非核苷类HIV-1逆转录酶抑制剂研究进展(续)

(接上期)

16 二芳基三嗪类

二芳基三嗪化合物(Diaryltriazine,DATA,31)是在提高ITU类化合物的稳定性时发现的第一个DATA类化合物[35]。随后经过自由能量子计算分析之后得到HIV-1 RT抑制剂的结构模型Het-NH-Ph-U[36],其中HET代表杂环,U代表不饱和的疏水性基团。以32为基本结构,人们合成大量的只含一个芳基的新型三嗪类衍生物,活性研究发现都有较好的抗HIV活性。DATA类已成为一大类潜在的活性极好的抗野生型和单突变型病毒株的HIV-1抑制剂,但该类化合物对双突变的病毒株(如L100I+K103N)的抑制能力却较低。

17 芳基嘧啶类(Diarylpyrimidine,DAPY)[36-38]

该类化合物的代表有TMC125(Dapivirine,Etravirine,R165335,33)、TMC-120(34)等,其中TMC125对单突变以及包括L100I+K103N在内的双突变的病毒株均有极强的抑制作用,且副作用小。

18三环苯并噻吩类(硫茚类)

从具有抗HIV-1 活性的NCI化合物储库中筛选出的化合物NSC-380292(35)是一个具有新的结构类型的潜在HIV-1 RT抑制剂[39],IC50=1.24 μmol/L,治疗指数为800。该化合物有2个手性中心(2a,7b),但只有2个异构体存在(R,R)和(S,S)。拆分后进行活性测定,(R,R)异构体活性较好(其IC50为0.8 μmol/L)。以NSC-380292为先导物合成了一系列衍生物,并对其光学纯的异构体进行了活性测定,发现其衍生物也均是(R,R)构型的活性优于(S,S)构型,且2-甲氧甲酰基取代对保持较高的活性有利。在所合成并进行了研究的一系列化合物中,化合物36活性最好,其(R,R)构型的IC50为0.5 μmol/L。

19 1,2,4-三唑类

该类中的第一个化合物37是通过利用以细胞为基础的HIV复制化验法而发现的[40],具有微摩尔级的抑制活性。随后,对55结构中在N-4位的取代苯环和侧链上取代氨基的苯环进行改造,得到了一些对野生型病毒株以及发生Y181C、K103N、L100I变异的耐药性病毒株有较好活性的化合物。其中化合物38具有较好的活性[41],其对野生型EC50为0.000 1 μmol/L;对Y181C-K103N双变异EC50为0.06 μmol/L,进一步对该类化合物进行改造,将三唑环上N-4位取代的苯环换成萘环或喹啉环,将其侧链上苯胺的苯环进行修饰,发现萘环取代比喹啉环取代活性好。许多改进的三唑类化合物正在临床评价之中。

20 四唑类

人们通过高通量筛选(high throughput screening,HTS)发现了与上述三唑类相似的四唑类化合物(Thiotetrazolyl acetanilides),具有抑制K103N/Y181C双变异株逆转录酶的活性。化合物39是该类化合物中最早被发现的,在抗K103N/Y181C双变异株的同时,还有抗野生型病毒株的作用,该系列化合物的结构改造筛选出的化合物40活性较好,其IC50(变异型K103N/Y181C)=28 nmol/L;EC50(变异型K103N/Y181C)=28 nmol/L;EC50(野生型)=28 nmol/L;且具有较好的理化性质,半衰期为90 min。该类化合物是极有潜力的抗变异株的抑制剂。构效关系研究表明:在该类化合物苯胺基的对位引入炔基有利于改善理化性质并提高活性[42]。

39 40

21天然产物

人们不仅借助人工合成发现新药,往往还从大自然中有所收获,从天然药物中寻找活性成分和直接应用天然药物以及中药配方治疗AIDS的研究正方兴未艾。目前从植物、海洋生物、微生物和动物来源的天然药物中已经发现了一些化合物对抑制HIV表现出良好势头。下文将综述该类天然产物。

吡喃香豆素类化合物是从马来西亚热带雨林植物Calophyllum lanigerum中分离而得的新型四环香豆素类化合物,其中化合物(+)Calanolide A(41)抗HIV-1活性最好[43],不仅对AZT耐药病毒株有抑制作用,对奈维雷平和TIBO类耐药病毒株也有抑制活性,现正在临床试验中。41和(-)Calanolide B(Costatolide,42)都具有抗HIV-1 RT活性,而其对映异构体(-)Calanolide A和(+)Calanolide B却无任何活性。活性好的还有以天然香豆素为先导化合物而合成的新型双吡喃香豆素Calanolid(43)和Inophyllum(44)两种结构类型的化合物[44]。这类化合物不仅结构新颖,且具有高度特异性的抗HIV-1 RT活性,它们不仅对抗艾滋病药物AZT耐药的病毒细胞株有抑制作用,对其他非核苷类HIV-1 RT抑制剂,如TIBO、Nevirapine及吡啶酮等产生耐药性的病毒株也有抑制作用。

从1990木脂素被首次报道具有抗HIV活性以来,确认和制备更有效、更安全的木脂素类化合物的抗HIV药物的研究不断深入。如芳基丁烷及芳基丁烯类木脂素,芳基丁内脂类木脂素等木脂素衍生物均得到研究,其中木脂素Anolignan A(45)与B(46)是具有抗HIV活性芳基丁烯类木脂素的代表[45],1994年它们从植物Anogeissus acuminata 中被首次分离出来,是较强的HIV-1 RT抑制剂,且这两个木脂素具有协同作用。

金丝桃素(Hypericin,47)是由贯叶金丝桃(Hypericum perzo-ratum)或三棱三叶金丝桃(H. triquetrifolium Turra)等金丝桃属植物的鲜花中提取的具有两分子大黄素结构的天然产物[46],体内外活性试验表明其具有抗HIV-1活性,与AZT联合使用有协同作用。

黄芩提取物及其主要成分黄芩苷(Baicalin,48)[47]及黄芩苷元(Baicalien,49)[48]在细胞培养中抑制HIV-1 RT和细胞病变(CPE),抑制病毒荧光抗原(FA),并可抑制HIV在H9细胞中复制。一般来讲,HIV-1感染的淋巴细胞、单核细胞和吞噬细胞是该病毒在体内生存和复制的宿主细胞,各种细胞上的蛋白受体都具有特异的糖基识别性,糖与苷元相连成苷,不仅有可能提高生物靶分子和HIV的亲和力,而且有利于改变溶解性及其在体内的转运。

许多硫酸酯化多糖如香菇多糖、地衣多糖、右旋多糖、裂褶菌多糖、木聚糖、海藻多糖、Curdlan等的硫酸酯都有明显抑制HIV-1的活性,其作用机制是通过干扰HIV-1对宿主细胞的黏附作用,抑制HIV-1 RT活性。从禾本科植物蓍竹(Indocalamus tesselatus)叶中提纯多糖,进行硫酸酯化所得硫酸酯化蓍叶多糖,研究证实它对HIV-1引起的MT-4细胞变性作用比蓍叶多糖本身具有更高的抑制活性,最低有效浓度(MEC)为5~10 μg/ml,EC50=2.5 mg/ml[49]。

开络内酯类化合物(Khellactone)对HIV显示一定的抑制活性,其中化合物有Suksdofin(50)和DKC(51),对HIV-1RT都有不同程度的抑制作用[50]。

与开络内酯类结构类似,Inophyllum类化合物也是另一类具有抗HIV-1活性的天然产物,代表物有Inophullum B(52)及Inophullum P(53)[51]。

一些生物碱也具有抗HIV-1活性,Psychotirine(54)是从植物Cephaelis ipecauanha (Brotero)根中提取的异喹啉类生物碱,其氧甲基化衍生物O-Methyl-psychotrine(55)比其母体54具有更好的抗HIV-1RT活性[52]。最近有研究发现中药常用的抗病毒药物大黄中小檗碱(56)和巴马汀(57)对重组HIV-1 RT有较高的抑制活性[53],两者在药物浓度为0.2 mg/ml时的抑制率高达99%以上。56的细胞毒性CC50为0.012 05 mg/ml,抗HIV-1活性EC50为0.006 45 mg/ml,治疗指数为1.87;57的毒性CC50为0.097 72 mg/ml,抗HIV-1活性EC50为0.021 38 mg/ml,治疗指数为5.47。

总之,NNRTIs虽可降低毒副作用,但长期临床应用也会产生耐药性,极大地限制了其临床应用。进一步研究表明,不同结构类型的NNRTIs所致HIV变异株各不相同,如双杂芳环呱嗪类化合物表现为HIV-1 RT 100位氨基酸变异产生抗药性,TIBO类则使其103位氨基酸变异,TSAO类又表现为138位氨基酸变异产生抗药性,双吡啶类会引起106位氨基酸发生变化,吡啶酮类抗药部位在181位。而HEPT类化合物的耐药株病毒主要表现在Leu100Ile位的亮氨酸变异为异亮氨酸,Lys103Asp位的赖氨酸变异为天门冬氨酸,Val106His位的缬氨酸变异为组氨酸。NNRTIs进一步研究重点应该转移到抗耐药性药物的设计与开发上,搞清楚突变部位和靶酶的三维结构以及作用机制显得尤为重要。

另外,笔者针对HIV-1 RT疏水性口袋的三维结构的柔韧性,用环己烷代替萘环的B环芳香环的芳香结构,设计了具有一定柔韧结构的HIV-1 RT抑制剂,发现其具有一定的抗耐药性[54]。

HIV-1逆转录酶是研究抗艾滋病药物选择性很好的靶酶,HIV-1 RT疏水性口袋的三维结构解析为从分子水平上设计各种NNRTIs提供了很好的条件,并为耐药性从氨基酸突变部位上找到根本原因[55]。HIV-1逆转录酶抑制剂作为目前临床上治疗艾滋病的鸡尾酒疗法配伍药物的重要组分,为广大艾滋病患者提供了新的临床治疗选择,是艾滋病治疗取得的重大进展。随着分子生物学的不断发展,结合计算机辅助模型、X射线衍射分析等手段,将进一步揭示酶促反应和药物作用的过程和细节,建立更完善、精确的模型指导高效、专一的HIV-1逆转录酶抑制剂的设计与合成。

(志谢:特别对杨涛、郑美林等同学在资料查阅和文献整理方面所做的工作表示感谢!)

[参考文献]

[1]Smith AJ, Scott WA. The influence of natural substrates and inhibitors on the nucleotide-dependent excision activity of HIV-1 reverse transcriptase in the infected cell [J]. Curr Pharm Design,2006,15(12):1827-1841.

[2]Basavapathruni A, Anderson KS. Developing novel nonnucleoside HIV-1 reverse transcriptase inhibitors: beyond the butterfly[J]. Curr Pharm Design,2006,15(12):1857-1865.

[3]Morales JC, Kool ET. Varied molecular interactions at the active sites of several DNA polymerases: nonpolar nucleoside isosteres as probes[J]. J Am Chem Soc,2000,6(122):1001-1007.

[4]Nicholas AB, Vivek KR, Marija P, et al. Synthesis of 2',3'-Dideoxynucleoside 5'-α-P-Borano-β, γ(difluoro methylene) triphosphates and their inhibition of HIV-1 reverse transcriptase [J]. J Med Chem,2005,48(7):2695-2700.

[5]Hargrave KD, Proudfoot JR, Grozinger KG, et al. Novel non-nucleoside inhibitors of HIV-1 reverse transcriptase. 1. tricyclic pyridobenzo- and dipyridodiazepinones [J]. J Med Chem,1991,34(7):2231-2241.

[6]Merluzzi VJ, Hargrave KD, Labadia M, et al. Inhibition of HIV-1 replication by a non-nucleoside reverse transcriptase inhibitor [J]. Science,1990, 250(12):1411-1413.

[7]Kohlstaedt LA, Wang J, Friedman JM, et al. Crystal structure at 3.5■resolution of HIV-1 reverse transcriptase complexed with an inhibitor [J]. Science,1992,256(6):1783-1790.

[8]Romero DL, Morge RA, Genin MJ, et al. Bis (heteroaryl) piperazine (BHAP) reverse transcriptase inhibitors: structure-activity relationships of novel substituted indole analogs and the identification of 1-[(5-methanesulfon amido-1H-indol-2-yl) carbonyl]-4-[3-[(1-methylethyl)amino]pyridinyl]piperazine mo nomethanesulfonate (U-90152S), a second-generation clinical candidate [J]. J Med Chem,1993,36(10):1505-1508.

[9]Romero DL, Morge RA, Biles C, et al. Discovery, synthesis, and bioactivity of bis (heteroaryl) piperazines. 1. a novel class of non-nucleoside HIV-1 reverse rranscriptase inhibitors [J]. J Med Chem,1994,37(7):999-1014.

[10]Romero DL, Olmsted RA, Poel TJ, et al. Targeting delavirdine/atevirdine resistant HIV-1: identification of (alkylamino) piperidine-containing bis(heteroaryl) piperazines as broad spectrum HIV-1 reverse transcriptase inhibitors [J]. J Med Chem,1996,39(19):3769-3789.

[11]Genin MJ, Poel TJ, Yagi Y, et al. Synthesis and bioactivity of novel bis (heteroaryl) piperazine (BHAP) reverse transcriptase inhibitors: structure-activity relationships and increased metabolic stability of novel substituted pyridine analogs [J]. J Med Chem,1996,39(26):5267-5275.

[12]Venkatachalam TK, Sudbeck EA, Mao M, et al. Anti-HIV activity of aromatic and heterocyclic thiazolyl thiourea compounds [J]. Bioorg Med Chem Lett,2001,11(4):523-528.

[13]Pedersen OS, Pedersen EB. The flourishing syntheses of non-nucleoside reverse transcriptase inhibitors [J]. Synthesis,2000,4:479-495.

[14]Miyasaka T, Tanaka H, Baba M, et al. A novel lead for specific anti-HIV-1 agents: 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine [J]. J Med Chem,1989,32(12):2507-2509.

[15]Robert WBJ, Valerie FB, Sharon YB, et al. Resistance to 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio) thymine derivatives is generated by mutations at multiple sites in the HIV-1 reverse transcriptase [J]. Virology,1995,210(1):186-193.

[16]孟歌,何严萍,陈芬儿.HEPT类逆转录酶三维定量构效关系[J].高等学校化学学报,2002,23(7):1910-1916.

[17]Meng G, Chen FE, De Clercq E, et al. Interactive study between two type of 6-naphthylmethyl HEPT analogs and HIV-1 reverse transcriptase inhibitors [J]. Chinese J Pro Engin,2003,31(1):24-28.

[18]Meng G, Chen FE, De Clercq E, et al. Nonnucleoside HIV-1 reverse transcriptase inhibitors: Part Ⅰ. synthesis and structure-activity relationship of 1-alkoxymethyl-5-alkyl-6-naphthylmethyl uracils as HEPT analogues [J]. Chem Pham Bull,2003,51(7):779-789.

[19]孟歌,陈芬儿.6-(1-萘甲基)取代HEPT HIV-1 RT抑制剂的定量构效关系[J].中国药物化学杂志,2003,13(5):254-259.

[20]孟歌,陈芬儿.HEPT类非核苷类HIV-1逆转录酶抑制剂研究进展[J].中国药物化学杂志,2004,14(1):57-66.

[21]Mai A, Artico M, Sbardella G, et al. Preparation and anti-HIV-1 activity of thio analogues of dichydroalkoxybenzy-loxopyrimidines [J]. J Med Chem,1995,38(17):3258-3263.

[22]Mai A, Artico M, Sbardella G, et al. Dihydro(alkylthio) (naphthylmethyl) oxopyrimidines: novel non-nucleoside reverse transcriptase inhibitors of the S-DABO series [J]. J Med Chem,1997,40(10):1447-1454.

[23]He YP, Chen FE, Yu XJ, et al. Nonnucleoside HIV-1 reverse transcriptase inhibitors; part 3. synthesis and antiviral activity of 5-alkyl-2-[(aryl and alkyloxylcarbonylmethyl)thio] -6-(1-naphthylmethyl)pyrimidin-4(3H)-ones [J]. Bioorg Chem,2004,32(6):536-548.

[24]Maruenda H, Johnson F. Design and synthesis of novel inhibitors of HIV-1 reverse transcriptase [J]. J Med Chem,1995,38(12):2145-2151.

[25]Pauwels R, Andries K, Desmyter J, et al. Potent and selective inhibition of HIV-1 replication in vitro by a novel series of TIBO derivatives [J]. Nature,1990,343(2):470-474.

[26]Merluzzi VJ, Hargrave KD, Labadia M, et al. Inhibition of HIV-1 replication by a non-nucleoside reverse transcriptase inhibitor [J]. Science,1990,250(12):1411-1413.

[27]Saari WS, Hoffman JM, Wai JS, et al. 2-Pyridinone derivatives: a new class of nonnucleoside, HIV-1-specific reverse transcriptase inhibitors [J]. J Med Chem,1991,34(9):2922-2925.

[28]Jan H, Marc R, Lucien MH, et al. Design, synthesis, and SAR of a novel pyrazinone series with non-nucleoside HIV-1 reverse transcriptase inhibitory activity [J]. J Med Chem,2005,48(6):1910-1918.

[29]Angelo R, Andrea S, Sara C, et al. Structure-based design, parallel synthesis, structure-activity relationship, and molecular modeling studies of thiocarbamates, new potent non-nucleoside HIV-1 reverse transcriptase inhibitor isosteres of phenethyl thiazolylthiourea derivatives [J]. J Med Chem,2005,48(11):3858-3873.

[30]Bonache MC, Chamorro C, Velázquez S, et al. Improving the antiviral efficacy and selectivity of HIV-1 reverse transcriptase inhibitor TSAO-T by the introduction of functional groups at the N-3 position[J]. J Med Chem,2005,48(21):6653-6660.

[31]Roberto R, Stefano R. Efficacy and resistance of recently developed non-nucleoside reverse transcriptase inhibitors for HIV-1 [J]. Future Medicine,2009,3(1):63-77.

[32]Tucker TJ, Sisko JT, Tynebor RM, et al. Discovery of 3-{5-[(6-Amino-1H-pyrazolo[3,4-b]pyridine-3-yl)-methoxy]-2-chlorophenoxy}-5-chlorobenzonitrile (MK-4965): a potent, orally bioavailable HIV-1 non-nucleoside reverse Transcriptase inhibitor with improved potency against key mutant viruses [J]. J Med Chem,2008,51(20):6503-6511.

[33]Sweeney ZK, Acharya S, Briggs A, et al. Discovery of triazolinone non-nucleoside inhibitors of HIV reverse transcriptase [J]. Bioorg Med Chem Lett,2008,18(15):4348-4351.

[34]Monforte AM, Rao A, Logoteta P, et al. Novel N1-substituted 1,3-dihydro-2H benzimidazol-2-ones as potent non-nucleoside reverse transcriptase inhibitors [J]. Bioorg Med Chem,2008,16(15):7429-7435.

[35]Ludovici DW, Kavash RW, Kukla MJ, et al. Evolution of anti-HIV drug candidates part 2: diaryltriazine (DATA) analogues [J]. Bioorg Med Chem Lett,2001,11(17):2229-2234.

[36]Thakur VV, Kim JT, Hamilton AD. Optimization of pyrimidinyl and triazinyl-amines as non-nucleoside inhibitors of HIV-1 reverse transcriptase [J]. Bioorg Med Chem Lett,2006,16(21):5664-5667.

[37]Ludovici DW, DeCorte BL, Kukla MJ, et al. Evolution of anti-HIV drug candidates part 3: diarylpyrimidine(DAPY) analogues [J]. Bioorg Med Chem Lett,2001,11(17):2235-2239.

[38]Etravirine A. R165335, TMC 125, TMC-125, TMC125 [J]. Drugs in R&D,2006,7(6):367-373.

[39]Krajewski K, Zhang YJ, Parrish D, et al. New HIV-1 reverse transcriptase inhibitors based on a tricyclicbenzothio-phene scaffold: synthesis, resolution, and inhibitory activity[J]. Bioorg Med Chem Lett,2006,16(11):3034-3038.

[40]Wang ZW, Wu BG, Kuhen KL, et al. Synthesis and biological evaluations of sulfanyltriazoles as novel HIV-1 non-nucleoside reverse transcriptase inhibitors [J]. Bioorg Med Chem Lett,2006,16(16):4174-4177.

[41]Martha DLR, Hong WK, Esmir G, et al. Trisubstituted triazoles as potent non-nucleoside inhibitors of the HIV-1 reverse transcriptase [J]. Bioorg Med Chem Lett,2006,16(17):4444-4449.

[42]Gagnon A, Amad MH, Bonneau PR. et al. Thiotetrazole alkynylacetanilides as potent and bioavailable non-nucleoside inhibitors of the HIV-1 wild type and K103N/Y181C double mutant reverse transcriptases [J]. Bioorg Med Chem Lett,2007,17(16):4437-4441.

[43]周春梅,王琳,张铭龙,等.抗人免疫缺陷病毒(HIV)天然产物Calanolide A及其类似物的合成[J].药学学报,1999,34(9):673-678.

[44]杨劲松.抗HIV活性香豆素类化合物的研究进展[J].华西药学杂志,2001,16(4):285-288.

[45]杨毅,张成路,王,等.木脂素抗艾滋病病毒研究[J].化学进展,2003,4(15):327-331.

[46]赵晶,张致平,陈鸿珊,等.金丝桃素与乙基金丝桃素的全合成及对人免疫缺陷病毒逆转录酶的抑制活性[J].药学学报,1998,33(1):67-71.

[47]赵晶,张致平,陈鸿珊,等.黄芩甙衍生物的合成及抗人免疫缺陷病毒活性研究[J].药学学报,1998,33(1):22-27.

[48]赵晶,张致平,陈鸿珊,等.黄芩甙元及其苄基衍生物的制备与抗人免疫缺陷病毒实验研究[J].药学学报,1997,32(2):140-143.

[49]陈春英,黄雪华,周井炎,等.硫酸酯化箬叶多糖的结构修饰及其抗艾滋病病毒活性[J].药学学报,1998,33(4):264-268.

[50]Xie L, Takeuchi Y, Cosentino LM, et al. Anti-AIDS agents. synthesis and structure-activity relationships of (3'R, 4'R)-(+)-cis-khellactone derivatives as novel potent anti-HIV agents [J]. J Med Chem,1999,42(14):2662-2672.

[51]Talor PB, Culp JS, Debouck C, et al. Kinetic and mutational analysis of human immunodeficiency virus type 1 reverse transcriptase inhibition by inophylums, a novel class of non-nucleoside inhibitors [J]. J Biol Chem,1994,269(8):6325-6331.

[52]Tan GT, Kinghorn AD, Hughes SH, et al. Psychotrine and its o-methyl ether are selective inhibitors of human immunodeficiency virus-1 reverse transcriptase [J]. J Biol Chem,1991,266(35):23529-23536.

[53]杨柳萌,王睿睿,李晶晶,等.四个小碱类化合物的体外抗HIV活性[J].中国天然药物,2007,5(3):225-228.

[54]Meng G, Kuang YY, Ji L, et al. Synthesis of 1-[(2-hydroxyethoxy)methyl]-6-(5,6,7,8-tetrahydronaphthylme-thyl-1) thymine as novel inhibitor against drug-resistant HIV mutants [J]. Synth Commun,2005,35(8):1095-1102.

[55]Meng G, Li ZY. Interaction model between a new HIV-1 RT inhibitor with its NNBP [C].昆明:第五届全国化学生物学学术会议,2007:83.

(收稿日期:2010-01-28)