Novel compound and application thereof

Abstract

The invention provides a novel compound and application thereof, and the compound can be used as a raw material for DNA nucleotide solid-phase synthesis, a synthesis raw material for oligonucleotide drugs or a raw material for siRNA drug synthesis, can be used for siRNA drug research, gene function research and / or screening of a whole gene pool, and especially can be used as a synthesis raw material for ASO drugs and a synthesis raw material for double-stranded siRNA drugs. The invention also relates to a novel nucleotide residue, and application of the novel nucleotide residue as an embedded group in ASO drug research, siRNA drug research, gene function research and / or whole gene pool screening.

Description

Technical Field

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

[0002] Small nucleic acid drugs, namely oligonucleotide drugs, are short-chain nucleic acids composed of a dozen to dozens of nucleotides in series. Nucleic acid monomers are the basic units that make up small nucleic acid drugs and are also the key raw materials for nucleic acid drugs. Due to reasons such as the easy degradation of the original small nucleic acid structure by nuclease in the body and poor pharmacokinetic properties, they generally cannot be directly used as drugs and need to be chemically modified at multiple nucleotide sites to improve their affinity, stability, metabolic properties, etc. Such modifications mainly include: base modification, modification of 2'-hydroxy ribose, modification on the phosphate backbone, modification of the ribose five-membered ring backbone, and combined modification for nucleic acid drugs, etc. By modifying and transforming nucleic acid monomers and then 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 uses. The compound can be used as a raw material for solid-phase synthesis of DNA nucleotides, a raw material for the synthesis of oligonucleotide drugs, and / or a raw material for the synthesis of siRNA drugs. It can also be used in the drug research of siRNA, gene function research, and / or screening of the entire gene library. In particular, it can be used as a raw material for the synthesis of ASO drugs and / or double-stranded siRNA drugs. Moreover, the present invention also relates to a novel nucleotide residue and its use as an embedding group in the research of ASO drugs, siRNA drug research, gene function research, and screening of the entire gene library. Experiments show that ASO drugs, siRNA double strands, and their conjugates embedded with the nucleotide residue of the present invention have obvious 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.), basically no off-target phenomenon, and / or low toxicity.

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

[0005]

[0006] Wherein,

[0007] 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

[0008] 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;

[0009] Z and Y 1 are each independently 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 phosphite amide group, or a hydrogen phosphate group, wherein each j is independently 1, 2, 3, 4, or 5;

[0010] 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;

[0011] 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;

[0012] Each R 2 , R 3b and R 4c are independently -NHR 1 or -N=CH-NR a R b ;

[0013] R 1 is an amino protecting group;

[0014] 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.

[0015] In some embodiments of the compounds of the present invention, Z and Y 1 are each independently 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) jC(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytriphenylmethyl, wherein each j, M, X, R x , R y , R c and R d has the meaning described in the present invention.

[0016] In some embodiments of the compounds described in the present invention, Z and Y 1 are each independently 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 1-10 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, wherein each j, M, X, R x , R y , R c and R d has the meaning described in the present invention.

[0017] In some embodiments of the compounds described in the present invention, Z and Y 1Each 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 alkyl C(=O)-, C 1-8 alkylmethyl, C 1-8 alkylsilyl, phenyl C 1-3 alkyl, HOC(=O)(CH2) j C(=O)-, M-C(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytriphenylmethyl, wherein each j, M, X, R x , R y , R c and R d has the meaning described in the present invention.

[0018] In some embodiments of the compounds described in the present invention, 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 1-6 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) jC(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytriphenylmethyl, wherein each j, M, X, R x , R y , R c and R d have the meanings described in the present invention.

[0019] In some embodiments of the compounds described in the present invention, X is Cl or Br.

[0020] In some embodiments of the compounds described in the present invention, each R x and R y are independently a hydroxyl protecting group.

[0021] In some embodiments of the compounds described in the present invention, 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 5-6 ring atoms, and the heterocyclic group composed of 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, propyl, methoxy, ethoxy, 1-propoxy and 2-propoxy.

[0022] In some embodiments of the compounds described in the present invention, 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-methoxybenzyl diphenylmethyl, triphenylmethyl, 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)-, wherein R 3 has the meaning described in the present invention.

[0023] In some embodiments of the compounds described in the present invention, each Ra and R b is 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 (such as methoxy, ethoxy, 1-propoxy, 2-propoxy, etc.) or R 4 C(=O)-, or R a 、R b and together with the N atom to which they are attached form pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl, and the pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl are independently optionally substituted by 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy, wherein R 4 has the meaning as defined in the present invention.

[0024] In some embodiments of the compounds of the present invention, each R a and R b is 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, 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 or R 4 C(=O)-, or R a 、Rb together with the N atom to which they are attached form a pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl group, and the pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl groups are independently optionally substituted by 1, 2, 3 or 4 substituents selected from deuterium, hydroxy, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy, wherein, R 4 has the meaning as defined in the present invention.

[0025] In some embodiments of the compounds of the present invention, each R 3 and R 4 independently is C 1-6 alkyl, halo C 1-6 alkyl, C 1-6 alkoxy, phenyl, halogen-substituted phenyl, C 1-6 alkylphenyl or benzyl.

[0026] In some embodiments of the compounds of the present invention, each R 3 and R 4 independently is C 1-4 alkyl, halo C 1-4 alkyl, C 1-4 alkoxy, phenyl, halogen-substituted phenyl, C 1-4 alkylphenyl or benzyl.

[0027] In some embodiments of the compounds of the present invention, each R 3 and R 4 independently is 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.

[0028] In some embodiments of the compounds of the present invention, each R x and R yIndependently being 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, cyanoC 1-6 alkyl, C 1-6 alkyl, C 1-6 alkylC(=O)-, C 1-6 alkylsilyl, phenyl, halogen-substituted phenyl, benzyl or phenethyl.

[0029] In some embodiments of the compounds of the present invention, each R x and R y Independently being 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 alkylC(=O)-, C 1-4 alkylsilyl, phenyl, halogen-substituted phenyl, benzyl or phenethyl.

[0030] In some embodiments of the compounds of the present invention, each R c and R dIndependently, it 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, 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 forms pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl, and the pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl are independently optionally substituted by 1, 2, 3 or 4 substituents selected from deuterium, hydroxy, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy.

[0031] In some embodiments of the compounds of the present invention, 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, 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, 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 by 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy.

[0032] In some embodiments of the compounds of the present invention, the compounds of the present invention have the structure shown in formula (I-a) or (I-b), or their stereoisomers, tautomers or acceptable salts,

[0033] wherein each L1, R and B independently has the meaning described in the present invention.

[0034] In some embodiments of the compounds of the present invention, it is one of the following compounds 1-25, or its stereoisomers, tautomers or acceptable salts,

[0035]

[0036] wherein, A1 is A2 is A3 is U is T is

[0037] On the other hand, the present invention provides a nucleotide residue having the structure shown in formula (II), or a stereoisomer, tautomer or acceptable salt of the structure shown in formula (II),

[0038]

[0039] 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, n2 is 2, 3, 4, 5, 6 or 7; or

[0040] 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;

[0041] 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.

[0042] In some embodiments of the nucleotide residue of the present invention, the nucleotide residue of the present invention is one of the following nucleotide residues (1)-(20), or its stereoisomer, tautomer or acceptable salt

[0043]

[0044] wherein A is G is C is U is T is

[0045] In some preferred embodiments of the nucleotide residue of the present invention, the nucleotide residue of the present invention is

[0046] On the other hand, the present invention provides the use of the nucleotide residue of the present invention as an intercalating group in the research of ASO drugs, siRNA drugs, gene function research and screening of the whole gene library.

[0047] In some embodiments of the use of the present invention, the nucleotide residue of the present invention is intercalated as an intercalating group 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. On the other hand, the present invention provides the use of the compound of the present invention 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, siRNA drugs, gene function research and / or screening of the whole gene library.

[0048] In some embodiments of the use of the present invention, the siRNA drug is a drug that inhibits the expression of the AGT gene.

[0049] 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, the sense strand consisting of 15-40 nucleotides, preferably consisting of 17-25 nucleotides, more preferably consisting of 18-23 nucleotides.

[0050] 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.

[0051] In some embodiments of the use of the present invention, the double-stranded region consists of 17 - 23 nucleotide base pairs.

[0052] In some embodiments of the use of the present invention, the sense strand and / or the antisense strand respectively contain 7, 6, 5, 4, 3, 2, 1, and 0 unmodified nucleotides.

[0053] In some embodiments of the use of the present invention, the siRNA drug further comprises a ligand.

[0054] In some embodiments of the use of the present invention, the ligand is conjugated at any position of the sense strand or the antisense strand.

[0055] In some embodiments of the use of the present invention, the ligand is conjugated at the 3'-end or 5'-end of the sense strand.

[0056] In some embodiments of the use of the present invention, the ligand is conjugated at the 3'-end or 5'-end of the antisense strand.

[0057] In some embodiments of the use of the present invention, the ligand is one or more GalNAc derivatives attached using a multivalent branched linker.

[0058] In some embodiments of the use of the present invention, the ligand is one or more GalNAc derivatives attached using a divalent, trivalent, or tetravalent branched linker.

[0059] In some embodiments of the use of the present invention, the ligand is a GalNAc derivative attached using a divalent, trivalent, or tetravalent branched linker.

[0060] On the other hand, the present invention relates to a double-stranded siRNA, its conjugate or salt, which comprises the sense strand 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 shown in the sense strand in Table A of the present invention.

[0061] In some embodiments, the double-stranded siRNA, its conjugate or salt of the present invention further comprises the antisense strands shown in SEQ ID NO:3 - SEQ ID NO:5, the length of the antisense strand does not exceed 25 nucleotides, and the sequence information of SEQ ID NO:3 - SEQ ID NO:5 is shown in the antisense strand in Table A of the present invention.

[0062] In some embodiments, the double-stranded siRNA, its conjugate or salt 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. For the sequence information of siRNA ID NO:1 to siRNA ID NO:3, see Table A of the present invention.

[0063] On the other hand, the present invention relates to a double-stranded siRNA, its conjugate or salt, which comprises a sense strand and an antisense strand, and at least one nucleotide residue of the present invention is embedded therein.

[0064] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the siRNA, its conjugate or salt is used to inhibit the expression of a pathogenic gene.

[0065] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the pathogenic gene is selected from the group consisting of AGT gene, HBV gene, INHBE gene, HSD17B13 gene and PNPLA3 gene.

[0066] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the nucleotide residue is used to enhance the inhibitory activity of the siRNA and / or reduce the off-target effect.

[0067] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the nucleotide residue is embedded in the antisense strand.

[0068] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the nucleotide residue is embedded at the 5th, 6th or 7th position at the 5' end of the antisense strand.

[0069] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the nucleotide residue is

[0070] In some embodiments, the sense strand in the double-stranded siRNA, its conjugate or salt of the present invention does not exceed 19, 20, 21, 22 or 23 nucleotides.

[0071] In some embodiments, the antisense strand in the double-stranded siRNA, its conjugate or salt of the present invention does not exceed 19, 20, 21, 22, 23, 24 or 25 nucleotides.

[0072] On the other hand, the present invention relates to a pharmaceutical composition comprising the double-stranded siRNA, its conjugate or its salt of the present invention, and a pharmaceutically acceptable carrier.

[0073] In another aspect, the present invention relates to the use of the double-stranded siRNA, its conjugate, its salt or the pharmaceutical composition described in the present invention in the preparation of a medicament for treating and / or preventing AGT-related diseases.

[0074] In some embodiments of the use described in the present invention, 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.

[0075] 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 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.

[0076] Detailed description of the present invention

[0077] Definitions and general terms

[0078] In the present invention, the term "comprising" or "including" is an open expression, that is, it includes the content specified in the present invention, but does not exclude other aspects.

[0079] 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. The "sense strand (or trailing strand)" refers to an RNAi strand that contains a sequence that is substantially complementary to the antisense strand. The term "substantially complementary" means completely complementary or at least partially complementary, for example, the antisense strand is completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can exist within the internal or terminal regions of the molecule, wherein the most tolerated mismatches exist within the terminal regions, such as within 5, 4, 3 or 2 nucleotides at the 5'- and / or 3'-ends of the RNAi.

[0080] It should be noted that "at least partially substantially complementary" between the antisense strand and the mRNA means that the antisense strand has a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest (such as the mRNA encoding hepatitis B virus). Alternatively, if a polynucleotide is substantially non-discontinuously complementary to a portion of the mRNA encoding hepatitis B virus, then the antisense strand is complementary to at least a portion of the hepatitis B virus mRNA.

[0081] In the present invention, the term "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a hepatitis B virus gene-encoding gene, including the mRNA that is an RNA processing product of the primary transcript.

[0082] In the present invention, the term "inhibiting the expression of hepatitis B virus gene" includes inhibition of the hepatitis B virus (abbreviated as HBV) gene at any level. 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%. Among them, the expression of the HBV gene can be evaluated based on the level of any variable related to the expression of the HBV gene. For example, the mRNA level of HBV or the HBV protein level. The inhibition can be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as the baseline level before administration, or the level measured in similar subjects, cells or samples that have not been treated or have been treated with a control (such as a control using only a buffer or a control without an active agent).

[0083] In the present invention, the "pharmaceutical composition" can refer to being used for the treatment of diseases and can also be used for in vitro cell culture experiments. When used for the treatment of diseases, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any one of the methods well-known in the pharmaceutical field. All methods include the step of combining the active ingredient with excipients constituting one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active siRNA with a liquid excipient, a finely divided solid excipient or both.

[0084] In the present invention, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal being treated therewith. Preferably, "pharmaceutically acceptable" as used in the present invention means approved by a federal regulatory agency or a national government or listed in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, particularly in humans.

[0085] In the present invention, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a particular target dosage form. Except to the extent that any conventional excipient is incompatible with the ASO or siRNA conjugate of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also contemplated within the scope of the present invention.

[0086] In the present invention, the term "treatment" refers to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or an adverse effect caused by the disease. "Treatment" as used in the present invention encompasses diseases in mammals, particularly in humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not been diagnosed with the disease; (b) inhibiting a disease, such as arresting the progression of the disease; or (c) alleviating a disease, such as relieving the symptoms associated with the disease. "Treatment" as used in the present invention encompasses any administration of a drug, RNAi reagent or siRNA to an individual to treat, cure, relieve, improve, alleviate or inhibit a disease in the individual, including but not limited to administering a drug containing the RNAi reagent, siRNA or siRNA conjugate of the present invention to an individual in need thereof.

[0087] The "RNAi reagent" as described in the present invention refers to a reagent containing an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of degrading or inhibiting the transcription and translation of a target messenger RNA (mRNA) in a sequence-specific manner. The RNAi reagent in the present invention can be manipulated by the RNA interference mechanism (i.e., by interacting with the components of the RNA interference pathway in mammalian cells (RNA-induced silencing complex or RISC) to induce RNA interference), or act through any other mechanism or pathway. RNAi reagents include but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs) and Dicer substrates.

[0088] In the context of the present invention, Bz represents benzoyl; MMTr represents 4'-methoxytrityl; DMTr represents 4',4'-dimethoxytrityl.

[0089] In the present invention, the terms "phosphate group", "phosphate moiety", and "phosphate bond" are used interchangeably and include mono-phosphate, di-phosphate, or tri-phosphate. The "phosphate group" in "phosphorothioate group" has the same meaning. Unless otherwise specified, the phosphate moiety between natural nucleotides is a di-phosphate moiety.

[0090] The term "oxo" refers to the =O group. For example, a carbon atom is connected to an oxygen atom by a double bond, where a keto or aldehyde group is formed.

[0091] As used in the present invention, "chemical modification" or "modification" means a structure that has a chemical difference 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 replacement of one chemical moiety with another.

[0092] The compounds of the present invention can be asymmetric, for example, having one or more stereoisomers. Unless otherwise specified, all stereoisomers are included, such as enantiomers and diastereomers. Compounds of the present invention containing an asymmetric carbon atom can be isolated in optically pure form or in racemic form. The optically pure form can be resolved from a racemic mixture or synthesized by using chiral starting materials or chiral reagents.

[0093] 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 an enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, where the resulting mixture of diastereomers 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, and then the diastereomers are resolved by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is usually accomplished by using chromatography employing a chiral stationary phase and optionally in combination with chemical derivatization methods (such as forming carbamates from amines).

[0094] The present invention also includes isotopically labeled compounds of the present invention that are the same as those described in the present invention, but in which one or more atoms are replaced by atoms having an atomic weight or mass number different from the atomic weight or mass number commonly 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, etc.

[0095] Unless otherwise specified, when a position is specifically designated as deuterium (D), that position should be understood to have a deuterium abundance greater than the natural abundance of deuterium (which is 0.015%) by at least 1000-fold (i.e., at least 10% deuterium incorporation). The deuterium in the compounds of the examples can have an abundance greater than the natural abundance of deuterium by at least 1000-fold, at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold, at least 6000-fold, or a higher abundance. The present invention also includes various deuterated forms of the compound of formula (I). Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can refer to relevant literature to synthesize deuterated forms of the compound of formula (I). Commercially available deuterated starting materials can be used in the preparation of deuterated forms of the compound of formula (I), or they can be synthesized using conventional techniques with deuterating reagents, including but not limited to deuterated borane, tetrahydrofuran solution of trideuterated borane, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane, etc.

[0096] The conjugating groups described in the present invention can enhance the delivery of a therapeutic agent to a specific target location (e.g., a specific organ or tissue) within a subject such as a human or an animal. In some embodiments of the present invention, the conjugating groups can enhance the targeted delivery of an expression inhibitory oligonucleotide. In some embodiments of the present invention, the conjugating groups can enhance the delivery of an expression inhibitory oligonucleotide to the liver.

[0097] The conjugating groups described in the present invention can be directly or indirectly linked to a compound, such as a therapeutic agent, e.g., an inhibitory oligonucleotide expressing agent, e.g., the 3' or 5' end of an inhibitory oligonucleotide expressing agent. In some embodiments of the present invention, the inhibitory oligonucleotide expressing agent includes one or more modified nucleotides. In some embodiments of the present invention, the inhibitory oligonucleotide expressing agent is an RNAi reagent, such as a double-stranded RNAi reagent comprising a sense strand and an antisense strand. In some embodiments of the present invention, the conjugating group disclosed in the present invention is linked to the 3' end of the sense strand of the double-stranded RNAi reagent. In some embodiments, the conjugating group disclosed in the present invention is linked to the inhibitory oligonucleotide reagent at the 3' end of the sense strand of the double-stranded RNAi reagent via a phosphate, phosphorothioate or phosphonate group.

[0098] The definitions and conventions for stereochemistry used in the present invention generally refer to the following references: 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 or chiral centers and thus exist in 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 prefixes D, L or R, S are used to denote the absolute configuration of the chiral center of the molecule. The prefixes d, l or (+), (-) are used to name the sign of the rotation of plane-polarized light by the compound, (-) or l means that the compound is levorotatory, and the prefix (+) or d means that the compound is dextrorotatory. The chemical structures of these stereoisomers are the same, but their spatial structures are different. A particular stereoisomer may be an enantiomer, and a mixture of isomers is usually called a racemic mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may result in no stereoselectivity or stereospecificity during a chemical reaction. The terms "racemic mixture" and "racemate" refer to a mixture of two enantiomers in equimolar amounts, lacking optical activity.

[0099] The term "tautomer" or "tautomeric form" refers to isomers of structures of different energies that can be interconverted via a low energy barrier. For example, proton tautomers (i.e., tautomers involving proton transfer) include interconversions via proton migration, such as the isomerization of keto - enol and imine - enamine.

[0100] The term "composition" refers to a mixture of a drug containing one or more of the compounds described in the present invention or their physiologically pharmaceutically acceptable salts or precursors with other chemical components, as well as other components such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of the composition is to facilitate administration to an organism, promote absorption of the active ingredient and thereby exert biological activity.

[0101] The term "pharmaceutically acceptable excipient" or "physiologically acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent or emulsifying agent that has been approved by the US Food and Drug Administration for use in humans or domestic animals.

[0102] Unless otherwise specified, the "compounds", "ligands", "nucleic acid conjugates", "double - stranded siRNA conjugates", "double - stranded siRNAs", "nucleic acids" of the present invention can each independently exist in the form of a salt, mixed salt or non - salt (such as free acid or free base). When in the form of a salt or mixed salt, it can be a pharmaceutically acceptable salt.

[0103] The term "acceptable salt" includes acceptable acid addition salts and pharmaceutically acceptable base addition salts. "Acceptable acid addition salts" refer to salts formed with inorganic acids or organic acids that can retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; organic acid salts include, but are not limited to, formate, acetate, 2,2 - dichloroacetate, trifluoroacetate, propionate, caproate, caprylate, caprate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p - toluenesulfonate, alginate, ascorbate, salicylate, 4 - aminosalicylate, naphthalenedisulfonate, etc. These salts can be prepared by methods known in the art.

[0104] "Pharmaceutically acceptable base addition salts" refers to salts formed with inorganic or organic bases that can 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, etc. 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, the following salts: primary amines, secondary amines, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0105] As described in the present invention, the compounds of the present invention may optionally be substituted by one or more substituents, such as in the general formula compounds above, or in specific examples, subclasses, and a class of compounds included in the present invention as in the examples. Generally, the term "substituted" means that one or more hydrogen atoms in the given structure are replaced by specific substituents. Unless otherwise indicated, an optional substituent group may have a substituent at each substitutable position of the group. When more than one position in the given structural formula can be substituted by one or more substituents selected from a specific group, the substituents may be the same or different at each position.

[0106] In addition, it should be noted that, unless otherwise explicitly indicated, the description methods "each... and... independently is", "... and... each independently is", and "... and... are respectively independent" used throughout the present invention can be interchanged and should be understood in a broad sense. It can either mean that among different groups, the specific options expressed between the same symbols do not affect each other, or it can also mean that within the same group, the specific options expressed between the same symbols do not affect each other.

[0107] In the present invention, the terms "optionally", "optional", or "option" generally mean that the subsequent event or condition may or may not occur, and this description includes the case where the event or condition occurs, as well as the case where the event or condition does not occur. For example, as described in the present invention, "-*O(CH2) n1 - and -*(CH2) n2-each independently optionally substituted with 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy” means -*O(CH2) n1 -and -*(CH2) n2 -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.

[0108] In each part of this specification, the substituents of the compounds disclosed in the present invention are disclosed according to the group type or range. It is specifically pointed out that the present invention includes each independent secondary combination of each member 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 independently disclosed.

[0109] As used herein, the term "alkyl" includes saturated straight-chain or branched-chain monovalent hydrocarbon radicals having 1 to 20 carbon atoms, wherein the alkyl groups may independently and optionally be substituted with one or more substituents described herein. In some embodiments, the alkyl groups contain 1 to 12 carbon atoms; in other embodiments, the alkyl groups contain 1 to 10 carbon atoms; in other embodiments, the alkyl groups contain 1 to 8 carbon atoms; in other embodiments, the alkyl groups contain 1 to 6 carbon atoms; in other embodiments, the alkyl groups contain 1 to 4 carbon atoms; in other embodiments, the alkyl groups contain 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)CH2CH3), 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.

[0110] The term "alkylene" refers to a saturated divalent or polyvalent hydrocarbon radical obtained by removing two hydrogen atoms from a saturated straight-chain or branched-chain hydrocarbon radical. Unless otherwise specified in detail, the alkylene radical contains 1 to 12 carbon atoms. In some embodiments, the alkylene radical contains 1 to 6 carbon atoms; in other embodiments, the alkylene radical contains 1 to 4 carbon atoms; in still other embodiments, the alkylene radical contains 1 to 3 carbon atoms; in yet other embodiments, the alkylene radical contains 1 to 2 carbon atoms. Examples of alkylene include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), isopropylidene (-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-hexylene (-CH(CH3)CH2CH2CH2CH2-), n-heptylene (-(CH2)7-), n-octylene (-(CH2)8-), n-nonylene (-(CH2)9-), decylene (-(CH2) 10 -), -(CH2) 11 -, -(CH2) 12 -, -(CH2) 13 -, -(CH2) 14 -, etc.

[0111] The term "heteroalkyl" as used in the present invention means the insertion of 1, 2, 3, 4, 5 or 6 heteroatoms in an alkyl group, the heteroatoms being selected from S, N, NH, O or P, and the sulfur atom may optionally be oxidized to an S-oxide. In some embodiments, the heteroalkyl group contains 1-12 carbon atoms; in some other embodiments, the heteroalkyl group contains 1-10 carbon atoms; in some other embodiments, the heteroalkyl group contains 1-8 carbon atoms; in some other embodiments, the heteroalkyl group contains 1-6 carbon atoms; in some other embodiments, the heteroalkyl group contains 1-4 carbon atoms; in some other embodiments, the heteroalkyl group contains 1-3 carbon atoms. Examples of heteroalkyl include, but are not limited to, CH3O-, CH3CH2O-, CH3CH2CH2O-, CH3CH2CH2CH2O-, CH3CH2OCH2-, CH3OCH2CH2-, CH3CH2CH2OCH2-, CH3CH2OCH2CH2-, CH3OCH2CH2CH2-, CH3S-, CH3CH2S-, CH3CH2CH2S-, CH3CH2CH2CH2S-, CH3CH2SCH2-, CH3SCH2CH2-, CH3CH2CH2SCH2-, CH3CH2SCH2CH2-, CH3SCH2CH2CH2-, CH3NH-, CH3CH2NH-, CH3CH2CH2NH-, CH3CH2CH2CH2NH-, CH3CH2NHCH2-, CH3NHCH2CH2-, CH3CH2CH2NHCH2-, CH3CH2NHCH2CH2-, CH3NHCH2CH2CH2-, etc.

[0112] The term "alkoxy" means that an alkyl group is linked to the remainder of the molecule through an oxygen atom, where the alkyl group has the meaning as described in the present invention. Unless otherwise specified in detail, the alkoxy group contains 1-12 carbon atoms. In some embodiments, the alkoxy group contains 1-8 carbon atoms; in some other embodiments, the alkoxy group contains 1-6 carbon atoms; in some other embodiments, the alkoxy group contains 1-4 carbon atoms; in still some other embodiments, the alkoxy group contains 1-3 carbon atoms. The alkoxy group may optionally be substituted by one or more substituents described in the present invention.

[0113] 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-1-butoxy (-OCH2CH2CH(CH3)2), 2-methyl-1-butoxy (-OCH2CH(CH3)CH2CH3), and the like.

[0114] The term "alkenyl" means a straight-chain or branched-chain monovalent, divalent or polyvalent hydrocarbon group containing 2 - 12 carbon atoms, or 2 - 8 carbon atoms, or 2 - 6 carbon atoms, or 2 - 4 carbon atoms, in which at least one position of C-C is a sp 2 double bond, and the alkenyl groups may be independently unsubstituted or substituted by one or more substituents described in the present invention, including "cis", "trans" or "Z", "E" isomers, and specific examples include, but are not limited to, vinyl (-CH=CH2), propenyl (-CH=CHCH3), allyl (-CH2CH=CH2), etc., and the alkenyl groups may be independently unsubstituted or substituted by one or more substituents described in the present invention.

[0115] The term "composed of M - M1 ring atoms" means that the cyclic group is composed of M - M1 ring atoms, and the ring atoms include carbon atoms and / or heteroatoms such as O, N, S, P, etc. For example, "heterocyclic group composed of 3 - 6 ring atoms" means it includes monocyclic heterocyclic groups composed of 3, 4, 5 or 6 ring atoms.

[0116] The term "heterocyclic group" refers to a non-aromatic, saturated or partially unsaturated monocyclic, bicyclic or tricyclic system containing 3 to 12 ring atoms, wherein at least one ring atom is selected from nitrogen, sulfur or oxygen atoms. Among them, the heterocyclic group may optionally be substituted by one or more substituents described in the present invention. Unless otherwise specified, the heterocyclic group may be a carbon-based or nitrogen-based group, and the -CH2- group may optionally be replaced by -C(=O)- or -C(=S)-. The sulfur atom of the ring may optionally be oxidized to the S-oxide. The nitrogen atom of the ring may optionally be oxidized to the N-oxide. In some embodiments, the heterocyclic group is a heterocyclic group composed of 3 to 12 ring atoms. In some embodiments, the heterocyclic group is a heterocyclic group composed of 5 to 10 ring atoms. In some embodiments, the heterocyclic group is a heterocyclic group composed of 3 to 6 ring atoms. In some embodiments, the heterocyclic group is a heterocyclic group composed of 4 to 6 ring atoms. In some embodiments, the heterocyclic group is a heterocyclic group composed of 5 to 6 ring atoms. In other embodiments, the heterocyclic group is a heterocyclic group composed of 4 atoms, which refers to a monovalent or polyvalent, saturated or partially unsaturated, non-aromatic monocyclic ring containing 4 ring atoms, wherein at least one ring atom is selected from nitrogen, sulfur and oxygen atoms. In other embodiments, the heterocyclic group is a heterocyclic group composed of 5 atoms, which refers to a monovalent or polyvalent, saturated or partially unsaturated, non-aromatic monocyclic ring containing 5 ring atoms, wherein at least one ring atom is selected from nitrogen, sulfur and oxygen atoms. In other embodiments, the heterocyclic group is a heterocyclic group composed of 6 atoms, 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 the fusion of heterocyclic groups with saturated or partially unsaturated rings or heterocycles. Examples of heterocycles include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, epoxypropyl, azepanyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, 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-indolylquinazyl and N-pyridylurea.Examples of the heterocyclic group also include 1,1-dioxothiomorpholinyl. Examples of the case where a carbon atom on the ring is substituted by oxo (=O) include, but are not limited to, pyrimidinedione group, 1,2,4-thiadiazol-5(4H)-one group, 1,2,4-oxadiazol-5(4H)-one group, 1H-1,2,4-triazol-5(4H)-one group, etc. Examples of the case where a carbon atom on the ring is substituted by the group =S include, but are not limited to, 1,2,4-oxadiazole-5(4H)-thione group, 1,3,4-oxadiazole-2(3H)-thione group, etc.

[0117] The term "heteroatom" means one or more of O, S, N, P, and Si, including any oxidized form of N, S, and P; the form of primary, secondary, tertiary amines, and quaternary ammonium salts; or the form in which the hydrogen on the nitrogen atom in the heterocycle 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, where R represents a substituent described in the present invention).

[0118] The terms "alkylsilyl" and "alkylsilane" mean that the hydrogen atoms in the silyl (-SiH3) group are independently substituted by one, two, or three alkyl groups respectively. In some embodiments, the alkylsilane is a lower alkylsilyl group formed by one, two, or three C 1-12 alkyl groups attached to the silicon atom. In other embodiments, the alkylsilyl is a lower alkylsilyl group formed by one, two, or three C 1-9 alkyl groups attached to the silicon atom. In other embodiments, the alkylsilyl is a lower alkylsilyl group formed by one, two, or three C 1-6 alkyl groups attached to the silicon atom. In other embodiments, the alkylsilyl is a lower alkylsilyl group formed by one, two, or three C 1-4 alkyl groups attached to the silicon atom. In still other embodiments, the alkylsilyl is a lower alkylsilyl group formed by one, two, or three C 1-3 alkyl groups attached to the silicon atom. Suitable alkylsilyl groups can be monoalkylsilyl, dialkylsilyl, or trialkylsilyl. Examples of alkylsilyl include, but are not limited to, trimethylsilyl (-Si(CH3)3), triethylsilyl (-Si(CH2CH3)3), tri-n-propylsilyl (-Si(CH2CH2CH3)3), and so on.

[0119] The term "hydroxy protecting group" refers to a labile chemical moiety that protects a hydroxy group from undesired reactions during one or more synthetic procedures. After the one or more synthetic procedures, the hydroxy protecting group can be selectively removed. Hydroxy protecting groups known in the art are generally described in T.H. Greene and P.G.M. 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, isopropoxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, 2-furyloxycarbonyl, 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-methoxybenzyl diphenylmethyl, triphenylmethyl (triphenylmethyl or trityl), tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, mesyl, tosyl, C 1-10 alkylsilyl (such as trimethylsilyl (TMS or -Si(CH3)3)), triethylsilyl, triisopropylsilyl, MMTr, DMTr or 4',4',4'-trimethoxytrityl, etc.

[0120] The term "amino protecting group" refers to a labile chemical moiety that protects an amino group from unwanted reactions during a synthetic procedure. After such one or more synthetic procedures, the amino protecting group as described herein can be selectively removed. Amino protecting groups known in the art are generally described in T.H. Greene and P.G.M. 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, 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, 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 meaning described in the present invention.

[0121] The term "solid support" specifically refers to any particle, bead or surface on which oligonucleotide synthesis can occur. For example, both inorganic solid supports and organic solid supports can be optionally used in the examples of the present invention. The inorganic solid support is preferably selected from silica gel and controlled-pore glass (CPG). The organic solid support is a resin, preferably a macroporous resin, more preferably highly cross-linked polystyrene, Tentagel (a graft copolymer composed of a low-cross-linked polystyrene matrix with polyethylene glycol (PEG or POE) grafted thereon), polyvinyl acetate (PVA), Poros - a copolymer of polystyrene / divinylbenzene, aminopolyethylene glycol and cellulose, etc. Preferred embodiments of the present invention utilize a CPG-based solid support. Many other commercially available solid supports also fall within the scope of the present invention.

[0122] Unless otherwise specified, in case of 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. Brief Description of the Drawings

[0123] Appendix Figure 1 Shows the inhibitory effect of the siRNA conjugate of the present invention and the vehicle control group (normal saline) on AGT in the hAGT transgenic mouse model experiment. Among them, siRNA1, siRNA2, and siRNA3 respectively represent siRNA conjugate ID NO 1-3, and the specific sequences corresponding thereto are shown in Table A. Among them, vehicle represents the vehicle.

[0124] Appendix Figure 2-1 Appendix Figure 2-2 Appendix Figure 2-3 Appendix Figure 2-4 And Appendix Figure 2-1 Appendix Figure 2-2 Appendix Figure 2-3 The siRNA1, siRNA2, and siRNA3 in Appendix Figure 2-4 The positive control in Appendix

[0125] Detailed Description of the Compounds of the Present Invention

[0126] The present invention provides a novel compound and its uses. The compound can be used as a raw material for the solid-phase synthesis of DNA nucleotides, a raw material for the synthesis of oligonucleotide drugs, and / or a raw material for the synthesis of siRNA drugs. It can also be used in the drug research of siRNA, the research of gene function, and / or the screening of the entire gene library. In particular, it can be used as a raw material for the synthesis of ASO drugs and / or double-stranded siRNA drugs. Moreover, the present invention also relates to a novel nucleotide residue and its use as an embedding group in the research of ASO drugs, the research of siRNA drugs, the research of gene function, and the screening of the entire gene library. Experiments show that the ASO drugs, siRNA double strands, and their conjugates embedded with the nucleotide residue of the present invention have obvious inhibitory activity and / or low toxicity against 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.).

[0127] On the one hand, the present invention provides a compound having a structure shown in formula (I), or a stereoisomer, tautomer, or acceptable salt of the structure shown in formula (I).

[0128]

[0129] Wherein each L1, R, Z, Y 1 and B have the meanings as described in the present invention.

[0130] In some embodiments of the compound of the present invention, 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.

[0131] In some embodiments of the compound of the present invention, L1 is -*CH2-, wherein -*CH2- is optionally substituted by 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.

[0132] In some embodiments of the compounds of the present invention, Z and Y 1 are each independently H, deuterium, a hydroxyl protecting group, HOC(=O)(CH2) j C(=O)-, M-C(=O)(CH2) j C(=O)-, a phosphate group, a phosphorothioate group, a phosphoroamidite group or a hydrogen phosphate group, where each j and M has the meaning described in the present invention.

[0133] Any phosphate group, phosphorothioate group, phosphoroamidite group or hydrogen phosphate group and other active phosphate groups that can enable the compounds of the present invention to participate in oligonucleotide synthesis belong to the content of the present invention. The phosphates described in the present invention include phosphodiesters and triesters; the phosphorothioates described include phosphodiesters and triesters; the hydrogen phosphate group refers to the group formed after the phosphate group forms a salt with a base.

[0134] In some embodiments of the compounds of 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.

[0135] In some embodiments of the compounds of the present invention, 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.

[0136] In some embodiments of the compounds of the present invention, each R 2 、R 3b and R 4c is independently -NHR 1 or -N=CH-NR a R b ,where each R 1 、R a and R b has the meaning described in the present invention.

[0137] In some embodiments of the compounds of the present invention, R 1 is an amino protecting group.

[0138] In some embodiments of the compounds of the present invention, each R a and R bIndependently H or an amino protecting group, or R a , R b and together with the N atom to which they are attached form a heterocyclic group composed of 5 - 6 ring atoms, and each of the heterocyclic groups composed of 5 - 6 ring atoms is independently 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.

[0139] In some embodiments of the compounds of the present invention, each j is independently 1, 2, 3, 4 or 5.

[0140] In some embodiments of the compounds of the present invention, Z and Y 1 are each independently 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)-, trityl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, wherein each j, M, X, R x , R y , R c and R d have the meanings as described in the present invention.

[0141] In some embodiments of the compounds of the present invention, Z and Y 1 are each independently 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 alkyl methyl, C 1-10 alkylsilyl, C6-10 Aryl C 1-4 alkyl, HOC(=O)(CH2) j C(=O)-, M-C(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, wherein each j, M, X, R x , R y , R c and R d have the meanings described in the present invention.

[0142] In some embodiments of the compounds described in the present invention, Z and Y 1 are each independently 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 alkyl C(=O)-, C 1-8 alkylmethyl, C 1-8 alkylsilyl, phenyl C 1-3 alkyl, HOC(=O)(CH2) j C(=O)-, M-C(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, wherein each j, M, X, R x , R y , R c C and R d have the meanings described in the present invention.

[0143] In some embodiments of the compounds described in the present invention, Z and Y 1Each 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 1-6 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 each j, M, X, R x , R y , R c and R d has the meaning described in the present invention.

[0144] In some embodiments of the compounds described in the present invention, X is Cl or Br.

[0145] In some embodiments of the compounds described in the present invention, each R x and R y is independently a hydroxyl protecting group.

[0146] In some embodiments of the compounds described in the present invention, each R c and R d is 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 5-6 ring atoms, and the heterocyclic group composed of 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, propyl, methoxy, ethoxy, 1-propoxy and 2-propoxy.

[0147] In some embodiments of the compounds described in the present invention, R 1is 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, methyl, tert-butyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, benzyl, p-methoxybenzyldiphenylmethyl, triphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, mesyl, tosyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, C 1-6 alkoxy (such as methoxy, ethoxy, 1-propoxy, 2-propoxy, etc.) or R 3 C(=O)-, wherein, R 3 has the meaning described in the present invention.

[0148] In some embodiments of the compounds described in the present invention, 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-methoxybenzyldiphenylmethyl, triphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, mesyl, tosyl, 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 the N atom to which they are attached together form a pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl group, and the pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl groups are independently optionally substituted by 1, 2, 3 or 4 substituents selected from deuterium, hydroxy, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy, wherein, R 4has the meaning as defined in the present invention.

[0149] In some embodiments of the compounds of the present invention, 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, 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, trityl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, mesyl, tosyl, trimethylsilyl, triethylsilyl, triisopropylsilyl or R 4 C(=O)-, or R a , R b and the N atom to which they are attached together form 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, wherein R 4 has the meaning as defined in the present invention.

[0150] In some embodiments of the compounds of the present invention, 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.

[0151] In some embodiments of the compounds of the present invention, 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.

[0152] In some embodiments of the compounds of the present invention, each R 3 and R 4Independently, it is 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.

[0153] In some embodiments of the compounds of the present invention, each R x and R y Independently, it is 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, 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.

[0154] In some embodiments of the compounds of the present invention, each R x and R y Independently, it is 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 1-4Alkylsilyl, phenyl, halogen-substituted phenyl, benzyl or phenethyl.

[0155] In some embodiments of the compounds of the present invention, 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 (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 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.

[0156] In some embodiments of the compounds of the present invention, each R c and R dIndependently, it is H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, 1-propoxy, 2-propoxy, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 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 or R 4 C(=O)-, or R a , R b Together with the N atom to which it is attached, it forms pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl, and the pyrrolidinyl, morpholinyl, piperidinyl and piperazinyl are independently optionally substituted by 1, 2, 3 or 4 substituents selected from deuterium, hydroxyl, cyano, F, Cl, Br, I, methoxy, ethoxy, 1-propoxy and 2-propoxy.

[0157] In some embodiments of the compounds of the present invention, the compounds of the present invention have the structure shown in formula (I-a) or (I-b), or their stereoisomers, tautomers or acceptable salts,

[0158] wherein each L1, R and B independently has the meaning described in the present invention.

[0159] In some embodiments of the compounds of the present invention, it is one of the following compounds 1-25, or its stereoisomers, tautomers or acceptable salts,

[0160]

[0161]

[0162] wherein, A1 is A2 is A3 is U is T is

[0163] On the other hand, the present invention provides a nucleotide residue having the structure shown in formula (II), or its stereoisomers, tautomers or acceptable salts of the structure shown in formula (II),

[0164]

[0165] Among them,

[0166] 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

[0167] 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;

[0168] 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.

[0169] In some embodiments of the nucleotide residue of the present invention, the nucleotide residue of the present invention is one of the following nucleotide residues (1)-(20), or its stereoisomer, tautomer or acceptable salt,

[0170]

[0171] Among them, A is G is C is U is T is

[0172] In some preferred embodiments of the nucleotide residue of the present invention, the nucleotide residue of the present invention is (i.e., Y), and the embedded in small nucleic acid drugs (such as siRNA and ASO, etc.) can not only significantly improve the gene silencing (such as AGT) activity of the drug, but also significantly reduce the off-target effect, thereby reducing the toxicity of the drug. That is, it contains the residue of the present invention (such as Small nucleic acids (such as siRNA, siRNA conjugates, ASO, etc.) can reduce the number of off-target genes that are downregulated or upregulated.

[0173] In another aspect, the present invention provides the use of the nucleotide residues described in the present invention as intercalating groups in the research of ASO drugs, siRNA drugs, gene function research, and / or screening of whole gene libraries.

[0174] In some embodiments of the use described in the present invention, the nucleotide residues are intercalated as intercalating groups on ASO drugs, on the sense strand of siRNA drugs, on the antisense strand of siRNA drugs, or on both the sense strand and the antisense strand of siRNA drugs.

[0175] In still another aspect, the present invention provides the use of the compounds described in 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 screening of whole gene libraries.

[0176] On the other hand, the present invention also provides a method for inhibiting the expression of a specific gene in cells in a patient, which includes administering to the patient an ASO drug, double-stranded siRNA, double-stranded siRNA conjugate, or a combination thereof that incorporates the nucleoside monomers described in the present invention, and the ASO drug, double-stranded siRNA, double-stranded siRNA conjugate, or a combination thereof can be a therapeutically effective amount.

[0177] In some embodiments of the use described in the present invention, the siRNA drug is a drug that inhibits the expression of the AGT gene.

[0178] In some embodiments of the use described in the present invention, the siRNA drug comprises a sense strand and an antisense strand capable of forming a double-stranded region, the sense strand is composed of 15 - 40 nucleotides, preferably composed of 17 - 25 nucleotides, and more preferably composed of 18 - 23 nucleotides.

[0179] In some embodiments of the use described in the present invention, the antisense strand is composed of 15 - 40 nucleotides.

[0180] In some embodiments of the use described in the present invention, the antisense strand is composed of 17 - 35 nucleotides.

[0181] In some embodiments of the use described in the present invention, the antisense strand is composed of 19 - 30 nucleotides.

[0182] In some embodiments of the use described in the present invention, the antisense strand is composed of 21 - 29 nucleotides.

[0183] In some embodiments of the use according to the present invention, the double-stranded region consists of 17-23 nucleotide base pairs.

[0184] In some embodiments of the use according to the present invention, the sense strand and / or the antisense strand respectively contain 7, 6, 5, 4, 3, 2, 1, and 0 unmodified nucleotides.

[0185] In some embodiments of the use according to the present invention, the siRNA drug further comprises a ligand.

[0186] In some embodiments of the use according to the present invention, the ligand is conjugated at any position of the sense strand or the antisense strand.

[0187] In some embodiments of the use according to the present invention, the ligand is conjugated at the 3'-end or 5'-end of the sense strand.

[0188] In some embodiments of the use according to the present invention, the ligand is conjugated at the 3'-end or 5'-end of the antisense strand.

[0189] In some embodiments of the use according to the present invention, the ligand is one or more GalNAc derivatives attached using a multivalent branched linker.

[0190] In some embodiments of the use according to the present invention, the ligand is one or more GalNAc derivatives attached using a divalent, trivalent, or tetravalent branched linker.

[0191] In some embodiments of the use according to the present invention, the ligand is a GalNAc derivative attached using a divalent, trivalent, or tetravalent branched linker.

[0192] On the other hand, the present invention relates to a double-stranded siRNA, its conjugate or salt, which comprises a sense strand 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 shown in the sense strand in Table A of the present invention.

[0193] In some embodiments, the double-stranded siRNA, its conjugate or salt according to the present invention further comprises an antisense strand shown in SEQ ID NO: 3 to SEQ ID NO: 5, the length of the antisense strand does not exceed 25 nucleotides, and the sequence information of SEQ ID NO: 3 to SEQ ID NO: 5 is shown in the antisense strand in Table A of the present invention.

[0194] In some embodiments, the double-stranded siRNA, its conjugate or salt 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, the length of the antisense strand does not exceed 25 nucleotides, and the sequence information of siRNA ID NO:1 to siRNA ID NO:3 can be found in Table A of the present invention.

[0195] On the other hand, the present invention relates to a double-stranded siRNA, its conjugate or salt, which comprises a sense strand and an antisense strand, and at least one nucleotide residue of the present invention is embedded therein.

[0196] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the siRNA, its conjugate or salt is used to inhibit the expression of a pathogenic gene.

[0197] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the pathogenic gene is selected from the AGT gene, the HBV gene, the INHBE gene, the HSD17B13 gene and the PNPLA3 gene.

[0198] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the nucleotide residue is used to improve the inhibitory activity of the siRNA and / or reduce the off-target effect.

[0199] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the nucleotide residue is embedded in the antisense strand.

[0200] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the nucleotide residue is embedded at the 5th, 6th or 7th position at the 5' end of the antisense strand.

[0201] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the nucleotide residue is

[0202] In some embodiments, the sense strand in the double-stranded siRNA, its conjugate or salt of the present invention does not exceed 19, 20, 21, 22 or 23 nucleotides.

[0203] In some embodiments, the antisense strand in the double-stranded siRNA, its conjugate or salt of the present invention does not exceed 19, 20, 21, 22, 23, 24 or 25 nucleotides.

[0204] On the other hand, the present invention relates to a pharmaceutical composition, which comprises the double-stranded siRNA, its conjugate or its salt of the present invention, and a pharmaceutically acceptable carrier.

[0205] In another aspect, the present invention relates to the use of the double-stranded siRNA, its conjugate, its salt or the pharmaceutical composition according to the present invention in the preparation of a drug for treating and / or preventing AGT-related diseases.

[0206] In some embodiments of the use according to the present invention, the AGT-related diseases are hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular lesions, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, ischemic heart disease, cardiomyocardial 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.

[0207] In some embodiments of the use according to the present invention, 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.

[0208] Formulations, administrations and methods of treating diseases of the ASO drugs, siRNA conjugates, nucleic acid conjugates of the present invention

[0209] The effective amount of the ASO drug, nucleic acid conjugate (such as siRNA conjugate or pharmaceutical composition) according to the present invention may vary with the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art according to various factors (such as through clinical trials). The factors include but are not limited to: the 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, due to the urgency of the treatment condition, several separate doses may be administered daily, for example, administered at a dose of four times a day, three times a day, two times a day, once a day or once every other day, or the several doses administered daily may be proportionally reduced.

[0210] Administration to a subject can be effected by any suitable means 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 and sublingual administration), with intravenous injection being preferred.

[0211] The pharmaceutical compositions disclosed in the present invention include formulations suitable for parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the pharmaceutical art. The amount of the active ingredient that may be combined with excipient substances to produce a single dosage form is generally the amount of the nucleic acid drug (such as siRNA drug or ASO drug) that produces a therapeutic effect. Generally, on a percentage basis, this amount is from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0212] In yet another aspect of the present invention, the present invention provides a method for inhibiting the expression or activity of a target gene (such as HBV (hepatitis B virus) gene, AGT (angiotensinogen) gene, LAP (lipoprotein (a)) gene, ANGPTL3 (angiopoietin-like 3) gene, APOC3 (apolipoprotein C-III) gene, etc.). According to an embodiment of the present invention, the method comprises: contacting the aforementioned ASO drug, siRNA, the aforementioned siRNA conjugate, or the aforementioned pharmaceutical composition with cells. As described above, the aforementioned ASO drugs, siRNAs, and siRNA conjugates can all inhibit the expression and replication of the target gene.

[0213] General synthetic methods of the compounds, double-stranded siRNAs, and double-stranded siRNA conjugates of the present invention

[0214] Generally, the compounds and nucleic acid conjugates of the present invention can be prepared by the methods described in the present invention, unless otherwise specified, wherein the definitions of the substituents are as shown in formulas (I) and (II). The following reaction schemes and examples are used to further illustrate the content of the present invention.

[0215] For the embodiments described below, unless otherwise indicated, all temperatures are in degrees Celsius (°C). The chromatographic column used is a silica gel column. The silica gel (200 - 300 mesh) was purchased from Qingdao Marine Chemical Factory, and all NH2CPG were purchased from Hebei Dina Xinke. Nuclear magnetic resonance spectra were recorded in CDC13, DMSO-d6, CD3OD, or acetone-d6 as solvents (in ppm), using TMS (0 ppm) or chloroform (7.25 ppm) as reference standards. When multiplets occur, the following abbreviations will be used: s (singlet), d (doublet), t (triplet), m (multiplet), br (broadened), dd (doublet of doublets), dt (doublet of triplets), br.s (broadened singlet), q (quartet). The coupling constant J is expressed in hertz (Hz).

[0216] Low-resolution mass spectrometry (MS) data were determined using a spectrometer of Agilent 6320 series LC-MS equipped with a G1312A binary pump and a G1316A TCC (column temperature maintained at 30 °C). A G1329A autosampler and a G1315B DAD detector were used for analysis, and an ESI source was used for the LC-MS spectrometer.

[0217] High-resolution mass spectrometry (MS) data were determined using a spectrometer of Agilent 6130 series LC-MS equipped with a G1311A quaternary pump and a G1316A TCC (column temperature maintained at 30 °C). A G1329A autosampler and a G1315D DAD detector were used for analysis, and an ESI source was used for the HR-MS spectrometer. Detailed implementation manners

[0218] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments 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 obtained by adjusting the synthesis according to the embodiments of the present invention or the conventional techniques in the art. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For those reagents or instruments not specified with the manufacturer, they are all conventional products that can be obtained commercially.

[0219] Synthesis method of the compounds of the present invention

[0220] The following synthetic schemes list the general experimental procedures for preparing the compounds disclosed in the present invention. Those skilled in the art can prepare the compounds of the present invention by making appropriate modifications to the methods or adjustments to the raw materials according to the actual situation.

[0221] The following abbreviations are used throughout the present invention:

[0222]

[0223] Synthesis of the nucleoside monomers DAW50072-a and DAW50072-b of the present invention

[0224] Step 1: Synthesis of DAW50072-1

[0225]

[0226] Dissolve 3-buten-2-ol (1.5 g, 20.39 mmol) and 4-dimethylaminopyridine (2.49 g, 20.39 mmol) in DCM (150 mL), and inject tert-butyldiphenylchlorosilane (6.73 g, 24.47 mmol) under stirring. React at room temperature overnight. Concentrate the reaction solution, and purify the residue by silica gel column chromatography (petroleum ether) to obtain a transparent oil DAW50072 (4.975 g, yield: 78.60%).

[0227] 1 H NMR (599 MHz, CDCl3) δ (ppm) 7.71–7.66 (m, 4H), 7.44–7.40 (m, 2H), 7.36 (dt, J = 7.9, 4.1 Hz, 4H), 5.86 (ddd, J = 17.1, 10.4, 5.4 Hz, 1H), 5.10 (dt, J = 17.2, 1.6 Hz, 1H), 4.95 (dt, J = 10.4, 1.5 Hz, 1H), 4.34–4.27 (m, 1H), 1.14 (d, J = 6.3 Hz, 3H), 1.07 (s, 9H).

[0228] Step 2: Synthesis of DAW50072-2

[0229]

[0230] Dissolve DAW50072-1 (4.975 g, 16.02 mmol) in DCM (100 mL), add m-chloroperoxybenzoic acid (5.20 g, 25.63 mmol) under stirring, and react at room temperature overnight. Concentrate the reaction solution under reduced pressure, and purify the residue by silica gel column chromatography (PE / EA (V / V) = 20 / 1) to obtain a transparent oil DAW50072-2 (4.53 g, yield: 86.30%).

[0231] 1 1H NMR (599 MHz, CDCl3) δ (ppm) 7.73 (ddd, J = 11.1, 8.0, 1.4 Hz, 4H), 7.46–7.37 (m, 6H), 3.67–3.61 (m, 1H), 3.05 (ddd, J = 5.9, 4.1, 2.8 Hz, 1H), 2.75–2.70 (m, 1H), 2.53 (dd, J = 5.0, 2.7 Hz, 1H), 1.12 (d, J = 6.4 Hz, 3H), 1.10 (s, 9H).

[0232] Step 3: Synthesis of DAW50072-3

[0233]

[0234] Dissolve DAW50072-2 (4.15 g, 12.71 mmol) and uracil (1.57 g, 13.98 mmol) in DMF (50 mL), cool down to -5 °C, add sodium hydride (0.15 g, 3.81 mmol) under stirring. After stirring for 5 min after addition, transfer to room temperature, then heat to 110 °C and react overnight. Stop the reaction, after cooling to room temperature, dilute with EA (100 mL). The resulting mixture is washed successively with saturated sodium bicarbonate (150 mL × 2) aqueous solution and saturated sodium chloride (150 mL) aqueous solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue is purified by silica gel column chromatography (DCM / methanol (V / V) = 20 / 1) to obtain white foamy solid DAW50072-3 (3.43 g, yield 61.53%).

[0235] MS (ESI, pos. ion) m / z: 439.2 [M + H] + .

[0236] Step 4: Synthesis of DAW50072-4

[0237]

[0238] Dissolve DAW50072-3 (3.43 g, 7.82 mmol) in THF (50 mL), add tetrabutylammonium fluoride (0.61 g, 2.35 mmol) under stirring, and react at 35 °C overnight. Concentrate the reaction solution, and the residue is purified by silica gel column chromatography (DCM / methanol (V / V) = 9 / 1) to obtain white foamy solid DAW50072-4 (1.374 g, yield 87.76%).

[0239] MS (ESI, pos. ion) m / z: 201.2 [M + H] + .

[0240] Step 5: Synthesis of DAW50072-a and DAW50072-b

[0241]

[0242] Under nitrogen protection, 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 the reaction was carried out overnight at room temperature. The reaction solution was concentrated under reduced pressure. The residue was dissolved in DCM (150 mL), then washed with saturated sodium chloride (100 mL×3). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and further purified by preparative chromatography column to obtain white foamy solid DAW50072-5-a (3.60 g, yield: 32.82%) and DAW50072-5-b (2.48 g, yield: 22.61%).

[0243] DAW50072-5-a: 1 H NMR (599 MHz, CDCl3) δ (ppm) 8.73 (s, 1H), 7.47–7.43 (m, 2H), 7.34 (t, J=5.9 Hz,

[0244] 4H), 7.29 (t, J=7.6 Hz, 2H), 7.23 (d, J=7.3 Hz, 1H), 6.91 (d, J=7.9 Hz, 1H), 6.83 (d, J=8.7 Hz, 4H), 5.53 (d, J=7.9 Hz, 1H), 3.79 (s, 6H), 3.70–3.57 (m, 4H), 2.66 (d, J=7.2 Hz, 1H), 1.18 (d, J=6.5 Hz, 3H).

[0245] DAW50072-5-b: 1 H NMR (599 MHz, CDCl3) δ (ppm) 9.13 (s, 1H), 7.47 (d, J=7.4 Hz, 2H), 7.36 (t, J=9.4

[0246] Hz, 4H), 7.29–7.25 (m, 2H), 7.21 (d, J = 7.3 Hz, 1H), 7.12 (d, J = 7.9 Hz, 1H), 6.82 (dd, J = 8.8, 1.0 Hz, 4H), 5.57 (d, J = 7.9 Hz, 1H), 3.87 (dd, J = 14.1, 1.5 Hz, 1H), 3.78 (d, J = 2.1 Hz, 6H), 3.64 (dd, J = 14.1, 7.8 Hz, 1H), 3.45 (dd, J = 8.9, 5.1 Hz, 2H), 2.96 (d, J = 3.8 Hz, 1H), 1.04 (d, J = 5.9 Hz, 3H).

[0247] Step 6: Synthesis of DAW50072-a

[0248]

[0249] Dissolve DAW50072-5-a (0.5 g, 0.99 mmol), 1H-tetrazole (86 mg, 1.20 mmol) and TEA (0.31 g, 3.02 mmol) in DCM (20 mL). While stirring, add bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.51 g, 1.68 mmol). The reaction mixture is stirred at room temperature for 3.5 h. After completion of the reaction, dilute with DCM (20 mL). The mixture is washed with saturated sodium chloride solution (40 mL × 2), dried over anhydrous sodium sulfate, and the solvent is concentrated. The residue is purified by C18 reverse-phase column chromatography (acetonitrile / water (V / V) = 70 / 30) to obtain white solid DAW50072-a (330 mg, yield 47.20%).

[0250] 1 1H NMR (599 MHz, CDCl3) δ (ppm) 8.00 (s, 1H), 7.40–7.37 (m, 2H), 7.31–7.23 (m, 6H), 7.20 (d, J = 7.3 Hz, 1H), 7.01 (d, J = 7.9 Hz, 1H), 6.81–6.77 (m, 4H), 5.41 (d, J = 7.9 Hz, 1H), 4.06 (dd, J = 13.9, 3.2 Hz, 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, 7.0 Hz, 3H), 2.52–2.45 (m, 2H), 1.35 (d, J = 6.5 Hz, 3H), 1.13 (d, J = 6.8 Hz, 6H), 0.99 (d, J = 6.8 Hz, 6H).

[0251] 31P NMR (243 MHz, CDCl3) δ 147.04 (s), 146.42 (s).

[0252] Step 7: Synthesis of DAW50072-b

[0253]

[0254] Dissolve DAW50072-5-b (0.5 g, 0.99 mmol), 1H-tetrazole (86 mg, 1.20 mmol) and TEA (0.31 g, 3.02 mmol) in DCM (20 mL). While stirring, add bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.51 g, 1.68 mmol). React at room temperature for 3.5 h. After completion of the reaction, dilute with DCM (20 mL), wash with saturated sodium chloride (40 mL × 2), dry over anhydrous sodium sulfate, and concentrate the solvent. Purify by C18 reverse-phase column chromatography, eluting with acetonitrile / water = 70 / 30 to obtain white solid DAW50072-b (350 mg, yield: 50.0%).

[0255] 1 H NMR (400 MHz, CDCl3) δ 8.61 (s, 1H), 7.47 (d, J = 7.4 Hz, 2H), 7.35 (dd, J = 8.8, 5.0 Hz, 4H), 7.31–7.24 (m, 2H), 7.21 (t, J = 7.1 Hz, 1H), 7.16 (d, J = 7.9 Hz, 1H), 6.82 (dd, J = 8.8, 1.7 Hz, 4H), 5.63 (d, J = 7.9 Hz, 1H), 4.24 (dd, J = 13.9, 2.5 Hz, 1H), 3.89–3.63 (m, 10H), 3.52 (ddd, J = 13.3, 10.9, 6.9 Hz, 3H), 2.41 (q, J = 6.4 Hz, 2H), 1.10 (t, J = 7.5 Hz, 12H), 0.96 (d, J = 6.4 Hz, 3H). MS (ESI, pos.ion) m / z: 703.32 [M+H] + . 31 P NMR (243 MHz, CDCl3) δ 149.35 (d, J = 237.3 Hz).

[0256] As is well known in the art, after the nucleoside monomers DAW50072-a and DAW50072-b are incorporated into oligonucleotides and deprotected during synthesis, they are respectively the nucleotide residues Y and N of the present invention.

[0257] The synthesis method of the antisense oligonucleotide of the present invention

[0258] It was completed according to the synthetic specification of 1 μmol theoretical yield. Weigh all the DNA phosphoramidite monomers, 2'-modified RNA phosphoramidite monomers, the phosphoramidite monomers of the present invention (i.e., the compounds described in the present invention), and auxiliary reagents of 1 μmol specification solid support CPG (purchased from Hebei Dina Xingke). All the phosphoramidite monomers are provided in a 0.1 M anhydrous acetonitrile solution. For oligonucleotides with phosphorothioate-modified phosphate backbones, a 0.1 M DDTT solution is used as the sulfurization reagent. A 5-ethylthio-1H-tetrazole acetonitrile solution (0.25 M) is used as the activator (purchased from Suzhou Kelema), and a dichloromethane solution of 3% trichloroacetic acid is used as the deprotection reagent, which are placed at the designated reagent positions corresponding to the DNA / RNA automatic synthesizer. Set the synthesis program and input the specified oligonucleotide base sequence. After checking without errors, start the cyclic oligonucleotide synthesis. The coupling time for each step is 6 minutes, and the sulfurization time is 6 minutes. After automatic cycling, an oligonucleotide containing solid support CPG is obtained.

[0259] Dry the obtained nucleotide containing solid support CPG with dry argon, then transfer it to a 2 mL EP tube, add 28% ammonia water solution (0.8 mL), and heat at 55 °C for 5 - 18 hours. Filter, wash the filter cake with water (0.5 mL), combine the filtrates, concentrate under reduced pressure, and obtain a white or yellow colloidal solid. After reverse-phase preparative purification, concentrate the preparation solution, pass through a gel column to remove excess salts, and obtain the oligonucleotide. The concentration of the obtained oligonucleotide is determined by a micro ultraviolet spectrophotometer (SPECTRO stat Nano). Mass spectrometry detection and analysis are completed on an Agilent 6530 LC-MS Q-Tof system. After the first-level scan, the nucleic acid molecular weight is calculated after deconvolution. The above method can be appropriately adjusted in combination with the synthetic methods well-known in the art, and the oligonucleotides of the present invention can be synthesized.

[0260] Synthesis of double-stranded siRNA and double-stranded siRNA conjugates of the present invention

[0261] 1. Synthesis of double-stranded siRNA without conjugated groups

[0262] The synthesis steps of the sense strand and antisense strand of the siRNA of the present invention are as follows:

[0263] It is completed according to the theoretical yield of 1 umol synthesis specification. Weigh all the 2'-modified RNA phosphoramidite monomers, the phosphoramidite monomers of the present invention (i.e., the compounds DAW50072-a and DAW50072-b described in the present invention), and auxiliary reagents of the solid support CPG of 1 umol specification (purchased from Hebei Dina Xingke). All the phosphoramidite monomers are provided in a 0.1M anhydrous acetonitrile solution. For oligonucleotides with a phosphorothioate-modified phosphate backbone, a 0.1M DDTT solution is used as the thioating reagent. A 5-ethylthio-1H-tetrazole acetonitrile solution (0.25M) is used as the activator (purchased from Suzhou Keloma), a 0.02M iodine pyridine / aqueous solution is used as the oxidant, and a 3% trichloroacetic acid dichloromethane solution is used as the deprotecting reagent, which are placed at the corresponding reagent designated positions of the KA-H8 model DNA / RNA automatic synthesizer. Set the synthesis program and input the specified oligonucleotide base sequence. After checking without errors, start the cyclic oligonucleotide synthesis. The coupling time for each step is 6 minutes, and the sulfuration time is 6 minutes. After automatic cycling, an oligonucleotide containing the solid support CPG is obtained.

[0264] Dry the obtained nucleotide containing the solid support CPG with dry argon, then transfer it to a 2 mL EP tube, add 28% ammonia aqueous solution (1.8 mL), and heat at 55 °C for 5 - 18 hours. Filter, wash the filter cake with water (0.5 mL), combine the filtrates, concentrate under reduced pressure, and obtain a white or yellow colloidal solid. After reverse-phase preparative purification, concentrate the preparation solution, pass through a gel column to remove excess salts, and obtain the oligonucleotide. The concentration of the obtained oligonucleotide is determined by a micro ultraviolet spectrophotometer (SPECTRO stat Nano). Mass spectrometry detection and analysis are completed on an Agilent 6530 LC-MS Q-Tof system. After the first-level scan, the nucleic acid molecular weight is calculated after deconvolution.

[0265] Annealing step:

[0266] Mix the sense strand of the double-stranded siRNA synthesized above with the antisense strand obtained above in an equimolar amount, heat to 95 °C, control the temperature for 10 min, and then slowly cool to room temperature. Subsequently, lyophilize to obtain the target double-stranded siRNA.

[0267] 2. Synthesis of siRNA conjugate:

[0268] The synthesis of the antisense strand is obtained by referring to the above synthesis method.

[0269] Synthesis of the sense strand: Replace the general solid support CPG with the GalNAc solid support prepared in the present invention, and prepare the sense strand connecting the conjugating group in the double-stranded siRNA conjugate of the present invention by referring to the above synthesis method.

[0270] Annealing step:

[0271] Mix the sense strand synthesized above with the antisense strand synthesized above in equimolar amounts, heat to 95°C, maintain the temperature for 10 min, and then slowly cool to room temperature. Subsequently, lyophilize to obtain the target siRNA conjugate. Some siRNAs or their conjugates of the present invention are shown in Table A.

[0272] Table A: Some siRNAs or their conjugates of the present invention

[0273]

[0274] Unless otherwise specified, in the context of the present invention, lowercase letters represent bases with a 2'-O-methyl modification at the 2'-position of the nucleotide ribose. For example, c, g, u, and a represent 2'-OMe (2'-O-methyl)C, 2'-OMe G, 2'-OMe U, and 2'-OMe A, respectively; the letter f to the right of an uppercase letter represents a base with a 2'-fluorine modification at the 2'-position of the nucleotide ribose. For example, Cf, Gf, Uf, and Af represent 2'-F (2'-fluorine)C, 2'-FG, 2'-F U, and 2'-FA, respectively; "s" represents a phosphorothioate linkage between the two nucleotide residues adjacent to "s" on the left and right. For example, "gsu" represents a phosphorothioate linkage between the g and u residues. Y represents N represents Tgn represents a thymine-diol nucleotide residue, and its structure is In the present invention, the 3' and 5' ends beside the O atom respectively represent that the O atom is connected to the 3' end (i.e., connected to the right nucleotide residue) and the 5' end (i.e., connected to the left nucleotide residue). Unless otherwise marked, it is default that the left O atom is connected to the left (i.e., the 3' end), and the right O atom is connected to the right (i.e., the 5' end).

[0275] The above synthesis scheme lists the general synthetic experimental steps for preparing the double-stranded siRNAs and their conjugates in the present invention. Those skilled in the art can prepare the double-stranded siRNAs and their conjugates described in the present invention by making appropriate modifications to the methods or adjustments to the raw materials according to the actual situation. Unless otherwise specified, the double-stranded siRNAs and their conjugates described in the present invention can be prepared by the methods described in the above synthesis scheme.

[0276] Cell activity and cytotoxicity tests of the nucleic acids or conjugates of the present invention

[0277] 1. Tests on the activity and cytotoxicity of the double-stranded siRNAs or conjugates of the present invention against hepatitis B virus

[0278] Testing method:

[0279] Hepatoma cells (Hep AD38) were cultured in DMEM medium containing 10% fetal bovine serum in a constant temperature incubator at 37 °C with 5% CO2. When the hepatoma cells were in the logarithmic growth phase and in good condition (70% confluence), they were used for the experiment. 1.5×10 4 cells were seeded into each well of a 96-well plate. After 24 h, different series of double-stranded siRNAs or their conjugates with a series of concentrations in the present invention were transfected according to the instructions of the RNAi MAX transfection reagent. Then, after incubation at 37 °C and 5% CO2 for 72 h, the HBsAg ELISA kit and CCK8 kit were used to detect the expression level of HBsAg and cytotoxicity, respectively.

[0280] The experimental results showed that the double-stranded siRNAs or their conjugates of the present invention embedded with the nucleotide residues of the present invention had good HBsA inhibitory activity, and the cytotoxicity of the double-stranded siRNAs or their conjugates of the present application was low.

[0281] 2. Experiment on HBV transgenic mice

[0282] Experimental method

[0283] Experimental animals: HBV-Tg transgenic mice, C57B / 6N-Tg(1.28HBV) / Vst, SPF-grade males, 6 - 8 weeks old, 16 - 20 g, purchased from Beijing Vital River Biotechnology Co., Ltd., animal production license number: SCXK(Jing)2019 - 0002. The animals were raised by Beijing Vital River Biotechnology Co., Ltd. and were housed individually in single cages.

[0284] Animal grouping: According to the quantitative detection results of mouse serum HBsAg (main) and HBeAg (subsidiary), the animals were randomly grouped in strata, with 5 animals in each group.

[0285] Preparation and administration of test articles: Calculate the required amount of drug (double-stranded siRNA or its conjugate) powder according to the purity of each test article. At a dosing concentration of 0.6 mg / mL, add the corresponding normal saline and shake well until a colorless and transparent liquid is obtained. The day of the first administration was defined as day 0. Each group of animals was administered once on day 0, and the subcutaneous administration volume was 5 mL / kg for all. If blood collection was required on the day of animal administration, the administration was carried out after blood collection.

[0286] Main observation indicators: Weigh and collect blood once on days 3, 7, 14, 21, 28, and 35, and separate the serum for the determination of HBsAg and HBeAg levels.

[0287] Determination of serum HBsAg and HBeAg levels: After blood collection, serum was separated and sent for inspection after dilution with PBS solution. 10 μL of serum was taken from each sample and diluted to 500 μL (diluted 50 times) with PBS solution. Serum HBsAg and HBeAg levels were determined using a hepatitis B e antigen assay kit and a hepatitis B surface antigen assay kit, respectively.

[0288] Experimental results of hepatitis B virus transgenic mice

[0289] The experimental results showed that the double-stranded siRNA or its conjugate of the present invention incorporating the nucleotide residues of the present invention had a good inhibitory effect on reducing HBsAg and also had a good inhibitory effect on reducing HBeAg.

[0290] 3. Activity and cytotoxicity testing of the siRNA or its conjugate of the present invention against AGT

[0291] Testing method:

[0292] Hep3B cells were cultured in DMEM medium containing 10% fetal bovine serum in a constant temperature incubator at 5% CO2 and 37 °C. When the cells were in the logarithmic growth phase and in good condition (70% confluence), transfection was carried out using a transfection reagent. The cell concentration was adjusted to 2.5×10 5 / mL, and 2×10 4 cells were seeded into each well of a 96-well plate, and different concentrations of the test siRNA and negative control were transfected according to the instructions of the lipofectamine RNAi MAX transfection reagent.

[0293] 1) After incubation at 37 °C and 5% CO2 for 24 h, the cells were collected, and the mRNA expression levels of AGT and GAPDH were detected using the QuickEasy Cell Direct RT-qPCR kit (Taqman). The relative quantification method was used to calculate the expression of the AGT gene in each sample, and finally the inhibition rate % = (relative quantification of the control - relative quantification of the sample) / relative quantification of the control × 100%, or a four-parameter fitting calculation was performed to calculate the IC 50 .

[0294] 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 by CCK8.

[0295] The experimental results showed that the siRNA and its conjugate of the present invention incorporating the nucleotide monomers of the present invention had a good knockdown effect on AGT mRNA. Among them, the experimental results of the inhibition rate of some siRNA conjugates against AGT are shown in Table B.

[0296] Table B: Inhibition rate of some siRNA conjugates of the present invention against AGT

[0297]

[0298]

[0299] 4. Evaluation of the knockdown activity of AGT siRNA conjugates using hAGT transgenic mice

[0300] To evaluate the in vivo activity of AGT siRNA conjugates, an AGT humanized mouse model (5 - 9 mice per group) was used. For drug administration, the baseline sera of the mice in each group were collected to detect body weight, ALT, and AGT protein content for grouping. On day D0, a single dose of 1 mg / kg or 3 mg / kg of GalNAc-siRNA or the vehicle saline was administered subcutaneously at the nape of the neck. Blood samples were collected on D0 / 4 / 7 / 14 / 21 / 28 / 35 / 49, and the human AGT protein concentration was measured using the Human AGT ELISA Kit (ab287170). The knockdown percentage was calculated by comparing the human AGT protein levels in the siRNA conjugate group and the vehicle group. The experimental results are shown in Figure 1 .

[0301] The experimental results show that the siRNA conjugates of the present invention after embedding the nucleotide residues of the present invention have a good knockdown effect on AGT mRNA in mice.

[0302] 5. Evaluation of the off-target effect of siRNA conjugates embedded with the nucleotide residues of the present invention using RNAseq technology

[0303] Human primary hepatocytes were resuscitated and plated onto 24-well plates at a cell density of 270,000 per well. At the same time of plating, the siRNA to be tested (2 concentrations, 3 replicates per concentration, and a cell control was set) was freely taken up into the cells. After 48 hours, the cells were collected, RNA was extracted, the concentration of total RNA was detected using NanoDrop One, the RNA integrity was analyzed, and RNAseq sequencing was performed using Illumina-NovaSeq. Among them, the experimental results of siRNA conjugate ID NO 1, siRNA conjugate ID NO 2, siRNA conjugate ID NO 3, and the positive control are shown in Figure 2-1 , Figure 2-2 , Figure 2-3 and Figure 2-4 . Among them, 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.

[0304] Experimental results show that the double-stranded siRNA conjugate of the present invention incorporating the nucleotide residues of the present invention can significantly reduce the number of down-regulated or up-regulated off-target genes. For example, the off-target effects of siRNA conjugates ID NO 1, siRNA conjugate ID NO 2, and siRNA conjugate ID NO 3 are significantly better than those of the positive control. Thus, the double-stranded siRNA conjugate of the present invention incorporating the nucleotide residues of the present invention can reduce the toxicity of siRNA drugs.

[0305] 6. Test on the activity and cytotoxicity of the single-stranded oligonucleic acid (ASO) of the present invention against hepatitis B virus

[0306] Testing method:

[0307] HepG2.2.15 cells were cultured in DMEM medium containing 10% fetal bovine serum in a constant temperature incubator at 5% CO2 and 37°C. When the cells were in the logarithmic growth phase and in good condition (70% confluence), transfection was carried out using a transfection reagent. The cell concentration was adjusted to 2.5×10 5 / mL, and 2*10 4 cells were seeded in each well of a 96-well plate. ASO was transfected according to the instructions of the RNAiMAX transfection reagent. After incubation at 37°C and 5% CO2 for 24 h, the fresh medium was replaced, and after continued culture for 72 h, the cell supernatant was collected to detect the expression level of HBsAg using an HBsAg Elisa kit, HBV DNA was detected by QPCR, cell cytotoxicity was detected by CCK8, and the cells were collected to detect the mRNA expression level using a QuickEasy Cell Direct RT-qPCR kit (Taqman).

[0308] Experimental results show that the oligonucleic acid of the present invention incorporating the nucleotide residues of the present invention has good inhibitory activity against HBsAg and low cytotoxicity.

[0309] 7. Test on the cell activity and cytotoxicity of the siRNA or its conjugate of the present invention against ANGPTL3

[0310] Testing method:

[0311] Hep3B cells were cultured in DMEM medium containing 10% fetal bovine serum in a constant temperature incubator at 5% CO2 and 37°C. When the cells were in the logarithmic growth phase and in good condition (70% confluence), transfection was carried out using a transfection reagent. The cell concentration was adjusted to 2.5×10 5 / mL, and 2*10 4Cells were transfected with siRNA according to the instructions of the RNAiMAX transfection reagent. After incubation at 37 °C and 5% CO2 for 24 h, the fresh medium was replaced. After continued culture for 24 h, the cell toxicity was detected by CCK8, and the cells were collected to detect the expression level of hANGPTL3 mRNA using the QuickEasy Cell Direct RT-qPCR kit (Taqman).

[0312] The experimental results show that the siRNA of the present invention or its conjugate incorporating the nucleotide residues of the present invention has good inhibitory activity against ANGPTL3.

[0313] 8. Cell viability and cytotoxicity tests of the siRNA or its conjugate of the present invention against APOC3

[0314] Testing method:

[0315] Hep3B cells were cultured in DMEM medium containing 10% fetal bovine serum in a constant temperature incubator at 5% CO2 and 37 °C. 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 4 cells were seeded in each well of a 96-well plate. The cells were transfected with siRNA according to the instructions of the RNAiMAX transfection reagent. After incubation at 37 °C and 5% CO2 for 24 h, the fresh medium was replaced. After continued culture for 24 h, the cell toxicity was detected by CCK8, and the cells were collected to detect the expression level of hAPOC3 mRNA using the QuickEasy Cell Direct RT-qPCR kit (Taqman).

[0316] The experimental results show that the siRNA of the present invention or its conjugate incorporating the nucleotide residues of the present invention has good inhibitory activity against APOC3.

[0317] 9. Cell viability and cytotoxicity tests of the siRNA or its conjugate of the present invention against LPA

[0318] In vitro knockdown activity screening:

[0319] Hek293 cells were cultured in DMEM medium containing 10% fetal bovine serum in a constant temperature incubator at 5% CO2 and 37°C. When the cells were in the logarithmic growth phase and in good condition (70% confluence), plasmid DNA (LPA_psiCHECK2 plasmid) was transfected into Hek293 cells using the transfection reagent Fugene HD. The transfected cells were seeded into a 96-well plate at a density of 10,000 cells per well, and the culture medium in each well was 100 μL. The cells were cultured in an incubator at 5% CO2 and 37°C overnight. Then, siRNA was transfected according to the RNAiMAX transfection reagent instruction manual. After incubation at 37°C and 5% CO2 for 24 h, the fresh medium was replaced. After continued culture for 24 h, the medium was discarded, and after lysing the cells, the Renilla luciferase signal was detected with reference to the dual-luciferase reporter assay kit instruction manual, and the ratio of the main reporter gene signal to the internal reference reporter gene signal in each well was calculated.

[0320] The experimental results showed that the siRNA or its conjugate of the present invention incorporating the nucleotide residues of the present invention had good inhibitory activity against LPA.

[0321] 10. Cell activity and cytotoxicity test of the siRNA or its conjugate of the present invention against PD-L1

[0322] Hep3B cells were cultured in DMEM medium containing 10% fetal bovine serum in a constant temperature incubator at 5% CO2 and 37°C. When the cells were in the logarithmic growth phase and in good condition (70% confluence), transfection was carried out using a transfection reagent. The cell concentration was adjusted to 2.5×10 5 / mL, and 2*10 4 cells were seeded into each well of a 96-well plate. siRNA was transfected according to the RNAi MAX transfection reagent instruction manual. After incubation at 37°C and 5% CO2 for 24 h, the fresh medium was replaced. After continued culture for 24 h, CCK8 was used to detect cell cytotoxicity, and the cells were collected and the hPD-L1 mRNA expression level was detected using the Quick Easy Cell Direct RT-qPCR kit (Taqman).

[0323] The experimental results showed that the siRNA and siRNA conjugate of the present invention incorporating the nucleotide residues of the present invention had good inhibitory effects against PD-L1.

[0324] 11. Cell activity and cytotoxicity test of the siRNA or its conjugate of the present invention against HSD17B13

[0325] Test method: Cos-7 cells were cultured in DMEM medium containing 10% fetal bovine serum in a constant temperature incubator at 37°C with 5% CO2. When the cells were in the logarithmic growth phase and in good condition (70% confluence), plasmid DNA (HSD17B13_psiCHECK2 plasmid) was transfected into Cos-7 cells using the transfection reagent FugeneHD. The transfected cells were seeded into a 96-well plate at a density of 10,000 cells per well, and the culture medium in each well was 100 μL. The cells were cultured in an incubator at 37°C with 5% CO2 overnight. Then, siRNA was transfected according to the RNAiMAX transfection reagent instruction manual. After incubation at 37°C with 5% CO2 for 24 h, the fresh medium was replaced. After continued culture for 24 h, the medium was discarded. After lysing the cells, the Renilla luciferase signal was detected with reference to the dual-luciferase reporter assay kit instruction manual, and the ratio of the main reporter gene signal to the internal reference reporter gene signal in each well was calculated.

[0326] The experimental results showed that the siRNA or its conjugate of the present invention incorporating the nucleotide residues of the present invention had good inhibitory activity against HSD17B13.

[0327] Although the present invention has been described in detail above with general descriptions, specific embodiments and experiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. 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), in, 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, 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 with 1, 2 or 3 substituents selected from deuterium, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methoxy and ethoxy; 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 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, further 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 are independently deuterium, H, CN, HO-, fluorine, chlorine, bromine, methyl, ethyl or methoxy; Each R 2 , R 3b and R 4c 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 Together with the N atom to which they are attached, they form a heterocyclic group consisting of 5-6 ring atoms, and each of the heterocyclic groups consisting of 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 are independently 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)-、MC(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, or Z and Y 1 each independently represents 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 Alkyl methyl, 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, or Z and Y 1 each independently represents 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 Alkyl methyl, 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, or Z and Y 1 each independently represents 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 Alkyl methyl, C 1-6 Alkylsilyl, benzyl, phenethyl, HOC(=O)(CH2) j C(=O)-、MC(=O)(CH2) j C(=O)-, triphenylmethyl, MMTr, DMTr, 4',4',4'-trimethoxytrityl, Wherein, X is Cl or Br; Each R x and R y are independently a hydroxy protecting group; Each R c and R d are independently H or an amino protecting group; or R c , R d Together with the N 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, 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, 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 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, 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 or R 4 C(=O)-, or R a , R b and together with the N atom to which they are attached form a pyrrolidinyl, morpholinyl, piperidinyl or piperazinyl, 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; Among them, each R 3 and R 4 Independently for C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, phenyl, halogen-substituted phenyl, C 1-6 Alkylphenyl or benzyl; or Each R 3 and R 4 Independently for C 1-4 Alkyl, halogenated C 1-4 Alkyl, C 1-4 Alkoxy, phenyl, halogen-substituted phenyl, C 1-4 Alkylphenyl or benzyl; or Each R 3 and R 4 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.

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, 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; or 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, allyloxycarbonyl, methyl, p-methoxybenzyldiphenylmethyl, tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, 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; 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, 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 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.

5. A compound according to any one of claims 1 to 4, having a structure as shown in formula (Ia) or (Ib), or a stereoisomer, tautomer or 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 to 5, which is one of the following compounds 1 to 25, or a stereoisomer, tautomer or acceptable salt thereof, in, A1 is A2 is A3 is U is T is 7. 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), in, 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 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; 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) to (20), or a stereoisomer, a tautomer or an acceptable salt thereof, in, A is G is C is U is T is Preferably, the nucleotide residues are 9. Use of the nucleotide residues according to claim 7 or 8 as intercalating groups in ASO drug research, siRNA drug research, gene function research and / or screening of a whole gene library.

10. The use according to claim 9, wherein the intercalating group is intercalated on the ASO drug, on the sense strand of the siRNA drug, on the antisense strand of the siRNA drug, or on the sense strand of the siRNA drug and the antisense strand of the siRNA drug.

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

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

13. The use according to any one of claims 9 to 12, wherein the siRNA drug comprises a sense strand and an antisense strand capable of forming a double-stranded region, the sense strand consisting of 15-40 nucleotides, preferably consisting of 17-25 nucleotides, more preferably consisting of 18-23 nucleotides; the antisense strand consisting of 15-40 nucleotides, preferably consisting of 17-35 nucleotides, more preferably consisting of 19-30 nucleotides, most preferably consisting 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 to 13, wherein the sense strand and / or antisense strand comprises 7, 6, 5, 4, 3, 2, 1 and 0 unmodified nucleotides, respectively.

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

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

17. A double-stranded siRNA, a conjugate or a salt thereof, comprising a sense strand and an antisense strand, and having at least one nucleotide residue according to any one of claims 7 or 8 embedded therein; Optionally, the siRNA, its conjugate or salt is used to inhibit the expression of disease-causing genes; Optionally, the pathogenic gene is selected from the group consisting of AGT gene, HBV gene, INHBE gene, HSD17B13 gene and PNPLA3 gene; Optionally, the nucleotide residues are used to enhance the inhibitory activity of the siRNA and / or reduce off-target effects; Optionally, the nucleotide residues are embedded in the antisense strand; Optionally, the nucleotide residue is embedded at position 5, 6 or 7 of the 5' end of the antisense strand.

18. A double-stranded siRNA, a conjugate or a salt thereof, characterized in that: It comprises a sense strand as shown in SEQ ID NO: 1; wherein the length of the sense strand does not exceed 23 nucleotides; Optionally, it further comprises an antisense strand shown in SEQ ID NO: 3 to SEQ ID NO: 5, wherein 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 to 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 comprising the double-stranded siRNA, its conjugate or salt thereof according to claim 19 , and a pharmaceutically acceptable carrier.

21. Use of the double-stranded siRNA, its conjugate and salt thereof according to any one of claims 18 to 19 or the pharmaceutical composition according to claim 20 in the preparation of a drug for treating and / or preventing AGT-related diseases; Preferably, the AGT-related disease is hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, 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.

22. The use according to claim 21, 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, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension and unstable hypertension.

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