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WO2025002208A1 - 一种核酸缀合物、制备方法及用途 - Google Patents

一种核酸缀合物、制备方法及用途 Download PDF

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Publication number
WO2025002208A1
WO2025002208A1 PCT/CN2024/101798 CN2024101798W WO2025002208A1 WO 2025002208 A1 WO2025002208 A1 WO 2025002208A1 CN 2024101798 W CN2024101798 W CN 2024101798W WO 2025002208 A1 WO2025002208 A1 WO 2025002208A1
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group
formula
integer
alkyl
nucleic acid
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PCT/CN2024/101798
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English (en)
French (fr)
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朱研
杨志伟
刘楠
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苏州时安生物技术有限公司
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Priority to CN202480001269.3A priority Critical patent/CN118974069A/zh
Publication of WO2025002208A1 publication Critical patent/WO2025002208A1/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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical

Definitions

  • the present invention belongs to the field of nucleic acid delivery, and specifically relates to a nucleic acid conjugate, a preparation method and a use thereof.
  • siRNA is a negatively charged macromolecule composed of two oligonucleotide chains. It cannot effectively target the target tissue in the body and cannot enter the cell autonomously. siRNA needs to rely on special delivery vectors to achieve the enrichment of siRNA in the target organ and allow it to enter the cell in order to exert the therapeutic effect of siRNA.
  • LNP Lipid nanoparticles
  • the drug Onpattro using LNP technology is already on the market.
  • shortcomings in the clinical application of LNP technology such as: the short shelf life of LNP preparations; immune responses are generated during application, and immunosuppressants such as dexamethasone are often required in combination, which complicates the clinical application of LNP.
  • asialoglycoprotein receptor is a receptor that is highly specifically expressed on the surface of hepatocytes, and has the characteristics of high abundance and high receptor recycling efficiency.
  • ASGPR asialoglycoprotein receptor
  • siRNA can be delivered to hepatocytes in a targeted manner, allowing siRNA to exert a gene silencing effect on the target gene. Therefore, the development of targeted ligands suitable for siRNA delivery is of great significance for the clinical application of siRNA.
  • the representative delivery molecule is L96 from Alnylam Pharmaceuticals (its structural formula is shown below), which has been used in four marketed siRNA drugs and has shown good delivery activity and selectivity in both in vitro and in vivo experiments.
  • the in vivo biological activity and efficacy of siRNA can be further improved, the complex CMC (chemical composition production and control) process and technology of the delivery molecule, and high production costs.
  • the delivery compound and the corresponding nucleic acid conjugate disclosed in the present invention use natural amino acids such as serine and threonine as the core connection skeleton to obtain a new type of GalNAc conjugate with a chemical structure.
  • natural amino acids such as serine and threonine
  • it has the advantages of simple molecular structure, cheap and easy to obtain raw materials, and easy development of CMC process, and at the same time has good oligonucleotide synthesis efficiency and in vivo biological activity.
  • a 0 represents a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells or
  • the ligands are preferably each independently selected from any one of the following: D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-mannofuranose, ⁇ -D-mannofuranose, ⁇ -D-mannopyranose, ⁇ -D-mannopyranose, ⁇ -D-glucose, ⁇ -D-glucose, ⁇ -D-glucose, ⁇ -D-glucose, ⁇ -D-glucose, ⁇ -D-furanose, ⁇ -D-fructofuranose, ⁇ -D-fructopyranose, ⁇ -D-galactopyranose, ⁇ -D- galactopyranose, ⁇ -D-galactofuranose, ⁇ -D-gal
  • b is an integer in the range of 1-4, preferably 1, 2 or 3, more preferably 1 or 3;
  • L1 and L2 independently represent a combination of one or more selected from the group consisting of formula A1-A11:
  • M represents a structural formula as shown in formula (A12):
  • m represents an integer of 0-6, preferably 0, 1 or 2; * represents a bond connected to G1 ,
  • X represents the formula (A14-1) or (A14-2) or the structural formula shown:
  • R 4 and R 5 are the same or different and are independently selected from H, fluorine, hydroxyl, C 1 -C 20 alkyl, C 1 -C 20 alkoxy, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, preferably H, fluorine, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl;
  • p is an integer of 1-6, preferably 1, 2 or 3 ; optionally, R 4 and R 5 are directly connected to form a three- to six-membered saturated carbocyclic ring;
  • R 1 is selected from H, fluorine, hydroxyl, cyano, C 1 -C 20 alkyl, C 1 -C 20 alkoxy, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, preferably selected from H, fluorine, hydroxyl, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl;
  • G1 and G2 each represent a phosphoramidite functional group or a hydroxyl protecting group having a structure represented by formula (G-1), provided that at least one of G1 and G2 has a structure represented by formula (G-1), and the hydroxyl protecting group is selected from any one of trityl, 4-methoxytrityl, 4,4'-bismethoxytrityl (DMTr) and 4,4',4'-trimethoxybenzyl, preferably 4,4'-bismethoxytrityl;
  • B1 is selected from substituted or unsubstituted C1 - C5 hydrocarbon groups, and optionally, B1 is methyl, ethyl or isopropyl;
  • B2 is selected from one of C1 - C5 alkyl, acetonitrile, propionitrile and butyronitrile, and optionally, B2 is cyanoethyl;
  • the present invention provides a nucleic acid conjugate, wherein the conjugate contains one or more structures represented by formula (II) connected to any position on the oligonucleotide sequence, preferably two, three or four structures represented by formula (II) connected in succession:
  • R p and R q are each H or have a structure represented by Formula A16, provided that at least one of R p and R q has a structure represented by Formula A16;
  • E 1 is OH, SH or BH 2 ,
  • a 0 , b, L 1 , L 2 , M, Z, X, and E are the same as those in formula (I), and are not described in detail herein.
  • the present invention provides a method for preparing the nucleic acid conjugate of the second aspect.
  • the present invention provides use of the nucleic acid conjugate of the present invention in preparing a medicament for treating and/or preventing a hepatic disease.
  • the present invention provides a method for treating a pathological condition or disease caused by the expression of a gene in a hepatocyte, the method comprising administering the nucleic acid conjugate of the second aspect to a patient suffering from the disease.
  • the present invention provides a kit comprising the nucleic acid conjugate of the present invention.
  • FIG. 1 is the result of the activity test of the siRNA conjugate in mice in Example 10; wherein ** in FIG. 1 represents P Less than 0.01, *** represents P less than 0.001.
  • the compound represented by the general formula (I) includes its tautomers, racemates, enantiomers, diastereomers, mixtures thereof and the like.
  • a hyphen that is not between two letters or symbols is used to indicate the position of the point of attachment of a substituent.
  • optionally substituted alkyl includes “alkyl” and “substituted alkyl” as defined below. It will be understood by those skilled in the art that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically unfeasible, and/or inherently unstable.
  • alkyl refers to straight and branched chains having a specified number of carbon atoms. When reference is made to an alkyl residue having a specific number of carbons, it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, “butyl” is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl.
  • Alkylene is a subset of alkyl and refers to residues identical to alkyl, but having two points of attachment.
  • alkenyl groups include, but are not limited to, vinyl; propenyl, such as prop-1-ene-1-yl, prop-1-ene-2-yl, prop-2-ene-1-yl (allyl), prop-2-ene-2-yl; butenyl, such as but-1-ene-1-yl, but-1-ene-2-yl, 2-methylprop-1-ene-1-yl, but-2-ene-1-yl, but-2-ene-2-yl, but-1,3-diene-1-yl, but-1,3-diene-2-yl, etc.
  • Alkenylene is a subset of alkenyl, referring to a residue identical to alkenyl but having two points of attachment.
  • alkynyl refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon triple bond obtained by losing two hydrogen atoms from adjacent carbon atoms of the parent alkyl group.
  • Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl, butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.
  • alkoxy refers to an alkyl group of the specified number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, etc.
  • Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through an oxygen bridge.
  • cycloalkyl refers to a non-aromatic carbocyclic ring, typically having 3 to 7 cyclic carbon atoms. The ring may be saturated or have one or more carbon-carbon double bonds. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl, as well as bridged and caged ring groups such as norbornane.
  • halogen substituent or “halo” refers to fluoro, chloro, bromo and iodo, and the term “halogen” includes fluorine, chlorine, bromine and iodine.
  • haloalkyl refers to an alkyl group with a specified number of carbon atoms that is substituted with one or more halogen atoms up to the maximum permitted number.
  • haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl and pentafluoroethyl.
  • Heterocyclic group refers to a stable 3 to 18-membered non-aromatic ring radical containing 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. Unless otherwise specified in the specification, heterocyclic groups are monocyclic, bicyclic, tricyclic or tetracyclic ring systems, which may include fused rings or bridged ring systems. The heteroatoms in the heterocyclic group may be optionally oxidized. One or more nitrogen atoms (if present) may be optionally quaternized. The heterocyclic group is partially saturated or fully saturated. The heterocyclic group may be connected to the rest of the molecule through any ring atom.
  • heterocyclic groups include, but are not limited to, dioxanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidine, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl,
  • hydroxy protecting groups can be used in the present disclosure.
  • protecting groups render chemical functional groups insensitive to specific reaction conditions and can be added to and removed from the functional group in a molecule without substantially damaging the remainder of the molecule.
  • Representative hydroxy protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, the above-mentioned document is incorporated herein by reference in its entirety.
  • the protecting group is stable under alkaline conditions, but can be removed under acidic conditions.
  • non-exclusive examples of hydroxyl protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (Mox).
  • non-exclusive examples of hydroxyl protecting groups that can be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl) and TMTr (4,4', 4"-trimethoxytrityl) and tert-butyldimethylsilyl (TBS or TBDMS).
  • Non-exclusive examples of hydroxyl protecting groups that can be used herein include alkyl acyl.
  • subject refers to any animal, such as a mammal or marsupial.
  • Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.
  • non-human primates e.g., rhesus monkeys or other types of macaques
  • mice pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.
  • “treat,” “alleviate,” or “ameliorate” are used interchangeably herein. These terms refer to an approach to obtaining beneficial or desired results, including but not limited to a therapeutic benefit.
  • “Therapeutic benefit” means eradication or amelioration of the underlying disorder being treated. Furthermore, a therapeutic benefit is obtained by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.
  • a conjugate or composition may be administered to a subject at risk for a particular disease, or to a subject reporting one or more pathological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.
  • a 0 represents a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells or
  • each K is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halogenated phenyl and alkylphenyl;
  • the ligands are preferably each independently selected from any one of the following: D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-mannofuranose, ⁇ -D-mannofuranose, ⁇ -D-mannopyranose, ⁇ -D-mannopyranose, ⁇ -D-glucose, ⁇ -D-glucose Glucose, ⁇ -D-glucofuranose, ⁇ -D-glucofuranose, ⁇ -D-fructofuranose, ⁇ -D-fructopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-
  • b is an integer in the range of 1-4, preferably 1, 2 or 3, more preferably 1 or 3;
  • L1 and L2 independently represent a combination of one or more selected from the group consisting of formula A1-A11:
  • M represents a structural formula as shown in formula (A12):
  • m represents an integer of 0-6, preferably 0, 1 or 2; * represents a bond connected to G1 ,
  • R2 and R3 are the same or different and are independently selected from H, C1 - C20 alkyl, C1 - C20 alkoxy, C2 - C20 alkenyl, C2-C20 alkynyl, preferably H, C1-C6 alkyl , C1 - C6 alkoxy , C2 - C6 alkenyl, C2 - C6 alkynyl;
  • Z is absent or represents C 1 -C 10 alkylene, preferably C 1 -C 3 alkylene;
  • X represents the formula (A14-1) or (A14-2) or the structural formula shown:
  • R 4 and R 5 are the same or different and are independently selected from H, fluorine, hydroxyl, C 1 -C 20 alkyl, C 1 -C 20 alkoxy, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, preferably H, fluorine, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl;
  • p is an integer of 1-6, preferably 1, 2 or 3 ; optionally, R 4 and R 5 are directly connected to form a three- to six-membered saturated carbocyclic ring;
  • R 1 is selected from H, fluorine, hydroxyl, cyano, C 1 -C 20 alkyl, C 1 -C 20 alkoxy, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, preferably selected from H, fluorine, hydroxyl, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl;
  • G1 and G2 each represent a phosphoramidite functional group or a hydroxyl protecting group having a structure represented by formula (G-1), provided that at least one of G1 and G2 has a structure represented by formula (G-1), and the hydroxyl protecting group is selected from any one of trityl, 4-methoxytrityl, 4,4'-bismethoxytrityl (DMTr) and 4,4',4'-trimethoxybenzyl, preferably 4,4'-bismethoxytrityl;
  • B1 is selected from substituted or unsubstituted C1 - C5 hydrocarbon groups, and optionally, B1 is methyl, ethyl or isopropyl; B2 is selected from one of C1 - C5 alkyl, acetonitrile, propionitrile and butyronitrile, and optionally, B2 is cyanoethyl.
  • L 1 or L 2 is independently selected from one or more of A1, A2, A4, A6, A7, A8, A9, A10. In some embodiments, L 1 or L 2 is independently selected from one or more of A1, A2, A4, A8, A9, A10. In some embodiments, wherein L 1 or L 2 is independently selected from one or more of A1, A4, A8, A9, A10. In some embodiments, the length of L 1 or L 2 is independently 1-25 atoms, and the length of L 1 or L 2 refers to the number of chain atoms on the longest straight chain. In some embodiments, the length of L 1 or L 2 is independently 1-20 atoms.
  • each j1 is independently an integer of 1-10, and each j2 is independently an integer of 1-10.
  • each j1 is independently an integer from 1 to 8
  • each j2 is independently an integer from 1 to 8.
  • A0 is an N-acetylgalactosamine (GalNAc) group as shown below, or a group formed by replacing all or part of its hydroxyl group with a KCOO- group, wherein each K is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halogenated phenyl and alkylphenyl:
  • GalNAc N-acetylgalactosamine
  • the compound has the structure (I-1) shown below:
  • L is A10, wherein j1 is an integer from 2 to 8, or A11, wherein j2 is an integer from 1 to 7.
  • m is 0, 1 or 2.
  • n 0, 1, 2, or 3.
  • the compounds of the present disclosure have any one of the structures shown below:
  • R p and R q are each H or have a structure represented by Formula A16, provided that at least one of R p and R q has a structure represented by Formula A16;
  • E 1 is OH, SH or BH 2 ,
  • a 0 represents a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells or
  • each K is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halogenated phenyl and alkylphenyl;
  • the ligands are preferably each independently selected from any one of the following: D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-mannofuranose, ⁇ -D-mannofuranose, ⁇ -D-mannopyranose, ⁇ -D-mannopyranose, ⁇ -D-glucosepyranose, ⁇ -D-glucosepyranose, ⁇ -D-glucosefuranose, ⁇ -D-glucosefuranose, ⁇ -D-fructofuranose, ⁇ -D-fructopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranose,
  • b is an integer in the range of 1-4, preferably 1, 2 or 3, more preferably 1 or 3;
  • L1 and L2 independently represent a combination of one or more selected from the group consisting of formula A1-A11:
  • j1 is an integer from 1 to 20;
  • j2 is an integer from 1 to 20;
  • R' is a C 1 -C 10 alkyl group
  • M represents a structural formula as shown in formula (A12):
  • m represents an integer of 0-6, preferably 0, 1 or 2; * represents a bond connected to R p ,
  • R 2 and R 3 are the same or different and are independently selected from H, C 1 -C 20 alkyl, C 1 -C 20 alkoxy, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, preferably H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl;
  • Z is absent or represents C 1 -C 10 alkylene, preferably C 1 -C 3 alkylene;
  • X represents the formula (A14-1) or (A14-2) or the structural formula shown:
  • R 4 and R 5 are the same or different and are independently selected from H, fluorine, hydroxyl, C 1 -C 20 alkyl, C 1 -C 20 alkoxy, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, preferably H, fluorine, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl p is an integer of 1-6, preferably 1, 2 or 3; optionally, R 4 and R 5 are directly connected to form a three- to six-membered saturated carbon ring;
  • R 1 is selected from H, fluorine, hydroxyl, cyano, C 1 -C 20 alkyl, C 1 -C 20 alkoxy, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, preferably selected from H, fluorine, hydroxyl, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl.
  • the present invention provides a nucleic acid conjugate, wherein the conjugate contains one or more structures represented by formula (II-A) linked to any position on the oligonucleotide sequence:
  • a represents an integer of 0-7, preferably an integer of 0-3, more preferably an integer of 0, 1, 2 or 3;
  • Rp and Rq are each H or have a structure represented by Formula A16, and at least one of them has a structure represented by Formula A16;
  • E 1 is OH, SH or BH 2 ,
  • a 0 , b, L 1 , L 2 , M, Z, X, and E are the same as those in formula (II), and are not described in detail herein.
  • L 1 or L 2 is independently selected from one or more of A1, A2, A4, A6, A7, A8, A9, A10. In some embodiments, L 1 or L 2 is independently selected from one or more of A1, A2, A4, A8, A9, A10. In some embodiments, wherein L 1 or L 2 is independently selected from one or more of A1, A4, A8, A9, A10. In some embodiments, the length of L 1 or L 2 is independently 1-25 atoms, and the length of L 1 or L 2 refers to the number of chain atoms on the longest straight chain. In some embodiments, the length of L 1 or L 2 is independently 1-20 atoms.
  • each j1 is independently an integer of 1-10, and each j2 is independently an integer of 1-10.
  • each j1 is independently an integer from 1 to 8
  • each j2 is independently an integer from 1 to 8.
  • A0 is an N-acetylgalactosamine (GalNAc) group as shown below, or all or part of its hydroxyl groups.
  • GalNAc N-acetylgalactosamine
  • conjugation refers to the covalent connection between two or more chemical moieties each having a specific function; accordingly, “conjugate” refers to a compound formed by covalent connection between the chemical moieties.
  • conjugate refers to a compound formed by covalently connecting one or more chemical moieties having a specific function to an oligonucleotide. In the following, the nucleic acid conjugate of the present disclosure is sometimes referred to as a "conjugate”.
  • nucleic acid conjugate of the present invention has any one of the following structures:
  • the oligonucleotide is selected from one of small interfering RNA, microRNA, anti-microRNA, microRNA antagonist, microRNA mimetic, decoy oligonucleotide, immunostimulant, G-quadrupole, alternative splicing, single-stranded RNA, antisense nucleic acid, nucleic acid aptamer, stem-loop RNA, mRNA fragment, and activating RNA; optionally, the oligonucleotide is a single-stranded oligonucleotide or a double-stranded oligonucleotide; optionally, the oligonucleotide is a single-stranded oligonucleotide, the P atom in formula (A16) is connected to the end of the single-stranded oligonucleotide, and the end of the single-stranded oligonucleotide refers to the first 4 nucleotides from
  • the oligonucleotide is a double-stranded oligonucleotide comprising a sense strand and an antisense strand
  • the P atom in formula (A16) is connected to the end of the double-stranded oligonucleotide
  • the end of the double-stranded oligonucleotide refers to the first 4 nucleotides from one end of the sense strand or the antisense strand
  • the P atom in formula (A16) is connected to the end of the sense strand or the antisense strand
  • the P atom in formula (A16) is connected to the 5' end of the antisense strand
  • the P atom in formula (A16) is connected to the 2', 3' or 5' position of the nucleotide in the nucleic acid conjugate by forming a phosphodiester bond.
  • the "target sequence” is a target mRNA.
  • the "target mRNA” refers to the mRNA corresponding to a gene abnormally expressed in hepatocytes, which can be either the mRNA corresponding to an overexpressed gene or the mRNA corresponding to an underexpressed gene. Since most diseases originate from the overexpression of mRNA, in the present disclosure, the target mRNA particularly refers to the mRNA corresponding to the overexpressed gene.
  • the target mRNA may be the mRNA corresponding to genes such as ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, HCV, etc.
  • the target mRNA may be the mRNA transcribed from the corresponding HBV gene, or the mRNA corresponding to the ANGPTL3 gene, or the mRNA corresponding to the APOC3 gene.
  • siRNA contains a nucleotide group as a basic structural unit, and the nucleotide group contains a phosphate group, a ribose group and a base.
  • the length of an active, i.e., functional siRNA is about 12-40 nucleotides, and in some embodiments, about 15-30 nucleotides.
  • Each nucleotide in the siRNA can be independently a modified or unmodified nucleotide. In order to increase stability, at least one nucleotide in the siRNA is a modified nucleotide.
  • the inventors of the present disclosure have found that the siRNA described in the following embodiments has higher activity and/or stability, and thus can be used as the invention object of the siRNA in the present disclosure.
  • each nucleotide in the siRNA in the siRNA conjugate of the present invention is independently a modified or unmodified nucleotide
  • the siRNA contains a sense strand and an antisense strand
  • the sense strand comprises a nucleotide sequence 1
  • the antisense strand comprises a nucleotide sequence 2
  • the lengths of the nucleotide sequence 1 and the nucleotide sequence 2 are both 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 nucleotides, and are at least partially reverse-complemented to form a complementary double-stranded region, and at least a portion of the nucleotide sequence 2 is complementary to a first nucleotide sequence
  • the first nucleotide sequence is a nucleotide sequence in the target mRNA.
  • these specific nucleotide differences do not significantly reduce the target gene inhibition ability of the siRNA conjugate, and these siRNA conjugates containing specific nucleotide differences are also within the scope of protection of the present disclosure.
  • nucleotide sequence 1 and the nucleotide sequence 2 are substantially reverse complementary, substantially completely reverse complementary, or completely reverse complementary.
  • the nucleotide sequence 1 differs from the first nucleotide sequence by no more than 1 nucleotide
  • the nucleotide sequence 2 differs from the nucleotide sequence B by no more than 1 nucleotide.
  • the nucleotide difference between the nucleotide sequence 2 and the nucleotide sequence B includes the difference in the position of the first nucleotide Z' on the nucleotide sequence 2 in the direction from the 5' end to the 3' end.
  • the last nucleotide Z on the nucleotide sequence 1 is a nucleotide complementary to Z'.
  • the sense strand further contains nucleotide sequence 3, and the antisense strand further contains nucleotide sequence 4, the lengths of nucleotide sequence 3 and nucleotide sequence 4 are equal and both are 1-4 nucleotides, the nucleotide sequence 3 is connected to the 5' end of the nucleotide sequence 1, and the nucleotide sequence 4 is connected to the 3' end of the nucleotide sequence 2, and the nucleotide sequence 4 is complementary to the second segment of nucleotide sequence, which refers to a nucleotide sequence in the target mRNA that is adjacent to the first segment of nucleotide sequence and has the same length as the nucleotide sequence 4.
  • the nucleotide sequence 3 and the nucleotide sequence 4 are substantially completely reverse complementary or completely reverse complementary. Therefore, the lengths of the sense strand and the antisense strand can be 19-23 nucleotides.
  • the siRNA of the present disclosure further comprises a nucleotide sequence 5, the length of which is 1 to 3 nucleotides, connected to the 3' end of the antisense strand, thereby constituting the 3' overhang of the antisense strand; in some embodiments, the length of the nucleotide sequence 5 is 1 or 2 nucleotides.
  • the length ratio of the sense strand to the antisense strand of the siRNA of the present disclosure can be 19/20, 19/21, 20/21, 20/22, 21/22, 21/23, 22/23, 22/24, 23/24 or 23/25.
  • the length of the nucleotide sequence 5 is 2 nucleotides, and in the direction from the 5' end to the 3' end, the nucleotide sequence 5 is 2 consecutive deoxythymine nucleotides, 2 consecutive uracil nucleotides, or complementary to the third segment of nucleotide sequence, and the third segment of sequence refers to a nucleotide sequence in the target mRNA that is adjacent to the first segment of nucleotide sequence, or adjacent to the second segment of nucleotide sequence, and has a length equal to that of the nucleotide sequence 5.
  • the ratio of the length of the sense strand and the antisense strand of the siRNA disclosed in the present invention is 19/21 or 21/23, in which case, the siRNA disclosed in the present invention has better hepatocyte mRNA silencing activity.
  • the nucleotides in the siRNA of the present disclosure are each independently a modified or unmodified nucleotide. In some embodiments, the siRNA of the present disclosure does not contain a modified nucleotide group; in some embodiments, the siRNA of the present disclosure contains a modified nucleotide group.
  • siRNA RNA RNA RNA cleavage modification
  • backbone modification also known as internucleotide linkage modification, such as phosphate group modification
  • ribose group modification and base modification for example, see Watts, J.K., G.F.Deleavey and M.J.Damha, Chemically modified siRNA: tools and applications.
  • Drug Discov Today, 2008.13(19-20): p.842-55 the entire contents of which are incorporated herein by reference).
  • modified nucleotide refers to a nucleotide whose ribose group is modified, such as a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group with other groups, or a nucleotide whose base is a modified base.
  • At least one nucleotide in the sense strand or the antisense strand is a modified nucleotide
  • at least one phosphate group is a phosphate group with a modified group.
  • at least a portion of the phosphate group and/or ribose group in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand is a phosphate group with a modified group and/or a ribose group with a modified group (or a modified phosphate group and/or a modified ribose group).
  • all nucleotides in the sense strand and/or the antisense strand are modified nucleotides.
  • each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.
  • the fluorinated nucleotide refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribose group of the nucleotide is substituted with fluorine, and has a structure represented by the following formula (17).
  • Non-fluorinated modified nucleotides refer to nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group.
  • each non-fluorinated modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group.
  • Nucleotides in which the hydroxyl group at the 2' position of the ribose group is replaced by a non-fluorinated group are well known to those skilled in the art, and these nucleotides can be selected from 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxynucleotides.
  • the 2'-alkoxy modified nucleotide is a methoxy modified nucleotide (2'-OMe), as shown in formula (18).
  • the 2'-substituted alkoxy modified nucleotide for example, can be a 2'-O-methoxyethyl modified nucleotide (2'-MOE), as shown in formula (19).
  • the 2'-amino modified nucleotide (2'-NH 2 ) is as shown in formula (20).
  • 2'-deoxynucleotide (DNA) is represented by formula (21).
  • Nucleotide analogs refer to groups that can replace nucleotides in nucleic acids but have structures different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine.
  • the nucleotide analogs can be, for example, isonucleotides, bridged nucleic acid (BNA) nucleotides or acyclic nucleotides.
  • BNA nucleotides refer to constrained or inaccessible nucleotides.
  • BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed"C3'-endosugar condensed bridge structure.
  • the bridge is usually incorporated into the 2'-, 4'-position of the ribose ring to provide a 2', 4'-BNA nucleotide, such as LNA, ENA, cET BNA, etc., wherein LNA is shown in formula (22), ENA is shown in formula (23), and cET BNA is shown in formula (24).
  • Acyclic nucleotides are a type of nucleotides formed by opening the sugar ring of a nucleotide, such as unlocked nucleic acid (UNA) nucleotides or glycerol nucleic acid (GNA) nucleotides, wherein UNA is shown in formula (25) and GNA is shown in formula (26).
  • UNA unlocked nucleic acid
  • GNA glycerol nucleic acid
  • R is selected from H, OH or alkoxy (O-alkyl).
  • Isonucleotides are compounds formed by a change in the position of the base on the ribose ring of a nucleotide, for example, compounds formed by the base moving from the 1'-position to the 2'-position or the 3'-position of the ribose ring, as shown in formula (27) or (28).
  • Base represents a base, such as A, U, G, C or T; R is selected from H, OH, F or the non-fluorine group as described above.
  • the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA.
  • each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, wherein the methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose moiety with a methoxy group.
  • fluorinated nucleotide In the above and below, “fluorinated nucleotide”, “2'-fluorinated nucleotide”, “nucleotide in which the 2'-hydroxyl group of the ribose group is substituted by fluorine” and “2'-fluororibose” have the same meaning, all referring to compounds in which the 2'-hydroxyl group of the nucleotide is replaced by fluorine and has a structure as shown in formula (17); “methoxy-modified nucleotide”, “2'-methoxy-modified nucleotide”, “nucleotide in which the 2'-hydroxyl group of the ribose group is substituted by methoxy” and “2'-methoxyribose” have the same meaning, all referring to compounds in which the 2'-hydroxyl group of the ribose group of the nucleotide is replaced by methoxy to form a structure as shown in formula (18).
  • the siRNA disclosed herein is a siRNA having the following modifications: both the sense strand and the antisense strand contain fluorinated modified nucleotides and non-fluorinated modified nucleotides, the fluorinated modified nucleotides are located in the aforementioned nucleotide sequence 1 and nucleotide sequence 2, the fluorinated modified nucleotides in the nucleotide sequence 1 are not more than 5, and, from the 5' end to the 3' end, the 7th, 8th, and 9th nucleotides in the nucleotide sequence 1 are fluorinated modified nucleotides; the fluorinated modified nucleotides in the nucleotide sequence 2 are not more than 7, and, from the 5' end to the 3' end, the 2nd, The nucleotides at positions 6, 14, and 16 are fluorinated modified nucleotides.
  • the siRNA of the present invention is a siRNA with the following modifications: in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence 1 in the sense strand of the siRNA are -fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; in the antisense strand, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence 2 are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are methoxy-modified nucleotides; in some embodiments, the siRNA of the present invention is a siRNA with the following modifications: or in the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of the nucleotide sequence 1 in the sense strand of the siRNA are fluorinated
  • the nucleotides are methoxy-modified nucleotides; in the antisense strand, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence 2 are fluorinated-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are methoxy-modified nucleotides; in some embodiments, the siRNA of the present invention is a siRNA with the following modifications: in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence 1 in the sense strand of the siRNA are fluorinated-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence 2 in the antisense strand of the siRNA
  • the nucleotide contains a phosphate group modification.
  • the phosphate group modification is, in one embodiment, a phosphorothioate modification as shown in the following formula (11), i.e., a non-bridging oxygen atom in a phosphodiester bond is substituted with a sulfur atom, thereby replacing the phosphodiester bond with a phosphorothioate diester bond.
  • this modification can stabilize the structure of the siRNA and maintain high specificity and high affinity for base pairing.
  • the phosphorothioate linkage is present in at least one of the group consisting of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the above.
  • the phosphorothioate linkage is present at all of the above positions except the 5' end of the sense strand.
  • the phosphorothioate linkage is present at all of the above positions except the 3' end of the sense strand.
  • the phosphorothioate linkage is present at at least one of the following positions:
  • the 5' terminal nucleotide of the antisense strand sequence of the siRNA molecule is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analog.
  • the 5'-phosphate nucleotide may have a structure as shown in formula (12):
  • R represents a group selected from the group consisting of H, OH, F and methoxy
  • Base represents a base selected from A, U, C, G or T.
  • the 5'-phosphate nucleotide or 5'-phosphate analog modified nucleotide is a nucleotide containing vinyl phosphate (E-vinylphosphonate, E-VP) as shown in formula (13), a 5'-phosphate modified nucleotide as shown in formula (12), or a 5'-thiophosphate modified nucleotide as shown in formula (15).
  • the double-stranded oligonucleotide is siRNA.
  • a method for preparing the nucleic acid conjugate disclosed in the present invention comprising oxidizing the phosphoramidite functional group in the compound of formula (I) of the present invention to a structure shown in formula (W), then combining with an oligonucleotide, and then cleaving and removing the protecting group to obtain the conjugate.
  • E1 is OH, SH or BH2 .
  • the preparation method of the nucleic acid conjugate disclosed in the present invention may include: under the conditions of phosphoramidite solid phase synthesis, according to the nucleotide types and sequence of the functional oligonucleotide, the nucleoside monomers are sequentially connected in the 3' to 5' direction, and the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization.
  • the oxidation reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 1-100 seconds, in some embodiments 5-50 seconds, and the oxidizing agent in some embodiments is iodine (in further embodiments, provided in the form of iodine water).
  • nucleic acid conjugate disclosed in the present invention in the preparation of a medicament for treating and/or preventing hepatic diseases.
  • a method for treating a pathological condition or disease caused by the expression of a gene in a hepatocyte comprising administering the nucleic acid conjugate disclosed in the present invention to a patient suffering from the disease.
  • a kit comprising the nucleic acid conjugate disclosed in the present invention.
  • N-benzyloxycarbonyl-L-serine (commercially available, purchased from Shanghai Titan Technology Co., Ltd.) (35.0 mmol, 8.4 g) was placed in a clean dry reaction bottle, 100 mL of dichloromethane was added, and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.5 equiv, 52.5 mmol, 19.9 g), compound 1-2 (1.1 equiv, 38.5 mmol, 16.1 g) and N,N-diisopropylethylamine (3.0 equiv, 105.0 mmol, 13.5 g) were added at room temperature, followed by stirring at room temperature for 1 hour.
  • N-benzyloxycarbonyl-L-serine (commercially available, purchased from Shanghai Titan Technology Co., Ltd.) (15.0 mmol, 3.58 g) was placed in a clean dry reaction bottle, 100 mL of dichloromethane was added, and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.5 equiv, 22.5 mmol, 8.53 g), compound 2-2 (1.1 equiv, 16.5 mmol, 3.31 g) and N,N-diisopropylethylamine (3.0 equiv, 45.0 mmol, 5.78 g) were added at room temperature, followed by stirring at room temperature for 1 hour.
  • compound 2-5 (1.1 equiv, 5.08 mmol, 3.0 g) and N,N-diisopropylethylamine (3.0 equiv, 13.86 mmol, 17.91 g) were added to the reaction system, and then stirring was continued at room temperature for 1 hour. After the reaction, 150 mL of dichloromethane was added to the reaction solution, and the mixture was washed with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated brine. The organic phase was dried, filtered and concentrated.
  • Example 2 According to the synthesis method of Example 2, a white solid I-1-5 (3.0 g, 2.68 mmol, 72% yield) was prepared from the starting material N-benzyl-4-hydroxypiperidine (commercially available, purchased from Shanghai Titan Technology Co., Ltd.). Compound I-1-5 molecular formula: C 57 H 78 O 15 N 6 P, molecular weight: 1119.5, LC-MS measured: 1142.4 (M+Na).
  • Example 1 According to the synthesis method of Example 1, a white solid I-1-2 (1.2 g, 1.05 mmol, 56% yield) was prepared from the starting material L-threonine (commercially available, purchased from Shanghai Titan Technology Co., Ltd.). Compound I-1-2 molecular formula: C 59 H 82 O 15 N 6 P, molecular weight: 1147.5, LC-MS measured: 1170.3 (M+Na).
  • Example 2 According to the synthesis method of Example 2, a white solid I-1-9 (3.02 g, 2.77 mmol, 83% yield) was prepared from the starting material 1-benzyloxycarbonyl-3-hydroxyazetidine (commercially available, purchased from Shanghai Titan Technology Co., Ltd.). Compound I-1-9 molecular formula: C 55 H 74 O 15 N 6 P, molecular weight: 1091.4, LC-MS measured: 1114.3 (M+Na).
  • white solid I-1-13 (2.32 g, 2.1 mmol, 81% yield) was prepared from the starting material N-benzyloxycarbonyl-DL-serine (commercially available, purchased from Shanghai Titan Technology Co., Ltd.).
  • white solid I-1-15 (2.1 g, 1.88 mmol, 78% yield) was prepared from the starting materials N-benzyl-4-hydroxypiperidine (commercially available, purchased from Shanghai Titan Technology Co., Ltd.) and N-benzyloxycarbonyl-DL-serine (commercially available, purchased from Shanghai Titan Technology Co., Ltd.).
  • N-benzyl-4-hydroxypiperidine commercially available, purchased from Shanghai Titan Technology Co., Ltd.
  • N-benzyloxycarbonyl-DL-serine commercially available, purchased from Shanghai Titan Technology Co., Ltd.
  • nucleoside monomers are connected one by one from the 3'-5' direction according to the nucleotide arrangement order.
  • the specially modified anti-off-target compound is placed in the seed region of the antisense chain (any position from the 4th to the 8th position starting from the 5' end), and the delivery monomer compound is freely set to be placed at the 3' end or the 5' end according to the common monomer.
  • Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. Synthesis conditions used for the sense chain and antisense chain.
  • the reagents used to synthesize siRNA conjugates were purchased from Suzhou Kelema Biotechnology Co., Ltd.
  • the single-strand synthesis reaction process is extended from 3'-5' direction and completed on a solid phase synthesizer. It includes four main reaction steps:
  • DMTr removal reaction Use dichloroacetic acid to remove the DMTr protecting group on the nucleotide to obtain the 5'-hydroxyl end;
  • Oxidation reaction Under the action of iodine, the phosphite triester obtained in the previous condensation reaction is converted into a more stable phosphate ester (i.e., trivalent phosphorus is oxidized to pentavalent phosphorus);
  • Capping reaction There may be a very small number of 5'-hydroxyl groups (less than 2%) that do not participate in the condensation reaction. Use acetic anhydride and 1-methylimidazole to react with them to form acetate caps that cannot participate in subsequent reactions, thereby preventing further reactions. This short fragment can be separated during purification.
  • the nucleic acid sequence connected to the solid phase carrier is cut, deprotected, purified, desalted, and then freeze-dried to obtain the sense chain and the antisense chain, wherein:
  • Aminolysis is carried out at 50°C in a constant temperature water bath for 16 hours. After 16 hours of aminolysis, the water bath is cooled to warm (25°C ⁇ 2°C), filtered with
  • a small sample of the crude product is sent to the analysis department for detection of the crude product LC-MS.
  • the detection method is as follows: Use Waters Acquity UPLC-LTQ LCMS (column: ACQUITY UPLC BEH C18) to detect the purity of the above-mentioned sense chain and antisense chain and analyze the molecular weight. The measured values are consistent with the theoretical values, see Table 1.
  • the purification and desalting conditions are as follows: purification using an ion exchange column and desalting using a HiPrepTM 26/10 Desalting gel column, followed by single-chain freeze-drying. After single-chain freeze-drying, sampling is required for LC-MS.
  • the obtained sense strand and antisense strand need to be annealed into a double strand.
  • Annealing operation is as follows: the sense chain and antisense chain obtained after purification are dissolved in water for injection respectively to prepare 0.1mg/mL-40mg/mL solution, mixed in equal molar ratio using Thermo Scientific Nanodrop Eight calibration, heated at 90°C for 5 minutes, and then slowly cooled naturally to form a double-stranded structure through hydrogen bonding, and samples are taken for testing of the SEC purity of the product, see Table 2, and the double-stranded sample is lyophilized.
  • mice Select 6-8 week old SPF grade female C57BL/6J mice, the weight of the mice was 20 ⁇ 2g.
  • the mice were weighed and observed before administration, and animals with uniform weight and normal condition were randomly divided into groups, with 4 mice in each group.
  • the mice in the experimental group were given the conjugate, and the mice in the vehicle group were given phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the conjugate was administered subcutaneously at a dose of 1 mg/kg per mouse. 20 days after administration, the animals were euthanized, and liver tissue was taken. According to conventional methods, the liver was cut into pieces and placed in RNALater (Invitrogen, AM7021M) for subsequent RNA extraction.
  • RNALater Invitrogen, AM7021M
  • the liver tissue was placed in a lysis solution (Changchun Zhiang Biotechnology Co., Ltd., MNTR/FX96) and ground (Shanghai Jingxin Industrial Development Co., Ltd., JXFSTPRP-48L) to extract total RNA, which was reverse transcribed into cDNA (Takara, 6210B), The expression level of target gene complement C5 mRNA was detected by fluorescence qPCR (Vazyme, Q711).
  • Reverse primer CGGCGTGTAAACAGGTTTGTC;
  • the results are expressed as the residual expression level of the siRNA administration group compared to the vehicle group (the vehicle group is 100%), and the siRNA sequences of the conjugates used for injection are shown in Table 1.
  • the conjugate SD003974 compared with the positive conjugate SD003317, the conjugate SD003974 showed comparable target gene complement C5 mRNA silencing activity; the conjugates SD004119, SD004122 and SD004125 showed significantly better complement C5 mRNA silencing activity than the conjugate SD003317 target gene.

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Abstract

本发明提供一种用于与寡核苷酸形成缀合物的化合物，该化合物具有如式(I)所示的结构。本公开还提供了相应的缀合物，以及所述缀合物在制备用于预防和/或治疗相关疾病的药物中的用途。本公开的缀合物有原料便宜、合成简单、工艺易于开发等优点，同时有着良好的寡核苷酸合成效率及小动物体内生物活性。

Description

一种核酸缀合物、制备方法及用途 技术领域
本公开属于核酸递送领域，具体涉及一种核酸缀合物、制备方法及用途。
背景技术
siRNA是由两条寡核苷酸链组成的带有负电荷的大分子，自身无法有效靶向至体内靶组织，也无法自主进入细胞内。siRNA需要借助特殊的递送载体来实现siRNA的靶器官富集，并使其进入细胞，以发挥siRNA的治疗作用。
脂质纳米粒(Lipid Nanoparticle,LNP)可以将siRNA包裹并有效实现肝靶向递送，采用LNP技术的药物Onpattro已经上市。然而LNP技术的临床应用仍然存在诸多不足，例如：LNP制剂有效期短；在应用中产生免疫反应，常需要协同使用地塞米松等免疫抑制剂，这使得LNP的临床应用复杂化。
通过细胞表面受体介导的内吞作用将药物靶向递送至细胞内部是一个有效的策略。去唾液酸糖蛋白受体(ASGPR)是在肝实质细胞表面高度特异性表达的受体，具有丰度高、受体循环利用效率高的特性。将能够特异性识别ASGPR的配体，如半乳糖、半乳糖胺、N-乙酰半乳糖胺(GalNAc)等单糖和多糖分子与siRNA共价偶联，可以将siRNA靶向递送至肝实质细胞内，使siRNA发挥对靶基因的基因沉默作用。因此，研发适用于siRNA递送的靶向配体，对于siRNA的临床应用具有重要意义。
目前全球范围内通过靶向ASGPR实现siRNA有效肝靶向递送的研究有了部分进展，代表性的递送分子有Alnylam Pharmaceuticals的L96(其结构式如下所示)，其已用于四款上市siRNA药物，在体外及体内实验中均表现出良好的递送活性和选择性；但是目前仍然存在着siRNA体内生物活性和功效还可以进一步提高及递送分子CMC(化学成分生产和控制)工艺和技术复杂、生产成本高等问题。
发明内容
本公开的递送化合物及相应的核酸缀合物以天然氨基酸如丝氨酸、苏氨酸等作为核心连接骨架，得到了一类全新化学结构的GalNAc缀合物。与现有递送技术相比有着分子结构简单、原料价廉易得、CMC工艺易于开发等优点，同时有着良好的寡核苷酸合成效率和体内生物活性。
在第一方面，本公开提供一种化合物，该化合物具有式(I)所示的结构：
其中，
A0代表对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体或者
代表所述配体中的羟基全部或部分地被KCOO-基团取代而形成的基团，其中，每个K独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；
其中所述配体优选地各自独立地选自下述中的任意一种：D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D-吡喃甘露糖、α-D-吡喃葡萄糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D- 吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳糖胺、N-乙酰基半乳糖胺、N-三氟乙酰基半乳糖胺、N-丙酰基半乳糖胺、N-正丁酰基半乳糖胺、N-异丁酰基半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6-O-三苯甲基-α-D-吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖、L-4-硫代核糖；
b为1-4范围内的整数，优选1、2或3，更优选1或3；
L1和L2彼此独立地代表选自式A1-A11基团中的一种或多种的连接组合：
其中，j1为1-20的整数；j2为1-20的整数；R’为C1-C10的烷基；
M代表如式(A12)所示的结构式：
其中，m代表0-6的整数，优选为0、1或2；*表示与G1相连的键，
Q代表式(A13)所示的结构式：
其中，与O相连的表示与G1相连的键；R2、R3相同或不同，且彼此独立地选自H、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基，优选H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；
Z不存在或代表C1-C10亚烷基，优选C1-C3亚烷基；
X代表式(A14-1)或(A14-2)或所示的结构式：
其中R4，R5相同或不同，且彼此独立地选自H、氟、羟基、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基，优选H、氟、羟基、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；p为1-6的整数，优选1、2或3；任选地，R4和R5直接相连形成三到六元饱和碳环；
E代表如式(A15)所示的结构式：
其中，代表C3-C18环烷基或C3-C18杂环基，优选代表C4-C8环烷基或C3-C8杂环基，更优选代表C3-C6杂环基，更优选四到六元含氮饱和环烷基，并且R1选自H、氟、羟基、氰基、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基，优选选自H、氟、羟基、氰基、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；
G1和G2各自代表具有式(G-1)所示结构的亚磷酰胺官能团或羟基保护基团，前提是G1和G2中至少一个具有式(G-1)所示的结构，所述羟基保护基团选自三苯甲基、4-甲氧基三苯甲基、4,4’-双甲氧基三苯甲基(DMTr)和4,4’,4’-三甲氧基苯甲基中的任意一种，优选4,4’-双甲氧基三苯甲基；
其中，B1选自取代或未取代的C1-C5的烃基，可选地，B1为甲基、乙基或异丙基；B2选自C1-C5的烷基、乙氰基、丙氰基和丁氰基中的一种，可选地，B2为氰乙基；
本文中，表示基团共价键连接的位点。
在第二方面，本发明提供了一种核酸缀合物，在所述缀合物中含有与寡核苷酸序列上的任意位置连接的一个或一个以上具有式(II)所示的结构，优选两个、三个或四个连续连接的具有式(II)所示的结构：
其中，Rp和Rq各自为H或具有式A16所示的结构，前提是Rp和Rq中至少一个具有式A16所示的结构；
其中，E1为OH、SH或BH2，
A0、b、L1、L2、M、Z、X、E等同式(I)，此处不再赘述。
在第三方面，本发明提供了第二方面核酸缀合物的制备方法。
在第四方面，本发明提供了本发明的核酸缀合物在制备用于治疗和/或预防肝源性疾病的药物中的用途。
在第五方面，本发明提供了一种治疗由肝细胞中基因的表达而引起的病理状况或疾病的方法，所述方法包括向患有该疾病的患者给予第二方面的核酸缀合物。
在第六方面，本发明提供了一种试剂盒，该试剂盒包含本发明的核酸缀合物。
附图说明
图1是实施例10中的小鼠体内测试siRNA缀合物的活性实验结果；其中图1中**代表P 小于0.01，***代表P小于0.001。
具体实施方式
以下对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
定义
需要说明的是，除非另外定义，本申请使用的技术术语或者科学术语应当为所属领域的技术人员所理解的通常意义。
在本公开中，通式(I)所示的化合物包括其互变异构体、外消旋体、对映异构体、非对映异构体、其混合物等形式。
如本文所使用的，不介于两个字母之间或两个符号之间的短横(“-”)是用于指示取代基连接点的位置。
如本文所使用的，“任选的”或“任选地”是指其后描述的事件或状况可以发生或不发生，并且所述描述包括事件或状况发生的情况和其中不发生的情况。例如，“任选的取代的烷基”包括下文定义的“烷基”和“取代烷基”。本领域技术人员将理解的是，对于包含一个或多个取代基的任何基团，这些基团不打算引入空间上不切实际、合成上不可行和/或内在不稳定的任何取代或取代型式。
如本文所使用的，“烷基”是指具有指定数量的碳原子的直链和支链。当提及具有特定数量的碳的烷基残基时，旨在涵盖具有该数量的碳的所有支链和直链形式；因此，例如，“丁基”意味着包括正丁基、仲丁基、异丁基和叔丁基。亚烷基是烷基的子集，指与烷基相同、但具有两个连接点的残基。
如本文所使用的，“烯基”是指具有至少一个碳-碳双键的不饱和支链或直链烷基，所述碳-碳双键是通过从母体烷基的相邻碳原子中各自失去一个氢原子而获得的。该基团可以处于双键的顺式或反式构型。典型的烯基基团包括但不限于：乙烯基；丙烯基，如丙-1-烯-1-基、丙-1-烯-2-基、丙-2-烯-1-基(烯丙基)、丙-2-烯-2-基；丁烯基，例如丁-1-烯-1-基、丁-1-烯-2-基、2-甲基丙-1-烯-1-基、丁-2-烯-1-基、丁-2-烯-2-基、丁-1,3-二烯-1-基、丁-1,3-二烯-2-基等等。亚烯基是烯基的一个子集，指与烯基相同、但具有两个连接点的残基。
如本文所使用的，“炔基”是指具有至少一个碳-碳三键的不饱和支链或直链烷基，所述碳-碳三键是通过从母体烷基的相邻碳原子中各自失去两个氢原子而获得的。典型的炔基基团包括但不限于：乙炔基；丙炔基，如丙-1-炔-1-基，丙-2-炔-1-基；丁炔基，例如丁-1-炔-1-基，丁-1-炔-3-基，丁-3-炔-1-基等。
如本文所使用的，“烷氧基”是指通过氧桥连接的指定数量碳原子的烷基，例如，甲氧基、乙氧基、丙氧基、异丙氧基、正丁氧基、仲丁氧基、叔丁氧基、戊氧基、2-戊氧基、异戊氧基、新戊氧基、己氧基、2-己氧基、3-己氧基、3-甲基戊氧基等。烷氧基通常具有1至10个、1至8个、1至6个，或1至4个通过氧桥连接的碳原子。
如本文所使用的，“环烷基”是指非芳香碳环，通常具有3至7个环状碳原子。环可以是饱和的，或具有一个或多个碳-碳双键。环烷基的实例包括环丙基、环丁基、环戊基、环戊烯基、环己基和环己烯基，以及桥联和笼状环基团，如降冰片烷(norbornane)。
如本文所使用的，“卤素取代基”或“卤”指氟代、氯代、溴代和碘代，术语“卤素”包括氟、氯、溴和碘。
如本文所使用的，“卤代烷基”是指具有指定数量的碳原子的烷基被一个或多个、直至最大允许数量的卤素原子取代。卤代烷基的实例包括但不限于三氟甲基、二氟甲基、2-氟乙基和五氟乙基。
“杂环基”是指稳定的3至18元非芳香族环基，其包含2-12个碳原子和选自氮、氧和硫的1-6个杂原子。除非说明书中另有说明，否则杂环基是单环、双环、三环或四环系统，可包括稠环或桥环系统。杂环基中的杂原子可以任选地被氧化。一个或多个氮原子(如果存在的话)任选地被季铵化。杂环基是部分饱和或完全饱和的。杂环基可以通过任何环原子连接至分子的其余部分。此类杂环基的实例包括但不限于：二噁烷基、噻吩基[1,3]二硫酰基(thienyl[1,3]dithianyl)、十氢异喹啉基、咪唑啉基、咪唑烷基、异噻唑烷基、异噁唑烷基、吗啉基、八氢吲哚基、八氢异吲哚基、2-氧杂哌嗪基、2-氧杂哌啶基、2-氧杂吡咯烷基、噁唑烷基、哌啶基、哌嗪基、4-哌啶酮基、吡咯烷基、吡唑烷基、奎宁环基、噻唑烷基、四氢呋喃基、三硫酰基(trithianyl)、四氢吡喃基、硫代吗啉基(thiomorpholinyl)、硫杂吗啉基(thiamorpholinyl)、1-氧代硫吗啉基(1oxo thiomorpholinyl)和1,1-二氧代硫吗啉基(1,1dioxo thiomorpholinyl)。
在本公开中可以使用各种羟基保护基团。一般来说，保护基团使化学官能团对特定的反应条件不敏感，并且可以在分子中的该官能团上附加以及去除，而在实质上并不损害分子的其余部分。代表性的羟基保护基团公开于Beaucage等人，Tetrahedron 1992,48,2223-2311，以及Greeneand Wuts， Protective Groups in Organic Synthesis，Chapter 2,2d ed，John Wiley&Sons，New York，1991中，以引用的方式将上述文献整体并入本文。在一些实施方式中，保护基团在碱性条件下稳定，但可以在酸性条件下脱除。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括二甲氧基三苯甲基(DMT)、单甲氧基三苯甲基、9-苯基黄嘌呤-9-基(Pixyl)和9-(对甲氧基苯基)黄嘌呤-9-基(Mox)。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括Tr(三苯甲基)、MMTr(4-甲氧基三苯甲基)、DMTr(4,4’-二甲氧基三苯甲基)和TMTr(4,4’,4”-三甲氧基三苯甲基)和叔丁基二甲基硅烷基(TBS或TBDMS)。本文可使用的羟基保护基的非排他性实例包括烃基酰基。
“受试者”一词，如本文所使用的，指任何动物，例如哺乳动物或有袋动物。本公开的受试者包括但不限于人类、非人灵长类(例如，恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
如本文所使用的，“治疗”、“减轻”、或“改善”可在此处互换使用。这些术语指的是获得有益的或期望的结果的方法，包括但不限于治疗益处。“治疗益处”意味着根除或改善被治疗的潜在障碍。此外，治疗益处通过根除或改善与潜在障碍相关的一个或多个生理症状，从而在受试者中观察到改善而获得，尽管受试者可能仍然受到潜在障碍的折磨。
如本文所使用的，“防止”和“预防”可互换使用。这些术语指获得有益或期望的结果的方法，包括但不限于预防性益处。为了获得“预防性益处”，可将缀合物或组合物给予有罹患特定疾病风险的受试者，或给予报告疾病的一种或多种病理症状的受试者，即便可能该疾病的诊断尚未作出。
根据本发明的第一方面，提供一种化合物，该化合物具有式(I)所示的结构：
其中，
A0代表对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体或者
代表所述配体中的羟基全部或部分地被KCOO-基团取代而形成的基团，其中，每个K独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；
其中所述配体优选地各自独立地选自下述中的任意一种：D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D-吡喃甘露糖、α-D-吡喃葡萄糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳糖胺、N-乙酰基半乳糖胺、N-三氟乙酰基半乳糖胺、N-丙酰基半乳糖胺、N-正丁酰基半乳糖胺、N-异丁酰基半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6-O-三苯甲基-α-D-吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖、L-4-硫代核糖；
b为1-4范围内的整数，优选1、2或3，更优选1或3；
L1和L2彼此独立地代表选自式A1-A11基团中的一种或多种的连接组合：

其中，j1为1-20的整数；j2为1-20的整数；R’为C1-C10的烷基；
M代表如式(A12)所示的结构式：
其中，m代表0-6的整数，优选为0、1或2；*表示与G1相连的键，
Q代表式(A13)所示的结构式：
其中，与O相连的表示与G1相连的键；R2、R3相同或不同，且彼此独立地选自H、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基，优选H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；
Z不存在或代表C1-C10亚烷基，优选C1-C3亚烷基；
X代表式(A14-1)或(A14-2)或所示的结构式：
其中R4，R5相同或不同，且彼此独立地选自H、氟、羟基、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基，优选H、氟、羟基、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；p为1-6的整数，优选1、2或3；任选地，R4和R5直接相连形成三到六元饱和碳环；
E代表如式(A15)所示的结构式：
其中，代表C3-C18环烷基或C3-C18杂环基，优选代表C4-C8环烷基或C3-C8杂环基，更优选代表C3-C6杂环基，更优选四到六元含氮饱和环烷基，并且R1选自H、氟、羟基、氰基、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基，优选选自H、氟、羟基、氰基、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；
G1和G2各自代表具有式(G-1)所示结构的亚磷酰胺官能团或羟基保护基团，前提是G1和G2中至少一个具有式(G-1)所示的结构，所述羟基保护基团选自三苯甲基、4-甲氧基三苯甲基、4,4’-双甲氧基三苯甲基(DMTr)和4,4’,4’-三甲氧基苯甲基中的任意一种，优选4,4’-双甲氧基三苯甲基；
其中，表示基团共价键连接的位点；
B1选自取代或未取代的C1-C5的烃基，可选地，B1为甲基、乙基或异丙基；B2选自C1-C5的烷基、乙氰基、丙氰基和丁氰基中的一种，可选地，B2为氰乙基。
在一些实施方式中，L1或L2彼此独立地选自A1、A2、A4、A6、A7、A8、A9、A10中的一种或多种的连接组合。在一些实施方式中，L1或L2彼此独立地选自A1、A2、A4、A8、A9、A10中的至少2个的连接组合。在一些实施方式中，其中L1或L2彼此独立地选自A1、A4、A8、A10中的至少2个的连接组合。在一些实施方式中，L1或L2的长度彼此独立地为1-25个原子，所述L1或L2的长度是指最长直链上的成链原子的个数。在一些实施方式中，L1或L2的长度彼此独立地为1-20个原子。
在一些实施方式中，每个j1彼此独立地为1-10的整数，每个j2彼此独立地为1-10的整数。
在一些实施方式中，每个j1彼此独立地为1-8的整数，每个j2彼此独立地为1-8的整数。
在一些实施方式中，A0为如下所示的N-乙酰基半乳糖胺(GalNAc)基团或其羟基全部或部分地被KCOO-基团取代而形成的基团，其中，每个K独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种：
在一些实施方式中，所述化合物具有以下所示结构(I-1)：
在一些实施方式中，L为A10，其中j1为2-8的整数，或者为A11，其中j2为1-7的整数。
在一些实施方式中，m为0、1或2。
在一些实施方式中，n为0、1、2或3。
在一些实施方式中，代表四到八元全碳或含氮饱和环。在一些实施方式中，为含有一或两个氮原子的四、五或六元饱和环烷基。
在一个具体实施方案中，本公开的化合物具有以下所示结构中的任意一种：


根据本发明的第二方面，提供一种核酸缀合物，在所述缀合物中含有与寡核苷酸序列上的任意位置连接的一个或一个以上具有式(II)所示的结构，优选两个、三个或四个连续连接的具有式(II)所示的结构：
其中，Rp和Rq各自为H或具有式A16所示的结构，前提是Rp和Rq中至少一个具有式A16所示的结构；
其中，E1为OH、SH或BH2，
A0代表对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体或者
代表所述配体中的羟基全部或部分地被KCOO-基团取代而形成的基团，其中，每个K独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；
其中所述配体优选地各自独立地选自下述中的任意一种：D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D-吡喃甘露糖、α-D-吡喃葡萄糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳糖胺、N-乙酰基半乳 糖胺、N-三氟乙酰基半乳糖胺、N-丙酰基半乳糖胺、N-正丁酰基半乳糖胺、N-异丁酰基半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6-O-三苯甲基-α-D-吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖、L-4-硫代核糖；
b为1-4范围内的整数，优选1、2或3，更优选1或3；
L1和L2彼此独立地代表选自式A1-A11基团中的一种或多种的连接组合：
其中，j1为1-20的整数；
j2为1-20的整数；
R’为C1-C10的烷基；
M代表如式(A12)所示的结构式：
其中，m代表0-6的整数，优选为0、1或2；*表示与Rp相连的键，
Q代表式(A13)所示的结构式：
其中，与O相连的表示与Rp相连的键；R2、R3相同或不同，且彼此独立地选自H、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基，优选H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；
Z不存在或代表C1-C10亚烷基，优选C1-C3亚烷基；
X代表式(A14-1)或(A14-2)或所示的结构式：
其中R4，R5相同或不同，且彼此独立地选自H、氟、羟基、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基，优选H、氟、羟基、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔 基；p为1-6的整数，优选1、2或3；任选地，R4和R5直接相连形成三到六元饱和碳环；
E代表如式(A15)所示的结构式：
其中，表示基团共价键连接的位点；代表C3-C18环烷基或C3-C18杂环基，优选代表C4-C8环烷基或C3-C8杂环基，更优选代表C3-C6杂环基，更优选四到六元含氮饱和环烷基，并且R1选自H、氟、羟基、氰基、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基，优选选自H、氟、羟基、氰基、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基。
在第三方面，本发明提供了一种核酸缀合物，其中所述缀合物含有与寡核苷酸序列上的任意位置连接的一个以上具有式(II-A)所示的结构：
其中，
a代表0-7的整数，优选0-3的整数，更优选0、1、2或3的整数；
Rp和Rq各自为H或具有式A16所示的结构，并且其中至少一个具有式A16所示的结构；
W的结构如下式所示：
其中，E1为OH、SH或BH2，
A0、b、L1、L2、M、Z、X、E等同式(II)，此处不再赘述。
在一些实施方式中，L1或L2彼此独立地选自A1、A2、A4、A6、A7、A8、A9、A10中的一种或多种的连接组合。在一些实施方式中，L1或L2彼此独立地选自A1、A2、A4、A8、A9、A10中的至少2个的连接组合。在一些实施方式中，其中L1或L2彼此独立地选自A1、A4、A8、A10中的至少2个的连接组合。在一些实施方式中，L1或L2的长度彼此独立地为1-25个原子，所述L1或L2的长度是指最长直链上的成链原子的个数。在一些实施方式中，L1或L2的长度彼此独立地为1-20个原子。
在一些实施方式中，每个j1彼此独立地为1-10的整数，每个j2彼此独立地为1-10的整数。
在一些实施方式中，每个j1彼此独立地为1-8的整数，每个j2彼此独立地为1-8的整数。
在一些实施方式中，A0为如下所示的N-乙酰基半乳糖胺(GalNAc)基团或其羟基全部或部 分地被KCOO-基团取代而形成的基团，其中，每个K独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种：
在本公开的上下文中，除非另有说明，“缀合”是指两个或多个各自具有特定功能的化学部分之间以共价连接的方式彼此连接；相应地，“缀合物”是指该各个化学部分之间通过共价连接而形成的化合物。进一步地，“核酸缀合物”表示一个或多个具有特定功能的化学部分共价连接至寡核苷酸上而形成的化合物。在下文中，有时也将本公开的核酸缀合物简称为“缀合物”。
在本发明的具体实施方案中，本发明的核酸缀合物具有以下所示结构中的任意一种：




其中Y为O或者S，为寡核苷酸。
在本公开的一些实施方式中，所述寡核苷酸选自小干扰RNA、微小RNA、抗微小RNA、微小RNA拮抗剂、微小RNA模拟物、诱饵寡核苷酸、免疫刺激物、G-四极子、可变剪接体、单链RNA、反义核酸、核酸适配体、茎环RNA、mRNA片段、激活RNA中的一种；可选地，所述寡核苷酸为单链寡核苷酸或者双链寡核苷酸；可选地，所述寡核苷酸为单链寡核苷酸，式(A16)中的P原子连接到所述单链寡核苷酸的端部，所述单链寡核苷酸的端部指所述单链寡核苷酸中从一端起算的前4个核苷酸；可选地，式(A16)中的P原子连接到所述单链寡核苷酸的末端；可选地，式(A16)中的P原子连接到所述单链寡核苷酸的3'末端；
可选地，所述寡核苷酸为双链寡核苷酸，所述双链寡核苷酸包含正义链和反义链，所述式(A16)中的P原子连接到所述双链寡核苷酸的端部，所述双链寡核苷酸的端部指所述正义链或所述反义链中从一端起算的前4个核苷酸；可选地，式(A16)中的P原子连接到所述正义链或所述反义链的末端；可选地，式(A16)中的P原子连接到所述反义链的5'末端；可选地，式(A16)中的P原子通过形成磷酸二酯键连接至所述核酸缀合物中的核苷酸的2'位、3'位或5'位。
在一些实施方式中，“靶序列”是靶mRNA。在本公开的上下文中，“靶mRNA”是指在肝细胞中异常表达的基因对应的mRNA，它既可以是过量表达的基因对应的mRNA，或者是表达不足的基因对应的mRNA。由于大部分疾病源于mRNA的过量表达，因此，在本公开中，靶mRNA尤其指过量表达的基因对应的mRNA。在本公开的一些实施方式中，相应于上述异常表达的基因，所述靶mRNA可以是ApoB、ApoC、ANGPTL3、PCSK9、SCD1、TIMP-1、Col1A1、FVII、STAT3、p53、HBV、HCV等基因对应的mRNA。在一些实施方式中，所述靶mRNA可以是由对应HBV基因转录而得的mRNA、或者ANGPTL3基因所对应的mRNA、或者APOC3基因所对应的mRNA。
本领域技术人员公知，siRNA含有核苷酸基团作为基本结构单元，所述核苷酸基团含有磷酸基团、核糖基团和碱基。通常具有活性的，即功能性的siRNA的长度约为12-40个核苷酸，在一些实施方式中约为15-30个核苷酸，所述siRNA中的每个核苷酸可以独立地是修饰或未修饰的核苷酸，为了增加稳定性，所述siRNA中至少一个核苷酸是修饰的核苷酸。
本公开的发明人发现，下面的实施方式中所述的siRNA具有较高的活性和/或稳定性，因而可以作为本公开中siRNA的发明目的。
在一些实施方式中，本公开的siRNA缀合物中的siRNA(以下，也称为本公开的siRNA)中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，该siRNA含有正义链和反义链，其中，所述正义链包含核苷酸序列1，所述反义链包含核苷酸序列2，所述核苷酸序列1和所述核苷酸序列2的长度均为10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25个核苷酸，并且至少部分地反向互补形成互补双链区，所述核苷酸序列2的至少一部分与第一段核苷酸序列互补，所述第一段核苷酸序列为靶mRNA中的一段核苷酸序列。
在一些实施方式中，所述核苷酸序列1与所述第一段核苷酸序列长度相等，且不超过3个核苷酸差异；所述核苷酸序列2与核苷酸序列B长度相等，且不超过3个核苷酸差异；所述核苷酸序列B为与所述第一段核苷酸序列完全反向互补的核苷酸序列。在不愿受到限制的情况下，这些特定的核苷酸差异并不会显著降低siRNA缀合物的靶基因抑制能力，而这些包含特定核苷酸差异的siRNA缀合物也在本公开的保护范围之内。
在一些实施方式中，所述核苷酸序列1和所述核苷酸序列2基本上反向互补、基本上完全反向互补或完全反向互补。
在一些实施方式中，所述核苷酸序列1与所述第一段核苷酸序列不多于1个核苷酸差异，和/或所述核苷酸序列2与所述核苷酸序列B不多于1个核苷酸差异。在一些实施方式中，所述核苷酸序列2与所述核苷酸序列B之间的核苷酸差异包括按照5'末端到3'末端的方向，所述核苷酸序列2上的第一个核苷酸Z'位置上的差异。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列1上的最后一个核苷酸Z是与Z'互补的核苷酸。
在一些实施方式中，所述正义链还含有核苷酸序列3，所述反义链还含有核苷酸序列4，所述核苷酸序列3和所述核苷酸序列4的长度相等且均为1-4个核苷酸，所述核苷酸序列3连接在所述核苷酸序列1的5'末端，并且所述核苷酸序列4连接在所述核苷酸序列2的3'末端，所述核苷酸序列4与第二段核苷酸序列互补，该第二段核苷酸序列是指靶mRNA中与所述第一段核苷酸序列相邻、且长度与所述核苷酸序列4相同的核苷酸序列。在一些实施方式中，所述核苷酸序列3和所述核苷酸序列4基本上完全反向互补或完全反向互补。因此，所述正义链和反义链的长度可以是19-23个核苷酸。
在一些实施方式中，本公开的siRNA还含有核苷酸序列5，所述核苷酸序列5的长度为1至3个核苷酸，连接在所述反义链的3'末端，从而构成所述反义链的3'突出端；在一些实施方式中，所述核苷酸序列5的长度为1或2个核苷酸。这样，在一些实施方式中，本公开的siRNA的正义链和反义链的长度之比可以是19/20、19/21、20/21、20/22、21/22、21/23、22/23、22/24、23/24或23/25。
在一个实施方式中，所述核苷酸序列5的长度为2个核苷酸，并且按照5'末端到3'末端的方向，所述核苷酸序列5为连续的2个脱氧胸腺嘧啶核苷酸、连续的2个尿嘧啶核苷酸、或者与第三段核苷酸序列互补，所述第三段序列是指靶mRNA中与所述第一段核苷酸序列相邻、或者和所述第二段核苷酸序列相邻，并且长度与所述核苷酸序列5相等的核苷酸序列。在一个实施方式中，本公开的siRNA的正义链和反义链的长度之比为19/21或21/23，此时，本公开的siRNA具有更好的肝细胞mRNA沉默活性。
在一些实施方式中，本公开的siRNA中的核苷酸各自独立地为修饰或未修饰的核苷酸。在一些实施方式中，本公开的siRNA不含修饰的核苷酸基团；在一些实施方式中，本公开的siRNA含有修饰的核苷酸基团。
目前，本领域存在多种可用于修饰siRNA的方式，包括骨架修饰(也称为核苷酸间连接修饰，如磷酸基团修饰)、核糖基团修饰及碱基修饰等(例如，请参见Watts，J.K.，G.F.Deleavey and M.J.Damha，Chemically modified siRNA:tools and applications.Drug Discov Today，2008.13(19-20)：p.842-55，以引用的方式将其整体内容并入本文)。
在本公开的上下文中，所使用的术语“修饰的核苷酸”是指核苷酸的核糖基被修饰，比如2'位羟基被其他基团取代形成的核苷酸或核苷酸类似物，或者核苷酸上的碱基是经修饰的碱基的核苷酸。
在本公开的一些实施方式中，所述正义链或所述反义链中的至少一个核苷酸为修饰的核苷酸，和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基，换句话说，所述正义链和所述反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基和/或核糖基的至少一部分为具有修饰基团的磷酸酯基和/或具有修饰基团的核糖基(或修饰的磷酸酯基和/或修饰的核糖基)。本公开的一些实施方式中，所述正义链和/或所述反义链中的全部核苷酸均为修饰的核苷酸。
在一些实施方式中，正义链和反义链中的每一个核苷酸独立地为氟代修饰的核苷酸或非氟代修饰的核苷酸。
氟代修饰的核苷酸指核苷酸的核糖基2'位的羟基被氟取代形成的核苷酸，其具有以下式(17)所示的结构。
非氟代修饰的核苷酸指核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物。在一些实施方式中，每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种。
核糖基2'位的羟基被非氟基团取代形成的核苷酸是本领域技术人员所公知的，这些核苷酸可以选自2'-烷氧基修饰的核苷酸、2'-经取代的烷氧基修饰的核苷酸、2'-烷基修饰的核苷酸、2'-经取代的烷基修饰的核苷酸、2'-氨基修饰的核苷酸、2'-经取代的氨基修饰的核苷酸、2'-脱氧核苷酸中的一种。
在一些实施方式中，2'-烷氧基修饰的核苷酸为甲氧基修饰的核苷酸(2'-OMe)，如式(18)所示。2'-经取代的烷氧基修饰的核苷酸，例如可以是2'-O-甲氧基乙基修饰的核苷酸(2'-MOE)，如式(19)所示。在一些实施方式中，2'-氨基修饰的核苷酸(2'-NH2)如式(20)所示。在一些 实施方式中，2'-脱氧核苷酸(DNA)如式(21)所示。
核苷酸类似物指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶的基团。在一些实施方式中，所述核苷酸类似物可以为如异核苷酸、桥联核酸(bridged nucleic acid，简称BNA)核苷酸或无环核苷酸。
BNA核苷酸是指受约束的或不能接近的核苷酸。BNA可以含有五元环、六元环、或七元环的具有“固定的”C3'-内切糖缩拢的桥联结构。通常将该桥掺入到该核糖环的2'-、4'-位处以提供一个2',4'-BNA核苷酸，如LNA、ENA、cET BNA等，其中，LNA如式(22)所示，ENA如式(23)所示，cET BNA如式(24)所示。
无环核苷酸是核苷酸的糖环被打开形成的一类核苷酸，如解锁核酸(UNA)核苷酸或甘油核酸(GNA)核苷酸，其中，UNA如式(25)所示，GNA如式(26)所示。
其中，R选自H、OH或烷氧基(O-烷基)。
异核苷酸是指核苷酸中碱基在核糖环上的位置发生改变而形成的化合物，例如，碱基从核糖环的1'-位移动至2'-位或3'-位而形成的化合物，如式(27)或(28)所示。
其中，Base表示碱基，例如A、U、G、C或T；R选自H、OH、F或者如上所述的非氟基团。
在一些实施方式中，核苷酸类似物选自异核苷酸、LNA、ENA、cET、UNA和GNA中的一种。在一些实施方式中，每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸，所述甲氧基修饰的核苷酸指核糖基的2'-羟基被甲氧基取代而形成的核苷酸。
在上文及下文中，“氟代修饰的核苷酸”、“2'-氟修饰的核苷酸”、“核糖基团的2'-羟基被氟取代的核苷酸”和“2'-氟代核糖基”意义相同，均指核苷酸的2'-羟基被氟取代而形成的具有如式(17)所示结构的化合物；“甲氧基修饰的核苷酸”、“2'-甲氧基修饰的核苷酸”、“核糖基团的2'-羟基被甲氧基取代的核苷酸”和“2'-甲氧基核糖基”意义相同，均指核苷酸核糖基团的2'-羟基被甲氧基取代，而形成如式(18)所示的结构。
在一些实施方式中，本公开的siRNA是具有以下修饰的siRNA：正义链和反义链均包含氟代修饰的核苷酸和非氟代修饰的核苷酸，所述氟代修饰的核苷酸位于前述的核苷酸序列1和核苷酸序列2中，所述核苷酸序列1中氟代修饰的核苷酸不多于5个，并且，按照5'末端到3'末端的方向，所述核苷酸序列1的第7、8、9位的核苷酸为氟代修饰的核苷酸；所述核苷酸序列2中氟代修饰的核苷酸不多于7个，并且，按照5'末端到3'末端的方向，所述核苷酸序列2的第2、 6、14、16位的核苷酸为氟代修饰的核苷酸。在一些实施方式中，本公开的siRNA是具有以下修饰的siRNA：按照5'末端到3'末端的方向，所述siRNA的正义链中所述核苷酸序列1的第7、8、9位的核苷酸为-氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为甲氧基修饰的核苷酸；在所述反义链中，所述核苷酸序列2的第2、6、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为甲氧基修饰的核苷酸；在一些实施方式中，本公开的siRNA是具有以下修饰的siRNA：或者按照5'末端到3'末端的方向，所述siRNA的正义链中所述核苷酸序列1的第5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为甲氧基修饰的核苷酸；在所述反义链中，所述核苷酸序列2的第2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为甲氧基修饰的核苷酸；在一些实施方式中，本公开的siRNA是具有以下修饰的siRNA：按照5'末端到3'末端的方向，所述siRNA的正义链中所述核苷酸序列1的第7、8和9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5'末端到3'末端的方向，所述siRNA的反义链中所述核苷酸序列2的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为甲氧基修饰的核苷酸。
在本公开所述siRNA的一些具体实施方式中，所述核苷酸含有磷酸基团修饰。在本公开的上下文中，磷酸基团修饰在一个实施方式中为如下式(11)所示的硫代磷酸(phosphorthioate)修饰，即，用一个硫原子取代磷酸二酯键中的非桥氧原子，从而以硫代磷酸二酯键替换磷酸二酯键。在一些实施方式中，该修饰能稳定siRNA的结构，保持碱基配对的高特异性和高亲和力。
根据本公开的一些实施方式，所述siRNA中，硫代磷酸酯基连接存在于由以下位置的组成的组中的至少一处：正义链或反义链任意一端的第一个和第二个核苷酸之间；正义链或反义链任意一端的第二个和第三个核苷酸之间；或上述的任意组合。在一些实施方式中，硫代磷酸酯基连接存在于除正义链5'末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯基连接存在于除正义链3'末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯基连接存在于以下位置中的至少一处：
所述正义链的5'末端端部第1个核苷酸和第2个核苷酸之间的连接；
所述正义链的5'末端端部第2个核苷酸和第3个核苷酸之间的连接；
所述正义链的3'末端端部第1个核苷酸和第2个核苷酸之间的连接；
所述正义链的3'末端端部第2个核苷酸和第3个核苷酸之间的连接；
所述反义链的5'末端端部第1个核苷酸和第2个核苷酸之间的连接；
所述反义链的5'末端端部第2个核苷酸和第3个核苷酸之间的连接；
所述反义链的3'末端端部第1个核苷酸和第2个核苷酸之间的连接；以及
所述反义链的3'末端端部第2个核苷酸和第3个核苷酸之间的连接。
按照本公开的一些实施方式，所述siRNA分子的反义链序列5'末端核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸。
在一些实施方式中，5'-磷酸核苷酸可具有式(12)所示结构：
同时，常用的所述5'-磷酸类似物修饰的核苷酸的种类是本领域技术人员公知的，例如，Anastasia Khvorova and Jonathan K.Watts,The chemical evolution of oligonucleotide therapies of clinical utility.Nature Biotechnology,2017,35(3):238-48中公开的如下如式(13)-(16)所示的4种核苷酸：
其中，R表示选自于由H、OH、F和甲氧基所组成的组的基团；
Base表示选自A、U、C、G或T的碱基。
在一些实施方式中，5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸为式(13)所示的含有乙烯基磷酸酯(E-vinylphosphonate，E-VP)的核苷酸、式(12)所示的含有5'-磷酸修饰的核苷酸或式(15)所示的含有5'-硫代磷酸修饰的核苷酸。
在本公开的一些实施方式中，所述双链寡核苷酸为siRNA。
根据本发明的第三方面，提供一种本发明公开的核酸缀合物的制备方法，包括本发明所述的式(I)化合物中的亚磷酰胺官能团氧化为式(W)所示的结构，然后与寡核苷酸结合，再进行切割和脱去保护基团，得到所述缀合物。
其中，表示基团共价键连接的位点；
E1为OH、SH或BH2。
可以采用任意合理的合成路线来制备本公开的核酸缀合物。
例如，本公开的核酸缀合物的制备方法可以包括：在亚磷酰胺固相合成的条件下，分别按照所述功能性寡核苷酸的核苷酸种类和顺序，按照3'到5'的方向将核苷单体依次连接，每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应。
在本公开的一些实施方式中，所述偶联反应在活化剂的存在下进行，所述活化剂例如选自1H-四氮唑、5-乙硫基1H-四氮唑、5-苄硫基1H-四氮唑中的一种或多种，在一些实施方式中为5-乙硫基1H-四氮唑。
在本公开的一些实施方式中，所述氧化反应条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为1-100秒，在一些实施方式中为5-50秒，氧化试剂在一些实施方式中为碘(在进一步实施方式中，以碘水的形式提供)。
根据本发明的第四方面，提供一种本发明公开的核酸缀合物在制备用于治疗和/或预防肝源性疾病的药物中的用途。
根据本发明的第五方面，提供一种治疗由肝细胞中基因的表达而引起的病理状况或疾病的方法，所述方法包括向患有该疾病的患者给予本发明公开的核酸缀合物。
根据本发明的第六方面，提供一种试剂盒，该试剂盒包本发明公开的的核酸缀合物。
以下实施例仅示例性地说明本发明，并非限制性。下述实施例中所用的原料、试剂等，如无特殊说明，均为市售产品。
实施例1化合物I-1-1的制备
1.1中间体1-1的制备
将化合物1-Cbz-4-羟甲基哌啶(市售，购买于上海泰坦科技股份有限公司)(40.1mmol,10.0g)置于洁净干燥反应瓶中，加入100mL吡啶，室温下加入4,4'-双甲氧基三苯甲基氯(1.2equiv,48.1mmol,16.3g)，随后室温继续搅拌1小时。反应后向反应液中加入150mL乙酸乙酯，并用150mL饱和碳酸氢钠溶液和150mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(石油醚/乙酸乙酯＝20/1-4/1),得到淡黄色油状物化合物1-1(21.7g,39.3mmol,98％收率)。化合物1-1分子式:C35H37O5N,分子量:551.3,LC-MS实测：552.4(M+H).1H NMR(400MHz,DMSO-d6):δ7.31– 7.22(m,9H),7.17(d,J＝8.6Hz,5H),6.82(d,J＝8.7Hz,4H),4.99(s,2H),3.93(d,J＝12.6Hz,2H),3.66(s,6H),2.78–2.61(m,4H),2.44(s,1H),1.62(d,J＝12.6Hz,2H),0.97(qd,J＝12.7,3.6Hz,2H).
1.2中间体1-2的制备
将化合物1-1(39.3mmol,21.7g)置于洁净干燥反应瓶中，加入150mL甲醇，室温氢气下加入钯碳(湿基,10％ Pd/C)(10％wt,2.2g)，随后室温继续搅拌12小时。反应后过滤除去钯碳，滤液浓缩，得到粗产品白色固体化合物1-2(16.1g,38.5mmol,98％收率)，未纯化直接投入下一步反应。化合物1-2分子式:C27H31O3N,分子量:417.2,LC-MS实测：418.4(M+H).
1.3中间体1-3的制备
将化合物N-苄氧羰基-L-丝氨酸(市售，购买于上海泰坦科技股份有限公司)(35.0mmol,8.4g)置于洁净干燥反应瓶中，加入100mL二氯甲烷，室温下加入苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(1.5equiv,52.5mmol,19.9g)、化合物1-2(1.1equiv,38.5mmol,16.1g)和N,N-二异丙基乙胺(3.0equiv,105.0mmol,13.5g),随后室温搅拌1小时。反应后向反应液中加入150mL二氯甲烷，并用150mL饱和碳酸氢钠溶液和150mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(石油醚/乙酸乙酯＝10/1-2/3),得到白色固体化合物1-3(14.9g,23.5mmol,67％收率)。化合物1-3分子式:C38H42O7N2,分子量:638.3,LC-MS实测：639.4(M+H).1H NMR(400MHz,DMSO-d6):δ7.42–7.26(m,9H),7.23(d,J＝8.7Hz,5H),6.89(d,J＝8.8Hz,4H),5.03–4.96(m,2H),4.79(dt,J＝25.7,5.4Hz,1H),4.55–4.48(m,1H),4.36(d,J＝10.7Hz,1H),4.01(dt,J＝24.8,8.8Hz,1H),3.73(s,6H),3.56(ddd,J＝18.7,12.7,5.8Hz,1H),3.43–3.38(m,1H),3.00(t,J＝12.3Hz,1H),2.85–2.79(m,2H),2.69(s,1H),2.56(d,J＝10.2Hz,1H),1.88(d,J＝21.0Hz,1H),1.69(dd,J＝36.5,11.3Hz,2H),1.19–0.98(m,2H).
1.4中间体1-4的制备
将化合物1-3(23.5mmol,14.9g)置于洁净干燥反应瓶中，加入100mL甲醇，室温氢气下加入钯碳(湿基,10％ Pd/C)(10％wt,1.5g)，随后室温继续搅拌12小时。反应后过滤除去钯碳，滤液浓缩，得到粗产品白色固体化合物1-4(11.6g,23.0mmol,98％收率)，未纯化直接投入下一步反应。化合物1-4分子式:C30H36O5N2,分子量:504.2,LC-MS实测：527.6(M+Na).1H NMR(400MHz,DMSO-d6):δ7.36(d,J＝7.5Hz,2H),7.31(t,J＝7.6Hz,2H),7.23(d,J＝8.8Hz,5H),6.89(d,J＝8.8Hz,4H),4.39(t,J＝12.2Hz,1H),3.97(d,J＝13.0Hz,1H),3.73(s,6H),3.69(t,J＝6.3Hz,1H),3.43–3.36(m,5H),3.01–2.92(m,2H),2.83(d,J＝6.1Hz,2H),1.84(dd,J＝11.6,8.1Hz,1H),1.72(d,J＝12.0Hz,2H),1.17–0.91(m,2H).
1.5中间体1-5的制备
将化合物5-(((2R,3R,4R,5R,6R)-3-乙酰氨基-4,5-二乙酰氧基-6-(乙酰氧基甲基)四氢-2H-吡喃-2-基)氧基)戊酸(市售，购买于诺甘林生物医药科技(苏州)有限公司)(9.3g,20.9mmol)置于洁净干燥 反应瓶中，加入100mL二氯甲烷，室温下加入苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(1.5equiv,31.3mmol,11.9g)，搅拌十分钟，随后向反应体系中加入化合物1-4(1.1equiv,23.0mmol,11.6g)和N,N-二异丙基乙胺(3.0equiv,62.7mmol,8.1g),随后室温继续搅拌1小时。反应后向反应液中加入150mL二氯甲烷，并用150mL饱和碳酸氢钠溶液和150mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(二氯甲烷/甲醇＝50/1-20/1),得到白色固体化合物1-5(15.4g,16.5mmol,79％收率)。化合物1-5分子式:C49H63O13N5,分子量:933.4,LC-MS实测：932.5(M-H).1HNMR(400MHz,DMSO-d6):δ7.93(dd,J＝24.8,8.3Hz,1H),7.80(dd,J＝8.8,5.3Hz,1H),7.36(d,J＝7.8Hz,2H),7.31(t,J＝7.6Hz,2H),7.23(d,J＝8.7Hz,5H),6.89(d,J＝8.8Hz,4H),5.21(d,J＝3.3Hz,1H),4.97(dd,J＝11.2,3.4Hz,1H),4.79(d,J＝4.7Hz,1H),4.47(dd,J＝8.0,3.6Hz,1H),4.37(d,J＝12.7Hz,1H),4.05–4.00(m,5H),3.87(dd,J＝20.2,10.3Hz,1H),3.73(s,6H),3.57–3.50(m,1H),3.14(qd,J＝7.3,4.3Hz,1H),2.99(t,J＝14.2Hz,1H),2.82(d,J＝3.8Hz,2H),2.10(s,5H),1.99–1.98(m,6H),1.89(s,3H),1.76(d,J＝9.2Hz,4H),1.25(dd,J＝12.6,6.5Hz,5H),1.17(t,J＝7.1Hz,3H).
1.6化合物I-1-1的制备
将化合物1-5(16.5mmol,15.4g)置于洁净干燥反应瓶中，加入50mL无水二氯甲烷。室温氩气保护下加入化合物2-氰乙基N,N,N’,N’-四异丙基亚磷酰二胺(2.0equiv,33.0mmol,9.9g)和4,5-二氰咪唑(1.5equiv,24.7mmol,2.9g)，室温下继续搅拌一小时。反应后向反应液中加入50mL二氯甲烷，并用100mL饱和碳酸氢钠溶液洗涤，干燥有机相，过滤并浓缩，所得粗产品经C18反相柱(规格：30μm；市售，购买于上海博蕴生物科技有限公司)(MeCN:H2O＝75％:25％)制备得到白色固体I-1-1(11.6g,10.2mmol,62％收率)。化合物I-1-1分子式:C58H80O15N6P,分子量:1133.5,LC-MS实测：1132.4(M-H).1H NMR(400MHz,DMSO-d6):δ8.22–8.04(m,1H),7.79(t,J＝8.2Hz,1H),7.35(d,J＝2.3Hz,2H),7.30(t,J＝7.6Hz,2H),7.22(d,J＝8.5Hz,5H),6.88(d,J＝8.7Hz,4H),5.21(d,J＝3.3Hz,1H),4.93(ddd,J＝18.3,11.7,4.3Hz,2H),4.49–4.45(m,1H),4.40–4.30(m,1H),3.99(p,J＝8.9Hz,4H),3.85(dt,J＝13.0,6.6Hz,2H),3.73(s,6H),3.71(s,2H),3.68–3.62(m,2H),3.49(ddd,J＝15.7,13.6,6.6Hz,2H),3.42–3.36(m,1H),3.00(t,J＝14.4Hz,1H),2.82–2.79(m,2H),2.77–2.72(m,1H),2.69–2.64(m,1H),2.60–2.54(m,1H),2.10(s,5H),1.98(dd,J＝8.7,3.5Hz,3H),1.89(s,4H),1.77–1.63(m,5H),1.50–1.37(m,4H),1.19–1.10(m,12H).31P NMR(162MHz,DMSO-d6):δ147.90,147.30.
实施例2化合物I-1-7的制备
2.1中间体2-1的制备
将化合物(R)-(+)-N-苄基-3-羟基吡咯烷(市售，购买于上海泰坦科技股份有限公司)(16.9mmol,3.0g)和咪唑(3.0equiv,50.7mmol,3.45g)置于洁净干燥反应瓶中，加入50mL乙腈，室温下缓慢加入叔丁基二甲基氯硅烷(1.3equiv,21.9mmol,3.31g)，随后室温继续搅拌12小时。反应后向反应液中加入100mL乙酸乙酯，并用100mL饱和碳酸氢钠溶液和100mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(石油醚/乙酸乙酯＝20/1-5/1),得到无色油状物化合物2-1(4.9g,16.8mmol,99％收率)。化合物2-1分子式:C17H29ONSi,分子量:291.2,LC-MS实测：292.4(M+H).
2.2中间体2-2的制备
将化合物2-1(16.8mmol,4.9g)置于洁净干燥反应瓶中，加入100mL甲醇，室温氢气下加入钯碳(湿基,10％ Pd/C)(10％wt,490.0mg)，随后室温继续搅拌12小时。反应后过滤除去钯碳，滤液浓缩，得到粗产品白色固体化合物2-2(3.31g,16.5mmol,98％收率)，未纯化直接投入下一步反应。化合物2-2分子式:C10H23ONSi,分子量:201.1,LC-MS实测：202.3(M+H).
2.3中间体2-3的制备
将化合物N-苄氧羰基-L-丝氨酸(市售，购买于上海泰坦科技股份有限公司)(15.0mmol,3.58g)置于洁净干燥反应瓶中，加入100mL二氯甲烷，室温下加入苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(1.5equiv,22.5mmol,8.53g)、化合物2-2(1.1equiv,16.5mmol,3.31g)和N,N-二异丙基乙胺(3.0equiv,45.0mmol,5.78g),随后室温搅拌1小时。反应后向反应液中加入150mL二氯甲烷，并用150mL饱和碳酸氢钠溶液和150mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(石油醚/乙酸乙酯＝10/1-1/3),得到白色固体化合物2-3(4.5g,10.65mmol,63％两步收率)。化合物2-3分子式:C21H34O5N2Si,分子量:422.2,LC-MS实测：423.3(M+H).1H NMR(400MHz,CDCl3):δ7.35–7.29(m,5H),5.96(dd,J＝14.3,8.3Hz,1H),5.10(s,2H),4.61–4.40(m,2H),3.85–3.68(m,2H),3.65–3.49(m,2H),3.41(d,J＝12.7Hz,1H),3.31(s,1H),1.95(qdd,J＝15.0,11.8,5.3Hz,2H),1.77(s,1H),0.86(s,9H),0.06(d,J＝3.1Hz,6H).
2.4中间体2-4的制备
将化合物2-3(10.65mmol,4.5g)置于洁净干燥反应瓶中，加入100mL吡啶，室温下加入4,4'-双甲氧基三苯甲基氯(1.2equiv,12.78mmol,4.32g)，随后室温继续搅拌12小时。反应后向反应液中加入150mL乙酸乙酯，并用150mL饱和碳酸氢钠溶液和150mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(石油醚/乙酸乙酯＝20/1-1/1),得到淡黄色油状物化合物2-4(7.56g,10.43mmol,98％收率)。化合物2-4分子式:C42H52O7N2Si,分子量:724.3,LC-MS实测：747.4(M+Na).1H NMR(400MHz,CDCl3):δ7.35–7.31(m,1H),7.28(d,J＝4.7Hz,4H),7.27–7.23(m,2H),7.23–7.14(m,5H),7.13–7.11(m,2H),6.80–6.78(m,1H),6.76(dd,J＝7.7,5.4Hz,4H),5.72(dd,J＝22.7,8.3Hz,1H),5.08–4.99(m,2H),4.69–4.59(m,1H),4.35–4.30(m,1H),3.73(dd,J＝4.5,3.7Hz,6H),3.65–3.44(m,2H),3.36–3.20(m,3H),1.86–1.81(m,1H),1.70(s,1H),0.80(d,J＝13.1Hz,9H),-0.02(dd,J＝14.9,4.2Hz,6H).
2.5中间体2-5的制备
将化合物2-4(10.43mmol,7.56g)置于洁净干燥反应瓶中，加入100mL甲醇，室温氢气下加入钯碳(湿基,10％ Pd/C)(10％wt,750.0mg)，随后室温继续搅拌12小时。反应后过滤除去钯碳，滤液浓缩，得到粗产品白色固体化合物2-5(6.0g,10.22mmol,98％收率)，未纯化直接投入下一步反应。化合物2-5分子式:C34H46O5N2Si,分子量:590.3,LC-MS实测：591.6(M+H).
2.6中间体2-6的制备
将化合物5-(((2R,3R,4R,5R,6R)-3-乙酰氨基-4,5-二乙酰氧基-6-(乙酰氧基甲基)四氢-2H-吡喃-2-基)氧基)戊酸(市售，购买于诺甘林生物医药科技(苏州)有限公司)(2.06g,4.62mmol)置于洁净干燥反应瓶中，加入100mL二氯甲烷，室温下加入苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(1.5equiv,6.93mmol,2.63g)，搅拌十分钟，随后向反应体系中加入化合物2-5(1.1equiv,5.08mmol,3.0g)和N,N-二异丙基乙胺(3.0equiv,13.86mmol,17.91g),随后室温继续搅拌1小时。反应后向反应液中加入150mL二氯甲烷，并用150mL饱和碳酸氢钠溶液和150mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(二氯甲烷/甲醇＝50/1-10/1),得到白色固体化合物2-6(3.58g,3.51mmol,76％收率)。化合物2-6分子式:C53H73O15N3Si,分子量:1019.3,LC-MS实测：1018.3(M-H).
2.7中间体2-7的制备
将化合物2-6(3.51mmol,3.58g)置于洁净干燥反应瓶中，加入50mL吡啶，室温下加入三乙胺氟化氢(5.0equiv,17.55mmol,2.97g)，随后室温继续搅拌12小时。反应后向反应液中加入100mL乙酸乙酯，并用100mL饱和碳酸氢钠溶液和100mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(二氯甲烷/甲醇＝50/1-10/1),得到淡黄色油状物化合物2-7(2.4g,2.65mmol,76％收率)。化合物2-7分子式:C47H59O15N3,分子量:905.3,LC-MS实测：904.4(M-H).1HNMR(400MHz,CDCl3):δ7.30(dd,J＝4.2,1.9Hz,1H),7.29–7.25(m,5H),7.19–7.15(m,4H),6.85–6.81(m,4H),5.40–5.34(m,1H),5.30(s,1H),5.26–5.19(m,1H),5.13(ddd,J＝14.5,10.9,3.3Hz,1H),4.85–4.78(m,1H),4.75–4.71(m,1H),4.68–4.61(m,1H),4.50(d,J＝11.5Hz,1H),4.18–4.11(m,2H),4.06–3.97(m,1H),3.90(dt,J＝10.6,6.8Hz,2H),3.80(s,6H),3.67–3.46(m,4H),3.25–3.19(m,2H),2.34–2.23(m,1H),2.17–2.15(m,3H),2.10–2.03(m,5H),1.98(dt,J＝12.9,2.4Hz,4H),1.76–1.61(m,4H),1.37(t,J＝7.3Hz,3H).
2.8化合物I-1-7的制备
将化合物2-7(2.65mmol,2.4g)置于洁净干燥反应瓶中，加入50mL无水二氯甲烷(市售，购买于上海泰坦科技股份有限公司)。室温氩气保护下加入化合物2-氰乙基N,N,N’,N’-四异丙基亚磷酰二胺(2.0equiv,5.3mmol,1.6g)和4,5-二氰咪唑(1.5equiv,3.97mmol,470.0mg)，室温下继续搅拌一小时。反应后向反应液中加入50mL二氯甲烷，并用100mL饱和碳酸氢钠溶液洗涤，干燥有机相，过滤并浓缩，所得粗产品经C18反相柱(规格：30μm；市售，购买于上海博蕴生物科技有限公司)(MeCN:H2O＝75％:25％)制备得到白色固体I-1-7(2.03g,1.84mmol,70％收率)。化合物I-1-7分子式:C56H76O15N6P,分子量:1105.5,LC-MS实测：1128.4(M+Na).1H NMR(400MHz,DMSO-d6):δ8.12–8.06(m,1H),7.78(dd,J＝9.1,2.0Hz,1H),7.35–7.28(m,4H),7.23–7.17(m,5H),6.88(d,J＝7.5Hz,4H),5.21(d,J＝3.3Hz,1H),4.97(dd,J＝11.2,3.0Hz,1H),4.86–4.75(m,1H),4.52(s,1H),4.47(d,J＝8.5Hz,1H),4.04–3.98(m,3H),3.91–3.83(m,1H),3.74(s,6H),3.70–3.60(m,3H),3.58–3.47(m,3H),3.39(t,J＝11.7Hz,2H),3.31(d,J＝4.1Hz,1H),3.19(qd,J＝8.7,3.8Hz,1H),3.02(dt,J＝19.9,6.8Hz,1H),2.78–2.70(m,1H),2.60(td,J＝5.8,1.8Hz,1H),2.09(s,5H),1.98(s,4H),1.89(s,3H),1.73(s, 3H),1.44(s,4H),1.20–0.88(m,14H).31P NMR(162MHz,DMSO-d6):δ148.22(s),146.82(t,J＝35.8Hz).
实施例3化合物I-1-5的制备
根据实施例2的合成方法，由起始原料N-苄基-4-羟基哌啶(市售，购买于上海泰坦科技股份有限公司)制备得到白色固体I-1-5(3.0g,2.68mmol,72％收率)。化合物I-1-5分子式:C57H78O15N6P,分子量:1119.5,LC-MS实测：1142.4(M+Na).1H NMR(400MHz,DMSO-d6):δ8.14–8.08(m,1H),7.80(d,J＝9.2Hz,1H),7.35(d,J＝7.6Hz,2H),7.29(t,J＝7.6Hz,2H),7.22(d,J＝7.5Hz,5H),6.87(d,J＝7.8Hz,4H),5.22(d,J＝3.3Hz,1H),5.02–4.96(m,2H),4.49(d,J＝8.4Hz,1H),4.02(tt,J＝7.9,3.8Hz,4H),3.88(dd,J＝19.8,8.9Hz,1H),3.74(s,6H),3.72–3.66(m,3H),3.63–3.54(m,3H),3.38(dd,J＝11.9,7.7Hz,3H),3.32–3.31(m,1H),3.19(t,J＝7.2Hz,1H),3.06(s,1H),2.77–2.74(m,2H),2.14–2.07(m,5H),1.98(s,3H),1.90(s,3H),1.75(s,4H),1.46(s,6H),1.27–1.05(m,13H).31P NMR(162MHz,DMSO-d6):δ148.24(s),145.47(dd,J＝15.7,11.5Hz).
实施例4化合物I-1-2的制备
根据实施例1的合成方法，由起始原料L-苏氨酸(市售，购买于上海泰坦科技股份有限公司)制备得到白色固体I-1-2(1.2g,1.05mmol,56％收率)。化合物I-1-2分子式:C59H82O15N6P,分子量:1147.5,LC-MS实测：1170.3(M+Na).1H NMR(400MHz,DMSO-d6):δ7.79(dt,J＝12.8,4.5Hz,1H),7.37–7.29(m,4H),7.22(d,J＝7.6Hz,5H),6.88(d,J＝7.7Hz,4H),5.21(d,J＝3.3Hz,1H),4.97(dd,J＝11.0,3.3Hz,1H),4.88(dt,J＝11.6,4.7Hz,1H),4.47(dd,J＝10.6,5.5Hz,1H),4.41–4.32(m,1H),4.16–4.08(m,1H),4.04–3.99(m,3H),3.92–3.82(m,1H),3.73(s,6H),3.70–3.63(m,2H),3.30–3.24(m,5H),2.90(dd,J＝10.7,5.2Hz,1H),2.82(dt,J＝9.3,5.6Hz,2H),2.76–2.66(m,1H),2.34–2.32(m,1H),2.20–2.14(m,2H),2.10(s,3H),2.01–1.96(m,4H),1.89–1.81(m,4H),1.77–1.65(m,5H),1.53–1.38(m,5H),1.23–0.94(m,17H).31P NMR(162MHz,DMSO-d6):δ148.02.
实施例5化合物I-1-9的制备
根据实施例2的合成方法，由起始原料1-苄氧羰基-3-羟基氮杂环丁烷(市售，购买于上海泰坦科技股份有限公司)制备得到白色固体I-1-9(3.02g,2.77mmol,83％收率)。化合物I-1-9分子式:C55H74O15N6P,分子量:1091.4,LC-MS实测：1114.3(M+Na).1H NMR(400MHz,DMSO-d6):δ8.09–8.02(m,1H),7.78(d,J＝9.1Hz,1H),7.37–7.29(m,4H),7.24–7.19(m,5H),6.89(d,J＝8.9Hz,4H),5.21(d,J＝3.4Hz,1H),4.97(dd,J＝11.2,3.1Hz,1H),4.77–4.61(m,1H),4.57–4.50(m,1H),4.47(dd,J＝8.5,2.6Hz,1H),4.24–4.06(m,2H),4.04–3.98(m,3H),3.92–3.81(m,1H),3.78(dd,J＝6.7,3.8Hz,1H),3.69(ddd,J＝9.9,5.5,2.7Hz,2H),3.61–3.51(m,2H),3.40–3.35(m,1H),3.16(q,J＝7.7Hz,1H),3.04–2.98(m,1H),2.80–2.74(m,2H),2.21–2.06(m,5H),1.98(s,3H),1.89(s,3H),1.74(d,J＝2.5Hz,3H),1.51–1.37(m,4H),1.18–1.10(m,12H),1.06(d,J＝6.7Hz,2H).31P NMR(162MHz,DMSO-d6):δ146.95(dd,J＝65.4,20.5Hz).
实施例6化合物I-1-8的制备
6.1中间体6-1的制备
将化合物丝氨醇(市售，购买于上海泰坦科技股份有限公司阿达玛斯)(54.9mmol,5.0g)置于洁净反应瓶中，加入90mL水溶解，室温下加入碳酸钠(3.4equiv,183.8mmol,19.5g)，冰水浴下，缓慢分批加入三光气(0.33equiv,18.3mmol,5.4g)，随后室温继续搅拌24小时。减压浓缩除去溶剂，加乙醇搅拌2h，抽滤并浓缩，所得粗产品硅胶柱层析分离纯化(二氯甲烷/甲醇＝100/1-9/1),得到白色固体化合物6-1(4.6g,39.3mmol,72％收率)。化合物6-1分子式:C4H7NO3,分子量:117.1,LC-MS实测：118.4(M+H).1H NMR(400MHz,DMSO-d6):δ7.58(s,1H),4.31(t,J＝8.6Hz,1H),4.05(dd,J＝8.5,5.0Hz,1H),3.78–3.72(m,1H),3.36(d,J＝5.0Hz,2H).
6.2中间体6-2的制备
将化合物6-1(47.0mmol,5.5g)置于洁净干燥反应瓶中，加入300mL二氯甲烷，加入DMAP(2.0equiv,93.9mmol,11.5g)，冰水浴下，缓慢分批加入TsCl(1.1equiv,51.7mmol,9.8g)，随后室温继续搅拌2小时。反应后反应液依次用100mL 1M HCl、100mL水和100mL饱和盐水洗涤，干燥，旋干得白色粉末6-2(12.3g,45.3mmol,97％收率)，粗产品未经纯化直接投入下一步反应。化合物6-2分子式:C11H13NO5S,分子量:271.1,LC-MS实测：272.4(M+H).
6.3中间体6-3的制备
将化合物6-2(45.5mmol,12.3g)置于洁净干燥反应瓶中，加入50mL四氢呋喃溶解，加入4-哌啶甲醇(3.0equiv,136.6mmol,15.7g)，加热到回流反应16h。反应液浓缩，所得粗产品硅胶柱层析分离纯化(二氯甲烷/甲醇＝100/1-9/1),得到无色糖浆状化合物6-3(7.9g,36.8mmol,81％收率)。化合物6-3分子式:C10H18N2O3,分子量:214.1,LC-MS实测：215.3(M+H).1H NMR(400MHz,DMSO-d6):δ7.61(s,1H),4.38(d,J＝5.3Hz,1H),4.33(t,J＝8.2Hz,1H),3.97(dd,J＝8.3,5.5Hz,1H),3.94–3.87(m,1H),3.22(t,J＝5.7Hz,2H),2.82(t,J＝10.5Hz,2H),2.37–2.28(m,2H),1.97–1.86(m,2H),1.59(d,J＝12.4Hz,2H),1.34–1.23(m,1H),1.10(pd,J＝12.3,3.9Hz,2H).
6.4中间体6-4的制备
将化合物6-3(23.3mmol,5.0g)置于洁净干燥反应瓶中，加入50mL吡啶，室温下加入4,4'-双甲氧基三苯甲基氯(1.2equiv,28.0mmol,9.5g)，随后室温继续搅拌1小时。反应后向反应液中加入150mL乙酸乙酯，分别用150mL饱和碳酸氢钠溶液和150mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(二氯甲烷/甲醇＝100/0-100/3),得到类白色泡沫状固体化合物6-4(8.2g,15.9mmol,68％收率)。化合物6-4分子式:C31H36N2O5,分子量:516.2,LC-MS实测：515.3(M-H).1H NMR(400MHz,DMSO-d6):δ7.61(s,1H),7.38–7.35(m,2H),7.30(t,J＝7.7Hz,2H),7.24–7.21(m,5H),6.88(d,J＝8.9Hz,4H),4.32(t,J＝8.2Hz,1H),3.96(dd,J＝8.2,5.4Hz,1H),3.89(dt,J＝13.2,6.8Hz,1H),3.73(s,6H),2.80(t,J＝9.2Hz,4H),2.37–2.27(m,2H),1.94(dd,J＝26.3,11.7Hz,2H),1.63(d,J＝12.3Hz,2H),1.56–1.50(m,1H),1.23–1.08(m,2H).
6.5中间体6-5的制备
将化合物6-4(15.9mmol,8.2g)置于洁净干燥反应瓶中，加入82mL乙醇溶解，室温下加入氢氧化钾(3.0equiv,47.6mmol,2.7g)的41mL水溶液，升温到85℃反应过夜。浓缩除去溶剂，向反应液中加入100mL乙酸乙酯，并用60mL饱和碳酸氢钠溶液洗涤，水相加50mL乙酸乙酯反萃，合并有机相，干燥，过滤并浓缩，得白色泡沫状固体6-5(7.6g,15.5mmol,98％收率),粗产品未经纯化直接投入下一步反应。化合物6-5分子式:C30H38N2O4,分子量:490.2,LC-MS实测：491.3(M+H).
6.6中间体6-6的制备
将化合物5-(((2R,3R,4R,5R,6R)-3-乙酰氨基-4,5-二乙酰氧基-6-(乙酰氧基甲基)四氢-2H-吡喃-2-基)氧基)戊酸(市售，购买于诺甘林生物医药科技(苏州)有限公司)(6.9g,15.5mmol)置于洁净干燥反应瓶中，加入80mL二氯甲烷，室温下加入苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(1.0equiv,15.5mmol,5.9g)，搅拌十分钟，随后向反应体系中加入化合物6-5(1.0equiv,15.5mmol,7.6g)和N,N-二异丙基乙胺(2.0equiv,31.0mmol,4.0g),随后室温继续搅拌1小时。反应后向反应液中加入150mL二氯甲烷，并用150mL饱和碳酸氢钠溶液和150mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(二氯甲烷/甲醇＝100/1 -10/1),得到白色泡沫状固体化合物6-6(7.7g,8.4mmol,54％收率)。化合物6-6分子式:C49H65N3O14,分子量:919.4,LC-MS实测：918.3(M-H).1HNMR(400MHz,DMSO-d6):δ7.81(dd,J＝9.2,3.1Hz,1H),7.45(d,J＝6.0Hz,1H),7.36(d,J＝7.3Hz,2H),7.30(t,J＝7.7Hz,2H),7.24–7.21(m,5H),6.88(d,J＝8.9Hz,4H),5.21(d,J＝3.4Hz,1H),4.97(dd,J＝11.2,3.0Hz,1H),4.48(dd,J＝8.4,1.4Hz,1H),4.04–3.98(m,3H),3.87(dd,J＝19.9,9.3Hz,2H),3.73(s,6H),3.71–3.67(m,1H),3.43–3.35(m,2H),2.84(dd,J＝17.8,8.8Hz,4H),2.10(s,3H),2.05(t,J＝6.7Hz,2H),1.98(d,J＝1.4Hz,4H),1.89(s,3H),1.77(s,3H),1.65(d,J＝9.9Hz,2H),1.53–1.41(m,5H),1.26–1.23(m,1H),1.14(dt,J＝22.4,9.2Hz,2H).
6.7化合物I-1-8的制备
将化合物6-6(2.2mmol,2.0g)置于洁净干燥反应瓶中，加入20mL无水二氯甲烷。室温氩气保护下加入化合物2-氰乙基N,N,N’,N’-四异丙基亚磷酰二胺(2.0equiv,4.4mmol,1.3g)和4,5-二氰咪唑(1.5equiv,3.3mmol,0.38g)，室温下继续搅拌一小时。反应后向反应液中加入20mL二氯甲烷，并用50mL饱和碳酸氢钠溶液洗涤，干燥有机相，过滤并浓缩，所得粗产品经C18反相柱(规格：30μm；市售，购买于上海博蕴生物科技有限公司)(MeCN:H2O＝75％:25％)制备得到白色固体I-1-8(1.1g,1.0mmol,45％收率)。化合物I-1-8分子式:C58H82O15N5P,分子量:1119.5,LC-MS实测：1118.4(M-H).1H NMR(400MHz,DMSO-d6):δ7.83(d,J＝9.2Hz,1H),7.36(d,J＝7.3Hz,2H),7.31(dd,J＝9.2,6.0Hz,2H),7.22(d,J＝8.6Hz,5H),6.88(dd,J＝8.8,1.5Hz,4H),5.21(d,J＝3.2Hz,1H),4.97(dd,J＝11.3,3.3Hz,1H),4.48(d,J＝8.4Hz,1H),4.06–3.96(m,4H),3.87(dd,J＝20.0,9.0Hz,1H),3.73(s,6H),3.72–3.69(m,2H),3.59–3.52(m,1H),2.81(d,J＝6.0Hz,3H),2.73(dt,J＝9.7,6.0Hz,2H),2.09(s,3H),2.07–2.03(m,2H),1.99–1.98(m,4H),1.88(d,J＝5.1Hz,4H),1.77(s,3H),1.69–1.59(m,2H),1.48–1.40(m,5H),1.23–1.11(m,12H).31P NMR(162MHz,DMSO-d6):δ146.45.
实施例7化合物I-1-13的制备
根据实施例2的合成方法，由起始原料N-苄氧羰基-DL-丝氨酸(市售，购买于上海泰坦科技股份有限公司)制备得到白色固体I-1-13(2.32g,2.1mmol,81％收率)。化合物I-1-13分子式:C56H76O15N6P,分子量:1105.5,LC-MS实测：1128.6(M+Na).1H NMR(400MHz,DMSO-d6):δ8.09–8.03(m,1H),7.75(dd,J＝9.1,2.0Hz,1H),7.32–7.25(m,4H),7.17(ddd,J＝13.7,8.2,2.3Hz,5H),6.85(d,J＝7.5Hz,4H),5.18(d,J＝3.3Hz,1H),4.94(dd,J＝11.2,3.0Hz,1H),4.83–4.72(m,1H),4.49–4.43(m,2H),4.01–3.95(m,3H),3.88–3.80(m,1H),3.71(s,6H),3.67–3.44(m,6H),3.36(t,J＝11.7Hz,2H),3.28(d,J＝4.1Hz,1H),3.16(qd,J＝8.7,3.8Hz,1H),2.99(dt,J＝19.9,6.8Hz,1H),2.75–2.67(m,1H),2.57(td,J＝5.8,1.8Hz,1H),2.06(s,5H),1.95(s,4H),1.86(s,3H),1.70(s,3H),1.41(s,3H),1.16–0.96(m,16H).31PNMR(162MHz,DMSO-d6):δ147.08,146.82,146.78,146.64.
实施例8化合物I-1-15的制备
根据实施例2的合成方法，由起始原料N-苄基-4-羟基哌啶(市售，购买于上海泰坦科技股份有限公司)以及N-苄氧羰基-DL-丝氨酸(市售，购买于上海泰坦科技股份有限公司)制备得到白色固体I-1-15(2.1g,1.88mmol,78％收率)。化合物I-1-15分子式:C57H78O15N6P,分子量:1119.5,LC-MS实测：1142.6(M+Na).1H NMR(400MHz,DMSO-d6):δ8.10–8.05(m,1H),7.76(d,J＝9.2Hz,1H),7.32(d,J＝7.6Hz,2H),7.25(t,J＝7.6Hz,2H),7.18(d,J＝7.5Hz,5H),6.84(d,J＝7.8Hz,4H),5.18(d,J＝3.3Hz,1H),4.98–4.92(m,2H),4.45(d,J＝8.4Hz,1H),4.01–3.95(m,4H),3.84(dd,J＝19.8,8.9Hz,1H),3.70(s,6H),3.69–3.63(m,3H),3.59–3.50(m,2H),3.34(dd,J＝11.9,7.7Hz,2H),3.28(d,J＝1.6Hz,1H),3.16(t,J＝7.2Hz,2H),2.73–2.70(m,2H),2.11–2.03(m,5H),1.95(s,3H),1.86(s,3H),1.72(s,4H),1.43(s,6H),1.12–1.09(m,15H).31P NMR(162MHz,DMSO-d6):δ145.56,145.49,145.46,145.39.
实施例9siRNA缀合物的制备
通过固相亚磷酰胺法，利用上述步骤制备的特殊修饰化合物及商业购买的常规修饰的单体(合成修饰核苷酸dT、Am、Cm、Gm、Um、Af、Cf、Gf、Uf的亚磷酰胺单体，均购自上海兆维科技发展有限公司)，按照核苷酸排布顺序自3’-5’方向逐一连接核苷单体。其中特殊修饰的防脱靶化合物置于反义链的种子区(从5’端开始数第4-8位任一位置)，其中递送单体化合物按照普通单体自由设定放在3’端或者5’端，每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化或硫化四步反应。正义链和反义链采用的合成条件。
仪器设备型号：MerMade 12 Oligonucleotide syntheizer固相合成仪，北京海精6mL合成柱，思拓凡SourceTM 15Q 4.6/100PE纯化柱。
合成siRNA缀合物所使用的试剂购自苏州柯乐玛生物技术有限公司。
合成简介如下：
单链合成反应过程是从3’-5’方向延伸，在固相合成仪上完成。包括四个主要的反应步骤：
a.脱DMTr反应：用二氯乙酸脱去核苷酸上的保护基团DMTr获得5’-羟基端；
b.偶联反应：保护的核苷酸亚磷酰胺单体，与活化剂乙硫基四氮唑混合，亚磷酰胺基团被活化，5’-羟基仍然被DMTr保护，与连接在固相载体上的5’-羟基发生缩合反应，生成亚磷酸三酯；
c.氧化反应：在氧化剂碘的作用下，上步缩合反应所得的亚磷酸三酯转变为更稳定的磷酸酯。(即将三价磷氧化成五价磷)；
d.硫化反应：在硫代试剂PADS(苯乙酰基二硫化物)的作用下，将上步缩合反应所得的亚磷酸三酯转变为硫代磷酸酯(根据序列设计选择氧化或硫代)。
e.盖帽反应：缩合反应中可能有极少数没有参加反应5’-羟基(少于2％)，用乙酸酐和1-甲基咪唑与其反应，形成不能参与后续反应的乙酸酯封端，阻止其后继续发生反应，这种短片段可以在纯化时分离掉。
重复进行以上四步的循环，直至合成完所需的序列，其主要的化学反应方程式如下
待最后一个核苷单体连接完成后，依次对固相载体上连接的核酸序列进行切割、脱保护、纯化、脱盐，随后冻干获得正义链和反义链，其中：
切割和脱保护条件如下：先配置好氨解溶液(氨水：乙醇＝3:1的混合溶液至体积为2mL)，固相载体加入上述反应瓶中，充分震荡均匀。在恒温水浴内50℃氨解16小时。氨解16小时后，水浴冷却至温(25℃±2℃)，用砂芯漏斗过滤，圆底烧瓶收集滤液，并用50％乙醇水溶液冲洗滤渣，收集滤液，旋转蒸发仪浓缩，再转移至玻璃瓶中，取粗品小样送分析部门检测粗品LC-MS。检测方法如下：使用Waters Acquity UPLC-LTQ LCMS(column：ACQUITY UPLC BEH C18)检测上述正义链和反义链纯度并分析分子量。实测值与理论值相符，见表1。
纯化和脱盐条件如下：利用离子交换色谱柱进行纯化并配合使用思拓凡HiPrepTM 26/10 Desalting凝胶柱脱盐，然后单链冻干。单链冻干后，需要取样测LC-MS。
最后需要将获得的正义链和反义链退火成双链。
退火操作如下：将纯化后获得的正义链和反义链分别溶于注射用水中，配制0.1mg/mL-40mg/mL的溶液，用Thermo Scientific Nanodrop Eight标定等摩尔比混合，90℃加热5分钟，再缓慢自然降温，使它们通过氢键形成双链结构，取样送检测产品的SEC纯度，见表2，将双链样品冻干。
表1递送分子缀合siRNA编号和序列信息
表2双链SEC-HPLC纯度
实施例10小鼠体内测试siRNA缀合物的活性
选择6-8周龄的SPF级雌性C57BL/6J小鼠，小鼠的体重为20±2g。给药前对上述小鼠称重并观察状态，选取体重均一、状态无异常的动物进行随机分组，每组4只，其中实验组小鼠给予缀合物，溶媒组小鼠给予磷酸盐缓冲盐水(PBS)，按照每只小鼠给予1mg/kg缀合物的剂量进行皮下给药。给药后20天，动物安乐死，取肝组织，按照常规方法，将肝脏切块并置于RNALater(Invitrogen，AM7021M)中，用于后续RNA提取。将肝脏组织放入裂解液(长春市志昂生物科技有限公司，MNTR/FX96)中研磨(上海净信实业发展有限公司，JXFSTPRP-48L)提取总RNA，反转录为cDNA(Takara，6210B)， 通过荧光法qPCR(Vazyme,Q711)检测靶基因补体C5 mRNA的表达水平。
目的基因引物：
正向引物：CCAGCCCAATCAAGTTCCTAGAG；
反向引物：CGGCGTGTAAACAGGTTTGTC；
内参基因GAPDH引物：
正向引物：TGCACCACCAACTGCTTAG；
反向引物：GATGCAGGGATGATGTTC；
结果以siRNA给药组相比于溶媒组(溶媒组为100％)的剩余表达水平表示，用于注射的缀合物的siRNA序列见表1。如图1和表3所示，与阳性缀合物SD003317相比，缀合物SD003974表现出了相当的靶基因补体C5 mRNA沉默活性；缀合物SD004119、SD004122和SD004125表现出了显著优于缀合物SD003317靶基因的补体C5 mRNA沉默活性。
表3受试缀合物给药20天后靶基因补体C5 mRNA相对剩余表达水平
上文及表1中提到的序列，各符号分别用于表示如下修饰的核苷酸
A＝腺苷-3’-磷酸酯；C＝胞苷-3’-磷酸酯；G＝鸟苷-3’-磷酸酯；U＝尿苷-3’-磷酸酯；dT＝胸腺嘧啶脱氧核苷酸；Am＝2’-O-甲基腺苷-3’-磷酸酯；Ams＝2’-O-甲基腺苷-3’-硫代磷酸酯；Cm＝2’-O-甲基胞苷-3’-磷酸酯；Cms＝2’-O-甲基胞苷-3’-硫代磷酸酯；Gm＝2’-O-甲基鸟苷-3’-磷酸酯；Gms＝2’-O-甲基鸟苷-3’-硫代磷酸酯；Um＝2’-O-甲基尿苷-3’-磷酸酯；Ums＝2’-O-甲基尿苷-3’-硫代磷酸酯；Af＝2’-氟腺苷-3’-磷酸酯；Afs＝2’-氟腺苷-3’-硫代磷酸酯；Cf＝2’-氟胞苷-3’-磷酸酯；Cfs＝2’-氟胞苷-3’-硫代磷酸酯；Gf＝2’-氟鸟苷-3’-磷酸酯；Gfs＝2’-氟鸟苷-3’-硫代磷酸酯；Uf＝2’-氟尿苷-3’-磷酸酯；Ufs＝2’-氟尿苷-3’-硫代磷酸酯
本文已公开了示例实施方式，并且虽然采用特定术语，但是它们仅以一般和描述性含义使用和说明，并非出于限制的目的。在一些情况下，如本领域技术人员自提交本申请起显而易见的是，结合特定实施方式描述的特性、特征和/或元件可以单独地使用或与结合其他实施方式描述的特性、特征和/或元件的组合使用，除非另有明确指示。因此，本领域技术人员应理解，可在不偏离权利要求书中阐述的本发明的精神和范围下作出形式和细节的各种变化。

Claims (24)

  1. 一种化合物，该化合物具有式(I)所示的结构：
    其中，
    A0代表对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体，或者代表所述配体中的羟基全部或部分地被KCOO-基团取代而形成的基团，其中，每个K独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；
    b为1-4范围内的整数；
    L1和L2彼此独立地代表选自式A1-A11基团中的一种或多种的连接组合：
    其中，j1为1-20的整数；j2为1-20的整数；R’为C1-C10的烷基；
    M代表如式(A12)所示的结构式：
    其中，m代表0-6的整数；*表示与G1相连的键，
    Q代表式(A13)所示的结构式：
    其中，与O相连的表示与G1相连的键；R2、R3相同或不同，且彼此独立地选自H、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基；
    Z不存在或代表C1-C10亚烷基；
    X代表式(A14-1)或(A14-2)或所示的结构式：
    其中R4，R5相同或不同，且彼此独立地选自H、氟、羟基、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基；p为1-6的整数；任选地，R4和R5直接相连形成三到六元饱和碳环；
    E代表如式(A15)所示的结构式：
    其中，代表C3-C18环烷基或C3-C18杂环基，并且R1选自H、氟、羟基、氰基、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基；
    G1和G2各自代表具有式(G-1)所示结构的亚磷酰胺官能团或羟基保护基团，前提是G1和G2中至少一个具有式(G-1)所示的结构，所述羟基保护基团选自三苯甲基、4-甲氧基三苯甲基、4,4’-双甲氧基三苯甲基(DMTr)和4,4’,4’-三甲氧基苯甲基中的任意一种；
    其中，B1选自取代或未取代的C1-C5的烃基；
    表示基团共价键连接的位点。
  2. 根据权利要求1所述的化合物，其中L1或L2彼此独立地选自A1、A2、A4、A6、A7、A8、A9、A10、A11中的一种或多种的连接组合；
    所述配体各自独立地选自下述中的任意一种：D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D-吡喃甘露糖、α-D-吡喃葡萄糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳糖胺、N-乙酰基半乳糖胺、N-三氟乙酰基半乳糖胺、N-丙酰基半乳糖胺、N-正丁酰基半乳糖胺、N-异丁酰基半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6-O-三苯甲基-α-D-吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖、L-4-硫代核糖；
    b为1、2或3；
    m为0、1或2；
    R2、R3彼此独立地选自H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；
    Z不存在或代表C1-C3亚烷基；
    R4、R5彼此独立地选自H、氟、羟基、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；p为1、2或3；
    代表C4-C8环烷基或C3-C8杂环基；并且R1选自H、氟、羟基、氰基、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；
    B1为甲基、乙基或异丙基；B2选自C1-C5的烷基、乙氰基、丙氰基和丁氰基中的一种。
  3. 根据权利要求2所述的化合物，其中b为1或3；
    代表C3-C6杂环基；
    A0为如下所示的N-乙酰基半乳糖胺(GalNAc)基团或其羟基全部或部分地被KCOO-基团取代而形成的基团，其中，每个K独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种：
    羟基保护基团为4,4’-双甲氧基三苯甲基；
    L1或L2的长度彼此独立地为1-25个原子，所述L1或L2的长度是指最长直链上的成链原子的个数；
    每个j1彼此独立地为1-10的整数，每个j2彼此独立地为1-10的整数。
  4. 根据权利要求3所述的化合物，其中代表四到六元含氮饱和环烷基；
    L1或L2的长度彼此独立地为1-20个原子；
    每个j1彼此独立地为1-8的整数，每个j2彼此独立地为1-8的整数。
  5. 根据权利要求2所述的化合物，其中L1或L2彼此独立地选自A1、A2、A4、A8、A9、A10、A11中的至少2个的连接组合。
  6. 根据权利要求2所述的化合物，其中L1或L2彼此独立地选自A1、A4、A8、A10、A11中的至少2个的连接组合。
  7. 根据权利要求1所述的化合物，其中所述化合物具有以下所示结构(I-1)：
    其中，
    L为A10，其中j1为2-8的整数，或者为A11，其中j2为1-7的整数；
    m为0、1或2；
    n为0、1、2或3；
    Q代表式(A13)所示的结构式：
    其中，表示基团共价键连接的位点；R2、R3彼此独立地选自H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；
    X代表式(A14-1)或(A14-2)或所示的结构式：
    其中R4，R5彼此独立地选自H、氟、羟基、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；p为1、2或3；任选地，R4和R5直接相连形成三到六元饱和碳环；
    代表四到八元全碳或含氮饱和环，
    R1选自H、氟、羟基、氰基、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；
    其中，中的羟基全部或部分地被KCOO-基团取代，其中，每个K独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种。
  8. 根据权利要求1所述的化合物，其中为含有一或两个氮原子的四、五或六元饱和环烷基。
  9. 根据权利要求1所述的化合物，其中所述化合物具有以下所示结构中的任意一种：


  10. 一种核酸缀合物，其中所述缀合物含有与寡核苷酸序列上的任意位置连接的一个或一个以上具有式(II)所示的结构：
    其中，
    Rp和Rq各自为H或具有式A16所示的结构，前提是Rp和Rq中至少一个具有式A16所示的结构；
    其中，表示基团共价键连接的位点；
    E1为OH、SH或BH2，
    A0代表对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体或者
    代表所述配体中的羟基全部或部分地被KCOO-基团取代而形成的基团，其中，每个K独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；
    b为1-4范围内的整数；
    L1和L2彼此独立地代表选自式A1-A11基团中的一种或多种的连接组合：
    其中，j1为1-20的整数；j2为1-20的整数；R’为C1-C10的烷基；
    M代表如式(A12)所示的结构式：
    其中，表示基团共价键连接的位点；m代表0-6的整数；*表示与Rp相连的键，
    Q代表式(A13)所示的结构式：
    其中，表示基团共价键连接的位点，与O相连的表示与Rp相连的键；R2、R3相同或不同，且彼此独立地选自H、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基，优选H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；
    Z不存在或代表C1-C10亚烷基；
    X代表式(A14-1)或(A14-2)或所示的结构式：

    其中R4，R5相同或不同，且彼此独立地选自H、氟、羟基、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基，优选H、氟、羟基、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；p为1-6的整数；任选地，R4和R5直接相连形成三到六元饱和碳环；
    E代表如式(A15)所示的结构式：
    其中，表示基团共价键连接的位点；代表C3-C18环烷基或C3-C18杂环基，并且R1选自H、氟、羟基、氰基、C1-C20烷基、C1-C20烷氧基、C2-C20烯基、C2-C20炔基。
  11. 根据权利要求10所述的核酸缀合物，其中所述缀合物含有与寡核苷酸序列上的任意位置连接的一个以上具有式(II-A)所示的结构：
    其中，
    a代表0-7的整数；
    W的结构如下式所示：
    其中，表示基团共价键连接的位点；E1为OH、SH或BH2。
  12. 根据权利要求11所述的核酸缀合物，其中a代表0、1、2或3的整数。
  13. 根据权利要求10或11所述的核酸缀合物，其中L1或L2彼此独立地选自A1、A2、A4、A6、A7、A8、A9、A10、A11中的一种或多种的连接组合；
    所述配体各自独立地选自下述中的任意一种：D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D-吡喃甘露糖、α-D-吡喃葡萄糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳糖胺、N-乙酰基半乳糖胺、N-三氟乙酰基半乳糖胺、N-丙酰基半乳糖胺、N-正丁酰基半乳糖胺、N-异丁酰基半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6-O-三苯甲基-α-D-吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖、L-4-硫代核糖；
    b为1、2或3；
    m为0、1或2；
    R2、R3彼此独立地选自H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；
    Z不存在或代表C1-C3亚烷基；
    R4，R5彼此独立地选自H、氟、羟基、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；p为1、2或3；
    代表C4-C8环烷基或C3-C8杂环基；并且R1选自H、氟、羟基、氰基、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；
    B1为甲基、乙基或异丙基；B2选自C1-C5的烷基、乙氰基、丙氰基和丁氰基中的一种。
  14. 根据权利要求13所述的核酸缀合物，其中b为1或3；
    代表C3-C6杂环基；
    A0为如下所示的N-乙酰基半乳糖胺(GalNAc)基团或其羟基全部或部分地被KCOO-基团取代而形成的基团，其中，每个K独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种：
    羟基保护基团为4,4’-双甲氧基三苯甲基；
    L1或L2的长度彼此独立地为1-25个原子，所述L1或L2的长度是指最长直链上的成链原子的个数；
    每个j1彼此独立地为1-10的整数，每个j2彼此独立地为1-10的整数。
  15. 根据权利要求14所述的核酸缀合物，其中代表四到六元含氮饱和环烷基；
    L1或L2的长度彼此独立地为1-20个原子；
    每个j1彼此独立地为1-8的整数，每个j2彼此独立地为1-8的整数。
  16. 根据权利要求10或11所述的核酸缀合物，其中L1或L2彼此独立地选自A1、A2、A4、A8、A9、A10中的至少2个的连接组合。
  17. 根据权利要求10或11所述的核酸缀合物，其中L1或L2彼此独立地选自A1、A4、A8、A10中的至少2个的连接组合。
  18. 根据权利要求10或11所述的核酸缀合物，其具有以下所示结构中的任意一种：





    其中Y为O或者S，为寡核苷酸。
  19. 根据权利要求10或11所述的核酸缀合物，其中，所述寡核苷酸选自小干扰RNA、微小RNA、抗微小RNA、微小RNA拮抗剂、微小RNA模拟物、诱饵寡核苷酸、免疫刺激物、G-四极子、可变剪接体、单链RNA、反义核酸、核酸适配体、茎环RNA、mRNA片段、激活RNA中的一种；可选地，所述寡核苷酸为单链寡核苷酸或者双链寡核苷酸；可选地，所述寡核苷酸为单链寡核苷酸，式(A16)中的P原子连接到所述单链寡核苷酸的端部，所述单链寡核苷酸的端部指所述单链寡核苷酸中从一端起算的前4个核苷酸；可选地，式(A16)中的P原子连接到所述单链寡核苷酸的末端；可选地，式(A16)中的P原子连接到所述单链寡核苷酸的3'末端；
    可选地，所述寡核苷酸为双链寡核苷酸，所述双链寡核苷酸包含正义链和反义链，所述式(A16)中的P原子连接到所述双链寡核苷酸的端部，所述双链寡核苷酸的端部指所述正义链或所述反义链中从一端起算的前4个核苷酸；可选地，式(A16)中的P原子连接到所述正义链或所述反义链的末端；可选地，式(A16)中的P原子连接到所述反义链的5'末端；可选地，式(A16)中的P原子通过形成磷酸二酯键连接至所述核酸缀合物中的核苷酸的2'位、3'位或5'位。
  20. 根据权利要求19所述的核酸缀合物，其中，所述双链寡核苷酸为siRNA。
  21. 权利要求10-20中任一项所述的核酸缀合物的制备方法，包括将权利要求1-9所述的式(I)化合物中的亚磷酰胺官能团氧化为式(W)所示的结构，然后与寡核苷酸结合，再进行切割和脱去保护基团，得到所述缀合物；
    其中，表示基团共价键连接的位点；
    E1为OH、SH或BH2。
  22. 权利要求10-20中任一项所述的核酸缀合物在制备用于治疗和/或预防肝源性疾病的药物中的用途。
  23. 一种治疗由肝细胞中基因的表达而引起的病理状况或疾病的方法，所述方法包括向患有该疾病的患者给予权利要求10-20中任一项所述的核酸缀合物。
  24. 一种试剂盒，该试剂盒包含权利要求10-20中任一项所述的核酸缀合物。
PCT/CN2024/101798 2023-06-30 2024-06-27 一种核酸缀合物、制备方法及用途 WO2025002208A1 (zh)

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