CN121358748A

CN121358748A - Oligonucleotide delivery ligands containing sugar

Info

Publication number: CN121358748A
Application number: CN202480038604.7A
Authority: CN
Inventors: 黄金宇; 邹昊; 刘俊凯
Original assignee: Darui Biomedical Technology Shanghai Co ltd
Current assignee: Darui Biomedical Technology Shanghai Co ltd
Priority date: 2023-06-12
Filing date: 2024-06-12
Publication date: 2026-01-16
Also published as: WO2024255761A1

Abstract

A compound of formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof is provided. The compounds of formula (I) are contained within nucleotides, either at the 5 'end and/or the 3' end, for delivery of double stranded RNA to extrahepatic tissues, 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

The present invention claims priority from chinese invention patent application, filed on 6/12/2023/CN 202310693838.2, incorporated herein by reference in its entirety.

Technical Field

The present invention is in the field of medicine, in particular to delivery vehicles with the delivery of double stranded RNA to extrahepatic tissues, such as the central nervous system or the eye, which are saccharides modified with hydrophobic groups, such as compounds of formula (X), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

Background

RNA interference is a phenomenon of efficient and specific degradation of target mRNA induced by double-stranded RNA (double-STRANDED RNA, DSRNA, also known as siRNA).

However, due to the presence of the blood brain barrier, it is difficult to deliver siRNA to the central nervous system to function in turn, which limits the use of siRNA. Some attempts in the art to deliver siRNA to the central nervous system have been made, for example WO2004094595A2 discloses the delivery of siRNA using a single lipid ligand (e.g. cholesterol or long chain alkane) at the end of the chain, WO2019217459A1 discloses the delivery of siRNA using a single lipid ligand inside the chain, WO2021092371A2 discloses a series of new lipid ligand structures.

There remains a need in the art to develop more hydrophobic groups to more effectively deliver siRNA to the central nervous system.

Disclosure of Invention

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

Wherein each group is as defined below.

In another aspect, the invention provides an oligonucleotide comprising one or more compounds of formula (II) or formula (III), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

Wherein each group is as defined below.

In another aspect, the invention provides an oligonucleotide comprising one, two or more delivery vehicles within, 5 'and/or 3' of the oligonucleotide, the delivery vehicles being saccharides modified with hydrophobic groups;

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

Wherein each group is as defined below.

In another aspect, the 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 substantially complementary to the sense strand and a target mRNA, wherein the sense strand and/or antisense strand comprises one or more compounds of formula (II) or formula (III) above, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

In another aspect, the 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 substantially complementary to the sense strand and target mRNA, wherein the sense strand and/or antisense strand comprises one, two or more delivery vehicles within, 5 'and/or 3' of the strand, the delivery vehicles being saccharides modified with hydrophobic groups;

Wherein each group is as defined below.

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

Wherein each group is as defined below.

In another aspect, the hydrophobic group provided herein (the P group in formula (I), (II) or (III)) can be attached to an oligonucleotide or double stranded RNA by hydroxyl or acetyl groups of the sugar moiety in the nucleotide.

In another aspect, the invention provides a vector comprising a nucleotide sequence encoding the aforementioned double stranded RNA.

In another aspect, the invention provides a cell comprising the aforementioned double stranded RNA or the aforementioned vector.

In another aspect, the 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.

In another aspect, the invention provides a kit comprising the aforementioned double stranded RNA, the aforementioned vector, or the aforementioned cell.

Detailed Description

Definition of the definition

Chemical definition

The definition of specific functional groups and chemical terms is described in more detail below.

When numerical ranges are listed, it is intended to include each and every value and subrange within the 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.

"C _1-6 alkyl" refers to a straight or branched saturated hydrocarbon group having 1 to 6 carbon atoms. "C _8-30 alkyl" refers to a straight or branched 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 are preferred. examples of C _1-6 alkyl groups include methyl (C ₁), ethyl (C ₂), n-propyl (C ₃), Isopropyl (C ₃), n-butyl (C ₄), tert-butyl (C ₄), sec-butyl (C ₄), Isobutyl (C ₄), n-pentyl (C ₅), 3-pentyl (C ₅), pentyl (C ₅), Neopentyl (C ₅), 3-methyl-2-butyl (C ₅), tert-amyl (C ₅) and n-hexyl (C ₆). 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 with a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted with one or more substituents, for example, with 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 (-CH ₂CH(CH₃)₂).

"C _2-10 alkenyl" refers to a straight or branched hydrocarbon group having 2 to 10 carbon atoms and at least one carbon-carbon double bond. "C _8-30 alkenyl" refers to a straight or branched 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 are preferred. Examples of C _2-6 alkenyl groups include vinyl (C ₂), 1-propenyl (C ₃), 2-propenyl (C ₃), 1-butenyl (C ₄), 2-butenyl (C ₄), butadienyl (C ₄), pentenyl (C ₅), Pentadienyl (C ₅), hexenyl (C ₆), 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 with heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The 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.

"C _2-10 alkynyl" refers to a straight or branched hydrocarbon group having 2 to 10 carbon atoms and at least one carbon-carbon triple bond. "C _8-30 alkynyl" refers to a straight or branched 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 are preferred. Examples of C _2-6 alkynyl include, but are not limited to, ethynyl (C ₂), 1-propynyl (C ₃), 2-propynyl (C ₃), 1-butynyl (C ₄), 2-butynyl (C ₄), pentynyl (C ₅), hexynyl (C ₆), 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 with heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"C _1-10 Alkylene", "C _2-10 alkenylene" and "C _2-10 alkynylene" refer to the removal of C _1-10 alkyl, respectively, The divalent group formed by the other hydrogen of C _2-10 alkenyl and C _2-10 alkynyl groups, and may be substituted or unsubstituted. 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 are preferred. Unsubstituted alkylene groups include, but are not limited to, methylene (-CH ₂ -), ethylene (-CH ₂CH₂ -), propylene (-CH ₂CH₂CH₂ -), butylene (-CH ₂CH₂CH₂CH₂ -), and, Pentylene (-CH ₂CH₂CH₂CH₂CH₂ -), hexylene (-CH ₂CH₂CH₂CH₂CH₂CH₂ -), and the like. Exemplary substituted alkylene groups, for example, alkylene groups substituted with one or more alkyl (methyl) groups, include, but are not limited to, substituted methylene (-CH (CH ₃)-、-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.

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

Thus, "C _1-6 haloalkyl" means that the above "C _1-6 alkyl" is substituted with one or more halo groups. In some embodiments, C _1-4 haloalkyl is particularly preferred, more preferably C _1-2 haloalkyl. Exemplary such 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 may be substituted at any available point of attachment, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"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 are particularly preferred, more preferably C _5-6 cycloalkyl. Cycloalkyl also includes ring systems in which the cycloalkyl ring is fused to one or more aryl or heteroaryl groups, where the point of attachment is on the cycloalkyl ring, and in such cases the number of carbons continues to represent the number of carbons in the cycloalkyl system. exemplary cycloalkyl groups include, but are not limited to, cyclopropyl (C ₃), cyclopropenyl (C ₃), cyclobutyl (C ₄), cyclobutenyl (C ₄), Cyclopentyl (C ₅), cyclopentenyl (C ₅), cyclohexyl (C ₆), cyclohexenyl (C ₆), Cyclohexadienyl (C ₆), cycloheptyl (C ₇), cycloheptenyl (C ₇), cycloheptadienyl (C ₇), Cycloheptatriene (C ₇), and the like. Cycloalkyl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"3-10 Membered heterocyclyl" refers to a group of a 3-10 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 a heterocyclic group containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as the valence permits. In some embodiments, a 4-10 membered heterocyclic group is preferred, which is a 4-10 membered non-aromatic ring system having a ring carbon atom and 1 to 5 ring heteroatoms, a 3-8 membered heterocyclic group is preferred, which is a 3-8 membered non-aromatic ring system having a ring carbon atom and 1 to 4 ring heteroatoms, a 3-6 membered heterocyclic group is preferred, which is a 3-6 membered non-aromatic ring system having a ring carbon atom and 1 to 3 ring heteroatoms, a 4-7 membered heterocyclic group is preferred, which is a 4-7 membered non-aromatic ring system having a ring carbon atom and 1 to 3 ring heteroatoms, and a 5-6 membered heterocyclic group is more preferred, which is a 5-6 membered non-aromatic ring system having a ring carbon atom and 1 to 3 ring heteroatoms. Heterocyclyl also includes ring systems in which the above heterocyclyl ring is fused to one or more cycloalkyl groups, wherein the point of attachment is on the cycloalkyl ring, or ring systems in which the above 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 cases the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, aziridine, oxetane, thiirane (thiorenyl). Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclic groups 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, oxathiolanyl (oxasulfuranyl), dithiolane (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 (thianyl). Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithiocyclohexenyl, dioxanyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, hexahydrotriazinyl (triazinanyl). Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepinyl, and thiepanyl. Exemplary 5-membered heterocyclic groups fused to a C ₆ aryl ring (also referred to herein as 5, 6-bicyclic heterocyclic groups) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinone groups, and the like. Exemplary 6-membered heterocyclyl groups fused to a C ₆ aryl ring (also referred to herein as 6, 6-bicyclic heterocyclyl) 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.

The term "hydrophobic group" refers broadly to any chemical group having an affinity for lipids. One way of characterizing 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 chemical species in the octanol phase to its concentration in the aqueous phase at equilibrium for a two-phase system. Typically, the hydrophobic moiety has a log k _ow of greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. In particular, according to the invention, the hydrophobic moiety is an R group in a compound of formula I.

Alkyl, alkenyl, alkynyl, and the like as defined herein are optionally substituted groups.

Exemplary substituents on carbon atoms include, but are not limited to, halo 、-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^bbC(＝O)R^aa、-NR^bbCO₂R^aa、-NR^bbC(＝O)N(R^bb)₂、-C(＝NR^bb)R^aa、-C(＝NR^bb)OR^aa、-OC(＝NR^bb)R^aa、-OC(＝NR^bb)OR^aa、-C(＝NR^bb)N(R^bb)₂、-OC(＝NR^bb)N(R^bb)₂、-NR^bbC(＝NR^bb)N(R^bb)₂、-C(＝O)NR^bbSO₂R^aa、-NR^bbSO₂R^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^bbP(＝O)(OR^cc)₂、-NR^bbP(＝O)(NR^bb)₂、-P(R^cc)₂、-P(R^cc)₃、-OP(R^cc)₂、-OP(R^cc)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 substituted with 0,1, 2, 3, 4, or 5R ^dd groups;

Or two geminal hydrogens on the carbon atom are substituted with a group ＝O、＝S、＝NN(R^bb)₂、＝NNR^bbC(＝O)R^aa、＝NNR^bbC(＝O)OR^aa、＝NNR^bbS(＝O)₂R^aa、＝NR^bb or = NOR ^cc;

Each of R ^aa is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R ^aa groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2, 3, 4, or 5R ^dd groups;

Each of R ^bb 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 groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2,3, 4, or 5R ^dd groups;

each of R ^cc is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R ^cc groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2, 3, 4, or 5R ^dd groups;

Each of R ^dd 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^ffC(＝O)R^ee、-NR^ffCO₂R^ee、-NR^ffC(＝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^ffC(＝NR^ff)N(R^ff)₂、-NR^ffSO₂R^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 with 0,1, 2,3, 4, or 5R ^gg groups, or two geminal R ^dd substituents may combine to form =o or =s;

Each of R ^ee 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 substituted with 0,1,2,3, 4, or 5R ^gg groups;

Each of R ^ff is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R ^ff groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2, 3, 4, or 5R ^gg groups;

R ^gg is each independently halogen, -CN, -NO ₂、-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), -CO ₂H、-CO₂(C_1-6 alkyl), -OC (=o) (C _1-6 alkyl), -OCO ₂(C_1-6 alkyl), -C (=o) NH ₂、-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), -NHCO ₂(C_1-6 alkyl), -NHC (=o) N (C _1-6 alkyl) ₂、-NHC(＝O)NH(C_1-6 alkyl), -NHC (=o) NH ₂、-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) NH ₂、-OC(＝NH)N(C_1-6 alkyl) ₂、-OC(NH)NH(C_1-6 alkyl, -OC (NH) NH ₂、-NHC(NH)N(C_1-6 alkyl) ₂、-NHC(＝NH)NH₂、-NHSO₂(C_1-6 alkyl), -SO ₂N(C_1-6 alkyl) ₂、-SO₂NH(C_1-6 alkyl, -SO ₂NH₂、-SO₂C_1-6 alkyl, -SO ₂OC_1-6 alkyl, -OSO ₂C_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) NH ₂、-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 haloalkyl, C ₂-C₆ alkenyl, C ₂-C₆ alkynyl, C ₃-C₇ cycloalkyl, C ₆-C₁₀ aryl, C ₃-C₇ heterocyclyl, C ₅-C₁₀ heteroaryl, or two geminal R ^gg substituents may combine to form =o or =s, wherein X ^- is a counterion.

Exemplary substituents on the nitrogen atom 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、-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 ^cc groups attached to a nitrogen atom combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2,3,4, or 5R ^dd groups, and wherein R ^aa、R^bb、R^cc and R ^dd are as described above.

Other definitions

The term "siRNA" herein is a class of double stranded RNA molecules that can mediate silencing of target RNAs (e.g., mRNA, e.g., transcripts of genes encoding proteins) complementary thereto. siRNA is typically double-stranded, comprising an antisense strand complementary to a 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 genes are also referred to as target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNAs (e.g., tRNA's) and viral RNAs can be targeted.

The term "antisense oligonucleotide", ASO (Antisense Oligonucleotides), is a single-stranded DNA or RNA sequence paired with a target gene consisting of 15-25 nucleotides, which specifically blocks the transcription or translation process of the target gene to achieve the purpose of gene regulation.

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

The term "complementary region" refers to a region on the antisense strand that is fully, substantially or essentially complementary to a target mRNA sequence. In cases where the complementary region is not perfectly complementary to the target sequence, the mismatch may be located in an internal or terminal region of the molecule. Typically, the most tolerated mismatch is located in the terminal region, e.g., within 5, 4, 3, 2 or 1 nucleotides of the 5 'and/or 3' end. The portion of the antisense strand that is most susceptible to mismatch is referred to as the "seed region". For example, in an siRNA comprising a strand of 19nt, the 19 th position (from 5 'to 3') may tolerate some mismatches.

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 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA at 50℃or 70℃for 12-16 hours. "complementary" sequences may also include or be formed entirely from non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides in terms of meeting the above requirements with respect to their ability to hybridize. Such non-Watson-Crick base pairs include, but are not limited to, G: U wobble base pairing or Hoogstein base pairing.

A polynucleotide that is "at least partially complementary," "substantially 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, if the sequence is substantially complementary to the non-interrupting portion of the PCSK 9-encoding mRNA, the polynucleotide is at least partially complementary to the PCSK9 mRNA. The terms "complementary," "fully complementary," "substantially complementary," and "substantially complementary" herein can be used with respect to base pairing between the sense strand and the antisense strand of an siRNA agent, or between the antisense strand and a target sequence of an siRNA agent.

"Substantially complementary" refers to the extent to which the sense strand need only be complementary to the antisense strand in order to maintain the overall double-stranded character of the molecule. In other words, while perfect complementarity is often desired, in some cases, particularly in the antisense strand, one or more, e.g., 6, 5, 4, 3, 2, or 1 mismatches (relative to the target mRNA) may be included, but the sense and antisense strands may still maintain the overall double stranded character of the molecule.

"Nucleoside" is a compound consisting of a purine or pyrimidine base, and ribose or deoxyribose, and "nucleotide" is a compound consisting of three substances, purine or pyrimidine base, ribose or deoxyribose, and phosphate, and "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) having a length of less than 100, 200, 300, or 400 nucleotides, for example.

"Base" is the basic constituent unit of synthetic nucleosides, nucleotides and nucleic acids, the constituent elements of which contain nitrogen, also known as "nitrogenous bases". Herein, unless otherwise indicated, capital letters A, U, T, G and C represent the base composition of nucleotides, adenine, uracil, thymine, guanine and cytosine, respectively.

"Modification" of a nucleotide as described herein includes, but is not limited to, methoxy modification, fluoro modification, phosphorothioate linkage, or conventional protecting group protection, and the like. For example, the fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide is substituted with fluoro, and the methoxy-modified nucleotide refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with methoxy.

"Modified nucleotides" herein include, but are not limited to, 2' -O-methyl modified nucleotides, 2' -fluoro modified nucleotides, 2' -deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides containing phosphorothioate groups, vinylphosphate modified nucleotides, locked nucleotides, 2' -amino-modified nucleotides, 2' -alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, unnatural bases containing nucleotides, and terminal nucleotides attached to cholesterol derivatives or dodecanoic didecanoyl amine groups, deoxyribonucleotides or conventional protecting group protection, and the like. For example, the 2 '-fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with fluorine. The 2 '-deoxy-modified nucleotide refers to a nucleotide formed by substituting a 2' -hydroxyl group of a ribosyl group with a methoxy group.

"Reactive phosphorus group" refers to a phosphorus-containing group contained in a nucleotide unit or in a nucleotide analog unit that can react with a hydroxyl or amine group contained in another molecule, especially in another nucleotide unit or in another nucleotide analog, by nucleophilic attack reaction. Typically, such a reaction produces an ester internucleoside linkage linking the first nucleotide unit or the first nucleotide analogue unit to the second nucleotide unit or the second nucleotide analogue unit. The reactive phosphorus group may be selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate ester mimics including, but not limited to, natural phosphates, phosphorothioates, phosphorodithioates, borane phosphates, borane phosphorothioates, phosphonates, halogen substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, phosphorothioates triesters, bisphosphates and triphosphates, preferably-P (OCH ₂CH₂CN)(N(iPr)₂).

"Protecting group" refers to any atom or group of atoms that is added to a molecule to prevent undesired chemical reactions of existing groups in the molecule. "protecting groups" may be labile chemical moieties known in the art that serve to protect reactive groups, such as hydroxyl, amino, and thiol groups, from undesired or untimely reactions during chemical synthesis. The protecting groups are typically used selectively and/or orthogonally to the protecting site during the reaction of the other reactive site, and can then be removed to leave the unprotected group intact or available for further reaction.

Non-limiting lists of protecting groups include benzyl, substituted benzyl, alkylcarbonyl and alkoxycarbonyl (e.g., t-Butoxycarbonyl (BOC), acetyl or isobutyryl), aralkylcarbonyl and aralkoxycarbonyl (e.g., benzyloxycarbonyl), substituted methyl ethers (e.g., methoxymethyl ether), substituted diethyl ether, substituted benzyl ether, tetrahydropyranyl ether, silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2- (trimethylsilyl) ethoxy ] methyl or t-butyldiphenylsilyl), esters (e.g., benzoates), carbonates (e.g., methoxymethyl carbonate), sulfonates (e.g., tosylate or mesylate), acyclic ketals (e.g., dimethylacetal), acyclic ketals (e.g., 1, 3-dioxane, 1, 3-dioxolane and those described herein), acyclic acetals, cyclic hemiacetals, cyclic dithioketals (e.g., those described herein), cyclic dithioketals (e.g., 1, 3-dioxane, 3-dioxolane), methyl, trimethyl benzene (e.g., 1, 3-dimethyl sulfide, 4 '-trimethylbenzene, tri-4' -methyl), methyl (e.g., tri-4, 4 '-methoxy) and (e.g., methyl) tri-4, 4' -phenyl) methyl (e.g., tri-methyl), 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), beta-Methoxyethoxymethyl Ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, piv-onyl (Piv), tetrahydropyranyl (THP), triphenylmethyl (Trt), methoxytrityl [ (4-methoxyphenyl) diphenylmethyl ] (MMT), dimethoxytrityl, [ bis- (4-methoxyphenyl) phenylmethyl (DMT), trimethylsilyl ether (TMS), t-butyldimethylsilyl ether (TBDMS), tri-isopropyl silyloxymethyl ether (TOM), tri-isopropyl silyl ether (TIPS), methyl ether, ethoxydiethyl Ether (EE) N, N-dimethyl formamidine and 2-Cyanoethyl (CE).

"Hydroxy protecting group" refers to a group that is capable of protecting a hydroxy group from chemical reaction and that can be removed under specific conditions to restore the hydroxy group. Mainly comprises a silane type protecting group, an acyl type protecting group or an ether type protecting group, preferably the following:

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-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), C (CH 25, C (OH) or C4-C (OH) or more preferably C4-C (OH) 35O, 3, C (CH) or C4-O3, more preferably C (OH) 35O (CH) or C4-O (CH) 35.

The term "pharmaceutically acceptable salts" as used herein means those carboxylate salts, amino acid addition salts of the compounds of the invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response and the like commensurate with a reasonable benefit/risk ratio, and effective for their intended use, including (if possible) zwitterionic forms of the compounds of the invention.

The present invention includes tautomers, which are functional group isomers that result from the rapid movement of an atom in a molecule at two positions. Compounds that exist in different tautomeric forms, one of the compounds is not limited to any particular tautomer, but is intended to encompass all tautomeric forms.

The compounds of the invention may include one or more asymmetric centers and thus may exist in a variety of stereoisomeric forms, for example, enantiomeric and/or diastereomeric forms. For example, the compounds of the 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. The isomers may be separated from the mixtures by methods known to those skilled in the art, including chiral High Pressure Liquid Chromatography (HPLC) and formation and crystallization of chiral salts, or the preferred isomers may be prepared by asymmetric synthesis.

The invention also includes isotopically-labelled compounds (isotopically-variant) which are identical to those recited in formula (I), but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as ²H、³H、¹³C、¹¹C、¹⁴C、¹⁵N、¹⁸O、¹⁷O、³¹P、³²P、³⁵S、¹⁸F and ³⁶ Cl, respectively. The compounds of the invention, prodrugs thereof, and pharmaceutically acceptable salts of the compounds or prodrugs thereof, which contain the isotopes described above and/or other isotopes of other atoms, are within the scope of this invention. Certain isotopically-labeled compounds of the present invention, for example those into which radioactive isotopes (e.g., ³ H and ¹⁴ C) are introduced, are useful in drug and/or substrate tissue distribution assays. Tritium, i.e., ³ H, and carbon-14, i.e., ¹⁴ C isotopes are particularly preferred because they are easy to prepare and detect. Further, substitution with heavier isotopes, such as deuterium, i.e., ² H, may be preferred in some circumstances because greater metabolic stability may afford therapeutic benefits such as increased in vivo half-life or reduced dosage requirements. Isotopically-labeled compounds of formula (I) of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes and/or examples and preparations below by substituting a readily available isotopically-labeled reagent for a non-isotopically-labeled reagent.

Compounds of the invention

The present invention relates in particular to an oligonucleotide comprising one or more compounds of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

Wherein, the

In the formula (I)Represents H, or represents the position of a phosphate or phosphorothioate linkage to an adjacent nucleotide;

x ₁ is selected from H, rs or

X ₂ is selected from OR ₁ OR

One of X ₁ and X ₂ is

R ₁ represents H, or represents the position of a phosphate or phosphorothioate linkage to another adjacent nucleotide;

in the formula (I) And R ₁ is not H at the same time;

Each of L ₁ and L ₂ is independently selected from the group consisting of a bond, C _1-10 alkylene, C _2-10 alkenylene, C _2-10 alkynylene, OC _1-10 alkylene, OC _1-10 alkenylene, OC _1-10 alkynylene, C (O) C _1-10 alkylene, said L ₁ and L ₂ being optionally substituted with 1, 2, 3, 4, 5, 6, 7, or 8R;

T is selected from the group consisting of a bond, -O-, -CH ₂-、-C(O)-、-OC(O)-、-M-、-O-M-、-CH₂ -M-, -C (O) -M-or-OC (O) -M-;

Wherein M is

A is a sugar, preferably a sugar that can be cleaved in an inclusion body or a lysosome, preferably a five-or six-membered sugar, more preferably a six-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;

P is a hydrophobic group, preferably C _8-30 alkyl, C _8-30 alkenyl or C _8-30 alkynyl, wherein non-adjacent 1,2, 3, 4,5, 6, 7, 8, 9, 10 carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the-CH ₂CH₂ -group may be replaced by-OC (O) -, -C (O) O-, -NHC (O) -or-C (O) NH-, or 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 being optionally substituted by 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, optionally deuterated until fully deuterated;

R _s is selected from H, D, halogen, C _1-6 alkyl, C _1-6 haloalkyl, 3-10 membered heterocyclyl or C _3-10 cycloalkyl, 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.

The 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:

Wherein, the

R _s' is selected from H, D, halogen, C _1-6 alkyl, C _1-6 haloalkyl, 3-10 membered heterocyclyl or C _3-10 cycloalkyl, optionally deuterated until fully deuterated;

The other groups are as defined in the foregoing.

The invention relates in particular to compounds of formula (II ') or (III'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

R ₁ and R ₂ are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group, or a solid support;

A is an acetylated sugar, preferably a sugar cleavable in an endosome or lysosome, preferably an acetylated five-or six-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;

Preferably, A is selected from

A is connected with L ₁ through an a end and is connected with a P group through a b end;

P, L ₁、L₂, T, rs', m, n, k are as defined above.

The invention specifically relates to 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 substantially complementary to the sense strand and target mRNA, wherein the sense strand and/or antisense strand comprises one or more compounds of formula (II) or formula (III) as described previously, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

In one embodiment,Represents a group H, and in another embodiment,Represents the position of the phosphate or phosphorothioate linkage to the adjacent nucleotide.

R ₁ and R ₂

In one embodiment, R ₁ represents H, and in another embodiment R ₁ represents a position of a phosphate or phosphorothioate linkage to another adjacent nucleotide.

In one embodiment, R ₁ is H; in another embodiment, R ₁ is selected from reactive phosphorus groups, preferably phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimics, such as natural phosphates, phosphorothioates, phosphorodithioates, borane phosphates, borane phosphorothioates, phosphonates, halogen substituted phosphonates and phosphates, phosphoramidates, phosphodiester, phosphotriester, phosphorothioate diester, phosphorothioate triester, diphosphate or triphosphate, preferably-P (OCH ₂CH₂CN)(N(iPr)₂); in another embodiment, R ₁ is selected from hydroxy protecting groups such as Trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, P-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), t-butoxycarbonyl (Boc), benzyl (Bn), P-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-P-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), phenylmethyl (Fmoc), 2, 2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C (O) CH ₂CH₂ C (O) OH, or 4,4' -dimethoxytrityl, preferably DMTr, in another embodiment R ₁ is a solid support.

In one embodiment, R ₂ is H; in another embodiment, R ₂ is selected from reactive phosphorus groups, preferably phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimics, such as natural phosphates, phosphorothioates, phosphorodithioates, borane phosphates, borane phosphorothioates, phosphonates, halogen substituted phosphonates and phosphates, phosphoramidates, phosphodiester, phosphotriester, phosphorothioate diester, phosphorothioate triester, diphosphate or triphosphate, preferably-P (OCH ₂CH₂CN)(N(iPr)₂); in another embodiment, R ₂ is selected from hydroxy protecting groups such as Trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, P-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), t-butoxycarbonyl (Boc), benzyl (Bn), P-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-P-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), phenylmethyl (Fmoc), 2, 2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C (O) CH ₂CH₂ C (O) OH, or 4,4' -dimethoxytrityl, preferably DMTr, in another embodiment R ₁ is a solid support.

X ₁ and X ₂

In one embodiment, X ₁ is H, in another embodiment X ₁ is Rs, in another embodiment X ₁ is

In one embodiment, X ₂ is OR ₁, in another embodiment, X ₂ is

L ₁ and L ₂

In one embodiment, L ₁ is a bond, in another embodiment L ₁ is a C _1-10 alkylene group, in another embodiment L ₁ is a C _1-6 alkylene group, in another embodiment L ₁ is a C _2-10 alkylene group, in another embodiment L ₁ is a C _2-10 alkynylene group, in another embodiment L ₁ is an OC _1-10 alkylene group, in another embodiment L ₁ is an OC _1-6 alkylene group, in another embodiment L ₁ is an OC _1-10 alkylene group, in another embodiment L ₁ is an OC _1-10 alkynylene group, in another embodiment L ₁ is a C (O) C _1-10 alkylene group, and in another embodiment L ₁ is a C (O) C _1-6 alkylene group.

In one particular embodiment, L ₁ is OCH ₂CH₂, and in another particular embodiment, L ₁ is O (CH ₂)₅.

In one embodiment, L ₁ is unsubstituted, in another embodiment L ₁ is substituted with 1R, in another embodiment L ₁ is substituted with 2R, in another embodiment L ₁ is substituted with 3R, in another embodiment L ₁ is substituted with 4R, in another embodiment L ₁ is substituted with 5R, in another embodiment L ₁ is substituted with 6R, in another embodiment L ₁ is substituted with 7R, and in another embodiment L ₁ is substituted with 8R.

In one embodiment, L ₂ is a bond, in another embodiment L ₂ is a C _1-10 alkylene group, in another embodiment L ₂ is a C _1-6 alkylene group, in another embodiment L ₂ is a C _2-10 alkylene group, in another embodiment L ₂ is a C _2-10 alkynylene group, in another embodiment L ₂ is an OC _1-10 alkylene group, in another embodiment L ₂ is an OC _1-6 alkylene group, in another embodiment L ₂ is an OC _1-10 alkylene group, in another embodiment L ₂ is an OC _1-10 alkynylene group, in another embodiment L ₂ is a C (O) C _1-10 alkylene group, and in another embodiment L ₂ is a C (O) C _1-6 alkylene group.

In one particular embodiment, L ₂ is OCH ₂, and in another particular embodiment, L ₂ is a bond.

In one embodiment, L ₂ is unsubstituted, in another embodiment L ₂ is substituted with 1R, in another embodiment L ₂ is substituted with 2R, in another embodiment L ₂ is substituted with 3R, in another embodiment L ₂ is substituted with 4R, in another embodiment L ₂ is substituted with 5R, in another embodiment L ₂ is substituted with 6R, in another embodiment L ₂ is substituted with 7R, and in another embodiment L ₂ is substituted with 8R.

T

In one embodiment, a T bond; in another embodiment, T is-O-; in another embodiment, T is-CH ₂ -; 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-O-M-, in another embodiment T is-CH ₂ -M-, in another embodiment T is-C (O) -M-, in another embodiment T is-OC (O) -M-.

In one embodiment, M isIn another embodiment, M isIn another embodiment, M isIn another embodiment, M is

A

In one embodiment, A is a sugar, in another embodiment A is a sugar that can be cleaved in the inclusion body or in the lysosome, in another embodiment A is a five-or six-membered sugar, preferably a six-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.

In a specific embodiment, A isA is linked to L ₁ via the a-terminus and to the P-group via the b-terminus, in another particular embodiment A isA is linked to L ₁ via the a-terminus and to the P-group via the b-terminus, in another particular embodiment A isA is linked to L ₁ via the a-terminus and to the P-group via the b-terminus, in another particular embodiment A isA is connected with L ₁ through an a end and is connected with a P group through a b end.

In one embodiment, A is an acetylated sugar, in another embodiment, an acetylated sugar that is cleavable in an inclusion or lysosome body, in another embodiment, an acetylated five-or six-membered sugar that is, for example, N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably, N-acetylgalactosamine or N-acetylglucosamine.

In a specific embodiment, A isA is linked to L ₁ via the a-terminus and to the P-group via the b-terminus, in another particular embodiment A isA is linked to L ₁ via the a-terminus and to the P-group via the b-terminus, in another particular embodiment A isA is linked to L ₁ via the a-terminus and to the P-group via the b-terminus, in another particular embodiment A is

A is connected with L ₁ through an a end and is connected with a P group through a b end.

P

In one embodiment, P is a hydrophobic group, and in another embodiment, P is a C _8-30 hydrocarbon group, such as C _8-30 alkyl, C _8-30 alkenyl or C _8-30 alkynyl, in another embodiment P is C _10-22 alkyl, e.g., 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 group, C ₂₂ alkyl, in another embodiment P is C _12-18 alkyl, such as C _14-16 alkyl, in another embodiment P is C _10-22 alkenyl.

In one embodiment, non-adjacent 1,2,3, 4, 5, 6, 7, 8, 9, 10 carbon atoms in P may be replaced by heteroatoms selected from O, S and N, or the-CH ₂CH₂ -group may be replaced by-OC (O) -, -C (O) O-, -NHC (O) -or-C (O) NH-, or substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring.

In one embodiment, P is unsubstituted, in another embodiment, P is substituted with 1R, in another embodiment, P is substituted with 2R, in another embodiment, P is substituted with 3R, in another embodiment, P is substituted with 4R, in another embodiment, P is substituted with 5R, in another embodiment, P is substituted with 6R, in another embodiment, P is substituted with 7R, in another embodiment, P is substituted with 8R, and in another embodiment, P is substituted with more R.

In a specific embodiment, P is- (CH ₂)_14-16CH₃).

P ₁、P₂、P₃ and P ₄

In one embodiment, P ₁ is H, and in another embodiment, P ₁ is a P group as defined above.

In one embodiment, P ₂ is H, and in another embodiment, P ₂ is a P group as defined above.

In one embodiment, P ₃ is H, and in another embodiment, P ₃ is a P group as defined above.

In one embodiment, P ₄ is CH ₃, and in another embodiment, P ₄ is a P group as defined above.

In one embodiment, one of P ₁、P₂、P₃、P₄ is a P group.

R

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 until fully deuterated.

R _s and R _s'

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 is C _1-6 alkyl, in another embodiment R _s is C _1-6 haloalkyl, in another embodiment R _s is a 3-10 membered heterocyclyl, in another embodiment R _s is C _3-10 cycloalkyl, in another embodiment R _s is optionally deuterated until fully deuterated.

In a specific embodiment, R _s is H.

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' is C _1-6 alkyl, in another embodiment R _s 'is 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 'is C _3-10 cycloalkyl, in another embodiment R _s' is optionally deuterated until fully deuterated.

In a specific embodiment, R _s' is cyclohexyl.

m

M is 0,1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

n

N is 0,1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

k

K=0, 1,2, 3, 4, 5 or 6.

Any one of the above embodiments or any combination thereof may be combined with any one of the other embodiments or any combination thereof. For example, any one of the aspects of A or any combination thereof can be combined withP、P₁-P₄、R₁、R₂、X₁、X₂、L₁、L₂、T、R、R_s、R_s'、m、n And k, etc., or any combination thereof. The invention is intended to include all such combinations, limited to the extent that they are not listed.

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

For example, siRNA targeting the PCSK9 gene may be expressed from a transcription unit inserted into a DNA or RNA vector. Expression may be transient (hours to weeks) or continuous (weeks to months or more), depending on the particular construct and target tissue or cell type used. The siRNA encoding nucleotide can be incorporated into a linear construct, a circular body, or a viral vector. The nucleotides of the siRNA may be integrated into the cell genome for stable expression or may be expressed extrachromosomally for stable inheritance. In general, siRNA expression vectors are typically DNA plasmids or viral vectors.

Viral vector systems comprising the coding sequence of the siRNA include, but are not limited to, (a) adenovirus vectors, (b) retrovirus vectors, (c) adeno-associated virus 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-dependent adenoviruses or entero-free adenoviruses.

The invention also provides a cell comprising an siRNA or vector according to the invention, wherein the siRNA or vector according to the invention is capable of being transcribed in the cell.

The invention relates to the following technical scheme:

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

Wherein, the

x ₁ is selected from H, rs or

X ₂ is selected from OR ₁ OR

One of X ₁ and X ₂ is

in the formula (I) And R ₁ is not H at the same time;

Wherein M is

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.

A2. The oligonucleotide of claim A1, wherein the compound of formula (I) is selected from the group consisting of a compound of formula (II) or formula (III), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

Wherein, the

Represents H, or represents the position of a phosphate or phosphorothioate linkage to an adjacent nucleotide;

And R ₁ is not H at the same time;

each of L ₁ and L ₂ is independently selected from the group consisting of a bond, C _1-10 alkylene, C _2-10 alkenylene, C _2-10 alkynylene, OC _1-10 alkylene, OC _1-10 alkenylene, OC _1-10 alkynylene, or C (O) C _1-10 alkylene, the L ₁ and L ₂ being optionally substituted with 1, 2, 3, 4, 5, 6, 7, or 8R;

Wherein M is

R _s is selected from H, D, halogen, C _1-6 alkyl or C _1-6 haloalkyl, optionally deuterated until 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.

A3. The oligonucleotide of claim A1 or A2, wherein,

And R ₁ is not H at the same time;

Each of L ₁ and L ₂ is independently selected from a bond, C _1-6 alkylene, OC _1-6 alkylene, or C (O) C _1-6 alkylene, said L ₁ and L ₂ being optionally substituted with 1,2, 3,4, or 5R;

Wherein 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, said C _10-22 alkyl or C _10-22 alkenyl optionally substituted with 1,2, 3,4 or 5R;

R is selected from H, D, halogen, C _1-6 alkyl or C _1-6 haloalkyl;

R _s is selected from H, D, halogen, C _1-6 alkyl or C _1-6 haloalkyl;

R _s' is selected from H, D, halogen, C _1-6 alkyl, C _1-6 haloalkyl, 3-10 membered heterocyclyl 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.

A4. the oligonucleotide according to any one of the claim A1 to A3, wherein,

And R ₁ is not H at the same time;

Each of L ₁ and L ₂ is independently selected from a bond or OC _1-6 alkylene, said L ₁ and L ₂ being optionally substituted with 1,2 or 3R;

T is selected from the group consisting of a bond, -O-, -CH ₂ -, -OC (O) -or-C (O) -;

A is selected from

P is selected from C _10-22 alkyl or C _10-22 alkenyl, said C _10-22 alkyl or C _10-22 alkenyl optionally substituted with 1,2 or 3R;

r is selected from H, D, halogen, C _1-4 alkyl;

R _s is selected from H or D;

R _s' is 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.

A5. the oligonucleotide according to any one of the claim A1 to A4, wherein,

Represents the position of a phosphate or phosphorothioate linkage to an adjacent nucleotide;

R ₁ is H, or represents the position of a phosphate or phosphorothioate linkage to another adjacent nucleotide;

L ₁ is OCH ₂CH₂ or O (CH ₂)₅;

L ₂ is OCH ₂ or a chemical bond;

t is selected from the group consisting of a bond, -O-, or-C (O) -;

A is selected from

p is- (CH ₂)_14-16CH₃);

R _s is H;

r _s' is cyclohexyl;

m is 0, 1 or 2;

n is 0 or 1;

k is 0 or 1.

A6. The oligonucleotide of any one of claims A1-A5, wherein the compound of formula (II) or formula (III) is selected from the group consisting of compounds of the following general formulas, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

Wherein, the

P ₁、P₂、P₃ is selected from H or a P group;

P ₄ is selected from CH ₃ or a P group;

One of P ₁、P₂、P₃、P₄ is a P group;

the variables are as defined in the technical schemes A1 to A5.

A7. The oligonucleotide of any one of claims 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:

Wherein, the Represents the position of the phosphate or phosphorothioate linkage to the adjacent nucleotide.

R ₁ is H, or represents the position of a phosphate or phosphorothioate linkage to another adjacent nucleotide.

A8. The oligonucleotide of any one of claims A1-A7, having 14 to 30 nucleotides.

A9. The oligonucleotide of any one of claims A1-A8, comprising at the 5' end a compound of formula (II) or formula (III) of any one of claims A1-A7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

A10. The oligonucleotide of any one of claims A1-A9, comprising at the 3' end a compound of formula (II) or formula (III) of any one of claims A1-A7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

A11. The oligonucleotide of any one of claims A1-a10, comprising at the 5 'and 3' ends, respectively, a compound of formula (II) or formula (III) of any one of claims A1-A7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

A12. The oligonucleotide of any one of claims A1-a11, comprising one or more compounds of formula (II) of any one of claims A1-A7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, within the oligonucleotide.

A13. An oligonucleotide comprising one, two or more delivery vehicles within, 5 'and/or 3' of the oligonucleotide, said delivery vehicles being saccharides modified with hydrophobic groups;

Wherein, the Represents the ligation site to the oligonucleotide;

p is a hydrophobic group;

A is a sugar moiety;

P and A are as defined in any of the technical schemes A1 to A5;

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

Preferably, the sugar modified with a hydrophobic group is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

Wherein, the Represents the ligation site to the oligonucleotide.

A14. The oligonucleotide of any one of claims A1-a13, which is an ASO or siRNA, preferably for inhibiting a gene expressed extrahepatic, more preferably for inhibiting a gene expressed in the Central Nervous System (CNS) and/or the eye.

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

A is an acetylated sugar, preferably a sugar cleavable in an endosome or lysosome, preferably an acetylated five-or six-membered sugar, e.g. N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably the five-or six-membered sugar is N-acetylgalactosamine or N-acetylglucosamine;

Preferably, A is selected from

P, L ₁、L₂, T, rs', m, n, k are as defined in any of claims A1 to A7.

A16. A compound of claim a15 wherein R ₁ and R ₂ are H.

A17. The compound of claim a15 or a16, wherein one of R ₁ and R ₂ is a reactive phosphorus group, preferably phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate or phosphate mimic, such as natural phosphate, phosphorothioate, phosphorodithioate, borane phosphate, borane phosphorothioate, phosphonate, halogen-substituted phosphonate and phosphate, phosphoramidate, phosphodiester, phosphotriester, phosphorothioate diester, phosphorothioate triester, diphosphate or triphosphate, preferably-P (OCH ₂CH₂CN)(N(iPr)₂).

A18. A compound according to any one of claims A15 to A17, wherein R ₁ and R ₂ 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-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMM), C (O) 37C (O) 35, or trimethoxy (DMTr) preferably DMT 4, DMT 4' -DMT.

A19. The compound of any one of claims a15-a18, wherein the compound of formula (II ') or (III') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

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 substantially complementary to the sense strand and a target mRNA, wherein the sense strand and/or antisense strand comprises one or more compounds of formula (II) or formula (III) as set forth in any one of claims A1-A7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

A21. The double stranded RNA of claim a20, wherein the compound of formula (II) or formula (III) is selected from the group consisting of compounds of the following general formulas, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

wherein the variables are as defined in technical schemes A1 to A7.

A22. The double stranded RNA of claim 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:

A23. The double stranded RNA of any one of claims a20-a22, wherein the sense strand comprises at the 5' end a compound of formula (II) or formula (III) of any one of claims A1-A7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

A24. The double stranded RNA of any one of claims a20-a23, wherein the sense strand comprises at the 3' end a compound of formula (II) or formula (III) of any one of claims A1-A7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

A25. The double stranded RNA of any one of claims a20-a24, wherein the sense strand comprises one compound of formula (II) or formula (III) of any one of claims A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 5 'and 3' ends, respectively.

A26. The double stranded RNA of any one of claims a20-a25, wherein the sense strand comprises one or more compounds of any one of claims A1-A7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, within an oligonucleotide.

A27. The double stranded RNA of any one of claims a20-a26, wherein the antisense strand comprises at the 5' end a compound of formula (II) or formula (III) of any one of claims A1-A7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

A28. The double stranded RNA of any one of claims a20-a27, wherein the antisense strand comprises at the 3' end a compound of formula (II) or formula (III) of any one of claims A1-A7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

A29. The double stranded RNA of any one of claims a20-a28, wherein the antisense strand comprises one compound of formula (II) or formula (III) of any one of claims A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 5 'and 3' ends, respectively.

A30. The double stranded RNA of any one of claims a20-a29, wherein the antisense strand comprises one or more compounds of any one of claims A1-A7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, within an oligonucleotide.

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 substantially complementary to the sense strand and target mRNA, wherein the sense strand and/or antisense strand comprises one, two or more delivery vectors at the internal, 5 'and/or 3' ends, the delivery vectors being saccharides modified with hydrophobic groups;

Wherein, the Represents the ligation site to the oligonucleotide;

p is a hydrophobic group;

A is a sugar moiety;

P and A are as defined in any of the technical schemes A1 to A5;

Wherein, the Represents the ligation site to the oligonucleotide.

A32. a vector comprising a nucleotide sequence encoding the double stranded RNA of any one of the preceding claims a20-a 31.

A33. A cell comprising the double stranded RNA of any one of claims a20-a31 or the vector of claim a 32.

A34. A pharmaceutical composition comprising the double stranded RNA of any one of claims a20-a31, the vector of claim a32, or the cell of claim a33, and optionally a pharmaceutically acceptable carrier or excipient.

A35. A kit comprising the double stranded RNA of any one of claims a20-a31, the vector of claim a32, or the cell of claim a 33.

The invention also relates to the following technical scheme:

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

And R ₁ is not H at the same time;

Wherein M is

A is N-acetylglucosamine;

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.

B2. the oligonucleotide of claim B1, wherein,

And R ₁ is not H at the same time;

Wherein M is

A is N-acetylgalactosamine;

R is selected from H, D, halogen, C _1-6 alkyl or C _1-6 haloalkyl;

R _s is selected from H, D, halogen, C _1-6 alkyl or C _1-6 haloalkyl;

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.

B3. The oligonucleotide of claim B1 or B2, wherein,

And R ₁ is not H at the same time;

Each L ₁ and L ₂ is independently selected from a bond or OC _1-6 alkylene, e.g., OCH ₂、OCH₂CH₂、(OCH₂CH₂)₂ or (OCH ₂CH₂)₃; the L ₁ and L ₂ are optionally substituted with 1,2 or 3R;

T is selected from the group consisting of a bond, -O-, -CH ₂ -, -OC (O) -or-C (O) -, preferably-C (O) -;

A is

R is selected from H, D, halogen, C _1-4 alkyl;

R _s is selected from H or D;

m is 0,1,2 or 3;

n is 0,1, 2 or 3;

k is 0,1,2 or 3.

B4. The oligonucleotide of any one of claims B1-B3, wherein the compound is selected from the group consisting of:

Wherein, the

Represents linking to the last nucleotide through a phosphate group, a phosphorothioate group;

Represents the linkage to the next nucleotide through a phosphate group, a phosphorothioate group.

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

Wherein, the

A is

L ₁、L₂, T, rs, m, n, k are as defined above.

B6. a compound of claim B5 wherein R ₁ and R ₂ are H;

Preferably, one of R ₁ and R ₂ is a reactive phosphorus group, preferably phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate or phosphate mimic, such as natural phosphate, phosphorothioate, phosphorodithioate, borane phosphate, borane phosphorothioate, phosphonate, halogen-substituted phosphonate and phosphate, phosphoramidate, phosphodiester, phosphotriester, phosphorothioate diester, phosphorothioate triester, diphosphate or triphosphate, preferably-P (OCH ₂CH₂CN)(N(iPr)₂);

r ₁ and R ₂ 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-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMM), C (O) 37C (O) 35, or trimethoxy (DMTr) preferably DMT 4, DMT 4' -DMT.

B7. A compound of claim 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:

B8. The oligonucleotide according to any one of the aspects B1 to B4, wherein the oligonucleotide is an siRNA for inhibiting a gene expressed in the liver.

List of specific Compounds

The numbering and structure of the compounds of the invention in the oligonucleotides is as follows, wherein the sequence from the compounds is 5' - >3To the point ofAnd (5) connection.

Specifically, according to the order of 5'- >3', such as the corresponding structure being located at the intermediate position of the nucleic acid strand,Represents the 3' carbon or corresponding position attached to the last nucleotide or nucleotide analogue by a phosphate group, phosphorothioate group or other linking group,Represents the attachment to the 5' carbon or corresponding position of the next nucleotide or nucleotide analogue via a phosphate group, phosphorothioate group or other linking group, e.g.the corresponding structure is located at a terminal position of the nucleic acid strand,Correspondingly, the attachment to the 3 'or 5' end of the nucleic acid strand via a phosphate group, phosphorothioate group or other attachment group.

Synthetic examples the following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

Abbreviations (abbreviations)

EXAMPLE 1 preparation of Compound BE1

1. Preparation of Compound 3

Compound 1 (1.00 g,2.03 mmol) was dissolved in DCM (30.0 mL) at 25℃and HATU (0.850 g,2.23 mmol) and DIEA (1.34 mL,8.11 mmol) were added and stirred for 0.5 h, then compound 2 (1.00 g,2.229 mmol) was added and the reaction stirred for 2.5 h at 25 ℃. LCMS showed mass values for the product. Thin layer chromatography (DCM/meoh=10/1) showed a new spot formation. The reaction solution is spin-dried to obtain a crude product. The crude product was purified by normal phase silica gel column (DCM/meoh=1/0-10/1) to give compound 3 as a white solid (1.70 g, yield 90.69%).

¹H 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)

2. Preparation of Compound BE1

Compound 3 (1.70 g,1.84 mmol) was dissolved in DCM (20.0 mL) at 25℃and DCI (0.160 g,1.38 mmol) and compound 4 (0.830 g,2.76 mmol) were added sequentially and the reaction stirred under nitrogen for 2 hours at 25 ℃. LCMS showed complete consumption of compound 3 and mass values for compound BE 1. Thin layer chromatography (DCM/meoh=10/1) showed a new spot formation. The reaction solution is spin-dried to obtain a crude product. The crude product was purified by normal phase silica gel column (dichloromethane/acetone=1/0-5/1) to give compound BE1 (1.02 g, yield 49.32%) as pale yellow oil.

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).

EXAMPLE 2 preparation of Compound BE2

1. Preparation of Compound 3

Compound 1 (1.0 g,2.23 mmol) was dissolved in DCM (20.0 mL) at 25℃and HATU (1.27 g,3.34 mmol) and DIEA (1.10 mL,6.68 mmol) were added. After the mixed liquid was stirred at 25℃for 0.5 hours, compound 2 (1.20 g,2.67 mmol) was added, and the mixed liquid was stirred for an additional 12 hours. TLC (T1: DCM/meoh=10/1) showed complete consumption of starting material. To this was added 200mL of methylene chloride. The mixture was washed with a saturated sodium hydrogencarbonate solution (30.0 mL. Times.3) and then with a saturated brine three times (30.0 mL. Times.3), and the organic phase was dried over anhydrous sodium sulfate and then dried under reduced pressure to give a crude product. The crude product was purified by column chromatography (DCM/meoh=1/0 to 20/1) to give compound 3 (1.43 g,1.70 mmol) as a yellow oil.

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)

2. Preparation of Compound BE2

Compound 3 (1.42 g,1.61 mmol) was dissolved in DCM (15.0 mL) at 25℃and molecular sieves were added followed by DCI (140 mg,1.21 mmol) and Compound 4 (730 mg,2.42 mmol) was added thereto under nitrogen atmosphere and the mixed liquor was stirred at 25℃for 1.0 h. TLC (DCM/ace=10/1) showed that a new spot was formed, to which was added 0.5mL of triethylamine, 20mL of dichloromethane, stirred with basic silica gel, and 4/1000 of triethylamine solution in mobile phase dichloromethane. Purification by column chromatography (DCM/ace=1/0 to 5/1) gave BE2 (900 mg, yield 51.64%) as a colourless oil.

¹H 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).

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

EXAMPLE 3 preparation of Compound BE3

1. Preparation of Compound 1

Compound 1 (1.17 g,2.18 mmol) was dissolved in DCM (20.0 mL) at 25℃and HATU (1.24 g,3.27 mmol) and DIEA (1.08 mL,6.53 mmol) were added. After the mixed liquid was stirred at 25℃for 0.5 hours, compound 2 (1.08 g,2.40 mmol) was added, and the mixed liquid was stirred for an additional 12 hours. LCMS showed product formation. TLC (T1: DCM/meoh=10/1) showed complete consumption of starting material. To this was added 100mL of methylene chloride. The mixture was washed three times with saturated sodium hydrogencarbonate solution (20.0 mL. Times.3) and saturated brine (20.0 mL. Times.3), and the organic phase was dried over anhydrous sodium sulfate and suspended under reduced pressure to give a crude product. The crude product was purified by column chromatography (DCM/meoh=1/0 to 20/1) to give compound 3 (1.67 g, yield 79.2%) as a yellow oil.

¹H 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)

2. Preparation of Compound BE3

Compound 3 (1.67 g,1.72 mmol) was dissolved in DCM (30.0 mL) at 25℃and DCI (0.150 g,1.29 mmol) and compound 4 (0.780 g,2.59 mmol) were added sequentially and the reaction stirred under nitrogen for 2 hours at 25 ℃. Thin layer chromatography (DCM/meoh=10/1) showed a new spot formation. The reaction solution is spin-dried to obtain a crude product. The crude product was purified by normal phase silica gel column (dichloromethane/acetone=1/0-5/1) to give compound BE3 (1.60 g, yield 79.40%) as pale yellow oil.

¹H 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),2.51-2.67(m,2H),2.14-2.17(m,3H),2.05-2.07(m,3H),1.99(s,6H),1.12-1.24(m,12H).

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

EXAMPLE 4 preparation of Compound BE4

1. Preparation of Compound 1c

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

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).

2. Preparation of Compound 1d

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

¹H 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,3H),1.89(s,3H),1.43(p,J＝7.1Hz,2H),1.23(s,26H),0.89–0.81(m,3H).

3. Preparation of Compound 1e

Compound 1d (5 g,8.54 mmol) was dissolved in DCM (50 mL) under nitrogen, the ice-water bath was cooled to 0-5℃and TMSOTF (2.28 g,10.250 mmol) was added dropwise. Then stirring at room temperature for 16 hours, after the reaction was completed, the reaction solution was added to a saturated sodium bicarbonate solution in portions at 0 to 5℃and the aqueous phase was extracted twice with methylene chloride, the organic phases were combined and washed once with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue, which was purified by silica gel column chromatography (eluent: methylene chloride/ethyl acetate) to give compound 1e (4.2 g,7.99mmol, 93.5%).

4. Preparation of Compound 1g

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

¹H 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).

5. Preparation of Compound 1h

After 1g (2.5 g,3.10 mmol) of the compound was dissolved in MeOH (20 mL) and the system was replaced with nitrogen three times, pd/C (0.566 g,0.1% wt%) was added and the system was replaced with hydrogen three times, and the reaction solution was stirred at room temperature under a hydrogen atmosphere for 16 hours. After completion of the reaction, the reaction mixture was filtered through celite to remove excess Pd/C, and the filtrate was concentrated under reduced pressure to give the oily compound (2.0 g,2.90mmol, 80.0%) for 1 h.

¹H 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),3.93–3.84(m,1H),3.80–3.73(m,1H),3.61–3.54(m,4H),3.53–3.49(m,4H),2.11(s,3H),2.00(s,3H),1.87(s,3H),1.49–1.38(m,2H),1.22(s,26H),0.89–0.82(m,3H).

6. Preparation of Compound 1j

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

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),3.18–3.09(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).

7. Preparation of Compound BE4

Compound 1j (552 mg,0.493 mmol) was dissolved in DCM (5 mL) under nitrogen, 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 in portions into a saturated sodium bicarbonate (20 mL) solution cooled to 0 to 5℃in advance, the aqueous phase was extracted twice with methylene chloride, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue, which was purified by silica gel column chromatography (eluent: 1% Et ₃ N ethyl acetate/1% Et ₃ N methylene chloride) to give compound BE4 (447 mg,0.338mmol, 68.8%).

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),2.71–2.65(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).

EXAMPLE 5 preparation of Compound BE5

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

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),6.91–6.86(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),3.95–3.81(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).

EXAMPLE 6 preparation of Compound BE6

The synthetic procedure for compound BE6 can BE found in example 4, in which compound 1b is replaced with stearic acid.

¹H 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).

EXAMPLE 7 Synthesis of siRNA

The siRNA of the present invention is prepared using a solid phase phosphoramidite method well known in the art. Specific methods are described in, for example, PCT publication Nos. WO2016081444 and WO2019105419, and are briefly described below.

1. Preparation of siRNA with ligand not connected to 3' -end of sense strand

1.1 Synthesis of sense strand (SS strand)

By the solid-phase phosphoramidite synthesis method, blank CPG solid-phase carriers are used as initial circulation, and nucleoside monomers are connected one by one from the 3'-5' direction according to the nucleotide arrangement sequence of the sense strand. Each nucleoside monomer attached involved four steps of deprotection, coupling, capping, oxidation or thio reactions to synthesize oligonucleotides on a scale of 5 umol. The synthesis conditions were as follows:

The nucleoside monomer was provided in 0.05mol/L acetonitrile, the conditions for each step were the same, i.e., the temperature was 25 ℃, 3% trichloroacetic acid-dichloromethane solution was used for deprotection, 3 times, 0.25 mol/L5-Ethylthiotetrazole (ETT) -acetonitrile solution was used for coupling, 2 times for coupling, 10% acetic anhydride-acetonitrile and pyridine/N-methylimidazole/acetonitrile (10:14:76, v/v/v), 2 times for capping, 0.05mol/L of iodine in tetrahydrofuran/pyridine/water (70/20/10, v/v/v) for oxidation, 2 times for oxidation, 0.2mol/L phenylacetyl disulfide (PADS) in acetonitrile/3-methylpyridine (1/1, v/v) for coupling, and 2 times for thio.

1.2 Synthesis of antisense strand (AS strand)

By the solid-phase phosphoramidite synthesis method, blank CPG solid-phase carriers are used as initial circulation, and nucleoside monomers are connected one by one from the 3'-5' direction according to the nucleotide arrangement sequence of the antisense strand. Each of the linked nucleoside monomers involved four steps of deprotection, coupling, capping, oxidation or thio reactions, 5umol of the antisense strand was synthesized under identical conditions as the sense strand.

1.3 Purification and annealing of oligonucleotides

1.3.1 Ammonolysis

Adding the synthesized solid phase carrier (sense strand or antisense strand) into a 5mL centrifuge tube, adding 3% diethylamine/ammonia water (v/v), reacting for 16 hours (or 8 hours) in a constant temperature water bath at 35 DEG (or 55 DEG), filtering, washing the solid phase carrier with ethanol/water three times, 1mL each time, centrifuging and concentrating the filtrate, and purifying the crude product.

1.3.2 Purification

Methods of purification and desalination are well known to those skilled in the art. For example, the column can be packed with strong anionic packing, the sodium chloride-sodium hydroxide system is used for eluting and purifying, the product is collected and is managed, the gel packing purification column can be used for desalting, and the eluting system is pure water.

1.3.3 Annealing

According to the specification, mixing the sense strand (SS strand) and the antisense strand (AS strand) in a molar ratio (SS strand/AS strand=1/1.05), heating to 70-95 ℃ in a water bath, maintaining for 3-5min, naturally cooling to room temperature, and freeze-drying the system to obtain the product.

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

in this context, the meanings of the abbreviations are as follows:

A. U, G and C represent natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide and cytosine ribonucleotide, respectively.

D indicates that the nucleotide adjacent to the right thereof is deoxyribonucleotide. For example dA, dT, dG and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide and cytosine deoxyribonucleotide, respectively.

M represents that the nucleotide adjacent to the left thereof is a 2' -OCH3 modified nucleotide. For example, am, um, gm and Cm represent 2' -OCH3 modified A, U, G and C.

F represents that the nucleotide adjacent to the left thereof is a 2' -F modified nucleotide. For example, af, uf, gf and Cf represent 2' -F modified A, U, G and C, respectively.

"S" means that two nucleotides adjacent to each other and/or the delivery vehicle are linked by phosphorothioate.

VP indicates that the nucleotide adjacent to the right is a vinyl phosphate modified nucleotide.

L96 represents a GalNAc delivery vector of the following structure, which is well known in the art, whereinThe position of attachment to the siRNA through a phosphate or phosphorothioate group is indicated, for example, by PCT publication Nos. WO2009073809 and WO2009082607.

The structure of GL34, LS3 and LS1 are as defined above.

The structure of GL34 when attached to the 3 'and 5' ends of a nucleic acid strand via a phosphate group, phosphorothioate group or other linking group is as follows:

the structure of LS3 when attached to the 3 'and 5' ends of the nucleic acid strand via a phosphate group, phosphorothioate group or other linking group is as follows:

The structure of LS1 when attached to the 3 'and 5' ends of a nucleic acid strand via a phosphate, phosphorothioate or other linking group is as follows:

EXAMPLE 8 demonstration of the efficacy of the Compounds of the invention in a C57BL/6 mouse model

C57BL/6 mice (males, 18 to 21g,6 to 8 weeks) were randomly grouped, and each animal was dosed by calculating the dose according to the body weight, and was dosed by subcutaneous injection in a single dose, and the siRNA conjugate was dosed in 1mg/mL solution (0.9% aqueous sodium chloride solution as solvent), specifically, before the experiment, the siRNA conjugate was dissolved and sized to the desired concentration and volume with 0.9% aqueous sodium chloride solution, and the dosing volume of physiological saline (control group) and the siRNA conjugate was 5mL/kg.

10Mg of liver was taken before dosing (indicated as day 0), and at days 7, 14, 21, 28, 35, 42, 56 and 70 after dosing, and placed in RNAlater ^TM solution, frozen at-80℃for detection of liver mTTR mRNA.

Detection of liver mTTR mRNA:

Cellular RNA extraction was performed using a nucleic acid extractor (Oriental, hangzhou, auto-pure 96), reverse transcription with reference to PRIMESCRIPT ^TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B), fluorescent quantitative PCR reaction (ABI, quantStudio 3) with reference to TaqMan ^TM FAST ADVANCED MASTER Mix (ABI, 4444965) 20. Mu.L system, and primers as shown in Table 1, according to the protocol of the high throughput tissue RNA extraction kit (well known medical science, FG 0412).

TABLE 1 primer information

Data statistics and analysis

Calculating a value of 2 ^-△△Ct and converting the value into a percentage to obtain a residual inhibition rate;

delta ct= [ (Ct experimental group gene of interest-Ct experimental group internal reference) - (Ct control group gene of interest-Ct control group internal reference) ].

Wherein the target gene is mTTR and the internal reference is mGAPDH.

Results of the long-acting test of the efficacy of the compounds of Table 2 in the C57BL/6 mouse model

EXAMPLE 9 demonstration of the efficacy of the Compounds of the invention in a C57BL/6 mouse model

C57BL/6 mice (males, 6-8 weeks) were randomly grouped and given a single dose of 7.5 μg per eye by double-sided intravitreal injection, specifically, prior to the experiment, the siRNA conjugate was dissolved and sized to the desired solution concentration and volume with phosphate buffer solution, and the dosing volume of the phosphate buffer solution and siRNA conjugate was 1.5 μl/eye.

Eyeballs were harvested on day 14 post-dose and isolated into 3 parts ① cornea+iris+ciliary body, ② retina, ③ retinal pigment epithelial cells (RPE) +choroid+sclera, and the isolated samples were immediately frozen in liquid nitrogen and then stored at-80 ℃ for detection of mTTR mRNA.

The subsequent experimental procedure and primers were the same as in example 8 above.

Results of the drug efficacy long-acting experiments of the compounds of Table 3 in the C57BL/6 mouse model

The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.

Claims

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

Wherein, the

In the formula (I)Represents H, or represents the position of a phosphate or phosphorothioate linkage to an adjacent nucleotide;

x ₁ is selected from H, rs or

X ₂ is selected from OR ₁ OR

One of X ₁ and X ₂ is

R ₁ represents H, or represents the position of a phosphate or phosphorothioate linkage to another adjacent nucleotide;

in the formula (I) And R ₁ is not H at the same time;

Each of L ₁ and L ₂ is independently selected from the group consisting of a bond, C _1-10 alkylene, C _2-10 alkenylene, C _2-10 alkynylene, OC _1-10 alkylene, OC _1-10 alkenylene, OC _1-10 alkynylene, C (O) C _1-10 alkylene, said L ₁ and L ₂ being optionally substituted with 1, 2, 3, 4, 5, 6, 7, or 8R;

T is selected from the group consisting of a bond, -O-, -CH ₂-、-C(O)-、-OC(O)-、-M-、-O-M-、-CH₂ -M-, -C (O) -M-or-OC (O) -M-;

Wherein M is

A is a sugar, preferably a sugar that can be cleaved in an inclusion body or a lysosome, preferably a five-or six-membered sugar, more preferably a six-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;

P is a hydrophobic group, preferably C _8-30 alkyl, C _8-30 alkenyl or C _8-30 alkynyl, wherein non-adjacent 1,2, 3, 4,5, 6, 7, 8, 9, 10 carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the-CH ₂CH₂ -group may be replaced by-OC (O) -, -C (O) O-, -NHC (O) -or-C (O) NH-, or 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 being optionally substituted by 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, optionally deuterated until fully deuterated;

R _s is selected from H, D, halogen, C _1-6 alkyl, C _1-6 haloalkyl, 3-10 membered heterocyclyl or C _3-10 cycloalkyl, 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.
The oligonucleotide of claim 1, wherein the compound of formula (I) is selected from the group consisting of a compound of formula (II) or formula (III), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

Wherein, the

Represents H, or represents the position of a phosphate or phosphorothioate linkage to an adjacent nucleotide;

R ₁ represents H, or represents the position of a phosphate or phosphorothioate linkage to another adjacent nucleotide;

And R ₁ is not H at the same time;

each of L ₁ and L ₂ is independently selected from the group consisting of a bond, C _1-10 alkylene, C _2-10 alkenylene, C _2-10 alkynylene, OC _1-10 alkylene, OC _1-10 alkenylene, OC _1-10 alkynylene, or C (O) C _1-10 alkylene, the L ₁ and L ₂ being optionally substituted with 1, 2, 3, 4, 5, 6, 7, or 8R;

T is selected from the group consisting of a bond, -O-, -CH ₂-、-C(O)-、-OC(O)-、-M-、-O-M-、-CH₂ -M-, -C (O) -M-or-OC (O) -M-;

Wherein M is

A is a sugar, preferably a sugar that can be cleaved in an inclusion body or a lysosome, preferably a five-or six-membered sugar, more preferably a six-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;

P is a hydrophobic group, preferably C _8-30 alkyl, C _8-30 alkenyl or C _8-30 alkynyl, wherein non-adjacent 1,2, 3, 4,5, 6, 7, 8, 9, 10 carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the-CH ₂CH₂ -group may be replaced by-OC (O) -, -C (O) O-, -NHC (O) -or-C (O) NH-, or 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 being optionally substituted by 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, optionally deuterated until fully deuterated;

R _s is selected from H, D, halogen, C _1-6 alkyl or C _1-6 haloalkyl, optionally deuterated until fully deuterated;

R _s' is selected from H, D, halogen, C _1-6 alkyl, C _1-6 haloalkyl, 3-10 membered heterocyclyl or C _3-10 cycloalkyl, optionally deuterated until 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.
The oligonucleotide of claim 1 or 2, wherein,

Represents H, or represents the position of a phosphate or phosphorothioate linkage to an adjacent nucleotide;

R ₁ represents H, or represents the position of a phosphate or phosphorothioate linkage to another adjacent nucleotide;

And R ₁ is not H at the same time;

Each of L ₁ and L ₂ is independently selected from a bond, C _1-6 alkylene, OC _1-6 alkylene, or C (O) C _1-6 alkylene, said L ₁ and L ₂ being optionally substituted with 1,2, 3,4, or 5R;

T is selected from the group consisting of a bond, -O-, -CH ₂-、-C(O)-、-OC(O)-、-M-、-O-M-、-CH₂ -M-, -C (O) -M-or-OC (O) -M-;

Wherein 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, said C _10-22 alkyl or C _10-22 alkenyl optionally substituted with 1,2, 3,4 or 5R;

R is selected from H, D, halogen, C _1-6 alkyl or C _1-6 haloalkyl;

R _s is selected from H, D, halogen, C _1-6 alkyl or C _1-6 haloalkyl;

R _s' is selected from H, D, halogen, C _1-6 alkyl, C _1-6 haloalkyl, 3-10 membered heterocyclyl 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.
The oligonucleotide of claim 1 to 3, wherein,

Represents H, or represents the position of a phosphate or phosphorothioate linkage to an adjacent nucleotide;

R ₁ represents H, or represents the position of a phosphate or phosphorothioate linkage to another adjacent nucleotide;

And R ₁ is not H at the same time;

Each of L ₁ and L ₂ is independently selected from a bond or OC _1-6 alkylene, said L ₁ and L ₂ being optionally substituted with 1,2 or 3R;

T is selected from the group consisting of a bond, -O-, -CH ₂ -, -OC (O) -or-C (O) -;

A is selected from

A is connected with L ₁ through an a end and is connected with a P group through a b end;

P is selected from C _10-22 alkyl or C _10-22 alkenyl, said C _10-22 alkyl or C _10-22 alkenyl optionally substituted with 1,2 or 3R;

r is selected from H, D, halogen, C _1-4 alkyl;

R _s is selected from H or D;

R _s' is 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.
The oligonucleotide of claim 1 to 4, wherein,

Represents the position of a phosphate or phosphorothioate linkage to an adjacent nucleotide;

R ₁ is H, or represents the position of a phosphate or phosphorothioate linkage to another adjacent nucleotide;

L ₁ is OCH ₂CH₂ or O (CH ₂)₅;

L ₂ is OCH ₂ or a chemical bond;

t is selected from the group consisting of a bond, -O-, or-C (O) -;

A is selected from

A is connected with L ₁ through an a end and is connected with a P group through a b end;

p is- (CH ₂)_14-16CH₃);

R _s is H;

r _s' is cyclohexyl;

m is 0, 1 or 2;

n is 0 or 1;

k is 0 or 1.
The oligonucleotide of any one of claims 1-5, wherein the compound of formula (II) or formula (III) is selected from the group consisting of compounds of the following general formulas, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

Wherein, the

P ₁、P₂、P₃ is selected from H or a P group;

P ₄ is selected from CH ₃ or a P group;

One of P ₁、P₂、P₃、P₄ is a P group;

The variables are as defined in claims 1 to 5.
The oligonucleotide of any one of claims 1-6, wherein the compound of formula (II) or formula (III) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

Wherein, the Represents the position of a phosphate or phosphorothioate linkage to an adjacent nucleotide;

R ₁ is H, or represents the position of a phosphate or phosphorothioate linkage to another adjacent nucleotide.
The oligonucleotide of any one of claims 1-7, having 14 to 30 nucleotides.
The oligonucleotide of any one of claims 1-8, comprising at the 5' end a compound of formula (II) or formula (III) of any one of claims 1-7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.
The oligonucleotide of any one of claims 1-9, comprising at the 3' end a compound of formula (II) or formula (III) of any one of claims 1-7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.
The oligonucleotide of any one of claims 1-10, comprising at the 5 'and 3' ends a compound of formula (II) or formula (III) according to any one of claims 1-7, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
An oligonucleotide comprising one, two or more delivery vehicles within, 5 'and/or 3' of the oligonucleotide, said delivery vehicles being saccharides modified with hydrophobic groups;

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

Wherein, the Represents the ligation site to the oligonucleotide;

p is a hydrophobic group;

A is a sugar moiety;

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 with a hydrophobic group is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

Wherein, the Represents the ligation site to the oligonucleotide.
The oligonucleotide of any one of claims 1-12, which is an ASO or siRNA, preferably for inhibiting genes expressed extrahepatic, more preferably for inhibiting genes expressed in the Central Nervous System (CNS) and/or the eye.
A compound of formula (II ') or (III'), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

R ₁ and R ₂ are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group, or a solid support;

A is an acetylated sugar, preferably a sugar cleavable in an endosome or lysosome, preferably an acetylated five-or six-membered sugar, e.g. N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably the five-or six-membered sugar is N-acetylgalactosamine or N-acetylglucosamine;

Preferably, A is selected from

A is connected with L ₁ through an a end and is connected with a P group through a b end;

p, L ₁、L₂, T, rs', m, n, k are as defined in any one of claims 1 to 7.
The compound of claim 14, wherein R ₁ and R ₂ are H.
The compound of claim 14 or 15, wherein one of R ₁ and R ₂ is a reactive phosphorus group, preferably phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate or phosphate mimic, such as natural phosphate, phosphorothioate, phosphorodithioate, boranyl phosphate, boranyl phosphorothioate, phosphonate, halogen substituted phosphonate and phosphate, phosphoramidate, phosphodiester, phosphotriester, phosphorothioate diester, phosphorothioate triester, diphosphate or triphosphate, preferably-P (OCH ₂CH₂CN)(N(iPr)₂).
The compound according to any one of claims 14 to 16, wherein R ₁ and R ₂ 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-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMM), C (O) 37C (O) 35, or trimethoxy (DMTr) preferably DMT 4, DMT 4' -DMT.
The compound of any one of claims 14-17, wherein the compound of formula (II ') or (III') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:
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 substantially complementary to the sense strand and a target mRNA, wherein the sense strand and/or antisense strand comprises one or more compounds of formula (II) or formula (III) as defined in any one of claims 1 to 7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
The double stranded RNA of claim 19, wherein the compound of formula (II) or formula (III) is selected from the group consisting of compounds of the following general formulas, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

wherein the variables are as defined in claims 1-7.
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:

Wherein, the Represents the position of a phosphate or phosphorothioate linkage to an adjacent nucleotide;

R ₁ is H, or represents the position of a phosphate or phosphorothioate linkage to another adjacent nucleotide.
The double stranded RNA of any one of claims 19-21, wherein the sense strand comprises one compound of formula (II) or formula (III) of any one of claims 1-7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, at the 5' end.
The double stranded RNA of any one of claims 19-22, wherein the sense strand comprises one compound of formula (II) or formula (III) of any one of claims 1-7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, at the 3' end.
The double stranded RNA of any one of claims 19-23, wherein the sense strand comprises one compound of formula (II) or formula (III) of any one of claims 1-7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 5 'and 3' ends, respectively.
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 substantially complementary to the sense strand and target mRNA, wherein the sense strand and/or antisense strand comprises one, two or more delivery vectors at the internal, 5 'and/or 3' ends, the delivery vectors being saccharides modified with hydrophobic groups;

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

Wherein, the Represents the ligation site to the oligonucleotide;

p is a hydrophobic group;

A is a sugar moiety;

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 with a hydrophobic group is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

Wherein, the Represents the ligation site to the oligonucleotide.
A cell comprising the double stranded RNA of any one of claims 19-25.
A pharmaceutical composition comprising the double stranded RNA of any one of claims 19-25, or the cell of claim 26, and optionally a pharmaceutically acceptable carrier or excipient.
A kit comprising the double stranded RNA of any one of claims 19-25, or the cell of claim 26.