新型哌啶取代DAPY类HIV-1非核苷逆转录酶抑制剂的设计、合成及活性研究
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摘要
艾滋病全称为获得性免疫缺陷综合征(AIDS),是一种全球流行,且严重威胁人类健康的传染性疾病,其主要病原体为人类免疫缺陷病毒1型(HIV-1)。目前抗艾滋病药物主要通过阻断或干扰HIV-1生命周期中某个环节发挥抑制作用,临床上多联合使用两种或两种以上不同靶点的抗HIV-1药物治疗AIDS患者,称为高效抗逆转录病毒疗法(HAART)。其中以逆转录酶为靶点的上市药物数量最多,应用最广,且主要分为两类：核苷类逆转录酶抑制剂(NRTIs)和非核苷类逆转录酶抑制剂(NNRTIs)。
HIV-1NNRTIs通过作用在距离DNA合成位点约10A的变构结合口袋(NNIBP),使RT构象发生变化,从而抑制HIV-1复制。NNRTIs具有高效、低毒、高特异性等特点,而且化学结构多样,具有50多种结构类型。其中二芳基嘧啶类(DAPY)化合物以其对野生型和临床常见耐药突变型HIV-1的强抑制作用,成为新一代的NNRTIs的典型代表。DAPY类化合物中依曲韦林(Etravirine, TMC125)和利匹韦林(Rilpivirine, TMC278)分别于2008年和2011年被美国FDA批准上市用于治疗AIDS,这引起药物化学家对DAPY类化合物进一步的研究和结构修饰的热情。
X射线晶体学和分子模拟研究发现,DAPY类化合物在结合口袋NNIBP中整体呈现出“U”型构象,可以分为一个疏水性作用区(左侧2,6-二甲基-4-氰基苯基部分),氢键作用区(右侧-NH-连接链或嘧啶环上N原子)和蛋白-溶剂交界作用区(右侧对胺基苯腈基团)。并且可以通过分子中-O-和-NH-化学键的自由旋转,灵活地调整自身构象来适应NNIBP结构变化或变异,对耐药突变型的RT仍能保持紧密的结合作用,所以DAPY类化合物能有效地抑制耐药突变病毒株。但是Etravirine水溶性较低,药物动力学性质较差,中枢神经系统药物浓度较低,长期应用会出现较严重的皮疹和皮肤反应。虽然Rilpivirine的抗病毒活性高于Etravirine,但是临床上抗HIV-1治疗失败率明显高于Etravirine。因此,研发新一代高效低毒、具有抗耐药性和良好药物动力学性质的NNRTI类药物是当前抗艾滋病药物研究的热点。
本论文基于对逆转录酶结合口袋结构的分析,以DAPY类化合物中N-苄基取代哌啶类化合物(20)和N-苯基取代哌啶类化合物(21)为先导化合物,保留左侧2,4,6-三取代苯基部分和右侧N-取代哌啶部分,利用生物电子等排体原理,将中间的嘧啶杂环替换为1,3,5-三嗪环,3-硝基/氨基吡啶环和2-氨基嘧啶环。同时新设计的三嗪-胺基哌啶类(系列Ⅰ)、3-硝基/氨基吡啶-胺基哌啶类(系列Ⅱ)和2-氨基嘧啶-胺基哌啶类化合物(系列Ⅲ)还做了以下结构变换：1)将左侧苯环上对位取代基(R1),中间的连接链(X)和杂环上取代基进行变换,以探讨更广泛的构效关系；2)右侧不仅继续使用N-苄基取代哌啶和N-苯基取代哌啶基团,也设计了取代苯甲酸和哌啶1位NH形成酰胺键,探讨N-苯甲酰胺类化合物的抗病毒活性；3)在右侧末端苄基的苯环上(邻、间和对位)引入多样化取代基；4)将原有的苄基中的苯环(Ph)用亲水性的芳杂环(Het)替换。新设计的上述三类化合物用计算机辅助药物设计软件进行分子对接研究表明,化合物具有和先导化合物类似的结合模式,和NNIBP形成疏水相互作用和氢键相互作用等,验证了设计的合理性。
此外,结合Etravirine和上述2-氨基嘧啶-胺基哌啶类化合物(系列Ⅲ)的药效团特征,利用分子杂合原理将N-苄基取代哌啶基团引入至Etravirine嘧啶环的6位,用以占据NNRTIs的入口通道(Entrance channel),设计了哌啶-4-胺基-DAPY类杂合体化合物(系列Ⅳ)。分子模拟研究发现新设计的杂合体化合物可以两种结合构象与NNIBP发生相互作用：1)左翼的2,4,6-三取代苯基与NNIBP的疏水亚口袋形成疏水相互作用和π-π堆积作用；右侧的4-胺基苯腈基团则位于Lys103,Tyr318和Val106处；新引入的N-苄基取代哌啶基团可以伸入并占据NNRTI的入口通道(模式1)。2)左翼的2,4,6-三取代苯基位置保持不变,N-苄基取代哌啶基团延伸至NNIBP的蛋白-溶剂交界区；新引入的4-胺基苯腈基团则可以伸入并占据NNRTI的入口通道(模式2)。在这两种结合模式中,化合物均有效地占据了NNRTI的入口通道,构建出了“多位点结合”的杂合体化合物。在进一步的研究中(系列V),N-苄基取代哌啶基团部分被缩短,使化合物呈现出类似于Etravirine的一种结合模式(模式1)。新引入的具有较大分子柔性和亲水性的脂肪杂环基团,利于该部分基团更好地伸入并占据NNRTI的入口通道。
根据逆合成分析和文献调研,本论文制定出目标化合物的合成路线,分别选用三聚氯氰、2,6-二氯-3-硝基吡啶和2-氨基-4,6-二氯嘧啶作为起始原料,然后分别与2,4,6-三取代苯胺／酚和4-氨基-1-Boc哌啶发生取代反应构建起S-3,S-7,B-3a,B-3b,B-7,M-4a和M-4b等关键中间体。经官能团转化和脱除Boc保护基后,再和适当的取代基相连接,得到系列Ⅰ-Ⅲ目标化合物；哌啶-4-胺基-DAPY类化合物(系列Ⅳ)则是先和对溴苯腈发生钯催化的偶联反应,再和4-氨基-1-Boc哌啶反应构建起M-6a,M-6b和M-10等关键中间体。所有目标化合物均经1H-NMR,IR和ESI-MS等分析方法进行结构确证,各个亚系列的代表化合物也利用13C-NMR进行分子骨架的确证。
所有目标化合物均在MT-4细胞中进行抗HIV活性筛选实验。结果显示,大多数化合物对野生型HIV-1(IIIB)具有纳摩尔级的抑制活性,部分化合物对常见耐药突变型HIV-1(K103N/Y181C)具有较强抑制作用,所有化合物对HIV-2(ROD)均无抑制作用,所以本论文所设计合成的化合物均属于HIV-1抑制剂。
三嗪-胺基哌啶类化合物(系列Ⅰ)中,N-苄基哌啶类化合物抗野生型HIV-1的EC5o值均小于20nM,抗K103N/Y181C耐药突变株的EC5o值大多数小于5μM。其中化合物TA-a5抗野生型HIV-1表现出最高的抑制活性和选择性(EC50=2.2nM,SI=45285),是阳性对照药物奈韦拉平(NVP)的89.5倍,地拉韦定(DLV)的15.6倍,依法韦仑(EFV)的2.9倍和齐多夫定(AZT)的2.7倍；同时化合物TN-a5表现出抗K103N/Y181C耐药突变株的抑制活性(EC50=0.12μM,SI=1500)是阳性药物EFV的4.6倍(NVP和DLV在同一实验中均失去抑制活性)。
3-硝基/氨基吡啶-胺基哌啶类化合物(系列Ⅱ)中,左侧NH连接的3-硝基化合物BD-el对野生型HIV-1表现出最高的抑制活性(EC50=5.1nM),是NVP的22倍,DLV的21倍,比EFV和AZT稍强；左侧O连接的3-硝基化合物BD-cl表现出较好的抗病毒活性(EC50=10nM),是NVP的11倍,DLV的11倍,比EFV和AZT稍弱,同时该化合物的选择性(SI≥14,126)和AZT相当。
2-氨基嘧啶-胺基哌啶类化合物(系列Ⅲ)中,多数化合物抗野生型HIV-1的ECso值小于10nM,比阳性对照药NVP和DLV高15-20倍,与EFV和AZT相当。但是该系列化合物对耐药突变株的抑制作用较弱,ECso值在微摩尔水平,低于EFV和AZT。
在哌啶-4-胺基-DAPY类化合物(系列Ⅳ)中,化合物MD-c5对野生型和耐药突变型HIV-1具有较强的抑制作用(EC50=0.038μM和0.95μM),但是该化合物细胞毒性较大,选择性指数较低(SI=112)。系列V化合物中DSC-a4抗HIV-1野生株和K103N/Y181C耐药突变病毒株的EC50值分别为7.8nM和0.65μM,比系列Ⅳ代表化合物MD-c5的活性有所提高。
此外,各系列活性代表化合物进行了HIV-1RT抑制实验,结果显示代表化合物均对HIV-1RT具有较高的亲和力和明显的抑制作用,所以本论文中所设计合成的化合物属于HIV-1非核苷类RT抑制剂。
综上所述,本论文以文献报道的DAPY类化合物为先导化合物,结合DAPY类化合物晶体结构分析、构效关系研究和药效团特征,分别利用“生物电子等排”和“分子杂合”药物设计原理,对先导化合物进行结构多样的骨架变换,设计出5个系列化合物,同时应用计算机辅助药物设计软件进行分子模拟验证设计的合理性。根据逆合成分析和文献调研,本论文共合成了107个结构全新的化合物,并对目标化合物进行了抗HIV活性筛选,其中部分化合物的抗野生株和K103N/Y181C耐药突变毒株的活性超过阳性对照药物NVP, DLV和EFV,具有进一步研究与开发价值。同时本论文还初步探讨了各系列目标化合物的构效关系,为进一步结构优化提供了重要信息。
Acquired immune deficiency syndrome (AIDS) which is mainly caused by human immunodeficiency virus type-1(HIV-1), remains to be one of the leading pandemic diseases worldwide. Currently, the highly active antiretroviral therapy (HAART) plays a crucial role in the treatment of AIDS which contains at least two kinds of anti-HIV-1drugs. Among the FDA approved anti-HIV-1drugs, the reverse transcriptase (RT) inhibitors are the most widely used in clinical, which can be mainly divided into two types:nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs).
Among the structurally diverse NNRTIs, diarylpyrimidine (DAPY) derivatives with unique antiviral potency, high specificity and low cytotoxicity have attracted considerable attention over the past few years. As the representatives of DAPYs, Etravirine (TMC125) and Rilpivirine (TMC278) were approved by US FDA in2008and2011respectively due to their superior activity against wild-type and NNRTI-resistant mutant strains of HIV-1.
X-ray crystallography and molecular modeling studies showed that the binding conformation of DAPYs resembled a horseshoe or "U" shape in the NNIBP, which can generate numerous conformational variants through the torsional flexibility of the-O-and-NH-linker to keep high binding affinity to the mutant RT. However, Etravirine has low water solubility and poor pharmacokinetic properties, and there will be serious rash after long-term use. Though Rilpivirine has higher antiviral activity, the failure rate of clinical treatment was significantly higher than that of Etravirine. Therefore, it is an urgent need to design and develop novel NNRTIs with low toxicity, good pharmacokinetic properties and high efficiency against drug resistant strains.
Based on the above analysis, triazine-amino-piperidine series (Series I),3-nitro/amino pyridine-amino-piperidine series (Series II) and2-amino pyrimidine-amino-piperidine series (Series III) were designed via bioisosterism principle. The newly designed compounds, which retained the left2,4,6-tri-substituted phenyl and the right substituted piperidine group, also made the following structural transformation:1) The para-substituent (R1) on the left phenyl, the substituent (R2) on the central ring and the linker (X) were designed structural diversely to generate many congeners;2) Not only the N-substituted benzyl/phenyl groups, and also the substituted benzoyl were introduced to the NH of piperidine;3) Diverse substituents were introduced to the ortho-, meta-and para-positions of the terminal phenyl;4) The terminal benzene ring (Ph) was replaced by the hydrophilic aromatic heterocycles (Het). Molecular simulation studies of the newly designed compounds using the software SYBYL-X (Surflex-Dock) indicated that the new compounds could bind in to NNIBP in similar mode as lead compounds, and could form hydrophobic interaction and hydrogen bond interaction.
Subsequently, a series of novel piperidinylamino-diarylpyrimidine (pDAPY) derivatives (Series IV) was designed through molecular hybridization strategy by introducing the N-benzyl substituted piperidine group to the6position of central pyrimidine ring. Docking studies showed that the novel hybrid compounds could bind into the NNIBP in two modes:1)2,4,6-tri-substituted phenyl formed hydrophobic interaction with aromatic amino acid residues; The4-cyanophenyl group occupied the groove lined by Pro236, Vall06and Leu234, and the newly introduced N-benzyl substituted piperidine group stretched out of the NNIBP through the entrance channel (Mode1).2) N-benzyl substituted piperidine group extended to the solvent/protein interface; And the4-cyanophenyl group was oriented into the entrance channel (Mode2). In both of the two modes, the entrance channel was occupied by N-benzyl substituted piperidine group or4-cyanophenyl group. Further optimization of pDAPY derivatives (shorten the N-benzyl substituted piperidine group) led to Series V, which were kept one binding mode as Etravirine and introduced more flexible and hydrophilic groups in order to occupy the entrance channel effectively.
The synthetic routes of target compounds were designed according to the retrosynthetic analysis and related literature. Firstly the starting materials reacted with2,4,6-trisubstituted aniline/phenol and4-amino-l-Boc piperidine respectively to construct the key intermediates S-3, S-7, B-3a, B-3b, B-7, M-4a and M-4b. After transformation of relevant functional groups and removal of Boc groups, the intermediates could connect with different substituents to get the target compounds, of which the structures were confirmed by1H-NMR,13C-NMR, IRand ESI-MS.
All the title compounds were tested in a cell-based antiviral assay against HIV (wild-type, ⅢB and a resistant mutant HIV-1strain, containing K103N/Y181C in the RT) and HIV-2(ROD) with nevirapine (NVP), delavirdine mesylate (DLV), efavirenz (EFV) and zidovudine (AZT) as reference drugs. Most of the new compounds were active against wild-type HIV-1with EC50values in the nanomolar concentration range, and some compounds were active against K103N/Y181C resistant mutant strains. However, none of the newly synthesized compounds were active against HIV-2(ROD) at a subtoxic concentration in MT-4cells.
Among the triazine-amino-piperidine derivatives (Series Ⅰ), compounds with the N-benzyl substituted piperidine group showed high activity against wild-type HIV-1with EC50values below20nM, and against K103N/Y181C resistant mutant strains with EC50values below5μM. Compound TA-a5showed the highest activity against wild-type HIV-1(EC50=2.2nM, SI=45285), which was89times higher than the reference drug NVP,15times higher than DLV,2.9times higher than EFV and2.7times higher than AZT. Compound TN-a5showed the highest activity against K103N/Y181C resistant mutant strains (EC50=0.12μM, SI=1500), which was4.6times higher than the reference drug EFV (NVP and DLV were not active in the same assay).
Among3-nitro/amino pyridine-amino-piperidine analogues (Series Ⅱ), compound BD-e2was the most potent compound with EC50value of5.1nM, which was22times higher than NVP,21times higher than DLV, and comparable to EFV and AZT. Compound BD-cl (EC50=10nM) showed exciting low cytotoxicity with a CC50value greater than146μM and high selectivity (SI>14126), which is comparable to AZT.
Most2-amino pyrimidine-amino-piperidine analogues (Series Ⅲ), showed high activity against wild-type HIV-1with EC50values below10nM, which were15-20times higher than NVP and DLV. Whereas, these compounds were less potent against K103N/Y181C resistant mutant strains with EC50values in the micromolar concentration range.
The hybrid compound MD-c5(Series Ⅳ) displayed high activity against wild-type and K103N/Y181C resistant mutant strains with EC50values0.038μM and0.95jiM respectively. Further optimization resulted in compound DSC-a4(Series Ⅴ) with higher activity against wild-type and K103N/Y181C resistant mutant strains (EC50=7.8nM and0.65μM) than compound MD-c5.
The most active compounds of each series were performed HIV-1RT inhibitory assays using commercial kit. The results indicated that the representative compounds showed high affinity to HIV-1RT, and inhibited the activity of RT in vitro with EC50values comparable to reference drug NVP.
In summary, based on the analysis of binding mode and SAR studies of the lead compounds,5series of compounds were designed using bioisosterism principle and molecular hybridization strategy. The newly designed compounds were docked into NNIBP and overlaped with lead compounds to elucidate the binding mode. All the target compounds were synthesized according to the retrosynthetic analysis and related literature, and screened for anti-HIV activity. Some compounds were confirmed to be highly active against wild-type and K103N/Y181C resistant mutant strains of HIV-1with lower EC50values than the reference drugs which have potential for further research. In addition, important structure activity relationship (SAR) information of each series in the thesis were discussed for further research.
HIV-1NNRTIs通过作用在距离DNA合成位点约10A的变构结合口袋(NNIBP),使RT构象发生变化,从而抑制HIV-1复制。NNRTIs具有高效、低毒、高特异性等特点,而且化学结构多样,具有50多种结构类型。其中二芳基嘧啶类(DAPY)化合物以其对野生型和临床常见耐药突变型HIV-1的强抑制作用,成为新一代的NNRTIs的典型代表。DAPY类化合物中依曲韦林(Etravirine, TMC125)和利匹韦林(Rilpivirine, TMC278)分别于2008年和2011年被美国FDA批准上市用于治疗AIDS,这引起药物化学家对DAPY类化合物进一步的研究和结构修饰的热情。
X射线晶体学和分子模拟研究发现,DAPY类化合物在结合口袋NNIBP中整体呈现出“U”型构象,可以分为一个疏水性作用区(左侧2,6-二甲基-4-氰基苯基部分),氢键作用区(右侧-NH-连接链或嘧啶环上N原子)和蛋白-溶剂交界作用区(右侧对胺基苯腈基团)。并且可以通过分子中-O-和-NH-化学键的自由旋转,灵活地调整自身构象来适应NNIBP结构变化或变异,对耐药突变型的RT仍能保持紧密的结合作用,所以DAPY类化合物能有效地抑制耐药突变病毒株。但是Etravirine水溶性较低,药物动力学性质较差,中枢神经系统药物浓度较低,长期应用会出现较严重的皮疹和皮肤反应。虽然Rilpivirine的抗病毒活性高于Etravirine,但是临床上抗HIV-1治疗失败率明显高于Etravirine。因此,研发新一代高效低毒、具有抗耐药性和良好药物动力学性质的NNRTI类药物是当前抗艾滋病药物研究的热点。
本论文基于对逆转录酶结合口袋结构的分析,以DAPY类化合物中N-苄基取代哌啶类化合物(20)和N-苯基取代哌啶类化合物(21)为先导化合物,保留左侧2,4,6-三取代苯基部分和右侧N-取代哌啶部分,利用生物电子等排体原理,将中间的嘧啶杂环替换为1,3,5-三嗪环,3-硝基/氨基吡啶环和2-氨基嘧啶环。同时新设计的三嗪-胺基哌啶类(系列Ⅰ)、3-硝基/氨基吡啶-胺基哌啶类(系列Ⅱ)和2-氨基嘧啶-胺基哌啶类化合物(系列Ⅲ)还做了以下结构变换：1)将左侧苯环上对位取代基(R1),中间的连接链(X)和杂环上取代基进行变换,以探讨更广泛的构效关系；2)右侧不仅继续使用N-苄基取代哌啶和N-苯基取代哌啶基团,也设计了取代苯甲酸和哌啶1位NH形成酰胺键,探讨N-苯甲酰胺类化合物的抗病毒活性；3)在右侧末端苄基的苯环上(邻、间和对位)引入多样化取代基；4)将原有的苄基中的苯环(Ph)用亲水性的芳杂环(Het)替换。新设计的上述三类化合物用计算机辅助药物设计软件进行分子对接研究表明,化合物具有和先导化合物类似的结合模式,和NNIBP形成疏水相互作用和氢键相互作用等,验证了设计的合理性。
此外,结合Etravirine和上述2-氨基嘧啶-胺基哌啶类化合物(系列Ⅲ)的药效团特征,利用分子杂合原理将N-苄基取代哌啶基团引入至Etravirine嘧啶环的6位,用以占据NNRTIs的入口通道(Entrance channel),设计了哌啶-4-胺基-DAPY类杂合体化合物(系列Ⅳ)。分子模拟研究发现新设计的杂合体化合物可以两种结合构象与NNIBP发生相互作用：1)左翼的2,4,6-三取代苯基与NNIBP的疏水亚口袋形成疏水相互作用和π-π堆积作用；右侧的4-胺基苯腈基团则位于Lys103,Tyr318和Val106处；新引入的N-苄基取代哌啶基团可以伸入并占据NNRTI的入口通道(模式1)。2)左翼的2,4,6-三取代苯基位置保持不变,N-苄基取代哌啶基团延伸至NNIBP的蛋白-溶剂交界区；新引入的4-胺基苯腈基团则可以伸入并占据NNRTI的入口通道(模式2)。在这两种结合模式中,化合物均有效地占据了NNRTI的入口通道,构建出了“多位点结合”的杂合体化合物。在进一步的研究中(系列V),N-苄基取代哌啶基团部分被缩短,使化合物呈现出类似于Etravirine的一种结合模式(模式1)。新引入的具有较大分子柔性和亲水性的脂肪杂环基团,利于该部分基团更好地伸入并占据NNRTI的入口通道。
根据逆合成分析和文献调研,本论文制定出目标化合物的合成路线,分别选用三聚氯氰、2,6-二氯-3-硝基吡啶和2-氨基-4,6-二氯嘧啶作为起始原料,然后分别与2,4,6-三取代苯胺／酚和4-氨基-1-Boc哌啶发生取代反应构建起S-3,S-7,B-3a,B-3b,B-7,M-4a和M-4b等关键中间体。经官能团转化和脱除Boc保护基后,再和适当的取代基相连接,得到系列Ⅰ-Ⅲ目标化合物；哌啶-4-胺基-DAPY类化合物(系列Ⅳ)则是先和对溴苯腈发生钯催化的偶联反应,再和4-氨基-1-Boc哌啶反应构建起M-6a,M-6b和M-10等关键中间体。所有目标化合物均经1H-NMR,IR和ESI-MS等分析方法进行结构确证,各个亚系列的代表化合物也利用13C-NMR进行分子骨架的确证。
所有目标化合物均在MT-4细胞中进行抗HIV活性筛选实验。结果显示,大多数化合物对野生型HIV-1(IIIB)具有纳摩尔级的抑制活性,部分化合物对常见耐药突变型HIV-1(K103N/Y181C)具有较强抑制作用,所有化合物对HIV-2(ROD)均无抑制作用,所以本论文所设计合成的化合物均属于HIV-1抑制剂。
三嗪-胺基哌啶类化合物(系列Ⅰ)中,N-苄基哌啶类化合物抗野生型HIV-1的EC5o值均小于20nM,抗K103N/Y181C耐药突变株的EC5o值大多数小于5μM。其中化合物TA-a5抗野生型HIV-1表现出最高的抑制活性和选择性(EC50=2.2nM,SI=45285),是阳性对照药物奈韦拉平(NVP)的89.5倍,地拉韦定(DLV)的15.6倍,依法韦仑(EFV)的2.9倍和齐多夫定(AZT)的2.7倍；同时化合物TN-a5表现出抗K103N/Y181C耐药突变株的抑制活性(EC50=0.12μM,SI=1500)是阳性药物EFV的4.6倍(NVP和DLV在同一实验中均失去抑制活性)。
3-硝基/氨基吡啶-胺基哌啶类化合物(系列Ⅱ)中,左侧NH连接的3-硝基化合物BD-el对野生型HIV-1表现出最高的抑制活性(EC50=5.1nM),是NVP的22倍,DLV的21倍,比EFV和AZT稍强；左侧O连接的3-硝基化合物BD-cl表现出较好的抗病毒活性(EC50=10nM),是NVP的11倍,DLV的11倍,比EFV和AZT稍弱,同时该化合物的选择性(SI≥14,126)和AZT相当。
2-氨基嘧啶-胺基哌啶类化合物(系列Ⅲ)中,多数化合物抗野生型HIV-1的ECso值小于10nM,比阳性对照药NVP和DLV高15-20倍,与EFV和AZT相当。但是该系列化合物对耐药突变株的抑制作用较弱,ECso值在微摩尔水平,低于EFV和AZT。
在哌啶-4-胺基-DAPY类化合物(系列Ⅳ)中,化合物MD-c5对野生型和耐药突变型HIV-1具有较强的抑制作用(EC50=0.038μM和0.95μM),但是该化合物细胞毒性较大,选择性指数较低(SI=112)。系列V化合物中DSC-a4抗HIV-1野生株和K103N/Y181C耐药突变病毒株的EC50值分别为7.8nM和0.65μM,比系列Ⅳ代表化合物MD-c5的活性有所提高。
此外,各系列活性代表化合物进行了HIV-1RT抑制实验,结果显示代表化合物均对HIV-1RT具有较高的亲和力和明显的抑制作用,所以本论文中所设计合成的化合物属于HIV-1非核苷类RT抑制剂。
综上所述,本论文以文献报道的DAPY类化合物为先导化合物,结合DAPY类化合物晶体结构分析、构效关系研究和药效团特征,分别利用“生物电子等排”和“分子杂合”药物设计原理,对先导化合物进行结构多样的骨架变换,设计出5个系列化合物,同时应用计算机辅助药物设计软件进行分子模拟验证设计的合理性。根据逆合成分析和文献调研,本论文共合成了107个结构全新的化合物,并对目标化合物进行了抗HIV活性筛选,其中部分化合物的抗野生株和K103N/Y181C耐药突变毒株的活性超过阳性对照药物NVP, DLV和EFV,具有进一步研究与开发价值。同时本论文还初步探讨了各系列目标化合物的构效关系,为进一步结构优化提供了重要信息。
Acquired immune deficiency syndrome (AIDS) which is mainly caused by human immunodeficiency virus type-1(HIV-1), remains to be one of the leading pandemic diseases worldwide. Currently, the highly active antiretroviral therapy (HAART) plays a crucial role in the treatment of AIDS which contains at least two kinds of anti-HIV-1drugs. Among the FDA approved anti-HIV-1drugs, the reverse transcriptase (RT) inhibitors are the most widely used in clinical, which can be mainly divided into two types:nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs).
Among the structurally diverse NNRTIs, diarylpyrimidine (DAPY) derivatives with unique antiviral potency, high specificity and low cytotoxicity have attracted considerable attention over the past few years. As the representatives of DAPYs, Etravirine (TMC125) and Rilpivirine (TMC278) were approved by US FDA in2008and2011respectively due to their superior activity against wild-type and NNRTI-resistant mutant strains of HIV-1.
X-ray crystallography and molecular modeling studies showed that the binding conformation of DAPYs resembled a horseshoe or "U" shape in the NNIBP, which can generate numerous conformational variants through the torsional flexibility of the-O-and-NH-linker to keep high binding affinity to the mutant RT. However, Etravirine has low water solubility and poor pharmacokinetic properties, and there will be serious rash after long-term use. Though Rilpivirine has higher antiviral activity, the failure rate of clinical treatment was significantly higher than that of Etravirine. Therefore, it is an urgent need to design and develop novel NNRTIs with low toxicity, good pharmacokinetic properties and high efficiency against drug resistant strains.
Based on the above analysis, triazine-amino-piperidine series (Series I),3-nitro/amino pyridine-amino-piperidine series (Series II) and2-amino pyrimidine-amino-piperidine series (Series III) were designed via bioisosterism principle. The newly designed compounds, which retained the left2,4,6-tri-substituted phenyl and the right substituted piperidine group, also made the following structural transformation:1) The para-substituent (R1) on the left phenyl, the substituent (R2) on the central ring and the linker (X) were designed structural diversely to generate many congeners;2) Not only the N-substituted benzyl/phenyl groups, and also the substituted benzoyl were introduced to the NH of piperidine;3) Diverse substituents were introduced to the ortho-, meta-and para-positions of the terminal phenyl;4) The terminal benzene ring (Ph) was replaced by the hydrophilic aromatic heterocycles (Het). Molecular simulation studies of the newly designed compounds using the software SYBYL-X (Surflex-Dock) indicated that the new compounds could bind in to NNIBP in similar mode as lead compounds, and could form hydrophobic interaction and hydrogen bond interaction.
Subsequently, a series of novel piperidinylamino-diarylpyrimidine (pDAPY) derivatives (Series IV) was designed through molecular hybridization strategy by introducing the N-benzyl substituted piperidine group to the6position of central pyrimidine ring. Docking studies showed that the novel hybrid compounds could bind into the NNIBP in two modes:1)2,4,6-tri-substituted phenyl formed hydrophobic interaction with aromatic amino acid residues; The4-cyanophenyl group occupied the groove lined by Pro236, Vall06and Leu234, and the newly introduced N-benzyl substituted piperidine group stretched out of the NNIBP through the entrance channel (Mode1).2) N-benzyl substituted piperidine group extended to the solvent/protein interface; And the4-cyanophenyl group was oriented into the entrance channel (Mode2). In both of the two modes, the entrance channel was occupied by N-benzyl substituted piperidine group or4-cyanophenyl group. Further optimization of pDAPY derivatives (shorten the N-benzyl substituted piperidine group) led to Series V, which were kept one binding mode as Etravirine and introduced more flexible and hydrophilic groups in order to occupy the entrance channel effectively.
The synthetic routes of target compounds were designed according to the retrosynthetic analysis and related literature. Firstly the starting materials reacted with2,4,6-trisubstituted aniline/phenol and4-amino-l-Boc piperidine respectively to construct the key intermediates S-3, S-7, B-3a, B-3b, B-7, M-4a and M-4b. After transformation of relevant functional groups and removal of Boc groups, the intermediates could connect with different substituents to get the target compounds, of which the structures were confirmed by1H-NMR,13C-NMR, IRand ESI-MS.
All the title compounds were tested in a cell-based antiviral assay against HIV (wild-type, ⅢB and a resistant mutant HIV-1strain, containing K103N/Y181C in the RT) and HIV-2(ROD) with nevirapine (NVP), delavirdine mesylate (DLV), efavirenz (EFV) and zidovudine (AZT) as reference drugs. Most of the new compounds were active against wild-type HIV-1with EC50values in the nanomolar concentration range, and some compounds were active against K103N/Y181C resistant mutant strains. However, none of the newly synthesized compounds were active against HIV-2(ROD) at a subtoxic concentration in MT-4cells.
Among the triazine-amino-piperidine derivatives (Series Ⅰ), compounds with the N-benzyl substituted piperidine group showed high activity against wild-type HIV-1with EC50values below20nM, and against K103N/Y181C resistant mutant strains with EC50values below5μM. Compound TA-a5showed the highest activity against wild-type HIV-1(EC50=2.2nM, SI=45285), which was89times higher than the reference drug NVP,15times higher than DLV,2.9times higher than EFV and2.7times higher than AZT. Compound TN-a5showed the highest activity against K103N/Y181C resistant mutant strains (EC50=0.12μM, SI=1500), which was4.6times higher than the reference drug EFV (NVP and DLV were not active in the same assay).
Among3-nitro/amino pyridine-amino-piperidine analogues (Series Ⅱ), compound BD-e2was the most potent compound with EC50value of5.1nM, which was22times higher than NVP,21times higher than DLV, and comparable to EFV and AZT. Compound BD-cl (EC50=10nM) showed exciting low cytotoxicity with a CC50value greater than146μM and high selectivity (SI>14126), which is comparable to AZT.
Most2-amino pyrimidine-amino-piperidine analogues (Series Ⅲ), showed high activity against wild-type HIV-1with EC50values below10nM, which were15-20times higher than NVP and DLV. Whereas, these compounds were less potent against K103N/Y181C resistant mutant strains with EC50values in the micromolar concentration range.
The hybrid compound MD-c5(Series Ⅳ) displayed high activity against wild-type and K103N/Y181C resistant mutant strains with EC50values0.038μM and0.95jiM respectively. Further optimization resulted in compound DSC-a4(Series Ⅴ) with higher activity against wild-type and K103N/Y181C resistant mutant strains (EC50=7.8nM and0.65μM) than compound MD-c5.
The most active compounds of each series were performed HIV-1RT inhibitory assays using commercial kit. The results indicated that the representative compounds showed high affinity to HIV-1RT, and inhibited the activity of RT in vitro with EC50values comparable to reference drug NVP.
In summary, based on the analysis of binding mode and SAR studies of the lead compounds,5series of compounds were designed using bioisosterism principle and molecular hybridization strategy. The newly designed compounds were docked into NNIBP and overlaped with lead compounds to elucidate the binding mode. All the target compounds were synthesized according to the retrosynthetic analysis and related literature, and screened for anti-HIV activity. Some compounds were confirmed to be highly active against wild-type and K103N/Y181C resistant mutant strains of HIV-1with lower EC50values than the reference drugs which have potential for further research. In addition, important structure activity relationship (SAR) information of each series in the thesis were discussed for further research.
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