Oligonucleotide delivery ligands comprising sugars

Abstract

Compounds of Formula (I), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof, are provided. The compound of formula (I) is contained inside the nucleotide, or at the 5 '-and / or 3'-terminus, for delivery of the double-stranded RNA to extrahepatic tissue, such as the central nervous system or the eye. Oligonucleotides, double-stranded RNAs, vectors, cells, pharmaceutical compositions and kits comprising the compounds of Formula (I) are also provided.

Description

Sugar-containing oligonucleotide delivery ligands

[0001] This application claims priority to Chinese invention patent application CN202310693838.2, filed June 12, 2023, which is incorporated herein by reference in its entirety. Field of the Invention

[0002] The present invention belongs to the field of medicine, and specifically relates to a delivery vector capable of delivering double-stranded RNA to extrahepatic tissues, such as the central nervous system or the eye. The delivery vector is a sugar modified with a hydrophobic group, such as a compound of formula (X), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. Background Art

[0003] RNA interference is a phenomenon in which double-stranded RNA (dsRNA, also known as siRNA) induces the efficient and specific degradation of target mRNA.

[0004] However, due to the presence of the blood-brain barrier, it is difficult to deliver siRNA to the central nervous system to function, which limits the application of siRNA. Some attempts have been made in this field to deliver siRNA to the central nervous system. For example, WO2004094595A2 discloses the use of a single lipid ligand (such as cholesterol or long-chain alkanes) at the end of the chain to deliver siRNA, WO2019217459A1 discloses the use of a single lipid ligand to deliver siRNA inside the chain, and WO2021092371A2 discloses a series of new lipid ligand structures.

[0005] There is still a need in this field to develop more hydrophobic groups to more effectively deliver siRNA to the central nervous system.

[0006] Summary of the Invention

[0007] In one aspect, the present invention provides an oligonucleotide comprising one or more compounds of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0008] wherein each group is as defined below.

[0009] In another aspect, the present invention provides an oligonucleotide comprising one or more compounds of formula (II) or formula (III), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0010] wherein each group is as defined below.

[0011] In another aspect, the present invention provides an oligonucleotide comprising one, two or more delivery vectors within the oligonucleotide, at the 5' end and / or at the 3' end, wherein the delivery vector is a sugar modified with a hydrophobic group;

[0012] Preferably, the sugar modified by the hydrophobic group is selected from the compound of formula (X), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0013] wherein each group is as defined below.

[0014] In another aspect, the present invention provides a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence that is sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or antisense strand comprises one or more compounds of formula (II) or formula (III) above, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0015] In another aspect, the present invention provides a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or antisense strand comprises one, two or more delivery vectors internally, at the 5' end and / or at the 3' end, and the delivery vector is a sugar modified with a hydrophobic group;

[0016] Preferably, the sugar modified by the hydrophobic group is selected from the compound of formula (X), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0017] wherein each group is as defined below.

[0018] In another aspect, the present invention provides a compound of formula (II') or (III'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0019] wherein each group is as defined below.

[0020] In another aspect, the hydrophobic group provided by the present invention (the P group in formula (I), (II) or (III)) can be linked to the oligonucleotide or double-stranded RNA through the hydroxyl group or acetyl group of the sugar portion of the nucleotide.

[0021] In another aspect, the present invention provides a vector comprising a nucleotide sequence encoding the aforementioned double-stranded RNA.

[0022] In another aspect, the present invention provides a cell containing the aforementioned double-stranded RNA or the aforementioned vector.

[0023] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned double-stranded RNA, the aforementioned vector, or the aforementioned cell, and optionally a pharmaceutically acceptable carrier or excipient.

[0024] In another aspect, the present invention provides a kit comprising the aforementioned double-stranded RNA, the aforementioned vector, or the aforementioned cell.

[0025] Detailed Description of the Invention

[0026] definition

[0027] Chemical definition

[0028] Definitions of specific functional groups and chemical terms are described in more detail below.

[0029] When a numerical range is listed, it is intended to include every value and sub-range within the stated range. For example, "C 1-6 "Alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-5 、C 2-4 、C 2-3 、C 3-6 、C 3-5 、C 3-4 、C 4-6 、C 4-5 and C 5-6 alkyl.

[0030] “C 1-6 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms. 8-30 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 30 carbon atoms. In some embodiments, C 8-25 Alkyl, C 10-22 Alkyl, C 8-20 Alkyl, C 1-10 Alkyl and C 1-6 Alkyl groups are preferred. 1-6 Examples of alkyl groups include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). The term “C 1-6"Alkyl" also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me(-CH3), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3), or i-Bu(-CH2CH(CH3)2).

[0031] “C 2-10 "Alkenyl" refers to a straight or branched chain hydrocarbon group having 2 to 10 carbon atoms and at least one carbon-carbon double bond. 8-30 "Alkenyl" refers to a straight or branched chain hydrocarbon group having 8 to 30 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 10-22 Alkenyl, C 2-10 Alkenyl, C 2-6 Alkenyl and C 2-4 Alkenyl is preferred. 2-6 Examples of alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. The term "C 2-6 "Alkenyl" also includes heteroalkenyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). An alkenyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0032] “C 2-10 "Alkynyl" refers to a straight or branched chain hydrocarbon group having 2 to 10 carbon atoms and at least one carbon-carbon triple bond. 8-30 "Alkynyl" refers to a straight or branched chain hydrocarbon group having 8 to 30 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 10-22 Alkynyl, C 2-10 Alkynyl, C 2-6 Alkynyl and C 2-4 Alkynyl is preferred. 2-6 Examples of alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. The term "C 2-6"Alkynyl" also includes heteroalkynyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups can be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0033] “C 1-10 Alkylene", "C 2-10 Alkenylene" and "C 2-10 "Alkynylidene" refers to the group with the C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 In some embodiments, C 2-8 Alkylene, C 3-7 Alkylene, C 1-6 Alkylene, C 4-6 Alkylene, C 1-4 Alkylene, C 2-4 Alkylene and C 1-3 Alkylene is preferred. Unsubstituted alkylene includes, but is not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and the like. Exemplary substituted alkylenes, for example, alkylenes substituted with one or more alkyl (methyl) groups, include, but are not limited to, substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3) 2- ), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), and the like.

[0034] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0035] Therefore, “C 1-6 "Haloalkyl" refers to the above-mentioned "C 1-6 "alkyl" is substituted with one or more halogen groups. In some embodiments, C 1-4 Halogenated alkyl is particularly preferred, more preferably C 1-2Haloalkyl. Exemplary haloalkyl groups include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like. The haloalkyl group can be substituted at any available point of attachment, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0036] “C 3-10 "Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, C 4-7 Cycloalkyl and C 3-6 Cycloalkyl is particularly preferred, more preferably C 5-6 Cycloalkyl. Cycloalkyl also includes a ring system in which the above-mentioned cycloalkyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such a case, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Exemplary cycloalkyls include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), and the like. The cycloalkyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0037] "3-10 membered heterocyclyl" refers to a group of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valence permits. In some embodiments, a 4-10 membered heterocyclyl is preferred, which is a 4-10 membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, a 3-8 membered heterocyclyl is preferred, which is a 3-8 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; a 3-6 membered heterocyclyl is preferred, which is a 3-6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; a 4-7 membered heterocyclyl is preferred, which is a 4-7 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; and a 5-6 membered heterocyclyl is more preferred, which is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes a ring system in which the above-mentioned heterocyclyl ring is fused to one or more cycloalkyl groups, wherein the point of attachment is on the cycloalkyl ring, or a ring system in which the above-mentioned heterocyclyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring; and in such a case, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Exemplary 3-membered heterocyclyls containing one heteroatom include, but are not limited to, aziridine, oxirane, and thiorenyl. Exemplary 4-membered heterocyclyls containing one heteroatom include, but are not limited to, azetidinyl, oxetane, and thiidine. Exemplary 5-membered heterocyclyls containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, hexahydrotriazinyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thienyl. Exemplary 5-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclyl) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinonyl, and the like.Exemplary 6-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl groups) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. The heterocyclyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0038] The term "hydrophobic group" refers broadly to any chemical group that has an affinity for lipids. One way to characterize the hydrophobicity of a hydrophobic group is by the octanol-water partition coefficient log K ow , where K ow is the ratio of the concentration of a chemical in the octanol phase to its concentration in the aqueous phase at equilibrium in a two-phase system. ow More than 1, more than 1.5, more than 2, more than 3, more than 4, more than 5 or more than 10. Specifically for the present invention, the hydrophobic portion is the R group in the compound of formula I.

[0039] Alkyl, alkenyl, alkynyl, etc., as defined herein, are optionally substituted groups.

[0040] Exemplary substituents on carbon atoms include, but are not limited to, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa 、-ON(R bb )2、-N(R bb )2、-N(R bb )3 + X - 、-N(OR cc )R bb 、-SH、-SR aa 、-SSR cc 、-C(=O)R aa 、-CO2H、-CHO、-C(OR cc )2, -CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NRbb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3、-OSi(R aa )3、-C(=S)N(R bb )2、-C(=O)SR aa 、-C(=S)SR aa 、-SC(=S)SR aa 、-SC(=O)SR aa 、-OC(=O)SR aa 、-SC(=O)OR aa 、-SC(=O)R aa 、-P(=O)2R aa 、-OP(=O)2R aa 、-P(=O)(R aa )2、-OP(=O)(R aa )2、-OP(=O)(OR cc )2、-P(=O)2N(R bb )2、-OP(=O)2N(R bb )2、-P(=O)(NR bb )2、-OP(=O)(NR bb )2、-NR bb P(=O)(OR cc )2、-NR bb P(=O)(NR bb )2、-P(R cc )2、-P(R cc )3、-OP(R cc )2、-OP(Rcc )3、-B(R aa )2、-B(OR cc )2, -BR aa (OR cc ), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0041] Or the two geminal hydrogen atoms on the carbon atom are replaced by groups =O, =S, =NN(R bb )2, =NNR bb C(=O)R aa 、=NNR bb C(=O)OR aa 、=NNR bb S(=O)2R aa 、=NR bb or = NOR cc replace;

[0042] R aa Each of R is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R aa The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0043] R bb Each of the following is independently selected from: hydrogen, -OH, -OR aa 、-N(R cc )2, -CN, -C(=O)R aa 、-C(=O)N(R cc )2, -CO2R aa 、-SO2R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc )2, -SO2R cc 、-SO2OR cc 、-SOR aa 、-C(=S)N(R cc )2, -C(=O)SR cc 、-C(=S)SR cc 、-P(=O)2R aa、-P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R bb The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0044] R cc Each of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R cc The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0045] R dd Each of the is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee 、-ON(R ff )2、-N(R ff )2,、-N(R ff )3 + X - 、-N(OR ee )R ff 、-SH、-SR ee 、-SSR ee 、-C(=O)R ee 、-CO2H、-CO2R ee 、-OC(=O)R ee 、-OCO2R ee 、-C(=O)N(R ff )2、-OC(=O)N(R ff )2、-NR ff C(=O)R ee 、-NR ff CO2R ee 、-NR ff C(=O)N(R ff )2、-C(=NR ff )OR ee 、-OC(=NR ff )R ee 、-OC(=NR ff )OR ee、-C(=NR ff )N(R ff )2、-OC(=NR ff )N(R ff )2、-NR ff C(=NR ff )N(R ff )2、-NR ff SO2R ee 、-SO2N(R ff )2, -SO2R ee 、-SO2OR ee 、-OSO2R ee 、-S(=O)R ee 、-Si(R ee )3、-OSi(R ee )3、-C(=S)N(R ff )2, -C(=O)SR ee 、-C(=S)SR ee 、-SC(=S)SR ee 、-P(=O)2R ee 、-P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR ee )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg Group substitution, or two geminal R dd Substituents may combine to form =O or =S;

[0046] R ee Each of R is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R gg group substitution;

[0047] R ff Each of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R ff The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R gg group substitution;

[0048] R ggEach of the independently: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 Alkyl, -ON(C 1-6 Alkyl)2, -N(C 1-6 Alkyl)2, -N(C 1-6 Alkyl)3 + X - 、-NH(C 1-6 Alkyl)2 + X - 、-NH2(C 1-6 alkyl) + X - 、-NH3 + X - 、-N(OC 1-6 Alkyl)(C 1-6 Alkyl), -N(OH)(C 1-6 Alkyl), -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 Alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 Alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 Alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 Alkyl, -C(=NH)N(C 1-6 Alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 Alkyl)2, -OC(NH)NH(C 1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6Alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 Alkyl), -SO2N(C 1-6 Alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2C 1-6 Alkyl, -SO2OC 1-6 Alkyl, -OSO2C 1-6 Alkyl, -SOC 1-6 Alkyl, -Si(C 1-6 alkyl)3, -OSi(C 1-6 alkyl)3, -C(=S)N(C 1-6 alkyl)2、C(=S)NH(C 1-6 alkyl), C(=S)NH2, -C(=O)S(C 1-6 alkyl), -C(=S)SC 1-6 Alkyl, -SC(=S)SC 1-6 Alkyl, -P(=O)2(C 1-6 alkyl), -P(=O)(C 1-6 alkyl)2, -OP(=O)(C 1-6 alkyl)2, -OP(=O)(OC 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C 10 Aryl, C3-C7 heterocyclic, C5-C 10 heteroaryl; or two geminal R gg Substituents may combine to form =O or =S; wherein X - For the counter ion.

[0049] Exemplary substituents on nitrogen atoms include, but are not limited to, hydrogen, -OH, -OR aa 、-N(R cc )2, -CN, -C(=O)R aa 、-C(=O)N(R cc )2, -CO2R aa 、-SO2R aa 、-C(=NR bb )R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc )2, -SO2R cc 、-SO2OR cc 、-SORaa 、-C(=S)N(R cc )2, -C(=O)SR cc 、-C(=S)SR cc 、-P(=O)2R aa 、-P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R attached to the nitrogen atom cc The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd Group substituted, and wherein R aa 、R bb 、R cc and R dd As mentioned above.

[0050] Other definitions

[0051] The term "siRNA" herein refers to a class of double-stranded RNA molecules that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a gene encoding a protein) that is complementary thereto. siRNA is typically double-stranded, comprising an antisense strand complementary to the target RNA and a sense strand complementary to the antisense strand. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA (e.g., tRNA) and viral RNA other than mRNA can also be targeted.

[0052] The term "antisense oligonucleotide" or ASO (Antisense Oligonucleotides) is a single-stranded DNA or RNA sequence consisting of 15-25 nucleotides that is paired with a target gene. It achieves the purpose of gene regulation by specifically blocking the transcription or translation process of the target gene.

[0053] The term "antisense strand" refers to a strand of an siRNA that includes a region that is completely, fully, or substantially complementary to a target sequence. The term "sense strand" refers to a strand of an siRNA that includes a region that is completely, fully, or substantially complementary to a region that is, as the term is defined herein, an antisense strand.

[0054] The term "complementary region" refers to a region on the antisense strand that is completely, fully or substantially complementary to the target mRNA sequence. In the case where the complementary region is not completely complementary to the target sequence, mispairing can be located in the interior or terminal regions of the molecule. Typically, the most tolerated mispairing is located in the terminal regions, for example, in 5' and / or 3' ends within 5, 4, 3, 2 or 1 nucleotide. The antisense strand portion that is most sensitive to mispairing is referred to as a "seed region." For example, in a siRNA comprising a 19nt chain, the 19th position (from 5' to 3') can tolerate some mispairing.

[0055] The term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50° C. or 70° C. for 12-16 hours. In terms of meeting the above requirements relative to their ability to hybridize, "complementary" sequences may also include or be completely formed from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0056] A polynucleotide that is "at least partially complementary," "sufficiently complementary," or "substantially complementary" to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest. For example, a polynucleotide is complementary to at least a portion of a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding PCSK9. The terms "complementary," "fully complementary," "sufficiently complementary," and "substantially complementary" as used herein can be used with respect to base pairing between the sense and antisense strands of an siRNA, or between the antisense strand of an siRNA agent and a target sequence.

[0057] "Fully complementary" refers to the extent to which the sense strand only needs to be complementary to the antisense strand in order to maintain the overall double-stranded nature of the molecule. In other words, while perfect complementarity is generally desired, in some cases, particularly in the antisense strand, one or more, for example, 6, 5, 4, 3, 2, or 1 mismatches (relative to the target mRNA) may be included, but the sense and antisense strands can still maintain the overall double-stranded nature of the molecule.

[0058] A "nucleoside" is a compound composed of a purine or pyrimidine base and ribose or deoxyribose; a "nucleotide" is a compound composed of a purine or pyrimidine base, ribose or deoxyribose, and phosphate; an "oligonucleotide" is a nucleic acid molecule (RNA or DNA) having, for example, fewer than 100, 200, 300, or 400 nucleotides in length.

[0059] "Bases" are the fundamental building blocks of nucleosides, nucleotides, and nucleic acids. They contain nitrogen and are also called "nitrogenous bases." Unless otherwise specified, the capital letters A, U, T, G, and C represent the bases of nucleotides, representing adenine, uracil, thymine, guanine, and cytosine, respectively.

[0060] The "modification" of nucleotides herein includes, but is not limited to, methoxy modification, fluorination, phosphorothioate linkage, or conventional protecting group protection. For example, the fluorination-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0061] " modified nucleotide " herein includes but is not limited to 2 '-O-methyl modified nucleotides, 2 '-fluoro modified nucleotides, 2 '-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, nucleotides comprising thiophosphate groups, vinyl phosphate modified nucleotides, locked nucleotides, 2 '-amino-modified nucleotides, 2 '-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, the non-natural bases comprising nucleotides and the terminal nucleotides, deoxyribonucleotides or conventional protecting group protections on a cholesterol derivative or a dodecanoic acid didecylamide group. For example, the 2 '-fluoro modified nucleotides refer to nucleotides in which the hydroxyl group at the ribose group 2 ' position is replaced by fluorine. The 2 '-deoxy-modified nucleotides refer to nucleotides in which the 2 '-hydroxyl group at the ribose group is replaced by methoxy and formed.

[0062] " reactive phosphorus group " refers to the phosphorus-containing group contained in the nucleotide unit or in the nucleotide analog unit, and it can react with the hydroxyl or the amido reaction contained in another molecule, especially in another nucleotide unit or in another nucleotide analog by nucleophilic attack reaction.Usually, such reaction produces the ester type internucleoside bond that the first nucleotide unit or the first nucleotide analog unit are connected with the second nucleotide unit or the second nucleotide analog unit.Reactive phosphorus group can be selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphate or phosphate analogs, include but not limited to: natural phosphate, thiophosphate, phosphorodithioate, borane phosphate, borane thiophosphate, phosphonate, halogen-substituted phosphonate and phosphate, phosphoramidate, phosphodiester, phosphotriester, thiophosphate diester, thiophosphate triester, diphosphate and triphosphate, preferably-P(OCH2CH2CN)(N(iPr)2).

[0063] A "protecting group" refers to any atom or group of atoms added to a molecule to prevent an existing group in the molecule from undergoing an undesirable chemical reaction. A "protecting group" may be an unstable chemical moiety known in the art that is used to protect reactive groups, such as hydroxyl, amino, and thiol groups, to prevent undesirable or inappropriate reactions during chemical synthesis. Protecting groups are typically used selectively and / or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group intact or available for further reactions.

[0064] A non-limiting list of protecting groups includes benzyl; substituted benzyl; alkylcarbonyl and alkoxycarbonyl (e.g., tert-butyloxycarbonyl (BOC), acetyl, or isobutyryl); arylalkylcarbonyl and arylalkoxycarbonyl (e.g., benzyloxycarbonyl); substituted methyl ethers (e.g., methoxymethyl ether); substituted ethyl ethers; substituted benzyl ethers; tetrahydropyranyl ethers; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy]methyl, or tert-butyldiphenylsilyl); esters (e.g., benzyl); esters); carbonates (e.g., methoxymethyl carbonate); sulfonates (e.g., tosylate or mesylate); acyclic ketals (e.g., dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); acyclic acetals; cyclic acetals (e.g., those described herein); acyclic hemiacetals; cyclic hemiacetals; cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiolane); orthoesters (e.g., those described herein); and triarylmethyl groups (e.g., trityl; monomethoxytrityl (MMTr); 4,4′-dimethoxytrityl (DMTr); 4,4′,4″-trimethoxytrityl (TMTr); and those described herein). Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethyl acetal (BOM), β-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), triphenyl methyl (Trt), methoxytrityl [(4-methoxyphenyl) diphenylmethyl] (MMT), dimethoxytrityl, [bis-(4-methoxyphenyl) phenylmethyl (DMT), trimethylsilyl ether (TMS), tert-butyldimethylsilyl ether (TBDMS), tri-iso-propylsilyloxymethyl ether (TOM), tri-isopropylsilyl ether (TIPS), methyl ether, ethoxyethyl ether (EE) N,N-dimethylformamidine and 2-cyanoethyl (CE).

[0065] A "hydroxyl protecting group" is a group that protects the hydroxyl group from chemical reactions and can be removed under specific conditions to restore the hydroxyl group. These groups primarily include silane-type protecting groups, acyl-type protecting groups, or ether-type protecting groups, with the following being preferred:

[0066] Trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc) , benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, more preferably -C(O)CH2CH2C(O)OH.

[0067] As used herein, the term "pharmaceutically acceptable salt" refers to those carboxylate salts, amino acid addition salts of the compounds of the present invention that are suitable for use in contact with patient tissues within the scope of sound medical judgment, do not produce undue toxicity, irritation, allergic response, etc., are commensurate with a reasonable benefit / risk ratio, and are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the present invention.

[0068] The present invention includes tautomers, which are functional isomers produced by the rapid movement of an atom in two positions in a molecule. Compounds that exist in different tautomeric forms are not limited to any specific tautomer, but are intended to cover all tautomeric forms.

[0069] The compounds of the present invention may include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, for example, enantiomers and / or diastereomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

[0070] The present invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described in formula (I) but for which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, for example 2 H. 3 H. 13 C. 11 C. 14 C. 15 N. 18 O. 17 O. 31 P. 32 P. 35 S. 18 F and 36 Cl. Compounds of the present invention containing the above-mentioned isotopes and / or other isotopes of other atoms, their prodrugs and pharmaceutically acceptable salts of the compounds or prodrugs are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, such as those in which radioactive isotopes (e.g. 3 H and 14 C) can be used in drug and / or substrate tissue distribution assays. 3 H and carbon-14, i.e. 14 C isotopes are particularly preferred because they are easy to prepare and detect. 2 H, because greater metabolic stability can provide therapeutic benefits, such as prolonged in vivo half-life or reduced dosage requirements, and thus may be preferred in some cases. Isotopically labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents when carrying out the processes disclosed in the following schemes and / or the Examples and Preparations.

[0071] Compounds of the present invention

[0072] The present invention specifically relates to an oligonucleotide comprising one or more compounds of formula (I), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0073] in,

[0074] In formula (I) represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0075] X1 is selected from H, Rs or

[0076] X2 is selected from OR1 or

[0077] One of X1 and X2 is

[0078] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0079] In formula (I) and R1 are not H at the same time;

[0080] Each L1 and L2 are independently selected from a chemical bond, C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, OC 1-10 Alkylene, OC 1-10 Alkenylene, OC 1-10 Alkynylidene, C(O)C 1-10 Alkylene; said L1 and L2 are optionally substituted by 1, 2, 3, 4, 5, 6, 7 or 8 R;

[0081] T is selected from a chemical bond, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M- or -OC(O)-M-;

[0082] Where M is

[0083] A is a sugar, which is preferably a sugar that can be cleaved in endosomes or lysosomes, preferably a penta- or hexa-membered sugar, more preferably a hexa-membered sugar, such as N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably N-acetylgalactosamine or N-acetylglucosamine;

[0084] P is a hydrophobic group, preferably C 8-30 Alkyl, C 8-30 Alkenyl or C8-30 Alkynyl, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH2CH2- group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring; the C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R;

[0085] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0086] R s Selected from H, D, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, 3-10 membered heterocyclic group or C 3-10 Cycloalkyl, which is optionally deuterated, up to fully deuterated;

[0087] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0088] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0089] k=0, 1, 2, 3, 4, 5 or 6.

[0090] The present invention specifically relates to an oligonucleotide, wherein the compound of formula (I) is selected from a compound of formula (II) or formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0091] in,

[0092] R s 'Selected from H, D, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, 3-10 membered heterocyclic group or C 3-10 Cycloalkyl, which is optionally deuterated, up to fully deuterated;

[0093] The other groups are as defined above.

[0094] The present invention specifically relates to a compound of formula (II') or (III'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0095] R1 and R2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;

[0096] A is an acetylated sugar, which is preferably a sugar that can be cleaved in endosomes or lysosomes, preferably an acetylated penta- or hexa-sugar, such as N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably N-acetylgalactosamine or N-acetylglucosamine;

[0097] Preferably, A is selected from

[0098] A is connected to L1 through the a-terminal end and to the P group through the b-terminal end;

[0099] P, L1, L2, T, Rs, Rs', m, n, k are as defined above.

[0100] The present invention specifically relates to a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand comprises a sequence that is sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or antisense strand comprises one or more compounds of the aforementioned formula (II) or formula (III), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0101] In one embodiment, represents H; in another embodiment, Indicates the position of the phosphate or phosphorothioate linkage to the adjacent nucleotide.

[0102] R1 and R2

[0103] In one embodiment, R1 represents H; in another embodiment, R1 represents the position of attachment to the phosphate or phosphorothioate of another adjacent nucleotide.

[0104] In one embodiment, R1 is H; in another embodiment, R1 is selected from a reactive phosphorus group, preferably a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimetic, such as a natural phosphate, a phosphorothioate, a phosphorodithioate, a boranephosphate, a boranephosphorothioate, a phosphonate, a halogen-substituted phosphonate and a phosphate, a phosphoramidate, a phosphodiester, a phosphotriester, a phosphorothioate diester, a phosphorothioate triester, a diphosphate or a triphosphate, preferably -P(OCH2CH2CN)(N(iPr)2); in another embodiment, R1 is selected from a hydroxy protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), Acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, preferably DMTr; in another embodiment, R1 is a solid support.

[0105] In one embodiment, R2 is H; in another embodiment, R2 is selected from a reactive phosphorus group, preferably a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimetic, such as a natural phosphate, a phosphorothioate, a phosphorodithioate, a boranephosphate, a boranephosphorothioate, a phosphonate, a halogen-substituted phosphonate and a phosphate, a phosphoramidate, a phosphodiester, a phosphotriester, a phosphorothioate diester, a phosphorothioate triester, a diphosphate or a triphosphate, preferably -P(OCH2CH2CN)(N(iPr)2); in another embodiment, R2 is selected from a hydroxy protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), Acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, preferably DMTr; in another embodiment, R1 is a solid support.

[0106] X1 and X2

[0107] In one embodiment, X1 is H; in another embodiment, X1 is Rs; in another embodiment, X1 is

[0108] In one embodiment, X2 is OR1; in another embodiment, X2 is

[0109] L1 and L2

[0110] In one embodiment, L1 is a chemical bond; in another embodiment, L1 is C 1-10 Alkylene; in another embodiment, L1 is C 1-6 Alkylene; in another embodiment, L1 is C 2-10 Alkenylene; in another embodiment, L1 is C 2-10Alkyne; In another embodiment, L1 is OC 1-10 Alkylene; in another embodiment, L1 is OC 1-6 Alkylene; in another embodiment, L1 is OC 1-10 Alkenylene; in another embodiment, L1 is OC 1-10 Alkyne; in another embodiment, L1 is C(O)C 1-10 Alkylene; in another embodiment, L1 is C(O)C 1-6 Alkylene.

[0111] In one specific embodiment, L1 is OCH2CH2; in another specific embodiment, L1 is O(CH2)5.

[0112] In one embodiment, L1 is unsubstituted; in another embodiment, L1 is substituted with 1 R; in another embodiment, L1 is substituted with 2 Rs; in another embodiment, L1 is substituted with 3 Rs; in another embodiment, L1 is substituted with 4 Rs; in another embodiment, L1 is substituted with 5 Rs; in another embodiment, L1 is substituted with 6 Rs; in another embodiment, L1 is substituted with 7 Rs; in another embodiment, L1 is substituted with 8 Rs.

[0113] In one embodiment, L2 is a chemical bond; in another embodiment, L2 is C 1-10 Alkylene; in another embodiment, L2 is C 1-6 Alkylene; in another embodiment, L2 is C 2-10 Alkenylene; in another embodiment, L2 is C 2-10 Alkyne; In another embodiment, L2 is OC 1-10 Alkylene; in another embodiment, L2 is OC 1-6 Alkylene; in another embodiment, L2 is OC 1-10 Alkenylene; in another embodiment, L2 is OC 1-10 Alkyne; in another embodiment, L2 is C(O)C 1-10 Alkylene; in another embodiment, L2 is C(O)C 1-6 Alkylene.

[0114] In one specific embodiment, L2 is OCH2; in another specific embodiment, L2 is a chemical bond.

[0115] In one embodiment, L2 is unsubstituted; in another embodiment, L2 is substituted with 1 R; in another embodiment, L2 is substituted with 2 Rs; in another embodiment, L2 is substituted with 3 Rs; in another embodiment, L2 is substituted with 4 Rs; in another embodiment, L2 is substituted with 5 Rs; in another embodiment, L2 is substituted with 6 Rs; in another embodiment, L2 is substituted with 7 Rs; in another embodiment, L2 is substituted with 8 Rs.

[0116] T

[0117] In one embodiment, T is a chemical bond; in another embodiment, T is -O-; in another embodiment, T is -CH2-; in another embodiment, T is -C(O)-; in another embodiment, T is -OC(O)-; in another embodiment, T is -M-; in another embodiment, T is -OM-; in another embodiment, T is -CH2-M-; in another embodiment, T is -C(O)-M-; in another embodiment, T is -OC(O)-M-.

[0118] In one embodiment, M is In another embodiment, M is In another embodiment, M is In another embodiment, M is

[0119] A

[0120] In one embodiment, A is a sugar; in another embodiment, A is a sugar that can be cleaved in an endosome or lysosome; in another embodiment, A is a penta- or hexa-membered sugar, preferably a hexa-membered sugar; in another embodiment, A is selected from N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, preferably N-acetylgalactosamine or N-acetylglucosamine.

[0121] In a specific embodiment, A is A is connected to L1 through the a-terminal and to the P group through the b-terminal; in another specific embodiment, A is A is connected to L1 through the a-terminal and to the P group through the b-terminal; in another specific embodiment, A is A is connected to L1 through the a-terminal and to the P group through the b-terminal; in another specific embodiment, A is A is connected to L1 through the a-terminal and to the P group through the b-terminal.

[0122] In one embodiment, A is an acetylated sugar; in another embodiment, A is an acetylated sugar, which is a sugar that can be cleaved in an inclusion body or a lysosome; in another embodiment, A is an acetylated penta- or hexa-sugar, which is, for example, N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably, the penta- or hexa-sugar is N-acetylgalactosamine or N-acetylglucosamine.

[0123] In a specific embodiment, A is A is connected to L1 through the a-terminal and to the P group through the b-terminal; in another specific embodiment, A is A is connected to L1 through the a-terminal and to the P group through the b-terminal; in another specific embodiment, A is A is connected to L1 through the a-terminal and to the P group through the b-terminal; In another specific embodiment, A is

[0124] A is connected to L1 through the a-terminal and to the P group through the b-terminal.

[0125] P

[0126] In one embodiment, P is a hydrophobic group; in another embodiment, P is C 8-30 Hydrocarbon groups, such as C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl; in another embodiment, P is C 10-22 Alkyl groups, such as C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 18 Alkyl, C 19 Alkyl, C 20 Alkyl, C 21 Alkyl, C 22 Alkyl; in another embodiment, P is C 12-18 Alkyl groups, such as C 14-16 Alkyl; in another embodiment, P is C 10-22 Alkenyl.

[0127] In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-adjacent carbon atoms in P may be replaced by heteroatoms selected from O, S, and N, or the -CH2CH2- group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)-, or -C(O)NH-, or the substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring.

[0128] In one embodiment, P is unsubstituted; in another embodiment, P is substituted with 1 R; in another embodiment, P is substituted with 2 Rs; in another embodiment, P is substituted with 3 Rs; in another embodiment, P is substituted with 4 Rs; in another embodiment, P is substituted with 5 Rs; in another embodiment, P is substituted with 6 Rs; in another embodiment, P is substituted with 7 Rs; in another embodiment, P is substituted with 8 Rs; in another embodiment, P is substituted with more Rs.

[0129] In a specific embodiment, P is -(CH2) 14-16 CH3.

[0130] P1, P2, P3, and P4

[0131] In one embodiment, P1 is H; in another embodiment, P1 is a P group as defined above.

[0132] In one embodiment, P2 is H; in another embodiment, P2 is a P group as defined above.

[0133] In one embodiment, P3 is H; in another embodiment, P3 is a P group as defined above.

[0134] In one embodiment, P4 is CH3; in another embodiment, P4 is a P group as defined above.

[0135] In one embodiment, one of P1, P2, P3, P4 is a P group.

[0136] R

[0137] In one embodiment, R is H; in another embodiment, R is D; in another embodiment, R is halogen; in another embodiment, R is C 1-6 Alkyl; in another embodiment, R is C 1-4 Alkyl; in another embodiment, R is C 1-6 haloalkyl; in another embodiment, R is optionally deuterated, up to fully deuterated.

[0138] R s and Rs '

[0139] In one embodiment, R s is H; in another embodiment, R s is D; in another embodiment, R s is halogen; in another embodiment, R s C 1-6 Alkyl; in another embodiment, R s C 1-6 haloalkyl; in another embodiment, R s is a 3-10 membered heterocyclyl; in another embodiment, R s C 3-10 Cycloalkyl; in another embodiment, R s Optionally deuterated, up to fully deuterated.

[0140] In a specific embodiment, R s For H.

[0141] In one embodiment, R s ' is H; in another embodiment, R s ' is D; in another embodiment, R s ' is halogen; in another embodiment, R s ' for C 1-6 Alkyl; in another embodiment, R s ' for C 1-6 haloalkyl; in another embodiment, R s ' is a 3-10 membered heterocyclyl, such as a 5-10 membered heterocyclyl; in another embodiment, R s ' for C 3-10 Cycloalkyl; in another embodiment, R s ' is optionally deuterated, up to fully deuterated.

[0142] In a specific embodiment, R s ' is a cyclohexyl group.

[0143] m

[0144] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0145] n

[0146] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0147] k

[0148] k=0, 1, 2, 3, 4, 5 or 6.

[0149] Any technical solution or any combination thereof in any of the above specific embodiments can be combined with any technical solution or any combination thereof in other specific embodiments. For example, any technical solution or any combination thereof in A can be combined with P, P1-P4, R1, R2, X1, X2, L1, L2, T, R, R s 、R s The present invention is intended to include combinations of all these technical solutions, which are not listed one by one due to space limitations.

[0150] The present invention also provides a vector comprising a nucleotide sequence encoding the siRNA of the present invention. The vector of the present invention is capable of amplifying or expressing the nucleotide sequence encoding the siRNA of the present invention linked thereto.

[0151] For example, siRNA targeting the PCSK9 gene can be expressed from a transcription unit inserted into a DNA or RNA vector. Expression can be short-lived (a few hours to a few weeks) or continuous (a few weeks to a few months or longer), depending on the specific construct and target tissue or cell type used. The coding nucleotides of the siRNA can be introduced into a linear construct, a circular plasmid or a viral vector. The nucleotides of the siRNA can be integrated into the cell genome for stable expression, or expressed in an extrachromosomal stable inheritance. In general, siRNA expression vectors are typically DNA plasmids or viral vectors.

[0152] Viral vector systems containing siRNA coding sequences include, but are not limited to: (a) adenoviral vectors; (b) retroviral vectors; (c) adeno-associated viral vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors; and (j) helper virus-dependent adenovirus or gut-free adenovirus.

[0153] The present invention also provides a cell containing the siRNA or vector of the present invention, wherein the siRNA or vector of the present invention can be transcribed in the cell.

[0154] The present invention specifically relates to the following technical solutions:

[0155] A1. An oligonucleotide comprising one or more compounds of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0156] in,

[0157] In formula (I) represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0158] X1 is selected from H, Rs or

[0159] X2 is selected from OR1 or

[0160] One of X1 and X2 is

[0161] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0162] In formula (I) and R1 are not H at the same time;

[0163] Each L1 and L2 are independently selected from a chemical bond, C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, OC 1-10 Alkylene, OC 1-10 Alkenylene, OC 1-10 Alkynylidene, C(O)C 1-10 Alkylene; said L1 and L2 are optionally substituted by 1, 2, 3, 4, 5, 6, 7 or 8 R;

[0164] T is selected from a chemical bond, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M- or -OC(O)-M-;

[0165] Where M is

[0166] A is a sugar, which is preferably a sugar that can be cleaved in endosomes or lysosomes, preferably a penta- or hexa-membered sugar, more preferably a hexa-membered sugar, such as N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably N-acetylgalactosamine or N-acetylglucosamine;

[0167] P is a hydrophobic group, preferably C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30Alkynyl, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH2CH2- group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring; the C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R;

[0168] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0169] R s Selected from H, D, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, 3-10 membered heterocyclic group or C 3-10 Cycloalkyl, which is optionally deuterated, up to fully deuterated;

[0170] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0171] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0172] k=0, 1, 2, 3, 4, 5 or 6.

[0173] A2. The oligonucleotide of technical solution A1, wherein the compound of formula (I) is selected from a compound of formula (II) or formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0174] in,

[0175] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0176] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0177] and R1 are not H at the same time;

[0178] Each L1 and L2 are independently selected from a chemical bond, C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, OC1-10 Alkylene, OC 1-10 Alkenylene, OC 1-10 Alkynylidene or C(O)C 1-10 Alkylene; said L1 and L2 are optionally substituted by 1, 2, 3, 4, 5, 6, 7 or 8 R;

[0179] T is selected from a chemical bond, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M- or -OC(O)-M-;

[0180] Where M is

[0181] A is a sugar, which is preferably a sugar that can be cleaved in endosomes or lysosomes, preferably a penta- or hexa-membered sugar, more preferably a hexa-membered sugar, such as N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably N-acetylgalactosamine or N-acetylglucosamine;

[0182] P is a hydrophobic group, preferably C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH2CH2- group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring; the C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R;

[0183] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0184] R s Selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0185] R s 'Selected from H, D, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, 3-10 membered heterocyclic group or C 3-10Cycloalkyl, which is optionally deuterated, up to fully deuterated;

[0186] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0187] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0188] k=0, 1, 2, 3, 4, 5 or 6.

[0189] A3. The oligonucleotide of technical solution A1 or A2, wherein

[0190] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0191] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0192] and R1 are not H at the same time;

[0193] Each L1 and L2 are independently selected from a chemical bond, C 1-6 Alkylene, OC 1-6 Alkylene or C(O)C 1-6 Alkylene; said L1 and L2 are optionally substituted by 1, 2, 3, 4 or 5 R;

[0194] T is selected from a chemical bond, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M- or -OC(O)-M-;

[0195] Where M is

[0196] A is selected from N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably N-acetylgalactosamine or N-acetylglucosamine;

[0197] P is selected from C 10-22 Alkyl or C 10-22 Alkenyl, the C 10-22 Alkyl or C 10-22 Alkenyl is optionally substituted with 1, 2, 3, 4 or 5 R;

[0198] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0199] R sSelected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0200] R s 'Selected from H, D, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, 3-10 membered heterocyclic group or C 3-10 Cycloalkyl;

[0201] m is 0, 1, 2, 3, 4 or 5;

[0202] n is 0, 1, 2, 3, 4 or 5;

[0203] k is 0, 1, 2, 3, 4 or 5.

[0204] A4. The oligonucleotide of any one of technical solutions A1-A3, wherein

[0205] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0206] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0207] and R1 are not H at the same time;

[0208] Each L1 and L2 are independently selected from a chemical bond or OC 1-6 Alkylene; said L1 and L2 are optionally substituted by 1, 2 or 3 R;

[0209] T is selected from a chemical bond, -O-, -CH2-, -OC(O)- or -C(O)-;

[0210] A is selected from

[0211] A is connected to L1 through the a-terminal end and to the P group through the b-terminal end;

[0212] P is selected from C 10-22 Alkyl or C 10-22 Alkenyl, the C 10-22 Alkyl or C 10-22 Alkenyl is optionally substituted with 1, 2 or 3 R;

[0213] R is selected from H, D, halogen, C 1-4 alkyl;

[0214] R s Selected from H or D;

[0215] R s 'Selected from H, D or C5-10 Cycloalkyl;

[0216] m is 0, 1, 2, or 3;

[0217] n is 0, 1, 2, or 3;

[0218] k is 0, 1, 2 or 3.

[0219] A5. The oligonucleotide of any one of technical solutions A1-A4, wherein

[0220] indicates the position of the phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0221] R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0222] L1 is OCH2CH2 or O(CH2)5;

[0223] L2 is OCH2 or a chemical bond;

[0224] T is selected from a chemical bond, -O- or -C(O)-;

[0225] A is selected from

[0226] A is connected to L1 through the a-terminal end and to the P group through the b-terminal end;

[0227] P is -(CH2) 14-16 CH3;

[0228] R s is H;

[0229] R s ' is cyclohexyl;

[0230] m is 0, 1, or 2;

[0231] n is 0 or 1;

[0232] k is 0 or 1.

[0233] A6. The oligonucleotide of any one of technical solutions A1-A5, wherein the compound of formula (II) or formula (III) is selected from the following compounds of the general formula, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0234] in,

[0235] P1, P2, P3 are selected from H or P groups;

[0236] P4 is selected from CH3 or P group;

[0237] One of P1, P2, P3, and P4 is a P group;

[0238] The variables are as defined in Technical Solutions A1-A5.

[0239] A7. The oligonucleotide of any one of technical solutions A1-A6, wherein the compound of formula (II) or formula (III) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0240] in, Indicates the position of the phosphate or phosphorothioate linkage to the adjacent nucleotide.

[0241] R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide.

[0242] A8. The oligonucleotide of any one of technical solutions A1-A7, which has 14 to 30 nucleotides.

[0243] A9. The oligonucleotide of any one of technical solutions A1-A8, which comprises a compound of formula (II) or formula (III) of any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 5' end.

[0244] A10. The oligonucleotide according to any one of technical solutions A1-A9, comprising a compound of formula (II) or formula (III) according to any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at its 3′ end.

[0245] A11. The oligonucleotide of any one of technical solutions A1-A10, which comprises a compound of formula (II) or formula (III) of any one of technical solutions A1-A7 at its 5' end and 3' end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0246] A12. The oligonucleotide of any one of technical solutions A1-A11, which comprises one or more compounds of formula (II) of any one of technical solutions A1-A7, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, inside the oligonucleotide.

[0247] A13. An oligonucleotide comprising one, two or more delivery vectors within the oligonucleotide, at the 5' end and / or at the 3' end, wherein the delivery vector is a sugar modified with a hydrophobic group;

[0248] Preferably, the sugar modified by the hydrophobic group is selected from the compound of formula (X), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0249] in, Indicates the position of attachment to the oligonucleotide;

[0250] P is a hydrophobic group;

[0251] A is the sugar moiety;

[0252] P and A are as defined in any one of technical solutions A1-A5;

[0253] Preferably, the hydrophobic group is attached to a hydroxyl or acetyl group of the sugar moiety;

[0254] Preferably, the sugar modified by the hydrophobic group is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0255] in, Indicates the position of attachment to the oligonucleotide.

[0256] A14. The oligonucleotide of any one of technical solutions A1-A13, which is an ASO or siRNA, is preferably used to inhibit genes expressed outside the liver, more preferably used to inhibit genes expressed in the central nervous system (CNS) and / or the eye.

[0257] A15. A compound of formula (II') or (III'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0258] R1 and R2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;

[0259] A is an acetylated sugar, preferably a sugar that can be cleaved in endosomes or lysosomes, preferably an acetylated penta- or hexa-sugar, wherein the penta- or hexa-sugar is, for example, N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose, or fucose, more preferably, the penta- or hexa-sugar is N-acetylgalactosamine or N-acetylglucosamine;

[0260] Preferably, A is selected from

[0261] A is connected to L1 through the a-terminal end and to the P group through the b-terminal end;

[0262] P, L1, L2, T, Rs, Rs', m, n, k are as defined in any one of technical solutions A1-A7.

[0263] A16. The compound of technical solution A15, wherein R1 and R2 are H.

[0264] A17. Compounds of technical solutions A15 or A16, wherein one of R1 and R2 is a reactive phosphorus group, preferably a phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate or phosphate mimetic, such as natural phosphates, thiophosphates, dithiophosphates, boranephosphates, boranethiophosphates, phosphonates, halogen-substituted phosphonates and phosphates, aminophosphorates, phosphate diesters, phosphate triesters, thiophosphate diesters, thiophosphate triesters, diphosphates or triphosphates, preferably -P(OCH2CH2CN)(N(iPr)2).

[0265] A18. The compound of any one of technical solutions A15-A17, wherein R1 and R2 are selected from protecting groups, preferably hydroxy protecting groups, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Fmoc), 1,2-dimethyl-2-oxo-1,2-dimethyl-3-oxo-1,2-dimethyl-4-oxo-1,2-dimethyl-5-oxo-1,2-dimethyl-6-oxo-1,2-dimethyl-7-oxo-1,2-dimethyl-8-oxo-1,2-dimethyl-9-oxo-1,2-dimethyl-1,2-dimethyl-1 alkyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, preferably DMTr.

[0266] A19. The compound of any one of technical solutions A15-A18, wherein the compound of formula (II') or (III') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0267] A20. A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence that is sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or antisense strand comprises one or more compounds of formula (II) or formula (III) as described in any one of technical solutions A1-A7, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0268] A21. The double-stranded RNA of technical solution A20, wherein the compound of formula (II) or formula (III) is selected from the following compounds of the general formula, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0269] Wherein, each variable is as defined in technical solutions A1-A7.

[0270] A22. The double-stranded RNA of technical solution A20 or A21, wherein the compound of formula (II) or formula (III) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0271] in, Indicates the position of the phosphate or phosphorothioate linkage to the adjacent nucleotide.

[0272] R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide.

[0273] A23. The double-stranded RNA of any one of technical solutions A20-A22, wherein the sense strand comprises a compound of formula (II) or formula (III) as described in any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 5' end.

[0274] A24. The double-stranded RNA of any one of technical solutions A20-A23, wherein the positive strand comprises a compound of formula (II) or formula (III) as described in any one of technical solutions A1-A7 at the 3' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0275] A25. The double-stranded RNA of any one of technical solutions A20-A24, wherein the positive strand comprises a compound of formula (II) or formula (III) described in any one of technical solutions A1-A7 at the 5' end and the 3' end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0276] A26. The double-stranded RNA of any one of technical solutions A20-A25, wherein the positive strand comprises one or more compounds of formula (II) described in any one of technical solutions A1-A7, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, inside the oligonucleotide.

[0277] A27. The double-stranded RNA of any one of technical solutions A20-A26, wherein the antisense strand comprises a compound of formula (II) or formula (III) as described in any one of technical solutions A1-A7 at the 5' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0278] A28. The double-stranded RNA of any one of technical solutions A20-A27, wherein the antisense strand comprises a compound of formula (II) or formula (III) as described in any one of technical solutions A1-A7 at the 3' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0279] A29. The double-stranded RNA of any one of technical solutions A20-A28, wherein the antisense strand comprises a compound of formula (II) or formula (III) described in any one of technical solutions A1-A7 at the 5' end and the 3' end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0280] A30. The double-stranded RNA of any one of technical solutions A20-A29, wherein the antisense strand comprises one or more compounds of formula (II) described in any one of technical solutions A1-A7, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, inside the oligonucleotide.

[0281] A31. A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand comprises one, two or more delivery vectors internally, at the 5' end and / or at the 3' end, wherein the delivery vector is a sugar modified with a hydrophobic group;

[0282] Preferably, the sugar modified by the hydrophobic group is selected from the compound of formula (X), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0283] in, Indicates the position of attachment to the oligonucleotide;

[0284] P is a hydrophobic group;

[0285] A is the sugar moiety;

[0286] P and A are as defined in any one of technical solutions A1-A5;

[0287] Preferably, the hydrophobic group is attached to a hydroxyl or acetyl group of the sugar moiety;

[0288] Preferably, the sugar modified by the hydrophobic group is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0289] in, Indicates the position of attachment to the oligonucleotide.

[0290] A32. A vector comprising a nucleotide sequence encoding the double-stranded RNA described in any one of the aforementioned technical solutions A20-A31.

[0291] A33. A cell containing the double-stranded RNA according to any one of technical solutions A20-A31 or the vector according to technical solution A32.

[0292] A34. A pharmaceutical composition comprising the double-stranded RNA as described in any one of technical solutions A20-A31, the vector as described in technical solution A32, or the cell as described in technical solution A33, and optionally a pharmaceutically acceptable carrier or excipient.

[0293] A35. A kit comprising the double-stranded RNA as described in any one of technical solutions A20-A31, the vector as described in technical solution A32, or the cell as described in technical solution A33.

[0294] The present invention also specifically relates to the following technical solutions:

[0295] B1. An oligonucleotide comprising one or more compounds of formula (IV), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0296] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0297] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0298] and R1 are not H at the same time;

[0299] Each L1 and L2 are independently selected from a chemical bond, C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, OC 1-10 Alkylene, OC 1-10 Alkenylene, OC 1-10 Alkynylidene or C(O)C 1-10 Alkylene; said L1 and L2 are optionally substituted by 1, 2, 3, 4, 5, 6, 7 or 8 R;

[0300] T is selected from a chemical bond, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M- or -OC(O)-M-;

[0301] Where M is

[0302] A is N-acetylglucosamine;

[0303] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0304] R s Selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0305] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0306] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0307] k=0, 1, 2, 3, 4, 5 or 6.

[0308] B2. The oligonucleotide of technical solution B1, wherein

[0309] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0310] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0311] and R1 are not H at the same time;

[0312] Each L1 and L2 are independently selected from a chemical bond, C 1-6 Alkylene, OC 1-6 Alkylene or C(O)C 1-6 Alkylene; said L1 and L2 are optionally substituted by 1, 2, 3, 4 or 5 R;

[0313] T is selected from a chemical bond, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M- or -OC(O)-M-;

[0314] Where M is

[0315] A is N-acetylgalactosamine;

[0316] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0317] R sSelected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0318] m is 0, 1, 2, 3, 4 or 5;

[0319] n is 0, 1, 2, 3, 4 or 5;

[0320] k is 0, 1, 2, 3, 4 or 5.

[0321] B3. The oligonucleotide of technical solution B1 or B2, wherein

[0322] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0323] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0324] and R1 are not H at the same time;

[0325] Each L1 and L2 are independently selected from a chemical bond or OC 1-6 Alkylene, for example, OCH2, OCH2CH2, (OCH2CH2)2 or (OCH2CH2)3; said L1 and L2 are optionally substituted with 1, 2 or 3 R;

[0326] T is selected from a chemical bond, -O-, -CH2-, -OC(O)- or -C(O)-, preferably -C(O)-;

[0327] A is

[0328] R is selected from H, D, halogen, C 1-4 alkyl;

[0329] R s Selected from H or D;

[0330] m is 0, 1, 2, or 3;

[0331] n is 0, 1, 2, or 3;

[0332] k is 0, 1, 2 or 3.

[0333] B4. The oligonucleotide of any one of technical solutions B1-B3, wherein the compound is selected from:

[0334] in,

[0335] Indicates that it is connected to the previous nucleotide through a phosphate group or a phosphorothioate group;

[0336] Indicates linkage to the next nucleotide via a phosphate group or a phosphorothioate group.

[0337] B5. A compound of formula (IV'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0338] in,

[0339] R1 and R2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;

[0340] A is

[0341] L1, L2, T, Rs, m, n, and k are as defined above.

[0342] B6. The compound of technical solution B5, wherein R1 and R2 are H;

[0343] Preferably, one of R1 and R2 is a reactive phosphorus group, preferably a phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate or phosphate mimetic, such as a natural phosphate, phosphorothioate, phosphorodithioate, boranophosphate, boranophosphothioate, phosphonate, halogen-substituted phosphonates and phosphates, phosphoramidate, phosphodiester, phosphotriester, phosphorothioate diester, phosphorothioate triester, diphosphate or triphosphate, preferably -P(OCH2CH2CN)(N(iPr)2);

[0344] R1 and R2 are selected from protecting groups, preferably hydroxy protecting groups, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p- Methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, preferably DMTr.

[0345] B7. The compound of technical solution B5 or B6, wherein the compound of formula (II') or (III') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0346] B8. The oligonucleotide of any one of technical solutions B1-B4, wherein the oligonucleotide is siRNA, used to inhibit genes expressed in the liver.

[0347] List of specific compounds

[0348] The numbering and structure of the compounds of the present invention in oligonucleotides are as follows, wherein the sequence of 5'-->3' from the compound to connect.

[0349] Specifically, according to the order of 5'–>3', if the corresponding structure is located in the middle of the nucleic acid chain, means that it is connected to the 3' carbon or corresponding position of the previous nucleotide or nucleotide analog through a phosphate group, a phosphorothioate group or other linking group, It means that it is connected to the 5' carbon or corresponding position of the next nucleotide or nucleotide analog through a phosphate group, a thiophosphate group or other linking group; if the corresponding structure is located at the terminal position of the nucleic acid chain, Correspondingly, it refers to the connection to the 3' or 5' end of the nucleic acid chain through a phosphate group, a phosphorothioate group or other linking group.

[0350] Synthesis Examples The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention.

[0351] abbreviation

[0352] Example 1. Preparation of compound BE1

[0353] 1. Preparation of Compound 3

[0354] Compound 1 (1.00 g, 2.03 mmol) was dissolved in DCM (30.0 mL) at 25°C. HATU (0.850 g, 2.23 mmol) and DIEA (1.34 mL, 8.11 mmol) were added and stirred for 0.5 h. Compound 2 (1.00 g, 2.229 mmol) was then added, and the reaction mixture was stirred at 25°C for 2.5 h. LCMS showed the mass of the product. Thin-layer chromatography (DCM / MeOH = 10 / 1) revealed the formation of new spots. The reaction mixture was spin-dried to obtain the crude product. The crude product was purified on a normal-phase silica gel column (DCM / MeOH = 1 / 0 to 10 / 1) to afford compound 3 (1.70 g, 90.69% yield) as a white solid.

[0355] 1 H NMR (400MHz, CD3OD) δ7.37-7.47(m,2H),7.16-7.34(m,7H),6.71-6.92(m,4H),5.22-5.37(m,1H),5.05-5. 15(m,1H),4.69-4.80(m,1H),3.55-4.24(m,28H),3.19-3.30(m,2H),2.09-2.15(m,3H),1.87-2.00(m,9H)

[0356] 2. Preparation of Compound BE1

[0357] Compound 3 (1.70 g, 1.84 mmol) was dissolved in DCM (20.0 mL) at 25°C. DCI (0.160 g, 1.38 mmol) and compound 4 (0.830 g, 2.76 mmol) were added sequentially. The reaction mixture was stirred at 25°C under a nitrogen atmosphere for 2 hours. LCMS showed complete consumption of compound 3 and the presence of a mass of compound BE1. Thin-layer chromatography (DCM / MeOH = 10 / 1) revealed the formation of a new spot. The reaction mixture was dried to give the crude product. The crude product was purified on a normal-phase silica gel column (dichloromethane / acetone = 1 / 0-5 / 1) to afford compound BE1 (1.02 g, 49.32% yield) as a pale yellow oil.

[0358] 1H NMR (400MHz, CDCl3) δ7.37-7.48(m,2H),7.23-7.36(m,7H),6.78-6.90(m,4H) ,5.17-5.28(m,1H),4.90-5.08(m,2H),4.04-4.36(m,6H),3.75-3.97(m,14H) ,3.54-3.71(m,9H),3.09-3.48(m,5H),2.51-2.67(m,2H),2.12-2.16(m,3H), 2.02-2.07(m,3H),1.96-2.00(m,3H),1.89-1.93(m,3H),1.11-1.22(m,12H).

[0359] Example 2. Preparation of compound BE2

[0360] 1. Preparation of Compound 3

[0361] Compound 1 (1.0 g, 2.23 mmol) was dissolved in DCM (20.0 mL) at 25°C, and HATU (1.27 g, 3.34 mmol) and DIEA (1.10 mL, 6.68 mmol) were added. The mixture was stirred at 25°C for 0.5 hours before compound 2 (1.20 g, 2.67 mmol) was added and the mixture was stirred for another 12 hours. TLC (Tl: DCM / MeOH = 10 / 1) showed complete consumption of the starting material. 200 mL of dichloromethane was added. The mixture was washed sequentially with saturated sodium bicarbonate solution (30.0 mL x 3) and saturated brine (30.0 mL x 3). The organic phase was dried over anhydrous sodium sulfate and then spin-dried under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 1 / 0 to 20 / 1) to obtain compound 3 (1.43 g, 1.70 mmol) as a yellow oil.

[0362] 1H NMR(400MHz,CD3OD)δ7.44(d,J=7.2Hz,2H),7.18-7.35(m,7H),6.87(t,J=8.0Hz ,4H),5.32(s,1H),5.04(d,J=11.6Hz,1H),4.66(dd,J=4.4,8.4Hz,1H),4.06-4. 29(m,6H),3.81-3.96(m,2H),3.78(s,6H),3.66-3.76(m,3H),3.47-3.65(m,6H) ,3.33-3.38(m,1H),3.15-3.25(m,2H),2.11(d,J=16.8Hz,3H),1.84-2.01(m,9H)

[0363] 2. Preparation of Compound BE2

[0364] Compound 3 (1.42 g, 1.61 mmol) was dissolved in DCM (15.0 mL) at 25°C. Several molecular sieves were added, followed by DCI (140 mg, 1.21 mmol). Compound 4 (730 mg, 2.42 mmol) was added under a nitrogen atmosphere, and the mixture was stirred at 25°C for 1.0 hour. TLC (DCM / ACE = 10 / 1) showed the formation of new spots. 0.5 mL of triethylamine and 20 mL of dichloromethane were added, and the sample was stirred with basic silica gel. A 4 / 1000 triethylamine solution was added to the mobile phase of dichloromethane. Purification by column chromatography (DCM / ACE = 1 / 0 to 5 / 1) afforded compound BE2 (900 mg, 51.64% yield) as a colorless oil.

[0365] 1 H NMR (400MHz, CDCl3) δ7.35-7.49(m,2H),7.24-7.34(m,7H),6.75-6.88(m,4H),5.26-5.38(m,1H),4.99-5.09(m,1H),4.85-4.95( m,1H),3.09-4.33(m,30H),2.54-2.68(m,2H),2.13-2.19(m,3H),2.02-2.06(m,3H),1.97-2.01(m,6H),1.24(d,J=14.4Hz,12H).

[0366] 31 P NMR (162MHz, CDCl3) δ149.32, 148.96, 14.16.

[0367] Example 3. Preparation of compound BE3

[0368] 1. Preparation of Compound 1

[0369] Compound 1 (1.17 g, 2.18 mmol) was dissolved in DCM (20.0 mL) at 25°C, and HATU (1.24 g, 3.27 mmol) and DIEA (1.08 mL, 6.53 mmol) were added. The mixture was stirred at 25°C for 0.5 hours before compound 2 (1.08 g, 2.40 mmol) was added. The mixture was stirred for another 12 hours. LCMS indicated the formation of the product. TLC (Tl: DCM / MeOH = 10 / 1) showed complete consumption of the starting material. 100 mL of dichloromethane was added. The mixture was washed sequentially with saturated sodium bicarbonate solution (20.0 mL x 3) and saturated brine (20.0 mL x 3). The organic phase was dried over anhydrous sodium sulfate and suspended to dryness under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 1 / 0 to 20 / 1) to afford compound 3 (1.67 g, 79.2% yield) as a yellow oil.

[0370] 1 H NMR (400MHz, CD3OD) δ7.44(d,J=7.6Hz,2H),7.16-7.34(m,7H),6.86(t,J=7.6Hz,4H),5.32(d,J=3.2Hz,1H),5. 05(dd,J=3.6,11.2Hz,1H),4.67(dd,J=3.6,8.0Hz,1H),4.21-4.26(m,1H),4.14-4.18(m,1H),4.07-4.13(m,4H ),3.94-4.06(m,3H),3.87-3.91(m,1H),3.78(s,6H),3.69-3.76(m,3H),3.55-3.66(m,13H),3.51-3.52(m,1H) ,3.13-3.29(m,2H),2.12(d,J=1.6Hz,3H),2.00-2.02(m,3H),1.99(s,1H),1.91-1.96(m,6H),1.20-1.28(m,2H)

[0371] 2. Preparation of Compound BE3

[0372] Compound 3 (1.67 g, 1.72 mmol) was dissolved in DCM (30.0 mL) at 25°C. DCI (0.150 g, 1.29 mmol) and compound 4 (0.780 g, 2.59 mmol) were added sequentially. The reaction mixture was stirred at 25°C under a nitrogen atmosphere for 2 hours. Thin-layer chromatography (DCM / MeOH = 10 / 1) revealed the formation of new spots. The reaction mixture was dried to obtain the crude product. The crude product was purified on a normal-phase silica gel column (dichloromethane / acetone = 1 / 0-5 / 1) to afford compound BE3 (1.60 g, 79.40% yield) as a pale yellow oil.

[0373] 1 H NMR (400MHz, CDCl3) δ7.38-7.47(m,2H),7.24-7.35(m,7H),6.78-6.90(m,4H),6. 54-6.69(m,1H),5.29-5.31(m,1H),4.97-5.06(m,1H),4.76-4.85(m,1H),4.10-4 .26(m,6H),3.76-4.06(m,15H),3.59-3.74(m,12H),3.10-3.54(m,5H),2.51-2.6 7(m,2H),2.14-2.17(m,3H),2.05-2.07(m,3H),1.99(s,6H),1.12-1.24(m,12H).

[0374] 31 P NMR (162MHz, CDCl3) δ149.16,148.87,148.81.

[0375] Example 4. Preparation of Compound BE4

[0376] 1. Preparation of Compound 1c

[0377] To a solution of compound 1b (20 g, 78.0 mmol) in DMF (100 mL) at room temperature were added DIEA (38.7 mL, 234 mmol) and HATU (32.6 g, 85.8 mmol). Half an hour later, compound 1a (16.8 g, 78.0 mmol) was added. The suspended reaction solution was stirred at room temperature for 16 hours. After completion of the reaction, water (400 mL) was added for dilution. The filter cake was collected by filtration and dried to yield compound 1c (30 g, 71.8 mmol, 92.1%).

[0378] 1H NMR (400MHz, DMSO-d6) δ7.61(d,J=8.3Hz,1H),6.38(d,J=6.5Hz,1H),4.56(t,J=5.6Hz,1H),4.48–4.34(m,3H),3.66–3.59 (m,1H),3.57–3.43(m,2H),3.43–3.37(m,1H),2.07(t,J=7.5Hz,2H),1.52–1.42(m,2H),1.24(s,26H),0.90–0.80(m,3H).

[0379] 2. Preparation of Compound 1d

[0380] Under a nitrogen atmosphere, compound 1c (20 g, 47.9 mmol) was suspended in dry pyridine (100 mL), cooled to 0°C in an ice-water bath, and then acetic anhydride (45 mL, 479 mmol) was added portionwise. After the addition was complete, the mixture was slowly warmed to room temperature and then stirred at room temperature for 16 hours. After the reaction was complete, the reaction solution was diluted with ethyl acetate, washed once with water, and the aqueous phase was extracted twice with ethyl acetate. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether) to obtain compound 1d (10.2 g, 17.4 mmol, 36.3%).

[0381] 1 H NMR (400MHz, DMSO-d6) δ7.84(d,J=9.2Hz,1H),5.65(d,J=8.8Hz,1H),5.27(d,J=3.1Hz,1H),5.07(dd,J=11.3,3.4Hz,1H),4.23–4.18(m,1H),4. 16–4.09(m,1H),4.07–3.96(m,2H),2.12(s,3H),2.02(s,3H),1.99(s,3 H),1.89(s,3H),1.43(p,J=7.1Hz,2H),1.23(s,26H),0.89–0.81(m,3H).

[0382] 3. Preparation of Compound 1e

[0383] Under a nitrogen atmosphere, compound 1d (5 g, 8.54 mmol) was dissolved in DCM (50 mL), cooled to 0-5°C in an ice-water bath, and TMSOTf (2.28 g, 10.250 mmol) was added dropwise. Stirring was then performed at room temperature for 16 hours. After completion of the reaction, the reaction solution was added portionwise to a saturated sodium bicarbonate solution at 0-5°C. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (eluent: dichloromethane / ethyl acetate) to obtain compound 1e (4.2 g, 7.99 mmol, 93.5%).

[0384] 4. Preparation of Compound 1g

[0385] To a solution of compound 1e (4.2 g, 7.99 mmol) in DCM (20 mL) was added compound 1f (1.73 g, 6.80 mmol) and TMSOTf (0.89 g, 4.00 mmol) at 0-5°C. The reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction, the reaction mixture was poured portionwise into a saturated sodium bicarbonate solution (40 mL) pre-cooled to 0-5°C. The organic phase was separated, and the aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / dichloromethane) to obtain compound 1g (2.83 g, 3.63 mmol, 45.4%).

[0386] 1 H NMR(400MHz,DMSO-d6)δ7.71(d,J=9.2Hz,1H),7.41–7.33(m,5H),5.21(d,J=3.3Hz,1H),5 .15(s,2H),4.98(dd,J=11.2,3.4Hz,1H),4.58(d,J=8.4Hz,1H),4.19(s,2H),4.08–3.97(m ,3H),3.94–3.85(m,1H),3.81–3.73(m,1H),3.64–3.55(m,3H),3.57–3.47(m,4H),2.10(s ,3H),2.03–1.94(m,5H),1.87(s,3H),1.49–1.38(m,2H),1.22(s,24H),0.88–0.82(m,3H).

[0387] 5. Preparation of Compound 1h

[0388] Compound 1g (2.5g, 3.10mmol) was dissolved in MeOH (20mL). After nitrogen purge three times, Pd / C (0.566g, 0.1% wt%) was added. The system was then purge three times with hydrogen. The reaction solution was stirred at room temperature under a hydrogen atmosphere for 16 hours. After the reaction was complete, the reaction solution was filtered through Celite to remove excess Pd / C, and the filtrate was concentrated under reduced pressure to obtain Compound 1h (2.0g, 2.90mmol, 80.0%) as an oil.

[0389] 1 H NMR (400MHz, DMSO-d6) δ7.73 (d, J=9.2Hz, 1H), 5.21 (d, J=3.4Hz, 1H), 4.99 (dd, J= 11.2,3.4Hz,1H),4.59(d,J=8.5Hz,1H),4.06–4.01(m,2H),4.01–3.98(m,2H),3.9 3–3.84(m,1H),3.80–3.73(m,1H),3.61–3.54(m,4H),3.53–3.49(m,4H),2.11(s,3 H),2.00(s,3H),1.87(s,3H),1.49–1.38(m,2H),1.22(s,26H),0.89–0.82(m,3H).

[0390] 6. Preparation of Compound 1j

[0391] To a solution of compound 1h (1 g, 1.45 mmol) in DCM (10 mL) were added DIEA (0.7 mL, 4.35 mmol), EDCI (0.42 g, 2.18 mmol), and HOBt (0.29 g, 2.18 mmol) in sequence. Half an hour later, compound 1i (0.65 g, 1.45 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction, the reaction solution was washed with water, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by reverse-phase flash column chromatography (eluent: acetonitrile / water) to obtain compound 1j (1.39 g, 1.24 mmol, 85.3%).

[0392] 1H NMR (400MHz, DMSO-d6) δ7.72(d,J=9.1Hz,1H),7.41–7.35(m,2H),7.31(t,J=7.5Hz,2H),7.28–7.20(m,5H),6.88(d,J=8. 6Hz,4H),5.21(d,J=3.4Hz,1H),4.99(dd,J=11.2,3.3Hz,1H),4.78–4.68(m,2H),4.61–4.55(m,1H),4.27–4.10(m,2H),4 .10(d,J=5.8Hz,1H),4.06–3.98(m,4H),3.95–3.85(m,2H),3.74(s,6H),3.62–3.55(m,4H),3.51–3.41(m,4H),3.18–3.0 9(m,1H),3.06–2.95(m,2H),2.10(s,3H),2.03–1.96(m,5H),1.86(s,3H),1.43(s,2H),1.23(s,26H),0.88–0.78(m,3H).

[0393] 7. Preparation of Compound BE4

[0394] Under a nitrogen atmosphere, compound 1j (552 mg, 0.493 mmol) was dissolved in DCM (5 mL), and compound 1k (193 mg, 0.640 mmol) and DCI (70 mg, 0.591 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction, the reaction solution was poured into a saturated sodium bicarbonate solution (20 mL) pre-cooled to 0-5°C. The aqueous phase was extracted twice with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (eluent: 1% Et3N in ethyl acetate / 1% Et3N in dichloromethane) to obtain compound BE4 (447 mg, 0.338 mmol, 68.8%).

[0395] 1H NMR (400MHz, DMSO-d6) δ7.72(d,J=9.2Hz,1H),7.42–7.35(m,2H),7.30(t,J=7.6Hz,2H),7.28–7.19(m,5H),6.88(d,J=8. 4Hz,4H),5.21(d,J=3.3Hz,1H),4.99(dd,J=11.2,3.4Hz,1H),4.63–4.55(m,1H),4.28–4.11(m,2H),4.06–3.95(m,5H),3 .95–3.85(m,1H),3.74(s,6H),3.62–3.51(m,8H),3.50–3.42(m,6H),3.20–2.92(m,2H),2.74(t,J=5.9Hz,1H),2.71–2.6 5(m,1H),2.10(s,3H),2.03–1.96(m,5H),1.86(s,3H),1.43(s,2H),1.22(s,26H),1.16–1.05(m,14H),0.88–0.80(m,3H).

[0396] Example 5. Preparation of compound BE5

[0397] To a solution of compound 1j (200 mg, 0.178 mmol) from Example 4 in DCM (2 mL) were added DIEA (0.18 mL, 1.071 mmol), succinic anhydride (107 mg, 1.07 mmol), and DMAP (5 mg, 0.045 mmol) in sequence. The reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction, the mixture was washed with saturated sodium bicarbonate solution, and the aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by reverse-phase flash column chromatography (eluent: acetonitrile / water) to provide compound BE5 (141 mg, 0.116 mmol, 64.1%).

[0398] 1H NMR(400MHz, DMSO-d6)δ7.79(d,J=9.8Hz,1H),7.42–7.34(m,2H),7.31(t,J=7.6Hz,2H),7.28–7.19(m,5H),6.91–6.8 6(m,4H),5.20(d,J=3.3Hz,2H),5.04–4.96(m,1H),4.67–4.56(m,2H),4.25–4.11(m,3H),4.06–3.96(m,4H),3.95–3.8 1(m,3H),3.74(s,6H),3.62–3.54(m,2H),3.52–3.42(m,5H),3.16–3.01(m,2H),3.01–2.95(m,1H),2.40–2.30(m,2H) ,2.10(s,3H),2.04–1.95(m,5H),1.86(s,3H),1.48–1.37(m,2H),1.21(s,24H),1.03–0.91(m,4H),0.88–0.80(m,3H).

[0399] Example 6. Preparation of Compound BE6

[0400] The synthesis steps of compound BE6 can be referred to Example 4, wherein stearic acid is used instead of compound 1b.

[0401] 1 H NMR (400MHz, DMSO-d6) δ7.41–7.35(m,2H),7.31(t,J=7.6Hz,2H),7.28–7.19(m,5H),6.88(d,J=8.5Hz,4H),5.20(d,J =3.3Hz,2H),4.99(dd,J=11.1,3.3Hz,1H),4.59(t,J=7.8Hz,1H),4.25–4.10(m,2H),4.09–3.97(m,5H),3.96–3.84(m ,3H),3.74(s,6H),3.63–3.55(m,2H),3.54–3.43(m,7H),3.17–3.09(m,1H),3.07–2.95(m,3H),2.47–2.40(m,2H),2. 10(s,3H),2.03–1.96(m,5H),1.86(s,3H),1.42(s,2H),1.25–1.18(m,26H),0.96(d,J=6.4Hz,4H),0.87–0.82(m,3H).

[0402] Example 7. Synthesis of siRNA

[0403] The siRNA of the present invention was prepared using the solid phase phosphoramidite method known in the art. Specific methods can be found in, for example, PCT Publication Nos. WO2016081444 and WO2019105419 and are briefly described below.

[0404] 1. Preparation of siRNA with no ligand attached to the 3' end of the sense strand

[0405] 1.1 Synthesis of the positive sense chain (SS chain)

[0406] Using solid-phase phosphoramidite synthesis, a blank CPG solid support is used as the starting cycle, and nucleoside monomers are linked one by one in the 3'-5' direction according to the order of the positive strand nucleotides. Each nucleoside monomer linking involves four steps: deprotection, coupling, capping, and oxidation or thiolation, resulting in a 5 μmol oligonucleotide. The synthesis conditions are as follows:

[0407] The nucleoside monomers were provided in a 0.05 mol / L acetonitrile solution. The reaction conditions for each step were the same, i.e., the temperature was 25°C. Deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution. The coupling reaction was performed twice using a 0.25 mol / L 5-ethylthiotetrazolium (ETT)-acetonitrile solution as the activator. Capping was performed twice using 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v). Oxidation was performed twice using 0.05 mol / L iodine in tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v). Thiosulfation was performed twice using 0.2 mol / L phenylacetyl disulfide (PADS) in acetonitrile / 3-methylpyridine (1 / 1, v / v).

[0408] 1.2 Synthesis of antisense strand (AS strand)

[0409] Using solid-phase phosphoramidite synthesis, a blank CPG solid support is used as the starting cycle. Nucleoside monomers are linked one by one in the 3'-5' direction according to the nucleotide arrangement of the antisense strand. Each ligation step involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for a 5 μmol oligonucleotide of the antisense strand are the same as those for the sense strand.

[0410] 1.3 Oligonucleotide purification and annealing

[0411] 1.3.1 Ammonolysis

[0412] Add the synthesized solid phase carrier (sense chain or antisense chain) to a 5 mL centrifuge tube, add 3% diethylamine / ammonia water (v / v), react in a constant temperature water bath at 35 degrees (or 55 degrees) for 16 hours (or 8 hours), filter, wash the solid phase carrier three times with ethanol / water, each time 1 mL, and centrifuge the filtrate to purify the crude product.

[0413] 1.3.2 Purification

[0414] Purification and desalination methods are well known in the art. For example, a column packed with a strong anion filler can be used, and a sodium chloride-sodium hydroxide system can be used for elution and purification, and the product can be collected and stored in a tube. A gel-filled purification column can be used for desalination, and the elution system can be pure water.

[0415] 1.3.3 Annealing

[0416] According to the instructions, the sense chain (SS chain) and the antisense chain (AS chain) were mixed at a molar ratio of (SS chain / AS chain = 1 / 1.05), heated in a water bath to 70-95 degrees, maintained for 3-5 minutes, cooled naturally to room temperature, and the system was lyophilized to obtain the product.

[0417] The siRNA sequences used in the present invention are as follows:

[0418] In this article, the meanings of the abbreviations are as follows:

[0419] A, U, G and C represent the natural ribonucleotides adenine, uracil, guanine and cytosine, respectively.

[0420] d indicates that the nucleotide adjacent to its right is a deoxyribonucleotide. For example, dA, dT, dG, and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and cytosine deoxyribonucleotide, respectively.

[0421] m indicates that the nucleotide adjacent to its left is a 2'-OCH3 modified nucleotide. For example, Am, Um, Gm, and Cm represent 2'-OCH3 modified A, U, G, and C.

[0422] f indicates that the nucleotide adjacent to its left is a 2'-F modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.

[0423] "s" indicates that the two adjacent nucleotides and / or delivery vectors are linked by phosphorothioate.

[0424] VP indicates that the adjacent nucleotide on the right is a vinyl phosphate-modified nucleotide.

[0425] L96 represents a GalNAc delivery vector of the following structure well known in the art, wherein The position of the siRNA is indicated by a phosphate group or a phosphorothioate group, as described in, for example, PCT Publication Nos. WO2009073809 and WO2009082607.

[0426] The structures of GL34, LS3 and LS1 are as defined above.

[0427] The structures of GL34 when connected to the 3' and 5' ends of the nucleic acid chain through a phosphate group, a phosphorothioate group, or other linking groups are as follows:

[0428] The structures of LS3 when connected to the 3' and 5' ends of the nucleic acid chain through a phosphate group, a phosphorothioate group, or other linking groups are as follows:

[0429] The structures of LS1 when connected to the 3' and 5' ends of the nucleic acid chain through a phosphate group, a phosphorothioate group, or other linking groups are as follows:

[0430] Example 8 Verification of the long-lasting efficacy of the compounds of the present invention in a C57BL / 6 mouse model

[0431] C57BL / 6 mice (male, 18-21 g, 6-8 weeks) were randomly divided into groups. The dosage for each animal was calculated based on body weight, and the siRNA conjugate was administered as a single subcutaneous injection in a 1 mg / mL solution (0.9% sodium chloride aqueous solution as the solvent). Specifically, before the experiment, the siRNA conjugate was dissolved in 0.9% sodium chloride aqueous solution and diluted to the desired concentration and volume. The administration volume of normal saline (control group) and siRNA conjugate was 5 mL / kg.

[0432] Before administration (recorded as day 0), and on days 7, 14, 21, 28, 35, 42, 56, and 70 after administration, 10 mg of liver was collected and placed in RNAlater TM Solution, frozen at -80℃, used for detection of liver mTTR mRNA.

[0433] Detection of liver mTTR mRNA:

[0434] Cell RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the protocol of the high-throughput tissue RNA extraction kit (Fanzhi Medical, FG0412); TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B) for reverse transcription; reference TaqMan TMFluorescence quantitative PCR (ABI, QuantStudio3) was performed using 20 μL of Fast Advanced Master Mix (ABI, 4444965). The primers are shown in Table 1.

[0435] Table 1: Primer information

[0436] Data statistics and analysis

[0437] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate;

[0438] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0439] The target gene is mTTR and the internal reference is mGAPDH.

[0440] Table 2 Results of long-term efficacy experiments of compounds in C57BL / 6 mouse model

[0441] Example 9 Verification of the long-lasting efficacy of the compounds of the present invention in a C57BL / 6 mouse model

[0442] C57BL / 6 mice (male, 6-8 weeks) were randomly divided into groups and administered a single dose of 7.5 μg per eye via bilateral intravitreal injection. Specifically, before the experiment, the siRNA conjugate was dissolved in phosphate buffer solution and diluted to the required solution concentration and volume. The administration volume of phosphate buffer solution and siRNA conjugate was 1.5 μL / eye.

[0443] On the 14th day after administration, the eyeballs were removed and separated into three parts: ① cornea + iris + ciliary body; ② retina; ③ retinal pigment epithelium (RPE) + choroid + sclera; the separated samples were immediately frozen in liquid nitrogen and then stored at -80°C for detection of mTTR mRNA.

[0444] The subsequent experimental steps and primers were the same as those in Example 8 above.

[0445] Table 3 Results of long-term efficacy experiments of compounds in C57BL / 6 mouse model

[0446] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

Claims

1. An oligonucleotide comprising one or more compounds of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, In formula (I) represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; X1 is selected from H, Rs or X2 is selected from OR1 or One of X1 and X2 is R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; In formula (I) and R1 are not H at the same time; Each L1 and L2 are independently selected from a chemical bond, C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, OC 1-10 Alkylene, OC 1-10 Alkenylene, OC 1-10 Alkynylidene, C(O)C 1-10 Alkylene; said L1 and L2 are optionally substituted by 1, 2, 3, 4, 5, 6, 7 or 8 R; T is selected from a chemical bond, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M- or -OC(O)-M-; Where M is A is a sugar, which is preferably a sugar that can be cleaved in an inclusion body or a lysosome, preferably a penta-sugar or a hexa-sugar, more preferably a hexa-sugar, such as N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably N-acetylgalactosamine or N-acetylglucosamine; P is a hydrophobic group, preferably C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH2CH2- group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring; the C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R; R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated; R s Selected from H, D, halogen, C 1-6 Alkyl, C 1-6 haloalkyl, 3-10 membered heterocyclic group or C 3-10 Cycloalkyl, which is optionally deuterated, up to fully deuterated; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; k=0, 1, 2, 3, 4, 5 or 6.

2. The oligonucleotide of claim 1, wherein The compound of formula (I) is selected from the compound of formula (II) or formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; and R1 are not H at the same time; Each L1 and L2 are independently selected from a chemical bond, C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, OC 1-10 Alkylene, OC 1-10 Alkenylene, OC 1-10 Alkynylidene or C(O)C 1-10 Alkylene; said L1 and L2 are optionally substituted by 1, 2, 3, 4, 5, 6, 7 or 8 R; T is selected from a chemical bond, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M- or -OC(O)-M-; Where M is A is a sugar, which is preferably a sugar that can be cleaved in an inclusion body or a lysosome, preferably a penta-sugar or a hexa-sugar, more preferably a hexa-sugar, such as N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably N-acetylgalactosamine or N-acetylglucosamine; P is a hydrophobic group, preferably C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH2CH2- group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring; the C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R; R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated; R s Selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated; R s 'Selected from H, D, halogen, C 1-6 Alkyl, C 1-6 haloalkyl, 3-10 membered heterocyclic group or C 3-10 Cycloalkyl, which is optionally deuterated, up to fully deuterated; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; k=0, 1, 2, 3, 4, 5 or 6.

3. The oligonucleotide according to claim 1 or 2, wherein represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; and R1 are not H at the same time; Each L1 and L2 are independently selected from a chemical bond, C 1-6 Alkylene, OC 1-6 Alkylene or C(O)C 1-6 Alkylene; said L1 and L2 are optionally substituted by 1, 2, 3, 4 or 5 R; T is selected from a chemical bond, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M- or -OC(O)-M-; Where M is A is selected from N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably N-acetylgalactosamine or N-acetylglucosamine; P is selected from C 10-22 Alkyl or C 10-22 Alkenyl, the C 10-22 Alkyl or C 10-22 Alkenyl is optionally substituted with 1, 2, 3, 4 or 5 R; R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 Haloalkyl; R s Selected from H, D, halogen, C 1-6 Alkyl or C 1-6 Haloalkyl; R s 'Selected from H, D, halogen, C 1-6 Alkyl, C 1-6 haloalkyl, 3-10 membered heterocyclic group or C 3-10 Cycloalkyl; m is 0, 1, 2, 3, 4 or 5; n is 0, 1, 2, 3, 4 or 5; k is 0, 1, 2, 3, 4 or 5.

4. The oligonucleotide according to any one of claims 1 to 3, wherein represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; and R1 are not H at the same time; Each L1 and L2 are independently selected from a chemical bond or OC 1-6 Alkylene; said L1 and L2 are optionally substituted by 1, 2 or 3 R; T is selected from a chemical bond, -O-, -CH2-, -OC(O)- or -C(O)-; A is selected from A is connected to L1 through the a-terminal and to the P group through the b-terminal; P is selected from C 10-22 Alkyl or C 10-22 Alkenyl, the C 10-22 Alkyl or C 10-22 Alkenyl is optionally substituted with 1, 2 or 3 R; R is selected from H, D, halogen, C 1-4 alkyl; R s Selected from H or D; R s 'Selected from H, D or C 5-10 Cycloalkyl; m is 0, 1, 2 or 3; n is 0, 1, 2 or 3; k is 0, 1, 2 or 3.

5. The oligonucleotide according to any one of claims 1 to 4, wherein indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; L1 is OCH2CH2 or O(CH2)5; L2 is OCH2 or a chemical bond; T is selected from a chemical bond, -O- or -C(O)-; A is selected from A is connected to L1 through the a-terminal and to the P group through the b-terminal; P is -(CH2) 14-16 CH3; R s is H; R s ' is cyclohexyl; m is 0, 1 or 2; n is 0 or 1; k is 0 or 1.

6. The oligonucleotide according to any one of claims 1 to 5, wherein The compound of formula (II) or formula (III) is selected from the following compounds of the general formula, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: in, P1, P2, P3 are selected from H or P groups; P4 is selected from CH3 or P group; One of P1, P2, P3, and P4 is a P group; The variables are as defined in claims 1-5.

7. The oligonucleotide according to any one of claims 1 to 6, wherein The compound of formula (II) or formula (III) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: in, indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide.

8. The oligonucleotide of any one of claims 1 to 7, having 14 to 30 nucleotides.

9. The oligonucleotide of any one of claims 1 to 8, comprising at the 5' end a compound of formula (II) or formula (III) of any one of claims 1 to 7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

10. The oligonucleotide of any one of claims 1 to 9, comprising at the 3' end a compound of formula (II) or formula (III) of any one of claims 1 to 7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

11. The oligonucleotide of any one of claims 1 to 10, which comprises a compound of formula (II) or formula (III) of any one of claims 1 to 7 at the 5' end and the 3' end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

12. An oligonucleotide comprising one, two or more delivery vectors within the oligonucleotide, at the 5' end and / or at the 3' end, wherein the delivery vector is a sugar modified with a hydrophobic group; Preferably, the sugar modified by the hydrophobic group is selected from the compound of formula (X), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, indicates the position of attachment to the oligonucleotide; P is a hydrophobic group; A is the sugar part; P and A are as defined in any one of claims 1 to 5; Preferably, the hydrophobic group is attached to a hydroxyl or acetyl group of the sugar moiety; Preferably, the sugar modified by the hydrophobic group is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: in, Indicates the position of attachment to the oligonucleotide.

13. The oligonucleotide according to any one of claims 1 to 12, which is an ASO or siRNA, preferably for inhibiting a gene expressed outside the liver, more preferably for inhibiting a gene expressed in the central nervous system (CNS) and / or the eye.

14. A compound of formula (II') or (III'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: R1 and R2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support; A is an acetylated sugar, which is preferably a sugar that can be cleaved in an inclusion body or a lysosome, and is preferably an acetylated penta- or hexa-sugar, and the penta- or hexa-sugar is, for example, N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, and more preferably the penta- or hexa-sugar is N-acetylgalactosamine or N-acetylglucosamine; Preferably, A is selected from A is connected to L1 through the a-terminal and to the P group through the b-terminal; P, L1, L2, T, Rs, Rs', m, n, k are as defined in any one of claims 1-7.

15. The compound of claim 14, wherein R1 and R2 are H.

16. The compound of claim 14 or 15, wherein One of R1 and R2 is a reactive phosphorus group, preferably a phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate or phosphate mimetic, such as natural phosphates, phosphorothioates, phosphorodithioates, boranophosphates, boranophosphorothioates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiester, phosphotriester, phosphorothioate diester, phosphorothioate triester, diphosphate or triphosphate, preferably -P(OCH2CH2CN)(N(iPr)2).

17. The compound according to any one of claims 14 to 16, wherein R1 and R2 are selected from protecting groups, preferably hydroxy protecting groups, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2, 2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), di-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, preferably DMTr.

18. The compound of any one of claims 14 to 17, wherein the compound of formula (II') or (III') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

19. A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand comprises a sequence that is sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand comprises one or more compounds of formula (II) or formula (III) as described in any one of claims 1 to 7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

20. The double-stranded RNA of claim 19, wherein the compound of formula (II) or formula (III) is selected from the following compounds of the general formula, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: in, The variables are as defined in claims 1-7.

21. The double-stranded RNA of claim 19 or 20, wherein the compound of formula (II) or formula (III) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: in, indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide.

22. The double-stranded RNA of any one of claims 19 to 21, wherein the sense strand comprises a compound of formula (II) or formula (III) according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 5' end.

23. The double-stranded RNA of any one of claims 19 to 22, wherein the sense strand comprises a compound of formula (II) or formula (III) according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 3' end.

24. The double-stranded RNA of any one of claims 19 to 23, wherein the sense strand comprises a compound of formula (II) or (III) according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 5' end and the 3' end, respectively.

25. A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand comprises one, two or more delivery vectors at the interior, 5' end and / or 3' end, the delivery vector being a sugar modified with a hydrophobic group; Preferably, the sugar modified by the hydrophobic group is selected from the compound of formula (X), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, indicates the position of attachment to the oligonucleotide; P is a hydrophobic group; A is the sugar part; P and A are as defined in any one of claims 1 to 5; Preferably, the hydrophobic group is attached to a hydroxyl or acetyl group of the sugar moiety; Preferably, the sugar modified by the hydrophobic group is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: in, Indicates the position of attachment to the oligonucleotide.

26. A cell comprising the double-stranded RNA of any one of claims 19 to 25.

27. A pharmaceutical composition comprising the double-stranded RNA according to any one of claims 19 to 25, or the cell according to claim 26, and optionally a pharmaceutically acceptable carrier or excipient.

28. A kit comprising the double-stranded RNA according to any one of claims 19 to 25, or the cell according to claim 26.

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