Compositions for conjugating oligonucleotides and carbohydrates

Abstract

The present invention provides novel compositions and linking configurations for oligonucleotides conjugated to ligands for targeted in vivo delivery of oligonucleotides.The present invention further provides the use of the obtained compounds and pharmaceutical compositions thereof in the preparation of medicaments effective for treating disease or condition.In a first aspect, the present invention relates to compounds as therapeutic agents, in which oligonucleotides are conjugated to at least one ligand, such as monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, polysaccharides or their derivatives, e.g. carbohydrate ligands, which can target the compounds to receptor cells in the liver, which can promote uptake by endocytosis, as discussed above.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of co-pending U.S. Provisional Patent Application No. 63 / 151,060, filed February 18, 2021, the entirety of which is incorporated herein by reference.

[0002] Technical Field The present invention relates to novel compositions and processes that can be used in conjugating carbohydrate ligands with oligonucleotides intended for biomedical applications. [Background technology]

[0003] 2. Background of the Invention Gene modulating, especially gene silencing oligonucleotide, has been the focus of many development research efforts, because these series of nucleotides are highly promising for treating or preventing many diseases and modulating physiological conditions.Examples of such oligonucleotides include short / small interfering RNA (siRNA), asymmetric short / small interfering RNA (aiRNA), antisense oligonucleotide (ASO) and microRNA (miRNA).

[0004] In many organisms, RNA interference (RNAi) functions in a gene-specific manner through short double-stranded RNA (dsRNA) duplexes, termed siRNAs. siRNAs have a well-defined structure of symmetric, short (usually 20-24 base pairs) dsRNA duplexes with phosphorylated 5' and hydroxylated 3' ends, forming two 3' overhangs of equal length. Gene modulation is mediated through the multiprotein RNA-induced silencing complex (RISC), which binds to, unwinds and incorporates the antisense siRNA strand from the siRNA duplex, and then recognizes and targets the complementary messenger RNA (mRNA) for cleavage, thereby decreasing that gene expression in a post-transcriptional manner.

[0005] Relatively new on the scene, aiRNA was developed to overcome off-target effects mediated by the sense strand of symmetrically constructed canonical siRNAs, as well as other off-target mechanisms of siRNAs (see PCT Patent Publication WO2009029688). aiRNAs are designed to contain short RNA duplexes in which the two RNA strands are not equal in length and are therefore "asymmetric." For example, aiRNAs may form a duplex that contains a first strand that is 18-23 nucleotides long and a second strand that is 12-17 nucleotides long, where the first strand may have a 3' overhang of 1-9 nucleotides and a 5' overhang of 0-8 nucleotides. aiRNA technology can be used in all areas where current siRNA or short hairpin RNA (shRNA) are applied, including biological research, R&D studies in biotechnology and pharmaceutical industries, as well as RNAi-based therapeutics.

[0006] Antisense technology is a highly selective gene silencing technology based on a concept originally proposed in 1978 (Zamecnik PC et al., 1978). In general, the principle behind ASO technology is that antisense oligonucleotides hybridize to target nucleic acids and modulate gene expression through post-transcriptional mechanisms. The mechanisms can be broadly classified as follows: (1) occupancy only without promoting RNA degradation, where ASO binding results in translation arrest, splicing inhibition, or induction of alternatively spliced ​​variants; or (2) occupancy-induced destabilization, where ASO binding promotes RNA degradation via endogenous enzymes such as ribonuclease H1 (RNase H1); and (3) increased translation, where ASOs can block upstream open reading frames (uORFs) or other inhibitory elements in the 5'UTR, increasing translation efficiency (Stanley T. Crooke et al., 2008; C. Frank Bennett, 2010; Richard G. Lee, 2013; Stanley T. Crooke, 2017). The typical structure of ASOs is a single-stranded deoxyribonucleotide sequence with a sulfur chemical modification known as phosphorothioate. After 40 years of research, antisense technology has been improved by various chemical modifications of single-stranded oligonucleotides.

[0007] miRNA molecules are usually derived from non-coding regions of RNA transcripts that fold back on themselves to form hairpins. After being processed from their precursors through various cellular mechanisms, mature miRNAs are small (approximately 22 nucleotide) RNA molecules found in plants, animals and some viruses that regulate gene expression by post-transcriptional silencing.

[0008] These and other nucleic acid-based therapeutics offer promising solutions for a variety of diseases involving non-druggable targets. However, despite advances in the application of oligonucleotides and oligonucleotide analogs as therapeutics, there remains a great need to enhance key pharmacological properties of these therapeutic oligonucleotides in areas such as serum stability, delivery to intended organs or cell populations, and uptake across cell membranes.

[0009] The preferred delivery of therapeutic oligonucleotides to cells in vivo, for example in the mammalian body, such as the human body, requires specific targeting and protection from the extracellular environment inside the body, including from proteins in serum.The method that researchers have used to achieve specific targeting is to conjugate targeting moieties to oligonucleotides to direct therapeutic oligonucleotides to desired target sites.

[0010] One way to improve specificity in delivery is by taking advantage of receptor-mediated endocytosis activity already present in the body. The mechanism of uptake involves molecules bound to cell membrane receptors passing through the membrane and moving into the cell via invagination of membrane structures or by fusion of the delivery system with the cell membrane. This process is initiated via activation of cell surface or membrane receptors following binding of a specific ligand to the receptor. Thus, by conjugating drug candidates to targeting moieties that target such cell surface receptor(s), native endocytosis pathways can be effectively borrowed for drug delivery. Many receptor-mediated endocytosis mechanisms are known and have been studied, including those that recognize sugars, peptides and proteins containing galactose, mannose, mannose-6-phosphate, such as transferrin, asialoglycoprotein, vitamin B12, insulin and epidermal growth factor (EGF). In particular, the asialoglycoprotein receptor (ASGP-R) is a highly abundant receptor on hepatocytes. ASGP-R shows 50 times higher affinity for N-acetyl-D-galactosylamine (GalNAc) than for D-Gal. However, it has been reported that when using this conjugation system, the design of the linker structure and various chemical properties of the linker moiety are important in determining the overall delivery efficiency, efficacy and safety of the conjugated oligonucleotide, as well as in affecting the stability and manufacturing challenges of various therapeutic oligonucleotides. Therefore, there remains a strong need for the design of new and effective receptor-specific ligand-binding nucleic acid complexes for various biomedical applications. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 2009 / 029688 Summary of the Invention [Means for solving the problem]

[0012] SUMMARY OF THE PRESENT APPLICATION In a first aspect, the present invention relates to a compound as a therapeutic agent, in which an oligonucleotide is conjugated to at least one ligand, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide or derivatives thereof, e.g., a carbohydrate ligand, that can target the compound to receptor cells in the liver that can facilitate uptake by endocytosis, as discussed above.

[0013] These ligand-linked compounds target one or more organs or cell types, for example, liver parenchymal cells in humans. In one embodiment, the compound comprises more than one carbohydrate ligand, preferably two or three. In another embodiment, the compound of the present invention comprises at least one (e.g., one, two or three or more) N-acetyl-galactosamine (GalNAc), N-Ac-glucosamine (GluNAc), galactose, lactose, or mannose (e.g., mannose-6-phosphate). In yet another embodiment, the compound of the present invention comprises at least one (e.g., one, two or three or more) ligand selected from the group consisting of GalNAc, cholesterol, tocopherol, biotin, cyanine dye, folic acid, RGDp, transferrin, anisamide, lactobionic acid, cRGD, hyaluronic acid, low molecular weight protamine, lipid derivative, peptide, cyclic peptide, and heterocycle.

[0014] In a second aspect, the present invention provides a ligand-binding compound having a novel structure:

[0015] Item 1. Structural formula (G-H1): [ka] [In the formula, R 111 , R 112 , R 113 are each independently H or R 119A ;R 111 , R112 , R 113 At least one of the 119A and; R 119A comprises at least one ligand capable of docking to a cell surface receptor; R 114 , R 117 , R 118 is H or alkyl, aryl, heteroaryl, haloalkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -Salkylphenyl, -alkyl-SH, -Shaloalkyl, halo, -OH, -SH, -NH2, -alkyl-NH2, -N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, -CO2H, -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), -CON one or more selected from the group consisting of H2, -NHC(O)(alkyl), -NHC(O)(phenyl), -N(alkyl)C(O)(alkyl), -N(alkyl)C(O)(phenyl), -C(O)alkyl, -C(O)alkylphenyl, -C(O)haloalkyl, -OC(O)alkyl, -SO2(alkyl), -SO2(phenyl), -SO2(haloalkyl), -SON2NH2, -SON2NH(alkyl), -SON2NH(phenyl), -NHSO2(alkyl), -NHSO2(phenyl), and -NHSO2(haloalkyl); R 115 , R 116 represents, independently at each occurrence, OH, a protecting group for OH, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, -OP(Z')(Z'')O-nucleoside, -OP(Z')(Z'')O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, an -O-nucleotide, a nucleoside, -OP(Z')(Z'')OR 119B -OP(Z''')(Z'''')O-oligonucleotide, or oligonucleotide; J 111 , J 112 , R 119Bis, independently for each occurrence, a spacer; Z', Z'', Z''' and Z'''' are, independently at each occurrence, O or S; n 111 , n 112 is, independently at each occurrence, 1, 2, 3, 4, 5, or 6; Oligonucleotides contain natural or chemically modified nucleotides / nucleosides. A compound having the formula:

[0016] Item 2. Structural formula (G-H1-01): [ka] [In the formula, J 112A is selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH=N and S(O)2; J 112B is selected from alkylene of 1 to 10 carbon atoms; R 115A is a solid support or H; R 116 is a protecting group for OH, OH, phosphate, phosphodiester, activated phosphate, activated phosphite, phosphoramidite, -OP(Z')(Z'')O-nucleoside, -OP(Z')(Z'')O-oligonucleotide, lipid, PEG, steroid, polymer, -O-nucleotide, nucleoside, -OP(Z')(Z'')OR 119B -OP(Z''')(Z'''')O-oligonucleotide, or oligonucleotide; If necessary, J 112B is selected from linear alkylenes of 1 to 10 carbon atoms. The compound according to item 1, having the formula:

[0017] Item 3. n 112 The compound according to item 1 or 2, wherein

[0018] Item 4. R 1164. The compound according to any one of items 1 to 3, wherein

[0019] Item 5. Structural formula (G-H1-02): [ka] 5. The compound according to claim 4, having the formula:

[0020] Item 6. Structural formula (G-H1-03): [ka] 5. The compound according to claim 4, having the formula:

[0021] Item 7. Structural formula (G-H1-04): [ka] [In the formula, R 119C is -C(O)-C5~C8 straight chain alkylene -NHCO-CH2- or -C(O)-C8~C 11 linear alkylene-; R 119L are independently selected from ligands capable of docking to a cell surface receptor; n 111L is selected from 1, 2, 3, 4 or 5] 5. The compound according to claim 4, having the formula:

[0022] Item 8. J 111 , J 112 , R 119B are independently selected from alkylene of 1 to 30 carbon atoms, one or more of the carbon atoms being C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylene, C6-C 10 Arylene, C3~C 18 Heterocyclylene, and C5-C 10Heteroarylene, optionally substituted with any one or more substituents from the group consisting of R 119B is not replaced as required or R 119D is replaced by; R 114 , R 117 , R 118 , R 119D is H or C1~C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1~C 10 Alkyl, -OC1~C 10 Alkylphenyl, -C1~C 10 Alkyl-OH, -OC1~C 10 Haloalkyl, -SC1~C 10 Alkyl, -SC1~C 10 Alkylphenyl, -C1~C 10 Alkyl-SH, -SC1~C 10 Haloalkyl, halo, -OH, -SH, -NH2, -C1~C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)OC1~C 10 Alkyl, -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 Alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)C(O)(phenyl), -C(O)C1~C 10 Alkyl, -C(O)C1~C 10Alkylphenyl, -C(O)C1~C 10 Haloalkyl, -OC(O)C1~C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl), and -NHSO2(C1-C 10 haloalkyl); n 111L is selected from 1, 2, 3, 4 or 5.

[0023] Item 9. The compound according to any one of items 1 to 8, wherein the spacer is an alkylene of 1 to 10 carbon atoms, one or more of the carbon atoms being optionally replaced with any one or more of the substituents from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH=N and S(O)2, and the spacer is optionally unsubstituted or substituted with at least one group selected from the group: H, or C1-C5 alkyl, -OC1-C5 alkyl.

[0024] Item 10. Branched groups are [ka] Selected from the group consisting of: each n is independently 1 to 20; 8. The compound according to item 7, wherein m is 2 to 6.

[0025] Item 11. Branched groups are [ka] 8. The compound according to item 7, selected from the group consisting of:

[0026] Item 12. Branched groups are [ka]

[0027] wherein each A1 is independently O, S, C=O, or NH;

[0028] each n is independently 1 to 20] 8. The compound according to item 7, selected from the group consisting of:

[0029] Item 13. Branched groups are [ka] 8. The compound according to item 7, selected from the group consisting of:

[0030] Item 14. Structural formula (G-H1-05) or (G-H1-06): [ka] [In the formula, each X' is independently selected from Table 1; each Z' is independently selected from Table 2;

[0031] Table 2 [Table 2]

[0032] Table 1 [Table 1]

[0033] R 119C is -C(O)-C5~C8 straight chain alkylene -NHCO-CH2- or -C(O)-C8~C 11 linear alkylene- The compound according to item 1, having the formula:

[0034] Item 15. Structural formula (G-H1-07) or (G-H1-08): [ka] [In the formula, R, R' are independently selected from the group consisting of a solid support, an oligonucleotide containing natural or chemically modified nucleotides / nucleosides, H and a protecting group; At least one of R and R' comprises an oligonucleotide formed by natural and / or chemically modified nucleotides / nucleosides; D is selected from Table 3; Each E is independently selected from Table 4;

[0035] Table 3 [Table 3] Z' is independently selected from Table 2;

[0036] Each L independently comprises a ligand moiety capable of docking to a cell-surface receptor.

[0037] Table 4 [Table 4] Item 15. The compound according to item 14, wherein

[0038] Item 16. D is -C(O)-C5-C8 linear alkylene -NHCO-CH2- or -C(O)-C8-C 11 Item 16. The compound according to item 15, wherein said alkylene- is selected from linear alkylene-.

[0039] Item 17. Structural formula (G-H1-09) or (G-H1-10): [ka] [ka] [In the formula, R, R' are independently selected from the group consisting of oligonucleotides containing natural or chemically modified nucleotides / nucleosides, H and protecting groups; At least one of R and R' comprises an oligonucleotide formed by natural and / or chemically modified nucleotides / nucleosides; A is independently O or S; X' is independently selected from Table 1; Z' is independently selected from Table 2; Each D is selected from Table 3; Each E is independently selected from Table 4; each L independently comprises a ligand moiety capable of docking to a cell-surface receptor. Item 15. The compound according to item 14, having the formula:

[0040] Item 18. The compound according to item 17, wherein A is O.

[0041] Item 19. The compound according to item 17, wherein A is S.

[0042] Item 20. The compound according to any one of items 1 to 19, wherein each ligand is independently selected from the group consisting of N-acetylgalactosamine (GalNAc), cholesterol, tocopherol, biotin, cyanine dyes, folic acid, RGDp, transferrin, anisamide, lactobionic acid, cRGD, hyaluronic acid, low molecular weight protamines, lipid derivatives, peptides, cyclic peptides, and heterocycles.

[0043] Item 21. Structural formula (G-H1-11): [ka] [In the formula, R 112B has the structure shown below: -branched group-(R 119B -R 119L ) n 111L having; R 119Lare independently selected from ligands capable of docking to a cell surface receptor; R 119B are independently selected from alkylene of 1 to 30 carbon atoms, where one or more carbon atoms are optionally selected from C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylene, C6-C 10 Arylene, C3~C 18 Heterocyclylene, and C5-C 10 Heteroarylene, R 119B is unsubstituted or R as appropriate. 119C is replaced by; R 114 , R 117 , R 118 , R 119C is H or C1~C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1~C 10 Alkyl, -OC1~C 10 Alkylphenyl, -C1~C 10 Alkyl-OH, -OC1~C 10 Haloalkyl, -SC1~C 10 Alkyl, -SC1~C 10 Alkylphenyl, -C1~C 10 Alkyl-SH, -SC1~C 10 Haloalkyl, halo, -OH, -SH, -NH2, -C1~C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)OC1~C 10Alkyl, -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 Alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)C(O)(phenyl), -C(O)C1~C 10 Alkyl, -C(O)C1~C 10 Alkylphenyl, -C(O)C1~C 10 Haloalkyl, -OC(O)C1~C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl), and -NHSO2(C1-C 10 haloalkyl); R, R' are independently selected from the group consisting of a solid support, an oligonucleotide containing natural or chemically modified nucleotides / nucleosides, H and a protecting group; At least one of R and R' comprises an oligonucleotide formed by natural and / or chemically modified nucleotides / nucleosides; n 111L is selected from 1, 2, 3, 4 or 5] 21. The compound according to any one of items 1 to 20, having the formula:

[0044] Item 22. Structural formula HC-1~HC-8: [ka] [ka] 5. The compound according to claim 4, having the formula:

[0045] Item 23. Structural formula HC-9: [ka] 5. The compound according to claim 4, having the formula:

[0046] Item 24. Structural formula HC-5: [ka] 5. The compound according to claim 4, having the formula:

[0047] Item 25. The compound according to any one of items 1 to 24, wherein the oligonucleotide is linked to the remainder of the compound via its 5'-end and / or 3'-end.

[0048] Item 26. The compound of item 25, wherein the oligonucleotide comprises a small interfering RNA (siRNA) duplex.

[0049] Item 27. The compound of item 25, wherein the oligonucleotide comprises an asymmetric interfering RNA (aiRNA) duplex.

[0050] Item 28. The aiRNA comprises an antisense strand and a sense strand, wherein the antisense strand is longer than the sense strand and has a length of 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides, and when duplexed with the sense strand, comprises a 3'-overhang of 1-9 nucleotides and a 5'-overhang of 0-8 nucleotides; 28. The compound according to item 27, wherein the sense strand has a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides and forms a double-stranded region with the antisense strand.

[0051] Item 29. The aiRNA comprises an antisense strand and a sense strand, the antisense strand being longer than the sense strand and having a length of 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides, and when duplexed with the sense strand, comprising a 3'-overhang of 1-9 nucleotides and a 5'-overhang of 1-8 nucleotides; 29. The compound according to item 28, wherein the sense strand has a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides and forms a double-stranded region with the antisense strand.

[0052] Item 30. The aiRNA comprises an antisense strand and a sense strand, the antisense strand being longer than the sense strand and having a length of 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides, and comprising a 3'-overhang of 1-9 nucleotides and a 5' blunt end when duplexed with the sense strand; 29. The compound according to item 28, wherein the sense strand has a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides and forms a double-stranded region with the antisense strand.

[0053] Item 31. The compound according to item 25, wherein the oligonucleotide comprises an antisense oligonucleotide (ASO).

[0054] Item 32. The compound according to item 25, wherein the oligonucleotide comprises a microRNA (miRNA).

[0055] Item 33. Structural formula (G-G1): [ka] [In the formula, R 127 comprises at least one ligand capable of docking to a cell surface receptor; R 123 , R 124 , R 125 , R 126is H, alkyl, aryl, heteroaryl, haloalkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -Salkylphenyl, -alkyl-SH, -Shaloalkyl, halo, -OH, -SH, -NH2, -alkyl-NH2, -N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, -CO2H, -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), -CONH2 , -NHC(O)(alkyl), -NHC(O)(phenyl), -N(alkyl)C(O)(alkyl), -N(alkyl)C(O)(phenyl), -C(O)alkyl, -C(O)alkylphenyl, -C(O)haloalkyl, -OC(O)alkyl, -SO2(alkyl), -SO2(phenyl), -SO2(haloalkyl), -SON2NH2, -SON2NH(alkyl), -SON2NH(phenyl), -NHSO2(alkyl), -NHSO2(phenyl), and -NHSO2(haloalkyl); R 121 , R 122 represents, independently at each occurrence, OH, a protecting group for OH, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, -OP(Z')(Z'')O-nucleoside, -OP(Z')(Z'')O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, an -O-nucleotide, a nucleoside, -OP(Z')(Z'')OR 128B -OP(Z''')(Z'''')O-oligonucleotide, or oligonucleotide; J 121 , J 122 , R 128B is, independently for each occurrence, a spacer; Z', Z'', Z''' and Z'''' are, independently at each occurrence, O or S; n 121 , n 122 is, independently at each occurrence, 1, 2, 3, 4, 5, or 6; The oligonucleotides contain natural or chemically modified nucleotides / nucleosides. A compound having the formula:

[0056] Item 34. Structural formula (G-G1-01): [ka] [In the formula, J 122A is selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH=N and S(O)2; J 122B is selected from alkylene of 1 to 10 carbon atoms; R 121A is selected from the group consisting of H and a solid support; R 122 is a protecting group for OH, OH, phosphate, phosphodiester, activated phosphate, activated phosphite, phosphoramidite, solid support, -OP(Z')(Z'')O-nucleoside, -OP(Z')(Z'')O-oligonucleotide, lipid, PEG, steroid, polymer, -O-nucleotide, nucleoside, -OP(Z')(Z'')OR 128B -OP(Z''')(Z'''')O-oligonucleotide, or oligonucleotide; If necessary, J 122B is selected from linear alkylenes of 1 to 10 carbon atoms. 34. The compound according to claim 33, having the formula:

[0057] Item 35. n 122 The compound according to claim 33 or 34, wherein

[0058] Item 36. R 122 36. The compound according to any one of items 33 to 35, wherein the compound comprises an oligonucleotide.

[0059] Item 37. Structural formula (G-G1-02): [ka] 37. The compound according to item 36, having the formula:

[0060] Item 38. Structural formula (G-G1-03):

[0061] [ka] 37. The compound according to item 36, having the formula:

[0062] Item 39. Structural formula (G-G1-04): [ka] [In the formula, J 123A is selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH═N, and S(O)2; R 128A has the structure shown below: -R 128C -branched group-(R 128B -R 128L ) n 121L or -R 128B -R 128L having; R 128L are independently selected from ligands capable of docking to a cell surface receptor; R 128B are independently selected from alkylene of 1 to 30 carbon atoms, where one or more carbon atoms are selected from C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylene, C6-C 10 Arylene, C3~C 18 Heterocyclylene, and C5-C 10 Heteroarylene, optionally substituted with any one or more substituents from the group consisting of R 128B is unsubstituted or R as appropriate. 128C is replaced by; R 128C is -C(O)-C5~C8 straight chain alkylene -NHCO-CH2- or -C(O)-C8~C 11 linear alkylene-, n 121L is selected from 1, 2, 3, 4 or 5; R 128L are independently selected from ligands capable of docking to a cell surface receptor; n 111L is selected from 1, 2, 3, 4 or 5; n 121 is 2; If necessary, R 128C is -C(O)-C5~C8 straight chain alkylene -NHCO-CH2- or -C(O)-C8~C 11 linear alkylene- 37. The compound according to claim 36, having the formula:

[0063] Item 40. The compound according to any one of items 33 to 39, wherein the spacer is an alkylene of 1 to 10 carbon atoms, one or more carbon atoms being optionally replaced with any one or more substituents from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH=N and S(O)2, and the spacer (saper) is optionally unsubstituted or substituted with at least one group selected from the group: H, C1 to C5 alkyl, -OC1 to C5 alkyl.

[0064] Item 41. Branched groups [ka] [ka] wherein each n is independently 1 to 20; and m is 2 to 6. 41. The compound according to item 40, selected from the group consisting of:

[0065] Item 42. Branched groups are [ka] [ka] 41. The compound according to item 40, selected from the group consisting of:

[0066] Item 43. Branched groups are

[0067] [ka] wherein each A1 is independently O, S, C=O, or NH; each n is independently 1 to 20] 41. The compound according to item 40, selected from the group consisting of:

[0068] Item 44. Branched groups are [ka] 41. The compound according to item 40, selected from the group consisting of:

[0069] Item 45. Structural formula (G-G1-05) or (G-G1-06): [ka] [In the formula, X' is independently selected from Table 1; Z' is independently selected from Table 2; R 128C is -C(O)-C5~C8 straight chain alkylene -NHCO-CH2- or -C(O)-C8~C 11 linear alkylene- 34. The compound according to claim 33, having the formula:

[0070] Item 46. Structural formula (G-G1-07) or (G-G1-08): [ka] [In the formula, R, R' are independently selected from an oligonucleotide containing natural or chemically modified nucleotides / nucleosides, H and a protecting group; At least one of R and R' comprises an oligonucleotide formed by natural and / or chemically modified nucleotides / nucleosides; Z' is independently selected from Table 2; R 128C is -C(O)-C5~C8 straight chain alkylene -NHCO-CH2- or -C(O)-C8~C 11 linear alkylene- 34. The compound according to claim 33, having the formula:

[0071] Item 47. Structural formula (G-G1-09) or (G-G1-10): [ka] [In the formula, R, R' are independently selected from an oligonucleotide containing natural or chemically modified nucleotides / nucleosides, H and a protecting group; At least one of R and R' comprises an oligonucleotide formed by natural and / or chemically modified nucleotides / nucleosides; A is O or S; D' is selected from Table 9; Each E is selected from Table 4; X' is independently selected from Table 1; Z' is independently selected from Table 2; each L independently comprises a ligand moiety capable of docking to a cell-surface receptor; n4 is independently selected from 1, 2, 3, and 4. 34. The compound according to claim 33, having the formula:

[0072] Table 9 [Table 9]

[0073] Item 48. The compound according to item 47, wherein A is O.

[0074] Item 49. The compound according to item 47, wherein A is S.

[0075] Item 50. The compound according to any one of items 33 to 49, wherein each ligand is independently selected from the group consisting of N-acetylgalactosamine (GalNAc), cholesterol, tocopherol, biotin, cyanine dyes, folic acid, RGDp, transferrin, anisamide, lactobionic acid, cRGD, hyaluronic acid, low molecular weight protamines, lipid derivatives, peptides, cyclic peptides, and heterocycles.

[0076] Item 51. Structural formula [ka] [In the formula, R 129 has the structure shown below: -branched group-(R 128B -R 128L ) n 121L having; R 128L are independently selected from ligands capable of docking to a cell surface receptor; R 128B are independently selected from alkylene of 1 to 30 carbon atoms, where one or more carbon atoms are selected from C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylene, C6-C 10 Arylene, C3~C 18 Heterocyclylene, and C5-C 10 Heteroarylene, optionally substituted with any one or more substituents from the group consisting of R 128B is unsubstituted or R as appropriate. 128C is replaced by; R, R' are independently selected from the group consisting of a solid support, an oligonucleotide containing natural or chemically modified nucleotides / nucleosides, H and a protecting group; At least one of R and R' comprises an oligonucleotide formed by natural and / or chemically modified nucleotides / nucleosides; n 121L is selected from 1, 2, 3, 4 or 5] 34. The compound according to claim 33, having the formula:

[0077] Item 52. Formula GC-1~GC-8: [ka] [ka] [ka] Item 37. The compound according to item 36, having the structure shown in

[0078] Item 53. Structural formula GC-9 [ka] 37. The compound according to claim 36, having the formula:

[0079] Item 54. Structural formula GC-5 [ka] 37. The compound according to claim 36, having the formula:

[0080] Item 55. The compound according to any of items 33 to 54, wherein the oligonucleotide is linked to the remainder of the compound via its 5'-end and / or 3'-end.

[0081] Item 56. The compound of item 55, wherein the oligonucleotide comprises a small interfering RNA (siRNA) duplex.

[0082] Item 57. The compound according to any one of items 33 to 54, wherein the oligonucleotide comprises an asymmetric interfering RNA (aiRNA) duplex.

[0083] Item 58. The aiRNA comprises an antisense strand and a sense strand, wherein the antisense strand is longer than the sense strand and has a length of 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides, and when duplexed with the sense strand, comprises a 3'-overhang of 1-9 nucleotides and a 5'-overhang of 0-8 nucleotides; 58. The compound according to item 57, wherein the sense strand has a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides and forms a double-stranded region with the antisense strand.

[0084] Item 59. The aiRNA comprises an antisense strand and a sense strand, wherein the antisense strand is longer than the sense strand and has a length of 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides, and when duplexed with the sense strand, comprises a 3'-overhang of 1-9 nucleotides and a 5'-overhang of 1-8 nucleotides; 59. The compound according to item 58, wherein the sense strand has a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides and forms a double-stranded region with the antisense strand.

[0085] Item 60. The aiRNA comprises an antisense strand and a sense strand, the antisense strand being longer than the sense strand and having a length of 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides, and when duplexed with the sense strand, comprising a 3'-overhang of 1-9 nucleotides and a 5' blunt end; 59. The compound according to item 58, wherein the sense strand has a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides and forms a double-stranded region with the antisense strand.

[0086] Item 61. The compound according to any one of items 33 to 54, wherein the oligonucleotide comprises an antisense oligonucleotide (ASO).

[0087] Item 62. The compound according to any one of items 33 to 54, wherein the oligonucleotide comprises a microRNA (miRNA).

[0088] Item 63. A small interfering RNA (siRNA) agent comprising the structural formula according to any one of items 1 to 55.

[0089] Item 64. An asymmetric interfering RNA (aiRNA) agent comprising the structural formula according to any one of items 1 to 55.

[0090] Item 65. An antisense oligonucleotide (ASO) agent comprising the structural formula according to any one of items 1 to 55.

[0091] Item 66. A microRNA (miRNA) agent comprising the structural formula according to any one of items 1 to 55.

[0092] Item 68. A pharmaceutical composition comprising a compound according to any one of items 1 to 60 or a drug according to any one of items 63 to 66, and a pharma- ceutically acceptable excipient, carrier, or diluent.

[0093] 6. Use of a compound according to any one of items 1 to 60 or an agent according to any one of items 63 to 66 in the preparation of a medicament effective for treating a disease or condition.

[0094] In a third aspect, the present invention features a compound comprising a carbohydrate ligand as provided in the second aspect above, wherein the presence of the carbohydrate ligand can increase the delivery of the compound to a target organ, such as the liver. Thus, the compound comprising a carbohydrate ligand can be useful for targeting genes associated with disease or undesirable conditions in a target organ. For example, the compound of the present invention comprising a carbohydrate ligand can target nucleic acids expressed by hepatitis viruses. In another example, the target genes include: Factor VII, Eg5, PCSK9, APOC3, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erkl / 2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, cyclin D gene, VEGF gene, EGFR gene, cyclin A gene, The mutation may be selected from the group consisting of a mutation in the cyclin E gene, the WNT-I gene, the beta-catenin gene, the c-MET gene, the PKC gene, the NFKB gene, the STAT3 gene, the survivin gene, the Her2 / Neu gene, the topoisomerase I gene, the topoisomerase II alpha gene, the p73 gene, a mutation in the p21(WAFl / CIPl) gene, a mutation in the p27(KIPl) gene, a mutation in the PPMlD gene, a mutation in the RAS gene, a mutation in the caveolin I gene, a mutation in the MIB I gene, a mutation in the MTAI gene, a mutation in the M68 gene, a mutation in a tumor suppressor gene, and a mutation in the p53 tumor suppressor gene.

[0095] In a further aspect, the present invention provides a pharmaceutical composition comprising a compound of the invention as provided in any of the above aspects and a pharma- ceutically acceptable excipient, carrier, or diluent.

[0096] In another aspect, the present invention features a method for delivering a compound to a specific target in a subject for therapeutic or diagnostic purposes.Therefore, the present invention provides a method for treating or preventing a disease or condition, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a compound of the present invention.The treatment or prevention of a disease or condition is carried out by partial or total silencing of a disease gene.The disease gene may be a patient's own gene or an externally derived microbial gene such as a virus.

[0097] The above and other objects, aspects, features, and advantages of the present invention will become more apparent from the following description and claims.

[0098] The objects and features of the present invention may be better understood with reference to the drawings and claims described below. The drawings illustrate the core ideas of the invention but do not necessarily determine all possible variations. In the drawings, like numerals are used to denote like parts throughout the various views. [Brief description of the drawings]

[0099] [Figure 1]FIG. 1 illustrates an exemplary structure of oligonucleotide-ligand conjugation. The conjugated interfering RNA duplex molecule comprises an antisense strand and a sense strand. In some embodiments, the oligonucleotide is an interfering RNA duplex molecule, and the ligand may be conjugated at the 3' end of the sense strand (such as structures 1.1-1.3, intermediate aiRNA, blunt-ended aiRNA, and siRNA), at the 3' end of the antisense strand (structure 2), at the 5' end of the sense strand (structure 3), or at both ends of the sense strand (structure 5), at both ends of the antisense strand (structure 4), at the 3' end of the antisense strand and the 5' end of the sense strand (structure 6), at the 3' end of the sense strand and the 3' end of the antisense strand (structure 7), or at the 3' end of the sense strand, the 3' end of the antisense strand, and the 5' end of the sense strand. In some embodiments, the oligonucleotide is an antisense oligonucleotide (ASO), and the ligand may be conjugated at the 3' end or / and 5' end of the antisense strand.

[0100] [Diagram 2] Figure 2 illustrates the ex vivo uptake β-catenin aiRNA results tested by QPCR. "Non-GalNAc" is unconjugated aiRNA. "GalNAc" is aiRNA conjugated with "His-cluster".

[0101] [Diagram 3] FIG. 3 illustrates the ex vivo uptake efficacy of mCat12 aiRNA conjugated with "His-cluster(3GalNAc)" and "Glu-cluster(3GalNAc)" in primary hepatocytes.

[0102] [Figure 4] Figure 4 illustrates the uptake efficacy of mCat12 aiRNA conjugated with "His-cluster(3GalNAc)" and "Glu-cluster(3GalNAc)" in vivo. aiRNA is administered subcutaneously at a dose of 20 mg / Kg.

[0103] [Diagram 5] Figure 5 illustrates the uptake efficacy of mCat12 aiRNA conjugated with "His-cluster(3GalNAc)" and "Glu-cluster(3GalNAc)" in vivo. aiRNA is administered subcutaneously at a dose of 2 mg / Kg.

[0104] [Figure 6] Figure 6 illustrates the ex vivo uptake β-catenin aiRNA results tested by QPCR. The aiRNA is conjugated with a "His-cluster". SS-intermediate represents aiRNA#1 containing a 5' overhang on the antisense strand. SS-3'blunt represents aiRNA#2 with blunt ends on the 3' sense strand and 5' antisense strand.

[0105] [Figure 7] Figure 7 illustrates the iv vivo uptake β-catenin aiRNA results. SS-intermediate represents aiRNA#1 containing a 5' overhang and a 3' overhang on the antisense strand. SS-3'blunt represents aiRNA#2 with blunt ends on the 3' sense strand and 5' antisense strand. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0106] Detailed Description of the Invention I. Definition Unless otherwise specified, technical terms are used according to conventional usage.Definitions of general terms in molecular biology can be found, for example, in J. Krebs et al. (eds.), Lewin's Genes XII, published by Jones and Bartlett Learning, 2017 (ISBN 9781284104493); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Anmol Publications Pvt. Ltd, 2011 (ISBN 9788126531783); and other similar technical references.

[0107] As used herein and in the claims, the singular forms "a," "an," or "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. It is further noted that the claims may be drafted to exclude any optional elements. In such cases, this statement is intended to serve as a basis for the use of exclusive language in the claims, such as "solely," "only," and the like, in connection with the recitation of the claim elements, or "negative" limitations, such as "wherein [the particular feature or element] is absent," or "except for [the particular feature or element]," or "wherein [the particular feature or element] is not present (not included, etc.)...."

[0108] In this specification, when dimensional measurements are given for portions, the values ​​are meant to describe the average for the required portion of that portion, i.e., the portion of the portion needed for the stated purpose, unless expressly stated or clear from the context. Any supplementary or excess portions are not meant to be included in the calculation of the value.

[0109] As used herein, the description of a numerical range for a variable is intended to convey that the invention can be practiced with the variable equal to any of the values ​​within that range. Thus, for a variable that is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end points of the range. Similarly, for a variable that is inherently continuous, the variable can be equal to any real value within the numerical range, including the end points of the range. By way of example and not of limitation, a variable described as having a value between 0 and 2 can take on values ​​of 0, 1 or 2 if the variable is inherently discrete, and can take on values ​​of 0.0, 0.1, 0.01, 0.001, or any other real value between >0 and <2 if the variable is inherently continuous.

[0110] As used herein, "about" means within plus or minus 10%. For example, "about 1" means "0.9 to 1.1", "about 2%" means "1.8% to 2.2%, "about 2% to 3%" means "1.8% to 3.3%, and "about 3% to about 4%" means "2.7% to 4.4%".

[0111] As used herein, the terms "spacer", "linker" and "link" are used to link two moieties of a compound, for example, alkylene of 1 to 10 carbon atoms, alkylene of 1 to 10 carbon atoms in which one or more carbon atoms are optionally replaced with any one or more substituents from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, S(O)2, alkylene of 1 to 10 carbon atoms that is unsubstituted or substituted with at least one group selected from the group consisting of H, C1-C5 alkyl, and -OC1-C5 alkyl.

[0112] A variety of hydroxyl protecting groups can be used in the present disclosure. In general, a protecting group renders a chemical functional group inert to a specific reaction condition and can be added to and removed from such functional group in a molecule without substantially damaging the remaining part of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage, et al., Tetrahedron 1992, 48, 2223-2311, as well as by Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4''-trimethoxytrityl).

[0113] As used herein, a dash ("-") that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -C 10 Alkyl-NH2 is C1-C 10 It is attached via an alkyl.

[0114] As used herein, "optionally" or "optionally" means that the event or circumstance described below may or may not occur, and the description includes the cases where the event or circumstance occurs and the cases where they do not occur. For example, "optionally substituted alkyl" encompasses both "alkyl" and "substituted alkyl" as defined below. With respect to any group that contains one or more substituents, it is understood by those skilled in the art that such groups are not intended to introduce any substitution or substitution pattern that is sterically infeasible, synthetically infeasible, and / or inherently unstable.

[0115] As used herein, "alkyl" refers to straight and branched chains having a specified number of carbon atoms, usually 1 to 20 carbon atoms, e.g., 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example, C1-C6 alkyl encompasses both straight and branched chain alkyls of 1 to 6 carbon atoms. When an alkyl residue having a specific number of carbons is named, all branched and straight chain versions having that number of carbons are intended to be encompassed; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and t-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl that has two points of attachment but refers to the same residues as alkyl.

[0116] As used herein, "alkenyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double bond derived by the removal of one hydrogen molecule from adjacent carbon atoms of a parent alkyl. The group may be in either cis or trans configuration about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl; and the like. In certain embodiments, alkenyl groups have from 2 to 20 carbon atoms, and in other embodiments from 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl referring to the same residues as alkenyl but with two points of attachment.

[0117] As used herein, "alkynyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon triple bond derived by the removal of two hydrogen molecules from adjacent carbon atoms of a parent alkyl. Typical alkynyl groups include, but are not limited to, ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl; and the like. In certain embodiments, alkynyl groups have 2-20 carbon atoms, and in other embodiments, 2-10, 2-8, or 2-6 carbon atoms. Alkynylene is a subset of alkynyl having two points of attachment but referring to the same residues as alkynyl.

[0118] As used herein, "alkoxy" refers to an alkyl group of designated number of carbon atoms attached through an oxygen bridge, such as, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentyloxy, etc. Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through an oxygen bridge.

[0119] As used herein, "aryl" refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. An aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon from 6 to 18 carbon atoms, and at least one of the rings in the ring system is fully unsaturated, i.e., contains a cyclic, delocalized (4n+2) π-electron system according to the Hückel theory. Aryl groups include, but are not limited to, groups such as phenyl, fluorenyl, and naphthyl. Arylene is a subset of aryl that has two points of attachment but refers to the same residues as aryl.

[0120] As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring, usually having 3 to 7 ring carbon atoms. The ring may be saturated or have one or more carbon-carbon double bonds. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl, as well as bridged and caged ring groups, such as norbornane.

[0121] As used herein, "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo, and the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0122] As used herein, "haloalkyl" refers to an alkyl as defined above, having a specified number of carbon atoms, substituted with one or more halogen atoms, up to the maximum number of halogen atoms permitted. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and penta-fluoroethyl.

[0123] "Heterocyclyl" refers to a stable 3- to 18-membered non-aromatic ring radical containing 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specifically stated in the specification, a heterocyclyl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system which may include fused or bridged ring systems. The heteroatoms in a heterocyclyl radical may be optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heterocyclyl radical is partially or fully saturated. The heterocyclyl may be attached to the remainder of the molecule through any atom of the ring(s). Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.

[0124] "Heteroaryl" refers to a radical derived from a 3-18 membered aromatic ring radical containing 2-17 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, a heteroaryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, where at least one ring in the ring system is fully unsaturated, i.e., contains a cyclic, delocalized (4n+2) pi-electron system according to the Hückel theory. Heteroaryl includes fused or bridged ring systems. The heteroatom(s) in the heteroaryl radical are optionally oxidized. The nitrogen atom(s), if present, are optionally quaternized. The heteroaryl is attached to the remainder of the molecule through any atom of the ring(s). Examples of heteroaryl include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzoindolyl, 1,3-benzodioxolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]iphenyl, and the like. midazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-Hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2- Oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyri pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohexyl These include thieno[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl, and thiophenyl (i.e., thienyl).

[0125] As used herein, the term "solid support" includes solid phase supports for the synthesis of oligonucleotides, such as CPGs.

[0126] As used herein, the terms "oligonucleotide" and "oligonucleotides" refer to a compound that contains multiple linked nucleosides. In certain embodiments, an "oligonucleotide" is a short single- or double-stranded DNA or RNA molecule, including antisense oligonucleotides (ASO), RNA interference (RNAi), and aptamer RNA. In certain embodiments, one or more of the multiple nucleosides are modified. In certain embodiments, the oligonucleotide contains one or more ribonucleosides (in the case of RNA) and / or deoxyribonucleosides (in the case of DNA). In some embodiments, the oligonucleotide is a single-stranded oligonucleotide. In some other embodiments, the oligonucleotide is a double-stranded interfering RNA, such as siRNA, aiRNA, shRNA, etc. In some embodiments, the oligonucleotide is a circRNA. In some embodiments, the oligonucleotide is an mRNA.

[0127] As used herein, the term "aiRNA" refers to an asymmetric interfering RNA duplex molecule that includes an antisense strand and a sense strand, the antisense strand being longer than the sense strand, consisting of 19-27 nucleotides, with at least one nucleotide 3' overhang, and 0-8 nucleotides at the 5' end; the antisense strand being at least 70% complementary to the target mRNA; the sense strand being 10-26 nucleotides, forming a double-stranded region with the antisense strand, the double-stranded region containing 0, 1 or 2 mismatch pair(s). Exemplary structures of aiRNA are described in US2009 / 0208564, which is incorporated by reference in its entirety.

[0128] As used herein, the term "intermediate-type" refers to an interfering RNA duplex molecule that includes an antisense strand and a sense strand, where the antisense strand is longer than the sense strand and includes both a 3' and a 5' overhang of at least one nucleotide.

[0129] As used herein, the term "blunt" refers to an interfering RNA duplex molecule that includes an antisense strand and a sense strand, and in which the RNA duplex molecule has at least one blunt end, preferably at the 3' end of the sense strand or the 5' end of the antisense strand.

[0130] As used herein, the term "modified oligonucleotide" means an oligonucleotide that contains at least one modified nucleotide.

[0131] As used herein, the term "modified nucleotide" means a nucleotide having at least one modified sugar moiety, modified internucleoside linkage, and / or modified nucleobase.

[0132] As used herein, the term "modified nucleoside" means a nucleoside having at least one modified sugar moiety and / or a modified nucleobase.

[0133] As used herein, the term "naturally occurring internucleoside linkage" means a 3'-5' phosphodiester linkage.

[0134] As used herein, the term "modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleotide bond. For example, a phosphorothioate linkage is a modified internucleotide linkage.

[0135] As used herein, the term "natural sugar moiety" means a sugar found in DNA (2-H) or RNA (2-OH).

[0136] As used herein, the term "modified sugar" refers to a substitution or change from a natural sugar. For example, a 2'-O-methoxyethyl modified sugar is a modified sugar.

[0137] As used herein, the term "bicyclic sugar" means a furosyl ring modified by bridging two non-geminal ring atoms. A bicyclic sugar is a modified sugar.

[0138] As used herein, the term "modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. For example, 5-methylcytosine is a modified nucleobase. "Unmodified nucleobase" refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0139] As used herein, "prevention" and "preventing" are used interchangeably. These terms refer to an approach to obtain advantageous or desired results, including, but not limited to, preventive benefits. In "preventive benefits," the conjugate or composition can be administered to a patient who is at risk of developing a particular disease, or to a patient who reports one or more of the physiological symptoms of the disease, even if the disease has not been diagnosed.

[0140] As used herein, the term "effective amount" of an active agent refers to an amount sufficient to induce a desired biological response.As will be appreciated by those skilled in the art, the effective amount of the compound of the present invention can vary depending on factors such as the desired biological end point, the pharmacokinetics of the compound, the disease to be treated, the mode of administration, and the patient.

[0141] As used herein, the term "treatment" of a disease or disorder or "treating" it refers to a method of alleviating, delaying, or ameliorating such a condition before or after its occurrence. Treatment may target one or more effects or symptoms of the disease and / or the underlying pathology. Treatment may be any alleviation, including but not limited to, complete disappearance of the disease or symptoms of the disease. When compared to an equivalent untreated control, the degree of such alleviation or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%, as measured by any standard technique.

[0142] As used herein, the term "subject" refers to any animal (e.g., mammal), including, but not limited to, humans, non-human primates, rodents, etc., that can be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably. As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of dsRNA and a pharma- ceutically acceptable carrier. As used herein, a "pharmacologically effective amount," "therapeutically effective amount," or simply an "effective amount" refers to an amount of RNA that is effective to produce the intended pharmacological, therapeutic, or prophylactic result. For example, if a given clinical treatment is deemed effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, then the therapeutically effective amount of a drug for treating that disease or disorder is the amount required to produce at least a 25% reduction in that parameter.

[0143] The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include pharmaceutically acceptable excipients, such as, but are not limited to, inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents, and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, while lubricants, if present, are generally magnesium stearate, stearic acid, or talc. If desired, tablets can be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.nce in human subjects.

[0144] Compound configuration

[0145] The compounds of the present invention, and salts thereof, may exist in their tautomeric form (for example, as an amide or imino ether). All such tautomeric forms are contemplated herein as part of the present invention.

[0146] All stereoisomers of the compounds of the present invention, including enantiomeric and diastereomeric forms (e.g., those that may exist due to asymmetric carbons on various substituents), are contemplated within the scope of the present invention. Individual stereoisomers of the compounds of the present invention may, for example, be substantially free of other isomers (e.g., as pure or substantially pure optical isomers with the specified activity), or may, for example, be racemic or admixed with all other or other selected stereoisomers. The chiral centers of the present invention may have the S or R configuration as defined by the IUPAC 1974 Recommendations. Racemic forms can be resolved by physical methods, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. Individual optical isomers can be obtained from racemates by any suitable method, including, but not limited to, conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization.

[0147] The compounds of the present invention are preferably isolated and purified after their preparation to obtain compositions containing an amount by weight equal to or greater than 95% (e.g., "substantially pure" Compound I), which are then used or formulated as described herein. In certain embodiments, the compounds of the present invention are greater than 99% pure.

[0148] All configurational isomers of the compounds of the present invention are contemplated, either in admixture or in pure or substantially pure form. The definition of the compounds of the present invention includes both cis (Z) and trans (E) alkene isomers, as well as cis and trans isomers of cyclic hydrocarbon or heterocyclic rings.

[0149] D-Amino Acids / L-Amino Acids

[0150] It is understood that the amino acids contained within the peptides or polypeptides described herein are of either the L- or D-configuration. II. Embodiment

[0151] The present invention provides a novel compound, comprising a novel linker composition for linking various components of the novel compound.The compound conjugates oligonucleotide with one or more targeting ligands.The oligonucleotide may be naturally occurring (isolated from nature or synthesized in laboratory) or chemically modified in at least one subunit.

[0152] In some embodiments, the oligonucleotide is a chemically modified oligonucleotide. In some embodiments, the chemically modified oligonucleotide comprises a backbone modification (or an internucleoside linkage modification, e.g., a phosphate group modification), a ribose group modification, or a base modification.

[0153] In certain embodiments, the oligonucleotide comprises at least one phosphorothioate internucleoside linkage, or at least one methylphosphonate internucleoside linkage, or at least one other modified internucleoside linkage, such as: [ka] has.

[0154] In certain embodiments, the oligonucleotide has at least one chemically modified nucleotide that includes a ribose modification. In certain embodiments, the 2' position of the modified ribose moiety is replaced with a group selected from OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where each R is independently C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. In some embodiments, the 2' position of the modified ribose moiety is replaced with a group selected from allyl, amino, azido, thio, O-allyl, O-C1-C6 alkyl, alkenyl, or alkynyl. 10 Alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), O-CH2-C(=O)-N(R m )(R n), or O-CH2-C(=O)-N(R1)-(CH2)2-N(R m )(R n ) wherein each R l , R m and R n are independently H or substituted or unsubstituted C1-C 10 In some embodiments, the modified ribose moiety is selected from the group of 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, 2'-OCH2CH3, 2'-OCH2CH2F and 2'-O(CH2)2OCH3 substituents. In some embodiments, the modified ribose moiety is substituted with a bicyclic sugar selected from the group of 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (cEt) and 4'-CH(CHOCH3)-O-2', 4'-C(CH3)(CH3)-O-2', 4'-CH2-N(OCH3)-2', 4'-CH2-ON(CH3)-2', 4'-CH2-N(R)-O-2', where R is H, C1-C12 alkyl, or a protecting group, 4'-CH2-C(H)(CH3)-2', and 4'-CH2-C-(=CH2)-2'. In some embodiments, the modified sugar moiety is selected from the group of 2'-O-methoxyethyl modified sugars (MOE), 4'-(CH2)-O-2' bicyclic sugars (LNA), 2'-deoxy-2'-fluoroarabinose (FANA), and methyl(methyleneoxy) (4'-CH(CH3)-O-2) bicyclic sugars (cEt). In certain embodiments, the oligonucleotide has a chemically modified nucleotide selected from the group consisting of 2'-methoxyethyl, 2'-OCH3, and 2'-fluoro.

[0155] The oligonucleotide may be conjugated to the remainder of the compound, or "backbone," at the 5' and / or 3' ends of the oligonucleotide. The conjugated oligonucleotide may be delivered as a single strand or may be hybridized to a substantially complementary oligonucleotide as part of a duplex. The substantially complementary oligonucleotide may or may not be conjugated in turn.

[0156] In one embodiment, the conjugated oligonucleotide forms part of an siRNA duplex (either the sense or antisense strand, or both). In a preferred embodiment, the conjugated oligonucleotide forms part of an aiRNA duplex (either the sense or antisense strand, or both). In another embodiment, the oligonucleotide conjugated according to the principles of the present invention is used as an antisense oligonucleotide (ASO). In yet another embodiment, the oligonucleotide conjugated according to the principles of the present invention is used as a microRNA (miRNA) molecule. An exemplary embodiment of a portion of a conjugated oligonucleotide as shown in Figure 1. In a duplex RNA molecule, preferably, the oligonucleotide may be conjugated at the 3' end of the sense strand to the remainder of the compound, or "backbone".

[0157] EMBODIMENT 1

[0158] In a first aspect, the oligonucleotide is conjugated to a backbone containing multiple moieties including termini, where clusters of more than one, e.g., 2-8 and preferably 3, ligands (e.g., GalNAc) are attached to the backbone directly or via one or more intermediate linkers at attachment points provided by residues derived from histidine residues.

[0159] In one embodiment, the compounds of the present invention have the structural formula as shown in (G-H1), (G-H1-01) to (G-H1-11).

[0160] In one embodiment, the compounds of the invention have the structures as shown in HC-1 through HC-9.

[0161] In one embodiment, where appropriate, the configuration of the compound is an R isomer or a mixture thereof. In one embodiment, the configuration of the compound refers to isomers of the chiral carbon atoms shown in the structural formula.

[0162] In the compounds of the invention, said naturally occurring or chemically modified oligonucleotide is linked via its 5'-terminus and / or its 3'-terminus to the remainder of the compound.

[0163] EMBODIMENT 2

[0164] In a second aspect, the oligonucleotide is conjugated to a backbone containing multiple moieties including termini, where clusters of more than one, e.g., 2-8 and preferably 3, ligands (e.g., GalNAc) are attached to the backbone directly or through one or more intermediate linkers at attachment points provided by moieties derived from glutamic acid residues.

[0165] In one embodiment, the compounds of the present invention have the structural formula as shown in (G-G1), (G-G1-01) through (G-G1-10).

[0166] In one embodiment, the compounds of the invention have structures as shown in GC-1 through GC-9.

[0167] In one embodiment, the configuration of the compound is an R isomer or racemate, as appropriate. In one embodiment, the configuration of the compound refers to isomers of the chiral carbon atoms shown in the structural formula. EXAMPLES

[0168] III. Examples synthesis In some embodiments, the compounds shown in formulas G-H1, G-H1-01 through G-H1-11, G-G1, G-G1-01 through G-H1-10, which include oligonucleotides conjugated to a backbone containing multiple moieties including termini, and clusters of more than one, e.g., 2-8 ligands (e.g., GalNAc), are synthesized by reacting a cluster backbone containing three or more reactive moieties with the ligands and oligonucleotides. Example 1 Synthesis of GC-05 (Glu(R)-cluster GalNAc)

[0169] The synthesis of GC-05 (Glu(R)-cluster GalNAc) is shown below in 5 steps:

[0170] Step 1: Synthetic route to intermediate N-2. [ka]

[0171] 30 g of compound N-1 was dissolved in 420 mL of 2M NaOH, and 30 mL of THF was added. The mixture was cooled to 0-5°C in an ice bath, and 35 g of CbzCl was added by slow dropwise addition. After the addition was completed, the mixture was stirred at room temperature for 1 hour, and the completed reaction was monitored by UPLC-MS (ultra performance liquid chromatography-mass spectrometry). The reaction solution was washed with MTBE (200 mL x 3), the aqueous phase was separated, EA was added, and after cooling, the pH was adjusted to 4 with 3M HCl, the EA phase was extracted, separated, and the aqueous phase was extracted again with 300 mL of EA, and the organic phases were combined, washed three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to give 51.58 g of compound N-2. MS (ESI) m / z 278.05 ([MH] - ).

[0172] Step 2: Synthetic route to intermediate M-3. [ka]

[0173] Synthesis of compound M-2

[0174] Under nitrogen atmosphere, 20 g of M-1 ((R)-3-hydroxy-methylbutyrate) was dissolved in 200 mL of DCM, 18 g of imidazole was added, and 56.2 g of TBDPSCl was added dropwise. After the addition was complete, the reaction was allowed to proceed for 2 hours at room temperature. TLC was used to monitor the reaction for completion. It was quenched by adding 200 mL of saturated ammonium chloride, the DCM layer was separated, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated and dried to give 73 g of crude M-2. MS(ESI) m / z 357.10 ([M+H] + )

[0175] Synthesis of compound M-3

[0176] An aqueous solution of sodium hydroxide was prepared by dissolving 13.6 g of sodium hydroxide in 80 mL of water, and 73 g of the M-2 crude product was dissolved in 500 mL of methanol, and then the aqueous solution of sodium hydroxide was added. After stirring at 35 °C for 15 h, the reaction was monitored for completion using TLC. Methanol was removed by concentration at 40 °C, 500 mL of ethyl acetate was added, the pH was adjusted by 2 M HCl, the EA layer was separated, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (PE:EA = 50:1 to 10:1), and 47.8 g of M-3 was obtained from the concentrated product. MS (ESI) m / z 341.10 ([MH] - ). 1 H-NMR(400MHz,CDCl3) δ7.66-7.69(m, 4H), 7.35-7.45(m,6H), 4.23-4.30(m,1H), 2.43-2.56(m, 2H), 1.14(d, 3H), 8.77(s, 9H).

[0177] Step 3: Synthetic route to intermediate S-04. [ka] [ka]

[0178] Synthesis of compound S-02

[0179] Under nitrogen atmosphere, 250g of compound S-01 was dissolved in 2L of anhydrous DCM, and 158g of TMSOTf was added by dropwise addition under ice bath. After the addition was completed, the temperature was raised to 40°C and stirred for 2 hours, and the reaction was completed by TLC monitoring. After cooling in an ice bath, 1.7L of saturated aqueous sodium bicarbonate solution was added dropwise, the organic phase was separated, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give 211g of yellow oily product S-02. ESI-MS m / z: [M+H] + =430.12

[0180] Synthesis of compound S-03

[0181] Under nitrogen atmosphere, 169g of compound S-02 was dissolved in 1.2L of anhydrous DCM, 56.6g of 5-hexene-1-alcohol was added, 57.38g of TMSOTf was added dropwise in ice-water bath, and after the addition was completed, the reaction was carried out at room temperature overnight. The completed reaction was monitored by TLC, cooled in ice-water bath, and saturated NaHCO3 was added dropwise to adjust the pH to 7-8, and then the organic phase was extracted and separated. The organic phase was washed once with saturated NaCl, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness, and purified by column chromatography, gradient eluted with DCM:MeOH=80:1-40:1, and the product eluate was collected and concentrated to dryness to obtain 94g of oily product S-03. ESI-MS m / z:[M+H] + =430.23.

[0182] Synthesis of compound S-04

[0183] Compound S-03 82g was weighed, acetonitrile 410mL, DCM 410mL and H2O 573mL were added, stirred for dissolution, cooled to 5-10℃ in ice bath, NaIO4 163g and RuCl3 2.37g were added to the reaction system. After stirring at room temperature for 2h, the reaction was completed and monitored by TLC. The reaction solution was filtered through Celite, the filtrate was combined, the aqueous phase was extracted and separated. The organic phase was washed three times with saturated NaHSO3 (200mL×3). The aqueous phase was combined, DCM 600mL was added, pH was adjusted to 2-3 with 2M HCl, DCM phase was extracted, separated, dried over anhydrous sodium sulfate, filtered and concentrated to give grey solid compound S-04 76g, ESI-MS m / z: [MH] - =446.17, 1H -NMR (400 MHz, DMSO-d6): d 11.97 (s, 1H, COOH); 7.79 (d, 1H, NH); 5.20 (d, 1H), 4.95 (dd, 1H); 4.48 (d, 1H); 4.05- 3.98 (m, 3.86 (dt, 1H); 3.74-3.65 (m, 1H, -OCH2-CH2); 3.45-3.37 (m, 1H, -OCH2-CH2); 2.19 (t, 2H, -CH2-COOH); 2.09 (s, 3H, -COCH3); 1.99 (s, 3H, -COCH3); 1.88 (s, 3H, -COCH3); 1.76 (s, 3H, -COCH3); 1.55-1.45 (m, 4H, 2x(-CH2)).

[0184] Step 4: Synthetic route to intermediate G-6. [ka]

[0185] Synthesis of compound G-2

[0186] Compound G-1 ((S)-N-Boc-glutamic acid methyl ester) 50g was dissolved in THF 500mL, NMM 25.2mL was added dropwise in ice water bath, and after stirring for 5 minutes, isobutyl chloroformate 26.6mL was added dropwise. After the addition was completed, it was stirred for 1 hour. Suction filtration was performed, the filtrate was collected, and NaBH 48.73g was added to the filtrate under ice water bath. After the addition was completed, the reaction was continued for 2 hours, a sample was taken to monitor the reaction for completion by TLC, water 250mL was added, and EA 500mL was added for extraction. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to obtain oily product G-2 45.35g. MS(ESI)m / z 248.19([M+H] + ).

[0187] Synthesis of compound G-3

[0188] Compound G-2 45.35g was dissolved in DCM 500mL, imidazole 31.2g was added, and TBDPSCl 91.1g was introduced by dropwise addition. After the dropwise addition was completed, the mixture was stirred at room temperature for 2 hours, and the completed reaction was monitored by TLC. Water 150mL was added to the reaction solution, and the DCM layer was extracted, separated, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to obtain an oily product. It was purified by silica gel column chromatography, gradient eluted with PE:EA=40:1 to 10:1, and concentrated to obtain a colorless and transparent oily product G-3 36.7g. MS(ESI)m / z 486.66([M+H] + ).

[0189] Synthesis of compound G-4

[0190] Compound G-3 46.15g was dissolved in DCM 500mL, TFA (trifluoroacetic acid) 70mL was added dropwise in ice water bath. After the addition was completed, the reaction was stirred at room temperature for 4 hours. The reaction was monitored by TLC, concentrated under reduced pressure, DCM 500mL was added dropwise to the residue, saturated sodium bicarbonate solution was added dropwise to adjust the pH to 8, the DCM layer was extracted, separated, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to dryness to give pale yellow oil G-4 36.7g. MS(ESI)m / z 386.51([M+H] + ).

[0191] Synthesis of compound G-5

[0192] Compound M-3 (31.9 g) was dissolved in DMF (300 mL), HBTU (42 g) and DIEA (23 mL) were added, stirred at room temperature for 10 min, then compound G-4 (35 g) was added, transferred to room temperature and stirred for 1 h. TLC was used to monitor the completion of the reaction. The reaction solution was extracted with saturated sodium bicarbonate (600 mL) and EA (400 mL). The EA layer was separated, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give an oil. The crude residue was purified by column chromatography (PE:EA=20:1 to 8:1) to produce white solid product G-5 (41 g). MS (ESI), m / z 710.33 ([M+H] + ).

[0193] Synthesis of compound G-6

[0194] Compound G-5 (33.3 g) was added to a mixture of MeOH (100 mL), THF (100 mL) and water (100 mL), lithium hydroxide monohydrate (5.92 g) was added, and reacted at room temperature for 8 hours. The reaction was monitored by TLC, and the reaction mixture was concentrated under reduced pressure, and then ethyl acetate (400 mL) was added to the concentrated residue, and the pH was adjusted to 4-5 using dilute hydrochloric acid solution, extracted, and the organic phase was separated, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain colorless, transparent, oily product G-6 (33.8 g). MS(ESI)m / z:694.35([M+H] + ).

[0195] Step 5: Synthetic route to compound GC-05. [ka] [ka] [ka] [ka]

[0196] Synthesis of compound HC-02

[0197] Compound HC-01 (trimethylolaminomethane) 100g was dissolved in DMSO 250mL, then 5M NaOH 16.5mL was added. In an ice-water bath, tert-butyl acrylate 400g was slowly introduced dropwise into the mixture. After the addition was completed, the reaction was completed after stirring at room temperature for 18 hours. Saturated sodium chloride solution 2L and ethyl acetate 500mL were added to the reaction solution, and the organic phase was separated after stirring. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain crude compound HC-02. It was purified by silica gel column chromatography and gradient eluted with petroleum ether:ethyl acetate=10:1 to 1:1. The product eluate was collected and concentrated to dryness to obtain compound HC-02 260g. MS(ESI)m / z 506.35([M+H] + ).

[0198] Synthesis of compound HC-03

[0199] 23.24g of intermediate N-2 was dissolved in 200mL of DMF, then 16g of DIPEA and 38g of HBTU were added, and after stirring at room temperature for 20 minutes, 50.47g of compound HC-02 dissolved in 50mL of DMF was slowly added dropwise. After the addition was completed, the mixture was stirred at room temperature for 1 hour, and the reaction was monitored by TLC for completion. 600mL of saturated sodium bicarbonate was added to the reaction solution, extracted with 300mL of ethyl acetate, the organic phase was separated, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography, gradient eluted with petroleum ether:ethyl acetate=8:1 to 4:1. The product eluate was collected and concentrated to dryness to give 43g of light yellow oil. MS(ESI)m / z 767.51([M+H] + ).

[0200] Synthesis of compound HC-04

[0201] 1280mL of formic acid was added to 160g of compound HC-03, stirred at room temperature for 6 hours, the reaction was monitored by TLC for completion, and the formic acid was evaporated under reduced pressure to obtain a light yellow oil. 300mL of toluene was added to the residue, and the toluene was evaporated under reduced pressure to obtain 111.85g of oily compound HC-04. 1 HNMR (400MHz, DMSO-d6) δ12.18(brs,3H),7.37-7.30(m,5H),7.20(t,1H),7.92(s,1H),5.00(s,2H),3.58-3.55(m,12H),2.97(q,2H),2.37-2.47(m, 6H),2.04(t,2H),1.46-1.36(m,4H),1.17-1.29(m,4H).MS(ESI)m / z 597.29([MH] - )

[0202] Synthesis of compound HC-05

[0203] Compound HC-04 110.72g was dissolved in DMF 800mL, DIPEA 107.48g and HBTU 253.28g were added. After the mixture was stirred at room temperature for 20 minutes, tert-butyl (3-aminopropyl) carbamate 101.3g dissolved in DMF 200mL was slowly introduced by dropwise addition. After the dropwise addition was completed, the mixture was stirred at room temperature for 1 hour, and the completed reaction was monitored by TLC. Saturated sodium bicarbonate aqueous solution 2L was added to the reaction solution, extracted with ethyl acetate 800mL, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography with gradient elution of dichloromethane:methanol = 80:1 to 20:1. The product eluate was collected and concentrated to dryness to obtain oily compound HC-05 220g. MS (ESI) m / z 1067.70 ([M+H] + ).

[0204] Synthesis of compound HC-06

[0205] Compound HC-05 (79.68 g) was dissolved in DCM (880 mL), cooled in an ice-water bath, and trifluoroacetic acid (190 g) was added dropwise. After the addition was complete, the mixture was stirred at room temperature for 4 h and the reaction was monitored for completion by UPLC-MS. DCM was evaporated under reduced pressure, toluene (600 mL) was added, and concentrated to dryness to give oily compound HC-06 (121.6 g). MS(ESI) m / z 767.61 ([M+H] + ).

[0206] Synthesis of compound HC-07

[0207] Compound S-04 90g was dissolved in DMF 265mL, DIPEA 72g was added, HBTU 52g was added, and compound HC-06 62g was dissolved in DMF 250mL, and after stirring for 20 minutes, DIPEA 72g was slowly added dropwise. Saturated sodium bicarbonate 1000mL was added to the reaction solution, ethyl acetate 500mL was added, the ethyl acetate layer was extracted, separated, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography, gradient eluted with dichloromethane:methanol = 50:1 to 10:1. The product eluate was collected and concentrated to dryness to obtain compound HC-07 75g as a white solid. MS (ESI) m / z 1028.46 ([(M+2) / 2] + ).

[0208] Synthesis of compound HC-08

[0209] Compound HC-07 (10 g) was dissolved in methanol (80 mL), 10% Pd / C (1 g) was added, and the air was replaced with hydrogen three times and stirred at room temperature for 8 hours. Pd / C was filtered off. The filtrate was concentrated and dried to give compound HC-08 (8.5 g) as an off-white solid. MS(ESI) m / z 961.37([(M+2) / 2] + ).

[0210] Synthesis of compound GC-01

[0211] 2.0g of intermediate G-6 was dissolved in 30mL of DMF, 1.41g of HBTU and 557mg of DIEA were added, stirred at room temperature for 20 minutes under nitrogen atmosphere, and 6.62g of compound HC-08 was added. After stirring at room temperature for 1 hour, a sample was taken to monitor the reaction for completion by TLC. 200mL of saturated sodium bicarbonate and 100mL of ethyl acetate were added to the reaction, and the organic phase was extracted and separated. The organic phase was washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography with gradient elution from DCM:MeOH=30:1 to 10:1. The product eluate was collected and concentrated to dryness to give 4.93g of compound GC-1 as a white solid. MS(ESI)m / z 866.72([(M+3) / 3] + ).

[0212] Synthesis of compound GC-02

[0213] Compound GC-01 (20.4 g) was dissolved in THF (200 mL), 1M TBAF-THF (tetrabutylammonium fluoride) solution (25 mL) was added, and the mixture was stirred at room temperature overnight. The reaction solution was concentrated and purified by column chromatography, eluted with gradient DCM:MeOH=50:1 to 10:1, and the product eluate was concentrated to obtain compound GC-02 (10.39 g) as a white solid. MS(ESI)m / z 1061.55([(M+2) / 2] + ).

[0214] Synthesis of compound GC-03

[0215] Under nitrogen atmosphere, 5.14g of compound GC-02 was dissolved in 40mL of anhydrous pyridine, and 4.43g of DMTrCl was added. After 0.5h of reaction at room temperature, a sample was taken to monitor the reaction for completion, and 10mL of methanol was added to quench the reaction. The reaction solution was concentrated and purified by column chromatography, eluting with gradient DCM:MeOH=50:1 to 10:1 to obtain 4.54g of compound GC-3 as a white solid. MS(ESI)m / z 1061.59([(M-302)+2) / 2] + ).

[0216] Synthesis of compound GC-04

[0217] Under nitrogen atmosphere, 3.5 g of compound GC-03 was dissolved in 30 mL of anhydrous pyridine, 10 mg of DMAP and 1.57 g of succinic anhydride were added, the mixture was stirred at room temperature for 72 h, and the completed reaction was monitored by TLC. The pyridine was concentrated to give the crude product, which was purified by silica gel column chromatography, gradient eluted with DCM:MeOH=50:1 to 10:1, and the product eluate was concentrated to give 3.61 g of compound GC-04 as a white solid. MS(ESI)m / z 1111.50([(M-302)+2) / 2] + ). 1 HNMR(400MHz,DMSO-d6) δ 7.89-7.82(m, 6H), 7.76-7.74(m, 4H), 7.38-7.36(m, 2H), 7.31-7.27(m, 2H), 7.24-7.16(m, 5H), 6.99(s, 1H), 6.88-6.81(m, 4H), 5.21(d, 2H), 4.98-4.95 (m, 3H), 4.50-4.47(m, 3H), 4.04-4.01(m, 10H), 3.91-3.86(m, 6H), 3.73-3.69(m, 12H), 3.54-3.52(m, 12H), 3.41-3.39(m, 4H), 3.04-3.02(m, 12H), 2.68-2.63(m, 9H), 2.29-2.26(m, 8H), 2.10(s, 9H), 2.06-2.02(m, 8H), 1.99(s, 9H), 1.89(s, 9H), 1.77(s, 9H), 1.52-1.44(m, 18H), 1.11(d, 3H).

[0218] Synthesis of compound GC-05

[0219] 246mg of compound GC-04 was dissolved in 10mL of anhydrous acetonitrile, 46mg of HATU and 27mg of DIPEA were added, and the mixture was shaken in a shaker for 10 minutes. 800mg of LCAA-CPG (100μmol / g) was added to the above reaction solution and shaken at room temperature overnight. On the second day, the reaction solution was filtered and the filter cake was washed with acetonitrile. 5mL of CapA (20%NMI-80%ACN) and 5mL of CapB (20%AC2O-30%lutidine-50%CAN) were added to the filter cake and shaken sequentially in a shaker at room temperature for 2 hours. The reaction solution was filtered, the filter cake was washed with acetonitrile, and dried under vacuum at room temperature for 2 hours to obtain 780mg of compound HC-13 with a measured loading of 45μmol / g. Example 2 Synthesis of HC-13 (His(R)-cluster GalNAc)

[0220] The synthesis of HC-13 (His(R)-cluster GalNAc) was show below (2 steps):

[0221] Step 1: Synthetic route to compound H-09 [ka]

[0222] Synthesis of compound H-02

[0223] 38.4 g of sodium hydroxide was dissolved in 400 mL of water, and 400 mL of THF was added. The mixture was cooled in an ice-water bath, and 50 g of LH-01 (histidine) was added to it and dissolved by stirring. 175 g of di-tert-butyl dicarbonate was added and stirred at room temperature for 4 hours. After the reaction was completed as monitored by TLC, the reaction mixture was filtered and concentrated under reduced pressure. 200 mL of MTBE was added to the concentrated residue, and washing and extraction were performed three times. The aqueous layer was separated, 500 mL of ethyl acetate was added, and the pH was adjusted to 2-4 with 3 M hydrochloric acid. The EA layer was separated, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and dried to give 98 g of a white solid. MS(ESI)m / z 356.25([M+H] + ).

[0224] Synthesis of compound H-03

[0225] Compound H-02 (110 g) was dissolved in anhydrous THF (880 mL) under nitrogen atmosphere. After cooling to 0-5°C in an ice bath, 1.1 L of borane tetrahydrofuran solution (1 M) was slowly added dropwise to the reaction solution and stirred at room temperature for 1 h. The reaction was monitored thoroughly by TLC. After the temperature of the reaction mixture was cooled to 0-10°C in an ice bath, the reaction was quenched by adding methanol (230 mL) dropwise. THF was concentrated, and ethyl acetate (800 mL) and saturated sodium chloride solution (300 mL) were added to the residue for extraction, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and dried to obtain compound H-03 crude product (106 g). MS (ESI) m / z 342.40 ([M+H] + ), 1 H-NMR(400MHz,DMSO-d6) δ 8.69(s,1H), 7.37(s,1H), 6.68(d,1H), 4.82(s,1H), 3.68-3.82(m,1H), 3.29-3.43(m,2H), 2.89(dd,1H), 2.54(d,1H), 1.57(s,9H),1.34(s,9H).

[0226] Synthesis of compound H-04

[0227] Compound H-03 (106g) was dissolved in dichloromethane (1L), imidazole (38g) was added, and TBDPSCl (128g) was added dropwise to the reaction solution. After the dropwise addition, the reaction was allowed to react at room temperature for 1 hour. TLC was used to monitor the completion of the reaction. Saturated sodium chloride (400mL) was added to the reaction solution, the DCM layer was extracted, washed with saturated sodium chloride, dried with sodium sulfate, concentrated, and purified by silica gel column chromatography with gradient elution, (petroleum ether:ethyl acetate=100:1 to 20:1). The product eluate was collected, condensed, and dried to obtain 106g of a white solid. 1 H-NMR (400MHz, DMSO-d6) δ 8.71(s, 1H), 7.63-7.65(m, 4H), 7.41-7.48(m,6H), 6.85(d, 1H), 3.91-4.02(m,1H), 3.61(d, 2H), 3.04(dd, 1H), 2.57-2.63(m, 1H), 1.58(s, 9H), 1.35(s,9H), 1.01(s, 9H).

[0228] Synthesis of compound H-05

[0229] Compound H-04 (80 g) was dissolved in glacial acetic acid (240 mL) and stirred at 80° C. overnight. After the reaction was completed as monitored by TLC, the mixture was cooled in an ice-water bath, and then ethyl acetate (400 mL) was added, and 4 M sodium hydroxide was added dropwise to adjust the pH to 8-9. The organic phase was separated, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, condensed, and dried to give a white solid (66 g). MS(ESI) m / z 480.27 ([M+H] + ).

[0230] Synthesis of compound H-06

[0231] Under nitrogen atmosphere, 37.8 g of compound H-05 was dissolved in 250 mL of DMF, 33.4 g of cesium carbonate was added, and the mixture was stirred at room temperature for 30 min. 14.5 g of methyl bromoacetate dissolved in 40 mL of DMF was added dropwise to the solution. After the dropwise addition, the mixture was stirred at room temperature for 0.5 h, and the reaction was monitored to be essentially complete using UPLC-MS. 300 mL of EA and 250 mL of saturated ammonium chloride were added to the reaction solution, and the EA layer was separated, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography with gradient elution (DCM:MeOH=100:1 to 50:1). The product eluent was collected, condensed, and dried to give 35 g of solid. MS(ESI) m / z 552.36 ([M+H] + ).

[0232] Synthesis of compound H-07

[0233] Compound H-06 (26 g) was dissolved in DCM (260 mL), the solution was cooled in an ice-water bath, and trifluoroacetic acid (45 mL) was added dropwise. After the addition, the reaction was allowed to warm to room temperature and stirred for 1 h, and the reaction was monitored for completion using UPLC-MS. TFA was concentrated, DCM (200 mL) was added to the residue, and saturated sodium bicarbonate was added dropwise to adjust the pH to 8-9. The DCM layer was separated, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and dried to give an oil (21.89 g). MS(ESI) m / z 452.30 ([M+H] + ).

[0234] Synthesis of compound H-08

[0235] Compound M-3 (17.53 g) was dissolved in DMF (105 mL), HBTU (21.21 g) and DIEA (12.2 mL) were added under nitrogen atmosphere, and the mixture was stirred at room temperature for 30 minutes. Then, compound H-07 (21 g) in DMF solution (100 mL) was added dropwise in 20 minutes. The mixture was stirred at room temperature for 1 hour, and the obtained sample was checked by TLC to monitor the completion of the reaction. EA (200 mL) and saturated sodium chloride (500 mL) were added to the reaction solution, and the organic phase was separated, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography with gradient elution (PE:EA=10:1 to 3:1). The product eluent was collected, condensed, and dried to obtain 20.2 g of oily substance. MS(ESI)m / z 776.50([M+H] + ).

[0236] Synthesis of compound H-09

[0237] Compound H-08 (19.66 g) was dissolved in THF (50 mL), methanol (100 mL) was added, and the mixture was stirred at room temperature to dissolve. Lithium hydroxide (3.2 g) was added and stirred at room temperature for 2 hours. The reaction was monitored for completion using TLC. Methanol and THF were removed under reduced pressure. Ethyl acetate (100 mL) was added to the concentrated residue, and the pH was adjusted to 3-4 using 2M HCl. The mixture was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and dried to give compound H-09 (13 g) as a white solid. 1 H-NMR (400MHz, DMSO-d6) δ7.85(d, 1H), 7.52-7.65(m, 9H), 7.34-7.47(m, 12H), 6.77(s, 1H), 4.72(s, 2H), 4.20-4.24(m, 1H), 4.09-4.14(m, 1H), 3.53-3.59(m, 2H), 2.80(dd, 1H), 2.63(dd, 1H), 2.39(dd, 1H), 2.24(dd, 1H), 9.67(d, 21H).MS(ESI)m / z 762.40([M+H] + ).

[0238] Step 2: Synthetic route for compound HC-13 [ka] [ka] [ka] [ka]

[0239] The synthesis method of compounds HC-02 to HC-08 was the same as the synthesis route for GC-05 (Glu(R)-cluster GalNAc).

[0240] Synthesis of compound HC-09

[0241] Compound H-09 3.9g was dissolved in DMF 40mL, HBTU 2.92g and DIPEA 2g were added and stirred at room temperature for 15 minutes. Intermediate compound HC-08 11.5g was dissolved in DMF 100mL, slowly added to the above system by stirring and stirred at room temperature for 30 minutes. TLC was used to monitor the completion of the reaction. Ethyl acetate 100mL and saturated sodium bicarbonate 100mL were added to the reaction solution to extract the EA layer. The organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography (dichloromethane:methanol=50:1 to 10:1). The product eluent was collected and dried under reduced pressure to obtain 10.5g of white solid. MS(ESI)m / z 889.44([(M+3) / 3] + ).

[0242] Synthesis of compound HC-10

[0243] Compound HC-09 10.1g (s10.1 g) was dissolved in THF 150mL, TBAF (1M in THF) 11.4mL was added, and stirred overnight at room temperature. After the reaction was completed as monitored by LC-MS, the solvent was removed by distillation under reduced pressure, and the crude product was purified by silica gel column chromatography with gradient elution (methylene chloride:methanol=50:1 to 10:1). The product eluate was collected, condensed, and dried to give 7.53g of a white solid. MS(ESI) m / z 1094.94([(M+2) / 2] + ).

[0244] Synthesis of compound HC-11

[0245] 3.9 g of compound HC-10 was dissolved in 33 mL of anhydrous pyridine, and 1.03 g of DMTrCl was added to the solution by stirring. After stirring at room temperature for 10 min, the reaction was monitored for completion using TLC. The reaction was quenched with 12 mL of methanol, and the solvent was removed by distillation under reduced pressure. Purification was performed by silica gel column chromatography through wet packing with gradient elution (dichloromethane:methanol=50:1 to 20:1). The product eluent was collected, condensed and dried to give 3.62 g of a pale yellow solid. MS(ESI)m / z 1094.95([(M-302)+2) / 2] + ).

[0246] Synthesis of compound HC-12

[0247] 3.6 g of compound HC-11 was dissolved in 36 mL of anhydrous pyridine, 20 mg of DMAP and 2.9 g of succinic anhydride were added, and the mixture was stirred overnight at room temperature under nitrogen atmosphere. The reaction was monitored for completion using TLC. Pyridine was removed by distillation under reduced pressure, and the product was purified by silica gel column chromatography (dichloromethane:methanol=50:1 to 10:1). The product eluate was collected, condensed, and dried to give 2 g of a white solid. 1HNMR(400MHz,DMSO-d6) δ 8.02-7.92(m, 10H), 7.43-7.38(m, 3H), 7.31-7.20(m, 8H), 7.06(s, 1H), 6.88-6.86(m, 4H), 6.64(s, 1H), 5.20(d, 3H), 4.98(dd, 3H), 4.55-4.49(m, 5H), 4.20-4.15(m, 2H), 4.05-3.97(m, 8H), 3.87(q, 3H), 3.73(s, 6H), 3.68-3.73(m, 3H), 3.55-3.52(m, 12H), 3.41-3.39(m, 4H), 3.04-3.01(m, 14H), 2.90(d, 2H), 2.77-2.59(m, 4H), 2.30-2.26(m, 8H), 2.10(s, 9H), 2.06-2.03(m, 8H), 1.99(s, 9H), 1.88(s, 9H), 1.77(s, 9H), 1.52-1.43(m, 20H), 1.27-1.20(m, 9H), 1.11(d, 3H),MS(ESI)m / z 1145.05([(M-302)+2) / 2] + ).

[0248] Synthesis of compound HC-13

[0249] 236mg of compound HC-12 was dissolved in 10mL of acetonitrile, 49.6mg of HATU and 29.8mg of DIPEA were added, and the mixture was shaken in a shaker for 10 minutes. 800mg of LCAA-CPG (96μmol / g) was added to the reaction solution and shaken overnight. The next day, the reaction solution was filtered and the filter cake was washed with acetonitrile. 5mL of CapA (20%NMI-80%CAN) and 5mL of CapB (20%AC2O-30%Lutidine-50%CAN) were added to the filter cake. The mixture was kept shaking in a shaker for 2 hours at room temperature, the reaction solution was filtered, the filter cake was washed with acetonitrile, and dried under vacuum at room temperature for 2 hours to obtain 780mg of compound HC-13 with a loading of 46μmol / g. Example 3 Synthesis of B-9 (His(R)-cluster GalNAc)

[0250] The synthesis of B-9 (His(R)-cluster GalNAc) is shown below (2 steps):

[0251] Step 1: Synthetic route for compound Y-03 [ka]

[0252] Synthesis of compound Y-01

[0253] Under nitrogen atmosphere, compound S-02 169g was dissolved in anhydrous DCM 1.2L, 1-benzoxyethanol 83.6g was added, TMSOTf 57.38g was added dropwise, and the reaction was carried out at room temperature overnight. The reaction was monitored for completion using TLC, cooled in an ice-water bath, and the pH was adjusted to 7-8 by adding saturated NaHCO3 dropwise, and then the organic phase was extracted and separated. The organic phase was washed once with saturated NaCl, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated, dried, and purified by column chromatography to give compound Y-01 182g. MS(ESI)m / z 482.16([M+H] + ).

[0254] Synthesis of compound Y-02

[0255] Compound Y-01 (80 g) was dissolved in methanol (600 mL) and 10% Pd / C (8 g) was added. After replacing the air with hydrogen three times, the reaction was completed with stirring at room temperature for 10 h. Palladium on carbon was filtered off and the filtrate was concentrated and dried to give a grey solid (59.86 g). MS(ESI) m / z 392.19 ([M+H] + ).

[0256] Synthesis of compound Y-03

[0257] Under nitrogen atmosphere, compound Y-02 (50.0 g) was dissolved in 500 mL of anhydrous CHCl, then 45 mL of DIPEA was added, and 40 g of N,N-diisopropyl chlorophosphoride was added, and the mixture was reacted at room temperature for 2 hours by stirring. The reaction was monitored for completion using TLC, and then concentrated and rapidly separated by column chromatography to obtain compound Y-03 46.02 g. MS(ESI)m / z 509.19([M+H] + ).

[0258] Step 2: Synthetic route to compound B-9 [ka] [ka] [ka] [ka]

[0259] Synthesis of Compound B-2

[0260] Compound SM-2 20.24g (purchased from PharmaBlock Sciences (Nanjing), Inc.) was dissolved in 200mL of DMF, then DIPEA 15.26g and HBTU 30.13g were added and stirred at room temperature for 20 minutes. Compound B-1 20.02g (purchased from PharmaBlock Sciences (Nanjing), Inc.) dissolved in DMF 50mL was added dropwise to the reaction solution. After the dropwise addition, the reaction was monitored for completion using TLC after stirring at room temperature for 1 hour. Saturated sodium bicarbonate 600mL was added to the reaction solution, then extracted with ethyl acetate 300mL, washed with saturated brine, dried with anhydrous sodium sulfate, concentrated, and purified by gradient elution (petroleum ether:ethyl acetate) on a silica gel column. The product eluate was collected, concentrated, and dried to obtain compound B-2 33.20g. MS (ESI) m / z 738.40 ([M+H] + ).

[0261] Synthesis of compound B-3

[0262] 33g of compound B-2 was dissolved in 400mL of 50% CH2Cl2 / acetonitrile, 180mL of activator (1M 4,5-dicyanimidazole / 1% N-methylimidazole) was added to the system, and 90g of Y-03 dissolved in 100mL of acetonitrile was added dropwise to the system. After stirring at room temperature for 30 minutes, 160mL of tert-butyl hydrogen peroxide / acetonitrile / water (10:87:3) was added to the reaction solution, and the completion of the reaction was monitored using TLC. The solvent was removed by distillation under reduced pressure and purified by silica gel column chromatography to obtain 77.45g of compound B-3. MS(ESI)m / z 1128.79([(M+2) / 2] + ).

[0263] Synthesis of compound B-4

[0264] Compound B-3 (60 g) was dissolved in methanol (600 mL) and 10% Pd / C (6 g) was added. After 3 cycles of hydrogen and stirring at room temperature for 8 hours, the reaction was complete. Palladium on carbon was filtered and the filtrate was concentrated and dried to give 51.54 g of a grey solid. MS(ESI)m / z MS(ESI)m / z 1061.74([(M+2) / 2] + ).

[0265] Synthesis of compound B-5

[0266] Compound H-09 16g was dissolved in DMF 160mL, HBTU 11.6g and DIPEA 5.9g were added and stirred at room temperature for 15 minutes. Intermediate compound B-4 46g was dissolved in DMF 400mL, then the solution was slowly added dropwise to the above system by stirring, stirred at room temperature for 30 minutes, and the completion of the reaction was monitored using UPLC-MS. Ethyl acetate 400mL and saturated sodium bicarbonate 400mL were added to the reaction solution, the EA layer was extracted, washed with saturated sodium chloride, dried with sodium sulfate, and purified by silica gel column chromatography. The product eluate was collected, condensed, and dried to obtain 42.1g of white solid. MS(ESI)m / z 956.20([(M+3) / 3] + ).

[0267] Synthesis of compound B-6

[0268] 40.0 g of intermediate compound B-5 was dissolved in 600 mL of anhydrous THF, and 45.6 mL of TBAF (1M in THF) was added at room temperature by stirring and stirred overnight. UPLC-MS was used to monitor the completion of the reaction. The solvent was removed under reduced pressure and the crude product was eluted by silica gel column chromatography with gradient elution (dichloromethane / methanol). The product eluent was collected, condensed and dried to give 23.10 g of a white solid. MS(ESI) m / z 797.28 ([(M+3) / 3] + ).

[0269] Synthesis of compound B-7

[0270] Under nitrogen atmosphere, compound B-6 (30.0 g) was dissolved in 300 mL of anhydrous pyridine, and 6.2 g of DMTrCl was added and stirred at room temperature for 30 min. The reaction was monitored for completion using TLC, concentrated, and separated by silica gel column chromatography to give a white crystalline solid (23.02 g). MS (ESI) m / z 797.38 ([(M-302+3) / 3] + ).

[0271] Synthesis of compound B-8

[0272] Under nitrogen atmosphere, 10 g of B-7 was dissolved in 100 mL of anhydrous pyridine, 100 mg of DMAP and 5.6 g of succinic anhydride were added, and the mixture was stirred at room temperature for 72 hours. After the reaction was completed, the pyridine was concentrated, and the product compound B-8 was purified by silica gel column chromatography to obtain 7.46 g. MS(ESI)m / z 830.33([(M-302+3) / 3] + ).

[0273] Synthesis of compound B-9

[0274] 3g of compound B-8 was dissolved in 30mL of acetonitrile, 650mg of HATU and 300mg of DIEA were added, and the mixture was shaken at room temperature for 10 minutes, and 10g of CPG-NH2 (96umol / g) was added. Then, the mixture was shaken overnight. After filtration and leaching with acetonitrile, 50mL of capA and 50mL of capB were added to the filter cake, and the mixture was shaken for 2 hours. After filtration and leaching with acetonitrile, the mixture was dried under vacuum for 3 hours, and 11.2g of compound B-9 was obtained with a loading of 45μmol / g. Example 4 Synthesis of SC-13 (His(R)-cluster GalNAc)

[0275] The synthesis of SC-13 (His(R)-cluster GalNAc) is shown below: [ka] [ka] [ka] [ka]

[0276] Synthesis of compound SC-02

[0277] The starting material was replaced with 3-amino-3-(2-hydroxyethyl)-1,5-pentanediol, and the synthesis method was the same as that of compound HC-02. MS(ESI) m / z 548.35 ([M+H] + ).

[0278] Synthesis of compound SC-03

[0279] Its synthesis method is the same as that of compound HC-03 in the His(R)-cluster GalNAc synthesis pathway. MS(ESI) m / z 809.66 ([M+H] + )

[0280] Synthesis of compound SC-04

[0281] Its synthesis method is the same as that of compound HC-04 in the His(R)-cluster GalNAc synthesis pathway. MS(ESI) m / z 639.30 ([MH] - ).

[0282] Synthesis of compound SC-05

[0283] Its synthesis method is the same as that of compound HC-05 in the His(R)-cluster GalNAc synthesis pathway. MS(ESI) m / z 1109.16 ([M+H] + ).

[0284] Synthesis of compound SC-06

[0285] The synthesis method is the same as that of compound HC-06 in the His(R)-cluster GalNAc synthesis pathway. MS(ESI) m / z 809.47 ([M+H] + ).

[0286] Synthesis of compound SC-07

[0287] Its synthesis method is the same as that of compound HC-07 in the His(R)-cluster GalNAc synthesis pathway. MS(ESI) m / z 1049.23 ([(M+2) / 2] + ).

[0288] Synthesis of compound SC-08

[0289] Its synthesis method is the same as that of compound HC-08 in the His(R)-cluster GalNAc synthesis pathway. MS(ESI) m / z 982.09 ([(M+2) / 2] + ).

[0290] Synthesis of compound SC-09

[0291] Its synthesis method is the same as that of compound HC-09 in the His(R)-cluster GalNAc synthesis pathway. MS(ESI) m / z 902.62 ([(M+3) / 3] + ).

[0292] Synthesis of compound SC-10

[0293] Its synthesis method is the same as compound HC-10 in the His(R)-cluster GalNAc synthesis pathway. MS(ESI) m / z 1115.52 ([(M+2) / 2] + ).

[0294] Synthesis of compound SC-11

[0295] Its synthesis method is the same as that of compound HC-11 in the His(R)-cluster GalNAc synthesis pathway. MS(ESI) m / z 1115.56 ([(M-302)+2) / 2] + ).

[0296] Synthesis of compound SC-12

[0297] Its synthesis method is the same as that of compound HC-12 in the His(R)-cluster GalNAc synthesis pathway. MS(ESI) m / z 1165.62 ([(M-302)+2) / 2] + ). 1 HNMR(400MHz,DMSO-d6) δ 8.01-7.88(m, 10H), 7.40-7.34(m, 3H), 7.29-7.18(m, 8H), 7.06(s, 1H), 6.78-6.82(m, 4H), 6.60(s, 1H), 5.18(d, 3H), 4.96(dd, 3H), 4.54-4.46(m, 5H), 4.20-4.15(m, 2H), 4.05-3.93(m, 8H), 3.82(q, 3H), 3.70(s, 6H), 3.65-3.71(m, 3H), 3.55-3.50(m, 12H), 3.41-3.34(m, 4H), 3.04-3.01(m, 14H), 2.87(d, 2H), 2.77-2.56(m, 4H), 2.30-2.26(m, 8H), 2.10(s, 9H), 2.06-2.01(m, 8H), 1.96(s, 9H), 1.94-1.90(m, 6H),1.85(s, 9H), 1.75(s, 9H), 1.52-1.43(m, 20H), 1.27-1.20(m, 9H), 1.11(d, 3H),MS(ESI)m / z 1145.05([(M-302)+2) / 2] + ).

[0298] Synthesis of compound SC-13

[0299] The synthesis method is the same as that of HC-13 in the His(R)-cluster GalNAc synthesis pathway. Example 5 Synthesis of conjugated oligonucleotides provided by the present invention

[0300] Naturally occurring or chemically modified oligonucleotides were synthesized by conventional methods, such as solid phase methods, and compound-conjugated oligonucleotides were synthesized by the exemplary methods shown below:

[0301] Preparation of oligoconjugate GC-05 (Glu-R-cluster GalNAc) [ka]

[0302] Solid Phase Synthesis Steps

[0303] Using phosphoramidite solid phase synthesis methods known in the art, GC-05 was used as a solid phase synthesis support, and nucleoside monomers were connected one by one in sequence from 3' to 5' direction by MerMade192 solid phase synthesizer. Each connection of nucleoside monomers involved four steps: deprotection, coupling, capping and oxidation, and the standard procedures of the above steps are known to those skilled in the art, and all monomer solutions were prepared in 0.1M acetonitrile solution.

[0304] The solid phase synthesis reagents were composed as follows: Wash: Acetonitrile Unblocking: 3% dichloroacetic acid in dichloromethane Activator: 0.25M 5-ethylthio-1H-tetrazole in acetonitrile Capping Reagent A: THF / Lutidine / Acetic Anhydride (8:1:1) Capping Reagent B: 15% NMI / THF, GL38 finish Oxidation reagent: 0.02M I2 in THF / pyridine / H2O Sulfurization reagent: 0.10M DDTT solution

[0305] As an example, the solid phase synthesis conditions for a 1 μmol synthesis scale are as follows: [Table 10]

[0306] Cleavage and deprotection steps

[0307] The oligo support obtained by the above solid phase synthesis step was added to a 1 mL centrifuge tube, 50-100 μL of concentrated ammonium hydroxide was added, and the mixture was incubated at 50-60°C for 10 hours. The liquid supernatant was removed by centrifugation, and two volumes of acetone-ethanol (80:20) solvent were added to the liquid supernatant to precipitate the white precipitate. The supernatant was removed by centrifugation at 10,000 g to obtain the precipitated product, and the precipitate was redissolved in 0.2 M sodium acetate solution.

[0308] Purification, desalting and lyophilization steps

[0309] Purification was carried out on an Avant 150 purification apparatus using an ion chromatography column with a volume of 1 mL (loaded with packing material Nano Q 30).

[0310] Detailed conditions are: Buffer A: 20 mM sodium phosphate-10% acetonitrile-water buffer solution (pH 7.5), Buffer B: 2.0 M NaCl-20 mM sodium phosphate-acetonitrile-water buffer solution (pH 7.5); Elution gradient: 0-50% buffer B, 100-50% buffer A It is.

[0311] The eluent was collected, combined, and finally used on a G25 Sephadex column for desalting; the OD260 concentration value of the desalted product solution was measured, the product content was calculated, and finally placed into a centrifuge tube for lyophilization to obtain a white freeze-dried product.

[0312] Detection: Reverse phase UPLC-MS tandem mass spectrometry was used for detection, purity was >90%, m / z [M-7 / 7] - , [M-8 / 8] - , [M-9 / ]- Mass spectrometry showed characteristic ion peaks of . In the synthesis example of AS-Oligo that mentioned GC-05-OLIGO, GC-05-OLIGO was referenced, and Unylinker-CPG (Glen Research) was used as the solid phase synthesis support for the synthesis.

[0313] In the synthesis example of Oligo-conjugated His-R-cluster GalNAc, GC-05-OLIGO was referenced and HC-13 was used as the solid-phase synthesis support for the synthesis.

[0314] In the synthesis example of Oligo-conjugated His-R-cluster GalNAc, GC-05-OLIGO was referenced and SC-13 was used as the solid phase synthesis support for the synthesis.

[0315] In the synthesis example of Oligo-conjugated His-R-cluster GalNAc, GC-05-OLIGO was referenced and B-9 was used as the solid phase synthesis support for the synthesis.

[0316] siRNA-GalNAc conjugates or aiRNA-GalNAc conjugates were prepared by annealing the Oligo-GalNAC conjugate obtained above with an antisense strand having its complementary sequence in a 1:1 molar ratio to obtain a double-stranded product. Activity Test material and method

[0317] Synthesis of conjugated aiRNA for testing: [ka] The compound HC-5 designed by the inventors is a triple cluster GalNAc based on a histidine linker conjugated at the 3' end of the sense strand of a double-stranded RNA such as aiRNA or siRNA, which is represented by the code "His-cluster" when used and described in the examples below. [ka] The compound GC-5 designed by the inventors is a triple cluster GalNAc based on a glutamine linker conjugated at the 3' end of the sense strand of a double-stranded RNA such as aiRNA or siRNA, which is represented by the code "Glu-cluster" when used and described in the examples below.

[0318] The sequences and structures of the oligonucleotides (aiRNA and siRNA) synthesized and used in the activity test examples are shown in the table below: Sequence of the aiRNA to be tested [Table 11]

[0319] The ex vivo delivery efficiency of the conjugate in the present invention was tested in liver cells by RT-qPCR. The procedure for primary mouse hepatocyte isolation is as follows: Part A: Perfusion (1) Perfusion with Buffer A (2) Perfusion with Buffer B (3) Remove the liver and place it in Buffer C. Buffer A: Add 93 mg (0.5 mM) EDTA to 500 mL of HBSS. Buffer B: Add 400 mg (0.8 mg / mL) of collagenase type I to 500 mL of DMEM. (Note: Collagenase is added at the time of perfusion.) Buffer C: Add 2 mg (2%) BSA to 100 mL of DMEM Part B: Isolation After perfusion, in the hood, place the liver into a 10 cm TC dish, open the liver sack, and rock the tissue with forceps to aid in dissociation. Filter through a 70um filter; wash the filter with buffer C and centrifuge at 50g for 5 minutes at 4°C. Discard the supernatant, gently resuspend in 50 ml of Buffer C (wash 1) and centrifuge at 50g for 5 minutes at 4°C. Discard the supernatant, gently resuspend in 50 ml of Buffer C (wash 2) and centrifuge at 50g for 5 minutes at 4°C. Discard the supernatant, gently resuspend in 50 ml of Buffer C (wash 3) and centrifuge at 50g for 5 minutes at 4°C. Discard the supernatant and resuspend in Thaw / Plant Medium. Count using trypan blue to assess viability / yield. Seed the cells on collagenase coated plates (Thermos Fisher, A1142802) by using mouse primary hepatocyte thawing medium (Thermos Fisher, CM3000). It is better to seed 1mL / well on a 24 well plate (24 will plate). After 3-4 hours, replace the medium with primary hepatocyte maintenance medium (thermofisher, CM4000).

[0320] Ex vivo self-delivery assay was performed without using transfection reagent (tested in 12-well plate, 100,000 cells / well, 48 hours incubation). The Oligo GalNAc conjugate concentration tested is marked in each example as below. The "mock" sample of Oligo GalNAc conjugate concentration is 0 nM. The expression level of targeted mRNA was detected by RT-qPCR.

[0321] For in vivo studies, all animals were acclimated in-house for at least 48 hours before the start of the study. Female C57BL / 6 mice aged 6-8 weeks were obtained from Charles River Laboratories and randomly assigned to each group. All animals were treated according to IACUC protocols. Mice were subcutaneously administered aiRNA duplexes or phosphate buffered saline (PBS) control at 20 mg / Kg or 2 mg / Kg in different experiments. Livers were harvested for efficacy analysis. Example 6 Ex vivo uptake of aiRNA by hepatocytes with and without conjugation

[0322] The "His-cluster"-conjugated mβ-catenin aiRNA (aiRNA#1) showed significant gene silencing activity in self-delivery ex vivo assays at 100 nM, 10 nM, and even 1 nM compared to unconjugated aiRNA, demonstrating that the GalNAc conjugates provided by the present invention have great potency for delivery of double-stranded RNAi agents such as aiRNA. The results were detected by QPCR as shown in Figure 2. Example 7 Ex vivo uptake of aiRNA by hepatocytes using GalNac conjugates

[0323] The mβ-catenin aiRNA (aiRNA#1) conjugated with two different GalNAc conjugates provided by the present invention was co-cultured with isolated mouse hepatocytes for 24 hours at 10 nM concentration. Both Glu-cluster (3GalNAc) and His-cluster (3GalNAc) showed very strong gene silencing in ex vivo self-delivery, and the GalNAc conjugate provided by the present invention has great potency for delivery of double-stranded RNAi agents such as aiRNA. Example 8 In vivo delivery efficacy of aiRNA conjugated with GalNac conjugates

[0324] The aiRNA conjugate mβ-catenin (aiRNA#1) conjugate containing "His-cluster" and "Glu-cluster" was subcutaneously injected into C57BL / 6J mice at 20 mg / kg. Liver samples were collected 2 days after injection and catenin expression levels were analyzed. As shown in FIG. 4, both the His-cluster conjugate and the Glu-cluster conjugate induced potent gene silencing activity in vivo. Similar to the ex vivo data collected previously, the Glu-cluster conjugate appears to be slightly more potent than the His-cluster in this test of the delivery of the aiRNA. Gene expression in the liver was also analyzed 2, 14, and 28 days after injection of the conjugated aiRNA targeting beta-catenin at a dose of 2 mg / kg. As shown in Figure 5, aiRNA conjugated via the glu-cluster or as a his-cluster induced potent and sustained gene silencing activity in vivo, demonstrating that the GalNAc conjugates provided by the present invention can highly efficiently deliver double-stranded RNAi in vivo. Example 9 Ex vivo uptake and in vivo delivery efficiency of aiRNA conjugated with GalNac conjugates

[0325] The structures of SS intermediate position aiRNA (aiRNA#1) and SS3'blunt end aiRNA (aiRNA#2) were tested ex vivo and in vivo as described above. SS intermediate position aiRNA is an exemplary intermediate interfering RNA duplex molecule, and SS3'blunt end aiRNA is an exemplary blunt end interfering RNA duplex molecule. Both types of aiRNA conjugated via the composition of the present invention showed strong gene silencing activity at 100nM, 10nM, and even 1nM in ex vivo self-delivery assays and in vivo (compared to control after 4 hours from a single dose of 23mg / kg sc). The results were tested by QPCR and shown in Figures 6 and 7. It is also surprising that the intermediate type showed even stronger activity than the blunt end configuration both ex vivo and in vivo. These data suggest that the novel linker composition provided by the present invention can be used to conjugate GalNac to various duplex RNA structures. Furthermore, the intermediate interfering RNA duplex / aiRNA-GalNac complex conjugated via the composition provided in the present invention can further improve gene silencing efficiency.

[0326] These results in Examples 5-9 clearly demonstrate that the GalNAc conjugate design based on the present invention can dramatically enhance the delivery efficiency of oligonucleotides both ex vivo and in vivo, and achieve great gene silencing efficacy for targeting various genes in liver cells. Furthermore, the linker composition provided in the present invention is based on amino acids in our body, which eliminates the safety risks of other types of linkers used in GalNAc conjugates.

[0327] It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

[0328] Throughout this application, various publications, patents, and / or patent applications are referenced in order to more fully describe the state of the art to which this invention pertains. The disclosures of these publications, patents, and / or patent applications are herein incorporated by reference in their entireties to the same extent as if each individual publication, patent, and / or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. Structural formula (GG1): 【Chemistry 85】 [In the formula, R 123 , R 124 , R 125 and R 126 are each independently H at each occurrence; R １２１ , R １２２ represents, independently at each occurrence, OH, a protecting group for OH, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, -OP(Z')(Z'')O-nucleoside, -OP(Z')(Z'')O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, -O-nucleotide, a nucleoside, -OP(Z')(Z'')O-R １２８Ｂ -OP(Z''')(Z'''')O-oligonucleotide, or an oligonucleotide; Z', Z'', Z''' and Z'''' are each independently at each occurrence O or S; J 121 , J 122 each independently at each occurrence are a spacer, said spacer being an alkylene of 1 to 10 carbon atoms, one or more carbon atoms being optionally replaced with one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH═N and S(O) 2 , said spacer being optionally substituted with at least one of C 1 -C 5 alkyl, or O—C 1 -C 5 alkyl; R 127 is J 123A -R 128A , wherein J 123A is selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH═N and S(O) 2 , and R 128A is -R 128C -branched group-(R 128B -R 128L ) n 121L or -R 128B -R 128L , wherein R 128C is -C(O)-C 5 -C 8 straight chain alkylene-NHCO-CH 2 -, -C(O)-C 8 -C 11 straight chain alkylene-, or 【Chemistry 201】 Selected from Table 9 of R 128B is independently selected from alkylene of 1 to 30 carbon atoms, wherein one or more carbon atoms are optionally replaced with one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH═N, S(O) 2 , C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocyclylene, and C 5 -C 10 heteroarylene; R 128L is independently selected from a ligand capable of docking to a cell surface receptor; said branching group independently at each occurrence: 【Chemistry 202】 wherein each A 1 is independently O, S, C═O, or NH; each n is independently 1 to 20, and each m is 2 to 6. selected from the group consisting of: n 121L is selected from the group consisting of 1, 2, 3, 4 and 5; n 121 is 1, 2, 3, 4, 5 or 6, preferably 1; n122 is 1; The oligonucleotides contain naturally occurring and / or chemically modified nucleotides / nucleosides. A compound having the formula:

2. R 127 is -C(O)NH-(CH 2 ) 6 -C(O)R 129 , wherein R 129 has the following structure: -Branched group-(R 128B -R 128L ) n 121L 2. The compound of claim 1 having the formula:

3. R １２２ The compound of claim 1 , wherein the compound comprises an oligonucleotide.

4. Structural formula (G-G1-09) or (G-G1-10): 【Chemistry 97】 [In the formula, R' is selected from oligonucleotides formed by naturally occurring and / or chemically modified nucleotides / nucleosides, H and protecting groups; R comprises an oligonucleotide formed by naturally occurring and / or chemically modified nucleotides / nucleosides; A is O or S; D' is selected from Table 9; each E is R 128B independently selected from alkylene of 1 to 30 carbon atoms, where one or more carbon atoms are optionally replaced with any one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH═N, S(O) 2 , C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocyclylene, and C 5 -C 10 heteroarylene; X' is a spacer J 122 which is an alkylene of 1 to 10 carbon atoms, where one or more carbon atoms are optionally replaced with any one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH═N and S(O) 2 , said spacer being optionally substituted with at least one of C 1 -C 5 alkyl, or —OC 1 -C 5 alkyl; Z' is as follows: 【Chemistry 203】 are independently selected from Table 2 of each L independently comprises a ligand moiety capable of docking to a cell-surface receptor; n4 is independently selected from 1, 2, 3 and 4.

2. The compound of claim 1 having the formula:

5. (i) A is O, or (ii) A is S; The compound according to claim 4.

6. The compound described in claim 1 or 4, wherein J 123A is C(O), R 128C and D' are each independently selected from the group consisting of NH-CH 2 -(CH 2 ) n -CH 2 -C(O), NH-CH 2 -(CH 2 ) n -CH 2 -NHC(O)-CH 2 -(CH 2 ) m -CH 2 -C(O) and NH-CH 2 -(CH 2 ) n -CH 2 -C(O)NH-CH 2 -(CH 2 ) m -CH 2 -C(O), wherein n is an integer from 0 to 10 and m is an integer from 0 to 10.

7. Structural formula 【Chemistry 204】 [In the formula, R １２９ has the structure shown below: -Branch base-(R １２８Ｂ -R １２８Ｌ ) ｎ １２１Ｌ having R' is selected from the group consisting of a solid support, an oligonucleotide containing natural or chemically modified nucleotides / nucleosides, H and a protecting group; 2. The compound of claim 1, wherein R comprises an oligonucleotide formed by naturally occurring and / or chemically modified nucleotides / nucleosides.

8. Each of E and R 128B is one of the following: 【Chemistry 205】 8. The compound according to any one of claims 1 to 7, selected from Table 4:

9. The branched group is 【Chemistry 206】 【Chemistry 207】 9. The compound according to any one of claims 1 to 8, selected from the group consisting of:

10. The compound of any one of claims 1 to 9, wherein each ligand is independently selected from the group consisting of N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), galactose, lactose, mannose, cholesterol, tocopherol, biotin, cyanine dyes, folic acid, RGDp, transferrin, anisamide, lactobionic acid, cRGD, hyaluronic acid, low molecular weight protamines, lipid derivatives, peptides, cyclic peptides, and heterocycles.

11. The compound according to any one of claims 1 to 10, wherein n 121 is 1 and n 121L is 3.

12. Formulas GC-1 to GC-9: 【Chemistry 99】 【Chemistry 100】 【Chemistry 101】 【Chemistry 102】 2. The compound of claim 1 having the structure shown in

13. 13. The compound of any one of claims 1 to 12, wherein the oligonucleotide is linked to the remainder of the compound via its 5' and / or 3' end.

14. 14. The compound of claim 13, wherein the oligonucleotide comprises a small interfering RNA (siRNA) duplex, an asymmetric interfering RNA (aiRNA) duplex, an antisense oligonucleotide (ASO) and a microRNA (miRNA).

15. the aiRNA comprises an antisense strand and a sense strand, (i) the antisense strand is longer than the sense strand, has a length of 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides, and when duplexed with the sense strand, contains a 3'-overhang of 1-9 nucleotides and a 5'-overhang of 0-8 nucleotides; The sense strand has a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides and forms a double-stranded region with the antisense strand; (ii) the antisense strand is longer than the sense strand, has a length of 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides, and when duplexed with the sense strand, contains a 3'-overhang of 1-9 nucleotides and a 5'-overhang of 1-8 nucleotides; the sense strand has a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides and forms a double-stranded region with the antisense strand; or (iii) the antisense strand is longer than the sense strand, has a length of 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides, and when duplexed with the sense strand, contains a 1-9 nucleotide 3'-overhang and a 5' blunt end; The sense strand has a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides and forms a double-stranded region with the antisense strand; 15. The compound of claim 14.

16. 13. A small interfering RNA (siRNA) agent comprising the structural formula of any one of claims 1 to 12.

17. 13. An asymmetric interfering RNA (aiRNA) agent comprising the structural formula of any one of claims 1 to 12.

18. 16. A pharmaceutical composition comprising a compound according to any one of claims 1 to 15 and a pharma- ceutically acceptable excipient, carrier, or diluent.

19. A composition comprising a compound according to any one of claims 1 to 15, or a composition comprising an agent according to any one of claims 16 to 17, or a pharmaceutical composition according to claim 18, for treating a disease or condition.

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