CN116333013A - 一种靶向配体 - Google Patents
一种靶向配体 Download PDFInfo
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- CN116333013A CN116333013A CN202211333934.8A CN202211333934A CN116333013A CN 116333013 A CN116333013 A CN 116333013A CN 202211333934 A CN202211333934 A CN 202211333934A CN 116333013 A CN116333013 A CN 116333013A
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- sirna
- nucleotides
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Abstract
本公开涉及基因工程技术领域,具体地,本公开涉及一种靶向配体。本发明提供的靶向配体与特异性小干扰RNA序列形成siRNA缀合物,靶向ANGPTL3,通过降解细胞中ANGPTL3基因的转录物,从而降低ANGPTL3蛋白表达量,因此本公开提供的靶向配体形成的siRNA缀合物可用于预防和/或治疗血脂异常疾病。
Description
本申请为分案申请,母案为PCT申请进入中国国家阶段的申请,母案申请号为2021800229023,PCT国际申请日为2021年09月30日,进入中国国家阶段日期为2022年10月18日,母案发明名称为“血管生成素样3的siRNA及其用途”。
技术领域
本公开涉及基因工程技术领域,具体地,本公开涉及一种新的可作为靶向配体的化合物。
背景技术
高脂血症又称血脂异常,是脂肪代谢或运转异常,使血浆脂质高于正常值的一种全身性疾病。血脂异常的临床表现主要包括两大方面:(1)脂质在真皮内沉积所引起的黄色瘤;(2)脂质在血管内皮沉积所引起的动脉粥样硬化,产生冠心病和周围血管病等。据调查,成人中血总胆固醇(TC)或甘油三酯(TG)升高者约占10%至20%,甚至儿童中也有近10%者血脂升高。现有的治疗血脂异常的药物主要有他汀类、胆固醇吸收抑制剂、树脂类、普罗步考、贝特类和烟酸及其衍生物。
血管生成素样蛋白3(ANGPTL3,NM_014495.4)是一种主要在肝脏细胞中表达的分泌蛋白。已有研究表明,血管生成素样蛋白3(ANGPTL3)是LDL-C、HDL-C和甘油三酯代谢的关键调节因子,具有多种潜在的作用节点,ANGPTL3的功能缺失突变可导致LDL-C、VLDL-C、HDL-C和甘油三酯(TG)降低,从而使基于GWAS的心血管疾病风险降低,且没有已知的遗传缺陷的不良表型。因此,抑制ANGPTL3的活性可以有效预防或治疗血脂异常。
现有技术中多采用ANGPTL3抗体抑制其活性。
申请号为CN201280038908.0的中国专利中公开了一种完全人源化抗体或人抗体的抗原结合片段,其与人血管生成素样蛋白3(hANGPTL3)特异性结合,并抑制或干扰其至少一种活性。该人抗hANGPTL3抗体可用于治疗与ANGPTL3相关的疾病或失调,如高脂血症、高脂蛋白血症和血脂异常,包括高甘油三酯血症、高胆固醇血症、乳糜微粒血症等。
申请号为CN201780026147.X的中国专利中公开了用于治疗患有家族性高胆固醇血症,包括HeFH和HoFH的患者的方法。通过施用治疗有效量的特异性结合ANGPTL3的抗体或其抗原结合片段,其与其他药物组合,降低患者的至少一个脂质参数。可用于治疗高胆固醇血症,以及高脂血症、高脂蛋白血症和血脂异常,包括高甘油三酯血症、乳糜微粒血症。
相比于抗体,siRNA药物具有候选靶点丰富、研发周期短、临床开发成功率高等优点。因此,在慢性病用药方面,更具有优势。但siRNA药物自身存在的缺陷(如:稳定性差、跨膜难等)限制了其临床应用。对siRNA进行结构改造,或者构建缀合物,均可以在一定程度上提高siRNA的成药性。对于构建缀合物的组成分子进行探索,是发展用于血脂异常疾病的siRNA药物的必经之路,也是亟待解决的问题。
发明内容
本公开旨在至少在一定程度上解决相关技术中的技术问题之一。为此,本公开的一个目的在于提供一种用于抑制ANGPTL3表达的siRNA,本公开的发明人通过设计合适的特异性小干扰RNA序列,并使其与靶向配体结合形成siRNA缀合物,靶向ANGPTL3,通过降解细胞中ANGPTL3基因的转录物,从而降低ANGPTL3蛋白表达量,因此本公开提供的siRNA可用于预防和/或治疗血脂异常疾病。
为此,本公开一方面提供一种siRNA。根据本公开的实施例,所述siRNA包括一条正义链和一条反义链,所述反义链包括与所述正义链互补配对的互补性区域,其中所述正义链选自与SEQ ID NO:1-SEQ ID NO:154中每一条链的核苷酸序列区别不多于5个核苷酸的核苷酸序列,所述反义链选自与SEQ ID NO:155-SEQ ID NO:308中每一条链的核苷酸序列区别不多于5个核苷酸的核苷酸序列。
本公开的发明人通过设计合适的小干扰RNA(siRNA)序列特异性减少肝细胞合成ANGPTL3,同时避免脱靶效应。siRNA通过形成沉默复合体(RNA-induced silencingcomplex,RISC),与靶标基因(ANGPTL3基因)的mRNA的序列互补配对,使靶标基因的mRNA降解从而抑制靶标基因的表达,继而降低LDL-C、VLDL-C、HDL-C和甘油三酯(TG)的水平。
根据本公开实施例的siRNA,还可以具有以下附加技术特征的至少之一:
本公开还提供一种siRNA,所述siRNA选自如下任一组中的任一对siRNA:
(1)能够特异性的靶向血管生成素样蛋白3基因序列的第60-80位核苷酸;优选的,所述的siRNA的正义链选自SEQ ID NO:10,反义链选自SEQ ID NO:165;
(2)能够特异性的靶向血管生成素样蛋白3基因序列的第107-133位核苷酸;优选的,所述的siRNA的正义链选自SEQ ID NO:17,反义链选自SEQ ID NO:171,或者所述siRNA的正义链选自SEQ ID NO:18,反义链选自SEQ ID NO:172;
(3)能够特异性的靶向血管生成素样蛋白3基因序列的第163-187位核苷酸;优选的,所述的siRNA的正义链选自SEQ ID NO:19,反义链选自SEQ ID NO:173;
(4)能够特异性的靶向血管生成素样蛋白3基因序列的第304-388位核苷酸,优选的,能够特异性的靶向血管生成素样蛋白3基因序列的第304-359位核苷酸;更优选的,所述的siRNA的正义链选自SEQ ID NO:27,反义链选自SEQ ID NO:181,
或者,所述的siRNA的正义链选自SEQ ID NO:29,反义链选自SEQ ID NO:183,
或者,所述的siRNA的正义链选自SEQ ID NO:31,反义链选自SEQ ID NO:185,
或者,所述的siRNA的正义链选自SEQ ID NO:32,反义链选自SEQ ID NO:186,
或者,所述的siRNA的正义链选自SEQ ID NO:35,反义链选自SEQ ID NO:189,
或者,所述的siRNA的正义链选自SEQ ID NO:36,反义链选自SEQ ID NO:190;
(5)能够特异性的靶向血管生成素样蛋白3基因序列的第430-459位核苷酸;优选的,所述的siRNA的正义链选自SEQ ID NO:43,反义链选自SEQ ID NO:197,
或者,所述的siRNA的正义链选自SEQ ID NO:44,反义链选自SEQ ID NO:198;
(6)能够特异性的靶向血管生成素样蛋白3基因序列的第1360-1430位核苷酸,优选的,能够特异性的靶向血管生成素样蛋白3基因序列的第1397-1430位核苷酸;更优选的,所述的siRNA的正义链选自SEQ ID NO:145,反义链选自SEQ ID NO:299,
或者,所述的siRNA的正义链选自SEQ ID NO:150,反义链选自SEQ ID NO:304,
或者,所述的siRNA的正义链选自SEQ ID NO:151,反义链选自SEQ ID NO:305,
或者,所述的siRNA的正义链选自SEQ ID NO:152,反义链选自SEQ ID NO:306,
或者,所述的siRNA的正义链选自SEQ ID NO:154,反义链选自SEQ ID NO:308。
根据本公开的实施例,所述siRNA包括至少一个被修饰的核苷酸;
任选地,所述修饰的核苷酸选自下列至少之一:
5'-硫代磷酸酯基的核苷酸、5-甲基化胞嘧啶核苷酸、2'-O-甲基修饰的核苷酸、2'-O-2-甲氧乙基修饰的核苷酸、2'-氟代修饰的核苷酸、3'-氮取代修饰的核苷酸、2'-脱氧-2'-氟修饰的核苷酸、2'-脱氧修饰的核苷酸、锁定的核苷酸、脱碱基核苷酸、2'-氨基修饰的核苷酸、吗啉代核苷酸、多肽核苷酸、氨基磷酸酯,以及包括非天然碱基的核苷酸。
根据本公开的实施例,所述互补性区域的长度至少为17bp;
任选地,所述互补性区域的长度为18-21bp;
任选地,所述互补性区域的长度为19bp。
根据本公开的实施例,所述siRNA中正义链和反义链的长度不多于25bp;
任选地,所述siRNA中正义链和反义链的长度为18-25bp;
任选地,所述siRNA中正义链和反义链的长度为21bp。
根据本公开的实施例,所述siRNA中正义链和反义链中的碱基可以是一一互补配对,也可以是错位几个碱基,但具有至少17bp的互补性区域。
本公开另一方面提供一种siRNA缀合物,所述siRNA缀合物包括前面所述的siRNA和靶向配体,其中,所述siRNA与所述靶向配体共价连接;
优选的,所述靶向配体连接于所述siRNA中正义链;
更优选的,所述靶向配体通过硫代磷酸酯键与所述siRNA中正义链的5’端连接。
根据本公开的实施例,所述靶向配体包括至少一个N-乙酰基-半乳糖胺。
根据本公开的实施例,所述靶向配体为GalNAC靶头化合物。
根据本公开的实施例,所述GalNAC靶头化合物为1043、1046、1048,其结构如下式所示:
根据本公开的实施例,所述靶向配体连接于所述siRNA中正义链。
本公开另一方面提供一种药物组合物。根据本公开的实施例,所述药物组合物包括前述的siRNA和/或前述的siRNA缀合物,任选地,所述药物组合物还包括药学上可接受的辅料。
由此,根据本公开实施例的药物组合物可以用于抑制细胞合成ANGPTL3,从而降低LDL-C、VLDL-C、HDL-C和甘油三酯(TG)的水平,以治疗预防和/或治疗高血脂症和高甘油三酯血症。
本公开又一方面提供一种化合物。
所述的化合物具有以下任一种结构:
以上化合物在脱羟基保护基后得到的化合物结构如下:
本发明又一方面提供了前述化合物在制备siRNA缀合物中的应用。
TO23、TO25、TO26的化合物先形成中间体再与siRNA共价连接,所述的中间体选自:
本公开同时提供以上中间体。
本公开又一方面提供了前述的化合物和/或中间体在制备药物或试剂盒中的用途,所述药物或试剂盒用于抑制ANGPTL3基因表达。
优选地,所述药物或试剂盒用于预防和/或治疗血脂异常疾病;进一步优选地,所述血脂异常疾病包括高血脂症和高甘油三酯血症。
本公开又一方面提供一种试剂盒。根据本公开的实施例,所述试剂盒包括所述的siRNA和/或siRNA缀合物。
由此,根据本公开实施例的试剂盒可以用于抑制细胞中ANGPTL3基因表达,从而降低LDL-C、VLDL-C、HDL-C和甘油三酯(TG)的水平,以治疗预防和/或治疗高血脂症和高甘油三酯血症。
本公开又一方面提供一种抑制受试者ANGPTL3基因表达的方法,所述方法包括:向受试者施用前述的siRNA和/或前述的siRNA缀合物,以抑制ANGPTL3基因的表达。
本公开又一方面提供一种抑制细胞中ANGPTL3基因表达的方法。根据本公开的实施例,所述方法包括:用所述的siRNA和/或所述的siRNA缀合物转染所述细胞,以抑制所述细胞中ANGPTL3基因的表达。
根据本公开实施例的抑制细胞中ANGPTL3基因表达的方法,利用siRNA形成沉默复合体,与靶标基因ANGPTL3基因的mRNA的序列互补配对,使靶标基因的mRNA降解从而抑制靶标基因的表达,继而降低LDL-C、VLDL-C、HDL-C和甘油三酯(TG)的水平。
根据本公开的实施例,所述细胞源自哺乳动物;
任选地,所述细胞源自人;
任选地,所述细胞为肝脏细胞。
利用本公开提供的siRNA,在人的肝脏细胞中形成沉默复合体,与ANGPTL3基因的mRNA的序列互补配对,使ANGPTL3基因的mRNA降解从而抑制其表达,继而降低LDL-C、VLDL-C、HDL-C和甘油三酯(TG)的水平。
本公开又一方面提供所述的siRNA和/或所述的siRNA缀合物在制备药物或试剂盒中的用途。根据本公开的实施例,所述药物或试剂盒用于抑制ANGPTL3基因表达。
利用本公开提供的siRNA制备药物或试剂盒,所述药物或试剂盒通过其中的siRNA以降低细胞中ANGPTL3基因的表达水平,从而预防和/或治疗血脂异常疾病。
根据本公开的实施例,所述药物或试剂盒用于预防和/或治疗血脂异常疾病;
任选地,所述血脂异常疾病包括高血脂症和高甘油三酯血症;
任选地,所述药物或试剂盒用于抑制细胞中ANGPTL3基因表达。
本公开又一方面提供一种预防和/或治疗血脂异常疾病的方法。根据本公开的实施例,所述方法包括:向受试者施用所述的siRNA和/或所述的siRNA缀合物。
根据本公开的实施例,所述血脂异常疾病包括高血脂症和高甘油三酯血症。
本公开的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本公开的实践了解到。
本发明的有益效果:
本发明提供的靶向配体在与siRNA形成缀合物后,缀合物在细胞模型和小鼠模型中都可以降低ANGPTL3的表达,相对于对照组可降低50%以上的表达量,最高可降低近90%,具有良好的临床应用前景。
附图说明
本公开的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解,其中:
图1显示了表2中部分siRNA以0.1nM浓度转染Hep 3B细胞后,利用实时定量PCR检测的细胞中ANGPTL3基因(图中简写为ANL3)的表达量结果;
图2显示了表2中部分siRNA以10nM浓度转染Hep 3B细胞后,利用实时定量PCR检测的细胞中ANGPTL3基因(图中简写为ANL3)的表达量结果;
图3显示了实施例3中合成的GalNAc-siRNA缀合物;
图4显示了实施例4中各个缀合物的活性测试结果(EC50值)。
具体实施方式
下面详细描述本公开的实施例。下面描述的实施例是示例性的,仅用于解释本公开,而不能理解为对本公开的限制。
“药学可接受的载体”在本领域中是公认的,包括适于将本公开的化合物施用于哺乳动物的药学可接受的材料、组合物或载体。所述载体包括参与携带主体物质或将其从一个器官或机体的一部分转移到另一个器官或机体的另一部分的液体或固体填充剂、稀释剂、赋形剂、溶剂或包封材料。各载体在与制剂中的其它成分相容和对患者无害的意义上必须是“可接受的”。可用作药学可接受的载体的材料的一些实例包括:糖类,如乳糖、葡萄糖和蔗糖;淀粉类,如玉米淀粉和马铃薯淀粉;纤维素及其衍生物,如羧甲基纤维素钠、乙基纤维素和醋酸纤维素、粉状西黄蓍胶、麦芽、明胶、滑石粉,赋形剂如可可脂和栓剂蜡类;油类,如花生油、棉子油、红花油、芝麻油、橄榄油、玉米油和豆油;二醇类,如丙二醇;多元醇类,如甘油、山梨醇、甘露醇和聚乙二醇;酯类,如油酸乙酯和月桂酸乙酯;琼脂;缓冲剂,如氢氧化镁和氢氧化铝;海藻酸;无热原的水;林格氏溶液;乙醇;磷酸盐缓冲液;和药物制剂中所用的其它无毒的可相容的物质。
在组合物中也可以存在润湿剂、乳化剂和润滑剂如十二烷基硫酸钠和硬脂酸镁,以及着色剂、释放剂、包衣剂、甜味剂、矫味剂和芳香剂、防腐剂和抗氧化剂。
本公开的药物组合物包括适于口服、鼻、局部、口含、舌下、直肠和/或胃肠外施用的那些。制剂可以方便地以单位剂型形式存在并且可以通过药学领域公知的任何方法来制备。可以与载体物质组合来制备单剂量形式的活性成分的量一般是产生治疗作用的化合物的量。一般而言,以百分之一为单位,该量为约1%至约99%活性成分,优选约5%至约70%,最优选约10至约30%。
术语“治疗”用于指获得期望的药理学和/或生理学效果。所述效果就完全或部分预防疾病或其症状而言可以是预防性的,和/或就部分或完全治愈疾病和/或疾病导致的不良作用而言可以是治疗性的。本文使用的“治疗”涵盖哺乳动物、特别是人的疾病,包括:(a)在容易患病但是尚未确诊得病的个体中预防疾病或病症发生;(b)抑制疾病,例如阻滞疾病发展;或(c)缓解疾病,例如减轻与疾病相关的症状。本文使用的“治疗”涵盖将药物或化合物给予个体以治疗、治愈、缓解、改善、减轻或抑制个体的疾病的任何用药,包括但不限于将含本文所述化合物的药物给予有需要的个体。
本公开提供一种用于抑制ANGPTL3表达的siRNA。根据本公开的实施例,所述siRNA包括一条正义链和一条反义链,所述反义链包括与所述正义链互补配对的互补性区域,其中所述正义链选自与SEQ ID NO:1-SEQ ID NO:154中每一条链的核苷酸序列区别不多于5个核苷酸的核苷酸序列,所述反义链选自与SEQ ID NO:155-SEQ ID NO:308中每一条链的核苷酸序列区别不多于5个核苷酸的核苷酸序列。
根据本公开的实施例,所述正义链除了包括表2中所示的SEQ ID NO:1-SEQ IDNO:154,还包括与表2中所示的正义链有1个、2个、3个、4个、5个核苷酸区别的连续核苷酸序列。
根据本公开的实施例,所述反义链除了包括表2中所示的SEQ ID NO:155-SEQ IDNO:308,还包括与表2中所示的反义链有1个、2个、3个、4个、5个核苷酸区别的连续核苷酸序列。
根据本公开的实施例,所述siRNA包括至少一个被修饰的核苷酸;
所述修饰的核苷酸选自下列至少之一:
5'-硫代磷酸酯基的核苷酸、5-甲基化胞嘧啶核苷酸、2'-O-甲基修饰的核苷酸、2'-O-2-甲氧乙基修饰的核苷酸、2'-氟代修饰的核苷酸、3'-氮取代修饰的核苷酸、2'-脱氧-2'-氟修饰的核苷酸、2'-脱氧修饰的核苷酸、锁定的核苷酸、脱碱基核苷酸、2'-氨基修饰的核苷酸、吗啉代核苷酸、多肽核苷酸、氨基磷酸酯,以及包括非天然碱基的核苷酸。
根据本公开的实施例,所述互补性区域的长度为18-21bp,例如为19bp。
根据本公开的实施例,所述siRNA中正义链和反义链的长度为18-25bp,例如为21bp。
根据本公开具体的实施例,所述siRNA中正义链和反义链的长度为21bp,所述正义链和反义链中的碱基一一互补,或者所述siRNA中正义链和反义链中具有19个连续的碱基互补,即所述互补性区域的长度为19bp。
根据本公开的实施例,用所述的siRNA转染肝脏细胞,以便抑制细胞中ANGPTL3基因的表达。
针对血管生成素样3(ANGPTL3)基因靶点,本公开的发明人设计合适的小干扰核酸(siRNA)序列,合成siRNA,利用转染试剂,将siRNA导入细胞内,形成沉默复合体(RNA-induce siliencing complex,RISC),特异性识别并靶向结合靶基因的mRNA序列,并在距离5’端10-11位剪辑之间切割mRNA,从而导致转录后基因沉默,调控血管生成素样3分泌蛋白表达。
根据本公开的实施例,所述siRNA与靶向配体通过共价键连接。
根据本公开的实施例,所述靶向配体包括至少一个N-乙酰基-半乳糖胺。
根据本公开的实施例,所述靶向配体连接于所述siRNA中正义链。
下面详细描述本公开的实施例。下面描述的实施例是示例性的,仅用于解释本公开,而不能理解为对本公开的限制。实施例中未注明具体技术或条件的,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。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
本实施例的部分合成路线可参考CN202110397429.9、CN202110008013.3;本申请的实施例以源引的方式加入上述两件专利申请。
实施例1体外细胞模型(Hep 3B细胞)测试小干扰核酸(siRNA)的活性
1)悬浮转染试剂配制:siRNA母液浓度为50μM,DEPC水稀释得10μMsiRNA体系,50μLOpti-MEM稀释得0.2μM siRNA体系,吹吸3-5次混匀(终浓度10nM)。50μL Opti-MEM稀释0.5μL 0.2μM的SiRNA得0.002μM的siRNA体系,吹吸3-5次混匀(终浓度0.1nM);50μL Opti-MEM稀释2μL RNAiMAX,吹吸3-5次混匀。分别混合转染试剂和小干扰核酸稀释液,吹吸3-5次混匀,室温下静置10min。
2)处理细胞:镜下观察Hep 3B细胞株汇合率>70%,进行细胞铺板,按2x105细胞/孔铺12孔板,每孔加入900μL含10%FBS DMEM培养基,将转染复合物加至12孔板中,置于37℃,5%CO2培养箱培养。
3)24h后,提取细胞的总RNA,通过实时定量PCR(Quantitative Real-Time PCR)检测细胞中ANGPTL3 mRNA序列的表达情况,其中用于扩增内参基因PPIB、ANGPTL3的PCR引物如表1所示:
表1:用于扩增内参基因PPIB、ANGPTL3的PCR引物序列
4)小干扰核酸对ANGPTL3表达水平的抑制率按如下等式计算:抑制率=[1-(实验组ANGPTL3 mRNA的表达量/实验组PPIB mRNA的表达量)/(阴性对照组ANGPTL3 mRNA的表达量/阴性对照组PPIB mRNA的表达量)]×100%。其中,各实验组为分别经小干扰核酸处理的细胞;阴性对照组(记为Blank)为未经任何小干扰核酸处理的细胞。
利用上述方法获得表2中154对siRNA分别以0.1nM和10nM的浓度转染Hep 3B细胞后,对ANGPTL3基因(NM_014495.4)表达的抑制率结果。
表2:154对靶向ANGPTL3的siRNA序列
附图1和2分别显示了表2中部分siRNA以0.1nM或10nM浓度转染Hep3B细胞后,利用实时定量PCR检测的细胞中ANGPTL3基因的表达量结果。表明附图中所示的siRNA不论是以0.1nM还是10nM浓度转染Hep 3B细胞,均能够明显降低ANGPTL3基因的表达。
实施例2GalNAc连接靶头的合成
一、GalNAc靶头1043的合成
按照以下方法,合成了TO-23及TP-23(1043靶头连接siRNA的前体)的一种非对映异构体。
1、中间体GN-17-01的合成
(1)在N2氛围下,将GC-1(12g,25.89mmol)溶于DCM(200mL)中,冰水浴降温至0-5℃,加入HBTU(11.78g,31mmol)和DIEA(10g,77.67mmol),搅拌10分钟;
(2)随后加入N-叔丁氧羰基-1,4-丁二胺(4.87g,25.89mmol),升温至25℃搅拌反应16小时,TLC显示原料基本消失;
(3)加入饱和的氯化铵溶液(100mL)淬灭,分液,DCM(100mL×2)萃取;
(4)合并有机相并用饱和食盐水(100mL)洗涤,无水Na2SO4干燥,过滤并浓缩。经柱层析纯化(DCM/MeOH=20/1)得白色固体化合物GN-17-01(15g,收率91%)。
2、中间体GN-17的合成
(1)将GN-17-01(15g,23.67mmol)溶于DCM(150mL)中,加入TFA(50mL),25℃搅拌1小时,TLC显示原料基本消失,浓缩;
(2)用乙腈(100mL×3)与TFA共沸除去多余TFA,得泡沫状固体GN-17(TFA盐,12.6g)。
3、中间体TO-23-01的合成
(1)在N2氛围下,将NC-4(2.6g,4.7mmol)溶于DCM(200mL)中,冰水浴降温至0-5℃,加入HATU(5.6g,14.83mmol)和DIEA(4.85g,37.6mmol)搅拌20分钟;
(2)随后加入GN-17(8.45g,15.5mmol),升温至25℃搅拌反应4小时。TLC检测,原料基本消失;
(3)加入饱和氯化铵溶液(50mL)淬灭,分液,DCM(100mL×2)萃取;
(4)合并有机相并用饱和食盐水(100mL)洗涤,无水Na2SO4干燥;
(5)过滤并浓缩得粗制品。经柱层析纯化(DCM/MeOH=10/1)得白色固体TO-23-01(6.3g,收率63.1%)。
4、化合物TO-23的合成
(1)向TO-23-01(6.3g,3.0mmol)的MeOH(100mL)溶液中加入10%Pd/C(600mg)和Pd(OH)2/C(600mg),H2置换3次,25℃搅拌反应3小时,TLC(DCM/MeOH=8/1)检测原料基本消失;
(2)过滤,浓缩得粗制品。经柱层析纯化(DCM/MeOH/TEA=10/1/0.1)得白色固体TO-23(4.5g,收率75%)。
1H NMR(400MHz,DMSO-d6)δ7.88-7.81(m,9H),7.14(s,1H),5.21(d,J=3.4Hz,3H),4.95(dd,J=11.2,3.4Hz,3H),4.53(d,J=8.5Hz,3H),4.07-3.97(m,9H),3.88(dt,J=11.0,9.0Hz,3H),3.77-3.71(m,3H),3.63-3.50(m,24H),3.49-3.41(m,8H),3.38-3.35(m,2H),3.08-2.98(m,12H),2.35–2.25(m,14H),2.10(s,9H),2.00(s,9H),1.89(s,9H),1.78(s,9H),1.40-1.33(s,12H).
MS(ESI):m/z[1/2M+H]+理论值1000.5,实测值1000.3。
5、化合物TP-23(1043靶头连接siRNA的前体)的合成
(1)N2氛围下,将TO-23(2.3g,1.15mmol)溶于干燥DCM(40mL)中,加入DIEA(0.86mL,5.2mmol),利用注射器缓慢滴加2-氰乙基-N,N-二异丙基氯代亚磷酰胺(0.46mL,2.1mmol)的干燥DCM(2mL)溶液。25℃反应1小时。TLC检测,原料基本消失;
(2)加入饱和NaHCO3(20mL)淬灭,分液,有机相用饱和NaHCO3(20mL)溶液,饱和食盐水(20mL)洗涤,无水MgSO4干燥,过滤浓缩得粗制品。经柱层析纯化(硅胶柱预先经1.5%TEA/DCM碱化,DCM/MeOH/TEA=15/1/0.1)得白色固体TP-23(1.8g,收率71.1%)。
1H NMR(400MHz,DMSO-d6)δ7.91-7.79(m,9H),7.15(s,1H),5.21(d,J=3.4Hz,3H),4.95(dd,J=11.2,3.4Hz,3H),4.53(d,J=8.5Hz,3H),4.06-3.97(m,9H),3.88(dt,J=11.1,8.9Hz,3H),3.78-3.66(m,6H),3.63-3.41(m,36H),3.07-2.98(m,12H),2.76(t,J=5.9Hz,2H),2.35-2.24(m,14H),2.10(s,9H),2.00(s,9H),1.89(s,9H),1.78(s,9H),1.40-1.33(m,12H),1.13(dd,J=6.7,4.1Hz,12H);
31P NMR(162MHz,DMSO-d6)δ147.81;
MS(ESI):m/z[1/2M+Na]+理论值1122.5,实测值1122.4。
二、GalNAc靶头1046的合成
按照以下方法,合成了TO25及TP-25(1046靶头连接siRNA的前体)的一种非对映异构体。
1、中间体NC-6-01的合成
(1)N2氛围下,向1000mL三口瓶中加入干燥的THF(300mL),冰浴降温至0-5℃搅拌,分批加入60% NaH(14g,354.8mmol),随后缓慢滴加2-氯乙氧基乙醇(40g,322.5mmol)的THF溶液(200mL),保温反应30分钟,再向反应瓶中滴加溴化苄(60.3g,354.8mmol),升至25℃搅拌16小时,TLC监测原料基本消耗完毕。
(2)缓慢滴加饱和氯化铵溶液(150mL)淬灭,分液,水相用EtOAc(100mL×2)萃取,合并有机相并用饱和食盐水(300mL)洗涤、无水Na2SO4干燥,过滤浓缩获得粗制产品。粗产品用硅胶柱层析纯化(石油醚/EtOAc=5/1)得淡黄色油状物化合物NC-6-01(53g,收率78%)。
MS(ESI):m/z[M+H]+理论值215.1,实测值215.1。
2、中间体NC-6-02的合成
(1)将乙二胺(196g,3.26mol)置于2000mL三口瓶中,加入乙腈(1000mL)、碳酸钾(90g,0.65mol)和碘化钠(60.6g,0.33mol)搅拌。随后将NC-6-01(70g,0.33mol)的乙腈(100mL)溶液缓慢滴加至反应瓶中,升温至60℃搅拌16小时,TLC检测原料基本消耗完毕。
(2)停止反应,浓缩,加入纯化水(300mL),浓盐酸调节pH至4-5,EtOAc(200mL×3)萃取三次,水相加入氢氧化钠固体调节pH至13-14,经DCM(200mL×3)萃取三次,合并有机相并用饱和食盐水(300mL)洗涤、无水Na2SO4干燥,过滤浓缩得淡黄色油状物NC-6-02(69.5g,87%)。
MS(ESI):m/z[M+H]+理论值239.2,实测值239.1。
3、中间体NC-6-03的合成
(1)将NC-6-02(69.5g,0.29mol)和溴乙酸叔丁酯(187g,0.96mol)加入到四氢呋喃(700mL)和纯化水(350mL)中,搅拌,冰水浴降温至5℃以下,加入碳酸钾(322g,2.34mol)。25℃搅拌反应14小时,TLC检测原料转化完全。
(2)反应液加入纯化水(300mL),静置分层,分出有机相,水相经EtOAc(200mL×2)萃取两次,合并有机相,加入饱和食盐水(500mL)洗涤、无水Na2SO4干燥,过滤浓缩得淡黄色油状物NC-6-03(201g)。
MS(ESI):m/z[M+H]+理论值581.4,实测值581.3。
4、中间体NC-6的合成
(1)将NC-6-03(23g,39.6mmol)溶于1,4-二氧六环(200mL)中,加入浓盐酸(40mL),升温至60℃反应2小时,TLC检测原料基本消耗完毕。
(2)浓缩,再次加入1,4-二氧六环(200mL)浓缩,得白色固体粗品,将粗品加入到乙酸乙酯(200mL)中打浆2小时,抽滤,收集滤饼,50℃真空烘干得得白色固体化合物NC-6(22.6g,96.9%)。
(3)MS(ESI):m/z[M+H]+理论值413.2,实测值413.1。
5、中间体TO-25-01的合成
(1)N2氛围下,将NC-6(1.5g,3.6mmol)、HBTU(4.5g,12.0mmol)和DIEA(4.75g,36mmol)加入到DCM(50mL)中搅拌30分钟,随后滴加GN-17(6.4g,12.0mmol)与DIEA(4.75g,36mmol)的DCM(50mL)溶液,25℃搅拌16小时,LCMS检测,原料基本消耗完毕。
(2)加入DCM(100mL)稀释,向反应液中加入1N盐酸溶液(80mL×2)洗涤,合并有机相,经饱和碳酸氢钠(100mL)洗涤、饱和食盐水(100mL)洗涤、无水Na2SO4干燥过滤浓缩获得粗制产品。粗产品经硅胶柱层析纯化(DCM/MeOH=7/1)得白色固体化合物TO-25-01(4.3g,收率60%)。
(3)MS(ESI):m/z[M/2+H]+理论值980.0,实测值979.9。
6、中间体TO-25的合成
(1)将TO-25-01(4.3g,2.2mmol)溶于甲醇(80mL)中,加入10%钯碳(1.0g),H2置换三次,25℃搅拌2小时,LCMS检测,原料基本消失。
(2)过滤,浓缩,加入DCM(20mL)溶解,缓慢滴加至MTBE(300mL)中,搅拌析晶30分钟,抽滤,得到白色固体化合物TO-25(3.7g,收率90%)。
1H NMR(400MHz,DMSO-d6)δ8.48(d,J=5.6Hz,1H),8.06(t,J=5.7Hz,2H),7.85(dd,J=11.7,6.8Hz,6H),5.21(d,J=3.3Hz,3H),4.95(dd,J=11.2,3.3Hz,3H),4.53(d,J=8.5Hz,3H),4.08-3.83(m,14H),3.75(p,J=4.8Hz,5H),3.68-3.26(m,28H),3.21-2.95(m,14H),2.30(q,J=7.9,6.7Hz,6H),1.94-1.78(m,,36H),1.41-1.38(m,12H);
MS(ESI):m/z[1/2M+H]+理论值934.9,实测值934.8。
7、TP-25的(1046靶头连接siRNA的前体)合成
(1)N2氛围下,将TO-25(700mg,0.37mmol)溶于干燥DCM(10mL)中,加入DIEA(0.31mL,1.9mmol),利用注射器缓慢滴加2-氰乙基-N,N-二异丙基氯代亚磷酰胺(0.19mL,0.74mmol)的干燥DCM(1mL)溶液,25℃反应30分钟,TLC检测,原料基本消失。
(2)加入饱和NaHCO3(10mL)淬灭,DCM(10mL)稀释,分液,有机相用饱和NaHCO3(10mL)溶液,饱和食盐水(10mL)洗涤,无水NaSO4干燥,过滤浓缩得粗制品。经柱层析纯化(硅胶柱预先经1.5%TEA/DCM碱化,DCM/MeOH/TEA=15/1/0.1)得白色固体TP-25(405mg,收率53%)。
1H NMR(400MHz,DMSO-d6)δ8.12(t,J=6.0Hz,2H),7.98-7.75(m,7H),5.21(d,J=3.4Hz,3H),4.96(dd,J=11.2,3.4Hz,2H),4.54(d,J=8.4Hz,2H),4.02(q,J=5.3,4.5Hz,9H),3.95-3.83(m,3H),3.82-3.50(m,23H),3.40-3.26(m,4H),3.12-2.94(m,27H),2.76-2.59(m,7H),2.29(t,J=6.7Hz,5H),2.11-1.78(m,38H),1.38(s,12H),1.16(d,J=7.5Hz,12H);
31P NMR(162MHz,DMSO-d6)δ147.97;
MS(ESI):m/z[1/2M+Na]+理论值1057.0,实测值1057.4。
三、GalNAc靶头1048的合成
按照以下方法,合成了TO26及TP-26(1048靶头连接siRNA的前体)的一种非对映异构体。
1、中间体GN-18-01的合成
(1)N2氛围下,将GC-2(20.1g,39.7mmol)溶于DCM(200mL)中,分批加入CDI(7.09g,73.7mmol),25℃搅拌3小时,随后将N-Boc-乙二胺(7.0g,43.7mmol)和三乙胺(12.05g,119.1mmol)加入反应液中,反应16小时,LCMS检测显示原料消失。
(2)加入饱和碳酸氢钠溶液(200mL)淬灭,分液,水相经DCM(100mL×3)萃取,合并有机相并使用饱和氯化铵溶液(200mL)、饱和氯化钠溶液(200mL)洗涤,无水Na2SO4干燥,过滤浓缩获得粗制产品。粗产品经甲基叔丁基醚(100mL)洗涤,油状产物浓缩得白色固体化合物GN-18-01(24.43g,收率95.1%)。
MS(ESI):m/z[M+H]+理论值650.3,实测值650.5。
2、中间体GN-18的合成
(1)将GN-18-01(45.52g,70mmol)分批加入到HCl/EtOAc溶液(2N,500mL)中,25℃搅拌2小时,LCMS检测显示原料消失。
(2)倾倒出溶剂,固体浓缩得粗制产品。粗产品经甲基叔丁基醚(200mL)打浆纯化,过滤,滤饼40℃真空干燥得白色固体GN-18(49.6g)。
MS(ESI):m/z[M+H]+理论值550.3,实测值550.5。
3、中间体TO-26-01的合成
(1)N2氛围下,将NC-6(1.5g,3.6mmol)、PyBOP(6.2g,12.0mmol)和DIEA(4.75g,36mmol)加入到DCM(50mL)中搅拌30分钟,随后滴加GN-18(6.6g,12.0mmol)与DIEA(4.75g,36mmol)的DCM(50mL)溶液,25℃搅拌16小时,LCMS检测,原料基本消耗完毕。
(2)加入DCM(100mL)稀释,向反应液中加入1N盐酸溶液(80mL×2)洗涤,合并有机相,经饱和碳酸氢钠(100mL)、饱和食盐水(100mL)洗涤、无水Na2SO4干燥过滤浓缩获得粗制产品。粗产品经硅胶柱层析纯化(DCM/MeOH=7/1)得白色固体化合物TO-26-01(4.7g,收率65%)。
MS(ESI):m/z[M/2+H]+理论值1004.0,实测值1004.2。
4、中间体TO-26的合成
(1)将TO-26-01(4.0g,2.0mmol)溶于甲醇(80mL)中,加入10%钯碳(1.0g),H2置换三次,25℃搅拌2小时,LCMS检测,原料基本消失。
(2)过滤,浓缩,加入DCM(20mL)溶解,缓慢滴加至MTBE(200mL)中,搅拌析晶30分钟,抽滤,得到白色固体化合物TO-26(3.5g,收率91%)。
1H NMR(400MHz,DMSO-d6)δ8.54(s,1H),8.14(s,2H),7.95-7.92(m,3H),7.84(d,J=7.8Hz,3H),5.21(d,J=3.4Hz,3H),4.97(dd,J=11.2,3.4Hz,3H),4.54(d,J=8.5Hz,3H),4.13-3.66(m,21H),3.60-3.44(m,37H),3.14(d,J=13.8Hz,15H),2.31(t,J=6.4Hz,6H),2.10(s,9H),2.00(s,9H),1.89(s,9H),1.77(s,9H).
MS(ESI):m/z[1/2M+H]+理论值958.9,实测值959.1。
5、TP-26(1048靶头连接siRNA的前体)的合成
(1)N2氛围下,将TO-26(900mg,0.47mmol)溶于干燥DCM(12mL)中,加入DIEA(0.39mL,0.44mmol),利用注射器缓慢滴加2-氰乙基-N,N-二异丙基氯代亚磷酰胺(277mg,1.17mmol)的干燥DCM(1mL)溶液,25℃反应30分钟,TLC检测,原料基本消失。
(2)加入饱和NaHCO3(10mL)淬灭,DCM(10mL)稀释,分液,有机相用饱和NaHCO3(10mL)溶液,饱和食盐水(10mL)洗涤,无水NaSO4干燥,过滤浓缩得粗制品。经柱层析纯化(硅胶柱预先经1.5%TEA/DCM碱化,DCM/MeOH/TEA=15/1/0.1)得白色固体TP-26(600mg,收率60%)。
1H NMR(400MHz,DMSO-d6)1H NMR(400MHz,DMSO-d6)δ8.15(s,2H),7.94-7.81(m,7H),5.22(d,J=3.4Hz,3H),4.97(dd,J=11.2,3.4Hz,3H),4.55(d,J=8.5Hz,3H),4.03(s,8H),3.88(dt,J=11.2,8.9Hz,3H),3.81-3.67(m,7H),3.64-3.46(m,30H),3.11(d,J=13.1Hz,19H),2.76(t,J=5.9Hz,3H),2.65-2.54(m,7H),2.31(t,J=6.6Hz,7H),2.11(s,9H),2.00(s,9H),1.89(s,9H),1.77(s,9H),1.13(d,J=6.8,12H).
31P NMR(162MHz,DMSO-d6)δ147.89;
MS(ESI):m/z 1/2[M-i-Pr2N]理论值1007.9,实测值1008.2。
实施例3体外构建偶联GalNAc靶头偶联的(修饰的)siRNA缀合物
以下RNAi剂双链体的反义链和正义链的寡核苷酸序列部分,以及靶向性配体与RNA的连接,均按照J.Org.Chem.2012,77,4566-4577;Curr.Protoc.Nucleic Acid Chem.,81,e107报道的亚磷酰胺偶联技术在用于寡核苷酸合成的固相上合成。靶向性配体1046、1048、1043均通过硫代磷酸酯键与siRNA正义链5’末端连接。
靶向性配体1046、1048、1043与siRNA连接的形式为脱羟基保护基的形式,具体如下:
靶向配体与siRNA连接后的结构如下所示:
合成的GalNAc-siRNA缀合物如图3所述,其中第二列的的缀合物结构包括三部分,例如,G1043-S2A2-A265的结构为:1043靶头通过硫代磷酸酯键与编号为A265的siRNA正义链5’末端连接,S2A2为对A265的siRNA的修饰类型,具体的修饰基团和修饰方式为:
核酸序列中,Ao表示腺嘌呤核苷,Uo表示尿嘧啶核苷,Go表示鸟嘌呤核苷,Co表示胞嘧啶核苷,直接相邻的核苷酸之间没有符号,表示以正常的磷酸酯键连接。
DNA:A G C T(A表示2’-脱氧腺嘌呤核苷,T表示2’-脱氧胸腺嘧啶核苷,G表示2’-脱氧鸟嘌呤核苷,C表示2’-脱氧胞嘧啶核苷);
2'-F:aF gF cF uF(aF表示2’-氟代腺嘌呤核苷,uF表示2’-氟代尿嘧啶核苷,gF表示2’-氟代鸟嘌呤核苷,cF表示2’-氟代胞嘧啶核苷);
2'-OMe:aM gM cM uM(aM表示2’-O-甲基腺嘌呤核苷,uM表示2’-O-甲基尿嘧啶核苷,gM表示2’-O-甲基鸟嘌呤核苷,cM表示2’-O-甲基胞嘧啶核苷);
*:表示以硫代磷酸酯键连接;
序列中的y、z代表靶头的位置。
实施例4:体外细胞模型(Hep 3B细胞)测试缀合物的活性
人肝癌Hep3B细胞(中国科学院上海细胞库),培养于添加10%胎牛血清(FBS)(Gibco,US)的DMEM(Gibco,US)中,于37℃,5% CO2条件下培养(il60,Thermo Fisher)。转染实验进行当天,使用0.25% Trysin(Gibco,US)消化细胞,计数并以450μL/孔、5万/孔的密度接种于24孔板。随后,以lipofectmine2000(Thermo Fisher)转染方式加入受试样品。按RNAiMAX试剂说明说书标准流程转染,siRNA终浓度为10nM/1nM/0.5nM/0.25nM/0.1nM/0.05nM/0.01nM。转染组以siNC为阴性对照,其序列为:
正义链(sense):5’-UUCUCCGAACGUGUCACGUTT-3’
反义链(antisense):5’-ACGUGACACGUUCGGAGAATT-3’。
24h后,提取细胞的总RNA,通过实时定量PCR(Quantitative Real-Time PCR)检测细胞中ANGPTL3 mRNA序列的表达情况,其中用于扩增内参基因PPIB、ANGPTL3的PCR引物如表1所示:
各个缀合物的活性测试结果(EC50值)见图4。
EC50值利用graphpad prism的非线性回归计算,表示抑制一半目标mRNA(ANGPTL3)表达量时的缀合物的用量。
结果可见,选取的缀合物在体外活性测试的实验中,表现出良好的降低ANGPTL3相对表达水平的结果。
实施例5:AAV-hANGPTL3小鼠模型的构建及给药测试
实验动物基本信息:
实验动物购自济南朋悦实验动物繁育有限公司,为SPF级动物。给药前对上述小鼠称重并观察状态,选取体重均一、状态无异常的动物进行后续实验。
饲养条件:非SPF级饲养条件。正常饲养条件下动物自由进食饮水。动物购进后,进行3-7天适应性培养后开始实验。
造模及给药:每只小鼠通过尾静脉注射2.5*10^11滴度的病毒溶液,100μL。7天后,对实验动物进行随机分组,每个受试物按5mg/kg的剂量进行皮下给药。给药后72小时,颈椎脱臼牺牲动物,取肝组织,进行RNA的提取与定量。
各个缀合物的结果见表3。
表3、各个缀合物的小鼠模型给药测试结果
结果可见,选取的缀合物在体内活性测试的实验中,也表现出良好的降低ANGPTL3相对表达水平的结果。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本公开的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本公开的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本公开的限制,本领域的普通技术人员在本公开的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (13)
3.权利要求1-2任一项所述的化合物在制备siRNA缀合物中的应用。
4.根据权利要求3所述的应用,其特征在于,所述的化合物作为靶向配体与siRNA连接。
5.根据权利要求4所述的应用,其特征在于,所述的siRNA缀合物中的siRNA包括一条正义链和一条反义链,所述反义链包括与所述正义链互补配对的互补性区域,其中所述正义链选自与SEQ ID NO:1-SEQ ID NO:154中每一条链的核苷酸序列区别不多于5个核苷酸的核苷酸序列,所述反义链选自与SEQ IDNO:155-SEQ ID NO:308中每一条链的核苷酸序列区别不多于5个核苷酸的核苷酸序列。
6.根据权利要求5所述的应用,其特征在于,所述siRNA包括至少一个被修饰的核苷酸;
所述修饰的核苷酸选自下列至少之一:
5'-硫代磷酸酯基的核苷酸、5-甲基化胞嘧啶核苷酸、2'-O-甲基修饰的核苷酸、2'-O-2-甲氧乙基修饰的核苷酸、2'-氟代修饰的核苷酸、3'-氮取代修饰的核苷酸、2'-脱氧-2'-氟修饰的核苷酸、2'-脱氧修饰的核苷酸、锁定的核苷酸、脱碱基核苷酸、2'-氨基修饰的核苷酸、吗啉代核苷酸、多肽核苷酸、氨基磷酸酯,以及包括非天然碱基的核苷酸。
7.根据权利要求6所述的应用,其特征在于,所述siRNA与所述靶向配体共价连接;所述靶向配体连接于所述siRNA中正义链;所述靶向配体通过硫代磷酸酯键与所述siRNA中正义链的5’端连接。
8.一种siRNA缀合物的制备方法,其特征在于,包括将权利要求1-2任一项所述的化合物作为靶向配体与siRNA共价连接。
11.权利要求1-2任一项所述的化合物和/或权利要求10所述的中间体在制备药物或试剂盒中的用途,其特征在于,所述药物或试剂盒用于抑制ANGPTL3基因表达。
12.根据权利要求11所述的应用,其特征在于,所述药物或试剂盒用于预防和/或治疗血脂异常疾病。
13.根据权利要求12所述的应用,其特征在于,所述血脂异常疾病包括高血脂症和高甘油三酯血症。
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