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WO2021249484A1 - 缀合基团及其缀合物 - Google Patents

缀合基团及其缀合物 Download PDF

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Publication number
WO2021249484A1
WO2021249484A1 PCT/CN2021/099405 CN2021099405W WO2021249484A1 WO 2021249484 A1 WO2021249484 A1 WO 2021249484A1 CN 2021099405 W CN2021099405 W CN 2021099405W WO 2021249484 A1 WO2021249484 A1 WO 2021249484A1
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acid
conjugate
expression
present disclosure
group
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PCT/CN2021/099405
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English (en)
French (fr)
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安可
孙飞
丁照中
陈曙辉
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南京明德新药研发有限公司
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Priority to CN202180041231.5A priority Critical patent/CN115702006A/zh
Priority to US18/001,429 priority patent/US20240033278A9/en
Publication of WO2021249484A1 publication Critical patent/WO2021249484A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • 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
    • 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
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present disclosure relates to a new type of conjugating group and its use.
  • the conjugating group disclosed herein can be attached to a compound (such as a therapeutic agent) to direct the compound to a target in the body.
  • a specific location e.g., a desired cell
  • the ability to efficiently deliver the compound to a specific location can limit or potentially eliminate undesirable consequences (such as off-target effects) that may be caused by the administration of the compound.
  • One method of facilitating the delivery of a compound such as a therapeutic agent to a desired location in the body is by linking or attaching the compound to a conjugating group.
  • oligonucleotides One type of therapeutic agent that can be targeted using a conjugating group is oligonucleotides. It has been shown that oligonucleotides comprising a nucleotide sequence that is at least partially complementary to a target nucleic acid will alter the function and activity of the target in vitro and in vivo. It has been shown that when delivered to a cell containing a target nucleic acid (such as mRNA), the oligonucleotide will modulate the expression of the target, resulting in a change in the transcription or translation of the target nucleic acid. In certain examples, oligonucleotides can reduce gene expression by inhibiting nucleic acid targets and/or triggering degradation of target nucleic acids.
  • a target nucleic acid such as mRNA
  • RNA interference is a biological process in which RNA or RNA-like molecules (such as chemically modified RNA molecules) can be degraded to silence gene expression. This post-transcriptional gene silencing process is considered to be an evolutionary conservative cell protection mechanism to prevent foreign gene expression.
  • Synthetic RNA and RNA-like molecules have been shown to trigger RNA interference in vivo.
  • Elbashir et al. (Nature 2000, 411, 494-98) describe RNAi induced by introducing a duplex of synthetic 21-nucleotide RNA molecules into cultured mammalian cells.
  • the types of synthetic RNA or RNA-like molecules that can trigger the RNAi response mechanism can include modified nucleotides and/or one or more non-phosphodiester linkages.
  • GalNAc a structural unit, has broad application prospects in delivering macromolecules to liver cells.
  • the present disclosure relates to a novel conjugating group that can be attached to a compound (such as a therapeutic agent), which can be used to direct the compound to a target in the body.
  • a compound such as a therapeutic agent
  • the conjugation groups disclosed herein can target expression-inhibiting oligonucleotides (such as RNAi reagents) to liver cells to regulate gene expression.
  • the conjugating group disclosed herein can be used for a variety of purposes when linked to an expression-inhibiting oligonucleotide, including use in therapy, diagnosis, target verification, and genome development.
  • the composition including the conjugating group disclosed herein can mediate the expression of target nucleic acid sequence in liver cells (such as hepatocytes) when linked to expression inhibitory oligonucleotides, which can be used for the treatment of cells and tissues. Or a disease or condition that the organism's gene expression or activity responds to.
  • the present disclosure provides a conjugating group having the structure of formula (I)
  • n is selected from an integer of 8-12.
  • the conjugating group may have the following structure:
  • the present disclosure provides a conjugate comprising the conjugating group according to the first aspect of the present disclosure and a therapeutic agent connected to the conjugating group.
  • the therapeutic agent in the above-mentioned conjugate is an expression inhibitory oligonucleotide.
  • the expression-inhibiting oligonucleotide in the above-mentioned conjugate is an RNAi agent.
  • the RNAi agent in the aforementioned conjugate includes one or more modified nucleotides.
  • the RNAi agent in the above-mentioned conjugate is a double-stranded siRNA comprising a sense strand and an antisense strand.
  • the double-stranded siRNA in the above-mentioned conjugate is connected to a conjugating group at the 5'end of its sense strand.
  • the expression-inhibiting oligonucleotide in the above-mentioned conjugate is connected to the conjugating group via a phosphate group, a phosphorothioate group, or a phosphonate group.
  • the phosphorothioate portion of the aforementioned conjugate or expression inhibitory oligonucleotide includes (R)- and (S)-enantiomers, diastereomers, And/or its racemic mixture.
  • the above-mentioned present disclosure also provides salts of conjugates.
  • the salt as described above is selected from base addition salts, acid addition salts, and combinations thereof.
  • the above-mentioned base addition salt is selected from sodium, potassium, calcium, ammonium, organic amine, magnesium salt and combinations thereof
  • the acid addition salt is selected from inorganic acid salts, organic acid salts and combinations thereof .
  • the above-mentioned inorganic acid is selected from hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid , Phosphorous acid and combinations thereof
  • organic acids are selected from acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, ortho Phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and combinations thereof.
  • the present disclosure provides a compound having the structure of formula (II) or formula (III)
  • n is selected from an integer of 8-12.
  • the compound may have the following structure
  • the present disclosure provides a pharmaceutical composition comprising the conjugate according to the second aspect of the present disclosure and a pharmaceutically acceptable carrier or excipient.
  • the present disclosure provides a method for inhibiting the expression of a target nucleic acid in a subject in need thereof, which comprises administering to the subject the conjugate according to the second aspect of the present disclosure or according to the first aspect of the present disclosure. Four steps of the pharmaceutical composition.
  • the target nucleic acid in the method is a nucleic acid from a virus.
  • the virus may be, for example, a virus that causes liver disease, such as hepatitis B virus.
  • the present disclosure provides a method of treating a disease, which includes the step of administering a conjugate according to the second aspect of the present disclosure or a pharmaceutical composition according to the fourth aspect of the present disclosure to a subject.
  • the present disclosure provides the use of the conjugate according to the second aspect of the present disclosure or the pharmaceutical composition according to the fourth aspect of the present disclosure in the preparation of a medicament for the treatment of diseases.
  • the present disclosure provides a conjugate according to the second aspect of the present disclosure or a pharmaceutical composition according to the fourth aspect of the present disclosure for use in the treatment of diseases.
  • the disease is a viral infection.
  • the disease is liver disease.
  • the disease is hepatitis B.
  • the conjugating group described in the present disclosure can enhance the delivery of a therapeutic agent to a specific target location (e.g., a specific organ or tissue) in a subject such as a human or an animal.
  • a specific target location e.g., a specific organ or tissue
  • the conjugating group can enhance the targeted delivery of expression-inhibiting oligonucleotides.
  • the conjugating group can enhance the delivery of the expression inhibitory oligonucleotide to the liver.
  • the conjugating group described in the present disclosure can be directly or indirectly connected to a compound, such as a therapeutic agent, for example, an expression inhibitory oligonucleotide, for example, the 3'or 5'end of an expression inhibitory oligonucleotide .
  • the expression-inhibiting oligonucleotide includes one or more modified nucleotides.
  • the expression inhibitory oligonucleotide is an RNAi agent, such as a double-stranded RNAi agent comprising a sense strand and an antisense strand.
  • the conjugating group disclosed herein is attached to the 5'end of the sense strand of the double-stranded RNAi agent. In some embodiments, the conjugating group disclosed herein is linked to the expression-inhibiting oligonucleotide agent at the 5'end of the sense strand of the double-stranded RNAi agent via a phosphate, phosphorothioate, or phosphonate group.
  • connection in the present disclosure, when referring to the connection between two molecules, means that two molecules are connected by a covalent bond or two molecules are connected by a non-covalent bond (for example, a hydrogen bond or an ionic bond).
  • the "oligonucleotide” described in the present disclosure is a nucleotide sequence containing 10-50 nucleotides or nucleotide base pairs.
  • the oligonucleotide has a nucleobase sequence that is at least partially complementary to a target nucleic acid expressed in a cell or a coding sequence in a target gene.
  • the nucleotides can be optionally modified.
  • the oligonucleotide after the oligonucleotide is delivered to the cell expressing the gene, the oligonucleotide can inhibit the expression of the potential gene, and is referred to as an "expression inhibitory oligomer" in the present disclosure.
  • Glycolic acid which can inhibit gene expression in vitro or in vivo.
  • Oligonucleotides include but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hair Clip RNA (shRNA), ribozyme, interfering RNA molecule, and Dicer enzyme substrate.
  • RNAi reagent refers to a reagent containing RNA or RNA-like (such as chemically modified RNA) oligonucleotide molecules capable of degrading or inhibiting translation of messenger RNA (mRNA) transcripts of target mRNA in a sequence-specific manner.
  • RNAi reagents described in the present disclosure can be manipulated by RNA interference mechanisms (ie, by interacting with a component of the RNA interference pathway of mammalian cells (RNA-induced silencing complex or RISC) to induce RNA interference), or by any other mechanism or pathway kick in.
  • RNAi reagents described in the present disclosure are mainly manipulated through RNA interference mechanisms, the disclosed RNAi reagents are not limited or restricted to any specific action pathway or mechanism.
  • RNAi reagents include but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) , And Dicer substrate.
  • the RNAi reagents described in the present disclosure include oligonucleotides having strands that are at least partially complementary to the targeted mRNA.
  • the RNAi agent described herein is double-stranded and includes an antisense strand and a sense strand that is at least partially complementary to the antisense strand.
  • RNAi reagents may include modified nucleotides and/or one or more non-phosphodiester linkages.
  • the RNAi agents described herein are single-stranded.
  • single-stranded oligonucleotide in the present disclosure refers to a single-stranded oligonucleotide having a sequence that is at least partially complementary to the target mRNA, which is capable of hydrogen bonding under mammalian physiological conditions (or equivalent in vitro environment) Hybridize with target mRNA.
  • the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.
  • the short interfering RNA (siRNA) described in the present disclosure is a type of RNA molecule with a length of 20-25 base pairs, similar to miRNA, and operates in the RNA interference (RNAi) pathway, which interferes with the complementation of the nucleotide sequence The translation of the mRNA of a specific gene leads to mRNA degradation.
  • the short interfering RNA (siRNA) described in the present disclosure includes double-stranded siRNA (including sense strand and antisense strand) and single-stranded siRNA (only antisense strand).
  • the "silence”, “reduction”, “inhibition”, “down-regulation” or “knockdown” mentioned in the present disclosure when referring to a given gene, means that it is related to a second cell, cell population or tissue that has not been treated in this way.
  • gene expression decreases, such as from the level of RNA transcribed from the gene or from the cell transcribing the gene, Measurement of the level of polypeptides, proteins, or protein subunits translated from mRNA in cell populations, tissues, or objects.
  • sequence or “nucleotide sequence” in the present disclosure refers to the sequence or sequence of nucleobases or nucleotides described by a sequence of letters designated by standard nucleotides.
  • the HBV gene described in the present disclosure refers to a gene whose DNA sequence is as shown in Genbank registration number NC_003977.1.
  • Genbank registration number NC_003977.1 is the complete genome of HBV.
  • double-stranded siRNA analogs can target the X open reading frame (X opening reading frame, X ORF) of HBV.
  • double-stranded siRNA analogs can target the S ORF of HBV.
  • double-stranded siRNA analogs can target the PORF of HBV.
  • modified sequences include, but are not limited to, methoxy modification, fluoro modification, phosphorothioate linkage, and the like.
  • sequences described in the present disclosure may include the "further modified sequences” listed in Table 1 below.
  • capital letters C, G, U, and A represent the base composition of nucleotides.
  • Lowercase letters c, g, u, and a respectively indicate that the nucleotides represented by their corresponding capital letters are modified by methoxy; underscores indicate that the nucleotides represented by capital letters are modified by fluorine; the interval " ⁇ " indicates the same as the interval " ⁇ "
  • Two adjacent nucleotide residues on the left and right are connected by phosphorothioate groups.
  • "a ⁇ g” means that the residues a and g are connected by phosphorothioate groups.
  • the fluoro-modified nucleotides described in the present disclosure refer to nucleotides formed by substituting fluorine for the hydroxyl group at the 2'position of the ribose group of the nucleotide, and the methoxy-modified nucleotides refer to the 2'-hydroxyl group of the ribose group. Nucleotides formed by substitution with methoxy groups.
  • complementary has a well-known meaning to those skilled in the art, that is, in a double-stranded nucleic acid molecule, bases on one strand are paired with bases on the other strand in a complementary manner.
  • the purine base adenine (A) is always paired with the pyrimidine base uracil (U); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G).
  • Each base pair includes a purine and a pyrimidine.
  • the two chains are considered to be complementary to each other, and it can be inferred from the sequence of the complementary chain The sequence of the chain.
  • the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. This disclosure contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomers Body-enriched mixtures, all of these mixtures are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All these isomers and their mixtures are included in the scope of the present disclosure.
  • enantiomers or “optical isomers” refer to stereoisomers that are mirror images of each other.
  • diastereomer refers to a stereoisomer in which a molecule has two or more chiral centers and the relationship between the molecules is non-mirror mirror image.
  • wedge-shaped solid line keys And wedge-shaped dashed key Represents the absolute configuration of a three-dimensional center, with a straight solid line key And straight dashed key Indicates the relative configuration of the three-dimensional center, using wavy lines Represents a wedge-shaped solid line key Or wedge-shaped dashed key Or use wavy lines Represents a straight solid line key And/or straight dashed key
  • the terms “enriched in one isomer”, “enriched in isomers”, “enriched in one enantiomer” or “enriched in enantiomers” refer to one of the isomers or pairs of
  • the content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or 96% or greater, or 97% or greater, or 98% or greater, or 99% or greater, or 99.5% or greater, or 99.6% or greater, or 99.7% or greater, or 99.8% or greater, or greater than or equal 99.9%.
  • the term “isomer excess” or “enantiomeric excess” refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80% .
  • optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one wants to obtain an enantiomer of a compound of the present disclosure, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure The desired enantiomer.
  • the molecule when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), it forms a diastereomeric salt with a suitable optically active acid or base, and then passes through a conventional method known in the art The diastereoisomers are resolved, and then the pure enantiomers are recovered.
  • the separation of enantiomers and diastereomers is usually accomplished through the use of chromatography, which employs a chiral stationary phase and is optionally combined with chemical derivatization (for example, the formation of amino groups from amines). Formate).
  • the compounds of the present disclosure may contain unnatural proportions of atomic isotopes on one or more of the atoms constituting the compound.
  • compounds can be labeled with radioisotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C).
  • deuterium can be substituted for hydrogen to form deuterated drugs.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon.
  • deuterated drugs have reduced toxic side effects and increased drug stability. , Enhance the efficacy, prolong the biological half-life of drugs and other advantages. All changes in the isotopic composition of the compounds of the present disclosure, whether radioactive or not, are included in the scope of the present disclosure.
  • salt refers to a salt of the compound of the present disclosure, which is prepared from a compound with a specific substituent found in the present disclosure and a relatively non-toxic acid or base.
  • a base addition salt can be obtained by contacting the compound with a sufficient amount of base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salt or similar salts.
  • the acid addition salt can be obtained by contacting the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, the organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.) , And salts of organic acids such as glucuronic acid. Certain specific compounds of the present disclosure contain basic and
  • the salt of the present disclosure can be synthesized from a parent compound containing an acid radical or a base by conventional chemical methods.
  • such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of both.
  • the compounds of the present disclosure can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and those well known to those skilled in the art Equivalent alternatives, and preferred implementations include but are not limited to the embodiments of the present disclosure.
  • the solvent used in the present disclosure is commercially available.
  • the ratios of solvents used in column chromatography and thin-layer silica gel chromatography of the present disclosure are all volume ratios.
  • the compound is used according to the conventional naming principle in the field or The software is named, and the commercially available compounds use the supplier catalog name.
  • Step A 11-Dodecyn-1-ol (25 g, 137.14 mmol) and triethylamine (16.65 g, 164.56 mmol) were dissolved in dichloromethane (250 mL) and methanesulfonyl chloride (18.85) was added at 0°C. G, 164.56 mmol). The mixture was stirred at 0 degrees Celsius for 2 hours. The reaction solution was diluted with water (400 mL) and extracted with 800 mL of dichloromethane (400 mL ⁇ 2). The combined organic phase was washed with 400 ml of water (200 ml ⁇ 2) and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 2-2.
  • Step B The compound represented by formula 2-3 (20 g, 67.26 mmol) was dissolved in N,N-dimethylformamide (200 mL) and sodium hydride (60% purity, 4.04 g, 100.89 mmol) was added at 0°C. ), and then add the compound represented by formula 2-2 (19.27 g, 73.99 mmol). The mixture was stirred at 25 degrees Celsius for 16 hours. The reaction solution was quenched with water (1 liter), and extracted with 1.6 liters of dichloromethane (800 mL ⁇ 2). The combined organic phase was washed with 800 ml (800 ml ⁇ 1) saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 2-4.
  • sodium hydride 50% purity, 4.04 g, 100.89 mmol
  • Step C The compound represented by formula 2-4 (48 g, 103.98 mmol) was dissolved in methanol (870 mL) and a methanol solution of hydrogen chloride (4 mol per liter, 400 mL, 1.6 mol) was added. The mixture was stirred at 30 degrees Celsius for 2 hours. A methanol solution of hydrogen chloride (4 moles per liter, 350 ml, 1.4 moles) was added to the reaction solution. The mixture was stirred at 30 degrees Celsius for 16 hours. The reaction solution was concentrated under reduced pressure, and 200 mL (100 mL ⁇ 2) of chloroform was added and concentrated under reduced pressure until a white solid appeared.
  • Step D To the compound represented by formula 2-5 (23 g, 80.58 mmol) and sodium hydroxide (322.31 mg, 8.06 mmol) in a mixture of dimethyl sulfoxide (70 mL) and water (6 mL) Add tert-butyl acrylate (22.72 g, 177.28 mmol), and stir at 25 degrees Celsius for 16 hours under the protection of nitrogen. The reaction solution was diluted with water (500 mL), and extracted with 1 liter of ethyl acetate (500 mL ⁇ 2). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product.
  • Step E To a dichloromethane (250 ml) solution of the compound represented by formula 2-6 (24.5 g, 45.22 mmol) was added triethylamine (9.15 g, 90.45 mmol) and succinic anhydride (6.79 g, 67.83) Millimoles) and stirred at 20 degrees Celsius for 16 hours. Dichloromethane (1 liter) and hydrochloric acid (1 mole per liter, 1 liter) were added to the reaction solution, the separated organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 2-7.
  • Step F The compound represented by formula 2-7 (27.4 g, 42.69 mmol) was dissolved in formic acid (140 ml), and the mixture was stirred at 20 degrees Celsius for 16 hours under the protection of nitrogen. The reaction solution was concentrated under reduced pressure, and 300 ml (150 ml ⁇ 2) of toluene was added and concentrated under reduced pressure to obtain 2-8.
  • Step G The compound represented by formula 2-8 (22.6 g, 42.67 mmol), N,N-diisopropylethylamine (33.09 g, 256.03 mmol) and O-(7-azabenzotriazole- 1-yl)-N,N,N,N-tetramethylurea hexafluorophosphate (51.92g, 136.55mmol) dissolved in N,N-dimethylformamide (250ml) was added to N-(3- Aminopropyl) tert-butyl carbamate (29.74 g, 170.69 mmol). The mixture was stirred at 20 degrees Celsius for 16 hours.
  • Step I The compound represented by formula 2-11 (22.15 g, 49.50 mmol), N,N-diisopropylethylamine (7.75 g, 60.00 mmol), 1-hydroxy-7-azabenzotriazole (6.12 g, 45.00 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphate (20.53 g, 54.00 mmol) Dissolve in N,N-dimethylformamide (90ml), add the compound represented by formula 2-10 (tris(trifluoroacetate), 15.6g, 15.00mmol) and N,N- A solution of diisopropylethylamine (21.32 g, 165.00 mmol) in N,N-dimethylformamide (120 mL).
  • Step J The compound represented by formula 2-12 (1.00 g, 0.50 mmol) and N-methyl-N,N,N-tri-n-octylammonium chloride (20.35 mg, 50.35 micromole) were dissolved in acetic acid (2.7 A solution of potassium permanganate (0.40 g, 2.52 mmol) in water (9 mL) was added dropwise to the mixed solution of n-pentane (6.3 mL) at 0 degrees Celsius. The mixture was stirred at 0 to 15 degrees Celsius for 2 hours.
  • Step K Add N,N-diisopropylethylamine (0.26 g, 1.99 Millimoles) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphate (0.23 g, 0.60 mmol). After the mixture was stirred, the compound represented by formula 2-14 (0.23 g, 0.55 mmol) was added. The mixture was stirred at 15 degrees Celsius for 16 hours. Add dichloromethane (50ml) and water (50ml) to the reaction solution, and use 50ml saturated sodium bicarbonate aqueous solution (50ml ⁇ 1), 50ml water (50ml ⁇ 1) and saturated salt for the organic phase after separation.
  • dichloromethane 50ml
  • water 50ml
  • Step L To the dichloromethane (8 ml) solution of the compound represented by formula 2-15 (0.80 g, 0.33 mmol), triethylamine (67.24 mg, 0.64 mmol), 4-N,N-dimethyl Aminopyridine (0.12 g, 1.00 mmol) and succinic anhydride (83.13 mg, 0.83 mmol). The mixture was stirred at 10 degrees Celsius for 16 hours.
  • the reaction solution was added with dichloromethane (50ml), water (30ml) and saturated brine (30ml), and the organic phase was separated into 30ml water (30ml ⁇ 1) and saturated brine 30ml (30ml ⁇ 1) Wash sequentially, dry with anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain a crude product.
  • Purified by p-HPLC separation column: Waters Xbridge C18 (specification: 150mm ⁇ 50mm, particle size: 10 ⁇ m); mobile phase: [water (10mM ammonium bicarbonate)-acetonitrile]; elution gradient: 27%-57%, 11min) D01 is obtained.
  • D is the residue after the chemical reaction of the small molecule fragment D01, which binds to the nucleic acid through a covalent bond, and its structure is shown in the following formula:
  • Step A Preparation of compound 3-2 Refer to the preparation of compound D01 in Example 1, replacing 2-1 with 3-1.
  • Step B The compound represented by formula 2-3 (35.70 g, 120.06 mmol) was dissolved in 2-methyltetrahydrofuran (285 ml), potassium tert-butoxide (17.51 g, 156.07 mmol) was added, and the mixture was stirred at 85 degrees Celsius for 2 Hour. Then, the compound represented by formula 3-2 (28.40 g, 114.34 mmol) was added. The mixture was stirred at 85 degrees Celsius for 12 hours. The reaction solution was added with water (400 mL), and extracted with 800 mL of dichloromethane (400 mL ⁇ 2). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain 3-3.
  • Step C The compound represented by formula 3-3 (35 g, 77.84 mmol) was dissolved in methanol (525 mL) and a methanol solution of hydrogen chloride (4 mol per liter, 175 mL, 750 mmol) was added. The mixture was stirred at 50 degrees Celsius for 12 hours. The reaction solution was poured into a mixture of potassium carbonate (80 g) and methanol (500 ml), filtered through a Buchner funnel and concentrated under reduced pressure. The resulting crude product was dissolved in methanol (240 ml), and sodium acetate (12.77 g, 155.68 mmol) and hydroxylamine hydrochloride (5.41 g, 77.84 mmol).
  • the mixture was stirred at 25 degrees Celsius for 0.5 hours.
  • the reaction solution was filtered through a Buchner funnel and concentrated under reduced pressure.
  • the resulting crude product was added to an aqueous sodium hydroxide solution (1 mole per liter, 500 ml), and extracted with 500 ml of dichloromethane (500 ml ⁇ 1).
  • the combined organic phase was washed successively with aqueous sodium hydroxide solution (1 mole per liter, 500 ml) and 500 ml (500 ml ⁇ 1) saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 3-4.
  • Step D to Step I Refer to each step of compound D01 in Example 1 respectively.
  • the combined organic phase was washed with 150 ml (150 ml ⁇ 1) of saturated aqueous sodium sulfite solution, and concentrated under reduced pressure.
  • the combined organic phase was washed with 500 ml (500 ml ⁇ 1) of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 3-11.
  • Step K Add N, N-diisopropylethylamine (0.71 g, 5.52 mmol), 1- Hydroxy-7-azabenzotriazole (1.50 g, 11.04 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluoro Phosphate (2.52 g, 6.63 mmol) and the compound represented by formula 2-14 (2.43 g, 5.80 mmol). After the mixture was stirred, N,N-diisopropylethylamine (2.14 g, 16.56 mmol) was added. The mixture was stirred at 25 degrees Celsius for 12 hours.
  • Step A To a solution of D-01 (100 mg, 39.88 ⁇ mol) in acetonitrile (0.7 mL) was added ammonia (30%, 1.4 mL). The mixture was stirred at 50 degrees Celsius for 12 hours. The reaction solution was filtered and purified by p-HPLC (separation column: Phenomenex Gemini-NX C18 (specification: 75mm ⁇ 30mm, particle size: 3 ⁇ m); mobile phase: [water (0.05% ammonia)-acetonitrile]; elution gradient: 14 %-42%, 7min) to get D-01-M.
  • the surface plasmon resonance technology is used to detect the binding ability of the compound to the human anti-asialoglycoprotein receptor (ASGPR), and the kinetic K D value of the compound is used as an indicator to evaluate the binding ability of the compound to ASGPR, thereby reflecting
  • the compound specifically targets the ability of liver cells to deliver nucleic acid molecules.
  • Asialoglycoprotein Receptor Protein Mouse, Recombinant(His Tag), Sino biological-50083-M07H-50 ⁇ g; Asialoglycoprotein Receptor Protein, Human, Recombinant(His Tag), Sino biological-10773-H07H-50 ⁇ g
  • NiHC 1500M Chip Xan Tec-SCNihc1500m0720
  • HEPES SIGMA-V900477
  • NaCl SIGMA-71376
  • Tween-20 Aldin-T104863
  • CaCl 2 SIGMA-C3306-250G
  • EDTA SIGMA-3609
  • NiCl 2 Energy-chemical-V830089
  • 10 ⁇ PBS Sangon-E607016-0500
  • Biacore 8k Series S CM5chip(GE Healthcare–BR100530) 96-well plate-250 ⁇ L(Greiner-650201); 384-well plate-200 ⁇ L(Greiner-781270); 96-well plate-1mL(Greiner-780201); 96 Microplatefoils(GE Healthcare-28975816); 384 Microplate foams (GE Healthcare-BR100577)
  • the chip is first treated with 350mM EDTA for 5min, 30 ⁇ l/min, running buffer rinse for 2min, and then combined with 40mM NiCl 2 for 2min, 30 ⁇ l/min, and then the protein diluted with running buffer to 5 ⁇ g/mL is labeled on the chip.
  • the flow rate is 10 ⁇ l/min, and the final labeling volume is 1500-2000RU
  • the compound was diluted 2-fold with a running buffer containing 2% DMSO in 9 concentrations, the initial concentration was 2 ⁇ M, and the final concentration of DMSO was 2%.
  • the data is analyzed by Biacore Insight Evaluation Software, kinetics is analyzed by 1:1 model, and affinity is analyzed by steady state affinity model.
  • test substance D-01-M showed good binding power in the SPR experiment.
  • the present invention exhibits an efficient delivery platform for oligo-nucleic acid molecules with high liver cell targeting: it can bind to the specific and highly expressed ASGPR protein on the surface of liver cells, and enter the endocytosis of the cell through endocytosis. Release into the cytoplasm and take effect.
  • the binding force constant of the delivery platform and ASGPR is better than that of the prior art. Demonstrating good tissue distribution and metabolic stability in vivo, it is expected to achieve more efficient liver-targeted molecular delivery and drug efficacy.
  • the use of the related conjugate of the present invention exhibits good activity for reducing HBsAg and exhibits long-term HBsAg inhibition efficacy.

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Abstract

本公开涉及一种可以与化合物(如治疗性化合物)连接的新型缀合基团，其可用于将化合物导向体内的靶标。本文所公开的缀合基团可以将表达抑制性寡聚核苷酸(如RNAi试剂)靶向肝脏细胞，以调节基因表达。本文所公开的缀合基团当与表达抑制性寡聚核苷酸连接时可以用于各种用途，包括用于治疗、诊断、靶标验证和基因组开发用途。包括本文所公开的缀合基团的组合物当与表达抑制性寡聚核苷酸连接时能够介导肝脏细胞(如肝细胞)中靶核酸序列的表达，这可以用于治疗对细胞、组织或生物体基因表达或活性响应的疾病或病症。

Description

缀合基团及其缀合物
本申请主张如下优先权：
CN202010522407.6，申请日2020年06月10日；
CN202011524307.3，申请日2020年12月21日。
技术领域
本公开涉及一种新型的缀合基团及其用途。本文所公开的缀合基团可以与化合物(如治疗剂)连接，以将化合物导向体内的靶标。
背景技术
许多化合物需要被递送至特定位置(例如，期望的细胞)以具有治疗作用或可用于诊断目的，特别是当试图在体内递送治疗性化合物的情况下。此外，向特定位置高效递送化合物的能力可以限制或潜在地消除可能是通过给予化合物而导致的不希望的后果(如脱靶作用)。促进诸如治疗剂的化合物递送至体内所需位置的一种方法是通过将化合物与缀合基团连接或连附。
可以使用缀合基团靶向的一类治疗剂是寡聚核苷酸。已经显示包括与靶核酸至少部分互补的核苷酸序列的寡聚核苷酸将改变体外和体内靶标的功能和活性。已经显示当递送至含有靶核酸(如mRNA)的细胞时，寡聚核苷酸将调节靶标的表达，导致靶核酸转录或翻译的改变。在某些示例中，通过抑制核酸靶标和/或触发靶核酸降解，寡聚核苷酸可以减少基因表达。
如果靶核酸是mRNA，则表达抑制性寡聚核苷酸可以调节mRNA靶标表达的一种机制是通过RNA干扰。RNA干扰是使RNA或RNA样分子(诸如化学修饰的RNA分子)通过降解能使基因表达沉默的生物过程。该转录后基因沉默过程被认为是防止外来基因表达的进化保守型细胞保护机制。
合成的RNA和RNA样分子已经显示将引发体内RNA干扰。例如，Elbashir等.(Nature2000，411，494-98)描述了通过在培养的哺乳动物细胞中导入合成的21-核苷酸RNA分子的双链体诱导的RNAi。可以触发RNAi反应机制的合成的RNA或RNA样分子的类型可以包括修饰的核苷酸和/或一种或多种非磷酸二酯连接。
Meier等(J.Mol.Biol.2000,300,857-65)报道了可以与肝脏细胞中高度表达的抗去唾液酸糖蛋白受体(ASGPR)紧密结合的乙酰胺基半乳糖(GalNAc)基团，及结合时的共晶结构。Khorev等(Bioorg.Med.Chem.2008,16,5216-31)报道了利用该基团实现荧光发色基团靶向递送至肝细胞。Prakash等(J.Med.Chem.2016,59,2718-33)和WO2009/073809分别报道了利用GalNAc运载平台递送反义核苷酸和siRNA至肝脏并实现相应的基因沉 默。因此，GalNAc这一结构单元在递送大分子至肝脏细胞中有着广阔的应用前景。
发明内容
本公开涉及一种可以与化合物(如治疗剂)连接的新型缀合基团，其可用于将化合物导向体内的靶标。本文所公开的缀合基团可以将表达抑制性寡聚核苷酸(如RNAi试剂)靶向肝脏细胞，以调节基因表达。
本文所公开的缀合基团当与表达抑制性寡聚核苷酸连接时可以用于各种用途，包括用于治疗、诊断、靶标验证和基因组开发用途。包括本文所公开的缀合基团的组合物当与表达抑制性寡聚核苷酸连接时能够介导肝脏细胞(如肝细胞)中靶核酸序列的表达，这可以用于治疗对细胞、组织或生物体基因表达或活性响应的疾病或病症。
因此，在第一方面，本公开提供了一种缀合基团，其具有式(I)的结构
Figure PCTCN2021099405-appb-000001
其中n选自8～12的整数。
在第一方面的一些实施方案中，缀合基团可以具有以下结构：
Figure PCTCN2021099405-appb-000002
在第二方面，本公开提供了一种缀合物，其包含根据本公开的第一方面的缀合基团和与缀合基团连接的治疗剂。
在第二方面的一些实施方案中，上述缀合物中的治疗剂是表达抑制性寡聚核苷酸。
在第二方面的一些实施方案中，上述缀合物中的表达抑制性寡聚核苷酸为RNAi试剂。
在第二方面的一些实施方案中，上述缀合物中的RNAi试剂包括一个或多个修饰的核苷酸。
在第二方面的一些实施方案中，上述缀合物中的RNAi试剂为包含正义链和反义链的双链siRNA。
在第二方面的一些实施方案中，上述缀合物中的双链siRNA在其正义链的5’末端与缀合基团连接。
在第二方面的一些实施方案中，上述缀合物中的表达抑制性寡聚核苷酸经由磷酸酯基团、硫代磷酸酯基团或膦酸酯基团与缀合基团连接。
在第二方面的一些实施方案中，上述缀合物或表达抑制性寡聚核苷酸的硫代磷酸酯部分包括(R)-和(S)-对映体、非对映异构体、和/或其外消旋混合物。
在第二方面的一些实施方案中，上述本公开还提供了缀合物的盐。
在第二方面的一些实施方案中，如上所述的盐选自碱加成盐、酸加成盐及其组合。
在第二方面的一些实施方案中，上述碱加成盐选自钠、钾、钙、铵、有机胺、镁盐及其组合，酸加成盐选自无机酸盐、有机酸盐及其组合。
在第二方面的一些实施方案中，上述无机酸选自盐酸、氢溴酸、硝酸、碳酸、碳酸氢 根、磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸及其组合，有机酸选自乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸、甲磺酸及其组合。
在第三方面，本公开提供了一种化合物，其具有式(II)或式(III)的结构
Figure PCTCN2021099405-appb-000003
其中m选自8～12的整数。
在第三方面的一些实施方案中，化合物可以具有以下结构
Figure PCTCN2021099405-appb-000004
Figure PCTCN2021099405-appb-000005
在第四方面，本公开提供了药物组合物，其包含根据本公开的第二方面的缀合物和药学上可接受的载体或赋形剂。
在第五方面，本公开提供了用于在有需要的受试者中抑制靶核酸表达的方法，其包括向受试者施用根据本公开的第二方面的缀合物或根据本公开的第四方面的药物组合物的步骤。
在第五方面的一些实施方案中，方法中的靶核酸是来自病毒的核酸。所述病毒可以是例如引起肝病的病毒，例如乙型肝炎病毒。
在第六方面，本公开提供了治疗疾病的方法，其包括对受试者施用根据本公开的第二方面的缀合物或根据本公开的第四方面的药物组合物的步骤。
在第七方面，本公开提供了根据本公开的第二方面的缀合物或根据本公开的第四方面的药物组合物在制备用于治疗疾病的药物中的用途。
在第八方面，本公开提供了根据本公开的第二方面的缀合物或根据本公开的第四方面的药物组合物，其用于治疗疾病。
在第七和第八方面的一些实施方案中，所述疾病是病毒感染。
在第七和第八方面的一些实施方案中，所述疾病是肝病。
在第七和第八方面的一些实施方案中，所述疾病是乙型肝炎。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照本领域普通技术人员所理解的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
本公开所述的缀合基团可以增强治疗剂向诸如人或动物的对象内特定靶位置(例如，特定器官或组织)的递送。在本公开的一些实施方式中，所述缀合基团可以增强表达抑制性寡聚核苷酸的靶向递送。在本公开的一些实施方式中，缀合基团可以增强表达抑制性寡聚核苷酸向肝脏的递送。
本公开所述的缀合基团可以直接或间接地连接至化合物，诸如治疗剂，例如，表达抑制性寡聚核苷酸，例如，表达抑制性寡聚核苷酸的3′或5′末端。在本公开的一些实施方式中，表达抑制性寡聚核苷酸包括一个或多个修饰的核苷酸。在本公开的一些实施方式中，表达抑制性寡聚核苷酸是RNAi试剂，诸如包含正义链和反义链的双链RNAi试剂。在本公开的一些实施方式中，本文所公开的缀合基团连接至双链RNAi试剂的正义链的5′末端。在一些实施方式中，本文所公开的缀合基团经由磷酸酯、硫代磷酸酯或膦酸酯基团在双链RNAi试剂正义链5′末端与表达抑制性寡聚核苷酸试剂连接。
本公开所述术语“连接”，当表示两个分子之间的联系时，指两个分子通过共价键连接或者两个分子经由非共价键(例如，氢键或离子键)关联。
本公开所述“寡聚核苷酸”是含有10～50个核苷酸或核苷酸碱基对的核苷酸序列。在本公开的一些实施方式中，寡聚核苷酸具有这样的核碱基序列，其与细胞内表达的靶核酸或靶基因中的编码序列至少部分互补。所述核苷酸可以任选被修饰。在本公开一些实施方式中，在将寡聚核苷酸递送至表达基因的细胞后，寡聚核苷酸能够抑制潜在基因的表达，并且在本公开中被称为“表达抑制性寡聚核苷酸”，其可以体外或体内抑制基因表达。“寡聚核苷酸”包括但不限于：单链寡核苷酸，单链反义寡核苷酸，短干扰RNA(siRNA)，双链RNA(dsRNA)，微RNA(miRNA)，短发夹RNA(shRNA)，核糖酶，干扰RNA分子，和Dicer酶底物。
本公开所述“RNAi试剂”指含有能够以序列特异性方式降解或抑制靶mRNA的信使RNA(mRNA)转录本翻译的RNA或RNA样(如化学修饰的RNA)寡核苷酸分子的试剂。本公开所述RNAi试剂可以通过RNA干扰机制(即，通过与哺乳动物细胞的RNA干扰通路组成部分(RNA诱导的沉默复合物或RISC)相互作用诱导RNA干扰)操纵，或通过任何其它机制或途径起作用。尽管本公开所述RNAi试剂主要通过RNA干扰机制操纵，但是公 开的RNAi试剂并不受限于或受约束于任何特定作用途径或机制。RNAi试剂包括但不限于：单链寡核苷酸，单链反义寡核苷酸，短干扰RNA(siRNA)，双链RNA(dsRNA)，微RNA(miRNA)，短发夹RNA(shRNA)，和Dicer底物。本公开所述的RNAi试剂包括寡核苷酸，所述寡核苷酸具有与靶向的mRNA至少部分互补的链。在本公开的一些实施方式中，本文所述RNAi试剂是双链的，并且包括反义链以及与反义链至少部分互补的正义链。RNAi试剂可以包括修饰的核苷酸和/或一个或多个非磷酸二酯连接。在一些实施方式中，本文所述的RNAi试剂是单链的。
本公开所述术语“单链寡核苷酸”指具有与靶mRNA至少部分互补的序列的单链寡聚核苷酸，其能够通过氢键在哺乳动物生理条件(或相当的体外环境)下与靶mRNA杂交。在本公开的一些实施方式中，单链寡核苷酸是单链反义寡核苷酸。
本公开所述短干扰RNA(siRNA)是一类RNA分子，长度为20～25个碱基对，类似于miRNA，并且在RNA干扰(RNAi)途径内操作，它干扰了与核苷酸序列互补的特定基因的mRNA的翻译，导致mRNA降解。本公开所述短干扰RNA(siRNA)包括双链siRNA(包括正义链和反义链)和单链siRNA(仅反义链)。
本公开所述“沉默”、“降低”、“抑制”、“下调”或“敲减”，当指代表达给定基因时，表示与还没有经这样处理的第二细胞、细胞群或组织相比，当用与本文所述缀合基团连接的寡聚核苷酸处理该细胞、细胞群、或组织时基因表达降低，如由从基因转录的RNA的水平或从转录基因的细胞、细胞群、组织或对象中mRNA翻译的多肽、蛋白质或蛋白质亚基的水平测量。
本公开所述“序列”或“核苷酸序列”表示使用标准核苷酸命名的一序列字母描述的核碱基或核苷酸的次序或顺序物。
本公开所述HBV基因是指DNA序列如Genbank注册号NC_003977.1所示的基因。如Genbank注册号NC_003977.1所示的基因是HBV的完整基因组。
在一些实施方案中，双链siRNA类似物可以靶向HBV的X开放阅读框(X opening reading frame，X ORF)。
在另外的实施方案中，双链siRNA类似物可以靶向HBV的S ORF。
在另外的实施方案中，双链siRNA类似物可以靶向HBV的P ORF。
本公开中所述核苷酸的“修饰”包括但不限于甲氧基修饰、氟代修饰、硫代磷酸酯基连接等。本公开所述的序列可以包括如下表1中“进一步修饰的序列”所列。
在本公开中，如无特别说明，大写字母C、G、U、A表示核苷酸的碱基组成。小写字母c、g、u、a分别表示其相应大写字母所代表的核苷酸被甲氧基修饰； 下划线表示大写字 母代表的核苷酸被氟代修饰；间隔号“·”表示与间隔号“·”左右相邻的两个核苷酸残基之间为硫代磷酸酯基连接。例如，“a·g”表示a和g残基之间通过硫代磷酸酯基连接。
本公开所述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，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。
在本公开中，“互补”具有本领域技术人员周知的含义，即，在双链核酸分子中，一条链的碱基与另一条链上的碱基以互补的方式相配对。嘌呤碱基腺嘌呤(A)始终与嘧啶碱基尿嘧啶(U)相配对；嘌呤碱基鸟嘌呤(C)始终与嘧啶碱基胞嘧啶(G)相配对。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的尿嘧啶配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此相互补的，以及从其互补链的序列中可以推断出该链的序列。
本公开的化合物可以存在特定的几何或立体异构体形式。本公开设想所有的这类化合物，包括(R)-和(S)-对映体、非对映异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本公开的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本公开的范围之内。
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，用楔形实线键
Figure PCTCN2021099405-appb-000006
和楔形虚线键
Figure PCTCN2021099405-appb-000007
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2021099405-appb-000008
和直形虚线键
Figure PCTCN2021099405-appb-000009
表示立体中心的相对构型，用波浪线
Figure PCTCN2021099405-appb-000010
表示楔形实线键
Figure PCTCN2021099405-appb-000011
或楔形虚线键
Figure PCTCN2021099405-appb-000012
或用波浪线
Figure PCTCN2021099405-appb-000013
表示直形实线键
Figure PCTCN2021099405-appb-000014
和/或直形虚线键
Figure PCTCN2021099405-appb-000015
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本公开某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。本公开的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本公开的化合物的所有同位素组成的变换，无论放射性与否，都包括在本公开的范围之内。
术语“盐”是指本公开化合物的盐，由本公开发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本公开的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本公开的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸、碳酸氢根、磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本公开的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本公开的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
本公开的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本公开的实施例。
本公开所使用的溶剂可经市售获得。
如无特殊说明，本公开柱层析、制备薄层硅胶色谱所用溶剂配比均为体积比。
缩略词清单
Ac 乙酰基
Boc 叔丁氧羰基
DMSO 二甲亚砜
DMT/DMTr 4,4’-二甲氧基三苯基甲基
dsRNA 双链核糖核酸
EC 50 半最大效应浓度
EDTA 乙二胺四乙酸二钠
i-Pr 异丙基
Me 甲基
Ms 甲烷磺酰基
Ph 苯基
p-HPLC 制备高效液相色谱，用于化合物的纯化
RNA 核糖核酸
RNAi 核糖核酸干扰技术
siRNA 小干扰核糖核酸
t-Bu 叔丁基
Tris 三羟甲基氨基甲烷
化合物依据本领域常规命名原则或者使用
Figure PCTCN2021099405-appb-000016
软件命名，市售化合物采用供应商目录名称。
具体实施方式
下面通过实施例对本公开进行详细描述，但并不意味着对本公开任何不利限制。本公开的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本公开的实施例。对本领域的技术人员而言，在不脱离本公开精神和范围的情况下针对本公开具体实施方式进行各种变化和改进将是显而易见的。
实施例1：D01的合成
Figure PCTCN2021099405-appb-000017
Figure PCTCN2021099405-appb-000018
Figure PCTCN2021099405-appb-000019
步骤A：11-十二炔-1-醇(25克，137.14毫摩尔)和三乙胺(16.65克，164.56毫摩尔)溶于二氯甲烷(250毫升)于0摄氏度加入甲烷磺酰氯(18.85克，164.56毫摩尔)。混合液在0摄氏度搅拌2小时。反应液用水(400毫升)稀释，二氯甲烷800毫升(400毫升×2)萃取。合并的有机相用水400毫升(200毫升×2)和饱和食盐水洗涤，无水硫酸钠干燥，过滤后减压浓缩得2-2。
步骤B：式2-3(20克，67.26毫摩尔)所示化合物溶于N，N-二甲基甲酰胺(200毫升)于0摄氏度加入氢化钠(60％纯度，4.04克，100.89毫摩尔)，接着加入式2-2(19.27克，73.99毫摩尔)所示化合物。混合液在25摄氏度搅拌16小时。反应液用水(1升)淬灭，并用二氯甲烷1.6升(800毫升×2)萃取。合并的有机相用饱和食盐水800毫升(800毫升×1)洗涤，无水硫酸钠干燥，过滤后减压浓缩得2-4。 1H NMR(400MHz，DMSO-d 6)：δ7.63-6.89(m，10H)，5.64-5.52(m，2H)，4.27-4.01(m，2H)，3.98-3.77(m，2H)，3.72-3.18(m，4H)，2.23-2.14(m，2H)，1.98-1.92(m，1H)，1.54-1.23(m，16H)。
步骤C：式2-4(48克，103.98毫摩尔)所示化合物溶于甲醇(870毫升)加入氯化氢甲醇溶液(4摩尔每升，400毫升，1.6摩尔)。混合液在30摄氏度搅拌2小时。向反应液加入氯化氢甲醇溶液(4摩尔每升，350毫升，1.4摩尔)。混合液在30摄氏度搅拌16小时。反应液减压浓缩，加入三氯甲烷200毫升(100毫升×2)减压浓缩直至出现白色固体。加入甲苯(130毫升)和石油醚(130毫升)，混合液在15摄氏度搅拌16小时。反应液经过布氏漏斗过滤，收集滤饼经真空干燥得白色固体。白色固体溶于二氯甲烷(50毫升)，加入氢氧化钠(6.59克，164.66毫摩尔)的水溶液(50毫升)，在20摄氏度搅拌1小时。 反应液用水(500毫升)稀释，并用二氯甲烷1升(500毫升×2)萃取。合并的有机相用无水硫酸钠干燥，过滤后减压浓缩得2-5。
步骤D：向式2-5(23克，80.58毫摩尔)所示化合物和氢氧化钠(322.31毫克，8.06毫摩尔)于二甲亚砜(70毫升)和水(6毫升)的混合液中加入丙烯酸叔丁酯(22.72克，177.28毫摩尔)，在氮气保护下于25摄氏度搅拌16小时。反应液用水(500毫升)稀释，并用乙酸乙酯1升(500毫升×2)萃取。合并的有机相用无水硫酸钠干燥，过滤后减压浓缩得粗品。经柱层析(SiO 2，石油醚/乙酸乙酯/乙醇(含0.1％氨水)＝36/3/1至16/3/1)纯化后得2-6。 1H NMR(400MHz，DMSO-d 6)：δ3.60-3.54(m，4H)，3.32(br s，5H)，3.15(s，5H)，2.74-2.66(m，1H)，2.40(t，J＝6.0Hz，4H)，2.18-2.11(m，2H)，1.58-1.38(m，22H)，1.34-1.23(m，12H)。
步骤E：向式2-6(24.5克，45.22毫摩尔)所示化合物的二氯甲烷(250毫升)溶液中加入三乙胺(9.15克，90.45毫摩尔)和丁二酸酐(6.79克，67.83毫摩尔)，于20摄氏度搅拌16小时。反应液加入二氯甲烷(1升)和盐酸(1摩尔每升，1升)，分液后的有机相用无水硫酸钠干燥，过滤后减压浓缩得2-7。 1H NMR(400MHz，CDCl 3)：δ6.49-6.37(m，1H)，3.72(s，2H)，3.70-3.57(m，8H)，3.37(t，J＝6.7Hz，2H)，2.69-2.51(m，4H)，2.50-2.36(m，4H)，2.22-2.13(m，2H)，1.96-1.90(m，1H)，1.57-1.47(m，4H)，1.46-1.40(m，18H)，1.40-1.31(m，2H)，1.30-1.21(m，10H)。
步骤F：式2-7(27.4克，42.69毫摩尔)所示化合物溶于甲酸(140毫升)，混合液在氮气保护下于20摄氏度搅拌16小时。反应液减压浓缩，加入甲苯300毫升(150毫升×2)减压浓缩得2-8。 1H NMR(400MHz，CDCl 3)：δ9.79-9.22(m，3H)，6.44-6.23(m，1H)，3.88-3.43(m，10H)，3.39-3.20(m，2H)，2.77-2.31(m，8H)，2.15-2.06(m，2H)，1.87(t，J＝2.6Hz，1H)，1.48-1.28(m，6H)，1.26-1.12(m，10H)。
步骤G：式2-8(22.6克，42.67毫摩尔)所示化合物，N，N-二异丙基乙胺(33.09克，256.03毫摩尔)和O-(7-氮杂苯并三唑-1-基)-N,N,N,N-四甲基脲六氟磷酸盐(51.92克，136.55毫摩尔)溶于N，N-二甲基甲酰胺(250毫升)加入N-(3-氨基丙基)氨基甲酸叔丁酯(29.74克，170.69毫摩尔)。混合液在20摄氏度搅拌16小时。反应液加入二氯甲烷(1升)和盐酸(1摩尔每升，1升)，分液后的有机相用水1升(1升×1)、碳酸氢钠水溶液1升(1升×1)和饱和食盐水1升(1升×1)依次洗涤，无水硫酸钠干燥，过滤后减压浓缩得粗品。经柱层析(SiO 2，石油醚/乙酸乙酯/乙醇＝40/3/1至10/3/1)纯化后得2-9。 1H NMR(400MHz，CDCl 3)：δ7.22-6.79(m，3H)，6.77-6.44(m，1H)，5.45-5.00(m，3H)，3.86-3.73(m，2H)，3.72-3.63(m，4H)，3.62-3.45(m，4H)，3.41-3.32(m，2H)，3.32- 3.20(m，6H)，3.19-3.03(m，6H)，2.56-2.47(m，4H)，2.47-2.39(m，4H)，2.21-2.12(m，2H)，1.95-1.90(m，1H)，1.70-1.57(m，6H)，1.56-1.47(m，4H)，1.46-1.38(m，29H)，1.30-1.25(m，10H)。
步骤H：式2-9(15克，15.03毫摩尔)所示化合物溶于二氯甲烷(114毫升)加入三氟乙酸(38毫升)，混合液于20摄氏度搅拌16小时。反应液减压浓缩，加入甲苯/乙腈＝3/1混合液600毫升(250毫升×3)减压浓缩得2-10。
步骤I：式2-11(22.15克，49.50毫摩尔)所示化合物，N，N-二异丙基乙胺(7.75克，60.00毫摩尔)，1-羟基-7-氮杂苯并三唑(6.12克，45.00毫摩尔)和O-(7-氮杂苯并三唑-1-基)-N,N,N,N-四甲基脲六氟磷酸盐(20.53克，54.00毫摩尔)溶于N，N-二甲基甲酰胺(90毫升)，向该混合液加入式2-10(三(三氟乙酸盐)，15.6克，15.00毫摩尔)所示化合物和N，N-二异丙基乙胺(21.32克，165.00毫摩尔)的N，N-二甲基甲酰胺(120毫升)溶液。混合液在20摄氏度搅拌16小时。反应液加入二氯甲烷(1.2升)和盐酸(1摩尔每升，1升)，分液后的有机相用水1升(1升×1)、碳酸氢钠水溶液1升(1升×1)和饱和食盐水1升(1升×1)依次洗涤，无水硫酸钠干燥，过滤后减压浓缩得粗品。经柱层析(SiO 2，二氯甲烷/甲醇＝100/1至10/1至二氯甲烷/乙醇＝1/1)纯化后得2-12。 1H NMR(400MHz，DMSO-d 6)：δ7.87-7.66(m，9H)，7.09(s，1H)，5.21(d，J＝3.4Hz，3H)，4.96(dd，J＝3.4，11.3Hz，3H)，4.48(d，J＝8.5Hz，3H)，4.06-3.98(m，9H)，3.91-3.82(m，3H)，3.74-3.66(m，3H)，3.58-3.46(m，12H)，3.31(br s，3H)，3.07-2.98(m，12H)，2.71(t，J＝2.6Hz，1H)，2.33-2.22(m，8H)，2.16-2.12(m，2H)，2.10(s，9H)，2.04(br t，J＝7.1Hz，6H)，1.99(s，9H)，1.89(s，9H)，1.81-1.74(m，9H)，1.54-1.39(m，22H)，1.32(br dd，J＝4.5，6.7Hz，2H)，1.24(s，10H)。
步骤J：式2-12(1.00克，0.50毫摩尔)所示化合物和N-甲基-N，N，N-三正辛基氯化铵(20.35毫克，50.35微摩尔)溶于乙酸(2.7毫升)和正戊烷(6.3毫升)的混合液，于0摄氏度向该混合液滴入高锰酸钾(0.40克，2.52毫摩尔)的水(9毫升)溶液。混合液在0至15摄氏度搅拌2小时。反应用亚硫酸氢钠(1.27克)淬灭，加入盐酸(2摩尔每升，5毫升)和水(30毫升)，并用三氯甲烷/异丙醇＝3/1混合液120毫升(40毫升×3)萃取。合并的有机相用无水硫酸钠干燥，过滤后减压浓缩，加入甲苯/乙腈＝1/1混合液180毫升(30毫升×6)减压浓缩得2-13。 1H NMR(400MHz，CD 3OD)：δ5.34(d，J＝2.9Hz，3H)，5.06(dd，J＝3.3，11.2Hz，3H)，4.56(d，J＝8.4Hz，3H)，4.19-4.06(m，9H)，4.04-3.98(m，3H)，3.87(td，J＝5.7，9.9Hz，4H)，3.72-3.64(m，9H)，3.57-3.50(m， 3H)，3.39(br t，J＝6.4Hz，2H)，3.22(q，J＝6.4Hz，12H)，2.51-2.40(m，9H)，2.21(br t，J＝7.3Hz，6H)，2.14(s，9H)，2.03(s，9H)，1.94(d，J＝7.9Hz，18H)，1.72-1.57(m，22H)，1.39(br s，12H)。
步骤K：向式2-13(1.00克，0.50毫摩尔)所示化合物的N，N-二甲基甲酰胺(10毫升)溶液加入N，N-二异丙基乙胺(0.26克，1.99毫摩尔)和O-(7-氮杂苯并三唑-1-基)-N,N,N,N-四甲基脲六氟磷酸盐(0.23克，0.60毫摩尔)。混合液搅拌后，加入式2-14(0.23克，0.55毫摩尔)所示化合物。混合液在15摄氏度搅拌16小时。反应液加入二氯甲烷(50毫升)和水(50毫升)，分液后的有机相用饱和碳酸氢钠水溶液50毫升(50毫升×1)、水50毫升(50毫升×1)和饱和食盐水50毫升(50毫升×1)依次洗涤，无水硫酸钠干燥，过滤后减压浓缩得粗品。经柱层析(SiO 2，二氯甲烷/甲醇(含0.1％三乙胺)＝20/1至10/1)纯化后得2-15。 1H NMR(400MHz，DMSO-d 6)：δ7.90-7.82(m，6H)，7.78(br d，J＝4.8Hz，3H)，7.40-7.26(m，10H)，6.91(br dd，J＝3.1，9.0Hz，4H)，5.26(d，J＝3.4Hz，3H)，5.03-4.99(m，3H)，4.53(d，J＝8.4Hz，3H)，4.43(br d，J＝3.8Hz，1H)，4.23-4.14(m，1H)，4.12-4.02(m，9H)，3.92(td，J＝9.0，11.0Hz，3H)，3.78(s，6H)，3.77-3.71(m，3H)，3.66-3.51(m，13H)，3.49-3.41(m，4H)，3.11-3.01(m，16H)，2.38-2.37(m，1H)，2.32(br s，9H)，2.14(s，9H)，2.08(br t，J＝6.9Hz，7H)，2.04(s，9H)，1.93(s，9H)，1.82(s，9H)，1.57-1.46(m，22H)，1.31-1.26(m，12H)。
步骤L：向式2-15(0.80克，0.33毫摩尔)所示化合物的二氯甲烷(8毫升)溶液依次加入三乙胺(67.24毫克，0.64毫摩尔)，4-N,N-二甲基氨基吡啶(0.12克，1.00毫摩尔)和丁二酸酐(83.13毫克，0.83毫摩尔)。混合液在10摄氏度搅拌16小时。反应液加入二氯甲烷(50毫升)，水(30毫升)和饱和食盐水(30毫升)，分液后的有机相用水30毫升(30毫升×1)和饱和食盐水30毫升(30毫升×1)依次洗涤，无水硫酸钠干燥，过滤后减压浓缩得粗品。经p-HPLC纯化(分离柱:Waters Xbridge C18(规格：150mm×50mm，粒径：10μm)；流动相:[水(10mM碳酸氢铵)-乙腈]；洗脱梯度：27％-57％，11min)得D01。 1H NMR(400MHz，DMSO-d 6)：δ7.96-7.69(m，9H)，7.33-7.09(m，10H)，6.90-6.78(m，4H)，5.21(d，J＝3.3Hz，3H)，4.97(dd，J＝3.3，11.2Hz，3H)，4.49(d，J＝8.4Hz，3H)，4.06-3.97(m，9H)，3.91-3.83(m，3H)，3.79-3.66(m，11H)，3.63-3.45(m，18H)，3.02(br d，J＝4.6Hz，14H)，2.46-2.37(m，4H)，2.35-2.14(m，12H)，2.10(s，9H)，2.04(br t，J＝7.0Hz，6H)，1.99(s，9H)，1.88(s，9H)，1.77(s，9H)，1.57-1.37(m，22H)，1.22(br s，12H)。
D为小分子片段D01进行化学反应后的残基，通过共价键与核酸结合，其结构如下式所示：
Figure PCTCN2021099405-appb-000020
实施例2：D02的合成
Figure PCTCN2021099405-appb-000021
Figure PCTCN2021099405-appb-000022
步骤A：化合物3-2的的制备参考实施例1化合物D01的制备，用3-1替换2-1。
步骤B：式2-3(35.70克，120.06毫摩尔)所示化合物溶于2-甲基四氢呋喃(285毫升)加入叔丁醇钾(17.51克，156.07毫摩尔)，混合液在85摄氏度搅拌2小时。接着加入式3-2(28.40克，114.34毫摩尔)所示化合物。混合液在85摄氏度搅拌12小时。反应液加入水(400毫升)，并用二氯甲烷800毫升(400毫升×2)萃取。合并的有机相用无水硫酸钠干燥，过滤后减压浓缩得3-3。
步骤C：式3-3(35克，77.84毫摩尔)所示化合物溶于甲醇(525毫升)加入氯化氢甲醇溶液(4摩尔每升，175毫升，750毫摩尔)。混合液在50摄氏度搅拌12小时。将反应液倒入碳酸钾(80克)和甲醇(500毫升)的混合液中，经过布氏漏斗过滤后减压浓缩，所得粗产品溶于甲醇(240毫升)，加入乙酸钠(12.77克，155.68毫摩尔)和羟胺盐酸盐(5.41克，77.84毫摩尔)。混合液在25摄氏度搅拌0.5小时。反应液经过布氏漏斗过滤 后减压浓缩，所得粗产品加入氢氧化钠水溶液(1摩尔每升，500毫升)，并用二氯甲烷500毫升(500毫升×1)萃取。合并的有机相用氢氧化钠水溶液(1摩尔每升，500毫升)和饱和食盐水500毫升(500毫升×1)依次洗涤，无水硫酸钠干燥，过滤后减压浓缩得3-4。 1H NMR(400MHz，CDCl 3)：δ5.81-5.66(m，1H)，4.98-4.79(m，2H)，3.50-3.37(m，4H)，3.36-3.30(m，2H)，3.24(br s，2H)，2.03-1.91(m，2H)，1.53-1.42(m，2H)，1.35-1.27(m，2H)，1.25-1.13(m，10H)。
步骤D～步骤I：分别参考实施例1化合物D01的各步骤制备。
步骤J：式3-10(20克，10.13毫摩尔)所示化合物和三水合三氯化钌(52.98毫克，202.61微摩尔)溶于二氯甲烷(60毫升)、乙腈(60毫升)和水(90毫升)的混合液，向该混合液缓慢加入高碘酸钠(10.83克，50.65毫摩尔)。混合液在25摄氏度搅拌3.5小时。反应液加入水(500毫升)，并用二氯甲烷/异丙醇＝3/1混合液1升(500毫升×2)萃取。合并的有机相用饱和亚硫酸钠水溶液150毫升(150毫升×1)洗涤，减压浓缩。粗产品加入饱和碳酸氢钠水溶液(500毫升)，用二氯甲烷1升(500毫升×2)洗涤，加入盐酸(1摩尔每升)至pH值等于3，并用二氯甲烷/异丙醇＝3/1混合液1.5升(500毫升×3)萃取。合并的有机相用饱和食盐水500毫升(500毫升×1)洗涤，无水硫酸钠干燥，过滤后减压浓缩得3-11。 1H NMR(400MHz，CD 3OD)：δ5.37-5.29(m,3H),5.06(dd,J＝3.4,11.3Hz,3H),4.61-4.51(m,3H),4.20-3.96(m,12H),3.92-3.82(m,3H),3.70-3.63(m,9H),3.57-3.49(m,3H),3.43-3.36(m,2H),3.28-3.14(m,12H),2.50-2.38(m,8H),2.33-2.24(m,3H),2.24-2.17(m,6H),2.17-2.11(m,9H),2.03-1.99(m,9H),1.98-1.90(m,18H),1.75-1.51(m,22H),1.38-1.27(m,10H)。
步骤K：向式3-11(11克，5.52毫摩尔)所示化合物的二氯甲烷(110毫升)溶液加入N，N-二异丙基乙胺(0.71克，5.52毫摩尔)、1-羟基-7-氮杂苯并三唑(1.50克，11.04毫摩尔)、O-(7-氮杂苯并三唑-1-基)-N,N,N,N-四甲基脲六氟磷酸盐(2.52克，6.63毫摩尔)和式2-14(2.43克，5.80毫摩尔)所示化合物。混合液搅拌后，加入N，N-二异丙基乙胺(2.14克，16.56毫摩尔)。混合液在25摄氏度搅拌12小时。反应液加入二氯甲烷(1升)和水(500毫升)，分液后的有机相用水500毫升(500毫升×1)和饱和食盐水500毫升(500毫升×1)依次洗涤，无水硫酸钠干燥，过滤后减压浓缩得粗品。经柱层析(SiO 2，二氯甲烷/甲醇(含0.5％三乙胺)＝30/1至10/1)纯化后得3-12。 1H NMR(400MHz，DMSO-d 6)：δ7.92-7.73(m,9H),7.38-7.16(m,10H),6.94-6.83(m,4H),5.22(d,J＝3.4Hz,3H),4.97(dd,J＝3.4,11.3Hz,3H),4.53-4.44(m,3H),4.44-4.35(m,1H),4.21-4.09(m,1H),4.03(s,8H),3.94-3.83(m,3H),3.77-3.68(m,9H),3.58-3.48(m,10H),3.43(br s,3H),3.36-3.28(m,4H),3.03(br s,14H),2.33-2.19(m,10H),2.11(s,9H),2.04(br t,J＝6.9Hz,7H),2.00(s, 9H),1.89(s,9H),1.81-1.74(m,9H),1.57-1.38(m,22H),1.37-1.17(m,10H)。
步骤L：参考实施例1化合物D01的制备得到D02。 1H NMR(400MHz,DMSO-d 6)δ＝7.94-7.70(m,9H),7.40-7.08(m,10H),6.95-6.79(m,4H),5.27-5.17(m,3H),4.98(dd,J＝3.3,11.2Hz,3H),4.50(d,J＝8.4Hz,3H),4.08-3.98(m,9H),3.92-3.85(m,3H),3.78-3.67(m,11H),3.59-3.49(m,12H),3.46-3.36(m,6H),2.95(br s,14H),2.48-2.38(m,4H),2.16(br s,12H),2.11(s,9H),2.05(br t,J＝6.8Hz,6H),2.00(s,9H),1.89(s,9H),1.83-1.72(m,9H),1.57-1.36(m,22H),1.31-1.12(m,10H).
实施例3：D-01-M的合成
Figure PCTCN2021099405-appb-000023
步骤A：向D-01(100毫克，39.88微摩尔)的乙腈(0.7毫升)溶液加入氨水(30％，1.4毫升)。混合液在50摄氏度搅拌12小时。反应液过滤后经p-HPLC纯化(分离柱:Phenomenex Gemini-NX C18(规格：75mm×30mm，粒径：3μm)；流动相:[水(0.05％氨水)-乙腈]；洗脱梯度：14％-42％，7min)得D-01-M。 1H NMR(400MHz，DMSO-d 6)：δ7.90-7.57(m，9H)，7.38-7.10(m，10H)，6.93-6.80(m，4H)，4.67-4.52(m，6H)，4.52-4.44(m，3H)，4.22-4.19(m，3H)，3.77-3.62(m，15H)，3.58-3.47(m，16H)，3.46-3.43(m，2H)，3.32-3.25(m，6H)，3.12-2.91(m，14H)，2.36-2.16(m，10H)，2.11-1.99(m，7H)，1.85-1.73(m，9H)，1.59-1.35(m，22H)，1.32-1.13(m，12H)。
实验例1 测试化合物与人源抗去唾液酸糖蛋白受体结合力
1.实验目的：
通过表面等离子共振技术(SPR)检测化合物与人源抗去唾液酸糖蛋白受体(ASGPR)结合力，以化合物的动力学K D值为指标，来评价化合物与ASGPR结合力强弱，从而反映该化合物特异性靶向肝细胞递送核酸分子的能力。
2.实验材料:
2.1蛋白：
Asialoglycoprotein Receptor Protein,Mouse,Recombinant(His Tag)，Sino biological-50083-M07H-50μg；Asialoglycoprotein Receptor Protein,Human,Recombinant(His Tag)，Sino biological-10773-H07H-50μg
2.2试剂：
NiHC 1500M Chip(Xan Tec-SCNihc1500m0720)；HEPES(SIGMA-V900477)；NaCl(SIGMA-71376)；Tween-20(Aladin-T104863)；CaCl 2(SIGMA-C3306-250G)；EDTA(SIGMA-3609)；NiCl 2(Energy-chemical-V830089)；10×PBS(Sangon-E607016-0500)
2.3耗材与仪器：
Biacore 8k；Series S CM5chip(GE Healthcare–BR100530)96孔板-250μL(Greiner-650201)；384孔板-200μL(Greiner-781270)；96孔板-1mL(Greiner-780201)；96 Microplate foils(GE Healthcare-28975816)；384 Microplate foils(GE Healthcare-BR100577)
3.实验步骤和方法：
3.1将两种蛋白分别用1×PBS溶解至0.25mg/mL，将待测化合物用DMSO溶解，准备running buffer(10mM HEPES,150mM NaCl,0.05％Tween 20,50mM CaCl 2,50μM EDTA)并用0.22μm的膜过滤。
3.2将NiHC 1500M Chip芯片dock进biacore,将系统切换到running buffer中。
3.3蛋白标记：芯片首先用350mM EDTA处理5min,30μl/min，running buffer冲洗2min，再用40mM NiCl 2结合2min,30μl/min,然后将用running buffer稀释成5μg/mL的蛋白标记到芯片上，流速为10μl/min，最终标记量为1500-2000RU
3.4仪器系统切换到含2％DMSO的running buffer中。
3.5化合物用含2％DMSO的running buffer 2倍梯度稀释9个浓度，起始浓度为2μM，DMSO的终浓度为2％.
3.6测试：每个cycle结合60s，解离180s,流速为50μl/min,化合物浓度从低到高开始测试，且化合物梯度测试前加4个cycle的空白(2％DMSO的running buffer)，并用1.5％–3.5％的DMSO(6个浓度)进行溶剂矫正。
3.7数据分析：
数据用Biacore Insight Evaluation Software进行分析，kinetics用1:1model进行分析，affinity用steady state affinity model分析。
样品 K D(mol/L) K a(L/(mol·s)) K d(1/s)
D-01-M 1.56×10 -8 1.08×10 6 1.68×10 -2
实验结论：
在本实验中，受试物D-01-M在SPR实验中表现出良好的结合力。
本发明展现出一种高效的具有高度肝细胞靶向性的寡聚核酸分子递送平台：它可以与肝细胞表面特异的、高度表达的ASGPR蛋白结合，通过细胞内吞作用进入细胞内吞体并释放至胞浆中起效。该递送平台与ASGPR的结合力常数优于现有技术。体内展现出良好的组织分布与代谢稳定性，有望实现更高效的肝靶向分子递送和药效。运用本发明的相关缀合物对于降低HBsAg展现出良好的的活性，并展现出长期抑制HBsAg效力。

Claims (20)

  1. 一种缀合基团，其具有式(I)的结构
    Figure PCTCN2021099405-appb-100001
    其中n选自8～12的整数。
  2. 根据权利要求1所述的缀合基团，其具有以下结构：
    Figure PCTCN2021099405-appb-100002
  3. 缀合物，其包含根据权利要求1或2所述的缀合基团和与所述缀合基团连接的治疗剂。
  4. 根据权利要求3所述的缀合物，其中所述治疗剂是表达抑制性寡聚核苷酸。
  5. 根据权利要求4所述的缀合物，其中所述表达抑制性寡聚核苷酸为RNAi试剂。
  6. 根据权利要求5所述的缀合物，其中所述RNAi试剂包括一个或多个修饰的核苷酸。
  7. 根据权利要求5或6所述的缀合物，其中所述RNAi试剂为包含正义链和反义链的双链siRNA。
  8. 根据权利要求7所述的缀合物，其中所述双链siRNA在其正义链的5’末端与所述缀合基团连接。
  9. 根据权利要求4-8中任一项所述的缀合物，其中所述表达抑制性寡聚核苷酸经由磷酸酯基团、硫代磷酸酯基团或膦酸酯基团与缀合基团连接。
  10. 一种化合物，其具有式(II)或式(III)的结构
    Figure PCTCN2021099405-appb-100003
    Figure PCTCN2021099405-appb-100004
    其中m分别独立地选自8～12的整数。
  11. 根据权利要求10所述的化合物，其具有以下结构
    Figure PCTCN2021099405-appb-100005
    Figure PCTCN2021099405-appb-100006
  12. 药物组合物，其包含根据权利要求3-9中任一项所述的缀合物和药学上可接受的载体或赋形剂。
  13. 用于在有需要的受试者中抑制靶核酸表达的方法，所述方法包括向所述受试者施用根据权利要求3-9中任一项的缀合物或根据权利要求12的药物组合物的步骤。
  14. 根据权利要求13的方法，其中所述靶核酸是来自病毒的核酸。
  15. 根据权利要求14的方法，其中所述靶核酸是来自乙型肝炎病毒的核酸。
  16. 治疗疾病的方法，其包括对所述受试者施用根据权利要求3-9中任一项所述的缀合物或根据权利要求12的药物组合物的步骤。
  17. 根据权利要求3-9中任一项所述的缀合物或根据权利要求12的药物组合物在制备用于治疗疾病的药物中的用途。
  18. 根据权利要求16所述的方法或根据权利要求17的用途，其中所述疾病是病毒感染。
  19. 根据权利要求16所述的方法或根据权利要求17的用途，其中所述疾病是肝病。
  20. 根据权利要求16所述的方法或根据权利要求17的用途，其中所述疾病是乙型肝炎。
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