苯酰胺类HIV潜伏激活剂的设计、合成及活性评价

发布时间：2018-03-27 13:06

本文选题：激活消灭　切入点：HDAC抑制剂　出处：《中国人民解放军军事医学科学院》2017年博士论文

【摘要】：基于人类免疫缺陷病毒(HIV)治疗方案中的“激活消灭”学说,本论文首先研究了苯酰胺类HIV潜伏激活剂(有效激活)的设计合成、活性评价;随即探究了配合潜伏激活剂联合用药的4-取代-1,5-二芳基胺类化合物(有效消灭)的活性及类药性等性质,旨在实现对病毒潜伏库先激活,后消灭的目的。一、苯酰胺类病毒潜伏激活剂的相关研究工作自1981年HIV在美国被首次发现,30多年间,已蔓延到各大洲,感染数千万人,造成社会负担及经济损失。高效抗逆转录病毒疗法(HAART)是现在临床上最有效的抗HIV/AIDS治疗方案,艾滋病患者在接受HAART治疗后可将血浆病毒载量降到不可检测的水平。但在停止HAART治疗后,其病毒载量将会迅速反弹,导致艾滋病患者必须终生服药。HIV的反弹主要是由于病毒潜伏在细胞的病毒库内所造成,且该潜伏库不能被HAART清除。HIV潜伏库的存在,是HIV难以被彻底清除的根本原因。基于“激活消灭”学说,我们首先设计合成了系列抗潜伏的化合物,以期激活潜伏的HIV。北卡大学李国雄课题组在研究中发现了一个很有特点的抗潜伏激活剂:苯酰胺类HDAC抑制剂Lead,它能在低浓度下激活潜伏HIV而无毒副作用,较目前已经进入临床研究的抗潜伏药物SAHA可能具有更优良的药效。因此,我们通过设计合成系列新衍生物,期望提高其抗潜伏活性,使之达到良好的激活效果。作为探索性研究,我们依据合理的设计思路,采用计算机辅助设计、生物电子等排原理、骨架跃迁及活性数据引导等多种方法相结合的手段,通过对合成方法的不断优化和对反应条件的不断改良,对前期研究中发现的抗潜伏先导化合物苯酰胺类HDAC抑制剂Lead的不同部位进行了修饰,包括对与锌离子结合的基团(ZBG)的环大小的改变、对与靶标袋状空腔相互作用的联二芳烃结构片段(Diaryl)的多种改变、对可识别蛋白靶标的亲脂性基团(CAP)和亲脂性链(Linker)的修饰,共合成了三大类全新化合物共48个,其结构均经过MS、1H-NMR确定。对与锌离子结合的功能基团(ZBG)的修饰考虑到该类抑制剂分子中ZBG与锌离子螯合形成的环的大小的不同,我们设想:如果改变螯合环的大小,或许可以改变ZBG与锌离子间结合的强度,从而对抑制剂的活性产生影响。因此,我们设计合成了三个系列的目标化合物I、II、III,其可分别与HDAC催化活性中心区域的锌离子形成七元、六元、五元螯合环,并评价了这些新化合物的抗潜伏活性,从而了解了ZBG与其抗潜伏活性之间的关系。对联二芳烃结构片段(Diaryl)的结构修饰我们设计合成了将B环替换为其他基团以增加小分子抑制剂与靶标的亲和力和分子表面的形状互补性的系列化合物,包括引入:四元饱和杂环、五元饱和杂环、六元芳香环以及对位连有取代基的六元芳香环。对亲脂性基团(CAP)和亲脂性链(Linker)的修饰先导物Lead的CAP部分很小,我们只引入了较小的基团,保持了分子原来较小CAP部分与靶标结合区域的结合模式,而对于Linker,我们通过生物等排体的替换来进行了修饰。我们又对这48个目标化合物分别在细胞水平和酶水平上进行了抗潜伏活性评价,5个化合物(5a、5b、29a、45a、45b)的抗潜伏活性与阳性对照SAHA相当,其中化合物5a的抗潜伏活性最好,且保持毒性较低的特点,优于阳性对照SAHA,具有进一步研究的意义。综上,此部分的研究对今后苯酰胺化合物的抗潜伏研究具有一定借鉴意义,同时也为研究其抗潜伏机制奠定了基础。目前,我们正在对合成的化合物5a的抗潜伏机制进行深入研究,以期寻找到高效低毒副作用的新型苯酰胺类HIV潜伏激活剂,最终实现激活病毒潜伏库的目的。二、4-取代-1,5-二芳基胺类病毒抑制剂的类药性相关研究工作基于“激活消灭”学说,在病毒库激活后,研究重点转变为病毒库的消灭。前期实验数据表明,我们已找到具有较好激活病毒库活性的苯酰胺类化合物,对于抗HIV治疗来说,“激活”步骤已经完成,治疗工作重点转移至“消灭”步骤,即病毒抑制剂的设计合成、活性测定及类药性评价。现行临床抗HIV药物主要分四大类:逆转录酶抑制剂、蛋白酶抑制剂、整合酶抑制剂、进入抑制剂。我们又发现了系列高活性的非核苷类逆转录酶抑制剂(HIV-NNRTIs),即4-取代-1,5-二芳基胺类化合物。实验数据表明,这类4-取代-1,5-二芳基胺类化合物的抗HIV活性优于临床用药,对其进一步的开发非常有必要。我们通过不断优化质谱条件和色谱条件,建立了一套完整的LC-MS/MS定量分析方法,其专属性强,灵敏度高,检测限低,分析速度非常快,可以对我们的系列4-取代-1,5-二芳基胺类化合物进行药物代谢方面的测试,也为我们后期开展体内药物代谢相关研究奠定了基础。为了丰富4-取代-1,5-二芳基胺类化合物的药动性质信息,我们选用人肝微粒体、人肝S9、人血浆分别对其中23个活性较好的4-取代-1,5-二芳基胺类化合物进行了体外药代稳定性的初步研究,完成了这部分化合物的体外药代动力学的评价工作,并分别获得了其在人肝微粒体、人肝S9、人血浆中的消除半衰期,从而为我们提供了这部分化合物的药代性质信息,也为我们的后期先导物优化提供了指导,我们也在不断推进后期的体内药代稳定性研究工作。目前,阶段性研究进展已整理成相关文章发表。
[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.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R914;R96

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