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题名: 泽泻醇A衍生物的合成与抗乙肝病毒活性研究
作者: 张泉
学位类别: 博士
答辩日期: 2009-05-19
授予单位: 中国科学院昆明植物研究所
授予地点: 昆明植物研究所
导师: 陈纪军
关键词: 抗乙型肝炎病毒活性 ; 原萜烷型三萜 ; 泽泻醇A ; 结构修饰 ; 构效关系
学位专业: 植物学
中文摘要: 本论文以抗乙型肝炎病毒(HBV)活性化合物泽泻醇A结构上的3-羰基(还原、成肟等)、11,23,24,25-羟基(酰化、脱水等)、13(17)-双键(环氧化)为结构修饰点,对其进行了较系统的结构修饰,合成了154个衍生物,测定了它们体外对HepG 2.2.15细胞分泌乙型肝炎病毒表面抗原(HBsAg)和e抗原(HBeAg)表达的抑制作用,以及对细胞的毒性,发现高效低毒的活性化合物41个,讨论和分析了其初步构效关系,为其深入研究和开发成抗乙型肝炎病毒药物提供了化学物质和药效学基础。 第一章 抗乙肝病毒活性化合物泽泻醇A的初步构效关系研究 本课题组从中药泽泻中首次发现原萜烷型四环三萜类化合物具有抗乙肝病毒活性,选择其中含量较大、可修饰点较多的泽泻醇A(1)为先导化合物,利用结构上的官能团3-羰基、11,23,24,25-羟基、13(17)-双键,对其进行了初步试探性修饰,合成了25个衍生物,对其体外抗乙肝病毒活性和细胞毒性进行了测定,发现活性化合物5个,其中化合物3a和6a对HBsAg和HBeAg的分泌表现出较强的抑制活性,其初步构效关系为: (1)11,23,24-位的羟基酯化能降低化合物的细胞毒性,但同时降低了对HBsAg分泌的抑制活性; (2)25(26)-位双键能显著提高化合物对HBsAg和HBeAg的抑制作用; (3)25(26)-位为双键时,空间位阻较小的11,23,24-位羟基的酰化基团对其抗HBV活性有利; (4)13(17)-位双键的环氧化能显著降低化合物的细胞毒性,且能保持衍生物对HBsAg和HBeAg的抑制作用; (5)3-位羰基修饰的衍生物7–10与先导化合物的活性相当,但其细胞毒性也较大,导致选择性指数均小于1.0,羰基对活性的影响还有待于进一步研究。初步构效关系为泽泻醇A的进一步修饰提供指导。 第二章 基于初步构效关系对泽泻醇A的进一步结构修饰 基于第一章中揭示的初步构效关系,11,23,24-位羟基酰化降低细胞毒性,25-位羟基脱水提高对HBsAg和HBeAg的抑制作用,13(17)-位双键环氧化降低毒性,所以本章着重对泽泻醇A结构中的11,23,24,25-位羟基和13(17)-位双键进行了修饰,合成42个泽泻醇A衍生物,并测定其抗HBV活性,发现17个活性化合物,其中化合物32对HBsAg和HBeAg的分泌表现出较强的抑制活性,并进一步讨论了它们的构效关系: (1)11,23,24-位的羟基酯化能降低化合物的细胞毒性,大部分衍生物同时降低了对HBsAg分泌的抑制活性,当酰化基团为丁酰基时(化合物11)对HBsAg(IC50 = 0.0074 mM,SI = 36)和HBeAg(IC50 = 0.0036 mM,SI = 75)均表现出较好的抑制活性; (2)11,23,24-位具有适当的酯链取代基时,25-位的羟基酰化可提高衍生物对HBsAg和HBeAg分泌的抑制作用; (3)25(26)位为双键时,空间位阻较小的11,23,24-位羟基的酰化基团对活性有利;然而空间位阻并不是唯一的影响因素,如位阻较大的烷氧乙酰基(或乙酰乙酰基)取代的衍生物40–42对HBsAg和HBeAg仍表现出较好的抑制作用; (4)13(17)位双键的环氧化能显著降低化合物的细胞毒性,不同底物对活性的影响不一致:a. 保持对HBsAg和HBeAg分泌抑制活性。化合物6a对HBsAg和HBeAg的抑制活性与化合物3a的相当(HBsAg:IC50 = 0.024 mM vs IC50 = 0.028 mM;HBeAg:IC50 = 0.028 mM vs IC50 = 0.029 mM)。b. 只保持对HBsAg的分泌抑制活性,而使失去对HBeAg的分泌抑制活性,如化合物50–52只对HBsAg的分泌有抑制活性,而对HBeAg的分泌没有表现出明显的抑制作用。c. 对于四酰化衍生物,同时降低对HBsAg和HBeAg分泌的抑制活性。化合物45(HBsAg:IC50 = 0.11 mM,SI > 7.3;HBeAg:IC50 > 1.6 mM)、47(HBsAg:IC50 = 0.25 mM,SI > 5.6;HBeAg:IC50 = 0.32 mM,SI > 4.4)对HBsAg和HBeAg的活性分别明显低于化合物32(HBsAg:IC50 = 0.028 mM,SI > 90;HBeAg:IC50 = 0.027 mM,SI > 93)、34(HBsAg:IC50 = 0.20 mM,SI > 11;HBeAg:IC50 = 0.26 mM,SI > 8.7)。 第三章 四酰化泽泻醇A的合成与抗乙肝病毒活性研究 第二章中揭示的构效关系,25-位羟基酰化提高衍生物对HBsAg和HBeAg分泌的抑制作用,所以本章着重设计合成了33个四酰化泽泻醇A衍生物,并测定其抗HBV活性,发现10个化合物具有抗乙肝病毒活性,其中3个高效低毒的衍生物A1、A23、A24,它们的初步构效关系为: (1)25-位羟基的酰化基团为乙酰基或烷氧乙酰时,能有效提高化合物的抗HBV活性。如化合物A1, A23和A24对HBsAg的IC50分别为0.0048 mM, 0.0044 mM, 0.014 mM,对HBeAg的IC50分别为0.011 mM, 0.012 mM, 0.018 mM,它们的细胞毒性均较低,故其选择性指数SI较大,分别为:SIHBsAg > 333, SIHBeAg > 145; SIHBsAg = 209, SIHBeAg = 77; SIHBsAg > 200, SIHBeAg > 156; (2)对于25-位羟基乙酰化的衍生物,11,23,24-位羟基的酰化基团较小时对其抗HBV活性有利; (3)A环对四乙酰泽泻醇A(A1)的抗HBV活性或许比较重要。A环修饰的化合物D(IC50 = 0.28 mM, SI > 7.5)对HBeAg的活性较化合物A1有明显降低,而且失去了对HBsAg的活性; (4)对于四酰化的衍生物,13(17)位双键环氧化同时降低对HBsAg和HBeAg的分泌抑制活性。化合物C1对HBsAg (IC50 = 0.046 mM, SI > 87) 和HBeAg (IC50 = 0.061 mM, SI > 66)的活性较四乙酰泽泻醇A(A1)有显著降低。 第四章 四乙酰泽泻醇A的构效关系研究 前章中发现化合物四乙酰化泽泻醇A(A1)对HBsAg和HBeAg的分泌表现出较强的抑制活性,本章较系统地研究了其构效关系,对化合物A1的11,23,24位、A环、D环进行了修饰,共合成化合物54个,并测定了其中36个化合物的抗乙肝病毒活性,发现活性化合物9个,其构效关系如下:(1)四乙酰泽泻醇A(A1)结构中的3-羰基对其抗HBV活性比较重要;(2)23,24-位酰化或许并不是活性所必需,当用丙酮叉替换时,能很好的保持化合物对HBeAg的活性,但使其对HBsAg的活性有一定程度的降低。 第五章 抗乙肝病毒活性化合物的研究进展 本章对具有抗乙肝病毒活性的核苷类化合物、小分子化合物、天然来源的化合物共计164个化合物进行了文献综述,根据其结构特点对它们的抗HBV活性进行了分类讨论。
英文摘要: This thesis described the chemical modification of alisol A on C-3-carbonyl, C-11, 23, 24, 25-hydroxyls, C-13(17)-carbon-carbon double bond and 154 derivatives were synthesized. Inhibitory activity to the secretion of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) of the derivatives using HepG 2.2.15 cell line and cytotoxicity to cells were evaluated in vitro, 41 high-efficiency, low-toxicity and active anti-hepatitis B virus (HBV) compounds were found and structure-activity relationships (SARs) were also discussed, which lay the basis of chemical substances and pharmaco-dynamics for its further study and developing into anti-HBV drugs. Chapter 1 Studies on preliminary structure-activity relationships of alisol A Our previous study revealed that protostane triterpenes possessed anti-HBV activity in vitro for the first time. Alisol A, a protostane-type triterpene, comprises 0.05–0.1 percent of rhizomes of Alisma orientalis Juzep. and carries C-3-carbonyl, four hydroxyl groups and C-13(17)-carbon-carbon double bond providing possibilities for more diverse library, was selected as lead compound. Twenty-five derivatives were synthsize to investigate their biological activities as potential anti-HBV agents. Five compounds were found to be active to HBV. Among them, compound 3a and 6a showed high activity against both HBsAg (0.028 mM, SI > 18; 0.024 mM, SI > 108, respectively) and HBeAg (0.029 mM, SI > 18; 0.028 mM, SI > 93, respectively) secretion. Preliminary structure-activity relationships were concluded as: (1) the acylation of hydroxyls at 11, 23, 24-position decreased cytotoxicity. However, inhibitory activity to the secretion of HBsAg also decreased; (2) 25(26)-carbon-carbon double bond enhanced the inhibitory activity against the secretin of HBsAg and HBeAg; (3) small acyl agents were appropriate for 25(26)-carbon-carbon double bond derivatives; (4) epoxide at 13(17)-position decreased the cytotoxicity and kept the suppressant properties on the secretion of HBsAg and HBeAg; (5) activity of derivatives 7–10 modified at 3-carbonyl was similar to that of alisol A, but they showed high cytotoxicity, leading to SI < 1.0. The effect of 3-carbonyl to anti-HBV activity needs further investigation. The preliminary structure-activity relationships provide guidance for further modifications of alisol A. Chapter 2 Further modifications of alisol A based on the preliminary structure-activity relationships Based on the preliminary structure-activity relationships, in this chapter, chemical modifications on 11,23,24,25-hydroxyls and 13(17)-carbon-carbon double bond of alisol A were carried out and forty-two alisol A derivatives were synthesized for evaluation of their anti-HBV activity. Seventeen compounds were found to be active to HBV. Among them, compound 32 showed high activities to the secretion of HBsAg (IC50 = 0.028 mM, SI > 90) and HBeAg (IC50 = 0.027 mM, SI > 93). Based on the results, the following conclusions could be made: (1) the acylation of hydroxyls at 11, 23, 24-position decreased cytotoxicity. However, inhibitory efficiency to the secretion of HBsAg and HBeAg also decreased for most of the derivatives except that compound 11 exhibited high inhibitory potency to the secretion of HBsAg (IC50 = 0.0074 mM, SI = 36) and HBeAg (IC50 = 0.0036 mM,SI = 75); (2) acylation of the hydroxyl at C-25 of alisol A increased anti-HBV activity for the derivatives linking appropriate groups at C-11,23,24; (3) for 25(26)-carbon-carbon double bond derivatives, small acyl agents at 11,23,24-positions were appropriate for anti-HBV activity. However, the size of acyl agents maybe not the only one factor, because the alkyloxyacetyl (or acetoxyacetyl) derivatives (40–42) also exhibited high potent anti-HBV activity. (4) epoxide group at C-13(17) decreased cytotoxicity and the role of epoxide functionality to anti-HBV activity depended on the substrate: a. In some analogues, epoxide group retained the inhibitory potency to the secretion of HBsAg and HBeAg: the activity of compound 6a was similar to that of compound 3a. (HBsAg: IC50 = 0.024 mM vs IC50 = 0.028 mM; HBeAg: IC50 = 0.028 mM vs IC50 = 0.029 mM). b. For other analogues, epoxide functionality retained the inhibitory potency to the secretion of HBsAg, but caused the loss of suppressant property on the secretion of HBeAg: compounds 50–52 showed high activity against the secretion of HBsAg, but could not inhibit the secretion of HBeAg. c. Finally, for tetra-acyl alisol A derivatives, epoxide group led to the decrease of suppressant property on the secretion of HBsAg and HBeAg: compounds 45 (HBsAg: IC50 = 0.11 mM, SI > 7.3; HBeAg: IC50 > 1.6 mM) and 47 (HBsAg: IC50 = 0.25 mM, SI > 5.6; HBeAg: IC50 = 0.32 mM, SI > 4.4) were less potent than compounds 32 (HBsAg: IC50 = 0.028 mM, SI > 90; HBeAg: IC50 = 0.027 mM, SI > 93) and 34 (HBsAg: IC50 = 0.20 mM, SI > 11; HBeAg: IC50 = 0.26 mM, SI > 8.7), respectively. Chapter 3 Studies on synthesis and anti-HBV activity of tetra-acylyl alisol A In previous chapter, SARs study revealed that acylation of the hydroxyl at C-25 of alisol A increased anti-HBV activity. Thirty-three tetra-acylyl alisol A were designed and synthesized for evaluation of their anti-HBV activity in this chapter. Ten derivatives were revealed to be active anti-HBV compounds. Of them, three compounds exhibited high activity against HBV. These results provided the following structure–activity relationships: (1) acetyloxyl, methoxyacetyloxyl and ethoxyacetyloxyl groups at C-25 of alisol A derivatives enhanced potency. Compounds A1, A23, A24 showed IC50 values of 0.0048 mM, 0.0044 mM, 0.014 mM, respectively, against HBsAg, and 0.011 mM, 0.012 mM, 0.018 mM against HBeAg with low cytotoxicity led high SI values of SIHBsAg > 333, SIHBeAg >145; SIHBsAg = 209, SIHBeAg = 77; SIHBsAg > 200, SIHBeAg > 156, respectively; (2) for C-25 acetyl alisol A derivatives, small acyloxy groups at C-11,23,24 may be preferred for anti-HBV activity; (3) A-ring of tetra-acetyl alisol A (A1) might be essential for potent anit-HBV activity. A-ring modified derivative D (IC50 = 0.28 mM, SI > 7.5) showed lower activity inhibiting HBeAg than that of compound A1 (IC50 = 0.011 mM, SI > 145) and lost suppressant activity on HBsAg; (4) for tetra-acyl alisol A derivatives, epoxide functionality at C-13(17) led to the decrease of suppressant property on the secretion of HBsAg and HBeAg. Compound C1 exhibited lower activtity inhibiting the secretion of HBsAg (IC50 = 0.046 mM, SI > 87) and HBeAg (IC50 = 0.061 mM, SI > 66) than that of compound A1. Chapter 4 Studies on structure-activity relationships of tetra-acetyl alisol A Tetra-acetyl alisol A was modified on 11,23,24-positions, A-ring and D-ring for its structure-activity relationships. Thirty-six compounds of the synthesized 54 compounds were evaluated for their anti-HBV activity. Of them, nine compounds showed high activity against HBV. Structure-activity relationships were also revealed as follows: (1) 3-carbonyl of tetra-acetyl alisol A was essential for its high anti-HBV activity; (2) acetylation of C-23,24-hydroxyls might not be necessary for its suppressant activity on HBsAg and HBeAg secretions. Substitution of acetyl at 23,24-positions with isopropylidenyl retained the inhibitory potency to the secretion of HBsAg, but decreased suppressant property on the secretion of HBeAg. Chapter 5 Progress in development of active anti-hepatitis B virus compounds Active nucleosides, small molecular compounds and natural compounds against hepatitis B viurs were reviewed based on literatures in this chapter. 164 active anti-HBV compounds were summerized and classified according to their chemical characteristics. The anti-HBV activities of active compounds were discussed.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/406
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泽泻醇A衍生物的合成与抗乙肝病毒活性研究.张泉[d].中国科学院昆明植物研究所,2009.20-25
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