A novel double-stranded siRNA, conjugates thereof and uses thereof

Abstract

The present application provides a novel double-stranded siRNA, conjugates thereof and uses thereof. The present application also provides uses of the double-stranded siRNA and conjugates thereof in the preparation of a medicament for treating and / or preventing hepatitis B disease. The present application relates to a novel compound, and uses of the compound in the raw material for DNA nucleotide solid-phase synthesis, the raw material for siRNA drug synthesis, the research of siRNA drug, the research of gene function and / or the screening of whole gene library, especially in the raw material for the synthesis of the double-stranded siRNA drug. Furthermore, the present application also relates to a novel nucleotide residue, and its application in the research of siRNA drug, the research of gene function and / or the screening of whole gene library, and its application as an oligonucleotide intercalating group.

Description

Technical Field

[0001] This invention belongs to the field of small nucleic acid drugs. The purpose of this invention is to provide a novel double-stranded siRNA, its conjugates, and their uses. This invention also provides the use of the said double-stranded siRNA and its conjugates in the preparation of drugs for the treatment and / or prevention of hepatitis B. Furthermore, this invention relates to a novel compound and its applications (uses) in DNA nucleotide solid-phase synthesis, oligonucleotide drug synthesis, siRNA drug synthesis, siRNA drug research, gene function research, and / or whole gene library screening, particularly its use as a synthetic raw material for the double-stranded siRNA drug of this invention. Moreover, this invention also relates to a novel nucleotide residue and its applications in siRNA drug research, gene function research, and / or whole gene library screening, as well as its use as an oligonucleotide intercalation group. Background Technology

[0002] Hepatitis B (HBV) is a serious infectious disease threatening the world, especially China. Currently, the two most widely recognized classes of hepatitis B treatment drugs are interferon and nucleoside analogs. However, these drugs have several drawbacks, such as the potential for drug resistance or limited use. For example, interferon is prone to adverse reactions, and nucleoside analogs have issues with drug resistance and relapse after discontinuation. Therefore, silencing viral gene expression at the gene level, blocking HBV generation and replication, and thus fundamentally reducing viral replication and infection of hepatocytes, would 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 like cancer) in a sequence-specific manner based on the RNA interference (RNAi) mechanism, thereby achieving the goal of treating the disease.

[0003] RNA interference (RNAi) is the phenomenon of silencing homologous gene expression at the mRNA level using double-stranded RNA (dsRNA) molecules. Also known as gene knockdown or gene silencing, RNAi is a typical post-transcriptional gene regulation method, also called post-transcriptional gene silencing (PTGS). The earliest reports of RNAi appeared in 1990, with two different research groups simultaneously reporting RNAi in transgenic plants. Subsequently, RNAi was observed in almost all eukaryotes, including nematodes, fruit flies, zebrafish, and mice. In 1999, Hamilton and Baulcombe detected RNA fragments of 21-25 nucleotides in length in plants experiencing RNAi; these fragments were proven essential for RNAi and were termed small interfering nucleic acids (siRNA). Double-stranded siRNA forms an RNA-induced silencing complex (RISC) with cell-derived related enzymes and proteins. During RNA interference, the sense strand of the double-stranded siRNA is excluded from the complex, and the antisense strand guides RISC to bind to the homologous site of the target mRNA. Then, the target mRNA is degraded by ribonuclease III in the complex, thereby shutting down the expression of the target gene.

[0004] Detailed description of the compounds of the present invention

[0005] This invention provides a novel targeting compound that can be used to prepare nucleic acid drugs for targeted delivery of oligonucleotides to the liver. The invention also provides a nucleotide conjugate, its preparation method, and its application, which exhibits high in vivo delivery efficiency, good stability, high gene expression inhibitory activity in hepatocytes, and / or low toxicity. Furthermore, this invention provides an siRNA reagent, its preparation method, and its application, which exhibits high in vivo delivery efficiency, good stability, high gene expression inhibitory activity in HBV, and / or low toxicity.

[0006] The purpose of this invention is to provide a novel double-stranded siRNA, its conjugates, its salts, and their uses, specifically relating to the use of the double-stranded siRNA and its conjugates in the preparation of medicaments for the treatment and / or prevention of hepatitis B.

[0007] Furthermore, this invention relates to a novel compound and its use as a raw material for solid-phase DNA nucleotide synthesis, a raw material for siRNA drug synthesis, siRNA drug research, gene function research, and / or screening of whole gene libraries, particularly its use as a raw material for the synthesis of the double-stranded siRNA drug described in this invention. Furthermore, this invention also relates to a novel nucleotide residue and its use in siRNA drug research, gene function research, and / or screening of whole gene libraries, as well as its application as an oligonucleotide intercalation group. The double-stranded siRNA and its conjugates described in this invention exhibit high gene expression inhibitory activity against HBV and / or low toxicity.

[0008] On one hand, the present invention relates to a compound having a structure as shown in formula (I), or a stereoisomer, tautomer, or acceptable salt of a compound as shown in formula (I).

[0009]

[0010] Among them, each R, Z, Z 1 X has the meaning as described in this invention.

[0011] In some embodiments of the compounds described in this invention, X is OR 1 F, benzyl, -CH2C(=O)OR 1a Or -P(OCH2CH2CN)N(i-Pr)2, where Pr is isopropyl, and each R 1 and R 1a It has the meaning as described in this invention. In some embodiments of the compounds described in this invention, each R... 1 R 1a R 1b R 1c R 1d and R 1e The methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl groups are independently and optionally substituted by 1, 2, 3, or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy, and 2-propoxy groups.

[0012] In some embodiments of the compounds described in this invention, Z and Z 1 Each is independently protected by H, deuterium, methyl, hydroxyl groups, or HOC(=O)(CH2). j C(=O)-、MC(=O)(CH2) jC(=O)-, phosphate ester group, thiophosphate ester group, phosphite amide group or hydrophosphate group, wherein each j and M has the meaning described in this invention.

[0013] Any Z that allows the compound described in this invention to participate in oligonucleotide synthesis 1 All functional groups are included in this invention, such as phosphate ester groups, thiophosphate ester groups, phosphite amide groups, or hydrophosphate groups. These active phosphate groups are all included in this invention and can be obtained by the preparation methods disclosed in the art for the compounds described in this invention. For example, the compounds of this application can be obtained by the preparation methods of phosphate diester method, phosphate triester method, and hydrophosphate method described in Section 3 of the master's thesis "Research on Chemical Synthesis of Nucleic Acids and Synthesis and Interference Activity Test of 4'-C-hydroxy Modified siRNA". Another example is the preparation method described in the literature Kyle W. Knouse et al. "Unlocking P(V): Reagents for chiral phosphorothioate synthesis" 2018, pages 6-9.

[0014] The phosphate esters described in this invention include diesters and triesters of phosphate; the thiophosphate esters include diesters and triesters of phosphate; and the hydrophosphate group refers to the group formed by the salt of a phosphate group and a base.

[0015] In some embodiments of the compounds described in this invention, M is a solid support, preferably a hydroxyl or amino-functionalized solid support, more preferably a resin or CPG, further preferably a macroporous resin or CPG, and even more preferably an aminomethyl resin, a hydroxyl resin, or -NHCPG.

[0016] In some embodiments of the compounds described in this invention, R is -NHR. 2 , hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, fluorine, chlorine, bromine, -CH2C(=O)OR 1b -O-phenyl-C(=O)OR 1c -N(-CH2C(=O)OR 1d )2、-NS(=O)2-R 1e Or -N = CH-NR a R b Among them, each R 2 R 1b R 1c R 1d R 1e R a and R b It has the meaning as described in this invention.

[0017] In some embodiments of the compounds described in this invention, R 2 It is an amino protecting group.

[0018] In some embodiments of the compounds described in this invention, each R a and R b Independently protected by H or amino groups, or R a R b Together with the N atoms to which they are attached, they form a heterocyclic group consisting of 5-6 ring atoms, each of which is independently and optionally substituted by 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, 1-propoxy and 2-propoxy.

[0019] In some embodiments of the compounds described in this invention, each j is independently 1, 2, 3, 4 or 5.

[0020] The condition is that the compound described in this invention is not

[0021] In some embodiments of the compounds described in this invention, Z and Z 1 Each of the following is independently represented as H, deuterium, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, p-methoxybenzyl diphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, C 1-12 Alkyl, C 1-12 Alkyl C(=O)-, C 1-12 Alkylsilyl, C 6-10 Aryl C 1-6 Alkyl group, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytriphenylmethyl, Among them, each j, M, X, R x R y R c and R d It has the meaning described in this invention.

[0022] In some embodiments of the compounds described in this invention, Z and Z 1 Each of the following is independently represented as H, deuterium, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, p-methoxybenzyl diphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, C 1-10 Alkyl, C 1-10 Alkyl C(=O)-, C 1-10 Alkylsilyl, C 6-10 Aryl C 1-4 Alkyl group, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytriphenylmethyl, Among them, each j, M, X, R x R y R c and R d It has the meaning described in this invention.

[0023] In some embodiments of the compounds described in this invention, Z and Z 1 Each of the following is independently represented as H, deuterium, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, p-methoxybenzyl diphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, C 1-8 Alkyl, C 1-8 Alkyl C(=O)-, C 1-8 Alkylsilyl, Phenyl C 1-3 Alkyl group, HOC(=O)(CH2)j C(=O)-、MC(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytriphenylmethyl, Among them, each j, M, X, R x R y R c and R d It has the meaning described in this invention.

[0024] In some embodiments of the compounds described in this invention, Z and Z 1 Each of the following is independently represented as H, deuterium, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, p-methoxybenzyl diphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, C 1-6 Alkyl, C 1-6 Alkyl C(=O)-, C 1-6 Alkylsilyl, benzyl, phenethyl, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytriphenylmethyl, Among them, each j, M, X, R x R y R c and R d It has the meaning described in this invention.

[0025] In some embodiments of the compounds described in this invention, X is Cl or Br.

[0026] In some embodiments of the compounds described in this invention, each R x and R y It is an independent hydroxyl protecting group.

[0027] In some embodiments of the compounds described in this invention, each R c and R d Independently protected by H or amino groups; or R c R dTogether with the N atoms to which they are attached, they form a heterocyclic group consisting of 5-6 ring atoms, which is independently and optionally substituted by 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methyl, ethyl, propyl, methoxy, ethoxy, 1-propoxy and 2-propoxy.

[0028] In some embodiments of the compounds described in this invention, R 2 The following are listed: 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, methyl, tert-butyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, benzyl, p-methoxybenzyl diphenylmethyl, triphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, C 1-6 Alkoxy, R 3 C(=O)-, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, wherein R 3 It has the meaning described in this invention.

[0029] In some embodiments of the compounds described in this invention, each R a and R b Independently, it is H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, 1-propoxy, 2-propoxy, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, Phenoxyacetyl, benzoyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, benzyl, p-methoxybenzyl diphenylmethyl, triphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl or R 4 C(=O)-, or Ra R b Together with the N atom to which they are attached, they form pyrrolidinyl, morpholinyl, piperidinyl, and piperazineyl groups, each of which is independently and optionally substituted by 1, 2, 3, or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy, and 2-propoxy, wherein R 4 It has the meaning described in this invention.

[0030] In some embodiments of the compounds described in this invention, each R 3 and R 4 Independently for C 1-6 Alkyl, Halogenated C 1-6 Alkyl, C 1-6 Alkoxy, phenyl, halogen-substituted phenyl, C 1-6 Alkylphenyl or benzyl.

[0031] In some embodiments of the compounds described in this invention, each R 3 and R 4 Independently for C 1-4 Alkyl, Halogenated C 1-4 Alkyl, C 1-4 Alkoxy, phenyl, halogen-substituted phenyl, C 1-4 Alkylphenyl or benzyl.

[0032] In some embodiments of the compounds described in this invention, each R 3 and R 4 Independently, methyl, ethyl, n-propyl, isopropyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, 2,2,2-trichloroethyl, methoxy, ethoxy, 1-propoxy, 2-propoxy, 1-butoxy, 2-methyl-l-propoxy, 2-butoxy, tert-butoxy, phenyl, halogen-substituted phenyl, C 1-3 Alkylphenyl or benzyl.

[0033] In some embodiments of the compounds described in this invention, each R x and R yIndependently, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, p-methoxybenzyl diphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, cyanoC 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkyl C(=O)-, C 1-6 Alkylsilyl, phenyl, halogen-substituted phenyl, benzyl or phenethyl.

[0034] In some embodiments of the compounds described in this invention, each R x and R y Independently, it is benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl p-Methoxybenzyl diphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, CNCH2-, CN(CH2)2-, CNCH(CH3)2CH2-, CN(CH2)3-, CNCH(CH3)2CH2CH2-, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, C 1-4 Alkyl C(=O)-, C 1-4 Alkylsilyl, phenyl, halogen-substituted phenyl, benzyl or phenethyl.

[0035] In some embodiments of the compounds described in this invention, each R c and R dIndependently, it is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, 1-propoxy, 2-propoxy, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxy Acetyl, benzoyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, benzyl, p-methoxybenzyl diphenylmethyl, triphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl or R 4 C(=O)-, or R c R d Together with the N atom to which it is attached, it forms a pyrrolidinyl, morpholinyl, piperidinyl, or piperazine group, wherein the pyrrolidinyl, morpholinyl, piperidinyl, and piperazine group are independently and optionally substituted by 1, 2, 3, or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy, and 2-propoxy.

[0036] In some embodiments of the compounds described in this invention, they have a structure as shown in formula (II), or a stereoisomer, tautomer, or acceptable salt of the compound shown in (II).

[0037] Among them, R, X, Z and Z 1 It has the meaning described in this invention.

[0038] In some embodiments of the compounds described in this invention, they have one of the following compounds, or a stereoisomer, tautomer, or an acceptable salt thereof.

[0039]

[0040]

[0041]

[0042] In another aspect, the present invention relates to a nucleotide residue having the structure described in formula (Ia) or Y9.

[0043]

[0044] Among them, R W1 and R W2It has the meaning described in this invention.

[0045] In some embodiments of the nucleotide residues described in this invention, R W1 -NHR 2a , hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, fluorine, chlorine, bromine, -CH2C(=O)OR 2b -O-phenyl-C(=O)OR 2c -N(-CH2C(=O)OR 2d )2、-NS(=O)2-R 2e Each R 2a R 2b R 2c R 2d and R 2e It has the meaning described in this invention.

[0046] In some embodiments of the nucleotide residues described in this invention, R W2 OR 2f F, benzyl or -CH2C(=O)OR 2g , where R 2f and R 2g It has the meaning described in this invention.

[0047] In some embodiments of the nucleotide residues described in this invention, each R 2b R 2c R 2d R 2e R 2f and R 2g It is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl.

[0048] In some embodiments of the nucleotide residues described in this invention, R 2a It can be H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0049] The nucleotide monomers described in this invention are not

[0050] In some embodiments of the nucleotide residues described in this invention, the nucleotide residues of this invention have one of the following structures from Y1 to Y15, wherein the structures of Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14 and Y15 are respectively as follows:

[0051]

[0052]

[0053] On the other hand, the present invention relates to a double-stranded siRNA, wherein the double-stranded siRNA comprises a sense strand and an antisense strand, each nucleotide in the double-stranded siRNA is independently modified or unmodified, and the double-stranded siRNA contains one or more N (e.g., 1, 2, 3, 4 or 5), wherein the N is the nucleotide residue described in the present invention.

[0054] Unless otherwise specified, the double-stranded siRNA of the present invention contains at least one N, which can be at any position in the double-stranded siRNA.

[0055] In some embodiments of the double-stranded siRNA described in this invention, the positive strand of the double-stranded siRNA contains at least one (e.g., 1, 2, 3, 4, or 5) N, which can be at any position on the positive strand.

[0056] In some embodiments of the double-stranded siRNA described in this invention, the antisense strand of the double-stranded siRNA contains at least one (e.g., 1, 2, 3, 4, or 5) N, which can be at any position on the antisense strand.

[0057] In some embodiments of the double-stranded siRNA described in this invention, the positive strand contains 0 or at least one (e.g., 1, 2, 3, 4 or 5) N, and the negative strand contains 0 or at least one (e.g., 1, 2, 3, 4 or 5) N, wherein the N can be at any position on the positive or negative strand.

[0058] In some embodiments of the double-stranded siRNA of the present invention, the double-stranded siRNA contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 N.

[0059] In some embodiments of the double-stranded siRNA of the present invention, the double-stranded siRNA contains 1, 2, 3, 4 or 5 N.

[0060] In some embodiments of the double-stranded siRNA described in this invention, N is nucleotide residues Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14 and Y15.

[0061] In some embodiments of the double-stranded siRNA described in this invention, N is selected from nucleotide residues Y1, Y2, Y3, or Y4.

[0062] In some embodiments of the double-stranded siRNA described in this invention, the antisense strand comprises a nucleotide sequence as shown in 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO:2), and the length of the antisense strand does not exceed 23 nucleotides.

[0063] In some embodiments of the double-stranded siRNA described in this invention, the antisense strand is no longer than 21 or 22 nucleotides.

[0064] In some embodiments of the double-stranded siRNA described in this invention, the positive strand contains a nucleotide sequence as shown in 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO:1), and the length of the positive strand does not exceed 23 nucleotides.

[0065] In some embodiments of the double-stranded siRNA described in this invention, the length of the antisense strand does not exceed 19, 20, 21 or 22 nucleotides.

[0066] In some embodiments of the double-stranded siRNA, its conjugates, or salts thereof of the present invention, 70%, 75%, 80%, 85%, 90%, or more of the nucleotides in the sense or antisense strand of the double-stranded siRNA are modified nucleotides.

[0067] In some embodiments of the double-stranded siRNA, its conjugates, or salts thereof described in this invention, 70%, 75%, 80%, 85%, 90%, or more of the nucleotides in the sense and antisense strands of the double-stranded siRNA are modified nucleotides.

[0068] In some embodiments of the double-stranded siRNA described in this invention, all nucleotides in the sense and antisense strands are modified nucleotides.

[0069] In some embodiments of the double-stranded siRNA described in this invention, the modification is selected from at least one of the following: 2'-methoxy modification (i.e., the hydroxyl group at the 2-position of the ribose is replaced by a methoxy group), 2'-methoxyethyl modification (i.e., the hydroxyl group at the 2-position of the ribose is replaced by a methoxyethyl group), 2'-fluoro modification (i.e., the hydroxyl group at the 2-position of the ribose is replaced by an F group), thiophosphate linkage, 2'-deoxy modification, 2'-amino modification (i.e., the hydroxyl group at the 2-position of the ribose is replaced by an amino group), locked nucleic acid modification, unlocked nucleic acid modification, ethylene glycol nucleic acid modification, and 5'-vinyl phosphate modification.

[0070] In some embodiments of the double-stranded siRNA described in this invention, the positive strand comprises a sequence in which 0 or at least one (e.g., 1, 2, 3, 4 or 5) N residues are replaced by 0, 1, 2, 3, 4 or 5 nucleotide residues, as shown in SEQ ID NO:1.

[0071] In some embodiments of the double-stranded siRNA described in this invention, the antisense strand comprises a sequence in which 0 or at least one (e.g., 1, 2, 3, 4 or 5) N residues are replaced by 0, 1, 2, 3, 4 or 5 nucleotide residues, as shown in SEQ ID NO:2.

[0072] In some embodiments of the double-stranded siRNA described in this invention, the N substitution occurs at nucleotides 1 to 19 at the 5' end of the sequence shown in SEQ ID NO:1 (i.e., the N substitution optionally occurs at nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 at the 5' end of the sequence shown in SEQ ID NO:1).

[0073] In some embodiments of the double-stranded siRNA described in this invention, the N substitution occurs at the 7th, 17th, or 19th nucleotide at the 5' end of the sequence shown in SEQ ID NO:1.

[0074] In some embodiments of the double-stranded siRNA described in this invention, the N substitution occurs at positions 1 to 21 of the 5' end of the sequence shown in SEQ ID NO:2 (i.e., the N substitution optionally occurs at nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 of the 5' end of the sequence shown in SEQ ID NO:2).

[0075] In some embodiments of the double-stranded siRNA described in this invention, the N substitution occurs at the 5th, 6th, 7th, 10th, 11th, 16th, 17th, or 18th nucleotide at the 5' end of the sequence shown in SEQ ID NO:2.

[0076] In some embodiments of the double-stranded siRNA described in this invention, the positive strand comprises a sequence in which 0 or at least one (e.g., 1, 2, 3, 4 or 5) N residues are replaced by 0, 1, 2, 3, 4 or 5 nucleotide residues in a sequence such as 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO:5).

[0077] In some embodiments of the double-stranded siRNA described in this invention, the antisense strand comprises a sequence in which 0 or at least one (e.g., 1, 2, 3, 4 or 5) N residues are replaced by 0, 1, 2, 3, 4 or 5 nucleotide residues in a sequence such as 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO:22).

[0078] In some embodiments of the double-stranded siRNA described in this invention, the N substitution occurs at nucleotides 1 to 19 at the 5' end of the sequence shown in SEQ ID NO:5 (i.e., the N substitution optionally occurs at nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 at the 5' end of the sequence shown in SEQ ID NO:5).

[0079] In some embodiments of the double-stranded siRNA described in this invention, the N substitution occurs at the 7th, 17th, 19th, or 21st nucleotide at the 5' end of the sequence shown in SEQ ID NO:5.

[0080] In some embodiments of the double-stranded siRNA described in this invention, the N substitution occurs at nucleotides 1 to 21 at the 5' end of the sequence shown in SEQ ID NO:22 (i.e., the N substitution optionally occurs at nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 at the 5' end of the sequence shown in SEQ ID NO:22).

[0081] In some embodiments of the double-stranded siRNA described in this invention, the N substitution occurs at the 5th, 6th, 7th, 10th, 11th, 16th, or 17th nucleotide at the 5' end of the sequence shown in SEQ ID NO:22.

[0082] In some embodiments of the double-stranded siRNA described in this invention, the positive strand comprises one of the following nucleotide sequences:

[0083] SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, wherein the length of the positive strand does not exceed 23 nucleotides, and detailed information of the positive strand sequence is shown in the positive strand in Table 1.

[0084] In some embodiments of the double-stranded siRNA described in this invention, the length of the positive strand does not exceed 19, 20, 21 or 22 nucleotides.

[0085] In some embodiments of the double-stranded siRNA described in this invention, the antisense strand comprises one of the following nucleotide sequences:

[0086] SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39; SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49, the antisense strand is no more than 23 nucleotides in length, and detailed information about the antisense strand sequence is shown in Table 1.

[0087] In some embodiments of the double-stranded siRNA described in this invention, the antisense strand is no longer than 21 or 22 nucleotides.

[0088] In some embodiments of the double-stranded siRNA described in this invention, the double-stranded siRNA is selected from one of siRNA ID NO:1 to siRNA ID NO:48, and the sequence details of siRNA ID NO:1 to siRNA ID NO:48 are shown in Table 1 of this invention.

[0089] On the other hand, the present invention also relates to a double-stranded siRNA conjugate or a salt thereof, which comprises the siRNA described in the present invention, wherein the double-stranded siRNA conjugate is formed by conjugating the double-stranded siRNA with a conjugating group.

[0090] In some embodiments of the double-stranded siRNA conjugate or its salt described in this invention, the 3' or 5' end of the sense or antisense strand of the double-stranded siRNA is conjugated with a conjugating group.

[0091] In some embodiments of the double-stranded siRNA conjugate or its salt described in this invention, the 3' end of the positive strand of the double-stranded siRNA is conjugated with a conjugating group.

[0092] In some embodiments of the double-stranded siRNA conjugate or its salt described in this invention, the 3' or 5' end of the positive strand of the double-stranded siRNA is conjugated to the conjugating group via a phosphate ester group, a thiophosphate ester group, or a phosphate group.

[0093] In some embodiments of the double-stranded siRNA conjugate or its salt described in this invention, the conjugating group includes GalNAc or its derivative.

[0094] In some embodiments of the double-stranded siRNA conjugate or its salts of the present invention, the conjugating group is GalNAc or its derivative connected by a divalent, trivalent or tetravalent branching linker.

[0095] In some embodiments of the double-stranded siRNA conjugate or its salt described in this invention, the conjugating group is L-96, DAW40007-4, or its stereoisomer, wherein the structures of the conjugating groups DAW40007-4 and L-96 are as follows:

[0096]

[0097] On the other hand, the present invention also relates to a pharmaceutical composition comprising the double-stranded siRNA described in this invention or the double-stranded siRNA conjugate described in this invention or a salt thereof, and a pharmaceutically acceptable carrier.

[0098] On the other hand, the present invention also relates to the use of the double-stranded siRNA conjugate, the double-stranded siRNA conjugate or its salt or the pharmaceutical composition thereof in the preparation of a medicament for the treatment and / or prevention of hepatitis B.

[0099] Furthermore, the present invention also relates to the application of the compounds (i.e., the nucleotide monomers described in the present invention) in the fields of raw materials for solid-phase synthesis of DNA nucleotides, raw materials for the synthesis of oligonucleotide drugs, raw materials for the synthesis of siRNA drugs, siRNA drug research, gene function research and / or screening of whole gene libraries.

[0100] In another aspect, the present invention also relates to the use of the aforementioned nucleotide residues as oligonucleotide intercalation groups, wherein the oligonucleotide is a nucleotide sequence containing 10 to 50 nucleotides or nucleotide base pairs, and the oligonucleotide is capable of inhibiting or blocking gene expression.

[0101] In some embodiments of the application of the nucleotide residues described in this invention as oligonucleotide intercalation groups, the gene is the HBV gene.

[0102] In some embodiments of the application of the nucleotide residues described in this invention as oligonucleotide intercalation groups, the oligonucleotide is siRNA, which includes a sense strand and an antisense strand.

[0103] In some embodiments of the application of the nucleotide residues described in this invention as oligonucleotide intercalation groups, the nucleotide residues are intercalated only into the positive strand of the siRNA.

[0104] In some embodiments of the application of the nucleotide residues described in this invention as oligonucleotide intercalation groups, the nucleotide residues are intercalated into the nucleotide at position 7, 17, or 19 of the 5' end of the positive strand.

[0105] In some embodiments of the application of the nucleotide residues described in this invention as oligonucleotide intercalation groups, the nucleotide residues are intercalated only into the antisense strand of the siRNA.

[0106] In some embodiments of the application of the nucleotide residues described in this invention as oligonucleotide intercalation groups, the nucleotide residues are intercalated into the nucleotides at positions 5, 6, 7, 10, 11, 16, 17, or 18 of the 5' end of the antisense strand.

[0107] In some embodiments of the application of the nucleotide residues described in this invention as oligonucleotide intercalation groups, the nucleotide residues are intercalated into the sense and antisense strands of the siRNA, the nucleotide residues are intercalated into the 7th, 17th, or 19th position at the 5' end of the sense strand, and the nucleotide residues are intercalated into the 5th, 6th, 7th, 10th, 11th, 16th, 17th, or 18th position of the antisense strand.

[0108] On the other hand, the present invention also provides a method for inhibiting the expression of a specific gene in hepatocytes in a patient, comprising administering to the patient the double-stranded siRNA, double-stranded siRNA conjugate or salt thereof, or a combination thereof, wherein the double-stranded siRNA, double-stranded siRNA conjugate or salt thereof, or a combination thereof may be in a therapeutically effective amount.

[0109] Detailed Description of the Invention

[0110] Definitions and general terms

[0111] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0112] In this invention, the term "small interfering RNA (siRNA)" refers to a class of double-stranded RNA molecules of 17 to 30 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts via the RISC pathway by forming an RNA-induced silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through known RNA interference (RNAi) processes, inhibiting the translation of mRNA into amino acids and its conversion into proteins. For example, siRNA can regulate (e.g., inhibit) the expression of hepatitis B virus in cells.

[0113] In this invention, the term "antisense strand (or guide strand)" refers to an siRNA strand that includes a region substantially complementary to a target sequence (such as a segment of a sequence in hepatitis B virus mRNA). "Sense strand (or follower strand)" refers to an siRNA strand containing a region substantially complementary to the antisense strand. The antisense strand or sense strand of the double-stranded siRNA described in this invention contains one or more (e.g., 2, 3, 4, or 5) nucleotide residues N, where N (e.g., Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, and Y15) has the structure described in this invention.

[0114] The term "substantially complementary" means fully complementary or at least partially complementary, such as the antisense strand being fully complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can exist within the molecule or in terminal regions, with the most tolerant mismatches occurring in terminal regions, such as within 5, 4, 3, or 2 nucleotides at the 5'- and / or 3'-terminus of either strand of the siRNA.

[0115] It should be noted that "at least partially substantially complementary" to mRNA means that the antisense strand has a polynucleotide substantially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding hepatitis B virus). Alternatively, if a polynucleotide is substantially non-discontinuously complementary to a portion of the hepatitis B virus mRNA, then the antisense strand is complementary to at least a portion of the hepatitis B virus mRNA.

[0116] In this invention, the term "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the hepatitis B virus gene, including mRNA that is a primary transcription product of RNA processing.

[0117] In this disclosure, a double-stranded siRNA analog refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, which have “sense” and “antisense” orientations relative to the target RNA. In this disclosure, “complementary” has the meaning known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand pair complementaryly with the bases of the other strand. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair comprises one purine and one pyrimidine. When adenine on one strand always pairs with uracil on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence.

[0118] In this invention, the term "inhibition of hepatitis B virus gene expression" includes inhibition of hepatitis B virus (HBV) gene expression at any level, such as at least partial inhibition of HBV gene expression, including inhibition of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. HBV gene expression can be evaluated based on the level of any variable associated with HBV gene expression, such as HBV mRNA levels or HBV protein levels. Inhibition can be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to a control level. A control level can be any type of control level used in the art, such as a baseline level before administration, or a level measured in similar subjects, cells, or samples that have never been treated or have been treated with a control (e.g., a control with only a buffer or a control without an active agent).

[0119] In this invention, "pharmaceutical composition" can refer to a drug for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients constituting one or more adjunct components. Typically, the composition is prepared by uniformly and sufficiently combining the active double-stranded siRNA with liquid excipients, finely pulverized solid excipients, or both.

[0120] The siRNA of this invention contains nucleotide groups as basic structural units. As is known to those skilled in the art, these nucleotide groups contain phosphate groups, ribose groups, and bases, which will not be elaborated further here. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, preferably an unmodified nucleotide. The double-stranded siRNA of this invention contains a sense strand and an antisense strand.

[0121] The term "embedding" as used in this invention means that an embedding group is linked to at least one nucleotide residue in a sequence, including replacing a nucleotide residue in the sequence with an embedding group (such as N).

[0122] The "intercalation group" described in this invention is a residue of a natural nucleotide base analog, unlike any publicly disclosed natural nucleotide base. Introducing it into a nucleic acid sequence can endow the sequence with certain functions (such as unpredictable activity). For example, N (such as Y1, Y2, Y3, Y4) described in this invention, when intercalated into an oligonucleotide sequence as an intercalation group, can inhibit gene expression, thereby producing unpredictable activity.

[0123] The "oligonucleotide intercalation group" mentioned in this invention refers to the intercalation group being linked to at least one nucleotide residue in the oligonucleotide, including replacing one nucleotide residue with an intercalation group N in the oligonucleotide.

[0124] The N-embedded sequence described in this invention refers to a sequence in which at least one nucleotide residue is linked to N, including sequences in which N replaces a nucleotide residue. The N-embedded sequence described in this invention can also be optionally modified, such as by methoxy modification, fluorination modification, or linkage with a thiophosphate group. The N-embedded sequences described in this invention include, but are not limited to: N-embedded siRNA, N-embedded sense strands, and N-embedded antisense strands.

[0125] The conjugation groups described in this invention include pharmaceutically acceptable conjugation groups. Generally, pharmaceutically acceptable conjugation groups contain pharmaceutically acceptable target molecules and optional linkers. Examples of exemplary conjugation groups, linkers, and target molecules can be found in the disclosures of WO2015006740A2 and CN114555188A. Exemplary conjugation groups include, but are not limited to, L96, NAG37, or DAW40007-4 as described in this invention.

[0126] Unless otherwise stated, “conjugation” means that two or more chemical parts, each having a specific function, are connected to each other by covalent linkage; correspondingly, “conjugated compound” means a compound formed by the covalent linkage of the chemical parts.

[0127] The double-stranded siRNA conjugate of this invention is a compound formed by linking double-stranded siRNA with a pharmaceutically acceptable conjugate group, and the double-stranded siRNA and the pharmaceutically acceptable conjugate group are covalently linked.

[0128] In this invention, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used herein means approved by a regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0129] In this invention, the term "pharmaceuticalally acceptable excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a specific target dosage form. The use of any conventional excipients, except those incompatible with the double-stranded siRNA or its conjugates or salts of this invention, for example, for any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0130] In this invention, the term "treatment" refers to the use of drugs to achieve desired pharmacological and / or physiological effects. These effects may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used in this invention, "treatment" encompasses diseases in mammals, particularly humans, including: (a) preventing the onset of disease or symptoms in individuals susceptible to disease but not yet diagnosed with the disease; (b) inhibiting disease, e.g., blocking disease progression; or (c) alleviating disease, e.g., reducing disease-related symptoms. As used in this invention, "treatment" encompasses any medication that administers a drug, RNAi reagent, or siRNA to an individual to treat, cure, alleviate, improve, reduce, or inhibit the individual's disease, including but not limited to administering a drug containing the RNAi reagent, siRNA, or siRNA conjugate described in this invention to an individual in need.

[0131] Unless otherwise specified, in the context of this invention, uppercase letters C, G, U, and A represent the base composition of natural nucleotides; lowercase letters c, g, u, and a respectively indicate that the ribose at the 2-position of the nucleotide represented by the corresponding uppercase letter is modified by a methoxy group, such as c, g, u, and a representing 2'-OMe(2'-O-methyl)C, 2'-OMeG, 2'-OMeU, and 2'-OMeA respectively; the uppercase letter f to the right indicates a fluorinated base at the 2-position of the ribose of the nucleotide, such as Cf, Gf, Uf, and Af representing 2'-F(2'-fluorine)C, 2'-FG, 2'-FU, and 2'-FA respectively; "s" indicates that the two nucleotide residues adjacent to "s" are linked by a thiophosphate group, for example, "gsu" indicates that the g and u residues are linked by a thiophosphate group; Agn is adenosine-glycolic acid (GNA); Tgn represents a thymine-diol nucleotide residue, the structure of which is Y in double-stranded siRNA represents Y1 represents Y2 represents Y3 indicates Y4 indicates

[0132] The fluorine modification at the 2-position of the ribose in this invention refers to the replacement of the hydroxyl group at the 2-position of the ribose with fluorine (F); the methoxy modification at the 2-position of the ribose refers to the replacement of the hydroxyl group at the 2-position of the ribose with a methoxy group.

[0133] In the context of this invention, Bz represents benzoyl; MMTr represents 4'-methoxytriphenylmethyl; and DMTr represents 4',4'-dimethoxytriphenylmethyl.

[0134] In this invention, the terms "phosphate ester group," "phosphate ester group," and "phosphate ester bond" are used interchangeably, including monophosphate ester group, diephosphate ester group, or triphosphate ester group. The term "phosphate ester group" in "thiophosphate ester group" has the same meaning. Unless otherwise specified, the internucleotide phosphate ester group is a diephosphate ester group. Unless otherwise specified, the N-substituted sequence in this invention refers to one N replacing one or more nucleotide residues, not one N replacing two or more nucleotide residues. If two nucleotide residues are substituted, then two Ns should each replace two nucleotide residues. For example, if the antisense strand contains a sequence as shown in SEQ ID NO: 22 where N replaces 0, 1, 2, 3, 4, or 5 nucleotide residues, it should be interpreted as the antisense strand containing a sequence as shown in SEQ ID NO: 22 where 0, 1, 2, 3, 4, or 5 Ns replace 0, 1, 2, 3, 4, or 5 nucleotide residues respectively.

[0135] The term "oxo" refers to the =O group. For example, a carbon atom is connected to an oxygen atom by a double bond, in which a ketone or aldehyde is formed.

[0136] As used in this invention, "chemical modification" or "modification" means a structure that is chemically different from its naturally occurring counterpart, including all alterations made by chemical means, such as the addition or removal of a chemical component, or the substitution of one chemical component for another.

[0137] The compounds of this invention can be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, for example, enantiomers and diastereomers. The compounds of this invention containing asymmetric carbon atoms can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0138] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the desired enantiomer in pure form. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., from amines to carbamates).

[0139] This invention also includes compounds identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into the compounds of this invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 2 H, 3 H, 11 C, 13C, 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0140] Unless otherwise stated, when a position is specifically designated as deuterium (D), that position should be understood as having a deuterium abundance of at least 1000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium incorporation). The natural abundance of deuterium in the example compounds can be at least 1000 times, at least 2000 times, at least 3000 times, at least 4000 times, at least 5000 times, at least 6000 times, or even higher. The invention also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of compounds of formula (I) with reference to relevant literature. When preparing the deuterated form of compound (I), commercially available deuterated starting materials can be used, or conventional techniques can be used to synthesize it with deuterated reagents, including but not limited to deuterated borane, trideuterated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane, etc.

[0141] The conjugation group described in this invention can enhance the delivery of therapeutic agents to specific target sites (e.g., specific organs or tissues) within a subject such as a human or animal. In some embodiments of this invention, the conjugation group can enhance the targeted delivery of repressive double-stranded siRNA. In some embodiments of this invention, the conjugation group can enhance the delivery of repressive double-stranded siRNA to the liver.

[0142] The conjugating group described in this invention can be directly or indirectly attached to a compound, such as a therapeutic agent, for example, an expression inhibitory oligonucleotide, for example, the 3' or 5' end of the expression inhibitory oligonucleotide. In some embodiments of this invention, the expression inhibitory oligonucleotide comprises one or more modified nucleotides. In some embodiments of this invention, the expression inhibitory oligonucleotide is an RNAi reagent, such as a double-stranded siRNA reagent comprising a sense strand and an antisense strand. In some embodiments of this invention, the conjugating group disclosed herein is attached to the 3' end of the sense strand of the double-stranded siRNA reagent. In some embodiments, the conjugating group disclosed herein is attached to the expression inhibitory oligonucleotide reagent at the 3' end of the sense strand of the double-stranded siRNA reagent via a phosphate ester, thiophosphate ester, or phosphonate group.

[0143] The definitions and conventions of stereochemistry used in this invention are generally referenced in the following literature: S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of this invention may contain asymmetric or chiral centers, and therefore exist as different stereoisomers. All stereoisomers of the compounds of this invention, including, but not limited to, diastereomers, enantiomers, transisomers, and mixtures thereof, such as racemic mixtures, constitute a part of this invention. Many organic compounds exist in optically active forms, i.e., they are capable of rotating the plane of plane-polarized light. In describing optically active compounds, the prefixes D, L, or R, S are used to indicate the absolute configuration of the chiral center of the molecule. The prefixes d, l, or (+), (-) are used to name compounds whose plane polarization is rotated. (-) or l indicates the compound is levorotatory, while (+) or d indicates it is dextrorotatory. These stereoisomers have the same chemical structure, but their stereostructures differ. Specific stereoisomers can be enantiomers, and mixtures of isomers are usually called enantiomeric mixtures. A 50:50 enantiomeric mixture is called a racemic mixture or racemate, which may result in a lack of stereoselectivity or stereodirection during chemical reactions. The terms "racemic mixture" and "racemate" refer to a mixture of two equimolar enantiomers that lack optical activity.

[0144] The term "tautomer" or "tautomer form" refers to isomers of structures with different energies that can interconvert through a low energy barrier. For example, proton tautomers (i.e., proton-transfer tautomers) include interconversions via proton migration, such as isomerization between keto-enols and imine-enamines.

[0145] The term "composition" refers to a mixture of a drug containing one or more of the compounds described in this invention or their physiologically pharmaceutically acceptable salts or prodrugs, along with other chemical components, such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of the composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0146] The terms “pharmaceutical-grade excipient” or “pharmaceutical-acceptable excipient” include, but are not limited to, any adjuvant, carrier, excipient, flow aid, sweetener, diluent, preservative, dye / coloring agent, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0147] Unless otherwise specified, the "compounds," "ligands," "nucleic acid conjugates," "double-stranded siRNA conjugates," "double-stranded siRNA," and "nucleic acids" of this invention may exist independently as salts, mixed salts, or non-salts (e.g., free acids or free bases). When present as salts or mixed salts, they may be pharmaceutically acceptable salts.

[0148] The term "acceptable salt" includes acceptable acid addition salts and pharmaceutically acceptable base addition salts. An "acceptable acid addition salt" is a salt formed with an inorganic or organic acid that retains the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylic acid salts, and naphthalene disulfonates. These salts can be prepared by methods known in the art.

[0149] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that maintain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts, with sodium salts being the most preferred. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0150] As described in this invention, the compounds of this invention may optionally be substituted with one or more substituents, such as the general formula compounds above, or the specific examples, subclasses, and classes of compounds included in this invention, as described in the embodiments. Generally, the term "substituted" means that one or more hydrogen atoms in the given structure are substituted by a specific substituent. Unless otherwise indicated, an optional substituent group may have one substituent substituted at each substituted position of the group. When more than one position in the given structural formula can be substituted by one or more substituents selected from a specific group, the substituents may be substituted at the same or different positions.

[0151] Additionally, it should be noted that, unless otherwise explicitly stated, the descriptive terms “each and each is independently”, “each and each is independently”, and “each and each is independently” used throughout this invention are interchangeable and should be interpreted broadly. They can mean either that the specific options expressed by the same symbols in different groups do not affect each other, or that the specific options expressed by the same symbols in the same group do not affect each other.

[0152] In this invention, the terms “optionally,” “optionally,” or “optionally” generally refer to events or conditions described below that may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0153] In various parts of this specification, the substituents of the compounds disclosed herein are disclosed according to the type or scope of the groups. In particular, the invention includes every independent secondary combination of the various members of these group types and scopes. For example, the term "C..." 1-6 "Alkyl" specifically refers to independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0154] As used in this invention, the term "alkyl" includes a monovalent hydrocarbon group consisting of a saturated straight-chain or branched chain of 1-20 carbon atoms, wherein the alkyl group may be independently and optionally substituted by one or more substituents described in this invention. Some embodiments have an alkyl group containing 1-12 carbon atoms, others have an alkyl group containing 1-10 carbon atoms, still others have an alkyl group containing 1-8 carbon atoms, still others have an alkyl group containing 1-6 carbon atoms, still others have an alkyl group containing 1-4 carbon atoms, and still others have an alkyl group containing 1-3 carbon atoms. Further examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (n-Bu, -CH2CH2CH2CH3), 2-methylpropyl or isobutyl (i-Bu, -CH2CH(CH3)2), 1-methylpropyl or sec-butyl (s-Bu, -CH(CH3)CH2C) H3), tert-butyl (t-Bu, -C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2 -Methyl-1-butyl (-CH2CH(CH3)CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3) 4-Methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-Methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-Methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-Dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-Dimethyl-2-butyl (-CH(CH3)C(CH3)3), n-Heptyl, n-Octyl, n-Nonyl, n-Decanyl, etc.

[0155] The term "alkoxy group" indicates that an alkyl group is attached to the remainder of the molecule by an oxygen atom, wherein the alkyl group has the meaning as described in this invention. Unless otherwise specified, the alkoxy group contains 1-12 carbon atoms. In some embodiments, the alkoxy group contains 1-8 carbon atoms; in other embodiments, the alkoxy group contains 1-6 carbon atoms; in still other embodiments, the alkoxy group contains 1-4 carbon atoms; and in yet another embodiment, the alkoxy group contains 1-3 carbon atoms. The alkoxy group may optionally be substituted by one or more substituents described in this invention.

[0156] Examples of alkoxy groups include, but are not limited to, methoxy (MeO, -OCH3), ethoxy (EtO, -OCH2CH3), 1-propoxy (n-PrO, n-propoxy, -OCH2CH2CH3), 2-propoxy (i-PrO, i-propoxy, -OCH(CH3)2), 1-butoxy (n-BuO, n-butoxy, -OCH2CH2CH2CH3), 2-methyl-l-propoxy (i-BuO, i-butoxy, -OCH2CH(CH3)2), 2-butoxy (s-BuO, s-butoxy, -OCH(CH3)CH2CH3), 2-methyl-2- Propoxy (t-BuO, t-butoxy, -OC(CH3)3), 1-pentoxy (n-pentoxy, -OCH2CH2CH2CH2CH3), 2-pentoxy (-OCH(CH3)CH2CH2CH3), 3-pentoxy (-OCH(CH2CH3)2), 2-methyl-2-butoxy (-OC(CH3)2CH2CH3), 3-methyl-2-butoxy (-OCH(CH3)CH(CH3)2), 3-methyl-l-butoxy (-OCH2CH2CH(CH3)2), 2-methyl-l-butoxy (-OCH2CH(CH3)CH2CH3), etc.

[0157] The term "alkenyl" refers to a straight-chain or branched monovalent, divalent, or polyvalent hydrocarbon group containing 2-12 carbon atoms, or 2-8 carbon atoms, or 2-6 carbon atoms, or 2-4 carbon atoms, wherein at least one position of C and D is sp. 2 The double bond, wherein the alkenyl group may be independently unsubstituted or substituted by one or more substituents described in this invention, including "cis", "trans" or "Z", "E" isomers, wherein specific examples include, but are not limited to, vinyl (-CH=CH2), propenyl (-CH=CHCH3), allyl (-CH2CH=CH2), etc., wherein the alkenyl group may be independently unsubstituted or substituted by one or more substituents described in this invention.

[0158] The term "composed of M-M1 ring atoms" indicates that the cyclic group is composed of M-M1 ring atoms, including carbon atoms and / or heteroatoms such as O, N, S, and P. For example, "heterocyclic group composed of 3-6 ring atoms" represents a monoheterocyclic group consisting of 3, 4, 5, or 6 ring atoms.

[0159] The term "heterocyclic group" refers to a non-aromatic, saturated or partially unsaturated monocyclic, bicyclic, or tricyclic system comprising 3 to 12 ring atoms, wherein at least one ring atom is selected from nitrogen, sulfur, or oxygen atoms. The heterocyclic group may optionally be substituted by one or more substituents described in this invention. Unless otherwise stated, the heterocyclic group may be carbocyclic or nitrogen-based, and the -CH2- group may optionally be replaced by -C(=O)- or -C(=S)-. The sulfur atom of the ring may optionally be oxidized to an S-oxide. The nitrogen atom of the ring may optionally be oxidized to an N-oxide. In some embodiments, the heterocyclic group is a heterocyclic group composed of 3-12 ring atoms; in some embodiments, the heterocyclic group is a heterocyclic group composed of 5-10 ring atoms; in some embodiments, the heterocyclic group is a heterocyclic group composed of 3-6 ring atoms; in some embodiments, the heterocyclic group is a heterocyclic group composed of 4-6 ring atoms; in some embodiments, the heterocyclic group is a heterocyclic group composed of 5-6 ring atoms; in other embodiments, the heterocyclic group is a heterocyclic group composed of 4 atoms, which refers to a monovalent or polyvalent, saturated or partially unsaturated, non-aromatic monocyclic ring containing 4 ring atoms, wherein at least one ring atom is selected from nitrogen, sulfur, and oxygen atoms. In still other embodiments, the heterocyclic group is a heterocyclic group composed of 5 atoms, which refers to a monovalent or polyvalent, saturated or partially unsaturated, non-aromatic monocyclic ring containing 5 ring atoms, wherein at least one ring atom is selected from nitrogen, sulfur, and oxygen atoms. In other embodiments, the heterocyclic group is a six-atom heterocyclic group, referring to a monovalent or polyvalent, saturated or partially unsaturated, non-aromatic monocyclic ring containing six ring atoms, wherein at least one ring atom is selected from nitrogen, sulfur, and oxygen atoms. "Heterocyclic group" also includes groups formed by the fusion of a heterocyclic group with a saturated or partially unsaturated ring or heterocyclic group. Examples of heterocycles include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiophenyl, piperidinyl, morpholinyl, thiomorpholinyl, thiooxaneyl, piperazinyl, homopiperazinyl, aziridine, oxacyclobutyl, thiohexacyclobutyl, homopiperazinyl, epioxypropyl, aziridineheptyl, oxacycloheptyl, thiohexacycloheptyl, oxazeptyl, diazacyclobutyl, thioazeptyl, 2-pyrrolidinyl, 3- Pyrrolinyl, dihydroindolyl, 2H-pyranyl, 4H-pyranyl, dioxacyclohexyl, 1,3-dioxapentyl, pyrazolinyl, dithiaalkyl, dithiamonyl, dihydrothienyl, pyrazolylimidazolinyl, imidazolinyl, 1,2,3,4-tetrahydroisoquinolinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, 3H-indolylquinazinyl, and N-pyridylurea.Examples of heterocyclic groups also include 1,1-dioxothiomorpholino, wherein examples of carbon atoms on the ring being substituted by oxy (=O) include, but are not limited to, pyrimidinidone, 1,2,4-thiadiazole-5(4H)-keto, 1,2,4-oxadiazole-5(4H)-keto, 1H-1,2,4-triazole-5(4H)-keto, etc., wherein examples of carbon atoms on the ring being substituted by the =S group include, but are not limited to, 1,2,4-oxadiazole-5(4H)-thioketone, 1,3,4-oxadiazole-2(3H)-thioketone, etc.

[0160] The term "heteroatom" refers to one or more O, S, N, P, and Si, including N, S, and P in any oxidation state; primary, secondary, tertiary amines, and quaternary ammonium salts; or forms in which the hydrogen atom on the nitrogen atom in the heterocycle is substituted, for example, N (like N in 3,4-dihydro-2H-pyrrole), NH (like NH in pyrrolidinyl), or NR (like NR in N-substituted pyrrolidinyl, where R represents the substituent described in this invention).

[0161] The terms "alkylsilyl" and "alkylsilyl" indicate that the hydrogen atoms in the silyl (-SiH3) group are independently replaced by one, two, or three alkyl groups. In some embodiments, the alkylsilyl group has one, two, or three carbon atoms. 1-12 The alkyl group is attached to a silicon atom to form a lower-order alkylsilyl group. In other embodiments, the alkylsilyl group has one, two, or three carbon atoms. 1-9 The alkyl group is attached to a silicon atom to form a lower-order alkylsilyl group. In other embodiments, the alkylsilyl group has one, two, or three carbon atoms. 1-6 The alkyl group is attached to a silicon atom to form a lower-order alkylsilyl group. In other embodiments, the alkylsilyl group has one, two, or three carbon atoms. 1-4 The alkyl group is attached to a silicon atom to form a lower-order alkylsilyl group. In some other embodiments, the alkylsilyl group has one, two, or three carbon atoms. 1-3 An alkyl group is formed by attaching an alkyl atom to a silicon atom to form a lower-order alkylsilyl group. Suitable alkylsilyl groups can be monoalkylsilyl, dialkylsilyl, or trialkylsilyl. Examples of alkylsilyl groups include, but are not limited to, trimethylsilyl (-Si(CH3)3), triethylsilyl (-Si(CH2CH3)3), tri-n-propylsilyl (-Si(CH2CH2CH3)3), etc.

[0162] The term "hydroxyl protecting group" refers to an unstable chemical moiety that protects the hydroxyl group from unwanted reactions during one or more synthetic procedures. The hydroxyl protecting group can be selectively removed after the one or more synthetic procedures. Hydroxyl protecting groups known in the art are generally described in TH Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, New York (1999). Examples of hydroxyl protecting groups in this invention include, but are not limited to, C 1-12 Alkyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl, isopropoxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, 2-furfuryloxycarbonyl, allyloxycarbonyl, acetyl (Ac or -C(O)CH3), formyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl (Bz or -C(O)C6H5), C 1-10 Alkyl (methyl, tert-butyl, etc.), 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, C 6-10 Aryl C 1-4 Alkyl groups (such as benzyl, phenethyl, etc.), p-methoxybenzyl diphenylmethyl, triphenylmethyl (triphenylmethyl or trirityl), tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, C 1-10 Alkyl silyl (such as trimethylsilyl (TMS or -Si(CH3)3)), triethylsilyl, triisopropylsilyl, MMTr, DMTr or 4',4',4'-trimethoxytriphenylmethyl, etc.

[0163] The term "amino protecting group" refers to an unstable chemical component that protects an amino group from unwanted reactions during a synthetic procedure. Following one or more synthetic procedures, the amino protecting group, as described herein, can be selectively removed. Amino protecting groups known in the art are generally described in TH Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, New York (1999). Examples of amino protecting groups include, but are not limited to, acetyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, and benzyloxycarbonyl.

[0164] The term "solid support" specifically refers to any particle, bead, or surface on which oligonucleotide synthesis can occur. For example, both inorganic and organic solid supports can be selected for use in embodiments of the invention. Inorganic solid supports are preferably selected from silica gel and controlled-pore glass (CPG). Organic solid supports are resins, preferably macroporous resins, more preferably highly cross-linked polystyrene, Tentagel (a graft copolymer of a low-crosslinked polystyrene matrix with polyethylene glycol (PEG or POE) grafted onto it), polyvinyl acetate (PVA), Poros-polystyrene / divinylbenzene copolymers, amino polyethylene glycol, and cellulose, etc. Preferred embodiments of the invention utilize CPG-based solid supports. Many other commercially available solid supports are also included in this invention.

[0165] The present invention relates to nucleic acid conjugates, formulations of nucleic acid conjugates, methods of administration, and methods of treating diseases.

[0166] The effective amount of the nucleic acid conjugates (such as siRNA conjugates or pharmaceutical compositions) described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, such as four times a day, three times a day, twice a day, once a day, or every other day, or the number of daily doses may be proportionally reduced.

[0167] The medication may be administered to the subject via 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 local administration (including oral and sublingual administration), preferably intravenous administration.

[0168] The pharmaceutical compositions disclosed in this invention comprise formulations suitable for parenteral administration. The formulations can be conveniently available in unit dosage forms and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with excipients to prepare a single-dose form is generally the amount of siRNA that produces the therapeutic effect. Generally, this amount, expressed in percent, is from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0169] In another aspect, the present invention provides a method for inhibiting the expression or activity of hepatitis B virus genes. According to an embodiment of the present invention, the method includes contacting cells with the aforementioned siRNA, the aforementioned siRNA conjugate, or the aforementioned pharmaceutical composition. As previously mentioned, both the aforementioned siRNA and siRNA conjugate can degrade hepatitis B virus mRNA, inhibiting HBV expression and replication. Therefore, the method of the present invention can be used to inhibit the expression or activity of hepatitis B virus genes. For example, it can inhibit the expression or activity of hepatitis B virus genes in animals; or it can inhibit the expression or activity of hepatitis B virus genes for non-disease treatment purposes, such as inhibiting the expression or activity of hepatitis B virus genes in vitro for subsequent research.

[0170] In another aspect, the present invention provides a method for preventing and / or treating diseases caused by hepatitis B virus. According to embodiments of the invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned siRNA, the aforementioned siRNA conjugate, or the aforementioned pharmaceutical composition. According to embodiments of the invention, this method can effectively prevent and / or treat diseases related to hepatitis B virus.

[0171] The general synthetic method of the compounds, double-stranded siRNA, and double-stranded siRNA conjugates described in this invention

[0172] Generally, the compounds and nucleic acid conjugates of the present invention can be prepared by the methods described in the present invention, unless otherwise specified, wherein the substituents are defined as shown in formulas (I) and (II). The following reaction schemes and examples are used to further illustrate the content of the present invention.

[0173] Unless otherwise specified, all temperatures described in the examples below are in degrees Celsius (°C). Silica gel columns were used; silica gel (200-300 mesh) was purchased from Qingdao Ocean Chemical Plant, and NH2CPG was purchased from Hebei Dina Xingke. Nuclear magnetic resonance spectroscopy was performed using CDCl3, DMSO-d6, CD3OD, or acetone-d6 as solvents (in ppm), with TMS (0 ppm) or chloroform (7.25 ppm) as reference standards. When multiplets are observed, the following abbreviations will be used: s (singlet), d (doublet), t (triplet), m (multiplet), br (broadened), dd (doublet of doublets), dt (doublet of triplets), br.s (broadened singlet), q (quartet). The coupling constant J is expressed in Hertz (Hz).

[0174] Low-resolution mass spectrometry (MS) data were determined using an Agilent 6320 series LC-MS spectrometer equipped with a G1312A binary pump and a G1316A TCC (column temperature maintained at 30°C). A G1329A autosampler and a G1315B DAD detector were used for analysis, and an ESI source was used in the LC-MS spectrometer.

[0175] High-resolution mass spectrometry (MS) data were determined using an Agilent 6130 series LC-MS spectrometer equipped with a G1311A quaternary pump and a G1316A TCC (column temperature maintained at 30°C). A G1329A autosampler and a G1315D DAD detector were used for analysis, and an ESI source was used in the HR-MS spectrometer.

[0176] The following abbreviations are used throughout this invention:

[0177] Detailed Implementation

[0178] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. In particular, the synthesis of small nucleic acids and nucleic acid conjugates can be obtained by adjusting the synthesis methods according to the embodiments of the present invention or conventional methods in the art. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0179] Method for synthesizing the compound described in this invention

[0180] The following synthetic scheme outlines the general experimental steps for preparing the compounds, double-stranded siRNA, and double-stranded siRNA conjugates disclosed in this invention. Those skilled in the art can modify the methods or adjust the raw materials as needed to obtain the compounds, double-stranded siRNA, and double-stranded siRNA conjugates described in this invention. Wherein, X, Z, and Z... 1 R and R have the meanings as described in this invention. Unless otherwise specified, the compounds described in this invention can be prepared by the methods described in the following synthetic schemes.

[0181] Synthesis Scheme 1:

[0182]

[0183] Compound (1-C) can be prepared according to the method described in Synthetic Scheme 1. First, compound (1-A) is protected with a hydroxyl protecting group (e.g., using DMT-TrCl reagent) to obtain compound (1-B). Compound (1-B) reacts with a suitable phosphoric acid reagent (e.g., bis(diisopropylamino)(2-cyanoethoxy)phosphine) or a solid support to obtain compound (1-C) containing an active phosphoric acid group or a solid support. The stereoisomers of compound (1-C) can also be prepared according to the method described in Synthetic Scheme 1.

[0184] Preparation Examples

[0185] In the following preparation examples, the inventors have described in detail the preparation process of the compounds of the present invention using some of the compounds as examples, wherein, For CPG.

[0186] Example 1: Synthesis of phosphoramide monomer 1

[0187]

[0188] Step 1: Synthesis of Compounds 1-2

[0189] Compound 1-1 (1.0 g, 3.43 mmol) was added to pyridine (15 mL) under nitrogen protection and cooled to -5 °C. 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (1.13 mL, 3.53 mmol) was added dropwise. After the addition was complete, the mixture was transferred to room temperature and stirred for 20 h. The solvent was removed by concentration under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA (V / V) = 2 / 3) to give a white, foamy solid (1.38 g, 75.3%). MS (ESI, pos.ion) m / z: 534.3 [M+H] + .

[0190] Step 2: Synthesis of compounds 1-3

[0191] Compounds 1-2 (1.08 g, 2.02 mmol) and N,N-dimethylformamide dimethyl acetal (0.48 g, 4.04 mmol) were added to toluene (15 mL), and the mixture was heated to 50 °C and stirred for 4 h. After the reaction was complete, the mixture was concentrated under reduced pressure to give a white, foamy solid (1.19 g, 100%). MS (ESI, pos.ion) m / z: 589.30 [M+H] + ;

[0192] 1 H NMR(600MHz,DMSO-d6)δ8.95(s,1H),8.15(s,1H),6.90(d,J=4.4Hz,1H),6.80(d ,J=4.5Hz,1H),6.49(d,J=5.7Hz,1H),4.58(dd,J=5.7,4.4Hz,1H),4.23–4.12(m ,3H),3.92(dd,J=13.2,2.5Hz,1H),3.25(s,3H),3.19(s,3H),1.06–1.04(m,7H) ,1.01(dd,J=7.2,2.0Hz,6H),0.96(dd,J=8.8,7.0Hz,8H),0.89(t,J=7.6Hz,7H).

[0193] Step 3: Synthesis of compounds 1-4

[0194] Compounds 1-3 (0.84 g, 1.43 mmol) were added to N,N-dimethylformamide (10 mL), and the mixture was cooled to 0 °C. Then, methyl iodoformane (0.41 g, 2.86 mmol) and sodium hydride (0.11 g, 2.86 mmol, 60%) were added sequentially, and the mixture was stirred at 0 °C for 1 h. The reaction mixture was poured into a saturated NH₄Cl aqueous solution (50 mL), extracted with EA (60 mL × 2), and the organic phase was concentrated. The residue was purified by silica gel column chromatography (EA / PE (V / V) = 1 / 1) to give a white solid (0.71 g, 83%). MS (ESI, pos.ion) m / z: 603.40 [M+H] + .

[0195] Step 4: Synthesis of compounds 1-5

[0196] Methanol (50 mL) and ammonia (1.37 g, 14.44 mmol) were added to compounds 1-4 (4.35 g, 7.22 mmol). The mixture was stirred at 50 °C for 5 h, cooled to room temperature, and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (DCM / MeOH (V / V) = 100 / 3) to give a white foamy solid (3.70 g, 93.61%). MS (ESI, pos.ion) m / z: 548.30 [M+H] + .

[0197] Step 5: Synthesis of compounds 1-6

[0198] Compounds 1-5 (3.70 g, 6.75 mmol) were dissolved in toluene (40 mL), and methanesulfonic anhydride (1.47 g, 8.44 mmol) was added. The mixture was heated at 100 °C for 30 min, followed by the addition of triethylamine (2.05 g, 20.25 mmol). The reaction mixture was kept at this temperature for 12 h, then cooled to room temperature. EA (150 mL) was added to the reaction solution. The organic phase was washed successively with water (50 mL) and saturated sodium chloride aqueous solution (50 mL), and dried over anhydrous sodium sulfate. The mixture was filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH (V / V) = 20 / 1) to give a yellow foamy solid (2.38 g, 56.30%). MS (ESI, negative ion) m / z: 624.30 [MH] - .

[0199] Step 6: Synthesis of compounds 1-7

[0200] Compounds 1-6 (2.18 g, 3.48 mmol) were dissolved in THF (25 mL). A tetrahydrofuran solution of TBAF (2.08 mL, 2.08 mmol, 1 mol / L) was added to the solution, and the reaction mixture was stirred at 35 °C for 22 h. After the reaction was complete, the solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography (DCM / MeOH (V / V) = 20 / 1) to give a pale yellow foamy solid (1.16 g, 86.87%). MS (ESI, pos.ion) m / z: 384.15 [M+H] + .

[0201] Step 7: Synthesis of compounds 1-8

[0202] Compounds 1-7 (1.23 g, 3.21 mmol) were dissolved in DCM (20 mL), and TEA (0.89 mL, 6.42 mmol) was added. The mixture was cooled in an ice bath, and then DMTrCl (1.41 g, 4.17 mmol) was added. After the addition was complete, the mixture was brought to room temperature and stirred for 12 h. The reaction mixture was diluted with DCM (100 mL), and the organic phase was washed successively with water (40 mL × 2) and saturated sodium chloride aqueous solution (40 mL). The phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH (V / V) = 50 / 1) to give a yellow foamy solid (1.46 g, 66.36%). MS (ESI, pos.ion) m / z: 686.30 [M+H] + .

[0203] Step 8: Synthesis of phosphonamide monomer 1

[0204] In a dry reaction flask, 1-8 (1.46 g, 2.13 mmol), 1H-tetrazole (0.18 g, 2.56 mmol), and DCM (15 mL) were added sequentially. After stirring at room temperature until homogeneous, TEA (0.59 mL, 4.26 mmol) was added. After the solid was completely dissolved, bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.96 g, 3.19 mmol) was added. The mixture was stirred at room temperature for 12 h under nitrogen protection. Then, 1H-tetrazole (0.15 g, 2.13 mmol), TEA (0.32 g, 3.19 mmol), and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.64 g, 2.13 mmol) were added. The reaction mixture was stirred at room temperature for another 4.5 h. After the reaction was complete, DCM (100 mL) was added to the reaction solution for dilution. The organic phase was washed successively with water (30 mL × 2) and saturated sodium chloride aqueous solution (30 mL), and dried over anhydrous sodium sulfate. The mixture was filtered, and the solvent was removed from the filtrate under reduced pressure to obtain a yellow oily liquid. The oily liquid was dissolved in DCM (4 mL), and PE (25 mL) was added. A viscous solid precipitated out. After stirring for 10 min, the supernatant was discarded; this process was repeated three times. The lower residue was purified by silica gel column chromatography (DCM / MeOH (V / V) = 100 / 1) to obtain a white foamy solid (0.80 g, 42.41%).

[0205] 1H NMR (400MHz, DMSO-d6) δ7.75(d,J=4.0Hz,1H),7.33(t,J=7.1Hz,2H),7.29–7.23(m,2H),7.18(dd,J=18.3,9.6H z,5H),6.81(d,J=8.5Hz,4H),6.64(dd,J=4.3,2.3Hz,1H),6.50–6.44(m,1H),4.78(dd,J=12.3,4.9Hz,1H),4.45 –4.29(m,2H),3.72(s,6H),3.52(d,J=12.3Hz,4H),3.07(d,J=7.5Hz,3H),3.04(s,2H),3.00(s,3H),2.77(t,J= 5.9Hz,1H),2.56(t,J=6.0Hz,1H),1.13(d,J=2.6Hz,3H),1.12–1.09(m,5H),1.08(s,2H),0.96(d,J=6.7Hz,2H);

[0206] 31 P NMR(162MHz,DMSO-d6)δ149.27(s).

[0207] Example 2: Synthesis of phosphoramide monomer 2

[0208]

[0209] Step 1: Synthesis of Compound 2-A

[0210] Compounds 1-4 (5.1 g, 8.46 mmol), tetrahydrofuran (50 mL), and a tetrahydrofuran solution of TBAF (9.48 mL, 9.48 mmol, 1 M) were reacted and stirred at room temperature for 12 h. The mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (MeOH / DCM (V / V) = 1 / 20) to give a white solid (2.4 g, 79%). MS (ESI, pos.ion) m / z: 361.30 [M+H] + .

[0211] Step 2: Synthesis of compound 2-B

[0212] Compound 2-A (4.83 g, 13.40 mmol) and anhydrous methanol (40 mL) were added sequentially to a dry reaction flask. After stirring thoroughly at room temperature, ammonia water (40 mL) was added, and the solid gradually dissolved. The mixture was stirred at room temperature for 5.5 h, and the solvent was removed by vacuum evaporation to obtain a white, foamy solid (4.09 g, 99.96%). MS (ESI, pos.ion) m / z: 306.00 [M+H] + .

[0213] Step 3: Synthesis of compound 2-C

[0214] Compound 2-B (4.0 g, 13.10 mmol) and 80% acetic acid (50 mL) were added sequentially to a dry reaction flask. The mixture was stirred at room temperature until most of the solid dissolved. Sodium nitrite (18.08 g, 262 mmol) was added in portions, and the reaction mixture was stirred at room temperature for 2 h, followed by stirring at 75 °C for 20 h. The solvent was removed by vacuum evaporation to give a pale yellow solid. The solid was dissolved in DCM (400 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid. Petroleum ether (300 mL) was added to the pale yellow solid, and the mixture was stirred at room temperature for 1 h. The mixture was then filtered, and the filter cake was dried under vacuum at room temperature for 2 h to give a pale yellow solid (3.9 g, 97.19%). MS (ESI, neg.ion) m / z: 305.3 [MH] - .

[0215] Step 4: Synthesis of compound 2-D

[0216] Compound 2-C (1.05 g, 3.43 mmol) and pyridine (20 mL) were added sequentially to a dry reaction flask and stirred at room temperature until completely dissolved. Then, DMTrCl (2.32 g, 6.86 mmol) was added, and the resulting mixture was stirred at room temperature for 13 h. The solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography (DCM / CH3OH (V / V) = 25 / 1) to give a pale yellow foamy solid (1.8 g, 86.26%). MS (ESI, negative ion) m / z: 607.25 [MH] - .

[0217] Step 5: Synthesis of phosphonamide monomer 2

[0218] Compound 2-D (1.06 g, 1.74 mmol), 1H-tetrazole (0.15 g, 2.09 mmol), DCM (20 mL), and TEA (0.28 g, 2.78 mmol) were added sequentially to a dry reaction flask. The mixture was stirred at room temperature until the solid gradually dissolved completely. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.84 g, 2.78 mmol) was slowly added to the flask using a syringe. The mixture was stirred at room temperature for 12 h. After the reaction was complete, DCM (60 mL) was added to the reaction solution for dilution. The solution was washed sequentially with saturated sodium bicarbonate aqueous solution (50 mL × 2) and saturated sodium chloride aqueous solution (50 mL). The solution was dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate by vacuum distillation. Add 1.5 mL of DCM to the obtained residue, stir to dissolve at room temperature, then slowly add 30 mL of petroleum ether. A large amount of viscous substance precipitates out. Let stand, and pour off the supernatant. Repeat the operation three times. The lower residue is purified by silica gel column chromatography (PE / EA(V / V) = 1 / 1) to obtain a white foamy solid (1.2 g, 85.18%). HRMS (m / z) calculated value C 43 H50N6O8P[M+H] + Test value: 809.3428;

[0219] 1 H NMR (400MHz, CDCl3) δ7.58-7.54(m,1H),7.45-7.38(m,2H),7.35–7.12(m,8H),7.07(dd,J=4.5,1.5Hz,1H ),6.87(dd,J=10.3,4.6Hz,1H),6.83-6.77(m,4H),4.71(t,J=5.4Hz,1H),4.62–4.43(m,2H),4.00–3.84( m,1H),3.81-3.78(m,6H),3.77–3.69(m,1H),3.66(d,J=5.7Hz,3H),3.63–3.46(m,3H),3.26(td,J=10.6, 3.3Hz,1H),2.63(t,J=6.2Hz,1H),2.34(dd,J=10.7,6.3Hz,1H),1.25–1.11(m,9H),1.04(d,J=6.8Hz,3H);

[0220] 31 P NMR (162MHz, CDCl3) δ150.05 (d, J=17.5Hz).

[0221] Example 3: Synthesis of phosphoramide monomer 3

[0222]

[0223] Step 1: Synthesis of Compound 3-1

[0224] Compounds 2-3 (3.12 g, 10.19 mmol) were dissolved in acetonitrile (30 mL), and TEA (2.06 g, 20.38 mmol) was added. TiPDSCl2 (3.86 g, 12.23 mmol) was then added dropwise. The mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure. The residue was diluted with DCM (150 mL). The resulting mixture was washed successively with water (30 mL × 2) and saturated sodium chloride aqueous solution (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH (V / V) = 50 / 1) to give a pale yellow viscous solid (3.50 g, 62.61%). MS (ESI, pos.ion) m / z: 549.30 [M+H] + .

[0225] Step 2: Synthesis of compound 3-2

[0226] Compound 3-1 (1.64 g, 2.99 mmol) was dissolved in DMF (20 mL), cesium carbonate (1.95 g, 5.98 mmol) was added, followed by iodomethane (0.51 g, 3.59 mmol). The reaction mixture was stirred overnight at room temperature. The solvent was removed by vacuum distillation, and the residue was diluted with EA (100 mL). The residue was then washed successively with water (30 mL × 2) and saturated sodium chloride aqueous solution (30 mL × 2), and dried over anhydrous sodium sulfate. The residue was filtered, and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (PE / EA (V / V) = 4 / 1) to give a pale yellow oily liquid (1.03 g, 61.24%).

[0227] MS(ESI,pos.ion)m / z:563.30[M+H] + .

[0228] Step 3: Synthesis of compound 3-3

[0229] Compound 3-2 (1.03 g, 1.83 mmol) was dissolved in THF (10 mL), and TBAF tetrahydrofuran solution (0.92 mL, 0.92 mmol, 1 M) was added. The reaction mixture was stirred at room temperature for 1.5 h, and then the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (DCM / MeOH (V / V) = 25 / 1) to give a white foamy solid (0.37 g, 63.12%).

[0230] Step 4: Synthesis of compounds 3-4

[0231] Compound 3-3 (0.37 g, 1.16 mmol), DCM (10 mL), and TEA (0.40 mL, 2.9 mmol) were added sequentially to a dry reaction flask and stirred until dissolved. DMTrCl (0.51 g, 1.51 mmol) was added, and the mixture was reacted at room temperature for 12 h. Then, DCM (50 mL) was added to the reaction solution. The organic phase was washed sequentially with water (50 mL) and saturated sodium chloride aqueous solution (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness. The residue was purified by silica gel column chromatography (PE / EA (V / V) = 2 / 3) to give a pale yellow foamy solid (0.67 g, 93.15%). MS (ESI, pos.ion) m / z: 645.30 [M+Na] + ;

[0232] 1 H NMR(400MHz, CDCl3)δ7.41-7.36(m,2H),7.35(s,1H),7.26(s,2H),7.24(s,2H),7 .21(t,J=3.3Hz,2H),7.16(dd,J=13.1,6.2Hz,1H),6.95(d,J=4.5Hz,1H),6.76(d ,J=4.4Hz,3H),6.73(s,2H),4.44(d,J=5.4Hz,1H),4.34-4.21(m,2H),3.73(s,6H ),3.67(s,3H),3.43(s,3H),3.40(d,J=2.7Hz,1H),3.28(dd,J=10.7,4.0Hz,1H).

[0233] Step 5: Synthesis of phosphoramide monomer 3

[0234] Compounds 3-4 (0.65 g, 1.04 mmol), 1H-tetrazole (0.087 g, 1.25 mmol), and DCM (15 mL) were added sequentially to a dry reaction flask. After stirring at room temperature until homogeneous, TEA (0.29 mL, 2.08 mmol) was added, and the solid dissolved completely. Then, bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.47 g, 1.56 mmol) was added. The reaction mixture was stirred at room temperature for 5 h under nitrogen protection. The solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography (PE / EA (V / V = 1 / 1) to give a white foamy solid (0.58 g, 67.52%). MS (ESI, pos.ion) m / z: 823.30 [M+H] + ;

[0235] 1H NMR(400MHz, DMSO-d6)δ8.18(d,J=3.9Hz,1H),7.34–7.27(m,2H),7.20(ddd,J=23.9,11.2,4.9Hz,7H),6.98–6 .91(m,1H),6.81(dd,J=8.7,3.2Hz,4H),6.73(d,J=4.4Hz,1H),4.62(dd,J=12.0,4.3Hz,1H),4.47–4.32(m,2H) ,3.80(dd,J=15.4,8.4Hz,1H),3.72(s,6H),3.61(s,2H),3.57(s,3H),3.54(s,1H),3.40(s,3H),3.32–3.24(m, 1H),3.14–3.02(m,1H),2.75(t,J=5.8Hz,1H),2.58–2.52(m,1H),1.11(t,J=6.0Hz,9H),0.98(d,J=6.6Hz,3H);

[0236] 31 P NMR(162MHz,DMSO-d6)δ149.53(s),149.26(s).

[0237] Example 4: Synthesis of phosphoramide monomer 4

[0238]

[0239] Replacing methyl iodide in the synthesis of phosphonamide monomer 3 (compound 3-2) with ethyl bromoethyl, and following the same synthesis method as for phosphonamide monomer 3, yielded a white, foamy solid (0.92 g, 60.52%). MS (ESI, pos.ion) m / z: 895.30 [M+H] + ;

[0240] 1H NMR (400MHz, DMSO-d6) δ8.24(d,J=2.4Hz,1H),7.32–7.27(m,2H),7.26–7.13(m,7H),7.02(dd,J=4.4,3.2Hz,1H),6.82(dd,J= 8.7,1.5Hz,4H),6.77(dd,J=4.5,2.1Hz,1H),4.76(s,2H),4.64(dd,J=6.4,4.7Hz,1H),4.48–4.35(m,2H),4.23–4.09(m,2H), 3.87–3.75(m,1H),3.72(d,J=1.0Hz,6H),3.62(s,2H),3.60–3.56(m,3H),3.55–3.50(m,1H),3.32–3.25(m,1H),3.14–3.01(m ,1H),2.76(t,J=5.9Hz,1H),2.55(t,J=5.7Hz,1H),1.19(td,J=7.1,5.2Hz,3H),1.12(t,J=5.9Hz,9H),0.98(d,J=6.7Hz,3H);

[0241] 31 P NMR (162MHz, DMSO-d6) δ149.47 (d, J = 6.3Hz);

[0242] HRMS (ESI): Theoretical value C 47 H 56 N6O 10 P[M+H] + Test value: 895.3796, Test value: 895.3814 [M+H] + .

[0243] Example 5: Synthesis of phosphoramide monomer 5

[0244]

[0245] Step 1: Synthesis of Compound 5-1

[0246] Compound 3-1 (1.0 g, 1.82 mmol), methyl 4-iodobenzoate (1.19 g, 4.55 mmol), potassium carbonate (0.63 g, 4.55 mmol), cuprous iodide (0.35 g, 1.82 mmol), and tert-amyl alcohol (30 mL) were added sequentially to a dry reaction flask. The mixture was stirred thoroughly at room temperature, and then N,N'-dimethyl-1,2-ethylenediamine (0.32 g, 3.64 mmol) was added. The reaction was carried out at 100 °C for 18 h under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, and EA (100 mL) was added to the reaction solution. The resulting mixture was washed with saturated sodium chloride aqueous solution (100 mL × 3) and dried over anhydrous sodium sulfate. The mixture was filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA (V / V) = 5 / 1) to give a colorless oil (0.51 g, 40.98%). MS(ESI,pos.ion)m / z:683.20[M+H] + ;

[0247] 1 H NMR(400MHz, CDCl3)δ8.22(d,J=8.6Hz,2H),7.77(s,1H),7.53(d,J=8.6Hz,2H), 7.10(d,J=4.5Hz,1H),6.91(d,J=4.5Hz,1H),4.36(dd,J=9.3,4.5Hz,1H),4.25(d d,J=11.8,4.5Hz,2H),4.15(d,J=4.4Hz,1H),4.01(dd,J=13.4,2.4Hz,1H),3.97 (s,3H),3.80(s,3H),1.13-1.04(m,13H),1.03-0.97(m,13H),0.96–0.87(m,2H).

[0248] Step 2: Synthesis of Compound 5-2

[0249] Compound 5-1 (1.04 g, 1.52 mmol) and THF (20 mL) were added sequentially to a dry reaction flask and stirred at room temperature until completely dissolved. A tetrahydrofuran solution of TBAF (0.61 mL, 0.61 mmol, 1 M) was then added, and the mixture was stirred at room temperature for 29 h. The solvent was removed by vacuum evaporation, yielding a yellow viscous substance. IPA (8 mL) was then added to the viscous substance, and the mixture was stirred at room temperature, gradually precipitating a solid. Stirring was continued at room temperature for 18 h, and the mixture was filtered. The resulting filter cake was washed with IPA (10 mL) and then dried under vacuum at 50 °C for 1 h to give a white solid (610 mg, 90.95%). MS (ESI, pos.ion) m / z: 441.05 [M+H] + ;

[0250] 1H NMR(600MHz,DMSO-d6)δ8.38(s,1H),8.12(d,J=8.5Hz,2H),7.73(d,J=8.4Hz ,2H),7.06(d,J=4.5Hz,1H),6.89(d,J=4.4Hz,1H),5.39(br,1H),4.95(t,J= 5.4Hz,1H),4.35(d,J=4.9Hz,1H),4.17-4.12(m,1H),4.11-4.06(dd,J=9.1, 4.1Hz,1H),3.91(s,3H),3.68–3.64(m,1H),3.61(s,3H),3.55–3.50(m,1H).

[0251] Step 3: Synthesis of compound 5-3

[0252] Compound 5-2 (255 mg, 0.58 mmol) and DCM (5 mL) were added sequentially to a dry reaction flask and stirred at room temperature until homogeneous. Then, DMAP (0.071 g, 0.58 mmol), TEA (0.18 g, 1.74 mmol), and DMTrCl (393.04 mg, 1.16 mmol) were added sequentially. The reaction mixture was heated to 40 °C and reacted for 4 h. Then, DMTrCl (0.20 g, 0.58 mmol) was added, and the reaction continued for another 21 h. After the reaction was complete, the reaction solution was cooled to room temperature, and the solvent was removed by vacuum distillation. The resulting residue was purified by silica gel column chromatography (PE / EA (V / V) = 2 / 1) to give a white, foamy solid (420 mg, 97.66%).

[0253] 1 H NMR (600MHz, CDCl3) δ8.23 (d, J = 8.6Hz, 2H), 7.60 (s, 1H), 7.50-7.47 (m, 2H), 7.46-7.42 (m, 2H), 7.34-7 .31(m,4H),7.28(d,J=6.5Hz,2H),7.23–7.19(m,1H),7.13(d,J=4.5Hz,1H),6.89(d,J=4.5Hz,1H),6.8 2(dd,J=8.8,1.4Hz,4H),4.57(d,J=5.5Hz,1H),4.43(dd,J=12.9,6.4Hz,1H),4.35(dt,J=6.5,3.4Hz,1 H), 3.99 (s, 3H), 3.78 (overlap, 9H), 3.53–3.50 (m, 1H), 3.38 (dd, J = 10.7, 3.9Hz, 1H), 2.74 (d, J = 7.6Hz, 1H).

[0254] Step 4: Synthesis of phosphoramide monomer 5:

[0255] Compound 5-3 (393 mg, 0.53 mmol), 1H-tetrazole (44.55 mg, 0.64 mmol), and DCM (30 mL) were added sequentially to a dry reaction flask. After stirring at room temperature until homogeneous, TEA (0.11 g, 1.06 mmol) was added. The solid was almost completely dissolved. Under nitrogen protection, the mixture was cooled to 0 °C, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (287.55 mg, 0.95 mmol) was added. The mixture was transferred to room temperature and stirred for 3.5 h. Then, bis(diisopropylamino)(2-cyanoethoxy)phosphine was added as a supplement. Propylamino(2-cyanoethoxy)phosphine (0.32 g, 1.06 mmol) was reacted with the resulting reaction mixture for 4 h. After the reaction was complete, the reaction solution was diluted with DCM (30 mL), and then washed successively with saturated sodium bicarbonate aqueous solution (40 mL × 2) and saturated sodium chloride aqueous solution (40 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA(V / V) = 3 / 1) to give a white solid (0.25 g, 50.11%). HRMS (ESI): Theoretical value C 51 H 55 N6O 10 P[M+H] + Test value: 943.3796;

[0256] 1 H NMR (600MHz, CDCl3) δ8.22(d,J=7.8Hz,2H),7.52(s,1H),7.48(d,J=7.3Hz,2H),7.42(t,J=7.0Hz,2H),7.33–7. 28(m,4H),7.27-7.24(m,2H),7.24-7.20(m,1H),7.13(d,J=3.4Hz,1H),6.88(dd,J=10.0,3.9Hz,1H),6.84-6.7 6(m,4H),4.74(dd,J=27.5,3.9Hz,1H),4.65–4.46(m,2H),3.98(s,3H),3.90-3.82(m,1H),3.78(s,6H),3.72–3 .49(m,7H),3.37–3.21(m,1H),2.66-2.62(m,1H),2.46–2.26(m,1H),1.28-1.17(m,9H),1.07(d,J=6.3Hz,3H);

[0257] 31P NMR (243MHz, CDCl3) δ150.01 (d, J=22.5Hz).

[0258] Example 6: Synthesis of phosphoramide monomer 6

[0259]

[0260] Step 1: Synthesis of Compound 6-1

[0261] Compounds 1-5 (2.2 g, 4.02 mmol) and DMF (30 mL) were added sequentially to a dry reaction flask. After complete dissolution by stirring at room temperature, ethyl bromoacetate (2.01 g, 12.06 mmol) was added, and the mixture was cooled to -10 °C. Sodium hydride (0.40 g, 10.05 mmol, 60%) was added in portions, and the mixture was stirred at this temperature for 30 min. The mixture was then transferred to room temperature and stirred for 3.5 h. After the reaction was complete, the reaction solution was diluted with EA (100 mL) and washed with saturated sodium chloride aqueous solution (100 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA (V / V) = 5 / 1) to give a white foamy solid (2.4 g, 83.00%). MS (ESI, pos.ion) m / z: 720.40 [M+H] + ;

[0262] 1 H NMR (600MHz, CDCl3) δ8.02(s,1H),6.98(d,J=4.8Hz,1H),6.60(d,J=4.8Hz,1H),4.69–4.45(m,4H),4.33–4.23(m,7H),4.14(d,J=4.6Hz ,1H),4.01(dd,J=13.6,2.7Hz,1H),3.81(s,3H),1.33(t,J=7.1Hz,6H),1.12–1.06(m,12H),1.00(t,J=6.9Hz,12H),0.94–0.87(m,4H).

[0263] Step 2: Synthesis of Compound 6-2

[0264] Compound 6-1 (2.0 g, 2.78 mmol) and THF (40 mL) were added sequentially to a dry reaction flask. The mixture was stirred at room temperature until the solid was completely dissolved. Then, a tetrahydrofuran solution of TBAF (0.83 mL, 0.83 mmol, 1 mol / L) was added. The resulting mixture was stirred at room temperature for 13.5 h. The solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (PE / EA(V / V) = 1 / 2) to give a white, foamy solid (0.7 g, 52.78%). MS (ESI, pos.ion) m / z: 478.20 [M+H] + ;

[0265] 1 H NMR (600MHz, CDCl3) δ7.94(s,1H),7.15(d,J=4.9Hz,1H),6.68(d,J=4.9Hz,1H),4.86(d,J=5.5Hz,1H),4.80(d,J=11.1Hz,1H),4.67-4.5 7(m,4H),4.52-4.51(m,1H),4.29(br.s,4H),3.93(d,J=12.7Hz,1H),3.83–3.77(m,1H),3.38(s,3H),2.95(s,1H),1.33(t,J=7.1Hz,6H).

[0266] Step 3: Synthesis of compound 6-3

[0267] Compound 6-2 (455 mg, 0.95 mmol), DMAP (0.058 g, 0.47 mmol), and DCM (20 mL) were added sequentially to a dry reaction flask and stirred at room temperature until completely dissolved. Then, TEA (0.29 g, 2.85 mmol) and DMTrCl (0.48 g, 1.42 mmol) were added, and the mixture was stirred at room temperature for 11 h. Finally, DMTrCl (0.48 g, 1.42 mmol) was added, and the reaction continued for another 12 h. After the reaction was complete, the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (PE / EA (V / V) = 1 / 1) to give a white, foamy solid (666 mg, 90%). MS (ESI, pos.ion) m / z: 780.30 [M+H] + .

[0268] Step 4: Synthesis of phosphoramide monomer 6

[0269] Compound 6-3 (0.468 g, 0.60 mmol), 1H-tetrazole (0.051 g, 0.72 mmol), and DCM (10 mL) were added sequentially to a dry reaction flask. The mixture was stirred at room temperature until most of the solid dissolved. Then, TEA (0.097 g, 0.96 mmol) was added, and the solid dissolved completely. The mixture was cooled to 0 °C, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.36 g, 1.2 mmol) was added. The reaction mixture was transferred to room temperature and stirred for 3 h. Then, bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.29 g, 0.96 mmol) was added, and the reaction was continued for 12 h. The reaction solution was diluted with DCM (60 mL) and washed sequentially with saturated sodium bicarbonate aqueous solution (60 mL × 2) and saturated sodium chloride aqueous solution (60 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA(V / V) = 1 / 1) to give a white, foamy solid (0.51 g, 86%). HRMS (ESI): Theoretical value C 51 H 63 N7O 11 P[M+H] + Test value: 980.4323;

[0270] 1 H NMR(600MHz, CDCl3)δ7.91(d,J=21.3Hz,1H),7.44(dd,J=13.7,7.3Hz,2H),7.35–7.30(m,4H),7.28-7.20(m,3H),7.00(dd,J=16.3 ,4.8Hz,1H),6.83–6.78(m,4H),6.60(t,J=4.8Hz,1H),4.79(dd,J=33.3,5.1Hz,1H),4.67–4.55(m,4H),4.54–4.40(m,2H),4.28(q, J=6.9Hz,4H),4.02–3.85(m,1H),3.80(dd,J=5.7,1.9Hz,6H),3.70–3.65(m,1H),3.64(d,J=15.3Hz,3H),3.62–3.49(m,3H),3.27( dd,J=10.6,3.6Hz,1H),2.65(t,J=6.4Hz,1H),2.37–2.29(m,1H),1.32(t,J=7.1Hz,6H),1.23-1.12(m,10H),1.03(d,J=6.8Hz,2H).

[0271] Example 7: Synthesis of phosphoramide monomer 7

[0272]

[0273] Step 1: Synthesis of Compound 7-1

[0274] Compounds 1-3 (2.1 g, 3.57 mmol) and anhydrous DMF (30 mL) were added sequentially to a dry reaction flask. The mixture was stirred at room temperature until completely dissolved. The temperature was lowered to -10 °C, and sodium hydride (0.20 g, 5.00 mmol, 60%) was added. After stirring for 2 min, BnBr (0.79 g, 4.64 mmol) was added, and the mixture was kept at this temperature and stirred for 30 min. The mixture was then transferred to room temperature and stirred for 5 h. After the reaction was complete, the reaction solution was diluted with EA (100 mL). The organic phase was washed with saturated sodium chloride aqueous solution (100 mL × 3), dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA (V / V) = 3 / 1) to give a white solid (1.62 g, 67%). MS (ESI, pos.ion) m / z: 679.20 [M+H] + ;

[0275] 1 H NMR (600MHz, CDCl3) δ8.85 (s, 1H), 8.11 (s, 1H), 7.54 (d, J = 7.3Hz, 2H), 7.34 (t, J = 7.4Hz, 2H),7.30-7.28(m,1H),7.04(d,J=4.5Hz,1H),6.89(d,J=4.5Hz,1H),5.14(q,J=12.3Hz,2 H),4.44(d,J=3.4Hz,1H),4.38–4.33(m,2H),4.28(d,J=13.3Hz,1H),4.03(d,J=12.9Hz, 1H),3.27(s,3H),3.24(s,3H),1.13-1.07(m,14H),1.04-0.99(m,7H),0.88–0.82(m,7H).

[0276] Step 2: Synthesis of Compound 7-2

[0277] Compound 7-1 (1.0 g, 1.47 mmol) and THF (16 mL) were added sequentially to a dry reaction flask and stirred at room temperature until completely dissolved. Then, a tetrahydrofuran solution of TBAF (0.29 mL, 0.29 mmol, 1 mol / L) was added, and the mixture was stirred at room temperature for 6 h. The solvent was removed under reduced pressure to obtain a white, foamy solid. The residue was purified by silica gel column chromatography (DCM / CH3OH (V / V) = 30 / 1) to give a white, foamy solid (0.58 g, 90.23%). MS (ESI, pos.ion) m / z: 436.80 [M+H] + .

[0278] Step 3: Synthesis of Compound 7-3

[0279] Compound 7-2 (0.64 g, 1.47 mmol), DMTrCl (1.0 g, 2.94 mmol), and DCM (15 mL) were added sequentially to a dry reaction flask. The mixture was stirred at room temperature until the solid completely dissolved. Then, DMAP (0.090 g, 0.73 mmol) and TEA (0.30 g, 2.94 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 4 h, followed by the addition of DMTrCl (0.20 g, 0.59 mmol), and the reaction was continued for another 16 h. After the reaction was complete, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA (V / V) = 1 / 2) to give a white, foamy solid (1.0 g, 92.30%). MS (ESI, pos.ion) m / z: 739.30 [M+H] + .

[0280] Step 4: Synthesis of phosphoramide monomer 7

[0281] Compound 7-3 (0.24 g, 0.32 mmol), 1H-tetrazole (0.027 g, 0.38 mmol), and DCM (6 mL) were added sequentially to a dry reaction flask. The mixture was stirred thoroughly at room temperature. TEA (0.065 g, 0.64 mmol) was added, and the solid was completely dissolved. The mixture was cooled to 0 °C, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.15 g, 0.51 mmol) was added. The mixture was stirred at this temperature for 5 min, followed by stirring at room temperature for 2 h. Then, bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.19 g, 0.64 mmol) was added, and stirring was continued for 3 h. The reaction solution was diluted with DCM (50 mL), washed sequentially with saturated sodium bicarbonate aqueous solution (40 mL × 2) and saturated sodium chloride aqueous solution (50 mL), and dried over anhydrous sodium sulfate. The solution was filtered, and the solvent was removed from the filtrate under reduced pressure to obtain a colorless oily substance. The crude product was purified by column chromatography (PE / EA(V / V) = 1 / 1) to give a white, foamy solid (0.23 g, 75.40%). HRMS (ESI): Theoretical value C 52 H 60 N8O7P[M+H] + Test value: 939.4323;

[0282] 1H NMR(600MHz, CDCl3)δ8.82(d,J=2.9Hz,1H),7.73(d,J=10.9Hz,1H),7.45–7.40(m,2H),7.33–7.30(m,4H),7.2 4–7.18(m,8H),7.05(dd,J=12.4,4.6Hz,1H),6.92(dd,J=8.0,4.6Hz,1H),6.81–6.72(m,4H),5.22(dd,J=27.5, 5.2Hz,1H),4.78–4.67(m,2H),4.65–4.52(m,2H),4.01-3.81(m,1H),3.79(t,J=4.2Hz,6H),3.73–3.57(m,3H) ,3.56-3.45(m,1H),3.28-3.21(m,7H),2.61–2.29(m,2H),1.18(dd,J=6.7,3.0Hz,10H),1.02(d,J=6.8Hz,2H);

[0283] 31 P NMR (243MHz, CDCl3) δ149.95(s), 149.54(s).

[0284] Example 8: Synthesis of phosphoramide monomer 8

[0285]

[0286] Replacing benzyl bromide in step 1 of Example 7 with ethyl bromoethyl, and following the same synthesis method as phosphoramidite monomer 7, yielded a white, foamy solid (0.22 g, 39.93%). HRMS (ESI): Theoretical value C 49 H 60 N8O9P[M+H] + Test value: 935.4221;

[0287] 1H NMR (600MHz, CDCl3) δ8.84(s,1H),7.95(d,J=43.5Hz,1H),7.41–7.20(m,10H),6.95(d,J=3.3Hz,1H),6.75(d,J=8.1Hz,4 H),5.42(d,J=3.5Hz,1H),4.69–4.56(m,2H),4.36–4.19(m,2H),4.15(d,J=7.4Hz,2H),4.00(d,J=77.5Hz,1H),3.78(d,J =4.6Hz, 6H), 3.65(s, 4H), 3.50(dd, J = 35.1, 8.1Hz, 1H), 3.24(d, J = 23.1Hz, 6H), 2.69(s, 1H), 2.41–2.32(m, 1H), 1.20(dd, J = 9.7, 6.4Hz, 12H), 1.05(d, J = 5.9Hz, 3H). Synthesis of compound L96-DMTr-CPG: Compound L96-DMTr-CPG was prepared according to the method described in patent application WO2014025805A1.

[0288]

[0289] Synthesis of compound DAW40007-3: Compound DAW40007-3 can be obtained by referring to the synthesis method in Example 2 of CN 119061009 A.

[0290]

[0291] As is known to those skilled in the art, compound L96-DMTr-CPG is deprotected after being linked to a double-stranded siRNA agent to obtain L96, and compound DAW40007-3 is deprotected after being linked to siRNA to obtain DAW40007-4.

[0292] Monomer 1 was obtained by the synthesis method of Example 1 in WO2024245381A1.

[0293]

[0294] As is known to those skilled in the art, after monomer 1 is inserted into a nucleotide, the amino protecting group on the monomer is deprotected, and the corresponding intercalation group is Y; after monomers 1-8 are inserted into a nucleotide, the amino protecting groups on the monomers are deprotected, and the corresponding intercalation groups are Y1-Y8, respectively.

[0295] Example 9: Synthesis of double-stranded siRNA and double-stranded siRNA conjugates

[0296] 1. Without attached conjugated groups Double chain siRNA synthesis

[0297] The synthesis steps for the sense and antisense strands of siRNA without conjugated groups are as follows:

[0298] The synthesis was performed according to the theoretical yield of 1 μmol. All 2'-modified RNA phosphoramidite monomers (unless otherwise specified in this invention) and auxiliary reagents of the 1 μmol solid-phase support CPG (purchased from Hebei Dinaxingke) were commercially available, and all phosphoramidite monomers were provided in 0.1 M anhydrous acetonitrile solution. For phosphate backbone thiolated oligonucleotides, 0.1 M DDTT solution was used as the thioretin. 5-Ethylthio-1H-tetrazole acetonitrile solution (0.25 M) was used as the activator (purchased from Suzhou Kelama), 0.02 M iodine in pyridine / water solution was used as the oxidant, and 3% trichloroacetic acid in dichloromethane solution was used as the deprotection reagent. These were placed in the designated reagent position on the KA-H8 model DNA / RNA automated synthesizer. The synthesis program was set and the specified oligonucleotide base sequence was entered. After verification, the oligonucleotide synthesis was started in cycles. Each coupling time was 6 minutes, and the thioretinization time was 6 minutes. After automatic cycling, oligonucleotides containing the solid-phase support CPG were obtained.

[0299] The nucleotides containing the solid-phase support CPG obtained above were dried by blowing with dry argon gas, then transferred to a 2 mL EP tube, and 1.8 mL of 28% ammonia solution was added. The mixture was heated at 55°C for 5–18 hours. After filtration, the filter cake was washed with water (0.5 mL), and the filtrates were combined and concentrated under reduced pressure to obtain a white or yellow gelatinous solid. After reverse-phase purification, the prepared solution was concentrated, passed through a gel column to remove excess salt, and oligonucleotides were obtained. The concentration of the obtained oligonucleotides was determined by a micro-ultraviolet spectrophotometer (SPECTRO statNano). Mass spectrometry analysis was performed on an Agilent 6530LC-MS Q-Tof system. After a single-stage scan, the molecular weight of the nucleic acid was calculated after deconvolution.

[0300] Annealing steps:

[0301] The positive strand of the double-stranded siRNA synthesized above was mixed with the negative strand synthesized above in an equimolar amount, heated to 95°C, and kept at that temperature for 10 min before being slowly cooled to room temperature. The resulting double-stranded siRNA was then lyophilized to obtain the target double-stranded siRNA.

[0302] 2. Synthesis of siRNA conjugates:

[0303] The antisense chain was synthesized by referring to the above-described methods for synthesizing the sense and antisense chains without attached conjugated groups.

[0304] Synthesis of the justice chain: The general solid support CPG is replaced with the conjugated group-solid support CPG prepared in this invention (e.g., ...). compoundThe sense strand of the double-stranded siRNA conjugate of this invention was prepared by referring to the antisense strand synthesis method (DAW40007-3).

[0305] Annealing steps:

[0306] The sense strand of the double-stranded siRNA conjugate obtained above was mixed with the antisense strand obtained above in an equimolar amount, heated to 95°C, and kept at that temperature for 10 min before being slowly cooled to room temperature. The resulting compound was then freeze-dried to obtain the target siRNA conjugate.

[0307] The double-stranded siRNAs synthesized above are shown in Table 1.

[0308] Table 1: Double-stranded siRNA synthesized in this invention

[0309]

[0310]

[0311] In Table 1, uppercase letters C, G, U, and A represent the base composition of natural nucleotides; lowercase letters c, g, u, and a represent nucleotides whose ribose 2-position is modified with a methoxy group, such as c, g, u, and a representing 2'-OMe (2'-O-methyl)C, 2'-OMe G, 2'-OMe U, and 2'-OMe A, respectively; the uppercase letter f to the right represents a nucleotide whose ribose 2-position is modified with fluorine, such as Cf, Gf, Uf, and Af representing 2'-F (2'-fluorine)C, 2'-FG, 2'-FU, and 2'-FA, respectively; "s" indicates that the two nucleotide residues adjacent to "s" are linked by a thiophosphate group, for example, "gsu" indicates that the g and u residues are linked by a thiophosphate group; Agn represents adenosine-ethylene glycol nucleic acid (GNA); Tgn represents thymine-diol nucleotide residues, whose structure is... Y represents Y1 represents Y2 represents Y3 indicates Y4 indicates

[0312] Example A: Cell viability and cytotoxicity test of the nucleic acid or conjugate of the present invention

[0313] Experimental steps:

[0314] Cell culture: The frozen HepAD38 cells were revived and cultured in culture medium (DMEM / F12 medium + 10% FBS + double antibiotics + tetracycline). The cells were used for experiments when they were in the logarithmic growth phase and in good condition (70-80% confluence).

[0315] Transfection administration: On day 0, healthy HepAD38 cells were digested with trypsin, adjusted to an appropriate density, and seeded into 96-well plates at 20,000 cells per well using culture medium (DMEM / F12 medium + 10% FBS + antibiotic-free). On day 1, after preparing siRNA to the appropriate concentration according to the final drug administration concentration, siRNA was transfected using RNAiMAX transfection reagent. First, siRNA and RNAiMAX transfection reagent were co-incubated in opti-MEM medium for 5 minutes. The specific preparations were as follows: Solution A and Solution B were prepared separately: Solution A contained 5 μL opti-MEM and 1 μL siRNA; Solution B contained 5 μL opti-MEM and 0.3 μL RNAiMAX. After incubating A+B solutions for 5 minutes, the old medium in the cell plate was discarded, and 11.3 μL of the A+B mixture was added to 88.7 μL (siRNA was equivalent to a 100-fold dilution in this step) of fresh maintenance medium (2% FBS + antibiotic-free + DMEM / F12 medium). The final drug administration concentration of siRNA was 20 nM and 0.05 nM. A certain number of control wells with solvent (H2O) were also prepared and transfected using the same procedure. On day 4, cell supernatant was collected, and 75 μl was used to detect HBsAg expression using an HBsAg detection kit (Shanghai Kehua). Cytotoxicity was detected using a CCK8 kit (DOJINDO).

[0316] Data analysis showed that the HBV surface antigen inhibition percentage was: %Inh. = (1 - absorbance of the drug well / absorbance of the solvent (H2O) control well) x 100, and the HBV surface antigen inhibition rate of each siRNA was calculated.

[0317] The double-stranded siRNA reagents of this invention exhibit good inhibitory activity against HBsA, and most siRNAs show significantly better HBsA inhibitory activity than the control VIR-2218. It is evident that the insertion of nucleotide residue N, especially Y1, Y2, Y3, and Y4, into the double-stranded siRNAs described in this application significantly enhances their activity. Furthermore, the double-stranded siRNAs described in this application are essentially non-toxic to cells. The HBsA inhibitory activity results of some of the double-stranded siRNAs of this invention are shown in Table 2.

[0318] Table 2: Inhibitory activity results of some double-stranded siRNAs in this invention

[0319]

[0320]

[0321]

[0322] Note: The double-stranded siRNAs in Table 1 of this invention are divided into two batches for testing, namely batch 1 and batch 2. The sequence of VIR-2218 (control) is: sense strand: 5'-gsusguGfcAfCfUfucgcuucaca-3', antisense strand: 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3'.

[0323] As is known in the art, the siRNA conjugate of the present invention is obtained by conjugating an siRNA drug with a GalNAc or its derivative. The main function of the conjugate is delivery. When the siRNA has good activity, the conjugate after connecting the conjugate can be expected to have similar activity. Those skilled in the art can select appropriate conjugate groups based on their knowledge in the art, such as L96 and DAW40007-4, etc., GalNAc or its derivatives.

[0324] Example B: Evaluation of anti-hepatitis B virus (HBV) activity using the PHH in vitro infection model

[0325] The experimental steps are as follows:

[0326] Transfection and drug administration: On day 0, the recovered PHH cells were adjusted to an appropriate density (6.67E+05 cells / ml) and seeded into 48-well cell culture plates, with 1.32E+05 cells per well. On day 1, PHH cells were infected with type D HBV (800 GE / cell). On day 2, the culture medium was replaced with fresh medium. On day 4, the cell culture medium was discarded, and the test substance was serially diluted to the required concentration, then administered with Lipofectamine. TM The test reagent was introduced into the cells via RNAiMAX transfection. The initial concentration of all test reagents was 500 pM, diluted 5-fold, and seven concentrations were set up in two replicates. Wells containing only the transfection reagent served as medium controls. The medium was replaced with fresh medium free of the compound 4-5 hours after transfection. The final concentration of DMSO in the medium was 2%. On day 6, the medium was replaced with fresh medium free of the compound. On day 8, the cell supernatant was collected, and cell viability was assessed using a CCK-8 assay.

[0327] Free drug uptake: On day 0, first dilute the test substance with enzyme-free water to the required concentration. The starting concentration is 500 nM, with a 5-fold dilution. A total of 7 concentrations are set, with 2 replicates per concentration. Add 22 μl to each well of the cell culture plate. Resuscitate the cryopreserved PHH, adjust the PHH to the appropriate density (6.67E+05 cells / ml), and add 1.32E+05 cells to each well. When plating the cells, the test substance is freely taken up into the cells. On day 1, change the culture medium and infect the PHH with D-type HBV (800 GE / cell). On day 2, change to fresh culture medium without virus and compounds. The final concentration of DMSO in the culture medium is 2%. On days 4 and 6, change to fresh culture medium without compounds. On day 8, collect the cell supernatant and detect cell viability using CCK-8.

[0328] Sample detection: Detect the content of HBV DNA in the cell culture supernatant by qPCR; detect the content of HBsAg and HBeAg in the cell culture supernatant by ELISA.

[0329] The experimental results show that: the HBsAg, HBeAg and DNA inhibitory activities of the compounds of the present invention containing the monomers of the present invention.

[0330] Example C: Experiment on HBV transgenic mice

[0331] 1. HBV-Tg transgenic mouse experiment

[0332] Experimental methods

[0333] Experimental animals: HBV-Tg transgenic mice, C57B / 6N-Tg(1.28HBV) / Vst, SPF-grade male, 6 - 8 weeks old, 16 - 20 g, purchased from Beijing Vitalstar Biotechnology Co., Ltd. Animal production license number: SCXK(Beijing)2019 - 0002. The animals are raised by Beijing Vitalstar Biotechnology Co., Ltd. and are housed individually in cages.

[0334] Animal grouping: According to the quantitative detection results of mouse serum HBsAg (main) and HBeAg (secondary), the animals are randomly stratified into groups, with 5 animals in each group.

[0335] Preparation and administration of test substances: Calculate the required amount of drug powder according to the purity of each test substance. At a dosing concentration of 0.6 mg / mL, add the corresponding normal saline and shake well until it becomes a colorless and transparent liquid. Define the day of the first administration as day 0. Each group of animals is administered once on day 0, and the subcutaneous administration volume is 5 mL / kg. If blood needs to be collected on the day of animal administration, the administration is carried out after blood collection.

[0336] Main observation indicators: Weighing and blood collection were performed once each on days 3, 7, 14, 21, 28 and 35, and serum was separated for HBsAg and HBeAg level determination.

[0337] Serum HBsAg and HBeAg level determination: After blood collection, serum was separated, diluted with PBS solution, and sent for testing. 10 μL of serum was taken from each sample and diluted to 500 μL (50-fold dilution) with PBS solution. Serum HBsAg and HBeAg levels were measured using a Hepatitis B e antigen assay kit (Maccura Biotechnology) and a Hepatitis B surface antigen assay kit (Maccura Biotechnology), respectively.

[0338] Results of HBV-Tg transgenic mouse experiments

[0339] The compounds of this invention containing the nucleotide residues of this invention have a significant inhibitory effect on HBsAg in HBV-Tg transgenic mice, and also have a good inhibitory effect on reducing HBeAg.

[0340] 2. Hepatitis B virus AAV mouse experiment

[0341] Experimental methods:

[0342] Preparation and grouping of rAAV8-1.3HBV / C57BL / 6 mouse model: A persistent HBV infection mouse model was established by intravenous injection of rAAV8-1.3HBV (type C) into the tail vein of C57 mice. The AAV virus injection dose was 1E+11vg / mouse. Six weeks after virus injection, blood samples were collected to detect the levels of HBeAg, HBsAg, and ALT in the serum. Based on the quantitative detection results of HBsAg (predominant) and HBeAg (secondary) in the mouse serum, 35 animals were stratified and randomly divided into groups of 5.

[0343] Preparation and administration of test samples: Calculate the required amount of drug powder based on the purity of each test sample. Add the corresponding physiological saline at a dosage concentration of 0.6 mg / mL, and shake to mix until a colorless and transparent liquid is obtained. Define the day of the first administration as day 0. All animals in each group were given a single subcutaneous dose on day 0, with a volume of 5 mL / kg. If blood collection is required on the day of administration, the administration should be performed after blood collection.

[0344] Main observation indicators: Weighing and blood collection were performed once each on days 3, 7, 14, 21 and 28, and serum was separated for HBsAg and HBeAg level determination.

[0345] Serum HBsAg and HBeAg level determination: After blood collection, serum was separated, diluted with PBS solution, and sent for testing. 10 μL of serum was taken from each sample and diluted to 500 μL (50-fold dilution) with PBS solution. Serum HBsAg and HBeAg levels were measured using a hepatitis B e antigen assay kit and a hepatitis B surface antigen assay kit, respectively.

[0346] Results of AAV mouse experiment with hepatitis B virus

[0347] The compounds of this invention containing the nucleotide residues of this invention have a significant inhibitory effect on HBsAg in AAV mice, and also have a good inhibitory effect on reducing HBeAg.

[0348] Example D: Toxicity test of the conjugate of this application administered subcutaneously to rats.

[0349] Experimental methods:

[0350] Rats were administered the drug five times over a total of 29 days. Clinical observation was conducted. Blood was collected from the abdominal aorta before anesthesia and dissection. Blood biochemistry was analyzed for ALT, AST, ALP, CK, UREA, CREA, TP, ALB, GLU, TBIL, DBIL, CHO, TG, Na, K, Cl, A / G, and GLB. Hematological parameters included WBC, RBC, HB, HCT, MCV, MCH, MCHC, RDW, PLT, MPV, WBC differential and percentage. Coagulation parameters included PT, APTT, and FIB. Additionally, histological virology was observed, including weighing of organs such as the aorta, esophagus, stomach, duodenum, cecum, colon, ileum, jejunum, rectum, lungs (including the main bronchus), mesenteric lymph nodes, pancreas, pituitary gland, eyeballs, thyroid / parathyroid glands, salivary glands, prostate, bladder, seminal vesicles, femur, sternum, skin and muscles at the administration sites (marked after the last administration), and any abnormal tissue observed grossly.

[0351] Statistical methods: SPSS 16.0 was used for statistical analysis. Means and standard deviations were calculated for each group's weight, blood biochemistry, hematology, organ weight, and coefficients. The following methods were used for statistical analysis: ① First, Levene's test was used to test data uniformity. If the data were uniform (P>0.05), one-way ANOVA was performed. If the Levene's test result was significant (p≤0.05), the Kruskal-Wallis test was performed. ② If the ANOVA result was significant (p≤0.05), the Dunnett parametric test was used for multiple comparisons. If the ANOVA result was not significant (p>0.05), the statistical analysis was terminated. ③ If the Kruskal-Wallis test result was significant (p≤0.05), the rank-transformed Dunnett test was used. If the Kruskal-Wallis test result was not significant (p>0.05), the statistical analysis was terminated. Clinical symptom observation, food intake, and anatomical observation data were not statistically processed.

[0352] Experimental conclusions: The experiments show that clinical observation, blood biochemistry, hematology, organ weight and histopathology results indicate that the siRNA conjugates of the present invention containing the nucleotide residues of the present invention have good safety.

[0353] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A compound having a structure as shown in formula (I) or (II), or a stereoisomer, tautomer, or acceptable salt of a compound as shown in formula (I) or (II), X is OR 1 F, benzyl, -CH2C(=O)OR 1a or-P(OCH2CH2CN)N(i-Pr)2; Each R 1 R 1a R 1b R 1c R 1d and R 1e Independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, wherein each of the methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl groups is independently and optionally substituted by 1, 2, 3, or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy, and 2-propoxy. Z and Z 1 Each is independently protected by H, deuterium, methyl, hydroxyl groups, or HOC(=O)(CH2). j C(=O)-、MC(=O)(CH2) j C(=O)-, phosphate ester group, thiophosphate ester group, phosphite amide group, or hydrophosphate group; or Z and Z 1 Each of the following is independently represented as H, deuterium, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, p-methoxybenzyl diphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, C 1-12 Alkyl, C 1-12 Alkyl C(=O)-, C 1-12 Alkylsilyl, C 6-10 Aryl C 1-6 Alkyl group, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytriphenylmethyl, or Z and Z 1 Each of the following is independently represented as H, deuterium, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, p-methoxybenzyl diphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, C 1-10 Alkyl, C 1-10 Alkyl C(=O)-, C 1-10 Alkylsilyl, C 6-10 Aryl C 1-4 Alkyl group, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytriphenylmethyl, or Z and Z 1 Each of the following is independently represented as H, deuterium, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, p-methoxybenzyl diphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, C 1-8 Alkyl, C 1-8 Alkyl C(=O)-, C 1-8 Alkylsilyl, Phenyl C 1-3 Alkyl group, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytriphenylmethyl, or Z and Z 1 Each of the following is independently represented as H, deuterium, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, p-methoxybenzyl diphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, C 1-6 Alkyl, C 1-6 Alkyl C(=O)-, C 1-6 Alkylsilyl, benzyl, phenethyl, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytriphenylmethyl, in, X is either Cl or Br; Each R x and R y Independently protected by a hydroxyl group; Each R c and R d Independently protected by H or amino groups; or R c R d Together with the N atoms to which they are attached, they form a heterocyclic group consisting of 5-6 ring atoms, which is independently and optionally substituted by 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methyl, ethyl, propyl, methoxy, ethoxy, 1-propoxy and 2-propoxy. M is a solid support, preferably a hydroxyl or amino functionalized solid support, more preferably a resin or CPG, further preferably a macroporous resin or CPG, and even more preferably an aminomethyl resin, a hydroxyl resin or -NHCPG. R is -NHR 2 , hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, fluorine, chlorine, bromine, -CH2C(=O)OR 1b -O-phenyl-C(=O)OR 1c -N(-CH2C(=O)OR 1d )2、-NHS(=O)2-R 1e Or -N = CH-NR a R b ; R 2 It is an amino protecting group, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; Each R a and R b Independently protected by H or amino groups, or R a R b Together with the N atoms to which they are attached, they form a heterocyclic group consisting of 5-6 ring atoms, each of which is independently and optionally substituted by 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, 1-propoxy and 2-propoxy. Each j is independently 1, 2, 3, 4 or 5; The condition is that the compound is not 2. The compound according to claim 1, wherein, R 2 The following are listed: 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, methyl, tert-butyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, benzyl, p-methoxybenzyl diphenylmethyl, triphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, C 1-6 Alkoxy, R 3 C(=O)-, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; Each R a and R b Independently, it is H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, 1-propoxy, 2-propoxy, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, Phenoxyacetyl, benzoyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, benzyl, p-methoxybenzyl diphenylmethyl, triphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl or R 4 C(=O)-, or R a R b Together with the N atoms to which they are attached, they form pyrrolidinyl, morpholinyl, piperidinyl, or piperazineyl groups, each of which is independently and optionally substituted by one, two, three, or four substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy, and 2-propoxy. Among them, each R 3 and R 4 Independently for C 1-6 Alkyl, Halogenated C 1-6 Alkyl, C 1-6 Alkoxy, phenyl, halogen-substituted phenyl, C 1-6 alkylphenyl or benzyl; or Each R 3 and R 4 Independently for C 1-4 Alkyl, Halogenated C 1-4 Alkyl, C 1-4 Alkoxy, phenyl, halogen-substituted phenyl, C 1-4 alkylphenyl or benzyl; or Each R 3 and R 4 Independently, methyl, ethyl, n-propyl, isopropyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, 2,2,2-trichloroethyl, methoxy, ethoxy, 1-propoxy, 2-propoxy, 1-butoxy, 2-methyl-l-propoxy, 2-butoxy, tert-butoxy, phenyl, halogen-substituted phenyl, C 1-3 Alkylphenyl or benzyl.

3. The compound according to claim 1, each R x and R y Independently, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, p-methoxybenzyl diphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, cyanoC 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkyl C(=O)-, C 1-6 Alkylsilyl, phenyl, halogen-substituted phenyl, benzyl, or phenethyl; or Each R x and R y Independently, it is benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl p-Methoxybenzyl diphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, CNCH2-, CN(CH2)2-, CNCH(CH3)2CH2-, CN(CH2)3-, CNCH(CH3)2CH2CH2-, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, C 1-4 Alkyl C(=O)-, C 1-4 Alkylsilyl, phenyl, halogen-substituted phenyl, benzyl or phenethyl; Each R c and R d Independently, H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, 1-propoxy, 2-propoxy, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxy Acetyl, benzoyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, benzyl, p-methoxybenzyl diphenylmethyl, triphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, trimethylsilyl, triethylsilyl or triisopropylsilyl, or R c R d Together with the N atoms to which they are attached, they form pyrrolidinyl, morpholinyl, piperidinyl, or piperazineyl groups, each of which is independently and optionally substituted by one, two, three, or four substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy, and 2-propoxy. Preferably, the compound has one of the following compounds, or a stereoisomer, tautomer, or an acceptable salt thereof.

4. A nucleotide residue having the structure described in formula (Ia) or Y9, R W1 -NHR 2a , hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, fluorine, chlorine, bromine, -CH2C(=O)OR 2b -O-phenyl-C(=O)OR 2c -N(-CH2C(=O)OR 2d )2、-NS(=O)2-R 2e ; R W2 OR 2f F, benzyl or -CH2C(=O)OR 2g ; Each R 2b R 2c R 2d R 2e R 2f and R 2g It can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl independently; R 2a It can be H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; The condition is that the nucleotide monomer is not Preferably, the nucleotide residues have one of the following structures from Y1 to Y15:

5. A double-stranded siRNA, wherein, The double-stranded siRNA comprises a sense strand and an antisense strand, wherein all nucleotides in the sense strand and the antisense strand are modified nucleotides, and the double-stranded siRNA contains one or more N, wherein the N is a nucleotide residue as described in claim 4. Preferably, N is selected from nucleotide residues Y1, Y2, Y3 or Y4 as described in claim 4; The antisense strand contains a nucleotide sequence as shown in 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO:2), and the length of the antisense strand does not exceed 23 nucleotides; Optionally, the positive strand comprises a nucleotide sequence as shown in 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO:1), and the length of the positive strand does not exceed 23 nucleotides; The modification is selected from at least one of the following: 2'-methoxy modification, 2'-methoxyethyl modification, 2'-fluorinated modification, thiophosphate linkage, 2'-deoxy modification, 2'-amino modification, locked nucleic acid modification, unlocked nucleic acid modification, ethylene glycol nucleic acid modification, and 5'-vinyl phosphate modification.

6. The double-stranded siRNA according to claim 5, wherein, The positive strand comprises a sequence in which 0, 1, 2, 3, 4 or 5 nucleotide residues are replaced by N in the sequence shown in SEQ ID NO:1; Preferably, the N substitution occurs at the nucleotides at positions 1 to 19 of the 5' end of the sequence shown in SEQ ID NO:1; preferably, the N substitution occurs at the nucleotides at positions 7, 17, or 19 of the 5' end of the sequence shown in SEQ ID NO:

1. Optionally, the antisense strand comprises a sequence in which 0, 1, 2, 3, 4, or 5 nucleotide residues are replaced by N in the sequence shown in SEQ ID NO:2; Preferably, the N substitution occurs at positions 1 to 21 of the 5' end of the sequence shown in SEQ ID NO:2; preferably, the N substitution occurs at the nucleotides at positions 5, 6, 7, 10, 11, 16, 17, or 18 of the 5' end of the sequence shown in SEQ ID NO:

2.

7. The double-stranded siRNA according to claim 5, wherein, The positive strand comprises a sequence in which 0, 1, 2, 3, 4, or 5 nucleotide residues are replaced by N in the sequence shown as 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO:5); preferably, the N substitution occurs at positions 1 to 19 of the 5' end of the sequence shown as SEQ ID NO:5; preferably, the N substitution occurs at position 7, 17, or 19 of the 5' end of the sequence shown as SEQ ID NO:5; Optionally, the antisense strand comprises a sequence in which 0, 1, 2, 3, 4 or 5 nucleotide residues are replaced by N in a sequence such as 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO:22); Preferably, the N substitution occurs at the nucleotides at positions 1 to 21 of the 5' end of the sequence shown in SEQ ID NO:22; preferably, the N substitution occurs at the nucleotides at positions 5, 6, 7, 10, 11, 16, or 17 of the 5' end of the sequence shown in SEQ ID NO:

22.

8. The double-stranded siRNA according to claim 5, wherein the sense strand comprises one of the following nucleotide sequences: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, wherein the length of the positive strand does not exceed 23 nucleotides; Optionally, the antisense strand comprises one of the following nucleotide sequences: SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39; SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49, wherein the length of the antisense strand does not exceed 23 nucleotides; Preferably, the double-stranded siRNA is selected from one of siRNAID NO:1 to siRNAID NO:

48.

9. A double-stranded siRNA conjugate or a salt thereof, comprising the siRNA according to any one of claims 5-8, wherein the double-stranded siRNA conjugate is formed by conjugating the double-stranded siRNA with a conjugating group; preferably, the 3' or 5' end of the sense strand or antisense strand of the double-stranded siRNA is conjugated with the conjugating group; more preferably, the 3' end of the sense strand of the double-stranded siRNA is conjugated with the conjugating group; Optionally, the 3' or 5' end of the positive strand of the double-stranded siRNA is conjugated to the conjugation group via a phosphate ester group, a thiophosphate ester group, or a phosphate group; Preferably, the conjugating group comprises GalNAc or a derivative thereof; more preferably, the conjugating group is GalNAc or a derivative thereof linked by divalent, trivalent, or tetravalent branching links; more preferably, the conjugating group is L-96, DAW40007-4, or a stereoisomer thereof, wherein... The structures of the conjugation groups DAW40007-4 and L-96 are as follows:

10. A pharmaceutical composition comprising the double-stranded siRNA of any one of claims 5-8 or the double-stranded siRNA conjugate of claim 9 or a salt thereof, and a pharmaceutically acceptable carrier.

11. Use of the double-stranded siRNA of any one of claims 5-8, the double-stranded siRNA conjugate of claim 9 or a salt thereof, or the pharmaceutical composition of claim 11 in the preparation of a medicament for the treatment and / or prevention of hepatitis B.

12. The application of the compound according to any one of claims 1-3 in the fields of raw materials for solid-phase synthesis of DNA nucleotides, raw materials for the synthesis of oligonucleotide drugs, raw materials for the synthesis of siRNA drugs, siRNA drug research, gene function research and / or screening of whole gene libraries.

13. The use of the nucleotide residue as an oligonucleotide intercalation group as described in claim 4, wherein, The oligonucleotide is a nucleotide sequence containing 10 to 50 nucleotides or nucleotide base pairs, and the oligonucleotide is capable of inhibiting or blocking gene expression. Preferably, the gene is the HBV gene. The oligonucleotide is siRNA, which includes a sense strand and an antisense strand; The nucleotide residues are inserted only into the positive strand of the siRNA; preferably, the nucleotide residues are inserted into the nucleotides at the 7th, 17th, or 19th position at the 5' end of the positive strand. Optionally, the nucleotide residues are inserted only into the antisense strand of the siRNA; preferably, the nucleotide residues are inserted into the nucleotides at positions 5, 6, 7, 10, 11, 16, 17, or 18 of the 5' end of the antisense strand. More preferably, the nucleotide residues are embedded in the sense and antisense strands of the siRNA, wherein the nucleotide residues are embedded in the nucleotide at position 7, 17 or 19 of the 5' end of the sense strand, and the nucleotide residues are embedded in the nucleotide at position 5, 6, 7, 10, 11, 16, 17 or 18 of the 5' end of the antisense strand.

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