CN1671384A - 治疗人和动物中微生物感染的方法 - Google Patents
治疗人和动物中微生物感染的方法 Download PDFInfo
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- CN1671384A CN1671384A CNA038185210A CN03818521A CN1671384A CN 1671384 A CN1671384 A CN 1671384A CN A038185210 A CNA038185210 A CN A038185210A CN 03818521 A CN03818521 A CN 03818521A CN 1671384 A CN1671384 A CN 1671384A
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Abstract
一种治疗患有基于微生物的感染的受试者的方法,包含对所述受试者施用一种化合物。在体外试验中,24小时后,与对照比较,所述化合物能降低微生物中ATP水平至少10%,而且在相同时间段不杀死哺乳动物细胞。通过以下步骤测量ATP水平的降低:(1)从试验位置取出细胞并将它们置于冰上;(2)通过在4℃离心收获细胞,并在ATP提取缓冲液中用玻珠打浆进行破裂;(3)通过在4℃离心除去细胞碎片,留下含有ATP的上清;(4)在4℃通过生物发光方法测定存在于上清中ATP的量。
Description
发明背景
在美国及全世界,基于微生物(Microbially-based)的感染仍然是一个主要的公共健康问题。例如,在美国及全球结核病仍然是一个重大的健康问题。结核病(TB)是世界上由于单一传染因素引起死亡的主要原因。据信大约18.6亿人或世界人口的32%被结核分枝杆菌(Mycobacteriumtuberculosis)(M.tb.)感染。每年大约有8百万新的活动性结核病例并有大约2百万人死亡。这转换成每小时200人每天5000人的死亡率。感染HIV的患者证明对M.tb.的易感性显著增加,比未感染HIV患者增加大约50倍(12,15)危险。类似地,在初次感染后,潜伏的TB进展到活动性疾病的比率更大,40%相比与HIV未感染个体中的大约5%。伴随着HIV全球性持续扩张,特别在亚洲及印度次大陆,TB的发病率和死亡率预计只会增长。
在对一种或多种标准一线药剂耐药的M.tb.菌株方面增加的发病率,加强了对鉴定新,新靶标和药物开发的需求。MDR-TB(多药耐受结核病)比药物敏感的TB治疗困难且昂贵,还与显著更高的死亡率相关。在缺乏有效的预防及治疗方法情况下,MDR-TB将成为一个持续增长并不能控制的问题。
存在改进的结核病药物的重大需求,所述药物具有降低的毒性,抗MDR-TB的活性,交替的作用机制,及抗潜伏性疾病的活性。尽管在过去的50年间,在结核病的预防与治疗方面取得了进展,但在能预期控制这种疾病之前,仍存在重大的障碍。当前护理策略的标准难以实施和维持,特别在低收入,非工业化的国家中,它们缺少财政资源或基础设施来支持有效的或包括一切的TB控制计划。MDR-TB的出现威胁着要逆转到目前为止在TB控制上取得的许多进展。
几种有希望的药物类型正在开发中,包括长效利福霉素类,喹诺酮类,噁唑烷酮类(oxazolidinones)和硝咪唑类。虽然如此,即使新的化合物到临床应用的成功率—大约0.5%—也存在发现并鉴定新的独特的的分枝杆菌靶标的开发需求。在过去三十年中,还没有开发具有新的作用机理的新的抗分枝杆菌药物。
发明的目的
本发明的一个目的是提供一种通过施用干扰微生物的中央能量代谢的化合物来治疗基于微生物感染的方法。
本发明进一步的目的包含施用抑制微生物中ATP合成和干扰这类微生物的细胞呼吸作用的化合物。
本发明进一步的目的包含施用将引起ATP(M)水平相对于对照下降至少10%的化合物。
本发明进一步的目的包含一种治疗具有基于微生物感染的受试者的方法,该方法包含对需要治疗的受试者施用一种有效量的化合物,其中所述化合物产生ATP合酶b亚单位的过量表达。
本发明进一步的目的是提供某些化合物,当被施用于具有微生物感染的人或动物时,所述化合物能通过上面描述的机理治疗感染。
附图简要说明
图1显示ATP合酶的一般结构与功能。
图2显示在处理4后小时对照与OSA处理(100μg/ml)的BCG的双向蛋白凝胶电泳分布图。
图3在OSA存在或不存在时,在BCG成体中编码ATP合酶(Rv1306)b亚单位的atpf与hsp(Rv0251c)的表达比较。
图4测定与未处理的对照比较,用OSA或二环己基碳二亚酰胺处理的BCG培养物中ATP水平的时程实验。
图5在暴露于已知的呼吸抑制剂及抗分枝杆菌药物OSA 5分钟之后,BCG中ATP浓度/CFU。
图6显示在低浓度的乙醇(0.05%)下,OSA抗结核分枝杆菌的抑制活性的增强。
图7显示暴露于BCG的早期对数期培养物10分钟后,OSA(100μg/ml),DCCD(100μg/ml)和TTFA(100μg/ml)对分枝菌酸合成的作用的比较。
发明详述
图1,来自Dimroth,et al.,″Operation of the F(0)motor of theATP synthase,″,(2000)1458:374-386,图示F1F0 ATP合酶(ATP酶)的结构。ATP酶使用来自质子动力的能量产生ATP。这种酶复合体由跨膜(F0)和胞质部分(F1)组成。质子通过F0成分的运动,被认为是可逆地与在F1上催化部位的ATP合成或水解偶联。在大肠杆菌(E.coli)中,F1和F0分别由下面的亚单位,α3β3γδε和a1b2c12组成。通常,虽然在原核和真核系统之间可能存在差别,但在线粒体和叶绿体中发现同源的亚单位。ATP合成由质子通过F0运动来驱动。然而,偶联的完整结构和机理还没有完全建立。
在大肠杆中b亚单位与另一个F0膜成分(亚单位c)的交联导致ATP水解和ATP驱动的质子泵的解偶联。相似地,在大肠杆菌中已发现一种影响F0的b亚单位,涉及单个氨基酸替代的解偶联突变,它破坏了全部酶功能。这种表型提示b亚单位在质子易位与催化的偶联中的功能性作用。b亚单位与F1成分的相互作用可能事实上是动态的(dynamic)和结构的。更为最近地,Struglics和同事们已经表明了线粒体的b亚单位被可逆性地磷酸化。作者提示这种磷酸化的生理作用可以控制F0-F1相互作用的稳定性并因此调节F0-F1传动器的能量偶联。如此,F0的b亚单位在ATP酶运转中将起结构和功能两方面作用。这种特定复合体的抑制可以通过与b亚单位或与导致ATP生成显著损害的F0的膜有关成分的直接相互作用而发生。
对这种可能的机理已经进行了有意义的研究,用n-辛烷磺酰基乙酰胺(OSA),一种□-磺酰基甲酰胺类化合物,其在体外具有对抗致病性分枝杆菌的强活性。在PCT申请No.PCT/US98/17830中公开了OSA,通过参考将其并入本申请。
应用双向蛋白质凝胶电泳和在所述化合物存在下过量表达的蛋白质的随后测序,试图鉴定在卡介菌(BCG)中的OSA的酶靶标。BCG中OSA的处理导致两个相对小的蛋白质(≈18kD):atpF基因编码的ATP合酶(F1F0ATP酶)的b亚单位和一个小热激蛋白,hsp(Rv0251c)的过量表达。RT-PCR揭示了hsp表达水平以及在更小程度上ATP合酶b亚单位的显著增加,一种与在双向凝胶上观察到的一致的带型。
为了评价是否这些结果可能代表一种对细胞损伤应激的一般化反应,在一种强抗分枝杆菌化合物,浅蓝菌素和另一种强抗TB化合物,异烟肼的存在下完成了双向蛋白分布图。浅蓝菌素和异烟肼处理都没有导致在BCG中蛋白质的过量表达,表明OSA是通过不同于浅蓝菌素或异烟肼的机理工作。
通过比较OSA处理的耻垢分枝杆菌(M.smegmatis)与BCG的双向蛋白质分布图,获得了另外的信息。以前,已经报导了耻垢分枝杆菌在大至溶解度限度(100μg/ml)的浓度下内在地对OSA耐受。两种BCG蛋白质的同系物是借助耻垢分枝杆菌基因组序列的BLAST检索找到的,所述耻垢分枝杆菌基因组序列可通过基因组研究所,Rockville,MD,(http://www.tigr.org)获得,并发现存在于耻垢分枝杆菌中。然而,在两者之间存在相异性区域。在这些两个分枝杆菌种之间,b亚单位看起来相当相似(63%相同,75%相似),然而,hsp更少相似(42%相同,54%相似)。b亚单位和hsp同系物估计的分子量分别为16和18kD。然而,在OSA处理的耻垢分枝杆菌中没有与这些分子量或pI′s一致的蛋白质被过量表达。
ATP合酶b亚单位的过量表达表明,在OSA的靶标途径中可能涉及ATP合酶,不论直接或间接。基于这些观察,进行了单一时点和时程实验以测定与一种已知非特异性ATP合酶抑制剂DCCD比较,在OSA存在下的ATP(M)水平。在所有测试的时点,经OSA和DCCD处理后,ATP(M)水平显著地降低。不但这种降低对两种化合物是可重复的,而且在暴露后短至5分钟内就非常快地发生。
为了测定这种作用是否是化合物特异的或是一般化应激反应的结果,测试了另外的抗分枝杆菌药物和呼吸抑制剂在相应时点影响ATP(M)水平的能力。一线抗分枝杆菌药物包括INH,RIF,STR,EMB和浅蓝菌素。呼吸抑制剂包括双香豆素(可选择的脱氢酶抑制剂),Rot(复合体I抑制剂)和TTFA(复合体II抑制剂)。以与OSA的水平可比较的水平(16倍于它们各自在BCG中的MIC′s)使用所有一线药物。有意义地,对测试的任何一线药物或呼吸抑制剂,在暴露后5分钟,没有检测到ATP(M)水平可察觉的下降。一个例外是TTFA,一种复合体II的特异性抑制剂,它证明了在5分钟时ATP(M)水平中度下降。这并不令人意外,因为这种抑制剂特异地定向为TCA循环整合部分的琥珀酸脱氢酶(复合体II)。没这种酶复合体,TCA循环严重受损。结果,需氧呼吸被损伤,减慢了ATP的产生。
因此,OSA摹拟了被很好地证明了的,非特异性ATP合酶抑制剂DCCD的作用。相比之下,其它的抗分枝杆菌药物不能引出类似作用。用下面的化合物进行了类似的研究:
这些化合物的检测揭示了与OSA显示的相似的结果。也就是说,它们显示了在相应的时点,与对照比较,降低ATP(M)水平的能力。
OSA诱导的ATP水平降低伴随着hsp(Rv0251c)的过量表达。不限制本发明的范围,这提示以下可能性:在分枝杆菌中,热激反应可能与能量感觉(sensing)/调节关联。Hsp(Rv0251c)编码一个159个氨基酸的相对小的蛋白质,它是Hsp20或□小热激蛋白-晶体蛋白家族的成员。最近,Stewart等(2002),证明hsp(作者命名为acr2)是分枝杆菌基因组中最可热诱导的基因。Hsp还与Rv0250c和Rv0249c一起被排列在一个表观操纵子中。hsp的调节涉及到热激阻抑物,HspR和一个ECF sigma因子σB。后者也在氧化或去污剂应激中被上调节并具有与结核分枝杆菌的α-晶体(acr)(14kDa抗原)显著的相似性(98个氨基酸41%相同)。热激反应是遍在的,并允许细胞在正常及有害的应激两种条件下存活。这种存活经常需要在基因表达中的全面变化。大多数热激蛋白被认为是分子陪伴,它有助于蛋白质折叠/降解及阻止蛋白质聚集。通常,热激蛋白有相对大的底物特异性。然而,渐显的证据确认了酶特异性伴侣蛋白的存在,它对特异性酶复合体的形成是必需的。酶特异性伴侣蛋白的例子包括酵母ATP10,ATP11和ATP12基因,它们编码ATP合酶组装所需的蛋白质。另外的酶特异性伴侣蛋白已经被鉴定,它们对细胞色素氧化酶,琥珀酸还原酶(复合体II)和NADH-辅酶Q氧化还原酶(复合体I)的形成来说是必需的。许多酶特异性伴侣蛋白属于hsp20类型的分子陪伴。另外,一些分子陪伴受氧化还原调节。分枝杆菌hsp的完整功能性角色在很大程度上是未知的。然而,存在这种热激蛋白可能在ATP合酶或呼吸链中其他相关复合体的酶特异性组装/调节中起作用的可能性。Hsp也可能代表了氧化还原调节的热激蛋白的一个分枝杆菌版本。
通过乙醇的活性增强进一步强化了在中央能量代谢中OSA介导的干扰的结论。已知分枝杆菌能利用低浓度的乙醇和其他短链醇作为碳源。乙醇是一种呼吸作用的底物,它被醇脱氢酶可逆地氧化为乙醛,伴随NAD的还原。随后乙醛的氧化生成乙酸,然后在依赖ATP的反应中乙酸被转化为乙酰辅酶A。乙酰辅酶A是中央代谢中的关键分子。乙酰辅酶A通过TCA循环的氧化驱动细胞能量的产生。因此,乙醇代谢及呼吸作用是互相联系的。以前的研究者已经证明,乙醇增加了哺乳动物线粒体中ATP合成的速度,这是通过呼吸链导致质子通量提高的NADH+H+产生增加的结果。加入乙醇后,ATP/O的比率增加,这表明在呼吸作用与ATP合成之间能量转换的增加。在这项研究中,乙醇与OSA共用相同的靶标是不太可能的。然而,乙醇提高的乙酰辅酶A和NADH+H+对事件中的细胞将是有害的,在呼吸链中的ATP合酶或其他成分受损,。在这种情况下,OSA和乙醇的增强作用是可能的。
ATP合成的抑制和细胞呼吸干扰将产生多重的下游作用。这些作用包括在其他大分子,如分枝菌酸的能量依赖性合成中的减少。以前,我们报告了OSA降低BCG中分枝菌酸的水平,对在这个生物合成途径中的中间体没有明显作用。这个观察与用已知的脂肪酸合酶抑制剂,硫乳霉素和浅蓝菌素观察到的霉素酸酯抑制的模式形成鲜明的对比。这些发现表明,由OSA和其他□-磺酰基甲酰胺类的分枝菌酸合成的抑制可能涉及脂肪酸合酶抑制以外的一种可选择性机理。
应用能选择性降低ATP水平的化合物,如OSA和化合物I-VIII,将有助于治疗两种患者:现在患有TB(包括MDR-TB)以及数百万的潜在的患者,他们隐藏着非活动性疾病,它可能因为免疫抑制或其他系统性疾病变为活动性疾病。
这类化合物也可以被用于对抗多种其他微生物,例如鸟孢子分枝杆菌(M.avium-intracellulare),麻风分枝杆菌(M.leprae),副结核分枝杆菌(M.paratuberculosis),溃疡分枝杆菌(M.ulcerans)和红球菌属(Rbodococcus),并可能用于人和动物,例如马,牛,绵羊,山羊及其他反刍动物。
根据本发明的治疗涉及给予治疗对象选择性降低微生物中ATP水平的化合物。包含这类化合物的任意药用组合物,可根据化合物,药用载体或疾病的需要,通过胃肠外(皮下,肌内,静脉内,腹膜内(intraoperitoneally),胸膜内,囊内,鞘内),局部,口服,直肠,鼻腔,或吸入途径施用。
优选在含有所述化合物及药学上可接受的载体的药用组合物中配制所述化合物。活性剂的浓度将依赖于它在载体中的溶解度,并且可以由本领域普通技术人员轻易地确定。类似地,在特定配方中使用的剂量将根据将被用于对抗的特定微生物确定。药用组合物可以包含其他成分,只要它们不使活性化合物的效力失效。药物载体是熟知的,并且本领域技术人员能够依靠施用的特定途径选择正确的载体。
治疗的剂量和持续时间将依赖于多种因素,包括药物的治疗指标,疾病类型,患者年龄,患者体重及毒性的耐受。通常将选择剂量达到血清浓度水平从大约1ng到100μg/ml,典型地从0.1μg/ml到10μg/ml。优选地,将基于它们达到在体外和体内模型及临床试验中显示有效的环境浓度的能力,选择最初剂量水平。由于上面的因素,用于特定受试者的特定药物的剂量和治疗持续时间可以由熟练的临床医生使用标准的药理学方法确定。通过分析活性化合物的血液或体液水平,测定化合物的活性或其在有关组织中的水平,或监测受试者的疾病状态,可以监测对治疗的反应。熟练的临床医生将根据由这些检测显示的对治疗的反应,调节剂量和治疗的持续时间。
当然,以低于杀死受试者的水平施用所述化合物,优选地,以低于将不可逆损害生命机能的水平施用。不排除以杀死患者的一些可以被再生的细胞(例如,子宫内膜细胞)水平的施用。
实施例
提供下面的实施例以举例说明,但不限制本发明的范围。
分枝杆菌及生长条件。在本研究中使用结核分枝杆菌(H37Rv)牛结核分枝杆菌(M.bovis)BCG(BCG,巴斯德菌株,ATCC 35734)和耻垢分枝杆菌(mc2 6 1-2c)。Lowenstein-Jensen琼脂斜面或Middlebrook 7H10琼脂平板(Difco,Detroit,Michigan)上维持菌株。对所有试验,BCG培养物在37℃下在旋转振荡器上生长至对数中期(OD=A600 0.3-0.4)。
化合物。n-辛烷磺酰基乙酰胺(OSA,Craig Townsend,Johns HopkinsUniversity,Baltimore,Maryland),二环己基碳二酰亚胺,ATP合酶特异性抑制剂(DCCD,ICN,Costa Mesa,California),噻吩甲酰三氟丙酮,呼吸性复合体II抑制剂(TTFA,ICN),鱼藤酮,呼吸性复合体I抑制剂(Rot,ICN)及浅蓝菌素,一种脂肪酸合酶抑制剂(Sigma-Aldrich,St.Louis,Missouri)的储备和工作溶液,用二甲基亚砜(DMSO,Sigma)配制。异烟肼(INH),链霉素(STR),和乙胺丁醇(EMB)(都来自Sigma)的储备溶液用无菌水制备。利福平(Sigma)的初始储备溶液用甲醇配制然后用无菌水稀释。
化合物I,II,IV,VI和VIII的制备
这些化合物的每一种的合成从3-磺酰基十一烯酸(″IX″)开始,3-磺酰基十一烯酸是按照J.Med.Chem.2000 43(17)3304中描述的方法被制备的。
化合物I
在惰性气体中,向火焰干燥的含有3mL无水二氯甲烷的圆底烧瓶中加入3-磺酰基十一烯酸IX,150mg,0.6mmol和1,1羰基二咪唑(CDI),(102mg,0.63mmol)。在室温搅拌混合物20分钟。然后加2-氯乙胺氢氯化物(73mg,0.63mmol),搅拌反应另外3小时。水性加工以令人满意的收率提供粗的酰胺X(145mg,88%)。使用酰胺X用于进一步化学的粗品。1H(CDCl3,300MHz)δ6.86(bs,1H),3.87(s,2H),3.67-3.65(m,4H),3.15(t,J=8.1Hz,2H),1.89-1.80(m,2H),1.40-1.26(m,14H),0.88(t,J=6.7Hz,3H)。将酰胺X(110mg,0.33mmol)溶解于1.5mL甲醇氢氧化钾(5%w/v)。室温下搅拌2小时后,用水稀释混合物并用乙酸乙酯抽提三次。用盐水洗有机层两次,干燥并在真空中浓缩。通过急骤柱色谱(1∶1己烷/EtOAc)纯化粗的噁唑啉提供70mg,73%得率的想要的产物I;溶点53-55℃;1H(CDCl3,400MHz)δ4.37(t,J=9.6Hz,2H),3.95(s,2H),3.95(t,J=9.4Hz,2H),3.22(t,J=8.2Hz,2H),1.90-1.79(m,2H),1.46-1.42(m,2H),1.30-1.23(m,12H),0.87(t,J=6.0Hz,3H)。
化合物II
在惰性气体中,向火焰干燥的含有3mL无水二氯甲烷的圆底烧瓶中加入3-磺酰基十一烯酸IX(150mg,0.6mmol)和CDI(102mg,0.63mmol)。在室温搅拌混合物20分钟,然后加异烟酰肼(86mg,0.63mmol),搅拌反应另外6.5小时。在真空中浓缩反应混合物并且通过急骤色谱(98%EtOAc/2%乙酸)纯化粗的酰肼II得到白色固体,122mg,56%。1H(DMSO-d6,400MHz)δ10.98(bs,1H),10.57(bs,1H),8.77(bs,2H),7.78(dxd,J1=1.2Hz,J2=5.4Hz,2H),4.21(s,2H),3.30(t,J=8Hz),1.76-1.68(m,2H),1.42-1.34(m,2H),1.30-1.23(m,12H),0.84(t,J=6.8Hz)。
化合物IV
在惰性气体中,向火焰干燥的含有3mL无水二氯甲烷的圆底烧瓶中加入3-磺酰基十一烯酸,158.4mg(0.6mmol)和1,1羰基二咪唑(CDI),115.7mg(0.72mmol)。在室温搅拌混合物20分钟。然后添加2-呋喃酰肼,90mg(0.72mmol)并搅拌反应另外3小时。用乙酸乙酯稀释反应混合物,用饱和的碳酸氢钠洗涤两次,用稀盐酸洗涤三次,并用饱和的NaCl洗涤一次。有机物用硫酸镁干燥并在减压下浓缩。将粗产物IV从EtOAc/己烷(3∶1)中重结晶,得到淡棕色粉末(147mg,66%);溶点130-131℃;1H(DMSO-d6,400MHz)δ10.53(s,1H),10.38(s,1H),7.90(dxd,J1=0.4Hz,J2=1.6Hz,1H),7.24(dxd,J1=0.6Hz,J2=3.4Hz),6.65(dxd,J1=1.6Hz,J2=3.6Hz),4.17(s,1H),3.28(t,J=7.8Hz,2H),1.75-1.67(m,2H),1.42-1.33(m,2H),1.30-1.23(m,12H),0.84(t,J=6.8Hz,3H)。
化合物VI
在惰性气体中,向火焰干燥的含有3mL无水二氯甲烷的圆底烧瓶中加入3-磺酰基十一烯酸(163mg,0.62mmol),CDI(113.9mg,0.74mmol)。
在室温搅拌混合物20分钟。然后添加三乙胺(TEA)(89μl,0.63mmol)和4-氯苯肼盐酸盐(115mg,0.62mmol),搅拌反应另外2小时。通过急骤色谱(40%EtOAc/60%己烷)纯化粗产物VI。(112mg,46%收率)。1H(DMSO-d6,400MHz)δ10.13(d,J=2.4Hz,1H),8.14(d,J=2.4Hz,1H),7.18-7.14(m,2H),6.78-6.72(m,2H),4.12(s,2H),3.24(t,J=7.8Hz,3H),1.74-1.66Hz(m,2H),1.40-1.32(m,2H),1.30-1.23(m,12H),0.84(t,J=6.8Hz,3H)。
化合物VIII
在惰性气体中,向火焰干燥的含有3mL无水二氯甲烷的圆底烧瓶中加入3-磺酰基十一烯酸(300mg,1.1mmol),CDI(214mg,1.3mmol)。在室温搅拌混合物20分钟。然后加TEA(154μl,1.1mmol)和甲基甘氨酸酯盐酸盐(138mg,1.1mmol),搅拌反应另外4小时。通过急骤色谱(50-100%EtOAc/己烷)纯化粗的酯VIII,得到白色固体(202mg,56%);溶点82-84℃;1H(CDCl3,400MHz)δ7.13(bs,1H),4.07(d,J=6Hz,2H),3.93(s,2H),3.77(s,3H),3.23(t,J=8Hz,2H),1.90-1.82(2H,m),1.46-1.40(m,2H),1.30-1.25(m,12H),0.87(t,J=6.8Hz,3H);13C(CDCl3,100MHz)。
化合物III的制备
在两步合成中,通过首先从(±)-α-亚甲基-γ-丁内酯-5-辛基-4-羧酸(C75)开始制备化合物7,制备了这种化合物。可以按照美国专利No.5,981,575中的阐述制备C75。
向C75(30mg,0.12mmol)的CH3CN(0.9mL)溶液中加入三(2-氧代-3-噁唑啉基)氧化膦(91.7mg,0.2mmol),乙醇胺(7.8μl,0.13mmol)和NEt3(0.04mL,0.3mmol),并且允许溶液在室温下搅拌30分钟。将混合物倒入NH4Cl(饱和)/1N HCl(10ml,3∶1)溶液中并用Et2O(3×15mL)抽提。合并的有机物被干燥(MgSO4),过滤,蒸发并用色谱分析(35%EtOAc/己烷),经急骤色谱(50%EtOAc/己烷-100%EtOAc/2%CH3CO2H)后得到化合物7(32mg,91%)。1H NMR(300MHz,CDCl3)δ0.86(t,J=6.9Hz,3H),1.24(s,10H),1.35-1.48(m,2H),1.64-1.75(m,2H),3.40-3.57(m,3H),3.74(t,J=5Hz,2H),4.73-4.79(dt,J=5.7,7Hz,1H),5.82(d,J=2Hz,1H),6.42(d,J=2Hz,1H)。
向7(44.9mg,0.15mmol)在CH2Cl2(0.7mL)的溶液中加入二甲胺基吡啶(DMAP,4mg,0.03mmol)和异氰酸烯丙酯(20□L,0.22mmol),并且允许溶液在室温搅拌1小时。将混合物倒入NH4Cl(饱和的,10mL),并用CH2Cl2(3×10mL)抽提。合并有机物,干燥(MgSO4)并蒸发以提供粗的15。急骤色谱(EtOAc)提供纯的15(19mg,33%)。1H NMR(300MHz,CDCl3)δ0.85(t,J=6Hz,3H),1.24(m,11H),1.35-1.48(m,1H),1.62-1.79(m,2H),3.38-3.40(m,1H),3.51-3.52(m,2H),3.78(t,J=5.2Hz,2H),4.20-4.21(m,2H),4.73-4.79(m,1H),4.96(bt,1H),5.09-5.20(m,2H),5.75-5.86(m,1H),5.79(d,J=2.3Hz,1H),6.38(d,J=2.3Hz,1H)。
化合物V和VII的制备
在用许多中间体的合成中制备这种化合物。在第一步中,如下制备了化合物23:
在-78℃,向LiHMDS(6.2mL,6.20mmol,1M在THF中)在THF(9.7mL)中的混合物中通过导管滴加THF(9.6mL)中的(±)-1(1.00g,5.75mmol),得到的溶液在-78℃搅拌30分钟。然后,在-78℃通过导管加入在THF(4mL)中的辛基triflate(1.63g,6.2mmol)。在-78℃搅拌2小时后,添加1NHCl(10mL)并且用Et2O(3×15mL)抽提溶液。合并的有机物被干燥(MgSO4),过滤并蒸发。急骤色谱(2%EtOAc/己烷)产生纯的23,为可分离的非对映异构体的2∶1-6∶1混合物(1.33g,81%)。1H NMR(300MHz,CDCL3)δ0.86(t,J=6.5Hz,3H),0.99(s,9H),1.24-1.26(m,12H),1.54(s,3H),1.72-1.84(m,2H),5.13(s,1H);13C NMR(75MHz,CDCl3)δ13.9,22.6,24.9,25.1,25.9,29.2,29.3,29.5,31.8,35.2,41.2,55.3,86.5,177.7。IR(NaCl)3443,2929,1829,1769cm-1;C16H30O2S的分析计算值:C,67.0;H,10.6;实测值:C,66.3;H,10.5。C16H30O2S+(M+)的HRMS(EI)m/z计算值286.1967观察值286.1969。
然后,制备化合物26:
向EtOH(14.1mL)中的23(650mg,2.27mmol)中加入NaOEt(2.1M)(2.16mL,4.54mmol)(刚刚由EtOH(4.0mL)中的Na金属(200mg,8.3mmol)制备),允许溶液在室温中搅拌。2小时后,将溶液倒入NH4Cl(饱和)/1N HCl(25mL,3∶1)中,混合物用Et2O(3×20mL)抽提。然后合并的有机物用水洗涤(3×25mL),干燥(MgSO4),过滤并蒸发以得到粗的25。在0℃向溶于CH2Cl2(26mL)中的25中添加NEt3(0.5mL,3.49mmol)和炔基氯化物(0.3mL,3.49mmol)。0℃40分钟后,加入NH4Cl(饱和的)(30mL)并用CH2Cl2抽提溶液。合并的有机物被干燥(MgSO4),过滤并蒸发。急骤色谱(5%EtOAc/己烷)得到纯的26(542mg,79%)。1H NMR(300MHz,CDCl3)δ0.87(t,J=6.9Hz,3H),1.22-1.27(m,15H),1.61(s,3H),1.75-1.84(m,2H),2.26(s,3H),4.18(q,J=7.1Hz,2H);13C NMR(75MHz,CDCl3)δ13.9,14.1,22.6,23.4,24.4,29.1,29.2,29.6,30.3,31.8,38.3,55.8,61.5,173.1,195.8。IR(NaCl)3430,1868,1693,1644cm-1;C15H28O3S的分析计算值:C,62.5;H,9.78;实测值:C,62.6;H,9.83。
从化合物26,制备化合物32
在-78℃,向甲苯(27mL)中的26(500mg,1.7mmol)中添加LiHMDS(4.3mL,4.3mmol,1.0M在THF中),允许溶液缓慢温热至-5℃。然后将溶液倒入1N HCl(40mL)并用EtOAc(3×25mL)抽提。将合并的有机物干燥(MgSO4),过滤并蒸发。急骤色谱(20%EtOAc/2%CH3CO2H/己烷)得到32(308mg,73%)。1H NMR(300MHz,CDCl3)(酮式互变异构体)δ0.86(t,J=6Hz,3H),1.19-1.24(m,10H),1.48-1.53(m,2H),1.65(s,3H),1.77-1.85(m,1H),1.94-2.01(m,1H),3.36(s,2H);1H NMR(300MHz,MeOD)(烯醇式互变异构体)0.87-0.89(m,3H),1.29(m,10H),3.29(s,3H),1.81-1.87(m,2H);13C NMR(75MHz,MeOD)(烯醇式互变异构体)δ14.7,23.8,26.4,27.1,30.5,30.6,30.8,33.2,39.8,61.3,103.1(m),189.8,197.8。IR(NaCl)3422,1593cm-1;C13H22O2S的分析计算值:C,64.4;H,9.15;实测值:C,64.3;H,9.10。
然后,制备化合物50
按照一般方法H,从32(60mg,0.25mmol)和叔丁基溴乙酸酯(73□L,0.49mmol),在急骤色谱(15%EtOAc/己烷)后,获得50(62mg,70%)。1HNMR(300MHz,CDCl3)δ0.86(t,J=7Hz,3H),1.24(s,12H),1.49(s,9H),1.68(s,3H),1.83-1.86(m,2H),4.43(s,2H),5.19(s,1H);13C NMR(75MHz,CDCl3)□δ14.0,22.6,25.2,26.3,28.1,29.2,29.3,29.5,31.8,38.9,59.7,68.5,83.4,102.1,165.2,185.5,193.4。C19H32O4S的分析计算值:C,64.0;H,9.05;实测值:C,64.1;H,9.08。
接下来,如下制备化合物53:
向溶解于CH2Cl2(1.4mL)中的50(65mg,0.18mmol)中添加三氟乙酸(TFA)(0.7mL)并且在室温搅拌溶液4小时。将溶剂蒸发并且色谱分析(20%EtOAc/2%CH3CO2H/己烷)粗的物质,得到纯的53(48mg,89%)。1HNMR(300MHz,CDCl3)δ0.86(t,J=6.9Hz,3H),1.24(s,11H),1.47-1.48(m,1H),1.68(s,3H),1.84-1.88(m,2H),4.62(s,2H),5.31(s,1H);13CNMR(75MHz,CDCl3)δ14.1,22.6,25.1,26.1,29.2,29.3,29.5,31.8,38.9,60.1,67.7,102.4,169.8,185.8,195.4。IR(NaCl)3442,1645cm-1;C15H24O4S的分析计算值0:C,59.9;H,8.05;实测值:C,60.0;H,8.09。
最后,从化合物53,制备化合物V。向53(100mg,0.33mmol)在CH2Cl2(1.61mL)中的冷的溶液(0℃)中添加1-[3-(二甲氨基)丙基]-3-乙基碳二亚胺氢氯化物(EDC)(128mg,0.43mmol),DMAP(6.0mg,0.05mmol)和2-呋喃甲酸酰肼(54mg,0.43mmol)。在0℃搅拌混合物30分钟,然后允许温热到室温并搅拌12小时。将溶液倒入NH4Cl(10ml,饱和)并用CH2Cl2(3×10ml)提取。将合并的有机物干燥(Na2SO4),过滤并蒸发,得到粗的化合物V。急骤色谱(10%EtOAc/己烷)得到纯的化合物V(91mg,68%)。1H NMR(400MHz,CDCl3)δ0.84(t,J=6.6Hz,3H),1.21(m,11H),1.43-1.47(m,1H),1.66(s,3H),1.81-1.86(m,2H),4.64(s,2H),5.42(s,1H),6.47(dd,J=1.6,3.6Hz,1H),7.16(d,J=4Hz,1H),7.45(m,1H),9.32(d,J=4Hz,1H),9.44(d,J=4Hz,1H);13C NMR(100MHz,CDCl3)δ14.0,22.6,25.3,26.0,29.2,29.3,29.5,31.7,38.8,59.7,69.1,103.0,112.3,116.5,145.1,145.4,156.4,164.2,184.8,193.9。
从化合物53还制备了化合物VII,如下:按照与制备化合物V(74mg,53%)所用的相同的一般方法,向53(100mg,0.33mmol)和4-氯苯肼盐酸盐(76.8mg,0.43mmol),在急骤色谱(50%EtOAc/己烷)后。1H NMR(300MHz,CDCl3)δ0.86(t,J=6Hz,3H),1.24(m,11H),1.46-1.54(m,1H),1.71(s,3H),1.82-1.90(m,2H),4.57(s,2H),5.39(s,1H),6.75(d,J=8.8Hz,2H),7.18(d,J=8.8Hz,2H),7.38(s,1H),8.09(s,1H);13C NMR(100MHz,CDCl3)□14.1,22.6,25.3,26.1,29.2,29.3,29.5,31.8,38.9,59.7,69.7,103.2,114.7,126.4,145.8,129.2,165.9,184.3,193.5。
IR(NaCl)2957,1695,1658,1609cm-1。
蛋白质测定:分枝杆菌和培养物。分别向20ml对照和处理过的培养物中加入DMSO(稀释剂),OSA(6.25和100μg/ml),浅蓝菌素(24μg/ml)或异烟肼(1.0μg/ml)。在同样条件下另外孵育24小时后,通过离心(13,000×g 30秒)收获2ml等份的细胞,去掉上清液,用1X PBS重复所述过程一次。将每个管中细胞内容物分到2到3个Eppendorf管中(每管大约3到5 OD单位),加入250μl含有3M尿素(Sigma),0.5%TritonX-100(Sigma),500mg二硫苏糖醇(DTT)(Gibco BRL,Life Technologies,Gaithersburg,Maryland)和500μl Pharmalyte(Pharmacia Biotech,Piscataway,New Jersey)的裂解液。随后,还添加苯甲基磺酰氟(PMSF)(Sigma)(100μg/ml)和亮抑蛋白酶肽(2μg/ml)(Sigma)。将含有细胞和裂解液的混合物于Biospec Mini-8小珠搅打器中使用200-300μm玻璃珠(Sigma)以最大速度小珠搅打60秒。每个样品重复这个过程两次,在振荡之间间歇30秒,放于冰上。将每个管的内容物离心(13,000×g)至少30秒并移去含有蛋白质的上清。在Shimadzu UC-1201分光光度计中,使用标准化的比色法(Coomassie Plus,Pierce,Rockford,Illinois)和BSA标准物(Pierce),测定蛋白质浓度。将定量过的样品分成等份样,并冷冻于-70℃。
蛋白质测定:时程。将储备液BCG(500ml)分成150ml等份,向各自对照和处理的培养物中加入DMSO或OSA(100μg/ml)二者之一,继之在37℃孵育和通气1小时。在添加化合物后的第一个4小时期间,以每小时的间歇及在16,24和48小时的额外时点,从每种培养物中取出等份(20ml)。通过低速离心收获细胞,在灭菌蒸馏水中洗涤一次,并冷冻于-70℃。
蛋白质检测:双向凝胶及潜在靶点的测序。将大约250μg每种蛋白质样品与含有8M尿素,0.5%Triton X-100,Pharmalyte 3-10,DTT及几粒溴酚兰(Sigma)的溶液混合(240μl整个体积/样品)。使用制造商的标准方案(Pharmacia Biotech),利用这种混合物使商业化制备的丙烯酰胺pH条(梯度4-7)在室温下再水化过夜。随后,按照标准化的方案(Pharmacia Biotech)将完全再水化的条带经受双向蛋白凝胶系统[第一向-20℃16小时,接着个体带分别在含有Tris-HCl(pH6.8),尿素(8M),甘油(30%)(Sigma),SDS(1mg/ml)(Sigma)和DTT或碘乙酰胺(Sigma)的溶液中平衡。]平衡后,将条带应用于商业化制备的丙烯酰胺凝胶(245×110×0.5mm,梯度8-18%,Pharmacia)并按制造商的标准流方案(Pharmacia)在15℃运行2小时。分子量标准(大小范围:14kD到200kD)购自Gibco BRL,Life Technologies,每块凝胶装载10μl。通过考马斯蓝(Sigma)染色凝胶过夜使蛋白质显色。用甲醇∶水∶乙酸(9∶9∶2,Sigma)洗涤2到3次除去过量的染料。将来自4块凝胶中的目的蛋白质混合并在50%乙腈中洗涤切割的凝胶片段两次。随后通过HarvardMicrochemistry(Cambridge,Massachusettes)进行蛋白质测序。
RNA提取。用1ml Trizol试剂(Invitrogen,Carlsbad,California)从用MSO(稀释剂)或OSA过夜处理的15ml BCG培养物中提取总RNA。随后,在微型玻珠打浆机中将细菌细胞匀浆化30秒(两次)并向细菌裂解液中加入三氯甲烷。总RNA用异丙醇沉淀,用乙醇洗涤并在蒸馏水(无DNA酶和RNA酶,Invitrogen)中重悬。总RNA用DNA酶I(Qiagen,Valencia,California)消化,并用RNeasy小试剂盒(Qiagen)纯化。使用2μg RNA和Super Script II,RNA酶H-反转录酶(Invrtrogen)反转录为cDNA。
PCR方案。在Perkin Elmer 2400热循环仪中进行PCR扩增。每个PCR反应含有2μl cDNA,2.5mM MgCl,0.2mM dNTP’s(Invitrogen)及2.5单位Taq聚合酶(Invitrogen)。扩增参数涉及95℃1分钟,60℃1.5分钟和72℃2分钟的30个循环。在72℃进行延伸10分钟。随后,将温度设置到4℃。通过琼脂糖凝胶电泳评价反应产物。
Southern杂交。通过与20XSSC Southern印迹将PCR产物转移至尼龙膜(Roche Diagnositics,Indianapolis,Indinana)上。随后,个体膜与基因特异性地高辛配基11-dUTP标记的PCR片段在42℃过夜杂交。然后除去探针,分别在室温和68℃,在低(2XSSC,0.1%SDS)和高(0.5%SSC,0.1%SDS)严格缓冲液中洗涤膜15分钟(两次)。用商业上可获得的化学发光试剂盒(Roche)检测地高辛标记的核酸。
ATP测定。将稀释剂或OSA(100μg/ml或16倍计算的MIC)加到120ml BCG培养物中。以与OSA使用的可比较的浓度(16倍它们各自的MIC’s)还测试了另外的抗分枝杆菌药物。这些包括化合物I-VIII的各一种,异烟肼(INH,1.6μg/ml),利福平(RIF,32μg/ml),链霉素(STR,32μg/ml),乙胺丁醇(EMB,32μg/ml)和两种浓度(1.5μg/ml和24μg/ml)的浅蓝菌素。测试的已知呼吸链抑制剂包括DCCD(100μg/ml),一种ATP合酶特异性抑制剂,TTFA(100μg/ml),一种呼吸复合体II特异性抑制剂,Rot(25μg/ml),一种呼吸复合体I特异性抑制剂,和双香豆素(DC,7μg/ml),一种可选择的脱氢酶抑制剂。
最初的单一或多个时点测定是通过在1,3和24小时取出培养物等份(30ml),并立即放在冰上进行的。所有随后的操作在4℃进行。在5,30和180分钟使用相同方法进行另外的时程。通过离心收获细胞并在ATP抽提缓冲液(100mM Tris,4mM EDTA,pH7.5)中使用200-300μm玻璃珠通过玻珠打浆以最大力量破裂总共2分钟。通过离心(13,000×g 15分钟)除去细胞碎片,并将含有ATP的上清液转移到干净的管中。使用商业上获得的ATP生物发光测定(Roche Diagnostics)进行处理的与对照的样品中ATP水平的测定。在Wallac Victor2TM发光计上测定相对光单元。通过将等份涂平板于M7H10-ADC琼脂,并在5%CO2,37℃孵育3周进行测定每种培养物的菌落计数(CFU’s/ml)。通过处理的和未处理的组的CFU计算ATP水平[M];然后通过将处理的值对对照值正态化计算相对[M]ATP。使用两因素t检验(2-tailed students’t-test)计算统计学上的显著性。
在乙醇存在下OSA的活性。使用标准BACTEC放射分析生长方法(44)的修改测定在乙醇,一种呼吸作用底物的存在下,OSA的体外活性。简要地,接种物由保存在Lowenstein-Jensen琼脂斜面(Difco,Detroit,Michigan)的结核分枝杆菌培养物,使用玻璃珠和商业上可获得的稀释液(Becton Dickinson,Sparks,Maryland)制备。分枝杆菌悬液与玻璃珠一起进行涡流并允许静置30分钟。调节上清至1.0McFarland标准并在每只BACTEC 12B瓶中接种(0.1ml)。将OSA加到个体瓶中至下面最终浓度:1.5μg/ml,3.0μg/ml,6.25μg/ml,12.5μg/ml和25.0μg/ml。用于组合试验的最终乙醇浓度为0.05%。还测试了链霉素(0.5μg/ml,1.0μg/ml和2.0μg/ml)与乙醇的组合以确定哪一种所观察到的对OSA的协同作用是化合物特异的或对可选择的抗分枝杆菌药物来是可概括化的。在37℃孵育全部瓶子,并每日记录每只瓶子的生长指数(GI)。
用OSA与其他呼吸链抑制剂及14C-乙酸脂质脉冲标记处理BCG。BCG(50mls)在37℃在M7H9-ADC-Tween(Difco,Detroit,Michigan)中需氧生长至早期对数期(OD=A6000.2)。在这时,将1μCi/ml[1,214C]乙酸(Amersham,Arlington Heights,Illinois)和稀释剂(DMSO)、OSA(100μg/ml),DCCD(100μg/ml)或TTFA(100μg/ml)之一加入各自培养物中并在相同条件下孵育10分钟。立即将培养物放于冰上并通过在4℃以3,000×g离心15分钟收获细胞。
分枝菌酸制备及分析。按出版物如Dobson,G,等,“Systematicanalysis of complex mycobacterial lipids”,在
Chemical Methods in Bacterial Systematics,p.237-265.M.Goodfellow和D.Minnikin(编辑),Academic Press,London(1985),和Minnikin,D.,等,“Extractionof mycobacterial mycolic acids and other long-chain compounds byan alkaline methanolysis procedure”,Journal of MicrobiologicalMethods,2:243-249(1984)中以前描述的,进行分枝菌酸提取。简要地,按照上面参考文献中已建立的方案,将极性与非极性可提取的脂类从等体积的细胞(60mg湿重)中除去。在75℃使所得含有结合分枝菌酸的脱脂细胞经受在甲醇(1ml),30%KOH(1ml)和甲苯(0.1ml)中的碱水解过夜并随后冷却至室温。然后用3.6%HCl将混合物酸化至pH 1并用乙醚抽提3次。合并的抽提物在氮气中干燥。通过混合二氯甲烷(1ml),一种催化剂溶液(1ml)(26)和碘甲烷(25ml)30分钟,离心并弃去上相,制备分枝菌酸的脂肪酸甲酯。在氮气中干燥下相。通过闪烁计数(Beckman LS6500多用途闪烁计数仪)测定14C-乙酸在分枝菌酸中的掺入以及值表示为未处理的对照的百分比。在BCG的早期对数期培养物中暴露10分钟后,OSA(100μg/ml),DCCD(100μg/ml)和TTFA(100μg/ml)对分枝菌酸合成的作用的比较显示在图7中。
结果
最初,在24小时暴露于BCG中OSA之后,通过检测双向凝胶电泳蛋白质分布图试图进行OSA特异性蛋白质靶点的定性与定量鉴定。以前的研究者成功地使用类似的方法鉴定结核分枝杆菌中异烟肼的酶靶点,如在Mdluli D.,等,“Inhibition of a Mycobacterium tuberculosisβ-ketoacyl ACP synthase by isoniazid”,Science,280:1607-1610(1998)中所描述。如图2(右)中所示,用OSA的处理导致大约分子量为17到18kD的两种相对小的蛋白质的明显过量表达。在对应的未处理的对照中,两种蛋白质都检测不到(图2,左)。过量表达以剂量依赖方式发生在OSA的MIC(6.25μg/ml)和大到16倍(16×)MIC的浓度(100μg/ml)下。使用35S-甲硫氨酸脉冲标记的分离的时程实验证明,两种蛋白质在OSA暴露后短至3.5小时过量表达。比较起来,用强抗分枝杆菌抑制剂浅蓝菌素或异烟肼处理BCG,在大到16X MIC的浓度下,没有导致任一蛋白质的过量表达。另外,在OSA处理的耻垢分枝杆菌中,两种蛋白质都不过量表达,耻垢分枝杆菌对这种化合物内在地耐受。
含有两种蛋白质中每一种的混合的双向凝胶片段的测序证明了更加突出的种类是一种小热激蛋白(hsp,Rv0251c),17,786道尔顿,等电点(pI)为5.0。第二种蛋白质被鉴定为由atpF基因(Rv1306)编码的ATP合酶的b亚单位,分子量为18,325道尔顿,pI为4.9。两种蛋白质的过量表达通过RT-PCR被证实(图3)。
在上述蛋白质资料的基础上,该资料提示通过与b亚单位相互作用与APT合酶的可能联系,进行了超过24小时的时程研究以检查与已知ATP合酶抑制剂DCCD比较,OSA(100μg/ml)对BCG中ATP水平的影响。如图4中所示,在暴露后1小时,相对于未处理的对照,在OSA处理的BCG中ATP(M)水平明显下降(46%)。这种趋势在3及24小时持续,ATP[M]分别降低54%和85%。在每个时点这些差异都有统计学上的显著性(p<0.02)。DCCD,一种ATP合酶抑制剂,也引起ATP[M]水平的显著降低,在全部时点降低95%(p=0.003)。
对化合物I-VIII的每种化合物都观察到相似的结果。
进行了随后实验以测定OSA对ATP[M]浓度的作用如何迅速地发生。如图5(A图)所示,在短至5分钟,相对于未处理的BCG,OSA显著降低处理的BCG中的ATP水平。这种差异具有统计学显著性(p=0.004)。还注意到用DCCD,一种特异性ATP合酶抑制剂在统计学上有显著性的下降(p=0.001)。用呼吸性复合体II抑制剂,TTFA处理,在对应的时点仅导致ATP中等降低。
为测定是否OSA对ATP水平的作用可能是BCG中一种一般化应激反应的结果,以与OSA所用的浓度可比较的浓度(16X它们各自的MIC’s,INH1.6μg/ml,RIF 32μg/ml,STR32μg/ml,EMB32μg/m及浅蓝菌素24μg/ml),试验了另外的抗分枝杆菌药物,例如INH,RIF,EMB和STR。如图5(B图8)中所示,在暴露后5分钟的对应时点,用标准抗分枝杆菌药物(16X它们各自的MIC’s)处理BCG,相对于未处理的对照,没有导致可察觉的APT水平差别。试验的另外的化合物包括双香豆素,一种可选择的脱氢酶抑制剂,鱼藤酮(Rot),对NADH脱氢酶(呼吸性复合体I)有特异性,和脂肪酸合酶抑制剂,浅蓝菌素。已注意到这组化合物没有可察觉的ATP水平下降(图5,C图)。
由于可能涉及ATP合酶及呼吸链的其他成分,在乙醇(0.05%)的存在下,用OSA进行了研究。乙醇是一种呼吸作用底物并已被众多研究者用于研究细胞的呼吸作用,例如,如在Beauvieux,M.P.,等,“Ethanolperfusion increases the yield of oxidative phosphorylation inisolated liver of fed rats,”Biochim.Biophys.Acta,570:135-140(2002)中所示。如图6所示,0.05%乙醇加强了OSA对生长抑制的作用,在结核分枝杆菌H37Rv中,降低MIC从6.25μg/ml到<1.5μg/ml。在乙醇与链霉素之间没有观察到活性的增强。以前我们报告了用OSA处理BCG导致分枝菌酸降低,而对这个途径中的中间体没有明显作用。ATP水平的显著降低可能潜在地对其它大分子,包括细胞壁的分枝菌酸有直接或间接的有害作用。为了研究ATP合成和呼吸在分枝菌酸产生中的作用,评价了ATP合酶抑制剂(DCCD)和呼吸性复合体II抑制剂(TTFA)并与BCG中OSA及未处理的对照比较。选择暴露后10分钟的短时间间隔以保证,分枝菌酸酯合成的抑制不是由于细胞死亡。如图6所示,与未处理的对照比较,全部分枝菌酸水平用DCCD(100μg/ml)降低79%,用TTFA(100μg/ml)降低46%,和用OSA(100μg/ml)降低43%。图6的A图显示,在无乙醇的标准BACTEC放射分析培养基中OSA的活性(在图例中浓度用μg/ml显示),而B图显示了使用相同浓度和补充有0.5%乙醇的培养基的OSA的活性。NC显示了未处理的对照的结果。
Claims (27)
1.一种治疗患有基于微生物的感染的受试者的方法,包括对需要治疗的受试者施用有效量的化合物,在体外试验中与对照比较所述化合物在24小时后能降低微生物中ATP水平至少10%,而在相同时段不杀死哺乳动物细胞,通过以下步骤测量所述ATP水平的降低:
(1)从测试位置取出细胞并将它们置于冰上;
(2)通过在4℃离心收获细胞,并在ATP提取缓冲液中用玻珠打浆进行破裂;
(3)通过在4℃离心除去细胞碎片,留下含有ATP的上清液;
(4)在4℃通过生物发光方法检测存在于上清液中的ATP数量;
其中所述化合物不是式R-SOn-Z-CO-Y,其中n为1或2,R为一种具有6-20碳原子的烃基,Z为一种烃连接部分,它可含有一个杂原子,以及Y选自-NH、-O-CH2-C6H5、-CO-CO-O-CH3,和-O-CH3。
2.权利要求1的方法,其中所述受试者是人。
3.权利要求1的方法,其中所述受试者是动物。
4.权利要求3的方法,其中所述受试者选自由马,牛,山羊和绵羊组成的组。
6.权利要求5的方法,其中所述化合物为
7.权利要求5的方法,其中所述化合物为
9.权利要求5的方法,其中所述化合物为
12.权利要求5的方法,其中所述化合物为
14.权利要求1的方法,其中所述受试者感染有选自由结核分枝杆菌(M.tuberculosis)、鸟孢子分枝杆菌(M.avium-intracellulare)、麻风分枝杆菌(M.leprae)、副结核分枝杆菌(M.para luberculosis)、溃疡分枝杆菌(M.ulcerans)和红球菌属(Rbodococcus)组成的组的微生物。
15.一种治疗患有基于微生物感染的受试者的方法,包括对需要治疗的受试者施用一种化合物,其中所述化合物产生ATP合酶b-亚单位的过量表达,并且更进一步其中所述化合物不是式R-SOn-Z-CO-Y,其中n为1或2;
R为一种具有6-20碳原子的烃基,Z为一种烃连接部分,它可含有一个杂原子,以及Y选自-NH、-O-CH2-C6H5、-CO-CO-O-CH3和-O-CH3。
16.权利要求15的方法,其中所述受试者是人。
17.权利要求15的方法,其中所述受试者是动物。
18.权利要求17的方法,其中所述受试者选自由马,牛和绵羊组成的组。
21.权利要求19的方法,其中所述化合物为
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US5908751A (en) * | 1996-04-26 | 1999-06-01 | Toyo Ink Mfg. Co., Ltd. | Method for detecting and/or determining ATP from microorganism cells in a sample |
DE69908819T2 (de) * | 1998-12-23 | 2004-05-06 | Theravance, Inc., South San Francisco | Glycopeptid-derivate und pharmazeutische zusammensetzungen, die sie enthalten |
US6448472B1 (en) * | 1999-02-05 | 2002-09-10 | Board Of Regents, The University Of Texas System | Genetic and epigenetic manipulation of ABC transporters and ectophosphatases for the conference of hormone and herbicide resistance |
WO2000052144A1 (en) * | 1999-03-03 | 2000-09-08 | Board Of Regents, The University Of Texas System | Genetic and epigenetic manipulation of abc transporters and ecto-phosphatases for the conference of drug resistance and for the loss of drug resistance in biological systems and methods for the detection of ecto-phosphatase inhibitors |
HN2000000051A (es) * | 1999-05-19 | 2001-02-02 | Pfizer Prod Inc | Derivados heterociclicos utiles como agentes anticancerosos |
US6376682B1 (en) * | 2000-02-01 | 2002-04-23 | Takama System, Ltd. | Compound with α-glucosidase inhibiting action and method for producing the same |
US6248790B1 (en) * | 2000-06-29 | 2001-06-19 | Parker Hughes Institute | Treatment of inflammation with 2,4,6-trihydroxy-alpha-rho-methoxyphenylacetophenone, or its pharmaceutically acceptable derivatives |
IL166054A0 (en) * | 2002-07-01 | 2006-01-15 | Univ Johns Hopkins | Novel compounds pharmaceutical compositions containing same and methods of use for same |
KR101087559B1 (ko) * | 2002-07-09 | 2011-11-29 | 더 존스 홉킨스 유니버시티 | 신규 화합물 및 이를 함유하는 약제학적 조성물 |
-
2003
- 2003-07-09 BR BRPI0312654A patent/BRPI0312654A2/pt not_active IP Right Cessation
- 2003-07-09 EP EP03763401A patent/EP1539147A4/en not_active Withdrawn
- 2003-07-09 CA CA002491573A patent/CA2491573A1/en not_active Abandoned
- 2003-07-09 CN CN200910225906A patent/CN101721412A/zh active Pending
- 2003-07-09 SG SG200703939-9A patent/SG149701A1/en unknown
- 2003-07-09 WO PCT/US2003/021469 patent/WO2004004712A1/en active Application Filing
- 2003-07-09 ZA ZA200500166A patent/ZA200500166B/xx unknown
- 2003-07-09 EA EA200500177A patent/EA200500177A1/ru unknown
- 2003-07-09 US US10/520,506 patent/US20060135568A1/en not_active Abandoned
- 2003-07-09 CN CNA038185210A patent/CN1671384A/zh active Pending
- 2003-07-09 JP JP2004520072A patent/JP4493494B2/ja not_active Expired - Fee Related
- 2003-07-09 KR KR1020057000353A patent/KR20050047519A/ko not_active Application Discontinuation
- 2003-07-09 AU AU2003248896A patent/AU2003248896B2/en not_active Expired - Fee Related
- 2003-07-09 MX MXPA05000361A patent/MXPA05000361A/es not_active Application Discontinuation
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2005
- 2005-01-03 IL IL16612205A patent/IL166122A0/xx unknown
Also Published As
Publication number | Publication date |
---|---|
KR20050047519A (ko) | 2005-05-20 |
SG149701A1 (en) | 2009-02-27 |
JP4493494B2 (ja) | 2010-06-30 |
BRPI0312654A2 (pt) | 2017-05-02 |
WO2004004712A1 (en) | 2004-01-15 |
MXPA05000361A (es) | 2005-09-20 |
AU2003248896B2 (en) | 2010-04-22 |
AU2003248896A1 (en) | 2004-01-23 |
CA2491573A1 (en) | 2004-01-15 |
ZA200500166B (en) | 2007-08-29 |
US20060135568A1 (en) | 2006-06-22 |
CN101721412A (zh) | 2010-06-09 |
JP2005533834A (ja) | 2005-11-10 |
EP1539147A4 (en) | 2007-04-25 |
IL166122A0 (en) | 2006-01-15 |
EP1539147A1 (en) | 2005-06-15 |
EA200500177A1 (ru) | 2005-12-29 |
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