WO2025031479A1 - Arn double brin contenant un analogue nucléotidique - Google Patents

Arn double brin contenant un analogue nucléotidique Download PDF

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Publication number
WO2025031479A1
WO2025031479A1 PCT/CN2024/110992 CN2024110992W WO2025031479A1 WO 2025031479 A1 WO2025031479 A1 WO 2025031479A1 CN 2024110992 W CN2024110992 W CN 2024110992W WO 2025031479 A1 WO2025031479 A1 WO 2025031479A1
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compound
formula
alkyl
double
stranded rna
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Chinese (zh)
Inventor
黄金宇
邹昊
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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 CN202480050856.1A priority Critical patent/CN121729422A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical

Definitions

  • the present invention belongs to the field of medicine, and in particular relates to double-stranded RNA with nucleotide analogs.
  • RNA interference is a phenomenon of efficient and specific degradation of target mRNA induced by double-stranded RNA (dsRNA). Incorporation of thermally unstable nucleotides (e.g., glycerol nucleic acid (GNA)) into the antisense strand seed region of double-stranded RNA helps to improve interference efficiency and reduce off-target toxicity, see, for example, PCT Publication No. WO2018098328A1.
  • dsRNA double-stranded RNA
  • siRNA molecules to improve the stability and effectiveness of siRNA molecules in vivo.
  • most siRNA drugs currently use a full modification mode, that is, all nucleotides of the siRNA double strand are modified. Commonly used modifications are 2'-F and 2'- OCH3 modifications.
  • the art needs to develop more modified nucleotides.
  • the present invention solves the above problems by providing a novel nucleotide analog.
  • 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 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 herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • the present invention provides a vector comprising a nucleotide sequence encoding a double-stranded RNA as described herein.
  • the present invention provides a cell containing the double-stranded RNA as described herein or the vector as described herein.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a double-stranded RNA molecule as described herein, and a pharmaceutically acceptable carrier or excipient.
  • the invention in another aspect, relates to a kit comprising a double-stranded RNA molecule as described herein.
  • the present invention relates to a method for inhibiting the expression of a target gene in a cell, comprising the step of introducing a double-stranded RNA molecule as described herein into the cell.
  • the present invention relates to a method for inhibiting the expression of a target gene in a cell, comprising expressing a double-stranded RNA molecule as described herein in said cell.
  • the present invention relates to a method for reducing off-target toxicity in a cell, comprising the step of introducing the double-stranded RNA molecule described herein into the cell.
  • the present invention relates to a method for reducing off-target toxicity in a cell, comprising expressing the double-stranded RNA molecule described herein in said cell.
  • nucleotide analogs of the present invention can be used to replace conventional 2'-F and 2'-OCH 3 modifications.
  • the resulting double-stranded RNA can also show reduced off-target toxicity.
  • 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 1-10 alkyl refers to a straight or branched saturated hydrocarbon group having 1 to 10 carbon atoms.
  • C 1-6 alkyl refers to a straight or branched saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C 1-6 alkyl, C 1-4 alkyl and C 1-2 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 2-6 alkenyl refers to a straight or branched hydrocarbon group having 2 to 6 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-6 alkenyl and C 2-4 alkenyl are preferred.
  • C 2-6 alkenyl examples include: vinyl (C 2 ), 1-propenyl (C 3 ), 2-propenyl (C 3 ), 1-butenyl (C 4 ), 2-butenyl (C 4 ), butadienyl (C 4 ), pentenyl (C 5 ), pentadienyl (C 5 ), hexenyl (C 6 ), and the like.
  • the term "C 2-6 alkenyl” also includes heteroalkenyl groups, in which one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus).
  • An alkenyl group can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • C 2-10 alkynyl refers to a straight or branched hydrocarbon group having 2 to 10 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds.
  • C 2-6 alkynyl refers to a straight or branched hydrocarbon group having 2 to 6 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 2-6 alkynyl and C 2-4 alkynyl are preferred.
  • C 2-6 alkynyl examples include, but are not limited to, ethynyl (C 2 ), 1-propynyl (C 3 ), 2-propynyl (C 3 ), 1-butynyl (C 4 ), 2-butynyl (C 4 ), pentynyl (C 5 ), hexynyl (C 6 ), 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.
  • Halo or "halogen” refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).
  • C 1-6 haloalkyl refers to the above-mentioned "C 1-6 alkyl” 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 may be substituted at any available attachment point, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • C 1-6 alkoxy refers to a -OR group wherein R is as defined above for “C 1-6 alkyl” and "C 1-6 haloalkyl”.
  • C 1-10 alkylene refers to a divalent group formed by removing another hydrogen of a C 1-10 alkyl group, 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.
  • 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 — ) , and the like.
  • substituted methylene groups —CH(CH 3 )—, —C(CH 3 ) 2 —
  • substituted ethylene groups —CH(CH 3 )
  • C2-10 alkenylene refers to a divalent group formed by removing another hydrogen of C2-10 alkenyl, and may be substituted or unsubstituted. In some embodiments, C2-8 alkenylene, C3-7 alkenylene, C2-6 alkenylene, C4-6 alkenylene, C1-4 alkenylene, C2-4 alkenylene are preferred.
  • C2-10 alkynylene refers to a divalent group formed by removing another hydrogen of a C2-10 alkynyl group, respectively, and may be substituted or unsubstituted. In some embodiments, C2-8 alkynylene, C3-7 alkynylene, C2-6 alkynylene, C4-6 alkynylene, C1-4 alkynylene, C2-4 alkynylene are preferred.
  • Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups and the like 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 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.
  • shRNA refers to short hairpin RNA.
  • shRNA includes two short inverted repeat sequences.
  • the shRNA cloned into the shRNA expression vector includes two short inverted repeat sequences separated by a stem-loop sequence in the middle, forming a hairpin structure controlled by the polIII promoter. Then 5-6 Ts are connected as the transcription terminator of RNA polymerase III.
  • 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 is also called “protecting group”, which refers to any atom or group of atoms added to a molecule to prevent the existing groups in the molecule from undergoing undesirable chemical reactions.
  • Protecting group can be an unstable chemical moiety known in the art, which 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 often 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-isopropylsilyloxy) methyl, [2-(trimethylsilyl)ethoxy]methyl, or tert-butyldiphenylsilyl); esters (e.
  • Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethylethyl Acetal (BOM), ⁇ -methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), triphenylmethyl (Trt), methoxytrityl [(4-methoxyphenyl) diphenylmethyl] (MMT), dimethoxytrityl, [bis-(4-methoxyphenyl) phenylmethyl (DMT), trimethylsilyl ether (TMS), tert-butyldimethylsilyl ether (TBDMS), tri-iso-propylsilyloxymethyl ether (
  • 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 oligonucleotides comprising one or more compounds of formula (I), or pharmaceutically acceptable salts, tautomers thereof or stereoisomers:
  • *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration;
  • 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
  • L 1 and L 2 are independently selected from C 1-10 alkylene, C 2-10 alkenylene or C 2-10 alkynylene, wherein 1, 2, 3, 4 or 5 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, preferably C 1-10 alkylene;
  • the L1 and L2 are optionally substituted by 1, 2, 3, 4 or 5 R;
  • Rs is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl;
  • R is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl
  • k is 1, 2, 3, 4 or 5;
  • Base is selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C; preferably, Base is selected from
  • the present invention also 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;
  • Base' is selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C; preferably, Base' is selected from
  • the present invention also 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:
  • each variable is as defined herein.
  • *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration.
  • *1 is the (S) configuration; in another embodiment, *1 is the (R) configuration; in another embodiment, *1 is a mixture of the (S) configuration and the (R) configuration.
  • *2 is the (S) configuration; in another embodiment, *2 is the (R) configuration; in another embodiment, *2 is a mixture of the (S) configuration and the (R) configuration.
  • R 1 represents H; in another embodiment, R 1 represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide.
  • R1 are not H at the same time.
  • R 1 represents a reactive phosphorus group, for example a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimetic, for example a natural phosphate, a phosphorothioate, a phosphorodithioate, a boranophosphate, a boranophosphorothioate, 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 );
  • R 1 represents a hydroxy protecting group, for example 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 (Boc),
  • R 1 represents a solid support.
  • R 2 represents H
  • R 2 represents a reactive phosphorus group, such as 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 boranophosphorothioate, 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 );
  • R2 represents a hydroxy protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDMS), tert-butyldimethyl ...
  • TMS trimethylsilyl
  • TES triethylsilyl
  • DMIPS dimethylisopropylsilyl
  • DEIPS diethylisopropylsilyl
  • TDMS tert-butyldimethyl
  • TDPS triisopropylsilyl
  • acetyl (Ac) chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), di-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM
  • R 1 represents a solid support.
  • L 1 is C 1-10 alkylene; in another embodiment, L 1 is C 1-6 alkylene; in another embodiment, L 1 is C 1-4 alkylene, preferably -CH 2 - or -CH 2 CH 2 -; in another embodiment, L 1 is C 2-10 alkenylene; in another embodiment, L 1 is C 2-10 alkynylene; in another embodiment, 1, 2, 3, 4 or 5 non-adjacent carbon atoms in the aforementioned C 1-10 alkylene, C 1-6 alkylene, C 1-4 alkylene, -CH 2 -, -CH 2 CH 2 -, C 2-10 alkenylene or C 2-10 alkynylene may be replaced by heteroatoms selected from O, S and N.
  • 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.
  • L 2 is C 1-10 alkylene; in another embodiment, L 2 is C 1-6 alkylene; in another embodiment, L 2 is C 1-4 alkylene, preferably -CH 2 - or -CH 2 CH 2 -; in another embodiment, L 2 is C 2-10 alkenylene; in another embodiment, L 2 is C 2-10 alkynylene; in another embodiment, 1, 2, 3, 4 or 5 non-adjacent carbon atoms in the aforementioned C 1-10 alkylene, C 1-6 alkylene, C 1-4 alkylene, -CH 2 -, -CH 2 CH 2 -, C 2-10 alkenylene or C 2-10 alkynylene may be replaced by heteroatoms selected from O, S and N.
  • L2 is unsubstituted; in another embodiment, L2 is substituted with 1 R; in another embodiment, L2 is substituted with 2 Rs; in another embodiment, L2 is substituted with 3 Rs; in another embodiment, L2 is substituted with 4 Rs; in another embodiment, L2 is substituted with 5 Rs.
  • 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, such as methyl; in another embodiment, R is C 1-6 haloalkyl; in another embodiment, R is C 1-4 haloalkyl.
  • 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 1-4 alkyl, such as methyl; in another embodiment, Rs is C 1-6 haloalkyl; in another embodiment, Rs is C 1-4 haloalkyl.
  • k is 1, 2, 3, 4 or 5.
  • Base is H; in another embodiment, Base is a modified or unmodified base or a leaving group, such as, preferably, modified or unmodified A, U, T, G and C.
  • Base' is H; in another embodiment, Base' is a modified or unmodified base or a leaving group, such as, preferably, modified or unmodified A, U, T, G and C.
  • Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is in another more specific embodiment, Base is in another more specific embodiment, Base is
  • Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is in another more specific embodiment, Base' is
  • 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 of R 1 can be combined with R 2 ,
  • 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 sustained (weeks to months or longer), depending on the specific construct used and the target tissue or cell type.
  • the encoding nucleotides of the siRNA can be introduced into a linear construct, a circular plasmid, or a viral vector.
  • the siRNA can be integrated into the cell genome for stable expression, or can be stably inherited and expressed outside the chromosome.
  • siRNA expression vectors are usually DNA plasmids or viral vectors.
  • Viral vector systems containing siRNA coding sequences include but are not limited to: (a) adenovirus vectors; (b) 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.
  • the present invention relates to an oligonucleotide comprising one or more compounds of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration;
  • 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
  • L 1 and L 2 are independently selected from C 1-10 alkylene, C 2-10 alkenylene or C 2-10 alkynylene, wherein 1, 2, 3, 4 or 5 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, preferably C 1-10 alkylene;
  • the L1 and L2 are optionally substituted by 1, 2, 3, 4 or 5 R;
  • Rs is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl;
  • R is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl
  • k is 1, 2, 3, 4 or 5;
  • Base is selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C; preferably, Base is selected from
  • the present invention relates to the above oligonucleotide, wherein L 1 and L 2 are independently selected from C 1-6 alkylene, C 2-6 alkenylene or C 2-6 alkynylene, preferably C 1-6 alkylene;
  • the L1 and L2 are 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
  • L 1 and L 2 are independently C 1-4 alkylene, preferably -CH 2 - or -CH 2 CH 2 -;
  • the L1 and L2 are optionally substituted by 1, 2 or 3 R;
  • R is selected from H, D, halogen or C 1-4 alkyl.
  • the present invention relates to the above-mentioned oligonucleotide, wherein:
  • *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration;
  • 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
  • L 1 and L 2 are independently selected from C 1-6 alkylene, C 2-6 alkenylene or C 2-6 alkynylene, preferably C 1-6 alkylene;
  • the L1 and L2 are optionally substituted by 1, 2, 3, 4 or 5 R;
  • Rs is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl;
  • R is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl
  • k 1, 2 or 3;
  • the present invention relates to the above-mentioned oligonucleotide, wherein:
  • *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration;
  • 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
  • L 1 and L 2 are independently C 1-4 alkylene, preferably -CH 2 -;
  • the L1 and L2 are optionally substituted by 1, 2 or 3 R;
  • Rs is selected from H, D, halogen or C 1-4 alkyl
  • R is selected from H, D, halogen or C 1-4 alkyl
  • k 1, 2 or 3;
  • the present invention relates to the above-mentioned oligonucleotide, wherein:
  • *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration;
  • 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
  • L 1 and L 2 are independently C 1-4 alkylene, preferably -CH 2 -;
  • Rs is H or C 1-4 alkyl, preferably H;
  • k 1, 2 or 3;
  • the present invention relates to the above oligonucleotide, wherein the compound of formula (I) is selected from the compound of formula (II) or (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • the compound of formula (I) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:
  • R 1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide
  • R1 are not H at the same time.
  • the present invention relates to the above-mentioned oligonucleotide having 14 to 30 nucleotides.
  • the present invention relates to the above-mentioned oligonucleotide, which comprises a compound of formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 5' end.
  • the present invention relates to the above-mentioned oligonucleotide, which comprises a compound of formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 3' end.
  • the present invention relates to the above-mentioned oligonucleotide, which comprises a compound of formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 5' end and the 3' end, respectively.
  • the present invention relates to the above-mentioned oligonucleotides, which comprise one or more compounds of formula (I) as described herein, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, within the oligonucleotide.
  • the present invention 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;
  • Base' is selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C; preferably, Base' is selected from
  • the present invention relates to compounds of the aforementioned formula (I'), wherein R1 and R2 are H.
  • the present invention relates to compounds of formula (I') as above, wherein 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, phosphorothioates, phosphorodithioates, boranophosphates, boranophosphorothioates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, phosphorothioatediesters, phosphorothioate triesters, diphosphates or triphosphates, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 ).
  • R 1 and R 2 are selected from reactive phosphorus groups, preferably phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics
  • the present invention relates to compounds of the above formula (I'), wherein 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), benzyl, p
  • the compound of formula (I') is selected from the compound of formula (II') or (III'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • Base' is selected from
  • the compound of formula (II') or (III') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:
  • the present invention relates to 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 herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • each variable is as defined herein.
  • 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:
  • the compound of formula (I) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:
  • R 1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide
  • R1 are not H at the same time.
  • the sense strand of the double-stranded RNA comprises a compound of Formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 5' end.
  • the sense strand of the double-stranded RNA comprises a compound of Formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 3' end.
  • the sense strand of the double-stranded RNA comprises a compound of formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 5' end and the 3' end, respectively.
  • the sense strand of the double-stranded RNA comprises one or more compounds of Formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, within the oligonucleotide.
  • the antisense strand of the double-stranded RNA comprises a compound of Formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 5' end.
  • the antisense strand of the double-stranded RNA comprises a compound of Formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 3' end.
  • the antisense strand of the double-stranded RNA comprises a compound of formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 5' end and the 3' end, respectively.
  • the antisense strand of the double-stranded RNA comprises one or more compounds of Formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, within the oligonucleotide.
  • the sense strand and the antisense strand of the double-stranded RNA each have 20 to 25 nucleotides.
  • one or more of the random sites of the double-stranded RNA may be a compound of formula (I), for example:
  • nucleotide 1 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 at the 5' end of the sense strand;
  • nucleotide 1 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 at the 5' end of the antisense strand.
  • the present invention relates to a double-stranded RNA, wherein the 2nd and 14th nucleotides at the 5' end of the antisense strand are not compounds of formula (I).
  • one or more of the following sites of the double-stranded RNA is a compound of formula (I):
  • one or more of the following sites of the double-stranded RNA is a compound of formula (I):
  • the present invention relates to double-stranded RNA, wherein the compound of formula (I) is located at position 2-8, such as position 2, 3, 4, 5, 6, 7 or 8, at the 5' end of the antisense strand.
  • the compound of formula (I) is located at position 5-7 of the 5' end of the antisense strand, for example, position 5, position 6 or position 7.
  • the compound of formula (I) is located at the 5th position of the 5' end of the antisense strand.
  • the compound of formula (I) is located at the 7th position of the 5' end of the antisense strand.
  • the double-stranded RNA exhibits reduced off-target toxicity compared to a double-stranded RNA having the same sequence but not comprising a compound of Formula (I) as described herein.
  • the antisense strand of the double-stranded RNA has a sequence that is sufficiently complementary to the sense strand and the target mRNA, and has the ability to induce degradation of the target mRNA.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a double-stranded RNA molecule as described herein, and a pharmaceutically acceptable carrier or excipient.
  • the invention relates to a kit comprising a double-stranded RNA molecule as described herein.
  • the invention relates to a cell containing a double-stranded RNA molecule as described herein.
  • the present invention relates to a method for inhibiting the expression of a target gene in a cell, comprising the step of introducing a double-stranded RNA molecule as described herein into the cell.
  • the present invention relates to a method for inhibiting the expression of a target gene in a cell, comprising expressing a double-stranded RNA molecule as described herein in the cell.
  • the present invention relates to a method for reducing off-target toxicity in a cell, comprising the step of introducing a double-stranded RNA molecule as described herein into the cell.
  • the present invention relates to a method for reducing off-target toxicity in a cell, comprising expressing a double-stranded RNA molecule as described herein in the cell.
  • Example 1 According to the method of Example 1 above, the following compounds CE2-P1, CE2-P2, CE3-P1, CE3-P2, CE4-P1 and CE4-P2 were prepared by replacing the uracil base of compound 1a in Example 1 with the corresponding base as the starting compound.
  • reaction solution was concentrated under reduced pressure, and the crude product was separated by preparative high-performance liquid chromatography (preparative-HPLC, column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: water-ACN; B%: 17%-57%, 5min) to obtain white solid compound E7 (53.0 mg, yield 17.08%, purity 78.94%).
  • siRNA of the present invention is prepared using the solid phase phosphoramidite method well known in the art.
  • the specific method can be found in, for example, PCT Publication Nos. WO2016081444 and WO2019105419, and is briefly described as follows.
  • the ligand L96 was linked to the CPG carrier using methods known in the art.
  • nucleoside monomers or nucleotide analog monomers including the compound of formula (I) of the present invention
  • connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or thiolation.
  • the conditions for the synthesis of oligonucleotides with a synthesis scale of 5umol are as follows:
  • the nucleoside monomer was provided with a 0.05 mol/L acetonitrile solution.
  • the reaction conditions for each step were the same, i.e., the temperature was 25 degrees.
  • the deprotection was performed using a 3% trichloroacetic acid-dichloromethane solution, and the deprotection was repeated 3 times.
  • the activating agent used in the coupling reaction was a 0.25 mol/L ETT-acetonitrile solution, and the coupling was repeated 2 times.
  • 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 2 times.
  • the oxidation was performed using 0.05 mol/L iodine/tetrahydrofuran/pyridine/water (70/20/10, v/v/v) and the oxidation was repeated 2 times.
  • the thiolation was performed using 0.2 mol/L PADS in acetonitrile/3-methylpyridine (1/1, v/v) and the thiolation was repeated 2 times.
  • nucleoside monomers or nucleotide analog 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, and the synthesis conditions of 5umol oligonucleotides of the antisense chain are the same as those of the sense chain.
  • a column filled with a strong anion filler can be used, a sodium chloride-sodium hydroxide system can be used for elution and purification, the product can be collected and piped, and a gel filler purification column can be used for desalination, and the elution system can be pure water.
  • siRNA conjugated with L96 was obtained in a similar manner.
  • the A, U, G and C distributions represent natural adenine, uracil, guanine and cytosine ribonucleotides.
  • m indicates that the adjacent nucleotide on its left is a 2'-OCH 3 modified nucleotide.
  • Am, Um, Gm and Cm represent 2'-OCH 3 modified A, U, G and C.
  • f indicates that the adjacent nucleotide 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.
  • d indicates that the adjacent nucleotide on the right is a 2'-deoxyribonucleotide (i.e., natural DNA).
  • dA, dT, dG, and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and cytosine deoxyribonucleotide.
  • s indicates that the two adjacent nucleotides and/or delivery vectors are linked by phosphorothioate.
  • 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.
  • GL6 represents a GalNAc delivery vector of the following structure, wherein Indicates the position where the phosphate or phosphorothioate group is attached to the siRNA
  • nucleotide analog monomers of the present invention are shown in Table 2.
  • oligonucleotide chains In the order of 5'–>3' oligonucleotide chains: It means that it is connected to the 3' carbon atom of the previous nucleotide in the oligonucleotide chain through a backbone group (such as a phosphate group or a thiophosphate group). Indicates that the next nucleotide in the oligonucleotide chain is linked to the 5' carbon atom of the next nucleotide through the backbone group.
  • a backbone group such as a phosphate group or a thiophosphate group
  • Isolate C57BL/6 wild-type mouse liver primary cells count, plate in 24-well plates, 900 ⁇ L/well, 8 ⁇ 10 4 cells/well; plate in 96-well plates, 100 ⁇ L/well, 1 ⁇ 10 4 cells/well. Then choose free uptake.
  • ⁇ 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 control gene is mGAPDH.
  • C57BL/6 wild-type mouse primary hepatocytes were selected, and the starting concentration of the compound was selected after the cells were plated in a 96-well plate. 10 nM, 10-fold dilution of 5 concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM) was used for 5-point IC50 activity screening of C57BL/6 wild-type mouse primary hepatocytes - free uptake. The experimental results are shown in Table 4.
  • the Huh7 cell line was digested, resuspended, and counted.
  • the cell resuspension was plated into a 96-well plate, 100 ⁇ L/well, 1 ⁇ 10 4 cells/well, and transfection was performed 18 hours later.
  • siRNA stock solution was diluted with Opti-MEM. Take 198 ⁇ L Opti-MEM and add 2 ⁇ L siRNA stock solution. The final siRNA concentration is shown below. Mix by pipetting and set aside.
  • RNAiMAX RNAiMAX
  • 1.3 ⁇ L RNAiMAX Thermo, 13778150
  • 15 ⁇ L of the prepared RNAi-MAX mixture and 15 ⁇ L of the diluted siRNA and pipette gently to mix without introducing bubbles let stand at room temperature for 10 minutes, add to 96-well plate, 10 ⁇ L/well. Incubate in 37°C, 5% CO2 incubator for 24 hours (no siRNA was added to the control group).
  • Cell RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the operating instructions of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0417-L).
  • Denaturation reaction mixture was prepared according to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B), single well Preparation volume: Oligo dT Primer 1 ⁇ L, dNTP Mixture 1 ⁇ L, template RNA 12.5 ⁇ L, incubate at 65°C in a conventional PCR instrument for 5 minutes, then quickly cool on ice for 2 minutes.
  • the preparation of the reverse transcription reaction solution was based on PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contained 4 ⁇ L of 5 ⁇ Prime Script II Buffer, 0.5 ⁇ L of RNase Inhibitor, and 1 ⁇ L of PrimeScript II RTase.
  • ⁇ 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 hApoc3 and the internal reference was hACTB.
  • Huh7 cell line was selected, and the starting concentration of the compound was 10 nM, and 11 concentration points of 3-fold gradient dilution (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, 0.000169 nM) were selected for cell line activity screening of the compounds of the present invention.
  • the experimental screening results are shown in Table 6.
  • Off-target plasmid The corresponding antisense chain off-target plasmid was designed according to the compound sequence.
  • the psiCHECK2 GSSM-5Hits recombinant plasmid was prepared by Sangon Biotech (Shanghai) Co., Ltd. and the recombinant plasmid was diluted to 1000 ng/ ⁇ L for use.
  • HEK293A cells (Nanjing Kebai, catalog number CBP60436) were used to plate 96-well plates with 100 ⁇ L of cell resuspension, with a cell amount of 8 ⁇ 10 3 cells/well.
  • the complete medium in the wells was firstly discarded and replaced with Opti-MEM medium 80 ⁇ L/well, and the cells were starved for about 1.5 h.
  • Plasmid mixture Single-well preparation volume: plasmid 0.01 ⁇ L/well, Opti-MEM 8.99 ⁇ L/well.
  • Lipo mixture dilute Lipo 2000 (Lipofectamine TM 2000 transfection reagent, Thermo, 11668019) with Opti-MEM, and let stand at room temperature for 5 minutes.
  • the specific amount of Lipo mixture is: Lipo 0.2 ⁇ L/well, Opti-MEM 9.8 ⁇ L/well.
  • the vacuum pump sucks away the original culture medium in the 96-well culture plate
  • the fluorescence activity was measured by an ELISA instrument, and the collected Renilla signal was normalized by the Firefly signal standard.
  • the inhibitory effect of siRNA was obtained by comparing the untreated results (residual inhibitory activity). The calculation process is as follows:
  • Ratio Renilla (Renilla luciferase)/Firefly (Firefly luciferase).
  • Ratio control is the average of the Ratio of two replicate wells of the control well (without siNRA). Calculate the Ratio siRNA /Ratio control of the two replicate wells separately, and then take the average value as the residual inhibition rate;
  • HEK293A cell line was selected, and the starting concentration of the compound was 40 nM, and 11 concentration points of 3-fold gradient dilution (40 nM, 13.3 nM, 4.44 nM, 1.48 nM, 0.493 nM, 0.164 nM, 0.0548 nM, 0.0182 nM, 0.00609 nM, 0.00203 nM, 0.000677 nM) were selected for screening of the off-target activity of the compound of the present invention, psiCHECK2 GSSM-5 Hits.
  • Table 7 The experimental screening results are shown in Table 7.

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Abstract

La présente invention concerne un ARN double brin contenant un analogue nucléotidique. L'ARN double brin de la présente invention présente une toxicité hors cible réduite.
PCT/CN2024/110992 2023-08-10 2024-08-09 Arn double brin contenant un analogue nucléotidique Ceased WO2025031479A1 (fr)

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