CN109134585A - 一类三萜与直链氨基衍生物的偶联物及其应用 - Google Patents
一类三萜与直链氨基衍生物的偶联物及其应用 Download PDFInfo
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- CN109134585A CN109134585A CN201811036290.XA CN201811036290A CN109134585A CN 109134585 A CN109134585 A CN 109134585A CN 201811036290 A CN201811036290 A CN 201811036290A CN 109134585 A CN109134585 A CN 109134585A
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- amino
- alkyl
- triterpene
- conjugate
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Abstract
本公开了结构式如下式所示的三萜与直链氨基衍生物的偶联物：
Description
技术领域
本发明涉及了一种三萜与直链氨基衍生物的偶联物及其应用。
背景技术
流感是由流感病毒(Influenza virus)引起的一种急性，传染性呼吸系统疾病。根据其内部核蛋白(NP)和基质蛋白(M)的抗原性不同，流感病毒可分为A型，B型，C型和D型。A型(又称甲型)流感病毒大规模流行可引起极高的发病率和死亡率，严重威胁着人类的健康(Virology Journal.2007，4，1-5)。A型流感病毒在二十世纪主要引起了三次大型流感，即1918年的H1N1，1957年的H2N2以及1968年的H3N2，共造成约5000万人死亡(EmergingInfectious Diseases.2006，12，9-14；Journal of the American Medical Association，2007，18，2025-2027)。2009年甲型流感也是由H1N1流感病毒引起(New England Journalof Medicine.2009，370，1335-1342)，其传播之迅速，引起了世界的关注。据统计，全世界平均每年有30-50万人死于流感(Southern Medical Journal.2007，57，1-60)。
迄今，FDA批准的抗流感药物主要有两类。第一类，达菲(Oseltamivir)和乐感清(Zanamivir)主要抑制流感病毒的神经氨酸酶(NA)，阻断流感病毒从感染细胞中释放出来(Nature Medicine.2004，10，82-87；Journal of the Americ an Chemical Society1997，119，681-690)。第二类，金刚烷胺(Amantadine)和金刚乙胺(Rimantadine)主要破坏流感病毒M2蛋白离子通道活性，能够抑制流感病毒的脱衣壳过程(Proceedings of the NationalAcademy of Sciences of the United States of America.2008，105，10967-10972)。然而，美国疾病预防控制中心抽样调查发现，2008/2009年的H3N2毒株和2009年大流行H1N1病毒中，100％的毒株对金刚烷类药物都具有耐药性；99.6％的季节性H1N1流感病毒对达菲具有耐药性
三萜类化合物是自然界中广泛存在的一类天然化合物，其结构包括A，B，C，D，E五个环，30个碳原子(Journal of the American Chemical Society，1996，35，8509-8509)。三萜类化合物由于其多种多样的生物及药理活性而引起越来越广泛的关注，如白桦脂酸及其衍生物已在临床试验中用作抗肿瘤和抗HIV的药物(U.S.Pat.Nos.5，679，828；6，689，767；6，369，109；U.S.App.Pub.No.2004/0204389)；齐墩果酸是保护肝脏防止化学试剂损伤和防治HIV感染的有效成份(Journal of Natural Products.1998，61，1090-1095)。北京大学周德敏教授课题组首次发现了自然界广泛存在的五环三萜天然产物与不同的环糊精偶联具有很强的抗流感病毒进入的活性，并对其机制进行了深入的研究(Europe an Journalof Medicinal Chemistry.2017，134，133-139；Biomaterials.2016，78，74-85)。三萜与直链氨基衍生物的偶联物对流感病毒的抑制作用则未见报道。
发明内容
本发明目的是提供一类三萜与直链氨基衍生物的偶联物，其化学结构式如下式所示：
其中，是单键或双键；
X和Y结合起来形成一个带有1-5个相同或不同取代基的五元环、六元环或七元环，其中所述取代基各自独立选自H，未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基，未取代的C1-C6烷氧基或被羟基、氨基或羧基取代的C1-C6烷氧基，卤素，羧基，羟基，硝基，氰基，巯基，C1-C6硫烷基或NHR9’，所述R9’是H，未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基；
R1选自-NH-(-CH2-)n-OH、-NH-(-CH2-)n-NH2、-NH-(-CH2-)n-COOH、-NH-(-CH2-)n-COOCH3，n＝1-12；
R2和R7各自独立选自H，卤素，羟基，氰基，硝基，巯基，C1-C6硫烷基，未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基，氨基，NR11’R12’，所述R11’和R12’各自独立选自未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基；
优选地，R2选自H、OH、SH或NH2，更优选为OH。
优选地，R7选自H、OH、NH2或SH，更优选为OH。
R3、R4、R5、R6和R8各自独立选自H，未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基；
优选地，R3、R4、R5、R6和R8各自独立选自甲基。
R9选自H，卤素，羟基，氰基，硝基，巯基，C1-C6硫烷基，羰基，肟基，未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基。
本发明提供了一类三萜与直链氨基衍生物的偶联物，其中X和Y结合起来形成一个带有1-5个相同或不同取代基的六元环，所述取代基各自独立选自H，未取代的C1-C3烷基或被羟基、氨基或羧基取代的C1-C3烷基，羧基，羟基，硝基，氰基或NHR9’，所述R9’是H，未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基。
所述X和Y结合起来形成一个带有1-5个相同或不同取代基的六元环的三萜与直链氨基衍生物的偶联物结构式如下：
其中R10，R11，R12，R13和R14各自独立选自H、OH、CH3、NHR9’,其中R9’是H，巯基，C1-C6硫烷基，未取代的C1-C3烷基或被羟基、氨基或羧基取代的C1-C3烷基。
进一步的R10，R11，R12，R13和R14各自独立选自H、OH、CH3、NH2。
进一步的R10，R11，R12，R13和R14各自独立选自H、OH、CH3。
进一步的R11和R12各自独立选自H或甲基。
进一步的R10是H。
本发明提供了一类三萜与直链氨基衍生物的偶联物，其中X和Y结合起来形成一个带有1-5个相同或不同取代基的五元环，所述取代基各自独立选自H，未取代的C1-C3烷基或被羟基、氨基或羧基取代的C1-C3烷基，羧基，羟基，硝基，氰基或NHR9’，其中R9’是H，未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基；
所述X和Y结合起来形成一个带有1-5个相同或不同取代基的五元环的三萜与直链氨基衍生物的偶联物结构式如下：
其中R10，R11，R12和R13各自独立选自H、OH、-C(CH3)＝CH2、NHR9’，其中R9’是H，巯基，C1-C6硫烷基，未取代的C1-C3烷基或被羟基、氨基或羧基取代的C1-C3烷基。
进一步的R10，R11，R12和R13各自独立选自H、OH、-C(CH3)＝CH2、NH2
进一步的R10是-C(CH3)＝CH2，R11，R12和R13各自独立选自H。
本发明另一目的是提供三萜与直链氨基衍生物的偶联物的具体结构，见下表：
本发明另一目的是将上述化合物应用在制备治疗或/或预防流感药物中的应用。
本发明的化合物能够用于预防或治疗流感，尤其是甲型流感；本发明化合物可阻断流感病毒进入细胞，但不仅局限于此机制。
本发明化合物可以以纯净化合物或化合物的混合物的形式给药，或者优选在药物赋形剂，稀释剂或载体中给药。
可以通过任何适当的途径来施用活性剂进行治疗。适当的施用途径可以包括口服，直肠、鼻、气雾或颗粒吸入剂，局部(包括含化和舌下)，经皮、阴道、膀胱内、伤口内和胃肠外(包括皮下、肌内、静脉内、胸骨内、膜内、硬膜外和真皮内)。
本发明也涉及组合物，包含本发明化合物，与一种或多种药学可接受的添加剂和任选的其他药物一起。药学可接受的添加剂可以是载体，稀释剂，佐剂和(或)赋形剂的形式，可以包括所有常规的溶剂、分散剂、填充剂、固体载体、包衣剂、抗真菌或抗菌剂、皮渗透剂、表面活性剂等张剂和吸收剂，和缓释或控释基质。活性剂可以以适合同时，分开或连续施用活性剂的组分的试剂盒的形式；在与组合物的其他成分相容和患者生理耐受的意义上，每种载体，稀释剂，佐剂和/或赋形剂必须是“药学可接受的”。该组合物可以方便地以单元剂型的形式存在，可以通过制药领域公知的方法来制备；这些方法包括将活性成分与载体相混合的步骤，其中载体是由一种或多种助剂组成的；一般地，制备该组合物，包括将活性成分与液体载体、稀释剂、佐剂和/或赋形剂或精细分离的固体载体或两者均匀和直接地混合，然后如果必要使产物成型。
适合口服的本发明的组合物可以是以每个都包含预定量的活性成分的分离单元例如胶囊，囊剂或片剂的形式存在；作为粉末或颗粒；作为水相或非水液体中的溶液或混悬液；或者作为水包油性液体乳剂或油包水性乳剂；活性成分也可以以大丸剂，药糖剂或糊剂的形式存在。
可以通过任选与一种或多种助剂压片或成模来制备片剂；可以通过在适当的机器中压制自由流动形式例如粉末或颗粒的活性成分来制备压制片，任选与粘合剂(例如惰性稀释剂、防腐剂、崩解剂、淀粉羟乙酸钠、交联聚维酮、交联羧甲基纤维素钠)，表面活性剂或分散剂混合。可以通过在适当的机器中将用惰性液体稀释剂湿润的粉末状化合物的混合物成型来制备模印片；任选可以将片剂包衣或刻痕，可以通过配制来缓释或控释活性成分，例如使用不同比例的羟丙基甲基纤维素来产生所需的释放性质；片剂任选可以具有肠溶衣，以在肠部分而不是胃中释放。
适合胃肠外施用的组合物包括水性和非水性等张无菌注射溶液，其可以包含抗氧化剂，缓冲剂，抑菌剂和使组合物与所预期的患者的血液等张的溶质；和水性和非水性无菌混悬液，其可以包括助悬剂和增稠剂。该组合物可以存在于单位剂量或多剂量的密封容器例如安瓿和管中，可以贮存在冷冻-干燥(冻干)条件下，仅需要在使用前加入无菌液体载体例如注射用水。可以由上述种类的无菌粉末，颗粒和片剂来制备无准备的注射溶液和混悬液。
适合局部施用于皮肤，即经皮施用的组合物可以包含溶解或悬浮在任何适当的载体或基质中的活性剂，可以是洗剂、凝胶、乳膏、糊剂、软膏等等的形式。适当的载体可以包括液状石蜡、丙二醇、蜡、聚氧乙烯和长链醇。也可以使用经皮装置例如贴剂，可以包含适当材料例如硝酸/乙酸纤维素，丙烯和聚碳酸酯制成的微孔膜。贴剂也可以包含适当的皮肤粘附性和基底材料。
本发明的活性化合物也可以以植入物的形式存在，其可以包含药物的聚合性装置，其中聚合物是生物相容性的和无毒性的。适当的聚合物可以包括水凝胶、硅酮、聚乙烯和生物可降解的聚合物。
本发明的化合物可以以持续(即控释)或缓释的形式施用；持续释放制剂是其中施用后活性成分在患者体内缓慢释放并在最小的时间里维持所需的药物浓度的制剂；持续释放制剂的制备是本领域技术人员公知的。剂型可以包括口服形式，植入物和经皮形式。对于缓释施用，活性成分可以作为例如，缓释颗粒悬浮或在脂质体内。
依据选择的化合物的特定活性，患者状况以及要处理的病症选择本发明化合物适合的剂量范围。本领域技术人员可以根据其普通知识和在本领域的经验适合的剂量范围；例如对于流感，人类适合的剂量范围可以为每人每天1-500mg，例如10-300mg，通常为30-150mg。
本发明的优点和技术效果如下：
1.本发明将三萜与直链氨基衍生物的偶联物，弥补了三萜类水溶性差导致的成药性差的缺点，大大增强了三萜类化合物的抗流感病毒活性；
2.本发明中的三萜与直链氨基衍生物的偶联物的合成方法采用了脱水缩合反应，生成酰胺键连接臂，相较于其他连接方式，不仅使反应变得快捷高效，还增强了化合物的生物兼容性；
3.本发明中的化合物以流感病毒进入细胞阶段为靶点，从源头上抑制了流感病毒感染，为抗流感病毒抑制剂的研究提供了依据。
附图说明
图1为加药时间点实验结果示意图；
图2为血凝抑制实验示意图。
具体实施方式
下面通过实施例对本发明作进一步详细说明，但本发明的保护范围不局限于所述内容，实施例中方法如无特殊说明均采用常规方法，使用试剂如无特殊说明，均为常规市售试剂或采用常规方法配置的试剂。
定义
术语“C1-C3烷基”是指含有一到三个碳原子的烷基，例如甲基，乙基，丙基等。
术语“C1-C6烷基”是指含有一到六个碳原子的直链或支链烷基，例如甲基，乙基，丙基，异丙基，正丁基，异丁基，叔丁基，戊基或己基等。
术语“三萜”是指由数个异戊二烯去掉羟基后首尾相连构成的物质，大部分为30个碳原子，少部分含27个碳原子的萜类化合物，例如齐墩果酸，白桦脂酸等。
术语“卤素”是指氟，氯，溴或碘。
术语“C1-C6硫烷基”是指其中一个氢原子被硫原子取代的C1-C6烷基。
实施例1：本三萜与直链氨基衍生物的偶联物的制备方法，可以采用下列技术路线制备：
其中，化合物M22的合成如下：
取1g齐墩果酸(OA)于50mL反应瓶中，溶解于20mL DMF，加入845mg TBTU和340mgDIEA，室温反应，12h后薄层检测，展开剂比例石油醚:乙酸乙酯＝3:1；反应完全，蒸干DMF，采用水/乙酸乙酯体系(水:乙酸乙酯体积比1:1)萃取3次，取有机相于MgSO4干燥后蒸除溶剂，重结晶(乙醇:水＝3:1)；得白色絮状固体M38 1582mg，产率92.8％，待用；
取1g化合物M38于50mL反应瓶中，15mL DMF溶解，加入217mg的4-氨基丁醇，再加入1.2eq的碳酸钠，室温反应，2h后薄层检测，展开剂比例石油醚:乙酸乙酯＝1:1；反应完全，将反应液20mL水洗2次，饱和NaCl洗一次，MgSO4干燥后柱分离；得白色固体M22 974mg，产率80％。
M22:1H NMR(400MHz,(CD3)2SO)δ:0.66,0.67,0.84,0.86,0.88,0.89,1.08(7×CH3),0.66—2.00(m,other aliphatic ring protons),2.78(d,J＝9.68Hz,1H),2.90—3.08(m,3H),3.38(q,J＝6Hz,2H),4.28(d,J＝5.16Hz,1H),4.37(t,J＝4.96Hz,1H),5.20(brs,1H),7.21(t,J＝4.96Hz,1H)13C NMR 100MHz,(CD3)2SO)δ:15.08,16.00,16.80,18.00,22.25,22.91,23.56,25.66,25.78,26.95,28.22,30.07,30.42,32.45,32.75,32.94,33.66,36.57,38.07,38.36,38.62,38.74,40.44,41.23,45.14,45.16,46.03,47.11,54.81,60.54,76.82,121.34,144.11,176.00。
化合物M26的合成如下：
取1，4-丁二胺892mg和碳酸钠1g于50mL反应瓶中，15mL DMF溶解，置于-10℃冷井中搅拌，用常压滴液漏斗缓慢滴加溶于DMF的M38 1g，3h后薄层检测，展开剂比例石油醚:乙酸乙酯＝1:1；反应完全，蒸干DMF，加入1N HCl 2mL使固体析出，真空抽滤，洗涤，得白色固体M26 931mg产率73.4％。
M26:1H NMR(400MHz,(CD3)2SO)δ:0.66,0.84,0.86,0.87,0.88,1.07(7×CH3),0.66—2.00(m,other aliphatic ring protons),2.77(d,J＝13.8Hz,1H),2.89-3.05(m,6H),5.20(s,1H),7.24(t,J＝5.4Hz,1H)8.31(S,1H)13C NMR(100MHz,(CD3)2SO)δ:15.08,16.00,16.81,18.01,22.27,22.92,23.56,25.66,26.59,26.95(2C),28.22,30.41,30.54,32.45,32.74,32.95,33.66,36.57,38.08,38.36,38.74,40.45,41.23,41.27,45.15,46.04,47.12,54.83,76.80,79.18,121.34,144.12,176.01。
化合物M35的合成如下：
取1g 6-氨基己酸于50mL反应瓶中，15mL甲醇溶解，置于-10℃的冷井中搅拌，用常压滴液漏斗缓慢滴加1.66mL的SOCl2，滴毕，移至室温反应，3h后薄层检测，展开剂比例二氯甲烷:甲醇＝10:1同时加一滴氨水；反应完全，直接蒸干甲醇，粗产物可直接用于下步反应，无需纯化，但要保证薄层上产物点单一，若产物点不单一，则需经柱层析纯化；得白色固体M42 1.69g，产率89.4％，待用；
取1g化合物M38于50mL反应瓶中，15mL DMF溶解，加入353mg的M42，再加入258mg的碳酸钠，室温反应，2h后薄层检测，展开剂比例石油醚:乙酸乙酯＝1:1；反应完全，将反应液20mL水洗2次，饱和NaCl洗一次，MgSO4干燥后柱分离。得白色固体M22 912mg，产率70.4％；
M35:1H NMR(600MHz,(CD3)2SO)δ:0.66,0.67,0.84,0.86,0.87,0.89,1.08(7×CH3),0.66—2.00(m,other aliphatic ring protons),2.27(t,J＝7.44Hz,2H),2.77(dd,J＝4.02,13.44Hz,1H),2.91—3.04(m,3H),3.57(s,3H),4.27(d,J＝5.16Hz,1H),5.20(t,J＝3.42Hz,2H),7.21(t,J＝5.58Hz,2H)13C NMR 100MHz,(CD3)2SO)δ:15.06,16.03,16.85,18.00,22.26,22.93,23.59,24.23,25.67,26.06,26.96,28.24,28.77,30.45,32.46,32.77,32.97,33.30,33.66,36.58,38.08,38.39,38.60,38.91,40.06,40.46,41.25,45.20,46.05,47.12,51.17,54.82,76.83,121.34,144.17,173.26,176.07。
化合物M31的合成如下：
取1g化合物M35，用混合溶剂(甲醇:四氢呋喃＝1:1)溶解，室温下加入2mL的1NNaOH，1h后薄层检测，展开剂比例石油醚:乙酸乙酯＝1:1；反应完全，蒸干溶剂，滴入2mmL1N HCl，析出大量固体，抽滤，干燥，得白色固体M31 555mg，产率97.5％；
M31:1H NMR(600MHz,(CD3)2SO)δ:0.66,0.67,0.84,0.86,0.87,0.88,1.08(7×CH3),0.66—2.00(m,other aliphatic ring protons),2.17(t,J＝7.38Hz,2H),2.78(dd,J＝3.9,13.32Hz,1H),2.91—3.04(m,3H),5.20(brs,1H),7.22(t,J＝5.58Hz,2H),11.98(s,1H)13C NMR 100MHz,(CD3)2SO)δ:15.08,16.05,16.86,18.01,22.27,22.94,23.60,24.30,25.68,26.17,26.97,28.25,28.86,30.46,32.47,32.77,32.98,33.67(2C),36.59,38.08,38.39,38.66,38.91,40.05,40.47,41.26,45.20,46.06,47.13,54.83,76.84,121.35,144.16,174.40,176.06。
其余三萜与直链氨基衍生物的偶联物的制备方法同上，不同在于三萜种类的不同及直链氨基衍生物的不同；其余化合物结构式和部分化合物的核磁共振1H及13C化学位移值如下：
M1:1H NMR(400MHz,(CD3)2SO)δ:0.67,0.84,0.86,0.87,0.89,1.08(7×CH3),0.67—2.00(m,other aliphatic ring protons),2.75(d,J＝9.28Hz,1H),2.94—3.02(m,2H),3.12—3.40(m,1H),3.33—3.36(m,2H),4.27(d,J＝5.16Hz,1H),4.59(t,J＝5.32Hz,1H),5.21(brs,1H),7.15(t,J＝5.56Hz,1H)13C NMR 100MHz,(CD3)2SO)δ:15.08,16.01,16.72,17.97,22.34,22.91,23.51,25.64,26.93(3C),28.21,30.41,32.36,32.68,32.89,33.61,36.55,38.36,40.54,41.23,41.54(2C),45.24,46.02,47.09,54.78,59.84,76.80,121.48,144.00,176.50。
M5:1H NMR(600MHz,(CD3)2SO)δ:0.66,0.67,0.84,0.86,0.88,0.89,1.08(7×CH3),0.66—2.00(m,other aliphatic ring protons),2.78(dd,J＝3.96,13.38Hz,1H),2.92—3.04(m,3H),3.36(dd,J＝6.6,11.76Hz,2H),4.29(d,J＝5.22Hz,1H),4.33(t,J＝5.16Hz,1H),5.20(brs,1H),7.20(t,J＝5.52Hz,1H)13C NMR 100MHz,(CD3)2SO)δ:15.07,16.04,16.85,18.00,22.26,22.94,23.59,25.38,25.67,26.62,26.96,28.24,29.20,30.45,32.47,32.63,32.77,32.97,33.67,36.58,38.08,38.39,38.83,38.91,40.06,40.46,41.25,45.19,46.06,47.12,54.83,60.69,76.83,121.33,144.18,176.02。
M9:1H NMR(600MHz,(CD3)2SO)δ:0.65,0.66,0.83,0.85,0.87,0.88,1.07(7×CH3),0.65—2.00(m,other aliphatic ring protons),2.54—2.56(m,2H),2.77(d,J＝9.54Hz,1H),2.87—3.05(m,4H),5.20(t,J＝4.92Hz,1H),7.24(t,J＝5.64Hz,1H 13C NMR100MHz,(CD3)2SO)δ:15.15,16.10,16.90,18.06,22.30,22.99,23.64,23.92,25.72,27.00,28.30,28.96,30.49,31.21,32.53,32.82,33.02,33.72,36.63,38.13,38.44,38.84,38.96,40.06,40.50,40.80,41.30,45.25,46.11,47.17,54.88,76.89,121.40,144.20,176.18。
M13:1H NMR(400MHz,(CD3)2SO)δ:0.65,0.67,0.84,0.86,0.89,1.08(7×CH3),0.65—2.00(m,other aliphatic ring protons),2.18(t,J＝7.4Hz,2H),2.78(d,J＝9.68Hz,1H),2.96—3.03(m,3H),3.32(d,J＝8.92Hz,2H),4.28(d,J＝5.08Hz,1H),5.21(brs,1H),7.29(t,J＝5.44Hz,1H),12.0(s,1H)13C NMR 100MHz,(CD3)2SO)δ:15.16,16.10,16.92,18.08,22.27,22.99,23.67,24.53,25.76,27.04,28.31,30.52,31.29,32.53,32.87,33.03,33.71,36.65,38.23,38.43,38.46,40.50,41.31,45.29,45.30,46.09,47.19,54.89,76.79,76.90,121.48,144.17,174.36,176.24。
M17:1H NMR(600MHz,(CD3)2SO)δ:0.65,0.67,0.84,0.86,0.87,0.89,1.08(7×CH3),0.65—2.00(m,other aliphatic ring protons),2.40—2.43(m,2H),2.49—2.51(m,2H),2.74(dd,J＝3.84,13.38Hz,1H),2.97—3.00(m,1H),3.15—3.21(m,1H),3.25—3.31(m,1H),3.57(s,3H),,4.28(d,J＝5.16Hz,1H),5.19(brs,1H),7.35(t,J＝5.58Hz,1H)13CNMR(100MHz,(CD3)2SO)δ:15.10,16.03,16.77,17.99,22.26,22.92,23.51,25.65,26.86,26.96,28.23,30.42,32.40,32.61,32.92,33.57,33.61,35.02,36.57,38.08,38.38,38.89,40.43,41.22,45.21,46.01,47.10,51.26,54.81,76.82,121.48,143.93,171.93,176.46。
M21:1H NMR(400MHz,(CD3)2SO)δ:0.67,0.84,0.86,0.88,0.89,1.08(7×CH3),0.67—2.00(m,other aliphatic ring protons),2.76(d,J＝9.84Hz,1H),2.96—3.04(m,2H),3.07—3.16(m,1H),3.38(q,J＝5.44Hz,2H),4.27(d,J＝5.12Hz,1H),4.41(t,J＝5.24Hz,1H),5.21(brs,1H),7.22(t,J＝5.56Hz,1H)13C NMR 100MHz,(CD3)2SO)δ:15.06,15.99,16.79,17.97,22.27,22.90,23.51,25.63,26.93,28.20,30.40,32.25,32.40,32.77,32.89,33.63,36.32,36.55,38.05,38.35,40.06,40.46(2C),41.21,45.17,46.04,47.08,54.78,58.84,76.80,121.43,144.01,176.21。
M23:1H NMR(400MHz,(CD3)2SO)δ:0.69,0.84,0.86,0.88,0.89.1.08(7×CH3),0.67—2.00(m,other aliphatic ring protons),2.78(d,J＝10.2Hz,1H),2.93—3.03(m,3H),3.17(d,J＝5.36Hz,1H),3.31—3.38(m,3H),4.28(d,J＝3.96Hz,1H),4.33(t,J＝5.04Hz,1H),5.20(s,1H),7.19(t,J＝5.24Hz,1H)13C NMR 100MHz,(CD3)2SO)δ:15.07,16.01,16.82,18.01,22.26,22.92,23.10,23.56,25.66,26.95,28.23,29.01,30.42,32.25,32.30,32.47,32.75,32.95,33.67,36.57,38.08,38.37,40.46,41.24,45.16,46.05,47.12,54.83,60.58,60.70,76.70,76.82,121.34,144.12,176.00。
M24:1H NMR(400MHz,CDCl3)δ:0.76,0.78,0.92,0.94,0.98,1.18(7×CH3),0.76—2.00(m,other aliphatic ring protons),2.66(d,J＝11.4Hz,1H),2.81(t,J＝6Hz,2H),3.11—3.21(m,2H),3.36—3.43(m,2H),5.40(s,1H)13C NMR(100MHz,CDCl3)δ:15.00,15.31,16.67,18.07,23.15,23.18,23.27,25.57,26.48,27.07,27.73,30.42,32.26,32.66(2C),33.85,36.74,38.35,38.51,39.18,40.44,40.80,41.47,41.69,46.23,46.31,47.35,55.01,78.43,122.79,144.00,179.73。
M27:1H NMR(600MHz,(CD3)2SO)δ:0.65,0.66,0.83,0.85,0.87,0.88,1.07(7×CH3),0.65—2.00(m,other aliphatic ring protons),2.54—2.56(m,2H),2.77(d,J＝9.54Hz,1H),2.87—3.05(m,4H),5.20(t,J＝4.92Hz,1H),7.24(t,J＝5.64Hz,1H)13C NMR100MHz,(CD3)2SO)δ:15.15,16.10,16.90,18.06,22.30,22.99,23.64,23.92,25.72,27.00,28.30,28.96,30.49,31.21,32.53,32.82,33.02,33.72,36.63,38.13,38.44,38.84,38.96,40.06,40.50,40.80,41.30,45.25,46.11,47.17,54.88,76.89,121.40,144.20,176.18。
M28:1H NMR(600MHz,(CD3)2SO)δ:0.64,0.67,0.83,0.87,0.87,0.89,1.08(7×CH3),0.64—2.00(m,other aliphatic ring protons),2.76(dd,J＝3.84,13.38Hz,1H),2.97—3.00(m,1H),3.56(dd,J＝3.54,17.28Hz,1H),3.73(dd,J＝6.0,17.28Hz,1H),4.28(d,J＝4.44Hz,1H),5.19(t,J＝3.36Hz,1H),7.57(t,J＝5.58Hz,1H),12.37(s,1H)13C NMR100MHz,(CD3)2SO)δ:15.12,16.05,16.60,18.00,22.45,22.95,23.50,25.65,26.85,26.97,28.24,30.42,32.39,32.48,32.91,33.62,36.58,38.10,38.39,38.88,40.47,40.90,41.24,45.14,46.05,47.15,54.82,76.83,121.48,143.97,171.48,176.68。
M32:1H NMR(600MHz,(CD3)2SO)δ:0.64,0.67,0.84,0.87,0.88,0.89,1.08(7×CH3),0.64—2.00(m,other aliphatic ring protons),2.76(d,J＝9.6Hz,1H),2.97—3.00(m,1H),3.59(s,3H),3.64(dd,J＝5.52Hz,17.04Hz,1H),3.80(dd,J＝6Hz,16.98Hz,1H),4.29(d,J＝5.16Hz,1H),5.17(t,J＝3.36Hz,1H),7.76(t,J＝5.7Hz,1H)13C NMR(150MHz,(CD3)2SO)δ:15.13,16.05,16.60,18.02,22.40,22.94,23.50,25.64,26.84,26.97,28.24,30.42,32.42,32.50,32.90,33.61,36.59,38.09,38.40,38.86,40.35,40.93,41.24,45.19,46.05,47.14,51.51,54.83,76.83,121.42,143.95,170.54,176.94。
M34:1H NMR(400MHz,(CD3)2SO)δ:0.65,0.67,0.84,0.86,0.88,0.89,1.08(7×CH3),0.65-2.00(m,other aliphatic ring protons),2.27(t,J＝7.24Hz,2H),2.76(d,J＝9.84Hz,1H),2.93—3.05(m,3H),3.57(s,3H),4.26(d,J＝5.16Hz,1H),5.20(s,1H),7.24(t,J＝5.52Hz,1H)13C NMR 100MHz,(CD3)2SO)δ:15.01,15.99,16.79,17.97,22.01,22.22,22.87,23.55,25.64,26.94,28.21,28.50,30.42,32.43,32.77,32.93,33.02(2C),33.63,36.55,38.06,38.31,38.35,38.88,40.44,41.22,45.19,46.02,47.10,51.11,54.79,76.80,121.36,144.08,173.21,176.08。
实施例2：本发明化合物抑制流感病毒进入细胞的生物活性评价方法
1、细胞病变(CPE)抑制试验
流感病毒感染细胞后会导致细胞病变，使得细胞活力降低；如果药物能够抑制流感病毒复制，则会降低细胞病变数量，提高细胞活力；具体来说：
(1)将犬肾上皮细胞(MDCK)以1:3的比例传代到白色的96孔板中，在37℃细胞培养箱中用含10％FBS的DMEM培养基培养24h；
(2)将流感病毒[A/WSN/33(H1N1)，感染复数(MOI)＝1]与100μM/L的待检化合物加入到100μl含有2μg/mL TPCK处理的胰酶，1％FBS的DMEM中，充分混匀；化合物的阴性对照为1％DMSO(稀释化合物所用的溶剂)；同时设立一组只加各化合物不加病毒实验组，用来检测化合物对细胞活力的影响；
(3)将96孔板中的MDCK细胞的培养基吸出，将混合有病毒和化合物的培养基加入到MDCK细胞中，37℃细胞培养箱中培养48h，每个样品三个复孔；
(4)用CellTiter-Glo荧光细胞活性检测试剂盒(Cat.G7571，Promega)检测细胞活力，首先将细胞和CellTiter-Glo检测试剂放于室温环境，待其温度平衡至室温，将100μl/孔的CellTiter-Glo检测试剂加入到细胞的培养上清中，震动2min后，避光静置10min，用仪器TecanInfinite M2000PROTM检测细胞活力；
(5)EC50的计算方法：首先对化合物进行浓度系列稀释，然后利用上述方法测定出细胞活力；化合物对细胞病变的保护率＝100×(1-(Test compound–MedianVirus1)/(Median Cells-Median Virus2)).其中Test compound表示只加待检化合物不加病毒组的细胞活力；Median Virus1表示加了待检化合物和病毒组的细胞活力；Median Cells表示只加入1％DMSO组的细胞活力；Median Virus2表示加入1％DMSO和病毒组的细胞活力。将化合物浓度和相应的保护率输入到软件Prism，即可计算EC50；此方法已被广泛应用于抗病毒药物筛选领域；
(6)CC50的计算方法:CellTiter-Glo也可以用来检测化合物对细胞的毒性；首先对化合物进行浓度系列稀释，然后将其加入到细胞中，方法同(2)-(4)，但不加入病毒，培养48h后，测定细胞活力；然后将对照组细胞活力(1％DMSO)定义为100％，将其他各化合物组细胞活力标准化，除以对照组1％DMSO的细胞活力，再乘以100％；将化合物的浓度和相应的标准化的细胞活力输入到软件Prism，即可计算出CC50；
实验结果显示：与齐墩果酸相比，上述化合物均表现出抗流感病毒活性，其中合物M17有非常好的抑制流感病毒活性，可以显著削弱病毒的感染性；M6、M13、M24、M25和M35虽然在抗流感病毒活性没有M17明显，但与OA相比，细胞毒性有了显著的降低；其它化合物毒性都非常弱。(见表1、2)
表1在50μM浓度下，各化合物对MDCK细胞的毒性检测
| 化合物 | OA | M1 | M2 | M3 | M4 | M5 | M6 |
| 细胞活力(％) | 73.9 | 101.2 | 85.3 | 107.3 | 98.4 | 102 | 87.4 |
| 化合物 | M7 | M8 | M9 | M10 | M11 | M12 | M13 |
| 细胞活力(％) | 104.1 | 101.5 | 87 | 97.7 | 98.6 | 93.3 | 109.8 |
| 化合物 | M14 | M15 | M16 | M17 | M18 | M19 | M20 |
| 细胞活力(％) | 99 | 95.5 | 86.4 | 100.3 | 87.6 | 71.6 | 71.7 |
| 化合物 | M21 | M22 | M23 | M24 | M25 | M26 | M27 |
| 细胞活力(％) | 100.5 | 96 | 90.7 | 96.4 | 94.9 | 87.5 | 73.4 |
| 化合物 | M28 | M29 | M30 | M31 | M32 | M33 | M34 |
| 细胞活力(％) | 92.8 | 86.5 | 104 | 72.2 | 77.5 | 80.4 | 109.5 |
| 化合物 | M35 | DMSO | |||||
| 细胞活力(％) | 82.8 | 100 |
表2在50μM浓度下，各化合物抗流感病毒活性，检测方法同表2
| 化合物 | OA | M1 | M2 | M3 | M4 | M5 | M6 |
| 病毒感染力(％) | 74.1 | 65.7 | 57.7 | 55.2 | 66.1 | 67.8 | 31.9 |
| 化合物 | M7 | M8 | M9 | M10 | M11 | M12 | M13 |
| 病毒感染力(％) | 56.8 | 70.4 | 64.3 | 57.9 | 71.4 | 68.3 | 38.3 |
| 化合物 | M14 | M15 | M16 | M17 | M18 | M19 | M20 |
| 病毒感染力(％) | 64 | 61.4 | 58.3 | 19.6 | 59.9 | 61 | 66.7 |
| 化合物 | M21 | M22 | M23 | M24 | M25 | M26 | M27 |
| 病毒感染力(％) | 70.8 | 73.6 | 71.3 | 54.3 | 50.2 | 71.2 | 57.1 |
| 化合物 | M28 | M29 | M30 | M31 | M32 | M33 | M34 |
| 病毒感染力(％) | 61.3 | 68.3 | 70.9 | 72 | 71.2 | 70.4 | 60.8 |
| 化合物 | M35 | DMSO | |||||
| 病毒感染力(％) | 45.6 | 99.9 |
通过CPE抑制试验化合物M17对流感病毒有着明显的抑制作用，强于阳性药物利巴韦林；CPE抑制试验表明M17的对流感病毒的EC50为37.4μM，而阳性药物达菲(磷酸奥司他韦，OSV-P)的EC50为42.2μM，利巴韦林(RBV)的EC50为53.6μM(见表3)；
表3 M17抑制流感病毒(WSN)的活性及其细胞毒性分析
2、加药时间点实验
用以分析化合物作用于病毒感染细胞的哪一阶段，具体步骤如下：
(1)将MDCK细胞传代到六孔板中，在37℃细胞培养箱中用含10％FBS的DMEM培养基培养24h；
(2)将A/WSN/33(H1N1)病毒(MOI＝1)稀释到不含血清的DMEM中，感染MDCK细胞；
(3)流感病毒从吸附到子代病毒粒子释放，其复制周期约为6-8h；故在以下时间段将药物加入到细胞培养基中：0–10，0–2，2–5，5–8或8–10h；
(4)感染10h后，用冰预冷的PBS清洗细胞一次，用200μl/孔的PIPA裂解液裂解细胞；用细胞刮将细胞刮下，吸入1.5mL EP管中，置于冰上15min；以12，000rpm 4℃离心10min，将上清液转移到另一个1.5mL EP管中；
(5)吸取30μl样品与等体积的2×蛋白上样缓冲液混合，100℃煮样10min；
(6)将煮好的样品各20μl加入到12％的蛋白质凝胶加样孔中，进行SDS-PAGE电泳；
(7)用免疫印迹法(Western blotting)检测流感病毒的NP蛋白的表达水平(以此来检测病毒在细胞内的复制情况)；同时以细胞蛋白GAPDH作为细胞内参(也可用于验证药物对细胞的毒性)；
通过上述加药时间点实验可以初步断定，M17作用于病毒进入细胞过程，且干扰了病毒与细胞受体之间的结合；(见表4和图1)
表4加药时间点实验表明M17作用于病毒复制的早期(0-2h)
3、血凝抑制试验
此方法用来检测药物是否影响病毒与细胞受体之间的结合，具体方法如下：
(1)制备1％(v/v)的鸡红细胞悬液
选1-2只健康鸡，将血液采集到等量抗凝液中混匀，置4℃冰箱保存，以800-1000rpm离心5分钟，用吸管吸去上清液和红细胞上层的白细胞薄膜，将沉淀的红细胞加生理盐水，慢慢混合均匀，再在离心机中800rpm离心5分钟，弃去上清液，再加生理盐水混匀，如此反复离心4-5次，最后一次离心后的红细胞，弃去上清液。放入4℃冰箱中可保存2-3天；使用时用1mL吸管吸取0.1mL红细胞，然后加入9.9mL的生理盐水，此即1％红细胞悬液；
(2)确定病毒的血凝效价，将WSN流感病毒以2倍梯度做倍比稀释，稀释液为PBS；
(3)将病毒液与1％红细胞悬液等体积(各50μl)混合加入到V底的96孔板中，置于微量振荡器上振荡1min，室温静置孵育30min；
(4)将反应板倾斜成45°，沉于孔底的红细胞沿着倾斜面向下呈线状流动者为沉淀，表明红细胞未被或不完全被病毒凝集；如果孔底的红细胞铺平孔底，凝成均匀薄层，表明红细胞被病毒所凝集。在确定流感病毒的血凝效价后，确定合适的病毒使用量；
(5)将药物、DMSO(阴性对照)或抗HA的特异性单克隆抗体(阳性对照)与病毒液混合后加入到细胞混悬液中，观察化合物对红细胞凝集有没有抑制效果。
通过上述血凝素实验可以初步断定，M17作用于病毒进入细胞过程，且干扰了病毒与细胞受体之间的结合(见图2)。
Claims (3)
1.结构式如下式所示的三萜与直链氨基衍生物的偶联物：
其中，是单键或双键；
X和Y结合起来形成一个带有1-5个相同或不同取代基的五元环、六元环或七元环，其中所述取代基各自独立选自H，未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基，未取代的C1-C6烷氧基或被羟基、氨基或羧基取代的C1-C6烷氧基，卤素，羧基，羟基，硝基，氰基，巯基，C1-C6硫烷基或NHR9’，所述R9’是H，未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基；
R1选自-NH-(-CH2-)n-OH、-NH-(-CH2-)n-NH2、-NH-(-CH2-)n-COOH、-NH-(-CH2-)n-COOCH3，n＝1-12；
R2和R7各自独立选自H，卤素，羟基，氰基，硝基，巯基，C1-C6硫烷基，未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基，氨基，NR11’R12’，所述R11’和R12’各自独立选自未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基；
R3、R4、R5、R6和R8各自独立选自H，未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基；
R9选自H，卤素，羟基，氰基，硝基，巯基，C1-C6硫烷基，羰基，肟基，未取代的C1-C6烷基或被羟基、氨基或羧基取代的C1-C6烷基。
2.根据权利要求2所述的三萜与直链氨基衍生物的偶联物，其特征在于，三萜与直链氨基衍生物的偶联物具体结构见下表：
3.权利要求1-3中任一项所述的三萜与直链氨基衍生物的偶联物在制备治疗或/或预防流感药物中的应用。
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