Ligands for delivery of siRNA to ocular and central nervous system

Abstract

The present invention provides a compound of formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. The compound of formula (I) is contained inside the nucleotide, or at the 5 '-and / or 3'-terminus, for increasing the ability of the double-stranded RNA to traverse the blood-brain barrier. The invention also relates to oligonucleotides, double-stranded RNAs, vectors, cells, pharmaceutical compositions and kits comprising the compounds of formula (I). # imgabs0 #

Description

Ligands for delivering SIRNA to the eye and central nervous system

[0001] This application claims priority to Chinese application No. 202211347517.9 filed on October 31, 2022, 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 hydrophobic group having the ability to enhance the delivery of double-stranded RNA across the blood-brain barrier and / or to the eye, such as the R group in formula (I), and a compound of formula (I) in which the hydrophobic group is linked to a nucleotide, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. Background Art

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

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

[0006] Summary of the Invention

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

[0008] wherein each group is as defined below.

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

[0010] wherein each group is as defined below.

[0011] 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 or more compounds of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0012] wherein each group is as defined below.

[0013] In another aspect, the hydrophobic group provided by the present invention (R group in formula (I)) can be linked to the siRNA through an existing linker structure, such as a biodegradable linker structure.

[0014] In another aspect, the present invention provides a vector comprising a nucleotide sequence encoding the aforementioned double-stranded RNA.

[0015] In another aspect, the present invention provides a cell containing the aforementioned double-stranded RNA or the aforementioned vector.

[0016] 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.

[0017] In another aspect, the present invention provides a kit comprising the aforementioned double-stranded RNA, the aforementioned vector, or the aforementioned cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG1 shows the reduction in the expression of SOD1 in the cervical spinal cord, thoracic spinal cord, cerebellum, brainstem, hippocampus and frontal cortex after puncture and injection of the siRNA of the present invention into SD rats through the intervertebral foramen.

[0019] FIG2 shows the reduction in TTR gene expression in the eyes of C57BL / 6 mice after bilateral intravitreal injection of the siRNA of the present invention.

[0020] Detailed Description of the Invention

[0021] definition

[0022] Chemical definition

[0023] Definitions of specific functional groups and chemical terms are described in more detail below.

[0024] 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.

[0025] “C 1-30 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 30 carbon atoms. In some embodiments, C 5-25 Alkyl, C 10-20 Alkyl, C 1-20 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).

[0026] “C 2-30 "Alkenyl" refers to a straight or branched chain hydrocarbon group having 2 to 30 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 10-25 Alkenyl, C 2-10 Alkenyl, C 2-6 Alkenyl and C 2-4 Alkenyl is preferred. 2-6 Examples of alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. The term "C 2-6"Alkenyl" also includes heteroalkenyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). An alkenyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0027] “C 2-30 "Alkynyl" refers to a straight or branched chain hydrocarbon group having 2 to 30 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 10-25 Alkynyl, C 2-10 Alkynyl, C 2-6 Alkynyl and C 2-4 Alkynyl is preferred. 2-6 Examples of alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. The term "C 2-6 "Alkynyl" also includes heteroalkynyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups can be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0028] “C 1-10 Alkylene", "C 2-10 Alkenylene" and "C 2-10 "Alkynylidene" refers to the group with the C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 In some embodiments, C 2-8 Alkylene, C 3-7 Alkylene, C 4-6 Alkylene, C 1-4 Alkylene, C 2-4 Alkylene and C 1-3Alkylene 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.

[0029] “C 0-10 "Alkylene" refers to the chemical bond and the above-mentioned "C 1-10 "Alkylene".

[0030] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0031] Therefore, “C 1-20 Halogenated alkyl, "C 1-6 Haloalkyl" and "C 1-4 "Haloalkyl" refers to the above-mentioned "C 1-20 Alkyl", "C 1-6 Alkyl" and "C 1-4 Alkyl", which is substituted by 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.

[0032] 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 Kow 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.

[0033] Alkyl, alkenyl, alkynyl, etc., as defined herein, are optionally substituted groups.

[0034] 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)NRbb 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(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;

[0035] 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;

[0036] 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;

[0037] 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;

[0038] 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;

[0039] R dd Each of the is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee 、-ON(R ff )2、-N(R ff )2,、-N(R ff )3 + X - 、-N(OR ee )R ff 、-SH、-SR ee 、-SSR ee 、-C(=O)R ee 、-CO2H、-CO2R ee 、-OC(=O)R ee 、-OCO2R ee 、-C(=O)N(R ff )2、-OC(=O)N(R ff )2、-NR ff C(=O)R ee 、-NR ff CO2R ee 、-NR ff C(=O)N(R ff )2、-C(=NR ff )OR ee 、-OC(=NR ff )R ee 、-OC(=NR ff )OR ee 、-C(=NR ff )N(R ff )2、-OC(=NR ff )N(R ff )2、-NR ff C(=NR ff )N(R ff )2、-NR ff SO2R ee 、-SO2N(R ff )2, -SO2R ee、-SO2OR ee 、-OSO2R ee 、-S(=O)R ee 、-Si(R ee )3、-OSi(R ee )3、-C(=S)N(R ff )2, -C(=O)SR ee 、-C(=S)SR ee 、-SC(=S)SR ee 、-P(=O)2R ee 、-P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR ee )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg Group substitution, or two geminal R dd Substituents may combine to form =O or =S;

[0040] 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;

[0041] R ff Each of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R ff The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R gg group substitution;

[0042] 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 1-6 Alkyl, -C(=NH)N(C 1-6 Alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 Alkyl)2, -OC(NH)NH(C 1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6 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(C1-6 alkyl)2、C(=S)NH(C 1-6 alkyl), C(=S)NH2, -C(=O)S(C 1-6 alkyl), -C(=S)SC 1-6 Alkyl, -SC(=S)SC 1-6 Alkyl, -P(=O)2(C 1-6 alkyl), -P(=O)(C 1- 6 alkyl) 2, -OP(=O)(C 1-6 alkyl)2, -OP(=O)(OC 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C 10 Aryl, C3-C7 heterocyclic, C5-C 10 heteroaryl; or two geminal R gg Substituents may combine to form =O or =S; wherein X - For the counter ion.

[0043] Exemplary substituents on nitrogen atoms include, but are not limited to, hydrogen, -OH, -OR aa 、-N(R cc )2, -CN, -C(=O)R aa 、-C(=O)N(R cc )2, -CO2R aa 、-SO2R aa 、-C(=NR bb )R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc )2, -SO2R cc 、-SO2OR cc 、-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 ccThe 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.

[0044] Other definitions

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] "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.

[0051] "shRNA" stands for short hairpin RNA. shRNA consists of two short inverted repeats. When cloned into an shRNA expression vector, the two short inverted repeats are separated by a stem-loop sequence, forming a hairpin structure controlled by a Pol III promoter. Five to six Ts are then attached to serve as a transcriptional terminator for RNA polymerase III.

[0052] 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.

[0053] "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.

[0054] 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.

[0055] " 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.

[0056] "Ligand moiety" refers to a chemical moiety conjugated to an siRNA that is capable of altering the distribution, targeting, or lifetime of the siRNA. In preferred embodiments, such a ligand provides enhanced affinity for a selected target (e.g., a molecule, a cell or cell type, a compartment (e.g., a cell or organ compartment, a tissue, an organ, or a region of the body) compared to, for example, an siRNA in the absence of such a ligand.

[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), 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 -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 a compound of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0068] in,

[0069] L1 and L2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;

[0070] R s Selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0071] m = 0, 1, 2, 3, 4, 5 or 6;

[0072] R is -C(O)-C 0-10 Alkylene-L-R1, -C(O)-C 2-10 Alkenylene-L-R1 or -C(O)-C 2-10 Alkynylidene-L-R1;

[0073] L is a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -NHC(O)-CH(OR1)CH2O-, -C(O)NH-CH(OR1)CH2O-, -OC(O)-CH(OR1)CH2O- , -C(O)O-CH(OR1)CH2O-, -NHC(O)-CH(R1)-, -C(O)NH-CH(R1)-, -OC(O)-CH(R1)-, -C(O) O-CH(R1)-, -CH(OR1)CH2O-, -O-CH(R1)CH2O-, -O-CH2CH(R1)O-, -O-CH(CH(OH)CH2OH)- , -O-CH(CH(NH2)CH2OH)-, -O-CH(CH2OH)CH(OH)-, -NH-CH(CH2OH)CH(OH)-, -O-CH2CH(OH)CH(OH)-, -O-CH2CH(NH2)CH(OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)-, -NHC(O)-CH2 -O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-O-CH2CH(OH)CH(OH)-, or -NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-;

[0074] R1 is independently C 1-30 Alkyl, C 2-30 Alkenyl or C 2-30 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;

[0075] Among them C 0-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 1-30 Alkyl, C 2-30 Alkenyl and C 2-30 The hydrogen atoms in the alkynyl group may be optionally replaced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more halogen, C 1-6 Alkyl or C 1-6The group is replaced by a haloalkyl group, which is optionally deuterated, up to full deuteration.

[0076] The present invention also relates to oligonucleotides comprising one or more compounds of formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0077] in,

[0078] represents H or a hydroxyl protecting group, or represents the position of attachment to the adjacent nucleotide;

[0079] L2 represents H or a solid support, or represents the position where it is connected to the adjacent nucleotide;

[0080] R s , m and R are as defined above.

[0081] The present invention also 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'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0082] in,

[0083] Indicates the position of attachment to the adjacent nucleotide;

[0084] L2 represents H or a hydroxyl protecting group, or represents the position of connection with the adjacent nucleotide;

[0085] R s , m and R are as defined above.

[0086] L1, L2 and

[0087] In one embodiment, L1 is H; in another embodiment, L1 is a reactive phosphorus group; in another embodiment, L1 is a hydroxy protecting group; in another embodiment, L1 is a solid support.

[0088] In one embodiment, L2 is H; in another embodiment, L2 is a reactive phosphorus group; in another embodiment, L2 is a hydroxyl protecting group; in another embodiment, L2 is a solid support; in another embodiment, L2 represents the position of attachment to the adjacent nucleotide.

[0089] In a more specific embodiment, one of L1 and L2 is -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; in another more specific embodiment, one of L1 and L2 is -C(O)CH2CH2C(O)OH.

[0090] In one embodiment, represents H; in another embodiment, represents a hydroxyl protecting group; in another embodiment, Indicates the position of attachment to the adjacent nucleotide.

[0091] R s

[0092] In one embodiment, R s is H; in another embodiment, R s is D; in another embodiment, R s is halogen; in another embodiment, R s C 1-6 Alkyl; in another embodiment, R s C 1-6 haloalkyl; in another embodiment, R s C 1-6 Alkyl or C 1-6 When alkyl is halogenated, the group is optionally deuterated until fully deuterated;

[0093] m

[0094] In one embodiment, m=0; in another embodiment, m=1; in another embodiment, m=2; in another embodiment, m=3; in another embodiment, m=4; in another embodiment, m=5; in another embodiment, m=6.

[0095] R

[0096] In one embodiment, R is -C(O)-C 0-10 Alkylene-L-R1; In another embodiment, R is -C(O)-C 2-10 Alkenylene-L-R1; in another embodiment, R is -C(O)-C 2-10 Alkynylidene-L-R1.

[0097] In a more specific embodiment, R is -C(O)-L-R1; in another more specific embodiment, R is -C(O)-C 2-8 Alkylene-L-R1; In another more specific embodiment, R is -C(O)-C 3-7Alkylene-L-R1; In another more specific embodiment, R is -C(O)-C 4-6 Alkylene-L-R1; In another more specific embodiment, R is -C(O)-C 1-3 Alkylene-L-R1.

[0098] L

[0099] In one embodiment, L is a chemical bond; in another embodiment, L is -NHC(O)-; in another embodiment, L is -C(O)NH-; in another embodiment, L is -OC(O)-; in another embodiment, L is -C(O)O-; in another embodiment, L is -SS-; in another embodiment, L is -NHC(O)O-; in another embodiment, L is -NHC(O)NH-; in another embodiment, L is -OC(O)O-; in another embodiment, L is -OC(O)NH-; in another embodiment, L is -NHC(O)-CH(OR1)CH2O-; in another embodiment , L is -C(O)NH-CH(OR1)CH2O-; in another embodiment, L is -OC(O)-CH(OR1)CH2O-; in another embodiment, L is -C(O)O-CH(OR1)CH2O-; in another embodiment, L is -NHC(O)-CH(R1)-; in another embodiment, L is -C(O)NH-CH(R1)-; in another embodiment, L is -OC(O)-CH(R1)-; in another embodiment, L is -C(O)O-CH(R1)-; in another embodiment, L is -CH(OR1)CH2O-; in another embodiment, L is -O-CH(R1) CHO-; in another embodiment, L is -O-CHCH(R1)O-; in another embodiment, L is -O-CH(CH(OH)CH2OH)-; in another embodiment, L is -O-CH(CH(NH2)CH2OH)-; in another embodiment, L is -O-CH(CH2OH)CH(OH)-; in another embodiment, L is -NH-CH(CH2OH)CH(OH)-; in another embodiment, L is -O-CHCH(OH)CH(OH)-; in another embodiment, L is -O-CHCH(NH2)CH(OH)-; in another embodiment, L is -NHC (O)-CH2-O-CH(CH(OH)CH2OH)-; in another embodiment, L is -NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-; in another embodiment, L is -NHC(O)-CH2-O-CH(CH2OH)CH(OH)-; in another embodiment, L is -NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-; in another embodiment, L is -NHC(O)-CH2-O-CH2CH(OH)CH(OH)-; in another embodiment, L is -NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-.

[0100] In more specific embodiments, L is a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -O-CH(CH(OH)CH2OH)-, -O-CH(CH(NH2)CH2OH)-, -O-CH(CH2OH)CH(OH)-, -NH-CH(CH2OH)CH(OH)-, -O-CH2CH(OH)CH(OH)-, -O-CH2CH (NH2)CH(OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)-, -NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH2OH)CH(O H)-, -NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-O-CH2CH(OH)CH(OH)-, or -NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-; in another update In a specific embodiment, L is a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -O-CH(CH(OH)CH2OH)-, -O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)-, or -NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-; in another more specific embodiment, In one embodiment, L is a chemical bond, -NHC(O)-, -SS-, -NHC(O)O-, -O-CH(CH(OH)CH2OH)-, -O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)-, or -NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-; in another more specific embodiment, L is a chemical bond, -NHC(O)-, -SS-, or -NHC(O)O-; in another more specific embodiment, L is -NHC(O)-.

[0101] In a more specific embodiment, L is -NHC(O)-CH(OR1)CHO-, -C(O)NH-CH(OR1)CHO-, -OC(O)-CH(OR1)CHO-, -C(O)O-CH(OR1)CHO-, -NHC(O)-CH(R1)-, -C(O)NH-CH(R1)-, -OC(O)-CH(R1)-, -C(O)O-CH(R1)-, -CH(OR1)CHO-, -O-CH(R1)CHO-, or -O-CHCH(R1)O-; in another more specific embodiment, L is -NHC(O)-CH(OR1)CHO-, -NHC(O)-CH(R1)-, or -CH(OR1)CHO-; in another more specific embodiment, L is -NHC(O)-CH(OR1)CHO-.

[0102] R1

[0103] In one embodiment, R1 is C 1-30 Alkyl; in another embodiment, R1 is C 2-30 In another embodiment, R1 is C 2-30 In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-adjacent carbon atoms in the group in R1 can be replaced by heteroatoms selected from O, S, and N; in another embodiment, the -CH2CH2- group in R1 can be replaced by -OC(O)-, -C(O)O-, -NHC(O)-, or -C(O)NH-; in another embodiment, the substituents on one or more carbon atoms in R1 can be connected to form a saturated or unsaturated ring.

[0104] In more specific embodiments, R1 is independently C 1-30 Alkyl or C 2-30 wherein non-adjacent 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH2CH2- group may be replaced by -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; in another more specific embodiment, R1 is independently C 5-25 Alkyl, C containing 1, 2, 3, 4, 5 or 6 double bonds 10-25 alkenyl, C in which 1, 2, 3, 4 or 5 carbon atoms are replaced by N heteroatoms and / or 1, 2 or 3 -CH2CH2- groups are replaced by -C(O)NH- 5-25 Alkyl, or one or more substituents on the carbon atom connected to form the C 5-25 Alkyl; in another more specific embodiment, R1 is selected from the following groups:

[0105] C6 alkyl, C8 alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 21 alkyl,

[0106] 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 L1 can be combined with L2, R s The present invention is intended to include combinations of all these technical solutions, which are not listed one by one due to space limitations.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] The present invention specifically relates to the following technical solutions:

[0112] Technical Solution 1. A compound of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0113] in,

[0114] L1 and L2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;

[0115] R s Selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0116] m = 0, 1, 2, 3, 4, 5 or 6;

[0117] R is -C(O)-C 0-10 Alkylene-L-R1, -C(O)-C 2-10 Alkenylene-L-R1 or -C(O)-C 2-10 Alkynylidene-L-R1;

[0118] L is a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -NHC(O)-CH(OR1)CH2O-, -C(O)NH-CH(OR1)CH2O-, -OC(O)-CH(OR1)CH2O- , -C(O)O-CH(OR1)CH2O-, -NHC(O)-CH(R1)-, -C(O)NH-CH(R1)-, -OC(O)-CH(R1)-, -C(O) O-CH(R1)-, -CH(OR1)CH2O-, -O-CH(R1)CH2O-, -O-CH2CH(R1)O-, -O-CH(CH(OH)CH2OH)- , -O-CH(CH(NH2)CH2OH)-, -O-CH(CH2OH)CH(OH)-, -NH-CH(CH2OH)CH(OH)-, -O-CH2CH(OH)CH(OH)-, -O-CH2CH(NH2)CH(OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)-, -NHC(O)-CH2 -O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-O-CH2CH(OH)CH(OH)-, or -NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-;

[0119] R1 is independently C 1-30 Alkyl, C 2-30 Alkenyl or C 2-30 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;

[0120] Among them C 0-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 1-30 Alkyl, C 2-30 Alkenyl and C 2-30 The hydrogen atoms in the alkynyl group may be optionally replaced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more halogen, C 1-6 Alkyl or C 1-6The group is replaced by a haloalkyl group, which is optionally deuterated, up to full deuteration.

[0121] Technical Solution 2. The compound of formula (I) of Technical Solution 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R is -C(O)-C 0-10 Alkylene-L-R1, preferably -C(O)-L-R1, preferably -C(O)-C 2-8 Alkylene-L-R1, more preferably -C(O)-C 3-7 Alkylene-L-R1, more preferably -C(O)-C 4-6 Alkylene-L-R1, more preferably -C(O)-C 1-3 Alkylene-L-R1.

[0122] Technical Solution 3. The compound of formula (I) of Technical Solution 1 or 2, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L is a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -O-CH(CH(OH)CH2OH)-, -O-CH(CH(NH2)CH2OH)-, -O-CH(CH2OH)CH(OH)-, -NH-CH(C H2OH)CH(OH)-, -O-CH2CH(OH)CH(OH)-, -O-CH2CH(NH2)CH(OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)-, -NHC(O)-CH2-O-CH (CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-O-CH2CH(OH) CH(OH)- or -NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-, preferably a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -O-CH(CH(OH)CH2OH)-, -O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)- or -NHC(O) )-CH2-O-CH(CH(NH2)CH2OH)-, preferably a chemical bond, -NHC(O)-, -SS-, -NHC(O)O-, -O-CH(CH(OH)CH2OH)-, -O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)- or -NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-, more preferably a chemical bond, -NHC(O)-, -SS- or -NHC(O)O-, more preferably -NHC(O)-.

[0123] Technical Solution 4. The compound of formula (I) of Technical Solution 1 or 2, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L is -NHC(O)-CH(OR1)CH2O-, -C(O)NH-CH(OR1)CH2O-, -OC(O)-CH(OR1)CH2O-, -C(O)O-CH(OR1)CH2O-, -NHC(O)-CH(R1)-, -C(O)NH-C H(R1)-, -OC(O)-CH(R1)-, -C(O)O-CH(R1)-, -CH(OR1)CH2O-, -O-CH(R1)CH2O-, -O-CH2CH(R1)O-, preferably -NHC(O)-CH(OR1)CH2O-, -NHC(O)-CH(R1)- or -CH(OR1)CH2O-, more preferably -NHC(O)-CH(OR1)CH2O-.

[0124] Technical Solution 5. The compound of formula (I) according to any one of Technical Solutions 1-4, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R1 is independently C 1-30 Alkyl or C 2-30 alkenyl, wherein non-adjacent 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH2CH2- group may be replaced by -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; preferably, R1 is independently C 5-25 Alkyl, C containing 1, 2, 3, 4, 5 or 6 double bonds 10-25 alkenyl, C in which 1, 2, 3, 4 or 5 carbon atoms are replaced by N heteroatoms and / or 1, 2 or 3 -CH2CH2- groups are replaced by -C(O)NH- 5-25 Alkyl, or one or more substituents on the carbon atom connected to form the C 5-25 Alkyl; preferably, R1 is selected from the following groups:

[0125] C6 alkyl, C8 alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 21 alkyl,

[0126] Technical Solution 6. The compound of formula (I) according to any one of Technical Solutions 1-5, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L1 and L2 are H.

[0127] Technical Solution 7. The compound of formula (I) according to any one of Technical Solutions 1-5, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein one of L1 and L2 is a reactive phosphorus group, preferably a phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate or phosphate mimetic, such as a natural phosphate, a thiophosphate, a dithiophosphate, a borane phosphate, a borane thiophosphate, a phosphonate, a halogen-substituted phosphonate and a phosphate, an aminophosphate, a phosphate diester, a phosphate triester, a thiophosphate diester, a thiophosphate triester, a diphosphate or a triphosphate, preferably -P(OCH2CH2CN)(N(iPr)2).

[0128] Technical Solution 8. The compound of formula (I) according to any one of Technical Solutions 1 to 5, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L1 and L2 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 -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, more preferably -C(O)CH2CH2C(O)OH.

[0129] Technical Solution 9. The compound of formula (I) according to any one of Technical Solutions 1 to 8, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, is selected from the following general formula:

[0130] Wherein each group is as defined in technical solutions 1-8.

[0131] Technical Solution 10. The compound of any one of Technical Solutions 1-9, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the compound is selected from the following:

[0132] Technical solution 11. An oligonucleotide comprising one or more compounds of formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0133] in,

[0134] represents H or a hydroxyl protecting group, or represents the position of attachment to the adjacent nucleotide;

[0135] L2 represents H or a solid support, or represents the position where it is connected to the adjacent nucleotide;

[0136] R s Selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0137] m = 0, 1, 2, 3, 4, 5 or 6;

[0138] R is a hydrophobic group;

[0139] Preferably,

[0140] represents H or a hydroxyl protecting group, or represents the position of attachment to the adjacent nucleotide;

[0141] L2 represents H or a solid support, or represents the position where it is connected to the adjacent nucleotide;

[0142] R s , m and R are as defined in any one of technical solutions 1-5.

[0143] Technical solution 12. The oligonucleotide of technical solution 11, wherein the compound of formula (I') is selected from the following compounds of the general formula, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0144] in,

[0145] represents H or a hydroxyl protecting group, or represents the position of attachment to the adjacent nucleotide;

[0146] L2 represents H or a solid support, or represents the position where it is connected to the adjacent nucleotide;

[0147] The other groups are as defined in technical solutions 1-5.

[0148] Technical solution 13. The oligonucleotide of technical solution 11, wherein the compound of formula (I') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, wherein the compound is selected from the following:

[0149] in One of them represents H or a hydroxyl protecting group, or represents the position of attachment to the adjacent nucleotide, and the other Represents H or a solid support, or indicates the position where it is linked to the adjacent nucleotide.

[0150] Technical Solution 14. The oligonucleotide according to any one of Technical Solutions 11-13, which has 14 to 30 nucleotides.

[0151] Technical Solution 15. The oligonucleotide according to any one of Technical Solutions 11-14, comprising a compound of formula (I') according to any one of Technical Solutions 11-13, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 5' end.

[0152] Technical Solution 16. The oligonucleotide according to any one of Technical Solutions 11-15, which comprises a compound of formula (I') according to any one of Technical Solutions 11-13, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at its 3' end.

[0153] Technical Solution 17. The oligonucleotide of any one of Technical Solutions 11-16, which comprises a compound of formula (I') of any one of Technical Solutions 11-13 at its 5' end and 3' end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0154] Technical Solution 18. The oligonucleotide according to any one of Technical Solutions 11-17, which comprises one or more compounds of formula (I') according to any one of Technical Solutions 11-13, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, inside the oligonucleotide.

[0155] Technical Solution 19. An oligonucleotide comprising two or more hydrophobic groups inside the oligonucleotide, at the 5' end and / or at the 3' end; preferably, the hydrophobic group is as defined as the R group in the compound of formula (I); preferably, the hydrophobic group is connected to the oligonucleotide via a linker, such as a biodegradable linker.

[0156] Technical Solution 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'), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0157] in,

[0158] represents H, or indicates the position of attachment to the adjacent nucleotide;

[0159] L2 represents H or a hydroxyl protecting group, or represents the position of connection with the adjacent nucleotide;

[0160] R s Selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0161] m = 0, 1, 2, 3, 4, 5 or 6;

[0162] R is a hydrophobic group;

[0163] Preferably,

[0164] represents H, or indicates the position of attachment to the adjacent nucleotide;

[0165] L2 represents H or a hydroxyl protecting group, or represents the position of connection with the adjacent nucleotide;

[0166] R s , m and R are as defined in any one of technical solutions 1-5.

[0167] Technical Solution 21. The double-stranded RNA of Technical Solution 20, wherein the compound of formula (I') is selected from the following compounds of the general formula, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0168] in,

[0169] represents H, or indicates the position of attachment to the adjacent nucleotide;

[0170] L2 represents H or a hydroxyl protecting group, or represents the position of connection with the adjacent nucleotide;

[0171] R s , m and R are as defined in Technical Solutions 1-5.

[0172] Technical Solution 22. The double-stranded RNA of Technical Solution 20, wherein the compound of formula (I') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, wherein the compound is selected from the following:

[0173] in One of them represents H, or represents the position of connection with the adjacent nucleotide, and the other represents H or a hydroxyl protecting group, or the position of attachment to the adjacent nucleotide.

[0174] Technical Solution 23. The double-stranded RNA of any one of Technical Solutions 20-22, wherein the positive strand comprises a compound of formula (I') of any one of Technical Solutions 11-13 at the 5' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0175] Technical Solution 24. The double-stranded RNA of any one of Technical Solutions 20-23, wherein the sense strand comprises a compound of formula (I') of any one of Technical Solutions 11-13, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 3' end.

[0176] Technical Solution 25. The double-stranded RNA of any one of Technical Solutions 20-24, wherein the sense strand comprises a compound of formula (I') of any one of Technical Solutions 11-13 at the 5' end and the 3' end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0177] Technical Solution 26. The double-stranded RNA of any one of Technical Solutions 20-25, wherein the positive strand comprises one or more compounds of formula (I') of any one of Technical Solutions 11-13, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, inside the oligonucleotide.

[0178] Technical Solution 27. The double-stranded RNA of any one of Technical Solutions 20-26, wherein the antisense strand comprises a compound of formula (I') of any one of Technical Solutions 11-13 at the 5' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0179] Technical Solution 28. The double-stranded RNA of any one of Technical Solutions 20-27, wherein the antisense strand comprises a compound of formula (I') of any one of Technical Solutions 11-13 at the 3' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0180] Technical Solution 29. The double-stranded RNA of any one of Technical Solutions 20-28, wherein the antisense strand comprises a compound of formula (I') of any one of Technical Solutions 11-13 at the 5' end and the 3' end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0181] Technical Solution 30. The double-stranded RNA of any one of Technical Solutions 20-29, wherein the antisense strand comprises one or more compounds of formula (I') of any one of Technical Solutions 11-13, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, inside the oligonucleotide.

[0182] Technical Solution 31. The double-stranded RNA of any one of Technical Solutions 20-30, wherein the two or more compounds of Formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof on the sense chain and / or antisense chain are separated by at least 5-30 nucleotides.

[0183] Technical Solution 32. The double-stranded RNA of any one of Technical Solutions 20-31, wherein the double-stranded RNA comprises two compounds of formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, which are located at any two of the following sites: the 5' end of the sense chain, the 3' end of the sense chain, the 5' end of the antisense chain and the 3' end of the antisense chain; preferably located at the 5' end of the sense chain and the 3' end of the sense chain.

[0184] Technical Solution 33. The double-stranded RNA of any one of Technical Solutions 20-32, wherein the double-stranded RNA comprises three compounds of formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, which are located at any three of the following sites: the 5' end of the sense chain, the 3' end of the sense chain, the 5' end of the antisense chain and the 3' end of the antisense chain; preferably located at the 5' end of the sense chain, the 3' end of the sense chain and the 3' end of the antisense chain.

[0185] Technical Solution 34. The double-stranded RNA of any one of Technical Solutions 20-33, wherein the double-stranded RNA comprises four compounds of Formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, which are located at the following sites: the 5' end of the sense chain, the 3' end of the sense chain, the 5' end of the antisense chain and the 3' end of the antisense chain.

[0186] Technical Solution 35. The double-stranded RNA of any one of Technical Solutions 20-33, further comprising a terminal phosphate protecting group or a prodrug protecting group coupled to the 5' end of the antisense strand, preferably a vinyl phosphate group or a prodrug protecting group represented by formula (X):

[0187] in,

[0188] X1 is selected from OH or

[0189] R a Selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, which is optionally deuterated, up to fully deuterated;

[0190] R b and R c Independently selected from H, C 1-6 Alkyl or C 1-6 haloalkyl, the R b and R c Can be optionally D, C 6-10 Aryl or 5-10 membered heteroaryl substitution, up to full deuteration;

[0191] X2 is a chemical bond connected to the first nucleotide at the 5' end of the antisense strand, preferably connected through a hydroxyl group;

[0192] X3 is independently selected from O or S;

[0193] T is selected from

[0194] Each R T1 Independently selected from H, D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2- 6 alkynyl groups or a chain comprising GalNAc, which is optionally deuterated, up to fully deuterated;

[0195] Each R T2 Independently selected from H, D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, which is optionally deuterated, up to fully deuterated;

[0196] Each R T3 Independently selected from H, D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, which is optionally deuterated, up to fully deuterated;

[0197] Each R T4 Independently selected from H, D, halogen, CN, C 1-6 Alkyl, C 1-6Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, which is optionally deuterated, up to fully deuterated;

[0198] m is 0, 1, 2, 3, 4 or 5;

[0199] n is 0, 1, 2, 3, 4 or 5;

[0200] p is 0, 1, 2, 3, 4, or 5;

[0201] X is selected from a chemical bond, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0202] R X1 Selected from H, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated;

[0203] L is -Ar-(CH2) 1-6 -O-, wherein each CH2 may be optionally substituted by R#, R# is selected from H, D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, which is optionally deuterated, up to fully deuterated;

[0204] Ar in L is connected to X, and the oxygen atom is connected to the phosphorus atom;

[0205] Ar is selected from C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl, the C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl may be optionally substituted with 1, 2, 3, 4 or 5 R*;

[0206] R* is selected from H, D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, which is optionally deuterated, up to fully deuterated;

[0207] wherein P1 is selected from a protecting group, preferably 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), 4,4'-dimethoxytrityl, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH, preferably -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH.

[0208] Technical solution 36. The double-stranded RNA of any one of technical solutions 20-35, which is selected from small interfering RNA (siRNA) and short hairpin RNA (shRNA), and is preferably used to inhibit genes expressed in the eye.

[0209] Technical solution 37. The double-stranded RNA of any one of technical solutions 20-36, wherein the sense strand comprises one of the following nucleotide sequences:

[0210] CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-LL6,

[0211] CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-LL7,

[0212] CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-LL8,

[0213] LL6s-CmsAmUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-LL7,

[0214] CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-dTdT-LL7,

[0215] CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-IbsIbsIbs IbsIbsIbs-LL7,

[0216] LL6s-IbsIbsIbs-CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-IbsIbsIbs-LL7,

[0217] Wherein LL6, LL7 and LL8 are selected from the following compounds:

[0218] And among them 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 indicates that it is connected to the 5' carbon or corresponding position of the next nucleotide or nucleotide analog through a phosphate group, a phosphorothioate group or other linking group; when the corresponding structure is at the terminal position of the nucleic acid chain, Correspondingly, this refers to attachment to hydrogen, terminal modifications, terminal protecting groups, or other structures that may be used at the ends of nucleic acid chains.

[0219] Technical solution 38. The double-stranded RNA of Technical solution 37, wherein the antisense strand comprises the following nucleotide sequence:

[0220] VPUmsUfsUmAmGmAfGmUfGfAmGmGmAmUfUmAfAmAmAmUmGmsAmsGm.

[0221] Technical Solution 39. A double-stranded RNA having a sense chain and an antisense chain, each chain having 14 to 30 nucleotides, the antisense chain comprising a sequence that is fully complementary to the sense chain and the target mRNA, wherein the sense chain and / or antisense chain comprises two or more hydrophobic groups inside, at the 5' end and / or at the 3' end; preferably, the hydrophobic group is as defined as the R group in the compound of formula (I); preferably, the hydrophobic group is connected to the sense chain and / or antisense chain via a linker, such as a biodegradable linker.

[0222] Technical solution 40. A vector comprising a nucleotide sequence encoding the double-stranded RNA described in any one of the aforementioned technical solutions 20-39.

[0223] Technical solution 41. A cell containing the double-stranded RNA as described in any one of Technical Solutions 20-39 or the vector as described in Technical Solution 40.

[0224] Technical Solution 42. A pharmaceutical composition comprising the double-stranded RNA as described in any one of Technical Solutions 20-39, the vector as described in Technical Solution 40, or the cell as described in Technical Solution 41, and optionally a pharmaceutically acceptable carrier or excipient.

[0225] Technical solution 43. A kit comprising the double-stranded RNA as described in any one of technical solutions 20-39, the vector as described in technical solution 40, or the cell as described in technical solution 41.

[0226] Technical Solution 44: 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 3' end of the sense strand is a compound of the following formula, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0227] And among them 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 indicates that it is connected to the 5' carbon or corresponding position of the next nucleotide or nucleotide analog through a phosphate group, a phosphorothioate group or other linking group; when the corresponding structure is at the terminal position of the nucleic acid chain, Correspondingly, this refers to attachment to hydrogen, terminal modifications, terminal protecting groups, or other structures that may be used at the ends of nucleic acid chains.

[0228] List of specific compounds

[0229] 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.

[0230] Specifically, according to the 5'->3' sequence, if the corresponding structure is located in the middle of the nucleic acid chain, means that it is connected to the 3' carbon or corresponding position of the previous nucleotide or nucleotide analog through a phosphate group, a phosphorothioate group or other linking group, It means that it is connected to the 5' carbon or corresponding position of the next nucleotide or nucleotide analog through a phosphate group, a thiophosphate group or other linking group; if the corresponding structure is located at the terminal position of the nucleic acid chain, Correspondingly, this refers to attachment to hydrogen, terminal modifications, terminal protecting groups, or other structures that may be used at the ends of nucleic acid chains.

[0231] Synthesis Example

[0232] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention.

[0233] abbreviation

[0234] Example 1: Preparation Example of Intermediate Compound

[0235] Example 1.1 Preparation of DL0066

[0236] Compound 1 was dissolved in DCM (20.0 mL) at room temperature. DCI (0.06 g, 0.502 mmol), 5A molecular sieves, and compound 2 (0.33 g, 1.105 mmol) were added to the solution. The nitrogen atmosphere was replaced three times, and the reaction mixture was stirred at 25°C for 1 hour. The reaction solution was cooled to 0°C in an ice-salt bath. 6 mL of saturated NaHCO₃ solution and 6 mL of saturated brine were added, followed by 50 mL of DCM. The organic phase was washed with a 1:1 mixture of saturated NaHCO₃ and saturated brine (20 mL x 3), then dried over Na₂SO₄ and spun down to dryness. The resulting crude product was purified by column chromatography (petroleum ether / ethyl acetate = 10 / 3 to 10 / 5) to obtain DL0066 as a white solid (620 mg, 53.89% yield, 96.00% purity). 1H NMR(400MHz,DMSO-d6)δ7.25-7.36(m,4H),7.13- 7.25(m,5H),7.01-7.03(m,1H),6.78-6.93(m,4H),5.30-5.32(m,1H),4.47-4.72(m,1H),4.24-4.34(m,1H ),4.12-4.15(m,1H),3.69-3.71(m,8H),3.48-3.59(m,3H),3.35-3.47(m,1H),3.13-3.25(m,1H),2.86-3.0 6(m,3H),2.70-2.78(m,2H),2.09-2.31(m,5H),1.86-1.96(m,2H),1.70-1.85(m,3H),1.47-1.51(m,6H),1. 22-1.42(m,11H),1.06-1.16(m,20H),0.93-0.95(m,4H),0.87-0.89(m,4H),0.82-0.85(m,7H),0.65(s,3H)

[0237] Example 1.2 Preparation of DL0067

[0238] Compound 1 (250 mg, 0.380 mmol) was dissolved in DCM (4.00 mL), and DIEA (0.377 mL, 2.28 mmol), DMAP (11.6 mg, 0.095 mmol), and compound 2 (228 mg, 2.28 mmol) were added. The reaction mixture was stirred at 25°C for 18 hours. LCMS detected a mass response of the product. TLC (PE / EA = 3 / 1) revealed a new spot. The reaction mixture was directly concentrated and purified using a basic silica gel normal phase column (SiO2, PE / EA = 1 / 0 to 3 / 1) to obtain DL0067 (87.0 mg, 28.65% yield, 94.84% purity) as a yellow oil. LCMS (ESI): m / z = 756.4 [MH] - ; 1H NMR (400MHz, CD3OD) δ7.36-7.38(m,2H),7.24-7.30(m,6H),7.16-7.22(m,1H),6.81-6.87(m, 4H),5.38-5.49(m,1H),4.24-4.29(m,1H),3.81-3.89(m,1H),3.77(d,J=2.0Hz,6H),3.67-3. 70(m,1H),3.54-3.58(m,1H),3.08-3.25(m,1H),2.57-2.59(m,4H),2.30-2.39(m,2H),2.21- 2.28(m,1H),2.07-2.16(m,1H),1.46-1.62(m,2H),1.22-1.35(m,24H),0.90(t,J=6.4Hz,3H).

[0239] Example 1.3 Preparation of DL0082

[0240] 1. Preparation of Intermediate 3

[0241] Compound 1 (2.00 g, 13.6 mmol) was dissolved in DCM (50.0 mL) at 25°C. DIEA (6.74 mL, 40.8 mmol), HATU (7.75 g, 20.4 mmol), and compound 2 (3.48 g, 13.6 mmol) were added sequentially. The reaction was stirred at 25°C for 12 hours. LCMS indicated product formation. TLC (DCM / MeOH = 10 / 1, PMA) showed that compound 2 was not completely reacted, with several new spots formed. Ethyl acetate (200 mL) was added, and the mixture was washed three times with brine (15.0 mL x 3). The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 10 / 1 to 5 / 1, PMA) to afford compound 3 (1.15 g, 21.95% yield) as a yellow oil. 1 H NMR (400MHz, CD3OD) δ3.79-3.91(m,2H),3.75-3.78(m,1H),3.57-3.69(m,4H),3.47-3.56(m,2H) ,3.27-3.34(m,1H),2.29-2.50(m,2H),1.53-1.66(m,2H),1.27-1.37(m,24H),0.84-0.95(m,3H)

[0242] 2. Preparation of Intermediate 4

[0243] Compound 3 (1.15 g, 2.98 mmol) was dissolved in pyridine (20.0 mL) at 25°C and the atmosphere was purged with nitrogen three times. A solution of DMTrCl (1.11 g, 3.28 mmol) in DCM (10.0 mL) was then added. The mixture was stirred at 25°C for 3 hours. LCMS indicated the formation of the product, with partial consumption of the starting material. TLC (DCM / MeOH = 10 / 1, PMA) indicated partial consumption of the starting material, with the formation of new spots. DCM (200 mL) was added, and the mixture was washed sequentially with saturated sodium bicarbonate solution (20.0 mL x 3) and saturated brine (20.0 mL x 3). The organic phase was concentrated under reduced pressure to yield the crude product. The crude product was purified by column chromatography (PE / EA = 3 / 1 to 1 / 1, PMA) to afford compound 4 (620 mg, 30.22% yield) as a yellow oil.

[0244] 3. Preparation of DL0082

[0245] Compound 4 (420 mg, 0.611 mmol) was dissolved in DCM (3.00 mL) at 25°C. DIEA (0.605 mL, 3.66 mmol), DMAP (18.6 mg, 0.153 mmol), and compound 5 (367 mg, 3.66 mmol) were added to the mixture in that order. The reaction mixture was stirred at 25°C for 3 hours. LCMS showed that the product was generated, but the starting material was not completely consumed. DCM (50.0 mL) was added to the mixture, and the mixture was washed three times with saturated brine (50.0 mL x 3). The organic phase was concentrated under reduced pressure to obtain the crude product compound. The crude product was purified by prep-HPLC (column: Waters Xbridge BEH C18 150*25mm*5um; mobile phase: TEAA-ACN; gradient: 55%-95% / 16min; flow rate: 15ml / min) to give DL0082 as a yellow oil (170mg, yield 35.34%, purity 97.26%).

[0246] 1H NMR(400MHz,CD3OD)δ7.43(d,J=8.0Hz,2H),7.18-7.34(m,7H),6.82-6.89(m,4H),4 .16-4.25(m,1H),3.92-4.13(m,2H),3.85(d,J=5.20Hz,1H),3.78(s,6H),3.66-3.76 (m,1H),3.53-3.64(m,2H),3.33-3.42(m,1H),3.28(s,1H),3.12-3.23(m,3H),2.48 -2.65(m,4H),2.19-2.43(m,2H),1.52(s,2H),1.25-1.35(m,27H),0.86-0.93(m,3H)

[0247] Example 1.4 Preparation of DL0084

[0248] 1. Preparation of Compound 3

[0249] Compound 1 (207 mg, 0.37 mmol) was dissolved in DCM (5.00 mL), and HATU (177 mg, 0.46 mmol) and DIEA (0.15 mL, 0.93 mmol) were added. The reaction was stirred at 25°C for half an hour, followed by the addition of compound 2 (140 mg, 0.31 mmol). The reaction was continued at 25°C for 16 hours. TLC (DCM / MeOH = 10 / 1) showed the formation of new spots. 10.0 mL of DCM and 10.0 mL of H₂O were added to the reaction mixture, and the layers were separated. Sodium bicarbonate solution (10.0 mL x 1) was added to the organic phase, which was then washed with saturated brine (10.0 mL x 1), dried over anhydrous sodium sulfate, filtered, and dried to afford the crude product, compound 3 (200 mg, 0.20 mmol, 65.10%), as a white solid.

[0250] 1 H NMR (400MHz, CD3OD) δ7.45(d,J=8.0Hz,2H),7.16-7.38(m,7H),6.82-6.90(m,4H),4.39(d,J=8.0Hz,1H),3.93-4.17(m,2H),3.8 0(s,7H),3.39-3.71(m,8H),3.10-3.31(m,2H),2.84-3.03(m,2H),1.46-1.70(m,4H),1.16-1.45(m,51H),0.91(t,J=8.0Hz,6H)

[0251] 2. Preparation of DL0084

[0252] Compound 3 was dissolved in DCM (7.0 mL), and DIEA (0.20 mL, 1.21 mmol), DMAP (9.9 mg, 0.08 mmol), and compound 4 (121 mg, 1.21 mmol) were added. The reaction was incubated at 25°C for 3 hours. LCMS indicated the presence of the desired product. The reaction mixture was dried, and the crude product was purified by prep-HPLC (column: 01-Waters Xbridge BEH C18 19×150 mm, 5 μm; conditions: TEAA-ACN; Begin B 65-95; gradient time: 15 minutes; 100% B hold time: 2 minutes; flow rate: 15 ml / min) to obtain the product DL0084 as a yellow oil (80.0 mg, yield: 36.27%).

[0253] 1 H NMR (400MHz, CDCl3) δ7.35(d,J=4.0Hz,2H),7.26-7.30(m,6H),7.09-7.25(m,1H),6.75(d,J=8.0Hz,4H),4.20-4.31(m,1H),3.75-4.16(m,5H),3. 71(s,6H),3.32-3.56(m,6H),3.05-3.29(m,4H),2.99(q,J=8.0Hz,2H),2 .50-2.60(m,4H),1.47(d,J=8.0Hz,4H),1.18(s,52H),0.77-0.84(m,6H)

[0254] Example 1.5 Preparation of DL0127

[0255] 1. Preparation of Compound 3

[0256] Compound 1 (1.00 g, 3.90 mmol) was dissolved in DCM (20 mL), and HATU (1.48 g, 3.90 mmol) and DIEA (3.86 mL, 23.3 mmol) were added. The reaction was stirred at 25°C for 30 minutes. Compound 2 (0.68 g, 4.68 mmol) was then added and the reaction was stirred at 25°C for 16 hours. LCMS showed the formation of the product. TLC (PE / EA = 1 / 1) revealed the formation of a new spot. 20 mL of water was added to the reaction solution, and the solution was extracted with DCM (15.0 mL x 2). The combined organic phases were washed with ammonium chloride solution (20.0 mL x 1), sodium bicarbonate solution (20.0 mL x 1), and saturated brine (20.0 mL x 1), dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain the crude product. The crude product was purified by column chromatography (PE:EA=1 / 0-1 / 1) to give compound 3 (2.24 g, 5.839 mmol, 149.73%) as a pale yellow solid.

[0257] 1 H NMR (400MHz, CD3OD) δ3.65 (s, 3H), 3.15-3.19 (m, 2H), 2.32 (t, J = 7.6Hz, 2H), 2.11-2.19 (m, 1H), 1 .59-1.68(m,2H),1.48-1.58(m,4H),1.37-1.40(m,2H),1.22-1.32(m,22H),0.89(t,J=6.8Hz,6H)

[0258] 2. Preparation of Compound 4

[0259] Compound 3 was dissolved in H₂O (1.00 mL) and THF (4.00 mL), followed by the addition of LiOH (218 mg, 5.21 mmol). The reaction was allowed to react at 25°C for 16 hours. LCMS indicated the disappearance of the starting material, while TLC (PE / EA = 1 / 1) indicated the formation of new spots. 10.0 mL of water was added to the reaction solution, and the solution was extracted with EtOAc (10.0 mL x 2). The aqueous phase was adjusted to pH ~6 with 3M HCl and extracted with EtOAc (10.0 mL x 2). The combined organic phases were washed with saturated brine (10.0 mL x 1), dried over anhydrous sodium sulfate, filtered, and spin-dried to afford compound 4 (220 mg, 0.595 mmol, 45.67%) as a white solid.

[0260] 1H NMR (400MHz, CD3OD) δ3.18(t,J=8.0Hz,2H),2.29(t,J=8.0Hz,2H),2.15(m,1H),1.60-1 .67(m,2H),1.48-1.59(m,4H),1.37-1.62(m,2H),1.17-1.35(m,22H),0.84-0.93(m,6H)

[0261] 3. Preparation of Compound 6

[0262] Compound 4 (213 mg, 0.57 mmol) was dissolved in DCM (5.00 mL), and HATU (219 mg, 0.57 mmol) and DIEA (0.22 mL, 1.335 mmol) were added. The reaction was stirred at 25°C for 30 minutes, followed by the addition of compound 5 (200 mg, 0.44 mmol). The reaction was stirred at 25°C for 16 hours. TLC (DCM / MeOH = 10 / 1) showed the formation of new spots. LCMS showed the disappearance of the starting material. The reaction solution was diluted with 10.0 mL of DCM, and sodium bicarbonate solution (10.0 mL x 2) was added to the reaction solution. The solution was washed with saturated brine (10.0 mL x 2), dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH=1 / 0-10 / 1) to give compound 6 (300 mg, 0.374 mmol, 84.15%) as a yellow oil. 1 H NMR (400MHz, CD3OD) δ7.41-7.47(m,2H),7.20-7.36(m,7H),6.84-6.92(m,4H),3.85-4.06(m,2H),3.80(d,J=4.0Hz,6H),3.72- 3.77(m,1H),3.42-3.71(m,5H),3.15-3.27(m,4H),2.13-2.43(m,3H),1.46-1.67(m,6H),1.20-1.38(m,23H),0.84-0.93(m,6H)

[0263] 4. Preparation of DL0127

[0264] Compound 6 (300 mg, 0.374 mmol) was dissolved in DCM (5.0 mL), and DIEA (0.371 mL, 2.247 mmol), DMAP (18.30 mg, 0.150 mmol), and compound 7 (224.84 mg, 2.247 mmol) were added. The reaction was incubated at 25°C for 16 hours. LCMS analysis indicated the presence of the desired product. The reaction mixture was dried, and the crude product was purified by prep-HPLC (column: Waters Xbridge BEH C18 100*25 mm*5 μm; conditions: TEAA-ACN; Begin B 55-95; gradient time: 15 minutes; 100% B hold time: 6 minutes; flow rate: 15 ml / min) to yield DL0127 (120 mg, 0.133 mmol, 35.56%) as a yellow oil.

[0265] 1 H NMR(400MHz,CD3OD)δ7.43(d,J=8.0Hz,2H),7.18-7.34(m,7H),6.86(dd,J=8.0,8.0Hz ,4H),4.16-4.25(m,1H),3.92-4.14(m,2H),3.84-3.90(m,1H),3.78-3.82(m,6H),3.5 2-3.76(m,3H),3.34-3.46(m,1H),3.17-3.22(m,4H),2.49-2.62(m,4H),2.22-2.41(m ,2H),2.10-2.17(m,1H),1.43-1.62(m,6H),1.25-1.35(m,24H),0.88(t,J=8.0Hz,6H)

[0266] Example 1.6 Preparation of DL0133

[0267] 1 Preparation of compound 3

[0268] Compound 2 (1.66 g, 8.26 mmol) was dissolved in DCM (50.0 mL) at 25°C. HATU (3.93 g, 10.3 mmol) and DIEA (3.42 mL, 20.7 mmol) were added sequentially. The reaction was stirred at 25°C for 0.5 h, followed by the addition of compound 1 (1.00 g, 6.89 mmol). The reaction was stirred at 25°C for 14 h. LCMS showed the presence of product by mass. Thin-layer chromatography (PE / EA = 3 / 1) revealed complete consumption of the reactants and the formation of new spots. The reaction was diluted with DCM (50.0 mL) and washed with saturated aqueous citric acid (10.0 mL x 3). The organic phase was washed sequentially with saturated aqueous NaHCO₃ (20.0 mL x 3) and then with saturated aqueous sodium chloride (20.0 mL x 3). The organic phase was then dried over Na₂SO₄ and spun down to yield the crude product. The crude product was purified by column chromatography (PE / EA=1 / 0-3 / 1) to give compound 3 (1.50 g) as a white solid. 1 H NMR (400MHz, CD3OD) δ3.65 (s, 3H), 3.11-3.20 (m, 2H), 2.33 (t, J = 7.2Hz, 2H), 2.16 (t, J = 7. 6Hz,2H),1.57-1.68(m,4H),1.51(q,J=7.2Hz,2H),1.25-1.40(m,19H),0.86-0.93(m,3H).

[0269] 2 Preparation of compound 4

[0270] Compound 3 (1.00 g, 3.05 mmol) was dissolved in a mixture of THF (5.00 mL) and H₂O (2.50 mL) at 25°C. LiOH (25.9 mg, 1.08 mmol) was added, and the reaction mixture was stirred at 25°C for 18 hours. Thin-layer chromatography (PE / EA = 3 / 1) showed complete consumption of the starting material and the formation of new spots. 1M HCl (1.00 mL) was added to the reaction mixture to adjust the acidity, followed by dichloromethane (20.0 mL) and separation. The organic phase was dried over Na₂SO₄ and evaporated to dryness to afford compound 4 (900 mg, 94.04% yield) as a white solid. 1H NMR(400MHz, CDCl3) δ3.26(q,J=6.8Hz,2H),2.37(t,J=7.2Hz,2H),2.13-2.20(m,2H),1.80-1.9 0(m,1H),1.58-1.71(m,4H),1.53(q,J=7.2Hz,2H),1.36-1.43(m,2H),1.21-1.32(m,16H),0.84- 0.92(m,3H).

[0271] 3 Preparation of compound 6

[0272] Compound 4 (167 mg, 0.534 mmol) was dissolved in DCM (10.0 mL) at 25°C. HATU (169 mg, 0.445 mmol) and DIEA (0.074 mL, 0.445 mmol) were added sequentially. The reaction was stirred at 25°C for 0.5 h, followed by the addition of compound 5 (200 mg, 0.445 mmol). The reaction was stirred at 25°C for 16 h. LCMS showed the presence of the product by mass. Thin-layer chromatography (DCM / MeOH = 10 / 1) revealed a new spot. The reaction was diluted with DCM (20.0 mL) and washed sequentially with saturated aqueous citric acid (5.00 mL x 3), saturated aqueous NaHCO₃ (5.00 mL x 3), and saturated aqueous sodium chloride (5.00 mL x 3). The organic phase was then dried over anhydrous Na₂SO₄ and spun down to yield the crude product. The crude product was purified by column chromatography (DCM / MeOH=1 / 0 to 10 / 1) to obtain a crude product of Compound 6 (460 mg). 1 H NMR (400MHz, CD3OD) δ7.42(dd,J=7.6,2.0Hz,2H),7.17-7.33(m,6H),6.80-6.92(m,4H),3.89-3.98(m,1H),3.78(s ,6H),3.36-3.75(m,8H),3.09-3.19(m,3H),2.11-2.40(m,4H),1.43-1.63(m,6H),1.28(s,18H),0.85-0.94(m,3H)

[0273] 4 Preparation of DL0133

[0274] Compound 6 (460 mg, 0.617 mmol) was dissolved in DCM (10.0 mL) at 25°C. DMAP (75.4 mg, 0.617 mmol), DIEA (0.102 mL, 0.617 mmol), and compound 7 (371 mg, 3.71 mmol) were added sequentially. The reaction mixture was stirred at 25°C for 2 hours. LCMS analysis indicated the presence of product. The reaction mixture was dried to obtain the crude product. The crude product was separated by prep-HPLC (column: Waters Xbridge BEH C18 250*50mm*10um; mobile phase: TEAA-ACN; B%: 55%-81% over 10 min; flow rate: 15 mL / min) to obtain DL0133 (180 mg, 34.52% yield, 98.01% purity) as a colorless oil. 1 H NMR(400MHz,CD3OD)δ7.43(d,J=7.6Hz,2H),7.17-7.34(m,7H),6.86(dd,J=7.8,5.9 Hz,4H),4.18-4.26(m,1H),3.93-4.15(m,2H),3.87(d,J=2.8Hz,1H),3.78(d,J=1.6H z,6H),3.33-3.69(m,4H),3.10-3.23(m,5H),2.51-2.62(m,4H),2.23-2.41(m,2H), 2.15(q,J=7.6Hz,2H),1.45-1.62(m,6H),1.21-1.39(m,22H),0.89(t,J=6.8Hz,3H).

[0275] Example 1.7 Preparation of DL0134

[0276] 1 Preparation of compound 3

[0277] Compound 2 (1.537 mL, 5.739 mmol) was dissolved in DCM (20.0 mL) at 25°C. HATU (3.27 g, 8.609 mmol) and DIEA (2.846 mL, 17.218 mmol) were added sequentially. The mixture was stirred at 25°C for 0.5 hours before compound 1 (1 g, 6.887 mmol) was added to the reaction system. The reaction mixture was stirred at 25°C for 12 hours. Thin-layer chromatography (PE / EA = 2 / 1) showed complete reaction of the starting material with the formation of new spots. The reaction mixture was diluted with dichloromethane (30 mL) and washed sequentially with saturated citric acid solution (15 mL x 3), saturated sodium bicarbonate solution (15 mL x 3), and saturated sodium chloride solution (15 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield the crude product, which was then purified by column chromatography (PE / EA = 50 / 50 to 40 / 60). The product 3 was obtained as a white solid (630 mg, yield 30.87%). 1 H NMR (400MHz, METHANOL-d4) δ3.65 (s, 3H), 3.16 (t, J = 7.03Hz, 2H), 2.33 (t, J = 7.40Hz, 2H),2.16(t,J=7.40Hz,2H),1.45-1.66(m,6H),1.25-1.36(m,22H),0.85-0.95(m,3H)

[0278] 2 Preparation of compound 4

[0279] Compound 3 (630 mg, 1.772 mmol) was dissolved in THF (10 mL) at 25°C. H₂O (5 mL) and KOH (149.13 mg, 2.658 mmol) were added and reacted at 25°C for 12 hours. TLC analysis (T1: PE / EA = 2 / 1) and T2: TLC analysis (DCM:MeOH = 10:1) showed the disappearance of the starting material and the formation of new spots. The reaction mixture was diluted with water (15 mL), the pH adjusted to 3-4 with 1 mol / L hydrochloric acid, and extracted three times with ethyl acetate (15 mL x 3). The reaction mixture was concentrated under reduced pressure to afford product 4 (610 mg, 100.80% yield) as a white solid. 1 H NMR (400MHz, METHANOL-d4) δ3.11-3.21 (m, 2H), 2.29 (t, J = 7.40Hz, 2H), 2.16 (t,J=7.40Hz,2H),1.43-1.68(m,6H),1.22-1.43(m,22H),0.83-0.96(m,3H)

[0280] 3 Preparation of compound 6

[0281] Compound 4 (227.92 mg, 0.667 mmol) was dissolved in DCM (10 mL) at 25°C, and HATU (317.18 mg, 0.834 mmol) and DIEA (0.368 mL, 2.224 mmol) were added. After reacting for 0.5 h, compound 5 was added and the reaction continued for 12 h. Thin-layer chromatography (DCM / MeOH = 10 / 1) showed complete reaction of the starting material with the formation of new spots. The reaction solution was diluted with dichloromethane (20 mL) and washed three times with saturated brine (10 mL x 3). The organic phase was concentrated under reduced pressure to yield the crude product. The crude product was purified by column chromatography (DCM / MeOH = 95 / 5 to 92 / 8) to yield product 6 (350 mg, 0.453 mmol, 81.41%) as a white solid. 1 H NMR (400MHz, METHANOL-d4) δ7.42 (dd, J=2.26, 7.78Hz, 2H), 7.18-7.35 (m, 7H) ,6.78-6.94(m,4H),3.85-4.04(m,2H),3.78(d,J=2.51Hz,6H),3.66-3.75(m, 1H),3.44-3.63(m,4H),3.33-3.42(m,1H),3.06-3.22(m,4H),2.26-2.42(m,2 H),2.15(q,J=7.19Hz,2H),1.44-1.62(m,6H),1.28(s,22H),0.87-0.92(m,3H)

[0282] 4. Preparation of DL0134

[0283] Compound 6 (350 mg, 0.453 mmol) was dissolved in DCM (10 mL) at 25 ° C, and DMAP (13.83 mg, 0.113 mmol), DIEA (0.449 mL, 2.716 mmol) and compound 7 (271.83 mg, 2.716 mmol) were added. The reaction solution was stirred at 25 ° C for 12 hours. LCMS showed that the raw material reaction was complete and product was generated. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by Prep-HPLC (chromatographic column: Waters Xbridge BEH C18 100*25mm*5um; mobile phase: TEAA-ACN; gradient: 55%-95% / 15min; flow rate: 15 ml / min). The colorless oily product DL0134 (216 mg, yield 54.64%, purity 95.94%) was obtained. 1H NMR(400MHz,METHANOL-d4)δ7.41-7.47(m,2H),7.19-7.34(m,7H),6.81-6.91(m,4H ),4.16-4.26(m,1H),3.84-4.14(m,3H),3.45-3.81(m,9H),3.35-3.43(m,1H),3.25 -3.28(m,1H),3.11-3.23(m,8H),2.55-2.61(m,2H),2.48-2.54(m,2H),2.20-2.39( m,2H),2.11-2.18(m,2H),1.43-1.64(m,6H),1.21-1.37(m,30H),0.87-0.92(m,3H)

[0284] Example 1.8 Preparation of DL0135

[0285] 1 Preparation of compound 3

[0286] Compound 1 (1.00 g, 3.90 mmol) was dissolved in DCM (20.0 mL), and HATU (1.78 g, 4.68 mmol) and DIEA (3.87 mL, 23.4 mmol) were added. The reaction was incubated at 25°C for 0.5 h. Compound 2 (0.92 g, 5.07 mmol) was then added, and the reaction was continued at 25°C for 16.5 h. Thin-layer chromatography (PE / EtOAc = 2 / 1) showed the formation of new spots. DCM (25.0 mL) and water (20.0 mL) were added to the reaction solution, and the reaction mixture was separated. The aqueous phase was extracted with DCM (15.0 mL). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain the crude product. The crude product was purified by column chromatography (PE / EtOAc=1 / 0 to 1 / 1) to give compound 3 as a white solid (1.20 g, yield 80.21%). 1 H NMR (400MHz, CD3OD) δ3.67 (s, 3H), 3.13-3.22 (m, 2H), 2.35 (t, J = 7.6Hz, 2H), 2 .18(t,J=7.6Hz,2H),1.47-1.71(m,6H),1.24-1.43(m,28H),0.87-0.97(m,3H)

[0287] 2 Preparation of compound 4

[0288] Compound 3 (740 mg, 1.93 mmol) was dissolved in THF (2.00 mL) and H₂O (6.00 mL). LiOH (324 mg, 7.72 mmol) was added and the reaction was incubated at 20°C for 16 hours. Thin-layer chromatography (PE / EA = 3 / 1, PMA) showed complete consumption of the starting material, with the formation of new spots. The reaction mixture was adjusted to pH 5 with 1M HCl and the product was extracted with dimethyltetrahydrofuran (30.0 mL x 3). The organic phase was evaporated to dryness under reduced pressure to afford Compound 4 (650 mg, 91.17% yield) as a white solid.

[0289] 3 Preparation of compound 6

[0290] Compound 4 (267 mg, 0.723 mmol) was dissolved in DCM (5.00 mL), and HATU (275 mg, 0.723 mmol) and DIEA (0.276 mL, 1.67 mmol) were added. The reaction was stirred at 25°C for 0.5 hours. Compound 5 (250 mg, 0.556 mmol) was then added, and the reaction was stirred at 25°C for 16 hours. Thin-layer chromatography (DCM / MeOH = 10 / 1) showed the formation of new spots. DCM (10.0 mL) was added to dilute the reaction solution, and sodium bicarbonate solution (10.0 mL x 2) was added to the reaction solution. The solution was washed with saturated brine (10.0 mL x 2), dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 1 / 0 to 10 / 1) to obtain compound 3 (390 mg, 87.54% yield) as a yellow oil.

[0291] 4 Preparation of DL0135

[0292] Compound 6 (390 mg, 0.487 mmol) was dissolved in DCM (7.00 mL), and DIEA (0.483 mL, 2.92 mmol), DMAP (23.8 mg, 0.195 mmol), and compound 7 (292 mg, 2.92 mmol) were added. The reaction was incubated at 25°C for 3 hours. LCMS indicated the presence of the desired product. The reaction mixture was dried, and the crude product was purified by prep-HPLC (column: Waters Xbridge BEH C18 100*25 mm*5 μm; conditions: TEAA-CAN; Begin B 55-95; gradient time: 15 minutes; 100% B hold time: 6 minutes; flow rate: 15 ml / min) to obtain DL0135 as a yellow oil (140 mg, yield 31.91%, purity 99.54%). 1H NMR(400MHz,CD3OD)δ7.39-7.48(m,2H),7.17-7.36(m,7H),6.80-6.92(m,4H ),4.18-4.26(m,1H),3.83-4.16(m,3H),3.78(m,6H),3.49-3.76(m,3H),3.3 3-3.45(m,1H),3.09-3.20(m,4H),2.50-2.64(m,4H),2.22-2.42(m,2H),2.1 5(q,J=8.0Hz,2H),1.42-1.65(m,6H),1.24-1.32(m,28H),0.84-0.96(m,3H)

[0293] Example 1.9 Preparation of DL0136

[0294] 1 Preparation of compound 3

[0295] Compound 2 (1305.00 mg, 4.587 mmol) was dissolved in DMF (50 mL) at 25°C. HATU (2616.43 mg, 6.881 mmol) and DIEA (2.275 mL, 13.762 mmol) were added sequentially. The mixture was stirred at 25°C for 0.5 hour, and then compound 1 (1000 mg, 5.505 mmol) was added to the reaction system. The reaction mixture was stirred at 25°C for 11.5 hours. Thin-layer chromatography (PE / EA = 2 / 1) showed complete reaction of the starting material, with the formation of new spots. The reaction mixture was diluted with ethyl acetate (50 mL) and washed sequentially with saturated citric acid solution (30 mL x 3), saturated sodium bicarbonate solution (30 mL x 3), and saturated sodium chloride solution (30 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product, which was then purified by column chromatography (PE / EA = 70 / 30 to 60 / 40). The product 3 was obtained as a white solid (1530 mg, yield 81.02%). 1 H NMR (400MHz, METHANOL-d4) δ3.65(s,3H),3.15(br d,J=6.38Hz,2H),2.33(br t,J=7.25Hz,2H),2.09-2.24(m,2H),1.42-1.72(m,6H),1.29(br s,30H),0.90(br s,3H)

[0296] 2 Preparation of compound 4

[0297] Compound 3 (1.53 g, 3.717 mmol) was dissolved in THF (20 mL) at 25°C. HO (10 mL) and LiOH (0.19 g, 4.460 mmol) were added and the reaction was stirred for 12 hours. Thin-layer chromatography (PE / EA = 2 / 1) showed the disappearance of the starting material and the formation of new spots. The reaction solution was concentrated under reduced pressure, dissolved in water (10 mL) and acetonitrile (5 mL), and lyophilized. This afforded product 4 as a white solid (1313 mg, 87.54% yield). 1 H NMR (400MHz, METHANOL-d4) δ3.16(t,J=6.94Hz,2H),2.29(t,J=7.38Hz,2H),2.16(t,J=7.50Hz,2H),1.45-1.63(m,6H),1.29(s,29H),0.90(br t,J=6.75Hz,3H)

[0298] 3 Preparation of compound 6

[0299] Compound 4 (300 mg, 0.754 mmol) was dissolved in DCM (10 mL) at 25°C, and HATU (406.40 mg, 1.069 mmol) and DIEA (0.416 mL, 2.515 mmol) were added. After 0.5 hour of reaction, compound 5 (282.64 mg, 0.629 mmol) was added and the reaction continued for 7.5 hours. Thin-layer chromatography (DCM / MeOH = 8 / 1) showed complete reaction of the starting material with the formation of new spots. The reaction solution was diluted with dichloromethane (10 mL) and washed sequentially with saturated citric acid solution (10 mL x 3), saturated sodium bicarbonate solution (10 mL x 3), and saturated sodium chloride solution (10 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield the crude product, which was then purified by column chromatography (DCM / MeOH = 95 / 5 to 94 / 6). Product 6 (358 mg, 68.71% yield) was obtained as a colorless oil. 1 H NMR (400MHz, METHANOL-d4) δ7.38-7.48(m,2H),7.20-7.35(m,7H),6.79-6.91(m,4H),3.86-4.08(m,2H),3.78(d,J=2.51Hz,6H),3 .36-3.74(m,6H),3.06-3.24(m,4H),2.23-2.43(m,2H),2.15(q,J=6.94Hz,2H),1.44-1.64(m,6H),1.28(s,30H),0.86-0.92(m,3H)

[0300] 4 Preparation of DL0136

[0301] Compound 6 (358 mg, 0.432 mmol) was dissolved in DCM (5 mL) at 25°C, and DMAP (13.19 mg, 0.108 mmol), DIEA (0.428 mL, 2.591 mmol), and compound 7 (259.23 mg, 2.591 mmol) were added. The reaction mixture was stirred at 25°C for 3 hours. Thin-layer chromatography (DCM / MeOH = 10 / 1) showed complete reaction of the starting material, with the formation of new spots. The reaction mixture was diluted with dichloromethane (10 mL) and washed three times with saturated NaHCO3 solution (10 mL x 3) and saturated NaCl solution (10 mL x 3). The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (DCM / MeOH = 95 / 5 to 90 / 10) to obtain the crude product. The crude product was purified by Prep-HPLC (column: Waters Xbridge BEH C18 100*25mm*5um; mobile phase: TEAA-ACN; gradient: 65%-95% / 15min; flow rate: 15ml / min). The product DL0136 (163mg, yield 40.63%, purity 97.57%) was obtained as a colorless oil. 1 H NMR(400MHz,METHANOL-d4)δ7.43(br d,J=8.03Hz,2H),7.18-7.34(m,7H),6.82-6.91(m,4H),4.08-4.26(m,2H ),3.84-4.04(m,3H),3.78(d,J=2.01Hz,6H),3.54-3.65(m,2H),3.35-3.4 5(m,1H),3.13-3.22(m,4H),2.49-2.65(m,4H),2.23-2.44(m,2H),2.16(q ,J=7.53Hz,2H),1.45-1.62(m,6H),1.25-1.36(m,33H),0.85-0.94(m,3H)

[0302] Example 1.10 Preparation of DL0137

[0303] 1. Preparation of Compound 3

[0304] Compound 2 (896 mg, 2.54 mmol) was dissolved in DCM (15.0 mL) at 25°C, and HATU (1205 mg, 3.17 mmol) and DIEA (1.05 mL, 6.34 mmol) were added sequentially. The mixture was stirred at 25°C for 0.5 hours, and then compound 1 (950 mg, 2.113 mmol) was added. The mixture was stirred at 25°C for 3 hours. LCMS showed product formation. TLC (DCM / MeOH = 10 / 1) indicated complete reaction. Dichloromethane (60.0 mL) was added to the reaction mixture, and the mixture was washed three times with saturated brine (10.0 mL x 3). The organic phase was dried over anhydrous sodium sulfate and then dried under reduced pressure to afford the crude product. The crude product was purified by column chromatography (PE / EA = 5 / 1 to 1 / 1) to afford compound 3 (1.3 g, 1.66 mmol, 78.37% yield) as a colorless oil. 1 H NMR(400MHz,CD3OD)δ7.78(d,J=7.6Hz,2H),7.57-7.69(m,2H),7.34-7.45(m ,4H),7.24-7.32(m,7H),7.18-7.21(m,1H),6.93-7.10(m,1H),6.79-6.89(m ,4H),4.29-4.39(m,2H),4.17(s,1H),3.86-4.01(m,1H),3.42-3.82(m,12H) ,3.33-3.40(m,1H),2.99-3.26(m,5H),2.13-2.43(m,2H),1.39-1.62(m,4H)

[0305] 2. Preparation of Compound 4

[0306] Compound 3 (650 mg, 0.828 mmol) was dissolved in MeCN (6.00 mL) at 25°C, and Et2NH (1.00 mL) was added. The mixture was stirred at 25°C for 3 hours. LCMS and TL (PE:EA = 1:1) showed that the reaction of the starting material was complete. The reaction mixture was directly concentrated under reduced pressure to dryness to obtain compound 4 (450 mg, 0.800 mmol, yield 96.57%).

[0307] 3. Preparation of Compound 6

[0308] Compound 5 (58.1 mg, 0.171 mmol) was dissolved in DMF (5.00 mL) at 25°C. HATU (70.3 mg, 0.185 mmol) and DIEA (0.070 mL, 0.427 mmol) were added sequentially. After stirring for 0.5 hours, compound 4 (80.0 mg, 0.142 mmol) was added, and the mixture was stirred at 25°C for 12 hours. TLC (DCM / MeOH = 10 / 1) showed the formation of new spots. Ethyl acetate (60.0 mL) was added to the reaction mixture, and the mixture was washed three times with saturated sodium bicarbonate solution (10.0 mL x 3) and saturated brine (10.0 mL x 3). The organic phase was dried over anhydrous sodium sulfate and then spin-dried under reduced pressure to obtain the crude compound. The crude product was purified by column chromatography (TLC) (DCM / MeOH = 10 / 1 to 5 / 1) to give compound 6 (120 mg, yield 95.34%) as a yellow oil. 1 H NMR (400MHz, CD3OD) δ7.39-7.46(m,2H),7.16-7.34(m,7H),6.81-6.91(m,4H),3.71-3.78(m,8H),3.43-3.70(m,5H),3.33-3.43(m,2H) ,3.18-3.23(m,4H),3.12-3.17(m,2H),2.11-2.42(m,4H),1.44-1.64(m,6H),1.32-1.40(m,6H),1.25-1.32(m,38H),0.85-0.93(m,3H)

[0309] 4. Preparation of Product DL0137

[0310] Compound 6 (120 mg, 0.136 mmol) and compound 7 (81.4 mg, 0.813 mmol) were added to DCM (3.00 mL) at 25°C, followed by DMAP (4.14 mg, 0.034 mmol) and DIEA (0.134 mL, 0.813 mmol). The mixture was stirred at 25°C for 12 hours. TLC (DCM / MeOH = 10 / 1, UV) showed complete reaction of the starting materials with the formation of new spots. Dichloromethane (60.0 mL) was added to the reaction mixture, and the mixture was washed three times with saturated sodium bicarbonate solution (10.0 mL x 3) and saturated brine (10.0 mL x 3). The organic phase was dried over anhydrous sodium sulfate and then spin-dried under reduced pressure to obtain the crude compound. The crude product was purified by column chromatography (DCM / MeOH = 10 / 1 to 8 / 1) to obtain a colorless oily crude compound. The crude product was purified by prep-HPLC (chromatographic column: Waters Xbridge BEH C18 150*25mm*5um; mobile phase: TEAA-ACN; gradient: 65%-95% / 15min; flow rate: 15ml / min) to obtain white solid compound DL0137 (45.0mg, yield 33.69%, purity 95.06%). 1 H NMR (400MHz, CD3OD) δ7.43 (d, J=8.0Hz, 2H), 7.16-7.34 (m, 7H), 6.84-6.87 (m,4H),3.85-4.26(m,4H),3.67-3.78(m,7H),3.56-3.65(m,2H),3.33-3. 46(m,1H),3.10-3.23(m,6H),2.48-2.63(m,4H),2.20-2.42(m,2H),2.16( q,J=7.6Hz,2H),1.45-1.63(m,6H),1.14-1.44(m,44H),0.85-0.94(m,3H)

[0311] Example 1.11 Preparation of DL0138

[0312] 1 Preparation of compound 3

[0313] Compound 2 (1.47 mL, 4.60 mmol) was dissolved in DCM (30.0 mL) at 25°C. HATU (2.10 g, 5.52 mmol) and DIEA (2.28 mL, 13.8 mmol) were added sequentially. The mixture was stirred for 0.5 h, followed by the addition of compound 1 (1.00 g, 5.52 mmol). The reaction mixture was stirred at 25°C for 16 h. Thin-layer chromatography (PE / EA = 3 / 1) showed complete consumption of the starting material and the formation of new spots. The reaction mixture was diluted with dichloromethane (30.0 mL) and washed sequentially with saturated aqueous citric acid (10.0 mL x 3), saturated aqueous NaHCO₃ (10.0 mL x 3), and saturated aqueous NaCl (10.0 mL x 3). The organic phase was then dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to yield the crude product. The crude product was purified by column chromatography (PE / EA=1 / 0 to 5 / 1) to give compound 3 as a white solid (1.30 g, yield 68.95%). 1 H NMR (400MHz, CD3OD) δ5.35-5.41(m,2H),3.65(s,3H),3.16(t,J=6.8Hz,2H),2.33(t,J=7.2Hz,2H),2.16(t,J=7 .6Hz,2H),1.98(d,J=4.8Hz,4H),1.56-1.68(m,4H),1.46-1.55(m,2H),1.27-1.40(m,22H),0.86-0.94(m,3H).

[0314] 2 Preparation of compound 4

[0315] Compound 3 (1.30 g, 3.17 mmol) was dissolved in a mixture of THF (5.00 mL) and H₂O (2.50 mL) at 25°C. LiOH (0.150 g, 6.35 mmol) was added, and the reaction mixture was stirred at 25°C for 18 hours. Thin-layer chromatography (PE / EA = 3 / 1) showed complete consumption of the starting material and the formation of new spots. 1M HCl (10.0 mL) was added to the reaction mixture to adjust the acidity, followed by dichloromethane (20.0 mL) and separation. The organic phase was dried over Na₂SO₄ and evaporated to dryness to afford compound 4 (800 mg, 63.72% yield) as a white solid. 1H NMR (400MHz, CDCl3) δ5.54-5.63(m,1H),5.38(dt,J=4.4,2.4Hz,2H),3.26(q,J=6.8Hz,2H),2.36(t,J=7. 2Hz,2H),2.12-2.20(m,2H),1.91-2.02(m,4H),1.47-1.72(m,6H),1.24-1.42(m,22H),0.84-0.93(m,3H).

[0316] 3 Preparation of compound 6

[0317] Compound 4 (253 mg, 0.641 mmol) was dissolved in DCM (9.00 mL) at 25°C. HATU (304 mg, 0.801 mmol) and DIEA (0.529 mL, 3.20 mmol) were added sequentially. The mixture was stirred for 0.5 h, followed by the addition of compound 5 (240 mg, 0.534 mmol). The reaction mixture was stirred at 25°C for 16 h. Thin-layer chromatography (DCM / MeOH = 10 / 1) showed complete consumption of the starting material and the formation of new spots. The reaction mixture was diluted with dichloromethane (30.0 mL) and washed sequentially with saturated aqueous NaHCO₃ (10.0 mL x 3) and saturated aqueous NaCl (10.0 mL x 3). The organic phase was then dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to yield the crude product. The crude product was purified by column chromatography (DCM / MeOH=1 / 0 to 10 / 1) to give compound 6 (300 mg, yield 67.92%) as a pale yellow oily liquid.

[0318] 4 Preparation of DL0138

[0319] Compound 6 (300 mg, 0.363 mmol) was dissolved in DCM (10.0 mL) at 25°C. DIEA (0.360 mL, 2.17 mmol), DMAP (17.7 mg, 0.145 mmol), and compound 7 (218 mg, 2.18 mmol) were added sequentially. The reaction mixture was stirred at 25°C for 2 hours. LCMS analysis indicated the presence of product. The reaction mixture was dried to obtain the crude product. The crude product was separated by preparative chromatography on a reverse-phase column (Waters Xbridge BEH C18 100 x 25 mm x 5 μm; mobile phase: TEAA-CAN; B%: 60%-95% over 10 min; flow rate: 15 ml / min) to obtain DL0138 (178 mg, 52.93% yield, 97.55% purity) as a colorless oil. 1H NMR (400MHz, CD3OD) δ7.41-7.48(m,2H),7.20-7.35(m,7H),6.84-6.93(m,4H),5.39(t,J=3.6Hz, 2H),3.93-4.26(m,3H),3.89(d,J=3.6Hz,1H),3.80(d,J=2.0Hz,6H),3.54-3.76(m,3H),3.35-3.4 8(m,1H),3.30(d,J=5.2Hz,1H),3.13-3.24(m,10H),2.49-2.63(m,4H),2.23-2.42(m,2H),2.17( q,J=7.6Hz,2H),1.98(d,J=4.8Hz,4H),1.46-1.64(m,6H),1.24-1.39(m,32H),0.88-0.95(m,3H).

[0320] Example 1.12 Preparation of DL0139

[0321] 1 Preparation of compound 3

[0322] Compound 1 (200 mg, 0.713 mmol), compound 2 (647 mg, 3.566 mmol), HOBt (144.55 mg, 1.070 mmol), EDCI (205.06 mg, 1.070 mmol), and DIEA (0.943 mL, 5.705 mmol) were added sequentially to DCM (5.00 mL) and reacted at 25°C for 14 hours. TLC (petroleum ether:ethyl acetate = 2:1) showed the disappearance of the starting compound 1 and the formation of new spots. The reaction solution was diluted with dichloromethane (50.0 mL) and washed with citric acid (10 mL x 3), sodium bicarbonate (10 mL x 3), and saturated sodium chloride (10 mL x 3). The organic phase was dried over anhydrous sodium sulfate and filtered to dryness to afford compound 3 (250 mg, 0.613 mmol, 86.00%) as a yellow solid. 1 H NMR (400MHz, CDCl3) δ5.39-5.47(m,4H),3.65-3.70(m,3H),3.25(q,J=6.8Hz,2H),2.65-2.70(m,2H),2.32(t,J=7.2Hz,2H),2 .15(t,J=7.6Hz,2H),1.94-2.03(m,4H),1.60-1.70(m,4H),1.52(q,J=7.2Hz,2H),1.26-1.38(m,16H),0.89(t,J=6.8Hz,3H).

[0323] 2 Preparation of compound 4

[0324] Compound 3 (330 mg, 0.810 mmol) was dissolved in THF (4.00 mL) and H₂O (2.00 mL). LiOH (67.9 mg, 1.62 mmol) was added and the mixture was allowed to react at 25°C for 14 hours. TLC (petroleum ether:ethyl acetate = 1:1) showed the disappearance of the starting material and the formation of new spots. The reaction mixture was cooled to 0°C and the pH was adjusted to 5 with 1M HCl. The aqueous phase was extracted with dimethyltetrahydrofuran (20 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and dried to afford compound 4 (350 mg, 0.889 mmol, 109.84%) as a brown solid. 1 H NMR (400MHz, CD3OD) δ5.32-5.46(m,4H),3.16(t,J=6.8Hz,2H),2.65(d,J=4.0Hz,2H),2.29(t,J=7.2Hz, 2H),2.13-2.21(m,2H),1.96-2.03(m,4H),1.48-1.67(m,6H),1.26-1.38(m,16H),0.90(t,J=6.8Hz,3H).

[0325] 3 Preparation of compound 6

[0326] Compound 4 (200 mg, 0.508 mmol), HATU (263 mg, 0.693 mmol), and DIEA (0.229 mL, 1.38 mmol) were added to DCM (3.00 mL) and reacted at 25°C for 30 minutes. Compound 5 (207 mg, 0.462 mmol) was then added and reacted at 25°C for 12 hours. LCMS showed 83% product formation. The reaction solution was diluted with dichloromethane (100 mL) and washed with saturated aqueous sodium bicarbonate (30 mL x 3) and saturated aqueous sodium chloride (30 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and spin-dried to afford the crude product, compound 6 (600 mg, 0.727 mmol), as a brown solid.

[0327] 4 Preparation of DL0139

[0328] Compound 6 (600 mg, 0.727 mmol) and compound 7 (436 mg, 4.36 mmol) were dissolved in DCM (6.00 mL). DIEA (0.721 mL, 4.36 mmol) and DMAP (22.2 mg, 0.182 mmol) were added. The atmosphere was purged with nitrogen three times and the reaction mixture was allowed to react at 25°C for 1 hour. LCMS showed the disappearance of the starting material and 84.2% product formation. The reaction mixture was directly spin-dried to obtain the crude product, which was then purified by Prep-HPLC (column: 01-Waters Xbridge BEH C18 19*150 mm; mobile phase: TEAA-ACN; gradient: 55%-95% / 15 min; flow rate: 15 ml / min). The product DL0139 (200 mg, 0.216 mmol, 29.73%) was obtained as a colorless oil. MS: m / z = 1971.7 (M+H) + ; 1 H NMR (400MHz, CD3OD) δ7.38-7.46(m,2H),7.18-7.35(m,7H),6.81-6.90(m,4H),5.32-5.45(m,4 H),4.16-4.26(m,1H),3.85-4.13(m,3H),3.66-3.81(m,7H),3.56-3.65(m,2H),3.35-3.46(m, 1H),3.14-3.22(m,9H),2.63-2.68(m,2H),2.54-2.61(m,2H),2.46-2.53(m,2H),2.23-2.41(m ,2H),2.10-2.19(m,2H),1.94-2.02(m,4H),1.45-1.61(m,6H),1.22-1.38(m,26H),0.85-0.93 (m,3H).

[0329] Example 1.13 Preparation of DL0140

[0330] 1 Preparation of compound 3

[0331] Compound 1 (500 mg, 1.52 mmol) and HATU (752 mg, 1.97 mmol) were dissolved in DCM (5.00 mL), and DIEA (1.50 mL, 9.13 mmol) was added. The reaction was allowed to proceed at 40°C for 0.5 h. Compound 2 (106 mg, 0.731 mmol) was then added. The reaction was allowed to proceed at 40°C for 16 h. TLC (PE / EtOAc = 2 / 1) showed the formation of new spots. 25 mL of DCM and 20 mL of water were added to the reaction solution, and the reaction mixture was separated. The aqueous phase was extracted with DCM (15 mL x 1). The combined organic phases were washed with saturated brine (20 mL x 1), dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain the crude product. The crude product was purified by column chromatography (PE / EtOAc = 1 / 0 to 1 / 1) to obtain compound 3 (520 mg, 1.14 mmol, 74.97%). 1 H NMR (400MHz, CD3OD) δ5.29-5.47(m,12H),3.67(s,3H),3.13-3.23(m,2H),2.80-2.95(m,10H),2.31-2.45(m,4H),2 .19-2.27(m,2H),2.06-2.16(m,2H),1.58-1.70(m,2H),1.46-1.57(m,2H),1.32-1.42(m,2H),0.99(t,J=7.6Hz,3H)

[0332] 2 Preparation of compound 4

[0333] Compound 3 (520 mg, 1.14 mmol) was dissolved in H₂O (2 mL) and THF (5 mL), and LiOH (191 mg, 4.56 mmol) was added. The reaction was allowed to react at 20°C for 16 hours. TLC (PE / EtOAc = 2 / 1) showed the formation of new spots. The reaction solution was spin-dried to dryness. 15 mL of DCM and 15 mL of water were added to the crude product, and the reaction mixture was separated. The aqueous phase was adjusted to pH 5 with 3M HCl. The aqueous phase was extracted with DCM (15 mL x 2). The organic phase was washed with saturated brine (20 mL x 1), dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain compound 4 (420 mg, 0.951 mmol, 83.34%) as a colorless oil. 1H NMR (400MHz, CD3OD) δ5.21-5.48(m,12H),3.16(t,J=7.2Hz,2H),2.78-2.94(m,10H),2.34-2.43(m,2H),2.29(t,J=7.2Hz, 2H),2.19-2.24(m,2H),2.04-2.15(m,2H),1.57-1.67(m,2H),1.46-1.56(m,2H),1.31-1.42(m,2H),0.97(t,J=7.6Hz,3H).

[0334] 3 Preparation of compound 6

[0335] Compound 4 (319 mg, 0.72 mmol) was dissolved in DCM (5 mL), and HATU (274 mg, 0.72 mmol) and DIEA (0.27 mL, 1.66 mmol) were added. The reaction was stirred at 25°C for half an hour. Compound 5 (250 mg, 0.556 mmol) was added, and the reaction was continued at 25°C for 16 hours. TLC (DCM / MeOH = 10 / 1) showed the formation of new spots. 10 mL of DCM and 15 mL of water were added to the reaction solution, and the reaction solution was separated. Sodium bicarbonate solution (10 mL x 2) was added to the combined organic phase, and the mixture was washed with saturated brine (10 mL x 2), dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 1 / 0-10 / 1) to obtain compound 6 as a yellow oil.

[0336] 4 Preparation of DL0140

[0337] Compound 6 (300 mg, 0.344 mmol) was dissolved in DCM (7 mL), and DIEA (0.34 mL, 2.06 mmol), DMAP (16.7 mg, 0.13 mmol), and compound 7 (206 mg, 2.06 mmol) were added. The reaction was incubated at 25°C for 3 hours. The reaction solution was spin-dried, and the crude product was purified by prep-HPLC (column: Waters Xbridge BEH C18 100*25mm*5um; conditions: TEAA-ACN; Begin B 55-95; gradient time: 15 minutes; 100% B hold time: 6 minutes; flow rate: 15 ml / min) to obtain the yellow oily product DL0140 (120 mg, yield 35.89%, purity 97.34%). 1H NMR(400MHz,CD3OD)δ7.43(d,J=8.0Hz,2H),7.17-7.36(m,7H),6.81-6.92(m,4H),5 .19-5.46(m,11H),4.16-4.29(m,1H),3.84-4.15(m,3H),3.76-3.80(m,6H),3.35-3 .73(m,4H),3.07-3.18(m,6H),2.76-2.93(m,10H),2.50-2.64(m,4H),2.16-2.44(m ,6H),2.03-2.13(m,2H),1.42-1.66(m,4H),1.32-1.38(m,6H),0.96(t,J=8.0Hz,3H)

[0338] Example 1.14 Preparation of DL0142

[0339] 1 Preparation of compound 2

[0340] Compound 1 (2.00 g, 10.9 mmol) was dissolved in THF (80.0 mL), and TBAB (0.71 g, 2.19 mmol), compound 1A (13.4 g, 43.9 mmol), and KOH (3.08 g, 54.8 mmol) were added. The reaction was allowed to react at 25°C for 16 hours. TLC (PE / EA = 10 / 1) showed the formation of new spots. 15.0 mL of water was added to the reaction solution, and the solution was extracted with EtOAc (15.0 mL x 2). The combined organic phases were washed with saturated brine (15.0 mL x 1), dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 0 to 10 / 1) to obtain product 2 (5.5 g, 8.71 mmol, 79.40%) as a colorless oil. 1 H NMR(400MHz,CD3OD)δ7.14-7.40(m,5H),4.41-4.55(m,2H),3.28-3.64(m,9H),0.26-1.08(m,56H),-0.58-0.00(m,6H)

[0341] 2 Preparation of compound 3

[0342] Compound 2 (5.50 g, 8.75 mmol) was dissolved in EtOH (70.0 mL), and Pd(OH)2 (0.20 g, 3.16 mmol) was added. The reaction was continued at 50°C under a H2 atmosphere (1 atm) for 16 hours. TLC (PE / EA = 10 / 1) showed the formation of new spots. The crude product was filtered and dried. The crude product was purified by column chromatography (PE / EtOAC = 1 / 0 to 10 / 1) to afford product 3 (2.45 g, 4.52 mmol, 51.9%) as a white solid. 1 H NMR (400MHz, CD3OD) δ3.51-3.64(m,5H),3.47(s,4H),1.51-1.62(m,4H),1.29(s,52H),0.90(t,J=8.0Hz,6H)

[0343] 3 Preparation of compound 4

[0344] Compound 3 (2.45 g, 4.52 mmol) was dissolved in DCM (15 mL) and DMF (30.0 mL). Pyridinium dichromate (PDC) (5.96 g, 15.8 mmol) was added and the reaction was incubated at 25°C for 16 hours. TLC (PE / EA = 3 / 1) indicated the formation of new spots. 40.0 mL of H2O was added to the reaction solution, which was then extracted with DCM (40.0 mL x 2), dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain the crude product. The crude product was purified by column chromatography (PE / EtOAC = 1 / 0 to 5 / 1) to afford product 4 (1.1 g, 1.982 mmol, 43.77%) as a white solid. 1 H NMR (400MHz, CDCl3) δ4.02-4.11(m,1H),3.77-3.87(m,1H),3.59-3.77(m, 3H),3.44-3.56(m,2H),1.53-1.61(m,4H),1.26(s,52H),0.86-0.95(m,6H)

[0345] 4 Preparation of compound 6

[0346] Compound 4 (665 mg, 1.198 mmol) was dissolved in DCM (15.0 mL), and HATU (592 mg, 1.55 mmol), DIEA (1.18 mL, 7.19 mmol), and compound 5 (28.7 mg, 0.19 mmol) were added. The reaction was allowed to react at 25°C for 16 hours. TLC (PE / EA = 3 / 1) showed the formation of new spots. The reaction solution was diluted with 15.0 mL of DCM. Monohydrated citric acid (15.0 mL x 1) and sodium bicarbonate solution (15.0 mL x 1) were added to the reaction solution. The product was washed with saturated brine (15.0 mL x 1), dried over anhydrous sodium sulfate, filtered, and dried to give a crude white solid, product 6 (510 mg, 0.748 mmol, 62.39%). 1 H NMR (400MHz, CDCl3) δ3.89 (dd, J=4.0, 5.6Hz, 1H), 3.76-3.81 (m, 1H), 3.60-3.70 (m, 5H), 3.39-3.56 (m, 3H ),3.29(q,J=8.0Hz,2H),2.33(t,J=8.0Hz,2H),1.52-1.72(m,12H),1.27-1.38(m,50H),0.87-0.93(m,6H)

[0347] 5 Preparation of compound 7

[0348] Compound 6 (510 mg, 0.748 mmol) was dissolved in THF (6.0 mL) and H₂O (3.0 mL), and LiOH (62.7 mg, 1.49 mmol) was added. The reaction was allowed to react at 25°C for 16 hours. TLC (PE / EA = 3 / 1) showed the formation of new spots. The reaction solution was dried by rotary evaporation, and 10.0 mL of water was added. The aqueous phase was adjusted to pH ~4 with 1 M HCl and extracted with dimethyltetrahydrofuran (10.0 mL x 2). The combined organic phases were washed with saturated brine (10.0 mL x 1), dried over anhydrous sodium sulfate, filtered, and dried by rotary evaporation to afford crude product 7 (460 mg, 0.55 mmol, 73.6%) as a white solid. 1 H NMR(400MHz,CD3OD)δ3.83-3.90(m,1H),3.36-3.65(m,6H),3.19-3.27(m,2H), 2.29(t,J=8.0Hz,2H),1.49-1.68(m,8H),1.23-1.37(m,54H),0.86-0.94(m,6H)

[0349] 6 Preparation of compound 9

[0350] Compound 7 (326 mg, 0.489 mmol) was dissolved in DCM (5.0 mL), and HATU (219 mg, 0.57 mmol) and DIEA (0.22 mL, 1.33 mmol) were added. The reaction solution was reacted at 25°C for 30 minutes. Compound 8 (200 mg, 0.44 mmol) was then added, and the reaction solution was reacted at 25°C for 16 hours. TLC (DCM / MeOH = 10 / 1) showed the formation of new spots. 10.0 mL of H2O was added to the reaction solution, and the reaction solution was extracted with DCM (10.0 mL x 2). Sodium bicarbonate solution (10.0 mL x 1) was added to the reaction solution, and the mixture was washed with saturated brine (10.0 mL x 1), dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain a crude product. The crude product was purified by column chromatography (DCM / MeOH = 1 / 0-5 / 1) to give a white solid crude product 9 (194 mg, 0.17 mmol, 39.7%). 1 H NMR (400MHz, CDCl3) δ7.38-7.46(m,1H),7.27-7.35(m,7H),7.18(d,J=8.0Hz,1H),6.84( d,J=8.0Hz,4H),4.75-4.80(m,1H),3.87(s,2H),3.77-3.85(m,6H),3.69-3.76(m,3H),3. 57-3.68(m,4H),3.37-3.56(m,5H),3.11-3.36(m,3H),2.72-2.81(m,1H),2.81(s,1H),2 .18-2.45(m,2H),1.66-1.75(m,4H),1.43-1.51(m,4H),1.26(s,54H),0.86-0.92(m,6H).

[0351] 7 Preparation of DL0142

[0352] Compound 9 (390 mg, 0.48 mmol) was dissolved in DCM (7.00 mL), and DIEA (0.16 mL, 0.98 mmol), DMAP (9.91 mg, 0.08 mmol), and compound 10 (98.28 mg, 0.982 mmol) were added. The reaction was incubated at 25°C for 3 hours. LCMS analysis indicated the presence of the desired product. The reaction mixture was dried, and the crude product was purified by prep-HPLC (column: Waters Xbridge BEH C18 19*150 mm; conditions: TEAA-ACN; Begin B 65-95; gradient time: 15 minutes; 100% B hold time: 2 minutes; flow rate: 15 ml / min) to yield DL0142 (106 mg, 0.08 mmol, 53.9%) as a white solid. 1 H NMR (400MHz, CDCl3) δ7.31-7.39(m,2H),7.21-7.27(m,5H),7.12-7.17(m,1H),6.89-6.96(m, 1H),6.73-6.79(m,4H),4.29-4.40(m,1H),3.94-4.04(m,1H),3.77-3.92(m,3H),3.70-3.76( m,6H),3.67(d,J=4.0Hz,1H),3.29-3.51(m,6H),3.04-3.28(m,5H),2.94(q,J=8.0Hz,3H),2. 47-2.59(m,4H),2.17-2.27(m,2H),1.43-1.61(m,8H),1.18-1.26(m,54H),0.78-0.85(m,6H)

[0353] Example 1.15 Preparation of DL0143

[0354] 1. Preparation of Compound 3

[0355] Compound 2 (2.00 g, 11.0 mmol) was dissolved in THF (60.0 mL) at 25°C. TBAB (0.71 g, 2.20 mmol), compound 1 (10.5 mL, 43.9 mmol), and KOH (3.08 g, 54.9 mmol) were added sequentially to the mixture. The reaction mixture was stirred at 25°C for 12 hours. Thin-layer chromatography (PE / EA = 10 / 1) showed complete reaction of the starting material, with the formation of new spots. The reaction solution was diluted with EA (50.0 mL) and washed sequentially with water (30.0 mL × 3), saturated citric acid solution (30.0 mL × 3), water (30.0 mL × 3), and saturated NaCl solution (30.0 mL × 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (PE / EA = 97 / 3 to 96 / 4) to give compound 3 (5.21 g, yield 91.48%) as a colorless oil. 1 H NMR (400MHz, CD3OD) δ7.21-7.38(m,5H),4.53(s,2H),3.43-3.65(m,9H),1.49-1.62(m,4H),1.25-1.37(m,36H),0.87-0.93(m,6H)

[0356] 2. Preparation of Compound 4

[0357] Compound 3 (2.77 g, 5.34 mmol) was added to EtOH (30.0 mL), followed by the addition of Pd(OH)2 (0.30 g, 0.214 mmol). The system was then degassed with hydrogen (14.696 psi) three times, and the reaction was allowed to proceed in an oil bath at 50°C for 12 hours. Thin-layer chromatography (PE / EA = 10 / 1) showed the disappearance of the starting material and the formation of new spots. The reaction mixture was filtered through celite, and the organic phase was concentrated under reduced pressure to afford the crude product. The crude product was purified by column chromatography (PE / EA = 95 / 5 to 94 / 6) to afford crude compound 4 (2.20 g) as a colorless oil. 1 H NMR (400MHz, CD3OD) δ3.42-3.62(m,9H),1.50-1.63(m,4H),1.27-1.39(m,36H),0.84-0.97(m,6H)

[0358] 3. Preparation of Compound 5

[0359] Compound 4 (700 mg, 1.63 mmol) was dissolved in DCM (10.0 mL) and DMF (5.00 mL) at 25°C, and PDC (2.15 g, 5.71 mmol) was added. The mixture was stirred at 25°C for 12 hours. Thin-layer chromatography (PE / EA = 3 / 1) showed the disappearance of the starting material and the formation of new spots. Water (50.0 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (100 mL x 3). The organic phase was concentrated under reduced pressure and dried to give the crude product. The crude product was purified by column chromatography (PE / EA = 5 / 1 to 3 / 1) to give Compound 5 (320 mg, 44.27% yield) as a white solid. 1 H NMR (400MHz, CDCl3) δ4.05 (dd, J = 3.2, 5.2Hz, 1H), 3.78-3.83 (m, 1H), 3.69-3.75 (m, 1H), 3.64 (t ,J=6.4Hz,2H),3.42-3.56(m,2H),1.52-1.70(m,4H),1.23-1.37(m,36H),0.89(t,J=6.4Hz,6H)

[0360] 4. Preparation of Compound 7

[0361] Compound 5 (320 mg, 0.723 mmol) was added to DCM (5.00 mL) at 25°C, followed by HATU (412 mg, 1.08 mmol) and DIEA (0.358 mL, 2.17 mmol). The mixture was stirred at 25°C for 0.5 hour, and then compound 6 (158 mg, 0.867 mmol) was added. The reaction mixture was stirred at 25°C for 12 hours. TLC (PE / EA = 3 / 1, PMA) showed complete consumption of the starting material, with the formation of new spots. Dichloromethane (100 mL) was added to the reaction mixture, and the mixture was washed three times with saturated sodium bicarbonate solution (15.0 mL x 3) and saturated brine (15.0 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=3 / 1 to 1 / 1, PMA) to give a colorless liquid compound 7 (200 mg, yield 48.55%). 1 H NMR (400MHz, CD3OD) δ3.86 (dd, J=3.2, 5.2Hz, 1H), 3.64-3.70 (m, 4H), 3.40-3.63 (m, 5H), 3.20 -3.24(m,2H),2.33(t,J=7.6Hz,2H),1.47-1.69(m,8H),1.22-1.43(m,38H),0.86-0.94(m,6H)

[0362] 5. Preparation of Compound 8

[0363] Compound 7 (200 mg, 0.351 mmol) was dissolved in THF (4.00 mL) and H₂O (2.00 mL) at 25°C, and LiOH (44.2 mg, 1.05 mmol) was added. The mixture was stirred at 25°C for 12 hours. Thin-layer chromatography (PE / EA = 3 / 1, PMA) showed complete consumption of the starting material, with the formation of new spots. The reaction mixture was adjusted to pH 5 with 1M HCl, and the product was extracted with dimethyltetrahydrofuran (30.0 mL x 3). The organic phase was dried under reduced pressure to afford crude compound 8 (200 mg) as a colorless oil. 1 H NMR(400MHz,CD3OD)δ3.83-3.89(m,1H),3.64-3.71(m,1H),3.39-3.63(m,5H),3.18-3.2 8(m,2H),2.29(t,J=7.6Hz,2H),1.50-1.68(m,8H),1.24-1.40(m,38H),0.86-0.94(m,6H)

[0364] 6. Preparation of Compound 10

[0365] Compound 8 (193 mg, 0.347 mmol) was added to DCM (5.00 mL) at 25°C, followed by HATU (165 mg, 0.433 mmol) and DIEA (0.143 mL, 0.867 mmol). The reaction mixture was stirred at 25°C for 0.5 hour, and then compound 9 (130 mg, 0.289 mmol) was added. Stirring was continued for 12 hours. LCMS showed the formation of the product. TLC (DCM / MeOH = 1 / 1) showed the formation of new spots. Dichloromethane (100 mL) was added to the reaction mixture, and the mixture was washed three times with saturated sodium bicarbonate solution (15.0 mL x 3) and saturated brine (15.0 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product compound. The crude product was purified by column chromatography (DCM / MeOH=10 / 1 to 8 / 1, PMA) to give a colorless liquid compound 10 (180 mg, yield 63.04%). 1H NMR(400MHz,CD3OD)δ7.69-7.87(m,1H),7.43(d,J=8.0Hz,1H),7.17-7.33(m,6H) ,7.07-7.15(m,1H),6.79-6.91(m,4H),3.83-4.05(m,2H),3.74-3.81(m,6H),3.6 3-3.73(m,3H),3.56-3.63(m,3H),3.40-3.56(m,6H),3.32-3.40(m,1H),3.15-3. 28(m,4H),2.14-2.46(m,2H),1.46-1.68(m,8H),1.28(s,34H),0.84-0.94(m,6H)

[0366] 7. Preparation of Compound DL0143

[0367] Compound 10 (180 mg, 0.182 mmol) was dissolved in DCM (5.00 mL) at 25°C, followed by the addition of compound 11 (109 mg, 1.09 mmol), DIEA (0.181 mL, 1.09 mmol), and DMAP (5.57 mg, 0.046 mmol). The reaction mixture was stirred at 25°C for 12 hours. LCMS indicated the formation of the product. The reaction mixture was directly suspended to dryness under reduced pressure to obtain the crude product, which was then separated by preparative chromatography on a reverse-phase column (column: 01-Waters Xbridge BEH C18 19*150 mm; mobile phase: TEAA-ACN; gradient: 75%-95% / 18 min; flow rate: 15 ml / min) to obtain compound DL0143 (110 mg, yield 55.49%, purity 99.49%) as a colorless oil. 1 H NMR(400MHz,CD3OD)δ7.43(d,J=7.6Hz,2H),7.18-7.33(m,7H),6.84-6.87(m,4 H),4.09-4.28(m,2H),3.83-4.03(m,4H),3.78(d,J=2.0Hz,7H),3.61-3.70(m, 2H),3.41-3.61(m,7H),3.33-3.41(m,1H),3.10-3.26(m,4H),2.51-2.66(m,4H ),2.18-2.44(m,2H),1.45-1.69(m,8H),1.20-1.43(m,41H),0.85-0.93(m,6H)

[0368] Example 1.16 Preparation of DL0144

[0369] 1. Preparation of Compound 3

[0370] Compound 1 (1.00 g, 2.59 mmol) was dissolved in DCM (16.0 mL) at 25°C. Pyridine (Py) (0.418 mL, 5.17 mmol) was added, and the temperature was lowered to 0°C. Compound 2 (0.630 g, 3.10 mmol) was then added, and the reaction mixture was stirred at 25°C for 2 hours. Thin-layer chromatography (PE / EA = 3 / 1, PE / EA = 10 / 1) showed that the starting material was not completely reacted, with new spots formed. The reaction mixture was diluted with DCM (50.0 mL) and washed with saturated aqueous NaHCO₃ (20.0 mL x 3). The organic phase was washed with saturated aqueous sodium chloride (20.0 mL x 3), then dried over Na₂SO₄ and spun down to dryness to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 0 to 10 / 1) to yield Compound 3 (800 mg, 55.94%) as a white solid. 1 H NMR (400MHz, CDCl3) δ8.22-8.34(m,2H),7.36-7.44(m,2H),5.44(d,J=4.8Hz,1H),4. 56-4.68(m,1H),2.43-2.55(m,2H),1.68-2.10(m,7H),1.44-1.54(m,4H),1.24-1.40 (m,4H),0.95-1.24(m,14H),0.92(d,J=6.4Hz,3H),0.87(dd,J=6.57,1.6Hz,6H),0.69(s,3H).

[0371] 2. Preparation of Compound 5

[0372] Compound 4 (200 mg, 1.10 mmol) was dissolved in DCM (10.0 mL) at 25°C. TEA (0.656 mL, 4.72 mmol) and compound 3 (434 mg, 0.787 mmol) were added sequentially. The reaction was stirred at 25°C for 16 hours. Thin-layer chromatography (PE / EA = 10 / 1, PE / EA = 3 / 1) showed complete consumption of the starting material and the formation of new spots. The reaction solution was diluted with DCM (50.0 mL) and washed with saturated aqueous NaHCO₃ (20.0 mL x 3). The organic phase was then washed with saturated aqueous sodium chloride (20.0 mL x 3) and dried over Na₂SO₄. The organic phase was then spin-dried to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 0 to 5 / 1) to obtain crude compound 5 (380 mg) as a white solid. 1H NMR (400MHz, CDCl3) δ5.35-5.41(m,1H),4.42-4.54(m,1H),3.68(s,3H),3.1 7(q,J=6.4Hz,2H),2.24-2.40(m,4H),1.92-2.05(m,2H),1.78-1.90(m,3H), 1.65(dt,J=15.2,7.2Hz,3H),1.46-1.54(m,6H),1.23-1.45(m,8H),0.97-1. 22(m,13H),0.92(d,J=6.4Hz,3H),0.87(dd,J=6.4,1.6Hz,6H),0.68(s,3H).

[0373] 3. Preparation of Compound 6

[0374] Compound 5 (380 mg, 0.681 mmol) was dissolved in a mixture of THF (5.00 mL) and H₂O (2.50 mL) at 25°C. LiOH (32.6 mg, 1.36 mmol) was added, and the reaction mixture was allowed to react at 25°C for 18 hours. Thin-layer chromatography (PE / EA = 3 / 1) showed complete consumption of the starting material and the formation of new spots. 1M HCl (10.0 mL) was added to the reaction mixture to adjust the acidity. DCM (20.0 mL) was then added, and the organic phase was collected, dried over Na₂SO₄, and spin-dried to afford Compound 6 (230 mg, 62.09% yield) as a white solid. 1 H NMR (400MHz, CDCl3) δ5.36-5.40(m,1H),4.45-4.56(m,1H),3.18(q,J=6.4Hz,2H),2.27-2.41(m,4H),1.96-2.05(m,2H),1.67(dt,J=15. 2,7.2Hz,3H),1.46-1.59(m,9H),1.29-1.44(m,8H),0.97-1.18(m,13H),0.92(d,J=6.4Hz,3H),0.87(dd,J=6.4,1.6Hz,6H),0.68(s,3H).

[0375] 4. Preparation of Compound 8

[0376] Compound 6 (232 mg, 0.427 mmol) was dissolved in DCM (10.0 mL) at 25°C. HATU (203 mg, 0.534 mmol) and DIEA (0.353 mL, 2.14 mmol) were added sequentially. The reaction was stirred at 25°C for 0.5 h, followed by the addition of compound 7 (160 mg, 0.356 mmol). The reaction was stirred at 25°C for 16 h. LCMS showed the presence of product. Thin-layer chromatography (DCM / MeOH = 10 / 1) revealed the formation of new spots. The reaction was diluted with DCM (20.0 mL) and washed sequentially with saturated aqueous citric acid (5.00 mL x 3), saturated aqueous NaHCO₃ (5.00 mL x 3), and saturated aqueous sodium chloride (5.00 mL x 3). The organic phase was then dried over anhydrous Na₂SO₄ and spun down to yield the crude product. The crude product was purified by column chromatography (DCM / MeOH=1 / 0-10 / 1) to obtain crude compound 8 (500 mg) as a colorless oily liquid. 1 H NMR (400MHz, CDCl3) δ7.27-7.34(m,5H),7.15-7.21(m,4H),6.81-6.87(m,4H),5.35-5.41(m,1H),4.6 3-4.80(m,1H),4.31-4.52(m,1H),3.86-3.94(m,2H),3.79-3.83(m,6H),3.57-3.74(m,7H),3.47-3.55 (m,1H),3.12-3.22(m,2H),2.24-2.41(m,4H),1.81-2.05(m,6H),1.45-1.59(m,11H),1.21-1.31(m,4 H),1.08-1.17(m,6H),0.95(d,J=5.2Hz,6H),0.90-0.94(m,3H),0.86-0.89(m,6H),0.66-0.71(m,3H).

[0377] 5. Preparation of DL0144

[0378] Compound 8 (500 mg, 0.308 mmol) was dissolved in DCM (10.0 mL) at 25°C. DIEA (0.305 mL, 1.85 mmol), DMAP (15.0 mg, 0.123 mmol), and compound 9 (185 mg, 1.85 mmol) were added sequentially. The reaction mixture was stirred at 25°C for 2 hours. LCMS showed the presence of product mass. The reaction mixture was spin-dried to obtain the crude product. The crude product was separated by reverse-phase chromatography (column: 01-Waters Xbridge BEH C18 19*150 mm; mobile phase: TEAA-ACN; B%: 75%-95% over 15 min; flow rate: 15 ml / min) to obtain compound DL0144 as a white solid (218 mg, yield 65.91%, purity 91.17%). 1 H NMR (400MHz, CDCl3) δ7.37-7.46 (m, 2H), 7.27-7.35 (m, 7H), 6.83 (d, J = 8.8Hz, 4H), 5.38 (s, 1H), 4.33-4. 72(m,2H),3.83-4.04(m,3H),3.79-3.81(m,6H),3.05-3.58(m,8H),2.96(q,J=7.2Hz,5H),2.49-2.69(m ,5H),2.23-2.40(m,5H),1.79-2.05(m,6H),1.42-1.62(m,11H),1.31-1.39(m,4H),1.26(t,J=7.2Hz,7H ),1.08-1.18(m,6H),0.97-1.07(m,6H),0.92(d,J=6.4Hz,3H),0.87(dd,J=6.4,1.6Hz,6H),0.68(s,3H).

[0379] Example 1.17 Preparation of DL0145

[0380] 1 Preparation of compound 3

[0381] Compound 2 (134 mg, 0.667 mmol) was dissolved in DCM (10.0 mL) at 25°C. HATU (380 mg, 1.00 mmol) and DIEA (0.331 mL, 2.00 mmol) were added sequentially. The mixture was stirred at 25°C for 0.5 hour, and then compound 1 (300 mg, 0.667 mmol) was added. The reaction mixture was stirred at 25°C for 12 hours. Thin-layer chromatography (DCM / MeOH = 10 / 1, PMA) and (DCM / MeOH = 10 / 1, PMA) showed complete reaction of the two starting materials, with the formation of new spots. Ethyl acetate (10.0 mL) was added, and the mixture was washed three times with brine (10 mL x 3). The organic phase was concentrated under reduced pressure to yield the crude product. The crude product was purified by column chromatography (DCM / MeOH=93 / 7 to 90 / 10) to give compound 3 (412 mg, yield 97.71%) as a colorless oil. 1 H NMR(400MHz,METHANOL-d4)δ7.39-7.51(m,2H),7.16-7.35(m,7H),6.86(dt,J=2.89,5.83Hz, 4H),3.79-4.04(m,2H),3.78(d,J=1.25Hz,6H),3.41-3.76(m,5H),3.32-3.38(m,1H),3.18(br d,J=6.27Hz,1H),2.94-3.14(m,1H),2.16-2.42(m,2H),1.52(br d,J=6.02Hz,2H),1.19-1.39(m,18H),0.87-0.92(m,3H)

[0382] 2 Preparation of DL0145

[0383] Compound 3 (200 mg, 0.317 mmol) was dissolved in DCM (3.00 mL) at 25°C. DIEA (0.314 mL, 1.90 mmol), DMAP (9.67 mg, 0.079 mmol), and compound 4 (31.7 mg, 0.317 mmol) were added sequentially to the mixture. The reaction mixture was stirred at 25°C for 3 hours. Thin-layer chromatography (DCM / MeOH = 10 / 1) indicated complete reaction of the starting material, with the formation of new spots. LCMS analysis also indicated complete reaction of the starting material. The reaction mixture was diluted with dichloromethane (10.0 mL) and washed three times with saturated NaHCO₃ solution (10.0 mL x 3) and saturated NaCl solution (10.0 mL x 3). The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (DCM / MeOH = 95 / 5 to 90 / 10) to obtain the crude product. The crude product was purified by Prep-HPLC (chromatographic column: Waters Xbridge BEH C18 100*25mm*5um; mobile phase: TEAA-ACN; gradient: 45%-95% / 16min; flow rate: 15ml / min) to give the colorless oily product DL0145 (84.0mg, yield 36.26%, purity 98.70%). 1 H NMR (400MHz, METHANOL-d4) δ7.43(br d,J=7.53Hz,2H),7.18-7.36(m,7H),6.82-6.93(m,4H),4.16-4.27(m,1H),4.11(d,J=5.52Hz,1H),3.97(br d,J=11.29Hz,1H),3.85(br s,1H),3.78(s,6H),3.43-3.75(m,3H),3.34-3.43(m,1H),3.10-3.28(m,5H),2.55-2.64(m,2H),2.52(d,J=6.02Hz,2H),2.33(br d,J=12.30Hz,2H),1.52(br s,2H),1.18-1.38(m,21H),0.83-0.95(m,3H)

[0384] Example 2: Synthesis of siRNA

[0385] 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.

[0386] 1. Preparation of siRNA with no ligand attached to the 3' end of the sense strand

[0387] 1.1 Synthesis of the positive sense chain (SS chain)

[0388] Using solid-phase phosphoramidite synthesis, a blank CPG solid support is used as the starting cycle, and nucleoside monomers are linked one by one in the 3'-5' direction according to the order of the positive strand nucleotides. Each nucleoside monomer linking involves four steps: deprotection, coupling, capping, and oxidation or thiolation, resulting in a 5 μmol oligonucleotide. The synthesis conditions are as follows:

[0389] 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).

[0390] 1.2 Synthesis of antisense strand (AS strand)

[0391] Using solid-phase phosphoramidite synthesis, a blank CPG solid support is used as the starting cycle. Nucleoside monomers are linked one by one in the 3'-5' direction according to the nucleotide arrangement of the antisense strand. Each ligation step involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for a 5 μmol oligonucleotide of the antisense strand are the same as those for the sense strand.

[0392] 1.3 Oligonucleotide purification and annealing

[0393] 1.3.1 Ammonolysis

[0394] 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.

[0395] 1.3.2 Purification

[0396] 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.

[0397] 1.3.3 Annealing

[0398] 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.

[0399] Test Examples

[0400] Example 3: In vivo activity testing (CNS delivery)

[0401] 1. Single-strand sequence information

[0402] In this article, the meanings of the abbreviations are as follows:

[0403] A, U, G and C represent the natural ribonucleotides adenine, uracil, guanine and cytosine, respectively.

[0404] 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.

[0405] i represents inosine ribonucleotide.

[0406] 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.

[0407] 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.

[0408] "s" indicates that the two adjacent nucleotides and / or delivery vectors are linked by phosphorothioate.

[0409] VP indicates that the adjacent nucleotide on the right is a vinyl phosphate-modified nucleotide.

[0410] Ib represents an inverted abasic deoxyribonucleotide, which can include the following three structures depending on its position / linking method in siRNA.

[0411] 2. Double-stranded sequences used

[0412] 3. Experimental Methods

[0413] Experimental animals: SD rats, male, 6-8 weeks old, 2-3 rats per group;

[0414] Experimental operation:

[0415] After the animal is anesthetized with isoflurane gas, it is placed on a constant temperature electric blanket. The skin on the buttocks and back is prepared and disinfected with iodine and 75% alcohol. Eye ointment is applied.

[0416] Accurately locate the L3-L5 level and incise the skin to expose the spine. Puncture through the intervertebral foramen. After the animal clearly flicks its tail (as a sign of successful puncture), slowly inject 30uL (0.9mg, 30mg / mL) of the corresponding compound. Leave the needle in place for 15-30s after injection, and then suture the skin.

[0417] Postoperative care of animals: Meloxicam and antibiotics were injected subcutaneously;

[0418] Fourteen days after injection, all animals were euthanized, and the cervical and thoracic spinal cords were removed. Brain tissue was harvested and the cerebellum, brainstem, hippocampus, and frontal cortex were separated and stored in RNAlater for subsequent SOD1 mRNA extraction and QPCR detection.

[0419] Methods for mRNA extraction and QPCR detection are well known in the art, and the primers used are as follows:

[0420] The residual inhibition rate was calculated using the following formula:

[0421] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate. ΔΔCt = [(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)].

[0422] The target gene was SOD1, the internal reference was GAPDH, and the control group was injected with artificial cerebrospinal fluid (aCSF).

[0423] 4. Experimental Results

[0424] The residual inhibition data are shown in the table below (also see Figure 1). DR005713, DR005714, DR005715, DR005717, DR005718, DR005716, and DR005735 all reduced SOD1 expression in the central nervous system. DR005716 and DR005735 demonstrated superior knockdown effects compared to the other sequences.

[0425] Example 4: In vivo activity assay (ocular delivery)

[0426] The sequence information used in this example is as follows:

[0427] Drug administration and isolation of ocular tissue

[0428] C57BL / 6 mice (male, 6-8 weeks) were randomly divided into groups and administered a single dose of 2 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 diluted to the desired solution concentration and volume. The administration volume of phosphate buffer solution and siRNA conjugate was 1.5 μL / eye.

[0429] On the 14th day after administration, the eyeballs were removed and separated into three parts: ① retinal pigment epithelium (RPE) + choroid + sclera; ② retina; ③ cornea + iris + ciliary body; the separated samples were immediately frozen in liquid nitrogen and then stored at -80℃ for detection of mTTR mRNA.

[0430] RNA extraction and detection

[0431] 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); reference was made to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B) for reverse transcription; reference TaqMan TM Fluorescence quantitative PCR reaction (ABI, QuantStudio3) was performed with 20 μL of Fast Advanced Master Mix (ABI, 4444965). The primers are shown in the table below.

[0432] Statistics

[0433] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate;

[0434] △△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)].

[0435] The target gene was mTTR, and the internal reference was mGAPDH.

[0436] The experimental results are shown in Figure 2. The results showed that DR005938, DR007050, and DR007871 could all reduce TTR gene expression in the eye, with the reduction effect being comparable to or better than that of the positive sequence DR005933.

[0437] 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. A compound of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, L1 and L2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support; R s Selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated; m = 0, 1, 2, 3, 4, 5 or 6; R is -C(O)-C 0-10 Alkylene-L-R1, -C(O)-C 2-10 Alkenylene-L-R1 or -C(O)-C 2-10 Alkynylidene-L-R1; L is a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -NHC(O)-CH(OR1)CH2O-, -C(O)NH-CH(OR1)CH2O-, -OC(O)-CH(OR1)CH2O- , -C(O)O-CH(OR1)CH2O-, -NHC(O)-CH(R1)-, -C(O)NH-CH(R1)-, -OC(O)-CH(R1)-, -C(O) O-CH(R1)-, -CH(OR1)CH2O-, -O-CH(R1)CH2O-, -O-CH2CH(R1)O-, -O-CH(CH(OH)CH2OH)- , -O-CH(CH(NH2)CH2OH)-, -O-CH(CH2OH)CH(OH)-, -NH-CH(CH2OH)CH(OH)-, -O-CH2CH(OH)CH(OH)-, -O-CH2CH(NH2)CH(OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)-, -NHC(O)-CH2 -O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-O-CH2CH(OH)CH(OH)-, or -NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-; R1 is independently C 1-30 Alkyl, C 2-30 Alkenyl or C 2-30 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; Among them C 0-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 1-30 Alkyl, C 2-30 Alkenyl and C 2-30 The hydrogen atoms in the alkynyl group may be optionally replaced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more halogen, C 1-6 Alkyl or C 1-6 The group is replaced by a haloalkyl group, which is optionally deuterated, up to full deuteration.

2. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R is -C(O)-C 0-10 Alkylene-L-R1, preferably -C(O)-L-R1, preferably -C(O)-C 2-8 Alkylene-L-R1, more preferably -C(O)-C 3-7 Alkylene-L-R1, more preferably -C(O)-C 4-6 Alkylene-L-R1, more preferably -C(O)-C 1-3 Alkylene-L-R1.

3. The compound of formula (I) according to claim 1 or 2, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: L is a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -O-CH(CH(O H)CH2OH)-, -O-CH(CH(NH2)CH2OH)-, -O-CH(CH2OH)CH(OH)-, -NH-CH(CH2OH)CH(OH)-, -O-CH2CH(OH)CH(OH)-, -O- CH2CH(NH2)CH(OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)-, -NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O- CH(CH2OH)CH(OH)-, -NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-O-CH2CH(OH)CH(OH)- or -NHC(O)-CH2-O-CH 2CH(NH2)CH(OH)-, preferably a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -O-CH(CH(OH)CH2OH)-, -O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)- or -NHC(O)-CH2-O-CH(C H(NH2)CH2OH)-, preferably a chemical bond, -NHC(O)-, -SS-, -NHC(O)O-, -O-CH(CH(OH)CH2OH)-, -O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)- or -NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-, more preferably a chemical bond, -NHC(O)-, -SS- or -NHC(O)O-, more preferably -NHC(O)-.

4. The compound of formula (I) according to claim 1 or 2, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L is -NHC(O)-CH(OR1)CH2O-, -C(O)NH-CH(OR1)CH2O-, -OC(O)-CH(OR1)CH2O-, -C(O)O-CH(OR1)CH2O-, -NHC(O)-CH(R1)-, -C(O)NH-CH(R1)-, -OC(O)-CH(R1)-, -C(O)O-CH(R1)-, -CH(OR1)CH2O-, -O-CH(R1)CH2O-, -O-CH2CH(R1)O-, preferably -NHC(O)-CH(OR1)CH2O-, -NHC(O)-CH(R1)- or -CH(OR1)CH2O-, more preferably -NHC(O)-CH(OR1)CH2O-.

5. A compound of formula (I) according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R1 is independently C 1-30 Alkyl or C 2-30 alkenyl, wherein non-adjacent 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH2CH2- group may be replaced by -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; preferably, R1 is independently C 5-25 Alkyl, C containing 1, 2, 3, 4, 5 or 6 double bonds 10-25 alkenyl, C in which 1, 2, 3, 4 or 5 carbon atoms are replaced by N heteroatoms and / or 1, 2 or 3 -CH2CH2- groups are replaced by -C(O)NH- 5-25 Alkyl, or one or more substituents on the carbon atom connected to form the C 5- 25 Alkyl; preferably, R1 is selected from the following groups: C6 alkyl, C8 alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 21 alkyl, 6. A compound of formula (I) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: One of L1 and L2 is a reactive phosphorus group, preferably a phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate or phosphate mimetic, such as natural phosphates, phosphorothioates, phosphorodithioates, boranophosphates, boranophosphorothioates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiester, phosphotriester, phosphorothioate diester, phosphorothioate triester, diphosphate or triphosphate, preferably -P(OCH2CH2CN)(N(iPr)2).

7. A compound of formula (I) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: L1 and L2 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 -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, more preferably -C(O)CH2CH2C(O)OH.

8. A compound of formula (I) according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, selected from the following general formula: wherein each group is as defined in claims 1-7.

9. The compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein The compound is selected from the following:

10. An oligonucleotide comprising one or more compounds of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, represents H or a hydroxyl protecting group, or represents the position of attachment to the adjacent nucleotide; L2 represents H or a solid support, or represents the position where it is connected to the adjacent nucleotide; R s Selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated; m = 0, 1, 2, 3, 4, 5 or 6; R is a hydrophobic group; Preferably, represents H or a hydroxyl protecting group, or represents the position of attachment to the adjacent nucleotide; L2 represents H or a solid support, or represents the position where it is connected to the adjacent nucleotide; R s , m and R are as defined in any one of claims 1-5.

11. The oligonucleotide of claim 10, wherein the compound of formula (I') is selected from the following compounds of the general formula, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: in, represents H or a hydroxyl protecting group, or represents the position of attachment to the adjacent nucleotide; L2 represents H or a solid support, or represents the position where it is connected to the adjacent nucleotide; The other groups are as defined in claims 1 to 5.

12. The oligonucleotide of claim 10, wherein the compound of formula (I') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, wherein: The compound is selected from the following: in One of them represents H or a hydroxyl protecting group, or represents the position of attachment to the adjacent nucleotide, and the other Represents H or a solid support, or indicates the position where it is linked to the adjacent nucleotide.

13. The oligonucleotide of any one of claims 10 to 12, having 14 to 30 nucleotides.

14. The oligonucleotide according to any one of claims 10 to 13, comprising at its 5' end a compound of formula (I') according to any one of claims 10 to 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

15. The oligonucleotide according to any one of claims 10 to 14, comprising at its 3' end a compound of formula (I') according to any one of claims 10 to 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

16. The oligonucleotide according to any one of claims 10 to 15, comprising one compound of formula (I') according to any one of claims 10 to 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at each of the 5' and 3' ends.

17. An oligonucleotide comprising two or more hydrophobic groups within the oligonucleotide, at the 5' end and / or at the 3' end; preferably, the hydrophobic groups are as defined for the R groups in the compound of formula (I); preferably, the hydrophobic groups are connected to the oligonucleotide via a linker, such as a biodegradable linker.

18. 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 the antisense strand comprises one or more compounds of formula (I'), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof: in, represents H, or indicates the position of attachment to the adjacent nucleotide; L2 represents H or a hydroxyl protecting group, or represents the position of connection with the adjacent nucleotide; R s Selected from H, D, halogen, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated; m = 0, 1, 2, 3, 4, 5 or 6; R is a hydrophobic group; Preferably, represents H, or indicates the position of attachment to the adjacent nucleotide; L2 represents H or a hydroxyl protecting group, or represents the position of connection with the adjacent nucleotide; R s , m and R are as defined in any one of claims 1-5.

19. The double-stranded RNA according to claim 18, wherein the compound of formula (I') is selected from the following compounds of the general formula, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: in, represents H, or indicates the position of attachment to the adjacent nucleotide; L2 represents H or a hydroxyl protecting group, or represents the position of connection with the adjacent nucleotide; R s , m and R are as defined in claims 1-5.

20. The double-stranded RNA of claim 18, wherein the compound of formula (I') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, wherein: The compound is selected from the following: in One of them represents H, or represents the position of connection with the adjacent nucleotide, and the other represents H or a hydroxyl protecting group, or the position of attachment to the adjacent nucleotide.

21. The double-stranded RNA according to any one of claims 18 to 20, wherein the sense strand comprises a compound of formula (I') according to any one of claims 10 to 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 5' end.

22. The double-stranded RNA according to any one of claims 18 to 21, wherein the sense strand comprises a compound of formula (I') according to any one of claims 10 to 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 3' end.

23. The double-stranded RNA according to any one of claims 18 to 22, wherein the sense strand comprises a compound of formula (I') according to any one of claims 10 to 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at each of the 5' and 3' ends.

24. The double-stranded RNA of any one of claims 18 to 23, wherein two or more compounds of formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, on the sense and / or antisense strands are separated by at least 5 to 30 nucleotides.

25. The double-stranded RNA of any one of claims 18 to 24, wherein the double-stranded RNA comprises two compounds of formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, located at any two of the following sites: the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand and the 3' end of the antisense strand; preferably at the 5' end of the sense strand and the 3' end of the sense strand.

26. The double-stranded RNA of any one of claims 18 to 25, wherein the double-stranded RNA comprises three compounds of formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, located at any three of the following sites: the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand and the 3' end of the antisense strand; preferably located at the 5' end of the sense strand, the 3' end of the sense strand and the 3' end of the antisense strand.

27. The double-stranded RNA of any one of claims 18 to 26, wherein the double-stranded RNA comprises four compounds of formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, located at the following positions: the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand and the 3' end of the antisense strand.

28. The double-stranded RNA according to any one of claims 18 to 26, further comprising a terminal phosphate protecting group or a prodrug protecting group coupled to the 5' end of the antisense strand, preferably a vinyl phosphate group or a prodrug protecting group represented by formula (X): in, X1 is selected from OH or R a Selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, which is optionally deuterated, up to fully deuterated; R b and R c Independently selected from H, C 1-6 Alkyl or C 1-6 haloalkyl, the R b and R c Can be optionally D, C 6-10 Aryl or 5-10 membered heteroaryl substitution, up to full deuteration; X2 is a chemical bond connected to the first nucleotide at the 5' end of the antisense strand, preferably connected through a hydroxyl group; X3 is independently selected from O or S; T is selected from Each R T1 Independently selected from H, D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2- 6 alkynyl groups or a chain comprising GalNAc, which is optionally deuterated, up to fully deuterated; Each R T2 Independently selected from H, D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, which is optionally deuterated, up to fully deuterated; Each R T3 Independently selected from H, D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, which is optionally deuterated, up to fully deuterated; Each R T4 Independently selected from H, D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, which is optionally deuterated, up to fully deuterated; m is 0, 1, 2, 3, 4 or 5; n is 0, 1, 2, 3, 4 or 5; p is 0, 1, 2, 3, 4, or 5; X is selected from a chemical bond, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -; R X1 Selected from H, C 1-6 Alkyl or C 1-6 haloalkyl, which is optionally deuterated, up to fully deuterated; L is -Ar-(CH2) 1-6 -O-, wherein each CH2 may be optionally substituted by R#, R# is selected from H, D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, which is optionally deuterated, up to fully deuterated; Ar in L is connected to X, and the oxygen atom is connected to the phosphorus atom; Ar is selected from C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl, the C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl may be optionally substituted with 1, 2, 3, 4 or 5 R*; R* is selected from H, D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, which is optionally deuterated, up to fully deuterated; wherein P1 is selected from a protecting group, preferably 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), 4,4'-dimethoxytrityl, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH, preferably -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH.

29. The double-stranded RNA according to any one of claims 18 to 28, which is selected from small interfering RNA (siRNA) and short hairpin RNA (shRNA), preferably for inhibiting a gene expressed in the eye.

30. A double-stranded RNA comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand comprises a sequence that is fully complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand comprises two or more hydrophobic groups internally, at the 5' end and / or at the 3' end; preferably, the hydrophobic groups are as defined for the R groups in the compound of formula (I); preferably, the hydrophobic groups are connected to the sense strand and / or the antisense strand via a linker, such as a biodegradable linker.

31. A cell comprising the double-stranded RNA of any one of claims 18 to 30.

32. A pharmaceutical composition comprising the double-stranded RNA according to any one of claims 18 to 30, or the cell according to claim 31, and optionally a pharmaceutically acceptable carrier or excipient.

33. A kit comprising the double-stranded RNA according to any one of claims 18 to 30, or the cell according to claim 31.

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