CN114073757A - 环肽化合物及其制备方法和抗病毒用途 - Google Patents
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Abstract
本发明公开了环肽化合物及其制备方法和抗病毒用途。该环肽化合物可为式Ⅰ所示的南平霉素D或式Ⅱ所示的泽尔科瓦霉素:
Description
技术领域
本发明属于医药生物领域中环肽化合物及其制备方法和抗病毒用途。
背景技术
流感病毒是一种正黏液病毒科病毒,传播速度快,传染性强,每年都会引起季节性流行,患者被病毒感染后,轻微者表现出流鼻涕、高热等症状,大多数患者还会引起呼吸道感染,表现出较为严重的肺炎症状,更严重的患者会出现心肾等多种脏器衰竭的情况,最终导致死亡。一些流感病毒对抗病毒药物已经产生了抗药性,限制了治疗效果,研发具有新作用机制的抗甲型流感病毒(H1N1)的药物迫在眉睫。丙型肝炎病毒(HCV)系黄病毒科肝炎病毒属,是感染慢性肝病的主要病因之一,感染者有很高的风险发展为肝硬化甚至肝癌。虽然抗丙型肝炎病毒的一线药物索非布韦等在丙肝的治疗中发挥了重大作用,但由于其价格昂贵,耐药等因素,依旧不能有效控制病毒感染,迫切需要价格合理,疗效更高的抗丙型肝炎病毒(HCV)药物。
植物内生放线菌由于与宿主植物的长期作用,可能具有独特的次级代谢途径,能够产生化学结构与活性多样性的产物。
发明内容
本发明所要解决的技术问题是如何抗流感病毒和/或丙型肝炎病毒。
为了解决上述技术问题,本发明提供了环肽化合物或其药学上可接受的盐在制备病毒抑制剂或抗病毒药物中的应用。
上述应用中,所述环肽化合物的结构式为式III:
南平霉素D的分子式为C35H43N9O9S,分子量为765;泽尔科瓦霉素的分子式为C36H45N9O9S,分子量为779。
本发明的环肽化合物或其药学上可接受的盐也属于本发明的保护范围。
本发明的环肽化合物可以以衍生自无机酸或有机酸的药学可接受的盐的形式使用。术语“药学上可接受的盐”指在可靠的医学判断范围内,适合用于与人类和低等动物的组织接触而不出现过度的毒性、刺激、过敏反应等,且与合理的效果/风险比相称的盐。药学可接受的盐是本领域公知的。例如,S.M.Berge,et al.,J.Pharmaceut ical Sciences,1977,66:1中对药学可接受的盐进行了详细描述。所述盐可通过使本发明化合物的游离碱官能度与合适的有机酸反应来制备。
为了解决上述技术问题,本发明提供了病毒抑制剂或抗病毒药物。
本发明所提供的病毒抑制剂或抗病毒药物含有所述环肽化合物或其药学上可接受的盐。
上文中,所述病毒抑制剂或抗病毒药物,除含有所述环肽化合物或其药学上可接受的盐外,还可含有药学上可接受的载体。其中,药学上可接受的载体以重量计可是所述病毒抑制剂或抗病毒药物总重量的0.1-99.9%。
其中,药学上可接受的载体包括微晶纤维素、羧甲基纤维素钠、低取代羟丙基纤维素、甘露醇、山梨醇、山梨酸或钾盐、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、维生素A、维生素C、维生素E、维生素D、氮酮、EDTA二钠、EDTA钙钠,一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、氯化钠、氯化钾、乳酸钠、木糖醇、麦芽糖、葡萄糖、果糖、右旋糖苷、甘氨酸、淀粉、蔗糖、乳糖、甘露糖醇、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、丙二醇、乙醇、土温60-80、司班-80、蜂蜡、羊毛脂、液体石蜡、十六醇、没食子酸酯类、琼脂、三乙醇胺、碱性氨基酸、尿素、尿囊素、碳酸钙、碳酸氢钙、表面活性剂、聚乙二醇、环糊精、β-环糊精、磷脂类材料、高岭土、滑石粉、硬脂酸钙、硬脂酸镁和微晶中的一种或以上。
本发明的病毒抑制剂或抗病毒药物,可以制备成任何要用剂型如片剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口服液、口含剂、颗粒剂、丸剂、散剂、膏剂、丹剂、混悬剂、溶液剂、注射剂、栓剂、软膏剂、硬膏剂、霜剂、喷雾剂、贴剂中的任意一种。
上述病毒抑制剂或抗病毒药物的活性成分可为所述环肽化合物或其药学上可接受的盐,上述病毒抑制剂或抗病毒药物的活性成分还可含有其他生物成分或非生物成分,其他活性成分本领域技术人员可根据抗病毒的效果确定。
本发明还提供了南平霉素D的制备方法。
本发明所提供的南平霉素D的制备方法包括用培养基培养北里孢菌,得到发酵产物,从所述发酵产物中得到南平霉素D,所述北里孢菌可为北里孢菌(Kitasatospora sp.)
CPCC204717,其在中国微生物菌种保藏管理委员会普通微生物中心的登记入册编号为CGMCC No.20381。
本发明还提供了泽尔科瓦霉素的制备方法。
本发明所提供的泽尔科瓦霉素的制备方法包括用培养基培养北里孢菌,得到发酵产物,从所述发酵产物中得到泽尔科瓦霉素,所述北里孢菌可为北里孢菌(Kitasatosporasp.)CPCC204717,其在中国微生物菌种保藏管理委员会普通微生物中心的登记入册编号为CGMCC No.20381。
上述南平霉素D的制备方法和泽尔科瓦霉素的制备方法中,所述培养基可为放线菌培养基(能培养放线菌的培养基),其可为固体培养基、半固体培养基或液体培养基。所述固体培养基选自:包括但不限于大米或以大米、小米、玉米、马铃薯为主要成分的固体培养基。所述放线菌培养基可为黄豆粉培养基、稻米培养基、马铃薯葡萄糖琼脂培养基或其他本领域专业人员熟知的放线菌发酵培养基等。所述黄豆粉培养基可由黄豆粉、可溶性淀粉和水制成,也可由黄豆粉、可溶性淀粉、无机盐和水制成,也可由黄豆粉、可溶性淀粉、无机盐、水、氮源和无机盐制成。所述稻米培养基可由稻米和水制成,也可由稻米、水和无机盐制成,也可由稻米、水和氮源制成,也可由稻米、水、氮源和无机盐制成。所述马铃薯葡萄糖琼脂培养基可由马铃薯、葡萄糖、琼脂和水制成,也可由马铃薯、葡萄糖、琼脂、水和无机盐制成,也可由马铃薯、碳源、琼脂、水和氮源制成,也可由马铃薯、碳源、琼脂、氮源和无机盐制成。所述其他本领域专业人员熟知的真菌发酵培养基可由某一种或几种速效、迟效碳源、某一种或几种速效、迟效氮源、水和/或无机盐等制成。碳源是微生物生长一类营养物,是含碳化合物,包括糖类、油脂、有机酸及有机酸酯和小分子醇等速效、迟效碳源。氮源是指提供微生物营养所需氮元素的物质,包括花生饼粉、黄豆饼粉、酵母粉、蛋白胨、氨水、铵盐和硝酸盐等速效、迟效氮源。
上述南平霉素D的制备方法和泽尔科瓦霉素的制备方法中,所述稻米可为大米或糙米。大米或糙米均是稻谷的制品。稻谷是指没有去除水稻谷壳的果实,稻谷由谷壳、果皮、种皮、外胚乳、糊粉层、胚乳和胚构成。糙米是指稻谷脱去谷壳,保留稻谷其它各部分的制品;大米是指仅保留胚乳,而将稻谷其余部分全部脱去的制品。
上述南平霉素D的制备方法和泽尔科瓦霉素的制备方法可为液体发酵制备南平霉素D和/或泽尔科瓦霉素。上述液体发酵制备南平霉素D和/或泽尔科瓦霉素中,所述培养基可为液体培养基,所述发酵产物可为液体发酵产物。
上述液体发酵制备南平霉素D和/或泽尔科瓦霉素中,从所述发酵产物中得到南平霉素D和/或泽尔科瓦霉素可包括(1)将所述液体发酵产物离心,得到上清液和菌丝体,所述上清液用大孔树脂进行固相萃取,得到上清液提取物,所述菌丝体用溶剂超声提取,得到菌丝体提取物;(2)将所述上清液提取物和所述菌丝体提取物合并,通过硅胶柱色谱分离,用溶剂进行梯度洗脱,经凝胶柱层析分离,HPLC制备分离得到南平霉素D和/或泽尔科瓦霉素。
上述液体发酵制备南平霉素D和/或泽尔科瓦霉素中,所述液体培养基可选自:包括但不限于A1液体培养基。
上述液体发酵制备南平霉素D和/或泽尔科瓦霉素中,步骤(1)的溶剂可选自:氯仿、二氯甲烷、乙酸乙酯、甲醇、乙醇等碳链≤4的醇类、丙酮、丁酮、或以上溶剂的混合溶液或以上溶剂与水的混合溶液;在本发明的一个实施例中为50%丙酮水溶液和/或丙酮。
上述液体发酵制备南平霉素D和/或泽尔科瓦霉素中,步骤(2)的溶剂溶液可选自:包括但不限于丙酮水溶液、碳链≤4的醇溶液、乙腈水溶液、或甲醇中一种或以上混合;在本发明的一个实施例中为由二氯甲烷和甲醇组成的溶液。
上述液体发酵制备南平霉素D和/或泽尔科瓦霉素中,所述凝胶柱层析分离中所用的介质包括但不限于Sephadex LH-20、Tosoh Toyopearl HW-40;在本发明的一个实施例中为Sephadex LH-20。所用的流动相选自氯仿、二氯甲烷、乙酸乙酯、甲醇、乙醇等碳链≤4的醇类、丙酮、丁酮、或以上溶剂的混合溶液或以上溶剂与水的混合溶液;在本发明的一个实施例中为,二氯甲烷-甲醇混合溶液以及甲醇。
上述液体发酵制备南平霉素D和/或泽尔科瓦霉素中,所述HPLC制备分离中所用的流动相可选自:丙酮水溶液、碳链≤4的醇溶液、乙腈水溶液,在本发明的一个实施例中为乙腈水溶液,所述HPLC制备分离中所用的色谱柱可包括但不限于C18色谱柱、C8色谱柱、C3色谱柱和苯基键合柱。
上述液体发酵制备南平霉素D和/或泽尔科瓦霉素中,所述硅胶柱色谱中所用的洗脱程序如下:1)先用100:1的二氯甲烷-甲醇溶液洗脱;2)接着用50:1的二氯甲烷-甲醇溶液洗脱;3)接着用20:1的二氯甲烷-甲醇溶液洗脱;收集用50:1的二氯甲烷-甲醇溶液洗脱出的液体,将该液体命名为馏分F2,和/或,收集用50:1的二氯甲烷-甲醇溶液洗脱出的液体和用20:1的二氯甲烷-甲醇溶液洗脱出的液体,将该液体命名为馏分F3至F6。上述液体发酵制备南平霉素D和/或泽尔科瓦霉素中,所述制备方法包括从所述馏分F2中分离得到泽尔科瓦霉素,从所述馏分F3至F6中分离得到南平霉素D与泽尔科瓦霉素。
上述液体发酵制备南平霉素D和/或泽尔科瓦霉素中,从所述馏分F2中分离得到泽尔科瓦霉素可包括将所述馏分F2进行凝胶柱层析,所述凝胶柱层析中所用的介质可为Sephadex LH-20,所用的流动相可为1:1的二氯甲烷-甲醇溶液;将用所述流动相洗脱出的液体,命名为馏分F2-4,进行HPLC制备分离得到泽尔科瓦霉素。所述HPLC制备分离中所用的流动相可为乙腈和水体积比为40:60的组成的液体。
上述液体发酵制备南平霉素D和/或泽尔科瓦霉素中,从所述馏分F3至F6中分离得到南平霉素D可包括将所述馏分F3至F6进行凝胶柱层析。所述凝胶柱层析中所用的介质可为Sephadex LH-20,所用的流动相可为甲醇。将用所述流动相洗脱出的液体,命名为馏分F3-2-F3-5,进行HPLC制备分离得到南平霉素D与泽尔科瓦霉素。所述HPLC制备分离中所用的流动相可为乙腈和水体积比为40:60组成的液体。
上述南平霉素D的制备方法和泽尔科瓦霉素的制备方法可为固体发酵制备南平霉素D和/或泽尔科瓦霉素。上述固体发酵制备南平霉素D和/或泽尔科瓦霉素中,所述培养基可为固体培养基,所述发酵产物可为固体发酵产物。
上述固体发酵制备南平霉素D和/或泽尔科瓦霉素中,从所述发酵产物中得到南平霉素D和/或泽尔科瓦霉素可包括用有机溶剂提取所述固体发酵产物,得到提取物;将所述提取物进行C18反相闪式柱色谱分离,用溶剂进行梯度洗脱,将洗脱得到的活性组分经凝胶柱层析分离,然后经HPLC制备分离得到南平霉素D和/或泽尔科瓦霉素。
上述固体发酵制备南平霉素D和/或泽尔科瓦霉素中,所述固体培养基可选自:包括但不限于大米或以大米、小米、玉米和/或马铃薯为主要成分的固体培养基。
上述固体发酵制备南平霉素D和/或泽尔科瓦霉素中,用有机溶剂提取所述固体发酵产物中,有机溶剂可选自:氯仿、二氯甲烷、乙酸乙酯、甲醇、乙醇等碳链≤4的醇类、丙酮、丁酮、或以上溶剂的混合溶液。在本发明的一个实施例中为乙酸乙酯。
上述固体发酵制备南平霉素D和/或泽尔科瓦霉素中,将所述提取物可进行C18反相闪式柱色谱分离,用溶剂进行梯度洗脱,所述溶剂可选自:包括但不限于丙酮水溶液、碳链≤4的醇溶液、乙腈水溶液、或甲醇水溶液中一种或以上混合。
上述固体发酵制备南平霉素D和/或泽尔科瓦霉素中,所述凝胶柱层析分离中所用的介质包括但不限于Sephadex LH-20、Tosoh Toyopearl HW-40;在本发明的一个实施例中为Sephadex LH-20与Tosoh Toyopearl HW-40F。所述凝胶柱层析分离中所用的流动相有机溶剂可选自:氯仿、二氯甲烷、乙酸乙酯、甲醇、乙醇等碳链≤4的醇类、丙酮、丁酮、水、或以上溶剂的混合溶液。在本发明的一个实施例中为甲醇水溶液。
上述固体发酵制备南平霉素D和/或泽尔科瓦霉素中,所述HPLC制备分离中所用的流动相可选自:丙酮水溶液、碳链≤4的醇溶液、乙腈水溶液,在本发明的一个实施例中为甲醇水溶液。所述HPLC制备分离中所用的色谱柱可包括但不限于C18色谱柱、C8色谱柱、C3色谱柱和苯基键合柱。
上述固体发酵制备南平霉素D和/或泽尔科瓦霉素中,所述C18反相闪式柱色谱分离中所用的流动相为甲醇水溶液,洗脱程序如下:1)先用10%的甲醇水溶液洗脱;2)接着用30%的甲醇水溶液洗脱;3)接着用50%的甲醇水溶液洗脱;4)接着用70%的甲醇水溶液洗脱,收集用70%的甲醇水溶液洗脱出的液体,将该液体命名为馏分F4。
上述固体发酵制备南平霉素D和/或泽尔科瓦霉素中,所述凝胶柱层析分离包括将所述馏分F4进行凝胶柱层析,所述凝胶柱层析中分离介质可为Sephadex LH-20,流动相可为80%的甲醇水溶液,将用所述流动相洗脱出的液体进行凝胶柱层析,所述凝胶柱层析中分离介质可为Toyopearl HW-40F,流动相可为70%甲醇-水溶液,收集洗脱出的液体,将该液体命名为馏分F4-4-3至F4-4-11。
上述固体发酵制备南平霉素D和/或泽尔科瓦霉素中,对所述馏分F4-4-3至F4-4-11进行HPLC制备分离得到南平霉素D和泽尔科瓦霉素。所述HPLC制备分离中所用的流动相均可为60%甲醇水溶液。
上述北里孢菌(Kitasatospora sp.)CPCC204717也属于本发明的保护范围。
本发明还提供了抗病毒的菌剂。
本发明所提供的抗病毒的菌剂含有所述北里孢菌(Kitasatospora sp.)CPCC204717或/和所述北里孢菌(Kitasatospora sp.)CPCC204717的代谢物。
上述菌剂的活性成分可为所述北里孢菌(Kitasatospora sp.)CPCC204717或/和所述北里孢菌(Kitasatospora sp.)CPCC204717的代谢物,上述菌剂的活性成分还可含有其他生物成分或非生物成分,上述菌剂的其他活性成分本领域技术人员可根据菌剂的抗病毒效果确定。
上文中,所述北里孢菌(Kitasatospora sp.)CPCC204717的代谢物可为所述北里孢菌(Kitasatospora sp.)CPCC204717的发酵产物。
上文中,所述病毒可为下述至少一种:1)正黏液病毒科病毒,2)流感病毒,3)甲型流感病毒,4)黄病毒科病毒,5)肝炎病毒。
上文中,所述抗病毒药物可为治疗或/和预防由所述病毒所致疾病的药物。
本发明中式I所示化合物南平霉素D抗甲型流感病毒(H1N1)以及抗丙型肝炎病毒(HCV)的EC50分别为0.3μM和3.2μM,其中抗甲型流感病毒(H1N1)病毒活性强于阳性药物利巴韦林近50倍,可用于甲型流感病毒(H1N1)以及丙型肝炎病毒(HCV)感染的治疗。南平霉素D的抗H1N1型甲型流感病毒以及丙型肝炎病毒(HCV)活性显著强于泽尔科瓦霉素(EC50分别为46.0μM和6.1μM)。南平霉素D和泽尔科瓦霉素为微生物发酵得到的天然产物,原料易得,因此可以作为抗甲型流感病毒(H1N1)以及丙型肝炎病毒(HCV)的先导化合物进行结构优化以开发活性更好的抗病毒药物。以南平霉素D作为活性成分,与药学上可接受的一种或多种载体、赋形剂或辅料配伍,可制成抗甲型流感病毒(H1N1)或抗丙型肝炎病毒(HCV)的药物组合物。南平霉素D及药物组合物可用于甲型流感病毒(H1N1)以及丙型肝炎病毒(HCV)的临床治疗。南平霉素D和/或泽尔科瓦霉素也可与已知药物组成复方制剂用于甲型流感病毒(H1N1)和丙型肝炎病毒(HCV)的治疗。
保藏说明
菌种名称:北里孢菌,拉丁名:Kitasatospora sp.,菌株编号:CPCC204717,保藏机构:中国微生物菌种保藏管理委员会普通微生物中心,保藏机构简称:CGMCC,地址:北京市朝阳区北辰西路1号院3号,保藏日期:2020年07月16日,保藏中心登记入册编号:CGMCCNo.20381。
附图说明
图1为式I所示化合物的高分辨电喷雾质谱。
图2为式I所示化合物的红外光谱。
图3为式I所示化合物的1H NMR谱。
图4为式I所示化合物的13C NMR谱。
图5为式I所示化合物的DEPT谱。
图6为式I所示化合物的1H-1H COSY谱。
图7为式I所示化合物的HSQC谱。
图8为式I所示化合物的HMBC谱。
图9为式I所示化合物的ROESY谱。
图10为式Ⅱ所示化合物的高分辨电喷雾质谱。
图11为式Ⅱ所示化合物的1H NMR谱。
图12为式Ⅱ所示化合物的13C NMR谱。
图13为式Ⅱ所示化合物的ROESY谱。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
本发明实施方案适用于从任何微生物,而不限于从北里孢菌发酵产物中制备南平霉素D(nanpingmycin D)及其衍生物。以下所列实施例是为帮助本领域技术人员更好的理解本发明,但不以任何方式限制本发明。
实施例1、北里孢菌(Kitasatospora sp.)CGMCC No.20381的分离鉴定
1.菌株的分离:
北里孢菌菌株CPCC 204717(=CGMCC No.20381)(Kitasatospora sp.CPCC204717)分离自蛇足石杉植株。蛇足石杉植株整棵植株采集后,次日带回实验室处理。流程如下:1)植株表面清洗:用缓流自来水冲洗植株表面;2)植株分割:用消过毒的枝剪将植株的地上部分和地下部分分割开,分别用于后续实验;3)植物样品表面消毒:首先,用无菌枝剪讲植物样品剪成大约10cm长短的节段,然后用依次用5%次氯酸钠浸泡3min,75%酒精浸泡1min,75%酒精漂洗30s,无菌水冲洗3次,然后,用无菌吸水纸将植物样品表面水分吸干;4)植物样品的粉碎:将表面消过毒的植物节段放入无菌粉碎仪处理45s-1min,使植物样品大部分呈碎屑状,用于后续菌种分离。
菌种分离方法:在超净工作台中取植物碎屑均匀分撒在添加了50ppm的重铬酸钾和30ppm的制霉菌素的ISP2(国际链霉菌计划2号培养基)分离培养基平板上,静置于28℃恒温培养箱,培养2周,挑取单菌落于新配置的ISP2平板上,划线纯化。把纯化得到的菌株CPCC204717纯菌种转接于ISP2斜面培养基上,28℃恒温培养7d后,长好后置4℃作短期保藏。
2.菌株的鉴定:
2.1形态特征
菌株CPCC204717在多数培养基上形成表面干燥的菌落,最大菌落直径达1.5mm;气生菌丝丰富,多呈絮状,白色至暗灰色,分化成长孢子链状,波曲;基内菌丝稀疏,不分枝,无断裂,呈灰褐色至黄褐色;能生成褐色可溶性色素。
2.2生理生化特征
实验参照徐丽华等主编的《放线菌系统学》相关方法操作。(徐丽华,李文均,刘志恒.放线菌系统学—原理、方法及实践.北京:科学出版社,2007:40-47,66-80,119-128.)菌株CPCC204717生长温度10-37℃,最适温度28—30℃。明胶液化、牛奶凝固并胨化、淀粉水解以及硝酸盐还原实验阳性;能够产生类黑色素、酪氨酸酶。能够利用葡萄糖、阿拉伯糖、果糖以及乳糖、甘露糖、纤维二糖、麦芽糖、淀粉、甘油、山梨醇、水杨苷、醋酸钠;不能同化蔗糖、鼠李糖、棉子糖、肌醇、甘露醇、松三糖、纤维素、卫矛醇、侧金盏醇以及七叶树素。
2.3序列测定及分析:提取菌株CPCC204717的基因组,PCR扩增其16S rRNA基因并进行测序,结果表明菌株CPCC204717的16S rRNA基因具有SEQ ID No.1的DNA,与菌株Kitasatospora aburaviensis strain RCS252(GenBank Accession No.MK942686.1)的16S rRNA基因相似性达99.66%。
根据菌株CPCC204717形态特征、生理生化特征和16S rRNA基因序列,确定菌株CPCC204717为北里孢菌属细菌(Kitasatospora sp.)。菌株CPCC204717已于2020年07月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.20381。下文简称北里孢菌CPCC204717。
实施例2、式I与式Ⅱ所示化合物的分离、制备及结构鉴定
1.种子液的制备
将北里孢菌CPCC204717孢子液接种于ISP4培养基平板[配方为:可溶性淀粉10g,K2HPO41g,MgSO4 1g,NaCl 1g,(NH4)2SO4 2g,CaCO3 2g,FeSO4 1mg,MnCl2 1mg,ZnSO4 1mg,琼脂15g,用去离子水定容至1L,121℃灭菌15分钟得到ISP4培养基]上,置于28℃恒温培养箱培养7-14天,用无菌铁铲取约1cm2的含菌琼脂块接种于100mL液体TCG发酵培养基[配方为:葡萄糖4g,酪蛋白5g,胰蛋白胨3g,用去离子水定容至1L,121℃灭菌15分钟得到液体TCG发酵培养基],在500mL三角瓶中,28℃,200r/min,摇瓶培养3天得到种子液。
2.从液体发酵产物中制备式I与式Ⅱ所示的化合物
2.1北里孢菌CPCC204717液体发酵产物的制备
取10mL步骤1的种子液转接于含有100mL A1培养基[配方为:黄豆粉15g,可溶性淀粉30g,K2HPO4 0.5g,FeSO4 0.5g,Na2S2O3 0.007μg KCl 0.3g,用去离子水定容至1L,pH=7.2,121℃灭菌15分钟得到A1液体发酵培养基]的500mL锥形瓶中,共23L,28℃,200r/min震荡培养5天;得到北里孢菌CPCC204717液体发酵产物。
2.2分离纯化式I与式Ⅱ所示的化合物
将2.1的北里孢菌CPCC204717液体发酵产物离心(6000rpm,10mim),得到上清液和菌丝体,上清液用大孔树脂进行固相萃取,先后用水、50%丙酮与丙酮进行洗脱,分别收集馏分,得到上清液提取物,具体方法如下:离心得到的上清液按每瓶300mL分装到500mL锥形瓶中,每瓶按10%(g/mL)的比例加入XAD7HP大孔树脂(Rohm&Haas,Amberlite XAD7HP型大孔树脂),置于摇床上,200rpm震荡12小时。然后将大孔树脂合并倒入层析柱内,先用蒸馏水洗脱,洗脱至流出液澄清,再加50%丙酮水溶液洗脱至流出液无颜色,再用丙酮洗脱至流出液无颜色。从50%丙酮水溶液洗脱程序开始不间断收集50%丙酮水溶液洗脱出的液体(简称50%丙酮水溶液洗脱部分的上清液提取物)。从丙酮洗脱程序开始不间断收集丙酮洗脱出的液体(简称丙酮洗脱部分的上清液提取物)。
离心得到的菌丝体加等体积的丙酮超声(40kHz,3h),得到菌丝体提取物。将50%丙酮水溶液洗脱部分的上清液提取物与丙酮洗脱部分的上清液提取物与菌丝体提取物合并,通过硅胶柱层析分离,硅胶柱层析中所用的介质为柱层析硅胶(200-300目,400g),所用的硅胶柱的规格为60×530mm(直径×长度),柱体积为1300mL。硅胶柱层析中所用的洗脱程序分如下三步:1)先用100:1的二氯甲烷-甲醇溶液(由二氯甲烷和甲醇组成的液体,该液体中二氯甲烷和甲醇的体积比为100:1)洗脱2500mL;2)接着用50:1的二氯甲烷-甲醇溶液(由二氯甲烷和甲醇组成的液体,该液体中二氯甲烷和甲醇的体积比为50:1)洗脱4500mL;3)接着用20:1的二氯甲烷-甲醇溶液(由二氯甲烷和甲醇组成的液体,该液体中二氯甲烷和甲醇的体积比为20:1)洗脱4500mL。从洗脱程序开始不间断收集洗脱出的液体,每份收集500mL洗脱液,共收集23份。收集的流出液经TLC检测、合并,得到7个馏分即馏分F1至F7。
将硅胶柱层析收集的馏分F2(第6份到第10份收集液,用50:1的二氯甲烷-甲醇溶液洗脱出的液体)通过旋转蒸发仪浓缩,将浓缩物过凝胶柱层析,流动相为二氯甲烷-甲醇溶液。凝胶柱层析中所用的分离介质是Sephadex LH-20,层析柱规格为34×1200mm(直径×长度),柱体积为850mL。洗脱程序如下:采用1:1的二氯甲烷-甲醇溶液(由二氯甲烷和甲醇组成的液体,该液体中二氯甲烷和甲醇的体积比为1:1)洗脱2010mL。从洗脱程序开始不间断收集洗脱出的液体,每管收集15mL,共计134管。收集的流出液经TLC检测、合并,得到8个馏分,即馏分F2-1至F2-8。馏分F2-4(即收集的第91管到第98管收集液)进行HPLC制备分离,以乙腈-水溶液(由乙腈和水组成的液体,该液体中乙腈和水的体积比为40:60)进行洗脱(流速4mL/min),收集保留时间为17.5min的洗脱峰,得到式Ⅱ所示的化合物。HPLC制备分离中所用的分离介质是Capcell C18 MG-Ⅱ,填料粒径为5μm,层析柱直径为10mm,长为250mm。
将硅胶柱层析收集的馏分F3至F6(即第11份至第21份收集液,用50:1的二氯甲烷-甲醇溶液洗脱出的液体和用20:1的二氯甲烷-甲醇溶液洗脱出的液体)合并后进行凝胶柱层析,流动相为甲醇。凝胶柱层析中所用的分离介质是Sephadex LH-20,层析柱规格为34×1200mm(直径×长度),柱体积为850mL。洗脱程序如下:采用甲醇洗脱2625mL(约3个柱体积)。从洗脱程序开始不间断收集洗脱出的液体,每管收集15mL,共收集175管。流出液经TLC检测、合并,得到8个馏分即馏分F3-1至F3-8。将收集的4个馏分F3-2至F3-5(第126管至第149管收集液)进行HPLC制备分离,以乙腈-水溶液(由乙腈和水组成的液体,该液体中乙腈和水的体积比为40:60)进行洗脱(流速4mL/min),收集保留时间为12.3min的洗脱峰,得到式I所示的化合物,收集保留时间为17.5min的洗脱峰,得到式Ⅱ所示的化合物。HPLC制备分离中所用的分离介质是Capcell C18 MG-Ⅱ,填料的粒径为5μm,层析柱直径为10mm,长为250mm。
3.从固体发酵产物中制备式I与式Ⅱ所示的化合物
3.1北里孢菌CPCC204717固体发酵产物的制备
将步骤1的种子液按10%的接种量,转接于大米固体发酵培养基[配方为:大米100g,蛋白胨0.3g,用去离子水100mL,121℃灭菌15分钟得到大米固体发酵培养基],共50瓶,28℃静置培养30天,得到北里孢菌CPCC204717固体发酵产物。
3.2分离纯化式I与式Ⅱ所示的化合物
收集3.1的北里孢菌CPCC204717固体发酵产物,加入乙酸乙酯超声提取3次(10L×3),每次60min,提取液经减压回收溶剂后,得到乙酸乙酯提取物。
将乙酸乙酯提取物经C18反相闪式柱(520g,60mm×440mm(直径×长度)色谱分离,柱体积为730mL。该色谱分离中所用的洗脱程序如下:1)采用10%的甲醇水溶液(由甲醇和水组成的液体,该液体中甲醇和水的体积比为10:90)洗脱3000mL;2)接着用30%的甲醇水溶液(由甲醇和水组成的液体,该液体中甲醇和水的体积比为30:70)洗脱2500mL;3)接着用50%的甲醇水溶液(由甲醇和水组成的液体,该液体中甲醇和水的体积比为50:50)洗脱2500mL;4)接着用70%的甲醇水溶液(由甲醇和水组成的液体,该液体中甲醇和水的体积比为70:30)洗脱3400mL。收集用70%的甲醇水溶液洗脱出的液体,将该液体命名为馏分F4。
将馏分F4经凝胶柱层析分离,流动相为80%的甲醇水溶液。凝胶柱层析中所用的分离介质是Sephadex LH-20,柱子规格为34×1200mm(直径×长度),柱体积为850mL。洗脱程序如下:采用80%的甲醇水溶液(由甲醇和水组成的液体,该液体中甲醇和水的体积比为80:20)洗脱2400mL,从洗脱程序开始不间断收集洗脱出的液体,每管收集15mL,共收集160管。流出液经TLC检测、合并得到12个洗脱馏分即馏分F4-1至F4-12。馏分F4-4(第80至第90管收集液)经凝胶柱层析分离,流动相为70%甲醇水溶液。凝胶柱层析中所用的分离介质是Toyopearl HW-40F,柱子规格为30×1200mm(直径×长度),柱体积为640mL。洗脱程序如下:采用70%甲醇水溶液(由甲醇和水组成的液体,该液体中甲醇和水的体积比为70:30)洗脱1920mL(3个柱体积),从洗脱程序开始不间断收集洗脱出的液体,每管收集15mL,得到128管,经TLC检测、合并,得到11个馏分,命名为馏分F4-4-1至F4-4-11。将F4-4-3至F4-4-11的9个馏分(第75至第128管收集液)进行HPLC色谱分离纯化(色谱柱参数为YMC-Pack Ph 5μm,10mm×250mm;流动相为60%甲醇水溶液(由色谱甲醇和水组成的液体,该液体中甲醇和水的体积比为60:40),流速4mL/min),收集保留时间为28.0min的洗脱峰,得到式I所示的化合物,收集保留时间为36.5min的洗脱峰,得到式Ⅱ所示的化合物。
4.式I与式Ⅱ所示的化合物的结构鉴定
4.1式Ⅰ所示化合物:南平霉素D的结构鉴定
白色粉末状,易溶于甲醇、氯仿、DMSO。高分辨电子喷雾质谱(HRESIMS)显示出准分子离子峰m/z 766.2967[M+H]+(C35H44O9N9S理论值为766.2977,图1),结合核磁共振波谱(NMR)数据,确定其分子式为C35H43O9N9S,不饱和度为20。式I所示化合物的IR光谱(图2)在3392、3299、1648、1539,1508cm-1显示出特征吸收峰,提示结构中存在氨基、羰基和苯环等官能团。式I所示化合物的1H NMR谱(CDCl3,图3,表1)在低场显示出6个芳香或烯氢质子信号δH8.10(s,H-17),6.98(d,H-22),6.42(d,H-26),7.02(t,H-27),6.78(d,H-28),5.19(q,H-8)以及7个活泼质子信号。在高场区显示出3个双峰甲基、2个单峰甲基、1个氮甲基、1个甲氧基信号,以及在1.54~5.50显示出9个亚甲基和次甲基质子信号。式I所示化合物的13C NMR谱(图4)显示出35个碳信号,DEPT谱(图5)表明这些碳分别为16个季碳(其中1个酮羰基碳δC200.5,7个酰胺羰基碳),9个次甲基碳[其中6个为sp2杂化碳,3个为含氮脂肪次甲基δC 52.5(C-20),48.6(C-3)和45.4(C-14)],3个亚甲基和7个甲基碳。以上NMR数据显示出肽类化合物的典型特征,包含7个酰胺羰基碳、6个α-氨基碳和7个氨基质子。
通过2DNMR谱(1H-1H COSY,HSQC,HMBC)(图6-8)数据分析,确定式I所示化合物中存在1个甘氨酰(Gly)、2个丙氨酰(Ala)、1个2-氨基-2-丁烯酰(Dhb)、1个N-甲基甘氨酰(Sar)、1个以噻唑酰(Tzl)形式出现的半胱氨酸和1个甲氧基取代的色氨酰(OMeTrp)、一个α-甲基脱氢苏氨酰(MeDht)结构单元。通过分析HMBC谱的远程C-H相关信号,进一步确定了各氨基酸残基及其连接方式。其中,NH-1和H2-1与C-1和C-2的相关信号表明结构中存在甘氨酰单元;NH-3与C-2,C-3和C-4,H-3与C-2和C-6,H3-4与C-3和C-6的相关信号,表明存在一个丙氨酰单元,与甘氨酸残基连接;NH-7与C-6、C-7和C-8,H3-9与C-8,H-8与C-10的相关信号,表明结构中存在一个2-氨基-2-丁烯酰单元,与丙氨酸残基连接;H-12a与C-13和C-10,H3-11与C-10和C-12的相关信号,表明存在一个N-甲基甘氨酰单元,与2-氨基-2-丁烯酸残基连接;NH-14与C-13、C-14,H3-15与C-14、C-16的相关表明存在丙氨酰单元,与N-甲基甘氨酸残基连接;H-17与C-16、C-18、C-19的相关,结合其化学位移表明结构中含有1,3-噻唑结构单元与丙氨酰单元连接;NH-20与C-19、C-20、C-31,H2-21与C-20、C-22、C-23、C-24,NH-22与C-23、C-24、C-29,H-26与C-24、C-28,H-28与C-24和C-26,H3-30与C-25的相关,表明分子中存在4-甲氧基色氨酰基单元,与1,3-噻唑酰基连接。在ROESY谱(图9)中,NH-22与H-28相关,OMe-30与H-26相关,进一步确证甲氧基连接在色氨酸的4位(C-25)。NH-32与C-31、C-32、C-34、C-36,H3-33与C-32、C-34、C-36,H3-35与C-34的相关,表明存在2-甲基脱氢苏氨酰单元,与色氨酰残基连接。最后,NH-1和H2-1与C-36的相关信号,表明2-甲基脱氢苏氨酰与甘氨酰残基连接。H3-9与NH-7的ROESY相关峰,表明C-7与C-8的双键构型为Z构型。因而确定了南平霉素D的平面结构为环[甘氨酰-丙氨酰-(2-氨基-2-丁烯酰)-(N-甲基甘氨酰)-丙氨酰-1,3-噻唑酰-4-甲氧基色氨酰-(2-甲基脱氢苏氨酰)]。
式I所示化合物的绝对构型利用高级Marfey’s方法以及量子化学NMR计算的方法确定。L-丙氨酸标准品的L和D-FDLA衍生物(m/z 384)的保留时间分别为28.2和34.3min。式I化合物酸水解产物中丙氨酸的L、D-FDLA衍生物(m/z 384)峰保留时间分别为34.2和28.2min,与标准品保留时间顺序相反。因此,结构中的丙氨酰单元确定为D型。L-4-甲氧基色氨酸标准品的L-和D-FDLA衍生物(m/z 529)保留时间分别为39.2和45.4min,式I化合物水解产物的4-甲氧基色氨酸的L、D-FDLA衍生物(m/z 592)峰保留时间分别为39.1和45.4min,与标准品保留时间一致。因此结构中的4-甲氧基色氨酸残基确定为L型。为确定丙氨酰-1,3-噻唑酰结构单元(Ala-Tzl)中丙氨酰的绝对构型,先将式I所示化合物进行臭氧氧化以释放出丙氨酰单元,然后将臭氧化产物经酸水解后,分别与L、D-FDLA进行衍生化反应。式I化合物臭氧化产物的L/D-FDLA-Ala衍生物m/z 384峰保留时间分别为34.2和28.2min,与L-丙氨酸标准品衍生物保留时间顺序相反。因此式I所示丙氨酰-1,3-噻唑酰结构单元中的丙氨酰结构单元确定为D型。
在化合物南平霉素D中,由于除α-甲基脱氢苏氨酰(α-Me-Dht)外的手性氨基酸的构型,如D-Ala、D-Ala-Tzl和L-4-OMe-Trp均已确定,因此该化合物的绝对构型仅存在两种可能,即3R,14R,20S,32S(异构体A)和3R,14R,20S,32R(异构体B)。为确定其绝对构型,对这两种异构体进行量子化学NMR计算(Willoughby,P.H.et al.Nat.Protoc.2014,9,643-660.)。利用校正平均误差(corrected mean absolute error,CMAE)对两种异构体NMR化学位移计算值和实验值进行比较分析,结果显示具有3R,14R,20S,32S构型的异体体A的13C和1H的校正平均误差值均小于具有3R,14R,20S,32R构型的异构体B(表2)。进一步利用DP4+方法(Grimblat,N.et al.J.Org.Chem.2015,80,12526-12534.)对计算值进行分析,结果显示,基于13C和1H数据计算,异构体A的可能性为100%(表3)。因此,南平霉素D的绝对构型确定为3R,14R,20S,32S。
表1.南平霉素D与泽尔科瓦霉素的1H和13C NMR数据(CDCl3)
表2南平霉素D两种异构体的NMR计算数据
a表示校正平均绝对误差,b表示最大绝对误差。
表3南平霉素D两种异构体的DP4+分析
式I所示化合物(南平霉素D,nanpingmycin D)的理化数据:白色粉末状。[α]20 D+86.5(c 1.00,MeOH);UV(MeOH)λmax(logε)222(4.78),282(3.69)nm;ECD(c 1.63×10-4M)λmax(Δε)205(+82.72),252(-8.89)nm;IR vmax:3392,3299,2921,2850,1648,1539,1508,1469,1254,1206,970,738cm-1;1H-NMR(CDCl3,600MHz)数据见表1,13C NMR(CDCl3,150MHz)数据见表1;HRESMS:m/z 766.2967[M+H]+(C35H44N9O9S理论值为766.2977)。式I所示化合物的结构式如下:
4.2式Ⅱ所示化合物:泽尔科瓦霉素(zelkovamycin)的结构鉴定
白色粉末状,易溶于甲醇、氯仿、DMSO。高分辨电子喷雾质谱(HRESIMS)显示出准分子离子峰m/z 780.3142[M+H]+(C36H46O9N9S计算值780.3134,图10),结合NMR数据确定其分子式为C36H45O9N9S。式Ⅱ所示化合物在CDCl3中测得的C谱和H谱(图11-12)数据与文献报道的zelkovamycin化合物一致,确定式II所示化合物与zelkovamycin实际上是同一个化合物。然而文献中zelkovamycin结构中的甲氧基取代在色氨酸的C-7位(即C-28),而式I化合物中甲氧基确定取代在中色氨酰单元的C-4位(即C-25)。从化合物的生源途径上考虑,式II所示化合物应当具有相同的色氨酸取代形式。在ROESY谱(图13)中,也观察到NH-22与H-28以及甲氧基质子与H-26的相关信号,证实了在化合物Ⅱ中甲氧基同样取代在色氨酸单元的C-4位,即C-25位。因此,将zelkovamyicn的平面结构修订为环[甘氨酰-(2-氨基)丁酰-(2-氨基-2-)丁烯酰-(N-甲基)甘氨酰-丙氨酰-1,3-噻唑酰-(4-甲氧基)色氨酰-(2-甲基)脱氢苏氨酰]。
式Ⅱ所示化合物的绝对构型利用高级Marfey’s反应以及量子化学NMR计算的方法确定。式Ⅱ化合物中,4-甲氧基色氨酰L-、D-FDLA衍生物(m/z 529)分别与L-4-甲氧基色氨酸标准品保留时间一致;2-氨基丁酰基L、D-FDLA衍生物(m/z 398)分别与L-2-氨基丁酸标准品的FDLA衍生物保留时间顺序相反。因此确定,4-甲氧基色氨酰和2-氨基丁酰单元分别为L和D构型。此外,将式Ⅱ化合物进行臭氧氧化然后进行酸水解、Marfey反应,确定丙氨酰-噻唑酰(Ala2-Tzl)中丙氨酰构型为D构型。从生源途径上考虑,式Ⅱ化合物与式I化合物中具有相同的甲基脱氢苏氨酸,因此推测二者具有相同的立体构型,因此式II化合物中C-32构型也确定为32S。据此,式Ⅱ所示化合物的绝对构型确定为3R,14R,20S,32S。
式II所示化合物(泽尔科瓦霉素,zelkovamycin)的理化数据:白色粉末状,易溶于甲醇、氯仿、DMSO。[α]20 D+86.0(c 1.00,MeOH);UV(MeOH)λmax(logε)220(4.68),283(3.63)nm;ECD(c 1.60×10-4M)λmax(Δε)205(+59.49),249(-5.82)nm;1H-NMR(CDCl3,600MHz)数据见表3,13C NMR(CDCl3,150MHz)数据见表3;HRESMS:m/z 780.3142[M+H]+(C36H46N9O9S理论值为780.3134)。式II所示化合物的结构式如下:
实施例3、南平霉素D(nanpingmycin D)与泽尔科瓦霉素(zelkovamycin)的抗病毒活性试验
1.抗H1N1甲型流感病毒实验方法
下述A/WSN/33(H1N1)重组流感病毒和293T-Gluc细胞(张永欣,等.中国医药生物技术,2017,12(3),207-214),公众可从申请人获得该生物材料,该生物材料只为重复本发明的相关实验所用,不可作为其它用途使用。
精确称取式I-Ⅱ所示化合物以及阳性对照药物利巴韦林,溶于DMSO中,配置成浓度为20mM的母液供活性测试用。
本实验以293T-Gluc细胞为病毒宿主,测定样品抑制病毒携带报告基因荧光素酶活性,计算到EC50和CC50。
EC50的测试方法:将293T-Gluc细胞以2.0×105个/mL浓度接种于96孔板中,每孔100μL(于100μL含10%FBS的DMEM培养液中培养)。5%CO2,37℃培养24h后,每孔加入1μL梯度稀释的待测化合物溶液,式I所示化合物在培养体系中的含量分别为10、2、0.5、0.1、0.05、0.02μM,式II所示化合物在培养体系中的含量分别为150、100、50、20、10、5μM,阳性药物在培养体系中的含量分别为80、50、40、20、10μM,同时设置空白对照(只加100μL DMEM培养基)和阴性对照(加入1μL DMSO)。孵育2h后利用A/WSN/33(H1N1)重组流感病毒感染细胞(MOI=0.3),继续培养24h后,每孔取10μL上清,于多功能酶标仪(Berthold Centro LB960)测定荧光素酶活性,计算各样品的抑制率并计算EC50(将病毒抑制50%所需的浓度),实验重复三个复孔。其中,A/WSN/33(H1N1)重组流感病毒的制备方法如下:在10cm细胞培养皿中以3:1比例接种1.8×106个293T细胞和0.6×106个MDCK细胞。培养24h后,转染甲型流感病毒(IAV)A/WSN/33(H1N1)的8质粒(pHW181-PB2,pHW182-PB1,pHW183-PA,pHW184-HA,pHW185-NP,pHW186-NA,pHW187-M,pHW188-NS),转染量为1.2μg,转染试剂为Lipofectamine2000,根据使用说明书,每皿使用40μl。转染6h后,更换为新鲜DMEM培养基。转染24h后加入终浓度为1μg/mL的TPCK-trypsin。48h后收上清,1000rpm离心5min去掉细胞碎片,用0.45μm滤膜过滤,分装成小份,得到A/WSN/33(H1N1)重组流感病毒,保存于-80℃冰箱。其中,甲型流感病毒(IAV)8质粒反向遗传系统获赠于Dr.Robert G.Webster,分别为:pHW181-PB2,pHW182-PB1,pHW183-PA,pHW184-HA,pHW185-NP,pHW186-NA,pHW187-M,pHW188-NS(Hoffmann,E.,G.Neumann,et al.A DNA transfection system forgeneration of influenza A virus from eight plasmids[J].Proc Natl AcadSci U SA,2000,97:6108-6113)。
CC50的测定:293T-Gluc细胞细胞接种于96孔板,每孔2.0×104个细胞,于100μL含10%FBS的DMEM培养液中培养。细胞铺板24h后每孔加入1μL梯度稀释的待测化合物溶液,使待测化合物(式I所示化合物、式II所示化合物或利巴韦林)在培养体系中的含量分别为式I所示化合物在培养体系中的含量分别为10、2、0.5、0.1、0.05、0.02μM,式II所示化合物在培养体系中的含量分别为150、100、50、20、10、5μM,利巴韦林在培养体系中的含量分别为80、50、40、20、10μM,同时设置空白对照(只加100μL DMEM培养基)和阴性对照(加入1μL DMSO),37℃孵育48h。取出96孔板,每孔加入10μL CCK-8,37℃继续孵育1-2h后,使用多功能酶标仪(Berthold Centro LB 960)检测各孔在450nm波长处的光吸收值,用以计算半数细胞毒性浓度CC50(致使50%细胞死亡的药物浓度)。实验重复三次。
2.抗丙型肝炎细胞(HCV)实验方法
下述丙型肝炎病毒J6/JFH(Peng,Z.-G..et al.Hepatology 2010,52,845-853),公众可从申请人获得该生物材料,该生物材料只为重复本发明的相关实验所用,不可作为其它用途使用。
精确称取式I-Ⅱ所示化合物,溶于DMSO中,配置成浓度为50mM的母液供活性测试用,阳性药物索非布韦配置成浓度为20mM的母液供活性测试用。
人肝癌细胞系Huh7.5细胞以3×104个/cm2的密度接种到96孔板中,每孔100μL(于100μL含10%FBS的DMEM培养液中培养)5%CO2,37℃培养24h后,待细胞贴壁后,用丙型肝炎病毒J6/JFH上清液以MOI=0.1病毒感染量感染正常Huh7.5细胞,同时加入相应药物或阳性药溶液,使药物(式I所示化合物或式II所示化合物)在培养体系中的含量分别为500、100、20、4、0.8、0.16、0.032、0.0064μM,索非布韦在培养体系中的含量分别为200、40、8、1.6、0.32、0.064、0.0128、0.00256μM,同时设置空白对照(只加等体积的培养基)和阴性对照(加入等体积的DMSO)。继续培养72小时后,吸弃培养上清,细胞用PBS漂洗一次,加入固定液,室温固定20分钟,吸弃固定液,加入0.3%Triton-100穿孔20分钟,加入封闭液室温封闭1小时,加入50μL用封闭液稀释的一抗(HCV Core和GAPDH)(HCV Core,丙型肝炎核心蛋白小鼠单克隆抗体,货号ab2740,购自英国Abcam公司。GAPDH,甘油醛-3-磷酸脱氢酶兔单克隆抗体,货号10494-1-AP,购自Proteintech公司),4℃孵育过夜,TBST漂洗四次,加入50μL抗体稀释液稀释的二抗(IRDye 680标记的山羊抗小鼠二抗,货号LI-COR Cat.#926-32220,IRDye 800CW标记的山羊抗兔二抗,货号LI-COR Cat.#926-32211,购自LI-CORBiosciences公司),室温避光孵育1小时,TBST漂洗四次后用Odyssey近红外双色荧光成像系统扫描成像。用GAPDH荧光信号值计算各浓度药物的抑制率,再用Reed&Muench法计算药物半数细胞毒浓度CC50,用HCV Core/GAPDH值计算各浓度药物的抑制率,再用Reed&Muench法计算半数抑制浓度EC50。实验重复三次。
1.实验结果
南平霉素D(nanpingmycin D)与泽尔科瓦霉素(zelkovamycin)的抗病毒活性如表2所示。南平霉素D对H1N1型甲型流感病毒A/WSN/33(H1N1)重组流感病毒具有较强的抑制作用,其半数有效抑制浓度(EC50)为0.30±0.05μM,强于阳性药物利巴韦林50倍,强于泽尔科瓦霉素150倍。此外,南平霉素D和泽尔科瓦霉素对丙型肝炎病毒J6/JFH均显示抗较好的HCV活性,EC50分别为3.2和6.1μM。
南平霉素D和泽尔科瓦霉素来自微生物发酵天然产物,相较合成产物易得、环保、低污染,因此具有开发为抗流感病毒和HCV等广谱抗病毒药物的应用前景。
表4.南平霉素D与泽尔科瓦霉素抗病毒活性结果
SI(选择指数):=CC50/EC50。nt表示未检测。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
SEQUENCE LISTING
<110> 中国医学科学院医药生物技术研究所
<120> 环肽化合物及其制备方法和抗病毒用途
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cactctggga caagccctgg aaacggggtc taataccgga tatgaccttc ctccgcatgg 180
gggttggtgt aaagctccgg cggtgcagga tgagcccgcg gcctatcagc ttgttggtgg 240
ggtaatggcc taccaaggcg acgacgggta gccggcctga gagggcgacc ggccacactg 300
ggactgagac acggcccaga ctcctacggg aggcagcagt ggggaatatt gcacaatggg 360
cgaaagcctg atgcagcgac gccgcgtgag ggatgacggc cttcgggttg taaacctctt 420
tcagcaggga agaagcgcaa gtgacggtac ctgcagaaga agcaccggct aactacgtgc 480
cagcagccgc ggtaatacgt agggtgcgag cgttgtccgg aattattggg cgtaaagagc 540
tcgtaggcgg cctgtcgcgt cggatgtgaa agcccggggc ttaaccccgg gtctgcattc 600
gatacgggca ggctagagtg tggtagggga gatcggaatt cctggtgtag cggtgaaatg 660
cgcagatatc aggaggaaca ccggtggcga aggcggatct ctgggccatt actgacgctg 720
aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg 780
ttgggaacta ggtgttggcg acattccacg tcgtcggtgc cgcagctaac gcattaagtt 840
ccccgcctgg ggagtacggc cgcaaggcta aaactcaaag gaattgacgg gggcccgcac 900
aagcagcgga gcatgtggct taattcgacg caacgcgaag aaccttacca aggcttgaca 960
tatgccggaa acatccagag atgggtgccc ccttgtggtc ggtatacagg tggtgcatgg 1020
ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttgt 1080
tctgtgttgc cagcatgcct ttcggggtga tggggactca caggagactg ccggggtcaa 1140
ctcggaggaa ggtggggacg acgtcaaatc atcatgcccc ttatgtcttg ggctgcacac 1200
gtgctacaat ggtcggtaca aagggctgcg atgccgcgag gcggagcgaa tcccaaaaag 1260
ccggcctcag ttcggattgg ggtctgcaac tcgaccccat gaagttggag ttgctagtaa 1320
tcgcagatca gcatgctgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcacg 1380
tcacgaaagt cggtaacacc cgaagccggt ggcctaaccc gtaaggggag gagccgtcga 1440
aggtgggacc agcgattggg acgaagtcg 1469
Claims (10)
3.根据权利要求1或2所述的应用,其特征在于:所述病毒为下述至少一种:
1)正黏液病毒科病毒,
2)流感病毒,
3)甲型流感病毒,
4)黄病毒科病毒,
5)肝炎病毒。
4.权利要求书1或2中所述的化合物或其药学上可接受的盐。
5.病毒抑制剂或抗病毒药物,其特征在于:所述病毒抑制剂或抗病毒药物含有权利要求1或2中所述的化合物或其药学上可接受的盐。
7.北里孢菌,其特征在于:所述北里孢菌为北里孢菌(Kitasatospora sp.)CPCC204717,其在中国微生物菌种保藏管理委员会普通微生物中心的登记入册编号为CGMCC No.20381。
8.抗病毒的菌剂,其特征在于:所述菌剂含有权利要求7所述的北里孢菌或/和所述北里孢菌的代谢物。
9.权利要求5所述的北里孢菌或权利要求6所述的菌剂在制备病毒抑制剂或抗病毒药物中的应用。
10.根据权利要求5所述的病毒抑制剂或抗病毒药物、权利要求8所述的菌剂或权利要求9所述的应用,其特征在于:所述病毒为下述至少一种:
1)正黏液病毒科病毒,
2)流感病毒,
3)甲型流感病毒,
4)黄病毒科病毒,
5)肝炎病毒。
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