格尔德霉素衍生物的设计、合成及抗肿瘤活性研究

发布时间：2018-06-05 07:27

本文选题：格尔德霉素 + 热休克蛋白90　；参考：《山东大学》2014年博士论文

【摘要】：格尔德霉素(Geldanamycin, GA)最初在1970年从吸水链霉菌(Streptomyces hygroscopicus)发酵液中分离得到,属于苯醌安莎霉素。GA具有抗菌、抗原虫、抗炎、抗肿瘤和抗病毒等生物活性。其抗肿瘤作用是近年来关注的热点之一。最初发现GA是蛋白酪氨酸激酶的非特异性抑制剂,后来发现热休克蛋白90(heat shock protein90, Hsp90)是其抗肿瘤作用靶点。GA通过竞争性结合Hsp90N末端ATP/ADP结构域,特异性地抑制Hsp90所必需的ATP酶活性、改变Hsp90构象,从而抑制Hsp90分子伴侣功能。Hsp90失活后,依赖Hsp90的众多蛋白,包括信号转导系统许多重要成员被泛素化和降解,产生抑制肿瘤细胞增殖、诱导细胞凋亡等效应。尽管GA有广谱抗肿瘤作用,但由于其水溶性较差,体内分布特异性低,尤其是肝毒性较强,严重影响了其新药开发。为了增加药效、提高成药性和降低毒性,药物化学家们长期对GA进行了结构优化,获得了数百个衍生物,其中17-AAG (Tanespimycin)、17-DMAG (Alvespimycin)、7-AG (IPI-493)和IPI-504(Retaspimycin-HCl)已进入临床试验。17-AAG对多种肿瘤有强的抑制活性,但是由于其昂贵的生产成本、专利期限和肝毒性等问题,施贵宝公司于2011年终止了17-AAG的III期临床试验；17-DMAG由其毒副作用,Kosan Biosciences公司于2008年暂停其I期临床试验；17-AG水溶性和生物利用度都很差,其I期临床试验也被迫暂停；只有IPI-504还在进行治疗KRAS突变的非小细胞肺癌的临床试验。因此,针对现有GA衍生物的毒性和成药性,设计合成GA衍生物,对开发以Hsp90为靶点的新颖抗肿瘤药物有重要意义。本论文分四章,第一章为前言进展,主要介绍Hsp90在肿瘤形成方面的最新研究进展,重点关注了该蛋白的抑制剂GA及其衍生物,主要包括三方面内容：1)Hsp90及其生物化学性质；2)Hsp90及其在肿瘤发生过程中的作用；3)GA衍生物化合物库的构建。第二章为GA荧光探针的设计、合成及其活性研究。随着对GA肝毒性的深入研究,越来越多的研究表明GA引起的氧化应激不依赖于Hsp90,直接通过蛋白质的氧化降解引起细胞损伤和死亡。目前认为GA引起的氧化应激主要包括两个方面：一是诱导细胞产生活性氧(Reactive Oxygen Species, ROS), GA结构上含有一个苯醌基团,该基团具有氧化性,在黄素酶的作用下发生单电子还原生成半醌阴离子,然后与氧分子反应生成超氧阴离子,这些超氧化物可以进一步转化为其他活性氧自由基。另一方面,研究发现GA在正常生理条件下可以和谷胱甘肽发生加成反应生成稳定的巯基化GA,降低细胞内GSH水平,破坏细胞内氧化还原平衡。为了验证我们的猜想,设计并合成了一系列双机制的H202荧光探针：1)验证GA是否诱导产生活性氧；2)探索GA在细胞内结合的位置。第三章和第四章主要是以GA为骨架进行结构修饰,设计合成7个系列、共146个化合物,并开展了体内外抗肿瘤活性和构效关系研究。在第三章,以GA为先导化合物,分别使用取代的芳基甲胺(A系列)、苯乙胺(B系列)和苯氧乙胺／苯丙胺(C系列)对其17-位进行结构修饰,合成了17-取代芳基甲胺基、17-苯乙胺基和17-取代苯氧乙胺基／苯丙胺基取代3个系列共67个GA衍生物,并对其进行了体内外活性评价和构效关系讨论。 17-取代芳基甲胺基-17-去甲氧基格尔德霉素(A系列)衍生物的设计思路是基于17-AAG侧链烯丙胺基的骨架迁越(scaffold hopping),将小π键(双键)替换为大π键(芳环),使用了苄胺基和杂环甲胺基,并考察了芳环上取代基对活性的影响。初步的构效关系研究结果表明,对于抑制MDA-MB-231细胞增殖,大多数17-苄胺基GA衍生物均呈现显著活性,且与17-AAG相当。但是,对于LNCaP细胞株,苯环上不同取代基对活性的影响非常大,且17-(4-氨基苄胺)-17-去甲氧基格尔德霉素(A21)对两株细胞株均呈现显著抑制作用。在GA的17-位引入杂环甲胺来替换取代的苄胺,得到了化合物A30-33。其中,六元杂环衍生物A32-33对两株细胞株都显示出优秀的抑制活性。尤其是17-((6-(三氟甲基吡啶-3-基)甲胺)-17-去甲氧基格尔德霉素(A33),对两株细胞株显示出与17-AAG相当的抑制作用。接着对代表性化合物A21和A32-33进行小鼠体内肝毒性评价,其中化合物A33注射组小鼠的转氨酶水平与溶媒组无显著性差异(P0.05),暗示化合物A33肝毒性较低。在进一步的体外抗Hsp90实验、细胞内抑制Hsp90实验及体内抑瘤实验中,A33的表现均优于17-AAG。因此,A33的抗肿瘤作用机制和成药性值得深入研究。鉴于A系列衍生物的突出抗肿瘤活性和个别化合物的较低肝脏毒性,并综合17-DMAG和17-AAG的设计思路,合成了26个17-苯乙胺基-17-去甲氧基格尔德霉素(B系列)衍生物。仍然,体外抗肿瘤活性筛选结果表明,B系列化合物的活性要弱于A系列。其中,17-(3-三氟甲基苯乙胺基)-17-去甲氧基格尔德霉素(B25)是B系列中活性最好的一个,其抗前列腺癌细胞的活性(IC50=0.27±0.11μM)优于GA(IC50=0.43±0.15μM)。肝毒性实验表明,B25组与溶媒组小鼠的AST和ALT水平相比,无显著性差异(P0.05)。综合考虑体外抗肿瘤活性和体外肝毒性结果,化合物B25是一个有潜力的抗前列腺癌先导物。另一方面,通过合成C系列化合物：17-取代苯氧乙胺3/苯丙胺基-17-去甲氧基格尔德霉素衍生物,初步考察取代苯环与GA的C-17取代基链长对GA衍生物抗肿瘤活性和毒性的影响。体外抗肿瘤活性筛选发现,C系列化合物整体的活性与B系列相比有所提高。第四章系统探讨取代芳酰基分别以4种二元胺为连接基团,对GA衍生物抗肿瘤和毒性的影响。在GA的17-位引入4种不同长度的二元胺连接基团,即4-氨甲基哌啶、1,4-丁二胺、1,6-己二胺和3,6-二氧杂-1,8-辛二胺,设计合成四个系列(D、E、F和G系列)共79个新型GA衍生物。起初,我们在GA的C-17位引入连接基团4-氨甲基哌啶、1,4-丁二胺和1,6-己二胺,合成了三个系列的化合物D1-7(4-氨甲基哌啶)、E1-7(1,4-丁二胺)和F1-7(1,6-己二胺),并考查了其对GA衍生物抗肿瘤活性和肝毒性的影响,发现连接基团越长活性越好,并且肝毒性越低。进而我们固定连接基团为1,6-己二胺,考查末端的芳酰基对活性及毒性的影响。通过生物活性筛选发现,末端芳酰基为3,4,5-三甲氧基肉桂酰基时,即17-(1,6-(3,4,5-三甲氧基肉桂酰基)已二胺)-17-去甲氧基格尔德霉素(F36),其对人乳腺癌MDA-MB-231细胞株具有较强的抑制活性,并且肝毒性较低。进一步在GA的C-17位引入更长的连接基团3,6-二氧杂-1,8-辛二胺,设计、合成了26个17-(3,6-二氧杂-8-N-(取代肉桂酰基)辛二胺)-17-去甲氧基格尔德霉素衍生物,并筛选了其对肿瘤细胞生长抑制活性。仍然,构效关系分析表明引入双甲氧基或羟基取代肉桂酰基,GA衍生物的细胞毒性显著降低,活性最好的化合物G21对细胞株MDA-MB-231的IC50也仅为1.5μM。因此,连接基团并非越长越有利于抗肿瘤活性。本学位论文研究结果表明：1)GA的C-17位连接基团为6个原子的1,6-己二胺时,抗肿瘤活性最好,且肝脏毒性较低；2)末端芳酰基为3,4,5-三甲氧基肉桂酰基时,抗肿瘤活性最好,且肝脏毒性较低。17-(1,6-(3,4,5-三甲氧基肉桂酰基)已二胺)-17-去甲氧基格尔德霉素(F36)的体内肝毒性显著低于17-AAG,且体内抗乳腺癌活性优于17-AAG,对于进一步开展GA的结构优化,发展以GA为母核的Hsp90抑制剂,有重要参考价值。
[Abstract]:Geldanamycin (GA) was originally isolated from the fermentation broth of Streptomyces hygromycma (Streptomyces hygroscopicus) in 1970. It belongs to the biologic activity of antibiosis, antigenic, anti-inflammatory, anti-tumor and antiviral activity, and its anti-tumor effect is one of the hot spots in recent years.
Initially, GA was found to be a nonspecific inhibitor of protein tyrosine kinase. Later, it was found that heat shock protein 90 (heat shock Protein90, Hsp90) was its anti-tumor target.GA through competitive binding of Hsp90N terminal ATP/ADP domain, specifically inhibiting the ATP enzyme activity necessary for Hsp90 and changing Hsp90 conformation, thus inhibiting Hsp90 molecular chaperone work. After the inactivation of.Hsp90, many proteins that depend on Hsp90, including many important members of the signal transduction system, are ubiquitinating and degraded, which can inhibit the proliferation of tumor cells and induce apoptosis.
Although GA has a broad spectrum of anti-tumor effects, but because of its poor solubility in water, low specific distribution in the body and strong liver toxicity, it has seriously affected the development of new drugs. In order to increase drug efficiency, increase drug resistance and reduce toxicity, drug chemists have optimized the structure of GA for a long time and obtained hundreds of derivatives, of which 17-AAG (Tanespimycin), 17-DMAG (Alvespimycin), 7-AG (IPI-493) and IPI-504 (Retaspimycin-HCl) have entered clinical trials with a strong inhibitory activity of.17-AAG on a variety of tumors. However, due to its expensive production costs, patent duration and liver toxicity, Shi Guibao terminated III clinical trial of 17-AAG in 2011; 17-DMAG is a toxic side effect, Kosan Bi. Osciences suspended its I clinical trial in 2008; 17-AG was poor in water solubility and bioavailability, and its I clinical trials were also suspended; only IPI-504 was still in clinical trials in the treatment of KRAS mutations in non small cell lung cancer. Therefore, a GA derivative was designed and synthesized for the toxicity and drug resistance of the existing GA derivatives. Sp90 is of great significance for novel anti-tumor drugs.
This thesis is divided into four chapters. The first chapter is the advance of the preface. It mainly introduces the latest research progress of Hsp90 in the formation of tumor, focusing on the inhibitor GA and its derivatives, mainly including three aspects: 1) Hsp90 and its biochemical properties; 2) Hsp90 and its role in the occurrence of swollen tumors; 3) GA derivative compound library Build.
The second chapter is the design, synthesis and activity of GA fluorescence probe. With the in-depth study of GA liver toxicity, more and more studies have shown that oxidative stress caused by GA is not dependent on Hsp90, which directly causes cell damage and death through oxidative degradation of protein. At present, the oxidative stress caused by GA mainly includes two aspects: one is The induced cells produce Reactive Oxygen Species (ROS), and the GA structure contains a quinone group. The group is oxidizing. The group has a single electron reduction to produce a semi quinone anion under the action of the flavin enzyme, and then reacts with the oxygen molecule to produce superoxide anion. These superoxides can be further converted to other reactive oxygen radicals. On the other hand, the study found that GA can react with glutathione in normal physiological conditions to generate a stable mercapto GA, reduce the GSH level in the cell and destroy the redox balance in the cell. In order to verify our conjecture, a series of double mechanism H202 fluorescence probes are designed and synthesized: 1) verify whether GA induces activity. Oxygen; 2) explore the location of GA in cell binding.
The third and fourth chapters are mainly composed of GA as the skeleton structure, designed and synthesized 7 series, a total of 146 compounds, and carried out the study of antitumor activity and structure-activity relationship in vivo and in vitro. In the third chapter, GA was used as the precursor compound, the substituted aryl methylamine (A series), phenylethylamine (B Series) and phenylethylamine / amphetamine (C Series) were used respectively. The structure modification of its 17- site was made to synthesize 17- substituted aryl methylamines, 17- phenylethylamines and 17- substituted phenoxy amine based / amphetamine groups to replace 3 series of 67 GA derivatives, and the activity evaluation and structure-activity relationship were discussed in vivo and in vivo.
The design idea of 17- replacing aryl methylamine -17- normethygromycin (A Series) derivatives is based on the skeleton migration (scaffold hopping) of the 17-AAG side chain alkenyl group (scaffold hopping), replacing the small pi bond (double bond) with the large pi bond (Fang Huan), using benzylamine and heterocyclic methylamines, and investigating the effect of the substituents on the aromatic ring. The results showed that most of the 17- benzyl GA derivatives were significantly active in inhibiting the proliferation of MDA-MB-231 cells, and the same as that of 17-AAG. However, for LNCaP cell lines, the effects of different substituents on the benzene ring were very large, and 17- (4- amino benzamide) -17- normethycin (A21) was found in two cell lines. At the 17- bit of GA, heterocyclic methylamine was introduced to replace substituted benzylamines, and compound A30-33. was obtained. The six membered heterocyclic derivative A32-33 showed excellent inhibitory activity to two cell lines, especially 17- ((6- (three fluoromethyl pyridyridine -3- based) methylamine) -17- normethycin (A33) and two cell lines. The inhibitory effect of 17-AAG was shown. Then the hepatotoxicity of the representative compounds, A21 and A32-33, was evaluated in mice. The aminotransferase level of the compound A33 injection group was not significantly different from that of the solvent group (P0.05), suggesting that the toxicity of the compound A33 was lower. In the further anti Hsp90 experiment in vitro, the intracellular inhibition of Hsp90 test and the inhibition of Hsp90 experiment in vitro, In vivo anti-tumor experiments, the performance of A33 is better than 17-AAG.. Therefore, the anti-tumor mechanism and drug resistance of A33 are worthy of further study.
In view of the protruding anti-tumor activity of A series derivatives and the lower liver toxicity of individual compounds, and combining the design ideas of 17-DMAG and 17-AAG, 26 17- benzyl ethylamine -17- normethoxicamycin (B Series) derivatives have been synthesized. The screening results of anti tumor activity in vitro show that the activity of B series compounds is weaker than that of A series. 17- (3- three fluoromethyl phenyl ethylamine) -17- normethygromycin (B25) is the best activity in the B series, and its activity against prostate cancer cells (IC50=0.27 + 0.11 M) is superior to GA (IC50=0.43 + 0.15 micron M). In vitro antitumor activity and in vitro hepatotoxicity, compound B25 is a potential anti prostate cancer precursor.
On the other hand, through the synthesis of C series compounds: 17- substituted phenoxy ethylamine 3/ phenylalanine -17- normethycin derivative, the effect of substituent phenyl ring and C-17 substituent chain length on the antitumor activity and toxicity of GA derivatives is preliminarily investigated. In vitro anti-tumor activity screening, the overall activity of C series compounds and B series phase The ratio is improved.
The fourth chapter systematically investigates the effect of substituted aromatic acyl groups with 4 two meta amines on the antitumor and toxic effects of GA derivatives. 4 different lengths of two meta amine connecting groups, namely 4- ammonia methyl piperidine, 1,4- butyl two amine, 1,6- hexamines and 3,6- two oxy -1,8- octyl two amines, are designed and synthesized in the 17- site of GA, and four series (D, E, F and G) are designed and synthesized. A total of 79 new GA derivatives.
At first, we introduced three series of compounds, D1-7 (4- methylation piperidine), E1-7 (1,4- butyl two amine) and F1-7 (1,6- hexane), in the C-17 site of GA, D1-7 (4- methylation piperidine), E1-7 (1,4- butyl two amine) and F1-7 (1,6- hexamines), and found that the longer the connecting group was, the better the activity of the connecting group. And the lower the liver toxicity, and then we fixed the connection group as 1,6- hexane, and examined the effect of the terminal aroyl on the activity and toxicity. Through biological activity screening, it was found that the terminal aryl group was 3,4,5- tri methoxy cinnamyl group, that is, 17- (1,6- (3,4,5- trimethoxyl cinnamyl), two amine) -17- normogromycin (F36), and Human breast cancer cell line MDA-MB-231 has strong inhibitory activity and low hepatotoxicity.
Further GA C-17 bit 3,6- two oxy -1,8- octyl two amine was introduced, and 26 17- (3,6- two oxo -8-N- (substituted cinnamyl) Xin Eran) -17- normethycin derivatives were synthesized and their inhibitory activity to tumor cells was screened. The structure activity relationship analysis showed that the dimethoxy or hydroxyl group was introduced. With the substituent of cinnamyl group, the cytotoxicity of GA derivatives decreased significantly. The best active compound, G21, was only 1.5 mu M. to the cell line of the cell line, and the longer the connection group was, the more it was beneficial to the antitumor activity.
The results of this thesis show that: 1) when the C-17 position group of GA is 6 atoms of 1,6- hexylamine, the antitumor activity is best and the liver toxicity is low; 2) when the terminal aryl group is 3,4,5- tri methoxy cinnamyl group, the antitumor activity is best, and the liver toxicity is lower.17- (1,6- (3,4,5- trtri methoxy cinnamyl) two amine) -17- normol The hepatotoxicity of F36 in vivo is significantly lower than that of 17-AAG, and the activity of anti breast cancer in vivo is better than that of 17-AAG. It has important reference value for the further development of GA structure and the development of Hsp90 inhibitor with GA as the parent nucleus.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R914.5;R965

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