WO2024255761A1 - Ligand d'administration d'oligonucléotides comprenant du sucre - Google Patents

Ligand d'administration d'oligonucléotides comprenant du sucre Download PDF

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WO2024255761A1
WO2024255761A1 PCT/CN2024/098675 CN2024098675W WO2024255761A1 WO 2024255761 A1 WO2024255761 A1 WO 2024255761A1 CN 2024098675 W CN2024098675 W CN 2024098675W WO 2024255761 A1 WO2024255761 A1 WO 2024255761A1
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group
formula
alkyl
compound
sugar
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Chinese (zh)
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黄金宇
邹昊
刘俊凯
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Shanghai Rona Therapeutics Co Ltd
Rona Bioscience Ltd
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Shanghai Rona Therapeutics Co Ltd
Rona Bioscience Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

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

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Abstract

L'invention concerne un composé représenté par la formule (I), ou un sel, un tautomère ou un stéréoisomère pharmaceutiquement acceptable de celui-ci. Le composé représenté par la formule (I) est contenu dans un nucléotide ou à l'extrémité 5' et/ou à l'extrémité 3', et est utilisé pour administrer de l'ARN double brin à des tissus extra-hépatiques, tels que le système nerveux central ou les yeux. L'invention concerne en outre un oligonucléotide comprenant le composé représenté par la formule (I), un ARN double brin, un vecteur, une cellule, une composition pharmaceutique et un kit.
PCT/CN2024/098675 2023-06-12 2024-06-12 Ligand d'administration d'oligonucléotides comprenant du sucre Pending WO2024255761A1 (fr)

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WO2004094595A2 (fr) * 2003-04-17 2004-11-04 Alnylam Pharmaceuticals Inc. Agents modifiés d'arni
CN105377887A (zh) * 2013-05-01 2016-03-02 Isis制药公司 用于调节载脂蛋白(a)表达的组合物和方法
CN110741087A (zh) * 2017-04-11 2020-01-31 阿布特斯生物制药公司 靶向组合物
CN112105625A (zh) * 2018-03-07 2020-12-18 赛诺菲 核苷酸前体、核苷酸类似物以及含其的寡聚化合物
CN112400018A (zh) * 2018-05-07 2021-02-23 阿尔尼拉姆医药品有限公司 肝外递送
WO2022084331A2 (fr) * 2020-10-20 2022-04-28 Sanofi Nouveaux ligands pour le récepteur d'asialoglycoprotéine
CN114945669A (zh) * 2019-11-06 2022-08-26 阿尔尼拉姆医药品有限公司 肝外递送

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

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