基于靶标的HIV-1非核苷类逆转录酶抑制剂的设计、合成及活性研究

发布时间：2018-03-07 09:31

本文选题：艾滋病　切入点：HIV-1逆转录酶　出处：《山东大学》2014年博士论文　论文类型：学位论文

【摘要】：人免疫缺陷病毒1型(HIV-1)是艾滋病的主要病原体。自从1981年发现以来,目前已经成为危害人类生命健康的重大传染性疾病。虽然高效抗逆转录疗法(Highly Active Antiretro viral Therapy, HAART)的实施是抗艾滋病治疗的一项重大突破,但是耐药性的出现及长期服药的毒性问题极大地限制了该疗法的应用,因此新型抗艾滋病药物的研发依然刻不容缓。 HIV-1非核苷类逆转录酶抑制剂(NNRTIs)是HAART疗法的重要组成部分。NNRTIs具有结构多样性,作用于HIV-1逆转录酶(Reverse Transcriptase, RT)的疏水性口袋。该类药物具有高效低毒、特异性强的优点,然而易产生耐药性的缺陷使该类药物迅速丧失临床效价。因此新型、高效、低毒、广谱抗耐药性的NNRTIs的研发是目前抗HIV药物研究的热点之一。由于NNRTIs结合口袋(Non-nucleoside Inhibitor-Binding Pocket, NNIBP)是在NNRTIs的存在下诱导产生的,因此完全基于HIV-1RT的三维结构进行全新抑制剂设计还存在较大困难。故选择研发前景较大的化合物为先导,在对其构效关系及结合模式分析的基础上,综合运用结构生物学信息、计算化学技术及传统药物化学策略进行先导化合物的优化,是当前发现新一代NNRTIs药物的有效途径。本论文对三类HIV-1RT非核苷类逆转录酶抑制剂进行了研究。一、硝基吡啶类DAPYs非核苷类逆转录酶抑制剂的研究二芳基嘧啶类衍生物(DAPYs)是一类非核苷类HIV-1逆转录酶抑制剂,因其高效低毒、抗耐药性的特点,近年来成为抗HIV药物研发的热点领域。2008年FDA批准上市的新一代NNRTIs Etravirine(TMC125)及2011年批准的Rilpivirine(TMC278)都属于DAPY类化合物。本论文基于DAPY类逆转录酶抑制剂的构效关系和DAPYs/HIV-1RT复合物晶体结构的分析,应用生物电子等排原理,将原本的嘧啶环替换为硝基吡啶环,设计了一系列硝基吡啶类DAPY系列衍生物(7a-7r),并对其进行了定向合成。该系列共合成了18个化合物,所有合成的化合物的结构均经波谱分析验证。对所合成的化合物应用MTT法进行了体外抗野生型HIV-1(ⅢB).HIV-2(ROD)及HIV-1突变毒株的细胞活性测试。其中,化合物7b显示出最佳的抗病毒活性(EC50=0.056μM,SI=1251).此外,化合物7k(EC50=0.034μM,SI=691),化合物7c(EC50=0.11μM,SI:339)和7h(EC50=0.17μM,SI=97)活性均比对照药物NVP和DLV高。选取活性较好的化合物7b、7c和7k进行了HIV-1逆转录酶的抑制活性测定,抑制活性分别为6.9μM.8.5μM和10.4μM,显示出了该类化合物对HIV-1RT选择性的抑制作用。利用DOCk分子对接方法,将活性最好的化合物7b和7k与野生型HIV-1RT和突变型的HIV-1RT分别进行分子模拟,并对该系列化合物与HIV-1RT的相互作用及构效关系进行了初步分析,为未来设计新型的高效低毒的DAPYs非核苷类逆转录酶抑制剂提供了有益的信息。二、噻唑烷酮类非核苷类逆转录酶抑制剂的研究噻唑烷酮类化合物是一类结构新颖的NNRTIs,经结构的修饰和改造,发现了很多具有很高抗HIV-1活性的噻唑烷酮类化合物,具有非常广阔的研发前景。硫代羧酰苯胺类衍生物(thiocarboxanilide)UC-781是一个非常有效的HIV-1RT抑制剂候选药物(EC50=0.002μ.M,SI=50000).UC-781可以降低病毒颗粒的感染力,被认为是预防HIV-1传播的很有希望的化合物。此外,该化合物具有良好的抗耐药谱,对多种突变毒株RT的抑制效果与对野生型HIV-1RT的接近,且可以在高剂量水平抑制NNRTIs耐药病毒株而无任何细胞毒性。以噻唑烷酮类NNRTIs和UC-781为先导物,借鉴两类NNRTIs的结构特征及与RT的结合模式,结合两类化合物的相似点,运用分子杂合的药物设计原理,合理地引入活性取代基,保留了先导化合物UC-781的异戊烯基基团,设计了一系列与靶点紧密结合的新型噻唑烷酮类系列化合物。并对目标化合物进行了定向合成,所有合成的化合物的结构均经波谱验证。应用MTT法对所合成的化合物进行了体外抗野生型HIV-1(ⅢB)、HIV-2(ROD)细胞活性测定。试验结果表明,部分噻唑烷酮类化合物表现出了一定的活性,但细胞毒性略高。将其中活性最好的化合物与HIV-1RT进行分析对接,对该类化合物的构效关系进行了初步分析,初步阐明了该类化合物与HIV-1RT的结合位点与构象,解释活性略低的可能原因,为进一步结构的优化提供新的思路。三、DABOs类非核苷类逆转录酶抑制剂二氢烷氧苄基嘧啶酮(Dihydro-alkylthio-benzyl-oxopyrimidines, DABOs)衍生物为其中较为典型的一类NNRTIs,由于其分子构象具有柔性和结合位点中具有适配性的特征,可有效地抑制野生型和耐药型病毒株的复制,成为目前抗HIV药物研究的重要方向。本论文基于DABO类抑制剂的构效关系,结合计算机对接模型,依据分子杂化的药物设计原理,以取代哌啶环来延长侧链的S-DABOs衍生物,通过变化哌啶环N原子上取代基、母环的5位、6位取代基来考察化合物各位点对活性的影响,并以未连有哌啶环的另一系列化合物作为对照共合成了两个系列化合物。为了从理论上验证设计思想的合理性,对设计的化合物进行了对接分析,分析结果表明目标分子的结构修饰具有理论上的合理性。最后根据虚拟筛选的结果,从高到低打分,确定并合成了36个目标化合物,并选取打分较高的化合物进行了HIV-1RT的活性测定。活性结果显示,化合物8a3-2、8a2-1和8b3-2对HIV-1RT的抑制活性分别为1.58μM、14.61μM和13.71μM,显示出了一定的HIV-1RT抑制活性,其中在虚拟筛选中打分最高的化合物8a3-2(IC50=1.58μM)的活性高于对照药奈韦拉平(IC5o=3.93μM),并与先导化合物TMC125(IC5o=1.05μM)类似,表现了较高的HIV-1RT抑制活性。总之,本论文以高效抗耐药的DAPYs、噻唑烷酮类和DABOs三类NNRTIs先导化合物为模板,在前人研究的基础上,结合构效关系结论及药效团特征,分别根据药物设计中的生物电子等排原理和分子杂化原理,对先导化合物进行了结构多样的骨架变换,并应用药物设计软件进行虚拟筛选。总共设计合成了四个系列结构全新的化合物。对目标化合物进行了抗HIV活性筛选,发现其中部分化合物的抗HIV-1(IIIB)活性达到或超过上市药物奈韦拉平与地拉韦定,具有进一步研究与开发价值。此外,还对目标化合物进行了计算机分子对接研究,根据其与HIV-1RT的结合模式对化合物的构效关系进行了初步分析,为进一步的研究提供了有利的信息。
[Abstract]:Human immunodeficiency virus type 1 (HIV-1) is the main pathogen of AIDS. Since discovered in 1981, has become the major infectious diseases endangering human life and health. Although effective antiretroviral therapy (Highly Active Antiretro viral Therapy, HAART) is a major breakthrough in the implementation of anti HIV treatment, but the emergence of drug resistance and toxicity problems the long-term medication greatly limits the application of the therapy, so the development of the new anti AIDS drugs is still urgent.
HIV-1 non nucleoside reverse transcriptase inhibitor (NNRTIs) is an important part of HAART therapy.NNRTIs has structural diversity, in the role of HIV-1 reverse transcriptase (Reverse Transcriptase, RT) of the hydrophobic pocket. The drugs with high efficiency and low toxicity, strong specificity, but drug-resistant defects make the drugs rapidly lost clinical value of model. Therefore, high efficiency, low toxicity, broad-spectrum drug resistance research of NNRTIs is one of the hot study of anti HIV drugs at present.
Due to the NNRTIs binding pocket (Non-nucleoside Inhibitor-Binding Pocket, NNIBP) is induced in the presence of NNRTIs, thus complete the three-dimensional structure of HIV-1RT based on a new inhibitor design still has many difficulties. The choice of R & D promising compounds as first, in the structure-activity relationship and based on the analysis of the model, the integrated use of the structure of biological information, optimization strategy of chemical technology and traditional medicine chemical compounds, is an effective way for a new generation of NNRTIs drug discovery at present. This paper focuses on three classes of HIV-1RT non nucleoside reverse transcriptase inhibitors were studied.
A study of nitropyridine DAPYs non nucleoside reverse transcriptase inhibitors
Two aryl pyrimidine derivatives (DAPYs) is a kind of HIV-1 non nucleoside reverse transcriptase inhibitors, because of its high efficiency and low toxicity, anti drug, anti HIV drugs in recent years become a hot spot in research field of.2008 FDA approved the listing of the new generation of NNRTIs Etravirine (TMC125) Rilpivirine and approved in 2011 (TMC278) belong to DAPY compounds. This paper analysis of structure effect relationship and DAPYs/HIV-1RT crystal structures of complexes of DAPY reverse transcriptase inhibitors based on the application of the principle of bioisosterism, originally a pyrimidine ring substituted for nitro pyridine ring derivatives, a series of nitro pyridine DAPY series design (7a-7r), and has carried on the synthesis. This series of 18 compounds were synthesized, all structures of the synthesized compounds were confirmed by spectral analysis.
The application of MTT compound prepared by the method of in vitro anti wild type HIV-1 (B III).HIV-2 (ROD) and HIV-1 mutant strain cell activity test. Among them, compound 7b shows the best antiviral activity (EC50=0.056 M, SI=1251). In addition, the compound 7K (EC50=0.034 M, SI=691, 7C) compounds (EC50=0.11 M, SI:339) and 7h (EC50=0.17 M, SI=97) activity was higher than that of the control drugs NVP and DLV. Choose good activity compounds 7b, 7C and 7K were determined by reverse transcriptase HIV-1 inhibitory activity, inhibitory activity of 6.9 M.8.5 M and 10.4 M, the inhibitory effect of the compounds on the selectivity of HIV-1RT display.
Using DOCk molecular docking method, the best activity of compound 7b and 7K with wild-type HIV-1RT and mutant HIV-1RT were molecular simulation, and the interaction of these compounds with HIV-1RT and structure-activity relationship were analyzed, provide useful information for the future design of high efficiency and low toxicity of new non nucleoside DAPYs reverse transcriptase inhibitors.
Two, a study of thiazolidone non nucleoside reverse transcriptase inhibitors
Thiazolidinones are a kind of novel structure of NNRTIs, the modification of the structure, found a lot of high anti HIV-1 activity of thiazolidinones, has very broad prospects for development. Thio carboxylic acylaniline derivative (thiocarboxanilide) UC-781 is a very effective drug candidate (HIV-1RT inhibitor EC50=0.002.M, SI=50000).UC-781 can reduce the infectious virus particles, considered compounds to prevent the spread of HIV-1 is very promising. In addition, the compound has good anti drug resistance, inhibition effect on a variety of mutant strain RT and the close of wild type HIV-1RT, and can inhibit the NNRTIs resistance at high dose levels virus without any cytotoxicity.
The thiazolidinone class NNRTIs and UC-781 as the lead compound, combined with the structure characteristics of two types of reference mode of NNRTIs and RT, with two kinds of compounds are similar, using the principle of molecular drug design heterozygous, reasonably introducing reactive substituents, retains the prenyl groups of lead compounds of UC-781, combined with a series of with the target of new thiazolidinones compound series is designed. And the target compounds were directed synthesis, structure of all the synthesized compounds were confirmed by spectral verification. By applying the MTT method against wild type HIV-1 in vitro of the synthesized compounds (B III), HIV-2 (ROD) cell activity assay. The test results that part of thiazolidinone compounds exhibited certain activity, but slightly higher. The cytotoxicity of compounds with HIV-1RT activity were analyzed for the best docking, the structure-activity relationship of these compounds were preliminary The binding sites and conformations of these compounds with HIV-1RT were preliminarily elucidated, and possible reasons for their low activity were explained, providing new ideas for further structural optimization.
Three, DABOs non nucleoside reverse transcriptase inhibitors
Two hydrogen alkoxy benzyl pyrimidine ketone derivatives (Dihydro-alkylthio-benzyl-oxopyrimidines, DABOs) for a class of NNRTIs which is more typical, because of its molecular conformation and binding sites with flexible adaptation features, can effectively inhibit the wild-type and drug-resistant strains of the virus replication, has become an important direction of the present study of anti HIV drugs.
The structure-activity relationship of DABO inhibitors based on the combination of computer docking model, based on the principle of drug design, molecular hybrid, S-DABOs derivatives to replace the piperidine ring to extend side chain, through the change of the piperidine ring substituent on the N atom, 5 ring, 6 substituents to examine all compounds on the activity the influence and not even a series of compound piperidine ring as the control of the two series of compounds were synthesized. In order to verify the rationality of design ideas from the theory, to design the compound for the docking analysis, structural modification analysis result of standard molecular eyesight have rationality in theory. According to virtual screening the results, from high to low scoring, and determine the 36 target compounds were synthesized, and selected the high scoring compounds were determined by HIV-1RT activity. The activity results showed that compounds 8a3-2,8a2-1 and 8b3-2 on H The inhibitory activity of IV-1RT were 1.58 M, 14.61 M and 13.71 M, showing a certain HIV-1RT inhibitory activity, which in the virtual screening scoring highest compound 8a3-2 (IC50=1.58 M) activity was higher than that of the control Po Nai Vee Lapin (IC5o=3.93 M), and TMC125 (IC5o=1.05 M) lead compounds similar that showed higher HIV-1RT inhibitory activity.
In a word, in this dissertation, resistance DAPYs, NNRTIs lead compounds of thiazolidinones and DABOs three as the template, on the basis of previous studies, combined with the conclusion of structure-activity relationship and pharmacophore features, respectively, according to the principle of bioisosterism and molecular hybridization principle in drug design, the framework of lead compound transform structure diversity, and virtual screening of drug design software. A total of four series of design structure of the new compounds were synthesized. The target compounds were screened for anti HIV activity, found that some of them were anti HIV-1 (IIIB) activity reached or exceeded the listed drug nevirapine and delavirdine, with further research and the development of value. In addition, the target compounds were studied by computer molecular docking, according to the binding mode with HIV-1RT on their structure-activity relationship were analyzed for Further research provides favorable information.

【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】：R96;R914.5

【共引文献】