Novel compound and use thereof

Abstract

The present invention provides a novel compound and a use thereof. The compound can be used as a raw material for the solid-phase synthesis of a DNA nucleotide, a raw material for the synthesis of an oligonucleotide drug, or a raw material for the synthesis of an siRNA drug, can be used for siRNA drug research, gene function research and / or whole genome library screening, and can be especially used as a raw material for the synthesis of an ASO drug or as a raw material for the synthesis of a double-stranded siRNA drug. The present invention further relates to a novel nucleotide residue and a use thereof as an intercalating group for ASO drug research, siRNA drug research, gene function research and / or whole genome library screening.

Description

A novel compound and its use Technical Field

[0001] The present invention belongs to the field of small nucleic acid drugs. The present invention aims to provide a novel compound and its use. The compound can be used as a raw material for solid-phase synthesis of DNA nucleotides, as a raw material for the synthesis of oligonucleotide drugs, and / or as a raw material for the synthesis of siRNA drugs. It can also be used for siRNA drug research, gene function research, and / or screening of whole gene libraries. In particular, it can be used as a raw material for the synthesis of ASO drugs and / or double-stranded siRNA drugs. The present invention also relates to a novel nucleotide residue and its use as an intercalating group in ASO drug research, siRNA drug research, gene function research, and / or screening of whole gene libraries. Background Art

[0002] Small nucleic acid drugs, also known as oligonucleotide drugs, are short nucleic acid chains composed of a dozen to dozens of nucleotides strung together. Nucleic acid monomers are the basic building blocks of small nucleic acid drugs and are also key raw materials for nucleic acid drugs. Due to the susceptibility of the original small nucleic acid structure to degradation by nucleases in the body and poor pharmacokinetic properties, they are generally not used directly as drugs. They require chemical modification of the nucleotides at multiple sites to improve their affinity, stability, and metabolic properties. Such modifications mainly include base modifications, modifications to the 2'-hydroxy ribose, modifications to the phosphate backbone, modifications to the ribose five-membered ring backbone, and combined modifications specific to nucleic acid drugs. By modifying and modifying nucleic acid monomers and thus achieving the modification and transformation of specific nucleic acid sequences, nucleic acid drugs with specific pharmacological activities can be designed and synthesized. Therefore, nucleic acid monomers play an important role in the research and development of small nucleic acid drugs. Summary of the Invention

[0003] The present invention provides a novel compound and its use. The compound can be used as a raw material for solid-phase synthesis of DNA nucleotides, as a raw material for the synthesis of oligonucleotide drugs, and / or as a raw material for the synthesis of siRNA drugs. It can also be used for siRNA drug research, gene function research, and / or screening of whole gene libraries. In particular, it can be used as a raw material for the synthesis of ASO drugs and / or double-stranded siRNA drugs. The present invention also relates to a novel nucleotide residue and its use as an intercalating group in ASO drug research, siRNA drug research, gene function research, and screening of whole gene libraries. Experiments have shown that ASO drugs, siRNA double strands and conjugates thereof embedded with the nucleotide residues of the present invention have significant inhibitory activity on the expression of target genes (such as HBV (hepatitis B virus) gene, AGT (angiotensinogen) gene, Lp(a) (lipoprotein(a)) gene, ANGPTL3 (angiopoietin-like 3) gene, APOC3 (apolipoprotein C-III) gene, PD-L1 (programmed cell death protein ligand 1) gene, HSD17B (17β-hydroxysteroid dehydrogenase) etc.), with basically no off-target phenomenon and / or low toxicity.

[0004] In one aspect, the present invention relates to a compound having a structure as shown in formula (I), or a stereoisomer, tautomer or acceptable salt of the structure as shown in formula (I),

[0005] in,

[0006] 1) L1 is -*O(CH2) n1 -or-*(CH2) n2 -, R is H, deuterium, fluorine, chlorine, bromine, iodine, methyl or ethyl, wherein -*O(CH2) n1 -and-*(CH2) n2 - are each independently optionally substituted with 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy, wherein n1 is 1, 2, 3, 4, 5, 6 or 7, and n2 is 2, 3, 4, 5, 6 or 7; or

[0007] 2) L1 is -*CH2-, R is H, deuterium, fluorine, chlorine, bromine, iodine, methyl or ethyl, wherein -*CH2- is optionally substituted with 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy;

[0008] Z and Y 1 are independently H, deuterium, a hydroxyl protecting group, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) jC(=O)-, a phosphate group, a thiophosphate group, a phosphite amide group, or a hydrogen phosphate group, wherein each j is independently 1, 2, 3, 4, or 5;

[0009] 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, further preferably an aminomethyl resin, a hydroxyl resin or -NHCPG;

[0010] B is Each R 1a 、R 1b 、R 2a 、R 2b 、R 3a 、R 4a and R 4b are independently deuterium, H, CN, HO-, fluorine, chlorine, bromine, methyl, ethyl or methoxy;

[0011] Each R 2 、R 3b and R 4c Independently -NHR 1 or -N=CH-NR a R b ;

[0012] R 1 is an amino protecting group;

[0013] Each R a and R b are independently H or an amino protecting group, or R a 、R b Together with the nitrogen atom to which they are attached, they form a heterocyclic group consisting of 5-6 ring atoms, and each heterocyclic group consisting of 5-6 ring atoms is optionally substituted by 1, 2, 3 or 4 substituents selected from deuterium, hydroxy, cyano, F, Cl, Br, I, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, 1-propoxy and 2-propoxy.

[0014] In some embodiments of the compounds described herein, Z and Y 1 are independently H, deuterium, C 1-12 Alkyl, C 1-12 Alkyl C(=O)-, C 1-12 Alkylmethyl, C 1-12 Alkylsilyl, C 6-10 Aryl C 1-6 Alkyl, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) jC(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, Among them, j, M, X, R x 、R y 、R c and R d has the meanings described in the present invention.

[0015] In some embodiments of the compounds described herein, Z and Y 1 and each is independently 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-methoxybenzyldiphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methylsulfonyl, p-toluenesulfonyl, C 1-10 Alkyl, C 1-10 Alkyl C(=O)-, C 1-10 Alkylmethyl, C 1-10 Alkylsilyl, C 6-10 Aryl C 1-4 Alkyl, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, Among them, j, M, X, R x 、R y 、R c and R d has the meanings described in the present invention.

[0016] In some embodiments of the compounds described herein, Z and Y 1and each is independently 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-methoxybenzyldiphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methylsulfonyl, p-toluenesulfonyl, C 1-8 Alkyl, C 1-8 Alkyl C(=O)-, C 1-8 Alkylmethyl, C 1-8 Alkylsilyl, phenyl C 1-3 Alkyl, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, Among them, j, M, X, R x 、R y 、R c and R d has the meanings described in the present invention.

[0017] In some embodiments of the compounds described herein, Z and Y 1 and each is independently 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-methoxybenzyldiphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methylsulfonyl, p-toluenesulfonyl, C 1-6 Alkyl, C 1-6 Alkyl C(=O)-, C 1-6 Alkylmethyl, C 1-6 Alkylsilyl, benzyl, phenethyl, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) jC(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, Among them, j, M, X, R x 、R y 、R c and R d has the meanings described in the present invention.

[0018] In some embodiments of the compounds described herein, X is Cl or Br.

[0019] In some embodiments of the compounds described herein, each R x and R y is independently a hydroxy protecting group.

[0020] In some embodiments of the compounds described herein, each R c and R d are independently H or an amino protecting group; or R c 、R d Together with the nitrogen atom to which they are attached, they form a heterocyclic group consisting of 5-6 ring atoms, and the heterocyclic group consisting of 5-6 ring atoms is optionally substituted by 1, 2, 3 or 4 substituents selected from deuterium, hydroxy, cyano, F, Cl, Br, I, methyl, ethyl, propyl, methoxy, ethoxy, 1-propoxy and 2-propoxy.

[0021] In some embodiments of the compounds described herein, R 1 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, 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-methoxybenzyldiphenylmethyl, trityl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, C 1-6 Alkoxy (such as methoxy, ethoxy, 1-propoxy, 2-propoxy, etc.) or R 3 C(=O)-, where R 3 has the meanings described in the present invention.

[0022] In some embodiments of the compounds described herein, each Ra and R b are independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, 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-methoxybenzyldiphenylmethyl, trityl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, C 1-6 Alkoxy (such as methoxy, ethoxy, 1-propoxy, 2-propoxy, etc.) or R 4 C(=O)-, or R a 、R b and together with the nitrogen atom to which they are attached form a pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl group, said pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl being independently optionally substituted with 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy groups, wherein R 4 has the meanings described in the present invention.

[0023] In some embodiments of the compounds described herein, each R a and R b are 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-methoxybenzyldiphenylmethyl, trityl, 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 、Rb and together with the nitrogen atom to which they are attached form a pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl group, said pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl being independently optionally substituted with 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy groups, wherein R 4 has the meanings described in the present invention.

[0024] In some embodiments of the compounds described herein, each R 3 and R 4 Independently C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, phenyl, halogen-substituted phenyl, C 1-6 Alkylphenyl or benzyl.

[0025] In some embodiments of the compounds described herein, each R 3 and R 4 Independently C 1-4 Alkyl, halogenated C 1-4 Alkyl, C 1-4 Alkoxy, phenyl, halogen-substituted phenyl, C 1-4 Alkylphenyl or benzyl.

[0026] In some embodiments of the compounds described herein, each R 3 and R 4 are 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-1-propoxy, 2-butoxy, tert-butoxy, phenyl, halogen-substituted phenyl, C 1-3 Alkylphenyl or benzyl.

[0027] In some embodiments of the compounds described herein, each R x and R yare 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-methoxybenzyldiphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methylsulfonyl, p-toluenesulfonyl, cyano C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkyl C(=O)-, C 1-6 alkylsilyl, phenyl, halogen-substituted phenyl, benzyl or phenethyl.

[0028] In some embodiments of the compounds described herein, each R x and R y are 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 methyl, p-methoxybenzyldiphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methylsulfonyl, p-toluenesulfonyl, CNCH2-, CN(CH2)2-, CNC(CH3)2CH2-, CN(CH2)3-, CNC(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.

[0029] In some embodiments of the compounds described herein, each R c and R dare independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, 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-methoxybenzyldiphenylmethyl, trityl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, C 1-6 Alkoxy (such as methoxy, ethoxy, 1-propoxy, 2-propoxy, etc.) or R 4 C(=O)-, or R a 、R b Together with the N atom to which it is attached, it forms a pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl group, and the pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl groups are independently optionally substituted with 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy groups.

[0030] In some embodiments of the compounds described herein, each R c and R d are 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-methoxybenzyldiphenylmethyl, trityl, 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 bTogether with the N atom to which it is attached, it forms a pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl group, and the pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl groups are independently optionally substituted with 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy groups.

[0031] In some embodiments of the compounds of the present invention, the compounds of the present invention have a structure as shown in Formula (Ia) or (Ib), or a stereoisomer, tautomer or acceptable salt thereof,

[0032] wherein each of L1, R and B independently has the meaning described herein.

[0033] In some embodiments of the compound described herein, it is one of the following compounds 1-25, or a stereoisomer, tautomer or acceptable salt thereof,

[0034] Among them, A1 is A2 is A3 is U is T is

[0035] On the other hand, the present invention provides a nucleotide residue having a structure as shown in formula (II), or a stereoisomer, tautomer or acceptable salt of the structure as shown in formula (II),

[0036] 1) L1 is -*O(CH2) n1 -or-*(CH2) n2 -, R is H, deuterium, fluorine, chlorine, bromine, iodine, methyl or ethyl, wherein -*O(CH2) n1 -and-*(CH2) n2 - are each independently optionally substituted with 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy, wherein n1 is 1, 2, 3, 4, 5, 6 or 7, and n2 is 2, 3, 4, 5, 6 or 7; or

[0037] 2) L1 is -*CH2-, R is H, deuterium, fluorine, chlorine, bromine, iodine, methyl or ethyl, wherein -*CH2- is optionally substituted with 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy;

[0038] B1 is Each R 1a 、R 1b 、R 2a、R 2b 、R 3a 、R 4a and R 4b are independently H, deuterium, CN, HO-, fluorine, chlorine, bromine, methyl, ethyl or methoxy.

[0039] In some embodiments of the nucleotide residues of the present invention, the nucleotide residues of the present invention are one of the following nucleotide residues (1)-(20), or stereoisomers, tautomers or acceptable salts thereof.

[0040] Among them, A is G is C is U is T is

[0041] In some preferred embodiments of the nucleotide residues of the present invention, the nucleotide residues of the present invention are

[0042] In another aspect, the present invention provides the use of the nucleotide residues of the present invention as intercalating groups in ASO drug research, siRNA drug research, gene function research and / or screening of whole gene libraries.

[0043] In some embodiments of the use of the present invention, the nucleotide residues of the present invention are embedded as intercalating groups in an ASO drug, a sense strand of an siRNA drug, an antisense strand of an siRNA drug, or both the sense strand and the antisense strand of an siRNA drug. In another aspect, the present invention provides uses of the compounds of the present invention as raw materials for solid-phase synthesis of DNA nucleotides, as raw materials for the synthesis of oligonucleotide drugs, as raw materials for the synthesis of siRNA drugs, in ASO drug research, siRNA drug research, gene function research, and / or in screening of whole gene libraries.

[0044] In some embodiments of the use of the present invention, the siRNA drug is a drug that inhibits the expression of the AGT gene.

[0045] In some embodiments of the use of the present invention, the siRNA drug comprises a sense strand and an antisense strand capable of forming a double-stranded region, and the sense strand consists of 15-40 nucleotides, preferably 17-25 nucleotides, and more preferably 18-23 nucleotides.

[0046] In some embodiments of the use of the present invention, the antisense strand consists of 15-40 nucleotides, preferably consists of 17-35 nucleotides, more preferably consists of 19-30 nucleotides, and most preferably consists of 21-29 nucleotides.

[0047] In some embodiments of the use of the present invention, the double-stranded region consists of 17-23 nucleotide base pairs.

[0048] In some embodiments of the use of the present invention, the sense strand and / or antisense strand comprises 7, 6, 5, 4, 3, 2, 1 and 0 unmodified nucleotides, respectively.

[0049] In some embodiments of the use of the present invention, the siRNA drug further comprises a ligand.

[0050] In some embodiments of the use of the present invention, the ligand is conjugated to any position of the sense strand or the antisense strand.

[0051] In some embodiments of the use of the present invention, the ligand is conjugated to the 3' end or the 5' end of the sense strand.

[0052] In some embodiments of the use of the present invention, the ligand is conjugated to the 3' end or the 5' end of the antisense strand.

[0053] In some embodiments of the uses of the present invention, the ligand is one or more GalNAc derivatives attached using a multivalent branched linkage.

[0054] In some embodiments of the uses described herein, the ligand is one or more GalNAc derivatives attached using a bivalent, trivalent, or tetravalent branched linkage.

[0055] In some embodiments of the uses of the present invention, the ligand is a GalNAc derivative attached using a divalent, trivalent, or tetravalent branched linkage.

[0056] On the other hand, the present invention relates to a double-stranded siRNA, a conjugate or a salt thereof, comprising a sense strand as shown in SEQ ID NO: 1; wherein the length of the sense strand does not exceed 23 nucleotides, and the sequence information of SEQ ID NO: 1 is detailed in the sense strand in Table A of the present invention.

[0057] In some embodiments, the double-stranded siRNA, conjugate or salt thereof of the present invention further comprises an antisense strand represented by SEQ ID NO: 3 to SEQ ID NO: 5, wherein the length of the antisense strand does not exceed 25 nucleotides. The sequence information of SEQ ID NO: 3 to SEQ ID NO: 5 is detailed in the antisense strand in Table A of the present invention.

[0058] In some embodiments, the double-stranded siRNA, conjugate, or salt thereof of the present invention comprises one of the double-stranded siRNAs shown in siRNA ID NO: 1 to siRNA ID NO: 3; wherein the length of the sense strand does not exceed 23 nucleotides, and the length of the antisense strand does not exceed 25 nucleotides. The sequence information of siRNA ID NO: 1 to siRNA ID NO: 3 is detailed in Table A of the present invention.

[0059] In another aspect, the present invention relates to a double-stranded siRNA, a conjugate or a salt thereof, which comprises a sense strand and an antisense strand and has at least one nucleotide residue described in the present invention embedded therein.

[0060] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof, the siRNA, its conjugate or salt is used to inhibit the expression of pathogenic genes.

[0061] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof, the pathogenic gene is selected from the group consisting of AGT gene, HBV gene, INHBE gene, HSD17B13 gene and PNPLA3 gene.

[0062] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof, the nucleotide residues are used to enhance the inhibitory activity of the siRNA and / or reduce off-target effects.

[0063] In some embodiments of the double-stranded siRNA, conjugate or salt thereof, the nucleotide residue is embedded in the antisense strand.

[0064] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof, the nucleotide residue is embedded at the 5th, 6th or 7th position of the 5' end of the antisense strand.

[0065] In some embodiments of the double-stranded siRNA, its conjugate or salt of the invention, the nucleotide residue is

[0066] In some embodiments, the sense strand of the double-stranded siRNA, conjugate or salt thereof of the present invention is no longer than 19, 20, 21, 22 or 23 nucleotides.

[0067] In some embodiments, the antisense strand in a double-stranded siRNA, conjugate or salt thereof of the present invention is no longer than 19, 20, 21, 22, 23, 24 or 25 nucleotides.

[0068] In another aspect, the present invention relates to a pharmaceutical composition comprising the double-stranded siRNA, its conjugate or salt thereof according to the present invention, and a pharmaceutically acceptable carrier.

[0069] In another aspect, the present invention relates to use of the double-stranded siRNA, its conjugate and salt thereof or the pharmaceutical composition of the present invention in the preparation of a medicament for treating and / or preventing AGT-related diseases.

[0070] In some embodiments of the use described herein, the AGT-related disease is hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vasculopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, ischemic heart disease, cardiomyopathy infarction, angina pectoris, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth restriction, fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis, non-alcoholic fatty liver, glucose intolerance, type 2 diabetes or metabolic syndrome.

[0071] In some embodiments of the use described in the present invention, the hypertension is selected from borderline hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency state, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension and unstable hypertension.

[0072] Detailed description of the present invention

[0073] Definitions and General Terms

[0074] In the present invention, the term "comprise" or "include" is an open expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.

[0075] In the present invention, the term "antisense strand (or guide strand)" includes a region that is substantially complementary to a target sequence, such as the mRNA of hepatitis B virus. "Sense strand (or follower strand)" refers to an RNAi strand that is substantially complementary to the antisense strand. The term "substantially complementary" refers to complete complementarity or at least partial complementarity, for example, the antisense strand is completely complementary to the target sequence or at least partially complementary. In the case of partial complementarity, mismatches can exist in the interior or terminal regions of the molecule, wherein the most tolerated mismatches exist in the terminal regions, for example, within 5, 4, 3 or 2 nucleotides of the 5'- and / or 3'-end of the RNAi.

[0076] It should be noted that "at least a portion of the antisense strand is substantially complementary to an mRNA" means that the antisense strand comprises a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding hepatitis B virus). Alternatively, if a polynucleotide is substantially non-interruptedly complementary to a portion of an mRNA encoding hepatitis B virus, then the antisense strand is complementary to at least a portion of the hepatitis B virus mRNA.

[0077] In the present invention, the term "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a gene encoding hepatitis B virus, including mRNA that is an RNA processing product of a primary transcription product.

[0078] In the present invention, the term "inhibiting the expression of hepatitis B virus genes" includes any level of inhibition of hepatitis B virus (HBV) genes, for example, at least partial inhibition of HBV gene expression, such as 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%. The expression of HBV genes can be evaluated based on the level of any variable related to HBV gene expression, for example, HBV mRNA level or HBV protein level. Inhibition can be evaluated by the reduction of 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 utilized in this area, for example, a baseline level before administration, or a level determined from a similar subject, cell or sample that has never been treated or treated with a control (e.g., a control or no active agent control using only a buffer).

[0079] In the present invention, "pharmaceutical composition" can refer to a composition used for treating a disease or for in vitro cell culture experiments. When used for treating a disease, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical art. All methods include the step of combining the active ingredient with an excipient that constitutes one or more auxiliary ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active siRNA with a liquid excipient, a finely divided solid excipient, or both.

[0080] In the present invention, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, the "pharmaceutically acceptable" herein means approved by federal regulatory agencies or national governments or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0081] As used herein, the term "pharmaceutically acceptable excipient" encompasses any solvent, solid excipient, diluent, or other liquid excipient, as appropriate for the particular intended dosage form. To the extent that any conventional excipient is incompatible with the ASO or siRNA conjugates of the present invention, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is contemplated by the present invention.

[0082] In the present invention, the term "treatment" refers to the use of a pharmacological and / or physiological effect to obtain the desired effect. The effect may be preventive in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. The "treatment" used in the present invention covers diseases in mammals, especially humans, including: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the development of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. The "treatment" used in the present invention covers any medication that administers a drug, RNAi agent 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 agent, siRNA or siRNA conjugate of the present invention to an individual in need.

[0083] As used herein, the term "RNAi agent" refers to an agent containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that are capable of degrading or inhibiting the transcription and translation of target messenger RNA (mRNA) in a sequence-specific manner. The RNAi agent herein may be manipulated via an RNA interference mechanism (i.e., by inducing RNA interference through interaction with the RNA interference pathway machinery of mammalian cells (RNA-induced silencing complex or RISC)), or may act via any other mechanism or pathway. RNAi agents include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrates.

[0084] In the context of the present invention, Bz represents benzoyl; MMTr represents 4'-methoxytrityl; DMTr represents 4',4'-dimethoxytriphenylmethyl.

[0085] In the present invention, "phosphate group," "phosphate group," and "phosphate bond" are used interchangeably to refer to monoesters, phosphodiesters, or phosphotriesters. The term "phosphate group" in "phosphorothioate group" also has the same meaning. Unless otherwise specified, a natural internucleotide phosphate group is a phosphodiester group.

[0086] The term "oxo" refers to a =0 group, for example, a carbon atom connected to an oxygen atom via a double bond, whereby a ketone or aldehyde group is formed.

[0087] As used herein, "chemically modified" or "modification" refers to a structure that is chemically different when compared to its naturally occurring counterpart, including all changes by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0088] The compounds of the present invention may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. Compounds of the present invention containing asymmetric carbon atoms can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

[0089] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the invention is desired, 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 pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), diastereomeric salts are formed with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and the pure enantiomers are recovered. In addition, separation of enantiomers and diastereomers is typically accomplished by using chromatography using a chiral stationary phase, optionally combined with chemical derivatization (e.g., carbamate formation from an amine).

[0090] The present invention also includes isotopically labeled compounds of the present invention that are identical to those described herein, but in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as2 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 et al.

[0091] Unless otherwise stated, when a position is particularly designated as deuterium (D), the position should be understood to have at least 1000 times the abundance of deuterium greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium incorporation). In the example, the compound has at least 1000 times the abundance of deuterium, at least 2000 times the abundance of deuterium, at least 3000 times the abundance of deuterium, at least 4000 times the abundance of deuterium, at least 5000 times the abundance of deuterium, at least 6000 times the abundance of deuterium or more abundant deuterium. The present invention also includes various deuterated forms of formula (I) compounds. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of formula (I) compounds with reference to relevant literature. In preparing deuterated forms of compounds of formula (I), commercially available deuterated starting materials may be used, or they may be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

[0092] The conjugated groups of the present invention can enhance the delivery of therapeutic agents to specific target locations (e.g., specific organs or tissues) within a subject such as a human or animal. In some embodiments of the present invention, the conjugated groups can enhance the targeted delivery of expression-inhibitory oligonucleotides. In some embodiments of the present invention, the conjugated groups can enhance the delivery of expression-inhibitory oligonucleotides to the liver.

[0093] The conjugated groups of the present invention can be directly or indirectly connected to a compound, such as a therapeutic agent, for example, an expression inhibitory oligonucleotide, for example, the 3' or 5' end of an expression inhibitory oligonucleotide. In some embodiments of the present invention, the expression inhibitory oligonucleotide comprises one or more modified nucleotides. In some embodiments of the present invention, the expression inhibitory oligonucleotide is an RNAi agent, such as a double-stranded RNAi agent comprising a sense strand and an antisense strand. In some embodiments of the present invention, the conjugated groups disclosed in the present invention are connected to the 3' end of the sense strand of the double-stranded RNAi agent. In some embodiments, the conjugated groups disclosed in the present invention are connected to the expression inhibitory oligonucleotide agent at the 3' end of the sense strand of the double-stranded RNAi agent via a phosphate, phosphorothioate or phosphonate group.

[0094] The definitions and conventions of stereochemistry used herein are generally those of 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 the present invention may contain asymmetric centers or chiral centers and therefore exist as different stereoisomers. All stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers, atropisomers, and mixtures thereof, such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound, the prefix D, L or R, S is used to indicate the absolute configuration of the molecule about its chiral center. The prefixes d, l, or (+), (-) are used to designate the sign of rotation of plane-polarized light in a compound. (-) or l means the compound is levorotatory, and the prefix (+) or d means the compound is dextrorotatory. These stereoisomers have the same chemical structure, but their stereostructures are different. Specific stereoisomers can be enantiomers, and a mixture of isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may result in a lack of stereoselectivity or stereospecificity during chemical reactions. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers that lacks optical activity.

[0095] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (i.e., prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerizations.

[0096] The term "composition" refers to a mixture of one or more compounds of the present invention, or their physiologically acceptable salts or precursors, with other chemical components, as well as other components such as physiologically acceptable carriers and excipients. The purpose of a composition is to facilitate administration to an organism, promote absorption of the active ingredient, and thereby exert its biological activity.

[0097] The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, 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 as acceptable for use in humans or domestic animals.

[0098] Unless otherwise specified, the "compound," "ligand," "nucleic acid conjugate," "double-stranded siRNA conjugate," "double-stranded siRNA," and "nucleic acid" of the present invention may independently exist in the form of a salt, a mixed salt, or a non-salt form (e.g., a free acid or a free base). When present in the form of a salt or a mixed salt, it may be a pharmaceutically acceptable salt.

[0099] The term "acceptable salt" includes acceptable acid addition salts and pharmaceutically acceptable base addition salts. "Acceptable acid addition salts" refers to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, octanoates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamate, pyroglutamate, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbate, salicylates, 4-aminosalicylates, and naphthalene disulfonates. These salts can be prepared by methods known in the art.

[0100] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, following salts: primary amines, secondary amines and tertiary amines, substituted amines, including natural 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, methylglucamine, 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.

[0101] As described herein, the compounds of the present invention may optionally be substituted with one or more substituents, as described above for the general formula compounds, or as described in the specific examples, subclasses, and classes of compounds encompassed by the present invention. Generally, the term "substituted" refers to the replacement of one or more hydrogen atoms in a given structure with a specified substituent. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group. When more than one position in a given formula can be substituted with one or more substituents selected from a specified group, the substituents may be the same or different at each position.

[0102] In addition, it should be noted that, unless otherwise explicitly stated, the descriptions used throughout the present invention of "each ... and ... are independently", "... and ... are each independently", and "... and ... are respectively independently" are interchangeable and should be understood in a broad sense. They can mean that in different groups, the specific options expressed by the same symbols do not affect each other, or that in the same group, the specific options expressed by the same symbols do not affect each other.

[0103] In the present invention, the term "optionally", "optional" or "optionally" generally means that the event or situation described later may but may not occur, and the description includes cases where the event or situation occurs, as well as cases where the event or situation does not occur. For example, the "-*O(CH2) n1 -and-*(CH2) n2- are each independently optionally substituted with 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy" represents -*O(CH2) n1 -and-*(CH2) n2 - are each independently unsubstituted or substituted with 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy, and the substituents may be the same or different.

[0104] In various parts of this specification, substituents of compounds disclosed herein are disclosed in terms of group types or ranges. It is specifically noted that the present invention includes every independent subcombination of the individual members of these group types and ranges. For example, the term "C 1-6 "Alkyl" specifically refers to methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl and C6 alkyl as disclosed independently.

[0105] The term "alkyl" as used herein includes saturated linear or branched monovalent hydrocarbon groups of 1 to 20 carbon atoms, wherein the alkyl group may be independently and optionally substituted with one or more substituents described herein. In some embodiments, the alkyl group contains 1 to 12 carbon atoms, in other embodiments, the alkyl group contains 1 to 10 carbon atoms, in other embodiments, the alkyl group contains 1 to 8 carbon atoms, in other embodiments, the alkyl group contains 1 to 6 carbon atoms, in other embodiments, the alkyl group contains 1 to 4 carbon atoms, and in other embodiments, the alkyl group contains 1 to 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-decyl, and the like.

[0106] The term "alkylene" refers to a saturated divalent or polyvalent hydrocarbon radical resulting from removing two carbon atoms from a saturated straight or branched hydrocarbon radical. Unless otherwise specified, an alkylene group contains 1 to 12 carbon atoms. In some embodiments, an alkylene group contains 1 to 6 carbon atoms; in other embodiments, an alkylene group contains 1 to 4 carbon atoms; in still other embodiments, an alkylene group contains 1 to 3 carbon atoms; and in still other embodiments, an alkylene group contains 1 to 2 carbon atoms. Examples of alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), isopropylene (-CH(CH3)CH2-), n-propylene (-CH2CH2CH2-), n-butylene (-CH2CH2CH2CH2-), sec-butylene (s-Bu, -CH(CH3)CH2CH2-), n-pentylene (-CH2CH2CH2CH2CH2-), 2-pentylene (-CH(CH3)CH2CH2CH2-), n-hexylene (-CH2CH2CH2CH2CH2CH2-), 2-hexyl (-CH(CH3)CH2CH2CH2CH2-), n-heptylene (-(CH2)7-), n-octylene (-(CH2)8-), n-nonylene (-(CH2)9-), hexylene (-(CH2) 10 -), -(CH2) 11 -、-(CH2) 12 -、-(CH2) 13 -、-(CH2) 14 -,etc.

[0107] The term "heteroalkyl" as used herein refers to an alkyl group having 1, 2, 3, 4, 5 or 6 heteroatoms inserted therein, wherein the heteroatoms are selected from S, N, NH, O or P, and the sulfur atom may be optionally oxidized to S-oxide. In some embodiments, the heteroalkyl group contains 1 to 12 carbon atoms, in other embodiments, the heteroalkyl group contains 1 to 10 carbon atoms, in other embodiments, the heteroalkyl group contains 1 to 8 carbon atoms, in other embodiments, the heteroalkyl group contains 1 to 6 carbon atoms, in other embodiments, the heteroalkyl group contains 1 to 4 carbon atoms, and in other embodiments, the heteroalkyl group contains 1 to 3 carbon atoms. Examples of heteroalkyl groups include, but are not limited to, CH3O-, CH3CH2O-, CH3CH2CH2O-, CH3CH2CH2CH2O-, CH3CH2OCH2-, CH3OCH2CH2OCH2-, CH3CH2OCH2CH2-, CH3OCH2CH2CH2-, CH3S-, CH3CH2S-, CH3CH2CH2S-, CH3CH2CH2CH2S-, CH3CH2SCH2-, CH3SCH2CH2-, CH3CH2CH2SCH2-, CH3CH2SCH2CH2-, CH3SCH2CH2CH2-, CH3NH-, CH3CH2NH-, CH3CH2CH2NH-, CH 3CH2CH2CH2NH-, CH3CH2NHCH2-, CH3NHCH2CH2-, CH3CH2CH2NHCH2-, CH3CH2NHCH2CH2-, CH3NHCH2CH2CH2-, etc.

[0108] The term "alkoxy" refers to an alkyl group attached to the remainder of a molecule through an oxygen atom, wherein the alkyl group has the meaning as described herein. 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 other embodiments, the alkoxy group contains 1-4 carbon atoms; and in yet other embodiments, the alkoxy group contains 1-3 carbon atoms. The alkoxy group may be optionally substituted with one or more substituents as described herein.

[0109] 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-1-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-pentyloxy (n-pentyloxy, -OCH2CH2CH2CH2CH3), 2-pentyloxy (-OCH(CH3)CH2CH2CH3), 3-pentyloxy (-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) and the like.

[0110] The term "alkenyl" refers to a linear or branched monovalent, divalent or polyvalent hydrocarbon radical containing 2 to 12 carbon atoms, or 2 to 8 carbon atoms, or 2 to 6 carbon atoms, or 2 to 4 carbon atoms, wherein at least one position of CC is sp 2 Double bond, wherein the alkenyl group can be independently unsubstituted or substituted with one or more substituents described in the present invention, including "cis", "trans" or "Z", "E" isomers, specific examples of which include, but are not limited to, vinyl (-CH=CH2), propenyl (-CH=CHCH3), allyl (-CH2CH=CH2) and the like, wherein the alkenyl group can be independently unsubstituted or substituted with one or more substituents described in the present invention.

[0111] The term "consisting of M-M1 ring atoms" means that the cyclic group is composed of M-M1 ring atoms, wherein the ring atoms include carbon atoms and / or heteroatoms such as O, N, S, and P. For example, "a heterocyclic group consisting of 3-6 ring atoms" means a monoheterocyclic group consisting of 3, 4, 5, or 6 ring atoms.

[0112] The term "heterocyclyl" refers to a non-aromatic, saturated or partially unsaturated monocyclic, bicyclic or tricyclic ring system containing 3-12 ring atoms, wherein at least one ring atom is selected from nitrogen, sulfur or oxygen atoms. The heterocyclyl group may be optionally substituted with one or more substituents described herein. Unless otherwise specified, the heterocyclyl group may be carbonyl or nitrogenyl, and the -CH2- group may be optionally replaced by -C(=O)- or -C(=S)-. The sulfur atom of the ring may be optionally oxidized to an S-oxide. The nitrogen atom of the ring may be optionally oxidized to an N-oxide. In some embodiments, the heterocyclyl group is a heterocyclyl group consisting of 3-12 ring atoms, in some embodiments, the heterocyclyl group is a heterocyclyl group consisting of 5-10 ring atoms, in some embodiments, the heterocyclyl group is a heterocyclyl group consisting of 3-6 ring atoms, in some embodiments, the heterocyclyl group is a heterocyclyl group consisting of 4-6 ring atoms, in some embodiments, the heterocyclyl group is a heterocyclyl group consisting of 5-6 ring atoms, in other embodiments, the heterocyclyl group is a heterocyclyl group consisting 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 other embodiments, the heterocyclyl group is a heterocyclyl group consisting 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 6-atom heterocyclic group, which refers to a monovalent or polyvalent, saturated or partially unsaturated, non-aromatic monocyclic ring containing 6 ring atoms, wherein at least one ring atom is selected from nitrogen, sulfur and oxygen atoms. "Heterocyclic group" also includes groups formed by condensing a heterocyclic group with a saturated or partially unsaturated ring or heterocyclic ring. Examples of heterocyclic rings include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, glycidyl, azepanyl, oxetanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3- pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolane, pyrazolinyl, dithianyl, dithiolanyl, dihydrothienyl, pyrazolidinylimidazolinyl, imidazolidinyl, 1,2,3,4-tetrahydroisoquinolinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, 3H-indolylquinolizinyl and N-pyridylurea.Examples of heterocyclic groups also include 1,1-dioxothiomorpholinyl, examples of which the carbon atoms on the ring are replaced by oxo (=O) include, but are not limited to, pyrimidinedione, 1,2,4-thiadiazole-5(4H)-one, 1,2,4-oxadiazole-5(4H)-one, 1H-1,2,4-triazole-5(4H)-one, etc., examples of which the carbon atoms on the ring are replaced by a group =S include, but are not limited to, 1,2,4-oxadiazole-5(4H)-thione, 1,3,4-oxadiazole-2(3H)-thione, etc.

[0113] The term "heteroatom" refers to one or more O, S, N, P and Si, including N, S and P in any oxidation state; in the form of primary, secondary, tertiary amines and quaternary ammonium salts; or in the form of a nitrogen atom in a heterocyclic ring in which the hydrogen is substituted, for example, N (such as N in 3,4-dihydro-2H-pyrrolyl), NH (such as NH in pyrrolidinyl) or NR (such as NR in N-substituted pyrrolidinyl, wherein R represents a substituent as described herein).

[0114] The terms "alkylsilyl" and "alkylsilyl" refer to silyl (-SiH3) groups in which the hydrogen atoms are independently replaced by 1, 2, or 3 alkyl groups. In some embodiments, the alkylsilyl group is 1, 2, or 3 C 1-12 In other embodiments, the alkylsilyl group is 1, 2 or 3 C 1-9 In other embodiments, the alkylsilyl group is 1, 2 or 3 C 1-6 In other embodiments, the alkylsilyl group is 1, 2 or 3 C 1-4 Alkyl groups are connected to silicon atoms to form lower alkylsilyl groups. In other embodiments, alkylsilyl groups are 1, 2 or 3 C 1-3 An alkyl group is a lower alkylsilyl group formed by connecting an alkyl group to a silicon atom. 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), and the like.

[0115] The term "hydroxy protecting group" refers to an unstable chemical moiety that protects the hydroxyl group from undesirable reactions during one or more synthesis procedures. After the one or more synthesis procedures, the hydroxy protecting group can be selectively removed. Hydroxy protecting groups as 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 hydroxy protecting groups of the present invention include, but are not limited to, C 1-10 alkylmethyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl, isopropyloxycarbonyl, 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 (such as benzyl, phenethyl, etc.), p-methoxybenzyldiphenylmethyl, triphenylmethyl (triphenylmethyl or trityl), tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, C 1-10 Alkylsilyl (such as trimethylsilyl (TMS or -Si(CH3)3)), triethylsilyl, triisopropylsilyl, MMTr, DMTr or 4',4',4'-trimethoxytrityl, etc.

[0116] The term "amino protecting group" refers to an unstable chemical moiety that protects an amino group from undesirable reactions during a synthetic procedure. After the one or more synthetic procedures, the amino protecting group as described herein can be selectively removed. Amino protecting groups as 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, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, phenyl Formyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, benzyl, p-methoxybenzyldiphenylmethyl, trityl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, C 1-6 Alkoxy (such as methoxy, ethoxy, 1-propoxy, 2-propoxy, etc.) or R 4 C(=O)-, etc., wherein R 4 has the meanings described in the present invention.

[0117] Term " solid phase support " specifically represents any particle, pearl or the surface on which oligonucleotide synthesis can occur.For example inorganic solid phase support and organic solid phase support, all can be selected for use in embodiments of the present invention.Inorganic solid phase support is preferably selected from silica gel and controlled pore glass beads (Controlled-pore glass is called for short CPG).Organic solid phase support is a resin, preferably macroporous resin, more preferably highly cross-linked polystyrene, Tentagel (graft copolymer made up of low cross-linked polystyrene matrix, polyethylene glycol (PEG or POE) is grafted thereon), polyvinyl acetate (PVA), copolymer, aminopolyethylene glycol and cellulose etc. of Bo Luosi (Poros)-polystyrene / divinylbenzene.Preferred embodiments of the present invention utilize solid phase support based on CPG.Many other purchasable solid phase supports all belong to the present invention.

[0118] Unless otherwise stated, in the event of any conflict or inconsistency between the sequence information in the specification of the present invention and the sequence information in the sequence listing (ST26 Sequence Listing), the sequence information described in the specification shall prevail. Description of the drawings:

[0119] Figure 1 shows the inhibitory effects of the siRNA conjugates of the present invention and a vehicle control group (normal saline) on AGT in an hAGT transgenic mouse model. siRNA1, siRNA2, and siRNA3 represent siRNA conjugate IDs 1-3, respectively, and their corresponding sequences are detailed in Table A. Vehicle represents the vehicle.

[0120] Figures 2-1, 2-2, 2-3, and 2-4 represent off-target effect plots for siRNA1, siRNA2, and siRNA3 conjugates, respectively, and the positive control. siRNA1, siRNA2, and siRNA3 in Figures 2-1, 2-2, and 2-3 correspond to siRNA conjugate ID No. 1, siRNA conjugate ID No. 2, and siRNA conjugate ID No. 3 in Table A, respectively. The positive control in Figure 2-4 corresponds to the positive control in Table A. "Down" represents knockdown; "Not sig" represents a significant difference; "Up" represents upregulation; and "vehicle" represents vehicle.

[0121] Detailed description of the compounds of the present invention

[0122] The present invention provides a novel compound and its use. The compound can be used as a raw material for solid-phase synthesis of DNA nucleotides, as a raw material for the synthesis of oligonucleotide drugs, and / or as a raw material for the synthesis of siRNA drugs. It can also be used for siRNA drug research, gene function research, and / or screening of whole gene libraries. In particular, it can be used as a raw material for the synthesis of ASO drugs and / or double-stranded siRNA drugs. The present invention also relates to a novel nucleotide residue and its use as an intercalating group in ASO drug research, siRNA drug research, gene function research, and screening of whole gene libraries. Experiments have shown that ASO drugs, siRNA double strands and conjugates thereof embedded with the nucleotide residues of the present invention have significant inhibitory activity and / or low toxicity on the expression of target genes (such as HBV (hepatitis B virus) gene, AGT (angiotensinogen) gene, Lp(a) (lipoprotein(a)) gene, ANGPTL3 (angiopoietin-like 3) gene, APOC3 (apolipoprotein C-III) gene), PD-L1 (programmed cell death protein ligand 1) gene, HSD17B (17β-hydroxysteroid dehydrogenase) etc.).

[0123] In one aspect, the present invention provides a compound having a structure as shown in formula (I), or a stereoisomer, tautomer or acceptable salt of the structure as shown in formula (I),

[0124] Among them, each L1, R, Z, Y 1 and B have the meanings as defined in the present invention.

[0125] In some embodiments of the compounds described herein, L1 is -*O(CH2) n1 -or-*(CH2) n2 -, R is H, deuterium, fluorine, chlorine, bromine, iodine, methyl or ethyl, wherein -*O(CH2) n1 -and-*(CH2) n2 - are each independently optionally substituted with 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy, wherein n1 is 1, 2, 3, 4, 5, 6 or 7, and n2 is 2, 3, 4, 5, 6 or 7.

[0126] In some embodiments of the compounds described herein, L1 is -*CH2-, wherein -*CH2- is optionally substituted with 1, 2, or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy, and ethoxy, and R is H, deuterium, fluorine, chlorine, bromine, iodine, methyl, or ethyl.

[0127] In some embodiments of the compounds described herein, Z and Y 1 are independently H, deuterium, a hydroxyl protecting group, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) j C(═O)—, a phosphate group, a thiophosphate group, a phosphite amide group or a hydrogen phosphate group, wherein each j and M has the meanings described in the present invention.

[0128] Any active phosphate group, such as a phosphate group, a thiophosphate group, a phosphoamido group, or a hydrogen phosphate group, that enables the compounds of the present invention to participate in oligonucleotide synthesis is encompassed by the present invention. Phosphates herein include phosphodiesters and triesters; thiophosphates include phosphodiesters and triesters; and hydrogen phosphate groups refer to salts formed by phosphate groups with bases.

[0129] In some embodiments of the compounds described in the present 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.

[0130] In some embodiments of the compounds described herein, B is Each R 1a 、R 1b 、R 2a 、R 2b 、R 3a 、R 4a and R 4b is independently deuterium, H, CN, HO-, fluorine, chlorine, bromine, methyl, ethyl or methoxy.

[0131] In some embodiments of the compounds described herein, each R 2 、R 3b and R 4c Independently -NHR 1 or -N=CH-NR a R b , where each R 1 、R a and R b has the meaning as described in the present invention.

[0132] In some embodiments of the compounds described herein, R 1 is an amino protecting group.

[0133] In some embodiments of the compounds described herein, each R a and R b are independently H or an amino protecting group, or R a 、R b Together with the nitrogen atom to which they are attached, they form a heterocyclic group consisting of 5-6 ring atoms, and each heterocyclic group consisting of 5-6 ring atoms is independently 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.

[0134] In some embodiments of the compounds described herein, each j is independently 1, 2, 3, 4, or 5.

[0135] In some embodiments of the compounds described herein, Z and Y 1 are independently H, deuterium, C 1-12 Alkyl, C 1-12 Alkyl C(=O)-, C 1-12 Alkylmethyl, C 1-12 Alkylsilyl, C 6-10 Aryl C 1-6 Alkyl, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) jC(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, Among them, j, M, X, R x 、R y 、R c and R d has the meanings described in the present invention.

[0136] In some embodiments of the compounds described herein, Z and Y 1 and each is independently 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-methoxybenzyldiphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methylsulfonyl, p-toluenesulfonyl, C 1-10 Alkyl, C 1-10 Alkyl C(=O)-, C 1-10 Alkylmethyl, C 1-10 Alkylsilyl, C 6-10 Aryl C 1-4 Alkyl, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, Among them, j, M, X, R x 、R y 、R c and R d has the meanings described in the present invention.

[0137] In some embodiments of the compounds described herein, Z and Y 1and each is independently 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-methoxybenzyldiphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methylsulfonyl, p-toluenesulfonyl, C 1-8 Alkyl, C 1-8 Alkyl C(=O)-, C 1-8 Alkylmethyl, C 1-8 Alkylsilyl, phenyl C 1-3 Alkyl, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, Among them, j, M, X, R x 、R y 、R c and R d has the meanings described in the present invention.

[0138] In some embodiments of the compounds described herein, Z and Y 1 and each is independently 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-methoxybenzyldiphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methylsulfonyl, p-toluenesulfonyl, C 1-6 Alkyl, C 1-6 Alkyl C(=O)-, C 1-6 Alkylmethyl, C 1-6 Alkylsilyl, benzyl, phenethyl, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) jC(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, Among them, j, M, X, R x 、R y 、R c and R d has the meanings described in the present invention.

[0139] In some embodiments of the compounds described herein, X is Cl or Br.

[0140] In some embodiments of the compounds described herein, each R x and R y is independently a hydroxy protecting group.

[0141] In some embodiments of the compounds described herein, each R c and R d are independently H or an amino protecting group; or R c 、R d Together with the nitrogen atom to which they are attached, they form a heterocyclic group consisting of 5-6 ring atoms, and the heterocyclic group consisting of 5-6 ring atoms is optionally substituted by 1, 2, 3 or 4 substituents selected from deuterium, hydroxy, cyano, F, Cl, Br, I, methyl, ethyl, propyl, methoxy, ethoxy, 1-propoxy and 2-propoxy.

[0142] In some embodiments of the compounds described herein, R 1 methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, 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-methoxybenzyldiphenylmethyl, trityl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, C 1-6 Alkoxy (such as methoxy, ethoxy, 1-propoxy, 2-propoxy, etc.) or R 3 C(=O)-, where R 3 has the meanings described in the present invention.

[0143] In some embodiments of the compounds described herein, each Ra and R b are independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, 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-methoxybenzyldiphenylmethyl, trityl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, C 1-6 Alkoxy (such as methoxy, ethoxy, 1-propoxy, 2-propoxy, etc.) or R 4 C(=O)-, or R a 、R b and together with the nitrogen atom to which they are attached form a pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl group, said pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl being independently optionally substituted with 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy groups, wherein R 4 has the meanings described in the present invention.

[0144] In some embodiments of the compounds described herein, each R a and R b are 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-methoxybenzyldiphenylmethyl, trityl, 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 、Rb and together with the nitrogen atom to which they are attached form a pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl group, said pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl being independently optionally substituted with 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy groups, wherein R 4 has the meanings described in the present invention.

[0145] In some embodiments of the compounds described herein, each R 3 and R 4 Independently C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, phenyl, halogen-substituted phenyl, C 1-6 Alkylphenyl or benzyl.

[0146] In some embodiments of the compounds described herein, each R 3 and R 4 Independently C 1-4 Alkyl, halogenated C 1-4 Alkyl, C 1-4 Alkoxy, phenyl, halogen-substituted phenyl, C 1-4 Alkylphenyl or benzyl.

[0147] In some embodiments of the compounds described herein, each R 3 and R 4 are 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-1-propoxy, 2-butoxy, tert-butoxy, phenyl, halogen-substituted phenyl, C 1-3 Alkylphenyl or benzyl.

[0148] In some embodiments of the compounds described herein, each R x and R yare 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-methoxybenzyldiphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methylsulfonyl, p-toluenesulfonyl, cyano C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkyl C(=O)-, C 1-6 alkylsilyl, phenyl, halogen-substituted phenyl, benzyl or phenethyl.

[0149] In some embodiments of the compounds described herein, each R x and R y are 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 methyl, p-methoxybenzyldiphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methylsulfonyl, p-toluenesulfonyl, CNCH2-, CN(CH2)2-, CNC(CH3)2CH2-, CN(CH2)3-, CNC(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.

[0150] In some embodiments of the compounds described herein, each R c and R dare independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, 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-methoxybenzyldiphenylmethyl, trityl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, C 1-6 Alkoxy (such as methoxy, ethoxy, 1-propoxy, 2-propoxy, etc.) or R 4 C(=O)-, or R a 、R b Together with the N atom to which it is attached, it forms a pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl group, and the pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl groups are independently optionally substituted with 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy groups.

[0151] In some embodiments of the compounds described herein, each R c and R d are 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-methoxybenzyldiphenylmethyl, trityl, 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 bTogether with the N atom to which it is attached, it forms a pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl group, and the pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl groups are independently optionally substituted with 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy groups.

[0152] In some embodiments of the compounds of the present invention, the compounds of the present invention have a structure as shown in Formula (Ia) or (Ib), or a stereoisomer, tautomer or acceptable salt thereof,

[0153] wherein each of L1, R and B independently has the meaning described herein.

[0154] In some embodiments of the compound described herein, it is one of the following compounds 1-25, or a stereoisomer, tautomer or acceptable salt thereof,

[0155] Among them, A1 is A2 is A3 is U is T is

[0156] On the other hand, the present invention provides a nucleotide residue having a structure as shown in formula (II), or a stereoisomer, tautomer or acceptable salt of the structure as shown in formula (II),

[0157] in,

[0158] 1) L1 is -*O(CH2) n1 -or-*(CH2) n2 -, R is H, deuterium, fluorine, chlorine, bromine, iodine, methyl or ethyl, wherein -*O(CH2) n1 -and-*(CH2) n2 - are each independently optionally substituted with 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy, wherein n1 is 1, 2, 3, 4, 5, 6 or 7, and n2 is 2, 3, 4, 5, 6 or 7; or

[0159] 2) L1 is -*CH2-, R is H, deuterium, fluorine, chlorine, bromine, iodine, methyl or ethyl, wherein -*CH2- is optionally substituted with 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy;

[0160] B1 is Each R1a 、R 1b 、R 2a 、R 2b 、R 3a 、R 4a and R 4b are independently H, deuterium, CN, HO-, fluorine, chlorine, bromine, methyl, ethyl or methoxy.

[0161] In some embodiments of the nucleotide residues of the present invention, the nucleotide residues of the present invention are one of the following nucleotide residues (1)-(20), or a stereoisomer, tautomer or acceptable salt thereof,

[0162] Among them, A is G is C is U is T is

[0163] In some preferred embodiments of the nucleotide residues of the present invention, the nucleotide residues of the present invention are (ie Y), Embedded into small nucleic acid drugs (such as siRNA and ASO, etc.), it can not only significantly improve the gene silencing activity of the drug (such as AGT), but also significantly reduce the off-target effect, thereby reducing the toxicity of the drug, that is, containing the residues of the present invention (such as Small nucleic acids (such as siRNA or siRNA conjugates or ASO, etc.) can reduce the number of downregulated or upregulated off-target genes.

[0164] In another aspect, the present invention provides use of the nucleotide residues of the present invention as intercalating groups in ASO drug research, siRNA drug research, gene function research and / or screening of whole gene libraries.

[0165] In some embodiments of the use of the present invention, the nucleotide residue is embedded as an intercalating group on the ASO drug, the sense strand of the siRNA drug, the antisense strand of the siRNA drug, or the sense strand of the siRNA drug and the antisense strand of the siRNA drug.

[0166] In another aspect, the present invention provides the use of the compounds of the present invention as raw materials for solid-phase synthesis of DNA nucleotides, as raw materials for the synthesis of oligonucleotide drugs, as raw materials for the synthesis of siRNA drugs, ASO drug research, siRNA drug research, gene function research and / or screening of whole gene libraries.

[0167] On the other hand, the present invention also provides a method for inhibiting the expression of a specific gene in cells in a patient's body, which comprises administering to the patient an ASO drug, double-stranded siRNA, double-stranded siRNA conjugate or a combination thereof embedded in the nucleoside monomer of the present invention, wherein the ASO drug, double-stranded siRNA, double-stranded siRNA conjugate or a combination thereof can be a therapeutically effective amount.

[0168] In some embodiments of the use of the present invention, the siRNA drug is a drug that inhibits the expression of the AGT gene.

[0169] In some embodiments of the use of the present invention, the siRNA drug comprises a sense strand and an antisense strand capable of forming a double-stranded region, and the sense strand consists of 15-40 nucleotides, preferably 17-25 nucleotides, and more preferably 18-23 nucleotides.

[0170] In some embodiments of the use of the present invention, the antisense strand consists of 15-40 nucleotides.

[0171] In some embodiments of the use of the present invention, the antisense strand consists of 17-35 nucleotides.

[0172] In some embodiments of the use of the present invention, the antisense strand consists of 19-30 nucleotides.

[0173] In some embodiments of the use of the present invention, the antisense strand consists of 21-29 nucleotides.

[0174] In some embodiments of the use of the present invention, the double-stranded region consists of 17-23 nucleotide base pairs.

[0175] In some embodiments of the use of the present invention, the sense strand and / or antisense strand comprises 7, 6, 5, 4, 3, 2, 1 and 0 unmodified nucleotides, respectively.

[0176] In some embodiments of the use of the present invention, the siRNA drug further comprises a ligand.

[0177] In some embodiments of the use of the present invention, the ligand is conjugated to any position of the sense strand or the antisense strand.

[0178] In some embodiments of the use of the present invention, the ligand is conjugated to the 3' end or the 5' end of the sense strand.

[0179] In some embodiments of the use of the present invention, the ligand is conjugated to the 3' end or the 5' end of the antisense strand.

[0180] In some embodiments of the uses of the present invention, the ligand is one or more GalNAc derivatives attached using a multivalent branched linkage.

[0181] In some embodiments of the uses described herein, the ligand is one or more GalNAc derivatives attached using a bivalent, trivalent, or tetravalent branched linkage.

[0182] In some embodiments of the uses of the present invention, the ligand is a GalNAc derivative attached using a divalent, trivalent, or tetravalent branched linkage.

[0183] On the other hand, the present invention relates to a double-stranded siRNA, a conjugate or a salt thereof, comprising a sense strand as shown in SEQ ID NO: 1; wherein the length of the sense strand does not exceed 23 nucleotides, and the sequence information of SEQ ID NO: 1 is detailed in the sense strand in Table A of the present invention.

[0184] In some embodiments, the double-stranded siRNA, conjugate or salt thereof of the present invention further comprises an antisense strand represented by SEQ ID NO: 3 to SEQ ID NO: 5, wherein the length of the antisense strand does not exceed 25 nucleotides. The sequence information of SEQ ID NO: 3 to SEQ ID NO: 5 is detailed in the antisense strand in Table A of the present invention.

[0185] In some embodiments, the double-stranded siRNA, conjugate, or salt thereof of the present invention comprises one of the double-stranded siRNAs shown in siRNA ID NO: 1 to siRNA ID NO: 3; wherein the length of the sense strand does not exceed 23 nucleotides, and the length of the antisense strand does not exceed 25 nucleotides. The sequence information of siRNA ID NO: 1 to siRNA ID NO: 3 is detailed in Table A of the present invention.

[0186] In another aspect, the present invention relates to a double-stranded siRNA, a conjugate or a salt thereof, which comprises a sense strand and an antisense strand and has at least one nucleotide residue described in the present invention embedded therein.

[0187] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof, the siRNA, its conjugate or salt is used to inhibit the expression of pathogenic genes.

[0188] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof, the pathogenic gene is selected from the group consisting of AGT gene, HBV gene, INHBE gene, HSD17B13 gene and PNPLA3 gene.

[0189] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof, the nucleotide residues are used to enhance the inhibitory activity of the siRNA and / or reduce off-target effects.

[0190] In some embodiments of the double-stranded siRNA, conjugate or salt thereof, the nucleotide residue is embedded in the antisense strand.

[0191] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof, the nucleotide residue is embedded at the 5th, 6th or 7th position of the 5' end of the antisense strand.

[0192] In some embodiments of the double-stranded siRNA, its conjugate or salt of the invention, the nucleotide residue is

[0193] In some embodiments, the sense strand of the double-stranded siRNA, conjugate or salt thereof of the present invention is no longer than 19, 20, 21, 22 or 23 nucleotides.

[0194] In some embodiments, the antisense strand in a double-stranded siRNA, conjugate or salt thereof of the present invention is no longer than 19, 20, 21, 22, 23, 24 or 25 nucleotides.

[0195] In another aspect, the present invention relates to a pharmaceutical composition comprising the double-stranded siRNA, its conjugate or salt thereof according to the present invention, and a pharmaceutically acceptable carrier.

[0196] In another aspect, the present invention relates to use of the double-stranded siRNA, its conjugate and salt thereof or the pharmaceutical composition of the present invention in the preparation of a medicament for treating and / or preventing AGT-related diseases.

[0197] In some embodiments of the use described herein, the AGT-related disease is hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vasculopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, ischemic heart disease, cardiomyopathy infarction, angina pectoris, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth restriction, fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis, non-alcoholic fatty liver, glucose intolerance, type 2 diabetes or metabolic syndrome.

[0198] In some embodiments of the use described in the present invention, the hypertension is selected from borderline hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency state, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension and unstable hypertension.

[0199] Preparation, administration and disease treatment methods of the ASO drug, siRNA conjugate and nucleic acid conjugate of the present invention

[0200] The effective amount of the ASO drug, nucleic acid conjugate (such as siRNA conjugate or pharmaceutical composition) of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, for example, at a dose of four times a day, three times a day, twice a day, once a day, or once every other day, or the administration of several doses per day may be proportionally reduced.

[0201] The drug can be administered to a subject by any suitable route known in the art, including but not limited to oral or parenteral routes, including intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration and topical administration (including buccal administration and sublingual administration), preferably intravenous administration.

[0202] The pharmaceutical composition disclosed in the present invention includes preparations suitable for parenteral administration. The preparation can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of the active ingredient that can be combined with excipients to prepare a single dose form is generally the amount of a nucleic acid drug (such as siRNA drug or ASO drug) that produces a therapeutic effect. Generally speaking, in percent units, this amount is about 1% to about 99% active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

[0203] In another aspect, the present invention provides a method for inhibiting the expression or activity of a target gene (e.g., the HBV (hepatitis B virus) gene, the AGT (angiotensinogen) gene, the LAP (lipoprotein(a)) gene, the ANGPTL3 (angiopoietin-like 3) gene, the APOC3 (apolipoprotein C-III) gene, etc.). According to an embodiment of the present invention, the method comprises contacting a cell with the aforementioned ASO drug, siRNA, siRNA conjugate, or pharmaceutical composition. As previously described, the aforementioned ASO drug, siRNA, and siRNA conjugate can inhibit the expression and replication of the target gene.

[0204] General synthesis methods of the compounds, double-stranded siRNA, and double-stranded siRNA conjugates of the present invention

[0205] Generally, the compounds and nucleic acid conjugates of the present invention can be prepared by the methods described herein, wherein, unless otherwise specified, the substituents are defined as shown in formulas (I) and (II). The following reaction schemes and examples are provided to further illustrate the present invention.

[0206] In the examples described below, all temperatures are in degrees Celsius (°C) unless otherwise indicated. Silica gel (200-300 mesh) was purchased from Qingdao Ocean Chemical Plant, and NH2CPG was purchased from Hebei Dinaxingke. Nuclear magnetic resonance spectra were obtained using CDC13, DMSO-d6, CD3OD, or acetone-d6 as solvents (in ppm), with TMS (0 ppm) or chloroform (7.25 ppm) as the reference standard. When multiple peaks are present, the following abbreviations are used: s (singlet), d (doublet), t (triplet), m (multiplet), br (broadened), dd (doublet of doublets), dt (doublet of triplets), br.s (broadened singlet), and q (quartet). Coupling constants, J, are expressed in Hertz (Hz).

[0207] Low-resolution mass spectrometry (MS) data were measured by an Agilent 6320 series LC-MS spectrometer equipped with a G1312A binary pump and a G1316A TCC (column temperature was maintained at 30 °C). A G1329A autosampler and a G1315B DAD detector were used for analysis, and an ESI source was applied to the LC-MS spectrometer.

[0208] High-resolution mass spectrometry (MS) data were determined by 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 applied to the HR-MS spectrometer. DETAILED DESCRIPTION

[0209] The scheme of the present invention will be explained below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. In particular, the synthesis of small nucleic acids and the synthesis of nucleic acid conjugates can be synthesized according to the embodiments of the present invention or routine adjustments in the art. If specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0210] Synthesis method of the compound of the present invention

[0211] The following synthesis scheme lists the general experimental steps for preparing the compounds disclosed in the present invention. Those skilled in the art can make appropriate modifications to the method or adjustment of the raw materials according to actual conditions to prepare the compounds of the present invention.

[0212] The following abbreviations are used throughout this invention:

[0213] Synthesis of nucleoside monomers DAW50072-a and DAW50072-b of the present invention

[0214] Step 1: Synthesis of DAW50072-1

[0215] Dissolve 3-buten-2-ol (1.5 g, 20.39 mmol) and 4-dimethylaminopyridine (2.49 g, 20.39 mmol) in DCM (150 mL). Add tert-butyldiphenylsilyl chloride (6.73 g, 24.47 mmol) with stirring and allow to react overnight at room temperature. The reaction solution is concentrated, and the residue is purified by silica gel column chromatography (petroleum ether) to obtain DAW50072 (4.975 g, yield: 78.60%) as a clear oil.

[0216] 1H NMR (599MHz, CDCl3) δ (ppm) 7.71–7.66 (m, 4H), 7.44–7.40 (m, 2H), 7.36 (dt, J = 7.9, 4.1Hz, 4H), 5.86 (ddd, J = 17.1, 10.4, 5.4 Hz,1H),5.10(dt,J=17.2,1.6Hz,1H),4.95(dt,J=10.4,1.5Hz,1H),4.34–4.27(m,1H),1.14(d,J=6.3Hz,3H),1.07(s,9H).

[0217] Step 2: Synthesis of DAW50072-2

[0218] DAW50072-1 (4.975 g, 16.02 mmol) was dissolved in DCM (100 mL) and m-chloroperbenzoic acid (5.20 g, 25.63 mmol) was added with stirring. The mixture was allowed to react at room temperature overnight. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (PE / EA (v / v) = 20 / 1) to obtain DAW50072-2 (4.53 g, yield: 86.30%) as a clear oil.

[0219] 1 H NMR (599MHz, CDCl3) δ (ppm) 7.73 (ddd, J=11.1, 8.0, 1.4Hz, 4H), 7.46–7.37 (m, 6H), 3.67–3.61 (m, 1H), 3.05 (dd d,J=5.9,4.1,2.8Hz,1H),2.75–2.70(m,1H),2.53(dd,J=5.0,2.7Hz,1H),1.12(d,J=6.4Hz,3H),1.10(s,9H).

[0220] Step 3: Synthesis of DAW50072-3

[0221] DAW50072-2 (4.15 g, 12.71 mmol) and uracil (1.57 g, 13.98 mmol) were dissolved in DMF (50 mL) and cooled to -5°C. Sodium hydride (0.15 g, 3.81 mmol) was added with stirring. After stirring for 5 minutes, the mixture was brought to room temperature and then heated to 110°C for overnight reaction. The reaction was stopped, cooled to room temperature, and diluted with EA (100 mL). The resulting mixture was washed sequentially with saturated aqueous sodium bicarbonate (150 mL x 2) and saturated aqueous sodium chloride (150 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / methanol (v / v) = 20 / 1) to obtain DAW50072-3 (3.43 g, 61.53% yield) as a white foamy solid.

[0222] MS (ESI, pos.ion) m / z: 439.2 [M+H] + .

[0223] Step 4: Synthesis of DAW50072-4

[0224] DAW50072-3 (3.43 g, 7.82 mmol) was dissolved in THF (50 mL), and tetrabutylammonium fluoride (0.61 g, 2.35 mmol) was added with stirring. The mixture was allowed to react at 35°C overnight. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (DCM / methanol (v / v) = 9 / 1) to obtain DAW50072-4 (1.374 g, 87.76% yield) as a white foamy solid.

[0225] MS (ESI, pos.ion) m / z: 201.2 [M+H] + .

[0226] Step 5: Synthesis of DAW50072-a and DAW50072-b

[0227] Under nitrogen, DAW50072-4 (4.37 g, 21.83 mmol), 4-dimethylaminopyridine (0.27 g, 2.18 mmol), and TEA (7.28 g, 71.94 mmol) were dissolved in THF (150 mL). DMTrCl (11.09 g, 32.74 mmol) was added with stirring and allowed to react at room temperature overnight. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in DCM (150 mL) and washed with saturated sodium chloride (100 mL x 3). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by preparative chromatography to obtain DAW50072-5-a (3.60 g, yield: 32.82%) and DAW50072-5-b (2.48 g, yield: 22.61%) as white foamy solids.

[0228] DAW50072-5-a: 1 H NMR (599MHz, CDCl3) δ (ppm) 8.73 (s, 1H), 7.47–7.43 (m, 2H), 7.34 (t, J = 5.9Hz, 4H), 7.29 (t, J = 7.6Hz, 2H), 7.23 (d, J = 7.3Hz, 1H), 6.91 (d, J=7.9Hz,1H),6.83(d,J=8.7Hz,4H),5.53(d,J=7.9Hz,1H),3.79(s,6H),3.70–3.57(m,4H),2.66(d,J=7.2Hz,1H),1.18(d,J=6.5Hz,3H).

[0229] DAW50072-5-b: 1 H NMR (599MHz, CDCl3) δ (ppm) 9.13 (s, 1H), 7.47 (d, J = 7.4Hz, 2H), 7.36 (t, J = 9.4Hz, 4H), 7.2 9–7.25(m,2H),7.21(d,J=7.3Hz,1H),7.12(d,J=7.9Hz,1H),6.82(dd,J=8.8,1.0Hz,4H), 5.57(d,J=7.9Hz,1H),3.87(dd,J=14.1,1.5Hz,1H),3.78(d,J=2.1Hz,6H),3.64(dd,J=14 .1,7.8Hz,1H),3.45(dd,J=8.9,5.1Hz,2H),2.96(d,J=3.8Hz,1H),1.04(d,J=5.9Hz,3H).

[0230] Step 6: Synthesis of DAW50072-a

[0231] DAW50072-5-a (0.5 g, 0.99 mmol), 1H-tetrazole (86 mg, 1.20 mmol), and TEA (0.31 g, 3.02 mmol) were dissolved in DCM (20 mL). Bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.51 g, 1.68 mmol) was added with stirring, and the reaction mixture was allowed to react at room temperature for 3.5 h. After completion of the reaction, the mixture was diluted with DCM (20 mL), washed with saturated sodium chloride solution (40 mL x 2), dried over anhydrous sodium sulfate, and the solvent was concentrated. The residue was purified by C18 reverse-phase column chromatography (acetonitrile / water (v / v) = 70 / 30) to obtain DAW50072-a (330 mg, 47.20% yield) as a white solid.

[0232] 1 H NMR (599MHz, CDCl3) δ (ppm) 8.00 (s, 1H), 7.40–7.37 (m, 2H), 7.31–7.23 (m, 6H), 7.20 (d, J = 7.3Hz, 1H),7.01(d,J=7.9Hz,1H),6.81–6.77(m,4H),5.41(d,J=7.9Hz,1H),4.06(dd,J=13.9,3.2Hz,1H) ,4.02–3.97(m,1H),3.90–3.85(m,1H),3.78(s,6H),3.73–3.68(m,2H),3.48(ddd,J=13.7,9.5,7 .0Hz,3H),2.52–2.45(m,2H),1.35(d,J=6.5Hz,3H),1.13(d,J=6.8Hz,6H),0.99(d,J=6.8Hz,6H).

[0233] 31 P NMR (243MHz, CDCl3) δ147.04(s), 146.42(s).

[0234] Step 7: Synthesize DAW50072-b

[0235] DAW50072-5-b (0.5 g, 0.99 mmol), 1H-tetrazole (86 mg, 1.20 mmol), and TEA (0.31 g, 3.02 mmol) were dissolved in DCM (20 mL). Bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.51 g, 1.68 mmol) was added with stirring and allowed to react at room temperature for 3.5 h. After completion of the reaction, the mixture was diluted with DCM (20 mL), washed with saturated sodium chloride (40 mL x 2), dried over anhydrous sodium sulfate, and the solvent was concentrated. Purification was performed by C18 reverse-phase column chromatography using acetonitrile / water = 70 / 30 as the eluent to afford DAW50072-b (350 mg, 50.0% yield) as a white solid.

[0236] 1H NMR (400MHz, CDCl3) δ8.61(s,1H),7.47(d,J=7.4Hz,2H),7.35(dd,J=8.8,5.0Hz,4H),7.31–7.24( m,2H),7.21(t,J=7.1Hz,1H),7.16(d,J=7.9Hz,1H),6.82(dd,J=8.8,1.7Hz,4H),5.63(d,J=7.9Hz, 1H),4.24(dd,J=13.9,2.5Hz,1H),3.89–3.63(m,10H),3.52(ddd,J=13.3,10.9,6.9Hz,3H),2.41( q,J=6.4Hz,2H),1.10(t,J=7.5Hz,12H),0.96(d,J=6.4Hz,3H).MS(ESI,pos.ion)m / z:703.32[M+H] + . 31 P NMR (243MHz, CDCl3) δ149.35 (d, J=237.3Hz).

[0237] It is well known in the art that the nucleoside monomers DAW50072-a and DAW50072-b are embedded in oligonucleotides during synthesis and then deprotected to form the nucleotide residues Y and N, respectively, described in the present invention.

[0238] The synthetic method of the antisense oligonucleotide of the present invention

[0239] The synthesis specification was completed according to the theoretical yield of 1 μmol. All DNA phosphoramidite monomers, 2'-modified RNA phosphoramidite monomers, phosphoramidite monomers of the present invention (i.e., compounds described in the present invention) and auxiliary reagents of the 1 μmol solid support CPG (purchased from Hebei Dina Xingke) were weighed, and all phosphoramidite monomers were provided in 0.1M anhydrous acetonitrile solution. For oligonucleotides with phosphate backbone thiolation modification, 0.1M DDTT solution was used as the thiolation reagent. 5-Ethylthio-1H-tetrazole acetonitrile solution (0.25M) was used as an activator (purchased from Suzhou Kelema), and 3% trichloroacetic acid in dichloromethane was used as a deprotection reagent. They were placed in the designated reagent position corresponding to the DNA / RNA automatic synthesizer. The synthesis program was set up and the specified oligonucleotide base sequence was entered. After checking that everything was correct, the cyclic oligonucleotide synthesis was started. The coupling time for each step was 6 minutes, and the thiolation time was 6 minutes. After automatic circulation, an oligonucleotide containing solid support CPG was obtained.

[0240] The CPG-containing nucleotides obtained above were dried with dry argon and then transferred to a 2 mL EP tube. 28% aqueous ammonia solution (0.8 mL) was added and heated at 55° C. for 5 to 18 hours. The mixture was filtered, the filter cake washed with water (0.5 mL), and the filtrates were combined and concentrated under reduced pressure to obtain a white or yellow colloidal solid. After reverse-phase preparative purification, the prepared solution was concentrated and passed through a gel column to remove excess salts to obtain the oligonucleotides. The concentration of the resulting oligonucleotides was determined using a micro-UV spectrophotometer (SPECTRO stat Nano). Mass spectrometry analysis was performed on an Agilent 6530 LC-MS Q-Tof system. After primary scanning, the nucleic acid molecular weight was calculated after deconvolution. The above method can be appropriately adjusted in combination with synthetic methods known in the art to synthesize the oligonucleotides of the present invention.

[0241] Synthesis of double-stranded siRNA and double-stranded siRNA conjugates of the present invention

[0242] 1. Synthesis of double-stranded siRNA without conjugation groups

[0243] The steps for synthesizing the siRNA sense strand and antisense strand of the present invention are as follows:

[0244] The synthesis was completed according to the theoretical yield of 1umol. Weigh all 2'-modified RNA phosphoramidite monomers, the phosphoramidite monomers of the present invention (i.e., compounds DAW50072-a and DAW50072-b described in the present invention) and auxiliary reagents on the 1umol solid phase support CPG (purchased from Hebei Dina Xingke). All phosphoramidite monomers were provided in 0.1M anhydrous acetonitrile solution. For oligonucleotides with phosphate backbone thiolation modification, 0.1M DDTT solution was used as the thiolation reagent. 5-Ethylthio-1H-tetrazole acetonitrile solution (0.25M) was used as the activator (purchased from Suzhou Kelema), 0.02M iodine in pyridine / water solution was used as the oxidant, and 3% trichloroacetic acid in dichloromethane was used as the deprotection reagent. They were placed in the designated reagent position corresponding to the KA-H8 model DNA / RNA automatic synthesizer. Set the synthesis program and enter the specified oligonucleotide base sequence. After checking that everything is correct, start the cyclic oligonucleotide synthesis. The coupling time for each step is 6 minutes and the thiolation time is 6 minutes. After automatic circulation, the oligonucleotide containing solid support CPG is obtained.

[0245] The CPG-containing nucleotides obtained above were blown dry with dry argon, then transferred to a 2 mL EP tube and 28% ammonia solution (1.8 mL) was added and heated at 55°C for 5 to 18 hours. Filter, wash the filter cake with water (0.5 mL), combine the filtrates, and concentrate under reduced pressure to obtain a white or yellow colloidal solid. After reverse phase preparative purification, the prepared solution was concentrated and passed through a gel column to remove excess salt to obtain the oligonucleotide. The concentration of the obtained oligonucleotide was determined by a micro-UV spectrophotometer (SPECTRO stat Nano). Mass spectrometry detection and analysis were performed on an Agilent 6530LC-MS Q-Tof system. After primary scanning, the nucleic acid molecular weight was calculated after deconvolution.

[0246] Annealing step:

[0247] The double-stranded siRNA sense strand synthesized above was mixed with the antisense strand synthesized above in equimolar amounts, heated to 95°C, maintained at this temperature for 10 minutes, and then slowly cooled to room temperature. The target double-stranded siRNA was then lyophilized.

[0248] 2. Synthesis of siRNA conjugates:

[0249] The synthesis of the antisense strand was obtained by referring to the above-mentioned synthesis method.

[0250] Synthesis of the sense strand: The universal solid support CPG was replaced with the GalNAc solid support prepared in the present invention, and the sense strand connected to the conjugation group in the double-stranded siRNA conjugate of the present invention was prepared with reference to the above-mentioned synthesis method.

[0251] Annealing step:

[0252] The synthesized sense strand was mixed with the synthesized antisense strand in equimolar amounts, heated to 95°C, maintained at this temperature for 10 minutes, and then slowly cooled to room temperature. The target siRNA conjugate was then lyophilized. Some siRNAs or their conjugates of the present invention are shown in Table A.

[0253] Table A: Some siRNAs or their conjugates of the present invention

[0254] Unless otherwise specified, in the context of the present invention, lowercase letters represent bases modified with methoxy at the 2nd position of the ribose of the nucleotide, such as c, g, u, and a, which represent 2'-OMe (2'-O-methyl) C, 2'-OMe G, 2'-OMe U, and 2'-OMe A, respectively; the capital letter f to the right represents a base modified with fluorine at the 2nd position of the ribose of the nucleotide, such as Cf, Gf, Uf, and Af, which represent 2'-F (2'-fluoro) C, 2'-FG, 2'-FU, and 2'-FA, respectively; "s" represents that the two nucleotide residues adjacent to "s" are connected by a thiophosphate group, for example, "gsu" represents that the g and u residues are connected by a thiophosphate group. Y represents N stands for Tgn represents a thymine-diol nucleotide residue, and its structure is The 3' and 5' ends next to the O atom in the present invention respectively indicate that the O atom is connected to the 3' end (i.e., the right nucleotide residue) and the 5' end (i.e., the left nucleotide residue). If there is no mark, it is assumed that the left O atom is connected to the left side (i.e., the 3' end) and the right O atom is connected to the right side (i.e., the 5' end).

[0255] The above synthesis schemes list general experimental steps for preparing the double-stranded siRNA and its conjugates of the present invention. Those skilled in the art can prepare the double-stranded siRNA and its conjugates of the present invention by making appropriate modifications to the methods or adjusting the raw materials according to actual circumstances. Unless otherwise specified, the double-stranded siRNA and its conjugates of the present invention can be prepared using the methods described in the above synthesis schemes.

[0256] Cellular activity and cytotoxicity testing of nucleic acids or conjugates of the invention

[0257] 1. Test of the activity and cytotoxicity of the double-stranded siRNA or conjugate of the present invention against hepatitis B virus

[0258] Test method:

[0259] Hepatoma cells (Hep AD38) were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2, 37°C incubator. Hepatoma cells were used for experiments when they were in the logarithmic growth phase and in good condition (70% confluence). 1.5×10 cells were seeded per well of a 96-well plate. 4 After 24 h, the cells were transfected with a series of double-stranded siRNA or its conjugates according to the instructions of RNAi MAX transfection reagent. Then, after incubation at 37°C and 5% CO2 for 72 h, HBsAg expression and cytotoxicity were detected using HBsAg ELISA kit and CCK8 kit, respectively.

[0260] The experimental results show that the double-stranded siRNA or its conjugate of the present invention embedded with the nucleotide residue of the present invention has good HBsA inhibitory activity, and the double-stranded siRNA or its conjugate of the present application has low cytotoxicity.

[0261] 2. Hepatitis B virus transgenic mouse experiments

[0262] Experimental methods

[0263] 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 Weitongda Biotechnology Co., Ltd., animal production certificate number: SCXK (Beijing) 2019-0002. The animals were raised by Beijing Weitongda Biotechnology Co., Ltd. and kept in individual cages.

[0264] Animal grouping: According to the quantitative detection results of mouse serum HBsAg (primary) and HBeAg (secondary), the animals were stratified and randomly divided into groups, with 5 animals in each group.

[0265] Test Article Preparation and Administration: Calculate the required dosage of drug (double-stranded siRNA or its conjugate) powder based on the purity of each test article. Add the appropriate saline solution at a dosing concentration of 0.6 mg / mL and vortex to mix until a colorless, transparent liquid is obtained. The day of the first dose is defined as Day 0. All groups of animals received a single subcutaneous dose of 5 mL / kg on Day 0. If blood sampling is required on the day of dosing, administer the drug after blood collection.

[0266] Main Outcome Measures: Patients were weighed and blood was collected once on the 3rd, 7th, 14th, 21st, 28th, and 35th day, and serum was separated for determination of HBsAg and HBeAg levels.

[0267] Serum HBsAg and HBeAg Levels: After blood collection, serum is separated and diluted with PBS solution before testing. 10 μL of serum from each sample is diluted to 500 μL with PBS solution (50-fold dilution). Serum HBsAg and HBeAg levels are measured using the Hepatitis B e Antigen Assay Kit and the Hepatitis B Surface Antigen Assay Kit, respectively.

[0268] Results of Hepatitis B Virus Transgenic Mouse Experiments

[0269] The experimental results show that the double-stranded siRNA of the present invention or its conjugate embedded with the nucleotide residue of the present invention has a good inhibitory effect on reducing HBsAg and also has a good inhibitory effect on reducing HBeAg.

[0270] 3. Activity and cytotoxicity test of siRNA or its conjugates against AGT

[0271] Test method:

[0272] Hep3B cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2, 37°C constant temperature incubator. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were transfected with transfection reagent. The cell concentration was adjusted to 2.5×10 5 / mL, and 2×10 4 Cells were transfected with different concentrations of test siRNA and negative control according to the instructions of lipofectamine RNAi MAX transfection reagent.

[0273] 1) After incubation at 37°C, 5% CO2 for 24 h, cells were collected and the mRNA expression levels of AGT and GAPDH were measured using the QuickEasy Cell Direct RT-qPCR kit (Taqman). The expression of the AGT gene in each sample was calculated using the relative quantification method. The final inhibition rate was calculated as % = (relative quantification of control - relative quantification of sample) / relative quantification of control × 100%, or the IC was calculated using a four-parameter fitting method. 50 .

[0274] 2) After incubation at 37°C and 5% CO2 for 72 h, the cell supernatant was collected and the AGT expression level was detected using an AGT Elisa kit, and the cytotoxicity was detected using CCK8.

[0275] The experimental results show that the siRNA embedded in the nucleotide monomer of the present invention and its conjugates have a good knockdown effect on AGT mRNA. The experimental results of the inhibition rate of some siRNA conjugates on AGT are shown in Table B.

[0276] Table B: Inhibition rate of some siRNA conjugates of the present invention on AGT

[0277] 4. Evaluation of AGT siRNA Conjugate Knockdown Activity Using hAGT Transgenic Mice

[0278] To evaluate the in vivo activity of the AGT siRNA conjugates, an AGT humanized mouse model (5-9 mice per group) was used. Baseline serum body weight, ALT, and AGT protein levels were measured in each group. A single dose of 1 mg / kg or 3 mg / kg of GalNAc-siRNA or saline (the vehicle) was administered subcutaneously at the nape of the neck on day 0. Blood samples were collected on days 0, 4, 7, 14, 21, 28, 35, and 49, and human AGT protein concentrations were measured using the Human AGT ELISA Kit (ab287170). The percentage of knockdown was calculated by comparing human AGT protein levels in the siRNA conjugate and vehicle groups. The results are shown in Figure 1.

[0279] The experimental results show that the siRNA conjugate of the present invention after embedding the nucleotide residue of the present invention has a good knockdown effect on AGT mRNA in mice.

[0280] 5. Using RNAseq technology to evaluate the off-target effects of siRNA conjugates embedded with nucleotide residues of the present invention

[0281] Human primary hepatocytes were revived and plated into 24-well plates at a cell density of 270,000 per well. Simultaneously with plating, the siRNA to be tested (2 concentrations in 3 replicates, with a cell control) was freely taken up by the cells. After 48 hours, the cells were harvested and RNA extracted. Total RNA concentration was measured using NanoDrop One, RNA integrity was analyzed, and RNA sequencing was performed using Illumina-NovaSeq. The results of siRNA conjugate ID NO 1, siRNA conjugate ID NO 2, siRNA conjugate ID NO 3, and the positive control experiments are shown in Figures 2-1, 2-2, 2-3, and 2-4, respectively. siRNA conjugate ID NO 1, siRNA conjugate ID NO 2, and siRNA conjugate ID NO 3 correspond to siRNA1, siRNA2, and siRNA3 in the figure, respectively.

[0282] The experimental results show that the double-stranded siRNA conjugates of the present invention embedded with the nucleotide residues of the present invention can significantly reduce the number of downregulated or upregulated off-target genes. For example, the off-target effects of siRNA conjugates ID NO 1, siRNA conjugates ID NO 2, and siRNA conjugates ID NO 3 are significantly better than those of the positive control. Therefore, the double-stranded siRNA conjugates of the present invention embedded with the nucleotide residues of the present invention can reduce the toxicity of siRNA drugs.

[0283] 6. Test of the activity and cytotoxicity of the single-stranded oligonucleotide (ASO) of the present invention against hepatitis B virus

[0284] Test method:

[0285] HepG2.2.15 cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2, 37°C constant temperature incubator. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were transfected with transfection reagent. The cell concentration was adjusted to 2.5×10 5 / mL, and 2*10 4 Cells were transfected with ASO according to the instructions of RNAiMAX transfection reagent. After incubation at 37°C and 5% CO2 for 24 hours, fresh culture medium was replaced and cultured for 72 hours. The cell supernatant was collected and HBsAg expression was detected using HBsAg Elisa kit, HBV DNA was detected by QPCR, and cytotoxicity was detected by CCK8. The cells were collected and the mRNA expression level was detected using QuickEasy Cell Direct RT-qPCR kit (Taqman).

[0286] The experimental results show that the oligonucleic acid of the present invention embedded with the nucleotide residue of the present invention has good inhibitory activity against HBsAg and low cytotoxicity.

[0287] 7. Cellular activity and cytotoxicity test of siRNA or its conjugates against ANGPTL3

[0288] Test method:

[0289] Hep3B cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2, 37°C constant temperature incubator. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were transfected with transfection reagent. The cell concentration was adjusted to 2.5×10 5 / mL, and 2*10 4 Cells were transfected with siRNA according to the instructions of RNAiMAX transfection reagent. After incubation at 37°C, 5% CO2 for 24 h, fresh culture medium was replaced and cultured for another 24 h. Cytotoxicity was detected by CCK8 and the cells were collected and the expression level of hANGPTL3 mRNA was detected using QuickEasy Cell Direct RT-qPCR kit (Taqman).

[0290] The experimental results show that the siRNA of the present invention or its conjugate embedded with the nucleotide residue of the present invention has good inhibitory activity against ANGPTL3.

[0291] 8. Cellular activity and cytotoxicity test of the siRNA or its conjugates against APOC3

[0292] Test method:

[0293] Hep3B cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2, 37°C incubator. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were transfected with a transfection reagent. The cell concentration was adjusted to 2.5×105 / mL and 2*10 cells were seeded into each well of a 96-well plate. 4 Cells were transfected with siRNA according to the instructions of RNAiMAX transfection reagent. After incubation at 37°C, 5% CO2 for 24 h, fresh culture medium was replaced and cultured for another 24 h. Cytotoxicity was detected by CCK8 and the cells were collected and the expression level of hAPOC3 mRNA was detected using QuickEasy Cell Direct RT-qPCR kit (Taqman).

[0294] The experimental results show that the siRNA of the present invention or its conjugate embedded with the nucleotide residue of the present invention has good inhibitory activity against APOC3.

[0295] 9. Cellular activity and cytotoxicity test of siRNA or its conjugates against LPA

[0296] In vitro knockdown activity screening:

[0297] HEK293 cells were cultured in DMEM supplemented with 10% fetal bovine serum in a 5% CO₂, 37°C incubator. Once the cells were in logarithmic growth phase and in good condition (70% confluence), plasmid DNA (LPA_psiCHECK2 plasmid) was transfected into the HEK293 cells using the Fugene HD transfection reagent. Transfected cells were seeded into 96-well plates at a density of 10,000 cells per well, with 100 μL of culture medium per well. The cells were incubated overnight in a 5% CO₂, 37°C incubator. siRNA was then transfected according to the RNAiMAX transfection reagent instructions. After incubation at 37°C, 5% CO₂ for 24 hours, the culture medium was replaced with fresh medium. After an additional 24 hours of culture, the culture medium was discarded, the cells were lysed, and the Renilla luciferase signal was measured according to the instructions for the Dual Fluorescence Reporter Assay Kit. The ratio of the primary reporter gene to the internal reference reporter gene signal in each well was calculated.

[0298] The experimental results show that the siRNA of the present invention or its conjugate embedded with the nucleotide residue of the present invention has good inhibitory activity against LPA.

[0299] 10. Cellular activity and cytotoxicity test of siRNA or its conjugates against PD-L1

[0300] Hep3B cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2, 37°C constant temperature incubator. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were transfected with transfection reagent. The cell concentration was adjusted to 2.5×10 5 / mL, and 2*10 4 Cells were transfected with siRNA according to the instructions of RNAi MAX transfection reagent. After incubation at 37°C, 5% CO2 for 24 h, fresh culture medium was replaced and cultured for another 24 h. Cytotoxicity was detected by CCK8 and the cells were collected and the expression level of hPD-L1 mRNA was detected using Quick Easy Cell Direct RT-qPCR kit (Taqman).

[0301] The experimental results show that the siRNA and siRNA conjugates of the present invention embedded with the nucleotide residues of the present invention have a good inhibitory effect on PD-L1.

[0302] 11. Cellular activity and cytotoxicity test of the siRNA or its conjugates against HSD17B13

[0303] Test Method: Cos-7 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum in a 5% CO2, 37°C incubator. When the cells were in logarithmic growth phase and in good condition (70% confluence), plasmid DNA (HSD17B13_psiCHECK2 plasmid) was transfected into the Cos-7 cells using the transfection reagent FugeneHD. The transfected cells were seeded at a density of 10,000 cells per well in a 96-well plate, with 100 μL of culture medium per well. The cells were incubated overnight in a 5% CO2, 37°C incubator. siRNA was then transfected according to the RNAiMAX transfection reagent instructions. After incubation at 37°C, 5% CO2 for 24 hours, the culture medium was replaced with fresh medium. After an additional 24 hours of culture, the culture medium was discarded, the cells were lysed, and the Renilla luciferase signal was measured according to the instructions for the Dual Fluorescence Reporter Assay Kit. The ratio of the primary reporter gene to the internal reference reporter gene signal in each well was calculated.

[0304] The experimental results show that the siRNA of the present invention or its conjugate embedded with the nucleotide residue of the present invention has good inhibitory activity against HSD17B13.

[0305] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A compound having a structure represented by formula (I), or a stereoisomer, tautomer or acceptable salt of the structure represented by formula (I), Among them, 1) L1 is -*O(CH2) n1 - or -*(CH2) n2 -, R is H, deuterium, fluorine, chlorine, bromine, iodine, methyl or ethyl, wherein -*O(CH2) n1 - and -*(CH2) n2 - are each independently optionally substituted by 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy, n1 is 1, 2, 3, 4, 5, 6 or 7, n2 is 2, 3, 4, 5, 6 or 7; or 2) L1 is -*CH2-, R is H, deuterium, fluorine, chlorine, bromine, iodine, methyl or ethyl, wherein -*CH2- is optionally substituted by 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy; Z and Y 1 each independently is H, deuterium, a hydroxyl protecting group, HOC(=O)(CH2) j C(=O)-, M-C(=O)(CH2) j C(=O)-, a phosphate group, a thiophosphate group, a phosphoroamidite group or a hydrogen phosphate group, where each j is independently 1, 2, 3, 4 or 5; 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; B is Each R 1a 、R 1b 、R 2a 、R 2b 、R 3a 、R 4a and R 4b is independently deuterium, H, CN, HO−, fluorine, chlorine, bromine, methyl, ethyl or methoxy; Each R 2 , R 3b and R 4c is independently -NHR 1 or -N=CH-NR a R b ; R 1 is an amino protecting group; Each R a and R b are independently H or an amino protecting group, or R a , R b and the N atom to which they are attached together form a heterocyclic group composed of 5 - 6 ring atoms, and the heterocyclic group composed of each 5 - 6 ring atoms is 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.

2. The compound according to claim 1, wherein Z and Y 1 each independently is H, deuterium, C 1-12 alkyl, C 1-12 alkyl C(=O)-, C 1-12 alkyl methyl, C 1-12 alkylsilyl, C 6-10 aryl C 1-6 alkyl, HOC(=O)(CH2) j C(=O)-, M-C(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, or Z and Y 1 Each independently is H, deuterium, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylylmethoxycarbonyl, 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-methoxybenzyldiphenylmethyl, 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 alkylmethyl, C 1-10 alkylsilyl, C 6-10 aryl C 1-4 alkyl, HOC(=O)(CH2) j C(=O)-, M-C(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytriphenylmethyl, or Z and Y 1 Each independently is H, deuterium, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylylmethoxycarbonyl, 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-methoxybenzyldiphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, mesyl, tosyl, C 1-8 alkyl, C 1-8 alkylC(=O)-, C 1-8 alkylmethyl, C 1-8 alkylsilyl, phenylC 1-3 alkyl, HOC(=O)(CH2) j C(=O)-, M-C(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, or Z and Y 1 Each independently is 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-methoxybenzyldiphenylmethyl, tetrahydrofuryl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, mesyl, tosyl, C 1-6 alkyl, C 1-6 alkyl C(=O)-, C 1-6 alkylmethyl, C 1-6 alkylsilyl, benzyl, phenethyl, HOC(=O)(CH2) j C(=O)-, M-C(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytriphenylmethyl, wherein X is Cl or Br; Each R x and R y are independently a hydroxyl protecting group; Each R c and R d are independently H or an amino protecting group; or R c , R d and the N atom to which they are attached together form a heterocyclic group composed of 5 to 6 ring atoms, and the heterocyclic group composed of 5 to 6 ring atoms is optionally substituted with 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.

3. The compound according to claim 1 or 2, wherein, R 1 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylylmethoxycarbonyl, 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, mesyl, tosyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, C 1-6 alkoxy or R 3 C(=O)-; Each R a and R b are independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylylmethoxycarbonyl, 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, mesyl, tosyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, C 1-6 alkoxy or R 4 C(=O)-, or R a , R b and the N atom to which they are attached together form pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl, and the pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl are independently optionally substituted with 1, 2, 3 or 4 substituents selected from deuterium, hydroxy, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy; wherein each R 3 and R 4 are independently C 1-6 alkyl, halo 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 are independently C 1-4 alkyl, halo 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 are independently methyl, ethyl, n-propyl, isopropyl, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, 2,2,2-trichloroethyl, methoxy, ethoxy, 1-propoxy, 2-propoxy, 1-butoxy, 2-methyl-1-propoxy, 2-butoxy, tert-butoxy, phenyl, halogen-substituted phenyl, C 1-3 alkylphenyl or benzyl.

4. The compound according to claim 2 or 3, wherein Each R x and R y are 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-methoxybenzyldiphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, mesyl, tosyl, cyano C 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 are independently benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylylmethoxycarbonyl, 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-methoxybenzyldiphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, mesyl, tosyl, CNCH2-, CN(CH2)2-, CNC(CH3)2CH2-, CN(CH2)3-, CNC(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 are independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, biphenylylmethoxycarbonyl, 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, mesyl, tosyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, C 1-6 alkoxy or R 4 C(=O)-, or R a and R b together with the N atom to which it is attached form pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl, and the pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl are independently optionally substituted with 1, 2, 3 or 4 substituents selected from deuterium, hydroxy, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy.

5. The compound according to any one of claims 1-4, which has a structure represented by formula (I-a) or (I-b), or a stereoisomer, a tautomer or an acceptable salt thereof, wherein each L1, R and B independently has the same meaning as L1, R and B shown in any one of claims 1-4.

6. The compound according to any one of claims 1-5, which is one of the following compounds 1-25, or a stereoisomer, tautomer or acceptable salt thereof, Among them, A1 is A2 is A3 is U is T is 7. A nucleotide residue having a structure represented by formula (II), or a stereoisomer, tautomer or acceptable salt of the structure represented by formula (II), Among them, 1) L1 is -*O(CH2) n1 - or -*(CH2) n2 -, R is H, deuterium, fluorine, chlorine, bromine, iodine, methyl or ethyl, wherein -*O(CH2) n1 - and -*(CH2) n2 - are each independently optionally substituted by 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy, wherein n1 is 1, 2, 3, 4, 5, 6 or 7 and n2 is 2, 3, 4, 5, 6 or 7; or 2) L1 is -*CH2-, R is H, deuterium, fluorine, chlorine, bromine, iodine, methyl or ethyl, wherein -*CH2- is optionally substituted by 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy; B1 is Each R 1a 、R 1b 、R 2a 、R 2b 、R 3a 、R 4a and R 4b is independently H, deuterium, CN, HO-, fluorine, chlorine, bromine, methyl, ethyl or methoxy.

8. The nucleotide residue according to claim 7, which is one of the following nucleotide residues (1)-(20), or a stereoisomer, tautomer or acceptable salt thereof, Among them, A is G is C is U is T is Preferably, the nucleotide residue is 10. Use of the nucleotide residue according to claim 7 or 8 as an embedding group in the research of ASO drugs, the research of siRNA drugs, the research of gene function and / or the screening of the whole gene library.

11. The use according to claim 9, wherein the embedding group is embedded on an ASO drug, on the sense strand of an siRNA drug, on the antisense strand of an siRNA drug or on the sense strand and the antisense strand of an siRNA drug.

12. Use of the compound according to any one of claims 1-6 as a raw material for solid-phase synthesis of DNA nucleotides, as a raw material for synthesis of oligonucleotide drugs, as a raw material for synthesis of siRNA drugs, in the research of ASO drugs, the research of siRNA drugs, the research of gene function and / or the screening of the whole gene library.

13. The use according to any one of claims 9-11, wherein the siRNA drug is a drug for inhibiting the expression of the AGT gene.

14. The use according to any one of claims 9-12, wherein the siRNA drug comprises a sense strand and an antisense strand capable of forming a double-stranded region, the sense strand consists of 15-40 nucleotides, preferably consists of 17-25 nucleotides, more preferably consists of 18-23 nucleotides; the antisense strand consists of 15-40 nucleotides, preferably consists of 17-35 nucleotides, more preferably consists of 19-30 nucleotides, and most preferably consists of 21-29 nucleotides; the double-stranded region consists of 17-23 nucleotide base pairs.

14. The use according to any one of claims 9-13, wherein the sense strand and / or the antisense strand respectively comprise 7, 6, 5, 4, 3, 2, 1 and 0 unmodified nucleotides.

15. Use according to any one of claims 9 - 14, wherein the siRNA drug further comprises a ligand, optionally, the ligand is conjugated at any position of the sense strand or the antisense strand; or the ligand is conjugated at the 3'-end or 5'-end of the sense strand; or the ligand is conjugated at the 3'-end or 5'-end of the antisense strand.

16. Use according to any one of claims 9 - 15, wherein the ligand is one or more GalNAc derivatives attached using a multivalent branched linker; or the ligand is one or more GalNAc derivatives attached using a divalent, trivalent or tetravalent branched linker; or the ligand is a GalNAc derivative attached using a divalent, trivalent or tetravalent branched linker.

17. A double-stranded siRNA, its conjugate or salt, which comprises a sense strand and an antisense strand, and at least one nucleotide residue according to any one of claims 7 or 8 is embedded therein; optionally, the siRNA, its conjugate or salt is used for inhibiting the expression of a pathogenic gene; optionally, the pathogenic gene is selected from the AGT gene, HBV gene, INHBE gene, HSD17B13 gene and PNPLA3 gene; optionally, the nucleotide residue is used for enhancing the inhibitory activity of the siRNA and / or reducing the off-target effect; optionally, the nucleotide residue is embedded in the antisense strand; optionally, the nucleotide residue is embedded at the 5th, 6th or 7th position at the 5'-end of the antisense strand.

18. A double-stranded siRNA, its conjugate or salt, characterized in that, It comprises the sense strand shown in SEQ ID NO:1; wherein, the length of the sense strand does not exceed 23 nucleotides; optionally, it further comprises the antisense strand shown in SEQ ID NO:3 - SEQ ID NO:5, and the length of the antisense strand does not exceed 25 nucleotides.

19. The double-stranded siRNA, its conjugate or salt according to claim 18, characterized in that, It comprises one of the double-stranded siRNAs shown in siRNA conjugate ID NO:1 - siRNA conjugate ID NO:3; wherein, the length of the sense strand does not exceed 23 nucleotides, and the length of the antisense strand does not exceed 25 nucleotides.

20. A pharmaceutical composition, which comprises the double-stranded siRNA, its conjugate or its salt according to claim 19, and a pharmaceutically acceptable carrier.

21. Use of the double-stranded siRNA, its conjugate and its salt according to any one of claims 18 - 19 or the pharmaceutical composition according to claim 20 in the preparation of a drug for treating and / or preventing AGT-related diseases.

22. The use according to claim 21, wherein, The AGT-related diseases are hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular lesion, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, ischemic heart disease, myocardial infarction, angina pectoris, stroke, nephropathy, renal failure, systemic sclerosis, intrauterine growth restriction, fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis, non-alcoholic fatty liver, glucose intolerance, type 2 diabetes or metabolic syndrome.

23. The use according to claim 22, wherein, The hypertension is selected from borderline hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, resistant hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, and labile hypertension.

24. The double-stranded siRNA, its conjugate, and its salt according to any one of claims 18-19 or the pharmaceutical composition according to claim 20 are used for treating and / or preventing AGT-related diseases in a patient.

25. According to the double-stranded siRNA, its conjugate, or its salt according to claim 24, or the pharmaceutical composition, the AGT-related diseases are hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, angiopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, ischemic heart disease, myocardial infarction, angina pectoris, stroke, nephrosis, renal failure, systemic sclerosis, intrauterine growth restriction, fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis, non-alcoholic fatty liver, glucose intolerance, type 2 diabetes, or metabolic syndrome.

26. The double-stranded siRNA, its conjugate or its salt, or the pharmaceutical composition according to claim 25, wherein, The hypertension is selected from borderline hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, resistant hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, and labile hypertension.

27. A method for treating and / or preventing AGT-related diseases in a patient, comprising administering to the patient an effective therapeutic amount of the double-stranded siRNA, its conjugate, or its salt according to any one of claims 18-19, or the pharmaceutical composition according to claim 20.

28. The method according to claim 27, wherein The AGT-related diseases are hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, angiopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, ischemic heart disease, myocardial infarction, angina pectoris, stroke, nephrosis, renal failure, systemic sclerosis, intrauterine growth restriction, fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis, non-alcoholic fatty liver, glucose intolerance, type 2 diabetes, or metabolic syndrome.

29. The method according to claim 28, wherein, The hypertension is selected from the group consisting of borderline hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, resistant hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, and labile hypertension.

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