WO2024002007A1 - 含有可降低脱靶毒性的核苷酸类似物的双链rna - Google Patents

含有可降低脱靶毒性的核苷酸类似物的双链rna Download PDF

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WO2024002007A1
WO2024002007A1 PCT/CN2023/102403 CN2023102403W WO2024002007A1 WO 2024002007 A1 WO2024002007 A1 WO 2024002007A1 CN 2023102403 W CN2023102403 W CN 2023102403W WO 2024002007 A1 WO2024002007 A1 WO 2024002007A1
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alkyl
haloalkyl
double
stranded rna
nucleotide
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PCT/CN2023/102403
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English (en)
French (fr)
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黄金宇
邹昊
郭洪利
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大睿生物医药科技(上海)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/167—Purine radicals with ribosyl as the 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention requires priority for the Chinese application CN202210744263.8 submitted on June 27, 2022, the Chinese application CN202210948347.3 submitted on August 8, 2022, and the Chinese application CN202211483699.2 submitted on November 24, 2022 rights, they are incorporated herein by reference in their entirety.
  • the invention belongs to the field of medicine, and specifically relates to double-stranded RNA with nucleotide analogs.
  • RNA interference is a phenomenon of efficient and specific degradation of target mRNA induced by double-stranded RNA (dsRNA).
  • dsRNA double-stranded RNA
  • Incorporating thermally unstable nucleotides such as glycerol nucleic acid (GNA)
  • GAA glycerol nucleic acid
  • the present invention solves the above problems by providing a new nucleotide analogue.
  • the invention provides a nucleotide dimer represented by formula (A):
  • the invention provides a double-stranded RNA molecule, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, and the antisense strand contains one or more nucleotide monomers represented by formula (III) or (IV):
  • the present invention provides a nucleic acid molecule having a nucleotide sequence comprising one or more as described herein The nucleotide dimer and/or the nucleotide monomer as described herein.
  • the invention provides a vector comprising a nucleotide sequence encoding the aforementioned double-stranded RNA.
  • the present invention provides cells containing the aforementioned double-stranded RNA or the aforementioned vector.
  • the invention in another aspect, relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a double-stranded RNA molecule as described herein, and a pharmaceutically acceptable carrier or excipient.
  • the invention in another aspect, relates to a kit comprising a double-stranded RNA molecule as described herein.
  • the invention in another aspect, relates to a method for inhibiting the expression of a target gene in a cell, comprising the step of introducing a double-stranded RNA molecule as described herein into the cell.
  • the invention in another aspect, relates to a method for inhibiting the expression of a target gene in a cell, comprising expressing in said cell a double-stranded RNA molecule as described herein.
  • the invention in another aspect, relates to a method for reducing off-target toxicity in a cell, comprising the step of introducing a double-stranded RNA molecule as described herein into the cell.
  • the invention in another aspect, relates to a method for reducing off-target toxicity in a cell, comprising expressing in said cell a double-stranded RNA molecule described herein.
  • the resulting double-stranded RNA displays one or more of enhanced stability, reduced off-target toxicity, and enhanced effectiveness.
  • 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 chain saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C 1-4 alkyl and C 1-2 alkyl are preferred. Examples of C 1-6 alkyl groups include: methyl (C 1 ), ethyl (C 2 ), n-propyl (C 3 ), isopropyl (C 3 ), n-butyl (C 4 ), tert-butyl base (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) substitution.
  • Alkyl groups may be optionally substituted with one or more substituents, for example, with 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 2-6 alkenyl refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-4 alkenyl is preferred. Examples of C 2-6 alkenyl groups include: vinyl (C 2 ), 1-propenyl (C 3 ), 2-propenyl (C 3 ), 1-butenyl (C 4 ), 2-butenyl (C 4 ), butadienyl (C 4 ), pentenyl (C 5 ), pentadienyl (C 5 ), hexenyl (C 6 ), etc.
  • C 2-6 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) substitution.
  • Alkenyl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • C 2-6 alkynyl refers to a straight group having 2 to 6 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. chain or branched hydrocarbon groups. In some embodiments, C 2-4 alkynyl is preferred. Examples of C 2-6 alkynyl groups include, but are not limited to: ethynyl (C 2 ), 1-propynyl (C 3 ), 2-propynyl (C 3 ), 1-butynyl (C 4 ), 2-Butynyl (C 4 ), pentynyl (C 5 ), hexynyl (C 6 ), etc.
  • C 2-6 alkynyl also includes heteroalkynyl 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) substitution.
  • An alkynyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • Halo or "halogen” refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).
  • C 1-6 haloalkyl refers to the above-mentioned "C 1-6 alkyl” which is substituted by one or more halogen groups.
  • C 1-4 haloalkyl is particularly preferred, with C 1-2 haloalkyl being more preferred.
  • Exemplary haloalkyl groups 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, etc.
  • Haloalkyl groups may be substituted at any available point of attachment, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • C 1-6 alkoxy refers to the -OR group, where R is as defined above for “C 1-6 alkyl” and "C 1-6 haloalkyl”.
  • C 3-10 cycloalkyl refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, C 4-7 cycloalkyl and C 3-6 cycloalkyl are particularly preferred, with C 5-6 cycloalkyl being more preferred. Cycloalkyl also includes ring systems in which the above-described cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such cases the number of carbons continues as indicated The number of carbons in a 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 ), cycloheptene group (C 7 ), cycloheptadienyl (C 7 ), cycloheptadienyl (C 7 ), etc.
  • a cycloalkyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • 3-10 membered heterocyclyl refers to a group of 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 valency permits.
  • 4-10 membered heterocyclyl is preferred, which is a 4-10 membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, 3-8 membered is preferred Heterocyclyl, which is a 3- to 8-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; preferably a 3-6-membered heterocyclyl, which is a 3- to 6-membered heterocyclic ring system having ring carbon atoms and 1 to 3 ring heteroatoms.
  • 3 to 6 membered non-aromatic ring system of atoms preferably 4 to 7 membered heterocyclyl, which is a 4 to 7 membered nonaromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; more preferably 5 to 6 membered Heterocyclyl, which is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms.
  • Heterocyclyl also includes ring systems in which the above-described heterocyclyl ring is fused with one or more cycloalkyl groups, wherein the point of attachment is on the cycloalkyl ring, or in which the above-described heterocyclyl ring is fused with one or more aryl groups or Heteroaryl fused ring systems wherein the point of attachment is on the heterocyclyl ring; and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system.
  • Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to: aziridinyl, oxirinyl, and thiorenyl.
  • Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to: azetidinyl, oxetanyl, and thietanyl.
  • Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to: tetrahydrofuryl, dihydrofuryl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2, 5-diketone.
  • Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to: dioxolyl, oxasulfuranyl, disulfuranyl, and oxalanyl.
  • Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl, and thiadiazolinyl.
  • Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridyl, and thianyl.
  • Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to: piperazinyl, morpholinyl, dithianyl, and dioxanyl.
  • Exemplary 6-membered heterocyclyl groups containing three heteroatoms include, but are not limited to: hexahydrotriazinyl (triazinanyl).
  • Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to: azepanyl, oxpanyl, and thipanyl.
  • Exemplary 5 fused with C 6 aryl ring Single-membered heterocyclyl groups (also referred to herein as 5,6-bicyclic heterocyclyl groups) include, but are not limited to: indolyl, isoindolyl, dihydrobenzofuranyl, dihydrobenzothiophene base, benzoxazolinone base, etc.
  • Exemplary 6-membered heterocyclyl fused to a C6 aryl ring include, but are not limited to: tetrahydroquinolyl, tetrahydroisoquinolyl, etc.
  • Heterocyclyl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • C 6-10 aryl refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6-10 ring carbon atoms and zero heteroatoms arranged in a cyclic Shared 6 or 10 ⁇ electrons) group.
  • an aryl group has six ring carbon atoms ("C 6 aryl”; e.g., phenyl).
  • an aryl group has ten ring carbon atoms ("C 10 aryl”; eg, naphthyl, eg, 1-naphthyl and 2-naphthyl).
  • Aryl also includes ring systems in which the aryl ring described above is fused to one or more cycloalkyl or heterocyclyl groups, and the point of attachment is on said aryl ring, in which case the number of carbon atoms continues to indicate The number of carbon atoms in the aryl ring system.
  • Aryl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • 5-14 membered heteroaryl refers to a 5-14 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms (e.g., having a 6, 10 or 14 ⁇ electrons), wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur.
  • the point of attachment may be a carbon or nitrogen atom as long as the valency permits.
  • Heteroaryl bicyclic systems may include one or more heteroatoms in one or both rings.
  • Heteroaryl also includes ring systems in which the heteroaryl ring described above is fused to one or more cycloalkyl or heterocyclyl groups, and the point of attachment is on the heteroaryl ring, in which case the carbon atom Number continues to represent the number of carbon atoms in the heteroaryl ring system.
  • 5-10 membered heteroaryl groups are preferred, which are 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms.
  • 5-6 membered heteroaryl groups are particularly preferred, which are 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms.
  • Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to: pyrrolyl, furyl, and thienyl.
  • Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to: imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl and isothiazolyl.
  • Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to: triazolyl, oxadiazolyl (eg, 1,2,4-oxadiazolyl), and thiadiazolyl.
  • Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to: tetrazolyl.
  • Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to: pyridyl.
  • Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to: pyridazinyl, pyrimidinyl, and pyrazinyl.
  • Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively.
  • Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to: azepantrienyl, oxetapyltrienyl, and thioheptantrienyl.
  • Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to: indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothienyl, isobenzothienyl, benzofuranyl , benzisofuryl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, Indazinyl and purinyl.
  • Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to: naphthyridinyl, pyridinyl, quinolinyl, isoquinolinyl, quinolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl .
  • Heteroaryl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, etc. are defined herein as optionally substituted groups.
  • Each R aa is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R aa groups are combined to form heterocyclyl or Heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd groups group replacement;
  • Each R cc is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R cc groups are combined to form a heterocycle or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;
  • Each R ee is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl Alkyl, heterocyclyl, aryl and heteroaryl are independently substituted by 0, 1, 2, 3, 4 or 5 R gg groups;
  • Each R ff is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R ff groups combine to form a heterocyclyl or a heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R gg group substitution;
  • siRNA refers to a class of double-stranded RNA molecules that can mediate silencing of a target RNA that is complementary to it (eg, mRNA, eg, the transcript of a gene encoding a protein).
  • siRNA is usually double-stranded, including an antisense strand that is complementary to the target RNA, and a sense strand that is complementary to the antisense strand.
  • mRNA is also referred to herein as the mRNA to be silenced.
  • Such genes are also called target genes.
  • the RNA to be silenced is an endogenous gene or a pathogen gene.
  • RNAs other than mRNA e.g., tRNA
  • viral RNA can also be targeted.
  • antisense strand refers to a strand of siRNA that contains a region that is completely, fully, or substantially complementary to the target sequence.
  • sense strand refers to a strand of siRNA that includes a region that is completely, fully, or substantially complementary to a region that is the antisense strand as the term is defined herein.
  • complementary region refers to a region on the antisense strand that is completely, fully or substantially complementary to the target mRNA sequence. In cases where the complementary region is not completely complementary to the target sequence, the mismatch can be located in the internal or terminal regions of the molecule. Typically, the most tolerated mismatches are in the terminal region, e.g., within 5, 4, 3, 2 or 1 nucleotide of the 5' and/or 3' end. The portion of the antisense strand that is most sensitive to mismatches is called the "seed region.” For example, in a siRNA containing a 19nt strand, some mismatches can be tolerated at position 19 (from 5' to 3').
  • 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 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA at 50°C or 70°C for 12-16 hours.
  • “complementary” sequences may also include or be formed entirely from non-Watson-Crick base pairs and/or from non-natural and modified nucleosides. Base pairs formed by acids. 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,””fullycomplementary,” or “substantially complementary” to messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the 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 the PCSK9 mRNA.
  • complementary may refer to the relationship between the sense strand and the antisense strand of the siRNA, or between the antisense strand of the siRNA agent and the target sequence. Used in base pairing.
  • “Sufficiently 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 character of the molecule. In other words, although perfect complementarity is usually required, in some cases, particularly in the antisense strand, one or more, such as 6, 5, 4, 3, 2 or 1, may be included. mismatch (relative to the target mRNA), but the sense and antisense strands can still maintain the overall double-stranded character of the molecule.
  • shRNA refers to short hairpin RNA.
  • shRNA consists of two short inverted repeats.
  • the shRNA cloned into the shRNA expression vector includes two short inverted repeat sequences, separated by a stem-loop sequence in the middle, forming a hairpin structure and controlled by the pol III promoter. Then 5-6 Ts are connected as the transcription terminator of RNA polymerase III.
  • Nucleoside is a compound composed of two substances: purine base or pyrimidine base, and ribose or deoxyribose.
  • Nucleoside is a compound composed of three substances: purine base or pyrimidine base, ribose or deoxyribose, and phosphate.
  • Olionucleotide refers to, for example, a nucleic acid molecule (RNA or DNA) having a length of less than 100, 200, 300 or 400 nucleotides.
  • Base is the basic unit for the synthesis of nucleosides, nucleotides and nucleic acids. Its constituent elements contain nitrogen, also known as “nitrogen-containing bases”.
  • the capital letters A, U, T, G and C represent the base composition of nucleotides, which are adenine, uracil, thymine, guanine and cytosine respectively.
  • Modification of nucleotides described herein includes, but is not limited to, methoxy modification, fluoro modification, phosphorothioate group connection or conventional protecting group protection, etc.
  • the fluoro-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group of the nucleotide is replaced by fluorine
  • the methoxy-modified nucleotide refers to the 2'-hydroxyl group of the ribosyl 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 containing phosphorothioate groups, vinyl phosphate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases containing nucleotides, and derivatives linked to cholesterol groups Terminal nucleotide, deoxyribonucleotide or conventional protecting group protection on the dodecanoic acid dodecylamide group.
  • the 2'-fluoro modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by fluorine.
  • the 2'-deoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.
  • Reactive phosphorus group means a phosphorus-containing group contained in a nucleotide unit or a nucleotide analog unit which can react by nucleophilic attack with a phosphorus-containing group contained in another molecule, especially another Reaction of a hydroxyl or amine group in a nucleotide unit or another nucleotide analogue. Typically, such a reaction results in an ester form linking said first nucleotide unit or said first nucleotide analog unit to said second nucleotide unit or said second nucleotide analog unit. Internucleoside bonds.
  • the reactive phosphorus group may be selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, including but not limited to: natural phosphates, thiophosphates, dithiophosphates Phosphates, borane phosphates, borane phosphorothioates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, phosphorothioate diesters, phosphorothioates Trysters, diphosphates and triphosphates, preferably -P( OCH2CH2CN )(N( iPr ) 2 ).
  • Protecting groups can be labile chemical moieties known in the art that serve to protect reactive groups, such as hydroxyl, amino, and thiol groups, to prevent undesirable or undesirable formation during chemical synthesis. reaction.
  • Protecting groups are typically used to protect sites selectively and/or orthogonally 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 (eg, tert-butoxycarbonyl (BOC), acetyl, or isobutyryl); arylalkylcarbonyl and arylalkoxycarbonyl (e.g., benzyloxycarbonyl); substituted methyl ether (e.g., methoxymethyl ether); substituted diethyl ether; substituted benzyl ether; tetrahydropyranyl ether; methyl Silyl group (for example, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-( ⁇ Methylsilyl)ethoxy]methyl or tert-butyldiphenylsilyl); esters (such as benzoate); carbonates (
  • non-cyclic ketals e.g. dimethylacetal
  • cyclic ketals e.g. 1,3-dioxane, 1,3-dioxopent cyclic acetals and those described herein
  • non-cyclic acetals e.g., those described herein
  • non-cyclic hemiacetals e.g., cyclic hemiacetals
  • cyclic disulfide ketals e.g., 1, 3-dithiane or 1,3-dithiolan
  • orthoesters e.g., those described herein
  • triarylmethyl groups e.g., trityl; monomethoxytrityl (MMTr); 4,4′-dimethoxytrityl (DMTr); 4,4′,4′′-trimethoxytrityl (TMTr); and those described herein).
  • the protecting group is selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethylacetal (BOM), ⁇ -Methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv ), tetrahydropyranyl (THP), triphenylmethyl (Trt), methoxytrityl[(4-methoxyphenyl)diphenylmethyl] (MMT), dimethoxy trityl, [bis-(4-methoxyphenyl)phenylmethyl (DMT), trimethylsilyl ether (TMS), tert-butyldimethylsilyl ether (TBDMS) , tri-iso-propylsilyloxymethyl ether (TO
  • 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.
  • Trimethylsilyl TMS
  • triethylsilyl TES
  • dimethylisopropylsilyl DMIPS
  • diethylisopropylsilyl DEIPS
  • tert-butyldimethylsilyl TDMS
  • tert-butyldiphenylsilyl TIPS
  • TIPS Trimethylsilyl
  • acetyl Ac
  • chloroacetyl dichloroacetyl, trichloroacetyl
  • trifluoroacetyl TSA
  • benzoyl p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc) , Benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzy
  • the term "pharmaceutically acceptable salts” means those carboxylate salts and amino acid addition salts of the compounds of the present invention which are suitable for contact with patient tissue within the scope of reliable medical judgment and will not produce undue toxicity, Irritation effects, allergic reactions, etc., commensurate with a reasonable benefit/risk ratio, are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the invention.
  • the present invention includes tautomers, which are functional group isomers resulting from the rapid movement of an atom in a molecule between two positions.
  • tautomers which are functional group isomers resulting from the rapid movement of an atom in a molecule between two positions.
  • Compounds exist in different tautomeric forms, and a said compound is not limited to any particular tautomeric form, but is intended to encompass all tautomeric forms.
  • the compounds of the present invention may contain one or more asymmetric centers and thus may exist in multiple stereoisomeric forms, for example, enantiomeric and/or diastereomeric 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 mixtures of stereoisomers, Includes racemic mixtures and mixtures enriched in one or more stereoisomers.
  • the 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 the preferred isomers may be separated by Prepared by asymmetric synthesis.
  • HPLC high pressure liquid chromatography
  • the present invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described in formula (I), except that one or more atoms are surrounded by atoms having an atomic mass or mass number different from that common in nature. replaced.
  • isotopes that may be incorporated 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 respectively. O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 Cl.
  • Inventive compounds containing the above-mentioned isotopes and/or other isotopes of other atoms The compounds, their prodrugs and pharmaceutically acceptable salts of said compounds or said prodrugs all fall within the scope of the present invention.
  • Certain isotopically labeled compounds of the present invention such as those incorporating radioactive isotopes (eg, 3 H and 14 C), may be used in drug and/or substrate tissue distribution assays. Tritium, ie 3 H, and carbon-14, ie 14 C isotopes are particularly preferred because they are easy to prepare and detect. Furthermore, substitution with heavier isotopes, such as deuterium, i.e.
  • the isotope-labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by replacing non-isotopes with readily available isotope-labeled reagents when performing the following processes and/or the processes disclosed in the Examples and Preparation Examples. Labeled reagents.
  • the present invention specifically relates to the nucleotide dimer represented by formula (A):
  • Q 1 and Q 2 are R 4 and the other is OL 2 ;
  • L 1 is H or P 1 , or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose sugar of another nucleotide or oligonucleotide, preferably P 1 ;
  • L 2 is H or P 2 , or a chemical bond to the phosphate P atom at the 5' end of the ribose sugar of another nucleotide or oligonucleotide, preferably P 2 ;
  • Y 1 is O, S or NR
  • Y 2 is O, S or chemical bond
  • R 1 and R 2 are independently selected from H, D, halogen, OH, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3- 10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-14 membered heteroaryl, which is optionally substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R'substitution;
  • R 3 is selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-14 membered heteroaryl, which is optionally substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R';
  • R 4 and R 5 are independently selected from H, D, OH, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy, preferably H, F or OMe.
  • P 1 is a hydroxyl protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), Tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, triisopropylsilyl Chloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2- Trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Bo
  • P 2 is a reactive phosphorus group, such as phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, such as natural phosphates, phosphorothioates, phosphorodithioates , borane phosphates, borane phosphorothioates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphoric diesters, phosphoric triesters, phosphorothioate diesters, phosphorothioate triesters , diphosphate or triphosphate, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 );
  • Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C;
  • R is selected from H, C 1-6 alkyl or C 1-6 haloalkyl
  • R' is selected from D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 3-10 yuan Heterocyclyl, C 6-10 aryl, 5-14 membered heteroaryl, -OR a , -OC(O)R a , -OC(O)OR b , -OC(O)NR a R b , - C(O)R a , -C(O)OR a , -C(O)NR a R b , -S(O) n R a , -S(O) n OR a , -S(O) n NR a R b , -OS(O) n R b , -NR a R b , -NR a C(O)R b , -NR a -C
  • R a and R b are independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-14 membered heteroaryl; or R a and R b and the nitrogen atoms to which they are connected form a 3-10 membered heterocyclyl;
  • n is selected from 1, 2, 3, 4 or 5;
  • n is independently selected from 1 or 2.
  • the invention also relates to a double-stranded RNA molecule, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides.
  • the antisense strand contains one or more nucleotide monomers represented by formula (III) or (IV):
  • the nucleotide monomer is selected from:
  • Base is selected from
  • Q1 is R4 and Q2 is OL2 ; in another embodiment, Q1 is OL2 and Q2 is R4 .
  • Y 1 is O; in another embodiment, Y 1 is S; in another embodiment, Y 1 is NR.
  • Y2 is O; in another embodiment, Y2 is S; in another embodiment, Y2 is a chemical bond.
  • L 1 is H; in another embodiment, L 1 is P 1 ; in another embodiment, L 1 is 2 linked to the ribose sugar of another nucleotide or oligonucleotide. ' or 3' end of the phosphate P atom chemical bond.
  • L2 is H; in another embodiment, L2 is P2 ; in another embodiment, L2 is 5 linked to the ribose sugar of another nucleotide or oligonucleotide. ' chemical bond with the phosphoric acid P atom.
  • R 1 is H; in another embodiment, R 1 is D; in another embodiment, R 1 is halogen; in another embodiment, R 1 is OH; in another In one embodiment, R 1 is CN; in another embodiment, R 1 is C 1-6 alkyl; in another embodiment, R 1 is C 1-4 alkyl; in another embodiment, R 1 is C 1-6 haloalkyl; in another embodiment, R 1 is C 1-4 haloalkyl; in another embodiment, R 1 is C 2-6 alkenyl; in another embodiment , R 1 is C 2-6 alkynyl; in another embodiment, R 1 is C 3-10 cycloalkyl; in another embodiment, R 1 is 3-10 membered heterocyclyl; in another embodiment , R 1 is C 6-10 aryl; in another embodiment, R 1 is 5-14 membered heteroaryl.
  • R 1 is unsubstituted; in another embodiment, R 1 is substituted with 1 R'; in another embodiment, R 1 is substituted with 2 R'; in another embodiment , R 1 is substituted with 3 R'; in another embodiment, R 1 is substituted with 4 R'; in another embodiment, R 1 is substituted with 5 R'; in another embodiment, R 1 is substituted with 6 R'; in another embodiment, R 1 is substituted with 7 R'; in another embodiment, R 1 is substituted with 8 R'; in another embodiment, R 1 is substituted with Multiple R' substitutions.
  • R2 is H; in another embodiment, R2 is D; in another embodiment, R2 is halogen; in another embodiment, R2 is OH; in another In one embodiment, R 2 is CN; in another embodiment, R 2 is C 1-6 alkyl; in another embodiment, R 2 is C 1-4 alkyl; in another embodiment, R 2 is C 1-6 haloalkyl; in another embodiment, R 2 is C 1-4 haloalkyl; in another embodiment, R 2 is C 2-6 alkenyl; in another embodiment , R 2 is C 2-6 alkynyl; in another embodiment, R 2 is C 3-10 cycloalkyl; in another embodiment, R 2 is 3-10 membered heterocyclyl; in another In one embodiment, R 2 is C 6-10 aryl; in another embodiment, R 2 is 5-14 membered heteroaryl.
  • R is unsubstituted; in another embodiment, R is substituted with 1 R'; in another embodiment, R is substituted with 2 R'; in another embodiment , R 2 is substituted with 3 R'; in another embodiment, R 2 is substituted with 4 R'; in another embodiment, R 2 is substituted with 5 R'; in another embodiment, R 2 is substituted with 6 R'; in another embodiment, R 2 is substituted with 7 R'; in another embodiment, R 2 is substituted with 8 R'; in another embodiment, R 2 is substituted with Multiple R' substitutions.
  • R 3 is H; in another embodiment, R 3 is C 1-6 alkyl; in another embodiment, R 3 is C 1-4 alkyl, such as Me; in another In one embodiment, R 3 is C 1-6 haloalkyl; in another embodiment, R 3 is C 1-4 haloalkyl; in another embodiment, R 3 is C 2-6 alkenyl; in In another embodiment, R 3 is C 2-6 alkynyl; in another embodiment, R 3 is C 3-10 cycloalkyl; in another embodiment, R 3 is 3-10 membered heterocycle group; in another embodiment, R 3 is C 6-10 aryl; in another embodiment, R 3 is 5-14 membered heteroaryl.
  • R 3 is unsubstituted; in another embodiment, R 3 is substituted with 1 R'; in another embodiment, R 3 is substituted with 2 R'; in another embodiment , R 3 is substituted with 3 R'; in another embodiment, R 3 is substituted with 4 R'; in another embodiment, R 3 is substituted with 5 R'; in another embodiment, R 3 is substituted with 6 R'; in another embodiment, R 3 is substituted with 7 R'; in another embodiment, R 3 is substituted with 8 R'; in another embodiment, R 3 is substituted with Multiple R' substitutions.
  • R 4 is H; in another embodiment, R 4 is D; in another embodiment, R 4 is OH; in another embodiment, R 4 is halogen; in another In another embodiment, R 4 is C 1-6 alkyl; in another embodiment, R 4 is C 1-4 alkyl; in another embodiment, R 4 is C 1-6 haloalkyl; in another In one embodiment, R 4 is C 1-4 haloalkyl; in another embodiment, R 4 is C 1-6 alkoxy; in another embodiment, R 4 is C 1-4 alkoxy. , such as OMe; in another embodiment, R 4 is C 1-6 haloalkoxy; in another embodiment, R 4 is C 1-4 haloalkoxy.
  • R 5 is H; in another embodiment, R 5 is D; in another embodiment, R 5 is OH; in another embodiment, R 5 is halogen, such as F; In another embodiment, R 5 is C 1-6 alkyl; in another embodiment, R 5 is C 1-4 alkyl; in another embodiment, R 5 is C 1-6 haloalkyl; in another embodiment, R 5 is C 1-4 haloalkyl; in another embodiment In one embodiment, R 5 is C 1-6 alkoxy; in another embodiment, R 5 is C 1-4 alkoxy, such as OMe; in another embodiment, R 5 is C 1-6 alkyl halo Oxy; in another embodiment, R 5 is C 1-4 haloalkoxy.
  • P 1 is a protecting group; in another embodiment, P 1 is a hydroxyl protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropyl silyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl ( TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxy Carbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert
  • P is a reactive phosphorus group, such as phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, such as natural phosphates, phosphorothioates , phosphorodithioates, borane phosphates, borane phosphorothioates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, phosphorothioate diesters , thiophosphoric triester, diphosphate or triphosphate, preferably -P(OCH 2 CH 2 CN) (N(iPr) 2 ).
  • Base is H; in another embodiment, Base is a modified or unmodified base or leaving group, such as preferably modified or unmodified A, U, T, G and C.
  • Base' is H; in another embodiment, Base' is a modified or unmodified base or leaving group, such as preferably modified or unmodified A, U, T, G and C.
  • Base is In another more specific implementation, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is in another more specific embodiment, Base is in another more In a specific implementation, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is in another more specific embodiment, Base is in another more specific embodiment, Base is in another more specific embodiment, Base is in another more specific embodiment, Base is in another more specific embodiment, Base is
  • Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is in another more specific embodiment, Base' is in another more specific embodiment, Base' is in another more specific embodiment, Base' is in another more specific embodiment, Base' is in another more specific embodiment, Base' is in another more specific embodiment, Base' is in another more specific embodiment, Base' is in another more specific embodiment, Base' is in another more specific embodiment, Base' is in another more specific embodiment, Base' is in another more specific embodiment, Base' is in another more specific embodiment, Base' is in another more specific embodiment, Base'
  • R is H; in another embodiment, R is C 1-6 alkyl; in another embodiment, R is C 1-6 haloalkyl.
  • R' is D; in another embodiment, R' is halogen; in another embodiment, R' is CN; in another embodiment, R' is C 1-6 alkane group; in another embodiment, R' is C 1-6 haloalkyl; in another embodiment In another embodiment, R' is C 2-6 alkenyl; in another embodiment, R' is C 2-6 alkynyl; in another embodiment, R' is C 3-10 cycloalkyl; in another In one embodiment, R' is a 3-10 membered heterocyclyl group; in another embodiment, R' is a C 6-10 aryl group; in another embodiment, R' is a 5-14 membered heteroaryl group; In another embodiment, R' is -OR a , such as OH; in another embodiment, R' is -OC(O)R a ; in another embodiment, R' is -OC(O) OR b ; in another embodiment, R' is -OC(O)NR a R b ; in another embodiment, R' is
  • each R a is independently selected from: H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 3 -10-membered heterocyclyl, C 6-10 aryl or 5-14-membered heteroaryl; or R a and R b and the nitrogen atoms to which they are connected form a 3-10-membered heterocyclyl;
  • Each R b is independently selected from: H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl , C 2-6 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-14 membered heteroaryl; or R a and R b and the nitrogen atoms to which they are connected form a 3-10 membered heterocyclyl.
  • m is 0; in another embodiment, m is 1; in another embodiment, m is 2; in another embodiment, m is 3; in another embodiment, m is 4; in another embodiment, m is 5.
  • n is 0; in another embodiment, n is 1; in another embodiment, n is 2; in another embodiment, n is 3; in another embodiment, n is 4; in another embodiment, n is 5.
  • GalNAc is a conjugation group represented by formula (X):
  • L G1 is a chemical bond, -CH 2 -, -CH 2 CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-( CH 2 NHC(O)) a -;
  • L G2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L G3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L G4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • A is a chemical bond, -CH 2 O- or -NHC(O)-;
  • A' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • B is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
  • R G1 and R G2 together form -CH 2 CH 2 O- or -CH 2 CH(R G )-O-, and R G3 is H;
  • R G1 and R G3 together form -C 1-2 alkylene-, and R G2 is H;
  • RG is -OR G ', -CH 2 OR G ' or -CH 2 CH 2 OR G ', wherein RG ' is H, a hydroxyl protecting group or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O )CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n1 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • GalNAc is a conjugated group of formula (I'):
  • L G1 is a chemical bond, -CH 2 -, -CH 2 CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-( CH 2 NHC(O)) a -;
  • L G2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L G3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L G4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • A is -CH 2 O- or -NHC(O)-;
  • A’ is a chemical bond, -C(O)NH- or -NHC(O)-;
  • R G1 and R G2 together form -CH 2 CH 2 O- or -CH 2 CH(R G )-O-, and R G3 is H;
  • R G1 and R G3 together form -C 1-2 alkylene-, and R G2 is H;
  • RG is -OR G ', -CH 2 OR G ' or -CH 2 CH 2 OR G ', wherein RG ' is H, a hydroxyl protecting group or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O )CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n1 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • GalNAc is a conjugation group of formula (X), wherein,
  • L G1 is a chemical bond, -CH 2 -, -CH 2 CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-( CH 2 NHC(O)) a -;
  • L G2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L G3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L G4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • A is a chemical bond, -CH 2 O- or -NHC(O)-;
  • A' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • B is a chemical bond, -CH 2 -, -M-, -CH 2 -M- or -C(O)-M-;
  • R G1 and R G2 together form -CH 2 CH 2 O- or -CH 2 CH(R G )-O-, and R G3 is H;
  • R G1 and R G3 together form -C 1-2 alkylene-, and R G2 is H;
  • RG is -OR G ', -CH 2 OR G ' or -CH 2 CH 2 OR G ', wherein RG ' is H, a hydroxyl protecting group or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O )CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n1 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • GalNAc is a conjugation group of formula (X), wherein:
  • L G1 is a chemical bond, -CH 2 -, -CH 2 CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-( CH 2 NHC(O)) a -;
  • L G2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L G3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L G4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • A is a chemical bond, -CH 2 O- or -NHC(O)-;
  • A' is -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • B is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
  • R G1 and R G2 together form -CH 2 CH 2 O- or -CH 2 CH(R G )-O-, and R G3 is H;
  • R G1 and R G3 together form -C 1-2 alkylene-, and R G2 is H;
  • RG is -OR G ', -CH 2 OR G ' or -CH 2 CH 2 OR G ', wherein RG ' is H, a hydroxyl protecting group or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O )CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n1 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • any technical solution or any combination thereof in any of the above specific embodiments may be combined with any technical solution or any combination thereof in other specific embodiments.
  • any technical solution of X or any combination thereof can be combined with Q1 , Q2 , Y1 , Y2 , L1 , L2 , R1 , R2 , R3 , R4 , R5 , Base and Base' and other technical solutions or any combination thereof.
  • the present invention is intended to include combinations of all these technical solutions, and due to space limitations, they will not be listed one by one.
  • the invention also provides a vector comprising a nucleotide sequence encoding the siRNA of the invention.
  • the vector of the present invention can amplify or express the nucleotide 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 sustained (weeks to months or longer), depending on the specific construct used and the target tissue or cell type.
  • the siRNA encoding nucleotides can be introduced into linear constructs, circular plasmids, or viral vectors.
  • the siRNA nucleotides can be integrated into the cell genome for stable expression, or can be stably inherited and expressed extrachromosomally.
  • siRNA expression vectors are usually DNA plasmids or viral vectors.
  • Viral vector systems containing siRNA coding sequences include, but are not limited to: (a) adenovirus vectors; (b) retroviral vectors; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vector; (f) polyomavirus vector; (g) papillomavirus vector; (h) picornavirus vector; (i) poxvirus vector; and (j) helper virus-dependent adenovirus or gutless adenovirus.
  • the invention also provides cells containing the siRNA or vector of the invention, wherein the siRNA or vector of the invention is capable of being transcribed in the cell.
  • Q 1 and Q 2 are R 4 and the other is OL 2 ;
  • L 1 is H or P 1 , or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose sugar of another nucleotide or oligonucleotide, preferably P 1 ;
  • L 2 is H or P 2 , or a chemical bond to the phosphate P atom at the 5' end of the ribose sugar of another nucleotide or oligonucleotide, preferably P 2 ;
  • Y 1 is O, S or NR
  • Y 2 is O, S or chemical bond
  • R 1 and R 2 are independently selected from H, D, halogen, OH, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3- 10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-14 membered heteroaryl, which is optionally substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R'substitution;
  • R 3 is selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-14 membered heteroaryl, which is optionally substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R';
  • R 4 and R 5 are independently selected from H, D, OH, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy, preferably H, F or OMe.
  • P 1 is a hydroxyl protecting group, preferably DMTr
  • P 2 is a reactive phosphorus group, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 );
  • Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C;
  • R is selected from H, C 1-6 alkyl or C 1-6 haloalkyl
  • R' is selected from D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 3-10 yuan Heterocyclyl, C 6-10 aryl, 5-14 membered heteroaryl, -OR a , -OC(O)R a , -OC(O)OR b , -OC(O)NR a R b , - C(O)R a , -C(O)OR a , -C(O)NR a R b , -S(O) n R a , -S(O) n OR a , -S(O) n NR a R b , -OS(O) n R b , -NR a R b , -NR a C(O)R b , -NR a -C
  • R a and R b are independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-14 membered heteroaryl; or R a and R b and the nitrogen atoms to which they are connected form a 3-10 membered heterocyclyl;
  • n is selected from 1, 2, 3, 4 or 5;
  • n is independently selected from 1 or 2.
  • nucleotide dimer of technical solution 1 which is the structure of formula (I) or (II):
  • each group is as defined in technical solution 1.
  • R 1 and R 2 are independently selected from H, D, halogen, OH, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optional Land is replaced by 1, 2, 3, 4, 5 or more R';
  • R' is selected from D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, -OR a , -OC(O)R a , -OC(O)OR b , -OC(O)NR a R b , -C(O)R a , -C(O)OR a , -C(O)NR a R b , -NR a R b , -NR a C(O)R b , -NR a -C(O)OR b or -NR a C(O)NR a R b ;
  • R a and R b are independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl;
  • n is selected from 1, 2, 3, 4 or 5;
  • R 1 and R 2 are independently selected from H, D, halogen, OH, CN, C 1-6 alkyl or C 1-6 haloalkyl, which is optionally substituted by 1, 2, 3 or more R';
  • R' is selected from D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, -OR a or -NR a R b ;
  • R a and R b are independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl;
  • n is selected from 1, 2 or 3;
  • Y 1 is O or NR
  • Y 2 is O, S or chemical bond
  • R is selected from H, C 1-6 alkyl or C 1-6 haloalkyl
  • Y 1 is O or NR
  • Y 2 is O, S or chemical bond
  • R is selected from H or C 1-6 alkyl
  • Y 1 is O
  • Y 2 is O.
  • nucleotide dimer according to any one of technical solutions 2-4, wherein,
  • R 3 is selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally replaced by 1, 2, 3, 4, 5 or more R'substitution;
  • R 3 is selected from H, C 1-6 alkyl or C 1-6 haloalkyl, which is optionally substituted by 1, 2, 3 or more R';
  • R 3 is C 1-4 alkyl, preferably Me.
  • R 4 and R 5 are independently selected from H, D, OH, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy;
  • R 4 and R 5 are independently selected from H, OH, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy, preferably H, F, OH or OMe;
  • R 4 is selected from H, OH or C 1-4 alkoxy, preferably H or OMe;
  • R 5 is selected from halogen, OH or C 1-4 alkoxy, preferably F or OMe.
  • Base and Base' are independently selected from
  • L 1 is H or P 1 , or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose sugar of another nucleotide or oligonucleotide, preferably P 1 ;
  • L 2 is H or P 2 , or a chemical bond to the phosphate P atom at the 5' end of the ribose sugar of another nucleotide or oligonucleotide, preferably P 2 ;
  • Y 1 is O, S or NR
  • Y 2 is O, S or chemical bond
  • R 1 and R 2 are independently selected from H, D, halogen, OH, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optional Land is replaced by 1, 2, 3, 4, 5 or more R';
  • R 3 is selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally replaced by 1, 2, 3, 4, 5 or more R'substitution;
  • R 4 and R 5 are independently selected from H, D, OH, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy, preferably H, F or OMe;
  • P 1 is a hydroxyl protecting group, preferably DMTr
  • P 2 is a reactive phosphorus group, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 );
  • Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C;
  • R is selected from H, C 1-6 alkyl or C 1-6 haloalkyl
  • R' is selected from D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, -OR a , -OC(O)R a , -OC(O)OR b , -OC(O)NR a R b , -C(O)R a , -C(O)OR a , -C(O)NR a R b , -NR a R b , -NR a C(O)R b , -NR a -C(O)OR b or -NR a C(O)NR a R b ;
  • R a and R b are independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl;
  • n is selected from 1, 2, 3, 4 or 5.
  • L 1 is H or P 1 , or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose sugar of another nucleotide or oligonucleotide, preferably P 1 ;
  • L 2 is H or P 2 , or a chemical bond to the phosphate P atom at the 5' end of the ribose sugar of another nucleotide or oligonucleotide, preferably P 2 ;
  • Y 1 is O or NR
  • Y 2 is O, S or chemical bond
  • R 1 and R 2 are independently selected from H, D, halogen, OH, CN, C 1-6 alkyl or C 1-6 haloalkyl, which is optionally substituted by 1, 2, 3 or more R';
  • R 3 is selected from H, C 1-6 alkyl or C 1-6 haloalkyl, which is optionally substituted by 1, 2, 3 or more R';
  • R 4 and R 5 are independently selected from H, D, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy, preferably H, F or OMe;
  • P 1 is a hydroxyl protecting group, preferably DMTr
  • P 2 is a reactive phosphorus group, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 );
  • Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C;
  • R is selected from H, C 1-6 alkyl or C 1-6 haloalkyl
  • R' is selected from D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, -OR a or -NR a R b ;
  • R a and R b are independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl;
  • n is selected from 1, 2 or 3.
  • L 1 is H or P 1 , or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose sugar of another nucleotide or oligonucleotide, preferably P 1 ;
  • L 2 is H or P 2 , or a chemical bond to the phosphate P atom at the 5' end of the ribose sugar of another nucleotide or oligonucleotide, preferably P 2 ;
  • Y 1 is O
  • Y 2 is O
  • R 1 and R 2 are independently selected from H, D, halogen, OH, CN, C 1-4 alkyl or C 1-4 haloalkyl;
  • R 3 is C 1-4 alkyl, preferably Me
  • R 4 is selected from H, OH or C 1-4 alkoxy, preferably H or OMe;
  • R 5 is selected from halogen, OH or C 1-4 alkoxy, preferably F or OMe;
  • P 1 is a hydroxyl protecting group, preferably DMTr
  • P 2 is a reactive phosphorus group, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 );
  • Base and Base' are independently selected from
  • n is selected from 1, 2 or 3.
  • nucleotide dimer of any one of technical solutions 1-10 selected from:
  • Base and Base' are as defined in any one of technical solutions 1-10, preferably
  • R5 is as defined in any one of technical solutions 1-10, preferably F or OMe.
  • a double-stranded RNA molecule, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and
  • the antisense strand contains one or more nucleotide monomers represented by formula (III) or (IV):
  • the nucleotide monomer is selected from:
  • Base is selected from
  • double-stranded RNA molecule of any one of technical solutions 12-15 wherein the double-stranded RNA has a melting temperature from about 40°C to about 80°C, preferably about 55°C to 67°C.
  • RNA molecule of any one of technical solutions 12-18, wherein the antisense strand has a sequence that is fully complementary to the sense strand and the target mRNA, and has the ability to induce degradation of the target mRNA.
  • RNA molecule of technical solutions 12-19 wherein the target mRNA is encoded by an endogenous gene or encoded by a pathogen gene.
  • the ligand includes one or more GalNAc.
  • a nucleic acid molecule the nucleotide sequence of which contains one or more nucleotide monomers as described in technical solution 12.
  • nucleic acid molecule of technical solution 23 wherein the nucleic acid is selected from the group consisting of DNA, RNA and DNA/RNA hybrids.
  • nucleic acid molecule of technical solution 24 which is single-stranded or double-stranded.
  • nucleic acid molecule of any one of technical solutions 23-25 wherein the nucleic acid molecule is selected from the group consisting of small interfering RNA (siRNA) and short hairpin RNA (shRNA).
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • Vector which contains a nucleotide sequence encoding the double-stranded RNA described in any one of the preceding technical solutions 12-22.
  • Cell which contains the double-stranded RNA as described in any one of technical solutions 12-22 or the vector as described in technical solution 27.
  • a pharmaceutical composition comprising the double-stranded RNA molecule as described in any one of technical solutions 12-22, and a pharmaceutically acceptable carrier or excipient.
  • a kit comprising the double-stranded RNA molecule as described in any one of technical solutions 12-22.
  • a method for inhibiting the expression of a target gene in a cell comprising the step of introducing the double-stranded RNA molecule according to any one of technical solutions 12-22 into the cell.
  • a method for inhibiting the expression of a target gene in a cell comprising expressing the double-stranded RNA molecule of any one of technical solutions 12-22 in the cell.
  • a method for reducing off-target toxicity in cells comprising converting the double-stranded RNA molecule described in any one of technical solutions 12-22 Steps to introduce the cells.
  • a method for reducing off-target toxicity in cells comprising expressing the double-stranded RNA molecule of any one of technical solutions 12-22 in the cells.
  • Dissolve compound 2 (95.0g, 222mmol) in toluene (1.50L) at 25°C, and add imidazole (30.2g, 443mmol), triphenylphosphine (116g, 443mmol) and iodine element (84.4g, 322mmol) in sequence.
  • the reaction solution was stirred at 100°C for 18 hours.
  • Add 20.0 mL of saturated NaHSO 3 solution to the reaction solution, and add 500 mL of water. The reaction solution is separated into layers.
  • the organic phase is washed with saturated NaCl solution (50.0 mL ⁇ 3), dried over anhydrous Na 2 SO 4 , and spin-dried to obtain the crude product.
  • reaction solution was cooled to 0°C, quenched by adding 50 mL saturated sodium carbonate solution, separated into layers, diluted with 300 mL DCM to the organic phase, washed with water (50 mL x 3) and saturated NaCl aqueous solution (50 mL x 1), and the organic phase was washed with anhydrous Na Dry over 2 SO 4 , filter, and spin dry to get the crude product.
  • the organic phase was washed with 20 mL of saturated NaHCO 3 aqueous solution and 20 mL of saturated NaCl aqueous solution.
  • the organic phase was dried over anhydrous Na 2 SO 4 , filtered, and spun to dryness to obtain the crude product, which was purified by reverse phase (acetonitrile/water: 20-80%, 30 min, 20 mL/min) to obtain yellow solid E1 (210 mg, 0.165 mmol, 46.57 %).
  • siRNA of the invention is prepared using the solid-phase phosphoramidite method, which is well known in the art. Specific methods can be found, for example, in PCT publication numbers WO2016081444 and WO2019105419, and are briefly described below.
  • a blank CPG solid-phase carrier or a solid-phase carrier connected with L96 is used as the starting cycle, and the nucleosides are connected one by one from the 3'-5' direction in the order of the sense strand nucleotides. monomer.
  • Each connected nucleoside monomer includes four-step reactions of deprotection, coupling, capping, oxidation or sulfation.
  • the synthesis conditions for an oligonucleotide with a synthesis scale of 5umol are as follows:
  • the nucleoside monomer is provided with a 0.05 mol/L acetonitrile solution.
  • the reaction conditions for each step are the same, that is, the temperature is 25°C.
  • the temperature is 25°C.
  • the capping agent used 10% acetic anhydride-acetonitrile and pyridine/N-methylimidazole/acetonitrile (10:14:76, v/v/ v), capping 2 times; oxidation using 0.05mol/L iodine/tetrahydrofuran/pyridine/water (70/20/10, v/v/v), oxidation 2 times; sulfide using 0.2mol/L PADS acetonitrile/ 3-methylpyridine (1/1, v/v), sulfide substituted 2 times.
  • a blank CPG solid-phase carrier is used as the initial cycle, and the nucleoside monomers or the core of the present invention are connected one by one from the 3'-5' direction according to the sequence of the antisense strand nucleotides. glycoside dimer.
  • Each connection of a nucleoside monomer or nucleotide dimer of the present invention includes a four-step reaction of deprotection, coupling, capping, oxidation or thiolation, oligonucleotide synthesis conditions of 5umol of the antisense strand and oligonucleotide synthesis conditions of the sense strand. same.
  • a strong anion packing column can be used, a sodium chloride-sodium hydroxide system can be used for elution and purification, and the products can be collected and tubed.
  • a gel packing purification column can be used for desalting, and the elution system is pure water.
  • siRNA sequences used in the present invention are as follows:
  • the A, U, G, and C distributions represent natural adenine ribonucleotides, uracil ribonucleotides, guanine ribonucleotides, and cytosine ribonucleotides.
  • m indicates that the adjacent nucleotide to its left is a 2'-OCH 3 modified nucleotide.
  • Am, Um, Gm and Cm represent 2'-OCH 3 modified A, U, G and C.
  • f indicates that the adjacent nucleotide to its left is a 2’-F modified nucleotide.
  • Af, Uf, Gf and Cf represent 2′-F modified A, U, G and C respectively.
  • s or "s-" means that two adjacent nucleotides and/or delivery vectors are connected through phosphorothioate.
  • L96 represents a GalNAc delivery vector of the following structure, which is well known in the art, wherein Represented by a phosphate group or phosphorothioate
  • the location where the ester group is attached to the siRNA can be found, for example, in PCT Publication Nos. WO2009073809 and WO2009082607.
  • ROR14 represents a nucleotide substitution of the structure described above, where Base can be any base, for example, ROR14-A represents Base as adenine.
  • ROR14-A is as follows: Among them, Base is adenine.
  • the target plasmid Design the corresponding antisense strand in the target plasmid based on the compound sequence.
  • the psiCHECK2GSCM recombinant plasmid was prepared by Sangon Bioengineering (Shanghai) Co., Ltd. and the recombinant plasmid was diluted to 1000ng/ ⁇ L for use.
  • Off-target plasmid The corresponding antisense chain off-target plasmid was designed based on the compound sequence.
  • the psiCHECK2GSSM-5Hits recombinant plasmid was prepared by Sangon Bioengineering (Shanghai) Co., Ltd. and the recombinant plasmid was diluted to 1000ng/ ⁇ L for later use.
  • HEK293A cells (Nanjing Kebai, Cat. No. CBP60436) were plated in 96-well plates with 100 ⁇ L of cell resuspension, and the cell volume was: 8 ⁇ 10 3 cells/well.
  • Plasmid mixture Single well preparation volume: plasmid 0.01 ⁇ L/well, Opti-MEM 8.99 ⁇ L/well.
  • Lipo mixing Dilute Lipo 2000 (Lipofectamine TM 2000 transfection reagent, Thermo, 11668019) with Opti-MEM, and let stand at room temperature for 5 minutes.
  • the specific preparation volume of Lipo mixture Lipo 0.2 ⁇ L/well, Opti-MEM 9.8 ⁇ L/well.
  • the vacuum pump sucks away the original culture medium in the 96-well culture plate
  • the fluorescence activity is measured by a microplate reader.
  • the collected Renilla signals are normalized by the Firefly signal standard.
  • the inhibitory effect of siRNA is compared with the unprocessed results (residual inhibitory activity). The calculation process is as follows:
  • Ratio Renilla (Renilla luciferase)/Firefly (firefly luciferase).
  • Residual inhibition rate the mean value of 2 duplicate wells (Ratio siRNA /Ratio control )*100%: where Ratio control is the mean value of the Ratio of the 2 duplicate wells of the control well (excluding siNRA), and the Ratio siRNA / Ratio control , and then the average is the remaining inhibition rate;
  • IC50 Half maximal inhibitory concentration
  • the HEK293A (Nanjing Kebai, Cat. No. CBP60436) cell line was selected for psiCHECK2-GSCM recombinant plasmid transfection.
  • the starting concentration of the selected compound was 10nM, and it was diluted 3 times to 11 concentration points (10nM, 3.33nM, 1.11nM, 0.37nM, 0.123nM, 0.041nM, 0.0136nM, 0.0045nM, 0.00152nM, 0.000508nM, 0.000169nM), the results of siRNA compound activity screening experiments are shown in Table 1.
  • HEK293A (Nanjing Kebai, Cat. No. CBP60436) cell line was selected for psiCHECK2-GSSM-5Hits recombinant plasmid transfection.
  • the starting concentration of the selected compound was 10nM, and it was diluted 3 times to 11 concentration points (10nM, 3.33nM, 1.11nM , 0.37nM, 0.123nM, 0.041nM, 0.0136nM, 0.0045nM, 0.00152nM, 0.000508nM, 0.000169nM), the results of siRNA compound activity screening experiments are shown in Table 2.
  • siRNA carrying ROR14 effectively reduced off-target activity while maintaining target activity.
  • C57BL/6 mice male, 18-21g, 6-8 weeks were randomly divided into groups according to Table 3, with 6 animals in each group.
  • the dosage of each animal was calculated according to body weight, and a single dose was administered by subcutaneous injection.
  • the siRNA compound was first prepared as a 1 mg/mL solution (0.9% sodium chloride aqueous solution as the solvent). Before the experiment, the siRNA compound was dissolved and diluted to the required solution concentration and volume with 0.9% sodium chloride aqueous solution. Physiological saline and The administration volume of siRNA compound was 5 mL/kg.
  • Blood was collected from the orbital venous plexus of mice before administration (recorded as day 0), and on days 14, 28, 42, and 56 after administration, and serum mTTR was detected using ELISA kit (Abcam, ab282297) at each time point. Protein; at the last experimental time point, take 10 mg of liver and place it in RNAlater solution to detect liver mTTR mRNA.
  • siRNA carrying ROR14 could reduce target gene expression in vivo for a long time.

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Abstract

本发明提供具有核苷酸类似物的双链RNA。本发明的双链RNA显示增强的稳定性、降低的脱靶毒性和增强的有效性中的一种或多种。

Description

含有可降低脱靶毒性的核苷酸类似物的双链RNA
本发明要求提交于2022年6月27日的中国申请CN202210744263.8、提交于2022年8月8日的中国申请CN202210948347.3、以及提交于2022年11月24日的中国申请CN202211483699.2的优先权，将它们以其整体引入本文作为参考。
发明领域
本发明属于医药领域，具体涉及具有核苷酸类似物的双链RNA。
背景技术
RNA干扰是一种由双链RNA(double-stranded RNA，dsRNA)诱发的靶标mRNA高效特异性降解的现象。在双链RNA的反义链种子区掺入热不稳定的核苷酸(例如甘油核酸(GNA))有助于提高干扰效率和降低脱靶毒性，参见例如PCT公开号WO2018098328A1。
因此，本领域需要开发一种新的核苷酸类似物，掺入双链RNA后有助于降低脱靶毒性。
发明内容
本发明通过提供一种新的核苷酸类似物解决了上述问题。
在一个方面中，本发明提供了式(A)所示的核苷酸双聚体：
其中各基团如下文所定义。
在另一个方面中，本发明提供了双链RNA分子或其药学上可接受的盐、互变异构体或立体异构体，其包含正义链和反义链，其中各链具有14至30个核苷酸，并且所述反义链包含一个或多个式(III)或(IV)所示的核苷酸单体：
其中，
所示核苷酸单体以5’＝>3’的顺序从至连接；
各基团如下文所定义。
在另一个方面中，本发明提供了核酸分子，所述核酸分子的核苷酸序列中包含一个或多个如本文 所述的核苷酸双聚体和/或如本文所述的核苷酸单体。
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，本发明涉及一种药物组合物，其包含如本文所述的双链RNA分子，和药学上可接受的载体或赋形剂。
在另一个方面中，本发明涉及一种试剂盒，其包含如本文所述的双链RNA分子。
在另一个方面中，本发明涉及一种用于抑制细胞中靶基因的表达的方法，包括将如本文所述的双链RNA分子引入该细胞的步骤。
在另一个方面中，本发明涉及一种用于抑制细胞中靶基因的表达的方法，包括在所述细胞中表达如本文所述的双链RNA分子。
在另一个方面中，本发明涉及一种用于降低细胞中脱靶毒性的方法，包括将本文所述的双链RNA分子引入该细胞的步骤。
在另一个方面中，本发明涉及一种用于降低细胞中脱靶毒性的方法，包括在所述细胞中表达本文所述的双链RNA分子。
本发明的核苷酸掺入dsRNA的反义链后，使得所得双链RNA显示增强的稳定性、降低的脱靶毒性和增强的有效性中的一种或多种。
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。在一些实施方案中，C1-4烷基和C1-2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)。
“C2-6烯基”是指具有2至6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，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。
“C2-6炔基”是指具有2至6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，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。
“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”，其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。
“C1-6烷氧基”是指-O-R基团，其中R如上文的“C1-6烷基”和“C1-6卤代烷基”所定义。
“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。
“C6-10芳基”是指具有6-10个环碳原子和零个杂原子的单环或多环的(例如，双环)4n+2芳族环体系(例如，具有以环状排列共享的6或10个π电子)的基团。在一些实施方案中，芳基具有六个环碳原子(“C6芳基”；例如，苯基)。在一些实施方案中，芳基具有十个环碳原子(“C10芳基”；例如，萘基，例如，1-萘基和2-萘基)。芳基还包括其中上述芳基环与一个或多个环烷基或杂环基稠合的环系统，而且连接点在所述芳基环上，在这种情况下，碳原子的数目继续表示所述芳基环系统中的碳原子数目。芳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。
“5-14元杂芳基”是指具有环碳原子和1-4个环杂原子的5-14元单环或双环的4n+2芳族环体系(例如，具有以环状排列共享的6、10或14个π电子)的基团，其中每个杂原子独立地选自氮、氧和硫。在含有一个或多个氮原子的杂芳基中，只要化合价允许，连接点可以是碳或氮原子。杂芳基双环系统在一个或两个环中可以包括一个或多个杂原子。杂芳基还包括其中上述杂芳基环与一个或多个环烷基或杂环基稠合的环系统，而且连接点在所述杂芳基环上，在这种情况下，碳原子的数目继续表示所述杂芳基环系统中的碳原子数目。在一些实施方案中，5-10元杂芳基是优选的，其为具有环碳原子和1-4个环杂原子的5-10元单环或双环的4n+2芳族环体系。在另一些实施方案中，5-6元杂芳基是特别优选的，其为具有环碳原子和1-4个环杂原子的5-6元单环或双环的4n+2芳族环体系。示例性的含有一个杂原子的5元杂芳基包括但不限于：吡咯基、呋喃基和噻吩基。示例性的含有两个杂原子的5元杂芳基包括但不限于：咪唑基、吡唑基、噁唑基、异噁唑基、噻唑基和异噻唑基。示例性的含有三个杂原子的5元杂芳基包括但不限于：三唑基、噁二唑基(例如，1,2,4-噁二唑基)和噻二唑基。示例性的含有四个杂原子的5元杂芳基包括但不限于：四唑基。示例性的含有一个杂原子的6元杂芳基包括但不限于：吡啶基。示例性的含有两个杂原子的6元杂芳基包括但不限于：哒嗪基、嘧啶基和吡嗪基。示例性的含有三个或四个杂原子的6元杂芳基分别包括但不限于：三嗪基和四嗪基。示例性的含有一个杂原子的7元杂芳基包括但不限于：氮杂环庚三烯基、氧杂环庚三烯基和硫杂环庚三烯基。示例性的5,6-双环杂芳基包括但不限于：吲哚基、异吲哚基、吲唑基、苯并三唑基、苯并噻吩基、异苯并噻吩基、苯并呋喃基、苯并异呋喃基、苯并咪唑基、苯并噁唑基、苯并异噁唑基、苯并噁二唑基、苯并噻唑基、苯并异噻唑基、苯并噻二唑基、茚嗪基和嘌呤基。示例性的6,6-双环杂芳基包括但不限于：萘啶基、喋啶基、喹啉基、异喹啉基、噌琳基、喹喔啉基、酞嗪基和喹唑啉基。杂芳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。
本文定义的烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳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。
“shRNA”是指短发夹RNA。shRNA包括两个短反向重复序列。克隆到shRNA表达载体中的shRNA包括两个短反向重复序列，中间由一茎环(loop)序列分隔的，组成发夹结构，由polⅢ启动子控制。随后再连上5-6个T作为RNA聚合酶Ⅲ的转录终止子。
“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”又称“保护基团”，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
本发明具体涉及式(A)所示的核苷酸双聚体：
其中，
Q1和Q2中一个为R4，另一个为O-L2；
L1是H或P1，或是连接至另一核苷酸或寡核苷酸的核糖的2’或3’端的磷酸P原子的化学键，优选为P1；
L2是H或P2，或是连接至另一核苷酸或寡核苷酸的核糖的5’端的磷酸P原子的化学键，优选为P2；
X选自-(CR1R2)m-或-CR1＝CR2-；
Y1是O、S或NR；
Y2是O、S或化学键；
R1和R2独立地选自H、D、卤素、OH、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、C3-10环烷基、3-10元杂环基、C6-10芳基或5-14元杂芳基，其任选地被1、2、3、4、5、6、7、8或多个R’取代；
R3选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、C3-10环烷基、3-10元杂环基、C6-10芳基或5-14元杂芳基，其任选地被1、2、3、4、5、6、7、8或多个R’取代；
R4和R5独立地选自H、D、OH、卤素、C1-6烷基、C1-6卤代烷基或C1-6烷氧基，优选H、F或OMe。
P1为羟基保护基，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)、4,4'-二甲氧基三苯甲基、-P(OCH2CH2CN)(N(iPr)2)或-C(O)CH2CH2C(O)OH，优选为DMTr；
P2为反应性磷基团，例如亚磷酰胺、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)；
Base和Base’独立地选自H、修饰或未修饰的碱基或离去基，优选修饰或未修饰的A、U、T、G和C；
R选自H、C1-6烷基或C1-6卤代烷基；
R’选自D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、C3-10环烷基、3-10元杂环基、C6-10芳基、5-14元杂芳基、-ORa、-OC(O)Ra、-O-C(O)ORb、-O-C(O)NRaRb、-C(O)Ra、-C(O)ORa、-C(O)NRaRb、-S(O)nRa、-S(O)nORa、-S(O)nNRaRb、-O-S(O)nRb、-NRaRb、-NRaC(O)Rb、-NRa-C(O)ORb、-NRa-S(O)nRb或-NRaC(O)NRaRb；
Ra和Rb独立地选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、C3-10环烷基、3-10元杂环基、C6-10芳基或5-14元杂芳基；或者Ra和Rb以及它们连接的氮原子形成3-10元杂环基；
m选自1、2、3、4或5；
n独立地选自1或2。
本发明还涉及一种双链RNA分子或其药学上可接受的盐、互变异构体或立体异构体，其包含正义链和反义链，其中各链具有14至30个核苷酸，并且所述反义链包含一个或多个式(III)或(IV)所示的核苷酸单体：
其中，
所示核苷酸单体以5’＝>3’的顺序从至连接；
各基团如上下文中所定义；
优选地，所述核苷酸单体选自：

其中，Base选自
Q1和Q2
在一个实施方案中，Q1为R4，Q2为O-L2；在另一个实施方案中，Q1为O-L2，Q2为R4。
X
在一个实施方案中，X为-(CR1R2)m-；在另一个实施方案中，X为-CR1＝CR2-。
在一个更具体的实施方案中，X为-CH2-；在另一个更具体的实施方案中，X为-CH(OH)-；在另一个更具体的实施方案中，X为-CH2-CH2-；在另一个更具体的实施方案中，X为-CH＝CH-。
Y1和Y2
在一个实施方案中，Y1为O；在另一个实施方案中，Y1为S；在另一个实施方案中，Y1为NR。
在一个实施方案中，Y2为O；在另一个实施方案中，Y2为S；在另一个实施方案中，Y2为化学键。
L1和L2
在一个实施方案中，L1为H；在另一个实施方案中，L1为P1；在另一个实施方案中，L1为连接至另一核苷酸或寡核苷酸的核糖的2’或3’端的磷酸P原子的化学键。
在一个实施方案中，L2为H；在另一个实施方案中，L2为P2；在另一个实施方案中，L2为连接至另一核苷酸或寡核苷酸的核糖的5’端的磷酸P原子的化学键。
R1和R2
在一个实施方案中，R1为H；在另一个实施方案中，R1为D；在另一个实施方案中，R1为卤素；在另一个实施方案中，R1为OH；在另一个实施方案中，R1为CN；在另一个实施方案中，R1为C1-6烷基；在另一个实施方案中，R1为C1-4烷基；在另一个实施方案中，R1为C1-6卤代烷基；在另一个实施方案中，R1为C1-4卤代烷基；在另一个实施方案中，R1为C2-6烯基；在另一个实施方案中，R1 为C2-6炔基；在另一个实施方案中，R1为C3-10环烷基；在另一个实施方案中，R1为3-10元杂环基；在另一个实施方案中，R1为C6-10芳基；在另一个实施方案中，R1为5-14元杂芳基。
在一个实施方案中，R1未被取代；在另一个实施方案中，R1被1个R’取代；在另一个实施方案中，R1被2个R’取代；在另一个实施方案中，R1被3个R’取代；在另一个实施方案中，R1被4个R’取代；在另一个实施方案中，R1被5个R’取代；在另一个实施方案中，R1被6个R’取代；在另一个实施方案中，R1被7个R’取代；在另一个实施方案中，R1被8个R’取代；在另一个实施方案中，R1被多个R’取代。
在一个实施方案中，R2为H；在另一个实施方案中，R2为D；在另一个实施方案中，R2为卤素；在另一个实施方案中，R2为OH；在另一个实施方案中，R2为CN；在另一个实施方案中，R2为C1-6烷基；在另一个实施方案中，R2为C1-4烷基；在另一个实施方案中，R2为C1-6卤代烷基；在另一个实施方案中，R2为C1-4卤代烷基；在另一个实施方案中，R2为C2-6烯基；在另一个实施方案中，R2为C2-6炔基；在另一个实施方案中，R2为C3-10环烷基；在另一个实施方案中，R2为3-10元杂环基；在另一个实施方案中，R2为C6-10芳基；在另一个实施方案中，R2为5-14元杂芳基。
在一个实施方案中，R2未被取代；在另一个实施方案中，R2被1个R’取代；在另一个实施方案中，R2被2个R’取代；在另一个实施方案中，R2被3个R’取代；在另一个实施方案中，R2被4个R’取代；在另一个实施方案中，R2被5个R’取代；在另一个实施方案中，R2被6个R’取代；在另一个实施方案中，R2被7个R’取代；在另一个实施方案中，R2被8个R’取代；在另一个实施方案中，R2被多个R’取代。
R3
在一个实施方案中，R3为H；在另一个实施方案中，R3为C1-6烷基；在另一个实施方案中，R3为C1-4烷基，例如Me；在另一个实施方案中，R3为C1-6卤代烷基；在另一个实施方案中，R3为C1-4卤代烷基；在另一个实施方案中，R3为C2-6烯基；在另一个实施方案中，R3为C2-6炔基；在另一个实施方案中，R3为C3-10环烷基；在另一个实施方案中，R3为3-10元杂环基；在另一个实施方案中，R3为C6-10芳基；在另一个实施方案中，R3为5-14元杂芳基。
在一个实施方案中，R3未被取代；在另一个实施方案中，R3被1个R’取代；在另一个实施方案中，R3被2个R’取代；在另一个实施方案中，R3被3个R’取代；在另一个实施方案中，R3被4个R’取代；在另一个实施方案中，R3被5个R’取代；在另一个实施方案中，R3被6个R’取代；在另一个实施方案中，R3被7个R’取代；在另一个实施方案中，R3被8个R’取代；在另一个实施方案中，R3被多个R’取代。
R4和R5
在一个实施方案中，R4为H；在另一个实施方案中，R4为D；在另一个实施方案中，R4为OH；在另一个实施方案中，R4为卤素；在另一个实施方案中，R4为C1-6烷基；在另一个实施方案中，R4为C1-4烷基；在另一个实施方案中，R4为C1-6卤代烷基；在另一个实施方案中，R4为C1-4卤代烷基；在另一个实施方案中，R4为C1-6烷氧基；在另一个实施方案中，R4为C1-4烷氧基，例如OMe；在另一个实施方案中，R4为C1-6卤代烷氧基；在另一个实施方案中，R4为C1-4卤代烷氧基。
在一个实施方案中，R5为H；在另一个实施方案中，R5为D；在另一个实施方案中，R5为OH；在另一个实施方案中，R5为卤素，例如F；在另一个实施方案中，R5为C1-6烷基；在另一个实施方案 中，R5为C1-4烷基；在另一个实施方案中，R5为C1-6卤代烷基；在另一个实施方案中，R5为C1-4卤代烷基；在另一个实施方案中，R5为C1-6烷氧基；在另一个实施方案中，R5为C1-4烷氧基，例如OMe；在另一个实施方案中，R5为C1-6卤代烷氧基；在另一个实施方案中，R5为C1-4卤代烷氧基。
P1和P2
在一个实施方案中，P1为保护基；在另一个实施方案中，P1为羟基保护基，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)、4,4'-二甲氧基三苯甲基、-P(OCH2CH2CN)(N(iPr)2)或-C(O)CH2CH2C(O)OH，优选DMTr。
在一个实施方案中，P2为反应性磷基团，例如亚磷酰胺、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)。
Base和Base’
在一个实施方案中，Base为H；在另一个实施方案中，Base为修饰或未修饰的碱基或离去基，例如优选修饰或未修饰的A、U、T、G和C。
在一个实施方案中，Base’为H；在另一个实施方案中，Base’为修饰或未修饰的碱基或离去基，例如优选修饰或未修饰的A、U、T、G和C。
在一个更具体的实施方案中，Base为在另一个更具体的实施方案中，Base为在另一个更具体的实施方案中，Base为在另一个更具体的实施方案中，Base为在另一个更具体的实施方案中，Base为在另一个更具体的实施方案中，Base为在另一个更具体的实施方案中，Base为在另一个更 具体的实施方案中，Base为在另一个更具体的实施方案中，Base为在另一个更具体的实施方案中，Base为
在一个更具体的实施方案中，Base’为在另一个更具体的实施方案中，Base’为在另一个更具体的实施方案中，Base’为在另一个更具体的实施方案中，Base’为在另一个更具体的实施方案中，Base’为在另一个更具体的实施方案中，Base’为在另一个更具体的实施方案中，Base’为在另一个更具体的实施方案中，Base’为在另一个更具体的实施方案中，Base’为在另一个更具体的实施方案中，Base’为
R
在一个实施方案中，R为H；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C1-6卤代烷基。
R’
在一个实施方案中，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CN；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C1-6卤代烷基；在另一个实施方案 中，R’为C2-6烯基；在另一个实施方案中，R’为C2-6炔基；在另一个实施方案中，R’为C3-10环烷基；在另一个实施方案中，R’为3-10元杂环基；在另一个实施方案中，R’为C6-10芳基；在另一个实施方案中，R’为5-14元杂芳基；在另一个实施方案中，R’为-ORa，例如OH；在另一个实施方案中，R’为-OC(O)Ra；在另一个实施方案中，R’为-O-C(O)ORb；在另一个实施方案中，R’为-O-C(O)NRaRb；在另一个实施方案中，R’为-C(O)Ra；在另一个实施方案中，R’为-C(O)ORa；在另一个实施方案中，R’为-C(O)NRaRb；在另一个实施方案中，R’为-S(O)nRa；在另一个实施方案中，R’为-S(O)nORa；在另一个实施方案中，R’为-S(O)nNRaRb；在另一个实施方案中，R’为-O-S(O)nRb；在另一个实施方案中，R’为-NRaRb；在另一个实施方案中，R’为-NRaC(O)Rb；在另一个实施方案中，R’为-NRa-C(O)ORb；在另一个实施方案中，R’为-NRa-S(O)nRb；在另一个实施方案中，R’为-NRaC(O)NRaRb。
其中，每个Ra独立地选自：H、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、C3-10环烷基、3-10元杂环基、C6-10芳基或5-14元杂芳基；或者Ra和Rb以及它们连接的氮原子形成3-10元杂环基；
每个Rb独立地选自：H、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、C3-10环烷基、3-10元杂环基、C6-10芳基或5-14元杂芳基；或者Ra和Rb以及它们连接的氮原子形成3-10元杂环基。
m
在一个实施方案中，m为0；在另一个实施方案中，m为1；在另一个实施方案中，m为2；在另一个实施方案中，m为3；在另一个实施方案中，m为4；在另一个实施方案中，m为5。
n
在一个实施方案中，n为0；在另一个实施方案中，n为1；在另一个实施方案中，n为2；在另一个实施方案中，n为3；在另一个实施方案中，n为4；在另一个实施方案中，n为5。
GalNAc
在一个实施方案中，GalNAc为式(X)所示的缀合基团：
其中，
表示与生物分子连接的位置；
Q独立地为H、
其中LG1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；
LG2为化学键或-CH2CH2C(O)-；
LG3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；
LG4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；
其中a＝0、1、2或3；
b＝1、2、3、4或5；
c＝1、2、3、4或5；
d＝1、2、3、4、5、6、7或8；
A为化学键、-CH2O-或-NHC(O)-；
A’为化学键、-C(O)NH-、-NHC(O)-或-O(CH2CH2O)e-；
其中e为1、2、3、4或5；
B为化学键、-CH2-、-C(O)-、-M-、-CH2-M-或-C(O)-M-；
其中M为
RG1和RG2一起形成-CH2CH2O-或-CH2CH(RG)-O-，并且RG3为H；
或者RG1和RG3一起形成-C1-2亚烷基-，并且RG2为H；
其中RG为-ORG’、-CH2ORG’或-CH2CH2ORG’，其中RG’为H、羟基保护基或固相载体，所述羟基保护基优选-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基；
m1＝0、1、2、3、4、5、6、7、8、9或10；
n1＝0、1、2、3、4、5、6、7、8、9或10。
在另一个实施方案中，GalNAc为式(I’)所示的缀合集团：
其中，
表示与生物分子连接的位置；
Q独立地为H、
其中LG1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；
LG2为化学键或-CH2CH2C(O)-；
LG3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；
LG4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；
其中a＝0、1、2或3；
b＝1、2、3、4或5；
c＝1、2、3、4或5；
d＝1、2、3、4、5、6、7或8；
A为-CH2O-或-NHC(O)-；
A’为化学键、-C(O)NH-或-NHC(O)-；
RG1和RG2一起形成-CH2CH2O-或-CH2CH(RG)-O-，并且RG3为H；
或者RG1和RG3一起形成-C1-2亚烷基-，并且RG2为H；
其中RG为-ORG’、-CH2ORG’或-CH2CH2ORG’，其中RG’为H、羟基保护基或固相载体，所述羟基保护基优选-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基；
m1＝0、1、2、3、4、5、6、7、8、9或10；
n1＝0、1、2、3、4、5、6、7、8、9或10。
在另一个实施方案中，GalNAc为式(X)所示的缀合基团，其中，
Q独立地为H、
其中LG1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；
LG2为化学键或-CH2CH2C(O)-；
LG3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；
LG4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；
其中a＝0、1、2或3；
b＝1、2、3、4或5；
c＝1、2、3、4或5；
d＝1、2、3、4、5、6、7或8；
A为化学键、-CH2O-或-NHC(O)-；
A’为化学键、-C(O)NH-、-NHC(O)-或-O(CH2CH2O)e-；
其中e为1、2、3、4或5；
B为化学键、-CH2-、-M-、-CH2-M-或-C(O)-M-；
其中M为
RG1和RG2一起形成-CH2CH2O-或-CH2CH(RG)-O-，并且RG3为H；
或者RG1和RG3一起形成-C1-2亚烷基-，并且RG2为H；
其中RG为-ORG’、-CH2ORG’或-CH2CH2ORG’，其中RG’为H、羟基保护基或固相载体，所述羟基保护基优选-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基；
m1＝0、1、2、3、4、5、6、7、8、9或10；
n1＝0、1、2、3、4、5、6、7、8、9或10。
在另一个实施方案中，GalNAc为式(X)所示的缀合基团，其中：
Q独立地为H、
其中LG1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；
LG2为化学键或-CH2CH2C(O)-；
LG3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；
LG4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；
其中a＝0、1、2或3；
b＝1、2、3、4或5；
c＝1、2、3、4或5；
d＝1、2、3、4、5、6、7或8；
A为化学键、-CH2O-或-NHC(O)-；
A’为-O(CH2CH2O)e-；
其中e为1、2、3、4或5；
B为化学键、-CH2-、-C(O)-、-M-、-CH2-M-或-C(O)-M-；
其中M为
RG1和RG2一起形成-CH2CH2O-或-CH2CH(RG)-O-，并且RG3为H；
或者RG1和RG3一起形成-C1-2亚烷基-，并且RG2为H；
其中RG为-ORG’、-CH2ORG’或-CH2CH2ORG’，其中RG’为H、羟基保护基或固相载体，所述羟基保护基优选-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基；
m1＝0、1、2、3、4、5、6、7、8、9或10；
n1＝0、1、2、3、4、5、6、7、8、9或10。
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，X的任一技术方案或其任意组合，可以与Q1、Q2、Y1、Y2、L1、L2、R1、R2、R3、R4、R5、Base和Base’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：
1.式(A)所示的核苷酸双聚体：
其中，
Q1和Q2中一个为R4，另一个为O-L2；
L1是H或P1，或是连接至另一核苷酸或寡核苷酸的核糖的2’或3’端的磷酸P原子的化学键，优选为P1；
L2是H或P2，或是连接至另一核苷酸或寡核苷酸的核糖的5’端的磷酸P原子的化学键，优选为P2；
X选自-(CR1R2)m-或-CR1＝CR2-；
Y1是O、S或NR；
Y2是O、S或化学键；
R1和R2独立地选自H、D、卤素、OH、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、C3-10环烷基、3-10元杂环基、C6-10芳基或5-14元杂芳基，其任选地被1、2、3、4、5、6、7、8或多个R’取代；
R3选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、C3-10环烷基、3-10元杂环基、C6-10芳基或5-14元杂芳基，其任选地被1、2、3、4、5、6、7、8或多个R’取代；
R4和R5独立地选自H、D、OH、卤素、C1-6烷基、C1-6卤代烷基或C1-6烷氧基，优选H、F或OMe。
P1为羟基保护基，优选为DMTr；
P2为反应性磷基团，优选-P(OCH2CH2CN)(N(iPr)2)；
Base和Base’独立地选自H、修饰或未修饰的碱基或离去基，优选修饰或未修饰的A、U、T、G和C；
R选自H、C1-6烷基或C1-6卤代烷基；
R’选自D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、C3-10环烷基、3-10元杂环基、C6-10芳基、5-14元杂芳基、-ORa、-OC(O)Ra、-O-C(O)ORb、-O-C(O)NRaRb、-C(O)Ra、-C(O)ORa、-C(O)NRaRb、-S(O)nRa、-S(O)nORa、-S(O)nNRaRb、-O-S(O)nRb、-NRaRb、-NRaC(O)Rb、-NRa-C(O)ORb、-NRa-S(O)nRb或-NRaC(O)NRaRb；
Ra和Rb独立地选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、C3-10环烷基、3-10元杂环基、C6-10芳基或5-14元杂芳基；或者Ra和Rb以及它们连接的氮原子形成3-10元杂环基；
m选自1、2、3、4或5；
n独立地选自1或2。
2.技术方案1的核苷酸双聚体，其为式(I)或(II)结构：
其中，各基团如技术方案1所定义。
3.技术方案2的核苷酸双聚体，其中，
X选自-(CR1R2)m-或-CR1＝CR2-；
R1和R2独立地选自H、D、卤素、OH、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被1、2、3、4、5或多个R’取代；
R’选自D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、-ORa、-OC(O)Ra、-O-C(O)ORb、-O-C(O)NRaRb、-C(O)Ra、-C(O)ORa、-C(O)NRaRb、-NRaRb、-NRaC(O)Rb、-NRa-C(O)ORb或-NRaC(O)NRaRb；
Ra和Rb独立地选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基；
m选自1、2、3、4或5；
优选地，
X选自-(CR1R2)m-或-CR1＝CR2-；
R1和R2独立地选自H、D、卤素、OH、CN、C1-6烷基或C1-6卤代烷基，其任选地被1、2、3或多个R’取代；
R’选自D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、-ORa或-NRaRb；
Ra和Rb独立地选自H、C1-6烷基或C1-6卤代烷基；
m选自1、2或3；
更优选地，
X选自-CH2-、-CH(OH)-、-CH2-CH2-或-CH＝CH-。
4.技术方案2或3的核苷酸双聚体，其中，
Y1为O或NR；
Y2为O、S或化学键；
R选自H、C1-6烷基或C1-6卤代烷基；
优选地，
Y1为O或NR；
Y2为O、S或化学键；
R选自H或C1-6烷基；
更优选地，
Y1为O；
Y2为O。
5.技术方案2-4中任一项的核苷酸双聚体，其中，
R3选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被1、2、3、4、5或多个R’取代；
优选地，
R3选自H、C1-6烷基或C1-6卤代烷基，其任选地被1、2、3或多个R’取代；
更优选地，
R3为C1-4烷基，优选为Me。
6.技术方案2-5中任一项的核苷酸双聚体，其中，
R4和R5独立地选自H、D、OH、卤素、C1-6烷基、C1-6卤代烷基或C1-6烷氧基；
优选地，
R4和R5独立地选自H、OH、卤素、C1-6烷基、C1-6卤代烷基或C1-6烷氧基，优选H、F、OH或OMe；
更优选地，
R4选自H、OH或C1-4烷氧基，优选为H或OMe；
R5选自卤素、OH或C1-4烷氧基，优选为F或OMe。
7.技术方案2-6中任一项的核苷酸双聚体，其中，
Base和Base’独立地选自
8.技术方案2-7中任一项的核苷酸双聚体，其中，
L1是H或P1，或是连接至另一核苷酸或寡核苷酸的核糖的2’或3’端的磷酸P原子的化学键，优选为P1；
L2是H或P2，或是连接至另一核苷酸或寡核苷酸的核糖的5’端的磷酸P原子的化学键，优选为P2；
X选自-(CR1R2)m-或-CR1＝CR2-；
Y1是O、S或NR；
Y2是O、S或化学键；
R1和R2独立地选自H、D、卤素、OH、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被1、2、3、4、5或多个R’取代；
R3选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被1、2、3、4、5或多个R’取代；
R4和R5独立地选自H、D、OH、卤素、C1-6烷基、C1-6卤代烷基或C1-6烷氧基，优选H、F或OMe；
P1为羟基保护基，优选为DMTr；
P2为反应性磷基团，优选-P(OCH2CH2CN)(N(iPr)2)；
Base和Base’独立地选自H、修饰或未修饰的碱基或离去基，优选修饰或未修饰的A、U、T、G和C；
R选自H、C1-6烷基或C1-6卤代烷基；
R’选自D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、-ORa、-OC(O)Ra、-O-C(O)ORb、-O-C(O)NRaRb、-C(O)Ra、-C(O)ORa、-C(O)NRaRb、-NRaRb、-NRaC(O)Rb、-NRa-C(O)ORb或-NRaC(O)NRaRb；
Ra和Rb独立地选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基；
m选自1、2、3、4或5。
9.技术方案2-8中任一项的核苷酸双聚体，其中，
L1是H或P1，或是连接至另一核苷酸或寡核苷酸的核糖的2’或3’端的磷酸P原子的化学键，优选为P1；
L2是H或P2，或是连接至另一核苷酸或寡核苷酸的核糖的5’端的磷酸P原子的化学键，优选为P2；
X选自-(CR1R2)m-或-CR1＝CR2-；
Y1为O或NR；
Y2为O、S或化学键；
R1和R2独立地选自H、D、卤素、OH、CN、C1-6烷基或C1-6卤代烷基，其任选地被1、2、3或多个R’取代；
R3选自H、C1-6烷基或C1-6卤代烷基，其任选地被1、2、3或多个R’取代；
R4和R5独立地选自H、D、卤素、C1-6烷基、C1-6卤代烷基或C1-6烷氧基，优选H、F或OMe；
P1为羟基保护基，优选为DMTr；
P2为反应性磷基团，优选-P(OCH2CH2CN)(N(iPr)2)；
Base和Base’独立地选自H、修饰或未修饰的碱基或离去基，优选修饰或未修饰的A、U、T、G和C；
R选自H、C1-6烷基或C1-6卤代烷基；
R’选自D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、-ORa或-NRaRb；
Ra和Rb独立地选自H、C1-6烷基或C1-6卤代烷基；
m选自1、2或3。
10.技术方案2-9中任一项的核苷酸双聚体，其中，
L1是H或P1，或是连接至另一核苷酸或寡核苷酸的核糖的2’或3’端的磷酸P原子的化学键，优选为P1；
L2是H或P2，或是连接至另一核苷酸或寡核苷酸的核糖的5’端的磷酸P原子的化学键，优选为P2；
X选自-(CR1R2)m-或-CR1＝CR2-，优选为-CH2-、-CH(OH)-、-CH2-CH2-或-CH＝CH-；
Y1为O；
Y2为O；
R1和R2独立地选自H、D、卤素、OH、CN、C1-4烷基或C1-4卤代烷基；
R3为C1-4烷基，优选为Me；
R4选自H、OH或C1-4烷氧基，优选为H或OMe；
R5选自卤素、OH或C1-4烷氧基，优选为F或OMe；
P1为羟基保护基，优选为DMTr；
P2为反应性磷基团，优选-P(OCH2CH2CN)(N(iPr)2)；
Base和Base’独立地选自
m选自1、2或3。
11.技术方案1-10中任一项的核苷酸双聚体，选自：
其中，Base和Base’如技术方案1-10中任一项所定义，优选为
R5如技术方案1-10中任一项所定义，优选为F或OMe。
12.一种双链RNA分子或其药学上可接受的盐、互变异构体或立体异构体，其包含正义链和反义链，其中各链具有14至30个核苷酸，并且所述反义链包含一个或多个式(III)或(IV)所示的核苷酸单体：
其中，
所示核苷酸单体以5’＝>3’的顺序从至连接；
各基团如技术方案1-11中任一项中所定义；
优选地，所述核苷酸单体选自：
其中，Base选自
13.技术方案12的双链RNA分子，其中所述正义链和反义链各具有20至25个核苷酸。
14.技术方案12或13的双链RNA分子，其中所述核苷酸单体位于该反义链5’端的第2-8位，优选第6或第7位，更优选第7位。
15.技术方案12-14中任一项的双链RNA分子，其中所述双链RNA相比于具有相同序列但不包含技术方案12所述的核苷酸单体的双链RNA，显示增强的稳定性。
16.技术方案12-15中任一项的双链RNA分子，其中所述双链RNA具有从约40℃至约80℃的解链温度，优选约55℃至67℃。
17.技术方案12-16中任一项的双链RNA分子，其中所述双链RNA相比于具有相同序列但不包含技术方案12所述的核苷酸单体的双链RNA，显示降低的脱靶毒性。
18.技术方案12-17中任一项的双链RNA分子，其中所述双链RNA相比于具有相同序列但不包含技术方案12所述的核苷酸单体的双链RNA，显示增强的有效性。
19.技术方案12-18中任一项的双链RNA分子，其中所述反义链具有与所述正义链和靶标mRNA充分互补的序列，并具有诱导靶标mRNA降解的能力。
20.技术方案12-19的双链RNA分子，其中所述靶标mRNA由内源性基因编码或由病原体基因编码。
21.技术方案12-20中任一项的双链RNA分子，其中所述正义链和/或反义链包含3’和/或5’悬端(over-hang)。
22.技术方案12-21中任一项的双链RNA分子，其中所述双链RNA进一步偶联至配体，优选地，该配体包含一个或多个GalNAc。
23.一种核酸分子，所述核酸分子的核苷酸序列中包含一个或多个如技术方案12所述的核苷酸单体。
24.技术方案23的核酸分子，其中所述核酸选自DNA、RNA和DNA/RNA杂合体。
25.技术方案24的核酸分子，所述核酸分子是单链的或双链的。
26.技术方案23-25中任一项的核酸分子，其中所述核酸分子选自小干扰RNA(siRNA)和短发夹RNA(shRNA)。
27.载体，其包含编码前述技术方案12-22中任一项所述的双链RNA的核苷酸序列。
28.细胞，其含有如技术方案12-22中任一项所述的双链RNA或如技术方案27所述的载体。
29.一种药物组合物，其包含如技术方案12-22中任一项所述的双链RNA分子，和药学上可接受的载体或赋形剂。
30.一种试剂盒，其包含如技术方案12-22中任一项所述的双链RNA分子。
31.一种用于抑制细胞中靶基因的表达的方法，包括将技术方案12-22中任一项所述的双链RNA分子引入该细胞的步骤。
32.一种用于抑制细胞中靶基因的表达的方法，包括在所述细胞中表达技术方案12-22中任一项所述的双链RNA分子。
33.一种用于降低细胞中脱靶毒性的方法，包括将技术方案12-22中任一项所述的双链RNA分子 引入该细胞的步骤。
34.一种用于降低细胞中脱靶毒性的方法，包括在所述细胞中表达技术方案12-22中任一项所述的双链RNA分子。
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。
实施例1 化合物E1的制备
1.化合物2的制备
在25℃下将化合物1(50.0g,263mmol)溶于DCM(800mL)，再依次加入咪唑(26.9g,394mmol)和TBDPSCl(75.2mL,289mmol)，反应液在25℃下搅拌18小时。薄层色谱(DCM/MeOH＝10/1,PE/EA＝3/1)显示反应物消耗完全且有新点生成。将反应液旋干得到粗品，粗品进行MPLC(PE/EA＝1/0-5/1)纯化得到无色油状液体化合物2(95.0g,收率84.31％)。
1H NMR(400MHz,CDCl3)δ7.70(dd,J＝7.6,1.6Hz,4H),7.35-7.46(m,6H),5.86(d,J＝3.6Hz,1H),4.61(dd,J＝4.8,4.0Hz,1H),4.15(td,J＝8.8,5.2Hz,1H),3.93-4.01(m,1H),3.81-3.92(m,2H),1.60(s,3H),1.39(s,3H),1.06(s,9H).
2.化合物3的制备
在25℃下将化合物2(95.0g,222mmol)溶于甲苯(1.50L)，依次加入咪唑(30.2g,443mmol)、三苯基膦(116g,443mmol)和碘单质(84.4g,322mmol)，反应液在100℃下搅拌18小时。薄层色谱(PE/EA＝5/1)显示反应物消耗完全且有新点生成。往反应液中加入20.0mL饱和NaHSO3溶液，加入500mL水，反应液分层，有机相用饱和NaCl溶液(50.0mL×3)洗涤，无水Na2SO4干燥，旋干得到粗品。粗品进行MPLC(PE/EA＝1/0-10/1)纯化得到无色油状液体化合物3(115g,收率96.35％)。
1H NMR(400MHz,CD3OD)δ7.64-7.72(m,4H),7.37-7.49(m,6H),5.95(d,J＝3.6Hz,1H),5.06(d,J＝3.6Hz,1H),4.42(d,J＝3.2Hz,1H),3.87(dd,J＝10.4,5.6Hz,1H),3.66(dd,J＝10.4,6.4Hz,1H),3.53(td,J＝6.0,3.2Hz,1H),1.44(s,3H),1.29(s,3H),1.02-1.07(m,9H).
3.化合物4的制备
在25℃下将化合物3(12.5g,23.2mmol)溶于MeOH(225mL)和EtOAc(25mL)的混合溶剂，加入TEA(6.45mL,46.4mmol)和Pd/C 10％(2.00g,18.8mmol)，反应液在氢气(14.696psi)氛围以及25℃条件下搅拌反应14小时。薄层色谱(PE/EA＝10/1)显示反应物消耗完全且有新点生成。将反应液过滤，滤液旋干得到粗品，粗品进行MPLC(PE/EA＝1/0-10/1)纯化得到无色油状液体化合物4(8.75g,收率83.06％)。
1H NMR(400MHz,CD3OD)δ7.64-7.72(m,4H),7.36-7.47(m,6H),5.80(d,J＝3.6Hz,1H),4.77(t,J＝4.4Hz,1H),4.25-4.32(m,1H),3.68-3.82(m,2H),2.00(dd,J＝13.6,4.8Hz,1H),1.78-1.87(m,1H),1.45(s,3H),1.30(s,3H),1.04(s,9H).
4.化合物5的制备
在25℃下将化合物4(35.0g,84.8mmol)溶于THF(500mL)，加入四乙基氟化铵(63.3g,424mmol)，反应液在25℃下搅拌18小时。薄层色谱(PE/EA＝10/1，DCM/MeOH＝10/1)显示反应物消耗完全且有新点生成。反应液真空浓缩得到粗品，粗品进行MPLC(DCM/MeOH＝1/0-10/1)纯化得到白色固体化合物5(12g,收率81.21％)。
1H NMR(400MHz,CD3OD)δ5.79(d,J＝3.6Hz,1H),4.77(t,J＝4.0Hz,1H),4.21-4.28(m,1H),3.70(dd,J＝12.0,3.6Hz,1H),3.54(dd,J＝12.0,4.8Hz,1H),1.95-2.02(m,1H),1.74(ddd,J＝13.2,10.8,4.8Hz,1H),1.46(s,3H),1.30(s,3H).
5.化合物6的制备
在25℃下将化合物5(12.0g,68.9mmol)溶于甲苯(500mL)，依次加入咪唑(9.38g,138mmol)、三苯基膦(36.1g,138mmol)和碘单质(26.2g,103mmol)，反应液在100℃下搅拌3小时。薄层色谱(PE/EA＝5/1)显示反应物消耗完全且有新点生成。往反应液中加入100mL饱和NaHSO3溶液，加入100mL水，反应液分层，有机相用饱和NaCl溶液(100mL×3)洗涤，无水Na2SO4干燥，旋干得到粗品。粗品进行MPLC(PE/EA＝1/0-10/1)纯化得到白色固体化合物6(16.6g,收率84.82％)。
1H NMR(400MHz,CDCl3)δ5.88(d,J＝3.6Hz,1H),4.77(t,J＝4.2Hz,1H),4.13-4.21(m,1H),3.25-3.38(m,2H),2.31(dd,J＝13.6,4.4Hz,1H),1.61-1.67(m,1H),1.52(s,3H),1.33(s,3H).
6.化合物7的制备
在25℃下将化合物6(40.0g,141mmol)溶于亚磷酸三甲酯(500mL)，反应液在120℃下搅拌11小时。薄层色谱(乙酸乙酯/丙酮＝3/1，PE/EA＝10/1)显示原料剩余且有新点生成。将反应液旋干得到粗品，粗品进行MPLC(乙酸乙酯/丙酮＝1/0-20/1)纯化得到淡黄色油状液体化合物7(8.90g,收率23.74％)以及回收原料白色固体化合物7(30.0g)。
1H NMR(400MHz,CDCl3)δ5.81(d,J＝4.0Hz,1H),4.74(t,J＝4.4Hz,1H),4.40-4.52(m,1H),3.76(dd,J＝10.8,0.8Hz,6H),2.23-2.35(m,2H),1.95-2.07(m,1H),1.63(ddd,J＝13.6,10.8,4.8Hz,1H),1.52(s,3H),1.32(s,3H).
7.化合物8的制备
化合物7(13g,48.830mmol)和Ac2O(23.036mL,244.150mmol)，H2SO4(2.615mL,48.830mmol)依次加入到AcOH(260mL)中。在25℃反应6小时。TLC(乙酸乙酯：丙酮＝3:1)检测到新点。反应液用冰水淬灭，用DCM(500mL x 3)萃取，无水Na2SO4干燥，过滤，减压浓缩得到粗品产物。粗产品通过柱层析TLC(乙酸乙酯：丙酮＝50：1-3:1)纯化得到黄色油状化合物8(7.9g,25.464mmol,52.15％)。
1H NMR(400MHz,CDCl3)δ6.10(d,J＝1.2Hz,1H),5.18(d,J＝5.2Hz,1H),4.58-4.72(m,1H),3.75-3.79(m,3H),3.71-3.74(m,3H)2.16-2.38(m,3H),2.07(s,3H),2.05(s,3H),1.96-2.04(m,1H).
8.化合物9的制备
化合物8(7.9g,25.464mmol)和化合物8A(6.09g,25.464mmol)加入到CH3CN(316mL)，在0℃然后缓慢滴加SnCl4(8.781mL,76.392mmol)，在25℃反应2小时。TLC(乙酸乙酯：丙酮＝10:1，PMA)显示原料化合物8消失，有新点生成。LCMS(RW0006-267-P1A)显示有31.3％的产物生成。反应液降温至0℃，用饱和NaHCO3水溶液调节pH＝8，然后水相用DCM萃取(200mL x 3)，有机相用无水Na2SO4干燥，过滤，旋干得到粗品，粗品通过柱层析纯化(乙酸乙酯：丙酮＝20:1-3:1)得到黄色油状化合物9(6.1g,12.464mmol,48.95％)。
1H NMR(400MHz,CDCl3)δ8.75-8.84(m,1H),8.17(s,1H),8.01-8.10(m,2H),7.59-7.70(m,1H),7.50-7.57(m,2H),6.07(s,1H),5.66-5.73(m,1H),4.98-5.12(m,1H),4.70-4.81(m,1H),3.71-3.82(m,12H),2.70-2.81(m,1H),2.34-2.52(m,2H),2.21-2.27(m,1H),2.16(s,3H),2.06(d,J＝4.4Hz,4H).
9.化合物10的制备
化合物9(6.1g,12.259mmol)溶解到吡啶(30mL)和水(20mL)中，在60℃反应12小时。TLC(DCM：MeOH＝10：1)显示原料消失。反应液直接旋干得到粗产品，粗品通过Prep-HPLC纯化(MeCN/H2O＝30/1-80/1；流速：30ml/分钟)纯化得到黄色油状化合物10(3.85g,8.098mmol,66.06％)。
10.化合物11的制备
化合物10(3.7g,7.783mmol)和化合物SM2(10.24g,15.566mmol)溶解在吡啶(37mL)中，在0℃时加入2,4,6-三异丙基苯磺酰氯(14.14g,46.698mmol)，0℃反应60分钟后N-甲基咪唑(5.11g,62.263mmol)缓慢滴加到反应液中。25℃反应13小时，LCMS(RW0006-284-P1C)发现产物。TLC(DCM：MeOH＝10:1，UV)有新点生成。反应液降温至0℃，加入50mL饱和碳酸钠溶液淬灭，分层，有机相加入300毫升DCM稀释，用水(50mL x 3)和饱和NaCl水溶液(50mL x 1)洗涤，有机相用无水Na2SO4干燥，过滤，旋干得到粗产品。粗产品通过柱层析纯化(DCM：MeOH＝2％-4％，TEA)得到棕色油状化合物11(2100mg,1.883mmol,24.20％)。
11.化合物12的制备
化合物11(2.4g,2.152mmol)溶解在MeOH(36mL)中，加入NH3/MeOH(12.299mL，7M)，在0℃反应3小时，LCMS(RW0006-290-P1A)显示有产物生成，反应液用150mLDCM稀释，用30mL水洗涤，有机相用无水Na2SO4干燥，过滤旋干得到粗品，粗品通过Prep-HPLC(色谱柱：01-Waters Xbridge BEH C18 19*150mm；流动相：TEAA-ACN；梯度：33％-58.5％/15min；流速：15ml/min；)纯化得到黄色固体化合物12(380mg,0.354mmol,16.45％)。
12.E1的制备
化合物12(380mg,0.354mmol)用乙腈带3遍，用DCM(6.0mL)溶解后依次加入5A分子筛，DCI(41.82mg,0.354mmol)和化合物13(426.95mg,1.417mmol)。氮气置换三次，在25℃反应1小时，LCMS(RW00006-291-P1A)显示有5.4％原料剩余。反应液滴加到20mL冰的饱和NaHCO3水溶液淬灭，加入30mLDCM稀释，分液，有机相用20mL饱和NaHCO3水溶液和20mL饱和NaCl水溶液洗涤。有机相用无水Na2SO4干燥，过滤，旋干得到粗品，粗品通过反相纯化(乙腈/水：20-80％，30min，20mL/min)得到黄色固体E1(210mg,0.165mmol,46.57％)。
1H NMR(400MHz,CDCl3)δ11.96-12.03(m,1H),10.07-10.20(m,1H),8.99-9.12(m,1H),8.62-8.80(m,1H),8.07-8.16(m,1H),8.01-8.05(m,2H),7.78(d,J＝1.2Hz,1H),7.60-7.66(m,1H),7.51-7.58(m,2H),7.21-7.26(m,2H),7.09-7.20(m,7H),6.66-6.75(m,4H),6.37-6.56(m,1H),5.75-6.11(m,3H),5.07-5.19(m,1H),4.60-4.74(m,1H),4.16-4.29(m,1H),3.81-3.89(m,1H),3.76-3.81(m,3H),3.70(d,J＝1.6Hz,7H),3.54-3.69(m,3H),3.05-3.15(m,1H),2.54-2.65(m,2H),2.03-2.53(m,5H),1.43-1.50(m,1H),1.14-1.21(m,11H),1.01-1.13(m,6H).
实施例2 siRNA的制备
使用本领域熟知的固相亚磷酰胺法制备本发明的siRNA。具体方法可参见例如PCT公开号WO2016081444和WO2019105419，并简述如下。
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固相载体或连接有L96的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：
核苷单体提供的是0.05mol/L的乙腈溶液，每一步反应的条件相同，即温度为25℃，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的碘/四氢呋喃/吡啶/水(70/20/10,v/v/v)，氧化2次；硫代使用0.2mol/L PADS的乙腈/3-甲基吡啶(1/1,v/v)，硫代2次。
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或本发明的核苷酸双聚体。每连接一个核苷单体或本发明的核苷酸双聚体都包含了脱保护、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。
3寡核苷酸的纯化与退火
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℃(或者55℃)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。
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。
3.3退火
根据表6将正义链(SS链)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℃，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分布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。
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”或“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。
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。
ROR14表示上文所述结构的核苷酸替代物，其中Base可以是任何碱基，例如ROR14-A表示Base为腺嘌呤。
具体地，ROR14-A结构如下：其中，Base为腺嘌呤。
实施例3 siRNA化合物psi-CHECK2在靶和脱靶活性检测
1.质粒制备：
在靶质粒：根据化合物序列设计相应的反义链在靶质粒，由生工生物工程(上海)股份有限公司制备psiCHECK2GSCM重组质粒并将重组质粒稀释至1000ng/μL备用。
脱靶质粒：根据化合物序列设计相应的反义链脱靶质粒，由生工生物工程(上海)股份有限公司制备psiCHECK2GSSM-5Hits重组质粒并将重组质粒稀释至1000ng/μL备用。
2.细胞转染：
用HEK293A细胞(南京科佰，货号CBP60436)，96孔板100μL细胞重悬液铺板，细胞量：8×103cells/孔。
第二天，首先将孔中的完全培养基吸弃，换成Opti-MEM培养基80μL/孔，饥饿处理1.5h左右。
质粒混合物：单孔配制量：质粒0.01μL/孔，Opti-MEM 8.99μL/孔。
Lipo混合：用Opti-MEM稀释Lipo 2000(LipofectamineTM 2000转染试剂，Thermo，11668019)，室温静置5分钟，Lipo混合物具体配制量：Lipo 0.2μL/孔，Opti-MEM 9.8μL/孔。
将已配制好的Lipo混合物22μL、化合物2.2μL、质粒混合物19.8μL，分别分装到对应的同一孔中：命名Well A，吹打混匀后室温孵育20分钟后进行共转染。最后将well A mixture加入每孔细胞中，20μL/孔，加上原有80μL Opti-MEM，每孔终体积为100μL。37℃5％CO2培养箱培养4h后，每孔补加100μL含20％胎牛血清的DMEM培养基。37℃5％CO2培养箱培养24h后检测。
3.结果检测：
在实验前先将混合好的Luciferase(Luciferase Assay System，Promega，E2940)进行复融，等平衡到室温后每管按1:1加入DMEM配制成底物I，现配现用。将Stop&Buffer进行复融，等平衡到室温后与Stop&Substrate 100:1配制成底物II，现 配现用。
真空泵吸去96孔培养板中原有的培养基；
每孔加入150μL底物I，在摇床上室温孵育10min；
取120μL底物I，转移到96孔酶标板上，在酶标仪(Tecan，Infinite 200)上读取Firefly化学发光值；
再向每孔加入60μL底物II，在摇床上室温孵育10min，在酶标仪上读取Renilla化学发光值。
4.数据分析处理
荧光活性由酶标仪测定，收集得到的Renilla信号由Firefly信号标准均一化，siRNA的抑制效果由未经过处理的结果比较得出(剩余抑制活性)，其计算过程见下：
均一化Ren/Fir比值：Ratio＝Renilla(海肾荧光素酶)/Firefly(萤火虫荧光素酶)。
剩余抑制率：2个复孔(RatiosiRNA/Ratiocontrol)*100％的均值：其中Ratiocontrol为对照孔(不含siNRA)2个复孔Ratio的均值，分别计算2个复孔的RatiosiRNA/Ratiocontrol，然后取均值即为剩余抑制率；
作图：利用Graphpad Prism作图
半数抑制浓度(Half maximal inhibitory concentration，IC50)：本次实验以Top和Bottom作图，IC50值根据公式Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))求得，其中Y＝50，X＝log(浓度)。
在靶活性检测实验选取HEK293A(南京科佰，货号CBP60436)细胞系进行psiCHECK2-GSCM重组质粒转染，选择化合物起始浓度为10nM，3倍稀释11个浓度点(10nM，3.33nM，1.11nM，0.37nM，0.123nM，0.041nM，0.0136nM，0.0045nM，0.00152nM，0.000508nM，0.000169nM)进行siRNA化合物活性筛选实验结果见表1。
表1 psi-CHECK2在靶活性检测实验结果
脱靶活性检测实验选取HEK293A(南京科佰，货号CBP60436)细胞系进行psiCHECK2-GSSM-5Hits重组质粒转染，选择化合物起始浓度为10nM，3倍稀释11个浓度点(10nM，3.33nM，1.11nM，0.37nM，0.123nM，0.041nM，0.0136nM，0.0045nM，0.00152nM，0.000508nM，0.000169nM)进行siRNA化合物活性筛选实验结果见表2。
表2 psi-CHECK2脱靶活性检测实验结果

结果显示，携带ROR14的siRNA在保持在靶活性的前提下，有效降低了脱靶活性。
实施例4 C57BL/6小鼠模型的化合物药效长效性验证
将C57BL/6小鼠(雄性，18～21g，6～8周)按照表3进行随机分组，每组6只动物，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化合物首先配置为以1mg/mL的溶液(0.9％氯化钠水溶液作为溶剂)，在实验前用0.9％氯化钠水溶液将siRNA化合物溶解且定容至所需溶液浓度和体积，生理盐水和siRNA化合物的给药体积为5mL/kg。
表3动物实验分组
分别于给药前(记为第0天)，及给药后第14、28、42和56天对小鼠眼眶静脉丛取血，在各个时间点ELISA试剂盒(Abcam,ab282297)检测血清mTTR蛋白；最后一个实验时间点取肝脏10mg放于RNAlater溶液中，检测肝脏mTTR mRNA。
表4 C57BL/6小鼠模型的化合物药效长效性验证结果
结果显示，携带ROR14的siRNA可以在体内长时间降低靶基因表达。

Claims (34)

  1. 式(A)所示的核苷酸双聚体：
    其中，
    Q1和Q2中一个为R4，另一个为O-L2；
    L1是H或P1，或是连接至另一核苷酸或寡核苷酸的核糖的2’或3’端的磷酸P原子的化学键，优选为P1；
    L2是H或P2，或是连接至另一核苷酸或寡核苷酸的核糖的5’端的磷酸P原子的化学键，优选为P2；
    X选自-(CR1R2)m-或-CR1＝CR2-；
    Y1是O、S或NR；
    Y2是O、S或化学键；
    R1和R2独立地选自H、D、卤素、OH、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、C3-10环烷基、3-10元杂环基、C6-10芳基或5-14元杂芳基，其任选地被1、2、3、4、5、6、7、8或多个R’取代；
    R3选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、C3-10环烷基、3-10元杂环基、C6-10芳基或5-14元杂芳基，其任选地被1、2、3、4、5、6、7、8或多个R’取代；
    R4和R5独立地选自H、D、OH、卤素、C1-6烷基、C1-6卤代烷基或C1-6烷氧基，优选H、F或OMe。
    P1为羟基保护基，优选为DMTr；
    P2为反应性磷基团，优选-P(OCH2CH2CN)(N(iPr)2)；
    Base和Base’独立地选自H、修饰或未修饰的碱基或离去基，优选修饰或未修饰的A、U、T、G和C；
    R选自H、C1-6烷基或C1-6卤代烷基；
    R’选自D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、C3-10环烷基、3-10元杂环基、C6-10芳基、5-14元杂芳基、-ORa、-OC(O)Ra、-O-C(O)ORb、-O-C(O)NRaRb、-C(O)Ra、-C(O)ORa、-C(O)NRaRb、-S(O)nRa、-S(O)nORa、-S(O)nNRaRb、-O-S(O)nRb、-NRaRb、-NRaC(O)Rb、-NRa-C(O)ORb、-NRa-S(O)nRb或-NRaC(O)NRaRb；
    Ra和Rb独立地选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、C3-10环烷基、3-10元杂环基、C6-10芳基或5-14元杂芳基；或者Ra和Rb以及它们连接的氮原子形成3-10元杂环基；
    m选自1、2、3、4或5；
    n独立地选自1或2。
  2. 权利要求1的核苷酸双聚体，其为式(I)或(II)结构：
    其中，各基团如权利要求1所定义。
  3. 权利要求2的核苷酸双聚体，其中，
    X选自-(CR1R2)m-或-CR1＝CR2-；
    R1和R2独立地选自H、D、卤素、OH、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被1、2、3、4、5或多个R’取代；
    R’选自D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、-ORa、-OC(O)Ra、-O-C(O)ORb、-O-C(O)NRaRb、-C(O)Ra、-C(O)ORa、-C(O)NRaRb、-NRaRb、-NRaC(O)Rb、-NRa-C(O)ORb或-NRaC(O)NRaRb；
    Ra和Rb独立地选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基；
    m选自1、2、3、4或5；
    优选地，
    X选自-(CR1R2)m-或-CR1＝CR2-；
    R1和R2独立地选自H、D、卤素、OH、CN、C1-6烷基或C1-6卤代烷基，其任选地被1、2、3或多个R’取代；
    R’选自D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、-ORa或-NRaRb；
    Ra和Rb独立地选自H、C1-6烷基或C1-6卤代烷基；
    m选自1、2或3；
    更优选地，
    X选自-CH2-、-CH(OH)-、-CH2-CH2-或-CH＝CH-。
  4. 权利要求2或3的核苷酸双聚体，其中，
    Y1为O或NR；
    Y2为O、S或化学键；
    R选自H、C1-6烷基或C1-6卤代烷基；
    优选地，
    Y1为O或NR；
    Y2为O、S或化学键；
    R选自H或C1-6烷基；
    更优选地，
    Y1为O；
    Y2为O。
  5. 权利要求2-4中任一项的核苷酸双聚体，其中，
    R3选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被1、2、3、4、5或多个R’取代；
    优选地，
    R3选自H、C1-6烷基或C1-6卤代烷基，其任选地被1、2、3或多个R’取代；
    更优选地，
    R3为C1-4烷基，优选为Me。
  6. 权利要求2-5中任一项的核苷酸双聚体，其中，
    R4和R5独立地选自H、D、OH、卤素、C1-6烷基、C1-6卤代烷基或C1-6烷氧基；
    优选地，
    R4和R5独立地选自H、OH、卤素、C1-6烷基、C1-6卤代烷基或C1-6烷氧基，优选H、F、OH或OMe；
    更优选地，
    R4选自H、OH或C1-4烷氧基，优选为H或OMe；
    R5选自卤素、OH或C1-4烷氧基，优选为F或OMe。
  7. 权利要求2-6中任一项的核苷酸双聚体，其中，
    Base和Base’独立地选自
  8. 权利要求2-7中任一项的核苷酸双聚体，其中，
    L1是H或P1，或是连接至另一核苷酸或寡核苷酸的核糖的2’或3’端的磷酸P原子的化学键，优选为P1；
    L2是H或P2，或是连接至另一核苷酸或寡核苷酸的核糖的5’端的磷酸P原子的化学键，优选为P2；
    X选自-(CR1R2)m-或-CR1＝CR2-；
    Y1是O、S或NR；
    Y2是O、S或化学键；
    R1和R2独立地选自H、D、卤素、OH、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被1、2、3、4、5或多个R’取代；
    R3选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被1、2、3、4、5或多个R’取代；
    R4和R5独立地选自H、D、OH、卤素、C1-6烷基、C1-6卤代烷基或C1-6烷氧基，优选H、F或OMe；
    P1为羟基保护基，优选为DMTr；
    P2为反应性磷基团，优选-P(OCH2CH2CN)(N(iPr)2)；
    Base和Base’独立地选自H、修饰或未修饰的碱基或离去基，优选修饰或未修饰的A、U、T、G和C；
    R选自H、C1-6烷基或C1-6卤代烷基；
    R’选自D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、-ORa、-OC(O)Ra、-O-C(O)ORb、-O-C(O)NRaRb、-C(O)Ra、-C(O)ORa、-C(O)NRaRb、-NRaRb、-NRaC(O)Rb、-NRa-C(O)ORb或-NRaC(O)NRaRb；
    Ra和Rb独立地选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基；
    m选自1、2、3、4或5。
  9. 权利要求2-8中任一项的核苷酸双聚体，其中，
    L1是H或P1，或是连接至另一核苷酸或寡核苷酸的核糖的2’或3’端的磷酸P原子的化学键，优选为P1；
    L2是H或P2，或是连接至另一核苷酸或寡核苷酸的核糖的5’端的磷酸P原子的化学键，优选为P2；
    X选自-(CR1R2)m-或-CR1＝CR2-；
    Y1为O或NR；
    Y2为O、S或化学键；
    R1和R2独立地选自H、D、卤素、OH、CN、C1-6烷基或C1-6卤代烷基，其任选地被1、2、3或多个R’取代；
    R3选自H、C1-6烷基或C1-6卤代烷基，其任选地被1、2、3或多个R’取代；
    R4和R5独立地选自H、D、卤素、C1-6烷基、C1-6卤代烷基或C1-6烷氧基，优选H、F或OMe；
    P1为羟基保护基，优选为DMTr；
    P2为反应性磷基团，优选-P(OCH2CH2CN)(N(iPr)2)；
    Base和Base’独立地选自H、修饰或未修饰的碱基或离去基，优选修饰或未修饰的A、U、T、G和C；
    R选自H、C1-6烷基或C1-6卤代烷基；
    R’选自D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基、C2-6炔基、-ORa或-NRaRb；
    Ra和Rb独立地选自H、C1-6烷基或C1-6卤代烷基；
    m选自1、2或3。
  10. 权利要求2-9中任一项的核苷酸双聚体，其中，
    L1是H或P1，或是连接至另一核苷酸或寡核苷酸的核糖的2’或3’端的磷酸P原子的化学键，优选为P1；
    L2是H或P2，或是连接至另一核苷酸或寡核苷酸的核糖的5’端的磷酸P原子的化学键，优选为P2；
    X选自-(CR1R2)m-或-CR1＝CR2-，优选为-CH2-、-CH(OH)-、-CH2-CH2-或-CH＝CH-；
    Y1为O；
    Y2为O；
    R1和R2独立地选自H、D、卤素、OH、CN、C1-4烷基或C1-4卤代烷基；
    R3为C1-4烷基，优选为Me；
    R4选自H、OH或C1-4烷氧基，优选为H或OMe；
    R5选自卤素、OH或C1-4烷氧基，优选为F或OMe；
    P1为羟基保护基，优选为DMTr；
    P2为反应性磷基团，优选-P(OCH2CH2CN)(N(iPr)2)；
    Base和Base’独立地选自
    m选自1、2或3。
  11. 权利要求1-10中任一项的核苷酸双聚体，选自：
    其中，Base和Base’如权利要求1-10中任一项所定义，优选为
    R5如权利要求1-10中任一项所定义，优选为F或OMe。
  12. 一种双链RNA分子或其药学上可接受的盐、互变异构体或立体异构体，其包含正义链和反义链，其中各链具有14至30个核苷酸，并且所述反义链包含一个或多个式(III)或(IV)所示的核苷酸单体：
    其中，
    所示核苷酸单体以5’＝>3’的顺序从至连接；
    各基团如权利要求1-11中任一项中所定义；
    优选地，所述核苷酸单体选自：
    其中，Base选自
  13. 权利要求12的双链RNA分子，其中所述正义链和反义链各具有20至25个核苷酸。
  14. 权利要求12或13的双链RNA分子，其中所述核苷酸单体位于该反义链5’端的第2-8位，优选第6或第7位，更优选第7位。
  15. 权利要求12-14中任一项的双链RNA分子，其中所述双链RNA相比于具有相同序列但不包含权利要求12所述的核苷酸单体的双链RNA，显示增强的稳定性。
  16. 权利要求12-15中任一项的双链RNA分子，其中所述双链RNA具有从约40℃至约80℃的解链温度，优选约55℃至67℃。
  17. 权利要求12-16中任一项的双链RNA分子，其中所述双链RNA相比于具有相同序列但不包含权利要求12所述的核苷酸单体的双链RNA，显示降低的脱靶毒性。
  18. 权利要求12-17中任一项的双链RNA分子，其中所述双链RNA相比于具有相同序列但不包含权利要求12所述的核苷酸单体的双链RNA，显示增强的有效性。
  19. 权利要求12-18中任一项的双链RNA分子，其中所述反义链具有与所述正义链和靶标mRNA充分互补的序列，并具有诱导靶标mRNA降解的能力。
  20. 权利要求12-19的双链RNA分子，其中所述靶标mRNA由内源性基因编码或由病原体基因编码。
  21. 权利要求12-20中任一项的双链RNA分子，其中所述正义链和/或反义链包含3’和/或5’悬端(over-hang)。
  22. 权利要求12-21中任一项的双链RNA分子，其中所述双链RNA进一步偶联至配体，优选地，该配体包含一个或多个GalNAc。
  23. 一种核酸分子，所述核酸分子的核苷酸序列中包含一个或多个如权利要求12所述的核苷酸单体。
  24. 权利要求23的核酸分子，其中所述核酸选自DNA、RNA和DNA/RNA杂合体。
  25. 权利要求24的核酸分子，所述核酸分子是单链的或双链的。
  26. 权利要求23-25中任一项的核酸分子，其中所述核酸分子选自小干扰RNA(siRNA)和短发夹RNA(shRNA)。
  27. 载体，其包含编码前述权利要求12-22中任一项所述的双链RNA的核苷酸序列。
  28. 细胞，其含有如权利要求12-22中任一项所述的双链RNA或如权利要求27所述的载体。
  29. 一种药物组合物，其包含如权利要求12-22中任一项所述的双链RNA分子，和药学上可接受的载体或赋形剂。
  30. 一种试剂盒，其包含如权利要求12-22中任一项所述的双链RNA分子。
  31. 一种用于抑制细胞中靶基因的表达的方法，包括将权利要求12-22中任一项所述的双链RNA分子引入该细胞的步骤。
  32. 一种用于抑制细胞中靶基因的表达的方法，包括在所述细胞中表达权利要求12-22中任一项所述的双链RNA分子。
  33. 一种用于降低细胞中脱靶毒性的方法，包括将权利要求12-22中任一项所述的双链RNA分子 引入该细胞的步骤。
  34. 一种用于降低细胞中脱靶毒性的方法，包括在所述细胞中表达权利要求12-22中任一项所述的双链RNA分子。
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