CN111961110A - 一种齐墩果酸c-3位糖偶联物其制备方法及抗流感病毒应用 - Google Patents

一种齐墩果酸c-3位糖偶联物其制备方法及抗流感病毒应用 Download PDF

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CN111961110A
CN111961110A CN202010981520.0A CN202010981520A CN111961110A CN 111961110 A CN111961110 A CN 111961110A CN 202010981520 A CN202010981520 A CN 202010981520A CN 111961110 A CN111961110 A CN 111961110A
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oleanolic acid
sugar conjugate
influenza virus
influenza
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CN111961110B (zh
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俞飞
邵亮
张继虹
杨帆
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Kunming University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
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Abstract

本发明提供了式(Ⅰ)结构的齐墩果酸‑C3位糖偶联物或其药学上可接受的盐及其制备方法：
Figure DDA0002687684350000011
本发明提供的化合物对流感病毒具有明显的抑制作用，可用于制备预防或治疗流感的药物；及阻止或抑制流感病毒进入细胞的药物。

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一种齐墩果酸C-3位糖偶联物其制备方法及抗流感病毒应用
技术领域
本发明涉及一类齐墩果酸-C3位糖偶联物、其制备方法，以及其在预防或治疗流感病毒中的应用，属于药物化学领域。
背景技术
流行性感冒简称为流感，是由流感病毒(Influenza virus,IV)引起的一种急性上呼吸道传染性疾病，具有传播速度快、传染性强、致病性强等特点，对人类的生命及健康构成了威胁(The pathology of influenza virus infections.Annual review ofpathology 2008；3；499-522)。在二十世纪，流感病毒主要引起了三次大型流行，即1918年的西班牙流感，1957年的亚洲流感以及1968年的香港流感，共造成约5000万人死亡(Influenza pandemic of the 20th centry. Emerging Infectious Diseases.2006；12；9-14)。甲型流感病毒根据病毒表面两种蛋白，神经氨酸酶(Neuraminidase)和血凝素(Hemagglutinin)抗原性的不同，又可分为多种亚型，例如H5N1、 H3N2等。目前已发现能在人类中流行的流感病毒和各种禽流感病毒都属于甲型流感病毒。在二十一世纪，2009年的流感也是由甲型H1N1流感病毒引起的(Origins and evolutionary genomics of theswine-orgin H1N1 influenza A epidemic.Nature.2009；459；1122-1125)，其传播之迅速，引起了世界的关注。据世界卫生组织估计，在非流感流行期，平均每年全世界仍有 5％-15％的人被感染，因流感导致的死亡人数在25-50万人之间(Resurrected pandemicinfluenza viruses.Annual Review Microbiology 2009；63；79-98)。
目前，经FDA批准临床使用的抗流感药物主要有两类。第一类药物是针对甲型流感病毒膜蛋白上的M2离子通道的抑制剂，主要包括金刚烷胺(Amantadine)和金刚乙胺(Rimantadine)。第二类药物主要通过抑制流感病毒的NA蛋白发挥抗病毒作用，包括奥司他韦(Oseltamivir)和扎那米韦(Zanamivir)(Anti-Influenza treatment:durg currentlyused and under development.Archivos de Bronconeumologia 2017；53；19-26)。另外，还有广谱抗病毒药物利巴韦林(Ribavirin)和帽依赖型内切核酸酶抑制剂巴罗沙韦(Baloxavir Marboxil)。利巴韦林可抑制甲型和乙型流感病毒的RNA聚合酶，干扰病毒合成。巴罗沙韦抑制病毒合成mRNA，进而阻断病毒的复制(Investigational antiviraltherapies for the treatment of influenza.Expert Opinion on InvestigationalDrugs.2009；28；481-488)。而随着抗流感病毒治疗药物的广泛应用，病毒耐药变异成为困扰临床应用的重大难题，因此开发新型抗流感病毒药物刻不容缓。
五环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，如白桦脂酸对HIV、A型流感病毒(H1N1)、单纯疱疹病毒(HSV)、呼吸道合胞体病毒(RSV)及柯萨奇病毒(CVB)等均具有不同程度的抑制作用(印楝对帕金森病大鼠模型神经保护作用的实验研究.项鑫)；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，且其衍生物对流感病毒有明显的抑制作用(Design,synthesis of oleanolic acid-saccharide conjugates using click chemistry methodology and study of theiranti-influenza activity.European Journal of Medicinal Chemistry.2019；182；111622)。
发明内容
本发明的一个目的是提供一种齐墩果酸-C3位糖偶联物及其药学上可接受的盐或它们的水合物，及其用于预防或治疗流感的用途。
本发明提供了一类齐墩果酸-C3位糖类衍生物，其化学结构式如下式所示：
Figure RE-GDA0002714461040000021
R1为H或苄基，
R2选自单糖、二糖或三糖及其衍生物，其通过1位与三氮唑连接。
本发明的另一个目的是提供如下具体结构的齐墩果酸-C3位糖偶联物，具体参见以下表1：
表1
Figure RE-GDA0002714461040000022
Figure RE-GDA0002714461040000031
Figure RE-GDA0002714461040000041
本发明化合物的制备路线包括但不仅限于以下路线：
Figure RE-GDA0002714461040000042
反应条件：(a)乙酸酐，4-二甲氨基吡啶，吡啶，室温反应过夜；(b)氢溴酸，二氯甲烷， 0℃至室温，反应过夜；(c)叠氮化钠，N,N-二甲基甲酰胺，室温反应过夜；(d)溴化苄，碳酸钠，N,N-二甲基甲酰胺，50℃反应6h；(e)3-溴丙炔，氢化钠，干燥四氢呋喃，室温反应6h；(f)五水硫酸铜，L-抗坏血酸钠，二氯甲烷/水，室温反应过夜；甲醇钠，甲醇，室温反应3h；钯碳，氢气，四氢呋喃，48h。
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、含化、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。
本发明的化合物中三氮唑模块具有大偶极矩、易与氢键供体形成氢键，且在生理条件下非常稳定，三氮唑模块同时作为一个作用显著的药效团，可以增加本发明化合物的抗病毒活性。
附图说明
图1显示齐墩果酸-C3位糖偶联物具有显著抗流感病毒活性，对流感病毒抑制率显著优于齐墩果酸。
图2显示化合物O5具有显著抗流感病毒活性，IC50＝12.45μM。
图3显示化合物O5能有效抑制流感病毒HA引起的血凝
图4显示部分化合物IC50、CC50及SI值。
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。
实施例1：本发明中齐墩果酸-C3位糖偶联物的制备方法，以制备化合物O1-O3为例，合成路线如下：
Figure RE-GDA0002714461040000071
O1-O3化合物的具体制备工艺如下：
(a)取10mmol葡萄糖(1800mg)于100mL反应瓶中，加入吡啶20mL溶解，此时将反应瓶置于冰浴中，加入催化剂DMAP，随后用常压滴液漏斗缓慢滴加溶于10mL的吡啶的100mmol乙酸酐(9.5mL)，滴毕后移至室温反应，12h后薄层检测，展开剂石油醚:乙酸乙酯＝1:1，CMC显色剂显色。蒸干溶剂后用水萃取，将有机相蒸干，得到2958mg化合物1，产率为87％；粗产物可直接用于后续反应，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；
(b)取上述化合物1于100mL反应瓶中，加入30mL二氯甲烷溶解，置于冰浴中，塞上胶皮塞子，用5mL针管吸取1mL的HBr溶液(约为3倍量)并将其缓慢注射加入至反应瓶中，之后将反应瓶放置于室温下搅拌反应至TLC检测反应完全。后处理加水摇晃使水相与有机相分层，投入无水碳酸钠反应掉过量氢溴酸后，再用水萃取一遍，蒸干有机相，得到2250 mg淡黄色油状液体化合物2，待用；
(c)将上一步反应得到的粗产物化合物2(约为3mmol)放入反应瓶中，加入20mLDMF 溶解，再加入254mg NaN3(3.9mmol)后室温下搅拌反应至TLC检测反应完全。蒸干溶剂后用水和二氯甲烷萃取，除去过量NaN3，有机相浓缩、硅胶柱层析分离(石油醚/乙酸乙酯，2/1 v/v)得到496mg白色固体产物化合物3，产率55％。
(d)将4560mg齐墩果酸(10mmol)溶于30mL DMF中，加入1079mg Na2CO3(13 mmol)，搅拌下再加入1.552mL的溴化苄(13mmol),混悬液置于50℃油浴下搅拌反应至TLC 检测反应完全。旋干溶剂用乙酸乙酯和水萃取后，滤液浓缩、硅胶柱层析分离(石油醚/乙酸乙酯，4/1v/v)得到4368mg白色固体产物化合物4，产率80％。
(e)将56mg NaH(2.3mmol)溶于20mL dry THF中，室温搅拌2h后，加入546mg化合物4(1mmol)，继续搅拌2h，再加入0.481mL的3-溴丙炔(1.5mmol),室温搅拌反应至TLC 检测反应完全。旋干溶剂用乙酸乙酯和水萃取后，滤液浓缩、硅胶柱层析分离(石油醚/乙酸乙酯，6/1v/v)得到176mg白色固体产物化合物5，产率30％。
(f)取1168mg的化合物5(1.2mmol)和360mg的化合物3(1mmol)溶于30mL的二氯甲烷：水＝1:1中，在搅拌下加入500mg五水硫酸铜(2mmol)和396mg抗坏血酸钠(2mmol)。室温搅拌反应至TLC检测反应完全。用水和二氯甲烷萃取，滤液浓缩、硅胶柱层析(石油醚/ 乙酸乙酯，6/1v/v、4/1v/v、2/1v/v)。得到450mg白色固体产物，产率51％。得到化合物 O1。
O1:1H NMR(600MHz,CDCl3):δ0.58,0.78,0.87,0.88,0.88,0.90,1.10(7×CH3),0.58-1.84(m),1.86(s,3H),2.02(s,3H),2.06(s,3H),2.07(s,3H),2.89(dd,J＝4.26,14.10Hz, 1H),2.96(dd,J＝4.20,11.70Hz,1H),3.97-4.00(m,1H),4.14(dd,J＝2.22,12.78Hz,1H), 4.27-4.30(m,1H),4.56(d,J＝12.66Hz,1H),4.76(d,J＝12.60Hz,1H),5.03(d,J＝12.54Hz, 1H),5.08(d,J＝12.54Hz,1H),5.23-5.29(m,1H),5.27(t,J＝3.48Hz,1H),5.40(t,J＝9.36Hz, 1H),5.47(t,J＝9.48Hz,1H),5.87(d,J＝9.42Hz,1H),7.33(s,5H),7.74(s,1H).13C NMR(150 MHz,CDCl3):δ15.42,16.57,16.97,18.34,20.32,20.66,20.68,20.83,22.69,23.14,23.53,23.76, 25.98,27.69,28.17,30.82,32.47,32.79,33.23,33.95,37.04,38.34,38.79,39.40,41.46,41.76, 45.94,46.84,47.66,55.67,61.66,62.96,66.04,67.78,70.22,72.89,75.14,85.72,86.72,120.67, 122.62,128.02,128.09(2C),128.53(2C),136.53,143.80,147.35,168.99,169.50,170.11,170.67,177.61.
(g)将150mg O1置于50mL反应瓶中，用20mL甲醇溶解，再加入80mg甲醇钠，室温下搅拌4h，薄层色谱检测，展开剂为石油醚:乙酸乙酯＝1:3；蒸干溶剂后向反应瓶中滴加1NHCl 10mL，抽滤，取滤纸上的固体进行干燥，得白色粉末O2 120mg，产率78％；
O2:1H NMR(600MHz,CD3OD):δ0.58,0.77,0.91,0.91,0.93,1.14(6×CH3),0.58-2.10 (m),2.90(dd,J＝4.32,13.86Hz,1H),2.98(dd,J＝4.26,11.70Hz,1H),3.50(t,J＝9.60Hz,1H), 3.54-3.59(m,2H),3.71(dd,J＝5.34,12.24Hz,1H),3.87-3.91(m,1H),4.51(d,J＝12.18Hz,1H), 4.73(d,J＝12.24Hz,1H),5.06(s,2H),5.24(t,J＝3.42Hz,1H),5.60(d,J＝9.18Hz,1H), 7.30-7.35(m,5H),8.16(s,1H).13C NMR(150MHz,CD3OD):δ16.08,17.12,17.86,19.49,23.73, 24.10,24.21,24.67,26.53,28.82,28.85,31.75,33.67,33.78,34.06,34.94,38.24,39.65,39.89, 40.74,42.97(2C),47.22,48.20,49.10,57.15,62.48,63.48,67.37,70.96,74.14,78.67,81.29,88.03, 89.72,124.09,124.37,129.33,129.51(2C),129.65(2C),137.81,145.04,146.97,179.20.
(h)将120mg化合物O2置于50mL反应瓶中，用20mL THF溶解，再加入少量Pd\C，用胶皮塞子塞住瓶口后，用水泵抽干空气，随后用气球吸满H2注入反应瓶中，随后每隔7 个小时补充H2，反应2天后用薄层色谱检测，展开剂为二氯甲烷:甲醇＝10:1；为除去催化剂 Pd\C，利用硅藻土的吸附能力将反应液用漏斗抽滤使其吸附在硅藻土上，将过滤后的无色反应液后蒸干留样，硅胶柱层析分离(二氯甲烷/甲醇，40/1v/v、30/1v/v、20/1v/v)，得到80mg 白色固体产物O3，产率75％。
其余齐墩果酸-C3位糖偶联物化合物制备方法同上，不同在于步骤a中糖类的不同；其余部分化合物的核磁共振1H及13C化学位移值如下：
O5：1H NMR(600MHz,CDCl3):δ0.60,0.79,0.89,0.70,0.90,0.92,1.12(7×CH3),0.59-1.80(m),1.88(s,3H),2.01(s,3H),2.05(s,3H),2.23(s,3H),2.90(dd,J＝4.08,13.80Hz, 1H),2.99(dd,J＝4.26,11.76Hz,1H),4.13-4.16(m,1H),4.18-4.24(m,2H),4.57(d,J＝12.60Hz, 1H),4.77(d,J＝12.60Hz,1H),5.04(d,J＝12.54Hz,1H),5.09(d,J＝12.54Hz,1H),5.24(dd,J ＝3.36,10.26Hz,1H),5.29(t,J＝3.72Hz,1H),5.54(d,J＝2.7Hz,1H),5.61(t,J＝9.96Hz,1H), 5.84(d,J＝9.42Hz,1H),7.34(s,5H),7.78(s,1H).13CNMR(150MHz,CDCl3):δ14.25,15.42, 16.56,16.96,18.34,20.40,20.62,20.78,20.82,22.71,23.12,23.52,23.75,25.97,27.68,28.14, 30.80,32.46,32.78,33.22,33.94,37.04,38.35,38.79,39.39,41.45,41.75,45.93,46.82,47.65, 55.66,61.29,62.94,66.02,66.97,67.76,71.03,86.27,86.68,120.83,122.61,128.01(2C), 128.08(2C),128.51,136.51,143.78,147.13,169.09,169.97,170.15,170.49,177.59
O6：1H NMR(600MHz,CD3OD):δ0.58,0.78,0.91,0.91,0.93,0.94,1.14(7×CH3),0.58-2.04(m),2.90(dd,J＝4.26,13.80Hz,1H),2.99(dd,J＝4.26,11.70Hz,1H),3.69(dd,J ＝3.24,9.54Hz,1H),3.73(dd,J＝5.16,11.58Hz,1H),3.77(dd,J＝6.84,11.58Hz,1H),3.84(t,J＝ 6.42Hz,1H),3.98(d,J＝2.64Hz,1H),4.13(t,J＝9.36Hz,1H),4.52(d,J＝12.24Hz,1H),4.74 (d,J＝12.24Hz,1H),5.06(s,2H),5.24(t,J＝3.36Hz,1H),5.57(d,J＝9.18Hz,1H),7.31-7.35(m, 5H),8.22(s,1H).13C NMR(150MHz,CD3OD):δ15.95,16.99,17.71,19.35,23.57,23.97,24.06, 24.52,26.41,28.67,28.72,31.60,33.54,33.62,33.91,34.79,38.09,39.48,39.74,40.58,42.81(2C), 47.06,48.03,48.93,56.98,62.37,63.42,67.21,70.36,71.48,75.28,79.97,87.94,90.26,123.65, 123.92,129.17,129.33(2C),129.50(2C),137.65,144.87,147.00,178.99.
O9：1H NMR(600MHz,CDCl3):δ0.51,0.69,0.77,0.80,0.82,0.84,1.03(7×CH3),0.58-1.84(m),1.91(s,3H),1.96(s,3H),2.01(s,3H),2.01(s,3H),2.02(s,3H),2.82(dd,J＝3.90, 13.86Hz,1H),2.88(dd,J＝4.20,11.70Hz,1H),3.93-3.96(m,1H),4.20(dd,J＝2.16,12.48Hz, 2H),4.29-4.32(m,1H),4.54(d,J＝12.60Hz,1H),4.77(d,J＝12.60Hz,1H),5.03(d,J＝12.54Hz, 1H),5.08(d,J＝12.60Hz,1H),5.28(brs,2H),5.34(t,J＝10.02Hz,1H),5.69(s,1H),6.15(s, 1H),7.33(s,5H),7.74(s,1H).13C NMR(150MHz,CDCl3):δ14.32,15.43,16.61,16.98,18.34, 20.63,20.74,20.81,20.87,22.64,23.14,23.53,23.77,25.99,27.70,28.24,30.82,32.48,32.75, 33.23,33.95,37.05,38.30,38.72,39.39,41.47,41.77,45.94,46.85,47.64,55.62,62.39,63.06, 65.07,66.04,69.03,70.96,84.77,86.62,121.28,122.59,128.02,128.09(2C),128.53(2C),136.54,143.82,146.89,169.19,169.76,169.95,170.71,177.63
O10：1H NMR(600MHz,CD3OD):δ0.58,0.77,0.91,0.91,0.93,1.14(6×CH3), 0.58-2.08(m),2.90(dd,J＝4.14,13.74Hz,1H),2.98(dd,J＝4.26,11.70Hz,1H),3.52-3.55(m,1H),3.71-3.76(m,2H),3.78(dd,J＝5.88,12.12Hz,1H),3.78(dd,J＝2.10,12.18Hz,1H),4.10 (s,1H),4.53(t,J＝12.00Hz,1H),4.72(d,J＝12.18Hz,1H),5.06(s,2H),5.24(t,J＝3.30Hz,1H), 6.01(s,1H),7.31-7.35(m,5H),8.22(s,1H).13C NMR(150MHz,CD3OD):δ16.06,17.11,17.84, 19.49,23.74,24.09,24.21,24.67,26.52,28.83,31.75,33.65,33.78,34.06,34.95,38.24,39.64, 39.90,40.74,42.98(2C),47.22,48.21,49.10,57.16,62.75,63.54,67.38,67.90,72.46,75.13,81.73, 87.84,87.94,88.48,124.09,124.84,129.33,129.52(2C),129.65(2C),137.82,145.04,146.29, 179.22.
O13：1H NMR(600MHz,CDCl3):δ0.59,0.78,0.88,0.89,0.92,1.11(6×CH3),0.59-1.80 (m),1.87(s,3H),2.01(s,3H),2.20(s,3H),2.91(dd,J＝3.06,13.80Hz,1H),2.97(dd,J＝4.02, 11.58Hz,1H),3.99(d,J＝12.90Hz,1H),4.15(d,J＝11.76Hz,1H),4.56(d,J＝12.66Hz,1H), 4.74(d,J＝12.72Hz,1H),5.01(d,J＝12.54Hz,1H),5.09(d,J＝12.60Hz,1H),5.29(s,2H),5.43 (t,J＝1.38Hz,1H),5.64(t,J＝9.84Hz,1H),5.83(d,J＝9.18Hz,1H),7.33(s,5H),7.81(s, 1H).13C NMR(150MHz,CDCl3):δ15.10,16.26,16.62,18.01,20.05,20.35,20.71,22.33,22.80, 23.18,23.44,25.65,27.34,27.80,30.45,32.12,32.46,32.91,33.61,36.67,38.00,38.41,39.05, 41.12,41.39,45.58,46.43,47.31,55.29,62.39,65.64,66.95,67.63,67.91,70.41,85.98,86.17, 120.55,122.28,127.67,127.75(2C),128.17(2C),136.19,143.39,146.57,168.69,169.57,169.87, 177.07
O14：1H NMR(600MHz,CD3OD):δ0.58,0.77,0.91,0.91,0.93,1.14(6×CH3),0.58-2.11 (m),2.90(dd,J＝4.14,13.80Hz,1H),2.98(dd,J＝4.20,11.64Hz,1H),3.70(dd,J＝3.30,9.42 Hz,1H),3.85(d,J＝12.60Hz,1H),3.95(s,1H),4.02(dd,J＝1.92,12.66Hz,1H),4.14(t,J＝ 9.30Hz,1H),4.51(d,J＝12.24Hz,1H),4.73(d,J＝12.24Hz,1H),5.06(s,2H),5.24(t,J＝3.30 Hz,1H),5.48(d,J＝9.12Hz,1H),7.30-7.35(m,5H),8.18(s,1H).13CNMR(150MHz,CD3OD): δ15.95,16.99,17.71,19.35,23.57,23.97,24.06,24.52,26.42,28.67,28.70,31.60,33.54,33.62, 33.90,34.79,38.08,39.49,39.72,40.58,42.81(2C),47.06,48.03,48.93,56.98,63.35,67.20,70.24, 70.74,71.46,74.83,87.75,90.49(2C),123.56,123.92,129.17,129.33(2C),129.50(2C),137.65, 144.87,178.99.
O17：1H NMR(600MHz,CDCl3):δ0.51,0.68,0.78,0.80,0.81,0.83,1.03(7×CH3),0.51-1.80(m),1.27(d,J＝6.18Hz,3H),1.91(s,3H),2.01(s,3H),2.01(s,3H),2.81(dd,J＝4.26, 13.98Hz,1H),2.97(dd,J＝4.26,11.70Hz,1H),3.73-3.77(m,1H),4.44(d,J＝12.54Hz,1H), 4.69(d,J＝12.48Hz,1H),4.95(d,J＝12.54Hz,1H),5.01(d,J＝12.54Hz,1H),5.11(t,J＝9.54 Hz,1H),5.16(dd,J＝3.12,10.20Hz,1H),5.20(t,J＝3.66Hz,1H),5.60(dd,J＝1.32,3.06Hz, 1H),6.05(d,J＝1.32Hz,1H),7.25(s,5H),7.65(s,1H).13C NMR(150MHz,CDCl3):δ14.21, 16.55,16.92,17.57,18.29,20.61,20.70,20.83,22.76,23.09,23.47,23.71,25.93,27.64,28.23, 29.39,31.99,32.42,33.17,33.90,36.98,38.26,38.75,39.34,41.41,41.71,45.88,46.78,47.61, 55.62,63.19,65.97,69.30,69.65,70.87,74.00,84.71,86.70,121.27,122.56,127.96,128.03(2C), 128.46(2C),136.48,143.72,146.61,169.21,169.89,169.96,177.52
O18：1H NMR(600MHz,CD3OD):δ0.58,0.77,0.91,0.92,0.93,1.14(6×CH3), 0.51-2.11(m),1.38(d,J＝6.00Hz,3H),2.90(dd,J＝4.26,13.80Hz,1H),2.98(dd,J＝4.32,11.70Hz,1H),3.50(t,J＝9.36Hz,1H),3.54-3.58(m,1H),3.69(dd,J＝3.18,9.30Hz,1H),4.10 (dd,J＝1.20,3.12Hz,1H),4.52(d,J＝12.42Hz,1H),4.74(t,J＝12.42Hz,1H),5.06(s,2H),5.24 (t,J＝3.42Hz,1H),6.00(d,J＝1.14,3.06Hz,1H),7.30-7.35(m,5H),8.18(s,1H).13C NMR (150MHz,CD3OD):δ16.12,17.15,17.88,18.26,19.50,23.72,24.15,24.22,24.67,26.61,28.83, 28.86,31.75,33.71,33.76,34.05,34.94,38.22,39.62,39.87,40.72,42.95(2C),47.22,48.16,49.06, 57.11,63.24,67.34,72.46,73.19,74.80,77.16,88.12,88.58,124.04,124.92,129.32,129.46(2C), 129.65(2C),137.79,145.02,145.88,179.09.
O21：1H NMR(600MHz,CDCl3):δ0.52,0.70,0.81,0.83,0.85,1.10(6×CH3),0.52-1.80 (m),1.96(s,3H),1.97(s,3H),2.00(s,3H),2.83(dd,J＝3.96,13.86Hz,1H),2,.90(dd,J＝4.20, 11.64Hz,1H),3.57(t,J＝11.28Hz,1H),4.02-4.06(m,3H),4.48(d,J＝12.78Hz,1H),4.68(d,J ＝12.78Hz,1H),4.97(d,J＝12.48Hz,1H),5.02(d,J＝12.54Hz,1H),5.21(t,J＝3.42Hz,1H), 5.36-5.41(m,1H),5.78(d,J＝8.82Hz,1H),7.27(s,5H),7.72(s,1H).13C NMR(150MHz, CDCl3):δ14.04,15.14,16.29,16.68,20.00,20.45,20.83,22.36,22.86,23.24,23.47,25.69,27.40, 27.86,30.52,32.19,32.52,32.95,33.67,36.74,38.06,38.45,39.12,41.19,41.46,45.65,46.50, 47.38,55.37,62.47,65.23,65.71,68.24,70.22,72.05,85.93,86.06,120.54,122.34,127.73, 127.83(2C),128.22(2C),136.25,143.47,146.81,168.64,169.53,169.68,170.81,177.15,
O22：1H NMR(600MHz,CD3OD):δ0.57,0.76,0.90,0.91,0.93,1.14(6×CH3), 0.58-2.08(m),2.90(dd,J＝4.32,13.86Hz,1H),2.98(dd,J＝4.20,11.70Hz,1H),3.47(t,J＝7.8Hz,1H),3.49(t,J＝9.06Hz,1H),3.70-3.66(m,1H),3.89(t,J＝9.12Hz,1H),4.01(dd,J＝5.46, 11.34Hz,1H),4.52(d,J＝12.30Hz,1H),4.72(d,J＝12.30Hz,1H),5.06(s,2H),5.24(t,J＝3.42 Hz,1H),5.50(d,J＝9.18Hz,1H),7.30-7.35(m,5H),8.11(s,1H).13C NMR(150MHz,CD3OD): δ16.08,17.12,17.86,19.49,23.75,24.11,24.21,24.67,26.55,28.82,31.75,33.67,33.78,34.05, 34.94,38.23,39.64,39.86,40.74,42.96(2C),47.22,48.19,49.08,57.14,63.41,67.37,69.98,70.77, 74.00,78.79,87.88,90.31(2C),124.08,124.24,129.33,129.50(2C),129.65(2C),137.81,145.03, 146.94,179.18.
O25：1H NMR(600MHz,CDCl3):δ0.55,0.74,0.84,0.86,0.88,1.07(6×CH3),0.55-1.80 (m),1.82(s,3H),1.95(s,3H),1.98(s,3H),2.00(s,3H),2.01(s,3H),2.06(s,3H),2.06(s,3H), 2.86(dd,J＝4.74,14.46Hz 1H),2.92(dd,J＝4.26,11.70Hz,1H),3.65-3.67(m,1H),3.89-3.94 (m,2H),4.02(dd,J＝2.04,12.60Hz,1H),4.06-4.09(m,1H),4.36(dd,J＝4.32,12.60Hz,1H), 4.47(d,J＝10.74Hz,1H),4.50(d,J＝12.72Hz,1H),4.53(d,J＝7.92Hz,1H),4.72(d,J＝12.78 Hz,1H),4.92(t,J＝8.04Hz,1H),5.00(d,J＝12.48Hz,1H),5.04(d,J＝7.14Hz,1H),5.06(d,J ＝4.32Hz,1H),5.13(t,J＝9.42Hz,1H),5.24(t,J＝3.54Hz,1H),5.35(t,J＝8.76Hz,1H),5.41 (t,J＝9.48Hz,1H),5.79(d,J＝9.3Hz,1H),7.30(s,5H),7.65(s,1H).13C NMR(150MHz, CDCl3):δ14.22,15.32,16.47,16.87,18.24,20.25,20.47,20.56,20.71,20.81,22.58,23.03,23.42, 23.66,25.88,27.59,28.06,30.71,32.37,32.68,33.13,33.84,36.93,38.23,38.67,39.30,41.36, 41.66,45.84,46.73,47.56,55.55,60.42,61.52,61.65,62.89,65.93,67.67,70.29,71.53,72.08,72.45,72.83,75.81,75.92,85.41,86.59,100.86,120.65,122.50,127.92,127.99(2C),128.42(2C), 136.42,143.69,147.11,169.08,169.12,169.35,169.62,170.22,170.24,170.52,177.48.
O26：1H NMR(600MHz,CD3OD):δ0.58,0.77,0.91,0.91,0.93,1.14(6×CH3),0.58-2.06 (m),2.90(dd,J＝4.20,13.80Hz,1H),2.99(dd,J＝4.26,11.70Hz,1H),3.27(t,J＝7.86Hz,1H), 3.34(t,J＝5.82Hz,1H),3.36-3.38(m,1H),3.40-3.41(m,1H),3.67(dd,J＝6.00,11.94Hz,1H), 3.72-3.75(m,2H),3.80(t,J＝9.24Hz,1H),3.89-3.90(m,1H),3.91-3.92(m,2H),3.97(t,J＝9.18 Hz,1H),4.47(d,J＝7.86Hz,1H),4.53(d,J＝12.36Hz,1H),4.74(d,J＝12.00Hz,1H),5.06(s, 2H),5.24(t,J＝3.42Hz,1H),5.67(d,J＝9.24Hz,1H),7.30-7.35(m,5H),8.16(s,1H).13C NMR (150MHz,CD3OD):δ15.95,16.98,17.71,19.34,23.56,23.97,24.05,24.51,26.41,28.66,28.70, 31.59,33.53,33.61,33.89,34.78,38.07,39.47,39.73,40.57,42.80(2C),47.06,48.02,48.92,56.96, 61.32,62.43,63.09,67.20,71.33,73.71,74.85,76.78,77.79,78.12,79.45,79.57,87.98,89.46, 104.53,123.90,124.44,129.17,129.33(2C),129.50(2C),137.63,144.87,146.53,178.98.
O29：1H NMR(600MHz,CDCl3):δ0.52,0.70,0.80,0.81,0.82,0.84,1.04(7×CH3),0.52-1.76(m),1.77(s,3H),1.95(s,3H),1.96(s,3H),1.96(s,3H),2.00(s,3H),2.04(s,3H),2.05 (s,3H),2.83(dd,J＝3.84,13.74Hz 1H),2.89(dd,J＝4.20,11.70Hz,1H),3.92(t,J＝9.24Hz, 2H),4.00(d,J＝12.90Hz,1H),4.09(t,J＝9.3Hz,1H),4.18(d,J＝10.14Hz,2H),4.41(d,J＝ 10.92Hz,1H),4.47(d,J＝12.72Hz,1H),4.69(d,J＝12.78Hz,1H),4.81(dd,J＝3.96,10.56Hz, 1H),4.96(d,J＝12.54Hz,1H),5.01(t,J＝9.96Hz,2H),5.20(brs,1H),5.30-5.33(m,2H), 5.38-5.41(m,2H),5.82(d,J＝9.30Hz,1H),7.28(s,5H),7.62(s,1H).13C NMR(150MHz, CDCl3):δ14.17,15.32,16.46,16.86,18.23,20.23,20.62,20.71,20.78,20.85,22.58,23.03,23.41, 23.66,25.87,27.58,28.06,29.32,30.71,32.36,32.67,33.13,33.84,36.93,38.22,38.66,39.29, 41.35,41.65,45.83,46.72,47.55,55.55,61.43,62.49,62.86,65.92,67.89,68.73,69.20,70.00, 70.80,72.39,75.21,75.27,85.11,86.59,95.88,120.64,122.50,127.91,127.99(2C),128.41(2C), 136.41,143.67,147.12,169.19,169.46,169.95,170.02,170.36,170.54,170.59,177.46
O30：1H NMR(600MHz,CD3OD):δ0.58,0.77,0.91,0.93,1.14(5×CH3),0.58-2.02(m), 2.90(dd,J＝4.08,13.74Hz,1H),2.99(dd,J＝4.20,11.64Hz,1H),3.28(d,J＝9.54Hz,1H),3.34 (dd,J＝3.72,9.72Hz,1H),3.64(d,J＝9.36Hz,1H),3.66-3.68(m,1H),3.70-3.73(m,2H),3.78 (t,J＝9.48Hz,1H),3.83-3.86(m,2H),3.87-3.91(m,2H),3.97(t,J＝9.18Hz,1H),4.51(d,J ＝12.24Hz,1H),4.73(d,J＝12.18Hz,1H),5.06(s,2H),5.24(m,2H),5.64(d,J＝9.18Hz,1H), 7.30-7.35(m,5H),8.16(s,1H).13C NMR(150MHz,CD3OD):δ15.95,16.98,17.71,19.34,23.56, 23.97,24.05,24.51,26.41,28.66,28.70,31.59,33.53,33.61,33.89,34.78,38.07,39.47,39.73, 40.57,42.80(2C),47.06,48.02,48.92,56.96,61.32,62.43,63.09,67.20,71.33,73.71,74.85,76.78, 77.79,78.12,79.45,79.57,87.98,89.46,104.53,123.90,124.44,129.17,129.33(2C),129.50(2C), 137.63,144.87,146.53,178.98.
O33：1H NMR(600MHz,CDCl3):δ0.57,0.75,0.85,0.87,0.89,1.09(6×CH3),0.57-1.92 (m),1.95(s,3H),2.03(s,3H),2.04(s,3H),2.04(s,3H),2.06(s,3H),2.08(s,3H),2.05(s,3H), 2.87(dd,J＝4.56,14.40Hz,1H),2.94(dd,J＝4.20,11.70Hz 1H),3.88-3.91(m,1H),3.93-3.97 (m,1H),4.07-4.09(m,2H),4.12-4.14(m,2H),4.45-4.48(m,1H),4.50-4.55(m,1H),4.74(d,J＝ 13.08Hz,1H),4.95(dd,J＝3.42,10.26Hz,1H),5.04(d,J＝18.06Hz,1H),5.10-5.13(m,2H), 5.26(t,J＝3.30Hz,1H),5.33-5.35(m,2H),5.37(t,J＝4.62Hz,1H),5.42(t,J＝9.42Hz,1H), 5.80(d,J＝9.12Hz,1H),7.31(s,5H),7.66(s,1H).13C NMR(150MHz,CDCl3):δ14.26,15.36, 16.51,16.90,18.27,20.32,20.58,20.69,20.72,21.13,22.61,23.07,23.46,23.70,25.91,27.62, 28.10,28.31,30.75,32.40,32.71,33.16,33.88,36.97,38.26,38.70,39.33,41.39,41.69,45.87, 46.77,47.59,55.58,60.46,60.85,61.79,62.93,65.96,66.61,69.03,70.40,70.95,72.54,72.78,75.75,75.83,86.62,87.73,101.20,120.66,122.53,127.96,128.03(2C),128.46(2C),136.46, 143.73,147.17,169.16,169.61,170.14,170.20,170.31,170.45,177.52
O34：1H NMR(600MHz,CD3OD):δ0.57,0.77,0.91,0.91,0.92,0.93,1.14(7×CH3),0.58-2.06(m),2.90(dd,J＝4.08,13.74Hz,1H),2.99(dd,J＝4.20,11.70Hz,1H),3.50(dd,J＝ 3.3,9.72Hz,1H),3.58(t,J＝9.78Hz,1H),3.60-3.62(m,1H),3.70-3.72(m,1H),3.72-3.74(m, 2H),3.78(d,J＝2.04Hz,1H),3.80-3.83(m,2H),3.90(d,J＝2.88Hz,2H),3.97(t,J＝9.24Hz, 1H),4.41(d,J＝7.74Hz,1H),4.52(d,J＝12.24Hz,1H),4.73(d,J＝12.24Hz,1H),5.06(d,J ＝2.28Hz,2H),5.24(t,J＝3.18Hz,1H),5.64(d,J＝9.30Hz,1H),7.30-7.35(m,5H),8.17(s,1H). 13C NMR(150MHz,CD3OD):δ15.91,16.95,17.69,19.33,23.57,23.93,24.05,24.51,26.36, 28.61,28.67,31.60,33.50,33.64,33.90,34.79,38.09,39.49,39.74,40.59,42.82(2C),44.70,47.07, 48.06,49.57,57.00,61.41,62.54,63.29,67.23,70.31,72.53,73.65,74.80,76.90,77.16,79.49, 79.56,87.81,89.32,105.08,123.95,124.19,129.19,129.38(2C),129.51(2C),137.67,144.89, 179.08.
O37：1H NMR(600MHz,CDCl3):δ0.60,0.78,0.87,0.88,0.90,0.92,1.11(7×CH3),0.60-1.83(m),1.85(s,3H),2.01(s,3H),2.01(s,3H),2.02(s,3H),2.03(s,3H),2.04(s,3H),2.04 (s,3H),2.07(s,3H),2.11(s,3H),2.16(s,3H),2.17(s,3H),2.89(dd,J＝4.08,13.80Hz,1H), 2.96(dd,J＝4.26,11.70Hz 1H),3.93-3.96(m,1H),3.97-3.99(m,1H),4.00-4.03(m,1H),4.06 (dd,J＝2.64,12.42Hz,1H),4.13(t,J＝9.96Hz,1H),4.18(dd,J＝3.72,12.72Hz,1H),4.25(dd, J＝3.84,13.08Hz,1H),4.31-4.36(m,2H),4.47-4.51(m,2H),4.55(d,J＝12.78Hz,1H), 4.76-4.78(m,2H),4.86(dd,J＝4.08,10.56Hz,1H),5.04(d,J＝12.54Hz,1H),5.06-5.10(m,2H), 5.28(t,J＝3.42Hz,1H),5.32(d,J＝4.08Hz,1H),5.35-5.40(m,2H),5.42-5.43(m,2H),5.46(t,J ＝9.06Hz,1H),5.89(d,J＝9.24Hz,1H),7.35(s,5H),7.68(s,1H).13C NMR(150MHz,CDCl3): δ14.27,15.43,16.57,16.98,18.35,20.37,20.72,20.74,20.83,20.94,20.96,21.05,22.71,22.82, 23.15,23.53,23.77,25.99,27.70,28.18,29.45,30.83,32.05,32.48,32.79,33.24,33.96,37.05, 38.34,38.78,39.41,41.47,41.77,45.95,46.85,47.67,55.67,61.45,62.30,62.74,63.01,66.05, 67.94,68.63,69.30,69.44,70.20,70.54,70.95,71.73,72.47,73.56,75.27,85.25,86.74,95.78, 96.23,120.74,122.62,128.03,128.10(2C),128.54(2C),136.54,143.81,147.27,169.38,169.61, 169.86,170.02,170.04,170.50,170.54,170.72,170.76,170.80,177.61.
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、抗流感病毒活性实验
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；具体方法如下：
(1)将犬肾上皮细胞(MDCK)以1:3的比例传代到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)将流感病毒的减毒病毒株[A/WSN/33(H1N1)，感染复数(MOI)＝1]与100μM的待检化合物加入到100μL含有0.25％TPCK处理的胰酶和1％FBS的DMEM中，充分混匀；化合物的阴性对照为1％的DMSO(稀释化合物所用的溶剂)，阳性对照为100μM的奥司他韦。
(3)将96孔板中的MDCK细胞的培养基吸出，将步骤(2)配置好的混合有病毒和化合物的培养基和另一组只加化合物不加病毒的实验组加入到96孔板中，将板放置在37℃细胞培养箱中培养36-46h。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，将每孔30μL的 CellTiter-Glo检测试剂加入到细胞的培养上清中，震动1min后，避光静置10min后，每孔取80μL加入到另一块96白板中，用酶标仪检测出细胞活力的吸光度值。
(5)抑制率的计算方法：使用CellTiter-Glo试剂盒测细胞活力值。首先将对照组细胞活力值(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％(相对细胞活力＝各组细胞的检测值/空白对照组细胞的检测值× 100％)。抑制率是指化合物起到抑制病毒生长的作用的比率(抑制率＝1－(提取物对细胞作用检测值－提取物对病毒作用检测值)/(空白组检测值－病毒对照检测值))。反映了化合物抗病毒活性。
(6)CC50的计算方法：使用CellTiter-Glo试剂盒测出的细胞活力值来计算化合物对细胞的毒性。首先将对照组细胞活力值(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。
(7)IC50的计算方法：重复步骤(1)，对基本无毒性且有明显抑制作用的某一化合物进行浓度稀释(2倍稀释)，之后将化合物与病毒均匀混合加入到已接种细胞的96孔板中，将板放置培养箱培养36-46h，然后利用步骤(4)方法测定出细胞活力。计算各化合物浓度和相应的抑制率率并输入到软件Prism，即可计算IC50；此方法已被广泛应用于抗病毒药物筛选领域； SI＝CC50/IC50,表示效果安全范围，指数越大安全范围越大。
实验结果显示：与齐墩果酸相比，本发明化合物表现出明显的抗流感病毒活性，其中 O5化合物显示出一定的抑制流感病毒活性，可以显著削弱病毒的感染性。在体外抑制A/WSN/33活性评价中，化合物O5表现出远高于齐墩果酸的流感病毒抑制活性，IC50为12.45μM。相关实验结果具体参见图1，图2和图4。
2、血凝抑制实验
利用血凝抑制实验探究本发明中的化合物是否阻止病毒与宿主细胞的结合，具体方法如下：
(1)将新鲜鸡血装入离心管中离心，时间为15min，转速为1000转；弃上清后加入生理盐水再次离心，时间与转速与上次相同；重复3-4次，留5mL上清液与45mL生理盐水混合配置成1％的鸡血红细胞悬液。
(2)测血凝效价。向V型血凝版中每孔加入50μL生理盐水，随后在第1孔加入50μL病毒，并从第1孔后开始倍比稀释；将已配置的1％鸡血红细胞悬液每孔各加入50μL，静止25min，将板倾斜，观察血凝板现象。能使红细胞完全凝集(100％凝集,++++)的抗原最高稀释度为该抗原的血凝效价,此效价为1个血凝单位(HAU)。注意对照孔应呈现完全不凝集(-),否则此次检验无效。
(3)根据血凝试验结果配制4HAU的病毒抗原,以完全血凝的病毒最高稀释倍数作为终点,终点稀释倍数除以4即为含4HAU的抗原的稀释倍数。例如,如果血凝的终点滴度为1:256,则4HAU抗原的稀释倍数应是1:64(256除以4)。
(4)在血凝板的第1-11孔加入25μL生理盐水,第12孔加入50μL生理盐水。之后往第1孔里加入2μL待测化合物和23μL生理盐水，从第1孔取25μL到下一孔梯度稀释，到第 11孔后将吸出的25μL丢弃，之后往第1-11孔补加25μL生理盐水。随后在第1-12孔均加入含4HAU混匀的病毒抗原液25μL,室温(20～25℃)静止30min。在1～12孔均加入25μL 的1％鸡红细胞悬液,轻轻震荡混匀,静止约25min，室温(20～25℃),对照红细胞将呈现纽扣状沉于孔底。
(5)重复上述步骤(4)，用HA抗体作为阳性对照。
实验结果显示：本发明中化合物O5显示出与HA抗体相似的能力，可有效抑制流感病毒诱导的鸡红细胞聚集。这表明O5和HA抗体具有相同的靶标HA蛋白，因此通过机制研究推断出O5可以阻断病毒和靶细胞之间的相互作用(图3)。

Claims (10)

1.式(Ⅰ)结构的齐墩果酸-C3位糖偶联物：
Figure FDA0002687684320000011
或其药学上可接受的盐，
其中，
R1为H或苄基，
R2选自单糖、二糖或三糖及其衍生物，其通过1位与三氮唑连接。
2.根据权利要求1所述的齐墩果酸-C3位糖偶联物，其中所述单糖为葡萄糖、甘露糖、鼠李糖、木糖、阿拉伯糖或半乳糖；所述二糖为麦芽糖、纤维二糖或乳糖；所述三糖为麦芽三糖。
3.根据权利要求1所述的齐墩果酸-C3位糖偶联物，其中所述单糖、二糖或三糖的衍生物是指单糖、二糖或三糖的1-10个羟基任选被乙酰氧基取代的衍生物。
4.根据权利要求1所述的齐墩果酸-C3位糖偶联物，其选自以下结构的化合物：
Figure FDA0002687684320000012
Figure FDA0002687684320000021
Figure FDA0002687684320000031
5.根据权利要求1-4中任一项所述的齐墩果酸-C3位糖偶联物的制备方法，其特征在于，通过点击化学反应连接齐墩果酸和叠氮糖，构建三氮唑连接基，从而快速引入糖基R2。
6.一种流感病毒抑制剂，其特征在于，其为根据权利要求1至4中的任一项所述的齐墩果酸-C3糖偶联物或其药学上可接受的盐。
7.一种药物组合物，其特征在于，其包括根据权利要求1至4中的任一项所述的齐墩果酸-C3糖偶联物或其药学上可接受的盐，以及药学上可接受的载体，其中所述药物组合物配制为通过以下途径中的一种或多种给药：口服，直肠，吸入，含化，舌下，经皮，阴道，膀胱内，伤口内和胃肠外。
8.根据权利要求7所述的药物组合物，其中所述药物组合物为喷雾剂，任选地用于口腔或鼻内喷雾给药或者室内或局部环境的灭菌和消毒。
9.根据权利要求1至4中的任一项所述的齐墩果酸-C3糖偶联物及其药学上可接受的盐在制备、根据权利要求6所述的流感病毒抑制剂或根据权利要求7-8中任一项所述的药物组合物在制备用于预防或治疗流感的药物中的应用。
10.权利要求1-4中任一项所述的齐墩果酸-C3位糖偶联物、权利要求5所述的制备方法、权利要求6所述的流感病毒抑制剂、权利要求7-8中任一项所述的药物组合物或权利要求9所述的用途，其中所述齐墩果酸-C3位糖偶联物为O5。
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