背景技术Background Art
乙型病毒性肝炎(又称乙型肝炎或乙肝)是严重威胁全球、特别是中国的一类传染病，目前全球公认的两大类乙肝防止药物为干扰素和核苷类似物，但是这两类药物存在使用后容易产生耐药性或使用受限等多种弊端，如干扰素容易产生不良反应、核苷类药物存在耐药性和停药后复发问题。因此，若能从基因水平沉默病毒的基因表达，阻断HBV的生成和复制，由此从根本上降低病毒代谢和对肝细胞的侵染，无疑将是最为理想的乙肝治疗手段。小干扰RNA(small interfering RNA，siRNA)可基于RNA干扰(RNA interference，RNAi)这一机制，以序列特异性的方式抑制或阻断任何感兴趣的目的基因(例如引发如癌症等疾病的基因)的表达，从而达到治疗疾病的目的。Hepatitis B virus (also known as hepatitis B or HBV) is a serious infectious disease threatening the world, especially China. Currently, the two major types of hepatitis B prevention drugs recognized worldwide are interferon and nucleoside analogs. However, these two types of drugs have many disadvantages such as easy to develop drug resistance or limited use after use. For example, interferon is prone to adverse reactions, and nucleoside drugs have drug resistance and recurrence after drug withdrawal. Therefore, if the gene expression of the virus can be silenced at the genetic level, the generation and replication of HBV can be blocked, thereby fundamentally reducing viral metabolism and infection of liver cells, it will undoubtedly be the most ideal treatment for hepatitis B. Small interfering RNA (siRNA) can inhibit or block the expression of any target gene of interest (such as genes that cause diseases such as cancer) in a sequence-specific manner based on the mechanism of RNA interference (RNAi), thereby achieving the purpose of treating the disease.
siRNA稳定化修饰及其递送系统是小RNA药物开发中的两个关键技术。siRNA stabilization modification and its delivery system are two key technologies in the development of small RNA drugs.
发明内容Summary of the invention
在一些实施方案中，本公开提供了一种能够抑制HBV基因表达的siRNA，该siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I含有核苷酸序列A，所述核苷酸序列A与SEQ ID NO：1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II含有核苷酸序列B，所述核苷酸序列B与SEQ ID NO：2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：In some embodiments, the present disclosure provides an siRNA capable of inhibiting HBV gene expression, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complemented to form a double-stranded region, wherein the nucleotide sequence I comprises a nucleotide sequence A, the nucleotide sequence A is equal in length to the nucleotide sequence shown in SEQ ID NO: 1, and differs by no more than 3 nucleotides, and the nucleotide sequence II comprises a nucleotide sequence B, the nucleotide sequence B is equal in length to the nucleotide sequence shown in SEQ ID NO: 2, and differs by no more than 3 nucleotides:
5′-CGUGUGCACUUCGCUUCAZ-3′(SEQ ID NO：1)；5′-CGUGUGCACUUCGCUUCAZ-3′ (SEQ ID NO: 1);
5′-Z′UGAAGCGAAGUGCACACG-3′(SEQ ID NO：2)，5′-Z′UGAAGCGAAGUGCACACG-3′ (SEQ ID NO: 2),
其中，Z为A，Z′为U；Among them, Z is A, Z′ is U;
并且，所述核苷酸序列A中包含位置对应于Z的核苷酸ZA，所述核苷酸序列B中包含位置对应于Z′的核苷酸Z′B，所述Z′B是所述反义链5′末端的第一个核苷酸。Furthermore, the nucleotide sequence A includes a nucleotide ZA corresponding to position Z, and the nucleotide sequence B includes a nucleotide Z′B corresponding to position Z′, and Z′B is the first nucleotide at the 5′ end of the antisense strand.
在一些实施方案中，本公开提供了一种药物组合物，所述药物组合物含有本公开的siRNA和药学上可接受的载体。In some embodiments, the present disclosure provides a pharmaceutical composition comprising the siRNA of the present disclosure and a pharmaceutically acceptable carrier.
在一些实施方案中，本公开提供了一种siRNA缀合物，所述siRNA缀合物含有本公开提供的siRNA以及缀合连接至该siRNA的缀合基团。In some embodiments, the present disclosure provides a siRNA conjugate, which contains the siRNA provided by the present disclosure and a conjugation group conjugated to the siRNA.
在一些实施方案中，本公开提供了本公开的siRNA和/或药物组合物和/或siRNA缀合物在制备用于治疗和/或预防由乙型肝炎病毒的感染引起的病理状况或疾病的药物中的用途。In some embodiments, the present disclosure provides use of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in the preparation of a medicament for treating and/or preventing a pathological condition or disease caused by infection with hepatitis B virus.
在一些实施方案中，本公开提供了一种治疗和/或预防由乙型肝炎病毒的感染引起的病理状况或疾病的方法，所述方法包括将有效量的本公开的siRNA和/或药物组合物和/或siRNA缀合物给予有需要的患者。In some embodiments, the present disclosure provides a method for treating and/or preventing pathological conditions or diseases caused by infection with hepatitis B virus, the method comprising administering an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure to a patient in need thereof.
在一些实施方案中，本公开提供了一种试剂盒，所述试剂盒含有本公开的siRNA和/或药物组合物和/或siRNA缀合物。In some embodiments, the present disclosure provides a kit containing the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1表示所测试siRNA缀合物在体外Tritosome中的稳定性半定量检测结果。FIG1 shows the semi-quantitative test results of the stability of the tested siRNA conjugates in Tritosome in vitro.
图2表示所测试siRNA缀合物在体外人血浆中的稳定性半定量检测结果。FIG2 shows the semi-quantitative test results of the stability of the tested siRNA conjugates in human plasma in vitro.
图3表示所测试siRNA缀合物在体外猴血浆中的稳定性半定量检测结果。FIG3 shows the semi-quantitative test results of the stability of the tested siRNA conjugates in monkey plasma in vitro.
图4表示缀合物20在体外的抑制活性结果。FIG4 shows the results of the inhibitory activity of conjugate 20 in vitro.
图5表示缀合物4在体外psiCHECK系统中IC50及脱靶效应的检测结果。FIG5 shows the detection results ofIC50 and off-target effects of conjugate 4 in the in vitro psiCHECK system.
图6表示缀合物4在小鼠中对HBV mRNA表达的抑制结果。FIG6 shows the results of inhibition of HBV mRNA expression by conjugate 4 in mice.
图7表示缀合物4在M-Tg模型上单次给药对HBsAg表达抑制作用的检测结果。FIG. 7 shows the results of the test on the inhibitory effect of a single administration of conjugate 4 on HBsAg expression in the M-Tg model.
图8表示缀合物4在M-Tg模型上单次给药对HBxmRNA表达抑制作用的检测结果。FIG8 shows the results of the detection of the inhibitory effect of a single administration of conjugate 4 on HBxmRNA expression in the M-Tg model.
具体实施方式DETAILED DESCRIPTION
以下对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。The specific embodiments of the present disclosure are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure.
定义definition
在本公开中，HBV基因是指DNA序列如Genbank注册号NC_003977.1所示的基因。In the present disclosure, the HBV gene refers to the gene whose DNA sequence is shown in Genbank Accession No. NC_003977.1.
在上文及下文中，如无特别说明，大写字母C、G、U、A、T表示核苷酸的碱基组成；小写字母d表示该字母d右侧相邻的一个核苷酸为脱氧核糖核苷酸；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸酯基连接；P1表示该P1右侧相邻的一个核苷酸为5′-磷酸核苷酸或5′-磷酸类似物修饰的核苷酸，尤指乙烯基磷酸酯修饰的核苷酸(以下实施例中以VP表示)、5′-磷酸核苷酸(以下实施例中以P表示)或5′-硫代磷酸酯修饰的核苷酸(以下实施例中以Ps表示)。In the above and below, unless otherwise specified, capital letters C, G, U, A, and T represent the base composition of nucleotides; lowercase letter d represents that the nucleotide adjacent to the right of letter d is a deoxyribonucleotide; lowercase letter m represents that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of letter f is a fluorine-modified nucleotide; lowercase letter s represents that the two nucleotides adjacent to the left and right of letter s are connected by thiophosphate groups; P1 represents that the nucleotide adjacent to the right of P1 is a 5′-phosphate nucleotide or a 5′-phosphate analogue-modified nucleotide, especially a vinyl phosphate-modified nucleotide (represented by VP in the following examples), a 5′-phosphate nucleotide (represented by P in the following examples), or a 5′-thiophosphate-modified nucleotide (represented by Ps in the following examples).
在上文及下文中，所述氟代修饰的核苷酸指核苷酸的核糖基2′位的羟基被氟取代形成的核苷酸，非氟代修饰的核苷酸指核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物，核苷酸类似物指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。如异核苷酸、桥联的核苷酸(bridged nucleic acid，简称BNA)或无环核苷酸。所述甲氧基修饰的核苷酸指核糖基的2′-羟基被甲氧基取代而形成的核苷酸。In the above and below, the fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2′ position of the ribose group of the nucleotide is replaced by fluorine, the non-fluoro-modified nucleotide refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2′ position of the ribose group of the nucleotide is replaced by a non-fluorine group, and the nucleotide analog refers to a group that can replace nucleotides in nucleic acids but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymine deoxyribonucleotide, such as isonucleotides, bridged nucleic acids (BNA for short) or acyclic nucleotides. The methoxy-modified nucleotide refers to a nucleotide in which the 2′-hydroxyl group of the ribose group is replaced by a methoxy group.
在本文的上下文中，“互补”或“反向互补”一词可互相替代使用，并具有本领域技术人员周知的含义，即，在双链核酸分子中，一条链的碱基与另一条链上的碱基以互补的方式相配对。在DNA中，嘌呤碱基腺嘌呤(A)始终与嘧啶碱基胸腺嘧啶(T)(或者在RNA中为尿嘧啶(U))相配对；嘌呤碱基鸟嘌呤(C)始终与嘧啶碱基胞嘧啶(G)相配对。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的胸腺嘧啶(或尿嘧啶)配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此相互补的，以及从其互补链的序列中可以推断出该链的序列。与此相应地，“错配”在本领域中意指在双链核酸中，对应位置上的碱基并未以互补的形式配对存在。In the context of this article, the terms "complementary" or "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one chain are paired with the bases on the other chain in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one chain is always paired with thymine (or uracil) on the other chain, and guanine is always paired with cytosine, the two chains are considered to be complementary to each other, and the sequence of the chain can be inferred from the sequence of its complementary chain. Correspondingly, "mismatch" means in the art that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary form.
在上文及下文中，如无特别说明，基本上反向互补是指所涉及的两段核苷酸序列之间存在不多于3个的碱基错配；实质上反向互补是指两段核苷酸序列之间存在不多于1个的碱基错配；完全互补是指两段核苷酸序列之间不存在碱基错配。In the above and below, unless otherwise specified, substantially reverse complementarity means that there are no more than 3 base mismatches between the two nucleotide sequences involved; substantially reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences; and fully complementary means that there is no base mismatch between the two nucleotide sequences.
在上文及下文中，一个核苷酸序列与另外一个核苷酸序列存在“核苷酸差异”，是指前者与后者相比，相同位置的核苷酸的碱基种类发生了改变，例如，在后者中一个核苷酸碱基为A时，在前者的相同位置处的对应核苷酸碱基为U、C、G或者T的情况下，认定为两个核苷酸序列之间在该位置处存在核苷酸差异。在一些实施方案中，以无碱基核苷酸或其等同物代替原位置的核苷酸时，也可认为在该位置处产生了核苷酸差异。In the above and below, "nucleotide difference" between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position in the former is changed compared with the latter. For example, when a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G or T, it is considered that there is a nucleotide difference at that position between the two nucleotide sequences. In some embodiments, when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, it can also be considered that a nucleotide difference occurs at that position.
在上文及下文中，特别是在描述本公开的缀合分子的制备方法或siRNA缀合物的制备方法时，除非特别说明，所述核苷单体(nucleoside monomer)指，根据欲制备的siRNA或siRNA缀合物中核苷酸的种类和顺序，亚磷酰胺固相合成中使用的修饰或未修饰的核苷亚磷酰胺单体(unmodified or modified RNA phosphoramidites，有时RNAphosphoramidites也称为Nucleoside phosphoramidites)。亚磷酰胺固相合成为本领域技术人员所公知的RNA合成中所用的方法。本公开所用的核苷单体均可商购得到。In the above and below, especially when describing the preparation method of the conjugated molecule of the present disclosure or the preparation method of the siRNA conjugate, unless otherwise specified, the nucleoside monomer (nucleoside monomer) refers to the modified or unmodified nucleoside phosphoramidite monomer (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites are also referred to as Nucleoside phosphoramidites) used in the phosphoramidite solid phase synthesis according to the type and order of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid phase synthesis is a method used in RNA synthesis known to those skilled in the art. The nucleoside monomers used in the present disclosure are all commercially available.
如本文所使用的，不介于两个字母之间或两个符号之间的短横(“-”)是用于指示取代基附着点的位置。例如：-C1-C10烷基-NH2通过C1-C10烷基而附着。As used herein, a hyphen ("-") that is not between two letters or symbols is used to indicate the position of the point of attachment of a substituent. For example: -C1 -C10 alkyl-NH2 is attached through the C1 -C10 alkyl.
如本文所使用的，“任选的”或“任选地”是指其后描述的事件或状况可以发生或不发生，并且所述描述包括事件或状况发生的情况和其中不发生的情况。例如，“任选的取代的烷基”包括下面定义的“烷基”和“取代烷基”。本领域技术人员将理解的是，对于包含一个或多个取代基的任何基团，这些基团不打算引入空间上不切实际、合成上不可行和/或内在不稳定的任何取代或取代型式。As used herein, "optional" or "optionally" means that the event or circumstance described thereafter may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, "optionally substituted alkyl" includes "alkyl" and "substituted alkyl" as defined below. It will be understood by those skilled in the art that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically unfeasible, and/or inherently unstable.
如本文所使用的，“烷基”是指具有指定数量的碳原子的直链和支链，所述数量通常为1到20个碳原子，例如1至10个碳原子，如1至8个或1至6个碳原子。例如，C1-C6烷基包含1至6个碳原子的直链和支链烷基。当对具有特定数量的碳的烷基残基进行命名时，旨在涵盖所有具有该数量的碳的支链和直链形式；因此，例如，“丁基”意味着包括正丁基、仲丁基、异丁基和叔丁基；“丙基”包括正丙基和异丙基。亚烷基是烷基的子集，指与烷基相同、但具有两个附着点的残基。As used herein, "alkyl" refers to straight and branched chains having a specified number of carbon atoms, typically 1 to 20 carbon atoms, e.g., 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example,C1 -C6 alkyl includes straight and branched chain alkyl groups of 1 to 6 carbon atoms. When an alkyl residue having a specific number of carbons is named, it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to residues that are the same as alkyl, but have two points of attachment.
如本文所使用的，“烯基”是指具有至少一个碳-碳双键的不饱和支链或直链烷基，所述碳-碳双键是通过从母体烷基的相邻碳原子中除去一个氢分子而获得的。该基团可以处于双键的顺式或反式构型。典型的烯基基团包括但不限于：乙烯基；丙烯基，如丙-1-烯-1-基、丙-1-烯-2-基、丙-2-烯-1-基(烯丙基)、丙-2-烯-2-基；丁烯基，例如丁-1-烯-1-基、丁-1-烯-2-基、2-甲基丙-1-烯-1-基、丁-2-烯-1-基、丁-2-烯-2-基、丁-1，3-二烯-1-基、丁-1，3-二烯-2-基等等。在某些实施方案中，烯基基团具有2到20个碳原子，而在其他实施方案中，具有2至10个、2至8个或2至6个碳原子。亚烯基是烯基的一个子集，指与烯基相同、但具有两个附着点的残基。As used herein, "alkenyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double bond, wherein the carbon-carbon double bond is obtained by removing a hydrogen molecule from the adjacent carbon atoms of the parent alkyl group. The group can be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to, vinyl; propenyl, such as prop-1-ene-1-yl, prop-1-ene-2-yl, prop-2-ene-1-yl (allyl), prop-2-ene-2-yl; butenyl, such as but-1-ene-1-yl, but-1-ene-2-yl, 2-methylprop-1-ene-1-yl, but-2-ene-1-yl, but-2-ene-2-yl, but-1,3-diene-1-yl, but-1,3-diene-2-yl, etc. In certain embodiments, the alkenyl group has 2 to 20 carbon atoms, and in other embodiments, has 2 to 10, 2 to 8 or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl and refers to residues that are identical to alkenyl but have two points of attachment.
如本文所使用的，“炔基”是指具有至少一个碳-碳三键的不饱和支链或直链烷基，所述碳-碳三键是通过从母体烷基的相邻碳原子中除去两个氢分子而获得的。典型的炔基基团包括但不限于：乙炔基；丙炔基，如丙-1-炔-1-基，丙-2-炔-1-基；丁炔基，例如丁-1-炔-1-基，丁-1-炔-3-基，丁-3-炔-1-基等。在某些实施方案中，炔基具有2到20个碳原子，而在其他实施方案中，具有2至10、2至8或2至6个碳原子。亚炔基是炔基的一个子集，指的是与炔基相同、但有两个附着点的残基。As used herein, "alkynyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon triple bond obtained by removing two hydrogen molecules from adjacent carbon atoms of the parent alkyl group. Typical alkynyl groups include, but are not limited to, ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc. In certain embodiments, the alkynyl group has 2 to 20 carbon atoms, and in other embodiments, has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to residues that are identical to alkynyl, but with two points of attachment.
如本文所使用的，“烷氧基”是指通过氧桥附着的指定数量碳原子的烷基，例如，甲氧基、乙氧基、丙氧基、异丙氧基、正丁氧基、仲丁氧基、叔丁氧基、戊氧基、2-戊氧基、异戊氧基、新戊氧基、己氧基、2-己氧基、3-己氧基、3-甲基戊氧基等。烷氧基通常具有1至10个、1至8个、1至6个，或1至4个通过氧桥附着的碳原子。As used herein, "alkoxy" refers to an alkyl group of the specified number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, etc. Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through an oxygen bridge.
如本文所使用的，“芳基”是指衍生自芳香族单环或多环烃环系统、通过从环碳原子中除去氢原子而形成的基团。所述芳香族单环或多环烃环系统仅含有氢和6至18个碳原子的碳，其中所述环系统中的至少一个环是完全不饱和的，即，其根据Hückel理论包含环状、离域的(4n+2)π-电子系统。芳基包括但不限于苯基、芴基和萘基等基团。亚芳基是芳基的一个子集，指与芳基相同，但具有两个附着点的残基。As used herein, "aryl" refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system formed by removing hydrogen atoms from ring carbon atoms. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbons of 6 to 18 carbon atoms, wherein at least one ring in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2)π-electron system according to the Hückel theory. Aryl includes, but is not limited to, radicals such as phenyl, fluorenyl, and naphthyl. Arylene is a subset of aryl and refers to residues identical to aryl, but having two points of attachment.
如本文所使用的，“环烷基”是指非芳香碳环，通常具有3至7个环状碳原子。环可以是饱和的，或具有一个或多个碳-碳双键。环烷基的实例包括环丙基、环丁基、环戊基、环戊烯基、环己基和环己烯基，以及桥联和笼状环基团，如降冰片烷(norbornane)。As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring, typically having 3 to 7 cyclic carbon atoms. The ring may be saturated or have one or more carbon-carbon double bonds. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl, as well as bridged and caged ring groups such as norbornane.
如本文所使用的，“卤素取代基”或“卤”指氟代、氯代、溴代和碘代，术语“卤素”包括氟、氯、溴和碘。As used herein, "halogen substituent" or "halo" refers to fluoro, chloro, bromo and iodo, and the term "halogen" includes fluorine, chlorine, bromine and iodine.
如本文所使用的，“卤代烷基”是指指定数量的碳原子被一个或多个、直至最大允许数量的卤素原子取代的如上述所定义的烷基。卤代烷基的实例包括但不限于三氟甲基、二氟甲基、2-氟乙基和五氟乙基。As used herein, "haloalkyl" refers to an alkyl group as defined above in which a specified number of carbon atoms is replaced by one or more, up to the maximum permitted number of halogen atoms. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl and pentafluoroethyl.
“杂环基”是指一个稳定的3-到18-元非芳香环基，包含2-12个碳原子和1-6个杂原子，所述杂原子选自氮、氧和硫。除非说明书中另有说明，杂环基是单环、双环、三环或四环系统，可包括稠环或桥环系统。杂环自由基中的杂原子可以任选地被氧化。一个或多个氮原子(如果存在的话)任选地被季铵化。杂环基是部分饱和或完全饱和的。杂环基可以通过环的任何原子附着至分子的其余部分。此类杂环基的实例包括但不限于：二噁烷基、噻吩基[1，3]二硫酰基、十氢异喹啉基、咪唑啉基、咪唑烷基、异噻唑烷基、异恶唑烷基、吗啉基、八氢吲哚基、八氢异吲哚基、2-氧杂哌嗪基、2-氧杂哌啶基、2-氧杂嘧啶基、恶唑烷基、哌啶基、哌嗪基、4-哌啶酮基、吡咯烷基、吡唑烷基、奎宁环基、噻唑烷基、四氢呋喃基、三硫酰基、四氢吡喃基、硫吗啉基、硫杂吗啉基、1-氧杂硫吗啉基和1，1-二氧杂硫吗啉基。"Heterocyclyl" refers to a stable 3- to 18-membered non-aromatic ring radical containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. Unless otherwise specified in the specification, the heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. The heteroatoms in the heterocyclic radical may be optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heterocyclyl is partially saturated or fully saturated. The heterocyclyl may be attached to the rest of the molecule through any atom of the ring. Examples of such heterocyclic groups include, but are not limited to, dioxanyl, thienyl[1,3]disulfanyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrimidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidine, thiazolidinyl, tetrahydrofuranyl, trisulfanyl, tetrahydropyranyl, thiomorpholinyl, thiomorpholinyl, 1-oxathiomorpholinyl, and 1,1-dioxathiomorpholinyl.
“杂芳基”指由3-至18-元芳香环自由基衍生而成的基团，其包含2个至17个碳原子和选自氮、氧和硫的1至6个杂原子。如本文所使用的，杂芳基可以是单环、双环、三环或四环系统，其中环系统中的至少一个环是完全不饱和的，即，根据Hückel理论，包含环状离域(4n+2)π-电子体系。杂芳基包括稠环或桥环系统。杂芳基中的杂原子被任选地氧化。一个或多个氮原子(如果存在的话)任选地被季铵化。杂芳基通过环中的任何原子附着至分子的其余部分。杂芳基的实例包括但不限于：氮杂环庚三烯基、吖啶基、苯并咪唑基、苯并吲哚基、1，3-苯并二恶唑基、苯并呋喃基、苯并恶唑基、苯并[d]噻唑基、苯并噻二唑基、苯并[b][1，4]二恶唑基、苯并[b][1，4]恶唑基、1，4-苯并二恶唑基、苯并萘并呋喃基、苯并二唑基、苯并二氧杂苯基、苯并吡喃基、苯并吡喃酮基、苯并呋喃基、苯并呋喃酮基、苯并噻吩基、苯并噻吩[3，2-d]嘧啶基、苯并三唑基、苯并[4，6]咪唑[1，2-a]吡啶基、咔唑基、噌啉基、环戊基[d]嘧啶基、6，7-二氢-5H-环戊基[4，5]噻吩[2，3-d]嘧啶基、5，6-二氢苯并[h]喹唑啉基、5，6-二氢苯并[h]辛诺林基、6，7-二氢-5H-苯并[6，7]环庚[1，2-c]哒嗪基、二苯并呋喃基、二苯并噻吩基、呋喃基、呋喃酮基、呋喃[3，2-c]吡啶基、5，6，7，8，9，10-六氢环庚烷[d]嘧啶基、5，6，7，8，9，10-六氢环辛酸[d]哒嗪基、5，6，7，8，9，10-六氢环辛酸[d]吡啶基、异噻唑基、吲唑基、咪唑基、吲哚基、异吲哚基、二氢吲哚基、异二氢氮茚基、异喹啉基、吲哚嗪基、异恶唑基、5，8-甲基-5，6，7，8-四氢喹唑啉基、萘啶酮基、1，6-萘啶酮基、恶二唑基、2-氧氮杂庚基、恶唑基、恶草酰基、5，6，6a，7，8，9，10，10a-八氢苯并[H]喹唑啉基、1-苯基-1H-吡咯基、吩嗪基、吩噻嗪基、吩恶嗪基、邻苯二甲酰基、蝶啶基、嘌呤基、吡咯基、吡唑基、吡唑并[3，4-d]嘧啶基、吡啶基、吡啶并[3，2-d]嘧啶基、吡啶并[3，4-d]嘧啶基、吡嗪基、嘧啶基、哒嗪基、吡咯基、喹唑啉基、喹喔啉基、喹啉基、异喹啉基、四氢喹啉基、5，6，7，8-四氢喹唑啉基、5，6，7，8-四氢苯并[4，5]噻吩[2，3-d]嘧啶基、6，7，8，9四氢-5H-环庚烷[4，5]噻吩[2，3-d]嘧啶基、5，6，7，8-四氢吡啶并[4，5-c]哒嗪基、噻唑基、噻二唑基、三唑基、四唑基、三嗪基、噻吩[2，3-d]嘧啶基、噻吩[3，2-d]嘧啶基、噻吩[2，3-c]普萘基和噻吩基。"Heteroaryl" refers to a group derived from a 3- to 18-membered aromatic ring radical containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, heteroaryl can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., according to the Hückel theory, containing a cyclic delocalized (4n+2)π-electron system. Heteroaryl includes fused or bridged ring systems. The heteroatoms in the heteroaryl are optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heteroaryl is attached to the rest of the molecule through any atom in the ring. Examples of heteroaryl groups include, but are not limited to, azepine, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxazolyl, benzo[b][1,4]oxazolyl, 1,4-benzodioxazolyl, benzonaphthofuranyl, benzodiazolyl, benzodioxaphenyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothiophenyl, benzothiophene[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopentyl[d]pyrimidinyl, 6,7-dihydro-5H-cyclopentyl[4,5]thienyl[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]octinolyl, 6,7-dihydro-5H-benzo[6,7]cycloheptyl[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothienyl, furanyl, furanonyl, furan[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloheptane[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocyclooctanoic acid[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocyclooctanoic acid[d]pyridinyl, isothiazolyl, indazolyl, imidazolyl, indolyl, isoindolyl, dihydroindolyl, isoindolyl, isoquinolinyl, indolizinyl, isoxazolyl, 5,8-methyl-5,6,7,8-tetrahydroquinazolinyl, naphthyridinonyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxazepineheptyl, oxazolyl, oxadiazolyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[H]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthaloyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido [3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thien[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cycloheptane[4,5]thien[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thien[2,3-d]pyrimidinyl, thien[3,2-d]pyrimidinyl, thien[2,3-c]propenyl and thienyl.
在本公开中可以使用各种羟基保护基团。一般来说，保护基团使化学官能度对特定的反应条件不敏感，并且可以在分子中的该官能度上附加以及去除，而不实质上损害分子的其余部分。代表性的羟基保护基团公开于Beaucage等人，Tetrahedron 1992，48，2223-2311，以及Greeneand Wuts，Protective Groups in Organic Synthesis，Chapter 2，2ded，John Wiley&Sons，New York，1991中，以引用的方式将上述文献整体并入本文。在一些实施方案中，保护基团在碱性条件下稳定，但可以在酸性条件下脱除。在一些实施方案中，本文可使用的羟基保护基的非排他性实例包括二甲氧基三苯甲基(DMT)、单甲氧基三苯甲基、9-苯基黄嘌呤-9-基(Pixyl)和9-(对甲氧基苯基)黄嘌呤-9-基(Mox)。在一些实施方案中，本文可使用的羟基保护基的非排他性实例包括Tr(三苯甲基)、MMTr(4-甲氧基三苯甲基)、DMTr(4，4’-二甲氧基三苯甲基)和TMTr(4，4’，4”-三甲氧基三苯甲基)。Various hydroxyl protecting groups can be used in the present disclosure. In general, the protecting group makes the chemical functionality insensitive to specific reaction conditions, and can be attached and removed on the functionality in the molecule without substantially damaging the rest of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2ded, John Wiley & Sons, New York, 1991, and the above-mentioned documents are incorporated herein by reference in their entirety. In some embodiments, the protecting group is stable under alkaline conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxy protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl).
“受试者”一词，如本文所使用的，指任何动物，例如哺乳动物或有袋动物。本公开的主题包括但不限于人类、非人灵长类(例如，恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。The term "subject", as used herein, refers to any animal, such as a mammal or marsupial. The subject matter of the present disclosure includes, but is not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.
如本文所使用的，“治疗方法”、“治疗”、“减轻”或“改善”可在此处互换使用。这些术语指的是获得有益的或期望的结果的方法，包括但不限于治疗益处。“治疗益处”意味着根除或改善被治疗的潜在障碍。此外，治疗益处通过根除或改善与潜在障碍相关的一个或多个生理症状，从而在患者中观察到改善而获得，尽管患者可能仍然受到潜在障碍的折磨。As used herein, "treatment," "treating," "alleviating," or "amelioration" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including, but not limited to, a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Furthermore, a therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient, although the patient may still be afflicted with the underlying disorder.
如本文所使用的，“防止”和“预防”可互换使用。这些术语指获得有益或期望的结果的方法，包括但不限于预防性益处。为了获得“预防性益处”，可将缀合物或组合物给予有罹患特定疾病风险的患者，或给予报告疾病的一种或多种病理症状的患者，即便可能该疾病的诊断尚未作出。As used herein, "prevent" and "prevention" are used interchangeably. These terms refer to an approach to obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," a conjugate or composition may be administered to a patient at risk for a particular disease, or to a patient reporting one or more pathological symptoms of a disease, even though a diagnosis of the disease may not have been made.
siRNAsiRNA
本公开提供了一种能够抑制乙型肝炎病毒基因表达的siRNA。The present disclosure provides a siRNA capable of inhibiting the expression of hepatitis B virus genes.
本公开的siRNA含有核苷酸基团作为基本结构单元，本领域技术人员公知，所述核苷酸基团含有磷酸基团、核糖基团和碱基，在此不再赘述。The siRNA disclosed herein contains a nucleotide group as a basic structural unit. It is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base, which will not be described in detail here.
CN102140458B公开了一种特异性抑制HBV基因的siRNA，并对该siRNA的多种化学修饰策略进行了研究。该研究发现，不同修饰策略会对siRNA的稳定性、生物活性及细胞毒性等指标产生截然不同的影响。在该研究中，证实了7种有效的修饰方式，与未经修饰的siRNA相比，其中一种修饰方式所得的siRNA在提高血液稳定性的同时，还保持了与未经修饰的siRNA基本相当的抑制活性。CN102140458B discloses a siRNA that specifically inhibits the HBV gene, and studies various chemical modification strategies for the siRNA. The study found that different modification strategies have completely different effects on indicators such as siRNA stability, biological activity, and cytotoxicity. In the study, 7 effective modification methods were confirmed. Compared with unmodified siRNA, the siRNA obtained by one of the modification methods not only improved blood stability, but also maintained an inhibitory activity that was basically equivalent to that of unmodified siRNA.
本公开的siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I含有核苷酸序列A，所述核苷酸序列A与SEQ ID NO：1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II含有核苷酸序列B，所述核苷酸序列B与SEQ ID NO：2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：The siRNA disclosed in the present invention contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I contains a nucleotide sequence A, and the nucleotide sequence A is equal in length to the nucleotide sequence shown in SEQ ID NO: 1, and has no more than 3 nucleotide differences, and the nucleotide sequence II contains a nucleotide sequence B, and the nucleotide sequence B is equal in length to the nucleotide sequence shown in SEQ ID NO: 2, and has no more than 3 nucleotide differences:
5′-UCCGUGUGCACUUCGCUUCAZ-3′(SEQ ID NO：1)；5′-UCCGUGUGCACUUCGCUUCAZ-3′ (SEQ ID NO: 1);
5′-Z′UGAAGCGAAGUGCACACG-3′(SEQ ID NO：2)，5′-Z′UGAAGCGAAGUGCACACG-3′ (SEQ ID NO: 2),
其中，Z为A，Z′为U；Among them, Z is A, Z′ is U;
并且，所述核苷酸序列A中包含位置对应于Z的核苷酸ZA，所述核苷酸序列B中包含位置对应于Z′的核苷酸Z′B，所述Z′B是所述反义链5′末端的第一个核苷酸。Furthermore, the nucleotide sequence A includes a nucleotide ZA corresponding to position Z, and the nucleotide sequence B includes a nucleotide Z′B corresponding to position Z′, and Z′B is the first nucleotide at the 5′ end of the antisense strand.
在上文与下文中，“位置对应”是指从核苷酸序列相同端起算，处于核苷酸序列中相同的位置。例如，核苷酸序列A的3′端第1个核苷酸是位置对应于SEQ ID NO：1的3′端第1个核苷酸的核苷酸。In the above and below, "corresponding position" means being at the same position in the nucleotide sequence, starting from the same end of the nucleotide sequence. For example, the first nucleotide at the 3' end of nucleotide sequence A is the nucleotide corresponding to the first nucleotide at the 3' end of SEQ ID NO:1.
在一些实施方案中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。In some embodiments, the sense strand comprises only nucleotide sequence I, and the antisense strand comprises only nucleotide sequence II.
在一些实施方案中，所述核苷酸序列A与SEQ ID NO：1所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列B与SEQ ID NO：2所示的核苷酸序列之间不多于1个核苷酸差异。In some embodiments, there is no more than 1 nucleotide difference between the nucleotide sequence A and the nucleotide sequence shown in SEQ ID NO: 1, and/or there is no more than 1 nucleotide difference between the nucleotide sequence B and the nucleotide sequence shown in SEQ ID NO: 2.
在一些实施方案中，所述核苷酸序列B与SEQ ID NO：2所示的核苷酸序列之间的核苷酸差异包括Z′B位置处的差异，且Z′B选自A、C或G。在一些实施方案中，所述核苷酸差异为Z′B位置处的差异，Z′B选自A、C或G，并且ZA是与Z′B互补的核苷酸。这些核苷酸差异并不会显著降低siRNA缀合物的靶基因抑制能力，而这些包含核苷酸差异的siRNA缀合物也在本公开的保护范围之内。In some embodiments, the nucleotide difference between the nucleotide sequence B and the nucleotide sequence shown in SEQ ID NO: 2 includes a difference at theZ'B position, andZ'B is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at theZ'B position,Z'B is selected from A, C or G, and ZA is a nucleotide complementary toZ'B . These nucleotide differences do not significantly reduce the target gene inhibition ability of the siRNA conjugate, and these siRNA conjugates containing nucleotide differences are also within the scope of protection of the present disclosure.
在一些实施方案中，所述核苷酸序列A和所述核苷酸序列B基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个的碱基错配；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配。In some embodiments, the nucleotide sequence A and the nucleotide sequence B are substantially reverse complementary, essentially reverse complementary, or completely reverse complementary; the substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences; the substantially reverse complementary means that there are no more than 1 base mismatch between the two nucleotide sequences; and the completely reverse complementary means that there are no mismatches between the two nucleotide sequences.
在一些实施方案中，核苷酸序列A是SEQ ID NO：3所示的核苷酸序列，核苷酸序列B是SEQ ID NO：4所示的核苷酸序列：In some embodiments, nucleotide sequence A is the nucleotide sequence shown in SEQ ID NO: 3, and nucleotide sequence B is the nucleotide sequence shown in SEQ ID NO: 4:
5′-CGUGUGCACUUCGCUUCAZA-3′(SEQ ID NO：3)；5′-CGUGUGCACUUCGCUUCAZA -3′ (SEQ ID NO: 3);
5′-Z′BUGAAGCGAAGUGCACACG-3′(SEQ ID NO：4)，5′-Z′B UGAAGCGAAGUGCACACG-3′ (SEQ ID NO: 4),
其中，所述Z′B是反义链5′末端的第一个核苷酸，ZA选自A、U、G或C，并且Z′B是与ZA互补的核苷酸；在一些实施方案中，ZA为A，Z′B为U；wherein saidZ′B is the first nucleotide at the 5′ end of the antisense strand,ZA is selected from A, U, G or C, andZ′B is a nucleotide complementary toZA ; in some embodiments,ZA is A andZ′B is U;
并且，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，反义链的长度为20-26个核苷酸。这样，本公开提供的siRNA正义链和反义链的长度比可以是19/20、19/21、19/22、19/23、19/24、19/25、19/26、20/20、20/21、20/22、20/23、20/24、20/25、20/26、21/20、21/21、21/22、21/23、21/24、21/25、21/26、22/20、22/21、22/22、22/23、22/24、22/25、22/26、23/20、23/21、23/22、23/23、23/24、23/25或23/26。在一些实施方案中，所述siRNA正义链和反义链的长度比为19/21、21/23或23/25。Furthermore, the sense strand and antisense strand have the same or different lengths, the sense strand has a length of 19-23 nucleotides, and the antisense strand has a length of 20-26 nucleotides. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure can be 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21/26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19/21, 21/23, or 23/25.
在一些实施方案中，所述正义链和反义链长度相同，所述核苷酸序列I还含有核苷酸序列III，所述核苷酸序列II还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV长度各自独立地为1-4个核苷酸；所述核苷酸序列III连接在核苷酸序列A的5’末端，所述核苷酸序列IV连接在核苷酸序列B的3’末端，所述核苷酸序列III和所述核苷酸序列IV长度相等。In some embodiments, the sense strand and the antisense strand are of the same length, the nucleotide sequence I further contains a nucleotide sequence III, the nucleotide sequence II further contains a nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV are each independently 1-4 nucleotides in length; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence A, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence B, and the nucleotide sequence III and the nucleotide sequence IV are of equal length.
所述核苷酸序列III和核苷酸序列IV可以互补或不互补，为了增加siRNA的稳定性，在一些实施方案中，核苷酸序列III和核苷酸序列IV至少部分互补；在一些实施方案中，核苷酸序列III和核苷酸序列IV 80％以上的碱基互补，或者90％以上的碱基互补；在一些实施方案中，核苷酸序列III和核苷酸序列IV实质上反向互补或完全反向互补；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配；在一些实施方案中，核苷酸序列III和核苷酸序列IV完全反向互补。由此，所述siRNA正义链和反义链等长，其长度比为20/20、21/21、22/22或23/23。在一些实施方案中，所述siRNA正义链和反义链的长度比为21/21或23/23。The nucleotide sequence III and nucleotide sequence IV may be complementary or non-complementary. In order to increase the stability of siRNA, in some embodiments, nucleotide sequence III and nucleotide sequence IV are at least partially complementary; in some embodiments, nucleotide sequence III and nucleotide sequence IV are more than 80% complementary in bases, or more than 90% complementary in bases; in some embodiments, nucleotide sequence III and nucleotide sequence IV are substantially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there is no more than one base mismatch between the two nucleotide sequences; completely reverse complementary means that there is no mismatch between the two nucleotide sequences; in some embodiments, nucleotide sequence III and nucleotide sequence IV are completely reverse complementary. Thus, the sense strand and antisense strand of the siRNA are of equal length, and the length ratio is 20/20, 21/21, 22/22 or 23/23. In some embodiments, the length ratio of the sense strand and antisense strand of the siRNA is 21/21 or 23/23.
在一些实施方案中，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为C，核苷酸序列IV的碱基为G；此时，正义链和反义链的长度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5’末端到3’末端的方向，核苷酸序列III的碱基组成为AC，核苷酸序列IV的碱基组成为GU；此时，正义链和反义链的长度比为21/21；或者，核苷酸序列III和IV的长度均为3个核苷酸，按照5’末端到3’末端的方向，核苷酸序列III的碱基组成为GAC，核苷酸序列IV的碱基组成为GUC；此时，正义链和反义链的长度比为22/22；或者，核苷酸序列III和IV的长度均为4个核苷酸，按照5’末端到3’末端的方向，核苷酸序列III的碱基组成为GGAC，核苷酸序列IV的碱基组成为GUCC；此时，正义链和反义链的长度比为23/23。在一些实施方案中，所述核苷酸序列III和核苷酸序列IV的长度为2个核苷酸，按照5’末端到3’末端的方向，核苷酸序列III的碱基组成为AC，核苷酸序列IV的碱基组成为GU；此时，正义链和反义链的长度比为21/21。In some embodiments, the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is C, and the base of the nucleotide sequence IV is G; in this case, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is AC, and the base composition of the nucleotide sequence IV is GU; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or, the length of the nucleotide sequence III and IV are both 3 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is GAC, and the base composition of the nucleotide sequence IV is GUC; in this case, the length ratio of the sense strand to the antisense strand is 22/22; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is GGAC, and the base composition of the nucleotide sequence IV is GUCC; in this case, the length ratio of the sense strand to the antisense strand is 23/23. In some embodiments, the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is AC, and the base composition of the nucleotide sequence IV is GU; at this time, the length ratio of the sense chain and the antisense chain is 21/21.
在一些实施方案中，核苷酸序列III和核苷酸序列IV的长度相同，并且完全反向互补，因此，给出了核苷酸序列III的碱基，核苷酸序列IV的碱基也就确定了。In some embodiments, nucleotide sequence III and nucleotide sequence IV are of the same length and are completely reverse complementary, so that when the base of nucleotide sequence III is given, the base of nucleotide sequence IV is also determined.
在一些实施方案中，所述正义链和反义链长度不同，所述核苷酸序列II还含有核苷酸序列V，核苷酸序列V的长度为1至3个核苷酸，连接在所述反义链的3’末端，构成反义链的3’悬垂末端。由此，本公开提供的siRNA正义链和反义链的长度比可以是19/20、19/21、19/22、20/21、20/22、20/23、21/22、21/23、21/24、22/23、22/24、22/25、23/24、23/25或23/26。在一些实施方案中，所述核苷酸序列V的长度为2个核苷酸，由此，本公开提供的siRNA正义链和反义链的长度比可以是19/21、21/23或23/25。In some embodiments, the sense strand and antisense strand are of different lengths, and the nucleotide sequence II further contains a nucleotide sequence V, the length of which is 1 to 3 nucleotides, connected to the 3' end of the antisense strand to form the 3' overhanging end of the antisense strand. Thus, the length ratio of the sense strand and antisense strand of the siRNA provided by the present disclosure can be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23, 21/24, 22/23, 22/24, 22/25, 23/24, 23/25 or 23/26. In some embodiments, the length of the nucleotide sequence V is 2 nucleotides, and thus, the length ratio of the sense strand and antisense strand of the siRNA provided by the present disclosure can be 19/21, 21/23 or 23/25.
所述核苷酸序列V中的每一个核苷酸可以是任意的核苷酸，为了便于合成并节约合成成本，所述核苷酸序列V为连续的2个胸腺嘧啶脱氧核糖核苷酸(TT)或连续的2个尿嘧啶核糖核苷酸(UU)；为了提高siRNA反义链与靶mRNA的亲和力，核苷酸序列V与靶mRNA的相应位置的核苷酸互补。因此，在一些实施方案中，本公开的siRNA的正义链和反义链的长度之比为19/21或21/23，此时，本公开的siRNA具有更好的mRNA沉默活性。Each nucleotide in the nucleotide sequence V can be any nucleotide. In order to facilitate synthesis and save synthesis costs, the nucleotide sequence V is 2 consecutive thymine deoxyribonucleotides (TT) or 2 consecutive uracil ribonucleotides (UU); in order to improve the affinity of the siRNA antisense strand to the target mRNA, the nucleotide sequence V is complementary to the nucleotides at the corresponding positions of the target mRNA. Therefore, in some embodiments, the ratio of the length of the sense strand and the antisense strand of the siRNA disclosed in the present invention is 19/21 or 21/23, at which time, the siRNA disclosed in the present invention has better mRNA silencing activity.
在一些实施方案中，所述siRNA的正义链含有如SEQ ID NO：3所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO：4所示的核苷酸序列：In some embodiments, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 3, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 4:
5’-CGUGUGCACUUCGCUUCAZA-3’(SEQ ID NO：3)；5'-CGUGUGCACUUCGCUUCAZA- 3' (SEQ ID NO: 3);
5’-Z′BUGAAGCGAAGUGCACACG-3’(SEQ ID NO：4)；5'-Z'B UGAAGCGAAGUGCACACG-3' (SEQ ID NO: 4);
其中，所述Z′B是反义链5′末端的第一个核苷酸，ZA选自A、U、G或C，并且Z′B是与ZA互补的核苷酸。Wherein,Z′B is the first nucleotide at the 5′ end of the antisense strand,ZA is selected from A, U, G or C, andZ′B is a nucleotide complementary toZA .
根据本公开一些具体的实施例，本公开所述siRNA为siHBV1siHBV1According to some specific embodiments of the present disclosure, the siRNA described in the present disclosure is siHBV1siHBV1
正义链：5′-CGUGUGCACUUCGCUUCAZ-3′(SEQ ID NO：1)，Sense strand: 5′-CGUGUGCACUUCGCUUCAZ-3′ (SEQ ID NO: 1),
反义链：5′-Z′UGAAGCGAAGUGCACACGGU-3′(SEQ ID NO：5)，Antisense strand: 5′-Z′UGAAGCGAAGUGCACACGGU-3′ (SEQ ID NO: 5),
其中，Z为A，Z′为U。Among them, Z is A and Z′ is U.
如前所述，本公开的siRNA中的核苷酸各自独立地为修饰或未修饰的核苷酸。在一些实施方案中，本公开的siRNA中的核苷酸为未经修饰的核苷酸；在一些实施方案中，本公开的siRNA中的部分或全部核苷酸为修饰的核苷酸，核苷酸基团上的这些修饰不会导致本公开的siRNA缀合物抑制乙肝病毒基因表达的功能明显削弱或丧失。As mentioned above, the nucleotides in the siRNA of the present disclosure are each independently modified or unmodified nucleotides. In some embodiments, the nucleotides in the siRNA of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNA of the present disclosure are modified nucleotides, and these modifications on the nucleotide groups will not cause the function of the siRNA conjugate of the present disclosure to inhibit the expression of the hepatitis B virus gene to be significantly weakened or lost.
在一些实施方案中，本公开的siRNA至少含有1个修饰的核苷酸。在本公开的上下文中，所使用的术语“修饰的核苷酸”是指核苷酸的核糖基2′位羟基被其他基团取代形成的核苷酸或核苷酸类似物，或者核苷酸上的碱基是经修饰的碱基的核苷酸。所述修饰的核苷酸不会导致siRNA抑制基因表达的功能明显削弱或丧失。例如，可以选择J.K.Watts，G.F.Deleavey，and M.J.Damha，Chemically modified siRNA：tools andapplications.Drug Discov Today，2008，13(19-20)：842-55中公开的修饰的核苷酸。In some embodiments, the siRNA of the present disclosure contains at least one modified nucleotide. In the context of the present disclosure, the term "modified nucleotide" used refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribose group of the nucleotide with other groups, or a nucleotide in which the base on the nucleotide is a modified base. The modified nucleotide will not cause the function of siRNA to inhibit gene expression to be significantly weakened or lost. For example, the modified nucleotide disclosed in J.K.Watts, G.F.Deleavey, and M.J.Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13 (19-20): 842-55 can be selected.
在一些实施方案中，本公开提供的siRNA的正义链或所述反义链中的至少一个核苷酸为修饰的核苷酸，和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基；换句话说，所述正义链和所述反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基和/或核糖基的至少一部分为具有修饰基团的磷酸酯基和/或具有修饰基团的核糖基。In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate group having a modified group; in other words, at least a portion of the phosphate groups and/or ribose groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand is a phosphate group having a modified group and/or a ribose group having a modified group.
在一些实施方案中，所述正义链和/或所述反义链中的全部核苷酸均为修饰的核苷酸。在一些实施方案中，本公开提供的siRNA的正义链和所述反义链中的每一个核苷酸独立地为氟代修饰的核苷酸或非氟代修饰的核苷酸。In some embodiments, all nucleotides in the sense strand and/or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the siRNA provided by the present disclosure is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.
本公开的发明人惊奇地发现，本公开所述的siRNA在动物实验中获得了血浆中稳定性和基因沉默效率的高度平衡。The inventors of the present disclosure surprisingly found that the siRNA described in the present disclosure achieved a high balance between stability in plasma and gene silencing efficiency in animal experiments.
在一些实施方案中，所述氟代修饰的核苷酸位于核苷酸序列A和核苷酸序列B中，并且，按照5′末端到3′末端的方向，所述核苷酸序列A的第7、8、9位的核苷酸为氟代修饰的核苷酸；按照5′末端到3′末端的方向，所述核苷酸序列B的第2、6、14、16位的核苷酸为氟代修饰的核苷酸。In some embodiments, the fluorinated modified nucleotides are located in nucleotide sequence A and nucleotide sequence B, and, in the direction from the 5′ end to the 3′ end, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence A are fluorinated modified nucleotides; in the direction from the 5′ end to the 3′ end, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence B are fluorinated modified nucleotides.
在一些实施方案中，所述氟代修饰的核苷酸位于核苷酸序列A和核苷酸序列B中，所述核苷酸序列A中氟代修饰的核苷酸不多于5个，并且，按照5′末端到3′末端的方向，所述核苷酸序列A的第7、8、9位的核苷酸为氟代修饰的核苷酸；所述核苷酸序列B中氟代修饰的核苷酸不多于7个，并且，所述核苷酸序列B的第2、6、14、16位的核苷酸为氟代修饰的核苷酸。In some embodiments, the fluorinated modified nucleotides are located in nucleotide sequence A and nucleotide sequence B, the number of fluorinated modified nucleotides in the nucleotide sequence A is no more than 5, and, in the direction from the 5′ end to the 3′ end, the 7th, 8th, and 9th nucleotides of the nucleotide sequence A are fluorinated modified nucleotides; the number of fluorinated modified nucleotides in the nucleotide sequence B is no more than 7, and the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence B are fluorinated modified nucleotides.
在一些实施方案中，按照5′末端到3′末端的方向，在所述正义链中，所述核苷酸序列A的第7、8、9位或者5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为非氟代修饰的核苷酸；按照5′末端到3′末端的方向，在所述反义链中，所述核苷酸序列B的第2、6、14、16位或者2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为非氟代修饰的核苷酸。In some embodiments, in the direction from the 5′ end to the 3′ end, in the sense strand, the nucleotides at positions 7, 8, 9 or positions 5, 7, 8, 9 of the nucleotide sequence A are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorinated modified nucleotides; in the direction from the 5′ end to the 3′ end, in the antisense strand, the nucleotides at positions 2, 6, 14, 16 or positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence B are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are non-fluorinated modified nucleotides.
在本公开的上下文中，“氟代修饰的核苷酸”指核苷酸的核糖基2′位的羟基被氟取代形成的核苷酸，其具有以下式(101)所示的结构。“非氟代修饰的核苷酸”指核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物。在一些实施方案中，每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种。In the context of the present disclosure, "fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and has a structure shown in the following formula (101). "Non-fluorinated modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group. In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group.
这些核糖基2′位的羟基被非氟基团取代形成的核苷酸是本领域技术人员所公知的，这些核苷酸可以选自2′-烷氧基修饰的核苷酸、2′-经取代的烷氧基修饰的核苷酸、2′-烷基修饰的核苷酸、2′-经取代的烷基修饰的核苷酸、2′-氨基修饰的核苷酸、2′-经取代的氨基修饰的核苷酸、2′-脱氧核苷酸中的一种。The nucleotides formed by replacing the hydroxyl group at the 2′ position of these ribose groups with non-fluorine groups are well known to those skilled in the art, and these nucleotides can be selected from 2′-alkoxy modified nucleotides, 2′-substituted alkoxy modified nucleotides, 2′-alkyl modified nucleotides, 2′-substituted alkyl modified nucleotides, 2′-amino modified nucleotides, 2′-substituted amino modified nucleotides, and 2′-deoxynucleotides.
在一些实施方案中，2′-烷氧基修饰的核苷酸为甲氧基修饰的核苷酸(2′-OMe)，如式(102)所示。2′-经取代的烷氧基修饰的核苷酸，例如可以是2′-O-甲氧基乙基修饰的核苷酸(2′-MOE)，如式(103)所示。2′-氨基修饰的核苷酸(2′-NH2)如式(104)所示。2′-脱氧核苷酸(DNA)如式(105)所示。In some embodiments, the 2′-alkoxy modified nucleotide is a methoxy modified nucleotide (2′-OMe), as shown in formula (102). The 2′-substituted alkoxy modified nucleotide, for example, can be a 2′-O-methoxyethyl modified nucleotide (2′-MOE), as shown in formula (103). The 2′-amino modified nucleotide (2′-NH2) is shown in formula (104). The 2′-deoxynucleotide (DNA) is shown in formula (105).
核苷酸类似物指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。在一些实施方案中，核苷酸类似物可以是如异核苷酸、桥联的核苷酸(bridgednucleicacid，简称BNA)或无环核苷酸。Nucleotide analogs refer to groups that can replace nucleotides in nucleic acids, but have structures different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxyribonucleotides. In some embodiments, nucleotide analogs can be, for example, isonucleotides, bridged nucleotides (BNAs) or acyclic nucleotides.
BNA是指受约束的或不能接近的核苷酸。BNA可以含有五元环、六元环、或七元环的具有“固定的”C3′-内切糖缩拢的桥联结构。通常将该桥掺入到该核糖的2′-、4′-位处以提供一个2′，4′-BNA核苷酸，如LNA、ENA、cET BNA等，其中，LNA如式(106)所示，ENA如式(107)所示，cET BNA如式(108)所示。BNA refers to a constrained or inaccessible nucleotide. BNA may contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endosugar condensed bridge structure. The bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2', 4'-BNA nucleotide, such as LNA, ENA, cET BNA, etc., wherein LNA is shown in formula (106), ENA is shown in formula (107), and cET BNA is shown in formula (108).
无环核苷酸是核苷酸的糖环被打开形成的一类核苷酸，如解锁核酸(UNA)或甘油核酸(GNA)，其中，UNA如式(109)所示，GNA如式(110)所示。Acyclic nucleotides are a type of nucleotides formed by opening the sugar ring of a nucleotide, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), wherein UNA is shown in formula (109) and GNA is shown in formula (110).
上述式(109)和式(110)中，R选自H、OH或烷氧基(O-烷基)。In the above formula (109) and formula (110), R is selected from H, OH or alkoxy (O-alkyl).
异核苷酸是指核苷酸中碱基在核糖环上的位置发生改变而形成的化合物，例如，碱基从核糖环的1′-位移动至2′-位或3′-位而形成的化合物，如式(111)或(112)所示。Isonucleotides refer to compounds formed by a change in the position of the base on the ribose ring of a nucleotide, for example, a compound formed by the base moving from the 1′-position to the 2′-position or the 3′-position of the ribose ring, as shown in formula (111) or (112).
上述式(111)-式(112)所示的化合物中，Base表示碱基，例如A、U、G、C或T；R选自H、OH、F或者如上所述的非氟基团。In the compounds represented by the above formula (111)-(112), Base represents a base, such as A, U, G, C or T; R is selected from H, OH, F or the non-fluorine group as described above.
在一些实施方案中，核苷酸类似物选自异核苷酸、LNA、ENA、cET、UNA和GNA中的一种。在一些实施方案中，每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸，在上文和下文中，所述甲氧基修饰的核苷酸指核糖基的2′-羟基被甲氧基取代而形成的核苷酸。In some embodiments, the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA. In some embodiments, each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, and in the above and below, the methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2′-hydroxyl group of the ribose group with a methoxy group.
在上文及下文中，“氟代修饰的核苷酸”、“2′-氟修饰的核苷酸”、“核糖基团的2′-羟基被氟取代的核苷酸”和“2′-氟代核糖基”意义相同，均指核苷酸的2′-羟基被氟取代，而形成的具有如式(207)所示结构的化合物；“甲氧基修饰的核苷酸”、“2′-甲氧基修饰的核苷酸”、“核糖基团的2′-羟基被甲氧基取代的核苷酸”和“2′-甲氧基核糖基”意义相同，均指核苷酸核糖基团的2′-羟基被甲氧基取代而形成的具有如式(208)所示结构的化合物。In the above and below, "fluorinated nucleotides", "2′-fluorinated nucleotides", "nucleotides in which the 2′-hydroxyl group of the ribose group is substituted by fluorine" and "2′-fluororibose" have the same meaning, all referring to compounds having a structure shown in formula (207) formed by the 2′-hydroxyl group of the nucleotide being replaced by fluorine; "methoxy-modified nucleotides", "2′-methoxy-modified nucleotides", "nucleotides in which the 2′-hydroxyl group of the ribose group is substituted by methoxy" and "2′-methoxyribose" have the same meaning, all referring to compounds having a structure shown in formula (208) formed by the 2′-hydroxyl group of the ribose group of the nucleotide being replaced by methoxy.
在一些实施方案中，本公开的siRNA是具有以下修饰的siRNA：按照5′末端到3′末端的方向，在所述正义链中，所述核苷酸序列A的第7、8、9位或者第5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为甲氧基修饰的核苷酸；在所述反义链中，所述核苷酸序列B的第2、6、14、16位或者第2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为甲氧基修饰的核苷酸。In some embodiments, the siRNA disclosed herein is a siRNA having the following modifications: in the direction from the 5′ end to the 3′ end, in the sense chain, the nucleotides at positions 7, 8, 9 or positions 5, 7, 8, 9 of the nucleotide sequence A are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense chain are methoxy-modified nucleotides; in the antisense chain, the nucleotides at positions 2, 6, 14, 16 or positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence B are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense chain are methoxy-modified nucleotides.
在一些实施方案中，本公开的siRNA是具有以下修饰的siRNA：按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第5、7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、8、9、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸；In some embodiments, the siRNA of the present disclosure is a siRNA with the following modifications: in the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8 and 9 of the nucleotide sequence A in the sense strand of the siRNA are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 9, 14 and 16 of the nucleotide sequence B in the antisense strand of the siRNA are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides;
或者，按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第5、7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸；Alternatively, in the direction from the 5' end to the 3' end, the 5th, 7th, 8th and 9th nucleotides of the nucleotide sequence A in the sense strand of the siRNA are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the siRNA sense strand are methoxy-modified nucleotides, and, in the direction from the 5' end to the 3' end, the 2nd, 6th, 14th and 16th nucleotides of the nucleotide sequence B in the antisense strand of the siRNA are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides;
或者，按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第7、8和9位的核苷酸为-氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸。Alternatively, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 of the nucleotide sequence A in the sense chain of the siRNA are -fluoro-modified nucleotides, and the nucleotides at the remaining positions of the siRNA sense chain are methoxy-modified nucleotides, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence B in the antisense chain of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions of the antisense chain of the siRNA are methoxy-modified nucleotides.
换句话说，该siRNA的磷酸-糖骨架中的核糖基分别具有如下修饰基团：按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第5、7、8和9位的糖基为2’-氟代核糖基，siRNA的正义链的其余位置核苷酸的糖基为2’-甲氧基核糖基，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、8、9、14和16位的糖基为2’-氟代核糖基，siRNA的反义链其余位置核苷酸的糖基为2’-甲氧基核糖基；In other words, the ribose groups in the phosphate-sugar backbone of the siRNA have the following modification groups respectively: in the direction from the 5' end to the 3' end, the sugar groups at positions 5, 7, 8 and 9 of the nucleotide sequence A in the sense strand of the siRNA are 2'-fluororibose groups, and the sugar groups at the remaining positions of the nucleotides in the sense strand of the siRNA are 2'-methoxyribose groups, and, in the direction from the 5' end to the 3' end, the sugar groups at positions 2, 6, 8, 9, 14 and 16 of the nucleotide sequence B in the antisense strand of the siRNA are 2'-fluororibose groups, and the sugar groups at the remaining positions of the nucleotides in the antisense strand of the siRNA are 2'-methoxyribose groups;
或者，按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第5、7、8和9位的糖基为2’-氟代核糖基，siRNA的正义链的其余位置核苷酸的糖基为2’-甲氧基核糖基，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、14和16位的糖基为2’-氟代核糖基，siRNA的反义链其余位置核苷酸的糖基为2’-甲氧基核糖基；Alternatively, in the direction from the 5' end to the 3' end, the sugar groups at positions 5, 7, 8 and 9 of the nucleotide sequence A in the sense strand of the siRNA are 2'-fluororibose groups, and the sugar groups at the remaining positions of the nucleotides in the sense strand of the siRNA are 2'-methoxyribose groups, and, in the direction from the 5' end to the 3' end, the sugar groups at positions 2, 6, 14 and 16 of the nucleotide sequence B in the antisense strand of the siRNA are 2'-fluororibose groups, and the sugar groups at the remaining positions of the nucleotides in the antisense strand of the siRNA are 2'-methoxyribose groups;
或者，按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第7、8和9位的糖基为2’-氟代核糖基，siRNA的正义链的其余位置核苷酸的糖基为2’-甲氧基核糖基，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、14和16位的糖基为2’-氟代核糖基，siRNA的反义链其余位置核苷酸的糖基为2’-甲氧基核糖基。Alternatively, in the direction from the 5' end to the 3' end, the sugar groups at positions 7, 8 and 9 of the nucleotide sequence A in the sense chain of the siRNA are 2'-fluororibose groups, and the sugar groups of the nucleotides at the remaining positions of the sense chain of the siRNA are 2'-methoxyribose groups, and, in the direction from the 5' end to the 3' end, the sugar groups at positions 2, 6, 14 and 16 of the nucleotide sequence B in the antisense chain of the siRNA are 2'-fluororibose groups, and the sugar groups of the nucleotides at the remaining positions of the antisense chain of the siRNA are 2'-methoxyribose groups.
在一些实施方案中，本公开提供的siRNA为siHBV2或siHBV3：In some embodiments, the siRNA provided by the present disclosure is siHBV2 or siHBV3:
siHBV2siHBV2
正义链：5′-CmGmUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′(SEQ ID NO：6)，Sense strand: 5′-CmGmUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′ (SEQ ID NO: 6),
反义链：5′-UmUfGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGmGmUm-3′(SEQ ID NO：7)，Antisense strand: 5′-UmUfGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGmGmUm-3′ (SEQ ID NO: 7),
siHBV3siHBV3
正义链：5′-CmGmUmGmUfGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′(SEQ ID NO：8)，Sense strand: 5′-CmGmUmGmUfGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′ (SEQ ID NO: 8),
反义链：5′-UmUfGmAmAmGfCmGfAfAmGmUmGmCfAmCfAmCmGmGmUm-3′(SEQ ID NO：9)，Antisense strand: 5′-UmUfGmAmAmGfCmGfAfAmGmUmGmCfAmCfAmCmGmGmUm-3′ (SEQ ID NO: 9),
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸。Among them, capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide.
具有上述修饰的siRNA不仅成本低，而且可使血液中的核糖核酸酶不易切割核酸，由此增加核酸的稳定性，使核酸具有更强的抵抗核酸酶水解的性能。The siRNA with the above modifications is not only low-cost, but also makes it difficult for ribonucleases in the blood to cut nucleic acids, thereby increasing the stability of the nucleic acids and making the nucleic acids more resistant to nuclease hydrolysis.
在一些实施方案中，本公开提供的siRNA的正义链和反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基中的至少一部分为具有修饰基团的磷酸酯基。在一些实施方案中，具有修饰基团的磷酸酯基为磷酸酯基中的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯基；在一些实施方案中，所述具有修饰基团的磷酸酯基为具有如式(1)所示结构的硫代磷酸酯基：In some embodiments, at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand of the siRNA provided by the present disclosure is a phosphate group having a modified group. In some embodiments, the phosphate group having a modified group is a thiophosphate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, the phosphate group having a modified group is a thiophosphate group having a structure as shown in formula (1):
这种修饰能稳定siRNA的双链结构，保持碱基配对的高特异性和高亲和力。This modification can stabilize the double-stranded structure of siRNA and maintain high specificity and high affinity of base pairing.
在一些实施方案中，本公开提供的siRNA中，硫代磷酸酯基连接存在于以下位置中的至少一处：正义链或反义链任意一端的第一个和第二个核苷酸之间；正义链或反义链任意一端的第二个和第三个核苷酸之间；或上述的任意组合。在一些实施方案中，硫代磷酸酯基连接存在于除正义链5′末端以外的全部上述位置处。在一些实施方案中，硫代磷酸酯基连接存在于除正义链3′末端以外的全部上述位置处。在一些实施方案中，硫代磷酸酯基连接存在于以下位置中的至少一处：In some embodiments, in the siRNA provided by the present disclosure, the phosphorothioate linkage is present in at least one of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the above. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 5′ end of the sense strand. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 3′ end of the sense strand. In some embodiments, the phosphorothioate linkage is present at at least one of the following positions:
所述正义链的5′末端端部第1个核苷酸和第2个核苷酸之间；Between the first nucleotide and the second nucleotide at the 5′ end of the sense strand;
所述正义链的5′末端端部第2个核苷酸和第3个核苷酸之间；Between the second nucleotide and the third nucleotide at the 5′ end of the sense strand;
所述正义链的3′末端端部第1个核苷酸和第2个核苷酸之间；Between the first nucleotide and the second nucleotide at the 3′ end of the sense strand;
所述正义链的3′末端端部第2个核苷酸和第3个核苷酸之间；Between the second nucleotide and the third nucleotide at the 3′ end of the sense strand;
所述反义链的5′末端端部第1个核苷酸和第2个核苷酸之间；Between the first nucleotide and the second nucleotide at the 5′ end of the antisense strand;
所述反义链的5′末端端部第2个核苷酸和第3个核苷酸之间；Between the second nucleotide and the third nucleotide at the 5′ end of the antisense strand;
所述反义链的3′末端端部第1个核苷酸和第2个核苷酸之间；以及between the first nucleotide and the second nucleotide at the 3′ end of the antisense strand; and
所述反义链的3′末端端部第2个核苷酸和第3个核苷酸之间。The 3' end of the antisense strand is between the second nucleotide and the third nucleotide.
在一些实施方案中，本公开提供的siRNA为siHBV4或siHBV5：In some embodiments, the siRNA provided by the present disclosure is siHBV4 or siHBV5:
siHBV4siHBV4
正义链：5′-CmsGmsUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′(SEQ ID NO：10)，Sense strand: 5′-CmsGmsUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′ (SEQ ID NO: 10),
反义链：5′-UmsUfsGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGmsGmsUm-3′(SEQ IDNO：11)，Antisense strand: 5′-UmsUfsGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGmsGmsUm-3′ (SEQ IDNO: 11),
siHBV5siHBV5
正义链：5′-CmsGmsUmGmUfGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′(SEQ ID NO：12)，Sense strand: 5′-CmsGmsUmGmUfGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′ (SEQ ID NO: 12),
反义链：5′-UmsUfsGmAmAmGfCmGfAfAmGmUmGmCfAmCfAmCmGmsGmsUm-3′(SEQ IDNO：13)，Antisense strand: 5′-UmsUfsGmAmAmGfCmGfAfAmGmUmGmCfAmCfAmCmGmsGmsUm-3′ (SEQ IDNO: 13),
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母左右两个核苷酸之间为硫代磷酸酯基连接。Among them, capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; the lowercase letter s indicates that the two nucleotides on the left and right of the letter are connected by a thiophosphate group.
在一些实施方案中，所述siRNA反义链的5’末端核苷酸为5’-磷酸核苷酸或5’-磷酸类似物修饰的核苷酸。In some embodiments, the 5' terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
常用的所述5’-磷酸核苷酸或5’-磷酸类似物修饰的核苷酸是本领域技术人员所公知的，如5’-磷酸核苷酸可具有如下结构：The commonly used 5'-phosphate nucleotides or 5'-phosphate analogue modified nucleotides are well known to those skilled in the art, such as 5'-phosphate nucleotides may have the following structure:
再如，Anastasia Khvorova and Jonathan K.Watts，The chemical evolutionof oligonucleotide therapies of clinical utility.Nature Biotechnology，2017，35(3)：238-48中公开了如下4种5’-磷酸类似物修饰的核苷酸：For example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48 disclose the following four 5'-phosphate analog-modified nucleotides:
其中，R选自H、OH、甲氧基、氟；Base表示碱基，选自A、U、C、G或T。Wherein, R is selected from H, OH, methoxy, and fluorine; Base represents a base, which is selected from A, U, C, G, or T.
在一些实施方案中，5′-磷酸核苷酸为式(2)所示的含有5′-磷酸修饰的核苷酸，5’-磷酸类似物修饰的核苷酸为含有乙烯基磷酸酯(5’-(E)-vinylphosphonate，E-VP)修饰的核苷酸，如式(3)所示，或者为硫代磷酸酯修饰的核苷酸，如式(5)所示。In some embodiments, the 5′-phosphate nucleotide is a nucleotide containing a 5′-phosphate modification as shown in formula (2), and the 5′-phosphate analog modified nucleotide is a nucleotide containing a vinyl phosphate (5′-(E)-vinylphosphonate, E-VP) modification as shown in formula (3), or a nucleotide modified with a thiophosphate as shown in formula (5).
在一些实施方案中，本公开提供的siRNA为siHBV6、siHBV7、siHBV8或siHBV9：In some embodiments, the siRNA provided by the present disclosure is siHBV6, siHBV7, siHBV8 or siHBV9:
siHBV6siHBV6
正义链：5′-CmGmUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′(SEQ ID NO：6)，Sense strand: 5′-CmGmUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′ (SEQ ID NO: 6),
反义链：5′-P1-UmUfGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGmGmUm-3′(SEQ ID NO：14)，Antisense strand: 5′-P1-UmUfGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGmGmUm-3′ (SEQ ID NO: 14),
siHBV7siHBV7
正义链：5′-CmGmUmGmUfGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′(SEQ ID NO：8)，Sense strand: 5′-CmGmUmGmUfGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′ (SEQ ID NO: 8),
反义链：5′-P1-UmUfGmAmAmGfCmGfAfAmGmUmGmCfAmCfAmCmGmGmUm-3′(SEQ ID NO：15)，Antisense strand: 5′-P1-UmUfGmAmAmGfCmGfAfAmGmUmGmCfAmCfAmCmGmGmUm-3′ (SEQ ID NO: 15),
siHBVX8sI
正义链：5′-CmsGmsUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′(SEQ ID NO：10)，Sense strand: 5′-CmsGmsUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′ (SEQ ID NO: 10),
反义链：5′-P1-UmsUfsGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGmsGmsUm-3′(SEQ IDNO：16)，Antisense strand: 5′-P1-UmsUfsGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGmsGmsUm-3′ (SEQ IDNO: 16),
siHBVX9siHBVX9
正义链：5′-CmsGmsUmGmUfGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′(SEQ ID NO：12)，Sense strand: 5′-CmsGmsUmGmUfGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′ (SEQ ID NO: 12),
反义链：5′-P1-UmsUfsGmAmAmGfCmGfAfAmGmUmGmCfAmCfAmCmGmsGmsUm-3′(SEQ IDNO：17)，Antisense strand: 5′-P1-UmsUfsGmAmAmGfCmGfAfAmGmUmGmCfAmCfAmCmGmsGmsUm-3′ (SEQ IDNO: 17),
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母左右两个核苷酸之间为硫代磷酸酯基连接；大写字母P1表示该字母右侧相邻的一个核苷酸为5’-磷酸核苷酸或5’-磷酸类似物修饰的核苷酸。Among them, capital letters C, G, U, and A represent the base composition of the nucleotide; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorinated-modified nucleotide; lowercase letter s indicates that the two nucleotides on the left and right of the letter are connected by a thiophosphate group; capital letter P1 indicates that the nucleotide adjacent to the right of the letter is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
本公开的发明人意外发现，本公开提供的siRNA不仅具有显著增强的血浆和溶酶体稳定性，还保留很高的基因抑制活性。The inventors of the present disclosure unexpectedly discovered that the siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosomal stability, but also retains high gene inhibition activity.
本公开提供的siRNA可以通过本领域常规的siRNA制备方法(例如固相合成和液相合成的方法)得到。其中，固相合成已经有商业化订制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的siRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。The siRNA provided by the present disclosure can be obtained by conventional siRNA preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis already has commercial customization services. The modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.
药物组合物Pharmaceutical composition
本公开提供了一种药物组合物，所述药物组合物含有如上所述的siRNA作为活性成分和药学上可接受的载体。The present disclosure provides a pharmaceutical composition, which contains the siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.
所述药学上可接受的载体可以是siRNA给药领域常规使用的载体，例如但不限于磁性纳米粒(magnetic nanoparticles，如基于Fe3O4或Fe2O3的纳米粒)、碳纳米管(carbonnanotubes)、介孔硅(mesoporous silicon)、磷酸钙纳米粒(calcium phosphatenanoparticles)、聚乙烯亚胺(polyethylenimine，PEI)、聚酰胺型树形高分子(polyamidoamine(PAMAM)dendrimer)、聚赖氨酸(poly(L-lysine)，PLL)、壳聚糖(chitosan)、1，2-二油酰基-3-三甲铵丙烷(1，2-dioleoyl-3-trimethylammonium-propane，DOTAP)、聚D型或L型乳酸/羟基乙酸共聚物(poly(D&L-lactic/glycolic acid)copolymer，PLGA)、聚(氨乙基乙撑磷酸酯)(poly(2-aminoethyl ethylene phosphate)，PPEEA)和聚(甲基丙烯酸-N，N-二甲氨基乙酯)(poly(2-dimethylaminoethylmethacrylate)，PDMAEMA)以及它们的衍生物中的一种或多种。The pharmaceutically acceptable carrier can be a carrier conventionally used in the field of siRNA administration, such as, butnot limited to, magnetic nanoparticles (such as nanoparticles based onFe3O4 orFe2O3 ), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine ), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly (D&L-lactic/glycolic acid) copolymer (PLGA), poly (2-aminoethyl ethylene phosphate ... Phosphate), PPEEA) and poly(methacrylate-N,N-dimethylaminoethyl ester) (poly(2-dimethylaminoethylmethacrylate), PDMAEMA) and one or more of their derivatives.
在一些实施方案中，所述药物组合物中，对siRNA和药学上可接受的载体的含量没有特别要求，在一些实施方案中，siRNA与药学上可接受的载体的重量比可以为1∶(1-500)，在一些的实施方案中，上述重量比为1∶(1-50)。In some embodiments, there are no special requirements for the content of siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition. In some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carrier can be 1: (1-500). In some embodiments, the above weight ratio is 1: (1-50).
在一些实施方案中，所述药物组合物中，还可以包含药学上可接受的其它辅料，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种。例如，所述药学上可接受的其它辅料可以包括pH值缓冲液、保护剂和渗透压调节剂中的至少一种。In some embodiments, the pharmaceutical composition may further include other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds conventionally used in the art. For example, the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.
所述pH值缓冲液可以为pH值7.5-8.5的三羟甲基胺基甲烷盐酸盐缓冲液和/或pH值5.5-8.5的磷酸盐缓冲液，例如可以为pH值5.5-8.5的磷酸盐缓冲液。The pH buffer may be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH value of 7.5-8.5 and/or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5.
所述保护剂可以为肌醇、山梨醇、蔗糖、海藻糖、甘露糖、麦芽糖、乳糖和葡萄糖中的至少一种。以所述药物组合物的总重量为基准，所述保护剂的含量可以为0.01-30重量％。The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.
所述渗透压调节剂可以为氯化钠和/或氯化钾。所述渗透压调节剂的含量使所述药物组合物的渗透压为200-700毫渗摩尔/千克。根据所需渗透压，本领域技术人员可以容易地确定所述渗透压调节剂的含量。The osmotic pressure regulator can be sodium chloride and/or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 mOsmole/kg. According to the desired osmotic pressure, the content of the osmotic pressure regulator can be easily determined by those skilled in the art.
在一些实施方案中，所述药物组合物可以为液体制剂，例如注射液；也可以为冻干粉针剂，实施给药时与液体辅料混合，配制成液体制剂。所述液体制剂可以但不限于用于皮下、肌肉或静脉注射给药，也可以但不限于通过喷雾给药到肺脏、或通过喷雾经肺脏给药到其它脏器组织(如肝脏)。在一些实施方案中，所述药物组合物用于静脉注射给药。In some embodiments, the pharmaceutical composition may be a liquid preparation, such as an injection; or a lyophilized powder injection, which is mixed with a liquid excipient during administration to prepare a liquid preparation. The liquid preparation may be, but is not limited to, administered subcutaneously, intramuscularly, or intravenously, or administered to the lungs by spraying, or administered to other organs (such as the liver) through the lungs by spraying. In some embodiments, the pharmaceutical composition is administered by intravenous injection.
在一些实施方案中，所述药物组合物可以为脂质体制剂的形式。在一些实施方案中，所述脂质体制剂中使用的药学上可接受的载体包含含胺的转染化合物(下文也可将其称为有机胺)、辅助脂质和/或聚乙二醇化脂质。其中，所述有机胺、辅助脂质和聚乙二醇化脂质可分别选自于CN103380113A(通过引用的方式将其整体并入本文)中所描述的含胺的转染化合物或其药学上可接受的盐或衍生物、辅助脂质和聚乙二醇化脂质中的一种或多种。In some embodiments, the pharmaceutical composition can be in the form of a liposome preparation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome preparation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), an auxiliary lipid and/or a pegylated lipid. Wherein, the organic amine, the auxiliary lipid and the pegylated lipid can be selected from one or more of the amine-containing transfection compound or its pharmaceutically acceptable salt or derivative, the auxiliary lipid and the pegylated lipid described in CN103380113A (which is incorporated herein by reference in its entirety).
在一些实施方案中，所述有机胺可为CN103380113A中描述的如式(201)所示的化合物或其药学上可接受的盐：In some embodiments, the organic amine may be a compound described in CN103380113A as shown in formula (201) or a pharmaceutically acceptable salt thereof:
其中：in:
X101和X102各自独立地是O、S、N-A或C-A，其中A是氢或C1-C20烃链；X101 andX102 are each independently O, S, NA or CA, wherein A is hydrogen or a C1-C20 hydrocarbon chain;
Y和Z各自独立地是C＝O、C＝S、S＝O、CH-OH或SO2；Y and Z are each independently C=O, C=S, S=O, CH-OH or SO2 ;
R101、R102、R103、R104、R105、R106和R107各自独立地是氢，环状或无环的、被取代的或未被取代的、支链或直链脂族基团，环状或无环的、被取代的或未被取代的、支链或直链杂脂族基团，被取代的或未被取代的、支链或直链酰基，被取代的或未被取代的、支链或直链芳基，被取代的或未被取代的、支链或直链杂芳基；R101 ,R102 ,R103 , R104,R105 ,R106andR107 are each independently hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic group, a substituted or unsubstituted, branched or straight-chain acyl group, a substituted or unsubstituted, branched or straight-chain aryl group, a substituted or unsubstituted, branched or straight-chain heteroaryl group;
x是1-10的整数；x is an integer from 1 to 10;
n是1-3的整数，m是0-20的整数，p是0或1；其中，如果m＝p＝0，则R102是氢；n is an integer of 1-3, m is an integer of 0-20, and p is 0 or 1; wherein, if m＝p＝0, R102 is hydrogen;
并且，如果n或m中的至少一个是2，那么R103和在式(201)中的氮形成如式(202)或式(203)所示的结构：And, if at least one of n or m is 2, then R103 and the nitrogen in formula (201) form a structure as shown in formula (202) or formula (203):
其中，g、e和f各自独立地是1-6的整数，“HCC”代表烃链，且每个＊N代表式(201)中的氮原子。wherein g, e and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (201).
在一些实施方案中，R103是多胺。在其它实施方案中，R103是缩酮。在一些实施方案中，在式(201)中的R101和R102中的每一个独立地是任意的被取代的或未被取代的、支链或直链烷基或烯基，所述烷基或烯基具有3至约20个碳原子，诸如8至约18个碳原子，和0至4个双键，诸如0至2个双键。In some embodiments, R103 is a polyamine. In other embodiments, R103 is a ketal. In some embodiments, each of R101 and R102 in formula (201) is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl having 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.
在一些实施方案中，如果n和m中的每一个独立地具有1或3的值，那么R103可以是下述式(204)-式(213)中的任一个：In some embodiments, if each of n and m independently has a value of 1 or 3, then R103 can be any one of the following formulas (204) to (213):
其中，式(204)-式(213)中，g、e和f各自独立地是1-6的整数，每个“个“，地代表烃链，且每个＊显示R103与在式(201)中的氮原子的可能连接点，其中在任意＊位置上的每个H可以被替换以实现与在式(201)中的氮原子的连接。Wherein, in formula (204)-formula (213), g, e and f are each independently an integer of 1-6, each "one" represents a hydrocarbon chain, and each * shows a possible connection point between R103 and the nitrogen atom in formula (201), wherein each H at any * position can be replaced to achieve connection with the nitrogen atom in formula (201).
其中，式(201)所示的化合物可以根据CN103380113A中的描述制备。Among them, the compound represented by formula (201) can be prepared according to the description in CN103380113A.
在一些实施方案中，所述有机胺为如式(214)所示的有机胺和/或如式(215)所示的有机胺：In some embodiments, the organic amine is an organic amine as shown in formula (214) and/or an organic amine as shown in formula (215):
所述辅助脂质为胆固醇、胆固醇的类似物和/或胆固醇的衍生物；The auxiliary lipid is cholesterol, a cholesterol analog and/or a cholesterol derivative;
所述聚乙二醇化脂质为1，2-二棕榈酰胺-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)]-2000。The PEGylated lipid is 1,2-dipalmitamide-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.
在一些实施方案中，所述药物组合物中，所述有机胺、所述辅助脂质和所述聚乙二醇化脂质三者之间的摩尔比为(19.7-80)∶(19.7-80)∶(0.3-50)，例如可以为(50-70)∶(20-40)∶(3-20)。In some embodiments, in the pharmaceutical composition, the molar ratio of the organic amine, the auxiliary lipid and the pegylated lipid is (19.7-80):(19.7-80):(0.3-50), for example, it can be (50-70):(20-40):(3-20).
在一些实施方案中，由本公开的siRNA与上述含胺的转染试剂形成的药物组合物颗粒具有约30nm至约200nm的平均直径，通常为约40nm至约135nm，更通常地，该脂质体颗粒的平均直径是约50nm至约120nm、约50nm至约100nm、约60nm至约90nm或约70nm至约90nm，例如，该脂质体颗粒的平均直径是约30、40、50、60、70、75、80、85、90、100、110、120、130、140、150或160nm。In some embodiments, the pharmaceutical composition particles formed by the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, more typically, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160 nm.
在一些实施方案中，由本公开的siRNA与上述含胺的转染试剂形成的药物组合物中，siRNA与全部脂质(例如有机胺、辅助脂质和/或聚乙二醇化脂质)的重量比(重量/重量比)在从约1∶1至约1∶50、从约1∶1至约1∶30、从约1∶3至约1∶20、从约1∶4至约1∶18、从约1∶5至约1∶17、从约1∶5至约1∶15、从约1∶5至约1∶12、从约1∶6至约1∶12或从约1∶6至约1∶10的范围内，例如，本公开的siRNA与全部脂质的重量比为约1∶5、1∶6、1∶7、1∶8、1∶9、1∶10、1∶11、1∶12、1∶13、1∶14、1∶15、1∶16、1∶17或1∶18。In some embodiments, in the pharmaceutical composition formed by the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent, the weight ratio (weight/weight ratio) of siRNA to total lipids (e.g., organic amines, helper lipids and/or pegylated lipids) is in the range of from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10, for example, the weight ratio of the siRNA of the present disclosure to total lipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17 or 1:18.
在一些实施方案中，所述药物组合物在销售时各组分可以独立存在，在使用时可以液体制剂的形式存在。在一些实施方案中，本公开提供的siRNA与上述药学上可接受的载体形成的药物组合物可以按照已知的各种方法制备，只是用本公开提供的siRNA替代现有siRNA即可；在一些实施方案中，可以按照如下方法制备：In some embodiments, the components of the pharmaceutical composition may exist independently when sold, and may exist in the form of a liquid preparation when used. In some embodiments, the pharmaceutical composition formed by the siRNA provided by the present disclosure and the above-mentioned pharmaceutically acceptable carrier may be prepared according to various known methods, except that the siRNA provided by the present disclosure is substituted for the existing siRNA; in some embodiments, it may be prepared according to the following method:
将有机胺、辅助脂质和聚乙二醇化脂质按照上述摩尔比悬浮于醇中并混匀得到脂质溶液；醇的用量使得到的脂质溶液的总质量浓度为2-25mg/mL，例如可以为8-18mg/mL。所述醇选自药学上可接受的醇，诸如在室温附近为液体的醇，例如，乙醇、丙二醇、苯甲醇、甘油、聚乙二醇200，聚乙二醇300，聚乙二醇400中的一种或多种，例如可以为乙醇。The organic amine, the auxiliary lipid and the PEGylated lipid are suspended in alcohol according to the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol used is such that the total mass concentration of the obtained lipid solution is 2-25 mg/mL, for example, 8-18 mg/mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid at room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, one or more, for example, ethanol.
将本公开提供的siRNA溶解于缓冲盐溶液中，得到siRNA水溶液。缓冲盐溶液的浓度为0.05-0.5M，例如可以为0.1-0.2M，调节缓冲盐溶液的pH至4.0-5.5，例如可以为5.0-5.2，缓冲盐溶液的用量使siRNA的浓度不超过0.6mg/mL，例如可以为0.2-0.4mg/mL。所述缓冲盐选自可溶性醋酸盐、可溶性柠檬酸盐中的一种或多种，例如可以为醋酸钠和/或醋酸钾。The siRNA provided by the present disclosure is dissolved in a buffered saline solution to obtain an siRNA aqueous solution. The concentration of the buffered saline solution is 0.05-0.5M, for example, 0.1-0.2M, and the pH of the buffered saline solution is adjusted to 4.0-5.5, for example, 5.0-5.2, and the amount of the buffered saline solution is such that the concentration of the siRNA does not exceed 0.6mg/mL, for example, 0.2-0.4mg/mL. The buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, sodium acetate and/or potassium acetate.
将脂质溶液和siRNA水溶液混合，将混合后得到的产物在40-60℃孵育至少2分钟，例如可以为5-30分钟，得到孵育后的脂质体制剂。脂质溶液和siRNA水溶液的体积比为1∶(2-5)，例如可以为1∶4。The lipid solution and the siRNA aqueous solution are mixed, and the mixed product is incubated at 40-60° C. for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome preparation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1:(2-5), for example, 1:4.
将孵育后的脂质体制剂浓缩或稀释，去除杂质，除菌，得到本公开提供的药物组合物，其理化参数为pH值为6.5-8，包封率不低于80％，粒径为40-200nm，多分散指数不高于0.30，渗透压为250-400mOsm/kg；例如理化参数可以为pH值为7.2-7.6，包封率不低于90％，粒径为60-100nm，多分散指数不高于0.20，渗透压为300-400mOsm/kg。The incubated liposome preparation is concentrated or diluted, impurities are removed, and sterilization is performed to obtain the pharmaceutical composition provided by the present disclosure, wherein the physicochemical parameters are a pH value of 6.5-8, an encapsulation rate of not less than 80%, a particle size of 40-200 nm, a polydispersity index of not more than 0.30, and an osmotic pressure of 250-400 mOsm/kg; for example, the physicochemical parameters may be a pH value of 7.2-7.6, an encapsulation rate of not less than 90%, a particle size of 60-100 nm, a polydispersity index of not more than 0.20, and an osmotic pressure of 300-400 mOsm/kg.
其中，浓缩或稀释可以在去除杂质之前、之后或同时进行。去除杂质的方法可以采用现有各种方法，例如可以使用切相流系统、中空纤维柱，在100KDa条件下超滤，超滤交换溶液为pH7.4的磷酸盐缓冲液(PBS)。除菌的方法可以采用现有各种方法，例如可以在0.22μm滤器上过滤除菌。The concentration or dilution can be performed before, after or simultaneously with the removal of impurities. The impurity removal method can adopt various existing methods, such as using a tangential flow system, a hollow fiber column, ultrafiltration under 100KDa conditions, and the ultrafiltration exchange solution is a phosphate buffered saline (PBS) with a pH of 7.4. The sterilization method can adopt various existing methods, such as filtration sterilization on a 0.22μm filter.
第一种siRNA缀合物The first siRNA conjugate
在一个方面，本公开提供了一种siRNA缀合物，所述siRNA缀合物包含上述的siRNA以及连接至该siRNA的缀合基团。In one aspect, the present disclosure provides a siRNA conjugate, comprising the above-mentioned siRNA and a conjugation group connected to the siRNA.
在本公开的上下文中，除非另有说明，“缀合”是指两个或多个各自具有特定功能的化学部分之间以共价连接的方式彼此连接；相应地，“缀合物”是指该各个化学部分之间通过共价连接而形成的化合物。进一步地，“指该各个化缀合物”表示一个或多个具有特定功能的化学部分共价连接至siRNA上而形成的化合物。在下文中，有时也将本公开的siRNA缀合物简称为“缀合物”。siRNA缀合物应根据上下文，理解为siRNA缀合物的总称，第一种siRNA缀合物或第二种siRNA缀合物。在本公开的上下文中，“缀合分子”应当理解为可通过反应缀合至siRNA，最终形成本公开的siRNA缀合物的化合物。In the context of the present disclosure, unless otherwise specified, "conjugation" refers to the connection between two or more chemical moieties, each having a specific function, in a covalently linked manner; accordingly, "conjugate" refers to a compound formed by covalently linking the individual chemical moieties. Further, "referring to the individual chemical conjugates" means a compound formed by covalently linking one or more chemical moieties having specific functions to siRNA. In the following, the siRNA conjugates of the present disclosure are sometimes referred to as "conjugates". The siRNA conjugates should be understood as a general term for siRNA conjugates, the first siRNA conjugate or the second siRNA conjugate, depending on the context. In the context of the present disclosure, "conjugated molecules" should be understood as compounds that can be conjugated to siRNA through a reaction to ultimately form the siRNA conjugates of the present disclosure.
本公开提供了第一种siRNA缀合物，所述siRNA缀合物包含上述的siRNA以及连接至该siRNA的缀合基团。一般来说，对于第一种siRNA缀合物，所述缀合基团包含药学上可接受的至少一个靶向基团和任选的接头(linker)，并且，所述siRNA、所述接头和所述靶向基团依次连接。在一种实施方案中，所述靶向基团为1-6个。在一种实施方案中，所述靶向基团为2-4个。所述siRNA分子可以非共价或共价缀合至所述缀合基团，例如可以共价缀合至所述缀合基团。siRNA与缀合基团的缀合位点可以在siRNA正义链的3’端或5’端，也可在反义链的5’端，还可以在siRNA的内部序列中。在一些实施方案中，所述siRNA与缀合基团的缀合位点在siRNA正义链的3’端。The present disclosure provides a first siRNA conjugate, the siRNA conjugate comprises the above-mentioned siRNA and a conjugated group connected to the siRNA. In general, for the first siRNA conjugate, the conjugated group comprises at least one pharmaceutically acceptable targeting group and an optional linker, and the siRNA, the linker and the targeting group are connected in sequence. In one embodiment, the targeting group is 1-6. In one embodiment, the targeting group is 2-4. The siRNA molecule can be non-covalently or covalently conjugated to the conjugated group, for example, it can be covalently conjugated to the conjugated group. The conjugation site of siRNA and the conjugated group can be at the 3' end or 5' end of the siRNA sense strand, or at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and the conjugated group is at the 3' end of the siRNA sense strand.
在一些实施方案中，所述缀合基团可以连接在核苷酸的磷酸基团、2’-位羟基或者碱基上。在一些实施方案中，所述缀合基团可以连接在3’-位羟基上，此时核苷酸之间采用2’-5’磷酸二酯键连接。当缀合基团连接在siRNA链的末端时，所述缀合基团通常连接在核苷酸的磷酸基团上；当缀合基团连接在siRNA的内部序列时，所述缀合基团通常连接在核糖糖环或者碱基上。各种连接方式可参考：Muthiah Manoharan et.al.siRNA conjugatescarrying sequentially assembled trivalent N-acetylgalactosamine linkedthrough nucleosides elicit robust gene silencing in vivo in hepatocytes.ACSChemical biology，2015，10(5)：1181-7。In some embodiments, the conjugate group can be connected to the phosphate group, 2'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugate group can be connected to the 3'-hydroxyl group, and the nucleotides are connected by a 2'-5' phosphodiester bond. When the conjugate group is connected to the end of the siRNA chain, the conjugate group is usually connected to the phosphate group of the nucleotide; when the conjugate group is connected to the internal sequence of the siRNA, the conjugate group is usually connected to the ribose sugar ring or the base. Various connection methods can be referred to: Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.ACS Chemical biology, 2015, 10(5): 1181-7.
在一些实施方案中，所述siRNA与缀合基团间可以通过酸不稳定的、或可还原的化学键相连，在细胞内涵体的酸性环境下，这些化学键可降解，从而使siRNA成为自由状态。对于不可降解的缀合方式，缀合基团可连接在siRNA的正义链，从而尽量降低缀合对siRNA活性的影响。In some embodiments, the siRNA and the conjugated group can be connected by acid-labile or reducible chemical bonds, which can be degraded in the acidic environment of the cell endosome, thereby making the siRNA free. For non-degradable conjugation methods, the conjugated group can be connected to the sense strand of the siRNA, thereby minimizing the effect of conjugation on the activity of the siRNA.
在一些实施方案中，所述药学上可接受的靶向基团指可以是siRNA给药领域常规使用的配体，例如WO2009082607A2中描述的各种配体，以引用的方式将其全部公开内容并入本文。In some embodiments, the pharmaceutically acceptable targeting group refers to a ligand conventionally used in the field of siRNA administration, such as various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.
在一些实施方案中，所述药学上可接受的靶向基团可以选自以下靶向分子或其衍生物形成的配体中的一种或多种：亲脂分子，例如胆固醇、胆汁酸、维生素(例如维生素E)、不同链长的脂质分子；聚合物，例如聚乙二醇；多肽，例如透膜肽；适配体；抗体；量子点；糖类，例如乳糖、聚乳糖、甘露糖、半乳糖、N-乙酰半乳糖胺(GalNAc)；叶酸(folate)；肝实质细胞表达的受体配体，例如去唾液酸糖蛋白、去唾液酸糖残基、脂蛋白(如高密度脂蛋白、低密度脂蛋白等)、胰高血糖素、神经递质(如肾上腺素)、生长因子、转铁蛋白等。In some embodiments, the pharmaceutically acceptable targeting group can be selected from one or more of the ligands formed by the following targeting molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acid, vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-permeable peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by hepatic parenchymal cells, such as asialoglycoproteins, asialosugar residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin, etc.
在一些实施方案中，所述的每个配体独立地选自一个能够与细胞表面受体结合的配体。在一些实施方案中，至少一个配体是能够与肝细胞表面受体结合的配体。在一些实施方案中，至少一个配体是能够与哺乳动物肝细胞表面受体结合的配体。在一些实施方案中，至少一个配体是能够与人肝细胞表面受体结合的配体。在一些实施方案中，至少一个配体是能够与肝表面去唾液酸糖蛋白受体(ASGPR)结合的配体。这些配体的种类为本领域技术人员所公知，其作用一般是与靶细胞表面的特异性受体相结合，介导与配体连接的siRNA递送至靶细胞。In some embodiments, each ligand is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a mammalian hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a liver surface asialoglycoprotein receptor (ASGPR). The types of these ligands are well known to those skilled in the art, and their function is generally to bind to a specific receptor on the surface of the target cell, mediating the delivery of the siRNA connected to the ligand to the target cell.
在一些实施方案中，所述药学上可接受的靶向基团可以是与哺乳动物肝细胞表面上的去唾液酸糖蛋白受体(ASGPR)结合的任意一种配体。在一种实施方案中，每个配体独立地为去唾液酸糖蛋白，例如去唾液酸血清类粘蛋白(asialoorosomucoid，ASOR)或去唾液酸胎球蛋白(asialofetuin，ASF)。在一种实施方案所述配体为糖或糖的衍生物。In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In one embodiment, each ligand is independently an asialoglycoprotein, such as asialo serum mucin (ASOR) or asialo fetuin (ASF). In one embodiment, the ligand is a sugar or a derivative of a sugar.
在一些实施方案中，至少一个配体是糖。在一些实施方案中，每个配体均是糖。在一些实施方案中，至少一个配体是单糖、多糖、修饰的单糖、修饰的多糖或糖衍生物。在一些实施方案中，至少一个所述配体可以是单糖，双糖或三糖。在一些实施方案中，至少有一个配体是修饰的糖。在一些实施方案中，每一个配体均为修饰的糖。在一些实施方案中，每个配体均独立地选自多糖、修饰的多糖、单糖、修饰的单糖、多糖衍生物或单糖衍生物。在一些实施方案中，每一个或至少一个配体选自由葡萄糖及其衍生物组、甘露聚糖及其衍生物、半乳糖及其衍生物、木糖及其衍生物、核糖及其衍生物、岩藻糖及其衍生物、乳糖及其衍生物、麦芽糖及其衍生物，阿拉伯糖及其衍生物、果糖及其衍生物和唾液酸组成的组。In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide or a sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, a disaccharide or a trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. In some embodiments, each ligand is independently selected from a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative or a monosaccharide derivative. In some embodiments, each or at least one ligand is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives and sialic acid.
在一些实施方案中，每个所述配体可独立地选自D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D-吡喃甘露糖、α-D-吡喃葡萄糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳糖胺、N-乙酰基半乳糖胺、N-三氟乙酰基半乳糖胺、N-丙酰基半乳糖胺、N-正丁酰基半乳糖胺、N-异丁酰基半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4，6-二脱氧-4-甲酰胺基-2，3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2，3，4-三-O-乙酰基-1-硫代-6-O-三苯甲基-α-D-吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3，4，6，7-四-O-乙酰基-2-脱氧-1，5-二硫代-α-D-吡喃葡庚糖苷乙酯、2，5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖或L-4-硫代核糖。所述配体的其它选择可参见例如CN105378082A的记载，以引用的方式将其全部公开内容并入本文。In some embodiments, each of the ligands can be independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine Glucose amine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2 , 3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-anhydro-D-allose nitrile, ribose, D-ribose, D-4-thioribose, L-ribose or L-4-thioribose. Other selections of the ligand can refer to, for example, the records of CN105378082A, and all of its disclosures are incorporated herein by reference.
在一些实施方案中，所述第一种siRNA缀合物中药学上可接受的靶向基团可以是半乳糖或N-乙酰半乳糖胺，其中，半乳糖或N-乙酰半乳糖胺分子可以是一价、二价、三价、四价。应当理解的是，这里所述的一价、二价、三价、四价分别指siRNA分子与含有作为靶向基团的半乳糖或N-乙酰半乳糖胺分子的缀合基团形成siRNA缀合物后，该siRNA缀合物中siRNA分子与半乳糖或N-乙酰半乳糖胺分子的摩尔比为1∶1、1∶2、1∶3或1∶4。在一些实施方案中，所述药学上可接受的靶向基团是N-乙酰半乳糖胺。在一些实施方案中，当本公开所述的siRNA与含有N-乙酰半乳糖胺的缀合基团缀合时，N-乙酰半乳糖胺分子是三价或四价。在一些实施方案中，当本公开所述的siRNA与含有N-乙酰半乳糖胺的缀合基团缀合时，N-乙酰半乳糖胺分子是三价。In some embodiments, the pharmaceutically acceptable targeting group in the first siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the monovalent, divalent, trivalent, and tetravalent described herein refer to the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate after the siRNA molecule and the conjugated group containing the galactose or N-acetylgalactosamine molecule as the targeting group form the siRNA conjugate, which is 1:1, 1:2, 1:3, or 1:4. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described in the present disclosure is conjugated to a conjugated group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described in the present disclosure is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.
当本公开所述的siRNA与缀合分子缀合时，缀合分子可经由合适的接头与siRNA分子相连，本领域技术人员可以根据靶向基团的具体类型选择合适的接头。这些缀合基团、接头、靶向基团的种类以及与siRNA的连接方式，可参见WO2015006740A2的公开内容，通过引用的方式将其整体内容并入本文。When the siRNA disclosed in the present invention is conjugated with a conjugated molecule, the conjugated molecule can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group. The types of these conjugated groups, linkers, targeting groups, and the connection mode with siRNA can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.
在一些实施方案中，当所述靶向基团为N-乙酰半乳糖胺时，合适的接头可以为如式(301)所示的结构：In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be a structure as shown in formula (301):
其中，in,
k为1-3的整数；k is an integer from 1 to 3;
LA为具有如式(302)所示结构的包含酰胺键的链状部分，每个所述LA在其两端分别与一个所述靶向基团和所述LC部分通过醚键相连接：LA is a chain portion containing an amide bond having a structure as shown in formula (302), and each of the LA is connected to one of the targeting groups and theLC portion via an ether bond at both ends thereof:
LB为具有如式(303)所示结构的包含N-酰基吡咯烷的链状部分，所述链状部分在其一端具有羰基并与所述LC部分通过酰胺键相连接，在另一端具有氧基并与所述siRNA通过磷酸酯键相连接：LB is a chain portion containing N-acylpyrrolidine having a structure as shown in formula (303), wherein the chain portion has a carbonyl group at one end and is connected to theLC portion via an amide bond, and has an oxygen group at the other end and is connected to the siRNA via a phosphate bond:
LC为基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的2-4价连接基团，所述LC经由氧原子与各个所述LA部分通过醚键相连接，并且经由氮原子与所述LB部分通过酰胺键相连接。LC is a 2-4 valent linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trihydroxymethylaminomethane, and is connected to eachLA part through an ether bond via an oxygen atom, and is connected to theLB part through anamide bond via a nitrogen atom.
在一些实施方案中，当n＝3，LC为基于三羟甲基氨基甲烷的4价连接基团时，由作为接头的-(LA)3三羟甲基氨基甲烷-LB-连接N-乙酰半乳糖胺分子和siRNA分子所形成的第一种siRNA缀合物，其结构如下式(304)所示：In some embodiments, when n=3,LC is a tetravalent linking group based on tris(hydroxymethylaminomethane), the first siRNA conjugate formed by linking an N-acetylgalactosamine molecule and an siRNA molecule using -(LA )3tris (hydroxymethylaminomethane)-LB- as a linker has a structure as shown in the following formula (304):
式中，双螺旋结构表示siRNA。In the formula, the double helix structure represents siRNA.
同样，siRNA与缀合分子的缀合位点可以在siRNA正义链的3′端或5′端，也可在反义链的5′端，还可以在siRNA的内部序列中。Likewise, the conjugation site between siRNA and the conjugation molecule can be at the 3' end or 5' end of the sense strand of the siRNA, at the 5' end of the antisense strand, or in the internal sequence of the siRNA.
在一些实施方案中，本公开所述siRNA的正义链3′末端通过接头-(LA)3三羟甲基氨基甲烷-LB-与三个N-乙酰半乳糖胺(GalNAc)分子共价缀合，得到siRNA分子与GalNAc分子的摩尔比为1∶3的第一种siRNA缀合物，下文也可将其称为(GalNAc)3-1-siRNA，其结构如下式(305)所示：In some embodiments, the 3′ end of the sense strand of the siRNA disclosed herein is covalently conjugated to three N-acetylgalactosamine (GalNAc) molecules via a linker -(LA )3 tris(hydroxymethylaminomethane)-LB- to obtain a first siRNA conjugate having a molar ratio of siRNA molecules to GalNAc molecules of 1:3, which may also be referred to as (GalNAc)3-1 -siRNA hereinafter, and has a structure as shown in the following formula (305):
其中，双螺旋结构表示所述siRNA，并且所述接头连接至所述siRNA的正义链3′末端。The double helix structure represents the siRNA, and the linker is connected to the 3′ end of the sense strand of the siRNA.
在一些实施方案中，当所述靶向基团为N-乙酰半乳糖胺时，合适的接头可以为如式(306)所示的结构：In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be a structure as shown in formula (306):
其中，in,
l为0-3的整数；l is an integer from 0 to 3;
＊表示接头上通过醚键与靶向基团连接的位点；* indicates the site on the linker that is connected to the targeting group via an ether bond;
#表示接头上通过磷酸酯键与siRNA连接的位点。# indicates the site on the linker that is connected to the siRNA via a phosphate bond.
在一些实施方案中，当l＝2时，所述第一种siRNA缀合物具有如式(307)所示的结构：In some embodiments, when l=2, the first siRNA conjugate has a structure as shown in formula (307):
其中，双螺旋结构表示所述siRNA，并且所述接头连接至所述siRNA的正义链3′末端。The double helix structure represents the siRNA, and the linker is connected to the 3′ end of the sense strand of the siRNA.
上述缀合物可以通过现有技术中已经详细描述的方法进行合成。例如，WO2015006740A2中详细描述了多种缀合物的制备方法。通过本领域技术人员熟知的方式，获得本公开的第一种siRNA缀合物。如WO2014025805A1中记载了式(305)所示结构的制备方法，Rajeev等人在ChemBioChem 2015，16，903-908中描述了式(307)所示结构的制备方法。The above conjugates can be synthesized by methods that have been described in detail in the prior art. For example, WO2015006740A2 describes in detail the preparation methods of various conjugates. The first siRNA conjugate of the present disclosure is obtained by a method well known to those skilled in the art. For example, WO2014025805A1 describes the preparation method of the structure shown in formula (305), and Rajeev et al. describe the preparation method of the structure shown in formula (307) in ChemBioChem 2015, 16, 903-908.
第二种siRNA缀合物Second siRNA conjugate
在一些实施方案中，本公开提供了第二种siRNA缀合物，该第二种siRNA缀合物具有如式(308)所示的结构：In some embodiments, the present disclosure provides a second siRNA conjugate having a structure as shown in formula (308):
其中：in:
n1为选自1-3的整数，n3为选自0-4的整数；n1 is an integer selected from 1-3, n3 is an integer selected from 0-4;
m1、m2和m3独立地为选自2-10的整数；m1, m2 and m3 are independently an integer selected from 2-10;
R10、R11、R12、R13、R14和R15各自独立地为H，或选自于由以下基团所组成的组：C1-C10烷基、C1-C10卤代烷基以及C1-C10烷氧基；R10 , R11 , R12 , R13 , R14 and R15 are each independently H, or selected from the group consisting of C1 -C10 alkyl, C1 -C10 haloalkyl and C1 -C10 alkoxy;
R3为式A59所示结构的基团：R3 is a group having a structure represented by formula A59:
其中，E1为OH、SH或BH2，Nu为本公开的siRNA；wherein E1 is OH, SH or BH2 , and Nu is the siRNA disclosed herein;
R2是长度为1-20个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，R2可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)。R2 is a straight chain alkylene group having a length of 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2 , C2 -C10 alkenylene, C2 -C10 alkynylene, C6 -C10 arylene, C3 -C18 heterocyclylene and C5 -C10 heteroarylene; and wherein R2 may optionally have any one or more substituents selected from the group consisting of: C1 -C10 alkyl, C6 -C10 aryl, C5 -C10 heteroaryl, C1 -C10 haloalkyl, -OC1 -C10 alkyl, -OC1 -C10 alkylphenyl, -C1 -C10 alkyl-OH, -OC1 -C10 haloalkyl, -SC1 -C -C1 -C10 alkyl), -C1 -C10 alkylphenyl, -C1 -C10 alkyl-SH, -C 1 -C10 haloalkyl, halogen substituent, -OH, -SH, -NH2 , -C1 -C10 alkyl-NH2 , -N(C1 -C10 alkyl)(C1 -C10 alkyl), -NH(C1 -C10 alkyl), cyano, nitro, -CO2H, -C(O)O(C1 -C10 alkyl), -CON(C1 -C10 alkyl)(C1 -C10 alkyl), -CONH(C1 -C 10 alkyl), -CONH2 , -NHC(O)(C1 -C10 alkyl), -NHC(O)(phenyl), -N(C1 -C10 alkyl)C(O)(C1 -C10 alkyl), -N(C1 -C 10 alkyl) -C10 alkyl)C(O)(phenyl), -C(O)C1 -C10 alkyl, -C(O)C1 -C10 alkylphenyl, -C(O)C1 -C10 haloalkyl, -OC(O)C1 -C10 alkyl, -SO2 (C1 -C10 alkyl), -SO2 (phenyl), -SO2 (C1 -C10 haloalkyl), -SO2 NH2 , -SO2 NH(C1 -C10 alkyl), -SO2 NH(phenyl), -NHSO2 (C1 -C10 alkyl), -NHSO2 (phenyl) and -NHSO2 (C1 -C10 haloalkyl).
每个L1是长度为1-70个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，L1可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)。Each L1 is a straight chain alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally replaced by one or more selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2 , C2 -C10 alkenylene, C2 -C10 alkynylene, C6 -C10 arylene, C3 -C18 heterocyclylene and C5 -C10 heteroarylene; and wherein L1 may optionally have any one or more substituents selected from the group consisting of the following groups: C1 -C10 alkyl, C6 -C10 aryl, C5 -C10 heteroaryl, C1 -C10 haloalkyl, -OC1 -C10 alkyl, -OC1 -C10 alkylphenyl, -C1 -C10 alkyl-OH, -OC1 -C10 haloalkyl, -SC1 -C -C10 alkyl), -S- C10 alkylphenyl, -C1 -C10 alkyl-SH, -S- C10 haloalkyl, halogen substituent, -OH, -SH, -NH2 , -C1 -C10 alkyl-NH2 , -N(C1 -C10 alkyl)(C1 -C10 alkyl), -NH(C1 -C10 alkyl), cyano, nitro, -CO2 H, -C(O)O(C1 -C10 alkyl), -CON(C1 -C10 alkyl)(C1 -C10 alkyl), -CONH(C1 -C10 alkyl), -CONH2 , -NHC(O)(C1 -C10 alkyl), -NHC(O)(phenyl), -N(C1 -C10 alkyl)C(O)(C1 -C 10 alkyl), -N(C1 -C10 alkyl) -C10 alkyl)C(O)(phenyl), -C(O)C1 -C10 alkyl, -C(O)C1 -C10 alkylphenyl, -C(O)C1 -C10 haloalkyl, -OC(O)C1 -C 10alkyl , -SO2 (C1 -C10 alkyl), -SO2 (phenyl), -SO2 (C1 -C10 haloalkyl), -SO2 NH2 , -SO2 NH(C1 -C10 alkyl), -SO2 NH(phenyl), -NHSO2 (C1 -C10 alkyl), -NHSO2 (phenyl) and -NHSO2 (C1 -C10 haloalkyl).
并且，在一些实施方案中，L1可选自于由A1-A26基团或其任意组合所组成的组，其中A1-A26的结构和定义如下所示：Furthermore, in some embodiments,L1 can be selected from the group consisting of A1-A26 groups or any combination thereof, wherein the structures and definitions of A1-A26 are as follows:
其中，j1为1-20的整数；j2为1-20的整数；Wherein, j1 is an integer from 1 to 20; j2 is an integer from 1 to 20;
R’为C1-C10的烷基；R' is a C1 -C10 alkyl group;
Ra选自式A27-A45基团中的一种：Ra is selected from one of the groups of formula A27-A45:
Rb为C1-C10的烷基；表示基团连接至分子其余部分的位点。Rb is a C1 -C10 alkyl group; Denotes the point at which a group is attached to the rest of a molecule.
技术人员会理解的是，尽管为了方便起见，L1被定义为线性烷基，但是它可能不是线性基团或者名称不同，例如由于上述替换和/或置换而产生的胺或烯基。为了本公开内容的目的，L1的长度是连接两个附着点的链中的原子数。为此目的，将替换所述直链亚烷基的碳原子而得到的环(如亚杂环基或亚杂芳基)计为一个原子。The skilled artisan will appreciate that althoughL is defined as a linear alkyl for convenience, it may not be a linear group or be named differently, such as an amine or alkenyl resulting from the above substitutions and/or replacements. For the purposes of this disclosure, the length ofL is the number of atoms in the chain connecting the two points of attachment. For this purpose, a ring resulting from the replacement of a carbon atom of the linear alkylene group (such as a heterocyclylene or heteroarylene) is counted as one atom.
M1表示靶向基团，其定义和可选择的范围与上述靶向基团相同。在一些实施方案中，每个M1独立地选自对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体中的一种。M1 represents a targeting group, and its definition and selectable range are the same as those of the above-mentioned targeting group. In some embodiments, eachM1 is independently selected from one of the ligands having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.
当M1为对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲和力的配体时，在一些实施方案中，n1可以是1-3的整数，n3可以是0-4的整数，保证所述缀合物中M1配体的个数至少为2；在一些实施方案中，n1+n3≥2，这样可以使得M1靶向基团的个数至少为3，使得M1靶向基团与肝表面去唾液酸糖蛋白受体更容易结合，进而促进所述缀合物通过内吞作用进入细胞。实验表明，当M1靶向基团的个数大于3个时，M1靶向基团与肝表面去唾液酸糖蛋白受体结合的容易程度增加并不明显，因此，从合成容易程度、结构/工艺成本和递送效率等多方面综合考虑，在一些实施方案中，n1为1-2的整数，n3为0-1的整数，且n1+n3＝2-3。WhenM1 is a ligand with affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, in some embodiments, n1 can be an integer of 1-3, and n3 can be an integer of 0-4, ensuring that the number ofM1 ligands in the conjugate is at least 2; in some embodiments, n1+n3≥2, so that the number ofM1 targeting groups can be at least 3, making it easier for theM1 targeting groups to bind to the asialoglycoprotein receptor on the surface of the liver, thereby promoting the conjugate to enter the cell through endocytosis. Experiments show that when the number ofM1 targeting groups is greater than 3, the ease of binding of theM1 targeting groups to the asialoglycoprotein receptor on the surface of the liver is not significantly increased. Therefore, considering the ease of synthesis, structure/process cost and delivery efficiency, in some embodiments, n1 is an integer of 1-2, n3 is an integer of 0-1, and n1+n3=2-3.
在一些实施方案中，m1、m2和m3独立地选自2-10的整数时，可以使多个M1靶向基团之间的空间位置适合M1靶向基团与肝表面去唾液酸糖蛋白受体的结合，为了使本公开提供的缀合物更为简单，更容易合成和/或降低成本，在一些实施方案中，m1、m2和m3各自独立地为2-5的整数，在一些实施方案中，m1＝m2＝m3。In some embodiments, when m1, m2 and m3 are independently selected from integers of 2-10, the spatial positions between the multipleM1 targeting groups can be made suitable for the binding of theM1 targeting group to the asialoglycoprotein receptor on the liver surface. In order to make the conjugate provided by the present disclosure simpler, easier to synthesize and/or reduce the cost, in some embodiments, m1, m2 and m3 are each independently an integer of 2-5. In some embodiments, m1=m2=m3.
本领域技术人员可以理解，当R10、R11、R12、R13、R14和R15各自独立地为H、C1-C10烷基、C1-C10卤代烷基、以及C1-C10烷氧基中的一种时，不会改变本文公开的缀合物的性质，均可以实现本公开的目的。在一些实施方案中，R10、R11、R12、R13、R14和R15各自独立地选自H、甲基和乙基。在一些实施方案中，R10、R11、R12、R13、R14和R15均为H。Those skilled in the art will appreciate that when R10 , R11 , R12 , R13 , R14 and R15 are each independently selected from H, C1 -C10 alkyl, C1 -C10 haloalkyl, and C1 -C10 alkoxy, the properties of the conjugate disclosed herein will not be changed, and the purpose of the present disclosure can be achieved. In some embodiments, R10 , R11 , R12 , R13 , R14 and R15 are each independently selected from H, methyl and ethyl. In some embodiments, R10 , R11 , R12 , R13 , R14 and R15 are all H.
根据本公开提供的第二种siRNA缀合物，R3为式A59所示结构的基团，其中，E1为OH、SH或BH2，基于制备原料易获取性的考虑，在一些实施方案中，E1为OH或SH。According to the second siRNA conjugate provided by the present disclosure, R3 is a group of the structure shown in formula A59, wherein E1 is OH, SH or BH2 . Based on the consideration of the availability of raw materials for preparation, in some embodiments, E1 is OH or SH.
在一些实施方案中，R2的选择是为了实现与含氮骨架上的N与A59的连接。在本公开的上下文中，“含氮骨架”是指连接有R10、R11、R12、R13、R14和R15的碳原子与N互相连接的链状结构。因此，R2可以是任何能够以适当方式将A59基团连接至含氮骨架上的N的连接基团。在一些实施方案中，在通过固相合成的工艺制备第二种siRNA缀合物的情况下，R2基团中需要同时含有与含氮骨架上的N连接的连接位点和与R3中的P相连接的连接位点。在一些实施方案中，R2中所述与含氮骨架上的N连接的位点与N形成酰胺键，所述与R3上的P连接的位点与P形成磷酸酯键。在一些实施方案中，R2可以是B5、B6、B5’或B6’：In some embodiments, R2 is selected to achieve connection with N on the nitrogen-containing skeleton and A59. In the context of the present disclosure, "nitrogen-containing skeleton" refers to a chain structure in which carbon atoms connected to R10 , R11 , R12 , R13 , R14 and R15 are connected to N. Therefore, R2 can be any connecting group that can connect the A59 group to N on the nitrogen-containing skeleton in an appropriate manner. In some embodiments, in the case of preparing the second siRNA conjugate by a solid phase synthesis process, the R2 group needs to contain both a connection site connected to N on the nitrogen-containing skeleton and a connection site connected to P in R3. In some embodiments, the site in R2 that is connected to N on the nitrogen-containing skeleton forms an amide bond with N, and the site that is connected to P on R3 forms a phosphate bond with P. In some embodiments, R2 can be B5, B6, B5' or B6':
其中，表示基团共价键连接的位点。in, Indicates the site of covalent attachment of a group.
q2的取值范围可以是1-10的整数，在一些实施方案中，q2为1-5的整数。The value range of q2 can be an integer from 1 to 10. In some embodiments, q2 is an integer from 1 to 5.
L1的作用是将M1靶向基团与含氮骨架上的N连接，为本公开的第二种siRNA缀合物提供肝靶向功能。在一些实施方案中，L1选自式A1-A26基团中的一种或多种的连接组合；在一些实施方案中，L1选自A1、A4、A5、A6、A8、A10、A11和A13中的一种或多种的连接组合；在一些实施方案中，L1选自A1、A4、A8、A10和A11中至少2个的连接组合；在一些实施方案中，L1选自A1、A8、A10中至少2个的连接组合。The role ofL1 is to connect theM1 targeting group to N on the nitrogen-containing skeleton, providing liver targeting function for the second siRNA conjugate disclosed in the present invention. In some embodiments,L1 is selected from a combination of one or more of the formula A1-A26 groups; in some embodiments,L1 is selected from a combination of one or more of A1, A4, A5, A6, A8, A10, A11 and A13; in some embodiments,L1 is selected from a combination of at least 2 of A1, A4, A8, A10 and A11; in some embodiments,L1 is selected from a combination of at least 2 of A1, A8, A10.
在一些实施方案中，L1的长度可以为3-25个原子，3-20个原子、4-15个原子或5-12个原子。在一些实施方案中，L1的长度为3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、35个、40个、45个、50个、55个、60个原子。In some embodiments,L1 can be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms in length. In some embodiments, L1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atoms in length.
在一些实施方案中，j1为2-10的整数，在一些实施方案中，j1为3-5的整数。j2为2-10的整数，在一些实施方案中，j2为3-5的整数。R’为C1-C4的烷基，在一些实施方案中，R’为甲基、乙基和异丙基中的一种。Ra为A27、A28、A29、A30和A31中的一种，在一些实施方案中，Ra为A27或A28。Rb为C1-C5的烷基，在一些实施方案中，Rb为甲基、乙基、异丙基和丁基中的一种。在一些实施方案中，在式A1-A26中各自对j1、j2、R’、Ra、Rb进行选择，以实现M1靶向基团与含氮骨架上的N连接，并使M1靶向基团之间的空间位置更适合M1靶向基团与肝表面去唾液酸糖蛋白受体结合。In some embodiments, j1 is an integer of 2-10, and in some embodiments, j1 is an integer of 3-5. j2 is an integer of 2-10, and in some embodiments, j2 is an integer of 3-5. R' is a C1-C4 alkyl group, and in some embodiments, R' is one of methyl, ethyl and isopropyl. Ra is one of A27, A28, A29, A30 and A31, and in some embodiments, Ra is A27 or A28. Rb is a C1 -C5 alkyl group, and in some embodiments, Rb is one of methyl, ethyl, isopropyl and butyl. In some embodiments, j1, j2, R', Ra, and Rb are selected in formulas A1-A26 to achieve the connection of the M1 targeting group to the N on the nitrogen-containing skeleton, and to make the spatial position between the M1 targeting groups more suitable for the M1 targeting group to bind to the liver surface asialoglycoprotein receptor.
在一些实施方案中，本公开的第二siRNA缀合物具有式(403)、(404)、(405)、(406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421)或(422)所示的结构：In some embodiments, the second siRNA conjugate of the present disclosure has a structure shown in formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422):
在一些实施方案中，式A59中的P可以连接到Nu代表的siRNA序列中任何可能的位置，例如，式A59中的P可以连接到Nu代表的siRNA正义链或反义链的任何一个核苷酸上；在一些实施方案中，式A59中的P连接到Nu代表的siRNA正义链的任何一个核苷酸上。在一些实施方案中，式A59中的P连接到Nu代表的siRNA正义链或反义链的端部；在一些实施方案中，式A59中的P连接到Nu代表的siRNA正义链的端部。Nu代表的siRNA的端部指Nu代表的siRNA正义链或所述反义链中从其一端起算的前4个核苷酸。在一些实施方案中，式A59中的P连接到Nu代表的siRNA正义链或反义链的末端；在一些实施方案中，式A59中的P连接到Nu代表的siRNA正义链的3′末端。在连接至Nu代表的siRNA的正义链的上述位置的情况下，第二种siRNA缀合物进入细胞后，在解旋时，可以释放出单独的siRNA反义链，以阻断HBV的mRNA翻译蛋白质的过程，抑制乙型肝炎病毒(hepatitis B virus，HBV)基因表达。In some embodiments, P in formula A59 can be connected to any possible position in the siRNA sequence represented by Nu, for example, P in formula A59 can be connected to any nucleotide of the sense strand or antisense strand of the siRNA represented by Nu; in some embodiments, P in formula A59 is connected to any nucleotide of the sense strand of the siRNA represented by Nu. In some embodiments, P in formula A59 is connected to the end of the sense strand or antisense strand of the siRNA represented by Nu; in some embodiments, P in formula A59 is connected to the end of the sense strand of the siRNA represented by Nu. The end of the siRNA represented by Nu refers to the first 4 nucleotides from one end of the sense strand or antisense strand of the siRNA represented by Nu. In some embodiments, P in formula A59 is connected to the end of the sense strand or antisense strand of the siRNA represented by Nu; in some embodiments, P in formula A59 is connected to the 3′ end of the sense strand of the siRNA represented by Nu. When connected to the above position of the positive strand of the siRNA represented by Nu, after the second siRNA conjugate enters the cell, when unwinding, the separate siRNA antisense strand can be released to block the process of HBV mRNA translation into protein and inhibit the expression of hepatitis B virus (HBV) genes.
式A59中的P可以连接到Nu代表的siRNA中的核苷酸上任何可能的位置，例如，核苷酸的5′位、核苷酸的2′位、核苷酸的3′位或核苷酸的碱基上。在一些实施方案中，式A59中的P可通过形成磷酸二酯键连接至Nu代表的siRNA中的核苷酸的2′位、3′位或5′位。在一些实施方案中，式A59中的P连接在Nu代表的siRNA正义链3′末端核苷酸的3′羟基脱氢后形成的氧原子上，或者式A59中的P通过取代Nu代表的siRNA正义链中的一个核苷酸的2′-羟基中的氢与核苷酸连接，或者式A59中的P通过取代Nu代表的siRNA正义链5′末端核苷酸的5′羟基中的氢与核苷酸连接。The P in formula A59 can be connected to any possible position on the nucleotide in the siRNA represented by Nu, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide, or the base of the nucleotide. In some embodiments, the P in formula A59 can be connected to the 2' position, 3' position, or 5' position of the nucleotide in the siRNA represented by Nu by forming a phosphodiester bond. In some embodiments, the P in formula A59 is connected to the oxygen atom formed after dehydrogenation of the 3' hydroxyl group of the 3' terminal nucleotide of the siRNA sense strand represented by Nu, or the P in formula A59 is connected to the nucleotide by replacing the hydrogen in the 2'-hydroxyl group of a nucleotide in the siRNA sense strand represented by Nu, or the P in formula A59 is connected to the nucleotide by replacing the hydrogen in the 5' hydroxyl group of the 5' terminal nucleotide of the siRNA sense strand represented by Nu.
本公开所述siRNA或siRNA缀合物中，每个相邻核苷酸之间由磷酸二酯键或硫代磷酸二酯键连接，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子带有负电荷，它可以以羟基或巯基的形式存在，羟基或巯基中的氢离子也可以部分或全部被阳离子取代。所述阳离子可以是任意的阳离子，如金属阳离子、铵离子NH4+、有机铵阳离子中的一种。出于提高溶解性考虑，在一种实施方案中，所述阳离子选自碱金属离子、三级胺形成的铵阳离子和季铵阳离子中的一种或多种。碱金属离子可以是K+和/或Na+，三级胺形成的阳离子可以是三乙胺形成的铵离子和/或N，N-二异丙基乙胺形成的铵离子。因此，本公开所述siRNA或第一种或第二种siRNA缀合物可以至少部分以盐的形式存在。在一种方式中，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子至少部分与钠离子结合，本公开所述siRNA或第一种或第二种siRNA缀合物以钠盐或部分钠盐的形式存在。In the siRNA or siRNA conjugate disclosed in the present invention, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate diester bond, and the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the phosphorothioate diester bond carries a negative charge, which can exist in the form of a hydroxyl group or a sulfhydryl group, and the hydrogen ion in the hydroxyl group or the sulfhydryl group can also be partially or completely replaced by a cation. The cation can be any cation, such as a metal cation, an ammonium ionNH4+ , or an organic ammonium cation. In order to improve solubility, in one embodiment, the cation is selected from one or more of an alkali metal ion, an ammonium cation formed by a tertiary amine, and a quaternary ammonium cation. The alkali metal ion can be K+ and/or Na+ , and the cation formed by the tertiary amine can be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N, N-diisopropylethylamine. Therefore, the siRNA or the first or second siRNA conjugate disclosed in the present invention can exist at least partially in the form of a salt. In one embodiment, the non-bridging oxygen atoms or sulfur atoms in the phosphodiester bond or the phosphorothioate diester bond are at least partially bound to sodium ions, and thesiRNA or the first or second siRNA conjugate described in the present disclosure exists in the form of a sodium salt or a partial sodium salt.
本领域技术人员清楚知晓的是，可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的siRNA中。制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。所有修饰的核苷单体均可以商购得到或者采用已知方法制备得到。It is well known to those skilled in the art that the modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications. The method of preparing nucleoside monomers with corresponding modifications and the method of introducing the modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or prepared by known methods.
第二种siRNA缀合物的制备Preparation of the second siRNA conjugate
可以采用任意合理的合成路线制备第二种siRNA缀合物。The second siRNA conjugate can be prepared using any reasonable synthetic route.
在一些实施方案中，第二种siRNA缀合物可以采用如下方法制备，该方法包括在亚磷酰胺固相合成的条件下，分别按照siRNA正义链和反义链的核苷酸种类和顺序，按照3′到5′的方向将核苷单体依次连接，每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；分离出siRNA的正义链和反义链，退火，其中，Nu代表的siRNA为上述本公开的siRNA；In some embodiments, the second siRNA conjugate can be prepared by the following method, which includes, under the conditions of phosphoramidite solid phase synthesis, sequentially connecting nucleoside monomers in the direction of 3′ to 5′ according to the nucleotide types and order of the siRNA sense strand and antisense strand, respectively, and the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; separating the sense strand and antisense strand of the siRNA, and annealing, wherein the siRNA represented by Nu is the siRNA disclosed above;
并且，该方法还包括在偶联反应条件和偶联试剂存在下，将式(321)所示的化合物与核苷单体或连接在固相载体上的核苷酸序列接触，使式(321)所示的化合物经偶联反应连接至核苷酸序列。下文中，式(321)所示的化合物也称作缀合分子。Furthermore, the method further comprises contacting the compound represented by formula (321) with a nucleoside monomer or a nucleotide sequence connected to a solid support under coupling reaction conditions and in the presence of a coupling reagent, so that the compound represented by formula (321) is connected to the nucleotide sequence through a coupling reaction. Hereinafter, the compound represented by formula (321) is also referred to as a conjugated molecule.
其中：in:
R4为能够结合至Nu代表siRNA的部分。在一些实施方案中，R4为能够通过共价键结合至Nu代表的siRNA的部分。在一些实施方案中，R4为能够经反应而通过磷酸二酯键缀合至Nu代表的siRNA的任意官能团的部分；R4 is a moiety capable of binding to the siRNA represented by Nu. In some embodiments, R4 is a moiety capable of binding to the siRNA represented by Nu via a covalent bond. In some embodiments, R4 is a moiety capable of reacting to conjugate to any functional group of the siRNA represented by Nu via a phosphodiester bond;
每个S1独立地是M1中全部活性羟基被YCOO-基团取代而形成的基团，其中，每个Y独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；Each S1 is independently a group formed by replacing all active hydroxyl groups in M1 with YCOO- groups, wherein each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halogenated phenyl and alkylphenyl;
n1、n3、m1、m2、m3、R10、R11、R12、R13、R14、R15、L1、M1各自的定义和可选择的范围如前所述。The definitions and selectable ranges of n1, n3, m1, m2, m3, R10 , R11 , R12 , R13 , R14 , R15 , L1 , and M1 are as described above.
R4的选择是为了实现与含氮骨架上的N的连接，并且为合成式(308)的siRNA缀合物提供合适的反应位点。在一些实施方案中，R4中包括R2连接基团或经保护的R2连接基团，以及可通过反应与siRNA形成A59所示结构的官能团。R4 is selected to achieve connection with N on the nitrogen-containing backbone and to provide a suitable reaction site for synthesizing the siRNA conjugate of formula (308). In some embodiments,R4 includes anR2 linking group or a protectedR2 linking group and a functional group that can react with siRNA to form the structure shown in A59.
在一些实施方案中，R4含包含可与Nu代表的siRNA或核苷单体上的基团形成亚磷酸酯的第1官能团以及可与羟基或氨基反应形成共价键的第2官能团或者含有由所述共价键连接的固相载体。在一些实施方案中，所述第1官能团为亚磷酰胺、羟基或被保护的羟基。在一些实施方案中，所述第2官能团为亚磷酰胺、羧酸或羧酸盐。在一些实施方案中，所述第2官能团为经由共价键连接至分子其他部分的固相载体，所述共价键由羟基或氨基形成。在一些实施方案中，所述固相载体经由磷酸酯键、羧酸酯键或酰胺键连接。在一些实施方案中，所述固相载体为树脂。In some embodiments, R4 contains a first functional group that can form a phosphite with a group on the siRNA or nucleoside monomer represented by Nu and a second functional group that can react with a hydroxyl or amino group to form a covalent bond, or contains a solid support connected by the covalent bond. In some embodiments, the first functional group is a phosphoramidite, a hydroxyl or a protected hydroxyl. In some embodiments, the second functional group is a phosphoramidite, a carboxylic acid or a carboxylate. In some embodiments, the second functional group is a solid support connected to the rest of the molecule via a covalent bond, and the covalent bond is formed by a hydroxyl or amino group. In some embodiments, the solid support is connected via a phosphate bond, a carboxylate bond or an amide bond. In some embodiments, the solid support is a resin.
在一些实施方案中，所述第1官能团含有羟基、-ORk或式(C3)所示的基团；所述第2官能团含有式(C1)、(C2)、(C3)、(C1’)或(C3’)所示的结构：In some embodiments, the first functional group contains a hydroxyl group, -ORk , or a group represented by formula (C3); the second functional group contains a structure represented by formula (C1), (C2), (C3), (C1') or (C3'):
式中，q1为1-4的整数，X为O或NH，M+为阳离子，Rk为羟基保护基团，SPS表示固相载体，表示基团共价连接的位点。In the formula,q1 is an integer of 1-4, X is O or NH, M+ is a cation,Rk is a hydroxyl protecting group, SPS represents a solid phase support, Indicates the site to which a group is covalently attached.
在一些实施方案中，所述第1官能团含有亚磷酰胺官能团，如式(C3)所示，该亚磷酰胺基团可以与核苷酸上的任意位置的羟基，如2′位羟基或3′位羟基发生偶联反应形成亚磷酸酯，并经氧化或硫化形成式A59所示的磷酸二酯键或硫代磷酸酯键，将缀合分子缀合至siRNA。此时，即使所述第2官能团并不存在，式(321)所示的化合物也能够缀合至核苷酸，不影响式(308)所示siRNA缀合物的获得。在此情况下，在经由亚磷酰胺固相合成等方法获得siRNA的正义链或反义链后，使式(321)所示的化合物与核苷酸序列中末端核苷酸上的羟基反应，并在后续的氧化或硫化过程中形成磷酸二酯键连接或硫代磷酸酯连接，将式(321)所示的化合物缀合至siRNA。In some embodiments, the first functional group contains a phosphoramidite functional group, as shown in formula (C3), and the phosphoramidite group can react with a hydroxyl group at any position on the nucleotide, such as the 2′ hydroxyl group or the 3′ hydroxyl group, to form a phosphite, and then oxidize or sulfurize to form a phosphodiester bond or a phosphorothioate bond as shown in formula A59, and conjugate the conjugate molecule to the siRNA. At this time, even if the second functional group does not exist, the compound shown in formula (321) can be conjugated to the nucleotide, and does not affect the acquisition of the siRNA conjugate shown in formula (308). In this case, after obtaining the sense strand or antisense strand of the siRNA by a method such as phosphoramidite solid phase synthesis, the compound shown in formula (321) is reacted with the hydroxyl group on the terminal nucleotide in the nucleotide sequence, and a phosphodiester bond or a phosphorothioate bond is formed in the subsequent oxidation or sulfurization process, and the compound shown in formula (321) is conjugated to the siRNA.
在一些实施方案中，所述第1官能团含有被保护的羟基。在一些实施方案中，所述第2官能团包含可与固相载体反应的基团，所述反应提供包含固相载体的缀合分子。在一些实施方案中，所述第2官能团含有羧基、羧酸盐或亚磷酰胺，如式(C1)、(C2)或(C3)所示，当所述第2官能团包含羧基或羧酸盐时，式(321)所示的化合物与固相载体，例如树脂上的羟基或氨基进行酯化反应或酰胺化反应，形成经羧酸酯键连接或经酰胺键连接的包含固相载体的缀合分子。当所述第2官能团包含亚磷酰胺官能团时，式(321)所示的化合物与通用固相载体，例如树脂上的羟基发生偶联反应，并经氧化形成经磷酸二酯键连接的包含固相载体的缀合分子。随后，以上述连接固相载体后的产物作为起始，按照亚磷酰胺固相合成方法依次连接核苷单体，获得连接有缀合基团的siRNA的正义链或反义链。在亚磷酰胺固相合成过程中，所述第1官能团发生脱保护，随后在偶联反应条件下与核苷单体上的亚磷酰胺基团发生偶联。In some embodiments, the first functional group contains a protected hydroxyl group. In some embodiments, the second functional group contains a group that can react with a solid phase carrier, and the reaction provides a conjugated molecule containing a solid phase carrier. In some embodiments, the second functional group contains a carboxyl group, a carboxylate or a phosphoramidite, as shown in formula (C1), (C2) or (C3). When the second functional group contains a carboxyl group or a carboxylate group, the compound shown in formula (321) undergoes an esterification reaction or an amidation reaction with a solid phase carrier, such as a hydroxyl group or an amino group on a resin, to form a conjugated molecule containing a solid phase carrier connected via a carboxylate bond or via an amide bond. When the second functional group contains a phosphoramidite functional group, the compound shown in formula (321) undergoes a coupling reaction with a universal solid phase carrier, such as a hydroxyl group on a resin, and is oxidized to form a conjugated molecule containing a solid phase carrier connected via a phosphodiester bond. Subsequently, starting from the product after the solid phase support is connected, the nucleoside monomers are connected in sequence according to the phosphoramidite solid phase synthesis method to obtain the sense strand or antisense strand of the siRNA connected with the conjugated group. During the phosphoramidite solid phase synthesis process, the first functional group is deprotected and then coupled with the phosphoramidite group on the nucleoside monomer under the coupling reaction conditions.
在一些实施方案中，所述第1官能团含有羟基或被保护的羟基；所述第2官能团含有经羧酸酯键连接的固相载体或经酰胺键连接的固相载体、或者经磷酸酯键连接的固相载体，如式(C1’)或(C3’)所示。此时，由式(321)所示的化合物代替固相载体作为起始，按照亚磷酰胺固相合成方法依次连接核苷单体，获得连接有缀合基团的siRNA的正义链或反义链。在一些实施方案中，羧酸盐可以表示为-，羧酸-M+，其中，M+是阳离子，例如选自金属阳离子、铵阳离子NH4+、有机铵阳离子中的一种。在一种实施方案中，所述金属离子选自碱金属离子中的一种，如K+或Na+。出于提高溶解性、使反应顺利进行的考虑，在一些实施方案中，有机铵离子为三级胺形成的铵阳离子或季铵阳离子，如，三乙胺形成的铵离子或N，N-二异丙基乙胺形成的铵离子。在一些实施方案中，羧酸盐是三乙胺羧酸盐或N，N-二异丙基乙胺羧酸盐。In some embodiments, the first functional group contains a hydroxyl group or a protected hydroxyl group; the second functional group contains a solid phase carrier connected via a carboxylate bond or a solid phase carrier connected via an amide bond, or a solid phase carrier connected via a phosphate bond, as shown in formula (C1') or (C3'). At this time, the compound shown in formula (321) is used as a starting point instead of the solid phase carrier, and the nucleoside monomers are sequentially connected according to the phosphoramidite solid phase synthesis method to obtain the sense chain or antisense chain of the siRNA connected with the conjugated group. In some embodiments, the carboxylate can be represented as -, carboxylicacid- M+ , wherein M+ is a cation, for example, one selected from a metal cation, an ammonium cation NH4+ , and an organic ammonium cation. In one embodiment, the metal ion is selected from one of the alkali metal ions, such as K+ or Na+ . In order to improve solubility and make the reaction proceed smoothly, in some embodiments, the organic ammonium ion is an ammonium cation formed by a tertiary amine or a quaternary ammonium cation, such as an ammonium ion formed by triethylamine or an ammonium ion formed by N, N-diisopropylethylamine. In some embodiments, the carboxylate is triethylamine carboxylate or N,N-diisopropylethylamine carboxylate.
在一些实施方案中，R4含有式(B9)、(B10)、(B9’)、(B10’)、(B11)、(B12)、(B11’)或(B12’)所示的结构：In some embodiments,R4 comprises a structure represented by formula (B9), (B10), (B9'), (B10'), (B11), (B12), (B11') or (B12'):
其中，q1为1-4的整数，q2为1-10的整数，X为O或NH，M+为阳离子，Rk为羟基保护基团，SPS表示固相载体，表示基团共价键连接的位点。在一些实施方案中，q1为1或2。在一些实施方案中，q2为1-5的整数。在一些实施方案中，R4含有式(B9)或(B10)所示的结构。在一些实施方案中，R4含有式(B11)或(B12)所示的结构。whereinq1 is an integer of 1-4,q2 is an integer of 1-10, X is O or NH, M+ is a cation,Rk is a hydroxyl protecting group, SPS represents a solid phase support, In some embodiments, q1 is 1 or 2. In some embodiments, q2 is an integer from 1 to 5. In some embodiments, R4 contains the structure shown in formula (B9) or (B10). In some embodiments, R4 contains the structure shown in formula (B11) or (B12).
在一些实施方案中，Rk是Tr(三苯甲基)、MMTr(4-甲氧基三苯甲基)、DMTr(4，4’-双甲氧基三苯甲基)、TMTr(4，4’，4’-三甲氧基苯甲基)中的一种或多种。在一些实施方案中，Rk可以是DMTr，即4，4’-双甲氧基三苯甲基(4，4’-dimethoxytrityl)。In some embodiments,Rk is one or more of Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-bismethoxytrityl), TMTr (4,4',4'-trimethoxybenzyl). In some embodiments,Rk can be DMTr, i.e. 4,4'-bismethoxytrityl (4,4'-dimethoxytrityl).
L1的定义如前所述。The definition ofL1 is as described above.
在一些实施方案中，L1被用于将M1靶向基团连接至含氮骨架上的N原子，从而为第二种siRNA缀合物提供肝靶向功能。在一些实施方案中，L1包含A1-A26中的任一个或其组合。In some embodiments,L1 is used to connect theM1 targeting group to the N atom on the nitrogen-containing backbone, thereby providing liver targeting function to the second siRNA conjugate. In some embodiments,L1 comprises any one or a combination of A1-A26.
根据上述描述，本领域技术人员容易理解的是，相较于本领域公知的亚磷酰胺固相合成方法而言，可通过上述第1官能团以及任选的第2官能团，获得将缀合分子连接至核苷酸序列的任意可能的位置的第二种siRNA缀合物，例如，缀合分子连接至核苷酸序列的端部，缀合分子连接至核苷酸序列的末端。相应地，除非另有说明，以下涉及缀合制备的描述中，当提及“脱保护”、“偶联”、“盖帽”、“氧化”、“硫化”等反应时，应当理解为本领域公知的亚磷酰胺核酸固相合成方法中所涉及的反应条件和试剂也同样适用于这些反应。示例性的反应条件和试剂将在后文详细描述。在一些实施方案中，每个S1独立地是M1。在一些实施方案中，每个S1独立地是M1中至少一个活性羟基被羟基保护基团保护而形成的基团。在一些实施方案中，每个S1独立地是M1中任何存在的活性羟基全部被羟基保护基团保护而形成的基团。在一些实施方案中，任何本领域技术人员已知的羟基保护基团均可被用于保护M1中的活性羟基。在一些实施方案中，被保护的羟基可以式YCOO-表示，其中，每个Y独立地选自于由C1-C10烷基和C6-C10芳基所组成的组，所述C1-C10烷基和C6-C10芳基任选地被一个或多个取代基取代，所述取代基选自于由卤素和C1-C6烷基所组成的组。在一些实施方案中，每个Y独立地选自于由以下基团所组成的组：甲基、三氟甲基、二氟甲基、单氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤苯基，以及C1-C6烷基苯基。According to the above description, it is easy for a person skilled in the art to understand that, compared with the phosphoramidite solid phase synthesis method known in the art, a second siRNA conjugate that connects the conjugated molecule to any possible position of the nucleotide sequence can be obtained through the above-mentioned first functional group and the optional second functional group, for example, the conjugated molecule is connected to the end of the nucleotide sequence, and the conjugated molecule is connected to the end of the nucleotide sequence. Accordingly, unless otherwise specified, in the following description of conjugation preparation, when referring to reactions such as "deprotection", "coupling", "capping", "oxidation", and "sulfurization", it should be understood that the reaction conditions and reagents involved in the phosphoramidite nucleic acid solid phase synthesis method known in the art are also applicable to these reactions. Exemplary reaction conditions and reagents will be described in detail later. In some embodiments, each S1 is independently M1. In some embodiments, each S1 is independently a group formed by at least one active hydroxyl group in M1 being protected by a hydroxyl protecting group. In some embodiments, each S1 is independently a group formed by all active hydroxyl groups present in M1 being protected by a hydroxyl protecting group. In some embodiments, any hydroxyl protecting group known to those skilled in the art can be used to protect the active hydroxyl group inM1 . In some embodiments, the protected hydroxyl group can be represented by the formula YCOO-, wherein each Y is independently selected from the group consisting of C1-C10 alkyl andC6 -C10 aryl, and theC1 -C10 alkyl andC6 -C10 aryl are optionally substituted with one or more substituents selected from the group consisting of halogen andC1 -C6 alkyl. In some embodiments, each Y is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, andC1 -C6 alkylphenyl.
在一些实施方案中，每个S1各自独立地选自于由式A46-A54所组成的组：In some embodiments, eachS1 is independently selected from the group consisting of Formulae A46-A54:
在一些实施方案中，S1为式A49或A50。In some embodiments,S1 is formula A49 or A50.
在一些实施方案中，每个Y独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；出于简化本公开的缀合分子的目的，在一些实施方案中，Y为甲基。In some embodiments, each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl and alkylphenyl; for the purpose of simplifying the conjugated molecules of the present disclosure, in some embodiments, Y is methyl.
如前所述，第二种siRNA缀合物的制备方法还包括以下步骤：合成siRNA的另一链(例如，当上述步骤合成了连接有缀合基团的siRNA正义链时，还包括按照固相合成方法合成siRNA的反义链，反之亦然)，分离正义链和反义链，以及退火。具体地，在分离步骤中，连接至核苷酸序列和/或缀合分子的固相载体被切割下来，同时必要的保护基团被脱除(此时，式(321)所示的化合物中的各S1基团转化为对应的M1靶向基团)，获得连接有缀合基团的siRNA正义链(或反义链)以及对应的反义链(或正义链)，正义链与反义链退火形成双链RNA结构，获得第二种siRNA缀合物。As mentioned above, the preparation method of the second siRNA conjugate further includes the following steps: synthesizing the other strand of siRNA (for example, when the above step synthesizes the siRNA sense strand connected to the conjugated group, it also includes synthesizing the antisense strand of siRNA according to the solid phase synthesis method, and vice versa), separating the sense strand and the antisense strand, and annealing. Specifically, in the separation step, the solid phase carrier connected to the nucleotide sequence and/or the conjugated molecule is cut off, and the necessary protective groups are removed (at this time, eachS1 group in the compound shown in formula (321) is converted into a correspondingM1 targeting group), and the siRNA sense strand (or antisense strand) connected to the conjugated group and the corresponding antisense strand (or sense strand) are obtained, and the sense strand and the antisense strand are annealed to form a double-stranded RNA structure to obtain the second siRNA conjugate.
在一些实施方案中，所述第二种siRNA缀合物的制备方法包含以下步骤：在偶联反应条件和偶联试剂存在下，将式(321)所示的化合物与正义链或反义链的3′端的第一个核苷单体接触，使式(321)所示的化合物连接上序列中第一个核苷酸，在亚磷酰胺固相合成的条件下，按照期望的正义链或反义链核苷酸种类和顺序，按照3′到5′的方向将核苷单体依次连接，合成siRNA的正义链或反义链；其中，式(321)所示的化合物为R4中含有第1官能团和第2官能团，第1官能团含有被保护的羟基，第2官能团具有如式(C1’)或(C3’)所示结构的式(321)化合物，与第一个核苷单体连接前，式(321)所示的化合物经过脱保护；每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；得到连接有缀合分子的核酸的正义链或反义链；在亚磷酰胺固相合成的条件下，按照反义链或正义链核苷酸种类和顺序，按照3′到5′的方向将核苷单体依次连接，合成核酸的反义链或正义链；每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；脱除保护基并与固相载体切割，分离纯化获得核酸的正义链和反义链，退火。In some embodiments, the preparation method of the second siRNA conjugate comprises the following steps: contacting the compound represented by formula (321) with the first nucleoside monomer at the 3′ end of the sense chain or antisense chain under coupling reaction conditions and in the presence of a coupling reagent, so that the compound represented by formula (321) is connected to the first nucleotide in the sequence, and under phosphoramidite solid phase synthesis conditions, according to the desired sense chain or antisense chain nucleotide type and sequence, the nucleoside monomers are sequentially connected in the direction from 3′ to 5′ to synthesize the sense chain or antisense chain of siRNA; wherein the compound represented by formula (321) is R4 contains a first functional group and a second functional group, the first functional group contains a protected hydroxyl group, and the second functional group has a compound of formula (321) with a structure as shown in formula (C1') or (C3'). Before being connected to the first nucleoside monomer, the compound shown in formula (321) is deprotected; the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; the sense chain or antisense chain of the nucleic acid connected with the conjugate molecule is obtained; under the conditions of phosphoramidite solid phase synthesis, the nucleoside monomers are sequentially connected in the direction from 3' to 5' according to the type and sequence of nucleotides in the antisense chain or sense chain to synthesize the antisense chain or sense chain of the nucleic acid; the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; the protecting group is removed and cut with the solid phase carrier, and the sense chain and antisense chain of the nucleic acid are separated and purified to obtain, and annealed.
在一些实施方案中，第二种siRNA缀合物的制备方法包含以下步骤：按照该双链siRNA中正义链或反义链的核苷酸种类和顺序，按照3′到5′的方向将核苷单体依次连接，合成正义链和反义链，每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应，得到连接在固相载体上的正义链和连接在固相载体上的反义链；在偶联反应条件和偶联试剂存在下，将式(321)所示的化合物与连接在固相载体上的正义链或连接在固相载体上的反义链接触，将式(321)所示的化合物连接至正义链或反义链，其中，式(321)化合物是R4中含有第1官能团，第1官能团为亚磷酰胺基团的式(321)化合物；脱除保护基并与固相载体切割，分别分离纯化，Nu代表的siRNA的正义链或反义链，退火，其中，所述siRNA的正义链或反义链上连接有缀合基团。In some embodiments, the preparation method of the second siRNA conjugate comprises the following steps: according to the nucleotide type and order of the sense strand or antisense strand in the double-stranded siRNA, the nucleoside monomers are sequentially connected in the 3′ to 5′ direction to synthesize the sense strand and the antisense strand, and the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization to obtain the sense strand connected to the solid phase support and the antisense strand connected to the solid phase support; under coupling reaction conditions and in the presence of a coupling reagent, the compound represented by formula (321) is contacted with the sense strand connected to the solid phase support or the antisense strand connected to the solid phase support, and the compound represented by formula (321) is connected to the sense strand or the antisense strand, wherein the compound of formula (321) is R4 contains a first functional group, wherein the first functional group is a compound of formula (321) which is a phosphoramidite group; removing the protecting group and cutting with the solid phase carrier, separating and purifying respectively, and annealing the sense chain or antisense chain of the siRNA represented by Nu, wherein the sense chain or antisense chain of the siRNA is connected to a conjugated group.
在一些实施方案中，式A59中的P连接至siRNA中的正义链的3′末端，第二种siRNA缀合物的制备方法包括：In some embodiments, P in formula A59 is linked to the 3′ end of the sense strand in the siRNA, and the method for preparing the second siRNA conjugate comprises:
(1)脱除式(321)化合物(其中，式(321)化合物为R4中含有第1官能团和第2官能团，第1官能团含有被保护的羟基ORk，第2官能团具有如式(C1’)或(C3’)所示结构的化合物)中的羟基保护基团Rk；在偶联反应条件和偶联试剂存在下，将脱保护得到的产物与核苷单体接触，得到通过缀合分子连接至固相载体的核苷单体；(1) removing the hydroxyl protecting group Rk in the compound of formula (321) (wherein the compound of formula (321) is a compound wherein R 4 contains a first functional group and a second functional group, the first functional group contains a protected hydroxyl group OR k,and the second functional group has a structure as shown in formula (C1') or (C3')); contacting the deprotected product with a nucleoside monomer under coupling reaction conditions and in the presence of a coupling reagent to obtain a nucleoside monomer connected to a solid phase carrier via a conjugated molecule;
(2)以该通过缀合分子连接至固相载体的核苷单体起始，按照3′-5′的方向通过亚磷酰胺固相合成方法合成siRNA的正义链；(2) starting with the nucleoside monomer connected to the solid phase carrier via the conjugated molecule, synthesizing the positive strand of the siRNA via a phosphoramidite solid phase synthesis method in the 3′-5′ direction;
(3)通过亚磷酰胺固相合成方法，合成siRNA的反义链；(3) synthesizing the antisense strand of siRNA by phosphoramidite solid phase synthesis method;
(4)分离出siRNA的正义链和反义链并退火，获得第二种siRNA缀合物。(4) Separating the sense strand and antisense strand of siRNA and annealing them to obtain a second siRNA conjugate.
其中，在步骤(1)中，脱除式(321)化合物中的保护基团Rk的方法包括在脱保护条件下，将式(321)化合物与脱保护试剂接触。脱保护条件包括温度为0-50℃，在一些实施方案中为15-35℃，反应时间为30-300秒，在一些实施方案中为50-150秒，脱保护试剂可以选自三氟乙酸、三氯乙酸、二氯乙酸、一氯乙酸中的一种或多种，在一些实施方案中为二氯乙酸。脱保护试剂与式(321)化合物的摩尔比为10∶1-1000∶1，在一些实施方案中为50∶1-500∶1。Wherein, in step (1), the method for removing the protecting group Rk in the compound of formula (321) comprises contacting the compound of formula (321) with a deprotecting agent under deprotecting conditions. The deprotecting conditions include a temperature of 0-50° C., in some embodiments 15-35° C., a reaction time of 30-300 seconds, in some embodiments 50-150 seconds, and the deprotecting agent can be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid, in some embodiments dichloroacetic acid. The molar ratio of the deprotecting agent to the compound of formula (321) is 10:1-1000:1, in some embodiments 50:1-500:1.
所述偶联反应条件和偶联试剂可使用任何适合于上述偶联反应的条件和试剂。在一些实施方案中，可使用与所采用的固相合成方法中的偶联反应相同的条件与试剂。The coupling reaction conditions and coupling reagents may use any conditions and reagents suitable for the above coupling reaction. In some embodiments, the same conditions and reagents as those used in the coupling reaction in the solid phase synthesis method may be used.
在一些实施方案中，所述偶联反应的条件包括反应温度为0-50℃，在一些实施方案中为15-35℃。式(321)化合物与核苷单体的摩尔比为1∶1-1∶50，在一些实施方案中为1∶2-1∶5；式(321)化合物和偶联试剂的摩尔比可以1∶1-1∶50，在一些实施方案中为1∶3-1∶10，反应时间为200-3000秒，在一些实施方案中为500-1500秒。偶联试剂选自1H-四氮唑、5-乙硫基1H-四氮唑、5-苄硫基1H-四氮唑中的一种或多种，在一些实施方案中为5-乙硫基1H-四氮唑。所述偶联反应可在有机溶剂中进行，所述有机溶剂选自无水乙腈、无水DMF、无水二氯甲烷中的一种或多种，在一些实施方案中为无水乙腈。相对于式(321)化合物，所述有机溶剂的用量为3-50L/mol，在一些实施方案中为5-20L/mol。In some embodiments, the conditions of the coupling reaction include a reaction temperature of 0-50°C, in some embodiments 15-35°C. The molar ratio of the compound of formula (321) to the nucleoside monomer is 1:1-1:50, in some embodiments 1:2-1:5; the molar ratio of the compound of formula (321) to the coupling reagent can be 1:1-1:50, in some embodiments 1:3-1:10, and the reaction time is 200-3000 seconds, in some embodiments 500-1500 seconds. The coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole, in some embodiments 5-ethylthio 1H-tetrazole. The coupling reaction can be carried out in an organic solvent, and the organic solvent is selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, in some embodiments anhydrous acetonitrile. The amount of the organic solvent used is 3-50 L/mol, and in some embodiments, 5-20 L/mol, relative to the compound of formula (321).
在步骤(2)中，通过亚磷酰胺核酸固相合成的方法，利用上述步骤制备的通过缀合分子连接至固相载体的核苷单体起始，按照3′-5′的方向合成第二种siRNA缀合物的正义链S。此时，缀合基团连接至所得到的正义链的3′末端。In step (2), the sense strand S of the second siRNA conjugate is synthesized in the 3′-5′ direction by phosphoramidite nucleic acid solid phase synthesis, starting from the nucleoside monomer prepared in the above step and connected to the solid phase carrier through the conjugation molecule. At this time, the conjugation group is connected to the 3′ end of the obtained sense strand.
步骤(2)和(3)中所述固相合成的其它条件，包括核苷单体脱保护条件，脱保护试剂种类和用量，偶联反应条件，偶联试剂的种类和用量，盖帽反应的条件，盖帽试剂的种类和用量，氧化反应条件，氧化试剂种类和用量，硫化反应条件，硫化试剂和用量采用本领域中常规使用的各种试剂、用量和条件。Other conditions of the solid phase synthesis described in steps (2) and (3), including nucleoside monomer deprotection conditions, deprotection reagent type and amount, coupling reaction conditions, coupling reagent type and amount, capping reaction conditions, capping reagent type and amount, oxidation reaction conditions, oxidation reagent type and amount, sulfurization reaction conditions, sulfurization reagent and amount adopt various reagents, amounts and conditions conventionally used in the art.
例如，在一些实施方案中，步骤(2)和(3)中所述固相合成可使用如下条件：For example, in some embodiments, the solid phase synthesis in steps (2) and (3) may use the following conditions:
核苷单体脱保护条件包括温度为0-50℃，在一些实施方案中为15-35℃，反应时间为30-300秒，在一些实施方案中为50-150秒，脱保护试剂可以选自三氟乙酸、三氯乙酸、二氯乙酸、一氯乙酸中的一种或多种，在一些实施方案中为二氯乙酸。脱保护试剂与固相载体上4，4′-二甲氧基三苯甲基保护基的的摩尔比为2∶1-100∶1，在一些实施方案中为3∶1-50∶1。The nucleoside monomer deprotection conditions include a temperature of 0-50° C., in some embodiments 15-35° C., a reaction time of 30-300 seconds, in some embodiments 50-150 seconds, a deprotection agent can be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid, in some embodiments dichloroacetic acid. The molar ratio of the deprotection agent to the 4,4′-dimethoxytrityl protecting group on the solid phase carrier is 2:1-100:1, in some embodiments 3:1-50:1.
偶联反应条件包括温度为0-50℃，在一些实施方案中为15-35℃，固相载体上连接的核酸序列与核苷单体的摩尔比为1∶1-1∶50，在一些实施方案中为1∶5-1∶15；固相载体上连接的核酸序列和偶联试剂的摩尔比为1∶1-1∶100，在一些实施方案中为1∶50-1∶80，反应时间和偶联试剂的选择与前述相同。The coupling reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a molar ratio of the nucleic acid sequence connected to the solid phase support to the nucleoside monomer of 1:1-1:50, in some embodiments 1:5-1:15; a molar ratio of the nucleic acid sequence connected to the solid phase support to the coupling reagent of 1:1-1:100, in some embodiments 1:50-1:80, and the reaction time and the selection of the coupling reagent are the same as described above.
盖帽反应条件包括温度为0-50℃，在一些实施方案中为15-35℃，反应时间为5-500秒，在一些实施方案中为10-100秒，盖帽试剂的选择与前述相同。盖帽试剂的总量与固相载体上连接的核酸序列的摩尔比为1∶100-100∶1，在一些实施方案中为1∶10-10∶1。在盖帽试剂使用等摩尔量的乙酸酐与N-甲基咪唑的情况下，乙酸酐、N-甲基咪唑以及固相载体上连接的核酸序列的摩尔比为1∶1∶10-10∶10∶1，在一些实施方案中为1∶1∶2-2∶2∶1。The capping reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 5-500 seconds, in some embodiments 10-100 seconds, and the selection of the capping reagent is the same as above. The molar ratio of the total amount of the capping reagent to the nucleic acid sequence connected to the solid phase carrier is 1:100-100:1, in some embodiments 1:10-10:1. In the case where the capping reagent uses equimolar amounts of acetic anhydride and N-methylimidazole, the molar ratio of acetic anhydride, N-methylimidazole and the nucleic acid sequence connected to the solid phase carrier is 1:1:10-10:10:1, in some embodiments 1:1:2-2:2:1.
氧化反应条件包括温度为0-50℃，在一些实施方案中为15-35℃，反应时间为1-100秒，在一些实施方案中为5-50秒，氧化试剂在一些实施方案中为碘(在一些实施方案中，以碘水的形式提供)。氧化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔比可以为1∶1-100∶1，在一些实施方案中为5∶1-50∶1。在一些实施方案中，所述氧化反应在四氢呋喃∶水∶吡啶＝3∶1∶1-1∶1∶3的混合溶剂中进行。硫化反应条件包括温度为0-50℃，在一些实施方案中为15-35℃，反应时间为50-2000秒，在一些实施方案中为100-1000秒，硫化试剂在一些实施方案中为氢化黄原素。硫化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔比可以为10∶1-1000∶1，在一些实施方案中为10∶1-500∶1。在一些实施方案中，所述硫化反应在乙腈∶吡啶＝1∶3-3∶1的混合溶剂中进行。The oxidation reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 1-100 seconds, in some embodiments 5-50 seconds, and the oxidizing agent is iodine in some embodiments (in some embodiments, provided in the form of iodine water). The molar ratio of the oxidizing agent to the nucleic acid sequence connected to the solid phase support in the coupling step can be 1:1-100:1, in some embodiments 5:1-50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran: water: pyridine = 3:1:1-1:1:3. The sulfurization reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 50-2000 seconds, in some embodiments 100-1000 seconds, and the sulfurizing agent is hydrogenated xanthan in some embodiments. The molar ratio of the sulfurizing agent to the nucleic acid sequence connected to the solid phase support in the coupling step can be 10:1-1000:1, in some embodiments 10:1-500:1. In some embodiments, the sulfurization reaction is carried out in a mixed solvent of acetonitrile:pyridine=1:3-3:1.
在将所有核苷单体连接之后，退火之前，该方法还包括分离出siRNA的正义链和反义链。分离的方法为本领域技术人员所公知，一般包括将合成得到的核苷酸序列从固相载体上切割下来，脱除碱基上、磷酸基上和配体上的保护基团，纯化和脱盐。After all nucleoside monomers are connected and before annealing, the method further includes separating the sense strand and the antisense strand of the siRNA. The separation method is well known to those skilled in the art and generally includes cutting the synthesized nucleotide sequence from the solid phase support, removing the protecting groups on the base, the phosphate group and the ligand, and purifying and desalting.
将合成得到的核苷酸序列从固相载体上切割下来，并脱除碱基上、磷酸基上和配体上的保护基团可按照siRNA合成中常规的切割和脱保护方法进行。例如，将得到的连接有固相载体的核苷酸序列与浓氨水接触；在脱保护的过程中，A46-A54基团的保护基团YCOO-转化为羟基，S1基团转化为相应的M1基团，生成式(308)所示的缀合物。其中，所述浓氨水可以是25-30重量％的氨水，浓氨水的用量与目标siRNA序列相比可以为0.2ml/μmol-0.8ml/μmol。The synthesized nucleotide sequence is cut from the solid phase support, and the protective groups on the base, phosphate group and ligand are removed according to the conventional cutting and deprotection methods in siRNA synthesis. For example, the obtained nucleotide sequence connected to the solid phase support is contacted with concentrated ammonia water; during the deprotection process, the protective group YCOO- of the A46-A54 group is converted into a hydroxyl group, and theS1 group is converted into the correspondingM1 group, generating a conjugate shown in formula (308). The concentrated ammonia water can be 25-30% by weight of ammonia water, and the amount of concentrated ammonia water used can be 0.2ml/μmol-0.8ml/μmol compared with the target siRNA sequence.
在所合成的核苷酸序列上存在至少一个2′-TBDMS保护时，所述方法还包括将脱除了固相载体的核苷酸序列与三乙胺三氢氟酸盐接触，以脱除该2′-TBDMS保护。此时，所得到的目标siRNA序列中具有游离的2′-羟基的相应核苷。三乙胺三氢氟酸盐纯品的用量与目标siRNA序列相比为0.4ml/μmol-1.0ml/μmol。这样即可得到式(308)的siRNA缀合物。When there is at least one 2′-TBDMS protection on the synthesized nucleotide sequence, the method further comprises contacting the nucleotide sequence from which the solid phase carrier has been removed with triethylamine trihydrofluoride to remove the 2′-TBDMS protection. At this time, the corresponding nucleoside with free 2′-hydroxyl in the obtained target siRNA sequence. The amount of pure triethylamine trihydrofluoride used is 0.4 ml/μmol-1.0 ml/μmol compared with the target siRNA sequence. In this way, the siRNA conjugate of formula (308) can be obtained.
纯化和脱盐的方法是本领域技术人员熟知的。例如，可利用制备型离子色谱纯化柱，通过NaBr或NaCl的梯度洗脱，完成核酸的纯化；产品收集合并后，可采用反相色谱纯化柱进行脱盐。The methods of purification and desalting are well known to those skilled in the art. For example, a preparative ion chromatography purification column can be used to elute the nucleic acid by gradient elution with NaBr or NaCl; after the product is collected and combined, a reverse phase chromatography purification column can be used for desalting.
这样得到的第二种siRNA缀合物中，核苷酸之间的磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子基本与钠离子结合，第二种siRNA缀合物基本以钠盐形式存在。可以采用熟知的离子交换方法，用氢离子和/或其他阳离子取代所述钠离子，得到其他形式的第二种siRNA缀合物。所述阳离子如前所述。In the second siRNA conjugate thus obtained, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or thiophosphodiester bond between the nucleotides is basically combined with the sodium ion, and the second siRNA conjugate is basically present in the form of a sodium salt. The well-known ion exchange method can be used to replace the sodium ion with hydrogen ions and/or other cations to obtain other forms of the second siRNA conjugate. The cation is as described above.
在合成过程中，可随时对核酸序列的纯度和分子量进行检测，更好地把控合成质量，此类检测方法为本领域技术人员所公知。例如，可通过离子交换色谱检测核酸纯度，并通过液质联用色谱测定分子量。During the synthesis process, the purity and molecular weight of the nucleic acid sequence can be tested at any time to better control the synthesis quality. Such detection methods are well known to those skilled in the art. For example, the purity of the nucleic acid can be tested by ion exchange chromatography, and the molecular weight can be determined by liquid chromatography-mass spectrometry.
退火的方法也是本领域技术人员熟知的。例如，可简单地将所合成的正义链(S链)与反义链(AS链)以等摩尔比混合在注射用水中加热至70-95℃，随后室温冷却，使其通过氢键形成双链结构。这样即可得到第二种siRNA缀合物。Annealing methods are also well known to those skilled in the art. For example, the synthesized sense strand (S strand) and antisense strand (AS strand) can be simply mixed in an equal molar ratio in water for injection and heated to 70-95° C., and then cooled to room temperature to form a double-stranded structure through hydrogen bonding. In this way, a second siRNA conjugate can be obtained.
在获得本公开的缀合物后，在一些实施方案中，还可利用例如液质联用色谱等方法，通过分子量检测等方式对所合成的第二种siRNA缀合物进行表征，确定所合成的siRNA缀合物为目标设计的第二种siRNA缀合物，且所合成的siRNA的序列与欲合成的siRNA的序列相符，例如为表2中所列的序列之一。After obtaining the conjugate of the present invention, in some embodiments, the synthesized second siRNA conjugate can be characterized by molecular weight detection and the like using methods such as liquid chromatography-mass spectrometry to determine that the synthesized siRNA conjugate is the target designed second siRNA conjugate, and that the sequence of the synthesized siRNA is consistent with the sequence of the siRNA to be synthesized, for example, one of the sequences listed in Table 2.
式(321)所示的化合物可以通过以下制备方法得到：该方法包括在有机溶剂中，在酯化反应条件下，以及在碱和成酯催化剂存在下，将式(313)所示的化合物与环状酸酐接触，离子交换，分离得到式(321)所示的化合物：The compound represented by formula (321) can be obtained by the following preparation method: the method comprises contacting the compound represented by formula (313) with a cyclic acid anhydride in an organic solvent under esterification reaction conditions and in the presence of a base and an esterification catalyst, ion exchange, and separation to obtain the compound represented by formula (321):
其中，n1、n3、m1、m2、m3、R10、R11、R12、R13、R14、R15、L1、S1各自的定义和可选择的范围如前所述；Wherein, the definitions and selectable ranges of n1, n3, m1, m2, m3, R10 , R11 , R12 , R13 , R14 , R15 , L1 and S1 are as described above;
R6为提供式(321)中R4的基团。在一些实施方案中，R6具有式(A61)所示的结构：R6 is a group that provides R4 in formula (321). In some embodiments, R6 has a structure shown in formula (A61):
其中，Ri为能够实现与含氮骨架上的N连接、与RkO连接并且连接有一个游离羟基的任意基团，Rk为羟基保护基团。此时，所获得的是R4中含有作为羟基保护基团的第1官能团和第2官能团，所述第2官能团含有如式(C1)或(C2)所示结构的式(321)化合物。Wherein, Ri is any group that can achieve connection with N on the nitrogen-containing skeleton, connect with Rk O and connect with a free hydroxyl group, and Rk is a hydroxyl protecting group. In this case, what is obtained is a compound of formula (321) containing a first functional group and a second functional group as a hydroxyl protecting group in R4 , wherein the second functional group contains a structure as shown in formula (C1) or (C2).
所述酯化反应条件包括反应温度为0-100℃，反应时间为8-48小时，在一些实施方案中，所述酯化反应条件为反应温度为10-40℃，反应时间为20-30小时。The esterification reaction conditions include a reaction temperature of 0-100° C. and a reaction time of 8-48 hours. In some embodiments, the esterification reaction conditions include a reaction temperature of 10-40° C. and a reaction time of 20-30 hours.
在一些实施方案中，所述有机溶剂包含环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种。在一些实施方案中，所述环氧类溶剂为二氧六环和/或四氢呋喃，所述醚类溶剂为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂为二氯甲烷、三氯甲烷和1，2-二氯乙烷中的一种或多种。在一些实施方案中，所述有机溶剂为二氯甲烷。相对于所述式(313)所示的化合物，所述有机溶剂的用量为3-50L/mol，在一些实施方案中为5-20L/mol。In some embodiments, the organic solvent comprises one or more of an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran, the ether solvent is ether and/or methyl tert-butyl ether, and the halogenated alkane solvent is one or more of dichloromethane, trichloromethane and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. Relative to the compound shown in the formula (313), the amount of the organic solvent is 3-50L/mol, and in some embodiments is 5-20L/mol.
在一些实施方案中，所述环状酸酐为丁二酸酐、戊二酸酐、己二酸酐或庚二酸酐中的一种，在一些实施方案中为丁二酸酐。所述环状酸酐与所述式(313)所示的化合物的摩尔比为1∶1-10∶1，在一些实施方案中为2∶1-5∶1。In some embodiments, the cyclic anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride or pimelic anhydride, and in some embodiments, succinic anhydride. The molar ratio of the cyclic anhydride to the compound represented by formula (313) is 1:1-10:1, and in some embodiments, 2:1-5:1.
所述成酯催化剂可以是任何对该酯化反应起到催化作用的催化剂，例如该催化剂可以是4-二甲氨基吡啶。所述催化剂与式(313)所示的化合物的摩尔比为1∶1-10∶1，在一些实施方案中为2∶1-5∶1。The esterification catalyst can be any catalyst that catalyzes the esterification reaction, for example, the catalyst can be 4-dimethylaminopyridine. The molar ratio of the catalyst to the compound represented by formula (313) is 1:1-10:1, and in some embodiments is 2:1-5:1.
在一些实施方案中，所述碱可以是任意的无机碱，有机碱或者它们的结合。考虑溶解性和产物稳定性，所述碱可以是例如三级胺类有机碱。在一些实施方案中，所述三级胺类有机碱为三乙胺或N，N-二异丙基乙胺。所述三级胺类有机碱与式(313)所示的化合物的摩尔比为1∶1-20∶1，在一些实施方案中为3∶1-10∶1。In some embodiments, the base can be any inorganic base, organic base or a combination thereof. Considering solubility and product stability, the base can be, for example, a tertiary amine organic base. In some embodiments, the tertiary amine organic base is triethylamine or N, N-diisopropylethylamine. The molar ratio of the tertiary amine organic base to the compound shown in formula (313) is 1:1-20:1, and in some embodiments is 3:1-10:1.
所述离子交换作用是将式(321)化合物转化为期望的羧酸或羧酸盐的形式，离子交换的方法为本领域技术人员所公知，可以使用合适的离子交换溶液和交换条件，得到前述阳离子为M+的缀合分子，在此不做详述。在一些实施方案中，所述离子交换反应使用三乙胺磷酸盐溶液进行，所述三乙胺磷酸盐溶液的浓度为0.2-0.8M，在一些实施方案中为0.4-0.6M，相对于式(313)化合物，所述三乙胺磷酸盐溶液的用量为3-6L/mol，在一些实施方案中为4-5L/mol。The ion exchange reaction is to convert the compound of formula (321) into the desired carboxylic acid or carboxylate form. The method of ion exchange is well known to those skilled in the art. Suitable ion exchange solutions and exchange conditions can be used to obtain the conjugated molecule in which the cation is M+ , which will not be described in detail here. In some embodiments, the ion exchange reaction is carried out using a triethylamine phosphate solution, the concentration of the triethylamine phosphate solution is 0.2-0.8M, in some embodiments, 0.4-0.6M, relative to the compound of formula (313), the amount of the triethylamine phosphate solution is 3-6L/mol, in some embodiments, 4-5L/mol.
可使用任何合适的分离方法从反应混合物中分离式(321)化合物。在一些实施方案中，可通过蒸发除去溶剂、随后通过色谱方法分离式(321)化合物，例如，可使用如下色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用含1wt‰三乙胺的二氯甲烷∶甲醇＝100∶18-100∶20梯度洗脱；或者(2)反相纯化：C18、C8反相填料，使用甲醇∶乙腈＝0.1∶1-1∶0.1梯度洗脱。在一些实施方案中，可以直接除去溶剂得到式(321)化合物粗产品，该粗产品可以直接用于后续反应。Any suitable separation method can be used to separate the compound of formula (321) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following chromatographic conditions can be used for separation: (1) normal phase purification silica gel: 200-300 mesh silica gel filler, using dichloromethane containing 1wt‰ triethylamine: methanol = 100: 18-100: 20 gradient elution; or (2) reverse phase purification: C18, C8 reverse phase filler, using methanol: acetonitrile = 0.1: 1-1: 0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (321), which can be directly used in subsequent reactions.
在一些实施方案中，式(321)化合物的制备方法还进一步包括在缩合反应条件下，在有机溶剂中，在缩合剂和三级胺类有机碱的存在下，将上述离子交换反应得到的产物进一步与含有氨基或羟基的固相载体进行接触。此时，所获得的是R4中含有第1官能团和第2官能团，第1官能团含有羟基保护基团，第2官能团含有如式(C1’)所示结构的式(321)化合物。In some embodiments, the preparation method of the compound of formula (321) further comprises contacting the product obtained by the above ion exchange reaction with a solid phase carrier containing amino or hydroxyl groups in an organic solvent under condensation reaction conditions in the presence of a condensing agent and a tertiary amine organic base. At this time, what is obtained is a compound of formula (321) in whichR4 contains the first functional group and the second functional group, the first functional group contains a hydroxyl protecting group, and the second functional group contains a structure as shown in formula (C1').
所述固相载体为固相合成siRNA中所用的载体中的一种，其中的一些为本领域技术人员所公知。例如，所述固相载体可以选自含有活性羟基或氨基官能团的固相载体，在一些实施方案中，所述固相载体为氨基树脂或羟基树脂。为了便于后续进行核酸固相合成，所述氨基或羟基树脂在一些实施方案中具有如下参数：粒径100-400目(mesh)，表面氨基或羟基载量为0.2-0.5mmol/g。所述式(321)所示的化合物与固相载体的用量比为10-400μmol化合物/每克固相载体(μmol/g)。在一些实施方案中，所述式(321)所示的化合物与固相载体的用量比为50-200μmol/g。The solid phase carrier is one of the carriers used in the solid phase synthesis of siRNA, some of which are well known to those skilled in the art. For example, the solid phase carrier can be selected from a solid phase carrier containing an active hydroxyl or amino functional group. In some embodiments, the solid phase carrier is an amino resin or a hydroxy resin. In order to facilitate the subsequent solid phase synthesis of nucleic acids, the amino or hydroxy resin has the following parameters in some embodiments: a particle size of 100-400 mesh, and a surface amino or hydroxy loading of 0.2-0.5 mmol/g. The amount ratio of the compound represented by the formula (321) to the solid phase carrier is 10-400 μmol compound/gram of solid phase carrier (μmol/g). In some embodiments, the amount ratio of the compound represented by the formula (321) to the solid phase carrier is 50-200 μmol/g.
所述有机溶剂可以是本领域技术人员已知的任何合适的溶剂或混合溶剂。在一些实施方案中，所述有机溶剂为乙腈、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种。在一些实施方案中，所述环氧类溶剂为二氧六环和/或四氢呋喃，所述醚类溶剂为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂为二氯甲烷、三氯甲烷和1，2-二氯乙烷中的一种或多种。在一些实施方案中，所述有机溶剂为乙腈。相对于式(321)化合物，所述有机溶剂的用量为20-200L/mol，在一些实施方案中为50-100L/mol。The organic solvent can be any suitable solvent or mixed solvent known to those skilled in the art. In some embodiments, the organic solvent is acetonitrile, epoxy solvents, ether solvents, halogenated alkane solvents, dimethyl sulfoxide, N, N-dimethylformamide and N, N-diisopropylethylamine, one or more. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran, the ether solvent is ether and/or methyl tert-butyl ether, and the halogenated alkane solvent is one or more of dichloromethane, trichloromethane and 1,2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. Relative to the compound of formula (321), the amount of the organic solvent is 20-200L/mol, and in some embodiments is 50-100L/mol.
所述缩合剂可以是六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮和/或O-苯并三氮唑-四甲基脲六氟磷酸酯，在一些实施方案中为O-苯并三氮唑-四甲基脲六氟磷酸酯。所述缩合剂与式(321)所示的化合物的摩尔比为1∶1-20∶1，在一些实施方案中为1∶1-5∶1。The condensing agent can be benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-benzoxazole 4 (3H)-one and/or O-benzotriazole-tetramethyluronium hexafluorophosphate, and in some embodiments, O-benzotriazole-tetramethyluronium hexafluorophosphate. The molar ratio of the condensing agent to the compound represented by formula (321) is 1:1-20:1, and in some embodiments, 1:1-5:1.
在一些实施方案中，所述三级胺类有机碱为三乙胺和/或N，N-二异丙基乙胺，在一些实施方案中为N，N-二异丙基乙胺；所述三级胺类有机碱与式(321)所示的化合物的摩尔比为1∶1-20∶1，在一些实施方案中为1∶1-5∶1。In some embodiments, the tertiary amine organic base is triethylamine and/or N,N-diisopropylethylamine, and in some embodiments it is N,N-diisopropylethylamine; the molar ratio of the tertiary amine organic base to the compound represented by formula (321) is 1:1-20:1, and in some embodiments it is 1:1-5:1.
在一些实施方案中，式(321)化合物的制备方法还可以包括将得到的缩合产物在盖帽反应条件下，在有机溶剂中，与盖帽试剂和酰化催化剂接触，分离得到式(321)所示的化合物。所述盖帽反应的作用在于除去任何尚未反应完全的活性反应官能团，以避免在后续反应中产生不必要的副产物。所述盖帽反应的条件包括反应温度为0-50℃，在一些实施方案中为15-35℃，反应的时间为1-10h，在一些实施方案中为3-6h。盖帽试剂可以使用siRNA固相合成中所使用的盖帽试剂，siRNA固相合成中所使用的盖帽试剂为本领域技术人员所公知。In some embodiments, the preparation method of the compound of formula (321) may further include contacting the obtained condensation product with a capping agent and an acylation catalyst in an organic solvent under capping reaction conditions, and separating to obtain the compound shown in formula (321). The function of the capping reaction is to remove any active reaction functional groups that have not yet reacted completely to avoid the generation of unnecessary by-products in subsequent reactions. The conditions of the capping reaction include a reaction temperature of 0-50°C, in some embodiments 15-35°C, and a reaction time of 1-10h, in some embodiments 3-6h. The capping agent can use the capping agent used in siRNA solid phase synthesis, and the capping agent used in siRNA solid phase synthesis is well known to those skilled in the art.
在一些实施方案中，所述盖帽试剂由盖帽试剂1(cap1)和盖帽试剂2(cap2)组成，其中，盖帽试剂A为N-基甲基咪唑，在一些实施方案中以N-甲基咪唑的吡啶/乙腈混合溶液形式提供，其中，吡啶与乙腈的体积比为1∶10-1∶1，在一些实施方案中为1∶3-1∶1，吡啶与乙腈的总体积与N-甲基咪唑的体积为1∶1-10∶1，在一些实施方案中为3∶1-7∶1。所述盖帽试剂B为乙酸酐，在一些实施方案中以乙酸酐的乙腈溶液形式提供，其中，乙酸酐和乙腈的体积为1∶1-1∶10，在一些实施方案中为1∶2-1∶6。In some embodiments, the capping reagent consists of capping reagent 1 (cap1) and capping reagent 2 (cap2), wherein capping reagent A is N-methylimidazole, and in some embodiments, it is provided in the form of a pyridine/acetonitrile mixed solution of N-methylimidazole, wherein the volume ratio of pyridine to acetonitrile is 1:10-1:1, in some embodiments, 1:3-1:1, and the total volume of pyridine and acetonitrile to the volume of N-methylimidazole is 1:1-10:1, in some embodiments, 3:1-7:1. Capping reagent B is acetic anhydride, and in some embodiments, it is provided in the form of an acetonitrile solution of acetic anhydride, wherein the volume of acetic anhydride and acetonitrile is 1:1-1:10, in some embodiments, 1:2-1:6.
在一些实施方案中，所述N-甲基咪唑的吡啶/乙腈混合溶液的体积与式(321)化合物的质量之比为5ml/g-50ml/g，在一些实施方案中为15ml/g-30ml/g。所述乙酸酐的乙腈溶液的体积与式(321)化合物的质量之比为0.5ml/g-10ml/g，在一些实施方案中为1ml/g-5ml/g。In some embodiments, the volume ratio of the pyridine/acetonitrile mixed solution of N-methylimidazole to the mass ratio of the compound of formula (321) is 5ml/g-50ml/g, and in some embodiments, 15ml/g-30ml/g. The volume ratio of the acetonitrile solution of acetic anhydride to the mass ratio of the compound of formula (321) is 0.5ml/g-10ml/g, and in some embodiments, 1ml/g-5ml/g.
在一些实施方案中，盖帽试剂使用等摩尔量的乙酸酐与N-甲基咪唑。所述有机溶剂为乙腈、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种。在一些实施方案中，所述有机溶剂为乙腈。相对于式(321)化合物，所述有机溶剂的用量为10-50L/mol，在一些实施方案中为5-30L/mol。In some embodiments, the capping agent uses equimolar amounts of acetic anhydride and N-methylimidazole. The organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, halogenated alkane solvents, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In some embodiments, the organic solvent is acetonitrile. Relative to the compound of formula (321), the amount of the organic solvent is 10-50 L/mol, and in some embodiments, 5-30 L/mol.
所述酰化催化剂可以选自任何可用于成酯缩合或成酰胺缩合的催化剂，例如碱性杂环化合物。在一些实施方案中，所述酰化催化剂为4-二甲氨基吡啶。所述催化剂与式(321)所示的化合物的质量之比为0.001∶1-1∶1，在一些实施方案中为0.01∶1-0.1∶1。The acylation catalyst can be selected from any catalyst that can be used for esterification or amide condensation, such as a basic heterocyclic compound. In some embodiments, the acylation catalyst is 4-dimethylaminopyridine. The mass ratio of the catalyst to the compound represented by formula (321) is 0.001:1-1:1, and in some embodiments, 0.01:1-0.1:1.
可使用任何合适的分离方法从反应混合物中分离式(321)化合物。在一些实施方案中，可通过以有机溶剂充分洗涤，并过滤，去除未反应的反应物、过量的盖帽试剂及其它杂质，得到式(321)化合物，所述有机溶剂选自乙腈、二氯甲烷、甲醇，在一些实施方案中为乙腈。Any suitable separation method can be used to separate the compound of formula (321) from the reaction mixture. In some embodiments, the compound of formula (321) can be obtained by washing with an organic solvent and filtering to remove unreacted reactants, excess capping reagents and other impurities, wherein the organic solvent is selected from acetonitrile, dichloromethane, methanol, and in some embodiments, acetonitrile.
在一些实施方案中，式(321)所示缀合分子的制备方法包括在有机溶剂中，在偶联反应条件下，以及在偶联试剂存在下，将式(313)所示的化合物与亚磷酰二胺接触，分离得到式(321)所示的化合物。此时，所获得的是R4中含有第1官能团和第2官能团，第1官能团含有羟基保护基团，第2官能团含有如式(C3)所示结构的式(321)化合物。In some embodiments, the preparation method of the conjugated molecule shown in formula (321) includes contacting the compound shown in formula (313) with phosphorodiamidite in an organic solvent under coupling reaction conditions and in the presence of a coupling reagent, and separating to obtain the compound shown in formula (321). At this time, what is obtained is a compound of formula (321) in whichR4 contains the first functional group and the second functional group, the first functional group contains a hydroxyl protecting group, and the second functional group contains a structure as shown in formula (C3).
在一些实施方案中，偶联反应条件包括温度为0-50℃，例如为15-35℃，式(313)化合物与亚磷酰二胺的摩尔比可以为1∶1-1∶50，例如为1∶5-1∶15；式(313)化合物和偶联试剂的摩尔比可以为1∶1-1∶100，例如为1∶50-1∶80；反应时间可以为200-3000秒，例如为500-1500秒。所述亚磷酰二胺例如可使用双(二异丙基氨基)(2-氰基乙氧基)膦，其可商购获得或按照本领域中公知的方法合成获得。偶联试剂选自1H-四氮唑、5-乙硫基1H-四氮唑、5-苄硫基1H-四氮唑中的一种或多种，例如为5-乙硫基1H-四氮唑。所述偶联反应可在有机溶剂中进行，所述有机溶剂选自无水乙腈、无水DMF、无水二氯甲烷中的一种或多种，例如为无水乙腈。在一些实施方案中，相对于式(313)化合物，所述有机溶剂的用量为3-50L/mol，例如可以为5-20L/mol。通过进行该偶联反应，式(313)化合物中的羟基与亚磷酰二胺反应形成亚磷酰胺基团。在一些实施方案中，可以直接除去溶剂得到式(321)化合物粗产品，该粗产品可以直接用于后续反应。In some embodiments, the coupling reaction conditions include a temperature of 0-50°C, such as 15-35°C, a molar ratio of the compound of formula (313) to the phosphorodiamidite of 1:1-1:50, such as 1:5-1:15; a molar ratio of the compound of formula (313) to the coupling reagent of 1:1-1:100, such as 1:50-1:80; and a reaction time of 200-3000 seconds, such as 500-1500 seconds. The phosphorodiamidite can be, for example, bis(diisopropylamino)(2-cyanoethoxy)phosphine, which can be commercially available or synthesized according to methods known in the art. The coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, and 5-benzylthio 1H-tetrazole, such as 5-ethylthio 1H-tetrazole. The coupling reaction can be carried out in an organic solvent, and the organic solvent is selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, such as anhydrous acetonitrile. In some embodiments, the amount of the organic solvent is 3-50 L/mol, for example, 5-20 L/mol, relative to the compound of formula (313). By carrying out the coupling reaction, the hydroxyl group in the compound of formula (313) reacts with the phosphorodiamidite to form a phosphoramidite group. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (321), which can be directly used in subsequent reactions.
在一些实施方案中，式(321)化合物的制备方法还进一步包括以下步骤：在偶联反应条件下，在有机溶剂中，以及在偶联试剂存在下，将分离得到的产物进一步与含有羟基的固相载体进行接触。随后，经盖帽反应、氧化反应，分离得到式(321)化合物。此时，所获得的是R4中含有第1官能团和第2官能团，第1官能团含有羟基保护基团，第2官能团具有如式(C3’)所示结构的式(321)化合物。In some embodiments, the preparation method of the compound of formula (321) further comprises the following steps: under coupling reaction conditions, in an organic solvent, and in the presence of a coupling agent, the separated product is further contacted with a solid phase carrier containing a hydroxyl group. Subsequently, the compound of formula (321) is separated through a capping reaction and an oxidation reaction. At this time, what is obtained is a compound of formula (321) in whichR4 contains a first functional group and a second functional group, the first functional group contains a hydroxyl protecting group, and the second functional group has a structure as shown in formula (C3').
在一些实施方案中，所述固相载体为本领域中公知的可用于核酸固相合成的固相载体，例如，可以是经脱保护反应后的市售的通用固相载体(UnyLinkerTM300 Oligonucleotide Synthesis Support，Kinovate Life Sciences公司，结构如式B80所示)：In some embodiments, the solid phase carrier is a solid phase carrier known in the art that can be used for solid phase synthesis of nucleic acids, for example, it can be a commercially available general solid phase carrier ( UnyLinkerTM 300 Oligonucleotide Synthesis Support, Kinovate Life Sciences, the structure is shown in formula B80):
脱保护反应为本领域技术人员所公知。在一些实施方案中，脱保护条件包括温度为0-50℃，例如为15-35℃；反应时间为30-300秒，例如为50-150秒。脱保护试剂可以选自三氟乙酸、三氯乙酸、二氯乙酸、一氯乙酸中的一种或多种，在一些实施方案中，脱保护试剂为二氯乙酸。脱保护试剂与固定相上的-DMTr(4，4′-二甲氧基三苯甲基)保护基的摩尔比为2∶1-100∶1，例如为3∶1-50∶1。通过进行所述脱保护，在所述固相载体表面上获得具有反应活性的游离羟基，便于进行下一步的偶联反应。Deprotection reaction is well known to those skilled in the art. In some embodiments, the deprotection conditions include a temperature of 0-50°C, for example, 15-35°C; a reaction time of 30-300 seconds, for example, 50-150 seconds. The deprotection reagent can be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid. In some embodiments, the deprotection reagent is dichloroacetic acid. The molar ratio of the deprotection reagent to the -DMTr (4,4'-dimethoxytrityl) protecting group on the stationary phase is 2:1-100:1, for example, 3:1-50:1. By performing the deprotection, reactive free hydroxyl groups are obtained on the surface of the solid phase carrier, which is convenient for the next coupling reaction.
偶联反应条件以及偶联试剂的选择可如上所述。通过进行该偶联反应，脱保护反应中形成的游离羟基与亚磷酰胺基团反应形成亚磷酸酯连接。The coupling reaction conditions and the selection of coupling reagents can be as described above.By carrying out the coupling reaction, the free hydroxyl group formed in the deprotection reaction reacts with the phosphoramidite group to form a phosphite linkage.
在一些实施方案中，盖帽反应条件包括温度为0-50℃，例如为15-35℃，反应时间为5-500秒，例如为10-100秒，所述盖帽反应在盖帽试剂存在下进行。盖帽试剂的选择和用量可如上所述。In some embodiments, the capping reaction conditions include a temperature of 0-50°C, such as 15-35°C, a reaction time of 5-500 seconds, such as 10-100 seconds, and the capping reaction is performed in the presence of a capping agent. The selection and dosage of the capping agent can be as described above.
氧化反应条件包括温度为0-50℃，例如可以为15-35℃，反应时间为1-100秒，例如可以为5-50秒，氧化试剂例如可以为碘(在一些实施方案中，以碘水的形式提供)。在一些实施方案中，氧化试剂与亚磷酸酯基团的摩尔比为1∶1-100∶1，例如可以为5∶1-50∶1。在一些实施方案中，所述氧化反应在四氢呋喃∶水∶吡啶＝3∶1∶1-1∶1∶3的混合溶剂中进行。The oxidation reaction conditions include a temperature of 0-50° C., such as 15-35° C., a reaction time of 1-100 seconds, such as 5-50 seconds, and an oxidizing agent, such as iodine (provided in the form of iodine water in some embodiments). In some embodiments, the molar ratio of the oxidizing agent to the phosphite group is 1:1-100:1, such as 5:1-50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran: water: pyridine = 3:1:1-1:1:3.
在一些实施方案中，R6为式B7或B8基团中的一种，In some embodiments, R6 is one of the groups of formula B7 or B8,
其中q2的定义如前所述，where q2 is defined as above,
此时，式(313)所示的化合物可以通过以下制备方法得到：在有机溶剂中，在成酰胺反应条件下，以及在成酰胺反应缩合剂和三级胺类有机碱存在下，将式(314)所示的化合物与式(A-1)所示的化合物或式(A-2)化合物接触，随后进行分离：At this time, the compound represented by formula (313) can be obtained by the following preparation method: in an organic solvent, under amide formation reaction conditions, and in the presence of an amide formation reaction condensation agent and a tertiary amine organic base, the compound represented by formula (314) is contacted with the compound represented by formula (A-1) or the compound represented by formula (A-2), and then separated:
其中，n1、n3、m1、m2、m3、R10、R11、R12、R13、R14、R15、L1、S1、q2和Rk各自的定义和可选择的范围如前所述。Wherein, the definitions and selectable ranges of n1, n3, m1, m2, m3,R10 ,R11 ,R12 ,R13 ,R14 ,R15 ,L1 ,S1 ,q2 andRk are as described above.
所述成酰胺反应条件可包括反应温度为0-100℃，反应时间为1-48小时，在一些实施方案中，所述成酰胺反应条件为反应温度为10-40℃，反应时间为2-16小时。The amide formation reaction conditions may include a reaction temperature of 0-100° C. and a reaction time of 1-48 hours. In some embodiments, the amide formation reaction conditions include a reaction temperature of 10-40° C. and a reaction time of 2-16 hours.
在一些实施方案中，所述有机溶剂为醇类溶剂、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种。所述醇类溶剂在一些实施方案中为甲醇、乙醇、丙醇中的一种或多种，在一些实施方案中为乙醇。所述环氧类溶剂在一些实施方案中为为二氧六环和/或四氢呋喃。所述醚类溶剂在一些实施方案中为为乙醚和/或甲基叔丁基醚。所述卤代烷类溶剂在一些实施方案中为为二氯甲烷、三氯甲烷和1，2-二氯乙烷中的一种或多种。在一些实施方案中，所述有机溶剂为二氯甲烷。相对于式(314)化合物，有机溶剂用量为3-50L/mol，在一些实施方案中为3-20L/mol。In some embodiments, the organic solvent is one or more of an alcohol solvent, an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N, N-dimethylformamide and N, N-diisopropylethylamine. The alcohol solvent is one or more of methanol, ethanol, and propanol in some embodiments, and ethanol in some embodiments. The epoxy solvent is dioxane and/or tetrahydrofuran in some embodiments. The ether solvent is ether and/or methyl tert-butyl ether in some embodiments. The halogenated alkane solvent is one or more of dichloromethane, trichloromethane and 1,2-dichloroethane in some embodiments. In some embodiments, the organic solvent is dichloromethane. Relative to the compound of formula (314), the amount of organic solvent used is 3-50L/mol, and in some embodiments, 3-20L/mol.
在一些实施方案中，所述成酰胺反应缩合剂为六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮、4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐、2-乙氧基-1-乙氧碳酰基-1，2-二氢喹啉(EEDQ)或O-苯并三氮唑-四甲基脲六氟磷酸酯，在进一步的实施方案中为3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮。所述成酰胺反应缩合剂与式(314)所示的化合物的摩尔比可以为1∶1-10∶1，在一些实施方案中为2.5∶1-5∶1。In some embodiments, the amide forming reaction condensation agent is benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-benzoxazole 4 (3H)-one, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) or O-benzotriazole-tetramethyluronium hexafluorophosphate, and in a further embodiment, 3-diethoxyphosphoryl-1,2,3-benzoxazole 4 (3H)-one. The molar ratio of the amide forming reaction condensation agent to the compound represented by formula (314) can be 1:1-10:1, and in some embodiments, 2.5:1-5:1.
在一些实施方案中，所述三级胺类有机碱为三乙胺或N，N-二异丙基乙胺，在一些实施方案中为N，N-二异丙基乙胺。所述三级胺类有机碱与式(314)所示的化合物的摩尔比为3∶1-20∶1，在一些实施方案中为5∶1-10∶1。In some embodiments, the tertiary amine organic base is triethylamine or N, N-diisopropylethylamine, and in some embodiments, it is N, N-diisopropylethylamine. The molar ratio of the tertiary amine organic base to the compound represented by formula (314) is 3:1-20:1, and in some embodiments, it is 5:1-10:1.
式(A-1)和式(A-2)化合物可通过任何适当的方式制备。例如，当Rk为DMTr基团时，可通过甘油酸钙与DMTrCl反应制备式(A-1)化合物；类似地，可先将3-氨基-1，2-丙二醇与环状酸酐接触，随后再与DMTrCl反应制备式(A-2)化合物，所述环状酸酐可以是碳原子数为4-13、在一些实施方案中为4-8的环状酸酐。本领域技术人员容易理解的是，所述环状酸酐的选择对应于(A-2)化合物中q2的不同值，例如，当所述环状酸酐为丁二酸酐时，q2＝1，当所述环状酸酐为戊二酸酐时，q2＝2，以此类推。The compounds of formula (A-1) and formula (A-2) can be prepared by any suitable means. For example, when Rk is a DMTr group, the compound of formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl; similarly, 3-amino-1,2-propanediol can be first contacted with a cyclic anhydride and then reacted with DMTrCl to prepare the compound of formula (A-2), wherein the cyclic anhydride can be a cyclic anhydride having 4-13 carbon atoms, and in some embodiments, 4-8 carbon atoms. It is easy for a person skilled in the art to understand that the selection of the cyclic anhydride corresponds to different values of q2 in the compound (A-2), for example, when the cyclic anhydride is succinic anhydride, q2 =1, when the cyclic anhydride is glutaric anhydride, q2 =2, and so on.
在一些变型中，也可通过使式(314)所示的化合物依次与所述环状酸酐、3-氨基-1，2-丙二醇和DMTrCl反应，制备式(313)化合物。本领域技术人员容易理解的是，这些变型不会影响式(313)化合物的结构与功能，并且这些变型是本领域技术人员在上述方法的基础上容易实现的。In some variations, the compound of formula (313) can also be prepared by reacting the compound of formula (314) with the cyclic anhydride, 3-amino-1,2-propylene glycol and DMTrCl in sequence. It is easy for a person skilled in the art to understand that these variations will not affect the structure and function of the compound of formula (313), and these variations are easy to implement by a person skilled in the art based on the above method.
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(313)化合物。在一些实施方案中，可通过蒸发除去溶剂、随后通过色谱方法分离式(313)化合物，例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用石油醚∶乙酸乙酯∶二氯甲烷∶N，N-二甲基甲酰胺＝1∶1∶1∶0.5-1∶1∶1∶0.6梯度洗脱；以及(2)反相纯化：C18、C8反相填料，使用甲醇∶乙腈＝0.1∶1-1∶0.1梯度洗脱。在一些实施方案中，可以直接除去溶剂得到式(313)化合物粗产品，该粗产品可以直接用于后续反应。Similar to the above, any suitable separation method can be used to separate the compound of formula (313) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two chromatographic conditions can be used for separation: (1) normal phase purification silica gel: 200-300 mesh silica gel filler, using petroleum ether: ethyl acetate: dichloromethane: N, N-dimethylformamide = 1:1:1:0.5-1:1:1:0.6 gradient elution; and (2) reverse phase purification: C18, C8 reverse phase filler, using methanol: acetonitrile = 0.1:1-1:0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (313), which can be directly used in subsequent reactions.
在一些实施方案中，式(314)所示的化合物可以通过以下制备方法得到：该方法包括在有机溶剂中，在脱保护反应条件下，将式(315)所示的化合物与卤代乙酸接触，随后进行分离：In some embodiments, the compound represented by formula (314) can be obtained by the following preparation method: the method comprises contacting the compound represented by formula (315) with a haloacetic acid in an organic solvent under deprotection reaction conditions, followed by separation:
其中，R7选自式(330)、(331)、(332)或(333)所示的基团，在一些实施方案中，R7的结构如式(330)所示：Wherein,R7 is selected from the group represented by formula (330), (331), (332) or (333). In some embodiments, the structure ofR7 is as represented by formula (330):
n1、n3、m1、m2、m3、R10、R11、R12、R13、R14、R15、L1、S1各自的定义和可选择的范围如前所述。The definitions and selectable ranges of n1, n3, m1, m2, m3, R10 , R11 , R12 , R13 , R14 , R15 , L1 and S1 are as described above.
所述卤代乙酸可选自二氯乙酸、三氯乙酸、一氯乙酸和三氟乙酸中的一种或多种，在一些实施方案中为二氯乙酸。The halogenated acetic acid may be selected from one or more of dichloroacetic acid, trichloroacetic acid, monochloroacetic acid and trifluoroacetic acid, and in some embodiments is dichloroacetic acid.
所述脱保护反应条件包括反应温度为0-100℃，反应时间为0.1-24小时，在一些实施方案中为反应温度为10-40℃，反应时间为0.5-16小时。The deprotection reaction conditions include a reaction temperature of 0-100° C. and a reaction time of 0.1-24 hours. In some embodiments, the reaction temperature is 10-40° C. and the reaction time is 0.5-16 hours.
在一些实施方案中，所述有机溶剂为环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种。所述环氧类溶剂在一些实施方案中为二氧六环和/或四氢呋喃，所述醚类溶剂在一些实施方案中为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂在一些实施方案中为二氯甲烷、三氯甲烷和1，2-二氯乙烷中的一种或多种，在一些实施方案中，所述有机溶剂为二氯甲烷。相对于式(315)化合物，有机溶剂用量为3-50L/mol，在一些实施方案中为5-20L/mol。In some embodiments, the organic solvent is one or more of an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. The epoxy solvent is dioxane and/or tetrahydrofuran in some embodiments, the ether solvent is ether and/or methyl tert-butyl ether in some embodiments, the halogenated alkane solvent is one or more of dichloromethane, trichloromethane and 1,2-dichloroethane in some embodiments, and the organic solvent is dichloromethane. Relative to the compound of formula (315), the amount of organic solvent used is 3-50 L/mol, and in some embodiments is 5-20 L/mol.
所述卤代乙酸与所述式(315)所示的化合物的摩尔比为5∶1-100∶1，在一些实施方案中为10∶1-50∶1。The molar ratio of the halogenated acetic acid to the compound of formula (315) is 5:1-100:1, and in some embodiments, 10:1-50:1.
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(314)化合物。在一些实施方案中，可通过蒸发除去溶剂、随后通过色谱方法分离式(314)化合物，例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用二氯甲烷∶甲醇＝100∶30-100∶40梯度洗脱；以及(2)反相纯化：C18、C8反相填料，使用甲醇∶乙腈＝0.1∶1-1∶0.1梯度洗脱。在一些实施方案中，可以直接除去溶剂得到式(314)化合物粗产品，该粗产品可以直接用于后续反应。Similar to the above, any suitable separation method can be used to separate the compound of formula (314) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two chromatographic conditions can be used for separation: (1) normal phase purification silica gel: 200-300 mesh silica gel filler, using dichloromethane: methanol = 100: 30-100: 40 gradient elution; and (2) reverse phase purification: C18, C8 reverse phase filler, using methanol: acetonitrile = 0.1: 1-1: 0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (314), which can be directly used in subsequent reactions.
式(315)所示的化合物可以通过以下制备方法得到：该方法包括在有机溶剂中，在成酰胺反应缩合剂和三级胺类有机碱存在下，在缩合反应条件下，将式(317)所示的化合物与式(316)所示的化合物接触，随后进行分离：The compound represented by formula (315) can be obtained by the following preparation method: the method comprises contacting the compound represented by formula (317) with the compound represented by formula (316) in an organic solvent in the presence of an amide-forming reaction condensation agent and a tertiary amine organic base under condensation reaction conditions, and then separating:
其中，n1、n3、m1、m2、m3、R7、R10、R11、R12、R13、R14、R15、L1、S1各自的定义和可选择的范围如前所述。Here, n1, n3, m1, m2, m3, R7 , R10 , R11 , R12 , R13 , R14 , R15 , L1 , and S1 have the same definitions and selectable ranges as described above.
式(316)化合物可使用例如J.Am.Chem.Soc.2014，136，16958-16961中所公开的化合物，或者，式(316)化合物可由本领域技术人员通过各种方法制备，例如，可参照美国专利US8106022B2实施例1中所公开的方法制备某些式(316)化合物，以引用的方式将以上文献的全部内容整体并入本文。The compound of formula (316) can be prepared, for example, using the compounds disclosed in J.Am.Chem.Soc.2014, 136, 16958-16961, or the compound of formula (316) can be prepared by a person skilled in the art by various methods. For example, certain compounds of formula (316) can be prepared by referring to the method disclosed in Example 1 of U.S. Patent No. 8106022B2. The entire contents of the above documents are incorporated herein by reference in their entirety.
在一些实施方案中，所述缩合反应条件包括反应温度为0-100℃，反应时间为0.1-24小时，在一些实施方案中为反应温度为10-40℃，反应时间为0.5-16小时。In some embodiments, the condensation reaction conditions include a reaction temperature of 0-100° C. and a reaction time of 0.1-24 hours. In some embodiments, the reaction temperature is 10-40° C. and the reaction time is 0.5-16 hours.
所述式(316)所示的化合物与所述式(317)所示的化合物的摩尔比可以为2∶1-10∶1，在一些实施方案中为2.5∶1-5∶1。The molar ratio of the compound represented by formula (316) to the compound represented by formula (317) can be 2:1-10:1, and in some embodiments, 2.5:1-5:1.
在一些实施方案中，所述有机溶剂为乙腈、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种，所述环氧类溶剂在一些实施方案中为二氧六环和/或四氢呋喃，所述醚类溶剂在一些实施方案中为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂在一些实施方案中为二氯甲烷、三氯甲烷和1，2-二氯乙烷中的一种或多种，在一些实施方案中，所述有机溶剂为乙腈。相对于式(317)化合物，所述有机溶剂的用量为3-50L/mol，在一些实施方案中为5-20L/mol。In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, halogenated alkane solvents, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine, the epoxy solvent is dioxane and/or tetrahydrofuran in some embodiments, the ether solvent is ether and/or methyl tert-butyl ether in some embodiments, the halogenated alkane solvent is one or more of dichloromethane, trichloromethane and 1,2-dichloroethane in some embodiments, and the organic solvent is acetonitrile. Relative to the compound of formula (317), the amount of the organic solvent is 3-50 L/mol, and in some embodiments is 5-20 L/mol.
所述成酰胺反应缩合剂为六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT)、O-苯并三氮唑-四甲基脲六氟磷酸酯或4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐，在一些实施方案中为4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐。所述成酰胺反应缩合剂与式(317)所示的化合物的摩尔比为2∶1-10∶1，在一些实施方案中为2.5∶1-5∶1。The amide forming reaction condensation agent is benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-benzoxazole 4(3H)-one (DEPBT), O-benzotriazole-tetramethyluronium hexafluorophosphate or 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, and in some embodiments, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride. The molar ratio of the amide forming reaction condensation agent to the compound shown in formula (317) is 2:1-10:1, and in some embodiments, it is 2.5:1-5:1.
所述三级胺类有机碱为N-甲基吗啉、三乙胺或N，N-二异丙基乙胺，在一些实施方案中为N-甲基吗啉；所述三级胺类有机碱与式(317)所示的化合物的摩尔比为3∶1-20∶1，在一些实施方案中为5∶1-10∶1。The tertiary amine organic base is N-methylmorpholine, triethylamine or N,N-diisopropylethylamine, and in some embodiments it is N-methylmorpholine; the molar ratio of the tertiary amine organic base to the compound represented by formula (317) is 3:1-20:1, and in some embodiments it is 5:1-10:1.
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(315)化合物。在一些实施方案中，可通过蒸发除去溶剂、随后通过色谱方法分离式(315)化合物例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用二氯甲烷∶甲醇＝100∶5-100∶7梯度洗脱；以及(2)反相纯化：C18、C8反相填料，使用甲醇∶乙腈＝0.1∶1-1∶0.1梯度洗脱。在一些实施方案中，可以直接除去溶剂得到式(315)化合物粗产品，该粗产品可以直接用于后续反应。Similar to the above, any suitable separation method can be used to separate the compound of formula (315) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two chromatographic conditions can be used for separation: (1) normal phase purification silica gel: 200-300 mesh silica gel filler, using dichloromethane: methanol = 100: 5-100: 7 gradient elution; and (2) reverse phase purification: C18, C8 reverse phase filler, using methanol: acetonitrile = 0.1: 1-1: 0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (315), which can be directly used in subsequent reactions.
在一些实施方案中，式(317)化合物与足量的一种式(316)化合物一次反应即生成期望的式(315)化合物，此时，各个S1-L1部分彼此相同。在一些实施方案中，可根据需要，通过使式(317)化合物分批与不同的式(316)化合物，即L1和/或S1不同的式(316)化合物发生反应，使得生成的式(315)化合物中含有两种以上的S1和/或L1。例如，对于1eq的式(317)化合物，可先使其与2eq的第一式(316)化合物接触，在式(317)化合物中的两个末端伯胺基团上连接第一S1-L1部分，随后，使其继续与(n3+n1-1)eq的第二式(316)化合物接触(n3和n1的定义和取值范围如前所述)，在式(317)化合物中的(n3+n1-1)个仲胺基团上连接第二S1-L1部分。In some embodiments, the compound of formula (317) reacts with a sufficient amount of a compound of formula (316) once to generate the desired compound of formula (315), in which case the variousS1 -L1 moieties are identical to each other. In some embodiments, the compound of formula (317) can be reacted in batches with different compounds of formula (316), i.e., compounds of formula (316) with differentL1 and/orS1 , so that the generated compound of formula (315) contains two or moreS1 and/orL1, as required. For example, for 1 eq of the compound of formula (317), it can be first contacted with 2 eq of the first compound of formula (316), and the firstS1 -L1 moiety can be connected to the two terminal primary amine groups in the compound of formula (317). Subsequently, it can be contacted with (n3+n1-1) eq of the second compound of formula (316) (the definition and value range of n3 and n1 are as described above), and the secondS1 -L1 moiety can be connected to the (n3+n1-1) secondary amine groups in the compound of formula (317).
在一些实施方案中，式(317)所示的化合物可以通过以下制备方法得到：该方法包括在有机溶剂存在下，在脱保护反应条件下，将式(318)所示的化合物与甲胺水溶液接触，随后进行分离：In some embodiments, the compound represented by formula (317) can be obtained by the following preparation method: the method comprises contacting the compound represented by formula (318) with a methylamine aqueous solution in the presence of an organic solvent under deprotection reaction conditions, followed by separation:
其中，n1、n3、m1、m2、m3、R7、R10、R11、R12、R13、R14、R15各自的定义和可选择的范围如前所述。Here, the definitions and selectable ranges of n1, n3, m1, m2, m3, R7 , R10 , R11 , R12 , R13 , R14 and R15 are as described above.
所述脱保护反应条件包括反应温度为0-150℃，反应时间为5-72小时，在一些实施方案中为反应温度为20-80℃，反应时间为10-30小时。The deprotection reaction conditions include a reaction temperature of 0-150° C. and a reaction time of 5-72 hours. In some embodiments, the reaction temperature is 20-80° C. and the reaction time is 10-30 hours.
所述有机溶剂选自醇，在一些实施方案中为甲醇、乙醇和异丙醇中的一种，在一些实施方案中为甲醇；相对于式(318)化合物，所述有机溶剂的用量为1-20L/mol，在一些实施方案中为1.5-10L/mol。The organic solvent is selected from alcohol, in some embodiments, it is one of methanol, ethanol and isopropanol, in some embodiments, it is methanol; relative to the compound of formula (318), the amount of the organic solvent used is 1-20 L/mol, in some embodiments, it is 1.5-10 L/mol.
所述甲胺水溶液的浓度可以为30-40质量％，甲胺与式(318)所示的化合物的摩尔比可以为10∶1-500∶1，在一些实施方案中为50∶1-200∶1。The concentration of the methylamine aqueous solution may be 30-40% by mass, and the molar ratio of methylamine to the compound represented by formula (318) may be 10:1-500:1, and in some embodiments, 50:1-200:1.
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(317)化合物。在一些实施方案中，可通过蒸发除去溶剂、随后通过色谱方法分离式(317)化合物例如，可使用如下两种色谱条件进行分离：正相纯化硅胶：(1)200-300目硅胶填料，使用二氯甲烷∶甲醇∶氨水(25wt％)＝1∶1∶0.05-1∶1∶0.25梯度洗脱；以及(2)反相纯化：C18、C8反相填料，使用甲醇∶乙腈＝0.1∶1-1∶0.1梯度洗脱。在一些实施方案中，可以直接除去溶剂得到式(317)化合物粗产品，该粗产品可以直接用于后续反应。Similar to the above, any suitable separation method can be used to separate the compound of formula (317) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two chromatographic conditions can be used for separation: normal phase purification silica gel: (1) 200-300 mesh silica gel filler, using dichloromethane: methanol: ammonia water (25wt%) = 1: 1: 0.05-1: 1: 0.25 gradient elution; and (2) reverse phase purification: C18, C8 reverse phase filler, using methanol: acetonitrile = 0.1: 1-1: 0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (317), which can be directly used in subsequent reactions.
在一些实施方案中，式(318)所示的化合物可以通过以下制备方法得到：该方法包括在有机溶剂存在下，在取代反应条件下，将式(319)所示的化合物与三苯基氯甲烷(TrCl)、二苯基乙苯基氯甲烷、苯基二乙苯基氯甲烷或三乙苯基氯甲烷、在一些实施方案中为三苯基氯甲烷(TrCl)接触，随后进行分离：In some embodiments, the compound represented by formula (318) can be obtained by the following preparation method: the method comprises contacting the compound represented by formula (319) with triphenylmethane (TrCl), diphenylethylphenylmethane, phenyldiethylphenylmethane or triethylphenylmethane, in some embodiments, triphenylmethane (TrCl) in the presence of an organic solvent under substitution reaction conditions, and then separating:
其中，n1、n3、m1、m2、m3、R10、R11、R12、R13、R14、R15各自的定义和可选择的范围如前所述。Here, the definitions and selectable ranges of n1, n3, m1, m2, m3, R10 , R11 , R12 , R13 , R14 and R15 are as described above.
所述取代反应条件可包含反应温度为0-100℃，反应时间为5-72小时，在一些实施方案中，反应条件包含反应温度为10-40℃，反应时间为10-30小时。The substitution reaction conditions may include a reaction temperature of 0-100° C. and a reaction time of 5-72 hours. In some embodiments, the reaction conditions include a reaction temperature of 10-40° C. and a reaction time of 10-30 hours.
三苯基氯甲烷(TrCl)、二苯基乙苯基氯甲烷、苯基二乙苯基氯甲烷或三乙苯基氯甲烷可商购得到，三苯基氯甲烷(TrCl)、二苯基乙苯基氯甲烷、苯基二乙苯基氯甲烷或三乙苯基氯甲烷与式(319)所示的化合物的摩尔比可以为1∶1-10∶1，在一些实施方案中为1∶1-3∶1。Triphenylmethane chloride (TrCl), diphenylethylphenylmethane chloride, phenyldiethylphenylmethane chloride or triethylphenylmethane chloride is commercially available, and the molar ratio of triphenylmethane chloride (TrCl), diphenylethylphenylmethane chloride, phenyldiethylphenylmethane chloride or triethylphenylmethane chloride to the compound represented by formula (319) can be 1:1-10:1, and in some embodiments, 1:1-3:1.
所述有机溶剂可以为环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种。所述环氧类溶剂在一些实施方案中为二氧六环和/或四氢呋喃，所述醚类溶剂在一些实施方案中为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂在一些实施方案中为二氯甲烷、三氯甲烷和1，2-二氯乙烷中的一种或多种；在一些实施方案中，所述有机溶剂为二氯甲烷。相对于式(319)化合物，所述有机溶剂的用量可以为3-50L/mol，在一些实施方案中为5-20L/mol。The organic solvent may be one or more of an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran, in some embodiments, the ether solvent is ether and/or methyl tert-butyl ether, in some embodiments, the halogenated alkane solvent is one or more of dichloromethane, trichloromethane and 1,2-dichloroethane; in some embodiments, the organic solvent is dichloromethane. Relative to the compound of formula (319), the amount of the organic solvent may be 3-50 L/mol, in some embodiments, 5-20 L/mol.
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(318)化合物。在一些实施方案中，可通过蒸发除去溶剂、随后通过色谱方法分离式(318)化合物例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用甲醇∶二氯甲烷＝0.01∶1-0.5∶1梯度洗脱；或者使用甲醇∶二氯甲烷∶乙酸乙酯∶石油醚＝0.1∶1∶1∶1-1∶1∶1∶1梯度洗脱。以及(2)反相纯化：C18、C8反相填料，使用甲醇∶乙腈＝0.1∶1-1∶0.1梯度洗脱。在一些实施方案中，可以直接除去溶剂得到式(318)化合物粗产品，该粗产品可以直接用于后续反应。Similar to the above, any suitable separation method can be used to separate the compound of formula (318) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two chromatographic conditions can be used for separation: (1) Normal phase purification silica gel: 200-300 mesh silica gel filler, using methanol: dichloromethane = 0.01: 1-0.5: 1 gradient elution; or using methanol: dichloromethane: ethyl acetate: petroleum ether = 0.1: 1: 1: 1-1: 1: 1 gradient elution. And (2) Reverse phase purification: C18, C8 reverse phase filler, using methanol: acetonitrile = 0.1: 1-1: 0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (318), which can be directly used in subsequent reactions.
在一些实施方案中，式(319)所示的化合物可以通过以下制备方法得到：该方法包括在有机溶剂中，在取代反应条件下，将式(320)所示的化合物与三氟乙酸乙酯接触，随后进行分离：In some embodiments, the compound represented by formula (319) can be obtained by the following preparation method: the method comprises contacting the compound represented by formula (320) with ethyl trifluoroacetate in an organic solvent under substitution reaction conditions, and then separating:
其中，n1、n3、m1、m2、m3、R10、R11、R12、R13、R14、R15各自的定义和可选择的范围如前所述。Here, the definitions and selectable ranges of n1, n3, m1, m2, m3, R10 , R11 , R12 , R13 , R14 and R15 are as described above.
在一些实施方案中，所述有机溶剂为乙腈、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种。在一些实施方案中，所述环氧类溶剂为二氧六环和/或四氢呋喃，在一些实施方案中，所述醚类溶剂为乙醚和/或甲基叔丁基醚，在一些实施方案中，所述卤代烷类溶剂为二氯甲烷、三氯甲烷和1，2-二氯乙烷中的一种或多种，在一些实施方案中，所述有机溶剂为乙腈。相对于式(320)化合物，所述有机溶剂的用量为1-50L/mol，在一些实施方案中为1-20L/mol。In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, halogenated alkane solvents, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran, in some embodiments, the ether solvent is ether and/or methyl tert-butyl ether, in some embodiments, the halogenated alkane solvent is one or more of dichloromethane, trichloromethane and 1,2-dichloroethane, in some embodiments, the organic solvent is acetonitrile. Relative to the compound of formula (320), the amount of the organic solvent is 1-50 L/mol, in some embodiments, 1-20 L/mol.
所述取代反应条件可包括反应温度为0-100℃，反应时间为5-72小时，在一些实施方案中，所述取代反应条件包括为反应温度为10-40℃，反应时间为10-30小时。The substitution reaction conditions may include a reaction temperature of 0-100° C. and a reaction time of 5-72 hours. In some embodiments, the substitution reaction conditions include a reaction temperature of 10-40° C. and a reaction time of 10-30 hours.
式(320)化合物可商购获得，或者由本领域技术人员使用已知的方法获得。例如，当m1＝m2＝m3＝3，n1＝1，n3＝2，且R10、R11、R12、R13、R14、R15均为H时，式(320)化合物可自阿法埃莎公司商购获得。The compound of formula (320) is commercially available or can be obtained by a person skilled in the art using known methods. For example, when m1=m2=m3=3, n1=1, n3=2, andR10 ,R11 ,R12 ,R13 ,R14 , andR15 are all H, the compound of formula (320) can be commercially available from Alfa Aesar.
所述三氟乙酸乙酯与式(320)所示的化合物的摩尔比为2∶1-10∶1，在一些实施方案中为3∶1-5∶1。The molar ratio of ethyl trifluoroacetate to the compound of formula (320) is 2:1-10:1, and in some embodiments, 3:1-5:1.
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(319)化合物。在一些实施方案中，可通过蒸发除去溶剂、随后通过色谱方法分离式(319)化合物例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用甲醇∶二氯甲烷＝0.01∶1-0.5∶1梯度洗脱；或者使用甲醇∶二氯甲烷∶乙酸乙酯∶石油醚＝0.1∶1∶1∶1-1∶1∶1∶1梯度洗脱，以及(2)反相纯化：C18、C8反相填料，使用甲醇∶乙腈＝0.1∶1-1∶0.1梯度洗脱。在一些实施方案中，可以直接除去溶剂得到式(319)化合物粗产品，该粗产品可以直接用于后续反应。Similar to the above, any suitable separation method can be used to separate the compound of formula (319) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two chromatographic conditions can be used for separation: (1) normal phase purification silica gel: 200-300 mesh silica gel filler, using methanol: dichloromethane = 0.01: 1-0.5: 1 gradient elution; or using methanol: dichloromethane: ethyl acetate: petroleum ether = 0.1: 1: 1: 1-1: 1: 1: 1 gradient elution, and (2) reverse phase purification: C18, C8 reverse phase filler, using methanol: acetonitrile = 0.1: 1-1: 0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (319), which can be directly used in subsequent reactions.
本公开的第一种或第二种siRNA缀合物也可以与药学上可接受的其它辅料联用，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种，详情可参见上文关于本公开的药物组合物的描述。The first or second siRNA conjugate of the present disclosure may also be used in combination with other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds conventionally used in the art. For details, see the above description of the pharmaceutical composition of the present disclosure.
本公开的siRNA、含该siRNA的药物组合物、第一种siRNA缀合物以及第二种siRNAsiRNA, pharmaceutical composition containing the siRNA, first siRNA conjugate and second siRNA disclosed herein缀合物的应用Application of conjugates
在一些实施方案中，本公开提供了本公开提供的siRNA、药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物在制备用于治疗和/或预防由所述乙型肝炎病毒的感染引起的病理状况或疾病的药物中的用途。In some embodiments, the present disclosure provides use of the siRNA, pharmaceutical composition, first siRNA conjugate, and second siRNA conjugate provided by the present disclosure in the preparation of a medicament for treating and/or preventing a pathological condition or disease caused by infection with the hepatitis B virus.
按照本公开的一种实施方案，本公开提供了一种治疗乙型肝炎病毒的感染引起的病理状况或疾病的方法，该方法包括向患者给予本公开提供的siRNA、药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物。According to one embodiment of the present disclosure, the present disclosure provides a method for treating pathological conditions or diseases caused by infection with hepatitis B virus, the method comprising administering to a patient the siRNA, pharmaceutical composition, first siRNA conjugate and second siRNA conjugate provided by the present disclosure.
按照本公开另外一种实施方案，本公开提供了一种抑制感染慢性乙型肝炎病毒的肝炎细胞中乙型肝炎病毒基因表达的方法，该方法包括将本公开提供的siRNA、药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物与所述感染慢性乙型肝炎病毒的肝炎细胞接触。According to another embodiment of the present disclosure, the present disclosure provides a method for inhibiting the expression of hepatitis B virus genes in hepatitis cells infected with chronic hepatitis B virus, the method comprising contacting the hepatitis cells infected with chronic hepatitis B virus with the siRNA, pharmaceutical composition, first siRNA conjugate and second siRNA conjugate provided by the present disclosure.
所述由乙型肝炎病毒的感染引起的病理状况或疾病选自慢性肝病、肝炎、肝纤维化疾病和肝增生性疾病。The pathological condition or disease caused by infection with the hepatitis B virus is selected from chronic liver disease, hepatitis, liver fibrosis and liver proliferative disease.
通过将本公开的siRNA和/或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物给予有需要的患者，可以通过RNA干扰的机制达到治疗乙肝的目的。因此，本公开的siRNA和/或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物可用于预防和/或治疗乙肝、或用于制备用于预防和/或治疗乙肝的药物。By administering the siRNA and/or pharmaceutical composition, the first siRNA conjugate, and the second siRNA conjugate disclosed herein to a patient in need, the purpose of treating hepatitis B can be achieved through the mechanism of RNA interference. Therefore, the siRNA and/or pharmaceutical composition, the first siRNA conjugate, and the second siRNA conjugate disclosed herein can be used to prevent and/or treat hepatitis B, or to prepare a drug for preventing and/or treating hepatitis B.
本文所使用的术语“给药/给予”是指通过使得至少部分地将siRNA或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物定位于期望的位点以产生期望效果的方法或途径，将siRNA或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物放置入患者体内。适于本公开方法的给药途径包括局部给药和全身给药。一般而言，局部给药导致与患者整个身体相比将更多siRNA或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物递送至特定位点；而全身给药导致将所述siRNA或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物递送至患者的基本整个身体。考虑到本公开旨在提供预防和/或治疗乙肝的手段，优选能够将药物递送至肝脏的给药方式。The term "administration/administration" as used herein refers to placing siRNA or pharmaceutical composition, first siRNA conjugate and second siRNA conjugate into the patient's body by a method or approach that at least partially positions the siRNA or pharmaceutical composition, first siRNA conjugate and second siRNA conjugate at a desired site to produce a desired effect. Routes of administration suitable for the disclosed method include local administration and systemic administration. In general, local administration results in more siRNA or pharmaceutical composition, first siRNA conjugate and second siRNA conjugate being delivered to a specific site compared to the patient's entire body; while systemic administration results in the siRNA or pharmaceutical composition, first siRNA conjugate and second siRNA conjugate being delivered to essentially the entire body of the patient. Considering that the present disclosure is intended to provide a means for preventing and/or treating hepatitis B, a mode of administration that is capable of delivering the drug to the liver is preferred.
可通过本领域已知的任何合适途径向患者给药，所述途径包括但不仅限于：口服或胃肠外途径，包括静脉内给药、肌肉内给药、皮下给药、经皮给药、气道给药(气雾剂)、肺部给药、鼻部给药、直肠给药和局部给药(包括口腔含化给药和舌下给药)。给药频率可以是每天、每周、每两周、每三周、每个月或每年1次或多次。The drug can be administered to the patient by any suitable route known in the art, including but not limited to oral or parenteral routes, including intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including oral administration and sublingual administration). The frequency of administration can be once or more every day, every week, every two weeks, every three weeks, every month, or every year.
本公开所述的siRNA或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物的使用剂量可为本领域常规的剂量，所述剂量可以根据各种参数、尤其是患者的年龄、体重和性别来确定。可在细胞培养或实验动物中通过标准药学程序测定毒性和疗效，例如测定LD50(使50％的群体致死的剂量)和ED50(在量反应中指能引起50％最大反应强度的剂量，在质反应中指引起50％实验对象出现阳性反应时的剂量)。毒性和疗效之间的剂量比为治疗指数，可以用LD50/ED50的比值来表示。优选显示出高治疗指数的siRNA或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物。可基于由细胞培养分析和动物研究得到的数据得出人用剂量的范围。The dosage of the siRNA or pharmaceutical composition, the first siRNA conjugate, and the second siRNA conjugate disclosed in the present invention can be a conventional dosage in the art, and the dosage can be determined according to various parameters, especially the age, weight, and sex of the patient. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determining LD50 (the dose that causes 50% of the population to die) and ED50 (the dose that can cause 50% of the maximum reaction intensity in quantitative response, and the dose that causes 50% of the experimental subjects to have a positive reaction in qualitative response). The dose ratio between toxicity and efficacy is the therapeutic index, which can be expressed as the ratio of LD50/ED50. siRNA or pharmaceutical composition, the first siRNA conjugate, and the second siRNA conjugate that show a high therapeutic index are preferred. The range of human dosage can be derived based on data obtained from cell culture analysis and animal studies.
在给予本公开所述的药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物时，例如，对于雄性或雌性、6-12周龄、体重18-25g的C57BL/6J或C3H/HeNCrlVr小鼠，以所述药物组合物或siRNA缀合物中的siRNA的量计：(i)对于siRNA与药学上可接受的载体形成的药物组合物，其siRNA用量可以为0.001-50mg/kg体重，在进一步的实施方案中为0.01-10mg/kg体重，在更进一步的实施方案中为0.05-5mg/kg体重，在又进一步的实施方案中为0.1-3mg/kg体重；(ii)对于siRNA与药学上可接受的缀合分子形成的第一种和/或第二种siRNA缀合物，其siRNA用量可以为0.001-100mg/kg体重，在进一步的实施方案中为0.01-50mg/kg体重，在更进一步的实施方案中为0.05-20mg/kg体重，在又进一步的实施方案中为0.1-10mg/kg体重。在给予本公开所述的siRNA时，可优选上述用量。When administering the pharmaceutical composition, the first siRNA conjugate, and the second siRNA conjugate of the present disclosure, for example, for male or female C57BL/6J or C3H/HeNCrlVr mice aged 6-12 weeks and weighing 18-25 g, based on the amount of siRNA in the pharmaceutical composition or siRNA conjugate: (i) for a pharmaceutical composition formed by siRNA and a pharmaceutically acceptable carrier, the amount of siRNA can be 0.001-50 mg/kg body weight, and in a further embodiment, 0.01-10 mg/kg body weight; In a further embodiment, it is 0.05-5 mg/kg body weight, and in a further embodiment, it is 0.1-3 mg/kg body weight; (ii) for the first and/or second siRNA conjugates formed by siRNA and a pharmaceutically acceptable conjugate molecule, the siRNA dosage can be 0.001-100 mg/kg body weight, in a further embodiment, it is 0.01-50 mg/kg body weight, in a further embodiment, it is 0.05-20 mg/kg body weight, and in a further embodiment, it is 0.1-10 mg/kg body weight. When administering the siRNA described in the present disclosure, the above dosages may be preferred.
另外，通过将本公开的siRNA和/或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物导入感染慢性HBV的肝炎细胞，还可以通过RNA干扰的机制达到抑制感染慢性HBV的肝炎细胞中HBV基因的表达这一目的。在一些优选的实施方案中，所述细胞为HepG2.2.15细胞。In addition, by introducing the siRNA and/or pharmaceutical composition, the first siRNA conjugate and the second siRNA conjugate disclosed herein into hepatitis cells infected with chronic HBV, the purpose of inhibiting the expression of HBV genes in hepatitis cells infected with chronic HBV can also be achieved through the mechanism of RNA interference. In some preferred embodiments, the cells are HepG2.2.15 cells.
采用本公开提供的方法抑制HBV基因在细胞中表达，无论使用提供的siRNA还是药物组合物还是第一种siRNA缀合物以及第二种siRNA缀合物，siRNA用量一般是这样的量：其足以减少靶基因的表达，并导致在靶细胞表面处1pM至1μM、或0.01nM至100nM、或0.05nM至50nM或至约5nM的细胞外浓度。达到该局部浓度所需的量将随各种因素而变化，所述因素包括递送方法、递送部位、在递送部位和靶细胞或组织之间的细胞层的数目、递送是局部还是全身等。在递送部位处的浓度可以显著高于在靶细胞或组织的表面处的浓度。The method provided by the present disclosure is used to inhibit the expression of HBV genes in cells. Whether the siRNA provided is used, the pharmaceutical composition, the first siRNA conjugate, or the second siRNA conjugate, the amount of siRNA used is generally such an amount: it is sufficient to reduce the expression of the target gene and result in an extracellular concentration of 1pM to 1μM, or 0.01nM to 100nM, or 0.05nM to 50nM, or to about 5nM at the surface of the target cell. The amount required to achieve this local concentration will vary with various factors, including the delivery method, the delivery site, the number of cell layers between the delivery site and the target cell or tissue, whether the delivery is local or systemic, etc. The concentration at the delivery site can be significantly higher than the concentration at the surface of the target cell or tissue.
试剂盒Reagent test kit
本公开提供了一种试剂盒，该试剂盒含有有效量的本公开的siRNA、药物组合物、第一种siRNA缀合物和第二种siRNA缀合物中的至少一种。The present disclosure provides a kit containing an effective amount of at least one of the siRNA of the present disclosure, a pharmaceutical composition, a first siRNA conjugate, and a second siRNA conjugate.
在一些实施方案中，本文所述的试剂盒可在一个容器中提供修饰的siRNA。在一些实施方案中，本文所述的试剂盒可包含一个提供药学上可接受的赋形剂的容器。在一些实施方案中，所述试剂盒中还可包含其它成分，如稳定剂或防腐剂等。在一些实施方案中，本文所述的试剂盒可在不同于提供本文所述修饰的siRNA的容器以外的其它容器中包含至少一种其它治疗剂。在一些实施方案中，所述试剂盒可包含用于将修饰的siRNA与药学上可接受的载体和/或辅料或其它成分(若有的话)进行混合的说明书。In some embodiments, the kit described herein may provide the modified siRNA in one container. In some embodiments, the kit described herein may include a container providing a pharmaceutically acceptable excipient. In some embodiments, the kit may also include other ingredients, such as stabilizers or preservatives, etc. In some embodiments, the kit described herein may include at least one other therapeutic agent in other containers other than the container providing the modified siRNA described herein. In some embodiments, the kit may include instructions for mixing the modified siRNA with a pharmaceutically acceptable carrier and/or excipient or other ingredients (if any).
在本公开的试剂盒中，所述修饰的siRNA和药学上可接受的载体和/或辅料以及所述修饰的siRNA、药物组合物、第一种siRNA缀合物和/或第二种siRNA缀合物和/或缀合物，和/或药学上可接受的辅料可以任何形式提供，例如液体形式、干燥形式或冻干形式。在一些实施方案中，所述修饰的siRNA和药学上可接受的载体和/或辅料以及所述药物组合物和/或缀合物和任选的药学上可接受的辅料基本上纯净和/或无菌。在一些实施方案中，可在本公开的试剂盒中提供无菌水。In the kit of the present disclosure, the modified siRNA and pharmaceutically acceptable carrier and/or adjuvant and the modified siRNA, pharmaceutical composition, first siRNA conjugate and/or second siRNA conjugate and/or conjugate, and/or pharmaceutically acceptable adjuvant can be provided in any form, such as liquid form, dry form or lyophilized form. In some embodiments, the modified siRNA and pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or conjugate and optional pharmaceutically acceptable adjuvant are substantially pure and/or sterile. In some embodiments, sterile water can be provided in the kit of the present disclosure.
下面将通过实施例来进一步说明本公开，但是本公开并不因此而受到任何限制。The present disclosure will be further described below by way of examples, but the present disclosure is not limited thereby.
有益效果Beneficial Effects
在一些实施方案中，本公开提供的siRNA、药物组合物或siRNA缀合物可在体内具有更高的稳定性、更低的毒性和/或更高的活性。在一些实施方案中，本公开提供的siRNA、siRNA组合物或siRNA缀合物在体内显示出至少20％、30％、40％、50％、60％、70％、80％、90％或95％的HBV基因表达抑制率。在一些实施方案中，本公开提供的siRNA、siRNA组合物或siRNA缀合物在体内显示出至少20％、30％、40％、50％、60％、70％、80％、90％或95％的肝内HBV基因表达抑制率。在一些实施方案中，本公开提供的siRNA、siRNA组合物或siRNA缀合物在体内显示出至少20％、30％、40％、50％、60％、70％、80％、90％或95％的动物模型中肝内HBV基因表达抑制率。在一些实施方案中，本公开提供的siRNA、siRNA组合物或siRNA缀合物在体内显示出至少20％、30％、40％、50％、60％、70％、80％、90％或95％的HBV表面抗原表达抑制率。在一些实施方案中，本公开提供的siRNA、组合物或siRNA缀合物未显示出明显脱靶效应。脱靶效应可以是例如抑制非靶基因的基因正常表达。据认为，如果脱靶基因表达的结合/抑制与在靶基因效果相比低于50％、40％、30％、20％或10％时，该脱靶效应就是不显著的。In some embodiments, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure may have higher stability, lower toxicity and/or higher activity in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure shows at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% HBV gene expression inhibition rate in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure shows at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% HBV gene expression inhibition rate in the liver in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure shows at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% HBV gene expression inhibition rate in the liver in an animal model in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure shows at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% HBV surface antigen expression inhibition rate in vivo. In some embodiments, the siRNA, composition or siRNA conjugate provided by the present disclosure does not show obvious off-target effects. The off-target effect can be, for example, to inhibit the normal expression of genes of non-target genes. It is believed that if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20% or 10% compared to the on-target gene effect, the off-target effect is not significant.
在一些实施方案中，本公开提供的siRNA缀合物具有较好的体外抑制活性，0.1nM下抑制率高达95％。In some embodiments, the siRNA conjugates provided by the present disclosure have good in vitro inhibitory activity, with an inhibition rate of up to 95% at 0.1 nM.
在一些实施方案中，本公开提供的siRNA缀合物具有较好的体内抑制活性，1mg/kg下抑制率高达93.63％。In some embodiments, the siRNA conjugates provided by the present disclosure have good in vivo inhibitory activity, with an inhibition rate of up to 93.63% at 1 mg/kg.
在一些实施方案中，本公开提供的siRNA缀合物在具有优异的靶mRNA抑制效果的同时，还显示出低的脱靶效应。In some embodiments, the siRNA conjugates provided by the present disclosure have excellent target mRNA inhibition effects while also showing low off-target effects.
在一些实施方案中，本公开提供的siRNA缀合物在Tritosome中可维持长时间不降解，显示出很好的稳定性。In some embodiments, the siRNA conjugates provided by the present disclosure can be maintained in Tritosome for a long time without degradation, showing good stability.
在一些实施方案中，本公开提供的siRNA缀合物在人血浆中直至72h时仍未降解，显示出优异的在人血浆中的稳定性。In some embodiments, the siRNA conjugates provided by the present disclosure are not degraded in human plasma up to 72 hours, showing excellent stability in human plasma.
在一些实施方案中，本公开提供的siRNA缀合物在食蟹猴血浆中直至72h仍未降解，显示出优异的在猴血浆中的稳定性。In some embodiments, the siRNA conjugates provided by the present disclosure are not degraded in cynomolgus monkey plasma until 72 h, showing excellent stability in monkey plasma.
在一些实施方案中，单次给药3mg/kg的缀合物4对HBsAg的最大抑制率在95％以上，在长达56天的时间内，对HBsAg的抑制率在90％以上，在D85对HBV X mRNA抑制率为62％。In some embodiments, the maximum inhibition rate of HBsAg by a single administration of 3 mg/kg of conjugate 4 is above 95%, the inhibition rate of HBsAg is above 90% for up to 56 days, and the inhibition rate of HBV X mRNA is 62% at D85.
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明。Other features and advantages of the present disclosure will be described in detail in the following detailed description.
实施例Example
以下将通过实施例对本公开进行详细描述。除非特别说明，以下实施例中所用到的试剂、培养基均为市售商品，所用到的核酸电泳、real-time PCR等操作均参照MolecularCloning(Cold Spring Harbor Laboratory Press(1989))所记载的进行。The present disclosure will be described in detail below through examples. Unless otherwise specified, the reagents and culture media used in the following examples are all commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are all performed in accordance with Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).
若无其它说明，以下提供的试剂比例均按体积比(v/v)计算。Unless otherwise specified, the reagent ratios provided below are calculated by volume (v/v).
制备例1：缀合物4、18和19的制备Preparation Example 1: Preparation of Conjugates 4, 18 and 19
本制备例中，合成了缀合物4(以下，也称为L10-siHB1M1SVP缀合物)，计划合成缀合物18(以下，也称为L10-siHB1M1SP)和缀合物19(以下，也称为L10-siHB1M1SPs)。所述缀合物为L-9缀合分子分别与编号为siHB1M1SVP、siHB1M1SP或siHB1M1SPs的siRNA缀合后形成的缀合物。该缀合物中所缀合的siRNA的序列参见表2。In this preparation example, conjugate 4 (hereinafter, also referred to as L10-siHB1M1SVP conjugate) was synthesized, and conjugate 18 (hereinafter, also referred to as L10-siHB1M1SP) and conjugate 19 (hereinafter, also referred to as L10-siHB1M1SPs) were planned to be synthesized. The conjugates are conjugated with L-9 conjugated molecules and siRNAs numbered siHB1M1SVP, siHB1M1SP or siHB1M1SPs, respectively. The sequences of the siRNAs conjugated in the conjugates are shown in Table 2.
(1-1)L-10化合物的合成(1-1) Synthesis of Compound L-10
按照以下方法，合成了L-10化合物：Compound L-10 was synthesized according to the following method:
(1-1-1)缀合末端段GAL-5的合成(1-1-1) Synthesis of conjugated terminal segment GAL-5
(1-1-1a)GAL-2的合成(1-1-1a) Synthesis of GAL-2
将100.0g GAL-1(N-乙酰-D-半乳糖胺盐酸盐，CAS号：1772-03-8，购自宁波弘翔生化公司，463.8mmol)溶于1000ml无水吡啶，冰水浴下加入540ml乙酸酐(购自Enox公司，5565.6mmol)，室温搅拌反应1.5小时。将反应液倒入10L冰水中，减压抽滤，滤饼用2L冰水洗涤后，加乙腈/甲苯混合溶剂(体积比乙腈∶甲苯＝1∶1)至完全溶解，蒸干溶剂，得到白色固体产品GAL-2130.0g。100.0g GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS No.: 1772-03-8, purchased from Ningbo Hongxiang Biochemical Co., Ltd., 463.8mmol) was dissolved in 1000ml anhydrous pyridine, 540ml acetic anhydride (purchased from Enox Co., Ltd., 5565.6mmol) was added under ice-water bath, and stirred at room temperature for 1.5 hours. The reaction solution was poured into 10L ice water, filtered under reduced pressure, the filter cake was washed with 2L ice water, and acetonitrile/toluene mixed solvent (volume ratio of acetonitrile: toluene = 1:1) was added until it was completely dissolved, and the solvent was evaporated to obtain 130.0g of white solid product GAL-2.
(1-1-1b)GAL-3的合成(1-1-1b) Synthesis of GAL-3
将步骤(1-1-1a)中获得的GAL-2(35.1g，90.0mmol)溶解于213ml无水1，2-二氯乙烷中，在冰水浴且氮气保护条件下，加入24.0gTMSOTf(CAS号：27607-77-8，购自麦克林公司，108.0mmol)，室温反应过夜。Dissolve GAL-2 (35.1 g, 90.0 mmol) obtained in step (1-1-1a) in 213 ml of anhydrous 1,2-dichloroethane. Add 24.0 g of TMSOTf (CAS No.: 27607-77-8, purchased from MacLean, 108.0 mmol) in an ice-water bath under nitrogen protection and allow to react overnight at room temperature.
在反应液中加入400ml二氯甲烷稀释，以硅藻土过滤，再加入1L饱和碳酸氢钠水溶液，搅拌均匀，分出有机相，水相用二氯乙烷萃取两次，每次300ml，合并有机相，分别用300ml饱和碳酸氢钠水溶液和300ml饱和食盐水洗涤，分出有机相，无水硫酸钠干燥，减压蒸干溶剂，得到浅黄色粘稠糖稀状产品GAL-326.9g。Add 400 ml of dichloromethane to the reaction solution to dilute it, filter it with diatomaceous earth, add 1 L of saturated sodium bicarbonate aqueous solution, stir it evenly, separate the organic phase, extract the aqueous phase with dichloroethane twice, 300 ml each time, combine the organic phases, wash them with 300 ml of saturated sodium bicarbonate aqueous solution and 300 ml of saturated brine respectively, separate the organic phase, dry it over anhydrous sodium sulfate, and evaporate the solvent under reduced pressure to obtain 26.9 g of light yellow viscous syrupy product GAL-3.
(1-1-1c)GAL-4的合成(1-1-1c) Synthesis of GAL-4
将步骤(1-1-1b)中获得的GAL-3(26.9g，81.7mmol)溶于136ml无水1，2-二氯乙烷中，加入干燥的分子筛粉末30g，再加入9.0g 5-己烯-1-醇(CAS号：821-41-0，购自Adamas-beta公司，89.9mmol)，室温下搅拌30分钟，冰浴和氮气保护下加入9.08g TMSOTf(40.9mmol)，室温下搅拌反应过夜。过滤除去分子筛粉末，滤液中加入300ml二氯甲烷稀释，以硅藻土过滤，再加入500ml饱和碳酸氢钠水溶液搅拌10分钟洗涤，分出有机相，水相用300ml二氯乙烷萃取一次，合并有机相并分别用300ml饱和碳酸氢钠水溶液和300ml饱和食盐水洗涤，分出有机相，无水硫酸钠干燥，减压蒸干溶剂，得到黄色糖稀状产品GAL-441.3g，不进行纯化直接进行下一步氧化反应。GAL-3 (26.9 g, 81.7 mmol) obtained in step (1-1-1b) was dissolved in 136 ml of anhydrous 1,2-dichloroethane and dried 30g molecular sieve powder, then add 9.0g 5-hexen-1-ol (CAS No.: 821-41-0, purchased from Adamas-beta, 89.9mmol), stir at room temperature for 30 minutes, add 9.08g TMSOTf (40.9mmol) under ice bath and nitrogen protection, stir and react at room temperature overnight. Filter to remove Molecular sieve powder, add 300ml of dichloromethane to the filtrate to dilute, filter with diatomaceous earth, add 500ml of saturated sodium bicarbonate aqueous solution, stir for 10 minutes to wash, separate the organic phase, extract the aqueous phase once with 300ml of dichloroethane, combine the organic phases and wash them with 300ml of saturated sodium bicarbonate aqueous solution and 300ml of saturated brine respectively, separate the organic phase, dry over anhydrous sodium sulfate, evaporate the solvent under reduced pressure to obtain 1.3g of yellow syrupy product GAL-44, which is directly carried out to the next oxidation reaction without purification.
(1-1-1d)GAL-5的合成(1-1-1d) Synthesis of GAL-5
将按照步骤(1-1-1c)中描述的方法得到的GAL-4(14.9g，34.7mmol)溶于77ml二氯甲烷和77ml乙腈的混合溶剂中，分别加入103ml去离子水和29.7g高碘酸钠(CAS号：7790-28-5，购自阿拉丁公司，138.8mmol)，冰水浴下搅拌10分钟，加入三氯化钌(CAS号：14898-67-0，购自安耐吉公司，238mg，1.145mmol)，室温反应过夜。反应液加入300ml水稀释搅拌，加饱和碳酸氢钠调pH约为7.5，分出并弃去有机相，水相用二氯甲烷萃取三次，每次200ml，弃去有机相。水相用柠檬酸固体调节pH约为3，用二氯甲烷萃取三次，每次200ml，合并有机相，无水硫酸钠干燥，减压蒸干溶剂，得到白色泡沫状固体产品GAL-5 6.85g。1H NMR(400MHz，DMSO)δ12.01(br，1H)，7.83(d，J＝9.2Hz，1H)，5.21(d，J＝3.2Hz，1H)，4.96(dd，J＝11.2，3.2Hz，1H)，4.49(d，J＝8.4Hz，1H)，4.07-3.95(m，3H)，3.92-3.85(m，1H)，3.74-3.67(m，1H)，3.48-3.39(m，1H)，2.20(t，J＝6.8Hz，2H)，2.11(s，3H)，2.00(s，3H)，1.90(s，3H)，1.77(s，3H)，1.55-1.45(m，4H)。GAL-4 (14.9 g, 34.7 mmol) obtained by the method described in step (1-1-1c) was dissolved in a mixed solvent of 77 ml of dichloromethane and 77 ml of acetonitrile, and 103 ml of deionized water and 29.7 g of sodium periodate (CAS No.: 7790-28-5, purchased from Aladdin, 138.8 mmol) were added respectively, stirred for 10 minutes under an ice-water bath, and ruthenium trichloride (CAS No.: 14898-67-0, purchased from Anaiji, 238 mg, 1.145 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. The reaction solution was diluted and stirred with 300 ml of water, saturated sodium bicarbonate was added to adjust the pH to about 7.5, the organic phase was separated and discarded, and the aqueous phase was extracted three times with dichloromethane, 200 ml each time, and the organic phase was discarded. The pH of the aqueous phase was adjusted to about 3 with solid citric acid, and extracted three times with dichloromethane, 200 ml each time. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 6.85 g of a white foamy solid product GAL-5.1 H NMR (400MHz, DMSO) δ 12.01 (br, 1H), 7.83 (d, J = 9.2Hz, 1H), 5.21 (d, J = 3.2Hz, 1H), 4.96 (dd, J = 11.2, 3.2Hz, 1H), 4.49 (d, J = 8.4Hz, 1H), 4.07-3.95 (m, 3H), 3 .92-3.85(m, 1H), 3.74-3.67(m, 1H), 3.48-3.39(m, 1H), 2.20(t, J=6.8Hz, 2H), 2.11(s, 3H), 2.00(s, 3H), 1.77(s, 3H), 1.55-1.45(m, 4H ).
(1-1-2)M-11-T3的合成：(1-1-2) Synthesis of M-11-T3:
将J-0(1.883g，10mmol，商购自阿法埃莎公司)溶于25ml乙腈中，加入三乙胺(4.048g，40mmol)冰水浴冷却至0℃，加入三氟乙酸乙酯(5.683g，40mmol)，室温下反应22h，减压蒸干溶剂，真空油泵发泡干燥18h，得到5.342g粗品固体M-11-T3，不经进一步纯化地直接用于后续反应。MS m/z：C15H22F9N4O3，[M+H]+，理论：477.35，实测：477.65。J-0 (1.883 g, 10 mmol, purchased from Alfa Aesar) was dissolved in 25 ml of acetonitrile, triethylamine (4.048 g, 40 mmol) was added, and the mixture was cooled to 0°C in an ice-water bath, and ethyl trifluoroacetate (5.683 g, 40 mmol) was added. The mixture was reacted at room temperature for 22 h, and the solvent was evaporated under reduced pressure and dried by a vacuum oil pump for 18 h to obtain 5.342 g of a crude solid M-11-T3, which was directly used in subsequent reactions without further purification. MS m/z: C15 H22 F9 N4 O3 , [M+H]+, theoretical: 477.35, measured: 477.65.
(1-1-3)M-11-T3-Tr的合成：(1-1-3) Synthesis of M-11-T3-Tr:
将M-11-T3粗品(5.342g，10mmol)溶于50ml二氯甲烷，向反应液中加入TrCl(3.345g，12mmol)和三乙胺(1.518g，15mmol)，室温下搅拌反应20h，用饱和碳酸氢钠洗涤反应液2次，每次20ml，20ml饱和食盐水洗涤1次，有机相用无水硫酸钠干燥，过滤后减压蒸干有机溶剂，真空油泵发泡干燥过夜，得到粗品固体M-11-T3-Tr 7.763g。MS m/z：C34H36F9N4O3，[M+Na]+，理论：741.25，实测：741.53。粗品固体M-11-T3-Tr不经纯化地继续用于下一步M-18-Tr的合成。The crude product of M-11-T3 (5.342 g, 10 mmol) was dissolved in 50 ml of dichloromethane, TrCl (3.345 g, 12 mmol) and triethylamine (1.518 g, 15 mmol) were added to the reaction solution, and the reaction was stirred at room temperature for 20 h. The reaction solution was washed twice with saturated sodium bicarbonate, 20 ml each time, and once with 20 ml of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the organic solvent was evaporated under reduced pressure. The crude solid M-11-T3-Tr was obtained by bubbling and drying overnight. 7.763 g. MS m/z: C34 H36 F9 N4 O3 , [M+Na]+, theoretical: 741.25, measured: 741.53. The crude solid M-11-T3-Tr was used for the synthesis of M-18-Tr in the next step without purification.
(1-1-4)M-18-Tr的合成：(1-1-4) Synthesis of M-18-Tr:
将步骤(1-1-3)中获得的M-11-T3-Tr粗品(7.763g，10mmol)溶于100ml甲醇，再加入100ml甲胺水溶液(40质量％)，在50℃搅拌反应23h，过滤除去不溶颗粒物，减压蒸干溶剂，加入200ml体积比为1∶1的二氯甲烷∶甲醇混合溶剂，用50ml饱和碳酸氢钠洗涤，水相再用二氯甲烷(DCM)萃取3次，每次50ml，合并有机相，用无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜，用200-300目正相硅胶柱纯化，石油醚装柱，以1wt％三乙胺中和硅胶酸性，以二氯甲烷∶甲醇∶氨水(25wt％)＝1∶1∶0.05-1∶1∶0.25梯度洗脱，收集产物洗脱液，减压蒸干溶剂，真空油泵发泡干燥得到纯品M-18-Tr 2.887g。1H NMR(400MHz，DMSO)δ7.47-7.39(m，6H)，7.32-7.24(m，6H)，7.19-7.12(m，3H)，2.60-2.47(m，4H)，2.46-2.19(m，13H)，1.70-1.55(m，4H)，1.40(p，J＝6.8Hz，2H)。MS m/z：C28H39N4，[M+H]+，理论：431.65，实测：432.61。The crude product of M-11-T3-Tr (7.763 g, 10 mmol) obtained in step (1-1-3) was dissolved in 100 ml of methanol, and then 100 ml of methylamine aqueous solution (40 mass%) was added, and the mixture was stirred at 50° C. for 23 h, and the insoluble particles were filtered out, and the solvent was evaporated under reduced pressure, and 200 ml of a 1:1 dichloromethane: methanol mixed solvent was added, and the mixture was washed with 50 ml of saturated sodium bicarbonate, and the aqueous phase was extracted with dichloromethane (DCM) for 3 hours. times, 50 ml each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the solvent was evaporated under reduced pressure, vacuum oil pump dried overnight, purified with a 200-300 mesh normal phase silica gel column, loaded with petroleum ether, neutralized the acidity of the silica gel with 1wt% triethylamine, and eluted with a gradient of dichloromethane: methanol: ammonia water (25wt%) = 1: 1: 0.05-1: 1: 0.25, collected the product eluate, evaporated the solvent under reduced pressure, and vacuum oil pump dried to obtain 2.887 g of pure M-18-Tr.1 H NMR (400 MHz, DMSO) δ 7.47-7.39 (m, 6H), 7.32-7.24 (m, 6H), 7.19-7.12 (m, 3H), 2.60-2.47 (m, 4H), 2.46-2.19 (m, 13H), 1.70-1.55 (m, 4H), 1.40 (p, J=6.8 Hz, 2H). MS m/z: C28 H39 N4 , [M+H]+, theory: 431.65, found: 432.61.
(1-1-5)L-5-Tr的合成：(1-1-5) Synthesis of L-5-Tr:
将步骤(1-1-4)中获得的M-18-Tr(2.02g，4.69mmol)与步骤(1-1-1)中获得的GAL-5(6.93g，15.48mmol)混合溶于47ml乙腈，加入N-甲基吗啉(3.13g，30.96mmol)和4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM，4.28g，15.48mmol)，室温搅拌反应2h。以200ml二氯甲烷稀释反应液，100ml饱和碳酸氢钠溶液洗涤有机相，100ml饱和食盐水洗涤有机相，有机相用无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品。200-300目正相硅胶柱纯化，石油醚装柱，以1wt％三乙胺中和硅胶酸性，以二氯甲烷∶甲醇＝100∶5-100∶7梯度洗脱，收集产物洗脱液，减压蒸干得到纯品L-5-Tr 7.49g。1H NMR(400MHz，DMSO)δ7.83-7.10(m，4H)，7.67-7.60(m，1H)，7.44-7.34(m，6H)，7.33-7.24(m，6H)，7.20-7.15(m，3H)，5.22(s，3H)，4.97(d，J＝11.3Hz，3H)，4.49(d，J＝8.4Hz，3H)，4.06-3.07(m，9H)，3.95-3.83(m，3H)，3.77-3.64(m，3H)，3.45-3.35(m，3H)，3.12-2.87(m，8H)，2.30-2.15(m，3H)，2.11-1.98(m，22H)，1.95-1.84(m，11H)，1.81-1.61(m，14H)，1.54-1.36(m，14H)。MSm/z：C85H119N7O30，[M+H]+，理论：1718.81，实测：1718.03。The M-18-Tr (2.02 g, 4.69 mmol) obtained in step (1-1-4) and the GAL-5 (6.93 g, 15.48 mmol) obtained in step (1-1-1) were mixed and dissolved in 47 ml of acetonitrile, and N-methylmorpholine (3.13 g, 30.96 mmol) and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 4.28 g, 15.48 mmol) were added, and the mixture was stirred at room temperature for 2 h. The reaction solution was diluted with 200 ml of dichloromethane, and the organic phase was washed with 100 ml of saturated sodium bicarbonate solution and 100 ml of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain a crude product. Purify on a 200-300 mesh normal phase silica gel column, pack with petroleum ether, neutralize the acidity of silica gel with 1wt% triethylamine, elute with a gradient of dichloromethane: methanol = 100: 5-100: 7, collect the product eluate, evaporate to dryness under reduced pressure to obtain 7.49 g of pure L-5-Tr.1 H NMR (400MHz, DMSO) δ7.83-7.10 (m, 4H), 7.67-7.60 (m, 1H), 7.44-7.34 (m, 6H), 7.33-7.24 (m, 6H), 7.20-7.15 (m, 3H), 5.22 (s, 3H), 4.97 (d, J = 11.3 Hz, 3H), 4.49 (d, J = 8.4 Hz, 3H), 4.06-3.0 7 (m, 9H), 3.95-3.83 (m, 3H), 3.77-3.64 (m, 3H), 3.45-3.35 (m, 3H), 3.12-2.87 (m, 8H), 2.30-2.15 (m, 3H), 2.11-1.98 (m, 22H), 1.95-1.84 (m, 11H), 1.81-1.61 (m, 14H), 1.54-1.36 (m, 14H). MS m/z: C85 H119 N7 O30 , [M+H]+, theory: 1718.81, found: 1718.03.
(1-1-6)L-8的合成：(1-1-6) Synthesis of L-8:
将步骤(1-1-5)中得到的L-5-Tr(5.94g，3.456mmol)溶于69ml二氯甲烷，再加入二氯乙酸(13.367g，103.67mmol)，室温下反应2h，加入100ml二氯甲烷稀释反应液，再加饱和碳酸氢钠溶液洗涤调节pH＝7-8之间，水相以二氯甲烷萃取6次，每次30ml，合并有机相，用无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品。纯化使用200-300目正相硅胶，以10wt％三乙胺中和硅胶酸性，1wt‰三乙胺平衡柱子，以二氯甲烷∶甲醇＝100∶30-100∶40梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品L-84.26g。1H NMR(400MHz，DMSO)δ7.84(d，J＝9.0Hz，3H)，7.27-7.23(m，1H)，7.13-7.18(m，1H)，5.22(d，J＝3.1Hz，3H)，4.97(dd，J＝11.3，3.1Hz，3H)，4.48(d，J＝8.4Hz，3H)，4.09-3.98(m，9H)，3.88(dd，J＝19.3，9.3Hz，3H)，3.75-3.66(m，3H)，3.44-3.38(m，3H)，3.17-3.30(m，4H)，3.10-2.97(m，4H)，2.35-2.20(m，6H)，2.15-2.08(m，9H)，2.07-1.98(m，13H)，1.94-1.87(m，9H)，1.81-1.74(m，9H)，1.65-1.42(m，18H)。MSm/z：C85H119N7O30，[M+H]+，理论：1477.59，实测：1477.23。The L-5-Tr (5.94 g, 3.456 mmol) obtained in step (1-1-5) was dissolved in 69 ml of dichloromethane, and then dichloroacetic acid (13.367 g, 103.67 mmol) was added. The reaction was allowed to react at room temperature for 2 h. 100 ml of dichloromethane was added to dilute the reaction solution, and then saturated sodium bicarbonate solution was added to wash and adjust the pH to between 7 and 8. The aqueous phase was extracted with dichloromethane for 6 times, 30 ml each time, and the organic phases were combined, dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain a crude product. Purification was performed using 200-300 mesh normal phase silica gel, 10 wt% triethylamine was used to neutralize the acidity of the silica gel, 1 wt‰ triethylamine was used to balance the column, and dichloromethane: methanol = 100: 30-100: 40 gradient elution was performed, and the product eluate was collected, and the solvent was evaporated under reduced pressure to obtain 4.26 g of pure L-8.1 H NMR (400MHz, DMSO) δ7.84 (d, J=9.0Hz, 3H), 7.27-7.23 (m, 1H), 7.13-7.18 (m, 1H), 5.22 (d, J=3.1Hz, 3H), 4.97 (dd, J=11.3, 3.1Hz, 3H), 4.48 (d, J=8.4Hz, 3H), 4 .09-3.98(m,9H),3.88(dd,J＝19.3,9.3Hz , 3H), 3.75-3.66 (m, 3H), 3.44-3.38 (m, 3H), 3.17-3.30 (m, 4H), 3.10-2.97 (m, 4H), 2.35-2.20 (m, 6H), 2.15-2.08 (m, 9H), 2.07-1.98 (m, 13H), 1.94-1.87 (m, 9H), 1.81-1.74 (m, 9H), 1.65-1.42 (m, 18H). MS m/z: C85 H119 N7 O30 , [M+H]+, theory: 1477.59, found: 1477.23.
(1-1-7a)A-1的合成(1-1-7a) Synthesis of A-1
将DMTrCl(4，4′-双甲氧基三苯甲基氯，38.12g，112.5mmol)溶于450ml无水吡啶中，加入DL-甘油酸钙水合物(12.88g，45.0mmol)，在45℃反应22h，将反应液过滤，滤饼用200ml DCM淋洗，滤液减压浓缩至干，剩余物用500ml二氯甲烷重新溶解，0.5M三乙胺磷酸盐(pH＝7-8)洗涤2次，每次200ml，水相以二氯甲烷萃取2次，每次200ml，合并有机相，用无水硫酸钠干燥，过滤，减压蒸干溶剂，200-300目正相硅胶柱纯化，以石油醚∶乙酸乙酯∶二氯甲烷∶甲醇＝1∶1∶1∶0.35-1∶1∶1∶0.55梯度洗脱，收集产物洗脱液，减压蒸干溶剂，500ml二氯甲烷重新溶解，以200ml0.5M三乙胺磷酸盐洗涤1次，水相用二氯甲烷萃取2次，每次200ml，合并有机相，无水硫酸钠干燥，过滤，减压蒸干溶剂，真空油泵减压下干过夜，得到白色固体产品A-120.7g。1H NMR(400MHz，DMSO-d6)δ7.46(ddd，J＝6.5，2.3，1.1Hz，1H)，7.40-7.28(m，7H)，6.89-6.81(m，4H)，4.84(d，J＝5.0Hz，1H)，4.36-4.24(m，1H)，4.29(s，6H)，3.92(dd，J＝12.4，7.0Hz，1H)，3.67(dd，J＝12.3，7.0Hz，1H)，2.52(q，J＝6.3Hz，6H)，1.03(t，J＝6.3Hz，9H)。MS m/z：C24H23O6，[M-H]-，理论：407.15，实测：406.92。Dissolve DMTrCl (4,4′-bismethoxytrityl chloride, 38.12 g, 112.5 mmol) in 450 ml of anhydrous pyridine, add DL-calcium glycerate hydrate (12.88 g, 45.0 mmol), react at 45°C for 22 h, filter the reaction solution, and purify the filter cake with 200 ml The product was washed with DCM and the filtrate was concentrated to dryness under reduced pressure. The residue was redissolved in 500 ml of dichloromethane and washed twice with 0.5 M triethylamine phosphate (pH = 7-8), 200 ml each time. The aqueous phase was extracted twice with dichloromethane, 200 ml each time. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The product was purified on a 200-300 mesh normal phase silica gel column by gradient elution with petroleum ether: ethyl acetate: dichloromethane: methanol = 1:1:1:0.35-1:1:1:0.55. The product eluate was collected and the solvent was evaporated under reduced pressure. The product was redissolved in 500 ml of dichloromethane and washed once with 200 ml of 0.5 M triethylamine phosphate. The aqueous phase was extracted twice with dichloromethane, 200 ml each time. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The product was dried overnight under reduced pressure of a vacuum oil pump to obtain 20.7 g of a white solid product A-1.1 H NMR (400 MHz, DMSO-d6) δ 7.46 (ddd, J = 6.5, 2.3, 1.1 Hz, 1H), 7.40-7.28 (m, 7H), 6.89-6.81 (m, 4H), 4.84 (d, J = 5.0 Hz, 1H), 4.36-4.24 (m, 1H), 4.29 (s, 6H), 3.92 (dd, J = 12.4, 7.0 Hz, 1H), 3.67 (dd, J = 12.3, 7.0 Hz, 1H), 2.52 (q, J = 6.3 Hz, 6H), 1.03 (t, J = 6.3 Hz, 9H). MS m/z: C24 H23 O6 , [MH]-, theory: 407.15, found: 406.92.
(1-1-7b)L-7的合成：(1-1-7b) Synthesis of L-7:
将步骤(1-1-6)中获得的L-8(2.262g，1.532mmol)和步骤(1-1-7a)中获得的A-1(2.342g，4.596mmol)混合，溶于16ml二氯甲烷，加入3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT)(1.375g，4.596mmol)，再加入二异丙基乙胺(1.188g，9.191mmol)，25℃下搅拌反应2h，用10ml饱和碳酸氢钠洗涤有机相，水相以二氯甲烷萃取3次，每次10ml，以10ml饱和食盐水洗涤有机相，水相以二氯甲烷萃取2次，每次10ml，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜，得到粗品4.900g。柱纯化使用120g200-300目正相硅胶，以20ml三乙胺中和硅胶酸性，以含1wt％三乙胺的石油醚平衡柱子，以石油醚∶乙酸乙酯∶二氯甲烷∶N，N-二甲基甲酰胺＝1∶1∶1∶0.5-1∶1∶1∶0.6梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品L-72.336g。1H NMR(400MHz，DMSO)δ7.90-7.78(m，4H)，7.75-7.64(m，1H)，7.38-7.18(m，9H)，6.91-6.83(m，4H)，5.25-5.10(m，4H)，4.97(dd，J＝11.2，3.2Hz，3H)，4.48-4.30(m，4H)，4.02(s，9H)，3.93-3.84(m，3H)，3.76-3.66(m，9H)，3.45-3.35(m，3H)，3.24-2.98(m，10H)，2.30-2.20(m，2H)，2.11-1.88(m，31H)，1.80-1.40(m，28H)。MS m/z：C90H128N7O35，[M-DMTr]+，理论：1564.65，实测：1564.88。The L-8 (2.262 g, 1.532 mmol) obtained in step (1-1-6) and the A-1 (2.342 g, 4.596 mmol) obtained in step (1-1-7a) were mixed, dissolved in 16 ml of dichloromethane, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT) (1.375 g, 4.596 mmol) was added, and then diisopropylethylamine (1.188 g, 9.191 mmol) was added. The reaction was stirred at 25°C for 2 h, and the organic phase was washed with 10 ml of saturated sodium bicarbonate, and the aqueous phase was extracted 3 times with dichloromethane, 10 ml each time, and the organic phase was washed with 10 ml of saturated brine, and the aqueous phase was extracted 2 times with dichloromethane, 10 ml each time. The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure, and vacuum oil pump was used to dry overnight to obtain 4.900 g of a crude product. Column purification was performed using 120 g of 200-300 mesh normal phase silica gel, 20 ml of triethylamine was used to neutralize the acidity of the silica gel, the column was balanced with petroleum ether containing 1 wt% triethylamine, and the gradient elution was performed with petroleum ether: ethyl acetate: dichloromethane: N, N-dimethylformamide = 1: 1: 1: 0.5-1: 1: 1: 0.6. The product eluate was collected and the solvent was evaporated under reduced pressure to obtain 2.336 g of pure product L-7.1 H NMR (400 MHz, DMSO) δ 7.90-7.78 (m, 4H), 7.75-7.64 (m, 1H), 7.38-7.18 (m, 9H), 6.91-6.83 (m, 4H), 5.25-5.10 (m, 4H), 4.97 (dd, J = 11.2, 3.2 Hz, 3H), 4.48-4.30 ( m, 4H), 4.02 (s, 9H), 3.93-3.84 (m, 3H), 3.76-3.66 (m, 9H), 3.45-3.35 (m, 3H), 3.24-2.98 (m, 10H), 2.30-2.20 (m, 2H), 2.11-1.88 (m, 31H), 1.80-1.40 (m, 28H). MS m/z: C90 H128 N7 O35 , [M-DMTr]+, theory: 1564.65, found: 1564.88.
(1-1-8)L-9缀合分子的合成：(1-1-8) Synthesis of L-9 conjugated molecules:
将步骤(1-1-7b)中获得的L-7(2.300g，1.26mmol)、丁二酸酐(0.378g，3.78mmol)和4-二甲氨基吡啶(DMAP，0.462g，3.78mmol)混合溶于13ml二氯甲烷，再加入二异丙基乙胺(DIEA，0.814g，6.30mmol)，25℃下搅拌24h，5ml 0.5M三乙胺磷酸盐洗涤反应液，水相以二氯甲烷萃取3次，每次5ml，合并有机相减压蒸干得到2.774g粗品。柱纯化使用60g 200-300目正相硅胶，以1wt％三乙胺中和硅胶酸性，以二氯甲烷平衡柱子，以含1wt‰三乙胺的二氯甲烷∶甲醇＝100∶18-100∶20梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品L-9缀合分子1.874g。1H NMR(400MHz，DMSO)δ8.58(d，J＝4.2Hz，1H)，7.94-7.82(m，3H)，7.41-7.29(m，5H)，7.22(d，J＝8.1Hz，5H)，6.89(d，J＝8.3Hz，4H)，5.49-5.37(m，1H)，5.21(d，J＝3.0Hz，3H)，4.97(d，J＝11.1Hz，3H)，4.49(d，J＝8.2Hz，3H)，4.02(s，9H)，3.88(dd，J＝19.4，9.4Hz，3H)，3.77-3.65(m，9H)，3.50-3.39(m，6H)，3.11-2.90(m，5H)，2.61-2.54(m，4H)，2.47-2.41(m，2H)，2.26-2.17(m，2H)，2.15-1.95(m，22H)，1.92-1.84(m，9H)，1.80-1.70(m，10H)，1.65-1.35(m，17H)，1.31-1.19(m，4H)，0.96(t，J＝7.1Hz，9H)。MS m/z：C94H132N7O38，[M-DMTr]+，理论：1664.72，实测：1665.03。The L-7 (2.300 g, 1.26 mmol) obtained in step (1-1-7b), succinic anhydride (0.378 g, 3.78 mmol) and 4-dimethylaminopyridine (DMAP, 0.462 g, 3.78 mmol) were mixed and dissolved in 13 ml of dichloromethane, and diisopropylethylamine (DIEA, 0.814 g, 6.30 mmol) was added. The mixture was stirred at 25°C for 24 h, and the reaction solution was washed with 5 ml of 0.5 M triethylamine phosphate. The aqueous phase was extracted with dichloromethane 3 times, 5 ml each time, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain 2.774 g of a crude product. Column purification used 60g of 200-300 mesh normal phase silica gel, neutralized the acidity of silica gel with 1wt% triethylamine, balanced the column with dichloromethane, and eluted with a gradient of dichloromethane containing 1wt‰ triethylamine: methanol = 100:18-100:20, collected the product eluate, and evaporated the solvent under reduced pressure to obtain1.874g of pure L-9 conjugated molecule. NMR (400MHz, DMSO) δ8.58 (d, J=4.2Hz, 1H), 7.94-7.82 (m, 3H), 7.41-7.29 (m, 5H), 7.22 (d, J=8.1Hz, 5H), 6.89 (d, J=8.3Hz, 4H), 5.49-5.37 (m, 1H), 5.21 (d, J= 3.0Hz, 3H), 4.97 (d, J=11.1Hz, 3H), 4.49 (d, J=8.2Hz, 3H), 4.02 (s, 9H), 3.88 (dd, J=19.4, 9 .4Hz, 3H), 3.77-3.65(m, 9H), 3.50-3.39(m, 6H), 3.11-2.90(m, 5H), 2.61-2.54(m, 4H), 2.47-2.41(m, 2H), 2.26-2.17(m, 2H), 2.15-1.95(m, 22H), 1 .92-1.84 (m, 9H), 1.80-1.70 (m, 10H), 1.65-1.35 (m, 17H), 1.31-1.19 (m, 4H), 0.96 (t, J=7.1Hz, 9H). MS m/z:C94H132N7O38, [M- DMTr]+, theory: 1664.72,found : 1665.03.
(1-1-9)L-10化合物的合成：(1-1-9) Synthesis of compound L-10:
此步骤中，通过将L-9缀合分子连接至固相载体，制备了L-10化合物。In this step, the L-10 compound was prepared by linking the L-9 conjugate molecule to a solid support.
将步骤(1-1-8)中获得的L-9缀合分子(0.233g，0.1126mmol)、O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU，0.064g，0.1689mmol)和二异丙基乙胺(DIEA，0.029g，0.2252mmol)混合，溶于19ml乙腈，室温搅拌5分钟，向反应液中加入氨甲基树脂(0.901g，100-200目，氨基载量400μmol/g，购自南开和成公司)，25℃下进行摇床反应，转速220转/分钟，反应15h后过滤，滤饼以DCM淋洗2次，每次30ml，乙腈淋洗3次，每次30ml，30ml乙醚淋洗1次，真空油泵干燥2h，随后再按照表1中示出的投料配比加入原料(CapA、CapB、4-二甲氨基吡啶(DMAP)和乙腈)进行盖帽反应。25℃下置于摇床上，转速200转/分钟，反应5h，反应液过滤，滤饼用乙腈淋洗3次，每次30ml，抽滤至干，真空油泵减压下干燥过夜，得到L-10化合物(即，连接固相载体的L-9缀合分子)1.100g，载量90.8μmol/g。The L-9 conjugated molecule (0.233 g, 0.1126 mmol) obtained in step (1-1-8), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 0.064 g, 0.1689 mmol) and diisopropylethylamine (DIEA, 0.029 g, 0.2252 mmol) were mixed and dissolved in 19 ml of acetonitrile and stirred at room temperature for 5 minutes. Aminomethyl resin (0.901 g, 100-200 mesh) was added to the reaction solution. , amino loading 400μmol/g, purchased from Nankai Hecheng Company), shaker reaction at 25°C, speed 220 rpm, filter after reaction for 15h, filter cake rinsed with DCM twice, 30ml each time, acetonitrile 3 times, 30ml each time, 30ml ether 1 time, vacuum oil pump dried for 2h, then added raw materials (CapA, CapB, 4-dimethylaminopyridine (DMAP) and acetonitrile) according to the feed ratio shown in Table 1 for capping reaction. Place on a shaker at 25°C, speed 200 rpm, react for 5h, filter the reaction liquid, rinse the filter cake with acetonitrile 3 times, 30ml each time, filter to dry, vacuum oil pump under reduced pressure overnight to obtain L-10 compound (i.e., L-9 conjugated molecule connected to a solid phase carrier) 1.100g, loading 90.8μmol/g.
表1盖帽反应投料配比Table 1 Capping reaction feed ratio
其中，CapA和CapB为盖帽试剂溶液，CapA为20体积％N-甲基咪唑的吡啶/乙腈混合溶液，吡啶与乙腈的体积比为3∶5；CapB为20体积％乙酸酐的乙腈溶液。CapA and CapB are capping reagent solutions, CapA is a 20 volume % N-methylimidazole mixed solution in pyridine/acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; CapB is a 20 volume % acetic anhydride solution in acetonitrile.
(1-2)合成缀合物4、18和19的正义链(1-2) Synthesis of the Sense Strand of Conjugates 4, 18, and 19
缀合物4、18和19的正义链序列相同，故其制备方法也相同。The sense strand sequences of conjugates 4, 18 and 19 are the same, so their preparation methods are also the same.
通过固相亚磷酰胺法，利用上述步骤制备的L-10化合物起始循环，按照正义链核苷酸排布顺序自3′-5′方向逐一连接核苷单体。每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化或硫化四步反应。其中，两个核苷酸之间采用磷酸酯连接时，连接后一个核苷单体时，包括脱保护、偶联、盖帽、氧化四步反应。两个核苷酸之间采用硫代磷酸酯连接时，连接后一个核苷单体时，包括保护、偶联、盖帽、硫化四步反应。合成条件给定如下：The solid phase phosphoramidite method is used to start the cycle using the L-10 compound prepared in the above steps, and the nucleoside monomers are connected one by one from the 3′-5′ direction according to the arrangement order of the positive chain nucleotides. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. Among them, when phosphate is used to connect two nucleotides, the four steps of deprotection, coupling, capping and oxidation are included when connecting the next nucleoside monomer. When thiophosphate is used to connect two nucleotides, the four steps of protection, coupling, capping and sulfurization are included when connecting the next nucleoside monomer. The synthesis conditions are given as follows:
核苷单体以0.1M浓度的乙腈溶液提供，每一步的脱保护反应的条件相同，即温度为25℃，反应时间为70秒，脱保护试剂为二氯乙酸的二氯甲烷溶液(3％v/v)，二氯乙酸与固相载体上4，4′-二甲氧基三苯甲基保护基的摩尔比为5∶1。The nucleoside monomer is provided in a 0.1 M acetonitrile solution. The conditions of the deprotection reaction in each step are the same, i.e., the temperature is 25° C., the reaction time is 70 seconds, the deprotection reagent is a dichloroacetic acid solution in dichloromethane (3% v/v), and the molar ratio of dichloroacetic acid to the 4,4′-dimethoxytrityl protecting group on the solid phase carrier is 5:1.
每一步偶联反应条件均相同，包括温度为25℃，固相载体上连接的核酸序列与核苷单体的摩尔比为1∶10，固相载体上连接的核酸序列和偶联试剂的摩尔比为1∶65，反应时间为600秒，偶联试剂为5-乙硫基-1H-四氮唑的0.5M乙腈溶液。The coupling reaction conditions for each step were the same, including a temperature of 25° C., a molar ratio of the nucleic acid sequence connected to the solid phase support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence connected to the solid phase support to the coupling reagent of 1:65, a reaction time of 600 seconds, and a coupling reagent of 0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazole.
每一步盖帽条件均相同，包括温度为25℃，反应时间为15秒。盖帽试剂溶液为摩尔比为1∶1的CapA和CapB的混合溶液，盖帽试剂与固相载体上连接的核酸序列的摩尔比为乙酸酐∶N-甲基咪唑∶固相载体上连接的核酸序列＝1∶1∶1。The capping conditions in each step are the same, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution is a mixed solution of CapA and CapB in a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence connected to the solid phase carrier is acetic anhydride: N-methylimidazole: nucleic acid sequence connected to the solid phase carrier = 1:1:1.
每一步氧化反应条件相同，包括温度为25℃，反应时间为15秒，氧化试剂为浓度为0.05M的碘水。碘与偶联步骤中固相载体上连接的核酸序列的摩尔比为30∶1。反应在四氢呋喃∶水∶吡啶＝3∶1∶1的混合溶剂中进行。The oxidation reaction conditions in each step were the same, including a temperature of 25° C., a reaction time of 15 seconds, and an oxidizing agent of 0.05 M iodine water. The molar ratio of iodine to the nucleic acid sequence connected to the solid phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran: water: pyridine = 3:1:1.
每一步硫化反应的条件相同，包括温度为25℃，反应时间为300秒，硫化试剂为氢化黄原素。硫化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔比为120∶1。反应在乙腈∶吡啶＝1∶1的混合溶剂中进行。The conditions of each step of the sulfurization reaction are the same, including a temperature of 25°C, a reaction time of 300 seconds, and a sulfurization reagent of hydrogenated xanthan. The molar ratio of the sulfurization reagent to the nucleic acid sequence connected to the solid phase support in the coupling step is 120:1. The reaction is carried out in a mixed solvent of acetonitrile:pyridine=1:1.
切割和脱保护条件如下：将合成的连接有载体的核苷酸序列加入浓度为25wt％的氨水中，氨水用量为0.5ml/μmol，在55℃反应16h，除去液体，真空浓缩至干。The cleavage and deprotection conditions are as follows: add the synthesized nucleotide sequence connected to the carrier into 25 wt% ammonia water, the amount of ammonia water used is 0.5 ml/μmol, react at 55° C. for 16 h, remove the liquid, and concentrate to dryness in vacuo.
纯化与脱盐：利用制备型离子色谱纯化柱(Source 15Q)，通过NaCl的梯度洗脱，完成核酸的纯化。具体而言为：洗脱剂A：20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9∶1(体积比)；洗脱剂B：1.5M氯化钠，20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9∶1(体积比)；洗脱梯度：洗脱剂A：洗脱剂B＝100∶0-50∶50梯度洗脱。收集产品洗脱液后合并，采用反相色谱纯化柱进行脱盐，具体条件包括采用葡聚糖凝胶柱进行脱盐，填料为葡聚糖凝胶G25，以去离子水洗脱。Purification and desalting: The nucleic acid was purified by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent A: 20 mM sodium phosphate (pH 8.1), solvent water/acetonitrile = 9:1 (volume ratio); eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), solvent water/acetonitrile = 9:1 (volume ratio); elution gradient: eluent A: eluent B = 100:0-50:50 gradient elution. The product eluates were collected and combined, and desalted using a reverse phase chromatography purification column. The specific conditions included desalting using a Sephadex column, the filler was Sephadex G25, and elution was performed with deionized water.
检测：使用离子交换色谱(IEX-HPLC)检测纯度，使用液质联用(LC-MS)分析分子量。Detection: Purity was determined by ion exchange chromatography (IEX-HPLC) and molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS).
对于缀合物4的正义链分子量，理论值为7485.3，实测值为7484.4。实测值与理论值相符，表明所合成的是3′末端缀合了L-9缀合分子的正义链S。The theoretical value of the molecular weight of the sense chain of conjugate 4 is 7485.3, and the measured value is 7484.4. The measured value is consistent with the theoretical value, indicating that the synthesized sense chain S is conjugated with the L-9 conjugated molecule at the 3' end.
(1-3)合成缀合物4、18和19的反义链(1-3) Synthesis of Antisense Strands of Conjugates 4, 18, and 19
(1-3A)缀合物4反义链的制备(1-3A) Preparation of the antisense strand of conjugate 4
通过固相亚磷酰胺法，利用通用固相载体(UnyLinkerTMloadedSolid Supports，Kinovate Life Sciences公司)起始循环，合成缀合物4的反义链AS。固相合成方法中的脱保护、偶联、盖帽、氧化或硫化反应条件，切割和脱保护，纯化与脱盐条件与合成正义链相同。Through the solid phase phosphoramidite method, using a universal solid phase carrier (UnyLinkerTM loaded Solid Supports, Kinovate Life Sciences) were used as the starting cycle to synthesize the antisense chain AS of conjugate 4. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid phase synthesis method were the same as those for synthesizing the sense chain.
检测：纯度采用离子交换色谱(IEX-HPLC)进行检测；分子量采用液质联用(LC-MS)进行分析，理论值为7161.7，实测值为7160.9。实测值与理论值相符，表明所合成的是具有目标序列的反义链AS。其中，乙烯基磷酸酯修饰的2′-甲氧基修饰尿嘧啶核苷单体(VP-Um)按照以下方法合成：Detection: Purity was detected by ion exchange chromatography (IEX-HPLC); molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS), the theoretical value was 7161.7, and the measured value was 7160.9. The measured value was consistent with the theoretical value, indicating that the synthesized antisense chain AS with the target sequence. Among them, the vinyl phosphate-modified 2′-methoxy-modified uridine nucleoside monomer (VP-Um) was synthesized according to the following method:
(1-3-1)VP-U-2的合成(1-3-1) Synthesis of VP-U-2
按照以下方法，合成了VP-U-2分子：The VP-U-2 molecule was synthesized according to the following method:
将2′-甲氧基修饰的尿嘧啶核苷(2′-OMe-U，51.30g，91.6mmol)，叔丁基二苯基氯硅烷(TBDPSCl，50.35g，183.2mmol)，咪唑(12.47g，183.2mmol)混合溶于450ml N，N-二甲基甲酰胺(DMF)，室温下搅拌反应20h。蒸除DMF，用600ml二氯甲烷溶解后加300ml饱和碳酸氢钠洗涤，水相再用二氯甲烷(DCM)萃取3次，每次300ml，合并有机相，用5％草酸洗涤至水相pH＜5，蒸发溶剂至干后获得VP-U-1粗品直接用于随后VP-U-2的合成。2′-methoxy modified uridine nucleoside (2′-OMe-U, 51.30 g, 91.6 mmol), tert-butyldiphenylsilyl chloride (TBDPSCl, 50.35 g, 183.2 mmol), imidazole (12.47 g, 183.2 mmol) were mixed and dissolved in 450 ml N, N-dimethylformamide (DMF), and stirred at room temperature for 20 h. DMF was evaporated, dissolved in 600 ml dichloromethane, and washed with 300 ml saturated sodium bicarbonate, and the aqueous phase was extracted with dichloromethane (DCM) for 3 times, 300 ml each time, and the organic phases were combined and washed with 5% oxalic acid until the pH of the aqueous phase was less than 5. After evaporating the solvent to dryness, the crude VP-U-1 was directly used for the subsequent synthesis of VP-U-2.
将VP-U-1粗品用100ml二氯甲烷溶解后，外加冰浴搅拌10分钟，再加入预先在4℃冰箱冷藏好的450ml 2％对甲苯磺酸溶液(溶剂为体积比3∶7的甲醇-二氯甲烷混合溶剂)，反应10分钟。再加入200ml饱和碳酸氢钠淬灭反应，有机相加入饱和碳酸氢钠水溶液洗涤至pH＝8。合并水相，用二氯甲烷萃取2次，每次200ml，合并有机相，再用200ml饱和食盐水洗涤一次，蒸发溶剂至干。200-300目正相硅胶柱纯化，石油醚装柱，以石油醚∶乙酸乙酯∶二氯甲烷∶甲醇＝1∶1∶1∶0.05-1∶1∶1∶0.25梯度洗脱，收集产物洗脱液，减压蒸干溶剂，真空油泵发泡干燥得到纯品VP-U-2共40.00g。1HNMR(400MHz，DMSO-d6)δ7.96(d，J＝7.8Hz，1H)，7.64(dtd，J＝5.1，4.0，2.2Hz，4H)，7.41-7.30(m，6H)，6.79(d，J＝4.7Hz，1H)，5.73(d，J＝7.6Hz，1H)，4.94(t，J＝7.0Hz，1H)，4.12(td，J＝4.6，3.9Hz，IH)，4.05(dd，J＝4.8，4.0Hz，1H)，3.96(t，J＝4.7Hz，1H)，3.68(ddd，J＝11.8，7.0，4.6Hz，1H)，3.57-3.46(m，1H)，3.39(s，3H)，1.05(s，8H)。MS m/z：C26H33N2O6Si，[M+H]+，理论：497.21，实测：497.45。After the crude VP-U-1 was dissolved in 100 ml of dichloromethane, it was stirred in an ice bath for 10 minutes, and then 450 ml of 2% p-toluenesulfonic acid solution (the solvent was a methanol-dichloromethane mixed solvent with a volume ratio of 3:7) pre-refrigerated in a 4°C refrigerator was added, and the reaction was allowed to proceed for 10 minutes. 200 ml of saturated sodium bicarbonate was added to quench the reaction, and the organic phase was washed with a saturated sodium bicarbonate aqueous solution until pH = 8. The aqueous phases were combined, extracted twice with dichloromethane, 200 ml each time, and the organic phases were combined, and then washed once with 200 ml of saturated brine, and the solvent was evaporated to dryness. Purification was performed on a 200-300 mesh normal phase silica gel column, loaded with petroleum ether, and gradient eluted with petroleum ether: ethyl acetate: dichloromethane: methanol = 1:1:1:0.05-1:1:1:0.25, the product eluate was collected, the solvent was evaporated under reduced pressure, and the pure product VP-U-2 was obtained by foaming and drying with a vacuum oil pump, totaling 40.00 g. 1HNMR (400MHz, DMSO-d6) δ7.96 (d, J=7.8Hz, 1H), 7.64 (dtd, J=5.1, 4.0, 2.2Hz, 4H), 7.41-7.30 (m, 6H), 6.79 (d, J=4.7Hz, 1H), 5.73 (d, J=7.6Hz, 1H), 4.94 (t , J=7.0Hz, 3H), 3.14 (m, 1H), 3.76 (s, 5H), 1.22 (m, 1H), 1.57 (s, 4H), 1.42 (m, 3H), 1.22 (s, 5H), 1.43 (m, 1H),1.20 (m, 3H), 1.44 (s, 7H). MS m/z:C26H33N2O6Si , [M+ H]+, theory: 497.21,found : 497.45.
(1-3-2)VP-U-4的合成：(1-3-2) Synthesis of VP-U-4:
将VP-U-2(19.84g，40.0mmol)，二环己基碳二亚胺(DCC，16.48g，80.0mmol)，吡啶(4.20g，53.2mmol)，三氟乙酸(6.61g，53.2mmol)混合溶于200ml二甲基亚砜(DMSO)，室温下搅拌反应20h。另取亚甲基二磷酸四乙酯(21.44g，74.4mmol)溶于120mlTHF，冰浴降温，在冰浴温度下加入t-BuOK(11.36g，101.2mmol)，先在冰浴温度下反应10min，再升至室温反应0.5h，然后加入至前述反应液中，约1h加完，冰浴温度下反应1h，再升至室温反应18h。加水淬灭反应，水相以二氯甲烷提取3次，每次200ml。合并有机相，用200ml饱和食盐水水洗一次后蒸发溶剂至干。用200-300目正相硅胶柱纯化，石油醚装柱，以石油醚∶乙酸乙酯＝1∶1-1∶4梯度洗脱，收集产物洗脱液，减压蒸干溶剂，真空油泵发泡干燥得到纯品VP-U-4共14.00g。1H NMR(400MHz，DMSO-d6)δ7.96(d，J＝7.8Hz，1H)，7.64(dtd，J＝5.1，4.0，2.2Hz，4H)，7.41-7.30(m，6H)，6.82-6.71(m，2H)，5.90(ddd，J＝25.9，15.0，1.0Hz，1H)，5.73(d，J＝7.6Hz，1H)，4.36-4.21(m，3H)，4.18(t，J＝4.9Hz，1H)，4.05(ddq，J＝9.7，8.5，6.9Hz，2H)，3.87(t，J＝4.8Hz，1H)，3.39(s，3H)，1.32(td，J＝6.9，0.7Hz，6H)，1.05(s，8H)。MS m/z：C31H42N2O8PSi，[M+H]+，理论：629.24，实测：629.51。VP-U-2 (19.84 g, 40.0 mmol), dicyclohexylcarbodiimide (DCC, 16.48 g, 80.0 mmol), pyridine (4.20 g, 53.2 mmol), trifluoroacetic acid (6.61 g, 53.2 mmol) were mixed and dissolved in 200 ml of dimethyl sulfoxide (DMSO), and stirred at room temperature for 20 h. Tetraethyl methylene diphosphate (21.44 g, 74.4 mmol) was dissolved in 120 ml of THF, cooled in an ice bath, and t-BuOK (11.36 g, 101.2 mmol) was added at ice bath temperature, first reacted at ice bath temperature for 10 min, then heated to room temperature for 0.5 h, and then added to the above reaction solution, and the addition was completed in about 1 h, and reacted at ice bath temperature for 1 h, and then heated to room temperature for 18 h. Water was added to quench the reaction, and the aqueous phase was extracted with dichloromethane 3 times, 200 ml each time. Combine the organic phases, wash once with 200 ml of saturated brine, and evaporate the solvent to dryness. Purify with a 200-300 mesh normal phase silica gel column, load the column with petroleum ether, and elute with a gradient of petroleum ether: ethyl acetate = 1: 1-1: 4. Collect the product eluate, evaporate the solvent under reduced pressure, and dry with a vacuum oil pump to obtain a total of 14.00 g of pure VP-U-4. 1H NMR (400 MHz, DMSO-d6) δ7.96 (d, J = 7.8 Hz, 1H), 7.64 (dtd, J = 5.1, 4.0, 2.2 Hz, 4H), 7.41-7.30 (m, 6H), 6.82-6.71 (m, 2H), 5.90 (ddd, J = 25.9, 15.0, 1.0 Hz, 1H), 5.73 (d, J = 2 =7.6 Hz, 1H), 4.36-4.21 (m, 3H), 4.18 (t, J=4.9 Hz, 1H), 4.05 (ddq, J=9.7, 8.5, 6.9 Hz, 2H), 3.87 (t, J=4.8 Hz, 1H), 3.39 (s, 3H), 1.32 (td, J=6.9, 0.7 Hz, 6H), 1.05 (s, 8H). MS m/z: C31 H42 N2 O8 PSi, [M+H]+, theory: 629.24, found: 629.51.
(1-3-3)VP-U-5的合成：(1-3-3) Synthesis of VP-U-5:
将VP-U-4(14.00g，22.29mmol)溶于100ml四氢呋喃，加入三乙胺三氢氟酸(17.96g，111.45mmol)，室温搅拌20h反应完全。直接蒸发溶剂至干，再用二氯甲烷溶解随后蒸干2次，每次使用50ml二氯甲烷，得到粗品。用200-300目正相硅胶柱纯化，石油醚装柱，以石油醚∶乙酸乙酯∶二氯甲烷∶甲醇＝1∶1∶1∶0.05-1∶1∶1∶0.25梯度洗脱，收集产物洗脱液，减压蒸干溶剂，真空油泵发泡干燥得到纯品VP-U-5共6.70g。1H NMR(400MHz，DMSO-d6)δ7.96(d，J＝7.8Hz，1H)，6.77(dd，J＝15.0，6.2Hz，1H)，5.99-5.82(m，2H)，5.73(d，J＝7.6Hz，1H)，5.27(d，J＝5.1Hz，1H)，5.10(dd，J＝5.3，4.7Hz，1H)，4.29(ddq，J＝9.8，8.6，7.0Hz，2H)，4.17(ddd，J＝6.2，5.2，1.0Hz，1H)，4.12-3.98(m，3H)，3.39(s，2H)，1.32(td，J＝6.9，0.6Hz，6H)。MS m/z：C15H24N2O8P，[M+H]+，理论：391.13，实测：391.38。Dissolve VP-U-4 (14.00 g, 22.29 mmol) in 100 ml tetrahydrofuran, add triethylamine trihydrofluoride (17.96 g, 111.45 mmol), stir at room temperature for 20 h to complete the reaction. Evaporate the solvent directly to dryness, dissolve it in dichloromethane and evaporate it to dryness twice, using 50 ml dichloromethane each time to obtain a crude product. Purify it with a 200-300 mesh normal phase silica gel column, load it with petroleum ether, and elute it with a gradient of petroleum ether: ethyl acetate: dichloromethane: methanol = 1:1:1:0.05-1:1:1:0.25. Collect the product eluate, evaporate the solvent under reduced pressure, and dry it with a vacuum oil pump to obtain a total of 6.70 g of pure VP-U-5. 1H NMR (400MHz, DMSO-d6) δ7.96 (d, J=7.8Hz, 1H), 6.77 (dd, J=15.0, 6.2Hz, 1H), 5.99-5.82 (m, 2H), 5.73 (d, J=7.6Hz, 1H), 5.27 (d, J=5.1Hz, 1H), 5.10 (dd, J=5 .3, 4.7Hz, 1H), 4.29 (ddq, J=9.8, 8.6, 7.0Hz, 2H), 4.17 (ddd, J=6.2, 5.2, 1.0Hz, 1H), 4.12-3.98 (m, 3H), 3.39 (s, 2H), 1.32 (td, J=6.9, 0.6Hz, 6H). MS m/ z:C15H24N2O8P , [M+ H]+, theory: 391.13,found : 391.38.
(1-3-4)VP-U-6的合成：(1-3-4) Synthesis of VP-U-6:
在氩气保护条件下向10ml无水二氯甲烷中加入VP-U-5(391mg，1.0mmol)、三氟乙酸吡啶盐(0.232g，1.2mmol)、N-甲基咪唑(0.099g，1.2mmol)，双(二异丙基氨基)(2-氰基乙氧基)膦(0.452g，1.5mmol)，室温搅拌反应5小时。蒸除溶剂至干，柱层析纯化(200-300目正相硅胶，二氯甲烷∶乙腈(含0.5wt％三乙胺)＝3∶1-1∶3梯度洗脱)，收集产物洗脱液，浓缩除去溶剂，得到目标产物VP-U-6共508mg。31P NMR(161MHz，DMSO-d6)δ150.34，150.29，17.07，15.50。MS m/z：C24H41N4O9P2，[M+H]+，理论：591.23，实测：591.55。表明VP-U-6是目标产物VP-Um，作为核苷单体参与RNA链合成。Under argon protection, VP-U-5 (391 mg, 1.0 mmol), pyridinium trifluoroacetate (0.232 g, 1.2 mmol), N-methylimidazole (0.099 g, 1.2 mmol), bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.452 g, 1.5 mmol) were added to 10 ml of anhydrous dichloromethane, and the mixture was stirred at room temperature for 5 hours. The solvent was evaporated to dryness, and the mixture was purified by column chromatography (200-300 mesh normal phase silica gel, dichloromethane: acetonitrile (containing 0.5 wt% triethylamine) = 3:1-1:3 gradient elution), the product eluate was collected, and the solvent was concentrated to obtain the target product VP-U-6, a total of 508 mg. 31P NMR (161 MHz, DMSO-d6) δ 150.34, 150.29, 17.07, 15.50. MS m/z: C24 H41 N4 O9 P2 , [M+H]+, theoretical: 591.23, measured: 591.55. This indicates that VP-U-6 is the target product VP-Um, and participates in RNA chain synthesis as a nucleoside monomer.
(1-3B)缀合物18的反义链的制备(1-3B) Preparation of the Antisense Strand of Conjugate 18
缀合物18的反义链与缀合物4的反义链的区别仅在于5′-末端第一个核苷酸修饰不同。按照固相亚磷酰胺法制备反义链时，最后连接的核苷单体为2′-甲氧基修饰尿嘧啶核苷单体(Um)，再经脱保护、偶联、盖帽、氧化四步反应将CPR-I单体(苏州吉玛，货号Cat#13-2601-XX)连接至反义链5′末端，形成5′-磷酸酯修饰。The difference between the antisense strand of conjugate 18 and the antisense strand of conjugate 4 is only the difference in the first nucleotide modification at the 5′-terminus. When the antisense strand is prepared by the solid phase phosphoramidite method, the last nucleoside monomer connected is a 2′-methoxy-modified uridine nucleoside monomer (Um), and then the CPR-I monomer (Suzhou Jima, Cat#13-2601-XX) is connected to the 5′ end of the antisense strand through four steps of deprotection, coupling, capping, and oxidation to form a 5′-phosphate modification.
合成中，使用的通用固相载体，脱保护、偶联、盖帽、氧化或硫化反应条件，切割和脱保护，纯化与脱盐条件与合成正义链相同，预期能够制得具有5′-磷酸酯修饰的缀合物18反义链。During the synthesis, the universal solid phase support, deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection, purification and desalting conditions used are the same as those for synthesizing the sense chain, and it is expected that the antisense chain of conjugate 18 with 5′-phosphate modification can be prepared.
(1-3C)缀合物19的反义链的制备(1-3C) Preparation of the antisense strand of conjugate 19
采用与缀合物18反义链相同的合成工艺，区别在于连接CPR-I单体时，以硫化反应条件代替上述氧化反应条件，预期能够制得具有5′-硫代磷酸酯修饰的缀合物19反义链。The same synthesis process as the antisense chain of conjugate 18 is used, except that when connecting the CPR-I monomer, the above-mentioned oxidation reaction conditions are replaced by sulfurization reaction conditions. It is expected that the antisense chain of conjugate 19 with 5′-phosphorothioate modification can be prepared.
(1-4)合成缀合物4、18、19(1-4) Synthesis of conjugates 4, 18, and 19
对于缀合物4，将S链与AS链分别溶于注射用水中，得到40mg/mL的溶液，以等摩尔比混合，50℃加热15min，室温冷却后，使它们通过氢键形成双链结构。使用超纯水(Milli-Q超纯水仪自制，电阻率18.2MΩ＊cm(25℃))将缀合物稀释至浓度为0.2mg/mL后，利用液质联用仪(LC-MS，Liquid Chromatography-Mass Spectrometry，购于Waters公司，型号：LCTPremier)进行分子量检测。实测值与理论值一致，说明所合成的缀合物4是目标设计的带有L-9缀合分子的双链核酸序列。For conjugate 4, the S chain and the AS chain were dissolved in water for injection to obtain a 40 mg/mL solution, mixed in an equimolar ratio, heated at 50°C for 15 min, and cooled at room temperature to form a double-stranded structure through hydrogen bonds. The conjugate was diluted to a concentration of 0.2 mg/mL using ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2 MΩ*cm (25°C)), and the molecular weight was detected using a liquid chromatography-mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCTPremier). The measured value was consistent with the theoretical value, indicating that the synthesized conjugate 4 was the target designed double-stranded nucleic acid sequence with L-9 conjugated molecules.
对于缀合物18和19，按照上述方法进行退火，预期能够合成缀合物18和19。上述缀合物4、18和19的结构均如式(403)所示。For conjugates 18 and 19, annealing is performed according to the above method, and it is expected that conjugates 18 and 19 can be synthesized. The structures of the above conjugates 4, 18 and 19 are all shown in formula (403).
制备例2：缀合物1-3、5-9与对比缀合物1的制备Preparation Example 2: Preparation of Conjugates 1-3, 5-9 and Comparative Conjugate 1
采用与制备例1相同的方法，预期能够制得题述缀合物，不同的是：1)所述siRNA分别为表2中所示的对应于缀合物1-3、5-9及对比缀合物1的序列；2)当目标序列中含有未修饰的核苷酸时，切割与脱保护条件中，在氨水处理后，相对于单链核酸的量，用0.4ml/μmolN-甲基吡咯烷酮溶解产品，随后加入0.3ml/μmol三乙胺和0.6ml/μmol三乙胺三氢氟酸盐，以脱除核糖上的2′-TBDMS保护。The title conjugate is expected to be prepared by the same method as in Preparation Example 1, except that: 1) the siRNAs are the sequences corresponding to conjugates 1-3, 5-9 and comparative conjugate 1 shown in Table 2; 2) when the target sequence contains unmodified nucleotides, in the cleavage and deprotection conditions, after ammonia treatment, the product is dissolved with 0.4 ml/μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, and then 0.3 ml/μmol triethylamine and 0.6 ml/μmol triethylamine trihydrofluoride are added to remove the 2′-TBDMS protection on the ribose.
题述缀合物中所缀合的siRNA的序列参见表2。See Table 2 for the sequences of the siRNAs conjugated in the subject conjugates.
表2：siRNA缀合物Table 2: siRNA conjugates
制备例3：P10-siHB1M1SVP(缀合物10)的制备Preparation Example 3: Preparation of P10-siHB1M1SVP (Conjugate 10)
(3-1)P-10化合物的合成(3-1) Synthesis of P-10 compound
按照以下方法，合成了P-10化合物：The P-10 compound was synthesized according to the following method:
(3-1-1)GAL5-C4-1的合成(3-1-1) Synthesis of GAL5-C4-1
向40ml N，N-二甲基甲酰胺中加入按照上述(1-1-1)中描述的方法得到的GAL-5(13.43g，30.0mmol)、4-氨基酸叔丁酯盐酸盐(5.87g，30.0mmol)、O-苯并三氮唑-四甲基脲六氟磷酸酯(13.65g，36.0mmol)和二异丙基乙胺(11.63g，90.0mmol)，溶解均一后室温搅拌反应5小时。向反应液中加入300ml饱和碳酸氢钠水溶液，用乙酸乙酯萃取3次，每次200ml，合并有机相，用200ml饱和食盐水洗涤一次，分出有机相，再用无水硫酸钠干燥，减压蒸除溶剂至干得到30.3g油状物粗品GAL5-C4-1，直接进行下一步反应。GAL-5 (13.43 g, 30.0 mmol), 4-amino acid tert-butyl ester hydrochloride (5.87 g, 30.0 mmol), O-benzotriazole-tetramethyluronium hexafluorophosphate (13.65 g, 36.0 mmol) and diisopropylethylamine (11.63 g, 90.0 mmol) obtained by the method described in (1-1-1) were added to 40 ml of N, N-dimethylformamide, and the mixture was stirred at room temperature for 5 hours after being uniformly dissolved. 300 ml of saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the mixture was extracted with ethyl acetate 3 times, 200 ml each time, and the organic phases were combined and washed once with 200 ml of saturated brine, and the organic phase was separated and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to dryness to obtain 30.3 g of crude oil GAL5-C4-1, which was directly subjected to the next step of reaction.
(3-1-2)GAL5-C4-2的合成(3-1-2) Synthesis of GAL5-C4-2
将步骤(3-1-1)中获得的GAL5-C4-1粗品(30.3g，30mmol)溶于180ml甲酸中，室温搅拌反应16小时。蒸发溶剂至干，柱层析纯化(200-300目正相硅胶，二氯甲烷∶甲醇＝100∶18-100∶20梯度洗脱)，收集反应洗脱液，浓缩除去溶剂，得到目标产物GAL5-C4-2共14.84g。The crude GAL5-C4-1 obtained in step (3-1-1) (30.3 g, 30 mmol) was dissolved in 180 ml formic acid and stirred at room temperature for 16 hours. The solvent was evaporated to dryness, and the mixture was purified by column chromatography (200-300 mesh normal phase silica gel, dichloromethane: methanol = 100: 18-100: 20 gradient elution), and the reaction eluate was collected and concentrated to remove the solvent to obtain the target product GAL5-C4-2, a total of 14.84 g.
(3-1-3)P-6的合成：(3-1-3) Synthesis of P-6:
将按照步骤(1-1-4)中描述的方法得到的M-18-Tr(2.02g，4.69mmol)与将步骤(3-1-2)中获得的GAL5-C4-2(8.24g，15.48mmol，由两批产物合并获得)混合溶于47ml乙腈，再加入N-甲基吗啉(3.13g，30.96mmol)，最后加入4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM，4.28g，15.48mmol)，室温搅拌反应2h。以20ml二氯甲烷稀释反应液，10ml饱和碳酸氢钠溶液洗涤有机相，10ml饱和食盐水洗涤有机相，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品，200-300目正相硅胶柱纯化，石油醚装柱，以1wt％三乙胺中和硅胶酸性，以二氯甲烷∶甲醇＝100∶5-100∶7梯度洗脱，收集产物洗脱液，减压蒸干得到纯品P-6共8.27g。M-18-Tr (2.02 g, 4.69 mmol) obtained according to the method described in step (1-1-4) and GAL5-C4-2 (8.24 g, 15.48 mmol, obtained by combining two batches of products) obtained in step (3-1-2) were mixed and dissolved in 47 ml of acetonitrile, and N-methylmorpholine (3.13 g, 30.96 mmol) was added. Finally, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 4.28 g, 15.48 mmol) was added, and the reaction was stirred at room temperature for 2 h. The reaction solution was diluted with 20 ml of dichloromethane, and the organic phase was washed with 10 ml of saturated sodium bicarbonate solution and 10 ml of saturated brine. The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain a crude product, which was purified by a 200-300 mesh normal phase silica gel column, loaded with petroleum ether, and the acidity of the silica gel was neutralized with 1 wt % triethylamine. The product was eluted with a gradient of dichloromethane: methanol = 100: 5-100: 7, and the product eluate was collected and evaporated under reduced pressure to obtain 8.27 g of pure P-6.
(3-1-4)P-7的合成：(3-1-4) Synthesis of P-7:
将按照上述(3-1-3)中得到的P-6(6.82g，3.456mmol)溶于69ml二氯甲烷，再加入二氯乙酸(13.367g，103.67mmol)，室温下反应2h。加入100ml二氯甲烷稀释反应液，再加入饱和碳酸氢钠溶液洗涤调节pH＝7-8之间，水相以二氯甲烷萃取6次，每次30ml，合并有机相，无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品。用200-300目正相硅胶纯化，以10wt％三乙胺中和硅胶酸性，以1wt‰三乙胺平衡柱子，二氯甲烷∶甲醇＝100∶30-100∶40梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到P-7共4.82g。MS m/z：C78H127N10O33，[M+H]+，理论：1732.91，实测：1735.73。P-6 (6.82 g, 3.456 mmol) obtained in (3-1-3) above was dissolved in 69 ml of dichloromethane, and then dichloroacetic acid (13.367 g, 103.67 mmol) was added, and the reaction was allowed to react at room temperature for 2 h. 100 ml of dichloromethane was added to dilute the reaction solution, and then a saturated sodium bicarbonate solution was added to wash and adjust the pH to between 7 and 8. The aqueous phase was extracted with dichloromethane for 6 times, 30 ml each time, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain a crude product. Purification was performed using 200-300 mesh normal phase silica gel, and the acidity of the silica gel was neutralized with 10 wt% triethylamine, and the column was balanced with 1 wt‰ triethylamine, and the gradient elution was performed with dichloromethane: methanol = 100: 30-100: 40. The product eluate was collected, and the solvent was evaporated under reduced pressure to obtain a total of 4.82 g of P-7. MS m/z: C78 H127 N10 O33 , [M+H]+, theory: 1732.91, found: 1735.73.
(3-1-5)P-8的合成：(3-1-5) Synthesis of P-8:
将P-7(2.653g，1.532mmol)和A-1(2.342g，4.596mmol)混合溶于16ml二氯甲烷，加入3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT)(1.375g，4.596mmol)，再加入二异丙基乙胺(1.188g，9.191mmol)，25℃下搅拌反应2h。用10ml饱和碳酸氢钠洗涤有机相，水相以二氯甲烷萃取3次，每次10ml，10ml饱和食盐水洗涤有机相，水相以二氯甲烷萃取2次，每次10ml，合并有机相，用无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜得到粗品。柱纯化使用120g200-300目正相硅胶，以20ml三乙胺中和硅胶酸性，以含1wt％三乙胺的石油醚平衡柱子，以石油醚∶乙酸乙酯∶二氯甲烷∶N，N-二甲基甲酰胺＝1∶1∶1∶0.5-1∶1∶1∶0.6梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品P-8共2.793g。P-7 (2.653 g, 1.532 mmol) and A-1 (2.342 g, 4.596 mmol) were mixed and dissolved in 16 ml of dichloromethane, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT) (1.375 g, 4.596 mmol) was added, and then diisopropylethylamine (1.188 g, 9.191 mmol) was added, and the mixture was stirred at 25°C for 2 h. The organic phase was washed with 10 ml of saturated sodium bicarbonate, and the aqueous phase was extracted with dichloromethane 3 times, 10 ml each time, and the organic phase was washed with 10 ml of saturated saline, and the aqueous phase was extracted with dichloromethane 2 times, 10 ml each time, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure, and vacuum oil pump was used to dry overnight to obtain a crude product. Column purification was performed using 120 g of 200-300 mesh normal phase silica gel. The acidity of the silica gel was neutralized with 20 ml of triethylamine. The column was balanced with petroleum ether containing 1 wt% of triethylamine. The gradient elution was performed with petroleum ether: ethyl acetate: dichloromethane: N, N-dimethylformamide = 1:1:1:0.5-1:1:1:0.6. The product eluate was collected and the solvent was evaporated under reduced pressure to obtain 2.793 g of pure P-8.
(3-1-6)P-9的合成：(3-1-6) Synthesis of P-9:
将P-8(490mg，0.231mm0l)、丁二酸酐(69mg，0.693mmol)和4-二甲氨基吡啶(DMAP，68mg，0.554mmol)混合溶于2.3ml二氯甲烷，再加入二异丙基乙胺(DIPEA，149mg，1.155mmol)，25℃下搅拌反应21h。50ml二氯甲烷稀释反应液，再加入100ml0.5M三乙胺磷酸盐洗涤反应液，水相以二氯甲烷萃取3次，每次10ml，合并有机相，减压蒸干得到粗品。柱纯化使用80g200-300目正相硅胶，以1wt％三乙胺中和硅胶酸性，以二氯甲烷平衡柱子，以含1wt‰三乙胺的二氯甲烷∶甲醇＝100∶18-100∶20梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品P-9缀合分子共200mg。MS m/z：C106H153N10O41，[M-DMTr]+，理论：1921.05，实测：1920.97。P-8 (490 mg, 0.231 mmol), succinic anhydride (69 mg, 0.693 mmol) and 4-dimethylaminopyridine (DMAP, 68 mg, 0.554 mmol) were mixed and dissolved in 2.3 ml of dichloromethane, and diisopropylethylamine (DIPEA, 149 mg, 1.155 mmol) was added, and the mixture was stirred at 25°C for 21 h. The reaction solution was diluted with 50 ml of dichloromethane, and 100 ml of 0.5 M triethylamine phosphate was added to wash the reaction solution. The aqueous phase was extracted with dichloromethane 3 times, 10 ml each time, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain a crude product. Column purification used 80g 200-300 mesh normal phase silica gel, neutralized the acidity of silica gel with 1wt% triethylamine, balanced the column with dichloromethane, eluted with dichloromethane containing 1wt‰ triethylamine: methanol = 100:18-100:20 gradient, collected the product eluate, and evaporated the solvent under reduced pressure to obtain a total of 200mg of pure P-9 conjugated molecules. MS m/z: C106 H153 N10 O41 , [M-DMTr]+, theoretical: 1921.05, measured: 1920.97.
(3-1-7)P-10的合成(3-1-7) Synthesis of P-10
通过与制备例1中步骤(1-1-9)相同的方法，制备P-10。不同的是以P-9缀合分子代替L-9缀合分子，得到连接固相载体的P-9缀合分子。P-10 was prepared by the same method as step (1-1-9) in Preparation Example 1, except that the L-9 conjugated molecule was replaced by the P-9 conjugated molecule to obtain the P-9 conjugated molecule connected to the solid phase carrier.
(3-2)合成P10-siHB1M1SVP缀合物(3-2) Synthesis of P10-siHB1M1SVP conjugate
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物10，不同的是以P-10化合物代替L-10化合物起始正义链合成。预期可以得到P10-siHB1M1SVP缀合物，其结构如式(404)所示。Conjugate 10 was prepared by the same method as steps (1-2), (1-3A), and (1-4) in Preparation Example 1, except that P-10 was used instead of L-10 compound to initiate the synthesis of the positive chain. It is expected that a P10-siHB1M1SVP conjugate can be obtained, and its structure is shown in Formula (404).
制备例4：R5-siHB1M1SVP缀合物(缀合物11)的制备Preparation Example 4: Preparation of R5-siHB1M1SVP conjugate (Conjugate 11)
(4-1)R-5化合物的合成(4-1) Synthesis of R-5 compound
按照以下方法，合成了R-5化合物：The R-5 compound was synthesized according to the following method:
(4-1-1)GAL-C7-1的合成(4-1-1) Synthesis of GAL-C7-1
将按照步骤(1-1-1b)中描述的方法得到的GAL-3(26.4g，80.2mmol)溶于134ml无水1，2-二氯乙烷中，加入分子筛粉末60g，再加入7-辛烯-1-醇(11.3g，88.2mmol)，室温下搅拌反应10分钟，冰浴和氮气保护下加入三氟甲基磺酸三甲基硅酯(8.9g，40.1mmol)，室温搅拌反应24小时。过滤除去分子筛粉末，滤液中加入500ml饱和碳酸氢钠水溶液洗涤，分出有机相，水相用100ml二氯甲烷萃取一次，合并有机相并用250ml饱和食盐水洗涤一次，分出有机相，用无水硫酸钠干燥，减压蒸除溶剂至干得到黄色糖稀状产品GAL-C7-1 33.3g，不进行纯化直接进行下一步氧化反应。GAL-3 (26.4 g, 80.2 mmol) obtained according to the method described in step (1-1-1b) was dissolved in 134 ml of anhydrous 1,2-dichloroethane and added 60g of molecular sieve powder was added, and 7-octen-1-ol (11.3g, 88.2mmol) was added, and the mixture was stirred at room temperature for 10 minutes. Trimethylsilyl trifluoromethanesulfonate (8.9g, 40.1mmol) was added under ice bath and nitrogen protection, and the mixture was stirred at room temperature for 24 hours. Filter and remove Molecular sieve powder, add 500 ml of saturated sodium bicarbonate aqueous solution to the filtrate for washing, separate the organic phase, extract the aqueous phase once with 100 ml of dichloromethane, combine the organic phases and wash once with 250 ml of saturated brine, separate the organic phase, dry it with anhydrous sodium sulfate, and evaporate the solvent to dryness under reduced pressure to obtain 33.3 g of yellow syrupy product GAL-C7-1, which is directly carried out to the next oxidation reaction without purification.
(4-1-2)GAL-C7-2的合成(4-1-2) Synthesis of GAL-C7-2
将按照步骤(4-1-1)中得到的GAL-C7-1(33.3g，72.8mmol)溶于160ml二氯甲烷和160ml乙腈的混合溶剂中，分别加入216ml水和高碘酸钠固体(62.3g，291.2mmol)，冰水浴下搅拌10分钟，加入催化剂三氯化钌(498mg，2.4mmol)自然升至室温搅拌反应23小时。反应液加入200ml水稀释搅拌，加饱和碳酸氢钠调节pH值为7.5，分掉有机相，水相再用二氯甲烷萃取三次，弃去有机相，水相用柠檬酸固体调节pH约为3，用二氯甲烷萃取三次，每次200ml，合并有机相，无水硫酸钠干燥，减压蒸除溶剂后柱层析(200-300目正相硅胶，二氯甲烷∶甲醇＝100∶18-100∶20梯度洗脱)纯化得到白色泡沫状固体产品GAL-C7-222.4g。MS m/z：C21H32NO11，[M+H]+，理论：476.50，实测：475.94。Dissolve GAL-C7-1 (33.3 g, 72.8 mmol) obtained in step (4-1-1) in a mixed solvent of 160 ml of dichloromethane and 160 ml of acetonitrile, add 216 ml of water and solid sodium periodate (62.3 g, 291.2 mmol) respectively, stir under an ice-water bath for 10 minutes, add catalyst ruthenium trichloride (498 mg, 2.4 mmol), naturally warm to room temperature and stir to react for 23 hours. The reaction solution was diluted with 200 ml of water and stirred, and saturated sodium bicarbonate was added to adjust the pH value to 7.5. The organic phase was separated, and the aqueous phase was extracted three times with dichloromethane. The organic phase was discarded, and the aqueous phase was adjusted to pH about 3 with solid citric acid, and extracted three times with dichloromethane, 200 ml each time. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. After column chromatography (200-300 mesh normal phase silica gel, dichloromethane: methanol = 100: 18-100: 20 gradient elution) was performed to purify the white foam solid product GAL-C7-222.4 g. MS m/z: C21 H32 NO11 , [M+H]+, theoretical: 476.50, found: 475.94.
(4-1-3)R-1的合成：(4-1-3) Synthesis of R-1:
将按照步骤(1-1-4)中描述的方法得到的M-18-Tr(2.02g，4.69mmol)与GAL-C7-2(7.36g，15.48mmol)混合溶于47ml乙腈，再加入N-甲基吗啉(3.13g，30.96mmol)，最后加入4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM，4.28g，15.48mmol)，室温搅拌反应2h。以200ml二氯甲烷稀释反应液，100ml饱和碳酸氢钠溶液洗涤有机相，100ml饱和食盐水洗涤有机相，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品，200-300目正相硅胶柱纯化，石油醚装柱，以1wt％三乙胺中和硅胶酸性，二氯甲烷∶甲醇＝100∶5-100∶7梯度洗脱，收集产物洗脱液，减压蒸干得到纯品R-1 7.82g。Mix M-18-Tr (2.02 g, 4.69 mmol) obtained according to the method described in step (1-1-4) and GAL-C7-2 (7.36 g, 15.48 mmol) and dissolve in 47 ml of acetonitrile, then add N-methylmorpholine (3.13 g, 30.96 mmol), and finally add 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 4.28 g, 15.48 mmol), and stir the reaction at room temperature for 2 h. The reaction solution was diluted with 200 ml of dichloromethane, and the organic phase was washed with 100 ml of saturated sodium bicarbonate solution and 100 ml of saturated brine. The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain a crude product, which was purified by a 200-300 mesh normal phase silica gel column, loaded with petroleum ether, and the acidity of the silica gel was neutralized with 1 wt% triethylamine. The product was eluted with a gradient of dichloromethane: methanol = 100: 5-100: 7, and the product eluate was collected and evaporated to dryness under reduced pressure to obtain 7.82 g of pure R-1.
(4-1-4)R-2的合成：(4-1-4) Synthesis of R-2:
将R-1(6.23g，3.456mmol)溶于69ml二氯甲烷，再加入二氯乙酸(13.367g，103.67mmol)，室温下反应2h。加入100ml二氯甲烷稀释反应液，再加饱和碳酸氢钠溶液洗涤调节pH＝7-8之间，水相以二氯甲烷萃取6次，每次30ml，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品。200-300目正相硅胶，以10wt％三乙胺中和硅胶酸性，以1wt‰三乙胺平衡柱子，二氯甲烷∶甲醇＝100∶30-100∶40梯度洗脱，减压蒸干溶剂得到纯品R-24.49g。R-1 (6.23 g, 3.456 mmol) was dissolved in 69 ml of dichloromethane, and then dichloroacetic acid (13.367 g, 103.67 mmol) was added, and the reaction was allowed to react at room temperature for 2 h. 100 ml of dichloromethane was added to dilute the reaction solution, and then saturated sodium bicarbonate solution was added to wash and adjust the pH to between 7 and 8. The aqueous phase was extracted with dichloromethane for 6 times, 30 ml each time, and the organic phases were combined and dried with anhydrous sodium sulfate. After filtering, the solvent was evaporated under reduced pressure to obtain a crude product. 200-300 mesh normal phase silica gel, 10 wt% triethylamine was used to neutralize the acidity of the silica gel, and 1 wt‰ triethylamine was used to balance the column, and dichloromethane: methanol = 100: 30-100: 40 gradient elution was performed, and the solvent was evaporated under reduced pressure to obtain 4.49 g of pure R-2.
(4-1-5)R-3的合成：(4-1-5) Synthesis of R-3:
将R-2(2.391g，1.532mmol)和A-1(2.342g，4.596mmol)混合溶于16ml二氯甲烷，加入3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT)(1.375g，4.596mmol)，再加入二异丙基乙胺(1.188g，9.191mmol)，25℃下搅拌反应2h。用10ml饱和碳酸氢钠洗涤有机相，水相以二氯甲烷萃取3次，每次10ml，以10ml饱和食盐水洗涤有机相，水相以二氯甲烷萃取2次，每次10ml，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜得到粗品。柱纯化使用120g 200-300目正相硅胶，以20ml三乙胺中和硅胶酸性，以含1wt％三乙胺的石油醚平衡柱子，石油醚∶乙酸乙酯∶二氯甲烷∶N，N-二甲基甲酰胺＝1∶1∶1∶0.5-1∶1∶1∶0.6梯度洗脱，减压蒸干溶剂得到纯品R-3 2.642g。R-2 (2.391 g, 1.532 mmol) and A-1 (2.342 g, 4.596 mmol) were mixed and dissolved in 16 ml of dichloromethane, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT) (1.375 g, 4.596 mmol) was added, and then diisopropylethylamine (1.188 g, 9.191 mmol) was added, and the mixture was stirred at 25°C for 2 h. The organic phase was washed with 10 ml of saturated sodium bicarbonate, the aqueous phase was extracted with dichloromethane 3 times, 10 ml each time, the organic phase was washed with 10 ml of saturated saline, the aqueous phase was extracted with dichloromethane 2 times, 10 ml each time, the organic phases were combined and dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure after filtration, and the crude product was obtained by bubbling with a vacuum oil pump overnight. Column purification was performed using 120 g of 200-300 mesh normal phase silica gel. The acidity of the silica gel was neutralized with 20 ml of triethylamine. The column was equilibrated with petroleum ether containing 1 wt% of triethylamine. The gradient elution was performed with petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.5-1:1:1:0.6. The solvent was evaporated under reduced pressure to obtain 2.642 g of pure R-3.
(4-1-6)R-4的合成：(4-1-6) Synthesis of R-4:
将R-3(795mg，0.4074mmol)、丁二酸酐(82mg，0.8148mmol)和4-二甲氨基吡啶(DMAP，100mg，0.8148mmol)混合溶于4ml二氯甲烷，再加入二异丙基乙胺(DIPEA，100mg，0.8148mmol)，25℃下搅拌反应18h。5ml 0.5M三乙胺磷酸盐洗涤反应液，水相以二氯甲烷萃取3次，每次5ml，合并有机相减压蒸干得到粗品。柱纯化使用30g 200-300目正相硅胶，以1wt％三乙胺中和硅胶酸性，以二氯甲烷平衡柱子，含1wt‰三乙胺的二氯甲烷∶甲醇＝100∶18-100∶20梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品R-4缀合分子505mg。R-3 (795 mg, 0.4074 mmol), succinic anhydride (82 mg, 0.8148 mmol) and 4-dimethylaminopyridine (DMAP, 100 mg, 0.8148 mmol) were mixed and dissolved in 4 ml of dichloromethane, and diisopropylethylamine (DIPEA, 100 mg, 0.8148 mmol) was added, and the mixture was stirred at 25°C for 18 h. The reaction solution was washed with 5 ml of 0.5 M triethylamine phosphate, and the aqueous phase was extracted with dichloromethane 3 times, 5 ml each time, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain a crude product. Column purification was performed using 30 g of 200-300 mesh normal phase silica gel. The acidity of the silica gel was neutralized with 1 wt% triethylamine. The column was equilibrated with dichloromethane. Gradient elution was performed with dichloromethane containing 1 wt‰ triethylamine: methanol = 100:18-100:20. The product eluate was collected and the solvent was evaporated under reduced pressure to obtain 505 mg of pure R-4 conjugated molecule.
(4-1-7)R-5的合成：(4-1-7) Synthesis of R-5:
通过与制备例1中步骤(1-1-9)相同的方法，制备R-5。不同的是以R-4缀合分子代替L-9缀合分子，得到连接固相载体的R-4缀合分子。R-5 was prepared by the same method as step (1-1-9) in Preparation Example 1, except that the L-9 conjugated molecule was replaced by the R-4 conjugated molecule to obtain the R-4 conjugated molecule connected to the solid phase carrier.
(4-2)合成R5-siHB1M1SVP缀合物(4-2) Synthesis of R5-siHB1M1SVP conjugate
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物11，不同的是以R-5化合物代替L-10化合物起始正义链合成。预期可以得到R5-siHB1M1SVP缀合物，其结构如式(407)所示。Conjugate 11 was prepared by the same method as steps (1-2), (1-3A), and (1-4) in Preparation Example 1, except that R-5 compound was used instead of L-10 compound to initiate the synthesis of the positive chain. It is expected that R5-siHB1M1SVP conjugate can be obtained, and its structure is shown in Formula (407).
制备例5：LA5-siHB1M1SVP缀合物(缀合物12)的制备Preparation Example 5: Preparation of LA5-siHB1M1SVP conjugate (Conjugate 12)
按照以下工艺路线，预期能够合成LA-5化合物：According to the following process route, it is expected that LA-5 compound can be synthesized:
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物12，不同的是以LA-5化合物代替L-10化合物起始正义链合成。预期可以得到LA5-siHB1M1SVP缀合物，其结构如式(412)所示。Conjugate 12 was prepared by the same method as steps (1-2), (1-3A), and (1-4) in Preparation Example 1, except that LA-5 compound was used instead of L-10 compound to initiate the synthesis of the positive chain. It is expected that LA5-siHB1M1SVP conjugate can be obtained, and its structure is shown in Formula (412).
制备例6：LB5-siHB1M1SVP缀合物(缀合物13)的制备Preparation Example 6: Preparation of LB5-siHB1M1SVP conjugate (Conjugate 13)
(6-1)LB-5化合物的合成(6-1) Synthesis of LB-5 compound
按照以下方法，合成了LB-5化合物：The LB-5 compound was synthesized according to the following method:
(6-1-1)LB-1的合成：(6-1-1) Synthesis of LB-1:
将按照步骤(1-1-6)中描述的方法得到的L-8(5.0g，3.386mmol)、己二酸酐(870mg，6.772mmol)和4-二甲氨基吡啶(DMAP，827mg，6.772mmol)混合溶于130ml二氯甲烷，再加入二异丙基乙胺(DIPEA，2.2g，16.931mmol)，25℃下搅拌反应4h。加入70ml二氯甲烷稀释反应液，以0.5M三乙胺磷酸盐洗涤反应液，水相以二氯甲烷萃取4次，每次10ml，合并有机相减压蒸干得到粗品。柱纯化使用120g200-300目正相硅胶，以1wt％三乙胺中和硅胶酸性，以二氯甲烷平衡柱子，石油醚∶乙酸乙酯∶二氯甲烷∶甲醇＝1∶1∶1∶0.2-1∶1∶1∶1梯度洗脱，减压蒸干溶剂得到纯品LB-1 4.267g。L-8 (5.0 g, 3.386 mmol), adipic anhydride (870 mg, 6.772 mmol) obtained according to the method described in step (1-1-6), and 4-dimethylaminopyridine (DMAP, 827 mg, 6.772 mmol) were mixed and dissolved in 130 ml of dichloromethane, and diisopropylethylamine (DIPEA, 2.2 g, 16.931 mmol) was added, and the mixture was stirred at 25°C for 4 h. 70 ml of dichloromethane was added to dilute the reaction solution, and the reaction solution was washed with 0.5 M triethylamine phosphate, and the aqueous phase was extracted with dichloromethane 4 times, 10 ml each time, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain a crude product. Column purification was performed using 120 g of 200-300 mesh normal phase silica gel. The acidity of the silica gel was neutralized with 1 wt % triethylamine. The column was balanced with dichloromethane. Gradient elution was performed with petroleum ether: ethyl acetate: dichloromethane: methanol = 1:1:1:0.2-1:1:1:1. The solvent was evaporated under reduced pressure to obtain 4.267 g of pure product LB-1.
(6-1-2)LB-2的合成：(6-1-2) Synthesis of LB-2:
将按照步骤(6-1-1)中描述的方法得到的LB-1(4.697g，2.753mmol，由两批次产物合并而得)、3-氨基-1，2-丙二醇(313mg，3.442mmol)、4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM，953mg，3.442mmol)和N-甲基吗啉(700mg，6.884mmol)先后加入30ml乙腈和3ml甲醇的混合液中，室温搅拌反应过夜。蒸发溶剂至干，柱层析(200-300目正相硅胶，二氯甲烷∶甲醇＝1∶0.07-1∶0.5梯度洗脱)纯化，收集产物洗脱液，浓缩除去溶剂，得到目标产物LB-2 3.27g。LB-1 (4.697 g, 2.753 mmol, obtained by combining two batches of products) obtained according to the method described in step (6-1-1), 3-amino-1,2-propanediol (313 mg, 3.442 mmol), 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 953 mg, 3.442 mmol) and N-methylmorpholine (700 mg, 6.884 mmol) were added to a mixture of 30 ml of acetonitrile and 3 ml of methanol, and stirred at room temperature for overnight reaction. The solvent was evaporated to dryness, and the mixture was purified by column chromatography (200-300 mesh normal phase silica gel, dichloromethane: methanol = 1: 0.07-1: 0.5 gradient elution), and the product eluate was collected and concentrated to remove the solvent to obtain 3.27 g of the target product LB-2.
(6-1-3)LB-3的合成：(6-1-3) Synthesis of LB-3:
将LB-2(2.27g，1.353mmol)用14ml无水吡啶溶解。再加入4，4′-双甲氧基三苯甲基氯(688mg，2.03mmol)室温下搅拌反应过夜。加150ml甲醇淬灭，蒸发溶剂至干。柱层析(200-300目正相硅胶，二氯甲烷∶甲醇＝1∶0.05-1∶0.2梯度洗脱)纯化，收集产物洗脱液，浓缩除去溶剂，得到目标产物LB-3 1.647g。Dissolve LB-2 (2.27 g, 1.353 mmol) in 14 ml of anhydrous pyridine. Add 4,4′-bismethoxytrityl chloride (688 mg, 2.03 mmol) and stir the reaction at room temperature overnight. Add 150 ml of methanol to quench, and evaporate the solvent to dryness. Purify by column chromatography (200-300 mesh normal phase silica gel, dichloromethane: methanol = 1: 0.05-1: 0.2 gradient elution), collect the product eluate, and concentrate to remove the solvent to obtain the target product LB-3 1.647 g.
(6-1-4)LB-4的合成：(6-1-4) Synthesis of LB-4:
将LB-3(822mg，0.415mmol)、丁二酸酐(83g，0.83mmol)和4-二甲氨基吡啶(DMAP，102mg，0.83mmol)混合溶于4ml二氯甲烷，再加入DIPEA(270mg，2.075mmol)，25℃下搅拌反应过夜。0.5M三乙胺磷酸盐洗涤反应液3次，水相以二氯甲烷萃取3次，每次2ml，合并有机相减压蒸干得到粗品。柱纯化使用200-300目正相硅胶，以5wt％三乙胺中和硅胶酸性，以石油醚平衡柱子，用含1wt‰三乙胺的二氯甲烷∶甲醇＝100∶5-100∶20梯度洗脱，减压蒸干溶剂得到纯品LB-4缀合分子787mg。LB-3 (822 mg, 0.415 mmol), succinic anhydride (83 g, 0.83 mmol) and 4-dimethylaminopyridine (DMAP, 102 mg, 0.83 mmol) were mixed and dissolved in 4 ml of dichloromethane, and DIPEA (270 mg, 2.075 mmol) was added, and the reaction was stirred at 25°C overnight. The reaction solution was washed 3 times with 0.5 M triethylamine phosphate, and the aqueous phase was extracted 3 times with dichloromethane, 2 ml each time, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain a crude product. Column purification used 200-300 mesh normal phase silica gel, 5 wt% triethylamine was used to neutralize the acidity of the silica gel, and the column was balanced with petroleum ether, and the gradient elution was carried out with dichloromethane containing 1 wt‰ triethylamine: methanol = 100: 5-100: 20, and the solvent was evaporated under reduced pressure to obtain 787 mg of pure LB-4 conjugated molecule.
(6-1-5)LB-5的合成：(6-1-5) Synthesis of LB-5:
通过与制备例1中步骤(1-1-9)相同的方法，制备LB-5。不同的是以LB-4缀合分子代替L-9缀合分子，得到连接固相载体的LB-4缀合分子。LB-5 was prepared by the same method as step (1-1-9) in Preparation Example 1, except that the L-9 conjugated molecule was replaced by the LB-4 conjugated molecule to obtain the LB-4 conjugated molecule connected to the solid phase carrier.
(6-2)合成LB5-siHB1M1SVP缀合物(6-2) Synthesis of LB5-siHB1M1SVP conjugate
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物13，不同的是以LB-5化合物代替L-10化合物起始正义链合成。预期可以得到LB5-siHB1M1SVP缀合物，其结构如式(413)所示。Conjugate 13 was prepared by the same method as steps (1-2), (1-3A), and (1-4) in Preparation Example 1, except that LB-5 was used instead of L-10 compound to initiate the synthesis of the positive chain. It is expected that LB5-siHB1M1SVP conjugate can be obtained, and its structure is shown in Formula (413).
制备例7：V8-siHB1M1SVP缀合物(缀合物14)的合成Preparation Example 7: Synthesis of V8-siHB1M1SVP conjugate (Conjugate 14)
按照以下工艺路线，预期能够合成V-8化合物：According to the following process route, it is expected that V-8 compounds can be synthesized:
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物14，不同的是以V-8化合物代替L-10化合物起始正义链合成。预期可以得到V8-siHB1M1SVP缀合物，其结构如式(414)所示。Conjugate 14 was prepared by the same method as steps (1-2), (1-3A), and (1-4) in Preparation Example 1, except that V-8 compound was used instead of L-10 compound to initiate the synthesis of the positive chain. It is expected that a V8-siHB1M1SVP conjugate can be obtained, and its structure is shown in Formula (414).
制备例8：W8-siHB1M1SVP缀合物(缀合物15)的制备Preparation Example 8: Preparation of W8-siHB1M1SVP conjugate (Conjugate 15)
(8-1)W-8化合物的合成(8-1) Synthesis of W-8 compound
按照以下方法，合成了W-8化合物：The W-8 compound was synthesized according to the following method:
(8-1-1)W-1的合成：(8-1-1) Synthesis of W-1:
将W-0(2.024g，10mmol)溶于25ml乙腈中，再加三乙胺(4.048g，40mmol)，冰水浴冷却至0℃左右，加入三氟乙酸乙酯(5.683g，40mmol)，室温下反应22h。减压蒸干溶剂，真空油泵发泡干燥18h，得到5.835g粗品固体W-1。Dissolve W-0 (2.024 g, 10 mmol) in 25 ml of acetonitrile, add triethylamine (4.048 g, 40 mmol), cool to about 0°C in an ice-water bath, add ethyl trifluoroacetate (5.683 g, 40 mmol), and react at room temperature for 22 h. Evaporate the solvent under reduced pressure, and dry it with a vacuum oil pump for 18 h to obtain 5.835 g of crude solid W-1.
(8-1-2)W-2的合成：(8-1-2) Synthesis of W-2:
将W-1粗品(5.835g，10mmol)溶于50ml二氯甲烷，向反应液中加入TrCl(3.345g，12mmol)和三乙胺(1.518g，15mmol)，室温下搅拌反应20h。用20ml饱和碳酸氢钠洗涤反应液2次，用20ml饱和食盐水洗涤1次，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干有机溶剂，真空油泵发泡干燥过夜，得到粗品固体W-2 8.012g。不经处理，进行下一步脱保护反应。The crude product of W-1 (5.835 g, 10 mmol) was dissolved in 50 ml of dichloromethane, TrCl (3.345 g, 12 mmol) and triethylamine (1.518 g, 15 mmol) were added to the reaction solution, and the reaction was stirred at room temperature for 20 h. The reaction solution was washed twice with 20 ml of saturated sodium bicarbonate and once with 20 ml of saturated brine. The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the organic solvent was evaporated under reduced pressure. The crude solid W-2 (8.012 g) was obtained by bubbling and drying with a vacuum oil pump overnight. The next step of deprotection reaction was carried out without treatment.
(8-1-3)W-3的合成：(8-1-3) Synthesis of W-3:
将W-2粗品(8.012g，10mmol)溶于100ml甲醇，再加入100ml甲胺水溶液(40wt％)，在50℃下搅拌反应23h。过滤除去不溶颗粒物，减压蒸干溶剂，加入200ml体积比为1∶1的DCM-甲醇混合溶剂，以50ml饱和碳酸氢钠洗涤有机相，水相再用二氯甲烷萃取3次，每次50ml，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜，200-300目正相硅胶柱纯化，石油醚装柱，以1wt％三乙胺中和硅胶酸性，以二氯甲烷∶甲醇∶氨水(25wt％)＝1∶1∶0.05-1∶1∶0.25梯度洗脱，收集产物洗脱液，减压蒸干溶剂，真空油泵发泡干燥得到纯品W-3 3.062g。The crude product of W-2 (8.012 g, 10 mmol) was dissolved in 100 ml of methanol, and then 100 ml of methylamine aqueous solution (40 wt%) was added, and the reaction was stirred at 50 ° C for 23 h. The insoluble particles were filtered off, the solvent was evaporated under reduced pressure, 200 ml of DCM-methanol mixed solvent with a volume ratio of 1:1 was added, and the organic phase was washed with 50 ml of saturated sodium bicarbonate. The aqueous phase was extracted with dichloromethane for 3 times, 50 ml each time, and the organic phases were combined and dried with anhydrous sodium sulfate. After filtering, the solvent was evaporated under reduced pressure, and vacuum oil pump was used for foaming and drying overnight. Purification was performed on a 200-300 mesh normal phase silica gel column, and petroleum ether was used as the column. The acidity of the silica gel was neutralized with 1 wt% triethylamine, and the gradient elution was performed with dichloromethane: methanol: ammonia water (25 wt%) = 1: 1: 0.05-1: 1: 0.25. The product eluate was collected, the solvent was evaporated under reduced pressure, and the pure product W-3 3.062 g was obtained by foaming and drying with a vacuum oil pump.
(8-1-4)W-4的合成：(8-1-4) Synthesis of W-4:
将W-3(0.675g，1.517mmol)与GAL-C7-2(2.60g，5.46mmol)混合溶于47ml乙腈，再加二异丙基乙胺(1.57g，12.14mmol)，最后加入3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT，1.816g，6.04mmol)，室温搅拌反应2.5h。以100ml二氯甲烷稀释反应液，80ml饱和碳酸氢钠溶液洗涤有机相，80ml饱和食盐水洗涤有机相，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品，200-300目正相硅胶柱纯化，石油醚装柱，以1wt％三乙胺中和硅胶酸性，以二氯甲烷∶甲醇＝100∶5-100∶7梯度洗脱，收集产物洗脱液，减压蒸干得到纯品W-4 1.610g。Mix W-3 (0.675 g, 1.517 mmol) and GAL-C7-2 (2.60 g, 5.46 mmol) and dissolve in 47 ml of acetonitrile, then add diisopropylethylamine (1.57 g, 12.14 mmol), and finally add 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT, 1.816 g, 6.04 mmol), and stir at room temperature for 2.5 h. The reaction solution was diluted with 100 ml of dichloromethane, and the organic phase was washed with 80 ml of saturated sodium bicarbonate solution and 80 ml of saturated brine. The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain a crude product, which was purified by a 200-300 mesh normal phase silica gel column, loaded with petroleum ether, and the acidity of the silica gel was neutralized with 1 wt% triethylamine. The product was eluted with a gradient of dichloromethane: methanol = 100: 5-100: 7, and the product eluate was collected and evaporated to dryness under reduced pressure to obtain 1.610 g of pure W-4.
(8-1-5)W-5的合成：(8-1-5) Synthesis of W-5:
将W-4(1.61g，0.886mmol)溶于125ml二氯甲烷，再加入二氯乙酸(3.5ml，42.43mmol)，室温下反应1h。加入150ml吡啶中和反应液，减压蒸干溶剂得粗品。200-300目正相硅胶，10wt％三乙胺中和硅胶酸性，1wt‰三乙胺平衡柱子，二氯甲烷∶甲醇＝100∶30-100∶40梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品W-5 1.26g。Dissolve W-4 (1.61 g, 0.886 mmol) in 125 ml of dichloromethane, add dichloroacetic acid (3.5 ml, 42.43 mmol), and react at room temperature for 1 h. Add 150 ml of pyridine to neutralize the reaction solution, and evaporate the solvent under reduced pressure to obtain a crude product. 200-300 mesh normal phase silica gel, 10 wt% triethylamine to neutralize the acidity of silica gel, 1 wt‰ triethylamine to balance the column, dichloromethane: methanol = 100: 30-100: 40 gradient elution, collect the product eluate, and evaporate the solvent under reduced pressure to obtain 1.26 g of pure W-5.
(8-1-6)W-6的合成：(8-1-6) Synthesis of W-6:
将W-5(1.25g，0.793mmol)和按照步骤(1-1-7a)中描述的方法得到的A-1(1.21g，2.38mmol)混合溶于12ml二氯甲烷，加入3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT，0.712g，2.38mmol)，再加入二异丙基乙胺(0.615g，4.76mmol)，25℃下搅拌反应3h。用80ml饱和碳酸氢钠洗涤有机相，水相以二氯甲烷萃取3次，每次10ml，合并有机相并以10ml饱和食盐水洗涤，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜得到粗品。柱纯化使用185g200-300目正相硅胶，20ml三乙胺中和硅胶酸性，以含1wt％三乙胺的石油醚平衡柱子，以石油醚∶乙酸乙酯∶二氯甲烷∶N，N-二甲基甲酰胺＝1∶1∶1∶0.1-1∶1∶0.7梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品W-6 1.57g。W-5 (1.25 g, 0.793 mmol) and A-1 (1.21 g, 2.38 mmol) obtained by the method described in step (1-1-7a) were mixed and dissolved in 12 ml of dichloromethane, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT, 0.712 g, 2.38 mmol) were added, and then diisopropylethylamine (0.615 g, 4.76 mmol) was added, and the reaction was stirred at 25°C for 3 h. The organic phase was washed with 80 ml of saturated sodium bicarbonate, and the aqueous phase was extracted with dichloromethane 3 times, 10 ml each time, and the organic phases were combined and washed with 10 ml of saturated brine, and the organic phases were combined and dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure, and vacuum oil pump was dried overnight to obtain a crude product. Column purification used 185 g 200-300 mesh normal phase silica gel, 20 ml triethylamine to neutralize the acidity of the silica gel, balanced the column with petroleum ether containing 1 wt% triethylamine, and gradient eluted with petroleum ether: ethyl acetate: dichloromethane: N, N-dimethylformamide = 1:1:1:0.1-1:1:0.7. The product eluate was collected and the solvent was evaporated under reduced pressure to obtain 1.57 g of pure W-6.
(8-1-7)W-7的合成：(8-1-7) Synthesis of W-7:
将W-6(1.238g，0.63mmol)、丁二酸酐(0.189g，1.89mmol)和4-二甲氨基吡啶(DMAP，0.231g，1.89mmol)混合溶于7ml二氯甲烷，再加入DIEA(0.407g，3.15mmol)，25℃下搅拌反应24h。以5ml 0.5M三乙胺磷酸盐洗涤反应液，水相以二氯甲烷萃取3次，每次5ml，合并有机相减压蒸干得到粗品。柱纯化使用30g 200-300目正相硅胶，以1wt％三乙胺中和硅胶酸性，二氯甲烷平衡柱子，以含1wt‰三乙胺的二氯甲烷∶甲醇＝100∶18-100∶20梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品W-7缀合分子1.033g。MS m/z：C101H146N7O38，[M-DMTr]+，理论：1763.92，实测：1763.21。W-6 (1.238 g, 0.63 mmol), succinic anhydride (0.189 g, 1.89 mmol) and 4-dimethylaminopyridine (DMAP, 0.231 g, 1.89 mmol) were mixed and dissolved in 7 ml of dichloromethane, and DIEA (0.407 g, 3.15 mmol) was added, and the mixture was stirred at 25°C for 24 h. The reaction solution was washed with 5 ml of 0.5 M triethylamine phosphate, and the aqueous phase was extracted with dichloromethane 3 times, 5 ml each time, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain a crude product. Column purification used 30 g of 200-300 mesh normal phase silica gel, and the acidity of the silica gel was neutralized with 1 wt% triethylamine, and the column was balanced with dichloromethane, and the gradient elution was carried out with dichloromethane containing 1 wt‰ triethylamine: methanol = 100: 18-100: 20, and the product eluate was collected, and the solvent was evaporated under reduced pressure to obtain 1.033 g of pure W-7 conjugated molecule. MS m/z:C101H146N7O38, [M- DMTr]+, theory: 1763.92,found : 1763.21.
(8-1-8)W-8的合成：(8-1-8) Synthesis of W-8:
通过与制备例1中步骤(1-1-9)相同的方法，制备W-8。不同的是以W-7缀合分子代替L-9缀合分子，得到连接固相载体的W-7缀合分子。W-8 was prepared by the same method as step (1-1-9) in Preparation Example 1, except that the L-9 conjugated molecule was replaced by the W-7 conjugated molecule to obtain the W-7 conjugated molecule connected to the solid phase carrier.
(8-2)合成W8-siHB1M1SVP缀合物(8-2) Synthesis of W8-siHB1M1SVP conjugate
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物15，不同的是以W-8化合物代替L-10化合物起始正义链合成。预期可以得到W8-siHB1M1SVP缀合物，其结构如式(415)所示。Conjugate 15 was prepared by the same method as steps (1-2), (1-3A), and (1-4) in Preparation Example 1, except that W-8 was used instead of L-10 compound to initiate the synthesis of the positive chain. It is expected that a W8-siHB1M1SVP conjugate can be obtained, and its structure is shown in Formula (415).
制备例9：X8-siHB1M1SVP缀合物(缀合物16)的制备Preparation Example 9: Preparation of X8-siHB1M1SVP conjugate (Conjugate 16)
按照以下工艺路线，预期能够合成X-8化合物：According to the following process route, it is expected that X-8 compound can be synthesized:
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物16，不同的是以X-8化合物代替L-10化合物起始正义链合成。预期可以得到X8-siHB1M1SVP缀合物，其结构如式(421)所示。Conjugate 16 was prepared by the same method as steps (1-2), (1-3A), and (1-4) in Preparation Example 1, except that X-8 was used instead of L-10 to initiate the synthesis of the positive chain. It is expected that X8-siHB1M1SVP conjugate can be obtained, and its structure is shown in Formula (421).
制备例10：Z5-siHB1M1SVP缀合物(缀合物17)的制备Preparation Example 10: Preparation of Z5-siHB1M1SVP conjugate (Conjugate 17)
(10-1)Z-5化合物的合成(10-1) Synthesis of Z-5 compound
按照以下方法，合成了Z-5化合物：Compound Z-5 was synthesized according to the following method:
(10-1-1)Z-1的合成：(10-1-1) Synthesis of Z-1:
将按照步骤(8-1-3)中描述的方法得到的W-3(1.50g，3.37mmol)与按照步骤(3-1-2)中描述的方法得到的GAL5-C4-2(7.18g，13.48mmol)混合溶于34ml二氯甲烷，再加入二异丙基乙胺(3.48g，26.96mmol)，最后加入3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT，4.04g，13.48mmol)，室温搅拌反应4.5h。以100ml二氯甲烷稀释反应液，80ml饱和碳酸氢钠溶液洗涤有机相，80ml饱和食盐水洗涤有机相，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品，200-300目正相硅胶柱纯化，石油醚装柱，以1wt％三乙胺中和硅胶酸性，以二氯甲烷∶甲醇＝30∶1-15∶1梯度洗脱，收集产物洗脱液，减压蒸干得到纯品Z-13.97g。MS m/z：C98H143N10O33，[M+H]+，理论：1987.98，实测：1987.90。W-3 (1.50 g, 3.37 mmol) obtained according to the method described in step (8-1-3) and GAL5-C4-2 (7.18 g, 13.48 mmol) obtained according to the method described in step (3-1-2) were mixed and dissolved in 34 ml of dichloromethane, and diisopropylethylamine (3.48 g, 26.96 mmol) was added. Finally, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT, 4.04 g, 13.48 mmol) was added, and the reaction was stirred at room temperature for 4.5 h. The reaction solution was diluted with 100 ml of dichloromethane, the organic phase was washed with 80 ml of saturated sodium bicarbonate solution, the organic phase was washed with 80 ml of saturated brine, the organic phases were combined and dried with anhydrous sodium sulfate, filtered and the solvent was evaporated under reduced pressure to obtain a crude product, which was purified by a 200-300 mesh normal phase silica gel column, loaded with petroleum ether, and the acidity of the silica gel was neutralized with 1 wt% triethylamine, and the gradient elution was carried out with dichloromethane: methanol = 30: 1-15: 1, the product eluate was collected, and the pure product Z-13.97 g was evaporated under reduced pressure. MS m/z: C98 H143 N10 O33 , [M+H]+, theoretical: 1987.98, measured: 1987.90.
(10-1-2)Z-2的合成：(10-1-2) Synthesis of Z-2:
将Z-1(3.97g，2.00mmol)溶于250ml二氯甲烷，再加入二氯乙酸(10.941g，84.85mmol)，室温下反应1h。加入吡啶中和反应液至中性，减压蒸干溶剂得粗品。220g 200-300目正相硅胶装柱，10％吡啶中和硅胶酸性，1‰吡啶平衡柱子，二氯甲烷∶甲醇＝10∶1-2∶1梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品Z-2 3.49g。MS m/z：C79H129N10O33，[M+H]+，理论：1746.94，实测：1746.90。Dissolve Z-1 (3.97 g, 2.00 mmol) in 250 ml of dichloromethane, add dichloroacetic acid (10.941 g, 84.85 mmol), and react at room temperature for 1 h. Add pyridine to neutralize the reaction solution to neutrality, and evaporate the solvent under reduced pressure to obtain a crude product. 220 g of 200-300 mesh normal phase silica gel column, 10% pyridine to neutralize the acidity of silica gel, 1‰ pyridine to balance the column, dichloromethane: methanol = 10: 1-2: 1 gradient elution, collect the product eluate, and evaporate the solvent under reduced pressure to obtain 3.49 g of pure Z-2. MS m/z: C79 H129 N10 O33 , [M+H]+, theoretical: 1746.94, measured: 1746.90.
(10-1-3)Z-3的合成：(10-1-3) Synthesis of Z-3:
将Z-2(3.49g，2.0mmol)和按照步骤(1-1-7a)中描述的方法得到的A-1(3.06g，6.0mmol)混合溶于30ml二氯甲烷，加入3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT，1.80g，6.0mmol)，再加入二异丙基乙胺(1.55g，12.0mmol)，25℃下搅拌反应3h。100ml二氯甲烷稀释反应液，用饱和碳酸氢钠洗涤有机相2次，每次30ml，水相以10二氯甲烷萃取，合并有机相并以50ml饱和食盐水洗涤，合并有机相无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜得到粗品。柱纯化使用200g 200-300目正相硅胶，20ml三乙胺中和硅胶酸性，以含1wt％三乙胺的石油醚平衡柱子，二氯甲烷∶甲醇＝25∶1-15∶1梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品Z-3 2.2g。MS m/z：C103H151N10O38，[M+H]+，理论：2136.02，实测：2136.20。Z-2 (3.49 g, 2.0 mmol) and A-1 (3.06 g, 6.0 mmol) obtained by the method described in step (1-1-7a) were mixed and dissolved in 30 ml of dichloromethane, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT, 1.80 g, 6.0 mmol) were added, and then diisopropylethylamine (1.55 g, 12.0 mmol) was added, and the mixture was stirred at 25°C for 3 h. The reaction solution was diluted with 100 ml of dichloromethane, and the organic phase was washed twice with saturated sodium bicarbonate, 30 ml each time, and the aqueous phase was extracted with 10% dichloromethane, the organic phases were combined and washed with 50 ml of saturated brine, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure, and vacuum oil pump was dried overnight to obtain a crude product. Column purification used 200g 200-300 mesh normal phase silica gel, 20ml triethylamine to neutralize the acidity of silica gel, balanced the column with petroleum ether containing 1wt% triethylamine, gradient elution with dichloromethane: methanol = 25: 1-15: 1, collected the product eluate, and evaporated the solvent under reduced pressure to obtain 2.2g of pure product Z-3. MS m/z: C103 H151 N10 O38 , [M+H]+, theoretical: 2136.02, found: 2136.20.
(10-1-4)Z-4的合成：(10-1-4) Synthesis of Z-4:
将Z-3(2.10g，0.983mmol)溶解在含有DIEA(0.635g，4.915mmol)的14.8ml二氯甲烷中，加入4-二甲氨基吡啶(DMAP，240mg，1.966mmol)搅拌澄清后，加入丁二酸酐(197mg，1.966mmol)，25℃下搅拌反应18h。加入50ml二氯甲烷稀释反应液，以80ml 0.5M三乙胺磷酸盐洗涤有机相，水相以二氯甲烷萃取2次，每次50ml，合并有机相减压蒸干得到粗品。柱纯化使用188g 200-300目正相硅胶，以1wt％三乙胺中和硅胶酸性，二氯甲烷平衡柱子，以含1wt‰三乙胺的二氯甲烷∶甲醇＝10∶1-3∶1梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品Z-4缀合分子1.95g。MS m/z：C107H155N10O41，[M+H]+，理论：1935.07，实测：1935.29。Z-3 (2.10 g, 0.983 mmol) was dissolved in 14.8 ml of dichloromethane containing DIEA (0.635 g, 4.915 mmol), 4-dimethylaminopyridine (DMAP, 240 mg, 1.966 mmol) was added, and after stirring to clarify, succinic anhydride (197 mg, 1.966 mmol) was added, and the mixture was stirred at 25°C for 18 h. 50 ml of dichloromethane was added to dilute the reaction solution, and the organic phase was washed with 80 ml of 0.5 M triethylamine phosphate, and the aqueous phase was extracted twice with dichloromethane, 50 ml each time, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain a crude product. Column purification used 188g of 200-300 mesh normal phase silica gel, neutralized the acidity of silica gel with 1wt% triethylamine, balanced the column with dichloromethane, eluted with dichloromethane containing 1wt‰ triethylamine: methanol = 10:1-3:1 gradient, collected the product eluate, and evaporated the solvent under reduced pressure to obtain 1.95g of pure Z-4 conjugated molecule. MS m/z: C107 H155 N10 O41 , [M+H]+, theoretical: 1935.07, found: 1935.29.
(10-1-5)Z-5的合成(10-1-5) Synthesis of Z-5
通过与制备例1中步骤(1-1-9)相同的方法，制备Z-5。不同的是以Z-4缀合分子代替L-9缀合分子，得到连接固相载体的Z-4缀合分子。Z-5 was prepared by the same method as step (1-1-9) in Preparation Example 1, except that the L-9 conjugated molecule was replaced by the Z-4 conjugated molecule to obtain the Z-4 conjugated molecule connected to the solid phase carrier.
(10-2)合成Z5-siHB1M1SVP缀合物(10-2) Synthesis of Z5-siHB1M1SVP conjugate
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物17，不同的是以Z-5化合物代替L-10化合物起始正义链合成。预期可以得到Z5-siHB1M1SVP缀合物，其结构如式(422)所示。Conjugate 17 was prepared by the same method as steps (1-2), (1-3A), and (1-4) in Preparation Example 1, except that Z-5 compound was used instead of L-10 compound to initiate the synthesis of the positive chain. It is expected that a Z5-siHB1M1SVP conjugate can be obtained, and its structure is shown in Formula (422).
制备例11：缀合物20的制备Preparation Example 11: Preparation of Conjugate 20
本制备例合成了缀合物20(以下，也称为FIN-siHB1M1SVP缀合物)。该缀合物中所缀合的siRNA的序列参见表2。In this preparation example, conjugate 20 (hereinafter also referred to as FIN-siHB1M1SVP conjugate) was synthesized. The sequence of the siRNA conjugated in this conjugate is shown in Table 2.
(11-1)FIN-2缀合分子的合成(11-1) Synthesis of FIN-2 conjugated molecules
参照Rajeev等人，ChemBioChem2015，16，903-908中描述的制备方法，按照以下工艺路线，合成了FIN-2缀合分子：Referring to the preparation method described in Rajeev et al., ChemBioChem 2015, 16, 903-908, the FIN-2 conjugated molecule was synthesized according to the following process route:
(11-1-1)PRO-10的合成(11-1-1) Synthesis of PRO-10
(11-1-1a)PRO-7的合成(11-1-1a) Synthesis of PRO-7
将2.93gPRO-6(L-羟基脯氨酸，CAS号：51-35-4，购自安耐吉公司，22.4mmol)溶于22.5ml1，4-dioxane(1，4-二氧六环，CAS号：123-91-1)中，加入34ml 10％(w/w)Na2CO3的水溶液，呈悬浊液状态，将6.95g Fmoc-Cl(氯甲酸-9-芴基甲酯，CAS号：28920-43-6，购自安耐吉公司，26.8mmol)溶于34ml 1，4-二氧六环，冰浴下加入到上述悬浊液中，自然升至室温反应过夜。将反应液倒入150ml冰水中，用甲基叔丁基醚萃取三次，每次100ml，弃去有机相，水相用浓HCl调节至pH≤5，用100ml乙酸乙酯萃取两次，合并有机相，无水硫酸钠干燥，减压蒸干溶剂得到白色泡沫状固体产品PRO-7 7.83g。1H NMR(400MHz，DMSO-d6)δ7.91(t，J＝7.2Hz，2H)，7.67(d，J＝7.5Hz，2H)，7.48-7.39(m，2H)，7.38-7.27(m，2H)，5.17(s，1H)，4.27(s，2H)，4.23-4.11(m，2H)，3.55-3.41(m，3H)，2.31-2.10(m，1H)，2.08-1.88(m，1H)。HRMS(ESI)m/z理论C20H19NO5[M-H]-352.1190，实测352.1033。2.93g PRO-6 (L-hydroxyproline, CAS No.: 51-35-4, purchased from Anage, 22.4mmol) was dissolved in 22.5ml 1,4-dioxane (1,4-dioxane, CAS No.: 123-91-1), and 34ml 10% (w/w) Na2CO3aqueous solution was added to form a suspension state. 6.95g Fmoc-Cl (9-fluorenylmethyl chloroformate, CAS No.: 28920-43-6, purchased from Anage, 26.8mmol) was dissolved in 34ml 1,4-dioxane and added to the above suspension under ice bath, and the temperature was naturally raised to room temperature for reaction overnight. The reaction solution was poured into 150 ml of ice water, extracted three times with methyl tert-butyl ether, 100 ml each time, the organic phase was discarded, the aqueous phase was adjusted to pH ≤ 5 with concentrated HCl, extracted twice with 100 ml of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 7.83 g of white foamy solid product PRO-7.1 H NMR (400 MHz, DMSO-d6 ) δ 7.91 (t, J=7.2 Hz, 2H), 7.67 (d, J=7.5 Hz, 2H), 7.48-7.39 (m, 2H), 7.38-7.27 (m, 2H), 5.17 (s, 1H), 4.27 (s, 2H), 4.23-4.11 (m, 2H), 3.55-3.41 (m, 3H), 2.31-2.10 (m, 1H), 2.08-1.88 (m, 1H). HRMS (ESI) m/z theory for C20 H19 NO5 [MH]− 352.1190, found 352.1033.
(11-1-1b)PRO-8的合成(11-1-1b) Synthesis of PRO-8
将7.83g PRO-7(22.2mmol)溶于80mlTHF(CAS号：109-99-9)中，油浴加热到65℃，回流状态下加入36.6ml 2mol/L的BH3-Me2S的THF溶液(CAS号13292-87-0，购自百灵威公司，73.2mmol)，继续回流反应3小时。倒出反应液，用甲醇溶解剩余固体，搅拌下加入甲醇至反应液无气体放出并继续搅拌30分钟，减压蒸除溶剂后用石油醚提纯三次后得白色固体产物PRO-8 7.1g。1H NMR(400MHz，DMSO-d6)δ7.91(t，J＝6.7Hz，2H)，7.67(d，J＝7.2Hz，2H)，7.49-7.39(m，2H)，7.38-7.26(m，2H)，5.18(dd，J＝6.1，3.8Hz，1H)，4.28(s，2H)，4.23-4.13(m，2H)，3.55-3.38(m，2H)，2.32-2.11(m，1H)，2.08-1.89(m，1H)。HRMS(ESI)m/z理论C20H21NO4[M+Na]+362.1368，实测362.1012。7.83 g PRO-7 (22.2 mmol) was dissolved in 80 ml THF (CAS No.: 109-99-9), heated to 65°C in an oil bath, and 36.6 ml 2 mol/L BH3 -Me2 S THF solution (CAS No. 13292-87-0, purchased from J&K Company, 73.2 mmol) was added under reflux, and the reflux reaction was continued for 3 hours. The reaction solution was poured out, and the remaining solid was dissolved with methanol. Methanol was added under stirring until the reaction solution no longer released gas and continued to stir for 30 minutes. The solvent was evaporated under reduced pressure, and then purified with petroleum ether three times to obtain 7.1 g of a white solid product PRO-8.1 H NMR (400MHz, DMSO-d6 ) δ7.91 (t, J=6.7Hz, 2H), 7.67 (d, J=7.2Hz, 2H), 7.49-7.39 (m, 2H), 7.38-7.26 (m, 2H), 5.18 (dd, J=6.1, 3.8Hz, 1H), 4.28 (s, 2H), 4 .23-4.13(m, 2H), 3.55-3.38(m, 2H), 2.32-2.11(m, 1H), 2.08-1.89(m, 1H). HRMS (ESI) m/z theoretical C20 H21 NO4 [M+Na]+ 362.1368, measured 362.1012.
(11-1-1c)PRO-9的合成(11-1-1c) Synthesis of PRO-9
将7.1g PRO-8(21mmol)溶于100ml吡啶中，加入14.2g DMTr-Cl(4，4′-双甲氧基三苯甲基氯，42mmol)，室温下搅拌反应5小时。减压蒸除溶剂，粗品用乙酸乙酯溶解后过滤除去盐类杂质，减压蒸除溶剂后硅胶柱纯化，硅胶柱预先用吡啶碱化后DCM溶解粗品上样，先用含1％(v/v)吡啶的DCM洗脱DMTr-Cl，随后用乙酸乙酯洗脱产物，收集产物洗脱液，减压蒸干溶剂，得白色固体产物PRO-9 8.2g；HRMS(ESI)m/z理论C41H39NO6[M+Na]+664.2675，实测664.2348；C18 RP-HPLC(批号JJS160324-1)纯度94.20％。7.1g PRO-8 (21mmol) was dissolved in 100ml pyridine, and 14.2g DMTr-Cl (4,4′-bis(methoxytrityl) chloride, 42mmol) was added, and the mixture was stirred at room temperature for 5 hours. The solvent was evaporated under reduced pressure, and the crude product was dissolved in ethyl acetate and filtered to remove salt impurities. The solvent was evaporated under reduced pressure and then purified by silica gel column. The silica gel column was alkalized with pyridine in advance and then the crude product was dissolved in DCM and loaded. DMTr-Cl was first eluted with DCM containing 1% (v/v) pyridine, and then the product was eluted with ethyl acetate. The product eluate was collected and the solvent was evaporated under reduced pressure to obtain 8.2g of white solid product PRO-9; HRMS (ESI) m/z theoretical C41 H39 NO6 [M+Na]+ 664.2675, measured 664.2348; C18 RP-HPLC (batch number JJS160324-1) purity 94.20%.
(11-1-1d)PRO-10的合成(11-1-1d) Synthesis of PRO-10
将8.2g PRO-9(12.8mmol)溶于64ml DMF(N，N-二甲基甲酰胺)中，加入40ml哌啶(384mmol)，室温下搅拌反应30分钟。反应液倒入300ml冰水中，乙酸乙酯萃取三次，每次150ml，合并有机相，用200ml饱和食盐水洗涤后，有机相以无水硫酸钠干燥，减压蒸除溶剂后硅胶柱纯化，硅胶柱预先用吡啶碱化后DCM溶解粗品上样，先用含1％(v/v)吡啶的DCM洗脱Fmoc，随后用乙酸乙酯洗脱产物，收集产物洗脱液，减压蒸干溶剂，得白色固体产物PRO-10 4.65g。1H NMR(400MHz，DMSO-d6)δ7.40(d，J＝7.2Hz，2H)，7.35-7.18(m，7H)，6.93-6.84(m，4H)，4.56(d，J＝3.9Hz，1H)，4.12(s，1H)，3.74(s，6H)，3.46-3.37(m，1H)，2.88(ddd，J＝18.5，10.0，5.5Hz，2H)，2.75(dd，J＝8.7，5.8Hz，1H)，2.62(dd，J＝11.0，2.7Hz，1H)，1.74-1.65(m，1H)，1.40(ddd，J＝12.9，8.5，5.9Hz，1H)；HRMS(ESI)m/z理论C26H29NO4[M+Na]+442.1994，实测442.1999；C18 RP-HPLC(批号JJS160329-1)纯度97.07％。8.2g PRO-9 (12.8mmol) was dissolved in 64ml DMF (N, N-dimethylformamide), and 40ml piperidine (384mmol) was added. The reaction mixture was stirred at room temperature for 30 minutes. The reaction solution was poured into 300ml ice water, extracted with ethyl acetate three times, 150ml each time, and the organic phases were combined and washed with 200ml saturated brine. The organic phases were dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure and then purified on a silica gel column. The silica gel column was alkalized with pyridine in advance and then the crude product was dissolved in DCM and loaded. Fmoc was first eluted with DCM containing 1% (v/v) pyridine, and then the product was eluted with ethyl acetate. The product eluate was collected and the solvent was evaporated under reduced pressure to obtain 4.65g of white solid product PRO-10.1 H NMR (400MHz, DMSO-d6 )δ7.40 (d, J=7.2Hz, 2H), 7.35-7.18 (m, 7H), 6.93-6.84 (m, 4H), 4.56 (d, J=3.9Hz, 1H), 4.12 (s, 1H), 3.74 (s, 6H), 3.46-3.37 (m, 1H), 2.88 (ddd, J=18.5 , 10.0, 5.5Hz, 2H), 2.75 (dd, J=8.7, 5.8Hz, 1H), 2.62 (dd, J=11.0, 2.7Hz, 1H), 1.74-1.65 (m, 1H), 1.40 (ddd, J=12.9, 8.5, 5.9Hz, 1H); HRMS (ESI) m/z theory C26 H29 NO4 [M+Na]+ 442.1994, found 442.1999; C18 RP-HPLC (batch number JJS160329-1) purity 97.07%.
(11-1-2)FIN-1的合成(11-1-2) Synthesis of FIN-1
将按照(1-1-1)中描述的方法得到的GAL-5(4.5g，10mmol)溶于40ml DMF中，依次加入3.9g DIPEA(N，N-二异丙基乙胺，CAS号：7087-68-5，购自阿拉丁公司，30mmol)和3.8gHBTU(苯并三氮唑-N，N，N′，N′-四甲基脲六氟磷酸盐，CAS号：94790-37-2，商购自阿拉丁公司，11mmol)，室温下搅拌10分钟，将步骤(11-1-1d)中获得的PRO-10(4.2g，10mmol)溶于40mlDMF中，随后加入到上述反应液中，反应液中加入无水硫酸钠干燥，室温搅拌2小时。将反应液倒入120ml冰水中，用乙酸乙酯萃取三次，每次60ml，合并有机相，分别用20ml水、20ml饱和食盐水洗涤，分出有机相并以无水硫酸钠干燥，减压蒸除溶剂，硅胶柱纯化，硅胶柱预先用吡啶碱化后上样，用含1体积％三乙胺和1体积％甲醇的二氯甲烷(DCM)溶液洗脱，收集产物洗脱液，减压蒸干溶剂，得到浅黄色泡沫状固体产品FIN-1 6.5g。1H NMR(400MHz，DMSO-d6)δ7.83(d，J＝9.2Hz，1H)，7.32(t，J＝6.6Hz，4H)，7.20(td，J＝8.9，3.5Hz，5H)，6.93-6.84(m，4H)，5.21(d，J＝3.2Hz，1H)，5.04-4.90(m，2H)，4.49(s，1H)，4.40(d，J＝4.4Hz，0.8H)，4.31(d，J＝5.0Hz，0.2H)，4.15(s，1H)，4.03(s，3H)，3.93(s，1H)，3.74(s，7H)，3.59(dt，J＝12.0，6.0Hz，1H)，3.50-3.40(m，1H)，3.39-3.25(m，3H)，3.13(dd，J＝8.9，5.2Hz，1H)，3.00(dq，J＝9.3，5.3，4.3Hz，1H)，2.22(s，2H)，2.07(s，3H)，1.99(s，3H)，1.90(s，4H)，1.74(s，3H)，1.50(s，3H)，1.36(s，1H)。C18 RP-HPLC(批号LJ160422)纯度95.45％。GAL-5 (4.5 g, 10 mmol) obtained according to the method described in (1-1-1) was dissolved in 40 ml of DMF, and 3.9 g of DIPEA (N, N-diisopropylethylamine, CAS No.: 7087-68-5, purchased from Aladdin, 30 mmol) and 3.8 g of HBTU (benzotriazole-N, N, N′, N′-tetramethyluronium hexafluorophosphate, CAS No.: 94790-37-2, commercially available from Aladdin, 11 mmol) were added in sequence. The mixture was stirred at room temperature for 10 minutes. PRO-10 (4.2 g, 10 mmol) obtained in step (11-1-1d) was dissolved in 40 ml of DMF and then added to the above reaction solution. Anhydrous sodium sulfate was added to the reaction solution for drying, and the mixture was stirred at room temperature for 2 hours. The reaction solution was poured into 120 ml of ice water, extracted with ethyl acetate three times, each time with 60 ml, the organic phases were combined, washed with 20 ml of water and 20 ml of saturated brine respectively, the organic phase was separated and dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the silica gel column was purified. The silica gel column was alkalized with pyridine in advance and then loaded with the sample, eluted with a dichloromethane (DCM) solution containing 1% by volume of triethylamine and 1% by volume of methanol, the product eluate was collected, and the solvent was evaporated under reduced pressure to obtain 6.5 g of light yellow foam solid product FIN-1.1 H NMR (400 MHz, DMSO-d6 )δ7.83 (d, J=9.2Hz, 1H), 7.32 (t, J=6.6Hz, 4H), 7.20 (td, J=8.9, 3.5Hz, 5H), 6.93-6.84 (m, 4H), 5.21 (d, J=3.2Hz, 1H), 5.04-4.90 (m, 2H), 4.49 (s, 1H), 4.40 (d, J=4.4Hz, 0.8H), 4.31 (d, J=5.0Hz, 0.2H), 4.15 (s, 1H), 4.03 (s, 3H), 3.93 (s, 1H) ), 3.74 (s, 7H), 3.59 (dt, J = 12.0, 6.0 Hz, 1H), 3.50-3.40 (m, 1H), 3.39-3.25 (m, 3H), 3.13 (dd, J = 8.9, 5.2 Hz, 1H), 3.00 (dq, J = 9.3, 5.3, 4.3 Hz, 1H), 2.22 (s, 2H), 2.07 (s, 3H), 1.99 (s, 3H), 1.90 (s, 4H), 1.74 (s, 3H), 1.50 (s, 3H), 1.36 (s, 1H). C18 RP-HPLC (batch number LJ160422) purity 95.45%.
(11-1-3)FIN-2的合成(11-1-3) Synthesis of FIN-2
将步骤(11-1-2)中获得的FIN-1(3.0g，3.53mmol)与乙腈共沸除水，减压抽干，溶于10ml DMF，氮气保护下加入2.13g PA(双(二异丙基氨基)(2-氰基乙氧基)膦，购自Adamas公司，商品编号11356B，7.06mmol)、346mg四唑(CAS号：288-94-8，购自阿拉丁公司，4.94mmol)，室温下搅拌反应，补加10ml DMF，继续搅拌反应1小时。减压蒸除溶剂后以硅胶柱色谱纯化，硅胶柱预先用吡啶碱化后DCM溶解粗品上样，乙酸乙酯洗脱，收集产物洗脱液，减压蒸除溶剂，得无色糖浆状粗品4.5g。粗品用50体积％乙腈水溶液溶解至完全溶解，用C-18，330g，中压纯化柱纯化样品，柱子先用1体积％吡啶的乙腈溶液碱化，梯度洗脱收集产品峰，减压蒸除溶剂得白色粉末产品FIN-2缀合分子2.2g。31p NMR(162MHz，CDCl3)δ148.04，147.94，147.62，147.19，磷谱纯度92％；C18 RP-HPLC纯度90.54％。The FIN-1 (3.0 g, 3.53 mmol) obtained in step (11-1-2) was azeotropically dehydrated with acetonitrile, dried under reduced pressure, dissolved in 10 ml of DMF, and 2.13 g of PA (bis(diisopropylamino)(2-cyanoethoxy)phosphine, purchased from Adamas, product number 11356B, 7.06 mmol) and 346 mg of tetrazole (CAS number: 288-94-8, purchased from Aladdin, 4.94 mmol) were added under nitrogen protection, stirred at room temperature, and 10 ml of DMF was added, and the stirring reaction was continued for 1 hour. After the solvent was evaporated under reduced pressure, it was purified by silica gel column chromatography. The silica gel column was pre-basified with pyridine and then the crude product was dissolved in DCM and loaded, eluted with ethyl acetate, and the product eluate was collected. The solvent was evaporated under reduced pressure to obtain 4.5 g of a colorless syrupy crude product. The crude product was dissolved in a 50% by volume acetonitrile aqueous solution until completely dissolved, and C-18, 330 g, The sample was purified by medium pressure purification column, the column was first alkalized with 1 volume % pyridine in acetonitrile solution, the product peak was collected by gradient elution, and the solvent was evaporated under reduced pressure to obtain 2.2 g of white powder product FIN-2 conjugated molecule.31 p NMR (162 MHz, CDCl3 ) δ 148.04, 147.94, 147.62, 147.19, phosphorus spectrum purity 92%; C18 RP-HPLC purity 90.54%.
(11-2)FIN-2缀合分子连接到固相载体(11-2) Linking FIN-2 conjugated molecules to solid phase carriers
采用核酸固相合成方法，将步骤(11-1-3)中得到的FIN-2缀合分子，通过三次循环，连接到通用固相载体(UnyLinkerTMloadedSolid Supports)上，从而实现缀合基团(FIN_FIN_FIN)连接在RNA正义链的3′末端。The FIN-2 conjugated molecule obtained in step (11-1-3) was linked to a universal solid phase carrier (UnyLinkerTM loaded Solid Supports), thereby connecting the conjugated group (FIN_FIN_FIN) to the 3′ end of the RNA sense strand.
参照Rajeev等人，ChemBioChem 2015，16，903-908中描述的制备方法进行上述连接，具体而言，首先，由上述通用固相载体开始，脱除固相载体上的羟基保护基团，在偶联反应条件和偶联试剂存在下与FIN-2缀合分子接触发生偶联，经盖帽反应和氧化反应后，获得连接至固相载体的FIN缀合分子；脱除该连接至固相载体的FIN缀合分子上的羟基保护基团DMTr，与FIN-2缀合分子接触发生偶联，进行盖帽反应和氧化反应，并再重复一次上述脱保护-偶联-盖帽-氧化步骤，连接第三个FIN-2缀合分子，获得连接在固相载体上的的缀合基团(FIN_FIN_FIN)。The above connection is performed with reference to the preparation method described in Rajeev et al., ChemBioChem 2015, 16, 903-908. Specifically, first, starting from the above-mentioned universal solid phase carrier, the hydroxyl protecting group on the solid phase carrier is removed, and the FIN-2 conjugate molecule is contacted under coupling reaction conditions and in the presence of a coupling reagent for coupling, and after a capping reaction and an oxidation reaction, a FIN conjugate molecule connected to the solid phase carrier is obtained; the hydroxyl protecting group DMTr on the FIN conjugate molecule connected to the solid phase carrier is removed, and the FIN-2 conjugate molecule is contacted for coupling, and a capping reaction and an oxidation reaction are performed, and the above-mentioned deprotection-coupling-capping-oxidation steps are repeated once, and the third FIN-2 conjugate molecule is connected to obtain a conjugate group (FIN_FIN_FIN) connected to the solid phase carrier.
上述反应中，所述的脱保护、偶联、盖帽、氧化的反应条件、溶剂和试剂用量与前述步骤(1-2)中描述的核酸固相合成方法相同。In the above reaction, the reaction conditions, solvents and reagent amounts of the deprotection, coupling, capping and oxidation are the same as those of the nucleic acid solid phase synthesis method described in the above step (1-2).
(11-3)缀合物20的合成(11-3) Synthesis of Conjugate 20
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备题述缀合物，不同的是：1)以步骤(11-2)得到的化合物起始正义链合成；2)缀合的siRNA具有表2中所示的对应于缀合物20的序列。The title conjugate was prepared by the same method as steps (1-2), (1-3A), and (1-4) in Preparation Example 1, except that: 1) the positive chain synthesis was initiated with the compound obtained in step (11-2); 2) the conjugated siRNA had the sequence corresponding to conjugate 20 shown in Table 2.
利用液质联用仪(LC-MS，Liquid Chromatography-Mass Spectrometry，购于Waters公司，型号：LCT Premier)进行分子量检测。其结果，实测值与理论值相符，从而确定所合成的缀合物是目标设计的化合物，其结构如式(307)所示。The molecular weight was measured by liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). As a result, the measured value was consistent with the theoretical value, thereby confirming that the synthesized conjugate was the target designed compound, and its structure was shown in formula (307).
在上述本公开的缀合物制备完成后，使用标准手段冻干为固体粉末保存备用。在使用时，可使用例如注射用水将其重新溶解为所需浓度的溶液使用。After the preparation of the conjugate disclosed above is completed, it is freeze-dried into solid powder using standard means and stored for later use. When used, it can be re-dissolved into a solution of desired concentration using, for example, water for injection.
实验例1本实验说明本公开的siRNA缀合物的稳定性Experimental Example 1 This experiment illustrates the stability of the siRNA conjugate disclosed in the present invention
(实验例1-1)siRNA缀合物在体外溶酶体裂解液中的稳定性(Experimental Example 1-1) Stability of siRNA conjugates in in vitro lysosomal lysates
经溶酶体裂解液处理的测试样品制备：将缀合物4(以siRNA浓度为20μM的0.9％氯化钠水溶液形式提供，每组6μl)分别与27.2μL柠檬酸钠水溶液(pH5.0)、4.08μL去离子水和2.72μL Tritosomes(商购自Xenotech公司，货号R0610LT，批号1610069，终浓度为0.2mU/μL)混匀。37℃恒温孵育。分别在0h、5min、15min、30min、1h、2h、4h、8h取出5μl样本，分别加入到15μL9M的尿素中变性，随后加入4μl 6×上样缓冲液(索莱宝公司，货号20160830)，立即冷冻于-80℃冰箱终止反应。0小时表示，将待测样品与溶酶体裂解液混匀后，立即取出的时刻。Preparation of test samples treated with lysosomal lysate: Conjugate 4 (provided in the form of 0.9% sodium chloride aqueous solution with a siRNA concentration of 20 μM, 6 μl per group) was mixed with 27.2 μL sodium citrate aqueous solution (pH 5.0), 4.08 μL deionized water and 2.72 μL Tritosomes (commercially available from Xenotech, product number R0610LT, batch number 1610069, final concentration 0.2 mU/μL). Incubate at 37°C. Take out 5 μl of sample at 0h, 5min, 15min, 30min, 1h, 2h, 4h, and 8h, respectively, and add them to 15 μL 9M urea for denaturation, then add 4 μl 6× loading buffer (Solerbo, product number 20160830), and immediately freeze in a -80°C refrigerator to terminate the reaction. 0 hours indicates the time when the sample to be tested is taken out immediately after being mixed with the lysosomal lysis solution.
未经溶酶体裂解液处理的参比样品制备：取等摩尔量的缀合物4(20μM)1.5μl与7.5μL柠檬酸钠水溶液(pH 5.0)、1μL去离子水混匀，加入30μL 9M的尿素溶液变性，随后加入8μL 6×上样缓冲液混匀，立即冷冻于-80℃冰箱终止反应。参比样品在电泳图中标记为Con。Preparation of reference sample without lysosomal lysate treatment: 1.5 μl of equimolar conjugate 4 (20 μM) was mixed with 7.5 μL sodium citrate aqueous solution (pH 5.0) and 1 μL deionized water, and then 30 μL 9M urea solution was added for denaturation, followed by 8 μL 6× loading buffer and mixing, and immediately frozen in a -80°C refrigerator to terminate the reaction. The reference sample was marked as Con in the electrophoresis diagram.
配制16重量％的非变性聚丙烯酰胺凝胶，上述测试样品及参比样品各取20μl上样至凝胶，在20mA恒流条件下电泳10min后，继续在40mA恒流条件下电泳30min。电泳结束后，将凝胶置于摇床上，用Gelred染料(BioTium公司，货号13G1203)染色10min。凝胶成像观察并拍照，结果如图1所示。A 16 wt% non-denaturing polyacrylamide gel was prepared, and 20 μl of each of the test sample and reference sample was loaded onto the gel. After electrophoresis for 10 min at a constant current of 20 mA, electrophoresis was continued for 30 min at a constant current of 40 mA. After the electrophoresis, the gel was placed on a shaker and stained with Gelred dye (BioTium, product number 13G1203) for 10 min. The gel was imaged and photographed, and the results are shown in FIG1 .
图1显示了所测试siRNA缀合物在体外溶酶体中的稳定性半定量检测结果。结果显示，本公开的缀合物在溶酶体中可维持长时间不降解，显示出很好的稳定性。Figure 1 shows the semi-quantitative test results of the stability of the tested siRNA conjugates in lysosomes in vitro. The results show that the conjugates disclosed in the present invention can be maintained in lysosomes for a long time without degradation, showing good stability.
(实验例1-2)siRNA缀合物在人血浆中的稳定性(Experimental Example 1-2) Stability of siRNA conjugates in human plasma
将缀合物4(以siRNA浓度为20μM的0.9％氯化钠水溶液形式提供，12μl)以及对比序列1(20μM，12μl)分别与108μL90％人血浆(Human plasma，PBS稀释)混匀。37℃恒温孵育。分别在0、2、4、6、8、24、48、72小时取出10μL样本，立即进行液氮速冻，于-80℃冰箱中冻存。待各时间点取样完毕后，将上述冻存样品分别以1×PBS(pH7.4)稀释5倍后每一样品取10μL备用。同时，取等摩尔量的siRNA(2μM，2μl)或siRNA缀合物(siRNA浓度为2μM，2μl)，与8μl1×PBS(pH7.4)混匀，制备成10μL未经人血浆处理的样品，记为Con。Conjugate 4 (provided in the form of 0.9% sodium chloride aqueous solution with a siRNA concentration of 20 μM, 12 μl) and comparison sequence 1 (20 μM, 12 μl) were mixed with 108 μL 90% human plasma (Human plasma, PBS dilution). Incubate at 37°C. Take out 10 μL samples at 0, 2, 4, 6, 8, 24, 48, and 72 hours, immediately freeze in liquid nitrogen, and freeze in a -80°C refrigerator. After sampling at each time point, dilute the above frozen samples 5 times with 1×PBS (pH7.4) and take 10 μL of each sample for standby use. At the same time, take an equal molar amount of siRNA (2 μM, 2 μl) or siRNA conjugate (siRNA concentration is 2 μM, 2 μl), mix it with 8 μl 1×PBS (pH7.4), and prepare a 10 μL sample that has not been treated with human plasma, recorded as Con.
配制20重量％的非变性聚丙烯酰胺凝胶，将上述备用样品中每一组的全部样品与4μL上样缓冲液(20mM EDTA，36重量％甘油，0.06重量％溴酚蓝的水溶液)混合，然后上样至前述凝胶，在80mA恒流条件下电泳60分钟。电泳结束后，用1×Sybr Gold染料(Invitrogen，Cat.11494)染色15分钟后成相，结果如图2所示。A 20 wt% non-denaturing polyacrylamide gel was prepared, and all samples in each group of the above-mentioned reserve samples were mixed with 4 μL of loading buffer (20 mM EDTA, 36 wt% glycerol, 0.06 wt% bromophenol blue in water), and then loaded onto the above-mentioned gel, and electrophoresed at a constant current of 80 mA for 60 minutes. After the electrophoresis, the samples were stained with 1× Sybr Gold dye (Invitrogen, Cat. 11494) for 15 minutes and then phased, and the results are shown in FIG2 .
对比序列1：Compare sequence 1:
正义链：CCUUGAGGCAUACUUCAAA(SEQ ID NO：29)Sense strand: CCUUGAGGCAUACUUCAAA (SEQ ID NO: 29)
反义链：UUUGAAGUAUGCCUCAAGGUU(SEQ ID NO：30)Antisense strand: UUUGAAGUAUGCCUCAAGGUU (SEQ ID NO: 30)
图2示出了所测试缀合物在体外人血浆中的稳定性半定量检测结果。FIG2 shows the results of semi-quantitative testing of the stability of the tested conjugates in human plasma in vitro.
由图2的结果可以看出，本公开的缀合物在人血浆中直至72h时仍未降解，显示出优异的在人血浆中的稳定性。As can be seen from the results of FIG. 2 , the conjugate of the present disclosure is not degraded in human plasma until 72 h, showing excellent stability in human plasma.
(实验例1-3)siRNA缀合物在猴血浆中的稳定性(Experimental Example 1-3) Stability of siRNA conjugates in monkey plasma
在另外的实验中，采用与实验例1-2相同的方法检测缀合物4在猴血浆(Monkeyplasma，购自鸿泉生物，HQ70082，PBS稀释)中的稳定性，结果如图3所示。In another experiment, the same method as in Experimental Example 1-2 was used to detect the stability of conjugate 4 in monkey plasma (Monkey plasma, purchased from Hongquan Biotechnology, HQ70082, diluted with PBS). The results are shown in FIG3 .
图3出了所测试缀合物在体外猴血浆中的稳定性半定量检测结果。FIG3 shows the semi-quantitative results of the stability of the tested conjugates in monkey plasma in vitro.
由图3的结果可以看出，本公开的siRNA缀合物在食蟹猴血浆中直至72h仍未降解，显示出优异的在猴血浆中的稳定性。As can be seen from the results of FIG3 , the siRNA conjugate of the present disclosure was not degraded in cynomolgus monkey plasma until 72 h, showing excellent stability in monkey plasma.
实验例2本实验例说明本公开的siRNA缀合物在体外(in vitro)的抑制活性(实验例2-1)体外psiCHECK系统中的在靶活性Experimental Example 2 This experimental example illustrates the inhibitory activity of the siRNA conjugate disclosed in the present invention in vitro (Experimental Example 2-1) On-target activity in the in vitro psiCHECK system
本实验例中所使用的HEK293A细胞由北京大学分子医学研究所核酸技术实验室提供，用含有20％的胎牛血清(FBS，Hyclone公司)及0.2体积％的青链霉素双抗(Penicillin-Streptomycin，Gibco，Invitrogen公司)的DMEM完全培养基(Hyclone公司)培养细胞，于37℃在含5％CO2/95％空气的培养箱中培养。HEK293A cells used in this experiment were provided by the Nucleic Acid Technology Laboratory, Institute of Molecular Medicine, Peking University. The cells were cultured in DMEM complete medium (Hyclone) containing 20% fetal bovine serum (FBS, Hyclone) and 0.2% penicillin-streptomycin (Gibco, Invitrogen) at 37°C in an incubator containing 5%CO2 /95% air.
本实验例考察了缀合物20在体外psiCHECK系统中的在靶活性(on-targetactivity)，即测定了缀合物20靶向完全匹配目标序列(其核苷酸序列与所述缀合物反义链的全长核苷酸序列完全互补)的活性。This experimental example investigated the on-target activity of conjugate 20 in the in vitro psiCHECK system, i.e., the activity of conjugate 20 targeting a fully matched target sequence (whose nucleotide sequence is completely complementary to the full-length nucleotide sequence of the antisense strand of the conjugate) was determined.
根据Kumico Ui-Tei et.al.，Functional dissection of siRNA sequence bysystematic DNA substitution：modified siRNA with a DNA seed arm is a powerfultool for mammalian gene silencing with significantly reduced off-targeteffect.Nucleic Acids Research，2008.36(7)，2136-2151描述的方法，构建检测质粒，与待评价的siRNA缀合物共转染至HEK293A细胞中，通过双萤光素酶报告基因的表达水平，来反应siRNA缀合物的在靶活性及脱靶效应。具体步骤如下：According to the method described in Kumico Ui-Tei et al., Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect. Nucleic Acids Research, 2008. 36 (7), 2136-2151, a detection plasmid was constructed and co-transfected with the siRNA conjugate to be evaluated into HEK293A cells. The on-target activity and off-target effect of the siRNA conjugate were reflected by the expression level of the dual luciferase reporter gene. The specific steps are as follows:
[1]构建检测质粒[1] Construction of detection plasmid
采用psiCHECKTM-2(PromegaTM)质粒构建在靶质粒，该质粒含有一个目标序列，该目标序列与待测缀合物中的反义链的所有21个核苷酸序列完全互补。将目标序列克隆到psiCHECKTM-2质粒的Xho I/Not I位点。The target plasmid was constructed using psiCHECK™ -2 (Promega™ ) plasmid, which contains a target sequence that is completely complementary to all 21 nucleotide sequences of the antisense strand in the conjugate to be tested. The target sequence was cloned into the Xho I/Not I site of the psiCHECK™ -2 plasmid.
[2]转染[2] Transfection
在96孔板中，根据LipofectamineTM2000(Invitrogen公司)的使用说明，分别共转染siRNA缀合物和上述质粒，其中每孔转染质粒10ng，使用LipofectamineTM2000 0.2μL。缀合物的终浓度(以siRNA的浓度计算)依次为0.1nM、0.05nM和0.01nM。各组以无缀合物处理为对照。每组3个复孔。In a 96-well plate, according to the instructions of LipofectamineTM 2000 (Invitrogen), the siRNA conjugate and the above plasmid were co-transfected, wherein each well was transfected with 10 ng of plasmid and 0.2 μL of LipofectamineTM 2000. The final concentrations of the conjugate (calculated based on the concentration of siRNA) were 0.1 nM, 0.05 nM and 0.01 nM, respectively. Each group was treated with no conjugate as a control. Three replicate wells were used for each group.
NC为吉玛公司与目的基因序列无同源性的通用阴性对照B01001。NC is the universal negative control B01001 produced by Genetron Health that has no homology with the target gene sequence.
[3]检测[3] Detection
共转染24小时后，使用双萤光素酶报告基因检测试剂盒(Dual luciferasereporter gene assay kit，Promega公司，cat.E2940)，根据使用说明书裂解HEK293A细胞，检测双萤光素酶报告基因的表达水平。以海肾萤光素酶蛋白水平相对于萤火虫萤光素酶蛋白水平进行标准化。结果如图4所示。After 24 hours of cotransfection, the expression level of the dual luciferase reporter gene was detected using a dual luciferase reporter gene assay kit (Promega, cat. E2940) according to the instruction manual for lysis of HEK293A cells. The Renilla luciferase protein level was standardized relative to the firefly luciferase protein level. The results are shown in Figure 4.
结果表明，缀合物20具有较好的体外抑制活性。The results showed that conjugate 20 had good inhibitory activity in vitro.
实验例2-2体外psiCHECK系统中IC50的测定及脱靶检测Experimental Example 2-2 Determination ofIC50 and off-target detection in the in vitro psiCHECK system
本实验例例考察了缀合物4在体外psiCHECK系统中的IC50及脱靶效应。This experimental example investigated theIC50 and off-target effects of conjugate 4 in the in vitro psiCHECK system.
根据Kumico Ui-Tei et.al.，Functional dissection of siRNA sequence bysystematic DNA substitution：modified siRNA with a DNA seed arm is a powerfultool for mammalian gene silencing with significantly reduced off-targeteffect.Nucleic Acids Research，2008.36(7)，2136-2151描述的方法，构建检测质粒，与待测缀合物共转染至HEK293A细胞中，通过双萤光素酶报告基因的表达水平，来反应缀合物的在靶活性及脱靶效应。具体步骤如下：According to the method described in Kumico Ui-Tei et al., Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect. Nucleic Acids Research, 2008. 36 (7), 2136-2151, a detection plasmid was constructed and co-transfected with the conjugate to be tested into HEK293A cells. The on-target activity and off-target effect of the conjugate were reflected by the expression level of the dual luciferase reporter gene. The specific steps are as follows:
[1]构建检测质粒[1] Construction of detection plasmid
采用psiCHECKTM-2(PromegaTM)质粒构建了4种重组质粒，其中GSCM表示在靶质粒，PSCM、GSSM、PSSM表示脱靶质粒：Four recombinant plasmids were constructed using psiCHECKTM -2 (PromegaTM ) plasmid, where GSCM represents the on-target plasmid, and PSCM, GSSM, and PSSM represent the off-target plasmids:
(1)GSCM，含有一个目标序列，该目标序列与缀合物4中的反义链的所有21个核苷酸序列完全互补；(1) GSCM, containing a target sequence that is completely complementary to all 21 nucleotides of the antisense strand in conjugate 4;
(2)PSCM，含有一个目标序列，该目标序列与缀合物4中的反义链的所有21个核苷酸序列完全一致；(2) PSCM, containing a target sequence that is completely identical to all 21 nucleotides of the antisense strand in conjugate 4;
(3)GSSM，含有一个目标序列，该目标序列与待测siRNA中反义链的5’端起1-8位核苷酸序列完全互补，该目标序列的剩余部分与待测siRNA中反义链5’端起9-21位的核苷酸序列相对应，其序列完全不互补，即待测siRNA中反义链5’端起9-21位中任一位置的核苷酸为G、C、A或U时，目标序列相应位置的核苷酸分别为T、A、C或G。(3) GSSM, comprising a target sequence that is completely complementary to the nucleotide sequence at positions 1-8 from the 5' end of the antisense strand in the test siRNA, and the remaining portion of the target sequence corresponds to the nucleotide sequence at positions 9-21 from the 5' end of the antisense strand in the test siRNA, and the sequences are completely non-complementary, that is, when the nucleotide at any position from positions 9-21 from the 5' end of the antisense strand in the test siRNA is G, C, A or U, the nucleotide at the corresponding position of the target sequence is T, A, C or G, respectively.
(4)PSSM，含有一个目标序列，该目标序列与待测siRNA中正义链的5’端起1-8位核苷酸序列完全互补，该目标序列的剩余部分与待测siRNA中正义链5’端起9-19位的核苷酸序列相对应，其序列完全不互补，即待测siRNA中正义链5’端起9-19位中任一位置的核苷酸为G、C、A或U时，目标序列相应位置的核苷酸分别为T、A、C或G。为了与GSSM靶序列等长，目标序列的3’末端依次加入核苷酸A、C。(4) PSSM, containing a target sequence, which is completely complementary to the nucleotide sequence at positions 1-8 from the 5' end of the sense strand in the siRNA to be tested, and the remaining part of the target sequence corresponds to the nucleotide sequence at positions 9-19 from the 5' end of the sense strand in the siRNA to be tested, and the sequences are completely non-complementary, that is, when the nucleotide at any position from positions 9-19 from the 5' end of the sense strand in the siRNA to be tested is G, C, A or U, the nucleotide at the corresponding position of the target sequence is T, A, C or G. In order to be equal in length to the GSSM target sequence, nucleotides A and C are added to the 3' end of the target sequence in sequence.
将目标序列克隆到psiCHECKTM-2质粒的Xho I/Not I位点。The target sequence was cloned into the Xho I/Not I site of the psiCHECK™ -2 plasmid.
[2]转染[2] Transfection
在96孔板中，根据LipofectamineTM2000(Invitrogen公司)的使用说明，分别共转染缀合物和上述每一种质粒，其中每孔转染质粒10ng，使用LipofectamineTM2000 0.2μL，siRNA缀合物终浓度(以siRNA的量计)自0.1nM起始，倍比稀释至0.0001nM，一种质粒对应11组siRNA浓度，每组3个复孔。In a 96-well plate, according to the instructions of Lipofectamine™ 2000 (Invitrogen), the conjugate and each of the above plasmids were co-transfected, wherein 10 ng of plasmid was transfected into each well, 0.2 μL of Lipofectamine™ 2000 was used, the final concentration of siRNA conjugate (in terms of siRNA amount) started from 0.1 nM and was diluted to 0.0001 nM in multiple ratios, one plasmid corresponded to 11 groups of siRNA concentrations, and each group had 3 replicate wells.
[3]检测[3] Detection
将HEK293A细胞培养24小时后，使用双萤光报告基因检测试剂盒(Dualluciferase reporter gene assay kit，Promega公司，cat.E2940)，根据使用说明书裂解细胞，检测双萤光报告基因的表达水平。每一特定浓度的缀合物测试组以无缀合物处理组为对照。以海肾萤光素酶蛋白水平(Ren)相对于萤火虫萤光素酶蛋白水平(Fir)进行标准化。After HEK293A cells were cultured for 24 hours, dual luciferase reporter gene assay kit (Promega, cat. E2940) was used to lyse cells according to the instruction manual to detect the expression level of dual fluorescent reporter gene. Each conjugate test group of a specific concentration was compared with the non-conjugate treatment group. The Renilla luciferase protein level (Ren) was standardized relative to the firefly luciferase protein level (Fir).
根据采用不同siRNA浓度所测得的活性结果，利用Graphpad 5.0软件log(inhibitor)vs.response-Variable slope功能来拟合剂量-效应曲线，根据剂量-效应曲线计算待测siRNA靶向GSCM的IC50值，计算方法如下：According to the activity results measured by different siRNA concentrations, the dose-effect curve was fitted using the log (inhibitor) vs. response-Variable slope function of Graphpad 5.0 software. The IC50 value of the siRNA to be tested targeting GSCM was calculated according to the dose-effect curve. The calculation method is as follows:
式中：Where:
Y是残留mRNA的表达水平，Y is the expression level of residual mRNA,
X为转染siRNA浓度的对数值，X is the logarithmic value of the transfected siRNA concentration,
Bot是稳态期底部的Y值，Bot is the Y value at the bottom of the steady state period,
Top是稳态期顶部的Y值，Top is the Y value at the top of the steady state period,
LogIC50是当Y在底部到顶部之间一半时的X值，而HillSlope则是曲线的斜率。结果如图5所示。LogIC50 is the value of X when Y is halfway between the bottom and the top, and HillSlope is the slope of the curve. The results are shown in Figure 5.
由图5可知，缀合物4在具有优异靶mRNA抑制效果的同时，还显示出低的脱靶效应。As shown in FIG5 , conjugate 4 has excellent target mRNA inhibition effect while also showing low off-target effect.
实验例3本实验例说明本公开的缀合物在小鼠中对HBV mRNA表达的抑制在本实验例中，对缀合物4在HBV转基因小鼠C57BL/6J-Tg(Alb1HBV)44Bri/J中对HBV mRNA表达量的抑制效率进行了考察。Experimental Example 3 This experimental example illustrates the inhibition of HBV mRNA expression by the conjugate of the present invention in mice. In this experimental example, the inhibitory efficiency of conjugate 4 on HBV mRNA expression in HBV transgenic mice C57BL/6J-Tg(Alb1HBV)44Bri/J was investigated.
C57BL/6J-Tg(Alb1HBV)44Bri/J小鼠购自北京大学医学部实验动物科学部，使用乙型肝炎病毒表面抗原诊断试剂盒(酶联免疫法)(上海科华生物)检测小鼠血清HBsAg含量，选取S/COV＞10的小鼠，随机分组(均为雌性)，每组4只小鼠，分别进行编号，并增加生理盐水NS对照组。所有动物根据体重计算药量，采用皮下注射方式单次给药，分别以1mg/kg以及0.1ml/kg的不同剂量给予缀合物4(以0.2mg/ml以及0.02mg/ml的0.9％氯化钠水溶液形式提供)，给药体积为5ml/kg。给药后第7天将动物处死，收集肝脏，用RNA later(SigmaAldrich公司)保存；用组织匀浆仪匀浆肝组织，再用Trizol根据总RNA提取的标准操作步骤提取得到总RNA。C57BL/6J-Tg(Alb1HBV)44Bri/J mice were purchased from the Department of Experimental Animal Science, Peking University Health Science Center. The hepatitis B virus surface antigen diagnostic kit (enzyme-linked immunosorbent assay) (Shanghai Kehua Biotechnology) was used to detect the HBsAg content in mouse serum. Mice with S/COV>10 were selected and randomly divided into groups (all female), with 4 mice in each group, numbered separately, and a normal saline NS control group was added. The drug dose for all animals was calculated according to body weight, and the conjugate 4 (provided in the form of 0.2 mg/ml and 0.02 mg/ml of 0.9% sodium chloride aqueous solution) was administered at different doses of 1 mg/kg and 0.1 ml/kg, respectively, with a dosing volume of 5 ml/kg. The animals were killed on the 7th day after administration, and the livers were collected and preserved with RNA later (SigmaAldrich); the liver tissue was homogenized with a tissue homogenizer, and total RNA was extracted with Trizol according to the standard operating procedures for total RNA extraction.
采用实时荧光定量PCR检测肝组织中HBV mRNA的表达量，具体地：使用ImProm-IITM反转录试剂盒(Promega公司)按其说明书将提取的总RNA逆转录为cDNA，接着用荧光定量PCR试剂盒(北京康为世纪生物科技有限公司)检测siRNA对肝组织中的HBV mRNA表达的抑制效率。在该荧光定量PCR法中，以β-肌动蛋白(β-actin)基因作为内参基因，使用针对HBV的引物和针对β-肌动蛋白的引物分别对HBV和β-肌动蛋白进行检测。Real-time fluorescence quantitative PCR was used to detect the expression of HBV mRNA in liver tissue. Specifically, the extracted total RNA was reverse transcribed into cDNA using the ImProm-IITM reverse transcription kit (Promega) according to its instructions, and then the fluorescence quantitative PCR kit (Beijing Kangwei Century Biotechnology Co., Ltd.) was used to detect the inhibitory efficiency of siRNA on HBV mRNA expression in liver tissue. In this fluorescence quantitative PCR method, the β-actin gene was used as the internal reference gene, and primers for HBV and primers for β-actin were used to detect HBV and β-actin, respectively.
检测引物的序列参见表3。See Table 3 for the sequences of the detection primers.
表3检测引物的序列Table 3 Sequences of detection primers
在该荧光定量PCR法中，HBVmRNA的表达量用HBV X基因表达剩余量表示，按如下等式计算：In this fluorescence quantitative PCR method, the expression level of HBV mRNA is represented by the residual expression level of HBV X gene, which is calculated according to the following equation:
HBV X基因表达剩余量＝(测试组HBV X基因的拷贝数/测试组β-actin的拷贝数)/(对照组HBV X基因的拷贝数/对照组β-actin的拷贝数)×100％，图中标识为HBV X/β-actin mRNA表达量。HBV X gene expression residual amount = (test group HBV X gene copy number / test group β-actin copy number) / (control group HBV X gene copy number / control group β-actin copy number) × 100%, marked as HBV X / β-actin mRNA expression amount in the figure.
随后根据下式计算缀合物对mRNA抑制率：The mRNA inhibition rate of the conjugate was then calculated according to the following formula:
缀合物对mRNA的抑制率＝(1-HBV X基因表达剩余量)×100％，The inhibition rate of the conjugate on mRNA = (1-HBV X gene expression remaining amount) × 100%,
其中，对照组为本实验中施以NS的对照组小鼠，各测试组为分别施以不同siRNA缀合物的给药组小鼠。结果示于图6中。The control group is the control group mice administered with NS in this experiment, and each test group is the administration group mice administered with different siRNA conjugates. The results are shown in FIG6 .
由图6的结果可见，上述本公开的缀合物在1mg/kg的给药量下，对于靶mRNA的抑制率达93.63％；在更低浓度(0.1mg/kg)的给药量下，仍具有77.05％的抑制率，均显示出良好的抑制效果。As shown in the results of FIG6 , the conjugate disclosed above has an inhibition rate of 93.63% for target mRNA at a dosage of 1 mg/kg; at a lower dosage (0.1 mg/kg), it still has an inhibition rate of 77.05%, both showing good inhibitory effects.
实验例4本实验说明本公开的siRNA缀合物在M-Tg模型上单次给药对HBsAg和HBVmRNA的抑制作用Experimental Example 4 This experiment illustrates the inhibitory effect of a single administration of the siRNA conjugate disclosed herein on HBsAg and HBV mRNA in an M-Tg model
将HBV转基因(M-TgHBV)小鼠(购自上海市公共卫生中心动物部)按血清HBsAg含量随机分成3组(每组6只，均为雄性)，分别为生理盐水(NS)对照组、缀合物41mg/kg和3mg/kg组。所有动物根据体重计算药量，给药体积为10ml/kg，皮下单次给药。在给药前(记为D0)与给药后第7、14、21、28、42、56、70、85天对小鼠眼眶静脉丛取血，在各时间点检测血清HBsAg水平。HBV transgenic (M-TgHBV) mice (purchased from the Animal Department of Shanghai Public Health Center) were randomly divided into 3 groups (6 in each group, all male) according to the serum HBsAg content, namely the normal saline (NS) control group, the conjugate 41mg/kg and 3mg/kg groups. The drug dose was calculated for all animals according to their body weight, and the administration volume was 10ml/kg, with a single subcutaneous administration. Blood was collected from the orbital venous plexus of mice before administration (denoted as D0) and on days 7, 14, 21, 28, 42, 56, 70, and 85 after administration, and serum HBsAg levels were detected at each time point.
眼眶取血每次约0.5ml，离心后血清不少于200μl。利用HBsAg CLIA试剂盒(安图生物，CL0310)检测血清中HBsAg的含量。About 0.5 ml of blood was collected from the orbit each time, and the serum was no less than 200 μl after centrifugation. The HBsAg CLIA kit (Antu Biotechnology, CL0310) was used to detect the HBsAg content in the serum.
标准化的血清HBsAg水平＝(给药后测试组HBsAg含量/给药前测试组HBsAg含量)×100％。Normalized serum HBsAg level = (HBsAg content of the test group after administration/HBsAg content of the test group before administration) x 100%.
HBsAg抑制率＝(1-给药后HBsAg含量/给药前HBsAg含量)量sAg％。HBsAg inhibition rate = (1-HBsAg content after administration/HBsAg content before administration) HBsAg%.
其中，HBsAg含量用每毫升(ml)血清含多少当量(UI)HBsAg表示。The HBsAg content is expressed as the number of HBsAg equivalents (UI) per milliliter (ml) of serum.
HBV mRNA的表达量检测同实验例3。The detection of HBV mRNA expression was the same as in Experimental Example 3.
以下图7和图8示出了上述测试siRNA缀合物在在M-Tg模型上单次给药对HBsAg和HBV X mRNA表达抑制作用的检测结果。The following Figures 7 and 8 show the test results of the inhibitory effect of the above-mentioned test siRNA conjugates on the expression of HBsAg and HBV X mRNA in the M-Tg model after a single administration.
从图7和图8的结果可以看出：单次给药3mg/kg的缀合物4对HBsAg的最大抑制率在95％以上，在长达56天的时间内，对HBsAg的抑制率在90％以上，在D85对HBV X mRNA抑制率为62％。From the results of Figures 7 and 8, it can be seen that the maximum inhibition rate of HBsAg by a single administration of 3 mg/kg of conjugate 4 was above 95%, the inhibition rate of HBsAg was above 90% for up to 56 days, and the inhibition rate of HBV X mRNA was 62% at D85.
实验数据均以表示，数据分析采用Graphpad prism5.0统计分析软件。首先对数据进行正态分布及方差齐性检验。符合正态分布(p＞0.20)及方差齐(p＞0.10)：多组间比较采用单因素方差分析的LSD法进行多重比较，p＜0.05认为有统计学意义；不符合正态分布或方差不齐：多组间比较采用非参数检验的Kruskal-Wallis H方法，如果Kruskal-wallis H检验结果显著(p＜0.05)，再将数据进行秩转换后，进行多组间两两比较，p＜0.05认为有统计学意义。图中以＊表示显著性差异。The experimental data are Indicates that the data were analyzed using Graphpad prism5.0 statistical analysis software. First, the data were tested for normal distribution and homogeneity of variance. If the data were in accordance with normal distribution (p>0.20) and homogeneity of variance (p>0.10), the LSD method of one-way analysis of variance was used for multiple comparisons among multiple groups, and p<0.05 was considered statistically significant; if the data were not in accordance with normal distribution or the variance was unequal, the Kruskal-Wallis H method of nonparametric test was used for multiple group comparisons. If the Kruskal-Wallis H test result was significant (p<0.05), the data were rank-transformed and then compared between multiple groups, and p<0.05 was considered statistically significant. Significant differences are indicated by * in the figure.
以上详细描述了本公开的实施方案，但是，本公开并不限于上述实施方案中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。The embodiments of the present disclosure are described in detail above; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.
另外需要说明的是，在上述具体实施方案中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合。为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.
此外，本公开的各种不同的实施方案之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.
以引用的方式并入Incorporated by Reference
本说明书中提及的所有出版物、专利以及专利申请均以引用的方式并入本文，其程度与每一单独的出版物、专利以及专利申请均专门并且单独地以引用的方式并入本文的程度相同。All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711249342 | 2017-12-01 | ||
| CN2017112493427 | 2017-12-01 | ||
| CN2017114870136 | 2017-12-29 | ||
| CN201711487013 | 2017-12-29 | ||
| PCT/CN2018/118106WO2019105403A1 (en) | 2017-12-01 | 2018-11-29 | Nucleic acid, composition and conjugate containing the same, preparation method and use thereof |
| Publication Number | Publication Date |
|---|---|
| CN110945131A CN110945131A (en) | 2020-03-31 |
| CN110945131Btrue CN110945131B (en) | 2024-05-28 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201880049191.7AActiveCN110945131B (en) | 2017-12-01 | 2018-11-29 | Nucleic acid, composition containing nucleic acid, conjugate, preparation method and application |
| Country | Link |
|---|---|
| CN (1) | CN110945131B (en) |
| TW (1) | TWI878211B (en) |
| WO (1) | WO2019105403A1 (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11492620B2 (en) | 2017-12-01 | 2022-11-08 | Suzhou Ribo Life Science Co., Ltd. | Double-stranded oligonucleotide, composition and conjugate comprising double-stranded oligonucleotide, preparation method thereof and use thereof |
| US11414661B2 (en) | 2017-12-01 | 2022-08-16 | Suzhou Ribo Life Science Co., Ltd. | Nucleic acid, composition and conjugate containing nucleic acid, preparation method therefor and use thereof |
| KR20250107294A (en) | 2017-12-01 | 2025-07-11 | 쑤저우 리보 라이프 사이언스 컴퍼니, 리미티드 | Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof |
| WO2019105435A1 (en) | 2017-12-01 | 2019-06-06 | 苏州瑞博生物技术有限公司 | Nucleic acid, composition and conjugate containing nucleic acid, preparation method and use |
| JP7365052B2 (en) | 2017-12-01 | 2023-10-19 | スーチョウ リボ ライフ サイエンス カンパニー、リミテッド | Nucleic acids, compositions and complexes containing the nucleic acids, and preparation methods and uses |
| HRP20250408T1 (en) | 2017-12-29 | 2025-06-06 | Suzhou Ribo Life Science Co., Ltd. | Conjugates and preparation and use thereof |
| JP2021533800A (en) | 2018-08-21 | 2021-12-09 | スーチョウ リボ ライフ サイエンス カンパニー、リミテッドSuzhou Ribo Life Science Co., Ltd. | Nucleic acid, drug compositions and complexes containing the nucleic acid, and their use |
| JP7376952B2 (en) | 2018-09-30 | 2023-11-09 | スーチョウ リボ ライフ サイエンス カンパニー、リミテッド | siRNA complex and its preparation method and use |
| CN115698288A (en)* | 2020-04-08 | 2023-02-03 | 北京瑞博开拓医药科技有限公司 | Nucleic acid, pharmaceutical composition containing the nucleic acid, siRNA conjugate, preparation method and use |
| CN113528516A (en)* | 2020-04-17 | 2021-10-22 | 北京瑞博开拓医药科技有限公司 | Nucleic acid, pharmaceutical composition and siRNA conjugate containing the nucleic acid, and preparation method and use |
| CN111575279A (en)* | 2020-04-27 | 2020-08-25 | 江苏为真生物医药技术股份有限公司 | Method for capturing extrahepatic vesicle or circulating tumor cell by using ASGPR (adenosine triphosphate) small molecule ligand specificity |
| TW202214301A (en)* | 2020-06-16 | 2022-04-16 | 大陸商上海拓界生物醫藥科技有限公司 | Carbohydrate molecular cluster and preparation method and medical use thereof |
| CN114621951B (en)* | 2020-12-10 | 2025-05-30 | 施能康生物科技有限公司 | Nucleic acid targeting hepatitis B virus and its use |
| CN114632089B (en)* | 2020-12-16 | 2024-08-13 | 北京瑞博开拓医药科技有限公司 | Application of compound containing acylglycosylamine group in preparation of medicine for treating novel coronavirus pneumonia and treatment method of disease |
| CN119731322A (en)* | 2023-04-21 | 2025-03-28 | 苏州时安生物技术有限公司 | Oligonucleotide and preparation method and application thereof |
| CN120114470A (en)* | 2024-01-13 | 2025-06-10 | 张川 | A pharmaceutical composition for inhibiting NFKBIZ gene expression and its application |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005116204A1 (en)* | 2004-05-11 | 2005-12-08 | Rnai Co., Ltd. | Polynucleotide causing rna interfere and method of regulating gene expression with the use of the same |
| CN102140458A (en)* | 2010-01-29 | 2011-08-03 | 苏州瑞博生物技术有限公司 | SiRNA (Small interference ribonucleic acid) as well as medicine composition and pharmaceutical application thereof |
| CN102344477A (en)* | 2010-07-27 | 2012-02-08 | 苏州瑞博生物技术有限公司 | Nucleotide and / or oligonucleotide and preparation method thereof |
| CN103890000A (en)* | 2011-06-21 | 2014-06-25 | 阿尔尼拉姆医药品有限公司 | Angiopoietin-like 3 (ANGPTL3) iRNA compositions and methods of use thereof |
| WO2015006740A2 (en)* | 2013-07-11 | 2015-01-15 | Alnylam Pharmaceuticals, Inc. | Oligonucleotide-ligand conjugates and process for their preparation |
| CN105392488A (en)* | 2013-05-01 | 2016-03-09 | Isis制药公司 | Compositions and methods for modulating apolipoprotein c-iii expression |
| WO2016206626A1 (en)* | 2015-06-26 | 2016-12-29 | 苏州瑞博生物技术有限公司 | Sirna, pharmaceutical composition and conjugate which contain sirna, and uses thereof |
| CN107075516A (en)* | 2014-08-20 | 2017-08-18 | 阿尔尼拉姆医药品有限公司 | Modified dsRNA reagents |
| CN107250362A (en)* | 2014-11-17 | 2017-10-13 | 阿尔尼拉姆医药品有限公司 | Apolipoprotein C3 (APOC3) iRNA compositions and methods of use thereof |
| CN108064313A (en)* | 2015-03-17 | 2018-05-22 | 箭头药业股份有限公司 | Compositions and methods for inhibiting gene expression of factor XII |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030206887A1 (en)* | 1992-05-14 | 2003-11-06 | David Morrissey | RNA interference mediated inhibition of hepatitis B virus (HBV) using short interfering nucleic acid (siNA) |
| EP2405921A4 (en)* | 2009-01-26 | 2013-05-22 | Protiva Biotherapeutics Inc | Compositions and methods for silencing apolipoprotein c-iii expression |
| CN102140460B (en)* | 2010-01-29 | 2012-12-12 | 苏州瑞博生物技术有限公司 | SiRNA (Small interference ribonucleic acid) as well as medicine composition and pharmaceutical application thereof |
| CN102140459B (en)* | 2010-01-29 | 2013-04-03 | 苏州瑞博生物技术有限公司 | SiRNA (Small interference ribonucleic acid) as well as medicine composition and pharmaceutical application thereof |
| CN104922141B (en)* | 2015-05-28 | 2016-05-25 | 厦门成坤生物技术有限公司 | A kind of siRNA composition that is used for the treatment of virus B hepatitis |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005116204A1 (en)* | 2004-05-11 | 2005-12-08 | Rnai Co., Ltd. | Polynucleotide causing rna interfere and method of regulating gene expression with the use of the same |
| CN102140458A (en)* | 2010-01-29 | 2011-08-03 | 苏州瑞博生物技术有限公司 | SiRNA (Small interference ribonucleic acid) as well as medicine composition and pharmaceutical application thereof |
| CN102344477A (en)* | 2010-07-27 | 2012-02-08 | 苏州瑞博生物技术有限公司 | Nucleotide and / or oligonucleotide and preparation method thereof |
| CN103890000A (en)* | 2011-06-21 | 2014-06-25 | 阿尔尼拉姆医药品有限公司 | Angiopoietin-like 3 (ANGPTL3) iRNA compositions and methods of use thereof |
| CN105392488A (en)* | 2013-05-01 | 2016-03-09 | Isis制药公司 | Compositions and methods for modulating apolipoprotein c-iii expression |
| WO2015006740A2 (en)* | 2013-07-11 | 2015-01-15 | Alnylam Pharmaceuticals, Inc. | Oligonucleotide-ligand conjugates and process for their preparation |
| CN107075516A (en)* | 2014-08-20 | 2017-08-18 | 阿尔尼拉姆医药品有限公司 | Modified dsRNA reagents |
| CN107250362A (en)* | 2014-11-17 | 2017-10-13 | 阿尔尼拉姆医药品有限公司 | Apolipoprotein C3 (APOC3) iRNA compositions and methods of use thereof |
| CN108064313A (en)* | 2015-03-17 | 2018-05-22 | 箭头药业股份有限公司 | Compositions and methods for inhibiting gene expression of factor XII |
| WO2016206626A1 (en)* | 2015-06-26 | 2016-12-29 | 苏州瑞博生物技术有限公司 | Sirna, pharmaceutical composition and conjugate which contain sirna, and uses thereof |
| Title |
|---|
| Advanced siRNA Designs Further Improve In Vivo Performance of GalNAc-siRNA Conjugates;Donald J. Foster等;《Mol Ther》;20180104;第26卷(第3期);第711页图2C* |
| Publication number | Publication date |
|---|---|
| WO2019105403A1 (en) | 2019-06-06 |
| CN110945131A (en) | 2020-03-31 |
| TW201925473A (en) | 2019-07-01 |
| TWI878211B (en) | 2025-04-01 |
| Publication | Publication Date | Title |
|---|---|---|
| CN110945131B (en) | Nucleic acid, composition containing nucleic acid, conjugate, preparation method and application | |
| CN110997917B (en) | Nucleic acid, composition containing nucleic acid, conjugate, preparation method and application | |
| CN110944675B (en) | Nucleic acid, composition containing nucleic acid, conjugate, preparation method and application | |
| CN110945130B (en) | Nucleic acid, composition and conjugate containing the nucleic acid, preparation method and use thereof | |
| CN110945132B (en) | Nucleic acid, composition and conjugate containing the nucleic acid, preparation method and use thereof | |
| CN111050807B (en) | Nucleic acid, composition and conjugate containing the nucleic acid, preparation method and use thereof | |
| TWI791696B (en) | Double-stranded oligonucleotides, pharmaceutical compositions and conjugates containing them, uses and kits thereof | |
| CN111655849B (en) | Nucleic acid, pharmaceutical composition and conjugate containing the nucleic acid and use thereof | |
| CN113795280B (en) | Nucleic acid, pharmaceutical composition and conjugate, preparation method and use thereof | |
| WO2020135581A1 (en) | Nucleic acid, composition and conjugate containing nucleic acid, preparation method therefor and use thereof | |
| CN113795582B (en) | Nucleic acid, pharmaceutical composition and conjugate, and preparation method and application thereof | |
| CN113330117B (en) | Nucleic acid, composition and conjugate containing the nucleic acid, preparation method and use thereof | |
| CN111973618A (en) | Nucleic acid, pharmaceutical composition and siRNA conjugate, preparation method and application | |
| CN113614230B (en) | Nucleic acid, pharmaceutical composition and conjugate, and preparation method and application thereof | |
| HK40019795A (en) | Nucleic acid, composition and conjugate containing nucleic acid, preparation method therefor and use thereof | |
| HK40019791A (en) | Nucleic acid, composition and conjugate containing nucleic acid, preparation method and use | |
| HK40019837A (en) | Nucleic acid, composition and conjugate containing the same, preparation method and use thereof | |
| HK40019842A (en) | Nucleic acid, composition and conjugate containing same, and preparation method and use |
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | Address after:215300, No. 168, Feng Feng Road, Yushan Town, Kunshan, Jiangsu, Suzhou Applicant after:Suzhou Ruibo Biotechnology Co.,Ltd. Address before:215300, No. 168, Feng Feng Road, Yushan Town, Kunshan, Jiangsu, Suzhou Applicant before:SUZHOU RIBO LIFE SCIENCE Co.,Ltd. | |
| CB02 | Change of applicant information | ||
| REG | Reference to a national code | Ref country code:HK Ref legal event code:DE Ref document number:40019837 Country of ref document:HK | |
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TG01 | Patent term adjustment | ||
| TG01 | Patent term adjustment |