【作者】 王博；

【导师】 程卯生；

【作者基本信息】 沈阳药科大学 ， 药物化学， 2012， 博士

【摘要】 甾体皂苷以其良好的生物活性,尤其是抗肿瘤活性获得了广泛的关注。由于皂苷在植物中的含量较低,并且分离纯化过程相对复杂,因此近年来以廉价易得的皂苷元为起始原料,通过对皂苷分子中的糖链和(或)皂苷元的结构改造,定向的半合成了大量的皂苷衍生物,同时也得到了若干具有良好抗肿瘤应用前景的候选化合物。本文首先对甾体皂苷进行了概述,根据皂苷元的结构不同可以分成螺甾皂苷、呋甾皂苷和胆甾皂苷三种,并指出了他们存在化学上实现简单相互转化的可能；随后又综述了薯蓣皂素这一在自然界中分布最为广泛的螺甾皂苷元的抗肿瘤作用及其衍生物的研究进展。由于甾体皂苷的抗肿瘤活性是皂苷元和糖链两个部分协同作用的结果,因此本文的第一部分工作是以薯蓣皂素为原料,探讨简便的制备天然活性呋甾皂苷的方法,为甾体皂苷类化合物从苷元上进行结构改造提供更加简便的途径；第二部分工作是对薯蓣皂素进行糖苷化反应,获得一系列非天然的新型螺甾皂苷化合物,以探讨糖链对螺甾皂苷的抗肿瘤活性的影响；第三部分工作是对薯蓣皂素氨基葡萄糖苷分子中的氨基进行抗肿瘤活性基团的修饰,以探讨糖链上的结构修饰对螺甾皂苷的抗肿瘤活性的影响。针对两种重要的天然抗肿瘤呋甾皂苷methyl protodioscin和icogenin的合成过程繁琐、不利于衍生物制备的缺点,本文发展了用三氟化硼-乙醚/醋酐高效打开螺甾烷E-环的方法,并以薯蓣皂素为起始原料,通过该E-环开环反应联合脱保护后的E-环自动合环的方法构建了呋甾皂苷的F-环开链结构,以7步反应、24%总收率优化制备得到了天然产物methyl protodioscin;并在反应过程中发现通过控制水解条件,该E-环开环反应可用于高效的合成胆甾烷、22-OH和22-OMe呋甾烷。在后续的实验中,又发展了用三氟化硼-乙醚/三氟乙酸酐/冰醋酸高效打开螺甾烷F-环的方法。通过该F-环开环反应,以薯蓣皂素为起始原料,以3步反应简便的合成了methyl protodioscin和icogenin的苷元——甲基原薯蓣皂素；在验证了该方法对糖苷键的兼容性后,应用该方法成功实现了对天然呋甾皂苷icogenin的高效合成。针对糖链结构的变化对薯蓣皂素糖苷分子抗肿瘤活性影响的研究,本文将一种常见于具有抗肿瘤作用的三萜皂苷中的二糖模块,即a-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl模块,引入到薯蓣皂素的3-位羟基,并将6种不同的糖分子分别引入该二糖模块中的阿拉伯糖4-位羟基或鼠李糖4-位羟基,得到了6个线状糖链衍生物和6个分支状糖链衍生物。这12个化合物的体外细胞毒活性筛选结果表明,化合物65对A549细胞具有较强的抑制活性。继而,通过细胞形态学观察、特异性染色、DNA片段化、流式细胞术以及蛋白免疫印迹法等方法,具体证明了该化合物是通过死亡受体途径诱导A549细胞发生凋亡从而抑制A549细胞的活力。对于已知皂苷结构中的糖链用其他基团进行修饰也是一种常见的皂苷衍生化方法。根据文献调研发现,薯蓣皂素氨基葡萄糖苷分子中的氨基取代基会对该化合物的抗肿瘤作用产生重要影响,据此本论文设计并合成了氨基取代基为肉桂酰基、脲和缩氨基硫脲的三个系列、共36个新型薯蓣皂素氨基葡萄糖苷衍生物。结合它们对多种肿瘤细胞的体外细胞毒活性测试结果,总结了该类化合物的构效关系,发现当取代基为肉桂酰基时其活性明显优于取代基为脲和缩氨基硫脲的活性；肉桂酰基衍生物中苯环上取代基的取代位置、数量和电性都能显著影响该化合物的活性和选择性。初步研究了其中一个活性较好的化合物91c对人外周血单核细胞的毒性和抑制MCF-7细胞活力的作用机制。研究结果表明,化合物91c对人外周血单核细胞具有一定毒性；而其抑制MCF-7细胞活力的初步机制是诱导MCF-7细胞发生凋亡,而不是程序性坏死或自噬。更多还原

【Abstract】 Steroidal saponins draw much attention due to their diverse biological activities, especially their excellent anti-tumor activity. Due to the low content in plants and the complexity in isolation and purification, abundant saponin derivatives were semi-synthesized via modifications on the sugar chain and/or the aglycon of saponin molecular starting from commercially available aglycon, meanwhile several candidates bearing good anti-tumor property were obtained.First, we summarized that steroidal saponin can be subdivided into spirostanol saponin, furostanol saponin and cholesteric saponin, and the possibility of a concise chemical conversion was figured out. Then derivative preparation and anti-tumor activity of diosgenin, which is the most widespreading spirostanol aglycon in nature, were reviewed generally. The anti-tumor activity of steroidal saponin resulted from the collaboration of its aglycon and sugar moiety, therefore this paper should be divided into three parts. In the first part, the simple preparation of natural furostanol saponin from diosgenin was developed, which provided an easier method to modify the aglycon of steroidal saponin. The second part was related to the glycosylation of diosgenin, yielding a series of unnatural steroidal saponin, to discuss the influence of sugar moiety on its parent’s anti-tumor activity. In the third part, several bioactive groups were introduced into the amino group of diosgenyl glucosaminide, which showed that modification on the sugar moiety could affect the anti-tumor activity of steroidal saponin.The total synthesis of the two important furostanol saponin methyl protodioscin and icogenin was firstly reviewed, and their inefficiency was figured out. Therefore, we have developed a highly efficient E-ring opening reaction of spirostanol saponin by BF3-Et2O/Ac2O. Starting from diosgenin and applying this E-ring opening reaction, methyl protodioscin was synthesized in seven steps with a total yield of24%. During the synthesis of methyl protodioscin, we found that this E-ring opening reaction could be applied for the efficient synthesis of cholestane,22-OH furostan and22-OMe furostan. Consequently, we have also developed a direct F-ring opening reaction of spirostanol saponin by BF3-Et2O/TFA-HOAc. Applying this F-ring opening reaction, methyl protodiosgenin, which is the mutual aglycon of methyl protodioscin and icogenin, was prepared in three steps starting from diosgenin. After stability of glycosidic bond under the F-ring opening reaction condition was confirmed, icogenin was synthesized concisely starting from diosgenin by this F-ring opening reaction.A common disaccharide moiety from cytotoxic triterpenoidal saponin, namely a-L-rhamnopyranosyl-(1→2)-a-L-arabinopyranosyl was introduced to the3-OH of diosgenin, and glycosylation of the4-OH in both arabinose residue and rhamnose residue using six sugar donors provided six derivatives with branched sugar chain and six derivatives with linear sugar chain. In the in vitro cytotoxicity screening, compound65was found to have a strong inhibition on A549cells, and this compound was demonstrated to induce apoptosis in A549cells via extrinsic pathway through cell morphology observation, cell staining, DNA fragmentation assay, flow cytometric analysis and western blot.It is a common derivation of saponin by modifying its sugar moiety with bioactive groups. The substituents on the amino groups of diosgenyl glucosaminides could affect their anti-tumor effect significantly according to the previou research. According to this conclusion, three series of diosgenyl glucosaminides bearing cinnamoyl, urea and thiosemicarbazone groups on their amino groups were designed and synthesized, giving thirty-four saponin derivatives. The structure-activity relationships were summarized after the in vitro cytotoxicity screening, and we found that cinnamoyl groups were preferred comparing to the urea and thiosemicarbazone group, meanwhile the amount, position and style of substituent on the benzene ring in the cinnamoyl group could affect the anti-tumor activity and selectivity of its parent significantly. The toxicity on PBMC and mechanism inhibiting MCF-7cells of compound91c were studied. Compound91c show toxicity on PBMC, and the action mechanism research of compound91indicated that this compound induces apoptosis in MCF-7cells, but autophagy and necroptosis did not occur in the treated MCF-7cells.更多还原