CN1751057A - 可用于治疗由黄病毒科病毒如丙型肝炎及牛病毒性腹泻病毒引起的感染的双环糖类化合物 - Google Patents
可用于治疗由黄病毒科病毒如丙型肝炎及牛病毒性腹泻病毒引起的感染的双环糖类化合物 Download PDFInfo
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Abstract
本发明公开双环糖类在治疗丙型肝炎病毒感染中的应用。针对DNA病毒、逆转录病毒以及人类和动物RNA病原体的重要家族黄病毒科病毒在体内测试了不同的双环糖类化合物。观察到对牛病毒性腹泻病毒(BVDV)具有显著的活性。由于瘟病毒如BVDV同丙型肝炎病毒(HCV)具有许多相似之处,因此预期通常的双环糖类以及更具体地优选的双环糖类可用于治疗丙型肝炎病毒感染。
Description
技术领域
本发明大体而言涉及可用于治疗由黄病毒科病毒引起的感染的双环糖类化合物。更具体而言,涉及可用于治疗或改善由丙型肝炎病毒、牛病毒性腹泻病毒、猪瘟病毒、西尼罗河病毒以及登革病毒引起的感染的这种化合物。
背景技术
丙型肝炎于1975年被最初确认为单独的疾病体,其时,发现大多数与输血相关的肝炎病例并非仅由当时已知的两种肝炎病毒——甲型肝炎病毒及乙型肝炎病毒——所导致。这种疾病被称作“非甲非已型肝炎”,并且发现其可传染至黑猩猩。然而,直到1989年该非甲非乙肝炎病毒的克隆及测序才得以首次报道,且该病毒被重新命名为“丙型肝炎病毒”(HCV)。紧接着进行了HCV抗体试验,而且筛查这种抗体仍为诊断的主要方法。
丙型肝炎病毒与黄病毒科病毒具有相同的病毒学及遗传学特征。其基因组组构与黄病毒属以及瘟病毒的相似,且与这些病毒尤其是瘟病毒共有少量的序列同一性。任一这些病毒组都组成黄病毒科的独立的属:黄病毒属、瘟病毒属以及丙肝病毒属(hepacivirus)。丙型肝炎病毒是一种直径约为50nm的球形包膜病毒。HCV的基因组为单链线性正义RNA。其为不分节段的。5’非编码(NC)区由约340个核苷酸组成。紧邻的下游为约9000个核苷酸的单个大的可读框(ORF)。其最末为由约100个核苷酸组成的3’NC区。HCV的基因组高度不均一。该基因组最为高度保守的区域为5’NC区部分以及终端3’NC区部分。ORF的最为高度保守的区域为衣壳基因。相比之下,该基因组最不均一的部分为编码包膜蛋白的基因。基于其遗传异质性,HCV品系可被分为大组(major group),该大组被称作病毒型或病毒基因型(并被暂时分为独立的物种)。各型中,HCV隔离种群(isolate)被分为许多亚型。最后,各个隔离种群由病毒基因组的异质群体组成,这些异质群体包括密切相关但不相同的病毒的“准群”(quasispecies)或“群”(swarm)。HCV的一些基因型显示出地理局限性,而其他的则分布于全世界。HCV更为广泛的遗传分析揭示,将隔离种群区分为型、亚型以及隔离种群在一定程度上为人为的产物,而病毒有可能作为遗传多样性的连续体存在。HCV遗传多样性的结果导致一种具有能够逃避宿主免疫监视的病毒,这使得重复暴露的个体慢性感染的比例高(80%以上)以及对再次感染缺乏免疫力。慢性以及缺乏可靠的免疫力可能都源于序列不同的病毒准群的小种群的出现(www.HEPNET.com,the Hepatitis Information Network;Challand R.,Young R.J.(1997)Antiviral Chemotherapy.Biochemical & MedicinalChemistry Series.Spektrum Academic Publishers,Oxford,pp.87-92;CannA.J.(1997)Principles of Molecular Virology.Second Edition.AcademicPress,San Diego,pp.230-235)。其他重要的导致未得以满足的医学需要的黄病毒科病毒为西尼罗河病毒以及导致登革热的病毒。
因此,存在强烈的医学需求以发现并开发出新的能够用于治疗黄病毒科病毒感染而同目前可用的治疗相比副作用更少且更小而疗效更好的生物活性分子。
发明内容
已发现某些具有以下通式的双环糖类对由黄病毒科病毒包括丙型肝炎病毒、牛病毒性腹泻病毒、猪瘟病毒、西尼罗河病毒以及登革病毒导致的感染具有活性:
其中,R1为芳基或苄基,R2及R3为烷基或芳基,R4为芳基且X为O、N或S。本申请中记载典型的、当前优选的双环糖类,但本领域技术人员易知其他双环糖类化合物亦可用于治疗由黄病毒科病毒导致的感染。本发明还包括这些化合物的药物学可接受的盐。
附图说明
图1为本发明双环糖类的化学结构,其被指定为式A。
图2为化合物A1及化合物A2的合成路线的图示。
图3为化合物A3的合成路线的图示。
具体实施方式
黄病毒科是一种重要的人类及动物RNA病毒病原体(Rice CM.1996.Flaviviridae:the viruses and their replication.In:Fields BN,Knipe DM,Howley PM,eds.Fields virology.Philadelphia:Lippincott-Raven Publishers.Pp 931-960)。黄病毒科目前确认的三个属在传播、宿主范围及发病机制上表现出显著的不同。该经典的黄病毒的成员为黄热病病毒、登革病毒以及瘟病毒,如牛病毒性腹泻(BVDV)以及猪瘟病毒(CSFV)。该科最近得以表征的成员为常见及独有的人类病原体——丙型肝炎病毒(HCV)。黄病毒属是具有正义RNA基因组极性的单链RNA病毒。其他具有正义RNA的病毒科包括小RNA病毒科(Picornaviridae)、披膜病毒科(Togaviridae)、杯状病毒科(Caliciviridae)以及冠状病毒科(Coronaviridae)。
本发明化合物可以其本来的形式或盐形式应用。当其中化合物的碱性或酸性足以生成稳定的无毒的酸式盐或碱式盐时,适宜以盐形式给药该化合物。药物学可接受的盐的实例为由能生成生理学可接受的阴离子的酸生成的有机酸加成盐,例如乙酸盐、抗坏血酸盐、安息香酸盐、柠檬酸盐、etoglutarate、甘油磷酸盐、丙二酸盐、甲磺酸盐、琥珀酸盐以及酒石酸盐。也可生成适宜的无机盐,包括碳酸氢盐、碳酸盐、盐酸盐、硝酸盐以及硫酸盐。
本发明的化合物可方便地以含有该化合物以及适宜的赋形剂的药物组合物的形式给药,该组合物可用于抵抗病毒感染。视该制剂是用于治疗内部感染或外部感染而定,本发明的化合物和组合物可非胃肠道给药、局部给药、阴道内给药、口服给药或直肠给药。
用于非胃肠道给药时,活性化合物或其盐的溶液可在水中制备,任选与无毒的表面活性剂混合。还可在甘油、液体聚乙二醇、三醋精、及其混合物以及油中制备分散体。
本发明可用的剂量可通过比较它们的体内活性来确定。将有效剂量外推至人类的方法为本领域所周知。
该化合物可方便地以单元剂量的形式给药,例如每个单元剂量形式含有0.1-2000mg、适宜地100-1000mg、最适宜地100-500mg的活性成分。预期的剂量可便利地存在于单一剂量中或为以适宜的间隔给药如每日二、三、四次或更多分次剂量的分剂量形式。该分次剂量本身还可再分,例如分为许多离散的宽松地间隔的给药,如由吹药器多次吸入或通过多次向眼中滴入施用。
对于内部感染,该组合物可以以游离碱计算约1-30mg/kg哺乳动物体重、优选为1-10mg/kg哺乳动物体重的剂量水平口服或非胃肠道给药。
给药此处所公开的化合物及组合物的精确方案将必然地取决于接受治疗的个体的需要、治疗的类型以及显而易见还取决于主治医师的判断。本发明的化合物可给药于需要治疗的动物。在多数情况下其为人类,但对牲畜以及宠物的治疗同样也被明确地认为属于本发明的范围之内。
实施例
方法及材料
式A化合物的合成
该化合物如下合成:
1.化合物A1及化合物A2的合成
化合物A1和化合物A2的合成如图2所示。
化合物1.1的合成
在RT(室温)下向2,3,4,6-四-O-苯甲基-D-葡萄糖(10.0g,18.5mmol)在CH2Cl2(125ml)及DMF(6.25ml)中的溶液内逐滴加入至草酰溴溶液(2.5ml在CH2Cl2中的10M溶液,1.35eq)。同时伴随强烈的气体生成。在Ar气氛下于室温将反应混合物搅拌60分钟。然后将反应混合物倒入冰水(125ml)中。相分离后,用冰水(2×125ml)洗涤有机相。用MgSO4干燥后,过滤并真空蒸发,得到黄色油状化合物1.1(图1),其无需进一步纯化即可用于以下的反应步骤。
分子式:C34H35BrO5
分子量:603.55
Rf:0.53(环己烷/乙酸乙酯85∶15)
1H-NMR(500MHz,CDCl3):[δ(ppm);J(Hz)]7.37(3H,m),7.33(5H,m),7.31(5H,m),7.28(5H,m),7.15(2H,m),6.43(1H,d,J=3.7),4.98(1H,d,J=5.0),4.83(2H,dd,app.t,J=10.9),4.58(1H,d,J=12.1),4.50(1H,d,J=10.7),4.46(2H,d,J=12.1),4.06(1H,m),4.03(1H,dd,app.t,J=9.2),3.80(1H,m),3.78(1H,m),3.76(1H,d,J=4.6),3.65(1H,dd,J=11.0,2.0),3.54(1H,dd,J=9.2,3.7)
化合物1.2的合成
向冷却至0℃的化合物1.1(理论量18.5mmol)在无水Et2O(250ml)中的溶液内缓缓加入苄基氯化镁(在Et2O中1M-opl.150ml,8eq)。将混合物在0℃下搅拌1小时,然后将温度缓缓升至室温。于室温下搅拌过夜后,将反应混合物倒入H2O(500ml)以及AcOH中,然后分离个相。将有机相用3×500ml饱和NaHCO3溶液以及250ml饱和NaCl溶液洗涤。用MgSO4干燥,过滤并真空蒸发,得到粗产物。将其用柱色谱(60-230目二氧化硅,梯度:甲苯∶环己烷8∶2,甲苯,环己烷∶乙酸乙酯9∶1),得到6.47g无色油状化合物1.2(57%,两步)。
分子式:C41H42O5
分子量:614.78
Rf:0.15(环己烷/二乙醚9∶1)
[α]D 20=+85.3°;[α]365 20=+88.1°(c=0.60,在氯仿中)
IR(KBr):(cm-1)2862,2360,1604,1496,1454,1360,1209,1085,1028,735,697,668
ES-MS:632=[M+NH4]+
1H-NMR(500MHz,CDCl3):[δ(ppm);J(Hz)]7.36(5H,m),7.34(5H,m),7.31(5H,m),7.29(5H,m),7.26(2H,m),7.22(3H,m),4.96(1H,d,J=11.0),4.95(1H,d,J=11.0),4.91(1H,d,J=11.0),4.84(1H,d,J=10.8),4.69(1H,d,J=11.0),4.62(1H,d,J=10.8),4.59(1H,d,J=12.2),4.52(1H,d,J=12.2),3.74(1H,dd,app.t,J=9.0),3.69(1H,m),3.68(1H,m),3.66(1H,dd,app.t,J=9.3),3.52(1H,ddd,J=18.3,9.2,2.3),3.37(1H,dd,app.t,J=9.0),3.36(1H,m),3.17(1H,dd,J=14.3,2.0),2.75(1H,dd,J=14.3,8.8)
APT-NMR(125MHz,CDCl3):δ(ppm)138.9(C),138.7(C),138.5(C),138.3(C),138.2(C),129.7(CH),128.6(CH),128.6(CH),128.5(CH),128.4(CH),128.2(CH),128.0(CH),127.9(CH),127.8(CH),127.7(CH),127.6(CH),126.2(CH),87.5(CH),81.8(CH),80.1(CH),79.0(CH),78.7(CH),75.7(CH2),75.2(CH2),75.1(CH2),73.5(CH2),69.0(CH2),38.0(CH2)
化合物1.3的合成
在室温下向化合物1.2(6.0g,9.76mmol)在无水EtOH(240ml)中的溶液内加入Pd/C(600mg,10摩尔%)。将反应混合物在Parr设备中于4个大气压的H2气下振荡5分钟。用硅藻土(celite)过滤并真空浓缩得到2.62克黄白色泡沫。其用柱色谱(60-230目,CH2Cl2∶MeOH 9∶1)纯化得到2.46克白色泡沫状化合物1.3(99%)。
分子式:C13H18O5
分子量:254.28
Rf:0.14(二氯甲烷/甲醇9∶1)
IR(KBr):(cm-1)3381,2922,2360,2341,1641,1603,1496,1454,1379,1308,1226,1079,1031,897,754,701,668
ES-MS:272=[254+NH4]+
1H-NMR(500MHz,CDCl3):[δ(ppm);J(Hz)]7.29(2H,d,J=7.0),7.22(2H,dd,app.t,J=7.3),7.14(1H,m),3.75(1H,dd,J=11.9,2.4),3.60(1H,dd,J=11.8,5.4),3.35(1H,m),3.32(1H,m),3.25(1H,dd,app.t,J=9.4),3.15(1H,m),3.12(1H,m),3.09(1H,dd,app.t,J=9.3),2.69(1H,dd,J=14.5,8.5)
APT-NMR(125MHz,CD3OD):δ(ppm)139.1(C),129.4(CH),127.6(CH),125.6(CH),80.4(CH),80.1(CH),78.6(CH),73.7(CH),70.6(CH),61.6(CH2),37.4(CH2)
化合物1.4的合成
室温下,向化合物1.3(1.0g,3.93mmol)的二甲基甲酰胺(38.6ml)中的溶液内相继加入苯甲醛二甲缩醛(benzaldehyde dimethyl acetal)(708μl,1.2eq)以及D(+)-10-樟脑磺酸(274mg,0.3eq)。将反应混合物在Ar气氛下于室温搅拌2小时。以EtOAc(150ml)稀释,由此开始精制。然后用1N NaOH溶液(2×150ml)、饱和NaHCO3溶液(2×100ml)以及饱和NaCl溶液(2×100ml)洗涤。用MgSO4干燥,过滤并真空蒸发得到1.52g白色固体残余物。以柱色谱纯化(60-230目二氧化硅,CH2Cl2∶MeOH 99∶1)得到940mg白色固体化合物1.4(70%)。
分子式:C20H22O5
分子量:342.39
Rf:0.20(环己烷∶乙酸乙酯6∶4)
熔点:43-44℃
[a]D 20=-6.9°;[α]365 20=-10.7(c=0.60,在氯仿中)
IR(KBr):(cm-1)3478,3031,2871,2360,1604,1497,1454,1385,1317,1299,1271,1212,1124,1099,1077,998,973,919,673,699,668,655,625,552,510
ES-MS:343=[M+H]+
1H-NMR(500MHz,CDCl3):[δ(ppm);J(Hz)]7.49(2H,m),7.38(3H,m),7.31(2H,m),7.28(2H,m),7.25(1H,m),5.51(1H,s),4.28(1H,dd,J=10.5,4.8),3.74(1H,dd,app.t,J=8.7),3.68(1H,dd,app.t,J=10.0),3.58(1H,ddd,J=9.6,8.2,2.6),3.43(1H,dd,app.t,J=9.2),3.39(1H,m),3.38(1H,dd,J=10.5,4.0),3.18(1H,dd,J=14.4,2.5),2.93(1H,brs),2.79(1H,dd,J=14.4,7.9),2.69(1H,brs)
APT-NMR(125MHz,CDCl3):δ(ppm)138.0(C),137.1(C),129.8(CH),129.4(CH),128.4(CH),128.2(CH),126.4(CH),126.3(CH),101.9(CH),81.1(CH),80.3(CH),75.5(CH),73.8(CH),70.1(CH),68.9(CH2),37.9(CH2)
化合物A1的合成
向冷却至0℃的化合物1.4(100mg,0.292mmol)的溶液中加入NaH(51mg 60%分散体,4eq)。然后将混合物在Ar气氛下于0℃搅拌30分钟。然后逐滴缓缓加入正丙基溴(133μl,5eq)。在0℃下10分钟后,于室温下继续搅拌过夜。TLC分析后,加入另外的2eq NaH以及1eqn-PrBr。在室温下搅拌4小时后,将反应混合物倒入H2O(25ml),然后以3×30ml Et2O萃取。用50ml饱和NaCl溶液洗涤合并的有机层,并用MgSO4干燥。过滤并真空浓缩得到144mg白色晶体残余物。以柱色谱(230-400目二氧化硅,戊烷∶乙醚9∶1)纯化后,得到白色晶体产物化合物A1(117mg,94%)。
分子式:C26H34O5
分子量:426.55
Rf:0.25(戊烷∶乙醚9∶1)
熔点:76-77℃
[α]D 20=-41.4°;[α]365 20=-127.6°(c=1.02,在氯仿中)
IR(KBr):(cm-1)2963(m),2918(m),2873(m),1454(m),1369(m),1121(s),1104(s),1089(s),1030(m),1008(m),968(m),951(m),748(s),697(s),652(m)
EI-MS:(m/z)43(86),91(100),115(39),149(26),176(17),217(5),251(3),277(32),208(1),335(3),366(2),426(8)[M+],427(2)[M++1]
1H-NMR(500MHz,CDCl3):[δ(ppm);J(Hz)]7.48-7.46(2H,m),7.38-7.33(3H,m),7.30-7.20(5H,m),5.52(1H,s),4.24(1H,dd,J=10.4,5.0),3.92(1H,dt,J=8.8,6.6),3.86(1H,dt,J=9.3,6.6),3.65(1H,dd,appt,J=10.4),3.64(1H,dt,J=9.3,6.8),3.57-3.48(4H,m),3.29(1H,ddd,app dt,J=10.0,5.0),3.15(1H,dd,14.3,2.0),3.08(1H,dd,J=9.3,8.3),2.70(1H,dd,J=14.3,8.7),1.62(4H,m),0.98(3H,t,J=7.4),0.93(3H,t,J=7.4)
APT-NMR(125MHz,CDCl3):δ(ppm)139.5(C),138.5(C),130.5(CH),129.7(CH),129.1(CH),129.0(CH),127.1(CH),126.9(CH),101.8(CH),84.4(CH),83.2(CH),82.6(CH),81.6(CH),76.1(CH2),75.7(CH2),71.1(CH),69.8(CH2),39.1(CH2),24.5(CH2),24.5(CH2),11.6(CH3),11.6(CH3)
化合物A2的合成
向冷却至0℃的化合物1.4(100mg,0.292mmol)的溶液中加入NaH(51mg 60%分散体,4eq.)。在Ar气氛下于0℃将混合物搅拌30分钟。然后逐滴缓缓加入正丁基溴(157μl,5eq)。于0℃下搅拌10分钟后,于室温下继续搅拌过夜。TLC分析后,加入另外的2eq NaH以及1eq n-BuBr。室温下搅拌5小时后,将反应混合物倒入H2O(25ml)中,用met 3×30mlEt2O萃取溶液。用50ml饱和NaCl溶液洗涤合并的有机相,并用MgSO4干燥。过滤并真空浓缩得到168mg黄白色残余物。用柱色谱(230-400目二氧化硅,戊烷∶乙醚92∶8)纯化得到125mg白色晶体化合物A2(94%)。
分子式:C28H38O5
分子量:454.60
Rf:0.24(戊烷∶乙醚92∶8)
熔点:69-70℃
[α]D 20=-38.7°;[α]365 20=-120.8°(c=0.98,在氯仿中)
IR(KBr):(cm-1)2963(s),2929(s),2873(s),1454(m),1375(m),1172(m),1092(s),1030(m),1008(m),968(m),748(m),697(s)
EI-MS:(m/z)57(75),91(100),129(49),177(12),189(19),235(2),291(36),307(4),363(2),454(5)[M+]
1H-NMR(500MHz,CDCl3):[δ(ppm);J(Hz)]7.49-7.47(2H,m),7.38-7.34(3H,m),7.31-7.21(5H,m),5.52(1H,s),4.24(1H,dd,J=10.4,5.0),3.96(1H,dt,J=8.9,6.6),3.90(1H,dt,J=9.4,6.6),3.71-3.64(2H,m),3.59(1H,dt,J=8.9,6.6),3.55-3.48(3H,m),3.29(1H,ddd,app dt,J=9.5,5.0),3.15(1H,dd,J=14.3,1.9),3.08(1H,dd,app t,J=8.8),2.97(1H,dd,J=14.3,8.7),1.65-1.55(4H,m),1.49-1.35(4H,m),0.96(3H,t,J=7.4),0.90(3H,t,J=7.4)
APT-NMR(125MHz,CDCl3):δ(ppm)140.0(C),139.0(C),131.0(CH),130.2(CH),129.6(CH),129.5(CH),127.6(CH),127.4(CH),102.4(CH),84.9(CH),83.7(CH),83.1(CH),82.1(CH),74.7(CH2),74.3(CH2),70.4(CH),64.5(CH2),39.6(CH2),34.0(CH2),33.9(CH2),20.8(CH2),20.7(CH2),15.4(CH3),15.3(CH3)
2.
化合物A3的合成
化合物A3的合成如图3所示。
化合物2.1的合成
同(β)-D-葡萄糖五乙酸酯(24.6g,63.0mmol)中加入溴化氢的乙酸溶液(33重量%,100ml)。立刻出现咖啡色。将反应混合物在氩气氛下于室温搅拌30分钟。然后用甲苯(4×50ml)以真空恒沸蒸馏除去溶剂得到棕绿色固体化合物2.1。该粗产物无需进一步纯化即可用于以下的反应步骤中。
分子式:C14H19O9Br
分子量:411.20
Rf:0.46(环己烷/乙酸乙酯1∶1)
IR(KBr):2962,2360,2342,1748,1435,1369,1218,1162,1112,1079,1042,911,752,668,601,563cm-1
ES-MS:433=[410+Na]+,435=[412+Na+]
1H-NMR(500MHz,CDCl3):[δ(ppm);J(Hz)]6.61(1H,d,J=4.0),5.56(1H,dd,app.t,J=9.7),5.16(1H,dd,app.t,J=9.7),4.84(1H,dd,J=10.0,4.0),4.33(1H,m),4.30(1H,m),4.13(1H,dd,J=12.3,1.5),2.11(3H,s),2.10(3H,s),2.05(3H,s),2.03(3H,s)
13C-NMR(125MHz,CDCl3):δ(ppm)170.37,169.70,169.64,169.31,86.34,71.91,70.39,69.94,66.94,60.76,20.48,20.48,20.38,20.38
化合物2.2的合成
通过插管向冷却至0℃的苯基溴化镁(200ml在二乙醚中的3M溶液,600mmol,9.5eq)在无水二乙醚(500ml)中的溶液内加入溴化物化合物1.1(理论值63.0mmol)在无水二乙醚(500ml)中的溶液。在氩气氛下于室温搅拌该反应混合物72小时。然后将该反应混合物倒入水(2000ml)中,并加入乙酸(200ml)以溶解镁盐。分离这2层,并将有机层用水(3×500ml)洗涤。在减压下浓缩合并的水相得到浅棕色固体残余物。将残余物溶于吡啶(500ml)中。在0℃下缓缓加入醋酐(340ml)。加入DMAP(200mg,1.64mmol)后,在氩气氛下于室温继续搅拌20小时。接着,将反应混合物在减压下浓缩,然后用甲苯(1×250ml)恒沸蒸馏,并加入二乙醚(31)。将得到有机层用饱和NaHCO3溶液(2×11)、1N HCl溶液(2×11)以及水(2×11)洗涤。用MgSO4干燥,并在减压下浓缩,得到25.1g浅棕色晶体。将其通过自2-丙醇中重结晶得到16.1g白色晶体化合物2.2(63%)。
分子式:C20H24O9
分子量:408.40
Rf:0.42(环己烷/乙酸乙酯1∶1)
熔点:149-150C
IR(KBr):2956,1753,1433,1368,1224,1104,1036,978,916,764,738,702,603cm-1
ES-MS:431=[408+Na]+
1H-NMR(500MHz,CDCl3):[δ(ppm);J(Hz)]7.39(5H,m),5.24(1H,dd,app.t,J=9.4),5.24(1H,dd,app.t,J=9.8),5.14(1H,dd,app.t,J=9.8),4.40(1H,d,J=9.9),4.30(1H,dd,J=17.2,4.7),4.16(1H,dd,J=12.2,1.5),3.85(1H,m),2.09(3H,s),2.06(3H,s),2.01(3H,s),1.80(3H,s)
13C-NMR(125MHz,CDCl3):δ(ppm)170.60,170.25,169.36,168.70,136.01,128.75,128.28,126.96,80.08,75.94,74.06,72.44,68.39,62.17,20.61,20.48,20.21
化合物1.3的合成
向四乙酸酯化合物2.2(16.08g,39.4mmol)在四氢呋喃(232ml)与甲醇(232ml)混合物中的溶液内加入无水碳酸钾(1.36g,9.84mmol,0.25eq)。将该混合物在氩气氛下于室温搅拌3小时。加入硅胶(40ml)并在减压下除去溶剂。用柱色谱(二氯甲烷/甲醇85/15)纯化产物化合物1.3得到9.50g产物化合物2.3(99%)。
分子式:C12H16O5
分子量:240.26
Rf:0.12(二氯甲烷/甲醇9∶1)
IR(KBr):3368,2919,2360,1636,1496,1455,1082,1042,891,764,701,595cm-1
ES-MS:258=[240+NH4]+,263=[240+Na]+
1H-NMR(500MHz,CDCl3):[δ(ppm);J(Hz)]7.44(2H,d,J=7.1),7.35(2H,dd,app.t,J=7.6),7.30(1H,m),4.15(1H,d,J=9.4),3.90(1H,dd,J=12.1,1.6),3.72(1H,dd,J=12.0,5.2),3.51(1H,dd,app.t,J=8.7),3.45(1H,dd,app.t,J=9.4),3.43(3H,m),3.40(1H,dd,app.t,J=9.2)
13C-NMR(125MHz,CDCl3):δ(ppm)139.30,127.43,82.41,80.70,78.23,74.98,70.40,61.41
化合物2.4的合成
在氩气氛下向四醇化合物2.3(1.15g,4.79mmol)在无水乙腈(3ml)中的溶液内加入樟脑磺酸(279mg,1.20mmol,0.25eq)以及苯甲醛二甲缩醛(1.44ml,9.58mmol,2eq)。将反应混合物在室温下搅拌3小时。然后加入三乙胺(0.337ml,2.40mmol)中和该混合物。减压下浓缩该反应混合物得到2.70g浅黄色油。用柱色谱(CH2Cl2/iPrOH 1/1)纯化得到1.53g白色固体化合物2.4(97%)。
分子式:C19H20O5
分子量:328.36
Rf:0.27(环己烷/乙酸乙酯1∶1)
熔点:114-115℃
[α]D 20=+9.3°;[α]365 20=+10.0°(c=1.13,在氯仿中)
IR(KBr):3433,2874,2357,1651,1496,1455,1385,1313,1272,1211,1109,1029,1009,913,765,733,700cm-1
ES-MS:346=[328+NH4]+
1H-NMR(500MHz,CDCl3):[δ(ppm);J(Hz)]7.53(2H,m),7.40(5H,m),7.39(3H,m),5.59(1H,s),4.37(1H,dd,J=10.3,5.9),4.30(1H,d,J=9.3),3.91(1H,dd,app.t,J=8.6),3.79(1H,dd,app.t,J=10.3),3.67(1H,dd,app.t,J=9.3),3.65(1H,m),3.63(1H,m)
13C-NMR(125MHz,CDCl3):δ(ppm)137.50,136.84,129.14,128.65,128.52,128.20,127.29,126.12,101.73,82.41,80.90,75.43,74.60,70.60,68.70
化合物2.5的合成
向冰冷的二醇化合物2.4(500mg;1.523mmol)在无水吡啶(15ml)中的溶液内相继加入DMAP(20mg;0.15mmol;0.1eq.)以及Ac2O(5ml)。移除冷却并将该反应混合物在Ar气氛下于室温搅拌18小时。然后在减压下用甲苯恒沸除去吡啶以及Ac2O。将残余物用柱色谱(Merckkieselgel;环己烷/EtOAc:85/15)纯化。得到收率为95%的化合物2.5(595mg;1.443mmol)。
分子式:C23H24O5
分子量:412.4
Rf:0.21(环己烷/EtOAc:85/15)
熔点:>150℃升华
[α]D 20:-78.46°(c=1.010;CHCl3)
IR(KBr-片,膜):(cm-1)3065(w);3033(w);2948(w);2873(w);2863(w);1749(s);1370(m);1238(s);1212(s);1105(s);1063(m);1031(m);999(m);767(m);701(m)
MS(m/z):43(100);91(17);105(26);107(13);189(9);219(11);352(3);369(<1)
1H-NMR(500MHz;CD3COCD3):[δ(ppm);J(Hz)]7.46(2H;m);7.39(2H;m);7.34(6H;m);5.69(1H;s);5.42(1H;dd(app.t);J=9.5);5.19(1H;dd(app.t);J=9.5);4.67(1H;d;J=9.8);4.31(1H;dd;J=4.6,9.9);4.98(1H;dd(app.t);J=9.4);3.87(1H;dd(app.t);J=10.0);3.82(1H;ddd;J=4.6,9.5,10.0);1.95(3H;s);1.76(3H;s)
APT(125MHz;CD3COCD3):δ(ppm)20.3(CH3);20.7(CH3);69.1(CH2);71.7(CH);73.8(CH);74.3(CH);79.6(CH);81.2(CH);102.1(CH);127.2(CH);128.2(CH);128.8(CH);129.0(CH);129.3(CH);129.6(CH);138.3(C);138.7(C);169.2(C);170.3(C)
化合物2.6的合成
将化合物2.5(400mg;0.970mmol)溶于干燥甲苯(5ml)中,并逐滴加入Petasis试剂(Cp2TiMe2)在甲苯(8.15ml;4.074mmol;4.2eq.)中的0.5M溶液。将反应混合物遮光并在Ar气氛下于70℃加热60小时。然后将该反应混合物在减压下浓缩,并用柱色谱(Merck kieselgel;环己烷/CH2Cl2/EtOAc:50/50/1)纯化残余物。
化合物A3的合成
向烯醇醚2.6(320mg;0.783mmol)在无水EtOAc(17ml)中的溶液中加入无水Et3N(1.7ml)。然后加入Pd/C(10重量%钯;320mg),并将反应混合物置于H2气氛下。在室温下搅拌22小时后,通过以硅藻土过滤而除去催化剂,并用EtOAc洗涤。用柱色谱(Merck kieselgel;戊烷/CH2Cl2/乙醚:50/50/1)纯化浓缩滤液后得到的残余物。得到收率为82%的化合物A3(265mg;0.642mmol)。
分子式:C25H32O5
分子量:0.22(戊烷CH2Cl2/ether:50/50/1)
熔点:98-100℃
[α]D 20:-31.02°(c=1.100;CHCl3)
IR(KBr-disc,film):(cm-1)3066(w);3035(w);2972(s);2925(m);2902(m);2869(m);1454(m);1380(m);1170(m);1106(s);1076(s);1028(s);1001(m);764(m);748(m);700(s)
MS(m/z):43(100);91(62);105(69);107(56);115(20);149(88);196(4);238(12);263(7);369(<1);412(<1;M+o)
1H-NM[R(500MHz;CD3COCD3):[δ(ppm);J(Hz)]7.51(2H;m);7.27-7.43(8H;m);5.65(1H;s);4.25(1H;d;J=9.2);4.23(1H;dd;J=5.0,10.2);4.01(1H;h;J=6.1);3.76(1H;dd(app.t);J=10.1);3.66(1H;dd(app.t);J=9.2);3.62(1H;dd(app.t);J=9.2);3.55(1H;ddd;J=5.0,9.1,9.9);3.28(1H;dd;J=8.3,9.2);3.19(1H;h;J=6.1);1.14(6H;2d(app.t);J=5.9);0.96(3H;d;J=6.1);0.44(3H;d;J=6.1)
APT(125MHz;CD3COCD3):δ(ppm)21.9(CH3);22.7(CH3);23.0(CH3);23.6(CH3);69.4(CH2);71.6(CH);72.8(CH);73.3(CH);80.9(CH);81.5(CH);83.1(CH);83.8(CH);101.7(CH);126.9(CH);128.6(CH);128.8(CH);128.8(CH);129.4(CH);139.3(C);140.7(C)
化合物生物活性筛选
以各种病原性病毒如人免疫缺损病毒(HIV)、单纯疱疹病毒(HSV)、牛痘病毒(VV)、水痘带状疱疹病毒(VZV)以及人类巨细胞病毒(CMV)对化合物进行筛选。对于除CMV外的所有病毒,测定EC50(能够以50%的抑制率抑制人CEM细胞培养物中的HIV诱导的致细胞病变(cytopathicity)、人胚胎成纤维细胞E6SM细胞培养物中的HSV以及VV诱导的致细胞病变、以及人类胚胎肺HEL细胞培养物中VZV诱导的噬斑形成的有效的化合物浓度)。对于测定表达为IC50的对CMV的抗病毒活性而言,将生长在96孔微板中的人类胚胎肺成纤维细胞(HEL)用每孔20PFU病毒进行感染。
在37℃下温育2小时后,以0.1ml的含受试化合物的系列稀释液的培养基补充该受感染的细胞。在第7天,在用吉姆萨氏溶液对细胞染色后用显微方法计数该噬斑。最小抗病毒浓度表达为以50%抑制率抑制病毒诱导的噬斑形成所需的剂量。
还以黄病毒对该化合物进行筛选。由于没有足够的体内试验可针对HCV进行筛选的事实,因为牛病毒性腹泻病毒(BVDV)与丙型肝炎病毒具有很多相似之处,因此我们选择其进行筛选。用BVDV的Pe515菌株在Madin Darby牛肾细胞(MDBK细胞)上评价抗病毒活性。抗病毒活性以及细胞毒性都通过MTS方法测定。EC50为减少病毒诱导的致细胞病变50%所需的浓度。MTC(最小毒性浓度)被定义为导致细胞代谢减少≥20%的浓度。
还通过鼠类白血病细胞(L1210/0)、鼠类乳腺癌细胞(FM3A)以及人类T一淋巴细胞(Molt4/C8,CEM/0)检验了该化合物的抗肿瘤活性。
基于NCCLS文件M7-A4,Vol.17 No.2,M27-A,Vol 17 No.9以及M38-P,Vol 18 No.13,应用自动读数温育器(reader-incubator)BioscreenC Analyser(Labsystems,Finland)开发出进行抗细菌以及抗真菌筛选的微量稀释方法(microdilution method)。其通过垂直光路光度测定(光密度)连续地测定生长、处理数据并提供打印结果。我们选择为初级靶标(primary target)的细菌为:金黄色葡萄球菌(Staphylococcus aureusATCC29213)、粪肠球菌(Enterococcus faecalis ATCC 29212)、大肠杆菌(Escherichia coli ATCC 25922)以及绿脓杆菌(Pseudomonas aeruginosaATCC 27853)。白色假丝酵母(Candida albicans ATCC 24433)以及新型隐球菌(Cryptococcus neoformans ATCC 90112)被选作酵母靶标。皮肤真菌须癣毛癣菌(Trichophyton mentagrophytes ATCC 9533)以及侵袭性霉菌烟曲霉(Aspergillus fumigatus ATCC 2895)被选作霉菌靶标。优化温育所必需的所有参数可在Biolink软件(Labsystems,Finland)中设定。所有细菌筛选都在35℃下温育16小时。对假丝酵母以及隐球菌筛选所用的温育参数分别为在35℃下24和48小时,侵袭性真菌烟曲霉为在30℃下温育3天,皮肤真菌须癣毛癣菌为在30℃下温育5天。使用以阳离子调节的Mueller-Hinton肉汤(Oxoid,Belgium)作为细菌菌种生长培养基。推荐合成培养基用于真菌易感性试验:RPMI 1640,含谷氨酰胺、不含碳酸氢盐、且含pH指示剂(Oxoid)并补充1%葡萄糖。将25μl的10倍浓度的化合物吸入每个孔中。向每25μl试验化合物中加入225μl菌种生长培养基。使用通过Biolink软件自动测定的生长曲线下面积作为抗细菌以及抗酵母筛选的测量工具。我们应用终端OD测定于针对致病霉菌的筛选。在试验安排中包含每种微生物的参照抗生素用于内部质量控制。
结果与讨论
在第一组筛选中(表1),针对HIV-1、HIV-2、HSV-1、HSV-2、VV、CMV以及VZV测定双环糖类的抗病毒活性。仅化合物A3对VZV显示出活性。
表1-针对HIV-1、HIV-2、HSV-1、HSV-2、VV、CMV
以及VZV的筛选结果
化合物 | aEC50(μg/ml) | |||||||
HIV-1(IIIB)(CEM) | HIV-2(ROD)(CEM) | HSV-1(KOS)(E6SM) | HSV-2(G)(E6SM) | VV(E6SM) | CMVDavis,D-169(HEL) | VZV(HEL) | ||
OKA | YS | |||||||
化合物A1化合物A2化合物A3 | >20>20>20 | >4>20>4 | >16>16>8 | >16>16>8 | >16>16ND | >20>20>5 | NDND1.1 | NDNDND |
a50%以50%的抑制率抑制的人淋巴细胞CEM细胞培养物中的HIV诱导的致细胞病变(cytopathicity)、人胚胎成纤维细胞E6SM细胞培养物中的HSV以及VV诱导的致细胞病变、以及人类胚胎肺HEL细胞培养物中CMV以及VZV诱导的噬斑形成的有效的化合物浓度。
表2-针对MDBK细胞中的BVDV的筛选结果
化合物 | EC50(μg/ml)BVDV | MTC(μg/ml)DBK |
化合物A1化合物A2化合物A3利巴韦林 | 23/4/3.27/53/54<0.840 | >100/>100/>100>100/>100/>100>100- |
为检测在牛肾(MDBK)细胞中的抗牛病毒性腹泻病毒(BVDV-Pe515菌株)活性,进行另一系列的病毒筛选(表2)。化合物A1的EC50分别为23、4以及3.2μg/ml。化合物A2的EC50分别为7、53以及54μg/ml。两种化合物的复制试验都是三个独立的试验。化合物A3的EC50<0.8μg/ml。利巴韦林是目前用于治疗HCV导致的感染的极好的标准。由表2中的结果可清楚看出,化合物A1、A2以及A3比利巴韦林有效得多。
当以化合物A1、A2以及A3处理时,MTC未达到MDBK细胞的最高浓度(100μg/ml)。
针对L1210/0、FM3A/0、Molt4/C8以及CEM/0筛选化合物A1的抗肿瘤活性。未发现化合物A1对所有受试细胞系具有任何抗肿瘤活性。化合物2对受试细胞系具有中等的抗肿瘤活性(表3)。
表3-针对L1210/0、FM3A/0、Molt4/C8以及CEM/0的抗肿瘤筛选结果
化合物 | IC50(μg/ml) | |||
L1210/0 | FM3A/0 | Molt4/C8 | CEM/0 | |
化合物A1化合物A2化合物A3 | >20082±7N.A. | >20022±0N.A. | >20019±1N.A. | >20027±2N.A. |
针对四种标准细菌筛选抗细菌活性:金黄色葡萄球菌、粪肠球菌、大肠杆菌以及绿脓杆菌(表4)。没有化合物对所选微生物显示出显著的抗细菌活性。两种化合物的最小抑制浓度为高于25μg/ml。
表4-对革兰氏阳性菌和革兰氏阴性菌的抗细菌筛选结果
化合物 | MIC(μg/ml) | |||
金黄色葡萄球菌ATCC 29213 | 粪肠球菌ATCC 29212 | 大肠杆菌ATCC 25922 | 绿脓杆菌ATCC 27853 | |
化合物A1化合物A2化合物A3 | >25>25>25 | >25>25>25 | >25>25>25 | >25>25>25 |
针对两种致病酵母以及霉菌筛选抗真菌活性(表5)。没有化合物对所选微生物显示出显著的抗真菌作用。两种化合物的最小抑制浓度为高于251μg/ml。
表5-针对致病真菌的筛选结果
化合物 | MIC(μg/ml) | |||
白色假丝酵母ATCC 24433 | 新型隐球菌ATCC 90112 | 须癣毛癣菌ATCC 9533 | 烟曲霉ATCC 2895 | |
化合物A1化合物A2化合物A3 | >25>25>25 | >25>25>25 | >25>25>25 | >25>25>25 |
结论
在第一组对DNA病毒或逆转录病毒的筛选中,没有观察到显著的抗病毒活性。然而,在第二组针对BVDV(RNA病毒)的筛选中,我们清楚地发现化合物A1以及化合物A3具有显著的抗病毒活性,而化合物A2具有中等活性。其他目前所测试的双环糖类对BVDV不具有活性。此外,未发现显著的抗肿瘤或抗微生物活性。由于BVDV以及HCV具有许多相似之处,化合物A1以及化合物A3以及可能的其他双环糖类都可能对HCV具有强大的且选择性的抗病毒性能。除化合物A1外,化合物A2及化合物A3同样对其他黄病毒如西尼罗河病毒及登革病毒具有活性。从筛选可知,与为目前用于治疗由如HCV导致的感染的极好标准的利巴韦林相比,化合物A1、A2以及A3对黄病毒科病毒具有更强的抗病毒活性。
尽管参考本发明优选的具体实施方案对本发明进行描述,但应明白,由于其中可进行各种变化和改进而不超出如所附权利要求书所定义的本发明的全部范围,因此本发明的范围不仅限于上述优选的具体实施方案。
Claims (7)
2.如权利要求1所述的方法,其中R1选自苯基和苄基,R2和R3选自丙基、异丙基和丁基,R4为苯基以及X为O,或其药物学可接受的盐。
3.如权利要求1所述的方法,其中所述感染为丙型肝炎。
4.如权利要求1所述的方法,其中所述感染为牛病毒性腹泻。
5.一种治疗正义RNA病毒感染的方法,其包括给药有效量的权利要求1中的化合物的步骤。
6.如权利要求4所述的方法,其中所述感染是丙型肝炎。
7.如权利要求3所述的方法,其中所述感染是牛病毒性腹泻。
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