Novel compositions for conjugating oligonucleotides and carbohydrates

Abstract

The present invention provides compositions and linkage configurations for oligonucleotides conjugated to carbohydrate ligands for targeted in vivo delivery of oligonucleotides. These ligand-conjugated compounds target one or more organs or cell types, such as liver parenchymal cells, which facilitate endocytic uptake. The compounds have the structural formula (S-HG1):R 206 -[A1] a -[A2] b -[A3] c -R 205 (S-HG1) and R 205 , R 206 represents, independently at each occurrence, H, 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(M')(M'')O-nucleoside, -OP(M')(M'')O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, an -O-nucleotide, a nucleoside, -OP(M')(M'')OR 201 -OP(M''')(M'''')O-oligonucleotide, or oligonucleotide; M', M'', M''' and M'''' are each independently at each occurrence O or S; A1, A2 and A3 are each independently at each occurrence selected from (S-1H) or (S-1G). JPEG2025526994000120.jpg30123

Description

[Technical Field]

[0001] Technical Field The present invention relates to novel compositions and processes that can be used in conjugating carbohydrate ligands to oligonucleotides intended for biomedical applications. [Background technology]

[0002] Background of the Invention Gene modulation, especially gene silencing oligonucleotide, has been the focus of many research and development efforts, because these series of nucleotides are highly promising for treating or preventing many diseases and for 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).

[0003] In many organisms, RNA interference (RNAi) functions in a gene-specific manner through short double-stranded RNA (dsRNA) duplexes called siRNAs. siRNAs have a well-defined structure: symmetric, short (usually 20–24 base pairs) dsRNA duplexes with a phosphorylated 5' end and a hydroxylated 3' end, 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, then recognizes and targets the complementary messenger RNA (mRNA) for cleavage, thereby reducing gene expression in a post-transcriptional manner.

[0004] Relatively new to the field, aiRNA was developed to overcome the off-target effects mediated by the sense strand of symmetrically constructed canonical siRNAs, as well as other off-target mechanisms of siRNA (see PCT Patent Publication WO2009029688). aiRNAs are designed to contain short RNA duplexes in which the two RNA strands are unequal in length and are therefore "asymmetric." For example, aiRNAs can form duplexes containing 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 fields where current siRNAs or short hairpin RNAs (shRNAs) are applied, including biological research, R&D studies in the biotechnology and pharmaceutical industries, and RNAi-based therapeutics.

[0005] 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 a post-transcriptional mechanism. The mechanisms can be broadly classified as follows: (1) occupancy alone without promoting RNA degradation, in which ASO binding results in translation arrest, splicing inhibition, or induction of alternatively spliced variants; (2) occupancy-induced destabilization, in which ASO binding promotes RNA degradation via endogenous enzymes such as ribonuclease H1 (RNase H1); and (3) increased translation, in which 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 an ASO is a single-stranded deoxyribonucleotide sequence with a sulfur chemical modification known as a phosphorothioate. After 40 years of research, antisense technology has been improved through various chemical modifications of single-stranded oligonucleotides.

[0006] miRNA molecules are usually derived from the 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.

[0007] 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 significant need to enhance the important pharmacological properties of these therapeutic oligonucleotides in areas such as serum stability, delivery to intended organs or cell populations, and uptake across cell membranes.

[0008] The preferred delivery of therapeutic oligonucleotides to cells in vivo, for example, in a mammalian body such as a human body, requires specific targeting and protection from the extracellular environment inside the body, including from proteins in serum. A method that researchers have used to achieve specific targeting is to conjugate a targeting moiety to the oligonucleotide to direct the therapeutic oligonucleotide to the desired target site.

[0009] One way to improve specificity in delivery is by exploiting receptor-mediated endocytosis already present in the body. The uptake mechanism involves molecules bound to cell membrane receptors passing through the membrane and moving into the cell via invagination of membrane structures or 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. Therefore, conjugating drug candidates to targeting moieties that target such cell surface receptor(s) can effectively harness the body's native endocytic pathway for drug delivery. Many receptor-mediated endocytosis mechanisms are known and have been studied, including those that recognize sugars containing galactose, mannose, and mannose-6-phosphate, peptides, and proteins, 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 exhibits 50-fold higher affinity for N-acetyl-D-galactosylamine (GalNAc) than for D-Gal. However, when using this conjugation system, the design of the linker structure and various chemical properties of the linker moiety have been reported to be 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]

[0010] [Patent Document 1] International Publication No. 2009 / 029688 Summary of the Invention [Means for solving the problem]

[0011] SUMMARY OF THE INVENTION In a first aspect, the present invention relates to a compound as a therapeutic agent, wherein the oligonucleotide is conjugated to at least one ligand, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide or derivative thereof, e.g., a carbohydrate ligand, which can target the compound to receptor cells in the liver where it can facilitate endocytic uptake, as discussed above.

[0012] These ligand-conjugated compounds target one or more organs or cell types, for example, liver parenchymal cells in humans. In one embodiment, the compound contains more than one carbohydrate ligand, preferably two or three. In another embodiment, the compound of the present invention contains 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 contains 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 derivatives, peptides, cyclic peptides, and heterocycles.

[0013] In a second aspect, the present invention provides a ligand-binding compound having a novel structure:

[0014] Item 1. Structural formula (S-HG1): R 206 -[A1] a -[A2] b -[A3] c -R 205 (S-HG1) [In the formula, R 205 , R 206represents, independently at each occurrence, H, 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(M')(M'')O-nucleoside, -OP(M')(M'')O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, an -O-nucleotide, a nucleoside, -OP(M')(M'')OR 201 -OP(M''')(M'''')O-oligonucleotide, -X-OP(M')(M'')O-oligonucleotide, -Z-OP(M')(M'')O-oligonucleotide, or oligonucleotide; M', M'', M''' and M'''' are each independently at each occurrence O or S; A1, A2 and A3, at each occurrence, are each independently (S-1H) or (S-1G): [ka] Selected from; R 202A -R 202 -R 202L and; R 217A -R 217 -R 217L and; R 202L , R 217L is one ligand that can dock independently to a cell surface receptor for each occurrence; R 202 , R 217 , R 201 are each independently selected at each occurrence from alkylene of 3 to 30 carbon atoms, wherein 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 and optionally substituted with any one or more substituents from the group consisting of heteroarylene, R 202, R 217 , R 201 each independently is optionally unsubstituted or R 209 and optionally R 202 , R 201 is independently selected at each occurrence from alkylene of 3 to 15 carbon atoms, wherein one or more carbon atoms are optionally replaced by one or more substituents from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O; R1, R2, R 204 , R 207 , R 208 , R 213 , R 214 , R 215 , R 216 , R 209 is independently at each occurrence H, alkyl, aryl, heteroaryl, haloalkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -Salkylphenyl, -alkyl-SH, -Shaloalkyl, halo, -OH, -SH, -NH, -alkyl-NH, -N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, -COH, -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), -C one or more selected from the group consisting of ONH2, -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); n 201 , n 211 is, independently for each occurrence, 1, 2, 3, 4, 5, or 6; J 201 , J 202 , J 211 , J212 is, independently at each occurrence, absent or a spacer; a, b, and c are each independently at each occurrence an integer from 0 to 5, and the sum of a, b, and c is an integer from 1 to 10; The oligonucleotides may contain naturally occurring or chemically modified nucleotides / nucleosides. A compound having the formula:

[0015] Item 2. The compound according to item 1, wherein the sum of a, b and c is an integer of 1 or 3.

[0016] Item 3. Structural formula (S-H1): [ka] [In the formula, R1 is one or more selected from the group consisting of H, C1-C5 alkyl, aryl, heteroaryl, C1-C5 haloalkyl, -C1-C5 alkyl-OH, -C1-C5 alkyl-SH, -C1-C5 alkyl-NH2, -CO2H, -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), -CONH2, -C(O)alkyl, -C(O)alkylphenyl, -C(O)haloalkyl, -SO2(alkyl), -SO2(haloalkyl), -SON2NH2, -SON2NH(alkyl), and -SON2NH(phenyl); n 202 is selected from 1 to 10, preferably 1 to 3. Item 1. The compound according to item 1, having the formula:

[0017] Item 4. Structural formula (S-H1-01): [ka] [In the formula, J 202A 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 202B is selected from alkylene of 1 to 10 carbon atoms, optionally selected from straight chain alkylene of 1 to 10 carbon atoms; R 205A is a solid support or a group containing H; Carbons marked with the symbol "*" are chiral carbon atoms.] Item 4. The compound according to item 3, having the formula:

[0018] Item 5. n 202 The compound according to item 3 or 4, wherein is 1 or 3.

[0019] Item 6. R 206 5. The compound according to any one of items 3 to 4, wherein

[0020] Item 7. Structural formula (S-H1-02): [ka] Item 7. The compound according to item 6, having the formula:

[0021] Item 8. J 201 , J 202 is independently selected at each occurrence 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 and optionally substituted by any one or more substituents from the group consisting of heteroarylene, J 201 , J 202 are each independently optionally unsubstituted or R 209 Item 4. The compound according to item 3, substituted by:

[0022] Item 9. J 201 , J 202 is independently selected at each occurrence from alkylene of 1 to 10 carbon atoms, wherein one or more carbon atoms are optionally replaced by 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; 201 , J 202 are each independently optionally unsubstituted or substituted with at least one group selected from the group consisting of H, C1-C5 alkyl, and -OC1-C5 alkyl.

[0023] Item 10. n 201 is 1 and n 202 Item 10. The compound according to item 9, wherein R is 3.

[0024] Item 11. Structural formula (S-H1-03), (S-H1-04), (S-H1-05) or (S-H2): [ka] [In the formula, A is O or S; X is independently selected from Table 1, or alkylene of 1 to 10 carbon atoms, wherein 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, and S(O)2; X1 is independently selected from Table 2; J 202 , Z is selected independently for each occurrence from Table 3; If necessary, J 202 Z is independently selected at each occurrence from alkylene of 1 to 10 carbon atoms, wherein one or more carbon atoms are optionally replaced with 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; 201, J 202 are each independently optionally unsubstituted or substituted with at least one group selected from the group: H, C1-C5 alkyl, -OC1-C5 alkyl; R, R' are each independently selected from the group consisting of naturally occurring and / or chemically modified oligonucleotides, protecting groups for H and OH; at least one of R and R' comprises an oligonucleotide formed by naturally occurring and / or chemically modified nucleotides / nucleosides; R 202 is selected from straight chain alkylene of 3 to 15 carbon atoms, wherein one or more carbon atoms are optionally replaced with one or more substituents from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O; R 202 is optionally unsubstituted or R 209 is replaced by; If necessary, R 202 is selected from -C3 to C8 straight chain alkylene-. Item 4. The compound according to item 3, having the formula:

[0025] Table 1: [Table 1]

[0026] Table 2 [Table 2]

[0027] Table 3: [Table 3]

[0028] Item 12. Structural formula (S-H1-06), (S-H1-07), (S-H1-08) or (S-H2-01): [ka] [ka] Item 12. The compound according to item 11, having the formula:

[0029] Item 13. Structural formula (S-H1-09), (S-H1-10), (S-H1-11) or (S-H2-02): [ka] Item 12. The compound according to item 11, having the formula:

[0030] Item 14. Structural formula (S-H1-12), (S-H1-13), (S-H1-14) or (S-H2-03): [ka] Item 12. The compound according to item 11, having the formula:

[0031] Item 15. Structural formula (S-H1-15), (S-H1-16) or (S-H2-04): [ka] [ka] [In the formula, R, R' are each independently selected from the group consisting of naturally occurring or chemically modified oligonucleotides, protecting groups for H and OH; At least one of R and R' comprises an oligonucleotide formed by natural and / or chemically modified nucleotides / nucleosides; each A is independently O or S; each Q is independently selected from the group consisting of absent, amide, ether, triazole, carbonate, carbamate, phosphate, phosphonate, phosphorothioate, sulfate, disulfide, ester, thioester, alkylamine, cyclic alkylamine, alkyne, cyclic alkyne, alkene, cyclic alkene, lactone, and lactam linkage; each X is independently selected from Table 1, or alkylene of 1 to 10 carbon atoms, wherein 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, and S(O)2; each Y is independently selected from Table 4, or a straight chain alkylene of 3 to 15 carbon atoms, wherein 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; Y is optionally unsubstituted or R 207 is replaced by; each Z is independently selected from Table 3; each L independently comprises a ligand moiety capable of docking to a cell surface receptor] Item 1. The compound according to item 1, having the formula:

[0032] Table 4: [Table 4]

[0033] Item 16. 16. The compound according to any one of items 11 to 15, wherein each A is O.

[0034] Item 17. 16. The compound according to any one of items 11 to 15, wherein at least one A is S.

[0035] Item 18. 18. The compound of any one of items 1 to 17, 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.

[0036] Item 19. 18. The compound according to any one of items 1 to 17, wherein the ligand is N-acetylgalactosamine (GalNAc).

[0037] Item 20. Structures represented by formulas HS-1 to HS-8 and HS-10: [ka] Item 13. The compound according to item 12, having the formula:

[0038] Item 21. HS-9, HS-14 shown structure: [ka] Item 13. The compound according to item 12, having the formula:

[0039] Item 22. The structure shown for HS-5 and HS-13: [ka] Item 13. The compound according to item 12, having the formula:

[0040] Item 23. Structural formula (S-G1): [ka] [In the formula, R2 is one or more selected from the group consisting of H, C1-C5 alkyl, aryl, heteroaryl, C1-C5 haloalkyl, -C1-C5 alkyl-OH, -C1-C5 alkyl-SH, -C1-C5 alkyl-NH2, -CO2H, -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), -CONH2, -C(O)alkyl, -C(O)alkylphenyl, -C(O)haloalkyl, -SO2(alkyl), -SO2(haloalkyl), -SON2NH2, -SON2NH(alkyl), and -SON2NH(phenyl); n 212 is selected from 1 to 10, preferably 1 to 3.

[0041] Item 24. Structural formula (S-G1-01): [ka] [In the formula, J 212A 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 212B is selected from alkylene of 1 to 10 carbon atoms; optionally selected from straight chain alkylene of 1 to 10 carbon atoms; R 205 is a solid support or a group containing H; Carbons marked with the symbol "*" are chiral carbon atoms.] 24. The compound according to item 23, having the formula:

[0042] Item 25. n 212 is selected from 1 or 3.

[0043] Item 26. R 206 25. The compound according to any one of items 23 to 24, wherein said compound comprises an oligonucleotide.

[0044] Item 27. Structural formula (S-G1-02): [ka] 27. The compound according to item 26, having the formula:

[0045] Item 28. J 211 , J 212 is independently selected at each occurrence 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 and optionally substituted by any one or more substituents from the group consisting of heteroarylene, J 211 , J 212 are each independently optionally unsubstituted or R 209 24. The compound according to item 23, substituted by:

[0046] Item 29. J 211 , J 212 are independently selected from alkylene of 1 to 10 carbon atoms, wherein one or more carbon atoms are optionally replaced with 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; 211 , J 212 are each independently optionally unsubstituted or substituted with at least one group selected from the group consisting of H, C1-C5 alkyl, and -OC1-C5 alkyl.

[0047] Item 30. n 211 is 1 and n 212 30. The compound according to any of items 29, wherein is 1 or 3.

[0048] Item 31. Structural formula (S-G1-03), (S-G1-04), (S-G1-05) or (S-G2): [ka] [In the formula, A is O or S; X is independently selected from Table 1, or alkylene of 1 to 10 carbon atoms, wherein 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, and S(O)2; X1 is independently selected from Table 2; J 212 , Z is independently selected for each occurrence from Table 3; If necessary, J 212 are independently selected from alkylene of 1 to 10 carbon atoms, wherein one or more carbon atoms are optionally replaced with 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; 212 is optionally unsubstituted or substituted by at least one group selected from the group: H, C1-C5 alkyl, -OC1-C5 alkyl; R, R' are each independently selected from the group consisting of naturally occurring and / or chemically modified oligonucleotides, protecting groups for H and OH; At least one of R and R' comprises an oligonucleotide formed by natural and / or chemically modified nucleotides / nucleosides; R 217 is selected from straight chain alkylene of 3 to 15 carbon atoms, wherein one or more carbon atoms are optionally replaced with one or more substituents from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O; R 217 is optionally unsubstituted or R209 is replaced by; If necessary, R 217 is selected from -C3 to C8 straight chain alkylene-. 29. The compound according to claim 28, having the formula:

[0049] Item 32. Structural formula (S-G1-06), (S-G1-07), (S-G1-08) or (S-G2-01): [ka] [ka] 32. The compound according to item 31, having the formula:

[0050] Item 33. Structural formula (S-G1-09), (S-G1-10), (S-G1-11) or (S-G2-02): [ka] 32. The compound according to item 31, having the formula:

[0051] Item 34. Structural formula (S-G1-12), (S-G1-13), (S-G1-14) or (S-G2-03): [ka] 32. The compound according to item 31, having the formula:

[0052] Item 35. Structural formula (S-G1-15), (S-G1-16) or (S-G2-04): [ka] [In the formula, R, R' are each independently selected from the group consisting of naturally occurring or chemically modified oligonucleotides, protecting groups for H and OH; at least one of R and R' comprises a naturally occurring or chemically modified oligonucleotide; each A is independently O or S; each Q is independently selected from the group consisting of absent, amide, ether, triazole, carbonate, carbamate, phosphate, phosphonate, phosphorothioate, sulfate, disulfide, ester, thioester, alkylamine, cyclic alkylamine, alkyne, cyclic alkyne, alkene, cyclic alkene, lactone, and lactam linkage; each X is independently selected from Table 1, or alkylene of 1 to 10 carbon atoms, wherein 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, and S(O)2; each Y is independently selected from Table 4, or a straight chain alkylene of 3 to 15 carbon atoms, wherein 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; Y is optionally unsubstituted or R 207 is replaced by; each Z is independently selected from Table 3; each Z'' is independently selected from Table 5; each L independently comprises a ligand moiety capable of docking to a cell surface receptor; each of n1, n2, and n3 is independently selected from 1, 2, 3, or 4; Item 1. The compound according to item 1, having the formula:

[0053] Table 5: [Table 5]

[0054] Item 36. 36. The compound according to any one of items 31 to 35, wherein each A is O.

[0055] Item 37. 36. The compound according to any one of items 31 to 35, wherein at least one A is S.

[0056] Item 38. 38. The compound according to any of items 23 to 37, 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.

[0057] Item 39. 38. The compound according to any one of items 23 to 37, wherein the ligand is N-acetylgalactosamine (GalNAc).

[0058] Item 40. The structures shown in formulas GS-1 to GS-8, GS-10: [ka] [ka] 32. The compound according to item 31, having the formula:

[0059] Item 41. The structures shown in formula GS-9, GS-14: [ka] 32. The compound according to item 31, having the formula:

[0060] Item 42. The structures shown in formula GS-5, GS-13: [ka] 32. The compound according to item 31, having the formula:

[0061] Item 43. 43. The compound according to any of items 1 to 42, wherein the naturally occurring or chemically modified oligonucleotide is linked to the remainder of the compound via its 5' and / or 3' end.

[0062] Item 44. 44. The compound of claim 43, wherein the oligonucleotide comprises a small interfering RNA (siRNA) duplex.

[0063] Item 45. 44. The compound of claim 43, wherein the oligonucleotide comprises an asymmetric interfering RNA (aiRNA) duplex.

[0064] Item 46. 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 to 9 nucleotides and a 5'-overhang of 0 to 8 nucleotides; 46. The compound of item 45, wherein the sense strand has a length of 10, 11, 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.

[0065] Item 47. 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 to 9 nucleotides and a 5'-overhang of 1 to 8 nucleotides; 47. The compound of item 46, wherein the sense strand has a length of 10, 11, 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.

[0066] Item 48. 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 to 9 nucleotides and a 5' blunt end; 47. The compound of item 46, wherein the sense strand has a length of 10, 11, 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.

[0067] Item 49. 44. The compound according to item 43, wherein the oligonucleotide comprises an antisense oligonucleotide (ASO).

[0068] Item 50. 44. The compound of item 43, wherein the oligonucleotide comprises a microRNA (miRNA).

[0069] Item 51. 51. A small interfering RNA (siRNA) agent duplex comprising the structural formula of any of items 1 to 50.

[0070] Item 52. 51. An asymmetric interfering RNA (aiRNA) agent comprising the structural formula of any one of items 1 to 50.

[0071] Item 53. 51. An antisense oligonucleotide (ASO) agent comprising the structural formula of any one of items 1 to 50.

[0072] Item 54. 51. A microRNA (miRNA) agent comprising the structural formula of any one of items 1 to 50.

[0073] Item 55. 55. A pharmaceutical composition comprising a compound according to items 1 to 50 or an agent according to any one of items 51 to 54, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0074] Item 56. 54. Use of a compound according to any one of items 1 to 50 or an agent according to any one of items 51 to 54 in the preparation of a medicament effective for treating a disease or condition.

[0075] 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.Therefore, a compound comprising a carbohydrate ligand can be useful for targeting genes associated with diseases or undesirable conditions in a target organ.For example, a 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(WAF1 / CIP1) gene, a mutation in the p27(KIP1) 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.

[0076] In a fourth aspect, the present invention provides compounds having novel structures.

[0077] Item 57. Structural formula (G-P1): [ka] [In the formula, R0 is one or more selected from the group consisting of H, C1-C5 alkyl, aryl, heteroaryl, C1-C5 haloalkyl, -C1-C5 alkyl-OH, -C1-C5 alkyl-SH, -C1-C5 alkyl-NH2, -CO2H, -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), -CONH2, -C(O)alkyl, -C(O)alkylphenyl, -C(O)haloalkyl, -SO2(alkyl), -SO2(haloalkyl), -SON2NH2, -SON2NH(alkyl), and -SON2NH(phenyl); R 202A comprises at least one ligand capable of docking to a cell surface receptor; R 204 , R 207 , R 208are independently H, alkyl, aryl, heteroaryl, haloalkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -Salkylphenyl, -alkyl-SH, -Shaloalkyl, halo, -OH, -SH, -NH, -alkyl-NH, -N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, -COH, -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), -CONH 2, one or more selected from the group consisting of -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); n 201 is selected from 1, 2, 3, 4, 5 or 6; R 206 is selected from OH or a protecting group for OH; J 201 , J 202 is, independently for each occurrence, a spacer; R 205B is -C2~C 10 alkynylene-CN; R 205C , R 205D are independently selected from -C1-C6-alkyl, or R 205C and R 205D together form a 5- or 6-membered ring, and optionally R 205C , R 205D is substituted and, if necessary, R 205C , R 205D contains one additional heteroatom selected from N and O] A compound having the formula:

[0078] Item 58. J 201 , J 202 are each independently selected from alkylene of 3 to 30 carbon atoms, wherein 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 to C 10 Alkenylene, C2-C 10 Alkynylene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 and optionally substituted by any one or more substituents from the group consisting of heteroarylene, J 201 , J 202 is optionally unsubstituted or R 209 58. The compound according to item 57, substituted by:

[0079] Item 59. Structural formula (G-P2): [ka] [In the formula, J 202A 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 202B is selected from alkylene of 1 to 10 carbon atoms. 58. The compound according to item 57, having the formula:

[0080] Item 60. Structural formula (G-P3): [ka] 59. The compound according to claim 58, having the formula:

[0081] Item 61. Structural formula (G-P4): [ka] 58. The compound according to item 57, having the formula:

[0082] Item 62. R 205B is -C2-C5 alkynylene-CN; R 205C , R 205D are independently selected from -C1-C6-alkyl; R 202A But -R 202C -branched group-(R 202B -R 202L ) n 111L or -R 202B -R 202L and; R 202L are independently selected from ligands capable of docking to a cell surface receptor; R 202B is selected from alkylene of 1 to 30 carbon atoms, wherein 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 optionally substituted by one or more substituents selected from the group consisting of heteroarylene, and R202 B is optionally unsubstituted or R 207 is replaced by; n 111L is selected from 1, 2 or 3.

[0083] Item 63. R 202C is -C(O)-C5 to C8 straight chain alkylene -NHCO-CH2- or -C(O)-C8 to C 11 63. The compound according to item 62, wherein the alkylene- is selected from linear alkylene-.

[0084] Item 64. The branched group is [ka] [In the formula, each A1 is independently O, S, C=O, or NH; each n is independently 1 to 20] 63. The compound according to item 62, selected from the group consisting of:

[0085] Item 65. The ligand is [ka] and R A is a protecting group for H or OH.

[0086] Item 66. Structural formula (G-P5): [ka] 62. The compound according to item 61, having the formula:

[0087] Item 67. Structural formula (G-P6): [ka] 62. The compound according to item 61, having the formula:

[0088] Item 68. Structural formula (G-P7): [ka] [In the formula, R3 is one or more selected from the group consisting of H, C1-C5 alkyl, aryl, heteroaryl, C1-C5 haloalkyl, -C1-C5 alkyl-OH, -C1-C5 alkyl-SH, -C1-C5 alkyl-NH2, -CO2H, -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), -CONH2, -C(O)alkyl, -C(O)alkylphenyl, -C(O)haloalkyl, -SO2(alkyl), -SO2(haloalkyl), -SON2NH2, -SON2NH(alkyl), -SON2NH(phenyl); R 217A comprises at least one ligand capable of docking to a cell surface receptor; R 213 , R 214 , R 215 , R 216 is 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, one or more selected from the group consisting of -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 212 is selected from OH or a protecting group for OH; J 211 , J 212 is, independently for each occurrence, a spacer; n 211 is, independently for each occurrence, 1, 2, 3, 4, 5, or 6; J 211 , J 212 is, independently for each occurrence, a spacer; R 211B is -C2~C 10 alkynylene-CN; R 211C , R 211D are independently selected from -C1-C6-alkyl, or R 211C and R 211D together form a 5- or 6-membered ring, and optionally R 211C , R 211D is substituted and, if necessary, R 211C , R 211D contains one additional heteroatom selected from N and O] A compound having the formula:

[0089] Item 69. J 211 , J 212 are each independently selected from alkylene of 3 to 30 carbon atoms, wherein 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 to C 10 Alkenylene, C2-C 10 Alkynylene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 and optionally substituted by any one or more substituents from the group consisting of heteroarylene, J 211 , J 212 is optionally unsubstituted or R 209 69. The compound according to item 68, substituted by:

[0090] Item 70. Structural formula (G-P8): [ka] [In the formula, J 212A 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 212B is selected from alkylene of 1 to 10 carbon atoms. 69. The compound according to item 68, having the formula:

[0091] Item 71. Structural formula (G-P9): [ka] 69. The compound according to item 69, having the formula:

[0092] Item 72. Structural formula (G-P10): [ka] 69. The compound according to item 68, having the formula:

[0093] Item 73. R 211B is -C2-C5 alkynylene-CN; R 211C , R 211D are independently selected from -C1-C6-alkyl; R 217 But -R 217C -branched group-(R 217B -R 217L ) n 211L or -R 217B -R 217L and; R 217L are independently selected from ligands capable of docking to a cell surface receptor; R 217B is selected from alkylene of 1 to 30 carbon atoms, wherein 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 10Arylene, C3-C 18 Heterocyclylene and C5-C 10 and optionally substituted with any one or more substituents from the group consisting of heteroarylene, R 217B is optionally unsubstituted or R 213 is replaced by; n 211L is selected from 1, 2 or 3.

[0094] Item 74. R 217C is -C(O)-C5 to C8 straight chain alkylene -NHCO-CH2- or -C(O)-C8 to C 11 74. The compound according to item 73, wherein the alkylene- is selected from linear alkylene-.

[0095] Item 75. The branched group is [ka] [In the formula, each A1 is independently O, S, C=O, or NH; each n is independently 1 to 20] 74. The compound according to item 73, selected from the group consisting of:

[0096] Item 76. The ligand is [ka] and R A is a protecting group for H or OH.

[0097] Item 77. Structural formula (G-P11): [ka] 69. The compound according to item 68, having the formula:

[0098] Item 78. Structural formula (G-P12): [ka] 69. The compound according to item 68, having the formula:

[0099] In another aspect, the invention features a compound as provided in the fourth aspect above that can be used as an intermediate linker for linking oligonucleotides and ligands to synthesize serial and / or cluster ligand conjugations, in particular, that can be used as an intermediate compound for synthesizing serial ligand conjugations.

[0100] In another aspect, the compounds as provided in the fourth aspect above can be used to directly conjugate ligand conjugates to the "backbone" at the 5' and / or 3' ends of the oligonucleotide. In some embodiments, the compounds as provided in the fourth aspect above can be used to directly conjugate ligand conjugates to the 5' end of any strand of the oligonucleotide.

[0101] 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 pharmaceutically acceptable excipient, carrier, or diluent.

[0102] 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. Thus, 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 the disease or condition is carried out by partial or total silencing of the disease gene. The disease gene may be a patient's own gene or an externally derived microbial gene such as a virus.

[0103] The above and other objects, aspects, features, and advantages of the present invention will become more apparent from the following description and claims.

[0104] 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 dictate all possible variations. In the drawings, like numerals are used to denote like parts throughout the various views. [Brief explanation of the drawings]

[0105] [Figure 1] Figure 1 illustrates exemplary structures 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 to the 3'-end of the sense strand (such as structures 1.1-1.3, intermediate aiRNA, blunt-ended aiRNA, and siRNA), the 3'-end of the antisense strand (structure 2), the 5'-end of the sense strand (structure 3), or both ends of the sense strand (structure 5), both ends of the antisense strand (structure 4), the 3'-end of the antisense strand and the 5'-end of the sense strand (structure 6), the 3'-end of the sense strand and the 3'-end of the antisense strand (structure 7), or 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 to the 3'-end and / or 5'-end of the antisense strand.

[0106] [Figure 2] Figure 2 illustrates the ex vivo uptake β-catenin aiRNA results tested by QPCR. "Non-GalNAc" is unconjugated aiRNA. "Glu(R)-seq(3GalNAc)" is aiRNA conjugated with the GalNAc conjugate composition "GS-5."

[0107] [Figure 3] Figure 3 illustrates the ex vivo uptake efficacy of mCat12 aiRNA conjugated to "His(R)-seq(3GalNAc)", "His(S)-seq(3GalNAc)", and "Glu-seq(3GalNAc)" in primary hepatocytes.

[0108] [Figure 4] FIG. 4 illustrates the ex vivo uptake efficacy of TTR aiRNA conjugated with "His-Seq(3GalNAc)" in primary hepatocytes. DETAILED DESCRIPTION OF THE INVENTION

[0109] Detailed Description of the Invention I. Definition Unless otherwise specified, technical terms are used according to conventional usage.The definition of general terms in molecular biology can be found in, for example, 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 reference books.

[0110] As used in this specification and 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 element. In such cases, this statement is intended to serve as a basis for the use in the claims of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements, or for "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 (e.g., not included)...."

[0111] As used herein, the recitation 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 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, or 0.0, 0.1, 0.01, 0.001, or any other real value >0 and <2 if the variable is inherently continuous.

[0112] 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%".

[0113] As used herein, the terms "spacer," "linker," and "link" are used to connect two portions of a compound, e.g., a compound 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, C2-C10 alkenylene, C2-C10 alkynylene, C6-C10 arylene, C3-C18 heterocyclylene, and C5-C10 heteroarylene, and J201, J202 are each independently optionally substituted or H, alkyl, aryl, heteroaryl, haloalkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -OHaloalkyl, -S(a) alkyl, -S alkylphenyl, -alkyl-SH, -S haloalkyl, 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), -SO2 It is an alkylene of 1 to 30 carbon atoms substituted with one or more from the group consisting of NH, -SONH(alkyl), -SONH(phenyl), -NHSO(alkyl), -NHSO(phenyl), and -NHSO(haloalkyl).

[0114] A variety of hydroxyl protecting groups can be used in the present disclosure. Generally, a protecting group renders a chemical functional group inert to specific reaction conditions and can be added to and removed from such a functional group in a molecule without substantially damaging the remainder of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage, et al., Tetrahedron 1992, 48, 2223-2311, and 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).

[0115] 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.

[0116] As used herein, "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and 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 containing one or more substituents, it will be understood by those skilled in the art that such group is not intended to introduce any substitution or substitution pattern that is sterically infeasible, synthetically infeasible, and / or inherently unstable.

[0117] 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, 1 to 6, 1 to 6, or 1 to 3 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 residue as alkyl.

[0118] 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 a 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), and 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, and 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.

[0119] 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 and prop-2-yn-1-yl; butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, and but-3-yn-1-yl; and the like. In certain embodiments, alkynyl groups have 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl having two points of attachment but referring to the same residues as alkynyl.

[0120] As used herein, "alkoxy" refers to an alkyl group of the indicated 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.

[0121] 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. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon atoms from 6 to 18 carbon atoms, and at least one ring within 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.

[0122] As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring, typically having 3 to 7 ring carbon atoms. The ring may be saturated or may 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.

[0123] As used herein, "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo, and the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0124] 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 pentafluoroethyl.

[0125] "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 herein, 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 rest 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.

[0126] "Heteroaryl" refers to a radical derived from a 3- to 18-membered aromatic ring radical containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, a heteroaryl radical may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, in which at least one ring in the ring system is fully unsaturated, i.e., contains a cyclic, delocalized (4n+2) π-electron system according to Hückel theory. Heteroaryl includes fused or bridged ring systems. The heteroatom(s) in the heteroaryl radical are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. A heteroaryl is attached to the rest 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]i 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]pyridazinyl,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 Examples include 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).

[0127] As used herein, the term "solid support" includes a solid phase carrier for the synthesis of oligonucleotides, such as CPGs.

[0128] As used herein, the terms "oligonucleotide" and "oligonucleotides" refer to a compound containing multiple linked nucleosides. In certain embodiments, an "oligonucleotide" is a short single- or double-stranded RNA or DNA molecule, including antisense oligonucleotides (ASOs), RNA interference (RNAi), and aptamer RNAs. In certain embodiments, one or more of the multiple nucleosides are modified. In certain embodiments, an 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, or shRNA. In some embodiments, the oligonucleotide is a circRNA. In some embodiments, the oligonucleotide is an mRNA.

[0129] As used herein, the term "aiRNA" refers to an asymmetric interfering RNA duplex molecule, comprising an antisense strand and a sense strand, wherein the antisense strand is 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 is at least 70% complementary to the target mRNA; the sense strand consists of 10-26 nucleotides, and forms a double-stranded region together with the antisense strand, wherein the double-stranded region contains 0, 1 or 2 mismatch pairs(s). Exemplary structures of aiRNA are described in US2009 / 0208564, the entire contents of which are incorporated by reference.

[0130] As used herein, the term "intermediate" refers to an interfering RNA duplex molecule that comprises an antisense strand and a sense strand, wherein the antisense strand is longer than the sense strand and comprises both a 3' overhang and a 5' overhang of at least one nucleotide.

[0131] As used herein, the term "blunt" refers to an interfering RNA duplex molecule that comprises 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.

[0132] As used herein, the term "modified oligonucleotide" means an oligonucleotide that includes at least one modified nucleotide.

[0133] As used herein, the term "modified nucleotide" means a nucleotide having at least one modified sugar moiety, modified internucleoside linkage, and / or modified nucleobase.

[0134] As used herein, the term "modified nucleoside" means a nucleoside having at least one modified sugar moiety and / or a modified nucleobase.

[0135] As used herein, the term "naturally occurring internucleoside linkage" means a 3'-5' phosphodiester linkage.

[0136] 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.

[0137] As used herein, the term "natural sugar moiety" means a sugar found in DNA (2-H) or RNA (2-OH).

[0138] 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.

[0139] 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.

[0140] 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).

[0141] As used herein, "prevention" and "preventing" are used interchangeably. These terms refer to an approach to obtaining advantageous or desired results, including, but not limited to, prophylactic benefit. In a "prophylactic benefit," the conjugate or composition can be administered to a patient at risk of developing a particular disease, or to a patient who reports one or more physiological symptoms of the disease, even if the disease has not been diagnosed.

[0142] 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 endpoint, the pharmacokinetics of the compound, the disease to be treated, the mode of administration, and the patient.

[0143] As used herein, the term "treatment" of or "treating" a disease or disorder 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 elimination 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.

[0144] 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 pharmaceutically acceptable carrier. As used herein, a "pharmacologically effective amount," a "therapeutically effective amount," or simply an "effective amount" refers to an amount of RNA that is effective to produce the intended pharmacological, therapeutic, or preventive result. For example, if a given clinical treatment is determined to be effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, 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.

[0145] 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 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 in human subjects.

[0146] Compound configuration

[0147] Compounds of the present invention, and salts thereof, may exist in their tautomeric form (for example, as an amide or imino ether), and all such tautomeric forms are contemplated herein as part of the present invention.

[0148] All stereoisomers of the compounds of the present invention, including enantiomeric and diastereomeric forms (e.g., those that may exist due to asymmetric carbon atoms 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 having the specified activity), or may, for example, be racemic or admixed with all other or 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.

[0149] 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.

[0150] 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 encompasses both cis (Z) and trans (E) alkene isomers, as well as cis and trans isomers of cyclic hydrocarbon or heterocyclic rings.

[0151] D-amino acids / L-amino acids

[0152] 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

[0153] The present invention provides a novel compound, comprising a novel linker composition for connecting various components of the novel compound.The compound conjugates oligonucleotide with one or more targeting ligands.The oligonucleotide can be naturally occurring (isolated from nature or synthesized in laboratory), or can be chemically modified in at least one subunit.

[0154] 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.

[0155] 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] It has.

[0156] In certain embodiments, an oligonucleotide has at least one chemically modified nucleotide comprising 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, NH, NHR, NR, or CN, where each R is independently C-C 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-C-C 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 ) and 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'-OCH, 2'-OCHCH, 2'-OCHCHF, and 2'-O(CH)OCH 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 consisting 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.

[0157] In certain embodiments, oligonucleotide has at least one chemically modified nucleobase.In certain embodiments, at least one chemically modified nucleobase is 5-methylcytosine (5-Me-C), inosine base, tritiated base, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine base, 6- Selected from the group consisting of azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.In certain embodiments, the modified nucleobase in the molecule of the present invention is 5-methylcytosine.In certain embodiments, the modified nucleobase is 5-methyluracil.

[0158] Any stabilizing modification known to those skilled in the art can be used to improve the stability of oligonucleotide molecules.In oligonucleotide molecules, chemical modifications can be introduced into phosphate backbone (for example, phosphorothioate linkage), sugar (for example, locked nucleic acid, glycerol nucleic acid, cEt, 2'-MOE, 2'-fluorouridine, 2'-O-methyl), and / or base (for example, 2'-fluoropyrimidine).

[0159] The oligonucleotide may be conjugated to the remainder, or "backbone," of the compound at the 5' and / or 3' end of the oligonucleotide. The conjugated oligonucleotide may be delivered as a single strand or hybridized to a substantially complementary oligonucleotide as part of a duplex. The substantially complementary oligonucleotide may or may not be conjugated in turn.

[0160] 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, an oligonucleotide conjugated in accordance with the principles of the present invention is used as an antisense oligonucleotide (ASO). In yet another embodiment, an oligonucleotide conjugated in accordance with 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, or "backbone," of the compound.

[0161] Embodiment 1

[0162] In a first aspect, the oligonucleotide is conjugated, either directly or through one or more intermediate linkers, to a backbone containing multiple moieties including consecutive motifs along the backbone of more than one, e.g., 2-8 and preferably 3, ligands (e.g., GalNAc), at attachment points provided by residues derived from histidine residues.

[0163] In one embodiment, the compounds of the invention have the structural formula as shown in (S-H1), (S-H2), (S-H1-01) through (S-H1-16), and (S-H2-01) through (S-H2-04).

[0164] In one embodiment, the compounds of the invention have a structure as shown in Table 6.

[0165] In one embodiment, as needed, the configuration of the compound is the R isomer, the S isomer, or a mixture thereof. In one embodiment, the configuration of the compound is a mixture of the R isomer and the S isomer. In a preferred embodiment, the configuration of the compound is the R isomer. In one embodiment, the configuration of the compound refers to the isomer of the chiral carbon atom shown in structural formula S-H1-01.

[0166] In the compounds of the present invention, said naturally occurring or chemically modified oligonucleotide is linked to the remainder of the compound via its 5' end and / or its 3' end.

[0167] Embodiment 2

[0168] In a second aspect, the oligonucleotide is conjugated, either directly or through one or more intermediate linkers, to a backbone containing multiple moieties including consecutive motifs along the backbone of more than one, e.g., 2-8 and preferably 3, ligands (e.g., GalNAc), at attachment points provided by moieties derived from glutamic acid residues.

[0169] In one embodiment, the compounds of the invention have the structural formula as shown in (S-G1), (S-G2), (S-G1-01) through (S-G1-16), and (S-G2-01) through (S-G2-04).

[0170] In one embodiment, the compounds of the invention have a structure as shown in Table 6.

[0171] In one embodiment, the configuration of the compound is optionally the R isomer, the S isomer, or a racemate. In one embodiment, the configuration of the compound is a mixture of the R isomer and the S isomer. In a preferred embodiment, the configuration of the compound is the R isomer. In one embodiment, the configuration of the compound refers to the isomer of the chiral carbon atom shown in structural formula S-G1-01.

[0172] In the compounds of the present invention, a naturally occurring or chemically modified oligonucleotide is linked via its 5' end and / or its 3' end to the remainder of the compound.

[0173] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Example]

[0174] III. Working Examples synthesis In some embodiments, compounds of formulas (S-H1), (S-H1-01)) through (S-H1-16), (S-G1), (S-G1-01)) through (S-G1-16) comprising oligonucleotides conjugated to a backbone containing multiple moieties comprising more than one, e.g., 2-8, consecutive motifs along the backbone of ligands (e.g., GalNAc) are synthesized by reacting a consecutive backbone containing three or more reactive moieties with the ligands and oligonucleotides.

[0175] [Table 9] Example 1 Synthesis of G-12 (Glu(R)-GalNAc)

[0176] The synthesis of G-12 (Glu(R)-GalNAc-sequential) is shown below in four steps:

[0177] Step 1: Synthetic route to compound M-3. [ka]

[0178] Synthesis of compound M-2

[0179] Under a nitrogen atmosphere, 20 g of M-1 ((R)-3-hydroxymethylbutyrate) 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. After quenching with 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] + )

[0180] Synthesis of compound M-3

[0181] An aqueous solution of sodium hydroxide was prepared by dissolving 13.6 g of sodium hydroxide in 80 mL of water. 73 g of crude M-2 was dissolved in 500 mL of methanol, and then the aqueous solution of sodium hydroxide was added. After stirring at 35 °C for 15 hours, the reaction was monitored for completion using TLC. Methanol was removed by concentration at 40 °C, 500 mL of ethyl acetate was added, and the pH was adjusted to 3 with 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). 47.8 g of M-3 was obtained from the concentrated product. MS (ESI) m / z 341.10 ([M-H] - ). 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).

[0182] Step 2: Synthetic route to compound N-4 [ka]

[0183] Compound N-3 (5-hydroxy-amylamine) (15.0 g) was dissolved in 150 mL of water, 36.6 g of NaHCO3 was added, and 33.5 g of CbzCl was added dropwise under an ice bath. After the dropwise addition, the mixture was stirred at room temperature for 3 hours. TLC was used to monitor the completion of the reaction. 100 mL of water was added to the reaction solution, and the mixture was extracted with 200 mL of EA, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The mixture was then separated by silica gel column chromatography (DCM:MeOH=30:1). The product components were collected and concentrated to a white solid (23.5 g). ESI-MS m / z: [M+H] + =238.16.

[0184] Step 3: Synthetic route for compound S-07 [ka]

[0185] Synthesis of compound S-02

[0186] 250 g of compound S-01 (N-acetylgalactosamine) was dissolved in 2000 mL of DCM, and 157.5 g of TMSOTf was added dropwise. After the dropwise addition, the reaction was carried out at 40°C for 6 hours under a nitrogen atmosphere, and the reaction completion was monitored using TLC. The pH was adjusted to 8-9 with saturated sodium bicarbonate, extracted, and the organic phase was separated, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 211 g of a yellow, transparent oil. ESI-MS M / Z: [M + H] + = 330.12.

[0187] Synthesis of compound S-06

[0188] 25.2 g of compound S-02 was dissolved in 200 mL of DCM, 20.0 g of compound N-4 was added, and 6.93 g of TMSOTf was added dropwise. After the dropwise addition, the reaction was carried out at room temperature overnight. Completion of the reaction was monitored using TLC. 100 mL of saturated sodium bicarbonate solution was added to the reaction mixture, and the organic phase was separated, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM:MeOH = 100:1 to 50:1). The product components were collected and concentrated under reduced pressure to give 28.9 g of compound S-06. ESI-MS m / z: [M + H] + = 567.21.

[0189] Synthesis of compound S-07

[0190] 16.5 g of compound S-06 was dissolved in 150 mL of MeOH, 1.65 g of Pd / C was added, and the reaction was carried out at room temperature for 4 hours after replacing the air with hydrogen three times. TLC was used to monitor the completion of the reaction. The reaction mixture was filtered under vacuum and concentrated under reduced pressure to give 12.5 g of a white foamy solid. ESI-MS m / z: [M + H]+ = 433.21.

[0191] Step 4: Synthetic route to compound G-12 [ka] [ka]

[0192] Synthesis of compound G-2

[0193] 50 g of compound G-1 ((S)-N-Boc-glutamic acid methyl ester) was dissolved in 500 mL of THF, and 25.2 mL of NMM was added dropwise in an ice-water bath. After stirring for 5 minutes, 26.6 mL of isobutyl chloroformate was added dropwise. After the addition was completed, the mixture was stirred for 1 hour. Suction filtration was performed, and the filtrate was collected. 48.73 g of NaBH was added to the filtrate under an ice-water bath. After the addition was completed, the reaction was continued for 2 hours, and a sample was taken to monitor the completion of the reaction by TLC. 250 mL of water was added, and 500 mL of EA was added for extraction. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to obtain 45.35 g of oily product G-2. MS (ESI) m / z 248.19 ([M+H] + ).

[0194] Synthesis of compound G-3

[0195] Compound G-2 (45.35 g) was dissolved in 500 mL of DCM, and 31.2 g of imidazole was added. 91.1 g of TBDPSCl was added dropwise. After the addition was complete, the mixture was stirred at room temperature for 2 hours, and the reaction was monitored by TLC for completion. 150 mL of water was added to the reaction mixture, and the DCM layer was extracted, separated, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to give an oily product. The product was purified by silica gel column chromatography using a gradient elution of PE:EA = 40:1 to 10:1, and concentrated to give 36.7 g of a colorless, clear oily product G-3. MS (ESI) m / z 486.66 ([M+H] + ).

[0196] Synthesis of compound G-4

[0197] Compound G-3 (46.15 g) was dissolved in 500 mL of DCM, and 70 mL of TFA (trifluoroacetic acid) was added dropwise in an ice-water bath. After the addition was complete, the reaction was stirred at room temperature for 4 hours. The reaction was monitored for completion by TLC, concentrated under reduced pressure, and 500 mL of DCM 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 36.7 g of a pale yellow oil, G-4. MS (ESI) m / z 386.51 ([M+H] + ).

[0198] Synthesis of compound G-5

[0199] 31.9 g of compound M-3 was dissolved in 300 mL of DMF, 42 g of HBTU and 23 mL of DIEA were added, and the mixture was stirred at room temperature for 10 minutes. Then, 35 g of compound G-4 was added, and the mixture was transferred to room temperature and stirred for 1 hour. TLC was used to monitor the completion of the reaction. The reaction solution was extracted with 600 mL of saturated sodium bicarbonate and 400 mL of EA. 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 a white solid product G-5 (41 g). MS (ESI), m / z 710.33 ([M+H] + ).

[0200] Synthesis of compound G-6

[0201] 33.3 g of compound G-5 was added to a mixture of 100 mL of MeOH, 100 mL of THF, and 100 mL of water, and 5.92 g of lithium hydroxide monohydrate was added. The reaction was allowed to proceed at room temperature for 8 hours. The reaction was monitored by TLC. The reaction mixture was concentrated under reduced pressure, and then 400 mL of ethyl acetate was added to the concentrated residue. 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 33.8 g of colorless, transparent oily product G-6. MS (ESI) m / z: 694.35 ([M+H] + ).

[0202] Synthesis of compound G-7

[0203] Compound G-6 (18.3 g) was dissolved in 150 mL of DMF, and HBTU (13.0 g) and DIEA (6.52 mL) were added. After stirring at room temperature for 15 minutes, a solution of compound S-07 (11.4 g) in DMF (100 mL) was added dropwise. After the dropwise addition, the mixture was allowed to reach room temperature and stirred for 1 hour. The completion of the reaction was monitored using TLC. Saturated sodium bicarbonate (400 mL) was added to the reaction solution, and the mixture was extracted with EA (200 mL). The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain an oily product. The crude residue was purified by column chromatography (PE:EA = 1:1 to 1:3) to produce a white solid (18.9 g). MS (ESI), m / z: 1110.5 ([M + H] + ).

[0204] Synthesis of compound G-8

[0205] 18.6 g of compound G-7 was dissolved in 150 mL of THF, and 50.3 mL of 1.0 M TBAF was added. The reaction was allowed to proceed overnight at room temperature. Completion of the reaction was monitored using TLC, and the reaction mixture was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 50:1 to 15:1). The product components were collected, concentrated, and dried to give 7.5 g of a white solid. MS (ESI), m / z: 634.35 ([M + H] + ).

[0206] Synthesis of compound G-9

[0207] 5.91 g of compound G-8 was dissolved in 60 mL of anhydrous pyridine, and 6.01 g of DMTrCl was added and reacted at room temperature for 30 minutes. Completion of the reaction was monitored using TLC. The reaction was quenched with 10 mL of methanol and concentrated under reduced pressure. The product was purified by column chromatography (DCM:MeOH = 100:1 to 50:1). The product components were collected and concentrated to give 5.07 g of a dry white solid. MS (ESI), m / z: 634.44 ([M - 302 + H] + ).

[0208] Synthesis of compound G-10

[0209] 3.00 g of compound G-9 was dissolved in 30 mL of anhydrous acetonitrile, and 2.04 mL of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphate-diamine (CTPPA) and 9 mL of 0.5 M tetrazole-acetonitrile solution were added. The mixture was allowed to react at room temperature for 2 hours under a nitrogen atmosphere. The reaction was monitored by UPLC-MS. After concentration under reduced pressure, 50 mL of DCM was added to dissolve the compound, and 30 mL of water was added. The DCM layer was extracted and separated. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 4.74 g of an oily product. Purification by column chromatography yielded 1.3 g of a white solid. 1 H NMR (400 MHz, DMSO) δ 7.85-7.74 (m, 2H), 7.72-7.62 (m, 1H), 7.41-7.35 (m, 2H), 7.33-7.27 (m, 2H), 7.26-7.18 (m, 5H), 6.88 (dd, 4H), 5.22 (d, 1H), 4.97 (dd, 1H), 4.49 (d, 1H), 4.31-4.18 (m, 1H), 4.06-3.98 (m, 3H), 3.94-3.82 (m, 2H), 3.74 (s, 6H), 3.72-3.65 (m, 2H), 3.65-3.48 (m, 3H), 3.44-3.37 (m, 1H), 3.04-2.94 (m, 2H), 2.91-2.81 (m, 2H), 2.77-2.69 (m, 1H), 2.64-2.58 (m, 1H), 2.36-2.19 (m, 2H), 2.10 (s, 3H), 2.06-1.95 (m, 5H), 1.90 (s, 3H), 1.77 (s, 3H), 1.64-1.54 (m, 1H), 1.51-1.42 (m, 2H), 1.40-1.32 (m, 2H), 1.29-1.22 (m, 3H), 1.20-1.08 (m, 14H), 1.06-1.01 (m, 1H). 31 P-NMR(d6-DMSO):145.79; ESI-MS m / z: [M+Na] +=1158.56

[0210] Synthesis of compound G-11

[0211] 700 mg of compound G-9 was dissolved in 7 mL of anhydrous pyridine, and 10 mg of DMAP and 749 mg of succinic anhydride were added. The mixture was allowed to react at room temperature for 24 hours under a nitrogen atmosphere. After the reaction was completed, monitored by LC-MS, the reaction mixture was concentrated under reduced pressure to an oily substance. The product was purified by silica gel column chromatography (DCM:MeOH = 100:1 to 20:1). The product components were collected and concentrated under reduced pressure to give 0.74 g of compound G-11. 1 H NMR (400 MHz, CDCl3) δ 7.5-7.25(m, 8H), 6.85-6.70(m, 5H), 5.34-5.25(m, 2H),4.69-4.67(m, 1H), 4.13-4.11(m, 3H), 3.80(s, 6H), 3.51-3.45(m, 1H), 2.93-2.87(m, 7H), 2.03-1.97 (m, 10H), 1.32-1.28(m, 8H), 1.24-1.21(m, 12H), 0.91-0.85(m, 3H), 0.09-0.02(m, 4H).MS(ESI) m / z: 734.45 ([(M-302) + H] + ).

[0212] Synthesis of compound G-12

[0213] Compound G-11 (209 mg), LCAA-CPG (96 μmol / g, 1.4 g), HATU (86.8 mg), and DIEA (52.0 mg) were added to a 50 mL centrifuge tube, and the mixture was dissolved in 10 mL of anhydrite acetonitrile and then placed in a shaker and shaken overnight. The reaction mixture was filtered, washed with acetonitrile, and dried by suction filtration. The carrier was poured into a 50 mL centrifuge tube, and 5 mL of CapA (20% NMI-80% ACN) and 5 mL of CapB (20% AC2O-30% lutidine-50% ACN) were added, respectively. After shaking for 2 hours at room temperature, the reaction mixture was filtered, washed with acetonitrile, and dried under vacuum overnight to obtain 1.32 g of compound G-12 with a loading of 62 μmol / g. Example 2 Synthesis of H-17 (His(R)-consecutive-GalNAc)

[0214] The synthesis of H-17 (His(R)-sequential-GalNAc) is shown below (4 steps):

[0215] Step 1: Synthetic route for compound S-05 [ka]

[0216] Under a nitrogen atmosphere, 800 mL of DCM and 57 g of 5-bromo-1-pentanol were added to 120 g of crude compound S-02. 28.5 mL of TMSOTf was added dropwise to the reaction solution. After the addition, the reaction solution was stirred overnight at 25°C. A sample was obtained and used to monitor the completion of the reaction by TLC. The reaction was quenched with 1 L of saturated sodium bicarbonate, and the DCM 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:EA = 10:1 to 2:1). The product eluate was collected, concentrated, and dried to give 98 g of compound S-05. MS (ESI) m / z 497.07, 498.00 ([M+H] + ). 1H-NMR (400MHz, CDCl3) δ 5.43(d, 1H), 5.36(d, 1H), 5.31(dd, 1H), 4.72(d,1H), 4.10-4.20(m, 2H), 3.89-3.97(m, 3H), 3.47-3.52(m, 1H), 3.41(t, 2H), 2.15(s, 3H), 2.05(s, 3H), 2.01(s, 3H), 1.97(s, 3H),1.84-1.91(m, 2H), 1.59-1.65(m, 2H), 1.47-1.54(m, 2H).

[0217] Step 2: Synthetic route for compound M-3

[0218] For the synthesis method, see the synthesis of compound M-3 in the G-12 (Glu(R)-continuous GalNAc) synthetic pathway.

[0219] Step 3: Synthetic route for compound H-05 [ka]

[0220] Synthesis of compound H-02

[0221] 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 H-01 (L-histidine) was added thereto 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] + ).

[0222] Synthesis of compound H-03

[0223] 110 g of compound H-02 was dissolved in 880 mL of anhydrous THF under a 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 hour. 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 230 mL of methanol dropwise. The THF was concentrated, and the residue was extracted with 800 mL of ethyl acetate and 300 mL of saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and dried to give 106 g of crude compound H-03. 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).

[0224] Synthesis of compound H-04

[0225] 106 g of compound H-03 was dissolved in 1 L of dichloromethane, 38 g of imidazole was added, and 128 g of TBDPSCl was added dropwise to the reaction solution. After the addition, the reaction was allowed to proceed at room temperature for 1 hour. The completion of the reaction was monitored using TLC. 400 mL of saturated sodium chloride was added to the reaction solution, and the DCM layer was extracted, washed with saturated sodium chloride, dried over 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 106 g of a white solid. 1H-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).

[0226] Synthesis of compound H-05

[0227] 80 g of compound H-04 was dissolved in 240 mL of glacial acetic acid and stirred overnight at 80°C. After the reaction was completed as monitored by TLC, the mixture was cooled in an ice-water bath, and then 400 mL of ethyl acetate was added. 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 66 g of a white solid. MS (ESI) m / z 480.27 ([M+H] + ).

[0228] Step 4: Synthetic route to compound H-17 [ka] [ka]

[0229] Synthesis of compound H-10

[0230] Under a nitrogen atmosphere, 58 g of compound S-05 was dissolved in 300 mL of anhydrous DMF, and 98.6 g of anhydrous cesium carbonate was added. 78 g of compound H-05 in 200 mL of DMF was added dropwise to the solution. After the dropwise addition, the mixture was stirred at room temperature for 1 hour. After the reaction was completed as monitored by UPLC-MS, the reaction mixture was filtered under reduced pressure, the filter cake was washed with 400 mL of EA, and then 1.2 L of saturated ammonium chloride was added to the filtrate. The EA layer was extracted, separated, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography with gradient elution (DCM:MeOH = 80:1 to 20:1). The product eluate was collected, concentrated, and dried to give 62.74 g of compound H-10. MS (ESI) m / z 895.53 ([M+H] + ).

[0231] 1 H-NMR(400MHz,CDCl3) δ7.62-7.66(m,4H), 7.44(s, 1H), 7.36-7.42(m, 6H), 6.58(s, 1H), 5.95(d, 1H), 5.35-5.39(m, 2H), 5.22(d, 1H), 4.79(d, 1H), 4.12-4.19(m, 2H), 3.89-3.93(m, 2H), 3.80-3.85(m, 2H), 3.65-3.69(m, 2H), 3.39-3.45(m, 1H), 2.83-2.87(m, 2H), 2.13(s, 3H), 2.04(s, 3H), 1.99(s, 3H), 1.90(s, 3H),1.68-1.76(m,2H), 1.54-1.62(m,2H), 1.41(s, 9H), 1.26-1.32(m, 2H),1.07(s, 9H).

[0232] Synthesis of compound H-11

[0233] 62 g of compound H-10 was dissolved in 600 mL of DCM, the solution was cooled to 0-10°C in an ice bath, and 103 mL of TFA was added dropwise to the reaction solution. After the addition, the mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by UPLC-MS, trifluoroacetic acid was removed, 500 mL of DCM was added to the residue, and the pH was then adjusted to 8-9 with saturated sodium bicarbonate. The DCM layer was extracted, separated, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and dried to give 58 g of compound H-11. MS (ESI) m / z 795.50 ([M+H] + ).

[0234] Synthesis of compound H-12

[0235] 13 g of compound M-3 was dissolved in 150 mL of DMF, and 8.23 mL of DIEA and 15.53 g of HBTU were added. After stirring at room temperature for 30 minutes, 25 g of compound H-11 dissolved in 100 mL of DMF was added dropwise to the solution. The reaction mixture was allowed to react at room temperature for 2 hours, and the reaction completion was monitored using TLC. 300 mL of EA and 600 mL of 10% ammonium chloride were added to the reaction solution, and the organic phase 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 30:1). The product eluate was collected, concentrated, and dried to give 19 g of compound H-12. MS (ESI) m / z 1119.69 ([M+H] + ).

[0236] Synthesis of compound H-13

[0237] 22 g of compound H-12 was dissolved in 200 mL of anhydrous THF, 59 mL of TBAF (1 M THF solution) was added, and the mixture was stirred at room temperature for 23 hours to complete the reaction. The mixture was concentrated and purified by silica gel column chromatography using gradient elution (DCM:MeOH = 20:1 to 8:1). The product eluate was collected, concentrated, and dried to give 10 g of compound H-13. MS (ESI) m / z 643.42 ([M+H] + ).

[0238] Synthesis of compound H-14

[0239] Under a nitrogen atmosphere, 10 g of compound H-13 was dissolved in 110 mL of anhydrous pyridine, and 10.6 g of DMTrCl was added. After stirring at room temperature for 20 minutes, the starting material was completely consumed as detected by UPLC-MS. The reaction solution was quenched with methanol (40 mL), concentrated, and purified by silica gel column chromatography using gradient elution (DCM:MeOH = 100:1 to 50:1). The product eluate was collected, concentrated, and dried to give 9.3 g of compound H-14. MS (ESI) m / z 945.56 ([M+H] + ).

[0240] Synthesis of compound H-15

[0241] Under a nitrogen atmosphere, 3 g of compound H-14 was dissolved in 40 mL of anhydrous acetonitrile, and then 2 g of CTPPA and 6.4 mL of 0.5 M tetrazole-acetonitrile solution were added. The mixture was stirred at room temperature for 1.5 hours, and a sample was obtained to monitor the completion of the reaction by UPLC-MS. After concentrating the acetonitrile, the resulting residue was extracted with 50 mL of DCM and 30 mL of water. The DCM layer was then extracted, separated, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and dried to obtain the crude product of compound H-15. 1.5 g of compound H-15 was obtained as a white solid by purifying the crude product by column chromatography. 1H-NMR(400MHz,DMSO-d6) δ8.04(t, 1H), 7.80(d, 1H), 7.47(s, 1H), 7.15-7.42(m, 9H), 6.80-6.94(m,4H), 6.66(s, 1H), 5.75(s, 1H), 5.22(d, 1H), 4.96(dd, 1H), 4.72-4.86(m, 1H), 4.48(d, 1H), 4.06-4.24(m, 3H), 4.02(s, 3H), 3.83-3.93(m, 2H), 3.77-3.83(m, 2H), 3.73(s, 6H), 3.64-3.71(m, 1H), 3.37-3.53(m, 3H), 2.87-2.95(m, 2H), 2.81-2.87(m, 1H), 2.76(dd, 1H), 2.53-2.66(m, 2H), 2.38(dd, 1H), 2.10(s, 3H), 2.01-2.08(m, 1H), 1.98(s, 3H), 1.89(s, 3H), 1.76(s, 3H), 1.57-1.62(m, 2H), 1.44-1.51(m, 2H), 1.28(d, 3H), 1.14-1.22(d, 12H). ESI-MS(ESI)m / z 1167.54 ([M+Na] + ). 31 P-NMR(d6-DMSO): 147.50;

[0242] Synthesis of compound H-16

[0243] Under a nitrogen atmosphere, 1.5 g of compound H-14 was dissolved in 15 mL of anhydrous pyridine, and DMAP (20 mg) and 1.58 g of succinic anhydride were added. After 25 h of reaction at room temperature, the reaction was monitored for completion using UPLC-MS. Pyridine was removed by concentration, and the residue was purified by silica gel column chromatography with gradient elution (DCM:MeOH = 40:1 to 20:1). A portion of the collected product was concentrated and dried to give 1.37 g of compound H-16. MS (ESI) m / z 1045.57 ([M+H] + ). 1H-NMR(400MHz,DMSO-d6) δ7.96(d, 1H), 7.88(d, 1H), 7.44(s, 1H), 7.34-7.41(m, 2H), 7.18-7.31(m, 7H), 6.82-6.89(m, 4H), 6.64(s, 1H), 5.22(d, 1H), 5.04-5.09(m, 1H), 4.97(dd, 1H), 4.50(d, 1H), 4.11-4.23(m, 1H), 4.02(s, 3H), 3.83-3.93(m, 2H), 3.79(t, 2H), 3.73(s, 6H), 3.65-3.72(m, 2H), 3.36-3.42(m, 2H), 2.90-2.94(m, 1H), 2.82-2.85(m, 1H), 2.72-2.79(dd, 1H), 2.56-2.61(dd, 1H), 2.35-2.45(dd, 2H), 2.27-2.32(dd, 2H), 2.10(s, 3H), 1.98(s, 3H), 1.89(s, 3H), 1.76(s, 3H), 1.56-1.64(m, 2H), 1.43-1.50(m, 2H), 1.12-1.20(m, 2H), 1.13(d, 3H).

[0244] Synthesis of compound H-17

[0245] 180 mg of compound H-16 was dissolved in 10 mL of acetonitrile, and then 75 mg of HATU and 42 mg of DIEA were added. The mixture was shaken in a shaker for 10 minutes. LCAA-CPG- (96 μmol / g) was added to the mixture and allowed to react overnight at room temperature in a shaker. The reaction solution was filtered, washed with acetonitrile, and the filter cake was dried under vacuum and poured into a 50 mL centrifuge tube. 5 mL of Cap A (20% NMI-80% ACN) and 5 mL of Cap B (20% AC2O-30% lutidine-50% ACN) were then added to the centrifuge tube. After shaking the mixture for 2 hours, the solvent was removed by filtration, washed with acetonitrile, and dried under vacuum at room temperature for 2 hours to obtain 1.17 g of compound H-17 with a loading of 47 μmol / g. Example 3 Synthesis of H-23 (His(S)-consecutive-GalNAc)

[0246] The synthesis of H-23 (His(S)-sequential-GalNAc) is shown below (2 steps):

[0247] Step 1: Synthetic route to compound M-6 [ka]

[0248] Synthesis of compound M-6

[0249] Methyl (R)-3-hydroxybutanoate was replaced with methyl (S)-3-hydroxybutanoate. For the synthesis method, see M-3 synthesis in G-12 (Glu(R)-contiguous GalNAc).

[0250] Step 2: Synthetic route to compound H-23 [ka]

[0251] Synthesis of compound H-18

[0252] For the synthesis method, see the synthesis of compound H-12 in the H-17 (His(R)-sequential-GalNAc) synthesis route. MS (ESI) m / z 1119.72 ([M+H] + ).

[0253] Synthesis of compound H-19

[0254] For the synthesis method, see the synthesis of compound H-13 in the H-17 (His(R)-sequential-GalNAc) synthesis route. MS (ESI) m / z 643.46 ([M+H] + ).

[0255] Synthesis of compound H-20

[0256] For the synthesis method, see the synthesis of compound H-14 in the H-17 (His(R)-sequential-GalNAc) synthesis route. MS (ESI) m / z 945.62 ([M+H] + ).

[0257] Synthesis of compound H-21

[0258] For the synthesis method, please refer to the synthesis of compound H-15 in the H-17 (His(R)-sequential-GalNAc) synthesis route. Due to the special chemical properties of H-15, it was identified by mass spectrometry as MS(ESI) m / z 1062.43 ([M-(i-Pr)2N+HO] + ) shows the characteristic structural fragment peaks. 1 H-NMR(400MHz,DMSO-d6) δ7.92(t, 1H), 7.73(d, 1H), 7.36(s, 1H), 7.05-7.33(m, 9H), 6.72-6.84(m, 4H), 6.55(s, 1H), 5.63(s, 1H), 5.16(d, 1H), 4.83(dd, 1H), 4.62-4.73(m, 1H), 4.36(d, 1H), 3.08-4.14(m, 3H), 3.91(s, 3H), 3.73-3.80(m, 2H), 3.63-3.73(m, 2H), 3.63(s, 6H), 3.54-3.66(m, 1H), 3.27-3.43(m, 3H), 2.77-2.85(m, 2H), 2.71-2.75(m, 1H), 2.66(dd, 1H), 2.43-2.58(m, 2H), 2.29(dd, 1H), 2.10(s, 3H), 2.01-2.07(m, 1H), 1.91(s, 3H), 1.86(s, 3H), 1.71(s, 3H), 1.47-1.58(m, 2H), 1.39-1.48(m, 2H), 1.26(d, 3H), 1.08-1.1.20(d, 12H)

[0259] Synthesis of compound H-22

[0260] For the synthesis method, see the synthesis of compound H-16 in the H-17 (His(R)-sequential-GalNAc) synthetic pathway.

[0261] MS(ESI) m / z 1045.66 ([M+H] + ). 1 H-NMR(400MHz,DMSO-d6) δ7.96(d, 1H), 7.88(d, 1H), 7.44(s, 1H), 7.34-7.41(m, 2H), 7.18-7.31(m, 7H), 6.82-6.89(m, 4H), 6.64(s, 1H), 5.22(d, 1H), 5.04-5.09(m, 1H), 4.97(dd, 1H), 4.50(d, 1H), 4.11-4.23(m, 1H), 4.02(s, 3H), 3.83-3.93(m, 2H), 3.79(t, 2H), 3.73(s, 6H), 3.65-3.72(m, 2H), 3.36-3.42(m, 2H), 2.90-2.94(m, 1H), 2.82-2.85(m, 1H), 2.72-2.79(dd, 1H), 2.56-2.61(dd, 1H), 2.35-2.45(dd, 2H), 2.27-2.32(dd, 2H), 2.10(s, 3H), 1.98(s, 3H), 1.89(s, 3H), 1.76(s, 3H), 1.56-1.64(m, 2H), 1.43-1.50(m, 2H), 1.12-1.20(m, 2H), 1.13(d, 3H).

[0262] Synthesis of compound H-23

[0263] For synthesis method, see the synthesis of compound H-17 in the H-17 (His(R)-sequential-GalNAc) synthesis pathway. H-23 at a loading of 52 μmol / g. Example 4 Synthesis of GS-13-1 (Glu(R)-consecutive-GalNAc)

[0264] The synthesis of GS-13-1 (Glu(R)-sequential-GalNAc) is shown below. [ka] [ka]

[0265] Synthesis of compound GS-13-11

[0266] The starting material was changed to N-Boc-L-glutamic acid benzyl ester, and the synthesis method was the same as that of H-10. MS(ESI) m / z 752.30 ([M+H] + ).

[0267] Synthesis of compound GS-13-10

[0268] The synthesis method was the same as that of H-11. MS(ESI) m / z 652.30 ([M+H] + ).

[0269] Synthesis of compound GS-13-9

[0270] The synthesis method was the same as that of H-12. MS(ESI) m / z 976.43 ([M+H] + ).

[0271] Synthesis of compound GS-13-8

[0272] GS-13-9 was dissolved in anhydrous methanol, 10% (w / w) palladium on carbon was added, and the air was replaced with hydrogen three times. The mixture was stirred at room temperature until the starting material was completely consumed. The palladium on carbon was then removed by filtration, and the filtrate was dried and used directly in the next step. MS(ESI) m / z 884.40 ([M−H] - ).

[0273] Synthesis of compound GS-13-7

[0274] Equal amounts of GS-13-8 and GS-13-10 were dissolved in anhydrous dichloromethane, and DMAP (0.1 equivalents), DCC (2.0 equivalents), and DIEA (2.0 equivalents) were added. The mixture was stirred at room temperature under a nitrogen atmosphere until the starting materials were completely consumed. The insoluble matter was removed by filtration, the reaction solution was washed with purified water, and the organic phase was concentrated to dryness. The crude product was then purified by silica gel column chromatography (DCM:MeOH=10:1) and dried to give a white solid. MS (ESI) m / z 1519.60 ([M+H] + ).

[0275] Synthesis of compound GS-13-6

[0276] The synthesis method was the same as that of GS-13-8. MS(ESI) m / z 1427.61 ([M−H] - ).

[0277] Synthesis of compound GS-13-5

[0278] The synthesis method was the same as that of GS-13-7, and the starting materials were GS-13-6 and GS-13-14. MS(ESI) m / z 1066.01 ([(M+2) / 2] + ).

[0279] Synthesis of compound GS-13-4

[0280] The synthesis method was the same as that of H-13. MS(ESI) m / z 889.41 ([(M+2) / 2] + ).

[0281] Synthesis of compound GS-13-3

[0282] The synthesis method was the same as that of H-14. MS(ESI) m / z 1040.92 ([(M+2) / 2] + ).

[0283] Synthesis of compound GS-13-2

[0284] The synthesis method was the same as that of H-16. MS(ESI) m / z 1091.15 ([M+2) / 2] + ).

[0285] Synthesis of compound GS-13-1

[0286] The synthesis method was the same as that of H-17, and the loading was 50 μmol / g. Example 5 Synthesis of HS-13-1 (His(R)-consecutive-GalNAc) [ka] [ka]

[0287] Synthesis of compound HS-13-11

[0288] The starting material was changed to N-Boc-3-L-histidine benzyl ester, and the synthesis method was the same as that of H-10. MS(ESI) m / z 761.30 ([M+H] + ).

[0289] Synthesis of compound HS-13-10

[0290] The synthesis method was the same as that of H-11. MS(ESI) m / z 661.30 ([M+H] + ).

[0291] Synthesis of compound HS-13-9

[0292] The synthesis method was the same as that of H-12. MS(ESI) m / z 985.46 ([M+H] + ).

[0293] Synthesis of compound HS-13-8

[0294] HS-13-9 was dissolved in anhydrous methanol, 10% (w / w) palladium on carbon was added, and the air was replaced with hydrogen three times. The mixture was stirred at room temperature until the starting material was completely consumed. The palladium on carbon was then removed by filtration, and the filtrate was dried and used directly in the next step. MS(ESI) m / z 893.40 ([M−H] - ).

[0295] Synthesis of compound HS-13-7

[0296] Equal amounts of HS-13-8 and HS-13-10 were dissolved in anhydrous dichloromethane, and DMAP (0.1 equivalents), DCC (2.0 equivalents), and DIEA (2.0 equivalents) were added. The mixture was stirred at room temperature under a nitrogen atmosphere until the starting materials were completely consumed. The insoluble matter was removed by filtration, the reaction solution was washed with purified water, and the organic phase was concentrated to dryness. The crude product was then purified by silica gel column chromatography (DCM:MeOH = 10:1) and dried to give a white solid. MS (ESI) m / z 1537.60 ([M+H] + ).

[0297] Synthesis of compound HS-13-6

[0298] The synthesis method was the same as that of HS-13-8. MS(ESI) m / z 1445.60 ([M−H] - ).

[0299] Synthesis of compound HS-13-5

[0300] The synthesis method was the same as that of HS-13-7, and the starting materials were HS-13-6 and HS-13-14. MS(ESI) m / z 1079.52 ([(M+2) / 2] + ).

[0301] Synthesis of compound HS-13-4

[0302] The synthesis method was the same as that of H-13. MS(ESI) m / z 903.44 ([(M+2) / 2] + ).

[0303] Synthesis of compound HS-13-3

[0304] The synthesis method was the same as that of H-14. MS(ESI) m / z 1054.45 ([(M+2) / 2] + ).

[0305] Synthesis of compound HS-13-2

[0306] The synthesis method was the same as that of H-16. MS(ESI) m / z 1104.66 ([M+2) / 2] + ).

[0307] Synthesis of compound HS-13-1

[0308] The synthesis method was the same as that of H-17, and the loading was 50 μmol / g. Example 6 Synthesis of conjugated oligonucleotides provided by the present invention

[0309] 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: Preparation of oligoconjugate G-12 (Glu(R)-consecutive-GalNAc) [ka]

[0310] Solid Phase Synthesis Steps

[0311] Using a phosphoramidite solid-phase synthesis method known in the art, G-12 was used as a solid-phase synthesis support, and a series of commands was performed on a MerMade192 solid-phase synthesizer, in which the linking position of compound G-10 was set to the 3' end of the series and the number of sequences was also set.

[0312] Each connection of nucleoside monomers involved four steps: deprotection, coupling, capping, and oxidation, and standard procedures for the above steps are known to those skilled in the art. G-10 solution was prepared in 0.1 M acetonitrile solution.

[0313] 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 Oxidizing reagent: 0.02M I2 in THF / pyridine / H2O Sulfuration reagent: 0.10M DDTT solution

[0314] For example, the solid phase synthesis conditions for a 1 μmol synthesis scale are as follows: [Table 10]

[0315] Cleavage and deprotection steps

[0316] 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 a white precipitate. The supernatant was removed by centrifugation at 10,000 g to obtain the precipitated product, which was then redissolved in 0.2 M sodium acetate solution.

[0317] Purification, desalting and lyophilization steps

[0318] Purification was carried out on an Avant 150 purification system using an ion chromatography column (loaded with packing material Nano Q 30) with a volume of 1 mL.

[0319] The 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 to 50% Buffer B, 100 to 50% Buffer A is.

[0320] The eluate was collected and combined, and finally used on a G25 Sephadex column for desalting; the OD260 concentration value of the desalted product solution was measured, and the product content was calculated, and finally placed in a centrifuge tube for lyophilization to obtain a white freeze-dried product.

[0321] Detection: Reverse phase UPLC-MS tandem mass spectrometry was used for detection, purity was greater than 90%, m / z [M-7 / 7] - , [M-8 / 8] - , [M-9 / 9] - The characteristic ion peaks of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,

[0322] In the example of the synthesis of AS-Oligo, G-12-OLIGO was used, and Unylinker-CPG (manufactured by Glen Research) was used as the solid-phase synthesis support for the synthesis.

[0323] In the synthesis example of the oligo-conjugated His-R-contiguous-GalNAc, G-12-OLIGO was used as the reference and H-17 was used as the solid-phase synthesis support for the synthesis. The only difference was that H-15 was prepared in 0.1 M acetonitrile solution, and the sequence was set up on a solid-phase synthesizer.

[0324] In the synthesis example of the oligo-conjugated His-S-contiguous-GalNAc, G-12-OLIGO was used as the reference and H-23 was used as the solid-phase synthesis support for the synthesis. The only difference was that H-21 was prepared in 0.1 M acetonitrile solution, and the sequence was set up on a solid-phase synthesizer.

[0325] siRNA-GalNAc conjugates or aiRNA-GalNAc conjugates were prepared by annealing the above-obtained Oligo-GalNAC conjugates with their complementary antisense strands at a 1:1 molar ratio to obtain double-stranded products. Activity test material and method

[0326] Synthesis of conjugated aiRNA for testing: [ka] Compounds HS-9, HS-5, and HS-6 are designed tri-contiguous 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, and are represented by the codes "His-seq(3GalNAc)," "His(R)-seq(3GalNAc)," and "His(S)-seq(3GalNAc)" when used and described in the Examples below. Compounds GS-9 and GS-5 are designed tri-contiguous 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, and are represented by the codes "Glu-seq(3GalNAc)" and "Glu(R)-seq(3GalNAc)" when used and described in the Examples below.

[0327] The sequences and structures of the oligonucleotides (aiRNA and siRNA) synthesized and used in the activity testing examples are shown in Table 7 below. [Table 7]

[0328] Unless otherwise specifically stated, the ex vivo delivery efficiency of the conjugates 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) of EDTA to 500 mL of HBSS. Buffer B: Add 400 mg of collagenase type I (0.8 mg / mL) to 500 mL of DMEM. Buffer C: Add 2 mg (2%) BSA to 100 mL of DMEM. Part-B: Isolation After perfusion, in the hood, place the liver in a 10 cm TC dish, open the liver sack, and shake 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 and gently resuspend in 50 ml of Buffer C (Wash 1) and centrifuge at 50 g for 5 minutes at 4°C. Discard the supernatant and gently resuspend in 50 ml of Buffer C (Wash 2) and centrifuge at 50 g for 5 minutes at 4°C. Discard the supernatant and gently resuspend in 50 ml of Buffer C (Wash 3) and centrifuge at 50 g for 5 minutes at 4°C. Discard the supernatant and resuspend in thawing / seeding medium. Count using trypan blue to assess viability / yield. Seed the cells onto collagenase-coated plates (Thermos Fisher, A1142802) using mouse primary hepatocyte thawing medium (Thermos Fisher, CM3000). It is best to seed 1 mL per well of a 24-well plate. After 3-4 hours, replace the medium with primary hepatocyte maintenance medium (thermofisher, CM4000).

[0329] Ex vivo self-delivery assays were performed without the use of transfection reagents (free uptake) (tested in 12-well plates at 100,000 cells / well for 48 hours of incubation). The tested Oligo GalNAc conjugate concentrations are marked below in each example. The expression level of targeted mRNA was detected by RT-qPCR. Example 7 Ex vivo uptake of aiRNA by hepatocytes with and without conjugation

[0330] The "Glu(R)-seq(3GalNAc)"-conjugated mβ-catenin aiRNA (aiRNA#1) exhibited significant gene silencing activity at 10 nM and even 1 nM in self-delivery ex vivo assays compared with unconjugated aiRNA, demonstrating that the GalNAc conjugates provided by the present invention have great efficacy for the delivery of double-stranded RNAi agents such as aiRNA. The results were detected by QPCR as shown in Figure 2. Example 8 Ex vivo uptake of aiRNA by hepatocytes using GalNac conjugates

[0331] Two different GalNAc conjugated mβ-catenin aiRNAs (aiRNA#2) provided by the present invention were co-cultured with isolated mouse hepatocytes for 24 hours at concentrations of 303 nM, 92 nM, and 28 nM. The results were tested by QPCR and are shown in Figure 3. Glu-seq(3GalNAc), His(R)-seq(3GalNAc), and His(S)-seq(3GalNAc) all exhibited highly potent gene silencing upon ex vivo self-delivery, demonstrating that the GalNAc conjugates provided by the present invention are highly effective for the delivery of double-stranded RNAi agents such as aiRNA. Example 9 Ex vivo uptake of mouse TTR-targeting aiRNA by hepatocytes using consecutive histidine-GalNAc conjugates

[0332] The gene silencing activity of the newly designed mouse TTR-targeting aiRNA "His-seq(3GalNAc)" conjugates, as shown in Table 8 below, was tested in freshly isolated mouse primary hepatocytes in the absence of transfection agents (free uptake). Ex vivo free uptake-mediated gene silencing evaluates the receptor-mediated intracellular transport enabled by the GalNAc conjugates provided by the present invention.

[0333] 50 μL of aiRNA conjugate (793-804) and 950 μL of cell culture medium (Thermofisher, CM4000) containing approximately 100,000 primary mouse hepatocytes were added to wells of a 12-well plate. Cells were incubated at 37°C and 5% CO2 for 24 hours. RNA was purified, and mRNA levels were determined by RT-qPCR. Values are plotted as fractions relative to untreated control cells. All aiRNA conjugates were tested at a concentration of 0.3 nM. GAPDH served as an internal standard. [Table 8]

[0334] The results are shown in Figure 4. All aiRNA conjugates via the conjugate composition of the present invention exhibited potent gene silencing activity in the ex vivo self-delivery assay at 0.3 nM, indicating that the GalNAc conjugates provided by the present invention have great potency for the delivery of various double-stranded RNAi agents.

[0335] The results in all activity test examples clearly demonstrate that the GalNAc conjugate design based on the present invention can dramatically enhance the delivery efficiency of oligonucleotides ex vivo and achieve high gene silencing efficacy for targeting various genes in liver cells. Furthermore, the linker composition provided in the present invention is based on amino acids found in our body, which eliminates the safety risks of other types of linkers used in GalNAc conjugates.

[0336] 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 exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

[0337] 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 (S-HG1): <h2 style=";text-align:left;direction:ltr">1<h2 style=";text-align:left;direction:ltr"> ２０６ <h2 style=";text-align:left;direction:ltr"> -[A11<h2 style=";text-align:left;direction:ltr"> ａ <h2 style=";text-align:left;direction:ltr"> -[A2]<h2 style=";text-align:left;direction:ltr"> ｂ <h2 style=";text-align:left;direction:ltr"> -[A3]<h2 style=";text-align:left;direction:ltr"> ｃ <h2 style=";text-align:left;direction:ltr"> 11<h2 style=";text-align:left;direction:ltr"> ２０５ <h2 style=";text-align:left;direction:ltr"> (5, 1, 2) A compound having the formula: R ２０５ , R ２０６ represents independently at each occurrence H, 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(M')(M'')O-nucleoside, -OP(M')(M'')O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, -O-nucleotide, a nucleoside, -OP(M')(M'')O-R ２０１ -OP(M''')(M'''')O-oligonucleotide, or oligonucleotide; M', M'', M''', and M'''' are each independently at each occurrence O or S; A1, A2 and A3 are each independently at each occurrence (S-1H) or (S-1G): 【Hua 62】 Selected from: R ２０２Ａ is -R ２０２ -R ２０２Ｌ and R ２１７Ａ is -R ２１７ -R ２１７Ｌ and R ２０２Ｌ , R ２１７Ｌ is one ligand that can dock to a cell surface receptor independently for each occurrence; R ２０２ , R ２１７ , R ２０１ is each independently selected at each occurrence from alkylene of 3 to 30 carbon atoms, wherein one or more carbon atoms are selected from 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 and optionally substituted with any one or more substituents from the group consisting of heteroarylene, R ２０２ , R ２１７ , R ２０１ are each independently optionally unsubstituted or R ２０９ and optionally substituted by R ２０２ , R 217 , R ２０１ is independently selected at each occurrence from alkylene of 3 to 15 carbon atoms, wherein one or more carbon atoms are optionally replaced by one or more substituents from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O; R ２０４ , R ２０７ , R ２０８ , R ２１３ , R ２１４ , R ２１５ , R ２１６ is, independently for each occurrence, H; R 1 , R 2 , R ２０９ is independently at each occurrence H, alkyl, aryl, heteroaryl, haloalkyl, —Oalkyl, —Oalkylphenyl, -alkyl-OH, —Ohaloalkyl, —Salkyl, —Salkylphenyl, -alkyl-SH, —Shaloalkyl, halo, —OH, —SH, —NH ２ , -alkyl-NH ２ , -N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, -CO ２ H, —C(O)Oalkyl, —CON(alkyl)(alkyl), —CONH(alkyl), —CONH ２ , —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, —SO ２ (alkyl), —SO ２ (phenyl), -SO ２ (haloalkyl), —SO ２ NH ２ , -SO ２ NH(alkyl), -SO ２ NH(phenyl), -NHSO ２ (alkyl), -NHSO ２ (phenyl), and —NHSO ２ (haloalkyl); n ２０１ , n ２１１ is independently 1 for each occurrence; J ２０１ , J ２０２ , J ２１１ , J ２１２ are each independently selected at each occurrence from alkylene of 1 to 30 carbon atoms, wherein one or more carbon atoms are optionally replaced by 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 , 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; and J 201 , J 202 , J 211 , J 212 are each independently unsubstituted or optionally substituted, or selected from H, alkyl, aryl, heteroaryl, haloalkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -Salkylphenyl, -alkyl-SH, -Shaloalkyl, halo, -OH, -SH, -NH 2 , -alkyl-NH 2 , -N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, -CO 2 H, -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), -CONH 2 , -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, -SO 2 (alkyl), —SO 2 (phenyl), —SO 2 (haloalkyl), —SO 2 NH 2 , —SO 2 NH(alkyl), —SO 2 NH(phenyl), —NHSO 2 (alkyl), —NHSO 2 (phenyl), and —NHSO 2 (haloalkyl); a, b, and c are each independently at each occurrence an integer from 0 to 5, and the sum of a, b, and c is an integer from 1 to 10; The oligonucleotides contain naturally occurring or chemically modified nucleotides / nucleosides. compound.

2. The compound has structural formula (S-H1): 【Chemistry 63】 wherein R １ is H, C １ ~C ５ Alkyl, aryl, heteroaryl, C １ ~C ５ Haloalkyl, —C １ ~C ５ Alkyl-OH, -C １ ~C ５ Alkyl-SH, -C １ ~C ５ Alkyl-NH ２ , -CO ２ H, —C(O)Oalkyl, —CON(alkyl)(alkyl), —CONH(alkyl), —CONH ２ , —C(O)alkyl, —C(O)alkylphenyl, —C(O)haloalkyl, —SO ２ (alkyl), —SO ２ (haloalkyl), —SO ２ NH ２ , -SO ２ NH(alkyl), and -SO ２ NH (phenyl); n ２０２ is selected from 1 to 10, preferably 1 to 3; The compound of claim 1.

3. n ２０２ The compound of claim 2, wherein is 3.

4. J ２０１ , J ２０２ is independently selected at each occurrence from alkylene of 1 to 10 carbon atoms, wherein one or more carbon atoms are selected from C(O), NH, O, S, OP(O)O, OP(S)O, CH═N, and S(O) ２ and J is optionally substituted with any one or more substituents from the group consisting of ２０１ , J ２０２ are each independently optionally unsubstituted or a group: H, or C １ ~C ５ Alkyl, —OC １ ~C ５ 10. The compound of claim 1, substituted with at least one group selected from alkyl.

5. The compound has structural formula (S-H1-06), (S-H1-07), or (S-H1-08): 【Hua 67】 【Chemistry 68】 wherein A is O or S; X is independently selected from Table 1 below, or alkylene of 1 to 10 carbon atoms, wherein 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, and S(O) 2 ; 【Table 1】 X 1 is independently selected from Table 2 below; 【Table 2】 J 202 is independently selected from Table 3 below; 【Table 3】 Optionally, J 202 is independently selected from alkylene of 1 to 10 carbon atoms, wherein 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, and S(O) 2 , and each J 202 is independently optionally unsubstituted or substituted with at least one group selected from the group: H, or C 1 -C 5 alkyl, —OC 1 -C 5 alkyl; R, R' are each independently selected from the group consisting of naturally occurring and / or chemically modified oligonucleotides, H, and protecting groups for OH; at least one of R and R' comprises an oligonucleotide formed by naturally occurring and / or chemically modified nucleotides / nucleosides; R 202 is selected from straight chain alkylene of 3 to 15 carbon atoms, wherein 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; R 202 is optionally unsubstituted or H, alkyl, haloalkyl, -Oalkyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -alkyl-SH, -Shaloalkyl, halo, -OH, -SH, -NH 2 , -alkyl-NH 2 , -N(alkyl)(alkyl), -NH(alkyl), cyano, nitro, -CO 2 H, -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), -CONH 2 , -NHC(O)(alkyl), -NHC(O)(phenyl), -N(alkyl)C(O)(alkyl), -C(O)alkyl, -C(O)haloalkyl, -OC(O)alkyl, -SO 2 (alkyl), -SO 2 (haloalkyl), -SO 2 NH 2 , -SO 2 NH(alkyl), -NHSO 2 (alkyl), and -NHSO 2 (haloalkyl); Optionally, R 202 is —C 3 -C 8 straight chain alkylene- or selected from Table 4 below: 【Table 4】 The compound of claim 2.

6. The compound has structural formula (S-H1-09), (S-H1-10), or (S-H1-11): 【Chemical Formula 69】 6. The compound of claim 5 having the formula:

7. Structural formula (S-H1-12), (S-H1-13) or (S-H1-14): 【Chemistry 70】 [Chemical Formula 71] 6. The compound of claim 5 having the formula:

8. The compound has structural formula (S-G1): 【Chemistry 75】 wherein R ２ is H, C １ ~C ５ Alkyl, aryl, heteroaryl, C １ ~C ５ Haloalkyl, —C １ ~C ５ Alkyl-OH, -C １ ~C ５ Alkyl-SH, -C １ ~C ５ Alkyl-NH ２ , -CO ２ H, —C(O)Oalkyl, —CON(alkyl)(alkyl), —CONH(alkyl), —CONH ２ , —C(O)alkyl, —C(O)alkylphenyl, —C(O)haloalkyl, —SO ２ (alkyl), —SO ２ (haloalkyl), —SO ２ NH ２ , -SO ２ NH(alkyl), and -SO ２ NH (phenyl); n ２１２ is selected from 1 to 10, preferably 1 to 3; The compound of claim 1.

9. n ２１２ The compound of claim 8, wherein is 3.

10. J ２１１ , J ２１２ is independently selected at each occurrence from alkylene of 1 to 10 carbon atoms, wherein one or more carbon atoms are selected from C(O), NH, O, S, OP(O)O, OP(S)O, CH═N, and S(O) ２ and J is optionally substituted with any one or more substituents from the group consisting of ２１１ , J ２１２ are each independently optionally unsubstituted or a group: H, or C １ ~C ５ Alkyl, —OC １ ~C ５ 9. The compound of claim 8, substituted with at least one group selected from alkyl.

11. The compound has structural formula (S-G1-06), (S-G1-07), or (S-G1-08): 【Chemistry 80】 wherein A is O or S; X is independently selected from Table 1 below, or alkylene of 1 to 10 carbon atoms, wherein 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, and S(O) 2 ; 【Table 1】 X 1 is independently selected from Table 2 below; 【Table 2】 J 212 is independently selected from Table 3 below; 【Table 3】 optionally, J 212 is independently selected from alkylene of 1 to 10 carbon atoms, wherein 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, and S(O) 2 , and J 212 is optionally unsubstituted or substituted with at least one group selected from the group: H, or C 1 -C 5 alkyl, —OC 1 -C 5 alkyl; R, R' are each independently selected from the group consisting of naturally occurring and / or chemically modified oligonucleotides, H, and protecting groups for OH; At least one of R and R' comprises an oligonucleotide formed by natural and / or chemically modified nucleotides / nucleosides; R 217 is selected from straight chain alkylene of 3 to 15 carbon atoms, wherein 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; R 217 is optionally unsubstituted or selected from H, alkyl, haloalkyl, -Oalkyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -alkyl-SH, -Shaloalkyl, halo, -OH, -SH, -NH 2 , -alkyl-NH 2 , -N(alkyl)(alkyl), -NH(alkyl), cyano, nitro, -CO 2 H, -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), -CONH 2 , -NHC(O)(alkyl), -NHC(O)(phenyl), -N(alkyl)C(O)(alkyl), -C(O)alkyl, -C(O)haloalkyl, -OC(O)alkyl, -SO 2 (alkyl), -SO 2 (haloalkyl), -SO 2 NH 2 , -SO 2 NH(alkyl), -NHSO 2 (alkyl), and -NHSO 2 (haloalkyl); Optionally, R 217 is —C 3 -C 8 straight chain alkylene- or selected from Table 4 below: 【Table 4】 The compound of claim 8.

12. The compound has structural formula (S-G1-09), (S-G1-10), or (S-G1-11): 【Chemistry 81】 12. The compound of claim 11 having the formula:

13. The compound has structural formula (S-G1-12), (S-G1-13), or (S-G1-14): 【Chemistry 82】 12. The compound of claim 11 having the formula:

14. The compound of claim 2 or 8, wherein R 206 comprises an oligonucleotide.

15. The compound of any one of claims 5 to 7, 11 to 13, wherein each A is O.

16. The compound of any one of claims 5 to 7, 11 to 13, wherein at least one A is S.

17. 14. The compound of any one of claims 1 to 13, wherein each ligand is independently selected from the group consisting of N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GlcNAc), 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.

18. The compound according to any one of claims 1 to 13, wherein the ligand is N-acetylgalactosamine (GalNAc).

19. The compound has the formula HS-1 to HS-9, GS-1 to GS-9: 【Chemical 73】 【Chemical 74】 【Chemistry 13-1】 【Chemistry 85】 【Chemistry 26-1】 2. The compound of claim 1 having the structure:

20. 2. The compound of claim 1, wherein the compound has the structure of formula HS-5, HS-9, GS-5, GS-9.

21. The compound according to any one of claims 1 to 13, 19 to 20, wherein the naturally occurring or chemically modified oligonucleotide is linked to the remainder of the compound via its 5'-end and / or 3'-end.

22. 22. The compound of claim 21 , wherein the oligonucleotide comprises a small interfering RNA (siRNA) duplex, an asymmetric interfering RNA (aiRNA) duplex, an antisense oligonucleotide (ASO), or a microRNA (miRNA).

23. 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 to 9 nucleotides and a 5'-overhang of 0 to 8 nucleotides; 23. The compound of claim 22, wherein the sense strand has a length of 10, 11, 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.

24. The compound described in claim 23, wherein (i) the antisense strand of the aiRNA, when duplexed with the sense strand, comprises a 5'-overhang of 1 to 8 nucleotides, or (ii) the antisense strand of the aiRNA, when duplexed with the sense strand, comprises a 5' blunt end.

25. A pharmaceutical composition comprising a compound according to claims 1 to 13, 19 to 20 and a pharmaceutically acceptable excipient, carrier or diluent.

26. A composition comprising a compound of claims 1-13, 19-20, or a pharmaceutical composition comprising a compound of claims 1-13, 19-20 and a pharmaceutically acceptable excipient, carrier, or diluent, for treating a disease or condition.

27. The compound of claim 2 or 8, wherein n 202 and n 212 are 1, R 206 is selected from OH or a protecting group for OH, and R 205 is a phosphoramidite.

28. Structural formulas (G-P5), (G-P6), (G-P11), and (G-P12): 【Chemistry 92】 【Chemistry 93】 【Chemistry 100】 【Chemistry 101】 28. The compound of claim 27, having the formula:

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