Bicyclic abasic nucleic acid analogs and oligomeric compounds produced therefrom

Abstract

The present invention relates to bicyclic abasic nucleic acid analogs and oligomeric compounds produced thereby, more particularly to bicyclic abasic nucleic acid analogs and monomeric compounds of bicyclic abasic nucleic acid analogs that can be incorporated into one or two termini of oligomeric compounds to deliver the oligomeric compounds in vivo to cells requiring specific targeting, and can also deliver to target cells in extrahepatic tissues such as the eye and CNS in addition to liver-related tissues.

Description

[Technical Field]

[0001] The present invention relates to bicyclic abasic nucleic acid analogs and oligomeric compounds produced therefrom, and more particularly to bicyclic abasic nucleic acid analogs and bicyclic abasic nucleic acid analog monomers that can be used to incorporate into one or two termini of oligomeric compounds. [Background technology]

[0002] RNA interference, or "RNAi," technology is an effective means of disease therapy known in the art. The RNAi mechanism begins with the Dicer enzyme-mediated generation of longer non-coding RNAs from RNA molecules, which are then loaded into the RNA-induced silencing complex (RISC), where the sense strand is discarded and the antisense or guide strand hybridizes to a fully or partially complementary mRNA sequence, inducing mRNA silencing via Ago2-mediated degradation or translational repression. Advances in RNAi technology and delivery methods have led to increasingly positive results for RNAi-based therapy, which represents a promising therapeutic approach for diseases. However, such technology is hindered from widespread application due to the inherent metabolic problems inherent to natural RNA, such as the susceptibility of natural oligonucleotides to degradation by cellular and extracellular nucleases in vivo and the inherent metabolic challenges of natural RNA, such as targeting and in vivo stability. Therefore, in conventional techniques, surrounding nucleotides are modified, and these modifications are particularly useful for improving nuclease resistance, binding affinity, targeting, and in vivo stability.

[0003] The development of various chemical modifications applied to RNAi oligonucleotides has made significant progress in overcoming the inherent metabolic problems of natural RNA, and such chemical modifications of RNAi oligonucleotides can have a crucial facilitating effect on the potential of fully utilizing such therapeutic regimens, improving their pharmacokinetic and pharmacodynamic properties. For example, Choung et al. reported using 2'-OMe (2'-O-methyl), 2'-F (2'-fluoro), and phosphorothioate-modified nucleotides and their various combinations to achieve serum stability, and further including other modifications of the nucleotide, such as where the furanose moiety of the nucleoside contains a bridge connecting two atoms on the furanose to form a bicyclic ring system. Bicyclic nucleosides are variously called bicyclic nucleic acids or locked nucleic acids, referred to as BNA and LNA, respectively, and furanose-opened forms such as UNA and GNA, to enhance thermal stability. It has also been reported that 5'-terminal phosphate ester groups have been incorporated into oligonucleotides to enhance the interaction between specific nucleic acid inhibitor molecules and Ago2. For example, phosphate mimetics at the 5' end of the antisense strand have also been a recent research direction to improve the silencing effect of RNAi. However, all of these modified nucleotide derivatives retain the base moiety and still require a base pairing process. Recently, in order to enhance the silencing effect of oligonucleotides and improve the stability of oligonucleotides, abasic nucleotides and reverse abasic nucleotides applied to 5' or 3' oligonucleotides have been adopted, for example, WO2022162155.

[0004] Although the development and improvement of delivery methods through chemical modification have made great progress in overcoming the inherent metabolic problems of natural RNA, there remains a need in the field for RNAi agents with enhanced in vivo efficacy and stability suitable for therapeutic administration. In particular, research into abasic nucleotides is still in its infancy, and it is desirable to provide further oligonucleotides incorporating novel abasic nucleic acid analogs that have improved stability against anti-cellular nucleases, longer in vivo efficacy, and higher affinity for nucleic acid targets. Summary of the Invention

[0005] The present invention relates to bicyclic abasic nucleic acid analogs and oligomeric compounds produced thereby, more particularly to bicyclic abasic nucleic acid analog monomeric compounds, and to bicyclic abasic nucleic acid analog monomeric compounds that can be incorporated into one or two termini of oligomeric compounds, and these oligomeric compounds can be applied to deliver to extrahepatic tissues such as ophthalmic and CNS target cells in addition to targeting extracellular liver-related tissues.

[0006] According to one aspect of the present invention, there is provided a compound of formula (I) or a stereoisomer thereof, [ka] A1 and A2 are each independently selected from O, S, SO, SO2, NR2 and CR3R4, and R2, R3 and R4 are each independently selected from hydrogen, halogen, sulfonyl group, sulfinyl group, substituted or unsubstituted C1-C6 alkyl group, substituted or unsubstituted C3-C6 cycloalkyl group, substituted or unsubstituted C2-C6 alkenyl group, substituted or unsubstituted C2-C6 alkynyl group, substituted or unsubstituted C5-C6 alkyl group, substituted or unsubstituted C5-C6 cycloalkyl ... alkyl group, substituted or unsubstituted C5-C6 alkyl group, substituted or unsubstituted C5-C6 alkyl group, substituted or 12 an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, or a substituted or unsubstituted 5- to 12-membered heterocyclyl group; one of T1 and T2 is a protecting group, Z, L, or -ZL, wherein Z represents a targeting group or a lipophilic group, and the lipophilic group is optionally linked to one of A1 or A2 via a linker; L represents a carrier spacer or comprises a carrier moiety linked by a spacer; the other of T1 and T2 is an active phosphorus group, a protecting group, and T1 and T2 are not simultaneously a protecting group; R1 is independently a halogen, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted C2-C6 alkenyl group, a substituted or unsubstituted C2-C6 alkynyl group, a substituted or unsubstituted C5-C 12 an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, or a substituted or unsubstituted 5- to 12-membered heterocyclyl group; n is an integer from 0 to 6, wherein formula (I) is [ka] It does not include the structure:

[0007] In some embodiments, A1 is O. In some embodiments, A2 is O. In some embodiments, A1 and A2 are each independently O. In some embodiments, A1 and A2 are each independently S. In some embodiments, A1 and A2 are each independently NR2. In some embodiments, R2 is hydrogen or CH3. In some embodiments, A1 is different from A2. In some embodiments, A1 is O and A2 is S. In some embodiments, A1 is O and A2 is NR2. In some embodiments, A1 is S and A2 is NR2.

[0008] In one embodiment, the protecting group of one of T1 and T2 above is different depending on A1 or A2, and corresponds to a hydroxy protecting group, a mercapto protecting group, and an amino protecting group, respectively.

[0009] In one embodiment, one protecting group of T1 and T2 is a hydroxy protecting group, and in one embodiment, the hydroxy protecting group is each independently an acetyl group, a tert-butyl group, a tert-butoxymethyl group, a methoxymethyl group, a tetrahydropyranyl group, a 1-ethoxyethyl group, a 1-(2-chloroethoxy)ethyl group, a 2-trimethylsilylethyl group, a p-chlorophenyl group, a 2,4-dinitrophenyl group, a benzyl group, a benzoyl group, a p-phenylbenzoyl group, a 2,6-dichlorobenzyl group, a diphenylmethyl group, a p-nitrobenzyl group, a trimethylsilyl group, a triethylsilyl group, a tert-butyldimethylsilyl group, a tert-butyldiphenyl group, a methylsilyl ... In one embodiment, preferred hydroxy protecting groups are each independently selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and 4,4'-dimethoxytrityl.

[0010] In one embodiment, one of the protecting groups of T1 and T2 is a mercapto protecting group, and in one embodiment, the mercapto protecting group is each independently an acetyl group, a tert-butyl group, a tert-butoxymethyl group, a methoxymethyl group, a tetrahydropyranyl group, a 1-ethoxyethyl group, a 1-(2-chloroethoxy)ethyl group, a 2-trimethylsilylethyl group, a p-chlorophenyl group, a 2,4-dinitrophenyl group, a benzyl group, a benzoyl group, a p-phenylbenzoyl group, a 2,6-dichlorobenzyl group, a diphenylmethyl group, a p-nitrobenzyl group, a trimethylsilyl group, a triethylsilyl group, a tert-butyl group, a methylsilyl ... In one embodiment, preferred mercapto protecting groups are each independently selected from benzyl and 4,4'-dimethoxytrityl groups.

[0011] In one embodiment, one of the protecting groups T1 and T2 is an amino protecting group, and in one embodiment, the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butyloxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrophenyl, 2-nitrobenzenesulfonyl, phthalimide, and dithiosuccinyl.

[0012] In some embodiments, one of T1 and T2 is a lipophilic group, wherein the lipophilicity of the lipophilic group can be measured by log Kow, and log Kow is greater than 0, preferably the lipophilic group log Kow 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 some embodiments, the lipophilic group is a saturated or unsaturated C4-C 30 It is selected from the group comprising hydrocarbon chains, aliphatic rings, aromatics, fatty acid groups or groups derived from fatty acids, steroid derived groups and any fat-soluble vitamin group.

[0013] 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 chains (e.g., linear 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., linear C 16 In one embodiment, the lipophilic group is a saturated linear C 14 , C 16 or C 18 The lipophilic group is optionally further substituted with a functional group selected from the group consisting of a hydroxy group, an amine, a carboxylic acid, a sulfonate ester, a phosphate ester, a thiol, an azide group, and an alkyne. These functional groups can be used to attach the lipophilic group to A1 or A2.

[0014] In another embodiment, the lipophilic group comprises any saturated or unsaturated fatty acid with a hydrocarbon chain having 4 to 28 carbon atoms and may contain one or two carboxy groups. In one embodiment, the lipophilic group is derived from a saturated fatty acid with a hydrocarbon chain having 8 to 24 carbon atoms. In a specific embodiment, the lipophilic group is derived from a saturated fatty acid, wherein the fatty acid is selected from the group consisting of octanoic acid, capric acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid, and tetracosanoic acid. In a specific embodiment, the lipophilic group is derived from an unsaturated fatty acid with a hydrocarbon chain having 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-isooleic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid, and erucic acid.

[0015] In certain embodiments, the lipophilic group is selected from cholesterol, vitamin E (tocopherol), or bile acids.

[0016] In certain embodiments, the lipophilic group is selected from docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosanoic acid (DCA), sterol cholesterol (bile), tocopherol succinate (TS), and lithocholic acid (LA), retinoic acid (vitamin A acid).

[0017] The lipophilic group can be covalently attached to A1 or A2 by direct attachment in the form of a covalent bond. Alternatively, the lipophilic group can be covalently attached to A1 or A2 via a linker.

[0018] In one embodiment, one of T1 and T2 is a lipophilic group, which is further covalently attached to A1 or A2 via a linker. In certain embodiments, the lipophilic group is covalently attached to A1 or A2 via one or more linkers.

[0019] 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-ol, 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. In some embodiments, 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 click reaction product (e.g., a triazole obtained by an azide-alkyne cycloaddition reaction), or a carbamate. In some embodiments, the phosphodiester is a thiophosphodiester or an oxophosphodiester.

[0020] In some embodiments, the linker comprises a cleavable group. At least one linker is a redox-cleavable linker (e.g., a reductively cleavable linker such as 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 monosaccharides or oligosaccharides, and combinations thereof.

[0021] In some embodiments, the linker may 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.

[0022] 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.

[0023] In some embodiments, the targeting group may be selected from one or more ligands including polymers, saccharides, ligands for receptors expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands, or derivatives thereof.

[0024] In some embodiments, at least one or each of the targeting groups is selected from ligands capable of binding to mammalian hepatocyte surface receptors.

[0025] In some embodiments, each of the targeting groups is independently a ligand that has affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes.

[0026] In some embodiments, each of the targeting groups is independently selected from 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-glucosidase, Furanose, 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, Nn-butyrylgalactosamine, N-isobutyrylgalactosamine thosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-ethanolacyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-tri-O The ligand comprises one or more selected from the group consisting of 4-acetyl-1-thio-6-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-glucoheptapyranoside ethyl ester, 2,5-anhydro-D-allonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

[0027] In some embodiments, at least one or each of the targeting groups is a ligand comprising galactose or N-acetylgalactosamine.

[0028] The targeting group can be linked to one of A1 or A2 via a suitable conjugation joint. Those skilled in the art can select a suitable conjugation joint for binding depending on the specific type of targeting group.

[0029] In certain embodiments, the targeting group is linked to one of A1 or A2, [ka] The compound fragment is selected from one of the following:

[0030] In some embodiments, one of T1 and T2 is a carrier spacer. In some embodiments, the carrier spacer is a dicarboxylic acid-derived group, an exemplary structure is [ka] wherein R is absent, an ether, polyethylene glycol, oxygen, sulfur, nitrogen, an alkylene group, an alkenyl group, an alkynyl group, an aryl group, an aralkyl group, a heteroalkyl group, or a heteroaryl group. In one embodiment, the carrier spacer is a polyalkylene glycol phosphate ester / phosphonate, and in one embodiment, the carrier spacer is preferably a C3-C6 alkyl dicarboxylate group. In one embodiment, the carrier spacer is preferably a succinyl group, [ka] In some embodiments, one of T1 and T2 is a carrier moiety linked by a spacer, wherein the carrier moiety includes a solid-phase carrier moiety and a liquid-phase carrier moiety. In some embodiments, the solid-phase carrier is selected from controlled-pore glass (CPG), polystyrene, silica gel, macroporous cross-linked polystyrene, poly(vinyl methacrylate alcohol copolymer), silicon-tipped glass, cellulose, polystyrene beads, polypropylene sheets, non-porous silicon beads, polyacrylamide, or polyacrylate. In some embodiments, the solid-phase carrier moiety is long-chain alkylamine controlled-pore glass (LCAA-CPG).

[0031] In certain embodiments, one of T1 and T2 is a targeting group or a lipophilic group and further comprises a carrier moiety linked by a spacer, in some more specific embodiments, wherein the carrier moiety is linked via the spacer to a carboxy group of a linker or conjugation joint in the targeting group or lipophilic group.

[0032] In one embodiment, the other of T1 and T2 is an activated phosphorus group, the activated phosphorus group being: [ka] It has the structure wherein M1 is H, a substituted or unsubstituted C1-C6 alkyl group, OR5, SR5, OH, SH or NR6R7, M2 is OH, SH, OR5' or NR6'R7', where each R5, R6, R7, R5', R6' or R7' is independently hydrogen, a substituted or unsubstituted C1-C6 alkyl group, a sulfonyl group, and m is 0 or 1.

[0033] In certain embodiments, M1 is selected from a methyl group, an ethyl group, a propyl group, and an isopropyl group.

[0034] In certain embodiments, M1 is selected from OR5, where R5 is selected from a substituted or unsubstituted methyl group, an ethyl group, a propyl group, and an isopropyl group.

[0035] In one embodiment, M1 is selected from a methylsulfonylamido group.

[0036] In certain embodiments, M2 is selected from N(CH(CH3)2)2.

[0037] In certain embodiments, each R5, R6, or R7 is independently a substituted alkyl group, wherein the substituent is selected from a cyano group, a halogen, a hydroxy group, and an amino group.

[0038] In one embodiment, M is O(CH)CN, M is N(CH(CH)), and m is 0.

[0039] In some embodiments, the other of T1 and T2 is an activated phosphorus group, and the activated phosphorus group is selected from diisopropylcyanoethoxyphosphoramidite, diisopropylmethylphosphoramidite, diisopropylethylphosphoramidite, and H-phosphonate.

[0040] 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, which is optionally linked to one of A1 or A2 via a linker.

[0041] In certain embodiments, one of T1 and T2 is diisopropylcyanoethoxyphosphoramidite and the other is a 4,4'-dimethoxytrityl group.

[0042] In certain embodiments, one of T1 and T2 is a diisopropylcyanoethoxyphosphoramidite and the other is a targeting group.

[0043] In certain embodiments, one of T1 and T2 is diisopropylcyanoethoxyphosphoramidite and the other is a lipophilic group.

[0044] In certain embodiments, each R1 is independently selected from halogen, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C2-C6 alkenyl group, and a substituted or unsubstituted C2-C6 alkynyl group.

[0045] In certain embodiments, n is 0.

[0046] In certain embodiments, the compounds provided herein have the configuration of Formula (IIa) or Formula (IIb): [ka] A1, A2, T1, T2, R1 and n are as defined above.

[0047] In certain embodiments, the compounds provided herein have the configuration of formula (IIa-1) or formula (IIb-1): [ka] A1, A2, T1, T2, R1 and n are as defined above.

[0048] According to another aspect of the present invention, there is provided an oligomeric compound comprising at least one 5'-end and / or 3'-end monomer having formula (III) or a stereoisomer thereof:

[0049] [ka] A1 and A2 are each independently selected from O, S, SO, SO2, NR2 and CR3R4, and R2, R3 and R4 are each independently selected from hydrogen, halogen, sulfonyl group, sulfinyl group, substituted or unsubstituted C1-C6 alkyl group, substituted or unsubstituted C3-C6 cycloalkyl group, substituted or unsubstituted C2-C6 alkenyl group, substituted or unsubstituted C2-C6 alkynyl group, substituted or unsubstituted C5-C6 alkyl group, substituted or unsubstituted C5-C6 cycloalkyl ... alkyl group, substituted or unsubstituted C5-C6 alkyl group, substituted or unsubstituted C5-C6 alkyl group, substituted or 12 an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, or a substituted or unsubstituted 5- to 12-membered heterocyclyl group; one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, said lipophilic group being linked to one of A1 or A2, optionally via a linker, and the other of T3 and T4 is an internucleoside linking group connecting said formula (III) monomer or a stereoisomer thereof to the oligomeric compound; R1 is independently a halogen, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted C2-C6 alkenyl group, a substituted or unsubstituted C2-C6 alkynyl group, a substituted or unsubstituted C5-C 12 an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, or a substituted or unsubstituted 5- to 12-membered heterocyclyl group; n is an integer from 0 to 6, The oligomeric compound optionally further comprises a targeting group.

[0050] In certain embodiments, oligomeric compounds are provided wherein A1 is O. In certain embodiments, oligomeric compounds are provided wherein A2 is O. In certain embodiments, oligomeric compounds are provided wherein A1 and A2 are each independently O. In certain embodiments, oligomeric compounds are provided wherein A1 and A2 are each independently S. In certain embodiments, oligomeric compounds are provided wherein A1 and A2 are each independently NR2. In certain embodiments, oligomeric compounds are provided wherein R2 is hydrogen or CH3. In certain embodiments, oligomeric compounds are provided wherein A1 is different from A2. In certain embodiments, oligomeric compounds are provided wherein A1 is O and A2 is S. In certain embodiments, oligomeric compounds are provided wherein A1 is O and A2 is NR2. In certain embodiments, oligomeric compounds are provided wherein A1 is S and A2 is NR2.

[0051] In certain embodiments, provided oligomeric compounds are provided wherein the other of T3 and T4 is an internucleoside linking group connecting a monomer of Formula (III) or a stereoisomer thereof to the 5'-terminus and / or 3'-terminus of the oligomeric compound, wherein the internucleoside linking group is selected from a phosphorus-containing linking group or a non-phosphorus-containing linking group.

[0052] In certain embodiments, provided are oligomeric compounds wherein the phosphorus-containing internucleoside linking group is [ka] wherein X is H, a substituted or unsubstituted C1-C6 alkyl group, OR8, SR8', OH, SH, or NR9R 10 Y represents O or S, z may be 0 or 1, and R, R', R or R 10 are independently hydrogen, a substituted or unsubstituted C1-C6 alkyl group, or a sulfonyl group; [ka] each independently represent a moiety linked to one of A1 or A2 herein and a moiety linked to an adjacent nucleotide.

[0053] In certain embodiments, the phosphorus-containing internucleoside linking groups are independently phosphodiester linking groups, phosphotriester linking groups, phosphorothioate linking groups, phosphorodithioate linking groups, alkylphosphonate linking groups, aminophosphonate linking groups, phosphonate linking groups, phosphinate linking groups, thiophosphoramidate linking groups, and phosphoramidate linking groups.

[0054] In certain embodiments, the internucleoside linking groups are independently alkylphosphonate linking groups, phosphodiester internucleoside linking groups, or phosphorothioate internucleoside linking groups.

[0055] In certain embodiments, in the provided oligomeric compounds, one protecting group of T3 and T4 above is different depending on A1 or A2 and corresponds to a hydroxy protecting group, a mercapto protecting group, and an amino protecting group, respectively.

[0056] In certain embodiments, in provided oligomeric compounds, one protecting group of T3 and T4 is a hydroxy protecting group, and in one embodiment, in provided oligomeric compounds, 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 ... In one embodiment, in provided oligomeric compounds, preferred hydroxy protecting groups are each independently selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and 4,4'-dimethoxytrityl.

[0057] In certain embodiments, in provided oligomeric compounds, one protecting group of T3 and T4 is a mercapto protecting group, and in one embodiment, in provided oligomeric compounds, the mercapto 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, and triethylsilyl. , tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethoxycarbonyl, methanesulfonyl, tosyl, trifluoromethanesulfonyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, or substituted 9-phenylxanthin-9-yl; in one embodiment, in provided oligomeric compounds, preferred mercapto protecting groups are each independently selected from benzyl and 4,4'-dimethoxytrityl.

[0058] In certain embodiments, provided oligomeric compounds are provided in which one protecting group of T3 and T4 is an amino protecting group, and in one embodiment, provided oligomeric compounds are provided in which the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butyloxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrophenyl, 2-nitrobenzenesulfonyl, phthalimido, and dithiosuccinyl.

[0059] In some embodiments, oligomeric compounds are provided wherein one of T3 and T4 is hydrogen and the other of T3 and T4 is an internucleoside linking group connecting a Formula (III) monomer or a stereoisomer thereof to the 5'-terminus and / or 3'-terminus of the oligomeric compound.

[0060] In provided oligomeric compounds, one of T3 and T4 is a lipophilic group, wherein in some embodiments, the lipophilicity of the lipophilic group can be measured by log Kow, where log Kow is greater than 0, and preferably the lipophilic group log Kow 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 some embodiments, provided oligomeric compounds, the lipophilic group is a saturated or unsaturated C4-C 30 It is selected from the group comprising hydrocarbon chains, aliphatic rings, aromatics, fatty acid groups or groups derived from fatty acids, steroid derived groups and any fat-soluble vitamin group.

[0061] In certain embodiments, in the provided oligomeric compounds, the lipophilic group is a saturated or unsaturated C 10 -C 25 Hydrocarbon chains (e.g., C 10 -C 25 In some embodiments, the oligomeric compounds provided herein include a lipophilic group selected from the group consisting of saturated and unsaturated C 10 -C 18 Hydrocarbon chains (e.g., linear C6-C 18 In one embodiment, the oligomeric compounds provided have lipophilic groups that are saturated or unsaturated C 14 , C 16 or C 18 Hydrocarbon chains (e.g., linear C 16 In one embodiment, the oligomeric compounds provided herein are lipophilic groups, which are saturated linear C 14 , C 16 or C 18The lipophilic group is optionally further substituted with a functional group selected from the group consisting of a hydroxy group, an amine, a carboxylic acid, a sulfonate ester, a phosphate ester, a thiol, an azide group, and an alkyne. These functional groups can be used to attach the lipophilic group to A1 or A2.

[0062] In another embodiment, oligomeric compounds are provided in which the lipophilic group comprises any saturated or unsaturated fatty acid with a hydrocarbon chain having 4 to 28 carbon atoms and optionally one or two carboxy groups. In one embodiment, oligomeric compounds are provided in which the lipophilic group is derived from a saturated fatty acid with a hydrocarbon chain having 8 to 24 carbon atoms. In particular embodiments, oligomeric compounds are provided in which the lipophilic group is derived from an unsaturated fatty acid, wherein the fatty acid is selected from the group consisting of octanoic acid, capric acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid, and tetracosanoic acid. In certain embodiments, provided are oligomeric compounds wherein the lipophilic group is derived from an unsaturated fatty acid, and the fatty acid is selected from the group consisting of myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, trans-isooleic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid, and erucic acid.

[0063] In certain embodiments, provided are oligomeric compounds in which the lipophilic group is selected from cholesterol, vitamin E (tocopherol), or bile acids.

[0064] In certain embodiments, provided oligomeric compounds are provided wherein the lipophilic group is selected from docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosanoic acid (DCA), sterol cholesterol (bile), tocopherol succinate (TS), and lithocholic acid (LA), retinoic acid (vitamin A acid).

[0065] The lipophilic group can be covalently attached to A1 or A2 by direct attachment in the form of a covalent bond. Alternatively, the lipophilic group can be covalently attached to A1 or A2 via a linker.

[0066] In one embodiment, oligomeric compounds are provided in which one of T3 and T4 is a lipophilic group, and the lipophilic group of T3 or T4 is further covalently attached to A1 or A2 via a linker. In certain embodiments, oligomeric compounds are provided in which the lipophilic group is covalently attached to A1 or A2 via one or more linkers.

[0067] In another embodiment, the oligomeric compound is provided, wherein the linker is a hydrocarbon chain linker or a polyethylene glycol (PEG) linker. In another embodiment, the oligomeric compound is provided, 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-ol, a hydroxyproline, a hydroxyprolinol, an amino-alkyl-phosphorothioate linker, an amino-PEG-phosphorothioate linker, an α-carboxylate-amino-alkyl phosphorothioate linker, and an α-carboxylate-amino-PEG-phosphorothioate linker. In some embodiments, 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 click reaction product (e.g., a triazole obtained by an azide-alkyne cycloaddition reaction), or a carbamate. In another embodiment, oligomeric compounds are provided wherein the phosphodiester linker is a thiophosphodiester, an oxophosphodiester.

[0068] In some embodiments, oligomeric compounds are provided in which the linker comprises a cleavable group. At least one linker is a redox-cleavable joint, e.g., a reduction-cleavable joint such as a disulfide group, an acid-cleavable joint (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase-cleavable joint (e.g., an ester group), a phosphatase-cleavable joint (e.g., a phosphate ester), or a peptidase-cleavable joint (e.g., a peptide bond). In other embodiments, at least one linker is a biocleavable joint selected from the group consisting of DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, mannose functionalized mono- or oligosaccharides, and combinations thereof.

[0069] In some embodiments, the linker in the provided oligomeric compound may 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.

[0070] In some embodiments, the 5'-end and / or 3'-end of the oligomeric compound further comprise one or more targeting groups. In some embodiments, the oligomeric compound is provided with one of T3 and T4 representing a covalent bond, and the oligomeric compound is linked to the targeting group in the form of a covalent bond via one of T3 and T4 in formula (III) or its stereoisomer. In some embodiments, the oligomeric compound is provided with the 5'-end and / or 3'-end nucleotide linked to a targeting group. The targeting group may be a ligand commonly used in the field of siRNA administration.

[0071] In some embodiments, provided oligomeric compounds include one or more ligands, including polymers, saccharides, ligands for receptors expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands, or derivatives thereof.

[0072] In some embodiments, provided oligomeric compounds are provided wherein at least one or each of the targeting groups is selected from a ligand capable of binding to a mammalian hepatocyte surface receptor.

[0073] In some embodiments, provided oligomeric compounds are provided wherein each of the targeting groups is independently a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes.

[0074] In some embodiments, provided are oligomeric compounds in which each of the targeting groups is independently selected from 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-glucose, and β-D-glucose. Copyranose, 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, Nn-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-carboxamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-ethanolacyl-α-neuraminic acid, 5-thio-β-D-glucopyrano The sugar is one selected from the group consisting of 2,3,4-tri-O-acetyl-1-thio-6-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-glucoheptapyranoside ethyl ester, 2,5-anhydro-D-allonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

[0075] In some embodiments, provided oligomeric compounds are provided wherein at least one or each of the targeting groups is a galactose or N-acetylgalactosamine-containing ligand.

[0076] In the provided oligomeric compounds, the targeting group can be linked to one of A1 or A2 via a suitable conjugation joint. Those skilled in the art can select a suitable conjugation joint depending on the specific type of targeting group.

[0077] In certain embodiments, provided are oligomeric compounds in which the targeting group is linked to one of A1 or A2, [ka] The compound fragment is selected from one of the following:

[0078] In certain embodiments, each R1 is independently selected from halogen, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C2-C6 alkenyl group, and a substituted or unsubstituted C2-C6 alkynyl group.

[0079] In certain embodiments, n is 0 in provided oligomeric compounds.

[0080] In certain embodiments, provided oligomeric compounds include 5'- and / or 3'-terminal monomers of at least one configuration as shown in formula (IVa) or formula (IVb), [ka] A1, A2, T3, T4, R1 and n are as defined above.

[0081] In certain embodiments, provided oligomeric compounds include 5'- and / or 3'-terminal monomers of at least one configuration as shown in formula (IVa-1) or formula (IVb-1), [ka] A1, A2, T3, T4, R1 and n are as defined above.

[0082] In certain embodiments, provided are oligomeric compounds in which 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 connecting 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.

[0083] In certain embodiments, provided are oligomeric compounds in which 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 connecting 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.

[0084] In certain embodiments, oligomeric compounds are provided in which 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 each independently an internucleoside linking group connecting a homologous or different (III) or stereoisomer thereof, Formula (IVa), Formula (IVb), (Iva-1) or Formula (IVb-1) monomer to the 5'-end and 3'-end of the oligomeric compound.

[0085] In certain embodiments, the oligomeric compound is a single-stranded oligonucleotide. In certain embodiments, the single-stranded oligonucleotide is a general antisense oligonucleotide (also known as ASO), a ribozyme, or an aptamer. In certain embodiments, the oligomeric compound is a double-stranded ribonucleic acid (dsRNA) reagent, and such compounds are double-stranded ribonucleic acids well known in the art, where one or both strands are the oligomeric compounds disclosed herein.

[0086] In certain embodiments, a double-stranded ribonucleic acid (dsRNA) reagent is provided, both of which comprise 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 partially or fully complementary to a nucleic acid target gene, and at least one of the sense strand and the antisense strand comprises at least one 5'-end and / or 3'-end monomer having Formula (III) or a stereoisomer thereof, Formula (IVa), Formula (IVb), Formula (IVa-1) or Formula (IVb-1), and is an oligomeric compound provided above, wherein the double-stranded ribonucleic acid (dsRNA) reagent optionally further comprises an independent targeting group.

[0087] In certain embodiments, double-stranded ribonucleic acid (dsRNA) reagents are provided, wherein the sense strand is an oligomeric compound provided by 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, double-stranded ribonucleic acid (dsRNA) reagents are provided, wherein the sense strand is an oligomeric compound provided by a 5'-terminal monomer of formula (III) or a stereoisomer thereof, formula (IVa), formula (IVb), formula (IVa-1), or formula (IVb-1) herein. In one embodiment, double-stranded ribonucleic acid (dsRNA) reagents are provided, wherein the sense strand is an oligomeric compound provided by 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) reagent is provided, wherein the sense strand is an oligomeric compound provided by the 5'-end and 3'-end monomers of homologous or different Formula (III) herein or a stereoisomer thereof, Formula (IVa), Formula (IVb), Formula (IVa-1) or Formula (IVb-1).

[0088] In certain embodiments, double-stranded ribonucleic acid (dsRNA) reagents are provided, wherein the antisense strand is an oligomeric compound provided by 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, double-stranded ribonucleic acid (dsRNA) reagents are provided, wherein the antisense strand is an oligomeric compound provided by a 3'-terminal monomer comprising formula (III) or a stereoisomer thereof, formula (IVa), formula (IVb), formula (IVa-1), or formula (IVb-1) herein. In one embodiment, double-stranded ribonucleic acid (dsRNA) reagents are provided, wherein the antisense strand is an oligomeric compound provided by a 5'-terminal monomer comprising formula (III) or a stereoisomer thereof, formula (IVa), formula (IVb), formula (IVa-1), or formula (IVb-1) herein.

[0089] In one embodiment, a double-stranded ribonucleic acid (dsRNA) reagent is provided, optionally further comprising an independent targeting group, wherein any one of the 5'-terminal and / or 3'-terminal nucleotides in the double-stranded ribonucleic acid (dsRNA) reagent further comprises one or more targeting groups, which may be ligands commonly used in the field of siRNA administration.

[0090] In some embodiments, in the provided double-stranded ribonucleic acid (dsRNA) reagents, the targeting group may be selected from one or more ligands, including polymers, sugars, ligands for receptors expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands, or derivatives thereof.

[0091] In some embodiments, provided double-stranded ribonucleic acid (dsRNA) reagents are provided wherein at least one or each of the targeting groups is selected from a ligand capable of binding to a mammalian hepatocyte surface receptor.

[0092] In some embodiments, provided are double-stranded ribonucleic acid (dsRNA) reagents wherein each of the targeting groups is independently a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes.

[0093] In certain embodiments, the oligomeric compound or double-stranded ribonucleic acid (dsRNA) has each strand comprising from 8 to 40 nucleotides in length.

[0094] In certain embodiments, there is provided the use of an oligomeric compound or double-stranded ribonucleic acid (dsRNA) reagent in the manufacture of a medicament for inhibiting gene expression, wherein said inhibiting gene expression comprises contacting said oligomeric compound with one or more cells, tissues, or animals.

[0095] In certain embodiments, a method of silencing gene expression is provided, the method comprising contacting a cell with a reagent comprising an oligomeric compound or double-stranded ribonucleic acid (dsRNA) described herein, wherein each strand of the oligomeric compound and the double-stranded ribonucleic acid (dsRNA) reagent each comprises 8 to 40 nucleotides in length, and the antisense strand of the oligomeric compound or the double-stranded ribonucleic acid (dsRNA) reagent is complementary to a target RNA.

[0096] 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 to inhibit 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 a reagent comprising an oligomeric compound or double-stranded ribonucleic acid (dsRNA) provided by a terminal monomer having Formula III, Formula (IVa), Formula (IVb), Formula (IVa-1), or Formula (IVb-1). These genes may be genes expressed in external tissues, such as ophthalmic and CNS target cell genes, in addition to liver tissue expression. DETAILED DESCRIPTION OF THE INVENTION

[0097] In certain embodiments, bicyclic abasic nucleic acid analogs are provided that can be used as nucleotide monomers to be incorporated into one or two termini of oligomeric compounds. The present specification further provides intermediates and methods for producing 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 into which they are incorporated, such as nuclease resistance. In certain embodiments, the oligomeric compounds and compositions provided herein are intended to hybridize with a portion of a target RNA, thereby disrupting the normal function of the target RNA. The oligomeric compounds are also intended to be used as primers and probes in diagnostic applications.

[0098] According to one aspect of the present invention, there is provided a compound of formula (I) or a stereoisomer thereof, [ka] A1 and A2 are each independently selected from O, S, SO, SO2, NR2 and CR3R4, and R2, R3 and R4 are each independently selected from hydrogen, halogen, sulfonyl group, sulfinyl group, substituted or unsubstituted C1-C6 alkyl group, substituted or unsubstituted C3-C6 cycloalkyl group, substituted or unsubstituted C2-C6 alkenyl group, substituted or unsubstituted C2-C6 alkynyl group, substituted or unsubstituted C5-C6 alkyl group, substituted or unsubstituted C5-C6 cycloalkyl ... alkyl group, substituted or unsubstituted C5-C6 alkyl group, substituted or unsubstituted C5-C6 alkyl group, substituted or 12 an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, or a substituted or unsubstituted 5- to 12-membered heterocyclyl group; one of T1 and T2 is a protecting group, Z, L, or -ZL, wherein Z represents a targeting group or a lipophilic group, and the lipophilic group is optionally linked to one of A1 or A2 via a linker; L represents a carrier spacer or comprises a carrier moiety linked by a spacer; the other of T1 and T2 is an active phosphorus group, a protecting group, and T1 and T2 are not simultaneously a protecting group; R1 is independently a halogen, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted C2-C6 alkenyl group, a substituted or unsubstituted C2-C6 alkynyl group, a substituted or unsubstituted C5-C 12 an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, or a substituted or unsubstituted 5- to 12-membered heterocyclyl group; n is an integer from 0 to 6, wherein formula (I) is [ka] It does not include the structure:

[0099] In some embodiments, A1 is O.

[0100] In some embodiments, A2 is O.

[0101] In some embodiments, A1 and A2 are each independently O, and the compound provided has the structure shown in formula (I-1) or a stereoisomer thereof: [ka] Here, T1, T2, R1 and n are as defined above.

[0102] In some embodiments, A1 and A2 are each independently S, and the compound provided has the structure shown in formula (I-2) or a stereoisomer thereof: [ka] Here, T1, T2, R1 and n are as defined above.

[0103] In some embodiments, A1 and A2 are each independently NR2, and the provided compound has the structure shown in formula (I-3) or a stereoisomer thereof: [ka] wherein T1, T2, R1, and n are as defined above, and R2 independently represents hydrogen, halogen, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted C2-C6 alkenyl group, a substituted or unsubstituted C2-C6 alkynyl group, a substituted or unsubstituted C5-C 12 It is selected from an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, and a substituted or unsubstituted 5- to 12-membered heterocyclyl group.

[0104] In one embodiment, A1 is O and A2 is S, and the provided compound has the structure shown in formula (I-4) or a stereoisomer thereof: [ka] Here, T1, T2, R1 and n are as defined above.

[0105] In one embodiment, A1 is O, A2 is NR2, and the provided compound has the structure shown in formula (I-5) or a stereoisomer thereof: [ka] where T1, T2, R1, and n are as defined above, and R2 is hydrogen, halogen, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted C2-C6 alkenyl group, a substituted or unsubstituted C2-C6 alkynyl group, a substituted or unsubstituted C5-C 12 It is selected from an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, and a substituted or unsubstituted 5- to 12-membered heterocyclyl group.

[0106] In one embodiment, A1 is S and A2 is NR2, and the provided compound has the structure shown in formula (I-6) or a stereoisomer thereof: [ka] where T1, T2, R1, and n are as defined above, and R2 is hydrogen, halogen, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted C2-C6 alkenyl group, a substituted or unsubstituted C2-C6 alkynyl group, a substituted or unsubstituted C5-C 12 It is selected from an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, and a substituted or unsubstituted 5- to 12-membered heterocyclyl group.

[0107] In certain embodiments, R2 is hydrogen or CH3.

[0108] As used herein, the term "substituted or unsubstituted" means that no hydrogen atoms in the parent compound are replaced with other substituents, or that one or more hydrogen atoms are replaced with a substituent. As used herein, the terms "substituent" and "substituent group" are meant to include groups that are typically added to other groups or parent compounds to enhance desired properties or provide other desired effects. Substituents may be protected or unprotected and can be added to one available site or many available sites in the parent compound. Substituents may be further substituted with other substituents and can be directly linked to the parent compound or linked to the parent compound via a linking group such as an alkyl group or a hydrocarbon group.

[0109] Suitable substituents herein include halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)R aa ), carboxy group (-C(O)OR aa ), aliphatic groups, alicyclic groups, alkoxy groups, substituted oxy (-OR aa ), aryl groups, aralkyl groups, heterocyclyl groups, heteroaryl groups, heteroarylalkyl groups, amino groups (-N(R bb )(R cc )), imino group (=NR bb ), amide group (-C(O)N(R bb )(R cc or -N(R bb )C(O)R aa , azide group (-N3), nitro group (-NO2), cyano group (-CN), ureido group (-N(R bb )C(O)N(R bb )(R cc )), thioureido group (-N(R bb )C(S)N(R bb )(R cc )), guanidino group (-N(R bb )C(=NR bb )N(R bb )(R cc )), amidine group (-C(=NR bb )N(R bb )(R cc Or 1N(R bb )C(=NR bb )(R aa )), a thiocarboxy group or a mercapto group (-SR bb ), sulfinyl group (-S(O)R bb ), sulfonyl group (-S(O)R bb ) and sulfonamido groups (-S(O)N(R bb )(R cc or -N(R bb )S(O)2R bb ), where each R aa , R bb and ccare independently H, an optionally linked chemical functionality or another substituent, and preferably the list includes, but is not limited to, H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclyl, and heteroarylalkyl groups. It will be understood by those skilled in the art of medicinal and organic chemistry that the total number of these substituents is reasonably limited by the desired properties of a given compound.

[0110] The term "protecting group," as used herein, refers to a labile chemical moiety known in the art to protect reactive groups (including, but not limited to, hydroxy, amino, and thiocarboxy groups) from undesired reactions during synthetic procedures. Protecting groups are typically used selectively to protect a site during the course of a reaction of another reactive site and are then removed to leave the unprotected group intact or available for further reaction.

[0111] In one embodiment, as should be understood in the art, one of the protecting groups of T1 and T2 varies depending on A1 or A2 and corresponds to a hydroxy protecting group, a mercapto protecting group, and an amino protecting group, respectively, i.e., when A1 or A2 is oxygen, one of the protecting groups of T1 and T2 is a hydroxy protecting group; when A1 or A2 is sulfur, sulfoxide (SO), sulfone (SO2), one of the protecting groups of T1 and T2 is a mercapto protecting group; when A1 or A2 is nitrogen, one of the protecting groups of T1 and T2 is an amino protecting group.

[0112] In one embodiment, one protecting group of T1 and T2 is a hydroxy protecting group, and in one embodiment, the hydroxy protecting group is each independently an acetyl group, a tert-butyl group, a tert-butoxymethyl group, a methoxymethyl group, a tetrahydropyranyl group, a 1-ethoxyethyl group, a 1-(2-chloroethoxy)ethyl group, a 2-trimethylsilylethyl group, a p-chlorophenyl group, a 2,4-dinitrophenyl group, a benzyl group, a benzoyl group, a p-phenylbenzoyl group, a 2,6-dichlorobenzyl group, a diphenylmethyl group, a p-nitrobenzyl group, a trimethylsilyl group, a triethylsilyl group, a tert-butyldimethylsilyl group, a tert-butyldiphenyl group, a methylsilyl ... In one embodiment, preferred hydroxy protecting groups are each independently selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and 4,4'-dimethoxytrityl.

[0113] In one embodiment, one of the protecting groups of T1 and T2 is a mercapto protecting group, and in one embodiment, the mercapto protecting group is each independently an acetyl group, a tert-butyl group, a tert-butoxymethyl group, a methoxymethyl group, a tetrahydropyranyl group, a 1-ethoxyethyl group, a 1-(2-chloroethoxy)ethyl group, a 2-trimethylsilylethyl group, a p-chlorophenyl group, a 2,4-dinitrophenyl group, a benzyl group, a benzoyl group, a p-phenylbenzoyl group, a 2,6-dichlorobenzyl group, a diphenylmethyl group, a p-nitrobenzyl group, a trimethylsilyl group, a triethylsilyl group, a tert-butyl group, a methylsilyl ... The mercapto-protecting group is selected from the group consisting of a methyldimethylsilyl group, a tert-butyldiphenylsilyl group, a triphenylsilyl group, a triisopropylsilyl group, benzoyl formate, a chloroacetyl group, a trichloroacetyl group, a trifluoroacetyl group, a pivaloyl group, a 9-fluorenylmethoxycarbonyl group, a methanesulfonyl group, a tosyl group, a trifluoromethanesulfonyl group, a trityl group, a monomethoxytrityl group, a dimethoxytrityl group, a trimethoxytrityl group, and a substituted 9-phenylxanthin-9-yl group. In one embodiment, preferred mercapto-protecting groups are independently selected from a benzyl group and a 4,4'-dimethoxytrityl group. As known in the art, when the mercapto group is further oxidized to a sulfoxide (SO) or a sulfone (SO), the above-mentioned protecting groups are similarly applied to the sulfoxide (SO) and the sulfone (SO).

[0114] In one embodiment, one of the protecting groups T1 and T2 is an amino protecting group, and in one embodiment, the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butyloxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrophenyl, 2-nitrobenzenesulfonyl, phthalimide, and dithiosuccinyl.

[0115] In the present invention, "optional," "optionally," and "selective" generally refer to the presence or absence of the group or functional group in question. A non-limiting example of an "optionally lipophilic group linked to A1 or A2 via a linker" can be expressed as the lipophilic group being directly linked to the attached atom or group in the form of a covalent bond, or the lipophilic group being required to be linked to the attached atom or group via one or more linkers.

[0116] In some embodiments, one of T1 and T2 is a lipophilic group. The terms "lipophilic" or "lipophilic group" broadly refer to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic group is by measuring the octanol-water partition coefficient log Kow, where Kow is the ratio of the concentration of a chemical in the octanol phase to the concentration in the aqueous phase at equilibrium in a two-phase system. The octanol-water partition coefficient is a property of a substance measured in a laboratory. However, it can also be predicted by using coefficients attributable to the structural components of a chemical, which are calculated using first principles or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001)), the entire contents of which are incorporated herein by reference). It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment over water (i.e., its hydrophilic / lipophilic balance). The lipophilic group-conjugated structure exposes the lipophilic group (e.g., fatty acid) and allows the lipophilic group to interact with albumin and / or fatty acid receptor or transporter proteins, thereby providing an oligonucleotide with a long in vivo half-life. It is understood in the art that lipophilic groups generally have a log Kow measurement greater than 0. In certain embodiments, the lipophilicity of a lipophilic moiety is measured by the octanol-water partition coefficient, log Kow, and the lipophilic moiety may have a log Kow greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. The lipophilicity of a molecule can be varied relative to the functional groups it carries; for example, the addition of functional groups such as hydroxyl groups, amines, carboxylic acids, sulfonate esters, phosphate esters, thiols, azide groups, and alkynes to the terminus of the lipophilic group can increase or decrease the partition coefficient (e.g., log Kow) value of the lipophilic moiety, and these functional groups can further attach lipophilic groups to the compounds described herein.

[0117] In certain embodiments, the lipophilic group is a saturated or unsaturated C4-C 30These include hydrocarbon chains, aliphatic rings, aromatic groups, fatty acid or fatty acid derived groups, steroid derived groups and any fat-soluble vitamin group.

[0118] 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., linear 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., linear C 16 In one embodiment, the lipophilic group is a saturated linear C 14 Alkyl group, C 16 Alkyl group or C 18 It is an alkyl group, and examples of the structure are: [ka] The lipophilic group is optionally further substituted with a functional group selected from the group consisting of a hydroxy group, an amine, a carboxylic acid, a sulfonate ester, a phosphate ester, a thiol, an azide group, and an alkyne. These functional groups can be used to attach the lipophilic group to A1 or A2.

[0119] In another embodiment, the lipophilic group is selected from groups derived from dicarboxylic fatty acids. In another embodiment, the lipophilic group comprises any saturated or unsaturated fatty acid with a hydrocarbon chain having 4 to 28 carbon atoms and may contain one or two carboxy groups. In one embodiment, the lipophilic group is a group derived from a saturated fatty acid with a hydrocarbon chain having 8 to 24 carbon atoms. In certain embodiments, the lipophilic group is a group derived from saturated fatty acids, wherein the fatty acids are butyric acid (CH3(CH2)2COOH), valeric acid, hexanoic acid, heptanoic acid, octanoic acid, capric 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 In another embodiment, the lipophilic group is a group derived from an unsaturated fatty acid, the unsaturated fatty acid being one of those shown in Table 1, and the fatty acid is preferably selected from the group consisting of myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, trans-isooleic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid, and erucic acid.

[0120] [Table 1]

[0121] 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. A "cholesterol derivative" refers to a compound derived from cholesterol, for example, by substitution, addition, or removal of substituents.

[0122] In another embodiment, the lipophilic group is selected from cholesterol, vitamin E (tocopherol), or bile acids.

[0123] In another embodiment, the lipophilic group is selected from the unsaturated fats docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), the saturated fatty acid docosanoic acid (DCA), the sterol cholesterol (bile), tocopherol succinate (TS) and lithocholic acid (LA), retinoic acid (vitamin A acid).

[0124] In another embodiment, the lipophilic group is an aromatic moiety. In this context, the term "aromatic" or "aromatic" refers broadly to monoaromatic and polyaromatic hydrocarbons. An aryl group is a C6-C6 alkyl group containing 1 to 3 aromatic rings, which may be optionally substituted. 14 These include, but are not limited to, aryl moieties, "aralkyl groups" or "arylalkyl groups" comprising an aryl group covalently bonded to an alkyl group, either of which may independently be optionally substituted or unsubstituted, and "heteroaryl groups." As used herein, the term "heteroaryl group" refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms, having 6, 10, or 14 pi-electrons shared in a cyclic arrangement, and having, in addition to carbon atoms, from 1 to about 3 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).

[0125] In one embodiment, the lipophilic group of the present invention can be directly covalently attached to A1 or A2 via a covalent bond. In one non-limiting embodiment, the fragment of the combination of A1 and T1 and / or the combination of A2 and T2 is: [ka] including, but not limited to, the structure: [ka] represents a single bond or a double bond, p and q each independently represent an integer of 5 to 25, and the sum of p and q is 25 or less. In some more specific embodiments, the fragments of the combination of A1 and T1 and / or the combination of A2 and T2 are [ka] Including, but not limited to:

[0126] In certain embodiments, the lipophilic group can be covalently attached to A1 or A2 via a linker, and the linkers can be one or a combination of more than one.

[0127] One of T1 and T2 of the present invention is a lipophilic group, which further comprises a linker and is covalently attached to A1 or A2 via one or more linkers.

[0128] 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-ol, a hydroxyproline, a hydroxyprolinol, an amino-alkyl-phosphorothioate linker, an amino-PEG-phosphorothioate linker, an α-carboxylate-amino-alkyl phosphorothioate linker, and an α-carboxylate-amino-PEG-phosphorothioate linker.

[0129] 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 click reaction product (e.g., a triazole via an azide-alkyne cycloaddition reaction), or a carbamate. In another embodiment, in the provided oligomeric compounds, the phosphodiester linker is a thiophosphodiester (non-limiting examples include [ka] ), oxophosphodiesters (non-limiting examples include: [ka] )

[0130] In one embodiment, the linker is a hydrocarbon chain linker or a polyethylene glycol (PEG) linker. In the case of a hydrocarbon chain linker, the linker contains 2 to 20 carbons, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbons. In the case of a polyethylene glycol (PEG) linker, the linker contains 1 to 20 ethylene glycol subunits, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ethylene glycol repeats.

[0131] In one embodiment, the linker is an amide bond linker.

[0132] In some embodiments, the linker comprises a cleavable group. In one embodiment, the linker comprises a cleavable group that can be pH-sensitive, such as a disulfide group. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. The pH of endosomes is more acidic, ranging from 5.5 to 6.0, and the pH of lysosomes is even more acidic, at about 5.0. Some linkers have a linking group that is cleaved at a preferred pH, thereby releasing the iRNA agent from an intracellular ligand (e.g., a targeting or cell-permeable ligand such as cholesterol) or entering a desired compartment of the cell.

[0133] In one embodiment, at least one of the linkers is a redox-cleavable joint (e.g., a reductively cleavable joint such as a disulfide group), an esterase-cleavable joint (e.g., an ester group), a phosphatase-cleavable joint (e.g., a phosphate ester), or a peptidase-cleavable joint (e.g., a peptide bond). One class of cleavable linking groups is redox-cleavable linking groups that cleave upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide bond (-SS-).

[0134] Ester-based linking groups are cleaved by enzymes such as intracellular esterases and amidases. 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)-.

[0135] Phosphate-based linking groups are cleaved by agents that decompose or hydrolyze phosphate esters. Examples of agents that cleave phosphate ester groups within cells include enzymes such as intracellular phosphatases. Examples of phosphate-based linking groups are -OP(O)(OH)-O- and -SP(S)(OH)-O-.

[0136] Peptide-based linking groups are cleaved by enzymes such as intracellular peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to produce oligopeptides (e.g., 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. A peptide bond is a special type of amide bond formed between amino acids to produce peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to produce peptides and proteins, and do not include the entire amide functionality.

[0137] In other embodiments, at least one linker is a biocleavable joint selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0138] In one embodiment, the α-carboxylate-amino-alkyl-phosphorothioate linker is [ka] It has the structure [ka] represents a linking moiety between the lipophilic group and one of A1 or A2, where m is preferably an integer from 2 to 12, more 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.

[0139] In one embodiment, the amino-PEG-phosphorothioate linker is [ka] It has the structure [ka] represents a linking moiety between the lipophilic group and one of A1 or A2, where 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.

[0140] and the α-carboxylate-amino-PEG-phosphorothioate linker is [ka] is.

[0141] In one embodiment, the amino-alkyl-phosphorothioate linker is [ka] It has the structure [ka] represents a linking moiety between the lipophilic group and one of A1 or A2, where m is preferably an integer of 2 to 12, and more preferably an integer of 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.

[0142] In one embodiment, the linker is selected from the group consisting of (i) an amino-alkyl-phosphorothioate linker, (ii) an α-carboxylate-amino-alkyl-phosphorothioate linker, (iii) an amino-PEG-phosphorothioate linker, and (iv) an α-carboxylate-amino-PEG-phosphorothioate linker, all of which are as defined in the formula provided above.

[0143] In one embodiment, the present invention provides conformationally restricted linkers, for example based on hydroxyproline, e.g., [ka] and [ka] represents a linking moiety between the lipophilic group and one of A1 or A2, where 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.

[0144] In one embodiment, the present invention provides conformationally restricted linkers, for example based on hydroxyprolinol, such as [ka] and [ka] represents the linking moiety between the lipophilic group and one of A1 or A2.

[0145] In one embodiment, the present invention provides a linker, for example an amino-alkyl-ol bond, e.g., [ka] and [ka] represents a linking moiety between the lipophilic group and one of A1 or A2, where 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.

[0146] In one embodiment, the present invention provides a linker, e.g., a phosphatidylcholine linker, e.g., [ka] and [ka] represents the linking moiety between the lipophilic group and one of A1 or A2.

[0147] In some embodiments, the linker may 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.

[0148] For further discussion of lipophilic groups and linkers, see PCT Publication No. WO2019 / 217459, filed July 7, 2019, entitled "Extrahepatic Delivery," PCT Publication No. WO2019 / 215333, filed May 10, 2019, entitled "Oligonucleotide Conjugates Comprising 7'-5'-α-Anobicyclosugar Nucleosides," and Nucleic Acids Research, 2019, Vol. 47, No. 31082-1096, entitled "Various Lipid Couplings for In Vivo Delivery of Functional Extrahepatic siRNA," the contents of which are incorporated herein by reference in their entireties.

[0149] In one embodiment, the present invention provides non-limiting examples of compounds in which a lipophilic group is covalently attached to A1 or A2 via a linker, [ka]

[0150] 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. The targeting group can alter the distribution, target or lifetime, endosomolytic properties, improved transport, hybridization and specificity properties of the siRNA reagent incorporated therein, for example, various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.

[0151] In some embodiments, the targeting group may be a ligand formed by one or more targeting molecules or derivatives thereof selected from the group consisting of polymers such as polyethylene glycol, sugars such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc), asialoglycoproteins, asialoglycoresidues, lipoproteins (high density lipoproteins, low density lipoproteins, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, receptor ligands expressed by hepatocytes such as aptamers for transferrin, antibodies, polypeptides such as quantum dots, membrane-permeable peptides, or small molecule ligands.

[0152] In some embodiments, at least one or each of the targeting groups is selected from ligands capable of binding to mammalian hepatocyte surface receptors. In some embodiments, each of the targeting groups is independently a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each of the targeting groups is independently an asialoglycoprotein- or sugar-containing ligand. In some embodiments, each of the targeting groups is independently an asialoglycoprotein-containing ligand, such as asialoorosomucoid (ASOR) or asialofetuin (ASF).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-glucopyranose, β-D-glucopyranose, α-D-glucose, Cofuranose, 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, Nn-butyrylgalactosamine, N-isobutyrylgalactosamine Lactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-ethanolacyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4- The ligand contains one selected from the group consisting of tri-O-acetyl-1-thio-6-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-glucoheptapyranoside ethyl ester, 2,5-anhydro-D-allonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

[0153] In some embodiments, at least one or each of the targeting groups is a ligand comprising galactose or N-acetylgalactosamine.

[0154] In some embodiments, the targeting group comprises one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a suitable tether by a bivalent or trivalent branched joint.

[0155] [ka] where: 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 duplicated units therein may be homologous or different; 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 absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q5C , Q 6A , Q 6B , Q 6C each occurrence independently represents absent, an alkylene group, or a substituted alkylene group, in which one or more methylene groups are optionally interrupted or terminated by one or more of O, S, S(O), SO, NH, C(R')=C(R''), C≡C, or C(O); 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 absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO or a heterocyclyl group; 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 (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R' and R'' are each independently H, CH3, OH, SH, NH2, an alkyl group, an alkenyl group, or an alkynyl group; Ra is H or an amino acid side chain, and m is 1 or 2.

[0156] The targeting group-conjugate compounds of formula (I) or their stereoisomers and iRNA reagents of the present invention can be prepared, for example, by direct reaction using phosphoramidites having reactive functional groups. The targeting group can be linked to one of A1 or A2 via an appropriate conjugation joint, and those skilled in the art can select an appropriate conjugation joint depending on the specific type of targeting group. For example, trans-4 carboxyproline, aminoalkyl alcohol derivatives, etc. form phosphate esters via reactive functional groups. The following exemplary fragments are conjugated with degradable structures such as esters, amides, and phosphate esters via appropriate conjugation joints: [ka] The targeting group is shown in the non-limiting structure: [ka] For conjugation joints, types of ligands, tethers, branched joints, and methods for binding targeting groups to oligonucleotides, see WO2009073809, WO2014179620, WO2014179627, and WO2015006740A2, the entire contents of which are incorporated herein by reference. In some embodiments, the targeting group can be linked by an acid-labile or reducible chemical bond, which can be degraded in the acidic environment of a cellular endosome.

[0157] The term "conjugation joint" or "linker" refers to two moieties that connect compounds, e.g., two moieties that covalently link compounds. Other saccharide conjugates and joints that find application in the present invention include those described in disclosures WO2014 / 179620 and WO2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0158] In certain embodiments, the targeting group is linked to one of A1 or A2, [ka] The compound fragment is selected from one of the following:

[0159] In some 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 (formula (III) herein and its stereoisomers or fragments) of the oligonucleotide to be synthesized is pre-loaded by linking it to a joint such as a reactive amino group on the carrier via a cleavable spacer such as a succinyl group, and then loaded onto a column for subsequent nucleotide docking. The carrier spacer serves as the attachment point for the oligonucleotide during the synthesis process, and the non-nucleoside or nucleoside in the oligonucleotide is covalently bound to the carrier spacer via the 3'-terminal carboxyl group, remains bound throughout the chain assembly process, and is then cleaved and deprotected. In oligonucleotide synthesis, the spacer is generally selected to avoid steric hindrance and be cleavable, and the oligonucleotide is attached thereto, and is used to position the oligonucleotide and the synthesis process away from the carrier. This method allows for greater flexibility and more space for synthesis. More specifically, the carrier moiety bound to the oxygen atom of the carboxy group at the 2'- or 3'-position of the ribose at the 3'-terminus of the non-nucleoside or nucleoside at the end of the oligonucleotide (e.g., A1 or A2 in formula (I) and its stereoisomers described herein) via a carrier spacer is shown below: [ka] Sp represents a carrier spacer.

[0160] Any carrier spacer (Sp) applied in the field of oligonucleotide synthesis may be incorporated herein. Commonly used examples are: [ka] wherein R is absent, an ether, a polyethylene glycol, oxygen, sulfur, nitrogen, an alkylene group, an alkenyl group, an alkynyl group, an aryl group, an aralkyl group, a heteroalkyl group, or a heteroaryl group; [ka] is a linking moiety to A1 or A2 in formula (I) at one end, and the carboxyl group at the other end may further be linked to a carrier, and R is more preferably absent, oxygen, sulfur, nitrogen, an alkylene group, an alkenyl group, or an alkynyl group, and another example is the polyalkylene glycol phosphate ester / phosphonate disclosed in patent CN103233003, and in one embodiment, the spacer is [ka] In one embodiment, the carrier spacer is preferably a succinyl group, [ka] In some embodiments, the spacer dicarboxylic acid is further derivatized, an example of which is further PEGylated: [ka] P is an integer of 5 to 120. The carboxy group at the other end can further be linked to a carrier moiety to form a covalent bond.

[0161] In certain embodiments, one of T1 and T2 is a carrier moiety linked by a spacer, wherein the carrier moiety comprises a solid phase carrier moiety and a soluble carrier moiety.

[0162] The support generally includes a joint, for example, a long-chain alkylamine may be used, a non-limiting example is binding to the silicon of the solid support via a propylamino joint, and LCAA-CPG is commonly used in the field. In some embodiments, the long-chain alkylamine and the solid support are further attached via a biodegradable joint such as acetylglycerol.

[0163] [ka]

[0164] In certain embodiments, examples of solid 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 supports include controlled pore glass (CPG), polystyrene, silica gel, macroporous cross-linked polystyrene, poly(vinyl methacrylate alcohol copolymer), silicon-tipped glass, cellulose, polystyrene beads, polypropylene sheets, non-porous silicon beads, polyacrylamide, or polyacrylate.

[0165] Recently, soluble supports have been applied to important advances in oligonucleotide synthesis that are amenable to large-scale solution-phase synthesis. In certain embodiments, non-limiting examples of soluble supports include: [ka] is.

[0166] The compositions of spacers, solid supports, joints, and their linking methods are generally referred to as solid-phase supports in the art, and their configurations are also known, as described in "Current Protocols in Nucleic Acid Chemistry," 2000, Chapter 3, Section 3.1, "Solid-Phase Supports for Oligonucleotide Synthesis." For further descriptions of these spacers, supports, joints, and their linking methods, please refer to the disclosures in "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.

[0167] In some embodiments, one of T1 and T2 is a targeting group or a lipophilic group, and is further linked to a carrier moiety via a spacer. In particular, when a compound of formula (I) or a stereoisomer thereof described herein is used at the 3' end of an oligonucleotide, an exemplary structure is [ka] and [ka] represents a moiety linked to A1 or A2 herein, and in some more specific embodiments, where the spacer links the linker to the targeting group or lipophilic group, particularly where the terminal hydroxyl group in the linker is attached to the spacer. [ka] In one embodiment, the other of T1 and T2 is an activated phosphorus group, the activated phosphorus group being: [ka] It has the structure wherein M1 is H, a substituted or unsubstituted C1-C6 alkyl group, OR5, SR5, OH, SH or NR6R7, M2 is OH, SH, OR5' or NR6'R7', where each R5, R6, R7, R5', R6' or R7' is independently hydrogen, a substituted or unsubstituted C1-C6 alkyl group, a sulfonyl group, and m is 0 or 1.

[0168] In some embodiments, each R, R, or R is independently a substituted C-C alkyl group, and the substituents can be selected from those generally consistent with alkyl substitution in the art. In some embodiments, each R, R, or R is independently a substituted alkyl group, and the substituents are preferably selected from a cyano group, a halogen atom, a hydroxyl group, and an amino group.

[0169] In one embodiment, M1 is selected from substituted or unsubstituted C1-C6 alkyl groups, preferably methyl, ethyl, propyl, and isopropyl groups, where the substituents are preferably selected from cyano, halogen, hydroxy, and amino groups.

[0170] In one embodiment, M1 is selected from OR5, where R5 is selected from a substituted or unsubstituted methyl group, ethyl group, propyl group, or isopropyl group, preferably a substituted methyl group, ethyl group, propyl group, or isopropyl group, and more preferably R5 is a cyanoethyl group, i.e., M1 is a cyanoethoxy group.

[0171] In one embodiment, M1 is NR6R7, where R6 and R7 are independently selected from hydrogen and a sulfonyl group, more preferably M1 is a methylsulfonylamido group.

[0172] In some embodiments, M2 is selected from NR6'R7', where R6' or R7' is independently a C1-C6 alkyl group. The C1-C6 alkyl group is selected from a methyl group, an ethyl group, a propyl group, and an isopropyl group. In some embodiments, M2 is selected from N(CH(CH3)2)2.

[0173] In one embodiment, M is O(CH)CN, M is N(CH(CH)), and m is 0.

[0174] In one embodiment, M is a methyl group, M is N(CH(CH) and m is 0.

[0175] In one embodiment, M is methanesulfonamide, M is N(CH(CH)), and m is 0.

[0176] In certain embodiments, the active phosphorus group is [ka] wherein m, M2, R5, R6, and R7 are as defined above.

[0177] In certain embodiments, the activated phosphorus group is a phosphoramidite. In certain embodiments, the activated 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)). A preferred solid phase synthesis involves the use of phosphoramidites (p III ) as a reactive phosphite, and then converting the intermediate phosphite compound to a phosphate or thiophosphate (p V) to generate phosphodiester or phosphorothioate internucleoside linkages. Additional activated phosphates and phosphites are disclosed in Beaucage and Iyer, Tetrahedron, 1992, 48(12), 2223-2311.

[0178] In some 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 some embodiments, A1 and A2 are preferably selected from O, and exemplary structures are [ka] is.

[0179] In some embodiments, one of T1 and T2 is diisopropylethyl phosphoramidite and the other is a 4,4'-dimethoxytrityl group. Further, in some embodiments, A1 and A2 are preferably selected from O.

[0180] 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.

[0181] 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.

[0182] In certain embodiments, when n is not 0, it means that the bicyclic structure has 1 to 6 independent R1 group substituents. It should be understood that in the bicyclic abasic compounds herein, these substituents do not include natural or modified nucleobases. In certain embodiments, R1 is independently selected from halogen, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C2-C6 alkenyl group, and a substituted or unsubstituted C2-C6 alkynyl group. More preferably, R1 is independently selected from fluorine, chlorine, bromine, iodine, methyl, ethyl, isopropyl, propyl, 3-propenyl, ethynyl, and 3-propynyl groups.

[0183] In certain embodiments, n is 0, representing an absence of R 1 group substitution on the ring structure.

[0184] In certain embodiments, the compounds provided herein have the configuration of Formula (IIa) or Formula (IIb): [ka] A1, A2, T1, T2, R1 and n are as described herein.

[0185] In some preferred embodiments, in Formula (IIa) or Formula (IIb), A1 and A2 are each independently selected from O; one of T1 and T2 is a protecting group, a targeting group, and a lipophilic group, the lipophilic group being optionally linked to one of A1 or A2 via a linker; the other of T1 and T2 is an activated phosphorus group; and n is 0. In some more preferred embodiments, in Formula (IIa) or Formula (IIb), A1 and A2 are each independently selected from O; one of T1 and T2 is a hydroxy protecting group and a lipophilic group, the lipophilic group being optionally linked to one of A1 or A2 via a linker; the other of T1 and T2 is an activated phosphorus group; n is 0; and the hydroxy protecting group is selected from an acetyl group, a benzyl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, and a 4,4'-dimethoxytrityl group; and the lipophilic group is C14 , C 16 or C 18 In some more preferred embodiments, the activated phosphorus is selected from diisopropyl cyanoethoxy phosphoramidite, diisopropyl methyl phosphoramidite, diisopropyl ethyl phosphoramidite, and H-phosphonate.

[0186] In some other preferred embodiments, in Formula (IIa) or Formula (IIb), A1 and A2 are each independently selected from O, one of T1 and T2 is Z, L, or -ZL, where Z represents a targeting group or a lipophilic group, which lipophilic group is optionally linked to one of A1 or A2 via a linker, L represents a carrier spacer or comprises a carrier moiety linked by a spacer, the other of T1 and T2 is a hydroxy protecting group, and n is 0. In some more preferred embodiments, the hydroxy protecting group is selected from an acetyl group, a benzyl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, and a 4,4'-dimethoxytrityl group.

[0187] In certain embodiments, the compounds provided herein have the configuration of formula (IIa-1) or formula (IIb-1): [ka] A1, A2, T1, T2, R1, and n are as described herein, and the configuration thereof corresponds to that of isosorbide or isomanitol, which are well known in the art. In some preferred embodiments, in Formula (IIa-1) or Formula (IIb-1), A1 and A2 are each independently selected from O; one of T1 and T2 is a protecting group, a targeting group, and a lipophilic group, the lipophilic group being optionally linked to one of A1 or A2 via a linker; 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 each independently selected from O; one of T1 and T2 is a hydroxy protecting group and a lipophilic group, the lipophilic group being optionally linked to one of A1 or A2 via a linker; the other of T1 and T2 is an activated phosphorus group; n is 0; the hydroxy protecting group is selected from an acetyl group, a benzyl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, and a 4,4'-dimethoxytrityl group; and the lipophilic group is C 14 , C 16 or C 18 In some more preferred embodiments, the activated phosphorus is selected from diisopropyl cyanoethoxy phosphoramidite, diisopropyl methyl phosphoramidite, diisopropyl ethyl phosphoramidite, and H-phosphonate.

[0188] In some other preferred embodiments, in Formula (IIa-1) or Formula (IIb-1), A1 and A2 are each independently selected from O; one of T1 and T2 is Z, L, or -ZL; Z represents a targeting group or a lipophilic group, which is optionally linked to one of A1 or A2 via a linker; L represents a carrier spacer or comprises a carrier moiety linked by a spacer; the other of T1 and T2 is a hydroxy-protecting group; and n is 0. In some more preferred embodiments, the hydroxy-protecting group is selected from an acetyl group, a benzyl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, and a 4,4'-dimethoxytrityl group.

[0189] In certain embodiments, the compounds of formula (IIa) or formula (IIa-1) provided herein have the exemplary configuration shown below: [ka]

[0190] In certain embodiments, the compounds of formula (IIb) or formula (IIb-1) provided herein have the exemplary configurations shown below: [ka]

[0191] In the present invention, a compound having a specific stereoconfiguration can be obtained from a starting material by synthetic chiral control performed by a person skilled in the art, or can be obtained from a starting material by chiral resolution technology. Alternatively, the compound can be synthesized from a starting material having a specific stereoconfiguration that is commonly found in industrial production, such as isosorbide, isomanitol, or the like, having a specific stereoconfiguration.

[0192] The features of two or more such compounds provided by the present invention may be combined in any manner and are within the scope of the present invention if such features are not mutually inconsistent. In one embodiment, the compounds provided herein have the following specific structure: [ka] [ka] [ka] [ka]

[0193] The compounds described in the present invention, such as those represented by formula (I) or its stereoisomers, (IIa), (IIb), (IIa-1), and (IIb-1), and any combination of the technical features of two or more such compounds, can be applied to oligonucleotides, and can be applied to the 5'-end, 3'-end, or both ends of an oligonucleotide. The oligonucleotides include, but are not limited to, single-stranded antisense oligonucleotides (ASOs), miRNAs, and double-stranded ribose nucleotides (dsRNAs). In some embodiments, the compounds are applied to the 3'-end of the antisense strand of a double-stranded ribose nucleotide (dsRNA) oligonucleotide. In some embodiments, the compounds are applied to the 5'-end of the antisense strand of a double-stranded ribose nucleotide (dsRNA) oligonucleotide. In some embodiments, the compounds are applied to the 5'-end and / or 3'-end of the sense strand of a double-stranded ribose nucleotide (dsRNA) oligonucleotide.

[0194] According to one aspect of the present invention, there is provided an oligomeric compound comprising at least one 5'-end and / or 3'-end monomer having the structure shown in formula (III) or a stereoisomer thereof:

[0195] [ka] A1 and A2 are each independently selected from O, S, SO, SO2, NR2 and CR3CR4, and R2, R3 and R4 are each independently selected from hydrogen, halogen, sulfonyl group, sulfinyl group, substituted or unsubstituted C1-C6 alkyl group, substituted or unsubstituted C3-C6 cycloalkyl group, substituted or unsubstituted C2-C6 alkenyl group, substituted or unsubstituted C2-C6 alkynyl group, substituted or unsubstituted C5-C6 alkyl group, substituted or unsubstituted C5-C6 cycloalkyl ... 12 an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, or a substituted or unsubstituted 5- to 12-membered heterocyclyl group; one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, said lipophilic group being linked to one of A1 or A2, optionally via a linker, and the other of T3 and T4 is an internucleoside linking group connecting the formula (III) monomer or a stereoisomer thereof to the oligomeric compound; R1 is independently a halogen, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted C2-C6 alkenyl group, a substituted or unsubstituted C2-C6 alkynyl group, a substituted or unsubstituted C5-C 12 an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, or a substituted or unsubstituted 5- to 12-membered heterocyclyl group; n is an integer from 0 to 6, The oligomeric compound optionally further comprises a targeting group.

[0196] In certain embodiments, A 1 is O in the oligomeric compounds provided.

[0197] In certain embodiments, A2 is O in provided oligomeric compounds.

[0198] In some embodiments, oligomeric compounds are provided, wherein A and A are each independently O, and the oligomeric compounds comprise at least one 5'-end and / or 3'-end monomer having the structure shown in formula (III-1) or a stereoisomer thereof: [ka] where T3, T4, R1, and n are as defined above for the oligomeric compound.

[0199] In certain embodiments, provided are oligomeric compounds in which A 1 and A 2 are each independently S.

[0200] In certain embodiments, in provided oligomeric compounds, A and A are each independently NR, and R is selected from the group consisting of hydrogen, halogen, a substituted or unsubstituted C-C alkyl group, a substituted or unsubstituted C-C cycloalkyl group, a substituted or unsubstituted C-C alkenyl group, a substituted or unsubstituted C-C alkynyl group, a substituted or unsubstituted C-C alkyl ... 12 In some embodiments, the oligomeric compounds are provided wherein R2 is hydrogen or CH3.

[0201] In some embodiments, oligomeric compounds are provided in which A1 is different from A2. In some embodiments, oligomeric compounds are provided in which A1 is O and A2 is S. In some embodiments, oligomeric compounds are provided in which A1 is O and A2 is NR2, and the oligomeric compounds include at least one 5'-end and / or 3'-end monomer having the structure shown in formula (III-2) or a stereoisomer thereof: [ka] wherein T3, T4, R1, R2, and n are as defined above for the oligomeric compounds, and in some preferred embodiments, R2 is hydrogen or CH3. In some embodiments, the oligomeric compounds provided herein are such that A1 is S and A2 is NR2.

[0202] As used herein, the terms "internucleoside linkage" or "internucleoside linking group" are intended to include various internucleoside linking groups known in the art, and in such embodiments, any internucleoside linkage can be used to link nucleosides or analogs together. The two main internucleoside linking groups are defined by the presence or absence of a phosphorus atom and include, but are not limited to, phosphodiester, phosphotriester, phosphorothioate, phosphorodithioate, alkylphosphonate, aminophosphonate, phosphonate, phosphinate, thiophosphoramidate, and phosphoramidate linking groups, as well as non-phosphorus-containing internucleoside linking groups such as thiodiester (-OC(O)-S-), thiocarbonylcarbamate (-OC(O)(NH)-S-), siloxane (-O-Si(H)-O-), N,N'-dimethylhydrazine (-CH-N(CH)-N(CH)-), formacetyl, and methyleneimino (-CH-N(CH)-O-CH-). Internucleoside linkages further include neutral non-ionic internucleoside linkages, and as used herein, the term "neutral internucleoside linkage" is intended to include non-ionic 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'), methyleneformacetal (3'-O-CH2-O-5'), and thioformacetal (3'-S-CH2-O-5'). Additional neutral internucleoside linkages include nonionic linkages, including siloxanes (dialkylsiloxanes), carboxylate esters, formamides, sulfides, sulfonates, and amides (see, e.g., "Carbohydrate Modifications in Antisense Research"; YS Sanghvi and PD Cook, eds., ACS Symposium Series 580; Chapters 3 and 4, pp. 40-65).Additional neutral internucleoside linkages include non-ionic linkages containing mixed N, O, S, and CH moieties, where a phosphorus atom is not always present. In some preferred embodiments, the alkylphosphonate linking group is selected from C1-C6 alkylphosphonate linking groups.

[0203] In certain embodiments, provided are oligomeric compounds wherein the phosphorus-containing internucleoside linking group is [ka] wherein X is H, a substituted or unsubstituted C1-C6 alkyl group, OR8, SR8', OH, SH, or NR9R 10 Y represents O or S, z may be 0 or 1, and R, R', R or R 10 are independently hydrogen, a substituted or unsubstituted C1-C6 alkyl group, or a sulfonyl group; [ka] each independently represent a moiety that links to one of A1 or A2 herein, and a moiety that links to a linking nucleotide.

[0204] Modified linkages compared to natural phosphodiester linkages are used to alter (generally enhance) the nuclease resistance of oligomeric compounds. In certain embodiments, modified linkages can produce internucleoside linkages with chiral atoms, either as racemic mixtures or as single enantiomers. Exemplary chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for producing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0205] In certain embodiments, provided are oligomeric compounds 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 linked to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group connecting a Formula (III) monomer or a stereoisomer thereof to the 5'-terminus and / or 3'-terminus of the oligomeric compound, wherein the internucleoside linking group is independently an alkylphosphonate linking group, a phosphodiester internucleoside linking group, or a phosphorothioate internucleoside linking group.

[0206] When T4 is an internucleoside linking group, oligomeric compounds comprising at least one monomer of formula (III) or a stereoisomer thereof include, but are not limited to, the following: [ka] wherein Olig represents an oligonucleotide moiety in which the indicated Formula (III) monomer or a stereoisomer thereof is attached to the 5'-end and / or 3'-end of the oligomeric compound, respectively, A1, A2, R1, and n are as previously described, T3 is 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 wherein the oligomeric compound optionally further comprises a targeting group.

[0207] In certain embodiments, in the provided oligomeric compounds, the protecting group of one of T1 and T2 is different depending on A1 or A2, i.e., when A1 or A2 is oxygen, the protecting group of one of T1 and T2 is a hydroxy protecting group, i.e., when A1 or A2 is sulfur, sulfoxide (SO), sulfone (SO2), the protecting group of one of T1 and T2 is a mercapto protecting group, i.e., when A1 or A2 is nitrogen, the protecting group of one of T1 and T2 is an amino protecting group.

[0208] In certain embodiments, in provided oligomeric compounds, one protecting group of T3 and T4 is a hydroxy protecting group, and in one embodiment, in provided oligomeric compounds, 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 ... In one embodiment, in provided oligomeric compounds, preferred hydroxy protecting groups are each independently selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and 4,4'-dimethoxytrityl.

[0209] In certain embodiments, in provided oligomeric compounds, one protecting group of T3 and T4 is a mercapto protecting group, and in one embodiment, in provided oligomeric compounds, the mercapto 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, and triethylsilyl. , tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethoxycarbonyl, methanesulfonyl, tosyl, trifluoromethanesulfonyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, or substituted 9-phenylxanthin-9-yl. In one embodiment, in the provided oligomeric compounds, preferred mercapto-protecting groups are each independently selected from benzyl and 4,4'-dimethoxytrityl. When A1 or A2 is SO or SO2, it is understood that the same applies to the mercapto-protecting group.

[0210] In certain embodiments, provided oligomeric compounds are provided in which one protecting group of T3 and T4 is an amino protecting group, and in one embodiment, provided oligomeric compounds are provided in which the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butyloxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrophenyl, 2-nitrobenzenesulfonyl, phthalimido, and dithiosuccinyl.

[0211] In some embodiments, in the provided oligomeric compounds, one of T3 and T4 is a lipophilic group, where the lipophilicity of the lipophilic group can be measured by log Kow, where log Kow is greater than 0. The terms "lipophilicity" or "lipophilic group" broadly refer to any compound or chemical moiety that has an affinity for lipids. These coefficients are calculated using first principles or empirical methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001)), the entire contents of which are incorporated herein by reference. The definition has the same scope as the bicyclic abasic compounds described herein. It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment over water (i.e., its hydrophilic / lipophilic balance). The lipophilic group-conjugated structure exposes the lipophilic group (e.g., fatty acid) and allows the lipophilic group to interact with albumin and / or fatty acid receptor or transporter proteins, thereby providing an oligonucleotide with a long in vivo half-life. In some examples, the lipophilic group generally has a log Kow measurement greater than 0. In certain embodiments, the lipophilicity of the lipophilic moiety is measured by the octanol-water partition coefficient, log Kow, and the lipophilic moiety may have a log Kow greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. The lipophilicity of a molecule can be varied by the functional groups it carries; for example, the addition of functional groups such as hydroxyl groups, amines, carboxylic acids, sulfonate esters, phosphate esters, thiols, azide groups, and alkynes to the terminus of the lipophilic moiety can increase or decrease the partition coefficient (e.g., log Kow) value of the lipophilic moiety, and these functional groups can further attach the lipophilic group to the monomers of the oligomeric compounds described herein.

[0212] In the provided oligomeric compounds, the definition of the lipophilic group similarly has the same scope of definition as the bicyclic abasic compounds described above, for example, but not limited to, the following:

[0213] In certain embodiments, provided oligomeric compounds include lipophilic groups selected from saturated or unsaturated C4-C 30 Groups containing hydrocarbon chains, aliphatic rings, aromatic groups, fatty acid groups or groups derived from fatty acids, steroid-derived groups and any fat-soluble vitamin group.

[0214] In certain embodiments, in the provided oligomeric compounds, 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., linear 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., linear C 16 In one embodiment, the lipophilic group is a saturated linear C 14 Alkyl group, C 16 Alkyl group or C 18 The lipophilic group is an optional functional group further 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.

[0215] In another embodiment, the provided oligomeric compounds have lipophilic groups selected from fatty acid-derived groups of dicarboxylic acids. In another embodiment, the lipophilic group comprises any saturated or unsaturated fatty acid with a hydrocarbon chain having 4 to 28 carbon atoms and may contain one or two carboxy groups. In one embodiment, the lipophilic group is a saturated fatty acid-derived group with a hydrocarbon chain having 8 to 24 carbon atoms. In certain embodiments, the lipophilic group is a group derived from saturated fatty acids, wherein the fatty acids are butyric acid (CH3(CH2)2COOH), valeric acid, hexanoic acid, heptanoic acid, octanoic acid, capric 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 In another embodiment, the lipophilic group is a group derived from an unsaturated fatty acid, the unsaturated fatty acid being one of those shown in Table 1, and the fatty acid is preferably selected from the group consisting of myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, trans-isooleic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid, and erucic acid.

[0216] In another embodiment, the provided oligomeric compound has 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. A "cholesterol derivative" refers to a compound derived from cholesterol, for example, by substitution, addition, or removal of a substituent.

[0217] In certain embodiments, provided are oligomeric compounds in which the lipophilic group is selected from cholesterol, vitamin E (tocopherol), or bile acids.

[0218] In certain embodiments, provided oligomeric compounds are provided in which the lipophilic group is selected from the unsaturated fatty acids docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), the saturated fatty acid docosanoic acid (DCA), the sterol cholesterol (bile), tocopherol succinate (TS) and lithocholic acid (LA), retinoic acid (vitamin A acid).

[0219] In another embodiment, the lipophilic group is an aromatic moiety.

[0220] In some embodiments, in the provided oligomeric compounds, the lipophilic group of the present invention can be directly attached to A1 or A2 via a covalent bond. In some embodiments, the fragments of the combination of A1 and T3 and / or the combination of A2 and T4 are: [ka] including, but not limited to, the non-limiting structure: [ka] represents a single bond or a double bond, and p and q each independently represent an integer of 5 to 25, and the sum of p and q does not exceed 25. In some further specific embodiments, the fragment of the combination of A1 and T3 and / or the combination of A2 and T4 is [ka] Including, but not limited to:

[0221] It is understood that any lipophilic group, linker, and linkage to the bicyclic abasic compound via a linker defined above, and the described properties or structures thereof, are equally applicable to the scope of the definition of the lipophilic group, linker, and linkage method thereof in the oligomeric compound herein.

[0222] In some embodiments, in the provided oligomeric compounds, the lipophilic group can also be covalently linked to A1 or A2 via a linker, and the above linkers can be one or a combination of two or more. As used herein, in the provided oligomeric compounds, the lipophilic group linker has the same lipophilic group linker as the bicyclic abasic nucleic acid analog compound used above, and the properties or structures described therein also apply to the lipophilic group linker of the oligomeric compounds herein.

[0223] One of T3 and T4 of the present invention is a lipophilic group, and the lipophilic group further comprises a linker and is covalently bound to A1 or A2 via one or more linkers. As a non-limiting example, 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-ol, 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. In some embodiments, 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 click reaction product (e.g., a triazole obtained by an azide-alkyne cycloaddition reaction), or a carbamate.

[0224] In some embodiments, the linker comprises a cleavable group, such as a pH-sensitive, redox-cleavable joint, an esterase-cleavable joint (e.g., an ester group), a phosphatase-cleavable joint (e.g., a phosphate ester), or a peptidase-cleavable joint (e.g., a peptide bond), a biocleavable joint, a hydroxyproline, a linker bonded by an amino-alkyl-ol, etc. For further discussion of lipophilic groups and linkers, see PCT Publication No. WO 2019 / 217459, filed July 7, 2019, entitled "Extrahepatic Delivery," PCT Publication No. WO 2019 / 215333, filed May 10, 2019, entitled "Oligonucleotide Conjugates Comprising 7'-5'-α-Anobicyclosugar Nucleosides," and Nucleic Acids Reference may be made to "Various lipid couplings for in vivo delivery of functional extrahepatic siRNA" in Research, 2019, Vol. 47, No. 31082-1096, the contents of which are also incorporated by reference in their entirety into the oligomeric compounds of this specification.

[0225] When the compound T1 or T2 of the present specification is a lipophilic group, the compounds described herein can be applied to the oligonucleotide field. As is well known in the art, depending on the nature or structure of the lipophilic group, the compounds can be applied to extrahepatic delivery systems, including, but not limited to, the CNS field, ophthalmology, and muscle. Because free oligonucleotides cannot cross the blood-brain barrier (BBB), delivery of oligonucleotides to the central nervous system (CNS) poses particular challenges. One method of delivering oligonucleotides to the CNS is intrathecal delivery. However, to achieve the desired therapeutic effect, oligonucleotides must also be effectively internalized into target cells in the CNS. Previous studies have shown that lipophilic groups are usually conjugated to specific positions within oligonucleotides, such as single nucleobases or 2-position sugar rings, to facilitate the internalization of oligonucleotides into cells of neuronal origin. Conjugating lipophilic groups to the monomers of the present specification and applying them to oligomeric compounds can also achieve delivery and distribution to the CNS. Lipophilic diseases that have been widely applied 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's disease.

[0226] Lipid groups are used to target oligonucleotides to corresponding CNS targets: APP in early-onset familial Alzheimer's disease, ATXN2 in spinocerebellar ataxia 2 and ALS, and C9orf72 in amyotrophic lateral sclerosis and frontotemporal dementia, TARDBP in ALS, MAPT (Tau) in frontotemporal dementia, and HTT in Huntington's disease, SNCA in Parkinson's disease, FUS in ALS, ATXN3 in spinocerebellar ataxia 3, ATXN1 in SCA1, SCA7 and SCA8 genes, ATN1 in DRPLA, MeCP2 in XLMR, PRNP in prion diseases, Lafora disease, DMPK in DM1 (CNS and skeletal muscle), and TTR in hATTR (CNS, eye, and systemic).

[0227] Lipid-based oligonucleotides are used to treat the corresponding ophthalmological diseases, including, but not limited to, age-related macular degeneration (AMD) (dry and wet), birdshot chorioretinopathy, dominant retinitis pigmentosa, 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 target cataract, and PPP3C target dry eye syndrome.

[0228] In certain embodiments, provided oligomeric compounds are provided wherein one of T3 and T4 is hydrogen and the other of T3 and T4 is an internucleoside linking group connecting a Formula III monomer or a stereoisomer thereof to the 5'-terminus and / or 3'-terminus of the oligomeric compound.

[0229] In some embodiments, the oligomeric compound further comprises a targeting group. In some embodiments, in the oligomeric compound, one of T3 and T4 is a covalent bond, and the oligomeric compound is linked to the targeting group by a covalent bond via one of T3 and T4 in formula (III) or a stereoisomer thereof: [ka] [ka] represents a portion of a monomer of formula (III), and in some other embodiments, the oligomeric compound is linked to the 5'-terminal and / or 3'-terminal nucleotide at one end via an oligomeric compound comprising formula (III) or a stereoisomer thereof at the other end. [ka] The targeting group may be a ligand commonly used in the field of siRNA administration, and as used herein, the targeting group of the provided oligomeric compound has the targeting group of the bicyclic abasic nucleic acid analog compound described above, and the properties or structures described therein are similarly applied to the targeting group of the oligomeric compound of the present invention. The targeting group can change the distribution, target or life span, endosomolytic properties, improved transport, hybridization and specific properties of the siRNA reagent incorporated therein, for example, various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.

[0230] In some embodiments, in oligomeric compounds, the targeting group may be a ligand formed by one or more targeting molecules or derivatives thereof selected from the group consisting of polymers such as polyethylene glycol, sugars such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc), asialoglycoproteins, asialoglycoresidues, lipoproteins (high density lipoproteins, low density lipoproteins, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, receptor ligands expressed by hepatocytes such as aptamers for transferrin, antibodies, polypeptides such as quantum dots, membrane-permeable peptides, or small molecule ligands.

[0231] In some embodiments, in the oligomeric compound, at least one or each of the targeting groups is selected from ligands capable of binding to mammalian hepatocyte surface receptors. In some embodiments, each of the targeting groups is independently a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each of the targeting groups is independently an asialoglycoprotein- or sugar-containing ligand. In some embodiments, each of the targeting groups is independently an asialoglycoprotein-containing ligand, such as asialoorosomucoid (ASOR) or asialofetuin (ASF).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-glucopyranose, β-D-glucopyranose, α-D-glucose, Cofuranose, 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, Nn-butyrylgalactosamine, N-isobutyrylgalactosamine Lactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-ethanolacyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4- The ligand contains one selected from the group consisting of tri-O-acetyl-1-thio-6-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-glucoheptapyranoside ethyl ester, 2,5-anhydro-D-allonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

[0232] In some embodiments, in the oligomeric compound, at least one or each of the targeting groups is a galactose or N-acetylgalactosamine containing ligand.

[0233] In certain embodiments, in the oligomeric compound, the targeting group is linked to one of A1 or A2, [ka] The compound fragment is selected from one of the following:

[0234] The targeting group can be linked to one of A1 or A2 via a suitable conjugation joint, and those skilled in the art can select an appropriate conjugation joint depending on the specific type of targeting group. For details of these conjugation joints, types of ligands, tethers, branched joints, and methods for binding targeting groups to oligonucleotides, see WO2009073809, WO2014179620, WO2014179627, and WO2015006740A2, the entire contents of which are incorporated herein by reference. In some embodiments, the targeting group can be linked via an acid-labile or reducible chemical bond, and these chemical bonds can be decomposed in the acidic environment of inclusion bodies.

[0235] In certain embodiments, the oligomeric compound further comprises one or more targeting groups and is linked to the oligomeric compound by a T3 covalent bond or to the terminal nucleotide at the other end of the oligomeric compound, non-exemplary linking methods are as follows: [ka] wherein T3' is a covalent bond, and Olig, A1, A2, T3, R1 and n are as defined above.

[0236] The oligomeric compounds described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations, which in terms of absolute stereochemistry can be defined as (R) or (S), α or β, such as for sugar anomers, or (D) or (L), such as for amino acids. The oligomeric compounds provided herein include all such possible isomers, as well as their racemic and optionally pure forms.

[0237] In certain embodiments, provided oligomeric compounds include 5'- and / or 3'-terminal monomers of at least one configuration as shown in formula (IVa) or formula (IVb), [ka] A1, A2, T3, T4, R1 and n are as defined above.

[0238] In some preferred embodiments, A1 and A2 are each independently selected from O; 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 connecting the monomer of Formula (IVa) or Formula (IVb) above to the oligomeric compound, wherein n is 0; and the oligomeric compound optionally further comprises a targeting group.

[0239] In some further preferred embodiments, A1 and A2 are each independently selected from O; one of T3 and T4 is H, a hydroxy 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 connecting the oligomeric compound of formula (IVa) or formula (IVb) above, wherein n is 0; and the oligomeric compound optionally further comprises a targeting group. In some further preferred embodiments, wherein the hydroxy protecting group is selected from an acetyl group, a benzyl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, and a 4,4'-dimethoxytrityl group, and the lipophilic group is C 14 , C 16 or C 18 The internucleoside linking group is selected from an alkylphosphonate linking group, a phosphodiester internucleoside linking group, or a phosphorothioate internucleoside linking group.

[0240] In certain embodiments, provided oligomeric compounds include 5'- and / or 3'-terminal monomers of at least one configuration as shown in formula (IVa-1) or formula (IVb-1), [ka] A1, A2, T3, T4, R1 and n are as defined above.

[0241] In some preferred embodiments, A1 and A2 are each independently selected from O; 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 connecting the monomer of Formula (IVa-1) or Formula (IVb-1) to the oligomeric compound, wherein n is 0; and the oligomeric compound optionally further comprises a targeting group.

[0242] In some further preferred embodiments, A1 and A2 are each independently selected from O; one of T3 and T4 is H, a hydroxy 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 connecting the monomer of Formula (IVa-1) or Formula (IVb-1) to the oligomeric compound, wherein n is 0; and the oligomeric compound optionally further comprises a targeting group. In some further preferred embodiments, wherein the hydroxy protecting group is selected from an acetyl group, a benzyl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, and a 4,4'-dimethoxytrityl group, and the lipophilic group is C 14 , C 16 or C 18 The internucleoside linking group is selected from an alkylphosphonate linking group, a phosphodiester internucleoside linking group, or a phosphorothioate internucleoside linking group.

[0243] In certain embodiments, oligomeric compounds provided herein include, but are not limited to, examples having the following configurations:

[0244] [ka] wherein Olig represents an oligonucleotide moiety linking the above monomers to the 5'-end and / or 3'-end of the oligomeric compound, respectively; A1, A2, R1 and n are as defined above; T3 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 oligomeric compound optionally further comprises a targeting group.

[0245] In certain embodiments, in the provided oligomeric compounds, one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, wherein the lipophilic group is linked to one of A1 or A2, optionally via a linker, and the other of T3 and T4 is an internucleoside linking group connecting a monomer included in the provided oligomeric compound to the 5'-terminus of the oligomeric compound. It is understood that the oligomeric compound comprises at least one 5'-terminal monomer provided herein (e.g., as shown in Formula (III) or its stereoisomer, Formula (IVa), Formula (IVb), (IVa-1), or (IVb-1)).

[0246] In certain embodiments, in the provided oligomeric compounds, 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 connecting a monomer included in the provided oligomeric compound to the 3'-terminus of the oligomeric compound. It is understood that the oligomeric compound comprises at least one 3'-terminal monomer provided herein (e.g., as shown in Formula (III) or its stereoisomer, Formula (IVa), Formula (IVb), (Iva-1), or (IVb-1)).

[0247] In certain embodiments, in the provided oligomeric compounds, one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, the lipophilic group being linked to one of A1 or A2, optionally via a linker, and the other of T3 and T4 is a homologous or different internucleoside linking group that independently connects a monomer contained in the provided oligomeric compound to the 5'-end and the 3'-end of the oligomeric compound, respectively. It is understood that the oligomeric compound comprises at least one homologous or different monomer provided herein (e.g., as shown in Formula (III) or its stereoisomer, Formula (IVa), Formula (IVb), (IVa-1), or (IVb-1)) at the 5'-end and the 3'-end of the oligomeric compound, respectively.

[0248] Generally, the oligomeric compound comprises, at any one of its 5'-end and / or 3'-end, independently one homologous or different monomeric region as shown in formula (III) or a stereoisomer thereof, formula (IVa), formula (IVb), (IVa-1), or (IVb-1). In some other cases, the oligomeric compound comprises, at any one of its 5'-end and / or 3'-end, independently at least two consecutive homologous or different monomeric regions as shown in formula (III) or a stereoisomer thereof, formula (IVa), formula (IVb), (IVa-1), or (IVb-1).

[0249] Exemplary, but not limited to, linking the monomers herein to the 5'-end and / or 3'-end of an oligomeric compound include: [ka] A2 and A2' represent the same or different monomer variables as described above, R1 and R1' represent the same or different monomer variables as described above, and Olig represents the oligonucleotide moiety that links the above monomers to the 5'-end and / or 3'-end of the oligomeric compound, respectively.

[0250] In certain embodiments, when n is not 0, it means that the bicyclic structure has 1 to 6 independent R1 group substituents. It should be understood that in the bicyclic abasic compounds herein, these substituents do not include natural or modified nucleobases. In certain embodiments, R1 is independently selected from halogen, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C2-C6 alkenyl group, and a substituted or unsubstituted C2-C6 alkynyl group. More preferably, R1 is independently selected from fluorine, chlorine, bromine, iodine, methyl, ethyl, isopropyl, propyl, 3-propenyl, ethynyl, and 3-propynyl groups.

[0251] In certain embodiments, n is 0, representing an absence of R 1 group substitution on the ring structure.

[0252] The features of two or more such oligomeric compounds provided by the present invention can be arbitrarily combined, and are included within the scope of the present invention, provided that such features are not mutually inconsistent. Furthermore, the bicyclic abasic compounds described in the present invention, such as compounds of formulae (I), (IIa), (IIb), (IIa-1), and (IIb-1), and any combination of the features of two or more such compounds, can be applied to 5'-end and / or 3'-end oligonucleotide monomers, and therefore, unless inconsistent, the technical features defined for the bicyclic abasic compounds described herein equally apply to the oligomeric compounds herein.

[0253] In certain embodiments, in an oligomeric compound, fragments are provided that comprise at least one monomer as shown below:

[0254] [ka]

[0255] In certain embodiments, the oligomeric compounds provided herein have the following specific structures:

[0256] [ka] [ka]

[0257] Here, Olig represents the oligonucleotide moiety that attaches the above monomers to the 5'-end and / or 3'-end of the oligomeric compound, respectively.

[0258] In certain embodiments, the oligomeric compound is a single-stranded oligonucleotide, such as an antisense oligonucleotide (also known as an ASO), a ribozyme, or an aptamer, as are well known in the art. In certain embodiments, the oligomeric compound is a double-stranded RNAi compound (siRNA), such as a double-stranded ribonucleic acid (also known as a dsRNA), as are well known in the art, wherein one or both strands are an oligomeric compound disclosed herein.

[0259] In certain embodiments, at least a portion of the nucleobase sequence of the oligomeric compound is partially or fully complementary to 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.

[0260] In certain embodiments, a double-stranded ribonucleic acid (dsRNA) reagent is provided, both 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, and at least one of the sense strand and the antisense strand is an oligomeric compound provided by the preceding sentence, comprising at least one 5'-terminal and / or 3'-terminal monomer having Formula (III) herein or a stereoisomer thereof, Formula (IVa), Formula (IVb), Formula (IVa-1), or Formula (IVb-1), and wherein the double-stranded ribonucleic acid (dsRNA) reagent optionally further comprises an independent targeting group.

[0261] In certain embodiments, double-stranded ribonucleic acid (dsRNA) reagents are provided, wherein the sense strand is an oligomeric compound provided by a 5'-terminal monomer of formula (III) or a stereoisomer thereof, formula (IVa), formula (IVb), formula (IVa-1), or formula (IVb-1) herein. In certain embodiments, double-stranded ribonucleic acid (dsRNA) reagents are provided, wherein the sense strand is an oligomeric compound provided by a 3'-terminal monomer of formula (III) or a stereoisomer thereof, formula (IVa), formula (IVb), formula (IVa-1), or formula (IVb-1) herein. In certain embodiments, double-stranded ribonucleic acid (dsRNA) reagents are provided, wherein the sense strand is an oligomeric compound provided by a 5'-terminal and a 3'-terminal monomer of formula (III) or a stereoisomer thereof, formula (IVa), formula (IVb), formula (IVa-1), or formula (IVb-1) herein.

[0262] In certain embodiments, double-stranded ribonucleic acid (dsRNA) reagents are provided, wherein the antisense strand is an oligomeric compound provided by 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 certain embodiments, double-stranded ribonucleic acid (dsRNA) reagents are provided, wherein the antisense strand is an oligomeric compound provided by a 3'-terminal monomer of formula (III) or a stereoisomer thereof, formula (IVa), formula (IVb), formula (IVa-1), or formula (IVb-1) herein. In certain embodiments, double-stranded ribonucleic acid (dsRNA) reagents are provided, wherein the antisense strand is an oligomeric compound provided by a 5'-terminal monomer of formula (III) or a stereoisomer thereof, formula (IVa), formula (IVb), formula (IVa-1), or formula (IVb-1) herein.

[0263] In certain embodiments, a double-stranded ribonucleic acid (dsRNA) reagent is provided, optionally further comprising an independent targeting group, which should be understood to be in addition to the targeting groups present in the oligomeric compound monomers herein: [ka] [ka] represents a monomer moiety of formula (III), which may further comprise a targeting group at other positions in the double-stranded ribonucleic acid (dsRNA) reagent. In some embodiments, the 5'- and / or 3'-terminal nucleotide in the double-stranded ribonucleic acid (dsRNA) reagent further comprises one or more targeting groups or linking groups, it being understood that these targeting groups may be conjugated to the 5'- and / or 3'-terminal nucleotide of either strand in the double-stranded ribonucleic acid (dsRNA) reagent. [ka] The targeting group can be a ligand commonly used in the field of siRNA administration.As used herein, these targeting groups can be selected from those having the above-mentioned oligomeric compounds and those having the same bicyclic abasic nucleic acid analogue compounds, and the targeting group of the provided double-stranded ribonucleic acid (dsRNA) reagent, its described properties or structure, can be applied to the targeting group of the oligomeric compounds and bicyclic abasic nucleic acid analogue compounds of this specification as well.

[0264] In some embodiments, in the provided double-stranded ribonucleic acid (dsRNA) reagents, the targeting group may be selected from one or more ligands including the following targeting molecules or derivatives thereof: polymers, saccharides, ligands for receptors expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands.

[0265] In some embodiments, provided double-stranded ribonucleic acid (dsRNA) reagents are provided wherein at least one or each of the targeting groups is selected from a ligand capable of binding to a mammalian hepatocyte surface receptor.

[0266] In some embodiments, provided are double-stranded ribonucleic acid (dsRNA) reagents wherein each of the targeting groups is independently a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes.

[0267] In some embodiments, in the provided double-stranded ribonucleic acid (dsRNA) reagent, each of the targeting groups independently comprises an asialoglycoprotein or a sugar. The targeting groups can be linked to the 5'-terminal and / or 3'-terminal nucleotide via a suitable conjugation joint, and those skilled in the art can select a suitable conjugation joint depending on the specific type of targeting group.

[0268] In some embodiments, one or more targeting or binding groups described in the dsRNA reagent are conjugated to the sense strand.The targeting ligands can alter the distribution, targeting or lifetime, endosomolytic properties, improve trafficking, hybridization, and specificity of the dsRNA reagent into which they are incorporated, and include, by way of non-limiting example, 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, and the like. Examples of suitable chemoattractants include: acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folate, vitamin B12, biotin, RGD peptides, RGD peptidomimetics, or aptamers; other examples include dyes, intercalators (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, etc. cleases or chelating agents (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, camphor, menthol, 1,3-propylene glycol, heptadecyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine), and peptide conjugates (e.g., Antennapedia Peptide Examples of suitable ribonucleases include: tides, Tat peptides), alkylating agents, phosphate, amino groups, mercapto groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino groups, alkyl groups, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), carriers / adsorption promoters (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole complexes, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl groups, HRP, or AP.In some embodiments, the targeting or binding group comprises a member selected from N-acetyl-galactosamine (GalNAc), a lipophilic molecule.

[0269] In certain embodiments, the targeting group, after attachment, is [ka] is selected from one of the compounds:

[0270] The targeting groups herein can be conjugated to the internucleoside linking groups with the monomeric compounds or oligonucleotides herein via reactive functional groups, for example, GLO generally indicates having an oxothioate internucleoside linking group, and GLS generally indicates having a phosphorothioate internucleoside linking group.

[0271] In certain embodiments, the present invention provides oligomeric compounds comprising any of a variety of lengths. In certain embodiments, in an oligomeric compound or double-stranded ribonucleic acid (dsRNA), any one of the single strand, sense strand, and / or antisense strand each comprises 8 to 40 nucleotides in length. For example, in certain embodiments, the nucleotide length of the oligomeric compound 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 nucleotides. For example, in certain embodiments, the present invention provides a method for treating a nucleotide sequence comprising the steps of: 1) providing a nucleotide sequence having a length of 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 8-25, 8-26, 8-27, 8-28, 8-29, 8-30, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27, 9-28, 9-29, 8-30, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-29, 9-30, 9-31, 9-32, 9-33, 9-34, 9-35, 9-36, 9-37, 9-38, 9-39, 9-40, 9-41, 9-42, 9-43, 9-44, 9-45, 9-46, 9-47, 9-48, 9-49, 9-50, 9-51, 9-52, 9-53, 9-54, 9-55, 9 ~17, 9~18, 9~19, 9~20, 9~21, 9~22, 9~23, 9~24, 9~25, 9~26, 9~27, 9~28, 9~29, 9~30, 10~11, 10~12, 10~13, 10~14, 10~15, 10~16, 10~17, 10~18, 10~19, 10~20, 10~21, 10~22, 10~23, 10~24, 10~25, 10~26, 10~27, 10~28, 10~29, 10~30, 10~31, 10~32, 10~33, 10~34, 10~35, 10~36, 10~37, 10~38, 10~39, 10~40, 10~41, 10~42, 10~43, 10~44, 10~45, 10~46, 10~47, 10~48, 10~49, 10~50, 10~51, 10~52, 10~53, 10~54, 10~55, 10~56, 10~57, 10~58, 10~59, 10~60, 10~61, 10~62, 10~63, 10~64, 10~65, 10~66, 10~67, 10~68, 10~69, 10~70, 10~71, 1 9, 10-30, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24, 11-25, 11-26, 11-27, 11-28, 11-29, 11-30, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22 , 12-23, 12-24, 12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23, 13-24, 13-25, 13-26, 13-27, 13-28, 13-29, 13-30, 14-15, 14-16, 14-17, 14-18, 14-19,14-20, 14-21, 14-22, 14-23, 14-24, 14-25, 14-26, 14-27, 14-28, 14-29, 14-30, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15-28, 15-29, 15-30, 16-17, 16-18, 16-19, 16-20, 16-21, 16-22, 16-23, 16-24, 1 6-25, 16-26, 16-27, 16-28, 16-29, 16-30, 17-18, 17-19, 17-20, 17-21, 17-22, 17-23, 17-24, 17-25, 17-26, 17-27, 17-28, 17-29, 17-30, 18-19, 18-20, 18-21, 18-22, 18-23, 18-24, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 19-20, 19-21, 19-22, 19- 23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 20-21, 20-22, 20-23, 20-24, 20-25, 20-26, 20-27, 20-28, 20-29, 20-30, 21-22, 21-23, 21-24, 21-25, 21-26, 21-27, 21-28, 21-29, 21-30, 22-23, 22-24, 22-25, 22-26, 22-27, 22-28, 22-29, 22-30 , 23-24, 23-25, 23-26, 23-27, 23-28, 23-29, 23-30, 24-25, 24-26, 24-27, 24-28, 24-29, 24-30, 25-26, 25-27, 25-28, 25-29, 25-30, 26-27, 26-28, 26-29, 26-30, 27-28, 27-29, 27-30, 28-29, 28-30, or 9-30 nucleosides. In embodiments limiting the number of nucleosides in the oligomeric compound or oligonucleotide, whether to a range or a specific number, the oligomeric compound or oligonucleotide may further include other substituents. For example, an oligonucleotide containing 8 to 30 nucleosides does not include an oligonucleotide having 31 nucleosides, unless otherwise indicated.Such oligonucleotides may further comprise, for example, one or more conjugates, terminal groups, or other substituents, and the monomers herein are generally considered to be terminal groups that the oligonucleotide further comprises. In certain embodiments, terminal groups include, but are not limited to, terminal nucleosides. In such embodiments, the terminal groups are modified differently from the terminal nucleosides of the oligonucleotide, thereby distinguishing such terminal groups from the nucleosides of the oligonucleotide.

[0272] In certain embodiments, the oligomeric compounds provided by the present invention contain one or more modifications in the remaining portions of the provided monomeric compounds. Such modifications include, but are not limited to, sugar modifications or substitutions with sugar substitutes, 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 ribonucleic acids disclosed herein.

[0273] In certain embodiments, a method for silencing gene expression is provided, comprising contacting a cell with an oligomeric compound or a reagent comprising double-stranded ribonucleic acid (dsRNA) as provided above, wherein the sense strand and antisense strand of the oligomeric compound and the double-stranded ribonucleic acid (dsRNA) reagent are both 8 to 40 nucleotides in length, and the antisense strand of the oligomeric compound or double-stranded ribonucleic acid (dsRNA) reagent is complementary to a 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 to silence its function. In certain embodiments, the method further comprises detecting the level of the target RNA. In certain embodiments, methods of inhibiting gene expression are provided, comprising contacting one or more cells or tissues with an oligomeric compound or double-stranded ribonucleic acid (dsRNA) comprising a terminal monomer of Formula (III) or a stereoisomer thereof, Formula (IVa), Formula (IVb), Formula (IVa-1) or Formula (IVb-1).

[0274] In the context of the present invention, the term "oligomeric compound" refers to a polymer having at least one region capable of hybridizing with a nucleic acid molecule. The term "oligomeric compound" includes oligonucleotides, oligonucleotide analogs, and oligonucleosides and nucleotide mimetics, and / or mixed polymers containing nucleic acid and non-nucleic acid components. The term "oligomeric compound" also includes polymers containing linked monomeric subunits, where the monomeric subunits include non-nucleic acid components such as nucleosides, modified nucleosides, nucleoside analogs, nucleoside mimetics, and coupling groups. In certain embodiments, mixtures of monomeric subunits (e.g., but not limited to) provide oligomeric compounds with enhanced properties for applications such as therapeutics and diagnostics. The bicyclic abasic compounds provided by the present invention are classified as non-nucleic acid components due to the absence of nucleobases. The monomer subunits can be linked by naturally occurring phosphodiester internucleoside linkages or any of the internucleoside linkages disclosed herein, such as, but not limited to, phosphorothioate internucleoside linkages or mixtures thereof.

[0275] Oligonucleotide: As used herein, the term "oligonucleotide" refers to a polymeric form of different nucleotides ranging from 2 to 2500 nucleotides, and oligonucleotides may be single-stranded or double-stranded. The term "oligonucleotide" includes oligonucleotides consisting of naturally occurring nucleoside bases, sugars, and covalent internucleoside linkages, as well as oligonucleotides containing one or more non-naturally occurring moieties. Such non-naturally occurring oligonucleotides are generally more desirable than naturally occurring forms due to their desirable properties, such as improved cellular integration, enhanced affinity for nucleic acid targets, and improved stability in the presence of nucleases, e.g., modifications to the base or sugar moiety. In certain embodiments, oligonucleotides have 500 to 1500 nucleotides, typically used in gene therapy, for example. In certain embodiments, oligonucleotides are single-stranded or double-stranded and have 7 to 100 nucleotides. In certain embodiments, oligonucleotides are single-stranded or double-stranded and have 15 to 100 nucleotides. In another embodiment, the oligonucleotide is single-stranded or double-stranded and has 15 to 50 nucleotides, typically, for example, where the oligonucleotide is a nucleic acid inhibitor molecule. In another embodiment, the oligonucleotide is single-stranded or double-stranded and has 25 to 40 nucleotides, typically, for example, where the oligonucleotide is a nucleic acid inhibitor molecule. In a further embodiment, the oligonucleotide is single-stranded or double-stranded and has 19 to 40 or 19 to 25 nucleotides, typically, for example, where the oligonucleotide is a double-stranded nucleic acid inhibitor molecule and forms a duplex of at least 18 to 25 base pairs. In another embodiment, the oligonucleotide is single-stranded and has 15 to 25 nucleotides, typically, for example, where the oligonucleotide is a single-stranded or double-stranded RNAi inhibitor molecule. Generally, the oligonucleotide has one or more phosphorus-containing internucleotide linkage groups, as described herein.In other embodiments, the internucleotide linking group is a non-phosphorus-containing linkage, as described herein. The oligomeric compounds herein are oligonucleotides in nature and further comprise a terminal monomer, as described herein.

[0276] Generally, oligomeric compounds comprise a backbone of linked monomeric subunits, and the linkages between the linked monomeric subunits, sugar moieties or substitutes, and heterocyclic base moieties may be independently modified. The linked sugar units may or may not contain heterocyclic bases and can be replaced with mimetics such as peptide nucleic acid monomers. The ability to modify or substitute some or all of the monomers in each monomer of an oligomeric compound can result in a large number of possible motifs.

[0277] Oligomeric compounds are generally prepared in a conventional linear form, linked or otherwise made circular, and may contain branches. Oligomeric compounds may form double-stranded constructs, e.g., hybridize to form the two strands of a double-stranded composition. The double-stranded composition may be linked or separated, and may contain overhangs at the ends.

[0278] As known in the art, the term "nucleoside" in the context of the present invention refers to a combination of a base and a sugar. The base portion of a nucleoside is called a nucleobase and is generally a heterocyclic base portion. The two most common classes of heterocyclic bases are purines and pyrimidines. For example, suitable natural nucleobases include purine and pyrimidine bases such as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U). Suitable modified nucleobases include diaminopurine and its derivatives, alkylated purines or pyrimidines, acylated purines or pyrimidines, thiolated purines or pyrimidines, etc. It is understood that in the context of the present invention, a bicyclic abasic nucleic acid analog having a 5'-end and / or 3'-end monomer does not have a base of such a nucleoside in its bicyclic substituent R1.

[0279] As is known in the art, the term "nucleotide" in the context of the present invention refers to a nucleoside further comprising a phosphate group covalently attached to the nucleoside sugar moiety. In the case of pentofuranosyl-containing nucleosides, the phosphate group can be attached to the 2', 3', or 5' hydroxyl moiety of the sugar. When forming oligonucleotides, the phosphate groups are covalently linked to adjacent nucleosides to form a linear polymeric compound. Cyclic structures can be obtained by hybridization or covalent bond formation by attachment to corresponding termini of the linear polymeric structure; however, open linear structures are generally preferred. In oligonucleotide structures, the phosphate groups generally form the internucleoside linkages of oligonucleotides; the common internucleoside linkage in RNA and DNA is a 3'-5' phosphodiester bond.

[0280] As used herein, a "terminal group" refers to one or more atoms or groups attached to one or both of the 3'-terminus or 5'-terminus of an oligonucleotide. In certain embodiments, the terminal group is a conjugate group. In certain embodiments, the terminal group comprises one or more additional nucleosides, commonly referred to as 3'- or 5'-terminus terminal nucleotides. As used herein, the term "5'-terminal nucleotide or 3'-terminal nucleotide" refers to a nucleotide located at the 5'- or 3'-terminus of an oligonucleotide or oligomeric compound.

[0281] As used herein, the term "phosphoramidite" refers to a nitrogen-containing trivalent phosphorus derivative. Examples of suitable phosphoramidites are described herein.

[0282] As used herein, the term "siRNA" refers to short interfering RNA or silencing RNA. siRNA is a class of double-stranded RNA molecules that can be 20-25 (or even shorter) base pairs in length, similar to microRNAs (miRNAs) that function in the RNA interference (RNAi) pathway. siRNAs interfere with the expression of specific genes that have nucleotide sequences complementary to the siRNA by degrading the mRNA after transcription, thereby preventing translation. siRNAs silence gene expression in cells by inducing messenger RNA (mRNA) cleavage by the RNA-induced silencing complex (RISC).

[0283] As used herein, unless otherwise indicated or modified, the term "duplex" refers to two single oligomeric compounds that hybridize to one another. Such double-stranded compounds may have one or more non-hybridizing nucleosides (dangling) at one or both ends of one or both strands and / or one or more internal non-hybridizing nucleosides (mismatches), provided that sufficient complementarity exists to maintain hybridization under physiologically relevant conditions.

[0284] As used herein, the term "self-complementary" or "hairpin" refers to a single oligomeric compound that includes a double-stranded region formed by self-hybridization of the oligomeric compound.

[0285] As used herein, the term "single-stranded" refers to an oligomeric compound that does not hybridize to its complementary sequence and does not have sufficient self-complementarity to form a hairpin structure under physiologically relevant conditions. A single-stranded compound can combine with its complementary sequence to form a double-stranded or partially double-stranded compound.

[0286] Therapeutic formulations of dsRNA reagents or target gene antisense polynucleotide reagents can be prepared for storage by mixing molecules or compounds having the desired purity and, optionally, pharmaceutically acceptable carriers, excipients, or stabilizers [Remington's Pharmaceutical Sciences 21st edition (2006)] in the form of a lyophilized formulation or aqueous solution. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the amounts and concentrations used, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., benzyl dimethylstearyl ammonium chloride hydrate, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, p-hydroxybenzoic acid esters such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues), and the like. ), 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 non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0287] Administration method In some embodiments, the tissue to which the compound is administered is a tissue where a disease or condition associated with the target gene exists or may manifest, non-limiting examples of which are the liver or kidney. Direct tissue administration can be achieved by direct injection or other means. While many orally delivered compounds naturally enter and pass through the liver and kidney, some embodiments of the therapeutic methods of the present invention include orally administering one or more target gene dsRNA reagents to a subject. The dsRNA reagent or target gene antisense polynucleotide agent can be administered once, alone or in combination with other therapeutic agents, or they can be administered multiple times. When administered multiple times, the target gene dsRNA reagent or target gene antisense polynucleotide agent can be administered by different routes. For example, and not intended to be limiting, the first (or first few) administrations can be administered subcutaneously, and one or more additional administrations can be administered orally and / or systemically.

[0288] In embodiments of the present invention in which systemic administration of a target gene dsRNA reagent or a target gene antisense polynucleotide agent is desired, the target gene dsRNA reagent or the target gene antisense polynucleotide reagent can be prepared for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable preparations can be in unit dosage forms such as ampoules or multi-dose containers, with or without added preservatives. The target gene dsRNA reagent formulation (also called a pharmaceutical composition) can take the form of a suspension, solution, or emulsion in an oily or aqueous carrier, and can contain formulatory agents such as suspending agents, stabilizers, and / or dispersing agents.

[0289] Formulations 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, alcoholic / aqueous solutions, emulsions, or suspensions containing saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose solution, glucose and sodium chloride solution, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose solution), and the like. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, can also be present. Other forms of administration, such as intravenous administration, result in relatively low doses. If a subject does not respond adequately to the initial dose, a higher dose can be used (or the dose can be effectively increased by a different, more localized delivery route) within the limits of patient tolerance. If necessary, multiple doses per day can be administered to achieve appropriate systemic or local levels of one or more target gene dsRNA reagents or target gene antisense polynucleotide reagents to achieve an appropriate reduction in target gene activity.

[0290] In other embodiments, the methods of the present invention involve the use of a delivery vehicle, e.g., a biocompatible microparticle, nanoparticle, or implant suitable for implantation into the intended recipient, etc. PCT Publication WO 95 / 24929 (incorporated herein by reference) describes an exemplary biodegradable implant that can be used in accordance with the methods, which describes a biocompatible, biodegradable polymer matrix that includes a biopolymer.

[0291] 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 reagents or target gene antisense polynucleotide reagents to a subject. In some embodiments, the matrix may be biodegradable. The matrix polymer may be a natural or synthetic polymer. The polymer can be selected based on the desired period of release, typically on the order of a few hours to a year or more. Release periods ranging from a few hours to 3-12 months are typically available. The polymer is optionally in the form of a hydrogel capable of absorbing up to about 90% of its weight in water and is optionally crosslinked with multivalent ions or other polymers.

[0292] Typically, in some embodiments of the present invention, target gene dsRNA reagents or target gene antisense polynucleotide reagents can be delivered by diffusion or degradation of the polymer matrix using biodegradable implants. Exemplary synthetic polymers for such use are known in the art. Using methods known in the art, biodegradable and non-biodegradable polymers can be used to deliver target gene dsRNA reagents or target gene antisense polynucleotide reagents. Bioadhesive polymers, such as bioerodible hydrogels (H.S.Sawhney, C.P.Pathak, and J.A. Hubbell in Macromolecules, 1993, 26, 581-587), can also be used to deliver target gene dsRNA reagents or target gene antisense polynucleotide reagents to treat diseases or conditions associated with target genes. Other suitable delivery systems include timed-release, delayed-release, or sustained-release delivery systems. Such systems can avoid repeated administration of target gene dsRNA reagents or target gene antisense polynucleotide agents, thereby increasing convenience for patients and medical professionals. Many types of release delivery systems are available and known to those skilled 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, some of which are also adaptable for implantation, can be used.

[0293] The use of long-term sustained release implants is suitable for the preventive treatment of subjects and for subjects at risk of developing recurrent target gene-related diseases or conditions.As used herein, long-term release refers to the construction and arrangement of implants to deliver therapeutic levels of target gene dsRNA reagents or target gene antisense polynucleotide reagents for at least 10 days, 20 days, 30 days, 60 days, 90 days, 6 months, 1 year or more.Long-term sustained release implants are known to those skilled in the art and include some of the release systems mentioned above.

[0294] Effective dose In some aspects, the methods of the invention involve contacting cells with an effective amount of a dsRNA reagent or antisense polynucleotide reagent to reduce gene expression in the contacted cells. Certain embodiments of the methods of the invention involve administering to a subject an amount of a dsRNA reagent or antisense polynucleotide agent effective to effectively reduce gene expression and treat the associated disease or condition in the subject. An "effective amount" for reducing expression and / or treating the associated disease or disorder refers to an amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of a dsRNA reagent or antisense polynucleotide agent for treating the associated disease or condition can be (i) the amount necessary to slow or halt the progression of the disease or condition, or (ii) an amount that reverses, reduces, or eliminates one or more symptoms of the disease or condition. In some aspects of the invention, an effective amount is the amount of a dsRNA reagent or antisense polynucleotide agent that, when administered to a subject in need of treatment for the associated disease or condition, results in a therapeutic response in preventing and / or treating the disease or condition. According to some aspects of the invention, an effective amount is an amount of a dsRNA or antisense polynucleotide reagent of the invention that, when combined or used in conjunction with another therapeutic treatment for the relevant disease or condition, results in a therapeutic response in preventing and / or treating the disease or condition. In some embodiments of the invention, the biological effect of treating a subject with a dsRNA or antisense polynucleotide reagent of the invention can be an improvement and / or complete elimination of symptoms caused by the relevant disease or condition. In some embodiments of the invention, the biological effect is complete elimination of the relevant disease or condition, as evidenced, for example, by a diagnostic test showing that the subject does not have the relevant disease or condition. In some embodiments, an effective amount is an amount that results in a desired response, such as, for example, an amount that reduces the relevant disease or condition in cells, tissues, and / or subjects with the disease or condition.Some embodiments of the present invention include methods of determining the efficacy of a target gene dsRNA reagent or antisense polynucleotide reagent of the present invention administered to a subject to treat a disease or condition associated with a target gene by assessing and / or monitoring one or more "physiological characteristics" of the disease or condition associated with the target gene in the subject.

[0295] It should be understood that gene silencing can be achieved in any cell in which the target gene is expressed, either constitutively or by genome engineering, and can be determined by any appropriate measurement. In some embodiments of the present invention, administration of the dsRNA reagent of the present invention reduces target gene expression 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%. In some embodiments of the present invention, administration of the dsRNA reagent of the present invention reduces target gene expression by 5% to 10%, 5% to 25%, 10% to 50%, 10% to 75%, 25% to 75%, 25% to 100%, or 50% to 100%.

[0296] The dsRNA and antisense polynucleotide reagents of the present invention are delivered in pharmaceutical compositions at a dose sufficient to express the target gene. In certain embodiments of the present invention, the dose of the dsRNA or antisense polynucleotide agent is 0.01 to 200.0 milligrams per kilogram of recipient body weight per day, typically 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, or 4 to 15 mg / kg body weight per day, inclusive. For example, a single dose of a dsRNA reagent or antisense polynucleotide reagent may be administered in amounts of 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 , 2mg / kg, 2.1mg / kg, 2.2mg / 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, 4.1 mg / 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 / k g, 6.3mg / kg, 6.4mg / kg, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg, 6.9mg / kg, 7mg / kg, 7.1mg / kg, 7.2mg / kg, 7.3m g / 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 / k g, 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 / It can be administered in amounts ranging from 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 of body weight.

[0297] When determining the delivery dose 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 to be delivered depends on various factors, including concurrent treatment, dosage, and individual subject parameters, including age, physical condition, body size and weight.These are factors known to those skilled in the art and can be solved by conventional experimentation.In some embodiments, the maximum dose based on sound medical judgment, i.e., the maximum safe dose, can be used.

[0298] In some embodiments, the methods of the present invention can include administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of a reagent or antisense polynucleotide reagent to a subject. In some cases, a dose of a pharmaceutical compound can be administered to a subject at least daily, every other day, weekly, biweekly, monthly, etc., and can be administered once a day or more times a day, for example, two, three, four, five, or more times in a 24-hour period. The pharmaceutical compositions of the present invention can be administered once a day, or the dsRNA reagent or antisense polynucleotide reagent can be administered in two, three, or more subdoses at appropriate intervals throughout the day, or can be delivered using continuous infusion or via a sustained-release formulation. In some embodiments of the methods of the present invention, the pharmaceutical compositions of the present invention are administered to a subject at least once a day, once a week, once a month, or once a year.

[0299] Certain embodiments of the present invention include the use of pharmaceutical compositions comprising a dsRNA or antisense polynucleotide agent and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising a dsRNA or antisense polynucleotide agent can be used in the methods of the present invention to downregulate gene expression and activity in cells and treat associated diseases or conditions. Such pharmaceutical compositions can be formulated based on the delivery method. Non-limiting examples of formulations for delivery methods include compositions formulated for subcutaneous delivery, systems via parenteral delivery, intravenous (IV) delivery, intrathecal delivery, and direct delivery to the brain. The dsRNA or antisense polynucleotide agent can be administered to cells using one or more methods, including topical (e.g., via a transdermal patch), pulmonary (e.g., by inhalation or insufflation of a powder or aerosol), nebulizer, intratracheal, intranasal, epidermal, transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, subcutaneous administration by an implanted device, or intracranial, intrathecal, or intraventricular administration by intraparenchymal administration. The dsRNA reagent or antisense polynucleotide agent can also be delivered directly to the target tissue, such as directly to the liver or directly to the kidney. "Delivering" a "dsRNA reagent" or "antisense polynucleotide reagent" to a cell can be understood to include delivering the dsRNA reagent or antisense polynucleotide agent, directly expressing the dsRNA reagent in the cell, expressing the dsRNA reagent from an encoding vector delivered in the cell, or any suitable means for causing the dsRNA reagent or antisense polynucleotide reagent to appear in the cell, respectively. The preparation and use of formulations and means for delivering inhibitory RNA are known and commonly used in the art.

[0300] In some embodiments, the composition further comprises one or more additional therapeutic agents. The compositions of the present invention may comprise one or more dsRNA reagents and, optionally, one or more pharmaceutically acceptable carriers, delivery agents, targeting agents, detectable labels, etc. A non-limiting example of a targeting agent that can be utilized by some embodiments of the methods of the present invention is an agent that allows the dsRNA reagent of the present invention to be introduced and / or enter the cells to be treated. The selection of a targeting agent depends on factors such as the nature of the associated disease or condition and the type of target cell. In a non-limiting example, some embodiments of the present invention may require targeting and / or entry of the dsRNA reagent into hepatocytes. It should be understood that in some embodiments of the methods of the present invention, the therapeutic agent includes a dsRNA reagent having only a delivery agent without any additional linking element, for example, a delivery agent containing N-acetylgalactosamine (GalNAc). For example, in some embodiments of the present invention, a dsRNA reagent may be linked to a delivery compound comprising GalNAc, included in a composition containing a pharmaceutically acceptable carrier, and administered to a cell or subject without any detectable label or targeting agent or the like linked to the dsRNA reagent.

[0301] When the dsRNA reagent of the present invention is administered together with and / or linked to one or more delivery agents, targeting agents, labeling agents, etc., those skilled in the art can recognize, select, and use the appropriate reagent for use in the method of the present invention. In certain methods of the present invention, labeling reagents can be used to determine the location of the dsRNA reagent in cells and tissues, and can be used to determine the location of cells, tissues, or organs to which a therapeutic composition containing a dsRNA reagent is administered in the method of the present invention. Means for attaching and using labeling reagents, such as enzyme labels, dyes, and radioactive labels, are known in the art. It should be understood that in some embodiments of the compositions and methods of the present invention, labeling reagents bind to one or both of the sense polynucleotide and the antisense polynucleotide contained in the dsRNA reagent.

[0302] In some embodiments, the composition is packaged in a reagent kit, container, packaging, dispenser, pre-filled syringe, or vial. Reagent kits containing one or more target gene dsRNA reagents and / or target gene antisense polynucleotide reagents, as well as instructions for use in the methods of the present invention, are also within the scope of the present invention. The reagent kits 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 diseases or conditions associated with target genes. Reagent kits containing one or more target gene dsRNA reagents, target gene sense polynucleotides, and target gene antisense polynucleotide reagents can be manufactured for use in the therapeutic methods of the present invention. The components of the reagent kits of the present invention can be packaged in aqueous media or lyophilized form. The reagent kits of the present invention may include a separate carrier for tightly containing 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 series of container devices can contain one or more compounds, such as a target gene) dsRNA reagent and / or a target gene sense or antisense polynucleotide reagent. The second container device or series of container devices can contain a targeting agent, labeling agent, delivery agent, etc., included as part of the target gene) dsRNA reagent and / or target gene antisense polynucleotide reagent administered in embodiments of the therapeutic methods of the invention.

[0303] The reagent kits of the present invention may further include instructions, which typically take written form and provide directions for administering the treatment embodied by the reagent kit and for making decisions based on that treatment.

[0304] Cells, subjects and controls The methods of the present invention can be used in conjunction with cells, tissues, organs, and / or subjects. In some embodiments of the present invention, the subject is a human or a vertebrate mammal, including, but not limited to, a dog, cat, horse, cow, goat, mouse, rat, and primate such as a monkey. Thus, the present invention can be used to treat a disease or condition associated with a target gene in human and non-human subjects.

[0305] In some aspects of the invention, the subject may be a farm animal, a zoo animal, a domestic animal, or a non-domestic animal, and the methods of the invention can be used in veterinary prophylactic and therapeutic methods. In some embodiments of the invention, the subject is a human, and the methods of the invention can be used in human prophylactic and therapeutic methods.

[0306] Non-limiting examples of subjects to which the present invention can be applied include subjects diagnosed with, suspected of having, or at risk for 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 levels of target gene expression and / or activity are described elsewhere herein. The methods of the present invention can be applied to subjects who, upon treatment, have been diagnosed with a disease or condition, are associated with higher than desired target gene expression and / or activity, or are believed to be at risk for having or developing a disease or condition associated with higher than desired target gene expression and / or activity. In some embodiments of the present invention, the disease or condition associated with higher than desired target gene expression and / or activity is an acute disease or condition, and in certain embodiments of the present invention, the disease or condition associated with higher than desired target gene expression and / or activity is a chronic disease or condition.

[0307] In another non-limiting example, the target gene dsRNA reagents of the present invention are administered to treat a disease or disorder caused by or associated with target gene activation, or a disease or disorder whose symptoms or progression are responsive to target gene inactivation. The term "target gene-associated disease" includes diseases, disorders, or conditions that would benefit from reducing target gene expression.

[0308] Cells to which the methods of the present invention can be applied include in vitro, in vivo, and ex vivo cells. Cells may be in a subject, in culture, and / or in suspension, or in any other suitable state or condition. Cells to which the methods of the present invention can be applied may be liver cells, hepatocytes, cardiac cells, pancreatic cells, cardiovascular cells, kidney cells, or other types of vertebrate cells, including human and non-human mammalian cells. In certain aspects of the present invention, cells to which the methods of the present invention can be applied are healthy, normal cells that are not known to be diseased cells. In certain embodiments of the present invention, the methods and compositions of the present invention are applied to liver cells, hepatocytes, cardiac cells, pancreatic cells, cardiovascular cells, and / or kidney cells. While in certain aspects of the present invention, the control cells are normal cells, it should be understood that cells with a disease or condition may also be used as control cells in certain cases, such as comparing treated cells with a disease or condition to untreated cells with the disease or condition.

[0309] According to the methods of the present invention, the activity level of a target gene polypeptide can be determined and compared to a control level of target gene polypeptide activity. The control can take various forms. It can be a single cutoff value, such as a median or mean value. It can be established based on comparative groups, for example, a group with normal levels of target gene polypeptide and / or target gene polypeptide activity and a group with elevated levels of target gene polypeptide and / or target gene polypeptide activity. Other non-limiting examples of comparative groups can be a population with one or more symptoms or a diagnosis of a disease or condition associated with the target gene and a population without one or more symptoms or a diagnosis of the disease or condition, or a group of subjects administered an siRNA treatment of the present invention and a group of subjects not administered an siRNA treatment of the present invention. Typically, the control can be based on apparently healthy normal individuals or apparently healthy cells of an appropriate age group. It should be understood that in addition to a predetermined value, a control according to the present invention can also be a material sample tested in parallel with the experimental material. Examples include samples from a control population or control samples produced by manufacturing for parallel testing with the experimental samples. In some embodiments of the invention, a control can include a cell or subject that has not been contacted or treated with a target gene dsRNA reagent of the invention, in which case the control level of target gene polypeptide and / or target gene polypeptide activity can be compared to the level of target gene polypeptide and / or target gene polypeptide activity in a cell or subject that has been contacted with a target gene dsRNA reagent or target gene antisense polynucleotide reagent of the invention.

[0310] In some embodiments of the present invention, the control level can be a target gene polypeptide level determined for a subject, where target gene polypeptide levels determined for the same subject at different time points are compared to the control level. In a non-limiting example, the level of the target gene is determined in a biological sample obtained from a subject who has not received treatment with a target gene of the present invention. In some embodiments, the biological sample is a serum sample. The target gene polypeptide level measured from a sample obtained from the subject can be used as the subject's baseline or control value. In the treatment methods of the present invention, after administering one or more target gene dsRNA reagents to the subject, one or more additional serum samples can be obtained from the subject, and the target gene polypeptide level in the subsequent sample or samples can be compared to the subject's control / baseline level. Such comparisons can be used to assess the onset, progression, or regression of a disease or condition associated with the target gene in the subject. For example, a higher level of target gene) polypeptide in a baseline sample obtained from a subject than the level obtained from the same subject after administering to the subject a target gene dsRNA reagent or a target gene) antisense polynucleotide reagent of the present invention indicates regression of the target gene-associated disease or condition and indicates the effectiveness of administering a target gene dsRNA reagent of the present invention to treat the target gene-associated disease or condition.

[0311] In certain embodiments of the present invention, one or more values ​​of target gene polypeptide and / or target gene polypeptide activity level determined for a subject can be used as a control value, which is then used to compare the target gene polypeptide and / or target gene activity level in the same subject, thereby making it possible to evaluate changes in the "baseline" target gene polypeptide activity in the subject. Thus, when an initial level is used as the control level for that subject, the initial target gene polypeptide level and / or initial target gene polypeptide activity level can be used as an indication and / or determination of the level in the subject of methods and compounds of the present invention that can reduce the target gene polypeptide and / or target gene polypeptide activity in the subject.

[0312] Using the methods of the present invention, a target gene dsRNA reagent and / or a target gene antisense polynucleotide reagent of the present invention can be administered to a subject. The effectiveness of administration and treatment of such dsRNAi reagents of the present invention can be evaluated as follows: after administration and treatment, the level of target gene polypeptide in a serum sample obtained from the subject 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 target gene (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 level of target gene polypeptide in a control serum sample). It should be understood that both the level of target gene polypeptide and the level of target gene polypeptide activity are correlated with the level of target gene expression. A specific embodiment of the method of the present invention includes administering a target gene dsRNA and / or a target gene antisense reagent of the present invention to a subject in an amount that effectively suppresses target gene expression, thereby reducing the level of target gene polypeptide and reducing the level of target gene polypeptide activity in the subject.In some embodiments of the methods of the invention, contacting a cell with an siRNA reagent of the invention (also referred to herein as treating) results in suppression 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%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 11 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100%, e.g., below the level of detection of the test.

[0313] Some embodiments of the present invention involve determining the presence, absence, and / or amount (also referred to herein as level) of a target gene polypeptide from one or more biological samples obtained from one or more subjects. Such measurements can be used to assess the effectiveness of a treatment method 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 the administration of a target gene dsNA reagent and / or a target gene antisense agent of the present invention. A decrease of at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more in the level of target gene polypeptide in a serum sample obtained from the subject after administration and treatment, 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 a non-contact control level (e.g., the level of the target gene polypeptide in a control serum sample), indicates a level of effectiveness of the treatment administered to the subject.

[0314] In some embodiments of the present invention, physiological characteristics of a target gene-associated disease or condition determined for a subject can be used as a control result, and the results of determining physiological characteristics of the same subject at different time points can be compared with the control result. In a non-limiting example, the pathological characteristic of hemolysis is measured from a subject not receiving treatment with the target gene of the present invention, which is used as the subject's baseline or control value. In the treatment methods of the present invention, after one or more administrations of a target gene dsRNA reagent to the subject, blood cells are compared with the subject's control / baseline level, respectively. Such comparisons can be used to assess the onset, progression, or regression of a target gene-associated disease or condition in a subject. For example, if the baseline blood cells obtained from the subject have a higher thrombus count than those measured from the same subject after administration of the target gene dsRNA reagent or target gene antisense polynucleotide reagent of the present invention to the subject, this indicates regression of the target gene-associated disease or condition and indicates the effectiveness of administering the target gene dsRNA reagent of the present invention to treat the target gene-associated disease or condition.

[0315] Some embodiments of the present invention involve determining the presence, absence, and / or changes in physiological characteristics of a disease or condition associated with a target gene, for example, using methods such as, but not limited to, (1) measuring blood cells in a subject, (2) assessing physiological characteristics of one or more biological samples obtained from one or more subjects, or (3) physical examination of the subject, which measurements can be used to assess the effectiveness of the therapeutic methods of the present invention.

[0316] Mismatch As known to those skilled in the art, the effectiveness of dsRNA can tolerate mismatches, especially when the mismatches are located in the terminal regions of the dsRNA. Some mismatches are better tolerated, for example, mismatches with wobble base pairs G:U and A:C are better tolerated (Du et al., 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).

[0317] Complementarity As used herein, unless otherwise specified, the term "complementarity" when used to describe the relationship between a first nucleotide sequence (e.g., a target gene dsRNA reagent sense strand or a target gene mRNA) and a second nucleotide sequence (e.g., a target gene dsRNA reagent antisense strand or a single-stranded antisense polynucleotide) refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising the second nucleotide sequence (forming inter-base pair hydrogen bonds under physiological conditions in mammals (or similar conditions in vitro)) and form a double helix or double helix structure under specific conditions. Other conditions, such as physiologically relevant conditions that may be encountered in vivo, may also be applicable. Those skilled in the art can determine the optimal set of conditions for testing the complementarity of two sequences based on the ultimate application of the hybridizing nucleotides. Complementary sequences may include Watson-Crick base pairs or non-Watson-Crick base pairs, and may include natural or modified nucleotides or nucleotide mimics, at least to the extent required for such hybridization. The sequence identity or complementarity is not relevant to the modifications.

[0318] For example, a complementary sequence within a target gene dsRNA described herein includes base pairing across the entire length of one or two nucleotide sequences between an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence. Such sequences can be referred to herein as "fully complementary" to each other. In embodiments in which two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, it should be understood that such overhangs are not considered mismatches as determined herein based on complementarity. For example, a target gene dsRNA reagent may include an oligonucleotide having a length of 19 nucleotides and another oligonucleotide having a length of 20 nucleotides, where the longer oligonucleotide comprises a 19-nucleotide sequence that is fully complementary to the shorter oligonucleotide, and for purposes described herein, this case can be referred to as "fully complementary." Therefore, as used herein, "fully complementary" refers to hybridization of all (100%) of the bases in a contiguous sequence of a first polynucleotide with the same number of bases in a contiguous sequence of a second polynucleotide. The contiguous sequence can include all or part of the first or second nucleotide sequence.

[0319] As used herein, the term "essentially complementary" refers to a hybridization pair of nucleobase sequences in which at least about 85% (but not all) of the bases in a contiguous sequence of a first polynucleotide hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. When the two sequences contain one or more mismatched base pairs upon hybridization, e.g., at least 1, 2, 3, 4, or 5 mismatched base pairs, the term "essentially complementary" can be used to refer to the first sequence forming a duplex of 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, such as silencing gene expression of a target gene via 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 a contiguous sequence of a first polynucleotide hybridize to the same number of bases in a contiguous sequence of a second polynucleotide. In some embodiments, "partially complementary" refers to 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 a contiguous sequence of a first polynucleotide hybridize to the same number of bases in a contiguous sequence of a second polynucleotide.

[0320] The terms "complementary," "fully complementary," "essentially complementary," and "partially complementary," as used herein, can be used to refer to base matching between the sense and antisense strands of a dsRNA reagent, base matching between the antisense strand of a dsRNA reagent and a target mRNA sequence, or base matching between a single-stranded antisense oligonucleotide and a target mRNA sequence. It should be understood that the term "antisense strand of a dsRNA reagent" can refer to the same sequence as an "antisense polynucleotide reagent."

[0321] Modifications 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. Nucleic acids in certain embodiments of the present invention can be synthesized and / or modified by methods known in the art, see, for example, "Current protocols in Nucleic Acid Chemistry," Beaucage, SLet et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications that can be present in certain embodiments of the dsRNA reagent of the present invention include, for example, (a) terminal modifications such as 5'-end modifications (phosphorylation, conjugation, inverted linkage, etc.), 3'-end modifications (conjugation, DNA nucleotide, inverted linkage, etc.), (b) base modifications such as stable bases, unstable bases, or base substitutions that base pair with an expanded partner library, deleted bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2'- or 4'-position) or sugar substitutions, and (d) backbone modifications, including modifications or substitutions that include phosphodiester bonds. Specific examples of RNA compounds that can be used in certain embodiments of the dsRNA reagent, antisense polynucleotide and sense polynucleotide of the present invention include, but are not limited to, RNA that contains modified backbone or does not contain natural internucleotide bond.As a non-limiting example, RNA with backbone modification may not have phosphorus atom in backbone.RNA that does not have phosphorus atom in its internucleoside backbone can be called oligonucleoside.In certain embodiments of the present invention, modified RNA has phosphorus atom in its internucleotide backbone.

[0322] The term "RNA molecule" or "RNA" or "ribonucleic acid molecule" should be understood to include not only RNA molecules expressed or found in nature, but also RNA analogs and derivatives containing one or more ribonucleotide / ribonucleoside analogs or derivatives described herein or known in the art. The terms "ribonucleoside" and "ribonucleotide" are used interchangeably herein. RNA molecules can be modified in the nucleobase 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 double strands.

[0323] The following examples are provided to illustrate specific implementations of the present invention and are not intended to limit the scope of the invention. Those skilled in the art will appreciate that the present invention is applicable to a variety of compositions and methods.

[0324] As used herein, "substituents" are an intended aspect of the present invention. Those skilled in the art of medicinal chemistry and organic chemistry will understand the generality of such substituents. In the present invention, "substituents" should satisfy the principle that the total number of substituents can form a chemical bond with the attached atom or group.

[0325] The term "hydrocarbon group" includes straight-chain, branched-chain, and cyclic groups of any degree of saturation. In some specific cases, including C-, O-, and H-containing groups, such hydrocarbon groups may be interrupted by one or more heteroatoms of nitrogen, oxygen, sulfur, and phosphorus, and may be further mono- or polysubstituted with one or more substituents.

[0326] As used herein, the term "alkyl group" refers to a straight- or branched-chain saturated hydrocarbon group having from 1 to about 24 carbon atoms. When appearing herein, a number range, such as a "C1-C6 alkyl group," 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., and may include up to 6 carbon atoms. Unless otherwise specified, the number of carbon atoms in the alkyl group, increased by further substitution with other atoms or groups, is generally not included within the count, and the same applies to other counts herein. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl, and the like. The term "alkyl group" also encompasses cases where no range of carbon atoms is specified, for example, the term "alkyl group" refers to C1-C6 alkyl groups. 10 (e.g., C1-C6). A "substituted alkyl group" refers to an alkyl moiety that has a substituent. As used herein, a "lower alkyl group" refers to an alkyl moiety having 1 to about 6 carbon atoms.

[0327] As used herein, the term "alkenyl group" refers to a straight- or branched-chain hydrocarbon group with at least one carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and dienes such as 1,3-butadiene. Alkenyl groups typically contain from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms, with from 2 to about 6 carbon atoms being more preferred. As used herein, alkenyl groups may optionally contain one or more additional substituents.

[0328] As used herein, the term "alkynyl group" refers to a straight- or branched-chain hydrocarbon group with 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 from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms, with from 2 to about 6 carbon atoms being more preferred. As used herein, alkynyl groups may optionally contain one or more further substituents.

[0329] As used herein, the term "acyl group" refers to a group having the general formula -C(O)-X, formed by removal of a hydroxy group from an organic acid, where X is generally aliphatic, alicyclic, or aromatic. Examples include aliphatic carbonyl groups, aromatic carbonyl groups, aliphatic sulfonyl groups, aromatic sulfinyl groups, aliphatic sulfinyl groups, aromatic phosphate esters, aliphatic phosphate esters, and the like. As used herein, acyl groups may optionally include further substituents.

[0330] The term "cycloalkyl group" refers to a ring system in which the ring is aliphatic. The ring system may include one or more rings in which at least one ring is aliphatic. Preferred alicyclic compounds include rings having from about 5 to about 9 carbon atoms in the ring. As used herein, cycloalkyl groups may optionally include further substituents.

[0331] As used herein, the term "alkoxy group" refers to a group formed between an alkyl group and an oxygen atom, where the oxygen atom is used to attach 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-pentyloxy, neopentyloxy, and n-hexyloxy groups. As used herein, an alkoxy group may optionally include further substituents.

[0332] As used herein, the term "aminoalkyl group" refers to an amino-substituted C-C 12 refers to an alkyl group. The alkyl portion of the group forms a covalent bond to 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 moieties.

[0333] As used herein, the terms "aryl group" and "aromatic" refer to monocyclic or polycyclic carbocyclic 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+2 π-electrons, where n is an integer. The aromatic ring may be formed from 5, 6, 7, 8, 9, or more than 9 atoms. The term "aromatic" is intended to include carbocyclic aryl groups (e.g., phenyl groups) and heterocycloaryl (or "heteroaryl groups" or "heteroaromatic") groups (e.g., pyridine). The terms include monocyclic or fused-ring polycyclic rings, i.e., rings that share adjacent pairs of carbon atoms. A "substituted aromatic group" refers to an aromatic group further bearing one or more substituents. Examples of aryl groups include, but are not limited to, phenyl groups, naphthyl groups, tetrahydronaphthyl groups, indanyl groups, and idenyl groups. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in the ring or rings. As used herein, aryl groups may optionally include further substituents.

[0334] As used herein, the terms "aralkyl group" and "arylalkyl group" refer to C-C 12 Aralkyl refers to an aromatic group covalently bonded to an alkyl group. The alkyl portion of the resulting aralkyl group (or arylalkyl group) forms a covalent bond with the parent molecule. Examples include, but are not limited to, benzyl, phenylethyl, and the like. As used herein, aralkyl groups may optionally include additional substituents attached to the alkyl group, the aryl group, or both of the forming groups.

[0335] As used herein, the terms "heteroaryl group" and "heteroaromatic" refer to a group containing a monocyclic or polycyclic aromatic ring, ring system, or fused ring system, in which at least one of the rings is aromatic and contains one or more heteroatoms. Heteroaryl groups are also intended to include fused ring systems, including systems in which one or more of the fused rings does not contain a heteroatom. Heteroaryl groups generally contain one 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, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, and the like. A heteroaryl group can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or a heteroatom. As used herein, a heteroaryl group may optionally include further substituents.

[0336] As used herein, the term "heteroarylalkyl group" refers to a covalently bonded C-C 12 "Heteroaryl" refers to a heteroaryl group, as defined above, further comprising an alkyl group. The alkyl portion of the resulting heteroarylalkyl group can form a covalent bond with the parent molecule. Examples include, but are not limited to, pyridylmethyl, pyridylethyl, napthyridinylpropyl, and the like. As used herein, heteroarylalkyl groups may optionally include further substituents on either or both of the heteroaryl or alkyl group portions.

[0337] As used herein, the term "halo" or "halogen" refers to an atom selected from fluorine, chlorine, bromine, and iodine.

[0338] As used herein, the term "heterocyclyl group" refers to a monocyclic or polycyclic ring system that contains at least one heteroatom and is unsaturated, partially saturated, or fully saturated, and thus includes heteroaryl groups. Heterocycle is also intended to include fused ring systems, in which one or more of the fused rings contain at least one heteroatom, and the other rings may contain one or more heteroatoms, or may contain no heteroatoms. Heterocyclyl groups generally contain at least one atom selected from sulfur, nitrogen, or oxygen. Examples of heterocyclyl 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 heterocyclyl group refers to the total number of n atoms in the ring system, and does not include atoms or groups outside the ring system. As used herein, heterocyclyl groups may optionally include further substituents.

[0339] 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 linked, and is intended to include single and mixed ring systems independently selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic, and heteroarylalkyl groups. Such monocyclic and polycyclic structures may contain rings that each have the same degree of saturation or each have independently different degrees of saturation (including fully saturated, partially saturated, or fully unsaturated). Each ring may contain ring atoms selected from C, N, O, and S to obtain heterocyclic and rings with only C ring atoms; these rings may also exist as mixed motifs, such as benzimidazole, in which one ring has only carbon ring atoms while the fused ring has two nitrogen atoms. The monocyclic or polycyclic structures may be further substituted with substituents such as, for example, phthalimide having two =0 groups attached to one of its rings. The monocyclic or polycyclic structures may be attached to the parent molecule using a variety of strategies, including direct attachment through ring atoms, attachment through a substituent or bifunctional linking moiety.

[0340] The term "oxo" refers to the (=O) group.

[0341] As used herein, the term "sulfonyl group" (alone or as part of another group) refers to a group of formula RSO2-, where R is hydrogen, an alkyl group, a substituted alkyl group, an aryl group, or a substituted aryl group. As used herein, the term "sulfonamido group" (alone or as part of another group) refers to a group of formula RSO2-NH-, where R is hydrogen, an alkyl group, a substituted alkyl group, an aryl group, or a substituted aryl group. Some non-limiting exemplary sulfonamido groups include CH3-SO2-N(H)-.

[0342] The terms "alicyclic" or "alicyclic group" refer to a cyclic ring system assembly in which the ring is an aliphatic ring. The ring system assembly may include one or more rings in which at least one ring is an aliphatic ring. Preferred aliphatic rings include rings having about 5 to about 9 carbon atoms in the ring. As used herein, aliphatic rings optionally include other substituents. As used herein, the term "aliphatic" refers to a straight- or branched-chain hydrocarbon group containing up to 24 carbon atoms, where the degree of saturation between any two carbon atoms is a single, double, or triple bond. Aliphatic groups preferably contain 1 to about 24 carbon atoms, more typically 1 to about 12 carbon atoms, and more preferably 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. Examples of such heteroatom-interrupted aliphatic groups include, but are not limited to, polyalkoxy groups, such as polyalkylene glycols, polyamines, and polyimines. As used herein, an aliphatic group optionally includes other substituents. A spacer is attached to an oligonucleotide to separate the oligonucleotide from the substrate or support during the synthesis process. This method allows for greater flexibility and more space for synthesis, and is easy to cleave upon completion of synthesis.

[0343] As used herein, the term "carboxy" (alone or as part of another group) refers to a group of formula -COOH.

[0344] Linking groups or bifunctional linking moieties, such as those known in the art, can be used to attach chemical functional groups, conjugate groups, reporter groups, and other groups to selective sites in a parent compound, such as an oligomeric compound. Generally, bifunctional linking moieties comprise a hydrocarbon moiety with two functional groups. One of the functional groups is selected to attach to a parent molecule or target compound, while the other is selected to attach to essentially any selected group, such as a chemical functional group or conjugate group. In some embodiments, the linking group comprises a chain structure or polymer of 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 an amino group, a hydroxy group, a carboxylic acid, a thiol, unsaturation (e.g., a double or triple bond), or the like. 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-aminocaproic acid (AHEX or AHA). Other linking groups include substituted C-C 10 Alkyl groups, substituted or unsubstituted C2-C 10 Alkenyl group, or substituted or unsubstituted C2-C 10 including, but not limited to, alkynyl groups, where a non-limiting list of preferred substituents includes hydroxy, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl groups.

[0345] As used herein, the term "ether" refers to the product of replacing the hydrogen in the hydroxy group of an alcohol or phenol with a hydrocarbon group, and has the general formula RO-R', where R and R' may be the same or different. Both identical ethers are called symmetrical ethers, also called simple ethers or monoethers, and both different ethers are called asymmetrical ethers, also called mixed ethers.

[0346] As used herein, [ka] or [ka] The term generally refers to a linkage to an adjacent group or moiety.

[0347] About: As used herein, the term "about" or "approximately" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. In certain embodiments, unless otherwise stated or clear from the context, the term "about" or "approximately" refers to a range of values ​​that falls 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 stated reference value in any direction (greater or less), excluding cases where such value exceeds 100% of possible values.

[0348] Some abbreviations used herein: the abbreviation "Ac" refers to an acetyl group, the abbreviation "Bn" refers to a benzyl group, the abbreviation "Bz" refers to a benzoyl group, the abbreviation "DMTrCl" refers to 4,4-dimethoxytrityl chloride, the abbreviation "DMTr" refers to a 4,4-dimethoxytrityl group, the abbreviation "THP" refers to a tetrahydropyranyl group, the abbreviation "TBDMS" refers to a tert-butyldimethylsilyl group, the abbreviation "TIPDS" refers to a tetraisopropyldimethylsilyl group, and the abbreviation "DTBS" refers to a di(tert-butyl)silyl group. [Example]

[0349] Specific Examples The following examples are provided to illustrate specific implementations of the present invention and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that the present invention can be applied in a variety of compositions and methods.

[0350] Example 1 Synthesis of phosphoramidite compound 2 [ka]

[0351] DMTrCl (232 g, 684 mmol, 1.0 equiv.) in pyridine (400 mL) was added to a solution of compound A and isomannitol (100 g, 684 mmol, 1.0 equiv.) in pyridine (600 mL), and the mixture was stirred at 25 °C for 16 h. 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) and extracted with dichloromethane (500 mL × 2). The combined organic phase was washed with brine (500 mL), dried over NaSO, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (DCM / MeOH = 100 / 1 to 50 / 1, 0.1% EtN) to give compound B (150 g, 48.9% yield) as a yellow solid. 1 H NMR: EC4783-404-P1B1_C (400 MHz, DMSO-d6) δ ppm 7.46 (br d, J=7.63 Hz, 2 H) 7.28 - 7.37 (m, 6 H) 7.19 - 7.25 (m, 1 H) 6.90 (br d, J=7.88 Hz, 4 H) 4.70 (d, J=6.50 Hz, 1 H) 3.99 - 4.09 (m, 6 H) 3.88 - 3.96 (m, 2 H) 3.83 (br dd, J=7.82, 6.94 Hz, 1 H) 3.74 (s, 6 H) 3.41 (br t, J=8.13 Hz, 1 H) 3.05 (t, J=8.44 Hz, 1 H) 2.85 (br t, J=7.50 Hz, 1 H).

[0352] Under N2 atmospheric pressure, 2H-tetrazole (0.45 M, 436 mL, 1.1 equiv.) was added dropwise to a solution of compound B (80.0 g, 178 mmol, 1.0 equiv.) in dichloromethane (5.0 mL) at 25°C, followed by dropwise addition of a solution of compound C (2-cyanoethyldiisopropylchlorophosphoramidite, 80.6 g, 267 mmol, 85.0 mL, 1.5 equiv.) in dichloromethane (200 mL). The reaction mixture was stirred at 25°C for 1.0 hour, and LC-MS showed that compound B was completely consumed, and a main peak having the desired mass was detected. The resulting reaction mixture was cooled to -20 °C, poured into ice-cold saturated NaHCO (500 mL), extracted with dichloromethane (500 mL × 3), and the combined organic layers were washed with NaHCO / brine (1:1, 300 mL / 300 mL), dried over NaSO, and concentrated in vacuo (35 °C) to give a residue (100 mL). The residue was purified by column chromatography (AlO, DCM / MeOH (100 / 1 to 50 / 1, 0.1% EtN) to give compound 2 (77 g, 119 mmol, 66.5% yield) as a white solid. 1 H NMR: EC4783-423-P1B1_C (400 MHz, DMSO-d6) δ ppm 7.22 (br d, J=7.50 Hz, 2 H) 7.05 - 7.14 (m, 6 H) 6.96 - 7.02 (m, 1 H) 6.67 (br dd, J=8.82, 1.81 Hz, 4 H) 3.95 - 4.07 (m, 2 H) 3.73 - 3.83 (m, 1 H) 3.62 - 3.72 (m, 2 H) 3.48 - 3.53 (m, 6 H) 3.27 - 3.37 (m, 3 H) 3.11 (s, 6 H) 2.82 (td, J=8.54, 2.31 Hz, 1 H) 2.47 - 2.63 (m, 3 H) 2.28 (br d, J=1.63 Hz, 3 H) 0.82 - 1.00 (m, 13 H).

[0353] Synthesis of phosphoramidite compound 1- [ka] To a solution of compound B (500 mg, 1.11 mmol, 1.0 equiv) in dichloromethane (5.0 mL) was added compound D (methyl diisopropyl chlorophosphoramidite, 607 mg, 3.34 mmol, 3.0 equiv) and triethylamine (432 mg, 3.34 mmol, 582 μL, 3.0 equiv) at 0-5°C, and the mixture was stirred at 25°C for 1.0 h. The resulting reaction mixture was cooled to -20°C and poured into a cold NaHCO3 (5.0 mL) solution at 0-5°C. The combined organic layer was extracted with dichloromethane (5.0 mL × 2), washed with cold NaHCO3 / brine (1:1, 5.0 mL / 5.0 mL) at 0-5°C, dried over Na2SO4, and concentrated in vacuo to give 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. 1 H NMR: EC10615-49-P1N (400 MHz, DMSO-d6) δ ppm 7.44 (br d, J=7.63 Hz, 2 H), 7.31 (br t, J=7.94 Hz, 6 H), 7.18 - 7.26 (m, 1 H), 6.89 (br d, J=8.00 Hz, 4 H), 4.08 - 4.13 (m, 1 H), 3.95 - 4.03 (m, 1 H), 3.84 - 3.93 (m, 1 H), 3.77 - 3.83 (m, 1 H), 3.74 (s, 6 H), 3.43 - 3.53 (m, 3 H), 3.38 (br d, J=6.75 Hz, 1 H), 2.94 - 3.04 (m, 1 H), 2.70 - 2.85 (m, 1 H), 1.09 - 1.15 (m, 12 H), 1.07 (br s, 3 H).

[0354] Synthesis of phosphoramidite compound 3- [ka]

[0355] A 100 mL flask was charged with 5 mL of DMF and Compound A isomannitol (0.5 g, 1.0 eq) at room temperature under nitrogen gas. NaH (0.18 g, 1.3 eq) was added in portions at -10 °C, and the mixture was stirred at -10 °C to 0 °C for 30 min. 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 min. The reaction mixture was then heated to 50 °C and stirred overnight. TLC (petroleum ether: EtOAc = 1:1, Rf = 0.5) detected the mixture, and although Compound A isomannitol was not completely consumed, a new spot formed, indicating that the reaction had been repeated. The two reaction mixtures were combined, poured into saturated NH4Cl (100 mL), and extracted with EtOAc (50 mL × 3). The combined organic layer was washed with saturated NaCl solution (50 mL × 2), dried over Na2SO4, and concentrated under reduced pressure. The crude Lipd-02-1A product was purified on a silica gel column eluted with petroleum ether: EtOAc (100:0 to 5:1) to give a white solid, Lipd-02-1A (0.72 g, 28% yield). MS (M+H) = 371.3.

[0356] At room temperature, a 100 mL flask was charged with 5.6 mL of CHCl and Lipd-02-1A (0.7 g, 1.0 eq) under nitrogen gas. Tetrazole (0.15 g, 1.1 eq, 0.45 M in CHCN) was added dropwise at room temperature, followed by the dropwise addition of a solution of 3-((bis(diisopropylamino)phosphonooxy)propionitrile (0.88 g, 1.5 eq) in CHCl (1 mL) to the above reaction mixture, which was then stirred at room temperature for 3 hours. A new spot was detected by TLC (petroleum ether: EtOAc = 2:1, Rf = 0.8), indicating the partial consumption of Lipd-02-1A. The resulting mixture was cooled to 0 °C, poured into saturated NaHCO3 solution (50 mL), and extracted with CHCl2 (50 mL × 3). The combined organic layers were washed with saturated NaCl solution (50 mL), dried over NaSO4, and concentrated under reduced pressure. The crude product of compound 3 was purified on a silica gel column 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. 1 H NMR (400 MHz, CDCl3): δ ppm 4.47-4.54 (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). 31 P NMR (400 MHz, CDCl3) :δ ppm 148.94, 149.03. MS (M+H) = 571.5.

[0357] Compounds 21 and 22 can be obtained in a similar manner, mainly substituting 1-iodotetradecane or 1-iodooctadecane for 1-iodohexadecane. [ka] Compound 21: 1H NMR (400 MHz, CDCl3): δ ppm 4.49-4.55 (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). 31 P NMR (400 MHz, CDCl3) :δ ppm 148.94, 149.03. MS (M+H) = 543.5. Compound 22: 1 H NMR (400 MHz, CDCl3) :δ 4.49-4.55 (m, 2H), 4.33-4.35 (m, 1H), 3.62-4.04 (m, 10H), 3.42-3.45 (m, 1H), 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). 31 P NMR (400 MHz, CDCl3) δ ppm 148.94, 149.03. MS (M+H) = 599.5.

[0358] Preparation of other nucleoside phosphoramidites can be accomplished according to procedures described herein and in the art, for example, but not limited to, published US Pat. No. 4,266,220 and WO 02 / 36743.

[0359] Example 2 Synthesis of oligomeric compounds The oligomeric compounds used in accordance with the present invention can be conveniently and conventionally prepared by well-known solid-phase synthesis techniques. Equipment for this synthesis, including, for example, Mermade 12 (LGC), is sold by several suppliers. Alternatively, any other equipment known in the art for such synthesis can be used. It is well known to use similar techniques to prepare oligonucleotides, such as alkylated derivatives and those with phosphorothioate linkages.

[0360] Oligomeric Compounds: Unsubstituted and substituted phosphodiester (O) oligomeric compounds (including, but not limited to, oligonucleotides) can be synthesized on an automated DNA synthesizer, Mermade 12 (LGC), using standard phosphoramidite chemistry followed by iodine oxidation.

[0361] In a specific embodiment, phosphorothioate internucleoside linkages (S) were synthesized in a manner similar to that of phosphodiester internucleoside linkages, except that thiohybridization was achieved by oxidation of the phosphite ester linkage with 10% w / v 3,H-1,2-benzodithiol-3-one-1,1-dioxide in acetonitrile. The time for the thiohybridization reaction step was increased to 180 seconds and continued through the normal capping step. After treatment in concentrated aqueous ammonia at 55°C (12-16 hours), cleavage, and deblocking from the CPG column, the oligomeric compounds were recovered by precipitation in 1 M NHOAc with three volumes of ethanol.

[0362] Phosphinate internucleoside linkages were prepared as described in US 5,508,270. Alkyl phosphate internucleoside linkages may be prepared as described in US 4,469,863 or by the phosphoramidite method described herein. 3'-deoxy-3'-methylenephosphonate internucleoside linkages were prepared as described in US 5,610,289 or 5,625,050. Phosphoramidite internucleoside linkages were prepared as described in US 5,256,775 or 5,366,878. Alkyl phosphorothioate internucleoside linkages were prepared as described in published WO 94 / 17093 and WO 94 / 02499. 3'-Deoxy-3'-amino phosphoramidate internucleoside linkages were prepared as described in US 5,476,925. Phosphate triester internucleoside linkages were prepared as described in US 5,023,243, and methods for preparing some phosphate internucleoside linkages can be found in Beilstein J Org Chem. 2017;13:1368-1387.

[0363] Non-phosphorus-containing internucleoside linkage(s) include, but are 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, prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240, and 5,610,289.

[0364] The double-stranded ribonucleic acid (dsRNA) reagent was obtained by mixing two complementary strands (sense strand and antisense strand) in a 1:1 molar ratio to form a duplex.

[0365] The duplexes in Table 2 were generally synthesized on an oligonucleotide synthesizer using a mature solid-phase synthesis method based on phosphoramidite chemistry to synthesize the sense and antisense strand sequences of siRNA. The oligonucleotide chain extension was achieved through a four-step cycle: deprotection, condensation, capping, and oxidation or sulfurization for each nucleotide addition. Synthesis was performed on a solid support made of controlled-pore glass (CPG, 1000A). While common monomeric phosphoramidites are commercially available, the monomeric compounds described herein can be incorporated into the oligonucleotide chain instead of the monomeric phosphoramidites. If the monomeric compounds described herein can be attached to the 3'-terminus instead of the monomeric phosphoramidite, they are attached to the CPG solid support during use. If attached to the 5'-terminus, the phosphoramidite can be used in the final coupling reaction and further coupled to the target group, if desired. Trichloroacetic acid (TCA) in 3% dichloromethane was used to deprotect the 4,4'-dimethoxytrityl protecting group (DMT). 5-Ethylthio-1H-tetrazolyl was used as the activating agent. I2 in THF / Py / HO and phenylacetyl disulfide (PADS) in pyridine / MeCN were used for the oxidation and sulfurization reactions, respectively. After the final solid-phase synthesis step, the solid-support-bound oligomer was cleaved and the protecting groups were removed by treatment with a 1:1 volume of 20 wt% aqueous methylamine and 28% ammonium hydroxide solution. To synthesize the oligomeric compounds for use in in vitro screening, the crude mixture was concentrated. The remaining solid was dissolved in 1.0 M NaOAc and ice-cold EtOH was added to precipitate the single-stranded product as the sodium salt, which was used for annealing without further purification. To synthesize siRNA for use in in vivo testing, the crude single-stranded product was further purified by ion-pair reverse-phase HPLC (IP-RP-HPLC). The purified single-stranded oligonucleotide product from IP-RP-HPLC was converted to the sodium salt by dissolving it in 1.0 M NaOAc and precipitating it by the addition of ice-cold EtOH.Sense and antisense strand oligonucleotides were annealed by equimolar complementation in water to form double-stranded siRNA products.

[0366] Example 3 Synthesis of targeting groups [ka] To a solution of intermediate compound 4 (275 g, 660 mmol, 1.00 eq.), a propylenediamine derivative, in dichloromethane (2.75 L) was added triethylamine (133 g, 1.32 mol, 2.00 eq.), followed by dropwise addition of Cbz-Cl (169 g, 990 mmol, 1.50 eq.). The reaction mixture was stirred at 25 °C for 2 h, and LCMS analysis showed complete conversion of the propylenediamine derivative. The reaction mixture was washed sequentially with saturated NaHCO3 (800 mL) and saturated brine (500 mL), and the organic phase was dried over anhydrous Na2SO4. After filtering off 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 give compound 5 (290 g, 527 mmol, 75.7% yield) as a colorless oil. 1 H NMR (400 MHz in DMSO-d6): δ ppm 7.23 - 7.40 (m, 5 H), 5.00 - 5.12 (m, 2 H), 3.86 - 3.95 (m, 2 H), 3.23 - 3.39 (m, 6 H), 2.55 - 2.67 (m, 2 H), 1.56 - 1.64 (m, 2 H), 1.31 - 1.46 (m, 27 H). MS (ESI) [M+H] + m / z: 551.6.

[0367] Compound 5 (145 g, 263 mmol, 1.00 eq) was added with HCOOH (2.9 L) and the solution was stirred at 60 °C for 12 h. LCMS showed complete conversion of compound 5. 1.5 L of toluene and 1.5 L of acetonitrile were added to the reaction mixture and concentrated under reduced pressure to approximately 500 mL. Subsequently, toluene / acetonitrile (1:1, v / v, approximately 750 mL) was added and concentrated to approximately 500 mL. Next, acetonitrile (approximately 1000 mL) was added and concentrated to dryness. The crude product was triturated with 700 mL of acetonitrile at 60 °C for 2 h and filtered. The solid was collected and dried to give compound 6 (105 g, quantitative) as a white solid. 1 H NMR (400 MHz in DMSO-d6): δ ppm 7.26 - 7.40 (m, 5 H), 5.02 - 5.10 (m, 2 H), 3.89 - 4.00 (m, 2 H), 3.36 - 3.45 (m, 4 H), 3.24 - 3.34 (m, 2 H), 2.59 - 2.72 (m, 2 H), 1.40 (s, 2 H). MS (ESI) [M+H] + m / z: 383.0.

[0368] 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), O-benzotriazolyl-N,N,N',N'-tetramethyluronium (TBTU) (327 g, 1.02 mol, 3.90 eq.) and triethylamine (212 g, 2.09 mol, 8.00 eq.) were added and the mixture was allowed to react at 25 °C for 1 h. LCMS analysis showed complete conversion of compound 6. The reaction mixture was added to 4000 mL of water, and impurities were removed with methyl tert-butyl ether (2000 mL portions). The remaining aqueous phase was extracted with dichloromethane (3000 mL portions). The dichloromethane phase was washed successively with 10% aqueous citric acid (2000 mL each, divided into two portions), saturated NaHCO (2.0 L each, divided into two portions), and saturated brine (2.0 L), and then dried over anhydrous NaSO. The filtrate was filtered and concentrated under reduced pressure to give compound 8 (260 g, 159 mmol, 60.9% yield) as a white solid. 1H NMR (400 MHz in DMSO-d6): δ ppm 7.99 - 8.08 (m, 2 H), 7.93 (br d, J=5.50 Hz, 1 H), 7.79 - 7.86 (m, 3 H), 7.26 - 7.39 (m, 5 H), 5.22 (d, J=3.13 Hz, 3 H), 4.95 - 5.08 (m, 5 H), 4.54 (br d, J=8.38 Hz, 3 H), 4.03 (s, 9 H), 3.81 - 3.93 (m, 5 H), 3.76 (br d, J=4.88 Hz, 3 H), 3.44 - 3.62 (m, 10 H), 3.34 - 3.43 (m, 6 H), 3.24 (br d, J=6.13 Hz, 7 H), 3.02 - 3.09 (m, 4 H), 2.40 - 2.47 (m, 2 H), 2.10 (s, 9 H), 1.99 (s, 9 H), 1.89 (s, 9 H), 1.77 (s, 9 H), 1.57 - 1.68 (m, 2 H)。MS (ESI) [M+H] + m / z: 816.4。

[0369] A 2 L hydrogenation reactor was inerted with argon gas, and dry Pd / C (9 g) was carefully added. MeOH (50 mL) was added to wet the Pd / C. Then, under an argon atmosphere, 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. The mixture was degassed and replaced with a hydrogen atmosphere by adding H2 three times, and stirred at 25 °C for 10 h. LCMS showed complete conversion of compound 8. The Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure to give compound 9 (80 g, 90.2% yield). 1H NMR (400 MHz in DMSO-d6): δ ppm 9.12 (br s, 2 H), 8.50 (br t, J=5.19 Hz, 1 H), 8.10 (br t, J=5.50 Hz, 2 H), 7.85 - 7.91 (m, 3 H), 5.22 (d, J=3.25 Hz, 3 H), 4.95 - 5.01 (m, 3 H), 4.52 - 4.58 (m, 3 H), 4.03 (s, 9 H), 3.84 - 3.93 (m, 3 H), 3.75 - 3.83 (m, 3 H), 3.39 - 3.61 (m, 16 H), 3.23 - 3.32 (m, 6 H), 3.15 - 3.18 (m, 3 H), 2.97 - 3.05 (m, 2 H), 2.54 - 2.61 (m, 2 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 9 H), 1.77 - 1.80 (m, 9 H), 1.70 - 1.76 (m, 2 H). MS (ESI) [M+H] + m / z: 749.3.

[0370] 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.). The solution was stirred at 25 °C for 1 h, and LCMS showed complete conversion of compound 9 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 mixture, and stirring was continued at 25 °C for 1 h. LCMS showed complete conversion of compound 11. The reaction was quenched by slow addition of saturated NH4Cl (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 phase was washed with a 1:1 (v / v) mixture of saturated NaHCO3 (aq) and saturated brine (3.0 L), dried over anhydrous Na2SO4, 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 filtered under vacuum, and the solid was collected and dried under vacuum to give compound 13 (207 g, 72.8% yield) as a white solid. 1H NMR (400 MHz in DMSO-d6): δ ppm 8.05 (br d, J=2.00 Hz, 2 H), 7.82 (br d, J=7.38 Hz, 3 H), 5.21 (br s, 3 H), 4.98 (br d, J=10.26 Hz, 3 H), 4.72 (br s, 1 H), 4.54 (br d, J=7.88 Hz, 3 H), 4.03 (br s, 9 H), 3.74 - 3.94 (m, 9 H), 3.45 - 3.71 (m, 12 H), 3.40 (br s, 6 H), 3.24 (br s, 7 H), 3.07 (br d, J=14.13 Hz, 5 H), 2.91 - 3.01 (m, 1 H), 2.24 - 2.44 (m, 5 H), 2.20 (br s, 1 H), 2.10 (s, 9 H), 1.96 - 2.04 (m, 9 H), 1.89 (br s, 9 H), 1.74 - 1.81 (m, 9 H), 1.51 - 1.73 (m, 6 H), 1.07 - 1.36 (m, 3 H). MS (ESI) [M+H] + m / z: 848.0。

[0371] To a solution of compound 13 (200 g, 118 mmol, 1.00 eq.) and tetrazolediisopropylammonium (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 was stirred at 40° C. for 2 hours, and LCMS showed complete conversion of compound 13. The reaction mixture 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 resulting 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 under vacuum. The product was dissolved in dichloromethane (1.0 L) and concentrated to dryness. This was repeated four times to remove residual methyl tert-butyl ether, affording GLPA15 (164 g, 73.3% yield). 1 H NMR (400 MHz in DMSO-d6): δ ppm 8.05 (br d, J = 6.50 Hz, 2 H), 7.81 (br d, J=9.01 Hz, 3 H), 5.22 (d, J=3.25 Hz, 3 H), 4.98 (dd, J=11.26, 3.25 Hz, 3 H), 4.55 (br d, J=8.50 Hz, 3 H), 4.03 (s, 9 H), 3.64 - 3.97 (m, 12 H), 3.55 - 3.63 (m, 6 H), 3.50 (br s, 5 H), 3.40 (br d, J=6.13 Hz, 6 H), 3.17 - 3.30 (m, 9 H), 3.07 (br d, J=14.26 Hz, 4 H), 2.76 (t, J=5.82 Hz, 2 H), 2.18 - 2.47 (m, 6 H), 2.10 (s, 9 H), 1.99 (s, 9 H), 1.89 (s, 9 H), 1.78 (s, 9 H), 1.52 - 1.74 (m, 6 H), 1.12 - 1.19 (m, 12 H). 31P NMR (DMSO-d6): ppm δ 145.25. MS (ESI) [M+H] + m / z: 1895.7.

[0372] Other GalNAc phosphoramidite compounds (GLPAn) can similarly be obtained using methods similar to those described herein or well known in the art after using reasonably corresponding intermediates and can be attached as targeting groups to the compounds or oligomers and duplexes herein at appropriate positions.

[0373] [ka] In one study, a method for attaching a targeting group containing GalNAc (also referred to herein as a GalNAc delivery compound) to the 5'-end of a sense strand was provided, involving the use of GalNAc phosphoramidite (GLPAn) in the final coupling step of solid-phase synthesis, and the attachment to the 5'-end of the sense strand was achieved using a process similar to that used during such synthesis processes, such as oligonucleotide chain extension (i.e., adding a nucleotide to the 5'-end of the sense strand). In some studies, a method for attaching a targeting group containing GalNAc to the 3'-end of a sense strand involves the use of a solid support (CPG) containing GLPAn. In some studies, a method for attaching a targeting group containing GalNAc to the 3'-end of a sense strand involves attaching the targeting group to a CPG solid support via an ester bond and using the resulting CPG with the attached targeting group during synthesis of the sense strand, whereby the GalNAc targeting group is attached to the 3'-end of the sense strand. Similarly, targeting groups may be linked to monomers comprising the oligomeric compounds of the present invention by methods such as the phosphoramidite method.

[0374] Example 4. Preparation of a solid support containing a monomer of the present invention [ka] [ka] represents the support portion of macroporous aminomethyl polyethylene resin A 50 L glass kettle was protected with nitrogen gas. Dichloromethane (19.50 kg) was added to the kettle and stirring was initiated. The temperature was controlled at 20-30°C, and DMTr-imann (1.47 kg) was added to the kettle. Triethylamine (1.50 kg), 4-dimethylaminopyridine (0.164 kg), and succinic anhydride (1.34 kg) were added to the reaction kettle. The system was kept at 20-30°C and reacted for 18 hours. After sampling, the reaction was terminated. After the reaction was completed, saturated sodium bicarbonate solution (22.50 kg) was added to the system. The system was stirred for 10-20 minutes and allowed to stand until the layers separated. The lower organic phase was decanted, and the upper aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was further decanted and concentrated by rotary evaporation until no further fractions remained, producing 1.83 kg of a gray to white solid.

[0375] N,N-dimethylformamide (23.50 kg) was added to a 100 L glass kettle and stirring was initiated. The temperature was controlled at 20-30°C, and under nitrogen gas protection, the product from the previous step, O-benzotriazole-tetramethyluronium hexafluorophosphate (0.33 kg), and N,N-diisopropylethylamine (0.13 kg) were added to the 100 L glass kettle via a solids feed hopper. After addition was complete, the mixture was stirred for 10-30 minutes and then discharged into a 50 L galvanized barrel for use. Macroporous aminomethyl resin (3.25 kg) (purchased from Tianjin Nankai Hecheng Technology Co., Ltd., lot number HA2X1209, loading 0.48 mmol / g) was added to the 100 L solid-phase synthesis reactor via the solid-injection hopper. The temperature was controlled at 20-30°C, and N,N-dimethylformamide (21.00 kg + 21.00 kg) and the reaction solution prepared for use in the galvanized barrel from the previous step were added to the reactor. The reaction mixture was reacted at a constant temperature until the solid loading reached ≥ 250 μmol / g. The loading was monitored by UV. The mixture was pressure-filtered with nitrogen gas, and the filter cake was rinsed three times with N,N-dimethylformamide (26.00 kg + 26.10 kg + 26.00 kg). The filter cake was then evaporated in the reactor. Capsule A (4.40 kg + 4.42 kg + 4.30 kg) and Capsule B (4.40 kg + 4.40 kg + 4.47 kg) were added to an 80 L glass kettle and stirred for 3-8 min before use. This procedure was repeated three times to cap the kettle. Acetonitrile (18.00 kg + 18.00 kg + 18.00 kg + 17.50 kg + 17.50 kg) was added to the kettle, and nitrogen gas was bubbled through for 10-30 min before pressure filtration. This procedure was repeated four times. The filter cake was purged with nitrogen gas in the kettle for 2-4 h, then transferred to a 50 L filter press tank and dried at a controlled temperature of 15-30 °C. After drying, a yellow to white solid product weighing 3.516 kg was obtained.

[0376] Example 5. In vivo evaluation of oligomeric compounds containing monomers of the present invention Oligomeric compounds containing the monomers of the present invention can be obtained using the methods described herein or methods known to those skilled in the art, and the sequence structures of the oligomeric compounds are shown in Table 2 for siRNA duplexes targeting FXII.

[0377] As shown in Table 2, the duplexes were shown to contain a sense strand and an antisense strand. Chemical modifications were represented by uppercase letters indicating 2'-fluoro-modified nucleotides and lowercase letters indicating 2'-methoxy-modified nucleotides. An "*", when present in an oligonucleotide, should be understood to indicate that these monomers are linked to each other by a thiophosphodiester nucleoside bond, while the absence of an "*" between two monomers indicates that they are linked to each other by an oxophosphodiester nucleoside bond. Invab was an inverted abasic, and GLS15* and GLO15 were each targeting groups described herein.

[0378] [Table 2]

[0379] "imann" when present in an oligomeric compound or at the end of dsRNA [ka] and "imann" is an oligomeric compound or dsRNA that further binds to a targeting group. When [ka] It is expressed as:

[0380] Example 6. In vivo testing of FXII double-stranded ribonucleic acid (also called dsRNA) To evaluate the in vivo activity of FXII double-stranded ribonucleic acid (also called dsRNA), 6-week-old, specific pathogen-free female C57BL / 6 mice purchased from Shanghai Slack Laboratory Animal Co., Ltd. were used.

[0381] a) Compound Solvent: PBS, Test compounds: AD00823, AD00824, AD00825, AD00826, AD00835, AD00836, AD00841, AD00827, AD00449.

[0382] b) Experimental design Animal experiments were conducted using four C57BL / 6 female mice in each group. The dose was 1 mg / kg, administered SC once on day 0, and blood samples were taken on days 7, 14, and 21 to detect plasma FXII protein levels.

[0383] c) Definition of experimental days: The day on which the mice were first administered was defined as experimental day 0, the day before as day -1, and the day after as day 1, and the number of days was calculated based on this.

[0384] d) All compounds to be measured were prepared as a 5 mg / mL stock solution in PBS before administration. A small amount of the 5 mg / mL stock solution was taken and diluted 20 times, and the OD value was detected by a Nanodrop machine. After converting the OD value to obtain the actual concentration of the stock solution, an appropriate amount was taken and diluted to the working concentration (0.2 mg / mL) for administration.

[0385] e) All mice (C57BL / 6, female, 6 weeks old) underwent a 6-day adaptation period before being dosed by subcutaneous injection on day 0 as described above.

[0386] f) On days 7, 14, and 21, plasma was collected from all mice by submandibular vein blood collection and used to detect FXII protein levels.

[0387] Sample detection analysis ELISA reagent kit was applied to detect FXII protein levels in mouse plasma.

[0388] FXII protein levels in mouse plasma were detected using an ELISA reagent kit (Molecular Innovations, IMSFXIIKTT). Briefly, plasma samples collected from EDTA-K2 tubes were diluted 30,000-fold and then added to a detection plate coated with a capture antibody. The detection antibody and an HRP-coupled secondary antibody were then added sequentially, followed by color development with TMB and absorbance readings at 450 nm. The standard curve was fitted using a four-parameter method, and the FXII protein content of each sample was calculated by substituting the OD values ​​of the detection samples and multiplying by the dilution factor to obtain the FXII protein concentration in the original plasma. The data results are shown in Table 3.

[0389] [Table 3]

[0390] The double-stranded ribonucleic acid (also called dsRNA) reagents AD00824 and AD00826 have oligomeric compounds simultaneously containing the imann monomer compound of the present specification at the 5'-end, 3'-end, or both ends of the sense strand, and both showed better silencing effects compared to double-stranded ribonucleic acid (also called dsRNA) reagents such as AD00823 and AD00825 that do not contain or contain other nucleotide modifications.When the double-stranded ribonucleic acid (also called dsRNA) sense strand contains the monomer described herein at the 5'-end and / or 3'-end, it has a better anti-nuclease cleavage effect, so the sense strand has a stabilizing effect and improves the in vivo action effect of the antisense strand. Similarly, the same results were observed for AD00827 vs AD00841.

[0391] On the other hand, for double-stranded ribonucleic acid (also known as dsRNA) reagents, AD00835, which contains the iMann monomer compound of the present specification in the antisense strand, compared to AD00836 (containing invab), and activity data within the same action time actually demonstrated addition to the 5'-antisense strand end, and the antisense blocked antinuclease cleavage by the iMann monomer compound of the present specification, killing the activity, indicating that the compounds of the present specification have higher antinuclease cleavage activity and higher stability than invab in oligomeric compounds.

[0392] Example 7 In vivo testing of FXII double-stranded ribonucleic acid (also called dsRNA) Oligomeric compounds containing the monomers of the present invention can be obtained using the methods described herein or methods known to those skilled in the art, and the sequence structures of the oligomeric compounds are shown in Table 4 for siRNA duplexes targeting FXII.

[0393] As shown in Table 4, the duplexes were shown to contain a sense strand and an antisense strand. Chemical modifications were represented by uppercase letters indicating 2'-fluoro-modified nucleotides and lowercase letters indicating 2'-methoxy-modified nucleotides. An "*", when present in the oligonucleotide, should be understood to indicate that these monomers are linked to each other by a thiophosphodiester nucleoside bond, while the absence of an "*" between two monomers indicates that they are linked to each other by an oxophosphodiester nucleoside bond. Invab was reverse abasic.

[0394] [Table 4] When present in an oligomeric compound or at the end of dsRNA, "imann" is [ka] and when the oligomeric compound or dsRNA is further linked to a targeting group, "imann" is [ka] When present in an oligomeric compound or at the end of a dsRNA, "Stab8" is [ka] and when the oligomeric compound or dsRNA is further attached to a targeting group linker, "Stab8" is [ka] When present in an oligomeric compound or at the end of a dsRNA, "Stab9" is [ka] and when the oligomeric compound or dsRNA is further conjugated to a targeting group, "Stab9" is [ka] was expressed as:

[0395] To evaluate the in vivo activity of FXII double-stranded ribonucleic acid (also called dsRNA), 6-week-old female C57BL / 6 mice were used.

[0396] a) Compound The solvent was PBS, and the test compounds were AD00734, AD00737, AD01970, and AD01971.

[0397] b) Experimental design Animal experiments were conducted using four C57BL / 6 female mice in each group, with a dose of 0.5 mg / kg or 1 mg / kg administered SC once on day 0. Blood samples were taken on days 7, 14, 21, and 28 to detect plasma FXII protein levels.

[0398] c) Definition of experimental days: The day on which the mice were first administered was defined as experimental day 0, the day before as day -1, and the day after as day 1, and the number of days was calculated based on this.

[0399] d) All compounds to be measured were prepared as a 5 mg / mL stock solution in PBS before administration. A small amount of the 5 mg / mL stock solution was taken and diluted 20 times, and the OD value was detected by a Nanodrop machine. After converting the OD value to obtain the actual concentration of the stock solution, an appropriate amount was taken and diluted to the working concentration (0.2 mg / mL) for administration.

[0400] e) All mice (C57BL / 6, female, 6 weeks old) underwent a 6-day adaptation period before being dosed by subcutaneous injection on day 0 as described above.

[0401] f) On days 7, 14, 21, and 28, plasma was collected from all mice by submandibular vein blood collection and used to detect FXII protein levels.

[0402] Sample detection analysis FXII protein levels in mouse plasma were detected using an ELISA reagent kit (Molecular Innovations, IMSFXIIKTT). Briefly, plasma samples collected from EDTA-K2 tubes were diluted 30,000-fold and then added to a detection plate coated with a capture antibody. The detection antibody and an HRP-coupled secondary antibody were then added sequentially, followed by color development with TMB and absorbance readings at 450 nm. The standard curve was fitted using a four-parameter method, and the FXII protein content of each sample was calculated by substituting the OD values ​​of the detection samples and multiplying by the dilution factor to obtain the FXII protein concentration in the original plasma. The data results are shown in Table 5.

[0403] [Table 5]

[0404] Example 8. In vivo evaluation of oligomeric compounds containing monomers of the present invention Oligomeric compounds containing the monomers of the present invention can be obtained using the methods described herein or methods known to those skilled in the art, and the sequence structures of the oligomeric compounds are shown in Table 6 as siRNA duplexes targeting SOD1.

[0405] As shown in Table 6, the duplexes were shown to contain a sense strand and an antisense strand. Chemical modifications were represented by uppercase letters indicating 2'-fluoro-modified nucleotides and lowercase letters indicating 2'-methoxy-modified nucleotides. An "*", when present in the oligonucleotide, should be understood to indicate that these monomers are linked to each other by a thiophosphodiester nucleoside bond, while the absence of an "*" between two monomers indicates that they are linked to each other by an oxophosphodiester nucleoside bond. Invab was reverse abasic.

[0406] [Table 6]

[0407] When present in an oligomeric compound or at the end of dsRNA, "imann" is [ka] and when the oligomeric compound or dsRNA is further linked to a targeting group, "imann" is [ka] Lipd01 is expressed as [ka] Lipd02 is expressed as [ka] Lipd03 is expressed as [ka] and C16 is C 16 H 33 (C16)*(imann) indicates that C16 and imann are bonded to each other via a thiophosphodiester nucleoside bond. [ka] VPu is [ka] was expressed as:

[0408] To evaluate the in vivo activity of SOD1 double-stranded ribonucleic acid (also known as dsRNA), mice were administered intravenously (ICV). Male C57 mice (~20 g) were randomly assigned to the following groups: aCSF group (vehicle 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 compound was dissolved in artificial cerebrospinal fluid (aCSF) and diluted to a 50 mg / mL injection solution, then administered at a dose of 2 μL per mouse. The specific steps were as follows: Mice were anesthetized via intraperitoneal injection of zoletil and xylazine. After the anesthesia took effect, the mouse's head was fixed in a stereotaxic apparatus and the drug was injected into the lateral ventricle of the mouse using a 10 μL microsyringe (Hamilton). The injection time was 10 minutes. After the injection was complete, the needle was left in place for 5 minutes before being slowly removed and the skin on the head was sutured. After the mouse woke up, it was returned to its cage and continued to be kept.

[0409] On the 8th day after injection, each mouse was anesthetized intraperitoneally and injected with saline. Then, the prefrontal cortex (PFC), striatum, hippocampus, and cerebellum tissues were quickly extracted and stored in a refrigerator at -80°C. Total RNA was extracted using the RNeasy Kit (QIAGEN) according to the manufacturer's instructions, and cDNA was reverse transcribed. Expression of the target gene SOD1 was detected by qPCR using FastStart Universal SYBR Green Master (ROX) (Roche). -ΔΔCt Quantitative analysis was performed using the method, and the results are shown in Table 7.

[0410] [Table 7]

[0411] Dose-response experimental design Male C57 mice (~20 g) were randomly assigned to one of the following groups: aCSF group (vehicle control, 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), or AD00509-12 group (0.1 mg, 0.033 mg, 0.01 mg). The mouse lateral ventricle injection method was as described above. Eight days after injection, each mouse was anesthetized intraperitoneally and injected with saline. Then, the prefrontal cortex (PFC), striatum, hippocampus, and cerebellum tissues were rapidly extracted and stored in a -80°C refrigerator. Subsequently, total RNA was extracted using the RNeasy Kit (QIAGEN) according to the manufacturer's instructions, and cDNA was reverse transcribed. The expression of the target gene SOD1 was then detected by qPCR using FastStart Universal SYBR Green Master (ROX) (Roche). -ΔΔCt Quantitative analysis was performed using the method, and the results are shown in Table 8.

[0412] [Table 8]

[0413] equivalent While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily appreciate that various other means and / or structures for performing the functions and / or results and / or obtaining one or more advantages described herein, and variations and / or modifications thereof, each of which 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 the actual parameters, dimensions, materials, and / or configurations will depend on the specific application taught by the present invention. Those skilled in the art will recognize, or be able to determine using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, the above-described examples are presented by way of example only, and within the scope of the appended claims and their equivalents, it should be understood that the invention may be practiced otherwise than as specifically described and claimed. The present invention relates to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials and / or methods, if such features, systems, articles, materials and / or methods are not mutually inconsistent, is also included within the scope of the present invention.

[0414] All definitions and definitions used herein should be understood as definitions in reference dictionaries, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0415] Where no numerical limitation is used in the specification and claims, it should be understood to be "at least one" unless expressly specified to the contrary.

[0416] The term "and / or" as used in the specification and claims should be understood to mean "one or two" of the elements in that combination, i.e., such elements appearing in combination in some cases and separately in other cases. Other elements may optionally be present other than those specifically labeled with "and / or," whether associated with the specifically labeled element or not, unless expressly specified to the contrary.

[0417] All references, patents and patent applications, and publications cited or referred to in this application are hereby incorporated by reference in their entirety.

Claims

1. A compound having the structure shown in formula (I) or a stereoisomer thereof, 【Chemistry 1】 A １ and A ２ are each independently O, S, SO, SO ２ , N.R. ２ and CR ３ R ４ Selected from R ２ , R ３ and ４ are each independently hydrogen, halogen, a sulfonyl group, a sulfinyl group, a substituted or unsubstituted C １ -C ６ alkyl group, substituted or unsubstituted C ３ -C ６ Cycloalkyl groups, substituted or unsubstituted C ２ -C ６ Alkenyl group, substituted or unsubstituted C ２ -C ６ Alkynyl group, substituted or unsubstituted C ５ -C １２ an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, or a substituted or unsubstituted 5- to 12-membered heterocyclyl group; T １ and T ２ is a protecting group, Z, L and -Z-L, wherein Z represents a targeting group or a lipophilic group, and the lipophilic group is optionally connected to A via a linker. １ or A ２ L represents a carrier spacer or comprises a carrier moiety linked by a spacer; T １ and T ２ The other is an active phosphorus group, a protecting group, and T １ and T ２ is not a protecting group at the same time, R １ are each independently halogen, substituted or unsubstituted C １ -C ６ alkyl group, substituted or unsubstituted C ３ -C ６ Cycloalkyl groups, substituted or unsubstituted C ２ -C ６ Alkenyl group, substituted or unsubstituted C ２ -C ６ Alkynyl group, substituted or unsubstituted C ５ -C １２ an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, or a substituted or unsubstituted 5- to 12-membered heterocyclyl group; n is an integer from 0 to 6, wherein formula (I) is 【Chemistry 2】 does not include the structure compound.

2. A １ is O, The compound of claim 1.

3. A ２ is O, A compound according to any one of claims 1 to 2.

4. A １ and A ２ are each independently S; A １ and A ２ are each independently NR ２ where R ２ is hydrogen or CH ３ and A １ Is O, A ２ is S and A １ Is O, A ２ is NR ２ and Or, A １ is S, A ２ is NR ２ That is, The compound of claim 1.

5. Said T １ and T ２ one of which is a carrier spacer or a carrier moiety linked by a spacer, said carrier spacer preferably being a dicarboxylic acid-derived group; 【Transformation 3】 R is absent, an ether, polyethylene glycol, oxygen, sulfur, nitrogen, an alkylene group, an alkenyl group, an alkynyl group, an aryl group, an aralkyl group, a heteroalkyl group, or a heteroaryl group, and the carrier spacer is preferably C ３ -C ６ the alkyl dicarboxylate group, more preferably a succinic acid group, and the support is preferably poly(vinyl alcohol methacrylate copolymer), silicon chip glass, cellulose, polystyrene beads, polypropylene sheet, non-porous silicon beads, polyacrylamide or polyacrylate, and more preferably long-chain alkylamine controlled pore glass (LCAA-CPG); The compound of claim 1.

6. T １ and T ２ one protecting group is a hydroxy protecting group, and the hydroxy protecting groups are each independently selected from the group consisting of an acetyl group, a tert-butyl group, a tert-butoxymethyl group, a methoxymethyl group, a tetrahydropyranyl group, a 1-ethoxyethyl group, a 1-(2-chloroethoxy)ethyl group, a 2-trimethylsilylethyl group, a p-chlorophenyl group, a 2,4-dinitrophenyl group, a benzyl group, a benzoyl group, a p-phenylbenzoyl group, a 2,6-dichlorobenzyl group, a diphenylmethyl group, a p-nitrobenzyl group, a trimethylsilyl group, a triethylsilyl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, a trimethylsilyl group, a triethylsilyl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, a methyl ... triphenylsilyl group, triisopropylsilyl group, benzoyl formate, chloroacetyl group, trichloroacetyl group, trifluoroacetyl group, pivaloyl group, 9-fluorenylmethoxycarbonyl group, methanesulfonyl group, tosyl group, trifluoromethanesulfonyl group, trityl group, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group or substituted 9-phenylxanthin-9-yl; preferred hydroxy protecting groups are each independently selected from acetyl group, benzyl group, tert-butyldimethylsilyl group, tert-butyldiphenylsilyl group and 4,4'-dimethoxytrityl group; T １ and T ２ is a mercapto-protecting group, and the mercapto-protecting groups are each independently selected from the group consisting of an acetyl group, a tert-butyl group, a tert-butoxymethyl group, a methoxymethyl group, a tetrahydropyranyl group, a 1-ethoxyethyl group, a 1-(2-chloroethoxy)ethyl group, a 2-trimethylsilylethyl group, a p-chlorophenyl group, a 2,4-dinitrophenyl group, a benzyl group, a benzoyl group, a p-phenylbenzoyl group, a 2,6-dichlorobenzyl group, a diphenylmethyl group, a p-nitrobenzyl group, a trimethylsilyl group, a triethylsilyl group, and a tert-butyldimethylsilyl group. a tert-butyldiphenylsilyl group, a triphenylsilyl group, a triisopropylsilyl group, benzoyl formate, a chloroacetyl group, a trichloroacetyl group, a trifluoroacetyl group, a pivaloyl group, a 9-fluorenylmethoxycarbonyl group, a methanesulfonyl group, a tosyl group, a trifluoromethanesulfonyl group, a trityl group, a monomethoxytrityl group, a dimethoxytrityl group, a trimethoxytrityl group, or a substituted 9-phenylxanthin-9-yl; preferred mercapto-protecting groups are each independently selected from a benzyl group and a 4,4'-dimethoxytrityl group; T １ and T ２ wherein one protecting group is an amino protecting group, and the amino protecting group is independently selected from a 2-trimethylsilylethoxycarbonyl group (Teoc), a 1-methyl-1-(4-biphenyl)ethoxycarbonyl group (Bpoc), a tert-butoxycarbonyl group (BOC), an allyloxycarbonyl group (Alloc), a 9-fluorenylmethoxycarbonyl group (Fmoc), a benzyloxycarbonyl group (Cbz), a formyl group, an acetyl group, a trihaloacetyl group, a benzoyl group, a nitrophenyl group, a 2-nitrobenzenesulfonyl group, a phthalimide group, and a dithiosuccinyl group; The compound of claim 1.

7. T １ and T ２ is a lipophilic group, wherein the lipophilicity of the lipophilic group is measured by log Kow, and log Kow is greater than 0, preferably the lipophilic group log Kow is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10; The compound of claim 1.

8. The lipophilic group may be a saturated or unsaturated C ４ -C ３０ selected from the group comprising hydrocarbon chains, aliphatic rings, aromatic groups, fatty acid groups or groups derived from fatty acids, steroid-derived groups and any fat-soluble vitamin group; The compound of claim 7.

9. The lipophilic group is a saturated or unsaturated C １０ -C １８ The hydrocarbon chain includes any saturated or unsaturated fatty acid, cholesterol, vitamin E (tocopherol) or bile acid, which may contain one or two carboxy groups, and more preferably a saturated linear C １４ , C １６ or C １８ alkyl groups, octanoic acid, capric acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid and tetracosanoic acid, myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, trans-isooleic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid and erucic acid, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosanoic acid (DCA), sterol cholesterol (bile), tocopherol succinate (TS) and lithocholic acid (LA), retinoic acid (vitamin A acid), The compound according to any one of claims 7 to 8.

10. The lipophilic group is connected to A via a linker. １ or A ２ capable of covalently bonding to The compound according to any one of claims 7 to 9.

11. 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-ol, 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, an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide bond, a click reaction product or a carbamate; The compound of claim 10.

12. 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 mono- or oligosaccharides of DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, mannose and combinations thereof; The compound of claim 10.

13. T １ and T ２ is a targeting group, The compound of claim 1.

14. The targeting group may be selected from one or more ligands including polymers, saccharides, ligands for receptors expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands, or derivatives thereof. The compound of claim 13.

15. Each said targeting group 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 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, and α-D-glucose. copyranose, 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-isobutyric acid, 2-Deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-ethanolacyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-tri-O-acetyl-1-thio-6-O-tri a ligand containing one or more selected from the group consisting of methyl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptapyranoside ethyl ester, 2,5-anhydro-D-allonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose, and more preferably a ligand containing galactose or N-acetylgalactosamine; 15. The compound of claim 14.

16. The targeting group is A １ or A ２ Connect to one of the 【Chemistry 4】 14. The compound of claim 13, wherein the compound is selected from one of the compound fragments:

17. T １ and T ２ The other is an active phosphorus group, and the active phosphorus group is 【Transformation 5】 It has the structure Here, M １ is H, substituted or unsubstituted C １ -C ６ Alkyl group, OR ５ , S.R. ５ , OH, SH or NR ６ R ７ and M ２ OH, SH, OR ５ ' or NR ６ 'R ７ ', and each R ５ , R ６ , R ７ , R ５ ', R ６ ' or R ７ ' are independently hydrogen, substituted or unsubstituted C １ -C ６ an alkyl group or a sulfonyl group, and m is 0 or 1; A compound according to any one of claims 1 to 16.

18. Said M １ is a methyl group, an ethyl group, a propyl group, an isopropyl group, OR ５ , methanesulfonamide group, R ５ is selected from a substituted or unsubstituted methyl group, an ethyl group, a propyl group, and an isopropyl group; 18. The compound of claim 17.

19. R ５ , R ６ or R ７ are independently substituted alkyl groups, and the substituents are selected from a cyano group, a halogen atom, a hydroxy group, and an amino group; The compound according to any one of claims 17 to 18.

20. Said M ２ is N(CH(CH ３ ) ２ ) ２ Selected from The compound according to any one of claims 17 to 19.

21. M １ is O(CH ２ ) ２ CN and M ２ is N(CH(CH ３ ) ２ ) ２ and m is 0.

18. The compound of claim 17.

22. T １ and T ２ the other is an activated phosphorus group, and the activated phosphorus group is selected from diisopropyl cyanoethoxy phosphoramidite, diisopropyl methyl phosphoramidite, diisopropyl ethyl phosphoramidite, and H-phosphonate; A compound according to any one of claims 1 to 16.

23. T １ and T ２ one of which is diisopropylcyanoethoxyphosphoramidite and the other is a 4,4'-dimethoxytrityl group, a targeting group, or a lipophilic group; or １ and T ２ one of which 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, A compound according to any one of claims 1 to 22.

24. R １ are each independently halogen, substituted or unsubstituted C １ -C ６ alkyl group, substituted or unsubstituted C ２ -C ６ Alkenyl group, substituted or unsubstituted C ２ -C ６ alkynyl groups, A compound according to any one of claims 1 to 23.

25. n is 0; A compound according to any one of claims 1 to 24.

26. The compound has the configuration of formula (IIa) or formula (IIb): 【Transformation 6】 A １ , A ２ , T １ , T ２ , R １ and n is as defined in claims 1 to 25. A compound according to any one of claims 1 to 25.

27. The compounds provided herein have the configuration of formula (IIa-1) or formula (IIb-1): 【Transformation 7】 A １ , A ２ , T １ , T ２ , R １ and n is as defined in claims 1 to 25. A compound according to any one of claims 1 to 26.

28. It has the following specific structure: 【Chemistry 8-1】 【Chemistry 8-2】 【Chemistry 8-3】 【Chemistry 8-4】 A compound according to any one of claims 1 to 27.

29. An oligomeric compound comprising at least one 5'-end and / or 3'-end monomer having the structure shown in formula (III) or a stereoisomer thereof, 【Chemistry 9】 A １ and A ２ are each independently O, S, SO, SO ２ , N.R. ２ and CR ３ R ４ Selected from R ２ , R ３ and ４ are each independently hydrogen, halogen, a sulfonyl group, a sulfinyl group, a substituted or unsubstituted C １ -C ６ alkyl group, substituted or unsubstituted C ３ -C ６ Cycloalkyl groups, substituted or unsubstituted C ２ -C ６ Alkenyl group, substituted or unsubstituted C ２ -C ６ Alkynyl group, substituted or unsubstituted C ５ -C １２ an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, or a substituted or unsubstituted 5- to 12-membered heterocyclyl group; T ３ and T ４ is H, a protecting group, a lipophilic group, and an optional covalent bond, said lipophilic group being optionally connected to A via a linker. １ or A ２ and T ３ and T ４ is an internucleoside linking group linking the monomer of formula (III) or a stereoisomeric form thereof to an oligomeric compound, R １ are each independently halogen, substituted or unsubstituted C １ -C ６ alkyl group, substituted or unsubstituted C ３ -C ６ Cycloalkyl groups, substituted or unsubstituted C ２ -C ６ Alkenyl group, substituted or unsubstituted C ２ -C ６ Alkynyl group, substituted or unsubstituted C ５ -C １２ an aryl group, a substituted or unsubstituted 5- to 12-membered heteroaryl group, or a substituted or unsubstituted 5- to 12-membered heterocyclyl group; n is an integer from 0 to 6, The oligomeric compound optionally further comprises a targeting group. Oligomeric compounds.

30. A １ is O, 30. The oligomeric compound of claim 29.

31. A ２ is O, 31. The oligomeric compound according to any one of claims 29 to 30.

32. A １ and A ２ are each independently S, and A １ and A ２ are each independently NR ２ and A １ Is O, A ２ is S and A １ Is O, A ２ is NR ２ or A １ is S, A ２ is NR ２ where R ２ is hydrogen, a methylsulfonyl group, or CH ３ That is, 30. The oligomeric compound of claim 29.

33. T ３ and T ４ the other is an internucleoside linking group connecting the monomer to the 5'-terminus and / or 3'-terminus of the oligomeric compound, wherein the internucleoside linking group is selected from a phosphorus-containing linking group or a non-phosphorus-containing linking group; 33. The oligomeric compound according to any one of claims 29 to 32.

34. The phosphorus-containing internucleoside linking group is 【Chemistry 10】 wherein X is H, substituted or unsubstituted C １ -C ６ Alkyl group, OR ８ , S.R. ８ ', OH, SH or NR ９ R １０ Y represents O or S, z may be 0 or 1, R ８ , R ８ ', R ９ or R １０ are independently hydrogen, substituted or unsubstituted C １ -C ６ alkyl group, sulfonyl group, 【Chemistry 11】 are each independently A １ or A ２ and a portion that links to an adjacent nucleotide, 34. The oligomeric compound of claim 33.

35. The phosphorus-containing internucleoside linking groups are independently phosphodiester linking groups, phosphotriester linking groups, phosphorothioate linking groups, phosphorodithioate linking groups, alkylphosphonate linking groups, aminophosphonate linking groups, phosphonate linking groups, phosphinate linking groups, thiophosphoramidate linking groups, and phosphoramidate linking groups.

34. The oligomeric compound of claim 33.

36. Said T ３ and T ４ one protecting group is a hydroxy protecting group, and the hydroxy protecting groups are each independently selected from the group consisting of an acetyl group, a tert-butyl group, a tert-butoxymethyl group, a methoxymethyl group, a tetrahydropyranyl group, a 1-ethoxyethyl group, a 1-(2-chloroethoxy)ethyl group, a 2-trimethylsilylethyl group, a p-chlorophenyl group, a 2,4-dinitrophenyl group, a benzyl group, a benzoyl group, a p-phenylbenzoyl group, a 2,6-dichlorobenzyl group, a diphenylmethyl group, a p-nitrobenzyl group, a trimethylsilyl group, a triethylsilyl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, a trimethylsilyl group, a triethylsilyl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, a methyl ... triphenylsilyl group, triisopropylsilyl group, benzoyl formate, chloroacetyl group, trichloroacetyl group, trifluoroacetyl group, pivaloyl group, 9-fluorenylmethoxycarbonyl group, methanesulfonyl group, tosyl group, trifluoromethanesulfonyl group, trityl group, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group or substituted 9-phenylxanthin-9-yl; preferred hydroxy protecting groups are each independently selected from acetyl group, benzyl group, tert-butyldimethylsilyl group, tert-butyldiphenylsilyl group and 4,4'-dimethoxytrityl group; T ３ and T ４ is a mercapto-protecting group, and the mercapto-protecting groups are each independently selected from the group consisting of an acetyl group, a tert-butyl group, a tert-butoxymethyl group, a methoxymethyl group, a tetrahydropyranyl group, a 1-ethoxyethyl group, a 1-(2-chloroethoxy)ethyl group, a 2-trimethylsilylethyl group, a p-chlorophenyl group, a 2,4-dinitrophenyl group, a benzyl group, a benzoyl group, a p-phenylbenzoyl group, a 2,6-dichlorobenzyl group, a diphenylmethyl group, a p-nitrobenzyl group, a trimethylsilyl group, a triethylsilyl group, and a tert-butyldimethylsilyl group. a tert-butyldiphenylsilyl group, a triphenylsilyl group, a triisopropylsilyl group, benzoyl formate, a chloroacetyl group, a trichloroacetyl group, a trifluoroacetyl group, a pivaloyl group, a 9-fluorenylmethoxycarbonyl group, a methanesulfonyl group, a tosyl group, a trifluoromethanesulfonyl group, a trityl group, a monomethoxytrityl group, a dimethoxytrityl group, a trimethoxytrityl group, or a substituted 9-phenylxanthin-9-yl; preferred mercapto-protecting groups are each independently selected from a benzyl group and a 4,4'-dimethoxytrityl group; T ３ and T ４ wherein one protecting group is an amino protecting group, and the amino protecting group is independently selected from a 2-trimethylsilylethoxycarbonyl group (Teoc), a 1-methyl-1-(4-biphenyl)ethoxycarbonyl group (Bpoc), a tert-butoxycarbonyl group (BOC), an allyloxycarbonyl group (Alloc), a 9-fluorenylmethoxycarbonyl group (Fmoc), a benzyloxycarbonyl group (Cbz), a formyl group, an acetyl group, a trihaloacetyl group, a benzoyl group, a nitrophenyl group, a 2-nitrobenzenesulfonyl group, a phthalimide group, and a dithiosuccinyl group; 36. The oligomeric compound according to any one of claims 29 to 35.

37. Said T ３ and T ４ One of the is hydrogen, and T ３ and T ４ the other is an internucleoside linking group that connects the monomer of formula (III) or a stereoisomer thereof to the 5'-end and / or 3'-end of the oligomeric compound; 36. The oligomeric compound according to any one of claims 29 to 35.

38. T ３ and T ４ is selected from a lipophilic group, wherein the lipophilicity of said lipophilic group is measured by log Kow, and log Kow is greater than 0, preferably the lipophilic group log Kow is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10; 36. The oligomeric compound according to any one of claims 29 to 35.

39. The lipophilic group may be a saturated or unsaturated C ４ -C ３０ selected from the group comprising hydrocarbon chains, aliphatic rings, aromatic groups, fatty acid groups or groups derived from fatty acids, steroid-derived groups and any fat-soluble vitamin group; 39. The oligomeric compound of claim 38.

40. The lipophilic group may be a saturated or unsaturated C １０ -C １８ The hydrocarbon chain may include any saturated or unsaturated fatty acid, cholesterol, vitamin E (tocopherol) or bile acid, which may contain one or two carboxy groups, and more preferably a saturated linear C １６ alkyl groups, octanoic acid, capric acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid and tetracosanoic acid, myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, trans-isooleic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid and erucic acid, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosanoic acid (DCA), sterol cholesterol (bile), tocopherol succinate (TS) and lithocholic acid (LA), retinoic acid (vitamin A acid), 40. The oligomeric compound according to any one of claims 38 to 39.

41. The lipophilic group is connected to A via a linker. １ or A ２ capable of covalently bonding to 40. The oligomeric compound according to any one of claims 37 to 39.

42. 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-ol, 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, an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide bond, a click reaction product or a carbamate; 42. The oligomeric compound of claim 41.

43. 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 mono- or oligosaccharides of DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, mannose and combinations thereof; 42. The oligomeric compound of claim 41.

44. the 5'-end and / or 3'-end of said oligomeric compound further comprises one or more targeting groups; 36. The oligomeric compound according to any one of claims 29 to 35.

45. Said T ３ and T ４ represents a covalent bond, and the oligomeric compound is ３ and T ４ linked to the targeting group in the form of a covalent bond via one of 36. The oligomeric compound according to any one of claims 29 to 35.

46. The targeting group may be selected from one or more ligands including polymers, saccharides, ligands for receptors expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands, or derivatives thereof.

46. ​​The oligomeric compound according to any one of claims 44 to 45.

47. Each said targeting group 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 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, and α-D-glucose. copyranose, 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-isobutyric acid, 2-Deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-ethanolacyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-tri-O-acetyl-1-thio-6-O-tri a ligand containing one or more selected from the group consisting of methyl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptapyranoside ethyl ester, 2,5-anhydro-D-allonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose, and more preferably a ligand containing galactose or N-acetylgalactosamine; 47. The oligomeric compound of claim 46.

48. The targeting group is A １ or A ２ Connect to one of the 【Chemistry 12】 is selected from one of the compound fragments 46. ​​The oligomeric compound according to any one of claims 44 to 45.

49. comprising a 5'-terminal and / or 3'-terminal monomer of at least one configuration shown in formula (IVa) or formula (IVb), 【Chemistry 13】 A １ , A ２ , T ３ , T ４ , R １ and n is as defined in claims 29 to 48.

49. The oligomeric compound according to any one of claims 29 to 48.

50. comprising a 5'-terminal and / or 3'-terminal monomer of at least one configuration shown in formula (IVa-1) or formula (IVb-1), 【Chemistry 14】 A １ , A ２ , T ３ , T ４ , R １ and n is as defined in any one of claims 29 to 48.

49. The oligomeric compound according to any one of claims 29 to 48.

51. The R １ are each independently halogen, substituted or unsubstituted C １ -C ６ alkyl group, substituted or unsubstituted C ２ -C ６ Alkenyl group, substituted or unsubstituted C ２ -C ６ alkynyl groups, 51. The oligomeric compound according to any one of claims 29 to 50.

52. n is 0; 52. The oligomeric compound according to any one of claims 29 to 51.

53. T ３ and T ４ is H, a protecting group, a lipophilic group, and an optional covalent bond, said lipophilic group being optionally connected to A via a linker. １ or A ２ and T ３ and T ４ the other is an internucleoside linking group connecting a monomer according to any one of claims 29 to 52 to the 5'-end of an oligomeric compound; 53. The oligomeric compound according to any one of claims 29 to 52.

54. T ３ and T ４ is H, a protecting group, a lipophilic group, and an optional covalent bond, said lipophilic group being optionally connected to A via a linker. １ or A ２ and T ３ and T ４ The other is an internucleoside linking group connecting a monomer according to any one of claims 29 to 52 to the 3'-end of an oligomeric compound.

53. The oligomeric compound according to any one of claims 29 to 52.

55. T ３ and T ４ is H, a protecting group, a lipophilic group, and an optional covalent bond, said lipophilic group being optionally connected to A via a linker. １ or A ２ and T ３ and T ４ and the other is, independently of each other, an internucleoside linking group connecting the homologous or different monomers according to any one of claims 29 to 52 to the 5'-end and 3'-end of the oligomeric compound.

53. The oligomeric compound according to any one of claims 29 to 52. 【Request Item 56】 【Chemistry 15-1】 【Chemistry 15-2】 It has a specific structure: where Olig represents the oligonucleotide moiety that attaches said monomer to the 5'-end and / or 3'-end of the oligomeric compound, respectively; 56. The oligomeric compound according to any one of claims 29 to 55.

57. The oligomeric compound is a single-stranded oligonucleotide, including an antisense oligonucleotide (also called an ASO), a ribozyme, or an aptamer.

57. The oligomeric compound according to any one of claims 29 to 56.

58. 57. A double-stranded ribonucleic acid (dsRNA) reagent 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, and 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) reagent optionally further comprises an independent targeting group. Double-stranded ribonucleic acid (dsRNA) reagents.

59. The sense strand is an oligomeric compound defined by a 5'-terminal monomer, an oligomeric compound provided by a 3'-terminal monomer, or an oligomeric compound provided by 5'-terminal and 3'-terminal monomers according to any one of claims 29 to 52 and 56.

59. The double-stranded ribonucleic acid (dsRNA) reagent of claim 58.

60. the antisense strand is an oligomeric compound provided by a 5'-end and / or a 3'-end monomer according to any one of claims 29 to 52, 56, preferably the double-stranded ribonucleic acid (dsRNA) reagent is an oligomeric compound provided by a 3'-end monomer according to any one of claims 29 to 52, 56 or an oligomeric compound provided by a 5'-end monomer according to any one of claims 29 to 52, 56; 59. The double-stranded ribonucleic acid (dsRNA) reagent of claim 58.

61. the 5'-terminal and / or 3'-terminal nucleotide of any one strand further comprises one or more targeting groups; 61. The double-stranded ribonucleic acid (dsRNA) reagent of any one of claims 58 to 60.

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

63. 62. Use of an oligomeric compound or double-stranded ribonucleic acid (dsRNA) reagent according to any one of claims 29 to 61 in the manufacture of a medicament for inhibiting gene expression.

64. The drug is used for intrahepatic and extrahepatic administration, and the use of the drug for extrahepatic administration includes targeting central nervous system (CNS) genes selected from APP, ATXN2, C9orf72, TARDBP, HTT, SNCA, FUS, ATXN3, ATXN1, SCA7, SCA8, ATN1, MeCP2, prion disease-related genes PRNP, DMPK, and TTR, and targeting groups for ocular tissues including AMD, CFB, MYOC, ROCK2, ADRB2, CA2, CRYGC, and PPP3C; 64. The use according to claim 63.

65. The application of the drug in CNS diseases includes Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar diseases, prion diseases and Lafora's disease, as well as ophthalmological diseases such as age-related macular degeneration (AMD) (dry and wet), birdshot chorioretinopathy, dominant retinitis pigmentosa, Fuch's dystrophy, hereditary and sporadic glaucoma and Stargardt's disease, target glaucoma, cataract, dry eye syndrome.

64. The use according to claim 63.

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