發(fā)布時(shí)間：2018-06-05 07:27

  本文選題：格爾德霉素 + 熱休克蛋白90��； 參考：《山東大學(xué)》2014年博士論文

【摘要】：格爾德霉素(Geldanamycin, GA)最初在1970年從吸水鏈霉菌(Streptomyces hygroscopicus)發(fā)酵液中分離得到,屬于苯醌安莎霉素。GA具有抗菌、抗原蟲、抗炎、抗腫瘤和抗病毒等生物活性。其抗腫瘤作用是近年來(lái)關(guān)注的熱點(diǎn)之一。 最初發(fā)現(xiàn)GA是蛋白酪氨酸激酶的非特異性抑制劑,后來(lái)發(fā)現(xiàn)熱休克蛋白90(heat shock protein90, Hsp90)是其抗腫瘤作用靶點(diǎn)。GA通過(guò)競(jìng)爭(zhēng)性結(jié)合Hsp90N末端ATP/ADP結(jié)構(gòu)域,特異性地抑制Hsp90所必需的ATP酶活性、改變Hsp90構(gòu)象,從而抑制Hsp90分子伴侶功能。Hsp90失活后,依賴Hsp90的眾多蛋白,包括信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)許多重要成員被泛素化和降解,產(chǎn)生抑制腫瘤細(xì)胞增殖、誘導(dǎo)細(xì)胞凋亡等效應(yīng)。 盡管GA有廣譜抗腫瘤作用,但由于其水溶性較差,體內(nèi)分布特異性低,尤其是肝毒性較強(qiáng),嚴(yán)重影響了其新藥開(kāi)發(fā)。為了增加藥效、提高成藥性和降低毒性,藥物化學(xué)家們長(zhǎng)期對(duì)GA進(jìn)行了結(jié)構(gòu)優(yōu)化,獲得了數(shù)百個(gè)衍生物,其中17-AAG (Tanespimycin)、17-DMAG (Alvespimycin)、7-AG (IPI-493)和IPI-504(Retaspimycin-HCl)已進(jìn)入臨床試驗(yàn)。17-AAG對(duì)多種腫瘤有強(qiáng)的抑制活性,但是由于其昂貴的生產(chǎn)成本、專利期限和肝毒性等問(wèn)題,施貴寶公司于2011年終止了17-AAG的III期臨床試驗(yàn)；17-DMAG由其毒副作用,Kosan Biosciences公司于2008年暫停其I期臨床試驗(yàn)；17-AG水溶性和生物利用度都很差,其I期臨床試驗(yàn)也被迫暫停；只有IPI-504還在進(jìn)行治療KRAS突變的非小細(xì)胞肺癌的臨床試驗(yàn)。因此,針對(duì)現(xiàn)有GA衍生物的毒性和成藥性,設(shè)計(jì)合成GA衍生物,對(duì)開(kāi)發(fā)以Hsp90為靶點(diǎn)的新穎抗腫瘤藥物有重要意義。 本論文分四章,第一章為前言進(jìn)展,主要介紹Hsp90在腫瘤形成方面的最新研究進(jìn)展,重點(diǎn)關(guān)注了該蛋白的抑制劑GA及其衍生物,主要包括三方面內(nèi)容：1)Hsp90及其生物化學(xué)性質(zhì)；2)Hsp90及其在腫瘤發(fā)生過(guò)程中的作用；3)GA衍生物化合物庫(kù)的構(gòu)建。 第二章為GA熒光探針的設(shè)計(jì)、合成及其活性研究。隨著對(duì)GA肝毒性的深入研究,越來(lái)越多的研究表明GA引起的氧化應(yīng)激不依賴于Hsp90,直接通過(guò)蛋白質(zhì)的氧化降解引起細(xì)胞損傷和死亡。目前認(rèn)為GA引起的氧化應(yīng)激主要包括兩個(gè)方面：一是誘導(dǎo)細(xì)胞產(chǎn)生活性氧(Reactive Oxygen Species, ROS), GA結(jié)構(gòu)上含有一個(gè)苯醌基團(tuán),該基團(tuán)具有氧化性,在黃素酶的作用下發(fā)生單電子還原生成半醌陰離子,然后與氧分子反應(yīng)生成超氧陰離子,這些超氧化物可以進(jìn)一步轉(zhuǎn)化為其他活性氧自由基。另一方面,研究發(fā)現(xiàn)GA在正常生理?xiàng)l件下可以和谷胱甘肽發(fā)生加成反應(yīng)生成穩(wěn)定的巰基化GA,降低細(xì)胞內(nèi)GSH水平,破壞細(xì)胞內(nèi)氧化還原平衡。為了驗(yàn)證我們的猜想,設(shè)計(jì)并合成了一系列雙機(jī)制的H202熒光探針：1)驗(yàn)證GA是否誘導(dǎo)產(chǎn)生活性氧；2)探索GA在細(xì)胞內(nèi)結(jié)合的位置。 第三章和第四章主要是以GA為骨架進(jìn)行結(jié)構(gòu)修飾,設(shè)計(jì)合成7個(gè)系列、共146個(gè)化合物,并開(kāi)展了體內(nèi)外抗腫瘤活性和構(gòu)效關(guān)系研究。在第三章,以GA為先導(dǎo)化合物,分別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)對(duì)其17-位進(jìn)行結(jié)構(gòu)修飾,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個(gè)系列共67個(gè)GA衍生物,并對(duì)其進(jìn)行了體內(nèi)外活性評(píng)價(jià)和構(gòu)效關(guān)系討論。 17-取代芳基甲胺基-17-去甲氧基格爾德霉素(A系列)衍生物的設(shè)計(jì)思路是基于17-AAG側(cè)鏈烯丙胺基的骨架遷越(scaffold hopping),將小π鍵(雙鍵)替換為大π鍵(芳環(huán)),使用了芐胺基和雜環(huán)甲胺基,并考察了芳環(huán)上取代基對(duì)活性的影響。初步的構(gòu)效關(guān)系研究結(jié)果表明,對(duì)于抑制MDA-MB-231細(xì)胞增殖,大多數(shù)17-芐胺基GA衍生物均呈現(xiàn)顯著活性,且與17-AAG相當(dāng)。但是,對(duì)于LNCaP細(xì)胞株,苯環(huán)上不同取代基對(duì)活性的影響非常大,且17-(4-氨基芐胺)-17-去甲氧基格爾德霉素(A21)對(duì)兩株細(xì)胞株均呈現(xiàn)顯著抑制作用。在GA的17-位引入雜環(huán)甲胺來(lái)替換取代的芐胺,得到了化合物A30-33。其中,六元雜環(huán)衍生物A32-33對(duì)兩株細(xì)胞株都顯示出優(yōu)秀的抑制活性。尤其是17-((6-(三氟甲基吡啶-3-基)甲胺)-17-去甲氧基格爾德霉素(A33),對(duì)兩株細(xì)胞株顯示出與17-AAG相當(dāng)?shù)囊种谱饔�。接著�?duì)代表性化合物A21和A32-33進(jìn)行小鼠體內(nèi)肝毒性評(píng)價(jià),其中化合物A33注射組小鼠的轉(zhuǎn)氨酶水平與溶媒組無(wú)顯著性差異(P0.05),暗示化合物A33肝毒性較低。在進(jìn)一步的體外抗Hsp90實(shí)驗(yàn)、細(xì)胞內(nèi)抑制Hsp90實(shí)驗(yàn)及體內(nèi)抑瘤實(shí)驗(yàn)中,A33的表現(xiàn)均優(yōu)于17-AAG。因此,A33的抗腫瘤作用機(jī)制和成藥性值得深入研究。 鑒于A系列衍生物的突出抗腫瘤活性和個(gè)別化合物的較低肝臟毒性,并綜合17-DMAG和17-AAG的設(shè)計(jì)思路,合成了26個(gè)17-苯乙胺基-17-去甲氧基格爾德霉素(B系列)衍生物。仍然,體外抗腫瘤活性篩選結(jié)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-去甲氧基格爾德霉素(B25)是B系列中活性最好的一個(gè),其抗前列腺癌細(xì)胞的活性(IC50=0.27±0.11μM)優(yōu)于GA(IC50=0.43±0.15μM)。肝毒性實(shí)驗(yàn)表明,B25組與溶媒組小鼠的AST和ALT水平相比,無(wú)顯著性差異(P0.05)。綜合考慮體外抗腫瘤活性和體外肝毒性結(jié)果,化合物B25是一個(gè)有潛力的抗前列腺癌先導(dǎo)物。 另一方面,通過(guò)合成C系列化合物：17-取代苯氧乙胺3/苯丙胺基-17-去甲氧基格爾德霉素衍生物,初步考察取代苯環(huán)與GA的C-17取代基鏈長(zhǎng)對(duì)GA衍生物抗腫瘤活性和毒性的影響。體外抗腫瘤活性篩選發(fā)現(xiàn),C系列化合物整體的活性與B系列相比有所提高。 第四章系統(tǒng)探討取代芳�；謩e以4種二元胺為連接基團(tuán),對(duì)GA衍生物抗腫瘤和毒性的影響。在GA的17-位引入4種不同長(zhǎng)度的二元胺連接基團(tuán),即4-氨甲基哌啶、1,4-丁二胺、1,6-己二胺和3,6-二氧雜-1,8-辛二胺,設(shè)計(jì)合成四個(gè)系列(D、E、F和G系列)共79個(gè)新型GA衍生物。 起初,我們?cè)贕A的C-17位引入連接基團(tuán)4-氨甲基哌啶、1,4-丁二胺和1,6-己二胺,合成了三個(gè)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),并考查了其對(duì)GA衍生物抗腫瘤活性和肝毒性的影響,發(fā)現(xiàn)連接基團(tuán)越長(zhǎng)活性越好,并且肝毒性越低。進(jìn)而我們固定連接基團(tuán)為1,6-己二胺,考查末端的芳�；鶎�(duì)活性及毒性的影響。通過(guò)生物活性篩選發(fā)現(xiàn),末端芳�；鶠�3,4,5-三甲氧基肉桂�；鶗r(shí),即17-(1,6-(3,4,5-三甲氧基肉桂�；�)已二胺)-17-去甲氧基格爾德霉素(F36),其對(duì)人乳腺癌MDA-MB-231細(xì)胞株具有較強(qiáng)的抑制活性,并且肝毒性較低。 進(jìn)一步在GA的C-17位引入更長(zhǎng)的連接基團(tuán)3,6-二氧雜-1,8-辛二胺,設(shè)計(jì)、合成了26個(gè)17-(3,6-二氧雜-8-N-(取代肉桂�；�)辛二胺)-17-去甲氧基格爾德霉素衍生物,并篩選了其對(duì)腫瘤細(xì)胞生長(zhǎng)抑制活性。仍然,構(gòu)效關(guān)系分析表明引入雙甲氧基或羥基取代肉桂�；�,GA衍生物的細(xì)胞毒性顯著降低,活性最好的化合物G21對(duì)細(xì)胞株MDA-MB-231的IC50也僅為1.5μM。因此,連接基團(tuán)并非越長(zhǎng)越有利于抗腫瘤活性。 本學(xué)位論文研究結(jié)果表明：1)GA的C-17位連接基團(tuán)為6個(gè)原子的1,6-己二胺時(shí),抗腫瘤活性最好,且肝臟毒性較低；2)末端芳�；鶠�3,4,5-三甲氧基肉桂�；鶗r(shí),抗腫瘤活性最好,且肝臟毒性較低。17-(1,6-(3,4,5-三甲氧基肉桂�；�)已二胺)-17-去甲氧基格爾德霉素(F36)的體內(nèi)肝毒性顯著低于17-AAG,且體內(nèi)抗乳腺癌活性優(yōu)于17-AAG,對(duì)于進(jìn)一步開(kāi)展GA的結(jié)構(gòu)優(yōu)化,發(fā)展以GA為母核的Hsp90抑制劑,有重要參考價(jià)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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R914.5;R965

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 吳云飛;李震宇;王貞;徐洪蛟;武興康;魯春華;沈月毛;;17-[3,6-二氧雜-8-N-(取代肉桂�；�)辛二胺]-17-去甲氧基格爾德霉素新穎衍生物的合成[J];有機(jī)化學(xué);2014年09期

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