WO2025007907A1 - Ligand d'administration d'oligonucléotide contenant un peptide - Google Patents

Ligand d'administration d'oligonucléotide contenant un peptide Download PDF

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WO2025007907A1
WO2025007907A1 PCT/CN2024/103493 CN2024103493W WO2025007907A1 WO 2025007907 A1 WO2025007907 A1 WO 2025007907A1 CN 2024103493 W CN2024103493 W CN 2024103493W WO 2025007907 A1 WO2025007907 A1 WO 2025007907A1
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compound
formula
pharmaceutically acceptable
alkyl
oligonucleotide
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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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Priority to CN202480044493.0A priority Critical patent/CN121569035A/zh
Publication of WO2025007907A1 publication Critical patent/WO2025007907A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • 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 eye.
  • the delivery vector is a peptide modified by a hydrophobic group, such as a compound of formula (III), 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 eye to work, which limits the application of siRNA.
  • Some attempts have been made in the art to deliver siRNA to the eye 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 inside the chain to deliver siRNA, 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, two or more delivery vectors within the oligonucleotide, at the 5' end and/or at the 3' end, wherein the delivery vector is a peptide-based linker modified with a hydrophobic group;
  • the peptide-based linking group modified by a hydrophobic group is selected from a compound of formula (III), 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 the antisense strand comprises one or more compounds of formula (I) above, 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 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, the delivery vector being a peptide-based linker modified with a hydrophobic group;
  • the peptide-based linking group modified by a hydrophobic group is selected from a compound of formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • the present invention provides a compound of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • 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 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. In some embodiments, C 10-22 alkenyl, C 14-20 alkenyl, C 16-18 alkenyl are preferred.
  • the alkenyl 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.
  • 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. In some embodiments, C10-22 alkynyl, C14-20 alkynyl, C16-18 alkynyl are preferred.
  • the alkynyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • C 0-10 alkylene refers to a divalent group formed by a chemical bond or by removing another hydrogen of a C 1-10 alkyl group, respectively, and may be substituted or unsubstituted. In some embodiments, 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.
  • the unsubstituted alkylene includes, but is not limited to, methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene (-CH 2 CH 2 CH 2 -), butylene (-CH 2 CH 2 CH 2 CH 2 - ), pentylene (-CH 2 CH 2 CH 2 CH 2 -), hexylene (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -), 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.
  • 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 Raa groups are combined to form a heterocyclyl or heteroaryl ring, wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • the heteroaryl groups are independently substituted with 0, 1, 2, 3, 4 or 5 R dd 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-Crick 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 for the synthesis of nucleosides, nucleotides and nucleic acids. Its constituent elements contain nitrogen, so it is also called “nitrogen-containing 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, neopentyl (V), benzyloxymethyl acetal (BOM), benzyloxymethyl ether (BOM ...
  • 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 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.
  • 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;
  • T1 is selected from a chemical bond, -C0-10alkylene- , -C0-10alkylene -O-, -C0-10alkylene -C(O)-, -C0-10alkylene -OC(O)- or -C0-10alkylene -M-;
  • A is a peptide-based linker that can be cleaved in an endosome or a lysosome;
  • T 2 is selected from a chemical bond, -C 0-10 alkylene-, -C 0-10 alkylene-OC 0-10 alkylene-, -C 0-10 alkylene-C(O)-C 0-10 alkylene-, -C 0-10 alkylene-OC(O)-C 0-10 alkylene- or -C 0-10 alkylene-C(O)OC 0-10 alkylene-; said T 2 is optionally substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R;
  • Rs is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl, which is optionally deuterated until fully deuterated;
  • k is selected from 0, 1, 2, 3, 4, 5 or 6.
  • the present invention specifically relates to a compound of formula (I'), 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;
  • P, T 1 , A, L, T 2 , Rs 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 sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and/or the antisense strand comprises one or more compounds of formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer 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 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
  • R 1 is selected from hydroxyl 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 (
  • R 1 is a solid support.
  • 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.
  • P is a hydrophobic group; in another embodiment, P is a C 8-30 hydrocarbon group, such as a C 8-30 alkyl group, a C 8-30 alkenyl group or a C 8-30 alkynyl group; in another embodiment, P is a C 10-22 alkyl group; 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 -( CH2 ) 14-20 - CH3 , for example -( CH2 ) 14 - CH3 , -( CH2 ) 15 - CH3 , -( CH2 ) 16 -CH3, -( CH2 ) 17 - CH3 , -( CH2 ) 18 - CH3 , -( CH2 ) 19 -CH3 or -( CH2 ) 20 - CH3 , preferably -( CH2 ) 14 - CH3 or -( CH2 ) 16 - CH3 .
  • T1 is a chemical bond; in another embodiment, T1 is -C0-10 alkylene-; in another embodiment, T1 is -C0-6 alkylene-, for example -( CH2 ) 1-6- , -( CH2 ) 1-4- or -CH2- ; in another embodiment, T1 is -C0-10 alkylene-O-; in another embodiment, T1 is -C0-10 alkylene-C(O)-, for example -C(O)-; in another embodiment, T1 is -C0-10 alkylene-OC(O)-, for example -OC(O)-; in another embodiment, T1 is -C0-10 alkylene-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 peptide-based linker; in another embodiment, A is a peptide-based linker that can be cleaved in an inclusion body or a lysosome; in another embodiment, A comprises a polypeptide, such as a tripeptide, a tetrapeptide or a pentapeptide, preferably a tetrapeptide.
  • A is In another embodiment, A is In another embodiment, A is In another embodiment, A is In another embodiment, A is In another embodiment, A is In another embodiment, A is In another embodiment, A is
  • L is -NH-; in another embodiment, L is -O-; in another embodiment, L is -CH2- ; in another embodiment, L is -C(O)-; in another embodiment, L is -C(O)O-.
  • T2 is a chemical bond; in another embodiment, T2 is -C0-10 alkylene-, for example -( CH2 ) z- ; in another embodiment, T2 is -C0-10 alkylene- OC0-10 alkylene-, for example -( CH2 ) z -O-; in another embodiment, T2 is -C0-10 alkylene-C(O) -C0-10 alkylene-, for example -( CH2 ) zC (O)-; in another embodiment, T2 is -C0-10 alkylene-OC(O) -C0-10 alkylene-, for example -( CH2 ) zOC (O)-; in another embodiment, T2 is -C0-10 alkylene-C(O) OC0-10 alkylene-, for example -( CH2 ) zC (O)O-.
  • T2 is unsubstituted; in another embodiment, T2 is substituted with 1 R; in another embodiment, T2 is substituted with 2 Rs; in another embodiment, T2 is substituted with 3 Rs; in another embodiment, T2 is substituted with 4 Rs; in another embodiment, T2 is substituted with 5 Rs; in another embodiment, T2 is substituted with 6 Rs; in another embodiment, T2 is substituted with 7 Rs; in another embodiment, T2 is substituted with 8 Rs; in another embodiment, T2 is substituted with more Rs.
  • z is selected from 0, 1, 2, 3, 4, 5 or 6.
  • R is H; in another embodiment, R is D; in another embodiment, R is halogen; in another embodiment, R is C 1-6 alkyl; in another embodiment, R is C 1-4 alkyl; in another embodiment, R is C 1-6 haloalkyl; in another embodiment, R is optionally deuterated, up to fully deuterated.
  • Rs is H; in another embodiment, Rs is D; in another embodiment, Rs is halogen; in another embodiment, Rs is C 1-6 alkyl; in another embodiment, Rs is C 1-6 haloalkyl; in another embodiment, Rs is optionally deuterated, up to fully deuterated.
  • Rs is H.
  • k is selected from 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, R1 , R2 , L, T1 , T2 , R, Rs , Rs ' and k etc.
  • the present invention is intended to include all combinations of 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.
  • Viral vector systems containing siRNA coding sequences include but are not limited to: (a) adenovirus vectors; (b) retrovirus vectors; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors; and (j) helper virus-dependent adenovirus or gut-free adenovirus.
  • 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.
  • An oligonucleotide comprising one or more compounds of formula (I), 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
  • 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;
  • T1 is selected from a chemical bond, -C0-10alkylene- , -C0-10alkylene -O-, -C0-10alkylene -C(O)-, -C0-10alkylene -OC(O)- or -C0-10alkylene -M-;
  • A is a peptide-based linker that can be cleaved in an endosome or a lysosome;
  • L is selected from -NH-, -O-, -CH2- , -C(O)- or -C(O)O-;
  • T2 is a chemical bond, -C0-10 alkylene-, -C0-10 alkylene- OC0-10 alkylene-, -C0-10 alkylene-C(O)-C0-10 alkylene- , or -C0-10 alkylene-C(O) OC0-10 alkylene-; said T2 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, C 1-6 alkyl or C 1-6 haloalkyl, which is optionally deuterated until fully deuterated;
  • k is selected from 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
  • P is a hydrophobic group, preferably a C 8-30 alkyl group, a C 8-30 alkenyl group or a C 8-30 alkynyl group ; is optionally substituted with 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
  • T 1 is selected from -OC(O)-, -C(O)- or -(CH 2 ) 1-6 -;
  • A is a peptide-based linker that can be cleaved in an inclusion body or a lysosome, wherein the peptide-based linker comprises a polypeptide, such as a tripeptide, a tetrapeptide or a pentapeptide, preferably a tetrapeptide;
  • L is selected from -NH-, -O- or -C(O)O-;
  • T 2 is a chemical bond, -(CH 2 ) z , -(CH 2 ) z -O-, -(CH 2 ) z C(O)-, -(CH 2 ) z OC(O)- or -(CH 2 ) z C(O)O-;
  • z is selected from 0, 1, 2, 3, 4, 5 or 6, preferably 5;
  • Rs is selected from H, C 1-6 alkyl or C 1-6 haloalkyl
  • k is selected from 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
  • P is selected from C 10-22 alkyl or C 10-22 alkenyl; the C 10-22 alkyl and C 10-22 alkenyl are optionally substituted by 1, 2 or 3 R;
  • R is selected from H, halogen or C 1-4 alkyl
  • T 1 is selected from -OC(O)-, -C(O)- or -(CH 2 ) 1-4 -;
  • A is a peptide-based linker that can be cleaved in an inclusion body or a lysosome, wherein the peptide-based linker comprises a polypeptide, such as a tripeptide, a tetrapeptide or a pentapeptide, preferably a tetrapeptide;
  • A is selected from
  • L is selected from -NH-, -O- or -C(O)O-;
  • T2 is selected from -( CH2 ) z- , -( CH2 ) z -C(O)- or -( CH2 ) z -O-;
  • z is selected from 0, 1, 2, 3, 4, 5 or 6, preferably 5;
  • Rs is selected from H or C 1-4 alkyl
  • k is selected from 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
  • P is -(CH 2 ) 14-20 -CH 3 , for example -(CH 2 ) 14 -CH 3 or -(CH 2 ) 16 -CH 3 ;
  • T1 is selected from -C(O)- or -CH2- ;
  • z is selected from 0, 1, 2, 3, 4 or 5, preferably 5;
  • R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide.
  • oligonucleotide of any one of technical solutions 1-7 which comprises a compound of formula (I) of any one of technical solutions 1-6 at the 5' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
  • oligonucleotide of any one of technical solutions 1-8 which comprises a compound of formula (I) of any one of technical solutions 1-6 at the 3' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
  • oligonucleotide of any one of technical solutions 1-9 which comprises a compound of formula (I) of any one of technical solutions 1-6 at the 5' end and the 3' end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
  • oligonucleotide according to any one of technical solutions 1 to 10, which comprises one or more compounds of formula (I) according to any one of technical solutions 1 to 6, 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 peptide-based linker modified with a hydrophobic group;
  • the peptide-based linking group modified by a hydrophobic group is selected from a compound of formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • P is a hydrophobic group
  • A is a peptide-based linker
  • P is connected to A through T 1 or directly;
  • P, T1 and A are as defined in any one of technical solutions 1-4;
  • A is selected from
  • the compound of formula (III) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:
  • oligonucleotide according to any one of technical solutions 1 to 12, wherein the oligonucleotide is an ASO or siRNA, preferably used to inhibit genes expressed outside the liver, and more preferably used to inhibit genes expressed in the eye.
  • R 1 and R 2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;
  • P, T 1 , A, L, T 2 , Rs and k are as defined in Technical Solutions 1-4.
  • R 1 and R 2 are selected from reactive phosphorus groups, preferably phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, such as natural phosphates, thiophosphates, dithiophosphates, borane phosphates, borane thiophosphates, phosphonates, halogen-substituted phosphonates and phosphates, aminophosphorates, phosphodiesters, phosphotriesters, thiophosphodiesters, thiophosphothioates, diphosphates or triphosphates, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 ).
  • reactive phosphorus groups preferably phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, such as natural phosphates, thiophosphates, dithiophosphates, borane phosphates, borane thi
  • R1 and R2 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), benzyloxycarbonyl (Cb
  • 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 or more compounds of formula (I) as described in any one of technical solutions 1 to 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • each variable is defined as in any one of technical solutions 1-4.
  • each variable is defined as in any one of technical solutions 1-4;
  • the compound of formula (I) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:
  • R1 is H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide.
  • 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, the delivery vector being a peptide-based linker modified with a hydrophobic group;
  • the peptide-based linking group modified by a hydrophobic group is selected from a compound of formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • P is a hydrophobic group
  • A is a peptide-based linker
  • P is connected to A through T 1 or directly;
  • P, T1 and A are as defined in any one of technical solutions 1-4;
  • A is selected from
  • a pharmaceutical composition comprising the double-stranded RNA as described in any one of Technical Solutions 20-31, or the cell as described in Technical Solution 32, and optionally a pharmaceutically acceptable carrier or excipient.
  • a kit comprising the double-stranded RNA as described in any one of Technical Solutions 20-31, or the cells as described in Technical Solution 32.
  • 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, or 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.
  • Stearic acid 1a (5.00 g, 17.6 mmol) was added to DCM (100 mL) at 25°C, and compound 1b (2.43 g, 21.1 mmol) and EDCI (4.04 g, 21.1 mmol) were added in sequence.
  • the reaction solution was stirred at 25°C for 18 hours. After the reaction was complete, the reaction solution was concentrated in vacuo to obtain a crude product.
  • the crude product was purified by normal phase silica gel column (eluent: petroleum ether/ethyl acetate) to obtain white solid compound 1c (5.00 g, yield 74.56%).
  • the white solid compound BE8 can be obtained using the same synthesis steps.
  • 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.
  • the nucleoside monomers are connected one by one from the 3'-5' direction.
  • 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, and 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 3 times.
  • the activating agent used in the coupling reaction was a 0.25 mol/L 5-ethylthiotetrazolyl (ETT)-acetonitrile solution, and the coupling was repeated 2 times.
  • ETT 5-ethylthiotetrazolyl
  • the capping reaction used 10% acetic anhydride-acetonitrile and pyridine/N-methylimidazole/acetonitrile (10:14:76, v/v/v) and the capping was repeated 2 times.
  • the oxidation reaction used 0.05 mol/L iodine in tetrahydrofuran/pyridine/water (70/20/10, v/v/v) and the oxidation was repeated 2 times.
  • the thiolation reaction used 0.2 mol/L phenylacetyl disulfide (PADS) in acetonitrile/3-methylpyridine (1/1, v/v) and the thiolation was repeated 2 times.
  • PADS phenylacetyl disulfide
  • the nucleoside monomers are connected one by one from the 3'-5' direction according to the arrangement order of the antisense chain nucleotides.
  • the monomers all include four steps of deprotection, coupling, capping, oxidation or thiolation.
  • the synthesis conditions of 5umol oligonucleotide of the antisense chain are the same as those of the sense chain.
  • the purification and desalination methods are well known to those skilled in the art.
  • a column filled with a strong anion filler 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.
  • 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.
  • i inosine ribonucleotide
  • 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.
  • IB or Ib represents an inverted abasic deoxyribonucleotide, which may include the following three structures depending on its position/connection mode in siRNA.
  • Uhd and L322 represent nucleotide monomers of the following structure, which are well known in the art, and can be found in, for example, PCT Publication No. WO2021092371, and their specific structures are as follows.
  • 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.
  • the siRNA conjugate was administered in a 5 mg/mL solution (phosphate buffer solution as solvent); 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 two parts: 1 retina; 2 retinal pigment epithelium (RPE) + choroid + sclera; the separated samples were immediately frozen in liquid nitrogen, then stored at -80°C and sent on dry ice for detection of mTTR mRNA.
  • RPE retinal pigment epithelium
  • 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 was mTTR and the internal reference was mGAPDH.
  • RNAlater RNAlater
  • Tissue RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the protocol of the high-throughput tissue RNA extraction kit (Shanghai Fushen Biotechnology Co., Ltd., FSF0035-TS); reverse transcription was performed using the PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B); and fluorescence quantitative PCR reaction (ABI, QuantStudio3) was performed using the 20 ⁇ L system of TaqMan TM Fast Advanced Master Mix (ABI, 4444965). The primers are shown in the table below.
  • 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 was rSOD1 and the internal reference was rGAPDH.

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Abstract

L'invention concerne un composé de formule (I), ou un sel, un tautomère ou un stéréoisomère pharmaceutiquement acceptable de celui-ci. Le composé de formule (I) est contenu à l'intérieur d'un oligonucléotide ou à l'extrémité 5' et/ou à l'extrémité 3', et est utilisé pour administrer de l'ARN double brin à un tissu extra-hépatique, tel que l'oeil. L'invention concerne également un oligonucléotide, un ARN double brin, un vecteur, une cellule, une composition pharmaceutique et un kit comprenant le composé de formule (I).
PCT/CN2024/103493 2023-07-04 2024-07-04 Ligand d'administration d'oligonucléotide contenant un peptide Ceased WO2025007907A1 (fr)

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Citations (4)

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CN110741087A (zh) * 2017-04-11 2020-01-31 阿布特斯生物制药公司 靶向组合物
WO2022028462A1 (fr) * 2020-08-04 2022-02-10 上海拓界生物医药科技有限公司 Arnsi modifié ayant une activité hors cible réduite
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CN114681621A (zh) * 2015-05-19 2022-07-01 萨勒普塔医疗公司 肽寡核苷酸缀合物
CN110741087A (zh) * 2017-04-11 2020-01-31 阿布特斯生物制药公司 靶向组合物
WO2022028462A1 (fr) * 2020-08-04 2022-02-10 上海拓界生物医药科技有限公司 Arnsi modifié ayant une activité hors cible réduite
WO2024093907A1 (fr) * 2022-10-31 2024-05-10 大睿生物医药科技(上海)有限公司 Ligand pour administrer de l'arnsi à l'oeil et au système nerveux central

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PAUNOVSKA, K. ET AL.: "Drug Delivery Systems for RNA Therapeutics", NATURE REVIEWS, vol. 23, 4 January 2022 (2022-01-04), pages 265 - 280, XP038002085, DOI: 10.1038/s41576-021-00439-4 *

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