發(fā)布時(shí)間：2018-06-07 08:45

  本文選題：組蛋白去乙�；�(HDAC) + HDAC抑制劑��； 參考：《山東大學(xué)》2014年博士論文

【摘要】：組蛋白的乙酰化和去乙�；揎検潜碛^遺傳學(xué)研究中重要的研究領(lǐng)域,主要是通過組蛋白乙�；D(zhuǎn)移酶(histone acetyltraqsferase, HAT)和組蛋白去乙酰化酶(histone deacetylase, HDAC)共同調(diào)控的。其中組蛋白去乙�；磻�(yīng)由HDAC催化,該酶主要包括Zn2+依賴性的和NAD+依賴性的兩種類型。其中Zn2+依賴性的HDAC在許多癌癥類型中表達(dá)異常,并與腫瘤的發(fā)生和發(fā)展密切相關(guān)。HDAC的小分子抑制劑能夠?qū)е履[瘤細(xì)胞凋亡、分化、生長(zhǎng)抑制和血管生成抑制。目前vorinostat(suberoylanilide hydroxamic acid, SAHA)和romidepsin已被FDA批準(zhǔn)上市用于治療皮膚T細(xì)胞淋巴瘤(cutaneous T cell lymphoma, CTCL),并有多種HDAC抑制劑在臨床試驗(yàn)階段對(duì)血液瘤顯示良好的治療效果。因此HDAC抑制劑已成為抗腫瘤藥物的重要研究領(lǐng)域。 根據(jù)HDAC2與SAHA的共晶結(jié)合方式,HDAC抑制劑包含Zn2+螯合基團(tuán)(ZBG)、連接區(qū)域(Linker)和酶表面識(shí)別區(qū)域(Cap)等三個(gè)部分。在前期工作中我們?cè)O(shè)計(jì)合成了一系列噻二唑類HDAC抑制劑,其中化合物6m具有與SAHA相當(dāng)?shù)囊置富钚�。初步生物活性研究發(fā)現(xiàn)6m對(duì)10種腫瘤細(xì)胞的增值抑制作用也與SAHA相當(dāng),還能夠引起腫瘤細(xì)胞凋亡、抑制腫瘤遷移并產(chǎn)生細(xì)胞周期抑制。為進(jìn)一步提高抗腫瘤活性,我們從以下三個(gè)方面對(duì)噻二唑類HDAC抑制劑進(jìn)行結(jié)構(gòu)修飾和改造：第一,根據(jù)前期構(gòu)效關(guān)系研究,Linker區(qū)域相對(duì)固定,一方面在噻二唑環(huán)5-位苯基上引入取代基得到A系列化合物,另一方面根據(jù)藥物設(shè)計(jì)學(xué)的生物電子等排原理,以萘環(huán)、吡啶環(huán)、呋喃環(huán)和噻吩環(huán)等替代苯基得到B系列化合物；第二,根據(jù)HDAC抑制劑的臨床研究進(jìn)展,用對(duì)癌癥顯示療效生物2-氨基苯胺甲酰基、N-羥基苯甲酰胺基和N-羥基肉桂酰胺基等Zn2+螯合基團(tuán)替代異羥肟酸基得到C系列化合物；第三,考慮到Cap區(qū)1,3,4-噻二唑雜環(huán)可被其它雜環(huán)替換,根據(jù)生物電子等排原理將噻二唑改為噻唑和吡唑雜環(huán)得到D系列化合物。 在目標(biāo)化合物的合成中,A和B系列化合物以芳基甲酸為原料,以三氯氧磷為脫水劑經(jīng)一鍋法反應(yīng)生成1,3,4-噻二唑衍生物,后與庚二酸單甲酯或辛二酸單甲酯縮合得到含噻二唑雜環(huán)的羧酸酯,最后異羥肟酸化得到目標(biāo)化合物；在C系列化合物合成中,可水解已有的噻二唑雜環(huán)羧酸酯,所得羧酸與Boc保護(hù)鄰苯二胺縮合,最后脫除Boc保護(hù)基得到化合物C1；其它目標(biāo)化合物C2-C4的合成主要以不同原料合成含有1,3,4-噻二唑的羧酸酯,最后轉(zhuǎn)化為異羥肟酸；D系列化合物的合成參考A系列和B系列合成方法,參照文獻(xiàn)合成噻唑或毗唑雜環(huán),再經(jīng)縮合和異羥肟酸化制得。本論文共合成新化合物143個(gè),其中包括新目標(biāo)化合物71個(gè)和新中間體72個(gè)。所有新化合物的結(jié)構(gòu)均經(jīng)過核磁共振氫譜、碳譜或質(zhì)譜等手段進(jìn)行確證。 所有目標(biāo)化合物均進(jìn)行了初步生物活性評(píng)價(jià)。其中多數(shù)A系列化合物和B系列化合物的抑酶活性與SAHA相當(dāng)甚至活性更高。A系列化合物的構(gòu)效關(guān)系說明：(1)向噻二唑5位苯基引入位阻越小的取代基,抑酶活性越好；(2)苯環(huán)取代基一般以引入鄰位取代的效果最好；(3)給電子取代基衍生物抑酶活性較吸電子取代顯著增強(qiáng)。B系列化合物中除萘環(huán)取代使HDAC抑制活性明顯下降甚至喪失外,吡啶環(huán)、呋喃環(huán)和噻吩環(huán)等芳雜環(huán)取代物的抑酶活性都與SAHA相當(dāng)或高于SAHA。在抗腫瘤細(xì)胞增殖方面,A系列化合物的A31效果最好,對(duì)所選測(cè)的6株腫瘤細(xì)胞的抗增殖活性均高于6m和SAHA;B系列化合物中B12的抑瘤效果最好,活性與6m和SAHA相當(dāng)。A系列和B系列化合物的抗增殖活性均顯示,當(dāng)Linker鏈長(zhǎng)n=5時(shí),對(duì)腫瘤細(xì)胞增殖的抑制活性不明顯；n=6時(shí)顯示相對(duì)較好的增殖抑制活性。 C系列化合物在活性評(píng)價(jià)中未達(dá)到相應(yīng)異羥肟酸衍生物的抗增殖活性水平,其中N-羥基苯甲酰胺衍生物和N-羥基肉桂酰胺衍生物的抗增殖活性較6m和SAHA明顯下降；2-氨基苯胺甲酰衍生物的抗增殖活性與6m和SAHA處于同一個(gè)數(shù)量級(jí),但是沒有達(dá)到6m和SAHA的活性水平。 將1,3,4-噻二唑基改造為噻唑基和吡唑基的D系列目標(biāo)化合物(D1、D2和ID3)對(duì)所選用的10株腫瘤細(xì)胞均顯示優(yōu)良的抗增殖活性,且活性明顯高于6m和SAHA。除了對(duì)K562慢性粒細(xì)胞白血病細(xì)胞系的活性提高明顯外,對(duì)其它9株實(shí)體瘤細(xì)胞抑制效果均比6m和SAHA提高2-6倍。 綜上所述,本論文針對(duì)噻二唑類HDAC抑制劑進(jìn)行了深入的結(jié)構(gòu)改造。相關(guān)構(gòu)效關(guān)系研究表明,1,3,4-噻二唑環(huán)的5-位苯基引入鄰位和位阻小的取代基有利于提高抑酶和抑瘤活性；將苯基替換為其它雜環(huán)或替換異羥肟酸基團(tuán)均未發(fā)現(xiàn)抑瘤活性更高的化合物,有時(shí)還導(dǎo)致活性喪失。我們?cè)谘芯恐幸馔獾陌l(fā)現(xiàn),將噻二唑雜環(huán)替換為噻唑或吡唑的D系列目標(biāo)化合物,其抗腫瘤活性較6m和SAHA大幅提高。針對(duì)該系列化合物的深入研究,將為今后發(fā)現(xiàn)高效、低毒的抗腫瘤藥物提供新的方向。
[Abstract]:The acetylation and deacetylation modification of histone is an important research field in epigenetics, mainly through the co regulation of histone acetyltransferase (histone acetyltraqsferase, HAT) and histone deacetylase (histone deacetylase, HDAC), in which the histone deacetylation reaction is catalyzed by HDAC, the main package of this enzyme Including two types of Zn2+ dependent and NAD+ dependent, Zn2+ dependent HDAC expresses abnormality in many cancer types, and small molecular inhibitors of.HDAC, closely related to the occurrence and development of tumors, can lead to tumor cell apoptosis, differentiation, growth inhibition and angiogenesis inhibition. Currently, vorinostat (suberoylanilide hydroxamic a) CID, SAHA) and romidepsin have been approved by FDA for the treatment of T cell lymphoma of the skin (cutaneous T cell lymphoma, CTCL), and a variety of HDAC inhibitors have shown good therapeutic effects on hematoma in clinical trials. Therefore HDAC inhibitors have become an important research area of antitumor drugs.
According to the eutectic binding method of HDAC2 and SAHA, HDAC inhibitors include Zn2+ chelating group (ZBG), connection region (Linker) and enzyme surface identification region (Cap). In the previous work, we designed and synthesized a series of thiazolazole HDAC inhibitors, in which the compound 6m has the equivalent enzyme inhibition activity with SAHA. Preliminary biological activity studies have been made. The inhibitory effect of 6m on 10 tumor cells is also similar to that of SAHA. It can also induce tumor cell apoptosis, inhibit tumor migration and produce cell cycle inhibition. In order to further improve the antitumor activity, we repair and reconstruct the thiotriazole HDAC inhibitors from the following three aspects: first, according to the prophase structure-activity relationship study On the one hand, the Linker region is relatively fixed, on the one hand, the substituent base is introduced to the A series compound on the thiothiazole ring 5- site phenyl group. On the other hand, the naphthalene ring, pyridine ring, furan ring and thiophene ring are replaced by naphthalene rings, pyridine rings, furan rings and thiophene rings to obtain the B series compounds on the basis of the biological electron emission principle of the drug design, and second, according to the clinical research progress of HDAC inhibitors, the A series compounds are used. Cancer shows the therapeutic organisms 2- amino aniline formyl, N- hydroxy benzamidyl group and N- hydroxyl cinnamamide group instead of hydroxamic acid group to obtain C series compounds. Third, 1,3,4- thiazolyl heterocyclic rings can be replaced by other heterocyclic rings, and thiazole and pyrazole heterocyclic rings are changed to thiazole and pyrazole heterocyclic rings according to the principle of bioelectron emission in Cap region. D series of compounds were obtained.
In the synthesis of target compounds, A and B series compounds are made of aryl formic acid as raw materials, and 1,3,4- thiothiazole derivatives are produced by one pot reaction with three oxychloride as dehydrating agent. Then, the carboxylic acid esters containing thiothiolate or monomethyl dimethicarate are condensed to obtain thiothiol heterocyclic acid esters. Finally, the target compounds are obtained by acidification of hydroxyl oxime, and C seriation. In the synthetic synthesis, the existing thiazolyl heterocyclic carboxylates can be hydrolyzed. The carboxylic acid and Boc protect the phthalic acid two amine, and then the Boc protective group is removed to get the compound C1. The synthesis of other target compounds C2-C4 is mainly synthesized by different raw materials, which contain 1,3,4- thiothiolates, and then converted to hydroxamic acid, and the synthesis of D series compounds. Referring to the synthesis of A series and B series, the synthesis of thiazole or vishzol heterocyclic rings by reference literature and then by condensation and hydroxamic acidification are made. 143 new compounds are synthesized in this paper, including 71 new target compounds and 72 new intermediates. All new compounds are confirmed by nuclear magnetic resonance spectroscopy, carbon spectra or mass spectrometry.
The initial biological activity of all the target compounds was evaluated. Most of the A series and B series compounds have the same activity with SAHA and even higher activity of.A series. (1) the smaller the substituent to the thiazolide 5 phenyl group, the better the suppressor activity, and (2) the substituent of the benzene ring is generally introduced. The effect of the substitutions on the neighborhood was the best, (3) the inhibitory activity of the substituent on the electron substituents was significantly enhanced by the substitution of the electron acceptor for the substitution of the naphthalene rings in the.B series. The inhibitory activities of the pyridine rings, furan rings and thiophene rings were both equivalent to or higher than that of the SAHA. in the antitumor activity of the pyridine ring, furan ring and thiophene ring. In cell proliferation, the A31 effect of A series compounds is best, the antiproliferative activity of the 6 selected tumor cells is higher than that of 6m and SAHA, and B12 in B series compounds is the best. The antiproliferative activity of the activity and the.A series of 6m and SAHA and the B series compounds are all shown, and the inhibitory activity to the proliferation of the tumor cells when the Linker n=5 is long chain length n=5. Sex was not obvious; n=6 showed relatively good proliferative inhibitory activity.
The anti proliferation activity of C series compounds did not reach the corresponding hydroxamic acid derivatives. The antiproliferative activity of N- hydroxy benzamide derivatives and N- hydroxyl cinnamamide derivatives decreased significantly than that of 6m and SAHA, and the anti proliferative activity of 2- amino aniline derivatives was in the same order of magnitude as 6m and SAHA. The activity levels of 6m and SAHA were not reached.
The D series target compounds (D1, D2 and ID3) of thiazolidic and pyrazole group (D1, D2 and ID3) showed excellent antiproliferative activity to the selected 10 tumor cells, and the activity was significantly higher than that of 6m and SAHA. in addition to the activity of K562 chronic granulocytic leukemia cell lines, and the inhibition effect on 9 other solid tumor cells. It is 2-6 times higher than 6m and SAHA.
To sum up, this paper has made an in-depth structural modification on thiothiazoles HDAC inhibitors. The related structure-activity relationship studies showed that the 5- based phenyl group of 1,3,4- thiothiazole ring introduced to the neighborhood and the small substituent group was beneficial to improve the inhibition of enzyme and tumor suppressor activity, and the substitution of phenyl group as other heterocyclic or hydroxamic acid group did not detect tumor suppressor. More active compounds sometimes lead to loss of activity. In our study, we discovered that the thiazole or pyrazole heterocyclic heterocyclic compounds were replaced by thiazole or pyrazole D series target compounds, and their antitumor activity was significantly higher than that of 6m and SAHA. Further research on this series of compounds will provide new effective and low toxic antitumor drugs for the future. Direction.
【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

【共引文獻(xiàn)】

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