WO2024255761A1 - 包含糖的寡核苷酸递送配体 - Google Patents

包含糖的寡核苷酸递送配体 Download PDF

Info

Publication number
WO2024255761A1
WO2024255761A1 PCT/CN2024/098675 CN2024098675W WO2024255761A1 WO 2024255761 A1 WO2024255761 A1 WO 2024255761A1 CN 2024098675 W CN2024098675 W CN 2024098675W WO 2024255761 A1 WO2024255761 A1 WO 2024255761A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
formula
alkyl
compound
sugar
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2024/098675
Other languages
English (en)
French (fr)
Inventor
黄金宇
邹昊
刘俊凯
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Rona Therapeutics Co Ltd
Rona Bioscience Ltd
Original Assignee
Shanghai Rona Therapeutics Co Ltd
Rona Bioscience Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Rona Therapeutics Co Ltd, Rona Bioscience Ltd filed Critical Shanghai Rona Therapeutics Co Ltd
Priority to CN202480038604.7A priority Critical patent/CN121358748A/zh
Publication of WO2024255761A1 publication Critical patent/WO2024255761A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • 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
    • 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

Definitions

  • the present invention belongs to the field of medicine, and specifically relates to a delivery vector capable of delivering double-stranded RNA to extrahepatic tissues, such as the central nervous system or the eye.
  • the delivery vector is a sugar modified by a hydrophobic group, such as a compound of formula (X), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
  • RNA interference is a phenomenon in which double-stranded RNA (dsRNA, also known as siRNA) induces efficient and specific degradation of target mRNA.
  • dsRNA double-stranded RNA
  • siRNA siRNA
  • siRNA due to the presence of the blood-brain barrier, it is difficult to deliver siRNA to the central nervous system to exert its effect, which limits the application of siRNA.
  • Some attempts have been made in the art to deliver siRNA to the central nervous system such as WO2004094595A2, which discloses the use of a single lipid ligand (such as cholesterol or long-chain alkane) at the end of the chain to deliver siRNA, WO2019217459A1, which discloses the use of a single lipid ligand to deliver siRNA inside the chain, and WO2021092371A2, which discloses a series of new lipid ligand structures.
  • the present invention provides an oligonucleotide comprising one or more compounds of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • the present invention provides an oligonucleotide comprising one or more compounds of formula (II) or formula (III), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:
  • the present invention provides an oligonucleotide comprising one, two or more delivery vectors within the oligonucleotide, at the 5' end and/or the 3' end, wherein the delivery vector is a sugar modified with a hydrophobic group;
  • the sugar modified by the hydrophobic group is selected from the compound of formula (X), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • the present invention provides a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence that is sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and/or antisense strand comprises one or more compounds of formula (II) or formula (III) above, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.
  • the present invention provides a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and/or the antisense strand comprises one, two or more delivery vectors at the interior, 5' end and/or 3' end, and the delivery vector is a sugar modified with a hydrophobic group;
  • the sugar modified by the hydrophobic group is selected from the compound of formula (X), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • the present invention provides a compound of formula (II') or (III'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • the hydrophobic group provided by the present invention can be linked to the oligonucleotide or double-stranded RNA through the hydroxyl group or acetyl group of the sugar part in the nucleotide.
  • the present invention provides a vector comprising a nucleotide sequence encoding the aforementioned double-stranded RNA.
  • the present invention provides a cell containing the aforementioned double-stranded RNA or the aforementioned vector.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned double-stranded RNA, the aforementioned vector, or the aforementioned cell, and optionally a pharmaceutically acceptable carrier or excipient.
  • the present invention provides a kit comprising the aforementioned double-stranded RNA, the aforementioned vector, or the aforementioned cell.
  • C 1-6 alkyl includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6, C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 alkyl.
  • C 1-6 alkyl refers to a straight or branched saturated hydrocarbon group having 1 to 6 carbon atoms.
  • C 8-30 alkyl refers to a straight or branched saturated hydrocarbon group having 1 to 30 carbon atoms. In some embodiments, C 8-25 alkyl, C 10-22 alkyl, C 8-20 alkyl, C 1-10 alkyl and C 1-6 alkyl are preferred.
  • C 1-6 alkyl examples include: methyl (C 1 ), ethyl (C 2 ), n-propyl (C 3 ), isopropyl (C 3 ) , n-butyl (C 4 ), tert-butyl (C 4 ), sec-butyl (C 4 ), isobutyl (C 4 ), n-pentyl (C 5 ), 3-pentyl (C 5 ), pentyl (C 5 ), neopentyl (C 5 ), 3-methyl-2-butyl (C 5 ), tert-pentyl (C 5 ) and n-hexyl (C 6 ).
  • C 1-6 alkyl also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus).
  • the alkyl group may be optionally substituted by one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • alkyl abbreviations include: Me(-CH 3 ), Et(-CH 2 CH 3 ), iPr(-CH(CH 3 ) 2 ), nPr(-CH 2 CH 2 CH 3 ), n-Bu(-CH 2 CH 2 CH 2 CH 3 ) or i-Bu(-CH 2 CH(CH 3 ) 2 ).
  • C 2-10 alkenyl refers to a straight or branched hydrocarbon group having 2 to 10 carbon atoms and at least one carbon-carbon double bond.
  • C 8-30 alkenyl refers to a straight or branched hydrocarbon group having 8 to 30 carbon atoms and at least one carbon-carbon double bond.
  • C 10-22 alkenyl, C2-10 alkenyl, C2-6 alkenyl and C2-4 alkenyl are preferred.
  • C2-6 alkenyl examples include vinyl ( C2 ), 1-propenyl ( C3 ), 2-propenyl ( C3 ), 1-butenyl ( C4 ), 2-butenyl ( C4 ), butadienyl ( C4 ), pentenyl ( C5 ), pentadienyl ( C5 ), hexenyl ( C6 ), and the like.
  • the term " C2-6 alkenyl” also includes heteroalkenyl groups in which one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus).
  • the alkenyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents or 1 substituent.
  • C2-10 alkynyl refers to a straight or branched hydrocarbon group having 2 to 10 carbon atoms and at least one carbon-carbon triple bond.
  • C8-30 alkynyl refers to a straight or branched hydrocarbon group having 8 to 30 carbon atoms, at least one carbon-carbon triple bond and optionally one or more carbon-carbon double bonds.
  • C10-22 alkynyl, C2-10 alkynyl, C2-6 alkynyl and C2-4 alkynyl are preferred.
  • C2-6 alkynyl examples include, but are not limited to, ethynyl ( C2 ), 1-propynyl ( C3 ), 2-propynyl ( C3 ), 1-butynyl ( C4 ), 2-butynyl ( C4 ), pentynyl ( C5 ), hexynyl ( C6 ), and the like.
  • the term " C2-6 alkynyl” also includes heteroalkynyl groups, wherein one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus).
  • the alkynyl group may be optionally substituted by one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents or 1 substituent.
  • C 1-10 alkylene refers to a divalent group formed by removing another hydrogen of C 1-10 alkyl, C 2-10 alkenyl and C 2-10 alkynyl, respectively, and may be substituted or unsubstituted.
  • C 2-8 alkylene, C 3-7 alkylene, C 1-6 alkylene, C 4-6 alkylene, C 1-4 alkylene, C 2-4 alkylene and C 1-3 alkylene are preferred.
  • Unsubstituted alkylene groups include, but are not limited to, methylene ( -CH2- ) , ethylene (-CH2CH2-) , propylene ( -CH2CH2CH2- ) , butylene (-CH2CH2CH2CH2-) , pentylene ( -CH2CH2CH2CH2- ) , hexylene (-CH2CH2CH2CH2CH2- ) , and the like .
  • substituted alkylene groups for example, substituted alkylene groups with one or more alkyl(methyl) groups, include, but are not limited to, substituted methylene groups (—CH(CH 3 )—, —C(CH 3 ) 2 —), substituted ethylene groups (—CH(CH 3 )CH 2 —, —CH 2 CH(CH 3 )—, —C(CH 3 ) 2 CH 2 —, —CH 2 C(CH 3 ) 2 — ), substituted propylene groups (—CH(CH 3 )CH 2 CH 2 —, —CH 2 CH(CH 3 )CH 2 —, —CH 2 CH 2 CH(CH 3 )—, —C(CH 3 ) 2 CH 2 CH 2 —, —CH 2 C(CH 3 ) 2 CH 2 —, —CH 2 CH 2 C(CH 3 ) 2 —), and the like.
  • substituted methylene groups —CH(CH 3 )—, —C(CH 3 ) 2 —
  • Halo or "halogen” refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).
  • C 1-6 haloalkyl means that the above "C 1-6 alkyl” is substituted by one or more halogen groups.
  • C 1-4 haloalkyl is particularly preferred, more preferably C 1-2 haloalkyl.
  • Exemplary haloalkyls include, but are not limited to: -CF 3 , -CH 2 F, -CHF 2 , -CHFCH 2 F, -CH 2 CHF 2 , -CF 2 CF 3 , -CCl 3 , -CH 2 Cl, -CHCl 2 , 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like.
  • the haloalkyl group can be substituted at any available attachment point, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • C3-10 cycloalkyl refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms.
  • C4-7 cycloalkyl and C3-6 cycloalkyl are particularly preferred, more preferably C5-6 cycloalkyl.
  • Cycloalkyl also includes a ring system in which the above cycloalkyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such a case, the number of carbons continues to refer to the number of carbons in the cycloalkyl system.
  • Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl (C 3 ), cyclopropenyl (C 3 ), cyclobutyl (C 4 ), cyclobutenyl (C 4 ), cyclopentyl (C 5 ), cyclopentenyl (C 5 ), cyclohexyl (C 6 ), cyclohexenyl (C 6 ) , cyclohexadienyl (C 6 ), cycloheptyl (C 7 ), cycloheptenyl (C 7 ), cycloheptadienyl (C 7 ), cycloheptatrienyl (C 7 ), etc.
  • the cycloalkyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • 3--10 membered heterocyclyl refers to a group of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus and silicon.
  • the point of attachment may be a carbon or nitrogen atom as long as the valence permits.
  • a 4-10 membered heterocyclyl is preferred, which is a 4 to 10 membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, a 3-8 membered heterocyclyl is preferred, which is a 3 to 8 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; a 3-6 membered heterocyclyl is preferred, which is a 3 to 6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; a 4-7 membered heterocyclyl is preferred, which is a 4 to 7 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; and a 5-6 membered heterocyclyl is more preferred, which is a 5 to 6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; and
  • Heterocyclyl also includes a ring system in which the above-mentioned heterocyclyl ring is fused to one or more cycloalkyl groups, wherein the point of attachment is on the cycloalkyl ring, or a ring system in which the above-mentioned heterocyclyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring; and in such a case, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system.
  • Exemplary 3-membered heterocyclyls containing one heteroatom include, but are not limited to: aziridine, oxirane, thiorenyl.
  • Exemplary 4-membered heterocyclyls containing one heteroatom include, but are not limited to: azetidine, oxetane and thiotane.
  • Exemplary 5-membered heterocyclyls containing one heteroatom include, but are not limited to: tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione.
  • Exemplary 5-membered heterocyclyls containing two heteroatoms include, but are not limited to: dioxolanyl, oxirane, thiorenyl.
  • Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl.
  • Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl.
  • Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl.
  • Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, hexahydrotriazine (triazinanyl).
  • Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepinyl, and thianyl.
  • Exemplary 5-membered heterocyclyl groups fused to a C6 aryl ring include, but are not limited to, dihydroindolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like.
  • Exemplary 6-membered heterocyclyl groups fused to a C6 aryl ring include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.
  • the heterocyclyl group may be optionally substituted with one or more substituents, for example, substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • hydrophobic group refers broadly to any chemical group that has an affinity for lipids.
  • One way to characterize the hydrophobicity of a hydrophobic group is by the octanol-water partition coefficient logKow , where Kow is the ratio of the concentration of a chemical in the octanol phase to its concentration in the aqueous phase when the two-phase system is at equilibrium.
  • the logKow of the hydrophobic moiety is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10.
  • the hydrophobic moiety is the R group in the compound of formula I.
  • Alkyl, alkenyl, alkynyl, etc. as defined herein are optionally substituted groups.
  • each of Raa is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two Ra groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;
  • each of R cc is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R cc groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R dd groups;
  • Each of R ee is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups;
  • each of Rff is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two Rff groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 Rgg groups;
  • siRNA refers to a class of double-stranded RNA molecules that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a gene encoding a protein) that is complementary thereto.
  • a target RNA e.g., mRNA, e.g., a transcript of a gene encoding a protein
  • siRNA is typically double-stranded, comprising an antisense strand complementary to the target RNA, and a sense strand complementary to the antisense strand.
  • mRNA is also referred to herein as mRNA to be silenced.
  • a gene is also referred to as a target gene.
  • the RNA to be silenced is an endogenous gene or a pathogen gene.
  • RNA (e.g., tRNA) and viral RNA other than mRNA can also be targeted.
  • antisense oligonucleotide or ASO (Antisense Oligonucleotides) refers to a single-stranded DNA or RNA sequence consisting of 15-25 nucleotides that is paired with a target gene. It achieves the purpose of gene regulation by specifically blocking the transcription or translation process of the target gene.
  • antisense strand refers to a strand of an siRNA that includes a region that is completely, fully or substantially complementary to a target sequence.
  • sense strand refers to a strand of an siRNA that includes a region that is completely, fully or substantially complementary to a region that is the antisense strand as defined herein.
  • complementary region refers to a region on the antisense strand that is completely, fully or substantially complementary to the target mRNA sequence.
  • mispairing can be located in the interior or terminal regions of the molecule.
  • the most tolerated mispairing is located in the terminal regions, for example, within 5, 4, 3, 2 or 1 nucleotides at 5' and/or 3' ends.
  • the antisense strand portion that is most sensitive to mispairing is referred to as a "seed region".
  • seed region For example, in a siRNA comprising a 19nt chain, the 19th position (from 5' to 3') can tolerate some mispairing.
  • complementary refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions.
  • stringent conditions may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50°C or 70°C for 12-16 hours.
  • “complementary" sequences may also include or be formed entirely from non-Watson-Cricket sequences. Base pairs and/or base pairs formed from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.
  • a polynucleotide that is "at least partially complementary,” “fully complementary,” or “substantially complementary” to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest.
  • mRNA messenger RNA
  • a polynucleotide is complementary to at least a portion of a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding PCSK9.
  • Fully complementary refers to the extent to which the sense strand only needs to be complementary to the antisense strand in order to maintain the overall double-stranded characteristics of the molecule.
  • perfect complementarity is generally required, in some cases, particularly in the antisense strand, one or more, such as 6, 5, 4, 3, 2 or 1 mismatches (relative to the target mRNA) may be included, but the sense strand and the antisense strand can still maintain the overall double-stranded characteristics of the molecule.
  • Nucleoside is a compound composed of two substances, a purine base or a pyrimidine base, and ribose or deoxyribose
  • nucleotide is a compound composed of three substances, a purine base or a pyrimidine base, ribose or deoxyribose, and phosphate
  • oligonucleotide refers to a nucleic acid molecule (RNA or DNA) with a length of, for example, less than 100, 200, 300 or 400 nucleotides.
  • Base is the basic unit of synthesis of nucleosides, nucleotides and nucleic acids. It contains nitrogen and is also called “nitrogenous base”.
  • capital letters A, U, T, G and C represent the base composition of nucleotides, which are adenine, uracil, thymine, guanine and cytosine respectively.
  • the "modification" of the nucleotides described herein includes, but is not limited to, methoxy modification, fluorine modification, phosphorothioate linkage, or conventional protecting group protection, etc.
  • the fluorine-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group of the nucleotide is replaced by fluorine
  • the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.
  • Modified nucleotides herein include, but are not limited to, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, nucleotides comprising thiophosphate groups, vinyl phosphate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases comprising nucleotides, and terminal nucleotides, deoxyribonucleotides or conventional protective groups connected to cholesterol derivatives or dodecanoic acid didecylamide groups.
  • the 2'-fluoro modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine.
  • the 2'-deoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.
  • Reactive phosphorus group refers to a phosphorus-containing group contained in a nucleotide unit or a nucleotide analog unit, which can react with a hydroxyl or amine group contained in another molecule, especially in another nucleotide unit or in another nucleotide analog, by a nucleophilic attack reaction. Typically, such a reaction produces an ester-type internucleoside bond connecting the first nucleotide unit or the first nucleotide analog unit to the second nucleotide unit or the second nucleotide analog unit.
  • the reactive phosphorus group can be selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, including but not limited to: natural phosphates, thiophosphates, dithiophosphates, borane phosphates, borane thiophosphates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, thiophosphorodiesters, thiophosphorothioates, diphosphates and triphosphates, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 ).
  • Protecting group refers to any atom or group of atoms added to a molecule to prevent an existing group in the molecule from undergoing an undesirable chemical reaction.
  • a “protecting group” may be an unstable chemical moiety known in the art that is used to protect reactive groups, such as hydroxyl, amino, and thiol groups, to prevent undesirable or inappropriate reactions during chemical synthesis.
  • Protecting groups are typically used selectively and/or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group intact or available for further reactions.
  • a non-limiting list of protecting groups includes benzyl; substituted benzyl; alkylcarbonyl and alkoxycarbonyl (e.g., tert-butyloxycarbonyl (BOC), acetyl or isobutyryl); arylalkylcarbonyl and arylalkoxycarbonyl (e.g., benzyloxycarbonyl); substituted methyl ethers (e.g., methoxymethyl ether); substituted ethyl ethers; substituted benzyl ethers; tetrahydropyranyl ethers; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy]methyl or tert-butyldiphenylsilyl); esters (e.g.,
  • Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethyl acetal (BOM), ⁇ -methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), triphenyl methyl (Trt), methoxytrityl [(4-methoxyphenyl) diphenylmethyl] (MMT), dimethoxytrityl, [bis-(4-methoxyphenyl) phenylmethyl (DMT), trimethylsilyl ether (TMS), tert-butyldimethylsilyl ether (TBDMS), tri-iso-propylsilyloxymethyl ether (TOM
  • Hydro protecting group refers to a group that can protect the hydroxyl group from chemical reactions and can be removed under specific conditions to restore the hydroxyl group. It mainly includes silane type protecting groups, acyl type protecting groups or ether type protecting groups, preferably the following:
  • Trimethylsilyl TMS
  • triethylsilyl TES
  • dimethylisopropylsilyl DMIPS
  • diethylisopropylsilyl DEIPS
  • tert-butyldimethylsilyl TDMS
  • tert-butyldiphenylsilyl TIPS
  • TIPS Trimethylsilyl
  • TES triethylsilyl
  • DMIPS dimethylisopropylsilyl
  • DEIPS diethylisopropylsilyl
  • TDMS tert-butyldimethylsilyl
  • TDPS tert-butyldiphenylsilyl
  • TIPS triisopropylsilyl
  • acetyl Ac
  • chloroacetyl dichloroacetyl
  • trichloroacetyl THF
  • benzoyl p-methoxybenzoyl, 9-fluorenylmethoxycarbony
  • pharmaceutically acceptable salt refers to those carboxylates, amino acid addition salts of the compounds of the present invention which are suitable for use in contact with patient tissues within the scope of sound medical judgment, do not produce undue toxicity, irritation, allergic response, etc., are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the present invention.
  • the present invention includes tautomers, which are functional isomers produced by rapid movement of an atom in two positions in a molecule.
  • tautomers which are functional isomers produced by rapid movement of an atom in two positions in a molecule.
  • Compounds that exist in different tautomeric forms are not limited to any specific tautomer, but are intended to cover all tautomeric forms.
  • the compounds of the present invention may include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, for example, enantiomers and/or diastereoisomeric forms.
  • the compounds of the present invention may be individual enantiomers, diastereomers or geometric isomers (e.g., cis and trans isomers), or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers.
  • Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.
  • HPLC high pressure liquid chromatography
  • the present invention also includes isotope-labeled compounds (isotope variants), which are equivalent to those described in formula (I), but one or more atoms are replaced by atoms whose atomic mass or mass number is different from the atomic mass or mass number commonly found in nature.
  • isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 Cl, respectively.
  • isotopically labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by replacing non-isotopically labeled reagents with readily available isotopically labeled reagents when carrying out the processes disclosed in the following schemes and/or the Examples and Preparations.
  • the present invention specifically relates to an oligonucleotide comprising one or more compounds of formula (I), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:
  • formula (I) represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide
  • X 1 is selected from H, Rs or
  • X 2 is selected from OR 1 or
  • R 1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide
  • Each L 1 and L 2 is independently selected from a chemical bond, a C 1-10 alkylene group, a C 2-10 alkenylene group, a C 2-10 alkynylene group, an OC 1-10 alkylene group, an OC 1-10 alkenylene group, an OC 1-10 alkynylene group, a C(O)C 1-10 alkylene group; said L 1 and L 2 are optionally substituted by 1, 2, 3, 4, 5, 6, 7 or 8 R;
  • T is selected from a chemical bond, -O-, -CH2- , -C(O)-, -OC(O)-, -M-, -OM-, -CH2 - M-, -C(O)-M-, or -OC(O)-M-;
  • A is a sugar, which is preferably a sugar that can be cleaved in an inclusion body or a lysosome, preferably a penta-sugar or a hexa-sugar, more preferably a hexa-sugar, such as N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably N-acetylgalactosamine or N-acetylglucosamine;
  • P is a hydrophobic group, preferably a C 8-30 alkyl, C 8-30 alkenyl or C 8-30 alkynyl group, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH 2 CH 2 - group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be connected to form a saturated or unsaturated ring; the C 8-30 alkyl, C 8-30 alkenyl or C 8-30 alkynyl group is optionally substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R;
  • R is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl, which is optionally deuterated up to fully deuterated;
  • Rs is selected from H, D, halogen, C1-6 alkyl, C1-6 haloalkyl, 3-10 membered heterocyclyl or C3-10 cycloalkyl, which is optionally deuterated until fully deuterated;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • k 0, 1, 2, 3, 4, 5 or 6.
  • the present invention specifically relates to an oligonucleotide, wherein the compound of formula (I) is selected from a compound of formula (II) or formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • R s ′ is selected from H, D, halogen, C 1-6 alkyl, C 1-6 haloalkyl, 3-10 membered heterocyclyl or C 3-10 cycloalkyl, which is optionally deuterated until fully deuterated;
  • the present invention specifically relates to a compound of formula (II') or (III'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • R 1 and R 2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;
  • A is an acetylated sugar, which is preferably a sugar that can be cleaved in an inclusion body or a lysosome, and is preferably an acetylated penta- or hexa-sugar, such as N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, and more preferably N-acetylgalactosamine or N-acetylglucosamine;
  • A is selected from
  • A is connected to L 1 through the a-terminal and to the P group through the b-terminal;
  • P, L 1 , L 2 , T, Rs, Rs′, m, n and k are as defined above.
  • the present invention specifically relates to a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand comprises a sequence that is fully complementary to the sense strand and the target mRNA, wherein the sense strand and/or the antisense strand comprises one or more compounds of the aforementioned formula (II) or formula (III), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.
  • R 1 represents H; in another embodiment, R 1 represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide.
  • R 1 is H; in another embodiment, R 1 is selected from a reactive phosphorus group, preferably a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimetic, such as a natural phosphate, a phosphorothioate, a phosphorodithioate, a boranephosphate, a boranephosphorothioate, a phosphonate, a halogen-substituted phosphonate and a phosphate, a phosphoramidate, a phosphodiester, a phosphotriester, a phosphorothioate diester, a phosphorothioate triester, a diphosphate or a triphosphate, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 ); in another embodiment, R 1 is selected from a hydroxy protecting group, such as trimethylsilyl (TMS),
  • R 2 is H; in another embodiment, R 2 is selected from a reactive phosphorus group, preferably a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimetic, such as a natural phosphate, a phosphorothioate, a phosphorodithioate, a boranophosphate, a boranophosphothioate, a phosphonate, a halogen-substituted phosphonate and a phosphate, a phosphoramidate, a phosphodiester, a phosphotriester, a phosphorothioate diester, a phosphorothioate triester, a diphosphate or a triphosphate, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 ); ...
  • a reactive phosphorus group preferably a phosphoramidite, an H-phosphonate, an alkyl
  • TMS trimethylsilyl
  • TES triethylsilyl
  • DMIPS dimethylisopropylsilyl
  • DEIPS diethylisopropylsilyl
  • TDMS tert-butyldimethylsilyl
  • TDPS tert-butyldiphenylsilyl
  • TIPS triisopropylsilyl
  • acetyl (Ac) chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl
  • R 1 is a solid support.
  • X1 is H; in another embodiment, X1 is Rs; in another embodiment, X1 is
  • X 2 is OR 1 ; in another embodiment, X 2 is
  • L 1 is a chemical bond; in another embodiment, L 1 is C 1-10 alkylene; in another embodiment, L 1 is C 1-6 alkylene; in another embodiment, L 1 is C 2-10 alkenylene; in another embodiment, L 1 is C 2-10 alkynylene; in another embodiment, L 1 is OC 1-10 alkylene; in another embodiment, L 1 is OC 1-6 alkylene; in another embodiment, L 1 is OC 1-10 alkenylene; in another embodiment, L 1 is OC 1-10 alkynylene; in another embodiment, L 1 is C(O)C 1-10 alkylene; in another embodiment, L 1 is C(O)C 1-6 alkylene.
  • L 1 is OCH 2 CH 2 ; in another specific embodiment, L 1 is O(CH 2 ) 5 .
  • L1 is unsubstituted; in another embodiment, L1 is substituted with 1 R; in another embodiment, L1 is substituted with 2 Rs; in another embodiment, L1 is substituted with 3 Rs; in another embodiment, L1 is substituted with 4 Rs; in another embodiment, L1 is substituted with 5 Rs; in another embodiment, L1 is substituted with 6 Rs; in another embodiment, L1 is substituted with 7 Rs; in another embodiment, L1 is substituted with 8 Rs.
  • L 2 is a chemical bond; in another embodiment, L 2 is C 1-10 alkylene; in another embodiment, L 2 is C 1-6 alkylene; in another embodiment, L 2 is C 2-10 alkenylene; in another embodiment, L 2 is C 2-10 alkynylene; in another embodiment, L 2 is OC 1-10 alkylene; in another embodiment, L 2 is OC 1-6 alkylene; in another embodiment, L 2 is OC 1-10 alkenylene; in another embodiment, L 2 is OC 1-10 alkynylene; in another embodiment, L 2 is C(O)C 1-10 alkylene; in another embodiment, L 2 is C(O)C 1-6 alkylene.
  • L 2 is OCH 2 ; in another specific embodiment, L 2 is a chemical bond.
  • L2 is unsubstituted; in another embodiment, L2 is substituted with 1 R; in another embodiment, L2 is substituted with 2 R; in another embodiment, L2 is substituted with 3 R; in another embodiment, L2 is substituted with 4 R; in another embodiment, L2 is substituted with 5 R; in another embodiment, L2 is substituted with 6 R; in another embodiment, In one embodiment, L 2 is substituted with 7 Rs; in another embodiment, L 2 is substituted with 8 Rs.
  • T is a chemical bond; in another embodiment, T is -O-; in another embodiment, T is -CH2- ; in another embodiment, T is -C(O)-; in another embodiment, T is -OC(O)-; in another embodiment, T is -M-; in another embodiment, T is -OM-; in another embodiment, T is -CH2- M-; in another embodiment, T is -C(O)-M-; in another embodiment, T is -OC(O)-M-.
  • M is In another embodiment, M is In another embodiment, M is In another embodiment, M is In another embodiment, M is
  • A is a sugar; in another embodiment, A is a sugar that can be cleaved in an inclusion body or a lysosome; in another embodiment, A is a penta- or hexa-sugar, preferably a hexa-sugar; in another embodiment, A is selected from N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, preferably N-acetylgalactosamine or N-acetylglucosamine.
  • a is A is connected to L1 through the a terminal and connected to the P group through the b terminal; in another specific embodiment, A is A is connected to L1 through the a terminal and connected to the P group through the b terminal; in another specific embodiment, A is A is connected to L1 through the a terminal and connected to the P group through the b terminal; in another specific embodiment, A is A is connected to L1 through the a terminal and connected to the P group through the b terminal; in another specific embodiment, A is A is connected to L1 through the a-terminal and to the P group through the b-terminal.
  • A is an acetylated sugar; in another embodiment, A is an acetylated sugar, which is a sugar that can be cleaved in an inclusion body or a lysosome; in another embodiment, A is an acetylated penta- or hexa-sugar, which is, for example, N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably, the penta- or hexa-sugar is N-acetylgalactosamine or N-acetylglucosamine.
  • A is A is connected to L1 through the a terminal and connected to the P group through the b terminal; in another specific embodiment, A is A is connected to L1 through the a terminal and connected to the P group through the b terminal; in another specific embodiment, A is A is connected to L 1 through the a-terminal and to the P group through the b-terminal; In another specific embodiment, A is
  • A is connected to L1 through the a-terminal and to the P group through the b-terminal.
  • P is a hydrophobic group; in another embodiment, P is a C 8-30 hydrocarbon group, such as a C 8-30 alkyl, a C 8-30 alkenyl or a C 8-30 alkynyl; in another embodiment, P is a C 10-22 alkyl group, such as a C 10 alkyl, a C 11 alkyl, a C 12 alkyl, a C 13 alkyl, a C 14 alkyl, a C 15 alkyl, a C 16 alkyl, a C 17 alkyl, a C 18 alkyl, a C 19 alkyl, a C 20 alkyl, a C 21 alkyl , a C 22 alkyl; in another embodiment, P is a C 12-18 alkyl group, such as a C 14-16 alkyl; in another embodiment, P is a C 10-22 alkenyl group.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10 non-adjacent carbon atoms in P may be replaced by heteroatoms selected from O, S and N, or a -CH2CH2- group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring.
  • P is unsubstituted; in another embodiment, P is substituted with 1 R; in another embodiment, P is substituted with 2 Rs; in another embodiment, P is substituted with 3 Rs; in another embodiment, P is substituted with 4 Rs; in another embodiment, P is substituted with 5 Rs; in another embodiment, P is substituted with 6 Rs; in another embodiment, P is substituted with 7 Rs; in another embodiment, P is substituted with 8 Rs; in another embodiment, P is substituted with more Rs.
  • P is -(CH 2 ) 14-16 CH 3 .
  • P1 is H; in another embodiment, P1 is a P group as defined above.
  • P3 is H; in another embodiment, P3 is a P group as defined above.
  • P 4 is CH 3 ; in another embodiment, P 4 is a P group as defined above.
  • one of P 1 , P 2 , P 3 , P 4 is a P group.
  • R is H
  • R s ' is H; in another embodiment, R s ' is D; in another embodiment, R s ' is halogen; in another embodiment, R s ' is C 1-6 alkyl; in another embodiment, R s ' is C 1-6 haloalkyl; in another embodiment, R s ' is 3-10 membered heterocyclyl, such as 5-10 membered heterocyclyl; in another embodiment, R s ' is C 3-10 cycloalkyl; in another embodiment, R s ' is optionally deuterated, up to fully deuterated.
  • R s ′ is cyclohexyl
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • k 0, 1, 2, 3, 4, 5 or 6.
  • any technical solution or any combination thereof in any of the above specific embodiments can be combined with any technical solution or any combination thereof in other specific embodiments.
  • any technical solution or any combination thereof in A can be combined with Any technical solution or any combination thereof of P, P1 - P4 , R1, R2 , X1 , X2 , L1 , L2 , T, R, Rs , Rs ', m, n and k etc.
  • the present invention is intended to include combinations of all these technical solutions, which are not listed one by one due to space limitations.
  • the present invention also provides a vector comprising a nucleotide sequence encoding the siRNA of the present invention.
  • the vector of the present invention can amplify or express the nucleotide sequence encoding the siRNA of the present invention connected thereto.
  • siRNA targeting the PCSK9 gene can be expressed from a transcription unit inserted into a DNA or RNA vector. Expression can be transient (within hours to weeks) or continuous (weeks to months or longer), depending on the specific construct used and the target tissue or cell type.
  • the coding nucleotides of the siRNA can be introduced into a linear construct, a circular plasmid or a viral vector.
  • the nucleotides of the siRNA can be integrated into the cell genome for stable expression, or expressed in a stable extrachromosomal inheritance.
  • siRNA expression vectors are typically DNA plasmids or viral vectors.
  • the present invention also provides a cell containing the siRNA or vector of the present invention, wherein the siRNA or vector of the present invention can be transcribed in the cell.
  • formula (I) represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide
  • X 1 is selected from H, Rs or
  • X 2 is selected from OR 1 or
  • R 1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide
  • Each L 1 and L 2 is independently selected from a chemical bond, a C 1-10 alkylene group, a C 2-10 alkenylene group, a C 2-10 alkynylene group, an OC 1-10 alkylene group, an OC 1-10 alkenylene group, an OC 1-10 alkynylene group, a C(O)C 1-10 alkylene group; said L 1 and L 2 are optionally substituted by 1, 2, 3, 4, 5, 6, 7 or 8 R;
  • T is selected from a chemical bond, -O-, -CH2- , -C(O)-, -OC(O)-, -M-, -OM-, -CH2 - M-, -C(O)-M-, or -OC(O)-M-;
  • A is a sugar, which is preferably a sugar that can be cleaved in an inclusion body or a lysosome, preferably a penta-sugar or a hexa-sugar, more preferably a hexa-sugar, such as N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably N-acetylgalactosamine or N-acetylglucosamine;
  • P is a hydrophobic group, preferably a C 8-30 alkyl, C 8-30 alkenyl or C 8-30 alkynyl group, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH 2 CH 2 - group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be connected to form a saturated or unsaturated ring; the C 8-30 alkyl, C 8-30 alkenyl or C 8-30 alkynyl group is optionally substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R;
  • R is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl, which is optionally deuterated until fully deuterated;
  • Rs is selected from H, D, halogen, C1-6 alkyl, C1-6 haloalkyl, 3-10 membered heterocyclyl or C3-10 cycloalkyl, which is optionally deuterated until fully deuterated;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • k 0, 1, 2, 3, 4, 5 or 6.
  • oligonucleotide of technical solution A1 wherein the compound of formula (I) is selected from a compound of formula (II) or formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • R 1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide
  • R 1 are not H at the same time
  • Each L 1 and L 2 is independently selected from a chemical bond, a C 1-10 alkylene group, a C 2-10 alkenylene group, a C 2-10 alkynylene group, an OC 1-10 alkylene group, an OC 1-10 alkenylene group, an OC 1-10 alkynylene group or a C(O)C 1-10 alkylene group; said L 1 and L 2 are optionally substituted by 1, 2, 3, 4, 5, 6, 7 or 8 R;
  • T is selected from a chemical bond, -O-, -CH2- , -C(O)-, -OC(O)-, -M-, -OM-, -CH2 - M-, -C(O)-M-, or -OC(O)-M-;
  • A is a sugar, which is preferably a sugar that can be cleaved in an inclusion body or a lysosome, preferably a penta-sugar or a hexa-sugar, more preferably a hexa-sugar, such as N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably N-acetylgalactosamine or N-acetylglucosamine;
  • P is a hydrophobic group, preferably a C 8-30 alkyl, C 8-30 alkenyl or C 8-30 alkynyl group, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH 2 CH 2 - group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be connected to form a saturated or unsaturated ring; the C 8-30 alkyl, C 8-30 alkenyl or C 8-30 alkynyl group is optionally substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R;
  • R is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl, which is optionally deuterated up to fully deuterated;
  • Rs is selected from H, D, halogen, C1-6 alkyl or C1-6 haloalkyl, which is optionally deuterated until fully deuterated;
  • R s ′ is selected from H, D, halogen, C 1-6 alkyl, C 1-6 haloalkyl, 3-10 membered heterocyclyl or C 3-10 cycloalkyl, which is optionally deuterated until fully deuterated;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • k 0, 1, 2, 3, 4, 5 or 6.
  • R 1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide
  • R 1 are not H at the same time
  • Each L 1 and L 2 is independently selected from a chemical bond, a C 1-6 alkylene group, an OC 1-6 alkylene group or a C(O)C 1-6 alkylene group; said L 1 and L 2 are optionally substituted by 1, 2, 3, 4 or 5 R;
  • T is selected from a chemical bond, -O-, -CH2- , -C(O)-, -OC(O)-, -M-, -OM-, -CH2 - M-, -C(O)-M-, or -OC(O)-M-;
  • A is selected from N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, more preferably N-acetylgalactosamine or N-acetylglucosamine;
  • P is selected from C 10-22 alkyl or C 10-22 alkenyl, wherein the C 10-22 alkyl or C 10-22 alkenyl is optionally substituted by 1, 2, 3, 4 or 5 R;
  • R is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl
  • Rs is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl;
  • R s ' is selected from H, D, halogen, C 1-6 alkyl, C 1-6 haloalkyl, 3-10 membered heterocyclyl or C 3-10 cycloalkyl;
  • n 0, 1, 2, 3, 4 or 5;
  • n 0, 1, 2, 3, 4 or 5;
  • k 0, 1, 2, 3, 4 or 5.
  • R 1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide
  • R 1 are not H at the same time
  • Each L 1 and L 2 is independently selected from a chemical bond or an OC 1-6 alkylene group; said L 1 and L 2 are optionally substituted by 1, 2 or 3 R;
  • T is selected from a chemical bond, -O-, -CH2- , -OC(O)- or -C(O)-;
  • A is selected from
  • A is connected to L 1 through the a-terminal and to the P group through the b-terminal;
  • P is selected from C 10-22 alkyl or C 10-22 alkenyl, wherein the C 10-22 alkyl or C 10-22 alkenyl is optionally substituted by 1, 2 or 3 R;
  • R is selected from H, D, halogen, C 1-4 alkyl
  • Rs is selected from H or D;
  • R s ' is selected from H, D or C 5-10 cycloalkyl
  • n 0, 1, 2 or 3;
  • n 0, 1, 2 or 3;
  • k 0, 1, 2 or 3.
  • R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide
  • L 1 is OCH 2 CH 2 or O(CH 2 ) 5 ;
  • L 2 is OCH 2 or a chemical bond
  • T is selected from a chemical bond, -O- or -C(O)-;
  • A is selected from
  • A is connected to L 1 through the a-terminal and to the P group through the b-terminal;
  • P is -(CH 2 ) 14-16 CH 3 ;
  • R s ' is cyclohexyl
  • n 0, 1 or 2;
  • n 0 or 1
  • k 0 or 1.
  • P 1 , P 2 , and P 3 are selected from H or P groups
  • P 4 is selected from CH 3 or a P group
  • P 1 , P 2 , P 3 , and P 4 is a P group
  • R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide.
  • A8 The oligonucleotide of any one of technical solutions A1-A7, which has 14 to 30 nucleotides.
  • the oligonucleotide of any one of technical solutions A1-A9 which comprises a formula (II) of any one of technical solutions A1-A7 at the 3' end or a compound of formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
  • An oligonucleotide according to any one of technical solutions A1-A10 which comprises a compound of formula (II) or formula (III) according to any one of technical solutions A1-A7 at its 5' end and 3' end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
  • the oligonucleotide of any one of technical solutions A1-A11 which comprises one or more compounds of formula (II) of any one of technical solutions A1-A7, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, inside the oligonucleotide.
  • An oligonucleotide comprising one, two or more delivery vectors within the oligonucleotide, at the 5' end and/or at the 3' end, wherein the delivery vector is a sugar modified with a hydrophobic group;
  • the sugar modified by the hydrophobic group is selected from the compound of formula (X), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • P is a hydrophobic group
  • A is the sugar part
  • P and A are as defined in any one of technical solutions A1-A5;
  • the hydrophobic group is attached to a hydroxyl or acetyl group of the sugar moiety
  • the sugar modified by the hydrophobic group is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:
  • the oligonucleotide of any one of technical solutions A1-A13, which is an ASO or siRNA, is preferably used to inhibit genes expressed outside the liver, and more preferably used to inhibit genes expressed in the central nervous system (CNS) and/or the eye.
  • A15 A compound of formula (II') or (III'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • R 1 and R 2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;
  • A is an acetylated sugar, which is preferably a sugar that can be cleaved in an inclusion body or a lysosome, and is preferably an acetylated penta- or hexa-sugar, and the penta- or hexa-sugar is, for example, N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose, glucuronic acid, neuraminic acid (sialic acid), xylose or fucose, and more preferably the penta- or hexa-sugar is N-acetylgalactosamine or N-acetylglucosamine;
  • A is selected from
  • A is connected to L 1 through the a-terminal and to the P group through the b-terminal;
  • P, L 1 , L 2 , T, Rs, Rs′, m, n, and k are as defined in any one of technical solutions A1 to A7.
  • R 1 and R 2 are a reactive phosphorus group, preferably a phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate or phosphate mimetic, such as natural phosphate, thiophosphate, dithiophosphate, boranephosphate, boranethiophosphate, phosphonate, halogen-substituted phosphonate and phosphate, aminophosphorate, phosphodiester, phosphotriester, thiophosphodiester, thiophosphothioates, diphosphate or triphosphate, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 ).
  • a reactive phosphorus group preferably a phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate or phosphate mimetic, such as natural phosphate, thiophosphate, dithiophosphate, boranephosphate, boranethiophosphate, phosphonate, halogen-substi
  • R 1 and R 2 are selected from protecting groups, preferably hydroxy protecting groups, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxy Carbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbon
  • TMS trimethylsilyl
  • TES triethyl
  • A20 A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence that is fully complementary to the sense strand and the target mRNA, wherein the sense strand and/or antisense strand comprises one or more compounds of formula (II) or formula (III) as described in any one of technical solutions A1-A7, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.
  • each variable is defined as in technical solutions A1-A7.
  • R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide.
  • A23 A double-stranded RNA according to any one of technical solutions A20-A22, wherein the positive strand comprises at the 5’ end a compound of formula (II) or formula (III) according to any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
  • A24 A double-stranded RNA according to any one of technical solutions A20-A23, wherein the positive strand comprises at the 3’ end a compound of formula (II) or formula (III) according to any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
  • A26 The double-stranded RNA of any one of technical solutions A20-A25, wherein the sense strand comprises one or more compounds of formula (II) described in any one of technical solutions A1-A7, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, inside the oligonucleotide.
  • A27 A double-stranded RNA according to any one of technical solutions A20-A26, wherein the antisense strand comprises at the 5’ end a compound of formula (II) or formula (III) according to any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
  • A28 A double-stranded RNA according to any one of technical solutions A20-A27, wherein the antisense strand comprises at the 3’ end a compound of formula (II) or formula (III) according to any one of technical solutions A1-A7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
  • A29 A double-stranded RNA according to any one of technical solutions A20-A28, wherein the antisense strand comprises a compound of formula (II) or formula (III) according to any one of technical solutions A1-A7 at the 5’ end and the 3’ end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
  • A30 The double-stranded RNA of any one of technical solutions A20-A29, wherein the antisense strand comprises one or more compounds of formula (II) described in any one of technical solutions A1-A7, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, inside the oligonucleotide.
  • A31 A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence that is sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and/or the antisense strand comprises one, two or more delivery vectors at the interior, 5' end and/or 3' end, and the delivery vector is a sugar modified with a hydrophobic group;
  • the sugar modified by the hydrophobic group is selected from the compound of formula (X), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • P is a hydrophobic group
  • A is the sugar part
  • P and A are as defined in any one of technical solutions A1-A5;
  • the hydrophobic group is attached to a hydroxyl or acetyl group of the sugar moiety
  • the sugar modified by the hydrophobic group is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:
  • A32 A vector comprising a nucleotide sequence encoding the double-stranded RNA described in any one of the aforementioned technical solutions A20-A31.
  • A33 A cell containing the double-stranded RNA as described in any one of technical solutions A20-A31 or the vector as described in technical solution A32.
  • a pharmaceutical composition comprising the double-stranded RNA as described in any one of technical solutions A20-A31, the vector as described in technical solution A32, or the cell as described in technical solution A33, and optionally a pharmaceutically acceptable carrier or excipient.
  • a kit comprising the double-stranded RNA as described in any one of technical solutions A20-A31, the vector as described in technical solution A32, or the cell as described in technical solution A33.
  • An oligonucleotide comprising one or more compounds of formula (IV), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • R 1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide
  • R 1 are not H at the same time
  • Each L 1 and L 2 is independently selected from a chemical bond, a C 1-10 alkylene group, a C 2-10 alkenylene group, a C 2-10 alkynylene group, an OC 1-10 alkylene group, an OC 1-10 alkenylene group, an OC 1-10 alkynylene group or a C(O)C 1-10 alkylene group; said L 1 and L 2 are optionally substituted by 1, 2, 3, 4, 5, 6, 7 or 8 R;
  • T is selected from a chemical bond, -O-, -CH2- , -C(O)-, -OC(O)-, -M-, -OM-, -CH2 - M-, -C(O)-M-, or -OC(O)-M-;
  • A is N-acetylglucosamine
  • R is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl, which is optionally deuterated up to fully deuterated;
  • Rs is selected from H, D, halogen, C1-6 alkyl or C1-6 haloalkyl, which is optionally deuterated until fully deuterated;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • k 0, 1, 2, 3, 4, 5 or 6.
  • R 1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide
  • R 1 are not H at the same time
  • Each L 1 and L 2 is independently selected from a chemical bond, a C 1-6 alkylene group, an OC 1-6 alkylene group or a C(O)C 1-6 alkylene group; said L 1 and L 2 are optionally substituted by 1, 2, 3, 4 or 5 R;
  • T is selected from a chemical bond, -O-, -CH2- , -C(O)-, -OC(O)-, -M-, -OM-, -CH2 - M-, -C(O)-M-, or -OC(O)-M-;
  • A is N-acetylgalactosamine
  • R is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl
  • Rs is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl;
  • n 0, 1, 2, 3, 4 or 5;
  • n 0, 1, 2, 3, 4 or 5;
  • k 0, 1, 2, 3, 4 or 5.
  • R 1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide
  • R 1 are not H at the same time
  • Each L 1 and L 2 is independently selected from a chemical bond or an OC 1-6 alkylene group, for example, OCH 2 , OCH 2 CH 2 , (OCH 2 CH 2 ) 2 or (OCH 2 CH 2 ) 3 ; said L 1 and L 2 are optionally substituted by 1, 2 or 3 R;
  • T is selected from a chemical bond, -O-, -CH 2 -, -OC(O)- or -C(O)-, preferably -C(O)-;
  • R is selected from H, D, halogen, C 1-4 alkyl
  • Rs is selected from H or D;
  • n 0, 1, 2 or 3;
  • n 0, 1, 2 or 3;
  • k 0, 1, 2 or 3.
  • R 1 and R 2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;
  • L 1 , L 2 , T, Rs, m, n and k are as defined above.
  • one of R 1 and R 2 is a reactive phosphorus group, preferably a phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate or phosphate mimetic, such as natural phosphates, phosphorothioates, phosphorodithioates, boranophosphates, boranophosphorothioates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiester, phosphotriester, phosphorothioatediester, phosphorothioatetriester, diphosphate or triphosphate, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 );
  • R1 and R 2 is selected from protecting groups, preferably hydroxy protecting groups, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl
  • oligonucleotides are as follows, wherein the sequence of 5'-->3' from the to connect.
  • the corresponding structure is located in the middle of the nucleic acid chain, It means that it is connected to the 3' carbon or corresponding position of the previous nucleotide or nucleotide analog through a phosphate group, a phosphorothioate group or other linking group, It means that it is connected to the 5' carbon or corresponding position of the next nucleotide or nucleotide analog through a phosphate group, a thiophosphate group or other linking group; if the corresponding structure is located at the terminal position of the nucleic acid chain, Correspondingly, it means that it is connected to the 3' or 5' end of the nucleic acid chain through a phosphate group, a phosphorothioate group or other linking group.
  • the mixture was washed three times with saturated sodium bicarbonate solution (30.0 mL x 3) and saturated brine (30.0 mL x 3) in sequence, and the organic phase was dried over anhydrous sodium sulfate and then dried under reduced pressure to obtain a crude product.
  • the mixture was washed three times with saturated sodium bicarbonate solution (20.0 mL x 3) and saturated brine (20.0 mL x 3) in sequence, and the organic phase was dried over anhydrous sodium sulfate and suspended to dryness under reduced pressure to obtain a crude product.
  • the synthesis steps of compound BE6 can refer to Example 4, wherein stearic acid is used to replace compound 1b.
  • the siRNA of the present invention is prepared using the solid phase phosphoramidite method well known in the art.
  • the specific method can be referred to, for example, PCT Publication Nos. WO2016081444 and WO2019105419, and is briefly described as follows.
  • nucleoside monomers are connected one by one from the 3'-5' direction according to the arrangement order of the positive chain nucleotides.
  • Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or thiolation, and the synthesis scale is 5umol of oligonucleotides.
  • the synthesis conditions are as follows:
  • the nucleoside monomer was provided in a 0.05 mol/L acetonitrile solution.
  • the reaction conditions for each step were the same, i.e., the temperature was 25 degrees.
  • a 3% trichloroacetic acid-dichloromethane solution was used for deprotection, and the deprotection was repeated three times.
  • the activator used in the coupling reaction was a 0.25 mol/L 5-ethylthiotetrazolyl (ETT)-acetonitrile solution, and the coupling was repeated twice.
  • ETT 5-ethylthiotetrazolyl
  • the capping reaction was performed using 10% acetic anhydride-acetonitrile and pyridine/N-methylimidazole/acetonitrile (10:14:76, v/v/v) and the capping was repeated twice.
  • the oxidation reaction was performed using 0.05 mol/L iodine in tetrahydrofuran/pyridine/water (70/20/10, v/v/v) and the oxidation was repeated twice.
  • the thiolation reaction was performed using 0.2 mol/L Phenylacetyl disulfide (PADS) in acetonitrile/3-methylpyridine (1/1, v/v) was thiolated twice.
  • PADS Phenylacetyl disulfide
  • nucleoside monomers are connected one by one from the 3'-5' direction according to the arrangement order of the antisense chain nucleotides.
  • Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or thiolation.
  • the synthesis conditions of 5umol oligonucleotides of the antisense chain are the same as those of the sense chain.
  • a column filled with strong anion fillers can be used, and a sodium chloride-sodium hydroxide system can be used for elution and purification, and the product can be collected and piped.
  • a gel filler purification column can be used for desalination, and the elution system is pure water.
  • siRNA sequences used in the present invention are as follows:
  • A, U, G and C represent the natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide and cytosine ribonucleotide, respectively.
  • d indicates that the adjacent nucleotide on the right is a deoxyribonucleotide.
  • dA, dT, dG, and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and cytosine deoxyribonucleotide, respectively.
  • m means that the adjacent nucleotide on its left side is a 2'-OCH3 modified nucleotide.
  • Am, Um, Gm and Cm represent 2'-OCH3 modified A, U, G and C.
  • f indicates that the adjacent nucleotide on its left side is a 2'-F modified nucleotide.
  • Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.
  • s indicates that the two adjacent nucleotides and/or delivery vectors are linked by phosphorothioate.
  • VP indicates that the adjacent nucleotide on the right is a vinyl phosphate-modified nucleotide.
  • L96 represents a GalNAc delivery vector of the following structure well known in the art, wherein Refer to the position of the siRNA linked via a phosphate group or a phosphorothioate group, for example, PCT Publication Nos. WO2009073809 and WO2009082607.
  • GL34 when connected to the 3' end and 5' end of the nucleic acid chain through a phosphate group, a thiophosphate group or other linking groups are as follows:
  • LS3 when connected to the 3' end and 5' end of the nucleic acid chain through a phosphate group, a thiophosphate group or other linking groups are as follows:
  • LS1 when connected to the 3' end and 5' end of the nucleic acid chain through a phosphate group, a thiophosphate group or other linking groups are as follows:
  • Example 8 Verification of the long-lasting efficacy of the compounds of the present invention in a C57BL/6 mouse model
  • C57BL/6 mice male, 18-21 g, 6-8 weeks were randomly divided into groups, and the dosage of each animal was calculated according to the body weight.
  • the siRNA conjugate was administered as a 1 mg/mL solution (0.9% sodium chloride aqueous solution as solvent) by subcutaneous injection; specifically, before the experiment, the siRNA conjugate was dissolved in 0.9% sodium chloride aqueous solution and fixed to the required concentration and volume, and the administration volume of normal saline (control group) and siRNA conjugate was 5 mL/kg.
  • liver Before administration (recorded as day 0), and on days 7, 14, 21, 28, 35, 42, 56, and 70 after administration, 10 mg of liver was collected and placed in RNAlater TM solution and stored at -80°C for detection of liver mTTR mRNA.
  • a nucleic acid extractor Auto-pure96, Hangzhou Aosheng
  • reverse transcription was performed with reference to the PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B)
  • fluorescence quantitative PCR reaction was performed with reference to the TaqMan TM Fast Advanced Master Mix (ABI, 4444965) 20 ⁇ L system.
  • the primers are shown in Table 1.
  • the 2 - ⁇ Ct value was calculated and converted into a percentage to obtain the residual inhibition rate
  • ⁇ Ct [(target gene in Ct experimental group-internal reference in Ct experimental group)-(target gene in Ct control group-internal reference in Ct control group)].
  • the target gene is mTTR and the internal reference is mGAPDH.
  • Example 9 Verification of the long-lasting efficacy of the compounds of the present invention in a C57BL/6 mouse model
  • C57BL/6 mice male, 6-8 weeks were randomly divided into groups and administered a single dose of 7.5 ⁇ g per eye by bilateral intravitreal injection; specifically, before the experiment, the siRNA conjugate was dissolved in phosphate buffer solution and diluted to the required solution concentration and volume, and the administration volume of phosphate buffer solution and siRNA conjugate was 1.5 ⁇ L/eye.
  • the eyeballs were removed and separated into three parts: 1 cornea + iris + ciliary body; 2 retina; 3 retinal pigment epithelium (RPE) + choroid + sclera; the separated samples were immediately frozen in liquid nitrogen and then stored at -80°C for detection of mTTR mRNA.
  • RPE retinal pigment epithelium

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Ophthalmology & Optometry (AREA)
  • Microbiology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

提供式(I)化合物，或其药学上可接受的盐、互变异构体或立体异构体。所述式(I)化合物包含在核苷酸内部，或者在5'端和/或3'端，用于将双链RNA递送至肝外组织，例如中枢神经系统或眼部。还提供包含式(I)化合物的寡核苷酸、双链RNA、载体、细胞、药物组合物和试剂盒。

Description

包含糖的寡核苷酸递送配体
本发明要求申请日为2023年6月12日、CN202310693838.2的中国发明专利申请的优先权，将其整体引入本文作为参考。
发明领域
本发明属于医药领域，具体涉及具有将双链RNA递送至肝外组织，例如中枢神经系统或眼部的递送载体，所述递送载体为被疏水性基团修饰的糖，例如式(X)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
背景技术
RNA干扰是一种由双链RNA(double-stranded RNA，dsRNA，也称siRNA)诱发的靶标mRNA高效特异性降解的现象。
然而，由于血脑屏障的存在，难以将siRNA递送到中枢神经系统继而发挥作用，这限制了siRNA的应用。本领域对于将siRNA递送到中枢神经系统进行了一些尝试，例如WO2004094595A2公开了在链末端使用单个脂质配体(例如胆固醇或长链烷烃)递送siRNA，WO2019217459A1公开了在链内部使用单个脂质配体递送siRNA，WO2021092371A2公开了一系列新的脂质配体结构。
本领域仍然需要开发更多的疏水性基团，以更有效地递送siRNA到中枢神经系统。
发明内容
在一个方面中，本发明提供了一种寡核苷酸，其包含一个或多个式(I)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中各基团如下文所定义。
在另一个方面中，本发明提供了一种寡核苷酸，其包含一个或多个式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中各基团如下文所定义。
在另一个方面中，本发明提供了一种寡核苷酸，其在寡核苷酸内部、5’端和/或3’端包含一个、两个或多个递送载体，所述递送载体为被疏水性基团修饰的糖；
优选地，所述被疏水性基团修饰的糖选自式(X)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中各基团如下文所定义。
在另一个方面中，本发明提供了一种双链RNA，其具有正义链和反义链，各链具有14至30个核苷酸，所述反义链包含与所述正义链和靶标mRNA充分互补的序列，其中所述正义链和/或反义链包含一个或多个上述式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
在另一个方面中，本发明提供了一种双链RNA，其具有正义链和反义链，各链具有14至30个核苷酸，所述反义链包含与所述正义链和靶标mRNA充分互补的序列，其中所述正义链和/或反义链在内部、5’端和/或3’端包含一个、两个或多个递送载体，所述递送载体为被疏水性基团修饰的糖；
优选地，所述被疏水性基团修饰的糖选自式(X)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中各基团如下文所定义。
在另一个方面中，本发明提供了式(II’)或(III’)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中各基团如下文所定义。
在另一个方面中，本发明提供的疏水性基团(式(I)、(II)或(III)中的P基团)可以通过核苷酸中糖部分的羟基或乙酰基上连接到寡核苷酸或双链RNA。
在另一个方面中，本发明提供了载体，其包含编码前述双链RNA的核苷酸序列。
在另一个方面中，本发明提供了细胞，其含有前述双链RNA或前述载体。
在另一个方面中，本发明提供了药物组合物，其包含前述双链RNA、前述载体、或前述细胞，以及任选的药学上可接受的载剂或赋形剂。
在另一个方面中，本发明提供了试剂盒，其包含前述双链RNA、前述载体、或前述细胞。
发明详述
定义
化学定义
下面更详细地描述具体官能团和化学术语的定义。
当列出数值范围时，既定包括每个值和在所述范围内的子范围。例如“C1-6烷基”包括C1、C2、C3、C4、C5、C6、C1-6、C1-5、C1-4、C1-3、C1-2、C2-6、C2-5、C2-4、C2-3、C3-6、C3-5、C3-4、C4-6、C4-5和C5-6烷基。
“C1-6烷基”是指具有1至6个碳原子的直链或支链饱和烃基团。“C8-30烷基”是指具有1至30个碳原子的直链或支链饱和烃基团。在一些实施方案中，C8-25烷基、C10-22烷基、C8-20烷基、C1-10烷基和C1-6烷基是优选的。C1-6烷基的例子包括：甲基(C1)、乙基(C2)、正丙基(C3)、异丙基(C3)、正丁基(C4)、叔丁基(C4)、仲丁基(C4)、异丁基(C4)、正戊基(C5)、3-戊基(C5)、戊基(C5)、新戊基(C5)、3-甲基-2-丁基(C5)、叔戊基(C5)和正己基(C6)。术语“C1-6烷基”还包括杂烷基，其中一或多个(例如，1、2、3或4个)碳原子被杂原子(例如，氧、硫、氮、硼、硅、磷)替代。烷基基团可以被一或多个取代基任选取代，例如，被1至5个取代基、1至3个取代基或1个取代基取代。常规烷基缩写包括：Me(-CH3)、Et(-CH2CH3)、iPr(-CH(CH3)2)、nPr(-CH2CH2CH3)、n-Bu(-CH2CH2CH2CH3)或i-Bu(-CH2CH(CH3)2)。
“C2-10烯基”是指具有2至10个碳原子和至少一个碳碳双键的直链或支链烃基团。“C8-30烯基”是指具有8至30个碳原子和至少一个碳碳双键的直链或支链烃基团。在一些实施方案中，C10-22烯基、 C2-10烯基、C2-6烯基和C2-4烯基是优选的。C2-6烯基的例子包括：乙烯基(C2)、1-丙烯基(C3)、2-丙烯基(C3)、1-丁烯基(C4)、2-丁烯基(C4)、丁二烯基(C4)、戊烯基(C5)、戊二烯基(C5)、己烯基(C6)，等等。术语“C2-6烯基”还包括杂烯基，其中一或多个(例如，1、2、3或4个)碳原子被杂原子(例如，氧、硫、氮、硼、硅、磷)替代。烯基基团可以被一或多个取代基任选取代，例如，被1至5个取代基、1至3个取代基或1个取代基取代。
“C2-10炔基”是指具有2至10个碳原子和至少一个碳-碳叁键的直链或支链烃基团。“C8-30炔基”是指具有8至30个碳原子、至少一个碳-碳叁键以及任选地一个或多个碳-碳双键的直链或支链烃基团。在一些实施方案中，C10-22炔基、C2-10炔基、C2-6炔基和C2-4炔基是优选的。C2-6炔基的例子包括但不限于：乙炔基(C2)、1-丙炔基(C3)、2-丙炔基(C3)、1-丁炔基(C4)、2-丁炔基(C4)，戊炔基(C5)、己炔基(C6)，等等。术语“C2-6炔基”还包括杂炔基，其中一或多个(例如，1、2、3或4个)碳原子被杂原子(例如，氧、硫、氮、硼、硅、磷)替代。炔基基团可以被一或多个取代基任选取代，例如，被1至5个取代基、1至3个取代基或1个取代基取代。
“C1-10亚烷基”、“C2-10亚烯基”和“C2-10亚炔基”分别是指除去C1-10烷基、C2-10烯基和C2-10炔基的另一个氢而形成的二价基团，并且可以是取代或未取代的。在一些实施方案中，C2-8亚烷基、C3-7亚烷基、C1-6亚烷基、C4-6亚烷基、C1-4亚烷基、C2-4亚烷基和C1-3亚烷基是优选的。未取代的所述亚烷基包括但不限于：亚甲基(-CH2-)、亚乙基(-CH2CH2-)、亚丙基(-CH2CH2CH2-)、亚丁基(-CH2CH2CH2CH2-)、亚戊基(-CH2CH2CH2CH2CH2-)、亚己基(-CH2CH2CH2CH2CH2CH2-)，等等。示例性的取代的所述亚烷基，例如，被一个或多个烷基(甲基)取代的所述亚烷基，包括但不限于：取代的亚甲基(-CH(CH3)-、-C(CH3)2-)、取代的亚乙基(-CH(CH3)CH2-、-CH2CH(CH3)-、-C(CH3)2CH2-、-CH2C(CH3)2-)、取代的亚丙基(-CH(CH3)CH2CH2-、-CH2CH(CH3)CH2-、-CH2CH2CH(CH3)-、-C(CH3)2CH2CH2-、-CH2C(CH3)2CH2-、-CH2CH2C(CH3)2-)，等等。
“卤代”或“卤素”是指氟(F)、氯(Cl)、溴(Br)和碘(I)。
因此，“C1-6卤代烷基”是指上述“C1-6烷基”被一个或多个卤素基团取代。在一些实施方案中，C1-4卤代烷基是特别优选的，更优选C1-2卤代烷基。示例性的所述卤代烷基包括但不限于：-CF3、-CH2F、-CHF2、-CHFCH2F、-CH2CHF2、-CF2CF3、-CCl3、-CH2Cl、-CHCl2、2,2,2-三氟-1,1-二甲基-乙基，等等。卤代烷基基团可以在任何可用的连接点上被取代，例如，1至5个取代基、1至3个取代基或1个取代基。
“C3-10环烷基”是指具有3至10个环碳原子和零个杂原子的非芳香环烃基团。在一些实施方案中，C4-7环烷基和C3-6环烷基是特别优选的，更优选C5-6环烷基。环烷基还包括其中上述环烷基环与一个或多个芳基或杂芳基稠合的环体系，其中连接点在环烷基环上，且在这样的情况中，碳的数目继续表示环烷基体系中的碳的数目。示例性的所述环烷基包括但不限于：环丙基(C3)、环丙烯基(C3)、环丁基(C4)、环丁烯基(C4)、环戊基(C5)、环戊烯基(C5)、环己基(C6)、环己烯基(C6)、环已二烯基(C6)、环庚基(C7)、环庚烯基(C7)、环庚二烯基(C7)、环庚三烯基(C7)，等等。环烷基基团可以被一或多个取代基任选取代，例如，被1至5个取代基、1至3个取代基或1个取代基取代。
“3-10元杂环基”是指具有环碳原子和1至5个环杂原子的3至10元非芳香环系的基团，其中，每个杂原子独立地选自氮、氧、硫、硼、磷和硅。在包含一个或多个氮原子的杂环基中，只要化合价允许，连接点可为碳或氮原子。在一些实施方案中，优选4-10元杂环基，其为具有环碳原子和1至5个环杂原子的4至10元非芳香环系；在一些实施方案中，优选3-8元杂环基，其为具有环碳原子和1至4个环杂原子的3至8元非芳香环系；优选3-6元杂环基，其为具有环碳原子和1至3个环杂原子的3至6元非芳香环系；优选4-7元杂环基，其为具有环碳原子和1至3个环杂原子的4至7元非芳香环系；更优选5-6元杂环基，其为具有环碳原子和1至3个环杂原子的5至6元非芳香环系。杂环基还包括其中上述杂环基环与一个或多个环烷基稠合的环体系，其中连接点在环烷基环上，或其中上述杂环基环与一个或多个芳基或杂芳基稠合的环体系，其中连接点在杂环基环上；且在这样的情况下，环成员的数目继续表示在杂环基环体系中环成员的数目。示例性的包含一个杂原子的3元杂环基包括但不限于：氮杂环丙烷基、氧杂环丙烷基、硫杂环丙烷基(thiorenyl)。示例性的含有一个杂原子的4元杂环基包括但不限于：氮杂环丁烷基、氧杂环丁烷基和硫杂环丁烷基。示例性的含有一个杂原子的5元杂环基包括但不限于：四氢呋喃基、二氢呋喃基、四氢噻吩基、二氢噻吩基、吡咯烷基、二氢吡咯基和吡咯基-2,5-二酮。示例性的包含两个杂原子的5元杂环基包括但不限于：二氧杂环戊烷基、氧 硫杂环戊烷基(oxasulfuranyl)、二硫杂环戊烷基(disulfuranyl)和噁唑烷-2-酮。示例性的包含三个杂原子的5元杂环基包括但不限于：三唑啉基、噁二唑啉基和噻二唑啉基。示例性的包含一个杂原子的6元杂环基包括但不限于：哌啶基、四氢吡喃基、二氢吡啶基和硫杂环己烷基(thianyl)。示例性的包含两个杂原子的6元杂环基包括但不限于：哌嗪基、吗啉基、二硫杂环己烷基、二噁烷基。示例性的包含三个杂原子的6元杂环基包括但不限于：六氢三嗪基(triazinanyl)。示例性的含有一个杂原子的7元杂环基包括但不限于：氮杂环庚烷基、氧杂环庚烷基和硫杂环庚烷基。示例性的与C6芳基环稠合的5元杂环基(在本文中也称作5,6-双环杂环基)包括但不限于：二氢吲哚基、异二氢吲哚基、二氢苯并呋喃基、二氢苯并噻吩基、苯并噁唑啉酮基，等等。示例性的与C6芳基环稠合的6元杂环基(本文还指的是6,6-双环杂环基)包括但不限于：四氢喹啉基、四氢异喹啉基，等等。杂环基基团可以被一或多个取代基任选取代，例如，被1至5个取代基、1至3个取代基或1个取代基取代。
术语“疏水性基团”广义上是指对脂质具有亲和力的任何化学基团。表征疏水性基团的疏水性的一种方式是通过辛醇-水分配系数logKow，其中Kow为二相系统在平衡时化学物质在辛醇相中的浓度与其在水相中的浓度的比率。通常，疏水性部分的logKow超过1、超过1.5、超过2、超过3、超过4、超过5或超过10。具体到本发明，疏水性部分即为式I化合物中的R基团。
本文定义的烷基、烯基和炔基等为任选取代的基团。
示例性的碳原子上的取代基包括但不局限于：卤素、-CN、-NO2、-N3、-SO2H、-SO3H、-OH、-ORaa、-ON(Rbb)2、-N(Rbb)2、-N(Rbb)3 +X-、-N(ORcc)Rbb、-SH、-SRaa、-SSRcc、-C(＝O)Raa、-CO2H、-CHO、-C(ORcc)2、-CO2Raa、-OC(＝O)Raa、-OCO2Raa、-C(＝O)N(Rbb)2、-OC(＝O)N(Rbb)2、-NRbbC(＝O)Raa、-NRbbCO2Raa、-NRbbC(＝O)N(Rbb)2、-C(＝NRbb)Raa、-C(＝NRbb)ORaa、-OC(＝NRbb)Raa、-OC(＝NRbb)ORaa、-C(＝NRbb)N(Rbb)2、-OC(＝NRbb)N(Rbb)2、-NRbbC(＝NRbb)N(Rbb)2、-C(＝O)NRbbSO2Raa、-NRbbSO2Raa、-SO2N(Rbb)2、-SO2Raa、-SO2ORaa、-OSO2Raa、-S(＝O)Raa、-OS(＝O)Raa、-Si(Raa)3、-OSi(Raa)3、-C(＝S)N(Rbb)2、-C(＝O)SRaa、-C(＝S)SRaa、-SC(＝S)SRaa、-SC(＝O)SRaa、-OC(＝O)SRaa、-SC(＝O)ORaa、-SC(＝O)Raa、-P(＝O)2Raa、-OP(＝O)2Raa、-P(＝O)(Raa)2、-OP(＝O)(Raa)2、-OP(＝O)(ORcc)2、-P(＝O)2N(Rbb)2、-OP(＝O)2N(Rbb)2、-P(＝O)(NRbb)2、-OP(＝O)(NRbb)2、-NRbbP(＝O)(ORcc)2、-NRbbP(＝O)(NRbb)2、-P(Rcc)2、-P(Rcc)3、-OP(Rcc)2、-OP(Rcc)3、-B(Raa)2、-B(ORcc)2、-BRaa(ORcc)、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代；
或者在碳原子上的两个偕氢被基团＝O、＝S、＝NN(Rbb)2、＝NNRbbC(＝O)Raa、＝NNRbbC(＝O)ORaa、＝NNRbbS(＝O)2Raa、＝NRbb或＝NORcc取代；
Raa的每个独立地选自烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基，或者两个Raa基团结合以形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代；
Rbb的每个独立地选自：氢、-OH、-ORaa、-N(Rcc)2、-CN、-C(＝O)Raa、-C(＝O)N(Rcc)2、-CO2Raa、-SO2Raa、-C(＝NRcc)ORaa、-C(＝NRcc)N(Rcc)2、-SO2N(Rcc)2、-SO2Rcc、-SO2ORcc、-SORaa、-C(＝S)N(Rcc)2、-C(＝O)SRcc、-C(＝S)SRcc、-P(＝O)2Raa、-P(＝O)(Raa)2、-P(＝O)2N(Rcc)2、-P(＝O)(NRcc)2、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基，或者两个Rbb基团结合以形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代；
Rcc的每个独立地选自氢、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基，或者两个Rcc基团结合以形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代；
Rdd的每个独立地选自：卤素、-CN、-NO2、-N3、-SO2H、-SO3H、-OH、-ORee、-ON(Rff)2、-N(Rff)2,、-N(Rff)3 +X-、-N(ORee)Rff、-SH、-SRee、-SSRee、-C(＝O)Ree、-CO2H、-CO2Ree、-OC(＝O)Ree、-OCO2Ree、-C(＝O)N(Rff)2、-OC(＝O)N(Rff)2、-NRffC(＝O)Ree、-NRffCO2Ree、-NRffC(＝O)N(Rff)2、-C(＝NRff)ORee、-OC(＝NRff)Ree、-OC(＝NRff)ORee、-C(＝NRff)N(Rff)2、-OC(＝NRff)N(Rff)2、-NRffC(＝NRff)N(Rff)2、-NRffSO2Ree、-SO2N(Rff)2、-SO2Ree、-SO2ORee、-OSO2Ree、-S(＝O)Ree、-Si(Ree)3、-OSi(Ree)3、-C(＝S)N(Rff)2、-C(＝O)SRee、-C(＝S)SRee、-SC(＝S)SRee、-P(＝O)2Ree、-P(＝O)(Ree)2、-OP(＝O)(Ree)2、-OP(＝O)(ORee)2、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基、杂芳基，其中，每个烷基、烯基、炔基、环 烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rgg基团取代，或者两个偕Rdd取代基可结合以形成＝O或＝S；
Ree的每个独立地选自烷基、卤代烷基、烯基、炔基、环烷基、芳基、杂环基和杂芳基，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rgg基团取代；
Rff的每个独立地选自氢、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基，或者两个Rff基团结合形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rgg基团取代；
Rgg的每个独立地是：卤素、-CN、-NO2、-N3、-SO2H、-SO3H、-OH、-OC1-6烷基、-ON(C1-6烷基)2、-N(C1-6烷基)2、-N(C1-6烷基)3 +X-、-NH(C1-6烷基)2 +X-、-NH2(C1-6烷基)+X-、-NH3 +X-、-N(OC1-6烷基)(C1-6烷基)、-N(OH)(C1-6烷基)、-NH(OH)、-SH、-SC1-6烷基、-SS(C1-6烷基)、-C(＝O)(C1-6烷基)、-CO2H、-CO2(C1-6烷基)、-OC(＝O)(C1-6烷基)、-OCO2(C1-6烷基)、-C(＝O)NH2、-C(＝O)N(C1-6烷基)2、-OC(＝O)NH(C1-6烷基)、-NHC(＝O)(C1-6烷基)、-N(C1-6烷基)C(＝O)(C1-6烷基)、-NHCO2(C1-6烷基)、-NHC(＝O)N(C1-6烷基)2、-NHC(＝O)NH(C1-6烷基)、-NHC(＝O)NH2、-C(＝NH)O(C1-6烷基)、-OC(＝NH)(C1-6烷基)、-OC(＝NH)OC1-6烷基、-C(＝NH)N(C1-6烷基)2、-C(＝NH)NH(C1-6烷基)、-C(＝NH)NH2、-OC(＝NH)N(C1-6烷基)2、-OC(NH)NH(C1-6烷基)、-OC(NH)NH2、-NHC(NH)N(C1-6烷基)2、-NHC(＝NH)NH2、-NHSO2(C1-6烷基)、-SO2N(C1-6烷基)2、-SO2NH(C1-6烷基)、-SO2NH2、-SO2C1-6烷基、-SO2OC1-6烷基、-OSO2C1-6烷基、-SOC1-6烷基、-Si(C1-6烷基)3、-OSi(C1-6烷基)3、-C(＝S)N(C1-6烷基)2、C(＝S)NH(C1-6烷基)、C(＝S)NH2、-C(＝O)S(C1-6烷基)、-C(＝S)SC1-6烷基、-SC(＝S)SC1-6烷基、-P(＝O)2(C1-6烷基)、-P(＝O)(C1-6烷基)2、-OP(＝O)(C1-6烷基)2、-OP(＝O)(OC1-6烷基)2、C1-6烷基、C1-6卤代烷基、C2-C6烯基、C2-C6炔基、C3-C7环烷基、C6-C10芳基、C3-C7杂环基、C5-C10杂芳基；或者两个偕Rgg取代基可结合形成＝O或＝S；其中，X-为反离子。
示例性的氮原子上取代基包括但不局限于：氢、-OH、-ORaa、-N(Rcc)2、-CN、-C(＝O)Raa、-C(＝O)N(Rcc)2、-CO2Raa、-SO2Raa、-C(＝NRbb)Raa、-C(＝NRcc)ORaa、-C(＝NRcc)N(Rcc)2、-SO2N(Rcc)2、-SO2Rcc、-SO2ORcc、-SORaa、-C(＝S)N(Rcc)2、-C(＝O)SRcc、-C(＝S)SRcc、-P(＝O)2Raa、-P(＝O)(Raa)2、-P(＝O)2N(Rcc)2、-P(＝O)(NRcc)2、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基，或者连接至氮原子的两个Rcc基团结合形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代，且其中Raa、Rbb、Rcc和Rdd如上所述。
其他定义
本文术语“siRNA”是一类双链RNA分子，其可以介导与其互补的靶RNA(例如mRNA，例如，编码蛋白质的基因的转录物)的沉默。siRNA通常是双链的，包括与靶RNA互补的反义链，和与该反义链互补的正义链。为方便起见，这样的mRNA在此也被称为有待被沉默的mRNA。这样的基因也称为靶基因。通常，有待被沉默的RNA是内源基因或病原体基因。另外，除了mRNA以外的RNA(例如tRNA)以及病毒RNA也可以被靶向。
术语“反义寡核苷酸”即ASO(Antisense Oligonucleotides)，是一段由15-25个核苷酸组成的靶基因配对的单链DNA或RNA序列，它通过特异性阻断靶基因的转录或者翻译过程，达到基因调控的目的。
术语“反义链”是指siRNA的这样一条链，所述链包含与靶序列完全、充分或基本互补的区域。术语“正义链”是指siRNA的这样一条链，所述链包括与作为在此定义的术语反义链的区域完全、充分或基本互补的区域。
术语“互补区域”是指反义链上与靶mRNA序列完全、充分或基本互补的区域。在互补区域与靶序列不完全互补的情况下，错配可以位于分子的内部或末端区域中。通常，最耐受的错配位于末端区域中，例如，在5’和/或3’端的5、4、3、2或1个核苷酸内。对错配最敏感的反义链部分被称为“种子区”。例如，在包含19nt的链的siRNA中，第19个位置(从5’向3’)可以耐受一些错配。
术语“互补”是指第一多核苷酸在某些条件例如严格条件下与第二多核苷酸杂交的能力。例如，严格条件可包括400mM NaCl、40mM PIPES pH 6.4、1mM EDTA在50℃或70℃下持续12-16小时。就满足以上相对于它们杂交的能力而言的要求来说，“互补”序列还可以包括或完全形成自非沃森-克里克 碱基对和/或从非天然的以及经修饰的核苷酸形成的碱基对。此类非沃森-克里克碱基对包括但不限于G:U摇摆碱基配对或Hoogstein碱基配对。
与信使RNA(mRNA)的“至少部分互补”、“充分互补”或“基本上互补”的多核苷酸是指与感兴趣的mRNA的连续部分基本互补的多核苷酸。例如，如果序列与编码PCSK9的mRNA的非中断部分基本上互补，则多核苷酸与PCSK9mRNA的至少部分互补。在此的术语“互补”、“完全互补”、“充分互补”和“基本上互补”可以相对于siRNA的正义链与反义链之间，或siRNA试剂的反义链与靶序列之间的碱基配对使用。
“充分互补”是指为了维持分子的整体双链特征，正义链仅需要与反义链互补的程度。换言之，虽然通常需要完美的互补性，但在一些情况下，特别是在反义链中，可以包括一个或多个，例如6个、5个、4个、3个、2个或1个的错配(相对于靶标mRNA)，但是正义链与反义链仍可以维持分子的整体双链特征。
“核苷”是由嘌呤碱或嘧啶碱、以及核糖或脱氧核糖两种物质组成的化合物，“核苷酸”则是由嘌呤碱或嘧啶碱、核糖或脱氧核糖以及磷酸三种物质组成的化合物，“寡核苷酸”是指例如具有少于100、200、300或400个核苷酸长度的核酸分子(RNA或DNA)。
“碱基”是合成核苷、核苷酸和核酸的基本组成单位，其组成元素中含有氮，也称“含氮碱基”。本文中，如无特别说明，大写字母A、U、T、G和C表示核苷酸的碱基组成，分别为腺嘌呤、尿嘧啶、胸腺嘧啶、鸟嘌呤和胞嘧啶。
本文中所述核苷酸的“修饰”包括但不限于甲氧基修饰、氟代修饰、硫代磷酸酯基连接或常规保护基保护等。例如，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。
本文中“修饰的核苷酸”包括但不限于2'-O-甲基修饰的核苷酸、2'-氟代修饰的核苷酸、2'-脱氧-修饰的核苷酸、肌苷核糖核苷酸、脱碱基核苷酸、反向无碱基脱氧核糖核苷酸、包含硫代磷酸酯基团的核苷酸、乙烯基磷酸酯修饰的核苷酸、锁核苷酸、2'-氨基-修饰的核苷酸、2'-烷基-修饰的核苷酸、吗啉代核苷酸、氨基磷酸酯、包含核苷酸的非天然碱基、以及连接到胆固醇基衍生物或十二烷酸二癸酰胺基团上的末端核苷酸、脱氧核糖核苷酸或常规保护基保护等。例如，所述2'-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2'-脱氧-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。
“反应性磷基团”是指包含在核苷酸单元中或核苷酸类似物单元中的含磷基团，其可以通过亲核攻击反应，与包含在另一个分子中、尤其是另一个核苷酸单元中或另一个核苷酸类似物中的羟基或胺基反应。通常，这样的反应产生将所述第一核苷酸单元或所述第一核苷酸类似物单元与所述第二核苷酸单元或所述第二核苷酸类似物单元连接的酯型核苷间键。反应性磷基团可选自亚磷酰胺，H-膦酸酯，烷基-膦酸酯，磷酸酯或磷酸酯模拟物，包括但不限于：天然磷酸酯、硫代磷酸酯、二硫代磷酸酯、硼烷磷酸酯、硼烷硫代磷酸酯、膦酸酯、卤素取代的膦酸酯和磷酸酯、氨基磷酸酯、磷酸二酯、磷酸三酯、硫代磷酸二酯、硫代磷酸三酯、二磷酸酯和三磷酸酯，优选-P(OCH2CH2CN)(N(iPr)2)。
“保护基”是指被添加到分子中以防止分子中现有基团进行不期望的化学反应的任何原子或原子团。“保护基”可为本领域已知的不稳定的化学部分，其用于保护反应性基团，例如羟基、氨基和硫醇基团，以防止在化学合成过程中发生不期望的或不合时宜的反应。保护基通常在其它反应性位点的反应期间选择性地和/或正交地用于保护位点，然后可以被去除以留下未受保护的基团保持原样或可用于进一步的反应。
保护基团的非限制性列表包括苄基；取代的苄基；烷基羰基和烷氧基羰基(例如，叔丁氧基羰基(BOC)、乙酰基或异丁酰基)；芳基烷基羰基和芳基烷氧基羰基(例如，苄基氧基羰基)；取代的甲基醚(例如甲氧基甲基醚)；取代的乙醚；取代的苄基醚；四氢吡喃基醚；甲硅烷基(例如，三甲基甲硅烷基、三乙基甲硅烷基、三异丙基甲硅烷基、叔丁基二甲基甲硅烷基、三-异丙基甲硅烷基氧基甲基、[2-(三甲基甲硅烷基)乙氧基]甲基或叔丁基二苯基甲硅烷基)；酯类(例如苯甲酸酯)；碳酸酯类(例如碳酸甲氧基甲基酯)；磺酸酯类(例如甲苯磺酸酯或甲磺酸酯)；非环缩酮(例如二甲基乙缩醛)；环缩酮(例如，1,3-二噁烷、1,3-二氧戊环以及本文所述的那些)；非环乙缩醛；环乙缩醛(例如，本文所述的那些)；非环半缩醛；环半缩醛；环二硫缩酮(例如，1,3-二噻烷或1,3-二硫戊环)；原酸酯(例如，本文所述的那些)以及三芳基甲基基团(例如，三苯甲基；单甲氧基三苯甲基(MMTr)；4,4′-二甲氧基三苯甲基(DMTr)； 4,4′,4″-三甲氧基三苯甲基(TMTr)；以及本文所述的那些)。优选的保护基团选自乙酰基(Ac)、苯甲酰基(Bzl)、苄基(Bn)、异丁酰基(iBu)、苯基乙酰基、苄基氧基甲基乙缩醛(BOM)、β-甲氧基乙氧基甲基醚(MEM)、甲氧基甲基醚(MOM)、对-甲氧基苄基醚(PMB)、甲基硫代甲基醚、新戊酰基(Piv)、四氢吡喃基(THP)、三苯基甲基(Trt)、甲氧基三苯甲基[(4-甲氧基苯基)二苯基甲基](MMT)、二甲氧基三苯甲基、[双-(4-甲氧基苯基)苯基甲基(DMT)、三甲基甲硅烷基醚(TMS)、叔丁基二甲基甲硅烷基醚(TBDMS)、三-异-丙基甲硅烷基氧基甲基醚(TOM)、三-异丙基甲硅烷基醚(TIPS)、甲基醚、乙氧基乙醚(EE)N,N-二甲基甲脒和2-氰基乙基(CE)。
“羟基保护基”是指能够避免羟基遭受化学反应，又可以在特定条件下脱除以恢复羟基的基团。主要包括硅烷型保护基、酰基型保护基或醚型保护基，优选以下：
三甲基硅基(TMS)、三乙基硅基(TES)、二甲基异丙基硅基(DMIPS)、二乙基异丙基硅基(DEIPS)、叔丁基二甲基硅基(TBDMS)、叔丁基二苯基硅基(TBDPS)、三异丙基硅基(TIPS)、乙酰基(Ac)、氯乙酰基、二氯乙酰基、三氯乙酰基、三氟乙酰基(TFA)、苯甲酰基、对甲氧基苯甲酰基、9-芴基甲氧基羰基(Fmoc)、烯丙氧羰基(Alloc)、2,2,2-三氯乙氧羰基(Troc)、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、苯甲基(Bn)、对甲氧基苄基(PMB)、烯丙基、三苯基甲基(Tr)、双对甲氧基三苯甲基(DMTr)、甲氧基甲基(MOM)、苯氧基甲基(BOM)、2,2,2-三氯乙氧基甲基、2-甲氧基乙氧基甲基(MEM)、甲硫基甲基(MTM)、对甲氧基苄氧基甲基(PMBM)、-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基，优选-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基，更优选-C(O)CH2CH2C(O)OH。
本文所用的术语“药学上可接受的盐”表示本发明化合物的那些羧酸盐、氨基酸加成盐，它们在可靠的医学判断范围内适用于与患者组织接触，不会产生不恰当的毒性、刺激作用、变态反应等，与合理的益处/风险比相称，就它们的预期应用而言是有效的，包括(可能的话)本发明化合物的两性离子形式。
本发明包括互变异构体，其为分子中某一原子在两个位置迅速移动而产生的官能团异构体。在不同的互变异构形式存在的化合物，一个所述化合物并不局限于任何特定的互变异构体，而是旨在涵盖所有的互变异构形式。
本发明化合物可包括一个或多个不对称中心，且因此可以存在多种立体异构体形式，例如，对映异构体和/或非对映异构体形式。例如，本发明化合物可为单独的对映异构体、非对映异构体或几何异构体(例如顺式和反式异构体)，或者可为立体异构体的混合物的形式，包括外消旋体混合物和富含一种或多种立体异构体的混合物。异构体可通过本领域技术人员已知的方法从混合物中分离，所述方法包括：手性高压液相色谱法(HPLC)以及手性盐的形成和结晶；或者优选的异构体可通过不对称合成来制备。
本发明还包括同位素标记的化合物(同位素变体)，它们等同于式(I)所述的那些，但一个或多个原子被原子质量或质量数不同于自然界常见的原子质量或质量数的原子所代替。可以引入本发明化合物中的同位素的实例包括氢、碳、氮、氧、磷、硫、氟和氯的同位素，分别例如2H、3H、13C、11C、14C、15N、18O、17O、31P、32P、35S、18F和36Cl。含有上述同位素和/或其它原子的其它同位素的本发明化合物、其前体药物和所述化合物或所述前体药物的药学上可接受的盐都属于本发明的范围。某些同位素标记的本发明化合物、例如引入放射性同位素(例如3H和14C)的那些可用于药物和/或底物组织分布测定。氚、即3H和碳-14、即14C同位素是特别优选的，因为它们容易制备和检测。进而，被更重的同位素取代，例如氘、即2H，由于代谢稳定性更高可以提供治疗上的益处，例如延长体内半衰期或减少剂量需求，因而在有些情况下可能是优选的。同位素标记的本发明式(I)化合物及其前体药物一般可以这样制备，在进行下述流程和/或实施例与制备例所公开的工艺时，用容易得到的同位素标记的试剂代替非同位素标记的试剂。
本发明化合物
本发明具体涉及一种寡核苷酸，其包含一个或多个式(I)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中，
式(I)中的表示H，或表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
X1选自H、Rs或
X2选自OR1或
X1和X2之一为
R1表示H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
式(I)中的和R1不同时为H；
每个L1和L2独立地选自化学键、C1-10亚烷基、C2-10亚烯基、C2-10亚炔基、OC1-10亚烷基、OC1-10亚烯基、OC1-10亚炔基、C(O)C1-10亚烷基；所述L1和L2任选地被1个、2个、3个、4个、5个、6个、7个或8个R取代；
T选自化学键、-O-、-CH2-、-C(O)-、-OC(O)-、-M-、-O-M-、-CH2-M-、-C(O)-M-或-OC(O)-M-；
其中M为
A为糖，所述糖优选为在内含体或溶酶体内可被切割的糖，优选为五元糖或六元糖，更优选为六元糖，例如N-乙酰半乳糖胺、半乳糖、N-乙酰葡萄糖胺、葡萄糖、甘露糖、葡萄糖醛酸、神经氨酸(唾液酸)、木糖或岩藻糖，更优选N-乙酰半乳糖胺或N-乙酰葡萄糖胺；
P为疏水性基团，优选为C8-30烷基、C8-30烯基或C8-30炔基，其中所述基团中不相邻的1、2、3、4、5、6、7、8、9、10个碳原子可以被选自O、S和N的杂原子替换，或者-CH2CH2-基团可以被-OC(O)-、-C(O)O-、-NHC(O)-或-C(O)NH-替换，或者一个或多个碳原子上的取代基可以连接成饱和或不饱和环；所述C8-30烷基、C8-30烯基或C8-30炔基任选地被1个、2个、3个、4个、5个、6个、7个、8个或更多个R取代；
R选自H、D、卤素、C1-6烷基或C1-6卤代烷基，其任选地被氘代，直至完全氘代；
Rs选自H、D、卤素、C1-6烷基、C1-6卤代烷基、3-10元杂环基或C3-10环烷基，其任选地被氘代，直至完全氘代；
m为0、1、2、3、4、5、6、7、8、9或10；
n为0、1、2、3、4、5、6、7、8、9或10；
k＝0、1、2、3、4、5或6。
本发明具体涉及一种寡核苷酸，其中，所述式(I)化合物选自式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中，
Rs’选自H、D、卤素、C1-6烷基、C1-6卤代烷基、3-10元杂环基或C3-10环烷基，其任选地被氘代，直至完全氘代；
其他各基团如前述中所定义。
本发明具体涉及式(II’)或(III’)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
R1和R2独立地选自H、反应性磷基团、羟基保护基或固相载体；
A为乙酰化的糖，所述糖优选为在内含体或溶酶体内可被切割的糖，优选为乙酰化的五元糖或六元糖，例如N-乙酰半乳糖胺、半乳糖、N-乙酰葡萄糖胺、葡萄糖、甘露糖、葡萄糖醛酸、神经氨酸(唾液酸)、木糖或岩藻糖，更优选N-乙酰半乳糖胺或N-乙酰葡萄糖胺；
优选地，A选自
A通过a端与L1相连，通过b端与P基团相连；
P、L1、L2、T、Rs、Rs’、m、n、k如前述所定义。
本发明具体涉及一种双链RNA，其具有正义链和反义链，各链具有14至30个核苷酸，所述反义链包含与所述正义链和靶标mRNA充分互补的序列，其中所述正义链和/或反义链包含一个或多个如前述式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
在一个实施方案中，表示H；在另一个实施方案中，表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置。
R1和R2
在一个实施方案中，R1表示H；在另一个实施方案中，R1表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置。
在一个实施方案中，R1为H；在另一个实施方案中，R1选自反应性磷基团，优选亚磷酰胺、H-膦酸酯、烷基-膦酸酯、磷酸酯或磷酸酯模拟物，例如天然磷酸酯、硫代磷酸酯、二硫代磷酸酯、硼烷磷酸酯、硼烷硫代磷酸酯、膦酸酯、卤素取代的膦酸酯和磷酸酯、氨基磷酸酯、磷酸二酯、磷酸三酯、硫代磷酸二酯、硫代磷酸三酯、二磷酸酯或三磷酸酯，优选-P(OCH2CH2CN)(N(iPr)2)；在另一个实施方案中，R1选自羟基保护基，例如三甲基硅基(TMS)、三乙基硅基(TES)、二甲基异丙基硅基(DMIPS)、二乙基异丙基硅基(DEIPS)、叔丁基二甲基硅基(TBDMS)、叔丁基二苯基硅基(TBDPS)、三 异丙基硅基(TIPS)、乙酰基(Ac)、氯乙酰基、二氯乙酰基、三氯乙酰基、三氟乙酰基(TFA)、苯甲酰基、对甲氧基苯甲酰基、9-芴基甲氧基羰基(Fmoc)、烯丙氧羰基(Alloc)、2,2,2-三氯乙氧羰基(Troc)、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、苯甲基(Bn)、对甲氧基苄基(PMB)、烯丙基、三苯基甲基(Tr)、双对甲氧基三苯甲基(DMTr)、甲氧基甲基(MOM)、苯氧基甲基(BOM)、2,2,2-三氯乙氧基甲基、2-甲氧基乙氧基甲基(MEM)、甲硫基甲基(MTM)、对甲氧基苄氧基甲基(PMBM)、-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基，优选DMTr；在另一个实施方案中，R1为固相载体。
在一个实施方案中，R2为H；在另一个实施方案中，R2选自反应性磷基团，优选亚磷酰胺、H-膦酸酯、烷基-膦酸酯、磷酸酯或磷酸酯模拟物，例如天然磷酸酯、硫代磷酸酯、二硫代磷酸酯、硼烷磷酸酯、硼烷硫代磷酸酯、膦酸酯、卤素取代的膦酸酯和磷酸酯、氨基磷酸酯、磷酸二酯、磷酸三酯、硫代磷酸二酯、硫代磷酸三酯、二磷酸酯或三磷酸酯，优选-P(OCH2CH2CN)(N(iPr)2)；在另一个实施方案中，R2选自羟基保护基，例如三甲基硅基(TMS)、三乙基硅基(TES)、二甲基异丙基硅基(DMIPS)、二乙基异丙基硅基(DEIPS)、叔丁基二甲基硅基(TBDMS)、叔丁基二苯基硅基(TBDPS)、三异丙基硅基(TIPS)、乙酰基(Ac)、氯乙酰基、二氯乙酰基、三氯乙酰基、三氟乙酰基(TFA)、苯甲酰基、对甲氧基苯甲酰基、9-芴基甲氧基羰基(Fmoc)、烯丙氧羰基(Alloc)、2,2,2-三氯乙氧羰基(Troc)、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、苯甲基(Bn)、对甲氧基苄基(PMB)、烯丙基、三苯基甲基(Tr)、双对甲氧基三苯甲基(DMTr)、甲氧基甲基(MOM)、苯氧基甲基(BOM)、2,2,2-三氯乙氧基甲基、2-甲氧基乙氧基甲基(MEM)、甲硫基甲基(MTM)、对甲氧基苄氧基甲基(PMBM)、-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基，优选DMTr；在另一个实施方案中，R1为固相载体。
X1和X2
在一个实施方案中，X1为H；在另一个实施方案中，X1为Rs；在另一个实施方案中，X1为
在一个实施方案中，X2为OR1；在另一个实施方案中，X2为
L1和L2
在一个实施方案中，L1为化学键；在另一个实施方案中，L1为C1-10亚烷基；在另一个实施方案中，L1为C1-6亚烷基；在另一个实施方案中，L1为C2-10亚烯基；在另一个实施方案中，L1为C2-10亚炔基；在另一个实施方案中，L1为OC1-10亚烷基；在另一个实施方案中，L1为OC1-6亚烷基；在另一个实施方案中，L1为OC1-10亚烯基；在另一个实施方案中，L1为OC1-10亚炔基；在另一个实施方案中，L1为C(O)C1-10亚烷基；在另一个实施方案中，L1为C(O)C1-6亚烷基。
在一个具体的实施方案中，L1为OCH2CH2；在另一个具体的实施方案中，L1为O(CH2)5。
在一个实施方案中，L1未被取代；在另一个实施方案中，L1被1个R取代；在另一个实施方案中，L1被2个R取代；在另一个实施方案中，L1被3个R取代；在另一个实施方案中，L1被4个R取代；在另一个实施方案中，L1被5个R取代；在另一个实施方案中，L1被6个R取代；在另一个实施方案中，L1被7个R取代；在另一个实施方案中，L1被8个R取代。
在一个实施方案中，L2为化学键；在另一个实施方案中，L2为C1-10亚烷基；在另一个实施方案中，L2为C1-6亚烷基；在另一个实施方案中，L2为C2-10亚烯基；在另一个实施方案中，L2为C2-10亚炔基；在另一个实施方案中，L2为OC1-10亚烷基；在另一个实施方案中，L2为OC1-6亚烷基；在另一个实施方案中，L2为OC1-10亚烯基；在另一个实施方案中，L2为OC1-10亚炔基；在另一个实施方案中，L2为C(O)C1-10亚烷基；在另一个实施方案中，L2为C(O)C1-6亚烷基。
在一个具体的实施方案中，L2为OCH2；在另一个具体的实施方案中，L2为化学键。
在一个实施方案中，L2未被取代；在另一个实施方案中，L2被1个R取代；在另一个实施方案中，L2被2个R取代；在另一个实施方案中，L2被3个R取代；在另一个实施方案中，L2被4个R取代；在另一个实施方案中，L2被5个R取代；在另一个实施方案中，L2被6个R取代；在另一个 实施方案中，L2被7个R取代；在另一个实施方案中，L2被8个R取代。
T
在一个实施方案中，T化学键；在另一个实施方案中，T为-O-；在另一个实施方案中，T为-CH2-；在另一个实施方案中，T为-C(O)-；在另一个实施方案中，T为-OC(O)-；在另一个实施方案中，T为-M-；在另一个实施方案中，T为-O-M-；在另一个实施方案中，T为-CH2-M-；在另一个实施方案中，T为-C(O)-M-；在另一个实施方案中，T为-OC(O)-M-。
在一个实施方案中，M为在另一个实施方案中，M为在另一个实施方案中，M为在另一个实施方案中，M为
A
在一个实施方案中，A为糖；在另一个实施方案中，A为在内含体或溶酶体内可被切割的糖；在另一个实施方案中，A为五元糖或六元糖，优选为六元糖；在另一个实施方案中，A选自N-乙酰半乳糖胺、半乳糖、N-乙酰葡萄糖胺、葡萄糖、甘露糖、葡萄糖醛酸、神经氨酸(唾液酸)、木糖或岩藻糖，优选N-乙酰半乳糖胺或N-乙酰葡萄糖胺。
在一个具体的实施方案中，A为A通过a端与L1相连，通过b端与P基团相连；在另一个具体的实施方案中，A为A通过a端与L1相连，通过b端与P基团相连；在另一个具体的实施方案中，A为A通过a端与L1相连，通过b端与P基团相连；在另一个具体的实施方案中，A为A通过a端与L1相连，通过b端与P基团相连。
在一个实施方案中，A为乙酰化的糖；在另一个实施方案中，A为乙酰化的糖，所述糖为在内含体或溶酶体内可被切割的糖；在另一个实施方案中，A为乙酰化的五元糖或六元糖，所述五元糖或六元糖是例如N-乙酰半乳糖胺、半乳糖、N-乙酰葡萄糖胺、葡萄糖、甘露糖、葡萄糖醛酸、神经氨酸(唾液酸)、木糖或岩藻糖，更优选所述五元糖或六元糖是N-乙酰半乳糖胺或N-乙酰葡萄糖胺。
在一个具体的实施方案中，A为A通过a端与L1相连，通过b端与P基团相连；在另一个具体的实施方案中，A为A通过a端与L1相连，通过b端与P基团相连；在另一个具体的实施方案中，A为A通过a端与L1相连，通过b端与P基团相连； 在另一个具体的实施方案中，A为
A通过a端与L1相连，通过b端与P基团相连。
P
在一个实施方案中，P为疏水性基团；在另一个实施方案中，P为C8-30烃基，例如C8-30烷基、C8-30烯基或C8-30炔基；在另一个实施方案中，P为C10-22烷基，例如C10烷基、C11烷基、C12烷基、C13烷基、C14烷基、C15烷基、C16烷基、C17烷基、C18烷基、C19烷基、C20烷基、C21烷基、C22烷基；在另一个实施方案中，P为C12-18烷基，例如C14-16烷基；在另一个实施方案中，P为C10-22烯基。
在一个实施方案中，P中不相邻的1、2、3、4、5、6、7、8、9、10个碳原子可以被选自O、S和N的杂原子替换，或者-CH2CH2-基团可以被-OC(O)-、-C(O)O-、-NHC(O)-或-C(O)NH-替换，或者一个或多个碳原子上的取代基可以连接成饱和或不饱和环。
在一个实施方案中，P未被取代；在另一个实施方案中，P被1个R取代；在另一个实施方案中，P被2个R取代；在另一个实施方案中，P被3个R取代；在另一个实施方案中，P被4个R取代；在另一个实施方案中，P被5个R取代；在另一个实施方案中，P被6个R取代；在另一个实施方案中，P被7个R取代；在另一个实施方案中，P被8个R取代；在另一个实施方案中，P被更多个R取代。
在一个具体的实施方案中，P为-(CH2)14-16CH3。
P1、P2、P3和P4
在一个实施方案中，P1为H；在另一个实施方案中，P1为上述定义的P基团。
在一个实施方案中，P2为H；在另一个实施方案中，P2为上述定义的P基团。
在一个实施方案中，P3为H；在另一个实施方案中，P3为上述定义的P基团。
在一个实施方案中，P4为CH3；在另一个实施方案中，P4为上述定义的P基团。
在一个实施方案中，P1、P2、P3、P4中之一为P基团。
R
在一个实施方案中，R为H；在另一个实施方案中，R为D；在另一个实施方案中，R为卤素；在另一个实施方案中，R为C1-6烷基；在另一个实施方案中，R为C1-4烷基；在另一个实施方案中，R为C1-6卤代烷基；在另一个实施方案中，R任选地被氘代，直至完全氘代。
Rs和Rs’
在一个实施方案中，Rs为H；在另一个实施方案中，Rs为D；在另一个实施方案中，Rs为卤素；在另一个实施方案中，Rs为C1-6烷基；在另一个实施方案中，Rs为C1-6卤代烷基；在另一个实施方案中，Rs为3-10元杂环基；在另一个实施方案中，Rs为C3-10环烷基；在另一个实施方案中，Rs任选地被氘代，直至完全氘代。
在一个具体的实施方案中，Rs为H。
在一个实施方案中，Rs’为H；在另一个实施方案中，Rs’为D；在另一个实施方案中，Rs’为卤素；在另一个实施方案中，Rs’为C1-6烷基；在另一个实施方案中，Rs’为C1-6卤代烷基；在另一个实施方案中，Rs’为3-10元杂环基，例如5-10元杂环基；在另一个实施方案中，Rs’为C3-10环烷基；在另一个实施方案中，Rs’任选地被氘代，直至完全氘代。
在一个具体的实施方案中，Rs’为环己基。
m
m为0、1、2、3、4、5、6、7、8、9或10。
n
n为0、1、2、3、4、5、6、7、8、9或10。
k
k＝0、1、2、3、4、5或6。
以上任一具体实施方案中的任一技术方案或其任意组合，可以与其它具体实施方案中的任一技术方案或其任意组合进行组合。例如，A的任一技术方案或其任意组合，可以与P、P1-P4、R1、R2、X1、X2、L1、L2、T、R、Rs、Rs’、m、n和k等的任一技术方案或其任意组合进行组合。本发明旨在包括所有这些技术方案的组合，限于篇幅，不再一一列出。
本发明还提供了载体，其包含编码本发明所述的siRNA的核苷酸序列。本发明的载体能够扩增或表达与其连接的编码本发明所述的siRNA的核苷酸。
例如，靶向PCSK9基因的siRNA可以从插入DNA或RNA载体中的转录单位表达。表达可以是短暂的(数小时至数星期内)或持续的(数星期至数个月或更久)，取决于所使用的特定建构体及靶组织或细胞类型。可以将siRNA的编码核苷酸引入线性建构体、环状质体或病毒载体中。siRNA的核苷酸可以被整合到细胞基因组中稳定表达，或者在染色体外稳定遗传而表达。一般来说，siRNA表达载体通常是DNA质粒或病毒载体。
包含siRNA的编码序列的病毒载体系统包括但不局限于：(a)腺病毒载体；(b)逆转录病毒载体；(c)腺伴随病毒载体；(d)单纯疱疹病毒载体；(e)SV40载体；(f)多瘤病毒载体；(g)乳头瘤病毒载体；(h)微小核糖核酸病毒载体；(i)痘病毒载体；以及(j)辅助病毒依赖性腺病毒或无肠腺病毒。
本发明还提供了细胞，其含有本发明所述的siRNA或载体，其中本发明所述的siRNA或载体能够在细胞中转录。
本发明具体涉及以下技术方案：
A1.寡核苷酸，其包含一个或多个式(I)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中，
式(I)中的表示H，或表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
X1选自H、Rs或
X2选自OR1或
X1和X2之一为
R1表示H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
式(I)中的和R1不同时为H；
每个L1和L2独立地选自化学键、C1-10亚烷基、C2-10亚烯基、C2-10亚炔基、OC1-10亚烷基、OC1-10亚烯基、OC1-10亚炔基、C(O)C1-10亚烷基；所述L1和L2任选地被1个、2个、3个、4个、5个、6个、7个或8个R取代；
T选自化学键、-O-、-CH2-、-C(O)-、-OC(O)-、-M-、-O-M-、-CH2-M-、-C(O)-M-或-OC(O)-M-；
其中M为
A为糖，所述糖优选为在内含体或溶酶体内可被切割的糖，优选为五元糖或六元糖，更优选为六元糖，例如N-乙酰半乳糖胺、半乳糖、N-乙酰葡萄糖胺、葡萄糖、甘露糖、葡萄糖醛酸、神经氨酸(唾液酸)、木糖或岩藻糖，更优选N-乙酰半乳糖胺或N-乙酰葡萄糖胺；
P为疏水性基团，优选为C8-30烷基、C8-30烯基或C8-30炔基，其中所述基团中不相邻的1、2、3、4、5、6、7、8、9、10个碳原子可以被选自O、S和N的杂原子替换，或者-CH2CH2-基团可以被-OC(O)-、-C(O)O-、-NHC(O)-或-C(O)NH-替换，或者一个或多个碳原子上的取代基可以连接成饱和或不饱和环；所述C8-30烷基、C8-30烯基或C8-30炔基任选地被1个、2个、3个、4个、5个、6个、7个、8个或更多个R取代；
R选自H、D、卤素、C1-6烷基或C1-6卤代烷基，其任选地被氘代，直至完全氘代；
Rs选自H、D、卤素、C1-6烷基、C1-6卤代烷基、3-10元杂环基或C3-10环烷基，其任选地被氘代，直至完全氘代；
m为0、1、2、3、4、5、6、7、8、9或10；
n为0、1、2、3、4、5、6、7、8、9或10；
k＝0、1、2、3、4、5或6。
A2.技术方案A1的寡核苷酸，其中，所述式(I)化合物选自式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中，
表示H，或表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
R1表示H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
和R1不同时为H；
每个L1和L2独立地选自化学键、C1-10亚烷基、C2-10亚烯基、C2-10亚炔基、OC1-10亚烷基、OC1-10亚烯基、OC1-10亚炔基或C(O)C1-10亚烷基；所述L1和L2任选地被1个、2个、3个、4个、5个、6个、7个或8个R取代；
T选自化学键、-O-、-CH2-、-C(O)-、-OC(O)-、-M-、-O-M-、-CH2-M-、-C(O)-M-或-OC(O)-M-；
其中M为
A为糖，所述糖优选为在内含体或溶酶体内可被切割的糖，优选为五元糖或六元糖，更优选为六元糖，例如N-乙酰半乳糖胺、半乳糖、N-乙酰葡萄糖胺、葡萄糖、甘露糖、葡萄糖醛酸、神经氨酸(唾液酸)、木糖或岩藻糖，更优选N-乙酰半乳糖胺或N-乙酰葡萄糖胺；
P为疏水性基团，优选为C8-30烷基、C8-30烯基或C8-30炔基，其中所述基团中不相邻的1、2、3、4、5、6、7、8、9、10个碳原子可以被选自O、S和N的杂原子替换，或者-CH2CH2-基团可以被-OC(O)-、-C(O)O-、-NHC(O)-或-C(O)NH-替换，或者一个或多个碳原子上的取代基可以连接成饱和或不饱和环；所述C8-30烷基、C8-30烯基或C8-30炔基任选地被1个、2个、3个、4个、5个、6个、7个、8个或更多个R取代；
R选自H、D、卤素、C1-6烷基或C1-6卤代烷基，其任选地被氘代，直至完全氘代；
Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基，其任选地被氘代，直至完全氘代；
Rs’选自H、D、卤素、C1-6烷基、C1-6卤代烷基、3-10元杂环基或C3-10环烷基，其任选地被氘代，直至完全氘代；
m为0、1、2、3、4、5、6、7、8、9或10；
n为0、1、2、3、4、5、6、7、8、9或10；
k＝0、1、2、3、4、5或6。
A3.技术方案A1或A2的寡核苷酸，其中，
表示H，或表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
R1表示H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
和R1不同时为H；
每个L1和L2独立地选自化学键、C1-6亚烷基、OC1-6亚烷基或C(O)C1-6亚烷基；所述L1和L2任选地被1个、2个、3个、4个或5个R取代；
T选自化学键、-O-、-CH2-、-C(O)-、-OC(O)-、-M-、-O-M-、-CH2-M-、-C(O)-M-或-OC(O)-M-；
其中M为
A选自N-乙酰半乳糖胺、半乳糖、N-乙酰葡萄糖胺、葡萄糖、甘露糖、葡萄糖醛酸、神经氨酸(唾液酸)、木糖或岩藻糖，更优选为N-乙酰半乳糖胺或N-乙酰葡萄糖胺；
P选自C10-22烷基或C10-22烯基，所述C10-22烷基或C10-22烯基任选地被1个、2个、3个、4个或5个R取代；
R选自H、D、卤素、C1-6烷基或C1-6卤代烷基；
Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基；
Rs’选自H、D、卤素、C1-6烷基、C1-6卤代烷基、3-10元杂环基或C3-10环烷基；
m为0、1、2、3、4或5；
n为0、1、2、3、4或5；
k为0、1、2、3、4或5。
A4.技术方案A1-A3中任一项的寡核苷酸，其中，
表示H，或表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
R1表示H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
和R1不同时为H；
每个L1和L2独立地选自化学键或OC1-6亚烷基；所述L1和L2任选地被1个、2个或3个R取代；
T选自化学键、-O-、-CH2-、-OC(O)-或-C(O)-；
A选自
A通过a端与L1相连，通过b端与P基团相连；
P选自C10-22烷基或C10-22烯基，所述C10-22烷基或C10-22烯基任选地被1个、2个或3个R取代；
R选自H、D、卤素、C1-4烷基；
Rs选自H或D；
Rs’选自H、D或C5-10环烷基；
m为0、1、2或3；
n为0、1、2或3；
k为0、1、2或3。
A5.技术方案A1-A4中任一项的寡核苷酸，其中，
表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
R1为H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
L1为OCH2CH2或O(CH2)5；
L2为OCH2或化学键；
T选自化学键、-O-或-C(O)-；
A选自
A通过a端与L1相连，通过b端与P基团相连；
P为-(CH2)14-16CH3；
Rs为H；
Rs’为环己基；
m为0、1或2；
n为0或1；
k为0或1。
A6.技术方案A1-A5中任一项的寡核苷酸，其中，所述式(II)或式(III)化合物选自以下通式化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中，
P1、P2、P3选自H或P基团；
P4选自CH3或P基团；
P1、P2、P3、P4中之一为P基团；
各变量如技术方案A1-A5中所定义。
A7.技术方案A1-A6中任一项的寡核苷酸，其中，所述式(II)或式(III)化合物选自以下化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中，表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置。
R1为H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置。
A8.技术方案A1-A7中任一项的寡核苷酸，其具有14至30个核苷酸。
A9.技术方案A1-A8中任一项的寡核苷酸，其在5’端包含一个技术方案A1-A7中任一项的式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
A10.技术方案A1-A9中任一项的寡核苷酸，其在3’端包含一个技术方案A1-A7中任一项的式(II) 或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
A11.技术方案A1-A10中任一项的寡核苷酸，其在5’端和3’端分别包含一个技术方案A1-A7中任一项的式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
A12.技术方案A1-A11中任一项的寡核苷酸，其在寡核苷酸内部包含一个或多个技术方案A1-A7中任一项的式(II)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
A13.寡核苷酸，其在寡核苷酸内部、5’端和/或3’端包含一个、两个或多个递送载体，所述递送载体为被疏水性基团修饰的糖；
优选地，所述被疏水性基团修饰的糖选自式(X)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中，表示与寡核苷酸的连接位置；
P为疏水性基团；
A为糖部分；
P和A如技术方案A1-A5中任一项所定义；
优选地，所述疏水性基团连接到糖部分的羟基或乙酰基上；
优选地，所述被疏水性基团修饰的糖选自以下化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中，表示与寡核苷酸的连接位置。
A14.技术方案A1-A13中任一项的寡核苷酸，所述寡核苷酸是ASO或siRNA，优选用于抑制在肝外表达的基因，更优选用于抑制在中枢神经系统(CNS)和/或眼部表达的基因。
A15.式(II’)或(III’)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
R1和R2独立地选自H、反应性磷基团、羟基保护基或固相载体；
A为乙酰化的糖，所述糖优选为在内含体或溶酶体内可被切割的糖，优选为乙酰化的五元糖或六元糖，所述五元糖或六元糖是例如N-乙酰半乳糖胺、半乳糖、N-乙酰葡萄糖胺、葡萄糖、甘露糖、葡萄糖醛酸、神经氨酸(唾液酸)、木糖或岩藻糖，更优选所述五元糖或六元糖是N-乙酰半乳糖胺或N-乙酰葡萄糖胺；
优选地，A选自
A通过a端与L1相连，通过b端与P基团相连；
P、L1、L2、T、Rs、Rs’、m、n、k如技术方案A1-A7中任一项所定义。
A16.技术方案A15的化合物，其中，R1和R2为H。
A17.技术方案A15或A16的化合物，其中，R1和R2之一为反应性磷基团，优选亚磷酰胺、H-膦酸酯、烷基-膦酸酯、磷酸酯或磷酸酯模拟物，例如天然磷酸酯、硫代磷酸酯、二硫代磷酸酯、硼烷磷酸酯、硼烷硫代磷酸酯、膦酸酯、卤素取代的膦酸酯和磷酸酯、氨基磷酸酯、磷酸二酯、磷酸三酯、硫代磷酸二酯、硫代磷酸三酯、二磷酸酯或三磷酸酯，优选-P(OCH2CH2CN)(N(iPr)2)。
A18.技术方案A15-A17中任一项的化合物，其中，R1和R2选自保护基，优选羟基保护基，例如三甲基硅基(TMS)、三乙基硅基(TES)、二甲基异丙基硅基(DMIPS)、二乙基异丙基硅基(DEIPS)、叔丁基二甲基硅基(TBDMS)、叔丁基二苯基硅基(TBDPS)、三异丙基硅基(TIPS)、乙酰基(Ac)、氯乙酰基、二氯乙酰基、三氯乙酰基、三氟乙酰基(TFA)、苯甲酰基、对甲氧基苯甲酰基、9-芴基甲氧基羰基(Fmoc)、烯丙氧羰基(Alloc)、2,2,2-三氯乙氧羰基(Troc)、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、苯甲基(Bn)、对甲氧基苄基(PMB)、烯丙基、三苯基甲基(Tr)、双对甲氧基三苯甲基(DMTr)、甲氧基甲基(MOM)、苯氧基甲基(BOM)、2,2,2-三氯乙氧基甲基、2-甲氧基乙氧基甲基(MEM)、甲硫基甲基(MTM)、对甲氧基苄氧基甲基(PMBM)、-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基，优选DMTr。
A19.技术方案A15-A18中任一项的化合物，其中所述式(II’)或(III’)化合物选自以下化合物，或其药学上可接受的盐、互变异构体或立体异构体：

A20.双链RNA，其具有正义链和反义链，各链具有14至30个核苷酸，所述反义链包含与所述正义链和靶标mRNA充分互补的序列，其中所述正义链和/或反义链包含一个或多个如技术方案A1-A7中任一项所述式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
A21.技术方案A20的双链RNA，其中所述式(II)或式(III)化合物选自以下通式化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中，各变量如技术方案A1-A7中所定义。
A22.技术方案A20或A21的双链RNA，其中所述式(II)或式(III)化合物选自以下化合物，或其药学上可接受的盐、互变异构体或立体异构体：

其中，表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置。
R1为H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置。
A23.技术方案A20-A22中任一项的双链RNA，其中所述正义链在5’端包含一个技术方案A1-A7中任一项所述式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
A24.技术方案A20-A23中任一项的双链RNA，其中所述正义链在3’端包含一个技术方案A1-A7中任一项所述式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
A25.技术方案A20-A24中任一项的双链RNA，其中所述正义链在5’端和3’端分别包含一个技术方案A1-A7中任一项所述式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
A26.技术方案A20-A25中任一项的双链RNA，其中所述正义链在寡核苷酸内部包含一个或多个技术方案A1-A7中任一项所述式(II)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
A27.技术方案A20-A26中任一项的双链RNA，其中所述反义链在5’端包含一个技术方案A1-A7中任一项所述式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
A28.技术方案A20-A27中任一项的双链RNA，其中所述反义链在3’端包含一个技术方案A1-A7中任一项所述式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
A29.技术方案A20-A28中任一项的双链RNA，其中所述反义链在5’端和3’端分别包含一个技术方案A1-A7中任一项所述式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
A30.技术方案A20-A29中任一项的双链RNA，其中所述反义链在寡核苷酸内部包含一个或多个技术方案A1-A7中任一项所述式(II)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
A31.双链RNA，其具有正义链和反义链，各链具有14至30个核苷酸，所述反义链包含与所述正义链和靶标mRNA充分互补的序列，其中所述正义链和/或反义链在内部、5’端和/或3’端包含一个、两个或多个递送载体，所述递送载体为被疏水性基团修饰的糖；
优选地，所述被疏水性基团修饰的糖选自式(X)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中，表示与寡核苷酸的连接位置；
P为疏水性基团；
A为糖部分；
P和A如技术方案A1-A5中任一项所定义；
优选地，所述疏水性基团连接到糖部分的羟基或乙酰基上；
优选地，所述被疏水性基团修饰的糖选自以下化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中，表示与寡核苷酸的连接位置。
A32.载体，其包含编码前述技术方案A20-A31中任一项所述的双链RNA的核苷酸序列。
A33.细胞，其含有如技术方案A20-A31中任一项所述的双链RNA或如技术方案A32所述的载体。
A34.药物组合物，其包含如技术方案A20-A31中任一项所述的双链RNA、如技术方案A32所述的载体、或如技术方案A33所述的细胞，以及任选的药学上可接受的载剂或赋形剂。
A35.试剂盒，其包含如技术方案A20-A31中任一项所述的双链RNA、如技术方案A32所述的载体、或如技术方案A33所述的细胞。
本发明还具体涉及以下技术方案：
B1.寡核苷酸，其包含一个或多个式(IV)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
表示H，或表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
R1表示H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
和R1不同时为H；
每个L1和L2独立地选自化学键、C1-10亚烷基、C2-10亚烯基、C2-10亚炔基、OC1-10亚烷基、OC1-10亚烯基、OC1-10亚炔基或C(O)C1-10亚烷基；所述L1和L2任选地被1个、2个、3个、4个、5个、6个、7个或8个R取代；
T选自化学键、-O-、-CH2-、-C(O)-、-OC(O)-、-M-、-O-M-、-CH2-M-、-C(O)-M-或-OC(O)-M-；
其中M为
A为N-乙酰葡萄糖胺；
R选自H、D、卤素、C1-6烷基或C1-6卤代烷基，其任选地被氘代，直至完全氘代；
Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基，其任选地被氘代，直至完全氘代；
m为0、1、2、3、4、5、6、7、8、9或10；
n为0、1、2、3、4、5、6、7、8、9或10；
k＝0、1、2、3、4、5或6。
B2.技术方案B1的寡核苷酸，其中，
表示H，或表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
R1表示H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
和R1不同时为H；
每个L1和L2独立地选自化学键、C1-6亚烷基、OC1-6亚烷基或C(O)C1-6亚烷基；所述L1和L2任选地被1个、2个、3个、4个或5个R取代；
T选自化学键、-O-、-CH2-、-C(O)-、-OC(O)-、-M-、-O-M-、-CH2-M-、-C(O)-M-或-OC(O)-M-；
其中M为
A是N-乙酰半乳糖胺；
R选自H、D、卤素、C1-6烷基或C1-6卤代烷基；
Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基；
m为0、1、2、3、4或5；
n为0、1、2、3、4或5；
k为0、1、2、3、4或5。
B3.技术方案B1或B2的寡核苷酸，其中，
表示H，或表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
R1表示H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
和R1不同时为H；
每个L1和L2独立地选自化学键或OC1-6亚烷基，例如，OCH2、OCH2CH2、(OCH2CH2)2或(OCH2CH2)3；所述L1和L2任选地被1个、2个或3个R取代；
T选自化学键、-O-、-CH2-、-OC(O)-或-C(O)-，优选为-C(O)-；
A为
R选自H、D、卤素、C1-4烷基；
Rs选自H或D；
m为0、1、2或3；
n为0、1、2或3；
k为0、1、2或3。
B4.技术方案B1-B3中任一项的寡核苷酸，其中，所述化合物选自：
其中，
表示通过磷酸酯基团、硫代磷酸酯基团连接至上一个核苷酸；
表示通过磷酸酯基团、硫代磷酸酯基团连接至下一个核苷酸。
B5.式(IV’)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
其中，
R1和R2独立地选自H、反应性磷基团、羟基保护基或固相载体；
A为
L1、L2、T、Rs、m、n、k如前述所定义。
B6.技术方案B5的化合物，其中，R1和R2为H；
优选地，R1和R2之一为反应性磷基团，优选亚磷酰胺、H-膦酸酯、烷基-膦酸酯、磷酸酯或磷酸酯模拟物，例如天然磷酸酯、硫代磷酸酯、二硫代磷酸酯、硼烷磷酸酯、硼烷硫代磷酸酯、膦酸酯、卤素取代的膦酸酯和磷酸酯、氨基磷酸酯、磷酸二酯、磷酸三酯、硫代磷酸二酯、硫代磷酸三酯、二磷酸酯或三磷酸酯，优选-P(OCH2CH2CN)(N(iPr)2)；
R1和R2选自保护基，优选羟基保护基，例如三甲基硅基(TMS)、三乙基硅基(TES)、二甲基异丙基硅基(DMIPS)、二乙基异丙基硅基(DEIPS)、叔丁基二甲基硅基(TBDMS)、叔丁基二苯基硅基(TBDPS)、三异丙基硅基(TIPS)、乙酰基(Ac)、氯乙酰基、二氯乙酰基、三氯乙酰基、三氟乙酰基(TFA)、苯甲酰基、对甲氧基苯甲酰基、9-芴基甲氧基羰基(Fmoc)、烯丙氧羰基(Alloc)、2,2,2-三氯乙氧羰基(Troc)、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、苯甲基(Bn)、对甲氧基苄基(PMB)、烯丙基、三苯基甲基(Tr)、双对甲氧基三苯甲基(DMTr)、甲氧基甲基(MOM)、苯氧基甲基(BOM)、2,2,2-三氯乙氧基甲基、 2-甲氧基乙氧基甲基(MEM)、甲硫基甲基(MTM)、对甲氧基苄氧基甲基(PMBM)、-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基，优选DMTr。
B7.技术方案B5或B6的化合物，其中所述式(II’)或(III’)化合物选自以下化合物，或其药学上可接受的盐、互变异构体或立体异构体：
B8.技术方案B1-B4中任一项的寡核苷酸，其中，所述寡核苷酸为siRNA，用于抑制在肝脏表达的基因。
具体化合物列表
本发明化合物在寡核苷酸中的编号和结构如下，其中，以5’–>3’的顺序从化合物的至连接。
具体而言，根据5’–>3’的顺序，如相应结构位于核酸链中间位置，表示通过磷酸酯基团、硫代磷酸酯基团或其他连接基团连接至上一个核苷酸或核苷酸类似物的3’碳或相应位置，表示通过磷酸酯基团、硫代磷酸酯基团或其他连接基团连接至下一个核苷酸或核苷酸类似物的5’碳或相应位置；如相应结构位于核酸链末端位置，相应地表示通过磷酸酯基团、硫代磷酸酯基团或其他连接基团连接至核酸链3’或5’末端。


合成实施例以下实施例用于例示本发明而非限制本发明的范围。
缩写
实施例1.化合物BE1的制备
1.化合物3的制备
在25℃下将化合物1(1.00g,2.03mmol)溶于DCM(30.0mL)，加入HATU(0.850g,2.23mmol)和DIEA(1.34mL,8.11mmol)，搅拌0.5小时，然后加入化合物2(1.00g,2.229mmol)，反应液在25℃下搅拌2.5小时。LCMS显示有产物的mass值。薄层色谱(DCM/MeOH＝10/1)显示有新点生成。将反应液旋干得到粗品。粗品进行正相硅胶柱(DCM/MeOH＝1/0-10/1)纯化得到白色固体化合物3(1.70g,收率90.69％)。
1H NMR(400MHz,CD3OD)δ7.37-7.47(m,2H),7.16-7.34(m,7H),6.71-6.92(m,4H),5.22-5.37(m,1H),5.05-5.15(m,1H),4.69-4.80(m,1H),3.55-4.24(m,28H),3.19-3.30(m,2H),2.09-2.15(m,3H),1.87-2.00(m,9H)
2.化合物BE1的制备
在25℃下化合物3(1.70g,1.84mmol)溶于DCM(20.0mL)，依次加入DCI(0.160g,1.38mmol)和化合物4(0.830g,2.76mmol)，反应液在氮气氛围下和25℃条件下搅拌2小时。LCMS显示化合物3消耗完全且有化合物BE1的mass值。薄层色谱(DCM/MeOH＝10/1)显示有新点生成。将反应液旋干得到粗品。粗品进行正相硅胶柱(二氯甲烷/丙酮＝1/0-5/1)纯化得到淡黄色油状化合物BE1(1.02g,收率49.32％)。
1H NMR(400MHz,CDCl3)δ7.37-7.48(m,2H),7.23-7.36(m,7H),6.78-6.90(m,4H),5.17-5.28(m,1H),4.90-5.08(m,2H),4.04-4.36(m,6H),3.75-3.97(m,14H),3.54-3.71(m,9H),3.09-3.48(m,5H),2.51-2.67(m,2H),2.12-2.16(m,3H),2.02-2.07(m,3H),1.96-2.00(m,3H),1.89-1.93(m,3H),1.11-1.22(m,12H).
实施例2.化合物BE2的制备
1.化合物3的制备
在25℃下将化合物1(1.0g,2.23mmol)溶于DCM(20.0mL)中,加入HATU(1.27g,3.34mmol)，DIEA(1.10mL,6.68mmol)。混合液体在25℃下搅拌0.5小时后加入化合物2(1.20g,2.67mmol)，混合液体继续搅拌12小时。TLC(T1:DCM/MEOH＝10/1)显示原料消耗完全。向其中加入200mL二氯甲烷。依次用饱和碳酸氢钠溶液(30.0mL x 3)和饱和食盐水洗涤三次(30.0mL x 3)，有机相用无水硫酸钠干燥后在减压条件下旋干得到粗产品。粗产品经柱层析纯化(DCM/MeOH＝1/0至20/1)得到黄色油状化合物3(1.43g,1.70mmol)。
1H NMR(400MHz,CD3OD)δ7.44(d,J＝7.2Hz,2H),7.18-7.35(m,7H),6.87(t,J＝8.0Hz,4H),5.32(s,1H),5.04(d,J＝11.6Hz,1H),4.66(dd,J＝4.4,8.4Hz,1H),4.06-4.29(m,6H),3.81-3.96(m,2H),3.78(s,6H),3.66-3.76(m,3H),3.47-3.65(m,6H),3.33-3.38(m,1H),3.15-3.25(m,2H),2.11(d,J＝16.8Hz,3H),1.84-2.01(m,9H)
2.化合物BE2的制备
在25℃下将化合物3(1.42g,1.61mmol)溶于DCM(15.0mL)中，加入分子筛若干，之后将DCI(140mg,1.21mmol)加入到其中，在氮气氛围下将化合物4(730mg,2.42mmol)加入到其中，混合液体在25℃下搅拌1.0小时。TLC(DCM/ACE＝10/1)显示有新点生成，向其中加入0.5mL三乙胺，加入二氯甲烷20毫升，用碱性硅胶拌样，流动相二氯甲烷加入4/1000的三乙胺溶液。经柱层析纯化(DCM/ACE＝1/0至5/1)得到无色油状化合物BE2(900mg,收率51.64％)。
1H NMR(400MHz,CDCl3)δ7.35-7.49(m,2H),7.24-7.34(m,7H),6.75-6.88(m,4H),5.26-5.38(m,1H),4.99-5.09(m,1H),4.85-4.95(m,1H),3.09-4.33(m,30H),2.54-2.68(m,2H),2.13-2.19(m,3H),2.02-2.06(m,3H),1.97-2.01(m,6H),1.24(d,J＝14.4Hz,12H).
31P NMR(162MHz,CDCl3)δ149.32,148.96,14.16.
实施例3.化合物BE3的制备
1.化合物1的制备
在25℃下将化合物1(1.17g,2.18mmol)溶于DCM(20.0mL)中,加入HATU(1.24g,3.27mmol)，DIEA(1.08mL,6.53mmol)。混合液体在25℃下搅拌0.5小时后加入化合物2(1.08g,2.40mmol)，混合液体继续搅拌12小时。LCMS显示产物生成。TLC(T1:DCM/MEOH＝10/1)显示原料消耗完全。向其中加入100mL二氯甲烷。依次用饱和碳酸氢钠溶液(20.0mL x 3)和饱和食盐水洗涤三次(20.0mL x 3),有机相用无水硫酸钠干燥后在减压条件下悬干得到粗产品。粗产品经柱层析纯化(DCM/MeOH＝1/0to 20/1)得到黄色油状化合物3(1.67g,收率79.2％)。
1H NMR(400MHz,CD3OD)δ7.44(d,J＝7.6Hz,2H),7.16-7.34(m,7H),6.86(t,J＝7.6Hz,4H),5.32(d,J＝3.2Hz,1H),5.05(dd,J＝3.6,11.2Hz,1H),4.67(dd,J＝3.6,8.0Hz,1H),4.21-4.26(m,1H),4.14-4.18(m,1H),4.07-4.13(m,4H),3.94-4.06(m,3H),3.87-3.91(m,1H),3.78(s,6H),3.69-3.76(m,3H),3.55-3.66(m,13H),3.51-3.52(m,1H),3.13-3.29(m,2H),2.12(d,J＝1.6Hz,3H),2.00-2.02(m,3H),1.99(s,1H),1.91-1.96(m,6H),1.20-1.28(m,2H)
2.化合物BE3的制备
在25℃下化合物3(1.67g,1.72mmol)溶于DCM(30.0mL)，依次加入DCI(0.150g,1.29mmol)和化合物4(0.780g,2.59mmol)，反应液在氮气氛围下和25℃条件下搅拌2小时。薄层色谱(DCM/MeOH＝10/1)显示有新点生成。将反应液旋干得到粗品。粗品进行正相硅胶柱(二氯甲烷/丙酮＝1/0-5/1)纯化得到淡黄色油状化合物BE3(1.60g,收率79.40％)。
1H NMR(400MHz,CDCl3)δ7.38-7.47(m,2H),7.24-7.35(m,7H),6.78-6.90(m,4H),6.54-6.69(m,1H),5.29-5.31(m,1H),4.97-5.06(m,1H),4.76-4.85(m,1H),4.10-4.26(m,6H),3.76-4.06(m,15H),3.59-3.74(m,12H),3.10-3.54(m,5H),2.51-2.67(m,2H),2.14-2.17(m,3H),2.05-2.07(m,3H),1.99(s,6H),1.12-1.24(m,12H).
31P NMR(162MHz,CDCl3)δ149.16,148.87,148.81.
实施例4.化合物BE4的制备
1.化合物1c的制备
室温下向化合物1b(20g，78.0mmol)的DMF(100mL)溶液中加入DIEA(38.7mL，234mmol)和HATU(32.6g，85.8mmol)。半小时后，加入化合物1a(16.8g，78.0mmol)。混悬反应液室温下搅拌16小时，反应完成后，加入水(400mL)稀释，过滤收集滤饼，干燥得到化合物1c(30g，71.8mmol，92.1％)。
1H NMR(400MHz,DMSO-d6)δ7.61(d,J＝8.3Hz,1H),6.38(d,J＝6.5Hz,1H),4.56(t,J＝5.6Hz,1H),4.48–4.34(m,3H),3.66–3.59(m,1H),3.57–3.43(m,2H),3.43–3.37(m,1H),2.07(t,J＝7.5Hz,2H),1.52–1.42(m,2H),1.24(s,26H),0.90–0.80(m,3H).
2.化合物1d的制备
氮气氛下，将化合物1c(20g，47.9mmol)混悬于干燥吡啶(100mL)中，冰水浴降温至0℃，然后分批加入乙酸酐(45mL，479mmol)。加完后，缓慢升至室温，然后室温下搅拌16小时。反应完成后，反应液用乙酸乙酯稀释，水洗涤一次，水相用乙酸乙酯再萃取两次，合并有机相后，有机相 用盐水洗涤，无水硫酸钠干燥后，过滤，真空减压浓缩得到残留物，残留物经硅胶柱层析纯化(洗脱液：乙酸乙酯/石油醚)得到化合物1d(10.2g，17.4mmol，36.3％)。
1H NMR(400MHz,DMSO-d6)δ7.84(d,J＝9.2Hz,1H),5.65(d,J＝8.8Hz,1H),5.27(d,J＝3.1Hz,1H),5.07(dd,J＝11.3,3.4Hz,1H),4.23–4.18(m,1H),4.16–4.09(m,1H),4.07–3.96(m,2H),2.12(s,3H),2.02(s,3H),1.99(s,3H),1.89(s,3H),1.43(p,J＝7.1Hz,2H),1.23(s,26H),0.89–0.81(m,3H).
3.化合物1e的制备
氮气氛下将化合物1d(5g，8.54mmol)溶于DCM(50mL)中，冰水浴降温到0至5℃，滴加TMSOTf(2.28g，10.250mmol)。然后室温搅拌16小时，反应完成后，0至5℃下将反应液分批加入到饱和碳酸氢钠溶液中，水相用二氯甲烷萃取两次，有机相合并后用饱和食盐水洗涤一次，无水硫酸钠干燥后，过滤，真空减压浓缩得到残留物，残留物经硅胶柱层析纯化(洗脱液：二氯甲烷/乙酸乙酯)得到化合物1e(4.2g，7.99mmol，93.5％)。
4.化合物1g的制备
在0至5℃下，向化合物1e(4.2g，7.99mmol)的DCM(20mL)溶液中加入化合物1f(1.73g，6.80mmol)和TMSOTf(0.89g，4.00mmol)。反应液室温搅拌16小时。反应完成后，将反应液分批倾入到预先降温到0至5℃的饱和碳酸氢钠(40mL)溶液中，分去有机相后，水相用二氯甲烷萃取两次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，过滤，滤液真空减压浓缩得到残留物，残留物经硅胶柱层析纯化(洗脱液：乙酸乙酯/二氯甲烷)得到化合物1g(2.83g，3.63mmol，45.4％)。
1H NMR(400MHz,DMSO-d6)δ7.71(d,J＝9.2Hz,1H),7.41–7.33(m,5H),5.21(d,J＝3.3Hz,1H),5.15(s,2H),4.98(dd,J＝11.2,3.4Hz,1H),4.58(d,J＝8.4Hz,1H),4.19(s,2H),4.08–3.97(m,3H),3.94–3.85(m,1H),3.81–3.73(m,1H),3.64–3.55(m,3H),3.57–3.47(m,4H),2.10(s,3H),2.03–1.94(m,5H),1.87(s,3H),1.49–1.38(m,2H),1.22(s,24H),0.88–0.82(m,3H).
5.化合物1h的制备
将化合物1g(2.5g，3.10mmol)溶于MeOH(20mL)中，氮气置换体系三次后，加入Pd/C(0.566g，0.1％wt％)，再将体系用氢气置换三次，反应液在氢气氛下室温搅拌16小时。反应完全后，将反应液通过硅藻土过滤除去多余的Pd/C，将滤液真空减压浓缩得到油状物化合物1h(2.0g，2.90mmol，80.0％)。
1H NMR(400MHz,DMSO-d6)δ7.73(d,J＝9.2Hz,1H),5.21(d,J＝3.4Hz,1H),4.99(dd,J＝11.2,3.4Hz,1H),4.59(d,J＝8.5Hz,1H),4.06–4.01(m,2H),4.01–3.98(m,2H),3.93–3.84(m,1H),3.80–3.73(m,1H),3.61–3.54(m,4H),3.53–3.49(m,4H),2.11(s,3H),2.00(s,3H),1.87(s,3H),1.49–1.38(m,2H),1.22(s,26H),0.89–0.82(m,3H).
6.化合物1j的制备
向化合物1h(1g，1.45mmol)的DCM(10mL)溶液中依次加入DIEA(0.7mL，4.35mmol)，EDCI(0.42g，2.18mmol)和HOBt(0.29g，2.18mmol)。半个小时后加入化合物1i(0.65g，1.45mmol)。反应混合溶液室温下搅拌16小时，反应完成后，反应液用水洗涤，水相用二氯甲烷萃取两次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，过滤后真空减压浓缩得到残留物，残留物经反相快速柱层析(洗脱液：乙腈/水)纯化得到化合物1j(1.39g，1.24mmol，85.3％)。
1H NMR(400MHz,DMSO-d6)δ7.72(d,J＝9.1Hz,1H),7.41–7.35(m,2H),7.31(t,J＝7.5Hz,2H),7.28–7.20(m,5H),6.88(d,J＝8.6Hz,4H),5.21(d,J＝3.4Hz,1H),4.99(dd,J＝11.2,3.3Hz,1H),4.78–4.68(m,2H),4.61–4.55(m,1H),4.27–4.10(m,2H),4.10(d,J＝5.8Hz,1H),4.06–3.98(m,4H),3.95–3.85(m,2H),3.74(s,6H),3.62–3.55(m,4H),3.51–3.41(m,4H),3.18–3.09(m,1H),3.06–2.95(m,2H),2.10(s,3H),2.03–1.96(m,5H),1.86(s,3H),1.43(s,2H),1.23(s,26H),0.88–0.78(m,3H).
7.化合物BE4的制备
氮气氛下，将化合物1j(552mg，0.493mmol)溶于DCM(5mL)中，室温下加入化合物1k(193mg，0.640mmol)和DCI(70mg，0.591mmol)。反应混合溶液室温下搅拌16小时。反应完成后，将反应液分批次倒入预先降温至0至5℃的饱和碳酸氢钠(20mL)溶液中，水相用二氯甲烷萃取两次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，过滤，真空减压浓缩得到残留物，残留物经硅胶柱层析(洗脱液：1％Et3N的乙酸乙酯/1％Et3N的二氯甲烷)纯化得到化合物BE4(447mg，0.338mmol，68.8％)。
1H NMR(400MHz,DMSO-d6)δ7.72(d,J＝9.2Hz,1H),7.42–7.35(m,2H),7.30(t,J＝7.6Hz,2H),7.28–7.19(m,5H),6.88(d,J＝8.4Hz,4H),5.21(d,J＝3.3Hz,1H),4.99(dd,J＝11.2,3.4Hz,1H),4.63–4.55(m,1H),4.28–4.11(m,2H),4.06–3.95(m,5H),3.95–3.85(m,1H),3.74(s,6H),3.62–3.51(m,8H),3.50–3.42(m,6H),3.20–2.92(m,2H),2.74(t,J＝5.9Hz,1H),2.71–2.65(m,1H),2.10(s,3H),2.03–1.96(m,5H),1.86(s,3H),1.43(s,2H),1.22(s,26H),1.16–1.05(m,14H),0.88–0.80(m,3H).
实施例5.化合物BE5的制备
向实施例4中化合物1j(200mg，0.178mmol)的DCM(2mL)溶液中依次加入DIEA(0.18mL，1.071mmol)，丁二酸酐(107mg，1.07mmol)和DMAP(5mg，0.045mmol)。反应液室温搅拌16小时，反应完成后，用饱和碳酸氢钠溶液洗涤，水相用二氯甲烷萃取两次，合并有机相，盐水洗涤，无水硫酸钠干燥，过滤，真空减压浓缩得到残留物，残留物经反相快速柱层析(洗脱液：乙腈/水)纯化得到化合物BE5(141mg，0.116mmol，64.1％)。
1H NMR(400MHz,DMSO-d6)δ7.79(d,J＝9.8Hz,1H),7.42–7.34(m,2H),7.31(t,J＝7.6Hz,2H),7.28–7.19(m,5H),6.91–6.86(m,4H),5.20(d,J＝3.3Hz,2H),5.04–4.96(m,1H),4.67–4.56(m,2H),4.25–4.11(m,3H),4.06–3.96(m,4H),3.95–3.81(m,3H),3.74(s,6H),3.62–3.54(m,2H),3.52–3.42(m,5H),3.16–3.01(m,2H),3.01–2.95(m,1H),2.40–2.30(m,2H),2.10(s,3H),2.04–1.95(m,5H),1.86(s,3H),1.48–1.37(m,2H),1.21(s,24H),1.03–0.91(m,4H),0.88–0.80(m,3H).
实施例6.化合物BE6的制备
化合物BE6的合成步骤可参考实施例4，其中以硬脂酸替换化合物1b。
1H NMR(400MHz,DMSO-d6)δ7.41–7.35(m,2H),7.31(t,J＝7.6Hz,2H),7.28–7.19(m,5H),6.88(d,J＝8.5Hz,4H),5.20(d,J＝3.3Hz,2H),4.99(dd,J＝11.1,3.3Hz,1H),4.59(t,J＝7.8Hz,1H),4.25–4.10(m,2H),4.09–3.97(m,5H),3.96–3.84(m,3H),3.74(s,6H),3.63–3.55(m,2H),3.54–3.43(m,7H),3.17–3.09(m,1H),3.07–2.95(m,3H),2.47–2.40(m,2H),2.10(s,3H),2.03–1.96(m,5H),1.86(s,3H),1.42(s,2H),1.25–1.18(m,26H),0.96(d,J＝6.4Hz,4H),0.87–0.82(m,3H).
实施例7.siRNA的合成
使用本领域熟知的固相亚磷酰胺法制备本发明的siRNA。具体方法可参考例如PCT公开号WO2016081444和WO2019105419，并简述如下。
1.正义链3’端未连接配体的siRNA的制备
1.1正义链(SS链)的合成
通过固相亚磷酰胺合成法，利用空白的CPG固相载体做为起始循环，按照正义链核苷酸排布顺序自3’-5’方向逐一连接核苷单体。每连接一个核苷单体都包含了脱保护、偶联、盖帽、氧化或硫代四步反应，合成规模为5umol的寡核酸。合成条件如下：
核苷单体以0.05mol/L的乙腈溶液提供，每一步反应的条件相同，即温度为25度，脱保护使用3％的三氯乙酸-二氯甲烷溶液，脱保护3次；偶联反应使用的活化剂为0.25mol/L的5-乙硫基四氮唑(ETT)-乙腈溶液，偶联2次；盖帽使用10％醋酐-乙腈和吡啶/N-甲基咪唑/乙腈(10:14:76,v/v/v)，盖帽2次；氧化使用0.05mol/L碘的四氢呋喃/吡啶/水(70/20/10,v/v/v)，氧化2次；硫代使用0.2mol/L 苯乙酰二硫化物(PADS)的乙腈/3-甲基吡啶(1/1,v/v)，硫代2次。
1.2反义链(AS链)的合成
通过固相亚磷酰胺合成法，利用空白的CPG固相载体做为起始循环，按照反义链核苷酸排布顺序自3’-5’方向逐一连接核苷单体。每连接一个核苷单体都包含了脱保护、偶联、盖帽、氧化或硫代四步反应，反义链的5umol的寡核酸合成条件和正义链的相同。
1.3寡核苷酸的纯化与退火
1.3.1氨解
将合成好的固相载体(正义链或者反义链)加入到5mL的离心管中，加入3％的二乙胺/氨水(v/v)，35度(或者55度)恒温水浴下反应16小时(或者8小时)，过滤，固相载体用乙醇/水洗涤三次，每次1mL，滤液离心浓缩后粗品进行纯化。
1.3.2纯化
纯化和脱盐的方法是本领域人员所熟知的。例如，可采用强阴离子填料装柱，氯化钠-氢氧化钠体系进行洗脱纯化，产品收集并管，可采用凝胶填料纯化柱进行脱盐，洗脱体系是纯水。
1.3.3退火
根据说明书将正义链(SS链)与反义链(AS链)以摩尔比(SS链/AS链＝1/1.05)混合，水浴锅加热至70-95度，保持3-5min，自然冷却至室温，将体系冻干得到产品。
本发明中使用的siRNA序列如下：
本文中，各缩写的意义如下：
A、U、G和C分别表示天然的腺嘌呤核糖核苷酸、尿嘧啶核糖核苷酸、鸟嘌呤核糖核苷酸和胞嘧啶核糖核苷酸。
d表示其右侧相邻的核苷酸是脱氧核糖核苷酸。例如dA、dT、dG和dC分别表示腺嘌呤脱氧核糖核苷酸、胸腺嘧啶脱氧核糖核苷酸、鸟嘌呤脱氧核糖核苷酸和胞嘧啶脱氧核糖核苷酸。
m表示其左侧相邻的核苷酸是2’-OCH3修饰的核苷酸。例如，Am、Um、Gm和Cm表示2’-OCH3修饰的A、U、G和C。
f表示其左侧相邻的核苷酸是2’-F修饰的核苷酸。例如，Af、Uf、Gf和Cf分别表示2’-F修饰的A、U、G和C。
“s”表示其左右相邻的两个核苷酸和/或递送载体通过硫代磷酸酯连接。
VP表示其右侧相邻的核苷酸是乙烯基磷酸酯修饰的核苷酸。
L96表示本领域熟知的以下结构的GalNAc递送载体，其中表示通过磷酸酯基团或硫代磷酸酯基团与siRNA连接的位置，可参见例如PCT公开号WO2009073809和WO2009082607。
GL34、LS3和LS1的结构如上文所定义。
GL34通过磷酸酯基团、硫代磷酸酯基团或其他连接基团连接至核酸链3’末端和5’末端时的结构分别如下：
LS3通过磷酸酯基团、硫代磷酸酯基团或其他连接基团连接至核酸链3’末端和5’末端时的结构分别如下：
LS1通过磷酸酯基团、硫代磷酸酯基团或其他连接基团连接至核酸链3’末端和5’末端时的结构分别如下：
实施例8在C57BL/6小鼠模型中验证本发明化合物的药效长效性
将C57BL/6小鼠(雄性，18～21g,6～8周)进行随机分组，每只动物根据体重计算给药剂量，采用皮下注射方式单次给药，siRNA缀合物以1mg/mL的溶液(0.9％氯化钠水溶液作为溶剂)给药；具体地，在实验前，用0.9％氯化钠水溶液将siRNA缀合物溶解且定容至所需浓度和体积，生理盐水(对照组)和siRNA缀合物的给药体积为5mL/kg。
分别于给药前(记为第0天)，及给药后第7、14、21、28、35、42、56和70天取肝脏10mg放于RNAlaterTM溶液中，-80℃冻存，用于检测肝脏mTTR mRNA。
检测肝脏mTTR mRNA：
按照高通量组织RNA提取试剂盒(凡知医疗，FG0412)的操作方案，使用核酸提取仪(杭州奥盛，Auto-pure96)进行细胞RNA提取；参考PrimeScriptTM II 1st Strand cDNA Synthesis Kit(Takara，6210B)反转录；参考TaqManTM Fast Advanced Master Mix(ABI，4444965)20μL体系进行荧光定量PCR反应(ABI，QuantStudio3)检测，引物见表1。
表1：引物信息
数据统计和分析
计算2-△△Ct值并换算成百分比以得到剩余抑制率；
△△Ct＝[(Ct实验组目的基因-Ct实验组内参)-(Ct对照组目的基因-Ct对照组内参)]。
其中目的基因为mTTR，内参为mGAPDH。
表2化合物在C57BL/6小鼠模型的药效长效性实验结果
实施例9在C57BL/6小鼠模型中验证本发明化合物的药效长效性
将C57BL/6小鼠(雄性，6～8周)进行随机分组，采用双侧眼玻璃体内注射方式单次给药，每只眼给药剂量为7.5μg；具体地，在实验前，用磷酸盐缓冲溶液将siRNA缀合物溶解且定容至所需溶液浓度和体积，磷酸盐缓冲溶液和siRNA缀合物的给药体积为1.5μL/眼。
给药后第14天摘取眼球，并分离为3部分：①角膜+虹膜+睫状体；②视网膜；③视网膜色素上皮细胞(RPE)+脉络膜+巩膜；分离样本立即放于液氮中急冻，之后储存于-80℃用于检测mTTR mRNA。
后续实验步骤及引物与上文实施例8相同。
表3化合物在C57BL/6小鼠模型的药效长效性实验结果
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (28)

  1. 寡核苷酸，其包含一个或多个式(I)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
    其中，
    式(I)中的表示H，或表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
    X1选自H、Rs或
    X2选自OR1或
    X1和X2之一为
    R1表示H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
    式(I)中的和R1不同时为H；
    每个L1和L2独立地选自化学键、C1-10亚烷基、C2-10亚烯基、C2-10亚炔基、OC1-10亚烷基、OC1-10亚烯基、OC1-10亚炔基、C(O)C1-10亚烷基；所述L1和L2任选地被1个、2个、3个、4个、5个、6个、7个或8个R取代；
    T选自化学键、-O-、-CH2-、-C(O)-、-OC(O)-、-M-、-O-M-、-CH2-M-、-C(O)-M-或-OC(O)-M-；
    其中M为
    A为糖，所述糖优选为在内含体或溶酶体内可被切割的糖，优选为五元糖或六元糖，更优选为六元糖，例如N-乙酰半乳糖胺、半乳糖、N-乙酰葡萄糖胺、葡萄糖、甘露糖、葡萄糖醛酸、神经氨酸(唾液酸)、木糖或岩藻糖，更优选N-乙酰半乳糖胺或N-乙酰葡萄糖胺；
    P为疏水性基团，优选为C8-30烷基、C8-30烯基或C8-30炔基，其中所述基团中不相邻的1、2、3、4、5、6、7、8、9、10个碳原子可以被选自O、S和N的杂原子替换，或者-CH2CH2-基团可以被-OC(O)-、-C(O)O-、-NHC(O)-或-C(O)NH-替换，或者一个或多个碳原子上的取代基可以连接成饱和或不饱和环；所述C8-30烷基、C8-30烯基或C8-30炔基任选地被1个、2个、3个、4个、5个、6个、7个、8个或更多个R取代；
    R选自H、D、卤素、C1-6烷基或C1-6卤代烷基，其任选地被氘代，直至完全氘代；
    Rs选自H、D、卤素、C1-6烷基、C1-6卤代烷基、3-10元杂环基或C3-10环烷基，其任选地被氘代，直至完全氘代；
    m为0、1、2、3、4、5、6、7、8、9或10；
    n为0、1、2、3、4、5、6、7、8、9或10；
    k＝0、1、2、3、4、5或6。
  2. 权利要求1的寡核苷酸，其中，所述式(I)化合物选自式(II)或式(III)化合物，或其药学上可接 受的盐、互变异构体或立体异构体：
    其中，
    表示H，或表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
    R1表示H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
    和R1不同时为H；
    每个L1和L2独立地选自化学键、C1-10亚烷基、C2-10亚烯基、C2-10亚炔基、OC1-10亚烷基、OC1-10亚烯基、OC1-10亚炔基或C(O)C1-10亚烷基；所述L1和L2任选地被1个、2个、3个、4个、5个、6个、7个或8个R取代；
    T选自化学键、-O-、-CH2-、-C(O)-、-OC(O)-、-M-、-O-M-、-CH2-M-、-C(O)-M-或-OC(O)-M-；
    其中M为
    A为糖，所述糖优选为在内含体或溶酶体内可被切割的糖，优选为五元糖或六元糖，更优选为六元糖，例如N-乙酰半乳糖胺、半乳糖、N-乙酰葡萄糖胺、葡萄糖、甘露糖、葡萄糖醛酸、神经氨酸(唾液酸)、木糖或岩藻糖，更优选N-乙酰半乳糖胺或N-乙酰葡萄糖胺；
    P为疏水性基团，优选为C8-30烷基、C8-30烯基或C8-30炔基，其中所述基团中不相邻的1、2、3、4、5、6、7、8、9、10个碳原子可以被选自O、S和N的杂原子替换，或者-CH2CH2-基团可以被-OC(O)-、-C(O)O-、-NHC(O)-或-C(O)NH-替换，或者一个或多个碳原子上的取代基可以连接成饱和或不饱和环；所述C8-30烷基、C8-30烯基或C8-30炔基任选地被1个、2个、3个、4个、5个、6个、7个、8个或更多个R取代；
    R选自H、D、卤素、C1-6烷基或C1-6卤代烷基，其任选地被氘代，直至完全氘代；
    Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基，其任选地被氘代，直至完全氘代；
    Rs’选自H、D、卤素、C1-6烷基、C1-6卤代烷基、3-10元杂环基或C3-10环烷基，其任选地被氘代，直至完全氘代；
    m为0、1、2、3、4、5、6、7、8、9或10；
    n为0、1、2、3、4、5、6、7、8、9或10；
    k＝0、1、2、3、4、5或6。
  3. 权利要求1或2的寡核苷酸，其中，
    表示H，或表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
    R1表示H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
    和R1不同时为H；
    每个L1和L2独立地选自化学键、C1-6亚烷基、OC1-6亚烷基或C(O)C1-6亚烷基；所述L1和L2任选地被1个、2个、3个、4个或5个R取代；
    T选自化学键、-O-、-CH2-、-C(O)-、-OC(O)-、-M-、-O-M-、-CH2-M-、-C(O)-M-或-OC(O)-M-；
    其中M为
    A选自N-乙酰半乳糖胺、半乳糖、N-乙酰葡萄糖胺、葡萄糖、甘露糖、葡萄糖醛酸、神经氨酸(唾液酸)、木糖或岩藻糖，更优选为N-乙酰半乳糖胺或N-乙酰葡萄糖胺；
    P选自C10-22烷基或C10-22烯基，所述C10-22烷基或C10-22烯基任选地被1个、2个、3个、4个或5个R取代；
    R选自H、D、卤素、C1-6烷基或C1-6卤代烷基；
    Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基；
    Rs’选自H、D、卤素、C1-6烷基、C1-6卤代烷基、3-10元杂环基或C3-10环烷基；
    m为0、1、2、3、4或5；
    n为0、1、2、3、4或5；
    k为0、1、2、3、4或5。
  4. 权利要求1-3中任一项的寡核苷酸，其中，
    表示H，或表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
    R1表示H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
    和R1不同时为H；
    每个L1和L2独立地选自化学键或OC1-6亚烷基；所述L1和L2任选地被1个、2个或3个R取代；
    T选自化学键、-O-、-CH2-、-OC(O)-或-C(O)-；
    A选自
    A通过a端与L1相连，通过b端与P基团相连；
    P选自C10-22烷基或C10-22烯基，所述C10-22烷基或C10-22烯基任选地被1个、2个或3个R取代；
    R选自H、D、卤素、C1-4烷基；
    Rs选自H或D；
    Rs’选自H、D或C5-10环烷基；
    m为0、1、2或3；
    n为0、1、2或3；
    k为0、1、2或3。
  5. 权利要求1-4中任一项的寡核苷酸，其中，
    表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
    R1为H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
    L1为OCH2CH2或O(CH2)5；
    L2为OCH2或化学键；
    T选自化学键、-O-或-C(O)-；
    A选自
    A通过a端与L1相连，通过b端与P基团相连；
    P为-(CH2)14-16CH3；
    Rs为H；
    Rs’为环己基；
    m为0、1或2；
    n为0或1；
    k为0或1。
  6. 权利要求1-5中任一项的寡核苷酸，其中，所述式(II)或式(III)化合物选自以下通式化合物，或其药学上可接受的盐、互变异构体或立体异构体：
    其中，
    P1、P2、P3选自H或P基团；
    P4选自CH3或P基团；
    P1、P2、P3、P4中之一为P基团；
    各变量如权利要求1-5中所定义。
  7. 权利要求1-6中任一项的寡核苷酸，其中，所述式(II)或式(III)化合物选自以下化合物，或其药学上可接受的盐、互变异构体或立体异构体：

    其中，表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
    R1为H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置。
  8. 权利要求1-7中任一项的寡核苷酸，其具有14至30个核苷酸。
  9. 权利要求1-8中任一项的寡核苷酸，其在5’端包含一个权利要求1-7中任一项的式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
  10. 权利要求1-9中任一项的寡核苷酸，其在3’端包含一个权利要求1-7中任一项的式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
  11. 权利要求1-10中任一项的寡核苷酸，其在5’端和3’端分别包含一个权利要求1-7中任一项的式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
  12. 寡核苷酸，其在寡核苷酸内部、5’端和/或3’端包含一个、两个或多个递送载体，所述递送载体为被疏水性基团修饰的糖；
    优选地，所述被疏水性基团修饰的糖选自式(X)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
    其中，表示与寡核苷酸的连接位置；
    P为疏水性基团；
    A为糖部分；
    P和A如权利要求1-5中任一项所定义；
    优选地，所述疏水性基团连接到糖部分的羟基或乙酰基上；
    优选地，所述被疏水性基团修饰的糖选自以下化合物，或其药学上可接受的盐、互变异构体或立体异构体：

    其中，表示与寡核苷酸的连接位置。
  13. 权利要求1-12中任一项的寡核苷酸，所述寡核苷酸是ASO或siRNA，优选用于抑制在肝外表达的基因，更优选用于抑制在中枢神经系统(CNS)和/或眼部表达的基因。
  14. 式(II’)或(III’)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
    R1和R2独立地选自H、反应性磷基团、羟基保护基或固相载体；
    A为乙酰化的糖，所述糖优选为在内含体或溶酶体内可被切割的糖，优选为乙酰化的五元糖或六元糖，所述五元糖或六元糖是例如N-乙酰半乳糖胺、半乳糖、N-乙酰葡萄糖胺、葡萄糖、甘露糖、葡萄糖醛酸、神经氨酸(唾液酸)、木糖或岩藻糖，更优选所述五元糖或六元糖是N-乙酰半乳糖胺或N-乙酰葡萄糖胺；
    优选地，A选自
    A通过a端与L1相连，通过b端与P基团相连；
    P、L1、L2、T、Rs、Rs’、m、n、k如权利要求1-7中任一项所定义。
  15. 权利要求14的化合物，其中，R1和R2为H。
  16. 权利要求14或15的化合物，其中，R1和R2之一为反应性磷基团，优选亚磷酰胺、H-膦酸酯、烷基-膦酸酯、磷酸酯或磷酸酯模拟物，例如天然磷酸酯、硫代磷酸酯、二硫代磷酸酯、硼烷磷酸酯、硼烷硫代磷酸酯、膦酸酯、卤素取代的膦酸酯和磷酸酯、氨基磷酸酯、磷酸二酯、磷酸三酯、硫代磷酸二酯、硫代磷酸三酯、二磷酸酯或三磷酸酯，优选-P(OCH2CH2CN)(N(iPr)2)。
  17. 权利要求14-16中任一项的化合物，其中，R1和R2选自保护基，优选羟基保护基，例如三甲基硅基(TMS)、三乙基硅基(TES)、二甲基异丙基硅基(DMIPS)、二乙基异丙基硅基(DEIPS)、叔丁基二甲基硅基(TBDMS)、叔丁基二苯基硅基(TBDPS)、三异丙基硅基(TIPS)、乙酰基(Ac)、氯乙酰基、二氯乙酰基、三氯乙酰基、三氟乙酰基(TFA)、苯甲酰基、对甲氧基苯甲酰基、9-芴基甲氧基羰基(Fmoc)、烯丙氧羰基(Alloc)、2,2,2-三氯乙氧羰基(Troc)、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、苯甲基(Bn)、对甲氧基苄基(PMB)、烯丙基、三苯基甲基(Tr)、双对甲氧基三苯甲基(DMTr)、甲氧基甲基(MOM)、苯氧基甲基(BOM)、2,2,2-三氯乙氧基甲基、2-甲氧基乙氧基甲基(MEM)、甲硫基甲基(MTM)、对甲氧基苄氧基甲基(PMBM)、-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基，优选DMTr。
  18. 权利要求14-17中任一项的化合物，其中所述式(II’)或(III’)化合物选自以下化合物，或其药学上可接受的盐、互变异构体或立体异构体：
  19. 双链RNA，其具有正义链和反义链，各链具有14至30个核苷酸，所述反义链包含与所述正义链和靶标mRNA充分互补的序列，其中所述正义链和/或反义链包含一个或多个如权利要求1-7中任一项所述式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
  20. 权利要求19的双链RNA，其中所述式(II)或式(III)化合物选自以下通式化合物，或其药学上可接受的盐、互变异构体或立体异构体：

    其中，各变量如权利要求1-7中所定义。
  21. 权利要求19或20的双链RNA，其中所述式(II)或式(III)化合物选自以下化合物，或其药学上可接受的盐、互变异构体或立体异构体：
    其中，表示与相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置；
    R1为H，或表示与另一相邻核苷酸的磷酸酯或硫代磷酸酯连接的位置。
  22. 权利要求19-21中任一项的双链RNA，其中所述正义链在5’端包含一个权利要求1-7中任一项所述式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
  23. 权利要求19-22中任一项的双链RNA，其中所述正义链在3’端包含一个权利要求1-7中任一 项所述式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
  24. 权利要求19-23中任一项的双链RNA，其中所述正义链在5’端和3’端分别包含一个权利要求1-7中任一项所述式(II)或式(III)化合物，或其药学上可接受的盐、互变异构体或立体异构体。
  25. 双链RNA，其具有正义链和反义链，各链具有14至30个核苷酸，所述反义链包含与所述正义链和靶标mRNA充分互补的序列，其中所述正义链和/或反义链在内部、5’端和/或3’端包含一个、两个或多个递送载体，所述递送载体为被疏水性基团修饰的糖；
    优选地，所述被疏水性基团修饰的糖选自式(X)化合物，或其药学上可接受的盐、互变异构体或立体异构体：
    其中，表示与寡核苷酸的连接位置；
    P为疏水性基团；
    A为糖部分；
    P和A如权利要求1-5中任一项所定义；
    优选地，所述疏水性基团连接到糖部分的羟基或乙酰基上；
    优选地，所述被疏水性基团修饰的糖选自以下化合物，或其药学上可接受的盐、互变异构体或立体异构体：
    其中，表示与寡核苷酸的连接位置。
  26. 细胞，其含有如权利要求19-25中任一项所述的双链RNA。
  27. 药物组合物，其包含如权利要求19-25中任一项所述的双链RNA、或如权利要求26所述的细胞，以及任选的药学上可接受的载剂或赋形剂。
  28. 试剂盒，其包含如权利要求19-25中任一项所述的双链RNA、或如权利要求26所述的细胞。
PCT/CN2024/098675 2023-06-12 2024-06-12 包含糖的寡核苷酸递送配体 Ceased WO2024255761A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202480038604.7A CN121358748A (zh) 2023-06-12 2024-06-12 包含糖的寡核苷酸递送配体

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202310693838 2023-06-12
CN202310693838.2 2023-06-12

Publications (1)

Publication Number Publication Date
WO2024255761A1 true WO2024255761A1 (zh) 2024-12-19

Family

ID=93851404

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/098675 Ceased WO2024255761A1 (zh) 2023-06-12 2024-06-12 包含糖的寡核苷酸递送配体

Country Status (2)

Country Link
CN (1) CN121358748A (zh)
WO (1) WO2024255761A1 (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004094595A2 (en) * 2003-04-17 2004-11-04 Alnylam Pharmaceuticals Inc. MODIFIED iRNA AGENTS
CN105377887A (zh) * 2013-05-01 2016-03-02 Isis制药公司 用于调节载脂蛋白(a)表达的组合物和方法
CN110741087A (zh) * 2017-04-11 2020-01-31 阿布特斯生物制药公司 靶向组合物
CN112105625A (zh) * 2018-03-07 2020-12-18 赛诺菲 核苷酸前体、核苷酸类似物以及含其的寡聚化合物
CN112400018A (zh) * 2018-05-07 2021-02-23 阿尔尼拉姆医药品有限公司 肝外递送
WO2022084331A2 (en) * 2020-10-20 2022-04-28 Sanofi Novel ligands for asialoglycoprotein receptor
CN114945669A (zh) * 2019-11-06 2022-08-26 阿尔尼拉姆医药品有限公司 肝外递送

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004094595A2 (en) * 2003-04-17 2004-11-04 Alnylam Pharmaceuticals Inc. MODIFIED iRNA AGENTS
CN105377887A (zh) * 2013-05-01 2016-03-02 Isis制药公司 用于调节载脂蛋白(a)表达的组合物和方法
CN110741087A (zh) * 2017-04-11 2020-01-31 阿布特斯生物制药公司 靶向组合物
CN112105625A (zh) * 2018-03-07 2020-12-18 赛诺菲 核苷酸前体、核苷酸类似物以及含其的寡聚化合物
CN112400018A (zh) * 2018-05-07 2021-02-23 阿尔尼拉姆医药品有限公司 肝外递送
CN114945669A (zh) * 2019-11-06 2022-08-26 阿尔尼拉姆医药品有限公司 肝外递送
WO2022084331A2 (en) * 2020-10-20 2022-04-28 Sanofi Novel ligands for asialoglycoprotein receptor

Also Published As

Publication number Publication date
CN121358748A (zh) 2026-01-16

Similar Documents

Publication Publication Date Title
JP7665509B2 (ja) ７’－５’－アルファ－アノマー二環式糖ヌクレオシドを含むオリゴヌクレオチドコンジュゲート
US20240343752A1 (en) Novel bicyclic nucleosides and oligomers prepared therefrom
WO2011005761A1 (en) Novel nucleic acid prodrugs and methods use thereof
EP3770257B1 (en) Bbb-passing lipid ligand of hetero nucleic acid
WO2024093907A1 (zh) 向眼部和中枢神经系统递送sirna的配体
CN116615542A (zh) 寡核苷酸的全身递送
TW202140043A (zh) 4’-o-亞甲基膦酸酯核酸及其類似物
US11414451B2 (en) Floxuridine synthesis
WO2024002006A1 (zh) 具有增强的稳定性的核苷酸替代物
WO2023131170A1 (zh) 具有核苷酸类似物的双链rna
WO2024255761A1 (zh) 包含糖的寡核苷酸递送配体
WO2025031479A1 (zh) 含有核苷酸类似物的双链rna
WO2024002007A1 (zh) 含有可降低脱靶毒性的核苷酸类似物的双链rna
CN118063535A (zh) 含氨基衍生结构的修饰核苷、寡聚核酸化合物及合成方法
WO2025007907A1 (zh) 包含肽的寡核苷酸递送配体
CN120904263B (zh) 具有增加寡核苷酸体内稳定性的2'核糖修饰的核苷酸化合物及其合成方法与应用
EP4541804A1 (en) Cyclic phosphonate-modified nucleotide
WO2025146138A1 (en) Ligands for extrahepatic delivery
HK40027749A (zh) 用於分子的跨膜递送的化合物和方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24822710

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE