Ligand-oligonucleotide conjugates and linkers therefor

Abstract

A ligand-oligonucleotide conjugate and a linker for the conjugate, the linker comprising a structure of Formula I or II:-NR1-C (O)-[C (R3R4)] n-C (O)-[NR2-C (RaRb)-C (O)] p-(Formula I), or-NR1-[C (O)-C (RaRb)-NR2] p-C (O)-[C (R3R4)] n-C (O)-(Formula II). The invention also relates to a preparation method and an intermediate of the conjugate.

Description

Ligand-oligonucleotide conjugate and linker for the conjugate

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on October 21, 2022, with application number 202211296452.X and application name “Ligand-oligonucleotide conjugate and linker for the conjugate”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to a ligand-oligonucleotide conjugate and a linker thereof, in particular a GalNAc-siRNA conjugate and a linker thereof. The present invention also relates to a preparation method of the ligand-oligonucleotide conjugate and the use of the linker. Background Art

[0003] RNA interference (RNAi) refers to antisense-mediated gene silencing achieved through the RNA-induced silencing complex (RISC) mechanism. Potential RNAi-based drug molecules include double-stranded RNA (siRNA) and shRNA. Although the mechanism of action of siRNA suggests its enormous potential in clinical applications and the pharmaceutical industry, its in vivo delivery still faces numerous challenges. After injection, naked siRNA is rapidly degraded by ribozymes in plasma or tissues. Furthermore, rapid renal clearance shortens the half-life of siRNA molecules in the bloodstream. Free siRNA has a large relative molecular mass (13,000–15,000) and carries a significant negative charge, making it difficult for it to cross cell membranes and exert its RNAi effect in the cytoplasm. Furthermore, the immunogenicity of free siRNA molecules in the bloodstream is a major limitation to its clinical application, causing severe immune responses during circulation.

[0004] Chemical modification of the base backbone of siRNA sequences can significantly enhance their stability against enzymatic degradation in the bloodstream; however, this enhanced chemical stability does not address the difficulty siRNAs face in penetrating cell membranes. Monoharan's team at Alnylam Pharmaceuticals covalently conjugated GalNAc trivalently to the 3' end of the sense strand of siRNA to create a GalNAc-siRNA conjugate. The intact GalNAc-siRNA conjugate enters the intracellular environment through clathrin-mediated endocytosis mediated by ASGPR, which is highly expressed on the surface of hepatocytes. As the pH within the endosomal lining decreases, the GalNAc-siRNA conjugate is released from the bound ASGPR. Subsequently, ASGPR recycles back to the cell surface, while the GalNAc-siRNA conjugate remains within the endosome. However, the mechanism of release of the GalNAc-siRNA conjugate from the endosome remains unclear. While the vast majority of free siRNA remains trapped within the endosome, a very small amount (<1%) is able to cross the endosomal lipid bilayer through unknown mechanisms to enter the cytoplasm and induce RNAi responses.

[0005] In addition to siRNA, GalNAc has also been used to deliver antisense oligonucleotides (ASOs) to hepatocytes. Prakash et al. demonstrated that using trivalent GalNAc to deliver second-generation gapmer antisense oligonucleotides enhanced their efficacy by 6-10-fold. When combined with a next-generation ASO design, efficacy was enhanced by approximately 60-fold, demonstrating robust liver-targeted delivery and efficacy enhancement (Prakash et al., Nucleic Acids Research, 2014, Vol. 42, No. 13, 8796–8807).

[0006] Currently, the clinical delivery methods of oligonucleotides are single and limited. It is still necessary to try more ligand-oligonucleotide conjugate structures to improve the performance of one or more aspects of the conjugate and ultimately improve the in vivo therapeutic effect of the conjugate.

[0007] Summary of the Invention

[0008] One aspect of the present invention provides a linker moiety, wherein the linker moiety is as shown in Formula I or II:

[0009] -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), or

[0010] -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II),

[0011] wherein each substituent and symbol have the meaning as defined herein.

[0012] Another aspect of the present invention provides a ligand-oligonucleotide conjugate comprising a linker moiety of the present invention.

[0013] Another aspect of the present invention provides an intermediate compound represented by formula III or IV:

[0014] (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -Q 2 (Formula III), or

[0015] (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-Q 2 (Formula IV),

[0016] wherein each substituent and symbol have the meaning as defined herein.

[0017] Another aspect of the present invention provides use of the above intermediate compound in preparing a ligand-oligonucleotide conjugate.

[0018] Another aspect of the present invention provides a method for preparing a ligand-oligonucleotide conjugate, comprising: providing any intermediate compound described above; providing an oligonucleotide having a terminal amino group at the 5' or 3' end; and covalently linking the intermediate compound to the oligonucleotide via the terminal amino group.

[0019] Another aspect of the present invention provides a linker represented by formula I or II:

[0020] -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), or

[0021] -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II),

[0022] wherein each substituent and symbol have the meaning as defined herein.

[0023] Another aspect of the present invention provides a linker unit represented by formula I' or II':

[0024] -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I'), or

[0025] -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II'),

[0026] wherein each substituent and symbol have the meaning as defined herein;

[0027] The left side of formula I' is connected to -NR1 - is Formula I, the left end of Formula II' is connected to -NR 1 - is Formula II.

[0028] Another aspect of the present invention provides use of the linker in preparing a ligand-oligonucleotide conjugate.

[0029] Another aspect of the present invention provides a pharmaceutical composition comprising any one of the ligand-oligonucleotide conjugates of the present invention and a pharmaceutically acceptable carrier.

[0030] Another aspect of the present invention provides a method for treating a disease, comprising administering to a subject a therapeutically effective amount of any one of the conjugates provided herein or a pharmaceutical composition comprising the conjugate.

[0031] Other aspects of the present invention will become apparent from the detailed description of the specification which follows. DETAILED DESCRIPTION

[0032] definition

[0033] The term "oligonucleotide" refers to a polydeoxyribonucleotide, a polyribonucleotide, or a polyribonucleotide / deoxyribonucleotide hybrid in this article, typically 15 to 30 nucleotides or nucleotide pairs in length, each nucleotide being natural or modified. The oligonucleotide can be double-stranded or single-stranded, including single-stranded and double-stranded DNA, single-stranded and double-stranded RNA (including shRNA, siRNA), and DNA / RNA hybrid molecules. In some embodiments of the invention, the oligonucleotide can be conjugated to another molecule, such as N-acetylgalactosamine (GalNAc) or its polymer (GalNAc cluster). In a preferred embodiment, the oligonucleotide for the present invention is selected from siRNA, shRNA, miRNA, and ASO.

[0034] The term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, and / or a modified nucleobase. Thus, the term modified nucleotide includes substitutions, additions, or removals of, for example, functional groups or atoms, to internucleoside linkages, sugar moieties, or nucleobases. Modifications suitable for use in the present invention include all types of modifications disclosed herein or known in the art. For example, the modified nucleotides are deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally constrained nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural base containing nucleotides, tetrahydropyran modified nucleotides, 1,5-deoxy modified nucleotides, A nucleotide modified with hydrohexitol, a cyclohexenyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a nucleotide comprising a 5'-methylphosphoester group, a nucleotide comprising a 5' phosphate or a 5' nucleotide phosphate analog, a nucleotide comprising vinyl phosphate, a nucleotide comprising adenosine-glycol nucleic acid (GNA), a nucleotide comprising the S-isomer of thymidine-glycol nucleic acid (GNA), a nucleotide comprising 2-hydroxymethyl-tetrahydrofuran-5-phosphate, a nucleotide comprising 2'-deoxythymidine-3' phosphate, a nucleotide comprising 2'-deoxyguanosine-3'-phosphate, or a terminal nucleotide linked to a cholesterol derivative and / or a dodecanoic acid bisdecanoylamino group. In preferred embodiments, the modified nucleotides are 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 3'-terminal deoxythymidine nucleotides (dT), locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates and / or non-natural base containing nucleotides.

[0035] Antisense oligonucleotide (ASO) is a single-stranded DNA or RNA complementary to a selected sequence. In the case of antisense DNA, it can be used to target specific complementary (coding or non-coding) RNA. If binding occurs, this hybrid can be degraded by RNAase H1. A typical example of ASO is gapmer, which has an internal "gap segment" with two external "wing segments" on its side, wherein the gap segment is composed of a plurality of nucleotides that support RNAse H cutting, and each wing segment is composed of one or more nucleotides that are chemically different from the nucleotides in the gap segment. For example, the 5' and 3' wing segments of gapmer are composed of 2'-MOE modified nucleotides, and the gap segment is composed of deoxyribonucleotides, and the connection between all nucleotides is optionally a phosphorothioate bond.

[0036] "siRNA" refers to a nucleic acid that forms double-stranded RNA that has the ability to reduce or inhibit the expression of a target gene when the siRNA and the target gene are present in the same cell. siRNAs are typically about 15 to about 30 base pairs in length, most typically about 19 to 25 base pairs in length, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs in length. 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs in length.

[0037] shRNA refers to short hairpin RNA, which includes two short inverted repeat sequences and an intermediate stem-loop structure connecting the two. The stem-loop may contain at least one unpaired nucleotide, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. In some embodiments, the stem-loop may be 10 or fewer nucleotides. In some embodiments, the stem-loop may be 8 or fewer unpaired nucleotides. In some embodiments, the stem-loop may be 4 to 10 unpaired nucleotides. In some embodiments, the stem-loop may be 4 to 8 nucleotides.

[0038] siRNA and shRNA are sometimes also collectively referred to as double-stranded RNA (dsRNA) herein. The two substantially complementary chains of dsRNA do not need but can also be covalently linked. The maximum number of base pairs is the number of nucleotides in the shortest chain of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the dsRNA can also include one or more nucleotide overhangs. For example, at least one chain includes a 3' overhang of at least one nucleotide, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14 or 15 nucleotides. For another example, at least one chain includes a 5' overhang of at least one nucleotide, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14 or 15 nucleotides. In other embodiments, the 3' end and the 5' end of a chain of the dsRNA both include an overhang of at least one nucleotide.

[0039] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a dsRNA. For example, a nucleotide overhang exists when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. The nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang may be present at the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the dsRNA.

[0040] As used herein, the term "blunt-ended" or "blunt-ended" with respect to dsRNA refers to the absence of unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., the absence of nucleotide overhangs. A dsRNA may be blunt at one or both ends. If both ends of a dsRNA are blunt-ended, the dsRNA is said to be blunt-ended. It should be noted that a "blunt-ended" dsRNA is a dsRNA with blunt ends at both ends, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such molecules are double-stranded throughout their entire length.

[0041] The term "antisense strand" refers to the strand of a dsRNA that contains a region that is substantially complementary to the target sequence. The term "sense strand" refers to the strand of a dsRNA that contains a region that is substantially complementary to the antisense strand region as defined herein. The term "substantially complementary region" refers to a region that is fully complementary or incompletely complementary. When the complementary region is not fully complementary to the target sequence, mismatches may be located in the interior or terminal regions of the molecule. Typically, the most tolerable mismatches are located in the terminal regions, for example, 5, 4, 3, or 2 at the 5' and / or 3' ends of the dsRNA.

[0042] The term "ligand" refers to a cell or tissue targeting agent that binds to a specified cell type (e.g., hepatocytes), such as a lectin, glycoprotein, lipid, or protein (e.g., an antibody). Exemplary targeting agents include thyrotropin, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetylgalactosamine (GalNAc), multivalent (e.g., divalent or trivalent) GalNAc, N-acetylglucosamine, multivalent mannose, multivalent trehalose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, cholesterol, steroids, bile acid, folate, vitamin B12, biotin, RGD peptide, and RGD peptide mimetics. In a preferred embodiment, the ligand is a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, or polysaccharide. For example, the ligand can be a derivative comprising GalNAc. In a preferred embodiment, the ligand comprises one or more N-acetylgalactosamine derivatives attached via a bivalent or trivalent branched linker.

[0043] The term "amino acid" refers to a molecule containing both an amino group and a carboxyl group. Suitable amino acids include, but are not limited to, the D- and L-isomers of naturally occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways. As used herein, the term amino acid includes, but is not limited to, α-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs. The term "α-amino acid" refers to a molecule containing both an amino group and a carboxyl group bound to a carbon designated as the α-carbon. The term "naturally occurring amino acid" refers to any of the 20 amino acids commonly found in peptides synthesized in nature, known by their single-letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. "Hydrophobic amino acids" include both small and large hydrophobic amino acids. "Small hydrophobic amino acids" include glycine, alanine, proline, and their analogs. "Large hydrophobic amino acids" include valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and their analogs. "Polar amino acids" include serine, threonine, asparagine, glutamine, cysteine, tyrosine, and their analogs. "Charged amino acids" include lysine, arginine, histidine, aspartic acid, glutamic acid, and their analogs.

[0044] The term "amino acid analog" refers to a molecule that is structurally similar to an amino acid and can replace an amino acid in the formation of a peptidomimetic macrocycle. Amino acid analogs include, but are not limited to, β-amino acids and amino acids in which either the amino or carboxyl group is substituted with a group of similar reactivity (e.g., a secondary or tertiary amine in place of a primary amine, or an ester in place of a carboxyl group). The term "β-amino acid" refers to a molecule containing both the amino and carboxyl groups in the β configuration.

[0045] The term "unnatural amino acid" refers to an amino acid that is not one of the twenty amino acids commonly found in peptides synthesized in nature. As used herein, the term "unnatural amino acid" includes α-substituted and α-disubstituted amino acids of natural amino acids, N-alkyl amino acids, lactic acid, halide derivatives such as trifluorotyrosine, p-chloro-phenylalanine, p-fluoro-phenylalanine, p-bromo-phenylalanine, p-NO2-phenylalanine, phenylglycine, sarcosine, penicillamine, D-2-methyltryptophan, phosphoserine, phosphothreonine, phosphotyrosine, pI-phenylalanine, L-allyl-glycine, β-alanine, β-aspartic acid, β-cyclohexylalanine, citrulline, homoserine, homocysteine, pyroglutamic acid, L-α-aminobutyric acid, L-γ-aminobutyric acid, L-α-aminoisobutyric acid, α-cyclohexylglycine, diaminobutyric acid, diaminopimelaneic acid, N-ε-dinitrophenyl-lysine, L-1-naphthylalanine, L-2-naphthylalanine, 3-(2-pyridinium chloride), 4-(2-aminobutyric acid), 5-(2-aminobutyric acid), 6-(2-aminobutyric acid), 7-(2-aminobutyric acid), 8-(2-aminobutyric acid), 9-(2-aminobutyric acid), 10-(2-aminobutyric acid), 11-(2-aminobutyric acid), 12-(2-aminobutyric acid), 13-(2-aminobutyric acid), 14-(2-aminobutyric acid), 15-(2-aminobutyric acid), 16-(2-aminobutyric acid), 17-(2-aminobutyric acid), 18-(2-aminobutyric acid), 19-(2-aminobuty 3-(3-pyridyl)-L-alanine, 3-(4-pyridyl)-L-alanine, N-ε-methyl-lysine, N,N-ε-dimethyl-lysine, N,N,N-ε-trimethyl-lysine, 3-mercaptopropionic acid, L-ε-aminocaproic acid, 7-aminoheptanoic acid, 6-aminocaproic acid, L-methionine sulfone, ornithine, L-norleucine, L-norvaline, p-nitro-L-phenylalanine amino acid, L-hydroxyproline, γ-glutamic acid, γ-aminobutyric acid, L-mercaptoproline, methyl derivatives of phenylalanine (Phe) (such as 4-methyl-Phe, pentamethyl-Phe, L-Phe(4-amino), L-Tyr(methyl), L-Phe(4-isopropyl), L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), L-diaminopropionic acid and L-Phe(4-benzyl)).

[0046] The term "amino acid side chain" refers to the moiety attached to the α-carbon in a natural or non-natural amino acid. For example, the amino acid side chain of alanine is a methyl group, the amino acid side chain of phenylalanine is a phenylmethylene group, the amino acid side chain of cysteine ​​is a mercaptomethylene group, the amino acid side chain of aspartic acid is a carboxymethylene group, the amino acid side chain of tyrosine is a 4-hydroxyphenylmethylene group, and so on. Other non-natural amino acid side chains are also included, for example, naturally occurring amino acid side chains (e.g., amino acid metabolites) or synthetically prepared amino acid side chains (e.g., citrulline side chain). Whether natural or non-natural amino acids, both L and D configurations are included, and accordingly, their side chains are also intended to include both L and D configurations.

[0047] The term "carboxyl protecting group" means a group intended to protect a carboxyl group, selected from methyl, substituted methyls, ethyl, 2-substituted ethyls, allyl, tert-butyl, alkoxyalkyls, alkoxyalkoxyalkyls, 2,6-dialkylphenyls, benzyl, substituted benzyls, silyls or stannyls; the substituted methyls are selected from 9-fluorenylmethyl, triisopropylsilylmethyl, cyclopropylmethyl, diphenylmethyl or triphenylmethyl; the 2-substituted ethyls are selected from 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(p-toluenesulfonyl)ethyl or 2-cyanoethyl; the alkoxyalkyls are selected from methoxymethyl, benzyloxymethyl or triisopropylsilyloxymethyl The alkoxyalkoxyalkyl group is selected from methoxyethoxymethyl; the 2,6-dialkylphenyl group is selected from 2,6-dimethylphenyl, 2,6-diisopropylphenyl or 2,6-di-tert-butyl-4-methoxyphenyl; the substituted benzyl group is selected from p-methylbenzyl, 2,4-dimethoxybenzyl, 2,6-dimethoxybenzyl, p-nitrobenzyl or o-nitrobenzyl; the silyl group is selected from trimethylsilyl, triethylsilyl, triisopropylsilyl or phenyldimethylsilyl; the stannyl group is selected from trimethylstannyl; preferably methyl, allyl, tert-butyl, benzyl, 2,4-dimethoxybenzyl, p-methylbenzyl, pentafluorophenyl or methoxyethoxymethyl.

[0048] The term "halo" or "halogen" refers to fluorine, chlorine, bromine or iodine or a radical thereof. When the number of halogens is not limited, it can be any suitable number, for example, monohalo, dihalo, trihalo; when the position of the halogen is not limited, it can be any suitable position, for example, a halophenyl group can be halogenated at the ortho position, para position, meta position or a combination thereof.

[0049] The term "alkyl" refers to a saturated straight or branched hydrocarbon chain. For an alkyl group having a specific number of carbon atoms, the term includes the corresponding n-alkyl group and its various isomeric forms (if any). For example, an alkyl group having 4 carbon atoms (C4 alkyl) includes n-butyl, isobutyl, sec-butyl, and tert-butyl. Exemplary C 1-10Alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, 4,4-dimethylpentyl, 5,5-dimethylhexyl, 6,6-dimethylheptyl, and the like.

[0050] The term "alkenyl" refers to a straight or branched hydrocarbon chain having one or more carbon-carbon double bonds. 2-10 Alkenyl groups include ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 3-hexenyl, 5-hexenyl, and the like.

[0051] The term "alkynyl" refers to a straight or branched hydrocarbon chain having one or more carbon-carbon triple bonds. 2-10 Alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 4-methyl-2-pentynyl, and the like.

[0052] The term "alkoxy" refers to an alkyl-O- group, wherein alkyl is as defined above. 1-10 Examples of "alkoxy" include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like. When halogenated, the alkoxy group may be substituted with 1 to 7, preferably 1 to 5, halogen atoms. Specific examples include difluoromethoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy, 4,4,4-trifluorobutoxy, and the like.

[0053] The term "cycloalkyl" refers to a cyclic saturated hydrocarbon group. 3-10 Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.2.1]octyl, and adamantyl.

[0054] The term "aryl" refers to aromatic monocyclic and polycyclic carbocyclic ring systems, wherein the individual carbocyclic rings in the polycyclic ring system are fused or connected to each other by single bonds. Suitable aryl groups include phenyl, naphthyl, 2,3-dihydro-1H-indenyl, and biphenyl. 6- to 14-membered aryl refers to aromatic ring systems that are 6-carbon monocyclic, 10-carbon bicyclic, 12-carbon biphenyl, or 14-carbon tricyclic.

[0055] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, the heteroatoms being selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S, respectively, if monocyclic, bicyclic, or tricyclic), wherein 0, 1, 2, 3, or 4 atoms of each ring are substituted with substituents. Examples of heteroaryl groups include pyridyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thienyl, quinolinyl, indolyl, thiazolyl, and the like.

[0056] The term "heterocyclyl" refers to a non-aromatic monocyclic, bicyclic or tricyclic ring system. Thus, a 5- to 14-membered heterocyclyl refers to a 5-8-membered monocyclic, 8-12-membered bicyclic or 11-14-membered tricyclic ring system having 1-3 heteroatoms if a monocyclic ring, 1-6 heteroatoms if a bicyclic ring, or 1-9 heteroatoms if a tricyclic ring, the heteroatoms being selected from O, N or S (e.g., carbon atoms and 1-3, 1-6 or 1-9 O, N or S heteroatoms, respectively, if a monocyclic, bicyclic or tricyclic ring) or other suitable heteroatoms (such as P or Si), wherein 0, 1, 2 or 3 atoms of each ring are substituted by substituents. Examples of 5- to 14-membered heterocyclyls include piperazinyl, pyrrolidinyl, dioxane, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl, etc.

[0057] The term "alkylthio" refers to a thiol group in which the hydrogen atom of the thiol group is replaced by an alkyl group. 1-3 The alkylthio group means a methylthio group, an ethylthio group, a propylthio group and an isopropylthio group.

[0058] The term "alkylphosphono" refers to a phosphono group in which one or both hydroxyl groups of the phosphono group are substituted with an alkyl group. 1-3 Examples of the alkylphosphonyl group include methylphosphonyl, ethylphosphonyl, dimethylphosphonyl, diethylphosphonyl, methylethylphosphonyl and the like.

[0059] The term "alkylphosphonooxy" refers to an alkylphosphono-O- group, wherein alkylphosphono is as defined above. For example, C 1-3 Examples of the alkylphosphonooxy group include methylphosphonooxy, ethylphosphonooxy, dimethylphosphonooxy, diethylphosphonooxy, methylethylphosphonooxy and the like.

[0060] The term "arylalkyl" refers to an alkyl group substituted by an aryl group, and the term "alkylaryl" refers to an aryl group substituted by an alkyl group, wherein alkyl and aryl are as defined above. Exemplary arylalkyl groups are phenyl, C 1-3 Alkyl, naphthyl C 1-3 Alkyl, biphenyl C 1- 6 alkyl, etc.

[0061] The term "heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group, and the term "alkylheteroaryl" refers to a heteroaryl group substituted with an alkyl group, wherein alkyl and heteroaryl are as defined above. Exemplary heteroarylalkyl groups include pyridinyl, C 1-3 Alkyl, indolyl C 1-3 Alkyl, quinolinyl C 1-3 Alkyl, isoquinolinyl C 1-3 Alkyl, thienyl C 1-3 Alkyl, thiaindenyl C 1-3 Alkyl, thiazolyl C 1-3 Alkyl, benzothiazolyl C 1-3 Alkyl, imidazolyl C 1-3 Alkyl, pyrimidinyl C 1-3 Alkyl, etc.

[0062] The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl group, and the term "alkylheterocyclyl" refers to a heterocyclyl group substituted by an alkyl group, wherein heterocyclyl refers to and alkyl is as defined above. Exemplary heterocyclylalkyls include furanyl, C 1-3 Alkyl, piperazinyl C 1-3 Alkyl, pyrrolidinyl C 1-3 Alkyl, dioxane C 1-3 Alkyl, morpholinyl C 1-3 Alkyl, tetrahydrofuranyl C 1-3 Alkyl, tetrahydropyrrolidinyl C 1-3 alkyl.

[0063] The term "TMSOTf" refers to trimethylsilyl trifluoromethanesulfonate.

[0064] The term "DCM" refers to dichloromethane.

[0065] The term "DIEA" refers to diisopropylethylamine.

[0066] The term "EA" refers to ethyl acetate.

[0067] The term "THF" refers to tetrahydrofuran.

[0068] The term "DMF" refers to N,N-dimethylformamide.

[0069] The term "HATU" refers to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0070] The term "PE" refers to petroleum ether.

[0071] The term "TFAPfp" refers to pentafluorophenyl trifluoroacetate.

[0072] The term "TFA" refers to trifluoroacetic acid.

[0073] The term "PI" refers to propidium iodide.

[0074] In some embodiments, the compounds disclosed herein contain one or more asymmetric centers and thus exist as racemates or racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. Unless otherwise expressly indicated, all such isomeric forms of these compounds are intended to be included herein. In some embodiments, the compounds disclosed herein also present as multiple tautomeric forms, in which case the compounds include all tautomeric forms of the compounds described herein (e.g., if the alkylation of the ring system results in alkylation at multiple positions, the present invention includes all such reaction products). Unless otherwise expressly indicated, all crystalline forms of the compounds are intended to be included herein.

[0075] The term "therapeutically effective amount" refers to an amount of a conjugate of the invention or composition thereof effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0076] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0077] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material, that participates in carrying or transporting the conjugate from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient.

[0078] The term "treatment" encompasses prevention, therapy, and cure. The patient receiving such treatment is generally any animal in need thereof, including primates (particularly humans) and other mammals such as horses, cattle, pigs, sheep, poultry, and pets.

[0079] Ligand-oligonucleotide conjugate

[0080] In one aspect, provided herein is a ligand-oligonucleotide conjugate comprising a linker moiety of Formula I or Formula II: 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(Ra R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II),

[0081] in:

[0082] R 1 、R 2 、R 3 、R 4 Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3- 10 Cycloalkyl, 6- to 14-membered aryl, 5- to 18-membered heteroaryl, 5- to 14-membered heterocyclyl, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, and mercapto, which are optionally replaced by R 5 Substituted, where R 5 Selected from C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl, 6- to 14-membered aryl, 5- to 18-membered heteroaryl, 5- to 14-membered heterocyclic group, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, C 1-3 alkylthio and mercapto groups;

[0083] R a 、R b Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-8 Cycloalkyl, 6 to 14 membered aryl C 1-3 Alkyl, 5 to 18 membered heteroaryl C 1-3 alkyl and 5 to 14 membered heterocyclic groups, which are optionally replaced by R c Substituted, where R c Selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C 1-3Alkylthio, indolyl, quinolyl, isoquinolyl, amide, pyrrolyl, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, C 1-10 Alkyl, C 1-10 Alkoxy, 6 to 14 membered aryl, C 3-8 Cycloalkyl, adamantyl, phosphono, phosphonooxy, C 1-3 Alkylphosphono, C 1-3 Alkylphosphonooxy, or R a and R b One of them, together with the C atom to which they are connected and the adjacent N atom, forms a 3-14 membered nitrogen-containing heterocyclic group, or R a 、R b Together with its co-connected C atom, it forms C 3-8 Cycloalkyl;

[0084] heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon;

[0085] n is 1 to 10, and each repeating unit is the same or different, for example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0086] p is 2 to 6 and each repeating unit is the same or different, for example, p can be 2, 3, 4, 5 or 6.

[0087] In a preferred embodiment, the ligand-oligonucleotide conjugate comprises a linker moiety of Formula I or II, wherein R a 、R b Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-8 Cycloalkyl, phenyl C 1- 3 alkyl, naphthyl C 1-3 Alkyl, 5 to 12 membered heteroaryl C 1-3 Alkyl and 5 to 14 membered heterocyclic C 1-3 Alkyl groups, which are optionally replaced by R c Substituted, where R c Selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C 1-3 Alkylthio, indolyl, quinolyl, isoquinolyl, amide, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, piperazinyl, pyrrolidinyl, dioxane, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl, C 1-10 Alkyl, C 1-10 Alkoxy, phenyl, benzyl, phenethyl, aminophenyl, carboxyphenyl, halogenated phenyl, biphenyl, C 3-8Cycloalkyl, adamantyl, phosphono, phosphonooxy, C 1-3 Alkylphosphono, C 1-3 Alkylphosphonooxy, or R a and R b One of them, together with the C atom to which they are connected and the adjacent N atom, forms a 3-14-membered nitrogen-containing heterocyclic group; R 1 、R 2 、R 3 、R 4 , heteroatoms, n and p are as defined above.

[0088] In a preferred embodiment, the ligand-oligonucleotide conjugate comprises a linker moiety of Formula I or II, wherein R a 、R b Independently selected from -H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, phenyl C 1-3 Alkyl, pyridyl C 1-3 Alkyl, indolyl C 1-3 Alkyl, quinolinyl C 1-3 Alkyl, isoquinolinyl C 1-3 Alkyl, thienyl C 1- 3-alkyl, thiaindenyl C 1-3 Alkyl, thiazolyl C 1-3 Alkyl, benzothiazolyl C 1-3 Alkyl, imidazolyl C 1-3 Alkyl, furyl C 1-3 Alkyl, pyrimidinyl C 1-3 Alkyl groups, which are optionally replaced by R c Substituted, where R c Selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C 1-3 Alkylthio, indolyl, quinolyl, isoquinolyl, amide, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, piperazinyl, pyrrolidinyl, dioxane, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl, pyridyl, C 1-10 Alkyl, C 1-10 Alkoxy, phenyl, benzyl, phenethyl, aminophenyl, carboxyphenyl, halogenated phenyl, biphenyl, C 3-8 Cycloalkyl, adamantyl, phosphono, phosphonooxy, C 1-3 Alkylphosphono, C 1-3 Alkylphosphonooxy, or R a and R b One of them, together with the C atom to which they are connected and the adjacent N atom, forms a 3-14-membered nitrogen-containing heterocyclic group; R1 、R 2 、R 3 、R 4 , heteroatoms, n and p are as defined above.

[0089] In a more preferred embodiment, the ligand-oligonucleotide conjugate comprises a linker moiety of Formula I or II, wherein R a 、R b independently selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-methyl-2-propynyl, 1,3-butadienyl, methoxy, ethoxy, propoxy, butoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenylmethylene, phenyldimethylene, pyridylmethylene, pyridyldimethylene, indolylmethylene, indolyldimethylene, quinolylmethylene, quinolyldimethylene, isoquinolylmethylene, isoquinolyldimethylene, thienylmethylene, thienyldimethylene, thiaindenylmethylene, thiaindenyldimethylene, thiazolylmethylene, thiazolyldimethylene, benzothiazolylmethylene, benzothiazolyldimethylene, imidazolylmethylene, imidazolyldimethylene, furylmethylene, furyldimethylene, pyrimidinylmethylene and pyrimidinyldimethylene, which are optionally replaced by R c Substituted, where R c is selected from the group consisting of hydroxy, thiol, carboxyl, amino, guanidino, halogen, methylenethio, dimethylenethio, indolyl, quinolyl, isoquinolyl, amide, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl, pyridyl, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, phenethyl, aminophenyl, carboxylphenyl, halophenyl, biphenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, phosphonyl, phosphonoxy, dimethylphosphonyl, diethylphosphonyl, dimethylphosphonoxy and diethylphosphonoxy, or R a and R b One of them, together with the C atom to which they are attached and the adjacent N atom, forms an imidazolyl, thiazolyl, pyrazolyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl or pyrrolyl group; R 1 、R 2 、R 3 、R 4 , heteroatoms, n and p are as defined above.

[0090] In a more preferred embodiment, the ligand-oligonucleotide conjugate comprises a linker moiety of Formula I or II, wherein R aYes -H,R b Selected from -H, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclopentyl, cyclohexyl, cyclohexyldimethylene, cyclohexylmethylene, cyclobutylmethylene, phenylmethylene, methylthiomethylene, 3-indolylmethylene, hydroxymethylene, amidomethylene, hydroxymethylmethylene, mercaptomethylene, amidomethylene, hydroxyphenylmethylene, hydroxyphenyldimethylene, carboxymethylene, carboxydimethylene, amino-n-butyl, guanidine dimethylene, guanidinotrimethylene, 4-imidazolylmethylene, isopropylphenylmethylene, 1-naphthylmethylene, 2-naphthylmethylene, indanyl, phenyldimethylene, fluorophenylmethylene, chlorophenylmethylene, trifluorophenylmethylene, dichlorophenylmethylene, pentafluorophenylmethylene, difluorophenylmethylene, thienylmethylene, thiandenylmethylene, quinolylmethylene, halogenated 3-indolylmethylene, diphenylmethylene, 3-pyridylmethylene and 4-pyridylmethylene, or R b Together with the C atom and the adjacent N atom to which it is connected, it forms a tetrahydropyrrolyl group; R 1 、R 2 、R 3 、

[0091] R 4 , heteroatoms, n and p are as defined above.

[0092] In some embodiments, the ligand-oligonucleotide conjugate comprises a linker moiety of Formula I or II, wherein R a and R b One of them is -H, and R a and R b The other is an amino acid side chain. The amino acids include natural amino acids and non-natural amino acids, including D and L configurations.

[0093] In R a and R b One of them is -H, and R a and R b In another embodiment where the amino acid side chain is a natural or unnatural amino acid, the unnatural amino acid includes but is not limited to the following structures:

[0094] Other unnatural amino acids include, but are not limited to, β-amino acid analogs. Examples of β-amino acid analogs include, but are not limited to, cyclic β-amino acid analogs; β-alanine; (R)-β-phenylalanine; (R)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid; (R)-3-amino-4-(1-naphthyl)-butyric acid; (R)-3-amino-4-(2,4-dichlorophenyl)butyric acid; (R)-3-amino-4-(2-chlorophenyl)-butyric acid; (R)-3-amino-4-(2-cyanophenyl)-butyric acid; (R)-3-amino-4-(2-fluorophenyl)-butyric acid; (R)-3-amino-4-(2-furyl)-butyric acid; (R)-3-amino-4-(2-tolyl)-butyric acid; (R)-3-amino-4-(2-naphthyl)-butyric acid (R)-3-amino-4-(2-thienyl)-butyric acid; (R)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-(3,4-dichlorophenyl)-butyric acid; (R)-3-amino-4-(3,4-difluorophenyl)-butyric acid; (R)-3-amino-4-(3-benzothienyl)-butyric acid; (R)-3-amino-4-(3-chlorophenyl)-butyric acid; (R)-3-amino-4-(3-cyanophenyl)-butyric acid; (R)-3-amino-4-(3-fluorophenyl)-butyric acid; (R)-3-amino-4-(3-methylphenyl)-butyric acid; (R)-3-amino-4-(3-pyridyl)-butyric acid; (R)-3-amino (R)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-(4-bromophenyl)-butyric acid; (R)-3-amino-4-(4-chlorophenyl)-butyric acid; (R)-3-amino-4-(4-cyanophenyl)-butyric acid; (R)-3-amino-4-(4-fluorophenyl)-butyric acid; (R)-3-amino-4-(4-iodophenyl)-butyric acid; (R)-3-amino-4-(4-tolyl)-butyric acid; (R)-3-amino-4-(4-nitrophenyl)-butyric acid; (R)-3-amino-4-(4-pyridyl)-butyric acid; (R)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid; ( (R)-3-amino-4-pentafluoro-phenylbutyric acid; (R)-3-amino-5-hexenoic acid; (R)-3-amino-5-hexenoic acid; (R)-3-amino-5-phenylvaleric acid; (R)-3-amino-6-phenyl-5-hexenoic acid; (S)-1,2,3,4-tetrahydroisoquinoline-3-acetic acid; (S)-3-amino-4-(1-naphthyl)-butyric acid; (S)-3-amino-4-(2,4-dichlorophenyl)-butyric acid; (S)-3-amino-4-(2-chlorophenyl)-butyric acid; (S)-3-amino-4-(2-cyanophenyl)-butyric acid; (S)-3-amino-4-(2-fluorophenyl)-butyric acid; (S)-3-amino-4-(2-furyl)-butyric acid;(S)-3-amino-4-(2-tolyl)-butyric acid; (S)-3-amino-4-(2-naphthyl)-butyric acid; (S)-3-amino-4-(2-thienyl)-butyric acid; (S)-3-amino- 4-(2-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-(3,4-dichlorophenyl)-butyric acid; (S)-3-amino-4-(3,4-difluorophenyl)-butyric acid; (S)-3-amino-4-(3-benzothienyl)-butyric acid; (S)-3-amino-4-(3-chlorophenyl)-butyric acid; (S)-3-amino-4-(3-cyanophenyl)-butyric acid; (S)-3-amino-4-(3-fluorophenyl)-butyric acid; (S)-3-amino-4-(3-tolyl)-butyric acid; (S)-3-amino-4-(3-pyridyl)-butyric acid; (S)-3-amino-4-(3-thienyl)-butyric acid; (S)-3-amino -4-(3-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-(4-bromophenyl)-butyric acid; (S)-3-amino-4-(4-chlorophenyl)-butyric acid; (S)-3-amino-4-(4-cyanophenyl)-butyric acid; (S)-3-amino-4-(4-fluorophenyl)-butyric acid; (S)-3-amino-4-(4-iodophenyl)-butyric acid; (S)-3-amino-4-(4-tolyl)-butyric acid; (S)-3-amino-4-(4-nitrophenyl)-butyric acid; (S)-3-amino-4-(4-pyridyl)-butyric acid; (S)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4- Pentafluoro-phenylbutyric acid; (S)-3-amino-5-hexenoic acid; (S)-3-amino-5-hexenoic acid; (S)-3-amino-5-phenylpentanoic acid; (S)-3-amino-6-phenyl-5-hexenoic acid; 1,2,5,6-tetrahydropyridine-3-carboxylic acid; 1,2,5,6-tetrahydropyridine-4-carboxylic acid; 3-amino-3-(2-chlorophenyl)-propionic acid; 3-amino-3-(2-thienyl)-propionic acid; 3-amino-3-(3-bromophenyl)-propionic acid; 3-amino-3-(4-chlorophenyl)-propionic acid; 3-amino-3-(4-methoxyphenyl)-propionic acid; 3-amino-4,4,4-trifluoro-butyric acid; 3-aminoadipic acid; D- β-Phenylalanine; β-Leucine; L-β-Homoalanine; γ-benzyl L-β-homoaspartate; δ-benzyl L-β-homoglutamate; L-β-homoisoleucine; L-β-homoleucine; L-β-homomethionine; L-β-homophenylalanine; L-β-homoproline; L-β-homotryptophan; L-β-homovaline; L-Nω-benzyloxycarbonyl-β-homolysine; Nω-L-β-homoarginine; O-benzyl-L-β-homohydroxyproline; O-benzyl-L-β-homoserine; O-benzyl-L-β-homothreonine; O-benzyl-L-β-homotyrosine; γ-trityl-L-β-homoasparagine; (R)-β-phenylalanine;L-β-homoaspartic acid γ-tert-butyl ester; L-β-homoglutamic acid δ-tert-butyl ester; L-Nω-β-homolysine; Nδ-trityl-L-β-homoglutamine; Nω-2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl-L-β-homoarginine; O-tert-butyl-L-β-homohydroxy-proline; O-tert-butyl-L-β-homoserine; O-tert-butyl-L-β-homothreonine; O-tert-butyl-L-β-homotyrosine; 2-aminocyclopentanecarboxylic acid; and 2-aminocyclohexanecarboxylic acid.

[0095] Other unnatural amino acids include, but are not limited to, analogs of alanine, valine, glycine, or leucine. Examples of amino acid analogs of alanine, valine, glycine, or leucine include, but are not limited to, the following: α-methoxyglycine; α-allyl-L-alanine; α-aminoisobutyric acid; α-methyl-leucine; β-(1-naphthyl)-D-alanine; β-(1-naphthyl)-L-alanine; β-(2-naphthyl)-D-alanine; β-(2-naphthyl)-L-alanine; β-(2-pyridyl)-D-alanine; β-(2-pyridyl)-L-alanine; β-(2-thienyl)-D-alanine; β-(2-thienyl)-L-alanine; β-(3-benzothienyl)-D-alanine; β-(3-benzothienyl)-L-alanine; β-(3-pyridyl) -D-Alanine; β-(3-pyridyl)-L-alanine; β-(4-pyridyl)-D-alanine; β-(4-pyridyl)-L-alanine; β-chloro-L-alanine; β-cyano-L-alanine; β-cyclohexyl-D-alanine; β-cyclohexyl-L-alanine; β-cyclopenten-1-yl-alanine; β-cyclopentyl-alanine; β-cyclopropyl-L-Ala-OH·dicyclohexylammonium salt; β-tert-butyl-D-alanine; β-tert-butyl-L-alanine; γ-aminobutyric acid; L-α,β-diaminopropionic acid; 2,4-dinitrophenylglycine; 2,5-dihydro-D-phenylglycine; 2-amino-4,4,4-trifluorobutyric acid; 2-fluoro-phenylglycine Aminoacid; 3-amino-4,4,4-trifluoro-butyric acid; 3-fluoro-valine; 4,4,4-trifluoro-valine; 4,5-dehydro-L-leu-OH·dicyclohexylammonium salt; 4-Fluoro-D-phenylglycine; 4-Fluoro-L-phenylglycine; 4-Hydroxy-D-phenylglycine; 5,5,5-trifluoro-leucine; 6-aminohexanoic acid; cyclopentyl-D-Gly-OH·dicyclohexylammonium salt; cyclopentyl-Gly-OH·dicyclohexylammonium salt; D-α,β-diaminopropionic acid; D-α-aminobutyric acid; D-α-tert-butylglycine; D-(2-thienyl)glycine; D-(3-thienyl)glycine; D-2-aminohexanoic acid; D-2-indanylglycine; D-allyl glycine·dicyclohexylammonium salt; D-cyclohexylglycine; D-norvaline; D-phenylglycine; β-aminobutyric acid; β-aminoisobutyric acid; (2-bromophenyl)glycine; (2-methoxyphenyl)glycine; (2-methylphenyl)glycine; (2-thiazolyl)glycine; (2-thienyl)glycine; 2-amino-3-(dimethylamino)-propionic acid; L-α,β-diaminopropionic acid; L-α-aminobutyric acid; L-α-tert-butylglycine; L-(3-thienyl)glycine; L-2-amino-3-(dimethylamino)-propionic acid; L-2-aminohexanoic acid dicyclohexyl-ammonium salt; L-2-indanylglycine; L-allylglycine·dicyclohexylammonium salt; L-cyclohexylglycine;L-phenylglycine; L-propargylglycine; L-norvaline; N-α-aminomethyl-L-alanine; D-α,γ-diaminobutyric acid; L-α,γ-diaminobutyric acid; β-cyclopropyl-L-alanine; (N-β-(2,4-dinitrophenyl))-L-α,β-diaminopropionic acid; (N-β-1-(4,4-dimethyl-2,6-dioxocyclohexylene-1-yl)ethyl )-D-α,β-diaminopropionic acid; (N-β-1-(4,4-dimethyl-2,6-dioxocyclohexane-1-ylidene)ethyl)-L-α,β-diaminopropionic acid; (N-β-4-methyltrityl)-L-α,β-diaminopropionic acid; (N-β-allyloxycarbonyl)-L-α,β-diaminopropionic acid; (N-γ-1-(4,4-dimethyl-2,6-dioxocyclohexane-1-ylidene)ethyl)-L-α,β-diaminopropionic acid (N-γ-1-(4,4-dimethyl-2,6-dioxocyclohexane-1-ylidene)ethyl)-L-α,γ-diaminobutyric acid; (N-γ-4-methyltrityl)-D-α,γ-diaminobutyric acid; (N-γ-4-methyltrityl)-L-α,γ-diaminobutyric acid; (N-γ-allyloxycarbonyl)-L-α,γ- diaminobutyric acid; D-α,γ-diaminobutyric acid; 4,5-dehydro-L-leucine; cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH; D-allylglycine; D-cyclohexylhomoalanine; L-1-pyrenylalanine; L-2-aminohexanoic acid; L-allylglycine; L-cyclohexylhomoalanine; and N-(2-hydroxy-4-methoxy-Bzl)-Gly-OH.

[0096] Other unnatural amino acids include, but are not limited to, analogs of arginine or lysine. Examples of amino acid analogs of arginine and lysine include, but are not limited to, the following: citrulline; L-2-amino-3-guanidinopropionic acid; L-2-amino-3-ureidopropionic acid; L-citrulline; Lys(Me)2-OH; Lys(N3)-OH; Nδ-benzyloxycarbonyl-L-ornithine; Nω-nitro-D-arginine; Nω-nitro-L-arginine; α-methyl-ornithine; 2,6-diaminopimelate; L-ornithine; (Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohexane-1-ylidene)ethyl)-D-ornithine; (Nδ-1-(4,4 -dimethyl-2,6-dioxo-cyclohexan-1-ylidene)ethyl)-L-ornithine; (Nδ-4-methyltrityl)-D-ornithine; (Nδ-4-methyltrityl)-L-ornithine; D-ornithine; L-ornithine; Arg(Me)(Pbf)-OH; Arg(Me)2-OH (asymmetric); Arg(Me)2-OH (symmetric); Lys(ivDde)-OH; Lys(Me)2-OH·HCl; Lys(Me)3-OH chloride; Nω-nitro-D-arginine; and Nω-nitro-L-arginine.

[0097] Other unnatural amino acids include, but are not limited to, analogs of aspartic acid or glutamic acid. Examples of amino acid analogs of aspartic acid and glutamic acid include, but are not limited to, the following: α-methyl-D-aspartic acid; α-methyl-glutamic acid; α-methyl-L-aspartic acid; γ-methylene-glutamic acid; (N-γ-ethyl)-L-glutamine; [N-α-(4-aminobenzoyl)]-L-glutamic acid; 2,6-diaminopimelate; L-α-aminosuberic acid; D-2-aminoadipic acid; D-α-aminosuberic acid; α-aminopimelate; iminodiacetic acid; L-2-aminoadipic acid; threo-β-methyl-aspartic acid; γ-carboxy-D-glutamic acid γ,γ-di-tert-butyl ester; γ-carboxy-L-glutamic acid γ,γ-di-tert-butyl ester; Glu(OAll)-OH; L-Asu(OtBu)-OH; and pyroglutamic acid.

[0098] Other unnatural amino acids include, but are not limited to, analogs of cysteine ​​and methionine. Examples of amino acid analogs of cysteine ​​and methionine include, but are not limited to, Cys(farnesyl)-OH, Cys(farnesyl)-OMe, α-methyl-methionine, Cys(2-hydroxyethyl)-OH, Cys(3-aminopropyl)-OH, 2-amino-4-(ethylthio)butanoic acid, buthionine, buthionine sulfoxide, ethionine, methionine methylsulfonium chloride, selenomethionine, cysteic acid, [2-(4-pyridyl)ethyl]-DL-penicillamine, [2-(4-pyridyl)ethyl]-L-cysteine, 4-methoxybenzyl-D-penicillamine, 4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D- Penicillamine, 4-methylbenzyl-L-penicillamine, benzyl-D-cysteine, benzyl-L-cysteine, benzyl-DL-homocysteine, carbamoyl-L-cysteine, carboxyethyl-L-cysteine, carboxymethyl-L-cysteine, diphenylmethyl-L-cysteine, ethyl-L-cysteine, methyl-L-cysteine, tert-butyl-D-cysteine, trityl-L-homocysteine, trityl-D-penicillamine, cystathionine, homocystine, L-homocystine, (2-aminoethyl)-L-cysteine, seleno-L-cystine, cystathionine, Cys(StBu)-OH, and acetylaminomethyl-D-penicillamine.

[0099] Other unnatural amino acids include, but are not limited to, analogs of phenylalanine and tyrosine.Examples of amino acid analogs of phenylalanine and tyrosine include β-methyl-phenylalanine, β-hydroxyphenylalanine, α-methyl-3-methoxy-DL-phenylalanine, α-methyl-D-phenylalanine, α-methyl-L-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 2,4-dichloro-phenylalanine, 2-(trifluoromethyl)-D-phenylalanine, 2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine, 2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine, 2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine, 2-cyano-L-phenylalanine, 2-fluoro-D-phenylalanine, 2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine, 2-Methyl-L-phenylalanine, 2-nitro-D-phenylalanine, 2-nitro-L-phenylalanine, 2,4,5-trihydroxy-phenylalanine, 3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine, 3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine, 3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine, 3,5,3'-triiodo-L-thyronine, 3,5-diiodo-D-tyrosine, 3,5-diiodo-L-tyrosine, 3,5-diiodo-L-thyronine, 3-(trifluoromethyl)-D-phenylalanine, 3 -(trifluoromethyl)-L-phenylalanine, 3-amino-L-tyrosine, 3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine, 3-chloro-D-phenylalanine, 3-chloro-L-phenylalanine, 3-chloro-L-tyrosine, 3-cyano-D-phenylalanine, 3-cyano-L-phenylalanine, 3-fluoro-D-phenylalanine, 3-fluoro-L-phenylalanine, 3-fluoro-tyrosine, 3-iodo-D-phenylalanine, 3-iodo-L-phenylalanine, 3-iodo-L-tyrosine, 3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine, 3-methyl-L-phenylalanine, 3-nitro-D-phenylalanine, 3-nitro-L-phenylalanine, 3-nitro-L-tyrosine, 4-(trifluoromethyl)-D- Phenylalanine, 4-(trifluoromethyl)-L-phenylalanine, 4-amino-D-phenylalanine, 4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine, 4-benzoyl-L-phenylalanine, 4-bis(2-chloroethyl)amino-L-phenylalanine, 4-bromo-D-phenylalanine, 4-bromo-L-phenylalanine, 4-chloro-D-phenylalanine, 4-chloro-L-phenylalanine, 4-cyano-D-phenylalanine, 4-cyano-L-phenylalanine, 4-fluoro-D-phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-D-phenylalanine, 4-iodo-L-phenylalanine, homophenylalanine, thyroxine, 3,3-diphenylalanine, thyronine, ethyl-tyrosine, and methyl-tyrosine.

[0100] Other unnatural amino acids include, but are not limited to, analogs of proline. Examples of amino acid analogs of proline include, but are not limited to, 3,4-dehydro-proline, 4-fluoro-proline, cis-4-hydroxy-proline, thiazolidine-2-carboxylic acid, and trans-4-fluoro-proline.

[0101] Other unnatural amino acids include, but are not limited to, analogs of serine and threonine. Examples of amino acid analogs of serine and threonine include, but are not limited to, 3-amino-2-hydroxy-5-methylhexanoic acid, 2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutanoic acid, 2-amino-3-methoxybutanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutanoic acid, and α-methylserine.

[0102] Other unnatural amino acids include, but are not limited to, analogs of tryptophan. Examples of amino acid analogs of tryptophan include, but are not limited to, the following: α-methyl-tryptophan; β-(3-benzothienyl)-D-alanine; β-(3-benzothienyl)-L-alanine; 1-methyl-tryptophan; 4-methyl-tryptophan; 5-benzyloxy-tryptophan; 5-bromo-tryptophan; 5-chloro-tryptophan; 5-fluoro-tryptophan; 5-hydroxy-tryptophan; 5-hydroxy-L-tryptophan; 5-methoxy-tryptophan; 5-methoxy-L-tryptophan; 5-methyl-tryptophan; 6-bromo- Tryptophan; 6-chloro-D-tryptophan; 6-chloro-tryptophan; 6-fluoro-tryptophan; 6-methyl-tryptophan; 7-benzyloxy-tryptophan; 7-bromo-tryptophan; 7-methyl-tryptophan; D-1,2,3,4-tetrahydro-norharman-3-carboxylic acid; 6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid; 7-azatryptophan; L-1,2,3,4-tetrahydro-norharman-3-carboxylic acid; 5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan.

[0103] In the present invention, the non-natural amino acid can be racemic. In the present invention, the non-natural amino acid used can be the D-isomer. In the present invention, the non-natural amino acid used can be the L-isomer. In the present invention, the non-natural amino acid can contain a chiral center with an R or S configuration. In some other embodiments, the amino group of the β-amino acid is replaced by a protecting group such as tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), tosyl. In some other embodiments, the carboxylic acid functional group of the β-amino acid is protected, for example, as its ester derivative. In some embodiments, salts of non-natural amino acids are used.

[0104] In any of the above embodiments, wherein R1 、R 2 、R 3 、R 4 Preferably, they are independently selected from -H, C 1-10 Alkyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl and 5 to 14 membered heterocyclic groups, which are optionally replaced by R 5 Substituted, where R 5 Selected from C 1-10 Alkyl, C 1-10 Alkoxy, C 3-10 cycloalkyl and 5- to 14-membered heterocyclyl.

[0105] In any of the above embodiments, wherein R 1 、R 2 、R 3 、R 4 More preferably, they are independently selected from -H, C 1-3 Alkyl, C 1-3 Alkoxy, C 3-6 Cycloalkyl, which are optionally replaced by R 5 Substituted, where R 5 Selected from C 1-10 Alkyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl, pyrrolyl, thienyl, thiazolyl, imidazolyl, pyridyl, furyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl.

[0106] In any of the above embodiments, wherein R 1 、R 2 、R 3 、R 4 More preferably, they are independently selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy and ethoxy.

[0107] In any of the above embodiments, wherein R 1 、R 2 、R 3 、R 4 Still more preferably, R is independently -H or methyl. 1 、R 2 、R 3 、R 4 All are -H.

[0108] In any of the above embodiments, preferably, n is 2 to 4, and each repeating unit is the same or different. More preferably, n is 2 or 3, and each repeating unit is the same or different. In some embodiments, n is 2, and each repeating unit is the same. In some embodiments, n is 3, and each repeating unit is the same. In some embodiments, n is 2, and each repeating unit is different. In some embodiments, n is 3, and each repeating unit is different.

[0109] In any of the above embodiments, preferably, p is 2 to 4, and each repeating unit is the same or different. More preferably, p is 2 or 3, and each repeating unit is the same or different. In some embodiments, p is 2, and each repeating unit is the same. In some embodiments, p is 3, and each repeating unit is the same. In some embodiments, p is 2, and each repeating unit is different. In some embodiments, p is 3, and each repeating unit is different.

[0110] In some embodiments, provided herein are ligand-oligonucleotide conjugates comprising a linker moiety represented by Formula I or Formula II: 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II),

[0111] where R 1 、R 2 、R 3 、R 4 is independently -H or methyl;

[0112] R a 、R b Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-8 Cycloalkyl, 6 to 14 membered aryl C 1-3 Alkyl, 5 to 18 membered heteroaryl C 1-3alkyl and 5 to 14 membered heterocyclic groups, which are optionally replaced by R c Substituted, where R c Selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C 1-3 Alkylthio, indolyl, quinolyl, isoquinolyl, amide, pyrrolyl, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, C 1-10 Alkyl, C 1-10 Alkoxy, 6 to 14 membered aryl, C 3-8 Cycloalkyl, adamantyl, phosphono, phosphonooxy, C 1-3 Alkylphosphono, C 1-3 Alkylphosphonooxy, or R a and R b One of the C atoms and the adjacent N atom to which it is commonly connected together form a 3-14 membered nitrogen-containing heterocyclic group;

[0113] The heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon, preferably nitrogen, oxygen and sulfur; n is 1 to 10, preferably 2 to 4, and is the same or different in each repeating unit; p is 2 to 6, preferably 2 to 4, and is the same or different in each repeating unit.

[0114] In some embodiments, provided herein are ligand-oligonucleotide conjugates comprising a linker moiety represented by Formula I or Formula II: 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II),

[0115] where R 1 、R 2 、R 3 、R 4 is independently -H or methyl;

[0116] where R a 、R b Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C2-10 Alkynyl, C 1-10 Alkoxy, C 3-8 Cycloalkyl, phenyl C 1-3 Alkyl, naphthyl C 1-3 Alkyl, 5 to 12 membered heteroaryl C 1-3 Alkyl and 5 to 14 membered heterocyclic C 1-3 Alkyl groups, which are optionally replaced by R c Substituted, where R c Selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C 1-3 Alkylthio, indolyl, quinolyl, isoquinolyl, amide, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, piperazinyl, pyrrolidinyl, dioxane, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl, C 1-10 Alkyl, C 1-10 Alkoxy, phenyl, benzyl, phenethyl, aminophenyl, carboxyphenyl, halogenated phenyl, biphenyl, C 3-8 Cycloalkyl, adamantyl, phosphono, phosphonooxy, C 1-3 Alkylphosphono, C 1-3 Alkylphosphonooxy, or R a and R b One of the C atoms and the adjacent N atom to which it is commonly connected together form a 3-14 membered nitrogen-containing heterocyclic group;

[0117] The heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon, preferably nitrogen, oxygen and sulfur; n is 1 to 10, preferably 2 to 4, and is the same or different in each repeating unit; p is 2 to 6, preferably 2 to 4, and is the same or different in each repeating unit.

[0118] In some embodiments, provided herein are ligand-oligonucleotide conjugates comprising a linker moiety represented by Formula I or Formula II: 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II),

[0119] where R 1 、R 2 、R 3 、R 4 is independently -H or methyl;

[0120] R a 、R b Independently selected from -H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, phenyl C 1-3 Alkyl, pyridyl C 1-3 Alkyl, indolyl C 1-3 Alkyl, quinolinyl C 1-3 Alkyl, isoquinolinyl C 1-3 Alkyl, thienyl C 1-3 Alkyl, thiaindenyl C 1-3 Alkyl, thiazolyl C 1-3 Alkyl, benzothiazolyl C 1-3 Alkyl, imidazolyl C 1-3 Alkyl, furyl C 1-3 Alkyl, pyrimidinyl C 1-3 Alkyl groups, which are optionally replaced by R c Substituted, where R c Selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C 1-3 Alkylthio, indolyl, quinolyl, isoquinolyl, amide, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, piperazinyl, pyrrolidinyl, dioxane, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl, pyridyl, C 1-10 Alkyl, C 1-10 Alkoxy, phenyl, benzyl, phenethyl, aminophenyl, carboxyphenyl, halogenated phenyl, biphenyl, C 3-8 Cycloalkyl, adamantyl, phosphono, phosphonooxy, C 1-3 Alkylphosphono, C 1-3 Alkylphosphonooxy, or R a and R b One of the C atoms and the adjacent N atom to which it is commonly connected together form a 3-14 membered nitrogen-containing heterocyclic group;

[0121] The heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon, preferably nitrogen, oxygen and sulfur; n is 1 to 10, preferably 2 to 4, and is the same or different in each repeating unit; p is 2 to 6, preferably 2 to 4, and is the same or different in each repeating unit.

[0122] In some embodiments, provided herein are ligand-oligonucleotide conjugates comprising a linker moiety represented by Formula I or Formula II: 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II),

[0123] where R 1 、R 2 、R 3 、R 4 is independently -H or methyl;

[0124] where R a 、R b independently selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-methyl-2-propynyl, 1,3-butadienyl, methoxy, ethoxy, propoxy, butoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenylmethylene, phenyldimethylene, pyridylmethylene, pyridyldimethylene, indolylmethylene, indolyldimethylene, quinolylmethylene, quinolyldimethylene, isoquinolylmethylene, isoquinolyldimethylene, thienylmethylene, thienyldimethylene, thiaindenylmethylene, thiaindenyldimethylene, thiazolylmethylene, thiazolyldimethylene, benzothiazolylmethylene, benzothiazolyldimethylene, imidazolylmethylene, imidazolyldimethylene, furylmethylene, furyldimethylene, pyrimidinylmethylene and pyrimidinyldimethylene, which are optionally replaced by R c Substituted, where R cis selected from the group consisting of hydroxy, thiol, carboxyl, amino, guanidino, halogen, methylenethio, dimethylenethio, indolyl, quinolyl, isoquinolyl, amide, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl, pyridyl, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, phenethyl, aminophenyl, carboxylphenyl, halophenyl, biphenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, phosphonyl, phosphonoxy, dimethylphosphonyl, diethylphosphonyl, dimethylphosphonoxy and diethylphosphonoxy, or R a and R b One of the C atoms to which it is commonly attached and the adjacent N atom together form an imidazolyl, thiazolyl, pyrazolyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl or pyrrolyl group;

[0125] n is 1 to 10, preferably 2 to 4, and each repeating unit is the same or different; p is 2 to 6, preferably 2 to 4, and each repeating unit is the same or different.

[0126] In some embodiments, provided herein are ligand-oligonucleotide conjugates comprising a linker moiety represented by Formula I or Formula II: 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II),

[0127] where R a 、R 1 、R 2 、R 3 、R 4 is independently -H or methyl;

[0128] where R bSelected from -H, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclopentyl, cyclohexyl, cyclohexyldimethylene, cyclohexylmethylene, cyclobutylmethylene, phenylmethylene, methylthiomethylene, 3-indolylmethylene, hydroxymethylene, amidomethylene, hydroxymethylmethylene, mercaptomethylene, amidomethylene, hydroxyphenylmethylene, hydroxyphenyldimethylene, carboxymethylene, carboxydimethylene, amino-n-butyl, guanidine dimethylene, guanidinotrimethylene, 4-imidazolylmethylene, isopropylphenylmethylene, 1-naphthylmethylene, 2-naphthylmethylene, indanyl, phenyldimethylene, fluorophenylmethylene, chlorophenylmethylene, trifluorophenylmethylene, dichlorophenylmethylene, pentafluorophenylmethylene, difluorophenylmethylene, thienylmethylene, thiandenylmethylene, quinolylmethylene, halogenated 3-indolylmethylene, diphenylmethylene, 3-pyridylmethylene and 4-pyridylmethylene, or R b Together with the C atom to which it is commonly connected and the adjacent N atom, it forms a tetrahydropyrrolyl group;

[0129] n is 1 to 10, preferably 2 to 4, and each repeating unit is the same or different; p is 2 to 6, preferably 2 to 4, and each repeating unit is the same or different.

[0130] In some embodiments, provided herein are ligand-oligonucleotide conjugates comprising a linker moiety represented by Formula I or Formula II: 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II),

[0131] where R a 、R 1 、R 2 、R 3 、R 4 R is independently -H or methyl; b is selected from -H, methyl, isopropyl and isobutyl, or R bThe C atom to which it is commonly attached and the adjacent N atom together form a tetrahydropyrrolyl group; n is 2 to 4, and each repeating unit is the same or different; p is 2 to 4, and each repeating unit is the same or different.

[0132] In some embodiments, provided herein are ligand-oligonucleotide conjugates comprising a linker moiety represented by Formula I or Formula II: 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II),

[0133] where R a 、R 1 、R 2 、R 3 、R 4 R is independently -H or methyl; b is selected from -H, methyl, isopropyl and isobutyl, or R b Together with the C atom and the adjacent N atom to which it is commonly attached, it forms a tetrahydropyrrolyl group; n is 2 or 3, and each repeating unit is the same or different; p is 2, and for the repeating unit where p is 2: in each repeating unit, R b isopropyl; in each repeating unit, R b is a methyl group; in a repeating unit, R b is isopropyl, and in another repeating unit, R b is a methyl group; in a repeating unit, R b is isobutyl, and in another repeating unit, R b is a methyl group; in a repeating unit, R b is isobutyl, and in another repeating unit, R b isopropyl; in a repeating unit, R b is isopropyl, and in another repeating unit, R b is hydrogen; in a repeating unit, R b is isopropyl, and in another repeating unit, R bTogether with the C atom and the adjacent N atom to which they are connected, they form a tetrahydropyrrolyl group; or in a repeating unit, R b is hydrogen, and in the other repeating unit, R b Together with the C atom to which it is commonly connected and the adjacent N atom, it forms a tetrahydropyrrolyl group.

[0134] In a preferred embodiment, the left end of the linker represented by Formula I or Formula II is connected to the ligand or used to be connected to the ligand; the right end is connected to the oligonucleotide or used to be connected to the oligonucleotide, preferably to an oligonucleotide with an amino group (e.g., an oligonucleotide with a terminal amino group at the 5' or 3' end) or used to be connected to an oligonucleotide with an amino group (e.g., an oligonucleotide with a terminal amino group at the 5' or 3' end).

[0135] In some embodiments, another aspect of the present invention provides a linker unit represented by formula I' or II': -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I'), or -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II'),

[0136] wherein each substituent and symbol have the meaning as defined herein;

[0137] The left side of formula I' is connected to -NR 1 - is Formula I, the left end of Formula II' is connected to -NR 1 - is Formula II.

[0138] In any of the above embodiments, preferably, the ligand in the ligand-oligonucleotide conjugate comprises one or more N-acetylgalactosamine derivatives attached via a bivalent or trivalent branched linker.

[0139] In a preferred embodiment, the ligand has the following structure: (GalNAc-Q 1 ) m CH m’ -,

[0140] Wherein, GalNAc represents N-acetylgalactosamine or its derivatives; in the present invention, the N-acetylgalactosamine or its derivatives preferably have the following structure:

[0141] Q 1 Indicates T 1 -(T 2 -T 3 -T 4 ) w , where T 1 and T 4 independently selected from absent, C(O), NH, O, S, OC(O), NHC(O), CH2, CH2NH, and CH2O; T 2 is selected from the group consisting of absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH2C(O), C(O)-CH2-NH, C(O) and CH=NO; T 3 One or more methylene groups are selected from the group consisting of: O, S, S(O), SO2, C(O) and C≡C; w is 0 to 20, preferably 1-5, and the repeating units are the same or different; said R o 、R o 'are independently selected from H, alkylcarbonyl, the alkyl group is as defined above; the R o ' is preferably acetyl; said R o Each independently preferably is H or acetyl, more preferably H.

[0142] m is 1 to 3, preferably 2 or 3, and each repeating unit is the same or different; m′ is 0 to 2, preferably 0 or 1, and m+m′ is 3.

[0143] In a preferred embodiment, the structure of the ligand is selected from:

[0144] In a more preferred embodiment, the structure of the ligand is:

[0145] In any of the above embodiments, preferably, the oligonucleotide in the ligand-oligonucleotide conjugate is selected from antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs) and microRNAs (miRNAs) or salts thereof, and the salt is preferably a sodium salt or a potassium salt.

[0146] In some embodiments, the 5' end of the oligonucleotide is attached to the linker portion, preferably to the right end of the linker portion. In some embodiments, the 3' end of the oligonucleotide is attached to the linker portion, preferably to the right end of the linker portion.

[0147] In some embodiments, the 5' end of the oligonucleotide is terminated by 3'-oligonucleotide-5'-OP(O)(OH)O-(CH2-Y) x -NH- is attached to the linker portion, wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different. In such an embodiment, preferably, the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(O)(OH)O-(CH2)6-NH-; further preferably, the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(O)(OH)O-(CH2)3-NH-; further preferably, the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(O)(OH)O-(CH2) 12 -NH- is attached to the linker portion; also preferably, the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH-.

[0148] In some embodiments, the 5' end of the oligonucleotide is terminated by 3'-oligonucleotide-5'-OP(S)(OH)O-(CH2-Y) x -NH- is attached to the linker portion, wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different. In such an embodiment, preferably, the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(S)(OH)O-(CH2)6-NH-; further preferably, the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(S)(OH)O-(CH2)3-NH-; further preferably, the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(S)(OH)O-(CH2) 12 -NH- is attached to the linker portion; also preferably, the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH-.

[0149] In some embodiments, the 3' end of the oligonucleotide may be 5'-oligonucleotide-3'-OP(O)(OH)O-(CH2-Y) x-NH- is attached to the linker portion, wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different. In such an embodiment, preferably, the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(O)(OH)O-(CH2)6-NH-; further preferably, the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(O)(OH)O-(CH2)3-NH-; further preferably, the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(O)(OH)O-(CH2) 12 -NH- is attached to the linker portion; also preferably, the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH-.

[0150] In some embodiments, the 3' end of the oligonucleotide may be 5'-oligonucleotide-3'-OP(S)(OH)O-(CH2-Y) x -NH- is attached to the linker portion, wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different. In such an embodiment, preferably, the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(S)(OH)O-(CH2)6-NH-; further preferably, the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(S)(OH)O-(CH2)3-NH-; further preferably, the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(S)(OH)O-(CH2) 12 -NH- is attached to the linker portion; also preferably, the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH-.

[0151] In a preferred embodiment, the oligonucleotide is a siRNA comprising a sense strand and an antisense strand, more preferably, one or more of the nucleotides in the siRNA are modified, the modification being a 2'-O-methyl modification and / or a 2'-fluoro modification. In some embodiments, the 3' end or the 5' end of the sense strand is attached to the linker moiety. In a preferred embodiment, the 5' end of the sense strand is attached to the linker moiety.

[0152] In a preferred embodiment, the 5' end of the sense strand is connected by 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH2-Y) x-NH- is attached to the linker portion, wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different. In a preferred embodiment, the 5' end of the sense strand is attached to the linker portion via 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH2)6-NH-; further preferably, the 5' end of the sense strand is attached to the linker portion via 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH2)3-NH-; further preferably, the 5' end of the sense strand is attached to the linker portion via 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH2) 12 -NH- is attached to the linker portion; also preferably, the 5' end of the sense strand is attached to the linker portion through 3'-siRNA sense strand -5'-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH-.

[0153] In a preferred embodiment, the 5' end of the sense strand is connected by 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH2-Y) x -NH- is attached to the linker portion, wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different. In a preferred embodiment, the 5' end of the sense strand is attached to the linker portion via 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH2)6-NH-; further preferably, the 5' end of the sense strand is attached to the linker portion via 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH2)3-NH-; further preferably, the 5' end of the sense strand is attached to the linker portion via 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH2) 12 -NH- is attached to the linker portion; also preferably, the 5' end of the sense strand is attached to the linker portion through 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH-.

[0154] In a preferred embodiment, the present invention provides a ligand-oligonucleotide conjugate represented by Formula V, Formula V', Formula VI, Formula VI', Formula V-1, Formula V'-1, Formula VI-1 or Formula VI'-1: (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b)-C(O)] p -NH-(CH2-Y) x -O- P(O)(OH)O-5'-oligonucleotide-3'(V), (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -NH-(CH2-Y) x -O- P(O)(OH)O-3'-oligonucleotide-5'(V'), (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-NH-(CH2-Y) x -O- P(O)(OH)O-5'-oligonucleotide-3'(VI); (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-NH-(CH2-Y) x -O- P(O)(OH)O-3'-oligonucleotide-5'(VI'), (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)]p -NH-(CH2-Y) x -O- P(S)(OH)O-5'-oligonucleotide-3'(V-1), (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -NH-(CH2-Y) x -O- P(S)(OH)O-3'-oligonucleotide-5'(V'-1), (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-NH-(CH2-Y) x -O- P(S)(OH)O-5'-oligonucleotide-3'(VI-1); (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-NH-(CH2-Y) x -O- P(S)(OH)O-3'-oligonucleotide-5'(VI'-1);

[0155] wherein each substituent and symbol has the corresponding meaning, value and numerical range as defined above, or its preferred meaning, value and numerical range; wherein the oligonucleotide is preferably siRNA, siRNA sense strand or ASO.

[0156] In a preferred embodiment, the present invention provides a ligand-oligonucleotide conjugate represented by formula VII, formula VII', formula VIII, formula VIII', VII-1, formula VII'-1, formula VIII-1, and formula VIII'-1: (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -NH-(CH2-Y) x -O- P(O)(OH)O-5'-siRNA sense strand-3'(VII), (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -NH-(CH2-Y) x -O- P(O)(OH)O-3'-siRNA sense strand-5'(VII'), (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-NH-(CH2-Y) x -O- P(O)(OH)O-5'-siRNA sense strand-3'(VIII), (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R4 )] n -C(O)-NH-(CH2-Y) x -O- P(O)(OH)O-3'-siRNA sense strand-5'(VIII'), (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -NH-(CH2-Y) x -O- P(S)(OH)O-5'-siRNA sense strand-3'(VII-1), (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -NH-(CH2-Y) x -O- P(S)(OH)O-3'-siRNA sense strand-5'(VII'-1), (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-NH-(CH2-Y) x -O- P(S)(OH)O-5'-siRNA sense strand-3'(VIII-1), (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R3 R 4 )] n -C(O)-NH-(CH2-Y) x -O- P(S)(OH)O-3'-siRNA sense strand-5'(VIII'-1);

[0157] wherein each substituent and symbol has the corresponding meaning, value and numerical range as defined herein above, or its preferred meaning, value and numerical range; wherein the siRNA antisense strand is connected to the siRNA sense strand by base pairing to form a double-stranded RNA.

[0158] In a preferred embodiment, the present invention provides a ligand-oligonucleotide conjugate represented by Formula IX, Formula IX', Formula X, Formula X', Formula IX-1, Formula IX'-1, Formula X-1, and Formula X'-1: (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -NH-(CH2-Y) x -O- P(O)(OH)O-5'-ASO-3'(IX), (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -NH-(CH2-Y) x -O- P(O)(OH)O-5'-ASO-3'(IX'), (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n-C(O)-NH-(CH2-Y) x -O- P(O)(OH)O-5’-ASO-3’(X), (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-NH-(CH2-Y) x -O- P(O)(OH)O-3’-ASO-5’(X’), (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -NH-(CH2-Y) x -O- P(S)(OH)O-5’-ASO-3’(IX-1), (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -NH-(CH2-Y) x -O- P(S)(OH)O-5’-ASO-3’(IX’-1), (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-NH-(CH2-Y)x -O- P(S)(OH)O-5'-ASO-3'(X-1), (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-NH-(CH2-Y) x -O- P(S)(OH)O-3'-ASO-5'(X'-1);

[0159] wherein each substituent and symbol has the corresponding meaning, value and numerical range as defined herein above, or its preferred meaning, value and numerical range.

[0160] In a preferred embodiment, provided herein is a ligand-oligonucleotide conjugate selected from the following formulae:

[0161] in indicates siRNA;

[0162] in,

[0163] Preferably:

[0164] Preferably:

[0165] Preferably:

[0166] Preferably:

[0167] Preferably,

[0168] Intermediate compounds

[0169] Another aspect of the present invention provides an intermediate compound having a structure shown in Formula III or IV:

[0170] (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -Q 2 (Formula III), or

[0171] (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-Q 2 (Formula IV),

[0172] Wherein, GalNAc represents N-acetylgalactosamine or its derivatives; in the present invention, the N-acetylgalactosamine or its derivatives preferably have the following structure:

[0173] Q 1 Indicates -T 1 -(T 2 -T 3 -T 4 ) w , where T 1 and T 4 independently selected from absent, C(O), NH, O, S, OC(O), NHC(O), CH2, CH2NH, and CH2O; T 2is selected from the group consisting of absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH2C(O), C(O)-CH2-NH, C(O) and CH=NO; T 3 is selected from one or more methylene groups which are absent and optionally interrupted or terminated by atoms or groups selected from O, S, S(O), SO2, C(O) and C≡C; w is 0 to 20, and the repeating units are the same or different; R o 、R o 'As defined above.

[0174] Q 2 represents an -OH or -O-carboxyl protecting group, wherein the carboxyl protecting group is preferably benzyl or pentafluorophenyl;

[0175] R 1 、R 2 、R 3 、R 4 Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3- 10 Cycloalkyl, 6- to 14-membered aryl, 5- to 18-membered heteroaryl, 5- to 14-membered heterocyclyl, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, and mercapto, which are optionally replaced by R 5 Substituted, where R 5 Selected from C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl, 6- to 14-membered aryl, 5- to 18-membered heteroaryl, 5- to 14-membered heterocyclic group, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, C 1-3 alkylthio and mercapto groups;

[0176] R a 、R b Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-8 Cycloalkyl, 6 to 14 membered aryl C 1-3 Alkyl, 5 to 18 membered heteroaryl C 1-3 alkyl and 5 to 14 membered heterocyclic groups, which are optionally replaced by R c Substituted, where R c Selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C 1-3Alkylthio, indolyl, quinolyl, isoquinolyl, amide, pyrrolyl, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, C 1-10 Alkyl, C 1-10 Alkoxy, 6 to 14 membered aryl, C 3-8 Cycloalkyl, adamantyl, phosphono, phosphonooxy, C 1-3 Alkylphosphono, C 1-3 Alkylphosphonooxy, or R a and R b One of them, together with the C atom to which they are connected and the adjacent N atom, forms a 3-14 membered nitrogen-containing heterocyclic group, or R a 、R b Together with its co-connected C atom, it forms C 3-8 Cycloalkyl;

[0177] heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon;

[0178] m is 1 to 3, and each repeating unit is the same or different; m' is 0 to 2, preferably 0 or 1, and m+m' is 3;

[0179] n is 1 to 10, and each repeating unit is the same or different;

[0180] p is 2 to 6, and each repeating unit is the same or different.

[0181] In a preferred embodiment, (GalNAc-Q 1 ) m CH m’ -The structure of the part is selected from:

[0182] In a more preferred embodiment, (GalNAc-Q 1 ) m CH m’ -The structure of the part is:

[0183] Therefore, in some embodiments, an intermediate compound is provided, which has a structure shown in Formula III or IV:

[0184] (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR2 -C(R a R b )-C(O)] p -Q 2 (Formula III), or

[0185] (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-Q 2 (Formula IV),

[0186] Among them (GalNAc-Q 1 ) m CH m’ -The structure of the part is:

[0187] Q 2 represents a -OH or -O carboxyl protecting group;

[0188] R 1 、R 2 、R 3 、R 4 Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3- 10 Cycloalkyl, 6- to 14-membered aryl, 5- to 18-membered heteroaryl, 5- to 14-membered heterocyclyl, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, and mercapto, which are optionally replaced by R 5 Substituted, where R 5 Selected from C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl, 6- to 14-membered aryl, 5- to 18-membered heteroaryl, 5- to 14-membered heterocyclic group, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, C 1-3 alkylthio and mercapto groups;

[0189] R a 、R b Independently selected from -H, C 1-10 Alkyl, C2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-8 Cycloalkyl, 6 to 14 membered aryl C 1-3 Alkyl, 5 to 18 membered heteroaryl C 1-3 alkyl and 5 to 14 membered heterocyclic groups, which are optionally replaced by R c Substituted, where R c Selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C 1-3 Alkylthio, indolyl, quinolyl, isoquinolyl, amide, pyrrolyl, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, C 1-10 Alkyl, C 1-10 Alkoxy, 6 to 14 membered aryl, C 3-8 Cycloalkyl, adamantyl, phosphono, phosphonooxy, C 1-3 Alkylphosphono, C 1-3 Alkylphosphonooxy, or R a and R b One of the C atoms and the adjacent N atom to which it is commonly connected together form a 3-14 membered nitrogen-containing heterocyclic group;

[0190] heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon;

[0191] n is 1 to 10, and each repeating unit is the same or different;

[0192] p is 2 to 6, and each repeating unit is the same or different.

[0193] In any embodiment of the above intermediate compound, in the structure shown in Formula III or IV, preferably, R a 、R b Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-8 Cycloalkyl, phenyl C 1-3 Alkyl, naphthyl C 1-3 Alkyl, 5 to 12 membered heteroaryl C 1-3 Alkyl and 5 to 14 membered heterocyclic C 1-3 Alkyl groups, which are optionally replaced by R c Substituted, where R c Selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C 1-3Alkylthio, indolyl, quinolyl, isoquinolyl, amide, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, piperazinyl, pyrrolidinyl, dioxane, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl, C 1-10 Alkyl, C 1-10 Alkoxy, phenyl, benzyl, phenethyl, aminophenyl, carboxyphenyl, halogenated phenyl, biphenyl, C 3-8 Cycloalkyl, adamantyl, phosphono, phosphonooxy, C 1-3 Alkylphosphono, C 1-3 Alkylphosphonooxy, or R a and R b One of the carbon atoms, the carbon atoms to which they are commonly attached, and the adjacent nitrogen atom together form a 3- to 14-membered nitrogen-containing heterocyclic group.

[0194] In any embodiment of the above intermediate compound, in the structure shown in formula III or IV, more preferably, R a 、R b Independently selected from -H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, phenyl C 1-3 Alkyl, pyridyl C 1-3 Alkyl, indolyl C 1-3 Alkyl, quinolinyl C 1-3 Alkyl, isoquinolinyl C 1-3 Alkyl, thienyl C 1- 3-alkyl, thiaindenyl C 1-3 Alkyl, thiazolyl C 1-3 Alkyl, benzothiazolyl C 1-3 Alkyl, imidazolyl C 1-3 Alkyl, furyl C 1-3 Alkyl, pyrimidinyl C 1-3 Alkyl groups, which are optionally replaced by R c Substituted, where R c Selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C 1-3 Alkylthio, indolyl, quinolyl, isoquinolyl, amide, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, piperazinyl, pyrrolidinyl, dioxane, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl, pyridyl, C 1-10 Alkyl, C 1-10 Alkoxy, phenyl, benzyl, phenethyl, aminophenyl, carboxyphenyl, halogenated phenyl, biphenyl, C 3-8 Cycloalkyl, adamantyl, phosphono, phosphonooxy, C 1-3Alkylphosphono, C 1-3 Alkylphosphonooxy, or R a and R b One of the carbon atoms, the carbon atoms to which they are commonly attached, and the adjacent nitrogen atom together form a 3- to 14-membered nitrogen-containing heterocyclic group.

[0195] In any embodiment of the above intermediate compound, in the structure shown in formula III or IV, more preferably, R a 、R b independently selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-methyl-2-propynyl, 1,3-butadienyl, methoxy, ethoxy, propoxy, butoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenylmethylene, phenyldimethylene, pyridylmethylene, pyridyldimethylene, indolylmethylene, indolyldimethylene, quinolylmethylene, quinolyldimethylene, isoquinolylmethylene, isoquinolyldimethylene, thienylmethylene, thienyldimethylene, thiaindenylmethylene, thiaindenyldimethylene, thiazolylmethylene, thiazolyldimethylene, benzothiazolylmethylene, benzothiazolyldimethylene, imidazolylmethylene, imidazolyldimethylene, furylmethylene, furyldimethylene, pyrimidinylmethylene and pyrimidinyldimethylene, which are optionally replaced by R c Substituted, where R c is selected from the group consisting of hydroxy, thiol, carboxyl, amino, guanidino, halogen, methylenethio, dimethylenethio, indolyl, quinolyl, isoquinolyl, amide, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl, pyridyl, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, phenethyl, aminophenyl, carboxylphenyl, halophenyl, biphenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, phosphonyl, phosphonoxy, dimethylphosphonyl, diethylphosphonyl, dimethylphosphonoxy and diethylphosphonoxy, or R a and R b One of the carbon atoms to which it is commonly attached and the adjacent nitrogen atom together form an imidazolyl, thiazolyl, pyrazolyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl or pyrrolyl group.

[0196] In any embodiment of the above intermediate compound, in the structure shown in formula III or IV, more preferably, R 1 、R 2 、R 3 、R 4Preferably, they are independently selected from -H, C 1-10 Alkyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl and 5 to 14 membered heterocyclic groups, which are optionally replaced by R 5 Substituted, where R 5 Selected from C 1-10 Alkyl, C 1-10 Alkoxy, C 3-10 cycloalkyl and 5- to 14-membered heterocyclyl.

[0197] In any embodiment of the above intermediate compound, in the structure shown in formula III or IV, more preferably, wherein R 1 、R 2 、R 3 、R 4 More preferably, they are independently selected from -H, C 1-3 Alkyl, C 1-3 Alkoxy, C 3-6 Cycloalkyl, which are optionally replaced by R 5 Substituted, where R 5 Selected from C 1-10 Alkyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl, pyrrolyl, thienyl, thiazolyl, imidazolyl, pyridyl, furyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl.

[0198] In any embodiment of the above intermediate compound, in the structure shown in formula III or IV, more preferably, wherein R 1 、R 2 、R 3 、R 4 More preferably, they are independently selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy and ethoxy.

[0199] In any embodiment of the above intermediate compound, in the structure shown in formula III or IV, more preferably, R 1 、R 2 、R 3 、R 4 More preferably, R is independently -H or methyl. In any embodiment of the above intermediate compound, in the structure shown in formula III or IV, more preferably, R 1 、R 2 、R 3 、R 4 All are -H.

[0200] In any embodiment of the above intermediate compound, preferably, n is 2 to 4, and each repeating unit is the same or different. More preferably, n is 2 or 3, and each repeating unit is the same or different. In some embodiments, n is 2, and each repeating unit is the same. In some embodiments, n is 3, and each repeating unit is the same. In some embodiments, n is 2, and each repeating unit is different. In some embodiments, n is 3, and each repeating unit is different.

[0201] In any embodiment of the above intermediate compound, preferably, p is 2 to 4, and each repeating unit is the same or different. More preferably, p is 2 or 3, and each repeating unit is the same or different. In some embodiments, p is 2, and each repeating unit is the same. In some embodiments, p is 3, and each repeating unit is the same. In some embodiments, p is 2, and each repeating unit is different. In some embodiments, p is 3, and each repeating unit is different.

[0202] In any embodiment of the above intermediate compound, in the structure shown in formula III or IV, more preferably, wherein R a Yes -H,R b Selected from -H, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclopentyl, cyclohexyl, cyclohexyldimethylene, cyclohexylmethylene, cyclobutylmethylene, phenylmethylene, methylthiomethylene, 3-indolylmethylene, hydroxymethylene, amidomethylene, hydroxymethylmethylene, mercaptomethylene, amidomethylene, hydroxyphenylmethylene, hydroxyphenyldimethylene, carboxymethylene, carboxydimethylene, amino-n-butyl, guanidine dimethylene, guanidinotrimethylene, 4-imidazolylmethylene, isopropylphenylmethylene, 1-naphthylmethylene, 2-naphthylmethylene, indanyl, phenyldimethylene, fluorophenylmethylene, chlorophenylmethylene, trifluorophenylmethylene, dichlorophenylmethylene, pentafluorophenylmethylene, difluorophenylmethylene, thienylmethylene, thiandenylmethylene, quinolylmethylene, halogenated 3-indolylmethylene, diphenylmethylene, 3-pyridylmethylene and 4-pyridylmethylene, or R b Together with the C atom to which it is commonly connected and the adjacent N atom, it forms a tetrahydropyrrolyl group.

[0203] In any embodiment of the above intermediate compound, in the structure shown in formula III or IV, more preferably, wherein R a 、R 1 、R 2 、R 3 、R 4 R is independently -H or methyl; bSelected from -H, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclopentyl, cyclohexyl, cyclohexyldimethylene, cyclohexylmethylene, cyclobutylmethylene, phenylmethylene, methylthiomethylene, 3-indolylmethylene, hydroxymethylene, amidomethylene, hydroxymethylmethylene, mercaptomethylene, amidomethylene, hydroxyphenylmethylene, hydroxyphenyldimethylene, carboxymethylene, carboxydimethylene, amino-n-butyl, guanidine dimethylene, guanidinotrimethylene, 4-imidazolylmethylene, isopropylphenylmethylene, 1-naphthylmethylene, 2-naphthylmethylene, indanyl, phenyldimethylene, fluorophenylmethylene, chlorophenylmethylene, trifluorophenylmethylene, dichlorophenylmethylene, pentafluorophenylmethylene, difluorophenylmethylene, thienylmethylene, thiandenylmethylene, quinolylmethylene, halogenated 3-indolylmethylene, diphenylmethylene, 3-pyridylmethylene and 4-pyridylmethylene, or R b The C atom to which it is commonly attached and the adjacent N atom together form a tetrahydropyrrolyl group; n is 1 to 10, preferably 2 to 4, and each repeating unit is the same or different; p is 2 to 6, preferably 2 to 4, and each repeating unit is the same or different.

[0204] In any embodiment of the above intermediate compound, in the structure shown in Formula III or IV, more preferably, R a 、R 1 、R 2 、R 3 、R 4 R is independently -H or methyl; b is selected from -H, methyl, isopropyl and isobutyl, or R b The C atom to which it is commonly attached and the adjacent N atom together form a tetrahydropyrrolyl group; n is 2 to 4, and each repeating unit is the same or different; p is 2 to 4, and each repeating unit is the same or different.

[0205] In any embodiment of the above intermediate compound, in the structure shown in Formula III or IV, more preferably, R a 、R 1 、R 2 、R 3 、R 4 R is independently -H or methyl; b is selected from -H, methyl, isopropyl and isobutyl, or R b Together with the C atom and the adjacent N atom to which it is commonly attached, it forms a tetrahydropyrrolyl group; n is 2 or 3, and each repeating unit is the same or different; p is 2, and for the repeating unit where p is 2: in each repeating unit, R b isopropyl; in each repeating unit, R bis a methyl group; in a repeating unit, R b is isopropyl, and in another repeating unit, R b is a methyl group; in a repeating unit, R b is isobutyl, and in another repeating unit, R b is a methyl group; in a repeating unit, R b is isobutyl, and in another repeating unit, R b isopropyl; in a repeating unit, R b is isopropyl, and in another repeating unit, R b is hydrogen; in a repeating unit, R b is isopropyl, and in another repeating unit, R b Together with the C atom and the adjacent N atom to which they are connected, they form a tetrahydropyrrolyl group; or in a repeating unit, R b is hydrogen, and in the other repeating unit, R b Together with the C atom to which it is commonly connected and the adjacent N atom, it forms a tetrahydropyrrolyl group.

[0206] In the present invention, the fragment-[NR 2 -

[0207] C(R a R b )-C(O)]-、-[C(O)-C(R a R b )-NR 2 ] - specifically, an amino acid residue or fragment, wherein the amino acid is as defined in the present invention;

[0208] The amino acid residue or fragment is a residue or fragment after the amino group of the amino acid loses a hydrogen and the carboxyl group loses -OH;

[0209] In any embodiment, preferably, -[NR 2 -C(R a R b )-C(O)]-are independently selected from

[0210] In any embodiment, preferably, -[C(O)-C(R a R b )-NR 2 ]-are independently selected from

[0211] The -[NR 2-C(R a R b )-C(O)] p -、-[C(O)-C(R a R b )-NR 2 ] p -, wherein p is preferably 2, i.e. -[NR 2 -C(R a R b )-C(O)]-[NR 2 -C(R a R b )-C(O)]-、-[C(O)-C(R a R b )-NR 2 ]-[C(O)-C(R a R b )-NR 2 ]-;

[0212] When p is 2, -[NR 2 -C(R a R b )-C(O)] p -(ie-[NR 2 -C(R a R b )-C(O)]-[NR 2 -C(R a R b )-C(O)]-) is preferably

[0213] When p is 2, -[C(O)-C(R a R b )-NR 2 ] p -(i.e. -[C(O)-C(R a R b )-NR 2 ]-[C(O)-C(R a R b )-NR 2 ]-) preferably

[0214] In some embodiments, the intermediate represented by formula III or IV is preferably:

[0215] Among them, R o 、R o', Q 2 The definitions are as above.

[0216] In a preferred embodiment, provided herein is a compound selected from the following formula:

[0217] Wherein Bn represents a benzyl protecting group, and Ac represents an acetoxy group.

[0218] Preparation method

[0219] Another aspect of the present invention provides a method for preparing a ligand-oligonucleotide conjugate, comprising: (a) providing any intermediate compound described herein; (b) providing an oligonucleotide having a terminal amino group at the 5' or 3' end of the oligonucleotide; (c) linking the intermediate compound to the oligonucleotide via the amino group; and optionally, (d) a deprotection step (preferably a deprotection step).

[0220] In some embodiments, in step (c), the intermediate compound is attached to the 5' end of the oligonucleotide. In some embodiments, in step (c), the intermediate compound is attached to the 3' end of the oligonucleotide. Typically, oligonucleotides are synthesized in a 3' to 5' direction on a solid support. When the intermediate compound is to be attached to the 3' end of an oligonucleotide, it is necessary to conjugate the intermediate compound with 3' nucleosides in advance and connect to a solid support, then synthesize the oligonucleotide by conventional synthesis methods (such as phosphoramidite method). However, conjugating in advance and being connected to a solid support increases the complexity of synthesis. In addition, using this method, conjugates will be present in the entire building-up process of oligonucleotides, during which degradation may occur, and the type of usable reactions and reagents may also be limited.

[0221] This document does not exclude the possibility of attaching the intermediate compound to the 3' end of the nucleotide, but preferably, the intermediate compound is attached to the 5' end of the oligonucleotide. When the intermediate compound is attached to the 5' end of the oligonucleotide, the oligonucleotide can be synthesized from the 3' to the 5' direction on a solid support according to conventional synthesis methods (e.g., the phosphoramidite method). The intermediate compound can be introduced into the conjugate together with the last (5'-most) nucleoside at the 5' end or after the oligonucleotide is cleaved from the solid support.

[0222] In some embodiments, when synthesizing oligonucleotides, a terminal amino group is introduced after the last (most 5') nucleoside at the 5' end, thereby making the synthesized oligonucleotide have a 5' terminal amino group. In such an embodiment, the synthesized oligonucleotide can have the structure shown in Formula XI, XI':

[0223] 3'-Oligonucleotide-5'-OP(O)(OH)O-(CH2-Y) x -NH2 (Formula XI)

[0224] 3'-Oligonucleotide-5'-OP(S)(OH)O-(CH2-Y) x -NH2 (Formula XI')

[0225] wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different.

[0226] In some embodiments, the structural formula of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(O)(OH)O-(CH2)6-NH2. In some embodiments, the structural formula of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(O)(OH)O-(CH2)3-NH2. In some embodiments, the structural formula of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(O)(OH)O-(CH2) 12 In some embodiments, the structure of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH2.

[0227] In some embodiments, the structural formula of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(S)(OH)O-(CH2)6-NH2. In some embodiments, the structural formula of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(S)(OH)O-(CH2)3-NH2. In some embodiments, the structural formula of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(S)(OH)O-(CH2) 12In some embodiments, the structure of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH2.

[0228] Reagents and methods for introducing a terminal amino group into the 5' end of a synthetic oligonucleotide are known in the art. Suitable 5'-terminal amino group introducing agents (also known as modifiers or linkers) include, but are not limited to, 5'-amino TFA (C6) (CAS#133975-85-6), 5'-amino TFA (C3) (CAS#853955-89-2), 5'-amino TFA (C12) (CAS#178925-51-4), and 5'-amino modifier 5-CEP (CAS#612548-86-4).

[0229] In some embodiments, to introduce an amino group at the 3'-terminus of an oligonucleotide, a terminal modifier is directly used as a starting solid support, thereby obtaining an oligonucleotide having a 3'-terminal amino group. In such embodiments, the synthesized oligonucleotide may have the structure shown in Formula XI-1 or XI-1':

[0230] 5'-Oligonucleotide-3'-OP(O)(OH)O-(CH2-Y) x -NH2 (Formula XI-1)

[0231] 5'-Oligonucleotide-3'-OP(S)(OH)O-(CH2-Y) x -NH2 (Formula XI-1')

[0232] wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different.

[0233] In some embodiments, the structural formula of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(O)(OH)O-(CH2)6-NH2. In some embodiments, the structural formula of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(O)(OH)O-(CH2)3-NH2. In some embodiments, the structural formula of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(O)(OH)O-(CH2) 12 In some embodiments, the structure of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH2.

[0234] In some embodiments, the structural formula of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(S)(OH)O-(CH2)6-NH2. In some embodiments, the structural formula of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(S)(OH)O-(CH2)3-NH2. In some embodiments, the structural formula of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(S)(OH)O-(CH2) 12 In some embodiments, the structure of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH2.

[0235] Reagents and methods for introducing a terminal amino group into the 3' end of a synthetic oligonucleotide are known in the art. Suitable 3' terminal amino group introducing agents (or terminal group modifiers) include, but are not limited to, 3'-PT-Amino-Modifier C6CPG.

[0236] In some embodiments, the oligonucleotide is a siRNA comprising a sense strand and an antisense strand, and the intermediate compound is connected to the sense strand of the siRNA. In some embodiments, in step (c), the intermediate compound is attached to the 3' end of the sense strand of the siRNA. In a preferred embodiment, in step (c), the intermediate compound is attached to the 5' end of the sense strand of the siRNA.

[0237] In some embodiments, when synthesizing the sense strand of an siRNA, a terminal amino group is introduced after the last (most 5') nucleoside at the 5' end, thereby providing the synthesized sense strand with a 5' terminal amino group. In such an embodiment, the synthesized sense strand of an siRNA may have the structure shown in Formula XII, XII':

[0238] 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH2-Y) x -NH2 (Formula XII)

[0239] 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH2-Y) x -NH2 (Formula XII')

[0240] wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different.

[0241] In some embodiments, the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH2)6-NH2. In some embodiments, the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH2)3-NH2. In some embodiments, the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH2) 12 In some embodiments, the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH2.

[0242] In some embodiments, the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH2)6-NH2. In some embodiments, the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH2)3-NH2. In some embodiments, the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH2) 12 In some embodiments, the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH2.

[0243] In some embodiments, when synthesizing the sense strand of an siRNA, in order to introduce an amino group at the 3'-terminus of the sense strand of the siRNA, a terminal group modifier is directly used as a starting solid support, thereby obtaining a sense strand of the siRNA having a 3'-terminal amino group. In such an embodiment, the synthesized sense strand of the siRNA may have the structure shown in Formula XII-1 or XII-1':

[0244] 5'-siRNA sense strand-3'-OP(O)(OH)O-(CH2-Y) x -NH2 (Formula XII)

[0245] 5'-siRNA sense strand-3'-OP(S)(OH)O-(CH2-Y) x -NH2 (Formula XII')

[0246] wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different.

[0247] In some embodiments, the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(O)(OH)O-(CH2)6-NH2. In some embodiments, the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(O)(OH)O-(CH2)3-NH2. In some embodiments, the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(O)(OH)O-(CH2) 12 In some embodiments, the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH2.

[0248] In some embodiments, the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(S)(OH)O-(CH2)6-NH2. In some embodiments, the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(S)(OH)O-(CH2)3-NH2. In some embodiments, the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(S)(OH)O-(CH2) 12 In some embodiments, the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH2.

[0249] In such an embodiment, the antisense strand of the siRNA can be synthesized by conventional synthesis and annealed to the sense strand through base pairing to form a double-stranded RNA.

[0250] In the present invention, the annealing or simulated annealing procedure is as follows: complementary strands (SS and AS) of acceptable purity (RP-HPLC purity >90%) are mixed in an equimolar ratio, the solution is heated to 90°C in a 70°C water bath, maintained at 90°C for 5 minutes, and slowly cooled to room temperature. A sample is then collected for SEC analysis of the double-strand annealing purity, and the ratio of SS or AS strands is fine-tuned to achieve a double-strand purity of greater than 90%. The siRNA is lyophilized and stored at -15°C to -25°C.

[0251] In a preferred embodiment, one or more of the nucleotides in the siRNA are modified. In a preferred embodiment, the modification is a 2'-O-methyl modification and / or a 2'-fluoro modification. In a preferred embodiment, the modification is a phosphorothioate modification in the backbone. In a preferred embodiment, the modification comprises a 2'-O-methyl modification, a 2'-fluoro modification, and a phosphorothioate modification in the backbone.

[0252] In some embodiments, the oligonucleotide is an ASO and the intermediate compound is attached to the 5' end or the 3' end of the ASO. In some embodiments, in step (c), the intermediate compound is attached to the 3' end of the ASO. In a preferred embodiment, in step (c), the intermediate compound is attached to the 5' end of the ASO.

[0253] In some embodiments, in the case of an ASO, a terminal amino group is introduced after the last (most 5') nucleoside at the 5' end, thereby making the ASO have a 5' terminal amino group. In such an embodiment, the synthesized ASO may have a structure shown in Formula XIII, XIII': 3'-ASO-5'-OP(O)(OH)O-(CH2-Y) x -NH2(Formula XIII) 3'-ASO-5'-OP(S)(OH)O-(CH2-Y) x -NH2 (Formula XIII')

[0254] wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different.

[0255] In some embodiments, the structure of the synthesized ASO is: 3'-ASO-5'-OP(O)(OH)O-(CH2)6-NH2. In some embodiments, the structure of the synthesized ASO is: 3'-ASO-5'-OP(O)(OH)O-(CH2)3-NH2. In some embodiments, the structure of the synthesized ASO is: 3'-ASO-5'-OP(O)(OH)O-(CH2)3-NH2. 12 In some embodiments, the structure of the synthesized ASO is: 3'-ASO-5'-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH2.

[0256] In some embodiments, the structure of the synthesized ASO is: 3'-ASO-5'-OP(S)(OH)O-(CH2)6-NH2. In some embodiments, the structure of the synthesized ASO is: 3'-ASO-5'-OP(S)(OH)O-(CH2)3-NH2. In some embodiments, the structure of the synthesized ASO is: 3'-ASO-5'-OP(S)(OH)O-(CH2) 12 In some embodiments, the structure of the synthesized ASO is: 3'-ASO-5'-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH2.

[0257] In some embodiments, in order to introduce an amino group at the 3'-terminus of the ASO, a terminal group modifier is directly used as a starting solid support, so that the resulting ASO has a 3'-terminal amino group. In such an embodiment, the synthesized ASO may have the structure shown in Formula XIII-1 or XIII-1': 5'-ASO-3'-OP(O)(OH)O-(CH2-Y) x -NH2(Formula XIII-1) 5'-ASO-3'-OP(S)(OH)O-(CH2-Y) x -NH2 (Formula XIII-1')

[0258] wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different.

[0259] In some embodiments, the structure of the synthesized ASO is: 5'-ASO-3'-OP(O)(OH)O-(CH2)6-NH2. In some embodiments, the structure of the synthesized ASO is: 5'-ASO-3'-OP(O)(OH)O-(CH2)3-NH2. In some embodiments, the structure of the synthesized ASO is: 5'-ASO-3'-OP(O)(OH)O-(CH2) 12 In some embodiments, the structure of the synthesized ASO is: 5'-ASO-3'-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH2.

[0260] In some embodiments, the structure of the synthesized ASO is: 5'-ASO-3'-OP(S)(OH)O-(CH2)6-NH2. In some embodiments, the structure of the synthesized ASO is: 5'-ASO-3'-OP(S)(OH)O-(CH2)3-NH2. In some embodiments, the structure of the synthesized ASO is: 5'-ASO-3'-OP(S)(OH)O-(CH2) 12In some embodiments, the structure of the synthesized ASO is: 5'-ASO-3'-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH2.

[0261] In a preferred embodiment, one or more of the nucleotides in the ASO are modified. In a preferred embodiment, the modification is selected from phosphorothioate (PSP), diaminomorpholino (PMO), 2'-methoxyethyl (2'-MOE) and 5-methylcytosine (5mC). In a preferred embodiment, the modification includes PSP, PMO, 2'-MOE and 5mC.

[0262] In step (c), the intermediate compound is linked to the oligonucleotide via the amino group to form the ligand-oligonucleotide conjugate, comprising reacting the free carboxyl group of the intermediate compound or a carboxyl protected form (e.g., an active ester form) thereof with the free terminal amino group of the oligonucleotide to covalently link the intermediate compound to the oligonucleotide.

[0263] The present invention also provides the use of any intermediate compound described herein in the preparation of a ligand-oligonucleotide conjugate. In some embodiments, the ligand-oligonucleotide conjugate is as defined herein above. In preferred embodiments, the ligand-oligonucleotide conjugate has a structure as shown in Formula V, V', VI, VI', VII, VII', VIII, VIII', IX, IX', X, X'; wherein each substituent and symbol has the corresponding meaning, value and numerical range as defined herein above, or its preferred meaning, value and numerical range.

[0264] Pharmaceutical composition

[0265] Another aspect of the present invention provides a pharmaceutical composition comprising any ligand-oligonucleotide conjugate described herein and a pharmaceutically acceptable carrier.

[0266] The conjugates of the present invention can be formulated for pharmaceutical use. Pharmaceutically acceptable compositions include a therapeutically effective amount of one or more of the conjugates described above, alone or in combination with one or more pharmaceutically acceptable carriers (additives), excipients and / or diluents.

[0267] The conjugates according to the invention may be formulated for administration in any convenient way by analogy with other drugs for use in human or veterinary medicine.

[0268] Treatment

[0269] In another aspect, provided herein is a method for treating a disease, comprising administering to a subject a therapeutically effective amount of any one of the conjugates provided herein or a pharmaceutical composition comprising the conjugate.

[0270] In one embodiment, the composition comprises a plurality of conjugate species. In another embodiment, the conjugate species have sequences that are non-overlapping and non-adjacent to another species relative to a naturally occurring target sequence. In another embodiment, the plurality of conjugate species are specific for different naturally occurring target genes. In another embodiment, the conjugate is allele-specific.

[0271] Embodiments of the present invention also relate to methods for inhibiting the expression of a target gene, comprising the step of administering any one of the above conjugates in an amount sufficient to inhibit the expression of the target gene.

[0272] In another aspect, the invention relates to a method of modulating the expression of a target gene in a cell, the method comprising providing to the cell a conjugate of the invention or a composition thereof.

[0273] In the present invention, the target gene of oligonucleotide, siRNA and ASO is selected from the group consisting of factor VII, Eg5, PCSK9, TPX2, apo(a), apoB, SAA, TTR, RSV, PDGFβ gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, h epciden, activated protein C, cyclin D gene, VEGF gene, EGFR gene, cyclin A gene, cyclin E gene, WNT-1 gene, β-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase IIα gene, p73 mutant gene, mutation in p21 (WAF1 / CIP1) gene, mutation in p27 (KIP1) gene, mutation in PPM 1D gene, mutation in RAS gene, mutation in caveolin I gene, mutation in MIB I gene, mutation in MTAI gene, mutation in M68 gene, mutation in tumor suppressor gene and mutation in p53 tumor suppressor gene.

[0274] The present invention is further illustrated by the following examples, which should not be construed as further limiting.The contents of all references, patent applications, and published patents cited throughout this application are expressly hereby incorporated by reference.

[0275] Example

[0276] Example 1

[0277] Step 1: Compound 2: Dissolve compound 1 (25 g, 64.21 mmol) in 1,2-dichloroethane (350 mL)

[0278] The mixture was stirred for 30 minutes (10 g of 4A molecular sieves were added). TMSOTf (17.13 g, 77.08 mmol) was then added dropwise to the reaction mixture. The mixture was allowed to react at 50°C for 4 hours under nitrogen. TLC confirmed the reaction was complete. The reaction mixture was cooled to room temperature, and 37.5 mL of triethylamine was slowly added dropwise to the reaction mixture. The mixture was then dried to obtain a brown liquid, Compound 2 (21.1 g crude product). This was used directly in the next step without purification. MS m / z (ESI): 330.1 [M+H] +

[0279] Step 2: Compound 3: Compound 2 (21.1 g, 64.07 mmol) and 5-hexenyl-1-ol (7.06 g, 70.49 mmol) were dissolved in 1,2-dichloroethane (350 mL). 20 g of 4A molecular sieves (excluding water) were added and stirred at room temperature for 30 min. TMSOTf (7.12 g, 32.04 mmol) was added dropwise to the reaction mixture under nitrogen at 0°C and allowed to react at room temperature for 3 h. The reaction mixture was poured into cold saturated aqueous NaHCO₃ (100 mL), extracted with DCM (100 mL × 3), washed with water (100 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated. After concentration, the crude product was slurried in petroleum ether (50 mL × 3) to remove impurities, yielding compound 3 (19.2 g, yield: 70%). The product was used directly in the next step without purification. MS m / z (ESI): 430.2 [M+H] +

[0280] Step 3: Compound 4: Dissolve compound 3 (35 g, 81.50 mmol) in dichloromethane / acetonitrile (1:1, 200 mL). Add sodium periodate (76.4 g, 357.2 mmol) in deionized water (120 mL) while cooling on ice. Stir for 15 min. Add RuCl3 (560 mg, 2.70 mmol) to the reaction mixture while cooling on ice. Slowly warm to room temperature and stir at room temperature for 1 h. Add sodium periodate (19.1 g, 89.3 mmol) to the reaction mixture and continue the reaction overnight. TLC monitoring indicates complete reaction of the starting material. 100 mL of water was added to the reaction mixture, filtered, and the filter cake was washed with dichloromethane (100 mL x 5). The filtrate was extracted with dichloromethane (100 mL x 5). The organic phase was discarded, and the aqueous phase was collected. The pH was adjusted to 3.5 with citric acid, and the aqueous phase was extracted with dichloromethane:methanol (10:1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Compound 4 (23.2 g, yield: 63.62%) was obtained. MS m / z (ESI): 448.2 [M+H]+

[0281] Step 4: Compound 5: Compound 4 (20 g, 44.7 mmol) was dissolved in DCM (400 mL) and DIEA (8.67 g, 67.08 mmol) was added. Pentafluorophenyl trifluoroacetate (19 g, 67.84 mmol) was added at 0°C and allowed to react at room temperature for 3 h. The reaction was monitored by TLC. The reaction solution was washed with 1 M potassium bisulfate aqueous solution (200 mL) and extracted with DCM (200 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried. Column purification (PE:EtOAc = 1:2) afforded compound 5 (22.9 g, yield 83.51%). MS m / z (ESI): 614.2 [M+H] +

[0282] Example 2

[0283] Step 1: Compound 7: In a reaction flask, add trimethylolamine (compound 6) (100 g, 0.825 mol), 1,4-dioxane (700 mL), mechanically stir, cool to 0-5 ° C in an ice-water bath, add 60% potassium hydroxide aqueous solution (1.3 g potassium hydroxide dissolved in 0.9 g water), stir at 0-5 ° C for 1 h, add acrylonitrile (130 g, 2.45 mol), naturally warm to room temperature (15-25 ° C), stir and react for 16 h. After the reaction is completed, filter, wash with dichloromethane (500 mL), concentrate and dry the solid to recover 21.5 g of raw trimethylolmethylamine, concentrate the filtrate under reduced pressure at 60 ° C, add water (500 mL) and dichloromethane (200 mL), extract and separate the liquids, extract the aqueous phase with dichloromethane (200 mL × 3), combine the organic phase dichloromethane layers, wash with saturated brine (100 mL), dry over anhydrous sodium sulfate and concentrate to obtain 161.7 g of crude product 7.

[0284] After dissolving 37 g of oxalic acid in methanol (100 mL), the mixture was added to the crude product of 7, stirred and dissolved, and the temperature was raised to 64 ° C. Ethyl acetate (2 L) was added, and the mixture was slowly cooled to room temperature (25 ° C) with stirring to crystallize. A large amount of white solid was precipitated, filtered, and washed with ethyl acetate (500 mL) to obtain 194.6 g of filter cake, which was dried in vacuo at 60 ° C for 4 h to obtain 149.2 g. The solid was dissolved in methanol (100 mL), heated to 70 ° C, and ethyl acetate (2 L) was added. The solution was slightly turbid and cooled to room temperature (25 ° C) to crystallize. Filtered, washed with ethyl acetate to obtain 212.2 g of filter cake, which was dried in vacuo at 60 ° C for 4 h to obtain 116 g of the dry product oxalate of compound 7. The oxalate salt was transferred to a flask, methanol (58 mL) was added, the temperature was raised to reflux to dissolve the clear solution, ethyl acetate (1.16 L) was added to precipitate the solid, refluxed for 1 h, cooled to room temperature (25 ° C) and stirred for 1 h, filtered, rinsed with ethyl acetate (116 mL) to obtain a wet fine product, which was freed with saturated sodium carbonate aqueous solution (300 mL), extracted with dichloromethane (300 mL), dried over anhydrous sodium sulfate and concentrated to obtain compound 7 (24.6 g, yield 21.19%).

[0285] Step 2: Compound 8: Add 7 (160 g, 0.57 mol) and ethanol / hydrogen chloride (480 g) to a reaction flask, stir, and reflux under nitrogen for 16 h. After the reaction is complete, filter to remove the solid, which is ammonium chloride. Concentrate the filtrate and neutralize it with aqueous sodium bicarbonate (200 mL) until neutral. Extract with dichloromethane (200 mL x 4), wash with saturated brine (200 mL x 1), dry over anhydrous sodium sulfate, and concentrate to obtain compound 8 (150 g, yield 62.43%) as an oil.

[0286] Step 3: Compound 9: Dissolve compound 8 (40 g, 94.9 mmol) in 1,4-dioxane (160 mL) and stir until clear. In a separate reaction flask, add NaCO (13.08 g, 123.37 mmol) dissolved in purified water (80 mL) and stir until clear. Add the prepared alkaline solution to the reaction mixture at 0-5°C and stir for 30 minutes.

[0287] Benzyl chloroformate (21.05 g, 123.39 mmol) was weighed and added dropwise to the system at 0-5°C. The temperature increased significantly, and the system gradually transformed into a white turbid liquid with flocculent particles. After addition, the reaction mixture was warmed to room temperature and stirred for 18 h. LC / MS monitoring was performed. The dioxane was removed from the reaction system, and the remaining aqueous phase was extracted with EA (80 mL x 2). The organic phases were combined, washed with saturated brine, and separated. The organic phase was dried over anhydrous sodium sulfate; filtered and dried to afford crude compound 9 (50 g, 94.8% yield) as a pale yellow oil. MS m / z (ESI): 556.3 [M+H] +

[0288] Step 4: Compound 10: Dissolve compound 9 (50 g, 89.99 mmol) in THF (80 mL) and MeOH (80 mL) and stir until clear. Add LiOH (21.55 g, 899.79 mmol) dissolved in purified water (140 mL) and add to the reaction system. Stir the reaction at room temperature overnight. Monitor by LC / MS. Remove THF and methanol from the system under reduced pressure. Add purified water (100 mL) to the concentrate and extract once with EA (200 mL). Separate the layers and take the aqueous phase. Adjust the pH of the aqueous phase to between 2 and 3 with concentrated hydrochloric acid. The solution becomes white and turbid. Cool to 0°C; no solid precipitates are evident. Extract the system with DCM (100 mL x 2). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and spin dry to obtain crude compound 10 (40.8 g, 96% yield) as a thick oil. MS m / z (ESI): 472.2 [M+H] +

[0289] Step 5: Compound 11: Dissolve compound 10 (40 g, 84.84 mmol) in DCM (400 mL) and stir until clear. Add DMF (4 mL) and replace the atmosphere with N2 three times. Add dichlorothionyl (62 mL) dropwise at room temperature under N2 protection. After addition, slowly raise the temperature to reflux and reflux for 3.0 h. Monitor by LC / MS (quench by adding MeOH). Remove the solvent by vacuum drying and add DCM (200 mL) once. Vortex until no solvent is discharged. Weigh to obtain crude compound 11 (43.03 g, 96% yield) as a yellow oil with a small amount of solids.

[0290] Step 6: Compound 12: tert-Butyloxycarbonylpropylenediamine (59.1 g, 339.2 mmol) was added to a three-necked flask, followed by DIEA (109.6 g, 847.9 mmol). Dissolve the mixture in DCM (360 mL) until clear, then replace the atmosphere with nitrogen three times. Cool the mixture to 0°C under nitrogen protection. Compound 11 (44.7 g, 84.85 mmol) was dissolved in DCM (300 mL) and added dropwise to the mixture under controlled temperature. With the addition, the mixture gradually became turbid, eventually becoming a cloudy white suspension. Stir for 30 minutes, then warm to room temperature and react overnight. LC / MS monitoring was performed. The solvent was removed under reduced pressure, and purified water (200 mL) was added. Extraction was then performed with EA (300 mL x 2). The combined organic phases were washed with 10% citric acid (200 mL x 2), 10% sodium carbonate solution (200 mL x 2), and saturated brine (200 mL) once. The organic phase was dried over anhydrous sodium sulfate, filtered, and dried to give a crude product of compound 12 (63.2 g, 67.22 mmol, yield 79%) as a pale yellow oil. MS m / z (ESI): 940.6 [M+H] +

[0291] Step 7: Compound 13: Dissolve compound 12 (30 g, 31.91 mmol) in MeOH (500 mL) and dissolve until clear. Add 20% Pd(OH)2 / C (8.2 g) and TFA (10 mL). Allow to react overnight at room temperature. Monitor with LC / MS. Add celite to filter the palladium-carbon mixture. Add excess solid sodium bicarbonate (in excess, if solid is present) and stir thoroughly. Filter. The filtrate is dried and dissolved in MeOH (20 mL). Purify by reverse-phase column chromatography (methanol:water = 5%-100%) to obtain compound 13 (24.7 g, 96.2% yield). MS m / z (ESI): 806.5 [M+H] +

[0292] Example 3

[0293] Step 1: A1: Boc-Val-Val-OH (2.82 g, 8.91 mmol) was dissolved in DMF (18 mL). Cesium carbonate (8.34 g, 25.61 mmol) was added, and benzyl bromide (4.22 g, 24.65 mmol) was added with stirring. The mixture was allowed to react at room temperature for 1.5 h. The reaction was monitored by LC / MS. Water was added and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (ethyl acetate / petroleum ether = 0-18%) to afford A1 (3.6 g, 99% yield). MS m / z (ESI): 407.3 [M+H] +

[0294] Step 2: A2: A1 (3.6 g, 8.86 mmol) was dissolved in dichloromethane (30 mL), and trifluoroacetic acid (6 mL) was added. The reaction was allowed to react at room temperature for 16 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated to afford the trifluoroacetic acid salt of A2 (3.7 g, 100% yield).

[0295] Step 3: A3: The trifluoroacetic acid salt of A2 (3.45 g, 8.26 mmol) was dissolved in dichloromethane (30 mL). Triethylamine (8.3 g, 82.02 mmol) and succinic anhydride (2.46 g, 24.58 mmol) were added and allowed to react at room temperature overnight. The reaction was monitored by LC / MS. The product was concentrated and purified by reverse phase column chromatography (methanol / water = 5%-95%) to afford compound A3 (3.2 g, 95% yield). MS m / z (ESI): 407.2 [M+H] +

[0296] Step 4: A4: A3 (1.00 g, 2.46 mmol) was dissolved in DMF (5 mL), and HATU (1.12 g, 2.95 mmol) and DIEA (3.17 mg, 24.60 mmol) were added. After stirring at room temperature for 15 min, compound 13 (1.88 g, 2.33 mmol) was added and stirred at room temperature overnight. The reaction was monitored by LC / MS. Water (100 mL) was added, and the mixture was extracted with dichloromethane (40 mL × 3 times). The organic phases were combined, washed once with saturated brine, added with anhydrous sodium sulfate, filtered, dried, and concentrated. Purification by column chromatography (dichloromethane:methanol = 0-20%) gave compound A4 (1.7 g, 58%). MS m / z (ESI): 497.9 [M / 2+H] +

[0297] Step 5: A5: A4 (1.60 g, 1.34 mmol) was dissolved in dichloromethane (50 mL), trifluoroacetic acid (10 mL) was added, and the mixture was allowed to react at room temperature for 2 h. The reaction was monitored by LC / MS. After the reaction was complete, the mixture was concentrated to afford the trifluoroacetate salt of compound A5 (1.12 g). MS m / z (ESI): 447.9 [M / 2+H] +

[0298] Step 6: A6: The trifluoroacetate salt of A5 (1.12 g, 1.34 mmol) was dissolved in dichloromethane (10 mL). DIEA (2.32 g, 17.98 mmol) was added. A dichloromethane solution (10 mL) of compound 5 (2.40 g, 3.91 mmol) was added with stirring in an ice bath. The mixture was then slowly warmed to room temperature and reacted for 18 h. LC / MS monitoring was performed. The mixture was diluted with 20 mL of dichloromethane, washed once with water and once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by reverse-phase column chromatography (acetonitrile:water = 5%-100%) afforded compound A6 (2.00 g, 68% yield). MS m / z (ESI): 1091.6 [M / 2+H] +

[0299] Step 7: A7: A6 (2.00 g, 0.92 mmol) was dissolved in tetrahydrofuran (35 mL), and 20% palladium hydroxide on carbon (0.40 g) was added. After replacing the system with hydrogen three times, the mixture was stirred at room temperature for 5 h. TLC confirmed the complete reaction of the starting material. The reaction solution was filtered through celite to remove the palladium hydroxide on carbon and concentrated. Purification by reverse phase column chromatography (acetonitrile / water = 5%-100%) gave compound A7 (0.35 g, yield: 18%). MS m / z (ESI): 1046.7 [M / 2+H] +

[0300] Example 4

[0301] Step 1: B1: Boc-Val-Pro-OH (5.34 g, 17 mmol) was dissolved in DMF (50 mL), and cesium carbonate (21.76 g, 66.8 mmol) was added. Benzyl bromide (10.88 g, 63.61 mmol) was added with stirring and allowed to react at room temperature for 1.5 h. The mixture was diluted with 200 mL of water and extracted with ethyl acetate (70 mL x 3). The organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification was performed by column chromatography (EA:PE = 0-50%) to obtain compound B1 (6.75 g, yield 98%). MS m / z (ESI): 405.3 [M+H] +

[0302] Step 2: B2: Dissolve B1 (6.75 g, 16.69 mmol) in dichloromethane (10 mL) and add trifluoroacetic acid (10 mL). After reacting at room temperature for 2 h, monitor with LC / MS. The reaction is complete and the product is concentrated to give the trifluoroacetic acid salt of compound B2 (5.08 g). MS m / z (ESI): 305.2 [M+H] +

[0303] Step 3: B3: The trifluoroacetic acid salt of B2 (5.08 g, 16.69 mmol) was dissolved in dichloromethane (50 mL), and triethylamine (7.48 mg, 73.92 mmol) was added. Succinic anhydride (2.96 g, 29.58 mmol) was added with stirring, and the mixture was allowed to react at room temperature overnight. The reaction was monitored by LC / MS. The product was concentrated and purified by reverse-phase column chromatography (methanol:water = 5%-100%) to afford compound B3 (6.38 g, 95%). MS m / z (ESI): 405.2 [M+H] +

[0304] Step 4: B4: Compound B3 (6.29 g, 15.55 mmol) was dissolved in DMF (30 mL), and DIEA (6.05 g, 46.82 mmol) was added. HATU (7.12 g, 18.73 mmol) was added with stirring. After stirring at room temperature for 15 min, a solution of compound 13 (12.58 g, 15.61 mmol) in DMF (10 mL) was added and the mixture was allowed to react overnight at room temperature. The reaction was monitored by LC / MS. Water (200 mL) was added, and the pH was adjusted to 6 with 1M HCl aqueous solution. The mixture was extracted with dichloromethane (70 mL x 3), washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by reverse phase column chromatography (acetonitrile / water = 5%-100%) afforded compound B4 (10.9 g, 58.8% yield). MS m / z (ESI): 496.9 [M / 2-100+H] +

[0305] Step 5: B5: Dissolve B4 (2 g, 1.68 mmol) in dichloromethane (20 mL), add trifluoroacetic acid (20 mL), and stir at room temperature for 2 h. Monitor the reaction by LC / MS. Concentrate to obtain the trifluoroacetate salt of compound B5 (1.40 g). MS m / z (ESI): 446.9 [M / 2+H] +

[0306] Step 6: B6: The trifluoroacetic acid salt of compound B5 (1.34 g, 1.5 mmol) was dissolved in pyridine (10 mL), and compound 5 (3.69 g, 6.01 mmol) was added. The mixture was reacted at 60°C for 3 h. The reaction was monitored by LC / MS. 100 mL of water was added, and the pH was adjusted to 2 with 2M aqueous hydrochloric acid. The mixture was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (acetonitrile / water = 5%-100%) to give compound B6 (0.50 g, yield 15%). MS m / z (ESI): 1090.7 [M / 2+H] +

[0307] Step 7: B7: Compound B6 (0.50 g, 0.23 mmol) was dissolved in tetrahydrofuran (20 mL), and 20% palladium hydroxide on carbon (0.10 g) was added. The system was purged with hydrogen three times, and then stirred at room temperature overnight under a hydrogen atmosphere. LC / MS monitoring of the reaction showed complete reaction of the starting material. 20% palladium hydroxide on carbon was removed by membrane filtration and concentrated. Reverse phase purification (acetonitrile / water = 5%-100%) gave compound B7 (50 mg, yield: 10%). MS m / z (ESI): 1045.6 [M / 2+H] +

[0308] Example 5

[0309] Step 1: C1: Boc-Ala-Ala-OH (5 g, 19.2 mmol) was dissolved in 50 mL of DMF, and cesium carbonate (13.5 g, 41.4 mmol) was added. Benzyl bromide (13.14 g, 76.8 mmol) was added with stirring and allowed to react at room temperature for 2 h. The reaction was monitored by TLC. The mixture was diluted with 200 mL of water and extracted three times with ethyl acetate (70 mL x 3). The organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (ethyl acetate / petroleum ether = 0-50%) to obtain C1 (6.1 g, 91% yield). MS m / z (ESI): 351.3 [M+H] +

[0310] Step 2: C2: Dissolve C1 (6.1 g, 17.4 mmol) in dichloromethane (50 mL), add trifluoroacetic acid (10 mL), and react at room temperature overnight. Monitor the reaction by TLC. After the reaction is complete, concentrate to obtain the trifluoroacetic acid salt of C2 (4.355 g, 100% yield).

[0311] Step 3: C3: The trifluoroacetic acid salt of C2 (4.355 g, 17.4 mmol) was dissolved in dichloromethane (50 mL), and triethylamine (17.59 g, 173.83 mmol) and succinic anhydride (3.48 g, 34.78 mmol) were added. The reaction was allowed to react at room temperature overnight. The reaction was monitored by LC / MS. The product was concentrated and purified by column chromatography (EA:PE = 1:5) to obtain the product C3 (4.98 g, 82% yield). MS m / z (ESI): 351.2 [M+H] +

[0312] Step 4: C4: C3 (3.0 g, 8.56 mmol) was dissolved in DMF (15 mL), and HATU (4.23 g, 11.12 mmol) and DIEA (3179 mg, 24.59 mmol) were added. After stirring at room temperature for 15 min, compound 13 (6.55 g, 8.13 mmol) was added and stirred at room temperature overnight. The reaction was monitored by LC / MS. Water (100 mL) was added, and the mixture was extracted with dichloromethane (50 mL × 3). The organic phases were combined, washed once with saturated brine, added with anhydrous sodium sulfate, filtered, dried, and concentrated. Purification by column chromatography (MeOH:DCM = 0-20%) gave compound C4 (2.51 g, 26%). MS m / z (ESI): 469.8 [M / 2-100+H] +

[0313] Step 5: Dissolve C4 (2.51 g, 2.2 mmol) in dichloromethane (50 mL) and add trifluoroacetic acid (10 mL). Allow to react at room temperature for 2 h. Monitor the reaction by TLC. After the reaction is complete, concentrate the mixture to afford the trifluoroacetate salt of compound C5 (1.84 g, 100% yield).

[0314] Step 6: C6: The trifluoroacetate salt of C5 (1.84 g, 2.2 mmol) was dissolved in dichloromethane (50 mL), and DIEA (2.86 g, 22.10 mmol) was added. A dichloromethane solution (20 mL) of compound 5 (4.21 g, 6.86 mmol) was added with stirring in an ice bath. The mixture was then slowly warmed to room temperature and reacted for 18 h. LC / MS monitoring was performed. The mixture was diluted with 20 mL of dichloromethane, washed once with water and once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by reverse-phase column chromatography (acetonitrile:water = 5%-100%) afforded compound C6 (0.50 g, 11% yield). MS m / z (ESI): 518.4 [M / 4+H]. +

[0315] Step 7: C7: C6 (0.50 g, 0.24 mmol) was dissolved in tetrahydrofuran (10 mL), and 20% palladium hydroxide on carbon (0.11 g) was added. The system was purged with hydrogen three times, and the mixture was stirred at room temperature for 5 h. After completion of the reaction, the reaction mixture was filtered through celite to remove the palladium hydroxide on carbon and concentrated. Crude compound C7 (0.11 g, 22%) was obtained. MS m / z (ESI): 1019.0 [M / 2+H] + ,511.0[M / 4+H] +

[0316] Example 6

[0317] Step 1: D2: Dissolve D1 (5 g, 22.50 mmol) in dichloromethane (50 mL), add DIEA (4.36 g, 33.74 mmol), and then add pentafluorophenyl trifluoroacetate (9.45 g, 33.74 mmol). The mixture was allowed to react at room temperature for 4 h. After completion of the reaction, the reaction solution was diluted with dichloromethane (10 mL) and washed with 1 M potassium bisulfate aqueous solution (50 mL x 2), saturated sodium bicarbonate aqueous solution (50 mL x 2), and water (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and dried by spin drying. Column purification (PE:EtOAc = 10:1) afforded D2 (6.4 g, yield: 73%). MS m / z (ESI): 389.0 [M+H] +

[0318] Step 2: D3: Compound 13 (5.0 g, 6.20 mmol) and Cbz-Val-Ala-OH (2.6 g, 8.06 mmol) were dissolved in 40 mL of dichloromethane. DIEA (2.41 g, 18.6 mmol) and HATU (3.54 g, 9.31 mmol) were added and reacted at 40°C for 16 h. After completion of the reaction, the reaction solution was diluted with 50 mL of dichloromethane and 50 mL of water was added. The layers were separated and the aqueous phase was extracted with dichloromethane (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and spin-dried. Column purification (DCM:MeOH = 10:1) gave compound D3 as a colorless viscous liquid (5.4 g, yield: 78%). MS m / z (ESI): 455.9 [M / 2-100+H] +

[0319] Step 3: D4: Dissolve D3 (2.8 g, 2.52 mmol) in 30 mL of methanol, add 20% Pd(OH)2 / C (0.5 g) and trifluoroacetic acid (0.86 g, 7.54 mmol), replace the atmosphere with a hydrogen balloon three times, and react at room temperature for 4 h under hydrogen. Filter through celite, wash the filter cake with methanol (30 mL x 3), and spin dry to obtain the trifluoroacetate salt of D4, which is used directly in the next step. MS m / z (ESI): 976.7 [M+H] +

[0320] Step 4: D5: The trifluoroacetic acid salt of D4 (2.46 g, 2.52 mmol) was dissolved in 30 mL of dichloromethane. DIEA (4.89 g, 37.84 mmol) and D2 (1.17 g, 3.01 mmol) were added at 0°C and allowed to react at room temperature for 16 h. After completion of the reaction, the reaction solution was diluted with dichloromethane (50 mL), and the organic phase was washed with 1 M KHSO4 (100 mL), saturated aqueous sodium bicarbonate (100 mL), and water (100 mL), respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, and spun down to obtain the crude product. Column purification (MeOH:DCM = 1:20) afforded the product D5 (2.67 g, yield: 89.8%) as a yellow oil. MS m / z (ESI): 490.9 [M / 2-100+H]. +

[0321] Step 5: D6: D5 (2.67 g, 2.26 mmol) was dissolved in TFA (15 mL) and stirred at room temperature for 1 h. The reaction solution was diluted with toluene (30 mL x 3) and dried to obtain the trifluoroacetic acid hydrochloride of D6, which was used directly in the next step. MS m / z (ESI): 880.6 [M+H] +

[0322] Step 6: D7: The trifluoroacetic acid salt of D6 (1.99 g, 2.26 mmol) was dissolved in dichloromethane (20 mL) and DIEA (11.69 g, 90.45 mmol) was added. A dichloromethane solution of compound 5 (4.3 g, 7.01 mmol) (20 mL) was added at 0°C and allowed to react at room temperature for 16 h. After completion of the reaction, the reaction solution was diluted with dichloromethane (50 mL), and the organic phase was washed with 1M KHSO4 (50 mL), saturated aqueous sodium bicarbonate (50 mL), and water (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and spun down to obtain the crude product. Column purification (MeOH:DCM = 1:10) gave product D7 (3.00 g, yield: 61%) as a white foamy solid. MS m / z (ESI): 1085.1 [M / 2+H] +

[0323] Step 7: D8: D7 (1.60 g, 0.74 mmol) was dissolved in THF (16 mL) and 20% Pd(OH)2 / C (600 mg). The mixture was purged with hydrogen three times and the reaction was carried out under hydrogen protection for 16 h. Filtered through celite, the filter cake was washed with THF (10 mL x 3), and the filtrate was dried to give D8 as a white foam, which was used directly in the next step. MS m / z (ESI): 1039.6 [M / 2+H] +

[0324] Step 8: D9: D8 (0.92 g, 0.443 mmol) was dissolved in dichloromethane (20 mL) and DIEA (0.17 g, 1.315 mmol) was added, followed by pentafluorophenyl trifluoroacetate (0.25 g, 0.89 mmol). The mixture was allowed to react at room temperature for 18 h. The reaction solution was diluted with dichloromethane (30 mL), and the organic phase was washed with 1 M KHSO4 (30 mL), saturated aqueous sodium bicarbonate (30 mL), and water (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and dried to obtain the crude product. Column purification (MeOH:DCM = 1:10) gave the product D9 (0.6 g, yield: 60%) as an oil. MS m / z (ESI): 1123.0 [M / 2+H] +

[0325] Example 7

[0326] Step 1: E2: E1 (5 g, 24.01 mmol) was dissolved in dichloromethane (50 mL), and DIEA (4.66 g, 36.02 mmol) was added. Pentafluorophenyl trifluoroacetate (10.09 g, 36.02 mmol) was then added at 0°C and allowed to react at room temperature for 16 h. After completion of the reaction, the reaction solution was diluted with dichloromethane (50 mL), and the organic phase was washed with 1 M potassium bisulfate aqueous solution (50 mL x 2), saturated sodium bicarbonate (50 mL x 2), and water (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and dried. Column purification (PE:EtOAc = 10:1) afforded E2 (6.4 g, yield: 71%) as a yellow solid. MS m / z (ESI): 375.0 [M+H] +

[0327] Step 2: E3: Compound 13 (5.38 g, 6.67 mmol) and Cbz-Gly-Pro-OH (2.66 g, 8.67 mmol) were dissolved in 50 mL of dichloromethane. DIEA (2.59 g, 20.0 mmol) and HATU (3.81 g, 10.0 mmol) were added and allowed to react at room temperature for 16 h. The reaction solution was diluted with 50 mL of dichloromethane and 50 mL of water was added. The layers were separated and the aqueous phase was extracted with dichloromethane (50 mL x 3). The combined organic phases were filtered over anhydrous sodium sulfate, dried, and purified by column chromatography (DCM:MeOH = 10:1) to afford E3 (6.7 g, 90% yield) as a colorless, viscous liquid. MS m / z (ESI): 994.7 [M-100+H]. + , 447.9[M / 2-100+H] +

[0328] Step 3: E4: Dissolve E3 (5.6 g, 5.11 mmol) in 60 mL of methanol, add 20% Pd(OH)2 / C (1 g), trifluoroacetic acid (1.75 g, 15.35 mmol), replace the atmosphere with hydrogen three times, and react at room temperature for 4 h. After completion of the reaction, filter through celite, wash the filter cake with methanol (30 mL x 3), and spin dry to obtain the trifluoroacetate salt of E4. Use it in the next step without purification. MS m / z (ESI): 960.6 [M+H] +

[0329] Step 4: E5: The trifluoroacetic acid salt of E4 (1 g, 1.04 mmol) was dissolved in 10 mL of dichloromethane. DIEA (2.02 g, 15.62 mmol) and E2 (0.43 g, 1.14 mmol) were added at 0°C and allowed to react at room temperature for 16 h. After completion of the reaction, the reaction solution was diluted with dichloromethane (20 mL). The organic phase was washed with 1 M KHSO4 (20 mL), saturated aqueous sodium bicarbonate (20 mL), and water (20 mL), dried over anhydrous sodium sulfate, filtered, and spun down to obtain the crude product. Column purification (MeOH:DCM = 1:20) afforded the product E5 (0.88 g, 73% yield) as a yellow oil. MS m / z (ESI): 475.9 [M / 2-100+H]. +

[0330] Step 5: E6: E5 (0.88 g, 0.765 mmol) was dissolved in TFA (4 mL) and stirred at room temperature for 1 h. The reaction solution was diluted with toluene (10 mL), dried by spin drying, and then diluted with toluene (10 mL x 3). The trifluoroacetic acid salt of E6 was obtained by spin drying and used directly in the next step. MS m / z (ESI): 850.5 [M+H] +

[0331] Step 6: E7: E6 trifluoroacetate (0.65 g, 0.765 mmol) was dissolved in dichloromethane (5 mL) and DIEA (3.95 g, 30.58 mmol) was added. A solution of compound 5 (1.45 g, 2.37 mmol) in dichloromethane (5 mL) was then added and the mixture was allowed to react at room temperature for 18 h. The reaction mixture was diluted with dichloromethane (20 mL), and the organic phase was washed with 1M KHSO4 (30 mL), saturated aqueous sodium bicarbonate (30 mL), and water (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and dried to obtain the crude product. Column purification (MeOH:DCM = 1:10) gave product E7 (0.71 g, yield: 43%) as a white, viscous solid. MS m / z (ESI): 1070.1 [M / 2+H] +

[0332] Step 7: E8: E7 (100 mg, 0.047 mmol) was dissolved in THF (1 mL) and 20% Pd(OH)2 / C (20 mg). The mixture was replaced with hydrogen three times and then reacted at room temperature for 4 h under a hydrogen atmosphere. Filtered through Celite, the filter cake was washed with THF (10 mL x 3), and the filtrate was dried to give E8 as a white foamy solid. MS m / z (ESI): 1024.6 [M / 2+H] +

[0333] Step 8: E9: E8 (460 mg, 0.225 mmol) was dissolved in DCM (5 mL) and DIEA (58.05 mg, 0.449 mmol) was added. The mixture was stirred over 4A molecular sieves at room temperature for 30 min. Pentafluorophenyl trifluoroacetate (94.35 mg, 0.337 mmol) was then added at room temperature and allowed to react for 3 h. The reaction mixture was diluted with icy DCM (5 mL). The organic phase was washed with icy 1M KHSO4 (5 mL x 2), icy saturated aqueous sodium bicarbonate (5 mL x 2), and icy saturated brine (5 mL x 2), dried over anhydrous sodium sulfate, and filtered to dryness to obtain E9 (0.28 g, yield: 55%) as a white foamy solid. MS m / z (ESI): 1107.6 [M / 2+H] +

[0334] Example 8

[0335] Step 1: E2': Dissolve E1 (4.14 g, 20 mmol) in dichloromethane (150 mL). Slowly add oxalyl chloride (13.8 g, 109 mmol) dropwise in an ice bath. Incubate the reaction at 0°C for 5 minutes, then at room temperature for 30 minutes. The reaction mixture is concentrated to afford E2' (4.5 g, 99% yield) as a colorless oil.

[0336] Step 2: F1: Compound 13 (1.00 g, 1.241 mmol) and Cbz-Gly-Val-OH (0.46 g, 1.489 mmol) were dissolved in 10 mL of DCM. DIEA (0.48 g, 3.722 mmol) and HATU (0.75 g, 1.985 mmol) were added and allowed to react at room temperature for 16 h. The reaction solution was diluted with 10 mL of DCM, 20 mL of water was added, and the layers were separated. The aqueous phase was extracted with DCM (10 mL x 3). The combined organic phases were filtered over anhydrous sodium sulfate, dried, and purified by column chromatography (DCM:MeOH = 10:1) to afford F1 (1.00 g, yield: 73%) as a white foamy solid. MS m / z (ESI): 1097.7 [M+H] +

[0337] Step 3: F2: Dissolve F1 (600 mg, 0.547 mmol) in 5 mL of MeOH, add 20% Pd(OH)2 / C (120 mg) and CF3COOH (187 mg, 1.642 mmol), purge with a hydrogen balloon three times, and react at room temperature for 16 h. Filter through celite, wash the filter cake with methanol (10 mL x 3), and spin dry to obtain the trifluoroacetate salt of F2. Used in the next step without purification. MS m / z (ESI): 431.9 [M / 2+H] +

[0338] Step 4: F3: F2 (526 mg, 0.547 mmol) was dissolved in 5 mL of DCM. DIEA (141.3 mg, 1.093 mmol) and E2' (161.08 mg, 0.711 mmol) were added at 0°C and allowed to react at room temperature for 16 h. The reaction mixture was diluted with DCM (10 mL), and the organic phase was washed with saturated aqueous sodium bicarbonate (10 mL) and water (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and spun down to obtain the crude product. Column purification (MeOH:DCM = 1:10) gave the product F3 (460 mg, 73% yield) as a yellow oil. MS m / z (ESI): 476.9 [M / 2-100+H]. +

[0339] Step 5: F4: F3 (400 mg, 0.347 mmol) was dissolved in TFA (3 mL) and stirred at room temperature for 1 h. The reaction solution was diluted with toluene (5 mL), dried by vortexing, and then diluted with toluene (5 mL x 3). The trifluoroacetic acid salt of F4 was obtained by vortexing and used directly in the next step. MS m / z (ESI): 426.9 [M / 2+H] +

[0340] Step 6: F5: F4 (295 mg, 0.346 mmol) was dissolved in dichloromethane (5 mL), and DIEA (1.78 g, 13.85 mmol) was added. Compound 5 (658.46 mg, 1.073 mmol) in dichloromethane (3 mL) was then added and allowed to react at room temperature for 18 h. The reaction solution was diluted with dichloromethane (10 mL), and the organic phase was washed with 1M KHSO4 (10 mL), saturated aqueous sodium bicarbonate (10 mL), and water (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and dried to obtain the crude product. Column purification (MeOH:DCM = 1:10) afforded the product F5 (333.2 mg, yield: 45%) as a viscous solid. MS m / z (ESI): 1071.6 [M / 2+H] +

[0341] Step 7: F6: F5 (300 mg, 0.139 mmol) was dissolved in THF (3 mL) and 20% Pd(OH)2 / C (40 mg). The mixture was replaced with hydrogen three times and then reacted at room temperature under a hydrogen atmosphere for 4 h. Filtered through celite, the filter cake was washed with THF (5 mL x 3), and the filtrate was dried to give F6 as a white foamy solid. MS m / z (ESI): 1026.1 [M / 2+H] +

[0342] Example 9

[0343] Step 1: G1: Compound 13 (5.0 g, 6.20 mmol) and Fmoc-Val-Val-OH (3.25 g, 7.44 mmol) were dissolved in 50 mL of DCM, and DIEA (2.4 g, 18.6 mmol) and HATU (3.75 g, 9.92 mmol) were added. The mixture was allowed to react at room temperature for 16 h. The reaction solution was diluted with 50 mL of DCM, and 50 mL of water was added. The layers were separated, and the aqueous phase was extracted with DCM (50 mL x 3). The organic phases were combined, filtered over anhydrous sodium sulfate, dried, and purified by column chromatography (DCM:MeOH = 10:1) to obtain G1 (5.0 g, yield: 70%). MS m / z (ESI): 513.8 [M / 2-100+H]. +

[0344] Step 2: G2: G1 (3.0 g, 2.44 mmol) was dissolved in DCM (10 mL) and diethylamine (5 mL) was added. The mixture was allowed to react at room temperature for 3 h. The solvent was dried and purified on an alkaline silica gel column (DCM:MeOH = 10:1) to obtain G2 (1.40 g, yield: 54%). MS m / z (ESI): 1004.7 [M+H] +

[0345] Step 3: G3: G2 (1.40 g, 1.39 mmol) was dissolved in 15 mL of DCM. DIEA (0.537 g, 4.17 mmol) and E2' (0.63 g, 2.78 mmol) were added at 0°C and allowed to react at room temperature for 16 h. The reaction mixture was diluted with DCM (20 mL), and the organic phase was washed with saturated sodium bicarbonate (20 mL) and water (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and spun down to obtain the crude product. Column purification (MeOH:DCM = 1:20) afforded the product G3 (1.00 g, 60% yield) as a pale yellow solid. MS m / z (ESI): 497.8 [M / 2-100+H]. +

[0346] Step 4: G4: G3 (1.00 g, 0.838 mmol) was dissolved in TFA (4 mL) and stirred at room temperature for 1 h. The reaction solution was diluted with toluene (10 mL), dried by spin drying, and then diluted with toluene (10 mL x 3). The trifluoroacetic acid salt of G4 was obtained by spin drying and used directly in the next step. MS m / z (ESI): 895.6 [M+H] +

[0347] Step 5: G5: The trifluoroacetic acid salt of G4 (0.748 mg, 0.824 mmol) was dissolved in dichloromethane (10 mL), and DIEA (4.32 g, 33.46 mmol) was added. A solution of compound 5 (1.59 g, 2.59 mmol) in dichloromethane (5 mL) was then added and allowed to react at room temperature for 2 h. The reaction solution was diluted with dichloromethane (30 mL), and the organic phase was washed with 1M KHSO4 (30 mL), saturated aqueous sodium bicarbonate (30 mL), and water (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and dried to obtain the crude product. Column purification (MeOH:DCM = 1:10) gave the product G5 (699 mg, yield: 38%) as a white foamy solid. MS m / z (ESI): 1091.6 [M / 2+H] +

[0348] Step 6: G6: G5 (699 mg, 0.033 mmol) was dissolved in THF (10 mL) and 20% Pd(OH)2 / C (35 mg). The mixture was replaced with hydrogen three times and allowed to react overnight at room temperature under a hydrogen atmosphere. Filter through celite, wash the filter cake with THF (10 mL x 3), and spin dry the filtrate to obtain G6 as a white foamy solid. MS m / z (ESI): 1047.1 [M / 2+H] +

[0349] Step 7: G7: Compound G6 (1.63 g, 0.779 mmol) was dissolved in DCM (10 mL) and DIEA (0.20 g, 1.558 mmol) was added. TFAPfp (0.33 g, 1.169 mmol) was then added at room temperature and allowed to react for 3 h. The reaction mixture was diluted with icy DCM (20 mL). The organic phase was washed with icy 1M KHSO4 (20 mL x 3), icy saturated aqueous sodium bicarbonate solution (20 mL x 3), and icy saturated brine (20 x 3 mL), dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain compound G7 (1.6 g, yield: 74%) as a white foamy solid. MS m / z (ESI): 1130.1 [M / 2+H] +

[0350] Example 10

[0351] Step 1: H1: 13 (5.0 g, 6.20 mmol) and Fmoc-Val-Leu-OH (3.35 g, 7.40 mmol) were dissolved in 50 mL of DCM. DIEA (2.4 g, 18.6 mmol) and HATU (3.75 g, 9.92 mmol) were added and reacted at room temperature for 16 h. The reaction solution was diluted with 50 mL of DCM, 50 mL of water was added, and the layers were separated. The aqueous phase was extracted with DCM (50 mL x 3). The combined organic phases were filtered over anhydrous sodium sulfate, dried, and purified by column chromatography (DCM:MeOH = 10:1) to afford H1 (5.5 g, 70% yield). MS m / z (ESI): 520.7 [M / 2-100+H]. +

[0352] Step 2: H2: H1 (3.0 g, 2.41 mmol) was dissolved in DCM (10 mL) and diethylamine (5 mL) was added. The mixture was allowed to react at room temperature for 3 h. The solvent was then dried and purified on an alkaline silica gel column (DCM:MeOH = 10:1) to afford H2 (1.9 g, yield: 77%). MS m / z (ESI): 1019.7 [M+H] +

[0353] Step 3: H3: H2 (1.90 g, 1.86 mmol) was dissolved in 15 mL of DCM. DIEA (0.719 mg, 5.58 mmol) and E2' (840 mg, 3.72 mmol) were added at 0°C and allowed to react at room temperature for 16 h. The reaction mixture was diluted with DCM (10 mL), and the organic phase was washed with saturated aqueous sodium bicarbonate (10 mL) and water (10 mL), dried over anhydrous sodium sulfate, filtered, and spun down to obtain the crude product. Column purification (MeOH:DCM = 1:20) afforded the product H3 (1.2 g, 53% yield). MS m / z (ESI): 505.2 [M / 2-100+H] +

[0354] Step 4: H4: H3 (1.0 g, 0.827 mmol) was dissolved in TFA (5 mL) and stirred at room temperature for 1 h. The reaction solution was diluted with toluene (5 mL), dried by vortexing, and then diluted with toluene (5 mL x 3). The trifluoroacetic acid salt of H4 was obtained by vortexing and used directly in the next step. MS m / z (ESI): 908.6 [M+H] +

[0355] Step 5: H5: The trifluoroacetic acid salt of H4 (750 mg, 0.827 mmol) was dissolved in dichloromethane (10 mL), and DIEA (4.28 g, 33.08 mmol) was added. A solution of compound 5 (1.57 g, 2.564 mmol) in dichloromethane (5 mL) was then added and allowed to react at room temperature for 18 h. The reaction solution was diluted with dichloromethane (30 mL), and the organic phase was washed with 1M KHSO4 (30 mL), saturated aqueous sodium bicarbonate (30 mL), and water (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and dried to obtain the crude product. Column purification (MeOH:DCM = 1:10) afforded the product H5 (691 mg, yield: 37%) as a white foamy solid. MS m / z (ESI): 1099.1 [M / 2+H] +

[0356] Step 6: H6: H5 (691 mg, 0.315 mmol) was dissolved in THF (10 mL) and 20% Pd(OH)2 / C (77 mg). The mixture was replaced with hydrogen three times under a hydrogen atmosphere at room temperature for 4 h. Filtered through celite, the filter cake was washed with THF (10 mL x 3), and the filtrate was dried to give H6 as a white foamy solid. MS m / z (ESI): 1054.1 [M / 2+H] +

[0357] Step 7: H7: Compound H6 (1.7 g, 0.807 mmol) was dissolved in DCM (10 mL) and DIEA (0.21 g, 1.614 mmol) was added. TFAPfp (0.45 g, 1.614 mmol) was then added at room temperature. The reaction mixture was allowed to react overnight at room temperature. The reaction mixture was then dissolved in DCM (10 mL) and DIEA (0.21 g, 1.614 mmol) was added. TFAPfp (0.45 g, 1.614 mmol) was then added at room temperature. The reaction mixture was allowed to react for 4 h at room temperature. The reaction mixture was diluted with icy DCM (15 mL). The organic phase was washed with icy 1M KHSO4 (15 mL x 3), icy saturated aqueous sodium bicarbonate solution (15 mL x 3), and icy saturated brine (15 x 3 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and dried to obtain a white foamy solid. The solid was dissolved again in icy DCM (15 mL). The organic phase was washed with icy 1M KHSO4 (15 mL x 2), icy saturated aqueous sodium bicarbonate solution (15 mL x 2), and ice water (15 x 2 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and spun down to afford compound H7 (1.4 g, yield: 35%) as a brownish-yellow foamy solid. MS m / z (ESI): 1137.1 [M / 2+H]. +

[0358] Example 11

[0359] Step 1: I1: Compound 13 (1.00 g, 1.241 mmol) and Fmoc-Ala-Ala-OH (0.57 g, 1.48 mmol) were dissolved in 10 mL of DCM. DIEA (0.48 g, 3.72 mmol) and HATU (0.75 g, 1.985 mmol) were added and reacted at room temperature for 16 h. The reaction solution was diluted with 10 mL of DCM, 20 mL of water was added, and the layers were separated. The aqueous phase was extracted with DCM (10 mL x 3). The combined organic phases were filtered over anhydrous sodium sulfate, dried, and purified by column chromatography (DCM:MeOH = 10:1) to afford I1 (1.2 g, 75% yield) as a white foamy solid.

[0360] Step 2: I2: I1 (600 mg, 0.513 mmol) was dissolved in DCM (4 mL) and diethylamine (2 mL) was added. The mixture was allowed to react at room temperature for 3 h. The solvent was then dried and purified on an alkaline silica gel column (DCM:MeOH = 10:1) to afford I2 (480 mg, yield: 98%). MS m / z (ESI): 948.6 [M / 2+H] +

[0361] Step 3: I3: I2 (480 mg, 0.506 mmol) was dissolved in 10 mL of dichloromethane. DIEA (196.3 mg, 1.519 mmol) and E2 (227.4 mg, 0.607 mmol) were added at 0°C and allowed to react at room temperature for 16 h. After completion of the reaction, the reaction solution was diluted with dichloromethane (20 mL). The organic phase was washed with 1 M KHSO4 (20 mL), saturated aqueous sodium bicarbonate (20 mL), and water (20 mL), dried over anhydrous sodium sulfate, filtered, and spun down to obtain the crude product. Column purification (MeOH:DCM = 1:20) afforded the product I3 (350 mg, 61% yield). MS m / z (ESI): 469.9 [M / 2-100+H]. +

[0362] Step 4: I4: I3 (350 mg, 0.307 mmol) was dissolved in TFA (3 mL) and stirred at room temperature for 1 h. The reaction solution was diluted with toluene (5 mL), dried by vortexing, and then diluted with toluene (5 mL x 3). The trifluoroacetic acid salt of I4 was obtained by vortexing and used directly in the next step. MS m / z (ESI): 419.9 [M / / 2+H] +

[0363] Step 5: I5: The trifluoroacetic acid salt of I4 (257 mg, 0.307 mmol) was dissolved in dichloromethane (5 mL) and DIEA (1.56 g, 12.267 mmol) was added. A solution of compound 5 (583.24 mg, 0.951 mmol) in dichloromethane (3 mL) was then added and allowed to react at room temperature for 18 h. The reaction solution was diluted with dichloromethane (10 mL), and the organic phase was washed with 1 M KHSO4 (10 mL), saturated aqueous sodium bicarbonate (10 mL), and water (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and dried to dryness to obtain the crude product. Column purification (MeOH:DCM = 1:10) afforded the product I5 (267.6 mg, yield: 41%). MS m / z (ESI): 1063.6 [M / 2+H] +

[0364] Step 6: I6: I5 (267.6 mg, 0.125 mmol) was dissolved in THF (4 mL) and 20% Pd(OH)2 / C (30 mg). The mixture was replaced with hydrogen three times and then reacted at room temperature under a hydrogen atmosphere for 4 h. Filtered through Celite, the filter cake was washed with THF (5 mL x 3), and the filtrate was dried to obtain I6 as a white foamy solid. MS m / z (ESI): 1018.9 [M / 2+H] +

[0365] Example 12

[0366] The sense and antisense strands of ASO and siRNA were synthesized in a solid phase using the phosphoramidite method. The 5' end of the sense strand of the siRNA was coupled to the 5' end of the nucleic acid using 5'-amino TFA (C6), i.e., phosphoramidite coupling using 5'-Amino-Modifier C6-TFA (monomer with CAS number: 133975-85-6). After the nucleic acid chain was synthesized and deprotected with ammonia, an oligonucleotide with an amino group (oligonucleotide with a 5' terminal amino group) was obtained. The sequences of the oligonucleotides used in the experimental examples are shown below.

[0367] The synthesis of oligonucleotides with an amino group at the 3' end can be carried out by using 3'-PT Amino-Modifier C6CPG as a starting solid support and solid phase synthesis by the phosphoramidite method. After the solid phase synthesis is completed, the oligonucleotide with an amino group (oligonucleotide with an amino group at the 3' end) is obtained through cleavage, deprotection, purification, etc.

[0368] Examples of oligonucleotide structures containing amino groups are as follows:

[0369] Oligonucleotide 1 with an amino group at the 5' end (wherein the sequence is shown in SEQ ID NO.1):

[0370] or

[0371] Oligonucleotide 2 with an amino group at the 5' end (the sequence of which is shown in SEQ ID NO.3):

[0372] Oligonucleotide 1 with an amino group at the 3' end (wherein the sequence is shown in SEQ ID NO.1):

[0373] or

[0374] Oligonucleotide 2 with an amino group at the 3' end (the sequence of which is shown in SEQ ID NO.3):

[0375] Wherein, m is a methoxy modification at the 2' position of the sugar ring, f is a fluorinated modification at the 2' position of the sugar ring, * is a thio modification of the phosphodiester bond, and invdA is an inverted deoxyadenosine (3'-3' linked nucleotide).

[0376] Comparative Example: Preparation of comparative conjugates

[0377] The comparative conjugates PC (without L96) and NC (without L96) were obtained by simulated annealing of the corresponding sense and antisense sequences;

[0378] Wherein, m is a methoxy modification at the 2' position of the sugar ring, f is a fluorinated modification at the 2' position of the sugar ring, and * is a thio modification at the phosphodiester bond;

[0379] PC means: Positive control; NC means: Negative control.

[0380] PC (3'L96) was prepared according to the method of US Pat. No. 10125369, wherein the structure of 3'L96 is as follows:

[0381] in, Indicates the position of the 3' linkage to the oligonucleotide.

[0382] The preparation of PC (5'L96) is described in J.Am.Chem.Soc.2014, 136, 16958-16961, wherein the structure of 5'L96 is as follows:

[0383] in, Indicates the position of the 5' linkage to the oligonucleotide.

[0384] AMG 890 is prepared according to patent CN116456990A / CN108368506A, and its structural formula is as follows:

[0385] Example 13. Preparation of ligand-oligonucleotide conjugates

[0386] 13.1. Compound A7 and HATU were dissolved in DMF, mixed, and DIEA was added. The mixture was vortexed at 30°C for 15 min. The mixture was then added to oligonucleotide 1 (dissolved in water) with an amino group at the 5' end. The mixture was mixed by sonication and vortexing until completely dissolved. The mixture was reacted at 25°C for 16 h. Ammonia was then added to the conjugate, and the mixture was reacted at 55°C for 30 min to obtain the target product mixture. The conjugate was prepared by HPLC, with the structural formula shown as CA1, and then simulated annealing with the corresponding antisense sequence to obtain the target compound ds-CA1.

[0387] 13.2. Compounds B7 and HATU were dissolved in DMF, mixed, and DIEA was added. The mixture was vortexed at 30°C for 15 min. The mixture was then added to oligonucleotide 1 (dissolved in water) with an amino group at the 5' end and mixed by sonication and vortexing until completely dissolved. The mixture was reacted at 25°C for 16 h. Ammonia was then added to the conjugate, and the reaction was continued at 55°C for 30 min to obtain the target product mixture. The conjugate was prepared by HPLC, with the structural formula shown as CB1, and then simulated annealing with the corresponding antisense sequence was performed to obtain the target compound ds-CB1.

[0388] 13.3. Compounds C7 and HATU were dissolved in DMF, mixed, and DIEA was added. The mixture was vortexed at 30°C for 15 min. The mixture was then added to oligonucleotide 1 (dissolved in water) with an amino group at the 5' end and mixed by sonication and vortexing until completely dissolved. The mixture was reacted at 25°C for 16 h. Ammonia was then added to the conjugate, and the reaction was continued at 55°C for 30 min to obtain the target product mixture. The conjugate was prepared by HPLC with the structural formula shown in CC1. The target compound ds-CC1 was then obtained by simulated annealing with the corresponding antisense sequence.

[0389] 13.4. Dissolve D9 in sodium phosphate buffer and add it to a solution of oligonucleotide 1 with an amino group at the 5' end (dissolved in PBS). Then, add the Py activator (pyridine). Mix by sonication and vortexing until completely dissolved. React at 25°C for 16 h. Then, add ammonia to the conjugate and react at 55°C for 30 min to obtain the target product mixture. Prepare the conjugate by HPLC (the structural formula is shown as CD1). Then, simulated annealing with the corresponding antisense sequence is performed to obtain the target compound ds-CD1.

[0390] 13.5. Dissolve E9 in sodium phosphate buffer and add it to a solution of oligonucleotide 1 with an amino group at the 5' end (dissolved in PBS). Then, add the Py activator and mix by sonication and vortexing until completely dissolved. Incubate the mixture at 25°C for 16 h. Then, add ammonia to the conjugate and incubate at 55°C for 30 min to obtain the target product mixture. The conjugate was prepared by HPLC with the structural formula shown in CE1. This was then simulated annealing with the corresponding antisense sequence to obtain the target compound ds-CE1.

[0391] 13.6. Compounds F6 and HATU were dissolved in DMF, mixed, and DIEA was added. The mixture was vortexed at 30°C for 15 min. The mixture was then added to oligonucleotide 1 (dissolved in water) with an amino group at the 5' end. The mixture was mixed by sonication and vortexing until completely dissolved. The mixture was reacted at 25°C for 16 h. Ammonia was then added to the conjugate, and the mixture was reacted at 55°C for 30 min to obtain the target product mixture. The conjugate was prepared by HPLC with the structural formula shown in CF1. The target compound ds-CF1 was then obtained by simulated annealing with the corresponding antisense sequence.

[0392] 13.7. Compound G6 and HATU were dissolved in DMF, mixed, and DIEA was added. The mixture was vortexed at 30°C for 15 min. The mixture was then added to oligonucleotide 1 (dissolved in water) with an amino group at the 5' end and mixed by sonication and vortexing until completely dissolved. The mixture was reacted at 25°C for 16 h. Ammonia was then added to the conjugate, and the mixture was reacted at 55°C for 30 min to obtain the target product mixture. The conjugate was prepared by HPLC with the structural formula shown in CG1. The target compound ds-CG1 was then obtained by simulated annealing with the corresponding antisense sequence.

[0393] 13.8. Compounds H6 and HATU were dissolved in DMF, mixed, and DIEA was added. The mixture was vortexed at 30°C for 15 min. The mixture was then added to oligonucleotide 1 (dissolved in water) with an amino group at the 5' end. The mixture was mixed by sonication and vortexing until completely dissolved. The mixture was reacted at 25°C for 16 h. Ammonia was then added to the conjugate, and the mixture was reacted at 55°C for 30 min to obtain the target product mixture. The conjugate was prepared by HPLC with the structural formula shown in CH1. The target compound ds-CH1 was then obtained by simulated annealing with the corresponding antisense sequence.

[0394] 13.9. Compound I6 and HATU were dissolved in DMF, mixed, and DIEA was added. The mixture was then vortexed at 30°C for 15 min. The mixture was then added to oligonucleotide 1 (dissolved in water) with an amino group at the 5' end and mixed by sonication and vortexing until completely dissolved. The mixture was reacted at 25°C for 16 h. Ammonia was then added to the conjugate, and the mixture was reacted at 55°C for 30 min to obtain the target product mixture. The conjugate was prepared by HPLC with the structural formula shown in CI1, and then simulated annealing with the corresponding antisense sequence was performed to obtain the target compound ds-CI1.

[0395] 13.10. Dissolve D9 in sodium phosphate buffer and add it to a solution of oligonucleotide 1 with an amino group at the 3' end (dissolved in PBS). Then, add the Py activator and mix by sonication and vortexing until completely dissolved. Incubate at 25°C for 16 h. Then, add ammonia to the conjugate and incubate at 55°C for 30 min to obtain the target product mixture. Prepare the conjugate by HPLC (the structural formula is shown as CD2). Then, simulated annealing with the corresponding antisense sequence is performed to obtain the target compound ds-CD2.

[0396] Compound G6 and HATU were dissolved in DMF, mixed, and DIEA was added. The mixture was vortexed at 30°C for 15 min. The mixture was then added to oligonucleotide 1 (dissolved in water) with an amino group at the 3' end. The mixture was mixed by sonication and vortexing until completely dissolved. The mixture was reacted at 25°C for 16 h. Ammonia was then added to the conjugate, and the mixture was reacted at 55°C for 30 min to obtain the target product mixture. The conjugate was prepared by HPLC with the structural formula shown in CG2. The target compound ds-CG2 was then obtained by simulated annealing with the corresponding antisense sequence.

[0397] Compound A7 (150 mg) was dissolved in DMF (2000 μL), and HATU and DIEA were added, respectively. The mixture was vortexed at 25°C for 15 min. The mixture was then added to oligonucleotide 2 (20 mg, 3000 μL water) with an amino group at the 5' end. The mixture was mixed by sonication and vortexing until completely dissolved. The mixture was reacted at 25°C for 16 h. Ac deprotection was performed, and ammonia (300 μL) was added. The mixture was reacted at 55°C for 30 min to obtain the target product mixture. The conjugate DA1 was prepared by HPLC and then simulated annealing with the corresponding antisense sequence to obtain the target compound ds-DA1.

[0398] Compound D9 (87 mg) was dissolved in DMF (300 μL) and added to oligonucleotide 2 (25 mg, dissolved in 1200 μL sodium carbonate buffer) with an amino group at the 5' end. The mixture was then sonicated and vortexed until completely dissolved. The mixture was reacted at 25°C for 16 h. Ac deprotection was performed, and ammonia (300 μL) was added. The mixture was reacted at 55°C for 30 min to obtain the target product mixture. The conjugate DB was prepared by HPLC and then simulated annealing with the corresponding antisense sequence to obtain the target compound ds-DB1.

[0399] Compound G7 (84 mg) was dissolved in DMF (300 μL) and added to oligonucleotide 2 (25 mg, dissolved in 1200 μL sodium carbonate buffer) with an amino group at the 5' end. The mixture was then sonicated and vortexed until completely dissolved. The mixture was reacted at 25°C for 16 h. Ac deprotection was performed, and ammonia (300 μL) was added. The mixture was reacted at 55°C for 30 min to obtain the target product mixture. The conjugate DC1 was prepared by HPLC and then simulated annealing with the corresponding antisense sequence to obtain the target compound ds-DC1.

[0400] 13.15. Compound H7 (86 mg) was dissolved in DMF (300 μL) and added to oligonucleotide 2 (25 mg, dissolved in 1200 μL sodium carbonate buffer) with an amino group at the 5' end. The mixture was then sonicated and vortexed until completely dissolved. The mixture was reacted at 25°C for 16 h. Ac deprotection was performed, and ammonia (300 μL) was added. The mixture was reacted at 55°C for 30 min to obtain the target product mixture. The conjugate DD1 was prepared by HPLC and then simulated annealing with the corresponding antisense sequence to obtain the target compound ds-DD1.

[0401] 13.16. Compound D9 (82 mg) was dissolved in DMF (300 μL) and added to oligonucleotide 2 (25 mg, dissolved in 1200 μL sodium carbonate buffer) with an amino group at the 3' end. The mixture was then sonicated and vortexed until completely dissolved. The mixture was reacted at 25°C for 16 h. Ac deprotection was performed, and ammonia (300 μL) was added. The mixture was reacted at 55°C for 30 min to obtain the target product mixture. The conjugate DB2 was prepared by HPLC and then simulated annealing with the corresponding antisense sequence to obtain the target compound ds-DB2.

[0402] Compound G7 (10 eq, 82 mg) was dissolved in DMF (300 μL) and added to the nucleic acid amino intermediate (25 mg, dissolved in 1200 μL sodium carbonate buffer). The mixture was then sonicated and vortexed until completely dissolved. The mixture was reacted at 25°C for 16 h. Ac deprotection was performed, and ammonia (200 μL) was added. The mixture was reacted at 55°C for 30 min to obtain the target product mixture. The conjugate DC2 was prepared by HPLC and then simulated annealing with the corresponding antisense sequence to obtain the target compound ds-DC2.

[0403] Experimental Example 1

[0404] Methods: The conjugates were self-delivered into freshly isolated mouse primary hepatocytes, and in vitro IC50 data were obtained by qPCR. In vitro experiments were performed in 24-well plates, with two technical replicates per concentration point for each conjugate. (A) Conjugate working solution concentration settings: Final free-uptake working solution concentrations for the in vitro conjugates (PC(3'L96), PC(5'L96), ds-CA1, and ds-CD1) were 2 nM, 1 nM, 0.5 nM, 0.25 nM, 125 pM, 62.5 pM, 31.3 pM, 15.6 pM, 7.81 pM, 3.91 pM, and 1.95 pM; final free-uptake working solution concentrations for the in vitro conjugates (PC(without L96) and NC(without L96)) were 1 nM, 0.5 nM, and 0.25 nM.

[0405] (B) Preparation and dilution of the conjugate: The conjugate was dissolved in DEPC water at a ratio of 50 μL DEPC HO per OD, and the concentration was measured using a Nanodrop 2000. Based on the measurement results, the conjugate was diluted to 266 ng / μL, recorded as 20 μM, and stored as a stock solution at -20°C.

[0406] (C) Free-uptake sample preparation: 10 μL / well of the conjugate of the corresponding concentration was directly added to the cell seeding plate.

[0407] (D) Isolation of primary mouse hepatocytes (PMH) without the use of Percoll. A sterile environment was maintained throughout the process. The survival rate of freshly isolated PMH was ≥85%. Primary mouse hepatocytes were seeded at a density of 8 x 10 cells per well in a 24-well plate pre-coated with type I collagen at a volume of 900 μL per well. 4 Live cells / well: 10 μL of the diluted conjugate was dispersed in 90 μL of Opti-MEM and added to the corresponding wells (n=2). The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours to allow for free uptake.

[0408] (E) Conventional real-time fluorescence quantitative PCR assay: Total RNA extraction from cells: Lyse cells and extract total RNA using a high-throughput nucleic acid extraction instrument-magnetic bead method. RNA concentration adjustment: Measure sample concentration using a nanophotometer and adjust all samples to the same concentration by adding water. Reverse transcription: Use a reverse transcription kit to remove genomic DNA and reverse transcribe all samples into cDNA. Conventional real-time fluorescence quantitative qPCR assay: Use a conventional qPCR kit (SYBR GREEN system) for relative quantification of cDNA samples. Perform quantitative analysis three times for each cDNA sample, and simultaneously generate a standard curve.

[0409] (E) Data processing and analysis: Calculation of fold difference: Fc = 2 ^-△△Cp. Graphing: IC analysis using Graphpad Prism 7's "[Inhibitor] vs. normalized response -- Variable slope" 50 .

[0410] (Y=100 / (1+(X^HillSlope) / (IC50^HillSlope)))

[0411] Experimental results / conclusions

[0412] 1. In vitro gene inhibition assays in the PMH control group showed that PC (without L96) and NC (without L96) were unable to enter cells and exert their effects under free uptake. They exhibited no inhibitory effect at final concentrations of 1, 0.5, and 0.25 nM, with the remaining inhibition efficiency fluctuating around 100%. This served as a negative QC in the entire assay system. Protein assay results in the PMH control group were consistent with those of the mRNA assay.

[0413] 2. Genetic level of free uptake of conjugate in PMH (IC 50 ), as shown in Table 1:

[0414] Table 1

[0415] Experimental Example 2

[0416] Methods: Subcutaneous administration was performed in C57BL / 6 mice, and liver activity was measured by qPCR. The delivery efficacy of the conjugates of the present invention was compared with that of a control using in vitro and in vivo data. Six animals were enrolled per dose for each conjugate in the in vivo experiments to meet the minimum number of animals required for statistical analysis.

[0417] The conjugate was dissolved in DEPC water at a ratio of 50 μL DEPC HO per OD, and the concentration was measured using a Nanodrop 2000. Based on the measurement results, the conjugate was diluted to 2,000 ng / μL as a stock solution and stored at -20°C.

[0418] Animal treatment: Animal preparation: After the animals arrive, they are observed and allowed to recover for one week. Dosing: Weigh the animals before dosing and administer the drug subcutaneously. Anatomy: After 72 hours, the animals are sacrificed and dissected. Two 1cm sections are taken from each animal. 3 The liver tissue was collected and placed in RNA Later preservation solution and stored at -80°C. Serum samples were collected by centrifugation with EDTA anticoagulation and stored at -80°C.

[0419] Conventional Real-Time Fluorescence Quantitative PCR Assay: Total RNA Extraction from Tissue: Tissue homogenate and extract total RNA using a high-throughput nucleic acid extraction instrument-magnetic bead method. RNA Concentration Adjustment: Sample concentration is measured using a nanophotometer and adjusted to the same concentration for all samples by adding water. Reverse Transcription: Genomic DNA is removed using a reverse transcription kit and all samples are reverse transcribed into cDNA. Conventional Real-Time Fluorescence Quantitative qPCR Assay: Relative quantification of cDNA samples is performed using a conventional qPCR kit (SYBR GREEN system). Quantitative analysis is performed in triplicate for each cDNA sample.

[0420] The results of the relative mRNA expression level detection of the conjugate in normal mice are shown in Table 2:

[0421] Table 2 Note: / indicates not tested

[0422] Experimental Example 3

[0423] Methods: Subcutaneous administration was performed in C57BL / 6 mice, and liver activity was measured by qPCR. In vivo data were used to compare the delivery efficacy of the conjugates of the present invention with that of a control. Six animals were enrolled per dose for each conjugate to meet the minimum group size for statistical analysis.

[0424] The conjugate was dissolved in PBS at a ratio of 50 μL PBS per OD, and the concentration was measured using a Nanodrop 2000. Based on the measurement results, the conjugate was diluted to 2,000 ng / μL as a stock solution and stored at -80°C.

[0425] Animal treatment: Animal preparation: After the animals arrive, they are observed and allowed to recover for one week. Dosing: Weigh the animals before dosing and administer 5 μL / g subcutaneously. Anatomy: After 72 hours, the animals are sacrificed and subjected to gross dissection. One blood aliquot is collected from each animal. After standing at room temperature for 20-30 minutes, the blood is centrifuged and packaged into approximately 40 μl of serum. The serum is then transferred to -80°C for storage for six months. Two 1 cm2 aliquots are collected from each animal. 3 The liver tissue was placed in RNA Later preservation solution at 4°C overnight and then stored at -80°C.

[0426] Conventional real-time fluorescence quantitative PCR detection: Total RNA extraction from tissue: Tissue homogenization, add 0.5ml Trizol and steel balls to the liver tissue, put it into a high-throughput tissue grinder, adjust the parameters to 30HZ, and grind until the liver is completely broken. Total RNA extraction from tissue: Tissue homogenization, use a high-throughput nucleic acid extractor-magnetic bead method to extract total RNA. RNA concentration adjustment: Use Nanodrop2000 to detect the sample concentration, and add water to adjust all samples to the same concentration. Reverse transcription: Use a reverse transcription kit to remove genomic DNA, and reverse transcribe all samples into cDNA. Conventional real-time fluorescence quantitative qPCR detection: Use a conventional qPCR kit (SYBR GREEN system) to perform relative quantification of cDNA samples. Repeat quantitative analysis for each cDNA sample 3 times.

[0427] Data processing and analysis: Calculate the fold difference: Fc = 2 ^-△△Cp. Graphing: Using Graphpad Prism 7

[0428] The results of the relative mRNA expression level detection of the conjugate in normal mice are shown in Table 3:

[0429] Table 3

[0430] Example 4

[0431] In vivo analysis of RNAi agent efficacy in transiently transgenic mice

[0432] 1. Animal Grouping: 5- to 6-week-old Balb / c mice were fed adaptively for at least 3 days after entering the animal facility. They were then randomly divided into groups based on body weight, with 6 mice per group. The day of grouping was defined as D-3.

[0433] 2. Administration: Administration was started on D-3, with a single subcutaneous injection of 3 mpk.

[0434] 3. HDI injection modeling: To evaluate the in vivo efficacy of LPA RNAi agents, transient transgenic mice were used. Wild-type Balb / c mice were injected with the APOA gene plasmid via hydrodynamic tail vein injection to serve as model mice. The day of injection was designated D0.

[0435] 4. LPA expression knockdown analysis: The apo(a) protein level in the serum of mice was detected using an LPA (abcam) ELISA kit. Blood was collected on D1 and 30 μL of serum was separated to detect apo(a) expression.

[0436] 5. Statistical Analysis: Experimental results are expressed as mean ± standard error (SEM). Comparisons between two groups were performed using the independent sample t-test. Single-variable comparisons between multiple groups were performed using one-way analysis of variance (ANOVA). Two-way ANOVA was used to compare two-variable comparisons between multiple groups. Graphs and data analysis were performed using Graphpad Prism 9. P < 0.05 was considered statistically significant.

[0437] Table 4. Relative expression levels of apo(a) in transiently transgenic mice

[0438] Experimental Example 5

[0439] The cytotoxicity of the conjugate to MHCC97 cells was detected by PI. The experiment was performed in 96-well plates. There were two technical replicates for each concentration point of each conjugate. PC, NC and MOCK were set for each plate of cell screening to calibrate the consistency between different plates.

[0440] The final concentration of the conjugate working solution and the negative and positive controls was 100 nM.

[0441] The conjugate was dissolved in DEPC water at a ratio of 50 μL DEPC HO per OD, and the concentration was measured using a Nanodrop 2000. Based on the measurement results, the conjugate was diluted to 2,000 ng / μL as a stock solution and stored at -80°C.

[0442] Cell seeding plate: 150 μL cell suspension / well, inoculated into 96-well plate, MHCC97 cell number: 4*10 3 Cells were cultured in a 37°C, 5% CO2 incubator for 24 hours before transfection.

[0443] Transfection: Disperse 5 μL of diluted conjugate in 20 μL Opti-MEM and 0.2 μL of RNAiMAX in 24.8 μL Opti-MEM. Incubate for 5 minutes, then mix with the conjugate dispersion and incubate for 10 minutes to obtain a transfection complex. Add the cells to the transfection complex for transfection.

[0444] PI staining: 48 hours after cell transfection, remove the original culture medium and add 200 μL PBS to each well for washing. Mix 1 mg / mL PI with PBS to a final concentration of 10 μg / mL, add 100 μL of the mixture to each cell well, place the culture plate in an incubator and incubate for 20 minutes, then photograph using a fluorescence microscope.

[0445] Data processing and analysis: The total number of cells and the number of PI-stained cells were counted based on the images, and the percentage of PI-stained cells was calculated. The percentage of conjugates was normalized to that of NC, where a higher percentage indicates a relatively higher toxicity.

[0446] The percentage of PI positive cells to all cells is shown in Table 5:

[0447] Table 5

Claims

1. A ligand-oligonucleotide conjugate comprising a linker moiety of formula I or II: 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), or -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II), in: R 1 、R 2 、R 3 、R 4 Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl, 6- to 14-membered aryl, 5- to 18-membered heteroaryl, 5- to 14-membered heterocyclyl, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, and mercapto, which are optionally replaced by R 5 Substituted, where R 5 Selected from C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl, 6- to 14-membered aryl, 5- to 18-membered heteroaryl, 5- to 14-membered heterocyclic group, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, C 1-3 alkylthio and mercapto groups; R a 、R b Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-8 Cycloalkyl, 6 to 14 membered aryl C 1-3 Alkyl, 5 to 18 membered heteroaryl C 1-3 alkyl and 5 to 14 membered heterocyclic groups, which are optionally replaced by R c Substituted, where R c Selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C 1-3 Alkylthio, indolyl, quinolyl, isoquinolyl, amide, pyrrolyl, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, C 1-10 Alkyl, C 1-10 Alkoxy, 6 to 14 membered aryl, C 3-8 Cycloalkyl, adamantyl, phosphono, phosphonooxy, C 1-3 Alkylphosphono, C 1-3 Alkylphosphonooxy, or R a and R b One of them, together with the C atom to which they are connected and the adjacent N atom, forms a 3-14 membered nitrogen-containing heterocyclic group, or R a 、R b Together with its co-connected C atom, it forms C 3-8 Cycloalkyl; heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon; n is 1 to 10, and each repeating unit is the same or different; p is 2 to 6, and each repeating unit is the same or different.

2. The ligand-oligonucleotide conjugate according to claim 1, wherein R a Yes -H,R b Selected from -H, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclopentyl, cyclohexyl, cyclohexyldimethylene, cyclohexylmethylene, cyclobutylmethylene, phenylmethylene, methylthiomethylene, 3-indolylmethylene, hydroxymethylene, amidomethylene, hydroxymethylmethylene, mercaptomethylene, amidomethylene, hydroxyphenylmethylene, hydroxyphenyldimethylene, carboxymethylene, carboxydimethylene, amino-n-butyl, guanidine dimethylene, guanidinotrimethylene, 4-imidazolylmethylene, isopropylphenylmethylene, 1-naphthylmethylene, 2-naphthylmethylene, indanyl, phenyldimethylene, fluorophenylmethylene, chlorophenylmethylene, trifluorophenylmethylene, dichlorophenylmethylene, pentafluorophenylmethylene, difluorophenylmethylene, thienylmethylene, thiandenylmethylene, quinolylmethylene, halogenated 3-indolylmethylene, diphenylmethylene, 3-pyridylmethylene and 4-pyridylmethylene, or R b Together with the C atom to which it is commonly connected and the adjacent N atom, it forms a tetrahydropyrrolyl group; Preferably, R 1 、R 2 、R 3 、R 4 Independently selected from -H, C 1-10 Alkyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl and 5 to 14 membered heterocyclic groups, which are optionally replaced by R 5 Substituted, where R 5 Selected from C 1-10 Alkyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl and 5- to 14-membered heterocyclyl; More preferably, R 1 、R 2 、R 3 、R 4 Independently selected from -H, C 1-3 Alkyl, C 1-3 Alkoxy, C 3-6 Cycloalkyl, which are optionally replaced by R 5 Substituted, where R 5 Selected from C 1-10 Alkyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl, pyrrolyl, thienyl, thiazolyl, imidazolyl, pyridyl, furyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl; More preferably, R 1 、R 2 、R 3 、R 4 independently selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, and ethoxy; Further preferably, R 1 、R 2 、R 3 、R 4 is independently -H or methyl.

3. The ligand-oligonucleotide conjugate according to any one of claims 1 to 2, wherein n is 2 to 4, and each repeating unit is the same or different; Preferably, n is 2 or 3, and each repeating unit is the same or different.

4. The ligand-oligonucleotide conjugate according to any one of claims 1 to 3, wherein p is 2 to 4, and each repeating unit is the same or different; Preferably, p is 2 or 3, and each repeating unit is the same or different.

5. The ligand-oligonucleotide conjugate according to claim 1, wherein R 1 、R 2 、R 3 、R 4 、R a All are -H, n is 2 or 3, p is 2, R b is selected from -H, methyl, isopropyl and isobutyl, or R b Together with the C atom to which it is commonly connected and the adjacent N atom, it forms a tetrahydropyrrolyl group.

6. The ligand-oligonucleotide conjugate according to claim 5, wherein for the repeating unit where p is 2: In each repeating unit, R b isopropyl; In each repeating unit, R b It is a methyl group; In a repeating unit, R b is isopropyl, and in another repeating unit, R b It is a methyl group; In a repeating unit, R b is isobutyl, and in another repeating unit, R b It is a methyl group; In a repeating unit, R b is isobutyl, and in another repeating unit, R b is isopropyl; In a repeating unit, R b is isopropyl, and in another repeating unit, R b It is hydrogen; In a repeating unit, R b is isopropyl, and in another repeating unit, R b Together with the C atom to which it is commonly attached and the adjacent N atom, it forms a tetrahydropyrrolyl group; or In a repeating unit, R b is hydrogen, and in the other repeating unit, R b Together with the C atom to which it is commonly connected and the adjacent N atom, it forms a tetrahydropyrrolyl group.

7. The ligand-oligonucleotide conjugate according to any one of claims 1 to 6, wherein the ligand comprises one or more N-acetylgalactosamine or derivatives thereof attached via a bivalent or trivalent branched linker.

8. The ligand-oligonucleotide conjugate according to claim 7, wherein the ligand has the following structure: (GalNAc-Q 1 ) m CH m’ -,in GalNAc represents N-acetylgalactosamine or its derivatives; the structure of N-acetylgalactosamine or its derivatives is as follows: Q 1 Indicates T 1 -(T 2 -T 3 -T 4 ) w , where T 1 and T 4 independently selected from absent, C(O), NH, O, S, OC(O), NHC(O), CH2, CH2NH, and CH2O; T 2 is selected from the group consisting of absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH2C(O), C(O)-CH2-NH, C(O) and CH=NO; T 3 One or more methylene groups are selected from the group consisting of: O, S, S(O), SO2, C(O) and C≡C; w is 0 to 20, and the repeating units are the same or different; said R o 、R o 'are independently selected from H, alkylcarbonyl; the R o ' is preferably acetyl; said R o Each is independently preferably H or acetyl, more preferably H; m is 1 to 3, and each repeating unit is the same or different; m′ is 0 to 2, and m+m′ is 3.

9. The ligand-oligonucleotide conjugate according to claim 8, wherein the structure of the ligand is selected from: Preferably, the structure of the ligand is:

10. The ligand-oligonucleotide conjugate according to any one of claims 1 to 9, wherein the oligonucleotide is selected from antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs) and microRNAs (miRNAs), or salts thereof. The ligand-oligonucleotide conjugate according to claim 10 , wherein the 5′ end or the 3′ end of the oligonucleotide is attached to the linker moiety.

12. The ligand-oligonucleotide conjugate according to claim 11, wherein the 5' end is connected to the oligonucleotide by 3'-oligonucleotide-5'-OP(O)(OH)O-(CH2-Y) x -NH- is attached to the linker moiety, wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different; Preferably, the 5' end is attached to the linker moiety via any one selected from: 3'-oligonucleotide-5'-OP(O)(OH)O-(CH2)6-NH-; 3'-oligonucleotide-5'-OP(O)(OH)O-(CH2)3-NH-; 3'-Oligonucleotide-5'-OP(O)(OH)O-(CH2) 12 -NH-; and 3'-oligonucleotide-5'-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH-; or, The 5' end is connected to 3'-oligonucleotide-5'-OP(S)(OH)O-(CH2-Y) x -NH- is attached to the linker moiety, wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different; Preferably, the 5' end is attached to the linker moiety via any one selected from: 3'-oligonucleotide-5'-OP(S)(OH)O-(CH2)6-NH-; 3'-oligonucleotide-5'-OP(S)(OH)O-(CH2)3-NH-; 3'-Oligonucleotide-5'-OP(S)(OH)O-(CH2) 12 -NH-; and 3'-Oligonucleotide-5'-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH-.

13. The ligand-oligonucleotide conjugate according to claim 10, wherein the oligonucleotide is a siRNA, comprising a sense strand and an antisense strand, and the 5' end or the 3' end of the sense strand is attached to the linker portion; Preferably, one or more of the nucleotides in the siRNA are modified, and the modification is a 2'-O-methyl modification and / or a 2'-fluoro modification.

14. A ligand-oligonucleotide conjugate selected from the group consisting of: in indicates siRNA; Preferably, 15. Compounds represented by formula III or IV: (GalNAc-Q 1 ) m CH m’ -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -Q 2 (Formula III), or (GalNAc-Q 1 ) m CH m’ -NR 1 -[C(O)-C(R a R b )-NR 2 p -C(O)-[C(R 3 R 4 )] n -C(O)-Q 2 (Formula IV)​ in: GalNAc represents N-acetylgalactosamine or its derivatives; the structure of N-acetylgalactosamine or its derivatives is: Q 1 Indicates -T 1 -(T 2 -T 3 -T 4 ) w , where T 1 and T 4 independently selected from absent, C(O), NH, O, S, OC(O), NHC(O), CH2, CH2NH, and CH2O; T 2 is selected from the group consisting of absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH2C(O), C(O)-CH2-NH, C(O) and CH=NO; T 3 One or more methylene groups are selected from the group consisting of: O, S, S(O), SO2, C(O) and C≡C; w is 0 to 20, and the repeating units are identical or different; Q 2 represents an -OH or -O-carboxyl protecting group; the carboxyl protecting group is preferably benzyl or pentafluorophenyl; R 1 、R 2 、R 3 、R 4 Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl, 6- to 14-membered aryl, 5- to 18-membered heteroaryl, 5- to 14-membered heterocyclyl, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, and mercapto, which are optionally replaced by R 5 Substituted, where R 5 Selected from C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl, 6- to 14-membered aryl, 5- to 18-membered heteroaryl, 5- to 14-membered heterocyclic group, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, C 1-3 alkylthio and mercapto groups; R a 、R b Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-8 Cycloalkyl, 6 to 14 membered aryl C 1-3 Alkyl, 5 to 18 membered heteroaryl C 1-3 alkyl and 5 to 14 membered heterocyclic groups, which are optionally replaced by R c Substituted, where R c Selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C 1-3 Alkylthio, indolyl, quinolyl, isoquinolyl, amide, pyrrolyl, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, C 1-10 Alkyl, C 1-10 Alkoxy, 6 to 14 membered aryl, C 3-8 Cycloalkyl, adamantyl, phosphono, phosphonooxy, C 1-3 Alkylphosphono, C 1-3 Alkylphosphonooxy, or R a and R b One of them, together with the C atom to which they are connected and the adjacent N atom, forms a 3-14-membered nitrogen-containing heterocyclic group; the R o 、R o 'are independently selected from H, alkylcarbonyl; the R o ' is preferably acetyl; said R o Each is independently preferably H or acetyl, more preferably H; heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon; m is 1 to 3, and each repeating unit is the same or different; m' is 0 to 2, preferably 0 or 1, and m+m' is 3; n is 1 to 10, and each repeating unit is the same or different; p is 2 to 6, and each repeating unit is the same or different.

16. The compound according to claim 15, wherein (GalNAc-Q 1 ) m CH m’ -The structure of the part is selected from: Preferably:

17. The compound according to any one of claims 15 to 16, wherein R a Yes -H,R b Selected from -H, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclopentyl, cyclohexyl, cyclohexyldimethylene, cyclohexylmethylene, cyclobutylmethylene, phenylmethylene, methylthiomethylene, 3-indolylmethylene, hydroxymethylene, amidomethylene, hydroxymethylmethylene, mercaptomethylene, amidomethylene, hydroxyphenylmethylene, hydroxyphenyldimethylene, carboxymethylene, carboxydimethylene, amino-n-butyl, guanidine dimethylene, guanidinotrimethylene, 4-imidazolylmethylene, isopropylphenylmethylene, 1-naphthylmethylene, 2-naphthylmethylene, indanyl, phenyldimethylene, fluorophenylmethylene, chlorophenylmethylene, trifluorophenylmethylene, dichlorophenylmethylene, pentafluorophenylmethylene, difluorophenylmethylene, thienylmethylene, thiandenylmethylene, quinolylmethylene, halogenated 3-indolylmethylene, diphenylmethylene, 3-pyridylmethylene and 4-pyridylmethylene, or R b Together with the C atom to which it is commonly connected and the adjacent N atom, it forms a tetrahydropyrrolyl group.

18. The compound according to claim 17, wherein for the repeating unit where p is 2: In each repeating unit, R b is isopropyl; In each repeating unit, R b It is a methyl group; In a repeating unit, R b is isopropyl, and in another repeating unit, R b It is a methyl group; In a repeating unit, R b is isobutyl, and in another repeating unit, R b It is a methyl group; In a repeating unit, R b is isobutyl, and in another repeating unit, R b isopropyl; In a repeating unit, R b is isopropyl, and in another repeating unit, R b It is hydrogen; In a repeating unit, R b is isopropyl, and in another repeating unit, R b Together with the C atom to which it is commonly attached and the adjacent N atom, it forms a tetrahydropyrrolyl group; or In a repeating unit, R b is hydrogen, and in the other repeating unit, R b Together with the C atom to which it is commonly connected and the adjacent N atom, it forms a tetrahydropyrrolyl group.

19. A compound selected from the following formula: Wherein Bn represents a benzyl protecting group, and Ac represents an acetoxy group.

20. A method for preparing the ligand-oligonucleotide conjugate according to any one of claims 1 to 14, comprising: (a) providing a compound according to any one of claims 15 to 19; (b) providing an oligonucleotide having a terminal amino group at the 5' or 3' end of the oligonucleotide; (c) linking the compound to the oligonucleotide via the amino group; Preferably, the oligonucleotide in step (b) is siRNA, which comprises a sense strand and an antisense strand, and the compound is linked to the 5' end of the sense strand.

21. Use of the compound according to any one of claims 15 to 19 in the preparation of a ligand-oligonucleotide conjugate.

22. The linker represented by formula I or II or the connecting unit represented by formula I' or II': -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II), -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I'), or -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)-(Formula II'), in: R 1 、R 2 、R 3 、R 4 Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl, 6- to 14-membered aryl, 5- to 18-membered heteroaryl, 5- to 14-membered heterocyclyl, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, and mercapto, which are optionally replaced by R 5 Substituted, where R 5 Selected from C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-10 Cycloalkyl, 6- to 14-membered aryl, 5- to 18-membered heteroaryl, 5- to 14-membered heterocyclic group, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, C 1-3 alkylthio and mercapto groups; R a 、R b Independently selected from -H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 1-10 Alkoxy, C 3-8 Cycloalkyl, 6 to 14 membered aryl C 1-3 Alkyl, 5 to 18 membered heteroaryl C 1-3 alkyl and 5 to 14 membered heterocyclic groups, which are optionally replaced by R c Substituted, where R c Selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C 1-3 Alkylthio, indolyl, quinolyl, isoquinolyl, amide, pyrrolyl, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furyl, C 1-10 Alkyl, C 1-10 Alkoxy, 6 to 14 membered aryl, C 3-8 Cycloalkyl, adamantyl, phosphono, phosphonooxy, C 1-3 Alkylphosphono, C 1-3 Alkylphosphonooxy, or R a and R b One of the C atoms and the adjacent N atom to which it is commonly connected together form a 3-14 membered nitrogen-containing heterocyclic group; heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon; n is 1 to 10, and each repeating unit is the same or different; p is 2 to 6, and each repeating unit is the same or different.

23. The joint or linking unit according to claim 22, wherein R a Yes -H,R b Selected from -H, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclopentyl, cyclohexyl, cyclohexyldimethylene, cyclohexylmethylene, cyclobutylmethylene, phenylmethylene, methylthiomethylene, 3-indolylmethylene, hydroxymethylene, amidomethylene, hydroxymethylmethylene, mercaptomethylene, amidomethylene, hydroxyphenylmethylene, hydroxyphenyldimethylene, carboxymethylene, carboxydimethylene, amino-n-butyl, guanidine dimethylene, guanidinotrimethylene, 4-imidazolylmethylene, isopropylphenylmethylene, 1-naphthylmethylene, 2-naphthylmethylene, indanyl, phenyldimethylene, fluorophenylmethylene, chlorophenylmethylene, trifluorophenylmethylene, dichlorophenylmethylene, pentafluorophenylmethylene, difluorophenylmethylene, thienylmethylene, thiandenylmethylene, quinolylmethylene, halogenated 3-indolylmethylene, diphenylmethylene, 3-pyridylmethylene and 4-pyridylmethylene, or R b Together with the C atom to which it is commonly connected and the adjacent N atom, it forms a tetrahydropyrrolyl group.

24. The linker or connecting unit according to claim 23, wherein for the repeating unit where p is 2: In each repeating unit, R b isopropyl; In each repeating unit, R b It is a methyl group; In a repeating unit, R b is isopropyl, and in another repeating unit, R b It is a methyl group; In a repeating unit, R b is isobutyl, and in another repeating unit, R b It is a methyl group; In a repeating unit, R b is isobutyl, and in another repeating unit, R b isopropyl; In a repeating unit, R b is isopropyl, and in another repeating unit, R b It is hydrogen; In a repeating unit, R b is isopropyl, and in another repeating unit, R b Together with the C atom to which it is commonly attached and the adjacent N atom, it forms a tetrahydropyrrolyl group; or In a repeating unit, R b is hydrogen, and in the other repeating unit, R b Together with the C atom to which it is commonly connected and the adjacent N atom, it forms a tetrahydropyrrolyl group.

25. Use of the linker or connecting unit according to any one of claims 22 to 24 in the preparation of a ligand-oligonucleotide conjugate.

26. A pharmaceutical composition comprising the ligand-oligonucleotide conjugate according to any one of claims 1 to 14, and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition according to claim 26, which is formulated for subcutaneous injection.