Oligonucleotide delivery ligands comprising sugars

Abstract

Provided are compounds of Formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. The compounds of Formula (I) are comprised within a nucleotide, or at the 5' end and / or 3' end, for delivery of double stranded RNA to extrahepatic tissues, such as the central nervous system or the eye. Also provided are oligonucleotides, double stranded RNAs, vectors, cells, pharmaceutical compositions, and kits comprising the compounds of Formula (I).

Description

[0001] This invention claims priority to Chinese Patent Application No. CN202310693838.2, filed on June 12, 2023, which is incorporated herein by reference in its entirety. Invention Field

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

[0003] RNA interference is a phenomenon in which target mRNA is efficiently and specifically degraded by double-stranded RNA (dsRNA, also known as siRNA).

[0004] However, the presence of the blood-brain barrier makes it difficult to deliver siRNA to the central nervous system to exert its effects, which limits the application of siRNA. Several attempts have been made in this field to deliver siRNA to the central nervous system. For example, WO2004094595A2 discloses the delivery of siRNA using a single lipid ligand (e.g., cholesterol or long-chain alkanes) at the chain end, WO2019217459A1 discloses the delivery of siRNA using a single lipid ligand inside the chain, and WO2021092371A2 discloses a series of novel lipid ligand structures.

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

[0006] In one aspect, the present invention provides an oligonucleotide comprising one or more compounds of formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0007]

[0008] The groups are defined below.

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

[0010]

[0011] The groups are defined below.

[0012] In another aspect, the present invention provides an oligonucleotide that includes one, two or more delivery vectors within the oligonucleotide, at its 5' end and / or 3' end, said delivery vector being a sugar modified with a hydrophobic group;

[0013] Preferably, the sugar modified with the hydrophobic group is selected from compounds of formula (X), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0014]

[0015] The groups are defined below.

[0016] In another aspect, the present invention provides a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand contains a sequence sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or antisense strand contains one or more compounds of formula (II) or formula (III) above, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0017] In another aspect, the present invention provides a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand contains a sequence fully complementary to the sense strand and the target mRNA, wherein the sense strand and / or antisense strand contains one, two or more delivery vectors internally, at the 5' end and / or the 3' end, the delivery vector being a sugar modified with a hydrophobic group;

[0018] Preferably, the sugar modified with the hydrophobic group is selected from compounds of formula (X), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0019]

[0020] The groups are defined below.

[0021] In another aspect, the present invention provides compounds of formula (II') or (III'), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0022]

[0023] The groups are defined below.

[0024] In another aspect, the hydrophobic group (P group in formula (I), (II) or (III)) provided by the present invention can be linked to an oligonucleotide or double-stranded RNA via a hydroxyl or acetyl group of the sugar moiety in the nucleotide.

[0025] In another aspect, the present invention provides a vector comprising a nucleotide sequence encoding the aforementioned double-stranded RNA.

[0026] In another aspect, the present invention provides a cell containing the aforementioned double-stranded RNA or the aforementioned vector.

[0027] In another aspect, the present invention provides pharmaceutical compositions comprising the aforementioned double-stranded RNA, the aforementioned vector, or the aforementioned cells, and optionally a pharmaceutically acceptable carrier or excipient.

[0028] In another aspect, the present invention provides a kit comprising the aforementioned double-stranded RNA, the aforementioned vector, or the aforementioned cells. Invention Details

[0030] definition

[0031] Chemical definition

[0032] The definitions of specific functional groups and chemical terms are described in more detail below.

[0033] When listing a range of values, it is assumed that each value and the subranges within that range are included. For example, "C 1-6 Alkyl groups include C1, C2, C3, C4, C5, C6, and C6. 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.

[0034] “C 1-6 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 6 carbon atoms. 8-30 "Alkyl" refers to a straight-chain 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 groups are preferred. C 1-6Examples of alkyl groups include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). The term "C" is used in conjunction with the preceding text. 1-6 "Alkyl" also includes heteroalkyl, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Common alkyl abbreviations include: Me(-CH3), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3) or i-Bu(-CH2CH(CH3)2).

[0035] “C 2-10 "Alkenyl" refers to a straight-chain or branched hydrocarbon group having 2 to 10 carbon atoms and at least one carbon-carbon double bond. 8-30 "Alkenyl" refers to a straight-chain 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 groups are preferred. C 2-6 Examples of alkenyl groups include: vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and so on. The term "C" is used in conjunction with these groups. 2-6 "Alkenyl" also includes heteroalkenyl groups, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkenyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0036] “C 2-10 "Alkyne" refers to a straight-chain or branched hydrocarbon group having 2 to 10 carbon atoms and at least one carbon-carbon triple bond. 8-30 "Alkyne" refers to a straight-chain 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 group, C 2-10 alkynyl group, C 2-6 alkynyl group and C 2-4The alkynyl group is preferred. C 2-6 Examples of alkynyl groups include, but are not limited to: ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentyynyl (C5), hexynyl (C6), etc. The term "C" is used in conjunction with other alkynyl groups. 2-6 "Alkyne" also includes heteroyne, wherein one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkynyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0037] “C 1-10 Alkylene, C 2-10 "Ideinyl" and "C" 2-10 "Iso-ynyl group" refers to the group with C removed. 1-10 Alkyl, C 2-10 alkenyl and C 2-10 The other hydrogen atom of the alkynyl group forms a divalent group, which can 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 Alkylenes are preferred. Unsubstituted alkylenes include, but are not limited to: methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and so on. Exemplary substituted alkylenes, for example, those substituted with one or more alkyl (methyl) groups, include, but are not limited to: substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3) 2- ), substituted propylidenes (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), etc.

[0038] "Halogen" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0039] Therefore, "C"1-6 "Halogenated alkyl" refers to the above "C 1-6 The alkyl group is replaced by one or more halogen groups. In some embodiments, C 1-4 Halogenated alkyl groups are particularly preferred, and C4 groups are more preferred. 1-2 Halogenated alkyl groups. Exemplary alkyl halogenated groups include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. The alkyl halogenated group can be substituted at any available connection point, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0040] “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 groups are particularly preferred, and C10 is more preferred. 5-6 Cycloalkyl groups. Cycloalkyl groups also include ring systems in which the aforementioned cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the linkage 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 (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cyclohepttrienyl (C7), etc. The cycloalkyl group may optionally be substituted with one or more substituents, for example, substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0041] "3-10 membered heterocyclic groups" refer to groups with a 3- to 10-membered non-aromatic ring system having a cyclic carbon atom and 1 to 5 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclic groups containing one or more nitrogen atoms, the linkage can be a carbon or nitrogen atom, provided that the valence allows. In some embodiments, a 4-10 membered heterocyclic group is preferred, which is a 4-10 membered non-aromatic ring system having a cyclic carbon atom and 1 to 5 cyclic heteroatoms; in some embodiments, a 3-8 membered heterocyclic group is preferred, which is a 3-8 membered non-aromatic ring system having a cyclic carbon atom and 1 to 4 cyclic heteroatoms; a 3-6 membered heterocyclic group is preferred, which is a 3-6 membered non-aromatic ring system having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; a 4-7 membered heterocyclic group is preferred, which is a 4-7 membered non-aromatic ring system having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; and a 5-6 membered heterocyclic group is preferred, which is a 5-6 membered non-aromatic ring system having a cyclic carbon atom and 1 to 3 cyclic heteroatoms. Heterocyclic groups also include ring systems in which the aforementioned heterocyclic ring is fused with one or more cycloalkyl groups, wherein the linking point is on the cycloalkyl ring, or ring systems in which the aforementioned heterocyclic ring is fused with one or more aryl or heteroaryl groups, wherein the linking point is on the heterocyclic ring; and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclic ring system. Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirropropyl, oxetane, and thiorenyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirrobutyl, oxetane, and thiorenyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to: tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolidinyl, and pyrrolidin-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to: dioxasulfuranyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to: piperazinyl, morpholinyl, disulfuranyl, and dioxalyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazinanyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirheptanyl, oxasulfuranyl, and thioheptanyl. Exemplary 5-membered heterocyclic groups fused with a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclic groups) include, but are not limited to: dihydroindolyl, isodihydroindolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinone, etc.Exemplary 6-membered heterocyclic groups fused with a C6 aryl ring (also referring to 6,6-bicyclic heterocyclic groups herein) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc. The heterocyclic group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0042] The term "hydrophobic group" broadly refers to any chemical group that has an affinity for lipids. One way to characterize the hydrophobicity of a hydrophobic group is through the octanol-water partition coefficient logK. ow K ow This represents the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the aqueous phase at equilibrium in a two-phase system. Typically, the logK of the hydrophobic portion... ow More than 1, more than 1.5, more than 2, more than 3, more than 4, more than 5, or more than 10. Specifically, in this invention, the hydrophobic part is the R group in the compound of formula I.

[0043] The alkyl, alkenyl, and ynyl groups defined in this article are optional substituted groups.

[0044] Exemplary substituents on carbon atoms include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa -ON(R) bb )2、-N(R bb )2、-N(R bb )3 + X - -N(OR) cc )R bb -SH, -SR aa -SSR cc -C(=O)R aa -CO2H, -CHO, -C(OR) cc )2、-CO2R aa -OC(=O)R aa -OCO2R aa -C(=O)N(R) bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa -NR bb CO2R aa -NR bb C(=O)N(R bb )2、-C(=NR bb )R aa -C(=NR) bb OR aa -OC(=NR)bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3、-OSi(R aa )3、-C(=S)N(R bb )2、-C(=O)SR aa 、-C(=S)SR aa 、-SC(=S)SR aa 、-SC(=O)SR aa 、-OC(=O)SR aa 、-SC(=O)OR aa 、-SC(=O)R aa 、-P(=O)2R aa 、-OP(=O)2R aa 、-P(=O)(R aa )2、-OP(=O)(R aa )2、-OP(=O)(OR cc )2、-P(=O)2N(R bb )2、-OP(=O)2N(R bb )2、-P(=O)(NR bb )2、-OP(=O)(NR bb )2、-NR bb P(=O)(OR cc )2、-NR bb P(=O)(NR bb )2、-P(R cc )2、-P(R cc )3、-OP(R cc )2、-OP(Rcc )3、-B(R aa 2. -B(OR) cc )2、-BR aa (OR cc ), alkyl, haloalkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, wherein each alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0045] Or the two hydrogen-bearing groups on the carbon atom: =O, =S, =NN(R) bb )2、=NNR bb C(=O)R aa =NNR bb C(=O)OR aa =NNR bb S(=O)2R aa =NR bb or = NOR cc replace;

[0046] R aa Each of them is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R aa Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0047] R bb Each 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, heterocyclic, aryl and heteroaryl, or two R bb Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0048] R cc Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R cc Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0049] R dd Each is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee -ON(R) ff )2、-N(R ff )2,、-N(R ff )3 + X - -N(OR) ee )R ff -SH, -SR ee -SSR ee -C(=O)R ee -CO2H, -CO2R ee -OC(=O)R ee -OCO2R ee -C(=O)N(R) ff )2、-OC(=O)N(R ff )2、-NR ff C(=O)R ee -NR ff CO2R ee -NR ff C(=O)N(R ff )2、-C(=NR ff OR ee -OC(=NR) ff )R ee -OC(=NR) ff OR ee-C(=NR) ff )N(R ff )2、-OC(=NR ff )N(R ff )2、-NR ff C(=NR ff )N(R ff )2、-NR ff SO2R ee -SO2N(R) ff )2、-SO2R ee -SO2OR ee -OSO2R ee -S(=O)R ee 、-Si(R ee 3. -OSi(R) ee 3. -C(=S)N(R) ff )2、-C(=O)SR ee -C(=S)SR ee -SC(=S)SR ee -P(=O)2R ee -P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR ee 2. Alkyl, haloalkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, wherein each alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently marked by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution, or two geminal radicals dd Substituents can combine to form =O or =S;

[0050] R ee Each is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl is independently surrounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution;

[0051] R ff Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R ff The groups combine to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution;

[0052] R ggEach of these is independently: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 Alkyl, -ON(C) 1-6 Alkyl)2, -N(C 1-6 Alkyl)2, -N(C 1-6 Alkyl)3 + X - -NH(C 1-6 Alkyl)2 + X - -NH2(C 1-6 alkyl) + X - -NH3 + X - -N(OC) 1-6 Alkyl)(C 1-6 Alkyl), -N(OH)(C 1-6 Alkyl groups, -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 Alkyl), -C(=O)(C 1-6 Alkyl group, -CO2H, -CO2(C 1-6 Alkyl), -OC (=O)(C 1-6 Alkyl), -OCO2(C 1-6 Alkyl groups, -C(=O)NH2, -C(=O)N(C 1-6 Alkyl)2、-OC(=O)NH(C 1-6 Alkyl), -NHC(=O)(C 1-6 alkyl), -N(C) 1-6 Alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 Alkyl), -NHC(=O)N(C 1-6 alkyl)2、-NHC(=O)NH(C 1-6 Alkyl groups, -NHC(=O)NH2, -C(=NH)O(C 1-6 Alkyl), -OC (=NH)(C 1-6 Alkyl group), -OC (=NH)OC 1-6 Alkyl group, -C(=NH)N(C 1-6 Alkyl)2、-C(=NH)NH(C 1-6 Alkyl groups, -C(=NH)NH2, -OC(=NH)N(C 1-6 Alkyl)2、-OC(NH)NH(C 1-6 Alkyl groups, -OC(NH)NH2, -NHC(NH)N(C 1-6Alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 alkyl), -SO2N(C 1-6 alkyl)2、-SO2NH(C 1-6 Alkyl groups, -SO2NH2, -SO2C 1-6 Alkyl, -SO2OC 1-6 Alkyl, -OSO2C 1-6 Alkyl, -SOC 1-6 Alkyl, -Si(C) 1-6 Alkyl)3、-OSi(C 1-6 Alkyl)3, -C(=S)N(C 1-6 Alkyl)2、C(=S)NH(C 1-6 Alkyl), C(=S)NH2, -C(=O)S(C 1-6 Alkyl), -C(=S)SC 1-6 Alkyl, -SC (=S)SC 1-6 Alkyl group, -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, C2-C6 alkenyl, C2-C6 ynyl, C3-C7 cycloalkyl, C6-C 10 Aryl, C3-C7 heterocyclic, C5-C 10 heteroaryl; or two ethryl groups gg Substituents can combine to form =O or =S; where X - It is a counterion.

[0053] 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 -SORaa -C(=S)N(R) cc )2、-C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc 2. Alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R atoms attached to a nitrogen atom. cc The groups combine to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution, wherein R aa R bb R cc and R dd As stated above.

[0054] Other definitions

[0055] The term "siRNA" in this article refers to a class of double-stranded RNA molecules that can mediate the silencing of a complementary target RNA (e.g., mRNA, such as transcripts of genes encoding proteins). siRNA is typically double-stranded, consisting of an antisense strand complementary to the target RNA and a sense strand complementary to that antisense strand. For convenience, such mRNA is also referred to herein as the 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. Additionally, RNAs other than mRNA (e.g., tRNA) and viral RNA can also be targeted.

[0056] The term "antisense oligonucleotide" (ASO) refers to a single-stranded DNA or RNA sequence consisting of 15-25 nucleotides that are paired with a target gene. It achieves gene regulation by specifically blocking the transcription or translation of the target gene.

[0057] The term "antisense strand" refers to a strand of siRNA that contains regions that are completely, sufficiently, or substantially complementary to the target sequence. The term "sense strand" refers to a strand of siRNA that includes regions that are completely, sufficiently, or substantially complementary to the regions defined herein as antisense strands.

[0058] The term "complementary region" refers to a region on the antisense strand that is completely, fully, or substantially complementary to the target mRNA sequence. In cases where the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the molecule or at the ends. Typically, the most tolerant mismatches are located in the end regions, for example, within 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends. The portion of the antisense strand most sensitive to mismatches is called the "seed region." For example, in a siRNA containing a 19-nt strand, the 19th position (from 5' to 3') can tolerate some mismatches.

[0059] The term "complementary" refers to the ability of a first polynucleotide to hybridize with a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400 mM NaCl, 40 mM PIPES at pH 6.4, and 1 mM EDTA at 50 or 70°C for 12–16 hours. In terms of meeting the above requirements regarding their hybridization ability, a "complementary" sequence may also include base pairs formed entirely from non-Watson-Crick base pairs and / or from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U swing base pairings or Hoogstein base pairs.

[0060] A polynucleotide that is “at least partially complementary,” “fully complementary,” or “substantially complementary” to messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest. For example, a polynucleotide is at least partially complementary to PCSK9 mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding PCSK9. The terms “complementary,” “fully complementary,” “fully complementary,” and “substantially complementary” can be used relative to base pairing between the sense and antisense strands of the siRNA, or between the antisense strand of the siRNA reagent and the target sequence.

[0061] "Perfect complementarity" means that in order to maintain the overall double-stranded character of the molecule, the sense strand only needs to be complementary to the antisense strand to a certain extent. In other words, although perfect complementarity is usually required, in some cases, especially in the antisense strand, there may be one or more mismatches (relative to the target mRNA), such as 6, 5, 4, 3, 2 or 1, but the sense and antisense strands can still maintain the overall double-stranded character of the molecule.

[0062] "Nucleoside" is a compound composed of two substances: a purine or pyrimidine base and ribose or deoxyribose. "Nucleotide" is a compound composed of three substances: a purine or pyrimidine base, ribose or deoxyribose, and phosphate. "Oligonucleotide" refers to nucleic acid molecules (RNA or DNA) with a length of less than 100, 200, 300, or 400 nucleotides.

[0063] A "base" is the basic building block for the synthesis of nucleosides, nucleotides, and nucleic acids. Its constituent elements include nitrogen, hence it is also called a "nitrogenous base." In this article, unless otherwise specified, the capital letters A, U, T, G, and C represent the base composition of nucleotides, namely adenine, uracil, thymine, guanine, and cytosine, respectively.

[0064] The term "modification" of nucleotides as used herein includes, but is not limited to, methoxy modification, fluorination modification, thiophosphate linkage, or conventional protecting group protection. For example, a fluorinated nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosome is replaced by fluorine, and a methoxy modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribosome is replaced by a methoxy group.

[0065] In this document, "modified nucleotides" include, but are not limited to, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-fluorine, nucleotides modified with 2'-deoxy-, inosine ribonucleotides, debased nucleotides, reverse abased deoxyribonucleotides, nucleotides containing a thiophosphate group, nucleotides modified with vinyl phosphate, locked nucleotides, nucleotides modified with 2'-amino, nucleotides modified with 2'-alkyl, morpholinonucleotides, aminophosphates, non-natural bases containing nucleotides, and terminal nucleotides linked to cholesterol derivatives or dodecanoic acid diecamide groups, deoxyribonucleotides, or those protected by conventional protecting groups. For example, a nucleotide modified with 2'-fluorine refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group is replaced by fluorine. A nucleotide modified with 2'-deoxy- refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group is replaced by a methoxy group.

[0066] "Reactive phosphorus group" refers to a phosphorus-containing group contained in a nucleotide unit or nucleotide analog unit that can react with a hydroxyl or amino group contained in another molecule, particularly in another nucleotide unit or another nucleotide analog, via a nucleophilic attack reaction. Typically, such a reaction produces an ester-type nucleoside bond linking the first nucleotide unit or the first nucleotide analog unit to the second nucleotide unit or the second nucleotide analog unit. The reactive phosphorus group can be selected from phosphorous amides, H-phosphonates, alkyl-phosphonates, phosphate esters, or phosphate ester analogs, including but not limited to: native phosphate esters, thiophosphate esters, dithiophosphate esters, boron phosphate esters, boron thiophosphate esters, phosphonates, halogen-substituted phosphonates and phosphate esters, aminophosphate esters, phosphate diesters, phosphate triesters, thiophosphate diesters, thiophosphate triesters, diphosphate esters, and triphosphate esters, preferably -P(OCH2CH2CN)(N(iPr)2).

[0067] A "protecting group" is any atom or group of atoms added to a molecule to prevent existing groups in the molecule from undergoing undesirable chemical reactions. A protecting group can be an unstable chemical motif known in the art, used to protect reactive groups, such as hydroxyl, amino, and thiol groups, from unwanted or untimely reactions during chemical synthesis. Protecting groups are typically used selectively and / or orthogonally to protect sites during reactions at other reactive sites, and can then be removed to leave unprotected groups intact or usable for further reactions.

[0068] A non-limiting list of protecting groups includes benzyl; substituted benzyl; alkyl carbonyl and alkoxy carbonyl (e.g., tert-butoxy carbonyl (BOC), acetyl, or isobutyryl); arylalkyl carbonyl and arylalkoxy carbonyl (e.g., benzyloxy carbonyl); substituted methyl ether (e.g., methoxymethyl ether); substituted diethyl ether; substituted benzyl ether; tetrahydropyranyl ether; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy)). Methyl or tert-butyldiphenylsilyl); esters (e.g., benzoates); carbonates (e.g., methoxymethyl carbonate); sulfonates (e.g., toluenesulfonates or methanesulfonates); noncyclic ketals (e.g., dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); noncyclic acetals; cycloacetals (e.g., those described herein); noncyclic hemiacetals; cyclic hemiacetals; cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiopentane); orthoesters (e.g., those described herein); and triarylmethyl groups. (For example, triphenylmethyl; monomethoxytriphenylmethyl (MMTr); 4,4′-dimethoxytriphenylmethyl (DMTr); 4,4′,4″-trimethoxytriphenylmethyl (TMTr); and those described herein). Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethylacetal (BOM), β-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methyl thiomethyl ether, new Vanoyl (Piv), Tetrahydropyranyl (THP), Triphenylmethyl (Trt), Methoxytriphenylmethyl[(4-methoxyphenyl)diphenylmethyl] (MMT), Dimethoxytriphenylmethyl, [bis-(4-methoxyphenyl)phenylmethyl (DMT), Trimethylsilyl ether (TMS), Tert-butyldimethylsilyl ether (TBDMS), Tri-isopropylsilyloxymethyl ether (TOM), Tri-isopropylsilyl ether (TIPS), Methyl ether, Ethoxyethyl ether (EE), N,N-dimethylformamidinium and 2-cyanoethyl (CE).

[0069] A "hydroxyl protecting group" is a group that prevents the hydroxyl group from undergoing chemical reactions and can be removed under specific conditions to restore the hydroxyl group. These mainly include silane-type protecting groups, acyl-type protecting groups, or ether-type protecting groups, with the following being preferred:

[0070] Trimethylsilyl (TMS), Triethylsilyl (TES), Dimethylisopropylsilyl (DMIPS), Diethylisopropylsilyl (DEIPS), Tert-butyldimethylsilyl (TBDMS), Tert-butyldiphenylsilyl (TBDPS), Triisopropylsilyl (TIPS), Acetyl (Ac), Chloroacetyl, Dichloroacetyl, Trichloroacetyl, Trifluoroacetyl (TFA), Benzoyl, p-Methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), Allyloxycarbonyl (Alloc), 2,2,2-Trichloroethoxycarbonyl (Troc), Benzyloxycarbonyl (Cbz), Tert-Butyloxycarbonyl (Boc) Benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzylmethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl, preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl, more preferably -C(O)CH2CH2C(O)OH.

[0071] As used herein, the term "pharmaceutically acceptable salt" refers to carboxylates and amino acid addition salts of the compounds of the present invention that are suitable for contact with patient tissues within the limits of reliable medical judgment, without producing undue toxicity, irritation, allergic reactions, etc., and are effective for their intended use in proportion to a reasonable benefit / risk ratio, including (where possible) zwitterionic forms of the compounds of the present invention.

[0072] This invention includes tautomers, which are functional group isomers resulting from the rapid movement of an atom in a molecule to two positions. A compound exists in different tautomer forms, and a compound is not limited to any particular tautomer, but is intended to encompass all tautomer forms.

[0073] The compounds of this invention may include one or more asymmetric centers and therefore may exist in a variety of stereoisomeric forms, such as enantiomers and / or diastereomers. For example, the compounds of this 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 rich in one or more stereoisomers. The isomers can be separated from the mixture by methods known to those skilled in the art, including chiral high-performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

[0074] The present invention also includes isotopically labeled compounds (isotopic variants) that are equivalent to those described in formula (I), but in which one or more atoms are replaced by atoms with atomic masses or mass numbers different from those commonly found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, respectively, for example... 2 H, 3 H, 13 C 11 C 14 C 15 N、 18 O、 17 O、 31 P, 32 P, 35 S, 18 F and 36 Cl. Other isotopes of the present invention containing the aforementioned isotopes and / or other atoms, their prodrugs, and pharmaceutically acceptable salts of said compounds or said prodrugs are all within the scope of this invention. Certain isotope-labeled compounds of the present invention, for example, those incorporating radioactive isotopes (e.g.,...) 3 H and 14 Those in category C) can be used for drug and / or substrate tissue distribution determination. Tritium, i.e. 3 H and carbon-14, i.e. 14 Carbon isotopes are particularly preferred because they are easy to prepare and detect. Subsequently, they are replaced by heavier isotopes, such as deuterium, i.e., 2 H, because higher metabolic stability can provide therapeutic benefits, such as prolonged in vivo half-life or reduced dosage requirements, may be preferred in some cases. Isotope-labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by using readily available isotope-labeled reagents instead of non-isotope-labeled reagents when performing the processes described below and / or the techniques disclosed in the examples and preparation examples.

[0075] Compounds of the present invention

[0076] This invention specifically relates to an oligonucleotide comprising one or more compounds of formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0077]

[0078] in,

[0079] In formula (I) It indicates H, or the position where it is linked to the phosphate or thiophosphate of an adjacent nucleotide;

[0080] X1 is selected from H, Rs, or

[0081] X2 is selected from OR1 or

[0082] One of X1 and X2 is

[0083] R1 represents H, or the position where it is attached to a phosphate ester or thiophosphate ester of another adjacent nucleotide;

[0084] In formula (I) Unlike R1, which is H at the same time;

[0085] Each L1 and L2 is independently selected from chemical bonds, C 1-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, OC 1-10 Alkylene, OC 1-10 Sub-alkenyl, OC 1-10 Ethyne group, C(O)C 1-10 Alkylene; wherein L1 and L2 are optionally substituted with 1, 2, 3, 4, 5, 6, 7 or 8 R;

[0086] T is selected from chemical bonds, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M-, or -OC(O)-M-;

[0087] Where M is

[0088] A is a sugar, which is preferably a sugar that can be cleaved in an integument or lysosome, preferably a pentagonal or hexagonal sugar, more preferably a hexagonal 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;

[0089] P is a hydrophobic group, preferably C. 8-30Alkyl, C 8-30 alkenyl or C 8-30 The alkynyl group, wherein the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, and 10th carbon atoms of the group that are not adjacent can be replaced by heteroatoms selected from O, S, and N, or the -CH2CH2- group can be replaced by -OC(O)-, -C(O)O-, -NHC(O)-, or -C(O)NH-, or the substituents on one or more carbon atoms can be linked to form a saturated or unsaturated ring; the C 8-30 Alkyl, C 8-30 alkenyl or C 8-30 The alkynyl group may optionally be substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R groups;

[0090] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0091] R s Selected from H, D, halogens, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, 3-10 membered heterocyclic groups or C 3-10 Cycloalkyl groups, optionally deuterated, up to and including complete deuteration;

[0092] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0093] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0094] k = 0, 1, 2, 3, 4, 5 or 6.

[0095] This invention specifically relates to an oligonucleotide, wherein the compound of formula (I) is selected from compounds of formula (II) or (III), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0096]

[0097] in,

[0098] R s Selected from H, D, halogens, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, 3-10 membered heterocyclic groups or C 3-10 Cycloalkyl groups, optionally deuterated, up to and including complete deuteration;

[0099] Other groups are as defined above.

[0100] This invention specifically relates to compounds of formula (II') or (III'), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0101]

[0102] R1 and R2 are independently selected from H, reactive phosphorus groups, hydroxyl protecting groups, or solid supports;

[0103] A is an acetylated sugar, which is preferably a sugar that can be cleaved in an integument or lysosome, and is preferably an acetylated pentaneous or hexaneous 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.

[0104] Preferably, A is selected from

[0105] A is connected to L1 through its a-terminus and to the P group through its b-terminus;

[0106] P, L1, L2, T, Rs, Rs', m, n, k are as defined above.

[0107] The present invention specifically relates to a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand contains a sequence that is fully complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand contains one or more compounds of formula (II) or formula (III) as described above, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0108]

[0109] In one implementation scheme H is represented; in another implementation, This indicates the position where the phosphate or thiophosphate of the adjacent nucleotide is attached.

[0110] R1 and R2

[0111] In one embodiment, R1 represents H; in another embodiment, R1 represents the position where it is linked to a phosphate ester or thiophosphate ester of another adjacent nucleotide.

[0112] In one embodiment, R1 is H; in another embodiment, R1 is selected from reactive phosphorus groups, preferably phosphoramide, H-phosphonate, alkyl-phosphonate, phosphate ester or phosphate ester analog, such as natural phosphate ester, thiophosphate ester, dithiophosphate ester, boron phosphate ester, boron thiophosphate ester, phosphonate ester, halogen-substituted phosphonate ester and phosphate ester, aminophosphate ester, phosphate diester, phosphate triester, thiophosphate diester, thiophosphate triester, diphosphate ester or triphosphate ester, preferably -P(OCH2CH2CN)(N(iPr)2); in another embodiment, R1 is selected from hydroxyl protecting groups, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS). Acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis(p-methoxytriphenylmethyl) (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzylmethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl, preferably DMTr; in another embodiment, R1 is a solid support.

[0113] In one embodiment, R2 is H; in another embodiment, R2 is selected from reactive phosphorus groups, preferably phosphoramide, H-phosphonate, alkyl-phosphonate, phosphate ester or phosphate ester analog, such as natural phosphate ester, thiophosphate ester, dithiophosphate ester, boron phosphate ester, boron thiophosphate ester, phosphonate ester, halogen-substituted phosphonate ester and phosphate ester, aminophosphate ester, phosphate diester, phosphate triester, thiophosphate diester, thiophosphate triester, diphosphate ester or triphosphate ester, preferably -P(OCH2CH2CN)(N(iPr)2); in another embodiment, R2 is selected from hydroxyl protecting groups, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS). Acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis(p-methoxytriphenylmethyl) (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzylmethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl, preferably DMTr; in another embodiment, R1 is a solid support.

[0114] X1 and X2

[0115] In one embodiment, X1 is H; in another embodiment, X1 is Rs; in yet another embodiment, X1 is

[0116] In one implementation, X2 is OR1; in another implementation, X2 is

[0117] L1 and L2

[0118] In one embodiment, L1 is a chemical bond; in another embodiment, L1 is a C 1-10 Alkylene; in another embodiment, L1 is C 1-6 Alkylene; in another embodiment, L1 is C 2-10 Ideonyl; in another embodiment, L1 is C 2-10Idemynyl; in another embodiment, L1 is OC 1-10 Alkylene; in another embodiment, L1 is OC 1-6 Alkylene; in another embodiment, L1 is OC 1-10 Ideonyl; in another embodiment, L1 is OC 1-10 Idemynyl; in another embodiment, L1 is C(O)C 1-10 Alkylene; in another embodiment, L1 is C(O)C 1-6 Alkylene.

[0119] In one specific implementation, L1 is OCH2CH2; in another specific implementation, L1 is O(CH2)5.

[0120] In one embodiment, L1 is not replaced; in another embodiment, L1 is replaced by 1 R; in another embodiment, L1 is replaced by 2 Rs; in another embodiment, L1 is replaced by 3 Rs; in another embodiment, L1 is replaced by 4 Rs; in another embodiment, L1 is replaced by 5 Rs; in another embodiment, L1 is replaced by 6 Rs; in another embodiment, L1 is replaced by 7 Rs; in another embodiment, L1 is replaced by 8 Rs.

[0121] In one embodiment, L2 is a chemical bond; in another embodiment, L2 is a C 1-10 Alkylene; in another embodiment, L2 is C 1-6 Alkylene; in another embodiment, L2 is C 2-10 Ideonyl; in another embodiment, L2 is C 2-10 Alkyne group; in another embodiment, L2 is OC 1-10 Alkylene; in another embodiment, L2 is OC 1-6 Alkylene; in another embodiment, L2 is OC 1-10 Ideonyl; in another embodiment, L2 is OC 1-10 Ethyne group; in another embodiment, L2 is C(O)C 1-10 Alkylene; in another embodiment, L2 is C(O)C 1-6 Alkylene.

[0122] In one specific implementation, L2 is OCH2; in another specific implementation, L2 is a chemical bond.

[0123] In one embodiment, L2 is not replaced; in another embodiment, L2 is replaced by 1 R; in another embodiment, L2 is replaced by 2 Rs; in another embodiment, L2 is replaced by 3 Rs; in another embodiment, L2 is replaced by 4 Rs; in another embodiment, L2 is replaced by 5 Rs; in another embodiment, L2 is replaced by 6 Rs; in another embodiment, L2 is replaced by 7 Rs; in another embodiment, L2 is replaced by 8 Rs.

[0124] T

[0125] In one embodiment, T is a chemical bond; in another embodiment, T is -O-; in another embodiment, T is -CH2-; in another embodiment, T is -C(O)-; in another embodiment, T is -OC(O)-; in another embodiment, T is -M-; in another embodiment, T is -OM-; in another embodiment, T is -CH2-M-; in another embodiment, T is -C(O)-M-; in another embodiment, T is -OC(O)-M-.

[0126] In one implementation, M is... In another implementation, M is In another implementation, M is In another implementation, M is

[0127] A

[0128] In one embodiment, A is a sugar; in another embodiment, A is a sugar that can be cleaved in integrons or lysosomes; in another embodiment, A is a pentaneous or hexaneous sugar, preferably a hexaneous 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.

[0129] In a specific implementation plan, A is... A is connected to L1 via its a-terminus and to the P group via its b-terminus; in another specific embodiment, A is... A is connected to L1 via its a-terminus and to the P group via its b-terminus; in another specific embodiment, A is... A is connected to L1 via its a-terminus and to the P group via its b-terminus; in another specific embodiment, A is... A is connected to L1 through its a-terminus and to the P group through its b-terminus.

[0130] In one embodiment, A is an acetylated sugar; in another embodiment, A is an acetylated sugar that can be cleaved in integrons or lysosomes; in yet another embodiment, A is an acetylated pentaneous or hexacanthose, 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.

[0131] In a specific implementation plan, A is... A is connected to L1 via its a-terminus and to the P group via its b-terminus; in another specific embodiment, A is... A is connected to L1 via its a-terminus and to the P group via its b-terminus; in another specific embodiment, A is... A is connected to L1 via its a-terminus and to the P group via its b-terminus; in another specific embodiment, A is...

[0132] A is connected to L1 through its a-terminus and to the P group through its b-terminus.

[0133] P

[0134] In one embodiment, P is a hydrophobic group; in another embodiment, P is C. 8-30 Hydrocarbon groups, such as C 8-30 Alkyl, C 8-30 alkenyl or C 8-30 Alkyne group; in another embodiment, P is C 10-22 Alkyl, such as C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 18 Alkyl, C 19 Alkyl, C 20 Alkyl, C 21 Alkyl, C 22 Alkyl; in another embodiment, P is C 12-18 Alkyl, such as C 14-16 Alkyl; in another embodiment, P is C 10-22 Alkenyl group.

[0135] In one embodiment, the 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 non-adjacent carbon atoms in P can be replaced by heteroatoms selected from O, S, and N, or the -CH2CH2- group can be replaced by -OC(O)-, -C(O)O-, -NHC(O)-, or -C(O)NH-, or the substituents on one or more carbon atoms can be linked to form a saturated or unsaturated ring.

[0136] In one embodiment, P is not replaced; in another embodiment, P is replaced by 1 R; in another embodiment, P is replaced by 2 Rs; in another embodiment, P is replaced by 3 Rs; in another embodiment, P is replaced by 4 Rs; in another embodiment, P is replaced by 5 Rs; in another embodiment, P is replaced by 6 Rs; in another embodiment, P is replaced by 7 Rs; in another embodiment, P is replaced by 8 Rs; in another embodiment, P is replaced by more than one R.

[0137] In one specific implementation, P is -(CH2). 14-16 CH3.

[0138] P1, P2, P3 and P4

[0139] In one embodiment, P1 is H; in another embodiment, P1 is a P group as defined above.

[0140] In one embodiment, P2 is H; in another embodiment, P2 is a P group as defined above.

[0141] In one embodiment, P3 is H; in another embodiment, P3 is a P group as defined above.

[0142] In one embodiment, P4 is CH3; in another embodiment, P4 is a P group as defined above.

[0143] In one embodiment, one of P1, P2, P3, and P4 is a P group.

[0144] R

[0145] In one embodiment, R is H; in another embodiment, R is D; in yet another embodiment, R is a halogen; in yet 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 Halogenated alkyl; in another embodiment, R is optionally deuterated, up to and including complete deuteration.

[0146] R s and Rs '

[0147] In one implementation, R s For H; in another implementation, R s For D; in another implementation, R s For halogen; in another embodiment, R s C 1-6 Alkyl; in another embodiment, R s C 1-6 Halogenated alkyl; in another embodiment, R s It is a 3-10 membered heterocyclic group; in another embodiment, R s C 3-10 cycloalkyl; in another embodiment, R s It can be selectively deuterated, until it is completely deuterated.

[0148] In one specific implementation plan, R s For H.

[0149] In one implementation, R s 'For H; in another implementation, R s 'For D; in another implementation, R s 'is halogen; in another embodiment, R s 'For C 1-6 Alkyl; in another embodiment, R s 'For C 1-6 Halogenated alkyl; in another embodiment, R s 'It is a 3-10 membered heterocyclic group, such as a 5-10 membered heterocyclic group; in another embodiment, R s 'For C 3-10 cycloalkyl; in another embodiment, R s 'Optionally replaced by deuterium, until completely deuterated.'

[0150] In one specific implementation plan, R s 'It is cyclohexyl.'

[0151] m

[0152] m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0153] n

[0154] n can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0155] k

[0156] k = 0, 1, 2, 3, 4, 5 or 6.

[0157] Any technical solution or any combination thereof in any of the above specific embodiments can be combined with any technical solution or any combination thereof in other specific embodiments. For example, any technical solution or any combination thereof of A can be combined with... P, P1-P4, R1, R2, X1, X2, L1, L2, T, R, R s R s This invention involves combinations of any technical solutions such as ', m, n, and k, or any combination thereof. The invention aims to include combinations of all these technical solutions; however, due to space limitations, they are not listed individually.

[0158] The present invention also provides a vector comprising a nucleotide sequence encoding the siRNA described herein. The vector of the present invention is capable of amplifying or expressing the nucleotides encoding the siRNA linked thereto.

[0159] For example, siRNA targeting the PCSK9 gene can be expressed from transcriptional units inserted into DNA or RNA vectors. Expression can be transient (lasting from hours to weeks) or persistent (lasting from weeks to months or longer), depending on the specific construct used and the target tissue or cell type. The coding nucleotides of the siRNA can be introduced into linear constructs, circular plasmids, or viral vectors. The siRNA nucleotides can be stably expressed by integration into the cellular genome or by stable extrachromosomal inheritance. Generally, siRNA expression vectors are typically DNA plasmids or viral vectors.

[0160] Viral vector systems containing coding sequences of 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) polyomavirus vectors; (g) papillomavirus vectors; (h) microRNA virus vectors; (i) poxvirus vectors; and (j) helper virus-dependent adenoviruses or enteroviruses.

[0161] The present invention also provides a cell containing the siRNA or vector described herein, wherein the siRNA or vector described herein is capable of transcription in the cell.

[0162] This invention specifically relates to the following technical solutions:

[0163] A1. An oligonucleotide comprising one or more compounds of formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0164]

[0165] in,

[0166] In formula (I) It indicates H, or the position where it is linked to the phosphate or thiophosphate of an adjacent nucleotide;

[0167] X1 is selected from H, Rs, or

[0168] X2 is selected from OR1 or

[0169] One of X1 and X2 is

[0170] R1 represents H, or the position where it is attached to a phosphate ester or thiophosphate ester of another adjacent nucleotide;

[0171] In formula (I) Unlike R1, which is H at the same time;

[0172] Each L1 and L2 is independently selected from chemical bonds, C 1-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, OC 1-10 Alkylene, OC 1-10 Sub-alkenyl, OC 1-10 Ethyne group, C(O)C 1-10 Alkylene; wherein L1 and L2 are optionally substituted with 1, 2, 3, 4, 5, 6, 7 or 8 R;

[0173] T is selected from chemical bonds, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M-, or -OC(O)-M-;

[0174] Where M is

[0175] A is a sugar, which is preferably a sugar that can be cleaved in an integument or lysosome, preferably a pentagonal or hexagonal sugar, more preferably a hexagonal 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;

[0176] P is a hydrophobic group, preferably C. 8-30 Alkyl, C 8-30 alkenyl or C 8-30The alkynyl group, wherein the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, and 10th carbon atoms of the group that are not adjacent can be replaced by heteroatoms selected from O, S, and N, or the -CH2CH2- group can be replaced by -OC(O)-, -C(O)O-, -NHC(O)-, or -C(O)NH-, or the substituents on one or more carbon atoms can be linked to form a saturated or unsaturated ring; the C 8-30 Alkyl, C 8-30 alkenyl or C 8-30 The alkynyl group may optionally be substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R groups;

[0177] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0178] R s Selected from H, D, halogens, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, 3-10 membered heterocyclic groups or C 3-10 Cycloalkyl groups, optionally deuterated, up to and including complete deuteration;

[0179] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0180] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0181] k = 0, 1, 2, 3, 4, 5 or 6.

[0182] A2. The oligonucleotide of technical solution A1, wherein the compound of formula (I) is selected from the compound of formula (II) or formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0183]

[0184] in,

[0185] It indicates H, or the position where it is linked to the phosphate or thiophosphate of an adjacent nucleotide;

[0186] R1 represents H, or the position where it is attached to a phosphate ester or thiophosphate ester of another adjacent nucleotide;

[0187] Unlike R1, which is H at the same time;

[0188] Each L1 and L2 is independently selected from chemical bonds, C 1-10 Alkylene, C 2-10 imidene group, C2-10 Ethyne group, OC 1-10 Alkylene, OC 1-10 Sub-alkenyl, OC 1-10 Ethyne or C(O)C 1-10 Alkylene; wherein L1 and L2 are optionally substituted with 1, 2, 3, 4, 5, 6, 7 or 8 R;

[0189] T is selected from chemical bonds, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M-, or -OC(O)-M-;

[0190] Where M is

[0191] A is a sugar, which is preferably a sugar that can be cleaved in an integument or lysosome, preferably a pentagonal or hexagonal sugar, more preferably a hexagonal 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;

[0192] P is a hydrophobic group, preferably C. 8-30 Alkyl, C 8-30 alkenyl or C 8-30 The alkynyl group, wherein the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, and 10th carbon atoms of the group that are not adjacent can be replaced by heteroatoms selected from O, S, and N, or the -CH2CH2- group can be replaced by -OC(O)-, -C(O)O-, -NHC(O)-, or -C(O)NH-, or the substituents on one or more carbon atoms can be linked to form a saturated or unsaturated ring; the C 8-30 Alkyl, C 8-30 alkenyl or C 8-30 The alkynyl group may optionally be substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R groups;

[0193] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0194] R s Selected from H, D, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0195] R s Selected from H, D, halogens, C 1-6 Alkyl, C 1-6Halogenated alkyl groups, 3-10 membered heterocyclic groups or C 3-10 Cycloalkyl groups, optionally deuterated, up to and including complete deuteration;

[0196] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0197] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0198] k = 0, 1, 2, 3, 4, 5 or 6.

[0199] A3. Oligonucleotides of technical solution A1 or A2, wherein,

[0200] It indicates H, or the position where it is linked to the phosphate or thiophosphate of an adjacent nucleotide;

[0201] R1 represents H, or the position where it is attached to a phosphate ester or thiophosphate ester of another adjacent nucleotide;

[0202] Unlike R1, which is H at the same time;

[0203] Each L1 and L2 is independently selected from chemical bonds, C 1-6 Alkylene, OC 1-6 Alkylene or C(O)C 1-6 Alkylene; wherein L1 and L2 are optionally substituted with 1, 2, 3, 4 or 5 R;

[0204] T is selected from chemical bonds, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M-, or -OC(O)-M-;

[0205] Where M is

[0206] 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;

[0207] P is selected from C 10-22 Alkyl or C 10-22 alkenyl, the C 10-22 Alkyl or C 10-22 The alkenyl group may optionally be substituted by one, two, three, four, or five R groups;

[0208] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0209] R s Selected from H, D, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0210] R s Selected from H, D, halogens, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, 3-10 membered heterocyclic groups or C 3-10 cycloalkyl;

[0211] m can be 0, 1, 2, 3, 4, or 5;

[0212] n is 0, 1, 2, 3, 4 or 5;

[0213] k can be 0, 1, 2, 3, 4, or 5.

[0214] A4. An oligonucleotide from any one of technical solutions A1-A3, wherein,

[0215] It indicates H, or the position where it is linked to the phosphate or thiophosphate of an adjacent nucleotide;

[0216] R1 represents H, or the position where it is attached to a phosphate ester or thiophosphate ester of another adjacent nucleotide;

[0217] Unlike R1, which is H at the same time;

[0218] Each L1 and L2 is independently selected from chemical bonds or OC. 1-6 Alkylene; L1 and L2 are optionally substituted with one, two or three Rs;

[0219] T is selected from chemical bonds, -O-, -CH2-, -OC(O)-, or -C(O)-;

[0220] A is selected from

[0221] A is connected to L1 through its a-terminus and to the P group through its b-terminus;

[0222] P is selected from C 10-22 Alkyl or C 10-22 alkenyl, the C 10-22 Alkyl or C 10-22 The alkenyl group may optionally be substituted by one, two, or three R groups;

[0223] R is selected from H, D, halogen, C 1-4 alkyl;

[0224] R s Selected from H or D;

[0225] R sSelected from H, D or C 5-10 cycloalkyl;

[0226] m can be 0, 1, 2, or 3;

[0227] n is 0, 1, 2, or 3;

[0228] k can be 0, 1, 2 or 3.

[0229] A5. An oligonucleotide from any one of technical solutions A1-A4, wherein,

[0230] Indicates the position where it is attached to the phosphate ester or thiophosphate ester of the adjacent nucleotide;

[0231] R1 is H, or indicates the position where it is linked to the phosphate ester or thiophosphate ester of another adjacent nucleotide;

[0232] L1 is OCH2CH2 or O(CH2)5;

[0233] L2 is OCH2 or a chemical bond;

[0234] T is selected from chemical bonds, -O-, or -C(O)-;

[0235] A is selected from

[0236] A is connected to L1 through its a-terminus and to the P group through its b-terminus;

[0237] P is -(CH2) 14-16 CH3;

[0238] R s For H;

[0239] R s 'For cyclohexyl;

[0240] m is 0, 1, or 2;

[0241] n is 0 or 1;

[0242] k is 0 or 1.

[0243] A6. An oligonucleotide of any one of technical solutions A1-A5, wherein the compound of formula (II) or formula (III) is selected from the following general formula compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0244]

[0245] in,

[0246] P1, P2, and P3 are selected from H or P groups;

[0247] P4 is selected from CH3 or a P group;

[0248] One of P1, P2, P3, and P4 is a P group;

[0249] Each variable is defined as described in technical solutions A1-A5.

[0250] A7. An oligonucleotide of any one of technical solutions A1-A6, wherein the compound of formula (II) or formula (III) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0251]

[0252] in, This indicates the position where the phosphate or thiophosphate of the adjacent nucleotide is attached.

[0253] R1 is H, or indicates the position where it is linked to a phosphate ester or thiophosphate ester of another adjacent nucleotide.

[0254] A8. An oligonucleotide of any one of technical solutions A1-A7, having 14 to 30 nucleotides.

[0255] A9. An oligonucleotide of any one of technical solutions A1-A8, wherein at its 5' end it comprises a compound of formula (II) or formula (III) of any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0256] A10. An oligonucleotide of any one of technical solutions A1-A9, wherein at its 3' end it comprises a compound of formula (II) or formula (III) of any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0257] A11. An oligonucleotide of any one of technical solutions A1-A10, wherein at the 5' end and the 3' end respectively, a compound of formula (II) or formula (III) of any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0258] A12. An oligonucleotide of any one of technical solutions A1-A11, wherein the oligonucleotide contains one or more compounds of formula (II) of any one of technical solutions A1-A7, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0259] A13. An oligonucleotide that contains one, two or more delivery vectors within the oligonucleotide, at its 5' end and / or 3' end, said delivery vector being a sugar modified with a hydrophobic group;

[0260] Preferably, the sugar modified with the hydrophobic group is selected from compounds of formula (X), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0261]

[0262] in, Indicates the linking position with the oligonucleotide;

[0263] P is a hydrophobic group;

[0264] A represents the sugar portion;

[0265] P and A are as defined in any of the technical solutions A1-A5;

[0266] Preferably, the hydrophobic group is attached to the hydroxyl or acetyl group of the sugar moiety;

[0267] Preferably, the sugar modified with the hydrophobic group is selected from the following compounds, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0268]

[0269] in, This indicates the linking position with the oligonucleotide.

[0270] A14. An oligonucleotide of any one of technical solutions A1-A13, wherein the oligonucleotide is ASO or siRNA, preferably for inhibiting genes expressed outside the liver, more preferably for inhibiting genes expressed in the central nervous system (CNS) and / or the eye.

[0271] A15. A compound of formula (II') or (III'), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0272]

[0273] R1 and R2 are independently selected from H, reactive phosphorus groups, hydroxyl protecting groups, or solid supports;

[0274] A is an acetylated sugar, which is preferably a sugar that can be cleaved in integrons or lysosomes, and is preferably an acetylated pentaneous or hexaneous sugar, such as N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, and more preferably N-acetylgalactosamine or N-acetylglucosamine;

[0275] Preferably, A is selected from

[0276] A is connected to L1 through its a-terminus and to the P group through its b-terminus;

[0277] P, L1, L2, T, Rs, Rs', m, n, k are as defined in any of the technical solutions A1-A7.

[0278] A16. The compound of technical solution A15, wherein R1 and R2 are H.

[0279] A17. A compound of technical solution A15 or A16, wherein one of R1 and R2 is a reactive phosphorus group, preferably a phosphorus amide, H-phosphonate, alkyl-phosphonate, phosphate ester or phosphate ester analog, such as natural phosphate ester, thiophosphate ester, dithiophosphate ester, borane phosphate ester, borane thiophosphate ester, phosphonate ester, halogen-substituted phosphonate ester and phosphate ester, aminophosphate ester, phosphate diester, phosphate triester, thiophosphate diester, thiophosphate triester, diphosphate ester or triphosphate ester, preferably -P(OCH2CH2CN)(N(iPr)2).

[0280] A18. A compound according to any one of technical solutions A15-A17, wherein R1 and R2 are selected from protecting groups, preferably hydroxyl protecting groups, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl Alloc, 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzylmethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl, preferably DMTr.

[0281] A19. A compound of any one of technical solutions A15-A18, wherein the compound of formula (II') or (III') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0282]

[0283]

[0284] A20. A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand contains a sequence fully complementary to the sense strand and the target mRNA, wherein the sense strand and / or antisense strand contains one or more compounds of formula (II) or formula (III) as described in any one of technical solutions A1-A7, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0285] A21. The double-stranded RNA of technical solution A20, wherein the compound of formula (II) or formula (III) is selected from the following general formula compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0286]

[0287] The variables are defined as described in technical solutions A1-A7.

[0288] A22. A double-stranded RNA of technical solution A20 or A21, wherein the compound of formula (II) or formula (III) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0289]

[0290]

[0291] in, This indicates the position where the phosphate or thiophosphate of the adjacent nucleotide is attached.

[0292] R1 is H, or indicates the position where it is linked to a phosphate ester or thiophosphate ester of another adjacent nucleotide.

[0293] A23. A double-stranded RNA of any one of technical solutions A20-A22, wherein the sense strand comprises at its 5' end a compound of formula (II) or formula (III) of any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0294] A24. A double-stranded RNA of any one of technical solutions A20-A23, wherein the sense strand comprises at its 3' end a compound of formula (II) or formula (III) of any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0295] A25. A double-stranded RNA of any one of technical solutions A20-A24, wherein the sense strand comprises a compound of formula (II) or formula (III) of any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 5' end and 3' end, respectively.

[0296] A26. A double-stranded RNA of any one of technical solutions A20-A25, wherein the sense strand comprises within an oligonucleotide one or more compounds of formula (II) of any one of technical solutions A1-A7, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0297] A27. A double-stranded RNA of any one of technical solutions A20-A26, wherein the antisense strand comprises at its 5' end a compound of formula (II) or formula (III) of any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0298] A28. A double-stranded RNA of any one of technical solutions A20-A27, wherein the antisense strand comprises at its 3' end a compound of formula (II) or formula (III) of any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0299] A29. A double-stranded RNA of any one of technical solutions A20-A28, wherein the antisense strand comprises a compound of formula (II) or formula (III) of any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 5' end and 3' end, respectively.

[0300] A30. A double-stranded RNA of any one of technical solutions A20-A29, wherein the antisense strand comprises within the oligonucleotide one or more compounds of formula (II) of any one of technical solutions A1-A7, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0301] A31. A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand contains a sequence fully complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand contains one, two or more delivery vectors internally, at the 5' end and / or the 3' end, the delivery vector being a sugar modified with a hydrophobic group;

[0302] Preferably, the sugar modified with the hydrophobic group is selected from compounds of formula (X), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0303]

[0304] in, Indicates the linking position with the oligonucleotide;

[0305] P is a hydrophobic group;

[0306] A represents the sugar portion;

[0307] P and A are as defined in any of the technical solutions A1-A5;

[0308] Preferably, the hydrophobic group is attached to the hydroxyl or acetyl group of the sugar moiety;

[0309] Preferably, the sugar modified with the hydrophobic group is selected from the following compounds, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0310]

[0311] in, This indicates the linking position with the oligonucleotide.

[0312] A32. A vector comprising a nucleotide sequence encoding a double-stranded RNA as described in any one of the preceding technical solutions A20-A31.

[0313] A33. A cell containing double-stranded RNA as described in any one of technical solutions A20-A31 or a vector as described in technical solution A32.

[0314] A34. A pharmaceutical composition comprising double-stranded RNA as described in any one of technical solutions A20-A31, a vector as described in technical solution A32, or a cell as described in technical solution A33, and optionally a pharmaceutically acceptable carrier or excipient.

[0315] A35. A kit comprising double-stranded RNA as described in any one of technical solutions A20-A31, a vector as described in technical solution A32, or cells as described in technical solution A33.

[0316] This invention also specifically relates to the following technical solutions:

[0317] B1. Oligonucleotides comprising one or more compounds of formula (IV), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0318]

[0319] It indicates H, or the position where it is linked to the phosphate or thiophosphate of an adjacent nucleotide;

[0320] R1 represents H, or the position where it is attached to a phosphate ester or thiophosphate ester of another adjacent nucleotide;

[0321] Unlike R1, which is H at the same time;

[0322] Each L1 and L2 is independently selected from chemical bonds, C 1-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, OC 1-10 Alkylene, OC 1-10 Sub-alkenyl, OC 1-10 Ethyne or C(O)C 1-10 Alkylene; wherein L1 and L2 are optionally substituted with 1, 2, 3, 4, 5, 6, 7 or 8 R;

[0323] T is selected from chemical bonds, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M-, or -OC(O)-M-;

[0324] Where M is

[0325] A is N-acetylglucosamine;

[0326] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0327] R s Selected from H, D, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0328] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0329] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0330] k = 0, 1, 2, 3, 4, 5 or 6.

[0331] B2. The oligonucleotide in technical solution B1, wherein,

[0332] It indicates H, or the position where it is linked to the phosphate or thiophosphate of an adjacent nucleotide;

[0333] R1 represents H, or the position where it is attached to a phosphate ester or thiophosphate ester of another adjacent nucleotide;

[0334] Unlike R1, which is H at the same time;

[0335] Each L1 and L2 is independently selected from chemical bonds, C 1-6 Alkylene, OC 1-6 Alkylene or C(O)C 1-6 Alkylene; wherein L1 and L2 are optionally substituted with 1, 2, 3, 4 or 5 R;

[0336] T is selected from chemical bonds, -O-, -CH2-, -C(O)-, -OC(O)-, -M-, -OM-, -CH2-M-, -C(O)-M-, or -OC(O)-M-;

[0337] Where M is

[0338] A is N-acetylgalactosamine;

[0339] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0340] R s Selected from H, D, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0341] m can be 0, 1, 2, 3, 4, or 5;

[0342] n is 0, 1, 2, 3, 4 or 5;

[0343] k can be 0, 1, 2, 3, 4, or 5.

[0344] B3. Oligonucleotides of technical solution B1 or B2, wherein,

[0345] It indicates H, or the position where it is linked to the phosphate or thiophosphate of an adjacent nucleotide;

[0346] R1 represents H, or the position where it is attached to a phosphate ester or thiophosphate ester of another adjacent nucleotide;

[0347] Unlike R1, which is H at the same time;

[0348] Each L1 and L2 is independently selected from chemical bonds or OC. 1-6 Alkylene, for example, OCH2, OCH2CH2, (OCH2CH2)2 or (OCH2CH2)3; wherein L1 and L2 are optionally substituted with one, two or three Rs;

[0349] T is selected from chemical bonds, -O-, -CH2-, -OC(O)- or -C(O)-, preferably -C(O)-;

[0350] A is

[0351] R is selected from H, D, halogen, C 1-4 alkyl;

[0352] R s Selected from H or D;

[0353] m can be 0, 1, 2, or 3;

[0354] n is 0, 1, 2, or 3;

[0355] k can be 0, 1, 2 or 3.

[0356] B4. An oligonucleotide of any one of technical solutions B1-B3, wherein the compound is selected from:

[0357]

[0358] in,

[0359] This indicates that the nucleotide is linked to the previous nucleotide via a phosphate ester group or a thiophosphate ester group.

[0360] This indicates that the nucleotide is linked to the next nucleotide via a phosphate ester group or a thiophosphate ester group.

[0361] B5. Compounds of formula (IV'), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0362]

[0363] in,

[0364] R1 and R2 are independently selected from H, reactive phosphorus groups, hydroxyl protecting groups, or solid supports;

[0365] A is

[0366] L1, L2, T, Rs, m, n, and k are as defined above.

[0367] B6. The compound of technical solution B5, wherein R1 and R2 are H;

[0368] Preferably, one of R1 and R2 is a reactive phosphorus group, preferably phosphoramide, H-phosphonate, alkyl-phosphonate, phosphate ester or phosphate ester analog, such as natural phosphate ester, thiophosphate ester, dithiophosphate ester, borane phosphate ester, borane thiophosphate ester, phosphonate ester, halogen-substituted phosphonate ester and phosphate ester, aminophosphate ester, phosphate diester, phosphate triester, thiophosphate diester, thiophosphate triester, diphosphate ester or triphosphate ester, preferably -P(OCH2CH2CN)(N(iPr)2);

[0369] R1 and R2 are selected from protecting groups, preferably hydroxyl protecting groups, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2, 2-Trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzylmethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl, preferably DMTr.

[0370] B7. The compound of technical solution B5 or B6, wherein the compound of formula (II') or (III') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0371]

[0372] B8. An oligonucleotide of any one of technical solutions B1-B4, wherein the oligonucleotide is siRNA, used to suppress genes expressed in the liver.

[0373] Specific list of compounds

[0374] The compound numbers and structures in oligonucleotides of the present invention are as follows, wherein the compounds are numbered from 5' to 3' in the order of 5'–>3'. to connect.

[0375] Specifically, according to the order 5'–>3', if the corresponding structure is located in the middle of the nucleic acid chain, This indicates that it is linked to the 3' carbon or corresponding position of the previous nucleotide or nucleotide analog via a phosphate ester group, a thiophosphate ester group, or other linking group. This indicates that the linker is attached to the 5' carbon or corresponding position of the next nucleotide or nucleotide analog via a phosphate ester group, thiophosphate group, or other linking group; if the corresponding structure is located at the end of the nucleic acid chain. Correspondingly, this indicates that the nucleic acid is linked to the 3' or 5' end of the nucleic acid chain via a phosphate group, a thiophosphate group, or other linking group.

[0376]

[0377]

[0378]

[0379] Synthesis Examples The following examples are used to illustrate the invention and not to limit the scope of the invention.

[0380] abbreviation

[0381]

[0382] Example 1. Preparation of compound BE1

[0383]

[0384] 1. Preparation of Compound 3

[0385]

[0386] Compound 1 (1.00 g, 2.03 mmol) was dissolved in DCM (30.0 mL) at 25 °C. HATU (0.850 g, 2.23 mmol) and DIEA (1.34 mL, 8.11 mmol) were added, and the mixture was stirred for 0.5 h. Then, compound 2 (1.00 g, 2.229 mmol) was added, and the reaction mixture was stirred at 25 °C for 2.5 h. LCMS showed the mass value of the product. Thin-layer chromatography (DCM / MeOH = 10 / 1) showed the formation of new spots. The reaction mixture was evaporated to dryness to obtain the crude product. The crude product was purified by normal-phase silica gel column chromatography (DCM / MeOH = 1 / 0-10 / 1) to give compound 3 (1.70 g, yield 90.69%) as a white solid.

[0387] 1 H NMR (400MHz, CD3OD) δ7.37-7.47(m,2H),7.16-7.34(m,7H),6.71-6.92(m,4H),5.22-5.37(m,1H),5.05-5. 15(m,1H),4.69-4.80(m,1H),3.55-4.24(m,28H),3.19-3.30(m,2H),2.09-2.15(m,3H),1.87-2.00(m,9H)

[0388] 2. Preparation of compound BE1

[0389]

[0390] Compound 3 (1.70 g, 1.84 mmol) was dissolved in DCM (20.0 mL) at 25 °C. DCI (0.160 g, 1.38 mmol) and compound 4 (0.830 g, 2.76 mmol) were added sequentially. The reaction mixture was stirred at 25 °C under a nitrogen atmosphere for 2 hours. LC-MS showed complete consumption of compound 3 and the presence of a mass value for compound BE1. Thin-layer chromatography (DCM / MeOH = 10 / 1) showed the formation of new spots. The reaction mixture was evaporated to dryness to obtain the crude product. The crude product was purified by normal-phase silica gel column chromatography (dichloromethane / acetone = 1 / 0-5 / 1) to give a pale yellow oily compound BE1 (1.02 g, yield 49.32%).

[0391] 1 H 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).

[0392] Example 2. Preparation of compound BE2

[0393]

[0394] 1. Preparation of Compound 3

[0395]

[0396] Compound 1 (1.0 g, 2.23 mmol) was dissolved in DCM (20.0 mL) at 25 °C, and HATU (1.27 g, 3.34 mmol) and DIEA (1.10 mL, 6.68 mmol) were added. The mixture was stirred at 25 °C for 0.5 hours, and then compound 2 (1.20 g, 2.67 mmol) was added. The mixture was stirred for another 12 hours. TLC (T1: DCM / MEOH = 10 / 1) showed complete consumption of the starting material. 200 mL of dichloromethane was added. The mixture was washed three times successively with saturated sodium bicarbonate solution (30.0 mL x 3) and saturated brine (30.0 mL x 3). The organic phase was dried over anhydrous sodium sulfate and then evaporated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 1 / 0 to 20 / 1) to give a yellow oily compound 3 (1.43 g, 1.70 mmol).

[0397] 1 H 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)

[0398] 2. Preparation of compound BE2

[0399]

[0400] Compound 3 (1.42 g, 1.61 mmol) was dissolved in DCM (15.0 mL) at 25 °C, and a certain amount of molecular sieve was added. Then, DCI (140 mg, 1.21 mmol) was added, followed by compound 4 (730 mg, 2.42 mmol) under a nitrogen atmosphere. The mixture was stirred at 25 °C for 1.0 h. TLC (DCM / ACE = 10 / 1) showed the formation of new spots. 0.5 mL of triethylamine was added, followed by 20 mL of dichloromethane. The mixture was stirred with alkaline silica gel, and the mobile phase consisted of dichloromethane mixed with 4 / 1000 triethylamine solution. The solution was purified by column chromatography (DCM / ACE = 1 / 0 to 5 / 1) to give a colorless oily compound BE2 (900 mg, yield 51.64%).

[0401] 1 H NMR (400MHz, CDCl3) δ7.35-7.49(m,2H),7.24-7.34(m,7H),6.75-6.88(m,4H),5.26-5.38(m,1H),4.99-5.09(m,1H),4.85-4.95( m,1H),3.09-4.33(m,30H),2.54-2.68(m,2H),2.13-2.19(m,3H),2.02-2.06(m,3H),1.97-2.01(m,6H),1.24(d,J=14.4Hz,12H).

[0402] 31 P NMR (162MHz, CDCl3) δ149.32, 148.96, 14.16.

[0403] Example 3. Preparation of compound BE3

[0404]

[0405] 1. Preparation of Compound 1

[0406]

[0407] Compound 1 (1.17 g, 2.18 mmol) was dissolved in DCM (20.0 mL) at 25 °C, and HATU (1.24 g, 3.27 mmol) and DIEA (1.08 mL, 6.53 mmol) were added. The mixture was stirred at 25 °C for 0.5 hours, and then compound 2 (1.08 g, 2.40 mmol) was added. The mixture was stirred for another 12 hours. LCMS showed product formation. TLC (T1: DCM / MEOH = 10 / 1) showed complete consumption of the starting material. 100 mL of dichloromethane was added. The mixture was washed three times successively with saturated sodium bicarbonate solution (20.0 mL x 3) and saturated brine (20.0 mL x 3). The organic phase was dried over anhydrous sodium sulfate and then suspended under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 1 / 0 to 20 / 1) to give a yellow oily compound 3 (1.67 g, yield 79.2%).

[0408] 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)

[0409] 2. Preparation of compound BE3

[0410]

[0411] Compound 3 (1.67 g, 1.72 mmol) was dissolved in DCM (30.0 mL) at 25 °C, followed by the addition of DCI (0.150 g, 1.29 mmol) and compound 4 (0.780 g, 2.59 mmol). The reaction mixture was stirred at 25 °C under a nitrogen atmosphere for 2 hours. Thin-layer chromatography (DCM / MeOH = 10 / 1) showed the formation of new spots. The reaction mixture was evaporated to dryness to obtain the crude product. The crude product was purified by normal-phase silica gel column chromatography (dichloromethane / acetone = 1 / 0-5 / 1) to give a pale yellow oily compound BE3 (1.60 g, yield 79.40%).

[0412] 1 H NMR (400MHz, CDCl3) δ7.38-7.47(m,2H),7.24-7.35(m,7H),6.78-6.90(m,4H),6. 54-6.69(m,1H),5.29-5.31(m,1H),4.97-5.06(m,1H),4.76-4.85(m,1H),4.10-4 .26(m,6H),3.76-4.06(m,15H),3.59-3.74(m,12H),3.10-3.54(m,5H),2.51-2.6 7(m,2H),2.14-2.17(m,3H),2.05-2.07(m,3H),1.99(s,6H),1.12-1.24(m,12H).

[0413] 31 P NMR (162MHz, CDCl3) δ149.16,148.87,148.81.

[0414] Example 4. Preparation of compound BE4

[0415]

[0416] 1. Preparation of compound 1c

[0417]

[0418] DIEA (38.7 mL, 234 mmol) and HATU (32.6 g, 85.8 mmol) were added to a DMF (100 mL) solution of compound 1b (20 g, 78.0 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 the reaction was complete, water (400 mL) was added to dilute the solution, the filter cake was collected, and dried to obtain compound 1c (30 g, 71.8 mmol, 92.1%).

[0419] 1 H 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).

[0420] 2. Preparation of compound 1d

[0421]

[0422] Under a nitrogen atmosphere, compound 1c (20 g, 47.9 mmol) was suspended in dry pyridine (100 mL), cooled to 0 °C in an ice-water bath, and then acetic anhydride (45 mL, 479 mmol) was added in portions. After the addition was complete, the mixture was slowly brought to room temperature and stirred at room temperature for 16 hours. After the reaction was complete, the reaction solution was diluted with ethyl acetate, washed once with water, and the aqueous phase was extracted twice more with ethyl acetate. The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain the residue. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether) to give compound 1d (10.2 g, 17.4 mmol, 36.3%).

[0423] 1 H NMR (400MHz, DMSO-d6) δ7.84(d,J=9.2Hz,1H),5.65(d,J=8.8Hz,1H),5.27(d,J=3.1Hz,1H),5.07(dd,J=11.3,3.4Hz,1H),4.23–4.18(m,1H),4. 16–4.09(m,1H),4.07–3.96(m,2H),2.12(s,3H),2.02(s,3H),1.99(s,3 H),1.89(s,3H),1.43(p,J=7.1Hz,2H),1.23(s,26H),0.89–0.81(m,3H).

[0424] 3. Preparation of compound 1e

[0425]

[0426] Compound 1d (5 g, 8.54 mmol) was dissolved in DCM (50 mL) under a nitrogen atmosphere and cooled to 0-5 °C in an ice-water bath. TMSOTf (2.28 g, 10.250 mmol) was added dropwise. The mixture was then stirred at room temperature for 16 hours. After the reaction was complete, the reaction solution was added in portions to a saturated sodium bicarbonate solution at 0-5 °C. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain the residue. The residue was purified by silica gel column chromatography (eluent: dichloromethane / ethyl acetate) to give compound 1e (4.2 g, 7.99 mmol, 93.5%).

[0427] 4. Preparation of 1g of compound

[0428]

[0429] Compound 1f (1.73 g, 6.80 mmol) and TMSOTf (0.89 g, 4.00 mmol) were added to a DCM (20 mL) solution of compound 1e (4.2 g, 7.99 mmol) at 0–5 °C. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was poured in portions into a saturated sodium bicarbonate (40 mL) solution pre-cooled to 0–5 °C. The organic phase was separated, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain the residue. The residue was purified by silica gel column chromatography (elution: ethyl acetate / dichloromethane) to give compound 1 g (2.83 g, 3.63 mmol, 45.4%).

[0430] 1 H NMR(400MHz,DMSO-d6)δ7.71(d,J=9.2Hz,1H),7.41–7.33(m,5H),5.21(d,J=3.3Hz,1H),5 .15(s,2H),4.98(dd,J=11.2,3.4Hz,1H),4.58(d,J=8.4Hz,1H),4.19(s,2H),4.08–3.97(m ,3H),3.94–3.85(m,1H),3.81–3.73(m,1H),3.64–3.55(m,3H),3.57–3.47(m,4H),2.10(s ,3H),2.03–1.94(m,5H),1.87(s,3H),1.49–1.38(m,2H),1.22(s,24H),0.88–0.82(m,3H).

[0431] 5. Preparation of compound 1h

[0432]

[0433] 1 g (2.5 g, 3.10 mmol) of the compound was dissolved in 20 mL of MeOH. After purging the system three times with nitrogen, Pd / C (0.566 g, 0.1% wt%) was added, and the system was then purged three times with hydrogen. The reaction solution was stirred at room temperature for 16 hours under a hydrogen atmosphere. After the reaction was complete, the reaction solution was filtered through diatomaceous earth to remove excess Pd / C. The filtrate was concentrated under vacuum to obtain an oily compound 1 h (2.0 g, 2.90 mmol, 80.0%).

[0434] 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),3.9 3–3.84(m,1H),3.80–3.73(m,1H),3.61–3.54(m,4H),3.53–3.49(m,4H),2.11(s,3 H),2.00(s,3H),1.87(s,3H),1.49–1.38(m,2H),1.22(s,26H),0.89–0.82(m,3H).

[0435] 6. Preparation of compound 1j

[0436]

[0437] To a DCM (10 mL) solution of compound 1h (1 g, 1.45 mmol), DIEA (0.7 mL, 4.35 mmol), EDCI (0.42 g, 2.18 mmol), and HOBt (0.29 g, 2.18 mmol) were added sequentially. After half an hour, compound 1i (0.65 g, 1.45 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction solution was washed with water, and the aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain the residue. The residue was purified by reversed-phase rapid column chromatography (elution: acetonitrile / water) to obtain compound 1j (1.39 g, 1.24 mmol, 85.3%).

[0438] 1H NMR (400MHz, DMSO-d6) δ7.72(d,J=9.1Hz,1H),7.41–7.35(m,2H),7.31(t,J=7.5Hz,2H),7.28–7.20(m,5H),6.88(d,J=8. 6Hz,4H),5.21(d,J=3.4Hz,1H),4.99(dd,J=11.2,3.3Hz,1H),4.78–4.68(m,2H),4.61–4.55(m,1H),4.27–4.10(m,2H),4 .10(d,J=5.8Hz,1H),4.06–3.98(m,4H),3.95–3.85(m,2H),3.74(s,6H),3.62–3.55(m,4H),3.51–3.41(m,4H),3.18–3.0 9(m,1H),3.06–2.95(m,2H),2.10(s,3H),2.03–1.96(m,5H),1.86(s,3H),1.43(s,2H),1.23(s,26H),0.88–0.78(m,3H).

[0439] 7. Preparation of compound BE4

[0440]

[0441] Under a nitrogen atmosphere, compound 1j (552 mg, 0.493 mmol) was dissolved in DCM (5 mL), and compound 1k (193 mg, 0.640 mmol) and DCI (70 mg, 0.591 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was poured in batches into a saturated sodium bicarbonate solution (20 mL) pre-cooled to 0-5 °C. The aqueous phase was extracted twice with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain the residue. The residue was purified by silica gel column chromatography (elution: 1% ethyl acetate of Et3N / 1% dichloromethane of Et3N) to obtain compound BE4 (447 mg, 0.338 mmol, 68.8%).

[0442] 1H NMR (400MHz, DMSO-d6) δ7.72(d,J=9.2Hz,1H),7.42–7.35(m,2H),7.30(t,J=7.6Hz,2H),7.28–7.19(m,5H),6.88(d,J=8. 4Hz,4H),5.21(d,J=3.3Hz,1H),4.99(dd,J=11.2,3.4Hz,1H),4.63–4.55(m,1H),4.28–4.11(m,2H),4.06–3.95(m,5H),3 .95–3.85(m,1H),3.74(s,6H),3.62–3.51(m,8H),3.50–3.42(m,6H),3.20–2.92(m,2H),2.74(t,J=5.9Hz,1H),2.71–2.6 5(m,1H),2.10(s,3H),2.03–1.96(m,5H),1.86(s,3H),1.43(s,2H),1.22(s,26H),1.16–1.05(m,14H),0.88–0.80(m,3H).

[0443] Example 5. Preparation of compound BE5

[0444]

[0445] To a DCM (2 mL) solution of compound 1j (200 mg, 0.178 mmol) from Example 4, DIEA (0.18 mL, 1.071 mmol), succinic anhydride (107 mg, 1.07 mmol), and DMAP (5 mg, 0.045 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was washed with saturated sodium bicarbonate solution, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain the residue. The residue was purified by reversed-phase rapid column chromatography (elution: acetonitrile / water) to obtain compound BE5 (141 mg, 0.116 mmol, 64.1%).

[0446] 1H NMR(400MHz, DMSO-d6)δ7.79(d,J=9.8Hz,1H),7.42–7.34(m,2H),7.31(t,J=7.6Hz,2H),7.28–7.19(m,5H),6.91–6.8 6(m,4H),5.20(d,J=3.3Hz,2H),5.04–4.96(m,1H),4.67–4.56(m,2H),4.25–4.11(m,3H),4.06–3.96(m,4H),3.95–3.8 1(m,3H),3.74(s,6H),3.62–3.54(m,2H),3.52–3.42(m,5H),3.16–3.01(m,2H),3.01–2.95(m,1H),2.40–2.30(m,2H) ,2.10(s,3H),2.04–1.95(m,5H),1.86(s,3H),1.48–1.37(m,2H),1.21(s,24H),1.03–0.91(m,4H),0.88–0.80(m,3H).

[0447] Example 6. Preparation of compound BE6

[0448]

[0449] The synthesis steps of compound BE6 can be found in Example 4, wherein stearic acid is used to replace compound 1b.

[0450] 1 H NMR (400MHz, DMSO-d6) δ7.41–7.35(m,2H),7.31(t,J=7.6Hz,2H),7.28–7.19(m,5H),6.88(d,J=8.5Hz,4H),5.20(d,J =3.3Hz,2H),4.99(dd,J=11.1,3.3Hz,1H),4.59(t,J=7.8Hz,1H),4.25–4.10(m,2H),4.09–3.97(m,5H),3.96–3.84(m ,3H),3.74(s,6H),3.63–3.55(m,2H),3.54–3.43(m,7H),3.17–3.09(m,1H),3.07–2.95(m,3H),2.47–2.40(m,2H),2. 10(s,3H),2.03–1.96(m,5H),1.86(s,3H),1.42(s,2H),1.25–1.18(m,26H),0.96(d,J=6.4Hz,4H),0.87–0.82(m,3H).

[0451] Example 7. Synthesis of siRNA

[0452] The siRNA of the present invention was prepared using the solid-phase phosphoramide method well known in the art. Specific methods can be found, for example, in PCT publications WO2016081444 and WO2019105419, and are briefly described below.

[0453] 1. Preparation of siRNA without ligands attached to the 3' end of the positive strand

[0454] 1.1 Synthesis of the Justice Chain (SS Chain)

[0455] The oligonucleotide was synthesized using a solid-phase phosphoramide method, employing a blank CPG solid-phase support as the starting cycle. Nucleoside monomers were sequentially linked from the 3'-5' direction according to the nucleotide arrangement of the positive strand. Each linkage of a nucleoside monomer involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthetic scale was 5 μmol. The synthetic conditions are as follows:

[0456] Nucleoside monomers were provided in 0.05 mol / L acetonitrile solution. The reaction conditions were identical for each step, i.e., a temperature of 25°C. Deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution. The coupling reaction was activated twice using a 0.25 mol / L 5-ethylthiotetrazole (ETT)-acetonitrile solution. Capping was performed twice using a 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazolium / acetonitrile mixture (10:14:76, v / v / v). Oxidation was performed twice using a 0.05 mol / L iodine-tetrahydrofuran / pyridine / water mixture (70 / 20 / 10, v / v / v). Thiolation was performed twice using a 0.2 mol / L phenylacetyl disulfide (PADS) mixture of acetonitrile / 3-methylpyridine (1 / 1, v / v).

[0457] 1.2 Synthesis of the antisense chain (AS chain)

[0458] The solid-phase phosphoramide synthesis method utilizes a blank CPG solid-phase support as the starting cycle, and nucleoside monomers are sequentially linked from the 3'-5' direction according to the nucleotide arrangement sequence of the antisense strand. Each linkage of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for 5 μmol oligonucleotides of the antisense strand are the same as those for the sense strand.

[0459] 1.3 Purification and Annealing of Oligonucleotides

[0460] 1.3.1 Ammonolysis

[0461] Add the synthesized solid support (sense or antisense chain) to a 5 mL centrifuge tube, add 3% diethylamine / ammonia (v / v), react in a constant temperature water bath at 35°C (or 55°C) for 16 hours (or 8 hours), filter, wash the solid support three times with ethanol / water, 1 mL each time, centrifuge and concentrate the filtrate, and then purify the crude product.

[0462] 1.3.2 Purification

[0463] Purification and desalting methods are well known to those skilled in the art. For example, a column packed with strong anion exchange material can be used for elution purification with a sodium chloride-sodium hydroxide system, and the product can be collected and piped. Desalting can be performed using a gel-packed purification column with pure water as the elution system.

[0464] 1.3.3 Annealing

[0465] According to the instructions, the sense chain (SS chain) and the antisense chain (AS chain) are mixed in a molar ratio (SS chain / AS chain = 1 / 1.05), heated in a water bath to 70-95 degrees Celsius, held for 3-5 minutes, and then naturally cooled to room temperature. The system is then freeze-dried to obtain the product.

[0466] The siRNA sequence used in this invention is as follows:

[0467]

[0468] The meanings of the abbreviations in this article are as follows:

[0469] A, U, G, and C represent natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide, and cytosine ribonucleotide, respectively.

[0470] The 'd' indicates that the nucleotide adjacent to its right is a deoxyribonucleotide. For example, dA, dT, dG, and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and cytosine deoxyribonucleotide, respectively.

[0471] The 'm' indicates that the nucleotide adjacent to it on the left is a nucleotide modified with 2'-OCH3. For example, Am, Um, Gm, and Cm represent A, U, G, and C modified with 2'-OCH3.

[0472] The 'f' indicates that the nucleotide adjacent to it on the left is a 2'-F modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.

[0473] “s” indicates that the two adjacent nucleotides and / or delivery carriers are linked by a phosphate thioester.

[0474] VP indicates that the nucleotide adjacent to its right is a vinyl phosphate modified nucleotide.

[0475] L96 represents a GalNAc delivery vector with the following structure well known in the art, wherein... The location indicated by the phosphate ester group or thiophosphate ester group linked to siRNA can be found, for example, in PCT publications WO2009073809 and WO2009082607.

[0476]

[0477] The structures of GL34, LS3, and LS1 are as defined above.

[0478] The structures of GL34 when linked to the 3' and 5' ends of a nucleic acid chain via phosphate ester groups, thiophosphate ester groups, or other linking groups are as follows:

[0479]

[0480] The structures of LS3 when linked to the 3' and 5' ends of a nucleic acid strand via phosphate ester groups, thiophosphate ester groups, or other linking groups are as follows:

[0481]

[0482] The structures of LS1 when linked to the 3' and 5' ends of a nucleic acid strand via phosphate ester groups, thiophosphate ester groups, or other linking groups are as follows:

[0483]

[0484] Example 8: Verification of the long-lasting efficacy of the compound of the present invention in a C57BL / 6 mouse model.

[0485] C57BL / 6 mice (male, 18–21 g, 6–8 weeks old) were randomly assigned to groups. The dosage for each animal was calculated based on its body weight. The medication was administered subcutaneously as a single dose. The siRNA conjugate was administered in a 1 mg / mL solution (0.9% sodium chloride aqueous solution as solvent). Specifically, before the experiment, the siRNA conjugate was dissolved in 0.9% sodium chloride aqueous solution and brought to the required concentration and volume. The administration volume of physiological saline (control group) and siRNA conjugate was 5 mL / kg.

[0486] Liver tissue samples (10 mg each) were collected before administration (day 0) and on days 7, 14, 21, 28, 35, 42, 56, and 70 after administration and placed in RNA later. TM The solution was frozen at -80°C for the detection of liver mTTR mRNA.

[0487] Detection of liver mTTR mRNA:

[0488] Following the operating protocol of the high-throughput tissue RNA extraction kit (Fanzhi Medical, FG0412), RNA was extracted from cells using a nucleic acid extractor (Hangzhou Aosheng, Auto-pure96); referencing PrimeScript... TM II. 1st Strand cDNA Synthesis Kit (Takara, 6210B) Reverse transcription; Refer to TaqMan TM A 20 μL system of Fast Advanced Master Mix (ABI, 4444965) was used for real-time PCR (ABI, QuantStudio3). Primers are shown in Table 1.

[0489] Table 1: Primer Information

[0490]

[0491] Data statistics and analysis

[0492] Calculate 2 -△△Ct The values ​​are then converted to percentages to obtain the residual inhibition rate;

[0493] △△Ct=[(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)].

[0494] The target gene is mTTR, and the internal reference is mGAPDH.

[0495] Table 2. Results of the long-term efficacy test of the compound in the C57BL / 6 mouse model.

[0496]

[0497] Example 9: Verification of the long-lasting efficacy of the compound of the present invention in a C57BL / 6 mouse model.

[0498] C57BL / 6 mice (male, 6–8 weeks old) were randomly assigned to groups and administered the drug via a single intravitreal injection in both eyes, with a dose of 7.5 μg per eye. Specifically, before the experiment, the siRNA conjugate was dissolved in phosphate buffer and brought to the required concentration and volume. The administration volume of phosphate buffer and siRNA conjugate was 1.5 μL per eye.

[0499] On day 14 after drug administration, the eyeballs were enucleated and separated into three parts: ① cornea + iris + ciliary body; ② retina; ③ retinal pigment epithelial cells (RPE) + choroid + sclera. The separated samples were immediately flash-frozen in liquid nitrogen and then stored at -80℃ for the detection of mTTR mRNA.

[0500] The subsequent experimental steps and primers are the same as in Example 8 above.

[0501] Table 3 shows the results of the long-term efficacy test of the compounds in the C57BL / 6 mouse model.

[0502]

[0503] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. An oligonucleotide comprising one or more compounds of formula (II), said compound (II) being selected from compounds of the following general formulas, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: (II-1) or (II-2); in, P1, P2, and P3 are selected from H or P groups; P4 is selected from CH3 or a P group; One of P1, P2, P3, and P4 is a P group; represents the position of the phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 is H, or indicates the position where it is linked to the phosphate ester or thiophosphate ester of another adjacent nucleotide; L1 is OCH2CH2 or O(CH2)5; L2 is OCH2 or a chemical bond; T is selected from -C(O)-; P is -(CH2) 14-16 CH3; R s is H; m is 0, 1, or 2; n is 0 or 1; k is 0 or 1.

2. The oligonucleotide of claim 1, wherein, The compound of formula (II) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: LS1, LS3, LS4, LS6, LS7, LS8 or LS9; in, Indicates the position where it is attached to the phosphate ester or thiophosphate ester of the adjacent nucleotide; R1 is H, or indicates the position where it is linked to a phosphate ester or thiophosphate ester of another adjacent nucleotide.

3. The oligonucleotide of claim 1 or 2, having 14 to 30 nucleotides.

4. The oligonucleotide of claim 1, wherein at its 3' end it comprises a compound of formula (II) of claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

5. The oligonucleotide of claim 1, comprising at its 5' end and 3' end a compound of formula (II) of claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

6. The oligonucleotide of claim 1, wherein the oligonucleotide is ASO or siRNA.

7. A compound of formula (II'), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof: (II’) R1 and R2 are independently selected from reactive phosphorus groups, hydroxyl protecting groups, or solid supports; A is selected from or ; A is connected to L1 through its a-terminus and to the P group through its b-terminus; The reactive phosphorus group is selected from phosphoramide, H-phosphonate, alkyl-phosphonate or phosphate ester, thiophosphate, dithiophosphate, borane phosphate, borane thiophosphate, phosphonate, halogen-substituted phosphonate, halogen-substituted phosphate ester, aminophosphate, phosphate diester, phosphate triester, thiophosphate diester, thiophosphate triester, diphosphate or triphosphate. The hydroxyl protecting group is selected from trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trimethylsilyl... Chloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzylmethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl; P, L1, L2, T, Rs, m, n, k are as defined in claim 1.

8. The compound of claim 7, wherein, One of R1 and R2 is a reactive phosphorus group, which is selected from thiophosphate, dithiophosphate, borane phosphate, borane thiophosphate, phosphonate, halogen-substituted phosphonate, halogen-substituted phosphate, aminophosphate, phosphate diester, phosphate triester, thiophosphate diester, thiophosphate triester, diphosphate, or triphosphate.

9. The compound of claim 7, wherein, The reactive phosphorus group is -P(OCH2CH2CN)(N(iPr)2).

10. The compound of claim 7, wherein, R1 and R2 are hydroxyl protecting groups.

11. The compound of claim 10, wherein, The hydroxyl protecting group is DMTr.

12. The compound of claim 7, wherein the compound of formula (II') is selected from the group consisting of compounds, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof: , , , , , , or .

13. A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand contains a sequence sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand contains one or more compounds of formula (II) as claimed in claim 1, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

14. The double-stranded RNA of claim 13, wherein the compound of formula (II) is selected from the group consisting of compounds, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof: LS1, LS3 LS4 LS6 LS7 LS8 or LS9; in, Indicates the position where it is attached to the phosphate ester or thiophosphate ester of the adjacent nucleotide; R1 is H, or indicates the position where it is linked to a phosphate ester or thiophosphate ester of another adjacent nucleotide.

15. The double-stranded RNA of claim 13, wherein the sense strand comprises at its 3' end a compound of formula (II) of claim 1 or 2, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

16. The double-stranded RNA of claim 13, wherein the sense strand comprises a compound of formula (II) of claim 1 or 2, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at its 5' end and 3' end, respectively.

17. A cell containing double-stranded RNA as described in any one of claims 13-16.

18. A pharmaceutical composition comprising double-stranded RNA as described in any one of claims 13-16, or cells as described in claim 17, and optionally a pharmaceutically acceptable carrier.

19. A pharmaceutical composition comprising double-stranded RNA as described in any one of claims 13-16, or cells as described in claim 17, and optionally a pharmaceutically acceptable excipient.

20. A kit comprising double-stranded RNA as described in any one of claims 13-16, or cells as described in claim 17.

21. Use of the oligonucleotide of any one of claims 1-6 in the preparation of a medicament for inhibiting genes expressed extrahepatically.

22. Use of the oligonucleotide of any one of claims 1-6 in the preparation of a medicament for inhibiting genes expressed in the central nervous system (CNS) and / or the eye.

Images