Oligonucleotide delivery ligands comprising peptides

Abstract

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

Description

Peptide-containing oligonucleotide delivery ligands

[0001] This application claims priority to Chinese invention patent application CN202310814387.3, filed on July 4, 2023, which is incorporated herein by reference in its entirety. Field of the Invention

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

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

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

[0005] There is still a need in the art to develop more hydrophobic groups to more effectively deliver siRNA to the eye.

[0006] Summary of the Invention

[0007] In one aspect, the present invention provides an oligonucleotide comprising one or more compounds of formula (I), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0008] wherein each group is as defined below.

[0009] In another aspect, the present invention provides an oligonucleotide comprising one, two or more delivery vectors within the oligonucleotide, at the 5' end and / or at the 3' end, wherein the delivery vector is a peptide-based linker modified with a hydrophobic group;

[0010] Preferably, the peptide-based linking group modified with a hydrophobic group is selected from a compound of formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0011] wherein each group is as defined below.

[0012] 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, and the antisense strand comprises a sequence that is sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or antisense strand comprises one or more compounds of formula (I) above, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0013] In another aspect, the present invention provides a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or antisense strand comprises one, two or more delivery vectors internally, at the 5' end and / or at the 3' end, the delivery vector being a peptide-based linker modified with a hydrophobic group;

[0014] Preferably, the peptide-based linking group modified with a hydrophobic group is selected from a compound of formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0015] wherein each group is as defined below.

[0016] In another aspect, the present invention provides a compound of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0017] wherein each group is as defined below.

[0018] In another aspect, the present invention provides a vector comprising a nucleotide sequence encoding the aforementioned double-stranded RNA.

[0019] In another aspect, the present invention provides a cell containing the aforementioned double-stranded RNA or the aforementioned vector.

[0020] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned double-stranded RNA, the aforementioned vector, or the aforementioned cell, and optionally a pharmaceutically acceptable carrier or excipient.

[0021] In another aspect, the present invention provides a kit comprising the aforementioned double-stranded RNA, the aforementioned vector, or the aforementioned cell.

[0022] Detailed Description of the Invention

[0023] definition

[0024] Chemical definition

[0025] Definitions of specific functional groups and chemical terms are described in more detail below.

[0026] When a numerical range is listed, it is intended to include every value and sub-range within the stated range. For example, "C 1-6 "Alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-5 、C 2-4 、C 2-3 、C 3-6 、C 3-5 、C 3-4 、C 4-6 、C 4-5 and C 5-6 alkyl.

[0027] “C 1-6 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms. 8-30 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 30 carbon atoms. In some embodiments, C 8-25 Alkyl, C 10-22 Alkyl, C 8-20 Alkyl, C 1-10 Alkyl and C 1-6 Alkyl groups are preferred. 1-6 Examples of alkyl groups include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). The term “C 1-6 "Alkyl" also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me(-CH3), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3), or i-Bu(-CH2CH(CH3)2).

[0028] “C 8-30"Alkenyl" refers to a straight or branched chain hydrocarbon group having 8 to 30 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 10-22 Alkenyl, C 14-20 Alkenyl, C 16-18 Alkenyl is preferred.Alkenyl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0029] “C 8-30 "Alkynyl" refers to a straight or branched chain hydrocarbon group having 8 to 30 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 10-22 Alkynyl, C 14-20 Alkynyl, C 16-18 Alkynyl is preferred.Alkynyl groups may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0030] “C 0-10 "Alkylene" refers to a chemical bond or the removal of C 1-10 In some embodiments, C 2-8 Alkylene, C 3-7 Alkylene, C 1-6 Alkylene, C 4-6 Alkylene, C 1-4 Alkylene, C 2-4 Alkylene and C 1-3 Alkylene is preferred. Unsubstituted alkylene includes, but is not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and the like. Exemplary substituted alkylenes, for example, alkylenes substituted with one or more alkyl (methyl) groups, include, but are not limited to, substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3) 2- ), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), and the like.

[0031] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0032] Therefore, “C 1-6 "Haloalkyl" refers to the above-mentioned "C 1-6 "alkyl" is substituted with one or more halogen groups. In some embodiments, C 1-4 Halogenated alkyl is particularly preferred, more preferably C 1-2 Haloalkyl. Exemplary haloalkyl groups include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like. The haloalkyl group can be substituted at any available point of attachment, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0033] The term "hydrophobic group" refers broadly to any chemical group that has an affinity for lipids. One way to characterize the hydrophobicity of a hydrophobic group is by the octanol-water partition coefficient log K ow , where K ow is the ratio of the concentration of a chemical in the octanol phase to its concentration in the aqueous phase at equilibrium in a two-phase system. ow More than 1, more than 1.5, more than 2, more than 3, more than 4, more than 5 or more than 10. Specifically for the present invention, the hydrophobic portion is the R group in the compound of formula I.

[0034] Alkyl, alkenyl, alkynyl, etc., as defined herein, are optionally substituted groups.

[0035] 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(Rbb )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(R cc )3、-B(R aa )2、-B(OR cc )2, -BR aa (OR cc ), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0036] Or the two geminal hydrogen atoms on the carbon atom are replaced by groups =O, =S, =NN(R bb )2, =NNR bb C(=O)R aa 、=NNR bb C(=O)OR aa 、=NNR bb S(=O)2R aa 、=NR bb or = NOR cc replace;

[0037] R aa Each of R is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R aa The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0038] R bb Each of the following is independently selected from: hydrogen, -OH, -OR aa 、-N(R cc )2, -CN, -C(=O)R aa 、-C(=O)N(R cc )2, -CO2R aa 、-SO2R aa 、-C(=NR cc )OR aa、-C(=NR cc )N(R cc )2、-SO2N(R cc )2, -SO2R cc 、-SO2OR cc 、-SOR aa 、-C(=S)N(R cc )2, -C(=O)SR cc 、-C(=S)SR cc 、-P(=O)2R aa 、-P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R bb The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0039] R cc Each of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R cc The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0040] R dd Each of the is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee 、-ON(R ff )2、-N(R ff )2,、-N(R ff )3 + X - 、-N(OR ee )R ff 、-SH、-SR ee 、-SSR ee 、-C(=O)R ee 、-CO2H、-CO2R ee 、-OC(=O)R ee 、-OCO2R ee 、-C(=O)N(R ff )2、-OC(=O)N(R ff )2、-NR ffC(=O)R ee 、-NR ff CO2R ee 、-NR ff C(=O)N(R ff )2、-C(=NR ff )OR ee 、-OC(=NR ff )R ee 、-OC(=NR ff )OR ee 、-C(=NR ff )N(R ff )2、-OC(=NR ff )N(R ff )2、-NR ff C(=NR ff )N(R ff )2、-NR ff SO2R ee 、-SO2N(R ff )2, -SO2R ee 、-SO2OR ee 、-OSO2R ee 、-S(=O)R ee 、-Si(R ee )3、-OSi(R ee )3、-C(=S)N(R ff )2, -C(=O)SR ee 、-C(=S)SR ee 、-SC(=S)SR ee 、-P(=O)2R ee 、-P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR ee )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg Group substitution, or two geminal R dd Substituents may combine to form =O or =S;

[0041] R ee Each of R is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R gg group substitution;

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

[0043] R gg Each of the independently: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 Alkyl, -ON(C 1-6 Alkyl)2, -N(C 1-6 Alkyl)2, -N(C 1-6 Alkyl)3 + X - 、-NH(C 1-6 Alkyl)2 + X - 、-NH2(C 1-6 alkyl) + X - 、-NH3 + X - 、-N(OC 1-6 Alkyl)(C 1-6 Alkyl), -N(OH)(C 1-6 Alkyl), -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 Alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 Alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 Alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6Alkyl, -C(=NH)N(C 1-6 Alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 Alkyl)2, -OC(NH)NH(C 1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6 Alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 Alkyl), -SO2N(C 1-6 Alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2C 1-6 Alkyl, -SO2OC 1-6 Alkyl, -OSO2C 1-6 Alkyl, -SOC 1-6 Alkyl, -Si(C 1-6 alkyl)3, -OSi(C 1-6 alkyl)3, -C(=S)N(C 1-6 alkyl)2、C(=S)NH(C 1-6 alkyl), C(=S)NH2, -C(=O)S(C 1-6 alkyl), -C(=S)SC 1-6 Alkyl, -SC(=S)SC 1-6 Alkyl, -P(=O)2(C 1-6 alkyl), -P(=O)(C 1-6 alkyl)2, -OP(=O)(C 1-6 alkyl)2, -OP(=O)(OC 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C 10 Aryl, C3-C7 heterocyclic, C5-C 10 heteroaryl; or two geminal R gg The substituents may combine to form =O or =S, wherein X- is a counterion.

[0044] Exemplary substituents on nitrogen atoms include, but are not limited to, hydrogen, -OH, -OR aa 、-N(R cc )2, -CN, -C(=O)R aa -C(=O)N(R cc )2, -CO2R aa 、-SO2R aa 、-C(=NR bb )Raa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc )2, -SO2R cc 、-SO2OR cc 、-SOR aa 、-C(=S)N(R cc )2, -C(=O)SR cc 、-C(=S)SR cc 、-P(=O)2R aa 、-P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R attached to the nitrogen atom cc The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substituted, and wherein R aa 、R bb 、R cc and R dd As mentioned above.

[0045] Other definitions

[0046] The term "siRNA" herein refers to a class of double-stranded RNA molecules that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a gene encoding a protein) that is complementary thereto. siRNA is typically double-stranded, comprising an antisense strand complementary to the target RNA and a sense strand complementary to the antisense strand. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA (e.g., tRNA) and viral RNA other than mRNA can also be targeted.

[0047] The term "antisense oligonucleotide" or ASO (Antisense Oligonucleotides) is a single-stranded DNA or RNA sequence consisting of 15-25 nucleotides that is paired with a target gene. It achieves the purpose of gene regulation by specifically blocking the transcription or translation process of the target gene.

[0048] The term "antisense strand" refers to a strand of an siRNA that includes a region that is completely, fully, or substantially complementary to a target sequence. The term "sense strand" refers to a strand of an siRNA that includes a region that is completely, fully, or substantially complementary to a region that is, as the term is defined herein, an antisense strand.

[0049] The term "complementary region" refers to a region on the antisense strand that is completely, fully or substantially complementary to the target mRNA sequence. In the case where the complementary region is not completely complementary to the target sequence, mispairing can be located in the interior or terminal regions of the molecule. Typically, the most tolerated mispairing is located in the terminal regions, for example, in 5' and / or 3' ends within 5, 4, 3, 2 or 1 nucleotide. The antisense strand portion that is most sensitive to mispairing is referred to as a "seed region." For example, in a siRNA comprising a 19nt chain, the 19th position (from 5' to 3') can tolerate some mispairing.

[0050] The term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50° C. or 70° C. for 12-16 hours. In terms of meeting the above requirements relative to their ability to hybridize, "complementary" sequences may also include or be completely formed from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0051] A polynucleotide that is "at least partially complementary," "sufficiently complementary," or "substantially complementary" to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest. For example, a polynucleotide is complementary to at least a portion of a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding PCSK9. The terms "complementary," "fully complementary," "sufficiently complementary," and "substantially complementary" as used herein can be used with respect to base pairing between the sense and antisense strands of an siRNA, or between the antisense strand of an siRNA agent and a target sequence.

[0052] "Fully complementary" refers to the extent to which the sense strand only needs to be complementary to the antisense strand in order to maintain the overall double-stranded nature of the molecule. In other words, while perfect complementarity is generally desired, in some cases, particularly in the antisense strand, one or more, for example, 6, 5, 4, 3, 2, or 1 mismatches (relative to the target mRNA) may be included, but the sense and antisense strands can still maintain the overall double-stranded nature of the molecule.

[0053] A "nucleoside" is a compound composed of a purine or pyrimidine base and ribose or deoxyribose; a "nucleotide" is a compound composed of a purine or pyrimidine base, ribose or deoxyribose, and phosphate; an "oligonucleotide" is a nucleic acid molecule (RNA or DNA) having, for example, fewer than 100, 200, 300, or 400 nucleotides in length.

[0054] "Bases" are the fundamental building blocks of nucleosides, nucleotides, and nucleic acids. They contain nitrogen and are also called "nitrogenous bases." Unless otherwise specified, the capital letters A, U, T, G, and C represent the bases of nucleotides, representing adenine, uracil, thymine, guanine, and cytosine, respectively.

[0055] The "modification" of nucleotides herein includes, but is not limited to, methoxy modification, fluorination, phosphorothioate linkage, or conventional protecting group protection. For example, the fluorination-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0056] " modified nucleotide " herein includes but is not limited to 2 '-O-methyl modified nucleotides, 2 '-fluoro modified nucleotides, 2 '-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, nucleotides comprising thiophosphate groups, vinyl phosphate modified nucleotides, locked nucleotides, 2 '-amino-modified nucleotides, 2 '-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, the non-natural bases comprising nucleotides and the terminal nucleotides, deoxyribonucleotides or conventional protecting group protections on a cholesterol derivative or a dodecanoic acid didecylamide group. For example, the 2 '-fluoro modified nucleotides refer to nucleotides in which the hydroxyl group at the ribose group 2 ' position is replaced by fluorine. The 2 '-deoxy-modified nucleotides refer to nucleotides in which the 2 '-hydroxyl group at the ribose group is replaced by methoxy and formed.

[0057] " reactive phosphorus group " refers to the phosphorus-containing group contained in the nucleotide unit or in the nucleotide analog unit, and it can react with the hydroxyl or the amido reaction contained in another molecule, especially in another nucleotide unit or in another nucleotide analog by nucleophilic attack reaction.Usually, such reaction produces the ester type internucleoside bond that the first nucleotide unit or the first nucleotide analog unit are connected with the second nucleotide unit or the second nucleotide analog unit.Reactive phosphorus group can be selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphate or phosphate analogs, include but not limited to: natural phosphate, thiophosphate, phosphorodithioate, borane phosphate, borane thiophosphate, phosphonate, halogen-substituted phosphonate and phosphate, phosphoramidate, phosphodiester, phosphotriester, thiophosphate diester, thiophosphate triester, diphosphate and triphosphate, preferably-P(OCH2CH2CN)(N(iPr)2).

[0058] A "protecting group" refers to any atom or group of atoms added to a molecule to prevent an existing group in the molecule from undergoing an undesirable chemical reaction. A "protecting group" may be an unstable chemical moiety known in the art that is used to protect reactive groups, such as hydroxyl, amino, and thiol groups, to prevent undesirable or inappropriate reactions during chemical synthesis. Protecting groups are typically used selectively and / or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group intact or available for further reactions.

[0059] A non-limiting list of protecting groups includes benzyl; substituted benzyl; alkylcarbonyl and alkoxycarbonyl (e.g., tert-butyloxycarbonyl (BOC), acetyl, or isobutyryl); arylalkylcarbonyl and arylalkoxycarbonyl (e.g., benzyloxycarbonyl); substituted methyl ethers (e.g., methoxymethyl ether); substituted ethyl ethers; substituted benzyl ethers; tetrahydropyranyl ethers; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy] methyl or tert-butyldiphenylsilyl); esters (e.g., benzoate); carbonates (e.g., methoxymethyl carbonate); sulfonates (e.g., tosylate or mesylate); acyclic ketals (e.g., dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); acyclic acetals; cyclic acetals (e.g., those described herein); acyclic hemiacetals; cyclic hemiacetals; cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiolane); orthoesters (e.g., those described herein), and triarylmethyl groups (e.g., trityl; monomethoxytrityl (MMTr); 4,4′-dimethoxytrityl (DMTr); 4,4′,4″-trimethoxytrityl (TMTr); and those described herein). Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethyl acetal (BOM), β-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, neopentyl (Vl), methylthiomethyl ether, ... Valeryl (Piv), tetrahydropyranyl (THP), triphenylmethyl (Trt), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMT), trimethylsilyl ether (TMS), tert-butyldimethylsilyl ether (TBDMS), tri-iso-propylsilyloxymethyl ether (TOM), tri-isopropylsilyl ether (TIPS), methyl ether, ethoxyethyl ether (EE) N,N-dimethylformamidine and 2-cyanoethyl (CE).

[0060] A "hydroxyl protecting group" is a group that protects the hydroxyl group from chemical reactions and can be removed under specific conditions to restore the hydroxyl group. These groups primarily include silane-type protecting groups, acyl-type protecting groups, or ether-type protecting groups, with the following being preferred:

[0061] Trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc) , benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, more preferably -C(O)CH2CH2C(O)OH.

[0062] As used herein, the term "pharmaceutically acceptable salt" refers to those carboxylate salts, amino acid addition salts of the compounds of the present invention that are suitable for use in contact with patient tissues within the scope of sound medical judgment, do not produce undue toxicity, irritation, allergic response, etc., are commensurate with a reasonable benefit / risk ratio, and are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the present invention.

[0063] The present invention includes tautomers, which are functional isomers produced by the rapid movement of an atom in two positions in a molecule. Compounds that exist in different tautomeric forms are not limited to any specific tautomer, but are intended to cover all tautomeric forms.

[0064] The compounds of the present invention may include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, for example, enantiomers and / or diastereomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

[0065] The present invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described in formula (I) but for which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, for example 2 H. 3 H. 13 C. 11 C. 14 C. 15 N. 18 O. 17 O. 31 P. 32 P. 35 S. 18 F and 36 Cl. Compounds of the present invention containing the above-mentioned isotopes and / or other isotopes of other atoms, their prodrugs and pharmaceutically acceptable salts of the compounds or prodrugs are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, such as those in which radioactive isotopes (e.g. 3 H and 14 C) can be used in drug and / or substrate tissue distribution assays. 3 H and carbon-14, i.e. 14 C isotopes are particularly preferred because they are easy to prepare and detect. 2 H, because greater metabolic stability can provide therapeutic benefits, such as prolonged in vivo half-life or reduced dosage requirements, and thus may be preferred in some cases. Isotopically labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents when carrying out the processes disclosed in the following schemes and / or the Examples and Preparations.

[0066] Compounds of the present invention

[0067] The present invention specifically relates to an oligonucleotide comprising one or more compounds of formula (I), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0068] in,

[0069] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0070] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0071] and R1 are not H at the same time;

[0072] P is a hydrophobic group, preferably C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH2CH2- group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring; the C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R;

[0073] T1 is selected from chemical bonds, -C 0-10 Alkylene-, -C 0-10 Alkylene-O-, -C 0-10 Alkylene-C(O)-, -C 0-10 Alkylene-OC(O)- or -C 0-10 Alkylene-M-;

[0074] Where M is

[0075] A is a peptide-based linker that can be cleaved in endosomes or lysosomes;

[0076] L is selected from -NH-, -O-, -CH2-, -C(O)- or -C(O)O-;

[0077] T2 is selected from chemical bonds, -C 0-10 Alkylene-, -C 0-10 Alkylene-OC 0-10 Alkylene-, -C 0-10 Alkylene-C(O)-C 0-10 Alkylene-, -C 0-10 Alkylene-OC(O)-C 0-10 Alkylene- or -C 0-10 Alkylene-C(O)OC 0-10 Alkylene-; said T2 is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R;

[0078] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0079] Rs is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0080] k is selected from 0, 1, 2, 3, 4, 5 or 6.

[0081] The present invention specifically relates to a compound of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0082] wherein R1 and R2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;

[0083] P, T1, A, L, T2, Rs and k are as defined above.

[0084] The present invention specifically relates to a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand comprises a sequence that is sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand comprises one or more compounds of formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0085] In one embodiment, represents H; in another embodiment, Indicates the position of the phosphate or phosphorothioate linkage to the adjacent nucleotide.

[0086] R1 and R2

[0087] In one embodiment, R1 represents H; in another embodiment, R1 represents the position of attachment to the phosphate or phosphorothioate of another adjacent nucleotide.

[0088] In one embodiment, R1 is H; in another embodiment, R1 is selected from a reactive phosphorus group, preferably a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimetic, such as a natural phosphate, a phosphorothioate, a phosphorodithioate, a boranephosphate, a boranephosphorothioate, a phosphonate, a halogen-substituted phosphonate and a phosphate, a phosphoramidate, a phosphodiester, a phosphotriester, a phosphorothioate diester, a phosphorothioate triester, a diphosphate or a triphosphate, preferably -P(OCH2CH2CN)(N(iPr)2); in another embodiment, R1 is selected from a hydroxy protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), Acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, preferably DMTr; in another embodiment, R1 is a solid support.

[0089] In one embodiment, R2 is H; in another embodiment, R2 is selected from a reactive phosphorus group, preferably a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimetic, such as a natural phosphate, a phosphorothioate, a phosphorodithioate, a boranephosphate, a boranephosphorothioate, a phosphonate, a halogen-substituted phosphonate and a phosphate, a phosphoramidate, a phosphodiester, a phosphotriester, a phosphorothioate diester, a phosphorothioate triester, a diphosphate or a triphosphate, preferably -P(OCH2CH2CN)(N(iPr)2); in another embodiment, R2 is selected from a hydroxy protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), Acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, preferably DMTr; in another embodiment, R1 is a solid support.

[0090] P

[0091] In one embodiment, P is a hydrophobic group; in another embodiment, P is C 8-30 Hydrocarbon groups, such as C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl; in another embodiment, P is C 10-22 Alkyl; in another embodiment, P is C 10-22 Alkenyl.

[0092] In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-adjacent carbon atoms in P may be replaced by heteroatoms selected from O, S, and N, or the -CH2CH2- group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)-, or -C(O)NH-, or the substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring.

[0093] In one embodiment, P is unsubstituted; in another embodiment, P is substituted with 1 R; in another embodiment, P is substituted with 2 Rs; in another embodiment, P is substituted with 3 Rs; in another embodiment, P is substituted with 4 Rs; in another embodiment, P is substituted with 5 Rs; in another embodiment, P is substituted with 6 Rs; in another embodiment, P is substituted with 7 Rs; in another embodiment, P is substituted with 8 Rs; in another embodiment, P is substituted with more Rs.

[0094] In a specific embodiment, P is -(CH2) 14-20 -CH3, for example -(CH2) 14 -CH3, -(CH2) 15 -CH3, -(CH2) 16 -CH3, -(CH2) 17 -CH3, -(CH2) 18 -CH3, -(CH2) 19 -CH3 or -(CH2) 20 -CH3, preferably -(CH2) 14 -CH3 or -(CH2) 16 -CH3.

[0095] T1

[0096] In one embodiment, T1 is a chemical bond; in another embodiment, T1 is -C 0-10 Alkylene-; In another embodiment, T1 is -C 0-6 Alkylene-, for example -(CH2) 1-6 -、-(CH2) 1-4 - or -CH2-; in another embodiment, T1 is -C 0-10 Alkylene-O-; in another embodiment, T1 is -C 0-10 Alkylene-C(O)-, for example -C(O)-; in another embodiment, T1 is -C 0-10 Alkylene-OC(O)-, for example -OC(O)-; in another embodiment, T1 is -C 0-10 Alkylene-M-.

[0097] In one embodiment, M is In another embodiment, M is In another embodiment, M is In another embodiment, M is

[0098] A

[0099] In one embodiment, A is a peptide-based linker; in another embodiment, A is a peptide-based linker that can be cleaved in endosomes or lysosomes; in another embodiment, A comprises a polypeptide, such as a tripeptide, tetrapeptide, or pentapeptide, preferably a tetrapeptide.

[0100] In one embodiment, A is In another embodiment, A is In another embodiment, A is In another embodiment, A is In another embodiment, A is

[0101] L

[0102] In one embodiment, L is -NH-; in another embodiment, L is -O-; in another embodiment, L is -CH2-; in another embodiment, L is -C(O)-; in another embodiment, L is -C(O)O-.

[0103] T2

[0104] In one embodiment, T2 is a chemical bond; in another embodiment, T2 is -C 0-10 Alkylene-, for example -(CH2) z -; In another embodiment, T2 is -C 0-10 Alkylene-OC 0-10 Alkylene-, for example -(CH2) z -O-; In another embodiment, T2 is -C 0-10 Alkylene-C(O)-C 0-10 Alkylene-, for example -(CH2) z C(O)-; In another embodiment, T2 is -C 0-10 Alkylene-OC(O)-C 0-10 Alkylene-, for example -(CH2) z OC(O)-; In another embodiment, T2 is -C 0-10 Alkylene-C(O)OC 0-10 Alkylene-, for example -(CH2) z C(O)O-.

[0105] In one embodiment, T2 is unsubstituted; in another embodiment, T2 is substituted with 1 R; in another embodiment, T2 is substituted with 2 Rs; in another embodiment, T2 is substituted with 3 Rs; in another embodiment, T2 is substituted with 4 Rs; in another embodiment, T2 is substituted with 5 Rs; in another embodiment, T2 is substituted with 6 Rs; in another embodiment, T2 is substituted with 7 Rs; in another embodiment, T2 is substituted with 8 Rs; in another embodiment, T2 is substituted with more Rs.

[0106] In one embodiment, z is selected from 0, 1, 2, 3, 4, 5 or 6.

[0107] R

[0108] In one embodiment, R is H; in another embodiment, R is D; in another embodiment, R is halogen; in another embodiment, R is C 1-6 Alkyl; in another embodiment, R is C 1-4 Alkyl; in another embodiment, R is C 1-6 haloalkyl; in another embodiment, R is optionally deuterated, up to fully deuterated.

[0109] Rs

[0110] In one embodiment, Rs is H; in another embodiment, Rs is D; in another embodiment, Rs is halogen; in another embodiment, Rs is C 1-6 Alkyl; in another embodiment, Rs is C 1-6 haloalkyl; in another embodiment, R s Optionally deuterated, up to fully deuterated.

[0111] In a specific embodiment, Rs is H.

[0112] k

[0113] In one embodiment, k is selected from 0, 1, 2, 3, 4, 5 or 6.

[0114] Any technical solution or any combination thereof in any of the above specific embodiments can be combined with any technical solution or any combination thereof in other specific embodiments. For example, any technical solution or any combination thereof in A can be combined with P, R1, R2, L, T1, T2, R, R s 、R s The present invention is intended to include all of these technical solutions, which are not listed one by one due to space limitations.

[0115] The present invention also provides a vector comprising a nucleotide sequence encoding the siRNA of the present invention. The vector of the present invention is capable of amplifying or expressing the nucleotide sequence encoding the siRNA of the present invention linked thereto.

[0116] For example, siRNA targeting the PCSK9 gene can be expressed from a transcription unit inserted into a DNA or RNA vector. Expression can be short-lived (a few hours to a few weeks) or continuous (a few weeks to a few months or longer), depending on the specific construct and target tissue or cell type used. The coding nucleotides of the siRNA can be introduced into a linear construct, a circular plasmid or a viral vector. The nucleotides of the siRNA can be integrated into the cell genome for stable expression, or expressed in an extrachromosomal stable inheritance. In general, siRNA expression vectors are typically DNA plasmids or viral vectors.

[0117] Viral vector systems containing siRNA coding sequences include, but are not limited to: (a) adenoviral vectors; (b) retroviral vectors; (c) adeno-associated viral vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors; and (j) helper virus-dependent adenovirus or gut-free adenovirus.

[0118] The present invention also provides a cell containing the siRNA or vector of the present invention, wherein the siRNA or vector of the present invention can be transcribed in the cell.

[0119] The present invention specifically relates to the following technical solutions:

[0120] 1. An oligonucleotide comprising one or more compounds of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0121] in,

[0122] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0123] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0124] and R1 are not H at the same time;

[0125] P is a hydrophobic group, preferably C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30Alkynyl, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH2CH2- group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring; the C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R;

[0126] T1 is selected from chemical bonds, -C 0-10 Alkylene-, -C 0-10 Alkylene-O-, -C 0-10 Alkylene-C(O)-, -C 0-10 Alkylene-OC(O)- or -C 0-10 Alkylene-M-;

[0127] Where M is

[0128] A is a peptide-based linker that can be cleaved in endosomes or lysosomes;

[0129] L is selected from -NH-, -O-, -CH2-, -C(O)- or -C(O)O-;

[0130] T2 is a chemical bond, -C 0-10 Alkylene-, -C 0-10 Alkylene-OC 0-10 Alkylene-, -C 0-10 Alkylene-C(O)-C 0-10 Alkylene- or -C 0-10 Alkylene-C(O)OC 0-10 Alkylene-; said T2 is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R;

[0131] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0132] Rs is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0133] k is selected from 0, 1, 2, 3, 4, 5 or 6.

[0134] 2. The oligonucleotide of technical solution 1, wherein

[0135] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0136] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0137] and R1 are not H at the same time;

[0138] P is a hydrophobic group, preferably C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl; the C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R;

[0139] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0140] T1 is selected from -OC(O)-, -C(O)- or -(CH2) 1-6 -;

[0141] A is a peptide-based linker that can be cleaved in endosomes or lysosomes, comprising a polypeptide, such as a tripeptide, a tetrapeptide, or a pentapeptide, preferably a tetrapeptide;

[0142] L is selected from -NH-, -O- or -C(O)O-;

[0143] T2 is a chemical bond, -(CH2) z 、-(CH2) z -O-, -(CH2) z C(O)-、-(CH2) z OC(O)- or -(CH2) z C(O)O-;

[0144] z is selected from 0, 1, 2, 3, 4, 5 or 6, preferably 5;

[0145] Rs is selected from H, C 1-6 Alkyl or C 1-6 alkyl halide;

[0146] k is selected from 0, 1, 2, 3, 4, 5 or 6.

[0147] 3. The oligonucleotide of technical solution 1 or 2, wherein

[0148] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0149] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0150] and R1 are not H at the same time;

[0151] P is selected from C 10-22 Alkyl or C 10-22 alkenyl; the C 10-22 Alkyl and C 10-22 Alkenyl is optionally substituted with 1, 2 or 3 R;

[0152] R is selected from H, halogen or C 1-4 alkyl;

[0153] T1 is selected from -OC(O)-, -C(O)- or -(CH2) 1-4 -;

[0154] A is a peptide-based linker that can be cleaved in endosomes or lysosomes, comprising a polypeptide, such as a tripeptide, a tetrapeptide, or a pentapeptide, preferably a tetrapeptide;

[0155] Preferably, A is selected from

[0156] L is selected from -NH-, -O- or -C(O)O-;

[0157] T2 is selected from -(CH2) z -、-(CH2) z -C(O)- or -(CH2) z -O-;

[0158] z is selected from 0, 1, 2, 3, 4, 5 or 6, preferably 5;

[0159] Rs is selected from H or C 1-4 alkyl;

[0160] k is selected from 0, 1, 2, 3, 4, 5 or 6.

[0161] 4. The oligonucleotide according to any one of technical solutions 1 to 3, wherein

[0162] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0163] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0164] and R1 are not H at the same time;

[0165] P is -(CH2) 14-20 -CH3, for example -(CH2) 14 -CH3 or -(CH2) 16 -CH3;

[0166] T1 is selected from -C(O)- or -CH 2- ;

[0167] A is selected from

[0168] L is -NH-;

[0169] T2 is -(CH2) z C(O)-;

[0170] z is selected from 0, 1, 2, 3, 4 or 5, preferably 5;

[0171] Rs is H;

[0172] k is 6.

[0173] 5. The oligonucleotide of any one of technical solutions 1-4, wherein the compound of formula (I) is selected from the compound of formula (II), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0174] in,

[0175] Each variable is defined as in any one of technical solutions 1-4.

[0176] 6. The oligonucleotide according to any one of technical solutions 1 to 5, wherein the compound of formula (I) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0177] in, indicates the position of the phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0178] R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide.

[0179] 7. The oligonucleotide according to any one of technical solutions 1 to 6, which has 14 to 30 nucleotides.

[0180] 8. The oligonucleotide according to any one of technical solutions 1 to 7, comprising a compound of formula (I) according to any one of technical solutions 1 to 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at its 5′ end.

[0181] 9. The oligonucleotide according to any one of technical solutions 1 to 8, comprising a compound of formula (I) according to any one of technical solutions 1 to 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at its 3′ end.

[0182] 10. The oligonucleotide according to any one of technical solutions 1 to 9, comprising a compound of formula (I) according to any one of technical solutions 1 to 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at its 5' end and 3' end respectively.

[0183] 11. The oligonucleotide according to any one of technical solutions 1 to 10, which comprises one or more compounds of formula (I) according to any one of technical solutions 1 to 6, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, inside the oligonucleotide.

[0184] 12. An oligonucleotide comprising one, two or more delivery vehicles within the oligonucleotide, at the 5' end and / or at the 3' end, wherein the delivery vehicle is a peptide-based linker modified with a hydrophobic group;

[0185] Preferably, the peptide-based linking group modified with a hydrophobic group is selected from a compound of formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0186] in, Indicates the position of attachment to the oligonucleotide;

[0187] P is a hydrophobic group;

[0188] A is a peptide-based linker;

[0189] P is connected to A through T1 or directly;

[0190] Preferably, P, T1 and A are as defined in any one of technical solutions 1-4;

[0191] Preferably, A is selected from

[0192] More preferably, the compound of formula (III) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0193] in, Indicates the position of attachment to the oligonucleotide.

[0194] 13. The oligonucleotide according to any one of technical solutions 1 to 12, wherein the oligonucleotide is an ASO or siRNA, preferably used to inhibit genes expressed outside the liver, more preferably used to inhibit genes expressed in the eye.

[0195] 14. A compound of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0196] wherein R1 and R2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;

[0197] P, T1, A, L, T2, Rs and k are as defined in technical solutions 1-4.

[0198] 15. The compound of technical solution 14, wherein R1 and R2 are H.

[0199] 16. The compound of claim 14 or 15, wherein R1 and R2 are selected from reactive phosphorus groups, preferably phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, such as natural phosphates, thiophosphates, dithiophosphates, boranephosphates, boranephosphorothioates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiester, phosphotriester, thiophosphate diester, thiophosphate triester, diphosphate or triphosphate, preferably -P(OCH2CH2CN)(N(iPr)2).

[0200] 17. The compound of any one of technical solutions 14-16, wherein R1 and R2 are selected from protecting groups, preferably hydroxy protecting groups, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, preferably DMTr.

[0201] 18. The compound of any one of technical solutions 14-17, wherein the compound of formula (I') is selected from the compound of formula (II'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0202] in,

[0203] Each variable is defined as in any one of technical solutions 14-17.

[0204] 19. The compound of any one of technical solutions 14-18, wherein the compound of formula (I') or (II') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0205] 20. A double-stranded RNA comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand comprises one or more compounds of formula (I) as described in any one of technical solutions 1 to 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0206] Wherein, each variable is defined as in any one of technical solutions 1-4.

[0207] 21. The double-stranded RNA of technical solution 20, wherein the compound of formula (I) is selected from the compound of formula (II), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0208] Wherein, each variable is defined as in any one of technical solutions 1-4;

[0209] Preferably, the compound of formula (I) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0210] in, indicates the position of the phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0211] R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide.

[0212] 22. A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence sufficiently complementary to the sense strand and a target mRNA, wherein the sense strand and / or antisense strand comprises one, two or more delivery vectors internally, at the 5' end and / or at the 3' end, the delivery vector being a peptide-based linker modified with a hydrophobic group;

[0213] Preferably, the peptide-based linking group modified with a hydrophobic group is selected from a compound of formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0214] in, Indicates the connection position with double-stranded RNA;

[0215] P is a hydrophobic group;

[0216] A is a peptide-based linker;

[0217] P is connected to A through T1 or directly;

[0218] Preferably, P, T1 and A are as defined in any one of technical solutions 1-4;

[0219] Preferably, A is selected from

[0220] 23. The double-stranded RNA of technical solution 22, wherein the compound of formula (III) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0221] in, Indicates the ligation position with double-stranded RNA.

[0222] 24. The double-stranded RNA of any one of technical solutions 20-23, wherein the positive strand comprises at the 5' end a compound of formula (I) according to any one of technical solutions 1-7 or formula (III) according to technical solution 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0223] 25. The double-stranded RNA of any one of technical solutions 20-24, wherein the positive strand comprises at the 3' end a compound of formula (I) according to any one of technical solutions 1-7 or formula (III) according to technical solution 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0224] 26. The double-stranded RNA of any one of technical solutions 20-25, wherein the positive strand comprises a compound of formula (I) according to any one of technical solutions 1-7 or formula (III) according to technical solution 12 at the 5' end and the 3' end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0225] 27. The double-stranded RNA of any one of technical solutions 20-26, wherein the positive strand comprises one or more compounds of formula (II) described in any one of technical solutions 1-7, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, inside the oligonucleotide.

[0226] 28. The double-stranded RNA of any one of technical solutions 20-27, wherein the antisense strand comprises at the 5' end a compound of formula (I) according to any one of technical solutions 1-7 or formula (III) according to technical solution 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0227] 29. The double-stranded RNA of any one of technical solutions 20-28, wherein the antisense strand comprises at the 3' end a compound of formula (I) according to any one of technical solutions 1-7 or formula (III) according to technical solution 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0228] 30. The double-stranded RNA of any one of technical solutions 20-29, wherein the antisense strand comprises a compound of formula (I) according to any one of technical solutions 1-7 or formula (III) according to technical solution 12 at the 5' end and the 3' end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0229] 31. The double-stranded RNA of any one of technical solutions 20-30, wherein the antisense strand comprises one or more compounds of formula (I) according to any one of technical solutions 1-7 or formula (III) according to technical solution 12, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, inside the oligonucleotide.

[0230] 32. A cell containing the double-stranded RNA according to any one of technical solutions 20-31.

[0231] 33. A pharmaceutical composition comprising the double-stranded RNA according to any one of technical solutions 20-31, or the cell according to technical solution 32, and optionally a pharmaceutically acceptable carrier or excipient.

[0232] 34. A kit comprising the double-stranded RNA according to any one of technical solutions 20-31, or the cell according to technical solution 32.

[0233] List of specific compounds

[0234] The numbering and structure of the compounds of the present invention in oligonucleotides are as follows, wherein the sequence of 5'-->3' from the compound to connect.

[0235] Specifically, according to the order of 5'–>3', if the corresponding structure is located in the middle of the nucleic acid chain, means that it is connected to the 3' carbon or corresponding position of the previous nucleotide or nucleotide analog through a phosphate group, a phosphorothioate group or other linking group, It means that it is connected to the 5' carbon or corresponding position of the next nucleotide or nucleotide analog through a phosphate group, a thiophosphate group or other linking group; if the corresponding structure is located at the terminal position of the nucleic acid chain, or Correspondingly, it refers to the connection to the 3' or 5' end of the nucleic acid chain through a phosphate group, a phosphorothioate group or other linking group.

[0236] Synthesis Example

[0237] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention.

[0238] abbreviation

[0239] Example 1 Preparation of Compound BE7

[0240] 1. Preparation of Compound 1c

[0241] Stearic acid 1a (5.00 g, 17.6 mmol) was added to DCM (100 mL) at 25°C, followed by compound 1b (2.43 g, 21.1 mmol) and EDCI (4.04 g, 21.1 mmol). The reaction was stirred at 25°C for 18 hours. After completion of the reaction, the reaction mixture was concentrated in vacuo to obtain the crude product. The crude product was purified on a normal phase silica gel column (eluent: petroleum ether / ethyl acetate) to afford compound 1c (5.00 g, 74.56% yield) as a white solid.

[0242] 1 H NMR (400MHz, CDCl3) δ2.77-2.91(m,4H),2.55-2.67(m,2H),1.69-1.82(m,2H),1.36-1.45(m,2H),1.22-1.35(m,26H),0.83-0.94(m,3H).

[0243] 2. Preparation of Compound 1e

[0244] Compound 1d (500 mg, 1.18 mmol) was added to DMF (10.0 mL) at 25°C, followed by compound 1c (541 mg, 1.42 mmol) and TEA (0.657 mL, 4.723 mmol). The reaction was stirred at 25°C for 18 hours. After the reaction was complete, the reaction solution was slowly added dropwise to 100 mL of water, resulting in the precipitation of a white solid. Filtering afforded compound 1e (280 mg, 34.37% yield) as a white solid.

[0245] 1 H NMR(400MHz,DMSO-d6)δ8.57-8.73(m,1H),8.08-8.36(m,4H),7.07-7.34(m,5H),4.57-4.67(m,2H),4.43-4.52(m,1H),3.87-4.01(m,2H ),3.59-3.82(m,6H),3.03-3.10(m,1H),2.76-2.87(m,1H),2.03-2.20(m,2H),1.39-1.53(m,2H),1.08-1.32(m,29H),0.79-0.92(m,3H).

[0246] 3. Preparation of Compound 1g

[0247] Compound 1e (200 mg, 0.290 mmol) was dissolved in DMSO (5.00 mL) at 25°C, followed by the addition of DIEA (0.192 mL, 1.16 mmol), HATU (121 mg, 0.319 mmol), and compound 1f (196 mg, 0.348 mmol). The reaction mixture was stirred at 25°C for 18 hours. After completion of the reaction, the reaction mixture was dried to afford crude compound 1g (300 mg) as a yellow oil, which was used directly in the next step without further purification.

[0248] 4. Preparation of Compound BE7

[0249] Compound 1g (300 mg, 0.243 mmol) was dissolved in DMF (5.00 mL) at 25°C. TEA (0.203 mL, 1.46 mmol), compound 1h (146 mg, 1.46 mmol), and DMAP (11.9 mg, 0.097 mmol) were added sequentially. The reaction solution was stirred at 25°C for 18 hours. After completion of the reaction, the mixture was concentrated under reduced pressure to obtain the crude product, which was then purified by reverse-phase column chromatography to afford compound BE7 (120 mg, 37.00% yield) as a white solid.

[0250] 1 H NMR (400MHz, CD3OD) δ7.39-7.47(m,2H),7.16-7.35(m,12H),6.81-6.92(m,4H),3.33-4.83(m,26H),2.95-3 .25(m,8H),2.45-2.63(m,4H),2.20-2.40(m,4H),1.46-1.67(m,6H),1.20-1.39(m,34H),0.84-0.94(m,3H).

[0251] Example 2 Preparation of Compound BE8

[0252] Using palmitic acid 2a instead of stearic acid as the starting compound in Example 1, the same synthetic steps were used to obtain white solid compound BE8.

[0253] 1H NMR (400MHz, CD3OD) δ7.39-7.47(m,2H),7.16-7.35(m,12H),6.81-6.92(m,4H),3.33-4.83 (m,26H),2.95-3.25(m,8H),2.45-2.63(m,4H),2.20-2.40(m,4H),1.46-1.67(m,6H),1.20- 1.39(m,34H),0.84-0.94(m,3H).

[0254] Example 3 Preparation of Compound BE9

[0255] Compound 2d (1430 mg, 1.19 mmol) was dissolved in DMF (20.0 mL) at 25°C. TEA (0.138 mL, 0.995 mmol), compound 3a (1429 mg, 4.76 mmol), and DCI (140 mg, 1.19 mmol) were added sequentially. The reaction was stirred at 25°C for 18 hours. After completion of the reaction, the reaction mixture was separated and purified by reverse-phase column chromatography to afford compound BE9 (310 mg, 18.59% yield) as a white solid.

[0256] 1 H NMR(400MHz,DMSO-d6)δ8.51-8.66(m,1H),8.24-8.38(m,1H),7.98-8.19( m,3H),7.66-7.78(m,1H),7.14-7.40(m,14H),6.78-6.94(m,4H),4.43-4. 63(m,3H),3.42-4.01(m,25H),2.63-3.16(m,9H),2.08-2.28(m,4H),1.34 -1.51(m,6H),1.16-1.31(m,26H),1.02-1.15(m,12H),0.80-0.88(m,3H). 31 P NMR(162MHz,DMSO-d6)δ147.96,147.83.

[0257] Example 4. Synthesis of siRNA

[0258] The siRNA of the present invention was prepared using the solid phase phosphoramidite method known in the art. Specific methods can be found in, for example, PCT Publication Nos. WO2016081444 and WO2019105419 and are briefly described below.

[0259] 1. Preparation of siRNA with no ligand attached to the 3' end of the sense strand

[0260] 1.1 Synthesis of the positive sense chain (SS chain)

[0261] Through solid phase phosphoramidite synthesis, using blank CPG solid phase support as the starting cycle,

[0262] Nucleoside monomers are linked one by one in the 3'-5' direction according to the order of the positive strand nucleotides. Each linking step involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis scale is 5 μmol of oligonucleotide. The synthesis conditions are as follows:

[0263] The nucleoside monomer was provided in a 0.05 mol / L acetonitrile solution. The reaction conditions for each step were the same, i.e., the temperature was 25 degrees Celsius. Deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution. The coupling reaction was performed twice using a 0.25 mol / L 5-ethylthiotetrazolium (ETT)-acetonitrile solution as the activator. Capping was performed twice using 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v). Oxidation was performed twice using 0.05 mol / L iodine in tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v). Thiolysis was performed twice using 0.2 mol / L phenylacetyl disulfide (PADS) in acetonitrile / 3-methylpyridine (1 / 1, v / v).

[0264] 1.2 Synthesis of antisense strand (AS strand)

[0265] Through solid phase phosphoramidite synthesis, using a blank CPG solid phase support as the starting cycle, the nucleoside monomers are connected one by one from the 3'-5' direction according to the arrangement order of the antisense chain nucleotides.

[0266] The monomers all include four steps of deprotection, coupling, capping, oxidation or thiolation. The synthesis conditions of 5 μmol oligonucleotide of the antisense chain are the same as those of the sense chain.

[0267] 1.3 Oligonucleotide purification and annealing

[0268] 1.3.1 Ammonolysis

[0269] Add the synthesized solid phase carrier (sense chain or antisense chain) to a 5 mL centrifuge tube, add 3% diethylamine / ammonia water (v / v), react in a constant temperature water bath at 35 degrees (or 55 degrees) for 16 hours (or 8 hours), filter, wash the solid phase carrier three times with ethanol / water, each time 1 mL, and centrifuge the filtrate to purify the crude product.

[0270] 1.3.2 Purification

[0271] Purification and desalination methods are well known in the art. For example, a column packed with a strong anion filler can be used, and a sodium chloride-sodium hydroxide system can be used for elution and purification, and the product can be collected and stored in a tube. A gel-filled purification column can be used for desalination, and the elution system can be pure water.

[0272] 1.3.3 Annealing

[0273] According to the instructions, the sense chain (SS chain) and the antisense chain (AS chain) were mixed at a molar ratio of (SS chain / AS chain = 1 / 1.05), heated in a water bath to 70-95 degrees, maintained for 3-5 minutes, cooled naturally to room temperature, and the system was lyophilized to obtain the product.

[0274] The siRNA sequences used in the present invention are as follows:

[0275] In this article, the meanings of the abbreviations are as follows:

[0276] A, U, G and C represent the natural ribonucleotides adenine, uracil, guanine and cytosine, respectively.

[0277] 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.

[0278] i represents inosine ribonucleotide.

[0279] m indicates that the nucleotide adjacent to its left is a 2'-OCH3 modified nucleotide. For example, Am, Um, Gm, and Cm represent 2'-OCH3 modified A, U, G, and C.

[0280] f indicates that the nucleotide adjacent to its left is a 2'-F modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.

[0281] "s" indicates that the two adjacent nucleotides and / or delivery vectors are linked by phosphorothioate.

[0282] VP indicates that the adjacent nucleotide on the right is a vinyl phosphate-modified nucleotide.

[0283] IB or Ib represents an inverted abasic deoxyribonucleotide, which may include the following three structures depending on its position / linking method in siRNA.

[0284] Uhd and L322 represent nucleotide monomers of the following structure, which are well known in the art, and can be found in, for example, PCT Publication No. WO2021092371. The specific structure is as follows.

[0285] Uhd, in the order of 5'–>3' from the to connect.

[0286] L322.

[0287] The structures of LS1, LS3, LS11 and LS10 are as described above.

[0288] Example 5: Verification of Compound Efficacy in C57BL / 6 Mouse Model (Ocular Administration)

[0289] Drug administration and isolation of ocular tissue

[0290] C57BL / 6 mice (male, 6-8 weeks) were randomly divided into groups and administered a single dose of 7.5 μg per eye via bilateral intravitreal injection. The siRNA conjugate was administered as a 5 mg / mL solution (phosphate buffer solution as the solvent). Specifically, before the experiment, the siRNA conjugate was dissolved in phosphate buffer solution and the volume was adjusted to the desired solution concentration and volume. The administration volume of phosphate buffer solution and siRNA conjugate was 1.5 μL / eye.

[0291] On the 14th day after administration, the eyeballs were removed and separated into two parts: ① retina; ② retinal pigment epithelium (RPE) + choroid + sclera; the separated samples were immediately frozen in liquid nitrogen, then stored at -80°C and sent on dry ice for detection of mTTR mRNA.

[0292] RNA extraction and detection

[0293] Cell RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the protocol of the high-throughput tissue RNA extraction kit (Fanzhi Medical, FG0412); refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B) for reverse transcription; reference TaqMan TM Fluorescence quantitative PCR (ABI, QuantStudio3) was performed using 20 μL of Fast Advanced Master Mix (ABI, 4444965). The primers are shown in the table below.

[0294] Statistics

[0295] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate;

[0296] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0297] The target gene was mTTR, and the internal reference was mGAPDH.

[0298] Experimental results

[0299] Example 6: Verification of Compound Efficacy in SD Rat Model (Central Nervous System Administration)

[0300] Drug administration and isolation of ocular tissue

[0301] Male SD rats (6-8 weeks) were randomly divided into vehicle control groups (n=3) and compound groups (n=5). Compounds were prepared in sterile artificial cerebrospinal fluid (aCSF) at a concentration of 30 mg / mL. After gas anesthesia, the animals were administered the corresponding compound via intrathecal injection (L4-L6 level) at a dose of 0.9 mg / rat (30 μL / rat).

[0302] On the 14th day after administration, the animals were euthanized by CO2, and the following tissues were quickly isolated and removed: lumbar spinal cord, thoracic spinal cord, cervical spinal cord, cerebellum, brainstem, hippocampus, striatum, frontal cortex, temporal cortex, liver, and kidney. After removal, the samples were placed in 5-10 times the volume of RNAlater and stored at 2-8°C overnight. They were then transferred to -80°C for storage for subsequent detection of rSOD1 mRNA.

[0303] RNA extraction and detection

[0304] Tissue RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the protocol of the High-Throughput Tissue RNA Extraction Kit (Shanghai Fushen Biotechnology, FSF0035-TS); TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B) for reverse transcription; reference TaqMan TM Fluorescence quantitative PCR (ABI, QuantStudio3) was performed using 20 μL of Fast Advanced Master Mix (ABI, 4444965). The primers are shown in the table below.

[0305] Statistics

[0306] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate;

[0307] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0308] The target gene was rSOD1, and the internal reference was rGAPDH.

[0309] Experimental results

[0310] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

Claims

1. An oligonucleotide comprising one or more compounds of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; and R1 are not H at the same time; P is a hydrophobic group, preferably C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH2CH2- group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring; the C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R; T1 is selected from chemical bonds, -C 0-10 Alkylene-, -C 0-10 Alkylene-O-, -C 0-10 Alkylene-C(O)-, -C 0-10 Alkylene-OC(O)- or -C 0-10 Alkylene-M-; Where M is A is a peptide-based linker that can be cleaved in an endosome or a lysosome; L is selected from -NH-, -O-, -CH2-, -C(O)- or -C(O)O-; T2 is selected from chemical bonds, -C 0-10 Alkylene-, -C 0-10 Alkylene-OC 0-10 Alkylene-, -C 0-10 Alkylene-C(O)-C 0-10 Alkylene-, -C 0-10 Alkylene-OC(O)-C 0-10 Alkylene- or -C 0-10 Alkylene-C(O)OC 0-10 Alkylene-; said T2 is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R; R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated; Rs is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated; k is selected from 0, 1, 2, 3, 4, 5 or 6.

2. The oligonucleotide of claim 1, wherein represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; and R1 are not H at the same time; P is a hydrophobic group, preferably C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl; the C 8-30 Alkyl, C 8-30 Alkenyl or C 8-30 Alkynyl is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R; R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 Haloalkyl; T1 is selected from -OC(O)-, -C(O)- or -(CH2) 1-6 -; A is a peptide-based linker that can be cleaved in an inclusion body or a lysosome, wherein the peptide-based linker comprises a polypeptide, such as a tripeptide, a tetrapeptide or a pentapeptide, preferably a tetrapeptide; L is selected from -NH-, -O- or -C(O)O-; T2 is selected from a chemical bond, -(CH2) z 、-(CH2) z -O-, -(CH2) z C(O)-、-(CH2) z OC(O)- or -(CH2) z C(O)O-; z is selected from 0, 1, 2, 3, 4, 5 or 6, preferably 5; Rs is selected from H, C 1-6 Alkyl or C 1-6 Haloalkyl; k is selected from 0, 1, 2, 3, 4, 5 or 6.

3. The oligonucleotide according to claim 1 or 2, wherein represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; and R1 are not H at the same time; P is selected from C 10-22 Alkyl or C 10-22 alkenyl; said C 10-22 Alkyl and C 10-22 Alkenyl is optionally substituted with 1, 2 or 3 R; R is selected from H, halogen or C 1-4 alkyl; T1 is selected from -OC(O)-, -C(O)- or -(CH2) 1-4 -; A is a peptide-based linker that can be cleaved in an inclusion body or a lysosome, wherein the peptide-based linker comprises a polypeptide, such as a tripeptide, a tetrapeptide or a pentapeptide, preferably a tetrapeptide; Preferably, A is selected from L is selected from -NH-, -O- or -C(O)O-; T2 is selected from -(CH2) z -、-(CH2) z -C(O)- or -(CH2) z -O-; z is selected from 0, 1, 2, 3, 4, 5 or 6, preferably 5; Rs is selected from H or C 1-4 alkyl; k is selected from 0, 1, 2, 3, 4, 5 or 6.

4. The oligonucleotide according to any one of claims 1 to 3, wherein represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; and R1 are not H at the same time; P is -(CH2) 14-20 -CH3, for example -(CH2) 14 -CH3 or -(CH2) 16 -CH3; T1 is selected from -C(O)- or -CH 2- ; A is selected from L is -NH-; T2 is -(CH2) z C(O)-; z is selected from 0, 1, 2, 3, 4 or 5, preferably 5; Rs is H; k is 6.

5. The oligonucleotide according to any one of claims 1 to 4, wherein The compound of formula (I) is selected from the compound of formula (II), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, The variables are as defined in any one of claims 1-4.

6. The oligonucleotide according to any one of claims 1 to 5, wherein The compound of formula (I) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: in, indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide.

7. The oligonucleotide of any one of claims 1 to 6, having 14 to 30 nucleotides.

8. An oligonucleotide according to any one of claims 1 to 7, comprising at the 5' end a compound of formula (I) according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

9. The oligonucleotide according to any one of claims 1 to 8, comprising at the 3' end a compound of formula (I) according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

10. The oligonucleotide according to any one of claims 1 to 9, which comprises one compound of formula (I) according to any one of claims 1 to 6 at each of the 5' end and the 3' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

11. An oligonucleotide according to any one of claims 1 to 10, comprising one or more compounds of formula (I) according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, within the oligonucleotide.

12. An oligonucleotide comprising one, two or more delivery vectors within the oligonucleotide, at the 5' end and / or at the 3' end, wherein the delivery vector is a peptide-based linker modified with a hydrophobic group; Preferably, the peptide-based linking group modified by a hydrophobic group is selected from a compound of formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, indicates the position of attachment to the oligonucleotide; P is a hydrophobic group; A is a peptide-based linker; P is connected to A through T1 or directly; Preferably, P, T1 and A are as defined in any one of claims 1 to 4; Preferably, A is selected from More preferably, the compound of formula (III) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: in, Indicates the position of attachment to the oligonucleotide.

13. The oligonucleotide according to any one of claims 1 to 12, which is an ASO or siRNA, preferably for inhibiting a gene expressed outside the liver, more preferably for inhibiting a gene expressed in the eye.

14. A compound of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, R1 and R2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support; P, T1, A, L, T2, Rs and k are as defined in claims 1-4.

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

16. The compound of claim 14 or 15, wherein R1 and R2 are selected from reactive phosphorus groups, preferably phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, such as natural phosphates, phosphorothioates, phosphorodithioates, boranophosphates, boranophosphorothioates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, phosphorothioate diesters, phosphorothioate triesters, diphosphates or triphosphates, preferably -P(OCH2CH2CN)(N(iPr)2).

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

18. The compound of any one of claims 14 to 17, wherein the compound of formula (I') is selected from the compound of formula (II'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, The variables are as defined in any one of claims 14-17.

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

20. A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand comprises a sequence that is sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand comprises one or more compounds of formula (I) as described in any one of claims 1 to 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, The variables are as defined in any one of claims 1-4.

21. The double-stranded RNA of claim 20, wherein the compound of formula (I) is selected from the compound of formula (II), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, Each variable is as defined in any one of claims 1 to 4; Preferably, the compound of formula (I) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: in, indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide.

22. A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand comprises one, two or more delivery vectors at the interior, 5' end and / or 3' end, the delivery vector being a peptide-based linker modified with a hydrophobic group; Preferably, the peptide-based linking group modified by a hydrophobic group is selected from a compound of formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, Indicates the connection position with double-stranded RNA; P is a hydrophobic group; A is a peptide-based linker; P is connected to A through T1 or directly; Preferably, P, T1 and A are as defined in any one of claims 1 to 4; Preferably, A is selected from 23. The double-stranded RNA of claim 22, wherein the compound of formula (III) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: in, Indicates the ligation position with double-stranded RNA.

24. The double-stranded RNA of any one of claims 20 to 23, wherein the sense strand comprises a compound of formula (I) according to any one of claims 1 to 7 or formula (III) according to claim 12 at the 5' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

25. The double-stranded RNA of any one of claims 20 to 24, wherein the sense strand comprises a compound of formula (I) according to any one of claims 1 to 7 or formula (III) according to claim 12 at the 3' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

26. The double-stranded RNA of any one of claims 20 to 25, wherein the sense strand comprises a compound of formula (I) according to any one of claims 1 to 7 or formula (III) according to claim 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 5' end and the 3' end, respectively.

27. The double-stranded RNA of any one of claims 20 to 26, wherein the sense strand comprises one or more compounds of formula (II) according to any one of claims 1 to 7, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, within the oligonucleotide.

28. The double-stranded RNA of any one of claims 20 to 27, wherein the antisense strand comprises a compound of formula (I) according to any one of claims 1 to 7 or formula (III) according to claim 12 at the 5' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

29. The double-stranded RNA of any one of claims 20 to 28, wherein the antisense strand comprises a compound of formula (I) according to any one of claims 1 to 7 or formula (III) according to claim 12 at the 3' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

30. The double-stranded RNA of any one of claims 20 to 29, wherein the antisense strand comprises a compound of formula (I) according to any one of claims 1 to 7 or formula (III) according to claim 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 5' end and the 3' end, respectively.

31. The double-stranded RNA of any one of claims 20 to 30, wherein the antisense strand comprises one or more compounds of formula (I) according to any one of claims 1 to 7 or formula (III) according to claim 12, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, within the oligonucleotide.

32. A cell comprising the double-stranded RNA of any one of claims 20 to 31.

33. A pharmaceutical composition comprising the double-stranded RNA of any one of claims 20-31, or the cell of claim 32, and optionally a pharmaceutically acceptable carrier or excipient.

34. A kit comprising the double-stranded RNA of any one of claims 20 to 31, or the cell of claim 32.

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