【作者】 王珺；

【导师】 刘新泳；

【作者基本信息】 山东大学 ， 药物化学， 2014， 博士

【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。更多还原

【Abstract】 The human immunodeficiency virus type1(HIV-1) is the main cause of the acquired immunodeficiency syndrome (AIDS), which was first identified in the Western world in1981. Since then, AIDS has developed into a worldwide pandemic of disastrous proportions. Considerable progress has been made in treating HIV-infected patients using highly active antiretroviral therapy (HAART) involving multidrug combinations. However, the increasing incidence of drug resistant viruses along with the drug toxicity among treated people calls for continuous efforts of developing anti-HIV-1drugs.HIV-1reverse transcriptase (RT) is one of the main targets for the action of anti-AIDS drugs. Drugs targeted at HIV RT can be divided into two categories:(ⅰ) nucleoside and nucleotide analogue RT inhibitors (NRTIs/NtRTIs), which, following activation to their triphosphate forms, compete with the RT substrate and also act as terminators of DNA synthesis after incorporation into the primer strand; and (ⅱ) nonnucleoside RT inhibitors (NNRTIs), including the approved drugs nevirapine, delavirdine, efavirenz and etravirine, which, although having wide structural variation, all bind at a similar site distal to the active site within RT. NNRTIs currently in clinical use have a low genetic barrier to resistance and therefore, the need for novel NNRTIs active against drug-resistant mutants selected by current therapies is of paramount importance.In recent year, in spite of the rapid growth of HFV-1RT3D-structural information, the difficulty in structure-based de novo design of NNRTIs scaffolds and docking based virtual screening approach lies in the following two aspects:i) The flexibility of NNIBP, formed by conformational changes in the RT on binding of the NNRTI ligand; ii) The NNRTI resistance mutations located in and around the NNIBP. Therefore, structure-based and ligand-based combined drug design methodology was carried out to facilitate both drug lead generation and lead optimization. And considerable cases illustrated the benefits for NNRTIs design of closely coupled traditional medicinal chemistry, structural biology, computational chemistry methodology, and many others.As a continuation of our efforts to discover and develop back-up analogs of DAPYs, novel substituted nitropyridine derivatives were designed via a structure-based core refining approach, synthesized and evaluated for their in vitro HIV-1activity in MT-4cells. Preliminary biological evaluation indicated that most of the compounds exhibited marked inhibitory activity against wild-type HIV-1IIIB. Most notably, the compound7b was identified as the most promising candidate in inhibiting HIV-1replication with an EC50value of0.056μM and a selective index (SI) of1251,7k (EC50=0.034μM, SI=691), compound7c (EC50=0.11μM, SI=339) and compound7h (EC50=0.17μM, SI=97) were more active than reference drugs NVP(EC50=0.23μM) and DLV(EC5o=0.51μM) against wt HIV-1(ⅢB). Some antivirally active compounds also showed moderate inhibitory activity against RT. Preliminary structure-activity relationships (SARs) and molecular modeling of these new analogs provide valuable avenues for future molecular optimization.Substituted2,3-diaryl-1,3-thiazolidin-4-one derivatives represented a new class of specific HIV-1NNRTIs. Thiocarboxanilide derivatives UC781,(N-[4-chloro-3-(3-methyl-2-butenyloxy)pheny1]-2-methyl-3-furancarbothiomide), is a extremely potent inhibitor of human immunodeficiency virus type1(HIV-1) reverse transcriptase (RT) with a favorable resistance spectrum. The EC50and SI of UC781inhibited of HIV-1replication in cell culture were0.002μM and50000, respectively. UC781targeted HIV-1RT and had extremely low and almost similar EC50values against wild-type HIV-1and a series of mutant viruses in their RT and restored the anti viral activity of AZT to AZT resistant HIV-1strains. Based on the general crystal structure of the HIV-1RT complexed with NNRTIs, which is like a "butterfly"type, and furthermore,2,3-diaryl-1,3-thiazolidin-4-one and UC-781are served as templates, and according to the general principle of bioisosteric replacement in medicinal chemistry, we designed and synthesized a series of novel2,3-diaryl-1,3-thiazolidin-4-one derivatives in which the2,6-dihalophenyl ring at C-2was retained as an essential moiety, whereas the pyridine ring was replaced by a suitably substituted benzene ring. All the newly compounds are screened for anti-HIV activity in vitro. The results showed that some compunds exhibited inhibition against HIV-1replication, Structure-activity relationship analysis revealed that the different substitute aromatic ring played an important role in affecting the anti-HIV activities. Structure-activity relation was discussed and some useful information was obtained in the design and development of new HIV-1NNRTIs.Among the most representative classes of NNRTIs, DABOs (dihydro-alkylthio-benzyl-oxopyrimidines) occupy a relevant position. Owing to their structures, DABOs can adapt to changes in the binding pocket due to mutations. So DABOs can inhibit the mutation of the target and also inhibit the emergence of drug resistance. Based on computer aided drug design (CADD), we have designed and synthesized novel DABOs derivatives:piperidine-linked S-DABO analogues. We have also used molecular docking to study the relationship between compounds and HIV-1RT, which provide clear guideline and actual activity predicitions for novel HIV-1RT inhibitors. The selected target molecules based on docking results of virtual screening were synthesized. The preliminary activity and cytotoxicity screening of the newly designed and synthesized target compounds S-DABOs for inhibition of HIV-1(strain IIIB), HIV-2(strain ROD) and HIV-1mutant virus strains are in progress.In summary, taking the DAPYs,2,3-Diaryl-1,3-thiazolidin-4-one derivatives and DABOs NNRTIs as leads, four series of novel NNRTIs were designed and synthesized in this dissertation according to bioisosterism principle. The new, simple, and convenient synthetic approaches to the title compounds were developed, or improved and optimized. Lastly, through biological evaluation, we find many high potent antiviral agents, which are worth further investigation and development. We hope that the knowledge and insight on the NNRTIs research learnt from the work will help a lot on the battle against the virus and benefit human health and life.更多还原