WO2024114776A1 - 双环脱碱基核酸类似物以及由它们制备的寡聚化合物 - Google Patents

双环脱碱基核酸类似物以及由它们制备的寡聚化合物 Download PDF

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WO2024114776A1
WO2024114776A1 PCT/CN2023/135759 CN2023135759W WO2024114776A1 WO 2024114776 A1 WO2024114776 A1 WO 2024114776A1 CN 2023135759 W CN2023135759 W CN 2023135759W WO 2024114776 A1 WO2024114776 A1 WO 2024114776A1
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group
acid
substituted
oligomeric compound
unsubstituted
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French (fr)
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舒东旭
邵鹏程
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Shanghai Argo Biopharmaceutical Co Ltd
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Shanghai Argo Biopharmaceutical Co Ltd
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Priority to JP2025531770A priority Critical patent/JP2025539467A/ja
Priority to EP23896918.2A priority patent/EP4628583A1/en
Priority to CR20250257A priority patent/CR20250257A/es
Priority to KR1020257021550A priority patent/KR20250116697A/ko
Priority to AU2023404700A priority patent/AU2023404700A1/en
Priority to IL321125A priority patent/IL321125A/en
Priority to PE2025001139A priority patent/PE20251853A1/es
Application filed by Shanghai Argo Biopharmaceutical Co Ltd filed Critical Shanghai Argo Biopharmaceutical Co Ltd
Priority to CN202380080330.3A priority patent/CN120225676A/zh
Publication of WO2024114776A1 publication Critical patent/WO2024114776A1/zh
Priority to DO2025000126A priority patent/DOP2025000126A/es
Priority to JOJO/P/2025/0131A priority patent/JOP20250131A1/ar
Priority to MX2025006436A priority patent/MX2025006436A/es
Anticipated expiration legal-status Critical
Priority to CONC2025/0008331A priority patent/CO2025008331A2/es
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Definitions

  • the present invention relates to bicyclic abasic nucleic acid analogs and oligomeric compounds prepared therefrom, and more particularly to bicyclic abasic nucleic acid analogs which can be incorporated into one or both ends of an oligomeric compound.
  • RNA interference or "RNAi” technology is already an effective means of disease therapy known in the art.
  • the RNAi mechanism is initiated by the production of longer non-coding RNAs mediated by the Dicer enzyme, which loads these RNA molecules into the RNA-induced silencing complex (RISC), where the sense strand is discarded and the antisense strand or guide strand is hybridized with a fully or partially complementary mRNA sequence, and then the silencing of the mRNA is induced via Ago2-mediated degradation or translation inhibition.
  • RISC RNA-induced silencing complex
  • Advances in RNAi technology and delivery methods have given RNAi-based therapies more and more positive results, and such therapies represent a promising treatment direction for a class of diseases.
  • RNAi oligonucleotides are crucial to fully exploit the potential of such therapeutic regimens, which can improve their pharmacokinetic and pharmacodynamic properties. For example, Choung et al.
  • abasic nucleotides and reverse abasic nucleotides have been used in 5' or 3' oligonucleotides to enhance the silencing effect of oligonucleotides and improve the stability of oligonucleotides, for example: WO2022162155.
  • RNAi agents with enhanced in vivo efficacy and stability suitable for administration for therapeutic purposes.
  • the research on abasic nucleotides is still in its initial stages. It is hoped that new abasic nucleic acid analogs can be further provided to be incorporated into oligonucleotides to improve stability against cellular nucleases, have longer in vivo pharmacodynamic effects, and have stronger affinity for nucleic acid targets.
  • the present invention relates to bicyclic abasic nucleic acid analogs and oligomeric compounds prepared therefrom, and more particularly to a bicyclic abasic nucleic acid analog monomer compound, and a bicyclic abasic nucleic acid analog monomer compound that can be incorporated into one or both ends of an oligomeric compound.
  • These oligomeric compounds can be used in addition to liver-related tissue target cells, and can also be used for delivery to extrahepatic tissues, such as ophthalmic and CNS target cells.
  • a 1 and A 2 are independently selected from the group consisting of O, S, SO, SO 2 , NR 2 and CR 3 R 4 , R 2 , R 3 and R 4 are independently selected from the group consisting of hydrogen, halogen, sulfonyl, sulfinyl, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 5 -C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;
  • T1 and T2 are a protecting group, Z, L or -ZL, wherein Z represents a targeting group or a lipophilic group, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and L represents a carrier spacer or a carrier part comprising a spacer link; and the other of T1 and T2 is an active phosphorus group or a protecting group; and T1 and T2 are not protecting groups at the same time;
  • R 1 is independently selected from halogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 5 -C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;
  • n is an integer from 0 to 6;
  • A1 is O. In certain embodiments, A2 is O. In certain embodiments, A1 and A2 are each independently O. In certain embodiments, A1 and A2 are each independently S. In certain embodiments, A1 and A2 are each independently NR2 . In certain embodiments, R2 is hydrogen or CH3 . In certain embodiments, A1 is different from A2 . In certain embodiments, A1 is O and A2 is S. In certain embodiments, A1 is O and A2 is NR2 . In certain embodiments, A1 is S and A2 is NR2 .
  • the protecting group of one of T1 and T2 is different from A1 or A2 , and corresponds to a hydroxyl protecting group, a thiol protecting group, and an amine protecting group, respectively.
  • one of the protecting groups of T1 and T2 is a hydroxy protecting group; in one embodiment, the hydroxy protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, Triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichlor
  • one of the protecting groups of T1 and T2 is a thiol protecting group; in one embodiment, the thiol protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl alkyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacet
  • one of the protecting groups of T1 and T2 is an amino protecting group; in one embodiment, the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrophenyl, acetyl, 2-nitrobenzenesulfonyl, phthalimido and dithiosuccinyl.
  • the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC
  • one of T1 and T2 is a lipophilic group, wherein the lipophilicity of the lipophilic group can be measured by logKow, and the logKow is greater than 0; preferably the logKow of the lipophilic group is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5 or greater than 10.
  • the lipophilic group is selected from a saturated or unsaturated C4- C30 hydrocarbon chain, an aliphatic ring, an aromatic, a fatty acid group or a fatty acid-derived group, a steroid-derived group, and any fat-soluble vitamin group.
  • the lipophilic group is a saturated or unsaturated C10- C25 hydrocarbon chain (e.g., C10 - C25 alkyl or alkenyl). In some embodiments, the lipophilic group is a saturated or unsaturated C10 - C18 hydrocarbon chain (e.g., a straight-chain C6 - C18 alkyl or alkenyl). In one embodiment, the lipophilic group is a saturated or unsaturated C14 , C16 or C18 hydrocarbon chain (e.g., a straight-chain C16 alkyl or alkenyl). In one embodiment, the lipophilic group is a saturated straight-chain C14 , C16 or C18 alkyl.
  • the lipophilic group is optionally further substituted by a group consisting of the following functional groups: 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 .
  • the lipophilic group comprises any saturated or unsaturated fatty acid with a hydrocarbon chain of 4 to 28 carbon atoms, and may contain one or two carboxyl groups. In one embodiment, the lipophilic group is a group derived from a saturated fatty acid with a hydrocarbon chain of 8 to 24 carbon atoms.
  • the lipophilic group is a group derived from a saturated fatty acid, wherein the fatty acid is selected from the group consisting of: octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid and lignoceric acid.
  • the lipophilic group is a group derived from an unsaturated fatty acid with a hydrocarbon chain of 8 to 24 carbon atoms, wherein the fatty acid is selected from the group consisting of: myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, trans-vaccenic acid, linoleic acid, trans-linoleic acid, alpha-linolenic acid, arachidonic acid and erucic acid.
  • the lipophilic group is selected from cholesterol, vitamin E (tocopherol) or bile acid.
  • the lipophilic group is selected from docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosanoic acid (DCA), sterol cholesterol (bile), tocopheryl succinate (TS) and lithocholic acid (LA), retinoic acid (vitamin A acid).
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • DCA docosanoic acid
  • bile sterol cholesterol
  • TS tocopheryl succinate
  • LA lithocholic acid
  • vitamin A acid retinoic acid
  • the lipophilic group may be covalently linked via direct attachment to A 1 or A 2.
  • the lipophilic group may be covalently linked to A 1 or A 2 via a linker.
  • one of T1 and T2 is a lipophilic group, which is further covalently linked to A1 or A2 via a linker.
  • the lipophilic group is covalently linked to A1 or A2 via one or more linkers.
  • the linker is a hydrocarbon linker or a polyethylene glycol (PEG) linker.
  • the linker is selected from the group consisting of: amide bond, phosphatidylcholine, hydrocarbon linker or polyethylene glycol (PEG) linker, amino-alkyl-alcohol, hydroxyproline, hydroxyproline alcohol, amino-alkyl-thiophosphate linker, amino-PEG-thiophosphate linker, ⁇ -carboxylate-amino-alkylthiophosphate linker and ⁇ -carboxylate-amino-PEG-thiophosphate linker.
  • the linker contains ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, click reaction product (e.g., triazole from azide-alkyne cycloaddition) or carbamate.
  • phosphodiester is thiophosphate diester and oxophosphate diester.
  • the linker includes a cleavable group.
  • At least the linker is a redox cleavable linker (such as a reduction 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).
  • a redox cleavable linker such as a reduction 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
  • At least one linker is a bio-cleavable linker selected from the group consisting of: functionalized monosaccharides or oligosaccharides of DNA, RNA, disulfides, amides, galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.
  • the linker can also be a cyclic group or a non-cyclic group.
  • the cyclic group is selected from the group consisting of: pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthalene.
  • the non-cyclic group is a part based on the main chain of serinol or the main chain of diethanolamine.
  • one of T1 and T2 is a targeting group
  • the targeting group may be a ligand conventionally used in the field of siRNA administration.
  • the targeting group can be selected from one or more of the ligands formed by the following targeting molecules or their derivatives: polymers, carbohydrates, receptor ligands expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands.
  • At least one or each of the targeting groups is selected from a group comprising a ligand capable of binding to a receptor on the surface of a mammalian hepatocyte.
  • each of the targeting groups independently comprises a ligand that has affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes.
  • ASGPR asialoglycoprotein receptor
  • each of the targeting groups is independently selected from D-pyranose, L-pyranose, D-arabinose, D-furanose, L-furanose, D-glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-furanose, ⁇ -D-furanose, ⁇ -D-pyranose, ⁇ -D-pyranose, ⁇ -D-glucose-sugar, ⁇ -D-pyranose.
  • At least one or each of the targeting groups is a ligand comprising galactose or N-acetylgalactosamine.
  • the targeting group may be connected to one of A1 or A2 via a suitable conjugated linker.
  • a suitable conjugated linker Those skilled in the art can select a suitable conjugated linker according to the specific type of the targeting group.
  • the targeting group is connected to one of A1 or A2 and is selected from one of the following compound fragments:
  • one of T1 and T2 is a carrier spacer.
  • the carrier spacer is a dicarboxylic acid derived group, with the example structure R is absent, ether, polyethylene glycol, oxygen, sulfur, nitrogen, alkylene, alkenyl, alkynyl, aryl, aralkyl, heteroalkyl or heteroaryl.
  • the carrier spacer is a polyalkylene glycol phosphate/phosphonate; in certain embodiments, the carrier spacer is preferably a C 3 -C 6 alkyl dicarboxylate group.
  • the carrier spacer is preferably a succinyl group
  • one of T1 and T2 is a carrier part linked by a spacer, wherein the carrier part includes a solid phase carrier part and a liquid phase carrier part.
  • the solid phase carrier is selected from controlled pore glass (CPG), polystyrene, silica gel, macroporous cross-linked polystyrene, polymethacrylic acid vinyl alcohol copolymer, silicon chip glass, cellulose, polystyrene beads, polypropylene sheets, non-porous silica beads, polyacrylamide or polyacrylate.
  • the solid phase carrier part is long chain alkylamine controlled pore glass (LCAA-CPG).
  • one of T1 and T2 is a targeting group or a lipophilic group and further comprises a carrier portion linked by a spacer.
  • the carrier portion is connected to a hydroxyl group in a linker in a targeting group or a lipophilic group or a conjugated linker through a spacer.
  • the other of T1 and T2 is an active phosphorus group, and the active phosphorus group has the following structure:
  • M 1 is H, substituted or unsubstituted C 1 -C 6 alkyl, OR 5 , SR 5 , OH, SH or NR 6 R 7 , M 2 is OH, SH, OR 5 ′ or NR 6′R 7 ′, each R 5 , R 6 , R 7 , R 5 ′, R 6 ′ or R 7 ′ is independently hydrogen, substituted or unsubstituted C 1 -C 6 alkyl, or sulfonyl; and m is 0 or 1.
  • M1 is selected from methyl, ethyl, propyl, and isopropyl.
  • M 1 is selected from OR 5
  • R 5 is selected from substituted or unsubstituted methyl, ethyl, propyl, isopropyl.
  • M 1 is selected from methanesulfonylamide.
  • M 2 is selected from N(CH(CH 3 ) 2 ) 2 .
  • each R 5 , R 6 or R 7 is independently a substituted alkyl, wherein the substituent is selected from cyano, halogen, hydroxyl, and amino.
  • M 1 is O(CH 2 ) 2 CN
  • M 2 is N(CH(CH 3 ) 2 ) 2
  • m is 0.
  • the other of T1 and T2 is an active phosphorus group, and the active phosphorus group is selected from diisopropyl cyanoethoxy phosphoramidite, diisopropyl methyl phosphoramidite, diisopropyl ethyl phosphoramidite and H-phosphonate.
  • one of T1 and T2 is a protecting group and the other is a carrier spacer, a carrier moiety linked via a spacer, a targeting group or a lipophilic group, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker.
  • one of T1 and T2 is diisopropylcyanoethoxyphosphoramidite and the other is 4,4'-dimethoxytrityl.
  • one of T1 and T2 is diisopropylcyanoethoxyphosphoramidite and the other is a targeting group.
  • one of T1 and T2 is diisopropylcyanoethoxyphosphoramidite and the other is a lipophilic group.
  • R 1 is each independently selected from halogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl.
  • n 0.
  • the compounds provided herein have the configuration of Formula (IIa) or Formula (IIb):
  • a 1 , A 2 , T 1 , T 2 , R 1 and n are as described above.
  • the compounds provided herein have the configuration of Formula (IIa-1) or Formula (IIb-1):
  • a 1 , A 2 , T 1 , T 2 , R 1 and n are as described above.
  • an oligomeric compound comprising at least one 5′-terminal and/or 3′-terminal monomer having formula (III) or a stereoisomer thereof:
  • a 1 and A 2 are independently selected from the group consisting of O, S, SO, SO 2 , NR 2 and CR 3 R 4 , R 2 , R 3 and R 4 are independently selected from the group consisting of hydrogen, halogen, sulfonyl, sulfinyl, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 5 -C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;
  • T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (III) or its stereoisomer to the oligomeric compound;
  • R 1 is independently selected from halogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 5 -C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;
  • n is an integer from 0 to 6;
  • the oligomeric compound optionally further comprises a targeting group.
  • a 1 is O. In certain embodiments, in the oligomeric compounds provided, A 2 is O. In certain embodiments, in the oligomeric compounds provided, A 1 and A 2 are each independently O. In certain embodiments, in the oligomeric compounds provided, A 1 and A 2 are each independently S. In certain embodiments, in the oligomeric compounds provided, A 1 and A 2 are each independently NR 2 . In certain embodiments, in the oligomeric compounds provided, R 2 is hydrogen or CH 3 . In certain embodiments, in the oligomeric compounds provided, A 1 is different from A 2 . In certain embodiments, in the oligomeric compounds provided, A 1 is O and A 2 is S. In certain embodiments, in the oligomeric compounds provided, A 1 is O and A 2 is NR 2 . In certain embodiments, in the oligomeric compounds provided, A 1 is S and A 2 is NR 2 .
  • the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (III) or its stereoisomer to the 5′-end and/or 3′-end of the oligomeric compound, and the internucleoside linking group is selected from a phosphorus-containing linking group or a phosphorus-free linking group.
  • the phosphorus-containing internucleoside linking group has a structural fragment as shown below:
  • X represents H, substituted or unsubstituted C 1 -C 6 alkyl, OR 8 , SR 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, sulfonyl, They independently represent the part connected to one of A1 or A2 herein, and the part connected to the adjacent nucleotide.
  • the phosphorus-containing internucleoside linking groups are independently: phosphodiester linking groups, phosphotriester linking groups, phosphorothioate linking groups, phosphorodithioate bond linking groups, alkylphosphonate linking groups, aminophosphonate linking groups, phosphonate linking groups, phosphinate linking groups, phosphinate linking groups, thiophosphoramidate linking groups and aminophosphoroester linking groups.
  • the internucleoside linking group is independently an alkylphosphonate linking group, a phosphodiester internucleoside linking group or a phosphorothioate internucleoside linking group.
  • the protecting group of one of T 3 and T 4 is different according to A 1 or A 2 , and corresponds to a hydroxyl protecting group, a thiol protecting group and an amine protecting group, respectively.
  • one of the protecting groups of T 3 and T 4 is a hydroxy protecting group; in one embodiment, in the oligomeric compound provided, the hydroxy protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, Triphenylsilyl, triisopropylsilyl,
  • one of the protecting groups of T 3 and T 4 is a thiol protecting group; in one embodiment, in the oligomeric compound provided, the thiol protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl alkyl, tert-butyldiphenylsilyl, triphenylsilyl, triiso
  • one of the protecting groups of T 3 and T 4 is an amino protecting group; in one embodiment, in the oligomeric compound provided, the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrophenyl, acetyl, 2-nitrobenzenesulfonyl, phthalimido and dithiosuccinyl.
  • the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-but
  • one of T3 and T4 is hydrogen, and the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (III) or its stereoisomer to the 5′-terminus and/or 3′-terminus of the oligomeric compound.
  • the oligomeric compound provided one of T3 and T4 is a lipophilic group, wherein the lipophilicity of the lipophilic group can be measured by logKow, and logKow is greater than 0; preferably, the logKow of the lipophilic group is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5 or greater than 10.
  • the lipophilic group is selected from a saturated or unsaturated C4 - C30 hydrocarbon chain, an aliphatic ring, an aromatic, a fatty acid group or a fatty acid-derived group, a steroid-derived group, and any fat-soluble vitamin group.
  • the lipophilic group is a saturated or unsaturated C 10- C 25 hydrocarbon chain (e.g., C 10 -C 25 alkyl or alkenyl). In some embodiments, in the oligomeric compounds provided, the lipophilic group is a saturated or unsaturated C 10 -C 18 hydrocarbon chain (e.g., a straight-chain C 6 -C 18 alkyl or alkenyl). In one embodiment, in the oligomeric compounds provided, the lipophilic group is a saturated or unsaturated C 14 , C 16 or C 18 hydrocarbon chain (e.g., a straight-chain C 16 alkyl or alkenyl).
  • the lipophilic group is a saturated straight-chain C 14 , C 16 or C 18 alkyl.
  • the lipophilic group is optionally further substituted by a group consisting of the following functional groups: hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide and alkyne. These functional groups can be used to attach the lipophilic group to A 1 or A 2 .
  • the lipophilic group comprises any saturated or unsaturated fatty acid with a hydrocarbon chain of 4 to 28 carbon atoms, and may contain one or two carboxyl groups. In one embodiment, in the oligomeric compound provided, the lipophilic group is a group derived from a saturated fatty acid with a hydrocarbon chain of 8 to 24 carbon atoms.
  • the lipophilic group is a group derived from a saturated fatty acid, wherein the fatty acid is selected from the group consisting of: octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid and lignoceric acid.
  • the lipophilic group is a group derived from an unsaturated fatty acid, wherein the fatty acid is selected from the group consisting of: myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, trans-isooleic acid, linoleic acid, trans-linoleic acid, ⁇ -linolenic acid, arachidonic acid and erucic acid.
  • the lipophilic group is selected from cholesterol, vitamin E (tocopherol) or bile acid.
  • 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).
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • DCA docosanoic acid
  • bile sterol cholesterol
  • TS tocopherol succinate
  • LA lithocholic acid
  • vitamin A acid retinoic acid
  • the lipophilic group may be covalently linked via direct attachment to A 1 or A 2.
  • the lipophilic group may be covalently linked to A 1 or A 2 via a linker.
  • one of T 3 and T 4 is a lipophilic group
  • the lipophilic groups of T 3 and T 4 are further covalently linked to A 1 or A 2 via a linker.
  • the lipophilic group is covalently linked to A 1 or A 2 via one or more linkers.
  • the linker is a hydrocarbon chain linker or a polyethylene glycol (PEG) linker.
  • the linker is selected from the group consisting of: amide bond, phosphatidylcholine, hydrocarbon linker or polyethylene glycol (PEG) linker, amino-alkyl-alcohol, hydroxyproline, hydroxyproline alcohol, amino-alkyl-thiophosphate linker, amino-PEG-thiophosphate linker, ⁇ -carboxylate-amino-alkylthiophosphate linker and ⁇ -carboxylate-amino-PEG-thiophosphate linker.
  • the linker contains ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, click reaction product (e.g., triazole from azide-alkyne cycloaddition) or carbamate.
  • the phosphodiester linker is a thiophosphate diester, an oxophosphate diester.
  • the linker in the oligomeric compound provided, includes a cleavable group.
  • At least the linker is a redox cleavable linker, such as a reduction cleavable linker, for example, a disulfide group, an acid cleavable linker (e.g., a hydrazone group, an ester group, an acetal group or a ketal group), an esterase cleavable linker (e.g., an ester group), a phosphatase cleavable linker (e.g., a phosphate ester) or a peptidase cleavable linker (e.g., a peptide bond).
  • a redox cleavable linker such as a reduction cleavable linker, for example, a disulfide group, an acid cleavable linker (e.g., a hydrazone group, an ester group, an ace
  • At least one linker is a bio-cleavable linker selected from the group consisting of: DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, mannose functionalized monosaccharide or oligosaccharide and combinations thereof.
  • the linker in the oligomeric compound provided, can also be a cyclic group or a non-cyclic group.
  • the cyclic group is selected from the group consisting of: pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthalene.
  • the non-cyclic group is a part based on the main chain of serinol or the main chain of diethanolamine.
  • the 5′-end and/or 3′-end of the oligomeric compound further comprises one or more targeting groups.
  • the oligomeric compound provided, one of T 3 and T 4 is represented as a covalent bond, and the oligomeric compound is connected to the targeting group in the form of a covalent bond through one of T 3 and T 4 in formula (III) or its stereoisomers.
  • the oligomeric compound provided, the 5′-end and/or 3′-end nucleotide is connected to the targeting group.
  • the targeting group can be a ligand conventionally used in the field of siRNA administration.
  • the targeting group in the provided oligomeric compounds, can be selected from one or more ligands formed by the following targeting molecules or their derivatives: polymers, carbohydrates, receptor ligands expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands.
  • At least one or each of the targeting groups is selected from a group comprising a ligand capable of binding to a receptor on the surface of a mammalian hepatic parenchymal cell.
  • each of the targeting groups independently comprises a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes.
  • ASGPR asialoglycoprotein receptor
  • 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, ⁇ -D-mannofuranose, ⁇ -D-mannofuranose, ⁇ -D-mannopyranose, ⁇ -D-mannopyranose, ⁇ -D-glucopyranose, ⁇ -D-pyranose glucose, ⁇ -D-furanose glucose, ⁇ -D-furanose glucose, ⁇ -D-fructofuranose, ⁇ -D-fructofuranose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galacto
  • At least one or each of the targeting groups is a ligand comprising galactose or N-acetylgalactosamine.
  • the targeting group can be connected to one of A1 or A2 via a suitable conjugated linker.
  • a suitable conjugated linker Those skilled in the art can select a suitable conjugated linker according to the specific type of the targeting group.
  • the targeting group is connected to one of A1 or A2 and is selected from one of the following compound fragments:
  • R 1 is each independently selected from halogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl.
  • oligomeric compounds are provided wherein n is 0.
  • the oligomeric compound provided comprises at least one 5′-terminal and/or 3′-terminal monomer having a configuration represented by formula (IVa) or formula (IVb):
  • a 1 , A 2 , T 3 , T 4 , R 1 and n are as described above.
  • the oligomeric compound provided comprises at least one 5′-terminal and/or 3′-terminal monomer having a configuration represented by formula (IVa-1) or formula (IVb-1):
  • a 1 , A 2 , T 3 , T 4 , R 1 and n are as described above.
  • one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects a monomer of formula (III) or its stereoisomers, formula (IVa), formula (IVb), (IVa-1) or formula (IVb-1) to the 5′-terminus of the oligomeric compound.
  • one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects a monomer of formula (III) or its stereoisomers, formula (IVa), formula (IVb), (IVa-1) or formula (IVb-1) to the 3′-terminus of the oligomeric compound.
  • one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond
  • the lipophilic group is optionally connected to one of A1 or A2 via a linker
  • the other of T3 and T4 independently connects a same or different (III) or its stereoisomer, formula (IVa), formula (IVb), (Iva-1) or formula (IVb-1) monomer to the internucleoside linking groups at the 5′-terminus and 3′-terminus of the oligomeric compound.
  • the oligomeric compound is a single-stranded oligonucleotide.
  • the single-stranded oligonucleotide is a conventional antisense oligonucleotide (also known as ASO), ribozyme or aptamer.
  • the oligomeric compound is a double-stranded ribonucleic acid (dsRNA) reagent, a double-stranded nucleoside nucleic acid well known in the art of such compounds, wherein one or two chains are oligomeric compounds as disclosed herein.
  • dsRNA double-stranded ribonucleic acid
  • double-stranded ribonucleic acid (dsRNA) agents each of which comprises: a sense strand and an antisense strand, wherein the sense strand and the antisense strand are fully or partially complementary, and the antisense strand is partially or fully complementary to a nucleic acid target gene; at least one of the sense strand and the antisense strand is an oligomeric compound as provided above, which comprises at least one 5′-terminal and/or 3′-terminal monomer having formula (III) or its stereoisomers, formula (IVa), formula (IVb), formula (IVa-1) or formula (IVb-1); and wherein the double-stranded ribonucleic acid (dsRNA) agent optionally further comprises an independent targeting group.
  • dsRNA double-stranded ribonucleic acid
  • a double-stranded ribonucleic acid (dsRNA) agent wherein the sense strand is an oligomeric compound provided by a 5′-terminal and/or 3′-terminal monomer comprising Formula (III) or its stereoisomers, Formula (IVa), Formula (IVb), Formula (IVa-1) or Formula (IVb-1) herein.
  • a double-stranded ribonucleic acid (dsRNA) agent is provided, wherein the sense strand is an oligomeric compound provided by a 5′-terminal monomer comprising Formula (III) or its stereoisomers, Formula (IVa), Formula (IVb), Formula (IVa-1) or Formula (IVb-1) herein.
  • a double-stranded ribonucleic acid (dsRNA) agent wherein the sense strand is an oligomeric compound provided by a 3′-terminal monomer comprising Formula (III) or its stereoisomers, Formula (IVa), Formula (IVb), Formula (IVa-1) or Formula (IVb-1) herein.
  • a double-stranded ribonucleic acid (dsRNA) agent is provided, wherein the sense strand is an oligomeric compound provided herein comprising the same or different 5′-terminal and 3′-terminal monomers of Formula (III) or its stereoisomers, Formula (IVa), Formula (IVb), Formula (IVa-1) or Formula (IVb-1).
  • a double-stranded ribonucleic acid (dsRNA) agent wherein the antisense strand is an oligomeric compound provided by a 5′-terminal and/or 3′-terminal monomer comprising a Formula (III) or a stereoisomer thereof, Formula (IVa), Formula (IVb), Formula (IVa-1) or Formula (IVb-1) herein.
  • dsRNA double-stranded ribonucleic acid
  • a double-stranded ribonucleic acid (dsRNA) agent wherein the antisense strand is an oligomeric compound provided by a 3′-terminal monomer comprising a Formula (III) or a stereoisomer thereof, Formula (IVa), Formula (IVb), Formula (IVa-1) or Formula (IVb-1) herein.
  • a double-stranded ribonucleic acid (dsRNA) agent is provided, wherein the antisense strand is an oligomeric compound provided by a 5′-terminal monomer comprising a Formula (III) or a stereoisomer thereof, Formula (IVa), Formula (IVb), Formula (IVa-1) or Formula (IVb-1) herein.
  • a double-stranded ribonucleic acid (dsRNA) agent is provided, optionally further comprising an independent targeting group, and further comprising one or more targeting groups on the 5'-terminal and/or 3'-terminal nucleotides of any one chain in the double-stranded ribonucleic acid (dsRNA) agent.
  • the targeting group can be a ligand conventionally used in the field of siRNA administration.
  • the targeting groups is selected from a ligand that can bind to a receptor on the surface of a mammalian hepatocyte.
  • each of the targeting groups independently comprises a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes.
  • ASGPR asialoglycoprotein receptor
  • each strand of the oligomeric compound or double-stranded ribonucleic acid (dsRNA) comprises 8-40 nucleotides in length, respectively.
  • the oligomeric compound or double-stranded ribonucleic acid (dsRNA) reagent provided is used in the preparation of a drug for inhibiting gene expression.
  • the inhibiting gene expression comprises contacting one or more cells, tissues or animals with the oligomeric compound.
  • a method for inhibiting gene expression comprising: contacting a cell with an oligomeric compound or a double-stranded ribonucleic acid (dsRNA) agent as described herein, wherein each strand of the oligomeric compound and the double-stranded ribonucleic acid (dsRNA) agent comprises 8-40 nucleotides in length, respectively, and the antisense strand of the oligomeric compound or the double-stranded ribonucleic acid (dsRNA) agent is complementary to the target RNA.
  • dsRNA double-stranded ribonucleic acid
  • the cell is in an animal. In certain embodiments, the cell is in a human. In certain embodiments, the target RNA is selected from mRNA, pre-mRNA and micro RNA. In certain embodiments, the target RNA is mRNA. In certain embodiments, the target RNA is human mRNA. In certain embodiments, the target RNA is cleaved, thereby inhibiting its function. In certain embodiments, the method further comprises detecting the level of the target RNA.
  • a method for inhibiting gene expression comprising: contacting one or more cells or tissues with an oligomeric compound or double-stranded ribonucleic acid (dsRNA) agent provided by a terminal monomer having Formula III, Formula (IVa), Formula (IVb), Formula (IVa-1) or Formula (IVb-1).
  • dsRNA ribonucleic acid
  • these genes can also be genes expressed in external tissues, such as target cell genes in ophthalmology and CNS.
  • bicyclic abasic nucleic acid analogs are provided, and can be used as nucleotide monomers to be incorporated into one or both ends of an oligomeric compound. Intermediates and methods for preparing these oligomeric compounds are also provided herein.
  • the modified bicyclic abasic nucleic acid analogs provided herein can be used to enhance one or more characteristics of the oligomeric compounds they are incorporated into, such as, for example, nuclease resistance.
  • the oligomeric compounds and compositions provided herein are expected to hybridize with a portion of a target RNA so that the target RNA loses normal function. The oligomeric compounds are expected to also be used as primers and probes in diagnostic applications.
  • a 1 and A 2 are independently selected from the group consisting of O, S, SO, SO 2 , NR 2 and CR 3 R 4 , R 2 , R 3 and R 4 are independently selected from the group consisting of hydrogen, halogen, sulfonyl, sulfinyl, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 5 -C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;
  • T1 and T2 are a protecting group, Z, L or -ZL, wherein Z represents a targeting group or a lipophilic group, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and L represents a carrier spacer or a carrier portion linked by a spacer; and the other of T1 and T2 is an active phosphorus group or a protecting group; and T1 and T2 are not protecting groups at the same time;
  • R 1 is independently selected from halogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 5 -C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;
  • n is an integer from 0 to 6;
  • a 1 is O.
  • A2 is O.
  • A1 and A2 are independently O, and the provided compound has the formula (I-1) or its stereoisomers, wherein T 1 , T 2 , R 1 and n are as described above.
  • A1 and A2 are independently S, and the provided compound has the formula (I-2) or a stereoisomer thereof, wherein T 1 , T 2 , R 1 and n are as described above.
  • a 1 and A 2 are independently NR 2 , and the provided compound has the formula (I-3) or a stereoisomer thereof, Wherein T 1 , T 2 , R 1 and n are as described above, and R 2 is independently selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 5 -C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl.
  • A1 is O
  • A2 is S
  • the provided compound has the formula (I-4) or its stereoisomers, wherein T 1 , T 2 , R 1 and n are as described above.
  • a 1 is O
  • a 2 is NR 2
  • the provided compound has the formula (I-5) or a stereoisomer thereof, wherein T 1 , T 2 , R 1 and n are as described above, and R 2 is selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 5 -C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl.
  • a 1 is S
  • a 2 is NR 2
  • the provided compound has the formula (I-6) or a stereoisomer thereof, wherein T 1 , T 2 , R 1 and n are as described above, and R 2 is selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 5 -C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl.
  • R 2 is hydrogen or CH 3 .
  • substituted or unsubstituted parent compound has hydrogen atoms that are not replaced by other substituents, or one or more hydrogen atoms are replaced by substituents.
  • substituted group is intended to include groups that are usually added to other groups or parent compounds to enhance desired properties or provide other desired effects.
  • the substituted group may be protected or unprotected, and may be added to an available site or many available sites in the parent compound.
  • the substituted group may also be further substituted by other substituted groups, and may be directly connected to the parent compound or connected to the parent compound through a linking group such as an alkyl or hydrocarbyl group.
  • each of Raa , R bb and R Cc is independently H, an optionally attached chemical functional group or another substituent group
  • a preferred list includes but is not limited to H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl.
  • protecting group refers to an unstable chemical moiety known in the art that protects reactive groups (including but not limited to hydroxyl, amino and thiol groups) from undesirable reactions during synthetic procedures. Protecting groups are often used selectively to protect sites during reactions at other reactive sites and can then be removed to leave unprotected groups as is or can be used for further reactions.
  • the protecting group of one of T 1 and T 2 is a hydroxyl protecting group, a thiol protecting group and an amine protecting group, respectively, depending on A 1 or A 2 ; that is, when A 1 or A 2 is oxygen, the protecting group of one of T 1 and T 2 is a hydroxyl protecting group; that is, when A 1 or A 2 is sulfur, sulfoxide (SO) or sulfone (SO 2 ), the protecting group of one of T 1 and T 2 is a thiol protecting group; that is, when A 1 or A 2 is nitrogen, the protecting group of one of T 1 and T 2 is an amino protecting group.
  • one of the protecting groups of T1 and T2 is a hydroxy protecting group; in one embodiment, the hydroxy protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, Triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichlor
  • one of the protecting groups of T1 and T2 is a thiol protecting group; in one embodiment, the thiol protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl alkyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacet
  • one of the protecting groups of T1 and T2 is an amino protecting group; in one embodiment, the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrophenyl, acetyl, 2-nitrobenzenesulfonyl, phthalimido and dithiosuccinyl.
  • the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC
  • a lipophilic group optionally connected to A1 or A2 via a linker may be directly linked to the attached atom or group in the form of a lipophilic group in the form of a covalent bond; or the lipophilic group may be linked to the attached atom or group via one or more linkers.
  • one of T 1 and T 2 is a lipophilic group.
  • lipophilicity or “lipophilic group” refers broadly to any compound or chemical part with affinity for lipids.
  • One way to characterize the lipophilicity of a lipophilic group can be determined by the octanol-water partition coefficient logKow, where Kow is the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the aqueous phase when the two-phase system is in equilibrium.
  • the octanol-water partition coefficient is a material property measured in the laboratory.
  • the structure to which the lipophilic group is conjugated exposes the lipophilic group (e.g., fatty acid), thereby allowing the lipophilic group to interact with albumin and/or fatty acid receptors or transporters, thereby providing an oligonucleotide with a long in vivo half-life.
  • the logKow of a group generally having lipophilicity is measured to be greater than 0.
  • the lipophilicity of the lipophilic portion as measured by the octanol-water partition coefficient logKow, may be greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10 logKow.
  • the lipophilicity of a molecule may vary relative to the functional groups it carries; for example, the addition of functional groups such as hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne at the end of the lipophilic group may increase or decrease the partition coefficient (e.g., logKow) value of the lipophilic portion; and the lipophilic group may also be further attached to the compounds shown herein through these functional groups.
  • functional groups such as hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne
  • the lipophilic group is a saturated or unsaturated C 4 -C 30 hydrocarbon chain, an aliphatic ring, an aromatic group, a fatty acid group or a fatty acid-derived group, a steroid-derived group, and any fat-soluble vitamin group.
  • the lipophilic group is a saturated or unsaturated C10- C25 hydrocarbon chain (e.g., C10- C25 alkyl or alkenyl). In some embodiments, the lipophilic group is a saturated or unsaturated C10 - C18 hydrocarbon chain (e.g., a straight-chain C10 - C18 alkyl or alkenyl). In one embodiment, the lipophilic group is a saturated or unsaturated C10 , C11 , C12 , C13 , C14 , C15 , C16 , C17 or C18 hydrocarbon chain (e.g., a straight-chain C16 alkyl or alkenyl); more preferably a straight-chain alkyl.
  • the lipophilic group is a saturated straight-chain C14 alkyl, C16 alkyl or C18 alkyl, and the structure is shown as follows:
  • the lipophilic group is optionally further substituted by a 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 .
  • the lipophilic group is selected from the group derived from the fatty acid of dicarboxylic acid.
  • the fatty acid comprises any saturated or unsaturated fatty acid with a hydrocarbon chain of 4 to 28 carbon atoms, and may contain one or two carboxyl groups.
  • the lipophilic group is a group derived from a saturated fatty acid with a hydrocarbon chain of 8 to 24 carbon atoms.
  • the lipophilic group is a group derived from a saturated fatty acid, wherein the fatty acid is selected from the group consisting of: butyric acid (CH 3 (CH 2 ) 2 COOH), valeric acid, caproic acid, heptanoic acid, octanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, heptacosanoic acid and octacosanoic acid
  • the lipophilic group is a group derived from an unsaturated fatty acid, and the unsaturated fatty acid is one of the ones presented in Table 1; the fatty acid is preferably selected from the group consisting of: myristic acid, palmitoleic acid, hexadecenoic acid, oleic acid, oleic acid, trans-oleic acid, linoleic acid, trans-linoleic acid, ⁇ -linolenic acid, arachidonic acid and erucic acid.
  • 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.
  • bile acids e.g., cholic acid, deoxycholic acid, and dehydrocholic acid
  • cortisone e.g., cholic acid, deoxycholic acid, and dehydrocholic acid
  • cortisone e.g., cholic acid, deoxycholic acid, and dehydrocholic acid
  • cortisone e.g., cholic acid, deoxycholic acid, and dehydrocholic acid
  • cortisone e.g.,
  • the lipophilic group is selected from cholesterol, vitamin E (tocopherol) or bile acid.
  • the lipophilic group is selected from the unsaturated fats docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), the saturated fatty acid docosanoic acid (DCA), sterol cholesterol (bile), tocopherol succinate (TS) and lithocholic acid (LA), retinoic acid (vitamin A acid).
  • DHA unsaturated fats docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • DCA saturated fatty acid docosanoic acid
  • sterol cholesterol sterol cholesterol
  • TS tocopherol succinate
  • LA lithocholic acid
  • vitamin A acid retinoic acid
  • the lipophilic group is an aromatic moiety.
  • aromatic or “aromatic” refers broadly to monoaromatic and polyaromatic hydrocarbons.
  • Aryl includes, but is not limited to, C 6 -C 14 aryl moieties comprising one to three aromatic rings, which may be optionally substituted; "aralkyl” or “arylalkyl” comprising an aryl group covalently linked to an alkyl group, any of which may be independently optionally substituted or unsubstituted; and "heteroaryl".
  • heteroaryl refers to a group having 5 to 14 ring atoms, preferably 5, 6, 9 or 10 ring atoms; having 6, 10 or 14 ⁇ electrons shared in the ring array, and having one to about three heteroatoms selected from the group consisting of nitrogen (N), oxygen (O) and sulfur (S) in addition to carbon atoms.
  • the lipophilic group of the present invention can be directly covalently linked to A 1 or A 2 via a covalent bond.
  • the combination of A 1 and T 1 and/or the combination of A 2 and T 2 include but are not limited to the following structures: in represents a single bond or a double bond, p or q independently represents an integer of 5-25, and the sum of p and q does not exceed 25.
  • the combination of A1 and T1 and/or the combination of A2 and T2 include but are not limited to the following examples:
  • the lipophilic group can be covalently linked to A1 or A2 via a linker, and the linker can be a combination of one or more.
  • one of T1 and T2 is a lipophilic group, which further comprises a linker and is covalently linked to A1 or A2 via one or more linkers.
  • the linker is selected from the group consisting of an amide bond, a phosphatidylcholine, a hydrocarbon linker or a polyethylene glycol (PEG) linker, an amino-alkyl-alcohol, a hydroxyproline, a hydroxyprolinol, an amino-alkyl-phosphorothioate linker, an amino-PEG-phosphorothioate linker, an ⁇ -carboxylate-amino-alkylphosphorothioate linker, and an ⁇ -carboxylate-amino-PEG-phosphorothioate linker.
  • PEG polyethylene glycol
  • the linker contains an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide bond, a product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.
  • the phosphodiester linker is a thiophosphate diester (non-limiting examples: ), phosphorothioate diesters (non-limiting examples: ).
  • the linker is a hydrocarbon chain linker or a polyethylene glycol (PEG) linker.
  • the linker includes 2-20 carbons, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons.
  • the linker includes 1-20 ethylene glycol subunits, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ethylene glycol repeats.
  • the linker is an amide bond linker.
  • the linker includes a cleavable group.
  • the linker includes a cleavable group that can be sensitive to pH, such as a disulfide group.
  • the pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH in the range of 5.5-6.0, and lysosomes have an even more acidic pH of about 5.0.
  • Some linkers will have a linking group that is cleaved at a preferred pH, thereby releasing the iRNA agent from a ligand (e.g., a targeting or cell permeability ligand, such as cholesterol) in the cell, or entering the desired compartment of the cell.
  • a ligand e.g., a targeting or cell permeability ligand, such as cholesterol
  • the linker is at least one of a redox cleavable linker (such as a reduction cleavable linker, e.g., a disulfide group).
  • a redox cleavable linker such as a reduction cleavable linker, e.g., a disulfide group.
  • cleavable linking group is a redox cleavable linking group that cleaves upon reduction or oxidation.
  • An example of a reduction cleavable linking group is a disulfide linking group (-S-S-).
  • Esterase cleavable linker e.g., ester group
  • Ester-based linking groups are cleaved by enzymes such as esterases and amidases in cells.
  • Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene.
  • Ester cleavable linking groups have the general formula -C(O)O- or -OC(O)-.
  • Phosphatases can cleave linkers (e.g., phosphates), and phosphate-based linking groups are cleaved by agents that degrade or hydrolyze phosphates.
  • agents that cleave phosphate groups in cells are enzymes in cells, such as phosphatases.
  • phosphate-based linking groups are -O-P(O)(OH)-O-, -S-P(S)(OH)-O-.
  • Peptide-based linking groups are cleaved by enzymes such as peptidases and proteases in cells.
  • Peptide-based cleavable linking groups are peptide bonds formed between amino acids to produce oligopeptides (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.
  • Peptide bonds are special types of amide bonds formed between amino acids to produce peptides and proteins.
  • Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to produce peptides and proteins, and do not include the entire amide functional group.
  • At least one linker is a biocleavable linker selected from the group consisting of functionalized monosaccharides or oligosaccharides of DNA, RNA, disulfides, amides, galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.
  • the ⁇ -carboxylate-amino-alkyl-phosphorothioate linker has the following structure:
  • m represents a linking portion to a lipophilic group and one of A1 or A2 , wherein m is preferably an integer from 2 to 12, preferably an integer from 4 to 10. In one embodiment, m is an integer from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In one embodiment, m is 6.
  • the amino-PEG-phosphorothioate linker has the following structure: represents a linking moiety to a lipophilic group and one of A1 or A2 , wherein m is preferably an integer from 1 to 8. In one embodiment, m is an integer of 1, 2, 3, 4, 5, 6, 7 or 8. And ⁇ -carboxylate-amino-PEG-phosphorothioate linker:
  • the amino-alkyl-phosphorothioate linker has the following structure: represents a linking portion to a lipophilic group and one of A1 or A2 ; wherein m is preferably an integer from 2 to 12. Preferably, it is an integer from 4 to 10. In one embodiment, m is an integer from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In one embodiment, m is 6.
  • the linker is selected from the group consisting of: (i) an amino-alkyl-phosphorothioate linker; (ii) an ⁇ -carboxylate-amino-alkyl-phosphorothioate linker; (iii) an amino-PEG-phosphorothioate linker, and (iv) an ⁇ -carboxylate-amino-PEG-phosphorothioate linker all as defined above in the formula provided.
  • the invention provides a conformationally restricted linker, for example based on hydroxyproline, for example,
  • m represents a linking portion to a lipophilic group and one of A1 or A2 , wherein m is preferably an integer from 1 to 8. In one embodiment, m is an integer of 2, 3, 4, 5, 6, 7 or 8.
  • the invention provides conformationally restricted linkers, such as those based on hydroxyprolinol, such as It represents a linking portion to a lipophilic group and one of A1 or A2 .
  • the present invention provides linkers such as amino-alkyl-alcohol linkages, for example, represents a linking portion to a lipophilic group and one of A1 or A2 , wherein m is preferably an integer from 1 to 8. In one embodiment, m is an integer of 1, 2, 3, 4, 5, 6, 7 or 8.
  • the present invention provides a linker such as phosphatidylcholine, for example,
  • the linker can also be a cyclic group or a non-cyclic group.
  • the cyclic group is selected from the group consisting of: pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthalene.
  • the non-cyclic group is based on the main chain of serinol or the main chain of diethanolamine.
  • lipophilic groups and linkers can be found in PCT Publication No. WO2019/217459, entitled “Extrahepatic Delivery,” filed on July 7, 2019, PCT Publication No. WO2019/215333, entitled “Oligonucleotide Conjugates Including 7′-5′- ⁇ -Anomeric Bicyclic Sugar Nucleosides,” filed on May 10, 2019, and Nucleic Acids Research, 2019, Vol. 47, No. 31082–1096, “Various Lipid Conjugates for Functional Extrahepatic In Vivo Nucleotide Delivery,” the contents of which are incorporated herein by reference in their entirety.
  • the present invention provides a non-limiting example of a compound formed by covalently linking a lipophilic group to A1 or A2 via a linker.
  • one of T1 and T2 is a targeting group, which may be a ligand conventionally used in the field of siRNA administration.
  • the targeting group may change the distribution, targeting or lifespan, endosomal dissolution properties, improved transport, hybridization and specificity of the siRNA agent into which it is incorporated, such as various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.
  • the targeting group can be selected from one or more of the following ligands formed by targeting molecules or their derivatives; such as polymers, such as polyethylene glycol; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); receptor ligands expressed by hepatic parenchymal cells, such as asialoglycoproteins, asialosugar residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin and other aptamers; antibodies; quantum dots; polypeptides, such as membrane-permeable peptides, or small molecule ligands.
  • polymers such as polyethylene glycol
  • carbohydrates such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc
  • At least one or each of the targeting groups is selected from a ligand that can bind to a mammalian hepatocyte surface receptor.
  • each of the targeting groups is independently a ligand that comprises an asialoglycoprotein receptor (ASGPR) affinity with the mammalian hepatocyte surface.
  • each of the targeting groups is independently a ligand that comprises an asialoglycoprotein or sugar.
  • each of the targeting groups is independently a ligand that comprises an asialoglycoprotein, such as asialo serum globulin (ASOR) or asialo fetuin (ASF).
  • ASOR asialo serum globulin
  • ASF asialo fetuin
  • each of the targeting groups independently comprises a D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-mannofuranose, ⁇ -D-mannofuranose, ⁇ -D-mannopyranose, ⁇ -D-mannopyranose, ⁇ -D-glucose-pyranose, ⁇ -D-glucose-pyranose.
  • At least one or each of the targeting groups is a ligand comprising galactose or N-acetylgalactosamine.
  • the targeting group comprises one or more "GalNAc” (N-acetylgalactosamine) derivatives attached via a suitable tether via a bivalent or trivalent branched linker.
  • GalNAc N-acetylgalactosamine
  • q2A , q2B , q3A , q3B , q4A , q4B , q5A, q5B, q5C, q6A, q6B and q6C each independently represent 0-20 , and the repeating units therein may be the same 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 independently represent: absent, CO, NH , O, S, OC (O), NHC(O), CH 2 , CH 2 NH or CH 2 O;
  • Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C , Q 6A , Q 6B , Q 6C each occurrence independently represents: absent, alkylene, substituted alkylene wherein one or more methylene groups may be interrupted or terminated by one or more of the following groups: O, S, S(O), SO 2 , NH, C(R′) ⁇ C(R′′), C ⁇ C or C(O);
  • L2A , L2B , L3A , L3B , L4A , L4B, L5A , L5B , L5C , L6A , L6B and L6C each independently represent at each occurrence: a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide , an oligosaccharide, or a polysaccharide;
  • a monosaccharide such as GalNAc
  • R' and R" are each independently H, CH3 , OH, SH, NH2 , alkyl, alkenyl or alkynyl;
  • Ra is H or an amino acid side chain; m is 1 or 2.
  • the targeting group-conjugated compound of formula (I) or its stereoisomer and iRNA agent of the present invention can be prepared by direct reaction with a reactive functional group, such as phosphoramidite.
  • the targeting group can be connected to one of A1 or A2 via a suitable conjugated linker, and those skilled in the art can select a suitable conjugated linker according to the specific type of the targeting group.
  • a suitable conjugated linker for example, trans-4-hydroxyproline, aminoalkyl alcohol derivatives, which form phosphates through reactive functional groups, the following exemplary fragments are conjugated with degradable structures such as esters, amides, and phosphates to form suitable conjugated links:
  • the types of conjugated linkers, ligands, tethers, branched linkers, and the connection mode of the targeting group to the oligonucleotide can be found in WO2009073809, WO2014179620, WO2014179627, and WO2015006740A2, the entire contents of which are incorporated herein by reference.
  • the targeting group can be connected by acid-labile or reducible chemical bonds, which are degradable in the acidic environment of the cell endosome.
  • conjugate linker refers to a linker that connects two parts of a compound, such as covalently binds two parts of a compound.
  • Other carbohydrate conjugates and linkers suitable for use in the present invention include those described in publications WO2014/179620 and WO2014/179627, the entire contents of each of which are incorporated herein by reference.
  • the targeting group is connected to one of A1 or A2 and is selected from one of the following compound fragments:
  • one of T1 and T2 is a carrier spacer.
  • the 3'-terminal non-nucleoside or nucleoside of the oligonucleotide to be synthesized (such as in formula (III) and its stereoisomers or fragments herein) is pre-loaded through a cleavable spacer such as a succinyl group, and then linked to a linker such as a reactive amino group in the carrier part, and then loaded into a column for subsequent nucleotide docking;
  • the carrier spacer serves as the attachment point of the oligonucleotide during the synthesis process, often covalently bonded to the carrier spacer through the 3'-terminal hydroxyl of the non-nucleoside or nucleoside in the oligonucleotide, and remains attached during the entire chain assembly process, and then they are cut off and deprotected.
  • the spacer is often selected to avoid steric hindrance and is cleavable, and the oligonucleotide is tethered to it, which is used to place the oligonucleotide and the synthesis process away from the carrier. This approach allows greater flexibility and more space for synthesis.
  • the carrier part connected to the oxygen atom of the hydroxyl group at the 2' or 3' position of the ribose at the non-nucleoside at the terminal end of the oligonucleotide or the 3' end of the nucleoside (such as A1 or A2 in the formula (I) and its stereoisomers described herein) through the carrier spacer can be more specifically exemplified as follows:
  • Sp represents a carrier spacer
  • the carrier spacers (Sp) used in the field of oligonucleotide synthesis can be summarized in this article.
  • Commonly used examples include dicarboxylic acid-derived groups, the structure shown R is absent, ether, polyethylene glycol, oxygen, sulfur, nitrogen, alkylene, alkenyl, alkynyl, aryl, aralkyl, heteroalkyl or heteroaryl, One end is connected to A1 or A2 in formula (I), and the other end carboxyl group can be further connected to the carrier.
  • R is more preferably absent, oxygen, sulfur, nitrogen, alkylene, alkenyl, or alkynyl.
  • Another example is polyalkylene glycol phosphate/phosphonate, as shown in the disclosed patent CN103233003.
  • the spacer is a C3 - C6 alkyl dicarboxylate group, and the structure shown is
  • the carrier spacer is preferably succinyl
  • the spacer dicarboxylic acid is further derivatized, examples of which are further PEGylated, P is an integer of 5 to 120.
  • the other end carboxyl group can be further linked to the carrier part to form a covalent bond.
  • one of T1 and T2 comprises a carrier portion linked by a spacer, wherein the carrier portion comprises a solid phase carrier portion and a soluble carrier portion.
  • the carrier generally contains a linker, such as a long-chain alkylamine, which is connected to the Si of the solid carrier through a propylamine linker, and LCAA-CPG, which is commonly used in the art, is shown as a non-limiting example.
  • a linker such as a long-chain alkylamine
  • LCAA-CPG which is commonly used in the art
  • the long-chain alkylamine is further attached to the solid carrier through a biodegradable linker such as acetylglycerol.
  • examples of solid phase carriers include inorganic porous carriers and organic resin carriers
  • examples of inorganic porous carriers include controlled pore glass (CPG)
  • examples of organic resin carriers include carriers made of polystyrene.
  • Some examples of solid phase carriers include controlled pore glass (CPG), polystyrene, silica gel, macroporous cross-linked polystyrene, polymethacrylate vinyl alcohol copolymer, silicon chip glass, cellulose, polystyrene beads, polypropylene sheets, non-porous silica beads, polyacrylamide or polyacrylate.
  • soluble carriers have been developed into an important oligonucleotide synthesis method, which can be used for large-scale liquid phase synthesis.
  • the soluble carriers are non-limiting examples:
  • the spacer, solid carrier, linker and their connection method are generally collectively referred to as solid phase support in the art, and their composition is also well known as described in Current Protocols in Nucleic Acid Chemistry, 2000, Chapter 3, Section 3.1 "Solid-Phase Supports for Oligonucleotide Synthesis".
  • Solid phase support for more information about these spacers, carriers, linkers and their linking methods, please refer to "Synthesis of oligonucleotides on a soluble support” Beilstein J. Org. Chem. 2017, 13, 1368-1387., WO2021/173615, CN103233003A, WO2010/131916, WO2022/005988A, and CN115003680A, the entire contents of which are incorporated herein by reference.
  • one of T1 and T2 is a targeting group or a lipophilic group, and is further linked to the carrier portion via a spacer.
  • the exemplary structure is,
  • [Corrected 12.12.2023 in accordance with Rule 26] represents a portion connected to A1 or A2 herein; in some more specific embodiments, the spacer is connected to a linker in a targeting group or a lipophilic group, and in particular, the terminal hydroxyl group in the linker is attached to the spacer.
  • the other of T1 and T2 is an active phosphorus group, and the active phosphorus group has the following structure:
  • M 1 is H, substituted or unsubstituted C 1 -C 6 alkyl, OR 5 , SR 5 , OH, SH or NR 6 R 7 , M 2 is OH, SH, OR 5 ′ or NR 6′R 7 ′, each R 5 , R 6 , R 7 , R 5 ′, R 6 ′ or R 7 ′ is independently hydrogen, substituted or unsubstituted C 1 -C 6 alkyl, or sulfonyl; and m is 0 or 1.
  • each R 5 , R 6 or R 7 is independently a substituted C 1 -C 6 alkyl, and the substituent can be selected from conventional alkyl substitutions in the art. In certain embodiments, each R 5 , R 6 or R 7 is independently a substituted alkyl, and the substituent is preferably selected from cyano, halogen, hydroxyl, and amino.
  • M1 is selected from substituted or unsubstituted C1 - C6 alkyl, wherein the substituted or unsubstituted C1 - C6 alkyl is preferably methyl, ethyl, propyl, isopropyl, wherein the substituent is preferably selected from cyano, halogen, hydroxyl, and amino.
  • M 1 is selected from OR 5 , R 5 is selected from substituted or unsubstituted methyl, ethyl, propyl, isopropyl; preferably substituted methyl, ethyl, propyl, isopropyl; more preferably R 5 is cyanoethyl, that is, M 1 is cyanoethoxy.
  • M 1 is NR 6 R 7 , R 6 and R 7 are independently selected from hydrogen and sulfonyl; more preferably, M 1 is methanesulfonyl.
  • M 2 is selected from NR 6 ′R 7 ′, R 6 ′ or R 7 ′ is independently C 1 -C 6 alkyl.
  • the C 1 -C 6 alkyl is selected from methyl, ethyl, propyl, isopropyl:
  • M 2 is selected from N(CH(CH 3 ) 2 ) 2 .
  • M 1 is O(CH 2 ) 2 CN
  • M 2 is N(CH(CH 3 ) 2 ) 2
  • m is 0.
  • M 1 is methyl
  • M 2 is N(CH(CH 3 ) 2 ) 2
  • m is 0.
  • M 1 is methanesulfonamide
  • M 2 is N(CH(CH 3 ) 2 ) 2
  • m is 0.
  • the reactive phosphorus group has the following substructure: m, M 2 , R 5 , R 6 and R 7 are as described above.
  • the reactive phosphorus group is a phosphoramidite. In certain embodiments, the reactive phosphorus group is selected from diisopropylcyanoethoxyphosphoramidite (-P(N(CH(CH 3 ) 2 ) 2 )O(CH 2 ) 2 CN). In certain embodiments, the reactive phosphorus group is selected from diisopropylmethylphosphoramidite (-P(N(CH(CH 3 ) 2 ) 2 )CH 3 ). In certain embodiments, the reactive phosphorus group is selected from diisopropylethylphosphoramidite (-P(N(CH(CH 3 ) 2 ) 2 )CH 2 CH 3 ).
  • Preferred synthesis solid phase synthesis utilizes phosphoramidite (p III ) as the reactive phosphite; the intermediate phosphite compound is subsequently oxidized to phosphate or thiophosphate (p V ) using known methods to produce phosphodiester or thiophosphate internucleoside linkages. Additional reactive phosphates and phosphites are disclosed by Beaucage and Iyer, Tetrahedron, 1992, 48(12), 2223-2311.
  • 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.
  • A1 and A2 are preferably selected from O, exemplary structures
  • one of T1 and T2 is diisopropylethylphosphoramidite, and the other is 4,4'-dimethoxytrityl.
  • A1 and A2 are preferably selected from O.
  • 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.
  • 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.
  • n when n is non-0, it means that there are 1-6 independent R1 group substituents on the bicyclic structure. It is understood that these substituents in the bicyclic abasic compounds herein do not include natural nucleobases or modified nucleobases.
  • R1 is independently selected from halogen, substituted or unsubstituted C1 - C6 alkyl, substituted or unsubstituted C2 - C6 alkenyl, substituted or unsubstituted C2 - C6 alkynyl.
  • R1 is independently selected from fluorine, chlorine, bromine, iodine, methyl, ethyl, isopropyl, propyl, 3-propenyl, ethynyl, 3-propynyl.
  • n is 0, indicating that there is no R 1 group substitution on the ring structure.
  • the compounds provided herein have the configuration of Formula (IIa) or Formula (IIb):
  • a 1 , A 2 , T 1 , T 2 , R 1 and n are as described herein.
  • A1 and A2 are independently selected from: O; one of T1 and T2 is a protecting group, a targeting group and a lipophilic group, and the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T1 and T2 is an active phosphorus group; n is 0.
  • A1 and A2 are independently selected from: O; one of T1 and T2 is a hydroxyl protecting group and a lipophilic group, the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T1 and T2 is an active phosphorus group; n is 0; wherein the hydroxyl protecting group is selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and 4,4'-dimethoxytrityl, the lipophilic group is selected from C14 , C16 or C18 straight-linked alkanes, and the active phosphorus is selected from phosphoramidites. In some more preferred embodiments, the active phosphorus is selected from diisopropylcyanoethoxyphosphoramidite, diisopropylmethylphosphoramidite, diisopropy
  • A1 and A2 are independently selected from: O, one of T1 and T2 is Z, L and -ZL, the Z represents a targeting group or a lipophilic group, the lipophilic group is optionally connected to one of A1 or A2 via a linker, L represents: a carrier spacer or a carrier part linked by a spacer; and the other of T1 and T2 is a hydroxyl protecting group; n is 0.
  • the hydroxyl protecting group is selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and 4,4'-dimethoxytrityl.
  • the compounds provided herein have the configuration of Formula (IIa-1) or Formula (IIb-1):
  • a 1 , A 2 , T 1 , T 2 , R 1 and n are as described herein, and their configurations are consistent with isosorbide or isomannide known in the art.
  • a 1 and A 2 are independently selected from: O; one of T 1 and T 2 is a protecting group, a targeting group and a lipophilic group, and the lipophilic group is optionally connected to one of A 1 or A 2 via a linker, and the other of T 1 and T 2 is an active phosphorus group; n is 0.
  • A1 and A2 are independently selected from: O; one of T1 and T2 is a hydroxyl protecting group and a lipophilic group, the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T1 and T2 is an active phosphorus group; n is 0; wherein the hydroxyl protecting group is selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and 4,4'-dimethoxytrityl, the lipophilic group is selected from C14 , C16 or C18 straight-linked alkanes, and the active phosphorus is selected from phosphoramidites. In some more preferred embodiments, the active phosphorus is selected from diisopropylcyanoethoxyphosphoramidite, diisopropylmethylphosphoramidite, diiso
  • A1 and A2 are independently selected from: O, one of T1 and T2 is Z, L and -ZL, the Z represents a targeting group or a lipophilic group, the lipophilic group is optionally connected to one of A1 or A2 via a linker, L represents: a carrier spacer or a carrier part linked by a spacer; and the other of T1 and T2 is a hydroxyl protecting group; n is 0.
  • the hydroxyl protecting group is selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and 4,4'-dimethoxytrityl.
  • the compound of formula (IIa) or formula (IIa-1) provided herein has the configuration example shown below:
  • the compounds provided herein have (IIb) or formula (IIb-1) have the configuration examples shown below:
  • the specific configuration compound can be obtained by obtaining the starting material through the chiral control of synthesis by those skilled in the art, or by obtaining the raw material through chiral resolution technology, or can be synthesized from the starting materials with specific configuration commonly used in industrial production, for example: from starting materials such as isosorbide and isomannide with specific configuration.
  • the compound provided herein has the following specific structure:
  • the compounds of the present invention such as formula (I) or its stereoisomers, (IIa), (IIb), (IIa-1), (IIb-1) and the technical features of two or more such compounds can be combined into compounds in any way, and can be applied to oligonucleotides, and further applied to the 5'-end, 3'-end or both ends of the oligonucleotides.
  • the oligonucleotides include but are not limited to single-stranded antisense oligonucleotides (ASOs), miRNAs, and double-stranded ribonucleotides (dsRNAs).
  • they are applied to the 3'-end of the antisense strand in the double-stranded ribonucleotides (dsRNAs) in the oligonucleotides. In some embodiments, they are applied to the 5'-end of the antisense strand in the double-stranded ribonucleotides (dsRNAs) in the oligonucleotides. In some embodiments, they are applied to the 5' and/or 3'-end of the sense strand in the double-stranded ribonucleotides (dsRNAs) in the oligonucleotides.
  • an oligomeric compound comprising at least one 5′-terminal and/or 3′-terminal monomer having formula (III) or a stereoisomer thereof:
  • a 1 and A 2 are independently selected from the group consisting of O, S, SO, SO 2 , NR 2 and CR 3 CR 4 ;
  • R 2 , R 3 and R 4 are independently selected from the group consisting of hydrogen, halogen, sulfonyl, sulfinyl, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 5 -C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;
  • T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, wherein the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (III) or a stereoisomer thereof to the oligomeric compound;
  • R 1 is independently selected from halogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 5 -C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;
  • n is an integer from 0 to 6;
  • the oligomeric compound optionally further comprises a targeting group.
  • a 1 is O.
  • A2 is O.
  • the oligomeric compound provided, A1 and A2 are independently O, and comprises at least one 5′-terminal and/or 3′-terminal monomer having formula (III-1) or its stereoisomers: wherein T 3 , T 4 , R 1 and n are as defined above for the oligomeric compound.
  • A1 and A2 are each independently S.
  • a 1 and A 2 are independently NR 2 ;
  • R 2 is selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 5 -C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocycle.
  • R 2 is hydrogen or CH 3 .
  • the oligomeric compound provided, A 1 is different from A 2 .
  • the oligomeric compound provided, A 1 is O, A 2 is S.
  • the oligomeric compound provided, A 1 is O, A 2 is NR 2 , which comprises at least one 5′-terminal and/or 3′-terminal monomer having a 5′-terminal and/or 3′-terminal monomer represented by formula (III-2) or a stereoisomer thereof: wherein T 3 , T 4 , R 1 , R 2 , and n are as defined above for the oligomeric compound; in some preferred embodiments, R 2 is hydrogen or CH 3 .
  • a 1 is S
  • a 2 is NR 2 .
  • internucleoside linkage or “internucleoside linking group” is intended to include various types of internucleoside linking groups known in the art, and in such embodiments any internucleoside linkage may be used to link nucleosides or analogs together.
  • 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, phosphonamidate, phosphonate, phosphinate, thiophosphoramidate, and phosphoramidate; as well as phosphorus-free internucleoside linking groups such as thiodiester (—OC(O)—S—), thionocarbamate (—OC(O)(NH)—S—), siloxane (—O—Si(H) 2 —O—), N,N′-dimethylhydrazine (—CH 2 —N(CH 3 )—N(CH 3 )—), formacetyl, and methyleneimino (—CH 2 —N(CH 3 )—O—CH 2 —).
  • Internucleoside linkages also include neutral nonionic internucleoside linkages, as the term “neutral internucleoside linkage” as used herein is intended to include nonionic internucleoside linkages.
  • Further neutral internucleoside linkages include nonionic linkages including siloxane (dialkylsiloxane), carboxylate, carboxamide, sulfide, sulfonate and amide (see, e.g., "Carbohydrate Modifications in Antisense Research”; YS Shanghvi and PD Cook, eds., ACS Symposium Series 580; Chapters 3 and 4, pp. 40-65).
  • Further neutral internucleoside linkages include nonionic linkages containing mixed N, O, S and CH2 components, wherein the phosphorus atom is not always present.
  • the alkylphosphonate linking group is selected from C1 - C6 alkylphosphonate linking groups.
  • the phosphorus-containing internucleoside linking group has a structural fragment as shown below:
  • X represents H, substituted or unsubstituted C 1 -C 6 alkyl, OR 8 , SR 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, sulfonyl, They independently represent the part connected to one of A1 or A2 herein, and the part connected to the connected nucleotide.
  • internucleoside bonds with chiral atoms can be prepared as racemic mixtures or separate enantiomers.
  • Representative chiral bonds include, but are not limited to, alkyl phosphonates and thiophosphates. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside bonds are well known to those skilled in the art.
  • one of T3 and T4 is H, a protecting group, a lipophilic group, and an optional covalent bond
  • the lipophilic group is optionally connected to one of A1 or A2 via a linker
  • the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (III) or its stereoisomer to the 5′-terminal and/or 3′-terminal group of the oligomeric compound
  • the internucleoside linking group is independently an alkyl phosphonate linking group, a phosphodiester internucleoside linking group, or a thiophosphate internucleoside linking group.
  • the oligomeric compound comprising at least one monomer represented by formula (III) or its stereoisomer is shown as follows:
  • Olig represents the oligonucleotide part that connects the monomer of formula (III) or its stereoisomer to the 5′-end and/or 3′-end of the oligomeric compound, respectively.
  • a 1 , A 2 , R 1 and n are as described above, T 3 is H, a protecting group, a lipophilic group, and an optional covalent bond, wherein the lipophilic group is optionally connected to A 1 via a linker, and the oligomeric compound optionally further comprises a targeting group.
  • the protecting group of one of T 3 and T 4 is different according to A 1 or A 2 , that is, when A 1 or A 2 is oxygen, the protecting group of one of T 1 and T 2 is a hydroxyl protecting group; that is, when A 1 or A 2 is sulfur, sulfoxide (SO), or sulfone (SO 2 ), the protecting group of one of T 1 and T 2 is a thiol protecting group; that is, when A 1 or A 2 is nitrogen, the protecting group of one of T 1 and T 2 is an amino protecting group.
  • a 1 or A 2 that is, when A 1 or A 2 is oxygen, the protecting group of one of T 1 and T 2 is a hydroxyl protecting group; that is, when A 1 or A 2 is sulfur, sulfoxide (SO), or sulfone (SO 2 ), the protecting group of one of T 1 and T 2 is a thiol protecting group; that is, when A 1 or A 2 is nitrogen, the protecting group of one of T 1 and T 2 is an amino protecting group
  • one of the protecting groups of T 3 and T 4 is a hydroxy protecting group; in one embodiment, in the oligomeric compound provided, the hydroxy protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, Triphenylsilyl, triisopropylsilyl,
  • one of the protecting groups of T 3 and T 4 is a thiol protecting group; in one embodiment, in the oligomeric compound provided, the thiol protecting groups are each independently selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl Alkyl, tert-butyldiphenylsilyl, triphenylsilyl, triiso
  • one of the protecting groups of T 3 and T 4 is an amino protecting group; in one embodiment, in the oligomeric compound provided, the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrophenyl, acetyl, 2-nitrobenzenesulfonyl, phthalimido and dithiosuccinyl.
  • the amino protecting groups are each independently selected from 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-but
  • oligomeric compounds are provided, one of T3 and T4 is a lipophilic group, wherein the lipophilicity of the lipophilic group can be determined by logKow, and logKow exceeds 0.
  • lipophilic or “lipophilic group” refers broadly to any compound or chemical moiety that has an affinity for lipids, and these coefficients are calculated using first principles or empirical methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41: 1407-21 (2001), which is incorporated herein by reference in its entirety, and its definition has the same scope as the bicyclic abasic compounds described herein.
  • the structure to which the lipophilic group is conjugated allows the lipophilic group (e.g., fatty acid) to be exposed, thereby allowing the lipophilic group to interact with albumin and/or fatty acid receptors or transporters, thereby providing Provide oligonucleotides with long in vivo half-life.
  • the logKow of the group generally having lipophilicity is determined to be greater than 0.
  • the lipophilicity of the lipophilic part measured by the octanol-water partition coefficient logKow, can be greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5 or greater than 10 logKow.
  • the lipophilicity of the molecule can vary relative to the functional groups it carries; for example, the addition of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide and alkyne functional groups at the end of the lipophilic part can increase or decrease the partition coefficient (e.g., logKow) value of the lipophilic part; and the lipophilic group can also be further attached to the monomers of the oligomeric compounds shown in this article through these functional groups.
  • the lipophilic group has the same definition as the bicyclic abasic compound described above. For example, but not limited to the following examples:
  • the oligomeric compounds provided have lipophilic groups containing saturated or unsaturated C 4 -C 30 hydrocarbon chains, aliphatic rings, aromatics, fatty acid groups or fatty acid-derived groups, steroid-derived groups and any fat-soluble vitamin groups.
  • the lipophilic group is a saturated or unsaturated C 10- C 25 hydrocarbon chain (e.g., C 10- C 25 alkyl or alkenyl). In some embodiments, the lipophilic group is a saturated or unsaturated C 10 -C 18 hydrocarbon chain (e.g., a straight-chain C 10 -C 18 alkyl or alkenyl). 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 chain (e.g., a straight-chain C 16 alkyl or alkenyl); more preferably a straight-chain alkyl.
  • the lipophilic group is a saturated straight-chain C 14 alkyl, C 16 alkyl or C 18 alkyl.
  • the lipophilic group is further selected from the optional functional groups of the group consisting of: hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azido and alkyne. These functional groups can be used to attach lipophilic groups to A1 or A2 .
  • the lipophilic group is selected from the group derived from the fatty acid of dicarboxylic acid.
  • the fatty acid comprises any saturated or unsaturated fatty acid with a hydrocarbon chain of 4 to 28 carbon atoms, and may contain one or two carboxyl groups.
  • the lipophilic group is a group derived from a saturated fatty acid with a hydrocarbon chain of 8 to 24 carbon atoms.
  • the lipophilic group is a group derived from a saturated fatty acid, wherein the fatty acid is selected from the group consisting of: butyric acid (CH 3 (CH 2 ) 2 COOH), valeric acid, caproic acid, heptanoic acid, octanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, heptacosanoic acid and octacosanoic acid
  • the lipophilic group is a group derived from an unsaturated fatty acid, and the unsaturated fatty acid is one of the ones presented in Table 1; the fatty acid is preferably selected from the group consisting of: myristic acid, palmitoleic acid, hexadecenoic acid, oleic acid, oleic acid, trans-oleic acid, linoleic acid, trans-linoleic acid, ⁇ -linolenic acid, arachidonic acid and erucic acid.
  • 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.
  • bile acids e.g., cholic acid, deoxycholic acid, and dehydrocholic acid
  • cortisone e.g., cholic acid, deoxycholic acid, and dehydrocholic acid
  • cortisone e.g., cholic acid, deoxycholic acid, and dehydrocholic acid
  • cortisone e.g., cholic acid, deoxycholic acid, and dehydrocholic acid
  • cortisone e.g.,
  • the lipophilic group is selected from cholesterol, vitamin E (tocopherol) or bile acid.
  • the lipophilic group is selected from the unsaturated fats docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), the saturated fatty acid docosanoic acid (DCA), sterol cholesterol (bile), tocopherol succinate (TS) and lithocholic acid (LA), retinoic acid (vitamin A acid).
  • DHA unsaturated fats docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • DCA saturated fatty acid docosanoic acid
  • sterol cholesterol sterol cholesterol
  • TS tocopherol succinate
  • LA lithocholic acid
  • vitamin A acid retinoic acid
  • the lipophilic group is an aromatic moiety.
  • the lipophilic group of the present invention can be directly connected to A1 or A2 via a covalent bond.
  • the combination of A1 and T3 and/or the combination of A2 and T4 include but are not limited to the following exemplary structures: in represents a single bond or a double bond, p or q independently represents an integer of 5-25, and the sum of p and q does not exceed 25.
  • the combination of A1 and T3 and/or the combination of A2 and T4 include but are not limited to the following examples:
  • any lipophilic group, linker and linkage to the bicyclic abasic compound defined in the above-described bicyclic abasic compound, and the properties or structures described therein are also applicable to the definition scope of the lipophilic group, linker and linkage method thereof in the oligomeric compound in this article.
  • the oligomeric compound provided can also be covalently linked to A1 or A2 via a linker, and the linker can be a combination of one or more.
  • the linker through which the lipophilic group of the provided oligomeric compound passes has the same lipophilic group linker as the bicyclic abasic nucleic acid analog compound used above, and the properties or structures described therein are also applicable to the lipophilic group linker of the oligomeric compound herein.
  • T3 and T4 of the present invention is a lipophilic group, which further comprises a linker and is covalently linked to A1 or A2 through one or more linkers.
  • the linker is selected from the group consisting of: an amide bond, phosphatidylcholine, a hydrocarbon linker or a polyethylene glycol (PEG) linker, an amino-alkyl-alcohol, hydroxyproline, hydroxyproline alcohol, an amino-alkyl-thiophosphate linker, an amino-PEG-thiophosphate linker, an ⁇ -carboxylate-amino-alkylthiophosphate linker, and an ⁇ -carboxylate-amino-PEG-thiophosphate linker.
  • the linker contains an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide bond, a product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition) or a carbamate.
  • a click reaction e.g., a triazole from an azide-alkyne cycloaddition
  • the linker comprises a cleavable group.
  • the linker includes a cleavable group, which may be pH-sensitive, a redox-cleavable linker, an esterase-cleavable linker (e.g., an ester group), a phosphatase-cleavable linker (e.g., a phosphate), or a peptidase-cleavable linker (e.g., a peptide bond), a bio-cleavable linker, a hydroxyproline, a hydroxyproline, an amino-alkyl-alcohol-linked linker.
  • a cleavable group which may be pH-sensitive, a redox-cleavable linker, an esterase-cleavable linker (e.g., an ester group), a phosphatase-cleavable linker (e.g., a phosphate), or a peptidase-cleavable link
  • WO2019/217459 entitled “Extrahepatic Delivery”, filed on July 7, 2019, and PCT Publication No. WO2019/215333, entitled “Oligonucleotide Conjugates Including 7′-5′- ⁇ -Anomeric Bicyclic Sugar Nucleosides”, filed on May 10, 2019, and Nucleic Acids Research, 2019, Vol. 47, No. 31082-1096 "Various Lipid Conjugates for Functional Extrahepatic In Vivo Nucleotide Delivery", the contents of which are also incorporated by reference in their entirety into the oligomeric compounds herein.
  • the compounds described herein and applied to the field of oligonucleotides can be applied to extrahepatic delivery systems according to the properties or structures of the lipophilic group, for example but not limited to the CNS field, ophthalmology field, muscle.
  • the delivery of oligonucleotides to the central nervous system (CNS) causes specific problems.
  • One way to deliver oligonucleotides to the CNS is by intrathecal delivery.
  • oligonucleotides also need to be effectively internalized into the target cells of the CNS to achieve the desired therapeutic effect.
  • Previous studies generally use lipophilic groups to conjugate specific positions in oligonucleotides such as single nucleobases, 2-position sugar rings, etc., to help oligonucleotides be internalized into cells of neuronal origin.
  • lipophilic groups are conjugated to monomers herein and applied to oligomeric compounds, which can also be used to start the delivery and distribution of the CNS field.
  • Lipophilic diseases for which the drug has been widely used in the CNS field include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar diseases, prion diseases, and Lafora's disease.
  • ALS amyotrophic lateral sclerosis
  • frontotemporal dementia Huntington's disease
  • Parkinson's disease spinocerebellar diseases
  • prion diseases and Lafora's disease.
  • the lipid groups are used in the application of oligonucleotides in the CNS field for corresponding targets: they target APP for early-onset familial Alzheimer's disease, ATXN2 for spinocerebellar ataxia 2 and ALS, and C9orf72 for amyotrophic lateral sclerosis and frontotemporal dementia, TARDBP for ALS, MAPT (Tau) for frontotemporal dementia and HTT for Huntington's disease, SNCA for Parkinson's disease, FUS for ALS, ATXN3 for spinocerebellar ataxia 3, ATXN1 for SCA1, genes for SCA7 and SCA8, ATN1 for DRPLA, MeCP2 for XLMR, prion diseases, recessive CNS disorders: PRNP for Lafora disease, DMPK for DM1 (CNS and skeletal muscle), and TTR for hATTR (CNS, eyes and systemic).
  • Lipid groups are used in oligonucleotides in the field of ophthalmology for corresponding diseases: These diseases include but are not limited to age-related macular degeneration (AMD) (dry and wet), birdshot chorioretinopathy, dominant pigmentary vision, retinitis, Fuch’s dystrophy, hereditary and sporadic glaucoma, and Stargardt’s disease.
  • AMD age-related macular degeneration
  • VEGF targeting wet (or exudative) AMD C3 and CFB targeting dry (or non-exudative) AMD
  • MYOC ROCK2
  • ADRB2 CA2 targeting glaucoma
  • CRYGC cataracts
  • PPP3C dry eye syndrome
  • an oligomeric compound wherein one of T3 and T4 is hydrogen and the other of T3 and T4 is an internucleoside linking group that links the monomer of Formula III or a stereoisomer thereof to the 5′-terminal and/or 3′-terminal group of the oligomeric compound.
  • the oligomeric compound further comprises a targeting group.
  • one of T 3 and T 4 is a covalent bond, and the oligomeric compound is connected to the targeting group in the form of a covalent bond through one of T 3 and T 4 in formula (III) or its stereoisomers, represents a monomeric portion of formula (III); in other embodiments, an oligomeric compound, which is connected to the 5'-terminal and/or 3'-terminal nucleotides at the other end of the oligomeric compound containing formula (III) or its stereoisomers at one end,
  • the targeting group may be a ligand conventionally used in the field of siRNA administration, as used herein, the oligomeric compound targeting group provided has a bicyclic abasic nucleic acid analog compound targeting group as described above, and the properties or structures described therein are also applicable to the targeting group of the oligomeric compound herein.
  • the targeting group may change the distribution, targeting or life span, endosomal dissolution properties, improved transport, hybridization and specific properties of the siRNA agent into which it is incorporated, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated into the oligomeric compound herein by reference.
  • the targeting group can be selected from one or more of the ligands formed by the following targeting molecules or their derivatives; such as polymers, such as polyethylene glycol; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); receptor ligands expressed by hepatic parenchymal cells, such as asialoglycoproteins, asialosugar residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin and other aptamers; antibodies; quantum dots; polypeptides, such as membrane-permeable peptides, or small molecule ligands.
  • targeting molecules or their derivatives such as polymers, such as polyethylene glycol; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetyl
  • each of the targeting groups is selected from a ligand that can bind to a receptor on the surface of a mammalian hepatocyte.
  • each of the targeting groups is independently a ligand that is affinity-containing with an asialoglycoprotein receptor (ASGPR) on the surface of a mammalian hepatocyte.
  • each of the targeting groups is independently a ligand that comprises an asialoglycoprotein or sugar.
  • each of the targeting groups is independently a ligand that comprises an asialoglycoprotein, such as an asialo serum globulin (ASOR) or an asialo globulin (ASF).
  • ASOR asialo serum globulin
  • ASF asialo globulin
  • each of the targeting groups independently comprises a D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-mannofuranose, ⁇ -D-mannofuranose, ⁇ -D-mannopyranose, ⁇ -D-mannopyranose, ⁇ -D-glucose-pyranose, ⁇ -D-glucose-pyranose.
  • At least one or each of the targeting groups is a ligand comprising galactose or N-acetylgalactosamine.
  • the targeting group is linked to one of A1 or A2 and is selected from one of the following compound fragments:
  • the targeting group can be connected to one of A1 or A2 via a suitable conjugated linker, and those skilled in the art can select a suitable conjugated linker according to the specific type of the targeting group.
  • the types of these conjugated linkers, ligands, tethers, branched linkers, and the connection mode of the targeting group to the oligonucleotide can be found in the disclosures of WO2009073809, WO2014179620, WO2014179627, and WO2015006740A2, which are incorporated herein by reference in their entirety.
  • the targeting group can be connected by acid-labile or reducible chemical bonds, which are degradable in the acidic environment of the cell endosome.
  • the oligomeric compound further comprises one or more targeting groups, and is connected to the oligomeric compound via a T3 covalent bond, or is connected to the other end nucleotide of the oligomeric compound.
  • T 3 ' is a covalent bond
  • Olig, A 1 , A 2 , T 3 , R 1 and n are as described above.
  • oligomeric compounds described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations which can be defined in terms of absolute stereochemistry as (R) or (S), ⁇ or ⁇ such as for sugar anomers, or as (D) or (L) such as for amino acids, etc. All such possible isomers as well as their racemic and optionally pure forms are included in the oligomeric compounds provided herein.
  • the oligomeric compound provided comprises at least one 5′-terminal and/or 3′-terminal monomer having a configuration represented by formula (IVa) or formula (IVb):
  • a 1 , A 2 , T 3 , T 4 , R 1 and n are as described above.
  • A1 and A2 are independently selected from: O; one of T3 and T4 is H, a protecting group, a lipophilic group, and optionally a covalent bond, the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (IVa) or formula (IVb) to the oligomeric compound; n is 0; the oligomeric compound optionally further comprises a targeting group.
  • A1 and A2 are independently selected from: O; one of T3 and T4 is H, a hydroxyl protecting group, a lipophilic group, and an optional covalent bond, the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects the formula (IVa) or formula (IVb) to the oligomeric compound; n is 0; the oligomeric compound optionally further comprises a targeting group.
  • the hydroxyl protecting group is selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and 4,4'-dimethoxytrityl
  • the lipophilic group is selected from C14 , C16 or C18 straight-linked alkanes
  • the internucleoside linking group is selected from an alkylphosphonate linking group, a phosphodiester internucleoside linking group or a thiophosphate internucleoside linking group.
  • the oligomeric compound provided comprises at least one 5′-terminal and/or 3′-terminal monomer having a configuration represented by formula (IVa-1) or formula (IVb-1):
  • a 1 , A 2 , T 3 , T 4 , R 1 and n are as described above.
  • A1 and A2 are independently selected from: O; one of T3 and T4 is H, a protecting group, a lipophilic group, and an optional covalent bond, the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (IVa-1) or formula (IVb-1) to the oligomeric compound; n is 0; the oligomeric compound optionally further comprises a targeting group.
  • A1 and A2 are independently selected from: O; one of T3 and T4 is H, a hydroxyl protecting group, a lipophilic group, and an optional covalent bond, the lipophilic group is optionally connected to one of A1 or A2 via a linker, and the other of T3 and T4 is an internucleoside linking group that connects the monomer of formula (IVa-1) or formula (IVb-1) to the oligomeric compound; n is 0; the oligomeric compound optionally further comprises a targeting group.
  • the hydroxyl protecting group is selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl and 4,4'-dimethoxytrityl
  • the lipophilic group is selected from C14 , C16 or C18 straight-linked alkanes
  • the internucleoside linking group is selected from alkylphosphonate linking groups, phosphodiester internucleoside linking groups or thiophosphate internucleoside linking groups.
  • oligomeric compounds provided herein include, but are not limited to, those having the following configurations:
  • Olig represents the oligonucleotide portion that connects the monomer to the 5′-end and/or 3′-end of the oligomeric compound, respectively;
  • a 1 , A 2 , R 1 and n are as described above;
  • T 3 is H, a protecting group, a lipophilic group, and an optional covalent bond; the lipophilic group is optionally connected to one of A 1 or A 2 via a linker; and the oligomeric compound optionally further comprises a targeting group.
  • one of T 3 and T 4 is H, a protecting group, a lipophilic group, and an optional covalent bond, the lipophilic group is optionally connected to one of A 1 or A 2 via a linker, and the other of T 3 and T 4 is a nucleoside linking group connected to the 5′-terminal of the oligomeric compound as a monomer included in the oligomeric compound provided above.
  • an oligomeric compound comprises at least one 5′-terminal monomer as provided herein (such as formula (III) or its stereoisomers, formula (IVa), formula (IVb), (IVa-1) or (IVb-1)).
  • one of T 3 and T 4 is H, a protecting group, a lipophilic group, and an optional covalent bond, the lipophilic group is optionally connected to one of A 1 or A 2 via a linker, and the other of T 3 and T 4 is a nucleoside linking group connected to the 3′-terminal of the oligomeric compound as a monomer included in the oligomeric compound provided above.
  • an oligomeric compound comprises at least one 3′-terminal monomer as provided herein (such as formula (III) or its stereoisomers, formula (IVa), formula (IVb), (Iva-1) or (IVb-1)).
  • one of T 3 and T 4 is H, a protecting group, a lipophilic group, and an optional covalent bond
  • the lipophilic group is optionally connected to one of A 1 or A 2 via a linker
  • the other of T 3 and T 4 independently connects a same or different monomer contained in the oligomeric compound provided above to the internucleoside linking group at the 5′-end and 3′-end of the oligomeric compound.
  • the oligomeric compound comprises at least one same or different monomer having a monomer as provided herein (such as formula (III) or its stereoisomer, formula (IVa), formula (IVb), (IVa-1) or (IVb-1)) at the 5′-end and 3′-end of the oligomeric compound, respectively.
  • the oligomeric compound comprises one identical or different monomer region of formula (III) or its stereoisomer, formula (IVa), formula (IVb), (IVa-1) or (IVb-1) at any terminal of the 5′-terminus and/or the 3′-terminus.
  • the oligomeric compound comprises at least two consecutive identical or different monomer regions of formula (III) or its stereoisomer, formula (IVa), formula (IVb), (IVa-1) or (IVb-1) at any terminal of the 5′-terminus and/or the 3′-terminus of the oligomeric compound.
  • the monomers herein are linked to the 5′-terminus and/or 3′-terminus of the oligomeric compound.
  • A2 and A2 ' represent the same or different monomer variable groups as described above
  • R1 and R1 ' represent the same or different monomer variable groups as described above
  • Olig represents the oligonucleotide part that connects the monomer to the 5'-end and/or 3'-end of the oligomeric compound respectively.
  • n when n is non-0, it means that there are 1-6 independent R1 group substituents on the bicyclic structure. It is understood that these substituents in the bicyclic abasic compounds herein do not include natural nucleobases or modified nucleobases.
  • R1 is independently selected from halogen, substituted or unsubstituted C1 - C6 alkyl, substituted or unsubstituted C2 - C6 alkenyl, substituted or unsubstituted C2 - C6 alkynyl.
  • R1 is independently selected from fluorine, chlorine, bromine, iodine, methyl, ethyl, isopropyl, propyl, 3-propenyl, ethynyl, 3-propynyl.
  • n is 0, indicating that there is no R 1 group substitution on the ring structure.
  • the bicyclic base compounds described in the present invention such as formula (I), (IIa), (IIb), (IIa-1), (IIb-1) and any combination of the features of two or more such compounds can be applied to 5'-terminal and/or 3'-terminal oligonucleotide monomers, therefore, the technical features defined for the bicyclic abasic compounds described herein are also applicable to the oligomeric compounds herein, as long as there is no contradiction.
  • a fragment comprising at least one monomer as shown below is provided:
  • the oligomeric compounds provided herein have the following specific structures:
  • Olig represents an oligonucleotide portion that connects the monomer to the 5′-end and/or 3′-end of the oligomeric compound, respectively.
  • the oligomeric compound is a single-stranded oligonucleotide, and single-stranded oligonucleotides well known in the art include antisense oligonucleotides (also known as ASOs), ribozymes or aptamers.
  • the oligomeric compound is a double-stranded RNAi compound (siRNA), a double-stranded nucleoside nucleic acid (also known as dsRNA) well known in the art, wherein one or both strands are oligomeric compounds as disclosed herein.
  • the nucleobase sequence of the oligomeric compound is complementary to a portion of the target nucleic acid or is completely complementary. 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.
  • double-stranded ribonucleic acid (dsRNA) agents each of which comprises: a sense strand and an antisense strand, wherein the sense strand is fully or partially complementary to the antisense strand, and the antisense strand is complementary to a nucleic acid target gene; at least one of the sense strand and the antisense strand is an oligomeric compound as provided above, at least one of which has a 5′-terminal and/or 3′-terminal monomer comprising formula (III) or its stereoisomers, formula (IVa), formula IV(b), formula (IVa-1) or formula (IVb-1) herein; and wherein the double-stranded ribonucleic acid (dsRNA) agent optionally further comprises an independent targeting group.
  • dsRNA double-stranded ribonucleic acid
  • a double-stranded ribonucleic acid (dsRNA) agent wherein the sense strand is an oligomeric compound provided by a 5′-terminal monomer comprising Formula (III) or a stereoisomer thereof, Formula (IVa), Formula IV (b), Formula (IVa-1), or Formula (IVb-1) herein.
  • a double-stranded ribonucleic acid (dsRNA) agent is provided, wherein the sense strand is an oligomeric compound provided by a 3′-terminal monomer comprising Formula (III) or a stereoisomer thereof, Formula (IVa), Formula IV (b), Formula (IVa-1), or Formula (IVb-1) herein.
  • a double-stranded ribonucleic acid (dsRNA) agent wherein the sense strand is an oligomeric compound provided by a 5′-terminal and 3′-terminal monomer comprising Formula (III) or a stereoisomer thereof, Formula (IVa), Formula IV (b), Formula (IVa-1), or Formula (IVb-1) herein.
  • dsRNA double-stranded ribonucleic acid
  • a double-stranded ribonucleic acid (dsRNA) agent wherein the antisense strand is an oligomeric compound provided by a 5′-terminal and/or 3′-terminal monomer comprising Formula (III) or its stereoisomers, Formula (IVa), Formula IV (b), Formula (IVa-1) or Formula (IVb-1) herein.
  • dsRNA double-stranded ribonucleic acid
  • a double-stranded ribonucleic acid (dsRNA) agent wherein the antisense strand is an oligomeric compound provided by a 3′-terminal monomer comprising Formula (III) or its stereoisomers, Formula (IVa), Formula IV (b), Formula (IVa-1) or Formula (IVb-1) herein.
  • a double-stranded ribonucleic acid (dsRNA) agent is provided, wherein the antisense strand is an oligomeric compound provided by a 5′-terminal monomer comprising Formula (III) or its stereoisomers, Formula (IVa), Formula IV (b), Formula (IVa-1) or Formula (IVb-1) herein.
  • a double-stranded ribonucleic acid (dsRNA) agent is provided, optionally further comprising an independent targeting group, and the independent targeting group can be understood as, in addition to the targeting group present in the oligomeric compound monomer herein,
  • the monomer part of formula (III) may also have a targeting group at other positions in the double-stranded ribonucleic acid (dsRNA) agent.
  • one or more targeting groups or linking groups are also included on the 5′-terminal and/or 3′-terminal nucleotides.
  • these targeting groups can be conjugated to the 5′-terminal and/or 3′-terminal nucleotides of any chain in the double-stranded ribonucleic acid (dsRNA) agent.
  • the targeting group may be a ligand conventionally used in the field of siRNA administration.
  • these targeting groups may be independently selected from the same targeting groups as the oligomeric compounds described above and as the bicyclic abasic nucleic acid analog compounds; the targeting groups of the double-stranded ribonucleic acid (dsRNA) reagents provided, the properties or structures described are also applicable to the targeting groups of the oligomeric compounds and as the bicyclic abasic nucleic acid analog compounds herein.
  • the targeting group in the double-stranded ribonucleic acid (dsRNA) reagent provided, can be selected from one or more ligands formed by the following targeting molecules or their derivatives: polymers, carbohydrates, receptor ligands expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands.
  • the targeting groups is selected from a ligand that can bind to a receptor on the surface of a mammalian hepatocyte.
  • each of the targeting groups independently comprises a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes.
  • ASGPR asialoglycoprotein receptor
  • each of the targeting groups independently comprises an asialoglycoprotein or sugar.
  • the targeting group can be connected to the 5′-terminal and/or 3′-terminal nucleotides via a suitable linker, and those skilled in the art can select a suitable conjugated linker according to the specific type of the targeting group.
  • the dsRNA agent wherein the one or more targeting groups or linking groups are conjugated to the sense strand.
  • the targeting ligand can alter the distribution, targeting or lifetime, endosomal dissolution properties, improved transport, hybridization and specificity properties of the dsRNA agent into which it is incorporated, such as, but not limited to, lectins, glycoproteins, lipids or proteins, thyroid stimulating hormone, melanocyte stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent trehalose, glycosylated polyamino acids , polygalactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide,
  • the targeting group is selected from one of the following compounds after being linked:
  • the targeting group herein can be conjugated with the monomer compound or oligonucleotide herein through a reactive functional group to form an internucleoside linking group.
  • GLO generally indicates an oxophosphate internucleoside linking group
  • GLS generally indicates a thiophosphate internucleoside linking group.
  • the present invention provides oligomeric compounds comprising any multiple length ranges.
  • any one of the single strand, sense strand and/or antisense strand comprises 8-40 nucleotides in length, respectively.
  • the oligomeric compound nucleotide length is selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40.
  • an oligonucleotide comprising 8-30 nucleosides does not include an oligonucleotide with 31 nucleosides, but unless otherwise specified, such an oligonucleotide may further comprise, for example, one or more conjugates, terminal groups or other substituents, and monomers herein are generally considered to be terminal groups that the oligonucleotide further comprises.
  • terminal groups include but are not limited to terminal group nucleosides. In such embodiments, terminal groups are modified differently from the terminal nucleosides of an oligonucleotide so that such terminal groups are distinguished from the nucleosides of an oligonucleotide.
  • the present invention provides oligomeric compounds in addition to providing monomeric compounds, and the remainder has one or more modifications.
  • modifications include but are not limited to sugar modifications or substitution with sugar surrogates, base modifications, internucleoside linkage modifications, and motifs.
  • modifications are described herein and many of them are known to those skilled in the art. All such modifications are suitable for oligomeric compounds and double-stranded RNA disclosed herein.
  • a method for inhibiting gene expression comprising: contacting a cell with an oligomeric compound or a double-stranded ribonucleic acid (dsRNA) reagent provided above, wherein the sense strand and the antisense strand in the oligomeric compound and the double-stranded ribonucleic acid (dsRNA) reagent both contain 8-40 nucleotides in length, and the antisense strand in the oligomeric compound or the double-stranded ribonucleic acid (dsRNA) reagent is complementary to the target RNA.
  • the cell is in an animal. In certain embodiments, the cell is in a human.
  • the target RNA is selected from mRNA, pre-mRNA, and micro RNA. In certain embodiments, the target RNA is mRNA. In certain embodiments, the target RNA is human mRNA. In certain embodiments, the target RNA is cleaved, thereby inhibiting its function. In certain embodiments, the method further comprises detecting the level of the target RNA. In certain embodiments, a method for inhibiting gene expression is provided, the method comprising contacting one or more cells or tissues with an oligomeric compound or double-stranded ribonucleic acid (dsRNA) comprising a terminal monomer having formula (III) or its stereoisomers, formula (IVa), formula IV(b), formula (IVa-1) or formula (IVb-1).
  • dsRNA oligomeric compound or double-stranded ribonucleic acid
  • oligomeric compound in the context of the present invention refers to a polymer having at least one region that can hybridize with a nucleic acid molecule.
  • oligomeric compound includes oligonucleotides, oligonucleotide analogs and oligonucleosides and nucleotide mimics and/or mixed polymers comprising nucleic acids and non-nucleic acid components.
  • oligomeric compound also includes polymers comprising connected monomer subunits, wherein the monomer subunits include nucleosides, modified nucleosides, nucleoside analogs, nucleoside mimics and non-nucleic acid components such as coupling groups.
  • a mixture of monomer subunits provides the following oligomeric compounds, which have enhanced properties for applications such as treatment and diagnosis.
  • the bicyclic abasic compounds provided by the present invention are classified as non-nucleic acid components due to the absence of nuclear bases.
  • the monomer subunits can be connected by naturally occurring phosphodiester nucleoside inter-bonding or by any multiple nucleoside inter-bonding disclosed herein, and the internucleoside bonding is such as but not limited to thiophosphorothioate nucleoside inter-bonding or its mixture.
  • Oligonucleotide refers to a polymeric form of nucleotides ranging from 2 to 2500 nucleotides, and the oligonucleotide can be single-stranded or double-stranded.
  • oligonucleotide includes oligonucleotides composed of naturally occurring nucleoside bases, sugars and covalent internucleoside linkages and oligonucleotides having one or more non-naturally occurring parts.
  • oligonucleotide is generally more ideal than naturally occurring forms due to their ideal properties, for example, improved cellular uptake, enhanced affinity for nucleic acid targets, and improved stability in the presence of nucleases, such as modifications to bases or sugar moieties.
  • the oligonucleotide has 500-1500 nucleotides, typically, for example, wherein the oligonucleotide is used in gene therapy.
  • the oligonucleotide is single-stranded or double-stranded and has 7-100 nucleotides.
  • the oligonucleotide is single-stranded or double-stranded and has 15-100 nucleotides.
  • the oligonucleotide is single-stranded or double-stranded, with 15-50 nucleotides, typically such as wherein the oligonucleotide is a nucleic acid inhibitor molecule. In another embodiment, the oligonucleotide is single-stranded or double-stranded, with 25-40 nucleotides, typically such as wherein the oligonucleotide is a nucleic acid inhibitor molecule.
  • the oligonucleotide is single-stranded or double-stranded and has 19-40 or 19-25 nucleotides, typically such as wherein the oligonucleotide is a double-stranded nucleic acid inhibitor molecule and forms a duplex of at least 18-25 base pairs.
  • the oligonucleotide is single-stranded and has 15-25 nucleotides, typically such as wherein the oligonucleotide nucleotide is a single-stranded or double-stranded RNAi inhibitor molecule.
  • the oligonucleotide has one or more phosphorus-containing internucleotide linking groups, as described herein.
  • the internucleotide linking group is a phosphorus-free bond, as described herein.
  • the oligonucleotides actually listed in the oligomeric compound herein further include terminal monomers as described herein.
  • oligomeric compounds comprise a backbone of linked monomeric subunits, the linkages of which, the sugar moieties or surrogates, and the heterocyclic base moieties can be independently modified.
  • the linked sugar units which may or may not contain heterocyclic bases, can be replaced with mimetics such as peptide nucleic acid monomers.
  • mimetics such as peptide nucleic acid monomers.
  • Oligomeric compounds are usually prepared with conventional linearity, are also connected or otherwise prepared into a bad shape, and may also contain branches. Oligomeric compounds can form double-stranded constructs, for example, double strands that hybridize to form double-stranded compositions. The double-stranded compositions can be connected or separated and may contain overhangs at the ends.
  • nucleoside in the context of the present invention refers to a base sugar combination.
  • the base portion of a nucleoside is called a nucleobase, typically a heterocyclic base portion.
  • the two most common categories of the heterocyclic base are purine and pyrimidine, for example: suitable natural nucleobases include purine bases 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 the present invention has a bicyclic abasic nucleic acid analog in a 5'-terminal and/or 3'-terminal monomer, and its bicyclic substituent R1 does not exist in the base of such a nucleoside.
  • nucleotide in the context of the present invention refers to a nucleoside further comprising a phosphate group covalently attached to a nucleoside sugar moiety.
  • the phosphate group can be attached to 2', 3' or 5' hydroxyl moieties of sugar.
  • the phosphate group covalently attaches nucleosides adjacent to each other to form a linear polymeric compound.
  • the corresponding ends of the linear polymeric structure can be connected to obtain a cyclic structure by hybridization or by forming a covalent bond, but a single open linear structure is usually desired.
  • the phosphate group usually forms an internucleoside of an oligonucleotide that connects the common internucleoside of RNA and DNA to be connected as 3' to 5' phosphodiester.
  • terminal group refers to one or more atoms or groups connected to one or both of the 3' end or 5' end of an oligonucleotide.
  • the terminal group is a conjugated group.
  • the terminal group comprises one or more additional nucleosides, commonly referred to as 3' end or 5' end-terminal nucleotides.
  • 5'-terminal nucleotide or 3'-terminal nucleotide refers to a nucleotide located at the 5'-end or 3'-end of an oligonucleotide or oligomeric compound.
  • phosphoramidite refers to a nitrogen-containing trivalent phosphorus derivative. Examples of suitable phosphoramidites are described herein.
  • siRNA refers to short interfering RNA or silencing RNA.
  • siRNA is a class of double-stranded RNA molecules that can be 20 to 25 (or shorter) base pairs in length, similar to microRNAs (miRNAs) that function in the RNA interference (RNAi) pathway.
  • miRNAs microRNAs
  • RNAi RNA interference pathway.
  • siRNA interferes with the expression of specific genes with nucleotide sequences complementary to the siRNA by degrading the mRNA after transcription, thereby preventing translation.
  • siRNA silences gene expression in cells by inducing the RNA-induced silencing complex (RISC) to cleave messenger RNA (mRNA).
  • RISC RNA-induced silencing complex
  • double-stranded refers to two separate oligomeric compounds that hybridize to each other.
  • double-stranded compounds may have one or more non-hybridizing nucleosides (overhangs) and/or one or more internal non-hybridizing nucleosides (mismatches) at one or both ends of one or both strands, provided that there is sufficient complementarity to maintain hybridization under physiologically relevant conditions.
  • self-complementary or “hairpin” refers to a single oligomeric compound comprising a duplex region formed by self-hybridization of the oligomeric compound.
  • single-stranded refers to an oligomeric compound that is not hybridized to its complementary sequence and does not have sufficient self-complementarity to form a hairpin structure under physiologically relevant conditions.
  • a single-stranded compound may be capable of binding to its complementary sequence thereby becoming a double-stranded or partially double-stranded compound.
  • Therapeutic formulations of dsRNA agents or target gene antisense polynucleotide agents can be prepared for storage by mixing the molecule or compound with the desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer [Remington's Pharmaceutical Sciences 21st edition, (2006)] in the form of a lyophilized formulation or an aqueous solution.
  • Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the doses and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, his
  • the tissue to which the compound is administered is a tissue in which a target gene-related disease or condition exists or may occur, and its non-limiting example is the liver or kidney.
  • Direct tissue administration can be achieved by direct injection or other means.
  • Many compounds delivered orally naturally enter and pass through the liver and kidney, and some embodiments of the method of the present invention include oral administration of one or more target gene dsRNA reagents to the subject.
  • dsRNA reagents or target gene antisense polynucleotide agents alone or in combination with other therapeutic agents, can be administered once, or they can be administered multiple times. If multiple administrations are given, target gene dsRNA reagents or target gene antisense polynucleotide agents can be administered by different routes.
  • the first (or first few) administration can be performed subcutaneously, and one or more additional administrations can be oral and/or systemic administration.
  • the target gene dsRNA agent or the target gene antisense polynucleotide agent can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.
  • injectable formulations can be present in unit dosage form, such as ampoules or multi-dose containers, with or without added preservatives.
  • Target gene dsRNA agent formulations (also referred to as pharmaceutical compositions) can be in the form of suspensions, solutions or emulsions in oily or aqueous carriers, and can contain preparatants, such as suspending agents, stabilizers and/or dispersants.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcohol/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral carriers include sodium chloride solution, Ringer's dextrose solution, glucose and sodium chloride solution, lactated Ringer's solution or fixed oil.
  • Intravenous excipients include fluid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose solution), etc.
  • Preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents and inert gases, etc.
  • Other forms of administration such as intravenous administration, will result in lower dosages. If the subject is not sufficiently responsive at the initial dose, a higher dose (or a different, more localized delivery route effectively increases the dose) can be used within the range allowed by patient tolerance. Multiple doses per day may be used as needed to achieve appropriate systemic or local levels of one or more target gene dsRNA agents or target gene antisense polynucleotide agents, and to achieve appropriate reduction in target gene activity.
  • the methods of the invention include the use of a delivery vehicle, such as a biocompatible microparticle, nanoparticle, or implant suitable for implantation into a recipient, such as a subject.
  • a delivery vehicle such as a biocompatible microparticle, nanoparticle, or implant suitable for implantation into a recipient, such as a subject.
  • exemplary biodegradable implants that can be used according to the methods are described in PCT Publication WO 95/24929 (incorporated herein by reference), which describes a biocompatible, biodegradable polymer matrix for containing a biomacromolecule.
  • both non-biodegradable and biodegradable polymer matrices can be used in the methods of the present invention to deliver one or more target gene dsRNA agents or target gene antisense polynucleotide agents to a subject.
  • the matrix can be biodegradable.
  • the matrix polymer can be a natural or synthetic polymer.
  • the polymer can be selected based on the time period of the desired release, usually on the order of a few hours to a year or more. Typically, release within a period of time between a few hours and three to twelve months can be used.
  • the polymer is optionally in the form of a hydrogel, which can absorb up to about 90% of its weight of water, and is also optionally cross-linked with multivalent ions or other polymers.
  • the target gene dsRNA agent or the target gene antisense polynucleotide agent can be delivered by diffusion or by degradation of the polymer matrix using a biodegradable implant in some embodiments of the present invention.
  • exemplary synthetic polymers for such use are well known in the art.
  • biodegradable polymers and non-biodegradable polymers can be used to deliver target gene dsRNA agents or target gene antisense polynucleotide agents.
  • Bioadhesive polymers such as bioerodible hydrogels (H.S.Sawhney, C.P.Pathak and J.A.Hubell in Macromolecules, 1993, 26, 581-587) can also be used to deliver target gene sRNA agents or target gene antisense polynucleotide agents to treat target gene related diseases or conditions.
  • Other suitable delivery systems may include timed release, delayed release or sustained release delivery systems. Such systems can avoid repeated administration of target gene dsRNA agents or target gene antisense polynucleotide agents, thereby improving the convenience of subjects and health care professionals.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. See, e.g., 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.
  • pump-based hardware delivery systems may be used, some of which are also suitable for implantation.
  • long-term sustained release implants can be applicable to the preventive treatment of objects and objects with the risk of recurrent target gene) related diseases or conditions.
  • long-term release refers to implant construction and arrangement to deliver target gene dsRNA reagents or target gene antisense polynucleotide reagents at least up to 10 days, 20 days, 30 days, 60 days, 90 days, six months, one year or longer time treatment levels.
  • Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the above-mentioned release systems.
  • the methods of the present invention include contacting a cell with an effective amount of a dsRNA agent or an antisense polynucleotide agent to reduce gene expression in the contacted cell.
  • Certain embodiments of the methods of the present invention include administering a dsRNA agent or an antisense polynucleotide agent to a subject in an amount that effectively reduces gene expression and treats a relevant disease or condition of the subject.
  • the "effective amount" used is the amount necessary or sufficient to achieve the desired biological effect.
  • the effective amount of a dsRNA agent or an antisense polynucleotide agent for treating a relevant disease or condition can be: (i) the amount required to slow down or stop the progression of the disease or condition; (ii) reverse, reduce or eliminate one or more symptoms of the disease or condition.
  • an effective amount is the amount of a dsRNA agent or an antisense polynucleotide agent that, when applied to a subject in need of treating a relevant disease or condition, results in a therapeutic response for the prevention and/or treatment of the disease or condition.
  • an effective amount is the amount of a therapeutic response that results in the prevention and/or treatment of the disease or condition when the dsRNA agent or antisense polynucleotide agent of the present invention is combined or co-administered with another therapeutic treatment for a relevant disease or condition.
  • the biological effect of treating a subject with a dsRNA agent or antisense polynucleotide agent of the invention can be an improvement and/or complete elimination of symptoms caused by a related disease or condition.
  • the biological effect is the complete elimination of a related disease or condition, as demonstrated, for example, by a diagnostic test indicating that the subject does not have a related disease or condition.
  • an effective amount is an amount that results in a desired response, such as a reduction in the amount of a related disease or condition in cells, tissues, and/or subjects with a disease or condition.
  • Some embodiments of the invention include methods for determining the efficacy of a target gene dsRNA agent or a target gene antisense polynucleotide agent of the invention administered to a subject to treat a target gene-related disease or condition, which is performed by evaluating and/or monitoring one or more "physiological characteristics" of a target gene-related disease or condition in a subject.
  • physiological characteristics of a target gene-related disease or condition are that many patients
  • gene silencing can be performed constitutively or by genome engineering in any cell expressing and determined by any suitable assay.
  • target gene expression is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.
  • target gene expression is reduced by 5% to 10%, 5% to 25%, 10% to 50%, 10% to 75%, 25% to 75%, 25% to 100% or 50% to 100%.
  • dsRNA agents and antisense polynucleotide agents are delivered in a pharmaceutical composition with a dosage sufficient for target gene expression.
  • the dosage of dsRNA agents or antisense polynucleotide agents is 0.01 to 200.0 mg per kilogram of recipient body weight per day, generally 1 to 50 mg/kg body weight, 5 to 40 mg/kg body weight, 10 to 30 mg/kg body weight, 1 to 20 mg/kg body weight, 1 to 10 mg/kg body weight, 4 to 15 mg/kg body weight, including end values.
  • each single administration of a dsRNA agent or an antisense polynucleotide agent can be in an amount of from about 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg , 3.4mg/kg, 3.5mg/kg, 3.6mg/kg, 3.7mg/kg, 3.8mg/kg, 3.9
  • dsRNA reagent of the present invention Various factors can be considered when determining the delivery dosage and time of dsRNA reagent of the present invention.
  • the absolute amount of the dsRNA reagent or antisense polynucleotide agent delivered will depend on various factors, including co-treatment, dosage number and individual object parameters, including age, physical condition, size and body weight. These are factors well known to those of ordinary skill in the art, and can be solved by routine experiments.
  • a maximum dose can be used, i.e., the highest safe dose according to reasonable medical judgment.
  • the methods of the present invention may include administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of an agent or antisense polynucleotide agent to a subject.
  • a dose of a drug compound may be administered to a subject at least daily, every other day, weekly, every other week, monthly, etc., and may be administered once a day or more than once a day, for example, 2, 3, 4, 5 or more times within a 24-hour cycle.
  • the pharmaceutical composition of the present invention may be administered once a day; or a dsRNA agent or antisense polynucleotide agent may be administered in two, three or more sub-doses at appropriate intervals throughout the day, or even delivered using continuous infusion or by a controlled release formulation.
  • the pharmaceutical composition of the present invention is administered to a subject once a day or more, once a week or more, once a month or more, or once a year or more.
  • Certain embodiments of the present invention include the use of a pharmaceutical composition containing a dsRNA agent or an antisense polynucleotide agent and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprising a dsRNA agent or an antisense polynucleotide agent can be used in the method of the present invention to reduce gene expression and activity in cells, and can be used to treat related diseases or conditions.
  • Such pharmaceutical compositions can be formulated based on the mode of delivery.
  • preparations for delivery modes are: compositions formulated for subcutaneous delivery, compositions formulated for systemic administration by parenteral delivery, compositions formulated for intravenous (IV) delivery, compositions formulated for intrathecal delivery, compositions formulated for direct delivery to the brain, etc.
  • the pharmaceutical composition of the present invention can be administered in one or more ways to deliver dsRNA agents or antisense polynucleotide agents to cells, for example: surface (e.g., by transdermal patch); lungs, for example, by inhalation or blowing into powder or aerosol, including by a nebulizer; intra-airway, intranasal, epidermal and transdermal, oral or parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, such as by implantation; or intracranial, such as by intraparenchymal; intrathecal or intraventricular administration.
  • dsRNA agents or antisense polynucleotide agents can also be delivered directly to target tissues, such as directly to the liver, directly to the kidneys, etc. It is understood that "delivering dsRNA agents” or “antisense polynucleotide agents” to cells includes delivering dsRNA agents or antisense polynucleotide agents, expressing dsRNA agents directly in cells, and expressing dsRNA agents from encoding vectors delivered to cells, or any suitable manner that causes dsRNA or antisense polynucleotide agents to appear in cells.
  • the preparation and use of formulations and means for delivering inhibitory RNA are well known in the art and commonly used.
  • the composition also includes one or more other therapeutic agents.
  • the composition of the present invention may include one or more dsRNA agents and optional one or more pharmaceutically acceptable carriers, delivery agents, targeting agents, detectable labels, etc., and the non-limiting example of the available targeting agent according to some embodiments of the inventive method is to guide the dsRNA agent of the present invention to and/or enter the agent of the cell to be treated.
  • the selection of the targeting agent will depend on the following elements: the nature of the relevant disease or illness, and the target cell type. In a non-limiting example, in some embodiments of the present invention, it may be necessary to target the dsRNA agent to and/or enter the hepatocyte.
  • the therapeutic agent includes only a dsRNA agent with a delivery agent, such as a delivery agent including N-acetylgalactosamine (GalNAc), without any additional connecting elements.
  • a delivery agent such as a delivery agent including N-acetylgalactosamine (GalNAc)
  • the dsRNA agent can be connected to the delivery compound including GalNAc, and is included in the composition containing a pharmaceutically acceptable carrier, and is administered to cells or objects without any detectable label or targeting agent connected to the dsRNA agent, etc.
  • Labeling agents can be used to determine the position of dsRNA agents in cells and tissues in certain methods of the present invention, and can be used to determine the cell, tissue or organ position of the therapeutic composition containing dsRNA agents that has been administered in the methods of the present invention.
  • the means of attaching and using labeling agents such as enzyme labels, dyes, radioactive labels, etc. are well known in the art. It should be understood that in some embodiments of the compositions and methods of the present invention, the labeling agent is connected to one or both of the sense polynucleotides and antisense polynucleotides contained in the dsRNA agent.
  • the composition is packaged in a medicine box, a container, a wrapper, a dispenser, a prefilled syringe or a vial.
  • a medicine box containing one or more target gene dsRNA reagents and/or target gene antisense polynucleotide reagents and instructions for use thereof in the method of the present invention is also within the scope of the present invention.
  • the medicine box of the present invention may contain one or more of the target gene dsRNA reagents, target gene sense polynucleotides and target gene antisense polynucleotide reagents that can be used to treat target gene related diseases or conditions.
  • a medicine box containing one or more target gene dsRNA reagents, target gene sense polynucleotides and target gene antisense polynucleotide reagents can be prepared for the treatment method of the present invention.
  • the components of the medicine box of the present invention can be packaged in aqueous medium or lyophilized form.
  • the medicine box of the present invention can include a carrier that is separated to contain one or more container devices or a series of container devices (e.g., test tubes, vials, flasks, bottles, syringes, etc.) in a closed manner therein.
  • the first container device or a series of container devices may contain one or more compounds, such as target gene) dsRNA reagents and/or target gene sense or antisense polynucleotide reagents.
  • the second container means or series of container means may contain targeting agents, labeling agents, delivery agents, etc., which may be included as part of the target gene) dsRNA agent and/or target gene antisense polynucleotide administered in embodiments of the treatment methods of the present invention.
  • the kit of the invention may also include instructions.
  • the instructions are generally in written form and will provide guidance for carrying out the treatment embodied by the kit and making decisions based on the treatment.
  • the methods of the present invention can be used in conjunction with cells, tissues, organs and/or objects.
  • the object is a human or vertebrate mammal, including but not limited to dogs, cats, horses, cows, goats, mice, rats and primates, such as monkeys. Therefore, the present invention can be used to treat target gene-related diseases or conditions in humans and non-human subjects.
  • the subject can be a farm animal, a zoo animal, a domesticated animal or a non-domesticated animal, and the methods of the invention can be used in veterinary prevention and treatment programs.
  • the subject is a human and the methods of the invention can be used in human prevention and treatment programs.
  • Non-limiting examples of subjects to which the present invention can be applied are subjects diagnosed with, suspected of having, or at risk of having a disease or condition associated with: higher than desired target gene expression and/or activity, also referred to as "elevated target gene expression levels".
  • higher than desired target gene expression and/or activity also referred to as "elevated target gene expression levels”.
  • diseases and conditions associated with target gene expression and/or activity higher than desired levels are described elsewhere herein.
  • the methods of the present invention can be applied to subjects who have been diagnosed with the disease or condition at the time of treatment, subjects associated with higher than desired target gene expression and/or activity, or subjects who are believed to be at risk of having or developing a disease or condition associated with higher than desired target gene expression and/or activity.
  • a disease or condition associated with higher than desired target gene expression and/or activity levels is an acute disease or condition; in certain aspects of the present invention, a disease or condition associated with higher than desired target gene expression and/or activity levels is a chronic disease or condition.
  • the target gene dsRNA agent of the present invention is administered to treat a disease or disorder caused by or associated with the activation of the target gene, or a disease or disorder whose symptoms or progression respond to the inactivation of the target gene.
  • target gene-related disease includes diseases, disorders or conditions that benefit from reduced expression of the target gene.
  • Cells to which the methods of the present invention can be applied include in vitro, in vivo, and ex vivo cells.
  • Cells can be in an object, in a 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 can be: liver cells, hepatocytes, heart cells, pancreatic cells, cardiovascular cells, kidney cells, or other types of vertebrate cells, including human and non-human mammalian cells.
  • cells to which the methods of the present invention can be applied are healthy normal cells that are not known to be disease cells.
  • control cells are normal cells, but it should be understood that cells with diseases or disorders can also be used as control cells in specific cases, such as when comparing the results of treated cells with diseases or disorders with untreated cells with diseases or disorders.
  • the level of target gene polypeptide activity can be determined and compared with the control level of target gene polypeptide activity.
  • the control can be a predetermined value, which can take a variety of forms. It can be a single cutoff value, such as a median or mean value. It can be established based on a comparison group, such as a group with a normal level of target gene polypeptide and/or target gene polypeptide activity and a group with an increased target gene polypeptide and/or target gene polypeptide activity level.
  • a comparison group can be a group with one or more symptoms or diagnoses of a target gene-related disease or condition and a group without one or more symptoms or diagnoses of a disease or condition; a group of subjects to which the siRNA treatment of the present invention has been applied and a group of subjects to which the siRNA treatment of the present invention has not been applied.
  • the control can be based on a normal individual or a cell that is obviously healthy in an appropriate age group. It should be understood that, in addition to the predetermined value, the control according to the present invention can be a material sample tested in parallel with the experimental material. Examples include samples from a control population or control samples produced by manufacturing for parallel testing with the experimental sample.
  • a control may include cells or objects that have not been contacted or treated with a target gene dsRNA agent of the present invention, in which case the control level of the target gene polypeptide and/or target gene polypeptide activity and the level of the target gene polypeptide and/or target gene polypeptide activity in cells or objects contacted with a target gene dsRNA agent or a target gene antisense polynucleotide agent of the present invention can be compared.
  • control level can be a target gene polypeptide level determined for the object, wherein the target gene polypeptide level determined for the same object at different times is compared with the control level.
  • the level of the target gene is determined in a biological sample obtained from an object that has never received the target gene therapy of the present invention.
  • the biological sample is a serum sample. The target gene polypeptide level measured in the sample obtained from the object can be used as the baseline or control value of the object.
  • one or more additional serum samples can be obtained from the object, and the target gene polypeptide level in the subsequent one or more samples can be compared with the control/baseline level of the object. Such comparisons can be used to assess the onset, progression or regression of target gene (target gene) related diseases or conditions in the object.
  • the level of target gene (target gene) polypeptide in the baseline sample obtained from the object is higher than the level obtained from the same object after the target gene dsRNA agent of the present invention or the target gene (target gene) antisense polynucleotide agent is given to the object, then the regression of the target gene related disease or condition and the efficacy of the target gene dsRNA agent of the present invention used to treat the target gene related disease or condition.
  • one or more of the target gene polypeptides and/or target gene polypeptide activity levels determined for a subject can be used as a control value and used to compare target gene polypeptides and/or target gene activity levels in the same subject later, thereby allowing the assessment of changes in "baseline" target gene polypeptide activity in the subject. Therefore, where the initial level is used as the control level for the subject, the initial target gene polypeptide level and/or the initial target gene polypeptide activity level can be used to show and/or determine that the methods and compounds of the present invention are capable of reducing the level of target gene polypeptides and/or target gene polypeptide activity in the subject in the subject.
  • the target gene dsRNA agents and/or target gene antisense polynucleotide agents of the present invention can be administered to a subject.
  • dsRNAi agents include the efficacy of the administration and treatment of the present invention can be assessed as follows: the level of the target gene polypeptide in a serum sample obtained from the subject after administration and treatment is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the pre-administration level of the target gene) polypeptide in a serum sample obtained from the subject at a previous time point, or compared to the non-contact control level (e.g., the target gene polypeptide level in a control serum sample).
  • the non-contact control level e.g., the target gene polypeptide level in a control serum sample.
  • the level of the target gene polypeptide and the level of target gene polypeptide activity are both related to the level of target gene expression.
  • Certain embodiments of the methods of the present invention include administering the target gene dsRNA and/or target gene antisense agent of the present invention to the subject in an amount effective to inhibit the expression of the target gene, thereby reducing the level of the target gene polypeptide in the subject and reducing the level of the target gene polypeptide activity.
  • contacting a cell with an siRNA agent of the present invention results in 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%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%
  • Some embodiments of the present invention include determining the presence, absence and/or amount (also referred to herein as level) of a target gene polypeptide in one or more biological samples obtained from one or more subjects.
  • the assay can be used to evaluate the efficacy of the therapeutic methods of the present invention.
  • the methods and compositions of the present invention can be used to determine the level of a target gene polypeptide in a biological sample obtained from a subject previously treated with a target gene dsNA reagent and/or a target gene antisense agent of the present invention.
  • the level of the target gene polypeptide in the 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, indicating the efficacy level of the treatment given to the subject.
  • the physiological characteristics of the target gene-related disease or condition determined for the object can be used as a control result, and the determination results of the physiological characteristics of the same object at different times are compared with the control results.
  • the pathological feature hemolysis is measured from an object that has never been treated with the target gene of the present invention, which can be used as a baseline or control value for the object.
  • the target gene dsRNA agent is applied to the object once or more in the treatment method of the present invention, the blood cells are compared with the control/baseline level of the object respectively. Such comparisons can be used to assess the onset, progression or regression of the target gene-related disease or condition in the object.
  • the baseline blood cells obtained from the object are higher than the thrombus measured from the same object after the target gene dsRNA agent or target gene antisense polynucleotide agent of the present invention is applied to the object, indicating the regression of the target-related disease or condition and indicating the efficacy of the target gene dsRNA agent of the present invention for treating the target gene-related disease or condition.
  • Some embodiments of the present invention include determining the presence, absence and/or change of physiological characteristics of a target gene-related disease or condition using methods such as, but not limited to, the following: (1) measuring blood cells of a subject; (2) evaluating physiological characteristics of one or more biological samples obtained from one or more subjects; (3) or performing a physical examination on a subject. This determination can be used to evaluate the efficacy of the treatment methods of the present invention.
  • mismatches are tolerated for efficacy of dsRNA, especially in the terminal regions of the dsRNA.
  • Certain mismatches are better tolerated, for example, mismatches with wobble base pairs G:U and A:C are better tolerated for efficacy (Du et el., A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res. 2005 Mar 21; 33(5): 1671-7. Doi: 10.1093/nar/gki312. Nucleic Acids Res. 2005; 33(11): 3698).
  • the term "complementarity/complementarity" when used to describe the correlation between a first nucleotide sequence (e.g., a target gene dsRNA agent sense strand or a target target gene mRNA) and a second nucleotide sequence (e.g., a target gene dsRNA agent antisense strand or a single-stranded antisense polynucleotide) refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising a second nucleotide sequence [forming hydrogen bonds between base pairs under mammalian physiological conditions (or similar conditions in vitro)] and to form a double helix or double helix structure under certain conditions.
  • Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimetics, as long as at least the above hybridization requirements are achieved. Sequence identity or complementarity is independent of modification.
  • the complementary sequence in the target gene dsRNA as described herein comprises an oligonucleotide or polynucleotide containing a first nucleotide sequence and an oligonucleotide or polynucleotide containing a second nucleotide sequence, and base pairing over the full length of one or two nucleotide sequences.
  • sequences may be referred to herein as "completely complementary" to each other. It should be understood that in embodiments in which two oligonucleotides are designed to form one or more single-stranded overhangs when hybridized, such overhangs are not considered as mismatches determined based on complementarity herein.
  • a target gene dsRNA agent comprises an oligonucleotide having a length of 19 nucleotides and another oligonucleotide having a length of 20 nucleotides, wherein the longer oligonucleotide comprises a sequence of 19 nucleotides that is completely complementary to the shorter oligonucleotide, and for purposes described herein, such a situation may be referred to as "completely complementary". Therefore, as used herein, “completely complementary” refers to that all (100%) bases in a continuous sequence of a first polynucleotide will hybridize with the same number of bases in a continuous sequence of a second polynucleotide.
  • a continuous sequence may comprise all or part of a first or second nucleotide sequence.
  • substantially complementary refers to a hybridization pair of a nucleobase sequence, wherein at least about 85% (but not all) of the bases in the continuous sequence of the first polynucleotide will hybridize with the same number of bases in the continuous sequence of the second polynucleotide.
  • the term "substantially complementary” can be used to refer to a duplex of up to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs (bp) of the first sequence relative to the second sequence, while retaining the ability to hybridize under conditions most relevant to its final application, for example, inhibiting target gene gene expression by the RISC pathway.
  • partially complementary can be used herein to refer to a hybridization pair of a nucleobase sequence, wherein at least 75% (but not all) of the bases in the continuous sequence of the first polynucleotide will hybridize with the same number of bases in the continuous sequence of the second polynucleotide.
  • partial complementarity means that at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bases in the contiguous sequence of the first polynucleotide will hybridize to the same number of bases in the contiguous sequence of the second polynucleotide.
  • complementary when used herein may be used to refer to base matching between the sense and antisense strands of a dsRNA agent, base matching between the antisense strand of a dsRNA agent and a sequence of a target mRNA, or base matching between a single-stranded antisense oligonucleotide and a target mRNA sequence.
  • antisense strand of a dsRNA agent may refer to the same sequence as an "antisense polynucleotide agent”.
  • the RNA of the gene RNAi agent is chemically modified to obtain enhanced stability and/or one or more other beneficial properties.
  • the nucleic acids in certain embodiments of the invention can be synthesized and/or modified by methods known in the art, for example, see “Current protocols in Nucleic Acid Chemistry,” Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y., USA, which is incorporated herein by reference.
  • Modifications that may be present in certain embodiments of the dsRNA agents of the invention include, for example: (a) terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, reverse connection, etc.), 3' terminal modifications (conjugation, DNA nucleotides, reverse connection, etc.); (b) base modifications, such as base substitutions with stable bases, destabilizing bases, or bases that are base-paired with an expanded partner library, deletions of bases (abasic nucleotides), or conjugated bases; (c) sugar modifications (e.g., at the 2' position or the 4' position) or replacement of sugars; and (d) backbone modifications, including modification or replacement of phosphodiester bonds.
  • terminal modifications such as 5' terminal modifications (phosphorylation, conjugation, reverse connection, etc.), 3' terminal modifications (conjugation, DNA nucleotides, reverse connection, etc.
  • base modifications such as base substitutions with stable bases, destabilizing bases, or bases that are base-paired with an expanded partner library, deletions of
  • RNAs with backbone modifications may not have phosphorus atoms in the backbone.
  • RNAs that do not have phosphorus atoms in their internucleoside backbones may be referred to as oligonucleosides.
  • modified RNAs have phosphorus atoms in their internucleoside backbones.
  • RNA molecule or “RNA” or “ribonucleic acid molecule” includes not only RNA molecules expressed or found in nature, but also analogs and derivatives of RNA, which include one or more ribonucleotides/ribonucleoside analogs or derivatives as described herein or known in the art.
  • ribonucleoside and “ribonucleotide” are used interchangeably herein.
  • RNA molecules can be modified in the core base structure or the ribose-phosphate backbone structure (e.g., as described below), and molecules containing ribonucleoside analogs or derivatives must retain the ability to form duplexes.
  • substituteduents are intended aspects of the present invention.
  • One of ordinary skill in the art of medicinal chemistry and organic chemistry will appreciate the versatility of such substituents.
  • a “substituent” should be sufficient to satisfy the principle that its total number will conform to the principle that a chemical bond can be formed with the atom or group to which it is attached in the present invention.
  • hydrocarbyl includes straight chain, branched and cyclic groups with any degree of saturation. In some special cases, including groups containing C, O, H, such hydrocarbyl groups may be interrupted by one or more heteroatoms of nitrogen, oxygen, sulfur and phosphorus, and may be further substituted by one or more substituents, either mono- or poly-substituted.
  • alkyl refers to a straight or branched saturated hydrocarbon group having 1 to about 24 carbon atoms.
  • a numerical range such as “C 1 -C 6 alkyl” generally refers to the number of carbon atoms in the alkyl group, which may contain only 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 6 carbon atoms. Unless otherwise specified, the number of carbon atoms added by further substitution of other atoms or groups in the general alkyl group is not included in the counting range, and the same description also exists in other counting methods herein.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl, etc.
  • alkyl also includes the case where the numerical range of carbon atoms is not specified, for example, the term “alkyl” can refer to a sub-range of C 1 -C 10 (e.g., C 1 -C 6 ).
  • Substituted alkyl refers to an alkyl portion with a substituent.
  • lower alkyl refers to an alkyl portion having 1 to about 6 carbon atoms.
  • alkenyl refers to a straight or branched hydrocarbon group of at least one carbon-carbon double bond.
  • alkenyl include, but are not limited to, vinyl, propenyl, butenyl, 1-methyl-2-butene-1-yl, diene, such as 1,3-butadiene, etc.
  • Alkenyl typically contains 2 to about 24 carbon atoms, more typically 2 to about 12 carbon atoms, with 2 to about 6 carbon atoms being more preferred.
  • Alkenyl as used herein may optionally contain one or more further substituted groups.
  • alkynyl refers to a straight or branched hydrocarbon group having at least one carbon-carbon triple bond.
  • alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, etc.
  • Alkynyl groups typically contain 2 to about 24 carbon atoms, more typically 2 to about 12 carbon atoms, with 2 to about 6 carbon atoms being more preferred.
  • Alkynyl groups as used herein may optionally contain one or more further substituent groups.
  • acyl refers to a group formed by removing a hydroxyl group from an organic acid and has the general formula -C(O)-X wherein X is typically aliphatic, alicyclic or aliphatic. Examples include aliphatic carbonyl, aromatic carbonyl, aliphatic sulfonyl, aromatic sulfinyl, aliphatic sulfinyl, aromatic phosphate, aliphatic phosphate, and the like. Acyl as used herein may optionally contain further substituent groups.
  • cycloalkyl refers to a ring system in which the ring is aliphatic.
  • the ring system may contain one or more rings in which at least one ring is aliphatic.
  • Preferred alicyclic compounds contain rings having from about 5 to about 9 carbon atoms in the ring.
  • cycloalkyl may optionally contain further substituent groups.
  • alkoxy refers to a group formed between an alkyl group and an oxygen atom, wherein the oxygen atom is used to connect the alkoxy group to the parent molecule.
  • alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, and n-hexoxy, etc.
  • Alkoxy groups as used herein may optionally contain further substituent groups.
  • aminoalkyl refers to a C 1 -C 12 alkyl group substituted with an amino group.
  • the alkyl portion of the group forms a covalent bond with the parent molecule.
  • the amino group may be located at any position and the aminoalkyl group may be substituted with further substituents at the alkyl and/or amino positions.
  • aryl and aromatic refer to monocyclic or polycyclic carbocyclic groups with one or more aromatic rings
  • aromatic group refers to a planar ring with a delocalized ⁇ electron system containing 4n+2 ⁇ electrons, where n is an integer.
  • the aromatic ring can be formed by 5, 6, 7, 8, 9 or more than 9 atoms.
  • aromatic is intended to include carbocyclic aryl (e.g., phenyl) and heterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g., pyridine).
  • aryl include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl (idenyl), etc.
  • Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings.
  • aryl may optionally include further substituent groups.
  • aralkyl and “arylalkyl” refer to an aromatic group covalently attached to a C 1 -C 12 alkyl group.
  • the alkyl portion of the resulting aralkyl (or arylalkyl) forms a covalent bond with the parent molecule. Examples include, but are not limited to, benzyl, phenethyl, etc.
  • Aralkyl groups as used herein may optionally contain further substituent groups attached to the alkyl group, the aryl group, or both to form the group.
  • heteroaryl and “heteroaromatic” refer to groups comprising monocyclic or polycyclic aromatic rings, ring systems or fused ring systems, wherein at least one of these rings is aromatic and contains one and or more heteroatoms. Heteroaryl is also intended to include fused ring systems including one or more systems in which the fused rings do not contain heteroatoms. Heteroaryl typically contains a ring atom selected from sulfur, nitrogen or oxygen.
  • heteroaryl examples include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, phenylthio, furanyl, quinolyl, isoquinolyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, etc.
  • Heteroaryl can be directly or connected to the parent molecule through a linking portion such as an aliphatic group or a heteroatom. As used herein, heteroaryl can optionally contain further substituent groups.
  • heteroarylalkyl refers to a heteroaryl group as defined previously further comprising a covalently linked C 1 -C 12 alkyl group.
  • the alkyl portion of the resulting heteroarylalkyl group is capable of forming a covalent bond with the parent molecule. Examples include, but are not limited to, pyridylmethyl, pyridylethyl, napthyridinylpropyl, and the like.
  • heteroarylalkyl groups may optionally contain further substituent groups on one or both of the heteroaryl or alkyl portions.
  • halo or halogen as used herein refers to an atom selected from fluorine, chlorine, bromine and iodine.
  • heterocyclic group refers to a monocyclic or polycyclic ring system containing at least one and heteroatom and being unsaturated, partially saturated or fully saturated, thus including heteroaryl groups.
  • Heterocyclic rings are also intended to include fused ring systems wherein one or more of the fused rings contain at least one heteroatom and the other rings may contain one or more heteroatoms or optionally contain no heteroatoms.
  • Heterocyclic groups typically contain at least one atom selected from sulfur, nitrogen or oxygen.
  • heterocyclic groups include [1,3] dioxolanyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, pyrazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, etc.
  • an "n" membered heterocyclic group is the sum of n atoms contained in the ring system, without protecting atoms or groups unexpected to the ring system.
  • a heterocyclic group may optionally contain further substituents.
  • the term "monocyclic or polycyclic structure” includes all ring systems selected from monocyclic ring systems or polycyclic ring systems in which the rings are fused or connected and is intended to include single and mixed ring systems selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl.
  • Such monocyclic and polycyclic structures may contain rings each having the same degree of saturation or each independently having 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 rings as well as rings containing only C ring atoms.
  • These rings may exist in mixed motifs such as, for example, benzimidazole, in which one ring has only carbon ring atoms and the fused ring has two nitrogen atoms.
  • the monocyclic or polycyclic structure may be connected to the parent molecule using a variety of strategies such as direct connection through ring atoms, through substituent groups or through bifunctional linking moieties.
  • sulfonyl refers to a radical of the formula RSO2- , wherein R is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl.
  • sulfonamido refers to a radical of the formula RSO2- NH-, wherein R is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl.
  • Some non-limiting exemplary sulfonamido groups include CH3 - SO2- N(H)-.
  • alicyclic refers to a cyclic ring system, wherein the ring is an aliphatic ring.
  • the ring system may contain one or more rings, wherein at least one ring is an aliphatic ring.
  • Preferred alicyclic rings include rings having about 5 to about 9 carbon atoms in the ring.
  • the alicyclic rings used herein optionally include other substituent groups.
  • aliphatic used herein refers to a straight or branched hydrocarbon group containing up to twenty-four carbon atoms, wherein the degree of saturation between any two carbon atoms is a single, double or triple bond.
  • the aliphatic group preferably contains 1 to about 24 carbon atoms, more usually 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.
  • the aliphatic group interrupted by heteroatoms includes, but is not limited to, polyalkoxy groups, such as polyalkylene glycols, polyamines and polyimines.
  • the aliphatic group used herein optionally includes other substituent groups.
  • a spacer, to which the oligonucleotide is attached, is used to place the oligonucleotide and the synthesis process away from the substrate or carrier. This allows for greater flexibility and more room for compositing, making it easier to cut when the composition is complete.
  • carboxy refers to a group of formula -COOH.
  • Linking groups or bifunctional linking moieties such as those known in the art can be used to connect chemical functional groups, conjugated groups, reporter groups and other groups to selective sites in parent compounds such as, for example, oligomeric compounds.
  • bifunctional linking moieties include a hydrocarbyl moiety with two functional groups. One of the functional groups is selected to be bound to the parent molecule or the compound of interest and the other is selected to be bound to substantially any selected group such as a chemical functional group or a conjugated group.
  • the linking group comprises a polymer of a chain structure or repeating units such as ethylene glycol or amino acid units.
  • bifunctional linking moieties examples include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups.
  • the bifunctional linking moiety comprises amino, hydroxyl, carboxylic acid, thiol, unsaturation (e.g., double bond or triple bond), etc.
  • Some non-limiting examples of bifunctional linking moieties include 8-amino-3,6-dioxaoctanoic acid (ADO), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide (SMCC), and 6-aminohexanoic acid (AHEX or AHA).
  • linking groups include, but are not limited to, substituted C 1 -C 10 alkyl, substituted or unsubstituted C 2 -C 10 alkenyl, or substituted or unsubstituted C 2 -C 10 alkynyl, wherein a non-limiting list of preferred substitution groups includes hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.
  • ether used in this article is a product in which the hydrogen in the hydroxyl group of an alcohol or phenol is replaced by a hydrocarbon group, and the general formula is R-O-R', where R and R' can be the same or different.
  • Ethers that are the same are called symmetrical ethers, also called simple ethers or monoethers; ethers that are not the same are called asymmetrical ethers, also called mixed ethers or mixed ethers.
  • Terminology used in this article or It generally refers to the connection between adjacent groups or parts.
  • the term “about” or “approximately” as applied to one or more values of interest refers to a value similar to the reference value. In certain embodiments, unless otherwise specified or otherwise apparent from the context, the term “about” or “approximately” refers to a range of values falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in either direction (greater than or less than) (unless such a numerical value will exceed 100% of the possible value).
  • the resulting mixture was cooled to 0°C and poured into a saturated NaHCO 3 solution (50 mL), and extracted with CH 2 Cl 2 (50 mL*3), and the combined organic layers were washed with a saturated NaCl solution (50 mL), dried over Na 2 SO 4 and concentrated under reduced pressure.
  • oligomeric compounds used in accordance with the present invention may be conveniently and routinely prepared by well-known solid phase synthesis techniques. Apparatus for such synthesis is sold by a number of suppliers including, for example, the Mermade 12 (LGC). Additionally or alternatively, any other apparatus known in the art for such synthesis may be employed. It is well known that oligonucleotides such as alkylated derivatives and those having phosphorothioate linkages are prepared using similar techniques.
  • 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 by iodine oxidation.
  • LGC DNA synthesizer Mermade 12
  • phosphorothioate internucleoside linkages are synthesized in a manner similar to phosphodiester internucleoside linkages, except that thiolation is achieved by oxidation of the phosphite linkage using 10% w/v 3,H-1,2-benzodithiol-3-one-1,1-dioxide in acetonitrile.
  • the thiolation step time is increased to 180 seconds and continued through the normal capping step.
  • the oligomeric compound is recovered by precipitation from 1M NH4OAc solution with 3 volumes more of ethanol after cleavage and deblocking from the CPG column in concentrated aqueous ammonia at 55°C (12-16h).
  • Phosphinate internucleoside linkages can be prepared as described in US5,508,270.
  • Alkyl phosphate internucleoside linkages can be prepared as described in US4,469,863, or by the phosphoramidite method described herein.
  • 3'-deoxy-3'-methylenephosphonate internucleoside linkages can be prepared as described in US5,610,289 or 5,625,050.
  • Phosphoramidite internucleoside linkages can be prepared as described in US5,256,775 or US5,366,878.
  • Alkyl thiophosphate internucleoside linkages can be prepared as described in published WO94/17093 and WO94/02499 publications.
  • 3'-deoxy-3'-aminophosphoramidate internucleoside linkages can be prepared as described in US5,476,925.
  • Phosphotriester internucleoside linkages are prepared as described in US5,023,243, and methods for preparing some phosphate internucleoside linkages can be found in Beilstein J Org Chem. 2017; 13:1368–1387.
  • Oligomeric compounds having one or more non-phosphorus-free internucleoside linkages including, but not limited to, methylenemethylimino-linked oligonucleosides, methylenedimethylhydrazine-linked oligonucleosides, methylenecarbonylamino-linked oligonucleosides, and methyleneaminocarbonyl-linked oligonucleosides, as well as mixed backbone oligomeric compounds having, for example, alternating O or S linkages, can be prepared as described in US5,378,825, US5,386,023, US5,489,677, US5,602,240, and US5,610,289.
  • Double-stranded ribonucleic acid (dsRNA) reagents are obtained by mixing each of the two complementary strands (sense strand and antisense strand) at a 1:1 molar ratio to form a duplex.
  • the duplexes in Table 2 are typically synthesized on an oligonucleotide synthesizer using a well-established solid-phase synthesis method based on phosphoramidite chemistry to synthesize the sense and antisense strands of siRNA.
  • Oligonucleotide chain growth is achieved through a 4-step cycle: deprotection, condensation, capping, and an oxidation or sulfurization step for each nucleotide added.
  • the synthesis is performed on a controlled pore glass (CPG) ) is carried out on a solid support made of.
  • CPG controlled pore glass
  • General monomeric phosphoramidites are purchased from commercial sources, and the monomeric compounds herein can be substituted as a monomeric phosphoramidite and introduced into the oligonucleotide chain.
  • the monomeric compound herein can be substituted as a monomeric phosphoramidite connected to the 3'-end, it is connected to the CPG solid support when used.
  • the phosphoramidite is used for the final coupling reaction, and the targeting group can be further coupled if necessary.
  • Trichloroacetic acid (TCA) in 3% dichloromethane is used for the deprotection of 4,4'-dimethoxytrityl protecting group (DMT). 5-Ethylthio-1H-tetrazole is used as an activator.
  • the crude single-stranded product is further purified by ion pair reversed-phase HPLC (IP-RP-HPLC).
  • IP-RP-HPLC ion pair reversed-phase HPLC
  • IP-RP-HPLC ion pair reversed-phase HPLC
  • O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) (327 g, 1.02 mol, 3.90 eq.) and triethylamine (212 g, 2.09 mol, 8.00 eq.) were added to a DMF (1.0 L) solution of compound 6 (100 g, 261 mmol) and intermediate-A (502 g, 915. mmol, 3.50 eq.). The reaction was carried out at 25°C for 1 hour. LCMS showed that the conversion of compound 6 was complete.
  • TBTU O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate
  • the reaction solution was added to 4000 mL of water, and extracted with methyl tert-butyl ether (2000 mL twice) to remove impurities.
  • the remaining aqueous phase was extracted with dichloromethane (3000 mL twice).
  • the dichloromethane phase was washed with 10% citric acid aqueous solution (2000 mL twice), saturated NaHCO 3 (2.0 L twice), and saturated brine (2.0 L) in sequence, and dried over anhydrous Na 2 SO 4 .
  • the filtrate was filtered and concentrated under reduced pressure to give white solid compound 8 (260 g, 159 mmol, yield 60.9%).
  • Triethylamine (67.8 g, 672 mmol, 4.00 eq.) was added 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), and the solution was stirred at 25° C. for 1 hour.
  • LCMS showed that compound 9 was completely converted into compound 11.
  • 4-Hydroxypiperidine (42.4 g, 420 mmol, 2.50 eq.) and TBTU (107 g, 335 mmol, 2.00 eq.) were added to the reaction solution, and stirring was continued at 25° C. for 1 hour.
  • the reaction solution was washed with a 1:1 mixture of saturated NaHCO 3 and saturated brine (2.0 L), dried over anhydrous Na 2 SO 4 , and the filtrate was concentrated and the obtained 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 tert-butyl ether, the solid was collected and dried in vacuo, the product was dissolved in dichloromethane (1.0 L) and concentrated to dryness, and the operation was repeated 4 times to remove residual tert-butyl ether to obtain GLPA15 (164 g, yield 73.3%).
  • GalNAc phosphoramidite compounds can also be obtained by using appropriate corresponding intermediates, using similar methods described herein or methods well known in the art, and can be attached as targeting groups to appropriate positions of the compounds described herein or oligomers and duplexes.
  • a method for connecting a targeting group comprising GalNAc (also referred to herein as a GalNAc delivery compound) to the 5'-end of a sense strand includes using a GalNAc phosphoramidite (GLPAn) in the last coupling step of solid phase synthesis, using such a synthesis process, such as the (i.e., adding nucleotides at the 5' end of the sense strand) process used when the oligonucleotide chain is extended, to connect it to the 5'-end of the sense strand.
  • the method of connecting a targeting group comprising GalNAc to the 3'-end of a sense strand includes using a solid support (CPG) comprising GLPAn.
  • CPG solid support
  • the method of connecting a targeting group comprising a targeting group to the 3'-end of a sense strand includes: connecting the targeting group to a CPG solid support via an ester bond, and using the resulting CPG with a connected targeting group when synthesizing the sense strand, which results in the GalNAc targeting group being connected to the 3'-end of the sense strand.
  • the targeting group can also be connected to a monomer containing an oligomeric compound of the present invention by means of a phosphoramidite method, etc.
  • the system was filtered with nitrogen, and the filter cake was rinsed three times with N,N-dimethylformamide (26.00kg+26.10kg+26.00kg), and the filter cake was left in the kettle.
  • CAP.A (4.40kg+4.42kg+4.30kg) and CAP.B (4.40kg+4.40kg+4.47kg) were added to the 80L glass kettle, stirred for 3-8min and set aside. This operation was repeated 3 times for capping, and acetonitrile (18.00kg+18.00kg+18.00kg+17.50kg+17.50kg) was added to the solid phase synthesis kettle, and nitrogen was bubbled for 10-30min before filtering. Repeat this operation four times.
  • the filter cake is purged with nitrogen in the solid phase synthesis reactor for 2 to 4 hours, it is transferred to a 50L filter press tank and the temperature is controlled at 15 to 30°C. Drying is continued to obtain a yellow to white solid product with a weight of 3.516 kg.
  • the oligomeric compound comprising the monomer of the present invention is obtained by the method described herein or known to those skilled in the art.
  • the sequence structure of the oligomeric compound and the siRNA duplex targeting FXII are shown in Table 2.
  • the duplex contains a sense strand and an antisense strand; chemical modifications are represented as: uppercase: 2'-fluoro modified nucleotides; lowercase: 2'-methoxy modified nucleotides; "*" should be understood that when present in an oligonucleotide, these monomers are interconnected by phosphorothioate diester nucleoside linkages, and the absence of "*" between the two monomers indicates that they are interconnected by phosphorothioate diester nucleoside linkages; Invab: inverted abasic, GLS15* and GLO15 are the targeting groups described in this article, respectively.
  • imann at the end of an oligomer or dsRNA is represented by: “imann” is expressed as when an oligomer or dsRNA is further linked to a targeting group.
  • FXII double-stranded nucleoside nucleic acid also known as dsRNA
  • female C57BL/6 mice, 6 weeks old, specific pathogen-free purchased from Shanghai Slack Laboratory Animal Co., Ltd. were used.
  • Test compounds AD00823, AD00824, AD00825, AD00826, AD00835, AD00836, AD00841, AD00827, AD00449.
  • All test compounds were prepared into 5 mg/mL stock solution with PBS before administration. A small amount of 5 mg/mL stock solution was diluted 20 times and the OD value was detected using Nanodrop. After the actual concentration of the stock solution was calculated according to the OD value, an appropriate amount was taken and diluted to the working solution concentration (0.2 mg/mL) for administration.
  • mice All mice (C57BL/6, female, 6 weeks) were given subcutaneous injections on day 0 after a 6-day acclimatization period as described above.
  • mice On days 7, 14, and 21, all mice were bled through the submandibular vein to collect plasma for FXII protein level detection. Sample detection and analysis
  • ELISA kit was used to detect the level of FXII protein in mouse plasma.
  • the ELISA kit (Molecular Innovations, IMSFXIIKTT) was used to detect the level of FXII protein in mouse plasma.
  • the plasma sample collected by EDTA-K2 was diluted 30,000 times and added to the detection plate coated with the capture antibody for incubation, and then the detection antibody and HRP-conjugated secondary antibody were added in sequence, and finally TMB was used for color development, and the absorbance at 450nm was read.
  • the standard curve was fitted using the four-parameter method, and the OD value of the test sample was substituted into the calculation to calculate the FXII protein content of each sample, and the FXII protein concentration in the original plasma was obtained by multiplying it by the dilution factor.
  • Table 3 The data results are shown in Table 3.
  • Double-stranded nucleoside nucleic acid (also known as dsRNA) reagents AD00824 and AD00826 have oligomeric compounds containing the imann monomer compound described herein at the 5' end, 3' end or both ends of the sense chain.
  • double-stranded nucleoside nucleic acid (also known as dsRNA) reagents such as AD00823 and AD00825 that do not contain or contain other nucleotides, they all show better silencing effects, indicating that when the monomer described herein is contained at the 5' end and/or 3' end of the sense chain of the double-stranded nucleoside nucleic acid (also known as dsRNA), it has a better anti-nuclease cleavage effect, so that the sense chain has a stability effect and improves the in vivo effect of the antisense body; the same results can be observed in AD00827VSAD00841.
  • dsRNA double-stranded nucleoside nucleic acid
  • AD00835 containing the imann monomer compound in the antisense chain is compared with AD00836 (containing invab). From the activity data within the same action time, this actually proves that when added to the 5'-antisense chain end, the antisense is blocked by the imann monomer compound in the present invention and the activity is killed, which indicates that the compound in the present invention has a higher anti-nuclease effect than invab in the oligomeric compound and has a higher stability.
  • the oligomeric compound comprising the monomer of the present invention is obtained by the method described herein or known to those skilled in the art.
  • the sequence structure of the oligomeric compound and the siRNA duplex targeting FXII are shown in Table 4.
  • the duplex comprises a sense strand and an antisense strand; chemical modifications are represented as follows: uppercase: 2'-fluoro modified nucleotides; lowercase: 2'-methoxy modified nucleotides; "*" should be understood as meaning that when present in an oligonucleotide, these monomers are interconnected by phosphorothioate diester nucleoside linkages, and the absence of "*" between the two monomers indicates that they are interconnected by phosphorothioate diester nucleoside linkages; Invab: inverted abasic.
  • imann at the end of an oligomer or dsRNA is represented by: “imann” is expressed as when an oligomer or dsRNA is further linked to a targeting group.
  • Sttab8 at the end of an oligomer or dsRNA is represented by: “Stab8” is expressed when an oligomer or dsRNA is further linked to a targeting group.
  • Sttab9 at the end of an oligomer or dsRNA is represented by: “Stab8” is expressed when an oligomer or dsRNA is further linked to a targeting group.
  • FXII double-stranded nucleoside nucleic acid also known as dsRNA
  • Test compounds AD00734, AD00737, AD01970, AD01971.
  • All test compounds were prepared into 5 mg/mL stock solution with PBS before administration. A small amount of 5 mg/mL stock solution was diluted 20 times and the OD value was detected using Nanodrop. After the actual concentration of the stock solution was calculated according to the OD value, an appropriate amount was taken and diluted to the working solution concentration (0.2 mg/mL) for administration.
  • mice All mice (C57BL/6, female, 6 weeks) were given subcutaneous injections on day 0 after a 6-day acclimatization period as described above.
  • mice On days 7, 14, 21, and 28, all mice were bled through the submandibular vein to collect plasma for the detection of FXII protein levels.
  • the ELISA kit (Molecular Innovations, IMSFXIIKTT) was used to detect the level of FXII protein in mouse plasma.
  • the plasma sample collected by EDTA-K2 was diluted 30,000 times and added to the detection plate coated with the capture antibody for incubation, and then the detection antibody and HRP-conjugated secondary antibody were added in sequence, and finally TMB was used for color development, and the absorbance at 450nm was read.
  • the standard curve was fitted using the four-parameter method, and the OD value of the test sample was substituted into the calculation to calculate the FXII protein content of each sample, and the FXII protein concentration in the original plasma was obtained by multiplying it by the dilution factor.
  • Table 5 The data results are shown in Table 5.
  • the oligomeric compound comprising the monomer of the present invention is obtained by the method described herein or known to those skilled in the art.
  • the sequence structure of the oligomeric compound is shown in Table 6 as a siRNA duplex targeting SOD1.
  • Table 6 shows that the duplex contains a sense strand and an antisense strand; chemical modifications are represented as: uppercase: 2'-fluoro modified nucleotides; lowercase: 2'-methoxy modified nucleotides; "*" should be understood that when present in an oligonucleotide, these monomers are interconnected by thiophosphate diester nucleoside linkages, and the absence of "*" between the two monomers indicates that they are interconnected by oxophosphate diester nucleoside linkages; Invab: inverted abasic.
  • mice were intracerebroventricularly (ICV) administered
  • aCSF group solvent control group
  • the specific steps are as follows: use Zotal + Xylazine hydrochloride to anesthetize the mouse by intraperitoneal injection.
  • mice After the anesthesia takes effect, fix the mouse head on the brain stereotaxic instrument and use a 10 ⁇ L microsyringe (Hamilton) to inject the drug into the mouse lateral ventricle.
  • the injection time is 10 minutes. After the injection, the needle is retained for 5 minutes, then the needle is slowly removed, and the head skin is sutured. After the mouse wakes up, it is returned to the cage and continued to be raised.
  • mice On the 8th day after injection, each mouse was intraperitoneally anesthetized and perfused with normal saline, and then the prefrontal cortex (PFC), striatum, hippocampus and cerebellum tissues of the mice were quickly extracted and stored in a -80°C refrigerator. Subsequently, total RNA was extracted and reverse transcribed into cDNA using RNeasy Kit (QIAGEN) according to the instructions, and then the expression of the target gene SOD1 was detected by qPCR using FastStart Universal SYBR Green Master (ROX) (Roche), and the 2 - ⁇ Ct method was used for quantitative analysis. The results are shown in Table 7.
  • the intracerebroventricular injection method of mice was as described above. On the 8th day after injection, each mouse was intraperitoneally anesthetized and perfused with normal saline, and then the prefrontal cortex (PFC), striatum, hippocampus and cerebellum tissues of the mice were quickly extracted and stored in a -80°C refrigerator.
  • PFC prefrontal cortex

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Abstract

本发明涉及双环脱碱基核酸类似物以及由它们制备的寡聚化合物，更具体涉及一种双环脱碱基核酸类似物，并且可用于并入到寡聚化合物的一个或者两个末端的双环脱碱基核酸类似物单体化合物；在体内将寡聚化合物递送至需要特异性靶向的细胞,除肝脏相关组织，还可以递送至肝外组织例如眼科以及CNS的靶细胞。

Description

双环脱碱基核酸类似物以及由它们制备的寡聚化合物 技术领域
本发明涉及可用于双环脱碱基核酸类似物以及由它们制备的寡聚化合物，更具体涉及双环脱碱基核酸类似物单体可用于并入到寡聚化合物的一个或者两个末端的双环脱碱基核酸类似物。
 背景技术
RNA干扰或“RNAi”技术已经是本领域已知的疾病疗法的有效手段。RNAi机制是由Dicer酶介导的来自更长的非编码RNA的产生来启动，将这些RNA分子加载到RNA诱导的沉默复合体(RISC)中，其中将正义链丢弃，并且将反义链或引导链与完全或部分互补的mRNA序列杂交，然后经由Ago2介导的降解或翻译抑制来诱导mRNA的沉默。在RNAi技术和递送方法的进展已经给予基于RNAi的疗法越来越多的积极结果，这类疗法代表一类有前途的疾病的治疗方向，然而这类技术由于天然低聚核苷酸易被体内细胞和细胞外核酸酶降解，导致靶向性和体内稳定性等天然RNA的固有代谢问题原因导致无法得到广泛应用。因此，现有技术中对围绕核苷酸修饰，特别是这些修饰以帮助改进核酸酶抗性、结合亲和力、靶向性和体内稳定性。
已经通过开发出各种化学修饰应用于RNAi寡核苷酸，在克服天然RNA的固有代谢问题等方面取得了极大进展，这种RNAi寡核苷酸的化学修饰对于完全利用这类的治疗方案潜力具有至关重要促进作用，其可改善其药代动力学和药效学特性。例如，Choung(庄)等人使用了2′-OMe(2'-O甲基)、2′-F(2′-氟)和硫代磷酸酯修饰的核苷酸以及他们之间不同组合在血清中的获得稳定；进一步包括对核苷酸其他修饰，例如其中该核苷的呋喃糖部分包含了连接呋喃糖十两个原子而形成双环环系统的桥，双环核苷具有各种名称，如双环核酸或锁核酸分别称为BNA和LNA；以及呋喃糖开环形式的UNA、GNA等方式以增强热稳定性；此外，已经报道，5′-末端磷酸酯基并入寡聚核苷酸中得以增强某些核酸抑制剂分子与Ago2之间的相互作用，例如：针对反义链5′端的磷酸模拟物也是近年的研究方向，以提高RNAi的沉默效果。然而，这些修饰核苷酸衍生物均保留碱基部分，仍需要参与碱基配对的过程。近年采用脱碱基核苷酸和反向脱碱基的应用于5′或者3′寡核苷酸，以增强寡核苷酸的沉默效果和提高寡核苷酸稳定性，例如：WO2022162155。
尽管已经通过化学修饰发展和改进的递送方法在克服天然RNA的固有代谢问题方面取得了极大进展，但是在本领域中仍然需要适用于治疗目的施用的具有增强的体内功效和稳定性的RNAi药剂，特别是脱碱基核苷酸的研究仍处于初始阶段，希望进一步提供新的脱碱基核酸类似物并入的低聚核苷酸类中，对抗细胞核酸酶提高有稳定性，具有更长的体内药效作用，并且对核酸靶具有更强的亲和力。
发明内容
本发明涉及可用于双环脱碱基核酸类似物以及由它们制备的寡聚化合物，更具体涉及一种双环脱碱基核酸类似物单体化合物，并且可用于并入到寡聚化合物的一个或者两个末端的双环脱碱基核酸类似物单体化合物，在这些寡聚化合物可以应用于除肝脏相关组织靶细胞外，还可以应用于递送至肝外组织，例如眼科以及CNS的靶细胞。
根据本发明的一个方面，提供了式(I)所示的化合物或其立体异构体：
A1和A2分别独立的选自：O、S、SO、SO2、NR2和CR3R4，R2、R3和R4分别独立的选自：氢、卤素、磺酰基、亚磺酰基、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基；
T1和T2之一为保护基、Z、L以及-Z-L，所述的Z表示为靶向基团或亲脂性基团，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，L表示为：载体间隔物或包含间隔物链接的载体部分；而T1和T2的另一个为活性磷基团、保护基；并且T1和T2不会同时为保护基；
R1分别独立地选自卤素、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基；
n为0-6的整数；
其中式(I)不包括如下结构：
在某些的实施方式中，A1为O。在某些的实施方式中，A2为O。在某些的实施方式中，A1和A2分别独立地为O。在某些的实施方式中，A1和A2分别独立地为S。在某些的实施方式中，A1和A2分别独立为NR2。在某些的实施方式中，R2为氢或者CH3。在某些的实施方式中，A1不同于A2。在某些的实施方式中，A1为O，A2为S。在某些的实施方式中，A1为O，A2为NR2。在某些的实施方式中，A1为S，A2为NR2。
在某些的实施方式中，所述的T1和T2之一的保护基根据A1或者A2不同，对应的分别是羟基保护基、巯基保护基以及胺基保护基。
在某些的实施方式中，T1和T2之一保护基是羟基保护基；在一个实施方式中，所述羟基保护基各自独立地选自乙酰基、叔丁基、叔丁氧基甲基、甲氧基甲基、四氢吡喃基、1-乙氧基乙基、1-(2-氯乙氧基)乙基、2-三甲基甲硅烷乙基、对氯苯基、2,4-二硝基苯基、苄基、苯甲酰基、对苯基苯甲酰基、2,6-二氯苄基、二苯基甲基、对硝基苄基、三甲基甲硅烷基、三乙基甲硅烷基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基、三苯甲硅烷基、三异丙基甲硅烷基、苯甲酰基甲酸酯、氯乙酰基、三氯乙酰基、三氟乙酰基、新戊酰基、9-芴甲氧羰基、甲磺酸基、甲苯磺酸基、三氟甲磺酸基、三苯甲基、单甲氧基三苯甲基、二甲氧三苯甲基、三甲氧基三苯甲基或取代的9-苯基黄嘌呤-9-基；在一个实施方式中，优选的羟基保护基各自独立地选自乙酰基、苄基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基以及4,4’-二甲氧三苯甲基。
在某些的实施方式中，T1和T2之一保护基是巯基保护基；在一个实施方式中，所述巯基保护基各自独立地选自乙酰基、叔丁基、叔丁氧基甲基、甲氧基甲基、四氢吡喃基、1-乙氧基乙基、1-(2-氯乙氧基)乙基、2-三甲基甲硅烷乙基、对氯苯基、2,4-二硝基苯基、苄基、苯甲酰基、对苯基苯甲酰基、2,6-二氯苄基、二苯基甲基、对硝基苄基、三甲基甲硅烷基、三乙基甲硅烷基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基、三苯甲硅烷基、三异丙基甲硅烷基、苯甲酰基甲酸酯、氯乙酰基、三氯乙酰基、三氟乙酰基、新戊酰基、9-芴甲氧羰基、甲磺酸基、甲苯磺酸基、三氟甲磺酸基、三苯甲基、单甲氧基三苯甲基、二甲氧三苯甲基、三甲氧基三苯甲基或取代的9-苯基黄嘌呤-9-基；在一个实施方式中，优选的巯基保护基各自独立地选自苄基、4,4’-二甲氧三苯甲基。
在某些的实施方式中，T1和T2之一保护基是氨基保护基；在一个实施方式中，所述氨基保护基各自独立地选自2-三甲基硅基乙氧基羰基(Teoc)、1-甲基-1-(4-联苯基)乙氧基羰基(Bpoc)、叔丁氧基羰基(BOC)、烯丙氧基羰基(Alloc)、9-芴基甲氧基羰基(Fmoc)、苄氧基羰基(Cbz)、甲酰基、乙酰基、三卤乙酰基、苯甲酰基、硝基苯基、乙酰基、2-硝基苯磺酰基、邻苯二甲酰亚胺基和二硫代丁二酰基。
在某些的实施方式中，T1和T2之一为亲脂性基团，其中亲脂性基团的亲脂性可以通过logKow测定，并且logKow超过0；优选地亲脂性基团logKow超过1，超过1.5，超过2，超过3，超过4，超过5或超过10。在一个某些的实施方式中，亲脂性基团选自含有饱和或不饱和的C4-C30烃链、脂肪环、芳香族、脂肪酸基团或脂肪酸衍生的基团、类固醇衍生的基团和任何脂溶性维生素基团。
在某些的实施方式中，亲脂性基团为饱和或不饱和C10-C25烃链(例如C10-C25烷基或烯基)。在一些实施例中，亲脂性基团为饱和或不饱和C10-C18烃链(例如直链C6-C18烷基或烯基)。在一个实施例中，亲脂性基团为饱和或不饱和的C14、C16或者C18烃链(例如直链C16烷基或烯基)。在一个实施例中，亲脂性基团为饱和的直链C14、C16或者C18烷基。亲脂性基团任选的被选自由以下官能团组成的组进一步取代：羟基、胺、羧酸、磺酸酯、磷酸酯、硫醇、叠氮基及炔烃。这些官能团可用于将亲脂性基团附接至A1或者A2中。
在另一个实施例中，亲脂性基团包含具有4到28个碳原子的烃链的任何饱和或不饱和脂肪酸，并且可以含有一个或两个羧基。在一个实施例中，所述亲脂性基团是具有8到24个碳原子的烃链的饱和脂肪酸衍生的基团。在某些实施例中，所述亲脂性基团是饱和脂肪酸衍生的基团，其中所述脂肪酸选自由以下组成的组：辛酸、癸酸、十二烷酸、十四烷酸、十六烷酸、十八烷酸、二十烷酸、二十二烷酸和二十四烷酸。在某些实施例中，所述亲脂性基团是具有8到24个碳原子的烃链的不饱和脂肪酸衍生的基团，其中所述脂肪酸选自由以下组成的组：肉豆蔻脑酸、棕榈油酸、十六碳烯酸、油酸、反油酸、反型异油酸、亚油酸、反亚油酸、α-亚麻酸、花生四烯酸和芥酸。
在某些的实施方式中，亲脂性基团选自胆固醇、维生素E(生育酚)或胆汁酸。
在某些的实施方式中，亲脂性基团选自二十二碳六烯酸酸(DHA)、二十碳五烯酸(EPA)、二十二烷酸(DCA)、甾醇胆固醇(胆汁)、生育酚琥珀酸酯(TS)和石胆酸(LA)、维甲酸(维生素A酸)。
亲脂性基团可经由直接附接于A1或者A2以共价键方式共价连接。可替代地，亲脂性基团可经由连接子与A1或者A2共价连接。
在一个实施例中，T1和T2之一为亲脂性基团，所述亲脂性基团进一步通过连接子与A1或者A2共价连接。在某些实施例中，亲脂性基团经由一个或多个连接子与A1或者A2共价连接。
在另一个实施例中，所述连接子是烃链连接子或聚乙二醇(PEG)连接子。在另一个实施例中，所述连接子选自由以下组成的组：酰胺键、磷脂酰胆碱、烃连接子或聚乙二醇(PEG)连接子、氨基-烷基-醇、羟脯氨酸、羟脯氨醇、氨基-烷基-硫代磷酸酯连接子、氨基-PEG-硫代磷酸酯连接子、α-羧酸盐-氨基-烷基硫代磷酸酯连接子和α-羧酸盐-氨基-PEG-硫代磷酸酯连接子。在一些实施例中，所述的连接子含有醚、硫醚、脲、碳酸酯、胺、酰胺、马来酰亚胺-硫醚、二硫化物、磷酸二酯、磺酰胺键、点击反应的产物(例如，来自叠氮化物-炔烃环加成的三唑)或氨基甲酸酯。在一些实施例中，磷酸二酯为硫代磷酸二酯和氧代磷酸二酯。
一些实施例中，所述连接子包括可裂解基团。至少连接子是氧化还原可裂解连接子(诸如还原可裂解连接子，例如二硫化物基团)、酸可裂解连接子(例如，腙基、酯基、缩醛基或缩酮基)、酯酶可裂解连接子(例如，酯基)、磷酸酶可裂解连接子(例如，磷酸酯)或肽酶可裂解连接子(例如，肽键)。在其他实施例中，至少一个连接子是生物可裂解连接子，该生物可裂解连接子选自由以下组成的组：DNA、RNA、二硫化物、酰胺、半乳糖胺、葡糖胺、葡萄糖、半乳糖、甘露糖的官能化单糖或寡糖及其组合。
一些实施例中，连接子还可以是环状基团或非环状基团。在一个实施例中，环状基团选自由以下组成的组：吡咯烷基、吡唑啉基、吡唑烷基、咪唑啉基、咪唑烷基、哌啶基、哌嗪基、[1,3]二氧戊环、噁唑烷基、异噁唑烷基、吗啉基、噻唑烷基、异噻唑烷基、喹喔啉基、哒嗪酮基、四氢呋喃基及十氢化萘。在一个实施例中，非环状基团为基于丝氨醇主链或二乙醇胺主链的部分。
在一些实施方式中，T1和T2之一为靶向基团，所述靶向基团可以是包含siRNA给药领域常规使用的配体。
在一些实施方式中，所述靶向基团可以选自包含以下靶向分子或其衍生物形成的配体中的一种或多种：聚合物、糖类、肝实质细胞表达的受体配体、抗体、量子点、多肽，或小分子配体。
在一些实施方式中，至少一个或每个所述靶向基团选自包含能够和哺乳动物肝实质细胞表面受体结合的配体。
在一些实施方式中，每个所述靶向基团独立地为包含与哺乳动物肝细胞表面的去唾液酸糖蛋白受体(ASGPR)亲和的配体。
在一些实施方式中，每个所述靶向基团独立地选自包含D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D吡喃甘露糖、α-D-吡喃葡萄-糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳-糖胺、N-乙酰半乳糖胺、N-三氟乙酰半乳糖胺、N-丙酰半乳糖胺、N-正丁酰半乳胺、N-异丁-36酰半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6O-三苯甲基-α-D吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖、L-4-硫代核糖中的一种或者几种的配体。
在一些实施方式中，至少一个或每个所述靶向基团为包含半乳糖或N-乙酰半乳糖胺的配体。
靶向基团可包括经由合适的缀合接头与A1或者A2之一相连。本领域技术人员可以根据靶向基团的具体类型选择合适的缀合接头连接。
在某些的实施方式中，靶向基团与A1或者A2之一相连选自以下化合物片段之一：
在某些的实施方式中，T1和T2之一为载体间隔物。在某些的实施方式中，所述的载体间隔物为二羧酸衍生的基团，示例结构R为不存在、醚、聚乙二醇、氧、硫、氮、亚烷基、烯基、炔基、芳基、芳烷基、杂烷基或杂芳基。在某些的实施方式中，所述的载体间隔物为聚亚烷基二醇磷酸酯/膦酸酯；在某些的实施方式中，所述的载体间隔物优选为C3-C6烷基二羧酸根基团。在某些的实施方式中，所述的载体间隔物优选为琥珀酰基，在某些的实施方式中，T1和T2之一为包含通过间隔物链接的载体部分，其中所述的载体部分包括固相载体部分和液相载体部分。在某些的实施方式中，固相载体选自控孔玻璃(CPG)、聚苯乙烯、硅胶、大孔交联聚苯乙烯、聚甲基丙烯酸乙烯醇共聚物、硅芯片玻璃、纤维素、聚苯乙烯珠、聚丙烯片、无孔硅珠、聚丙烯酰胺或聚丙烯酸酯。在某些的实施方式中，固相载体部分为长链烷胺控孔玻璃(LCAA-CPG)。
在某些的实施方式中，T1和T2之一为靶向基团或亲脂性基团并进一步包含通过间隔物链接的载体部分。在一些更具体的实施方式中，其中载体部分通过间隔物与靶向基团或亲脂性基团中的连接子或者缀合接头中的羟基相连。
在某些的实施方式中，T1和T2的另一个为活性磷基团，所述的活性磷基团具有如下结构：
其中M1为H、取代或未取代的C1-C6烷基、OR5、SR5、OH、SH或NR6R7，M2为OH、SH、OR5′或NR6′R7′，每个R5、R6、R7、R5′、R6′或R7′独立地为氢、取代或未取代的C1-C6烷基、磺酰基；m为0或者1。
在某些的实施方式中，M1选自甲基、乙基、丙基、异丙基。
在某些的实施方式中，M1选自OR5，R5选自取代或未取代的甲基、乙基、丙基、异丙基。
在某些的实施方式中，M1选自甲磺酰胺基。
在某些的实施方式中，M2选自N(CH(CH3)2)2。
在某些的实施方式中，每个R5、R6或R7独立地为取代烷基中，所述的取代基选自氰基、卤素、羟基、氨基。
在某些的实施方式中，其中M1为O(CH2)2CN，M2为N(CH(CH3)2)2，m为0。
在某些的实施方式中，T1和T2的另一个为活性磷基团，所述的活性磷基团选自二异丙基氰基乙氧基亚磷酰胺、二异丙基甲基亚磷酰胺、二异丙基乙基亚磷酰胺和H-膦酸酯。
在某些的实施方式中，T1和T2的一个是保护基，而另一个是载体间隔物、通过间隔物链接的载体部分、靶向基团或亲脂性基团，所述亲脂性基团任选地经由连接子与A1或者A2之一相连。
在某些的实施方式中，T1和T2的一个是二异丙基氰基乙氧基亚磷酰胺，而另一个是4,4’-二甲氧三苯甲基。
在某些实施方案中，T1和T2的一个是二异丙基氰基乙氧基亚磷酰胺，而另一个是靶向基团。
在某些实施方案中，T1和T2的一个是二异丙基氰基乙氧基亚磷酰胺，而另一个是亲脂性基团。
在某些实施方案中，R1分别独立地选自卤素、取代或未取代的C1-C6烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基。
在某些实施方案中，n为0。
在某些实施方案中，本文提供的化合物具有式(IIa)或者式(IIb)的构型：
A1、A2、T1、T2、R1以及n如前文所述。
在某些实施方案中，本文提供的化合物具有式(IIa-1)或者式(IIb-1)的构型：
A1、A2、T1、T2、R1以及n如前文所述。
根据本发明的另一个方面，提供了一种寡聚化合物，其包含至少一种具有式(III)所示或其立体异构体的5′-末端和/或者3′-末端单体：
A1和A2分别独立的选自：O、S、SO、SO2、NR2和CR3R4，R2、R3和R4分别独立的选自：氢、卤素、磺酰基、亚磺酰基、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基；
T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连，并且T3和T4中的另一个为将所述式(III)单体或其立体异构体连接到寡聚化合物的核苷间连接基团；
R1分别独立地选自卤素、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基；
n为0-6的整数；
所述的寡聚化合物任选地进一步包含靶向基团。
在某些的实施方式中，提供的寡聚化合物，A1为O。在某些的实施方式中，提供的寡聚化合物，A2为O。在某些的实施方式中，提供的寡聚化合物，A1和A2分别独立为O。在某些的实施方式中，提供的寡聚化合物，A1和A2分别独立为S。在某些的实施方式中，提供的寡聚化合物，A1和A2分别独立为NR2。在某些的实施方式中，提供的寡聚化合物，R2为氢或者CH3。在某些的实施方式中，提供的寡聚化合物，A1不同于A2。在某些的实施方式中，提供的寡聚化合物，A1为O，A2为S。在某些的实施方式中，提供的寡聚化合物，A1为O，A2为NR2。在某些的实施方式中，提供的寡聚化合物，A1为S，A2为NR2。
在某些的实施方式中，提供的寡聚化合物，T3和T4的另一个为将式(III)单体或其立体异构体连接到寡聚化合物5′-末端和/或3′-末端的核苷间连接基团，所述的核苷间连接基团选自含磷的链接基团或者不含磷的链接基团。
在某些的实施方式中，提供的寡聚化合物，所述的含磷核苷间连接基团具有如下所示的结构片段：X表示H、取代或未取代的C1-C6烷基、OR8、SR8′、OH、SH或NR9R10，Y表示O或者S，z可以是0或者1，R8、R8′、R9或R10独立地为氢、取代或未取代的C1-C6烷基、磺酰基，分别独立地表示与本文的A1或者A2之一相连部分，以及与相邻核苷酸相连部分。
在某些的实施方式中，含磷核苷间连接基团独立地为：磷酸二酯连接基团、磷酸三酯连接基团、硫代磷酸酯连接基团、二硫代磷酸酯键连接基团、烷基膦酸酯连接基团、氨基膦酸酯连接基团、膦酸酯连接基团、次膦酸酯连接基团、硫代氨基磷酸酯连接基团和氨基磷酸酯连接基团。
在某些的实施方式中，所述的核苷间连接基团独立地为烷基膦酸酯连接基团、磷酸二酯核苷间连接基团或硫代磷酸酯核苷间连接基团。
在某些的实施方式中，提供的寡聚化合物，所述的T3和T4之一的保护基根据A1或者A2不同，对应的分别是羟基保护基、巯基保护基以及胺基保护基。
在某些的实施方式中，提供的寡聚化合物，T3和T4之一保护基是羟基保护基；在一个实施方式中，提供的寡聚化合物，所述羟基保护基各自独立地选自乙酰基、叔丁基、叔丁氧基甲基、甲氧基甲基、四氢吡喃基、1-乙氧基乙基、1-(2-氯乙氧基)乙基、2-三甲基甲硅烷乙基、对氯苯基、2,4-二硝基苯基、苄基、苯甲酰基、对苯基苯甲酰基、2,6-二氯苄基、二苯基甲基、对硝基苄基、三甲基甲硅烷基、三乙基甲硅烷基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基、三苯甲硅烷基、三异丙基甲硅烷基、苯甲酰基甲酸酯、氯乙酰基、三氯乙酰基、三氟乙酰基、新戊酰基、9-芴甲氧羰基、甲磺酸基、甲苯磺酸基、三氟甲磺酸基、三苯甲基、单甲氧基三苯甲基、二甲氧三苯甲基、三甲氧基三苯甲基或取代的9-苯基黄嘌呤-9-基；在一个实施方式中，提供的寡聚化合物，优选的羟基保护基各自独立地选自乙酰基、苄基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基以及4,4’-二甲氧三苯甲基。
在某些的实施方式中，提供的寡聚化合物，T3和T4之一保护基是巯基保护基；在一个实施方式中，提供的寡聚化合物，所述巯基保护基各自独立地选自乙酰基、叔丁基、叔丁氧基甲基、甲氧基甲基、四氢吡喃基、1-乙氧基乙基、1-(2-氯乙氧基)乙基、2-三甲基甲硅烷乙基、对氯苯基、2,4-二硝基苯基、苄基、苯甲酰基、对苯基苯甲酰基、2,6-二氯苄基、二苯基甲基、对硝基苄基、三甲基甲硅烷基、三乙基甲硅烷基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基、三苯甲硅烷基、三异丙基甲硅烷基、苯甲酰基甲酸酯、氯乙酰基、三氯乙酰基、三氟乙酰基、新戊酰基、9-芴甲氧羰基、甲磺酸基、甲苯磺酸基、三氟甲磺酸基、三苯甲基、单甲氧基三苯甲基、二甲氧三苯甲基、三甲氧基三苯甲基或取代的9-苯基黄嘌呤-9-基；在一个实施方式中，提供的寡聚化合物，优选的巯基保护基各自独立地选自苄基、4,4’-二甲氧三苯甲基。
在某些的实施方式中，提供的寡聚化合物，T3和T4之一保护基是氨基保护基；在一个实施方式中，提供的寡聚化合物，所述氨基保护基各自独立地选自2-三甲基硅基乙氧基羰基(Teoc)、1-甲基-1-(4-联苯基)乙氧基羰基(Bpoc)、叔丁氧基羰基(BOC)、烯丙氧基羰基(Alloc)、9-芴基甲氧基羰基(Fmoc)、苄氧基羰基(Cbz)、甲酰基、乙酰基、三卤乙酰基、苯甲酰基、硝基苯基、乙酰基、2-硝基苯磺酰基、邻苯二甲酰亚胺基和二硫代丁二酰基。
在某些的实施方式中，提供的寡聚化合物，T3和T4的一个为氢，T3和T4的另一个为将式(III)单体或其立体异构体连接到寡聚化合物5′-末端和/或3′-末端的核苷间连接基团。
在某些的实施方式中，提供的寡聚化合物，T3和T4之一亲脂性基团，其中亲脂性基团的亲脂性可以通过logKow测定，logKow超过0；优选地亲脂性基团logKow超过1，超过1.5，超过2，超过3，超过4，超过5或超过10。在一个某些的实施方式中，提供的寡聚化合物中，亲脂性基团选自含有饱和或不饱和的C4-C30烃链、脂肪环、芳香族、脂肪酸基团或脂肪酸衍生的基团、类固醇衍生的基团和任何脂溶性维生素基团。
在某些的实施方式中，提供的寡聚化合物中，亲脂性基团为饱和或不饱和C10-C25烃链(例如C10-C25烷基或烯基)。在一些实施例中，提供的寡聚化合物中，亲脂性基团为饱和或不饱和C10-C18烃链(例如直链C6-C18烷基或烯基)。在一个实施例中，提供的寡聚化合物中，亲脂性基团为饱和或不饱和的C14、C16或者C18烃链(例如直链C16烷基或烯基)。在一个实施例中，提供的寡聚化合物中，亲脂性基团为饱和的直链C14、C16或者C18烷基。亲脂性基团任选的被选自由以下官能团组成的组进一步取代：羟基、胺、羧酸、磺酸酯、磷酸酯、硫醇、叠氮基及炔烃。这些官能团可用于将亲脂性基团附接至A1或者A2中。
在另一个实施例中，提供的寡聚化合物中，亲脂性基团包含具有4到28个碳原子的烃链的任何饱和或不饱和脂肪酸，并且可以含有一个或两个羧基。在一个实施例中，提供的寡聚化合物中，所述亲脂性基团是具有8到24个碳原子的烃链的饱和脂肪酸衍生的基团。在某些实施例中，提供的寡聚化合物中，所述亲脂性基团是饱和脂肪酸衍生的基团，其中所述脂肪酸选自由以下组成的组：辛酸、癸酸、十二烷酸、十四烷酸、十六烷酸、十八烷酸、二十烷酸、二十二烷酸和二十四烷酸。在某些实施例中，提供的寡聚化合物中，所述亲脂性基团是不饱和脂肪酸衍生的基团，其中所述脂肪酸选自由以下组成的组：肉豆蔻脑酸、棕榈油酸、十六碳烯酸、油酸、反油酸、反型异油酸、亚油酸、反亚油酸、α-亚麻酸、花生四烯酸和芥酸。
在某些的实施方式中，提供的寡聚化合物中，亲脂性基团选自胆固醇、维生素E(生育酚)或胆汁酸。
在某些的实施方式中，提供的寡聚化合物中，亲脂性基团选自二十二碳六烯酸酸(DHA)、二十碳五烯酸(EPA)、二十二烷酸(DCA)、甾醇胆固醇(胆汁)、生育酚琥珀酸酯(TS)和石胆酸(LA)、维甲酸(维生素A酸)。
亲脂性基团可经由直接附接于A1或者A2以共价键方式共价连接。可替代地，亲脂性基团可经由连接子与A1或者A2共价连接。
在一个实施例中，提供的寡聚化合物中，T3和T4之一为亲脂性基团，提供的寡聚化合物中，所述T3和T4的亲脂性基团进一步通过连接子与A1或者A2共价连接。在某些实施例中，提供的寡聚化合物中，亲脂性基团经由一个或多个连接子与A1或者A2共价连接。
在另一个实施例中，提供的寡聚化合物中，所述连接子是烃链连接子或聚乙二醇(PEG)连接子。在另一个实施例中，提供的寡聚化合物中，所述连接子选自由以下组成的组：酰胺键、磷脂酰胆碱、烃连接子或聚乙二醇(PEG)连接子、氨基-烷基-醇、羟脯氨酸、羟脯氨醇、氨基-烷基-硫代磷酸酯连接子、氨基-PEG-硫代磷酸酯连接子、α-羧酸盐-氨基-烷基硫代磷酸酯连接子和α-羧酸盐-氨基-PEG-硫代磷酸酯连接子。在一些实施例中，所述的连接子含有醚、硫醚、脲、碳酸酯、胺、酰胺、马来酰亚胺-硫醚、二硫化物、磷酸二酯、磺酰胺键、点击反应的产物(例如，来自叠氮化物-炔烃环加成的三唑)或氨基甲酸酯。在另一个实施例中，提供的寡聚化合物中，所述磷酸二酯连接子是硫代磷酸二酯、氧代磷酸二酯。
一些实施例中，提供的寡聚化合物中，所述连接子包括可裂解基团。至少连接子是氧化还原可裂解接头，诸如还原可裂解接头，例如二硫化物基团、酸可裂解接头(例如，腙基、酯基、缩醛基或缩酮基)、酯酶可裂解接头(例如，酯基)、磷酸酶可裂解接头(例如，磷酸酯)或肽酶可裂解接头(例如，肽键)。在其他实施例中，至少一个连接子是生物可裂解接头，该生物可裂解接头选自由以下组成的组：DNA，RNA，二硫化物，酰胺，半乳糖胺、葡糖胺、葡萄糖、半乳糖、甘露糖的官能化单糖或寡糖及其组合。
一些实施例中，提供的寡聚化合物中，连接子还可以是环状基团或非环状基团。在一个实施例中，环状基团选自由以下组成的组：吡咯烷基、吡唑啉基、吡唑烷基、咪唑啉基、咪唑烷基、哌啶基、哌嗪基、[1,3]二氧戊环、噁唑烷基、异噁唑烷基、吗啉基、噻唑烷基、异噻唑烷基、喹喔啉基、哒嗪酮基、四氢呋喃基及十氢化萘。在一个实施例中，非环状基团为基于丝氨醇主链或二乙醇胺主链的部分。
在某些的实施方式中，该寡聚化合物中5′-末端和/或3′-末端还包含一种或更多种靶向基团。在某些的实施方式中，提供的寡聚化合物，T3和T4中的之一表示为共价键，该寡聚化合物通过式(III)或其立体异构体中的T3和T4之一以共价键形式与靶向基团相连。在某些的实施方式中，提供的寡聚化合物，该5′-末端和/或3′-末端核苷酸与靶向基团相连。所述靶向基团可以是包含siRNA给药领域常规使用的配体。
在一些实施方式中，提供的寡聚化合物中，所述靶向基团可以选自包含以下靶向分子或其衍生物形成的配体中的一种或多种：聚合物、糖类、肝实质细胞表达的受体配体、抗体、量子点、多肽，或小分子配体。
在一些实施方式中，提供的寡聚化合物中，至少一个或每个所述靶向基团选自包含能够和哺乳动物肝实质细胞表面受体结合的配体。
在一些实施方式中，提供的寡聚化合物中，每个所述靶向基团独立地为包含与哺乳动物肝细胞表面的去唾液酸糖蛋白受体(ASGPR)亲和的配体。
在一些实施方式中，提供的寡聚化合物中，每个所述靶向基团独立地选自包含D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D吡喃甘露糖、α-D-吡喃葡萄-糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳-糖胺、N-乙酰半乳糖胺、N-三氟乙酰半乳糖胺、N-丙酰半乳糖胺、N-正丁酰半乳胺、N-异丁-36酰半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6O-三苯甲基-α-D吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖、L-4-硫代核糖中的一种。
在一些实施方式中，提供的寡聚化合物中，至少一个或每个所述靶向基团为包含半乳糖或N-乙酰半乳糖胺的配体。
提供的寡聚化合物中，靶向基团可经由合适的缀合接头与A1或者A2之一相连。本领域技术人员可以根据靶向基团的具体类型选择合适的缀合接头。
在某些的实施方式中，提供的寡聚化合物中，靶向基团与A1或者A2之一相连选自以下化合物片段之一：
在某些实施方案中，R1分别独立地选自卤素、取代或未取代的C1-C6烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基。
在某些实施方案中，提供的寡聚化合物，n为0。
在某些实施方案中，提供的寡聚化合物，包含至少一种式(IVa)或者式(IVb)所示的构型的5′-末端和/或者3′-末端单体：
A1、A2、T3、T4、R1以及n如前文所述。
在某些实施方案中，提供的寡聚化合物，包含至少一种式(IVa-1)或者式(IVb-1)所示的构型的5′-末端和/或者3′-末端单体：
A1、A2、T3、T4、R1以及n如前文所述。
在某些实施方案中，提供的寡聚化合物，T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连，并且T3和T4中的另一个为将一种式(III)或其立体异构体、式(IVa)、式(IVb)、(IVa-1)或式(IVb-1)单体连接到寡聚化合物5′-末端的核苷间连接基团。
在某些实施方案中，提供的寡聚化合物，T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连，并且T3和T4中的另一个为将一种式(III)或其立体异构体、式(IVa)、式(IVb)、(IVa-1)或式(IVb-1)单体连接到寡聚化合物3′-末端的核苷间连接基团。
在某些实施方案中，提供的寡聚化合物，T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连，并且T3和T4中的另一个分别独立地将一种相同或者不同的(III)或其立体异构体、式(IVa)、式(IVb)、(Iva-1)或式(IVb-1)单体连接到寡聚化合物5′-末端和3′-末端的核苷间连接基团。
在某些实施方案中，所述寡聚化合物是单链寡核苷酸。在某些实施方案中，所述单链寡核苷酸是常规的反义寡核苷酸(又称ASO)、核糖酶或适体。在某些实施方案中，所述寡聚化合物是双链核糖核酸(dsRNA)试剂，这类化合物本领域所熟知的双链核苷核酸，其中一条或两条链为如本文所公开的寡聚化合物。
在某些实施方案中，提供了双链核糖核酸(dsRNA)试剂，它们均包含：正义链和反义链，其中所述正义链和反义链完全或者部分互补，并且所述反义链与核酸靶基因部分或者完全互补；所述正义链和反义链的至少一者为如前文所提供的寡聚化合物，其包含至少一种具有式(III)或其立体异构体、式(IVa)、式(IVb)、式(IVa-1)或者式(IVb-1)的5′-末端和/或者3′-末端单体；并且其中所述双链核糖核酸(dsRNA)试剂任选地进一步包含独立的靶向基团。
在某些实施方案中，提供了双链核糖核酸(dsRNA)试剂，其中正义链为包含本文式(III)或其立体异构体、式(IVa)、式(IVb)、式(IVa-1)或者式(IVb-1)的5′-末端和/或者3′-末端单体所提供的寡聚化合物。在一个实施方案中，提供了双链核糖核酸(dsRNA)试剂，其中正义链为包含本文式(III)或其立体异构体、式(IVa)、式(IVb)、式(IVa-1)或者式(IVb-1)的5′-末端单体所提供的寡聚化合物。在一个实施方案中，提供了双链核糖核酸(dsRNA)试剂，其中正义链为包含本文式(III)或其立体异构体、式(IVa)、式(IVb)、式(IVa-1)或者式(IVb-1)的3′-末端单体所提供的寡聚化合物。在一个实施方案中，提供了双链核糖核酸(dsRNA)试剂，其中正义链为包含相同或者不同本文式(III)或其立体异构体、式(IVa)、式(IVb)、式(IVa-1)或者式(IVb-1)的5′-末端和3′-末端单体所提供的寡聚化合物。
在某些实施方案中，提供了双链核糖核酸(dsRNA)试剂，其中反义链为包含本文式(III)或其立体异构体、式(IVa)、式(IVb)、式(IVa-1)或者式(IVb-1)的5′-末端和/或者3′-末端单体提供的寡聚化合物。在一个实施方案中，提供了双链核糖核酸(dsRNA)试剂，其中反义链为包含本文式(III)或其立体异构体、式(IVa)、式(IVb)、式(IVa-1)或者式(IVb-1)的3′-末端单体提供的寡聚化合物。在一个实施方案中，提供了双链核糖核酸(dsRNA)试剂，其中反义链为包含本文式(III)或其立体异构体、式(IVa)、式(IVb)、式(IVa-1)或者式(IVb-1)的5′-末端单体提供的寡聚化合物。
在某些的实施方式中，提供了双链核糖核酸(dsRNA)试剂，任选地进一步包含独立的靶向基团，在双链核糖核酸(dsRNA)试剂中任意一条链的5′-末端和/或3′-末端核苷酸上还包含一种或更多种靶向基团。所述靶向基团可以是包含siRNA给药领域常规使用的配体。
在一些实施方式中，提供的双链核糖核酸(dsRNA)试剂中，所述靶向基团可以选自包含以下靶向分子或其衍生物形成的配体中的一种或多种：聚合物、糖类、肝实质细胞表达的受体配体、抗体、量子点、多肽，或小分子配体。
在一些实施方式中，提供的双链核糖核酸(dsRNA)试剂中，至少一个或每个所述靶向基团选自包含能够和哺乳动物肝实质细胞表面受体结合的配体。
在一些实施方式中，提供的双链核糖核酸(dsRNA)试剂中，每个所述靶向基团独立地为包含与哺乳动物肝细胞表面的去唾液酸糖蛋白受体(ASGPR)亲和的配体。
在某些实施方案中，寡聚化合物或双链核糖核酸(dsRNA)中，每一条链均分别包含8-40核苷酸长度。
在某些实施方案中，提供的寡聚化合物或双链核糖核酸(dsRNA)试剂在药物制备中的用途，所述药物用于抑制基因表达。在某些实施方案中，其中所述抑制基因表达包括用所述寡聚化合物接触一种或多种细胞、组织或动物。
在某些实施方案中，提供了抑制基因表达的方法，该方法包括：使细胞与包括具有本文所述的寡聚化合物或双链核糖核酸(dsRNA)试剂接触，其中该寡聚化合物以及该双链核糖核酸(dsRNA)试剂中的每一条链均分别包含8-40核苷酸长度，并且该寡聚化合物或该双链核糖核酸(dsRNA)试剂的反义链与靶RNA互补。
在某些实施方案中，细胞在动物中。在某些实施方案中，细胞在人中。在某些实施方案中，靶RNA选自mRNA、pre-mRNA和micro RNA。在某些实施方案中，靶RNA为mRNA。在某些实施方案中，靶RNA为人mRNA。在某些实施方案中，靶RNA被裂解，从而抑制其功能。在某些实施方案中，该方法进一步包括检测靶RNA的水平。在某些实施方案中，提供了抑制基因表达的方法，该方法包括：使一种或多种细胞或组织与包括具有式III、式(IVa)、式(IVb)、式(IVa-1)或者式(IVb-1)的末端单体提供的寡聚化合物或者双链核糖核酸(dsRNA)试剂接触。这些基因除肝脏组织表达外，还可以是外组织表达的基因，例如眼科以及CNS的靶细胞基因。
发明详述
在某些实施方案中，提供了具有双环脱碱基核酸类似物，并且作为核苷酸单体可用于并入到寡聚化合物的一个或者两个末端。本文还提供了用于制备这些寡聚化合物的中间体和方法。本文提供的修饰的双环脱碱基核酸类似物可用于增强它们所并入的寡聚化合物的一种或多种特性，诸如，例如核酸酶抗性。在某些实施方案中，本文提供的寡聚化合物和组合物预期与靶RNA的一部分杂交以使该靶RNA丧失正常功能。所述寡聚化合物预期还可在诊断应用中用作为引物和探针。
根据本发明的一个方面，提供了式(I)所示的化合物或其立体异构体：
A1和A2分别独立的选自：O、S、SO、SO2、NR2和CR3R4，R2、R3和R4分别独立的选自：氢、卤素、磺酰基、亚磺酰基、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基；
T1和T2之一为保护基、Z、L以及-Z-L，所述的Z表示为靶向基团或亲脂性基团，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，L表示为：载体间隔物或包含通过间隔物链接的载体部分；而T1和T2的另一个为活性磷基团、保护基；并且T1和T2不会同时为保护基；
R1分别独立地选自卤素、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基；
n为0-6的整数；
其中式(I)不包括如下结构：
在某些的实施方式中，A1为O。
在某些的实施方式中，A2为O。
在某些的实施方式中，A1和A2分别独立为O，提供的化合物具有式(I-1)所示或其立体异构体，其中T1、T2、R1以及n如前文所述。
在某些的实施方式中，A1和A2分别独立为S，提供的化合物具有式(I-2)所示或其立体异构体，其中T1、T2、R1以及n如前文所述。
在某些的实施方式中，A1和A2分别独立为NR2，提供的化合物具有式(I-3)所示或其立体异构体，其中T1、T2、R1以及n如前文所述，R2独立地选自：氢、卤素、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基。
在某些的实施方式中，A1为O，A2为S，提供的化合物具有式(I-4)所示或其立体异构体，其中T1、T2、R1以及n如前文所述。
在某些的实施方式中，A1为O，A2为NR2，提供的化合物具有式(I-5)所示或其立体异构体，其中T1、T2、R1以及n如前文所述，R2选自：氢、卤素、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基。
在某些的实施方式中，A1为S，A2为NR2，提供的化合物具有式(I-6)所示或其立体异构体，其中T1、T2、R1以及n如前文所述，R2选自：氢、卤素、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基。
在某些的实施方式中，R2为氢或者CH3。
如本文所用的术语“取代或未取代的”母体化合物中氢原子未被其他取代基取代，或者一个或者多个氢原子被取代基所代替。如本文所用的术语“取代基”和“取代基团”意在包括通常添加至其他基团或母体化合物来增强期望特性或提供其他期望效应的基团。取代基团可以被保护或者不被保护，并且可以被加至母体化合物中的一个可用位点或许多可用位点。取代基团也可以进一步被其他取代基团取代，并且可以与母体化合物直接相连或通过连接基团诸如烷基或烃基基团与母体化合物相连。
本文适合的取代基团包括但不限于：卤素、羟基、烷基、烯基、炔基、酰基(-C(O)Raa)、羧基(-C(O)O-Raa)、脂肪族基团、脂环族基团、烷氧基、取代的氧(-O-Raa)、芳基、芳烷基、杂环基、杂芳基、杂芳基烷基、氨基(-N(Rbb)(Rcc))、亚氨基(＝NRbb)、酰氨基(-C(O)N(Rbb)(Rcc)或一N(Rbb)C(O)Raa、叠氮基(-N3)、硝基(-NO2)、氰基(-CN)、脲基(ureido)(-N(Rbb)C(O)N(Rbb)(Rcc))、硫脲基(-N(Rbb)C(S)N(Rbb)(Rcc))、胍基(-N(Rbb)C(＝NRbb)N(Rbb)(Rcc))、脒基(-C(＝NRbb)N(Rbb)(Rcc)或一N(Rbb)C(＝NRbb)(Raa))、硫羟基或巯基(-SRbb)、亚磺酰基(-S(O)Rbb)、磺酰基(-S(O)2Rbb)以及磺酰氨基(sulfonamidyl)(-S(O)2N(Rbb)(Rcc)或-N(Rbb)S(O)2Rbb)。其中每个Raa、Rbb和Rcc独立地是H、任选地连接的化学官能团或另外的取代基团，优选列表包括但不限于H、烷基、烯基、炔基、脂肪族、烷氧基、酰基、芳基、芳烷基、杂芳基、脂环族、杂环和杂芳基烷基。在药用化学和有机化学领域中的普通技术人员理解这些取代基的总数适度地受预定化合物的期望特性限制。
如本文所用的术语“保护基”是指本领域已知的在合成程序中保护反应性基团(包括但不限于羟基、氨基和硫羟基基团)免于不期望的反应的不稳定的化学部分。保护基通常被选择地使用以便在其他反应位点的反应过程中保护位点，然后能够被移除以照原样留下未保护的基团或者可用于进一步反应。
在某些的实施方式中，本领域理解的是，根据T1和T2之一的保护基根据A1或者A2不同，对应的分别是羟基保护基、巯基保护基以及胺基保护基；即当A1或者A2为氧时，T1和T2之一的保护基是羟基保护基；即当A1或者A2为硫、亚砜(SO)、砜(SO2)时，T1和T2之一的保护基是巯基保护基，即当A1或者A2为氮时，T1和T2之一的保护基是氨基保护基。
在某些的实施方式中，T1和T2之一保护基是羟基保护基；在一个实施方式中，所述羟基保护基各自独立地选自乙酰基、叔丁基、叔丁氧基甲基、甲氧基甲基、四氢吡喃基、1-乙氧基乙基、1-(2-氯乙氧基)乙基、2-三甲基甲硅烷乙基、对氯苯基、2,4-二硝基苯基、苄基、苯甲酰基、对苯基苯甲酰基、2,6-二氯苄基、二苯基甲基、对硝基苄基、三甲基甲硅烷基、三乙基甲硅烷基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基、三苯甲硅烷基、三异丙基甲硅烷基、苯甲酰基甲酸酯、氯乙酰基、三氯乙酰基、三氟乙酰基、新戊酰基、9-芴甲氧羰基、甲磺酸基、甲苯磺酸基、三氟甲磺酸基、三苯甲基、单甲氧基三苯甲基、二甲氧三苯甲基、三甲氧基三苯甲基或取代的9-苯基黄嘌呤-9-基；在一个实施方式中，优选的羟基保护基各自独立地选自乙酰基、苄基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基以及4,4’-二甲氧三苯甲基。
在某些的实施方式中，T1和T2之一保护基是巯基保护基；在一个实施方式中，所述巯基保护基各自独立地选自乙酰基、叔丁基、叔丁氧基甲基、甲氧基甲基、四氢吡喃基、1-乙氧基乙基、1-(2-氯乙氧基)乙基、2-三甲基甲硅烷乙基、对氯苯基、2,4-二硝基苯基、苄基、苯甲酰基、对苯基苯甲酰基、2,6-二氯苄基、二苯基甲基、对硝基苄基、三甲基甲硅烷基、三乙基甲硅烷基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基、三苯甲硅烷基、三异丙基甲硅烷基、苯甲酰基甲酸酯、氯乙酰基、三氯乙酰基、三氟乙酰基、新戊酰基、9-芴甲氧羰基、甲磺酸基、甲苯磺酸基、三氟甲磺酸基、三苯甲基、单甲氧基三苯甲基、二甲氧三苯甲基、三甲氧基三苯甲基或取代的9-苯基黄嘌呤-9-基；在一个实施方式中，优选的巯基保护基各自独立地选自苄基、4,4’-二甲氧三苯甲基。本领域知晓的，当巯基进一步被氧化为亚砜(SO)、砜(SO2)，所述的保护基同样适用于亚砜(SO)、砜(SO2)。
在某些的实施方式中，T1和T2之一保护基是氨基保护基；在一个实施方式中，所述氨基保护基各自独立地选自2-三甲基硅基乙氧基羰基(Teoc)、1-甲基-1-(4-联苯基)乙氧基羰基(Bpoc)、叔丁氧基羰基(BOC)、烯丙氧基羰基(Alloc)、9-芴基甲氧基羰基(Fmoc)、苄氧基羰基(Cbz)、甲酰基、乙酰基、三卤乙酰基、苯甲酰基、硝基苯基、乙酰基、2-硝基苯磺酰基、邻苯二甲酰亚胺基和二硫代丁二酰基。
本发明“任选的”、“任选地”和“可选的”一般表示该基团或者官能团可以存在，或者不存在的。非限制性举例“任选地经由连接子与A1或者A2相连的亲脂性基团”可以表示为以亲脂性基团形式直接以共价键形式链接到所附着的原子或者基团中；或者该亲脂性基团需通过含有一个或者多个连接子链接到所附着的原子或者基团中。
在某些的实施方式中，T1和T2之一亲脂性基团。术语“亲脂性”或“亲脂性基团”广义上是指对脂质具有亲和力的任何化合物或化学部分。表征亲脂性基团的亲脂性一种方式可以通过辛醇-水分配系数logKow测定，其中Kow为二相系统在平衡时化学物质在辛醇相中的浓度与其在水相中的浓度的比率。辛醇-水分配系数为实验室测量的物质特性。然而，其也可通过使用归因于化学物质的结构组分的系数来预测，这些系数是使用第一原理或经验方法计算的(参见例如Tetko等人,J.Chem.Inf.Comput.Sci.41:1407-21(2001)，将其通过引用以其全文并入本文)。其提供物质偏好非水性或油性环境而不是水的倾向的热力学量度(即其亲水性/亲脂性平衡)。亲脂性基团缀合的结构使得亲脂性基团(例如，脂肪酸)暴露，从而允许亲脂性基团与白蛋白和/或脂肪酸受体或转运蛋白相互作用，从而提供具有长体内半衰期的寡核苷酸。本领域可以理解的是，一般具有亲脂性的基团其logKow测定超过0。在某些的实施方式中，亲脂部分的亲油性，由辛醇-水分配系数logKow测量，亲脂部分可以超过1，超过1.5，超过2，超过3，超过4，超过5或超过10的logKow。分子的亲脂性可以相对于其携带的官能团而变化；例如，在亲脂性基团末端添加羟基、胺、羧酸、磺酸酯、磷酸酯、硫醇、叠氮基及炔烃等官能团可以增加或减少亲脂部分的分配系数(例如logKow)值；并且还可以通过这些官能团进一步将亲脂性基团附接到本文中所示的化合物中。
在某些的实施方式中，亲脂性基团为含有饱和或不饱和的C4-C30烃链、脂肪环、芳香族、脂肪酸基团或脂肪酸衍生的基团、类固醇衍生的基团和任何脂溶性维生素基团。
在一个的实施方式中，亲脂性基团为饱和或不饱和C10-C25烃链(例如C10-C25烷基或烯基)。在一些实施例中，亲脂性基团为饱和或不饱和C10-C18烃链(例如直链C10-C18烷基或烯基)。在一个实施例中，亲脂性基团为饱和或不饱和的C10、C11、C12、C13、C14、C15、C16、C17或者C18烃链(例如直链C16烷基或烯基)；更优选的是直链烷基。在一个实施例中，亲脂性基团为饱和的直链C14烷基、C16烷基或C18烷基，结构示列具有如下片段：亲脂性基团任选地被选自由以下的官能团的组成的组进一步取代：羟基、胺、羧酸、磺酸酯、磷酸酯、硫醇、叠氮基及炔烃。这些官能团可用于将亲脂性基团附接至A1或者A2中。
在另一个实施例中，亲脂性基团选自二羧酸的脂肪酸衍生的基团。在另一个实施例中，脂肪酸包含具有4到28个碳原子的烃链的任何饱和或不饱和脂肪酸，并且可以含有一个或两个羧基。在一个实施例中，所述亲脂性基团是具有8到24个碳原子的烃链的饱和脂肪酸衍生的基团。在某些实施例中，所述亲脂性基团是饱和脂肪酸衍生的基团，其中所述脂肪酸选自由以下组成的组：丁酸(CH3(CH2)2COOH)、戊酸、己酸、庚酸、辛酸、癸酸、十一烷酸、十二烷酸、十三烷酸、十四烷酸、十五烷酸、十六烷酸、十七烷酸、十八烷酸、十九烷酸、二十烷酸、二十一烷酸、二十二烷酸、二十三烷酸、二十四烷酸、二十五烷酸、二十六烷酸、二十七烷酸和二十八烷酸(CH3(CH2)16COOH)。在另一个实施例中，所述亲脂性基团是不饱和脂肪酸衍生的基团，所述的不饱和的脂肪酸是表1中呈现的中的一种；所述脂肪酸选优选为自由以下组成的组：肉豆蔻脑酸、棕榈油酸、十六碳烯酸、油酸、反油酸、反型异油酸、亚油酸、反亚油酸、α-亚麻酸、花生四烯酸和芥酸。
表1不饱和的脂肪酸
在另一个实施例中，亲脂性基团为类固醇，诸如固醇。类固醇为含有全氢-1,2-环戊菲环系统的多环化合物。类固醇包括但不限于胆汁酸(例如胆酸、去氧胆酸及脱氢胆酸)、可的松、地高辛(digoxigenin)、睾酮、胆固醇及阳离子类固醇，诸如可的松。“胆固醇衍生物”是指例如通过取代、添加或移除取代基衍生自胆固醇的化合物。
在另一个的实施方式中，亲脂性基团选自胆固醇、维生素E(生育酚)或胆汁酸。
在另一个的实施方式中，亲脂性基团选自不饱和脂肪二十二碳六烯酸酸(DHA)、二十碳五烯酸(EPA)饱和脂肪酸二十二烷酸(DCA)、甾醇胆固醇(胆汁)、生育酚琥珀酸酯(TS)和石胆酸(LA)、维甲酸(维生素A酸)。
在另一个实施例中，亲脂性基团为芳香族部分。在此上下文中，术语“芳族”或“芳香族”泛指单芳烃及多芳烃。芳基包括但不限于包含一至三个芳环的C6-C14芳基部分，其可以任选地经取代；包含与烷基共价连接的芳基的“芳烷基”或“芳基烷基”，其中的任一个可独立地任选地经取代或未经取代；及“杂芳基”。如本文所用，术语“杂芳基”是指具有5至14个环原子，优选地5、6、9或10个环原子；具有在环阵列中共享的6、10或14个π电子，并且除碳原子外具有一至约三个选自由氮(N)、氧(O)及硫(S)组成的组的杂原子的基团。
在某些的实施方式中，本发明的亲脂性基团可以通过共价键直接与A1或者A2直接共价相连。非限制性的，在一个的实施方式中，是A1和T1组合和/或者A2和T2组合片段包括但是非限制性示列结构： 其中表示单键或者双键，p或q分别独立地表示5-25的整数，p与q之和不超过25。在一些更具体的实施例A1和T1组合和/或者A2和T2组合片段包括但是非限制性示例：
在某些的实施方式中，亲脂性基团可经由连接子与A1或者A2共价连接，并且所述的连接子可以是一个或者多个的组合。
本发明的T1和T2之一为亲脂性基团，所述亲脂性基团进一步包含连接子，并通过一个或者多个连接子与A1或者A2共价连接。
在一个实施例中，所述的连接子选自由以下组成的组：酰胺键、磷脂酰胆碱、烃连接子或聚乙二醇(PEG)连接子、氨基-烷基-醇、羟脯氨酸、羟脯氨醇、氨基-烷基-硫代磷酸酯连接子、氨基-PEG-硫代磷酸酯连接子、α-羧酸盐-氨基-烷基硫代磷酸酯连接子和α-羧酸盐-氨基-PEG-硫代磷酸酯连接子。
在一个实施例中，所述的连接子含有醚、硫醚、脲、碳酸酯、胺、酰胺、马来酰亚胺-硫醚、二硫化物、磷酸二酯、磺酰胺键、点击反应的产物(例如，来自叠氮化物-炔烃环加成的三唑)或氨基甲酸酯。在另一个实施例中，提供的寡聚化合物中，所述磷酸二酯连接子是硫代磷酸二酯(非限制示列：)、氧代磷酸二酯(非限制示列：)。
在一个实施例中，所述连接子是烃链连接子或聚乙二醇(PEG)连接子。对于烃链连接子，连接子包括2-20个碳，例如2个、3个、4个、5个、6个、7个、8个、9个或10个碳。对于聚乙二醇(PEG)连接子，连接子包括1-20个乙二醇亚单元，例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个乙二醇重复。
在一个实施例中，所述连接子是酰胺键连接子。
一些实施例中，所述连接子包括可裂解基团。在一个实施例中，所述连接子包括可裂解基团，可对pH敏感，如二硫化物基团。人类血清的pH为7.4，而平均细胞内pH略微较低，范围介于约7.1-7.3。内体具有在5.5-6.0范围内的更酸的pH，并且溶酶体具有约为5.0的甚至更酸的pH。一些连接子将具有在优选的pH下裂解的连接基团，从而自细胞内的配体(例如，靶向或细胞渗透性配体，诸如胆固醇)释放iRNA剂，或进入细胞的所需区室。
在一个实施例中，所述连接子至少连接子是氧化还原可裂解接头(诸如还原可裂解接头，例如二硫化物基团)、一类可裂解连接基团是氧化还原可裂解连接基团，其在还原或氧化时裂解。还原可裂解连接基团的实例为二硫化物连接基团(-S-S-)。
酯酶可裂解接头(例如，酯基)。基于酯的连接基团通过细胞中诸如酯酶及酰胺酶的酶来裂解。基于酯的可裂解连接基团的实例包括但不限于亚烷基、亚烯基及亚炔基的酯。酯可裂解连接基团具有通式-C(O)O-或-OC(O)-。
磷酸酶可裂解接头(例如，磷酸酯)，基于磷酸酯的连接基团通过降解或水解磷酸酯的试剂来裂解。裂解细胞中磷酸酯基的试剂的实例为细胞中的酶，诸如磷酸酶。基于磷酸酯的连接基团的实例为-为-O-P(O)(OH)-O-、-S-P(S)(OH)-O-。
或肽酶可裂解接头(例如，肽键)。基于肽的连接基团是通过细胞中诸如肽酶及蛋白酶的酶来裂解。基于肽的可裂解连接基团是在氨基酸之间形成以产生寡肽(例如二肽、三肽等)及多肽的肽键。基于肽的可裂解基团不包括酰胺基(-C(O)NH-)。酰胺基可在任何亚烷基、亚烯基或亚炔基之间形成。肽键是在氨基酸之间形成以产生肽及蛋白质的特殊类型的酰胺键。基于肽的裂解基团一般限于在产生肽及蛋白质的氨基酸之间形成的肽键(即酰胺键)，并且不包括整个酰胺官能团。
在其他实施例中，至少一个连接子是生物可裂解接头，该生物可裂解接头选自由以下组成的组：DNA、RNA、二硫化物、酰胺、半乳糖胺、葡糖胺、葡萄糖、半乳糖、甘露糖的官能化单糖或寡糖及其组合。
在一个实施例中，所述α-羧酸盐-氨基-烷基-硫代磷酸酯连接子具有如下结构：
表示与亲脂基团以及A1或A2之一的链接部分，其中m优选地是2到12的整数，优选地是4到10的整数。在一个实施例中，m是2、3、4、5、6、7、8、9、10、11或12的整数。在一个实施例中m是6。
在一个实施例中，所述氨基-PEG-硫代磷酸酯连接子具有如下结构：表示与亲脂基团以及A1或A2之一的链接部分，其中m优选地是1到8的整数。在一个实施例中，m是1、2、3、4、5、6、7或8的整数。并且α-羧酸盐-氨基-PEG-硫代磷酸酯连接子：
在一个实施例中，所述氨基-烷基-硫代磷酸酯连接子具有如下结构：表示与亲脂基团以及A1或A2之一的链接部分；其中m优选地是2到12的整数。优选地是4到10的整数。在一个实施例中，m是2、3、4、5、6、7、8、9、10、11或12的整数。在一个实施例中，m是6。
在一个实施例中，所述连接子选自由以下组成的组：(i)氨基-烷基-硫代磷酸酯连接子；(ii)α-羧酸盐-氨基-烷基-硫代磷酸酯连接子；(iii)氨基-PEG-硫代磷酸酯连接子，以及(iv)α-羧酸盐-氨基-PEG-硫代磷酸酯连接子所有如以上在提供的式中所定义。
在一个实施例中，本发明提供了例如基于羟脯氨酸而被构象限制的连接子，例如，
表示与亲脂基团以及A1或A2之一的链接部分，其中m优选地是1到8的整数。在一个实施例中，m是2、3、4、5、6、7或8的整数。
在一个实施例中，本发明提供了例如基于羟脯氨醇而被构象限制的连接子，例如，表示与亲脂基团以及A1或A2之一的链接部分。
在一个实施例中，本发明提供了例如氨基-烷基-醇连接的连接子，例如，表示与亲脂基团以及A1或A2之一的链接部分，其中m优选地是1到8的整数。在一个实施例中，m是1、2、3、4、5、6、7或8的整数。
在一个实施例中，本发明提供了例如磷脂酰胆碱的连接子，例如，
表示与亲脂基团以及A1或A2之一的链接部分。
一些实施例中，连接子还可以是环状基团或非环状基团。在一个实施例中，环状基团选自由以下组成的组：吡咯烷基、吡唑啉基、吡唑烷基、咪唑啉基、咪唑烷基、哌啶基、哌嗪基、[1,3]二氧戊环、噁唑烷基、异噁唑烷基、吗啉基、噻唑烷基、异噻唑烷基、喹喔啉基、哒嗪酮基、四氢呋喃基及十氢化萘。在一个实施例中，非环状基团为基于丝氨醇主链或二乙醇胺主链的部分。
关于亲脂性基团和连接子的更多论述可见于2019年7月7日提交的名称为“肝外递送”的PCT公开号WO2019/217459，2019年5月10日提交的名称为“包括7′-5′-α-异头双环糖核苷的寡核苷酸缀合物”的PCT公开号WO2019/215333，以及Nucleic Acids Research,2019,Vol.47,No.31082–1096《多种脂质偶联物用于功能性肝外体内核苷酸递送》，将其内容通过引用以其全文并入本文。
在一个实施例中，本发明提供了亲脂性基团通过连接子与A1或者A2共价连接的形成化合物非限制性示例，
在一些实施方式中，T1和T2之一为靶向基团，所述靶向基团可以是包含siRNA给药领域常规使用的配体。所述靶向基团可以改变它所并入的siRNA试剂的分布、靶向或寿命，内体溶解特性、改进运载、杂交以及特异性特性，例如WO2009082607A2中描述的各种配体，以引用的方式将其全部公开内容并入本文。
在一些实施方式中，所述靶向基团可以选自以下靶向分子或其衍生物形成的配体中的一种或多种；如聚合物，例如聚乙二醇；糖类，例如乳糖、聚乳糖、甘露糖、半乳糖、N-乙酰半乳糖胺(GalNAc)；肝实质细胞表达的受体配体，例如去唾液酸糖蛋白、去唾液酸糖残基、脂蛋白(如高密度脂蛋白、低密度脂蛋白等)、胰高血糖素、神经递质(如肾上腺素)、生长因子、转铁蛋白等适配体；抗体；量子点；多肽，例如透膜肽，或小分子配体。
在一些实施方式中，至少一个或每个所述靶向基团选自包含能够和哺乳动物肝实质细胞表面受体结合的配体。在一些实施方式中，每个所述靶向基团独立地为包含与哺乳动物肝细胞表面的去唾液酸糖蛋白受体(ASGPR)亲和的配体。在一些实施方式中，每个所述靶向基团独立地为包含去唾液酸糖蛋白或糖的配体。在一些实施方式中，每个所述靶向基团独立地为包含去唾液酸糖蛋白的配体，例如去唾液酸血清类枯蛋白(asialoorosomucoid,ASOR)或去唾液酸始球蛋白(asialofetuin，ASF)。在一些实施方式中，每个所述靶向基团独立地包含选自D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D吡喃甘露糖、α-D-吡喃葡萄-糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳-糖胺、N-乙酰半乳糖胺、N-三氟乙酰半乳糖胺、N-丙酰半乳糖胺、N-正丁酰半乳胺、N-异丁-36酰半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6O-三苯甲基-α-D吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖、L-4-硫代核糖中的一种的配体。
在一些实施方式中，至少一个或每个所述靶向基团为包含半乳糖或N-乙酰半乳糖胺的配体。
在一些实施方式中，靶向基团包括通过二价或三价分支接头经合适的系链结合的一个或多个“GalNAc”(N‐乙酰半乳糖胺)衍生物。
其中:
q2A、q2B、q3A、q3B、q4A、q4B、q5A、q5B、q5C、q6A、q6B和q6C每次出现独立地表示0-20，并且其中的重复单元可以相同或不同；
P2A、P2B、P3A、P3B、P4A、P4B、P5A、P5B、P5C、P6A、P6B、P6C、T2A、T2B、T3A、T3B、T4A、T4B、T4A、T5B、T5C、T6A、T6B、T6C每次出现各自独立地表示：不存在、CO、NH、O、S、OC(O)、NHC(O)、CH2、CH2NH或CH2O；
Q2A、Q2B、Q3A、Q3B、Q4A、Q4B、Q5A、Q5B、Q5C、Q6A、Q6B、Q6C每次出现独立地表示：不存在、亚烷基、取代的亚烷基，其中一个或多个亚甲基可以被如下一个或多个基团所中断或终止，O、S、S(O)、SO2、NH、C(R’)＝C(R”)、C≡C or C(O)；
R2A、R2B、R3A、R3B、R4A、R4B、R5A、R5B、R5C、R6A、R6B、R6C每次出现各自独立地表示：不存在、NH、O、S、C H2、C(O)O、C(O)N H、N H C H(R a)C(O)、-C(O)-C H(R a)-N H-、C O、C H＝N-O或杂环基；
L2A、L2B、L3A、L3B、L4A、L4B、L5A、L5B、L5C、L6A、L6B和L6C每次出现各自独立地表示：单糖(诸如GalNAc)、二糖、三糖、四糖、寡糖、或多糖；
R'和R”各自独立地是H、CH3、0H、SH、NH2、烷基、烯基或炔基；
Ra是H或氨基酸侧链；m为1或2。
本发明的靶向基团-缀合的式(I)化合物或其立体异构体以及iRNA试剂，可以通过使用具有反应性官能团，例如亚磷酰胺直接反应制备。靶向基团可经由合适的缀合接头与A1或者A2之一相连，本领域技术人员可以根据靶向基团的具体类型选择合适的缀合接头。例如反-4羟基脯氨酸、氨基烷基醇衍生物这些通过反应性官能团形成磷酸酯，以下示列性片段与可降解的结构如酯、酰胺、磷酸酯缀合成合适的缀合接头：
靶向基团非限制结构示意：
缀合接头、配体的种类、系链、分支接头，以及靶向基团与寡核苷酸的连接方式可参见WO2009073809、WO2014179620、WO2014179627、WO2015006740A2的公开内容，通过引用的方式将其整体内容并入本文。在一些实施方式中，所述靶向基团可以通过酸不稳定的或可还原的化学键相连，在细胞内涵体的酸性环境下，这些化学键可降解。
术语“缀合接头”或“连接子”是指连接化合物的两个部分，例如共价地结合化合物的两个部分。适用于本发明的其他糖类缀合物和接头包含那些在公开案WO2014/179620和WO2014/179627中描述的，其各自的全部内容通过引用在此并入本文。
在某些的实施方式中，靶向基团与A1或者A2之一相连选自以下化合物片段之一：
在某些的实施方式中，T1和T2之一为载体间隔物。本领域熟知的是在寡核苷酸合成中，将待合成的寡核苷酸的3'‐末端的非核苷或核苷(如本文式(III)及其立体异构体中或片段部分)通过可切割的间隔物如琥珀酰基，再与载体部分中的接头如反应性氨基链接，进行预先负载，然后装柱进行后续核苷酸的对接；载体间隔物充当合成过程中寡核苷酸的附连点，经常通过寡核苷酸中的非核苷或核苷的3'‐末端羟基共价结合到所述载体间隔物，并在整个链组装过程中保持附连，随后它们被切掉并去保护。寡核苷酸合成，间隔物经常选择避免空间位阻以及可切割，将寡核苷酸系连到其上，用于将所述寡核苷酸和合成过程放置得远离所述载体。这种方式允许更大的灵活性和更多的用于合成的空间。通过载体间隔物与寡核苷酸末端非核苷或核苷3'末端的核糖的2'位或3'位的羟基的氧原子(例如本文所述的式(I)及其立体异构体中的A1或者A2)连接的载体部分，更具体而言可以通过以下示列，
Sp表示载体间隔物。
在寡核苷酸合成领域应用的载体间隔物(Sp)均可归纳于本文中，经常使用的例子，包括二羧酸衍生的基团，所示的结构R为不存在、醚、聚乙二醇、氧、硫、氮、亚烷基、烯基、炔基、芳基、芳烷基、杂烷基或杂芳基，为一端与式(I)中A1或者A2链接处，另一端羧基可进一步与载体处相连，R更优选地为不存在、氧、硫、氮、亚烷基、烯基、炔基；另一个例子如聚亚烷基二醇磷酸酯/膦酸酯，如在公开的专利CN103233003所示；在某些的实施方式中，间隔物为C3-C6烷基二羧酸根基团，所示的结构在某些的实施方式中，所述的载体间隔物优选为琥珀酰基，在一些实施例中，间隔物二羧酸的进一步被衍生，衍生物的示列如进一步被PEG化，P为5～120的整数。另一端羧基可进一步与载体部分链接形成共价键。
在某些的实施方式中，T1和T2之一为包含通过间隔物链接的载体部分，其中所述的载体部分包括固相载体部分和可溶性载体部分。
载体上一般含有接头，例如可以长链烷基胺，非限制示列通过丙胺基接头被连接至固体载体的Si上，本领域经常被使用的的LCAA-CPG。在一些实施例中，长链烷基胺与固体载体通过生物可降解的接头如乙酰甘油进一步附接。
在某些的实施方式中，固相载体的例子包括无机多孔载体和有机树脂载体，无机多孔载体的例子包括可控多孔玻璃(CPG)，有机树脂载体的例子包括由聚苯乙烯制成的载体。一些固相载体的例子包括控孔玻璃(CPG)、聚苯乙烯、硅胶、大孔交联聚苯乙烯、聚甲基丙烯酸乙烯醇共聚物、硅芯片玻璃、纤维素、聚苯乙烯珠、聚丙烯片、无孔硅珠、聚丙烯酰胺或聚丙烯酸酯。
近年来，可溶性载体应用于发展成一种重要的寡核苷酸合成方法，可以用于大规模的液相合成。在某些的实施方式中，可溶性载体非限制举例：
间隔物、固体载体、接头以及其链接方式组成在本领域中一般也合称为固相支持物，其构成也是公知的如《核酸化学现代方法》(Current Protocols in Nucleic Acid Chemistry)，2000，第3章，第3.1节中的“用于寡核苷酸合成的”(Solid-Phase Supports for Oligonucleotide Synthesis)中所描述。关于更多这些间隔物、载体、接头以及其链接方式可参见于《Synthesis of oligonucleotides on a soluble support》Beilstein J.Org.Chem.2017,13,1368–1387.，WO2021/173615、CN103233003A、WO2010/131916、WO2022/005988A、CN115003680A中公开的内容，通过引用的方式将其整体内容并入本文。
[根据细则26改正 12.12.2023]
在某些的实施方式中，T1和T2之一为靶向基团或亲脂性基团，并进一步通过间隔物链接到载体的部分。特别是在本文所述的式(I)化合物或其立体异构体用于寡核苷酸3'端时，示列性的结构，
[根据细则26改正 12.12.2023]
表示与本文A1或者A2相连部分；在一些更具体的实施方式中，其中间隔物与靶向基团或亲脂性基团中的连接子相连，特别是连接子中的端位羟基与间隔物进行附件，
在某些的实施方式中，T1和T2的另一个为活性磷基团，所述的活性磷基团具有如下结构：
其中M1为H、取代或未取代的C1-C6烷基、OR5、SR5、OH、SH或NR6R7，M2为OH、SH、OR5′或NR6′R7′，每个R5、R6、R7、R5′、R6′或R7′独立地为氢、取代或未取代的C1-C6烷基、磺酰基；m为0或者1。
在某些的实施方式中，每个R5、R6或R7独立地为取代的C1-C6烷基，所述的取代基可以选择本领域常规符合烷基取代的取代基。在某些的实施方式中，每个R5、R6或R7独立地为取代烷基中，所述的取代基优选自氰基、卤素、羟基、氨基。
在某些的实施方式中，M1为选自取代或未取代的C1-C6烷基，取代或未取代的C1-C6烷基优先为甲基、乙基、丙基、异丙基。其中取代基优选自氰基、卤素、羟基、氨基。
在某些的实施方式中，M1选自OR5，R5选自取代或未取代的甲基、乙基、丙基、异丙基；优选为取代的基、乙基、丙基、异丙基；更优选的R5为氰基乙基，即M1为氰基乙氧基。
在某些的实施方式中，M1为NR6R7，R6和R7独立的选自氢、磺酰基；更优选地，M1为甲磺酰胺基。
在某些的实施方式中，M2选自NR6′R7′，R6′或R7′独立地为C1-C6烷基。所述的C1-C6烷基选自甲基、乙基、丙基、异丙基：在某些的实施方式中，M2选自N(CH(CH3)2)2。
在某些的实施方式中，其中M1为O(CH2)2CN，M2为N(CH(CH3)2)2，m为0。
在某些的实施方式中，在某些的实施方式中，其中M1为甲基，M2为N(CH(CH3)2)2，m为0。
在某些的实施方式中，在某些的实施方式中，其中M1为甲磺酰胺，M2为N(CH(CH3)2)2，m为0。
在某些实施方案中，活性磷基团具有如下亚结构： m、M2、R5、R6、R7如前文所述。
在某些的实施方式中，活性磷基团为亚磷酰胺。在某些实施方案中，活性磷基团选自二异丙基氰基乙氧基亚磷酰胺(-P(N(CH(CH3)2)2)O(CH2)2CN)。在某些实施方案中，反应性磷基团选自二异丙基甲基亚磷酰胺(-P(N(CH(CH3)2)2)CH3)。在某些实施方案中，反应性磷基团选自二异丙基乙基亚磷酰胺(-P(N(CH(CH3)2)2)CH2CH3)。在某些实施方案中，反应性磷基团选自H-膦酸酯(-P(＝O)(H)OH))。优选的合成固相合成利用亚磷酰胺(pIII)作为反应性亚磷酸盐；随后使用已知方法将中间体亚磷酸盐化合物氧化为磷酸盐或硫代磷酸盐(pV)以产生磷酸二酯或硫代磷酸酯核苷间连接。另外的活性磷酸盐和亚磷酸盐公开于Beaucage和Iyer,Tetrahedron,1992，48(12)，2223-2311中。
在某些的实施方式中，T1和T2的一个是保护基，而另一个是载体间隔物、通过间隔物链接的载体部分、靶向基团或亲脂性基团。进一步，在某些的实施方式中，A1和A2优选为选自O，示例性的结构
在某些的实施方式中，T1和T2的一个是二异丙基乙基亚磷酰胺，而另一个是4,4’-二甲氧三苯甲基。进一步，在某些的实施方式中，A1和A2优选为选自O。
在某些实施方案中，T1和T2的一个是二异丙基氰基乙氧基亚磷酰胺，而另一个是靶向基团。进一步，在某些的实施方式中，A1和A2优选为选自O。
在某些实施方案中，T1和T2的一个是二异丙基氰基乙氧基亚磷酰胺，而另一个是亲脂性基团。进一步，在某些的实施方式中，A1和A2优选为选自O。
在某些实施方案中，n为非0时，表示在双环结构上具有1-6个独立的R1基团取代基，可以理解的是本文双环脱碱基化合物中这些取代基并不包括天然核碱基或者经过修饰的核碱基。在某些实施方案中，R1分别独立地选自卤素、取代或未取代的C1-C6烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基。更优选的，R1分别独立地选自氟、氯、溴、碘、甲基、乙基、异丙基、丙基、3-丙烯基、乙炔基、3-丙炔基。
在某些实施方案中，n为0，表示在环结构上不存在R1基团取代。
在某些实施方案中，本文提供的化合物具有式(IIa)或者式(IIb)的构型：
A1、A2、T1、T2、R1以及n如本文所述。
在一些优选实施例中，式(IIa)或者式(IIb)中，A1和A2分别独立的选自：O；T1和T2之一为保护基、靶向基团以及亲脂性基团，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，而T1和T2的另一个为活性磷基团；n为0。在一些更优选实施例中，式(IIa)或者式(IIb)中，A1和A2分别独立的选自：O；T1和T2之一为羟基保护基以及亲脂性基团，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，而T1和T2的另一个为活性磷基团；n为0；其中羟基保护基选自乙酰基、苄基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基以及4,4’-二甲氧三苯甲基，亲脂性基团选自C14、C16或C18直连烷烃，活性磷选自亚磷酰胺。在一些更优选实施例中，活性磷选自二异丙基氰基乙氧基亚磷酰胺、二异丙基甲基亚磷酰胺、二异丙基乙基亚磷酰胺和H-膦酸酯。
在另一些优选实施例中，式(IIa)或者式(IIb)中A1和A2分别独立的选自：O，T1和T2之一为Z、L以及-Z-L，所述的Z表示为靶向基团或亲脂性基团，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，L表示为：载体间隔物或通过间隔物链接的载体部分；而T1和T2的另一个为羟基保护基；n为0。在一些更优先的实施例中，其中羟基保护基选自乙酰基、苄基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基以及4,4’-二甲氧三苯甲基。
在某些实施方案中，本文提供的化合物具有式(IIa-1)或者式(IIb-1)的构型：
A1、A2、T1、T2、R1以及n如本文所述，其构型同本领域所熟知的异山梨醇或者异甘露醇保持一致。在一些优选实施例中，式(IIa-1)或者式(IIb-1)中，A1和A2分别独立的选自：O；T1和T2之一为保护基、靶向基团以及亲脂性基团，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，而T1和T2的另一个为活性磷基团；n为0。在一些更优选实施例中，式(IIa-1)或者式(IIb-1)中，A1和A2分别独立的选自：O；T1和T2之一为羟基保护基以及亲脂性基团，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，而T1和T2的另一个为活性磷基团；n为0；其中羟基保护基选自乙酰基、苄基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基以及4,4’-二甲氧三苯甲基，亲脂性基团选自C14、C16或C18直连烷烃，活性磷选自亚磷酰胺。在一些更优选实施例中，活性磷选自二异丙基氰基乙氧基亚磷酰胺、二异丙基甲基亚磷酰胺、二异丙基乙基亚磷酰胺和H-膦酸酯。
在另一些优选实施例中，式(IIa-1)或者式(IIb-1)中A1和A2分别独立的选自：O，T1和T2之一为Z、L以及-Z-L，所述的Z表示为靶向基团或亲脂性基团，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，L表示为：载体间隔物或通过间隔物链接的载体部分；而T1和T2的另一个为羟基保护基；n为0。在一些更优先的实施例中，其中羟基保护基选自乙酰基、苄基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基以及4,4’-二甲氧三苯甲基。
在某些实施方案中，本文提供的化合物式(IIa)或式(IIa-1)具有以下所示的构型示例：
在某些实施方案中，本文提供的化合物具有(IIb)或式(IIb-1)具有以下所示的构型示例：
在本发明中，具体构型化合物可以通过本领域技术人员的合成手性控制获得起始原料，也可以通过手性拆分技术获得原料，也可以从工业生产中常见的具有特定构型起始原料合成，例如：来由具有具体构型的异山梨醇、异甘露醇等起始物料。
本发明提供的两个或更多个此类化合物的特征可以的任何组合，如果此类特征互不矛盾，则也包括在本发明的范围内。在一个实施方案中，本文提供的化合物具有以下具体结构：
本发明所述的化合物，例如式(I)或其立体异构体、(IIa)、(IIb)、(IIa-1)、(IIb-1)以及两个或更多个此类化合物的技术特征可以任何组合成化合物，可以应用于寡核苷酸中，进一步应用于寡核苷酸的5'-末端、3'-末端或者两个末端。所述的寡核苷酸包括但是不具限于单链反义寡核苷酸(ASO)、miRNA、双链核糖核苷酸(dsRNA)。在一些实施例中，应用于寡核苷酸中双链核糖核苷酸(dsRNA)中的反义链的3'-末端。在一些实施例中，应用于寡核苷酸中双链核糖核苷酸(dsRNA)中的反义链的5'-末端。在一些实施例中，应用于寡核苷酸中双链核糖核苷酸(dsRNA)中的正义链的5'和/或3'-末端。
根据本发明的一个方面，提供了一种寡聚化合物，其包含至少一种具有式(III)所示或其立体异构体的5′-末端和/或者3′-末端单体：
A1和A2分别独立的选自：O、S、SO、SO2、NR2和CR3CR4，R2、R3和R4分别独立的选自：氢、卤素、磺酰基、亚磺酰基、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基；
T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，并且T3和T4中的另一个为将式(III)单体或其立体异构体连接到寡聚化合物的核苷间连接基团；
R1分别独立地选自卤素、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基；
n为0-6的整数；
所述的寡聚化合物任选地进一步包含靶向基团。
在某些的实施方式中，提供的寡聚化合物，A1为O。
在某些的实施方式中，提供的寡聚化合物，A2为O。
在某些的实施方式中，提供的寡聚化合物，A1和A2分别独立为O，其包含至少一种具有式(III-1)所示或其立体异构体的5′-末端和/或者3′-末端单体： 其中T3、T4、R1、n如上文寡聚化合物所定义。
在某些的实施方式中，提供的寡聚化合物，A1和A2分别独立为S。
在某些的实施方式中，提供的寡聚化合物，A1和A2分别独立为NR2；R2选自：氢、卤素、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环。在某些的实施方式中，提供的寡聚化合物，R2为氢或者CH3。
在某些的实施方式中，提供的寡聚化合物，A1不同于A2。在某些的实施方式中，提供的寡聚化合物，A1为O，A2为S。在某些的实施方式中，提供的寡聚化合物，A1为O，A2为NR2，其包含至少一种具有式(III-2)所示或其立体异构体的5′-末端和/或者3′-末端单体：其中T3、T4、R1、R2、n如上文寡聚化合物所定义；在一些优选方案中，R2为氢或者CH3。在某些的实施方式中，提供的寡聚化合物，A1为S，A2为NR2。
如本文所用的术语“核苷间连接”或“核苷间连接基团”旨在包括本领域已知的各类核苷间连接基团，在此类实施方案中可使用任何核苷间键合将核苷或者类似物连接在一起。两类主要的核苷间连接基团由存在或不存在磷原子而定义，包括但不限于：磷酸二酯连接基团、磷酸三酯连接基团、硫代磷酸酯连接基团、二硫代磷酸酯键连接基团、烷基膦酸酯连接基团、氨基膦酸酯连接基团、膦酸酯连接基团、次膦酸酯连接基团、硫代氨基磷酸酯连接基团和氨基磷酸酯连接基团；以及不含磷核苷间连接基团诸如硫代二酯(-O-C(O)-S-)、硫羰氨基甲酸酯(-O-C(O)(NH)-S-)、硅氧烷(-O-Si(H)2-O-)、N,N′-二甲基肼(-CH2-N(CH3)-N(CH3)-)、甲酰基(formacetyl)和亚甲基亚氨基(-CH2-N(CH3)-O-CH2-)。核苷间连接还包括中性非离子核苷间连接，如本文所用术语“中性核苷间键合”旨在包括非离子的核苷间键合。中性核苷间键合包括但不限于磷酸三酯、甲基膦酸酯、MMI(3′-CH2-N(CH3)-O-5′)、酰胺-3(3′-CH2-C(＝O)-N(H)-5′)、酰胺-4(3′-CH2-N(H)-C(＝O)-5′)、亚甲基缩醛(formacetal)(3′-O-CH2-O-5′)和硫代亚甲基缩醛(thioformacetal)(3′-S-CH2-O-5′)。进一步的中性核苷间键合包括非离子键合包括硅氧烷(二烷基硅氧烷)、羧酸酯、甲酰胺、硫化物、磺酸酯和酰胺(参见例如:″Carbohydrate Modifications in Antisense Research″；Y.S.Sanghvi和P.D.Cook主编ACS SymposiumSeries580；第3和4章第40-65页)。进一步的中性核苷间键合包括含有混合N、O、S和CH2组成部分的非离子键合，其中磷原子并非总是存在。在一些优选方案中，烷基膦酸酯连接基团选自C1-C6烷基膦酸酯连接基团。
在某些的实施方式中，提供的寡聚化合物，所述的含磷核苷间连接基团具有如下所示的结构片段：X表示H、取代或未取代的C1-C6烷基、OR8、SR8′、OH、SH或NR9R10，Y表示O或者S，z可以是0或者1，R8、R8′、R9或R10独立地为氢、取代或未取代的C1-C6烷基、磺酰基，分别独立地表示与本文的A1或者A2之一相连部分，以及与相连核苷酸相连部分。
与天然磷酸二酯键合相比的修饰键合可用于改变(通常增强)寡聚化合物的核酸酶抗性。在某些实施方案中可作为外消旋混合物或单独的对映体制备具有手性原子的核苷间键合。代表性的手性键合包括但不限于膦酸烷基酯和硫代磷酸酯。制备含磷和不含磷的核苷间键合的方法对本领域的技术人员而言是熟知的。
在某些的实施方式中，提供的寡聚化合物，T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，T3和T4的另一个为将式(III)单体或其立体异构体连接到寡聚化合物的5′-末端和/或3′-末端基团的核苷间连接基团，所述的核苷间连接基团独立地为烷基膦酸酯连接基团、磷酸二酯核苷间连接基团或硫代磷酸酯核苷间连接基团。
举例但是不局限于，T4为核苷间连接基团时，包含至少一种式(III)所示单体或其立体异构体的寡聚化合物如下示列：
其中Olig表示将所示的式(III)单体或其立体异构体分别连接到寡聚化合物的5′-末端和/或3′-末端的寡核苷酸部分。A1、A2、R1以及n如前文所述，T3为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1相连的，所述的寡聚化合物任选地进一步包含靶向基团。
在某些的实施方式中，提供的寡聚化合物，所述的T3和T4之一的保护基根据A1或者A2不同，即当A1或者A2为氧时，T1和T2之一的保护基是羟基保护基；即当A1或者A2为硫、亚砜(SO)、砜(SO2)时，T1和T2之一的保护基是巯基保护基，即当A1或者A2为氮时，T1和T2之一的保护基是氨基保护基。
在某些的实施方式中，提供的寡聚化合物，T3和T4之一保护基是羟基保护基；在一个实施方式中，提供的寡聚化合物，所述羟基保护基各自独立地选自乙酰基、叔丁基、叔丁氧基甲基、甲氧基甲基、四氢吡喃基、1-乙氧基乙基、1-(2-氯乙氧基)乙基、2-三甲基甲硅烷乙基、对氯苯基、2,4-二硝基苯基、苄基、苯甲酰基、对苯基苯甲酰基、2,6-二氯苄基、二苯基甲基、对硝基苄基、三甲基甲硅烷基、三乙基甲硅烷基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基、三苯甲硅烷基、三异丙基甲硅烷基、苯甲酰基甲酸酯、氯乙酰基、三氯乙酰基、三氟乙酰基、新戊酰基、9-芴甲氧羰基、甲磺酸基、甲苯磺酸基、三氟甲磺酸基、三苯甲基、单甲氧基三苯甲基、二甲氧三苯甲基、三甲氧基三苯甲基或取代的9-苯基黄嘌呤-9-基；在一个实施方式中，提供的寡聚化合物，优选的羟基保护基各自独立地选自乙酰基、苄基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基以及4,4’-二甲氧三苯甲基。
在某些的实施方式中，提供的寡聚化合物，T3和T4之一保护基是巯基保护基；在一个实施方式中，提供的寡聚化合物，所述巯基保护基各自独立地选自乙酰基、叔丁基、叔丁氧基甲基、甲氧基甲基、四氢吡喃基、1-乙氧基乙基、1-(2-氯乙氧基)乙基、2-三甲基甲硅烷乙基、对氯苯基、2,4-二硝基苯基、苄基、苯甲酰基、对苯基苯甲酰基、2,6-二氯苄基、二苯基甲基、对硝基苄基、三甲基甲硅烷基、三乙基甲硅烷基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基、三苯甲硅烷基、三异丙基甲硅烷基、苯甲酰基甲酸酯、氯乙酰基、三氯乙酰基、三氟乙酰基、新戊酰基、9-芴甲氧羰基、甲磺酸基、甲苯磺酸基、三氟甲磺酸基、三苯甲基、单甲氧基三苯甲基、二甲氧三苯甲基、三甲氧基三苯甲基或取代的9-苯基黄嘌呤-9-基；在一个实施方式中，提供的寡聚化合物，优选的巯基保护基各自独立地选自苄基、4,4’-二甲氧三苯甲基。可以理解的是当A1或A2为SO、SO2时，巯基保护基同样适用。
在某些的实施方式中，提供的寡聚化合物，T3和T4之一保护基是氨基保护基；在一个实施方式中，提供的寡聚化合物，所述氨基保护基各自独立地选自2-三甲基硅基乙氧基羰基(Teoc)、1-甲基-1-(4-联苯基)乙氧基羰基(Bpoc)、叔丁氧基羰基(BOC)、烯丙氧基羰基(Alloc)、9-芴基甲氧基羰基(Fmoc)、苄氧基羰基(Cbz)、甲酰基、乙酰基、三卤乙酰基、苯甲酰基、硝基苯基、乙酰基、2-硝基苯磺酰基、邻苯二甲酰亚胺基和二硫代丁二酰基。
在某些的实施方式中，提供的寡聚化合物，T3和T4之一亲脂性基团，其中亲脂性基团的亲脂性可以通过logKow测定，logKow超过0。术语“亲脂性”或“亲脂性基团”广义上是指对脂质具有亲和力的任何化合物或化学部分，这些系数是使用第一原理或经验方法计算的(参见例如Tetko等人,J.Chem.Inf.Comput.Sci.41:1407-21(2001)，将其通过引用以其全文并入本文，其定义如本文所描述的双环脱碱基化合物具有一样的定义范围。其提供物质偏好非水性或油性环境而不是水的倾向的热力学量度(即其亲水性/亲脂性平衡)。亲脂性基团缀合的结构使得亲脂性基团(例如，脂肪酸)暴露，从而允许亲脂性基团与白蛋白和/或脂肪酸受体或转运蛋白相互作用，从而提供具有长体内半衰期的寡核苷酸。在一些实施例中，一般具有亲脂性的基团其logKow测定超过0。在某些的实施方式中，亲脂部分的亲油性，由辛醇-水分配系数logKow测量，亲脂部分可以超过1，超过1.5，超过2，超过3，超过4，超过5或超过10的logKow。分子的亲脂性可以相对于其携带的官能团而变化；例如，在亲脂部分末端添加羟基、胺、羧酸、磺酸酯、磷酸酯、硫醇、叠氮基及炔烃等官能团可以增加或减少亲脂部分的分配系数(例如logKow)值；并且还可以通过这些官能团进一步将亲脂性基团附接到本文中所示的寡聚化合物的单体中。
提供的寡聚化合物，亲脂性基团同样其定义如前文所描述的双环脱碱基化合物具有一样的定义范围。例如但不局限于以下举例：
某些的实施方式中，提供的寡聚化合物，亲脂性基团为含有饱和或不饱和的C4-C30烃链、脂肪环、芳香族、脂肪酸基团或脂肪酸衍生的基团、类固醇衍生的基团和任何脂溶性维生素基团。
在某些的实施方式中，提供的寡聚化合物，亲脂性基团为饱和或不饱和C10-C25烃链(例如C10-C25烷基或烯基)。在一些实施例中，亲脂性基团为饱和或不饱和C10-C18烃链(例如直链C10-C18烷基或烯基)。在一个实施例中，亲脂性基团为饱和或不饱和的C10、C11、C12、C13、C14、C15、C16、C17或者C18烃链(例如直链C16烷基或烯基)；更优选的是直链烷基。在一个实施例中，亲脂性基团为饱和的直链C14烷基、C16烷基或C18烷基。亲脂性基团进一步选自由以下组成的组的任选的官能团的：羟基、胺、羧酸、磺酸酯、磷酸酯、硫醇、叠氮基及炔烃。这些官能团可用于将亲脂性基团附接至A1或者A2中。
在另一个实施例中，提供的寡聚化合物，亲脂性基团选自二羧酸的脂肪酸衍生的基团。在另一个实施例中，脂肪酸包含具有4到28个碳原子的烃链的任何饱和或不饱和脂肪酸，并且可以含有一个或两个羧基。在一个实施例中，所述亲脂性基团是具有8到24个碳原子的烃链的饱和脂肪酸衍生的基团。在某些实施例中，所述亲脂性基团是饱和脂肪酸衍生的基团，其中所述脂肪酸选自由以下组成的组：丁酸(CH3(CH2)2COOH)、戊酸、己酸、庚酸、辛酸、癸酸、十一烷酸、十二烷酸、十三烷酸、十四烷酸、十五烷酸、十六烷酸、十七烷酸、十八烷酸、十九烷酸、二十烷酸、二十一烷酸、二十二烷酸、二十三烷酸、二十四烷酸、二十五烷酸、二十六烷酸、二十七烷酸和二十八烷酸(CH3(CH2)16COOH)。在另一个实施例中，所述亲脂性基团是不饱和脂肪酸衍生的基团，所述的不饱和的脂肪酸是表1中呈现的中的一种；所述脂肪酸选优选为自由以下组成的组：肉豆蔻脑酸、棕榈油酸、十六碳烯酸、油酸、反油酸、反型异油酸、亚油酸、反亚油酸、α-亚麻酸、花生四烯酸和芥酸。
在另一个实施例中，提供的寡聚化合物，亲脂性基团为类固醇，诸如固醇。类固醇为含有全氢-1,2-环戊菲环系统的多环化合物。类固醇包括但不限于胆汁酸(例如胆酸、去氧胆酸及脱氢胆酸)、可的松、地高辛(digoxigenin)、睾酮、胆固醇及阳离子类固醇，诸如可的松。“胆固醇衍生物”是指例如通过取代、添加或移除取代基衍生自胆固醇的化合物。
在某些的实施方式中，提供的寡聚化合物，亲脂性基团选自胆固醇、维生素E(生育酚)或胆汁酸。
在某些的实施方式中，提供的寡聚化合物，亲脂性基团选自不饱和脂肪二十二碳六烯酸酸(DHA)、二十碳五烯酸(EPA)饱和脂肪酸二十二烷酸(DCA)、甾醇胆固醇(胆汁)、生育酚琥珀酸酯(TS)和石胆酸(LA)、维甲酸(维生素A酸)。
在另一个实施例中，亲脂性基团为芳香族部分。
在一个某些的实施方式中，提供的寡聚化合物，本发明的亲脂性基团可以通过共价键直接与A1或者A2直接相连。在某些的实施方式中，是A1和T3组合和/或者A2和T4组合片段包括但是非限制性示列结构： 其中表示单键或者双键，p或q分别独立地表示5-25的整数，p与q之和不超过25。在一些跟个具体的实施例A1和T3组合和/或者A2和T4组合片段包括但是非限制性示例：
可以理解的是，如前文所描述的双环脱碱基化合物所定义任何亲脂性基团、连接子以及通过连接子与双环脱碱基化合物链接，其描述的性质或者结构同样适用于本文中的寡聚化合物中的亲脂性基团、连接子及其链接方式的的定义范围。
在一个某些的实施方式中，提供的寡聚化合物，亲脂性基团同样可经由连接子与A1或者A2共价连接，并且所述的连接子可以是一个或者多个的组合。如本文所用，提供的寡聚化合物亲脂性基团通过的连接子具有如前文所用的双环脱碱基核酸类似物化合物同样的亲脂性基团连接子，其描述的性质或者结构同样适用于本文中的寡聚化合物的亲脂性基团连接子。
本发明的T3和T4之一为亲脂性基团，所述亲脂性基团进一步包含连接子，并通过一个或者多个连接子与A1或者A2共价连接。非限制性举例：在一个实施例中，所述的连接子选自由以下组成的组：酰胺键、磷脂酰胆碱、烃连接子或聚乙二醇(PEG)连接子、氨基-烷基-醇、羟脯氨酸、羟脯氨醇、氨基-烷基-硫代磷酸酯连接子、氨基-PEG-硫代磷酸酯连接子、α-羧酸盐-氨基-烷基硫代磷酸酯连接子和α-羧酸盐-氨基-PEG-硫代磷酸酯连接子。在一些实施例中，所述的连接子含有醚、硫醚、脲、碳酸酯、胺、酰胺、马来酰亚胺-硫醚、二硫化物、磷酸二酯、磺酰胺键、点击反应的产物(例如，来自叠氮化物-炔烃环加成的三唑)或氨基甲酸酯。
一些实施例中，所述连接子包括可裂解基团。所述连接子包括可裂解基团，可对pH敏感、氧化还原可裂解接头、酯酶可裂解接头(例如，酯基)、磷酸酶可裂解接头(例如，磷酸酯)、或肽酶可裂解接头(例如，肽键)、生物可裂解接头、羟脯氨酸、羟脯氨酸、氨基-烷基-醇连接的连接子，关于亲脂性基团和连接子的更多论述可见于2019年7月7日提交的名称为“肝外递送”的PCT公开号WO2019/217459，2019年5月10日提交的名称为“包括7′-5′-α-异头双环糖核苷的寡核苷酸缀合物”的PCT公开号WO2019/215333，以及Nucleic Acids Research,2019,Vol.47,No.31082–1096《多种脂质偶联物用于功能性肝外体内核苷酸递送》，将其内容同样通过引用以其全文并入本文寡聚化合物中。
可以理解的是，当本文化合物T1或者T2为亲脂性基团时，本文所述的化合物以及应用于寡核苷酸领域，根据本领域熟知的，根据亲脂性基团性质的或者结构不同可以应用于肝外递送系统，举例但是不局限于CNS领域、眼科领域、肌肉。由于游离寡核苷酸无法穿过的血脑屏障(BBB)，寡核苷酸向中枢神经系统(CNS)的递送引起特定问题。将寡核苷酸递送至CNS中的一种方式是通过鞘内递送。然而，寡核苷酸也需要有效内化至CNS的靶细胞中以实现期望的治疗效果，先前的研究通常使用亲脂性基团缀合致寡核苷酸中的特定位置如单个核碱基、2-位糖环等，以帮助寡核苷酸细胞内内化至神经元起源的细胞中，本文中将亲脂性基团缀合本文中的单体并应用于寡聚化合物中同样可以启到CNS领域的递送和分配的作用。已经在CNS领域广泛应用的亲脂性疾病包括阿尔茨海默病、肌萎缩性侧索硬化(ALS)、额颞叶型痴呆、亨廷顿病、帕金森病、脊髓小脑病、朊病毒病以及拉福拉病。
脂性基团在寡核苷酸应用于CNS领域相应的靶点应用：其靶向早发性家族性阿尔茨海默病的APP、脊髓小脑共济失调2及ALS的ATXN2、以及肌萎缩侧索硬化症及额颞叶型痴呆的C9orf72，靶向ALS的TARDBP、额颞叶型痴呆的MAPT(Tau)及亨廷顿病的HTT，靶向帕金森病的SNCA、ALS的FUS，脊髓小脑共济失调3的ATXN3，SCA1的ATXN1，SCA7及SCA8的基因，DRPLA的ATN1，XLMR的MeCP2，朊病毒疾病、隐性CNS障碍：拉福拉病的PRNP、DM1(CNS及骨骼肌)的DMPK，及hATTR(CNS、眼及全身)的TTR。
脂性基团在寡核苷酸应用于眼科领域相应的疾病应用：这些疾病包括但不限于年龄相关性黄斑变性(AMD)(干性及湿性)、鸟枪弹样脉络膜视网膜病变、显性色素性视、网膜炎、富克氏营养不良(Fuch’s dystrophy)、遗传性及偶发性青光眼以及斯特格特氏病(stargardt’s disease)。例如：靶向湿性(或渗出性)AMD的VEGF，靶向干性(或非渗出性)AMD的C3、CFB，靶向青光眼的MYOC、ROCK2的、ADRB2、CA2，靶向白内障的CRYGC，靶向干眼综合征的PPP3C。
在某些的实施方式中，提供的寡聚化合物，T3和T4的一个为氢，T3和T4的另一个为将式III单体或其立体异构体连接到寡聚化合物的5′-末端和/或3′-末端基团的核苷间连接基团。
在一些实施方式中，寡聚化合物，还进一步包含靶向基团。在一些实施方式中，寡聚化合物，T3和T4之一为共价键，该寡聚化合物通过式(III)或其立体异构体中的T3和T4之一以共价键形式与靶向基团相连，表示式(III)单体部分；在另一些实施方式中，寡聚化合物，该寡聚化合物通过与一端含有式(III)或其立体异构体的寡聚化合物的另一端的5′-末端和/或3′-末端核苷酸相连,所述靶向基团可以是包含siRNA给药领域常规使用的配体，如本文所用，提供的寡聚化合物靶向基团具有如前文所述的双环脱碱基核酸类似物化合物靶向基团，其描述的性质或者结构同样适用于本文中的寡聚化合物的靶向基团。所述靶向基团可以改变它所并入的siRNA试剂的分布、靶向或寿命，内体溶解特性、改进运载、杂交以及特异性特性，例如WO2009082607A2中描述的各种配体，以引用的方式将其全部公开内容并入本文寡聚化合物中。
在一些实施方式中，寡聚化合物，所述靶向基团可以选自以下靶向分子或其衍生物形成的配体中的一种或多种；如聚合物，例如聚乙二醇；糖类，例如乳糖、聚乳糖、甘露糖、半乳糖、N-乙酰半乳糖胺(GalNAc)；肝实质细胞表达的受体配体，例如去唾液酸糖蛋白、去唾液酸糖残基、脂蛋白(如高密度脂蛋白、低密度脂蛋白等)、胰高血糖素、神经递质(如肾上腺素)、生长因子、转铁蛋白等适配体；抗体；量子点；多肽，例如透膜肽，或小分子配体。
在一些实施方式中，寡聚化合物，至少一个或每个所述靶向基团选自包含能够和哺乳动物肝实质细胞表面受体结合的配体。在一些实施方式中，每个所述靶向基团独立地为包含与哺乳动物肝细胞表面的去唾液酸糖蛋白受体(ASGPR)亲和的配体。在一些实施方式中，每个所述靶向基团独立地为包含去唾液酸糖蛋白或糖的配体。在一些实施方式中，每个所述靶向基团独立地为包含去唾液酸糖蛋白的配体，例如去唾液酸血清类枯蛋白(asialoorosomucoid,ASOR)或去唾液酸始球蛋白(asialofetuin，ASF)。在一些实施方式中，每个所述靶向基团独立地包含选自D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D吡喃甘露糖、α-D-吡喃葡萄-糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳-糖胺、N-乙酰半乳糖胺、N-三氟乙酰半乳糖胺、N-丙酰半乳糖胺、N-正丁酰半乳胺、N-异丁-36酰半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6O-三苯甲基-α-D吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖、L-4-硫代核糖中的一种的配体。
在一些实施方式中，寡聚化合物，至少一个或每个所述靶向基团为包含半乳糖或N-乙酰半乳糖胺的配体。
在某些的实施方式中，寡聚化合物，靶向基团与A1或者A2之一相连选自以下化合物片段之一示例：
靶向基团可经由合适的缀合接头与A1或者A2之一相连，本领域技术人员可以根据靶向基团的具体类型选择合适的缀合接头。这些缀合接头、配体的种类、系链、分支接头，以及靶向基团与寡核苷酸的连接方式可参见WO2009073809、WO2014179620、WO2014179627、WO2015006740A2的公开内容，通过引用的方式将其整体内容并入本文。在一些实施方式中，所述靶向基团可以通过酸不稳定的、或可还原的化学键相连，在细胞内涵体的酸性环境下，这些化学键可降解。
在某些的实施方式中，该寡聚化合物中还包含一种或更多种靶向基团，并且通过T3共价键与寡聚化合物相连，或者与寡聚化合物的另一端末端核苷酸相连，非示范性示列链接方式如下示列：T3′为共价键，Olig、A1、A2、T3、R1以及n如前文所述。
本文所述的寡聚化合物包含一个或多个不对称中心并且因而产生对映体、非对映体和其它立体异构构型就绝对立体化学而言这些构型可被定义为(R)或(S)、α或β诸如针对糖异头物或定义为(D)或(L)诸如针对氨基酸等。在本文提供的寡聚化合物中包括所有此类可能的同分异构体以及它们的外消旋形式和任选纯的形式。
在某些实施方案中，提供的寡聚化合物，包含至少一种式(IVa)或者式(IVb)所示的构型的5′-末端和/或者3′-末端单体：
A1、A2、T3、T4、R1以及n如前文所述。
在一些优选实施方案中，A1和A2分别独立的选自：O；T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，并且T3和T4中的另一个为将所述式(IVa)或者式(IVb)单体连接到寡聚化合物的核苷间连接基团；n为0；寡聚化合物任选地进一步包含靶向基团。
在一些更优选实施方案中，A1和A2分别独立的选自：O；T3和T4之一为H、羟基保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，并且T3和T4中的另一个为将所述式(IVa)或者式(IVb)连接到寡聚化合物的核苷间连接基团；n为0；寡聚化合物任选地进一步包含靶向基团。在一些更优选实施方案中，其中羟基保护基选自乙酰基、苄基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基以及4,4’-二甲氧三苯甲基，亲脂性基团选自C14、C16或C18直连烷烃，核苷间连接基团选自烷基膦酸酯连接基团、磷酸二酯核苷间连接基团或硫代磷酸酯核苷间连接基团。
在某些实施方案中，提供的寡聚化合物，包含至少一种式(IVa-1)或者式(IVb-1)所示的构型的5′-末端和/或者3′-末端单体：
A1、A2、T3、T4、R1以及n如前文所述。
在一些优选实施方案中，A1和A2分别独立的选自：O；T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，并且T3和T4中的另一个为将所述式(IVa-1)或者式(IVb-1)单体连接到寡聚化合物的核苷间连接基团；n为0；寡聚化合物任选地进一步包含靶向基团。
在一些更优选实施方案中，A1和A2分别独立的选自：O；T3和T4之一为H、羟基保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，并且T3和T4中的另一个为将所述式(IVa-1)或者式(IVb-1)单体连接到寡聚化合物的核苷间连接基团；n为0；寡聚化合物任选地进一步包含靶向基团。在一些更优选实施方案中，其中羟基保护基选自乙酰基、苄基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基以及4,4’-二甲氧三苯甲基，亲脂性基团选自C14、C16或C18直连烷烃，核苷间连接基团选自烷基膦酸酯连接基团、磷酸二酯核苷间连接基团或硫代磷酸酯核苷间连接基团。
在某些实施方案中，本文提供寡聚化合物包括但是不局限于具有以下的构型的示列：
其中Olig表示将所述的单体分别连接到寡聚化合物的5′-末端和/或3′-末端的寡核苷酸部分，A1、A2、R1以及n如前文所述，T3为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，寡聚化合物任选地进一步包含靶向基团。
在某些实施方案中，提供的寡聚化合物，T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，并且T3和T4中的另一个为如前文所提供寡聚化合物中包含的单体连接到寡聚化合物5′-末端的核苷间连接基团。可以理解的是，该种寡聚化合物，其包含至少一种具有如本文所提供的5′-末端单体(如式(III)或其立体异构体、式(IVa)、式(IVb)、(IVa-1)或(IVb-1)所示)。
在某些实施方案中，提供的寡聚化合物，T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，并且T3和T4中的另一个为如前文所提供寡聚化合物中包含的单体连接到寡聚化合物3′-末端的核苷间连接基团。可以理解的是，该种寡聚化合物，其包含至少一种具有如本文所提供的3′-末端单体(如式(III)或其立体异构体、式(IVa)、式(IVb)、(Iva-1)或(IVb-1)所示)。
在某些实施方案中，提供的寡聚化合物，T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，并且T3和T4中的另一个分别独立地将一种相同或者不同的如前文所提供寡聚化合物中包含的单体连接到寡聚化合物5′-末端和3′-末端的核苷间连接基团。可以理解的是，该种寡聚化合物，其分别在寡聚化合物的5′-末端和3′-末端包含至少一种相同或者不同的具有如本文所提供的单体(如式(III)或其立体异构体、式(IVa)、式(IVb)、(IVa-1)或(IVb-1)所示)。
一般情况下，该种寡聚化合物，其分别在其在5′-末端和/或3′-末端的任意一端端位独立地包含一个相同或者不同的式(III)或其立体异构体、式(IVa)、式(IVb)、(IVa-1)或(IVb-1)所示的单体区域。在另一些情况下，该种寡聚化合物，其分别在寡聚化合物的5′-末端和/或3′-末端的任意一端端位独立地包含至少2个连续相同或者不同的式(III)或其立体异构体、式(IVa)、式(IVb)、(IVa-1)或(IVb-1)所示的单体区域。
示例性但是不局限于，将与本文单体连接到寡聚化合物的5′-末端和/或3′-末端，A2和A2′表示相同或者不同的如前文所述的单体变量基团，R1与R1’表示相同或者不同的如前文所述的单体变量基团,Olig表示将所述的单体分别连接到寡聚化合物的5′-末端和/或3′-末端的寡核苷酸部分。
在某些实施方案中，n为非0时，表示在双环结构上具有1-6个独立的R1基团取代基，可以理解的是本文双环脱碱基化合物中这些取代基并不包括天然核碱基或者经过修饰的核碱基。在某些实施方案中，R1分别独立地选自卤素、取代或未取代的C1-C6烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基。更优选的，R1分别独立地选自氟、氯、溴、碘、甲基、乙基、异丙基、丙基、3-丙烯基、乙炔基、3-丙炔基。
在某些实施方案中，n为0，表示在环结构上不存在R1基团取代。
本发明提供的两个或更多个此类寡聚化合物的特征可以的任何组合，如果此类特征互不矛盾，则也包括在本发明的范围内。进一步，本发明所述的双环碱基化合物，例如式(I)、(IIa)、(IIb)、(IIa-1)、(IIb-1)以及两个或更多个此类化合物的特征可以的任何组合化合物可以应用于5′-末端和/或3′-末端寡核苷酸单体中，因此，如本文所描述的双环脱碱基化合物所定义技术特征同样均适用于本文寡聚化合物中，只要其不存在矛盾。
某些实施方案中，寡聚化合物中，提供了包含至少一种以下所示单体的片段：
在某些实施方案中，本文提供的寡聚化合物具有以下具体结构：
,其中Olig表示将所述的单体分别连接到寡聚化合物的5′-末端和/或3′-末端的寡核苷酸部分。
在某些实施方案中寡聚化合物为单链寡核苷酸，本领域所熟知的单链寡核苷酸有反义寡核苷酸(又称ASO)、核糖酶或适体。在某些实施方案中寡聚化合物为双链RNAi化合物(siRNA)，这类化合物本领域所熟知的双链核苷核酸(又称dsRNA)，其中一条或两条链为如本文所公开的寡聚化合物。
在某些实施方案中寡聚化合物的核碱基序列的至少一部分与靶核酸的一部分互补或者完全互补。在某些实施方案中与靶核酸100％互补。在某些实施方案中与靶核酸90％互补。在某些实施方案中与靶核酸80％互补。在某些实施方案中与靶核酸90％互补。在某些实施方案中与靶核酸70％互补。在某些实施方案中与靶核酸90％互补。在某些实施方案中与靶核酸60％互补。
在某些实施方案中，提供了双链核糖核酸(dsRNA)试剂，它们均包含：正义链和反义链，其中所述正义链与所述反义链完全或者部分互补，并且所述反义链与核酸靶基因互补；所述正义链和反义链的至少一者为如前文所提供的寡聚化合物，其至少一种具有包含本文式(III)或其立体异构体、式(IVa)、式IV(b)、式(IVa-1)或者式(IVb-1)的5′-末端和/或者3′-末端单体；并目其中所述双链核糖核酸(dsRNA)试剂任选地进一步包含独立的靶向基团。
在某些实施方案中，提供了双链核糖核酸(dsRNA)试剂，在其中正义链为包含本文式(III)或其立体异构体、式(IVa)、式IV(b)、式(IVa-1)或者式(IVb-1)的5′-末端单体所提供的寡聚化合物。在某些实施方案中，提供了双链核糖核酸(dsRNA)试剂，其中正义链为包含本文式(III)或其立体异构体、式(IVa)、式IV(b)、式(IVa-1)或者式(IVb-1)的3′-末端单体所提供的寡聚化合物。在某些实施方案中，提供了双链核糖核酸(dsRNA)试剂，其中正义链为包含本文式(III)或其立体异构体、式(IVa)、式IV(b)、式(IVa-1)或者式(IVb-1)的5′-末端和3′-末端单体所提供的寡聚化合物。
在某些实施方案中，提供了双链核糖核酸(dsRNA)试剂，其中反义链为包含本文式(III)或其立体异构体、式(IVa)、式IV(b)、式(IVa-1)或者式(IVb-1)的5′-末端和/或者3′-末端单体提供的寡聚化合物。在某些实施方案中，提供了双链核糖核酸(dsRNA)试剂，其中反义链为包含式(III)或其立体异构体、式(IVa)、式IV(b)、式(IVa-1)或者式(IVb-1)的3′-末端单体提供的寡聚化合物。在某些实施方案中，提供了双链核糖核酸(dsRNA)试剂，其中反义链为包含本文式(III)或其立体异构体、式(IVa)、式IV(b)、式(IVa-1)或者式(IVb-1)的5′-末端单体提供的寡聚化合物。
在某些的实施方式中，提供了双链核糖核酸(dsRNA)试剂，任选地进一步包含独立的靶向基团，独立的靶向基团可以理解的是，除本文寡聚化合物单体中存在的靶向基团外，表示式(III)单体部分，还可以在双链核糖核酸(dsRNA)试剂中其他位置具有靶向基团。在某些的实施方式中，在双链核糖核酸(dsRNA)试剂中，在5′-末端和/或3′-末端核苷酸上还包含一种或更多种靶向基团或连接基团，可以理解的是这些靶向基团可以缀合致双链核糖核酸(dsRNA)试剂中的任意一条链5′-末端和/或3′-末端的核苷酸上， 所述靶向基团可以是包含siRNA给药领域常规使用的配体。如本文所用，这些靶向基团可以是独立地选自具有与前文所述的寡聚化合物以及如双环脱碱基核酸类似物化合物同样的靶向基团；提供的双链核糖核酸(dsRNA)试剂的靶向基团，其描述的性质或者结构同样适用于本文中的寡聚化合物以及如双环脱碱基核酸类似物化合物的靶向基团。
在一些实施方式中，提供的双链核糖核酸(dsRNA)试剂中，所述靶向基团可以选自包含以下靶向分子或其衍生物形成的配体中的一种或多种：聚合物、糖类、肝实质细胞表达的受体配体、抗体、量子点、多肽，或小分子配体。
在一些实施方式中，提供的双链核糖核酸(dsRNA)试剂中，至少一个或每个所述靶向基团选自包含能够和哺乳动物肝实质细胞表面受体结合的配体。
在一些实施方式中，提供的双链核糖核酸(dsRNA)试剂中，每个所述靶向基团独立地为包含与哺乳动物肝细胞表面的去唾液酸糖蛋白受体(ASGPR)亲和的配体。
在一些实施方式中，提供的双链核糖核酸(dsRNA)试剂中，每个所述靶向基团独立地为包含去唾液酸糖蛋白或糖。靶向基团可经由合适的接头与5′-末端和/或3′-末端核苷酸相连，本领域技术人员可以根据靶向基团的具体类型选择合适的缀合接头。
在一些实施例中，该dsRNA试剂其中所述一种或更多种靶向基团或连接基团与所述正义链缀合。所述的靶向配体可以改变它所并入的dsRNA试剂的分布、靶向或寿命，内体溶解特性、改进运载、杂交以及特异性特性，例如：但是非限制性的实例，凝集素、糖蛋白、脂质或蛋白质，促甲状腺激素、促黑激素、凝集素、糖蛋白、表面活性蛋白A、粘蛋白碳水化合物、多价乳糖、多价半乳糖、N-乙酰基-半乳糖胺、N-乙酰基-葡糖胺多价甘露糖、多价海藻糖、糖基化的聚氨基酸、多价半乳糖、转铁蛋白、双膦酸盐、聚谷氨酸盐、聚天冬氨酸盐、一种脂质、胆固醇、一种类固醇、胆汁酸、叶酸盐、维生素B12、生物素、一种RGD肽、一种RGD肽模拟物或一种适体，其他实例包括染料、插入剂(例如吖啶)、交联剂(例如补骨脂素、丝裂霉素C)、卟啉(TPPC4、特沙弗林(texaphyrin)、噻啉(Sapphyrin))、多环芳香族烃(例如吩嗪、二氢吩嗪)、人工核酸内切酶或一种螯合剂(例如EDTA)、亲脂性分子，例如胆固醇、胆酸、金刚烷乙酸、1-芘丁酸、二氢睾酮、1，3-双-O(十六基)甘油、香叶氧基己基、十六基甘油、茨醇、薄荷醇、1，3-丙二醇、十七基、棕榈酸、肉豆蔻酸、O3-(油酰基)石胆酸、O3-(油酰基)胆烯酸、二甲氧基三苯甲基或吩噁嗪)以及肽结合物(例如触足蛋白肽、Tat肽)、烷化剂、磷酸盐、氨基、巯基、PEG(例如PEG-40K)、MPEG、[MPEG]2、聚氨基、烷基、被取代的烷基、放射性标记的标记物、酶、半抗原(例如生物素)、运载/吸附促进剂(例如阿司匹林、维生素E、叶酸)、合成核糖核酸酶(例如咪唑、双咪唑、组织胺、咪唑簇、吖啶-咪唑结合物、四氮杂大环的Eu3+络合物)、二硝基苯基、HRP或AP。在一些实施例中，所述靶向基团或连接基团包括选自：N-乙酰基-半乳糖胺(GalNAc)、亲脂性分子。
在某些的实施方式中，靶向基团相连后选自以下化合物之一：
可以理解的是，本文中靶向基团可以通过反应性官能团与本文中单体化合物或者寡核苷酸缀合成核苷间连接基团，例如GLO一般表示具有氧代磷酸酯核苷间连接基团，而用GLS一般表示具有硫代磷酸酯核苷间连接基团。
在某些实施方案中，本发明提供包含任何多种长度范围的寡聚化合物。在某些实施方案中，寡聚化合物或双链核糖核酸(dsRNA)中，单链、正义链和/或反义链任意一条链均分别包含8-40核苷酸长度。例如在某些实施方案中，寡聚化合物核苷酸长度选自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和40。例如在某些实施方案中本发明提供寡聚化合物核苷酸长度范围其包含由8至9、8至10、8至11、8至12、8至13、8至14、8至15、8至16、8至17、8至18、8至9、8至20、8至21、8至22、8至23、8至24、8至25、8至26、8至27、8至28、8至29、8至30、9至10、9至11、9至12、9至13、9至14、9至15、9至16、9至17、9至18、9至19、9至20、9至21、9至22、9至23、9至24、9至25、9至26、9至27、9至28、9至29、9至30、10至11、10至12、10至13、10至14、10至15、10至16、10至17、10至18、10至19、10至20、10至21、10至22、10至23、10至24、10至25、10至26、10至27、10至28、10至29、10至30、11至12、11至13、11至14、11至15、11至16、11至17、11至18、11至19、11至20、11至21、11至22、11至23、11至24、11至25、11至26、11至27、11至28、11至29、11至30、12至13、12至14、12至15、12至16、12至17、12至18、12至19、12至20、12至21、12至22、12至23、12至24、12至25、12至26、12至27、12至28、12至29、12至30、13至14、13至15、13至16、13至17、13至18、13至19、13至20、13至21、13至22、13至23、13至24、13至25、13至26、13至27、13至28、13至29、13至30、14至15、14至16、14至17、14至18、14至19、14至20、14至21、14至22、14至23、14至24、14至25、14至26、14至27、14至28、14至29、14至30、15至16、15至17、15至18、15至19、15至20、15至21、15至22、15至23、15至24、15至25、15至26、15至27、15至28、15至29、15至30、16至17、16至18、16至19、16至20、16至21、16至22、16至23、16至24、16至25、16至26、16至27、16至28、16至29、16至30、17至18、17至19、17至20、17至21、17至22、17至23、17至24、17至25、17至26、17至27、17至28、17至29、17至30、18至19、18至20、18至21、18至22、18至23、18至24、18至25、18至26、18至27、18至28、18至29、18至30、19至20、19至21、19至22、19至23、19至24、19至25、19至26、19至29、19至28、19至29、19至30、20至21、20至22、20至23、20至24、20至25、20至26、20至27、20至28、20至29、20至30、21至22、21至23、21至24、21至25、21至26、21至27、21至28、21至29、21至30、22至23、22至24、22至25、22至26、22至27、22至28、22至29、22至30、23至24、23至25、23至26、23至27、23至28、23至29、23至30、24至25、24至26、24至27、24至28、24至29、24至30、25至26、25至27、25至28、25至29、25至30、26至27、26至28、26至29、26至30、27至28、27至29、27至30、28至29、28至30或9至30个连接的核苷组成的寡核苷酸。在其中限制寡聚化合物或寡核苷酸的核苷数的实施方案无论是针对某一范围还是针对具体的数值寡聚化合物或寡核苷酸都可进一步包含另外的其它取代基。例如包含8-30个核苷的寡核苷酸不包括具有31个核苷的寡核苷酸，但是除非另外指明否则这种寡核苷酸可进一步包含例如一个或多个共轭物、末端基团或其它取代基，本文中的单体一般认为是寡核苷酸进一步包含的末端基团。在某些实施方案中末端基团包括但不限于末端基团核苷。在此类实施方案中末端基团与寡核苷酸的末端核苷不同地修饰从而将此类末端基团与寡核苷酸的核苷区别开来。
在某些实施方案中，本发明提供寡聚化合物除提供单体化合物之外，其余部分存在一个或者多个修饰。此类修饰包括但是不限于糖修饰或用糖替代物替代、碱基修饰、核苷间键合修饰以及基序。此类修饰在本文有所描述并且其中许多对本领域的技术人员而言都是已知的。所有此类修饰都适于本文所公开的寡聚化合物和双链核糖核酸。
在某些实施方案中，提供了抑制基因表达的方法，该方法包括：使细胞与包括具有上文所提供的寡聚化合物或双链核糖核酸(dsRNA)试剂接触，其中该寡聚化合物以及该双链核糖核酸(dsRNA)试剂中的正义链和反义链均包含8-40核苷酸长度，并且该寡聚化合物或双链核糖核酸(dsRNA)试剂中的反义链与靶RNA互补。在某些实施方案中，细胞在动物中。在某些实施方案中，细胞在人中。在某些实施方案中，靶RNA选自mRNA、pre-mRNA和micro RNA。在某些实施方案中，靶RNA为mRNA。在某些实施方案中，靶RNA为人mRNA。在某些实施方案中，靶RNA被裂解，从而抑制其功能。在某些实施方案中，该方法进一步包括检测靶RNA的水平。在某些实施方案中，提供了抑制基因表达的方法，该方法包括：使一种或多种细胞或组织与包括具有式(III)或其立体异构体、式(IVa)、式IV(b)、式(IVa-1)或者式(IVb-1)末端单体的寡聚化合物或者双链核糖核酸(dsRNA)接触。
本发明的上下文中的术语“寡聚化合物”指至少具有一个区域能与核酸分子杂交的聚合物。术语“寡聚化合物”包括寡核苷酸、寡核苷酸类似物和寡核苷以及核苷酸模拟物和/或包含核酸和非核酸成分的混合聚合物。术语“寡聚化合物”还包括包含连接的单体亚单位的聚合物，其中所述单体亚单位包括核苷、修饰的核苷、核苷类似物、核苷模拟物以及非核酸组分如偶联基团。在某些实施方案中，单体亚单位的混合物(例如但不限于所列的那些)提供下述寡聚化合物，所述寡聚化合物对于如治疗和诊断的应用具有增强的性质。本发明提供的双环脱碱基化合物由于不存在核碱基，被归类为非核酸成分。所述单体亚单位可通过天然存在的磷酸二酯核苷问键合或者通过此处公开的任何多个核苷问键合连接，核苷间键合例如但不限于硫代磷酯核苷间键合或其混合物。
寡核苷酸：如本文所用，如本文所用的术语“寡核苷酸”是指从2个到2500个核苷酸不等的核苷酸的聚合形式，寡核苷酸可以是单链的或双链的。该术语“寡核苷酸”包括由天然存在的核苷碱基、糖和共价核苷间连接构成的寡核苷酸以及具有一个或多个非天然存在部分的寡核苷酸。该种非天然存在的寡核苷酸由于其具有理想的性质而通常比天然存在的形式更理想，例如，细胞摄取提高，对核酸靶标的亲和力增强以及在核酸酶存在下的稳定性提高，例如对碱基或者糖部分的修饰。在某些实施方案中，寡核苷酸具有500-1500个核苷酸，通常例如其中所述寡核苷酸用于基因疗法中。在某些实施方案中，寡核苷酸是单链的或双链的并且具有7个-100个核苷酸。在某些实施方案中，寡核苷酸是单链的或双链的并且具有15个-100个核苷酸。在另一个实施方案中，寡核苷酸是单链的或双链的，具有15个-50个核苷酸，通常例如其中所述寡核苷酸是核酸抑制剂分子。在另一个实施方案中，寡核苷酸是单链的或双链的，具有25个-40个核苷酸，通常例如其中所述寡核苷酸是核酸抑制剂分子。在又另一个实施方案中，寡核苷酸是单链的或双链的并且具有19个-40个或19个-25个核苷酸，通常例如其中所述寡核苷酸是双链核酸抑制剂分子并且形成至少18个-25个碱基对的双链体。在其他实施方案中，寡核苷酸是单链的并且具有15个-25个核苷酸，通常例如其中所述寡核苷酸核苷酸是单链或者双链的RNAi抑制剂分子。通常，寡核苷酸有一个或多个含磷核苷酸间连接基团，如本文所述。在其他实施方案中，核苷酸间连接基团是不含磷的键，如本文所述。本文中的寡聚化合物实际上市的寡核苷酸进一步包含本文所述的末端单体。
通常，寡聚化合物包含连接的单体亚单位的主链，所述连接单体亚单位、糖部分或替代物和杂环碱基部分的键合可被独立地修饰。所述键合的糖单元，其可能包含或不包含杂环碱基，可以用模拟物例如肽核酸单体代替。在寡聚化合物每个单体进行修饰或取代部分或全部单体的能力可产生大量可能的基序。
寡聚化合物通常用常规线性制备，也被连接或以其它方式制备成坏状，并且还可包含分支。寡聚化合物可形成双链构建体，例如，杂交形成双链组合物的双链。该双链组合物可被连接或分离并可在末端包含突出(overhangs)。
如本领域所知，本发明的上下文中的术语“核苷”是指碱基糖组合。核苷的碱基部分称为核碱基，通常为杂环碱基部分。所述杂环碱基最常见的两种类别为嘌呤和嘧啶，例如：合适的天然核碱基包括嘌呤碱基和嘧啶碱基，例如腺嘌呤(A)、胸腺嘧啶(T)、胞嘧啶(C)、鸟嘌呤(G)或尿嘧啶(U)。合适的修饰的核碱基包括二氨基嘌呤和它的衍生物、烷基化的嘌呤或嘧啶、酰化的嘌呤或嘧啶、硫醇化的嘌呤或嘧啶等。可以理解的是，本文具有5′-末端和/或者3′-末端单体中双环脱碱基核酸类似物，其双环取代基R1并不存在这样的核苷的碱基。
如本领域所知，本发明的上下文中的术语“核苷酸”是指为进一步包含共价连接至核苷糖部分的磷酸基团的核苷。对于包含了呋喃戊糖的核苷，该磷酸基团可被联接至糖的2'、3’或5'羟基部分。在形成寡核苷酸时，该磷酸基团共价连接彼此相邻的核苷以形成线性聚合化合物。可以连接该线性聚合结构的相应末端以通过杂交或通过形成共价键得到环状结构，然而，通常希望单开放的线性结构。在寡核苷酸结构中，磷酸基团通常形成寡核苷酸的核苷间连接RNA和DNA的常见核苷间连接为3′至5′磷酸二酯联接。
如本文所用“末端基团”是指一个或多个连接到寡核苷酸3′端或5′端之一或两者的原子或者基团。在某些实施方案中末端基团为共轭基团。在某些实施方案中末端基团包含一个或多个另外的核苷，通常称为3’端或5’端-末端核苷酸。如本文所用的术语“5’-末端核苷酸或3’-末端核苷酸”是指位于寡核苷酸或者寡聚化合物的5’-末端或3’-末端的核苷酸。
如本文所用的术语“亚磷酰胺”是指含氮的三价磷衍生物。合适的亚磷酰胺的实例描述于本文中。
本文中使用的术语“siRNA”是指短干扰RNA或沉默RNA。siRNA是一类双链RNA分子，其长度可以是20至25(或更短)个碱基对，与在RNA干扰(RNAi)途径中起作用的微RNA(miRNA)类似。siRNA通过在转录之后降解mRNA来干扰具有与siRNA互补的核苷酸序列的特定基因的表达，从而防止翻译。siRNA在细胞中通过诱导RNA诱导的沉默复合物(RNA-induced silencing complex，RISC)切割信使RNA(mRNA)来沉默基因表达。
如本文所，除非另外指明或修正否则术语“双链”是指彼此杂交的两种单独的寡聚化合物。此类双链化合物可具有一条或两条链的一端或两端处的一个或多个非杂交核苷(悬垂)和/或一个或多个内部非杂交核苷(错配)，前提是存在足够的互补性以维持生理相关条件下的杂交。
如本文所用术语“自互补”或“发夹”是指包含通过寡聚化合物自我杂交而形成的双链体区域的单一寡聚化合物。
如本文所用术语“单链的”是指未与其互补序列杂交并且不具有足够的自互补性以在生理相关条件下形成发夹结构的寡聚化合物。单链化合物可以能够结合到其互补序列从而变成双链的或部分双链的化合物。
dsRNA试剂或靶基因反义多核苷酸试剂的治疗制剂可以通过将具有所需纯度的分子或化合物与任选的药学上可接受的载体、赋形剂或稳定剂[Remington's Pharmaceutical Sciences 21st edition,(2006)]以冻干制剂或水溶液的形式混合来制备用于储存。可接受的载体、赋形剂或稳定剂在所采用的剂量和浓度下对接受者是无毒的，并且包括缓冲剂，例如磷酸盐、柠檬酸盐和其他有机酸；抗氧化剂，包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；六甲铵氯化物；苯扎氯铵、苄索氯铵；苯酚、丁醇或苯甲醇；对羟基苯甲酸酯类，例如对羟基苯甲酸甲酯或丙酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；和间甲酚)；低分子量(少于约10个残基)多肽；蛋白质，例如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，如聚乙烯吡咯烷酮；氨基酸，例如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖和其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂如EDTA；蔗糖、甘露糖醇、海藻糖或山梨糖醇等糖类；形成盐的反离子，如钠；金属配合物(例如，锌-蛋白质配合物)；和/或非离子表面活性剂，例如或聚乙二醇(PEG)。
给药方式
在一些实施方案中，施用化合物的组织是其中存在或可能出现靶基因相关疾病或病症的组织，其非限制性实例是肝脏或肾脏。直接组织给药可以通过直接注射或其他方式实现。许多口服递送的化合物自然进入并通过肝脏和肾脏，本发明的治疗方法的一些实施方案包括向对象口服施用一种或更多种靶基因dsRNA试剂。dsRNA试剂或靶基因反义多核苷酸剂，单独或与其他治疗剂联合，可以施用一次，或者它们可以多次施用。如果多次给药，靶基因dsRNA试剂或靶基因反义多核苷酸剂可以通过不同途径给药。例如，虽然不打算限制，第一次(或前几次)给药可以通过皮下方式进行，并且一次或更多次额外给药可以是口服和/或全身给药。
对于其中希望全身性施用靶基因dsRNA试剂或靶基因反义多核苷酸剂的本发明实施方案，可以配制靶基因dsRNA试剂或靶基因反义多核苷酸剂用于通过注射例如通过推注或连续输注肠胃外施用。注射制剂可以以单位剂型存在，例如安瓿或多剂量容器，其添加或不添加防腐剂。靶基因dsRNA试剂制剂(也称为药物组合物)可采用油性或水性载体中的混悬液、溶液或乳液等形式，并且可含有配制剂，例如混悬剂、稳定剂和/或分散剂。
肠胃外给药的制剂包括无菌水溶液或非水溶液、混悬液和乳液。非水溶剂的例子是丙二醇、聚乙二醇、植物油如橄榄油和可注射的有机酯如油酸乙酯。水性载体包括水、酒精/水溶液、乳液或混悬液，包括盐水和缓冲介质。肠胃外载体包括氯化钠溶液、林格氏葡萄糖溶液、葡萄糖和氯化钠溶液、乳酸林格氏液或固定油。静脉内赋形剂包括流体和营养补充剂、电解质补充剂(例如基于林格氏葡萄糖溶液的那些)等。也可以存在防腐剂和其他添加剂，例如抗微生物剂、抗氧化剂、螯合剂和惰性气体等。其他形式的给药，例如静脉给药，将导致较低的剂量。如果对象在初始剂量下的反应不足，则可以在患者耐受性允许的范围内采用更高的剂量(或通过不同的、更局部的递送途径有效地提高剂量)。可以根据需要每天使用多次剂量以实现一种或更多种靶基因dsRNA试剂或靶基因反义多核苷酸试剂的适当全身或局部水平，并实现靶基因活性的适当降低。
在其他实施方案中，本发明的方法包括使用递送载体，例如生物相容性微粒、纳米颗粒或适合植入受体例如对象的植入物。PCT公开WO 95/24929(通过引用并入本文)中描述了可根据该方法使用的示例性可生物降解植入物，其描述了用于包含生物大分子的生物相容的、可生物降解的聚合物基质。
不可生物降解的和可生物降解的聚合物基质都可用于本发明的方法中，以将一种或更多种靶基因dsRNA试剂或靶基因反义多核苷酸试剂递送给对象。在一些实施方案中，基质可以是可生物降解的。基质聚合物可以是天然或合成聚合物。可以基于期望释放的时间段来选择聚合物，通常在几小时到一年或更长时间的数量级。通常，可以使用在几小时到三到十二个月之间的一段时间内的释放。聚合物任选地呈水凝胶形式，其可以吸收高达其重量约90％的水，并且还任选地与多价离子或其他聚合物交联。
通常，靶基因dsRNA试剂或靶基因反义多核苷酸试剂在本发明的一些实施方案中可以使用可生物降解的植入物通过扩散或通过聚合物基质的降解来递送。用于这种用途的示例性合成聚合物是本领域公知的。使用本领域已知的方法，可生物降解的聚合物和不可生物降解的聚合物可用于递送靶基因dsRNA试剂或靶基因反义多核苷酸试剂。生物黏附聚合物如可生物侵蚀的水凝胶(H.S.Sawhney,C.P.Pathak and J.A.Hubell in Macromolecules,1993,26,581-587)也可用于递送靶基因sRNA试剂或靶基因反义多核苷酸试剂，以治疗靶基因相关疾病或病症。其他合适的递送系统可以包括定时释放、延迟释放或持续释放递送系统。此类系统可避免重复施用靶基因dsRNA试剂或靶基因反义多核苷酸剂，从而提高对象和医疗保健专业人员的便利性。许多类型的释放递送系统是可用的并且是本领域普通技术人员已知的。见例如美国专利No.5,075,109、4,452,775、4,675,189、5,736,152、3,854,480、5,133,974和5,407,686。此外，可以使用基于泵的硬件输送系统，其中一些也适用于植入。
长期持续释放植入物的使用可以适用于对象的预防性治疗和具有发生复发性靶基因)相关疾病或病症的风险的对象。如本文所用，长期释放是指将植入物构建和布置成以至少长达10天、20天、30天、60天、90天、六个月、一年或更长时间递送治疗水平的靶基因dsRNA试剂或靶基因反义多核苷酸试剂。长期持续释放植入物是本领域普通技术人员众所公知的并且包括上述的一些释放系统。
有效量
在一些方面，本发明的方法包括将细胞与有效量的dsRNA试剂或反义多核苷酸试剂接触以减少所接触细胞中的基因表达。本发明方法的某些实施方案包括以有效降低基因表达和治疗对象的相关疾病或病症的量向对象施用dsRNA试剂或反义多核苷酸剂。就减少的表达和/或用于治疗相关疾病或病症而言，所使用的“有效量”是实现所需生物学效果所必需或足够的量。例如，治疗相关疾病或病症的dsRNA试剂或反义多核苷酸试剂的有效量可以是：(i)减缓或停止疾病或病症的进展所需的量；(ii)逆转、减少或消除疾病或病症的一种或更多种症状。在本发明的一些方面，有效量是当施用于需要治疗相关疾病或病症的对象时，导致疾病或病症的预防和/或治疗的治疗响应的dsRNA试剂或反义多核苷酸剂的量。根据本发明的一些方面，有效量是本发明的dsRNA试剂或反义多核苷酸试剂当与针对相关疾病或病症的另一种治疗性治疗组合或共同施用时，导致预防和/或治疗该疾病或病症的治疗响应的量。在本发明的一些实施方案中，用本发明的dsRNA试剂或反义多核苷酸试剂治疗对象的生物学效应可以是由相关疾病或病症引起的症状的改善和/或完全消除。在本发明的一些实施方案中，生物学效应是相关疾病或病症的完全消除，例如通过指示对象没有相关疾病或病症的诊断测试来证明。在一些实施方案中，有效量是导致所需响应的量，例如减少细胞、组织和/或患有疾病或病症的对象中的相关疾病或病症的量本发明的一些实施方案包括确定向对象施用的本发明的靶基因dsRNA试剂或靶基因反义多核苷酸试剂来治疗靶基因相关疾病或病症之功效的方法，其通过评估和/或监测对象中靶基因相关疾病或病症的一种或更多种“生理特征”来进行。靶基因相关疾病或病症的生理特征的非限制性实例是许多患者
应当认识到，基因沉默可以在表达的任何细胞中通过组成型或通过基因组工程进行，并通过任何合适的测定来确定。在本发明的一些实施方案中，通过施用本发明的dsRNA试剂，靶基因表达降低至少5％、6％、7％、8％、9％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或100％。在本发明的一些实施方案中，通过施用本发明的dsRNA试剂，靶基因表达减少5％至10％、5％至25％、10％至50％、10％至75％、25％至75％、25％至100％或50％至100％。
本发明中dsRNA试剂和反义多核苷酸试剂以足以靶基因表达的剂量在药物组合物中递送。在本发明的某些实施方案中，dsRNA试剂或反义多核苷酸剂的剂量为每千克接受者体重每天0.01至200.0毫克，一般为每天1至50mg/kg体重、5至40mg/kg体重、10至30mg/kg体重、1至20mg/kg体重、1至10mg/kg体重、4至15mg/kg体重，包括端值。例如，dsRNA试剂或反义多核苷酸试剂的每单次给药可以以从约0.01mg/kg、0.05mg/kg、0.1mg/kg、0.2mg/kg、0.3mg/kg、0.4mg/kg、0.5mg/kg、1mg/kg、1.1mg/kg、1.2mg/kg、1.3mg/kg、1.4mg/kg、1.5mg/kg、1.6mg/kg、1.7mg/kg、1.8mg/kg、1.9mg/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、4.1mg/kg、4.2mg/kg、4.3mg/kg、4.4mg/kg、4.5mg/kg、4.6mg/kg、4.7mg/kg、4.8mg/kg、4.9mg/kg、5mg/kg、5.1mg/kg、5.2mg/kg、5.3mg/kg、5.4mg/kg、5.5mg/kg、5.6mg/kg、5.7mg/kg、5.8mg/kg、5.9mg/kg、6mg/kg、6.1mg/kg、6.2mg/kg、6.3mg/kg、6.4mg/kg、6.5mg/kg、6.6mg/kg、6.7mg/kg、6.8mg/kg、6.9mg/kg、7mg/kg、7.1mg/kg、7.2mg/kg、7.3mg/kg、7.4mg/kg、7.5mg/kg、7.6mg/kg、7.7mg/kg、7.8mg/kg、7.9mg/kg、8mg/kg、8.1mg/kg、8.2mg/kg、8.3mg/kg、8.4mg/kg、8.5mg/kg、8.6mg/kg、8.7mg/kg、8.8mg/kg、8.9mg/kg、9mg/kg、9.1mg/kg、9.2mg/kg、9.3mg/kg、9.4mg/kg、9.5mg/kg、9.6mg/kg、9.7mg/kg、9.8mg/kg、9.9mg/kg、10mg/kg、11mg/kg、12mg/kg、13mg/kg、14mg/kg、15mg/kg、16mg/kg、17mg/kg、18mg/kg、19mg/kg、20mg/kg、21mg/kg、22mg/kg、23mg/kg、24mg/kg、25mg/kg、26mg/kg、27mg/kg、28mg/kg、29mg/kg、30mg/kg、31mg/kg、32mg/kg、33mg/kg、34mg/kg、35mg/kg、36mg/kg、37mg/kg、38mg/kg、39mg/kg、40mg/kg、41mg/kg、42mg/kg、43mg/kg、44mg/kg、45mg/kg、46mg/kg、47mg/kg、48mg/kg、49mg/kg至50mg/kg体重的量来施用。
在确定本发明的dsRNA试剂的递送剂量和时间时可以考虑多种因素。递送的dsRNA试剂或反义多核苷酸剂的绝对量将取决于多种因素，包括共同治疗、剂量数和个体对象参数，包括年龄、身体状况、体格大小和体重。这些是本领域普通技术人员众所公知的因素，并且可以通过常规实验解决。在一些实施方案中，可以使用最大剂量，即根据合理的医学判断的最高安全剂量。
在一些实施方案中，本发明的方法可包括向对象施用1、2、3、4、5、6、7、8、9、10或更多个剂量的试剂或反义多核苷酸试剂。在一些情况下，可以至少每天、每隔一天、每周、每隔一周、每月等向对象施用药物化合物的剂量，可以每天给药一次或每天给药多于一次，例如在一个24小时周期内给药2、3、4、5或更多次。本发明的药物组合物可以每天给药一次；或者dsRNA试剂或反义多核苷酸试剂可以在一天中以适当的间隔以两个、三个或更多个亚剂量给药，或者甚至使用连续输注或通过控释制剂递送。在本发明方法的一些实施方案中，将本发明的药物组合物每天一次或更多次、每周一次或更多次、每月一次或更多次或每年一次或更多次施用给对象。
本发明的某些实施方案包括含有dsRNA试剂或反义多核苷酸试剂和药学上可接受的载体的药物组合物的用途。包含dsRNA试剂或反义多核苷酸剂的药物组合物可用于本发明的方法中以降低细胞中的基因表达和活性，并可用于治疗相关疾病或病症。此类药物组合物可以基于递送方式来配制。用于递送方式的制剂的非限制性实例是：配制用于皮下递送的组合物、配制用于通过肠胃外递送全身给药的组合物、配制用于静脉内(IV)递送的组合物、配制用于鞘内递送的组合物、配制用于直接递送至脑中的组合物等。可以使用一种或更多种方式施用本发明的药物组合物以将dsRNA试剂或反义多核苷酸试剂递送到细胞中，例如：表面(例如，通过透皮贴剂)；肺部，例如通过吸入或吹入粉末或气雾剂，包括通过雾化器；气道内、鼻内、表皮和透皮、口服或肠胃外。肠胃外给药包括静脉内、动脉内、皮下、腹膜内或肌肉内注射或输注；表皮下，例如通过植入装置；或颅内，例如通过实质内；鞘内或心室内施用。dsRNA试剂或反义多核苷酸剂也可以直接递送至靶组织，例如直接递送至肝脏、直接递送至肾脏等。可以理解的是，“递送dsRNA试剂”或“反义多核苷酸试剂”到细胞中分别包括递送dsRNA试剂或反义多核苷酸剂、直接在细胞中表达dsRNA试剂以及从递送到细胞中的编码载体表达dsRNA试剂，或使得dsRNA或反义多核苷酸试剂出现在细胞中的任何合适的方式。制剂的制备和使用以及用于递送抑制性RNA的手段是本领域公知的和常规使用的。
在一些实施方案中，组合物还包含一种或更多种另外的治疗剂。本发明的组合物可包含一种或更多种dsRNA试剂和任选的一种或更多种药学上可接受的载体、递送剂、靶向剂、可检测标签等，根据本发明方法的一些实施方案可用的靶向剂的非限制性实例是将本发明的dsRNA试剂引导至和/或进入待治疗细胞的试剂。靶向剂的选择将取决于以下要素：相关疾病或病症的性质，以及靶细胞类型。在一个非限制性实例中，在本发明的一些实施方案中，可能需要将dsRNA试剂靶向至和/或进入肝细胞。应当理解，在本发明方法的一些实施方案中，治疗剂包含仅具有递送剂的dsRNA试剂，例如包含N-乙酰半乳糖胺(GalNAc)的递送剂，而没有任何附加的连接元件。例如，在本发明的一些方面，dsRNA试剂可以连接到包含GalNAc的递送化合物上，并且包含在含有药学上可接受载体的组合物中，并且在没有任何连接至dsRNA试剂的可检测标记或靶向剂等的情况下施用至细胞或对象。
在本发明的dsRNA试剂与一种或更多种递送剂、靶向剂、标记剂等一起施用和/或连接到其上的情况下，本领域技术人员能够了解并能够选择和使用适合的试剂用于本发明的方法中。在本发明的某些方法中可以使用标记试剂来确定dsRNA试剂在细胞和组织中的位置，并且可用于确定已在本发明的方法中施用的包含dsRNA试剂的治疗组合物的细胞、组织或器官位置。接附和使用标记试剂如酶标记、染料、放射性标记等的手段是本领域公知的。应当理解，在本发明的组合物和方法的一些实施方案中，标记试剂连接至dsRNA试剂中所包含的正义多核苷酸和反义多核苷酸之一或两者。
在一些实施方案中，组合物被包装在药盒、容器、包装物、分配器、预填充注射器或小瓶中。药盒包含一种或更多种靶基因dsRNA试剂和/或靶基因反义多核苷酸试剂及其在本发明方法中的使用说明的药盒也在本发明的范围内。本发明的药盒可包含可用于治疗靶基因相关疾病或病症的靶基因dsRNA试剂、靶基因正义多核苷酸和靶基因反义多核苷酸试剂中的一种或更多种。可以制备包含一种或更多种靶基因dsRNA试剂、靶基因正义多核苷酸和靶基因反义多核苷酸试剂的药盒以用于本发明的治疗方法。本发明药盒的组分可以以水性介质或冻干形式包装。本发明的药盒可以包含被分隔开以在其中封闭地收纳一个或更多个容器装置或一系列容器装置(例如试管、小瓶、烧瓶、瓶子、注射器等)的载体。第一容器装置或一系列容器装置可包含一种或更多种化合物，例如靶基因)dsRNA试剂和/或靶基因正义或反义多核苷酸试剂。第二容器装置或一系列容器装置可包含靶向剂、标记剂、递送剂等，其可作为在本发明的治疗方法的实施方案中施用的靶基因)dsRNA试剂和/或靶基因反义多核苷酸的一部分包括在内。
本发明的药盒还可包含说明书。说明书通常采用书面形式，并且将为执行由药盒体现的治疗和基于该治疗做出决定提供指导。
细胞、对象和对照
本发明的方法可以与细胞、组织、器官和/或对象结合使用。在本发明的一些方面，对象是人或脊椎动物哺乳动物，包括但不限于狗、猫、马、牛、山羊、小鼠、大鼠和灵长类动物，例如猴。因此，本发明可用于治疗人和非人对象的靶基因相关疾病或病症。
在本发明的一些方面，对象可以是农场动物、动物园动物、驯养动物或非驯养动物，并且本发明的方法可用于兽医预防和治疗方案。在本发明的一些实施方案中，对象是人并且本发明的方法可用于人预防和治疗方案。
可应用本发明的对象的非限制性实例是被诊断患有、怀疑患有或有风险患有与以下疾病或病症相关的疾病或病症的对象：高于期望的靶基因表达和/或活性，也称为“升高的靶基因表达水平”。与高于期望水平的靶基因表达和/或活性相关的疾病和病症的非限制性实例在本文别处描述。本发明的方法可应用于在治疗时已被诊断为患有该疾病或病症的对象、与高于期望的靶基因表达和/或活性相关的对象，或被认为处于患有或发展与高于期望的靶基因表达和/或活性相关的疾病或病症的风险中的对象。在本发明的一些方面，与高于期望的靶基因表达和/或活性水平相关的疾病或病症是急性疾病或病症；在本发明的某些方面，与高于期望的靶基因表达和/或活性水平相关的疾病或病症是慢性疾病或病症。
在另一个非限制性实例中，将本发明的靶基因dsRNA试剂施用以治疗指因靶基因被激活导致或与其相关的疾病或障碍，或其症状或进展响应于靶基因失活的疾病或障碍。术语“靶基因相关疾病”包括因靶基因表达降低而受益的疾病、障碍或病症。
可应用本发明方法的细胞包括体外、体内、离体细胞。细胞可以在对象中、在培养物中和/或混悬液中，或处于任何其他合适的状态或条件中。可以应用本发明的方法的细胞可以是：肝脏细胞(liver cell)、肝细胞(hepatocyte)、心脏细胞、胰腺细胞、心血管细胞、肾细胞或其他类型的脊椎动物细胞，包括人和非人哺乳动物细胞。在本发明的某些方面，可应用本发明方法的细胞是健康的正常细胞，其未知为疾病细胞。在本发明的某些实施方案中，将本发明的方法和组合物应用于肝脏细胞、肝细胞、心脏细胞、胰腺细胞、心血管细胞和/或肾细胞的细胞。在本发明的某些方面，对照细胞是正常细胞，但应当理解，具有疾病或病症的细胞也可以在特定情况下用作对照细胞，例如在比较具有疾病或病症的经处理细胞与具有疾病或病症的未处理细胞的结果等的情况下。
根据本发明的方法，可以确定靶基因多肽活性的水平并将其与靶基因多肽活性的对照水平进行比较。对照可以是预定值，其可以采取多种形式。它可以是单个截止值，例如中位数或平均值。它可以基于比较组来建立，例如在具有正常水平的靶基因多肽和/或靶基因多肽活性的组和具有增加的靶基因多肽和/或靶基因多肽活性水平的组中。比较组的另一个非限制性实例可以是具有靶基因相关疾病或病症的一种或更多种症状或诊断的群体与没有疾病或病症的一种或更多种症状或诊断的群体；已对其施用本发明的siRNA治疗的对象组与未对其施用本发明的siRNA治疗的对象组。通常，对照可以基于适当年龄组中的明显健康的正常个体或明显健康的细胞。应当理解，除了预定值之外，根据本发明的对照可以是与实验材料平行测试的材料样品。示例包括来自对照群体的样品或通过制造产生的对照样品，以用于与实验样品进行平行测试。在本发明的一些实施方案中，对照可包括未用本发明的靶基因dsRNA试剂接触或处理的细胞或对象，在这种情况下，可以比较靶基因多肽和/或靶基因多肽活性的对照水平以及与本发明的靶基因dsRNA试剂或靶基因反义多核苷酸试剂接触的细胞或对象中靶基因多肽和/或靶基因多肽活性的水平。
在本发明的一些实施方案中，对照水平可以是为对象确定的靶基因多肽水平，其中将在不同时间为同一对象确定的靶基因多肽水平与该对照水平进行比较。在一个非限制性实例中，在从未接受过本发明的靶基因治疗的对象获得的生物样品中确定靶基因的水平。在一些实施方案中，生物样品是血清样品。从对象获得的样品中测定的靶基因多肽水平可作为对象的基线或对照值。在本发明的治疗方法中向对象施用一次或更多次靶基因dsRNA试剂之后，可以从对象获得一个或更多个另外的血清样品，并且可以将随后的一个或更多个样品中的靶基因多肽水平与对象的对照/基线水平进行比较。此类比较可用于评估对象中靶基因)相关疾病或病症的发作、进展或消退。例如，从对象获得的基线样品中靶基因)多肽的水平高于在给予对象本发明的靶基因dsRNA试剂或靶基因)反义多核苷酸试剂后从同一对象获得的水平，则表示靶基因相关疾病或病症的消退并且表示施用的本发明的靶基因dsRNA试剂治疗靶基因相关疾病或病症产生的功效。
在本发明的某些方面，为对象确定的靶基因多肽和/或靶基因多肽活性水平中的一个或更多个值可以作为对照值，并用于稍后在同一对象中比较靶基因多肽和/或靶基因活性水平，从而允许评估对象中“基线”靶基因多肽活性的变化。因此，将初始水平用作该对象的对照水平的情况下，初始靶基因多肽水平和/或初始靶基因多肽活性水平可以用于显示和/或确定本发明的方法和化合物在对象中所能够降低对象中靶基因多肽和/或靶基因多肽活性的水平。
使用本发明的方法，可以将本发明的靶基因dsRNA试剂和/或靶基因反义多核苷酸试剂施用于对象。这样的dsRNAi试剂包括可以如下评估本发明的施用和治疗的功效：与先前时间点从对象获得的血清样品中靶基因)多肽的给药前水平相比，或与非接触对照水平(例如对照血清样品中的靶基因多肽水平相比，当施用和治疗后，从对象获得的血清样品中靶基因多肽的水平降低至少0.5％、1％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％、95％或更多。应当理解，靶基因多肽的水平和靶基因多肽活性的水平都与靶基因表达的水平相关。本发明方法的某些实施方案包括以有效抑制靶基因表达的量向对象施用本发明的靶基因dsRNA和/或靶基因反义试剂，从而降低对象中靶基因多肽的水平并降低靶基因多肽活性的水平。在本发明的方法的一些实施方案中，细胞与本发明的siRNA试剂的接触(在本文中也称为处理)导致细胞中靶基因表达抑制至少约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％或约100％，例如，至低于化验检测水平。
本发明的一些实施方案包括从一个或更多个对象获得的一个或更多个生物样品中确定靶基因多肽的存在、不存在和/或量(本文也称为水平)。该测定可用于评估本发明的治疗方法的功效。例如，本发明的方法和组合物可用于确定生物样品中靶基因多肽的水平，该生物样品获自先前用施用本发明的靶基因dsNA试剂和/或靶基因反义剂治疗的对象。与先前时间点从对象获得的血清样品中靶基因多肽的给药前水平相比，或与非接触对照水平(例如对照血清样品中的靶基因多肽水平)相比，当施用和治疗后，从对象获得的血清样品中靶基因多肽的水平降低至少0.5％、1％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％、95％或更多，则表明给予对象的治疗的功效水平。
在本发明的一些实施方案中，针对对象确定的靶基因相关疾病或病症的生理特征可以作为对照结果，并将同一对象在不同时间的生理特征的确定结果与对照结果进行比较。在一个非限制性实例中，病理特征溶血测量自从未给予本发明的靶基因治疗的对象，其可用作对象的基线或对照值。在本发明的治疗方法中向对象施用一次或更多次靶基因dsRNA试剂之后，血细胞分别与对象的对照/基线水平进行比较。此类比较可用于评估对象中靶基因相关疾病或病症的发作、进展或消退。例如，从对象获得的基线血细胞高于在对对象施用本发明的靶基因dsRNA试剂或靶基因反义多核苷酸试剂后从同一对象测定的血栓，则表示靶基相关疾病或病症的消退并且表示施用的本发明的靶基因dsRNA试剂治疗靶基因相关疾病或病症的功效。
本发明的一些实施方案包括使用例如但不限于以下方法确定靶基因关疾病或病症的生理特征的存在、不存在和/或变化：(1)测量对象的血细胞；(2)评估从一名或更多名对象获得的一份或更多份生物样品的生理特征；(3)或对对象进行身体检查。该测定可用于评估本发明的治疗方法的功效。
错配
本领域技术人员已知，对于dsRNA的功效而言，错配是可以容忍的，尤其是错配在dsRNA的末端区域内的情况。某些错配具有更好的耐受性，例如具有摆动碱基对G:U和A:C的错配对功效的耐受性更好(Du et el.,A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites.Nucleic Acids Res.2005 Mar 21；33(5):1671-7.Doi:10.1093/nar/gki312.Nucleic Acids Res.2005；33(11):3698)。
互补性
如本文所用，除非另有说明，否则术语“互补性/互补”当用于描述第一核苷酸序列(例如，靶基因dsRNA试剂正义链或靶标靶基因mRNA)与第二核苷酸序列(例如，靶基因dsRNA试剂反义链或单链反义多核苷酸)的相关性时，是指包含第一核苷酸序列的寡核苷酸或多核苷酸与包含第二核苷酸序列的寡核苷酸或多核苷酸杂交[在哺乳动物生理条件(或体外类似条件)下形成碱基对间氢键]、并且在某些条件下形成双螺旋或双螺旋结构的能力。其中也可以应用其他条件，例如在生物体内可能遇到的生理相关条件。技术人员将能够根据杂交核苷酸的最终应用确定最适合测试两个序列互补性的条件集。互补序列包括沃森-克里克碱基对或非沃森-克里克碱基对，并且包括天然或修饰的核苷酸或核苷酸模拟物，只要至少达到上述杂交要求的程度即可。序列同一性或互补性与修饰无关。
例如，在如本文所述的靶基因dsRNA内的互补序列包含含有第一核苷酸序列的寡核苷酸或多核苷酸与含有第二核苷酸序列的寡核苷酸或多核苷酸在一个或两个核苷酸序列的全长上的碱基配对。此类序列在本文中可被称为彼此“完全互补”。应当理解，在设计两个寡核苷酸以在杂交时形成一个或更多个单链突出端的实施方案中，这种突出端在本文中不被视为基于互补性确定的错配。例如，靶基因dsRNA试剂包含一个长度为19个核苷酸的寡核苷酸和另一个长度为20个核苷酸的寡核苷酸，其中较长的寡核苷酸包含与较短的寡核苷酸完全互补的19个核苷酸的序列，出于本文所述的目的，此种情况可以称为“完全互补”。因此，如本文所用，“完全互补”是指第一多核苷酸的连续序列中的所有(100％)碱基会与第二多核苷酸的连续序列中的相同数目的碱基杂交。连续序列可以包含第一或第二核苷酸序列的全部或部分。
如本文所用，术语“基本互补”是指在核碱基序列的杂交对中，第一多核苷酸的连续序列中的碱基的至少约85％(但不是全部)会与第二多核苷酸的连续序列中相同数目的碱基杂交。如果两个序列在杂交时包含一个或更多个错配碱基对，例如至少1、2、3、4或5个错配碱基对，则可以使用术语“基本上互补”来指第一序列相对于第二序列形成多达15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个碱基对(bp)的双链体，同时保留在与其最终应用最相关的条件下杂交的能力，例如，通过RISC途径抑制靶基因基因表达。术语“部分互补”在本文中可用于指核碱基序列的杂交对中，第一多核苷酸的连续序列中碱基的至少75％(但不是全部)会与第二多核苷酸的连续序列中相同数目的碱基杂交。在一些实施方案中，“部分互补”是指第一多核苷酸的连续序列中至少76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的碱基会与第二多核苷酸的连续序列中相同数量的碱基杂交。
术语“互补”、“完全互补”、“基本互补”和“部分互补”在本文中使用时可用于指dsRNA试剂的正义链与反义链之间的碱基匹配、dsRNA试剂的反义链与靶mRNA的序列之间的碱基匹配，或单链反义寡核苷酸与靶mRNA序列之间的碱基匹配。应当理解，术语“dsRNA试剂的反义链”可以指与“反义多核苷酸试剂”相同的序列。
关于修饰
在本发明的一些实施方案中，基因RNAi剂的RNA被化学修饰以获得增强的稳定性和/或一种或更多种其他有益特性。本发明的某些实施方案中的核酸可以通过本领域公知的方法合成和/或修饰，例如，见“Current protocols in Nucleic Acid Chemistry,"Beaucage,S.L.et al.(Eds.),John Wiley&Sons,Inc.,New York,N.Y.,USA，其作为参考在此并入本文。可以存在于本发明的dsRNA试剂的某些实施方案中的修饰包括例如：(a)末端修饰，例如5'端修饰(磷酸化、缀合、反向连接等)、3'端修饰(缀合、DNA核苷酸、反向连接等)；(b)碱基修饰，例如用稳定碱基、去稳定碱基或与扩展的配偶体库进行碱基配对的碱基替换、缺失碱基(无碱基核苷酸)或缀合碱基；(c)糖修饰(例如，在2'位置或4'位置)或糖的替换；以及(d)骨架修饰，包括磷酸二酯键的修饰或替换。在本发明的dsRNA试剂、反义多核苷酸和正义多核苷酸的某些实施方案中可用的RNA化合物的具体实例包括但不限于包含修饰骨架或没有天然核苷间键联的RNA。作为非限制性实例，具有骨架修饰的RNA在骨架中可以不具有磷原子。在其核苷间骨架中没有磷原子的RNA可称为寡核苷。在本发明的某些实施方案中，修饰的RNA在其核苷间骨架中具有磷原子。
应当理解，术语“RNA分子”或“RNA”或“核糖核酸分子”不仅包括在自然界中表达或发现的RNA分子，还包括RNA的类似物和衍生物，其包含一种或更多种如本文所述或本领域已知的核糖核苷酸/核糖核苷类似物或衍生物。术语“核糖核苷”和“核糖核苷酸”在本文中可互换使用。RNA分子可以在核碱基结构或核糖-磷酸骨架结构中进行修饰(例如，如下文所述)，并且包含核糖核苷类似物或衍生物的分子必须保留形成双链体的能力。
提供以下实施例以说明本发明实践的具体实例，其并不旨在限制本发明的范围。对本领域普通技术人员来说明显的是，本发明可应用于多种组合物和方法。
如本文所用“取代基”是本发明的预期方面。药物化学和有机化学领域的普通技术人员将理解此类取代基的通用性。在“取代基”应满足于本发明中其总数将符合与其附着原子或者基团可形成化学键存在的原理。
术语“烃基”包括具有任意饱和度的直链、支链和环状基团。在一些特殊情况下，包括包含C、O、H的基团，这样的烃基基团可包括一个或多个氮、氧、硫和磷的杂原子中断，并可进一步被一或多个取代基单或多取代。
如本文所用的术语“烷基”是指具有1个至约24个碳原子的直链或支链饱和的烃基。当它在本文出现时，诸如“C1-C6烷基”的数量范围意一般是指烷基中碳原子的数目，可以仅包含1个碳原子、2个碳原子、3个碳原子等，直至并且包括6个碳原子，除另有说明，一般烷基中进一步被其他原子或者基团取代所增加的碳原子的数目不在计数范围内，本文其他计数方式也存在同样的描述。烷基的实例包括但不限于甲基、乙基、丙基、丁基、异丙基、正己基、辛基、癸基、十二烷基等。尽管术语“烷基”还包括其中没有指定碳原子的数量范围的情况，举例来说，术语“烷基”可以是指C1-C10的子范围(例如C1-C6)。“取代的烷基”是指带有取代基的烷基部分。如本文所用的“低级烷基”是指具有1个至约6个碳原子的烷基部分。
如本文所用的术语“烯基”是指至少一个碳-碳双键的直链或支链烃基。烯基的实例包括但不限于乙烯基、丙烯基、丁烯基、1-甲基-2-丁烯-1-基、二烯，诸如1,3-丁二烯等。烯基通常包含2至约24个碳原子，更通常包含2至约12个碳原子，其中2至约6个碳原子是更优选的。如本文所用的烯基可任选地包含一个或更多个进一步的取代基团。
如本文所用的术语“炔基”是指至少一个碳-碳三键的直链或支链烃基。炔基的实例包括但不限于乙炔基、1-丙炔基、1-丁炔基等。炔基通常包含2至约24个碳原子更通常包含2至约12个碳原子，其中2至约6个碳原子是更优选的。如本文所用的炔基可任选地包含一个或更多个进一步的取代基团。
如本文所用的术语“酰基”是指通过从有机酸中移除羟基而形成的基团并具有通式-C(O)-X其中X通常为脂族、脂环族或族的。实例包括脂族羰基、芳族羰基、脂族磺酰基、芳族亚磺酰基、脂族亚磺酰基、芳族磷酸酯、脂族磷酸酯等。如本文所用的酰基可任选地包含进一步的取代基团。
术语“环烷基”是指其中环为脂族的环体系。该环体系可包含一个或多个环其中至少一个环是脂族的。优选的脂环族化合物包含环中具有约5至约9个碳原子的环。如本文所用环烷基可任选地包含进一步的取代基团。
如本文所用术语“烷氧基”是指在烷基和氧原子之间形成的基团，其中氧原子用于将烷氧基连接到母体分子。烷氧基的实例包括但不限于：甲氧基、乙氧基、丙氧基、异丙氧基、正丁氧基、仲丁氧基、叔丁氧基、正戊氧基、新戊氧基和正己氧基等。如本文所用的烷氧基可任选地包含进一步的取代基团。
如本文所用的术语“氨基烷基”是指氨基取代的C1-C12烷基。该基团的烷基部分与母体分子形成共价键。氨基可位于任何位置并且氨基烷基可在烷基和/或氨基部位被进一步的取代基团取代。
如本文所用的术语“芳基”和“芳族”是指具有一个或多个芳环的单环或多环的碳环系基团，如本文所用的术语“芳族基团”是指具有含有4n+2π电子的离域π电子体系的平面环，其中n是整数。芳环可以由5个、6个、7个、8个、9个或多于9个原子形成。术语“芳族”意图包括碳环芳基(例如苯基)和杂环芳基(或“杂芳基”或“杂芳族”)基团(例如吡啶)。所述术语包括单环或稠环多环，即共用相邻碳原子对的环。“取代的芳族基”是指进一步带有一个或多个取代基的芳族基团。芳基的实例包括但不限于苯基、萘基、四氢萘基、茚满基、茚基(idenyl)等。优选的芳基环体系在一个或多个环中具有约5至约20个碳原子。如本文所用的芳基可任选地包含进一步的取代基团。
如本文所用的术语“芳烷基”和“芳基烷基”是指共价连接到C1-C12烷基的芳族基团。所得的芳烷基(或芳基烷基)的烷基部分与母体分子形成共价键。实例包括但不限于苄基、苯乙基等。如本文所用的芳烷基可任选地包含连接到烷基、芳基或形成基团的两者的进一步的取代基团。
如本文所用的术语“杂芳基”和“杂芳族”是指包含单环或多环芳环、环体系或稠合环体系的基团其中这些环的至少一个为芳族的并包含一和或多个杂原子。杂芳基还意在包括稠合环体系包括其中稠环的一个或多个不含杂原子的体系。杂芳基通常包含一个选自硫、氮或氧的环原子。杂芳基的实例包括但不限于:吡啶基、吡嗪基、嘧啶基、吡咯基、吡唑基、咪唑基、噻唑基、噁唑基、异噁唑基、噻二唑基、噁二唑基、苯硫基、呋喃基、喹啉基、异喹啉基、苯并咪唑基、苯并噁唑基、喹噁啉基等。杂芳基可直接地或通过连接部分诸如脂族基团或杂原子连接到母体分子上。如本文所用的杂芳基可任选地包含进一步的取代基团。
如本文所用的术语“杂芳基烷基”是指进一步包含共价连接的C1-C12烷基的如之前定义的杂芳基。所得杂芳基烷基的烷基部分能够与母体分子形成共价键。实例包括但不限于吡啶基甲基、吡啶基乙基、萘啶基丙基(napthyridinylpropyl)等。如本文所用的杂芳基烷基可在杂芳基或烷基部分的一者或两者上任选地包含进一步的取代基团。
如本文所用的术语“卤”或“卤素”是指选自氟、氯、溴和碘的原子。
如本文所用的术语“杂环基团”是指包含至少一个和杂原子并为不饱和的、部分饱和的或完全饱和的单环或多环环体系因而包括杂芳基基团。杂环还意在包括稠合环体系其中稠合环的一个或多个包含至少一个杂原子并且其它环可包含一个或多个杂原子或任选地不含杂原子。杂环基团通常包含至少一个选自硫、氮或氧的原子。杂环基团的实例包括[1,3]二氧戊环基、吡咯烷基、吡唑啉基、吡唑烷基、咪唑啉基、咪唑烷基、哌啶基、吡嗪基、噁唑烷基、异噁唑烷基、吗啉基、噻唑烷基、异噻唑烷基、喹噁啉基、哒嗪基(pyridazinonyl)、四氢呋喃基等。如本文所用“n”元的杂环基团是只环系中含有n个原子的总和，不保护环系意外的原子或者基团。如本文所用的杂环基团可任选地包含进一步的取代基团。
如本文所用的术语“单环或多环结构”包括选自单环环体系或其中环为稠合的或相连的多环环体系的所有环体系并意在包括单独地选自脂基、脂环基、芳基、杂芳基、芳烷基、芳基烷基、杂环基、杂芳基、杂芳族基和杂芳基烷基的单个和混合环体系。此类单环和多环结构可包含每一个都具有相同饱和度或每一个独立地具有不同饱和度(包括完全饱和、部分饱和或完全不饱和)的环。每个环可包含选自C、N、O和S的环原子以得到杂环以及只含C环原子的环这些环可以混合基序存在诸如例如苯并咪唑其中一个环只具有碳环原子而稠合的环具有两个氮原子。单环或多环结构可进一步被取代基团取代诸如例如邻苯二甲酰亚胺其具有连接到其中一个环上的两个＝O基团。单环或多环结构可使用多种策略连接到母体分子上诸如通过环原子直接连接、通过取代基团或通过双官能连接部分连接。
术语“氧代”是指基团(＝O)。
本文中使用的术语“磺酰基”(单独或作为另一基团的一部分)是指式RSO2—的基团，其中R是氢、烷基、经取代烷基、芳基、或经取代芳基。本文中使用的术语“磺酰胺基”(单独或作为另一基团的一部分)是指式RSO2-NH-的基团，其中R是氢、烷基、经取代烷基、芳基、或经取代芳基。一些非限制性示例性磺酰胺基包CH3-SO2-N(H)-。
术语“脂环族的”或“脂环基”指环状的环系缔，其中该环为脂肪族环。该环系缔可包含一个或多个环，其中至少一个环为脂肪族环。优选的脂肪环包括环中具有约5至约9个碳原子的环。此处所用的脂肪环任选地包括其它取代基团。此处所用的术语“脂肪族”指最多包含二十四个碳原子的直链或支链烃基，其中任意两个碳原子之间的饱和度为单、双或三键。脂肪族基团优选包含1至约24个碳原子，更通常包含1至约12个碳原子，更优选包含1至约6个碳原子脂肪基团的直链或支链可被一个或多个杂原子包括氮、氧、硫和磷等所中断。所述被杂原子中断的脂肪族基团包括但不限于聚烷氧基，例如聚亚烷基二醇、聚胺和聚亚胺。此处所用的脂肪族基团任选地包括其它取代基团。间隔物，将寡核苷酸系连到其上，用于将所述寡核苷酸和合成过程放置得远离所述底物或者载体。这种方式允许更大的灵活性和更多的用于合成的空间，在完成合成时方便于切割。
本文中使用的术语“羧基”(单独或作为另一基团的一部分)是指式-COOH的基团。
连接基团或双官能连接部分诸如本领域已知的那些可用于将化学官能团、共轭基团、报告基团和其它基团连接到母体化合物诸如例如寡聚化合物中的选择性位点上。一般来讲双官能连接部分包括具有两个官能团的烃基部分。选择其中一个官能团以结合到母体分子或所关注的化合物上而选择另一个以结合到基本上任何选定的基团诸如化学官能团或共轭基团上。在一些实施方案中连接基包含链结构或重复单元诸如乙二醇或氨基酸单元的聚合物。通常用于双官能连接部分的官能团的实例包括但不限于用于与亲核基团反应的亲电体以及用于与亲电基团反应的亲核体。在一些实施方案中双官能连接部分包含氨基、羟基、羧酸、硫醇、不饱和度(例如双键或三键)等。双官能连接部分的一些非限制实例包括8-氨基-3,6-二氧杂辛酸(ADO)、4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺(SMCC)和6-氨基己酸(AHEX或AHA)。其它连接基团包括但不限于取代的C1-C10烷基、取代的或未取代的C2-C10烯基或者取代的或未取代的C2-C10炔基其中优选取代基团的非限制性列表包括羟基、氨基、烷氧基、羧基、苄基、苯基、硝基、硫醇、硫代烷氧基、卤素、烷基、芳基、烯基和炔基。
本文中使用的术语“醚”是醇或酚的羟基中的氢被烃基取代的产物，通式为R-O-R'，R和R’可以相同，也可以不同。两者相同的醚称为对称醚，也叫简单醚或单醚；两者不相同的醚称为不对称醚，也叫混合醚或混醚。
本文中使用的术语或者是一般是指于相邻基团或者部分的链接处。
约：如本文所用，如应用于所关注的一个或多个值的术语“约”或“大约”是指与所述的参考值类似的值。在某些实施方案中，除非另有说明或另外从上下文中明显看出，否则术语“约”或“大约”是指落入所述参考值在任一方向(大于或小于)上的25％、20％、19％、18％、17％、16％、15％、14％、13％、12％、11％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％或更少内的值的范围(除非这样的数值将超过可能值的100％)。
本文中一些所用缩写“Ac”指乙酰基；缩写“Bn＂指苄基；缩写"Bz"指苯甲酰基；缩写"DMTrCl”指4,4-二甲氧基三苯甲基氯；缩写"DMTr”指4,4-二甲氧基三苯甲基；缩写'TIIP"指四氢吡喃基；缩写"TBDMS”指叔丁基二甲基甲硅烷基；缩写："TIPDS"指四异丙基二甲硅烷基；及缩写“DTBS”指二(叔丁基)甲硅烷基。
 具体实施例
提供以下实施例以说明实施本发明的具体实例，并不旨在限制本发明的范围。如对本领域普通技术人员显而易见的，本发明将在多种组合物和方法中得到应用。
实施例1.
亚磷酰胺化合物2-的合成
将在吡啶(400毫升)中的DMTrCl(232克，684毫摩尔，1.0当量)，加入到化合物A异甘露醇(100克，684毫摩尔，1.0当量)的吡啶(600毫升)溶液中，将混合物在25℃下搅拌16小时。LC-MS显示化合物A被完全消耗，并且检测到一个具有所需质量的主峰。将所得反应混合物用水(500毫升)稀释，用二氯甲烷(500毫升*2)萃取，合并的有机相，用盐水(500毫升)洗涤，用Na2SO4干燥并在真空中浓缩以获得残留物。残余物用柱层析(DCM/MeOH＝100/1至50/1，0.1％Et3N)纯化，得到化合物B(150克，收率48.9％)的黄色固体。
1H NMR:EC4783-404-P1B1_C(400MHz,DMSO-d6)
δppm7.46(br d,J＝7.63Hz,2H)7.28-7.37(m,6H)7.19-7.25(m,1H)6.90(br d,J＝7.88Hz,4H)4.70(d,J＝6.50Hz,1H)3.99-4.09(m,6H)3.88-3.96(m,2H)3.83(br dd,J＝7.82,6.94Hz,1H)3.74(s,6H)3.41(br t,J＝8.13Hz,1H)3.05(t,J＝8.44Hz,1H)2.85(br t,J＝7.50Hz,1H).
在N2大气压下，在向化合物B(80.0克，178毫摩尔，1.0当量)的二氯甲烷(5.0毫升)溶液中，在25℃下滴加2H-四氮唑(0.45M，436毫升，1.1当量)，然后滴加化合物C(2-氰基乙基二异丙基氯代亚磷酰胺，80.6克，267毫摩尔，85.0毫升，1.5当量)中的二氯甲烷(200毫升)的溶液；将反应混合物在25℃下搅拌1.0小时；LC-MS显示化合物B被完全消耗，并且检测到一个具有所需质量的主峰。将所得反应混合物冷却至-20℃并倒入冰冷的坐下；NaHCO3(500毫升)，用二氯甲烷(500毫升*3)提取，合并的有机层用NaHCO3/盐水＝1：1(300毫升/300毫升)洗涤，用Na2SO4干燥，并在真空(35℃)中浓缩以获得残留物(100毫升)。残余物用柱层析法纯化(Al2O3，DCM/MeOH＝100/1至50/1，0.1％Et3N)，得到化合物2(77g，119mmol，收率66.5％)为白色固体。
1H NMR:EC4783-423-P1B1_C(400MHz,DMSO-d6)
δppm7.22(br d,J＝7.50Hz,2H)7.05-7.14(m,6H)6.96-7.02(m,1H)6.67(br dd,J＝8.82,1.81Hz,4H)3.95-4.07(m,2H)3.73-3.83(m,1H)3.62-3.72(m,2H)3.48-3.53(m,6H)3.27-3.37(m,3H)3.11(s,6H)2.82(td,J＝8.54,2.31Hz,1H)2.47-2.63(m,3H)2.28(br d,J＝1.63Hz,3H)0.82-1.00(m,13H).
亚磷酰胺化合物1-的合成
向化合物B(500毫克，1.11毫摩尔，1.0当量)在二氯甲烷(5.0毫升)的溶液中加入化合物D(甲基二异丙基氯代亚磷酰胺，607毫克，3.34毫摩尔，3.0当量)和三乙胺(432毫克，3.34毫摩尔，582微升，3.0等式)，在0-5℃下，混合物在25℃下搅拌1.0小时。将所得反应混合物冷却至-20℃并倒入冷NaHCO3(5.0毫升)溶液中(0-5℃)。用二氯甲烷(5.0毫升*2)萃取，将合并的有机层用冷的NaHCO3/盐水＝1：1(5.0mL/5.0mL)洗涤(0-5℃)，用Na2SO4干燥并在真空中浓缩以获得残留物。柱层析纯化残余物(碱性Al2O3，石油醚/乙酸乙酯＝10/1～5/1，0.1％Et3N)，得到化合物1(280mg，471μmol，产率42.3％)，为白色固体。
1H NMR:EC10615-49-P1N(400MHz,DMSO-d6)δppm7.44(br d,J＝7.63Hz,2H),7.31(br t,J＝7.94Hz,6H),7.18-7.26(m,1H),6.89(br d,J＝8.00Hz,4H),4.08-4.13(m,1H),3.95-4.03(m,1H),3.84-3.93(m,1H),3.77-3.83(m,1H),3.74(s,6H),3.43-3.53(m,3H),3.38(br d,J＝6.75Hz,1H),2.94-3.04(m,1H),2.70-2.85(m,1H),1.09-1.15(m,12H),1.07(br s,3H)。
亚磷酰胺化合物3-的合成
在室温下，在氮气下向100mL烧瓶中加入5mL DMF和化合物A异甘露醇(0.5g，1.0eq)。在-10℃下分批加入NaH(0.18g，1.3eq)，然后在-10℃～0℃下搅拌混合物30分钟。将1-碘代十六烷(1.57g，1.3当量)在DMF(3mL)中的溶液在约0℃下滴入上述反应混合物10分钟。然后将反应混合物加热至50℃并搅拌过夜。通过TLC(石油醚：EtOAc＝1∶1，Rf＝0.5)检测该混合物，表明尽管化合物A异甘露醇未完全消耗，但形成了新的斑点，重复反应。将两浴反应混合物合并并倒入饱和NH4Cl(100mL)溶液中，并用EtOAc(50mL*3)萃取，将合并的有机层用饱和NaCl溶液(50mL*2)洗涤，用Na2SO4干燥并减压浓缩。通过硅胶柱纯化Lipd-02-1A的粗品，并用(石油醚：EtOAc＝100:0至5:1)洗脱，得到白色固体Lipd-02-1A(0.72g，产率28％)。MS(M+H)＝371.3。
在室温下，在氮气下向100mL烧瓶中加入5.6mL CH2Cl2和Lipd-02-1A(0.7g，1.0eq)。室温下滴加四唑(0.15g，1.1eq，0.45M在CH3CN中)，然后将3-((双(二异丙基氨基)膦酰氧基)丙腈(0.88g，1.5eq)在CH2Cl2(1mL)中的溶液滴加到上述反应混合物中，并在室温下搅拌3小时。通过TLC(石油醚：EtOAc＝2:1，Rf＝0.8)检测混合物，这表明产生了新的斑点，Lipd-02-1A的一部分未消耗。将所得混合物冷却至0℃并倒入饱和NaHCO3溶液(50mL)中，并用CH2Cl2(50mL*3)萃取，合并的有机层用饱和.NaCl溶液(50mL)洗涤，用Na2SO4干燥并减压浓缩。通过硅胶柱纯化化合物3的粗品，并用(石油醚：EtOAc＝100:0至20:1；1％Et3N)洗脱，得到无色油状的化合物3(0.33g，产率30％)。
1H NMR(400MHz,CDCl3)：δppm4.47-4.54(m,2H),4.29-4.38(m,1H),3.58-4.05(m,10H),3.41-3.46(m,1H),2.62-2.69(m,2H),1.57-1.64(m,2H),1.25(s,26H),1.15-1.22(m,12H),0.87(t,J＝6.8,3H).
31P NMR(400MHz,CDCl3)：δppm148.94,149.03.
MS(M+H)＝571.5。
同样的方法可以获得化合物21和22，主要将1-碘代十六烷替换为1-碘代十四烷或者1-碘代十八烷。
化合物21：1H NMR(400MHz,CDCl3)：δppm4.49-4.55(m,2H),4.32-4.36(m,1H),3.62-4.04(m,10H),3.40-3.45(m,1H),2.64-2.70(m,2H),1.54-1.61(m,2H),1.23(s,22H),1.15-1.22(m,12H),0.88(t,J＝6.8,3H).
31P NMR(400MHz,CDCl3)：δppm148.94,149.03.
MS(M+H)＝543.5。
化合物22：1H NMR(400MHz,CDCl3)：δ4.49-4.55(m,2H),4.33-4.35(m,1H),3.62-4.04(m,10H),3.42-3.45(m,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).
31P NMR(400MHz,CDCl3)δppm148.94,149.03.
MS(M+H)＝599.5。
其他核苷亚磷酰胺的制备根据本文以及本领域所述的程序进行，诸如但不限于已公布的US,426,220和WO 02/36743。
实施例2
寡聚化合物的合成
根据本发明使用的寡聚化合物可通过熟知的固相合成技术便利且常规地制得。用于此合成的设备由多家供应商销售包括例如Mermade 12(LGC)。此外或作为另外一种选择可采用本领域已知的用于这种合成的任何其它装置。熟知的是使用类似的技术制备寡核苷酸诸如烷基化衍生物以及具有硫代磷酸酯键合的那些。
寡聚化合物:未取代的和取代的磷酸二酯(O)寡聚化合物(包括但不限于寡核苷酸)可使用标准亚磷酰胺化学通过碘氧化而在自动化DNA合成仪Mermade 12(LGC)上合成。
在某些实施方案中硫代磷酸酯核苷间键合(S)以类似于磷酸二酯核苷间键合的方式合成，不同的是:通过使用10％w/v的3,H-1,2-苯并二硫醇-3-酮-1,1-二氧化物的乙腈溶液氧化亚磷酸酯键合而实现硫杂化。将硫杂化反应步骤的时间增至180秒并通过正常加帽步骤继续。在55℃下的浓氨水中处理(12-16h)从CPG柱裂解并解封后通过用超过3倍体积的乙醇从1M的NH4OAc溶液中进行沉淀来回收寡聚化合物。
次磷酸酯(phosphinate)核苷间键合可如US5,508,270中所述而制备。烷基磷酸酯核苷间键合可如US4,469,863中所述而制备，也可以通过本文所述的亚磷酰胺法。3’-脱氧-3’-亚甲基膦酸酯核苷间键合可如US5,610,289或5,625,050中所述而制备。亚磷酰胺核苷间键合可如US5,256,775或美5,366,878中所述而制备。烷基硫代磷酸酯核苷间键合可如已公布的WO94/17093和WO94/02499公布中所述而制备。3′-脱氧-3′-氨基磷酰胺酯核苷间键合可如US5,476,925中所述而制备。磷酸三酯核苷间键合如US5,023,243中所述而制备，一些磷酸核苷间键的制备方法可见Beilstein J Org Chem.2017；13:1368–1387。
具有一个或多个不含磷的核苷间键合包括但不限于亚甲基甲基亚氨基连接的寡核苷、亚甲基二甲基肼连接的寡核苷、亚甲基羰基氨基连接的寡核苷以及亚甲基氨基羰基连接的寡核苷的寡聚化合物以及具有例如交替O或S键合的混合主链寡聚化合物可如US5,378,825、US5,386,023、US5,489,677、US5,602,240和US5,610,289中所述而制备。
双链核糖核酸(dsRNA)试剂通过以1∶1摩尔比混合两条互补链(正义链和反义链)中的每一条来形成双链体以获得。
表2中的双链体通常使用基于亚磷酰胺化学的成熟固相合成方法，在寡核苷酸合成仪上合成siRNA的正义和反义链序列。寡核苷酸链的增长是通过4步循环实现的：去保护、缩合、加帽和用于添加每个核苷酸的氧化或硫化步骤。合成是在由可控多孔玻璃(CPG,)制成的固体支持物上进行的。一般的单体亚磷酰胺购自商业来源，而本文中单体化合物可以替代的作为一种单体亚磷酰胺，而引入寡核苷酸链中。在本文中单体化合物可以替代的作为单体亚磷酰胺连接在的3'-末端的情况下，使用时使其连接在的CPG固体支持物。在连接在的5'-末端的情况下，将亚磷酰胺用于最后的偶联反应，必要时可进一步偶联靶向基团。将3％二氯甲烷中的三氯乙酸(TCA)用于4,4'-二甲氧基三苯甲基保护基(DMT)的脱保护。5-乙硫基-1H-四唑用作活化剂。THF/Py/H2O中的I2和吡啶/MeCN中的苯乙酰二硫化物(PADS)分别用于氧化和硫化反应。在最后的固相合成步骤之后，通过用1:1体积的20wt％甲胺水溶液和28％氢氧化铵溶液处理来切割固体载体结合的低聚物并去除保护基团。为了合成用于体外筛选的寡聚化合物，将粗混合物浓缩。将剩余的固体溶解在1.0M NaOAc中，加入冰冷的EtOH以沉淀出作为钠盐的单链产物，其无需进一步纯化即可用于退火。为了合成用于体内测试的siRNA，粗单链产物通过离子对反相HPLC(IP-RP-HPLC)进一步纯化。通过将来自IP-RP-HPLC的纯化单链寡核苷酸产物溶解在1.0M NaOAc中并通过添加冰冷的EtOH进行沉淀，将其转化为钠盐。在水中通过等摩尔互补进行正义链和反义链寡核苷酸的退火，以形成双链siRNA产物。
实施例3
靶向基团的合成
向丙二胺衍生物中间体化合物4(275g，660mmol，1.00eq.)的二氯甲烷(2.75L)溶液中加入三乙胺(133g，1.32mol，2.00eq.)，随后滴加入Cbz-Cl(169g，990mmol，1.50eq.)。反应液在25℃搅拌2小时，LCMS显示丙二胺衍生物完全转化。反应液依次用NaHCO3(800mL)饱和溶液、饱和食盐水(500mL)洗涤，有机相用无水Na2SO4干燥。过滤除去干燥剂后，滤液浓缩至干。该粗品经柱层析后(SiO2，石油醚(PE)/乙酸乙酯(EA)＝100/1至5/1，v/v)得到无色油状化合物5(290g，527mmol，产率75.7％)。1H NMR(400MHz in DMSO-d6):δppm7.23-7.40(m,5H),5.00-5.12(m,2H),3.86-3.95(m,2H),3.23-3.39(m,6H),2.55-2.67(m,2H),1.56-1.64(m,2H),1.31-1.46(m,27H).MS(ESI)[M+H]+m/z:551.6。
向化合物5(145g，263mmol，1.00eq)中加入HCOOH(2.9L)，该溶液在60℃下搅拌12小时，LCMS显示化合物5转化完全。向反应液中加入1.5L甲苯和1.5L乙腈，减压浓缩至约500mL，随后加入甲苯/乙腈(1:1，v/v，约750mL)并浓缩至约500mL,然后加入乙腈(约1000mL)并浓缩至干，粗品用700mL乙腈在60℃粉碎2小时，过滤。收集固体，干燥得白色固体化合物6(105g，定量)。1H NMR(400MHz in DMSO-d6):δppm 7.26-7.40(m,5H),5.02-5.10(m,2H),3.89-4.00(m,2H),3.36-3.45(m,4H),3.24-3.34(m,2H),2.59-2.72(m,2H),1.40(s,2H).MS(ESI)[M+H]+m/z:383.0。
向化合物6(100g，261mmol)和中间体-A(502g，915.mmol，3.50eq.)的DMF(1.0L)溶液中加入O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸(TBTU)(327g，1.02mol，3.90eq.)，三乙胺(212g，2.09mol，8.00eq.)，反应在25℃进行1小时，LCMS显示化合物6转化完成。将反应液加入到4000mL水中，并用甲基叔丁基醚(2000mL分两次)萃取除去杂质，剩余水相用二氯甲烷(3000mL分两次)萃取。二氯甲烷相依次用10％柠檬酸水溶液(2000mL分两次)、饱和NaHCO3(2.0L分两次)，饱和盐水(2.0L)洗涤,无水Na2SO4干燥。过滤得滤液，减压浓缩得到白色固体化合物8(260g，159mmol，产率60.9％)。1H NMR(400MHz in DMSO-d6):δppm 7.99-8.08(m,2H),7.93(br d,J＝5.50Hz,1H),7.79-7.86(m,3H),7.26-7.39(m,5H),5.22(d,J＝3.13Hz,3H),4.95-5.08(m,5H),4.54(br d,J＝8.38Hz,3H),4.03(s,9H),3.81-3.93(m,5H),3.76(br d,J＝4.88Hz,3H),3.44-3.62(m,10H),3.34-3.43(m,6H),3.24(br d,J＝6.13Hz,7H),3.02-3.09(m,4H),2.40-2.47(m,2H),2.10(s,9H),1.99(s,9H),1.89(s,9H),1.77(s,9H),1.57-1.68(m,2H)。MS(ESI)[M+H]+m/z:816.4。
用氩气惰性化2L氢化釜并小心加入干Pd/C(9g)，加入MeOH(50mL)润湿Pd/C，然后在氩气气氛下缓慢加入化合物8(90g，55.1mmol，1.00eq.)和三氟乙酸(6.29g，55.1mmol，1.00eq.)的MeOH(850mL)溶液。混合物经脱气/加H2三次置换为氢气气氛，在25℃搅拌10小时。LCMS显示化合物8转化完全，过滤除去Pd/C，滤液经减压浓缩得到化合物9(80g，产率90.2％)。1H NMR(400MHz in DMSO-d6):δppm9.12(br s,2H),8.50(br t,J＝5.19Hz,1H),8.10(br t,J＝5.50Hz,2H),7.85-7.91(m,3H),5.22(d,J＝3.25Hz,3H),4.95-5.01(m,3H),4.52-4.58(m,3H),4.03(s,9H),3.84-3.93(m,3H),3.75-3.83(m,3H),3.39-3.61(m,16H),3.23-3.32(m,6H),3.15-3.18(m,3H),2.97-3.05(m,2H),2.54-2.61(m,2H),2.10(s,9H),2.00(s,9H),1.89(s,9H),1.77-1.80(m,9H),1.70-1.76(m,2H).MS(ESI)[M+H]+m/z:749.3。
向化合物9(270g，168mmol，1.00eq.)和戊二酸酐(28.6g，252mmol，1.50eq)的二氯甲烷(2.7L)溶液中加入三乙胺(67.8g，672mmol，4.00eq)，该溶液在25℃搅拌1小时，LCMS显示化合物9完全转化为化合物11。向反应液中加入4-羟基哌啶(42.4g，420mmol，2.50eq.)和TBTU(107g，335mmol，2.00eq.)，并在25℃继续搅拌1小时。LCMS显示化合物11转化完全。缓慢加入饱和NH4Cl(3.0L)淬灭反应，分层，水相用二氯甲烷(2×1000mL)萃取并与先前的有机相合并。合并的有机相用饱和NaHCO3(aq)和饱和盐水的1:1(v/v)混合液(3.0L)洗涤，用无水Na2SO4干燥，过滤减压浓缩。粗品溶于1.5L二氯甲烷，滴加到甲基叔丁基醚(7.5L)中，半透明的白色沉淀在滴加过程中逐渐形成。真空过滤沉淀，收集固体真空干燥得到白色固体化合物13(207g，产率72.8％)。1H NMR(400MHz in DMSO-d6):δppm8.05(br d,J＝2.00Hz,2H),7.82(br d,J＝7.38Hz,3H),5.21(br s,3H),4.98(br d,J＝10.26Hz,3H),4.72(br s,1H),4.54(br d,J＝7.88Hz,3H),4.03(br s,9H),3.74-3.94(m,9H),3.45-3.71(m,12H),3.40(br s,6H),3.24(br s,7H),3.07(br d,J＝14.13Hz,5H),2.91-3.01(m,1H),2.24-2.44(m,5H),2.20(br s,1H),2.10(s,9H),1.96-2.04(m,9H),1.89(br s,9H),1.74-1.81(m,9H),1.51-1.73(m,6H),1.07-1.36(m,3H).MS(ESI)[M+H]+m/z:848.0。
向化合物13(200g，118mmol，1.00eq.)、四唑二异丙基铵(8.08g，47.2mmol，0.40eq.)的二氯甲烷(2.0L)溶液中加入3-双(二异丙基氨基)膦酰氧基丙腈(53.3g，177mmol，1.50eq.)，反应液在40℃搅拌2小时，LCMS显示化合物13转化完成。反应液用饱和NaHCO3和饱和食盐水的1:1混合液(2.0L)洗涤，经无水Na2SO4干燥，滤液浓缩后所得粗品溶于二氯甲烷(1.2L)，滴加到搅拌的甲基叔丁基醚(6.0L)中，过滤悬浊液，滤饼用基叔丁基醚淋洗，收集固体进行真空干燥，将产品溶于二氯甲烷(1.0L)并浓缩至干，重复操作4次以除去残留叔丁基醚，得到GLPA15(164g，产率73.3％)。1H NMR(400MHz in DMSO-d6):δppm8.05(br d,J＝6.50Hz,2H),7.81(br d,J＝9.01Hz,3H),5.22(d,J＝3.25Hz,3H),4.98(dd,J＝11.26,3.25Hz,3H),4.55(br d,J＝8.50Hz,3H),4.03(s,9H),3.64-3.97(m,12H),3.55-3.63(m,6H),3.50(br s,5H),3.40(br d,J＝6.13Hz,6H),3.17-3.30(m,9H),3.07(br d,J＝14.26Hz,4H),2.76(t,J＝5.82Hz,2H),2.18-2.47(m,6H),2.10(s,9H),1.99(s,9H),1.89(s,9H),1.78(s,9H),1.52-1.74(m,6H),1.12-1.19(m,12H).31P NMR(DMSO-d6):ppmδ145.25.MS(ESI)[M+H]+m/z:1895.7。
其他GalNAc亚磷酰胺化合物(GLPAn)也同样可以在使用合理对应的中间体后，采用本文类似或者本领域中熟知的方法获得，并作为靶向基团连接到本文化合物或者寡聚以及双链体合适的位置。
在某些研究中，提供了用于将包含GalNAc(本文也称为GalNAc递送化合物)的靶向基团连接到正义链的5'-末端的方法，其包括在固相合成的最后一个偶联步骤中使用GalNAc亚磷酰胺(GLPAn)，使用这样的合成工艺，例如在寡核苷酸链延长时使用的(即在正义链的5'末端添加核苷酸)工艺，以将其连接到正义链的5'-末端。在一些研究中，将包含GalNAc的靶向基团连接到正义链的3'-末端的方法包括使用包含GLPAn的固体支持物(CPG)。在一些研究中，将包含靶向基团连接到正义链的3'-末端的方法包括：将靶向基团通过酯键连接到CPG固体支持物上，并在合成正义链时使用带有连接的靶向基团的所得CPG，这导致GalNAc靶向基团连接在正义链的3'-末端。同样的，靶向基团也可以通过亚磷酰胺法等方式与含有本发明的寡聚化合物单体处相连。
实施例4.包含本发明单体的固体载体的制备
表示大孔胺甲基聚乙烯树脂载体部分
将50L玻璃釜在氮气保护下，向玻璃釜中加入二氯甲烷(19.50kg)，开动搅拌。控温20～30℃，向玻璃釜中加入DMTr-imann(1.47kg)，向反应釜中加入三乙胺(1.50kg)、4-二甲氨基吡啶(0.164kg)，以及加入丁二酸酐(1.34kg)。体系保温20～30℃反应18h后取样，结束反应。向反应完体系中加入饱和碳酸氢钠溶液(22.50kg)，搅拌10～20min后静置至分层，下层有机相转移至，上层水相用二氯甲烷萃取2次，合并有机相，采用无水硫酸钠干燥，过滤滤液，再转移旋蒸浓缩至无馏分，形成成固体灰色至类白色固体1.83kg。
在向100L玻璃釜中加入N,N-二甲基甲酰胺(23.50kg)，开动搅拌。控温20～30℃，氮气保护下，通过固体加料漏斗向上述100L玻璃釜中加入上步产物、O-苯并三氮唑-四甲基脲六氟磷酸盐(0.33kg)、加入N,N-二异丙基乙胺(0.13kg)，加料完毕搅拌10～30分钟后放出至50L镀锌桶待用。通过固体加料漏斗向上述100L固相合成釜中加入大孔胺甲基树脂(3.25kg)(购于天津南开和成科技有限公司，批号HA2X1209,负载量0.48mmol/g)，控温20～30℃，向固相合成釜中加入N,N-二甲基甲酰胺(21.00kg+21.00kg)，和上步镀锌桶待用反应液。体系于保温反应，，跟踪至固体载量≥250umol/g，载量检测方法为UV。体系用氮气压滤，滤饼用N,N-二甲基甲酰胺淋洗三次(26.00kg+26.10kg+26.00kg)，滤饼留在釜中。向80L玻璃釜中加入CAP.A(4.40kg+4.42kg+4.30kg)和CAP.B(4.40kg+4.40kg+4.47kg)，搅拌3～8min后待用。此操作重复进行3次进行盖帽，向固相合成釜中加入乙腈(18.00kg+18.00kg+18.00kg+17.50kg+17.50kg)，氮气鼓泡10～30min后压滤。重复此操作四次，滤饼在固相合成釜中使用氮气吹扫2～4h后转移至50L压滤罐，控温15～30℃，继续干燥，将烘干后黄色至白色固体产品，重量:3.516kg。
实施例5.包含本发明单体的寡聚化合物体内评价
包含本发明单体的的寡聚化合物用本文所述或者本领域技术人员所熟知的方法获得，寡聚化合物序列结构以FXII为靶的siRNA双链体如表2所示。
如表2表示了双链体包含正义链和反义链；化学修饰表示为：大写：2'-氟修饰核苷酸；小写：2'-甲氧基修饰核苷酸；“*”应理解，当存在于寡核苷酸中时，这些单体通过硫代磷酸二酯核苷链接键相互连接，两个单体之间的不存“*”表示通过氧代磷酸二酯核苷链接键相互连接；Invab：反向无碱基，GLS15*和GLO15分别是本文所述的靶向基团。
“imann”在寡聚合物或者dsRNA末端时表示为：“imann”在寡聚合物或者dsRNA进步连接靶向基团连接时表示为
实施例6.FXII双链核苷核酸(又称dsRNA)的体内测试
为了评估FXII双链核苷核酸(又称dsRNA)的体内活性，使用了雌性C57BL/6小鼠，6周龄，无特定病原体，购于上海斯莱克实验动物有限责任公司。
a)化合物
溶媒.PBS，受试化合物.AD00823、AD00824、AD00825、AD00826、AD00835、AD00836、AD00841、AD00827、AD00449。
b)实验方案
动物实验每组4只C57BL/6,雌性小鼠，给药剂量1(mg/kg)，给药频率SC,第0天给药1次，第7、14和21天进行采血，检测血浆：FXII蛋白水平
c)实验天数定义：以小鼠首次给药当天定义为实验第0天，往前一天为第-1天，往后一天为第1天，以此类推。
d)所有待测化合物在给药前用PBS配制成5mg/mL储液。取少量5mg/mL储液稀释20倍，使用Nanodrop上机检测OD值。根据OD值换算得到储液实际浓度后，取适量稀释至工作液浓度(0.2mg/mL)进行给药。
e)所有小鼠(C57BL/6,雌性，6周)经过6天适应期后，在第0天按上述进行皮下注射给药。
f)第7、14、21天，所有小鼠经颌下静脉采血收集血浆，用于FXII蛋白水平检测。样品检测分析
应用ELISA试剂盒检测小鼠血浆中FXII蛋白水平。
使用ELISA试剂盒(Molecular Innovations，IMSFXIIKTT)检测小鼠血浆中FXII蛋白水平。简言之，将EDTA-K2收集的血浆样本稀释30,000倍后加入到捕获抗体包被的检测板中孵育，然后依次加入检测抗体和HRP偶联的二抗，最后用TMB显色，读取450nm处吸光值。使用四参数法拟合标准曲线，并将检测样本OD值代入计算出每个样品的FXII蛋白含量，乘以稀释倍数即为原始血浆中FXII蛋白浓度。数据结果如表3所示。
表3小鼠血浆中FXII蛋白表达的剩余百分比(保留)NA表示未检测。
从双链核苷核酸(又称dsRNA)试剂AD00824和AD00826具有在正义链的5'端、3'端或者两端同时包含本文imann单体化合物的寡聚化合物，相对于不含或者含其他核苷酸双链核苷核酸(又称dsRNA)试剂如AD00823和AD00825，均表现出更好的沉默效果，说明在双链核苷核酸(又称dsRNA)正义链中在5'端和/或者3'含有本文所述的单体时具有更好的抗核酸酶切效果，使得正义链具有稳定性效果，提高反义物体内作用效果；同样在AD00827VSAD00841可以观察同样的结果。
而在双链核苷核酸(又称dsRNA)试剂，反义链中包含本文imann单体化合物的AD00835与AD00836(包含invab)相比，从相同作用时间内的活性数据看，这实际上证明了添加在5'-反义链末端，反义物被本文imann单体化合物阻断抗核酸酶切，并杀死活性，这表明本文化合物在寡聚化合物具有比invab更高的抗核酸酶切作用，具有更高的稳定性。
实施例7 FXII双链核苷核酸(又称dsRNA)的体内测试
包含本发明单体的的寡聚化合物用本文所述或者本领域技术人员所熟知的方法获得，寡聚化合物序列结构以FXII为靶的siRNA双链体如表4所示。
如表4表示了表示了双链体包含正义链和反义链；化学修饰表示为：大写：2'-氟修饰核苷酸；小写：2'-甲氧基修饰核苷酸；“*”应理解，当存在于寡核苷酸中时，这些单体通过硫代磷酸二酯核苷链接键相互连接，两个单体之间的不存“*”表示通过氧代磷酸二酯核苷链接键相互连接；Invab：反向无碱基。
“imann”在寡聚合物或者dsRNA末端时表示为：“imann”在寡聚合物或者dsRNA进步连接靶向基团连接时表示为“Stab8”在寡聚合物或者dsRNA末端时表示为：“Stab8”在寡聚合物或者dsRNA进步连接靶向基团连接时表示为“Stab9”在寡聚合物或者dsRNA末端时表示为：“Stab8”在寡聚合物或者dsRNA进步连接靶向基团连接时表示为
为了评估FXII双链核苷核酸(又称dsRNA)的体内活性，使用了雌性C57BL/6小鼠，6周龄。
a)化合物
溶媒.PBS，受试化合物.AD00734、AD00737、AD01970、AD01971。
b)实验方案
动物实验每组4只C57BL/6,雌性小鼠，给药剂量0.5or1mg/kg，给药频率SC,第0天给药1次，第7、14、21和28天进行采血，检测血浆：FXII蛋白水平。
c)实验天数定义：以小鼠首次给药当天定义为实验第0天，往前一天为第-1天，往后一天为第1天，以此类推。
d)所有待测化合物在给药前用PBS配制成5mg/mL储液。取少量5mg/mL储液稀释20倍，使用Nanodrop上机检测OD值。根据OD值换算得到储液实际浓度后，取适量稀释至工作液浓度(0.2mg/mL)进行给药。
e)所有小鼠(C57BL/6,雌性，6周)经过6天适应期后，在第0天按上述进行皮下注射给药。
f)第7、14、21、28天，所有小鼠经颌下静脉采血收集血浆，用于FXII蛋白水平检测。
样品检测分析
使用ELISA试剂盒(Molecular Innovations，IMSFXIIKTT)检测小鼠血浆中FXII蛋白水平。简言之，将EDTA-K2收集的血浆样本稀释30,000倍后加入到捕获抗体包被的检测板中孵育，然后依次加入检测抗体和HRP偶联的二抗，最后用TMB显色，读取450nm处吸光值。使用四参数法拟合标准曲线，并将检测样本OD值代入计算出每个样品的FXII蛋白含量，乘以稀释倍数即为原始血浆中FXII蛋白浓度。数据结果如表5所示。
表5.使用对应于表4中所示的序列、化学修饰和递送的化合物，FXII蛋白的相对表达水平剩余百分比.
实施例8.包含本发明单体的寡聚化合物体内评价
包含本发明单体的的寡聚化合物用本文所述或者本领域技术人员所熟知的方法获得，寡聚化合物序列结构以SOD1为靶的siRNA双链体如表6所示。
表6表示了双链体包含正义链和反义链；化学修饰表示为：大写：2'-氟修饰核苷酸；小写：2'-甲氧基修饰核苷酸；“*”应理解，当存在于寡核苷酸中时，这些单体通过硫代磷酸二酯核苷链接键相互连接，两个单体之间的不存“*”表示通过氧代磷酸二酯核苷链接键相互连接；Invab：反向无碱基。
“imann”在寡聚合物或者dsRNA末端时表示为：“imann”在寡聚合物或者dsRNA进步连接靶向基团连接时表示为Lipd01表示为Lipd02表示为Lipd03表示为C16表示为C16H33,(C16)*(imann)表示为C16与imann之间通过硫代磷酸二酯核苷链接键相互连接VPu表示为
为了评估SOD1双链核苷核酸(又称dsRNA)的体内活性，小鼠侧脑室(ICV)给药
将雄性C57小鼠(～20g)随机分组：aCSF组(溶剂对照组，n＝5)，AD00509-6组(n＝4)，AD00509-7组(n＝3)，AD00509-8组(n＝4)，AD00509-9组(n＝3)，AD00509-10组(n＝5)，AD00509-12组(n＝3)。对于每一个待测化合物，用人工脑脊液aCSF将其溶解并稀释成50mg/ml的注射液，然后按照2μL/只的剂量分别给药。具体步骤如下：应用舒泰+盐酸塞拉嗪进行腹腔注射麻醉小鼠，待麻醉生效后，将小鼠头颅固定于脑立体定位仪，使用10μL微量注射器(Hamilton)将药物注射到小鼠侧脑室，注射时间为10分钟，注射完留针5分钟，之后缓慢拔针，并缝合好头部皮肤，待小鼠清醒后放回笼盒继续饲养。
在注射后的第8天对每只小鼠腹腔麻醉后应用生理盐水进行灌注，之后快速提取小鼠的前额叶皮层(PFC)，纹状体，海马和小脑组织并保存于-80℃冰箱。随后，使用RNeasy Kit(QIAGEN)根据说明书提取总RNA并逆转录cDNA，之后应用FastStart Universal SYBR Green Master(ROX)(Roche)进行qPCR检测靶基因SOD1的表达，采用2-ΔΔCt法进行定量分析，结果如表7所示。
表7：各组织SOD1的表达的剩余百分比(保留)
剂量剂量反应实验方案
将雄性C57小鼠(～20g)随机分组：aCSF组(溶剂对照组，n＝5)，AD00509-8组(0.1mg,0.033mg,0.01mg)，AD00509-9组(0.1mg,0.033mg,0.01mg)，AD00509-12组(0.1mg,0.033mg,0.01mg)。小鼠侧脑室注射方法如上所述。在注射后的第8天对每只小鼠腹腔麻醉后应用生理盐水进行灌注，之后快速提取小鼠的前额叶皮层(PFC)，纹状体，海马和小脑组织并保存于-80℃冰箱。随后，使用RNeasy Kit(QIAGEN)根据说明书提取总RNA并逆转录cDNA，之后应用FastStart Universal SYBR Green Master(ROX)(Roche)进行qPCR检测靶基因SOD1的表达，采用2-ΔΔCt法进行定量分析；结果如表8所示。
表8：各组织SOD1的表达的剩余百分比(保留)
等同物
尽管本文已经描述和说明了本发明的几个实施例，但本领域普通技术人员很容易理解，用于执行此处描述的功能和/或获得结果和/或一个或更多个优点的多种其他手段和/或结构，以及这些变化和/或修改中的每一个都被认为在本发明的范围内。更一般地，本领域技术人员将容易理解，此处描述的所有参数、尺寸、材料和配置都是示例性的，并且实际参数、尺寸、材料和/或配置将取决于使用本发明教导的具体应用。本领域技术人员将认识到或能够仅使用常规实验来确定本文描述的本发明的特定实施例的许多等价物。因此，应当理解，前述实施例仅通过示例的方式呈现并且属于所附权利要求及其等效物的范围内，本发明可以以不同于具体描述和要求保护的方式实施。本发明针对在此描述的每个单独的特征、系统、物品、材料和/或方法。此外，两个或更多个此类特征、系统、物品、材料和/或方法的任何组合，如果此类特征、系统、物品、材料和/或方法相互不矛盾，则也包括在本发明的范围内。
如本文所定义和使用的所有定义应理解为对照字典定义、通过引用并入的文件中的定义和/或所定义术语的普通含义。
在说明书和权利要求中并未使用数量限定的情况，除非明确指出相反，否则应理解为“至少一个”。
说明书和权利要求书中使用的短语“和/或”应理解为表示如此结合的要素中的“一个或两个”，即这样的要素在某些情况下组合出现而在其他情况下分离出现。除了由“和/或”具体标识的要素之外，除非明确指出相反，否则可以可选地存在其他要素，无论与那些具体标识的要素相关或不相关。
本申请中引用或参考的所有参考文献、专利和专利申请和出版物均通过引用整体并入本文。

Claims (65)

  1. 一种化合物，所述的化合物具有式(I)所示或其立体异构体：
    A1和A2分别独立的选自：O、S、SO、SO2、NR2和CR3R4，R2、R3和R4分别独立的选自：氢、卤素、磺酰基、亚磺酰基、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基；
    T1和T2之一为保护基、Z、L以及-Z-L，所述的Z表示为靶向基团或亲脂性基团，所述亲脂性基团任选地经由连接子与A1或者A2之一相连的，L表示为：载体间隔物或通过间隔物链接的载体部分；而T1和T2的另一个为活性磷基团、保护基；并且T1和T2不会同时为保护基；
    R1分别独立地选自卤素、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基；
    n为0-6的整数；
    其中式(I)不包括如下结构：
  2. 如权利要求1所述的化合物，A1为O。
  3. 如权利要求1-2任一项所述的化合物，A2为O。
  4. 如权利要求1所述的化合物，A1和A2分别独立地为S；
    A1和A2分别独立为NR2，其中R2为氢或者CH3；
    A1为O，A2为S；A1为O，A2为NR2；
    或A1为S，A2为NR2。
  5. 如权利要求1所述的化合物，所述的T1和T2之一为载体间隔物或通过间隔物链接的载体部分；所述的载体间隔物优选为二羧酸衍生的基团，R为不存在、醚、聚乙二醇、氧、硫、氮、亚烷基、烯基、炔基、芳基、芳烷基、杂烷基或杂芳基；所述的载体间隔物优选为C3-C6烷基二羧酸根基团；更优先为琥珀酸基团；所述的载体优选为聚甲基丙烯酸乙烯醇共聚物、硅芯片玻璃、纤维素、聚苯乙烯珠、聚丙烯片、无孔硅珠、聚丙烯酰胺或聚丙烯酸酯；更优选为长链烷胺控孔玻璃(LCAA-CPG)。
  6. 如权利要求1所述的化合物，
    T1和T2之一保护基是羟基保护基，所述羟基保护基各自独立地选自乙酰基、叔丁基、叔丁氧基甲基、甲氧基甲基、四氢吡喃基、1-乙氧基乙基、1-(2-氯乙氧基)乙基、2-三甲基甲硅烷乙基、对氯苯基、2,4-二硝基苯基、苄基、苯甲酰基、对苯基苯甲酰基、2,6-二氯苄基、二苯基甲基、对硝基苄基、三甲基甲硅烷基、三乙基甲硅烷基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基、三苯甲硅烷基、三异丙基甲硅烷基、苯甲酰基甲酸酯、氯乙酰基、三氯乙酰基、三氟乙酰基、新戊酰基、9-芴甲氧羰基、甲磺酸基、甲苯磺酸基、三氟甲磺酸基、三苯甲基、单甲氧基三苯甲基、二甲氧三苯甲基、三甲氧基三苯甲基或取代的9-苯基黄嘌呤-9-基；优选的羟基保护基各自独立地选自乙酰基、苄基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基以及4,4’-二甲氧三苯甲基；
    T1和T2之一保护基是巯基保护基，所述巯基保护基各自独立地选自乙酰基、叔丁基、叔丁氧基甲基、甲氧基甲基、四氢吡喃基、1-乙氧基乙基、1-(2-氯乙氧基)乙基、2-三甲基甲硅烷乙基、对氯苯基、2,4-二硝基苯基、苄基、苯甲酰基、对苯基苯甲酰基、2,6-二氯苄基、二苯基甲基、对硝基苄基、三甲基甲硅烷基、三乙基甲硅烷基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基、三苯甲硅烷基、三异丙基甲硅烷基、苯甲酰基甲酸酯、氯乙酰基、三氯乙酰基、三氟乙酰基、新戊酰基、9-芴甲氧羰基、甲磺酸基、甲苯磺酸基、三氟甲磺酸基、三苯甲基、单甲氧基三苯甲基、二甲氧三苯甲基、三甲氧基三苯甲基或取代的9-苯基黄嘌呤-9-基；优选的巯基保护基各自独立地选自苄基、4,4’-二甲氧三苯甲基；
    T1和T2之一保护基是氨基保护基，所述氨基保护基各自独立地选自2-三甲基硅基乙氧基羰基(Teoc)、1-甲基-1-(4-联苯基)乙氧基羰基(Bpoc)、叔丁氧基羰基(BOC)、烯丙氧基羰基(Alloc)、9-芴基甲氧基羰基(Fmoc)、苄氧基羰基(Cbz)、甲酰基、乙酰基、三卤 乙酰基、苯甲酰基、硝基苯基、乙酰基、2-硝基苯磺酰基、邻苯二甲酰亚胺基和二硫代丁二酰基。
  7. 如权利要求1所述的化合物，T1和T2之一为亲脂性基团，其中亲脂性基团的亲脂性通过logKow测定，logKow超过0；优选地亲脂性基团logKow超过1，超过1.5，超过2，超过3，超过4，超过5或超过10。
  8. 如权利要求7所述的化合物，其中亲脂性基团选自含有饱和或不饱和的C4-C30烃链、脂肪环、芳香基、脂肪酸基团或脂肪酸衍生的基团、类固醇衍生的基团和任何脂溶性维生素基团。
  9. 如权利要求7-8任一项所述的化合物，亲脂性基团饱和或不饱和亲脂性基团为饱和或不饱和C10-C18烃链、包含具有4到28个碳原子的烃链的任何饱和或不饱和脂肪酸并且可以含有一个或两个羧基、胆固醇、维生素E(生育酚)或胆汁酸；更优选为：饱和的直链C14、C16或者C18烷基、辛酸、癸酸、十二烷酸、十四烷酸、十六烷酸、十八烷酸、二十烷酸、二十二烷酸和二十四烷酸、肉豆蔻脑酸、棕榈油酸、十六碳烯酸、油酸、反油酸、反型异油酸、亚油酸、反亚油酸、α-亚麻酸、花生四烯酸和芥酸、二十二碳六烯酸酸(DHA)、二十碳五烯酸(EPA)、二十二烷酸(DCA)、甾醇胆固醇(胆汁)、生育酚琥珀酸酯(TS)和石胆酸(LA)、维甲酸(维生素A酸)。
  10. 如权利要求7-9任一项所述的化合物，亲脂性基团可经由连接子与A1或者A2共价连接。
  11. 如权利要求10所述的化合物，所述连接子是选自由以下组成的组：酰胺键、磷脂酰胆碱、烃连接子或聚乙二醇(PEG)连接子、氨基-烷基-醇、羟脯氨酸、羟脯氨醇、氨基-烷基-硫代磷酸酯连接子、氨基-PEG-硫代磷酸酯连接子、α-羧酸盐-氨基-烷基硫代磷酸酯连接子和α-羧酸盐-氨基-PEG-硫代磷酸酯连接子、醚、硫醚、脲、碳酸酯、胺、酰胺、马来酰亚胺-硫醚、二硫化物、磷酸二酯、磺酰胺键、点击反应的产物或氨基甲酸酯。
  12. 如权利要求10所述的化合物，所述连接子是可降解连接子，选自由以下组成的组：氧化还原可裂解连接子、酸可裂解连接子、酯酶可裂解连接子、磷酸酶可裂解连接子或肽酶可裂解连接子；或者是生物可裂解连接子，选自由以下组成的组：DNA、RNA、二硫化物、酰胺、半乳糖胺、葡糖胺、葡萄糖、半乳糖、甘露糖的官能化单糖或寡糖及其组合。
  13. 如权利要求1所述的化合物，T1和T2之一为靶向基团。
  14. 如权利要求13所述的化合物，所述靶向基团可以选自包含以下靶向分子或其衍生物形成的配体中的一种或多种：聚合物、糖类、肝实质细胞表达的受体配体、抗体、量子点、多肽，或小分子配体。
  15. 如权利要求14所述的化合物，每个所述靶向基团独立地为包含与哺乳动物肝细胞表面的去唾液酸糖蛋白受体(ASGPR)亲和的配体，靶向基团优选为独立地选自包含D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D吡喃甘露糖、α-D-吡喃葡萄-糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳-糖胺、N-乙酰半乳糖胺、N-三氟乙酰半乳糖胺、N-丙酰半乳糖胺、N-正丁酰半乳胺、N-异丁-36酰半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6 O-三苯甲基-α-D吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖、L-4-硫代核糖中的一种或者几种的配体；更优选为包含半乳糖或N-乙酰半乳糖胺的配体。
  16. 如权利要求13所述的化合物，所述靶向基团与A1或者A2之一相连选自以下化合物片段之一：

  17. 如权利要求1-16任一项所述的化合物，T1和T2的另一个为活性磷基团，所述的活性磷基团具有如下结构：
    其中M1为H、取代或未取代的C1-C6烷基、OR5、SR5、OH、SH或NR6R7，M2为OH、SH、OR5′或NR6′R7′，每个R5、R6、R7、R5′、R6′或R7′独立地为氢、取代或未取代的C1-C6烷基、磺酰基；m为0或者1。
  18. 如权利要求17所述的化合物，所述的M1选自甲基、乙基、丙基、异丙基、OR5、甲磺酰胺基，R5选自取代或未取代的甲基、乙基、丙基、异丙基。
  19. 如权利要求17-18任一项所述的化合物，R5、R6或R7独立地为取代烷基，所述的取代基选自氰基、卤素、羟基、氨基。
  20. 如权利要求17-19任一项所述的化合物，所述的M2选自N(CH(CH3)2)2。
  21. 如权利要求17所述的化合物，其中M1为O(CH2)2CN，M2为N(CH(CH3)2)2，m为0。
  22. 如权利要求1-16任一项所述的化合物，T1和T2的另一个为活性磷基团，所述的活性磷基团选自二异丙基氰基乙氧基亚磷酰胺、二异丙基甲基亚磷酰胺、二异丙基乙基亚磷酰胺和H-膦酸酯。
  23. 如权利要求1-22任一项所述的化合物，T1和T2的一个是二异丙基氰基乙氧基亚磷酰胺，而另一个是4,4’-二甲氧三苯甲基、靶向基团、亲脂性基团；或者T1和T2的一个是保护基，而另一个是载体间隔物、通过间隔物链接的载体部分、靶向基团或亲脂性基团。
  24. 如权利要求1-23任一项所述的化合物，R1分别独立地选自卤素、取代或未取代的C1-C6烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基。
  25. 如权利要求1-24任一项所述的化合物，n为0。
  26. 如权利要求1-25任一项所述的化合物，所述的化合物具有式(IIa)或者式(IIb)的构型：
    A1、A2、T1、T2、R1以及n如权利要求1-25所定义。
  27. 如权利要求1-26任一项所述的化合物，本文提供的化合物具有式(IIa-1)或者式(IIb-1)的构型：
    A1、A2、T1、T2、R1以及n如如权利要求1-25所定义。
  28. 如权利要求1-27任一项所述的化合物，具有以下具体结构：
    N-为固相载体(LCAA-CPG)， N-为固相载体(LCAA-CPG)，
  29. 一种寡聚化合物，其包含至少一种具有式(III)所示或其立体异构体的5′-末端和/或者3′-末端单体：
    A1和A2分别独立的选自：O、S、SO、SO2、NR2和CR3R4，R2、R3和R4分别独立的选自：氢、卤素、磺酰基、亚磺酰基、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基；
    T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连，并且T3和T4中的另一个为将所述式(III)或其立体异构体单体连接到寡聚化合物的核苷间连接基团；
    R1分别独立地选自卤素、取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C5-C12芳基、取代或未取代的5-12元杂芳基、取代或未取代的5-12元杂环基；
    n为0-6的整数；
    所述的寡聚化合物任选地进一步包含靶向基团。
  30. 如权利要求29所述的寡聚化合物，A1为O。
  31. 如权利要求29-30任一项所述的寡聚化合物，A2为O。
  32. 如权利要求29所述的寡聚化合物，A1和A2分别独立地为S；A1和A2分别独立为NR2；A1为O，A2为S；A1为O，A2为NR2；A1或为S，A2为NR2；其中R2为氢、甲磺酰基或者CH3。
  33. 如权利要求29-32任一项所述的寡聚化合物，T3和T4的另一个为所述的单体连接到寡聚化合物5′-末端和/或3′-末端的核苷间连接基团，所述的核苷间连接基团选自含磷的链接基团或者不含磷的链接基团。
  34. 如权利要求33所述的寡聚化合物，所述的含磷核苷间连接基团具有所示的结构片段：X表示H、取代或未取代的C1-C6烷基、OR8、SR8′、OH、SH或NR9R10，Y表示O或者S，z可以是0或者1，R8、R8′、R9或R10独立地为氢、取代或未取代的C1-C6烷基、磺酰基，分别独立地表示与A1或者A2之一相连部分，以及与相连核苷酸相连部分。
  35. 如权利要求33所述的寡聚化合物，所述的含磷核苷间连接基团独立地为：磷酸二酯连接基团、磷酸三酯连接基团、硫代磷酸酯连接基团、二硫代磷酸酯键连接基团、烷基膦酸酯连接基团、氨基膦酸酯连接基团、膦酸酯连接基团、次膦酸酯连接基团、硫代氨基磷酸酯连接基团和氨基磷酸酯连接基团。
  36. 如权利要求29-35任一项所述的寡聚化合物，所述的T3和T4之一保护基是羟基保护基，所述羟基保护基各自独立地选自乙酰基、叔丁基、叔丁氧基甲基、甲氧基甲基、四氢吡喃基、1-乙氧基乙基、1-(2-氯乙氧基)乙基、2-三甲基甲硅烷乙基、对氯苯基、2,4-二硝基苯基、苄基、苯甲酰基、对苯基苯甲酰基、2,6-二氯苄基、二苯基甲基、对硝基苄基、三甲基甲硅烷基、三乙基甲硅烷基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基、三苯甲硅烷基、三异丙基甲硅烷基、苯甲酰基甲酸酯、氯乙酰基、三氯乙酰基、三氟乙酰基、新戊酰基、9-芴甲氧羰基、甲磺酸基、甲苯磺酸基、三氟甲磺酸基、三苯甲基、单甲氧基三苯甲基、二甲氧三苯甲基、三甲氧基三苯甲基或取代的9-苯基 黄嘌呤-9-基；优选的羟基保护基各自独立地选自乙酰基、苄基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基以及4,4’-二甲氧三苯甲基；
    T3和T4之一保护基是巯基保护基，所述巯基保护基各自独立地选自乙酰基、叔丁基、叔丁氧基甲基、甲氧基甲基、四氢吡喃基、1-乙氧基乙基、1-(2-氯乙氧基)乙基、2-三甲基甲硅烷乙基、对氯苯基、2,4-二硝基苯基、苄基、苯甲酰基、对苯基苯甲酰基、2,6-二氯苄基、二苯基甲基、对硝基苄基、三甲基甲硅烷基、三乙基甲硅烷基、叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基、三苯甲硅烷基、三异丙基甲硅烷基、苯甲酰基甲酸酯、氯乙酰基、三氯乙酰基、三氟乙酰基、新戊酰基、9-芴甲氧羰基、甲磺酸基、甲苯磺酸基、三氟甲磺酸基、三苯甲基、单甲氧基三苯甲基、二甲氧三苯甲基、三甲氧基三苯甲基或取代的9-苯基黄嘌呤-9-基；优选的巯基保护基各自独立地选自苄基、4,4’-二甲氧三苯甲基；
    T3和T4之一保护基是氨基保护基，所述氨基保护基各自独立地选自2-三甲基硅基乙氧基羰基(Teoc)、1-甲基-1-(4-联苯基)乙氧基羰基(Bpoc)、叔丁氧基羰基(BOC)、烯丙氧基羰基(Alloc)、9-芴基甲氧基羰基(Fmoc)、苄氧基羰基(Cbz)、甲酰基、乙酰基、三卤乙酰基、苯甲酰基、硝基苯基、乙酰基、2-硝基苯磺酰基、邻苯二甲酰亚胺基和二硫代丁二酰基。
  37. 如权利要求29-35任一项所述的寡聚化合物，所述的T3和T4的一个为氢，T3和T4的另一个为将式(III)单体或其立体异构体连接到寡聚化合物5′-末端和/或3′-末端的核苷间连接基团。
  38. 如权利要求29-35任一项所述的寡聚化合物，T3和T4之一选自亲脂性基团，其中所述的亲脂性基团的亲脂性通过logKow测定，logKow超过0；优选地亲脂性基团logKow超过1，超过1.5，超过2，超过3，超过4，超过5或超过10。
  39. 如权利要求38所述的寡聚化合物，亲脂性基团选自含有饱和或不饱和的C4-C30烃链、脂肪环、芳香基、脂肪酸基团或脂肪酸衍生的基团、类固醇衍生的基团和任何脂溶性维生素基团。
  40. 如权利要求38-39任一项所述的寡聚化合物，所述的亲脂性基团饱和或不饱和亲脂性基团为饱和或不饱和C10-C18烃链、包含具有4到28个碳原子的烃链的任何饱和或不饱和脂肪酸，并且可以含有一个或两个羧基、胆固醇、维生素E(生育酚)或胆汁酸；更优选为：饱和的直链C16烷基、辛酸、癸酸、十二烷酸、十四烷酸、十六烷酸、十八烷酸、二十烷酸、二十二烷酸和二十四烷酸、肉豆蔻脑酸、棕榈油酸、十六碳烯酸、油 酸、反油酸、反型异油酸、亚油酸、反亚油酸、α-亚麻酸、花生四烯酸和芥酸、二十二碳六烯酸酸(DHA)、二十碳五烯酸(EPA)、二十二烷酸(DCA)、甾醇胆固醇(胆汁)、生育酚琥珀酸酯(TS)和石胆酸(LA)、维甲酸(维生素A酸)。
  41. 如权利要求37-39任一项所述的寡聚化合物，亲脂性基团可经由连接子与A1或者A2共价连接。
  42. 如权利要求41所述的寡聚化合物，所述亲脂性基团所述连接子是选自由以下组成的组：酰胺键、磷脂酰胆碱、烃连接子或聚乙二醇(PEG)连接子、氨基-烷基-醇、羟脯氨酸、羟脯氨醇、氨基-烷基-硫代磷酸酯连接子、氨基-PEG-硫代磷酸酯连接子、α-羧酸盐-氨基-烷基硫代磷酸酯连接子和α-羧酸盐-氨基-PEG-硫代磷酸酯连接子、醚、硫醚、脲、碳酸酯、胺、酰胺、马来酰亚胺-硫醚、二硫化物、磷酸二酯、磺酰胺键、点击反应的产物或氨基甲酸酯。
  43. 如权利要求41所述的寡聚化合物，所述连接子是可降解连接子，选自由以下组成的组：氧化还原可裂解连接子、酸可裂解连接子、酯酶可裂解连接子、磷酸酶可裂解连接子或肽酶可裂解连接子；或者是生物可裂解连接子，选自由以下组成的组：DNA、RNA、二硫化物、酰胺、半乳糖胺、葡糖胺、葡萄糖、半乳糖、甘露糖的官能化单糖或寡糖及其组合。
  44. 权利要求29-35任一项所述的寡聚化合物，所述的寡聚化合物中5′-末端和/或3′-末端还包含一种或更多种靶向基团。
  45. 权利要求29-35任一项所述的寡聚化合物，所述的T3和T4中的之一表示为共价键，寡聚化合物通过T3和T4中的之一以共价键形式与靶向基团相连。
  46. 权利要求44-45任一项所述的寡聚化合物，所述靶向基团可以选自包含以下靶向分子或其衍生物形成的配体中的一种或多种：聚合物、糖类、肝实质细胞表达的受体配体、抗体、量子点、多肽，或小分子配体。
  47. 权利要求46所述的寡聚化合物，每个所述靶向基团独立地为包含与哺乳动物肝细胞表面的去唾液酸糖蛋白受体(ASGPR)亲和的配体，靶向基团优选为独立地选自包含D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D吡喃甘露糖、α-D-吡喃葡萄-糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳-糖胺、N-乙酰半乳糖胺、N-三氟乙酰半 乳糖胺、N-丙酰半乳糖胺、N-正丁酰半乳胺、N-异丁-36酰半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6 O-三苯甲基-α-D吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖、L-4-硫代核糖中的一种或者几种的配体；更优选为包含半乳糖或N-乙酰半乳糖胺的配体。
  48. 权利要求44-45任一项所述的寡聚化合物，所述靶向基团与A1或者A2之一相连选自以下化合物片段之一：
  49. 权利要求29-48任一项所述的寡聚化合物，包含至少一种式(IVa)或者式(IVb)所示的构型的5′-末端和/或者3′-末端单体：
    A1、A2、T3、T4、R1以及n如权利要求29-48所定义。
  50. 权利要求29-48任一项所述的寡聚化合物，包含至少一种式(IVa-1)或者式(IVb-1)所示的构型的5′-末端和/或者3′-末端单体：
    A1、A2、T3、T4、R1以及n如权利要求29-48任一项所定义。
  51. 权利要求29-50任一项所述的寡聚化合物，所述的R1分别独立地选自卤素、取代或未取代的C1-C6烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基。
  52. 权利要求29-51任一项所述的寡聚化合物，n为0。
  53. 权利要求29-52任一项所述的寡聚化合物，T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连，并且T3和T4中的另一个为将根据权利要求29-52任一项的单体连接到寡聚化合物5′-末端的核苷间连接基团。
  54. 权利要求29-52任一项所述的寡聚化合物，T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连，并且T3和T4中的另一个为将根据权利要求29-52任一项单体连接到寡聚化合物3′-末端的核苷间连接基团。
  55. 权利要求29-52任一项所述的寡聚化合物，T3和T4之一为H、保护基、亲脂性基团，以及任选的共价键，所述亲脂性基团任选地经由连接子与A1或者A2之一相连，并且T3和T4中的另一个分别独立地将一种相同或者不同的将根据权利要求29-52任一项单体连接到寡聚化合物5′-末端和3′-末端的核苷间连接基团。
  56. 权利要求29-55任一项所述的寡聚化合物，具有以下具体结构：

    ,其中Olig表示将所述的单体分别连接到寡聚化合物的5′-末端和/或3′-末端的寡核苷酸部分。
  57. 权利要求29-56任一项所述的寡聚化合物，所述寡聚化合物是单链寡核苷酸，包括反义寡核苷酸(又称ASO)、核糖酶或适体。
  58. 一种是双链核糖核酸(dsRNA)试剂，所述的双链核糖核酸(dsRNA)试剂包含：正义链和反义链，其中所述正义链与反义链完全或者部分互补，并且所述反义链与核酸靶基因互补；所述正义链和反义链的至少一者为根据权利要求29-56任一项所定义的寡聚化合物；并且其中所述双链核糖核酸(dsRNA)试剂任选地进一步包含独立的靶向基团。
  59. 权利要求58所述的双链核糖核酸(dsRNA)试剂，其中正义链为包含根据权利要求29-52、56任一项的5′-末端单体所定义的寡聚化合物、3′-末端单体所提供的寡聚化合物、或者5′-末端和3′-末端单体所提供的寡聚化合物。
  60. 权利要求58所述双链核糖核酸(dsRNA)试剂，其中其中反义链为包含根据权利要求29-52、56任一项的5′-末端和/或者3′-末端单体提供的寡聚化合物；优选地所述的双链核糖核酸(dsRNA)试剂，其中反义链为包含根据权利要求29-52、56的3′-末端单体提供的寡聚化合物，或者根据权利要求29-52、56任一项的5′-末端单体提供的寡聚化合物。
  61. 权利要求58-60任一项所述的双链核糖核酸(dsRNA)试剂中任意一条链的5′-末端和/或3′-末端核苷酸上还进一步包含一种或更多种靶向基团。
  62. 权利要求29-61任一项所述的寡聚化合物或双链核糖核酸(dsRNA)中，正义链或反义链每一条链均分别包含8-40核苷酸长度。
  63. 权利要求29-61任一项所述的寡聚化合物或双链核糖核酸(dsRNA)试剂在制备抑制基因表达的药物中的用途。
  64. 权利要求63所述的用途，所述的药物用于肝内施用和肝外施用，所述的药物用于肝外施用包括中枢神经(CNS)靶基因选自APP、ATXN2、C9orf72、TARDBP、HTT、SNCA、FUS、ATXN3、ATXN1、SCA7、SCA8、ATN1、MeCP2，朊病毒疾病、PRNP、DMPK、TTR，以及眼组织的靶基：AMD、CFB、MYOC、ROCK2、ADRB2、CA2、CRYGC、PPP3C。
  65. 权利要求63所述的用途，所述的药物应用在CNS疾病包括阿尔茨海默病、肌萎缩性侧索硬化(ALS)、额颞叶型痴呆、亨廷顿病、帕金森病、脊髓小脑病、朊病毒病以及拉福拉病；眼科领域疾病：年龄相关性黄斑变性(AMD)(干性及湿性)、鸟枪弹样脉络膜视网膜病变、显性色素性视、网膜炎、富克氏营养不良(Fuch’s dystrophy)、遗传性及偶发性青光眼以及斯特格特氏病(stargardt’s disease)、靶向青光眼、白内障、干眼综合征。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2026012491A1 (zh) * 2024-07-11 2026-01-15 北京福元医药股份有限公司 用于向眼部和中枢神经系统递送的包含亲脂性单体的化合物及其应用
WO2026021467A1 (en) * 2024-07-23 2026-01-29 Shanghai Argo Biopharmaceutical Co., Ltd. Stereospecific linkages double stranded rna agents and compositions

Citations (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US426220A (en) 1890-04-22 Seal-lock
US3854480A (en) 1969-04-01 1974-12-17 Alza Corp Drug-delivery system
US4452775A (en) 1982-12-03 1984-06-05 Syntex (U.S.A.) Inc. Cholesterol matrix delivery system for sustained release of macromolecules
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US4675189A (en) 1980-11-18 1987-06-23 Syntex (U.S.A.) Inc. Microencapsulation of water soluble active polypeptides
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US5075109A (en) 1986-10-24 1991-12-24 Southern Research Institute Method of potentiating an immune response
US5133974A (en) 1989-05-05 1992-07-28 Kv Pharmaceutical Company Extended release pharmaceutical formulations
US5256775A (en) 1989-06-05 1993-10-26 Gilead Sciences, Inc. Exonuclease-resistant oligonucleotides
WO1994002499A1 (en) 1992-07-27 1994-02-03 Hybridon, Inc. Oligonucleotide alkylphosphonothioates
WO1994017093A1 (en) 1993-01-25 1994-08-04 Hybridon, Inc. Oligonucleotide alkylphosphonates and alkylphosphonothioates
US5366878A (en) 1990-02-15 1994-11-22 The Worcester Foundation For Experimental Biology Method of site-specific alteration of RNA and production of encoded polypeptides
US5378825A (en) 1990-07-27 1995-01-03 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs
US5386023A (en) 1990-07-27 1995-01-31 Isis Pharmaceuticals Backbone modified oligonucleotide analogs and preparation thereof through reductive coupling
US5407686A (en) 1991-11-27 1995-04-18 Sidmak Laboratories, Inc. Sustained release composition for oral administration of active ingredient
WO1995024929A2 (en) 1994-03-15 1995-09-21 Brown University Research Foundation Polymeric gene delivery system
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5508270A (en) 1993-03-06 1996-04-16 Ciba-Geigy Corporation Nucleoside phosphinate compounds and compositions
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5610289A (en) 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5625050A (en) 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
US5736152A (en) 1995-10-27 1998-04-07 Atrix Laboratories, Inc. Non-polymeric sustained release delivery system
WO2002036743A2 (en) 2000-10-30 2002-05-10 Isis Pharmaceuticals, Inc. Antisense modulation of calreticulin expression
WO2009073809A2 (en) 2007-12-04 2009-06-11 Alnylam Pharmaceuticals, Inc. Carbohydrate conjugates as delivery agents for oligonucleotides
WO2010131916A2 (ko) 2009-05-14 2010-11-18 (주)바이오니아 siRNA 접합체 및 그 제조방법
US20110196141A1 (en) * 2009-09-07 2011-08-11 Council Of Scientific & Industrial Research Locked and unlocked 2'-o phosphoramidite nucleosides, process of preparation thereof and oligomers comprising the nucleosides
WO2014179627A2 (en) 2013-05-01 2014-11-06 Isis Pharmaceuticals, Inc. Compositions and methods for modulating hbv and ttr expression
WO2015006740A2 (en) 2013-07-11 2015-01-15 Alnylam Pharmaceuticals, Inc. Oligonucleotide-ligand conjugates and process for their preparation
US20160333341A1 (en) * 2013-10-25 2016-11-17 Regulus Therapeutics Inc. Microrna compounds and methods for modulating mir-21 activity
WO2019215333A1 (en) 2018-05-11 2019-11-14 Alpha Anomeric Sas Oligonucleotides conjugates comprising 7'-5'-alpha-anomeric-bicyclic sugar nucleosides
WO2019217459A1 (en) 2018-05-07 2019-11-14 Alnylam Pharmaceuticals, Inc. Extrahepatic delivery
WO2021173615A1 (en) 2020-02-24 2021-09-02 Integrated Dna Technologies, Inc. Oligonucleotide synthesis on solid support
WO2022005988A2 (en) 2020-06-30 2022-01-06 United States Of America, As Represented By The Secretary, Department Of Health And Human Services Solid support for synthesizing nucleic acid sequences and methods for making and using
CN114096540A (zh) * 2019-05-23 2022-02-25 斯特拉托斯基因公司 用于纳米孔测序的易位控制元件、报告子代码和用于易位控制的其他手段
WO2022162155A1 (en) 2021-01-30 2022-08-04 E-Therapeutics Plc Nucleic acids containing abasic nucleotides
CN115003680A (zh) 2020-01-29 2022-09-02 住友化学株式会社 制备核酸寡聚物的方法
WO2023138689A1 (zh) * 2022-01-24 2023-07-27 上海舶望制药有限公司 抑制LPA(Apo(a))蛋白表达的组合物和方法

Patent Citations (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US426220A (en) 1890-04-22 Seal-lock
US3854480A (en) 1969-04-01 1974-12-17 Alza Corp Drug-delivery system
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US4675189A (en) 1980-11-18 1987-06-23 Syntex (U.S.A.) Inc. Microencapsulation of water soluble active polypeptides
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US4452775A (en) 1982-12-03 1984-06-05 Syntex (U.S.A.) Inc. Cholesterol matrix delivery system for sustained release of macromolecules
US5075109A (en) 1986-10-24 1991-12-24 Southern Research Institute Method of potentiating an immune response
US5133974A (en) 1989-05-05 1992-07-28 Kv Pharmaceutical Company Extended release pharmaceutical formulations
US5256775A (en) 1989-06-05 1993-10-26 Gilead Sciences, Inc. Exonuclease-resistant oligonucleotides
US5366878A (en) 1990-02-15 1994-11-22 The Worcester Foundation For Experimental Biology Method of site-specific alteration of RNA and production of encoded polypeptides
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5378825A (en) 1990-07-27 1995-01-03 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs
US5386023A (en) 1990-07-27 1995-01-31 Isis Pharmaceuticals Backbone modified oligonucleotide analogs and preparation thereof through reductive coupling
US5610289A (en) 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5407686A (en) 1991-11-27 1995-04-18 Sidmak Laboratories, Inc. Sustained release composition for oral administration of active ingredient
WO1994002499A1 (en) 1992-07-27 1994-02-03 Hybridon, Inc. Oligonucleotide alkylphosphonothioates
WO1994017093A1 (en) 1993-01-25 1994-08-04 Hybridon, Inc. Oligonucleotide alkylphosphonates and alkylphosphonothioates
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
US5508270A (en) 1993-03-06 1996-04-16 Ciba-Geigy Corporation Nucleoside phosphinate compounds and compositions
WO1995024929A2 (en) 1994-03-15 1995-09-21 Brown University Research Foundation Polymeric gene delivery system
US5625050A (en) 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
US5736152A (en) 1995-10-27 1998-04-07 Atrix Laboratories, Inc. Non-polymeric sustained release delivery system
WO2002036743A2 (en) 2000-10-30 2002-05-10 Isis Pharmaceuticals, Inc. Antisense modulation of calreticulin expression
WO2009073809A2 (en) 2007-12-04 2009-06-11 Alnylam Pharmaceuticals, Inc. Carbohydrate conjugates as delivery agents for oligonucleotides
WO2009082607A2 (en) 2007-12-04 2009-07-02 Alnylam Pharmaceuticals, Inc. Targeting lipids
WO2010131916A2 (ko) 2009-05-14 2010-11-18 (주)바이오니아 siRNA 접합체 및 그 제조방법
CN103233003A (zh) 2009-05-14 2013-08-07 株式会社百奥尼 siRNA缀合物及其制备方法
US20110196141A1 (en) * 2009-09-07 2011-08-11 Council Of Scientific & Industrial Research Locked and unlocked 2'-o phosphoramidite nucleosides, process of preparation thereof and oligomers comprising the nucleosides
WO2014179620A1 (en) 2013-05-01 2014-11-06 Isis Pharmaceuticals, Inc. Conjugated antisense compounds and their use
WO2014179627A2 (en) 2013-05-01 2014-11-06 Isis Pharmaceuticals, Inc. Compositions and methods for modulating hbv and ttr expression
CN112921036A (zh) * 2013-05-01 2021-06-08 Ionis制药公司 用于调节载脂蛋白(a)表达的组合物和方法
WO2015006740A2 (en) 2013-07-11 2015-01-15 Alnylam Pharmaceuticals, Inc. Oligonucleotide-ligand conjugates and process for their preparation
US20160333341A1 (en) * 2013-10-25 2016-11-17 Regulus Therapeutics Inc. Microrna compounds and methods for modulating mir-21 activity
WO2019217459A1 (en) 2018-05-07 2019-11-14 Alnylam Pharmaceuticals, Inc. Extrahepatic delivery
WO2019215333A1 (en) 2018-05-11 2019-11-14 Alpha Anomeric Sas Oligonucleotides conjugates comprising 7'-5'-alpha-anomeric-bicyclic sugar nucleosides
CN114096540A (zh) * 2019-05-23 2022-02-25 斯特拉托斯基因公司 用于纳米孔测序的易位控制元件、报告子代码和用于易位控制的其他手段
CN115003680A (zh) 2020-01-29 2022-09-02 住友化学株式会社 制备核酸寡聚物的方法
WO2021173615A1 (en) 2020-02-24 2021-09-02 Integrated Dna Technologies, Inc. Oligonucleotide synthesis on solid support
WO2022005988A2 (en) 2020-06-30 2022-01-06 United States Of America, As Represented By The Secretary, Department Of Health And Human Services Solid support for synthesizing nucleic acid sequences and methods for making and using
WO2022162155A1 (en) 2021-01-30 2022-08-04 E-Therapeutics Plc Nucleic acids containing abasic nucleotides
WO2023138689A1 (zh) * 2022-01-24 2023-07-27 上海舶望制药有限公司 抑制LPA(Apo(a))蛋白表达的组合物和方法

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
"ACS Symposium", article "Carbohydrate Modifications in Antisense Research", pages: 40 - 65
"Current Protocols in Nucleic Acid Chemistry", 2000, article "Solid-Phase Supports for Oligonucleotide Synthesis"
"Diverse Lipid Conjugates for Functional Extra-Hepatic siRNA Delivery In Vivo", NUCLEIC ACIDS RESEARCH, vol. 47, no. 31082-1096, 2019, pages 31082 - 1096
"Synthesis of oligonucleotides on a soluble support", BEILSTEIN J. ORG. CHEM., vol. 13, 2017, pages 1368 - 1387
BEAUCAGEIYER, TETRAHEDRON, vol. 48, no. 12, 1992, pages 2223 - 2311
BEILSTEIN J. ORG. CHEM., vol. 13, 2017, pages 1368 - 1387
DU: "A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites", NUCLEIC ACIDS RES, vol. 33, no. 5, 21 March 2005 (2005-03-21), pages 1671 - 7, XP002624727, DOI: 10.1093/nar/gki312
H. S. SAWHNEYC. P. PATHAKJ. A. HUBELL, MACROMOLECULES, vol. 26, 1993, pages 581 - 587
NUCLEIC ACIDS RES, vol. 33, no. 11, 2005, pages 3698
See also references of EP4628583A1
TETKO ET AL., J. CHEM. INF. COMPUT. SCI., vol. 41, 2001, pages 1407 - 21

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2026012491A1 (zh) * 2024-07-11 2026-01-15 北京福元医药股份有限公司 用于向眼部和中枢神经系统递送的包含亲脂性单体的化合物及其应用
WO2026021467A1 (en) * 2024-07-23 2026-01-29 Shanghai Argo Biopharmaceutical Co., Ltd. Stereospecific linkages double stranded rna agents and compositions

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