TW202408581A - Novel compositions for conjugating oligonucleotides and carbohydrates - Google Patents

Abstract

The present invention provides a novel composition and a connection configuration for coconjugating an oligonucleotide with a ligand, which is used for in vivo targeted delivery of the oligonucleotide. The present invention also provides the use of the obtained compound and its drug composition in the preparation of a drug for effectively treating a disease or condition.

Description

Novel compositions for conjugating oligonucleotides and carbohydrates

The present invention relates to novel compositions and methods useful for conjugating carbohydrate ligands to oligonucleotides for biomedical applications.

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

RNA interference (RNAi) acts in a gene-specific manner in many organisms via short double-stranded RNA (dsRNA) duplexes, known as siRNA. siRNA has a well-defined symmetrical short (usually 20-24 base pairs) dsRNA double-stranded structure with a phosphorylated 5' end and a hydroxylated 3' end, forming two equal length 3' overhangs. Gene regulation is mediated through the multi-protein RNA-induced silencing complex (RISC), which binds, unwinds, and incorporates antisense siRNA strands from the siRNA duplex, which then recognizes and targets Cleavage of complementary messenger RNA (mRNA) thereby reducing its gene expression in a post-transcriptional manner.

A relatively new scenario is the development of aiRNAs to overcome off-target effects mediated by the symmetrical configuration of the sense strand of standard siRNA as well as other off-target mechanisms of siRNA (see PCT patent publication WO2009029688). aiRNAs are designed to contain double strands of short RNA, where the two RNA strands are not equal in length and are therefore 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, resulting in a first strand that may be 1-9 nucleotides in length. Double-stranded 'overhangs and 5' overhangs of 0-8 nucleotides. aiRNA technology can be used in all areas where siRNA or short hairpin RNA (shRNA) are currently being used, including biological research, research and development (R&D) in the biotechnology and pharmaceutical industries, and RNAi-based treatments.

Antisense technology is a highly selective gene silencing technology based on a concept first proposed in 1978 ( Zamecnik PC et al., 1978 ). In general, the principle behind ASO technology is that antisense oligonucleotides hybridize with target nucleic acids and regulate gene expression through post-transcriptional mechanisms. The mechanisms can be roughly divided into: (1) site occupancy without promoting RNA degradation, in which ASO binding leads to translation arrest, splicing inhibition or induction of alternative splicing variants, or (2) site occupancy-induced destabilization, in which ASO binding promotes RNA degradation by endogenous enzymes, such as ribonuclease H1 (RNase H1); and (3) translation enhancement: ASOs 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 a sulfur chemical modification (called phosphorothioate). Over 40 years of research, antisense technology has been improved through various chemical modifications of single-stranded oligonucleotides.

miRNA molecules are typically derived from noncoding regions of RNA transcripts that fold onto themselves to form a hairpin. After processing of their precursors by various cellular machinery, mature miRNAs found in plants, animals, and some viruses are small (approximately 22 nucleotides) RNA molecules that regulate gene expression by posttranscriptional silencing.

Therapeutics based on these and other nucleic acids offer promising treatment options for a variety of diseases, including non-druggable targets. However, despite advances in the use of oligonucleotides and oligonucleotide analogs as therapeutic options, there remains a significant need to enhance key pharmacological properties of these therapeutic oligonucleotides, such as in serum stability, Areas such as delivery to the intended organ or cell population and uptake across cell membranes.

The delivery of therapeutic oligonucleotides to cells in vivo, such as mammals, such as humans, requires specific targeting and protection from the extracellular environment in vivo, including proteins in serum. One method used by researchers to achieve specific targeting is to conjugate a targeting group to the oligonucleotide to target the therapeutic oligonucleotide to a desired target site.

One way to improve delivery specificity is to exploit receptor-mediated endocytic activity already present in the body. The uptake mechanism involves transmembrane movement of molecules bound to cell membrane receptors into the cell through invagination of the membrane structure or fusion with the cell membrane through a delivery system. This process is initiated by activating cell surface receptors or membrane receptors after specific ligands bind to the receptors. Therefore, by conjugating drug candidates with targeting groups that target such cell surface receptors, the innate endocytosis pathway can be effectively exploited for drug delivery. Many receptor-mediated endocytic systems are known and studied, including those that recognize sugars, including galactose, mannose, mannose-6-phosphate, peptides, and proteins such as transferrin , asialoglycoprotein, vitamin B12, insulin and epidermal growth factor (EGF). In particular, asialoglycoprotein receptor (ASGP-R, Asialoglycoprotein receptor) is a highly abundant receptor on hepatocytes. ASGP-R has a 50-fold higher affinity for N-acetyl-D-Galactosylamine (GalNAc) than for D-Gal. However, it has been reported that when using such conjugated conjugation systems, the linker structural design and various chemical properties of the linker moiety are important in determining the overall delivery efficiency, effectiveness, and safety of the conjugated oligonucleotide, as well as The impact on the stability and manufacturing challenges of various therapeutic oligonucleotides is critical.

Therefore, there is an urgent need to design new and efficient receptor-specific, ligand-conjugated nucleic acid complexes for various biomedical applications.

In a first aspect, the invention relates to a compound for use as a therapeutic agent, wherein the oligonucleotide is conjugated to at least one ligand, such as a carbohydrate ligand, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide or derivatives thereof, which can target the compound to receptor cells in the liver, thereby promoting endocytosis as described above.

These ligand-conjugated compounds target one or more organs or cell types, for example, the parenchymal cells of the human liver. In one embodiment, the compound includes more than one carbohydrate ligand, preferably two or three. In another embodiment, compounds of the invention include at least one (eg, one, two, or three or more) N-Acetyl-Galactosamine (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 present invention include at least one (eg, one, two or three or more) ligand, wherein the ligand is selected from the group consisting of: GalNAc, cholesterol, tocopherol, biological cyanine dyes, folic acid, RGDp, transferrin, anisidine, lactobionic acid, cRGD, hyaluronic acid, low molecular weight protamine, lipid derivatives, peptides, cyclic peptides and heterocycles.

In a second aspect, the present invention provides ligand conjugated compounds with novel structures:

Item 1, a compound with the structural formula (S-HG1): R ²⁰⁶ -[A1] _a -[A2] _b -[A3] _c -R ²⁰⁵ (S-HG1) where R ²⁰⁵ and R ²⁰⁶ are for each time Each independently appears to be H, OH, OH protecting group, phosphate group, phosphodiester group, activated phosphate group, activated phosphite group, phosphoramidite, solid carrier, -OP(M')(M" )O-nucleoside, -OP(M')(M")O-oligonucleotide, lipid, PEG, steroid, polymer, -O-nucleotide, nucleoside, -OP(M')(M ")OR ²⁰¹ -OP(M'")(M"")O-oligonucleotide, -X-OP(M')(M")O-oligonucleotide, -Z-OP(M') (M") O-oligonucleotide, or oligonucleotide; M', M", M'" and M"" are each independently O or S for each occurrence; A1, A2 and A3 are for each occurrence Occurrences are each independently selected from (S-1H) and (S-1G): (S-1H)、 (S-1G); R ^202A is -R ²⁰² -R ^202L ; R ^217A is -R ²¹⁷ -R ^217L ; R ^202L and R ^217L are independently a ligand capable of docking with cell surface receptors for each occurrence; Each R ²⁰² , R ²¹⁷ , R ²⁰¹ is independently selected for each occurrence from an alkylene group of 3 to 30 carbon atoms, one or more of which carbon atoms are optionally substituted by any one of the group consisting of Or multiple substituents: 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 (alkynylene), C ₆ -C ₁₀ arylene (arylene), C ₃ -C ₁₈ heterocyclylene (heterocyclylene) and C ₅ -C ₁₀ heteroarylene (heteroarylene) ), and wherein each R ²⁰² , R ²¹⁷ , R ²⁰¹ is independently optionally unsubstituted or replaced by R ²⁰⁹ ; optionally, R ²⁰² , R ²⁰¹ is independently selected for each occurrence from: 3 to 15 Alkylene of carbon atoms in which 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 and OP(S)O; R ₁ , R ₂ , R ²⁰⁴ , R ²⁰⁷ , R ²⁰⁸ , R ²¹³ , R ²¹⁴ , R ²¹⁵ , R ²¹⁶ , R ²⁰⁹ are independently selected for each occurrence one of the following or more: H, alkyl, aryl, heteroaryl, haloalkyl, -O alkyl, -O alkylphenyl, -alkyl-OH, -O haloalkyl, -S alkyl, -S alkyl phenyl, -alkylSH, -S haloalkyl, halo, -OH, -SH, -NH ₂ , -alkyl-NH ₂ , -N (alkyl) (alkyl), -NH (alkyl ), -N (alkyl) (alkylphenyl), -NH (alkylphenyl), cyano, nitro, -CO ₂ H, -C(O)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 are independently 1, 2, 3, 4, 5 or 6 for each occurrence; J ²⁰¹ , J ²⁰² , J ²¹¹ , J ²¹² are independently absent or spacer for each occurrence; a, b and c are independently integers from 0 to 5 for each occurrence, 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.

Item 2, a compound described in Item 1, wherein the sum of a, b and c is 1 or 3.

Item 3, the compound described in item 1, wherein the compound has a structural formula (S-H1): (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 -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); n ²⁰² is selected from 1-10, preferably 1-3.

Item 4: The compound described in Item 3 has the structural formula (S-H1-01): (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 an alkylene group having 1 to 10 carbon atoms, optionally selected from a straight chain alkylene group having 1 to 10 carbon atoms; R ^205A is a group comprising a solid phase support or H; the carbon atom marked with the symbol "*" is a chiral carbon atom.

The compound described in item 5, item 3 or item 4, wherein n ²⁰² is 1 or 3.

Item 6: The compound described in any one of Items 3-4, wherein R ²⁰⁶ comprises an oligonucleotide.

Item 7, the compound described in item 6, wherein the compound has a structural formula (S-H1-02): (S-H1-02).

Item 8, a compound described in Item 3, wherein: J ²⁰¹ and J ²⁰² are independently selected from an alkylene group of 1 to 30 carbon atoms for each occurrence, wherein one or more carbon atoms are optionally replaced by any one or more substituents 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 heterocycloyl and C ₅ -C ₁₀ heteroarylene, and wherein J ²⁰¹ and J ²⁰² are each independently optionally unsubstituted or substituted by R ²⁰⁹ .

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

Item 10, the compound described in item 9, wherein n ²⁰¹ is 1 and n ²⁰² is 1 or 3.

Item 11, the compound described in item 3, wherein the compound has a structural formula (S-H1-03), (S-H1-04), (S-H1-05) or (S-H2): (S-H1-03) (S-H1-04) (S-H1-05) (S-H2) Where, A is O or S, Or multiple substituents are substituted: 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 , Z is independently selected for each occurrence from Table 3; Optionally, J ²⁰² , Z is independently selected for each occurrence from an alkylene group of 1 to 10 carbon atoms, one or more of which carbon atoms are optionally Replaced by any one or more substituents consisting of: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N and S(O) ₂ , and where J ²⁰¹ , J ²⁰² are each independently optionally unsubstituted or substituted with at least one substituent selected from the following group: H, or C ₁ -C ₅ alkyl and -OC ₁ -C ₅ alkyl; R, R '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 a naturally occurring and/or chemically modified oligonucleotide; Oligonucleotides formed from nucleotides/nucleosides; R ²⁰² is selected from a linear alkylene group of 3 to 15 carbon atoms, one or more of which carbon atoms are optionally replaced by one of the group consisting of Or multiple substituents are substituted: C(O), NH, O, S, OP(O)O and OP(S)O, and wherein R ²⁰² is optionally unsubstituted or substituted by R ²⁰⁹ ; Optionally, R ²⁰² is selected from -C ₃ -C ₈ linear alkylene.

Table 1

Table 2

table 3

Item 12, the compound described in item 11, wherein the compound has a structural formula (S-H1-06), (S-H1-07), (S-H1-08) or (S-H2-01): (S-H1-06) (S-H1-07) (S-H1-08) (S-H2-01).

Item 13. The compound of Item 11, wherein the compound has the structural formula (S-H1-09), (S-H1-10), (S-H1-11) or (S-H2-02): (S-H1-09) (S-H1-10) (S-H1-11) (S-H2-02).

Item 14. The compound of Item 11, wherein the compound has the structural formula (S-H1-12), (S-H1-13), (S-H1-14) or (S-H2-03): (S-H1-12) (S-H1-13) (S-H1-14) (S-H2-03).

Item 15. The compound of Item 1, wherein the compound has the structural formula (S-H1-15), (S-H1-16) or (S-H2-04): (S-H1-15) (S-H1-16) (S-H2-04) 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' 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: absence, amide, ether, triazole, carbonate, carbamate, phosphate, phosphonate, phosphorothioate, sulfate, disulfide, ester, thioester, alkylamine, cycloalkylamine, alkyne, cycloalkyne, alkene, cycloalkene, lactone and lactamide bond; Each X is independently selected from Table 1, or an alkylene group of 1 to 10 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more substituents 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 of 3 to 15 carbon atoms, wherein one or more carbon atoms are optionally replaced by one or more substituents from the group consisting of: C(O), NH, O, S, OP(O)O and OP(S)O; and wherein Y is optionally unsubstituted or substituted by R ²⁰⁷ ; each Z is independently selected from Table 3; each L independently comprises a ligand group capable of pairing with a cell surface receptor.

Table 4

Item 16, a compound described in any one of Items 11-15, wherein each A is O.

Item 17, a compound described in any one of Items 11-15, wherein at least one A is O.

Item 18, a compound described in 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, lactobionic acid, cRGD, hyaluronic acid, low molecular weight protamine, lipid derivatives, peptides, cyclic peptides and heterocycles.

Item 19, a compound as described in any one of Items 1 to 17, wherein the ligand is N-acetylgalactosamine (GalNAc).

Item 20, a compound described in Item 12, wherein the compound has a structure as shown in HS-1 to HS-8 and HS-10: .

Item 21, the compound described in item 12, wherein the compound has the structure shown in HS-9 and HS-14: .

Item 22, the compound described in item 12, wherein the compound has the structure shown in HS-5 and HS-13: .

Item 23, the compound described in item 1, has the structural formula (S-G1): (S-G1) wherein R ₂ 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); n ²¹² is selected from 1-10, preferably 1-3.

The compound described in Item 24 and Item 23 has the structural formula (S-G1-01): (S-G1-01) Where: 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 an alkylene group of 1 to 10 carbon atoms, optionally selected from a straight-chain alkylene group of 1 to 10 carbon atoms; R ²⁰⁵ is a base containing a solid support or H; symbol "* The carbon atoms marked with "are chiral carbon atoms."

The compound of item 25, item 23 or item 24, wherein n ²¹² is selected from 1 and 3.

Item 26, the compound described in any one of items 23 to 24, wherein R ²⁰⁶ includes an oligonucleotide.

Item 27, the compound of Item 26, wherein the compound has the structural formula (S-G1-02): (S-G1-02).

Item 28, a compound described in Item 23, wherein: J ²¹¹ and J ²¹² are independently selected from an alkylene group of 1 to 30 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more substituents 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 heterocycloyl and C ₅ -C ₁₀ heteroarylene, and wherein J ²¹¹ and J ²¹² are each independently optionally unsubstituted or substituted by R ²⁰⁹ .

Item 29, the compound described in item 23, wherein: J ²¹¹ and J ²¹² are independently selected from alkylene groups of 1 to 10 carbon atoms, wherein one or more carbon atoms are optionally composed of any one of the following or Multiple substituents replace: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N and S(O) ₂ , and among them, J ²¹¹ and J ²¹² can each be independently Optionally unsubstituted or substituted with at least one substituent selected from the group consisting of: H, or C ₁ -C ₅ alkyl and -OC ₁ -C ₅ alkyl.

Item 30, the compound described in Item 29, wherein n ²¹¹ is 1, and n ²¹² is 1 or 3.

Item 31, the compound described in item 29, wherein the compound has a structural formula (S-G1-03), (S-G1-04), (S-G1-05) or (S-G2): (S-G1-03) (S-G1-04) (S-G1-05) (S-G2) Where, A is O or S, Or multiple substituents are substituted: 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 is independently selected from Table 3 for each occurrence; Optionally, J ²¹² is independently selected from an alkylene group of 1 to 10 carbon atoms, where one or more carbon atoms optionally consists of Any one or more substituents replace: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, and S(O) ₂ , and where J ²¹² is optionally not Substituted or substituted with at least one substituent selected from the following group: H, or C ₁ -C ₅ alkyl and -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 Glycoside; R ²¹⁷ is selected from a linear alkylene group of 3 to 15 carbon atoms, in which 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 wherein R ²¹⁷ is optionally unsubstituted or substituted by R ²⁰⁹ ; Optionally, R ²¹⁷ is selected from -C ₃ -C ₈ straight Chain alkylene.

Item 32. The compound of Item 31, wherein the compound has the structural formula (S-G1-06), (S-G1-07), (S-G1-08) or (S-G2-01): (S-G1-06) (S-G1-07) (S-G1-08) (S-G2-01).

Item 33. The compound described in Item 31, wherein the compound has a structural formula (S-G1-09), (S-G1-10), (S-G1-11) or (S-G2-02): (S-G1-09) (S-G1-10) (S-G1-11) (S-G2-02).

Item 34. The compound described in Item 31, wherein the compound has a structural formula (S-G1-12), (S-G1-13), (S-G1-14) or (S-G2-03): (S-G1-12) (S-G1-13) (S-G1-14) (S-G2-03).

Item 35, the compound described in item 1, wherein the compound has a structural formula (S-G1-15), (S-G1-16) or (S-G2-04): (S-G1-15) (S-G1-16) (S-G2-04) 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' One comprises a natural 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, Carbamates, phosphates, phosphonates, phosphorothioates, sulfates, disulfides, esters, thioesters, alkylamines, cycloalkylamines, alkynes, cycloalkynes, alkenes, cycloalkenes , lactone and lactam bonds; each Substituent substitution: 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 3 to 15 A straight-chain alkylene group of carbon atoms, in which 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 and OP(S)O; and wherein Y is optionally unsubstituted or substituted with R ²⁰⁷ ; each Z is independently selected from Table 3; each Z'' is independently selected from Table 5; and each L is independently included A ligand group capable of pairing with a cell surface receptor; and each n1, n2, n3 is independently selected from 1, 2, 3 and 4.

table 5

Item 36, the compound described in any one of Items 31 to 35, wherein each A is O.

Item 37: A compound described in any one of Items 31-35, wherein at least one A is O.

Item 38, the compound described in any one of Items 23 to 37, wherein each ligand is independently selected from the group consisting of: N-acetyl galactosamine (GalNAc), cholesterol, tocopherol, biotin, cyanine Dyes, folic acid, RGDp, transferrin, anisidamide, lactobionic acid, cRGD, hyaluronic acid, low molecular weight protamine, lipid derivatives, peptides, cyclic peptides and heterocycles.

Item 39: A compound as described in any one of Items 23-37, wherein the ligand is N-acetylgalactosamine (GalNAc).

Item 40, a compound described in Item 31, wherein the compound has a structure as shown in GS-1 to GS-8 and GS-10: .

Item 41, the compound described in Item 31, wherein the compound has a structure as shown in GS-9 or GS-14: .

Item 42 and the compound described in Item 31, wherein the compound has a structure as shown in GS-5 and GS-13: .

Item 43, the compound described in any one of items 1 to 42, wherein the naturally occurring and/or chemically modified oligonucleotide is connected to other parts of the compound through its 5' end and/or 3' end.

Item 44, a compound described in Item 43, wherein the oligonucleotide comprises a small interfering double-stranded RNA (siRNA).

Item 45, a compound described in Item 43, wherein the oligonucleotide comprises an asymmetric interfering double-stranded RNA (aiRNA).

Item 46, the compound of item 45, wherein the aiRNA includes an antisense strand and a sense strand, wherein the antisense strand is longer than the sense strand, and the length of the antisense strand is 19, 20, 21, 22 , 23, 24, 25, 26 or 27 nucleotides, and when duplexed with the sense strand the antisense strand includes a 3' overhang of 1-9 nucleotides and 0-8 cores The 5' overhang of the nucleotide; 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 is with the Antisense strands form a double-stranded zone.

Item 47, the compound of item 46, wherein the aiRNA includes an antisense strand and a sense strand, wherein the antisense strand is longer than the sense strand, and the length of the antisense strand is 19, 20, 21, 22 , 23, 24, 25, 26 or 27 nucleotides, and when duplexed with the sense strand the antisense strand includes a 3' overhang of 1-9 nucleotides and a core of 1-8 The 5' overhang of the nucleotide; 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 is with the Antisense strands form a double-stranded zone.

Item 48, a compound described in Item 46, wherein the aiRNA comprises an antisense strand and a sense strand, wherein the antisense strand is longer than the sense strand, the antisense strand is 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides in length, and when forming a double strand with the sense strand, the antisense strand comprises a 3' overhang of 1-9 nucleotides and a 5' blunt end; wherein the sense strand is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length and forms a double strand region with the antisense strand.

Item 49, the compound described in Item 43, wherein the oligonucleotide comprises an antisense oligonucleotide (ASO).

Item 50, the compound of item 43, wherein the oligonucleotide contains microRNA (miRNA).

Item 51. A small interfering RNA (siRNA) agent comprising the structural formula described in any one of Items 1-50.

Item 52. An asymmetric interfering RNA (aiRNA) agent comprising the structural formula described in any one of Items 1-50.

Item 53. An antisense oligonucleotide (ASO) agent comprising the structural formula described in any one of Items 1-50.

Item 54. A microRNA (miRNA) agent comprising the structural formula described in any one of Items 1-50.

Item 55. A pharmaceutical composition comprising the compound described in any one of Items 1-50 or the agent described in any one of Items 51-54, and a pharmaceutically acceptable excipient, carrier, or diluent.

Item 56. Use of a compound described in any one of Items 1 to 50 or an agent described in any one of Items 51 to 54 for preparing a medicament effective for treating a disease or condition.

In a third aspect, the present invention is characterized in that, as described in the second aspect above, the compound comprises a carbohydrate ligand, and the presence of the carbohydrate ligand can enhance the delivery of the compound to a target organ (e.g., liver). Therefore, the compound comprising a carbohydrate ligand can be used to target genes associated with a disease or an undesirable condition in a target organ. For example, the compound of the present invention comprises a carbohydrate ligand that can target a nucleic acid expressed by a hepatitis virus. In other examples, the target gene can be selected from the group consisting of: Factor VII, Eg5, PCSK9, APOC3, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erkl/2 gene, PCNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, cytokine D gene, VEGF gene, EGFR gene, cytokine A gene, cytokine E gene, WNT-I gene, β-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 (WAF1/CIPl) gene, mutations in the p27 (KIPl) gene, mutations in the PPM1D 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.

In the fourth aspect, the present invention provides compounds with new structures:

Item 57, a compound having the structural formula (G-P1): (G-P1) wherein: R ₀ is one or more selected from the group consisting of: H, C ₁ -C ₅ alkyl, aryl, heteroaryl, C ₁ -C ₅ halogenated alkyl, -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)halogenated alkyl, -SO ₂ (alkyl), -SO ₂ (halogenated alkyl), -SO ₂ NH ₂ , -SO ₂ NH(alkyl) and -SO ₂ NH(phenyl); R ^202A comprises at least one ligand capable of binding to a cell surface receptor; R ²⁰⁴ , R ²⁰⁷ , R ²⁰⁸ are independently selected from one or more of the following: H, alkyl, aryl, heteroaryl, halogenated alkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohalogenated alkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shalogenated alkyl, halogen, -OH, -SH, _-NH2 , -alkyl- _NH2 , -N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, _-CO2H , -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), _-CONH2 , -NHC(O)(alkyl), -NHC(O)(phenyl), -N(alkyl)C(O)(alkyl), -N(alkyl)C(O)(phenyl), -C(O)alkyl, -C(O)alkylphenyl, -C(O)halogenated alkyl, -OC(O)alkyl, _-SO2 R 205C and R _205D are _each independently selected from -C _{1 -C 6 alkyl} , R ^205C and R 205D together form a _five -membered or six-membered ring, optionally, R 205C and R ^205D are _both substituted _, optionally, R ^205C and R ^205D are each independently selected from -C ₁ -C _{6 alkyl} , R ^205C and R ^205D together form a five-membered or _six ^- membered ring, optionally, R ^205C and R ^{205D are} both substituted, optionally ^, R ^205C , R ^205D further contain a foreign atom selected from N and O.

Item 58, the compound described in item 57, wherein: J ²⁰¹ and J ²⁰² are each independently selected from an alkylene group of 3 to 30 carbon atoms, in which one or more carbon atoms are optionally selected from the group consisting of Replace any one or more substituents of the group: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, S(O) ₂ , C ₂ -C ₁₀ sub Alkenyl, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C ₃ -C ₁₈ heterocyclylene, and C ₅ -C ₁₀ heteroarylene, and wherein J ²⁰¹ and J ²⁰² can The elective site is not superseded or superseded by R ²⁰⁹ ;

The compound described in Item 59 and Item 57 has the structural formula (G-P2): (G-P2) Where: 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 of 1 to 10 carbon atoms.

Item 60, the compound described in Item 59 has the structural formula (G-P3): (G-P3).

Item 61, the compound described in Item 57, having the structural formula (G-P4): (G-P4).

Item 62, the compound described in Item 57, wherein: R ^205B is -C ₂ -C ₁₀ alkynylene -CN; R ^205C and R ^205D are each independently selected from -C ₁ -C ₆ alkyl; R ^202A is - R ^202C -branched base-(R ^202B -R ^202L ) _n ^111L or -R ^202B -R ^202L ; R ^202L is independently selected from a ligand capable of docking with cell surface receptors; R ^202B is selected from 1 to 30 carbons An alkylene group of atoms in which 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 heteroylene Cyclyl, 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, and 3.

Item 63. The compound described in Item 62, wherein R ^202C is selected from -C(O)-C ₅ -C ₈ linear alkylene-NHCO-CH ₂ - and -C(O)-C ₈ -C ₁₁ linear alkylene-.

Item 64, a compound described in Item 62, wherein the branching group is selected from the group consisting of: and ; wherein each A ₁ is independently O, S, C═O, or NH; and each n is independently 1 to 20.

Item 65, a compound described in Item 61, wherein the ligand is , wherein ^RA is a protecting group for H or OH.

The compound described in Item 66 and Item 61 has the structural formula (G-P5): (G-P5).

Item 67, the compound described in Item 61, has the structural formula (G-P6): (G-P6).

Item 68, a compound having the structural formula (G-P7): (G-P7) wherein: R ₃ is one or more selected from the group consisting of: H, C ₁ -C ₅ alkyl, aryl, heteroaryl, C ₁ -C ₅ halogenated alkyl, -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)halogenated alkyl, -SO ₂ (alkyl), -SO ₂ (halogenated alkyl), -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 R ²¹⁵ and R ²¹⁶ are independently selected from one or more of the following: H, alkyl, aryl, heteroaryl, halogenated alkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohalogenated alkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shalogenated alkyl, halogen, -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); R ²¹² is selected from OH and a protecting group for OH; J ²¹¹ and J ²¹² are independently spacers for each occurrence; n ²¹¹ is selected from 1, 2 _, 3, 4, 5 and 6; J ²¹¹ and J ²¹² are independently spacers for each occurrence; R R ^211C and R ^211D are each independently selected from -C _{1 -C 6} alkyl; R ^211C and R ^211D together form a five _- membered 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.

Item 69, the compound described in item 68, wherein: J ²¹¹ and J ²¹² are each independently selected from an alkylene group of 3 to 30 carbon atoms, in which one or more carbon atoms are optionally selected from the group consisting of Replace any one or more substituents: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, S(O) ₂ , C ₂ -C ₁₀ alkenylene base, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C ₃ -C ₁₈ heterocyclylene, and C ₅ -C ₁₀ heteroarylene, and where J ²¹¹ and J ²¹² are optional Land is not replaced or replaced by R ²⁰⁹ ;

Item 70, the compound described in Item 68, having the structural formula (G-P8): (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 an alkylene group having 1 to 10 carbon atoms.

Item 71, the compound described in Item 69, has the structural formula (G-P9): (G-P9).

Item 72, the compound described in Item 68 has the structural formula (G-P10): (G-P10).

Item 73, the compound described in 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 base - (R ^217B -R ^217L ) _n ^211L or -R ^217B -R ^217L ; R ^217L is independently selected from a ligand capable of docking with cell surface receptors; R ^217B is selected from 1 to 30 carbons An alkylene group of atoms in which 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 heteroylene Cyclyl, 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 and 3.

Item 74: The compound described in Item 73, wherein R ^217C is selected from -C(O)-C ₅ -C ₈ straight chain alkylene-NHCO-CH ₂ - and -C(O)-C ₈ -C ₁₁ straight chain alkylene-.

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

Item 76, the compound described in Item 68, wherein the ligand is , wherein ^RA is a protecting group for H or OH.

The compound described in Item 77 and Item 68 has the structural formula (G-P11): (G-P11).

Item 78, the compound described in Item 68, has the structural formula (G-P12): (G-P12).

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, and is used to synthesize sequential and/or cluster ligand conjugates; in particular, this type of compound can be used as an intermediate for synthesizing sequential ligand conjugates.

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

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

In another aspect, the invention features a method for delivering a compound to a specific target in a subject for therapeutic or diagnostic purposes. Accordingly, the invention provides a method of treating or preventing a disease or condition, wherein the method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a compound of the invention. Treat or prevent disease by silencing part or all of the disease gene. Disease genes may be the patient's own genes, or they may be microbial genes from the outside, such as viruses.

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

Invention details I. definition

Technical terms are used according to conventional usage unless otherwise stated. Definitions of common terms in molecular biology can be found, for example, in Lewin's Genes XII (2017 (ISBN 9781284104493)) by J. Krebs et al. Found in Molecular Biology and Biotechnology: a Comprehensive Desk Reference (2011 (ISBN 9788126531783)), edited by Robert A. Meyers (ed.); and other similar technical references.

As used in the specification and claims, the singular forms "a," "an," "an," and "the" include the 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 elements. In this regard, this statement is intended to serve as support for the use of exclusive terminology in claims, such as "only," "only," and similar terms with respect to the recitation of claim elements, or the use of "negative" limitations, such as "wherein [the specified feature or element] is not present," or "except for [the specified feature or element]," or "wherein [the specified feature or element] is not present (is included, etc.)..."

As used herein, descriptions of numerical ranges for variables are intended to indicate that the present invention may be implemented with the variable equal to any value within the range. Thus, for variables that are discrete in nature, the variable may be equal to any integer value within the numerical range, including the endpoints of the range. Similarly, for variables that are continuous in nature, the variable may 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 may take on the values 0, 1, or 2 if the variable is discrete in nature, and may take on the values 0.0, 0.1, 0.01, 0.001, or any other actual value >0 and <2 if the variable is continuous in nature.

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%".

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, 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, wherein J 201 , J 202, J 203, J ₂₀₄ , J 205, J 206, J 207, J 208, _J 209, J ²¹⁰ , J 211, J 212, J 213, J 214, J ₂₁₅ , J 216, J 217, J ₂₁₈ , J 219, J 220, J 221 ²⁰² are each independently optionally unsubstituted or substituted with one or more of the group consisting of: H, alkyl, aryl, heteroaryl, halogenated alkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohalogenated alkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shalogenated alkyl, halogen, -OH, -SH, _-NH2 , -alkyl- _NH2 , -N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, _-CO2H , -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), _-CONH2 , -NHC(O)(alkyl), -NHC(O)(phenyl), -N(alkyl)C(O)(alkyl), -N(alkyl)C(O)(phenyl), -C(O)alkyl, -C(O)alkylphenyl, -C(O)haloalkyl, -OC(O)alkyl, -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).

Various hydroxyl protecting groups can be used in the present disclosure. Generally speaking, protecting groups make chemical functional groups insensitive to specific reaction conditions, and can be attached to and removed from the functional groups of the molecule without significantly damaging the rest 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 of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under alkaline 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).

As used herein, a dash ("-") that is not between two letters or two symbols is used to indicate the position of the point of attachment of a substituent. For example: -C ₁ -C ₁₀ alkyl -NH ₂ is connected through C ₁ -C ₁₀ alkyl.

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 occur. For example, "optionally substituted alkyl" includes "alkyl" and "substituted alkyl" as defined below. It will be understood by those of ordinary skill in the art to which the present invention pertains that, for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically unfeasible, and/or inherently unstable.

As used herein, "alkyl" refers to straight and branched chains having a specified number of carbon atoms, typically 1 to 20 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 , 1 to 6, 1 to 5, or 1 to 3 carbon atoms. For example, C ₁ -C ₆ alkyl groups include straight and branched chain alkyl groups of 1 to 6 carbon atoms. When an alkyl residue with a specific number of carbon atoms is named, it is intended to cover all branched and straight-chain forms with that number of carbon atoms; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, iso-butyl Butyl and tert-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to the same residue as alkyl but with two points of attachment.

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 the 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, such as 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, but-1,3-dien-1-yl, but-1,3-dien-2-yl, etc. In certain embodiments, alkenyl has 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl and refers to a residue that is the same as alkenyl but has two points of attachment.

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

As used herein, "alkoxy" refers to an alkyl group having the specified number of carbon atoms connected through 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-methyl Pentoxy etc. Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected through an oxygen bridge.

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

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

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

As used herein, "haloalkyl" refers to an alkyl group as defined above having a specific number of carbon atoms, substituted with one or more, up to the maximum allowed number of halogen atoms. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.

"Heterocyclic group" refers to a stable 3- to 18-membered non-aromatic cyclic group containing 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, which may include a fused ring or a bridged ring system. The heteroatoms in the heterocyclic group may be optionally oxidized. One or more nitrogen atoms (if present) may be optionally quaternized. The heterocyclic group is partially saturated or fully saturated. The heterocyclic group may be connected to the rest 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.

"Heteroaryl" refers to a substituent derived from a 3 to 18 membered aromatic ring group, which contains 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, a heteroaryl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., according to Hückel theory, it contains cyclic, delocalized (4n+2)π-electron system. Heteroaryl groups include fused or bridged ring systems. Heteroatoms in heteroaryl groups may be optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. A heteroaryl group is attached to the rest of the molecule through any atom on the ring. Examples of heteroaryl groups include, but are not limited to: azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzene 1, 3-benzodioxolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzo[d]thiazolyl Benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl benzo[b][1,4]oxazinyl), 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzene Benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzo benzofuranonyl, benzothienyl (benzothiophenyl), benzothieno [3, 2-d] pyrimidinyl, benzotriazolyl ( benzotriazolyl), benzo[4,6]imidazo[1,2-a]pyridinyl (benzo [4, 6] imidazo [1, 2-a] pyridinyl), carbazolyl (carbazolyl), cinnolinyl ( 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-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cycloheptano[1,2 -c]pyridazinyl (6, 7-dihydro-5H-benzo [6, 7] cyclohepta [1, 2-c] pyridazinyl), dibenzofuranyl (dibenzofuranyl), dibenzothiophenyl (dibenzothiophenyl), Furanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydro Cycloctano[d]pyrimidinyl (5, 6, 7, 8, 9, 10-hexahydrocycloocta [d] pyrimidinyl), 5,6,7,8,9,10-hexahydrocyclooctano[d] Pyridazinyl (5, 6, 7, 8, 9, 10-hexahydrocycloocta [d] pyridazinyl), 5,6,7,8,9,10-hexahydrocyclooctano[d]pyridyl (5, 6 , 7, 8, 9, 10-hexahydrocycloocta [d] pyridinyl), isothiazolyl (isothiazolyl), imidazolyl (imidazolyl), indazolyl (indazolyl), indolyl (indolyl), isoindolyl (isoindolyl) , indolinyl, isoindolinyl, soquinolyl, indolizinyl, isoxazolyl, 5,8-methylene -5,6,7,8-tetrahydroquinazolinyl (5,8-methano-5,6,7,8-tetrahydroquinazolinyl), naphthyridinyl (naphthyridinyl), 1,6-naphthyridinonyl (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- 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 (pyrazinyl), pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinoline tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno [2,3-d]pyrimidinyl (5, 6, 7, 8-tetrahydrobenzo [4, 5] thieno [2, 3-d] pyrimidinyl), 6,7,8,9-tetrahydro-5H-cycloheptyl Alkano[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 (5, 6, 7, 8-tetrahydropyrido [4, 5-c] pyridazinyl), thiazolyl, thiadiazolyl (thiadiazolyl), triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno [3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl and thiophenyl/thienyl ).

As used herein, the term "solid phase support" includes solid phase supports used in oligonucleotide synthesis, such as CPG.

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

As used herein, the term "aiRNA" is an asymmetric interfering RNA double-stranded molecule that contains an antisense strand and a sense strand, where the antisense strand is longer than the sense strand, consists of 19-27 nucleotides, and includes at least one core The 3' overhang of the nucleotide and the 5' end of 0-8 nucleotides; wherein the antisense strand is at least 70% complementary to the target mRNA; wherein the sense strand consists of 10-26 nucleotides, A two-stranded region containing 0, 1 or 2 mispairs is formed with the antisense strand. Exemplary structures of aiRNA are described in US 2009/0208564, which is incorporated herein by reference in its entirety.

As used herein, the term "middle type" refers to an interfering RNA duplex that includes an antisense strand and a sense strand, where the antisense strand is longer than the sense strand, and the 3' overhang of the antisense strand is The 5' overhangs all contain at least one nucleotide.

As used herein, the term "blunt type" refers to an interfering RNA double-stranded molecule including an antisense strand and a sense strand, wherein the RNA double-stranded molecule has at least one blunt end, preferably 3' to the sense strand end or have a blunt end at the 5' end of the antisense strand.

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

As used herein, the term "modified nucleotide" refers to a nucleotide having at least one modified sugar group, a modified internucleoside linkage and/or a modified nucleobase.

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

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

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

As used herein, the term "natural sugar group" refers to sugars found in DNA (2-H) or RNA (2-OH).

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

As used herein, the term "bicyclic sugar" refers to a furosyl ring modified by bridging two non-geminal ring atoms. Bicyclic sugars are modified sugars.

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

As used herein, "prevention" and "preventing" are used interchangeably. These terms refer to an approach to obtaining a beneficial or desired result, including but not limited to a preventive benefit. For a "preventive benefit," the conjugate or composition may be administered to a patient at risk for developing a particular disease, or to a patient reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.

As used herein, the term "effective amount" of an active agent refers to an amount sufficient to induce a desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of a compound of the invention may vary depending on factors such as the desired biological endpoint, the pharmacokinetic properties of the compound, the disease being treated, the route of administration, and the patient.

As used herein, the terms "treating" or "treatment" of a disease or disorder refer to methods that reduce, delay, or ameliorate the condition before or after the disease or disorder occurs. Treatment may target one or more effects or symptoms of the disease and/or underlying pathology. Treatment may be any relief and may be, but is not limited to, complete elimination of the disease or symptoms of the disease. The degree of reduction or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to an equivalent untreated control group (via any standard technical measurement).

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

The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term expressly excludes cell culture media. For oral medications, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorants, and preservatives. Suitable inert diluents include sodium and calcium carbonates, sodium and calcium phosphates, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, while the lubricant, if any, is generally magnesium stearate, stearic acid or talc. If necessary, the tablets may be coated with materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the human gastrointestinal tract.

Compound configuration

The compounds of the present invention and their salts may exist in tautomeric forms (for example, as amides or imino ethers). All such tautomeric forms are considered part of this invention.

All stereoisomers of the compounds of the invention (e.g., those that may exist due to asymmetric carbons on various substituents), including enantiomers and diastereomers, are contemplated by the invention. within the range. For example, a single stereoisomer of a compound of the invention may be substantially free of other isomers (e.g., as a pure or substantially pure optical isomer having a particular activity), or may be present, for example, as a racemate or with All other or other selected stereoisomers are mixed. The chiral center of the present invention may have an S configuration or an R configuration as defined in the IUPAC 1974 recommendations. The racemic form can be resolved by physical methods, such as fractional crystallization, isolation or crystallization of non-stereomeric derivatives, or separation by chiral column chromatography. Individual optical isomers may be obtained from the racemate by any suitable method, including but not limited to conventional methods, for example, salt formation with an optically active acid followed by crystallization.

After preparation, the compounds of the 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), which is then used or formulated as described herein. composition. In certain embodiments, compounds of the invention are more than 99% pure.

All configurational isomers of the compounds of the present invention are either in the intended mixtures or in the intended pure or substantially pure form. The definition of the compounds of the present invention includes cis (Z) and trans (E) olefin isomers, as well as cis and trans isomers of cyclic hydrocarbons or heterocyclic rings.

D- Amino Acids /L- amino acids

Amino acids included in the peptides or polypeptides described herein will be understood to be in the L-configuration or the D-configuration. II. Implementation plan

The present invention provides novel compounds having novel linker moieties for linking the various components of the compound. The compound conjugates an oligonucleotide to one or more targeting ligands. Oligonucleotides can be naturally occurring (isolated from nature, or synthesized in the laboratory) or chemically modified in at least one subunit.

In some embodiments, the oligonucleotide is a chemically modified oligonucleotide. In some embodiments, chemically modified oligonucleotides include backbone modifications (or internucleoside bond modifications, such as phosphate modifications), ribosyl modifications, and base modifications.

In certain embodiments, the oligonucleotide has at least one phosphorothioate internucleoside linkage, or at least one methylphosphonate internucleoside linkage, or at least one other modified internucleoside linkage, such as:

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 group is replaced with a group selected from: OR, R, halo, SH, SR, NH ₂ , NHR, NR ₂ and CN, where each R is independently Ground is a C ₁ -C ₆ alkyl, alkenyl or alkynyl group, wherein the halo group is F, Cl, Br or I. In some embodiments, the 2' position of the modified ribose group is replaced with a group selected from: allyl, amine, azido, thio, O-allyl, OC ₁ -C ₁₀ Alkyl, OCF ₃ , OCH ₂ F, O(CH2) ₂ SCH ₃ , O(CH ₂ ) ₂ -ON(R _m )(R _n ), O-CH ₂ -C(=O)-N(R _m )(R _n ) and 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 substituted or unsubstituted C ₁ -C ₁₀ alkyl. In some embodiments, the modified ribose group is substituted with a group selected from the following group: 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 group is replaced with a bicyclic sugar selected from the following group: 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' (where R is H, C ₁ -C ₁₂ alkyl, or protecting group), 4'-CH ₂ —C(H)(CH ₃ )- 2' and 4'-CH ₂ —C—(═CH ₂ )-2'. In some embodiments, the modified ribose group is selected from the following group: 2'-O-methoxyethyl modified sugar (MOE), 4'-(CH ₂ )-O-2' bicyclic sugar ( LNA), 2'-deoxy-2'-fluoroarabinose (FANA) and methyl (methyleneoxy) (4'-CH(CH ₃ )—O-2) bicyclic sugar (cEt). In some embodiments, the oligonucleotide has a chemically modified nucleotide selected from the group consisting of 2'-methoxyethyl, 2'- _OCH3 , and 2'-fluoro.

In some embodiments, the oligonucleotide has at least one chemically modified nucleobase. In some embodiments, at least one chemically modified nucleobase is selected from the group consisting of 5-methylcytosine (5-Me-C), hypoxanthine nucleobase, trityl alkanobase, 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 ₃ ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogen (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 invention is 5-methylcytosine. In certain embodiments, the modified nucleobase is 5-methyluracil.

Any stabilizing modification known to one of ordinary skill in the art may be used to increase the stability of the oligonucleotide molecule. Within the oligonucleotide molecule, chemical modifications can be introduced into the phosphate backbone (e.g., phosphorothioate linkages), sugars (e.g., locked nucleic acids, glycerol nucleic acids, cEt, 2'-MOE, 2'-fluorouridine, 2' -O-methyl), and/or bases (e.g. 2'-fluoropyrimidine).

The oligonucleotide may be conjugated to the remainder of the compound or the "backbone" of the compound at the 5' end and/or the 3' end of the oligonucleotide. The conjugated oligonucleotide may be delivered as a single strand or hybridized with a substantially complementary oligonucleotide and delivered as part of a double strand. The substantially complementary oligonucleotide may be similarly conjugated or not conjugated.

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

Embodiment 1

In the first feature, the oligonucleotide is conjugated to a backbone comprising multiple components, wherein the backbone comprises a sequential motif having one or more (e.g., 2-8, preferably 3) ligands (e.g., GalNAc) arranged along the backbone, directly or through one or more intermediate linkers at the attachment point provided by a group derived from a histidine residue.

In one embodiment, compounds of the invention have compounds such as (S-H1), (S-H2), (S-H1-01)-(S-H1-16) and (S-H2-01))-( S-H2-04).

In one embodiment, a compound of the invention has the structure shown in Table 6.

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

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

Implementation Plan 2

In the second feature, the oligonucleotide is conjugated to a backbone comprising multiple components, wherein the backbone comprises a sequence pattern having one or more (e.g. 2-8, preferably 3) ligands (e.g. GalNAc) arranged along the backbone, directly or through one or more intermediate linkers at the attachment point provided by a group derived from a glutamine residue.

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

In one embodiment, the compound of the present invention has the structure shown in Table 6.

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

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

Table 6 No. structure No. structure GS-15 HS-15 GS-16 HS-16 GS-17 HS-17 GS-18 HS-18 GS-19 HS-19 GS-20 HS-20 GS-21 HS-21 GS-22 HS-22 GS-23 HS-23 GS-24 HS-24 GS-25 HS-25 GS-26 HS-26 GS-27 HS-27 GS-28 HS-28 GS-29 HS-29 HS-30 HS-31 HS-32 HS-33 III ． Example synthesis

In some embodiments, the compounds as shown in formula (S-H1), (S-H1-01) to (S-H1-16), (S-G1), (S-G1-01) to (S-G1-16), wherein the compounds have an oligonucleotide conjugated to a main chain comprising multiple components, wherein the main chain includes a sequence pattern having more than one (e.g., 2-8) ligands (e.g., GalNAc) arranged along the main chain, and are synthesized by reacting a sequence backbone comprising three or more reactive groups with the ligand and the oligonucleotide.

Abbreviation Reagent Abbreviation Reagents/Materials/Methods The ACN Acetonitrile DDTT N,N-dimethyl-N'-(3-thio-3H-1,2,4-disulfazol-5-yl)formamidine The _AC2O Acetic anhydride HBTU O-Benzotriazole-tetramethyluronium hexafluorophosphate DCM Dichloromethane HATU 2-(7-nitrobenzotriazole-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate DMAP 4-Dimethylaminopyridine TLC thin layer chromatography DMF dimethylformamide THF Tetrahydrofuran DMSO dimethyl sulfate TMSOT Trimethylsilyl trifluoromethanesulfonate DIPEA diisopropylethylamine MS Mass Spectrometry DIEA diisopropylethylamine NMI N-methylimidazole UPLC ultra high performance liquid chromatography NMM N-methylmorpholine PE Petroleum ether TBDPSCl Tert-butyldiphenylchlorosilane TFA Trifluoroacetate TBAF-THF Tetrabutylammonium fluoride EA Ethyl acetate ​    Example 1G-12 ( Glu(R)- sequence GaNAc )Synthesis

The synthesis method of G-12 (Glu(R)-GalNAc) is shown in the following 4 steps:

Step 1 : Synthesis route of compound M-3

Synthesis of Compound M-2

Under nitrogen protection, 20 g of M-1 ((R)-3-hydroxy-methylbutyrate) was dissolved in 200 mL of DCM, 18 g of imidazole was added, and 56.2 g of TBDPSCl was added dropwise. After the addition, the reaction was allowed to react at room temperature for 2 h. The reaction was monitored by TLC, and 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, and concentrated to dryness to obtain 73 g of crude M-2. MS (ESI) m/z 357.10 ([M+H] ⁺ ).

Synthetic compound M-3

13.6 g of sodium hydroxide was dissolved in 80 mL of water to prepare a sodium hydroxide aqueous solution. 73 g of crude M-2 was dissolved in 500 mL of methanol and then added to the above sodium hydroxide aqueous solution. Stir at 35°C for 15 h and monitor the reaction completion by TLC. Concentrate at 40°C to remove methanol, add 500 mL of ethyl acetate, adjust pH to 3 with 2 M HCl, separate the EA layer, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography (PE:EA=50:1-10:1). The concentrated product fraction obtained 47.8 g of M-3. MS (ESI) m/z 341.10 ([MH] ^- ). ¹ H-NMR (400MHz, CDCl ₃ ) δ7.66-7.69(m, 4H), 7.35-7.45(m,6H), 4.23-4.30(m,1H), 2.43-2.56(m, 2H), 1.14(d, 3H), 8.77(s, 9H)

Step 2 : Synthesis route of compound N-4 .

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

Step 3 : Synthesis route of compound S-07 .

Synthetic compounds S-02

250 g of compound S-01 (N-acetylgalactosamine) was dissolved in 2000 mL DCM, and TMSOTf (157.5 g) was added dropwise. After dripping, react at 40°C for 6 h under nitrogen protection, and use TLC to monitor the completion of the reaction. Adjust the pH of the reaction solution to 8-9 with saturated sodium bicarbonate, extract and separate the organic phase, wash once with saturated sodium chloride solution, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain 211 g of a yellow transparent oily substance. ESI-MS M/Z: [M +H] ⁺ = 330.12.

synthetic compounds S-06

25.2 g of compound S-02 was dissolved in 200 mL of DCM, 20.0 g of compound N-4 was added, and 6.93 g of TMSOTf was added dropwise. After the dropwise addition, the reaction was carried out at room temperature overnight. The completion of the reaction was monitored using TLC. Add 100 mL of saturated sodium bicarbonate solution to the reaction mixture, separate the organic phase, wash once with saturated sodium chloride solution, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and perform silica gel column chromatography (DCM: MeOH=100:1- 50:1) purification. 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.

synthetic compounds S-07

Dissolve 16.5 g of compound S-06 in 150 mL of MeOH, add 1.65 g of Pd/C, replace the atmosphere with hydrogen three times, and react at room temperature for 4 h. Monitor the reaction completion using TLC. Filter, reduce the pressure and concentrate the filtrate to obtain 12.5 g of a white foamy solid. ESI-MS m/z: [M+H] ⁺ = 433.21.

Step 3 : Synthetic route of compound G-12

Synthesis of compound G-2

G-1 ((S)-N-Boc-glutamate methyl ester) 50 g was dissolved in 500 mL THF, 25.2 mL NMM was added dropwise in an ice water bath, stirred for 5 minutes, and then 26.6 mL isobutyl chloroformate was added dropwise. After that, continue stirring for 1 hour. Filtrate with suction, collect the filtrate, and add 8.73 g NaBH4 to the filtrate in an ice-water bath. After the addition is completed, the reaction is continued for 2 hours. The sample is monitored by TLC to see if the reaction is complete. 250 mL of water is added, and 500 mL of EA is extracted. The organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 45.35 g of oil G-2. MS (ESI) m/z 248.19 ([M+H] ⁺ ).

Synthesis of compound G-3

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, the reaction was allowed to react at room temperature for 2 h. TLC was used to monitor the reaction completion. 150 mL of water was added to the reaction solution, and the DCM layer was extracted and separated. The layer was washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance. The product was purified by silica gel column chromatography with a gradient elution of PE:EA = 40:1-10:1. The concentrated product obtained 36.7 g of a colorless and transparent oily substance G-3. MS (ESI) m/z 486.66 ([M+H] ⁺ ).

Synthesis of compound G-4

Dissolve 46.15 g of compound G-3 in 500 mL DCM, and add 70 mL TFA (trifluoroacetic acid) dropwise under ice bath. After the addition, stir and react at room temperature for 4 h. Monitor the reaction by TLC, reduce the pressure and concentrate, add 500 mL DCM to the residue, add saturated sodium bicarbonate solution dropwise to adjust the pH to 8, extract and separate the DCM layer, wash once with saturated brine, dry over anhydrous sodium sulfate, concentrate and dry, and obtain 36.7 g of light yellow oil G-4. MS (ESI) m/z 386.51 ([M+H] ⁺ ).

Synthesis of compound G-5

Dissolve 31.9 g of compound M-3 in 300 mL of DMF, add 42 g of HBTU and 23 mL of DIEA, stir at room temperature for 10 minutes, then add 35 g of compound G-4, move to room temperature and stir for 1 hour. The reaction was monitored for completeness using TLC. Add 600 mL saturated sodium bicarbonate and 400 ml EA to the reaction solution for extraction. The EA layer was separated and 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] ⁺ ).

Synthesis of compound G-6

Add 33.3 g of compound G-5 to a mixed solvent of 100 mL MeOH, 100 mL THF and 100 mL water, add 5.92 g of lithium hydroxide monohydrate, and react at room temperature for 8 h. Use TLC to monitor the completion of the reaction. Concentrate the reaction solution under reduced pressure. Add 400 mL of ethyl acetate to the concentrated residue. Adjust the pH to 4-5 with dilute hydrochloric acid solution. Extract and separate the organic phase. Wash once with saturated brine and dry with anhydrous sodium sulfate. , concentrated under reduced pressure to obtain 33.8 g of colorless and transparent oil G-6. MS(ESI)m/z: 694.35 ([M+H] ⁺ ).

Synthesis of compound G-7

Dissolve 18.3 g of compound G-6 in 150 mL of DMF, add 13.0 g of HBTU and 6.52 mL of DIEA, stir at room temperature for 15 minutes, and then add dropwise a solution of 11.4 g of S-07 in 100 mL of DMF. After the dropping is completed, move to room temperature and stir for 1 hour. The reaction was monitored for completeness using TLC. Add 400 mL saturated sodium bicarbonate to the reaction solution, extract the organic phase with 200 mL EA, wash with saturated brine, dry over anhydrous sodium sulfate, and concentrate 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] ⁺ ).

Synthetic compound G-8

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

Synthetic compound G-9

5.91 g of compound G-8 was dissolved in 60 mL of anhydrous pyridine, and 6.01 g of DMTrCl was added. The reaction was allowed to react at room temperature for 30 min. TLC was used to monitor the reaction to be complete. 10 mL of methanol was added to the reaction solution to quench the reaction, and the solution was concentrated under reduced pressure. Column chromatography was used for separation (DCM:MeOH=100:1--50:1). The product components were collected and concentrated to obtain 5.07 g of a white solid. MS (ESI) m/z: 634.44 ([M-302+H] ⁺ ).

Synthetic compound G-10

Dissolve 3.00 g of compound G-9 in 30 mL of anhydrous acetonitrile, add 2.04 mL of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphitediamine (CTPPA), and add 9 mL of 0.5 M tetrazole acetonitrile solution, react at room temperature for 2 hours under nitrogen protection. The reaction was monitored almost completely using UPLC-MS. Concentrate under reduced pressure, add 50 mL DCM to dissolve, add 30 mL water, extract and separate the DCM layer, wash with saturated brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 4.74 g of oil. Purification by column chromatography yielded 1.3 g of 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 .

Synthesis of compound G-11

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 hours under nitrogen protection. Use UPLC-MS to monitor the completion of the reaction. Reduce the pressure and concentrate the reaction solution to obtain an oily substance. Purify by silica gel column chromatography (DCM:MeOH=100:1-20:1). Collect the product components and reduce the pressure and concentrate to obtain 0.74g 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] ⁺ ).

Synthetic compound G-12

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 were added to a 50 mL centrifuge tube, and 10 mL of anhydrous acetonitrile was added to dissolve the mixture, and then placed on a shaker for overnight. The reaction solution was filtered, rinsed with acetonitrile, and after drying, the carrier was poured into a 50 mL centrifuge tube, and 5 mL of CapA (20% NMI-80% ACN) and 5 mL of CapB (20% AC ₂ O-30% lutidine-50% ACN) were added respectively, and the reaction was shaken at room temperature for 2 hours. Filter, rinse with acetonitrile, and vacuum dry overnight. 1.32 g of G-12 was obtained, with a loading of 62 μmol/g. Example 2 Synthesis of H-17 ( His(R) -sequence GalNAc )

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

Step 1 : Synthetic route of compound S-05

Under nitrogen protection, add 800 mL DCM and 57 g bromopentanol to 120 g of the crude product of compound S-02. Add 28.5 mL TMSOTf dropwise. After the addition, stir at room temperature 25°C overnight. Take a sample and monitor the reaction completion by TLC. Add 1 L saturated sodium bicarbonate to quench the reaction, separate the DCM layer, wash once with saturated sodium chloride, dry over anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography, DCM:EA=10:1-2:1 gradient elution, collect the product part, and concentrate to obtain 98 g of compound S-05. MS (ESI) m/z 497.07, 498.00 ([M+H] ⁺ ). ¹ H-NMR (400MHz, 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).

Step 2: Synthetic route of compound M-3

For the synthesis method, see the synthesis of compound M-3 in the G-12 (Glu(R)-sequence GalNAc) synthesis route.

Step 2: Synthetic route of compound H-05

Synthetic compound H-02

Dissolve 38.4 g sodium hydroxide in 400 mL water, add 400 mL THF, cool the mixture in an ice water bath, add 50 g H-01 (L-histidine acid), stir and dissolve. Add 175 g of di-tert-butyl dicarbonate, complete the addition, and stir for 4 hours at room temperature. After TLC monitors that the reaction is complete, the reaction mixture is filtered, the filtrate is concentrated, and 200 mL MTBE is added to the concentrated residue for washing and extraction three times. Separate the water layer, add 500 mL of ethyl acetate and adjust to pH 2~4 with 3 M hydrochloric acid. Separate the EA layer and wash with saturated sodium chloride. Dry over anhydrous sodium sulfate, filter, and concentrate the filtrate to dryness to obtain 98 g of white solid. MS(ESI) m/z 356.25 ([M+H] ⁺ ).

Synthesis of compound H-03

Under nitrogen protection, 110 g H-02 was dissolved in 880 mL of anhydrous THF. The reaction solution 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 the addition was completed, the solution was stirred at room temperature for 1 hour, and the reaction was complete by TLC monitoring. The reaction solution was cooled to 0-10°C in an ice-water bath, and 230 mL of methanol was slowly added dropwise to quench the reaction. THF was concentrated, 800 mL of ethyl acetate and 300 mL of saturated brine were added to the residue, and the extract was separated. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to obtain 106 g of crude H-03 product, 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).

Synthetic compound H-04

106 g H-03 was dissolved in 1 L dichloromethane, 38 g imidazole was added, and 128 g TBDPSCl was added dropwise to the reaction solution. After the addition, the reaction was allowed to proceed at room temperature for 1 h, and the reaction was complete as monitored by TLC. 400 mL saturated sodium chloride was added to the reaction solution, and the DCM layer was extracted and separated, washed with saturated sodium chloride, dried with sodium sulfate, concentrated, and purified by silica gel column chromatography, with a gradient elution of petroleum ether: ethyl acetate = 100:1-20:1. The product eluate was collected and concentrated to obtain 106 g of a white solid. ¹ H-NMR (400 MHz, 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).

Synthetic compound H-05

Dissolve 80 g H-04 in 240 mL glacial acetic acid, stir and react at 80°C overnight. After TLC monitors that the reaction is complete, the mixture is cooled in an ice-water bath, then 400 mL of ethyl acetate is added, and 4 M sodium hydroxide is added dropwise to adjust the pH to 8-9. The organic phase is separated, washed once with saturated sodium chloride, and dried over anhydrous sodium sulfate. , concentrated to dryness to obtain 66 g of white solid. MS(ESI) m/z 480.27 ([M+H] ⁺ ).

Step 4: Synthetic route of compound H-17

Synthetic compound H-10

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, the mixture was stirred at room temperature for 1 h, and the reaction was monitored by UPLC-MS to be complete. The reaction mixture was filtered, and the filter cake was washed with 400 mL of EA. 1.2 L of saturated ammonium chloride was added to the filtrate, and the EA layer was extracted and separated, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography, with a gradient elution of DCM:MeOH=80:1-20:1. The product was collected and concentrated to obtain 62.74 g of compound H-10. MS (ESI) m/z 895.53 ([M+H] ⁺ ).

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

Synthesis of compound H-11

62 g of compound H-10 was dissolved in 600 mL of DCM. The solution was cooled to 0-10 degrees in an ice-water bath and 103 mL of TFA was added dropwise. After the addition, the mixture was stirred at room temperature for 2 hours. The reaction was monitored by UPLC-MS. 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 extracted and separated, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to obtain 58 g of compound H-11. MS (ESI) m/z 795.50 ([M+H] ⁺ ).

Synthesis of compound H-12

13 g of compound M-3 was dissolved in 150 mL of DMF, and 8.23 mL of DIEA and 15.53 g of HBTU were added. Stir at room temperature for 30 min, and add dropwise a solution of 25 g of compound H-11 in 100 mL of DMF. After the dripping was completed, the reaction was completed at room temperature for 2 hours, and TLC monitored the reaction to be complete. Add 300 mL EA and 600 mL 10% ammonium chloride to the reaction solution, separate the organic phase, wash once with saturated sodium chloride, dry over anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography, DCM: MeOH=100: Gradient elution was performed from 1 to 30:1, and the product was collected and concentrated to dryness to obtain 19 g of compound H-12. MS(ESI) m/z 1119.69 ([M+H] ⁺ ).

Synthetic compound H-13

22 g of compound H-12 was dissolved in 200 mL of anhydrous THF, and 59 mL of TBAF (1 M THF solution) was added. The mixture was stirred at room temperature for 23 h, and 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] ⁺ ).

Synthetic compound H-14

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, UPLC-MS detected that 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] ⁺ ).

Synthesis of compound H-15

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

Synthesis of compound H-16

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, UPLC-MS was used to monitor the completion of the reaction. Concentrate to remove pyridine, and the residue is purified by gradient elution with silica gel column chromatography (DCM: MeOH=40:1-20:1). The product was collected, concentrated and dried to obtain 1.37 g of compound H-16. MS(ESI)m/z 1045.57 ([M+H] ⁺ ). ¹ H-NMR(400MHz,DMSO-d6) δ7.96(d, 1H), 7.88(d, 1H), 7.44(s, 1H) , 7.34-7.41(m, 2H), 7.18-7.31(m, 7H), 6.82-6.89(m, 4H), 6.64(s, 1H), 5.22(d, 1H), 5.04-5.09(m, 1H) , 4.97(dd, 1H), 4.50(d, 1H), 4.11-4.23(m, 1H), 4.02(s, 3H), 3.83-3.93(m, 2H), 3.79(t, 2H), 3.73(s , 6H), 3.65-3.72(m, 2H), 3.36-3.42(m, 2H), 2.90-2.94(m, 1H), 2.82-2.85(m, 1H), 2.72-2.79(dd, 1H), 2.56 -2.61(dd, 1H), 2.35-2.45(dd, 2H), 2.27-2.32(dd, 2H), 2.10(s, 3H), 1.98(s, 3H), 1.89(s, 3H), 1.76(s , 3H), 1.56-1.64(m, 2H), 1.43-1.50(m, 2H), 1.12-1.20(m, 2H), 1.13(d, 3H).

Synthetic compound H-17

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 for 10 min, LCAA- CPG- (96 μmol/g) was added, and the mixture was shaken overnight at room temperature. The reaction solution was filtered, washed with acetonitrile, and the filter cake was vacuum dried and 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 filtered, washed with acetonitrile, and vacuum dried 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 )

The synthesis of H-23 (His(S)-order-GalNAc) is as follows (2 steps):

Step 1: Synthesis route of compound M-6

Synthetic compound M-6

Methyl (R)-3-hydroxybutyrate methyl ester was replaced with methyl (S)-3-hydroxybutyrate methyl ester. For the synthesis method, please refer to the synthesis of M-3 in the G-12 (Glu(R)-sequence GalNAc) synthesis route.

Step 2: Synthetic route of compound H-23

Synthesis of compound H-18

For the synthesis method, please 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] ⁺ ).

Synthesis of compound H-19

For the synthesis method, please 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] ⁺ ).

Synthetic compound H-20

For the synthesis method, please 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] ⁺ ).

Synthetic compound H-21

For the synthesis method, please 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 presents a unique structural fragment peak on the mass spectrum, MS(ESI) m/z 1062.43 ([M-(i-Pr) ₂ N+H ₂ O ] ⁺ ). ¹ H- NMR(400MHz,DMSO-d6) δ7.92(t, 1H), 7.73(d, 1H), 7.36(s, 1H), 7.05-7.33(m, 9H), 6.72-6.84(m, 4H), 6.55 (s, 1H), 5.63(s, 1H), 5.16(d, 1H), 4.83(dd, 1H), 4.62-4.73(m, 1H), 4.36(d, 1H), 3.08-4.14(m, 3H ), 3.91(s, 3H), 3.73-3.80(m, 2H), 3.63-3.73(m, 2H), 3.63(s, 6H), 3.54-3.66(m, 1H), 3.27-3.43(m, 3H ), 2.77-2.85(m, 2H), 2.71-2.75(m, 1H), 2.66(dd, 1H), 2.43-2.58(m, 2H), 2.29(dd, 1H), 2.10(s, 3H), 2.01-2.07(m, 1H), 1.91(s, 3H), 1.86(s, 3H), 1.71(s, 3H), 1.47-1.58(m, 2H), 1.39-1.48(m, 2H), 1.26( d, 3H), 1.08-1.1.20(d, 12H).

Synthetic compound H-22

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

MS (ESI) m/z 1045.66 ([M+H] ⁺ ). ¹ H-NMR (400 MHz, DMSO-d6) δ7.96 (d, 1H), 7.88 (d, 1H), 7.44 (s, 1H), 7.34-7.41 (m, 2H), 7.18-7.31 (m, 7H), 6.82-6.89 (m, 4H), 6.64 (s, 1H), 5.22 (d, 1H), 5.04-5.09 (m, 1H), 4.97 (dd, 1H), 4.50 (d, 1H), 4.11-4.23 (m, 1H), 4.02 (s, 3H), 3.83-3.93 (m, 2H), 3.79 (t, 7-1.48(m, 2H), 1.70-1.81(m, 2H), 1.69-1.87(m, 2H), 1.73-1.70(m, 2H), 1.82-1.84(m, 2H), 1.12-1.20(m, 2H), 1.64-1.81(m, 2H), 1.62-1.64(m, 2H), 1.14-1.20(m, 2H), 1.62-1.81(m, 2H), 1.64-1.83(m, 2H), 1.62-1.83(m, 2H), 1.64-1.82(m, 2H), 1.64-1.83(m, 2H), 1.13(d, 3H).

Synthesis of compound H-23

For the synthesis method, please refer to the synthesis of compound H-17 in the synthetic route of H-17 (His(R)-sequence-GalNAc). The H-23 loading capacity is 52 μmol/g. Example 4 : Synthesis of GS-13-1 ( Glu(R) -sequence -GalNAc )

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

Synthetic compound GS-13-11

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

Synthetic compound GS-13-10

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

Synthetic compound GS-13-9

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

Synthetic compound GS-13-8

GS-13-9 was dissolved in anhydrous methanol, and 10% (W/W) palladium carbon was added. The hydrogen was replaced three times, and the mixture was stirred at room temperature until the raw material reacted completely. The palladium carbon was removed by filtration, and the filtrate was dried and directly used for the next step of reaction. MS (ESI) m/z 884.40 ([MH] ^- ).

Synthetic compound GS-13-7

Dissolve equal amounts of GS-13-8 and GS-13-10 in anhydrous dichloromethane, add 0.1 equivalent of DMAP, 2.0 equivalent of DCC and 2.0 equivalent of DIEA, stir at room temperature under nitrogen protection until the raw material reaction is complete, and filter to remove. Insoluble matter, the reaction solution was washed with purified water, and the organic phase was concentrated to dryness. The crude product was subjected to silica gel column chromatography (DCM:MeOH=10:1), and dried to obtain a white solid. MS(ESI) m/z 1519.60 ([M+H] ⁺ ).

Synthetic compound GS-13-6

The synthesis method is the same as GS-13-8. MS(ESI)m/z1427.61 ([MH] ^- ).

Synthetic compound GS-13-5

The synthesis method is the same as GS-13-7, and the raw materials are GS-13-6 and GS-13-14. MS(ESI)m/z 1066.01([(M+2)/2] ⁺ ).

Synthetic compound GS-13-4

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

Synthetic compound GS-13-3

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

Synthetic compound GS-13-2

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

Synthetic compound GS-13-1

The synthesis method is the same as H-17, and the loading capacity is 50 μmol/g. Example 5 : Synthesis of HS-13-1 ( His(R) -sequence -GalNAc )

Synthetic compound HS-13-11

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

Synthetic compound HS-13-10

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

Synthetic compound HS-13-9

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

Synthetic compound HS-13-8

Dissolve HS-13-9 in anhydrous methanol, add 10% (W/W) palladium carbon, replace with hydrogen three times, stir at room temperature until the raw material reacts completely, filter to remove palladium carbon, spin dry the filtrate and directly put it into the next step. MS (ESI) m/z 893.40 ([MH] ^- )

Synthetic compound HS-13-7

Dissolve equal amounts of HS-13-8 and HS-13-10 in anhydrous dichloromethane, add 0.1 equivalent of DMAP, 2.0 equivalent of DCC and 2.0 equivalent of DIEA, stir at room temperature under nitrogen protection until the raw material reaction is complete, and filter to remove. Insoluble matter, the reaction solution was washed with purified water, and the organic phase was concentrated to dryness. The crude product was subjected to silica gel column chromatography (DCM:MeOH=10:1), and dried to obtain a white solid. MS(ESI) m/z 1537.60 ([M+H] ⁺ ).

Synthetic compound HS-13-6

The synthesis method is the same as HS-13-8. MS (ESI) m/z 1445.60 ([MH] ^- ).

Synthetic compound HS-13-5

The synthesis method is the same as HS-13-7, and the raw materials are HS-13-6 and HS-13-14. MS (ESI) m/z 1079.52 ([(M+2)/2] ⁺ ).

Synthetic compound HS-13-4

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

Synthetic compound HS-13-3

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

Synthetic compound HS-13-2

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

Synthetic compound HS-13-1

The synthesis method is the same as H-17, with a loading of 50 μmol/g. Example 6 : Synthesis of the conjugated oligonucleotide provided by the present invention

Naturally occurring or chemically modified oligonucleotides are synthesized using common methods such as solid-phase synthesis. And compound-conjugated oligonucleotides can be synthesized by an exemplary method as shown below: Preparation of Oligo -conjugated G-12 ( Glu(R) -sequence GalNAc )

Solid phase synthesis steps

Through the solid-phase synthesis method of phosphoramidite known in the art, using G-12 as the solid-phase synthesis carrier, run the sequence command on the MerMade192 solid-phase synthesizer: set the connection position of compound G-10 to 3' of the sequence End and set the number of connecting compounds G-10.

Each connection of a nucleoside monomer includes four steps: deprotection, coupling, capping and oxidation. The standard procedures of the above steps are known to ordinary technicians in the field, and the G-10 solution is prepared with a 0.1 M acetonitrile solution.

The solid phase synthesis reagents were configured as follows: Detergent: acetonitrile Deblocking agent: 3% dichloroacetic acid in dichloromethane Activating agent: 0.25 M 5-ethylthio-1H-tetrazole in acetonitrile Capping agent A: THF/dimethylpyridine/acetic anhydride (8:1:1) Capping agent B: 15% NMI/THF, GL38 finish Oxidizing agent: 0.02MI ₂ in THF/pyridine/H ₂ O Sulfiding agent: 0.10M DDTT solution

Taking the 1 μmol synthesis scale as an example, the solid-phase synthesis conditions are as follows: PO loop column parameters PO reagent column steps Reagent volume ( μL ) Reaction time ( s ) 3 x washes Detergent 200 μL 0s 2 x dissociation dissociating agent 150 μL 45s 2 x wash Detergent 200 μL 0s 2 x coupling Activator 85 μL 360s Amidite 70 μL 1 x wash Detergent 200 μL 0s 1 x capped Capping reagent A 75 μL 60s Capping reagent B 75 μL 1 x wash Detergent 200 μL 0s 1 x oxidized oxidizing agent 150 μL 60s 2 x washing Detergent 200 μL 0s PS loop column PS reagent column steps Reagent volume ( μL ) Reaction time ( s ) 3 x washes Detergent 200 μL 0s 2 x dissociation dissociating agent 150 μL 45s 2 x washing Detergent 200 μL 0s 2 x coupling Activator 85 μL 360s Amidite 70 μL 1 x wash Detergent 200 μL 0s 1 x vulcanized Vulcanizing agent 200 μL 200s 2 x washing Detergent 200 μL 0s 1 x capped Capping reagent A 75 μL 60s Capping reagent B 75 μL 2 x washing Detergent 200 μL 0s

Lysis and deprotection steps

Add the Oligo-support obtained in the above solid phase synthesis step to a 1 mL centrifuge tube, add 50~100 μl concentrated ammonia water, react at 50-60°C for 10 hours, centrifuge and absorb the supernatant, and add 2 times the volume to the supernatant. Use acetone-ethanol (80:20) solvent to precipitate a white precipitate. Centrifuge at 10,000 g to remove the supernatant to obtain the precipitated product. The precipitate is redissolved in 0.2 M sodium acetate solution.

Purification, desalination and freeze-drying steps

Purification was performed on an Avant 150 purification equipment using a 1 mL volume ion chromatography column (loaded with Nano Q 30).

The specific conditions are: Buffer A: 20 mM sodium phosphate-10% acetonitrile-water buffer solution (pH7.5), Buffer B: 2.0 M NaCl-20 mM sodium phosphate-acetonitrile-water buffer solution (pH7.5); elution gradient: buffer B 0 ~50%, buffer A 100~50%.

The product eluates were collected and combined, and finally desalinated using a G25 dextran gel column; the OD260 concentration of the desalted product solution was measured to calculate the product content, and finally placed in a centrifuge tube for freeze-drying to obtain a white freeze-dried product.

Detection: Reverse-phase UPLC-MS tandem mass spectrometry is used for detection. The purity is above 90%. The mass spectrum shows m/z [M-7/7] ^- , [M-8/8] ^- , [M-9/9] ^{-Characteristic} ion peaks.

The synthesis example of AS-Oligo can be found in G-12-OLIGO, which was synthesized using Unylinker-CPG (purchased from Glen Research) as a solid phase synthesis carrier.

For the synthesis example of Oligo conjugated to His-R-sequence-GalNAc, see G-12-OLIGO, which was synthesized using H-17 as a solid phase synthesis carrier. The only difference from the synthesis of G-12-OLIGO is that H-15 was prepared in a 0.1 M acetonitrile solution and the sequence was run on a solid phase synthesis instrument.

For the synthesis example of Oligo conjugation with His-S-sequence-GalNAc, see G-12-OLIGO, which is synthesized using H-23 as a solid-phase synthesis carrier. The only difference from the synthesis of G-12-OLIGO is that H-21 is configured in a 0.1 M acetonitrile solution and the sequence settings are run on a solid-phase synthesis instrument.

The preparation of siRNA-GalNAc conjugate or aiRNA-GalNAc conjugate is to bond the above-obtained Oligo-GalNAc conjugate with the antisense strand of its complementary sequence at a molar ratio of 1:1 to obtain a double-stranded product activity test material and method

Synthesized conjugated aiRN for testing: Compounds HS-9, HS-5 and HS-7 are three-sequence GalNAc designed based on histidine linkers, and are conjugated to the 3' end of the sense strand of double-stranded RNA (such as aiRNA or siRNA). In the following examples When used and described, 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 three-sequence GalNAc designed based on glutamine linkers, which are 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 implementations, They are represented by the numbers "Glu-seq (3GalNAc)" and "Glu(R)-seq (3GalNAc)" respectively.

The sequences and structures of the oligonucleotides (aiRNA and siRNA) synthesized and used to test the activity of the examples are shown in Table 7 below: Table 7 Tested aiRNA sequences targeting mouse β-catenin aiRNA# Sequential strand (5'-3') (SS) SEQ ID No. Antisense strand (5'-3') (AS) SEQ ID No. as/ss 1 A*G*UGgauucuGuaCUGU-L 1 A*a*ACAgUacAGaAuCCACU*G*G 2 17/21mer 2 A*G*UGgAuUcuGUaCUGU-L 3 17/21mer AGCU indicates 2'OMe-modified RNA, agcu indicates 2'F-modified RNA, *=PS, -L indicates GalNAc conjugate.

Unless otherwise specified, the in vitro delivery efficiency of the conjugates of the present invention was tested on hepatocytes by RT-qPCR. The steps for isolating primary mouse hepatocytes are as follows: Part A: Perfusion (1) Perfusion with buffer A (2) Perfusion 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: Separation •After perfusion, place the liver into a 10 cm TC dish, open the liver bag, and use forceps to shake the tissue to aid dissociation. • Filter through a 70 micron filter; wash filter with Buffer C and centrifuge at 50 g for 5 minutes at 4°C. • Discard the supernatant, resuspend gently in 50 ml Buffer C (Wash 1) and centrifuge at 50g for 5 min at 4°C. • Discard the supernatant, resuspend gently in 50 ml Buffer C (Wash 2) and centrifuge at 50g for 5 min at 4°C. • Discard the supernatant, resuspend gently in 50 ml Buffer C (Wash 3) and centrifuge at 50g for 5 min at 4°C. •Discard supernatant and resuspend in thaw/plating medium. • Count with trypan blue to assess viability/yield. •Cells were seeded on collagenase-coated plates (thermos fisher, A1142802) using mouse primary hepatocyte thawing medium (thermos fisher, CM3000). It is better to inoculate 1 ml/well in a 24-well plate. •After 3-4 hours, the medium was replaced with primary hepatocyte maintenance medium (thermofisher, CM4000).

In vitro self-delivery assays (free uptake) were performed without transfection agents (tested in 12-well plates, 100,000 cells/well, 48 h reaction). The concentration of the tested Oligo GalNAc conjugates is indicated in each example below. The expression of the targeted mRNA was detected by RT-qPCR. Example 7 : In vitro uptake of conjugated / unconjugated aiRNA by hepatocytes

Compared with the non-conjugated aiRNA, the mβ-catenin aiRNA (aiRNA#1) conjugated with "Glu(R)-seq (3GalNAc)" showed significant gene silencing activity in an in vitro self-delivery assay at 10 nM or even 1 nM, demonstrating that the GalNAc conjugate provided by the present invention has a great ability to deliver double-stranded RNAi agents such as aiRNA. The results of the QPCR test are shown in Figure 2. Example 8 : In vitro uptake of aiRNA conjugated with GalNAc conjugate by the liver

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, respectively, for 24 hours. The results of the QPCR test are shown in Figure 3. Glu-seq (3 GalNAc), His(R)-seq (3 GalNAc), and His(S)-seq (3 GalNAc) all showed very effective gene silencing in in vitro self-delivery, indicating that the GalNAc conjugates provided by the present invention have a strong effect on the delivery of double-stranded RNAi agents (such as aiRNA). Example 9 : In vitro uptake of aiRNA targeting mouse TTR conjugated with histidine sequence GalNAc conjugate by hepatocytes

In newly isolated mouse primary hepatocytes, the newly designed aiRNA conjugate targeting mouse TTR (conjugated with "His-seq (3GalNAc)") was tested without transfection agent (free uptake) ( (as shown in Table 8 below) gene silencing activity. Free uptake-mediated gene silencing in vitro evaluates the receptor-mediated intracellular transport that can be achieved by the GalNAc conjugates provided by the invention.

50 μL of aiRNA conjugate (793-804) and 950 μL of cell culture medium (thermofisher, CM4000) were added to a well of a 12-well plate containing approximately 100,000 primary mouse hepatocytes per well. The cells were incubated at 37°C and 5% CO ₂ for 24 h. RNA was purified and the amount of mRNA was detected by RT-qPCR. 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 aiRNA conjugate ID 5'-3' sequence SEQ ID No. HisT/793 SS C*a*GuGuUCUuGcUcUaUDDD 4 AS u*U*aUaGaGcAagaAcAcUg*U*u 5 HisT/794 SS C*a*GuGuUCUuGcUcUaUDDD 4 AS a*A*aUaGaGcAagaAcAcUg*U*u 6 HisT/795 SS c*a*guguUcUuGcucuauDDD 7 AS u*u*aUAGaGcAaGaAcAcUg*u*u 8 HisT/796 SS c*a*guUuUcUuGcucuauDDD 9 AS u*u*aUAGaGcAaGaAcAcUg*u*u 8 HisT/797 SS c*a*guguUcUuGcUcuauDDD 10 AS u*u*aUAGaGcAaGaAcAcUg*u*u 8 HisT/798 SS c*a*GuGuUcUugcucuauDDD 11 AS u*u*aUAGaGcAaGaAcAcUg*u*u 8 HisT/799 SS c*a*guguUcUuGcucuauDDD 7 AS a*a*aUAGaGcAaGaAcAcUg*u*u 12 HisT/800 SS c*a*guUuUcUuGcucuauDDD 9 AS a*a*aUAGaGcAaGaAcAcUg*u*u 12 HisT/801 SS c*a*guguUcUuGcUcuauDDD 10 AS a*a*aUAGaGcAaGaAcAcUg*u*u 12 HisT/802 SS c*a*GuGuUcUugcucuauDDD 11 AS a*a*aUAGaGcAaGaAcAcUg*u*u 12 HisT/803 SS C*a*GuGuUCUuGcUcUaUDDD 4 AS u*u*auaGaGcAagaAcAcUg*U*u 13 HisT/804 SS c*a*GuGuUCUuGcUcUaUDDD 4 AS a*A*a*U*aGaGcAagaAcAcUg*U*u 14 AGCU = 2'OMe modified RNA, agcu = 2'F modified RNA, * = PS, DDD = His-seq (3GalNAc)

The results are shown in Figure 4 . All aiRNA conjugates combined with the composition of the present invention showed strong gene silencing activity in the in vitro self-delivery test at a concentration of 0.3 nM, indicating that the GalNAc conjugates provided by the present invention are useful for delivering various Double-stranded RNAi agents are all highly potent.

The results in all activity test examples clearly show that the GalNAc conjugate designed based on the present invention can significantly improve the delivery efficiency of oligonucleotides in vitro and achieve huge gene silencing efficacy targeting different genes in hepatocytes. Furthermore, the linker composition provided in the present invention is based on our body's amino acids, which eliminates the safety risks of other types of linkers used in GalNAc conjugates.

It will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. Other embodiments of the present invention will be apparent to those skilled in the art by reference to the specification and practice disclosed herein. It is intended that the specification and examples be considered exemplary only, and that the following claims will set forth the true scope and spirit of the present invention.

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

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The purpose and features of the present invention may be better understood by referring to the following drawings and the scope of the patent application. The drawings illustrate the core idea of the present invention, but do not necessarily summarize all possible variations. In the drawings, similar numbers are used to indicate similar parts in each view.

FIG1 shows an exemplary structure of an oligonucleotide-ligand conjugation. A conjugated interfering RNA double-stranded molecule includes an antisense strand and a sense strand. In some embodiments, the oligonucleotide is an interfering RNA double-stranded molecule, and the ligand can be conjugated conjugated at the 3' end of the sense strand (e.g., structures 1.1-1.3, intermediate aiRNA, blunt-ended aiRNA, and siRNA), at the 3' end of the antisense strand (structure 2), at the 5' end of the sense strand (structure 3), or at both ends of the sense strand (structure 5), or at both ends of the antisense strand (structure 4), at the 3' end of the antisense strand and the 5' end of the sense strand (structure 6), at the 3' end of the sense strand and the 3' end of the antisense strand (structure 7), or at the 3' end of the sense strand, the 3' end of the antisense strand, and the 5' end of the sense strand. In some embodiments, the oligonucleotide is an antisense oligonucleotide (ASO), and the ligand can be conjugated to the 3' end or/and the 5' end of the antisense strand.

Figure 2 shows the results of in vitro uptake of β-Catenin aiRNA tested by QPCR. "Non-GalNAc" is aiRNA without conjugation. "Glu(R)-seq (3GalNAc)" is aiRNA conjugated to the GalNAc conjugate composition "GS-5".

Figure 3 shows the initial generation of mCat12 aiRNA conjugated to "His(R)-seq (3 GalNAc)", "His(S)-seq (3 GalNAc)" and "Glu-seq (3 GalNAc)" respectively. In vitro uptake effects in hepatocytes.

Figure 4 shows the in vitro uptake effect of TTR aiRNA conjugated to "His-Seq (3 GalNAc)" in primary hepatocytes.

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TW202408581A_111132148_SEQL.xml

Claims (75)

A compound with the structural formula (S-HG1): R ²⁰⁶ -[A1] _a -[A2] _b -[A3] _c -R ²⁰⁵ (S-HG1) where R ²⁰⁵ and R ²⁰⁶ are independent for each occurrence Ground is H, OH, protective group of OH, phosphate group, phosphodiester group, activated phosphate group, activated phosphite group, phosphoramidite, solid phase carrier, -OP(M')(M")O- Nucleoside, -OP(M')(M")O-oligonucleotide, lipid, PEG, steroid, polymer, -O-nucleotide, nucleoside, -OP(M')(M")OR 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 for each occurrence from (S-1H) and (S-1G): (S-1H)、 (S-1G); R ^202A is -R ²⁰² -R ^202L ; R ^217A is -R ²¹⁷ -R ^217L ; R ^202L and R ^217L are independently a ligand capable of docking with cell surface receptors for each occurrence; Each R ²⁰² , R ²¹⁷ , R ²⁰¹ is independently selected for each occurrence from an alkylene group of 3 to 30 carbon atoms, where one or more carbon atoms is optionally replaced by any one of the group consisting of or Multiple substituents: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, S(O) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C ₃ -C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene, and wherein each R ²⁰² , R ²¹⁷ , R ²⁰¹ can independently Optionally unsubstituted or substituted by R ²⁰⁹ ; optionally, R ²⁰² , R ²⁰¹ are each independently selected for each occurrence: alkylene of 3 to 15 carbon atoms, one or more of which are optional is replaced by one or more substituents from the group consisting of: C(O), NH, O, S, OP(O)O and 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 are independently selected from one or more of the following: H, alkyl, aryl, heteroaryl, haloalkyl, -O alkyl, -O alkylphenyl, -alkyl-OH, -O haloalkyl, -S alkyl, -S alkylphenyl, -alkylSH, -S haloalkyl, halo, -OH , -SH, -NH ₂ , -alkyl-NH ₂ , -N (alkyl) (alkyl), -NH (alkyl), -N (alkyl) (alkylphenyl), -NH (alkyl) phenyl), 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 (alkyl halo base); n ²⁰¹ , n ²¹¹ are independently 1, 2, 3, 4, 5 or 6 for each occurrence; J ²⁰¹ , J ²⁰² , J ²¹¹ , J ²¹² are independently absent or spacer for each occurrence (spacer); a, b and c are independently for each occurrence an integer from 0 to 5, and the sum of a, b and c is an integer from 1 to 10; the oligonucleotide comprises a naturally occurring or chemically modified of nucleotides/nucleosides. The compound as claimed in claim 1, wherein the sum of a, b and c is 1 or 3. The compound as claimed in claim 1, wherein the compound has the structural formula (S-H1): (S-H1) wherein R ₁ is one or more selected from the group consisting of H, C ₁ -C ₅ alkyl, aryl, heteroaryl, C ₁ -C ₅ halogenated alkyl, -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)halogenated alkyl, -SO ₂ (alkyl), -SO ₂ (halogenated alkyl), -SO ₂ NH ₂ , -SO ₂ NH(alkyl) and -SO ₂ NH(phenyl); n ²⁰² is selected from 1-10, preferably 1-3. The compound as described in claim 3 has the structural formula (S-H1-01): (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 an alkylene group of 1 to 10 carbon atoms, optionally selected from a straight chain alkylene group of 1 to 10 carbon atoms; R ^205A is a group comprising a solid phase support or H. The compound of claim 3 or claim 4, wherein n ²⁰² is 3. The compound of any one of claims 3-4, wherein R ²⁰⁶ comprises an oligonucleotide. The compound of claim 6, wherein the compound has the structural formula (S-H1-02): (S-H1-02). The compound of claim 3, wherein: J ²⁰¹ and J ²⁰² are independently selected from alkylene groups of 1 to 30 carbon atoms for each occurrence, wherein one or more carbon atoms are optionally replaced by any one or more substituents 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 and J 202 are independently selected from alkylene groups of 1 to 30 carbon atoms for each occurrence, wherein one or more carbon atoms are optionally replaced by any one or more substituents 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 ²⁰² are each independently optionally unsubstituted or substituted with one or more groups selected from the group consisting of: H, alkyl, aryl, heteroaryl, halogenated alkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohalogenated alkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shalogenated alkyl, halogen, -OH, -SH, _-NH2 , -alkyl- _NH2 , -N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, _-CO2H , -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), _-CONH2 , -NHC(O)(alkyl), -NHC(O)(phenyl), -N(alkyl)C(O)(alkyl), -N(alkyl)C(O)(phenyl), -C(O)alkyl, -C(O)alkylphenyl, -C(O)haloalkyl, -OC(O)alkyl, -SO ₂ (alkyl), -SO ₂ (phenyl), -SO ₂ (haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH(alkyl), -SO ₂ NH(phenyl), -NHSO ₂ (alkyl), -NHSO ₂ (phenyl) and -NHSO ₂ (haloalkyl). A compound as described in claim 3, wherein: J ²⁰¹ and J ²⁰² are independently selected from an alkylene group of 1 to 10 carbon atoms for each occurrence, wherein one or more carbon atoms are optionally replaced by any one or more substituents consisting of: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N and S(O) ₂ , and wherein J ²⁰¹ and J ²⁰² are each independently optionally unsubstituted or substituted by at least one substituent selected from the following group: H, C ₁ -C ₅ alkyl and -OC ₁ -C ₅ alkyl. A compound as described in claim 9, wherein ⁿ²⁰¹ is 1 and ⁿ²⁰² is 3. The compound as claimed in claim 3, wherein the compound has a structural formula (S-H1-03), (S-H1-04) or (S-H1-05): (S-H1-03) (S-H1-04) (S-H1-05) Where, A is O or S, X is independently selected from Table 1; X ₁ is independently selected from Table 2; J ²⁰² is independently selected from Table 3 for each occurrence; optionally, J ²⁰² is independently selected from an alkylene group of 1 to 10 carbon atoms, in which one or more carbon atoms are optionally replaced by any one or more substituents 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 selected from the following groups Substituted with: H, or C ₁ -C ₅ alkyl and -OC ₁ -C ₅ alkyl; R, R' are each independently selected from the group consisting of: naturally occurring and/or chemically modified oligonuclear nucleotide, 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 3 to 15 carbon atoms Linear alkylene groups in which 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 and OP(S)O, and wherein R ²⁰² is optionally unsubstituted or substituted with one or more groups selected from the group consisting of: H, alkyl, haloalkyl, -O alkyl, -alkyl -OH, -O haloalkyl, -S alkyl, -alkylSH, -S haloalkyl, halo, -OH, -SH, -NH ₂ , -alkyl-NH ₂ , -N (alkyl) ( Alkyl), -NH (alkyl), 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), -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. The compound of claim 11, wherein the compound has the structural formula (S-H1-06), (S-H1-07) or (S-H1-08): (S-H1-06) (S-H1-07) (S-H1-08). The compound as claimed in claim 11, wherein the compound has the structural formula (S-H1-09), (S-H1-10) or (S-H1-11): (S-H1-09) (S-H1-10) (S-H1-11). The compound as claimed in claim 11, wherein the compound has the structural formula (S-H1-12), (S-H1-13) or (S-H1-14): (S-H1-12) (S-H1-13) (S-H1-14). The compound as claimed in claim 1, wherein the compound has the structural formula (S-H1-15) or (S-H1-16): (S-H1-15) (S-H1-16) 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' 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: absence, amide, ether, triazole, carbonate, carbamate, phosphate, phosphonate, phosphorothioate, sulfate, disulfide, ester, thioester, alkylamine, cycloalkylamine, alkyne, cycloalkyne, alkene, cycloalkene, lactone and lactamide 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 contains a ligand group capable of binding to a cell surface receptor. The compound of any one of claims 11-15, wherein each A is O. A compound as described in any one of claims 11-15, wherein at least one A is O. The compound according to any one of claims 1-17, wherein each ligand is independently selected from the group consisting of: N-acetyl galactosamine (GalNAc), cholesterol, tocopherol, biotin, cyanine dye , folic acid, RGDp, transferrin, anisidine, lactobionic acid, cRGD, hyaluronic acid, low molecular weight protamine, lipid derivatives, peptides, cyclic peptides and heterocycles. A compound as described in any one of claims 1 to 17, wherein the ligand is N-acetylgalactosamine (GalNAc). The compound of claim 12, wherein the compound has the structure shown as HS-1 to HS-8: . A compound as described in claim 12, wherein the compound has a structure as shown in HS-9. The compound of claim 12, wherein the compound has a structure shown as HS-5. The compound as described in claim 1, wherein the compound has the structural formula (S-G1): (S-G1) wherein R ₂ is selected from one or more of the group consisting of H, C ₁ -C ₅ alkyl, aryl, heteroaryl _, C ₁ -C ₅ halogenated alkyl, -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)halogenated alkyl, -SO ₂ (alkyl), -SO ₂ (halogenated alkyl), -SO ₂ NH ₂ , -SO ₂ NH(alkyl) and -SO ₂ NH(phenyl); n ²¹² is selected from 1-10, preferably 1-3. The compound described in claim 23 has the structural formula (S-G1-01): (S-G1-01) Where: 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 an alkylene group of 1 to 10 carbon atoms, optionally selected from a linear alkylene group of 1 to 10 carbon atoms; R ²⁰⁵ is a group containing a solid support or H. A compound as described in claim 23 or claim 24, wherein ⁿ²¹² is 3. The compound of any one of claims 23-24, wherein R ²⁰⁶ comprises an oligonucleotide. The compound of claim 26, wherein the compound has the structural formula (S-G1-02): (S-G1-02). The compound of claim 23, wherein: J ²¹¹ , J ²¹² are independently selected for each occurrence from an alkylene group of 1 to 30 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more substituents 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 independently selected from an alkylene group of 1 to 30 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more substituents 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 ²¹² are each independently optionally unsubstituted or substituted with one or more groups selected from the group consisting of: H, alkyl, aryl, heteroaryl, halogenated alkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohalogenated alkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shalogenated alkyl, halogen, -OH, -SH, _-NH2 , -alkyl- _NH2 , -N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, _-CO2H , -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), _-CONH2 , -NHC(O)(alkyl), -NHC(O)(phenyl), -N(alkyl)C(O)(alkyl), -N(alkyl)C(O)(phenyl), -C(O)alkyl, -C(O)alkylphenyl, -C(O)haloalkyl, -OC(O)alkyl, -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). The compound of claim 23, wherein: J ²¹¹ , J ²¹² are independently selected for each occurrence from an alkylene group of 1 to 10 carbon atoms, wherein one or more carbon atoms are optionally composed of Any one or more substituents are substituted: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N and S(O) ₂ , and each of J ²¹¹ and J ²¹² Independently optionally unsubstituted or substituted with at least one substituent selected from the group consisting of: H, or C ₁ -C ₅ alkyl, -OC ₁ -C ₅ alkyl. The compound of claim 29, wherein n ²¹¹ is 1 and n ²¹² is 1 or 3. The compound of claim 29, wherein the compound has the structural formula (S-G1-03), (S-G1-04) or (S-G1-05): (S-G1-03) (S-G1-04) (S-G1-05) wherein A is O or S, X is independently selected from Table 1; X ₁ is independently selected from Table 2; J ²¹² is independently selected from Table 3; alternatively, J ²¹² is independently selected from an alkylene group of 1 to 10 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more substituents 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 substituent selected from the following group: H, or C ₁ -C ₅ alkyl and -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' comprises an oligonucleotide formed from natural and/or chemically modified nucleotides/nucleosides; R ²¹⁷ is selected from a linear alkylene group of 3 to 15 carbon atoms, wherein one or more carbon atoms are optionally replaced by one or more substituents from the group consisting of: C(O), NH, O, S, OP(O)O and 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, halo, -OH, -SH, -NH ₂ , -alkyl-NH ₂ , -N(alkyl)(alkyl), -NH(alkyl), cyano, nitro, -CO ₂ H, -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), -CONH ₂ , -NHC(O)(alkyl), -NHC(O)(phenyl), -N(alkyl)C(O)(alkyl), -C(O)alkyl, -C(O)haloalkyl, -OC(O)alkyl, -SO ₂ (alkyl), -SO ₂ (haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH(alkyl), -NHSO ₂ (alkyl) and -NHSO ₂ (haloalkyl); Optionally, R ²¹⁷ is selected from -C ₃ -C ₈ straight chain alkylene. The compound of claim 31, wherein the compound has the structural formula (S-G1-06), (S-G1-07) or (S-G1-08): (S-G1-06) (S-G1-07) (S-G1-08). The compound of claim 31, wherein the compound has the structural formula (S-G1-09), (S-G1-10) or (S-G1-11): (S-G1-09) (S-G1-10) (S-G1-11). The compound of claim 31, wherein the compound has the structural formula (S-G1-12), (S-G1-13) or (S-G1-14): (S-G1-12) (S-G1-13) (S-G1-14). The compound as claimed in claim 1, wherein the compound has the structural formula (S-G1-15) or (S-G1-16): (S-G1-15) (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' 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: absence, amide, ether, triazole, carbonate, carbamate, phosphate, phosphonate, phosphorothioate, sulfate, disulfide, ester, thioester, alkylamine, cycloalkylamine, alkyne, cycloalkyne, alkene, cycloalkene, lactone and lactamide 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 group capable of pairing with a cell surface receptor; and each n1, n2, n3 is independently selected from 1, 2, 3 and 4. The compound according to any one of claims 31-35, wherein each A is O. A compound as described in any of claims 31-35, wherein at least one A is O. The compound according to any one of claims 23-37, wherein each ligand is independently selected from the group consisting of: N-acetyl galactosamine (GalNAc), cholesterol, tocopherol, biotin, cyanine dye , folic acid, RGDp, transferrin, anisidine, lactobionic acid, cRGD, hyaluronic acid, low molecular weight protamine, lipid derivatives, peptides, cyclic peptides and heterocycles. A compound as described in any one of claims 23-37, wherein the ligand is N-acetylgalactosamine (GalNAc). The compound of claim 31, wherein the compound has the structure shown in GS-1 to GS-8: . The compound of claim 31, wherein the compound has a structure shown in GS-9. The compound of claim 31, wherein the compound has a structure shown as GS-5. The compound of any one of claims 1-42, wherein the naturally occurring and/or chemically modified oligonucleotide is connected to other parts of the compound through its 5' end and/or 3' end. The compound of claim 43, wherein the oligonucleotide comprises a small interfering RNA (siRNA) double strand, an asymmetric interfering RNA (aiRNA) double strand, an antisense oligonucleotide (ASO) or a microRNA (miRNA) ). A compound as described in claim 44, wherein the aiRNA comprises an antisense strand and a sense strand, wherein the antisense strand is longer than the sense strand, the antisense strand is 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides in length, and when forming a double strand with the sense strand, the antisense strand comprises a 3' overhang of 1-9 nucleotides and a 5' overhang of 0-8 nucleotides; wherein the sense strand is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length and forms a double strand region with the antisense strand. The compound of claim 45, wherein when the aiRNA antisense strand forms a double strand with the sense strand, it includes a 5' overhang of 1-8 nucleotides. The compound of claim 45, wherein when the aiRNA antisense strand and the sense strand form a double strand, a 5' blunt end is included. A small interfering RNA (siRNA) duplex comprising the structural formula as described in any one of claims 1-43. An asymmetric interfering RNA (aiRNA) agent comprising the structural formula described in any one of claims 1-43. An antisense oligonucleotide (ASO) agent comprising the structural formula as described in any one of claims 1-43. A microRNA (miRNA) agent comprising the structural formula as described in any one of claims 1-43. A pharmaceutical composition comprising a compound as described in any one of claims 1-46 or an agent as described in any one of claims 47-51, and a pharmaceutically acceptable excipient, carrier, or diluent agent. Use of a compound according to any one of claims 1 to 46 or an agent according to any one of claims 47 to 51 in the preparation of a medicament effective for treating a disease or condition. A compound having the structural formula (G-P1): (G-P1) wherein: R ₀ is one or more selected from the group consisting of: H, C ₁ -C ₅ alkyl, aryl, heteroaryl, C ₁ -C ₅ halogenated alkyl, -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)halogenated alkyl, -SO ₂ (alkyl), -SO ₂ (halogenated alkyl), -SO ₂ NH ₂ , -SO ₂ NH(alkyl) and -SO ₂ NH(phenyl); R ^202A comprises at least one ligand capable of binding to a cell surface receptor; R ²⁰⁴ , R ²⁰⁷ , R 208 ²⁰⁸ are independently selected from one or more of the following: H, alkyl, aryl, heteroaryl, halogenated alkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohalogenated alkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shalogenated alkyl, halogen, -OH, -SH, _-NH2 , -alkyl- _NH2 , -N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, _-CO2H , -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), _-CONH2 , -NHC(O)(alkyl), -NHC(O)(phenyl), -N(alkyl)C(O)(alkyl), -N(alkyl)C(O)(phenyl), -C(O)alkyl, -C(O)alkylphenyl, -C(O)halogenated alkyl, -OC(O)alkyl, _-SO2 R 205C and R _205D are _each independently selected _from -C _{1 -C 6 alkyl} or R ^205C and R 205D together form a five-membered or six-membered ring, optionally, R 205C and R ^205D are _both substituted _, optionally, R ^205C and R ^205D are each independently selected from -C ₁ -C _{6 alkyl} or R ^205C and R ^205D together form a five-membered or _six ^- membered ring, optionally, R ^205C and R ^{205D are} both substituted, optionally ^, R ^205C , R ^205D further contain a foreign atom selected from N and O. The compound of claim 57, wherein: J ²⁰¹ and J ²⁰² are each independently selected from an alkylene group of 3 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 heterocycloylene, and C ₅ -C ₁₀ heteroarylene, and wherein J 201 and J 202 are each independently selected from an alkylene group of 3 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 heterocycloylene, and C ₅ -C ₁₀ heteroarylene, and wherein ²⁰² is optionally unsubstituted or substituted with one or more groups selected from the group consisting of: H, alkyl, aryl, heteroaryl, halogenated alkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohalogenated alkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shalogenated alkyl, halogen, -OH, -SH, _-NH2 , -alkyl- _NH2 , -N(alkyl)(alkyl), -NH(alkyl), -N(alkyl)(alkylphenyl), -NH(alkylphenyl), cyano, nitro, _-CO2H , -C(O)Oalkyl, -CON(alkyl)(alkyl), -CONH(alkyl), _-CONH2 , -NHC(O)(alkyl), -NHC(O)(phenyl), -N(alkyl)C(O)(alkyl), -N(alkyl)C(O)(phenyl), -C(O)alkyl, -C(O)alkylphenyl, -C(O)haloalkyl, -OC(O)alkyl, -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). The compound described in claim 54 has the structural formula (G-P2): (G-P2) Where: 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 of 1 to 10 carbon atoms. A compound as described in claim 56, having the structural formula (G-P3): (G-P3). The compound as described in claim 54 has the structural formula (G-P4): (G-P4). The compound of claim 54, wherein: R ^205B is -C ₂ -C ₁₀ alkynylene-CN; R ^205C and 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 an alkylene group of 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 R ^202B is optionally substituted with ^{R 207} _; n ^111L is _{selected from 1 , 2} and 3 _. The compound of claim 59, wherein R ^202C is selected from -C(O)-C ₅ -C ₈ straight chain alkylene -NHCO-CH ₂ - and -C(O)-C ₈ -C ₁₁ straight Chain alkylene-. The compound of claim 59, wherein the branching group is selected from the group consisting of: and ; where each A ₁ is independently O, S, C=O, or NH; and each n is independently 1 to 20. The compound of claim 59, wherein the ligand is , where R ^A is a protecting group for H or OH. The compound as described in claim 59 has the structural formula (G-P5): (G-P5). The compound described in claim 59 has the structural formula (G-P6): (G-P6). A compound has the structural formula (G-P7): (G-P7) wherein: R ₃ is one or more selected from the group consisting of: H, C ₁ -C ₅ alkyl, aryl, heteroaryl, C ₁ -C ₅ halogenated alkyl, -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)halogenated alkyl, -SO ₂ (alkyl), -SO ₂ (halogenated alkyl), -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 R ²¹⁵ and R ²¹⁶ are independently selected from one or more of the following: H, alkyl, aryl, heteroaryl, halogenated alkyl, -Oalkyl, -Oalkylphenyl, -alkyl-OH, -Ohalogenated alkyl, -Salkyl, -Salkylphenyl, -alkylSH, -Shalogenated alkyl, halogen, -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); R ²¹² is selected from OH and a protecting group for OH; J ²¹¹ and J ²¹² are independently spacers for each occurrence; n ²¹¹ is selected from 1, 2 _, 3, 4, 5 and 6; J ²¹¹ and J ²¹² are independently spacers for each occurrence; R R ^211C and R ^211D are each independently selected from -C _{1 -C 6} alkyl; R ^211C and R ^211D together form a five _- membered 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. The compound of claim 65, wherein: J ²¹¹ , J ²¹² are each independently selected from an alkylene group of 3 to 30 carbon atoms, in which one or more carbon atoms are optionally selected from the group consisting of Replace any one or more substituents: C(O), NH, O, S, OP(O)O, OP(S)O, CH=N, S(O) ₂ , C ₂ -C ₁₀ alkenylene , C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C ₃ -C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene, and wherein J ²¹¹ , J ²¹² is optionally not Substituted or substituted with one or more groups selected from the group consisting of: H, alkyl, aryl, heteroaryl, haloalkyl, -Oalkyl, -Oalkylphenyl, -alkyl -OH, -O haloalkyl, -S alkyl, -S alkylphenyl, -alkylSH, -S haloalkyl, halo, -OH, -SH, -NH ₂ , -alkyl-NH ₂ , -N (alkyl) (alkyl), -NH (alkyl), -N (alkyl) (alkylphenyl), -NH (alkylphenyl), cyano, nitro, -CO ₂ H , -C(O)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). The compound as described in claim 65 has the structural formula (G-P8): (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 an alkylene group having 1 to 10 carbon atoms. The compound described in claim 66 has the structural formula (G-P9): (G-P9). The compound described in claim 65 has the structural formula (G-P10): (G-P10). The compound as described in claim 65, 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 -branching 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 an alkylene group of 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 R _{217B is} optionally substituted with ^{R 213} _; n ^{211L is} selected _{from 1 , 2} and 3. The compound of claim 70, wherein R ^217C is selected from -C(O)-C ₅ -C ₈ straight chain alkylene -NHCO-CH ₂ - and C(O) -C ₈ -C ₁₁ straight chain Alkylene-. The compound of claim 70, wherein the branching group is selected from the group consisting of: and ; wherein each A ₁ is independently O, S, C=O, or NH; and each n is independently 1 to 20. The compound of claim 65, wherein the ligand is , where R ^A is a protecting group for H or OH. The compound described in claim 65 has the structural formula (G-P11): (G-P11). The compound as described in claim 65 has the structural formula (G-P12): (G-P12).