【作者】 展鹏；

【导师】 刘新泳；

【作者基本信息】 山东大学 ， 药物化学， 2010， 博士

【摘要】 艾滋病(Acquired Immunodeficiency Syndrome, AIDS)的主要病原体是人免疫缺陷病毒1型(Human Immunodeficiency Virus type 1, HIV-1)。自从1981年被发现以来,艾滋病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 Antiretroviral Therapy, HAART)的实施是抗艾滋病治疗的一项重大突破,但是病毒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。因此研发新型的抗艾滋病药物依然是当前一项重大的科研任务。HIV-1非核苷类逆转录酶抑制剂(Non-nucleoside Rreverse Transcriptase Inhibitors, NNRTI)是HAART疗法的重要组成部分。NNRTI具有结构多样性,但都作用于HIV-1逆转录酶(Reverse Transcriptase, RT)的疏水性口袋。该类药物具有高效、低毒的优点,但是耐药毒株的蔓延使该类药物迅速丧失临床效价。因此新型、高效、低毒、广谱抗耐药的NNRTI的研发是目前抗HIV药物研究的重要方向之一。在目前的非核苷类抗艾滋病药物研究领域,由于NNRTI结合口袋(NNRTI Binding Pocket, NNIBP)本身不存在(需要NNRTI诱导产生)、结构柔韧性较强及组成的氨基酸极易突变等限制性因素,使精确预测NNRTI的结合模式、完全基于HIV-1 RT的三维结构进行全新抑制剂设计还存在较大困难。因此,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NNRTI药物的有效途径。三唑／四唑巯乙酰胺类化合物是通过高通量筛选(High-throughput Screening,HTS)得到的一类结构新颖的NNRTI。在细胞水平的活性测试中,大多数衍生物在亚微摩尔浓度就产生较强的抗HIV活性。体外实验发现,该类化合物不但对野生型RT具有较强的抑制活性,而且对K103N-Y181C双突变的HIV-1 RT也具有很高的活性,因此三唑／四唑巯乙酰胺类化合物是非常具有研发前景的一类NNRTI。本论文即是以此类NNRTI为先导化合物,在前期初步构效关系及结合模式分析的基础上,根据“生物电子等排”、“骨架跃迁”及“多靶点配体设计”等药物设计基本原理,对先导化合物中的“杂环支架及氢键作用区”进行广泛的结构变换,构建全新的结构骨架。并以新设计的“等排”、“跃迁”及多靶点“杂合”骨架为母核,优选先导化合物中的活性基团,对先导化合物中的“疏水作用区”及“开口区”进行系统的结构修饰,构建起结构多样的虚拟化合物库。通过基于分子对接的虚拟筛选,对预测活性较高的化合物实施定向合成,并经体外抗HIV-1活性实验,由此发现高效低毒、抗耐药性且具有自主知识产权的的新型NNRTI,为该类抗艾滋病药物的研发奠定基础。目标化合物的合理设计1)基于HIV-1 RT与NNRTI复合物的三维结构以及NNRTI药效团的构成要素(支架／氢键作用区、疏水作用区及“开口区”),在对三唑／四唑巯乙酰胺类先导化合物构效关系和分子模拟的基础上,按照“生物电子等排”原理,对五元芳杂环进行结构多样性的变换,构建了1,2,3-噻二唑、1,2,3-硒二唑、咪唑及新三唑等4类新型的唑类巯乙酰胺骨架,并在“开口区”引入多种含N、S原子的杂环基团,以增强该亲水区域的作用力。此外,尽管嗪类杂环作为唑类杂环的非经典的电子等排体,但是它们具有非常类似的电子排布及生物学功能。为了进一步考察杂环的种类对活性的影响,本论文还设计了全新的芳嗪类(如哒嗪、三嗪等)巯乙酰胺骨架。2)根据先导化合物的药效团特征及“骨架跃迁”(Scaffold Hopping)概念,对芳唑巯乙酰胺类NNRTI的“杂环支架及氢键作用区”作进一步改造,设计了杂环剖开或取代、且结合模式类似的“跃迁骨架”：①用磺酰胺基团代替唑类杂环,羰基氧原子模拟唑类杂环上的N、S等杂原子,作为氢键的受体。并在连接链引入新的构象限制性基团,保持与先导化合物具有类似的分子自由度及活性构象。据此,设计了氨基磺酰胺及磺酰胺吡咯烷“跃迁”骨架。②通过对先导化合物分子模拟,并结合具有类似结合模式的二芳基嘧啶类NNRTI的结构特征,在唑环上引入氨基、卤素等取代基,以增强该区域与结合位点氨基酸主链形成氢键或卤键的能力。设计了杂环上连有氨基或卤素的取代噻唑巯乙酰胺类结构骨架。3)多靶点抑制剂研究已成为当前新药研发的新策略,也是抗艾滋病药物研究的新热点。根据靶点的结构生物学信息及先导化合物的构效关系,利用“药效团整合”的方法,并借助计算机手段进行合理设计是发现新型双靶点HIV抑制剂的最有效途径。在对芳基二酮类HIV整合酶抑制剂及芳唑巯乙酰胺类NNRTI构效关系及结合模式分析的基础上,结合两类抑制剂结构的相似性,充分利用每类抑制剂中的可修饰位点,通过药效团整合的策略,构建新型的芳唑巯乙酰胺二酮结构骨架。4)以上述新设计的“等排”、“跃迁”及多靶点“杂合”骨架为母核,优选先导化合物中的活性基团,构建起结构多样的虚拟化合物库。借鉴基于分子对接的虚拟筛选结果来确定拟合成目标化合物的结构。目标化合物的合成通过对目标分子的结构进行逆合成分析,确定化学合成路线。目前已合成1,2,3-噻二唑、1,2,3-硒二唑、咪唑、三唑、哒嗪、三嗪、取代噻唑巯乙酰胺、氨基磺酰胺乙酰胺及含有二酮酸基团的芳唑巯乙酰胺等9大系列200余个结构全新的目标化合物,其制备方法未见文献报道,具有较高的创新性,已申请多项国家专利。已完成三嗪巯乙酰胺类NNRTI母核结构硫代三嗪酮的合成。目标化合物的活性筛选将设计合成的部分芳唑巯乙酰胺类衍生物进行了初步的抗HIV-1(ⅢB)及抗HIV-2 (ROD)舌性筛选。结果表明,在1,2,3-噻二唑及咪唑巯乙酰胺衍生物中,大多数目标化合物都具有较高的抗HIV-1活性。共有13个化合物的EC50值低于(或接近)上市药物奈韦拉平与地拉韦定(阳性对照药)；多数1,2,3-硒二唑巯乙酰胺衍生物活性较低,且细胞毒性较高；在新型三唑巯乙酰胺系列中,有多个化合物对野生型HIV及E138K、K103N及L100I耐药株具有显著的抑制活性,EC50处在微摩尔水平；在氯代噻唑球乙酰系列化合物中共有4个化合物(IX-B8a、IX-B8e、IX-B8f、IX-B8g)的EC50值达到微摩尔水平。而且化合物IX-B8e、IX-B8f、IX-B8g能有效地抑制耐药株(E138K、K103N、L100I);芳唑巯乙酰胺-二酮酸类化合物X-3A及X-3B对野生型HIV (ⅢB)毒株及多种耐药毒株(E138K、K103N、L100I、L100I)具有较好的抑制活性,单比先导化合物的活性有所降低；氨基噻唑巯乙酰胺系列及氨基磺酰胺系列化合物丧失抗病毒活性。所筛选的目标化合物对HIV-2(ROD)均无明显抑制活性。此外,一些具有较高活性的目标化合物对多种耐药毒株的活性筛选试验正在进行中,期待有新的发现。本论文还初步探讨了各类目标化合物抑制HIV-1(ⅢB)的构效关系。利用计算机辅助药物设计软件对发现的活性化合物进行了分子模拟(Dock)及3D-QSAR研究,佐证了构效关系结论,为进一步结构修饰提供了合理的指导。本论文对部分目标化合物中进行了抗流感病毒活性的随机筛选,结果发现,化合物IX-A8g和IX-A8h呈现出显著的抗流感病毒活性,而且对甲型H1N1流感病毒毒株有较高的选择性及较强的抑制作用。EC50值比几种对照药物低1-2个数量级,但是这两个化合物的细胞毒性较大,值得进一步结构修饰。总之,本论文以高效抗耐药的先导化合物四唑／三唑巯乙酰胺类NNRTI为模板,在作者前期初步结构修饰的基础上,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”、“骨架跃迁”及“多靶点配体设计”等原理,对先导化合物进行结构多样的骨架变换,并应用药物设计软件进行虚拟筛选,避免了合成的盲口性。本课题总共设计合成了9个系列共200余个结构全新的化合物。对其中的部分目标化合物进行了抗HIV活性筛选及抗流感病毒活性的随机筛选,发现了新型结构骨架的1,2,3-噻二唑巯乙酰胺类及咪唑巯乙酰胺类NNRTI,其中10余个化合物的抗HIV-1(ⅢB)活性达到或超过上市药物奈韦拉平与地拉韦定,并发现了2个对甲型H1N1流感病毒毒株有较高的选择性及较强抑制作用的氨基噻唑类苗头化合物,具有进一步研究与开发价值。更多还原

【Abstract】 The human immunodeficiency virus type 1 (HIV-1) is the main cause of the acquired immunodeficiency syndrome (AIDS), which was first identified in the Western world in 1981. 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-1 drugs.HIV-1 reverse 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 HIV-1 RT 3D-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.From high-throughput screening (HTS) of compounds library, several interesting sulfanyltriazole and sulfanyltetrazole-typed leads were identified as novel NNRTIs, which have simple, yet distinctively different chemical structure from the HIV inhibitors reported in the literature. Extensive structural modification and bioactivity research demonstrated that most of them showed submicromolar activity in a cell assay and significant in vitro activity on the WT or K103N-Y181C HIV-1 RT. With a suitable combination of substitution patterns on the aryl linked to the tetrazole/triazole core (hydrophobic interaction domain), the anilide aryl (the solvent exposed region), five-membered moiety and thioacetamide linker (scaffold and hydrogen bond interaction domain), it is possible to identify compounds which maintain the same intrinsic activity on the wild-type HIV-1 enzyme and the clinically relevant K103N mutant. Based on the above, we hypothesized that alternate, potentially better scaffolds could be designed.Bioisosteric replacement principle is an excellent tool of Medicinal Chemistry for lead optimization to produce the desired potency and selectivity and the requisite ADME profile for a marketable drug. On the basis of extensive SAR and molecular modeling studies of sulfanyltriazole and sulfanyltetrazole-typed NNRTIs, we replace the triazole or tetrazole five-membered core in the leads by other five-membered heterocycles (1,2,3-thiadiazole,1,2,3-selenadiazole, imidazole, triazole) and six-membered heterocycles (pyridazine, triazine) to obtain the novel scaffolds, i.e. Arylazolyl(azinyl)thioacetanilides based NNRTIs.In chemoinformatics, searching for compounds which are structurally diverse and share a biological activity is called scaffold hopping. On account of the structural similarity of NNRTIs families, scaffold hopping or chemotype switching via dismantlement and simplification of known NNRTIs is important since it can be used to obtain alternative structures with improved efficiency and unexpected side-effects. Based on the "scaffold hopping" concept and the pharmacophore characteristic of lead compounds, sulfamoylaminoacetamide and sulfamoyl pyrrolidine-2-carboxamide scaffold were designed, which exhibited similar binding conformation to tetra(tri)azole thioacetanilides. In addition, the SAR analysis and the similarity of molecular modeling of tetra(tri)azole thioacetanilides based NNRTIs and DAPY-based NNRTIs permitted the scaffold hopping to a novel series of substituted thiazole thioacetanilides NNRTIs.Multiple ligands are emerging in anti-HIV drug discovery strategies, using a single entity to inhibit multitargets could yield improved patient compliance, thus reducing the likelihood of drug resistance. The exploration of such multifunctional ligands has proven valuable for anti-HIV leads discovery. Our design began with the general knowledge that a diketoacid (DKA) group and an directly connected aromatic ring are the two indispensable structural features for the DKA class of HIV-1 integrase (IN) inhibitors. The key to multiple ligands design strategy would be to incorporate these two features into a tolerant region in the RT inhibitors. Molecular modelling has shown that the amide connected phenyl group of azole thioacetanilides-based NNRTIs is situated in an open area controlled by the P236 loop where structural modification could be tolerated. Based on these general knowledge and the multiple ligands design strategy, azolthioacetamidophenyl-2-hydroxy-4-oxobut-2-enoic acid-based RT/IN dual inhibitors were designed via incorporation of an IN pharmacophore to this tolerant region of the azole thioacetanilides NNRTIs. Virtual compounds library was constructed by introducing superior substituent group of lead compounds to the designed core:1,2,3-thiadiazole thioacetanilides, 1,2,3-selenadiazole thioacetanilides, imidazole thioacetanilides, triazole thioacetanilides, pyridazine thioacetanilides, triazine thioacetanilides, sulfamoylaminoacetamide and sulfamoyl pyrrolidine-2-carboxamide scaffold, substituted thiazole thioacetanilides and azolthioacetamidophenyl-2-hydroxy-4-oxobut-2-enoic acid-based scaffold. Virtual compounds were evaluated by docking, program, carried out using FlexX, which is a widely used docking algorithm in drug design whose ability in predicting a binding mode of the ligand very close to its X-ray structure has been widely described in literature.In this dissertation,206 title compounds belong to 9 novel scaffolds were designed and synthesized, starting from the commercially available starting material, and were structurally identified by IR, Mass spectroscopy,’H-NMR and/or 13C-NMR spectral analysis respectively. These compounds and the new synthesis approaches have not been reported in literature.The preliminary activity and cytotoxicity screening of the newly designed and synthesized target compounds 1,2,3-thiadiazole thioacetanilides,1,2,3-selenadiazole thioacetanilides and imidazole thioacetanilides were tested in MT-4 cells for inhibition of HIV-1 (strain IIIB) and HIV-2 (strain ROD). Bioactivity assay indicated that most of the title compounds showed good activities against HIV-1 and none of the compounds exhibited inhibitory activity against HIV-2.①In particular,10 compounds in 1,2,3-thiadiazole thioacetanilide (TTA) series showed anti-HIV-1 activities at sub-micromolar concentrations. Interestingly, the cytotoxicity of TTAs was generally low. Owing to the above reasons, their SI values were in many cases similar to that of the reference drug. In particular, compound I-A7c displayed the most potent anti-HIV-1 activity (EC50=36.4 nM), inhibiting HIV-1 replication in MT-4 cells more effectively than NVP (by 7-fold) and DLV (by 8-fold).②The results showed that some 1,2,3-selenadiazole thioacetanilide derivatives, such as II-7f (EC50=2.45μM), possess similar HIV-1 inhibitory activity compared with that of sulfanyltriazole series, but most of these derivatives exhibited decreased anti-HIV-1 specificity due to a significantly increased cytotoxicity. Although the pharmacological results are not very encouraging, this study provides useful information to further design new anti-HIV agents.③Most of the tested imidazole thioacetanilides derivatives inhibited HIV-1 replication in a lower micromolar concentration range. The most potent HIV-1 inhibitors were III-A4e (EC50=0.18μM, CC50=28.81μM, SI=162), andⅢ-A4b (EC50= 0.20μM, CC50=35.24μM, SI=170). The EC50 values of these two compounds were lower than those of one triazole lead compound (EC50=2.053μM) and of the reference drugs NVP and DLV. Other compounds,Ⅲ-A4c,Ⅲ-A4d,Ⅲ-C4e, andⅢ-A4a, also showed higher anti-HIV-1 potency (EC50=0.64,0.73,1.03, and 1.78μM, respectively) compared with the respect to that of the triazole lead compound, indicating that the imidazole is also an acceptable isosteric replacement for the triazole, tetrazole, or 1,2,3-thiadiazole in the lead compounds.④Several compounds in 1,2,4-series and chloro-substituted thiazole series (Ⅳ-7a,Ⅳ-7c,Ⅳ-7e,Ⅳ-7g,Ⅳ-71,Ⅳ-7m andⅨ-B8a,Ⅸ-B8e,Ⅸ-B8f,Ⅸ-B8g) proved to be active against HIV-Ⅰ(Ⅲb). These compounds also showed an appreciable activity against mutant HIV-1 strains (E138K、K103N、L1001).⑤All of the tested compounds in sulfamoylaminoacetamide series, sulfamoyl pyrrolidine-2-carboxamide series and amino-substituted thiazole series lost their anti-HIV activity.⑥Two azolthioacetamidophenyl-2-hydroxy-4-oxobut-2-enoic acid derivatives (Ⅹ-3A andⅩ-3B) retained moderate activity against wild-type HIV-1 and the mutant strains E138K, K103N and L1001. On the basis of the chemical structure and the fact that the target compounds inhibit HIV-1, but not HIV-2 replication, these molecules can be proposed to act as genuine HIV-1 NNRTIs. The preliminary structure-activity relationships (SAR) of the newly synthesized congeners are discussed. Molecular modeling (docking) and 3D-QSAR studies of some potent compounds in complex with HIV-1 RT are described, allowing rationalization of some SAR conclusions. Moreover, the anti-HIV activity against an NNRTI-resistant strain of the newly synthesized derivatives is in progress, from which we are expecting to get some useful information. Some selected target compounds (54 compounds) were randomly tested for their anti-influenza virus activity. Preliminary result showed that two amino-substituted thiazole derivatives (IX-A8g and IX-A8h) displayed potent and selective inhibitory activities against influenza A H1N1 subtype. And these compounds could be used as "hit" compounds to further design influenza inhibitors.In summary, taking the sulfanyltriazole and sulfanyltetrazole-typed NNRTIs as leads, night series of arylazolyl(azinyl)thioacetanilides-based NNRTIs were designed and synthesized in this dissertation according to "bioisosteric replacement" principle, "scaffold hopping" concept and "multiple ligands design" strategy, respectively. 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, the 1,2,3-thiadiazole/imidazole thioacetanilide scaffolds were identified as novel NNRTIs, amino-substituted thiazole derivatives were discovered as promising anti-influenza virus agents in random screening, which are worth further investigation and development. We hope that the knowledge and insight on the NNRTI 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