Novel compositions of conjugated oligonucleotides and carbohydrates

Abstract

The present invention provides composition linker configurations for conjugating oligonucleotides to carbohydrate ligands for targeted delivery of oligonucleotides in vivo. These ligand-conjugated compounds target one or more organ or cell types, such as parenchymal cells of the liver that promote endocytosis uptake. The compounds have the structural formula (S-HG1): R206-[A1] a-[A2] b-[A3] c-R205, R205, R206 for each occurrence independently of H, OH, a protecting group of OH, a phosphoric acid group, a phosphodieester group, an activated phosphoric acid group, an activated phosphite group, a phosphoramidite, a solid support,-OP (M ') (M '') O-nucleoside,-OP (M') (M'') O-oligonucleotide, a lipid, PEG, a steroid, a polymer,-O-nucleotide, nucleoside,-OP (M ') (M '') O-R201-OP (M''') (M '''') O-oligonucleotide, or oligonucleotide; m ', M' ', M' ''and M'' ''are each independently O or S for each occurrence; a1, A2 and A3 are each independently selected from (S-1H) or (S-1G) for each occurrence. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to new compositions and methods useful for conjugating carbohydrate ligands to oligonucleotides for biomedical applications. Background Art

[0002] Gene regulation, especially gene-silencing oligonucleotides, has been the focus of many research and development efforts because of the great promise of this string of nucleotides in treating or preventing many diseases and modulating physiological conditions. Examples of these oligonucleotides include short / small interfering RNA (siRNA), asymmetric short / small interfering RNA (aiRNA), antisense oligonucleotides (ASO), and microRNA (miRNA).

[0003] RNA interference (RNAi) functions in a gene-specific manner in many organisms through short double-stranded RNA (dsRNA) duplexes called siRNAs. siRNAs have a well-defined symmetric short (usually 20-24 base pairs) dsRNA duplex structure with phosphorylated 5'-ends and hydroxylated 3'-ends, forming two 3'-overhangs of equal length. Gene regulation is mediated by the multiprotein RNA-induced silencing complex (RISC), which binds, unwinds, and incorporates the antisense siRNA strand from the siRNA duplex and then recognizes and targets complementary messenger RNA (mRNA) for cleavage, thereby reducing its gene expression post-transcriptionally.

[0004] In a relatively new scenario, aiRNAs have been developed to overcome off-target effects mediated by the sense strand of standard siRNAs with symmetric configurations as well as other off-target mechanisms of siRNAs (see PCT patent publication WO2009029688). aiRNAs are designed to contain short RNA duplexes in which the two RNA strands are of unequal length and are thus called "asymmetric". For example, an aiRNA can include a first strand that is 18-23 nucleotides in length and a second strand that is 12-17 nucleotides in length, thereby forming a duplex with a 3'-overhang of 1-9 nucleotides and a 5'-overhang of 0-8 nucleotides in the first strand. The aiRNA technology can be used in all areas where current siRNA or short hairpin RNA (shRNA) is being applied, including biological research, research and development (R&D) in the biotechnology and pharmaceutical industries, and RNAi-based therapies.

[0005] Antisense technology is a highly selective gene silencing technology based on the concept initially proposed in 1978 (Zamecnik P.C. et al., 1978). Generally, the principle behind ASO technology is that antisense oligonucleotides hybridize with target nucleic acids and regulate gene expression through post-transcriptional mechanisms. Its mechanisms can be roughly divided into: (1) simply occupying the position without promoting RNA degradation, where the binding of ASO leads to translation stalling, splicing inhibition, or induction of alternative splicing variants, or (2) occupancy-induced destabilization, where the binding of ASO promotes the degradation of RNA through endogenous enzymes such as ribonuclease H1 (RNase H1); and (3) enhancing translation: ASO can block upstream open reading frames (uORFs) or other inhibitory elements in the 5’UTR region to improve translation efficiency (Stanley T. Crooke et al., 2008; C. Frank Bennett, 2010; Richard G. Lee, 2013; Stanley T. Crooke, 2017). The typical structure of ASO is a single-stranded deoxynucleotide sequence with sulfur chemical modifications (called phosphorothioates). After 40 years of research, antisense technology has been improved through various chemical modifications of single-stranded oligonucleotides.

[0006] miRNA molecules usually originate from the non-coding regions of RNA transcripts, and these non-coding regions of transcripts fold onto themselves to form hairpins. After being processed by various cellular mechanisms, the mature miRNA found in plants, animals, and some viruses is a small (about 22 nucleotides) RNA molecule that regulates gene expression through post-transcriptional silencing.

[0007] Therapies based on these and other nucleic acids offer promising treatment options for a variety of diseases, including non-drugable targets. However, despite the progress made in the application of oligonucleotides and oligonucleotide analogs as treatment options, there remains a great need to enhance the key pharmacological properties of these therapeutic oligonucleotides, such as in the areas of serum stability, delivery to the intended organ or cell population, and uptake across cell membranes.

[0008] The preferential delivery of therapeutic oligonucleotides to cells in the body, such as in mammals, like in the human body, requires specific targeting and protection from the extracellular environment in the body, including proteins in the serum. One method that researchers use to achieve specific targeting is to conjugate a targeting moiety to the oligonucleotide to target the therapeutic oligonucleotide to the desired target site.

[0009] One way to improve delivery specificity is to utilize receptor-mediated endocytic activities that already exist in vivo. The uptake mechanism involves molecules that bind to cell membrane receptors entering the cell through transmembrane movement via invagination of the membrane structure or through fusion of the delivery system with the cell membrane. This process is initiated by activation of cell surface or membrane receptors following binding of a specific ligand to the receptor. Thus, by conjugating a candidate drug with a targeting moiety that targets such cell surface receptors, the innate endocytic pathway can be effectively borrowed for drug delivery. Many receptor-mediated endocytic systems are known and studied, including those that recognize carbohydrates, including galactose, mannose, 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. The ASGP-R has a 50-fold higher affinity for N-acetyl-D-galactosamine (GalNAc) than for D-galactose. However, it has been reported that when using such conjugation systems, the linker structure design and various chemical properties of the linker moiety are crucial in determining the overall delivery efficiency, effectiveness, and safety of the conjugated oligonucleotides, as well as their impact on the stability and manufacturing challenges of various therapeutic oligonucleotides.

[0010] Therefore, there is an urgent need to design new and effective receptor-specific, ligand-conjugated nucleic acid complexes for various biomedical applications. Summary of the Invention

[0011] In a first aspect, the present invention relates to a compound as a therapeutic agent, wherein an oligonucleotide is conjugated to at least one ligand, such as a carbohydrate ligand, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide, or a derivative thereof, which can target the compound to receptor cells in the liver, thereby facilitating endocytic uptake as described above.

[0012] These ligand-conjugated compounds target one or more organs or cell types, e.g., the parenchymal cells of the human liver. In one embodiment, the compound comprises more than one carbohydrate ligand, preferably two or three. In another embodiment, the compounds of the invention comprise at least one (e.g., one, two, three or more) N-acetylgalactosamine (GalNAc, N-Acetyl-Galactosamine), N-acetyl-glucosamine (GlcNAc, N-Ac-Glucosamine), galactose, lactose or mannose (e.g., mannose-6-phosphate). In yet another embodiment, the compounds of the invention comprise at least one (e.g., one, two, three or more) ligand, wherein the ligand is 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 invention provides ligand-conjugated compounds having a novel structure:

[0014] Item 1, a compound having the structural formula (S-HG1): R 206 -[A1] a -[A2] b -[A3] c -R 205 (S-HG1) Wherein, R 205 、R 206 Each occurrence is independently H, 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, PEG, a steroid, a polymer, -O-nucleotide, a nucleoside, -OP(M')(M")O-R 201 -OP(M'")(M"")O-oligonucleotide, -X-OP(M')(M")O-oligonucleotide, -Z-OP(M')(M")O-oligonucleotide, or an oligonucleotide; M', M", M'" and M"" are each independently O or S for each occurrence; A1, A2 and A3 are each independently selected from (S-1H) or (S-1G) for each occurrence: R 202A is -R 202 -R 202L ; R 217A is -R 217 -R 217L ; R 202L 、R 217L For each occurrence independently is a ligand capable of docking to a cell surface receptor; Each R 202 、R 217 、R 201 For each occurrence independently is selected from alkylene groups having 3 to 30 carbon atoms, wherein one or more carbon atoms may optionally be replaced by 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 、C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocycloalkylene, and C 5 -C 10 heteroarylene, and wherein each R 202 、R 217 、R 201 is independently optionally unsubstituted or substituted by R 209 ; optionally, R 202 、R 201 For each occurrence is independently selected from: alkylene groups having 3 to 15 carbon atoms, wherein one or more carbon atoms may optionally be replaced by one or more substituents from the group consisting of: C(O), NH, O, S, OP(O)O, OP(S)O; R 1 、R 2 、R 204 、R 207 、R 208 、R 213 、R 214 、R 215 、R 216 、R 209 For each occurrence is independently selected from one or more of the group consisting of: H, alkyl, aryl, heteroaryl, haloalkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shaloalkyl, halogen substituents, -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 For each occurrence independently is 1, 2, 3, 4, 5 or 6; J 201 , J 202 , J 211 , J 212 For each occurrence independently is absent or a spacer; a, b and c for each occurrence independently are integers from 0 - 5, and the sum of a, b and c is an integer from 1 - 10; The oligonucleotide comprises naturally occurring or chemically modified nucleotides / nucleosides.

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

[0016] Item 3, the compound according to item 1, wherein the compound has the structural formula (S-H1): Wherein, R 1 Is selected from one or more of the group consisting of: H, C 1 -C 5 alkyl, aryl, heteroaryl, C 1 -C 5 haloalkyl, -C 1 -C 5 alkyl-OH, -C 1 -C5 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 - 10, preferably 1 - 3.

[0017] The compound according to item 4, item 3, having the structural formula (S - H1 - 01): Wherein: 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 having 1 to 10 carbon atoms, optionally selected from straight-chain alkylene having 1 to 10 carbon atoms; R 205A is a group containing a solid-phase carrier or H; The carbon atom marked with the symbol "*" is a chiral carbon atom.

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

[0019] The compound according to item 6, any one of items 3 - 4, wherein R 206 contains an oligonucleotide.

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

[0021] The compound according to item 8, item 3, wherein: J 201 、J 202For each occurrence, an alkylene independently selected from 1 to 30 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more substituents consisting of the following group: 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 heterocycloalkylene and C 5 -C 10 heteroarylene, and wherein J 201 , J 202 are each independently optionally unsubstituted or substituted by R 209 .

[0022] Item 9, the compound according to item 3, wherein: J 201 , J 202 For each occurrence, an alkylene independently selected from 1 to 10 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more substituents consisting of the following group: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, and S(O) 2 , and wherein J 201 , J 202 are each independently optionally unsubstituted or substituted by at least one group selected from the following group: H, or C 1 -C 5 alkyl, -OC 1 -C 5 alkyl.

[0023] Item 10, the compound according to item 9, wherein n 201 is 1, n 202 is 1 or 3.

[0024] Item 11, the compound according to item 3, wherein the compound has the structural formula (S-H1-03), (S-H1-04), (S-H1-05) or (S-H2): Wherein, A is O or S, X is independently selected from Table 1, or an alkylene of 1 to 10 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more substituents consisting of the following group: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, and S(O) 2 ; X 1 is independently selected from Table 2; J 202 and Z are each independently selected from Table 3 for each occurrence; Optionally, J 202 and Z are each independently selected from alkylene groups having 1 to 10 carbon atoms for each occurrence, where one or more of the carbon atoms may optionally be replaced by any one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, and S(O) 2 and where J 201 and J 202 are each independently optionally unsubstituted or substituted by at least one group selected from the group consisting of H, or C 1 -C 5 alkyl, -OC 1 -C 5 alkyl; R and R' are each independently selected from the group consisting of naturally occurring and / or chemically modified oligonucleotides, H, and OH protecting groups; at least one of R and R' comprises an oligonucleotide formed from natural and / or chemically modified nucleotides / nucleosides; R 202 is selected from straight-chain alkylene groups having 3 to 15 carbon atoms, where one or more of the carbon atoms may optionally be replaced by one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, and where R 202 is optionally unsubstituted or substituted by R 209 ; Optionally, R 202 is selected from -C 3 -C 8 straight-chain alkylene.

[0025] Table 1

[0026] Table 2

[0027] Table 3

[0028] Item 12, the compound according to item 11, wherein the compound has the structural formula (S-H1-06), (S-H1-07), (S-H1-08), or (S-H2-01):

[0029] Item 13, the compound according to item 11, wherein the compound has a structural formula (S-H1-09), (S-H1-10), (S-H1-11) or (S-H2-02):

[0030] Item 14, the compound according to item 11, wherein the compound has a structural formula (S-H1-12), (S-H1-13), (S-H1-14) or (S-H2-03):

[0031] Item 15, the compound according to item 1, wherein the compound has a structural formula (S-H1-15), (S-H1-16) or (S-H2-04): Wherein: R and R' are each independently selected from the group consisting of: naturally occurring or chemically modified oligonucleotides, H and OH protecting groups; At least one of R and R' comprises an oligonucleotide formed from 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, thiophosphate, sulfate, disulfide, ester, thioester, alkylamine, cycloalkylamine, alkyne, cycloalkyne, alkene, cycloalkene, lactone and lactam bond; Each X is independently selected from Table 1, or an alkylene group having 1 to 10 carbon atoms, wherein one or more carbon atoms may optionally be replaced by any one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH=N and S(O) 2 ; Each Y is independently selected from Table 4, or a straight-chain alkylene group having 3 to 15 carbon atoms, wherein one or more carbon atoms may optionally be replaced by one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O; and wherein Y may optionally be unsubstituted or substituted by R 207 Substituted; Each Z is independently selected from Table 3; Each L independently comprises a ligand moiety capable of pairing with a cell surface receptor.

[0032] Table 4

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

[0034] Item 17, the compound according to any one of Items 11 - 15, wherein at least one A is O.

[0035] Item 18, the compound according to any one of Items 1 - 17, wherein each ligand is independently selected from the group consisting of: N - acetylgalactosamine (GalNAc), cholesterol, tocopherol, biotin, cyanine dye, folic acid, RGDp, transferrin, anisamide, lactic acid, cRGD, hyaluronic acid, low - molecular - weight protamine, lipid derivatives, peptides, cyclic peptides, and heterocycles.

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

[0037] Item 20, the compound according to Item 12, wherein the compound has the structures shown as HS - 1 to HS - 8 and HS - 10:

[0038] Item 21, the compound according to Item 12, wherein the compound has the structures shown as HS - 9, HS - 14:

[0039] Item 22, the compound according to Item 12, wherein the compound has the structures shown as HS - 5, HS - 13:

[0040] Item 23, the compound according to Item 1, having the structural formula (S - G1): wherein R 2 is one or more selected from the group consisting of: 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 - 10, preferably 1 - 3.

[0041] The compound described in item 24, item 23 has the structural formula (S - G1 - 01): Wherein: 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 groups having 1 to 10 carbon atoms, optionally selected from straight-chain alkylene groups having 1 to 10 carbon atoms; R 205 is a group containing a solid-phase carrier or H; The carbon atom marked with the symbol "*" is a chiral carbon atom.

[0042] The compound described in item 25, item 23 or item 24, wherein n 212 is selected from 1 or 3.

[0043] The compound described in item 26, any one of items 23 - 24, wherein R 206 contains an oligonucleotide.

[0044] The compound described in item 27, item 26 has the structural formula (S - G1 - 02):

[0045] The compound described in item 28, item 23, wherein: J 211 , J 212 are independently selected from alkylene groups having 1 to 30 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more substituents selected from the group consisting of: C(O), NH, O, S, OP(O)O, OP(S)O, CH = N, S(O) 2 , C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C10 Arylene, C 3 -C 18 Heterocycloalkylene and C 5 -C 10 Heteroarylene, and wherein J 211 、J 212 Each independently is optionally unsubstituted or substituted by R 209 .

[0046] Item 29, the compound according to item 23, wherein: J 211 、J 212 Independently selected from alkylene having 1 to 10 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more substituents selected from the group consisting of: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N and S(O) 2 , and wherein J 211 、J 212 Each independently is optionally unsubstituted or substituted by at least one group selected from the group consisting of: H, or C 1 -C 5 Alkyl, -OC 1 -C 5 Alkyl.

[0047] Item 30, the compound according to item 29, wherein n 211 is 1, n 212 is 1 or 3.

[0048] Item 31, the compound according to item 29, wherein the compound has the structural formula (S-G1-03), (S-G1-04), (S-G1-05) or (S-G2): Wherein, A is O or S, X is independently selected from Table 1, or alkylene having 1 to 10 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more substituents selected from the group consisting of: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N and S(O) 2 ; X 1 Is independently selected from Table 2; J 212 、Z are independently selected from Table 3 for each occurrence; Optionally, J 212independently selected from C1-C10 alkylene groups, wherein one or more carbon atoms may optionally be replaced by any one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, and S(O) 2 , and wherein J 212 is optionally unsubstituted or substituted with at least one group selected from the group consisting of H, or C 1 -C 5 alkyl, -OC 1 -C 5 alkyl; R and R' are each independently selected from the group consisting of naturally occurring and / or chemically modified oligonucleotides, H, and OH protecting groups; at least one of R and R' comprises an oligonucleotide formed from natural and / or chemically modified nucleotides / nucleosides; R 217 is selected from straight-chain C3-C15 alkylene groups, wherein one or more carbon atoms may optionally be replaced by one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, and wherein R 217 is optionally unsubstituted or substituted with R 209 ; Optionally, R 217 is selected from -C 3 -C 8 straight-chain alkylene.

[0049] Item 32, the compound according to Item 31, wherein the compound has the structural formula (S-G1-06), (S-G1-07), (S-G1-08), or (S-G2-01):

[0050] Item 33, the compound according to Item 31, wherein the compound has the structural formula (S-G1-09), (S-G1-10), (S-G1-11), or (S-G2-02):

[0051] Item 34, the compound according to Item 31, wherein the compound has the structural formula (S-G1-12), (S-G1-13), (S-G1-14), or (S-G2-03):

[0052] Item 35, the compound according to Item 1, wherein the compound has the structural formula (S-G1-15), (S-G1-16), or (S-G2-04): Wherein: R and R' are each independently selected from the group consisting of: naturally occurring or chemically modified oligonucleotides, H, and OH protecting groups; At least one of R and R' comprises a naturally occurring and / 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, thiophosphate, sulfate, disulfide, ester, thioester, alkylamine, cycloalkylamine, alkyne, cycloalkyne, alkene, cycloalkene, lactone, and lactam bond; Each X is independently selected from Table 1, or an alkylene group having 1 to 10 carbon atoms, wherein one or more carbon atoms may optionally be replaced by any one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, and S(O); 2 ; Each Y is independently selected from Table 4, or a straight-chain alkylene group having 3 to 15 carbon atoms, wherein one or more carbon atoms may optionally be replaced by one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O; and wherein Y may optionally be unsubstituted or substituted by R; 207 substituted; Each Z is independently selected from Table 3; Each Z'' is independently selected from Table 5; and Each L independently comprises a ligand moiety capable of pairing with a cell surface receptor; and Each n1, n2, n3 is independently selected from 1, 2, 3, or 4.

[0053] Table 5

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

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

[0056] Item 38. A compound according to any one of items 23 - 37, wherein each ligand is independently selected from the group consisting of: N-acetylgalactosamine (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.

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

[0058] Item 40, a compound according to Item 31, wherein the compound has a structure as shown in GS-1 to GS-8 and GS-10:

[0059] Item 41, a compound according to Item 31, wherein the compound has a structure as shown in GS-9 and GS-14:

[0060] Item 42, a compound according to Item 31, wherein the compound has a structure as shown in GS-5 and GS-13:

[0061] Item 43, a compound according to any one of Items 1 - 42, wherein the naturally occurring and / or chemically modified oligonucleotide is linked to the other part of the compound through its 5'-end and / or 3'-end.

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

[0063] Item 45, a compound according to Item 43, wherein the oligonucleotide comprises an asymmetric interfering RNA (aiRNA) duplex.

[0064] Item 46, a compound according to Item 45, wherein the aiRNA comprises an antisense strand and a sense strand, wherein the antisense strand is longer than the sense strand, the length of the antisense strand is 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides, and when forming a duplex with the sense strand, the antisense strand includes a 3'-overhang of 1 - 9 nucleotides and a 5'-overhang of 0 - 8 nucleotides; wherein the length of the sense strand is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides, and forms a duplex region with the antisense strand.

[0065] Item 47, a compound according to Item 46, wherein the aiRNA comprises an antisense strand and a sense strand, wherein the antisense strand is longer than the sense strand, the length of the antisense strand is 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides, and when forming a duplex with the sense strand, the antisense strand includes a 3'-overhang of 1 - 9 nucleotides and a 5'-overhang of 1 - 8 nucleotides; The length of the sense strand is 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 compound according to item 46, wherein the aiRNA comprises an antisense strand and a sense strand, wherein the antisense strand is longer than the sense strand, the length of the antisense strand is 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides, and when forming a double strand with the sense strand, the antisense strand includes a 3'-overhang of 1-9 nucleotides and a 5'-blunt end; The length of the sense strand is 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. The compound according to item 43, wherein the oligonucleotide comprises an antisense oligonucleotide (ASO).

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

[0069] Item 51. A small interfering RNA (siRNA) agent comprises the structural formula according to any one of items 1-50.

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

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

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

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

[0074] Item 56. Use of the compound according to any one of items 1-50 or the agent according to any one of items 51-54 in the preparation of a drug for effectively treating a disease or disorder.

[0075] In a third aspect, the invention features, as described above in the second aspect, a compound comprising a carbohydrate ligand, and the presence of the carbohydrate ligand enhances the delivery of the compound to a target organ (e.g., the liver). Thus, a compound comprising a carbohydrate ligand can be used to target genes associated with a disease or an undesired condition in a target organ. For example, a compound of the invention comprising a carbohydrate ligand can target nucleic acids expressed by a hepatitis virus. In other instances, the target gene can be selected from the group consisting of: Factor VII, Eg5, PCSK9, APOC3, TPX2, apoB, SAA, TTR, RSV, PDGFbeta 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, cyclin E gene, WNT-I gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase IIα gene, mutations in the p73 gene, mutations in the p21 (WAFl / CIPl) gene, mutations in the p27 (KIPl) gene, mutations in the PPMlD gene, mutations in the RAS gene, mutations in the caveolin I gene, mutations in the MIB I gene, mutations in the MTAI gene, mutations in the M68 gene, mutations in tumor suppressor genes, and mutations in the p53 tumor suppressor gene.

[0076] In a fourth aspect, the invention provides a compound having a novel structure:

[0077] Item 57, a compound having the structural formula (G-P1): Wherein: R 0 is selected from one or more of the group consisting of: 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 2H, -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); R 202A comprises at least one ligand capable of pairing with a cell surface receptor; R 204 , R 207 , R 208 are independently selected from one or more of the group consisting of: H, alkyl, aryl, heteroaryl, haloalkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shaloalkyl, halogen substituents, -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 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 202Each occurrence is independently a spacer; R 205B is -C 2 -C 10 alkynylene -CN; R 205C 、R 205D are each independently selected from -C 1 -C 6 alkyl or R 205C and R 205D together form a five - or six - membered ring. Optionally, R 205C 、R 205D are both substituted. Optionally, R 205C 、R 205D further contain a heteroatom selected from N and O.

[0078] Item 58, the compound according to item 57, wherein: J 201 、J 202 are each independently selected from alkylene groups having 3 to 30 carbon atoms, wherein one or more carbon atoms are optionally replaced by 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 、C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocycloalkylene, and C 5 -C 10 heteroarylene, and wherein J 201 、J 202 are optionally unsubstituted or substituted by R 209 ;

[0079] Item 59, the compound according to item 57, having the structural formula (G - P2): wherein: 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 groups having 1 - 10 carbon atoms.

[0080] Item 60, the compound according to item 59, having the structural formula (G - P3):

[0081] Item 61. The compound described in item 57 has the structural formula (G-P4):

[0082] Item 62. The compound described in item 57, wherein: R 205B is -C 2 -C 10 alkynylene -CN; R 205C 、R 205D are each independently selected from -C 1 -C 6 alkyl; R 202A is —R 202C -branching group-(R 202B -R 202L ) n 111L or —R 202B -R 202L ; R 202L is independently selected from a ligand capable of docking with a cell surface receptor; R 202B is selected from alkylene having 1 to 30 carbon atoms, wherein one or more carbon atoms may optionally be replaced by 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 、C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocycloalkylene, and C 5 -C 10 heteroarylene, and wherein R 202B is optionally unsubstituted or substituted by R 207 ; n 111L is selected from 1, 2 or 3.

[0083] Item 63. The compound described in item 62, wherein R 202C is selected from the straight-chain alkylene -NHCO-CH 5 –C 8 - of -C(O)-C 2 -, or -C(O)-C 8 –C 11Linear alkane-.

[0084] Item 64. The compound according to item 62, wherein the branched group is selected from the group consisting of: where each A 1 is independently O, S, C═O, or NH; and each n is independently from 1 to 20.

[0085] Item 65. The compound according to item 61, wherein the ligand is wherein R A is a protecting group for H or OH.

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

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

[0088] Item 68. A compound having the structural formula (G-P7): wherein: R 3 is selected from one or more of the group consisting of: 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)O alkyl, -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); R 217A comprises at least one ligand capable of pairing with a cell surface receptor; R 213 、R 214 、R 215 、R 216 are independently selected from one or more of the group consisting of: H, alkyl, aryl, heteroaryl, haloalkyl, -O-alkyl, -O-alkylphenyl, -alkyl-OH, -O-haloalkyl, -S-alkyl, -S-alkylphenyl, -alkyl-SH, -S-haloalkyl, halogen substituents, -OH, -SH, -NH 2 、-alkyl-NH 2 、-N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, -CO 2 H, -C(O)O-alkyl, -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); R 212 is selected from OH or a protecting group for OH; J 211 、J 212 is independently a spacer for each occurrence; n 211 is selected from 1, 2, 3, 4, 5 or 6; J 211 、J 212 is independently a spacer for each occurrence; R 211B is -C 2 -C 10 alkynylene-CN; R 211C 、R 211D are each independently selected from -C 1 -C 6 alkyl or R 211C and R 211DTogether form a five- or six-membered ring, optionally, R 211C and R 211D are both substituted, optionally, R 211C and R 211D further contain a heteroatom selected from N and O.

[0089] Item 69, the compound according to item 68, wherein: J 211 and J 212 each independently selected from alkylene groups having 3 to 30 carbon atoms, wherein one or more carbon atoms are optionally replaced by 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 and C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocycloalkylene, and C 5 -C 10 heteroarylene, and wherein J 211 and J 212 are optionally unsubstituted or substituted by R 209 ;

[0090] Item 70, the compound according to item 68, having the structural formula (G-P8): wherein: 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 groups having 1 to 10 carbon atoms.

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

[0092] Item 72, the compound according to item 68, having the structural formula (G-P10):

[0093] Item 73, the compound according to item 68, wherein: R 211B is -C 2 -C 10 alkynylene-CN; R 211C and R 211D are each independently selected from -C 1 -C 6 alkyl; R 217 is –R 217C -branched group-(R 217B -R 217L ) n 211L or –R 217B -R 217L ; R 217L is independently selected from a ligand capable of docking with a cell surface receptor; R 217B is selected from alkylene groups having from 1 to 30 carbon atoms, wherein one or more of the carbon atoms may optionally be replaced by 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 , C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocycloalkylene, and C 5 -C 10 heteroarylene, and wherein R 217B is optionally unsubstituted or substituted by R 213 ; n 211L is selected from 1, 2 or 3.

[0094] Item 74, the compound according to item 73, wherein R 217C is selected from a straight-chain alkylene group of -C(O)-C 5 –C 8 -NHCO-CH 2 -, or -C(O)-C 8 –C 11 straight-chain alkane-.

[0095] Item 75, the compound according to item 73, wherein the branched group is selected from the group consisting of: wherein each A 1 is independently O, S, C=O, or NH; and each n is independently from 1 to 20.

[0096] Item 76, the compound according to item 68, wherein the ligand is wherein R A is a protecting group for H or OH.

[0097] Item 77, the compound according to item 68, having the structural formula (G-P11):

[0098] Item 78, the compound according to item 68, having the structural formula (G-P12):

[0099] On the other hand, the present invention is characterized in that the compound provided in the fourth aspect above can be used as an intermediate linker for connecting oligonucleotides and ligands for the synthesis of sequential and / or cluster ligand conjugates; in particular, such compounds can be used as intermediates for the synthesis of sequential ligand conjugates.

[0100] In another aspect, the compound provided in the fourth aspect above can be used to directly conjugate a ligand conjugate to the backbone of the 5'-end and / or 3'-end of an oligonucleotide. In some embodiments, the compound provided in the fourth aspect above can be used to directly conjugate a ligand conjugate to the 5'-end of any strand of an oligonucleotide.

[0101] In a further aspect, the present invention provides a pharmaceutical composition comprising the compound of the present invention provided in any of the above aspects and a pharmaceutically acceptable excipient, carrier or diluent.

[0102] In another aspect, the present invention is characterized in that a method for delivering a compound to a specific target in a subject's body for therapeutic or diagnostic purposes. Accordingly, the present invention provides a method for treating or preventing a disease or disorder, which method comprises administering to a subject in need an effective amount of a pharmaceutical composition comprising the compound of the present invention. Treating or preventing a disease by partially or completely silencing a disease gene. The disease gene may be the patient's own gene or a gene from an external microorganism such as a virus.

[0103] According to the following description and claims, the above and other objects, aspects, features and advantages of the present invention will become more apparent. Brief Description of the Drawings

[0104] The objects and features of the present invention can be better understood with reference to the following drawings and claims. The drawings illustrate the core ideas of the present invention but do not necessarily summarize all possible variations. In the drawings, like numbers are used to indicate like parts in the various views.

[0105] Figure 1 Exemplary structures of oligonucleotide-ligand conjugations are shown. A conjugated interfering RNA duplex molecule includes an antisense strand and a sense strand. In some embodiments, the oligonucleotide is an interfering RNA duplex molecule, and the ligand can be conjugated to the 3'-end of the sense strand (e.g., structures 1.1-1.3, intermediate aiRNA, blunt-end aiRNA, and siRNA), to the 3'-end of the antisense strand (structure 2), to the 5'-end of the sense strand (structure 3), or to both ends of the sense strand (structure 5), or to both ends of the antisense strand (structure 4), to the 3'-end of the antisense strand and the 5'-end of the sense strand (structure 6), to the 3'-end of the sense strand and the 3'-end of the antisense strand (structure 7), or to 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 can be conjugated to the 3'-end or / and 5'-end of the antisense strand.

[0106] Figure 2 Results of in vitro uptake of β-Catenin aiRNA tested by QPCR are shown. "Non-GalNAc" is aiRNA without conjugation. "Glu(R)-seq(3GalNAc)" is aiRNA conjugated with the GalNAc-conjugated composition "GS-5".

[0107] Figure 3 Results of in vitro uptake of mCat12 aiRNA conjugated with "His(R)-seq(3GalNAc)", "His(S)-seq(3GalNAc)", and "Glu-seq(3GalNAc)" in primary hepatocytes are shown.

[0108] Figure 4 Results of in vitro uptake of TTR aiRNA conjugated with "His-Seq(3GalNAc)" in primary hepatocytes are shown. DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS

[0109] Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in, for example, Lewin’s Genes XII (2017 (ISBN 9781284104493)) edited by J. Krebs et al. (eds.) published by Jones and Bartlett Learning; Molecular Biology and Biotechnology: a Comprehensive Desk Reference (2011 (ISBN 9788126531783)) edited by Robert A. Meyers (ed.) published by Anmol Publications Pvt. Ltd.; and other similar technical references.

[0110] As used in the specification and claims, the singular forms “a,” “an,” “the,” or “said” include plural forms unless the context clearly dictates otherwise. For example, the term “cell” includes a plurality of cells, including mixtures thereof. It is further noted that claims may be drafted to exclude any optional element. To this end, this statement is intended to support the recitation of exclusive terms in claims, such as “only,” “solely,” and similar terms related to the recitation of claim elements, or the use of “negative” limitations, such as “wherein [specific feature or element] is absent,” or “except for [specific feature or element],” or “wherein [specific feature or element] is not present (is included, etc)…”

[0111] As used herein, the description of a numerical range for a variable is intended to indicate that the invention can be practiced with the variable equal to any value 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 endpoints of the range. Similarly, for a variable that is inherently continuous, the variable can be equal to any actual value within the numerical range, including the endpoints of the range. As an example, and not by way of limitation, a variable described as having a value between 0 and 2 can take the values 0, 1, or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other actual 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 "linkage" are used to connect two parts of a compound, such as an alkylene group of 1 to 30 carbon atoms, where one or more carbon atoms are optionally replaced by 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 、C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocycloalkylene and C 5 -C 10 heteroarylene, where J 201 、J 202 are each independently optionally unsubstituted or substituted by one or more substituents from the group consisting of: H, alkyl, aryl, heteroaryl, haloalkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shaloalkyl, halogen substituents, -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).

[0114] A variety of hydroxyl protecting groups can be used in the present disclosure. Generally, a protecting group renders a chemical functional group insensitive to specific reaction conditions and can be attached to and removed from the functional group of a molecule without significantly disrupting the remainder of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, each 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 can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthine-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthine-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that can 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 two symbols is used to indicate the position of a substituent attachment point. For example: -C 1 -C 10 alkyl-NH 2 is attached via C 1 -C 10 alkyl.

[0116] As used herein, “optional” or “optionally” means that the event or circumstance described thereafter may or may not occur, and the description includes instances where the event or circumstance occurs and instances where it does not. For example, “optionally substituted alkyl” includes “alkyl” and “substituted alkyl” as defined hereinbelow. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitutions or substitution patterns that are spatially impracticable, synthetically infeasible, and / or inherently unstable.

[0117] As used herein, “alkyl” refers to straight and branched chains having a specified number of carbon atoms, typically from 1 to 20 carbon atoms, such as from 1 to 10 carbon atoms, such as from 1 to 8, from 1 to 6, from 1 to 5, or from 1 to 3 carbon atoms. For example, C 1-C 6 The alkyl group includes straight-chain and branched-chain alkyl groups having 1 to 6 carbon atoms. When naming an alkyl residue having a specific number of carbon atoms, it is intended to cover all branched-chain and straight-chain forms having that number of carbon atoms; thus, for example, "butyl" means including n-butyl, sec-butyl, isobutyl, and tert-butyl; "propyl" includes n-propyl and isopropyl. An alkylene group is a subset of the alkyl group and refers to a residue that is the same as the alkyl group but has two attachment points.

[0118] As used herein, "alkenyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond, which is obtained by removing a hydrogen molecule from adjacent carbon atoms of the parent alkyl group. The double bond of this group can be in the cis or trans configuration. Typical alkenyl groups include, but are not limited to: vinyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl; butenyl, for example but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, and so on. In certain embodiments, the alkenyl group has 2 to 20 carbon atoms, and in other embodiments, it has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. An alkenylene group is a subset of the alkenyl group and refers to a residue that is the same as the alkenyl group but has two attachment points.

[0119] As used herein, "alkynyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond, which is obtained by removing two hydrogen molecules from adjacent carbon atoms of the parent alkyl group. Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, for example but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc. In certain embodiments, the alkynyl group has 2 to 20 carbon atoms, and in other embodiments, it has 2 to 10, 2 to 8, or 2 to 6 carbons. An alkynylene group is a subset of the alkynyl group and refers to a residue that is the same as the alkynyl group but has two attachment points.

[0120] As used herein, "alkoxy" refers to an alkyl group having a specified number of carbon atoms connected by an oxygen bridge, 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. The alkoxy group generally has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by an oxygen bridge.

[0121] As used herein, "aryl" refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system formed by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only carbon and hydrogen, with 6 to 18 carbon atoms, where at least one ring in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n + 2)π-electron system according to Hückel's theory. Aryl groups include, but are not limited to, groups such as phenyl, fluorenyl, and naphthyl. Arylene is a subset of aryl and refers to a residue identical to aryl but having two attachment points.

[0122] As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring, typically having 3 to 7 ring carbon atoms. The ring can be saturated or have one or more carbon-carbon double bonds. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl, as well as bridged and cage-like groups such as norbornane.

[0123] As used herein, "halo substituent" 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 group having a specific number of carbon atoms as defined above, substituted by one or more, up to the maximum allowable number of halogen atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.

[0125] "Heterocyclic group" means a stable 3- to 18-membered non-aromatic ring group that contains 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specified in the specification, the heterocyclic group is a monocyclic, bicyclic, tricyclic, or tetracyclic system and may include fused ring or bridged ring systems. The heteroatoms in the heterocyclic group may optionally be oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heterocyclic group is partially saturated or fully saturated. The heterocyclic group can be attached to the remainder of the molecule through any atom on any ring. Examples of such heterocyclic groups 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, oxazolidiny, 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 group derived from an aromatic ring group of 3 to 18 ring atoms, which contains 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, heteroaryl can be a monocyclic, bicyclic, tricyclic, or tetracyclic system, wherein at least one ring in the ring system is completely unsaturated, i.e., according to Hückel's theory, it contains a cyclic, delocalized (4n + 2)π-electron system. Heteroaryl includes fused ring or bridged ring systems. The heteroatoms in heteroaryl can be optionally oxidized. One or more nitrogen atoms (if present) can be optionally quaternized. Heteroaryl is attached to the rest of the molecule through any atom on the ring. Examples of heteroaryl include, but are not limited to: azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzooxazolyl, 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]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl), 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, 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, 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]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl and thiophenyl / thienyl.,

[0127] As used herein, the term "solid support" includes solid supports for oligonucleotide synthesis, such as CPG.

[0128] As used herein, the terms "oligonucleotide" and "oligonucleotides" refer to compounds comprising multiple linked nucleosides. In certain embodiments, an "oligonucleotide" is a short single-stranded 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 comprises one or more ribonucleosides (such as in RNA) and / or deoxyribonucleosides (such as in DNA). In some embodiments, the oligonucleotide is a single-stranded oligonucleotide. In some other embodiments, the oligonucleotide is a double-stranded interfering RNA, such as siRNA, aiRNA, shRNA. In some embodiments, the oligonucleotide is circular RNA (circRNA). In some embodiments, the oligonucleotide is 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, consists of 19-27 nucleotides, and includes a 3'-overhang of at least one nucleotide and a 5'-end of 0-8 nucleotides; wherein the antisense strand is at least 70% complementary to the target mRNA; and wherein the sense strand consists of 10-26 nucleotides and forms a duplex region with the antisense strand that includes 0, 1, or 2 mismatched pairs. US Exemplary structures of aiRNA are described in US 2009 / 0208564, which is hereby incorporated by reference in its entirety.

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

[0131] As used herein, the term "blunt type" refers to an interfering RNA duplex molecule comprising an antisense strand and a sense strand, wherein the RNA duplex molecule has at least one blunt end, preferably a blunt end at the 3'-end of the sense strand or at the 5'-end of the antisense strand.

[0132] As used herein, the term "modified oligonucleotide" refers to an oligonucleotide comprising at least one modified nucleotide.

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

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

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

[0136] As used herein, the term "modified internucleoside bond" refers to a substitution or any alteration from a naturally occurring internucleoside bond. For example, a phosphorothioate bond is a modified internucleoside bond.

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

[0138] As used herein, the term "modified sugar moiety" refers to a substitution or alteration from a natural sugar. For example, a 2'-O-methoxyethyl-modified sugar is a modified sugar.

[0139] As used herein, the term "bicyclic sugar" refers to a furanosyl ring modified by bridging two non-bicyclic 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 adenine (A) and guanine (G) which are purine bases, and thymine (T), cytosine (C) and uracil (U) which are pyrimidine bases.

[0141] As used herein, "prevention" and "preventing" are used interchangeably. These terms refer to methods of obtaining a beneficial or desired result, including but not limited to prophylactic benefit. For "prophylactic benefit", the conjugate or composition may be administered to a patient at risk of developing a particular disease, or to a patient who has reported one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0142] As used herein, the term "effective amount" of an active agent refers to an amount sufficient to elicit a desired biological response. As will be understood by those of ordinary skill in the art, the effective amount of the compounds of the invention may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration and the patient.

[0143] As used herein, the term "treating" or "treatment" of a disease or disorder refers to a method of alleviating, delaying or improving such condition before or after the occurrence of the disease or disorder. Treatment can be directed at one or more effects or symptoms of the disease and / or underlying pathology. Treatment can be any alleviation, which can be, but is not limited to, complete elimination of the disease or disease symptoms. This degree of alleviation or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% (measured by any standard technique) compared to an equivalent untreated control group.

[0144] As used herein, the term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, rodents, etc., which will be the recipient of a particular treatment. Generally, the terms "subject" and "patient" can be used interchangeably. As used herein, "pharmaceutical composition" includes a pharmaceutically effective amount of dsRNA and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically effective amount", "therapeutically effective amount" or simply "effective amount" refers to the amount of RNA effective to produce the desired pharmacological, therapeutic or prophylactic result. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or disorder is reduced by at least 25%, then the therapeutically effective amount of a drug used to treat that disease or disorder is the amount required to reduce that parameter by at least 25%.

[0145] The term "pharmaceutically acceptable carrier" refers to a carrier used to administer a therapeutic agent. Such carriers include but are not limited to saline, buffered saline, glucose, water, glycerol, ethanol and combinations thereof. This term specifically excludes cell culture media. For oral drugs, pharmaceutically acceptable carriers include but are not limited to pharmaceutically acceptable excipients, such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders can include starch and gelatin, while lubricants (if any) are generally magnesium stearate, stearic acid or talc. If desired, tablets can be coated with materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the human gastrointestinal tract.

[0146] Compound configuration

[0147] The compounds of the present invention and their salts can exist in tautomeric forms (e.g., as amides or imino ethers). All such tautomeric forms are considered part of the present invention.

[0148] All stereoisomers of the compounds of the present invention (e.g., stereoisomers that may exist due to asymmetric carbons on various substituents), including enantiomeric forms and diastereomeric forms, are within the scope of the present invention. For example, a single stereoisomer of a compound of the present invention may be substantially free of other isomers (e.g., as a pure or substantially pure optical isomer with a specific activity), or may be, for example, in a racemic form or mixed with all other or other selected stereoisomers. The chiral centers of the present invention may have the S configuration or the R configuration as defined in the IUPAC 1974 recommendations. Racemic forms can be resolved by physical methods, such as fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. Separate optical isomers can be obtained from the racemate by any suitable method, including but not limited to conventional methods, such as forming salts with optically active acids and then crystallizing.

[0149] After preparation, the compounds of the present invention are preferably isolated and purified to obtain a composition containing an amount equal to or greater than 95% by weight (e.g., "substantially pure" Compound I), and then the composition is used or formulated as described herein. In certain embodiments, the compounds of the present invention have a purity exceeding 99%.

[0150] All configurational isomers of the compounds of the present invention are either the expected mixtures or the expected pure or substantially pure forms. The definition of the compounds of the present invention includes cis (Z) and trans (E) alkene isomers, as well as cis and trans isomers of cyclic hydrocarbons or heterocycles.

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

[0152] The amino acids contained in the peptides or polypeptides described herein will be understood to be in the L-configuration or the D-configuration. II. Embodiments

[0153] The present invention provides novel compounds having a novel linker moiety, wherein the linker is used to connect various components of the compound. The compound conjugates an oligonucleotide with one or more targeting ligands. The oligonucleotide can be naturally occurring (isolated from nature or synthesized in the laboratory) or 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 includes backbone modifications (or internucleoside bond modifications, such as phosphate group modifications), ribose group modifications, and base modifications.

[0155] In certain embodiments, the oligonucleotide has at least one phosphorothioate internucleoside bond, or at least one methylphosphonate internucleoside bond, or at least one other modified internucleoside bond, such as:

[0156] In certain embodiments, the oligonucleotide has at least one chemically modified nucleotide having a ribose modification. In certain embodiments, the 2'-position of the modified ribose moiety is replaced by a group selected from: OR, R, halogen substituents, SH, SR, NH 2 , NHR, NR 2 , or CN, where each R is independently C 1 -C 6 alkyl, alkenyl or alkynyl, where the halogen substituent is F, Cl, Br or I. In some embodiments, the 2'-position of the modified ribose moiety is replaced by a group selected from: allyl, amino, azido, thio, O-allyl, O-C 1 -C 10 alkyl, OCF 3 , OCH 2 F, O(CH2) 2 SCH 3 , O(CH 2 ) 2 -O-N(R m )(R n ), O-CH 2 -C(=O)-N(R m )(R n ), or O-CH 2 -C(=O)-N(R 1 )-(CH 2 ) 2 -N(R m )(R n ), where each R l , R m and R n is independently H or a substituted or unsubstituted C 1 -C 10 alkyl. In some embodiments, the modified ribose moiety has a substituent group selected from the group consisting of: 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH 3 , 2'-OCH 2 CH 3 , 2'-OCH 2 CH 2 F and 2'-O(CH2) 2 OCH 3. In some embodiments, the modified ribose moiety is replaced by a bicyclic sugar selected from the group consisting of: 4′-(CH 2 )—O-2′(LNA), 4′-(CH 2 )—S-2, 4′-(CH 2 )2—O-2′(ENA), 4′-CH(CH 3 )—O-2′(cEt), and 4′-CH(CH 2 OCH 3 )—O-2′, 4′-C(CH 3 )(CH 3 )—O-2′, 4′-CH 2 —N(OCH 3 )-2′, 4′-CH 2 —O—N(CH 3 )-2′, 4′-CH 2 —N(R)—O-2′ (wherein R is H, C 1 -C 12 alkyl, or a protecting group), 4′-CH 2 —C(H)(CH 3 )-2′, and 4′-CH 2 —C—(═CH 2 )-2′. In some embodiments, the modified ribose moiety is selected from the group consisting of: 2’-O-methoxyethyl-modified sugar (MOE), 4′-(CH 2 )—O-2′ bicyclic sugar (LNA), 2’-deoxy-2’-fluoroarabinose (FANA), and methyl (methylenoxy)(4′-CH(CH 3 )—O-2) bicyclic sugar (cEt). In some embodiments, the oligonucleotide has a chemical modification of a nucleotide selected from the group consisting of: 2’-methoxyethyl, 2’-OCH 3 , and 2’-fluoro.

[0157] In certain embodiments, the oligonucleotide has at least one chemically modified nucleobase. In certain embodiments, at least one chemically modified nucleobase is selected from the group consisting of: 5-methylcytosine (5-Me-C), hypoxanthine nucleobase, tritylated 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-CH 3Uracil, cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azauracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-mercapto, 8-alkylthio, 8-hydroxy, and other 8-substituted adenines and guanines, 5-halogen substituents (especially 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 molecules 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. Within the oligonucleotide molecule, chemical modifications can be introduced into the phosphate backbone (e.g., phosphorothioate bonds), the sugar (e.g., locked nucleic acid, glycerol nucleic acid, cEt, 2'-MOE, 2'-fluorouridine, 2'-O-methyl), and / or the base (e.g., 2'-fluoropyrimidine).

[0159] The oligonucleotide can be conjugated to the remainder of the compound or the "backbone" of the compound at the 5' end and / or 3' end of the oligonucleotide. The conjugated oligonucleotide can be delivered as a single strand or hybridized with a substantially complementary oligonucleotide and delivered as part of a duplex. The substantially complementary oligonucleotide can be similarly conjugated or unconjugated.

[0160] In one embodiment, the conjugated oligonucleotide forms part of an siRNA duplex (sense strand or antisense strand, or both). In a preferred embodiment, the conjugated oligonucleotide forms part of an aiRNA duplex (sense strand or antisense strand, or both). In another embodiment, the conjugated oligonucleotide conjugated according to the principles of the present invention is used as an antisense oligonucleotide (ASO). In yet another embodiment, the conjugated oligonucleotide conjugated according to the principles of the present invention is used as a microRNA (miRNA) molecule. Some exemplary embodiments of the conjugated oligonucleotide are as Figure 1 shown. For double-stranded RNA molecules, preferably, the oligonucleotide can be conjugated to the remainder of the compound or the "backbone" of the compound at the 3' end of the sense strand.

[0161] Embodiment 1

[0162] In a first feature, an oligonucleotide is conjugated to a backbone containing multiple components, wherein the backbone includes a sequential motif having more than one (such as 2 - 8, preferably 3) ligands (such as GalNAc) arranged along the backbone, directly or through one or more intermediate linkers, at a point of attachment provided by a residue derived from a histidine residue.

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

[0164] In one embodiment, the compounds of the present invention have the structures shown in Table 6.

[0165] In one embodiment, optionally, 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 example, the configuration of the compound refers to the isomer of the chiral carbon atom shown in the structural formula S - H1 - 01.

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

[0167] Embodiment 2

[0168] In a second feature, an oligonucleotide is conjugated to a backbone containing multiple components, wherein the backbone includes a sequential motif having more than one (such as 2 - 8, preferably 3) ligands (such as GalNAc) arranged along the backbone, directly or through one or more intermediate linkers, at a point of attachment provided by a residue derived from a glutamic acid residue.

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

[0170] In one embodiment, the compounds of the present invention have the structures shown in Table 6.

[0171] In one embodiment, optionally, the configuration of the compound is 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 example, the configuration of the compound refers to the isomers of the chiral carbon atoms shown in the structural formula S-G1-01.

[0172] In the compounds of the present invention, the naturally occurring or chemically modified oligonucleotides are linked to the rest of the compound through their 5'-end and / or 3'-end.

[0173] Table 6 III. Examples Synthesis

[0174] In some embodiments, compounds represented by formulas (S-H1), (S-H1-01) to (S-H1-16), (S-G1), (S-G1-01) to (S-G1-16), wherein the compound has an oligonucleotide conjugated to a backbone comprising a plurality of components, and the backbone includes an array pattern having more than one (such as 2-8) ligands (such as GalNAc) arranged along the backbone, are synthesized by reacting a sequence backbone comprising three or more reactive moieties with the ligands and the oligonucleotide.

[0175] Synthesis of Example 1 G-12 (Glu(R)-seqGalNAc)

[0176] The synthesis method of G-12 (Glu(R)-seqGalNAc) is shown in the following 4 steps:

[0177] Step 1: Synthetic route of compound M-3

[0178] Synthesize compound M-2

[0179] Under nitrogen protection, 20 g of M-1 ((R)-3-hydroxy-methyl butyrate) 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 carried out at room temperature for 2 h. The reaction was monitored by TLC until completion, 200 mL of saturated ammonium chloride was added to quench the reaction, the DCM layer was separated, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated to dryness, and 73 g of the crude product of M-2 was obtained. MS (ESI) m / z 357.10 ([M+H] + ).

[0180] Synthesis of compound M-3

[0181] 13.6 g of sodium hydroxide was dissolved in 80 mL of water to prepare an aqueous sodium hydroxide solution. After 73 g of the crude product of M-2 was dissolved in 500 mL of methanol, the above aqueous sodium hydroxide solution was added. The mixture was stirred at 35 °C for 15 h, and the reaction was monitored by TLC until completion. Methanol was removed by concentration at 40 °C, 500 mL of ethyl acetate was added, the pH was adjusted to 3 with 2M 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 - 10:1). The concentrated product was partially obtained to give 47.8 g of M-3. MS (ESI) m / z 341.10 ([M-H] - ). 1 1H-NMR (400 MHz, CDCl 3 ) δ 7.66 - 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 of compound N-4

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

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

[0185] Synthetic compound S-02

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

[0187] Synthetic 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 addition, the reaction was carried out overnight at room temperature. The completion of the reaction was monitored by TLC. 100 mL of saturated sodium bicarbonate solution was added to the reaction mixture, 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 - 50:1). The product fractions were collected and concentrated under reduced pressure to obtain 28.9 g of compound S-06. ESI-MS m / z: [M+H] + = 567.21

[0189] Synthetic 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 mixture was purged with hydrogen three times, and then reacted at room temperature for 4 h. The completion of the reaction was monitored by TLC. The mixture was filtered by suction, and the filtrate was concentrated under reduced pressure to obtain 12.5 g of a white foamy solid. ESI-MS m / z: [M+H] + = 433.21

[0191] Step 3: Synthetic route of compound G-12

[0192] Synthetic compound G-2

[0193] 50 g of G-1 ((S)-N-Boc-glutamic acid methyl ester) was dissolved in 500 mL of THF. 25.2 mL of NMM was added dropwise under an ice-water bath. After stirring for 5 min, 26.6 mL of isobutyl chloroformate was added dropwise. After the addition was complete, stirring was continued for 1 h. Filtration was performed by suction, and the filtrate was collected. 8.73 g of NaBH4 was added to the filtrate under an ice-water bath. After the addition was complete, the reaction was continued for 2 h. The reaction was monitored by TLC until completion. 250 mL of water was added, and extraction was performed with 500 mL of EA. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 45.35 g of an oily substance G-2. MS (ESI) m / z 248.19 ([M+H] + ).

[0194] Synthesis of compound G-3

[0195] 45.35 g of compound G-2 was dissolved in 500 mL of DCM. 31.2 g of imidazole was added, and 91.1 g of TBDPSCl was added dropwise. After the addition was complete, the reaction was carried out at room temperature for 2 h. The reaction was monitored by TLC until completion. 150 mL of water was added to the reaction solution, and the DCM layer was separated by extraction. It was washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance. Purification by silica gel column chromatography was performed with a gradient elution of PE:EA = 40:1 - 10:1, and the concentrated product was obtained as 36.7 g of a colorless and transparent oily substance G-3. MS (ESI) m / z 486.66 ([M+H] + ).

[0196] Synthesis of compound G-4

[0197] 46.15 g of compound G-3 was dissolved in 500 mL of DCM. 70 mL of TFA (trifluoroacetic acid) was added dropwise under an ice bath. After the addition was complete, the reaction was stirred at room temperature for 4 h. The reaction was monitored by TLC until completion. It was concentrated under reduced pressure. 500 mL of DCM was added to the residue, and the pH was adjusted to 8 by adding saturated sodium bicarbonate solution. The DCM layer was separated by extraction, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain 36.7 g of a pale yellow oily substance 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 stirred at room temperature for 1 hour. The reaction was monitored by TLC until completion. 600 mL of saturated sodium bicarbonate and 400 mL of EA were added to the reaction solution for extraction. The EA layer was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain an oily substance. The crude product was separated and purified by column chromatography (PE:EA = 20:1 - 8:1) to obtain 41 g of white solid G-5. 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 mixed solvent of 100 mL of MeOH, 100 mL of THF and 100 mL of water. 5.92 g of lithium hydroxide monohydrate was added, and the reaction was carried out at room temperature for 8 h. The reaction was monitored by TLC until completion. The reaction solution was concentrated under reduced pressure. 400 mL of ethyl acetate was added to the concentrated residue, and the pH was adjusted to 4 - 5 with dilute hydrochloric acid solution. The organic phase was separated by extraction, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 33.8 g of colorless transparent oily substance G-6. MS(ESI) m / z: 694.35([M+H] + )。

[0202] Synthesis of compound G-7

[0203] 18.3 g of compound G-6 was dissolved in 150 mL of DMF. 13.0 g of HBTU and 6.52 mL of DIEA were added, and the mixture was stirred at room temperature for 15 minutes. Then a 100 mL DMF solution of 11.4 g of S-07 was added dropwise. After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by TLC until completion. 400 mL of saturated sodium bicarbonate was added to the reaction solution, and the organic phase was separated by extraction with 200 mL of EA. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain an oily substance. The crude product was separated by column chromatography (PE:EA = 1:1 - 1:3) to obtain 18.9 g of white solid. 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. 50.3 mL of 1.0 M TBAF was added, and the reaction was carried out at room temperature overnight. The reaction was monitored by TLC until completion. The reaction solution was concentrated under reduced pressure and separated by column chromatography (DCM:MeOH = 50:1 - 15:1). The product fraction was collected and concentrated to dryness to obtain 7.5 g of 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, 6.01 g of DMTrCl was added, and the reaction was carried out at room temperature for 30 min. The reaction was monitored by TLC until completion. 10 mL of methanol was added to the reaction solution to quench the reaction, and the solution was concentrated under reduced pressure. Column chromatography separation (DCM:MeOH = 100:1 - 50:1) was performed. The product fraction was collected and concentrated to dryness to obtain 5.07 g of a 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, 2.04 mL of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (CTPPA) was added, and 9 mL of 0.5 M tetrazole acetonitrile solution was added. The reaction was carried out at room temperature for 2 h under nitrogen protection. The reaction was monitored by UPLC-MS until it was basically complete. The solution was concentrated under reduced pressure, dissolved in 50 mL of DCM, 30 mL of water was added, the DCM layer was separated by extraction, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 4.74 g of an oily substance. Column chromatography purification was carried out to obtain 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] Dissolve 700 mg of compound G-9 in 7 mL of anhydrous pyridine, add 10 mg of DMAP and 749 mg of succinic anhydride, and react at room temperature for 24 h under nitrogen protection. Monitor the completion of the reaction by UPLC-MS. Concentrate the reaction solution under reduced pressure to obtain an oily substance. Purify by silica gel column chromatography (DCM:MeOH = 100:1 - 20:1). Collect the product fraction and concentrate under reduced pressure to obtain 0.74 g of compound G-11. 1 H NMR (400 MHz, CDCl 3 ) 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] Add 209 mg of compound G-11, LCAA-CPG (96 μmol / g, 1.4 g), 86.8 mg of HATU, and 52.0 mg of DIEA to a 50 mL centrifuge tube, add 10 mL of anhydrous acetonitrile to dissolve the mixture, and then place it on a shaker and shake overnight. Filter the reaction solution, wash it with acetonitrile, and after drying by suction, pour the support into a 50 mL centrifuge tube, and add 5 mL of CapA (20% NMI - 80% ACN) and 5 mL of CapB (20% AC 2 O - 30% lutidine - 50% ACN) respectively, and react at room temperature with shaking for 2 h. Filter, wash with acetonitrile, and dry under vacuum overnight. Obtain 1.32 g of G-12 with a loading capacity of 62 μmol / g. Synthesis of Example 2 H-17 (His(R)-sequence GalNAc)

[0214] The synthesis method of H-17 (His(R)-sequence GalNAc) is as follows (4 steps):

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

[0216] Under nitrogen protection, 800 mL of DCM and 57 g of bromopentanol were added to 120 g of the crude product of compound S-02. 28.5 mL of TMSOTf was added dropwise. After the addition was complete, the mixture was stirred at room temperature (25 °C) overnight. A sample was taken and monitored by TLC until the reaction was complete. 1 L of saturated sodium bicarbonate was added to quench the reaction. 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 of DCM:EA = 10:1 - 2:1. The product fraction was collected and concentrated to dryness to obtain 98 g of compound S-05. MS(ESI) m / z 497.07, 498.00([M+H] + ). 1 H-NMR(400 MHz, CDCl 3 ) 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 of compound M-3

[0218] For the synthetic method, refer to the synthesis of compound M-3 in the synthetic route of G-12 (Glu(R)-sequence GalNAc).

[0219] Step 2: Synthetic route of compound H-05

[0220] Synthesize compound H-02

[0221] 38.4 g of sodium hydroxide was dissolved in 400 mL of water, 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 and stirred until dissolved. 175 g of di-tert-butyl dicarbonate was added. After addition, the mixture was stirred at room temperature for 4 h. After monitoring the reaction to completion by TLC, the reaction mixture was filtered by suction, the filtrate was concentrated, and 200 mL of MTBE was added to the concentrated residue for a total of 3 washes and extractions. 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 and washed with saturated sodium chloride. It was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness to obtain 98 g of a white solid. MS (ESI) m / z 356.25 ([M+H] + ).

[0222] Synthesis of compound H-03

[0223] Under nitrogen protection, 110 g of H-02 was dissolved in 880 mL of anhydrous THF. The temperature was cooled to 0 - 5 °C in an ice bath, and 1.1 L of borane tetrahydrofuran solution (1 M) was slowly added dropwise to the reaction solution. After addition, the mixture was stirred at room temperature for 1 h, and the reaction was monitored to completion by TLC. After the reaction solution was cooled to 0 - 10 °C in an ice bath, 230 mL of methanol was slowly added dropwise to quench the reaction. The THF was concentrated, 800 mL of ethyl acetate and 300 mL of saturated brine were added to the residue for extraction and liquid separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness to obtain 106 g of crude product of H-03, MS (ESI) m / z 342.40 ([M+H] + ), 1 H-NMR (400 MHz, 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 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 addition, the reaction was carried out at room temperature for 1 h, and the reaction was monitored to completion by TLC. 400 mL of saturated sodium chloride was added to the reaction solution, the DCM layer was separated by extraction, washed with saturated sodium chloride, dried over sodium sulfate, concentrated, and purified by silica gel column chromatography, petroleum ether:ethyl acetate = 100:1 - 20:1 gradient elution. The product eluate was collected and concentrated to dryness to obtain 106 g of a white solid. 1 H-NMR (400 MHz, In 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 H-04 was dissolved in 240 mL of glacial acetic acid, and the reaction was stirred at 80 °C overnight. After monitoring the reaction to completion by TLC, the mixture was cooled in an ice-water bath, then 400 mL of ethyl acetate was added, and 4 M sodium hydroxide was added dropwise to adjust the pH to 8 - 9. The organic phase was separated, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain 66 g of a white solid. MS(ESI) m / z 480.27 ([M+H] + ).

[0228] Step 4: Synthetic Route of Compound H-17

[0229] Synthesis of Compound H-10

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

[0231] 1 H-NMR(400 MHz, CDCl 3)δ 7.62 - 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 - water bath, and 103 mL of TFA was added dropwise. After the addition was complete, the mixture was stirred at room temperature for 2 h, and the reaction was monitored by UPLC - MS until completion. Trifluoroacetic acid was removed, 500 mL of DCM was added to the residue, and the pH was adjusted to 8 - 9 with saturated sodium bicarbonate. The DCM layer was separated by extraction, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain 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, 8.23 mL of DIEA and 15.53 g of HBTU were added. The mixture was stirred at room temperature for 30 min, and a 100 mL DMF solution of 25 g of compound H - 11 was added dropwise. After the addition was complete, the reaction was carried out at room temperature for 2 h, and the reaction was monitored by TLC until completion. 300 mL of EA and 600 mL of 10% ammonium chloride were added to the reaction solution. 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 of DCM:MeOH = 100:1 - 30:1. The product fraction was collected and concentrated to dryness to obtain 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 h until the reaction was completed. The mixture was concentrated and purified by gradient elution on a silica gel column (DCM:MeOH = 20:1 - 8:1). The product eluate was collected, concentrated, and dried to obtain 10 g of compound H-13. MS(ESI) m / z 643.42([M+H] + )。

[0238] Synthesis of compound H-14

[0239] Under nitrogen protection, 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, it was detected by UPLC-MS and no starting material remained. The reaction was quenched with 40 mL of methanol, concentrated, and purified by gradient elution on a silica gel column (DCM:MeOH = 100:1 - 50:1). The product eluate was collected, concentrated, and dried to obtain 9.3 g of compound H-14. MS(ESI) m / z 945.56([M+H] + )。

[0240] Synthesis of compound H-15

[0241] Under nitrogen protection, 3 g of compound H-14 was dissolved in 40 mL of anhydrous acetonitrile, then 2 g of CTPPA and 0.5 M tetrazole-acetonitrile (6.4 mL) solution were added, and the mixture was stirred at room temperature for 1.5 h. The reaction was monitored by sampling UPLC-MS until completion. The acetonitrile was concentrated, and the residue was extracted with 50 mL of DCM and 30 mL of water. The DCM layer was separated, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated and dried to obtain the crude product of H-15. The crude product was purified by column chromatography to obtain 1.5 g of white solid compound H-15. 1H-NMR(400MHz, DMSO-d6) δ 8.04 (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 nitrogen protection, 1.5 g of Compound H-14 was dissolved in 15 mL of anhydrous pyridine, and 20 mg of DMAP and 1.58 g of succinic anhydride were added. After reacting at room temperature for 25 h, the reaction completion was monitored by UPLC-MS. Pyridine was removed by concentration, and the residue was purified by gradient elution on a silica gel column (DCM:MeOH = 40:1 - 20:1). The product fraction was collected, concentrated and dried to obtain 1.37 g of Compound H-16. MS (ESI) m / z 1045.57 ([M+H] + ). 1H-NMR(400 MHz, DMSO-d6) δ 7.96 (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. 75 mg of HATU and 42 mg of DIEA were added. The mixture was shaken on a shaker for 10 min. LCAA-CPG-(96 μmol / g) was added, and the mixture was shaken at room temperature overnight. The reaction solution was filtered, washed with acetonitrile, and the filter cake was dried under vacuum. Then it was poured into a 50 mL centrifuge tube. 5 mL of Cap A (20% NMI - 80% ACN) and 5 mL of Cap B (20% AC 2 O - 30% lutidin - 50% ACN) were added to the centrifuge tube. After shaking for 2 h, the solvent was removed by filtration, washed with acetonitrile, and dried under vacuum at room temperature for 2 h to obtain 1.17 g of compound H-17 with a loading of 47 μmol / g. Example 3: Synthesis of H-23 (His(S)-sequence-GalNAc)

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

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

[0248] Synthesis of compound M-6

[0249] Replace methyl (R)-3-hydroxybutyrate with methyl (S)-3-hydroxybutyrate. For the synthesis method, refer to the synthesis of M-3 in the synthesis route of G-12 (Glu(R)-sequence GalNAc).

[0250] Step 2: Synthesis route of compound H-23

[0251] Synthesize compound H-18

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

[0253] Synthesize compound H-19

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

[0255] Synthesize compound H-20

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

[0257] Synthesize compound H-21

[0258] For the synthesis method, refer to the synthesis of compound H-15 in the synthesis route of H-17 (His(R)-sequence-GalNAc). Due to the unique chemical properties of H-21, it shows a unique structural fragment peak in the mass spectrum. MS(ESI) m / z 1062.43 ([M-(i-Pr) 2 N+H 2 O] + ) 1H-NMR(400MHz, DMSO-d6) δ 7.92 (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] Synthesize compound H-22

[0260] For the synthesis method, refer to the synthesis of compound H-16 in the synthesis route of H-17 (His(R)-sequence-GalNAc).

[0261] MS(ESI) m / z 1045.66 ([M+H] + ). 1H-NMR (400 MHz, DMSO-d6) δ 7.96 (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 the synthesis method, refer to the synthesis of compound H-17 in the synthesis route of H-17 (His(R)-sequence-GalNAc). The loading of H-23 is 52 μmol / g. Example 4: Synthesis of GS-13-1 (Glu(R)-sequence-GalNAc)

[0264] The synthesis of GS-13-1 (Glu(R)-sequence-GalNAc) is as follows:

[0265] Synthesis of compound GS-13-11

[0266] The starting material was changed to N-Boc-L-benzyl glutamate, and the synthesis method was the same as that for the preparation 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 for the synthesis of H-11. MS (ESI) m / z 652.30 ([M+H] + )

[0269] Synthetic compound GS-13-9

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

[0271] Synthetic compound GS-13-8

[0272] GS-13-9 is dissolved in anhydrous methanol, 10% (W / W) palladium-carbon is added, and the mixture is purged with hydrogen three times. Stir at room temperature until the raw materials react completely. Filter to remove the palladium-carbon, rotary evaporate the filtrate and directly input it into the next reaction. MS(ESI) m / z 884.40([M-H] - )。

[0273] Synthetic compound GS-13-7

[0274] Equivalent amounts of GS-13-8 and GS-13-10 are dissolved in anhydrous dichloromethane, 0.1 equivalent of DMAP, 2.0 equivalents of DCC and 2.0 equivalents of DIEA are added. Stir at room temperature under nitrogen protection until the raw materials react completely. Filter to remove the insoluble substances, wash the reaction solution with purified water, concentrate the organic phase to dryness, and purify the crude product by silica gel column chromatography (DCM:MeOH = 10:1) and dry to obtain a white solid. MS(ESI) m / z 1519.60([M+H] + )。

[0275] Synthetic compound GS-13-6

[0276] The synthesis method is the same as that of GS-13-8. MS(ESI) m / z1427.61([M-H] - )。

[0277] Synthetic compound GS-13-5

[0278] The synthesis method is the same as that of GS-13-7, and the raw materials are GS-13-6 and GS-13-14. MS(ESI) m / z1066.01([(M+2) / 2] + )。

[0279] Synthetic compound GS-13-4

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

[0281] Synthetic compound GS-13-3

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

[0283] Synthesize compound GS-13-2

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

[0285] Synthesize compound GS-13-1

[0286] The synthesis method is the same as that of H-17, with a loading of 50 μmol / g. Example 5: Synthesis of HS-13-1 (His(R)-sequence-GalNAc)

[0287] Synthesize compound HS-13-11

[0288] Replace the starting material with N-Boc-3-L-histidine benzyl ester, and the synthesis method is the same as that of H-10 preparation. MS(ESI) m / z761.30([M+H] + ).

[0289] Synthesize compound HS-13-10

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

[0291] Synthesize compound HS-13-9

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

[0293] Synthesize compound HS-13-8

[0294] Dissolve HS-13-9 in anhydrous methanol, add 10% (W / W) palladium on carbon, replace with hydrogen three times, stir at room temperature until the raw materials react completely, filter off the palladium on carbon, spin-dry the filtrate and directly put it into the next step. MS(ESI) m / z 893.40([M-H] - )

[0295] Synthesize compound HS-13-7

[0296] Equivalent amounts of HS-13-8 and HS-13-10 were dissolved in anhydrous dichloromethane, 0.1 equivalent of DMAP, 2.0 equivalents of DCC and 2.0 equivalents of DIEA were added, and the mixture was stirred at room temperature under nitrogen protection until the reaction of the starting materials was complete. The insoluble matter was removed by filtration, the reaction solution was washed with purified water, the organic phase was concentrated to dryness, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 10:1) and dried to obtain 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 synthesis. 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 synthesis. 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 synthesis. 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, with a loading of 50 μmol / g. Example 6: Synthesis of the conjugated oligonucleotides provided by the present invention

[0309] Natural or chemically modified oligonucleotides were synthesized by common methods such as solid-phase synthesis. And the compound conjugated oligonucleotides can be synthesized by the exemplary methods shown below: Preparation of Oligo-conjugated G-12 (Glu(R)-ordered GalNAc)

[0310] Solid-phase synthesis steps

[0311] Using the phosphoramidite solid-phase synthesis method known in the art, with G-12 as the solid-phase synthesis carrier, run the sequence command on the MerMade192 solid-phase synthesizer: set the attachment position of compound G-10 at the 3'-end of the sequence and set the number of compound G-10 to be attached.

[0312] Each attachment of a nucleoside monomer includes four steps: deprotection, coupling, capping, and oxidation. The standard procedures for the above steps are known to those of ordinary skill in the art, and a G-10 solution is prepared with a 0.1M acetonitrile solution.

[0313] The solid-phase synthesis reagents are prepared as follows: Detergent: Acetonitrile Deblock: 3% dichloroacetic acid in dichloromethane solution Activator: 0.25M 5-ethylthio-1H-tetrazole in acetonitrile solution Capping reagent A: THF / dimethylpyridine / acetic anhydride (8:1:1) Capping reagent B: 15% NMI / THF, GL38 finish Oxidizer: 0.02M I 2 in THF / pyridine / H 2 O Sulfurizing agent: 0.10M DDTT solution

[0314] Taking a 1 μmol synthesis scale as an example, the solid-phase synthesis conditions are as follows:

[0315] Cleavage and deprotection steps

[0316] Add the Oligo-support obtained in the above solid-phase synthesis step to a 1 mL centrifuge tube, add 50 - 100 μl of concentrated ammonia water, incubate at 50 - 60 °C for 10 h, centrifuge and aspirate the supernatant. Add 2 volumes of acetone-ethanol (80:20) solvent to the supernatant to precipitate a white solid. Centrifuge at 10,000 g to remove the supernatant to obtain the precipitated product, and redissolve the precipitate in 0.2M sodium acetate solution.

[0317] Purification, desalting, and lyophilization steps

[0318] Purification was carried out on an Avant 150 purification device using a 1 mL ion chromatography column (packed with Nano Q 30).

[0319] The specific conditions were as follows: 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: Buffer B 0 - 50%, Buffer A 100 - 50%.

[0320] After collecting the product eluate and combining them, desalting was finally carried out using a G25 Sephadex column; the OD260 concentration value of the desalted product solution was measured, the product content was calculated, and finally it was placed in a centrifuge tube for lyophilization to obtain a white lyophilized product.

[0321] Detection: Detection was carried out using reversed-phase UPLC-MS tandem mass spectrometry, and the purity was above 90%. The mass spectrum showed characteristic ion peaks at m / z [M - 7 / 7] - 、[M - 8 / 8] - 、[M - 9 / 9] -

[0322] For the synthesis example of AS-Oligo, refer to G-12-OLIGO, and it was synthesized using Unylinker-CPG (purchased from GlenResearch) as the solid-phase synthesis carrier.

[0323] For the synthesis example of Oligo conjugated with His-R-sequence-GalNAc, refer to G-12-OLIGO, and it was synthesized using H-17 as the solid-phase synthesis carrier. The difference from the synthesis of G-12-OLIGO was only that: H-15 was configured in a 0.1 M acetonitrile solution and the sequence was set on the solid-phase synthesis instrument.

[0324] For the synthesis example of Oligo conjugated with His-S-sequence-GalNAc, refer to G-12-OLIGO, and it was synthesized using H-23 as the solid-phase synthesis carrier. The difference from the synthesis of G-12-OLIGO was only that: H-21 was configured in a 0.1 M acetonitrile solution and the sequence was set on the solid-phase synthesis instrument.

[0325] The preparation of the siRNA-GalNAc conjugate or the aiRNA-GalNAc conjugate was to anneal the above-obtained Oligo-GalNAc conjugate with its complementary antisense strand at a molar ratio of 1:1 to obtain a double-stranded product. Activity test Materials and methods ​

[0326] Conjugated aiRNA for testing, synthesized: Compounds HS-9, HS-5, and HS-7 are tri-sequence GalNAcs designed based on a histidine linker, conjugated to the 3'-end of the sense strand of double-stranded RNA (such as aiRNA or siRNA). When used and described in the following examples, they are represented by the numbers "His-seq(3GalNAc)", "His(R)-seq(3GalNAc)", and "His(S)-seq(3GalNAc)", respectively. Compounds GS-9 and GS-5 are tri-sequence GalNAcs designed based on a glutamine linker, conjugated to the 3'-end of the sense strand of double-stranded RNA (such as aiRNA or siRNA). When used and described in the following examples, they are represented by the numbers "Glu-seq(3GalNAc)" and "Glu(R)-seq(3GalNAc)", respectively.

[0327] The sequences and structures of the oligonucleotides (aiRNA and siRNA) synthesized and used to test the activity in the examples are shown in Table 7 below: Table 7 aiRNA sequences targeting mouse β-Catenin for testing AGCU represents 2'-OMe modified RNA, agcu represents 2'-F modified RNA, * = PS, -L represents GalNAc conjugate.

[0328] Unless otherwise specifically stated, the in vitro delivery efficiency of the conjugates in the present invention was tested on hepatocytes by RT-qPCR. The steps for isolating primary mouse hepatocytes are as follows: Part A: Perfusion (1) Perfuse with Buffer A (2) Perfuse with Buffer B (3) Dissect the liver and place it in Buffer C Buffer A: Add 93 mg EDTA (0.5 mM) to 500 mL HBSS Buffer B: Add 400 mg type I collagenase (0.8 mg / mL) to 500 mL DMEM Buffer C: Add 2 mg BSA (2%) to 100 mL DMEM Part B: Isolation · After perfusion, place the liver in a 10 cm TC dish, open the liver bag, and shake the tissue with forceps to help dissociation. · Filter through a 70 μm filter; wash the filter with Buffer C and centrifuge at 4°C, 50 g for 5 minutes. · Discard the supernatant, gently resuspend in 50 ml of Buffer C (Wash 1), and centrifuge at 50 g for 5 minutes at 4°C. · Discard the supernatant, gently resuspend in 50 ml of Buffer C (Wash 2), and centrifuge at 50 g for 5 minutes at 4°C. · Discard the supernatant, 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 / plating medium. · Count using trypan blue to evaluate viability / yield. · Seed the cells on a collagenase-coated plate (Thermo Fisher, A1142802) using mouse primary hepatocyte thawing medium (Thermo Fisher, CM3000). It is preferably seeded at 1 ml / well in a 24-well plate. · After 3 - 4 hours, replace the medium with primary hepatocyte maintenance medium (Thermo Fisher, CM4000).

[0329] Perform an in vitro self-delivery assay (free uptake) without using a transfection agent (tested in a 12-well plate, 100,000 cells / well, 48-hour incubation). The concentrations of the tested Oligo GalNAc conjugates are indicated in each of the following examples. Detect the expression level of the targeted mRNA by RT-qPCR. Example 7: In vitro uptake of conjugated / unconjugated aiRNA by hepatocytes

[0330] The “Glu(R)-seq(3GalNAc)”-conjugated mβ-Catenin aiRNA (aiRNA#1) showed significant gene silencing activity in the in vitro self-delivery assay at 10 nM and even 1 nM compared to the unconjugated aiRNA, demonstrating the great potency of the GalNAc conjugate provided by the present invention in delivering double-stranded RNAi agents such as aiRNA. The QPCR test results are as Figure 2 shown. Example 8: In vitro uptake of aiRNA conjugated with GalNac conjugate by the liver

[0331] mβ-Catenin aiRNA (aiRNA#2) was conjugated with two different GalNAc conjugates provided by the present invention, and the two were co-cultured with isolated mouse hepatocytes at concentrations of 303 nM, 92 nM, and 28 nM for 24 hours respectively. The QPCR test results are as Figure 3As shown, Glu-seq(3GalNAc), His(R)-seq(3GalNAc), and His(S)-seq(3GalNAc) all showed very effective gene silencing in in vitro self-delivery, indicating that the GalNAc conjugates provided by the present invention have strong potency for delivering double-stranded RNAi agents (such as aiRNA). Example 9: In Vitro Uptake of aiRNA Targeting Mouse TTR Conjugated with Histidine-Sequence GalNAc Conjugates by Hepatocytes

[0332] In freshly isolated primary mouse hepatocytes, the gene silencing activity of newly designed aiRNA conjugates targeting mouse TTR (conjugated with "His-seq(3GalNAc)") (shown in Table 8 below) was tested without transfection agents (free uptake). Gene silencing mediated by in vitro free uptake evaluates the receptor-mediated intracellular transport achievable by the GalNAc conjugates provided by the present invention.

[0333] To a well of a 12-well plate containing approximately 100,000 primary mouse hepatocytes, 50 μL of aiRNA conjugate (793 - 804) and 950 μL of cell culture medium (thermofisher, CM4000) were added. The cells were incubated at 37 °C and 5% CO 2 2 for 24 h. RNA was purified, and the mRNA level was detected by RT-qPCR. The values are shown relative to untreated control cells. All aiRNA conjugates were tested at a concentration of 0.3 nM. GAPDH was used as an internal control. Table 8 aiRNA-Histidine-Sequence GalNAc Conjugates Targeting Mouse TTR AGCU = 2'-OMe modified RNA, agcu = 2'-F modified RNA, *=PS, DDD = His-seq(3GalNAc)

[0334] The results are as Figure 4 shown. All aiRNA conjugates conjugated with the compositions of the present invention showed strong gene silencing activity at a concentration of 0.3 nM in in vitro self-delivery assays, indicating that the GalNAc conjugates provided by the present invention have strong potency for delivering various double-stranded RNAi agents.

[0335] The results in all the active test examples clearly show that the GalNAc conjugates designed based on the present invention can significantly improve the in vitro delivery efficiency of oligonucleotides and achieve a great gene silencing effect for targeting different genes in hepatocytes. In addition, the linker composition provided in the present invention is based on the amino acids 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 to the present invention without departing from the scope or spirit of the invention. Other embodiments of the present invention will be apparent to those skilled in the art upon reference to the specification and practice of the present invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, and that the true scope and spirit of the present invention be set forth by the following claims.

[0337] Throughout this application, to more fully describe the state of the prior art relevant to the present invention, reference is made to various publications, patents, and / or patent applications. The disclosures of these publications, patents, and / or patent applications are incorporated herein by reference in their entirety as if each individual publication, patent, and / or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A compound having the structural formula (S-HG1): R 206 -[A1] a -[A2] b -[A3] c -R 205 (S-HG1) Wherein, R 205 、R 206 For each occurrence, independently, a protecting group, phosphate group, phosphodiester group, activated phosphate group, activated phosphite group, phosphoramidite, solid support, -OP(M')(M")O-nucleoside, -OP(M')(M")O-oligonucleotide, lipid, PEG, steroid, polymer, -O-nucleotide, nucleoside, -OP(M')(M")O-R 201 -OP(M'")(M"")O-oligonucleotide, or oligonucleotide; M', M", M'", and M"" are each independently O or S for each occurrence; A1, A2, and A3 are each independently selected from (S-1H) or (S-1G) for each occurrence: R 202A is -R 202 -R 202L ; R 217A is -R 217 -R 217L ; R 202L 、R 217L Each occurrence independently is a ligand capable of docking with a cell surface receptor; Each R 202 、R 217 、R 201 For each occurrence, independently selected from an alkylene group having 3 to 30 carbon atoms, wherein one or more carbon atoms may optionally be replaced by 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 、C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocycloalkylene, and C 5 -C 10 heteroarylene, and wherein each R 202 、R 217 、R 201 is independently optionally unsubstituted or substituted by R 209 ; optionally, R 202 、R 201 For each occurrence, each independently selected from: an alkylene group having 3 to 15 carbon atoms, wherein one or more carbon atoms may optionally be replaced by one or more substituents from the group consisting of: C(O), NH, O, S, OP(O)O, OP(S)O; R 1 、R 2 、R 204 、R 207 、R 208 、R 213 、R 214 、R 215 、R 216 、R 209 For each occurrence, independently selected from one or more of the group consisting of: H, alkyl, aryl, heteroaryl, haloalkyl, -O-alkyl, -O-alkylphenyl, -alkyl-OH, -O-haloalkyl, -S-alkyl, -S-alkylphenyl, -alkyl-SH, -S-haloalkyl, halogen substituents, -OH, -SH, -NH 2 、-alkyl-NH 2 、-N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, -CO 2 H, -C(O)O-alkyl, -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 independently 1, 2, 3, 4, 5 or 6 for each occurrence; J 201 、J 202 、J 211 、J 212 Each occurrence is independently either absent or a spacer; a, b, and c are each independently an integer from 0 to 5, and the sum of a, b, and c is an integer from 1 to 10; The oligonucleotide comprises naturally occurring or chemically modified nucleotides / nucleosides.

2. The compound according to claim 1, wherein the sum of a, b, and c is 1 or 3.

3. The compound according to claim 1, wherein the compound has the structural formula (S-H1): Wherein, R 1 selected from the group consisting of one or more of: 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 Selected from 1 - 10, preferably 1 - 3.

4. The compound according to claim 3, having the structural formula (S-H1-01): Wherein: J 202A 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 an alkylene group having 1 to 10 carbon atoms, optionally a straight-chain alkylene group having 1 to 10 carbon atoms; R 205A is a group containing a solid support or H.

5. The compound according to claim 3 or claim 4, wherein n 202 is 3.

6. The compound according to any one of claims 3-4, wherein R 206 comprises an oligonucleotide.

7. The compound according to claim 6, wherein the compound has the structural formula (S-H1-02):

8. The compound according to claim 3, Wherein: J 201 、J 202 For each occurrence of an alkylene independently selected from 1 to 30 carbon atoms, one or more of the carbon atoms may optionally be replaced by any one or more substituents selected from the group consisting of: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, S(O) 2 、C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocycloalkylene and C 5 -C 10 heteroarylene, and wherein J 201 、J 202 are each independently optionally unsubstituted or substituted by one or more groups selected from the group consisting of: H, alkyl, aryl, heteroaryl, haloalkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shaloalkyl, halogen substituents, -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).

9. The compound according to claim 3, Wherein: J 201 、J 202 For each occurrence of an alkylene group independently selected from 1 to 10 carbon atoms, one or more of the carbon atoms may optionally be replaced by any one or more substituents selected from the group consisting of: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, and S(O) 2 and wherein J 201 、J 202 are each independently optionally unsubstituted or substituted by at least one group selected from the group consisting of: H, or C 1 -C 5 alkyl, -OC 1 -C 5 alkyl.

10. The compound according to claim 9, wherein n 201 is 1, and n 202 is 3.

11. The compound according to claim 3, wherein the compound has the structural formula (S-H1-03), (S-H1-04), or (S-H1-05): Wherein, A is O or S, X is independently selected from Table 1; X 1 independently selected from Table 2; J 202 For each occurrence, independently selected from Table 3; optionally, J 202 independently selected from alkylene groups having 1 to 10 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH═N, and S(O) 2 , and wherein J 201 、J 202 are each independently optionally unsubstituted or substituted by at least one group selected from the group consisting of H, or C 1 -C 5 alkyl, -OC 1 -C 5 alkyl; R and R' are each independently selected from the group consisting of: naturally occurring and / or chemically modified oligonucleotides, H, and OH protecting groups; at least one of R and R' comprises an oligonucleotide formed from naturally and / or chemically modified nucleotides / nucleosides; R 202 a straight-chain alkylene of 3 to 15 carbon atoms, where 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, and where R 202 is optionally unsubstituted or substituted by one or more groups selected from the group consisting of: H, alkyl, haloalkyl, -Oalkyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -alkylSH, -Shaloalkyl, halogen substituents, -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 selected from -C 3 -C 8 linear alkylene groups.

12. The compound according to claim 11, wherein the compound has the structural formula (S-H1-06), (S-H1-07), or (S-H1-08):

13. The compound according to claim 11, wherein the compound has the structural formula (S-H1-09), (S-H1-10), or (S-H1-11):

14. The compound according to claim 11, wherein the compound has the structural formula (S-H1-12), (S-H1-13), or (S-H1-14):

15. The compound according to claim 1, wherein the compound has the structural formula (S-H1-15) or (S-H1-16): Wherein: R and R' are each independently selected from the group consisting of: naturally occurring or chemically modified oligonucleotides, H, and OH protecting groups; At least one of R and R' comprises an oligonucleotide formed from naturally 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, thiophosphate, sulfate, disulfide, ester, thioester, alkylamine, cycloalkylamine, alkyne, cycloalkyne, alkene, cycloalkene, lactone, and lactam bond; Each X is independently selected from Table 1; Each Y is independently selected from Table 4; Each Z is independently selected from Table 3; Each L independently comprises a ligand moiety capable of pairing with a cell surface receptor.

16. The compound according to any one of claims 11-15, wherein each A is O.

17. The compound according to any one of claims 11 - 15, wherein at least one A is O.

18. The compound according to any one of claims 1 - 17, wherein each ligand is independently selected from the group consisting of: N - acetylgalactosamine (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.

19. The compound according to any one of claims 1 - 17, wherein the ligand is N - acetylgalactosamine (GalNAc).

20. The compound according to claim 12, wherein the compound has the structure shown as HS - 1 to HS - 8:

21. The compound according to claim 12, wherein the compound has the structure shown as HS - 9.

22. The compound according to claim 12, wherein the compound has the structure shown as HS - 5.

23. The compound according to claim 1, wherein the compound has the structural formula (S - G1): wherein R 2 selected from one or more of the group consisting of: 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 Selected from 1 - 10, preferably 1 - 3.

24. The compound according to claim 23, having the structural formula (S - G1 - 01): wherein: J 212A 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 an alkylene group having 1 to 10 carbon atoms, optionally a straight-chain alkylene group having 1 to 10 carbon atoms; R 205 is a group containing a solid support or H.

25. The compound according to claim 23 or claim 24, wherein n 212 is 3.

26. The compound according to any one of claims 23-24, wherein R 206 comprises an oligonucleotide.

27. The compound according to claim 26, wherein the compound has the structural formula (S - G1 - 02):

28. The compound according to claim 23, wherein: J 211 and J 212 For each occurrence, an alkylene independently selected from 1 to 30 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more substituents selected from the group consisting of: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, S(O) 2 , C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocycloalkylene and C 5 -C 10 heteroarylene, and wherein J 211 , J 212 are each independently optionally unsubstituted or substituted by one or more groups selected from the group consisting of: H, alkyl, aryl, heteroaryl, haloalkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shaloalkyl, halogen substituents, -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).

29. The compound according to claim 23, wherein: J 211 、J 212 For each occurrence, an alkylene independently selected from 1 to 10 carbon atoms, wherein one or more carbon atoms may optionally be replaced by any one or more substituents selected from the group consisting of: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, and S(O) 2 , and wherein J 211 、J 212 are each independently optionally unsubstituted or substituted by at least one group selected from the group consisting of: H, or C 1 -C 5 alkyl, -OC 1 -C 5 alkyl The compound according to claim 29, wherein n 211 is 1, and n 212 is 1 or 3.

31. The compound according to claim 29, wherein the compound has the structural formula (S - G1 - 03), (S - G1 - 04), or (S - G1 - 05): wherein, A is O or S, X is independently selected from Table 1; X 1 independently selected from Table 2; J 212 independently selected from Table 3; Optionally, J 212 is independently selected from alkylene groups having 1 to 10 carbon atoms, wherein one or more of the carbon atoms are optionally replaced by any one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, and S(O) 2 , and wherein J 212 is optionally unsubstituted or substituted by at least one group selected from the group consisting of 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 OH protecting groups; at least one of R and R’ contains an oligonucleotide formed from naturally occurring and / or chemically modified nucleotides / nucleosides; R 217 a straight-chain alkylene group having from 3 to 15 carbon atoms, wherein one or more of the carbon atoms may optionally be replaced by one or more substituents selected from the group consisting of C(O), NH, O, S, OP(O)O, OP(S)O, and wherein R 217 is optionally unsubstituted or substituted by one or more groups selected from the group consisting of H, alkyl, haloalkyl, -Oalkyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -alkylSH, -Shaloalkyl, halogen substituents, -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 selected from -C 3 -C 8 linear alkylene group.

32. The compound according to claim 31, wherein the compound has the structural formula (S - G1 - 06), (S - G1 - 07), or (S - G1 - 08):

33. The compound according to claim 31, wherein the compound has the structural formula (S - G1 - 09), (S - G1 - 10), or (S - G1 - 11):

34. The compound according to claim 31, wherein the compound has the structural formula (S - G1 - 12), (S - G1 - 13), or (S - G1 - 14):

35. The compound according to claim 1, wherein the compound has the structural formula (S - G1 - 15) or (S - G1 - 16): wherein: R, R’ are each independently selected from the group consisting of: naturally occurring or chemically modified oligonucleotides, H, and OH protecting groups; at least one of R and R’ contains a naturally occurring and / 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, thiophosphate, sulfate, disulfide, ester, thioester, alkylamine, cycloalkylamine, alkyne, cycloalkyne, alkene, cycloalkene, lactone, and lactam bond; Each X is independently selected from Table 1; Each Y is independently selected from Table 4; Each Z is independently selected from Table 3; Each Z” is independently selected from Table 5; and Each L independently comprises a ligand moiety capable of pairing with a cell surface receptor; and Each n1, n2, n3 is independently selected from 1, 2, 3, or 4.

36. The compound according to any one of claims 31 - 35, wherein each A is O.

37. The compound according to any one of claims 31 - 35, wherein at least one A is O.

38. The compound according to any one of claims 23 - 37, wherein each ligand is independently selected from the group consisting of: N - acetylgalactosamine (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.

39. The compound according to any one of claims 23 - 37, wherein the ligand is N - acetylgalactosamine (GalNAc).

40. The compound according to claim 31, wherein the compound has the structure shown as GS - 1 to GS - 8:

41. The compound according to claim 31, wherein the compound has the structure shown as GS - 9.

42. The compound according to claim 31, wherein the compound has the structure shown as GS - 5.

43. The compound according to any one of claims 1 - 42, wherein the naturally occurring and / or chemically modified oligonucleotide is linked to other parts of the compound through its 5’ - end and / or 3’ - end.

44. The compound according to claim 43, wherein the oligonucleotide comprises a small interfering RNA (siRNA) duplex, an asymmetric interfering RNA (aiRNA) duplex, an antisense oligonucleotide (ASO), or a microRNA (miRNA).

45. The compound according to claim 44, wherein the aiRNA comprises an antisense strand and a sense strand, wherein the antisense strand is longer than the sense strand, the length of the antisense strand is 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides, and when forming a duplex with the sense strand, the antisense strand includes a 3' overhang of 1 - 9 nucleotides and a 5' overhang of 0 - 8 nucleotides; wherein the length of the sense strand is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides, and forms a duplex region with the antisense strand.

46. The compound according to claim 45, wherein when the aiRNA antisense strand forms a duplex with the sense strand, it includes a 5' overhang of 1 - 8 nucleotides.

47. The compound according to claim 45, wherein when the antisense strand of the aiRNA forms a double strand with the sense strand, it comprises a 5'-flat end.

48. A small interfering RNA (siRNA) duplex agent comprises the structural formula according to any one of claims 1-43.

49. An asymmetric interfering RNA (aiRNA) agent comprises the structural formula according to any one of claims 1-43.

50. An antisense oligonucleotide (ASO) agent comprises the structural formula according to any one of claims 1-43.

51. A microRNA (miRNA) agent comprises the structural formula according to any one of claims 1-43.

52. A pharmaceutical composition comprises the compound according to any one of claims 1-46 or the agent according to any one of claims 47-51 and a pharmaceutically acceptable excipient, carrier, or diluent.

53. Use of the compound according to any one of claims 1-46 or the agent according to any one of claims 47-51 in the preparation of a drug for effectively treating a disease or disorder.

54. A compound having the structural formula (G-P1): Wherein: R 0 selected from the group consisting of one or more of: 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); R 202A comprising at least one ligand capable of pairing with a cell surface receptor; R 204 、R 207 、R 208 are independently selected from one or more of the group consisting of: H, alkyl, aryl, heteroaryl, haloalkyl, -O-alkyl, -O-alkylphenyl, -alkyl-OH, -O-haloalkyl, -S-alkyl, -S-alkylphenyl, -alkyl-SH, -S-haloalkyl, halogen substituents, -OH, -SH, -NH 2 、-alkyl-NH 2 、-N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, -CO 2 H, -C(O)O-alkyl, -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 selected from 1, 2, 3, 4, 5 or 6; R 206 A protecting group selected from OH or OH; J 201 、J 202 Each occurrence independently is a spacer; R 205B is -C 2 -C 10 alkynylene -CN; R 205C 、R 205D are each independently selected from -C 1 -C 6 alkyl or R 205C and R 205D together form a five- or six-membered ring. Optionally, R 205C 、R 205D are both substituted. Optionally, R 205C 、R 205D further contains a heteroatom selected from N and O.

55. The compound according to claim 54, Wherein: J 201 and J 202 are each independently selected from alkylene groups having 3 to 30 carbon atoms, where one or more of the carbon atoms may optionally be replaced by 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 , C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocycloalkylene, and C 5 -C 10 heteroarylene, and where J 201 , J 202 is optionally unsubstituted or substituted by one or more groups selected from the group consisting of: H, alkyl, aryl, heteroaryl, haloalkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shaloalkyl, halogen substituents, -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).

56. The compound according to claim 54, having the structural formula (G-P2): Wherein: J 202A 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 An alkylene group having 1 to 10 carbon atoms, selected.

57. The compound according to claim 56, having the structural formula (G-P3):

58. The compound according to claim 54, having the structural formula (G-P4):

59. The compound according to claim 54, Wherein: R 205B is -C 2 -C 10 alkynylene -CN; R 205C 、R 205D each independently selected from -C 1 -C 6 -alkyl; R 202A is —R 202C -branching group-(R 202B -R 202L ) n 111L or —R 202B -R 202L ; R 202L independently selected from a ligand capable of docking with a cell surface receptor; R 202B An alkylene group having from 1 to 30 carbon atoms, wherein one or more carbon atoms may optionally be replaced by 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 , C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocycloalkylene, and C 5 -C 10 heteroarylene, and wherein R 202B is optionally unsubstituted or substituted by R 207 ; n 111L Selected from 1, 2, or 3.

60. The compound according to claim 59, wherein R 202C is selected from -C(O)-C 5 –C 8 linear alkylene-NHCO-CH 2 -, or -C(O)-C 8 –C 11 linear alkane-.

61. The compound according to claim 59, wherein the branching group is selected from the group consisting of: where each A 1 is independently O, S, C═O, or NH; and Each n is independently 1 to 20. The compound according to claim 59, wherein the ligand is wherein R A is a protecting group for H or OH.

63. The compound according to claim 59, having the structural formula (G-P5):

64. The compound according to claim 59, having the structural formula (G-P6):

65. A compound having the structural formula (G-P7): Wherein: R 3 selected from one or more of the group consisting of: 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); R 217A comprising at least one ligand capable of pairing with a cell surface receptor; R 213 、R 214 、R 215 、R 216 are independently selected from one or more of the group consisting of: H, alkyl, aryl, heteroaryl, haloalkyl, -O-alkyl, -O-alkylphenyl, -alkyl-OH, -O-haloalkyl, -S-alkyl, -S-alkylphenyl, -alkyl-SH, -S-haloalkyl, halogen substituents, -OH, -SH, -NH 2 、-alkyl-NH 2 、-N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, -CO 2 H, -C(O)O-alkyl, -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); R 212 A protecting group selected from OH or OH; J 211 、J 212 Each occurrence independently is a spacer; n 211 selected from 1, 2, 3, 4, 5 or 6; J 211 、J 212 For each occurrence independently, a spacer; R 211B is -C 2 -C 10 alkynylene -CN; R 211C and R 211D are each independently selected from -C 1 -C 6 alkyl or R 211C and R 211D together form a five- or six-membered ring. Optionally, R 211C and R 211D are both substituted. Optionally, R 211C and R 211D further contain a heteroatom selected from N and O.

66. The compound according to claim 65, Wherein: J 211 and J 212 each independently selected from alkylene groups having 3 to 30 carbon atoms, wherein one or more carbon atoms may optionally be replaced by 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 , C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocycloalkylene, and C 5 -C 10 heteroarylene, and wherein J 211 , J 212 may optionally be unsubstituted or substituted by one or more groups selected from the group consisting of: H, alkyl, aryl, heteroaryl, haloalkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohaloalkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shaloalkyl, halogen substituents, -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).

67. The compound according to claim 65, having the structural formula (G-P8): Wherein: J 212A 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 an alkylene group having 1 to 10 carbon atoms 68. The compound according to claim 66, having the structural formula (G-P9):

69. The compound according to claim 65, having the structural formula (G-P10):

70. The compound according to claim 65, Wherein: R 211B is -C 2 -C 10 alkynylene -CN; R 211C 、R 211D each independently selected from -C 1 -C 6 -alkyl; R 217 is –R 217C -branching group-(R 217B -R 217L ) n 211L or –R 217B -R 217L ; R 217L independently selected from a ligand capable of docking with a cell surface receptor; R 217B An alkylene group having 1 to 30 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more substituents of 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 heterocycloalkylene, and C 5 -C 10 heteroarylene, and wherein R 217B is optionally unsubstituted or substituted by R 213 ; n 211L Selected from 1, 2 or 3.

71. The compound according to claim 70, wherein R 217C is selected from -C(O)-C 5 –C 8 linear alkylene-NHCO-CH 2 -, or -C(O)-C 8 –C 11 linear alkane-.

72. The compound according to claim 70, wherein the branching group is selected from the group consisting of: where each A 1 is independently O, S, C═O, or NH; and Each n is independently 1 to 20. The compound according to claim 65, wherein the ligand is wherein R A is a protecting group for H or OH.

74. The compound according to claim 65, having the structural formula (G-P11):

75. The compound according to claim 65, having the structural formula (G-P12):

Images