WO2024083220A1 - 配体-寡核苷酸共轭物及用于该共轭物的接头 - Google Patents

配体-寡核苷酸共轭物及用于该共轭物的接头 Download PDF

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WO2024083220A1
WO2024083220A1 PCT/CN2023/125626 CN2023125626W WO2024083220A1 WO 2024083220 A1 WO2024083220 A1 WO 2024083220A1 CN 2023125626 W CN2023125626 W CN 2023125626W WO 2024083220 A1 WO2024083220 A1 WO 2024083220A1
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repeating unit
methylene
oligonucleotide
alkyl
amino
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PCT/CN2023/125626
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English (en)
French (fr)
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倪帅健
宋颖
景茜
刘子轩
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上海京新生物医药有限公司
浙江京新药业股份有限公司
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Publication of WO2024083220A1 publication Critical patent/WO2024083220A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound

Definitions

  • the present invention relates to a ligand-oligonucleotide conjugate and a connector thereof, in particular to a GalNAc-siRNA conjugate and a connector thereof.
  • the present invention also relates to a preparation method of the ligand-oligonucleotide conjugate and the use of the connector.
  • RNA interference refers to antisense-mediated gene silencing achieved by utilizing the mechanism of RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • Potential drug molecules based on RNAi include double-stranded RNA, such as siRNA and shRNA.
  • Free siRNA has a large relative molecular mass (13,000-15,000) and contains a large amount of negative charge, making it difficult to cross the cell membrane and exert RNAi effects in the cytoplasm.
  • the immunogenicity of free siRNA molecules in the blood is also one of the main reasons limiting its clinical application, causing severe immune responses during circulation in the body.
  • GalNAc-siRNA conjugates Chemical modification of the base backbone structure of siRNA sequences can significantly enhance their anti-enzymatic stability in blood circulation; however, the improvement of chemical stability has not solved the problem that siRNA is not easy to penetrate the cell membrane barrier.
  • the Monoharan team of Alnylam Pharmaceuticals covalently coupled GalNAc to the 3' end of the sense chain of siRNA in a trivalent state to obtain GalNAc-siRNA conjugates.
  • the complete GalNAc-siRNA conjugate can enter the intracellular environment through endocytosis mediated by ASGPR, which is highly expressed on the surface of hepatocytes and involves mesh proteins. As the pH in the endosome decreases, the GalNAc-siRNA conjugate is released from the bound ASPGR.
  • GalNAc-siRNA conjugates from endosomes is not clear.
  • the vast majority of free siRNAs are still captured in endosomes, while a very small amount ( ⁇ 1%) can enter the cytoplasm through the lipid bilayer membrane of the endosomal body and induce RNAi reactions through unknown mechanisms.
  • GalNAc is also used to deliver antisense oligonucleotides (ASOs) to hepatocytes.
  • ASOs antisense oligonucleotides
  • Prakash et al. demonstrated that the use of trivalent GalNAc to deliver second-generation gapmer antisense oligonucleotides enhanced their efficacy by 6-10 times. When combined with the next-generation ASO design, the efficacy was enhanced by about 60 times, showing a strong liver-targeted delivery and efficacy enhancement capability (Prakash et al., Nucleic Acids Research, 2014, Vol. 42, No. 13, 8796–8807).
  • linker portion wherein the linker portion is as shown in Formula I or II:
  • Another aspect of the present invention provides a ligand-oligonucleotide conjugate comprising a linker moiety of the present invention.
  • Another aspect of the present invention provides an intermediate compound shown in formula III or IV:
  • Another aspect of the present invention provides use of the above intermediate compound in preparing a ligand-oligonucleotide conjugate.
  • Another aspect of the present invention provides a method for preparing a ligand-oligonucleotide conjugate, comprising: providing any of the above intermediate compounds; providing an oligonucleotide having a terminal amino group at the 5' or 3' end; and covalently linking the intermediate compound to the oligonucleotide via the terminal amino group.
  • Another aspect of the present invention provides a linker represented by formula I or II:
  • Another aspect of the present invention provides a linking unit represented by formula I' or II':
  • the left end of Formula I' is connected to -NR 1 - to obtain Formula I, and the left end of Formula II' is connected to -NR 1 - to obtain Formula II.
  • Another aspect of the present invention provides use of the above linker in preparing a ligand-oligonucleotide conjugate.
  • Another aspect of the present invention provides a pharmaceutical composition comprising any one of the ligand-oligonucleotide conjugates of the present invention and a pharmaceutically acceptable carrier.
  • Another aspect of the present invention provides a method for treating a disease, comprising administering to a subject a therapeutically effective amount of any one of the conjugates provided herein or a pharmaceutical composition comprising the conjugate.
  • oligonucleotide refers to a polydeoxyribonucleotide, a polyribonucleotide or a polyribonucleotide/deoxyribonucleotide hybrid in this article, and is generally 15 to 30 nucleotides or nucleotide pairs in length, and each nucleotide may be natural or modified.
  • the oligonucleotide may be double-stranded or single-stranded, including single-stranded and double-stranded DNA, single-stranded and double-stranded RNA (including shRNA, siRNA) and DNA/RNA hybrid molecules.
  • the oligonucleotide may be conjugated to another molecule, such as N-acetylgalactosamine (GalNAc) or its polymer (GalNAc cluster).
  • GalNAc N-acetylgalactosamine
  • the oligonucleotide used in the present invention is selected from siRNA, shRNA, miRNA and ASO.
  • modified nucleotide refers to a nucleotide independently having a modified sugar moiety, a modified internucleotide bond and/or a modified nucleobase. Therefore, the term modified nucleotide includes substitution, addition or removal of, for example, a functional group or atom of an internucleoside bond, a sugar moiety or a nucleobase. Modifications suitable for use in the present invention include all types of modifications disclosed herein or known in the art.
  • the modified nucleotide is a deoxynucleotide, a 3' terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a configurationally restricted nucleotide, a restricted ethyl nucleotide, a baseless nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxy modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate
  • the modified nucleotide is a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a 3' terminal deoxythymidine nucleotide (dT), a locked nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, an amino phosphorothioate and/or a non-natural base containing nucleotide.
  • dT deoxythymidine nucleotide
  • Antisense oligonucleotides are single-stranded DNA or RNA that are complementary to a selected sequence. In the case of antisense DNA, they can be used to target a specific complementary (coding or noncoding) RNA. If binding occurs, this hybrid can be degraded by RNAse H1.
  • a typical example of an ASO is a gapmer, which has an internal "gap segment" flanked by two external "wing segments", where the gap segment consists of a number of nucleotides that support RNase H cleavage and each wing segment consists of one or more nucleotides in the ribonucleic acid molecule.
  • the gapmer may be composed of nucleotides that are chemically different from the nucleotides within the gap segment.
  • the 5' and 3' wing segments of the gapmer are composed of 2'-MOE modified nucleotides
  • the gap segment is composed of deoxyribonucleotides
  • optionally the linkages between all nucleotides are phosphorothioate bonds.
  • siRNA refers to a nucleic acid that forms double-stranded RNA that has the ability to reduce or inhibit target gene expression when the siRNA and target gene are present in the same cell.
  • siRNA is typically about 15 to about 30 base pairs in length, most typically about 19 to 25 base pairs in length, such as 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs in length.
  • shRNA refers to short hairpin RNA, which includes two short inverted repeat sequences and an intermediate stem-loop structure connecting the two.
  • the stem-loop may include at least one unpaired nucleotide, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.
  • the stem-loop may be 10 or less nucleotides.
  • the stem-loop may be 8 or less unpaired nucleotides.
  • the stem-loop may be 4 to 10 unpaired nucleotides.
  • the stem-loop may be 4 to 8 nucleotides.
  • siRNA and shRNA are sometimes collectively referred to as double-stranded RNA (dsRNA) herein.
  • dsRNA double-stranded RNA
  • the two substantially complementary chains of dsRNA do not need to be but can also be covalently linked.
  • the maximum number of base pairs is the number of nucleotides in the shortest chain of the dsRNA minus any overhangs present in the duplex.
  • the dsRNA can also include one or more nucleotide overhangs.
  • at least one chain includes a 3' overhang of at least 1 nucleotide, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14 or 15 nucleotides.
  • At least one chain includes a 5' overhang of at least 1 nucleotide, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14 or 15 nucleotides.
  • the 3' end and 5' end of a chain of the dsRNA both include an overhang of at least 1 nucleotide.
  • nucleotide overhang refers to at least one unpaired nucleotide that protrudes from the duplex structure of a dsRNA. For example, when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa, there is a nucleotide overhang. At the nucleotide overhang.
  • the nucleotide overhang may comprise or consist of nucleotide/nucleoside analogs, including deoxynucleotides/nucleosides.
  • the overhang may be on the sense strand, the antisense strand, or any combination thereof.
  • the nucleotides of the overhang may be present at the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the dsRNA.
  • the term "blunt end” or “blunt end” with respect to dsRNA refers to the absence of unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., no nucleotide overhangs.
  • One or both ends of a dsRNA may be flat. If both ends of a dsRNA are blunt, the dsRNA is said to be blunt-ended.
  • a "blunt-ended" dsRNA is a dsRNA with both ends being blunt, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such molecules are double-stranded over their entire length.
  • antisense strand refers to a strand in a dsRNA that includes a region that is substantially complementary to a target sequence.
  • sense strand refers to a strand in a dsRNA that includes a region that is substantially complementary to an antisense strand region as defined herein.
  • substantially complementary region refers to a region that is fully complementary or incompletely complementary. When the complementary region is not fully complementary to the target sequence, mismatches may be located in the interior or terminal regions of the molecule. Typically, the most tolerable mismatches are located in the terminal regions, such as 5, 4, 3, or 2 at the 5'- and/or 3' ends of the dsRNA.
  • ligand refers to a cell or tissue targeting agent that binds to a specified cell type (such as a hepatocyte), such as a lectin, glycoprotein, lipid or protein (such as an antibody).
  • exemplary targeting agents include thyrotropin, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetylgalactosamine (GalNAc), multivalent (such as divalent or trivalent) GalNAc, N-acetylglucosamine, multivalent mannose, multivalent trehalose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, cholesterol, steroids, bile acid, folate, vitamin B12, biotin, RGD peptide and RGD peptide mimetic.
  • the ligand is a carbohydrate, such as a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, a polysaccharide.
  • the ligand can be a derivative comprising GalNAc.
  • the ligand is comprised of one or more N-acetylgalactosamine derivatives attached via a divalent or trivalent branched linker.
  • amino acid refers to a molecule containing both an amino group and a carboxyl group. Suitable amino acids include, but are not limited to, the D- and L-isomers of naturally occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways.
  • amino acid as used herein includes, but is not limited to, ⁇ -amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.
  • ⁇ -amino acid refers to a molecule containing both an amino group and a carboxyl group bound to a carbon designated as the ⁇ -carbon.
  • Naturally occurring amino acid refers to any of the 20 amino acids commonly found in peptides synthesized in nature, known by their single-letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V.
  • Hydrophobic amino acids include small hydrophobic amino acids and large hydrophobic amino acids.
  • Mall hydrophobic amino acids are glycine, alanine, proline, and their analogs.
  • Large hydrophobic amino acids are valine, leucine, isoleucine, phenylalanine, methionine, Tryptophan and its analogs.
  • Poly amino acids are serine, threonine, asparagine, glutamine, cysteine, tyrosine and their analogs.
  • Charge amino acids are lysine, arginine, histidine, aspartic acid, glutamic acid and their analogs.
  • amino acid analog refers to a molecule that is structurally similar to an amino acid and can replace an amino acid in the formation of a peptidomimetic macrocycle.
  • Amino acid analogs include, but are not limited to, ⁇ -amino acids and amino acids in which the amino or carboxyl group is substituted with a group of similar reactivity (e.g., a secondary or tertiary amine is substituted for a primary amine, or an ester is substituted for a carboxyl group).
  • ⁇ -amino acid refers to a molecule containing both the amino and carboxyl groups in the ⁇ configuration.
  • unnatural amino acid refers to an amino acid that is not one of the twenty amino acids commonly found in peptides synthesized in nature.
  • the term “unnatural amino acid” includes ⁇ -substituted and ⁇ -disubstituted amino acids of natural amino acids, N-alkyl amino acids, lactic acid, halide derivatives such as trifluorotyrosine, p-chloro-phenylalanine, p-fluoro-phenylalanine, p-bromo-phenylalanine, p-NO 2 -phenylalanine, phenylglycine, sarcosine, penicillamine, D-2-methyltryptophan, phosphoserine, phosphothreonine, phosphotyrosine, pI-phenylalanine, L-allyl-glycine, ⁇ -alanine, ⁇ -aspartic acid, ⁇ -cyclohexylalanine, citrulline,
  • amino acid side chain refers to the moiety attached to the ⁇ -carbon in a natural or non-natural amino acid.
  • amino acid side chain of alanine is a methyl group
  • amino acid side chain of phenylalanine is a phenylmethylene group
  • amino acid side chain of cysteine is a mercaptomethylene group
  • amino acid side chain of aspartic acid is a carboxymethylene group
  • amino acid side chain of tyrosine is a 4-hydroxyphenylmethylene group, and the like.
  • non-natural amino acid side chains are also included, for example, naturally occurring amino acid side chains (e.g., amino acid metabolites) or synthetically prepared amino acid side chains (e.g., citrulline side chains). Whether natural or non-natural amino acids, both L and D configurations thereof are included, and accordingly, their side chains are also expected to include L and D side chains.
  • carboxyl protecting group means a group intended to protect a carboxyl group, selected from methyl, substituted methyls, ethyl, 2-substituted ethyls, allyl, tert-butyl, alkoxyalkyls, alkoxyalkoxyalkyls, 2,6-dialkylphenyls, benzyl, substituted benzyls, silyls or stannyls; the substituted methyls are selected from 9-fluorenylmethyl, triisopropylsilylmethyl, cyclopropylmethyl, diphenylmethyl or triphenylmethyl; the 2-substituted ethyls are selected from 2,2,2-trichloroethyl
  • the alkoxyalkyl group is selected from methoxymethyl, benzyloxymethyl or triisopropylsilyloxymethyl; the alkoxyalkoxyalkyl group is selected from methoxyethoxymethyl; the
  • halogen refers to fluorine, chlorine, bromine or iodine or a group thereof.
  • number of halogens is not limited, it can be any suitable number, such as monohalogen, dihalogen, trihalogen; when the position of the halogen is not limited, it can be any suitable position, for example, the halogenated phenyl can be halogenated at the ortho position, para position, meta position or a combination thereof.
  • alkyl refers to a saturated straight or branched hydrocarbon chain.
  • alkyl groups having a specific number of carbon atoms the term includes the corresponding n-alkyl group and its various isomeric forms (if any).
  • alkyl groups having 4 carbon atoms include n-butyl, isobutyl, sec-butyl and tert-butyl.
  • Exemplary C1-10 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, 4,4-dimethylpentyl, 5,5-dimethylhexyl, 6,6-dimethylheptyl, etc.
  • alkenyl refers to a straight or branched hydrocarbon chain having one or more carbon-carbon double bonds.
  • Exemplary C2-10 alkenyls include ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 3-hexenyl, 5-hexenyl, etc.
  • alkynyl refers to a straight or branched hydrocarbon chain having one or more carbon-carbon triple bonds.
  • exemplary C2-10 alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 4-methyl-2-pentynyl, etc.
  • alkoxy refers to an alkyl-O-group, wherein alkyl is as defined above.
  • C 1-10 alkoxy include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.
  • the alkoxy group may be substituted with 1 to 7, preferably 1 to 5, halogen atoms, and specific examples include difluoromethoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy, 4,4,4-trifluorobutoxy, and the like.
  • cycloalkyl refers to a cyclic saturated hydrocarbon group.
  • Examples of C3-10 cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.2.1]octyl and adamantyl.
  • aryl refers to aromatic monocyclic and polycyclic carbocyclic ring systems, wherein the individual carbocyclic rings in the polycyclic system are fused or connected to each other by single bonds. Suitable aryl groups include phenyl, naphthyl, 2,3-dihydro-1H-indenyl and biphenyl. 6 to 14 membered aryl refers to an aromatic ring system of a 6-carbon monocyclic ring, a 10-carbon bicyclic ring, a 12-carbon biphenyl ring or a 14-carbon tricyclic ring.
  • heteroaryl refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, the heteroatoms being selected from O, N or S (e.g., carbon atoms and 1-3, 1-6 or 1-9 O, N or S heteroatoms, respectively, if monocyclic, bicyclic or tricyclic), wherein 0, 1, 2, 3 or 4 atoms of each ring are substituted with substituents.
  • heteroaryl examples include pyridyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thienyl, quinolinyl, indolyl, thiazolyl, and the like.
  • heterocyclyl refers to a non-aromatic monocyclic, bicyclic or tricyclic ring system.
  • a 5- to 14-membered heterocyclyl refers to a 5-8-membered monocyclic, 8-12-membered bicyclic or 11-14-membered tricyclic ring system having 1-3 heteroatoms if a monocyclic ring, 1-6 heteroatoms if a bicyclic ring, or 1-9 heteroatoms if a tricyclic ring, the heteroatoms being selected from O, N or S (e.g., carbon atoms and 1-3, 1-6 or 1-9 O, N or S heteroatoms, respectively, if a monocyclic, bicyclic or tricyclic ring) or other suitable heteroatoms (such as P or Si), wherein 0, 1, 2 or 3 atoms of each ring are substituted by substituents.
  • Examples of 5- to 14-membered heterocyclyls include piperazinyl, pyrrolidinyl, dioxacyclohexyl, morpholinyl, tetrahydrofuranyl, tetrahydropyrrolidinyl, etc.
  • alkylthio refers to a mercapto group in which the hydrogen atom of the mercapto group is substituted by an alkyl group.
  • the C1-3 alkylthio group refers to a methylthio group, an ethylthio group, a propylthio group, and an isopropylthio group.
  • alkylphosphonyl refers to a phosphonyl group in which one or both hydroxyl groups of the phosphonyl group are substituted by an alkyl group.
  • examples of C1-3 alkylphosphonyl groups include methylphosphonyl, ethylphosphonyl, dimethylphosphonyl, diethylphosphonyl, methylethylphosphonyl, and the like.
  • alkylphosphonyloxy refers to an alkylphosphonyl-O-group, wherein the alkylphosphonyl group is as defined above.
  • examples of C1-3 alkylphosphonyloxy include methylphosphonyloxy, ethylphosphonyloxy, dimethylphosphonyloxy, diethylphosphonyloxy, methylethylphosphonyloxy, and the like.
  • arylalkyl refers to an alkyl substituted by an aryl group
  • alkylaryl refers to an aryl substituted by an alkyl group, wherein alkyl and aryl are as defined above.
  • exemplary arylalkyl groups are phenyl C 1-3 alkyl, naphthyl C 1-3 alkyl, biphenyl C 1-6 alkyl , and the like.
  • heteroarylalkyl refers to an alkyl group substituted by a heteroaryl group
  • alkylheteroaryl refers to a heteroaryl group substituted by an alkyl group, wherein the alkyl and heteroaryl groups are as defined above.
  • heteroarylalkyl groups include pyridyl C 1-3 alkyl, indolyl C 1-3 alkyl, quinolyl C 1-3 alkyl, isoquinolyl C 1-3 alkyl, thienyl C 1-3 alkyl, thioindenyl C 1-3 alkyl, thioindenyl C 1-3 alkyl, oxazolyl C 1-3 alkyl, benzothiazolyl C 1-3 alkyl, imidazolyl C 1-3 alkyl, pyrimidinyl C 1-3 alkyl and the like.
  • heterocyclylalkyl refers to an alkyl group substituted by a heterocyclyl group
  • alkylheterocyclyl refers to a heterocyclyl group substituted by an alkyl group, wherein the heterocyclyl group refers to and the alkyl group is as defined above.
  • heterocyclylalkyl groups include furanyl C 1-3 alkyl, piperazinyl C 1-3 alkyl, pyrrolidinyl C 1-3 alkyl, dioxane C 1-3 alkyl, morpholinyl C 1-3 alkyl, tetrahydrofuranyl C 1-3 alkyl, tetrahydropyrrolidinyl C 1-3 alkyl.
  • TMSOTf refers to trimethylsilyl trifluoromethanesulfonate.
  • DCM dichloromethane
  • DIEA diisopropylethylamine
  • EA refers to ethyl acetate
  • THF tetrahydrofuran
  • DMF N,N-dimethylformamide
  • HATU refers to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.
  • PE refers to petroleum ether
  • TFAPfp refers to pentafluorophenyl trifluoroacetate.
  • TFA trifluoroacetic acid
  • PI refers to propidium iodide
  • the compounds disclosed herein contain one or more asymmetric centers, thus existing as racemates or racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. Unless otherwise expressly stated, all such isomeric forms of these compounds are intended to be included herein.
  • the compounds disclosed herein are also presented as a variety of tautomeric forms, in which case the compounds include all tautomeric forms of the compounds described herein (e.g., if the alkylation of the ring system results in alkylation at multiple positions, the present invention includes all these reaction products). Unless otherwise expressly stated, all crystalline forms of the compounds are intended to be included herein.
  • terapéuticaally effective amount refers to an amount of a conjugate of the invention or composition thereof effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit/risk ratio applicable to any medical treatment.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms which are within the scope of sound medical judgment and suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition or vehicle that participates in carrying or delivering the conjugate from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient, manufacturing aid or solvent encapsulating material.
  • a pharmaceutically acceptable material, composition or vehicle that participates in carrying or delivering the conjugate from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient, manufacturing aid or solvent encapsulating material.
  • Each carrier must be in a suitable composition for the composition.
  • the other ingredients are "acceptable” in the sense that they are compatible and not injurious to the patient.
  • treatment encompasses prevention, therapy, and cure.
  • the patient receiving such treatment is generally any animal in need, including primates (particularly humans) and other mammals such as horses, cattle, pigs, sheep, poultry, and pets.
  • a ligand-oligonucleotide conjugate comprising a linker moiety as shown in Formula I or Formula II: -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)- (Formula II),
  • R 1 , R 2 , R 3 , R 4 are independently selected from -H, C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 1-10 alkoxy, C 3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro and mercapto, which are optionally substituted with R 5 , wherein R 5 is selected from C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 1-10 alkoxy, C 3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, C 1-3 alkylthio and mercapto
  • Ra , Rb are independently selected from -H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-8 cycloalkyl, 6 to 14 membered arylC1-3 alkyl, 5 to 18 membered heteroarylC1-3 alkyl and 5 to 14 membered heterocyclyl, which are optionally substituted with Rc , wherein Rc is selected from hydroxy, thiol, carboxyl, amino, guanidinyl, halogen, C1-3 alkylthio, indolyl, quinolyl, isoquinolyl, amido, pyrrolyl, thienyl, thiaindenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furanyl, C1-10 alkyl, C1-10 alkoxy, 6 to 14 membered aryl, C3-8 cycloalkyl, adamant
  • the heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon;
  • n is 1 to 10, and each repeating unit is the same or different, for example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • p is 2 to 6 and each repeating unit is the same or different, for example p may be 2, 3, 4, 5 or 6.
  • the ligand-oligonucleotide conjugate comprises a linker moiety as shown in Formula I or II, wherein Ra , Rb are independently selected from -H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-8 cycloalkyl , phenyl C1-3 alkyl, naphthyl C1-3 alkyl, 5 to 12 membered heteroaryl C1-3 alkyl and 5 to 14 membered heterocyclyl C1-3 alkyl, which are optionally substituted by Rc , wherein Rc is selected from hydroxyl, sulfhydryl, carboxyl, amino, guanidinyl, halogen, C1-3 alkylthio, indolyl, quinoline, linyl, isoquinolyl, amido, thienyl, thiandenyl, thiazolyl, benzothiazolyl, imidazolyl
  • the ligand-oligonucleotide conjugate comprises a linker portion as shown in Formula I or II, wherein Ra , Rb are independently selected from -H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy, C3-6 cycloalkyl, phenyl C1-3 alkyl, pyridyl C1-3 alkyl, indolyl C1-3 alkyl, quinolyl C1-3 alkyl , isoquinolyl C1-3 alkyl, thienyl C1-3 alkyl, thiandenyl C1-3 alkyl, thiazolyl C1-3 alkyl, benzothiazolyl C1-3 alkyl , imidazolyl C1-3 alkyl , furanyl C1-3 alkyl, pyrimidinyl C1-3 alkyl, which are optionally substituted by Rc , wherein Rc is selected from hydroxyl, thiol, carboxyl, amino,
  • the ligand-oligonucleotide conjugate comprises a linker portion as shown in Formula I or II, wherein Ra and Rb are independently selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, 1-butenyl, 2-butenyl, isobutylenyl, ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-methyl-2-propynyl, 1,3-butadienyl, methoxy, ethoxy, propoxy, butoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenylmethylene, phenyldimethylene, pyridylmethylene, pyridyldimethylene , ind
  • the ligand-oligonucleotide conjugate comprises a linker moiety as shown in Formula I or II, wherein Ra is -H and Rb is selected from -H, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclopentyl, cyclohexyl, cyclohexyl dimethylene, cyclohexyl methylene, cyclobutyl methylene, phenyl methylene, methylthio methylene, 3-indolyl methylene, hydroxy methylene, amidomethylene, hydroxymethyl methylene, mercapto methylene, amidomethylene, hydroxyphenyl methylene, hydroxyphenyl dimethylene, carboxy methylene, carboxy dimethylene, amino-n-butyl, guanidino R 1 , R
  • R 4 , heteroatoms, n and p are as defined above.
  • the ligand-oligonucleotide conjugate comprises a linker moiety as shown in Formula I or II, wherein one of Ra and Rb is -H, and the other of Ra and Rb is an amino acid side chain.
  • the amino acid includes natural amino acids and non-natural amino acids, including D and L configurations.
  • the amino acid is a natural or unnatural amino acid, wherein unnatural amino acids include, but are not limited to, the following structures:
  • ⁇ -amino acid analogs include, but are not limited to, the following: cyclic ⁇ -amino acid analogs; ⁇ -alanine; (R)- ⁇ -phenylalanine; (R)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid; (R)-3-amino-4-(1-naphthyl)-butyric acid; (R)-3-amino-4-(2,4-dichlorophenyl)-butyric acid; (R)-3-amino-4-(2-chlorophenyl)-butyric acid; (R)-3-amino-4-(2-cyanophenyl)-butyric acid; (R)-3-amino-4-(2-fluorophenyl)-butyric acid; (R)-3-amino-4-(2-furanyl)-butyric acid; (R)
  • amino acid analogs of alanine, valine, glycine, or leucine include, but are not limited to, the following: ⁇ -methoxyglycine; ⁇ -allyl-L-alanine; ⁇ -aminoisobutyric acid; ⁇ -methyl-leucine; ⁇ -(1-naphthyl)-D-alanine; ⁇ -(1-naphthyl)-L-alanine; ⁇ -(2-naphthyl)-D-alanine; ⁇ -(2-naphthyl)-L-alanine; ⁇ -(2-pyridyl)-D-alanine; ⁇ -(2-pyridyl)-L-alanine; ⁇ -(2-thienyl)-D-alanine; ⁇ -(2-thienyl)-D-alanine; ⁇ -(2-thienyl)-D-alanine; ⁇ -(2-thienyl
  • amino acid analogs of arginine and lysine include, but are not limited to, the following: citrulline; L-2-amino-3-guanidinopropionic acid; L-2-amino-3-ureidopropionic acid; L-citrulline; Lys(Me)2-OH; Lys(N3)-OH; N ⁇ -benzyloxycarbonyl-L-ornithine; N ⁇ -nitro-D-arginine; N ⁇ -nitro-L-arginine; ⁇ -methyl-ornithine; 2,6-diaminopimelic acid; L-ornithine; (N ⁇ -1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-D-ornithine; (N ⁇ -1-(4,4 -dimethyl-2,6-diox
  • amino acid analogs of aspartic acid and glutamic acid include, but are not limited to, analogs of aspartic acid or glutamic acid.
  • amino acid analogs of aspartic acid and glutamic acid include, but are not limited to, the following: ⁇ -methyl-D-aspartic acid; ⁇ -methyl-glutamic acid; ⁇ -methyl-L-aspartic acid; ⁇ -methylene-glutamic acid; (N- ⁇ -ethyl)-L-glutamine; [N- ⁇ -(4-aminobenzoyl)]-L-glutamic acid; 2,6-diaminopimelate; L- ⁇ -aminosuberic acid; D-2-aminoadipic acid; D- ⁇ -aminosuberic acid; ⁇ -aminopimelate; iminodiethyl acid; L-2-aminoadipic acid; threo- ⁇ -methyl-aspartic acid; ⁇ -carboxy-D-glutamic acid ⁇ , ⁇
  • cysteine and methionine include, but are not limited to, analogs of cysteine and methionine.
  • amino acid analogs of cysteine and methionine include, but are not limited to, Cys(farnesyl)-OH, Cys(farnesyl)-OMe, ⁇ -methyl-methionine, Cys(2-hydroxyethyl)-OH, Cys(3-aminopropyl)-OH, 2-amino-4-(ethylthio)butyric acid, buthionine, buthionine sulfoxide, ethionine, methionine methylsulfonium chloride, selenomethionine, cysteic acid, [2-(4-pyridyl)ethyl]-DL-penicillamine, [2-(4-pyridyl)ethyl]-L-cysteine, 4-methoxybenzyl-D-penicillamine, 4-methoxybenzyl-L
  • unnatural amino acids include, but are not limited to, analogs of phenylalanine and tyrosine.
  • amino acid analogs of phenylalanine and tyrosine include ⁇ -methyl-phenylalanine, ⁇ -hydroxyphenylalanine, ⁇ -methyl-3-methoxy-DL-phenylalanine, ⁇ -methyl-D-phenylalanine, ⁇ -methyl-L-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 2,4-dichloro-phenylalanine, 2-(trifluoromethyl)-D-phenylalanine, 2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine, 2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine, 2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine, 2-cyano-L-phen
  • phenylalanine 2-methyl-D-phenylalanine, 2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine, 2-nitro-L-phenylalanine, 2,4,5-trihydroxy-phenylalanine, 3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine, 3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine, 3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine, 3,5,3'-triiodo-L-thyronine, 3,5-diiodo-D-tyrosine, 3,5-diiodo-L-tyrosine, 3, 5-Diiodo-L-th
  • unnatural amino acids include, but are not limited to, analogs of proline.
  • amino acid analogs of proline include, but are not limited to, 3,4-dehydro-proline, 4-fluoro-proline, cis-4-hydroxy-proline, thiazolidine-2-carboxylic acid, and trans-4-fluoro-proline.
  • amino acids include, but are not limited to, analogs of serine and threonine.
  • amino acid analogs of serine and threonine include, but are not limited to, 3-amino-2-hydroxy-5-methylhexanoic acid, 2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutyric acid, 2-amino-3-methoxybutyric acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutyric acid, and ⁇ -methylserine.
  • amino acid analogs of tryptophan include, but are not limited to, analogs of tryptophan.
  • amino acid analogs of tryptophan include, but are not limited to, the following: ⁇ -methyl-tryptophan; ⁇ -(3-benzothienyl)-D-alanine; ⁇ -(3-benzothienyl)-L-alanine; 1-methyl-tryptophan; 4-methyl-tryptophan; 5-benzyloxy-tryptophan; 5-bromo-tryptophan; 5-chloro-tryptophan; 5-fluoro-tryptophan; 5-hydroxy-tryptophan; 5-hydroxy-L-tryptophan; 5-methoxy-tryptophan; 5-methoxy-L-tryptophan; 5-methyl-tryptophan; 6-bromo- Tryptophan; 6-chloro-D-tryptophan; 6-chloro-tryptophan; 6-fluoro-tryptophan; 6-methyl-tryptophan; 7-
  • the non-natural amino acid can be racemic.
  • the non-natural amino acid used can be a D-isomer.
  • the non-natural amino acid used can be an L-isomer.
  • the non-natural amino acid can contain a chiral center that is an R or S configuration.
  • the amino group of the ⁇ -amino acid is replaced by a protecting group such as tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), tosyl, etc.
  • the carboxylic acid functional group of the ⁇ -amino acid is protected, for example, as its ester derivative.
  • a salt of a non-natural amino acid is used.
  • R 1 , R 2 , R 3 , R 4 are preferably independently selected from -H, C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl and 5 to 14 membered heterocyclyl, they are optionally substituted by R 5 , wherein R 5 is selected from C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl and 5 to 14 membered heterocyclyl.
  • R 1 , R 2 , R 3 , and R 4 are more preferably independently selected from -H, C 1-3 alkyl, C 1-3 alkoxy, and C 3-6 cycloalkyl, which are optionally substituted by R 5 , wherein R 5 is selected from C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, pyrrolyl, thienyl, thiazolyl, imidazolyl, pyridyl, furanyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and tetrahydropyrrolidinyl.
  • R 1 , R 2 , R 3 , and R 4 are more preferably independently selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, and ethoxy.
  • R 1 , R 2 , R 3 , and R 4 are more preferably independently -H or methyl.
  • R 1 , R 2 , R 3 , and R 4 are all -H.
  • n is 2 to 4, and each repeating unit is the same or different. More preferably, n is 2 or 3, and each repeating unit is the same or different. In some embodiments, n is 2, and each repeating unit is the same. In some embodiments, n is 3, and each repeating unit is the same. In some embodiments, n is 2, and each repeating unit is different. In some embodiments, n is 3, and each repeating unit is different.
  • p is 2 to 4, and each repeating unit is the same or different. More preferably, p is 2 or 3, and each repeating unit is the same or different. In some embodiments, p is 2, and each repeating unit is the same. In some embodiments, p is 3, and each repeating unit is the same. In some embodiments, p is 2, and each repeating unit is different. In some embodiments, p is 3, and each repeating unit is different.
  • a ligand-oligonucleotide conjugate comprising a linker moiety as shown in Formula I or Formula II: -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)- (Formula II),
  • R 1 , R 2 , R 3 , and R 4 are independently -H or methyl
  • Ra , Rb are independently selected from -H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-8 cycloalkyl, 6 to 14 membered arylC1-3 alkyl, 5 to 18 membered heteroarylC1-3 alkyl and 5 to 14 membered heterocyclyl, which are optionally substituted with Rc , wherein Rc is selected from hydroxy, thiol, carboxyl, amino, guanidinyl, halogen, C1-3 alkylthio, indolyl, quinolyl, isoquinolyl, amido, pyrrolyl, thienyl, thiaindenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furanyl, C1-10 alkyl, C1-10 alkoxy, 6 to 14 membered aryl, C3-8 cycloalkyl, adamant
  • the heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon, preferably nitrogen, oxygen and sulfur; n is 1 to 10, preferably 2 to 4, and is the same or different in each repeating unit; p is 2 to 6, preferably 2 to 4, and is the same or different in each repeating unit.
  • a ligand-oligonucleotide conjugate comprising a linker moiety as shown in Formula I or Formula II: -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)- (Formula II),
  • R 1 , R 2 , R 3 , and R 4 are independently -H or methyl
  • Ra , Rb are independently selected from -H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-8 cycloalkyl, phenylC1-3 alkyl, naphthylC1-3 alkyl, 5 to 12 membered heteroarylC1-3 alkyl and 5 to 14 membered heterocyclylC1-3 alkyl, which are optionally substituted by Rc , wherein Rc is selected from hydroxy, thiol, carboxyl, amino, guanidinyl, halogen, C1-3 alkylthio, indolyl, quinolyl, isoquinolyl , amido, thienyl, thiazolyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furanyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofur
  • the heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon, preferably nitrogen, oxygen and sulfur; n is 1 to 10, preferably 2 to 4, and is the same or different in each repeating unit; p is 2 to 6, preferably 2 to 4, and is the same or different in each repeating unit.
  • a ligand-oligonucleotide conjugate comprising a linker moiety as shown in Formula I or Formula II: -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)- (Formula II),
  • R 1 , R 2 , R 3 , and R 4 are independently -H or methyl
  • Ra , Rb are independently selected from -H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy, C3-6 cycloalkyl, phenyl C1-3 alkyl, pyridyl C1-3 alkyl, indolyl C1-3 alkyl, quinolyl C1-3 alkyl, isoquinolyl C1-3 alkyl, thienyl C1-3 alkyl, thiaindenyl C1-3 alkyl, thiazolyl C1-3 alkyl, benzothiazolyl C1-3 alkyl, imidazolyl C1-3 alkyl, furanyl C1-3 alkyl, pyrimidinyl C1-3 alkyl, and they are optionally substituted by Rc , wherein Rc is selected from hydroxyl, thiol , carboxyl, amino, guanidinyl, halogen, C1-3 alkyl , thiocyanate ... R a and R b together with the
  • the heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon, preferably nitrogen, oxygen and sulfur; n is 1 to 10, preferably 2 p is 2 to 6, preferably 2 to 4, and each repeating unit is the same or different; p is 2 to 6, preferably 2 to 4, and each repeating unit is the same or different.
  • a ligand-oligonucleotide conjugate comprising a linker moiety as shown in Formula I or Formula II: -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)- (Formula II),
  • R 1 , R 2 , R 3 , and R 4 are independently -H or methyl
  • Ra and Rb are independently selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-methyl-2-propynyl, 1,3-butadienyl, methoxy, ethoxy, propoxy, butoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenylmethylene, phenyldimethylene, pyridylmethylene, pyridyldimethylene , indolylmethylene, indolyldimethylene, quinolylmethylene, quinolyldimethylene, isoquinolylmethylene,
  • a ligand-oligonucleotide conjugate comprising a linker moiety as shown in Formula I or Formula II: -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)- (Formula II),
  • Ra , R1 , R2 , R3 , and R4 are independently -H or methyl
  • R b is selected from -H, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclopentyl, cyclohexyl, cyclohexyl dimethylene, cyclohexyl methylene, cyclobutyl methylene, phenyl methylene, methylthio methylene, 3-indolyl methylene, hydroxy methylene, amidomethylene, hydroxymethyl methylene, mercapto methylene, amidomethylene, hydroxyphenyl methylene, hydroxyphenyl dimethylene, carboxy methylene, carboxy dimethylene, amino-n-butyl, guanidine R is selected from the group consisting of dimethylene, guanidinyltrimethylene, 4-imidazolylmethylene, isopropylphenylmethylene, 1-naph
  • a ligand-oligonucleotide conjugate comprising a linker moiety as shown in Formula I or Formula II: -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)- (Formula II),
  • Ra , R1 , R2 , R3 , and R4 are independently -H or methyl;
  • Rb is selected from -H, methyl, isopropyl, and isobutyl, or Rb together with the C atom to which it is commonly connected and the adjacent N atom form a tetrahydropyrrolyl group;
  • n is 2 to 4, and each repeating unit is the same or different; and
  • p is 2 to 4, and each repeating unit is the same or different.
  • a ligand-oligonucleotide conjugate comprising a linker moiety as shown in Formula I or Formula II: -NR 1 -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p -(Formula I), -NR 1 -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)- (Formula II),
  • Ra , R1 , R2 , R3 , and R4 are independently -H or methyl;
  • Rb is selected from -H, methyl, isopropyl, and isobutyl, or Rb, together with the C atom to which it is commonly attached and the adjacent N atom, forms a tetrahydropyrrolyl group;
  • n is 2 or 3, and each repeating unit is the same or different;
  • p is 2, and for the repeating unit in which p is 2: in each repeating unit, Rb is isopropyl; in each repeating unit, Rb is methyl; in one repeating unit, Rb is is isopropyl, and in another repeating unit, Rb is methyl; in one repeating unit, Rb is isobutyl, and in another repeating unit, Rb is methyl; in one repeating unit, Rb is isobutyl, and in another repeating unit, Rb is methyl; in one repeating unit, Rb is isobut
  • the left end of the linker represented by Formula I or Formula II is connected to the ligand or used to be connected to the ligand; the right end is connected to the oligonucleotide or used to be connected to the oligonucleotide, preferably to an oligonucleotide with an amino group (e.g., an oligonucleotide with a terminal amino group at the 5' or 3' end) or used to be connected to an oligonucleotide with an amino group (e.g., an oligonucleotide with a terminal amino group at the 5' or 3' end).
  • an amino group e.g., an oligonucleotide with a terminal amino group at the 5' or 3' end
  • another aspect of the present invention provides a linking unit represented by formula I' or II': -C(O)-[C(R 3 R 4 )] n -C(O)-[NR 2 -C(R a R b )-C(O)] p - (Formula I'), or -[C(O)-C(R a R b )-NR 2 ] p -C(O)-[C(R 3 R 4 )] n -C(O)- (Formula II'),
  • the left end of Formula I' is connected to -NR 1 - to obtain Formula I, and the left end of Formula II' is connected to -NR 1 - to obtain Formula II.
  • the ligand in the ligand-oligonucleotide conjugate comprises one or more N-acetylgalactosamine derivatives attached via a divalent or trivalent branched linker.
  • the ligand has the following structure: (GalNAc-Q 1 ) m CH m′ -,
  • GalNAc represents N-acetylgalactosamine or its derivatives; in the present invention, the N-acetylgalactosamine or its derivatives are preferably the following structures:
  • the structure of the ligand is selected from:
  • the structure of the ligand is:
  • the oligonucleotide in the ligand-oligonucleotide conjugate is selected from antisense oligonucleotide (ASO), small interfering RNA (siRNA), small hairpin RNA (shRNA) and micro RNA (miRNA) or a salt thereof, and the salt is preferably a sodium salt or a potassium salt.
  • ASO antisense oligonucleotide
  • siRNA small interfering RNA
  • shRNA small hairpin RNA
  • miRNA micro RNA
  • the 5' end of the oligonucleotide is attached to the linker portion, preferably to the right end of the linker portion. In some embodiments, the 3' end of the oligonucleotide is attached to the linker portion, preferably to the right end of the linker portion.
  • the 5' end of the oligonucleotide is attached to the linker moiety via 3'-oligonucleotide-5'-OP(O)(OH)O-(CH 2 -Y) x -NH-, wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different.
  • the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(O)(OH)O-(CH 2 ) 6 -NH-; further preferably, the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(O)(OH)O-(CH 2 ) 3 -NH-; further preferably, the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(O)(OH)O-(CH 2 ) 12 -NH-; further preferably, the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(O)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH-.
  • the 5' end of the oligonucleotide is attached to the linker moiety via 3'-oligonucleotide-5'-OP(S)(OH)O-( CH2 -Y) x -NH-, wherein Y represents O or is absent, x is 3 to 12 and each repeat unit is the same or different.
  • the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(S)(OH)O-(CH 2 ) 6 -NH-; further preferably, the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(S)(OH)O-(CH 2 ) 3 -NH-; further preferably, the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(S)(OH)O-(CH 2 ) 12 -NH-; further preferably, the 5' end is attached to the linker portion via 3'-oligonucleotide-5'-OP(S)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH-.
  • the 3' end of the oligonucleotide may be attached to the linker moiety via 5'-oligonucleotide-3'-OP(O)(OH)O-(CH 2 -Y) x -NH-, wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different.
  • the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(O)(OH)O-(CH 2 ) 6 -NH-; further preferably, the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(O)(OH)O-(CH 2 ) 3 -NH-; further preferably, the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(O)(OH)O-(CH 2 ) 12 -NH-; further preferably, the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(O)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH-.
  • the 3' end of the oligonucleotide may be attached to the linker moiety via 5'-oligonucleotide-3'-OP(S)(OH)O-(CH 2 -Y) x -NH-, wherein Y represents O or is absent, x is 3 to 12 and each repeating unit is the same or different.
  • the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(S)(OH)O-(CH 2 ) 6 -NH-; further preferably, the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(S)(OH)O-(CH 2 ) 3 -NH-; further preferably, the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(S)(OH)O-(CH 2 ) 12 -NH-; further preferably, the 3' end is attached to the linker portion via 5'-oligonucleotide-3'-OP(S)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH-.
  • the oligonucleotide is a siRNA, comprising a sense strand and an antisense strand, more preferably, one or more of the nucleotides in the siRNA are modified, and the modification is a 2'-O-methyl modification and/or a 2'-fluoro modification.
  • the 3' end or the 5' end of the sense strand is attached to the linker portion.
  • the 5' end of the sense strand is attached to the linker portion.
  • the 5' end of the sense strand is attached to the linker moiety via 3'-siRNA sense strand-5'-OP(O)(OH)O-( CH2 -Y) x -NH-, wherein Y represents O or is absent, x is 3 to 12 and each repeat unit is the same or different.
  • the 5' end of the sense strand is attached to the linker portion via 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH 2 ) 6 -NH-; preferably, the 5' end of the sense strand is attached to the linker portion via 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH 2 ) 3 -NH-; preferably, the 5' end of the sense strand is attached to the linker portion via 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH 2 ) 12 -NH-; preferably, the 5' end of the sense strand is attached to the linker portion via 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH-.
  • the 5' end of the sense strand is attached to the linker moiety via 3'-siRNA sense strand-5'-OP(S)(OH)O-( CH2 -Y) x -NH-, wherein Y represents O or is absent, x is 3 to 12 and each repeat unit is the same or different.
  • the 5' end of the sense strand is attached to the linker portion via 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH 2 ) 6 -NH-; preferably, the 5' end of the sense strand is attached to the linker portion via 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH 2 ) 3 -NH-; preferably, the 5' end of the sense strand is attached to the linker portion via 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH 2 ) 12 -NH-; preferably, the 5' end of the sense strand is attached to the linker portion via 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH-.
  • each substituent and symbol has the corresponding meaning, value and numerical range as defined herein above, or its preferred meaning, value and numerical range; wherein the oligonucleotide is preferably siRNA, siRNA sense strand or ASO.
  • each substituent and symbol has the corresponding meaning, value and numerical range as defined herein above, or its preferred meaning, value and numerical range; wherein the siRNA antisense strand is connected to the siRNA sense strand by base pairing to form a double-stranded RNA.
  • ligand-oligonucleotide conjugate selected from the following formulas:
  • Another aspect of the present invention provides an intermediate compound having a structure shown in Formula III or IV:
  • GalNAc represents N-acetylgalactosamine or its derivatives; in the present invention, the N-acetylgalactosamine or its derivatives are preferably the following structures:
  • Q 2 represents -OH or -O-carboxyl protecting group, wherein the carboxyl protecting group is preferably benzyl or pentafluorophenyl;
  • R 1 , R 2 , R 3 , R 4 are independently selected from -H, C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 1-10 alkoxy, C 3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro and mercapto, which are optionally substituted with R 5 , wherein R 5 is selected from C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 1-10 alkoxy, C 3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, C 1-3 alkylthio and mercapto
  • Ra , Rb are independently selected from -H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-8 cycloalkyl, 6 to 14 membered arylC1-3 alkyl, 5 to 18 membered heteroarylC1-3 alkyl and 5 to 14 membered heterocyclyl, which are optionally substituted with Rc , wherein Rc is selected from hydroxy, thiol, carboxyl, amino, guanidinyl, halogen, C1-3 alkylthio, indolyl, quinolyl, isoquinolyl, amido, pyrrolyl, thienyl, thiaindenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furanyl, C1-10 alkyl, C1-10 alkoxy, 6 to 14 membered aryl, C3-8 cycloalkyl, adamant
  • the heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon;
  • n 1 to 10, and each repeating unit is the same or different;
  • p 2 to 6, and each repeating unit is the same or different.
  • the structure of the (GalNAc-Q 1 ) m CH m′ -moiety in the above formula III or IV is selected from:
  • the structure of the (GalNAc-Q 1 ) m CH m′ -part in the structure represented by Formula III or IV is:
  • an intermediate compound which has a structure shown in Formula III or IV:
  • Q2 represents -OH or -O carboxyl protecting group
  • R 1 , R 2 , R 3 , R 4 are independently selected from -H, C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 1-10 alkoxy, C 3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro and mercapto, which are optionally substituted with R 5 , wherein R 5 is selected from C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 1-10 alkoxy, C 3-10 cycloalkyl, 6 to 14 membered aryl, 5 to 18 membered heteroaryl, 5 to 14 membered heterocyclyl, halogen, hydroxy, amino, guanidino, carboxyl, cyano, nitro, C 1-3 alkylthio and mercapto
  • Ra , Rb are independently selected from -H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-8 cycloalkyl, 6 to 14 membered arylC1-3 alkyl, 5 to 18 membered heteroarylC1-3 alkyl and 5 to 14 membered heterocyclyl, which are optionally substituted with Rc , wherein Rc is selected from hydroxy, thiol, carboxyl, amino, guanidinyl, halogen, C1-3 alkylthio, indolyl, quinolyl, isoquinolyl, amido, pyrrolyl, thienyl, thiaindenyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furanyl, C1-10 alkyl, C1-10 alkoxy, 6 to 14 membered aryl, C3-8 cycloalkyl, adamant
  • the heteroatoms are selected from nitrogen, oxygen, sulfur, phosphorus and silicon;
  • n 1 to 10, and each repeating unit is the same or different;
  • p 2 to 6, and each repeating unit is the same or different.
  • Ra , Rb are independently selected from -H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C3-8 cycloalkyl, phenyl C1-3 alkyl, naphthyl C1-3 alkyl, 5 to 12 membered heteroaryl C1-3 alkyl and 5 to 14 membered heterocyclyl C1-3 alkyl, which are optionally substituted by Rc, wherein Rc is selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C1-3 alkylthio , indolyl , quinolyl, isoquinolyl, amide, thienyl, thiazolyl, thiazolyl, benzothiazolyl, imidazolyl, pyridyl, furanyl, piperazinyl, pyrrolidinyl
  • Rc is selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C1-3 alkylthio, indolyl, quinolyl, is 1-10 alkoxy, phenyl, benzyl, phenethyl, aminophenyl, carboxyphenyl, halogenated phenyl, biphenyl, C 3-8 cycloalkane R a and R b together with the C atom to which they are commonly attached and the adjacent N atom form a 3- to 14 - membered nitrogen-containing heterocyclic group.
  • Ra , Rb are independently selected from -H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy, C3-6 cycloalkyl, phenyl C1-3 alkyl, pyridyl C1-3 alkyl, indolyl C1-3 alkyl, quinolyl C1-3 alkyl, isoquinolyl C1-3 alkyl, thienyl C1-3 alkyl, thiandenyl C1-3 alkyl, thiazolyl C1-3 alkyl, benzothiazolyl C1-3 alkyl, imidazolyl C1-3 alkyl, furanyl C1-3 alkyl, pyrimidinyl C1-3 alkyl, which are optionally substituted by Rc , wherein Rc is selected from hydroxyl, thiol, carboxyl, amino, guanidinyl, halogen, C1-3 alkyl,
  • Ra and Rb are independently selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, 1-butenyl, 2-butenyl, isobutylenyl, ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-methyl-2-propynyl, 1,3-butadienyl, methoxy, ethoxy, propoxy, butoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenylmethylene, phenyldimethylene, pyridylmethylene, pyridyldimethylene.
  • R 1 , R 2 , R 3 , R 4 are preferably independently selected from -H, C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl and 5 to 14-membered heterocyclyl, which are optionally substituted by R 5 , wherein R 5 is selected from C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl and 5 to 14-membered heterocyclyl.
  • R 1 , R 2 , R 3 , and R 4 are more preferably independently selected from -H, C 1-3 alkyl, C 1-3 alkoxy, and C 3-6 cycloalkyl, which are optionally substituted by R 5 , wherein R 5 is selected from C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, pyrrolyl, thienyl, thiazolyl, imidazolyl, pyridyl, furanyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and tetrahydropyrrolidinyl.
  • R 1 , R 2 , R 3 , and R 4 are more preferably independently selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, and ethoxy.
  • R 1 , R 2 , R 3 , and R 4 are more preferably independently -H or methyl. In any embodiment of the intermediate compound, in the structure of formula III or IV, more preferably, R 1 , R 2 , R 3 , and R 4 are all -H.
  • n is 2 to 4, and each repeating unit is the same or different. More preferably, n is 2 or 3, and each repeating unit is the same or different. In some embodiments, n is 2, and each repeating unit is the same. In some embodiments, n is 3, and each repeating unit is the same. In some embodiments, n is 2, and each repeating unit is different. In some embodiments, n is 3, and each repeating unit is different.
  • p is 2 to 4, and each repeating unit is the same or different. More preferably, p is 2 or 3, and each repeating unit is the same or different. In some embodiments, p is 2, and each repeating unit is the same. In some embodiments, p is 3, and each repeating unit is the same. In some embodiments, p is 2, and each repeating unit is different. In some embodiments, p is 3, and each repeating unit is different.
  • Ra is -H
  • Rb is selected from -H, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclopentyl, cyclohexyl, cyclohexyl dimethylene, cyclohexyl methylene, cyclobutyl methylene, phenyl methylene, methylthio methylene, 3-indolyl methylene, hydroxy methylene, amide methylene, hydroxymethyl methylene, mercapto methylene, amide methylene, hydroxyphenyl methylene, hydroxyphenyl dimethylene, carboxy methylene, carboxy methylene, R b, methyl, amino-n-butyl, guanidinyldimethylene, gu
  • Ra , R1 , R2 , R3 , and R4 are independently -H or methyl;
  • Rb is selected from -H, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclopentyl, cyclohexyl, cyclohexyl dimethylene, cyclohexyl methylene, cyclobutyl methylene, phenyl methylene, methylthio methylene, 3-indolyl methylene, hydroxy methylene, amidomethylene, hydroxymethyl methylene, mercapto methylene, amidomethylene, hydroxyphenyl methylene, hydroxyphenyl dimethylene, carboxy methylene, carboxy dimethylene, amino-n-butyl, guani
  • Ra , R1 , R2 , R3 , and R4 are independently -H or methyl;
  • Rb is selected from -H, methyl, isopropyl, and isobutyl, or Rb, together with the C atom to which it is commonly connected and the adjacent N atom, forms a tetrahydropyrrolyl group;
  • n is 2 to 4, and each repeating unit is the same or different; and
  • p is 2 to 4, and each repeating unit is the same or different.
  • Ra , R1 , R2 , R3 , and R4 are independently -H or methyl;
  • Rb is selected from -H, methyl, isopropyl and isobutyl, or Rb forms a tetrahydropyrrolyl group together with the C atom to which it is commonly connected and the adjacent N atom;
  • n is 2 or 3, and each repeating unit is the same or different;
  • p is 2, and for the repeating unit in which p is 2: in each repeating unit, Rb is isopropyl; in each repeating unit, Rb is methyl; in one repeating unit, Rb is isopropyl, and in another repeating unit, Rb is methyl; in one repeating unit, Rb is isobutyl, and in another repeating unit, Rb is methyl; in one repeating unit, Rb is isobutyl, and in another repeating unit, Rb is methyl; in one repeating unit, Rb is isobutyl, and
  • C( RaRb ) -C(O)]-, -[C(O)-C( RaRb ) -NR2 ]- may specifically be an amino acid residue or fragment, wherein the amino acid is as defined in the present invention
  • the amino acid residue or fragment is a residue or fragment after the amino group of the amino acid loses a hydrogen and the carboxyl group loses -OH;
  • -[NR 2 -C(R a R b )-C(O)]- is independently selected from
  • -[C(O)-C(R a R b )-NR 2 ]- is independently selected from
  • p is preferably 2, i.e., -[NR 2 -C(R a R b )-C(O)]-[NR 2 -C(R a R b )-C(O)]-, -[C(O)-C(R a R b )-NR 2 ]-[C(O)-C(R a R b )-NR 2 ]-;
  • the intermediate represented by formula III or IV is preferably:
  • R o , R o ', and Q 2 are defined as above.
  • Bn represents a benzyl protecting group
  • Ac represents an acetoxy group
  • Another aspect of the present invention provides a method for preparing a ligand-oligonucleotide conjugate, which comprises: (a) providing any intermediate compound described herein; (b) providing an oligonucleotide having a terminal amino group at the 5' or 3' end of the oligonucleotide; (c) connecting the intermediate compound to the oligonucleotide via the amino group; and optionally, (d) a deprotection step (preferably a deprotection step).
  • the intermediate compound in step (c), is attached to the 5' end. In some embodiments, in step (c), the intermediate compound is attached to the 3' end of the oligonucleotide.
  • oligonucleotides are synthesized in the 3' to 5' direction on a solid support.
  • the intermediate compound needs to be conjugated with the 3' nucleoside in advance and connected to the solid support, and then the oligonucleotide is synthesized by a conventional synthesis method (e.g., phosphoramidite method).
  • a conventional synthesis method e.g., phosphoramidite method
  • pre-conjugation and connection to a solid support increases the complexity of synthesis.
  • the conjugate will be present in the entire synthesis process of the oligonucleotide, during which degradation may occur, and the types of reactions and reagents that can be used may also be limited.
  • the intermediate compound is connected to the 5' end of the oligonucleotide.
  • the oligonucleotide can be synthesized from 3' to 5' on a solid support by conventional synthesis methods (e.g., phosphoramidite method).
  • the intermediate compound can be introduced into the conjugate together with the last (most 5') nucleoside at the 5' end or after the oligonucleotide is cleaved from the solid support.
  • a terminal amino group is introduced after the last (most 5') nucleoside at the 5' end, so that the synthesized oligonucleotide has a 5' terminal amino group.
  • the synthesized oligonucleotide may have a structure shown in formula XI, XI':
  • Y represents O or is absent
  • x is 3 to 12 and each repeating unit is the same or different.
  • the structural formula of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(O)(OH)O-(CH 2 ) 6 -NH 2 . In some embodiments, the structural formula of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(O)(OH)O-(CH 2 ) 3 -NH 2 . In some embodiments, the structural formula of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(O)(OH)O-(CH 2 ) 12 -NH 2 .
  • the structural formula of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(O)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH 2 .
  • the structural formula of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(S)(OH)O-(CH 2 ) 6 -NH 2 . In some embodiments, the structural formula of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(S)(OH)O-(CH 2 ) 3 -NH 2 . In some embodiments, the structural formula of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(S)(OH)O-(CH 2 ) 12 -NH 2 .
  • the structural formula of the synthesized oligonucleotide is: 3'-oligonucleotide-5'-OP(S)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH 2 .
  • Suitable 5' terminal amino group introducing agents include, but are not limited to, 5'-amino TFA (C6) (CAS#133975-85-6), 5'-amino TFA (C3) (CAS#853955-89-2), 5'-amino TFA (C12) (CAS#178925-51-4) and 5'-amino modifier-5-CEP (CAS#612548-86-4), etc.
  • a terminal group modifier is directly used as a starting solid support, so that the oligonucleotide obtained has a 3' terminal amino group.
  • the synthesized oligonucleotide may have a structure shown in Formula XI-1, XI-1':
  • Y represents O or is absent
  • x is 3 to 12 and each repeating unit is the same or different.
  • the structural formula of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(O)(OH)O-(CH 2 ) 6 -NH 2 . In some embodiments, the structural formula of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(O)(OH)O-(CH 2 ) 3 -NH 2 . In some embodiments, the structural formula of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(O)(OH)O-(CH 2 ) 12 -NH 2 .
  • the structural formula of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(O)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH 2 .
  • the structural formula of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(S)(OH)O-(CH 2 ) 6 -NH 2 . In some embodiments, the structural formula of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(S)(OH)O-(CH 2 ) 3 -NH 2 . In some embodiments, the structural formula of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(S)(OH)O-(CH 2 ) 12 -NH 2 .
  • the structural formula of the synthesized oligonucleotide is: 5'-oligonucleotide-3'-OP(S)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH 2 .
  • Suitable 3' terminal amino group introducing agents include, but are not limited to, 3'-PT-Amino-Modifier C6CPG, etc.
  • the oligonucleotide is a siRNA comprising a sense strand and an antisense strand
  • the intermediate compound is connected to the sense strand of the siRNA.
  • the intermediate compound is attached to the 3' end of the sense strand of the siRNA.
  • the intermediate compound is attached to the 5' end of the sense strand of the siRNA.
  • the synthesized siRNA sense strand may have a structure shown in Formula XII, XII':
  • Y represents O or is absent
  • x is 3 to 12 and each repeating unit is the same or different.
  • the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH 2 ) 6 -NH 2 . In some embodiments, the structural formula of the synthesized siRNA sense strand is: 3'- siRNA sense strand-5'-OP(O)(OH)O-(CH 2 ) 3 -NH 2 . In some embodiments, the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH 2 ) 12 -NH 2 . In some embodiments, the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(O)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH 2 .
  • the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH 2 ) 6 -NH 2 . In some embodiments, the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH 2 ) 3 -NH 2 . In some embodiments, the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH 2 ) 12 -NH 2 . In some embodiments, the structural formula of the synthesized siRNA sense strand is: 3'-siRNA sense strand-5'-OP(S)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH 2 .
  • the synthesized siRNA sense strand may have the structure shown in Formula XII-1, XII-1':
  • Y represents O or is absent
  • x is 3 to 12 and each repeating unit is the same or different.
  • the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(O)(OH)O-(CH 2 ) 6 -NH 2 . In some embodiments, the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(O)(OH)O-(CH 2 ) 3 -NH 2 . In some embodiments, the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(O)(OH)O-(CH 2 ) 12 -NH 2 . In some embodiments, the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(O)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH 2 .
  • the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(S)(OH)O-(CH 2 ) 6 -NH 2 . In some embodiments, the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(S)(OH)O-(CH 2 ) 3 -NH 2 . In some embodiments, the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(S)(OH)O-(CH 2 ) 12 -NH 2 . In some embodiments, the structural formula of the synthesized siRNA sense strand is: 5'-siRNA sense strand-3'-OP(S)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH 2 .
  • the antisense strand of the siRNA can be synthesized by conventional synthesis and annealed to the sense strand through base pairing to form a double-stranded RNA.
  • the annealing or simulated annealing operation is as follows: complementary chains (SS chain and AS chain) of qualified purity (RP-HPLC purity>90%) are mixed in an equal molar ratio, the solution is placed in a 70°C water bath, heated to 90°C, maintained at 90°C for 5 minutes, and slowly cooled to room temperature. Samples are taken for SEC detection of double-strand annealing purity, and the ratio of SS chain or AS chain is fine-tuned to make the double-strand purity reach more than 90%.
  • the siRNA is lyophilized and stored in - 15°C to -25°C.
  • one or more of the nucleotides in the siRNA are modified.
  • the modification is a 2'-O-methyl modification and/or a 2'-fluoro modification.
  • the modification is a phosphorothioate modification in the backbone.
  • the modification includes a 2'-O-methyl modification, a 2'-fluoro modification, and a phosphorothioate modification in the backbone.
  • the oligonucleotide is an ASO and the intermediate compound is attached to the 5' end or the 3' end of the ASO. In some embodiments, in step (c), the intermediate compound is attached to the 3' end of the ASO. In a preferred embodiment, in step (c), the intermediate compound is attached to the 5' end of the ASO.
  • the synthesized ASO may have a structure shown in Formula XIII, XIII': 3'-ASO-5'-OP(O)(OH)O-(CH 2 -Y) x -NH 2 (Formula XIII) 3'-ASO-5'-OP(S)(OH)O-(CH 2 -Y) x -NH 2 (Formula XIII')
  • Y represents O or is absent
  • x is 3 to 12 and each repeating unit is the same or different.
  • the structure of the synthesized ASO is: 3'-ASO-5'-OP(O)(OH)O-(CH 2 ) 6 -NH 2 . In some embodiments, the structure of the synthesized ASO is: 3'-ASO-5'-OP(O)(OH)O-(CH 2 ) 3 -NH 2 . In some embodiments, the structure of the synthesized ASO is: 3'-ASO-5'-OP(O)(OH)O-(CH 2 ) 12 -NH 2 . In some embodiments, the structure of the synthesized ASO is: 3'-ASO-5'-OP(O)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH 2 .
  • the structure of the synthesized ASO is: 3'-ASO-5'-OP(S)(OH)O-(CH 2 ) 6 -NH 2 . In some embodiments, the structure of the synthesized ASO is: 3'-ASO-5'-OP(S)(OH)O-(CH 2 ) 3 -NH 2 . In some embodiments, the structure of the synthesized ASO is: 3'-ASO-5'-OP(S)(OH)O-(CH 2 ) 12 -NH 2 . In some embodiments, the structure of the synthesized ASO is: 3'-ASO-5'-OP(S)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH 2 .
  • the synthesized ASO may have a structure shown in Formula XIII-1 or XIII-1': 5'-ASO-3'-OP(O)(OH)O-(CH 2 -Y) x -NH 2 (Formula XIII-1) 5'-ASO-3'-OP(S)(OH)O-(CH 2 -Y) x -NH 2 (Formula XIII-1')
  • Y represents O or is absent
  • x is 3 to 12 and each repeating unit is the same or different.
  • the structure of the synthesized ASO is: 5'-ASO-3'-OP(O)(OH)O-(CH 2 ) 6 -NH 2 . In some embodiments, the structure of the synthesized ASO is: 5'-ASO-3'-OP(O)(OH)O-(CH 2 ) 3 -NH 2 . In some embodiments, the structure of the synthesized ASO is: 5'-ASO-3'-OP(O)(OH)O-(CH 2 ) 12 -NH 2 . In some embodiments, the structure of the synthesized ASO is: 5'-ASO-3'-OP(O)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH 2 .
  • the structure of the synthesized ASO is: 5'-ASO-3'-OP(S)(OH)O-(CH 2 ) 6 -NH 2 . In some embodiments, the structure of the synthesized ASO is: 5'-ASO-3'-OP(S)(OH)O-(CH 2 ) 3 -NH 2 . In some embodiments, the structure of the synthesized ASO is: 5'-ASO-3'-OP(S)(OH)O-(CH 2 ) 12 -NH 2 . In some embodiments, the structure of the synthesized ASO is: 5'-ASO-3'-OP(S)(OH)O-(CH 2 ) 3 -O-(CH 2 ) 3 -NH 2 .
  • one or more of the nucleotides in the ASO are modified.
  • the modification is selected from phosphorothioate (PSP), diaminomorpholino (PMO), 2'-methoxyethyl (2'-MOE) and 5-methylcytosine (5mC).
  • the modification includes PSP, PMO, 2'-MOE and 5mC.
  • step (c) the intermediate compound is linked to the oligonucleotide via the amino group to form the ligand-oligonucleotide conjugate, comprising reacting the free carboxyl group of the intermediate compound or a carboxyl protected form (e.g., an active ester form) with the free terminal amino group of the oligonucleotide to covalently link the intermediate compound to the oligonucleotide.
  • a carboxyl protected form e.g., an active ester form
  • the present invention also provides the use of any intermediate compound described herein in the preparation of a ligand-oligonucleotide conjugate.
  • the ligand-oligonucleotide conjugate is as defined herein above.
  • the ligand-oligonucleotide conjugate has a structure as shown in Formula V, V', VI, VI', VII, VII', VIII, VIII', IX, IX', X, X'; wherein each substituent and symbol has the corresponding meaning, value and numerical range as defined herein above, or its preferred meaning, value and numerical range.
  • Another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising any ligand-oligonucleotide conjugate described herein and a pharmaceutically acceptable carrier.
  • compositions include a therapeutically effective amount of one or more of the conjugates described above, alone or in combination with one or more pharmaceutically acceptable carriers (additives), excipients and/or diluents.
  • conjugates according to the invention may be formulated for administration in any convenient way by analogy from other drugs for use in human or veterinary medicine.
  • provided herein is a method for treating a disease, comprising administering to a subject a therapeutically effective amount of any one of the conjugates provided herein or a pharmaceutical composition comprising the conjugate.
  • the composition includes a plurality of conjugate species.
  • the conjugate species have sequences that are non-overlapping and non-adjacent to another species relative to a naturally occurring target sequence.
  • the plurality of conjugate species are specific for different naturally occurring target genes.
  • the conjugate is allele specific.
  • Embodiments of the present invention also relate to a method for inhibiting the expression of a target gene, which comprises the step of administering any of the above conjugates in an amount sufficient to inhibit the expression of the target gene.
  • the invention in another aspect, relates to a method of regulating the expression of a target gene in a cell, the method comprising providing to said cell a conjugate of the invention or a composition thereof.
  • the target gene of oligonucleotide, siRNA and ASO is selected from the group consisting of factor VII, Eg5, PCSK9, TPX2, apo(a), apoB, SAA, TTR, RSV, PDGF ⁇ gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepciden, activated protein C, cyclin D gene, VEGF gene, EGFR gene, cyclin A gene, cell cycle protein 2 gene, Cyclin E gene, WNT-1 gene, ⁇ -catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene, topoisomerase II ⁇ gene, p73 mutant gene, mutation in p21 (WAF)
  • Step 1 Compound 2: Dissolve compound 1 (25 g, 64.21 mmol) in 1,2-dichloroethane (350 mL)
  • Step 2 Compound 3: Compound 2 (21.1 g, 64.07 mmol) and 5-hexenyl-1-ol (7.06 g, 70.49 mmol) were dissolved in 1,2-dichloroethane (350 mL), 4A molecular sieve 20 g (water removed) was added and stirred at room temperature for 30 min. TMSOTf (7.12 g, 32.04 mmol) was added dropwise to the reaction solution under nitrogen protection at 0 ° C, and reacted at room temperature for 3 h.
  • Step 3 Compound 4: Compound 3 (35 g, 81.50 mmol) was dissolved in dichloromethane/acetonitrile (1:1, 200 mL), and deionized water (120 mL) containing sodium periodate (76.4 g, 357.2 mmol) was added under ice bath conditions. Stir for 15 min. RuCl 3 (560 mg, 2.70 mmol) was added to the reaction solution under ice bath conditions, and the temperature was slowly raised to room temperature, and stirred at room temperature for 1 h. Sodium periodate (19.1 g, 89.3 mmol) was added to the reaction solution at room temperature and the reaction was continued overnight. TLC monitoring showed that the raw material was completely reacted.
  • Step 1 Compound 7: Add trimethylolamine (Compound 6) (100 g, 0.825 mol), 1,4-dioxane (700 mL) to a reaction bottle, stir mechanically, cool to 0-5°C in an ice-water bath, add 60% potassium hydroxide aqueous solution (1.3 g potassium hydroxide dissolved in 0.9 g water), stir at 0-5°C for 1 h, add acrylonitrile (130 g, 2.45 mol), naturally heat to room temperature (15-25°C) and stir for 16 h.
  • Trimethylolamine Compound 6
  • 1,4-dioxane 700 mL
  • Step 2 Compound 8: Add 7 (160 g, 0.57 mol) and ethanol/hydrogen chloride (480 g) to a reaction bottle, stir, protect with nitrogen, and heat and reflux for 16 h. After the reaction is complete, filter, the solid is ammonium chloride, concentrate the filtrate, add carbon The mixture was neutralized with aqueous sodium bicarbonate solution (200 mL) until neutral, extracted with dichloromethane (200 mL ⁇ 4), washed with saturated brine (200 mL ⁇ 1), dried over anhydrous sodium sulfate, and concentrated to dryness to obtain compound 8 (150 g, yield 62.43%) as an oil.
  • Step 3 Compound 9: Dissolve compound 8 (40 g, 94.9 mmol) in 1,4-dioxane (160 mL) and stir until dissolved. Add Na 2 CO 3 (13.08 g, 123.37 mmol) in purified water (80 mL) to another reaction bottle and stir until dissolved. Add the prepared alkaline solution to the above reaction system at 0-5°C and stir for 30 minutes.
  • Step 5 Compound 11: Dissolve compound 10 (40 g, 84.84 mmol) in DCM (400 mL) and stir to dissolve, add DMF (4 mL), and replace with N 2 for 3 times. Add dichlorothionyl (62 mL) dropwise under N 2 protection at room temperature. After the addition, slowly heat up to reflux and reflux for 3.0 h. Monitor by LC/MS (quench by adding MeOH to sample). Dry the solvent under reduced pressure and add DCM (200 mL) to entrain once. Rotate until no solvent flows out. Weigh to obtain a yellow crude compound 11 (43.03 g, yield 96%) with a small amount of solid oil.
  • Step 6 Compound 12: Add tert-butyloxycarbonyl propylene diamine (59.1g, 339.2mmol) to a three-necked flask, add DIEA (109.6g, 847.9mmol), and dissolve in DCM (360mL). Replace with nitrogen three times, and cool to 0°C under nitrogen protection. Compound 11 (44.7g, 84.85mmol) is dissolved in DCM (300mL) and added dropwise to the above system under temperature control. As the addition is continued, the system gradually becomes turbid, and finally becomes a turbid white suspension system. After stirring for 30min, the mixture is heated to room temperature and reacted overnight. LC/MS monitoring.
  • Step 1 A1: Boc-Val-Val-OH (2.82 g, 8.91 mmol) was dissolved in DMF (18 mL), cesium carbonate (8.34 g, 25.61 mmol) was added, and benzyl bromide (4.22 g, 24.65 mmol) was added under stirring and reacted at room temperature for 1.5 h. The reaction was monitored by LC/MS. Water was added, and ethyl acetate was extracted three times.
  • Step 2 A2: A1 (3.6 g, 8.86 mmol) was dissolved in dichloromethane (30 mL), trifluoroacetic acid (6 mL) was added, and the mixture was reacted at room temperature for 16 h. The reaction was monitored by TLC. After the reaction of the raw materials was complete, the mixture was concentrated to obtain the trifluoroacetate salt of A2 (3.7 g, yield 100%).
  • Step 5 A5: A4 (1.60 g, 1.34 mmol) was dissolved in dichloromethane (50 mL), trifluoroacetic acid (10 mL) was added, and the reaction was allowed to react at room temperature for 2 h. The reaction was monitored by LC/MS. After the reaction of the raw material was complete, it was directly concentrated to obtain the trifluoroacetate salt of compound A5 (1.12 g). MS m/z (ESI): 447.9 [M/2+H] +
  • Step 2 B2: B1 (6.75 g, 16.69 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (10 mL) was added, and the mixture was reacted at room temperature for 2 h, and then monitored by LC/MS. The raw material was reacted completely, and the mixture was directly concentrated to obtain the trifluoroacetate salt of compound B2 (5.08 g). MS m/z (ESI): 305.2 [M+H] +
  • Step 5 B5: B4 (2 g, 1.68 mmol) was dissolved in dichloromethane (20 mL), trifluoroacetic acid (20 mL) was added, and the mixture was stirred at room temperature for 2 h. The reaction was monitored by LC/MS. Concentration gave the trifluoroacetate salt of compound B5 (1.40 g). MS m/z (ESI): 446.9 [M/2+H] +
  • Step 1 C1: Boc-Ala-Ala-OH (5 g, 19.2 mmol) was dissolved in 50 mL DMF, cesium carbonate (13.5 g, 41.4 mmol) was added, benzyl bromide (13.14 g, 76.8 mmol) was added under stirring, and the mixture was reacted at room temperature for 2 h. The reaction was monitored by TLC.
  • Step 2 C2: C1 (6.1 g, 17.4 mmol) was dissolved in dichloromethane (50 mL), trifluoroacetic acid (10 mL) was added, and the reaction was allowed to proceed overnight at room temperature. The reaction was monitored by TLC. After the reaction of the raw materials was complete, the product C2 trifluoroacetate (4.355 g, yield 100%) was obtained by concentration.
  • Step 5 C5: C4 (2.51 g, 2.2 mmol) was dissolved in dichloromethane (50 mL), trifluoroacetic acid (10 mL) was added, and the mixture was reacted at room temperature for 2 h. The reaction was monitored by TLC. After the reaction of the raw material was complete, it was directly concentrated to obtain the trifluoroacetate salt of compound C5 (1.84 g, yield 100%).
  • Step 6 C6: The trifluoroacetate of C5 (1.84 g, 2.2 mmol) was dissolved in dichloromethane (50 mL), DIEA (2.86 g, 22.10 mmol) was added, and a dichloromethane solution (20 mL) of compound 5 (4.21 g, 6.86 mmol) was added under stirring under ice bath conditions, and then the temperature was slowly raised to room temperature for reaction for 18 h.
  • Step 7 C7: C6 (0.50 g, 0.24 mmol) was dissolved in tetrahydrofuran (10 mL), and 20% palladium hydroxide on carbon (0.11 g) was added. After hydrogen was replaced three times, the mixture was stirred at room temperature for 5 h. After the reaction was completed, the reaction solution was filtered through diatomaceous earth to remove palladium hydroxide on carbon and concentrated. The crude compound C7 (0.11 g, 22%) was obtained. MS m/z (ESI): 1019.0 [M/2+H] + , 511.0 [M/4+H] +
  • Step 2 D3: Compound 13 (5.0 g, 6.20 mmol) and Cbz-Val-Ala-OH (2.6 g, 8.06 mmol) were dissolved in 40 mL of dichloromethane, DIEA (2.41 g, 18.6 mmol) and HATU (3.54 g, 9.31 mmol) were added, and the mixture was reacted at 40 °C for 16 h. After the reaction, the reaction solution was diluted with 50 mL of dichloromethane, 50 mL of water was added, and the mixture was separated.
  • Step 3 D4: D3 (2.8 g, 2.52 mmol) was dissolved in 30 mL methanol, 20% Pd(OH) 2 /C (0.5 g), trifluoroacetic acid (0.86 g, 7.54 mmol) were added, and the gas was replaced three times with a hydrogen balloon. The reaction was carried out at room temperature for 4 h under hydrogen conditions. Filtered with diatomaceous earth, the filter cake was washed with methanol (30 mL ⁇ 3), and the trifluoroacetate of D4 was obtained by spin drying, which was directly used in the next step. MS m/z (ESI): 976.7 [M + H] +
  • Step 5 D6: D5 (2.67 g, 2.26 mmol) was dissolved in TFA (15 mL) and stirred at room temperature for 1 h. The reaction solution was diluted with toluene (30 mL ⁇ 3) and spin-dried to obtain trifluoroacetic acid hydrochloride of D6, which was used directly in the next step. MS m/z (ESI): 880.6 [M + H] +
  • Step 6 D7: The trifluoroacetate of D6 (1.99 g, 2.26 mmol) was dissolved in dichloromethane (20 mL) and DIEA (11.69 g, 90.45 mmol) was added. A dichloromethane solution (20 mL) of compound 5 (4.3 g, 7.01 mmol) was added at 0°C and reacted at room temperature for 16 h. After the reaction was completed, the reaction solution was diluted with dichloromethane (50 mL), and the organic phase was washed with 1M KHSO 4 (50 mL), saturated sodium bicarbonate aqueous solution (50 mL), and water (50 mL).
  • Step 7 D8: D7 (1.60 g, 0.74 mmol) was dissolved in THF (16 mL), 20% Pd(OH) 2 /C (600 mg), and the mixture was purged with hydrogen three times. The reaction was carried out under hydrogen protection for 16 h. Filtered with diatomaceous earth, the filter cake was washed with THF (10 mL ⁇ 3), and the filtrate was dried to obtain D8 as a white foam, which was used directly in the next step. MS m/z (ESI): 1039.6 [M/2+H] +
  • Step 8 D9: D8 (0.92 g, 0.443 mmol) was dissolved in dichloromethane (20 mL), DIEA (0.17 g, 1.315 mmol) was added, pentafluorophenyl trifluoroacetate (0.25 g, 0.89 mmol) was added, and the reaction was carried out at room temperature for 18 h.
  • the reaction solution was diluted with dichloromethane (30 mL), and the organic phase was washed with 1M KHSO 4 (30 mL), saturated sodium bicarbonate aqueous solution (30 mL), and water (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and dried to obtain a crude product.
  • Step 1 E2: E1 (5 g, 24.01 mmol) was dissolved in dichloromethane (50 mL) and DIEA (4.66 g, 36.02 mmol) was added. Pentafluorophenyl trifluoroacetate (10.09 g, 36.02 mmol) was added at 0°C and reacted at room temperature for 16 h. After the reaction, the reaction solution was diluted with dichloromethane (50 mL), and the organic phase was washed with 1 M potassium hydrogen sulfate aqueous solution (50 mL ⁇ 2), saturated sodium bicarbonate (50 mL ⁇ 2), and water (50 mL).
  • Step 2 E3: Compound 13 (5.38 g, 6.67 mmol), Cbz-Gly-Pro-OH (2.66 g, 8.67 mmol) were dissolved in 50 mL of dichloromethane, DIEA (2.59 g, 20.0 mmol) and HATU (3.81 g, 10.0 mmol) were added and reacted at room temperature for 16 h. The reaction solution was diluted with 50 mL of dichloromethane, 50 mL of water was added, and the liquid was separated.
  • Step 3 E4: E3 (5.6 g, 5.11 mmol) was dissolved in 60 mL methanol, 20% Pd(OH) 2 /C (1 g), trifluoroacetic acid (1.75 g, 15.35 mmol) were added, and the gas was replaced by hydrogen three times. The reaction was carried out at room temperature for 4 h under hydrogen conditions. After the reaction was completed, the mixture was filtered through diatomaceous earth, the filter cake was washed with methanol (30 mL ⁇ 3), and the trifluoroacetate of E4 was obtained by spin drying. It was used directly in the next step without purification. MS m/z (ESI): 960.6 [M + H] +
  • Step 5 E6: E5 (0.88 g, 0.765 mmol) was dissolved in TFA (4 mL) and stirred at room temperature for 1 h. The reaction solution was diluted with toluene (10 mL), dried by spin drying, and then diluted with toluene (10 mL ⁇ 3), and dried by spin drying to obtain the trifluoroacetate salt of E6, which was used directly in the next step. MS m/z (ESI): 850.5 [M + H] +
  • Step 6 E7: E6 trifluoroacetate (0.65 g, 0.765 mmol) was dissolved in dichloromethane (5 mL), DIEA (3.95 g, 30.58 mmol) was added, and a dichloromethane (5 mL) solution of compound 5 (1.45 g, 2.37 mmol) was added, and the reaction was carried out at room temperature for 18 h.
  • the reaction solution was diluted with dichloromethane (20 mL), and the organic phase was washed with 1M KHSO 4 (30 mL), saturated sodium bicarbonate aqueous solution (30 mL), and water (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and dried to obtain a crude product.
  • Step 7 E8: E7 (100 mg, 0.047 mmol) was dissolved in THF (1 mL), 20% Pd(OH) 2 /C (20 mg), replaced with hydrogen three times, and reacted at room temperature for 4 h under hydrogen protection. Filtered with diatomaceous earth, washed with THF (10 mL ⁇ 3), and the filtrate was dried to obtain E8 as a white foamy solid. MS m/z (ESI): 1024.6 [M/2 + H] +
  • Step 8 E9: E8 (460 mg, 0.225 mmol) was dissolved in DCM (5 mL) and DIEA (58.05 mg, 0.449 mmol) was added. The mixture was stirred at room temperature for 30 min over 4A molecular sieves. Pentafluorophenyl trifluoroacetate (94.35 mg, 0.337 mmol) was added at room temperature and reacted for 3 h at room temperature. The reaction solution was diluted with ice DCM (5 mL), and the organic phase was washed with ice 1M KHSO 4 (5 mL ⁇ 2), ice saturated sodium bicarbonate aqueous solution (5 mL ⁇ 2), and ice saturated brine (5 mL ⁇ 2). The organic phase was dried over anhydrous sodium sulfate, filtered and dried to obtain E9 (0.28 g, yield: 55%) as a white foam solid. MS m/z (ESI): 1107.6 [M/2+H] +
  • Step 1 E2': E1 (4.14 g, 20 mmol) was dissolved in dichloromethane (150 mL), and oxalyl chloride (13.8 g, 109 mmol) was slowly added dropwise under ice bath, and the reaction solution was reacted at 0°C for 5 min, and then returned to room temperature for 30 min. The reaction solution was concentrated to obtain E2' (4.5 g, yield: 99%) as a colorless oil.
  • Step 2 F1: Compound 13 (1.00 g, 1.241 mmol), Cbz-Gly-Val-OH (0.46 g 1.489 mmol) were dissolved in 10 mL DCM, DIEA (0.48 g, 3.722 mmol), HATU (0.75 g, 1.985 mmol) were added and reacted at room temperature for 16 h.
  • Step 3 F2: F1 (600 mg, 0.547 mmol) was dissolved in 5 mL MeOH, 20% Pd(OH) 2 /C (120 mg), CF 3 COOH (187 mg, 1.642 mmol) were added, and the mixture was ventilated with a hydrogen balloon for three times. The mixture was reacted at room temperature for 16 h under hydrogen conditions. The mixture was filtered through diatomaceous earth, the filter cake was washed with methanol (10 mL ⁇ 3), and the trifluoroacetate of F2 was obtained by spin drying. It was used directly in the next step without purification. MS m/z (ESI): 431.9 [M/2+H] +
  • Step 5 F4: F3 (400 mg, 0.347 mmol) was dissolved in TFA (3 mL) and stirred at room temperature for 1 h. The reaction solution was diluted with toluene (5 mL), dried by spin drying, and then diluted with toluene (5 mL ⁇ 3), and dried by spin drying to obtain the trifluoroacetate salt of F4, which was used directly in the next step.
  • Step 6 F5: F4 (295 mg, 0.346 mmol) was dissolved in dichloromethane (5 mL), DIEA (1.78 g, 13.85 mmol) was added, and a solution of compound 5 (658.46 mg, 1.073 mmol) in dichloromethane (3 mL) was added, and the reaction was carried out at room temperature for 18 h.
  • the reaction solution was diluted with dichloromethane (10 mL), and the organic phase was washed with 1M KHSO 4 (10 mL), saturated sodium bicarbonate aqueous solution (10 mL), and water (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and dried to obtain a crude product.
  • Step 7 F6: F5 (300 mg, 0.139 mmol) was dissolved in THF (3 mL), 20% Pd(OH) 2 /C (40 mg), replaced with hydrogen three times, and reacted at room temperature for 4 h under hydrogen protection. Filtered with diatomaceous earth, washed with THF (5 mL ⁇ 3), and the filtrate was dried to obtain F6 as a white foamy solid. MS m/z (ESI): 1026.1 [M/2 + H] +
  • Step 1 G1: Compound 13 (5.0 g, 6.20 mmol), Fmoc-Val-Val-OH (3.25 g 7.44 mmol) were dissolved in 50 mL DCM, DIEA (2.4 g, 18.6 mmol), HATU (3.75 g, 9.92 mmol) were added and reacted at room temperature for 16 h.
  • Step 4 G4: G3 (1.00 g, 0.838 mmol) was dissolved in TFA (4 mL) and stirred at room temperature for 1 h. The reaction solution was diluted with toluene (10 mL), dried by spin drying, and then diluted with toluene (10 mL ⁇ 3), and dried by spin drying to obtain the trifluoroacetate salt of G4, which was used directly in the next step.
  • Step 5 G5: The trifluoroacetate of G4 (0.748 mg, 0.824 mmol) was dissolved in dichloromethane (10 mL), DIEA (4.32 g, 33.46 mmol) was added, and a dichloromethane (5 mL) solution of compound 5 (1.59 g, 2.59 mmol) was added, and the reaction was carried out at room temperature for 2 h. The reaction solution was diluted with dichloromethane (30 mL), and the organic phase was washed with 1M KHSO 4 (30 mL), saturated sodium bicarbonate aqueous solution (30 mL), and water (30 mL).
  • Step 6 G6: G5 (699 mg, 0.033 mmol) was dissolved in THF (10 mL), 20% Pd(OH) 2 /C (35 mg), replaced with hydrogen three times, and reacted overnight at room temperature under hydrogen protection. Filtered with diatomaceous earth, washed with THF (10 mL ⁇ 3), and the filtrate was dried to obtain G6 as a white foamy solid. MS m/z (ESI): 1047.1 [M/2 + H] +
  • Step 7 G7: Compound G6 (1.63 g, 0.779 mmol) was dissolved in DCM (10 mL) and DIEA (0.20 g, 1.558 mmol) was added. TFAPfp (0.33 g, 1.169 mmol) was added at room temperature and reacted at room temperature for 3 h.
  • Step 1 H1: 13 (5.0 g, 6.20 mmol), Fmoc-Val-Leu-OH (3.35 g 7.40 mmol) were dissolved in 50 mL DCM, DIEA (2.4 g, 18.6 mmol), HATU (3.75 g, 9.92 mmol) were added and reacted at room temperature for 16 h.
  • Step 4 H4: H3 (1.0 g, 0.827 mmol) was dissolved in TFA (5 mL) and stirred at room temperature for 1 h. The reaction solution was diluted with toluene (5 mL), dried by spin drying, and then diluted with toluene (5 mL ⁇ 3), and dried by spin drying to obtain the trifluoroacetate salt of H4, which was used directly in the next step.
  • Step 5 H5: The trifluoroacetate of H4 (750 mg, 0.827 mmol) was dissolved in dichloromethane (10 mL), DIEA (4.28 g, 33.08 mmol) was added, and a dichloromethane (5 mL) solution of compound 5 (1.57 g, 2.564 mmol) was added, and the reaction was carried out at room temperature for 18 h.
  • the reaction solution was diluted with dichloromethane (30 mL), and the organic phase was washed with 1M KHSO 4 (30 mL), saturated sodium bicarbonate aqueous solution (30 mL), and water (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and dried to obtain a crude product.
  • Step 6 H6: H5 (691 mg, 0.315 mmol) was dissolved in THF (10 mL), 20% Pd(OH) 2 /C (77 mg), and hydrogen was replaced three times. The reaction was carried out at room temperature under hydrogen protection for 4 h. Filtered with diatomaceous earth, the filter cake was washed with THF (10 mL ⁇ 3), and the filtrate was dried by rotary evaporation to obtain H6 is a white foamy solid. MS m/z(ESI):1054.1[M/2+H] +
  • Step 7 H7: Compound H6 (1.7 g, 0.807 mmol) was dissolved in DCM (10 mL) and DIEA (0.21 g, 1.614 mmol) was added. TFAPfp (0.45 g, 1.614 mmol) was added at room temperature. The reaction was allowed to react overnight at room temperature. The reaction solution was dissolved in DCM (10 mL) and DIEA (0.21 g, 1.614 mmol) was added. TFAPfp (0.45 g, 1.614 mmol) was added at room temperature. The reaction was allowed to react for 4 h at room temperature.
  • reaction solution was diluted with ice DCM (15 mL), and the organic phase was washed with ice 1M KHSO 4 (15 mL ⁇ 3), ice saturated sodium bicarbonate aqueous solution (15 mL ⁇ 3), and ice saturated brine (15 ⁇ 3 mL), dried over anhydrous sodium sulfate, filtered, and dried to obtain a white foam solid.
  • Step 1 I1: Compound 13 (1.00 g, 1.241 mmol), Fmoc-Ala-Ala-OH (0.57 g, 1.48 mmol) were dissolved in 10 mL DCM, DIEA (0.48 g, 3.72 mmol), HATU (0.75 g, 1.985 mmol) were added and reacted at room temperature for 16 h.
  • Step 4 I4: I3 (350 mg, 0.307 mmol) was dissolved in TFA (3 mL) and stirred at room temperature for 1 h. The reaction solution was diluted with toluene (5 mL), dried by spin drying, and then diluted with toluene (5 mL ⁇ 3), and dried by spin drying to obtain the trifluoroacetate salt of I4, which was used directly in the next step. MS m/z (ESI): 419.9 [M//2+H] +
  • Step 5 The trifluoroacetate salt of I4 (257 mg, 0.307 mmol) was dissolved in dichloromethane (5 mL), DIEA (1.56 g, 12.267 mmol) was added, and a dichloromethane (3 mL) solution of compound 5 (583.24 mg, 0.951 mmol) was added, and the reaction was carried out at room temperature for 18 h.
  • the reaction solution was diluted with dichloromethane (10 mL), and the organic phase was washed with 1M KHSO 4 (10 mL), saturated sodium bicarbonate aqueous solution (10 mL), and water (10 mL).
  • Step 6 I6: I5 (267.6 mg, 0.125 mmol) was dissolved in THF (4 mL), 20% Pd(OH) 2 /C (30 mg), replaced with hydrogen three times, and reacted at room temperature for 4 h under hydrogen protection. Filtered with diatomaceous earth, washed with THF (5 mL ⁇ 3), and the filtrate was dried to obtain I6 as a white foamy solid. MS m/z (ESI): 1018.9 [M/2+H] +
  • the sense and antisense chains of ASO and siRNA were solid-phase synthesized by the phosphoramidite method.
  • the 5' end of the sense chain of siRNA can be coupled to the 5' end of the nucleic acid by adding a terminal amino group through 5'-amino TFA (C6), that is, by phosphoramidite coupling, using 5'-Amino-Modifier C6-TFA (monomer with cas number: 133975-85-6).
  • C6-amino TFA 5'-amino TFA
  • 5'-Amino-Modifier C6-TFA monomer with cas number: 133975-85-6.
  • oligonucleotides with an amino group at the 3' end can use 3'-PT Amino-Modifier C6CPG as the starting solid support and undergo solid phase synthesis by the phosphoramidite method. After the solid phase synthesis is completed, the oligonucleotide with an amino group (oligonucleotide with an amino group at the 3' end) is obtained through cleavage, deprotection, purification, etc.
  • Oligonucleotide 1 with an amino group at the 5' end (wherein the sequence is shown in SEQ ID NO.1):
  • Oligonucleotide 2 with an amino group at the 5' end (wherein the sequence is shown in SEQ ID NO.3):
  • Oligonucleotide 1 with an amino group at the 3' end (wherein the sequence is shown in SEQ ID NO.1):
  • Oligonucleotide 2 with an amino group at the 3' end (wherein the sequence is shown in SEQ ID NO.3):
  • m is a methoxy modification at the 2' position of the sugar ring
  • f is a fluorine modification at the 2' position of the sugar ring
  • * is a thio modification of the phosphodiester bond
  • invdA is an inverted deoxyadenosine (3'-3' linked nucleotide).
  • the comparative conjugates PC (without L96) and NC (without L96) were obtained by simulated annealing of the corresponding sense and antisense sequences;
  • m is the methoxy modification of the 2' position of the sugar ring
  • f is the fluorine modification of the 2' position of the sugar ring
  • * is the sulfur of the phosphodiester bond.
  • NC means Negative control
  • PC (3'L96) was prepared according to the method of US Pat. No. 10125369, wherein the structure of 3'L96 is as follows:
  • AMG 890 refers to patent CN116456990A/CN108368506A, and its structural formula is as follows:
  • Dissolve D9 in sodium phosphate buffer add it to the solution of oligonucleotide 1 with an amino group at the 5' end (dissolved in PBS), then add Py activator (pyridine), mix by ultrasound and vortex oscillator until completely dissolved, react at 25°C for 16 hours, then add ammonia water to the conjugate, react at 55°C for 30 minutes to obtain a target product mixture, prepare the conjugate by HPLC, the structural formula is shown in CD1, and then simulate annealing with the corresponding antisense sequence to obtain the target compound ds-CD1.
  • Dissolve D9 in sodium phosphate buffer add it to the solution of oligonucleotide 1 with 3' amino group (dissolved in PBS), then add Py activator, mix by ultrasound and vortex oscillator until completely dissolved, and react at 25°C. 16h, then add ammonia water to the conjugate, react at 55°C for 30min to obtain a target product mixture, and prepare the conjugate by HPLC with the structural formula shown in CD2, and then simulate annealing with the corresponding antisense sequence to obtain the target compound ds-CD2.
  • Compound G7 (10eq, 82mg) was dissolved in DMF (300uL), added to the nucleic acid amino intermediate (25mg, dissolved in 1200uL sodium carbonate buffer), and then mixed by ultrasound and vortex oscillator until completely dissolved. After reacting at 25°C for 16h, Ac deprotection was performed, ammonia water (200uL) was added, and the target product mixture was obtained after reacting at 55°C for 30min.
  • the conjugate DC2 was prepared by HPLC, and then simulated annealing with the corresponding antisense sequence was performed to obtain the target compound ds-DC2.
  • the conjugate molecules were self-delivered into freshly isolated mouse primary hepatocytes, and their in vitro IC50 data were obtained by qPCR.
  • the in vitro experiment used a 24-well plate, and each concentration point of each conjugate had two technical replicates.
  • (A) Conjugate Working solution concentration setting The final concentrations of the free-uptake working solution of the in vitro experimental conjugates (PC (3'L96), PC (5'L96), ds-CA1, ds-CD1) are: 2nM, 1nM, 0.5nM, 0.25nM, 125pM, 62.5pM, 31.3pM, 15.6pM, 7.81pM, 3.91pM, 1.95pM; the final concentrations of the free-uptake working solution of the in vitro experimental conjugates (PC (without L96), NC (without L96)) are: 1nM, 0.5nM, 0.25nM.
  • conjugates were dissolved in DEPC water at a ratio of 50 ⁇ L DEPC H 2 O per OD, and the concentration was measured using Nanodrop 2000. Based on the measurement results, the conjugates were diluted to 266 ng/ ⁇ L, recorded as 20 ⁇ M, as the stock solution and stored at -20°C.
  • (C) Free-uptake sample preparation Add 10 ⁇ L/well of the corresponding concentration of conjugate directly into the cell seeding plate.
  • RNA extraction from cells Lyse cells and use a high-throughput nucleic acid extractor-magnetic bead method to extract total RNA.
  • RNA concentration adjustment Use a nanophotometer to detect sample concentration and add water to adjust all samples to the same concentration.
  • Reverse transcription Use a reverse transcription kit to remove genomic DNA and reverse transcribe all samples into cDNA.
  • Conventional real-time fluorescence quantitative qPCR detection Use a conventional qPCR kit (SYBR GREEN system) to perform relative quantification of cDNA samples. Perform quantitative analysis three times for each cDNA sample and prepare a standard curve at the same time.
  • the in vitro gene inhibition effect shows that in the gene detection results of the control group in PMH, PC (without L96) and NC (without L96) cannot enter the cells to produce effects in the free uptake delivery mode. They have no inhibitory effect at the final concentrations of 1, 0.5, and 0.25nM, and the remaining inhibition efficiency fluctuates around 100%. It serves as a negative QC in the entire test system.
  • the protein detection results of the control group in PMH are consistent with the mRNA detection results.
  • the conjugate was dissolved in DEPC water at a ratio of 50 ⁇ L DEPC H 2 O per OD, and the concentration was measured using Nanodrop 2000. Based on the measurement results, the conjugate was diluted to 2,000 ng/ ⁇ L as a stock solution and stored at -20°C.
  • Animal treatment Animal preparation: After the animals arrived, they were observed and recovered for one week.
  • Drug administration The animals were weighed before administration and administered subcutaneously.
  • Anatomy The animals were killed after 72 hours, and gross dissection was performed. Two 1 cm3 liver tissues were taken from each animal and placed in RNA Later preservation solution and stored at -80°C. Whole blood was collected by EDTA anticoagulation and centrifuged to obtain serum samples, which were stored at -80°C.
  • RNA extraction from tissues Tissue homogenate, use high-throughput nucleic acid extractor-magnetic bead method to extract total RNA.
  • RNA concentration adjustment Use nanophotometer to detect sample concentration, add water to adjust all samples to the same concentration.
  • Reverse transcription Use reverse transcription kit to remove genomic DNA, and reverse transcribe all samples into cDNA.
  • Conventional real-time fluorescence quantitative qPCR detection Use conventional qPCR kit (SYBR GREEN system) to perform relative quantification of cDNA samples. Perform quantitative analysis 3 times for each cDNA sample.
  • the conjugate was dissolved in PBS at a ratio of 50 ⁇ L PBS per OD, and the concentration was measured using Nanodrop 2000. Based on the measurement results, the conjugate was diluted to 2,000 ng/ ⁇ L as a stock solution and stored at -80°C.
  • Animal treatment Animal preparation: After the animals arrive, they will be observed and recovered for one week.
  • Drug administration Weigh the animals before administration and administer 5 ⁇ L/g subcutaneously.
  • Anatomy The animals were killed 72 hours later, and a gross dissection was performed.
  • One blood sample was collected from each animal. After standing at room temperature for 20 to 30 minutes, the serum was centrifuged and divided into approximately 40ul, which was transferred to -80°C for storage for half a year.
  • Two 1cm3 liver tissues were taken from each animal, placed in RNA Later preservation solution, stored at 4°C overnight, and then transferred to -80°C for storage.
  • RNA extraction from tissues Tissue homogenization, add 0.5 ml Trizol and steel beads to the liver tissue, put it into a high-throughput tissue grinder, adjust the parameters to 30HZ, and grind until the liver is completely broken.
  • Total RNA extraction from tissues Tissue homogenization, use a high-throughput nucleic acid extractor-magnetic bead method to extract total RNA.
  • RNA concentration adjustment Use Nanodrop2000 to detect the sample concentration, and add water to adjust all samples to the same concentration.
  • Reverse transcription Use a reverse transcription kit to remove genomic DNA, and reverse transcribe all samples into cDNA.
  • Ordinary real-time fluorescence quantitative qPCR detection Use a conventional qPCR kit (SYBR GREEN system) to perform relative quantification of cDNA samples. Repeat the quantitative analysis for each cDNA sample 3 times.
  • Animal grouping 5-6 week-old Balb/c mice were fed adaptively for 3 days or more after entering the animal facility, and then randomly divided into groups based on body weight, with 6 mice in each group; the day of grouping was defined as D-3.
  • Administration started on D-3, with a single subcutaneous injection at a dose of 3 mpk.
  • LPA expression knockdown analysis The serum of mice was measured using an LPA (abcam) ELISA kit to detect the apo(a) protein level in the serum. Blood was collected on D1 and 30 ⁇ L of serum was separated to detect apo(a) expression.
  • test results are expressed as mean ⁇ standard error (Mean ⁇ SEM).
  • T-Test The independent sample T test (T-Test) was used to compare the two groups of samples, the one-way ANOVA test was used to compare the single variables of multiple groups, and the two-way ANOVA test was used to compare the two variables of multiple groups.
  • the drawing and data analysis software was Graphpad prism 9. P ⁇ 0.05 was considered to be significantly different.
  • the toxic effect of the conjugate on the MHCC97 cell line was detected by PI.
  • the experiment was carried out in a 96-well plate. Each concentration point of each conjugate had two technical replicates. PC, NC and MOCK were set for each plate of cell screening to calibrate the consistency between different plates.
  • the final concentration of the conjugate working solution and negative and positive controls was: 100 nM.
  • the conjugate was dissolved in DEPC water at a ratio of 50 ⁇ L DEPC H 2 O per OD, and the concentration was measured using Nanodrop 2000. Based on the measurement results, the conjugate was diluted to 2,000 ng/ ⁇ L as a stock solution and stored at -80°C.
  • Cell seeding 150 ⁇ L cell suspension/well, inoculated in 96-well plate, MHCC97 cell number: 4*10 3 cells/well. Cells were cultured in a 37°C, 5% CO 2 incubator for 24 hours before transfection.
  • Transfection Disperse 5 ⁇ L of diluted conjugate in 20 ⁇ L Opti-MEM, and disperse 0.2 ⁇ L RNAiMAX in 24.8 ⁇ L Opti-MEM. Incubate for 5 minutes, mix with the conjugate dispersion, incubate for 10 minutes to obtain the transfection complex, and add the cells to the transfection complex for transfection.
  • PI staining 48 hours after cell transfection, remove the original culture medium and add 200 ⁇ L PBS to each well for washing; mix 1 mg/mL PI with PBS to a final concentration of 10 ⁇ g/mL, add 100 ⁇ L of the mixture to each cell well, place the culture plate in the incubator and incubate for 20 minutes, then take pictures with a fluorescence microscope.

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Abstract

一种配体-寡核苷酸共轭物及用于该共轭物的接头，所述接头包含式I或II的结构：-NR 1-C(O)-[C(R 3R 4)] n-C(O)-[NR 2-C(R aR b)-C(O)] p-（式I），或-NR 1-[C(O)-C(R aR b)-NR 2] p-C(O)-[C(R 3R 4)] n-C(O)-（式II）。该共轭物的制备方法及中间体。

Description

配体-寡核苷酸共轭物及用于该共轭物的接头
本申请要求于2022年10月21日提交中国专利局、申请号为202211296452.X、申请名称为“配体-寡核苷酸共轭物及用于该共轭物的接头”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及配体-寡核苷酸共轭物及其接头，特别是GalNAc-siRNA共轭物及其接头。本发明还涉及配体-寡核苷酸共轭物的制备方法及接头的用途。
背景技术
RNA干扰(RNAi)是指通过利用RNA诱导的沉默复合物(RISC)的机制实现的反义介导的基因沉默。基于RNAi的潜在药物分子包括双链RNA，如siRNA和shRNA。尽管siRNA的作用机制表明其在临床应用及制药行业中有着巨大的应用前景，但其在体内的递送仍然面临着许多挑战。裸露的siRNA被注射进入体内后会在短时间内被血浆中或组织中的核酶降解。此外，肾脏快速清除缩短了siRNA分子在血液循环中的半衰期。游离的siRNA相对分子质量较大(13000～15000)，且含有大量的负电荷，难以自行穿越细胞膜并在胞浆内发挥RNAi效应。另外，血液中游离的siRNA分子的免疫原性也是限制其临床应用的主要原因之一，使其在体内循环过程中引起严重的免疫反应。
对siRNA序列的碱基骨架结构上基团进行化学修饰可以显著增强其在血液循环中的抗酶解稳定性；然而，化学稳定性的提高未能解决siRNA不易穿透细胞膜屏障的问题。Alnylam制药公司的Monoharan团队将GalNAc以三价态的方式共价偶联到siRNA的正义链3'末端得到GalNAc-siRNA共轭物。完整的GalNAc-siRNA共轭物可通过肝细胞表面高表达的ASGPR介导的网格蛋白参与的细胞内吞作用进入细胞内环境。随着内涵体中的pH下降，GalNAc-siRNA共轭物从结合的ASPGR中释放出来。随后，ASGPR循环回到细胞表面；而GalNAc-siRNA共轭物保留在内涵体中。然而，GalNAc-siRNA偶联物从内涵体内释放的机制并不明确。绝大多数游离siRNA仍被捕获在内涵体中，而极少量(<1％)能够通过未知机制穿过内涵体脂质双层膜进入细胞质并诱导RNAi反应。
除了siRNA，GalNAc也被用于将反义寡核苷酸(ASO)递送至肝细胞。Prakash等证实，利用三价GalNAc递送二代gapmer反义寡核苷酸使得其药效增强了6-10倍。当将其与下一代ASO设计组合时，药效增强约60倍，显示出强大的肝脏靶向递送和药效增强能力(Prakash et al.,Nucleic Acids Research,2014,Vol.42,No.13,8796–8807)。
目前，临床递送寡核苷酸的方式单一且有限，仍有必要尝试更多的配体-寡核苷酸共轭物结构以提高共轭物的一个或多个方面的性能，并最终提高共轭物的体内治疗效果。
发明内容
本发明的一个方面提供一种接头部分，所述接头部分如式I或II所示：
-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I)，或
-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II)，
其中各取代基、符号具有如本文所定义的含义。
本发明的另一个方面提供一种配体-寡核苷酸共轭物，其包含本发明的接头部分。
本发明的另一个方面提供式III或IV所示的中间体化合物：
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-Q2(式III)，或
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-Q2(式IV)，
其中各取代基、符号具有如本文所定义的含义。
本发明的另一个方面提供上述中间体化合物在制备配体-寡核苷酸共轭物中的用途。
本发明的另一个方面提供制备配体-寡核苷酸共轭物的方法，其包括：提供上述任一中间体化合物；提供5’或3’端具有末端氨基的寡核苷酸；以及将所述中间体化合物与所述寡核苷酸通过所述末端氨基共价连接。
本发明的另一个方面提供式I或II所示的接头：
-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I)，或
-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II)，
其中各取代基、符号具有如本文所定义的含义。
本发明的另一个方面提供式I’或II’所示的连接单元：
-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I’)，或
-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II’)，
其中各取代基、符号具有如本文所定义的含义；
式I’的左侧端连接-NR1-即为式I，式II’的左侧端连接-NR1-即为式II。
本发明的另一个方面提供上述接头在制备配体-寡核苷酸共轭物中的用途。
本发明的另一个方面提供一种药物组合物，其包含本发明的任一种配体-寡核苷酸共轭物，以及药学上可接受的载体。
本发明的另一个方面提供一种治疗疾病的方法，其包括向受试者施用治疗有效量的本文提供的任一种共轭物或包含该共轭物的药物组合物。
本发明的其他方面将从以下说明书的详细描述中看出。
具体实施方式
定义
术语“寡核苷酸”在本文中是指聚脱氧核糖核苷酸、聚核糖核苷酸或聚核糖核苷酸/脱氧核糖核苷酸杂合体，长度通常为15至30个核苷酸或核苷酸对，各个核苷酸可为天然的或经修饰的。寡核苷酸可以是双链或单链，包括单链和双链DNA、单链和双链RNA(包括shRNA、siRNA)以及DNA/RNA杂合分子。在本发明的一些实施方式中，寡核苷酸可以共轭至另一种分子，例如N-乙酰基半乳糖胺(GalNAc)或其多聚体(GalNAc簇)。在优选的实施例中，用于本发明的寡核苷酸选自siRNA、shRNA、miRNA和ASO。
术语“经修饰的核苷酸”是指独立地具有修饰的糖部分、经修饰的核苷酸间键合和/或经修饰的核碱基的核苷酸。因此，术语经修饰的核苷酸包括对核苷间键合、糖部分或核碱基的例如官能团或原子的取代、添加或去除。适用于本发明的修饰包括本文公开的或本领域已知的所有类型的修饰。例如，所述经修饰的核苷酸是脱氧核苷酸、3'末端脱氧胸腺嘧啶(dT)核苷酸、2'-O-甲基修饰的核苷酸、2'-氟修饰的核苷酸、2'-脱氧修饰的核苷酸、锁核苷酸、未锁核苷酸、构型受限核苷酸、受限乙基核苷酸、无碱基核苷酸、2'-氨基修饰的核苷酸、2'-O-烯丙基修饰的核苷酸、2'-C-烷基修饰的核苷酸、2'-羟基修饰的核苷酸、2'-甲氧基乙基修饰的核苷酸、2'-O-烷基修饰的核苷酸、吗啉基核苷酸、氨基磷酸酯、包含核苷酸的非天然碱基、四氢吡喃修饰的核苷酸、1,5-脱水己糖醇修饰的核苷酸、环己烯基修饰的核苷酸、包含5'-硫代磷酸酯基团的核苷酸、包含5'-磷酸甲酯基团的核苷酸、包含5'磷酸酯或5'的核苷酸磷酸盐类似物的核苷酸、包含乙烯基磷酸酯的核苷酸、包含腺苷-乙二醇核酸(GNA)的核苷酸、包含胸苷-乙二醇核酸(GNA)S-异构体的核苷酸、包含2-羟甲基-四氢呋喃-5-磷酸的核苷酸、包含2'-脱氧胸苷-3'磷酸酯的核苷酸、包含2'-脱氧鸟苷-3'-磷酸酯的核苷酸，或连接至胆固醇基衍生物和/或十二烷酸双癸酰氨基团的末端核苷酸。在优选的实施例中，所述经修饰的核苷酸是2'-脱氧-2'-氟修饰的核苷酸、2'-脱氧-修饰的核苷酸、3'末端脱氧胸腺嘧啶核苷酸(dT)、锁核苷酸、无碱基核苷酸、2'-氨基修饰的核苷酸、2'-烷基修饰的核苷酸、吗啉基核苷酸、氨基磷酸酯和/或包含核苷酸的非天然碱基。
反义寡核苷酸(ASO)是与所选序列互补的单链DNA或RNA。在反义DNA的情况下，可用于靶向特定的互补(编码或非编码)RNA。如果发生结合，这种杂合体可以被RNAase H1降解。ASO的一个典型例子是gapmer，其具有内部“缺口区段”，其侧接两个外部“翼区段”，其中缺口区段由多个支持RNA酶H切割的核苷酸组成，每个翼区段由一个或多个在化 学上不同于缺口区段内的核苷酸的核苷酸组成。例如，gapmer的5’和3’翼区段由2’-MOE修饰的核苷酸组成，缺口区段由脱氧核糖核苷酸组成，并且任选地在所有核苷酸之间的连接是硫代磷酸酯键。
“siRNA”指形成双链RNA的核酸，当siRNA与靶基因在同一细胞中存在时该双链RNA具有降低或抑制靶基因表达的能力。siRNA通常为约15至约30个碱基对长度，最通常为约19至25个碱基对长度，例如19、20、21、22、23、24或25个核苷酸对长度。例如，约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或36个碱基对的长度，例如约15至30、15至29、15至28、15至27、15至26、15至25、15至24、15至23、15至22、15至21、15至20、15至19、15至18、15至17、18至30、18至29、18至28、18至27、18至26、18至25、18至24、18至23、18至22、18至21、18至20、19至30、19至29、19至28、19至27、19至26、19至25、19至24、19至23、19至22、19至21、19至20、20至30、20至29、20至28、20至27、20至26、20至25、20至24、20至23、20至22、20至21、21至30、21至29、21至28、21至27、21至26、21至25、21至24、21至23或21至22个碱基对的长度。
shRNA指短发夹RNA，其包括两个短反向重复序列和连接两者的中间茎环结构。茎环可包含至少一个未配对的核苷酸，例如，可包含至少2个、至少3个、至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个、至少20个、至少23个或更多未配对的核苷酸。在一些实施例中，茎环可以是10个或更少的核苷酸。在一些实施例中，茎环可以是8个或更少的未配对核苷酸。在一些实施例中，茎环可以是4至10个未配对的核苷酸。在一些实施例中，茎环可为4至8个核苷酸。
siRNA和shRNA在本文有时也统称为双链RNA(dsRNA)。dsRNA的两条基本上互补的链不需要但也可以共价连接。碱基对的最大数量是dsRNA最短链中的核苷酸数量减去双链体中存在的任何突出端。除了双链体结构之外，dsRNA还可以包含一个或多个核苷酸突出端。例如，至少一条链包含至少1个核苷酸的3'突出端，例如，2、3、4、5、6、7、9、10、11、12、13、14或15个核苷酸。又例如，至少一条链包含至少1个核苷酸的5'突出端，例如2、3、4、5、6、7、9、10、11、12、13、14或15个核苷酸。在其他实施例中，dsRNA的一条链的3’末端和5’末端均包含至少1个核苷酸的突出端。
如本文所用，术语“核苷酸突出端”是指从dsRNA的双链体结构突出的至少一个未配对核苷酸。例如，当dsRNA一条链的3'末端延伸超出另一条链的5'末端时，反之亦然，则存 在核苷酸突出端。核苷酸突出端可包含核苷酸/核苷类似物或由其组成，包括脱氧核苷酸/核苷。突出端可以在正义链、反义链或其任何组合上。此外，突出端的核苷酸可存在于dsRNA的反义链或正义链的5’末端、3’末端或两端。
如本文所用，关于dsRNA的术语“平端”或“平末端”是指在dsRNA的给定末端没有未配对的核苷酸或核苷酸类似物，即没有核苷酸突出端。dsRNA的一端或两端可以是平的。如果dsRNA的两端都是平端，则称所述dsRNA是平端的。需要明确的是，“平端”dsRNA是两端都是平端的dsRNA，即分子的任一端都没有核苷酸突出端。大多数情况下，这样的分子在其整个长度上都是双链的。
术语“反义链”是指dsRNA中包含与靶序列基本互补的区域的链。术语“正义链”是指dsRNA中包含与本文定义的反义链区域基本互补的区域的链。术语“基本互补的区域”是指完全互补或不完全互补的区域。当互补的区域与靶序列不完全互补时，错配可能位于分子的内部或末端区域。通常，最可容忍的错配位于末端区域，例如在dsRNA的5'-和/或3'末端的5、4、3或2个。
术语“配体”是指与指定的细胞类型(如肝细胞)结合的细胞或组织靶向剂，例如凝集素、糖蛋白、脂质或蛋白质(例如抗体)。示例性的靶向剂包括促甲状腺激素、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(GalNAc)、多价(如二价或三价)GalNAc、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、胆固醇、类固醇、胆汁酸、叶酸盐、维生素B12、生物素、RGD肽和RGD肽模拟物。在优选的实施例中，该配体是一种碳水化合物，例如单糖、二糖、三糖、四糖、多糖。例如，该配体可以是包含GalNAc的衍生物。在优选的实施例中，配体是包含通过二价或三价支链接头附接的一种或多种N-乙酰半乳糖胺衍生物。
术语“氨基酸”是指同时含有氨基和羧基的分子。合适的氨基酸包括但不限于天然存在的氨基酸的D-和L-异构体，以及通过有机合成或其它代谢途径制备的非天然存在的氨基酸。本文所用的术语氨基酸包括但不限于α-氨基酸、天然氨基酸、非天然氨基酸和氨基酸类似物。术语“α-氨基酸”是指含有的氨基和羧基都结合到被指定为α-碳的碳上的分子。术语“天然存在的氨基酸”是指在自然界合成的肽中通常发现的20种氨基酸中的任一种，已知其单字母缩写为A、R、N、C、D、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y和V。“疏水性氨基酸”包括小疏水性氨基酸和大疏水性氨基酸。“小疏水性氨基酸”为甘氨酸、丙氨酸、脯氨酸及其类似物。“大疏水性氨基酸”为缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、甲硫氨酸、 色氨酸及其类似物。“极性氨基酸”为丝氨酸、苏氨酸、天冬酰胺、谷氨酰胺、半胱氨酸、酪氨酸及其类似物。“带电氨基酸”为赖氨酸、精氨酸、组氨酸、天冬氨酸、谷氨酸及其类似物。
术语“氨基酸类似物”是指在结构上类似于氨基酸并且在拟肽大环化合物的形成中可代替氨基酸的分子。氨基酸类似物包括但不限于β-氨基酸和其中氨基或羧基被相似反应性的基团取代(例如，用仲胺或叔胺取代伯胺，或用酯取代羧基)的氨基酸。术语“β-氨基酸”是指含有均为β构型的氨基和羧基的分子。
术语“非天然氨基酸”是指并非在自然界合成的肽中通常发现的二十种氨基酸之一的氨基酸。如本文所用，术语“非天然氨基酸”包括天然氨基酸的α-取代和α-双取代的氨基酸、N-烷基氨基酸、乳酸、卤化物衍生物，如三氟酪氨酸、对-氯-苯丙氨酸、对-氟-苯丙氨酸、对-溴-苯丙氨酸、p-NO2-苯丙氨酸、苯基甘氨酸、肌氨酸、青霉胺、D-2-甲基色氨酸、磷酸丝氨酸、磷酸苏氨酸、磷酸酪氨酸、p-I-苯丙氨酸、L-烯丙基-甘氨酸、β-丙氨酸、β-天冬氨酸、β-环己基丙氨酸、瓜氨酸、高丝氨酸、高半胱氨酸、焦谷氨酸、L-α-氨基丁酸、L-γ-氨基丁酸、L-α-氨基异丁酸、α-环己基甘氨酸、二氨基丁酸、二氨基庚二酸、N-ε-二硝基苯基-赖氨酸、L-1-萘基丙氨酸、L-2-萘基丙氨酸、3-(2-吡啶基)-L-丙氨酸、3-(3-吡啶基)-L-丙氨酸、3-(4-吡啶基)-L-丙氨酸、N-ε-甲基-赖氨酸、N,N-ε-二甲基-赖氨酸、N,N,N-ε-三甲基-赖氨酸、3-巯基丙酸、L-ε-氨基已酸、7-氨基庚酸、6-氨基己酸、L-蛋氨酸砜、鸟氨酸、L-正亮氨酸、L-正缬氨酸、p-硝基-L-苯丙氨酸、L-羟基脯氨酸、γ-谷氨酸、γ-氨基丁酸、L-巯基脯氨酸、苯丙氨酸(Phe)的甲基衍生物(如4-甲基-Phe、五甲基-Phe、L-Phe(4-氨基)、L-Tyr(甲基)、L-Phe(4-异丙基)、L-Tic(1,2,3,4-四氢异喹啉-3-羧酸)、L-二氨基丙酸和L-Phe(4-苄基))。
术语“氨基酸侧链”是指连接到天然或非天然氨基酸中的α-碳上的部分。例如，丙氨酸的氨基酸侧链是甲基，苯丙氨酸的氨基酸侧链是苯基亚甲基，半胱氨酸的氨基酸侧链是巯基亚甲基，天冬氨酸的氨基酸侧链是羧基亚甲基，酪氨酸的氨基酸侧链是4-羟基苯基亚甲基，等等。也包括其它非天然存在的氨基酸侧链，例如，自然产生的氨基酸侧链(例如，氨基酸代谢物)或合成制备的氨基酸侧链(例如瓜氨酸侧链)。无论是天然还是非天然氨基酸，均包括其L和D型构型，相应地，其侧链亦预期包括L和D型的侧链。
术语“羧基保护基团”表示旨在保护羧基的基团，选自甲基、取代的甲基类、乙基、2-取代的乙基类、烯丙基、叔丁基、烷氧基烷基类、烷氧基烷氧基烷基类、2,6-二烷基苯基类、苄基、取代的苄基类、硅烷基类或锡烷基类；所述取代的甲基类选自9-芴基甲基、三异丙基硅甲基、环丙基甲基、二苯基甲基或三苯基甲基；所述2-取代的乙基类选自2,2,2-三氯乙 基、2-(三甲基硅烷基)乙基、2-(对甲苯磺酰基)乙基或2-氰基乙基；所述烷氧基烷基类选自甲氧基甲基、苄氧基甲基或三异丙基硅氧基甲基；所述烷氧基烷氧基烷基类选自甲氧基乙氧基甲基；所述2,6-二烷基苯基类选自2,6-二甲基苯基、2,6-二异丙基苯基或2,6-二叔丁基-4-甲氧基苯基；所述取代的苄基类选自对甲基苄基、2,4-二甲氧基苄基、2,6-二甲氧基苄基、对硝基苄基或邻硝基苄基；所述硅烷基类选自三甲基甲硅烷基、三乙基甲硅烷基、三异丙基甲硅烷基或苯基二甲基甲硅烷基；所述锡烷基类选自三甲基甲锡烷基；优选甲基、烯丙基、叔丁基、苄基、2,4-二甲氧基苄基、对甲基苄基、五氟代苯基或甲氧乙氧甲基。
术语“卤代”或“卤素”是指氟、氯、溴或碘或其基团。当未限定卤代数量时，可以是任何合适的数量，例如一卤代、二卤代、三卤代；当未限定卤代位置时，可以是任何合适的位置，例如卤代苯基可以是在邻位、对位、间位或其组合的卤代。
术语“烷基”是指饱和的直链或支链烃链。对于具有特定数量的碳原子的烷基，该术语包含对应的正烷基及其各种异构体形式(如有)。例如，具有4个碳原子的烷基(C4烷基)包括正丁基、异丁基、仲丁基和叔丁基。示例性的C1-10烷基包括甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、戊基、异戊基、新戊基、1-乙基丙基、己基、异己基、1,1-二甲基丁基、2,2-二甲基丁基、3,3-二甲基丁基、2-乙基丁基、4,4-二甲基戊基，5,5-二甲基己基、6,6-二甲基庚基等。
术语“烯基”是指具有一个或多个碳-碳双键的直链或支链烃链。示例性的C2-10烯基包括乙烯基、1-丙烯基、2-丙烯基、2-甲基-1-丙烯基、1-丁烯基、2-丁烯基、3-丁烯基、3-甲基-2-丁烯基、1-戊烯基、2-戊烯基、3-戊烯基、4-戊烯基、4-甲基-3-戊烯基、1-己烯基、3-己烯基、5-己烯基等。
术语“炔基”是指具有一个或多个碳-碳叁键的直链或支链烃链。示例性的C2-10炔基包括乙炔基、1-丙炔基、2-丙炔基、1-丁炔基、2-丁炔基、3-丁炔基、1-戊炔基、2-戊炔基、3-戊炔基、4-戊炔基、1-己炔基、2-己炔基、3-己炔基、4-己炔基、5-己炔基、4-甲基-2-戊炔基等。
术语“烷氧基”是指烷基-O-基，其中烷基如上所定义。“C1-10烷氧基”的实例包括甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、仲丁氧基、叔丁氧基、戊基氧基、己基氧基等。当被卤代时，烷氧基可被1-7个，优选1-5个卤素原子取代，具体实例包括二氟甲氧基、三氟甲氧基、2,2,2-三氟乙氧基、4,4,4-三氟丁氧基等。
术语“环烷基”是指环状饱和烃基。C3-10环烷基的实例包括环丙基、环丁基、环戊基、环己基、环庚基、环辛基、二环[2.2.1]庚基、二环[2.2.2]辛基、二环[3.2.1]辛基和金刚烷基。
术语“芳基”是指芳族的单碳环和多碳环的环系，其中在多环系中的各个碳环稠合或通过单键彼此连接。合适的芳基包括苯基、萘基、2,3-二氢-1H-茚基和联苯基。6至14元芳基是指6碳单环、10碳双环、12碳联苯基或14碳三环的芳香环系。
术语“杂芳基”是指芳香族的5-8元单环、8-12元双环或11-14元三环的环系，其如果是单环则具有1-3个杂原子，如果是双环则具有1-6个杂原子，或者如果是三环则具有1-9个杂原子，所述杂原子选自O、N或S(例如，如果是单环、双环或三环，分别为碳原子和1-3、1-6或1-9个O、N或S杂原子)，其中各个环的0、1、2、3或4个原子被取代基取代。杂芳基的例子包括吡啶基、呋喃基、咪唑基、苯并咪唑基、嘧啶基、噻吩基、喹啉基、吲哚基、噻唑基等。
术语“杂环基”是指非芳香族的单环、双环或三环的环系。因此，5至14元杂环基是指5-8元单环、8-12元双环或11-14元三环的环系，其如果是单环则具有1-3个杂原子，如果是双环则具有1-6个杂原子，或者如果是三环则具有1-9个杂原子，所述杂原子选自O、N或S(例如，如果是单环、双环或三环，分别为碳原子和1-3、1-6或1-9个O、N或S杂原子)或其他合适的杂原子(如P或Si)，其中各个环的0、1、2或3个原子被取代基取代。5至14元杂环基的例子包括哌嗪基、吡咯烷基、二氧杂环己基、吗啉基、四氢呋喃基、四氢吡咯烷基等。
术语“烷基硫基”是指巯基的氢原子被烷基取代的巯基。例如，C1-3烷基硫基是指甲基硫基、乙基硫基、丙基硫基和异丙基硫基。
术语“烷基膦酰基”是指膦酰基的一个或两个羟基被烷基取代的膦酰基。例如，C1-3烷基膦酰基的例子包括甲基膦酰基、乙基磷酰基、二甲基磷酰基、二乙基磷酰基、甲基乙基磷酰基等。
术语“烷基膦酰氧基”是指烷基磷酰基-O-基，其中烷基磷酰基如上所定义。例如，C1-3烷基膦酰氧基的例子包括甲基膦酰氧基、乙基磷酰氧基、二甲基磷酰氧基、二乙基磷酰氧基、甲基乙基磷酰氧基等。
术语“芳基烷基”是指被芳基取代的烷基，术语“烷基芳基”是指被烷基取代的芳基，其中烷基和芳基如以上所定义。示例性的芳基烷基是苯基C1-3烷基、萘基C1-3烷基、联苯基C1- 6烷基等。
术语“杂芳基烷基”是指被杂芳基取代的烷基，术语“烷基杂芳基”是指被烷基取代的杂芳基，其中烷基和杂芳基如以上所定义。示例性的杂芳基烷基包括吡啶基C1-3烷基、吲哚基C1-3烷基、喹啉基C1-3烷基、异喹啉基C1-3烷基、噻吩基C1-3烷基、噻茚基C1-3烷基、噻 唑基C1-3烷基、苯并噻唑基C1-3烷基、咪唑基C1-3烷基、嘧啶基C1-3烷基等。
术语“杂环基烷基”是指被杂环基取代的烷基，术语“烷基杂环基”是指被烷基取代的杂环基，其中杂环基是指和烷基如以上所定义。示例性的杂环基烷基包括呋喃基C1-3烷基、哌嗪基C1-3烷基、吡咯烷基C1-3烷基、二氧杂环己基C1-3烷基、吗啉基C1-3烷基、四氢呋喃基C1-3烷基、四氢吡咯烷基C1-3烷基。
术语“TMSOTf”是指三氟甲磺酸三甲基硅酯。
术语“DCM”是指二氯甲烷。
术语“DIEA”是指二异丙基乙胺。
术语“EA”是指乙酸乙酯。
术语“THF”是指四氢呋喃。
术语“DMF”是指N,N-二甲基甲酰胺。
术语“HATU”是指2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯。
术语“PE”是指石油醚。
术语“TFAPfp”是指三氟乙酸五氟苯酯。
术语“TFA”是指三氟乙酸。
术语“PI”是指碘化丙啶。
在一些实施方式中，本文公开的化合物包含一个或多个不对称中心，因而作为外消旋体或外消旋混合物、单一对映异构体、单独的非对映异构体和非对映体混合物存在。除非另外明确地指出，本文意在包括这些化合物的所有这样的异构体形式。在一些实施方案中，本文公开的化合物也呈现为多种互变异构形式，在这些情况下，所述化合物包括本文所述化合物的所有互变异构形式(例如，如果环系的烷基化作用导致在多个位置发生烷基化，那么本发明包括所有这些反应产物)。除非另外明确地指出，本文意在包括所述化合物的所有晶形。
术语“治疗有效量”指一种本发明的共轭物或其组合物的在适用于任何医学治疗的合理效益/风险比下在动物中的细胞的至少一个亚群中有效产生某些所希望的治疗效果的量。
术语“药学上可接受的”指位于正确医学判断的范围内、适于与人类和动物的组织接触而无过量毒性、刺激、过敏反应或其他问题或并发症、与一个合理效益/风险比相称的那些化合物、材料、组合物和/或剂型。
术语“药学上可接受的载体”是指参与将所述共轭物从身体的一个器官或部位携载或运载到身体的另一个器官或部位的一种药学上可接受的材料、组合物或运载体，如一种液体或固体填充剂、稀释剂、赋形剂、制造助剂或溶剂包封材料。每种载体必须在与该组合物的 其他成分相容并且对患者无害的意义上是“可接受的”。
术语“治疗”涵盖预防、治疗以及治愈。接受这一治疗的患者通常是任何有需要的动物，包括灵长类动物(特别是人类)以及其他哺乳动物，如马、牛、猪、羊、家禽和宠物。
配体-寡核苷酸共轭物
在一个方面，本文提供了配体-寡核苷酸共轭物，其包含式I或式II所示的接头部分：
-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I)，
-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II)，
其中：
R1、R2、R3、R4独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3- 10环烷基、6至14元芳基、5至18元杂芳基、5至14元杂环基、卤素、羟基、氨基、胍基、羧基、氰基、硝基和巯基，它们任选地被R5取代，其中R5选自C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-10环烷基、6至14元芳基、5至18元杂芳基、5至14元杂环基、卤素、羟基、氨基、胍基、羧基、氰基、硝基、C1-3烷基硫基和巯基；
Ra、Rb独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-8环烷基、6至14元芳基C1-3烷基、5至18元杂芳基C1-3烷基和5至14元杂环基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、C1-3烷基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、吡咯基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、C1-10烷基、C1-10烷氧基、6至14元芳基、C3-8环烷基、金刚烷基、膦酰基、膦酰氧基、C1-3烷基膦酰基、C1-3烷基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成3至14元含氮杂环基，或者Ra、Rb与其共同连接的C原子一起形成C3-8环烷基；
杂原子选自氮、氧、硫、磷和硅；
n为1至10，并且每个重复单元相同或不同，例如n可为1、2、3、4、5、6、7、8、9或10；
p为2至6，并且每个重复单元相同或不同，例如p可为2、3、4、5或6。
在优选的实施方式中，配体-寡核苷酸共轭物包含式I或II所示的接头部分，其中Ra、Rb独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-8环烷基、苯基C1- 3烷基、萘基C1-3烷基、5至12元杂芳基C1-3烷基和5至14元杂环基C1-3烷基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、C1-3烷基硫基、吲哚基、喹 啉基、异喹啉基、酰胺基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、哌嗪基、吡咯烷基、二氧杂环己基、吗啉基、四氢呋喃基、四氢吡咯烷基、C1-10烷基、C1-10烷氧基、苯基、苯甲基、苯乙基、氨基苯基、羧基苯基、卤代苯基、联苯基、C3-8环烷基、金刚烷基、膦酰基、膦酰氧基、C1-3烷基膦酰基、C1-3烷基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成3至14元含氮杂环基；R1、R2、R3、R4、杂原子、n和p如以上所定义。
在优选的实施方式中，配体-寡核苷酸共轭物包含式I或II所示的接头部分，其中Ra、Rb独立地选自-H、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、C3-6环烷基、苯基C1-3烷基、吡啶基C1-3烷基、吲哚基C1-3烷基、喹啉基C1-3烷基、异喹啉基C1-3烷基、噻吩基C1- 3烷基、噻茚基C1-3烷基、噻唑基C1-3烷基、苯并噻唑基C1-3烷基、咪唑基C1-3烷基、呋喃基C1-3烷基、嘧啶基C1-3烷基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、C1-3烷基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、哌嗪基、吡咯烷基、二氧杂环己基、吗啉基、四氢呋喃基、四氢吡咯烷基、吡啶基、C1-10烷基、C1-10烷氧基、苯基、苯甲基、苯乙基、氨基苯基、羧基苯基、卤代苯基、联苯基、C3-8环烷基、金刚烷基、膦酰基、膦酰氧基、C1-3烷基膦酰基、C1-3烷基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成3至14元含氮杂环基；R1、R2、R3、R4、杂原子、n和p如以上所定义。
在更优选的实施方式中，配体-寡核苷酸共轭物包含式I或II所示的接头部分，其中Ra、Rb独立地选自-H、甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、乙烯基、丙烯基、1-丁烯基、2-丁烯基、异丁烯基、乙炔基、丙炔基、1-丁炔基、2-丁炔基、3-甲基-2-丙炔基、1,3-丁二烯基、甲氧基、乙氧基、丙氧基、丁氧基、环丙基、环丁基、环戊基、环己基、苯基亚甲基、苯基二亚甲基、吡啶基亚甲基、吡啶基二亚甲基、吲哚基亚甲基、吲哚基二亚甲基、喹啉基亚甲基、喹啉基二亚甲基、异喹啉基亚甲基、异喹啉基二亚甲基、噻吩基亚甲基、噻吩基二亚甲基、噻茚基亚甲基、噻茚基二亚甲基、噻唑基亚甲基、噻唑基二亚甲基、苯并噻唑基亚甲基、苯并噻唑基二亚甲基、咪唑基亚甲基、咪唑基二亚甲基、呋喃基亚甲基、呋喃基二亚甲基、嘧啶基亚甲基和嘧啶基二亚甲基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、亚甲基硫基、二亚甲基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、哌嗪基、吡咯烷基、二氧杂环己基、吗啉基、四氢呋喃基、四氢吡咯烷基、吡啶基、甲基、乙基、丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、苯基、苯甲基、苯 乙基、氨基苯基、羧基苯基、卤代苯基、联苯基、环丙基、环丁基、环戊基、环己基、金刚烷基、膦酰基、膦酰氧基、二甲基膦酰基、二乙基膦酰基、二甲基膦酰氧基和二乙基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成咪唑基、噻唑基、吡唑基、嘧啶基、吲哚基、喹啉基、异喹啉基或吡咯基；R1、R2、R3、R4、杂原子、n和p如以上所定义。
在更优选的实施方式中，配体-寡核苷酸共轭物包含式I或II所示的接头部分，其中Ra是-H，Rb选自-H、甲基、乙基、丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、环丙基、环戊基、环己基、环己基二亚甲基、环己基亚甲基、环丁基亚甲基、苯基亚甲基、甲基硫基亚甲基、3-吲哚基亚甲基、羟基亚甲基、酰胺基二亚甲基、羟基甲基次甲基、巯基亚甲基、酰胺基亚甲基、羟基苯基亚甲基、羟基苯基二亚甲基、羧基亚甲基、羧基二亚甲基、氨基正丁基、胍基二亚甲基、胍基三亚甲基、4-咪唑基亚甲基、异丙基苯基亚甲基、1-萘基亚甲基、2-萘基亚甲基、茚满基、苯基二亚甲基、氟代苯基亚甲基、氯代苯基亚甲基、三氟苯基亚甲基、二氯苯基亚甲基、五氟苯基亚甲基、二氟苯基亚甲基、噻吩基亚甲基、噻茚基亚甲基、喹啉基亚甲基、卤代3-吲哚基亚甲基、二苯基亚甲基、3-吡啶基亚甲基和4-吡啶基亚甲基，或者Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基；R1、R2、R3、
R4、杂原子、n和p如以上所定义。
在一些实施方式中，配体-寡核苷酸共轭物包含式I或II所示的接头部分，其中Ra和Rb中的一个是-H，并且Ra和Rb中的另一个是氨基酸侧链。所述氨基酸包括天然氨基酸和非天然氨基酸，包括D和L构型。
在Ra和Rb中的一个是-H，并且Ra和Rb中的另一个是氨基酸侧链的实施方式中，该氨基酸是天然或非天然氨基酸，其中非天然氨基酸包括但不限于以下结构：



其他非天然氨基酸包括但不限于β-氨基酸类似物。β-氨基酸类似物的实例包括但不限以下：环状β-氨基酸类似物；β-丙氨酸；(R)-β-苯丙氨酸；(R)-1,2,3,4-四氢-异喹啉-3-乙酸；(R)-3-氨基-4-(1-萘基)-丁酸；(R)-3-氨基-4-(2,4-二氯苯基)丁酸；(R)-3-氨基-4-(2-氯苯基)-丁酸；(R)-3-氨基-4-(2-氰基苯基)-丁酸；(R)-3-氨基-4-(2-氟苯基)-丁酸；(R)-3-氨基-4-(2-呋喃基)-丁酸；(R)-3-氨基-4-(2-甲苯基)-丁酸；(R)-3-氨基-4-(2-萘基)-丁酸；(R)-3-氨基-4-(2-噻吩基)-丁酸；(R)-3-氨基-4-(2-三氟甲苯基)-丁酸；(R)-3-氨基-4-(3,4-二氯苯基)丁酸；(R)-3-氨基-4-(3,4-二氟苯基)丁酸；(R)-3-氨基-4-(3-苯并噻吩基)-丁酸；(R)-3-氨基-4-(3-氯苯基)-丁酸；(R)-3-氨基-4-(3-氰基苯基)-丁酸；(R)-3-氨基-4-(3-氟苯基)-丁酸；(R)-3-氨基-4-(3-甲苯基)-丁酸；(R)-3-氨基-4-(3-吡啶基)-丁酸；(R)-3-氨基-4-(3-噻吩基)-丁酸；(R)-3-氨基-4-(3-三氟甲苯基)-丁酸；(R)-3-氨基-4-(4-溴苯基)-丁酸；(R)-3-氨基-4-(4-氯苯基)-丁酸；(R)-3-氨基-4-(4-氰基苯基)-丁酸；(R)-3-氨基-4-(4-氟苯基)-丁酸；(R)-3-氨基-4-(4-碘苯基)-丁酸；(R)-3-氨基-4-(4-甲苯基)-丁酸；(R)-3-氨基-4-(4-硝基苯基)-丁酸；(R)-3-氨基-4-(4-吡啶基)-丁酸；(R)-3-氨基-4-(4-三氟甲苯基)-丁酸；(R)-3-氨基-4-五氟-苯基丁酸；(R)-3-氨基-5-己烯酸；(R)-3-氨基-5-己炔酸；(R)-3-氨基-5-苯基戊酸；(R)-3-氨基-6-苯基-5-己烯酸；(S)-1,2,3,4-四氢-异喹啉-3-乙酸；(S)-3-氨基-4-(1-萘基)-丁酸；(S)-3-氨基-4-(2,4-二氯苯基)丁酸；(S)-3-氨基-4-(2-氯苯基)-丁酸；(S)-3-氨基-4-(2-氰基苯基)-丁酸；(S)-3-氨基-4-(2-氟苯基)-丁酸；(S)-3-氨基-4-(2-呋喃基)-丁酸；(S)-3-氨基-4-(2-甲苯基)-丁酸；(S)-3-氨基-4-(2-萘基)-丁酸；(S)-3-氨基-4-(2-噻吩基)-丁酸；(S)-3-氨基- 4-(2-三氟甲苯基)-丁酸；(S)-3-氨基-4-(3,4-二氯苯基)丁酸；(S)-3-氨基-4-(3,4-二氟苯基)丁酸；(S)-3-氨基-4-(3-苯并噻吩基)-丁酸；(S)-3-氨基-4-(3-氯苯基)-丁酸；(S)-3-氨基-4-(3-氰基苯基)-丁酸；(S)-3-氨基-4-(3-氟苯基)-丁酸；(S)-3-氨基-4-(3-甲苯基)-丁酸；(S)-3-氨基-4-(3-吡啶基)-丁酸；(S)-3-氨基-4-(3-噻吩基)-丁酸；(S)-3-氨基-4-(3-三氟甲苯基)-丁酸；(S)-3-氨基-4-(4-溴苯基)-丁酸；(S)-3-氨基-4-(4-氯苯基)-丁酸；(S)-3-氨基-4-(4-氰基苯基)-丁酸；(S)-3-氨基-4-(4-氟苯基)-丁酸；(S)-3-氨基-4-(4-碘苯基)-丁酸；(S)-3-氨基-4-(4-甲苯基)-丁酸；(S)-3-氨基-4-(4-硝基苯基)-丁酸；(S)-3-氨基-4-(4-吡啶基)-丁酸；(S)-3-氨基-4-(4-三氟甲苯基)-丁酸；(S)-3-氨基-4-五氟-苯基丁酸；(S)-3-氨基-5-己烯酸；(S)-3-氨基-5-己炔酸；(S)-3-氨基-5-苯基戊酸；(S)-3-氨基-6-苯基-5-己烯酸；1,2,5,6-四氢吡啶-3-甲酸；1,2,5,6-四氢吡啶-4-甲酸；3-氨基-3-(2-氯苯基)-丙酸；3-氨基-3-(2-噻吩基)-丙酸；3-氨基-3-(3-溴苯基)-丙酸；3-氨基-3-(4-氯苯基)-丙酸；3-氨基-3-(4-甲氧苯基)-丙酸；3-氨基-4,4,4-三氟-丁酸；3-氨基己二酸；D-β-苯丙氨酸；β-亮氨酸；L-β-高丙氨酸；L-β-高天冬氨酸γ-苄酯；L-β-高谷氨酸δ-苄酯；L-β-高异亮氨酸；L-β-高亮氨酸；L-β-高甲硫氨酸；L-β-高苯丙氨酸；L-β-高脯氨酸；L-β-高色氨酸；L-β-高缬氨酸；L-Nω-苄氧羰基-β-高赖氨酸；Nω-L-β-高精氨酸；O-苄基-L-β-高羟脯氨酸；O-苄基-L-β-高丝氨酸；O-苄基-L-β-高苏氨酸；O-苄基-L-β-高酪氨酸；γ-三苯甲基-L-β-高天冬酰胺；(R)-β-苯丙氨酸；L-β-高天冬氨酸γ-叔丁酯；L-β-高谷氨酸δ-叔丁酯；L-Nω-β-高赖氨酸；Nδ-三苯甲基-L-β-高谷氨酰胺；Nω-2,2,4,6,7-五甲基-二氢苯并呋喃-5-磺酰基-L-β-高精氨酸；O-叔丁基-L-β-高羟基-脯氨酸；O-叔丁基-L-β-高丝氨酸；O-叔丁基-L-β-高苏氨酸；O-叔丁基-L-β-高酪氨酸；2-氨基环戊烷羧酸；和2-氨基环己烷羧酸。
其他非天然氨基酸包括但不限于丙氨酸、缬氨酸、甘氨酸或亮氨酸的类似物。丙氨酸、缬氨酸、甘氨酸或亮氨酸的氨基酸类似物的实例包括但不限于以下：α-甲氧基甘氨酸；α-烯丙基-L-丙氨酸；α-氨基异丁酸；α-甲基-亮氨酸；β-(1-萘基)-D-丙氨酸；β-(1-萘基)-L-丙氨酸；β-(2-萘基)-D-丙氨酸；β-(2-萘基)-L-丙氨酸；β-(2-吡啶基)-D-丙氨酸；β-(2-吡啶基)-L-丙氨酸；β-(2-噻吩基)-D-丙氨酸；β-(2-噻吩基)-L-丙氨酸；β-(3-苯并噻吩基)-D-丙氨酸；β-(3-苯并噻吩基)-L-丙氨酸；β-(3-吡啶基)-D-丙氨酸；β-(3-吡啶基)-L-丙氨酸；β-(4-吡啶基)-D-丙氨酸；β-(4-吡啶基)-L-丙氨酸；β-氯-L-丙氨酸；β-氰基-L-丙氨酸；β-环己基-D-丙氨酸；β-环己基-L-丙氨酸；β-环戊烯-1-基-丙氨酸；β-环戊基-丙氨酸；β-环丙基-L-Ala-OH·二环己基铵盐；β-叔丁基-D-丙氨酸；β-叔丁基-L-丙氨酸；γ-氨基丁酸；L-α,β-二氨基丙酸；2,4-二硝基-苯基甘氨酸；2,5-二氢-D-苯基甘氨酸；2-氨基-4,4,4-三氟丁酸；2-氟-苯基甘氨酸；3-氨基-4,4,4-三氟-丁酸；3-氟-缬氨酸；4,4,4-三氟-缬氨酸；4,5-脱氢-L-leu-OH·二环己基铵盐；4-氟-D-苯基甘氨 酸；4-氟-L-苯基甘氨酸；4-羟基-D-苯基甘氨酸；5,5,5-三氟-亮氨酸；6-氨基己酸；环戊基-D-Gly-OH·二环己基铵盐；环戊基-Gly-OH·二环己基铵盐；D-α,β-二氨基丙酸；D-α-氨基丁酸；D-α-叔丁基甘氨酸；D-(2-噻吩基)甘氨酸；D-(3-噻吩基)甘氨酸；D-2-氨基己酸；D-2-茚满基甘氨酸；D-烯丙基甘氨酸·二环己基铵盐；D-环己基甘氨酸；D-正缬氨酸；D-苯基甘氨酸；β-氨基丁酸；β-氨基异丁酸；(2-溴苯基)甘氨酸；(2-甲氧基苯基)甘氨酸；(2-甲苯基)甘氨酸；(2-噻唑基)甘氨酸；(2-噻吩基)甘氨酸；2-氨基-3-(二甲氨基)-丙酸；L-α,β-二氨基丙酸；L-α-氨基丁酸；L-α-叔丁基甘氨酸；L-(3-噻吩基)甘氨酸；L-2-氨基-3-(二甲氨基)-丙酸；L-2-氨基己酸二环己基-铵盐；L-2-茚满基甘氨酸；L-烯丙基甘氨酸·二环己基铵盐；L-环己基甘氨酸；L-苯基甘氨酸；L-炔丙基甘氨酸；L-正缬氨酸；N-α-氨基甲基-L-丙氨酸；D-α,γ-二氨基丁酸；L-α,γ-二氨基丁酸；β-环丙基-L-丙氨酸；(N-β-(2,4-二硝基苯基))-L-α,β-二氨基丙酸；(N-β-1-(4,4-二甲基-2,6-二氧代亚环己-1-基)乙基)-D-α,β-二氨基丙酸；(N-β-1-(4,4-二甲基-2,6-二氧环己-1-亚基)乙基)-L-α,β-二氨基丙酸；(N-β-4-甲基三苯甲基)-L-α,β-二氨基丙酸；(N-β-烯丙氧羰基)-L-α,β-二氨基丙酸；(N-γ-1-(4,4-二甲基-2,6-二氧代环己-1-亚基)乙基)-D-α,γ-二氨基丁酸；(N-γ-1-(4,4-二甲基-2,6-二氧代环己-1-亚基)乙基)-L-α,γ-二氨基丁酸；(N-γ-4-甲基三苯甲基)-D-α,γ-二氨基丁酸；(N-γ-4-甲基三苯甲基)-L-α,γ-二氨基丁酸；(N-γ-烯丙氧羰基)-L-α,γ-二氨基丁酸；D-α,γ-二氨基丁酸；4,5-脱氢-L-亮氨酸；环戊基-D-Gly-OH；环戊基-Gly-OH；D-烯丙基甘氨酸；D-环己基高丙氨酸；L-1-芘基丙氨酸；L-2-氨基己酸；L-烯丙基甘氨酸；L-环己基高丙氨酸；和N-(2-羟基-4-甲氧基-Bzl)-Gly-OH。
其他非天然氨基酸包括但不限于精氨酸或赖氨酸的类似物。精氨酸和赖氨酸的氨基酸类似物的实例包括但不限于以下：瓜氨酸；L-2-氨基-3-胍基丙酸；L-2-氨基-3-脲基丙酸；L-瓜氨酸；Lys(Me)2-OH；Lys(N3)-OH；Nδ-苄氧羰基-L-鸟氨酸；Nω-硝基-D-精氨酸；Nω-硝基-L-精氨酸；α-甲基-鸟氨酸；2,6-二氨基庚二酸；L-鸟氨酸；(Nδ-1-(4,4-二甲基-2,6-二氧代-环己-1-亚基)乙基)-D-鸟氨酸；(Nδ-1-(4,4-二甲基-2,6-二氧-环己-1-亚基)乙基)-L-鸟氨酸；(Nδ-4-甲基三苯甲基)-D-鸟氨酸；(Nδ-4-甲基三苯甲基)-L-鸟氨酸；D-鸟氨酸；L-鸟氨酸；Arg(Me)(Pbf)-OH；Arg(Me)2-OH(不对称的)；Arg(Me)2-OH(对称的)；Lys(ivDde)-OH；Lys(Me)2-OH·HCl；Lys(Me)3-OH氯化物；Nω-硝基-D-精氨酸；和Nω-硝基-L-精氨酸。
其他非天然氨基酸包括但不限于天冬氨酸或谷氨酸的类似物。天冬氨酸和谷氨酸的氨基酸类似物的实例包括但不限于以下：α-甲基-D-天冬氨酸；α-甲基-谷氨酸；α-甲基-L-天冬氨酸；γ-亚甲基-谷氨酸；(N-γ-乙基)-L-谷氨酰胺；[N-α-(4-氨基苯甲酰基)]-L-谷氨酸；2,6-二氨基庚二酸；L-α-氨基辛二酸；D-2-氨基己二酸；D-α-氨基辛二酸；α-氨基庚二酸；亚氨基二乙 酸；L-2-氨基己二酸；苏-β-甲基-天冬氨酸；γ-羧基-D-谷氨酸γ,γ-二-叔丁酯；γ-羧基-L-谷氨酸γ,γ-二-叔丁酯；Glu(OAll)-OH；L-Asu(OtBu)-OH；和焦谷氨酸。
其他非天然氨基酸包括但不限于半胱氨酸和甲硫氨酸的类似物。半胱氨酸和甲硫氨酸的氨基酸类似物的实例包括但不限于Cys(法呢基)-OH、Cys(法呢基)-OMe、α-甲基-甲硫氨酸、Cys(2-羟乙基)-OH、Cys(3-氨丙基)-OH、2-氨基-4-(乙硫基)丁酸、丁硫氨酸、丁硫氨酸亚砜胺、乙硫氨酸、甲硫氨酸甲基锍氯化物、硒代甲硫氨酸、磺丙氨酸、[2-(4-吡啶基)乙基]-DL-青霉胺、[2-(4-吡啶基)乙基]-L-半胱氨酸、4-甲氧苄基-D-青霉胺、4-甲氧苄基-L-青霉胺、4-甲基苄基-D-青霉胺、4-甲基苄基-L-青霉胺、苄基-D-半胱氨酸、苄基-L-半胱氨酸、苄基-DL-高半胱氨酸、氨甲酰基-L-半胱氨酸、羧乙基-L-半胱氨酸、羧甲基-L-半胱氨酸、二苯基甲基-L-半胱氨酸、乙基-L-半胱氨酸、甲基-L-半胱氨酸、叔丁基-D-半胱氨酸、三苯甲基-L-高半胱氨酸、三苯甲基-D-青霉胺、胱硫醚、高胱氨酸、L-高胱氨酸、(2-氨基乙基)-L-半胱氨酸、硒代-L-胱氨酸、胱硫醚、Cys(StBu)-OH和乙酰氨甲基-D-青霉胺。
其他非天然氨基酸包括但不限于苯丙氨酸和酪氨酸的类似物。苯丙氨酸和酪氨酸的氨基酸类似物的实例包括β-甲基-苯丙氨酸、β-羟基苯丙氨酸、α-甲基-3-甲氧基-DL-苯丙氨酸、α-甲基-D-苯丙氨酸、α-甲基-L-苯丙氨酸、1,2,3,4-四氢异喹啉-3-甲酸、2,4-二氯-苯丙氨酸、2-(三氟甲基)-D-苯丙氨酸、2-(三氟甲基)-L-苯丙氨酸、2-溴-D-苯丙氨酸、2-溴-L-苯丙氨酸、2-氯-D-苯丙氨酸、2-氯-L-苯丙氨酸、2-氰基-D-苯丙氨酸、2-氰基-L-苯丙氨酸、2-氟-D-苯丙氨酸、2-氟-L-苯丙氨酸、2-甲基-D-苯丙氨酸、2-甲基-L-苯丙氨酸、2-硝基-D-苯丙氨酸、2-硝基-L-苯丙氨酸、2,4,5-三羟基-苯丙氨酸、3,4,5-三氟-D-苯丙氨酸、3,4,5-三氟-L-苯丙氨酸、3,4-二氯-D-苯丙氨酸、3,4-二氯-L-苯丙氨酸、3,4-二氟-D-苯丙氨酸、3,4-二氟-L-苯丙氨酸、3,4-二羟基-L-苯丙氨酸、3,4-二甲氧基-L-苯丙氨酸、3,5,3’-三碘-L-甲状腺原氨酸、3,5-二碘-D-酪氨酸、3,5-二碘-L-酪氨酸、3,5-二碘-L-甲状腺原氨酸、3-(三氟甲基)-D-苯丙氨酸、3-(三氟甲基)-L-苯丙氨酸、3-氨基-L-酪氨酸、3-溴-D-苯丙氨酸、3-溴-L-苯丙氨酸、3-氯-D-苯丙氨酸、3-氯-L-苯丙氨酸、3-氯-L-酪氨酸、3-氰基-D-苯丙氨酸、3-氰基-L-苯丙氨酸、3-氟-D-苯丙氨酸、3-氟-L-苯丙氨酸、3-氟-酪氨酸、3-碘-D-苯丙氨酸、3-碘-L-苯丙氨酸、3-碘-L-酪氨酸、3-甲氧基-L-酪氨酸、3-甲基-D-苯丙氨酸、3-甲基-L-苯丙氨酸、3-硝基-D-苯丙氨酸、3-硝基-L-苯丙氨酸、3-硝基-L-酪氨酸、4-(三氟甲基)-D-苯丙氨酸、4-(三氟甲基)-L-苯丙氨酸、4-氨基-D-苯丙氨酸、4-氨基-L-苯丙氨酸、4-苯甲酰基-D-苯丙氨酸、4-苯甲酰基-L-苯丙氨酸、4-双(2-氯乙基)氨基-L-苯丙氨酸、4-溴-D-苯丙氨酸、4-溴-L-苯丙氨酸、4-氯-D-苯丙氨酸、4-氯-L-苯丙氨酸、4-氰基-D-苯丙氨酸、4-氰基-L-苯丙氨酸、4-氟-D-苯丙氨酸、4-氟-L-苯丙氨酸、4-碘 -D-苯丙氨酸、4-碘-L-苯丙氨酸、高苯丙氨酸、甲状腺氨酸、3,3-二苯丙氨酸、甲状腺原氨酸、乙基-酪氨酸和甲基-酪氨酸。
其他非天然氨基酸包括但不限于脯氨酸的类似物。脯氨酸的氨基酸类似物的实例包括但不限于3,4-脱氢-脯氨酸、4-氟-脯氨酸、顺-4-羟基-脯氨酸、噻唑烷-2-甲酸和反-4-氟-脯氨酸。
其他非天然氨基酸包括但不限于丝氨酸和苏氨酸的类似物。丝氨酸和苏氨酸的氨基酸类似物的实例包括但不限于3-氨基-2-羟基-5-甲基己酸、2-氨基-3-羟基-4-甲基戊酸、2-氨基-3-乙氧丁酸、2-氨基-3-甲氧丁酸、4-氨基-3-羟基-6-甲基庚酸、2-氨基-3-苄氧丙酸、2-氨基-3-苄氧丙酸、2-氨基-3-乙氧丙酸、4-氨基-3-羟基丁酸和α-甲基丝氨酸。
其他非天然氨基酸包括但不限于色氨酸的类似物。色氨酸的氨基酸类似物的实例包括但不限于以下：α-甲基-色氨酸；β-(3-苯并噻吩基)-D-丙氨酸；β-(3-苯并噻吩基)-L-丙氨酸；1-甲基-色氨酸；4-甲基-色氨酸；5-苄氧基-色氨酸；5-溴-色氨酸；5-氯-色氨酸；5-氟-色氨酸；5-羟基-色氨酸；5-羟基-L-色氨酸；5-甲氧基-色氨酸；5-甲氧基-L-色氨酸；5-甲基-色氨酸；6-溴-色氨酸；6-氯-D-色氨酸；6-氯-色氨酸；6-氟-色氨酸；6-甲基-色氨酸；7-苄氧基-色氨酸；7-溴-色氨酸；7-甲基-色氨酸；D-1,2,3,4-四氢-去甲哈尔满-3-甲酸；6-甲氧基-1,2,3,4-四氢去甲哈尔满-1-甲酸；7-氮杂色氨酸；L-1,2,3,4-四氢-去甲哈尔满-3-甲酸；5-甲氧基-2-甲基-色氨酸；和6-氯-L-色氨酸。
本发明中，非天然氨基酸可以是外消旋的。本发明中，使用的非天然氨基酸可以是D型异构体。本发明中，使用的非天然氨基酸可以是L型异构体。本发明中，非天然氨基酸可包含为R或S构型的手性中心。在一些其它实施方案中，β-氨基酸的氨基基团被诸如叔丁氧羰基(BOC基团)、9-芴甲氧羰基(FMOC)、甲苯磺酰基等保护基团取代。在一些其它实施方案中，β-氨基酸的羧酸官能团例如作为其酯衍生物被保护。在一些实施方案中，使用非天然氨基酸的盐。
在以上任一实施方式中，其中R1、R2、R3、R4优选独立地选自-H、C1-10烷基、C1-10烷氧基、C3-10环烷基和5至14元杂环基，它们任选地被R5取代，其中R5选自C1-10烷基、C1-10烷氧基、C3-10环烷基和5至14元杂环基。
在以上任一实施方式中，其中R1、R2、R3、R4更优选独立地选自-H、C1-3烷基、C1-3烷氧基、C3-6环烷基，它们任选地被R5取代，其中R5选自C1-10烷基、C1-10烷氧基、C3-10环烷基、吡咯基、噻吩基、噻唑基、咪唑基、吡啶基、呋喃基、哌嗪基、吡咯烷基、二氧杂环己基、吗啉基、四氢呋喃基、四氢吡咯烷基。
在以上任一实施方式中，其中R1、R2、R3、R4更优选独立地选自-H、甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、甲氧基和乙氧基。
在以上任一实施方式中，其中R1、R2、R3、R4还更优选独立是-H或甲基。优选地，R1、R2、R3、R4均为-H。
在以上任一实施方式中，优选地，n为2至4，并且每个重复单元相同或不同。更优选地，n为2或3，并且每个重复单元相同或不同。在一些实施方式中，n为2，并且每个重复单元相同。在一些实施方式中，n为3，并且每个重复单元相同。在一些实施方式中，n为2，并且每个重复单元不同。在一些实施方式中，n为3，并且每个重复单元不同。
在以上任一实施方式中，优选地，p为2至4，并且每个重复单元相同或不同。更优选地，p为2或3，并且每个重复单元相同或不同。在一些实施方式中，p为2，并且每个重复单元相同。在一些实施方式中，p为3，并且每个重复单元相同。在一些实施方式中，p为2，并且每个重复单元不同。在一些实施方式中，p为3，并且每个重复单元不同。
在一些实施方式中，本文提供了配体-寡核苷酸共轭物，其包含式I或式II所示的接头部分：
-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I)，
-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II)，
其中R1、R2、R3、R4独立是-H或甲基；
Ra、Rb独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-8环烷基、6至14元芳基C1-3烷基、5至18元杂芳基C1-3烷基和5至14元杂环基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、C1-3烷基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、吡咯基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、C1-10烷基、C1-10烷氧基、6至14元芳基、C3-8环烷基、金刚烷基、膦酰基、膦酰氧基、C1-3烷基膦酰基、C1-3烷基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成3至14元含氮杂环基；
杂原子选自氮、氧、硫、磷和硅，优选地选自氮、氧、硫；n为1至10，优选地为2至4，并且每个重复单元相同或不同；p为2至6，优选地为2至4，并且每个重复单元相同或不同。
在一些实施方式中，本文提供了配体-寡核苷酸共轭物，其包含式I或式II所示的接头部分：
-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I)，
-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II)，
其中R1、R2、R3、R4独立是-H或甲基；
其中Ra、Rb独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-8环烷基、苯基C1-3烷基、萘基C1-3烷基、5至12元杂芳基C1-3烷基和5至14元杂环基C1-3烷基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、C1-3烷基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、哌嗪基、吡咯烷基、二氧杂环己基、吗啉基、四氢呋喃基、四氢吡咯烷基、C1-10烷基、C1-10烷氧基、苯基、苯甲基、苯乙基、氨基苯基、羧基苯基、卤代苯基、联苯基、C3-8环烷基、金刚烷基、膦酰基、膦酰氧基、C1-3烷基膦酰基、C1-3烷基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成3至14元含氮杂环基；
杂原子选自氮、氧、硫、磷和硅，优选地选自氮、氧、硫；n为1至10，优选地为2至4，并且每个重复单元相同或不同；p为2至6，优选地为2至4，并且每个重复单元相同或不同。
在一些实施方式中，本文提供了配体-寡核苷酸共轭物，其包含式I或式II所示的接头部分：
-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I)，
-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II)，
其中R1、R2、R3、R4独立是-H或甲基；
Ra、Rb独立地选自-H、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、C3-6环烷基、苯基C1-3烷基、吡啶基C1-3烷基、吲哚基C1-3烷基、喹啉基C1-3烷基、异喹啉基C1-3烷基、噻吩基C1-3烷基、噻茚基C1-3烷基、噻唑基C1-3烷基、苯并噻唑基C1-3烷基、咪唑基C1-3烷基、呋喃基C1-3烷基、嘧啶基C1-3烷基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、C1-3烷基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、哌嗪基、吡咯烷基、二氧杂环己基、吗啉基、四氢呋喃基、四氢吡咯烷基、吡啶基、C1-10烷基、C1-10烷氧基、苯基、苯甲基、苯乙基、氨基苯基、羧基苯基、卤代苯基、联苯基、C3-8环烷基、金刚烷基、膦酰基、膦酰氧基、C1-3烷基膦酰基、C1-3烷基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成3至14元含氮杂环基；
杂原子选自氮、氧、硫、磷和硅，优选地选自氮、氧、硫；n为1至10，优选地为2 至4，并且每个重复单元相同或不同；p为2至6，优选地为2至4，并且每个重复单元相同或不同。
在一些实施方式中，本文提供了配体-寡核苷酸共轭物，其包含式I或式II所示的接头部分：
-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I)，
-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II)，
其中R1、R2、R3、R4独立是-H或甲基；
其中Ra、Rb独立地选自-H、甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、乙烯基、丙烯基、1-丁烯基、2-丁烯基、异丁烯基、乙炔基、丙炔基、1-丁炔基、2-丁炔基、3-甲基-2-丙炔基、1,3-丁二烯基、甲氧基、乙氧基、丙氧基、丁氧基、环丙基、环丁基、环戊基、环己基、苯基亚甲基、苯基二亚甲基、吡啶基亚甲基、吡啶基二亚甲基、吲哚基亚甲基、吲哚基二亚甲基、喹啉基亚甲基、喹啉基二亚甲基、异喹啉基亚甲基、异喹啉基二亚甲基、噻吩基亚甲基、噻吩基二亚甲基、噻茚基亚甲基、噻茚基二亚甲基、噻唑基亚甲基、噻唑基二亚甲基、苯并噻唑基亚甲基、苯并噻唑基二亚甲基、咪唑基亚甲基、咪唑基二亚甲基、呋喃基亚甲基、呋喃基二亚甲基、嘧啶基亚甲基和嘧啶基二亚甲基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、亚甲基硫基、二亚甲基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、哌嗪基、吡咯烷基、二氧杂环己基、吗啉基、四氢呋喃基、四氢吡咯烷基、吡啶基、甲基、乙基、丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、苯基、苯甲基、苯乙基、氨基苯基、羧基苯基、卤代苯基、联苯基、环丙基、环丁基、环戊基、环己基、金刚烷基、膦酰基、膦酰氧基、二甲基膦酰基、二乙基膦酰基、二甲基膦酰氧基和二乙基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成咪唑基、噻唑基、吡唑基、嘧啶基、吲哚基、喹啉基、异喹啉基或吡咯基；
n为1至10，优选地为2至4，并且每个重复单元相同或不同；p为2至6，优选地为2至4，并且每个重复单元相同或不同。
在一些实施方式中，本文提供了配体-寡核苷酸共轭物，其包含式I或式II所示的接头部分：
-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I)，
-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II)，
其中Ra、R1、R2、R3、R4独立是-H或甲基；
其中Rb选自-H、甲基、乙基、丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、环丙基、环戊基、环己基、环己基二亚甲基、环己基亚甲基、环丁基亚甲基、苯基亚甲基、甲基硫基亚甲基、3-吲哚基亚甲基、羟基亚甲基、酰胺基二亚甲基、羟基甲基次甲基、巯基亚甲基、酰胺基亚甲基、羟基苯基亚甲基、羟基苯基二亚甲基、羧基亚甲基、羧基二亚甲基、氨基正丁基、胍基二亚甲基、胍基三亚甲基、4-咪唑基亚甲基、异丙基苯基亚甲基、1-萘基亚甲基、2-萘基亚甲基、茚满基、苯基二亚甲基、氟代苯基亚甲基、氯代苯基亚甲基、三氟苯基亚甲基、二氯苯基亚甲基、五氟苯基亚甲基、二氟苯基亚甲基、噻吩基亚甲基、噻茚基亚甲基、喹啉基亚甲基、卤代3-吲哚基亚甲基、二苯基亚甲基、3-吡啶基亚甲基和4-吡啶基亚甲基，或者Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基；
n为1至10，优选地为2至4，并且每个重复单元相同或不同；p为2至6，优选地为2至4，并且每个重复单元相同或不同。
在一些实施方式中，本文提供了配体-寡核苷酸共轭物，其包含式I或式II所示的接头部分：
-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I)，
-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II)，
其中Ra、R1、R2、R3、R4独立是-H或甲基；Rb选自-H、甲基、异丙基和异丁基，或者Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基；n为2至4，并且每个重复单元相同或不同；p为2至4，并且每个重复单元相同或不同。
在一些实施方式中，本文提供了配体-寡核苷酸共轭物，其包含式I或式II所示的接头部分：
-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I)，
-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II)，
其中Ra、R1、R2、R3、R4独立是-H或甲基；Rb选自-H、甲基、异丙基和异丁基，或者Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基；n为2或3，并且每个重复单元相同或不同；p为2，并且对于p为2的重复单元：在每个重复单元中，Rb是异丙基；在每个重复单元中，Rb是甲基；在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb是甲基；在一个重复单元中，Rb是异丁基，在另一个重复单元中，Rb是甲基；在一个重复单元中，Rb是异丁基，在另一个重复单元中，Rb是异丙基；在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb是氢；在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基；或在一个重复单元中， Rb是氢，在另一个重复单元中，Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基。
在优选的实施方式中，式I或式II所示的接头，其左侧端与配体连接或用于与配体连接；其右侧端与寡核苷酸连接或用于与寡核苷酸连接，优选与带氨基的寡聚核苷酸(例如5’或3’端具有末端氨基的寡核苷酸)连接或用于与带氨基的寡聚核苷酸(例如5’或3’端具有末端氨基的寡核苷酸)连接。
在一些实施方式中，本发明的另一个方面提供式I’或II’所示的连接单元：
-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I’)，或
-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II’)，
其中各取代基、符号具有如本文所定义的含义；
式I’的左侧端连接-NR1-即为式I，式II’的左侧端连接-NR1-即为式II。
在上述任一实施方式中，优选地，所述配体-寡核苷酸共轭物中的配体包含通过二价或三价支链接头附接的一种或多种N-乙酰半乳糖胺衍生物。
在优选的实施方式中，所述配体具有以下结构：(GalNAc-Q1)mCHm’-，
其中，GalNAc表示N-乙酰半乳糖胺或其衍生物；本发明中，所述N-乙酰半乳糖胺或其衍生物优选为如下结构：
Q1表示T1-(T2-T3-T4)w，其中T1和T4独立地选自不存在、C(O)、NH、O、S、OC(O)、NHC(O)、CH2、CH2NH和CH2O；T2选自不存在、NH、O、S、CH2、C(O)O、C(O)NH、NHCH2C(O)、C(O)-CH2-NH、C(O)和CH＝N-O；T3选自不存在和任选地被选自O、S、S(O)、SO2、C(O)和C≡C的原子或基团间杂或封端的一个或多个亚甲基；w为0至20，优选为1-5，并且该重复单元相同或不同；所述Ro、Ro’分别独立的选自H、烷基羰基，所述烷基如以上所定义；所述Ro’优选为乙酰基；所述Ro分别独立地优选为H或乙酰基，更优选为H。
m为1至3，优选为2或3，并且每个重复单元相同或不同；m’为0至2，优选为0或1，且m+m’为3。
在优选的实施方式中，其中所述配体的结构选自：

在更优选的实施方式中，其中所述配体的结构为：
在上述任一实施方式中，优选地，所述配体-寡核苷酸共轭物中的寡核苷酸选自反义寡核苷酸(ASO)、小干扰RNA(siRNA)、小发卡RNA(shRNA)和微RNA(miRNA)或其盐，所述盐优选为钠盐或钾盐。
在一些实施方式中，所述寡核苷酸的5’端附接至所述接头部分，优选地附接至所述接头部分的右侧端。在一些实施方式中，所述寡核苷酸的3’端附接至所述接头部分，优选地附接至所述接头部分的右侧端。
在一些实施方式中，所述寡核苷酸的5’端通过3’-寡核苷酸-5’-OP(O)(OH)O-(CH2-Y)x-NH-附接至所述接头部分，其中Y代表O或不存在，x为3至12并且每个重复单元相同或不 同。在这样的实施方式中，优选地，所述5’端通过3’-寡核苷酸-5’-OP(O)(OH)O-(CH2)6-NH-附接至所述接头部分；还优选地，所述5’端通过3’-寡核苷酸-5’-OP(O)(OH)O-(CH2)3-NH-附接至所述接头部分；还优选地，所述5’端通过3’-寡核苷酸-5’-OP(O)(OH)O-(CH2)12-NH-附接至所述接头部分；还优选地，所述5’端通过3’-寡核苷酸-5’-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH-附接至所述接头部分。
在一些实施方式中，所述寡核苷酸的5’端通过3’-寡核苷酸-5’-OP(S)(OH)O-(CH2-Y)x-NH-附接至所述接头部分，其中Y代表O或不存在，x为3至12并且每个重复单元相同或不同。在这样的实施方式中，优选地，所述5’端通过3’-寡核苷酸-5’-OP(S)(OH)O-(CH2)6-NH-附接至所述接头部分；还优选地，所述5’端通过3’-寡核苷酸-5’-OP(S)(OH)O-(CH2)3-NH-附接至所述接头部分；还优选地，所述5’端通过3’-寡核苷酸-5’-OP(S)(OH)O-(CH2)12-NH-附接至所述接头部分；还优选地，所述5’端通过3’-寡核苷酸-5’-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH-附接至所述接头部分。
在一些实施方式中，也可以是所述寡核苷酸的3’端通过5’-寡核苷酸-3’-OP(O)(OH)O-(CH2-Y)x-NH-附接至所述接头部分，其中Y代表O或不存在，x为3至12并且每个重复单元相同或不同。在这样的实施方式中，优选地，所述3’端通过5’-寡核苷酸-3’-OP(O)(OH)O-(CH2)6-NH-附接至所述接头部分；还优选地，所述3’端通过5’-寡核苷酸-3’-OP(O)(OH)O-(CH2)3-NH-附接至所述接头部分；还优选地，所述3’端通过5’-寡核苷酸-3’-OP(O)(OH)O-(CH2)12-NH-附接至所述接头部分；还优选地，所述3’端通过5’-寡核苷酸-3’-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH-附接至所述接头部分。
在一些实施方式中，也可以是所述寡核苷酸的3’端通过5’-寡核苷酸-3’-OP(S)(OH)O-(CH2-Y)x-NH-附接至所述接头部分，其中Y代表O或不存在，x为3至12并且每个重复单元相同或不同。在这样的实施方式中，优选地，所述3’端通过5’-寡核苷酸-3’-OP(S)(OH)O-(CH2)6-NH-附接至所述接头部分；还优选地，所述3’端通过5’-寡核苷酸-3’-OP(S)(OH)O-(CH2)3-NH-附接至所述接头部分；还优选地，所述3’端通过5’-寡核苷酸-3’-OP(S)(OH)O-(CH2)12-NH-附接至所述接头部分；还优选地，所述3’端通过5’-寡核苷酸-3’-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH-附接至所述接头部分。
在优选的实施方式中，所述寡核苷酸是siRNA，包含正义链和反义链，更优选地，所述siRNA中的核苷酸的一个或多个被修饰，所述修饰为2’-O-甲基修饰和/或2’-氟基修饰。在一些实施方式中，所述正义链的3’端或5’端附接至所述接头部分。在优选的实施方式中，所述正义链的5’端附接至所述接头部分。
在优选的实施方式中，所述正义链的5’端通过3’-siRNA正义链-5’-OP(O)(OH)O-(CH2-Y)x-NH-附接至所述接头部分，其中Y代表O或不存在，x为3至12并且每个重复单元相同或不同。在优选的实施方式中，所述正义链的5’端通过3’-siRNA正义链-5’-OP(O)(OH)O-(CH2)6-NH-附接至所述接头部分；还优选地，所述正义链的5’端通过3’-siRNA正义链-5’-OP(O)(OH)O-(CH2)3-NH-附接至所述接头部分；还优选地，所述正义链的5’端通过3’-siRNA正义链-5’-OP(O)(OH)O-(CH2)12-NH-附接至所述接头部分；还优选地，所述正义链的5’端通过3’-siRNA正义链-5’-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH-附接至所述接头部分。
在优选的实施方式中，所述正义链的5’端通过3’-siRNA正义链-5’-OP(S)(OH)O-(CH2-Y)x-NH-附接至所述接头部分，其中Y代表O或不存在，x为3至12并且每个重复单元相同或不同。在优选的实施方式中，所述正义链的5’端通过3’-siRNA正义链-5’-OP(S)(OH)O-(CH2)6-NH-附接至所述接头部分；还优选地，所述正义链的5’端通过3’-siRNA正义链-5’-OP(S)(OH)O-(CH2)3-NH-附接至所述接头部分；还优选地，所述正义链的5’端通过3’-siRNA正义链-5’-OP(S)(OH)O-(CH2)12-NH-附接至所述接头部分；还优选地，所述正义链的5’端通过3’-siRNA正义链-5’-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH-附接至所述接头部分。
在优选的实施方式中，本文提供式V、式V’、式VI、式VI’、式V-1、式V’-1、式VI-1或式VI’-1所示的配体-寡核苷酸共轭物：
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-NH-(CH2-Y)x-O-
P(O)(OH)O-5’-寡核苷酸-3’(V)，
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-NH-(CH2-Y)x-O-
P(O)(OH)O-3’-寡核苷酸-5’(V’)，
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-NH-(CH2-Y)x-O-
P(O)(OH)O-5’-寡核苷酸-3’(VI)；
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-NH-(CH2-Y)x-O-
P(O)(OH)O-3’-寡核苷酸-5’(VI’)，
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-NH-(CH2-Y)x-O-
P(S)(OH)O-5’-寡核苷酸-3’(V-1)，
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-NH-(CH2-Y)x-O-
P(S)(OH)O-3’-寡核苷酸-5’(V’-1)，
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-NH-(CH2-Y)x-O-
P(S)(OH)O-5’-寡核苷酸-3’(VI-1)；
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-NH-(CH2-Y)x-O-
P(S)(OH)O-3’-寡核苷酸-5’(VI’-1)；
其中各取代基和符号具有本文如上所定义的相应含义、数值和数值范围，或其优选含义、数值和数值范围；其中，寡核苷酸优选是siRNA、siRNA正义链或ASO。
在优选的实施方式中，本文提供式VII、式VII’、式VIII、式VIII’、VII-1、式VII’-1、式VIII-1、式VIII’-1所示的配体-寡核苷酸共轭物：
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-NH-(CH2-Y)x-O-
P(O)(OH)O-5’-siRNA正义链-3’(VII)，
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-NH-(CH2-Y)x-O-
P(O)(OH)O-3’-siRNA正义链-5’(VII’)，
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-NH-(CH2-Y)x-O-
P(O)(OH)O-5’-siRNA正义链-3’(VIII)，
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-NH-(CH2-Y)x-O-
P(O)(OH)O-3’-siRNA正义链-5’(VIII’),
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-NH-(CH2-Y)x-O-
P(S)(OH)O-5’-siRNA正义链-3’(VII-1)，
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-NH-(CH2-Y)x-O-
P(S)(OH)O-3’-siRNA正义链-5’(VII’-1)，
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-NH-(CH2-Y)x-O-
P(S)(OH)O-5’-siRNA正义链-3’(VIII-1)，
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-NH-(CH2-Y)x-O-
P(S)(OH)O-3’-siRNA正义链-5’(VIII’-1)；
其中各取代基和符号具有本文如上所定义的相应含义、数值和数值范围，或其优选含义、数值和数值范围；其中siRNA反义链通过碱基配对方式与siRNA正义链连接形成双链RNA。
在优选的实施方式中，本文提供式IX、式IX’、式X、式X’、式IX-1、式IX’-1、式X-1、式X’-1所示的配体-寡核苷酸共轭物：
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-NH-(CH2-Y)x-O-
P(O)(OH)O-5’-ASO-3’(IX)，
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-NH-(CH2-Y)x-O-
P(O)(OH)O-5’-ASO-3’(IX’)，
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-NH-(CH2-Y)x-O-
P(O)(OH)O-5’-ASO-3’(X),
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-NH-(CH2-Y)x-O-
P(O)(OH)O-3’-ASO-5’(X’)，
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-NH-(CH2-Y)x-O-
P(S)(OH)O-5’-ASO-3’(IX-1)，
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-NH-(CH2-Y)x-O-
P(S)(OH)O-5’-ASO-3’(IX’-1)，
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-NH-(CH2-Y)x-O-
P(S)(OH)O-5’-ASO-3’(X-1),
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-NH-(CH2-Y)x-O-
P(S)(OH)O-3’-ASO-5’(X’-1)；
其中各取代基和符号具有本文如上所定义的相应含义、数值和数值范围，或其优选含义、数值和数值范围。
在优选的实施方式中，本文提供了选自下式的配体-寡核苷酸共轭物：


















其中表示siRNA；
其中，
优选为：
优选为：
优选为：
优选为：
优选，



中间体化合物
本文的另一个方面提供了中间体化合物，其具有式III或IV所示的结构：
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-Q2(式III)，或
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-Q2(式IV)，
其中，GalNAc表示N-乙酰半乳糖胺或其衍生物；本发明中，所述N-乙酰半乳糖胺或其衍生物优选为如下结构：
Q1表示-T1-(T2-T3-T4)w，其中T1和T4独立地选自不存在、C(O)、NH、O、S、OC(O)、NHC(O)、CH2、CH2NH和CH2O；T2选自不存在、NH、O、S、CH2、C(O)O、 C(O)NH、NHCH2C(O)、C(O)-CH2-NH、C(O)和CH＝N-O；T3选自不存在和任选地被选自O、S、S(O)、SO2、C(O)和C≡C的原子或基团间杂或封端的一个或多个亚甲基；w为0至20，并且该重复单元相同或不同；Ro、Ro’定义如上文所述。
Q2表示-OH或-O-羧基保护基团，其中羧基保护基优选为苄基或五氟代苯基；
R1、R2、R3、R4独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3- 10环烷基、6至14元芳基、5至18元杂芳基、5至14元杂环基、卤素、羟基、氨基、胍基、羧基、氰基、硝基和巯基，它们任选地被R5取代，其中R5选自C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-10环烷基、6至14元芳基、5至18元杂芳基、5至14元杂环基、卤素、羟基、氨基、胍基、羧基、氰基、硝基、C1-3烷基硫基和巯基；
Ra、Rb独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-8环烷基、6至14元芳基C1-3烷基、5至18元杂芳基C1-3烷基和5至14元杂环基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、C1-3烷基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、吡咯基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、C1-10烷基、C1-10烷氧基、6至14元芳基、C3-8环烷基、金刚烷基、膦酰基、膦酰氧基、C1-3烷基膦酰基、C1-3烷基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成3至14元含氮杂环基，或者Ra、Rb与其共同连接的C原子一起形成C3-8环烷基；
杂原子选自氮、氧、硫、磷和硅；
m为1至3，并且每个重复单元相同或不同；m’为0至2，优选为0或1，且m+m’为3；
n为1至10，并且每个重复单元相同或不同；
p为2至6，并且每个重复单元相同或不同。
在优选的实施方式中，上述式III或IV中的(GalNAc-Q1)mCHm’-部分的结构选自：


在更优选的实施方式中，式III或IV所示的结构中的(GalNAc-Q1)mCHm’-部分的结构是：
因此，在一些实施方式中，提供了中间体化合物，其具有式III或IV所示的结构：
(GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-Q2(式III)，或
(GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-Q2(式IV)，
其中(GalNAc-Q1)mCHm’-部分的结构是：
Q2表示-OH或-O羧基保护基团；
R1、R2、R3、R4独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3- 10环烷基、6至14元芳基、5至18元杂芳基、5至14元杂环基、卤素、羟基、氨基、胍基、羧基、氰基、硝基和巯基，它们任选地被R5取代，其中R5选自C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-10环烷基、6至14元芳基、5至18元杂芳基、5至14元杂环基、卤素、羟基、氨基、胍基、羧基、氰基、硝基、C1-3烷基硫基和巯基；
Ra、Rb独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-8环烷基、6至14元芳基C1-3烷基、5至18元杂芳基C1-3烷基和5至14元杂环基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、C1-3烷基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、吡咯基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、C1-10烷基、C1-10烷氧基、6至14元芳基、C3-8环烷基、金刚烷基、膦酰基、膦酰氧基、C1-3烷基膦酰基、C1-3烷基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成3至14元含氮杂环基；
杂原子选自氮、氧、硫、磷和硅；
n为1至10，并且每个重复单元相同或不同；
p为2至6，并且每个重复单元相同或不同。
在上述中间体化合物的任一实施方式中，在式III或IV所示的结构中，优选地，Ra、Rb独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-8环烷基、苯基C1-3烷基、萘基C1-3烷基、5至12元杂芳基C1-3烷基和5至14元杂环基C1-3烷基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、C1-3烷基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、哌嗪基、吡咯烷基、二氧杂环己基、吗啉基、四氢呋喃基、四氢吡咯烷基、C1-10烷基、C1-10烷氧基、苯基、苯甲基、苯乙基、氨基苯基、羧基苯基、卤代苯基、联苯基、C3-8环烷 基、金刚烷基、膦酰基、膦酰氧基、C1-3烷基膦酰基、C1-3烷基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成3至14元含氮杂环基。
在上述中间体化合物的任一实施方式中，在式III或IV所示的结构中，更优选地，Ra、Rb独立地选自-H、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷氧基、C3-6环烷基、苯基C1-3烷基、吡啶基C1-3烷基、吲哚基C1-3烷基、喹啉基C1-3烷基、异喹啉基C1-3烷基、噻吩基C1- 3烷基、噻茚基C1-3烷基、噻唑基C1-3烷基、苯并噻唑基C1-3烷基、咪唑基C1-3烷基、呋喃基C1-3烷基、嘧啶基C1-3烷基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、C1-3烷基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、哌嗪基、吡咯烷基、二氧杂环己基、吗啉基、四氢呋喃基、四氢吡咯烷基、吡啶基、C1-10烷基、C1-10烷氧基、苯基、苯甲基、苯乙基、氨基苯基、羧基苯基、卤代苯基、联苯基、C3-8环烷基、金刚烷基、膦酰基、膦酰氧基、C1-3烷基膦酰基、C1-3烷基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成3至14元含氮杂环基。
在上述中间体化合物的任一实施方式中，在式III或IV所示的结构中，更优选地，Ra、Rb独立地选自-H、甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、乙烯基、丙烯基、1-丁烯基、2-丁烯基、异丁烯基、乙炔基、丙炔基、1-丁炔基、2-丁炔基、3-甲基-2-丙炔基、1,3-丁二烯基、甲氧基、乙氧基、丙氧基、丁氧基、环丙基、环丁基、环戊基、环己基、苯基亚甲基、苯基二亚甲基、吡啶基亚甲基、吡啶基二亚甲基、吲哚基亚甲基、吲哚基二亚甲基、喹啉基亚甲基、喹啉基二亚甲基、异喹啉基亚甲基、异喹啉基二亚甲基、噻吩基亚甲基、噻吩基二亚甲基、噻茚基亚甲基、噻茚基二亚甲基、噻唑基亚甲基、噻唑基二亚甲基、苯并噻唑基亚甲基、苯并噻唑基二亚甲基、咪唑基亚甲基、咪唑基二亚甲基、呋喃基亚甲基、呋喃基二亚甲基、嘧啶基亚甲基和嘧啶基二亚甲基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、亚甲基硫基、二亚甲基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、哌嗪基、吡咯烷基、二氧杂环己基、吗啉基、四氢呋喃基、四氢吡咯烷基、吡啶基、甲基、乙基、丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、苯基、苯甲基、苯乙基、氨基苯基、羧基苯基、卤代苯基、联苯基、环丙基、环丁基、环戊基、环己基、金刚烷基、膦酰基、膦酰氧基、二甲基膦酰基、二乙基膦酰基、二甲基膦酰氧基和二乙基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成咪唑基、噻唑基、吡唑基、嘧啶基、吲哚基、喹啉基、异喹啉基或吡咯基。
在上述中间体化合物的任一实施方式中，在式III或IV所示的结构中，更优选地，R1、R2、R3、R4优选独立地选自-H、C1-10烷基、C1-10烷氧基、C3-10环烷基和5至14元杂环基，它们任选地被R5取代，其中R5选自C1-10烷基、C1-10烷氧基、C3-10环烷基和5至14元杂环基。
在上述中间体化合物的任一实施方式中，在式III或IV所示的结构中，更优选地，其中R1、R2、R3、R4更优选独立地选自-H、C1-3烷基、C1-3烷氧基、C3-6环烷基，它们任选地被R5取代，其中R5选自C1-10烷基、C1-10烷氧基、C3-10环烷基、吡咯基、噻吩基、噻唑基、咪唑基、吡啶基、呋喃基、哌嗪基、吡咯烷基、二氧杂环己基、吗啉基、四氢呋喃基、四氢吡咯烷基。
在上述中间体化合物的任一实施方式中，在式III或IV所示的结构中，更优选地，其中R1、R2、R3、R4更优选独立地选自-H、甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、甲氧基和乙氧基。
在上述中间体化合物的任一实施方式中，在式III或IV所示的结构中，更优选地，R1、R2、R3、R4还更优选独立是-H或甲基。在上述中间体化合物的任一实施方式中，在式III或IV所示的结构中，更优选地，R1、R2、R3、R4均为-H。
在上述中间体化合物的任一实施方式中，优选地，n为2至4，并且每个重复单元相同或不同。更优选地，n为2或3，并且每个重复单元相同或不同。在一些实施方式中，n为2，并且每个重复单元相同。在一些实施方式中，n为3，并且每个重复单元相同。在一些实施方式中，n为2，并且每个重复单元不同。在一些实施方式中，n为3，并且每个重复单元不同。
在上述中间体化合物的任一实施方式中，优选地，p为2至4，并且每个重复单元相同或不同。更优选地，p为2或3，并且每个重复单元相同或不同。在一些实施方式中，p为2，并且每个重复单元相同。在一些实施方式中，p为3，并且每个重复单元相同。在一些实施方式中，p为2，并且每个重复单元不同。在一些实施方式中，p为3，并且每个重复单元不同。
在上述中间体化合物的任一实施方式中，在式III或IV所示的结构中，还更优选地，其中Ra是-H，Rb选自-H、甲基、乙基、丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、环丙基、环戊基、环己基、环己基二亚甲基、环己基亚甲基、环丁基亚甲基、苯基亚甲基、甲基硫基亚甲基、3-吲哚基亚甲基、羟基亚甲基、酰胺基二亚甲基、羟基甲基次甲基、巯基亚甲基、酰胺基亚甲基、羟基苯基亚甲基、羟基苯基二亚甲基、羧基亚甲基、羧基二亚 甲基、氨基正丁基、胍基二亚甲基、胍基三亚甲基、4-咪唑基亚甲基、异丙基苯基亚甲基、1-萘基亚甲基、2-萘基亚甲基、茚满基、苯基二亚甲基、氟代苯基亚甲基、氯代苯基亚甲基、三氟苯基亚甲基、二氯苯基亚甲基、五氟苯基亚甲基、二氟苯基亚甲基、噻吩基亚甲基、噻茚基亚甲基、喹啉基亚甲基、卤代3-吲哚基亚甲基、二苯基亚甲基、3-吡啶基亚甲基和4-吡啶基亚甲基，或者Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基。
在上述中间体化合物的任一实施方式中，在式III或IV所示的结构中，还更优选地，其中Ra、R1、R2、R3、R4独立是-H或甲基；Rb选自-H、甲基、乙基、丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、环丙基、环戊基、环己基、环己基二亚甲基、环己基亚甲基、环丁基亚甲基、苯基亚甲基、甲基硫基亚甲基、3-吲哚基亚甲基、羟基亚甲基、酰胺基二亚甲基、羟基甲基次甲基、巯基亚甲基、酰胺基亚甲基、羟基苯基亚甲基、羟基苯基二亚甲基、羧基亚甲基、羧基二亚甲基、氨基正丁基、胍基二亚甲基、胍基三亚甲基、4-咪唑基亚甲基、异丙基苯基亚甲基、1-萘基亚甲基、2-萘基亚甲基、茚满基、苯基二亚甲基、氟代苯基亚甲基、氯代苯基亚甲基、三氟苯基亚甲基、二氯苯基亚甲基、五氟苯基亚甲基、二氟苯基亚甲基、噻吩基亚甲基、噻茚基亚甲基、喹啉基亚甲基、卤代3-吲哚基亚甲基、二苯基亚甲基、3-吡啶基亚甲基和4-吡啶基亚甲基，或者Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基；n为1至10，优选地为2至4，并且每个重复单元相同或不同；p为2至6，优选地为2至4，并且每个重复单元相同或不同。
在上述中间体化合物的任一实施方式中，在式III或IV所示的结构中，还更优选地，Ra、R1、R2、R3、R4独立是-H或甲基；Rb选自-H、甲基、异丙基和异丁基，或者Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基；n为2至4，并且每个重复单元相同或不同；p为2至4，并且每个重复单元相同或不同。
在上述中间体化合物的任一实施方式中，在式III或IV所示的结构中，还更优选地，Ra、R1、R2、R3、R4独立是-H或甲基；Rb选自-H、甲基、异丙基和异丁基，或者Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基；n为2或3，并且每个重复单元相同或不同；p为2，并且对于p为2的重复单元：在每个重复单元中，Rb是异丙基；在每个重复单元中，Rb是甲基；在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb是甲基；在一个重复单元中，Rb是异丁基，在另一个重复单元中，Rb是甲基；在一个重复单元中，Rb是异丁基，在另一个重复单元中，Rb是异丙基；在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb是氢；在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基；或在一个重复单元中，Rb是氢，在另一 个重复单元中，Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基。
本发明中，所述配体-寡核苷酸共轭物、中间体化合物任一通式中的片段-[NR2-
C(RaRb)-C(O)]-、-[C(O)-C(RaRb)-NR2]-具体可为氨基酸的残基或片段，所述氨基酸如本发明所定义；
所述氨基酸的残基或片段为氨基酸的氨基失去一个氢、羧基失去-OH后的残基或片段；
在任一实施方式中，优选地，-[NR2-C(RaRb)-C(O)]-分别独立的选自
在任一实施方式中，优选地，-[C(O)-C(RaRb)-NR2]-分别独立的选自
所述配体-寡核苷酸共轭物、中间体化合物任一通式中的-[NR2-C(RaRb)-C(O)]p-、-[C(O)-C(RaRb)-NR2]p-，其中p优选为2，即-[NR2-C(RaRb)-C(O)]-[NR2-C(RaRb)-C(O)]-、-[C(O)-C(RaRb)-NR2]-[C(O)-C(RaRb)-NR2]-；
当p为2时，-[NR2-C(RaRb)-C(O)]p-(即-[NR2-C(RaRb)-C(O)]-[NR2-C(RaRb)-C(O)]-)优选为
当p为2时，-[C(O)-C(RaRb)-NR2]p-(即-[C(O)-C(RaRb)-NR2]-[C(O)-C(RaRb)-NR2]-)优选为
在一些实施方式中，所述式III或IV所示的中间体，优选为：


其中，Ro、Ro’、Q2定义如上文所述。
在优选的实施方式中，本文提供选自下式的化合物：

















其中Bn表示苄基保护基，Ac表示乙酰氧基。
制备方法
本发明的另一个方面提供一种制备配体-寡核苷酸共轭物的方法，其包括：(a)提供本文所述的任一中间体化合物；(b)提供寡核苷酸，所述寡核苷酸的5’或3’端具有末端氨基；(c)将所述中间体化合物与所述寡核苷酸通过所述氨基连接；以及任选地，(d)脱保护的步骤(优选为脱Ac的步骤)。
在一些实施方式中，在步骤(c)中，所述中间体化合物附接至所述寡核苷酸的5’ 端。在一些实施方式中，在步骤(c)中，所述中间体化合物附接至所述寡核苷酸的3’端。通常，在固相载体上在3’至5’方向上合成寡核苷酸。当所述中间体化合物要附接至寡核苷酸的3’端时，需要将中间体化合物预先与3’核苷共轭并连接至固相载体，再按常规合成方法(例如亚磷酰胺法)合成寡核苷酸。然而，预先共轭并连接至固相载体增加了合成的复杂性，此外，使用该方法，共轭物将存在于寡核苷酸的整个合成过程中，在此期间可能发生降解，也可能限制可使用的反应和试剂的种类。
本文不排除将所述中间体化合物连接至核苷酸的3’端，但优选地，将所述中间体化合物连接至寡核苷酸的5’端。当将中间体化合物连接至寡核苷酸的5’端时，寡核苷酸可按常规合成方法(例如亚磷酰胺法)在固相载体上由3’向5’方向合成。所述中间体化合物可以与5’端的最后一个(最5’)核苷一起或在寡核苷酸从固相载体上裂解之后引入共轭物。
在一些实施方式中，在合成寡核苷酸时，在5’端的最后一个(最5’)核苷之后，引入末端氨基，从而使得所合成的寡核苷酸具有5’末端氨基。在这样的实施方式中，所合成的寡核苷酸可具有式XI、XI’所示的结构：
3’-寡核苷酸-5’-OP(O)(OH)O-(CH2-Y)x-NH2(式XI)
3’-寡核苷酸-5’-OP(S)(OH)O-(CH2-Y)x-NH2(式XI’)
其中Y代表O或不存在，x为3至12并且每个重复单元相同或不同。
在一些实施方式中，所合成的寡核苷酸的结构式为：3’-寡核苷酸-5’-OP(O)(OH)O-(CH2)6-NH2。在一些实施方式中，所合成的寡核苷酸的结构式为：3’-寡核苷酸-5’-OP(O)(OH)O-(CH2)3-NH2。在一些实施方式中，所合成的寡核苷酸的结构式为：3’-寡核苷酸-5’-OP(O)(OH)O-(CH2)12-NH2。在一些实施方式中，所合成的寡核苷酸的结构式为：3’-寡核苷酸-5’-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH2。
在一些实施方式中，所合成的寡核苷酸的结构式为：3’-寡核苷酸-5’-OP(S)(OH)O-(CH2)6-NH2。在一些实施方式中，所合成的寡核苷酸的结构式为：3’-寡核苷酸-5’-OP(S)(OH)O-(CH2)3-NH2。在一些实施方式中，所合成的寡核苷酸的结构式为：3’-寡核苷酸-5’-OP(S)(OH)O-(CH2)12-NH2。在一些实施方式中，所合成的寡核苷酸的结构式为：3’-寡核苷酸-5’-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH2。
在合成的寡核苷酸的5’端引入末端氨基的试剂和方法是本领域已知的。合适的5’端末端氨基引入剂(或称改性剂或连接剂)包括但不限于5’-氨基TFA(C6)(CAS#133975-85-6)、5’-氨基TFA(C3)(CAS#853955-89-2)、5’-氨基TFA(C12)(CAS#178925-51-4)和5’-氨基改性剂-5-CEP(CAS#612548-86-4)等。
在一些实施方式中，为了在寡核苷酸的3’-末端引入氨基，直接采用端基修饰剂作为起始固体支撑物，从而得到的寡核苷酸具有3’末端氨基。在这样的实施方式中，所合成的寡核苷酸可具有式XI-1、XI-1’所示的结构：
5’-寡核苷酸-3’-OP(O)(OH)O-(CH2-Y)x-NH2(式XI-1)
5’-寡核苷酸-3’-OP(S)(OH)O-(CH2-Y)x-NH2(式XI-1’)
其中Y代表O或不存在，x为3至12并且每个重复单元相同或不同。
在一些实施方式中，所合成的寡核苷酸的结构式为：5’-寡核苷酸-3’-OP(O)(OH)O-(CH2)6-NH2。在一些实施方式中，所合成的寡核苷酸的结构式为：5’-寡核苷酸-3’-OP(O)(OH)O-(CH2)3-NH2。在一些实施方式中，所合成的寡核苷酸的结构式为：5’-寡核苷酸-3’-OP(O)(OH)O-(CH2)12-NH2。在一些实施方式中，所合成的寡核苷酸的结构式为：5’-寡核苷酸-3’-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH2。
在一些实施方式中，所合成的寡核苷酸的结构式为：5’-寡核苷酸-3’-OP(S)(OH)O-(CH2)6-NH2。在一些实施方式中，所合成的寡核苷酸的结构式为：5’-寡核苷酸-3’-OP(S)(OH)O-(CH2)3-NH2。在一些实施方式中，所合成的寡核苷酸的结构式为：5’-寡核苷酸-3’-OP(S)(OH)O-(CH2)12-NH2。在一些实施方式中，所合成的寡核苷酸的结构式为：5’-寡核苷酸-3’-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH2。
在合成的寡核苷酸的3’端引入末端氨基的试剂和方法是本领域已知的。合适的3’端末端氨基引入剂(或称端基修饰剂)包括但不限于3'-PT-Amino-Modifier C6CPG等。
在一些实施方式中，所述寡核苷酸是siRNA，其包含正义链和反义链，所述中间体化合物连接至所述siRNA的正义链。在一些实施方式中，在步骤(c)中，所述中间体化合物附接至所述siRNA的正义链的3’端。在优选的实施方式中，在步骤(c)中，所述中间体化合物附接至所述siRNA的正义链的5’端。
在一些实施方式中，在合成siRNA的正义链时，在5’端的最后一个(最5’)核苷之后，引入末端氨基，从而使得所合成的正义链具有5’末端氨基。在这样的实施方式中，所合成的siRNA正义链可具有式XII、XII’所示的结构：
3’-siRNA正义链-5’-OP(O)(OH)O-(CH2-Y)x-NH2(式XII)
3’-siRNA正义链-5’-OP(S)(OH)O-(CH2-Y)x-NH2(式XII’)
其中Y代表O或不存在，x为3至12并且每个重复单元相同或不同。
在一些实施方式中，所合成的siRNA正义链的结构式为：3’-siRNA正义链-5’-OP(O)(OH)O-(CH2)6-NH2。在一些实施方式中，所合成的siRNA正义链的结构式为：3’- siRNA正义链-5’-OP(O)(OH)O-(CH2)3-NH2。在一些实施方式中，所合成的siRNA正义链的结构式为：3’-siRNA正义链-5’-OP(O)(OH)O-(CH2)12-NH2。在一些实施方式中，所合成的siRNA正义链的结构式为：3’-siRNA正义链-5’-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH2。
在一些实施方式中，所合成的siRNA正义链的结构式为：3’-siRNA正义链-5’-OP(S)(OH)O-(CH2)6-NH2。在一些实施方式中，所合成的siRNA正义链的结构式为：3’-siRNA正义链-5’-OP(S)(OH)O-(CH2)3-NH2。在一些实施方式中，所合成的siRNA正义链的结构式为：3’-siRNA正义链-5’-OP(S)(OH)O-(CH2)12-NH2。在一些实施方式中，所合成的siRNA正义链的结构式为：3’-siRNA正义链-5’-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH2。
在一些实施方式中，在合成siRNA的正义链时，为了在siRNA的正义链的3’-末端引入氨基，直接采用端基修饰剂作为起始固体支撑物，从而得到的siRNA的正义链具有3’末端氨基。在这样的实施方式中，所合成的siRNA正义链可具有式XII-1、XII-1’所示的结构：
5’-siRNA正义链-3’-OP(O)(OH)O-(CH2-Y)x-NH2(式XII)
5’-siRNA正义链-3’-OP(S)(OH)O-(CH2-Y)x-NH2(式XII’)
其中Y代表O或不存在，x为3至12并且每个重复单元相同或不同。
在一些实施方式中，所合成的siRNA正义链的结构式为：5’-siRNA正义链-3’-OP(O)(OH)O-(CH2)6-NH2。在一些实施方式中，所合成的siRNA正义链的结构式为：5’-siRNA正义链-3’-OP(O)(OH)O-(CH2)3-NH2。在一些实施方式中，所合成的siRNA正义链的结构式为：5’-siRNA正义链-3’-OP(O)(OH)O-(CH2)12-NH2。在一些实施方式中，所合成的siRNA正义链的结构式为：5’-siRNA正义链-3’-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH2。
在一些实施方式中，所合成的siRNA正义链的结构式为：5’-siRNA正义链-3’-OP(S)(OH)O-(CH2)6-NH2。在一些实施方式中，所合成的siRNA正义链的结构式为：5’-siRNA正义链-3’-OP(S)(OH)O-(CH2)3-NH2。在一些实施方式中，所合成的siRNA正义链的结构式为：5’-siRNA正义链-3’-OP(S)(OH)O-(CH2)12-NH2。在一些实施方式中，所合成的siRNA正义链的结构式为：5’-siRNA正义链-3’-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH2。
在这样的实施方式中，siRNA的反义链可按常规合成方式合成并通过退火而与正义链通过碱基配对方式连接，以形成双链RNA。
本发明中，所述退火或模拟退火操作为：将纯度合格(RP-HPLC纯度>90％)的互补链(SS链和AS链)通过等摩尔比进行混合，将该溶液置于70℃的水浴锅中，加热至90℃，在90℃下保持5分钟，并缓慢冷却至室温。取样进行SEC检测其双链退火纯度，微调SS链或AS链的比例，使其双链纯度达到90％以上即可。将siRNA冻干并储存在- 15℃至-25℃。
在优选的实施方式中，所述siRNA中的核苷酸的一个或多个被修饰。在优选的实施方式中，所述修饰为2’-O-甲基修饰和/或2’-氟基修饰。在优选的实施方式中，所述修饰为骨架中的硫代磷酸修饰。在优选的实施方式中，所述修饰包括2’-O-甲基修饰、2’-氟基修饰和骨架中的硫代磷酸修饰。
在一些实施方式中，所述寡核苷酸是ASO，所述中间体化合物连接至所述ASO的5’端或3’端。在一些实施方式中，在步骤(c)中，所述中间体化合物附接至所述ASO的3’端。在优选的实施方式中，在步骤(c)中，所述中间体化合物附接至ASO的5’端。
在一些实施方式中，在ASO时，在5’端的最后一个(最5’)核苷之后，引入末端氨基，从而使得ASO具有5’末端氨基。在这样的实施方式中，所合成的ASO可具有式XIII、XIII’所示的结构：
3’-ASO-5’-OP(O)(OH)O-(CH2-Y)x-NH2(式XIII)
3’-ASO-5’-OP(S)(OH)O-(CH2-Y)x-NH2(式XIII’)
其中Y代表O或不存在，x为3至12并且每个重复单元相同或不同。
在一些实施方式中，所合成的ASO结构式为：3’-ASO-5’-OP(O)(OH)O-(CH2)6-NH2。在一些实施方式中，所合成的ASO的结构式为：3’-ASO-5’-OP(O)(OH)O-(CH2)3-NH2。在一些实施方式中，所合成的ASO的结构式为：3’-ASO-5’-OP(O)(OH)O-(CH2)12-NH2。在一些实施方式中，所合成的ASO的结构式为：3’-ASO-5’-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH2。
在一些实施方式中，所合成的ASO结构式为：3’-ASO-5’-OP(S)(OH)O-(CH2)6-NH2。在一些实施方式中，所合成的ASO的结构式为：3’-ASO-5’-OP(S)(OH)O-(CH2)3-NH2。在一些实施方式中，所合成的ASO的结构式为：3’-ASO-5’-OP(S)(OH)O-(CH2)12-NH2。在一些实施方式中，所合成的ASO的结构式为：3’-ASO-5’-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH2。
在一些实施方式中，在ASO时，为了在ASO的3’-末端引入氨基,直接采用端基修饰剂作为起始固体支撑物，从而得到的ASO具有3’末端氨基。在这样的实施方式中，所合成的ASO可具有式XIII-1、XIII-1’所示的结构：
5’-ASO-3’-OP(O)(OH)O-(CH2-Y)x-NH2(式XIII-1)
5’-ASO-3’-OP(S)(OH)O-(CH2-Y)x-NH2(式XIII-1’)
其中Y代表O或不存在，x为3至12并且每个重复单元相同或不同。
在一些实施方式中，所合成的ASO结构式为：5’-ASO-3’-OP(O)(OH)O-(CH2)6-NH2。在一些实施方式中，所合成的ASO的结构式为：5’-ASO-3’-OP(O)(OH)O-(CH2)3-NH2。在一 些实施方式中，所合成的ASO的结构式为：5’-ASO-3’-OP(O)(OH)O-(CH2)12-NH2。在一些实施方式中，所合成的ASO的结构式为：5’-ASO-3’-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH2。
在一些实施方式中，所合成的ASO结构式为：5’-ASO-3’-OP(S)(OH)O-(CH2)6-NH2。在一些实施方式中，所合成的ASO的结构式为：5’-ASO-3’-OP(S)(OH)O-(CH2)3-NH2。在一些实施方式中，所合成的ASO的结构式为：5’-ASO-3’-OP(S)(OH)O-(CH2)12-NH2。在一些实施方式中，所合成的ASO的结构式为：5’-ASO-3’-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH2。
在优选的实施方式中，所述ASO中的核苷酸的一个或多个被修饰。在优选的实施方式中，所述修饰选自硫代磷酸(PSP)、二氨基吗啉代(PMO)、2’-甲氧基乙基(2’-MOE)和5-甲基胞嘧啶(5mC)。在优选的实施方式中，所述修饰包括PSP、PMO、2’-MOE和5mC。
步骤(c)中，将所述中间体化合物与所述寡核苷酸通过所述氨基连接以形成所述配体-寡核苷酸共轭物，包括将所述中间体化合物的游离羧基或其羧基保护形式(例如活泼酯形式)与寡核苷酸的游离末端氨基反应以将中间体化合物与所述寡核苷酸共价连接。
本发明还提供了本文所述的任一中间体化合物在制备配体-寡核苷酸共轭物中的用途。在一些实施方式中，所述配体-寡核苷酸共轭物如本文以上所定义。在优选的实施方式中，所述配体-寡核苷酸共轭物具有如式V、V’、VI、VI’、VII、VII’、VIII、VIII’、IX、IX’、X、X’所示的结构；其中各取代基和符号具有本文如上所定义的相应含义、数值和数值范围，或其优选含义、数值和数值范围。
药物组合物
本发明的另一个方面提供一种药物组合物，其包含本文所述的任一配体-寡核苷酸共轭物以及药学上可接受的载体。
本发明的共轭物可以被配制用于药物用途。药学上可接受的组合物包括治疗有效量的以上所述共轭物中的一种或多种，单独使用或与一种或多种药学上可接受的载体(添加剂)、赋形剂和/或稀释剂配制在一起。
可以通过从其他药物类推将根据本发明的共轭物配制为用于以任何便利方式给药以在人类或兽医学中使用。
治疗方法
在另一个方面，本文提供一种治疗疾病的方法，其包括向受试者施用治疗有效量的本文提供的任一种共轭物或包含该共轭物的药物组合物。
在一个实施例中，该组合物包括多个共轭物种类。在另一个实施例中，该共轭物种类具有相对于一种天然存在的靶序列与另一个种类不重叠且不相邻的序列。在另一个实施例 中，该多个共轭物种类特异性针对不同的天然存在的靶基因。在另一个实施例中，该共轭物是等位基因特异性的。
本发明的实施例还涉及用于抑制一种靶基因的表达的方法。该方法包括以足够抑制该靶基因的表达的量给予在以上任一种共轭物的步骤。
另一方面，本发明涉及一种调节细胞中的一种靶基因的表达的方法，该方法包括向所述细胞提供一种本发明的共轭物或其组合物。
本发明中，寡核苷酸、siRNA、ASO的靶基因选自下组，该组由以下各项组成：因子VII、Eg5、PCSK9、TPX2、apo(a)、apoB、SAA、TTR、RSV、PDGFβ基因、Erb-B基因、Src基因、CRK基因、GRB2基因、RAS基因、MEKK基因、JNK基因、RAF基因、Erk1/2基因、PCNA(p21)基因、MYB基因、JUN基因、FOS基因、BCL-2基因、hepciden、活化蛋白C、细胞周期蛋白D基因、VEGF基因、EGFR基因、细胞周期蛋白A基因、细胞周期蛋白E基因、WNT-1基因、β-连环蛋白基因、c-MET基因、PKC基因、NFKB基因、STAT3基因、生存素基因、Her2/Neu基因、拓扑异构酶I基因、拓扑异构酶IIα基因、p73突变基因、在p21(WAF1/CIP1)基因中的突变、在p27(KIP1)基因中的突变、在PPM 1D基因中的突变、在RAS基因中的突变、在小窝蛋白I基因中的突变、在MIB I基因中的突变、在MTAI基因中的突变、在M68基因中的突变、在肿瘤抑制基因中的突变以及在p53肿瘤抑制基因中的突变。
本发明由以下实施例进一步展示，这些实例不应被视为进一步限制性的。贯穿本申请引用的所有参考文献、专利申请和公开的专利的内容清楚地特此通过引用而结合。
实施例
实施例1
第一步：化合物2：将化合物1(25g,64.21mmol)溶于1,2-二氯乙烷(350mL)
(加入4A分子筛10g)中搅拌30min，TMSOTf(17.13g,77.08mmol)滴加到反应液，氮气 保护下50℃反应4h。TLC检测反应完毕。反应冷却至室温，37.5mL三乙胺缓慢滴入反应液,旋干得棕色液体化合物2(21.1g粗品)。无纯化，直接用于下一步。MS m/z(ESI):330.1[M+H]+
第二步：化合物3：将化合物2(21.1g,64.07mmol)和5-己烯基-1-醇(7.06g,70.49mmol)溶于1,2-二氯乙烷(350mL)，加入4A分子筛20g(除水)室温搅拌30min。TMSOTf(7.12g,32.04mmol)在0℃氮气保护条件下滴加加到反应液，室温反应3h。反应液倒入到冷的饱和NaHCO3水溶液(100mL)，DCM(100mL×3)萃取，水洗(100mL×3)，无水硫酸钠干燥，过滤，浓缩。浓缩后粗产物加入石油醚打浆(50mL×3),洗去杂质，获得化合物3(19.2g，收率：70％)。无纯化，直接投入下一步反应。MS m/z(ESI):430.2[M+H]+
第三步：化合物4：将化合物3(35g,81.50mmol)溶于二氯甲烷/乙腈(1:1,200mL)，冰浴条件下加入溶有高碘酸钠(76.4g,357.2mmol)的去离子水(120mL)。搅拌15min。RuCl3(560mg,2.70mmol)在冰浴条件下加入反应液，缓慢升温至室温，室温搅拌1h。高碘酸钠(19.1g,89.3mmol)室温加入到反应液继续反应过夜。TLC监测显示原料反应完全。100mL水加入到反应液中，过滤，滤饼用二氯甲烷洗(100mL×5),滤液用二氯甲烷(100mL×5)萃取，丢弃有机相，收集水相，用柠檬酸调节pH到3.5，用二氯甲烷：甲醇(10：1)萃取水相，合并有机相，用无水硫酸钠干燥，过滤，浓缩。得化合物4(23.2g，收率：63.62％)。MS m/z(ESI):448.2[M+H]+
第四步：化合物5：将化合物4(20g,44.7mmol)溶于DCM(400mL)加入DIEA(8.67g,67.08mmol)，0℃下加入三氟乙酸五氟苯酯(19g,67.84mmol),室温反应3h。TLC监测反应，反应液用1M硫酸氢钾水溶液(200mL)水洗,DCM(200mL×2)萃取，合并有机相，无水硫酸钠干燥，过滤，旋干。柱纯化(PE:EtOAc＝1:2)得到化合物5(22.9g,收率83.51％)。MS m/z(ESI):614.2[M+H]+
实施例2
第一步：化合物7：于反应瓶中，加入三羟甲基甲胺(化合物6)(100g，0.825mol)，1,4-二氧六环(700mL)，机械搅拌，冰水浴降温0-5℃，加入氢氧化钾水溶液60％(1.3g氢氧化钾溶于0.9g水中)，0-5℃搅拌1h，加入丙烯腈(130g，2.45mol)加完自然升温至室温15-25℃搅拌反应16h。反应完毕后，过滤，二氯甲烷洗涤(500mL)，固体浓缩干燥回收原料三羟甲基甲胺21.5g，将滤液60℃减压浓缩干，加水(500mL)，二氯甲烷(200mL)，萃取分液，水相用二氯甲烷萃取(200mL×3)，合并有机相二氯甲烷层，用饱和食盐水(100mL)洗涤，无水硫酸钠干燥后浓缩，得7粗品161.7g。
用甲醇(100mL)将草酸37g溶解后，加入到7粗品中，搅拌溶清，升温至64℃，加入乙酸乙酯(2L)，搅拌下缓慢降温至室温25℃结晶，析出大量白色固体，抽滤，乙酸乙酯(500mL)洗涤，得到滤饼194.6g，60℃真空干燥4h，得149.2g，将固体溶解在甲醇(100mL)中，升温至70℃后加入乙酸乙酯(2L)，溶液略微浑浊，降温至室温25℃结晶，抽滤，乙酸乙酯洗涤，得到滤饼212.2g,60℃真空干燥4h得干品化合物7的草酸盐116g。将草酸盐转入烧瓶中，加甲醇(58mL)，升温回流溶清，加乙酸乙酯(1.16L)，析出固体，回流打浆1h，降温至室温25℃搅拌1h,过滤，乙酸乙酯(116mL)漂洗，得湿精品，用饱和碳酸钠水溶液(300mL)游离，用二氯甲烷(300mL)萃取，无水硫酸钠干燥后浓缩，得化合物7精品(24.6g，收率21.19％)。
第二步：化合物8：于反应瓶中，加入7(160g，0.57mol)，乙醇/氯化氢(480g),搅拌，氮气保护，升温回流反应16h。反应完后，过滤，固体为氯化铵，将滤液浓缩后，加碳 酸氢钠水溶液(200mL)中和至中性，用二氯甲烷萃取(200mL×4)，饱和食盐水洗涤(200mL×1)，无水硫酸钠干燥，浓缩干得化合物8(150g，收率62.43％)油状物。
第三步：化合物9：将化合物8(40g,94.9mmol)溶于1,4-二氧六环(160mL)，搅拌溶清。另取反应瓶加入Na2CO3(13.08g,123.37mmol)溶于纯化水(80mL)，搅拌溶清。0-5℃下将配好的碱溶液加入到上述反应体系中,搅拌反应30min。
称取氯甲酸苄酯(21.05g,123.39mmol)控温0-5℃下滴加到体系中。过程升温明显，体系逐渐转变为白色浑浊液，有类似絮状物。滴毕，反应升至室温，搅拌反应18h。LC/MS监测。反应体系旋去二氧六环，剩余水相加入EA(80mL×2)萃取。合并有机相，加入饱和食盐水洗，分液。有机相加入无水硫酸钠干燥；过滤，旋干，得淡黄色油状物化合物9粗品(50g,收率94.8％)。MS m/z(ESI):556.3[M+H]+
第四步：化合物10：将化合物9(50g,89.99mmol)溶于THF(80mL)和MeOH(80mL)，搅拌溶清，加入LiOH(21.55g,899.79mmol)溶于纯化水(140mL)，再加入反应体系中，反应室温搅拌过夜。LC/MS监测。体系减压除去THF和甲醇，浓缩物中加入纯化水(100mL)，EA(200mL)萃取一次，分液，取水相。水相用浓盐酸调pH＝2～3之间，体系变为白色浑浊液，降温0℃，无明显固体析出。体系加入DCM萃取(100mL×2)。合并有机相无水硫酸钠干燥，过滤，旋干得到稠状油状物化合物10粗品(40.8g,收率96％)。MS m/z(ESI):472.2[M+H]+
第五步：化合物11：将化合物10(40g,84.84mmol)溶于DCM(400mL)搅拌溶清，加入DMF(4mL)，N2置换3次。常温N2保护滴加二氯亚砜(62mL)。滴毕，缓慢升温至回流，回流反应3.0h。LC/MS监测(取样加入MeOH淬灭)。减压旋干溶剂，加入DCM(200mL)夹带一次。旋至无溶剂流出。称重，得到黄色带有少量固体油状物化合物11粗品(43.03g，收率96％)。
第六步：化合物12：将叔丁氧羰基丙二胺(59.1g,339.2mmol)加入到三口瓶中，加入DIEA(109.6g,847.9mmol)，DCM(360mL)溶清后，氮气置换3次，氮气保护下降温0℃。化合物11(44.7g,84.85mmol)溶于DCM(300mL)中，控温滴加到上述体系中。随着滴加，体系逐渐变浑浊，最后变成浑白色悬浊体系，搅拌30min后升至室温反应过夜。LC/MS监测。体系减压除去溶剂，加入纯化水(200mL)，再加入EA(300mL×2)萃取。合并有机相，加入10％柠檬酸(200mL×2)洗，10％碳酸钠溶液洗(200mL×2)，饱和食盐水(200mL)洗一次。有机相无水硫酸钠干燥，过滤，旋干，得到淡黄色油状物化合物12粗品(63.2g,67.22mmol)收率79％)。MS m/z(ESI):940.6[M+H]+
第七步：化合物13：将化合物12(30g,31.91mmol)溶于MeOH(500mL)溶清，加入20％Pd(OH)2/C(8.2g)，加入TFA(10mL)，室温反应过夜。LC/MS监测。加入硅藻土过滤钯炭。体系加入过量碳酸氢钠固体(过量，有固体存在)搅拌充分。过滤。滤液旋干，加入MeOH(20mL)溶解后，反相柱层析(甲醇：水＝5％-100％)纯化得到化合物13(24.7g,收率96.2％)。MS m/z(ESI):806.5[M+H]+
实施例3

第一步：A1：Boc-Val-Val-OH(2.82g，8.91mmol)溶于DMF(18mL)中，加入碳酸铯(8.34g，25.61mmol)，搅拌状态下加入溴化苄(4.22g，24.65mmol)室温反应1.5h。LC/MS监测反应。加水，乙酸乙酯萃取三次，合并有机相，饱和食盐水洗一次，无水硫酸钠干燥，过滤，浓缩，柱层析纯化(乙酸乙酯/石油醚＝0-18％)得到A1(3.6g，收率99％)。MS m/z(ESI):407.3[M+H]+
第二步：A2：A1(3.6g，8.86mmol)溶于二氯甲烷(30mL)中，加入三氟乙酸(6mL)，室温反应16h。TLC监测反应，原料反应完全后，浓缩得到A2的三氟乙酸盐(3.7g，收率100％)。
第三步：A3：A2的三氟乙酸盐(3.45g，8.26mmol)溶于二氯甲烷(30mL)中，加入三乙胺(8.3g，82.02mmol)，丁二酸酐(2.46g，24.58mmol)，室温反应过夜。LC/MS监测反应。浓缩，反相柱层析纯化(甲醇/水＝5％-95％)后得到化合物A3(3.2g，收率95％)。MS m/z(ESI):407.2[M+H]+
第四步：A4：A3(1.00g，2.46mmol)溶于DMF(5mL)中，加入HATU(1.12g,2.95mmol)，DIEA(3.17mg，24.60mmol)，室温搅拌15min后，加入化合物13(1.88g，2.33mmol)，室温搅拌过夜。LC/MS监测反应。加水(100mL)，二氯甲烷(40mL×3次)萃取，合并有机相，饱和食盐水洗一次，加无水硫酸钠，过滤，干燥，浓缩。柱层析(二氯甲烷：甲醇＝0-20％)纯化得到化合物A4(1.7g，58％)。MS m/z(ESI):497.9[M/2+H]+
第五步：A5：A4(1.60g，1.34mmol)溶于二氯甲烷(50mL)中，加入三氟乙酸(10mL)，室温反应2h。LC/MS监测反应。原料反应完全后，直接浓缩，得到化合物A5的三氟乙酸盐(1.12g)。MS m/z(ESI):447.9[M/2+H]+
第六步：A6：A5的三氟乙酸盐(1.12g，1.34mmol)溶于二氯甲烷(10mL)，加入DIEA(2.32g,17.98mmol)，冰浴条件下搅拌状态下加入化合物5(2.40g，3.91mmol)的二氯甲 烷溶液(10mL)，随后缓慢升温至室温反应18h。LC/MS监测。加二氯甲烷20mL稀释，水洗一次，饱和食盐水洗一次，无水硫酸钠干燥，过滤，浓缩。反相柱层析(乙腈：水＝5％-100％)纯化得到化合物A6(2.00g,收率68％)。MS m/z(ESI):1091.6[M/2+H]+
第七步：A7：A6(2.00g,0.92mmol)溶于四氢呋喃(35mL)中，加入20％氢氧化钯碳(0.40g)，氢气置换体系三次后，室温搅拌5h。TLC监测原料反应完全。反应液用硅藻土过滤除氢氧化钯碳，浓缩。反相柱层析纯化(乙腈/水＝5％-100％)，得到化合物A7(0.35g,收率:18％)。MS m/z(ESI):1046.7[M/2+H]+
实施例4

第一步：B1：Boc-Val-Pro-OH(5.34g，17mmol)溶于DMF(50mL)中，加入碳酸铯(21.76g，66.8mmol)，搅拌状态下加入溴化苄(10.88g，63.61mmol),室温反应1.5h。加水200mL稀释，乙酸乙酯(70mL×3)萃取，合并有机相，饱和食盐水洗一次，无水硫酸钠干燥，过滤，浓缩。柱层析(EA:PE＝0-50％)纯化。得到化合物B1(6.75g，收率98％)。MS m/z(ESI):405.3[M+H]+
第二步：B2：B1(6.75g，16.69mmol)溶于二氯甲烷(10mL)中，加入三氟乙酸(10mL)，室温反应2h后，LC/MS监测。原料反应完全，直接浓缩，得到化合物B2的三氟乙酸盐(5.08g)。MS m/z(ESI):305.2[M+H]+
第三步：B3：B2的三氟乙酸盐(5.08g，16.69mmol)溶于二氯甲烷(50mL)中，加入三乙胺(7.48mg,73.92mmol)，搅拌状态下加入丁二酸酐(2.96g，29.58mmol)，室温反应过夜。LC/MS监测反应。浓缩，反向柱层析纯化(甲醇：水＝5％-100％)后得到化合物B3(6.38g，95％)。MS m/z(ESI):405.2[M+H]+
第四步：B4：化合物B3(6.29g，15.55mmol)溶于DMF(30mL)中，加入 DIEA(6.05g，46.82mmol)，搅拌状态下加入HATU(7.12g,18.73mmol)，室温搅拌15min后，加入化合物13(12.58g,15.61mmol)的DMF(10mL)溶液，室温反应过夜。LC/MS监测反应。加水(200mL)，1M HCl水溶液调pH＝6，二氯甲烷(70mL×3)萃取，饱和食盐水洗涤一次，无水硫酸钠干燥，过滤，浓缩。反相柱层析(乙腈/水＝5％-100％)纯化，得到化合物B4(10.9g收率58.8％)。MS m/z(ESI):496.9[M/2-100+H]+
第五步：B5：B4(2g，1.68mmol)溶于二氯甲烷(20mL)中，加入三氟乙酸(20mL)，室温搅拌反应2h。LC/MS监测反应。浓缩，得到化合物B5的三氟乙酸盐(1.40g)。MS m/z(ESI):446.9[M/2+H]+
第六步：B6：化合物B5的三氟乙酸盐(1.34g,1.5mmol)溶于吡啶(10mL)中，加入化合物5(3.69g，6.01mmol)，60℃反应3h。LC/MS监测反应。加水100mL，2M盐酸水溶液调pH＝2，二氯甲烷萃取，饱和食盐水洗涤，无水硫酸钠干燥，过滤，浓缩，反相柱层析纯化(乙腈/水＝5％-100％)得到化合物B6(0.50g,收率15％)。MS m/z(ESI):1090.7[M/2+H]+
第七步：B7：化合物B6(0.50g，0.23mmol)溶于四氢呋喃(20mL)，加入20％氢氧化钯碳(0.10g)，氢气置换体系三次，后于氢气氛围下室温搅拌过夜。LC/MS监测反应，原料反应完全。滤膜过滤除去20％氢氧化钯碳，浓缩。反相纯化(乙腈/水＝5％-100％)，得到化合物B7(50mg，收率：10％)。MS m/z(ESI):1045.6[M/2+H]+
实施例5

第一步：C1：Boc-Ala-Ala-OH(5g，19.2mmol)溶于50mL DMF中，加入碳酸铯(13.5g,41.4mmol)，搅拌状态下加入溴化苄(13.14g,76.8mmol)，室温反应2h。TLC监测反应。加水200mL稀释，乙酸乙酯(70mL×3)萃取三次，合并有机相，饱和食盐水洗一次，无水硫酸钠干燥，过滤，浓缩，柱层析纯化(乙酸乙酯/石油醚＝0-50％)，得到C1(6.1g，收率91％)。MS m/z(ESI):351.3[M+H]+
第二步：C2：C1(6.1g，17.4mmol)溶于二氯甲烷(50mL)中，加入三氟乙酸(10mL)，室温反应过夜。TLC监测反应，原料反应完全后，浓缩得到产物C2的三氟乙酸盐(4.355g，收率100％)。
第三步：C3：C2的三氟乙酸盐(4.355g,17.4mmol)溶于二氯甲烷(50mL)中，加入三乙胺(17.59g，173.83mmol)，丁二酸酐(3.48g，34.78mmol)，室温反应过夜。LC/MS监 测反应。浓缩，柱纯化(EA:PE＝1:5)得产物C3(4.98g，收率82％)。MS m/z(ESI):351.2[M+H]+
第四步：C4：C3(3.0g，8.56mmol)溶于DMF(15mL)中，加入HATU(4.23g，11.12mmol)，DIEA(3179mg，24.59mmol)，室温搅拌15min后，加入化合物13(6.55g,8.13mmol)，室温搅拌过夜。LC/MS监测反应。加水(100mL)，二氯甲烷(50mL×3)萃取，合并有机相，饱和食盐水洗一次，加无水硫酸钠，过滤，干燥，浓缩。柱层析(MeOH：DCM＝0-20％)纯化得到化合物C4(2.51g，26％)。MS m/z(ESI):469.8[M/2-100+H]+
第五步：C5：C4(2.51g.2.2mmol)溶于二氯甲烷(50mL)中，加入三氟乙酸(10mL)，室温反应2h。TLC监测反应。原料反应完全后，直接浓缩，得到化合物C5的三氟乙酸盐(1.84g,收率100％)。
第六步：C6：C5的三氟乙酸盐(1.84g，2.2mmol)溶于二氯甲烷(50mL)，加入DIEA(2.86g,22.10mmol)，冰浴条件搅拌状态下加入化合物5(4.21g,6.86mmol)的二氯甲烷溶液(20mL)，随后缓慢升温至室温反应18h。LC/MS监测。加二氯甲烷20mL稀释，水洗一次，饱和食盐水洗一次，无水硫酸钠干燥，过滤，浓缩。反相柱层析(乙腈：水＝5％-100％)纯化得到化合物C6(0.50g,收率11％)。MS m/z(ESI):518.4[M/4+H]+
第七步：C7：C6(0.50g，0.24mmol),溶于四氢呋喃(10mL)中,加入20％氢氧化钯碳(0.11g),氢气置换体系三次后，室温搅拌5h。反应完毕后，反应液用硅藻土过滤除氢氧化钯碳，浓缩。得到化合物C7粗品(0.11g,22％)。MS m/z(ESI):1019.0[M/2+H]+，511.0[M/4+H]+
实施例6


第一步：D2：将D1(5g,22.50mmol)溶于二氯甲烷(50mL)加入DIEA(4.36g,33.74mmol)，加入三氟乙酸五氟苯酯(9.45g,33.74mmol),室温反应4h。反应结束后，反应液用二氯甲烷(10mL)稀释，用1M硫酸氢钾水溶液(50mL×2)水洗,饱和碳酸氢钠水溶液(50mL×2)，水(50mL)洗有机相，无水硫酸钠干燥，过滤，旋干。柱纯化(PE:EtOAc＝10:1)得D2(6.4g,收率：73％)。MS m/z(ESI):389.0[M+H]+
第二步：D3：将化合物13(5.0g,6.20mmol),Cbz-Val-Ala-OH(2.6g,8.06mmol)溶于40mL二氯甲烷中，加入DIEA(2.41g,18.6mmol),HATU(3.54g,9.31mmol)，在40℃反应16h。反应结束后，反应液用50mL二氯甲烷稀释，加入50mL水，分液，水相用二氯甲烷萃取(50mL×3)，合并有机相，无水硫酸钠干燥，过滤，旋干。柱纯化(DCM:MeOH＝10:1)得化合物D3为无色粘稠液体(5.4g,收率：78％)。MS m/z(ESI):455.9[M/2-100+H]+
第三步：D4：D3(2.8g,2.52mmol)溶于30mL甲醇,加入20％Pd(OH)2/C(0.5g),三氟乙酸(0.86g,7.54mmol)，氢气球置换气体三次，氢气条件下室温反应4h。硅藻土过滤，用甲醇(30mL×3)洗滤饼，旋干得D4的三氟乙酸盐，直接用于下一步。MS m/z(ESI):976.7[M+H]+
第四步：D5：D4的三氟乙酸盐(2.46g,2.52mmol)溶于30mL二氯甲烷,0℃下加入DIEA(4.89g,37.84mmol),D2(1.17g,3.01mmol)，室温反应16h。反应结束后，反应液用二 氯甲烷(50mL)稀释，分别用1M KHSO4(100mL)，饱和碳酸氢钠水溶液(100mL)，水(100mL)洗有机相，有机相无水硫酸钠干燥，过滤旋干得粗品。柱纯化(MeOH:DCM＝1:20)得产物D5(2.67g，收率：89.8％)为黄色油状物。MS m/z(ESI):490.9[M/2-100+H]+
第五步：D6：D5(2.67g,2.26mmol)溶于TFA(15mL)室温搅拌1h。反应液用甲苯(30mL×3)稀释，旋干得D6的三氟乙酸盐酸盐，直接用于下一步。MS m/z(ESI):880.6[M+H]+
第六步：D7：D6(1.99g,2.26mmol)的三氟乙酸盐溶于二氯甲烷(20mL)加入DIEA(11.69g,90.45mmol),0℃下加入化合物5(4.3g,7.01mmol)的二氯甲烷溶液(20mL)，室温反应16h。反应完毕后，反应液用二氯甲烷(50mL)稀释，用1M KHSO4(50mL)，饱和碳酸氢钠水溶液(50mL)，水(50mL)洗有机相，有机相用无水硫酸钠干燥，过滤旋干得粗品。柱纯化(MeOH:DCM＝1:10)得产物D7(3.00g,收率：61％)为白色泡沫固体。MS m/z(ESI):1085.1[M/2+H]+
第七步：D8：D7(1.60g,0.74mmol)溶于THF(16mL),20％Pd(OH)2/C(600mg)，氢气抽换气三次，氢气保护反应16h。硅藻土过滤，THF(10mL×3)洗滤饼，滤液旋干得D8为白色泡沫，直接用于下一步。MS m/z(ESI):1039.6[M/2+H]+
第八步：D9：D8(0.92g,0.443mmol)溶于二氯甲烷(20mL)加入DIEA(0.17g,1.315mmol)，加入三氟乙酸五氟苯酯(0.25g,0.89mmol)，室温反应18h，反应液用二氯甲烷(30mL)稀释，用1M KHSO4(30mL)，饱和碳酸氢钠水溶液(30mL)，水洗(30mL)有机相，无水硫酸钠干燥有机相，过滤旋干得粗品。柱纯化(MeOH:DCM＝1:10)得产物D9(0.6g,收率：60％)为油状物。MS m/z(ESI):1123.0[M/2+H]+
实施例7


第一步：E2：E1(5g,24.01mmol)溶于二氯甲烷(50mL)加入DIEA(4.66g，36.02mmol)，0℃下加入三氟乙酸五氟苯酯(10.09g,36.02mmol),室温反应16h。反应结束后，反应液用二氯甲烷(50mL)稀释，分别用1M硫酸氢钾水溶液(50mL×2),饱和碳酸氢钠(50mL×2)，水洗(50mL)有机相，无水硫酸钠干燥有机相，过滤，旋干。柱纯化(PE:EtOAc＝10:1)得E2(6.4g,收率：71％)为黄色固体。MS m/z(ESI):375.0[M+H]+
第二步：E3：化合物13(5.38g,6.67mmol)，Cbz-Gly-Pro-OH(2.66g,8.67mmol)溶于50mL二氯甲烷加入DIEA(2.59g,20.0mmol),HATU(3.81g,10.0mmol)室温反应16h。反应液用50mL二氯甲烷稀释，加入50mL水，分液，水相用二氯甲烷萃取(50mL×3)，合并有机相，无水硫酸钠过滤，旋干，柱纯化(DCM:MeOH＝10:1)得E3(6.7g,收率：90％)为无色粘稠液体。MS m/z(ESI):994.7[M-100+H]+，447.9[M/2-100+H]+
第三步：E4：E3(5.6g,5.11mmol)溶于60mL甲醇,加入20％Pd(OH)2/C(1g)，三氟乙酸(1.75g,15.35mmol)，氢气置换气体三次，氢气条件下室温反应4h。反应完毕后，硅藻土过滤，用甲醇(30mL×3)洗滤饼，旋干得E4的三氟乙酸盐。未纯化直接用于下一步。MS m/z(ESI):960.6[M+H]+
第四步：E5：E4(1g,1.04mmol)的三氟乙酸盐溶于10mL二氯甲烷，0℃下加入 DIEA(2.02g,15.62mmol)，E2(0.43g,1.14mmol)，室温反应16h。反应结束后，反应液用二氯甲烷(20mL)稀释，用1M KHSO4(20mL)，饱和碳酸氢钠水溶液(20mL)，水(20mL)洗有机相，无水硫酸钠干燥，过滤旋干得粗品。柱纯化(MeOH:DCM＝1:20)得产物E5(0.88g,收率73％)为黄色油状物。MS m/z(ESI):475.9[M/2-100+H]+
第五步：E6：E5(0.88g,0.765mmol)溶于TFA(4mL)室温搅拌1h。反应液用甲苯(10mL)稀释，旋干，再加甲苯(10mL×3)稀释，旋干得E6的三氟乙酸盐，直接用于下一步。MS m/z(ESI):850.5[M+H]+
第六步：E7：E6三氟乙酸盐(0.65g,0.765mmol)溶于二氯甲烷(5mL)加入DIEA(3.95g,30.58mmol),加入化合物5(1.45g,2.37mmol)的二氯甲烷(5mL)溶液，室温反应18h。反应液用二氯甲烷(20mL)稀释，用1M KHSO4(30mL)，饱和碳酸氢钠水溶液(30mL)，水(30mL)洗有机相，无水硫酸钠干燥有机相，过滤旋干得粗品。柱纯化(MeOH:DCM＝1:10)得产物E7(0.71g,收率：43％)为白色粘稠状固体。MS m/z(ESI):1070.1[M/2+H]+
第七步：E8：E7(100mg,0.047mmol)溶于THF(1mL),20％Pd(OH)2/C(20mg)，氢气置换三次，氢气保护室温反应4h。硅藻土过滤，THF(10mL×3)洗滤饼，滤液旋干得E8为白色泡沫状固体。MS m/z(ESI):1024.6[M/2+H]+
第八步：E9：E8(460mg,0.225mmol)溶于DCM(5mL)加入DIEA(58.05mg,0.449mmol)，4A分子筛室温搅拌30min,室温加入三氟乙酸五氟苯酯(94.35mg,0.337mmol),室温反应3h。反应液用冰的DCM(5mL)稀释，用冰1M KHSO4(5mL×2)，冰饱和碳酸氢钠水溶液(5mL×2)，冰饱和食盐水(5mL×2)洗有机相，无水硫酸钠干燥，过滤旋干得E9(0.28g,收率：55％)白色泡沫固体。MS m/z(ESI):1107.6[M/2+H]+
实施例8
第一步：E2’：E1(4.14g，20mmol)溶于二氯甲烷(150mL)，冰浴下缓慢滴加草酰氯(13.8g,109mmol)，反应液在0℃下反应5min，回到室温反应30min。反应液浓缩得E2’(4.5g，收率：99％)为无色的油状物。
第二步：F1：化合物13(1.00g,1.241mmol)，Cbz-Gly-Val-OH(0.46g 1.489mmol)溶于10mL DCM加入DIEA(0.48g,3.722mmol),HATU(0.75g,1.985mmol)室温反应16h。反应液用10mL DCM稀释，加入20mL水，分液，水相用DCM萃取(10mL×3)，合并有机相，无水硫酸钠过滤，旋干，柱纯化(DCM:MeOH＝10:1)得F1(1.00g,收率：73％)白色泡沫状固体。MS m/z(ESI):1097.7[M+H]+
第三步：F2：F1(600mg,0.547mmol)溶于5mL MeOH,加入20％Pd(OH)2/C(120mg)，CF3COOH(187mg,1.642mmol)，氢气球抽换气三次，氢气条件下室温反应16h。硅藻土过滤，用甲醇(10mL×3)洗滤饼，旋干得F2的三氟乙酸盐。未纯化，直接用于下一步。MS m/z(ESI):431.9[M/2+H]+
第四步：F3：F2(526mg,0.547mmol)溶于5mL DCM,0℃下加入DIEA(141.3mg,1.093mmol),E2’(161.08mg,0.711mmol)，室温反应16h。反应液用DCM(10mL)稀释，饱和碳酸氢钠水溶液(10mL)，水(10mL)洗有机相，无水硫酸钠干燥，过滤旋干得粗品。柱纯化(MeOH:DCM＝1:10)得产物F3(460mg,收率73％)为黄色油状物。MS m/z(ESI):476.9[M/2-100+H]+
第五步：F4：F3(400mg,0.347mmol)溶于TFA(3mL)室温搅拌1h。反应液用甲苯(5mL)稀释，旋干，再加甲苯(5mL×3)稀释，旋干得F4的三氟乙酸盐,直接用于下一步。MS m/z(ESI):426.9[M/2+H]+
第六步：F5：F4(295mg,0.346mmol)溶于二氯甲烷(5mL)，加入DIEA(1.78g,13.85mmol),加入化合物5(658.46mg,1.073mmol)的二氯甲烷(3mL)溶液，室温反应18h。反应液用二氯甲烷(10mL)稀释，用1M KHSO4(10mL)，饱和碳酸氢钠水溶液(10mL)，水(10mL)洗有机相，无水硫酸钠干燥有机相，过滤旋干得粗品。柱纯化(MeOH:DCM＝1:10)得产物F5(333.2mg,收率：45％)为粘稠状固体。MS m/z(ESI):1071.6[M/2+H]+
第七步：F6：F5(300mg,0.139mmol)溶于THF(3mL),20％Pd(OH)2/C(40mg)，氢气置换三次，氢气保护室温反应4h。硅藻土过滤，THF(5mL×3)洗滤饼，滤液旋干得F6为白色泡沫状固体。MS m/z(ESI):1026.1[M/2+H]+
实施例9

第一步：G1：化合物13(5.0g,6.20mmol)，Fmoc-Val-Val-OH(3.25g 7.44mmol)溶于50mL DCM加入DIEA(2.4g,18.6mmol)，HATU(3.75g,9.92mmol)室温反应16h。反应液用50mL DCM稀释，加入50mL水，分液，水相用DCM萃取(50mL×3)，合并有机相，无水硫酸钠过滤，旋干，柱纯化(DCM:MeOH＝10:1)得G1(5.0g,收率：70％)。MS m/z(ESI):513.8[M/2-100+H]+
第二步：G2：G1(3.0g,2.44mmol)溶于DCM(10mL)加入二乙胺(5mL),室温反应3h。旋干溶剂，碱化的硅胶柱纯化(DCM:MeOH＝10:1)得G2(1.40g,收率：54％)。MS m/z(ESI):1004.7[M+H]+
第三步：G3：G2(1.40g,1.39mmol)溶于15mL DCM,0℃下加入DIEA(0.537g, 4.17mmol),E2’(0.63g,2.78mmol)，室温反应16h。反应液用DCM(20mL)稀释，饱和碳酸氢钠水溶液(20mL)，水(20mL)洗有机相，无水硫酸钠干燥，过滤旋干得粗品。柱纯化(MeOH:DCM＝1:20)得产物G3(1.00g,收率60％)为淡黄色固体。MS m/z(ESI):497.8[M/2-100+H]+
第四步：G4：G3(1.00g,0.838mmol)溶于TFA(4mL)室温搅拌1h。反应液用甲苯(10mL)稀释，旋干，再加甲苯(10mL×3)稀释，旋干得G4的三氟乙酸盐,直接用于下一步。MS m/z(ESI):895.6[M+H]+
第五步：G5：G4(0.748mg,0.824mmol)的三氟乙酸盐溶于二氯甲烷(10mL)加入DIEA(4.32g,33.46mmol),加入化合物5(1.59g,2.59mmol)的二氯甲烷(5mL)溶液，室温反应2h。反应液用二氯甲烷(30mL)稀释，用1M KHSO4(30mL)，饱和碳酸氢钠水溶液(30mL)，水(30mL)洗有机相，无水硫酸钠干燥有机相，过滤旋干得粗品。柱纯化(MeOH:DCM＝1:10)得产物G5(699mg,收率：38％)为白色泡沫状固体。MS m/z(ESI):1091.6[M/2+H]+
第六步：G6：G5(699mg,0.033mmol)溶于THF(10mL),20％Pd(OH)2/C(35mg)，氢气置换三次，氢气保护室温反应过夜。硅藻土过滤，THF(10mL×3)洗滤饼，滤液旋干得G6为白色泡沫状固体。MS m/z(ESI):1047.1[M/2+H]+
第七步：G7：化合物G6(1.63g,0.779mmol)溶于DCM(10mL)加入DIEA(0.20g,1.558mmol),室温加入TFAPfp(0.33g,1.169mmol),室温反应3h。反应液用冰的DCM(20mL)稀释，用冰1M KHSO4(20mL×3)，冰饱和碳酸氢钠水溶液(20mL×3)，冰饱和食盐水(20×3mL)洗有机相，无水硫酸钠干燥，过滤，旋干得化合物G7(1.6g，收率：74％)为白色泡沫状固体。MS m/z(ESI):1130.1[M/2+H]+
实施例10

第一步：H1：13(5.0g,6.20mmol),Fmoc-Val-Leu-OH(3.35g 7.40mmol)溶于50mL DCM加入DIEA(2.4g,18.6mmol),HATU(3.75g,9.92mmol)室温反应16h。反应液用50mL DCM稀释，加入50mL水，分液，水相用DCM萃取(50mL×3)，合并有机相，无水硫酸钠过滤，旋干，柱纯化(DCM:MeOH＝10:1)得H1(5.5g,收率：70％)。MS m/z(ESI):520.7[M/2-100+H]+
第二步：H2：H1(3.0g,2.41mmol)溶于DCM(10mL)加入二乙胺(5mL),室温反应3h。旋干溶剂，碱化的硅胶柱纯化(DCM:MeOH＝10:1)得H2(1.9g,收率：77％)。MS m/z(ESI):1019.7[M+H]+
第三步：H3：H2(1.90g,1.86mmol)溶于15mL DCM，0℃下加入DIEA(0.719mg,5.58mmol),E2’(840mg,3.72mmol)，室温反应16h。反应液用DCM(10mL)稀释，饱和碳酸氢钠水溶液(10mL)，水(10mL)洗有机相，无水硫酸钠干燥，过滤旋干得粗品。柱纯化(MeOH:DCM＝1:20)得产物H3(1.2g,收率53％)。MS m/z(ESI):505.2[M/2-100+H]+
第四步：H4：H3(1.0g,0.827mmol)溶于TFA(5mL)室温搅拌1h。反应液用甲苯(5mL)稀释，旋干，再加甲苯(5mL×3)稀释，旋干得H4的三氟乙酸盐,直接用于下一步。MS m/z(ESI):908.6[M+H]+
第五步：H5：H4(750mg,0.827mmol)的三氟乙酸盐溶于二氯甲烷(10mL)加入DIEA(4.28g,33.08mmol),加入化合物5(1.57g,2.564mmol)的二氯甲烷(5mL)溶液，室温反应18h。反应液用二氯甲烷(30mL)稀释，用1M KHSO4(30mL)，饱和碳酸氢钠水溶液(30mL)，水(30mL)洗有机相，无水硫酸钠干燥有机相，过滤旋干得粗品。柱纯化(MeOH:DCM＝1:10)得产物H5(691mg,收率：37％)为白色泡沫状固体。MS m/z(ESI):1099.1[M/2+H]+
第六步：H6：H5(691mg,0.315mmol)溶于THF(10mL),20％Pd(OH)2/C(77mg)，氢气置换三次，氢气保护室温反应4h。硅藻土过滤，THF(10mL×3)洗滤饼，滤液旋干得 H6为白色泡沫状固体。MS m/z(ESI):1054.1[M/2+H]+
第七步：H7：化合物H6(1.7g,0.807mmol)溶于DCM(10mL)加入DIEA(0.21g,1.614mmol),室温加入TFAPfp(0.45g,1.614mmol),室温反应过夜。旋干反应液溶于DCM(10mL)加入DIEA(0.21g,1.614mmol),室温加入TFAPfp(0.45g,1.614mmol),室温反应4h。反应液用冰的DCM(15mL)稀释，用冰1M KHSO4(15mL×3)，冰饱和碳酸氢钠水溶液(15mL×3)，冰饱和食盐水(15×3mL)洗有机相，无水硫酸钠干燥，过滤旋干得白色泡沫固体。再次用冰的DCM(15mL)溶解固体，用冰1M KHSO4(15mL×2)，冰饱和碳酸氢钠水溶液(15mL×2)，冰水(15×2mL)洗有机相，无水硫酸钠干燥，过滤旋干得化合物H7(1.4g，收率：35％)棕黄色泡沫固体。MS m/z(ESI):1137.1[M/2+H]+
实施例11

第一步：I1：化合物13(1.00g,1.241mmol),Fmoc-Ala-Ala-OH(0.57g,1.48mmol)溶于10mL DCM加入DIEA(0.48g,3.72mmol),HATU(0.75g,1.985mmol)室温反应16h。反应液用10mL DCM稀释，加入20mL水，分液，水相用DCM萃取(10mL×3)，合并有机相，无水硫酸钠过滤，旋干，柱纯化(DCM:MeOH＝10:1)得白色泡沫固体I1(1.2g,收率：75％)。
第二步：I2：I1(600mg,0.513mmol)溶于DCM(4mL)加入二乙胺(2mL),室温反应3h，旋干溶剂，碱化的硅胶柱纯化(DCM:MeOH＝10:1)得I2(480mg,收率：98％)。MS m/z(ESI):948.6[M/2+H]+
第三步：I3：I2(480mg,0.506mmol)溶于10mL二氯甲烷，0℃下加入DIEA(196.3mg,1.519mmol),E2(227.4mg,0.607mmol)，室温反应16h。反应结束后，反应液用二氯甲烷(20mL)稀释，用1M KHSO4(20mL)，饱和碳酸氢钠水溶液(20mL)，水(20mL)洗有机相，无水硫酸钠干燥，过滤旋干得粗品。柱纯化(MeOH:DCM＝1:20)得产物I3(350mg,收率61％)。MS m/z(ESI):469.9[M/2-100+H]+
第四步：I4：I3(350mg,0.307mmol)溶于TFA(3mL)室温搅拌1h。反应液用甲苯(5mL)稀释，旋干，再加甲苯(5mL×3)稀释，旋干得I4的三氟乙酸盐,直接用于下一步。MS m/z(ESI):419.9[M//2+H]+
第五步：I5：I4的三氟乙酸盐(257mg,0.307mmol)溶于二氯甲烷(5mL)加入DIEA(1.56g,12.267mmol),加入化合物5(583.24mg,0.951mmol)的二氯甲烷(3mL)溶液，室温反应18h。反应液用二氯甲烷(10mL)稀释，用1M KHSO4(10mL)，饱和碳酸氢钠水溶液(10mL)，水(10mL)洗有机相，无水硫酸钠干燥有机相，过滤旋干得粗品。柱纯化(MeOH:DCM＝1:10)得产物I5(267.6mg,收率：41％)。MS m/z(ESI):1063.6[M/2+H]+
第六步：I6：I5(267.6mg,0.125mmol)溶于THF(4mL),20％Pd(OH)2/C(30mg)，氢气置换三次，氢气保护室温反应4h。硅藻土过滤，THF(5mL×3)洗滤饼，滤液旋干得I6为白色泡沫状固体。MS m/z(ESI):1018.9[M/2+H]+
实施例12
通过亚磷酰胺法固相合成ASO、siRNA的正义链和反义链，siRNA的正义链的5’端可通过5’-氨基TFA(C6)加入末端氨基，即通过亚磷酰胺偶联，采用5'-Amino-Modifier C6-TFA(cas号为：133975-85-6的单体)，偶联至核酸的5’末端，核酸链合成完毕氨水脱保护后，得到带氨基的寡聚核苷酸(5’端氨基的寡聚核苷酸)，用于实验例的寡核苷酸的序列如下所示。
3’端带氨基的寡聚核苷酸的合成，可以使用3'-PT Amino-Modifier C6CPG作为起始固体支撑物，通过亚磷酰胺法固相合成，固相合成完成后，经过裂解、脱保护、纯化等得到带氨基的寡聚核苷酸(3’端氨基的寡聚核苷酸)。
带氨基的寡聚核苷酸的结构示例如下：
5’端带氨基的寡聚核苷酸1(其中序列如SEQ ID NO.1所示)：
或
5’端带氨基的寡聚核苷酸2(其中序列如SEQ ID NO.3所示)：
3’端带氨基的寡聚核苷酸1(其中序列如SEQ ID NO.1所示)：
或
3’端带氨基的寡聚核苷酸2(其中序列如SEQ ID NO.3所示)：
其中，m为糖环2’位的甲氧基修饰，f为糖环2’位的氟代修饰，*为磷酸二酯键的硫代修饰,invdA是反向脱氧腺苷(3'-3'连接的核苷酸)。
对比例：对比共轭物的制备
通过将对应的正义链、反义序列进行模拟退火得到对比共轭物PC(无L96)、NC(无L96)；
其中，m为糖环2’位的甲氧基修饰，f为糖环2’位的氟代修饰，*为磷酸二酯键的硫 代修饰；
PC表示：Positive control,阳性对照；NC表示：Negative control,阴性对照。
PC(3’L96)参考美国专利US10125369的方法制备，其中3’L96结构如下：
其中，表示与寡核苷酸3’连接的位置。
PC(5’L96)的制备参考文献J.Am.Chem.Soc.2014,136,16958-16961，其中5’L96结构如下：
其中，表示与寡核苷酸5’连接的位置。
AMG 890的制备参考专利CN116456990A/CN108368506A，其结构式如下：
实施例13.配体-寡核苷酸共轭物的制备
13.1.将化合物A7和HATU分别溶解在DMF中，混合后加入DIEA，然后放入涡旋振荡器30℃下反应15min后，加入到5’端带氨基的寡聚核苷酸1(用水溶解)中，超声和涡旋振荡器混匀至完全溶解，25℃下反应16h，再向偶联物中加入氨水，55℃下反应30min， 得目标产物混合液，HPLC制备得到偶联物，结构式如CA1所示，然后与对应的反义序列进行模拟退火得到目标化合物ds-CA1。
13.2.将化合物B7和HATU分别溶解在DMF中，混合后加入DIEA，然后放入涡旋振荡器30℃下反应15min后，加入到5’端带氨基的寡聚核苷酸1(用水溶解)中，超声和涡旋振荡器混匀至完全溶解，25℃下反应16h，再向偶联物中加入氨水，55℃下反应30min，得目标产物混合液，HPLC制备得到偶联物，结构式如CB1所示，然后与对应的反义序列进行模拟退火得到目标化合物ds-CB1。

13.3.将化合物C7和HATU分别溶解在DMF中，混合后加入DIEA，然后放入涡旋振荡器30℃下反应15min后，加入到5’端带氨基的寡聚核苷酸1(用水溶解)中，超声和涡旋振荡器混匀至完全溶解，25℃下反应16h，再向偶联物中加入氨水，55℃下反应30min，得目标产物混合液，HPLC制备得到偶联物，结构式如CC1所示，然后与对应的反义序列进行模拟退火得到目标化合物ds-CC1。
13.4.将D9溶解在磷酸钠缓冲液中，将其加入到5’端带氨基的寡聚核苷酸1溶液中(用PBS溶解),然后再加入Py活化剂(吡啶)，超声和涡旋振荡器混匀至完全溶解，25℃下反应16h，再向偶联物中加入氨水，55℃下反应30min，得目标产物混合液，HPLC制备得到偶联物，结构式如CD1所示，然后与对应的反义序列进行模拟退火得到目标化合物ds-CD1。
13.5.将E9溶解在磷酸钠缓冲液中，将其加入到5’端带氨基的寡聚核苷酸1溶液中(用PBS溶解),然后再加入Py活化剂，超声和涡旋振荡器混匀至完全溶解，25℃下反应16h，再向偶联物中加入氨水，55℃下反应30min，得目标产物混合液，HPLC制备得到偶联物，结构式如CE1所示，然后与对应的反义序列进行模拟退火得到目标化合物ds-CE1。
13.6.将化合物F6和HATU分别溶解在DMF中，混合后加入DIEA，然后放入涡旋振荡器30℃下反应15min后，加入到5’端带氨基的寡聚核苷酸1(用水溶解)中，超声和涡旋振荡器混匀至完全溶解，25℃下反应16h，再向偶联物中加入氨水，55℃下反应30min，得目标产物混合液，HPLC制备得到偶联物，结构式如CF1所示，然后与对应的反义序列进 行模拟退火得到目标化合物ds-CF1。
13.7.将化合物G6和HATU分别溶解在DMF中，混合后加入DIEA，然后放入涡旋振荡器30℃下反应15min后，加入到5’端带氨基的寡聚核苷酸1(用水溶解)中，超声和涡旋振荡器混匀至完全溶解，25℃下反应16h，再向偶联物中加入氨水，55℃下反应30min，得目标产物混合液，HPLC制备得到偶联物，结构式如CG1所示，然后与对应的反义序列进行模拟退火得到目标化合物ds-CG1。
13.8.将化合物H6和HATU分别溶解在DMF中，混合后加入DIEA，然后放入涡旋振荡器30℃下反应15min后，加入到5’端带氨基的寡聚核苷酸1(用水溶解)中，超声和涡旋振荡器混匀至完全溶解，25℃下反应16h，再向偶联物中加入氨水，55℃下反应30min， 得目标产物混合液，HPLC制备得到偶联物，结构式如CH1所示，然后与对应的反义序列进行模拟退火得到目标化合物ds-CH1。
13.9.将化合物I6和HATU分别溶解在DMF中，混合后加入DIEA，然后放入涡旋振荡器30℃下反应15min后，加入到5’端带氨基的寡聚核苷酸1(用水溶解)中，超声和涡旋振荡器混匀至完全溶解，25℃下反应16h，再向偶联物中加入氨水，55℃下反应30min，得目标产物混合液，HPLC制备得到偶联物，结构式如CI1所示，然后与对应的反义序列进行模拟退火得到目标化合物ds-CI1。
13.10.将D9溶解在磷酸钠缓冲液中，将其加入到3’端带氨基的寡聚核苷酸1溶液中(用PBS溶解),然后再加入Py活化剂，超声和涡旋振荡器混匀至完全溶解，25℃下反应 16h，再向偶联物中加入氨水，55℃下反应30min，得目标产物混合液，HPLC制备得到偶联物，结构式如CD2所示，然后与对应的反义序列进行模拟退火得到目标化合物ds-CD2。
13.11.将化合物G6和HATU分别溶解在DMF中，混合后加入DIEA，然后放入涡旋振荡器30℃下反应15min后，加入到3’端带氨基的寡聚核苷酸1(用水溶解)中，超声和涡旋振荡器混匀至完全溶解，25℃下反应16h，再向偶联物中加入氨水，55℃下反应30min，得目标产物混合液，HPLC制备得到偶联物，结构式如CG2所示，然后与对应的反义序列进行模拟退火得到目标化合物ds-CG2。
13.12.将化合物A7(150mg)溶解在DMF(2000uL)中，分别加入HATU和DIEA，然后放入涡旋振荡器25℃下反应15min后，加入到5’端带氨基的寡聚核苷酸2(20mg， 3000uL水溶解)中，超声和涡旋振荡器混匀至完全溶解，25℃下反应16h后；进行Ac脱保护，加入氨水(300uL)，55℃下反应30min后得目标产物混合液，HPLC制备得到偶联物DA1，然后与对应的反义序列进行模拟退火得到目标化合物ds-DA1。
13.13.将化合物D9(87mg)溶解在DMF(300uL)中，将其加入到5’端带氨基的寡聚核苷酸2(25mg，1200uL碳酸钠缓冲液溶解)中,然后超声和涡旋振荡器混匀至完全溶解，25℃下反应16h后；进行Ac脱保护，加入氨水(300uL)，55℃下反应30min得目标产物混合液，HPLC制备得到偶联物DB，然后与对应的反义序列进行模拟退火得到目标化合物ds-DB1。
13.14.将化合物G7(84mg)溶解在DMF(300uL)中，将其加入到5’端带氨基的寡聚 核苷酸2(25mg，1200uL碳酸钠缓冲液溶解)中,然后超声和涡旋振荡器混匀至完全溶解，25℃下反应16h后；进行Ac脱保护，加入氨水(300uL)，55℃下反应30min后得目标产物混合液，HPLC制备得到偶联物DC1，然后与对应的反义序列进行模拟退火得到目标化合物ds-DC1。
13.15.将化合物H7(86mg)溶解在DMF(300uL)中，将其加入到5’端带氨基的寡聚核苷酸2(25mg，1200uL碳酸钠缓冲液溶解)中,然后超声和涡旋振荡器混匀至完全溶解，25℃下反应16h后；进行Ac脱保护，加入氨水(300uL)，55℃下反应30min后得目标产物混合液，HPLC制备得到偶联物DD1，然后与对应的反义序列进行模拟退火得到目标化合物ds-DD1。
13.16.将化合物D9(82mg)溶解在DMF(300uL)中，将其加入到3’端带氨基的寡聚 核苷酸2(25mg，1200uL碳酸钠缓冲液溶解)中,然后超声和涡旋振荡器混匀至完全溶解，25℃下反应16h后；进行Ac脱保护，加入氨水(300uL)，55℃下反应30min后得目标产物混合液，HPLC制备得到偶联物DB2，然后与对应的反义序列进行模拟退火得到目标化合物ds-DB2。
13.17.将化合物G7(10eq,82mg)溶解在DMF(300uL)中，将其加入到核酸氨基中间体(25mg，1200uL碳酸钠缓冲液溶解)中,然后超声和涡旋振荡器混匀至完全溶解，25℃下反应16h后；进行Ac脱保护，加入氨水(200uL)，55℃下反应30min后得目标产物混合液，HPLC制备得到偶联物DC2，然后与对应的反义序列进行模拟退火得到目标化合物ds-DC2。
实验例1
方法：共轭物分子自递送进入新鲜分离的小鼠原代肝细胞，qPCR检测获得其体外IC50数据。体外实验使用24孔板，每个共轭物的每个浓度点有两次技术重复。(A)共轭物 工作液浓度设置：体外实验共轭物(PC(3’L96)、PC(5’L96)、ds-CA1、ds-CD1)free-uptake工作液的终浓度为:2nM,1nM,0.5nM,0.25nM,125pM,62.5pM,31.3pM,15.6pM,7.81pM,3.91pM,1.95pM；体外实验共轭物(PC(无L96)、NC(无L96))free-uptake工作液的终浓度为:1nM,0.5nM,0.25nM。
(B)共轭物的制备和稀释:共轭物以50μL DEPC H2O每OD的比例用DEPC水溶解,并用Nanodrop 2000测定浓度。根据测量结果将共轭物稀释至266ng/μL,记为20μM,作为原液并保存于-20℃。
(C)Free-uptake样品制备:将相应浓度的共轭物10μL/孔直接添加到细胞接种板中即可。
(D)分离小鼠原代肝细胞(PMH)，过程中不使用percoll试剂。在此过程中保证无菌环境。新鲜分离的PMH的存活率为≥85％。将小鼠原代肝细胞按照900μL细胞悬液/孔，接种于预涂I型胶原的24孔板中，细胞数为8*104活细胞/孔。将10μL稀释共轭物分散在90μL Opti-MEM中，并将其添加到相应的孔中(n＝2)。在37℃、5％CO2培养箱中培养24小时，为自由摄取过程。
(E)普通实时荧光定量PCR检测:细胞总RNA提取：裂解细胞，使用高通量核酸提取仪-磁珠法提取总RNA。RNA浓度调整：使用纳米光度计检测样本浓度，加水调整所有样本至相同浓度。反转录：使用反转录试剂盒去除基因组DNA，并将所有样本反转录成cDNA。普通实时荧光定量qPCR检测:使用常规qPCR试剂盒(SYBR GREEN system)对cDNA样本进行相对定量。对每个cDNA样本进行3次重复定量分析，并同时制作标准曲线。
(E)数据处理与分析：计算差异倍数：Fc＝2^-△△Cp.绘图：使用Graphpad Prism 7的“[Inhibitor]vs.normalized response--Variable slope”分析IC50。
(Y＝100/(1+(X^HillSlope)/(IC50^HillSlope)))
实验结果/结论
1、体外基因抑制效果显示，在PMH中对照组的基因检测结果，PC(无L96)和NC(无L96)不能在自由摄取的递送模式下进入细胞产生作用，其在1,0.5,0.25nM的终浓度下都未产生抑制效果，剩余抑制效率都在100％左右浮动。在整个试验系统中作为阴性QC。在PMH中的对照组的蛋白检测结果与mRNA检测结果一致。
2、共轭物在PMH中自由摄取的基因水平(IC50)，如表1所示：
表1
实验例2
方法：普通C57BL/6小鼠皮下给药，qPCR检测肝脏组织中活性。通过体内外实验数据，比较本发明的共轭物与对照品的递送效果的强弱。体内实验每个共轭物的每个剂量需要6只动物，以满足统计学分析的最低单组数量。
共轭物以50μL DEPC H2O每OD的比例用DEPC水溶解,并用Nanodrop 2000测定浓度。根据测量结果将共轭物稀释至2,000ng/μL,作为原液并保存于-20℃。
动物处理：动物准备：动物到场后，一周时间观察及状态恢复。给药：给药前称量动物体重，皮下给药。解剖取材：72小时后处死动物，大体解剖，每只动物取2份1cm3的肝脏组织，置于RNA Later保存液中，-80℃保存。EDTA抗凝收集全血离心获得血清样本，-80℃保存。
普通实时荧光定量PCR检测:组织总RNA提取：组织匀浆，使用高通量核酸提取仪-磁珠法提取总RNA。RNA浓度调整：使用纳米光度计检测样本浓度，加水调整所有样本至相同浓度。反转录：使用反转录试剂盒去除基因组DNA，并将所有样本反转录成cDNA。普通实时荧光定量qPCR检测:使用常规qPCR试剂盒(SYBR GREEN system)对cDNA样本进行相对定量。对每个cDNA样本进行3次重复定量分析。
共轭物在普通小鼠体内mRNA相对表达水平检测结果如表2所示：
表2

注：/表示未测试
实验例3
方法：普通C57BL/6小鼠皮下给药，qPCR检测肝脏组织中活性。通过体内实验数据，比较本发明的共轭物与对照品的递送效果的强弱。每个共轭物的每个剂量需要6只动物，以满足统计学分析的最低单组数量。
共轭物以50μL PBS每OD的比例用PBS溶解,并用Nanodrop 2000测定浓度。根据测量结果将共轭物稀释至2,000ng/μL,作为原液并保存于-80℃。
动物处理：动物准备：动物到场后，一周时间观察及状态恢复。给药：给药前称量动物体重，按5μL/g皮下给药。解剖取材：72小时后处死动物，大体解剖，每只动物收集1份血液，室温静置20～30min后离心分装血清，约40ul，转入-80℃保存，保存时间期限为半年；每只动物取2份1cm3的肝脏组织，置于RNA Later保存液中，4℃过夜，然后转入-80℃保存。
普通实时荧光定量PCR检测:组织总RNA提取：组织匀浆，向肝组织中加入0.5ml Trizol和钢珠，放入高通量组织研磨仪中，参数调整为30HZ，研磨至肝脏完全破碎即可。组织总RNA提取：组织匀浆，使用高通量核酸提取仪-磁珠法提取总RNA。RNA浓度调整：使用Nanodrop2000检测样本浓度，加水调整所有样本至相同浓度。反转录：使用反转录试剂盒去除基因组DNA，并将所有样本反转录成cDNA。普通实时荧光定量qPCR检测:使用常规qPCR试剂盒(SYBR GREEN system)对cDNA样本进行相对定量。对每个cDNA样本进行3次重复定量分析。
数据处理与分析：计算差异倍数：Fc＝2^-△△Cp.绘图：使用Graphpad Prism 7
共轭物在普通小鼠体内mRNA相对表达水平检测结果如表3所示：
表3
实施例4
瞬时转基因小鼠中RNAi剂功效的体内分析
1.动物分组：5～6周龄Balb/c小鼠，小鼠进入动物设施后，适应性饲养3天及以上，随后以体重为主要分组指标，进行随机分组，每组6只；分组当天定义为D-3。
2.给药：于D-3开始给药，单次皮下注射，给药剂量为3mpk。
3.HDI注射造模：为了评价LPA RNAi剂的体内功效，使用瞬时转基因小鼠，通过流体动力学尾静脉注射方法，向野生型小鼠Balb/c注射APOA基因的质粒，作为造模鼠。注射当天为D0。
4.LPA表达敲低分析：使用LPA(abcam)的ELISA试剂盒测定小鼠的血清来检测血清中的apo(a)蛋白质水平，于D1采血，分离血清30μL检测apo(a)表达。
5.统计分析：试验结果以平均值±标准误差(Mean±SEM)表示。两组样本之间比较采用独立样本T检验(T-Test)，多组单变量比较采用单因素方差分析(One-way ANOVA)检验，多组双变量比较采用双因素方差分析(Two-way ANOVA)检验。作图和数据分析软件为Graphpad prism 9。P<0.05为具有显著性差异。
表4.瞬时转基因小鼠中的apo(a)相对表达水平
实验例5
通过PI检测共轭物对细胞MHCC97毒性作用，使用96孔板进行试验，每个共轭物的每个浓度点有两次技术重复，每板细胞筛选均需设置PC，NC和MOCK以此校准不同板间一致性。
共轭物工作液和阴性和阳性对照的终浓度为:100nM。
共轭物以50μL DEPC H2O每OD的比例用DEPC水溶解,并用Nanodrop 2000测定浓度。根据测量结果将共轭物稀释至2,000ng/μL,作为原液并保存于-80℃。
细胞种板：150μL细胞悬液/孔，接种于96孔板中，MHCC97细胞数：4*103细胞/孔。细胞在37℃、5％CO2培养箱中培养24小时后转染。
转染：将5μL稀释共轭物分散在20μL Opti-MEM中，0.2μL RNAiMAX分散在24.8μL Opti-MEM中孵育5分钟后，与共轭物分散液混匀，孵育10分钟，得转染复合物，将细胞加入转染复合物中转染。
PI染色：细胞转染48h后，去除原有培养基，每孔加入200μL PBS清洗一次；将1mg/mL PI与PBS混合配置，终浓度为10μg/mL，每细胞孔中加入100μL混合液，将培养板放入培养箱孵育20min后用荧光显微镜拍照。
数据处理与分析：根据图片计数细胞总数和PI染色的细胞数，计算PI染色的细胞数 百分比，将共轭物百分比相对于NC归一化，其中百分比越高表示毒性相对越高；
PI阳性细胞数占全部细胞百分数如表5所示：
表5

Claims (27)

  1. 一种配体-寡核苷酸共轭物，其包含式I或II所示的接头部分：
    -NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I)，或
    -NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II)，
    其中：
    R1、R2、R3、R4独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-10环烷基、6至14元芳基、5至18元杂芳基、5至14元杂环基、卤素、羟基、氨基、胍基、羧基、氰基、硝基和巯基，它们任选地被R5取代，其中R5选自C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-10环烷基、6至14元芳基、5至18元杂芳基、5至14元杂环基、卤素、羟基、氨基、胍基、羧基、氰基、硝基、C1-3烷基硫基和巯基；
    Ra、Rb独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-8环烷基、6至14元芳基C1-3烷基、5至18元杂芳基C1-3烷基和5至14元杂环基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、C1-3烷基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、吡咯基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、C1-10烷基、C1-10烷氧基、6至14元芳基、C3-8环烷基、金刚烷基、膦酰基、膦酰氧基、C1-3烷基膦酰基、C1-3烷基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成3至14元含氮杂环基，或者Ra、Rb与其共同连接的C原子一起形成C3-8环烷基；
    杂原子选自氮、氧、硫、磷和硅；
    n为1至10，并且每个重复单元相同或不同；
    p为2至6，并且每个重复单元相同或不同。
  2. 根据权利要求1所述的配体-寡核苷酸共轭物，其中Ra是-H，Rb选自-H、甲基、乙基、丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、环丙基、环戊基、环己基、环己基二亚甲基、环己基亚甲基、环丁基亚甲基、苯基亚甲基、甲基硫基亚甲基、3-吲哚基亚甲基、羟基亚甲基、酰胺基二亚甲基、羟基甲基次甲基、巯基亚甲基、酰胺基亚甲基、羟基苯基亚甲基、羟基苯基二亚甲基、羧基亚甲基、羧基二亚甲基、氨基正丁基、胍基二亚甲基、胍基三亚甲基、4-咪唑基亚甲基、异丙基苯基亚甲基、1-萘基亚甲基、2-萘基亚甲基、茚满基、苯基二亚甲基、氟代苯基亚甲基、氯代苯基亚甲基、三氟苯基亚甲基、二氯苯基亚甲 基、五氟苯基亚甲基、二氟苯基亚甲基、噻吩基亚甲基、噻茚基亚甲基、喹啉基亚甲基、卤代3-吲哚基亚甲基、二苯基亚甲基、3-吡啶基亚甲基和4-吡啶基亚甲基，或者Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基；
    优选，R1、R2、R3、R4独立地选自-H、C1-10烷基、C1-10烷氧基、C3-10环烷基和5至14元杂环基，它们任选地被R5取代，其中R5选自C1-10烷基、C1-10烷氧基、C3-10环烷基和5至14元杂环基；
    更优选，R1、R2、R3、R4独立地选自-H、C1-3烷基、C1-3烷氧基、C3-6环烷基，它们任选地被R5取代，其中R5选自C1-10烷基、C1-10烷氧基、C3-10环烷基、吡咯基、噻吩基、噻唑基、咪唑基、吡啶基、呋喃基、哌嗪基、吡咯烷基、二氧杂环己基、吗啉基、四氢呋喃基、四氢吡咯烷基；
    进一步优选，R1、R2、R3、R4独立地选自-H、甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、甲氧基和乙氧基；
    更进一步优选，R1、R2、R3、R4独立是-H或甲基。
  3. 根据权利要求1至2任一项所述的配体-寡核苷酸共轭物，其中n为2至4，并且每个重复单元相同或不同；
    优选，n为2或3，并且每个重复单元相同或不同。
  4. 根据权利要求1至3任一项所述的配体-寡核苷酸共轭物，其中p为2至4，并且每个重复单元相同或不同；
    优选，p为2或3，并且每个重复单元相同或不同。
  5. 根据权利要求1所述的配体-寡核苷酸共轭物，其中R1、R2、R3、R4、Ra均为-H，n为2或3，p为2，Rb选自-H、甲基、异丙基和异丁基，或者Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基。
  6. 根据权利要求5所述的配体-寡核苷酸共轭物，其中对于p为2的重复单元：
    在每个重复单元中，Rb是异丙基；
    在每个重复单元中，Rb是甲基；
    在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb是甲基；
    在一个重复单元中，Rb是异丁基，在另一个重复单元中，Rb是甲基；
    在一个重复单元中，Rb是异丁基，在另一个重复单元中，Rb是异丙基；
    在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb是氢；
    在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基；或
    在一个重复单元中，Rb是氢，在另一个重复单元中，Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基。
  7. 根据权利要求1至6任一项所述的配体-寡核苷酸共轭物，其中所述配体包含通过二价或三价支链接头附接的一种或多种N-乙酰半乳糖胺或其衍生物。
  8. 根据权利要求7所述的配体-寡核苷酸共轭物，其中所述配体具有以下结构：(GalNAc-Q1)mCHm’-，其中
    GalNAc表示N-乙酰半乳糖胺或其衍生物；所述N-乙酰半乳糖胺或其衍生物结构如下：
    Q1表示T1-(T2-T3-T4)w，其中T1和T4独立地选自不存在、C(O)、NH、O、S、OC(O)、NHC(O)、CH2、CH2NH和CH2O；T2选自不存在、NH、O、S、CH2、C(O)O、C(O)NH、NHCH2C(O)、C(O)-CH2-NH、C(O)和CH＝N-O；T3选自不存在和任选地被选自O、S、S(O)、SO2、C(O)和C≡C的原子或基团间杂或封端的一个或多个亚甲基；w为0至20，并且该重复单元相同或不同；所述Ro、Ro’分别独立的选自H、烷基羰基；所述Ro’优选为乙酰基；所述Ro分别独立地优选为H或乙酰基，更优选为H；
    m为1至3，并且每个重复单元相同或不同；m’为0至2，且m+m’为3。
  9. 根据权利要求8所述的配体-寡核苷酸共轭物，其中所述配体的结构选自：


    优选，所述配体的结构为：
  10. 根据权利要求1至9任一项所述的配体-寡核苷酸共轭物，其中所述寡核苷酸选自反义寡核苷酸(ASO)、小干扰RNA(siRNA)、小发卡RNA(shRNA)和微RNA(miRNA)或其盐。
  11. 根据权利要求10所述的配体-寡核苷酸共轭物，其中所述寡核苷酸的5’端或3’端附接至所述接头部分。
  12. 根据权利要求11所述的配体-寡核苷酸共轭物，其中所述5’端通过3’-寡核苷酸-5’-OP(O)(OH)O-(CH2-Y)x-NH-附接至所述接头部分，其中Y代表O或不存在，x为3至12并且每个重复单元相同或不同；
    优选，所述5’端通过选自以下的任一个附接至所述接头部分：
    3’-寡核苷酸-5’-OP(O)(OH)O-(CH2)6-NH-；
    3’-寡核苷酸-5’-OP(O)(OH)O-(CH2)3-NH-；
    3’-寡核苷酸-5’-OP(O)(OH)O-(CH2)12-NH-；和
    3’-寡核苷酸-5’-OP(O)(OH)O-(CH2)3-O-(CH2)3-NH-；
    或者，
    所述5’端通过3’-寡核苷酸-5’-OP(S)(OH)O-(CH2-Y)x-NH-附接至所述接头部分，其中Y代表O或不存在，x为3至12并且每个重复单元相同或不同；
    优选，所述5’端通过选自以下的任一个附接至所述接头部分：
    3’-寡核苷酸-5’-OP(S)(OH)O-(CH2)6-NH-；
    3’-寡核苷酸-5’-OP(S)(OH)O-(CH2)3-NH-；
    3’-寡核苷酸-5’-OP(S)(OH)O-(CH2)12-NH-；和
    3’-寡核苷酸-5’-OP(S)(OH)O-(CH2)3-O-(CH2)3-NH-。
  13. 根据权利要求10所述的配体-寡核苷酸共轭物，其中所述寡核苷酸是siRNA，包含正义链和反义链，所述正义链的5’端或3’端附接至所述接头部分；
    优选，所述siRNA中的核苷酸的一个或多个被修饰，所述修饰为2’-O-甲基修饰和/或2’-氟基修饰。
  14. 选自下式的配体-寡核苷酸共轭物：


















    其中表示siRNA；
    优选，



  15. 式III或IV所示的化合物：
    (GalNAc-Q1)mCHm’-NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-Q2(式III)，或
    (GalNAc-Q1)mCHm’-NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-Q2(式IV)，
    其中：
    GalNAc表示N-乙酰半乳糖胺或其衍生物；所述N-乙酰半乳糖胺或其衍生物结构为：
    Q1表示-T1-(T2-T3-T4)w，其中T1和T4独立地选自不存在、C(O)、NH、O、S、OC(O)、NHC(O)、CH2、CH2NH和CH2O；T2选自不存在、NH、O、S、CH2、C(O)O、C(O)NH、NHCH2C(O)、C(O)-CH2-NH、C(O)和CH＝N-O；T3选自不存在和任选地被选自O、S、S(O)、SO2、C(O)和C≡C的原子或基团间杂或封端的一个或多个亚甲基；w为0至20，并且该重复单元相同或不同；
    Q2表示-OH或-O-羧基保护基团；所述羧基保护基优选为苄基或五氟代苯基；
    R1、R2、R3、R4独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-10环烷基、6至14元芳基、5至18元杂芳基、5至14元杂环基、卤素、羟基、氨基、胍基、羧基、氰基、硝基和巯基，它们任选地被R5取代，其中R5选自C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-10环烷基、6至14元芳基、5至18元杂芳基、5至14元杂环基、卤素、羟基、氨基、胍基、羧基、氰基、硝基、C1-3烷基硫基和巯基；
    Ra、Rb独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-8环烷基、6至14元芳基C1-3烷基、5至18元杂芳基C1-3烷基和5至14元杂环基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、C1-3烷基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、吡咯基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、C1-10烷基、C1-10烷氧基、6至14元芳基、C3-8环烷基、金刚烷基、膦酰基、膦酰氧基、C1-3烷基膦酰基、C1-3烷基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成3至14元含氮杂环基；所述Ro、Ro’分别独立的选自H、烷基羰基；所述Ro’优选为乙酰基；所述Ro分别独立地优选为H或乙酰基，更优选为H；
    杂原子选自氮、氧、硫、磷和硅；
    m为1至3，并且每个重复单元相同或不同；m’为0至2，优选为0或1，且m+m’为3；
    n为1至10，并且每个重复单元相同或不同；
    p为2至6，并且每个重复单元相同或不同。
  16. 根据权利要求15所述的化合物，其中式III或IV所示的结构中的(GalNAc-Q1)mCHm’-部分的结构选自：

    优选为：
  17. 根据权利要求15至16任一项所述的化合物，其中Ra是-H，Rb选自-H、甲基、乙基、丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、环丙基、环戊基、环己基、环己基二亚甲基、环己基亚甲基、环丁基亚甲基、苯基亚甲基、甲基硫基亚甲基、3-吲哚基亚甲基、羟基亚甲基、酰胺基二亚甲基、羟基甲基次甲基、巯基亚甲基、酰胺基亚甲基、羟基苯基亚甲基、羟基苯基二亚甲基、羧基亚甲基、羧基二亚甲基、氨基正丁基、胍基二亚甲基、胍基三亚甲基、4-咪唑基亚甲基、异丙基苯基亚甲基、1-萘基亚甲基、2-萘基亚甲基、茚满基、苯基二亚甲基、氟代苯基亚甲基、氯代苯基亚甲基、三氟苯基亚甲基、二氯苯基亚甲基、五氟苯基亚甲基、二氟苯基亚甲基、噻吩基亚甲基、噻茚基亚甲基、喹啉基亚甲基、卤 代3-吲哚基亚甲基、二苯基亚甲基、3-吡啶基亚甲基和4-吡啶基亚甲基，或者Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基。
  18. 根据权利要求17所述的化合物，其中对于p为2的重复单元：
    在每个重复单元中，Rb是异丙基；
    在每个重复单元中，Rb是甲基；
    在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb是甲基；
    在一个重复单元中，Rb是异丁基，在另一个重复单元中，Rb是甲基；
    在一个重复单元中，Rb是异丁基，在另一个重复单元中，Rb是异丙基；
    在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb是氢；
    在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基；或
    在一个重复单元中，Rb是氢，在另一个重复单元中，Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基。
  19. 选自下式的化合物：


















    其中Bn表示苄基保护基，Ac表示乙酰氧基。
  20. 一种制备权利要求1-14任一项配体-寡核苷酸共轭物的方法，其包括：
    (a)提供权利要求15至19任一项所述的化合物；
    (b)提供寡核苷酸，所述寡核苷酸的5’或3’端具有末端氨基；
    (c)将所述化合物与所述寡核苷酸通过所述氨基连接；
    优选，步骤(b)中的寡核苷酸为siRNA，其包含正义链和反义链，所述化合物连接至所述正义链的5’端。
  21. 权利要求15至19任一项所述的化合物在制备配体-寡核苷酸共轭物中的用途。
  22. 式I或II所示的接头或式I’或II’所示的连接单元：
    -NR1-C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I)，
    -NR1-[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II)，
    -C(O)-[C(R3R4)]n-C(O)-[NR2-C(RaRb)-C(O)]p-(式I’)，或
    -[C(O)-C(RaRb)-NR2]p-C(O)-[C(R3R4)]n-C(O)-(式II’)，
    其中：
    R1、R2、R3、R4独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-10环烷基、6至14元芳基、5至18元杂芳基、5至14元杂环基、卤素、羟基、氨基、胍基、羧基、氰基、硝基和巯基，它们任选地被R5取代，其中R5选自C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-10环烷基、6至14元芳基、5至18元杂芳基、5至14元杂环基、卤素、羟基、氨基、胍基、羧基、氰基、硝基、C1-3烷基硫基和巯基；
    Ra、Rb独立地选自-H、C1-10烷基、C2-10烯基、C2-10炔基、C1-10烷氧基、C3-8环烷基、6至14元芳基C1-3烷基、5至18元杂芳基C1-3烷基和5至14元杂环基，它们任选地被Rc取代，其中Rc选自羟基、巯基、羧基、氨基、胍基、卤素、C1-3烷基硫基、吲哚基、喹啉基、异喹啉基、酰胺基、吡咯基、噻吩基、噻茚基、噻唑基、苯并噻唑基、咪唑基、吡啶基、呋喃基、C1-10烷基、C1-10烷氧基、6至14元芳基、C3-8环烷基、金刚烷基、膦酰基、膦酰氧基、C1-3烷基膦酰基、C1-3烷基膦酰氧基，或者Ra和Rb中的一个与其共同连接的C原子及相邻的N原子一起形成3至14元含氮杂环基；
    杂原子选自氮、氧、硫、磷和硅；
    n为1至10，并且每个重复单元相同或不同；
    p为2至6，并且每个重复单元相同或不同。
  23. 根据权利要求22所述的接头或连接单元，其中Ra是-H，Rb选自-H、甲基、乙基、丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、环丙基、环戊基、环己基、环己基二亚甲基、环己基亚甲基、环丁基亚甲基、苯基亚甲基、甲基硫基亚甲基、3-吲哚基亚甲基、羟基亚甲基、酰胺基二亚甲基、羟基甲基次甲基、巯基亚甲基、酰胺基亚甲基、羟基苯基亚甲基、羟基苯基二亚甲基、羧基亚甲基、羧基二亚甲基、氨基正丁基、胍基二亚甲基、胍基三亚甲基、4-咪唑基亚甲基、异丙基苯基亚甲基、1-萘基亚甲基、2-萘基亚甲基、茚满基、苯基二亚甲基、氟代苯基亚甲基、氯代苯基亚甲基、三氟苯基亚甲基、二氯苯基亚甲基、五氟苯基亚甲基、二氟苯基亚甲基、噻吩基亚甲基、噻茚基亚甲基、喹啉基亚甲基、卤代3-吲哚基亚甲基、二苯基亚甲基、3-吡啶基亚甲基和4-吡啶基亚甲基，或者Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基。
  24. 根据权利要求23所述的接头或连接单元，其中对于p为2的重复单元：
    在每个重复单元中，Rb是异丙基；
    在每个重复单元中，Rb是甲基；
    在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb是甲基；
    在一个重复单元中，Rb是异丁基，在另一个重复单元中，Rb是甲基；
    在一个重复单元中，Rb是异丁基，在另一个重复单元中，Rb是异丙基；
    在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb是氢；
    在一个重复单元中，Rb是异丙基，在另一个重复单元中，Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基；或
    在一个重复单元中，Rb是氢，在另一个重复单元中，Rb与其共同连接的C原子及相邻的N原子一起形成四氢吡咯基。
  25. 权利要求22至24任一项所述的接头或连接单元在制备配体-寡核苷酸共轭物中的用途。
  26. 一种药物组合物，其包含权利要求1至14任一项所述的配体-寡核苷酸共轭物，以及药学上可接受的载体。
  27. 根据权利要求26所述的药物组合物，其被配制为皮下注射剂。
PCT/CN2023/125626 2022-10-21 2023-10-20 配体-寡核苷酸共轭物及用于该共轭物的接头 WO2024083220A1 (zh)

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US10125369B2 (en) * 2012-12-05 2018-11-13 Alnylam Pharmaceuticals, Inc. PCSK9 iRNA compositions and methods of use thereof
CN113543791A (zh) * 2018-12-20 2021-10-22 维尔生物科技有限公司 组合hbv疗法
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