以防止脑损伤后反应性胶质增生和帮助脑功能恢复，这些成果为进一步研究开发抗肿瘤药物提供依据。通过抑菌实验，发现缩菌肽A和灵菌红素对对白念珠菌和苏云金杆菌有较强的抑制活性，并对植物病原菌如西瓜枯萎病菌、棉花黄萎病菌、葡萄座腔病菌和棉花枯萎病菌有有较强的抑制活性，1, 2, 10-三羟基菲在浓度为100μg/ml金黄色葡萄球菌、大肠杆菌和白念珠菌有较强的抑制作用，从土壤筛选得到能够转化的菌株，通过生理生化检测、形态特征分析，16S rRNA序列或18S rRNA序列分析，确定为洋葱伯克霍尔德氏菌SE-1，粘质沙雷氏菌S823，枝孢霉IS547和假单胞菌DH（Pseudomonas sp）。通过DEAE-52离子交换层析、Sephadex G-200凝胶层析及非变性凝胶电泳等方法分离提纯了菌株SE-1产生的7-羟化酶，其比活为：37.22 U/mg，采用SDS-聚丙烯酰胺凝胶电泳检测得该酶的分子质量约为80 kDa，通过MS-MS技术，测定了部分蛋白质序列，这些成果为研究开发抗菌药物和病害防治提供科学依据。