CN1371367A - 表现出杀精子活性的苯乙基-5-溴吡啶基硫脲(pbt)和二氢烷氧基苄基氧代嘧啶(dabo)衍生物 - Google Patents
表现出杀精子活性的苯乙基-5-溴吡啶基硫脲(pbt)和二氢烷氧基苄基氧代嘧啶(dabo)衍生物 Download PDFInfo
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- CN1371367A CN1371367A CN99815250A CN99815250A CN1371367A CN 1371367 A CN1371367 A CN 1371367A CN 99815250 A CN99815250 A CN 99815250A CN 99815250 A CN99815250 A CN 99815250A CN 1371367 A CN1371367 A CN 1371367A
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- thiocarbamide
- ethyl
- pyrimidine
- methylthiomethyl
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Abstract
表现出杀精子活性以及抗病毒活性的新的苯乙基-5-溴吡啶基硫脲(PBT)和二氢烷氧基苄基氧代嘧啶(DABO)衍生物。可将这些新的化合物掺入到避孕组合物中以提供杀精子或精子固定活性。
Description
发明领域
本发明涉及新的PBT和DABO衍生物。在一个特定实施方案中,本发明涉及表现出杀精子活性的新的PBT和DABO衍生物。
发明背景
人免疫缺陷病毒(HIV)-获得性免疫缺陷综合征(AIDS)的病原体是导致育龄妇女死亡的增长最迅速的病因。在全世界所有HIV感染中,异性间的传播占90%,并且在美国导致新感染HIV的比例不断增加。到2000年为止,有一千三百万妇女感染了HIV,而在全世界范围内总共有四千万感染HIV的个体。在缺乏有效的预防性抗HIV疫苗或抗逆转录病毒治疗的情况下,人们需要用来抑制粘膜和产期HIV传播的由女性控制的阴道杀微生物剂。
有人担心,在全球范围内使用洗涤剂型杀精子药例如壬苯醇醚-9(N-9)实际上可能会增加HIV传播的危险。N-9已经作为不需处方即可以出售的杀精子凝胶剂、泡沫剂、霜剂、海绵擦拭剂、薄膜剂、和发泡片剂使用了30年以上。因为据表明N-9在体外能灭活HIV,所以目前它成为唯一考虑的用于在妇女中防止性传播HIV感染的局部用杀微生物剂。然而,使用N-9的主要缺点是其对上皮细胞有去污剂型作用。将N-9作为阴道用避孕药频繁使用会增加子宫颈阴道感染、刺激、或溃疡的危险性。因为连续使用N-9可改变阴道菌丛以及增加机会感染的危险性,所以它可能增加子宫颈外上皮和子宫颈内粘膜对HIV感染的易感性。此外,最近的临床试验表明,当作为综合预防措施的一部分来提供以预防HIV/AIDS的异性传播时,含有N-9的阴道避孕药对HIV/AIDS和其它STDs的传播没有任何作用。参见例如Roddy等人,1998,N.Eng.J.Med.339:504-510;Hira等人,1997,Int.J.STD AIDS 8:243-250)。
与目前市售的阴道杀微生物剂相比,没有洗涤剂型膜毒性的新的、有效、安全、且由女性控制使用的局部用杀微生物剂应该有临床优点。生理受精取决于射出的精子游动、结合透明带、以及穿透卵子的能力。每一这些活动性都主要取决于精子的运动性。因此,给所选的抗逆转录病毒药物例如非核苷类抑制剂(NNIs)加上杀精子功能可以是抑制HIV的异性阴道传播以及防止受孕的有效手段。
发明简述
本发明提供了新的非核苷类抑制剂(NNIs)苯乙基-5-溴吡啶基硫脲(PBT)和二氢烷氧基苄基氧代嘧啶(DABO)衍生物。这些化合物可用作精子固定剂或避孕剂。据本申请人所知,PBT和DABO衍生物在现有技术中没有研究过,并且没有认识到它们具有任何避孕活性例如杀精子或精子固定活性。
本发明提供了具有化学结构(I)的卤代PBT衍生物或其可药用盐:其中R、R1、R2、R3、和R4独立地为氢、F、Cl、Br、或I,并且R、R1、R2、R3、和R4当中至少有一个是F、Cl、Br、或I。
本发明还提供了包含式(III)的新的DABO衍生物化合物或其可药用盐:其中R1和R2相同或不同,并且是氢、卤素、烷基、链烯基、羟基、烷氧基、烷硫基、硫羟基、膦基、ROH、或RNH基团,其中R是烷基;Y是S或O;且R3是烷基、链烯基、芳基、芳烷基、ROH、或RNH2基团,其中R是烷基。
本发明还提供了在哺乳动物中抑制受孕的方法。所述方法包含用杀精子有效量的本发明新的卤代PBT衍生物或新的DABO衍生物与哺乳动物精子接触。
本发明另一方面是抑制精子运动的方法。所述方法包含用精子运动抑制有效量的本发明卤代PBT衍生物或DABO衍生物与精子接触。
本发明还提供了包含杀精子有效量的杀精子剂和可药用载体、稀释剂或赋形剂的组合物。所述杀精子剂包括本发明卤代PBT衍生物或DABO衍生物。
本发明还提供了包含本发明卤代PBT衍生物或DABO衍生物的杀精子产品。
通过阅读下述附图、详细说明、实施例和权利要求书,本发明的其它方面对于本领域技术人员来说将变得显而易见。
附图的简要说明
附图1是下述化合物的化学结构:N-[2-(2,5-二甲氧基苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲(D-PBT);N-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲(F-PBT);5-异丙基-2-[(甲硫基甲基)硫基]-6-(苄基)嘧啶-4-(1H)-酮(S-DABO);和N-[2-(2-吡啶基)乙基]-N’-[2-溴吡啶基)]硫脲(trovirdine,一种PETT衍生物)。
附图2表示的是trovirdine、D-PBT、F-PBT、S-DABO、和N-9对精子运动的浓度-依赖性抑制。在测定培养基中,将精子的高运动性部分与浓度递增的(31.2μM-500μM)trovirdine、D-PBT、F-PBT、S-DABO、和N-9或1%DMSO一起培养3小时,并通过CASA评估能运动的精子的百分比。每一数据点代表3-4个独立实验的平均值。
附图3表示的是在F-PBT存在下时间-依赖性精子固定。在测定培养基中,在1mM F-PBT或仅1%DMSO存在下将能运动的精子在37℃培养。以5或10分钟的时间间隔通过CASA测定精子的运动性。每一数据点代表两个独立实验的平均值±SD。
附图4表示的是F-PBT对精子运动参数的影响。在测定培养基中,在1mM F-PBT存在下培养精子的运动部分,并用在″Materialsand Methods″中描述的Hamilton Thorne-IVOS version 10 CASA测定时间-依赖性运动特征。值:对于VCL、VAP、VSL是μm/s;对于ALH是μm;对于MOT、STR、LIN是%;对于BCF是Hz。
附图5A表示的是D-PBT、F-PBT、S-DABO、和N-9对精子顶体膜完整性的影响。在或不在递增浓度的三种NNIs和N-9存在下将能运动的精子预培养6小时。通过FITC-豌豆(Pisum sativum)凝集素结合测定法测定的顶体完整精子的百分比以三个独立实验的平均值±SD表示。
附图5B-5E是精子的激光扫描同焦荧光图像。所示精子进行了三种标记,即用FITC-豌豆凝集素标记顶体(绿色)、用TOTO-3碘化物标记DNA(蓝色)、用尼罗红标记膜脂质(红色)。在顶体完整精子中,精子头的顶体区域表现出均匀的亮绿色荧光。在顶体发生反应的精子中,绿色荧光不存在或者局限在精子头的中纬线部分。附图5B表示的是仅暴露于0.5%DMSO的精子;附图5C表示的是暴露于500μMF-PBT的精子;附图5D表示的是暴露于500μMS-DABO的精子。附图5B、5C、或5D没有一个表现出培养6小时后精子的顶体反应增加。附图5E表示的是暴露于500μM N-9的精子,并仅表示了顶体发生反应的精子(原始放大倍数为×1000)。
发明详述定义
除非另外指出,否则在本申请中使用的所有科学和技术术语都具有其在本领域内常用的含义。在本申请中使用的下述词语或短语具有指定含义。
如在本文中使用的“DABO”是指二氢烷氧基苄基氧代嘧啶化合物。
如在本文中使用的“S-DABO”是指化合物5-异丙基-2-[(甲硫基甲基)硫基]-6-(苄基)嘧啶-4-(1H)-酮类。
如在本文中使用的“PBT”是指化合物苯乙基-5-溴吡啶基硫脲。
如在本文中使用的“F-PBT”是指化合物N-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲。
如在本文中使用的“D-PBT”是指化合物N-[2-(2,5-二甲氧基苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲。
如在本文中使用的“N-9”是指杀病毒/杀精子剂壬苯醇醚-9。
如在本文中使用的“逆转录酶(RT)”是指具有与HIV-1 RT类似的NNI结合点的酶。
如在本文中使用的HIV逆转录酶(HIV-RT)的“非核苷类抑制剂(NNI)”是指能结合到HIV-RT的变构位点上从而非竞争性地抑制HIV-RT活性的化合物。HIV-RT的非核苷类抑制剂的实例包括但不限于S-DABO、F-PBT、和D-PBT。
如在本文中使用的术语“类似物”或“衍生物”可互换地表示在结构和功能上与另一物质相关的化学物质。类似物或衍生物含有从其它物质修饰的结构,并保持其它物质的功能,在本申请中,是保持与NNI-RT结合位点相互作用的能力。类似物或衍生物不是必须、但是可以由其它物质合成得到。例如,DABO类似物或衍生物是指在结构上与DABO相关、但不是必须从DABO制得的化合物。
如在本文中使用的“可药用盐”是指保持母化合物的所需生物活性、并且不产生任何不良毒性作用的盐。可药用盐的实例包括但不限于(a)与无机酸形成的酸加成盐,所述无机酸有例如盐酸、氢溴酸、硫酸、磷酸、硝酸等;和与有机酸形成的盐,所述有机酸有例如乙酸、草酸、酒石酸、琥珀酸、马来酸、富马酸、葡糖酸、柠檬酸、苹果酸、抗坏血酸、苯甲酸、鞣酸、扑酸、藻酸、聚谷氨酸、萘磺酸、萘二磺酸、聚半乳糖醛酸;(b)与多价金属阳离子例如锌离子、钙离子、铋离子、钡离子、镁离子、铝离子、铜离子、钴离子、镍离子、镉离子等形成的盐;或(c)与由N,N’-二苄基乙二胺或乙二胺形成的有机阳离子形成的盐;或(d)(a)与(b)或(c)的组合,例如锌鞣酸盐等。优选的酸加成盐是三氟乙酸盐和乙酸盐。
如在本文中使用的“可药用载体”包括当与本发明化合物混合在一起时使得化合物能保持其生物活性例如抑制精子活动的能力、并且与个体的免疫系统不发生反应的任何物质。其实例包括但不限于任何标准药物载体,例如磷酸盐缓冲盐水溶液、水、乳液例如油/水乳液、和各种类型的湿润剂。用于气雾剂或非胃肠道给药的优选稀释剂是磷酸盐缓冲盐水或标准(0.9%)盐水。通过公知的常规方法配制含有这样的载体的组合物(参见,例如Remington’s PharmaceuticalSciences,第43章,14th Ed.,Mack Publishing Col,Easton Pa.18042,USA)。
化合物
在具有改善的抗HIV活性的治疗剂的发展史中,合成了新的HIV-1逆转录酶(RT)的NNIs。在1998年3月17日提交的美国专利申请09/040538公开了逆转录酶的非核苷类抑制剂—结合HIV-RT袋的NNI复合物,及使用它们的方法,该申请引入本发明以作参考。
我们出乎意料地发现,其中一些NNIs具有杀精子和/或精子固定活性,这使得它们可用作避孕活性剂和精子固定产品以及方法。具有杀精子活性的NNIs包括PBT和DABO的新衍生物。本发明PBT和DABO衍生物可特别用于形成可减少性传播疾病传播、尤其是HIV传播的避孕产品。将精子与这些杀精子NNIs接触可抑制精子的运动性并达到所需的杀精子和/或受孕抑制效果。
表现出显著杀精子活性的PBT衍生物包括具有化学结构(I)的卤代PBT衍生物或其可药用盐:其中R、R1、R2、R3、和R4独立地为氢、F、Cl、Br、和I,并且R、R1、R2、R3、和R4当中至少有一个是F、Cl、Br、或I。
优选地,在结构(I)中,R、R1、R2、R3、和R4当中有一个是F或Cl。一些、但不是全部合适的本发明卤代PBT衍生物列举如下:
N-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲
N-[2-(2-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲
N-[2-(3-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲
N-[2-(3-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲
N-[2-(4-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲
N-[2-(4-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲。
本发明PBT衍生物可如实施例中所述,和如Vig等人在BIOORG.MED.CHEM.,6:1789-1797(1998)中所述合成得到。简言之,将2-氨基-5-溴吡啶与1,1-硫代羰基二咪唑缩合以生成硫代羰基衍生物前体。再进一步与适当卤代的苯乙胺反应以生成目标卤代PBT衍生物。
表现出显著杀精子活性的DABO衍生物包括具有下示化学结构(III)的DABO衍生物或其可药用盐:其中R1和R2相同或不同,并且是氢、卤素、烷基、链烯基、羟基、烷氧基、烷硫基、硫羟基、膦基、ROH、或RNH基团,其中R是烷基。优选地,一个或多个R1和R2是具有1-3个碳原子(C1-C3)的烷基,例如甲基(Me)、乙基(Et)、或异丙基(i-Pr)。优选地,R1是烷基、链烯基、ROH、或RNH2。R2优选为卤素、烷基、或C1-C3烷氧基;
Y是S或O,优选为S。R3是烷基、链烯基、芳基、芳烷基、ROH、或RNH基团,其中R是烷基,并且优选为C1-C3烷基。
有些、但不是全部合适的本发明DABO衍生物化合物包括下述化合物(a)-(d)或其可药用盐:
(a)5-甲基-2-[(甲硫基甲基)硫基]-6-苄基-嘧啶-4-1H-酮,
(b)5-乙基-2-[(甲硫基甲基)硫基]-6苄基-嘧啶-4-1H-酮,
(c)5-异丙基-2-[(甲硫基甲基)硫基]-6-苄基-嘧啶-4-1H-酮,和
(d)5-异丙基-2-[(甲硫基甲基)硫基]-6-(3,5-二甲基苄基)-嘧啶-4-1H-酮。
DABO衍生物可如实施例中所述,和如Vig等人,BIOORG MED CHEMLETTERS,8:1461-1466(1998)所述制得。
试剂和条件:a)R2CHBrCOOEt/Zn/THF,b)HCl(aq),c)(H2N)2CS/Na/EtOH,d)DMF,K2CO3,氯甲基甲基硫化物,15h。
简言之,由市售苯基乙腈制得2-烷基-4-(苯基)-3-氧代丁酸乙酯1a-d。在乙醇钠存在下将该β-酮基酯与硫脲缩合,以生成相应的硫尿嘧啶2a-d。化合物(1a-d和2a-d)是按照现有技术中的方法制得的(Danel,K.等人,Acta Chemica Scandinavica,1997,51,426-430;Mai,A.等人,J.Med.Chem.,1997,40,1447-1454;Danel,K.等人,J.Med.Chem.,1998,41,191-198)。
然后在碳酸钾存在下将硫尿嘧啶与甲基氯甲基硫化物在N,N-二甲基甲酰胺(DMF)中反应,以中等产率获得了化合物3a-d。将硫尿嘧啶化合物2(1mmol)、甲基氯甲基硫化物(1mmol)、和碳酸钾(1mmol)在无水DMF(5ml)中的混合物于室温搅拌过夜。用水(50ml)处理后,用乙酸乙酯萃取该溶液(3×50ml)。合并萃取液,用饱和NaCl洗涤(2×50ml),干燥(MgSO4),过滤并真空浓缩,获得了粗产物3a-d,通过柱色谱法纯化(洗脱剂为己烷∶乙酸乙酯)。
本申请实施例中描述了一些PBT衍生物和DABO衍生物的纯度和物理化学特征,Vig等人,BIOORG.MED.CHEM.,6:1789-1797(1998);和Vig等人,BIOORG MED CHEM LETTERS,8:1461-1466(1998)中对此作了更详细报道。
组合物、产品和方法
本发明PBT和DABO衍生物可配制成杀精子用组合物。这样的组合物可特别用于哺乳动物,即用乳腺分泌的乳汁哺育其幼子的任何高级脊椎动物,例如人、兔和猴子。本发明组合物还可用作精子固定组合物。预计本发明可由人在大多数实际应用中使用。
本发明组合物含有一种或多种如上所公开的杀精子PBT和DABO衍生物和其可药用盐。杀精子PBT和DABO衍生物的含量一般为实现所需杀精子和抗病毒保护结果所必需的量。对于具体组合物,该量可随需要而变。杀精子PBT和DABO衍生物的含量一般为占避孕用组合物总重量的约0.025-0.5%重量。杀精子PBT和DABO衍生物的含量优选为占组合物总重量的约0.05-0.5%重量、更优选为0.05-0.25%重量。
本发明组合物可不仅含有杀精子PBT和DABO衍生物,而且随着具体需要还含有可药用载体、稀释剂或赋形剂,即用于将杀精子NNIs适当地递送给和/或保持在与精子接触的位置上以提供所需杀精子和/或抗病毒保护活性的物质。
用于施用杀精子剂的药物组合物中的一种有益组分是如在US5595980中描述的聚合递送组分,该文献引入本发明以作参考。已经发现,这样的聚合递送组分能提高所施用的杀精子剂的有效性和减轻阴道刺激。
除了聚合组分以外,本发明避孕组合物的平衡部分、即一般占组合物约0.1-99.8%、经常约50-99.8%重量的部分可任选包含一种或多种美容组分。这样的美容组分是本领域技术人员已知的,并且在本领域中经常称为稀释剂、溶剂和辅料。美容组分一般包括例如水、乙醇、异丙醇、甘油、丙二醇、山梨醇和其它高分子量醇。此外,本发明避孕组合物可含有少量、例如占避孕组合物重量约0.1-5%重量的其它添加剂,例如稳定剂、表面活性剂、薄荷醇、桉树油、其它精油、香料等。聚氧化乙烯20脱水山梨醇一月桂酸酯是用于本发明组合物的优选稳定剂。美容组分、其它添加剂的选择和用量、以及混合操作可依据本领域众所周知的技术进行。
本发明杀精子活性组分和含有所述组分的避孕组合物可通过本领域技术人员已知的手段递送到哺乳动物阴道中。递送本发明组合物的典型形式包括例如霜剂、洗剂、凝胶剂、泡沫剂,阴道间装置例如海绵、避孕套包括女用避孕套、栓剂和薄膜。此外,本发明避孕化合物和组合物可用作个人用养护产品例如避孕套润滑剂等。除了本发明组合物以外。这样的润滑剂还可包含已知的常用组分,例如:湿润剂,例如甘油、山梨醇、甘露醇、二元醇和二元醇醚;缓冲剂,例如葡萄糖酸-d-内酯;杀菌剂或灭菌剂,例如葡萄糖酸洗必太;防腐剂,例如对羟基苯甲酸甲酯;粘度调节剂,例如羟乙基纤维素等;其它辅料,例如着色剂和香料。本领域技术人员可认识到,递送形式的物理性质例如粘度可广泛变化。例如,凝胶形式的本发明组合物的粘度例如150000厘泊可基本上高于洗液形式的本发明组合物的粘度例如100厘泊。关于这样的递送形式的材料、组分、比例和操作的进一步详细内容可依据本领域众所周知的技术来选择。
本发明避孕组合物优选以能有效地固定精子例如存在于阴道中的精子和/或抑制精子穿透子宫颈粘液的剂量施用到接触精子的位置上,例如施用到哺乳动物的阴道中。典型剂量为约0.0001-0.001克组合物/千克哺乳动物体重。
还可以使用如在US 5069906中描述的阴道间装置来帮助施用杀精子活性组分或含有所述组分的避孕组合物,该文献引入本发明以作参考。
在以上述组合物形式施用杀精子活性组分时,还可以将组合物配制成能迅速地释放和/或延时释放杀精子剂药物的制剂。US 4707362中描述了提供迅速和延时释放的这样的制剂,该文献引入本发明以作参考。
还可将本发明杀精子NNIs掺入到杀精子产品例如阴道插入物、避孕套或其它这种装置中,这样当使用这样的产品时,可递送杀精子NNI与精子接触。
用下述实施例更详细地描述本发明,但这些实施例不应理解为是对本发明的限制。
实施例
为了开发既能预防HIV传播又能控制生育的阴道杀微生物避孕药,我们合成了新的HIV-1逆转录酶(RT)非核苷类抑制剂(NNIs),并测定了其抗HIV和杀精子的双重功能。
用作HIV RT抑制剂的新NNIs是基于计算机模型合成的,其中在该计算机模型中用9个有独立晶体结构的RT-NNI复合物构建一个复合结合袋。[插入物指引]新的NNIs包括:N-[2-(2,5-二甲氧基苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲(D-PBT)、N-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲(F-PBT)、和5-异丙基-2-[(甲硫基甲基)硫基]-6-(苄基)-嘧啶-4-(1H)-酮(S-DABO)。
如附图1所示,NNI trovirdine可看作由硫脲基连接在一起的两个化学基团。该分子的一半是由形成分子内氢键键合杂环的吡啶基硫脲部分组成。该分子的另一半是通过乙基接头与硫代羰基隔开的吡啶环。使用trovirdine的计算机docking操作处理显示了可用于在吡啶环和乙基接头周围并且靠近trovirdine的5-溴位点处掺入较大官能团的多个位点。因此,加入战略设计的官能团以产生对于HIV RT的NNI结合袋有潜在较高亲和力的新衍生物。通过用2,5-二甲氧基苯基部分(D-PBT)或在邻位有氟原子的苯基(F-DBT)替换trovirdine的2-吡啶基环合成了两个新的PBT衍生物(D-PBT和F-PBT)。通过强效HEPT衍生物的模型化研究和通过在胸腺嘧啶环的C-5位置加入异丙基(S-DABO)完成了DABO的衍生化。
通过使用感染HIV的人外周血液单核细胞(PBMCs)测定病毒RT活性和p24抗原生成(作为病毒复制的标记物),比较这三种新的NNI(D-PBT、F-DBT和S-DABO)与trovirdine和杀病毒剂/杀精子剂壬苯醇醚-9(N-9)的抗HIV活性。分别通过计算机辅助精子分析(CASA)和同焦激光扫描显微镜检查(CLSM)来分析对精子运动运动学和精子膜完整性的影响。使用MTT测定法测试NNI与N-9抗正常人子宫颈外和子宫颈内上皮细胞的生长抑制作用。所有这三种NNIs都是纯化的重组HIV RT的强效抑制剂,并且以毫微摩尔浓度消除了HIV在PBMC中的复制(IC50<1nM),而N-9与trovirdine的IC50值分别为2.2μM和0.007μM。两种NNIs-F-PBT与S-DABO还表现出浓度-和时间-依赖性的杀精子活性。I.材料和方法A.化学合成
所用的所有化学品都是得自Aldrich Chemical Company(Milwaukee,Wisconsin)。所有反应都是在氮气氛下进行的。使用EM Science硅胶60和一种下述溶剂进行柱色谱法:乙酸乙酯、甲醇、氯仿、己烷、或二氯甲烷。核磁共振(NMR)光谱是在Varian(Palo Alto,CA)300 MHz仪器(Mercury 2000型)记录的,并且化学位移(δ)是以相对于内标物四甲基硅烷(0ppm)的百万分之分数(ppm)报告的。13C NMR光谱是使用质子去偶技术在同一仪器上在CDCl3中于75MHz记录的。所报告的关于13C NMR的化学位移是参照在77ppm的氯仿三重峰。熔点是用Mel-Temp 3.0(Laboratory Devices Inc.,Holliston,MA)熔化仪器测定的,并且未经校正。UV光谱是用Beckmann(Fullerton,CA)型DU 7400 UV/Vis分光计记录的,其中使用通路长度为1cm的小池和甲醇溶剂。用FT-Nicolet(Madison,WI)型Protege 460仪器记录傅里叶变换红外光谱。质谱分析是用Hewlett-Packard(Palo Alto,CA)Matrix Assisted LaserDescription飞行时间(MALDI-TOF)分光计(G2025A型)以分子离子检测方式进行的(所用基质是氰基羟基肉桂酸)。有些样本是用Finnigan(Madison,WI)MAT 95仪器分析的。元素分析是通过Atlantic Microlabs(Norcross,GA)进行的。1.Trovirdine的化学合成
依据文献中记载的方法合成Trovirdine(N-[2-(2-吡啶基)乙基]-N’-[2-溴吡啶基)]硫脲)。参见Cantrell等人,J MED CHEM39:4261-4274(1996)。2.D-PBT的化学合成
按照Vig等人在BIOORG.MED.CHEM.,6:1789-1797(1998)中描述的方法合成化合物D-PBT([N-[2-(2,5-二甲氧基苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲)。
简言之,如反应方案1所示合成D-PBT。反应方案1所得D-PBT的纯度和性质如下所示:
白色固体(2g,67%);mp 133-138℃;UV(MeOH)λmax:202,205,231,276和300nm;IR(KBr圆片)ν3209,3152,3078,3028,2951,2831,1595,1533,1468,1306,1227,1095,1059,1022,862,825,796,707cm-1;1H NMR(CDCl3)δ 11.24(br s,1H),9.30(brs,1H),8.10-8.09(d,1H),7.65(dd,1H),6.82-6.76(m,4H),4.03-3.97(q,2H),3.77(s,3H),3.76(s,3H),3.00-2.96(t,2H);13C NMR(CDCl3)δ178.7,153.1,151.8,151.7,146.5,140.9,128.1,117.7,113.3,112.6,111.2,110.9,55.7,55.5,45.6,和29.9;MALDI-TOF MS:实测值394.0(M-1),396.0(M+1),计算值395.0;元素分析(C16H18BrN3O2S)C,H,N,S,Br。3.F-PBT的化学合成
按照Vig等人在BIOORG.MED.CHEM.,6:1789-1797(1998)中描述的方法合成化合物F-PBT(N-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲)。
更具体来说,依据反应方案2合成F-PBT。简言之,将2-氨基-5-溴吡啶与1,1-硫代羰基二咪唑缩合,以生成硫代羰基衍生物前体。进一步与适当取代的苯乙胺反应,当与2-氟苯乙胺反应时,生成目标产物F-PBT。反应方案2
其中:i)1,1-硫代羰基二咪唑,乙腈,室温,12小时;和
ii)DMF,2-氟苯乙胺,100℃,15小时。
具体采用下述操作来合成F-PBT:在室温将硫代羰基二咪唑(8.90g,50mmol)和2-氨基-5-溴吡啶(8.92g,50mmol)加到50mL无水乙腈中。将该反应混合物搅拌12小时,过滤沉淀,用冷的乙腈洗涤(2×25mL),并真空干燥,获得了11.40g(80%)化合物A。向化合物A(0.55当量)在二甲基甲酰胺(15mL)内的悬浮液中加入适当胺-2-氟苯乙胺(0.50当量)。将该反应混合物加热至100℃并搅拌15小时。将该反应混合物倒入冰冷的水中,将该悬浮液搅拌30分钟。将产物过滤,用水洗涤,干燥,通过柱色谱法进一步纯化,以良好产率获得了目标化合物。
所得N-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲(F-PBT)的纯度和性质如下所示:
产率:71%;mp 156-157℃;UV(MeOH)λmax:209,256,274和305nm;IR(KBr)ν3446,3234,3163,3055,2935,1672,1595,1560,1531,1466,1390,1362,1311,1265,1227,1169,1136,1089,1003,864,825,756cm-1;1H NMR(CDCl3)δ11.36(br s,1H),9.47(br s,1H),8.05-8.04(d,1H),7.72-7.68(dd,1H),7.30-7.03(m,4H),6.87-6.84(d,1H),4.06-3.99(q,2H),3.10-3.05(t,2H);13C NMR(CDCl3)δ179.1,163.1,151.7,146.2,141.1,131.2,131.1,128.5,128.4,124.1,115.5,115.2,113.6,112.2,45.8和28.2;19F NMR(CDCl3)δ-42.58&-42.55(d);MaldiTof,实测值:355.0(M+1),计算值:354.0;元素分析(C14H13BrFN3S)C,H,N,S。4.S-DABO的化学合成
按照Vig等人在BIOORG MED CHEM LETTERS,8:1461-1466(1998)中描述的方法合成S-DABO,即5-异丙基-2-[(甲硫基甲基)硫基]-6-(苄基)-嘧啶-4-(1H)-酮(S-DABO)。
其中:a)R2CHBrCOOEt/Zn/THF,
b)HCl(aq),
c)(H2N)2CS/Na/EtOH,
d)DMF,K2CO3,氯甲基甲基硫化物,15小时。
简言之,由市售苯基乙腈制得2-异丙基-4-(苯基)-3-氧代丁酸乙酯。在乙醇钠存在下将该β-酮基酯与硫脲缩合,以生成硫尿嘧啶。2-异丙基-4-(苯基)-3-氧代丁酸乙酯和硫尿嘧啶化合物是按照现有技术中的方法制得的(Danel,K.等人,Acta Chemica Scandinavica,1997,51,426-430;Mai,A.等人,J.Med.Chem.,1997,40,1447-1454;Danel,K.等人,J.Med.Chem.,1998,41,191-198)。然后在碳酸钾存在下将硫尿嘧啶与甲基氯甲基硫化物在N,N-二甲基甲酰胺(DMF)中反应,以中等产率获得了化合物3a-d。将硫尿嘧啶化合物2(1mmol)、甲基氯甲基硫化物(1mmol)、和碳酸钾(1mmol)在无水DMF(5ml)中的混合物于室温搅拌过夜。用水(50ml)处理后,用乙酸乙酯(3×50ml)萃取该溶液。合并萃取液,用饱和NaCl洗涤(2×50ml),干燥(MgSO4),过滤并真空浓缩,获得了粗产物3a-d,通过硅胶60柱色谱法纯化(洗脱剂为己烷∶乙酸乙酯)。
所得5-异丙基-2-[(甲硫基甲基)硫基]-6-苄基-嘧啶-4-1H-酮(S-DABO)的纯度和性质如下所示:
产率57%;mp 116-117℃;1H NMR(CDCl3):δ1.22(d,6H),2.07(s,3H),3.03(q,1H),3.88(s,2H),4.21(s,2H),7.24-7.13(m,5H),12.43(s,1H);13C NMR(CDCl3):δ15.4(SCH3),19.6(CH3),28.0(CH),36.3(CH2Ph),40.9(SCH2),125.3(C-5),138.3-126.3(Ph),155.5(C-6),161.1(C-4),164.5(C-2);CI-MS 321.1(M+1)。
B.体外测定抗HIV活性
在CCRF-CEM细胞中繁殖HIV-1的HTLVIIIB株,用MT-2细胞滴定从受感染细胞的无细胞上清液获得的病毒贮存物。收集无细胞上清液,并以1ml等分试样在-70℃冷冻。用MT-2细胞测定病毒贮存物的效价,在该实验中使用的病毒的细胞病变作用是HIV-1对MT-2细胞的典型作用。
为了在体外分析测试药物-D-PBT、F-PBT、S-DABO、trovirdine、和N-9(IGEPAL CO-630;Rhone Poulenc,Cranbury,NJ)的抗HIV-1活性,将得自HIV-1阴性供体的正常外周血液单核细胞(PBMCs)在RPMI 1640培养基(Gibco-BRL,Grand Island,NY)中培养72小时,其中所述培养基含有20%(v/v)热灭活的胎牛血清、3%白介素、2.2mM L-谷氨酰胺、25mM HEPES、2g/L NaHCO3、50μg/ml庆大霉素、和4μg/ml植物凝集素,然后在潮湿的5%CO2气氛中于37℃在1小时吸附时间内以0.1的感染复数暴露于HIV-1。在二甲亚砜(DMSO)中制备NNIs、trovirdine、D-PBT、F-PBT、和S-DABO的贮存液(10mM),将N-9在培养基中稀释。将细胞在96-孔微量滴定板(100μl/孔;2×106个细胞/ml,一式三份孔)中在和不在不同浓度(0.001μM-100μM)抗HIV剂存在下培养7天。未感染对照组的细胞以相同方式处理,只是在制备过程中省去病毒感染操作。在感染后第7天从各孔中取出等分试样的培养物上清液,并按照在下述文献中描述的方法进行RT测定和p24抗原测定:Erice等人,ANTIMICROB AG CHEMOTHER,37:835-838(1993);Uckun等人,ANTIMICROB AGENTS CHEMOTHER,42:383-388(1998);和Zarling等人,Nature 347:92-95(1990)。
该p24酶免疫测定是通过商业渠道获得的未改进动力学测定(Coulter Corporation/Immunotech,Inc.,Westbrook,ME)。该测定使用抗HIV核心蛋白的涂敷在微孔条上的鼠单克隆抗体,存在于测试培养物上清液样本中的抗原将结合到该微孔带上。将该平板在ELISA读数器(Molecular Devices,Sunnyvale,CA)上于650nm读数,使用由Coulter/Immunotech,Inc.提供的已知标准物计算p24水平,以ng/ml表示。通过比较用测试物处理的受感染细胞的p24值与未处理的受感染细胞(即病毒对照)的p24值来计算病毒抑制百分比。
使用无细胞Quan-T-RT系统(Amersham Corp.,ArlingtonHeights,IL)测定化合物D-PBT、F-PBT、S-DABO、和trovirdine抗纯化的重组HIV RT的RT抑制活性,该系统利用的是闪烁亲近测定原理,参见Bosworth N,和Towers P,闪烁亲近测定法,NATURE,341:167-168(1989)。在该测定中,通过生物素/链霉抗生物素蛋白连接将DNA/RNA引物/模板结合到SPA玻珠上。该引物DNA是已经退火到聚(rA)模板上的16-mer寡聚(T)。将该引物/模板结合到用链霉抗生物素蛋白涂敷的SPA玻珠上。通过逆转录将3H-TTP掺入到该引物内。简言之,将在RT测定缓冲液(49.5mM Tris-HCl,pH8.0,80mM KCl,10mM MgCl2,10mM DTT,2.5mM EGTA,0.05%Nonidet-P-40)中稀释至终浓度为0.5 Ci/样本的3H-TTP加到结合在SPA玻珠上的退火DNA/RNA中。把测试化合物以0.001μM-100μM的浓度加到该反应混合物中。加入10mU重组RT,并在37℃培养1小时,获得了由于掺入3H-TTP而延伸的引物。通过加入0.2ml 120mM EDTA来中止反应。用Beckman LS 7600仪器(Beckman Instruments,Fullerton,CA)将样本在开放窗口中计数。
通过比较得自用测试药物处理的受感染细胞的p24抗原值或RT活性与得自未处理受感染细胞的p24抗原值或RT活性来计算病毒复制的抑制百分比。化合物的抗HIV活性以IC50值表示,所述IC50值是由剂量-响应曲线计算的,并且定义为在HIV-1感染的PBMCs中使HIV-1 RT活性或p24抗原生成降低50%时药物的浓度。使用如Uckun等人在ANTIMICROB AGENTS CHEMOTHER,42:383-388(1998)中所公开的微量培养四唑鎓测定法(MTA)平行测定不同处理对细胞存活力的影响,该方法使用的是2,3-二(2-甲氧基-4-硝基-5-磺苯基)-5-[(苯基氨基)羰基]-2H-四唑鎓氢氧化物(XTT)。
C.测定精子固定活性(SLA)
为了测定与N-9相比trovirdine、D-PBT、F-PBT、S-DABO、和ZDV的杀精子作用,通过不连续(90-45%)Percoll梯度(ConceptionTechnologies,San Diego,CA)离心和如D’Cruz等人在BIOL REPROD,54:1217-1228(1996)中描述的“游泳”法制备合并的供体精子(n=6)的高运动性部分。所有供体精子标本都是在获得同意后按照HughesInstitute Institutional Review Board的指导获得的。将能运动的精子(≥10×106/ml)悬浮在1ml Biggers,Whitten,andWhittingam’s培养基(BWW)中,该培养基含有25mM HEPES(IrvineScientific,Santa Ana,CA)和0.3%BSA,并且其中存在和不存在测试物在1%DMSO中的系列两倍稀释物(500μM-31.2μM)。在DMSO中制备NNIs的贮存液(100mM),并在测定培养基中稀释以产生所需浓度。将相应体积的DMSO(1%)加到对照管中。将N-9在BWW-0.3%BSA(pH7.4)中稀释以产生所需浓度(31.2μM-500μM)。在37℃培养3小时后,按照在下述文献中描述的方法通过CASA测定能运动的精子的百分比:D’Cruz等人,BIOL REPROD,58:1515-1526,(1998);和D’Cruz等人,MOL HUM REPROD,4:683-693(1998)。将该运动性百分比与能运动精子的假处理对照悬浮液的运动性百分比比较。测试化合物的杀精子活性以EC50表示(使能运动的精子的比例下降50%时培养基中化合物的终浓度)。
为了测试在培养期间杀精子NNIs对SIA的影响,在1mM F-PBT、S-DABO(在1%DMSO中)存在下或仅在用作赋形剂对照的1%DMSO存在下,在BWW-0.3%BSA中将能运动的精子部分(107/ml)在37℃培养。以5或10分钟的时间间隔,将一式两份的等分试样(4-μl)转移到两个20μm微细胞室(Conception Technologies)中,并通过CASA在60分钟期间内测定精子的运动性。
D.精子运动学参数
对于CASA,在37℃将4μl每份精子悬浮液加到在计数室中的两个20-μm微细胞室中。对于每个室,用Hamilton Thorne IntegratedVisual Optical System(IVOS),version 10仪器(Hamilton ThorneResearch Inc.,Danvers,Mass.)扫描至少5-8个区域以进行分析。每个区域记录30秒。在30个读框上以30/秒的读框速度设置计算机校准。其它设置如下:最小对比度8;最小尺寸6;低-尺寸门,1.0;高-尺寸门,2.9;低-强度门,0.6;高-强度门,1.4;相衬照明;低路径速度为10μm/s,阈平直度为80%;扩大因数,1.95。
测定的精子运动学参数包括运动(MOT)和渐进性(PRG)运动精子的数目;曲线速度(VCL;给定精子在一定时间内移动的总距离除以流逝的时间的一种量度);平均路径速度(VAP;消除精子头摆动的空间平均路程),直线速度(VSL;从足迹开始到终点的直线距离除以所用时间),交叉拍打频率(BCF;精子头横越精子平均路径的频率),侧面头移位的幅度(ALH;精子头摆动的平均宽度),和派生物,平直度(STR=VSL/VAP×100)和直线性(LIN=VSL/VCL×100;精子足迹偏离直线)。记录各个独立细胞足迹的数据并分析。对于每一等分试样,分析至少20个能运动的精子。
E.同焦激光扫描显微镜检查(CLSM)
通过CLSM测定用和不用递增浓度的(31.2μM-500μM)三种NNIs(S-DABO、F-PBT和D-PBT)以及与之比较的N-9处理6小时后具有完整顶体的精子的百分比。用乙醇渗透,然后用三种荧光标记物FITC-豌豆凝集素、TOTO-3碘化物、和尼罗红(Molecular Probes,Eugene,Oreg.)将风干的精子涂片染色,之所以使用这三种标记物是因为它们的目标不同(分别给顶体、核和膜脂质染色)。用装配着氪/氩混合气体激光器(激发线488、568和647nm)、并固定在Nikon Eclipse E800系列立式显微镜上的BioRad MRC-1024激光扫描同焦显微镜(BioRadLaboratories,Hercules,CA)检测样本。用乙醇渗透后,分别用598/40nm、522 DF32、和680 DF32发射/滤器同时检测荧光素、TOTO-3碘化物、和尼罗红从顶体区域、核、和精子质膜上发射的荧光。使用Nikon 100×(NA 1.35)物镜和Kalman收集滤器获得同焦图像。将数字化图像保存在Jaz磁盘(Iomega Corporation,Roy,UT)上,并用带Adobe Photoshop软件(Adobe Systems,Mountain View,CA)的Lasersharp(Bio-Rad)处理。用Fuji Pictrography 3000(FujiPhoto Film Co.,Tokyo,Japan)彩色打印机打印最终的图像。
F.细胞增殖测定
从Clonetics(San Diego,CA)获得正常人子宫颈外上皮细胞(CrEC 4627)和子宫颈内上皮细胞(CrEC-En 4312),并在T-150cm2组织培养瓶(Corning Corp.,Corning,NY)内在小导气管上皮细胞基本培养基(Clonetics)中繁殖,该培养基补充有50μg/ml BPE、0.5μg/ml氢化可的松、0.5μg/ml hEGF、0.1μg/ml视黄酸、10μg/ml转铁蛋白、5μg/ml胰岛素、5μg/ml肾上腺素、和0.5mg/mlBSA-FAF。为了测定与N-9相比F-PBT和S-DABO的生长抑制作用,我们使用如Narla等人在CLIN CANCER RES,4:1405-1414(1998)中描述的基于MTT(3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四唑鎓溴化物)的比色测定法来定量测定细胞增殖。
简言之,用0.125%(w/v)胰蛋白酶-0.02%EDTA(GIBCO)从指数期维持培养物中收获细胞,并离心(300g×5分钟)。悬浮并计数后,将细胞以100μl的体积按一式三份的方式加到96-孔组织培养板中。培养3小时和24小时后,弃去培养基,并用含有进行过系列两倍稀释的药物的100ml新鲜培养基替换,其中对于N-9,是将其在培养基中稀释以产生3.9μM-250μM的浓度,对于F-PBT和S-DABO,是在培养基中稀释以生产250μM-4mM的浓度。将NNIs在DMSO中重新配制以使其浓度为100mM。在无菌PBS中制备N-9的贮存液。具有仅含0.25%DMSO的培养基的对照孔用作对照。将培养板培养24小时,然后向各孔中加入10μl MTT溶液(5mg/ml,在PBS中)。仅含培养基和MTT的孔用作各平板的对照。将四唑鎓/甲反应在37℃进行4小时,然后向所有孔中加入100μl增溶缓冲液(10%十二烷基硫酸钠,在0.1%HCl中),并充分混合以将深蓝色甲晶体溶解。
在37℃培养过夜后,用96-孔多扫描自动读数器在540nm测定光密度(OD),使用该增溶缓冲液作为空白。为了将OD540值转化成各孔中活细胞的数目,把该OD540值与所产生的关于各细胞系的标准OD540-对-细胞数目曲线上的OD540值比较。用下述公式计算存活百分比:%存活=活细胞数目[测试]/活细胞数目[对照]×100。所有测定都是以一式三份进行,并且结果以IC50值表示。IC50定义为使细胞存活减少50%所需的浓度。
G.统计学分析
结果以各独立测试的平均值或平均值+SD值表示。通过方差单向分析和Dunnett’s多重比较检验分析测试组之间差异的统计显著性。使用线性回归分析来得到两个测定的参数之间的相关值。<0.05的p值认为有统计显著性。采用非线性回归分析,使用GraphPadPRISM 2.0版本软件程序(San Diego,CA)来从浓度作用曲线中得到IC50和EC50值。
II.结果
A.表
通过下面两个表来讨论实验结果:
表1.三种新NNIs对感染HIV的PBMC中的纯化的重组HIV-RT的酶活性、p24抗原生成、以及人精子运动性的影响。化合物 抗HIV活性 杀精子活性
rRTa HTLVIIIB EC50(μM)d
IC50rRT(μM)b IC50p24(μM)cD-PBT 0.1 <0.001 >500F-PBT 0.4 <0.001 147S-DABO 6.1 <0.001 202Trovirdine 0.8 0.007 >500N-9 Nde 2.2 81a重组HIV逆转录酶bIC50 RT=将HIV-RT活性抑制50%的药物浓度cIC50 p24=将HIV-p24抗原生成抑制50%的药物浓度dEC50=将精子运动性抑制50%的药物浓度eND=未测定
表2.通过MTT测定法定量测定的F-PBT和N-9对正常人子宫颈外和子宫颈内上皮细胞的细胞生长的影响。处理 精子 子宫颈外上皮细胞a 子宫颈内上皮细胞a
EC50(μM)b IC50(μM)c SId IC50(μM) SIF-PBT 147 >1000 >6.8 >1000 >6.8S-DABO 202 >4000 >19.8 >4000 >19.8N-9 81 15 0.18 11 0.13a用5种不同浓度的(250μM-4000μM)F-PBT和S-DABO处理24小时后测试细胞增殖bEC50=将精子运动性抑制50%的药物浓度cIC50=将细胞生长抑制50%的药物浓度dSI=等于IC50与EC50比值的选择性指数
B.新NNIs的抗HIV活性
在使用纯化的重组HIV RT的无细胞测定中测定如附图1所示的三种新NNI衍生物以及trovirdine关于RT抑制活性的作用(作为IC50[rRT]列出),并通过体外测定在感染HTLVIIIB的PBMCs中测定它们的抗HIV活性(IC50[p24])。如附图1所示,正如通过在感染HIV的人PBMCs中测定p24抗原生成所表明的那样,所有这三种NNIs都表现出很强的抗HIV活性,其IC50值低于1nM。在相同实验条件下,迄今为止所报道的最有效的NNI-Trovirdine的效力至少低7倍。而且,在抑制重组HIV RT方面,D-PBT与F-PBT的效力比trovirdine强至少2-8倍。所观察到的由这三种NNIs所致的RT活性和p24生成下降并不是由于它们具有任何细胞毒性作用,这是因为即使在最高测试浓度下PBMC的细胞存活力也没有受到影响(IC50[MTA]>100μM)。这三种新NNIs的抗HIV活性(IC50[p24])比洗涤剂型杀微生物剂N-9(IC50=2.2μM)高至少2000倍。
C.新NNIs的杀精子活性
接下来测定了与N-9相比三种NNIs(S-DABO、D-PBT、和F-PBT)和trovirdine对人精子功能的影响。将人精子的高运动性部分暴露于trovirdine或D-PBT没有影响精子运动性,即使在高达500μM浓度下也是如此(见附图2和表1)。此外,使用CASA的精子运动运动学证实,trovirdine或D-PBT处理没有改变精子运动参数,例如渐进性运动(PRG)、足迹速度(VCL)、路径速度(VAP)、直线速度(VLS)、游动模式的平直度(STR)、精子足迹的直线性(LIN)、拍打频率(BCF)、和侧面精子头移位的幅度(ALH)。与trovirdine或D-PBT相反,在PBT中苯基环的邻位引入氟原子,或者在S-DABO胸腺嘧啶环的C-5位引入异丙基,产生了浓度依赖性杀精子活性。由F-PBT获得的147μM的EC50值(95%CI:94-330μM)在N-9的杀精子范围内(EC50值81μM;95%CI:41-410μM)(见附图2和表1)。而且,通过CASA测定的精子固定动力学表明,暴露于化合物F-PBT后,在培养时间与渐进精子运动性损失之间有直线关系(相关系数0.811,p<0.005)(见附图3)。暴露于F-PBT后,使能渐进运动的精子的运动性下降50%所需的时间是10分钟(95%CI:8-12分钟)。与之相比,对照样本中的精子运动性在60分钟的监测期间内保持稳定(与基准相比为95%±3%)。
F-PBT诱导的时间依赖性精子运动性损失(PRG)还伴有存活精子运动特征的显著改变,特别是在足迹速度(VCL)、路径速度(VAP)、和直线速度(VSL)方面的改变。用F-PBT处理的精子的代表性精子运动学参数与时间的关系如附图4所示。VSL与VCL或者VSL与VAP的下降幅度相类似。因此,关于精子足迹直线性的值(LIN)与游动模式平直度的值(STR)保持相对恒定。而且,当能运动的精子的比例在运动性损失的直线相期间下降时,拍打频率(BCF)、和侧面精子头移位的幅度(ALH)相对稳定。在60分钟的培养期间,对照精子的精子运动参数没有表现出显著改变。
D.杀精子NNIs没有类似于洗涤剂的膜毒性
使用最广泛的阴道杀精子剂壬苯醇醚-9是通过对精子质膜的洗涤剂型作用而固定精子。因为其膜破坏特性,据表明连续使用N-9会破坏子宫颈阴道上皮,引起急性组织炎性响应,并增加通过异性传播感染HIV的可能性。不引起任何非-特定膜毒性(这种毒性是洗涤剂型避孕药的特征)的杀精子剂将具有临床方面的优点。
因此,通过将精子三重染色(用FITC-豌豆凝集素给顶体染色,用TOTO-3碘化物给核DNA染色,用尼罗红给膜脂质染色)使用CLSM测试F-PBT与S-DABO对精子头顶体膜完整性的影响(作为膜破坏标志)。通过CLSM检查用FITC-凝集素、TOTO-3、和尼罗红染色的精子,结果显示了非常清楚的被FITC-凝集素染色的顶体(绿色)、被TOTO-3染色的核(蓝色)、和被尼罗红染色的膜(红色)(见附图5)。通过用同焦显微镜检查发现,培养6小时后,尽管在F-PBT和S-DABO存在下精子被完全固定,但是与显示出顶体染色完全缺失的N-9相比,有>90%的被处理精子显示出顶体染色(见附图5A)。因此,化合物F-PBT的杀精子活性没有引起或伴有膜破坏作用。在仅暴露于赋形剂(即1%DMSO)(如附图5B所示)或500μMF-PBT(如附图5C所示)或S-DABO(如附图5D所示)6小时的精子中,有一半以上的精子头(即顶体区域)显示出均匀、明亮的绿色荧光,这意味着顶体保持完整。与之相比,在相同条件下暴露于500μM N-9的精子(如附图5E所示)由于膜完整性被破坏以及缺失顶体膜而没有显示出任何绿荧光。F-PBT与S-DABO的这些性质与目前使用的杀精子剂N-9的活性特征形成了鲜明对照,N-9是通过类似于精子剂破坏精子膜并因此削弱精子功能的能力来发挥作用的。
E.与N-9相比F-PBT与S-DABO的选择性杀精子活性
使用测定细胞增殖和存活力的MTT测定法在体外测试与N-9相比F-PBT与S-DABO对融合的单层正常人子宫颈外和子宫颈内上皮细胞的细胞毒性。将细胞暴露于剂量范围介于3.9μM-4mM之间的这些化合物3或24小时。将通过MTT测定的关于F-PBT与S-DABO以及与之相比的N-9的浓度-响应细胞存活曲线与通过CASA测定的杀精子活性进行比较。在MTT测定中,N-9对子宫颈外上皮细胞和子宫颈内上皮细胞表现出显著的细胞毒性,平均IC50值分别为15μM和11μM。与之相比,对于子宫颈外上皮细胞和子宫颈内上皮细胞,F-PBT与S-DABO剂量存活曲线的IC50值分别>4mM(S-DABO)和>1mM(F-PBT)(见表2)。因此,N-9仅在细胞毒性浓度下有杀精子能力(EC50值:81μM;选择性指数:对子宫颈外上皮细胞和子宫颈内上皮细胞分别是0.18和0.13),而F-PBT与S-DABO对这些细胞表现出高选择性指数(SI:对于子宫颈外上皮细胞和子宫颈内上皮细胞分别是>6.8和>19.8)。因此,F-PBT与S-DABO抗这些生殖道细胞的活性非常低。这些实验证实了F-PBT与S-DABO的杀精子活性与非特异性细胞毒性无关。III.实验结果讨论
A.抗HIV活性
现有技术中的模型化实验已经揭示了产生更有效NNIs的几个潜在配基衍生化位点。将PETT衍生物trovirdine修饰,通过用2,5-二甲氧基苯基替换吡啶基环,D-PBT(N-[2-(2,5-二甲氧基苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲),或者用在邻位有氟原子的苯基环替换吡啶基环,F-PBT(N-[2-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲),获得了更有效的抗HIV药物。生物测定证实了这两种NNIs比trovirdine-迄今为止所报道的其中一种最有效的PETT衍生物更有活性(至少7倍),这两种NNIs抗HIV活性的IC50[p24]值低于1nM。计算机docking操作还表明,在S-DABO胸腺嘧啶环的5-位加上异丙基可使得对位于S-DABO结合袋的相对疏水环境有更高亲和力。DABO衍生物5-异丙基-2-[(甲硫基甲基)硫基]-6-(苄基)-嘧啶-4-(1H)-酮(S-DABO)具有很强的抗HIV活性,其抑制HIV复制的IC50值低于1nM。因此,所有这三种NNIs都比N-9强>2000倍。
据发现,NNIs结合到靠近聚合酶位点的HIV-1 RT的特异变构位点上,并通过改变RT的构象或移动性而干扰逆转录酶,从而非竞争性地抑制了该酶。与NNIs接触的多个氨基酸残基具有相当高的柔性,从结构到结构发生改变,可以被恰当的NNI替换。这些残基包括Tyr180、Tyr181、Tyr318、Try319、Phe227、Leu234、Trp229、Pro95、和Glu138(来自RT的p51亚基)。本发明所研究的新PBT和DABO衍生物的提高的抗HIV活性很可能是由于它们较大的分子表面使其有利于结合NNI袋、以及具有更强的亲脂性(由于用较大的二甲氧基苯基替换苯基环所致)、更良好的疏水接触(由于在胸腺嘧啶环的C-5位加上异丙基取代基所致)、和与目标氨基酸残基-RT(44,45)的Pro95与Trp229更紧密地接触(由于在未取代苯基环的的间位/邻位加上甲氧基取代基或者在邻位加上氟所致)。此外,这改变了构象,构象改变又影响邻近氨基酸残基、特别是Tyr183和Tyr188的位置,从而可导致HIV-1 RT失活。docking研究显示,D-PBT的2-甲氧基提供了与氨基酸残基Pro195和Trp229的紧密接触。这启示我们,将分别是独一无二的所有结构优点组合起来可获得更有效的RT抑制剂。
B.杀精子活性
联合采用精子运动运动学与同焦激光扫描显微镜检查证实了F-PBT与S-DABO以浓度和时间依赖方式使精子运动停止,并且与洗涤剂型杀精子剂N-9不同,这二者不影响精子质膜和顶体膜的完整性。此外,与在杀精子剂量下对人子宫颈外和子宫颈内上皮细胞有细胞毒性的N-9相比,F-PBT与S-DABO选择性地杀精子。因此,子宫颈外上皮细胞似乎易受洗涤剂型杀精子剂N-9所致的细胞毒性伤害。获得本发明新NNIs的杀精子功能意味着,将这些NNIs进一步修饰可获得甚至更有效的具有抗HIV和杀精子双重活性、并且对生殖道上皮细胞的细胞毒性降低的双功能NNIs。
用作具有强效杀精子活性的双重功能抗HIV剂的新NNIs的合成代表着开发用于抑制异性阴道HIV传播的新杀微生物剂的重要步骤。这些有希望的结果举例说明了如下所述的双重功能NNIs:这些NNIs表现出独特的临床潜力,有希望成为由新女性控制的局部杀病毒阴道避孕药的活性成分,以供有通过异性阴道传播获得HIV的高度危险性的女性使用。
本文描述的本发明可变更以包括供选择替代的实施方案。所有这些明显的供选择替代方案都包括在如下面权利要求书所保护的本发明实质和范围内。
本申请引用了多个出版物。这些出版物的公开内容全文引入本申请作为参考,以更全面地描述本申请所属领域的现有技术状况。
Claims (34)
2.权利要求1的化合物,其中R、R1、R2、R3、和R4当中至少有一个是F或Cl。
3.权利要求2的化合物,其中所述化合物选自:
N-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(2-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(3-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(3-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(4-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(4-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;和
它们的可药用盐。
4.权利要求3的化合物,其中所述化合物选自N-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲及其可药用盐。
5.抑制哺乳动物受孕的方法,所述方法包含用杀精子有效量的权利要求1的化合物与哺乳动物精子接触。
6.权利要求5的方法,其中所述化合物选自:
N-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(2-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(3-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(3-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(4-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(4-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;和
它们的可药用盐。
7.权利要求6的方法,其中所述化合物选自N-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲及其可药用盐。
8.抑制精子运动的方法,所述方法包含用精子运动抑制有效量的权利要求1的化合物与精子接触。
9.权利要求8的方法,其中所述化合物选自:
N-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(2-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(3-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(3-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(4-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(4-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;和
它们的可药用盐。
10.权利要求9的方法,其中所述化合物选自N-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲及其可药用盐。
11.一种组合物,它包含杀精子有效量的权利要求1的化合物和可药用载体、稀释剂或赋形剂。
12.权利要求11的组合物,其中所述化合物选自:
N-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(2-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(3-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(3-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(4-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;
N-[2-(4-氯苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲;和
它们的可药用盐。
13.权利要求12的组合物,其中所述化合物选自N-[2-(2-氟苯乙基)]-N’-[2-(5-溴吡啶基)]硫脲及其可药用盐。
14.权利要求11的组合物,其中所述药物载体提供杀精子剂的快速和延时释放。
15.权利要求11的组合物,其中所述药物赋形剂选自阴道内插入物和避孕套。
16.一种杀精子产品,它包含权利要求1的化合物。
17.权利要求13的产品,其中所述产品选自阴道插入物和避孕套。
19.下式所示权利要求18的化合物或其可药用盐:
其中R1是Me、Et、或i-Pr,且R2是H或Me。
20.权利要求19的化合物,其中所述化合物选自:
5-甲基-2-[(甲硫基甲基)硫基]-6-苄基-嘧啶-4-1H-酮;
5-乙基-2-[(甲硫基甲基)硫基]-6-苄基-嘧啶-4-1H-酮;
5-异丙基-2-[(甲硫基甲基)硫基]-6-苄基-嘧啶-4-1H-酮;
5-异丙基-2-[(甲硫基甲基)硫基]-6-(3,5-二甲基苄基)嘧啶-4-1H-酮;和
它们的可药用盐。
21.权利要求20的化合物,其中所述化合物选自5-异丙基-2-[(甲硫基甲基)硫基]-6-(苄基)-嘧啶-4-(1H)-酮及其可药用盐。
22.一种抑制哺乳动物受孕的方法,所述方法包含用杀精子有效量的权利要求18的化合物与哺乳动物精子接触。
23.权利要求22的方法,其中所述化合物选自:
5-甲基-2-[(甲硫基甲基)硫基]-6-苄基-嘧啶-4-1H-酮;
5-乙基-2-[(甲硫基甲基)硫基]-6苄基-嘧啶-4-1H-酮;
5-异丙基-2-[(甲硫基甲基)硫基]-6-苄基-嘧啶-4-1H-酮;
5-异丙基-2-[(甲硫基甲基)硫基]-6-(3,5-二甲基苄基)嘧啶-4-1H-酮;和
它们的可药用盐。
24.权利要求23的方法,其中所述化合物选自5-异丙基-2-[(甲硫基甲基)硫基]-6-(苄基)-嘧啶-4-(1H)-酮及其可药用盐。
25.一种抑制精子运动的方法,所述方法包含用精子运动抑制有效量的权利要求18的化合物与精子接触。
26.权利要求25的方法,其中所述化合物选自:
5-甲基-2-[(甲硫基甲基)硫基]-6-苄基-嘧啶-4-1H-酮;
5-乙基-2-[(甲硫基甲基)硫基]-6-苄基-嘧啶-4-1H-酮;
5-异丙基-2-[(甲硫基甲基)硫基]-6-苄基-嘧啶-4-1H-酮;
5-异丙基-2-[(甲硫基甲基)硫基]-6-(3,5-二甲基苄基)嘧啶-4-1H-酮;和
它们的可药用盐。
27.权利要求26的方法,其中所述化合物选自5-异丙基-2-[(甲硫基甲基)硫基]-6-(苄基)-嘧啶-4-(1H)-酮及其可药用盐。
28.一种组合物,它包含杀精子有效量的权利要求18的化合物和可药用载体、稀释剂或赋形剂。
29.权利要求28的组合物,其中所述化合物选自:
5-甲基-2-[(甲硫基甲基)硫基]-6-苄基-嘧啶-4-1H-酮;
5-乙基-2-[(甲硫基甲基)硫基]-6-苄基-嘧啶-4-1H-酮;
5-异丙基-2-[(甲硫基甲基)硫基]-6-苄基-嘧啶-4-1H-酮;
5-异丙基-2-[(甲硫基甲基)硫基]-6-(3,5-二甲基苄基)-嘧啶-4-1H-酮;和
它们的可药用盐。
30.权利要求29的组合物,其中所述化合物选自5-异丙基-2-[(甲硫基甲基)硫基]-6-(苄基)-嘧啶-4-(1H)-酮及其可药用盐。
31.权利要求28的组合物,其中所述药物载体提供杀精子剂的快速和延时释放。
32.权利要求28的组合物,其中所述药物赋形剂选自阴道插入物和避孕套。
33.一种杀精子产品,它包含权利要求18的化合物。
34.权利要求33的产品,其中所述产品选自阴道插入物和避孕套。
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TW200600492A (en) | 2004-05-18 | 2006-01-01 | Achillion Pharmaceuticals Inc | Substituted aryl acylthioureas and related compounds; inhibitors of viral replication |
WO2009102736A1 (en) | 2008-02-12 | 2009-08-20 | Dow Agrosciences Llc | Pesticidal compositions |
CN101628996B (zh) * | 2008-07-18 | 2012-05-30 | 绿神(北京)乳胶业有限公司 | 一种乳胶避孕套的制备方法 |
CN103491782B (zh) * | 2011-02-09 | 2016-02-24 | 陶氏益农公司 | 杀虫组合物及其相关方法 |
CA2841450C (en) | 2011-07-12 | 2019-04-23 | Dow Agrosciences Llc | Pesticidal compositions and processes related thereto |
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ZA892407B (en) | 1988-03-31 | 1989-11-29 | Mitsubishi Chem Ind | 6-substituted acyclopyrimidine nucleoside derivative and antiviral agent containing the same as active ingredient thereof |
US5686428A (en) * | 1989-04-07 | 1997-11-11 | Aktiebolaget Astra | Pharmaceutical composition |
HU205917B (en) * | 1989-09-29 | 1992-07-28 | Mitsubishi Chem Ind | Process for producing 6-substituted pyrimidine derivatives and antiviral pharmaceutical compositions containing them as active components |
US5503978A (en) * | 1990-06-11 | 1996-04-02 | University Research Corporation | Method for identification of high affinity DNA ligands of HIV-1 reverse transcriptase |
US5593993A (en) * | 1991-08-02 | 1997-01-14 | Medivir Ab | Method for inhibition of HIV related viruses |
IL102548A (en) * | 1991-08-02 | 1998-08-16 | Medivir Ab | Use of painting derivatives in the preparation of drugs for VIH inhibition and treatment of SDIA and new compounds |
JPH0725770A (ja) * | 1993-07-09 | 1995-01-27 | Mitsubishi Chem Corp | 経皮吸収性抗ウイルス剤 |
DE69414708T2 (de) * | 1993-08-24 | 1999-04-15 | Medivir Ab | Verbindungen und methoden zur inhibition von hiv und verwandten viren |
US5998411A (en) * | 1998-03-17 | 1999-12-07 | Hughes Institute | Heterocyclic nonnucleoside inhibitors of reverse transcriptase |
IT1305313B1 (it) * | 1998-07-17 | 2001-05-04 | Colla Paolo | 3,4 - diidro- 6- benzil-4-oxopirimidine sostituite e relativo processodi produzione e impiego nella terapia delle infezioni da hiv-1. |
US6136335A (en) * | 1998-12-31 | 2000-10-24 | Hughes Institute | Phenethyl-5-bromopyridylthiourea (PBT) and dihydroalkoxybenzyloxopyrimidine (DABO) derivatives exhibiting spermicidal activity |
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- 1999-12-29 WO PCT/US1999/031160 patent/WO2000039095A2/en active IP Right Grant
- 1999-12-29 CN CN99815250A patent/CN1371367A/zh active Pending
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103193771A (zh) * | 2013-03-29 | 2013-07-10 | 中国人民解放军军事医学科学院生物工程研究所 | 链状酰胺类ccr5受体抑制剂的药物新用途 |
CN103193771B (zh) * | 2013-03-29 | 2015-08-05 | 中国人民解放军军事医学科学院生物工程研究所 | 链状酰胺类ccr5受体抑制剂的药物新用途 |
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EP1140848A2 (en) | 2001-10-10 |
ATE306475T1 (de) | 2005-10-15 |
DE69927710D1 (de) | 2006-02-23 |
NO20012817L (no) | 2001-08-29 |
CA2362492A1 (en) | 2000-07-06 |
US6136335A (en) | 2000-10-24 |
KR20010082772A (ko) | 2001-08-30 |
EP1140848B1 (en) | 2005-10-12 |
BR9916620A (pt) | 2001-09-25 |
DE69927710T2 (de) | 2006-07-20 |
IL143508A0 (en) | 2002-04-21 |
JP2002533444A (ja) | 2002-10-08 |
MXPA01006746A (es) | 2002-04-24 |
WO2000039095B1 (en) | 2001-06-21 |
EP1557197A1 (en) | 2005-07-27 |
WO2000039095A2 (en) | 2000-07-06 |
WO2000039095A3 (en) | 2000-11-09 |
AU2396800A (en) | 2000-07-31 |
HUP0104904A2 (hu) | 2002-04-29 |
NO20012817D0 (no) | 2001-06-07 |
US6376504B1 (en) | 2002-04-23 |
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