Bicyclic dealkalized nucleic acid analogs and oligomeric compounds made therefrom

Abstract

The present invention relates to bicyclic dealkalized nucleic acid analogs and oligomeric compounds prepared therefrom, and more particularly, to a bicyclic dealkalized nucleic acid analogue, and to a bicyclic dealkalized nucleic acid analogue monomeric compound that can be incorporated into one or both ends of an oligomeric compound; the oligomeric compounds are delivered in vivo to cells requiring specific targeting, in addition to liver-related tissues, and may also be delivered to extrahepatic tissues such as ophthalmic and CNS target cells.

Description

Bicyclic abasic nucleic acid analogs and oligomeric compounds prepared therefrom Technical Field

[0001] The present invention relates to bicyclic abasic nucleic acid analogs and oligomeric compounds prepared therefrom, and more particularly to bicyclic abasic nucleic acid analogs that can be incorporated into one or both ends of an oligomeric compound. Background Art

[0002] RNA interference or "RNAi" technology is already an effective means of disease therapy known in the art. The RNAi mechanism is initiated by the production of longer non-coding RNAs mediated by the Dicer enzyme, which loads these RNA molecules into the RNA-induced silencing complex (RISC), where the sense strand is discarded and the antisense strand or guide strand is hybridized to a fully or partially complementary mRNA sequence, and then silencing of the mRNA is induced via Ago2-mediated degradation or translation inhibition. Advances in RNAi technology and delivery methods have given RNAi-based therapies an increasing number of positive results, and such therapies represent a promising treatment direction for a class of diseases. However, such technologies have not been widely used due to the inherent metabolic problems of natural RNA such as targeting and in vivo stability caused by the susceptibility of natural oligonucleotides to degradation by cellular and extracellular nucleases in the body. Therefore, the prior art focuses on nucleotide modifications, particularly those modifications that help improve nuclease resistance, binding affinity, targeting and in vivo stability.

[0003] By developing various chemical modifications for RNAi oligonucleotides, significant progress has been made in overcoming the inherent metabolic problems of natural RNA. Such chemical modifications of RNAi oligonucleotides are crucial for fully utilizing the potential of this type of therapeutic approach, as they can improve their pharmacokinetic and pharmacodynamic properties. For example, Choung et al. used 2′-OMe (2′-O methyl), 2′-F (2′-fluoro), and phosphorothioate-modified nucleotides, as well as various combinations thereof, to achieve stability in serum. Further modifications include nucleotides in which the furanose moiety of the nucleoside contains a bridge connecting the two furanose atoms to form a bicyclic ring system. Bicyclic nucleosides are variously referred to as bicyclic nucleic acids (BNA) and locked nucleic acids (LNA), respectively. Furthermore, furanose ring-opening forms such as UNA and GNA enhance thermal stability. Furthermore, it has been reported that the incorporation of a 5′-terminal phosphate group into oligonucleotides can enhance the interaction between certain nucleic acid inhibitor molecules and Ago2. For example, the incorporation of phosphate mimetics at the 5′ end of the antisense strand has also been a recent research direction to enhance the silencing effect of RNAi. However, these modified nucleotide derivatives all retain the base portion and still need to participate in the base pairing process. In recent years, abasic nucleotides and reverse abasic nucleotides have been used in 5' or 3' oligonucleotides to enhance the silencing effect and improve the stability of oligonucleotides, for example: WO2022162155.

[0004] Although great progress has been made in overcoming the inherent metabolic problems of natural RNA through the development of chemical modifications and improved delivery methods, there is still a need in the art for RNAi agents with enhanced in vivo efficacy and stability suitable for therapeutic administration. In particular, research on abasic nucleotides is still in its initial stages. It is hoped that new abasic nucleic acid analogs can be further provided to be incorporated into oligonucleotides to improve stability against cellular nucleases, have longer in vivo pharmacodynamics, and have stronger affinity for nucleic acid targets.

[0005] Summary of the Invention

[0006] The present invention relates to bicyclic abasic nucleic acid analogs and oligomeric compounds prepared therefrom, and more particularly to a monomeric bicyclic abasic nucleic acid analog compound, and a monomeric bicyclic abasic nucleic acid analog compound that can be incorporated into one or both ends of an oligomeric compound. These oligomeric compounds can be applied to target cells in liver-related tissues as well as to extrahepatic tissues, such as ophthalmic and CNS target cells.

[0007] According to one aspect of the present invention, there is provided a compound represented by formula (I) or a stereoisomer thereof:

[0008] A1 and A2 are independently selected from the group consisting of O, S, SO, SO2, NR2 and CR3R4, and R2, R3 and R4 are independently selected from the group consisting of hydrogen, halogen, sulfonyl, sulfinyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;

[0009] One of T1 and T2 is a protecting group, Z, L, or -ZL, wherein Z represents a targeting group or a lipophilic group, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and L represents a carrier spacer or a carrier portion comprising a spacer linker; and the other of T1 and T2 is an active phosphorus group or a protecting group; and T1 and T2 are not simultaneously protecting groups;

[0010] R1 is independently selected from halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;

[0011] n is an integer from 0 to 6;

[0012] Wherein formula (I) does not include the following structure:

[0013] In certain embodiments, A1 is O. In certain embodiments, A2 is O. In certain embodiments, A1 and A2 are each independently O. In certain embodiments, A1 and A2 are each independently S. In certain embodiments, A1 and A2 are each independently NR2. In certain embodiments, R2 is hydrogen or CH3. In certain embodiments, A1 is different from A2. In certain embodiments, A1 is O and A2 is S. In certain embodiments, A1 is O and A2 is NR2. In certain embodiments, A1 is S and A2 is NR2.

[0014] In certain embodiments, the protecting group of one of T1 and T2 is different depending on A1 or A2, and corresponds to a hydroxyl protecting group, a thiol protecting group, and an amine protecting group, respectively.

[0015] In certain embodiments, one of the protecting groups of T1 and T2 is a hydroxy protecting group; in one embodiment, the hydroxy protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldi Phenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyloxycarbonyl, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl or substituted 9-phenylxanthin-9-yl; In one embodiment, the preferred hydroxy protecting groups are each independently selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and 4,4'-dimethoxytrityl.

[0016] In certain embodiments, one of the protecting groups of T1 and T2 is a thiol protecting group; in one embodiment, the thiol protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyl ... Butyldimethylsilyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyloxycarbonyl, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl or substituted 9-phenylxanthine-9-yl; in one embodiment, the preferred thiol protecting groups are each independently selected from benzyl, 4,4'-dimethoxytrityl.

[0017] In certain embodiments, one of the protecting groups of T1 and T2 is an amino protecting group; in one embodiment, the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrophenyl, acetyl, 2-nitrobenzenesulfonyl, phthalimido and dithiosuccinyl.

[0018] In certain embodiments, one of T1 and T2 is a lipophilic group, wherein the lipophilicity of the lipophilic group can be measured by logKow, and the logKow is greater than 0; preferably, the logKow of the lipophilic group is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5 or greater than 10. In certain embodiments, the lipophilic group is selected from a group containing saturated or unsaturated C 4- C 30 hydrocarbon chains, aliphatic rings, aromatic groups, fatty acid groups or fatty acid-derived groups, steroid-derived groups and any fat-soluble vitamin groups.

[0019] In certain embodiments, the lipophilic group is a saturated or unsaturated C 10- C 25 Hydrocarbon chains (e.g. C 10 -C 25 In some embodiments, the lipophilic group is a saturated or unsaturated C 10 -C 18 Hydrocarbon chain (e.g. straight chain C6-C 18 In one embodiment, the lipophilic group is a saturated or unsaturated C 14 、C 16 or C 18 Hydrocarbon chains (e.g. straight chain C 16 In one embodiment, the lipophilic group is a saturated straight chain C 14 、C 16 or C 18 The lipophilic group is optionally further substituted with a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. These functional groups can be used to attach the lipophilic group to A1 or A2.

[0020] In another embodiment, the lipophilic group comprises any saturated or unsaturated fatty acid with a hydrocarbon chain of 4 to 28 carbon atoms and may contain one or two carboxyl groups. In one embodiment, the lipophilic group is a group derived from a saturated fatty acid with a hydrocarbon chain of 8 to 24 carbon atoms. In certain embodiments, the lipophilic group is a group derived from a saturated fatty acid, wherein the fatty acid is selected from the group consisting of: octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid, and tetracosanoic acid. In certain embodiments, the lipophilic group is a group derived from an unsaturated fatty acid with a hydrocarbon chain of 8 to 24 carbon atoms, wherein the fatty acid is selected from the group consisting of: myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, trans-vaccenic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid, and erucic acid.

[0021] In certain embodiments, the lipophilic group is selected from cholesterol, vitamin E (tocopherol), or bile acid.

[0022] In certain embodiments, the lipophilic group is selected from docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosanoic acid (DCA), sterol cholesterol (bile), tocopheryl succinate (TS) and lithocholic acid (LA), tretinoin (vitamin A acid).

[0023] The lipophilic group may be covalently linked via direct attachment to A1 or A2. Alternatively, the lipophilic group may be covalently linked to A1 or A2 via a linker.

[0024] In one embodiment, one of T1 and T2 is a lipophilic group, which is further covalently linked to A1 or A2 via a linker. In certain embodiments, the lipophilic group is covalently linked to A1 or A2 via one or more linkers.

[0025] In another embodiment, the linker is a hydrocarbon chain linker or a polyethylene glycol (PEG) linker. In another embodiment, the linker is selected from the group consisting of: an amide bond, a phosphatidylcholine, a hydrocarbon linker or a polyethylene glycol (PEG) linker, an amino-alkyl-alcohol, a hydroxyproline, a hydroxyprolinol, an amino-alkyl-thiophosphate linker, an amino-PEG-thiophosphate linker, an α-carboxylate-amino-alkylthiophosphate linker, and an α-carboxylate-amino-PEG-thiophosphate linker. In some embodiments, the linker contains an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide bond, a product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate. In some embodiments, the phosphodiester is a thiophosphate diester and an oxophosphate diester.

[0026] In some embodiments, the linker includes a cleavable group. At least one linker is a redox cleavable linker (such as a reduction cleavable linker, e.g., a disulfide group), an acid cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase cleavable linker (e.g., an ester group), a phosphatase cleavable linker (e.g., a phosphate ester), or a peptidase cleavable linker (e.g., a peptide bond). In other embodiments, at least one linker is a biocleavable linker selected from the group consisting of functionalized monosaccharides or oligosaccharides of DNA, RNA, disulfides, amides, galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0027] In some embodiments, the linker can also be a cyclic group or an acyclic group. In one embodiment, the cyclic group is selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthalene. In one embodiment, the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone.

[0028] In some embodiments, one of T1 and T2 is a targeting group, which may be a ligand commonly used in the field of siRNA administration.

[0029] In some embodiments, the targeting group can be selected from one or more ligands comprising the following targeting molecules or their derivatives: polymers, carbohydrates, receptor ligands expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands.

[0030] In some embodiments, at least one or each of the targeting groups is selected from a group comprising a ligand capable of binding to a surface receptor of a mammalian hepatocyte.

[0031] In some embodiments, each of the targeting groups independently comprises a ligand that has affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes.

[0032] In some embodiments, each of the targeting groups is independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucose-sugar, β-D-glucose-sugar. , α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2 -amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-trimethoxy-1,2-dimethylamino-L-glucopyranose The ligands include one or more of the following: -O-acetyl-1-thio-6O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside heptanoside ethyl ester, 2,5-anhydro-D-allose nitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

[0033] In some embodiments, at least one or each of the targeting groups is a ligand comprising galactose or N-acetylgalactosamine.

[0034] The targeting group may be connected to one of A1 or A2 via a suitable conjugation linker. Those skilled in the art can select a suitable conjugation linker according to the specific type of the targeting group.

[0035] In certain embodiments, the targeting group is linked to one of A1 or A2 and is selected from one of the following compound fragments:

[0036] In certain embodiments, one of T1 and T2 is a carrier spacer. In certain embodiments, the carrier spacer is a dicarboxylic acid derived group, with the structure R is absent, ether, polyethylene glycol, oxygen, sulfur, nitrogen, alkylene, alkenyl, alkynyl, aryl, aralkyl, heteroalkyl or heteroaryl. In certain embodiments, the carrier spacer is a polyalkylene glycol phosphate / phosphonate; in certain embodiments, the carrier spacer is preferably a C3-C6 alkyl dicarboxylate group. In certain embodiments, the carrier spacer is preferably a succinyl group, In certain embodiments, one of T1 and T2 comprises a carrier portion linked by a spacer, wherein the carrier portion comprises a solid phase carrier portion and a liquid phase carrier portion. In certain embodiments, the solid phase carrier is selected from controlled pore glass (CPG), polystyrene, silica gel, macroporous cross-linked polystyrene, poly(vinyl methacrylate) copolymer, silicon chip glass, cellulose, polystyrene beads, polypropylene sheets, non-porous silica beads, polyacrylamide or polyacrylate. In certain embodiments, the solid phase carrier portion is long chain alkylamine controlled pore glass (LCAA-CPG).

[0037] In certain embodiments, one of T1 and T2 is a targeting group or a lipophilic group and further comprises a carrier portion linked by a spacer. In some more specific embodiments, the carrier portion is connected to a hydroxyl group in a linker in the targeting group or the lipophilic group or a conjugated linker via a spacer.

[0038] In certain embodiments, the other of T1 and T2 is an active phosphorus group, and the active phosphorus group has the following structure:

[0039] Wherein M1 is H, substituted or unsubstituted C1-C6 alkyl, OR5, SR5, OH, SH or NR6R7, M2 is OH, SH, OR5′ or NR6′R7′, each R5, R6, R7, R5′, R6′ or R7′ is independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or sulfonyl; m is 0 or 1.

[0040] In certain embodiments, M1 is selected from methyl, ethyl, propyl, and isopropyl.

[0041] In certain embodiments, M1 is selected from OR5, and R5 is selected from substituted or unsubstituted methyl, ethyl, propyl, and isopropyl.

[0042] In certain embodiments, M1 is selected from methanesulfonylamide.

[0043] In certain embodiments, M2 is selected from N(CH(CH3)2)2.

[0044] In certain embodiments, each R5, R6 or R7 is independently a substituted alkyl group, wherein the substituent is selected from cyano, halogen, hydroxyl, and amino.

[0045] In certain embodiments, wherein M1 is O(CH2)2CN, M2 is N(CH(CH3)2)2, and m is 0.

[0046] In certain embodiments, the other of T1 and T2 is a reactive phosphorus group, and the reactive phosphorus group is selected from diisopropylcyanoethoxyphosphoramidite, diisopropylmethylphosphoramidite, diisopropylethylphosphoramidite and H-phosphonate.

[0047] In certain embodiments, one of T1 and T2 is a protecting group and the other is a carrier spacer, a carrier moiety linked via a spacer, a targeting group, or a lipophilic group, which is optionally linked to one of A1 or A2 via a linker.

[0048] In certain embodiments, one of T1 and T2 is diisopropylcyanoethoxyphosphoramidite and the other is 4,4'-dimethoxytrityl.

[0049] In certain embodiments, one of T1 and T2 is diisopropylcyanoethoxyphosphoramidite and the other is a targeting group.

[0050] In certain embodiments, one of T1 and T2 is diisopropylcyanoethoxyphosphoramidite and the other is a lipophilic group.

[0051] In certain embodiments, R1 is independently selected from halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl.

[0052] In certain embodiments, n is 0.

[0053] In certain embodiments, the compounds provided herein have the configuration of Formula (IIa) or Formula (IIb):

[0054] A1, A2, T1, T2, R1 and n are as described above.

[0055] In certain embodiments, the compounds provided herein have the configuration of Formula (IIa-1) or Formula (IIb-1):

[0056] A1, A2, T1, T2, R1 and n are as described above.

[0057] According to another aspect of the present invention, there is provided an oligomeric compound comprising at least one 5′-terminal and / or 3′-terminal monomer having formula (III) or a stereoisomer thereof:

[0058] A1 and A2 are independently selected from the group consisting of O, S, SO, SO2, NR2 and CR3R4, and R2, R3 and R4 are independently selected from the group consisting of hydrogen, halogen, sulfonyl, sulfinyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;

[0059] One of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (III) or its stereoisomer to the oligomeric compound;

[0060] R1 is independently selected from halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;

[0061] n is an integer from 0 to 6;

[0062] The oligomeric compound optionally further comprises a targeting group.

[0063] In certain embodiments, in the oligomeric compounds provided, A1 is O. In certain embodiments, in the oligomeric compounds provided, A2 is O. In certain embodiments, in the oligomeric compounds provided, A1 and A2 are each independently O. In certain embodiments, in the oligomeric compounds provided, A1 and A2 are each independently S. In certain embodiments, in the oligomeric compounds provided, A1 and A2 are each independently NR2. In certain embodiments, in the oligomeric compounds provided, R2 is hydrogen or CH3. In certain embodiments, in the oligomeric compounds provided, A1 is different from A2. In certain embodiments, in the oligomeric compounds provided, A1 is O and A2 is S. In certain embodiments, in the oligomeric compounds provided, A1 is O and A2 is NR2. In certain embodiments, in the oligomeric compounds provided, A1 is S and A2 is NR2.

[0064] In certain embodiments, the oligomeric compound provided, the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (III) or its stereoisomer to the 5′-end and / or 3′-end of the oligomeric compound, and the internucleoside linking group is selected from a phosphorus-containing linking group or a phosphorus-free linking group.

[0065] In certain embodiments, in the provided oligomeric compound, the phosphorus-containing internucleoside linking group has a structural fragment as shown below: X represents H, substituted or unsubstituted C1-C6 alkyl, OR8, SR8', OH, SH or NR9R 10 , Y represents O or S, z can be 0 or 1, R8, R8', R9 or R 10 are independently hydrogen, substituted or unsubstituted C1-C6 alkyl, sulfonyl, They independently represent the part connected to one of A1 or A2 herein, and the part connected to the adjacent nucleotide.

[0066] In certain embodiments, the phosphorus-containing internucleoside linking groups are independently: phosphodiester linking groups, phosphotriester linking groups, phosphorothioate linking groups, phosphorodithioate bond linking groups, alkylphosphonate linking groups, aminophosphonate linking groups, phosphonate linking groups, phosphinate linking groups, phosphinate linking groups, thiophosphoramidate linking groups and phosphoramidate linking groups.

[0067] In certain embodiments, the internucleoside linking group is independently an alkylphosphonate linking group, a phosphodiester internucleoside linking group, or a phosphorothioate internucleoside linking group.

[0068] In certain embodiments, in the oligomeric compound provided, the protecting group of one of T3 and T4 is different depending on A1 or A2, and corresponds to a hydroxyl protecting group, a thiol protecting group, and an amine protecting group, respectively.

[0069] In certain embodiments, in the oligomeric compound provided, one of the protecting groups of T3 and T4 is a hydroxy protecting group; in one embodiment, in the oligomeric compound provided, the hydroxy protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl , tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyloxycarbonyl, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl or substituted 9-phenylxanthine-9-yl; in one embodiment, the oligomeric compound provided, preferably the hydroxyl protecting groups are each independently selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and 4,4'-dimethoxytrityl.

[0070] In certain embodiments, in the oligomeric compound provided, one of the protecting groups of T3 and T4 is a thiol protecting group; in one embodiment, in the oligomeric compound provided, the thiol protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, alkyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyloxycarbonyl, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl or substituted 9-phenylxanthine-9-yl; in one embodiment, the oligomeric compound provided, preferably the thiol protecting group is each independently selected from benzyl, 4,4'-dimethoxytrityl.

[0071] In certain embodiments, in the oligomeric compound provided, one of the protecting groups of T3 and T4 is an amino protecting group; in one embodiment, in the oligomeric compound provided, the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrophenyl, acetyl, 2-nitrobenzenesulfonyl, phthalimido and dithiosuccinyl.

[0072] In certain embodiments, the oligomeric compound is provided wherein one of T3 and T4 is hydrogen and the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (III) or a stereoisomer thereof to the 5′-terminus and / or 3′-terminus of the oligomeric compound.

[0073] In certain embodiments, the oligomeric compound provided, one of T3 and T4 is a lipophilic group, wherein the lipophilicity of the lipophilic group can be measured by logKow, logKow is greater than 0; preferably, the logKow of the lipophilic group is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5 or greater than 10. In certain embodiments, the oligomeric compound provided, the lipophilic group is selected from a group containing saturated or unsaturated C 4- C 30 hydrocarbon chains, aliphatic rings, aromatic groups, fatty acid groups or fatty acid-derived groups, steroid-derived groups and any fat-soluble vitamin groups.

[0074] In certain embodiments, the oligomeric compounds provided herein have lipophilic groups that are saturated or unsaturated C 10- C 25 Hydrocarbon chains (e.g. C 10 -C 25 In some embodiments, the oligomeric compounds provided herein have lipophilic groups that are saturated or unsaturated C 10 -C 18 Hydrocarbon chain (e.g. straight chain C6-C 18 In one embodiment, the oligomeric compound provided herein has a lipophilic group that is a saturated or unsaturated C 14 、C 16 or C 18 Hydrocarbon chains (e.g. straight chain C 16 In one embodiment, the oligomeric compound provided herein has a lipophilic group that is a saturated straight chain C 14 、C 16 or C 18The lipophilic group is optionally further substituted with a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. These functional groups can be used to attach the lipophilic group to A1 or A2.

[0075] In another embodiment, the oligomeric compound provided herein comprises any saturated or unsaturated fatty acid having a hydrocarbon chain of 4 to 28 carbon atoms and may contain one or two carboxyl groups. In one embodiment, the oligomeric compound provided herein comprises a lipophilic group derived from a saturated fatty acid having a hydrocarbon chain of 8 to 24 carbon atoms. In certain embodiments, the oligomeric compound provided herein comprises a lipophilic group derived from a saturated fatty acid, wherein the fatty acid is selected from the group consisting of octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid, and tetracosanoic acid. In certain embodiments, the oligomeric compound provided herein comprises a lipophilic group derived from an unsaturated fatty acid, wherein the fatty acid is selected from the group consisting of myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, trans-vaccenic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid, and erucic acid.

[0076] In certain embodiments, in the provided oligomeric compounds, the lipophilic group is selected from cholesterol, vitamin E (tocopherol) or bile acid.

[0077] In certain embodiments, the oligomeric compounds provided herein have a lipophilic group selected from docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosanoic acid (DCA), sterol cholesterol (bile), tocopheryl succinate (TS) and lithocholic acid (LA), retinoic acid (vitamin A acid).

[0078] The lipophilic group may be covalently linked via direct attachment to A1 or A2. Alternatively, the lipophilic group may be covalently linked to A1 or A2 via a linker.

[0079] In one embodiment, in the oligomeric compound provided, one of T3 and T4 is a lipophilic group, and in the oligomeric compound provided, the lipophilic group of T3 or T4 is further covalently linked to A1 or A2 via a linker. In certain embodiments, in the oligomeric compound provided, the lipophilic group is covalently linked to A1 or A2 via one or more linkers.

[0080] In another embodiment, in the oligomeric compound provided, the linker is a hydrocarbon chain linker or a polyethylene glycol (PEG) linker. In another embodiment, in the oligomeric compound provided, the linker is selected from the group consisting of: an amide bond, a phosphatidylcholine, a hydrocarbon linker or a polyethylene glycol (PEG) linker, an amino-alkyl-alcohol, hydroxyproline, hydroxyprolinol, an amino-alkyl-thiophosphate linker, an amino-PEG-thiophosphate linker, an α-carboxylate-amino-alkylthiophosphate linker, and an α-carboxylate-amino-PEG-thiophosphate linker. In some embodiments, the linker contains an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide bond, a product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate. In another embodiment, in the oligomeric compound provided, the phosphodiester linker is a thiophosphate diester or an oxophosphate diester.

[0081] In some embodiments, in the oligomeric compounds provided, the linker comprises a cleavable group. At least the linker is a redox cleavable linker, such as a reduction cleavable linker, for example, a disulfide group, an acid cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase cleavable linker (e.g., an ester group), a phosphatase cleavable linker (e.g., a phosphate ester), or a peptidase cleavable linker (e.g., a peptide bond). In other embodiments, at least one linker is a biocleavable linker selected from the group consisting of: DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, mannose functionalized monosaccharide or oligosaccharide, and combinations thereof.

[0082] In some embodiments, in the oligomeric compounds provided, the linker can also be a cyclic group or an acyclic group. In one embodiment, the cyclic group is selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthalene. In one embodiment, the acyclic group is a portion based on a serinol backbone or a diethanolamine backbone.

[0083] In certain embodiments, the 5′-end and / or 3′-end of the oligomeric compound further comprises one or more targeting groups. In certain embodiments, the oligomeric compound provided, one of T3 and T4 is represented as a covalent bond, and the oligomeric compound is covalently bonded to the targeting group through one of T3 and T4 in formula (III) or its stereoisomers. In certain embodiments, the oligomeric compound provided, the 5′-end and / or 3′-end nucleotide is connected to the targeting group. The targeting group can be a ligand conventionally used in the field of siRNA administration.

[0084] In some embodiments, in the provided oligomeric compounds, the targeting group can be selected from one or more ligands formed by the following targeting molecules or their derivatives: polymers, carbohydrates, receptor ligands expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands.

[0085] In some embodiments, in the oligomeric compound provided, at least one or each of the targeting groups is selected from a ligand that can bind to a receptor on the surface of a mammalian hepatocyte.

[0086] In some embodiments, in the oligomeric compound provided, each targeting group independently comprises a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes.

[0087] In some embodiments, in the oligomeric compound provided, each of the targeting groups is independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyl- 36-Acylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose , one of 2,3,4-tri-O-acetyl-1-thio-6O-trityl-α-D-pyranoglucopyranoside methyl ester, 4-thio-β-D-pyranoglucopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-pyranoglucopyranoside ethyl ester, 2,5-anhydro-D-allosenitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

[0088] In some embodiments, in the oligomeric compounds provided, at least one or each targeting group is a ligand comprising galactose or N-acetylgalactosamine.

[0089] In the provided oligomeric compounds, the targeting group can be linked to either A1 or A2 via a suitable conjugation linker. Those skilled in the art can select a suitable conjugation linker based on the specific type of the targeting group.

[0090] In certain embodiments, in the oligomeric compounds provided, the targeting group is linked to one of A1 or A2 and is selected from one of the following compound fragments:

[0091] In certain embodiments, R1 is independently selected from halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl.

[0092] In certain embodiments, provided oligomeric compounds have n as 0.

[0093] In certain embodiments, provided oligomeric compounds comprise at least one 5′-terminal and / or 3′-terminal monomer having a configuration represented by formula (IVa) or formula (IVb):

[0094] A1, A2, T3, T4, R1 and n are as described above.

[0095] In certain embodiments, provided oligomeric compounds comprise at least one 5′-terminal and / or 3′-terminal monomer having a configuration represented by formula (IVa-1) or formula (IVb-1):

[0096] A1, A2, T3, T4, R1 and n are as described above.

[0097] In certain embodiments, an oligomeric compound is provided wherein one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects a monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula (IVb), (IVa-1) or Formula (IVb-1) to the 5′-terminus of the oligomeric compound.

[0098] In certain embodiments, an oligomeric compound is provided wherein one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects a monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula (IVb), (IVa-1) or Formula (IVb-1) to the 3′-terminus of the oligomeric compound.

[0099] In certain embodiments, an oligomeric compound is provided, wherein one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 independently connects one of the same or different (III) or its stereoisomers, formula (IVa), formula (IVb), (Iva-1) or formula (IVb-1) monomers to the internucleoside linking groups at the 5′-terminus and 3′-terminus of the oligomeric compound.

[0100] In certain embodiments, the oligomeric compound is a single-stranded oligonucleotide. In certain embodiments, the single-stranded oligonucleotide is a conventional antisense oligonucleotide (also known as ASO), ribozyme or aptamer. In certain embodiments, the oligomeric compound is a double-stranded ribonucleic acid (dsRNA) reagent, a double-stranded nucleoside nucleic acid well known in the art, wherein one or both strands are oligomeric compounds as disclosed herein.

[0101] In certain embodiments, double-stranded ribonucleic acid (dsRNA) agents are provided, each comprising: a sense strand and an antisense strand, wherein the sense strand and the antisense strand are fully or partially complementary, and the antisense strand is partially or fully complementary to a nucleic acid target gene; at least one of the sense strand and the antisense strand is an oligomeric compound as provided above, comprising at least one 5′-terminal and / or 3′-terminal monomer having formula (III) or its stereoisomers, formula (IVa), formula (IVb), formula (IVa-1) or formula (IVb-1); and wherein the double-stranded ribonucleic acid (dsRNA) agent optionally further comprises an independent targeting group.

[0102] In certain embodiments, double-stranded ribonucleic acid (dsRNA) agents are provided, wherein the sense strand is an oligomeric compound provided herein comprising a 5′-terminal and / or 3′-terminal monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula (IVb), Formula (IVa-1), or Formula (IVb-1). In one embodiment, double-stranded ribonucleic acid (dsRNA) agents are provided, wherein the sense strand is an oligomeric compound provided herein comprising a 5′-terminal monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula (IVb), Formula (IVa-1), or Formula (IVb-1). In one embodiment, double-stranded ribonucleic acid (dsRNA) agents are provided, wherein the sense strand is an oligomeric compound provided herein comprising a 3′-terminal monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula (IVb), Formula (IVa-1), or Formula (IVb-1). In one embodiment, a double-stranded ribonucleic acid (dsRNA) agent is provided, wherein the sense strand is an oligomeric compound provided herein comprising the same or different 5′-terminal and 3′-terminal monomers of Formula (III) or its stereoisomers, Formula (IVa), Formula (IVb), Formula (IVa-1), or Formula (IVb-1).

[0103] In certain embodiments, a double-stranded ribonucleic acid (dsRNA) agent is provided, wherein the antisense strand is an oligomeric compound comprising a 5′-terminal and / or 3′-terminal monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula (IVb), Formula (IVa-1), or Formula (IVb-1) herein. In one embodiment, a double-stranded ribonucleic acid (dsRNA) agent is provided, wherein the antisense strand is an oligomeric compound comprising a 3′-terminal monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula (IVb), Formula (IVa-1), or Formula (IVb-1) herein. In one embodiment, a double-stranded ribonucleic acid (dsRNA) agent is provided, wherein the antisense strand is an oligomeric compound comprising a 5′-terminal monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula (IVb), Formula (IVa-1), or Formula (IVb-1) herein.

[0104] In certain embodiments, a double-stranded RNA (dsRNA) reagent is provided, optionally further comprising an independent targeting group, and further comprising one or more targeting groups on the 5'-terminal and / or 3'-terminal nucleotides of any strand in the double-stranded RNA (dsRNA) reagent. The targeting group can be a ligand conventionally used in the field of siRNA administration.

[0105] In some embodiments, in the double-stranded ribonucleic acid (dsRNA) reagent provided, the targeting group can be selected from one or more ligands formed by the following targeting molecules or their derivatives: polymers, carbohydrates, receptor ligands expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands.

[0106] In some embodiments, in the double-stranded ribonucleic acid (dsRNA) reagent provided, at least one or each targeting group is selected from a ligand that can bind to a receptor on the surface of a mammalian hepatocyte.

[0107] In some embodiments, in the double-stranded RNA (dsRNA) reagent provided, each of the targeting groups independently comprises a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes.

[0108] In certain embodiments, each strand of the oligomeric compound or double-stranded ribonucleic acid (dsRNA) comprises 8-40 nucleotides in length.

[0109] In certain embodiments, the oligomeric compound or double-stranded ribonucleic acid (dsRNA) reagent provided is used in the preparation of a drug for inhibiting gene expression. In certain embodiments, wherein the inhibiting gene expression comprises contacting one or more cells, tissues or animals with the oligomeric compound.

[0110] In certain embodiments, a method of inhibiting gene expression is provided, the method comprising: contacting a cell with an oligomeric compound or a double-stranded ribonucleic acid (dsRNA) agent as described herein, wherein each strand of the oligomeric compound and the double-stranded ribonucleic acid (dsRNA) agent comprises 8-40 nucleotides in length, respectively, and the antisense strand of the oligomeric compound or the double-stranded ribonucleic acid (dsRNA) agent is complementary to the target RNA.

[0111] In certain embodiments, the cell is in an animal. In certain embodiments, the cell is in a human. In certain embodiments, the target RNA is selected from mRNA, pre-mRNA, and micro RNA. In certain embodiments, the target RNA is mRNA. In certain embodiments, the target RNA is human mRNA. In certain embodiments, the target RNA is cleaved, thereby inhibiting its function. In certain embodiments, the method further comprises detecting the level of the target RNA. In certain embodiments, a method for inhibiting gene expression is provided, the method comprising: contacting one or more cells or tissues with an oligomeric compound or double-stranded ribonucleic acid (dsRNA) reagent provided by a terminal monomer comprising Formula III, Formula (IVa), Formula (IVb), Formula (IVa-1), or Formula (IVb-1). These genes, in addition to liver tissue expression, can also be genes expressed in external tissues, such as target cell genes in ophthalmology and CNS.

[0112] Detailed Description of the Invention

[0113] In certain embodiments, bicyclic abasic nucleic acid analogs are provided and can be used as nucleotide monomers to be incorporated into one or both ends of an oligomeric compound. Also provided herein are intermediates and methods for preparing these oligomeric compounds. The modified bicyclic abasic nucleic acid analogs provided herein can be used to enhance one or more properties of the oligomeric compounds to which they are incorporated, such as, for example, nuclease resistance. In certain embodiments, the oligomeric compounds and compositions provided herein are expected to hybridize with a portion of a target RNA so that the target RNA loses its normal function. The oligomeric compounds are expected to also be used as primers and probes in diagnostic applications.

[0114] According to one aspect of the present invention, there is provided a compound represented by formula (I) or a stereoisomer thereof:

[0115] A1 and A2 are independently selected from the group consisting of O, S, SO, SO2, NR2 and CR3R4, and R2, R3 and R4 are independently selected from the group consisting of hydrogen, halogen, sulfonyl, sulfinyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;

[0116] One of T1 and T2 is a protecting group, Z, L, or -ZL, wherein Z represents a targeting group or a lipophilic group, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and L represents a carrier spacer or a carrier portion linked via a spacer; and the other of T1 and T2 is an active phosphorus group or a protecting group; and T1 and T2 are not simultaneously protecting groups;

[0117] R1 is independently selected from halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;

[0118] n is an integer from 0 to 6;

[0119] Wherein formula (I) does not include the following structure:

[0120] In certain embodiments, A1 is O.

[0121] In certain embodiments, A2 is O.

[0122] In certain embodiments, A1 and A2 are each independently O, and the provided compound has the formula (I-1) or a stereoisomer thereof, wherein T1, T2, R1 and n are as described above.

[0123] In certain embodiments, A1 and A2 are each independently S, and the provided compound has the formula (I-2) or a stereoisomer thereof, wherein T1, T2, R1 and n are as described above.

[0124] In certain embodiments, A1 and A2 are independently NR2, and the provided compound has the formula (I-3) or a stereoisomer thereof, wherein T1, T2, R1 and n are as described above, and R2 is independently selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C6 alkyl, 12 aryl, substituted or unsubstituted 5- to 12-membered heteroaryl, or substituted or unsubstituted 5- to 12-membered heterocyclic group.

[0125] In certain embodiments, A1 is O, A2 is S, and the provided compound has the formula (I-4) or a stereoisomer thereof, wherein T1, T2, R1 and n are as described above.

[0126] In certain embodiments, A1 is O, A2 is NR2, and the provided compound has the formula (I-5) or a stereoisomer thereof, wherein T1, T2, R1 and n are as described above, and R2 is selected from hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 aryl, substituted or unsubstituted 5- to 12-membered heteroaryl, or substituted or unsubstituted 5- to 12-membered heterocyclic group.

[0127] In certain embodiments, A1 is S, A2 is NR2, and the provided compound has the formula (I-6) or a stereoisomer thereof, wherein T1, T2, R1 and n are as described above, and R2 is selected from hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 aryl, substituted or unsubstituted 5- to 12-membered heteroaryl, or substituted or unsubstituted 5- to 12-membered heterocyclic group.

[0128] In certain embodiments, R2 is hydrogen or CH3.

[0129] As used herein, the term "substituted or unsubstituted" means that a hydrogen atom in a parent compound is not replaced by other substituents, or one or more hydrogen atoms are replaced by substituents. As used herein, the terms "substituent" and "substituting group" are intended to include groups that are typically added to other groups or parent compounds to enhance desired properties or provide other desired effects. Substituting groups may be protected or unprotected, and may be added to one or many available sites in the parent compound. Substituting groups may also be further substituted by other substituting groups, and may be directly attached to the parent compound or attached to the parent compound through a linking group such as an alkyl or hydrocarbon group.

[0130] Suitable substituting groups herein include, but are not limited to: halogen, hydroxy, alkyl, alkenyl, alkynyl, acyl (-C(O)R aa ), carboxyl (-C(O)O-R aa ), aliphatic group, alicyclic group, alkoxy, substituted oxygen (-O-R aa ), aryl, aralkyl, heterocyclic group, heteroaryl, heteroarylalkyl, amino (-N(R bb )(R cc )), imino (=NR bb ), amido (-C(O)N(R bb )(R cc ) or -N(R bb )C(O)R[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​) or -N(R bb )S(O)2R bb ). Each R aa 、R bb and R cc is independently H, an optionally attached chemical functional group or another substituent group, a preferred list including but not limited to H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. One of ordinary skill in the art of medicinal chemistry and organic chemistry understands that the total number of such substituents is appropriately limited by the desired properties of the intended compound.

[0131] As used herein, the term "protecting group" refers to an unstable chemical moiety known in the art that protects reactive groups (including but not limited to hydroxyl, amino, and thiol groups) from undesirable reactions during synthetic procedures. Protecting groups are often used selectively to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group as is or available for further reactions.

[0132] In certain embodiments, it is understood in the art that the protecting group of one of T1 and T2 is different according to A1 or A2, and the corresponding ones are hydroxyl protecting groups, sulfhydryl protecting groups and amino protecting groups, respectively; that is, when A1 or A2 is oxygen, the protecting group of one of T1 and T2 is a hydroxyl protecting group; that is, when A1 or A2 is sulfur, sulfoxide (SO), or sulfone (SO2), the protecting group of one of T1 and T2 is a sulfhydryl protecting group; that is, when A1 or A2 is nitrogen, the protecting group of one of T1 and T2 is an amino protecting group.

[0133] In certain embodiments, one of the protecting groups of T1 and T2 is a hydroxy protecting group; in one embodiment, the hydroxy protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldi Phenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyloxycarbonyl, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl or substituted 9-phenylxanthin-9-yl; In one embodiment, the preferred hydroxy protecting groups are each independently selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and 4,4'-dimethoxytrityl.

[0134] In certain embodiments, one of the protecting groups of T1 and T2 is a thiol protecting group; in one embodiment, the thiol protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyl ... Butyldimethylsilyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyloxycarbonyl, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl or substituted 9-phenylxanthine-9-yl; In one embodiment, the preferred thiol protecting groups are each independently selected from benzyl and 4,4'-dimethoxytrityl. As known in the art, when the thiol group is further oxidized to sulfoxide (SO) or sulfone (SO2), the protecting groups are also applicable to sulfoxide (SO) and sulfone (SO2).

[0135] In certain embodiments, one of the protecting groups of T1 and T2 is an amino protecting group; in one embodiment, the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrophenyl, acetyl, 2-nitrobenzenesulfonyl, phthalimido and dithiosuccinyl.

[0136] The terms "optional," "optionally," and "optional" herein generally indicate that the group or functional group may or may not be present. For example, a "lipophilic group optionally linked to A1 or A2 via a linker" may be a lipophilic group that is directly covalently linked to the atom or group to which it is attached; or the lipophilic group may be linked to the atom or group to which it is attached via one or more linkers.

[0137] In certain embodiments, one of T1 and T2 is a lipophilic group. The term "lipophilicity" or "lipophilic group" refers broadly to any compound or chemical moiety with affinity for lipids. One way to characterize the lipophilicity of a lipophilic group can be determined by the octanol-water partition coefficient logKow, where Kow is the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the aqueous phase when the two-phase system is in equilibrium. The octanol-water partition coefficient is a material characteristic measured in the laboratory. However, it can also be predicted by using coefficients attributed to the structural components of the chemical substance, which are calculated using first principles or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41: 1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure (i.e., its hydrophilicity / lipophilicity balance) of the tendency of a substance to prefer non-aqueous or oily environments rather than water. The structure to which the lipophilic group is conjugated exposes the lipophilic group (e.g., fatty acid), thereby allowing the lipophilic group to interact with albumin and / or fatty acid receptors or transporters, thereby providing an oligonucleotide with a long in vivo half-life. It is understood in the art that a group generally having lipophilicity has a logKow determination exceeding 0. In certain embodiments, the lipophilicity of the lipophilic moiety, as measured by the octanol-water partition coefficient logKow, may be greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10 logKow. The lipophilicity of a molecule can vary relative to the functional groups it carries; for example, the addition of functional groups such as hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne at the end of the lipophilic group can increase or decrease the partition coefficient (e.g., logKow) value of the lipophilic moiety; and lipophilic groups can also be further attached to the compounds shown herein via these functional groups.

[0138] In certain embodiments, the lipophilic group is a saturated or unsaturated C 4- C 30 hydrocarbon chains, aliphatic rings, aromatic groups, fatty acid groups or fatty acid-derived groups, steroid-derived groups and any fat-soluble vitamin groups.

[0139] In one embodiment, the lipophilic group is a saturated or unsaturated C 10- C 25 Hydrocarbon chains (e.g. C 10- C 25 In some embodiments, the lipophilic group is a saturated or unsaturated C 10 -C 18 Hydrocarbon chains (e.g. straight chain C 10 -C 18 In one embodiment, the lipophilic group is a saturated or unsaturated C 10、C 11 、C 12 、C 13 、C 14 、C 15 、C 16 、C 17 or C 18 Hydrocarbon chains (e.g. straight chain C 16 In one embodiment, the lipophilic group is a saturated straight chain C 14 Alkyl, C 16 Alkyl or C 18 Alkyl, the structure shown has the following fragments: The lipophilic group is optionally further substituted with a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide and alkyne. These functional groups can be used to attach the lipophilic group to A1 or A2.

[0140] In another embodiment, the lipophilic group is selected from a group derived from a fatty acid of a dicarboxylic acid. In another embodiment, the fatty acid comprises any saturated or unsaturated fatty acid having a hydrocarbon chain of 4 to 28 carbon atoms and may contain one or two carboxyl groups. In one embodiment, the lipophilic group is a group derived from a saturated fatty acid having a hydrocarbon chain of 8 to 24 carbon atoms. In certain embodiments, the lipophilic group is a group derived from a saturated fatty acid, wherein the fatty acid is selected from the group consisting of butanoic acid (CH3(CH2)2COOH), pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, heptacosanoic acid and octacosanoic acid (CH3(CH2)2COOH) 16 COOH). In another embodiment, the lipophilic group is a group derived from an unsaturated fatty acid, wherein the unsaturated fatty acid is one of the fatty acids presented in Table 1; the fatty acid is preferably selected from the group consisting of: myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, trans-vaccenic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid, and erucic acid.

[0141] Table 1 Unsaturated fatty acids

[0142] In another embodiment, the lipophilic group is a steroid, such as a sterol. Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentaphenanthrene ring system. Steroids include, but are not limited to, bile acids (e.g., cholic acid, deoxycholic acid, and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cationic steroids such as cortisone. "Cholesterol derivatives" refer to compounds derived from cholesterol, for example, by substitution, addition, or removal of substituents.

[0143] In another embodiment, the lipophilic group is selected from cholesterol, vitamin E (tocopherol) or bile acid.

[0144] In another embodiment, the lipophilic group is selected from the unsaturated fatty acids docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), the saturated fatty acid docosanoic acid (DCA), sterol cholesterol (bile), tocopheryl succinate (TS) and lithocholic acid (LA), retinoic acid (vitamin A acid).

[0145] In another embodiment, the lipophilic group is an aromatic moiety. In this context, the term "aromatic" or "aromatic" refers to monoaromatic and polyaromatic hydrocarbons. Aryl groups include but are not limited to C6-C6 containing one to three aromatic rings. 14 Aryl moieties, which may be optionally substituted; "aralkyl" or "arylalkyl" comprising an aryl group covalently linked to an alkyl group, either of which may independently be optionally substituted or unsubstituted; and "heteroaryl". As used herein, the term "heteroaryl" refers to a group having 5 to 14 ring atoms, preferably 5, 6, 9 or 10 ring atoms; having 6, 10 or 14 π electrons shared in the ring array, and having one to about three heteroatoms selected from the group consisting of nitrogen (N), oxygen (O) and sulfur (S) in addition to carbon atoms.

[0146] In certain embodiments, the lipophilic group of the present invention can be directly covalently linked to A1 or A2 via a covalent bond. In one non-limiting embodiment, the combination of A1 and T1 and / or the combination of A2 and T2 fragments include but are not limited to the following structures: in represents a single bond or a double bond, p or q independently represents an integer of 5-25, and the sum of p and q does not exceed 25. In some more specific embodiments, the combination of A1 and T1 and / or the combination of A2 and T2 include but are not limited to the following examples:

[0147] In certain embodiments, the lipophilic group can be covalently linked to A1 or A2 via a linker, and the linker can be a combination of one or more.

[0148] In the present invention, one of T1 and T2 is a lipophilic group, which further comprises a linker and is covalently linked to A1 or A2 via one or more linkers.

[0149] In one embodiment, the linker is selected from the group consisting of an amide bond, a phosphatidylcholine, a hydrocarbon linker or a polyethylene glycol (PEG) linker, an amino-alkyl-alcohol, a hydroxyproline, a hydroxyprolinol, an amino-alkyl-phosphorothioate linker, an amino-PEG-phosphorothioate linker, an α-carboxylate-amino-alkylphosphorothioate linker, and an α-carboxylate-amino-PEG-phosphorothioate linker.

[0150] In one embodiment, the linker comprises an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide bond, a product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate. In another embodiment, an oligomeric compound is provided wherein the phosphodiester linker is a phosphorothioate diester (non-limiting examples: ), phosphorothioate diesters (non-limiting examples: ).

[0151] In one embodiment, the linker is a hydrocarbon chain linker or a polyethylene glycol (PEG) linker. For hydrocarbon chain linkers, the linker comprises 2-20 carbon atoms, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. For polyethylene glycol (PEG) linkers, the linker comprises 1-20 ethylene glycol subunits, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ethylene glycol repeats.

[0152] In one embodiment, the linker is an amide bond linker.

[0153] In some embodiments, the linker includes a cleavable group. In one embodiment, the linker includes a cleavable group that is sensitive to pH, such as a disulfide group. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers will have a linking group that cleaves at a preferred pH, thereby releasing the iRNA agent from the intracellular ligand (e.g., a targeting or cell permeability ligand, such as cholesterol), or entering the desired compartment of the cell.

[0154] In one embodiment, at least one of the linkers is a redox cleavable linker (such as a reduction cleavable linker, e.g., a disulfide group). One type of cleavable linking group is a redox cleavable linking group that cleaves upon reduction or oxidation. An example of a reduction cleavable linking group is a disulfide linking group (-SS-).

[0155] Esterase cleavable linkers (e.g., ester groups). Ester-based linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -OC(O)-.

[0156] Phosphatases can cleave linkers (e.g., phosphates), and phosphate-based linking groups are cleaved by agents that degrade or hydrolyze phosphates. Examples of agents that cleave phosphate groups in cells are enzymes in cells, such as phosphatases. Examples of phosphate-based linking groups are -OP(O)(OH)-O-, -SP(S)(OH)-O-.

[0157] Or peptidase cleavable linker (for example, peptide bond). Peptide-based linking groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to produce oligopeptides (for example, dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C (O) NH-). Amide groups can be formed between any alkylene, alkenylene or alkynylene groups. Peptide bonds are special types of amide bonds formed between amino acids to produce peptides and proteins. Peptide-based cleavable groups are generally limited to the peptide bonds (i.e., amide bonds) formed between the amino acids that produce peptides and proteins, and do not include the entire amide functional group.

[0158] In other embodiments, at least one linker is a biocleavable linker selected from the group consisting of functionalized monosaccharides or oligosaccharides of DNA, RNA, disulfides, amides, galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0159] In one embodiment, the α-carboxylate-amino-alkyl-phosphorothioate linker has the following structure:

[0160] represents a linking portion to a lipophilic group and one of A1 or A2, wherein m is preferably an integer from 2 to 12, preferably an integer from 4 to 10. In one embodiment, m is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In one embodiment, m is 6.

[0161] In one embodiment, the amino-PEG-phosphorothioate linker has the following structure: represents a linker moiety to a lipophilic group and one of A1 or A2, wherein m is preferably an integer from 1 to 8. In one embodiment, m is an integer of 1, 2, 3, 4, 5, 6, 7 or 8. And α-carboxylate-amino-PEG-phosphorothioate linker:

[0162] In one embodiment, the amino-alkyl-phosphorothioate linker has the following structure: represents a linking portion to a lipophilic group and one of A1 or A2; wherein m is preferably an integer from 2 to 12. Preferably, it is an integer from 4 to 10. In one embodiment, m is an integer from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In one embodiment, m is 6.

[0163] In one embodiment, the linker is selected from the group consisting of: (i) amino-alkyl-phosphorothioate linkers; (ii) α-carboxylate-amino-alkyl-phosphorothioate linkers; (iii) amino-PEG-phosphorothioate linkers, and (iv) α-carboxylate-amino-PEG-phosphorothioate linkers all as defined above in the provided formulas.

[0164] In one embodiment, the present invention provides a conformationally restricted linker, for example, based on hydroxyproline, for example,

[0165] represents a linking portion to a lipophilic group and one of A1 or A2, wherein m is preferably an integer from 1 to 8. In one embodiment, m is an integer of 2, 3, 4, 5, 6, 7 or 8.

[0166] In one embodiment, the present invention provides conformationally restricted linkers, such as those based on hydroxyprolinol, e.g., represents a linking portion to a lipophilic group and one of A1 or A2.

[0167] In one embodiment, the present invention provides linkers such as amino-alkyl-alcohol linkages, for example, represents a linking portion to a lipophilic group and one of A1 or A2, wherein m is preferably an integer from 1 to 8. In one embodiment, m is an integer of 1, 2, 3, 4, 5, 6, 7 or 8.

[0168] In one embodiment, the present invention provides a linker such as phosphatidylcholine, for example,

[0169] represents a linking portion to a lipophilic group and one of A1 or A2.

[0170] In some embodiments, the linker can also be a cyclic group or an acyclic group. In one embodiment, the cyclic group is selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthalene. In one embodiment, the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone.

[0171] Further discussion of lipophilic groups and linkers can be found in PCT Publication No. WO2019 / 217459, entitled “Extrahepatic Delivery,” filed on July 7, 2019, PCT Publication No. WO2019 / 215333, entitled “Oligonucleotide Conjugates Comprising 7′-5′-α-Anomeric Bicyclic Sugar Nucleosides,” filed on May 10, 2019, and Nucleic Acids Research, 2019, Vol. 47, No. 31082–1096, “Various Lipid Conjugates for Functional Extrahepatic In Vivo Nucleotide Delivery,” the contents of which are incorporated herein by reference in their entirety.

[0172] In one embodiment, the present invention provides a non-limiting example of a compound formed by covalently linking a lipophilic group to A1 or A2 via a linker.

[0173] In some embodiments, one of T1 and T2 is a targeting group, which can be a ligand commonly used in the field of siRNA administration. The targeting group can modify the distribution, targeting, or lifespan of the siRNA agent into which it is incorporated, as well as its endosomal solubility properties, improve transport, hybridization, and specificity, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.

[0174] In some embodiments, the targeting group can be selected from one or more of the following ligands formed by targeting molecules or their derivatives; such as polymers, such as polyethylene glycol; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); receptor ligands expressed by hepatocytes, such as asialoglycoproteins, asialoglycosidic residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as epinephrine), growth factors, transferrin and other aptamers; antibodies; quantum dots; polypeptides, such as membrane-permeable peptides, or small molecule ligands.

[0175] In some embodiments, at least one or each of the targeting groups is selected from a ligand that can bind to a mammalian hepatocyte surface receptor. In some embodiments, each of the targeting groups is independently a ligand that has affinity for an asialoglycoprotein receptor (ASGPR) on the surface of a mammalian hepatocyte. In some embodiments, each of the targeting groups is independently a ligand that comprises an asialoglycoprotein or sugar. In some embodiments, each of the targeting groups is independently a ligand that comprises an asialoglycoprotein, such as an asialo serum serotonin (ASOR) or an asialo globulin (ASF). In some embodiments, each of the targeting groups independently comprises a D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, Sugar, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine , 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3, A ligand selected from the group consisting of 4-tri-O-acetyl-1-thio-6O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside heptanoside ethyl ester, 2,5-anhydro-D-allosenitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

[0176] In some embodiments, at least one or each of the targeting groups is a ligand comprising galactose or N-acetylgalactosamine.

[0177] In some embodiments, the targeting group comprises one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a suitable tether via a bivalent or trivalent branched linker.

[0178] in:

[0179] q 2A ,q 2B ,q 3A ,q 3B ,q 4A ,q 4B ,q 5A ,q 5B ,q 5C ,q 6A ,q 6B and q 6C Each occurrence independently represents 0-20, and the repeating units therein may be the same or different;

[0180] P 2A 、P 2B 、P 3A 、P 3B 、P 4A 、P 4B 、P 5A 、P 5B 、P 5C 、P 6A 、P 6B 、P 6C 、T 2A 、T 2B 、T 3A 、T 3B 、T 4A 、T 4B 、T 4A 、T 5B 、T 5C 、T 6A 、T 6B 、T 6C Each occurrence independently represents: absence, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CHO;

[0181] Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C , Q 6A , Q 6B , Q 6C Each occurrence independently represents: absent, alkylene, substituted alkylene, wherein one or more methylene groups may be interrupted or terminated by one or more of the following groups: O, S, S(O), SO2, NH, C(R')=C(R"), C≡C or C(O);

[0182] R 2A 、R 2B 、R 3A 、R 3B 、R 4A 、R 4B 、R 5A 、R 5B 、R 5C 、R 6A 、R 6B 、R 6C Each occurrence independently represents: absence, NH, O, S, C H2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO or a heterocyclic group;

[0183] L 2A 、L 2B 、L 3A 、L 3B 、L 4A 、L 4B 、L 5A 、L 5B 、L 5C 、L 6A 、L 6B and L 6C Each occurrence independently represents: a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide;

[0184] R' and R" are each independently H, CH3, OH, SH, NH2, alkyl, alkenyl or alkynyl;

[0185] Ra is H or an amino acid side chain; m is 1 or 2.

[0186] The targeting group-conjugated compound of formula (I) or its stereoisomer and iRNA agent of the present invention can be prepared by direct reaction with a reactive functional group, such as a phosphoramidite. The targeting group can be connected to one of A1 or A2 via a suitable conjugation linker. Those skilled in the art can select a suitable conjugation linker based on the specific type of the targeting group. For example, trans-4-hydroxyproline and aminoalkyl alcohol derivatives form phosphates through reactive functional groups. The following exemplary fragments are conjugated to degradable structures such as esters, amides, and phosphates to form suitable conjugation linkers:

[0187] Schematic diagram of the non-restricted structure of the targeting group:

[0188] The types of conjugated linkers, ligands, tethers, branched linkers, and methods of connecting the targeting group to the oligonucleotide can be found in WO2009073809, WO2014179620, WO2014179627, and WO2015006740A2, the entire contents of which are incorporated herein by reference. In some embodiments, the targeting group can be connected via acid-labile or reducible chemical bonds that are degradable in the acidic environment of the endosome.

[0189] The term "conjugate linker" or "linker" refers to a compound that connects two parts, such as covalently binds two parts of a compound. Other carbohydrate conjugates and linkers suitable for use in the present invention include those described in publications WO2014 / 179620 and WO2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0190] In certain embodiments, the targeting group is linked to one of A1 or A2 and is selected from one of the following compound fragments:

[0191] In certain embodiments, one of T1 and T2 is a carrier spacer. It is well known in the art that in oligonucleotide synthesis, the 3'-terminal non-nucleoside or nucleoside of the oligonucleotide to be synthesized (such as in formula (III) and its stereoisomers or fragments) is pre-loaded through a cleavable spacer such as a succinyl group, and then linked to a linker such as a reactive amino group in the carrier part, and then loaded into a column for docking of subsequent nucleotides; the carrier spacer serves as the attachment point of the oligonucleotide during the synthesis process, often covalently bonded to the carrier spacer through the 3'-terminal hydroxyl group of the non-nucleoside or nucleoside in the oligonucleotide, and remains attached during the entire chain assembly process, after which they are cut off and deprotected. In oligonucleotide synthesis, spacers are often selected to avoid steric hindrance and are cleavable, and the oligonucleotide is tethered to it to place the oligonucleotide and the synthesis process away from the carrier. This approach allows greater flexibility and more space for synthesis. The carrier moiety connected to the oxygen atom of the hydroxyl group at the 2' or 3' position of the ribose of the non-nucleoside at the terminal end of the oligonucleotide or the 3' terminal end of the nucleoside (such as A1 or A2 in the formula (I) and its stereoisomers described herein) through the carrier spacer can be more specifically shown as follows:

[0192] Sp represents a carrier spacer.

[0193] The carrier spacers (Sp) used in the field of oligonucleotide synthesis can be summarized in this article. Commonly used examples include dicarboxylic acid-derived groups, the structure shown R is absent, ether, polyethylene glycol, oxygen, sulfur, nitrogen, alkylene, alkenyl, alkynyl, aryl, aralkyl, heteroalkyl, or heteroaryl, One end is connected to A1 or A2 in formula (I), and the other end carboxyl group can be further connected to the carrier. R is more preferably absent, oxygen, sulfur, nitrogen, alkylene, alkenyl, or alkynyl. Another example is polyalkylene glycol phosphate / phosphonate, as shown in the disclosed patent CN103233003. In some embodiments, the spacer is a C3-C6 alkyl dicarboxylate group, and the structure shown is In certain embodiments, the carrier spacer is preferably succinyl, In some embodiments, the spacer dicarboxylic acid is further derivatized, for example by further PEGylation. P is an integer ranging from 5 to 120. The other end carboxyl group can be further linked to the carrier part to form a covalent bond.

[0194] In certain embodiments, one of T1 and T2 comprises a carrier portion linked by a spacer, wherein the carrier portion comprises a solid phase carrier portion and a soluble carrier portion.

[0195] The support generally contains a linker, such as a long-chain alkylamine, for example, a non-limiting example of a propylamine linker connected to the Si of the solid support, such as LCAA-CPG, which is commonly used in the art. In some embodiments, the long-chain alkylamine is further attached to the solid support via a biodegradable linker such as acetylglycerol.

[0196] In certain embodiments, examples of solid phase supports include inorganic porous supports and organic resin supports, examples of inorganic porous supports include controlled pore glass (CPG), and examples of organic resin supports include supports made of polystyrene. Some examples of solid phase supports include controlled pore glass (CPG), polystyrene, silica gel, macroporous cross-linked polystyrene, poly(vinyl methacrylate) copolymer, silicon chip glass, cellulose, polystyrene beads, polypropylene sheets, non-porous silica beads, polyacrylamide or polyacrylate.

[0197] In recent years, soluble carriers have been developed into an important oligonucleotide synthesis method that can be used for large-scale liquid phase synthesis. In certain embodiments, soluble carriers include, but are not limited to:

[0198] The spacer, solid support, linker and their linking mode are generally collectively referred to as solid phase supports in the art, and their composition is also well known as "Solid-Phase Supports for Oligonucleotide Synthesis" in Current Protocols in Nucleic Acid Chemistry, 2000, Chapter 3, Section 3.1. For more information on these spacers, supports, linkers and their linking modes, please refer to "Synthesis of oligonucleotides on a soluble support" Beilstein J. Org. Chem. 2017, 13, 1368–1387., WO2021 / 173615, CN103233003A, WO2010 / 131916, WO2022 / 005988A, and CN115003680A, the entire contents of which are incorporated herein by reference.

[0199] [Corrected 12.12.2023 according to Rule 26] In certain embodiments, one of T1 and T2 is a targeting group or a lipophilic group, and is further linked to the carrier portion via a spacer. In particular, when the compound of formula (I) or its stereoisomer as described herein is used at the 3' end of the oligonucleotide, the exemplary structure is, [0199.1] [Corrected 12.12.2023 according to Rule 26] represents a portion connected to A1 or A2 herein; in some more specific embodiments, the spacer is connected to the linker in the targeting group or the lipophilic group, in particular, the terminal hydroxyl group in the linker is attached to the spacer,

[0200] In certain embodiments, the other of T1 and T2 is an active phosphorus group, and the active phosphorus group has the following structure:

[0201] Wherein M1 is H, substituted or unsubstituted C1-C6 alkyl, OR5, SR5, OH, SH or NR6R7, M2 is OH, SH, OR5′ or NR6′R7′, each R5, R6, R7, R5′, R6′ or R7′ is independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or sulfonyl; m is 0 or 1.

[0202] In certain embodiments, each R5, R6 or R7 is independently a substituted C1-C6 alkyl, and the substituent can be selected from conventional alkyl substituted substituents in the art. In certain embodiments, each R5, R6 or R7 is independently a substituted alkyl, and the substituent is preferably selected from cyano, halogen, hydroxyl, and amino.

[0203] In certain embodiments, M1 is selected from substituted or unsubstituted C1-C6 alkyl, preferably methyl, ethyl, propyl, isopropyl, wherein the substituent is preferably selected from cyano, halogen, hydroxyl, and amino.

[0204] In certain embodiments, M1 is selected from OR5, and R5 is selected from substituted or unsubstituted methyl, ethyl, propyl, and isopropyl; preferably substituted methyl, ethyl, propyl, and isopropyl; more preferably, R5 is cyanoethyl, that is, M1 is cyanoethoxy.

[0205] In certain embodiments, M1 is NR6R7, R6 and R7 are independently selected from hydrogen and sulfonyl; more preferably, M1 is methanesulfonyl.

[0206] In certain embodiments, M2 is selected from NR6′R7′, R6′ or R7′ is independently C1-C6 alkyl. The C1-C6 alkyl is selected from methyl, ethyl, propyl, isopropyl. In certain embodiments, M2 is selected from N(CH(CH3)2)2.

[0207] In certain embodiments, wherein M1 is O(CH2)2CN, M2 is N(CH(CH3)2)2, and m is 0.

[0208] In certain embodiments, in certain embodiments, wherein M1 is methyl, M2 is N(CH(CH3)2)2, and m is 0.

[0209] In certain embodiments, in certain embodiments, wherein M1 is methanesulfonamide, M2 is N(CH(CH3)2)2, and m is 0.

[0210] In certain embodiments, the reactive phosphorus group has the following substructure: m, M2, R5, R6, and R7 are as described above.

[0211] In certain embodiments, the reactive phosphorus group is a phosphoramidite. In certain embodiments, the reactive phosphorus group is selected from diisopropylcyanoethoxyphosphoramidite (-P(N(CH(CH3)2)2)O(CH2)2CN). In certain embodiments, the reactive phosphorus group is selected from diisopropylmethylphosphoramidite (-P(N(CH(CH3)2)2)CH3). In certain embodiments, the reactive phosphorus group is selected from diisopropylethylphosphoramidite (-P(N(CH(CH3)2)2)CH2CH3). In certain embodiments, the reactive phosphorus group is selected from H-phosphonate (-P(=O)(H)OH)). Preferred synthesis solid phase synthesis utilizes phosphoramidite (p III ) as a reactive phosphite; the intermediate phosphite compound is subsequently oxidized to a phosphate or thiophosphate (p V ) to produce phosphodiester or phosphorothioate internucleoside linkages. Additional reactive phosphates and phosphites are disclosed in Beaucage and Iyer, Tetrahedron, 1992, 48(12), 2223-2311.

[0212] In certain embodiments, one of T1 and T2 is a protecting group, and the other is a carrier spacer, a carrier moiety linked by a spacer, a targeting group, or a lipophilic group. Further, in certain embodiments, A1 and A2 are preferably selected from O, exemplary structures

[0213] In certain embodiments, one of T1 and T2 is diisopropylethylphosphoramidite, and the other is 4,4'-dimethoxytrityl. Further, in certain embodiments, A1 and A2 are preferably selected from O.

[0214] In certain embodiments, one of T1 and T2 is diisopropylcyanoethoxyphosphoramidite, and the other is a targeting group. Further, in certain embodiments, A1 and A2 are preferably selected from O.

[0215] In certain embodiments, one of T1 and T2 is diisopropylcyanoethoxyphosphoramidite, and the other is a lipophilic group. Further, in certain embodiments, A1 and A2 are preferably selected from O.

[0216] In certain embodiments, when n is non-zero, it indicates that there are 1-6 independent R1 group substituents on the bicyclic structure. It is understood that these substituents in the bicyclic abasic compounds herein do not include natural nucleobases or modified nucleobases. In certain embodiments, R1 is independently selected from halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl. More preferably, R1 is independently selected from fluorine, chlorine, bromine, iodine, methyl, ethyl, isopropyl, propyl, 3-propenyl, ethynyl, 3-propynyl.

[0217] In certain embodiments, n is 0, indicating that there is no R1 group substitution on the ring structure.

[0218] In certain embodiments, the compounds provided herein have the configuration of Formula (IIa) or Formula (IIb):

[0219] A1, A2, T1, T2, R1 and n are as described herein.

[0220] In some preferred embodiments, in formula (IIa) or (IIb), A1 and A2 are independently selected from: O; one of T1 and T2 is a protecting group, a targeting group, and a lipophilic group, wherein the lipophilic group is optionally linked to one of A1 or A2 via a linker, and the other of T1 and T2 is an active phosphorus group; and n is 0. In some more preferred embodiments, in formula (IIa) or (IIb), A1 and A2 are independently selected from: O; one of T1 and T2 is a hydroxyl protecting group and a lipophilic group, wherein the lipophilic group is optionally linked to one of A1 or A2 via a linker, and the other of T1 and T2 is an active phosphorus group; and n is 0; wherein the hydroxyl protecting group is selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and 4,4'-dimethoxytrityl, and the lipophilic group is selected from C 14 、C 16 or C 18 The straight-linked alkane, the active phosphorus is selected from phosphoramidites. In some more preferred embodiments, the active phosphorus is selected from diisopropylcyanoethoxyphosphoramidite, diisopropylmethylphosphoramidite, diisopropylethylphosphoramidite and H-phosphonate.

[0221] In other preferred embodiments, in Formula (IIa) or Formula (IIb), A1 and A2 are independently selected from the group consisting of O, one of T1 and T2 is Z, L, and -ZL, wherein Z represents a targeting group or a lipophilic group, wherein the lipophilic group is optionally linked to one of A1 or A2 via a linker, and L represents a carrier spacer or a carrier moiety linked via a spacer; the other of T1 and T2 is a hydroxyl protecting group; and n is 0. In some more preferred embodiments, the hydroxyl protecting group is selected from the group consisting of acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and 4,4'-dimethoxytrityl.

[0222] In certain embodiments, the compounds provided herein have the configuration of Formula (IIa-1) or Formula (IIb-1):

[0223] A1, A2, T1, T2, R1, and n are as described herein, and their configurations are consistent with isosorbide or isomannide known in the art. In some preferred embodiments, in Formula (IIa-1) or Formula (IIb-1), A1 and A2 are independently selected from: O; one of T1 and T2 is a protecting group, a targeting group, and a lipophilic group, the lipophilic group optionally being linked to one of A1 or A2 via a linker; and the other of T1 and T2 is an active phosphorus group; and n is 0. In some more preferred embodiments, in formula (IIa-1) or formula (IIb-1), A1 and A2 are independently selected from: O; one of T1 and T2 is a hydroxyl protecting group and a lipophilic group, the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T1 and T2 is an active phosphorus group; n is 0; wherein the hydroxyl protecting group is selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and 4,4'-dimethoxytrityl, and the lipophilic group is selected from C 14 、C 16 or C 18 The straight-linked alkane, the active phosphorus is selected from phosphoramidites. In some more preferred embodiments, the active phosphorus is selected from diisopropylcyanoethoxyphosphoramidite, diisopropylmethylphosphoramidite, diisopropylethylphosphoramidite and H-phosphonate.

[0224] In other preferred embodiments, in Formula (IIa-1) or Formula (IIb-1), A1 and A2 are independently selected from the group consisting of O, one of T1 and T2 is Z, L, and -ZL, wherein Z represents a targeting group or a lipophilic group, wherein the lipophilic group is optionally linked to one of A1 or A2 via a linker, L represents a carrier spacer or a carrier moiety linked via a spacer, and the other of T1 and T2 is a hydroxyl protecting group; and n is 0. In some more preferred embodiments, the hydroxyl protecting group is selected from the group consisting of acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and 4,4'-dimethoxytrityl.

[0225] In certain embodiments, the compounds of Formula (IIa) or Formula (IIa-1) provided herein have the configuration examples shown below:

[0226] In certain embodiments, the compounds provided herein have (IIb) or formula (IIb-1) have the configuration exemplified below:

[0227] In the present invention, compounds of specific configurations can be obtained by obtaining starting materials through chiral control of synthesis by those skilled in the art, or by chiral resolution techniques, or can be synthesized from starting materials with specific configurations commonly used in industrial production, for example, starting materials such as isosorbide and isomannide with specific configurations.

[0228] Any combination of the features of two or more of these compounds provided herein is also included within the scope of the present invention if such features do not contradict each other. In one embodiment, the compounds provided herein have the following specific structure:

[0229] The compounds of the present invention, such as formula (I) or its stereoisomers, (IIa), (IIb), (IIa-1), (IIb-1) and the technical features of two or more such compounds can be combined into compounds in any manner, which can be applied to oligonucleotides, and further applied to the 5'-end, 3'-end or both ends of the oligonucleotides. The oligonucleotides include but are not limited to single-stranded antisense oligonucleotides (ASOs), miRNAs, and double-stranded ribonucleotides (dsRNAs). In some embodiments, they are applied to the 3'-end of the antisense strand in the double-stranded ribonucleotides (dsRNAs) in the oligonucleotides. In some embodiments, they are applied to the 5'-end of the antisense strand in the double-stranded ribonucleotides (dsRNAs) in the oligonucleotides. In some embodiments, they are applied to the 5' and / or 3'-end of the sense strand in the double-stranded ribonucleotides (dsRNAs) in the oligonucleotides.

[0230] According to one aspect of the present invention, there is provided an oligomeric compound comprising at least one 5′-terminal and / or 3′-terminal monomer having formula (III) or a stereoisomer thereof:

[0231] A1 and A2 are independently selected from the group consisting of O, S, SO, SO2, NR2 and CR3CR4, and R2, R3 and R4 are independently selected from the group consisting of hydrogen, halogen, sulfonyl, sulfinyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;

[0232] One of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, wherein the lipophilic group is optionally linked to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that links the monomer of formula (III) or a stereoisomer thereof to the oligomeric compound;

[0233] R1 is independently selected from halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;

[0234] n is an integer from 0 to 6;

[0235] The oligomeric compound optionally further comprises a targeting group.

[0236] In certain embodiments, in the provided oligomeric compounds, A1 is O.

[0237] In certain embodiments, in the oligomeric compounds provided, A2 is O.

[0238] In certain embodiments, the oligomeric compound provided, A1 and A2 are each independently O, comprising at least one 5′-terminal and / or 3′-terminal monomer having formula (III-1) or a stereoisomer thereof: wherein T3, T4, R1, and n are as defined above for the oligomeric compound.

[0239] In certain embodiments, in the provided oligomeric compounds, A1 and A2 are each independently S.

[0240] In certain embodiments, in the oligomeric compound provided, A1 and A2 are independently NR2; R2 is selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 In some embodiments, in the oligomeric compound provided, R2 is hydrogen or CH3.

[0241] In certain embodiments, the oligomeric compound provided, A1 is different from A2. In certain embodiments, the oligomeric compound provided, A1 is O, A2 is S. In certain embodiments, the oligomeric compound provided, A1 is O, A2 is NR2, which comprises at least one 5'-terminal and / or 3'-terminal monomer having formula (III-2) or a stereoisomer thereof: wherein T3, T4, R1, R2, and n are as defined above for the oligomeric compound; in some preferred embodiments, R2 is hydrogen or CH3. In certain embodiments, the oligomeric compound provided herein, A1 is S, and A2 is NR2.

[0242] As used herein, the term "internucleoside linkage" or "internucleoside linking group" is intended to include various types of internucleoside linking groups known in the art, and in such embodiments, any internucleoside linkage can be used to link nucleosides or analogs together. Two major types of internucleoside linking groups are defined by the presence or absence of a phosphorus atom, including but not limited to: phosphodiester linking groups, phosphotriester linking groups, phosphorothioate linking groups, phosphorodithioate bond linking groups, alkylphosphonate linking groups, aminophosphonate linking groups, phosphonate linking groups, phosphinate linking groups, thiophosphoramidate linking groups, and phosphoramidate linking groups; as well as non-phosphorus-containing internucleoside linking groups such as thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-), siloxane (-O-Si(H)2-O-), N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-), formacetyl, and methyleneimino (-CH2-N(CH3)-O-CH2-). Internucleoside linkages also include neutral nonionic internucleoside linkages, as used herein, the term "neutral internucleoside linkage" is intended to include nonionic internucleoside linkages. Neutral internucleoside linkages include, but are not limited to, phosphotriester, methylphosphonate, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), methylene acetal (formacetal) (3'-O-CH2-O-5') and thioformacetal (thioformacetal) (3'-S-CH2-O-5'). Further neutral internucleoside linkages include nonionic linkages including siloxanes (dialkylsiloxanes), carboxylates, formamides, sulfides, sulfonates, and amides (see, for example, "Carbohydrate Modifications in Antisense Research"; YS Sanghvi and PDCook, eds., ACS Symposium Series 580; Chapters 3 and 4, pp. 40-65). Further neutral internucleoside linkages include nonionic linkages containing mixed N, O, S, and CH2 components, wherein phosphorus atoms are not always present. In some preferred embodiments, the alkyl phosphonate linking group is selected from a C1-C6 alkyl phosphonate linking group.

[0243] In certain embodiments, in the provided oligomeric compound, the phosphorus-containing internucleoside linking group has a structural fragment as shown below: X represents H, substituted or unsubstituted C1-C6 alkyl, OR8, SR8', OH, SH or NR9R 10 , Y represents O or S, z can be 0 or 1, R8, R8', R9 or R10 are independently hydrogen, substituted or unsubstituted C1-C6 alkyl, sulfonyl, They independently represent the part connected to one of A1 or A2 herein, and the part connected to the connected nucleotide.

[0244] Modified linkages compared to native phosphodiester linkages can be used to alter (usually enhance) the nuclease resistance of oligomeric compounds. In certain embodiments, internucleoside linkages with chiral atoms can be prepared as racemic mixtures or individual enantiomers. Representative chiral linkages include, but are not limited to, alkyl phosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0245] In certain embodiments, in the provided oligomeric compound, one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (III) or its stereoisomer to the 5′-terminal and / or 3′-terminal group of the oligomeric compound, and the internucleoside linking group is independently an alkyl phosphonate linking group, a phosphodiester internucleoside linking group, or a thiophosphate internucleoside linking group.

[0246] For example, but not limited to, when T4 is an internucleoside linking group, the oligomeric compound comprising at least one monomer represented by formula (III) or a stereoisomer thereof is shown below:

[0247] wherein Olig represents an oligonucleotide moiety that links the monomer of formula (III) or a stereoisomer thereof to the 5′-terminus and / or 3′-terminus of the oligomeric compound, respectively. A1, A2, R1, and n are as described above, T3 represents H, a protecting group, a lipophilic group, and optionally a covalent bond, wherein the lipophilic group is optionally linked to A1 via a linker, and the oligomeric compound optionally further comprises a targeting group.

[0248] In certain embodiments, in the oligomeric compound provided, the protecting group of one of T3 and T4 is different depending on A1 or A2, that is, when A1 or A2 is oxygen, the protecting group of one of T1 and T2 is a hydroxyl protecting group; that is, when A1 or A2 is sulfur, sulfoxide (SO), or sulfone (SO2), the protecting group of one of T1 and T2 is a thiol protecting group; that is, when A1 or A2 is nitrogen, the protecting group of one of T1 and T2 is an amino protecting group.

[0249] In certain embodiments, in the oligomeric compound provided, one of the protecting groups of T3 and T4 is a hydroxy protecting group; in one embodiment, in the oligomeric compound provided, the hydroxy protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl , tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyloxycarbonyl, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl or substituted 9-phenylxanthine-9-yl; in one embodiment, the oligomeric compound provided, preferably the hydroxyl protecting groups are each independently selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and 4,4'-dimethoxytrityl.

[0250] In certain embodiments, in the oligomeric compound provided, one of the protecting groups of T3 and T4 is a thiol protecting group; in one embodiment, in the oligomeric compound provided, the thiol protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, Alkyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyloxycarbonyl, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl or substituted 9-phenylxanthine-9-yl; In one embodiment, the oligomeric compound provided, preferably the thiol protecting group is independently selected from benzyl, 4,4'-dimethoxytrityl. It is understood that when A1 or A2 is SO, SO2 ， Thiol protecting groups are also suitable.

[0251] In certain embodiments, in the oligomeric compound provided, one of the protecting groups of T3 and T4 is an amino protecting group; in one embodiment, in the oligomeric compound provided, the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrophenyl, acetyl, 2-nitrobenzenesulfonyl, phthalimido and dithiosuccinyl.

[0252] In certain embodiments, oligomeric compounds are provided in which one of T3 and T4 is a lipophilic group, wherein the lipophilicity of the lipophilic group can be measured by logKow, which is greater than 0. The term "lipophilic" or "lipophilic group" refers broadly to any compound or chemical moiety that has an affinity for lipids, and these coefficients are calculated using first principles or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41: 1407-21 (2001), which is incorporated herein by reference in its entirety, and its definitions have the same scope as the bicyclic abasic compounds described herein. It provides a thermodynamic measure of the tendency of a substance to prefer a non-aqueous or oily environment rather than water (i.e., its hydrophilicity / lipophilicity balance). The structure to which the lipophilic group is conjugated allows the lipophilic group (e.g., fatty acid) to be exposed, thereby allowing the lipophilic group to interact with albumin and / or fatty acid receptors or transporters, thereby providing Provide oligonucleotides with long in vivo half-lives. In some embodiments, the group generally having lipophilicity has a logKow determination of greater than 0. In certain embodiments, the lipophilicity of the lipophilic portion, as measured by the octanol-water partition coefficient logKow, can be greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10 logKow. The lipophilicity of a molecule can vary relative to the functional groups it carries; for example, the addition of functional groups such as hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne at the end of the lipophilic portion can increase or decrease the partition coefficient (e.g., logKow) value of the lipophilic portion; and lipophilic groups can also be further attached to the monomers of the oligomeric compounds described herein through these functional groups.

[0253] In the oligomeric compounds provided, the lipophilic group is also defined in the same manner as the bicyclic abasic compounds described above. For example, but not limited to, the following examples are provided:

[0254] In certain embodiments, the oligomeric compound provided has a lipophilic group containing a saturated or unsaturated C 4- C 30hydrocarbon chains, aliphatic rings, aromatic groups, fatty acid groups or fatty acid-derived groups, steroid-derived groups and any fat-soluble vitamin groups.

[0255] In certain embodiments, the oligomeric compound provided herein has a lipophilic group that is a saturated or unsaturated C 10- C 25 Hydrocarbon chains (e.g. C 10- C 25 In some embodiments, the lipophilic group is a saturated or unsaturated C 10 -C 18 Hydrocarbon chains (e.g. straight chain C 10 -C 18 In one embodiment, the lipophilic group is a saturated or unsaturated C 10 、C 11 、C 12 、C 13 、C 14 、C 15 、C 16 、C 17 or C 18 Hydrocarbon chains (e.g. straight chain C 16 In one embodiment, the lipophilic group is a saturated straight chain C 14 Alkyl, C 16 Alkyl or C 18 The lipophilic group is further selected from the group consisting of an optional functional group consisting of a hydroxyl group, an amine group, a carboxylic acid group, a sulfonate group, a phosphate group, a thiol group, an azide group, and an alkyne group. These functional groups can be used to attach the lipophilic group to A1 or A2.

[0256] In another embodiment, the oligomeric compound provided, the lipophilic group is selected from the group derived from the fatty acid of dicarboxylic acid. In another embodiment, the fatty acid comprises any saturated or unsaturated fatty acid with a hydrocarbon chain of 4 to 28 carbon atoms, and may contain one or two carboxyl groups. In one embodiment, the lipophilic group is a group derived from a saturated fatty acid with a hydrocarbon chain of 8 to 24 carbon atoms. In certain embodiments, the lipophilic group is a group derived from a saturated fatty acid, wherein the fatty acid is selected from the group consisting of butanoic acid (CH3(CH2)2COOH), pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, heptacosanoic acid and octacosanoic acid (CH3(CH2)2COOH) 16COOH). In another embodiment, the lipophilic group is a group derived from an unsaturated fatty acid, wherein the unsaturated fatty acid is one of the fatty acids presented in Table 1; the fatty acid is preferably selected from the group consisting of: myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, trans-vaccenic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid, and erucic acid.

[0257] In another embodiment, the oligomeric compound provided is a lipophilic group that is a steroid, such as a sterol. Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentaphenanthrene ring system. Steroids include, but are not limited to, bile acids (e.g., cholic acid, deoxycholic acid, and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cationic steroids, such as cortisone. "Cholesterol derivatives" refer to compounds derived from cholesterol, for example, by substitution, addition, or removal of substituents.

[0258] In certain embodiments, in the oligomeric compounds provided, the lipophilic group is selected from cholesterol, vitamin E (tocopherol), or bile acid.

[0259] In certain embodiments, the oligomeric compounds provided, the lipophilic group is selected from the unsaturated fatty acids docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), the saturated fatty acid docosanoic acid (DCA), sterol cholesterol (bile), tocopheryl succinate (TS) and lithocholic acid (LA), retinoic acid (vitamin A acid).

[0260] In another embodiment, the lipophilic group is an aromatic moiety.

[0261] In certain embodiments, the oligomeric compounds provided herein may be directly linked to A1 or A2 via a covalent bond. In certain embodiments, the combination of A1 and T3 and / or the combination of A2 and T4 may include, but are not limited to, the following structures: in represents a single bond or a double bond, p or q independently represents an integer of 5-25, and the sum of p and q does not exceed 25. In some specific embodiments, the combination of A1 and T3 and / or the combination of A2 and T4 include but are not limited to the following examples:

[0262] It is understood that any lipophilic group, linker, and linkage to the bicyclic abasic compound defined in the above-described bicyclic abasic compound, and the properties or structures described therein are also applicable to the definition scope of the lipophilic group, linker, and linkage method thereof in the oligomeric compound in this article.

[0263] In certain embodiments, in the provided oligomeric compounds, the lipophilic group can also be covalently linked to A1 or A2 via a linker, and the linker can be a combination of one or more. As used herein, the linker through which the lipophilic group of the provided oligomeric compound is linked has the same lipophilic group linker as the bicyclic abasic nucleic acid analog compound used above, and the properties or structures described therein are also applicable to the lipophilic group linker of the oligomeric compound herein.

[0264] One of T3 and T4 of the present invention is a lipophilic group, which further comprises a linker and is covalently linked to A1 or A2 through one or more linkers. Non-limiting examples: In one embodiment, the linker is selected from the group consisting of: an amide bond, a phosphatidylcholine, a hydrocarbon linker or a polyethylene glycol (PEG) linker, an amino-alkyl-alcohol, hydroxyproline, hydroxyprolinol, an amino-alkyl-thiophosphate linker, an amino-PEG-thiophosphate linker, an α-carboxylate-amino-alkylthiophosphate linker, and an α-carboxylate-amino-PEG-thiophosphate linker. In some embodiments, the linker contains an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide bond, a product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0265] In some embodiments, the linker comprises a cleavable group. The linker includes a cleavable group, which may be pH-sensitive, a redox-cleavable linker, an esterase-cleavable linker (e.g., an ester group), a phosphatase-cleavable linker (e.g., a phosphate), or a peptidase-cleavable linker (e.g., a peptide bond), a bio-cleavable linker, a hydroxyproline, a hydroxyproline, an amino-alkyl-alcohol-linked linker, and more discussion of lipophilic groups and linkers can be found in PCT Publication No. WO2019 / 217459, entitled "Extrahepatic Delivery," filed on July 7, 2019, and PCT Publication No. WO2019 / 215333, entitled "Oligonucleotide Conjugates Including 7'-5'-α-Anomeric Bicyclic Sugar Nucleosides," filed on May 10, 2019, and Nucleic Acids Research, 2019, Vol. 47, No. 31082–1096, "Various Lipid Conjugates for Functional Extrahepatic In Vivo Nucleotide Delivery," the contents of which are also incorporated by reference in their entirety into the oligomeric compounds herein.

[0266] It is understood that when compound T1 or T2 herein is a lipophilic group, the compounds described herein and applied to the field of oligonucleotides, as is well known in the art, can be applied to extrahepatic delivery systems according to the properties or structures of the lipophilic group, for example but not limited to the field of CNS, ophthalmology, muscle. Due to the blood-brain barrier (BBB) ​​that free oligonucleotides cannot pass through, the delivery of oligonucleotides to the central nervous system (CNS) causes specific problems. One way to deliver oligonucleotides to the CNS is by intrathecal delivery. However, oligonucleotides also need to be effectively internalized into the target cells of the CNS to achieve the desired therapeutic effect. Previous studies typically use lipophilic groups to conjugate specific positions in oligonucleotides such as single core bases, 2-position sugar rings, etc., to help oligonucleotides be internalized into cells of neuronal origin within the cell, and herein, lipophilic groups are conjugated to monomers herein and applied to oligomeric compounds, which can also be used to initiate the delivery and distribution of CNS fields. Lipophilic diseases for which the drug has been widely used in the CNS field include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar diseases, prion diseases, and Lafora disease.

[0267] The lipid groups are used in the application of oligonucleotides in the CNS field for corresponding targets: they target APP for early-onset familial Alzheimer's disease, ATXN2 for spinocerebellar ataxia 2 and ALS, and C9orf72 for amyotrophic lateral sclerosis and frontotemporal dementia; they target TARDBP for ALS, MAPT (Tau) for frontotemporal dementia and HTT for Huntington's disease; they target SNCA for Parkinson's disease, FUS for ALS, ATXN3 for spinocerebellar ataxia 3, ATXN1 for SCA1, genes for SCA7 and SCA8, ATN1 for DRPLA, MeCP2 for XLMR, prion diseases, recessive CNS disorders: PRNP for Lafora disease, DMPK for DM1 (CNS and skeletal muscle), and TTR for hATTR (CNS, eyes and systemic).

[0268] Lipid groups are used in oligonucleotides for the treatment of diseases in the field of ophthalmology, including but not limited to age-related macular degeneration (AMD) (wet and dry), birdshot chorioretinopathy, dominant pigmentary vision, retinitis, Fuch's dystrophy, hereditary and sporadic glaucoma, and Stargardt's disease. For example, VEGF targets wet (or exudative) AMD, C3 and CFB target dry (or non-exudative) AMD, MYOC, ROCK2, ADRB2, and CA2 target glaucoma, CRYGC targets cataracts, and PPP3C targets dry eye syndrome.

[0269] In certain embodiments, oligomeric compounds are provided wherein one of T3 and T4 is hydrogen and the other of T3 and T4 is an internucleoside linker that connects the monomer of Formula III or a stereoisomer thereof to the 5'-terminal and / or 3'-terminal group of the oligomeric compound.

[0270] In some embodiments, the oligomeric compound further comprises a targeting group. In some embodiments, the oligomeric compound, one of T3 and T4 is a covalent bond, and the oligomeric compound is covalently bonded to the targeting group via one of T3 and T4 in formula (III) or its stereoisomers. represents a monomeric portion of formula (III); in other embodiments, an oligomeric compound, the oligomeric compound being linked to the 5′-terminal and / or 3′-terminal nucleotides at one end of an oligomeric compound containing formula (III) or a stereoisomer thereof, The targeting group may comprise a ligand commonly used in the field of siRNA administration. As used herein, the oligomeric compound targeting group provided herein comprises a bicyclic abasic nucleic acid analog compound targeting group as described above, and the properties or structures described therein are also applicable to the targeting group of the oligomeric compound herein. The targeting group may alter the distribution, targeting, or lifespan of the siRNA agent into which it is incorporated, as well as its endosomal solubility properties, improved transport, hybridization, and specificity. Examples include the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated by reference into the oligomeric compound herein.

[0271] In some embodiments, the oligomeric compound, the targeting group can be selected from one or more of the following ligands formed by targeting molecules or their derivatives; such as polymers, such as polyethylene glycol; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); receptor ligands expressed by hepatocytes, such as asialoglycoproteins, asialoglycosidic residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as epinephrine), growth factors, transferrin and other aptamers; antibodies; quantum dots; polypeptides, such as membrane-permeable peptides, or small molecule ligands.

[0272] In some embodiments, in the oligomeric compound, at least one or each of the targeting groups is selected from a ligand that can bind to a receptor on the surface of a mammalian hepatocyte. In some embodiments, each of the targeting groups is independently a ligand that has affinity for an asialoglycoprotein receptor (ASGPR) on the surface of a mammalian hepatocyte. In some embodiments, each of the targeting groups is independently a ligand that comprises an asialoglycoprotein or a sugar. In some embodiments, each of the targeting groups is independently a ligand that comprises an asialoglycoprotein, such as an asialo serum globulin (ASOR) or an asialo globulin (ASF). In some embodiments, each of the targeting groups independently comprises a D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, Sugar, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine , 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3, A ligand selected from the group consisting of 4-tri-O-acetyl-1-thio-6O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside heptanoside ethyl ester, 2,5-anhydro-D-allosenitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

[0273] In some embodiments, in the oligomeric compound, at least one or each targeting group is a ligand comprising galactose or N-acetylgalactosamine.

[0274] In certain embodiments, in the oligomeric compound, the targeting group is linked to one of A1 or A2 and is selected from one of the following compound fragments:

[0275] The targeting group can be connected to one of A1 or A2 via a suitable conjugated linker, and those skilled in the art can select a suitable conjugated linker according to the specific type of the targeting group. The type of these conjugated linkers, ligands, tethers, branched linkers, and the connection mode of the targeting group and oligonucleotide can be found in the disclosures of WO2009073809, WO2014179620, WO2014179627, and WO2015006740A2, which are incorporated herein by reference. In some embodiments, the targeting group can be connected by acid-labile or reducible chemical bonds, which are degradable in the acidic environment of the endosome.

[0276] In certain embodiments, the oligomeric compound further comprises one or more targeting groups, which are linked to the oligomeric compound via a T3 covalent bond, or to the other terminal nucleotide of the oligomeric compound. Non-exemplary linkages are shown below: T3′ is a covalent bond, and Olig, A1, A2, T3, R1 and n are as described above.

[0277] The oligomeric compounds described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations. These configurations can be defined in terms of absolute stereochemistry as (R) or (S), α or β, such as for sugar anomers, or as (D) or (L), such as for amino acids, etc. All such possible isomers, as well as their racemic and optionally pure forms, are included in the oligomeric compounds provided herein.

[0278] In certain embodiments, provided oligomeric compounds comprise at least one 5′-terminal and / or 3′-terminal monomer having a configuration represented by formula (IVa) or formula (IVb):

[0279] A1, A2, T3, T4, R1 and n are as described above.

[0280] In some preferred embodiments, A1 and A2 are independently selected from: O; one of T3 and T4 is H, a protecting group, a lipophilic group, and an optional covalent bond, the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (IVa) or formula (IVb) to the oligomeric compound; n is 0; the oligomeric compound optionally further comprises a targeting group.

[0281] In some more preferred embodiments, A1 and A2 are independently selected from: O; one of T3 and T4 is H, a hydroxyl protecting group, a lipophilic group, and optionally a covalent bond, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects the formula (IVa) or formula (IVb) to the oligomeric compound; n is 0; the oligomeric compound optionally further comprises a targeting group. In some more preferred embodiments, wherein the hydroxyl protecting group is selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and 4,4'-dimethoxytrityl, and the lipophilic group is selected from C 14 、C 16 or C 18 The straight-linked alkane, the internucleoside linking group is selected from an alkylphosphonate linking group, a phosphodiester internucleoside linking group or a thiophosphate internucleoside linking group.

[0282] In certain embodiments, provided oligomeric compounds comprise at least one 5′-terminal and / or 3′-terminal monomer having a configuration represented by formula (IVa-1) or formula (IVb-1):

[0283] A1, A2, T3, T4, R1 and n are as described above.

[0284] In some preferred embodiments, A1 and A2 are independently selected from: O; one of T3 and T4 is H, a protecting group, a lipophilic group, and an optional covalent bond, the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (IVa-1) or formula (IVb-1) to the oligomeric compound; n is 0; the oligomeric compound optionally further comprises a targeting group.

[0285] In some more preferred embodiments, A1 and A2 are independently selected from: O; one of T3 and T4 is H, a hydroxyl protecting group, a lipophilic group, and optionally a covalent bond, the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (IVa-1) or formula (IVb-1) to the oligomeric compound; n is 0; the oligomeric compound optionally further comprises a targeting group. In some more preferred embodiments, the hydroxyl protecting group is selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and 4,4'-dimethoxytrityl, and the lipophilic group is selected from C 14 、C 16 or C 18The straight-linked alkane, the internucleoside linking group is selected from an alkylphosphonate linking group, a phosphodiester internucleoside linking group or a thiophosphate internucleoside linking group.

[0286] In certain embodiments, oligomeric compounds provided herein include, but are not limited to, those having the following configurations:

[0287] Wherein Olig represents the oligonucleotide portion that connects the monomer to the 5′-terminus and / or 3′-terminus of the oligomeric compound, respectively; A1, A2, R1 and n are as described above; T3 is H, a protecting group, a lipophilic group, and an optional covalent bond; the lipophilic group is optionally connected to one of A1 or A2 via a linker; and the oligomeric compound optionally further comprises a targeting group.

[0288] In certain embodiments, in the oligomeric compound provided, one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is a monomer included in the oligomeric compound as provided above, connected to the 5'-terminal internucleoside linking group of the oligomeric compound. It is understood that such an oligomeric compound comprises at least one 5'-terminal monomer as provided herein (such as Formula (III) or its stereoisomers, Formula (IVa), Formula (IVb), (IVa-1) or (IVb-1)).

[0289] In certain embodiments, in the oligomeric compound provided, one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is a monomer included in the oligomeric compound as provided above, connected to the internucleoside linker of the oligomeric compound 3'-terminus. It is understood that such an oligomeric compound comprises at least one monomer having a 3'-terminal monomer as provided herein (such as Formula (III) or its stereoisomers, Formula (IVa), Formula (IVb), (Iva-1) or (IVb-1)).

[0290] In certain embodiments, in the oligomeric compound provided, one of T3 and T4 is H, a protecting group, a lipophilic group, and an optional covalent bond, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 independently connects a same or different monomer contained in the oligomeric compound provided above to the internucleoside linking group at the 5′-terminus and 3′-terminus of the oligomeric compound. It is understood that such an oligomeric compound comprises at least one same or different monomer having a structure as provided herein (such as Formula (III) or its stereoisomers, Formula (IVa), Formula (IVb), (IVa-1) or (IVb-1)) at the 5′-terminus and 3′-terminus of the oligomeric compound, respectively.

[0291] In general, the oligomeric compound comprises one identical or different monomer region represented by formula (III) or its stereoisomer, formula (IVa), formula (IVb), (IVa-1) or (IVb-1) at either end of the 5′-terminus and / or the 3′-terminus. In other cases, the oligomeric compound comprises at least two consecutive identical or different monomer regions represented by formula (III) or its stereoisomer, formula (IVa), formula (IVb), (IVa-1) or (IVb-1) at either end of the 5′-terminus and / or the 3′-terminus of the oligomeric compound.

[0292] By way of example and not limitation, the monomers herein are linked to the 5′-terminus and / or 3′-terminus of the oligomeric compound. A2 and A2' represent the same or different monomer variable groups as described above, R1 and R1' represent the same or different monomer variable groups as described above, and Olig represents the oligonucleotide portion that connects the monomer to the 5'-end and / or 3'-end of the oligomeric compound, respectively.

[0293] In certain embodiments, when n is non-zero, it indicates that there are 1-6 independent R1 group substituents on the bicyclic structure. It is understood that these substituents in the bicyclic abasic compounds herein do not include natural nucleobases or modified nucleobases. In certain embodiments, R1 is independently selected from halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl. More preferably, R1 is independently selected from fluorine, chlorine, bromine, iodine, methyl, ethyl, isopropyl, propyl, 3-propenyl, ethynyl, 3-propynyl.

[0294] In certain embodiments, n is 0, indicating that there is no R1 group substitution on the ring structure.

[0295] Any combination of the features of two or more such oligomeric compounds provided by the present invention is also included in the scope of the present invention if such features are not contradictory. Further, the bicyclic base compounds described in the present invention, such as formula (I), (IIa), (IIb), (IIa-1), (IIb-1) and any combination of the features of two or more such compounds can be applied to 5'-terminal and / or 3'-terminal oligonucleotide monomers. Therefore, the technical features defined for the bicyclic abasic compounds described herein are also applicable to the oligomeric compounds herein, as long as there is no contradiction.

[0296] In certain embodiments, oligomeric compounds include fragments comprising at least one monomer as shown below:

[0297] In certain embodiments, the oligomeric compounds provided herein have the following specific structure:

[0298] , wherein Olig represents an oligonucleotide portion that connects the monomer to the 5′-terminus and / or 3′-terminus of the oligomeric compound, respectively.

[0299] In certain embodiments, the oligomeric compound is a single-stranded oligonucleotide, such as antisense oligonucleotides (also known as ASOs), ribozymes, or aptamers, as are well known in the art. In certain embodiments, the oligomeric compound is a double-stranded RNAi compound (siRNA), such as double-stranded nucleoside nucleic acids (also known as dsRNA), wherein one or both strands are oligomeric compounds as disclosed herein.

[0300] In certain embodiments, at least a portion of the nucleobase sequence of the oligomeric compound is complementary or fully complementary to a portion of the target nucleic acid. In certain embodiments, it is 100% complementary to the target nucleic acid. In certain embodiments, it is 90% complementary to the target nucleic acid. In certain embodiments, it is 80% complementary to the target nucleic acid. In certain embodiments, it is 90% complementary to the target nucleic acid. In certain embodiments, it is 70% complementary to the target nucleic acid. In certain embodiments, it is 90% complementary to the target nucleic acid. In certain embodiments, it is 60% complementary to the target nucleic acid.

[0301] In certain embodiments, double-stranded ribonucleic acid (dsRNA) agents are provided, each comprising: a sense strand and an antisense strand, wherein the sense strand is fully or partially complementary to the antisense strand, and the antisense strand is complementary to a nucleic acid target gene; at least one of the sense strand and the antisense strand is an oligomeric compound as provided above, at least one of which has a 5′-terminal and / or 3′-terminal monomer comprising Formula (III) or its stereoisomers, Formula (IVa), Formula IV(b), Formula (IVa-1) or Formula (IVb-1) herein; and wherein the double-stranded ribonucleic acid (dsRNA) agent optionally further comprises an independent targeting group.

[0302] In certain embodiments, double-stranded ribonucleic acid (dsRNA) agents are provided, wherein the sense strand is an oligomeric compound provided herein comprising a 5′-terminal monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula IV(b), Formula (IVa-1), or Formula (IVb-1). In certain embodiments, double-stranded ribonucleic acid (dsRNA) agents are provided, wherein the sense strand is an oligomeric compound provided herein comprising a 3′-terminal monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula IV(b), Formula (IVa-1), or Formula (IVb-1). In certain embodiments, double-stranded ribonucleic acid (dsRNA) agents are provided, wherein the sense strand is an oligomeric compound provided herein comprising a 5′-terminal and 3′-terminal monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula IV(b), Formula (IVa-1), or Formula (IVb-1).

[0303] In certain embodiments, double-stranded ribonucleic acid (dsRNA) agents are provided, wherein the antisense strand is an oligomeric compound comprising a 5′-terminal and / or 3′-terminal monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula IV (b), Formula (IVa-1), or Formula (IVb-1) herein. In certain embodiments, double-stranded ribonucleic acid (dsRNA) agents are provided, wherein the antisense strand is an oligomeric compound comprising a 3′-terminal monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula IV (b), Formula (IVa-1), or Formula (IVb-1) herein. In certain embodiments, double-stranded ribonucleic acid (dsRNA) agents are provided, wherein the antisense strand is an oligomeric compound comprising a 5′-terminal monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula IV (b), Formula (IVa-1), or Formula (IVb-1) herein.

[0304] In certain embodiments, a double-stranded ribonucleic acid (dsRNA) agent is provided, optionally further comprising an independent targeting group. The independent targeting group can be understood to be, in addition to the targeting group present in the oligomeric compound monomer herein, The monomer portion of formula (III) may also have a targeting group at other positions in the double-stranded ribonucleic acid (dsRNA) agent. In certain embodiments, the double-stranded ribonucleic acid (dsRNA) agent further comprises one or more targeting groups or linking groups on the 5′-terminal and / or 3′-terminal nucleotides. It is understood that these targeting groups can be conjugated to the 5′-terminal and / or 3′-terminal nucleotides of any chain in the double-stranded ribonucleic acid (dsRNA) agent. The targeting group may be a ligand commonly used in the field of siRNA administration. As used herein, these targeting groups may be independently selected from the same targeting groups as those described above for the oligomeric compounds and bicyclic abasic nucleic acid analog compounds; the properties or structures described for the double-stranded ribonucleic acid (dsRNA) reagents provided are also applicable to the targeting groups of the oligomeric compounds and bicyclic abasic nucleic acid analog compounds herein.

[0305] In some embodiments, in the double-stranded ribonucleic acid (dsRNA) reagent provided, the targeting group can be selected from one or more ligands formed by the following targeting molecules or their derivatives: polymers, carbohydrates, receptor ligands expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands.

[0306] In some embodiments, in the double-stranded ribonucleic acid (dsRNA) reagent provided, at least one or each targeting group is selected from a ligand that can bind to a receptor on the surface of a mammalian hepatocyte.

[0307] In some embodiments, in the double-stranded RNA (dsRNA) reagent provided, each of the targeting groups independently comprises a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes.

[0308] In some embodiments, in the provided double-stranded RNA (dsRNA) reagents, each of the targeting groups independently comprises an asialoglycoprotein or sugar. The targeting group can be linked to the 5′-terminal and / or 3′-terminal nucleotide via a suitable linker. Those skilled in the art can select a suitable conjugation linker based on the specific type of targeting group.

[0309] In some embodiments, the dsRNA agent wherein the one or more targeting groups or linking groups are conjugated to the sense strand. The targeting ligand can alter the distribution, targeting or lifespan, endosomal solubility properties, improved transport, hybridization and specificity properties of the dsRNA agent into which it is incorporated, such as, but not limited to, lectins, glycoproteins, lipids or proteins, thyroid stimulating hormone, melanocyte stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent trehalose, glycosylated polyamino acids , polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an aptamer, other examples include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., such as phenazine, dihydrophenazine), artificial endonucleases or a chelating agent (such as EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, camphenol, menthol, 1,3-propylene glycol, heptadecanol, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine) and peptide conjugates (such as antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino groups, alkyl groups, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), delivery / adsorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl groups, HRP, or AP. In some embodiments, the targeting group or linking group comprises a group selected from: N-acetyl-galactosamine (GalNAc), a lipophilic molecule.

[0310] In certain embodiments, the targeting group is selected from one of the following compounds after attachment:

[0311] It is understood that the targeting group herein can be conjugated to the monomer compound or oligonucleotide herein through a reactive functional group to form an internucleoside linking group. For example, GLO generally indicates an oxophosphate internucleoside linking group, while GLS generally indicates a thiophosphate internucleoside linking group.

[0312] In certain embodiments, the present invention provides oligomeric compounds comprising any of a variety of length ranges. In certain embodiments, in an oligomeric compound or double-stranded ribonucleic acid (dsRNA), any one of the single-stranded, sense, and / or antisense strands comprises 8-40 nucleotides in length, respectively. For example, in certain embodiments, the oligomeric compound nucleotide length is selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 9, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 1 6, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30,15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 29, 27 to 30, 28 to 29, 28 to 30, or 9 to 30 linked nucleosides. For example, an oligonucleotide comprising 8-30 nucleosides does not include an oligonucleotide having 31 nucleosides, but unless otherwise specified, such an oligonucleotide may further comprise, for example, one or more conjugates, terminal groups, or other substituents, and monomers herein are generally considered to be terminal groups further comprising oligonucleotides. In certain embodiments, terminal groups include, but are not limited to, terminal group nucleosides. In such embodiments, the terminal groups are modified differently from the terminal nucleosides of the oligonucleotides to distinguish such terminal groups from the nucleosides of the oligonucleotides.

[0313] In certain embodiments, the present invention provides oligomeric compounds that, in addition to providing monomeric compounds, have one or more modifications in the remainder. Such modifications include, but are not limited to, sugar modifications or substitutions with sugar surrogates, base modifications, internucleoside linkage modifications, and motifs. Such modifications are described herein and many of them are known to those skilled in the art. All such modifications are suitable for the oligomeric compounds and double-stranded RNAs disclosed herein.

[0314] In certain embodiments, a method for inhibiting gene expression is provided, the method comprising: contacting a cell with an oligomeric compound or double-stranded ribonucleic acid (dsRNA) reagent provided above, wherein the sense strand and the antisense strand in the oligomeric compound and the double-stranded ribonucleic acid (dsRNA) reagent each comprise 8-40 nucleotides in length, and the antisense strand in the oligomeric compound or double-stranded ribonucleic acid (dsRNA) reagent is complementary to the target RNA. In certain embodiments, the cell is in an animal. In certain embodiments, the cell is in a human. In certain embodiments, the target RNA is selected from mRNA, pre-mRNA, and microRNA. In certain embodiments, the target RNA is mRNA. In certain embodiments, the target RNA is human mRNA. In certain embodiments, the target RNA is cleaved, thereby inhibiting its function. In certain embodiments, the method further comprises detecting the level of the target RNA. In certain embodiments, a method of inhibiting gene expression is provided, the method comprising contacting one or more cells or tissues with an oligomeric compound or double-stranded ribonucleic acid (dsRNA) comprising a terminal monomer having Formula (III) or its stereoisomers, Formula (IVa), Formula IV(b), Formula (IVa-1) or Formula (IVb-1).

[0315] The term "oligomeric compound" in the context of the present invention refers to a polymer having at least one region that can hybridize with a nucleic acid molecule. The term "oligomeric compound" includes oligonucleotides, oligonucleotide analogs and oligonucleosides and nucleotide mimics and / or mixed polymers comprising nucleic acid and non-nucleic acid components. The term "oligomeric compound" also includes polymers comprising connected monomeric subunits, wherein the monomeric subunits include nucleosides, modified nucleosides, nucleoside analogs, nucleoside mimics and non-nucleic acid components such as coupling groups. In certain embodiments, a mixture of monomeric subunits (such as, but not limited to, those listed) provides the following oligomeric compounds, which have enhanced properties for applications such as treatment and diagnosis. The bicyclic abasic compounds provided by the present invention are classified as non-nucleic acid components due to the absence of nucleobases. The monomeric subunits can be connected by naturally occurring phosphodiester internucleoside linkages or by any multiple internucleoside linkages disclosed herein, internucleoside linkages such as, but not limited to, thiophosphate internucleoside linkages or mixtures thereof.

[0316] Oligonucleotide: As used herein, the term "oligonucleotide" as used herein refers to a polymeric form of nucleotides ranging from 2 to 2500 nucleotides, which can be single-stranded or double-stranded. The term "oligonucleotide" includes oligonucleotides composed of naturally occurring nucleobases, sugars, and covalent internucleoside linkages, as well as oligonucleotides having one or more non-naturally occurring portions. Such non-naturally occurring oligonucleotides are often more desirable than naturally occurring forms because they have desirable properties, such as improved cellular uptake, enhanced affinity for nucleic acid targets, and increased stability in the presence of nucleases, such as modifications to the base or sugar moieties. In certain embodiments, the oligonucleotide has 500-1500 nucleotides, typically such as where the oligonucleotide is used in gene therapy. In certain embodiments, the oligonucleotide is single-stranded or double-stranded and has 7-100 nucleotides. In certain embodiments, the oligonucleotide is single-stranded or double-stranded and has 15-100 nucleotides. In another embodiment, the oligonucleotide is single-stranded or double-stranded and has 15-50 nucleotides, typically such as where the oligonucleotide is a nucleic acid inhibitor molecule. In another embodiment, the oligonucleotide is single-stranded or double-stranded and has 25-40 nucleotides, typically such as where the oligonucleotide is a nucleic acid inhibitor molecule. In another embodiment, the oligonucleotide is single-stranded or double-stranded and has 19-40 or 19-25 nucleotides, typically such as where the oligonucleotide is a double-stranded nucleic acid inhibitor molecule and forms a duplex of at least 18-25 base pairs. In other embodiments, the oligonucleotide is single-stranded and has 15-25 nucleotides, typically such as where the oligonucleotide nucleotide is a single-stranded or double-stranded RNAi inhibitor molecule. Typically, the oligonucleotide has one or more phosphorus-containing internucleotide linking groups, as described herein. In other embodiments, the internucleotide linking group is a phosphorus-free bond, as described herein. The oligomeric compounds herein are actually oligonucleotides that further comprise terminal monomers as described herein.

[0317] Typically, oligomeric compounds comprise a backbone of linked monomeric subunits, wherein the linkages linking the monomeric subunits, the sugar moieties or surrogates, and the heterocyclic base moieties can be independently modified. The linked sugar units, which may or may not contain heterocyclic bases, can be replaced with mimetics such as peptide nucleic acid monomers. The ability to modify or replace some or all of the monomers in each oligomeric compound allows for a large number of possible motifs.

[0318] Oligomeric compounds are typically prepared using conventional linear methods, but may also be ligated or otherwise prepared into a truncated form and may also contain branches. Oligomeric compounds may form double-stranded constructs, for example, by hybridizing to form double strands of a double-stranded composition. The double-stranded composition may be ligated or separated and may contain overhangs at the ends.

[0319] As is known in the art, the term "nucleoside" in the context of the present invention refers to a base sugar combination. The base portion of a nucleoside is called a core base, typically a heterocyclic base portion. The two most common categories of the heterocyclic base are purines and pyrimidines, for example: suitable natural core bases include purine bases and pyrimidine bases, such as adenine (A), thymine (T), cytosine (C), guanine (G) or uracil (U). Suitable modified core bases include diaminopurine and its derivatives, alkylated purines or pyrimidines, acylated purines or pyrimidines, thiolated purines or pyrimidines, etc. It is understood that herein there is a bicyclic abasic nucleic acid analog with a 5'-terminal and / or 3'-terminal monomer, and its bicyclic substituent R1 does not exist in the base of such a nucleoside.

[0320] As known in the art, term " nucleotide " in the context of the present invention refers to the nucleoside that further comprises the phosphate group that is covalently connected to the nucleoside sugar moiety.For the nucleoside that has comprised pentofuranosyl, this phosphate group can be coupled to 2 ', 3 ' or 5 ' hydroxyl moiety of sugar.When forming oligonucleotide, this phosphate group covalently connects the nucleoside adjacent to each other to form linear polymeric compound.Can connect the corresponding end of this linear polymeric structure to obtain cyclic structure by hybridization or by forming covalent bond, yet, usually wish single open linear structure.In oligonucleotide structure, phosphate group usually forms the nucleoside of oligonucleotide and connects the common nucleoside of RNA and DNA and is connected for 3 ' to 5 ' phosphodiester connection.

[0321] As used herein, "terminal group" refers to one or more atoms or groups attached to one or both of the 3' end or the 5' end of an oligonucleotide. In certain embodiments, the terminal group is a conjugated group. In certain embodiments, the terminal group comprises one or more additional nucleosides, commonly referred to as the 3' end or the 5' end-terminal nucleotide. As used herein, the term "5'-terminal nucleotide or 3'-terminal nucleotide" refers to a nucleotide located at the 5'-end or the 3'-end of an oligonucleotide or oligomeric compound.

[0322] As used herein, the term "phosphoramidite" refers to a nitrogen-containing trivalent phosphorus derivative. Examples of suitable phosphoramidites are described herein.

[0323] The term "siRNA" as used herein refers to short interfering RNA or silencing RNA. siRNA is a type of double-stranded RNA molecule that can be 20 to 25 (or shorter) base pairs in length, similar to microRNAs (miRNAs) that function in the RNA interference (RNAi) pathway. siRNA interferes with the expression of specific genes with nucleotide sequences complementary to the siRNA by degrading mRNA after transcription, thereby preventing translation. siRNA silences gene expression in cells by inducing RNA-induced silencing complex (RISC) to cut messenger RNA (mRNA).

[0324] As used herein, unless otherwise indicated or amended, the term "double-stranded" refers to two separate oligomeric compounds that hybridize to each other. Such double-stranded compounds may have one or more non-hybridizing nucleosides (overhangs) and / or one or more internal non-hybridizing nucleosides (mismatches) at one or both ends of one or both strands, provided that there is sufficient complementarity to maintain hybridization under physiologically relevant conditions.

[0325] As used herein, the term "self-complementary" or "hairpin" refers to a single oligomeric compound comprising a duplex region formed by self-hybridization of the oligomeric compound.

[0326] As used herein, the term "single-stranded" refers to an oligomeric compound that is not hybridized to its complementary sequence and does not have sufficient self-complementarity to form a hairpin structure under physiologically relevant conditions. A single-stranded compound may be capable of binding to its complementary sequence thereby becoming a double-stranded or partially double-stranded compound.

[0327] Therapeutic formulations of dsRNA agents or target gene antisense polynucleotide agents can be prepared for storage by mixing the molecule or compound having the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers [Remington's Pharmaceutical Sciences 21st edition, (2006)] in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the doses and concentrations employed and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as or polyethylene glycol (PEG).

[0328] Dosage

[0329] In some embodiments, the tissue to which the compound is administered is a tissue in which a target gene-related disease or condition is present or may occur, a non-limiting example of which is the liver or kidney. Direct tissue administration can be achieved by direct injection or other means. Many compounds delivered orally naturally enter and pass through the liver and kidney, and some embodiments of the therapeutic methods of the present invention include oral administration of one or more target gene dsRNA reagents to the subject. dsRNA reagents or target gene antisense polynucleotide agents, alone or in combination with other therapeutic agents, can be administered once, or they can be administered multiple times. If administered multiple times, target gene dsRNA reagents or target gene antisense polynucleotide agents can be administered by different routes. For example, although not intended to limit, the first (or first several) administration can be performed subcutaneously, and one or more additional administrations can be oral and / or systemic administration.

[0330] For embodiments of the present invention in which systemic administration of a target gene dsRNA agent or a target gene antisense polynucleotide agent is desired, the target gene dsRNA agent or the target gene antisense polynucleotide agent can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable formulations can be in unit dosage form, such as ampoules or multi-dose containers, with or without preservatives. Target gene dsRNA agent formulations (also referred to as pharmaceutical compositions) can be in the form of suspensions, solutions, or emulsions in oily or aqueous carriers, and can contain preparatants, such as suspending agents, stabilizers, and / or dispersants.

[0331] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose solution, glucose and sodium chloride solution, lactated Ringer's solution or fixed oils. Intravenous excipients include fluid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose solution), etc. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, etc., may also be present. Other forms of administration, such as intravenous administration, will result in lower dosages. If the subject's response under the initial dose is insufficient, a higher dosage (or effectively increasing the dosage by different, more localized delivery routes) can be adopted within the scope allowed by patient tolerance. Multiple doses per day can be used as needed to achieve appropriate systemic or local levels of one or more target gene dsRNA agents or target gene antisense polynucleotide agents and to achieve appropriate reduction in target gene activity.

[0332] In other embodiments, the methods of the present invention include the use of a delivery vehicle, such as a biocompatible microparticle, nanoparticle, or implant suitable for implantation into a recipient, such as a subject. Exemplary biodegradable implants that can be used according to this method are described in PCT Publication WO 95 / 24929 (incorporated herein by reference), which describes a biocompatible, biodegradable polymer matrix for containing biomacromolecules.

[0333] Both non-biodegradable and biodegradable polymer matrices can be used in the methods of the present invention to deliver one or more target gene dsRNA agents or target gene antisense polynucleotide agents to a subject. In some embodiments, the matrix can be biodegradable. The matrix polymer can be a natural or synthetic polymer. The polymer can be selected based on the time period of the desired release, typically on the order of a few hours to a year or longer. Typically, release over a period of time between a few hours and three to twelve months can be used. The polymer is optionally in the form of a hydrogel that can absorb up to about 90% of its weight in water and is optionally cross-linked with multivalent ions or other polymers.

[0334] Typically, target gene dsRNA reagent or target gene antisense polynucleotide reagent can be delivered by diffusion or by degradation of a polymer matrix using a biodegradable implant in some embodiments of the present invention. Exemplary synthetic polymers for such purposes are well known in the art. Using methods known in the art, biodegradable polymers and non-biodegradable polymers can be used for delivering target gene dsRNA reagent or target gene antisense polynucleotide reagent. Bioadhesive polymers such as bioerodible hydrogels (HS Sawhney, CP Pathak and JA Hubell in Macromolecules, 1993, 26, 581-587) can also be used for delivering target gene sRNA reagent or target gene antisense polynucleotide reagent to treat target gene related diseases or illness. Other suitable delivery systems can include timed release, delayed release or sustained release delivery systems. Such systems can avoid repeated administration of target gene dsRNA reagent or target gene antisense polynucleotide agent, thereby improving the convenience of object and healthcare professionals. Many types of release delivery systems are available and known to those of ordinary skill in the art. See, for example, U.S. Patent Nos. 5,075,109, 4,452,775, 4,675,189, 5,736,152, 3,854,480, 5,133,974, and 5,407,686. Additionally, pump-based hardware delivery systems may be used, some of which are also suitable for implantation.

[0335] The use of long-term sustained release implants can be applicable to the preventative treatment of objects and objects with the risk of recurrent target gene) related diseases or conditions. As used herein, long-term release refers to that implants are constructed and arranged to deliver target gene dsRNA reagents or target gene antisense polynucleotide reagents at least up to 10 days, 20 days, 30 days, 60 days, 90 days, six months, one year or longer treatment levels. Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the above-mentioned release systems.

[0336] effective dose

[0337] In some aspects, the methods of the present invention include contacting cells with an effective amount of a dsRNA agent or an antisense polynucleotide agent to reduce gene expression in the contacted cells. Certain embodiments of the methods of the present invention include administering a dsRNA agent or an antisense polynucleotide agent to a subject in an amount that effectively reduces gene expression and treats the relevant disease or condition of the subject. In terms of reduced expression and / or for treating relevant diseases or conditions, the "effective amount" used is the amount necessary or sufficient to achieve the desired biological effect. For example, the effective amount of a dsRNA agent or an antisense polynucleotide agent for treating a relevant disease or condition can be: (i) the amount required to slow down or stop the progression of the disease or condition; (ii) reversing, reducing or eliminating one or more symptoms of the disease or condition. In some aspects of the present invention, an effective amount is the amount of a dsRNA agent or an antisense polynucleotide agent that results in a therapeutic response to the prevention and / or treatment of the disease or condition when applied to a subject in need of treating a relevant disease or condition. According to some aspects of the invention, an effective amount is the amount of a dsRNA agent or antisense polynucleotide agent of the invention that, when combined or co-administered with another therapeutic treatment for a related disease or condition, results in a therapeutic response that prevents and / or treats the disease or condition. In some embodiments of the invention, the biological effect of treating a subject with a dsRNA agent or antisense polynucleotide agent of the invention can be an improvement and / or complete elimination of symptoms caused by the related disease or condition. In some embodiments of the invention, the biological effect is the complete elimination of the related disease or condition, as demonstrated, for example, by a diagnostic test indicating that the subject does not have the related disease or condition. In some embodiments, an effective amount is an amount that results in a desired response, such as a reduction in the amount of the related disease or condition in cells, tissues, and / or subjects with the disease or condition. Some embodiments of the invention include methods for determining the efficacy of a target gene dsRNA agent or target gene antisense polynucleotide agent of the invention administered to a subject to treat a target gene-related disease or condition by evaluating and / or monitoring one or more "physiological characteristics" of the target gene-related disease or condition in the subject. Non-limiting examples of physiological characteristics of target gene-related diseases or conditions are that many patients have

[0338] It should be appreciated that gene silencing can be performed constitutively or by genomic engineering in any cell expressing and determined by any suitable assay. In some embodiments of the invention, target gene expression is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% by administering a dsRNA agent of the invention. In some embodiments of the invention, target gene expression is reduced by 5% to 10%, 5% to 25%, 10% to 50%, 10% to 75%, 25% to 75%, 25% to 100% or 50% to 100% by administering a dsRNA agent of the invention.

[0339] In the present invention, dsRNA agents and antisense polynucleotide agents are delivered in a pharmaceutical composition at a dosage sufficient for target gene expression. In certain embodiments of the present invention, the dosage of the dsRNA agent or antisense polynucleotide agent is 0.01 to 200.0 mg per kilogram of recipient body weight per day, generally 1 to 50 mg / kg body weight, 5 to 40 mg / kg body weight, 10 to 30 mg / kg body weight, 1 to 20 mg / kg body weight, 1 to 10 mg / kg body weight, 4 to 15 mg / kg body weight, including end values. For example, each single administration of a dsRNA agent or antisense polynucleotide agent can be at a dosage of from about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2 mg / kg, 2.1 mg / kg, 2.2 mg / kg, or more. / kg, 2.3mg / kg, 2.4mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3.0mg / kg, 3.1mg / kg, 3.2mg / kg, 3.3mg / kg, 3.4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8mg / kg, 3.9mg / kg, 4mg / kg, 4.1mg / kg, 4.2mg / kg, 4.3mg / kg, 4.4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7mg / kg, 4.8mg / kg, 4.9mg / kg, 5mg / kg, 5.1mg / kg, 5.2mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5mg / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg, 5.9mg / kg, 6mg / kg, 6.1mg / kg, 6.2mg / kg, 6.3mg / kg, 6.4mg / kg, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg g, 6.9mg / kg, 7mg / kg, 7.1mg / kg, 7.2mg / kg, 7.3mg / kg, 7.4mg / kg, 7.5mg / kg, 7.6mg / kg, 7.7mg / kg, 7.8mg / kg, 7.9mg / kg, 8mg / kg, 8.1mg / kg, 8.2mg / kg, 8.3mg / kg, 8.4mg / kg, 8.5mg / kg, 8.6mg / kg, 8.7mg / kg, 8.8mg / kg, 8.9mg / kg, 9mg / kg, 9.1mg / kg, 9.2mg / kg, 9.3mg / kg, 9.4mg / kg, 9.5mg / kg, 9.6mg / kg, 9.7mg / kg, 9.8mg / kg, 9.9mg / kg, 10mg / kg, 11mg / kg, 12mg / kg, 13mg / kg , 14mg / kg, 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg, 21mg / kg, 22mg / kg, 23mg / kg, 24mg / kg, 25mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg, 30mg / kg, 31mg / kg, 32mg / kg, 33mg / kg, 34mg / kg, 35mg / kg, 36mg / kg, 37mg / kg, 38mg / kg, 39mg / kg, 40mg / kg, 41mg / kg, 42mg / kg, 43mg / kg, 44mg / kg, 45mg / kg, 46mg / kg, 47mg / kg, 48mg / kg, 49mg / kg to 50mg / kg body weight.

[0340] In determining the delivery dosage and time of the dsRNA reagent of the present invention, various factors can be considered. The absolute amount of the dsRNA reagent or antisense polynucleotide agent delivered will depend on various factors, including co-treatment, dosage number and individual subject parameters, including age, physical condition, size and body weight. These are factors well known to those of ordinary skill in the art and can be solved by routine experiments. In some embodiments, a maximum dose can be used, i.e., the highest safe dose according to reasonable medical judgment.

[0341] In some embodiments, the methods of the present invention may include administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of an agent or antisense polynucleotide agent to a subject. In some cases, a dose of the pharmaceutical compound may be administered to a subject at least daily, every other day, weekly, every other week, monthly, etc., and may be administered once a day or more than once a day, for example, 2, 3, 4, 5 or more times within a 24-hour cycle. The pharmaceutical composition of the present invention may be administered once a day; or the dsRNA agent or antisense polynucleotide agent may be administered in two, three or more sub-doses at appropriate intervals throughout the day, or even using continuous infusion or delivered by a controlled release formulation. In some embodiments of the methods of the present invention, the pharmaceutical composition of the present invention is administered to a subject once a day or more, once a week or more, once a month or more, or once a year or more.

[0342] Certain embodiments of the present invention include the use of pharmaceutical compositions containing dsRNA agents or antisense polynucleotide agents and pharmaceutically acceptable carriers. Pharmaceutical compositions comprising dsRNA agents or antisense polynucleotide agents can be used in the methods of the present invention to reduce gene expression and activity in cells, and can be used to treat related diseases or conditions. Such pharmaceutical compositions can be formulated based on the mode of delivery. Non-limiting examples of formulations for delivery modes are: compositions formulated for subcutaneous delivery, compositions formulated for systemic administration by parenteral delivery, compositions formulated for intravenous (IV) delivery, compositions formulated for intrathecal delivery, compositions formulated for direct delivery to the brain, etc. The pharmaceutical compositions of the present invention can be administered using one or more modes to deliver dsRNA agents or antisense polynucleotide agents to cells, for example: surface (e.g., by transdermal patch); lungs, for example, by inhalation or blowing into powder or aerosol, including by a nebulizer; intra-airway, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, for example, by implantation; or intracranial, for example, by intraparenchymal; intrathecal or intraventricular administration. dsRNA agents or antisense polynucleotide agents can also be delivered directly to target tissues, for example, directly to the liver, directly to the kidneys, etc. It will be understood that "delivering a dsRNA agent" or "antisense polynucleotide agent" to a cell includes delivering a dsRNA agent or antisense polynucleotide agent, expressing the dsRNA agent directly in the cell, and expressing the dsRNA agent from an encoding vector delivered to the cell, or any suitable means for causing the dsRNA or antisense polynucleotide agent to appear in the cell. The preparation and use of the formulations and the means for delivering inhibitory RNA are well known in the art and commonly used.

[0343] In some embodiments, composition also comprises one or more other therapeutic agents. The composition of the present invention can comprise one or more dsRNA reagents and optional one or more pharmaceutically acceptable carriers, delivery agents, targeting agents, detectable labels, etc., and the non-limiting examples of available targeting agents according to some embodiments of the inventive method are reagents that the dsRNA reagents of the present invention are guided to and / or enter cells to be treated. The selection of targeting agents will depend on the following key factors: the nature of the relevant disease or illness, and the target cell type. In a non-limiting example, in some embodiments of the present invention, it may be necessary to target the dsRNA reagent to and / or enter hepatocytes. It should be understood that in some embodiments of the inventive method, therapeutic agent comprises a dsRNA reagent only with a delivery agent, such as a delivery agent comprising N-acetylgalactosamine (GalNAc), without any additional connecting elements. For example, in some aspects of the present invention, a dsRNA reagent can be connected to a delivery compound comprising GalNAc, and is included in a composition containing a pharmaceutically acceptable carrier, and is administered to a cell or object without any detectable label or targeting agent connected to the dsRNA reagent.

[0344] In the case where the dsRNA reagent of the present invention is administered together with one or more delivery agents, targeting agents, labeling agents, etc. and / or is connected thereto, those skilled in the art will be able to understand and be able to select and use suitable reagents for use in the methods of the present invention. In certain methods of the present invention, labeling agents can be used to determine the position of the dsRNA reagent in cells and tissues, and can be used to determine the cell, tissue or organ position of the therapeutic composition comprising the dsRNA reagent administered in the methods of the present invention. The means of attaching and using labeling agents such as enzyme labels, dyes, radioactive labels, etc. are well known in the art. It should be understood that in some embodiments of the compositions and methods of the present invention, the labeling agent is connected to one or both of the sense polynucleotides and antisense polynucleotides contained in the dsRNA reagent.

[0345] In some embodiments, the composition is packaged in a medicine box, container, packaging, dispenser, prefilled syringe or bottle. A medicine box comprising one or more target gene dsRNA reagents and / or target gene antisense polynucleotide reagents and their instructions for use in the methods of the present invention is also within the scope of the present invention. The medicine box of the present invention may include one or more target gene dsRNA reagents, target gene sense polynucleotides and target gene antisense polynucleotide reagents that can be used to treat target gene related diseases or conditions. A medicine box comprising one or more target gene dsRNA reagents, target gene sense polynucleotides and target gene antisense polynucleotide reagents can be prepared for use in the therapeutic methods of the present invention. The components of the medicine box of the present invention can be packaged in aqueous medium or lyophilized form. The medicine box of the present invention can include a carrier that is separated to enclose one or more container devices or a series of container devices (e.g., test tubes, vials, flasks, bottles, syringes, etc.) therein. The first container device or a series of container devices can include one or more compounds, such as target gene) dsRNA reagents and / or target gene sense or antisense polynucleotide reagents. The second container means or series of container means may contain targeting agents, labeling agents, delivery agents, etc., which may be included as part of the target gene (dsRNA) reagents and / or target gene antisense polynucleotides administered in embodiments of the treatment methods of the present invention.

[0346] The kit of the present invention may also include instructions. The instructions are typically in written form and will provide guidance for performing the treatment embodied by the kit and for making decisions based on the treatment.

[0347] Cells, subjects, and controls

[0348] The methods of the present invention can be used in conjunction with cells, tissues, organs, and / or subjects. In some aspects of the present invention, the subject is a human or a vertebrate mammal, including but not limited to dogs, cats, horses, cows, goats, mice, rats, and primates, such as monkeys. Thus, the present invention can be used to treat target gene-related diseases or conditions in both human and non-human subjects.

[0349] In some aspects of the invention, the subject can be a farm animal, a zoo animal, a domesticated animal, or a non-domesticated animal, and the methods of the invention can be used in veterinary prevention and treatment regimens. In some embodiments of the invention, the subject is a human and the methods of the invention can be used in human prevention and treatment regimens.

[0350] Non-limiting examples of subjects to which the present invention can be applied are subjects diagnosed with, suspected of having, or at risk of having a disease or condition associated with higher than desired target gene expression and / or activity, also referred to as "elevated target gene expression levels." Non-limiting examples of diseases and conditions associated with higher than desired target gene expression and / or activity are described elsewhere herein. The methods of the present invention can be applied to subjects who have been diagnosed with the disease or condition at the time of treatment, subjects associated with higher than desired target gene expression and / or activity, or subjects who are believed to be at risk of having or developing a disease or condition associated with higher than desired target gene expression and / or activity. In some aspects of the invention, the disease or condition associated with higher than desired target gene expression and / or activity levels is an acute disease or condition; in certain aspects of the invention, the disease or condition associated with higher than desired target gene expression and / or activity levels is a chronic disease or condition.

[0351] In another non-limiting example, the target gene dsRNA agent of the present invention is administered to treat a disease or disorder caused by or associated with the activation of the target gene, or a disease or disorder whose symptoms or progression respond to the inactivation of the target gene. The term "target gene-associated disease" includes diseases, disorders, or conditions that benefit from reduced expression of the target gene.

[0352] The cell that can be applied the method of the present invention includes in vitro, in vivo, in vitro cell.Cell can be in object, in culture and / or in suspension, or in any other suitable state or condition.The cell that can be applied the method of the present invention can be: liver cell (liver cell), liver cell (hepatocyte), heart cell, pancreatic cell, cardiovascular cell, kidney cell or other types of vertebrate cells, including human and non-human mammalian cells.In some aspects of the present invention, the cell that can be applied the method of the present invention is healthy normal cell, which is not known to be disease cell.In some embodiments of the present invention, the method and composition of the present invention are applied to the cell of liver cell, liver cell, heart cell, pancreatic cell, cardiovascular cell and / or kidney cell.In some aspects of the present invention, control cell is normal cell, but it should be understood that the cell with disease or illness can also be used as control cell in specific cases, for example, when comparing the treated cell with disease or illness and the result of untreated cell with disease or illness etc.

[0353] According to the method of the present invention, the level of target gene polypeptide activity can be determined and compared with a control level of target gene polypeptide activity. The control can be a predetermined value, which can take many forms. It can be a single cutoff value, such as a median or mean value. It can be established based on a comparison group, for example, in a group with normal levels of target gene polypeptide and / or target gene polypeptide activity and a group with increased target gene polypeptide and / or target gene polypeptide activity levels. Another non-limiting example of a comparison group can be a group with one or more symptoms or diagnoses of a target gene-related disease or condition and a group without one or more symptoms or diagnoses of a disease or condition; a group of subjects to whom the siRNA of the present invention has been administered and a group of subjects to whom the siRNA of the present invention has not been administered. Typically, the control can be based on apparently healthy normal individuals or apparently healthy cells in an appropriate age group. It should be understood that, in addition to the predetermined value, the control according to the present invention can be a material sample tested in parallel with the experimental material. Examples include samples from a control group or control samples produced by manufacturing for parallel testing with the experimental sample. In some embodiments of the present invention, a control may include cells or subjects that have not been contacted or treated with a target gene dsRNA agent of the present invention. In this case, the control level of the target gene polypeptide and / or target gene polypeptide activity can be compared with the level of the target gene polypeptide and / or target gene polypeptide activity in cells or subjects contacted with a target gene dsRNA agent or a target gene antisense polynucleotide agent of the present invention.

[0354] In some embodiments of the present invention, the control level can be a target gene polypeptide level determined for a subject, wherein the target gene polypeptide level determined for the same subject at different times is compared to the control level. In one non-limiting example, the target gene level is determined in a biological sample obtained from a subject that has never received the targeted gene therapy of the present invention. In some embodiments, the biological sample is a serum sample. The target gene polypeptide level measured in the sample obtained from the subject can serve as the baseline or control value for the subject. After administering one or more targeted gene dsRNA agents to a subject in the treatment methods of the present invention, one or more additional serum samples can be obtained from the subject, and the target gene polypeptide level in the subsequent one or more samples can be compared to the control / baseline level of the subject. Such comparisons can be used to assess the onset, progression, or regression of a target gene (target gene)-related disease or condition in a subject. For example, if the level of a target gene (target gene) polypeptide in a baseline sample obtained from a subject is higher than the level obtained from the same subject after administering the target gene dsRNA agent of the present invention or the target gene (target gene) antisense polynucleotide agent to the subject, then this indicates regression of the target gene-related disease or condition and indicates the efficacy of the administered target gene dsRNA agent of the present invention in treating the target gene-related disease or condition.

[0355] In certain aspects of the present invention, one or more values ​​of the target gene polypeptide and / or target gene polypeptide activity level determined for a subject can be used as a control value and used to compare the target gene polypeptide and / or target gene activity level in the same subject at a later time, thereby allowing the assessment of changes in the "baseline" target gene polypeptide activity in the subject. Therefore, when the initial level is used as the control level for the subject, the initial target gene polypeptide level and / or the initial target gene polypeptide activity level can be used to demonstrate and / or determine that the methods and compounds of the present invention are capable of reducing the level of the target gene polypeptide and / or target gene polypeptide activity in the subject in the subject.

[0356] Using the methods of the present invention, the target gene dsRNA agents and / or target gene antisense polynucleotide agents of the present invention can be administered to a subject. Such dsRNAi agents include those that can be assessed for efficacy of administration and treatment of the invention as follows: the level of the target gene polypeptide in a serum sample obtained from the subject after administration and treatment is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the pre-administration level of the target gene polypeptide in a serum sample obtained from the subject at a previous time point, or compared to the level of the target gene polypeptide in a non-contact control (e.g., a control serum sample). It is understood that both the level of the target gene polypeptide and the level of target gene polypeptide activity are correlated with the level of target gene expression. Certain embodiments of the methods of the invention comprise administering to a subject a target gene dsRNA and / or a target gene antisense agent of the invention in an amount effective to inhibit the expression of the target gene, thereby reducing the level of the target gene polypeptide in the subject and reducing the level of the target gene polypeptide activity. In some embodiments of the methods of the invention, contacting a cell with an siRNA agent of the invention (also referred to herein as treatment) results in inhibition of target gene expression in the cell by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100%, for example, to below the level of detection in an assay.

[0357] Some embodiments of the present invention include determining the presence, absence, and / or amount (also referred to herein as level) of a target gene polypeptide in one or more biological samples obtained from one or more subjects. This assay can be used to evaluate the efficacy of the therapeutic methods of the present invention. For example, the methods and compositions of the present invention can be used to determine the level of a target gene polypeptide in a biological sample obtained from a subject previously treated with a target gene dsNA reagent and / or a target gene antisense agent of the present invention. When the level of the target gene polypeptide in a serum sample obtained from the subject after administration and treatment is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the pre-dose level of the target gene polypeptide in a serum sample obtained from the subject at a previous time point, or compared to a non-contact control level (e.g., the target gene polypeptide level in a control serum sample), it indicates the efficacy level of the treatment administered to the subject.

[0358] In some embodiments of the present invention, the physiological characteristics of the target gene-related disease or condition determined for the object can be used as a control result, and the determination results of the physiological characteristics of the same object at different times are compared with the control results. In a non-limiting example, the pathological feature hemolysis is measured from an object that has never been treated with the target gene therapy of the present invention, which can be used as the baseline or control value of the object. After the target gene dsRNA reagent is applied to the object once or more in the treatment method of the present invention, the blood cells are compared with the control / baseline levels of the object respectively. Such comparisons can be used to assess the onset, progression or regression of the target gene-related disease or condition in the object. For example, the baseline blood cells obtained from the object are higher than the thrombus measured from the same object after the target gene dsRNA reagent or target gene antisense polynucleotide reagent of the present invention is applied to the object, indicating the regression of the target gene-related disease or condition and indicating the efficacy of the target gene dsRNA reagent of the present invention in treating the target gene-related disease or condition.

[0359] Some embodiments of the present invention include determining the presence, absence, and / or changes in physiological characteristics of a target gene associated with a disease or condition using, for example, but not limited to, the following methods: (1) measuring blood cells of a subject; (2) evaluating physiological characteristics of one or more biological samples obtained from one or more subjects; (3) or performing a physical examination of the subject. This determination can be used to evaluate the efficacy of the treatment methods of the present invention.

[0360] mismatch

[0361] It is known to those skilled in the art that mismatches are tolerable for the efficacy of dsRNA, especially in the terminal regions of the dsRNA. Certain mismatches are better tolerated, for example, mismatches with wobble base pairs G:U and A:C are better tolerated (Du et el., A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res. 2005 Mar 21; 33(5): 1671-7. Doi: 10.1093 / nar / gki312. Nucleic Acids Res. 2005; 33(11): 3698).

[0362] Complementarity

[0363] As used herein, unless otherwise indicated, the term "complementarity" when used to describe the relatedness of a first nucleotide sequence (e.g., a target gene dsRNA agent sense strand or a target gene mRNA) to a second nucleotide sequence (e.g., a target gene dsRNA agent antisense strand or a single-stranded antisense polynucleotide) refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize [form inter-base pair hydrogen bonds under mammalian physiological conditions (or similar conditions in vitro)] with an oligonucleotide or polynucleotide comprising the second nucleotide sequence and to form a duplex or duplex structure under certain conditions. Other conditions may also apply, such as physiologically relevant conditions that may be encountered in an organism. A skilled person will be able to determine the set of conditions that is most suitable for testing the complementarity of two sequences based on the ultimate application of the hybridizing nucleotides. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimetics, as long as at least the above hybridization requirements are met. Sequence identity or complementarity is independent of modification.

[0364] For example, the complementary sequence in the target gene dsRNA as described herein comprises an oligonucleotide or polynucleotide containing a first nucleotide sequence and an oligonucleotide or polynucleotide containing a second nucleotide sequence that base-pairs over the full length of one or two nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. It should be understood that in embodiments in which two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs are not considered herein as mismatches determined based on complementarity. For example, a target gene dsRNA reagent comprises an oligonucleotide of 19 nucleotides in length and another oligonucleotide of 20 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 19 nucleotides that is fully complementary to the shorter oligonucleotide. For purposes described herein, this situation may be referred to as "fully complementary." Therefore, as used herein, "fully complementary" refers to that all (100%) bases in the continuous sequence of the first polynucleotide will hybridize with the same number of bases in the continuous sequence of the second polynucleotide. The continuous sequence may comprise all or part of the first or second nucleotide sequence.

[0365] As used herein, the term "substantially complementary" refers to a hybridization pair of nucleobase sequences in which at least about 85% (but not all) of the bases in the contiguous sequence of a first polynucleotide hybridize to the same number of bases in the contiguous sequence of a second polynucleotide. If the two sequences contain one or more mismatched base pairs, such as at least 1, 2, 3, 4, or 5 mismatched base pairs, when hybridized, the term "substantially complementary" can be used to refer to a first sequence that forms a duplex of up to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs (bp) relative to the second sequence while retaining the ability to hybridize under conditions most relevant to its ultimate application, for example, inhibition of target gene expression by the RISC pathway. The term "partially complementary" can be used herein to refer to a hybridization pair of nucleobase sequences in which at least 75% (but not all) of the bases in the contiguous sequence of a first polynucleotide hybridize to the same number of bases in the contiguous sequence of a second polynucleotide. In some embodiments, "partially complementary" means that at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bases in the contiguous sequence of the first polynucleotide will hybridize to the same number of bases in the contiguous sequence of the second polynucleotide.

[0366] The terms "complementary," "fully complementary," "substantially complementary," and "partially complementary" as used herein may refer to base matching between the sense and antisense strands of a dsRNA agent, base matching between the antisense strand of a dsRNA agent and the sequence of a target mRNA, or base matching between a single-stranded antisense oligonucleotide and the target mRNA sequence. It should be understood that the term "antisense strand of a dsRNA agent" can refer to the same sequence as an "antisense polynucleotide agent."

[0367] About modification

[0368] In some embodiments of the present invention, the RNA of the gene RNAi agent is chemically modified to obtain enhanced stability and / or one or more other beneficial properties. The nucleic acids in certain embodiments of the present invention can be synthesized and / or modified by methods well known in the art, for example, see "Current protocols in Nucleic Acid Chemistry," Beaucage, SL et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications that may be present in certain embodiments of the dsRNA agents of the present invention include, for example: (a) terminal modifications, such as 5' end modifications (phosphorylation, conjugation, reverse connection, etc.), 3' end modifications (conjugation, DNA nucleotides, reverse connection, etc.); (b) base modifications, such as base substitution with stable bases, destabilizing bases, or base pairing with an expanded partner library, deletion of bases (abasic nucleotides), or conjugation of bases; (c) sugar modifications (e.g., at the 2' position or the 4' position) or replacement of sugars; and (d) backbone modifications, including modification or replacement of phosphodiester bonds. Specific examples of RNA compounds available in certain embodiments of the dsRNA reagents, antisense polynucleotides and sense polynucleotides of the present invention include, but are not limited to, RNAs comprising modified backbones or without natural internucleoside linkages. As non-limiting examples, RNAs with backbone modifications may not have phosphorus atoms in the backbone. RNAs without phosphorus atoms in their internucleoside backbones may be referred to as oligonucleosides. In certain embodiments of the present invention, modified RNAs have phosphorus atoms in their internucleoside backbones.

[0369] It should be understood that the term "RNA molecule" or "RNA" or "ribonucleic acid molecule" includes not only RNA molecules expressed or found in nature, but also analogs and derivatives of RNA, which include one or more ribonucleotides / ribonucleoside analogs or derivatives as described herein or known in the art. The terms "ribonucleoside" and "ribonucleotide" are used interchangeably herein. RNA molecules can be modified in the core base structure or the ribose-phosphate backbone structure (e.g., as described below), and molecules containing ribonucleoside analogs or derivatives must retain the ability to form duplexes.

[0370] The following examples are provided to illustrate specific examples of the practice of the present invention and are not intended to limit the scope of the invention. It will be apparent to one of ordinary skill in the art that the present invention can be applied to a variety of compositions and methods.

[0371] As used herein, "substituents" are contemplated aspects of the present invention. One of ordinary skill in the art of medicinal chemistry and organic chemistry will appreciate the versatility of such substituents. A "substituent" should be sufficient to satisfy the principle of the present invention that its total number will be consistent with the existence of a chemical bond to the atom or group to which it is attached.

[0372] The term "hydrocarbyl" includes straight chain, branched chain and cyclic groups with any degree of saturation. In some special cases, groups containing C, O, and H are included. Such hydrocarbyl groups may be interrupted by one or more heteroatoms of nitrogen, oxygen, sulfur and phosphorus, and may be further substituted or substituted with one or more substituents.

[0373] The term "alkyl" as used herein refers to a straight or branched saturated hydrocarbon group having 1 to about 24 carbon atoms. When it appears herein, a numerical range such as "C1-C6 alkyl" generally refers to the number of carbon atoms in the alkyl group, which may contain only 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 6 carbon atoms. Unless otherwise specified, the number of carbon atoms added by further substitution of other atoms or groups in the alkyl group is not included in the counting range, and the same description also exists in other counting methods herein. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl, etc. Although the term "alkyl" also includes cases where the numerical range of carbon atoms is not specified, for example, the term "alkyl" may refer to C1-C 10 "Substituted alkyl" refers to an alkyl moiety having a substituent. As used herein, "lower alkyl" refers to an alkyl moiety having 1 to about 6 carbon atoms.

[0374] As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon group having at least one carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, 1-methyl-2-butene-1-yl, dienes such as 1,3-butadiene, and the like. Alkenyl groups typically contain 2 to about 24 carbon atoms, more typically 2 to about 12 carbon atoms, with 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally contain one or more further substituted groups.

[0375] As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon group having at least one carbon-carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically contain 2 to about 24 carbon atoms, more typically 2 to about 12 carbon atoms, with 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally contain one or more further substituent groups.

[0376] As used herein, the term "acyl" refers to a group formed by removing a hydroxyl group from an organic acid and has the general formula -C(O)-X, wherein X is typically aliphatic, alicyclic, or isocyclic. Examples include aliphatic carbonyl, aromatic carbonyl, aliphatic sulfonyl, aromatic sulfinyl, aliphatic sulfinyl, aromatic phosphate, aliphatic phosphate, and the like. Acyl groups as used herein may optionally contain further substituent groups.

[0377] The term "cycloalkyl" refers to a ring system in which the ring is aliphatic. The ring system may comprise one or more rings, at least one of which is aliphatic. Preferred alicyclic compounds comprise rings having from about 5 to about 9 carbon atoms in the ring. As used herein, a cycloalkyl group may optionally contain further substituent groups.

[0378] As used herein, the term "alkoxy" refers to a group formed between an alkyl group and an oxygen atom, wherein the oxygen atom is used to connect the alkoxy group to the parent molecule. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, and n-hexoxy. As used herein, the alkoxy group may optionally contain further substituents.

[0379] As used herein, the term "aminoalkyl" refers to a C1-C 12 Alkyl. The alkyl portion of the group forms a covalent bond with the parent molecule. The amino group may be located at any position and the aminoalkyl group may be substituted with further substituents at the alkyl and / or amino positions.

[0380] As used herein, the terms "aryl" and "aromatic" refer to monocyclic or polycyclic carbocyclic ring groups having one or more aromatic rings. As used herein, the term "aromatic group" refers to a planar ring having a delocalized π electron system containing 4n+2π electrons, where n is an integer. The aromatic ring can be formed by 5, 6, 7, 8, 9 or more than 9 atoms. The term "aromatic" is intended to include carbocyclic aromatic groups (e.g., phenyl) and heterocyclic aromatic groups (or "heteroaryl" or "heteroaromatic") groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic rings, i.e., rings that share adjacent pairs of carbon atoms. "Substituted aromatic" refers to an aromatic group further carrying one or more substituents. Examples of aryl include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, etc. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. As used herein, aryl may optionally include further substituents.

[0381] As used herein, the terms "aralkyl" and "arylalkyl" refer to a group covalently attached to a C1-C 12 The aromatic group of an alkyl group. The alkyl portion of the resulting aralkyl (or arylalkyl) forms a covalent bond with the parent molecule. Examples include, but are not limited to, benzyl, phenethyl, etc. Aralkyl groups as used herein may optionally contain further substituent groups attached to the alkyl group, the aryl group, or both to form the group.

[0382] As used herein, the terms "heteroaryl" and "heteroaromatic" refer to groups comprising monocyclic or polycyclic aromatic rings, ring systems, or fused ring systems, wherein at least one of these rings is aromatic and contains one or more heteroatoms. Heteroaryl is also intended to include fused ring systems, including systems in which one or more of the fused rings do not contain heteroatoms. Heteroaryl groups typically contain a ring atom selected from sulfur, nitrogen, or oxygen. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, phenylthio, furyl, quinolyl, isoquinolyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, etc. Heteroaryl groups can be attached to the parent molecule directly or via a linking portion such as an aliphatic group or a heteroatom. As used herein, heteroaryl groups can optionally contain further substituent groups.

[0383] As used herein, the term "heteroarylalkyl" refers to a C1-C 12 The heteroaryl group of the alkyl group is as defined above. The alkyl portion of the resulting heteroarylalkyl group is capable of forming a covalent bond with the parent molecule. Examples include, but are not limited to, pyridylmethyl, pyridylethyl, naphthyridinylpropyl, and the like. As used herein, heteroarylalkyl groups may optionally contain further substituents on one or both of the heteroaryl or alkyl portions.

[0384]

[0046] The term "halo" or "halogen" as used herein refers to an atom selected from fluorine, chlorine, bromine and iodine.

[0385] As used herein, the term "heterocyclic group" refers to a monocyclic or polycyclic ring system containing at least one heteroatom and being unsaturated, partially saturated, or fully saturated, thus including heteroaryl groups. Heterocyclic is also intended to include fused ring systems in which one or more of the fused rings contains at least one heteroatom and the other rings may contain one or more heteroatoms or optionally no heteroatoms. Heterocyclic groups typically contain at least one atom selected from sulfur, nitrogen, or oxygen. Examples of heterocyclic groups include [1,3]dioxolanyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, pyrazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and the like. As used herein, an "n"-membered heterocyclic group is the sum of the n atoms in the ring system, without protecting atoms or groups outside the ring system. As used herein, heterocyclic groups may optionally contain further substituents.

[0386] As used herein, the term "monocyclic or polycyclic structure" includes all ring systems selected from monocyclic ring systems or polycyclic ring systems in which the rings are fused or connected and is intended to include single and mixed ring systems selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic, and heteroarylalkyl. Such monocyclic and polycyclic structures may contain rings each having the same degree of saturation or each independently having a different degree of saturation (including fully saturated, partially saturated, or fully unsaturated). Each ring may contain ring atoms selected from C, N, O, and S to provide heterocyclic rings as well as rings containing only C ring atoms. These rings may exist in mixed motifs such as, for example, benzimidazole, where one ring has only carbon ring atoms and the fused ring has two nitrogen atoms. The monocyclic or polycyclic structure may be further substituted with substituent groups such as, for example, phthalimide, which has two =O groups attached to one of the rings. The monocyclic or polycyclic structures can be linked to the parent molecule using a variety of strategies such as direct linkage through a ring atom, through a substituent group, or through a bifunctional linking moiety.

[0387] The term "oxo" refers to the group (=0).

[0388] The term "sulfonyl" as used herein (alone or as part of another group) refers to a radical of the formula RSO2-, wherein R is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl. The term "sulfonamido" as used herein (alone or as part of another group) refers to a radical of the formula RSO 2- NH-, wherein R is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl. Some non-limiting exemplary sulfonamide groups include CH3-SO 2-N(H)-.

[0389] The term "alicyclic" or "alicyclic group" refers to a cyclic ring system wherein the ring is an aliphatic ring. The ring system may comprise one or more rings, at least one of which is an aliphatic ring. Preferred alicyclic rings include those having from about 5 to about 9 carbon atoms in the ring. Alicyclic rings, as used herein, may optionally include other substituents. The term "aliphatic," as used herein, refers to a straight or branched chain hydrocarbon group containing up to 24 carbon atoms, wherein the degree of saturation between any two carbon atoms is a single, double, or triple bond. Aliphatic groups preferably contain from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms, and more preferably from 1 to about 6 carbon atoms. The straight or branched chain of the aliphatic group may be interrupted by one or more heteroatoms, including nitrogen, oxygen, sulfur, and phosphorus. Aliphatic groups interrupted by heteroatoms include, but are not limited to, polyalkoxy groups, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups, as used herein, may optionally include other substituents. Spacers, to which the oligonucleotide is attached, are used to position the oligonucleotide and the synthesis process away from the substrate or support. This allows for greater flexibility and more space for compositing, making it easier to cut when the composition is complete.

[0390] As used herein, the term "carboxy" (alone or as part of another group) refers to a group of formula -COOH.

[0391] Linking groups or bifunctional linking moieties such as those known in the art can be used to link chemical functional groups, conjugated groups, reporter groups and other groups to selective sites in parent compounds such as, for example, oligomeric compounds. Generally speaking, bifunctional linking moieties include a hydrocarbyl moiety with two functional groups. One of the functional groups is selected to be bound to the parent molecule or compound of interest and the other is selected to be bound to substantially any selected group such as a chemical functional group or a conjugated group. In some embodiments, the linking group comprises a polymer of a chain structure or repeating units such as ethylene glycol or amino acid units. Examples of functional groups commonly used in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, the bifunctional linking moiety comprises amino, hydroxyl, carboxylic acid, thiol, unsaturation (e.g., double or triple bonds), etc. Some non-limiting examples of bifunctional linking moieties include 8-amino-3,6-dioxaoctanoic acid (ADO), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C1-C 10 Alkyl, substituted or unsubstituted C2-C 10 Alkenyl or substituted or unsubstituted C2-C 10A non-limiting list of preferred substituent groups for alkynyl groups includes hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halo, alkyl, aryl, alkenyl, and alkynyl.

[0392] As used herein, the term "ether" refers to a product in which the hydrogen of the hydroxyl group of an alcohol or phenol is replaced by a hydrocarbon group. The general formula is RO-R', where R and R' can be the same or different. Ethers in which the two are identical are called symmetrical ethers, also known as simple ethers or monoethers; ethers in which the two are different are called asymmetric ethers, also known as mixed ethers or mixed ethers.

[0393] Terminology used in this article or It generally refers to the connection between adjacent groups or parts.

[0394] About: As used herein, the term "about" or "approximately" as applied to one or more values ​​of interest refers to a value similar to a stated reference value. In certain embodiments, unless otherwise indicated or otherwise apparent from the context, the term "about" or "approximately" refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in either direction (greater than or less than) (unless such a value would exceed 100% of the possible value).

[0395] As used herein, the abbreviations "Ac" refer to acetyl; "Bn" refers to benzyl; "Bz" refers to benzoyl; "DMTrCl" refers to 4,4-dimethoxytrityl chloride; "DMTr" refers to 4,4-dimethoxytrityl; "TIIP" refers to tetrahydropyranyl; "TBDMS" refers to tert-butyldimethylsilyl; "TIPDS" refers to tetraisopropyldisilyl; and "DTBS" refers to di(tert-butyl)silyl. Specific embodiments

[0396] The following examples are provided to illustrate specific examples of implementing the present invention and are not intended to limit the scope of the present invention. As will be apparent to one of ordinary skill in the art, the present invention will find application in a variety of compositions and methods.

[0397] Example 1.

[0398] Synthesis of phosphoramidite compound 2-

[0399] DMTrCl (232 g, 684 mmol, 1.0 eq) in pyridine (400 ml) was added to a solution of compound A isomannide (100 g, 684 mmol, 1.0 eq) in pyridine (600 ml), and the mixture was stirred at 25°C for 16 hours. LC-MS showed that compound A was completely consumed, and a main peak with the desired mass was detected. The resulting reaction mixture was diluted with water (500 ml), extracted with dichloromethane (500 ml * 2), and the combined organic phases were washed with brine (500 ml), dried over Na2SO4 and concentrated in vacuo to obtain a residue. The residue was purified by column chromatography (DCM / MeOH=100 / 1 to 50 / 1, 0.1% Et3N) to give compound B (150 g, 48.9% yield) as a yellow solid.

[0400] 1 H NMR:EC4783-404-P1B1_C(400MHz,DMSO-d6)

[0401] δppm7.46(br d,J=7.63Hz,2H)7.28-7.37(m,6H)7.19-7.25(m,1H)6.90(br d,J=7.88Hz,4H)4.70(d,J=6.50Hz,1H)3.99-4.09(m,6H)3.88-3.96(m,2H)3.83(br dd,J=7.82,6.94Hz,1H)3.74(s,6H)3.41(br t,J=8.13Hz,1H)3.05(t,J=8.44Hz,1H)2.85(br t,J=7.50Hz,1H).

[0402] To a solution of compound B (80.0 g, 178 mmol, 1.0 eq) in dichloromethane (5.0 mL) under N2 atmosphere was added dropwise 2H-tetrazole (0.45 M, 436 mL, 1.1 eq) at 25°C, followed by the addition of a solution of compound C (2-cyanoethyldiisopropylchlorophosphoramidite, 80.6 g, 267 mmol, 85.0 mL, 1.5 eq) in dichloromethane (200 mL); the reaction mixture was stirred at 25°C for 1.0 h; LC-MS showed that compound B was completely consumed, and a major peak with the desired mass was detected. The resulting reaction mixture was cooled to -20°C and poured into an ice-cold oven; NaHCO 3 (500 mL), extracted with dichloromethane (500 mL * 3), and the combined organic layers were washed with NaHCO 3 / brine = 1: 1 (300 mL / 300 mL), dried over Na 2 SO 4, and concentrated in vacuo (35°C) to obtain a residue (100 mL). The residue was purified by column chromatography (Al 2 O 3, DCM / MeOH = 100 / 1 to 50 / 1, 0.1% Et 3 N) to obtain compound 2 (77 g, 119 mmol, yield 66.5%) as a white solid.

[0403] 1 H NMR:EC4783-423-P1B1_C(400MHz,DMSO-d6)

[0404] δppm7.22(br d,J=7.50Hz,2H)7.05-7.14(m,6H)6.96-7.02(m,1H)6.67(br dd,J=8.82,1.81Hz,4H)3.95-4.07(m,2H)3.73-3.83(m,1H)3.62-3.72(m,2H)3.48-3.53(m ,6H)3.27-3.37(m,3H)3.11(s,6H)2.82(td,J=8.54,2.31Hz,1H)2.47-2.63(m,3H)2.28(br d,J=1.63Hz,3H)0.82-1.00(m,13H).

[0405] Synthesis of phosphoramidite compound 1-

[0406] To a solution of compound B (500 mg, 1.11 mmol, 1.0 eq) in dichloromethane (5.0 mL) were added compound D (methyldiisopropylchlorophosphoramidite, 607 mg, 3.34 mmol, 3.0 eq) and triethylamine (432 mg, 3.34 mmol, 582 μL, 3.0 eq) at 0-5°C. The mixture was stirred at 25°C for 1.0 hour. The resulting reaction mixture was cooled to -20°C and poured into a cold NaHCO₃ (5.0 mL) solution (0-5°C). Extraction was performed with dichloromethane (5.0 mL x 2). The combined organic layers were washed with cold NaHCO₃ / brine = 1:1 (5.0 mL / 5.0 mL) (0-5°C), dried over Na₂SO₄, and concentrated in vacuo to obtain a residue. The residue was purified by column chromatography (basic Al2O3, petroleum ether / ethyl acetate = 10 / 1 to 5 / 1, 0.1% Et3N) to give compound 1 (280 mg, 471 μmol, yield 42.3%) as a white solid.

[0407] 1 H NMR: EC10615-49-P1N (400MHz, DMSO-d6) δppm7.44 (br d, J=7.63Hz, 2H), 7.31 (br t, J=7.94Hz, 6H), 7.18-7.26 (m, 1H), 6.89 (br d,J=8.00Hz,4H),4.08-4.13(m,1H),3.95-4.03(m,1H),3.84-3.93(m,1H),3.77-3.83(m,1H),3.74(s,6H),3.43-3.53(m,3H),3.38(br d,J=6.75Hz,1H),2.94-3.04(m,1H),2.70-2.85(m,1H),1.09-1.15(m,12H),1.07(br s,3H).

[0408] Synthesis of phosphoramidite compound 3-

[0409] At room temperature, under nitrogen, a 100 mL flask was charged with 5 mL of DMF and Compound A isomannide (0.5 g, 1.0 eq). NaH (0.18 g, 1.3 eq) was added portionwise at -10°C, and the mixture was stirred at -10°C to 0°C for 30 minutes. A solution of 1-iodohexadecane (1.57 g, 1.3 eq) in DMF (3 mL) was added dropwise to the reaction mixture at approximately 0°C for 10 minutes. The reaction mixture was then heated to 50°C and stirred overnight. TLC (petroleum ether:EtOAc = 1:1, Rf = 0.5) indicated that although Compound A isomannide was not completely consumed, a new spot had formed, and the reaction was repeated. The two reaction mixtures were combined and poured into a saturated NH4Cl solution (100 mL), extracted with EtOAc (50 mL * 3). The combined organic layers were washed with a saturated NaCl solution (50 mL * 2), dried over Na2SO4, and concentrated under reduced pressure. The crude product of Lipd-02-1A was purified by silica gel column and eluted with (petroleum ether: EtOAc = 100:0 to 5:1) to give Lipd-02-1A (0.72 g, 28% yield) as a white solid. MS (M+H) = 371.3.

[0410] At room temperature, 5.6 mL of CH2Cl2 and Lipd-02-1A (0.7 g, 1.0 eq) were added to a 100 mL flask under nitrogen. Tetrazole (0.15 g, 1.1 eq, 0.45 M in CH3CN) was added dropwise at room temperature, and then a solution of 3-((bis(diisopropylamino)phosphinoyloxy)propionitrile (0.88 g, 1.5 eq) in CH2Cl2 (1 mL) was added dropwise to the reaction mixture and stirred at room temperature for 3 hours. The mixture was detected by TLC (petroleum ether: EtOAc = 2: 1, Rf = 0.8), which showed that a new spot was generated and a portion of Lipd-02-1A was not Consumption. The resulting mixture was cooled to 0°C and poured into a saturated NaHCO3 solution (50 mL), extracted with CH2Cl2 (50 mL*3), and the combined organic layers were washed with a saturated NaCl solution (50 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude product of compound 3 was purified by silica gel column and eluted with (petroleum ether: EtOAc = 100:0 to 20:1; 1% Et3N) to give compound 3 (0.33 g, 30% yield) as a colorless oil.

[0411] 1H NMR (400MHz, CDCl3): δppm4.47-4.54(m,2H),4.29-4.38(m,1H),3.58-4.05(m,10H),3.41-3.46(m ,1H),2.62-2.69(m,2H),1.57-1.64(m,2H),1.25(s,26H),1.15-1.22(m,12H),0.87(t,J=6.8,3H).

[0412] 31 P NMR (400MHz, CDCl3): δppm148.94,149.03.

[0413] MS (M+H) = 571.5.

[0414] The same method can be used to obtain compounds 21 and 22, mainly by replacing 1-iodohexadecane with 1-iodotetradecane or 1-iodooctadecane.

[0415] Compound 21: 1 H NMR (400MHz, CDCl3): δppm4.49-4.55(m,2H),4.32-4.36(m,1H),3.62-4.04(m,10H),3.40-3.45(m ,1H),2.64-2.70(m,2H),1.54-1.61(m,2H),1.23(s,22H),1.15-1.22(m,12H),0.88(t,J=6.8,3H).

[0416] 31 P NMR (400MHz, CDCl3): δppm148.94,149.03.

[0417] MS (M+H) = 543.5.

[0418] Compound 22: 1 H NMR (400MHz, CDCl3): δ4.49-4.55(m,2H),4.33-4.35(m,1H),3.62-4.04(m,10H),3.42-3.45(m,1 H),2.59-2.70(m,2H),1.58-1.65(m,2H),1.25(s,30H),1.17-1.20(m,12H),0.88(t,J=6.8,3H).

[0419] 31 P NMR (400MHz, CDCl3) δppm148.94,149.03.

[0420] MS (M+H) = 599.5.

[0421] Preparation of other nucleoside phosphoramidites was performed according to procedures described herein and in the art, such as, but not limited to, published US Pat. No. 426,220 and WO 02 / 36743.

[0422] Example 2

[0423] Synthesis of oligomeric compounds

[0424] Oligomeric compounds used according to the present invention can be conveniently and routinely prepared by the solid phase synthesis technique known to all. The equipment used for this synthesis is sold by a plurality of suppliers and includes, for example, Mermade 12 (LGC). In addition or alternatively, any other device known in the art for this synthesis can be adopted. It is well known that similar techniques are used to prepare oligonucleotides such as alkylated derivatives and those with phosphorothioate linkages.

[0425] Oligomeric Compounds: Unsubstituted and substituted phosphodiester (O) oligomeric compounds (including but not limited to oligonucleotides) can be synthesized using standard phosphoramidite chemistry by iodine oxidation on an automated DNA synthesizer, Mermade 12 (LGC).

[0426] In certain embodiments, phosphorothioate internucleoside linkages (S) are synthesized in a manner similar to phosphodiester internucleoside linkages, except that thiolation is achieved by oxidation of the phosphite linkage using a 10% w / v solution of 3,H-1,2-benzodithiol-3-one-1,1-dioxide in acetonitrile. The thiolation reaction time is increased to 180 seconds and the capping step is continued as normal. The oligomeric compound is recovered by precipitation from a 1M NHOAc solution using more than 3 volumes of ethanol after cleavage and deblocking from a CPG column in concentrated aqueous ammonia at 55°C (12-16 hours).

[0427] Phosphinate internucleoside linkages can be prepared as described in US Pat. No. 5,508,270. Alkyl phosphate internucleoside linkages can be prepared as described in US Pat. No. 4,469,863, or by the phosphoramidite method described herein. 3'-deoxy-3'-methylenephosphonate internucleoside linkages can be prepared as described in US Pat. No. 5,610,289 or 5,625,050. Phosphoramidite internucleoside linkages can be prepared as described in US Pat. No. 5,256,775 or 5,366,878. Alkyl phosphorothioate internucleoside linkages can be prepared as described in published WO 94 / 17093 and WO 94 / 02499. 3'-deoxy-3'-aminophosphoramidate internucleoside linkages can be prepared as described in US Pat. No. 5,476,925. Phosphotriester internucleoside linkages were prepared as described in US Pat. No. 5,023,243. Methods for preparing some phosphate internucleoside linkages can be found in Beilstein J Org Chem. 2017; 13: 1368–1387.

[0428] Oligomeric compounds having one or more non-phosphorus-containing internucleoside linkages including, but not limited to, methylenemethylimino-linked oligonucleosides, methylenedimethylhydrazine-linked oligonucleosides, methylenecarbonylamino-linked oligonucleosides, and methyleneaminocarbonyl-linked oligonucleosides, as well as mixed backbone oligomeric compounds having, for example, alternating O or S linkages, can be prepared as described in US Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240, and 5,610,289.

[0429] Double-stranded ribonucleic acid (dsRNA) reagents are obtained by mixing each of the two complementary strands (sense strand and antisense strand) at a 1:1 molar ratio to form a duplex.

[0430] The duplexes in Table 2 are typically synthesized on an oligonucleotide synthesizer using a well-established solid-phase synthesis method based on phosphoramidite chemistry to synthesize the sense and antisense strands of siRNA. Oligonucleotide chain growth is achieved through a four-step cycle: deprotection, condensation, capping, and oxidation or sulfurization steps for each nucleotide addition. Synthesis is performed on a controlled pore glass (CPG) ). Monomeric phosphoramidites are typically purchased from commercial sources, but in this case, the monomeric compound can be used as a monomeric phosphoramidite and introduced into the oligonucleotide chain. In cases where the monomeric compound is used as a monomeric phosphoramidite attached to the 3'-terminus, it is attached to a CPG solid support. In cases where it is attached to the 5'-terminus, the phosphoramidite is used in the final coupling reaction and, if necessary, further coupling with a targeting group. 3% trichloroacetic acid (TCA) in dichloromethane is used for deprotection of the 4,4'-dimethoxytrityl protecting group (DMT). 5-Ethylthio-1H-tetrazole is used as an activator. I2 in THF / Py / H2O and phenylacetyl disulfide (PADS) in pyridine / MeCN are used for oxidation and sulfurization reactions, respectively. Following the final solid-phase synthesis step, the solid support-bound oligomer is cleaved and the protecting groups removed by treatment with a 1:1 volume of 20 wt% aqueous methylamine and 28% ammonium hydroxide solution. The oligomeric compound for in vitro screening is synthesized by a crude mixture that is concentrated. The remaining solid is dissolved in 1.0 M NaOAc, ice-cold EtOH is added to precipitate the single-stranded product as a sodium salt, which can be used for annealing without further purification. In order to synthesize the siRNA for in vivo testing, the crude single-stranded product is further purified by ion pair reversed-phase HPLC (IP-RP-HPLC). The purified single-stranded oligonucleotide product from IP-RP-HPLC is dissolved in 1.0 M NaOAc and precipitated by adding ice-cold EtOH, and converted into a sodium salt. Annealing of sense strand and antisense strand oligonucleotide is carried out in water by equimolar complementation to form a double-stranded siRNA product.

[0431] Example 3

[0432] Synthesis of targeting groups

[0433] To a solution of the propylenediamine derivative intermediate compound 4 (275 g, 660 mmol, 1.00 eq.) in dichloromethane (2.75 L) was added triethylamine (133 g, 1.32 mol, 2.00 eq.), followed by the dropwise addition of Cbz-Cl (169 g, 990 mmol, 1.50 eq.). The reaction mixture was stirred at 25°C for 2 hours. LCMS indicated complete conversion of the propylenediamine derivative. The reaction mixture was washed sequentially with a saturated solution of NaHCO₃ (800 mL) and then saturated brine (500 mL), and the organic phase was dried over anhydrous Na₂SO₄. After filtering to remove the desiccant, the filtrate was concentrated to dryness. The crude product was purified by column chromatography (SiO₂, petroleum ether (PE) / ethyl acetate (EA) = 100 / 1 to 5 / 1, v / v) to afford compound 5 (290 g, 527 mmol, 75.7% yield) as a colorless oil. 1H NMR(400MHz in DMSO-d6): δppm7.23-7.40(m,5H),5.00-5.12(m,2H),3.86-3.95(m,2H),3.23-3.3 9(m,6H),2.55-2.67(m,2H),1.56-1.64(m,2H),1.31-1.46(m,27H).MS(ESI)[M+H] + m / z:551.6.

[0434] To compound 5 (145 g, 263 mmol, 1.00 eq) was added HCOOH (2.9 L), and the solution was stirred at 60°C for 12 hours. LCMS showed complete conversion of compound 5. 1.5 L of toluene and 1.5 L of acetonitrile were added to the reaction solution, which was concentrated under reduced pressure to approximately 500 mL. Toluene / acetonitrile (1:1, v / v, approximately 750 mL) was then added and concentrated to approximately 500 mL. Acetonitrile (approximately 1000 mL) was then added and concentrated to dryness. The crude product was triturated with 700 mL of acetonitrile at 60°C for 2 hours and filtered. The solid was collected and dried to yield compound 6 (105 g, quantitative) as a white solid. 1 H NMR (400MHz in DMSO-d6): δppm 7.26-7.40(m,5H),5.02-5.10(m,2H),3.89-4.00(m,2H),3.36-3.45(m,4H),3.24-3.34(m,2H),2.59-2.72(m,2H),1.40(s,2H).MS(ESI)[M+H] + m / z:383.0.

[0435] To a solution of compound 6 (100 g, 261 mmol) and intermediate-A (502 g, 915 mmol, 3.50 eq.) in DMF (1.0 L) were added O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) (327 g, 1.02 mol, 3.90 eq.) and triethylamine (212 g, 2.09 mol, 8.00 eq.). The reaction was allowed to proceed at 25°C for 1 hour. LCMS indicated complete conversion of compound 6. The reaction solution was added to 4000 mL of water and extracted with methyl tert-butyl ether (2000 mL in two portions) to remove impurities. The remaining aqueous phase was extracted with dichloromethane (3000 mL in two portions). The dichloromethane phase was washed sequentially with 10% aqueous citric acid (2000 mL in two portions), saturated NaHCO3 (2.0 L in two portions), and saturated brine (2.0 L), and then dried over anhydrous Na2SO4. The filtrate was filtered and concentrated under reduced pressure to give white solid compound 8 (260 g, 159 mmol, yield 60.9%). 1H NMR (400MHz in DMSO-d6): δppm 7.99-8.08(m,2H),7.93(br d,J=5.50Hz,1H),7.79-7.86(m,3H),7.26-7.39(m,5H),5.22(d,J=3.13Hz,3H),4.95-5.08(m,5H),4.54(br d,J=8.38Hz,3H),4.03(s,9H),3.81-3.93(m,5H),3.76(br d,J=4.88Hz,3H),3.44-3.62(m,10H),3.34-3.43(m,6H),3.24(br d,J=6.13Hz,7H),3.02-3.09(m,4H),2.40-2.47(m,2H),2.10(s,9H),1.99(s,9H),1.89(s,9H),1.77(s,9H),1.57-1.68(m,2H). MS(ESI)[M+H] + m / z:816.4.

[0436] A 2 L hydrogenation reactor was inerted with argon and dry Pd / C (9 g) was carefully added. MeOH (50 mL) was added to wet the Pd / C, and then a solution of compound 8 (90 g, 55.1 mmol, 1.00 eq.) and trifluoroacetic acid (6.29 g, 55.1 mmol, 1.00 eq.) in MeOH (850 mL) was slowly added under an argon atmosphere. The mixture was degassed and replaced with a hydrogen atmosphere by adding H2 three times and stirred at 25°C for 10 hours. LCMS showed complete conversion of compound 8. The Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure to provide compound 9 (80 g, 90.2% yield). 1 H NMR (400MHz in DMSO-d6): δppm9.12(br s,2H),8.50(br t,J=5.19Hz,1H),8.10(br t,J=5.50Hz,2H),7.85-7.91(m,3H),5.22(d,J=3.25Hz,3H),4.95-5.01(m,3H),4.52 -4.58(m,3H),4.03(s,9H),3.84-3.93(m,3H),3.75-3.83(m,3H),3.39-3.61(m,16H) ,3.23-3.32(m,6H),3.15-3.18(m,3H),2.97-3.05(m,2H),2.54-2.61(m,2H),2.10(s ,9H),2.00(s,9H),1.89(s,9H),1.77-1.80(m,9H),1.70-1.76(m,2H).MS(ESI)[M+H]+ m / z:749.3.

[0437] To a solution of compound 9 (270 g, 168 mmol, 1.00 eq.) and glutaric anhydride (28.6 g, 252 mmol, 1.50 eq.) in dichloromethane (2.7 L) was added triethylamine (67.8 g, 672 mmol, 4.00 eq.), and the solution was stirred at 25° C. for 1 hour. LCMS showed that compound 9 was completely converted to compound 11. 4-Hydroxypiperidine (42.4 g, 420 mmol, 2.50 eq.) and TBTU (107 g, 335 mmol, 2.00 eq.) were added to the reaction solution, and stirring was continued at 25° C. for 1 hour. LCMS showed that compound 11 was completely converted. The reaction was quenched by slowly adding saturated NH 4 Cl (3.0 L), the layers were separated, and the aqueous phase was extracted with dichloromethane (2×1000 mL) and combined with the previous organic phase. The combined organic phases were washed with a 1:1 (v / v) mixture (3.0 L) of saturated NaHCO (aq) and saturated brine, dried over anhydrous Na SO, filtered, and concentrated under reduced pressure. The crude product was dissolved in 1.5 L of dichloromethane and added dropwise to methyl tert-butyl ether (7.5 L). A translucent white precipitate gradually formed during the addition. The precipitate was vacuum filtered, and the collected solid was dried under vacuum to give Compound 13 (207 g, 72.8% yield) as a white solid. 1 H NMR (400MHz in DMSO-d6): δppm8.05(br d,J=2.00Hz,2H),7.82(br d,J=7.38Hz,3H),5.21(br s,3H),4.98(br d,J=10.26Hz,3H),4.72(br s,1H),4.54(br d,J=7.88Hz,3H),4.03(br s,9H),3.74-3.94(m,9H),3.45-3.71(m,12H),3.40(br s,6H),3.24(br s,7H),3.07(br d,J=14.13Hz,5H),2.91-3.01(m,1H),2.24-2.44(m,5H),2.20(br s,1H),2.10(s,9H),1.96-2.04(m,9H),1.89(br s,9H),1.74-1.81(m,9H),1.51-1.73(m,6H),1.07-1.36(m,3H).MS(ESI)[M+H] + m / z:848.0.

[0438] To a solution of compound 13 (200 g, 118 mmol, 1.00 eq.) and diisopropylammonium tetrazolium (8.08 g, 47.2 mmol, 0.40 eq.) in dichloromethane (2.0 L) was added 3-bis(diisopropylamino)phosphonooxypropionitrile (53.3 g, 177 mmol, 1.50 eq.), and the reaction solution was stirred at 40°C for 2 hours. LCMS showed that the conversion of compound 13 was complete. The reaction solution was washed with a 1:1 mixture of saturated NaHCO3 and saturated brine (2.0 L), dried over anhydrous Na2SO4, and the filtrate was concentrated. The crude product was dissolved in dichloromethane (1.2 L) and added dropwise to stirred methyl tert-butyl ether (6.0 L). The suspension was filtered, the filter cake was rinsed with methyl tert-butyl ether, and the solid was collected and dried in vacuo. The product was dissolved in dichloromethane (1.0 L) and concentrated to dryness. The operation was repeated four times to remove residual tert-butyl ether to obtain GLPA15 (164 g, yield 73.3%). 1 H NMR (400MHz in DMSO-d6): δppm8.05(br d,J=6.50Hz,2H),7.81(br d,J=9.01Hz,3H),5.22(d,J=3.25Hz,3H),4.98(dd,J=11.26,3.25Hz,3H),4.55(br d,J=8.50Hz,3H),4.03(s,9H),3.64-3.97(m,12H),3.55-3.63(m,6H),3.50(br s,5H),3.40(br d,J=6.13Hz,6H),3.17-3.30(m,9H),3.07(br d,J=14.26Hz,4H),2.76(t,J=5.82Hz,2H),2.18-2.47(m,6H),2.10(s,9H),1 .99(s,9H),1.89(s,9H),1.78(s,9H),1.52-1.74(m,6H),1.12-1.19(m,12H). 31 P NMR(DMSO-d6):ppmδ145.25.MS(ESI)[M+H] + m / z:1895.7.

[0439] Other GalNAc phosphoramidite compounds (GLPAn) can also be obtained by using appropriate corresponding intermediates using similar methods herein or methods well known in the art, and can be linked as targeting groups to appropriate positions of the compounds herein or oligomers and duplexes.

[0440] In some studies, a method for connecting a targeting group comprising GalNAc (also referred to herein as a GalNAc delivery compound) to the 5'-end of a sense strand is provided, comprising using a GalNAc phosphoramidite (GLPAn) in the last coupling step of solid phase synthesis, using a synthesis process such as that used when the oligonucleotide chain is extended (i.e., adding nucleotides at the 5' end of the sense strand) to connect it to the 5'-end of the sense strand. In some studies, a method for connecting a targeting group comprising GalNAc to the 3'-end of a sense strand comprises using a solid support (CPG) comprising GLPAn. In some studies, a method for connecting a targeting group comprising GalNAc to the 3'-end of a sense strand comprises: connecting the targeting group to a CPG solid support via an ester bond, and using the resulting CPG with the targeting group connected when synthesizing the sense strand, resulting in the GalNAc targeting group being connected to the 3'-end of the sense strand. Similarly, the targeting group can also be connected to a monomer containing an oligomeric compound of the present invention by means of the phosphoramidite method, etc.

[0441] Example 4. Preparation of a solid support comprising the monomer of the present invention

[0442] Represents the macroporous aminomethyl polyethylene resin support part

[0443] Place a 50L glass kettle under nitrogen protection, add dichloromethane (19.50kg) to the glass kettle, and start stirring. Control the temperature at 20-30°C, add DMTr-imann (1.47kg) to the glass kettle, add triethylamine (1.50kg), 4-dimethylaminopyridine (0.164kg), and add succinic anhydride (1.34kg) to the reactor. Keep the system warm at 20-30°C for 18 hours, then take a sample and terminate the reaction. Add saturated sodium bicarbonate solution (22.50kg) to the reaction system, stir for 10-20 minutes, and then let it stand until it separates. Transfer the lower organic phase to, extract the upper aqueous phase twice with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, filter the filtrate, and then transfer to rotary evaporation to concentrate until there is no fraction, forming 1.83kg of solid gray to off-white solid.

[0444] Add N,N-dimethylformamide (23.50 kg) to a 100 L glass kettle and start stirring. Control the temperature at 20-30°C. Under nitrogen protection, add the product from the previous step, O-benzotriazole-tetramethyluronium hexafluorophosphate (0.33 kg), and N,N-diisopropylethylamine (0.13 kg) to the 100 L glass kettle via a solid addition funnel. After stirring for 10-30 minutes, pour the mixture into a 50 L galvanized bucket for later use. Add macroporous amine methyl resin (3.25 kg) (purchased from Tianjin Nankai Hecheng Technology Co., Ltd., batch number HA2X1209, loading 0.48 mmol / g) to the 100 L solid phase synthesis reactor via a solid addition funnel. Control the temperature at 20-30°C. Add N,N-dimethylformamide (21.00 kg + 21.00 kg) and the reaction solution from the previous galvanized bucket for later use. The system was kept warm and the reaction was monitored until the solid loading reached ≥250 μmol / g. UV loading was used for detection. The system was filtered under nitrogen pressure, and the filter cake was rinsed three times with N,N-dimethylformamide (26.00 kg + 26.10 kg + 26.00 kg), leaving the filter cake in the reactor. CAP.A (4.40 kg + 4.42 kg + 4.30 kg) and CAP.B (4.40 kg + 4.40 kg + 4.47 kg) were added to an 80-L glass reactor and stirred for 3-8 minutes before use. This operation was repeated three times to cap the reactor. Acetonitrile (18.00 kg + 18.00 kg + 18.00 kg + 17.50 kg + 17.50 kg) was added to the solid-phase synthesis reactor and nitrogen bubbled for 10-30 minutes before filter pressure. Repeat this operation four times, and transfer the filter cake to a 50L filter press after nitrogen purge in the solid phase synthesis reactor for 2 to 4 hours, control the temperature at 15 to 30°C, and continue drying to obtain a yellow to white solid product with a weight of 3.516 kg.

[0445] Example 5. In vivo evaluation of oligomeric compounds comprising monomers of the present invention

[0446] The oligomeric compound comprising the monomer of the present invention is obtained by the method described herein or known to those skilled in the art. The sequence structure of the oligomeric compound is shown in Table 2 as a siRNA duplex targeting FXII.

[0447] As shown in Table 2, the duplex comprises a sense strand and an antisense strand; chemical modifications are represented as follows: uppercase: 2'-fluoro modified nucleotides; lowercase: 2'-methoxy modified nucleotides; "*" should be understood as meaning that when present in an oligonucleotide, these monomers are interconnected by phosphorothioate diester nucleoside linkages, and the absence of "*" between the two monomers indicates that they are interconnected by phosphorothioate diester nucleoside linkages; Invab: inverted abasic, GLS15* and GLO15 are the targeting groups described herein, respectively.

[0448] "imann" at the end of an oligomer or dsRNA is represented as: "imann" is indicated when an oligomer or dsRNA is further linked to a targeting group.

[0449] Example 6. In vivo testing of FXII double-stranded nucleoside nucleic acid (also known as dsRNA)

[0450] To evaluate the in vivo activity of FXII double-stranded nucleoside nucleic acid (also known as dsRNA), female C57BL / 6 mice, 6 weeks old, specific pathogen-free, purchased from Shanghai Slake Laboratory Animal Co., Ltd., were used.

[0451] a) Compounds

[0452] Vehicle: PBS, Test compounds: AD00823, AD00824, AD00825, AD00826, AD00835, AD00836, AD00841, AD00827, AD00449.

[0453] b) Experimental plan

[0454] Animal experiment: Each group consisted of 4 C57BL / 6 female mice. The dose was 1 mg / kg. The administration frequency was SC. The drug was administered once on day 0. Blood was collected on days 7, 14, and 21 to measure the plasma FXII protein level.

[0455] c) Definition of experimental days: The day of the first administration of the mice was defined as day 0 of the experiment, the day before that was day -1, the day after that was day 1, and so on.

[0456] d) All test compounds were prepared into 5 mg / mL stock solutions in PBS prior to administration. A small amount of the 5 mg / mL stock solution was diluted 20-fold and the OD value was measured using a Nanodrop analyzer. The actual concentration of the stock solution was calculated based on the OD value, and an appropriate amount was diluted to the working solution concentration (0.2 mg / mL) for administration.

[0457] e) All mice (C57BL / 6, female, 6 weeks) were acclimated for 6 days and then administered subcutaneously on day 0 as described above.

[0458] f) On days 7, 14, and 21, all mice were bled through the submandibular vein to collect plasma for FXII protein level detection. Sample detection and analysis

[0459] The FXII protein level in mouse plasma was detected by ELISA kit.

[0460] FXII protein levels in mouse plasma were measured using an ELISA kit (Molecular Innovations, IMSFXIIKTT). Briefly, plasma samples collected with EDTA-K2 were diluted 30,000-fold and added to a capture antibody-coated assay plate for incubation. Detection antibody and HRP-conjugated secondary antibody were then added sequentially. Finally, TMB was used for color development, and absorbance was read at 450 nm. A four-parameter method was used to fit the standard curve, and the OD values ​​of the test samples were substituted into the standard curve to calculate the FXII protein content of each sample. Multiplying by the dilution factor yielded the original plasma FXII protein concentration. The results are shown in Table 3.

[0461] Table 3 Remaining percentage (retention) of FXII protein expression in mouse plasma NA means not detected.

[0462] Double-stranded nucleoside nucleic acid (also known as dsRNA) reagents AD00824 and AD00826 have oligomeric compounds containing the imann monomer compound described herein at the 5' end, 3' end, or both ends of the sense strand. Compared with double-stranded nucleoside nucleic acid (also known as dsRNA) reagents such as AD00823 and AD00825 that do not contain or contain other nucleotides, they all show better silencing effects. This indicates that when the monomer described herein is contained at the 5' end and / or 3' end of the sense strand of the double-stranded nucleoside nucleic acid (also known as dsRNA), it has better resistance to nuclease cleavage, which makes the sense strand stable and improves the in vivo effect of the antisense. The same results can be observed in AD00827VSAD00841.

[0463] In the double-stranded nucleoside nucleic acid (also known as dsRNA) reagent, AD00835, whose antisense chain contains the imann monomer compound herein, is compared with AD00836 (containing invab). Based on the activity data within the same exposure time, this actually proves that when added to the 5'-end of the antisense chain, the antisense is blocked by the imann monomer compound herein in terms of nuclease resistance and kills its activity. This indicates that the compound herein has a higher nuclease resistance than invab among oligomeric compounds and has higher stability.

[0464] Example 7 In vivo testing of FXII double-stranded nucleoside nucleic acid (also known as dsRNA)

[0465] The oligomeric compound comprising the monomer of the present invention is obtained by the method described herein or known to those skilled in the art. The sequence structure of the oligomeric compound is shown in Table 4 as a siRNA duplex targeting FXII.

[0466] As shown in Table 4, the duplex comprises a sense strand and an antisense strand; chemical modifications are indicated as follows: uppercase: 2'-fluoro modified nucleotides; lowercase: 2'-methoxy modified nucleotides; "*" should be understood as meaning that when present in an oligonucleotide, these monomers are linked to each other via phosphorothioate diester nucleoside linkages, and the absence of "*" between the two monomers indicates that they are linked to each other via oxophosphodiester nucleoside linkages; Invab: inverted abasic.

[0467] "imann" at the end of an oligomer or dsRNA is represented as: "imann" is indicated when an oligomer or dsRNA is further linked to a targeting group. "Stab8" at the end of an oligomer or dsRNA is represented as: "Stab8" is indicated when an oligomer or dsRNA is further linked to a targeting group. "Stab9" at the end of an oligomer or dsRNA is represented as: "Stab8" is indicated when an oligomer or dsRNA is further linked to a targeting group.

[0468] To evaluate the in vivo activity of FXII double-stranded nucleoside nucleic acid (also known as dsRNA), female C57BL / 6 mice, 6 weeks of age, were used.

[0469] a) Compounds

[0470] Vehicle: PBS, Test compounds: AD00734, AD00737, AD01970, AD01971.

[0471] b) Experimental plan

[0472] Animal experiments were conducted with 4 C57BL / 6 female mice in each group. The mice were given a dose of 0.5 or 1 mg / kg, and the administration frequency was SC, once on day 0. Blood was collected on days 7, 14, 21, and 28 to detect plasma FXII protein levels.

[0473] c) Definition of experimental days: The day of the first administration of the mice was defined as day 0 of the experiment, the day before that was day -1, the day after that was day 1, and so on.

[0474] d) All test compounds were prepared into 5 mg / mL stock solutions in PBS prior to administration. A small amount of the 5 mg / mL stock solution was diluted 20-fold and the OD value was measured using a Nanodrop analyzer. The actual concentration of the stock solution was calculated based on the OD value, and an appropriate amount was diluted to the working solution concentration (0.2 mg / mL) for administration.

[0475] e) All mice (C57BL / 6, female, 6 weeks) were acclimated for 6 days and then administered subcutaneously on day 0 as described above.

[0476] f) On days 7, 14, 21, and 28, all mice were bled through the submandibular vein to collect plasma for the detection of FXII protein levels.

[0477] Sample testing and analysis

[0478] An ELISA kit (Molecular Innovations, IMSFXIIKTT) was used to measure FXII protein levels in mouse plasma. Briefly, plasma samples collected with EDTA-K2 were diluted 30,000-fold and added to a capture antibody-coated assay plate for incubation. The detection antibody and HRP-conjugated secondary antibody were then added sequentially. Finally, TMB was used for color development, and the absorbance at 450 nm was read. A four-parameter method was used to fit the standard curve, and the OD values ​​of the test samples were substituted into the standard curve to calculate the FXII protein content of each sample. Multiplying by the dilution factor yielded the original plasma FXII protein concentration. The results are shown in Table 5.

[0479] Table 5. Relative expression levels of FXII protein using compounds corresponding to the sequences, chemical modifications, and delivery shown in Table 4.

[0480] Example 8. In vivo evaluation of oligomeric compounds comprising monomers of the present invention

[0481] The oligomeric compound comprising the monomer of the present invention is obtained by the method described herein or known to those skilled in the art. The sequence structure of the oligomeric compound is shown in Table 6 as a siRNA duplex targeting SOD1.

[0482] Table 6 shows that the duplex contains a sense strand and an antisense strand; chemical modifications are represented as follows: uppercase: 2'-fluoro modified nucleotides; lowercase: 2'-methoxy modified nucleotides; "*" should be understood as meaning that when present in an oligonucleotide, these monomers are linked to each other by phosphorothioate diester nucleoside linkages, and the absence of "*" between the two monomers indicates that they are linked to each other by phosphorothioate diester nucleoside linkages; Invab: inverted abasic.

[0483] "imann" at the end of an oligomer or dsRNA is represented as: "imann" is indicated when an oligomer or dsRNA is further linked to a targeting group. Lipd01 is expressed as Lipd02 is expressed as Lipd03 is expressed as C16 is represented by C16 H 33 ,(C16)*(imann) means that C16 and imann are connected to each other through a phosphorothioate diester nucleoside linkage. VPu is expressed as

[0484] To evaluate the in vivo activity of SOD1 double-stranded nucleoside nucleic acid (dsRNA), mice were intracerebroventricularly (ICV) administered

[0485] Male C57 mice (~20 g) were randomly divided into the following groups: aCSF group (solvent control group, n=5), AD00509-6 group (n=4), AD00509-7 group (n=3), AD00509-8 group (n=4), AD00509-9 group (n=3), AD00509-10 group (n=5), and AD00509-12 group (n=3). Each test compound was dissolved in artificial cerebrospinal fluid (aCSF) and diluted to a 50 mg / ml injection solution, and then administered at a dose of 2 μL per mouse. The specific steps are as follows: use Zotai + xylazine hydrochloride to anesthetize the mouse by intraperitoneal injection. After the anesthesia takes effect, fix the mouse skull on the brain stereotaxic instrument and use a 10μL microsyringe (Hamilton) to inject the drug into the mouse's lateral ventricle. The injection time is 10 minutes. After the injection, the needle is retained for 5 minutes, and then the needle is slowly removed. The head skin is sutured and the mouse is returned to the cage for continued feeding after it wakes up.

[0486] On the 8th day after injection, each mouse was anesthetized intraperitoneally and perfused with normal saline. The prefrontal cortex (PFC), striatum, hippocampus, and cerebellum tissues of the mice were quickly extracted and stored in a -80°C refrigerator. Subsequently, total RNA was extracted and reverse transcribed into cDNA using the RNeasy Kit (QIAGEN) according to the instructions. The expression of the target gene SOD1 was detected by qPCR using FastStart Universal SYBR Green Master (ROX) (Roche). -ΔΔCt The quantitative analysis was performed and the results are shown in Table 7.

[0487] Table 7: Remaining percentage of SOD1 expression in each tissue (retention)

[0488] Dose-response experimental protocol

[0489] Male C57 mice (~20 g) were randomly divided into: aCSF group (solvent control group, n=5), AD00509-8 group (0.1 mg, 0.033 mg, 0.01 mg), AD00509-9 group (0.1 mg, 0.033 mg, 0.01 mg), and AD00509-12 group (0.1 mg, 0.033 mg, 0.01 mg). The intracerebroventricular injection method of mice was as described above. On the 8th day after injection, each mouse was anesthetized intraperitoneally and perfused with normal saline. The prefrontal cortex (PFC), striatum, hippocampus and cerebellum tissues of the mice were quickly extracted and stored in a -80°C refrigerator. Subsequently, total RNA was extracted and cDNA was reverse transcribed using the RNeasy Kit (QIAGEN) according to the instructions. The expression of the target gene SOD1 was then detected by qPCR using FastStart Universal SYBR Green Master (ROX) (Roche). 2 -ΔΔCt The results were shown in Table 8.

[0490] Table 8: Remaining percentage of SOD1 expression in each tissue (retention)

[0491] equivalent

[0492] Although several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily appreciate that a variety of other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages, as well as each of these variations and / or modifications, are considered to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on the specific application in which the teachings of the present invention are used. Those skilled in the art will recognize or be able to ascertain, using only routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Therefore, it should be understood that the foregoing embodiments are presented by way of example only and are within the scope of the appended claims and their equivalents, and that the present invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, provided that such features, systems, articles, materials, and / or methods are not mutually inconsistent, is also within the scope of the present invention.

[0493] All definitions, as defined and used herein, should be understood against dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0494] Where no quantity limitation is used in the specification and claims, it should be understood as "at least one" unless explicitly stated to the contrary.

[0495] The phrase "and / or" as used in the specification and claims should be understood to mean "one or both" of the elements so conjoined, i.e., such elements are present in combination in some cases and separately in other cases. In addition to the elements specifically identified by "and / or," other elements may optionally be present, whether related or unrelated to those specifically identified elements, unless explicitly stated to the contrary.

[0496] All references, patents and patent applications and publications cited or referenced in this application are hereby incorporated by reference in their entirety.

Claims

1. A compound having the formula (I) or a stereoisomer thereof: A1 and A2 are independently selected from the group consisting of O, S, SO, SO2, NR2 and CR3R4, and R2, R3 and R4 are independently selected from the group consisting of hydrogen, halogen, sulfonyl, sulfinyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl; One of T1 and T2 is a protecting group, Z, L, or -ZL, wherein Z represents a targeting group or a lipophilic group, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and L represents a carrier spacer or a carrier portion linked via a spacer; and the other of T1 and T2 is an active phosphorus group or a protecting group; and T1 and T2 cannot be protecting groups at the same time; R1 is independently selected from halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl; n is an integer from 0 to 6; Wherein formula (I) does not include the following structure:

2. The compound according to claim 1, wherein A1 is O.

3. The compound according to any one of claims 1 to 2, wherein A2 is O.

4. The compound according to claim 1, wherein A1 and A2 are each independently S; A1 and A2 are each independently NR2, wherein R2 is hydrogen or CH3; A1 is O, A2 is S; A1 is O, A2 is NR2; Or A1 is S and A2 is NR2.

5. The compound according to claim 1, wherein one of T1 and T2 is a carrier spacer or a carrier portion linked by a spacer; the carrier spacer is preferably a dicarboxylic acid-derived group , R is absent, ether, polyethylene glycol, oxygen, sulfur, nitrogen, alkylene, alkenyl, alkynyl, aryl, aralkyl, heteroalkyl or heteroaryl; the carrier spacer is preferably a C3-C6 alkyl dicarboxylate group; more preferably a succinic acid group; the carrier is preferably poly(vinyl methacrylate) copolymer, silicon chip glass, cellulose, polystyrene beads, polypropylene sheets, non-porous silicon beads, polyacrylamide or polyacrylate; more preferably long-chain alkylamine controlled pore glass (LCAA-CPG).

6. The compound according to claim 1, One of the protecting groups of T1 and T2 is a hydroxy protecting group, and the hydroxy protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilane alkyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyloxycarbonyl, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl or substituted 9-phenylxanthin-9-yl; preferred hydroxy protecting groups are each independently selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and 4,4'-dimethoxytrityl; One of the protecting groups of T1 and T2 is a thiol protecting group, and the thiol protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethyl Silyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyloxycarbonyl, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl or substituted 9-phenylxanthin-9-yl; preferred thiol protecting groups are each independently selected from benzyl, 4,4'-dimethoxytrityl; One of the protecting groups of T1 and T2 is an amino protecting group, and the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihalogen Acetyl, benzoyl, nitrophenyl, acetyl, 2-nitrobenzenesulfonyl, phthalimido and dithiosuccinyl.

7. The compound of claim 1, wherein one of T1 and T2 is a lipophilic group, wherein the lipophilicity of the lipophilic group is determined by logKow, and logKow is greater than 0; preferably, the lipophilic group has a logKow greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5 or greater than 10.

8. The compound of claim 7, wherein the lipophilic group is selected from a saturated or unsaturated C4-C 30 Hydrocarbon chains, aliphatic rings, aromatic groups, fatty acid groups or fatty acid-derived groups, steroid-derived groups and any fat-soluble vitamin groups.

9. The compound according to any one of claims 7 to 8, wherein the lipophilic group is a saturated or unsaturated C 10 -C 18 hydrocarbon chain, any saturated or unsaturated fatty acid containing a hydrocarbon chain having 4 to 28 carbon atoms and may contain one or two carboxyl groups, cholesterol, vitamin E (tocopherol) or bile acid; more preferably: saturated straight chain C 14 、C 16 or C 18 Alkyl, Caprylic, Decanoic, Dodecanoic, Tetradecanoic, Hexadecanoic, Octadecanoic, Eicosanoic, Docosanoic and Tetracosanoic Acids, Myristoleic, Palmitoleic, Hexadecenoic, Oleic, Elaidic, Trans-Veneric, Linoleic, Elaidic, Alpha-Linolenic, Arachidonic and Erucic Acids, Docosahexaenoic and Eicosapentaenoic Acids (DHA), Eicosapentaenoic and Docosanoic Acids (DCA), Sterol Cholesterol (Bile), Tocopheryl Succinate (TS) and Lithocholic Acid (LA), Retinoic Acid (Vitamin A).

10. The compound according to any one of claims 7 to 9, wherein the lipophilic group is covalently linked to A1 or A2 via a linker.

11. The compound of claim 10, wherein the linker is selected from the group consisting of an amide bond, a phosphatidylcholine, a hydrocarbon linker or a polyethylene glycol (PEG) linker, an amino-alkyl-alcohol, hydroxyproline, hydroxyprolinol, an amino-alkyl-phosphorothioate linker, an amino-PEG-phosphorothioate linker, an α-carboxylate-amino-alkylphosphorothioate linker and an α-carboxylate-amino-PEG-phosphorothioate linker, an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide bond, a product of a click reaction, or a carbamate.

12. The compound of claim 10, wherein the linker is a degradable linker selected from the group consisting of a redox-cleavable linker, an acid-cleavable linker, an esterase-cleavable linker, a phosphatase-cleavable linker, or a peptidase-cleavable linker; or a biocleavable linker selected from the group consisting of functionalized monosaccharides or oligosaccharides of DNA, RNA, disulfides, amides, galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof. The compound according to claim 1 , wherein one of T1 and T2 is a targeting group.

14. The compound of claim 13, wherein the targeting group can be selected from one or more ligands comprising the following targeting molecules or their derivatives: polymers, carbohydrates, receptor ligands expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands.

15. The compound of claim 14, wherein each of the targeting groups is independently a ligand having affinity for an asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes, and the targeting group is preferably independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D- -galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyl-36-ylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-tri-O-acetyl-1-thio-6 The ligand is one or more of the following: O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside heptanoside ethyl ester, 2,5-anhydro-D-allose nitrile, ribose, D-ribose, D-4-thioribose, L-ribose, L-4-thioribose; more preferably, the ligand comprises galactose or N-acetylgalactosamine.

16. The compound according to claim 13, wherein the targeting group connected to one of A1 or A2 is selected from one of the following compound fragments:

17. The compound according to any one of claims 1 to 16, wherein the other of T1 and T2 is an active phosphorus group, and the active phosphorus group has the following structure: Wherein M1 is H, substituted or unsubstituted C1-C6 alkyl, OR5, SR5, OH, SH or NR6R7, M2 is OH, SH, OR5′ or NR6′R7′, each R5, R6, R7, R5′, R6′ or R7′ is independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or sulfonyl; m is 0 or 1.

18. The compound according to claim 17, wherein M1 is selected from methyl, ethyl, propyl, isopropyl, OR5, and methanesulfonyl, and R5 is selected from substituted or unsubstituted methyl, ethyl, propyl, and isopropyl.

19. The compound according to any one of claims 17 to 18, wherein R5, R6 or R7 is independently a substituted alkyl group, wherein the substituent is selected from cyano, halogen, hydroxyl, and amino.

20. The compound of any one of claims 17-19, wherein M2 is selected from N(CH(CH3)2)2.

21. The compound of claim 17, wherein M1 is O(CH2)2CN, M2 is N(CH(CH3)2)2, and m is 0.

22. The compound according to any one of claims 1 to 16, wherein the other of T1 and T2 is a reactive phosphorus group, and the reactive phosphorus group is selected from diisopropylcyanoethoxyphosphoramidite, diisopropylmethylphosphoramidite, diisopropylethylphosphoramidite and H-phosphonate.

23. The compound of any one of claims 1 to 22, wherein one of T1 and T2 is diisopropylcyanoethoxyphosphoramidite and the other is 4,4'-dimethoxytrityl, a targeting group, or a lipophilic group; or one of T1 and T2 is a protecting group and the other is a carrier spacer, a carrier moiety linked via a spacer, a targeting group, or a lipophilic group.

24. The compound of any one of claims 1 to 23, wherein R1 is independently selected from halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, or substituted or unsubstituted C2-C6 alkynyl.

25. The compound of any one of claims 1 to 24, wherein n is 0.

26. The compound according to any one of claims 1 to 25, wherein the compound has the configuration of formula (IIa) or formula (IIb): A1, A2, T1, T2, R1 and n are as defined in claims 1-25.

27. The compound of any one of claims 1 to 26, wherein the compound provided herein has the configuration of Formula (IIa-1) or Formula (IIb-1): A1, A2, T1, T2, R1 and n are as defined in claims 1-25.

28. The compound according to any one of claims 1 to 27, having the following specific structure: N- is a solid phase carrier (LCAA-CPG), N- is a solid phase carrier (LCAA-CPG), 29. An oligomeric compound comprising at least one 5′-terminal and / or 3′-terminal monomer having formula (III) or a stereoisomer thereof: A1 and A2 are independently selected from the group consisting of O, S, SO, SO2, NR2 and CR3R4, and R2, R3 and R4 are independently selected from the group consisting of hydrogen, halogen, sulfonyl, sulfinyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl; One of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (III) or its stereoisomer to the oligomeric compound; R1 is independently selected from halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C5-C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl; n is an integer from 0 to 6; The oligomeric compound optionally further comprises a targeting group. The oligomeric compound according to claim 29 , wherein A 1 is O.

31. The oligomeric compound according to any one of claims 29 to 30, wherein A2 is O.

32. The oligomeric compound of claim 29, wherein A1 and A2 are each independently S; A1 and A2 are each independently NR2; A1 is O, A2 is S; A1 is O, A2 is NR2; A1 is S, A2 is NR2; wherein R2 is hydrogen, methylsulfonyl or CH3.

33. The oligomeric compound according to any one of claims 29 to 32, wherein the other of T3 and T4 is an internucleoside linking group connecting the monomer to the 5′-terminus and / or 3′-terminus of the oligomeric compound, and the internucleoside linking group is selected from a phosphorus-containing linking group or a phosphorus-free linking group.

34. The oligomeric compound of claim 33, wherein the phosphorus-containing internucleoside linking group has the structural fragment shown: X represents H, substituted or unsubstituted C1-C6 alkyl, OR8, SR8', OH, SH or NR9R 10 , Y represents O or S, z can be 0 or 1, R8, R8', R9 or R 10 are independently hydrogen, substituted or unsubstituted C1-C6 alkyl, sulfonyl, They independently represent the portion connected to one of A1 or A2, and the portion connected to the connected nucleotide.

35. The oligomeric compound of claim 33, wherein the phosphorus-containing internucleoside linking groups are independently selected from the group consisting of: a phosphodiester linking group, a phosphotriester linking group, a phosphorothioate linking group, a phosphorodithioate bond linking group, an alkylphosphonate linking group, an aminophosphonate linking group, a phosphonate linking group, a phosphinate linking group, a thiophosphoramidate linking group, and an aminophosphoroate linking group.

36. The oligomeric compound according to any one of claims 29 to 35, wherein one of the protecting groups of T3 and T4 is a hydroxy protecting group, and the hydroxy protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethyl Silyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyloxycarbonyl, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl or substituted 9-phenylxanthin-9-yl; preferred hydroxy protecting groups are each independently selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and 4,4'-dimethoxytrityl; One of the protecting groups of T3 and T4 is a thiol protecting group, and the thiol protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethyl Silyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyloxycarbonyl, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl or substituted 9-phenylxanthin-9-yl; preferred thiol protecting groups are each independently selected from benzyl, 4,4'-dimethoxytrityl; One of the protecting groups of T3 and T4 is an amino protecting group, and the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrophenyl, acetyl, 2-nitrobenzenesulfonyl, phthalimido and dithiosuccinyl.

37. The oligomeric compound according to any one of claims 29 to 35, wherein one of T3 and T4 is hydrogen, and the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (III) or its stereoisomer to the 5'-terminus and / or 3'-terminus of the oligomeric compound.

38. The oligomeric compound of any one of claims 29 to 35, wherein one of T3 and T4 is selected from a lipophilic group, wherein the lipophilicity of the lipophilic group is determined by logKow, and the logKow is greater than 0; preferably the logKow of the lipophilic group is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5 or greater than 10.

39. The oligomeric compound of claim 38, wherein the lipophilic group is selected from a saturated or unsaturated C4-C 30 Hydrocarbon chains, aliphatic rings, aromatic groups, fatty acid groups or fatty acid-derived groups, steroid-derived groups and any fat-soluble vitamin groups.

40. The oligomeric compound according to any one of claims 38 to 39, wherein the saturated or unsaturated lipophilic group is a saturated or unsaturated C 10 -C 18 hydrocarbon chain, any saturated or unsaturated fatty acid containing a hydrocarbon chain having 4 to 28 carbon atoms, and may contain one or two carboxyl groups, cholesterol, vitamin E (tocopherol) or bile acid; more preferably: saturated straight chain C 16 Alkyl, Caprylic, Decanoic, Dodecanoic, Tetradecanoic, Hexadecanoic, Octadecanoic, Eicosanoic, Docosanoic and Tetracosanoic Acids, Myristoleic, Palmitoleic, Hexadecenoic, Oleic, Elaidic, Trans-Veneric, Linoleic, Elaidic, Alpha-Linolenic, Arachidonic and Erucic Acids, Docosahexaenoic and Eicosapentaenoic Acids (DHA), Eicosapentaenoic and Docosanoic Acids (DCA), Sterol Cholesterol (Bile), Tocopheryl Succinate (TS) and Lithocholic Acid (LA), Retinoic Acid (Vitamin A). The oligomeric compound according to any one of claims 37 to 39 , wherein the lipophilic group is covalently linked to A1 or A2 via a linker.

42. The oligomeric compound of claim 41 , wherein the lipophilic group, the linker, is selected from the group consisting of an amide bond, a phosphatidylcholine, a hydrocarbon linker or a polyethylene glycol (PEG) linker, an amino-alkyl-alcohol, hydroxyproline, hydroxyprolinol, an amino-alkyl-phosphorothioate linker, an amino-PEG-phosphorothioate linker, an α-carboxylate-amino-alkylphosphorothioate linker and an α-carboxylate-amino-PEG-phosphorothioate linker, an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide bond, a product of a click reaction, or a carbamate.

43. The oligomeric compound of claim 41 , wherein the linker is a degradable linker selected from the group consisting of a redox-cleavable linker, an acid-cleavable linker, an esterase-cleavable linker, a phosphatase-cleavable linker, or a peptidase-cleavable linker; or a biocleavable linker selected from the group consisting of functionalized monosaccharides or oligosaccharides of DNA, RNA, disulfides, amides, galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

44. The oligomeric compound according to any one of claims 29 to 35, further comprising one or more targeting groups at the 5'-end and / or the 3'-end of the oligomeric compound.

45. The oligomeric compound according to any one of claims 29 to 35, wherein one of T3 and T4 is represented by a covalent bond, and the oligomeric compound is connected to the targeting group in the form of a covalent bond via one of T3 and T4.

46. ​​The oligomeric compound according to any one of claims 44-45, wherein the targeting group can be selected from one or more ligands formed by the following targeting molecules or their derivatives: polymers, carbohydrates, receptor ligands expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands.

47. The oligomeric compound of claim 46, wherein each of the targeting groups is independently a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes, and the targeting groups are preferably independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D- D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyl-36-galactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-tri-O-acetyl-1-thio-6 The ligand is one or more of the following: O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside heptanoside ethyl ester, 2,5-anhydro-D-allose nitrile, ribose, D-ribose, D-4-thioribose, L-ribose, L-4-thioribose; more preferably, the ligand comprises galactose or N-acetylgalactosamine.

48. The oligomeric compound according to any one of claims 44-45, wherein the targeting group connected to one of A1 or A2 is selected from one of the following compound fragments:

49. The oligomeric compound of any one of claims 29 to 48, comprising at least one 5′-terminal and / or 3′-terminal monomer having a configuration represented by formula (IVa) or formula (IVb): A1, A2, T3, T4, R1 and n are as defined in claims 29-48.

50. The oligomeric compound according to any one of claims 29 to 48, comprising at least one 5′-terminal and / or 3′-terminal monomer having a configuration represented by formula (IVa-1) or formula (IVb-1): A1, A2, T3, T4, R1 and n are as defined in any one of claims 29-48.

51. The oligomeric compound according to any one of claims 29-50, wherein R1 is independently selected from halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, or substituted or unsubstituted C2-C6 alkynyl.

52. The oligomeric compound of any one of claims 29 to 51, wherein n is 0.

53. The oligomeric compound of any one of claims 29-52, wherein one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, the lipophilic group being optionally linked to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that links the monomer according to any one of claims 29-52 to the 5′-terminus of the oligomeric compound.

54. The oligomeric compound of any one of claims 29-52, wherein one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, the lipophilic group being optionally linked to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that links the monomer according to any one of claims 29-52 to the 3′-terminus of the oligomeric compound.

55. The oligomeric compound of any one of claims 29-52, wherein one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, the lipophilic group is optionally linked to one of A1 or A2 via a linker, and the other of T3 and T4 independently connects a same or different internucleoside linking group that connects the monomer according to any one of claims 29-52 to the 5′-terminus and the 3′-terminus of the oligomeric compound.

56. The oligomeric compound according to any one of claims 29 to 55, having the following specific structure: , wherein Olig represents an oligonucleotide portion that connects the monomer to the 5′-terminus and / or 3′-terminus of the oligomeric compound, respectively.

57. The oligomeric compound of any one of claims 29 to 56, which is a single-stranded oligonucleotide, including an antisense oligonucleotide (also known as an ASO), a ribozyme, or an aptamer.

58. A double-stranded ribonucleic acid (dsRNA) agent, comprising: a sense strand and an antisense strand, wherein the sense strand is fully or partially complementary to the antisense strand, and the antisense strand is complementary to a nucleic acid target gene; at least one of the sense strand and the antisense strand is an oligomeric compound as defined in any one of claims 29 to 56; and wherein the double-stranded ribonucleic acid (dsRNA) agent optionally further comprises an independent targeting group.

59. The double-stranded ribonucleic acid (dsRNA) agent of claim 58, wherein the sense strand is an oligomeric compound defined as comprising a 5′-terminal monomer according to any one of claims 29-52, 56, an oligomeric compound provided by a 3′-terminal monomer, or an oligomeric compound provided by both a 5′-terminal and a 3′-terminal monomer.

60. The double-stranded ribonucleic acid (dsRNA) agent of claim 58, wherein the antisense strand is an oligomeric compound comprising a 5′-terminal and / or 3′-terminal monomer according to any one of claims 29-52 and 56; preferably, the double-stranded ribonucleic acid (dsRNA) agent, wherein the antisense strand is an oligomeric compound comprising a 3′-terminal monomer according to any one of claims 29-52 and 56, or an oligomeric compound comprising a 5′-terminal monomer according to any one of claims 29-52 and 56.

61. The double-stranded ribonucleic acid (dsRNA) agent of any one of claims 58 to 60, further comprising one or more targeting groups on the 5'-terminal and / or 3'-terminal nucleotides of any one strand.

62. The oligomeric compound or double-stranded ribonucleic acid (dsRNA) of any one of claims 29 to 61, wherein each of the sense strand and the antisense strand comprises 8 to 40 nucleotides in length, respectively.

63. Use of the oligomeric compound or double-stranded ribonucleic acid (dsRNA) agent according to any one of claims 29 to 61 in the preparation of a drug for inhibiting gene expression.

64. The use of claim 63, wherein the drug is for intrahepatic administration and extrahepatic administration, and the drug for extrahepatic administration comprises a central nervous system (CNS) target gene selected from APP, ATXN2, C9orf72, TARDBP, HTT, SNCA, FUS, ATXN3, ATXN1, SCA7, SCA8, ATN1, MeCP2, prion disease, PRNP, DMPK, TTR, and a target gene of eye tissue: AMD, CFB, MYOC, ROCK2, ADRB2, CA2, CRYGC, PPP3C.

65. The use of claim 63, wherein the drug is used in CNS diseases including Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar disease, prion disease and Lafora disease; and ophthalmological diseases including age-related macular degeneration (AMD) (dry and wet), birdshot chorioretinopathy, dominant pigmentary vision, retinitis, Fuch's dystrophy, hereditary and sporadic glaucoma and Stargardt's disease, targeted glaucoma, cataracts, and dry eye syndrome.