本文選題：激活消滅　切入點(diǎn)：HDAC抑制劑　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年博士論文

【摘要】：基于人類免疫缺陷病毒(HIV)治療方案中的“激活消滅”學(xué)說,本論文首先研究了苯酰胺類HIV潛伏激活劑(有效激活)的設(shè)計合成、活性評價;隨即探究了配合潛伏激活劑聯(lián)合用藥的4-取代-1,5-二芳基胺類化合物(有效消滅)的活性及類藥性等性質(zhì),旨在實(shí)現(xiàn)對病毒潛伏庫先激活,后消滅的目的。一、苯酰胺類病毒潛伏激活劑的相關(guān)研究工作自1981年HIV在美國被首次發(fā)現(xiàn),30多年間,已蔓延到各大洲,感染數(shù)千萬人,造成社會負(fù)擔(dān)及經(jīng)濟(jì)損失。高效抗逆轉(zhuǎn)錄病毒療法(HAART)是現(xiàn)在臨床上最有效的抗HIV/AIDS治療方案,艾滋病患者在接受HAART治療后可將血漿病毒載量降到不可檢測的水平。但在停止HAART治療后,其病毒載量將會迅速反彈,導(dǎo)致艾滋病患者必須終生服藥。HIV的反彈主要是由于病毒潛伏在細(xì)胞的病毒庫內(nèi)所造成,且該潛伏庫不能被HAART清除。HIV潛伏庫的存在,是HIV難以被徹底清除的根本原因。基于“激活消滅”學(xué)說,我們首先設(shè)計合成了系列抗?jié)摲幕衔?以期激活潛伏的HIV。北卡大學(xué)李國雄課題組在研究中發(fā)現(xiàn)了一個很有特點(diǎn)的抗?jié)摲せ顒?苯酰胺類HDAC抑制劑Lead,它能在低濃度下激活潛伏HIV而無毒副作用,較目前已經(jīng)進(jìn)入臨床研究的抗?jié)摲幬颯AHA可能具有更優(yōu)良的藥效。因此,我們通過設(shè)計合成系列新衍生物,期望提高其抗?jié)摲钚?使之達(dá)到良好的激活效果。作為探索性研究,我們依據(jù)合理的設(shè)計思路,采用計算機(jī)輔助設(shè)計、生物電子等排原理、骨架躍遷及活性數(shù)據(jù)引導(dǎo)等多種方法相結(jié)合的手段,通過對合成方法的不斷優(yōu)化和對反應(yīng)條件的不斷改良,對前期研究中發(fā)現(xiàn)的抗?jié)摲葘?dǎo)化合物苯酰胺類HDAC抑制劑Lead的不同部位進(jìn)行了修飾,包括對與鋅離子結(jié)合的基團(tuán)(ZBG)的環(huán)大小的改變、對與靶標(biāo)袋狀空腔相互作用的聯(lián)二芳烴結(jié)構(gòu)片段(Diaryl)的多種改變、對可識別蛋白靶標(biāo)的親脂性基團(tuán)(CAP)和親脂性鏈(Linker)的修飾,共合成了三大類全新化合物共48個,其結(jié)構(gòu)均經(jīng)過MS、1H-NMR確定。對與鋅離子結(jié)合的功能基團(tuán)(ZBG)的修飾考慮到該類抑制劑分子中ZBG與鋅離子螯合形成的環(huán)的大小的不同,我們設(shè)想:如果改變螯合環(huán)的大小,或許可以改變ZBG與鋅離子間結(jié)合的強(qiáng)度,從而對抑制劑的活性產(chǎn)生影響。因此,我們設(shè)計合成了三個系列的目標(biāo)化合物I、II、III,其可分別與HDAC催化活性中心區(qū)域的鋅離子形成七元、六元、五元螯合環(huán),并評價了這些新化合物的抗?jié)摲钚?從而了解了ZBG與其抗?jié)摲钚灾g的關(guān)系。對聯(lián)二芳烴結(jié)構(gòu)片段(Diaryl)的結(jié)構(gòu)修飾我們設(shè)計合成了將B環(huán)替換為其他基團(tuán)以增加小分子抑制劑與靶標(biāo)的親和力和分子表面的形狀互補(bǔ)性的系列化合物,包括引入:四元飽和雜環(huán)、五元飽和雜環(huán)、六元芳香環(huán)以及對位連有取代基的六元芳香環(huán)。對親脂性基團(tuán)(CAP)和親脂性鏈(Linker)的修飾先導(dǎo)物L(fēng)ead的CAP部分很小,我們只引入了較小的基團(tuán),保持了分子原來較小CAP部分與靶標(biāo)結(jié)合區(qū)域的結(jié)合模式,而對于Linker,我們通過生物等排體的替換來進(jìn)行了修飾。我們又對這48個目標(biāo)化合物分別在細(xì)胞水平和酶水平上進(jìn)行了抗?jié)摲钚栽u價,5個化合物(5a、5b、29a、45a、45b)的抗?jié)摲钚耘c陽性對照SAHA相當(dāng),其中化合物5a的抗?jié)摲钚宰詈?且保持毒性較低的特點(diǎn),優(yōu)于陽性對照SAHA,具有進(jìn)一步研究的意義。綜上,此部分的研究對今后苯酰胺化合物的抗?jié)摲芯烤哂幸欢ń梃b意義,同時也為研究其抗?jié)摲鼨C(jī)制奠定了基礎(chǔ)。目前,我們正在對合成的化合物5a的抗?jié)摲鼨C(jī)制進(jìn)行深入研究,以期尋找到高效低毒副作用的新型苯酰胺類HIV潛伏激活劑,最終實(shí)現(xiàn)激活病毒潛伏庫的目的。二、4-取代-1,5-二芳基胺類病毒抑制劑的類藥性相關(guān)研究工作基于“激活消滅”學(xué)說,在病毒庫激活后,研究重點(diǎn)轉(zhuǎn)變?yōu)椴《編斓南麥纭Ｇ捌趯?shí)驗數(shù)據(jù)表明,我們已找到具有較好激活病毒庫活性的苯酰胺類化合物,對于抗HIV治療來說,“激活”步驟已經(jīng)完成,治療工作重點(diǎn)轉(zhuǎn)移至“消滅”步驟,即病毒抑制劑的設(shè)計合成、活性測定及類藥性評價�，F(xiàn)行臨床抗HIV藥物主要分四大類:逆轉(zhuǎn)錄酶抑制劑、蛋白酶抑制劑、整合酶抑制劑、進(jìn)入抑制劑。我們又發(fā)現(xiàn)了系列高活性的非核苷類逆轉(zhuǎn)錄酶抑制劑(HIV-NNRTIs),即4-取代-1,5-二芳基胺類化合物。實(shí)驗數(shù)據(jù)表明,這類4-取代-1,5-二芳基胺類化合物的抗HIV活性優(yōu)于臨床用藥,對其進(jìn)一步的開發(fā)非常有必要。我們通過不斷優(yōu)化質(zhì)譜條件和色譜條件,建立了一套完整的LC-MS/MS定量分析方法,其專屬性強(qiáng),靈敏度高,檢測限低,分析速度非�？�,可以對我們的系列4-取代-1,5-二芳基胺類化合物進(jìn)行藥物代謝方面的測試,也為我們后期開展體內(nèi)藥物代謝相關(guān)研究奠定了基礎(chǔ)。為了豐富4-取代-1,5-二芳基胺類化合物的藥動性質(zhì)信息,我們選用人肝微粒體、人肝S9、人血漿分別對其中23個活性較好的4-取代-1,5-二芳基胺類化合物進(jìn)行了體外藥代穩(wěn)定性的初步研究,完成了這部分化合物的體外藥代動力學(xué)的評價工作,并分別獲得了其在人肝微粒體、人肝S9、人血漿中的消除半衰期,從而為我們提供了這部分化合物的藥代性質(zhì)信息,也為我們的后期先導(dǎo)物優(yōu)化提供了指導(dǎo),我們也在不斷推進(jìn)后期的體內(nèi)藥代穩(wěn)定性研究工作。目前,階段性研究進(jìn)展已整理成相關(guān)文章發(fā)表。
[Abstract]:Based on the human immunodeficiency virus (HIV) in the treatment of the "active elimination" theory, this paper studied the benzamide HIV latent activator (activate) synthesis, activity evaluation; then explores with latent activator combination of 4- substituted -1,5- two aryl amine compounds (effective eradication) activity and drug like properties, to realize the latent virus database is first activated after eradication. A benzamide latent virus activation related research agent since 1981 HIV was first found in the United States for more than 30 years, has spread to all continents, with tens of millions of people, causing the social burden and efficient economic losses. Anti retroviral therapy (HAART) is now the most clinically effective anti HIV/AIDS therapy, AIDS patients after receiving HAART therapy the plasma viral load decreased to undetectable levels. But in the end HAART After treatment, the viral load will lead to the rapid rebound in AIDS patients need lifelong medication.HIV rebound is mainly due to the latent virus caused by the virus in the cell library, and the library is not clear.HIV HAART latent latent reservoir exists, is the fundamental reason why HIV is difficult to be completely removed. On the basis of the theory of "destroy" activation first, we design the anti latent compound series was synthesized in order to activate latent HIV. North Carolina research group of Li Guoxiong University in the study found a very characteristic of the anti latent activator: benzamides HDAC inhibitor Lead, it can at a low concentration activated latent HIV and non-toxic side effects, drug resistance potential SAHA may have better efficacy than the current has entered the clinical study. Therefore, we designed and synthesized series of new derivatives, in order to improve the anti latent activity, so as to achieve a good activation effect. For For exploratory research, we based on the reasonable design ideas, using computer aided design, the principle of bioisosterism, combining skeleton transitions and the activity data of a variety of methods such as guide means, through continuous optimization of synthesis methods and modification of the reaction conditions, the different parts found in the previous research of anti latent lead compounds benzamide inhibitors of HDAC Lead were modified, including the binding of zinc ions with the group (ZBG) the ring size changes, and the target of the pouch cavity interaction of two aromatic fragments (Diaryl) of various changes of lipophilic groups can identify target protein (CAP) and lipophilic chain (the modification, Linker) were synthesized in three categories a total of 48 new compounds, and their structures were determined by MS and 1H-NMR. The functional groups in combination with zinc ion (ZBG) modified by taking into account the type of inhibitor molecules in ZBG The formation of zinc ion and chelate ring size is different, if we change the size of the chelate ring, may change the ZBG and zinc ion binding strength, and thus affects the inhibitor activity. Therefore, we designed and synthesized three series of target compounds I, II, III, the zinc ion with the catalytic activity of HDAC in the center region of the formation of seven yuan, six yuan, five yuan of chelate ring, and evaluate these new compounds of anti latent activity, so as to understand the relationship between ZBG and its anti latent activity. Two fragments of antithetical couplet aromatic structure (Diaryl) modification we designed and synthesized B ring was replaced by other in order to increase the affinity and molecular groups of small molecule inhibitors and the target surface shape of the complementary series of compounds, including the introduction of four yuan: five yuan saturated heterocycles, unsaturated heterocyclic aromatic ring, six yuan and six yuan even para substituents The aromatic ring. The lipophilic group (CAP) and lipophilic chain (Linker) CAP part of the modified precursor of Lead is very small, we only introduce smaller groups, maintaining the original small molecular and CAP target binding mode region, and for Linker, we pass bioisoterism substitution to the body we have modified. Of the 48 target compounds at the cellular level and the enzyme level of anti latent activity evaluation, 5 compounds (5a, 5b, 29a, 45A, 45B) anti latent activity and positive control SAHA, compound 5A anti latent activity is best, and keep the characteristic of low toxicity the SAHA is better than the positive control, the significance of further research. In summary, has certain reference significance of anti latent research this part of the study on the future benzamide compound, but also for the study of the anti latent mechanism of the foundation. At present, we are on The mechanism of anti latent synthetic compounds 5A in-depth research, in order to find new benzamides HIV efficient low toxicity latent activator, and ultimately activate the latent virus library. In two, 4- -1,5- two substituted aryl amine inhibitor drug like related research work on the basis of the theory of "destroy" activation, activation in the virus database, focuses on the transformation for the virus. The early elimination of the experimental data show that we have found with benzamide compounds with good activation activity for the virus, anti HIV therapy, "activate" steps have been completed, the treatment work focus to "destroy" steps, design and synthesis of virus inhibitors determination of drug like activity, and evaluation. The current clinical anti HIV drugs are mainly divided into four categories: reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, entry inhibitors. We found A series of highly active non nucleoside reverse transcriptase inhibitors (HIV-NNRTIs), namely 4- -1,5- two substituted aryl amine compounds. The experimental data show that this kind of 4- to replace the anti HIV activity of superior clinical medication -1,5- two aryl amine compounds, it is necessary for its further development. We through continuous optimization of mass spectrometry and chromatographic separation conditions a method, a complete set of LC-MS/MS quantitative, its strong specificity, high sensitivity, low detection limit, the analysis speed is very fast, can we replace the -1,5- two series 4- aryl amine compounds for drug metabolism test, also laid the foundation for our later research to drug metabolism in vivo. Rich 4- to replace -1,5- two aryl amine compounds on the pharmacokinetics of the nature of the information, we selected human liver microsomes, liver S9, plasma respectively, of which 23 good activity -1,5- two 4- substituted aryl Amine compounds were studied in vitro pharmacokinetic stability, complete the evaluation of this compound in vitro pharmacokinetics, and were obtained from the human liver microsomes in human liver, S9, in human plasma elimination half-life, thereby providing information pharmacokinetic properties of the complexes differentiation for us, too provides guidance for optimization of our late leader, we also continue to promote the in vivo pharmacokinetic study on stability of work. At present, the progress of research have been organized into the relevant article.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R914;R96

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