WO2023241587A1 - 环膦酸酯修饰的核苷酸 - Google Patents

环膦酸酯修饰的核苷酸 Download PDF

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Publication number
WO2023241587A1
WO2023241587A1 PCT/CN2023/099987 CN2023099987W WO2023241587A1 WO 2023241587 A1 WO2023241587 A1 WO 2023241587A1 CN 2023099987 W CN2023099987 W CN 2023099987W WO 2023241587 A1 WO2023241587 A1 WO 2023241587A1
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alkyl
oligonucleotide
haloalkyl
independently selected
halogen
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PCT/CN2023/099987
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English (en)
French (fr)
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黄金宇
邹昊
刘俊凯
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大睿生物医药科技(上海)有限公司
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Publication of WO2023241587A1 publication Critical patent/WO2023241587A1/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical

Definitions

  • the invention belongs to the field of medicine, and specifically relates to double-stranded RNA having a cyclic phosphonate structure.
  • RNA interference is a phenomenon of efficient and specific degradation of target mRNA induced by double-stranded RNA (dsRNA, also known as siRNA).
  • dsRNA double-stranded RNA
  • the 5'-phosphate of the guide strand (antisense strand) in siRNA can stabilize the complex formed by the guide strand and Ago2 by forming electrostatic interactions with the cationic amino acid residues near the interface of the Ago2 protein MID and PIWI domains. Therefore, siRNA
  • the 5'-phosphate of the guide strand is critical for RNAi-based gene silencing. However, the 5'-phosphate can be cleaved by endogenous phosphatases, thereby losing the 5' terminal phosphate group, causing a significant reduction in siRNA activity.
  • siRNA small interfering RNA
  • WO2011139702A2 and WO2013033230A1 disclose a nucleotide containing 5'-vinyl phosphonate (referred to as E-VP or VP).
  • E-VP 5'-vinyl phosphonate
  • 5'-E-VP can replace 5'-phosphate to bind to Ago2 and bind to internal It is highly resistant to native phosphatases, thereby affecting the activity and/or stability of siRNA.
  • WO2017214112A1 discloses a 5'-cyclo-phosphonate-containing nucleotide.
  • 5'-E-VP is connected to the sugar ring through a CC single bond, which can still rotate freely, resulting in the phosphonic acid group being in different positions relative to the sugar ring (see the figure below, where The ap position is the conformation in which unmodified 5'-phosphate binds to the Ago2 protein).
  • the inventors unexpectedly discovered that restricting the rotation of the terminal phosphonic acid through a rigid ring structure and locking it in the Ago2 binding conformation is beneficial to further improving siRNA activity.
  • the invention relates to an oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, the 5' end of the oligonucleotide comprising the formula (I) The structure shown:
  • oligonucleotide means linked to the remainder of the oligonucleotide, preferably to a modified or unmodified nucleoside;
  • X and Y are each independently selected from CR a R b or (CR a R b ) 2 , preferably CR a R b ;
  • Z is CR a ;
  • R a and R b are independently selected from H, D, halogen, OH, NH 2 , CN, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy group, C 2-6 alkenyl or C 2-6 alkynyl; the R a and R b are optionally further substituted by 1, 2, 3, 4 or 5 independently selected R#;
  • R 1 is selected from O or S
  • R 2 and R 3 are independently selected from OH, SH, NH 2 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy; the R 2 and R3 optionally further substituted by 1, 2, 3, 4 or 5 independently selected R#;
  • R# is selected from H, D, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl;
  • Each of the above defined groups is optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms until completely deuterated.
  • the structure of the present invention contains a four-membered carbon ring. This relatively rigid structure enables the oligonucleotide of the present invention (especially the phosphonate group at its 5' end) to better interact with Ago2 protein binds, thereby increasing the activity of the oligonucleotide.
  • the compounds of the present invention can lock the phosphonic acid in an ap conformation that is highly similar to unmodified 5'-phosphate-bound Ago2, in which the phosphonate group is perfectly aligned with Arg812, Lys570, Lys566 and Lys533 Matching, multiple salt bridge bonds and hydrogen bonds are formed, and one hydrogen bond is also formed with Tyr529 and Cys526.
  • the base (uracil) in the spirocyclic phosphonic acid compound forms one hydrogen bond each with the backbone amide of Thr526 and Gly524. The combination of these structures stabilizes the binding of the compound of the invention to the Ago2 protein.
  • the sugar ring fuses highly rigid aromatic rings (including five-membered aromatic rings and six-membered aromatic rings), locking the 5' terminal phosphonate in a specific conformation. It highly simulates the ap conformation of natural 5'-phosphate.
  • the phosphonate group perfectly matches Arg812, Lys570, Lys566 and Lys533, forming multiple salt bridge bonds and hydrogen bonds. It also forms one with Tyr529 and Cys526 each. hydrogen bonding.
  • the base uracil on the sugar ring also forms a hydrogen bond with the backbone amide of Thr526 and Gly524. The combination of these structures stabilizes the binding of the compound of the invention to the Ago2 protein.
  • C 1-6 alkyl includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1 -2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5, C 3-4 , C 4-6 , C 4-5 and C 5 -6 alkyl.
  • C 1-6 alkyl refers to a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C 1-4 alkyl and C 1-2 alkyl are preferred. Examples of C 1-6 alkyl groups include: methyl (C 1 ), ethyl (C 2 ), n-propyl (C 3 ), isopropyl (C 3 ), n-butyl (C 4 ), tert-butyl base (C 4 ), sec-butyl (C 4 ), isobutyl (C 4 ), n-pentyl (C 5 ), 3-pentyl (C 5 ), pentyl (C 5 ), neopentyl ( C 5 ), 3-methyl-2-butan group (C 5 ), tert-amyl group (C 5 ) and n-hexyl group (C 6 ).
  • C 1-6 alkyl also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, Phosphorus) substitution.
  • Alkyl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • alkyl abbreviations include: Me(-CH 3 ), Et(-CH 2 CH 3 ), iPr(-CH(CH 3 ) 2 ), nPr(-CH 2 CH 2 CH 3 ), n-Bu(-CH 2 CH 2 CH 2 CH 3 ) or i-Bu(-CH 2 CH(CH 3 ) 2 ).
  • C 2-6 alkenyl refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-4 alkenyl is preferred. Examples of C 2-6 alkenyl groups include: vinyl (C 2 ), 1-propenyl (C 3 ), 2-propenyl (C 3 ), 1-butenyl (C 4 ), 2-butenyl (C 4 ), butadienyl (C 4 ), pentenyl (C 5 ), pentadienyl (C 5 ), hexenyl (C 6 ), etc.
  • C 2-6 alkenyl also includes heteroalkenyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, Phosphorus) substitution.
  • Alkenyl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • C 2-6 alkynyl refers to a straight or branched chain 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 2-4 alkynyl is preferred. Examples of C 2-6 alkynyl groups include, but are not limited to: ethynyl (C 2 ), 1-propynyl (C 3 ), 2-propynyl (C 3 ), 1-butynyl (C 4 ), 2-Butynyl (C 4 ), pentynyl (C 5 ), hexynyl (C 6 ), etc.
  • C 2-6 alkynyl also includes heteroalkynyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, Phosphorus) substitution.
  • An alkynyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • Halo or "halogen” refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).
  • C 1-6 haloalkyl refers to the above-mentioned "C 1-6 alkyl” which is substituted by one or more halogen groups.
  • C 1-4 haloalkyl is particularly preferred, with C 1-2 haloalkyl being more preferred.
  • Exemplary haloalkyl groups include, but are not limited to: -CF 3 , -CH 2 F, -CHF 2 , -CHFCH 2 F, -CH 2 CHF 2 , -CF 2 CF 3 , -CCl 3 , -CH 2 Cl , -CHCl 2 , 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc.
  • Haloalkyl groups may be substituted at any available point of attachment, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • C 1-6 alkoxy refers to the -OR group, where R is as defined above for “C 1-6 alkyl”.
  • C 1-6 haloalkoxy refers to the -OR group, where R is as defined above for "C 1-6 haloalkyl”.
  • C 5-6 cycloalkyl refers to a non-aromatic cyclic hydrocarbon group having 5 to 6 ring carbon atoms and zero heteroatoms. Cycloalkyl also includes ring systems in which the above-described cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such cases the number of carbons continues as indicated The number of carbons in a cycloalkyl system.
  • Exemplary cycloalkyl groups include, but are not limited to: cyclopentyl (C 5 ), cyclopentenyl (C 5 ), cyclohexyl (C 6 ), cyclohexenyl (C 6 ), cyclohexadiene base (C 6 ), etc.
  • a cycloalkyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • 5-6 membered heterocyclyl refers to a group of 5 to 6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, Sulfur, boron, phosphorus and silicon.
  • the point of attachment may be a carbon or nitrogen atom as long as the valency permits.
  • Heterocyclyl also includes ring systems in which the above-described heterocyclyl ring is fused with one or more cycloalkyl groups, wherein the point of attachment is on the cycloalkyl ring, or in which the above-described heterocyclyl ring is fused with one or more aryl groups or Heteroaryl fused ring systems wherein the point of attachment is on the heterocyclyl ring; and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system.
  • Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to: tetrahydrofuryl, dihydrofuryl, tetrahydrothiophenyl, dihydrothiophenyl, Pyrrolidyl, dihydropyrrolyl and pyrrolyl-2,5-dione.
  • Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to: dioxolyl, oxasulfuranyl, disulfuranyl, and oxalanyl. Azolidin-2-one.
  • Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl, and thiadiazolinyl.
  • Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridyl, and thianyl.
  • Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to: piperazinyl, morpholinyl, dithianyl, and dioxanyl.
  • Exemplary 6-membered heterocyclyl groups containing three heteroatoms include, but are not limited to: hexahydrotriazinyl (triazinanyl). Heterocyclyl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl and the like are defined herein as optionally substituted groups.
  • Each R aa is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R aa groups are combined to form heterocyclyl or Heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd groups group replacement;
  • Each R cc is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R cc groups are combined to form a heterocycle or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;
  • Each R ee is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl Alkyl, heterocyclyl, aryl and heteroaryl are independently substituted by 0, 1, 2, 3, 4 or 5 R gg groups;
  • Each R ff is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R ff groups combine to form a heterocyclyl or a heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R gg group substitution;
  • siRNA refers to a class of double-stranded RNA molecules that can mediate silencing of a target RNA that is complementary to it (eg, mRNA, eg, the transcript of a gene encoding a protein).
  • siRNA is usually double-stranded, including an antisense strand that is complementary to the target RNA, and a sense strand that is complementary to the antisense strand.
  • mRNA is also referred to herein as the mRNA to be silenced.
  • Such genes are also called target genes.
  • the RNA to be silenced is an endogenous gene or a pathogen gene.
  • RNAs other than mRNA e.g., tRNA
  • viral RNA can also be targeted.
  • antisense strand refers to a strand of siRNA that contains a region that is completely, fully, or substantially complementary to the target sequence.
  • sense strand refers to a strand of siRNA that includes a region that is completely, fully, or substantially complementary to a region that is the antisense strand as the term is defined herein.
  • complementary region refers to a region on the antisense strand that is completely, fully or substantially complementary to the target mRNA sequence. In cases where the complementary region is not completely complementary to the target sequence, the mismatch can be located in the internal or terminal regions of the molecule. Typically, the most tolerated mismatches are in the terminal region, e.g., within 5, 4, 3, 2 or 1 nucleotide of the 5' and/or 3' end. The portion of the antisense strand that is most sensitive to mismatches is called the "seed region.” For example, in a siRNA containing a 19nt strand, some mismatches can be tolerated at position 19 (from 5' to 3').
  • complementary refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions.
  • stringent conditions may include 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA at 50°C or 70°C for 12-16 hours.
  • “complementary” sequences may also include or be formed entirely from non-Watson-Crick base pairs and/or from non-natural and modified nucleosides. Base pairs formed by acids. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.
  • a polynucleotide that is "at least partially complementary,” “fully complementary,” or “substantially complementary” to messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest.
  • a polynucleotide is complementary to at least a portion of a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of the PCSK9 mRNA.
  • the terms “complementary,” “completely complementary,” “sufficiently complementary,” and “substantially complementary” as used herein may refer to the relationship between the sense strand and the antisense strand of the siRNA, or between the antisense strand of the siRNA agent and the target sequence. Used in base pairing.
  • “Sufficiently complementary” refers to the extent to which the sense strand only needs to be complementary to the antisense strand in order to maintain the overall double-stranded character of the molecule. In other words, although perfect complementarity is usually required, in some cases, particularly in the antisense strand, one or more, such as 6, 5, 4, 3, 2 or 1, may be included. mismatch (relative to the target mRNA), but the sense and antisense strands can still maintain the overall double-stranded character of the molecule.
  • shRNA refers to short hairpin RNA.
  • shRNA consists of two short inverted repeats.
  • the shRNA cloned into the shRNA expression vector includes two short inverted repeat sequences, separated by a stem-loop sequence in the middle, forming a hairpin structure and controlled by the pol III promoter. Then 5-6 Ts are connected as the transcription terminator of RNA polymerase III.
  • Nucleoside is a compound composed of two substances: purine base or pyrimidine base, and ribose or deoxyribose.
  • Nucleoside is a compound composed of three substances: purine base or pyrimidine base, ribose or deoxyribose, and phosphate.
  • Olionucleotide refers to, for example, a nucleic acid molecule (RNA or DNA) having a length of less than 100, 200, 300, or 400 nucleotides, and may be single- or double-stranded.
  • Base is the basic unit for the synthesis of nucleosides, nucleotides and nucleic acids. Its constituent elements contain nitrogen, also known as “nitrogen-containing bases”.
  • nitrogen-containing bases also known as “nitrogen-containing bases”.
  • the capital letters A, U, T, G, and C represent the base composition of nucleotides, which are adenine, uracil, and Thymine, guanine and cytosine.
  • Modification of nucleotides described herein includes, but is not limited to, methoxy modification, fluoro modification, phosphorothioate group connection or conventional protecting group protection, etc.
  • the fluoro-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group of the nucleotide is replaced by fluorine
  • the methoxy-modified nucleotide refers to the 2'-hydroxyl group of the ribosyl group.
  • Modified nucleotides herein include, but are not limited to, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine Ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, nucleotides containing phosphorothioate groups, vinyl phosphate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases containing nucleotides, and derivatives linked to cholesterol groups Terminal nucleotide, deoxyribonucleotide or conventional protecting group protection on the dodecanoic acid dodecylamide group.
  • the 2'-fluoro modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by fluorine.
  • the 2'-deoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.
  • Reactive phosphorus group means a phosphorus-containing group contained in a nucleotide unit or a nucleotide analog unit which can react by nucleophilic attack with a phosphorus-containing group contained in another molecule, especially another Reaction of a hydroxyl or amine group in a nucleotide unit or another nucleotide analogue. Typically, such a reaction results in an ester form linking said first nucleotide unit or said first nucleotide analog unit to said second nucleotide unit or said second nucleotide analog unit. Internucleoside bonds.
  • the reactive phosphorus group may be selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, including but not limited to: natural phosphates, thiophosphates, dithiophosphates Phosphates, borane phosphates, borane phosphorothioates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, phosphorothioate diesters, phosphorothioates Trysters, diphosphates and triphosphates, preferably -P( OCH2CH2CN )(N( iPr ) 2 ).
  • Protecting groups can be labile chemical moieties known in the art that serve to protect reactive groups, such as hydroxyl, amino, and thiol groups, to prevent undesirable or undesirable formation during chemical synthesis. reaction.
  • Protecting groups are typically used to protect sites selectively and/or orthogonally during reactions at other reactive sites and can then be removed to leave the unprotected group intact or available for further reactions.
  • a non-limiting list of protecting groups includes benzyl; substituted benzyl; alkylcarbonyl and alkoxycarbonyl (eg, tert-butoxycarbonyl (BOC), acetyl, or isobutyryl); arylalkylcarbonyl and arylalkoxycarbonyl (e.g., benzyloxycarbonyl); substituted methyl ether (e.g., methoxymethyl ether); substituted diethyl ether; substituted benzyl ether; tetrahydropyranyl ether; methyl Silyl group (for example, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(Trimethylsilyl)ethoxy]methyl or tert-butyldiphenylsilyl); Esters (such as benzoate); Carbonates (such as me
  • Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethyl acetyl Aldehyde (BOM), ⁇ -methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, Pivaloyl (Piv), tetrahydrogen Pyranyl (THP), triphenylmethyl (Trt), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), dimethoxytrityl base, [bis-(4-methoxyphenyl)phenylmethyl (DMT), trimethylsilyl ether (TMS), tert-butyldimethylsilyl ether (TBDMS), tri-iso -Propyls
  • Hydro protecting group refers to a group that can protect the hydroxyl group from chemical reactions and can be removed under specific conditions to restore the hydroxyl group.
  • Trimethylsilyl TMS
  • triethylsilyl TES
  • dimethylisopropylsilyl DMIPS
  • diethylisopropylsilyl DEIPS
  • tert-butyldimethylsilyl TDMS
  • tert-butyldiphenylsilyl TIPS
  • TIPS Trimethylsilyl
  • acetyl Ac
  • chloroacetyl dichloroacetyl, trichloroacetyl
  • trifluoroacetyl TSA
  • benzoyl p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc) , Benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzy
  • the term "pharmaceutically acceptable salts” means those carboxylate salts and amino acid addition salts of the compounds of the present invention which are suitable for contact with patient tissue within the scope of reliable medical judgment and will not produce undue toxicity, Irritation effects, allergic reactions, etc., commensurate with a reasonable benefit/risk ratio, are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the invention.
  • the present invention includes tautomers, which are functional group isomers resulting from the rapid movement of an atom in a molecule between two positions.
  • tautomers which are functional group isomers resulting from the rapid movement of an atom in a molecule between two positions.
  • Compounds exist in different tautomeric forms, and a said compound is not limited to any particular tautomeric form, but is intended to encompass all tautomeric forms.
  • the compounds of the present invention may contain one or more asymmetric centers and thus may exist in multiple stereoisomeric forms, for example, enantiomeric and/or diastereomeric forms.
  • the compounds of the present invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, Includes racemic mixtures and mixtures enriched in one or more stereoisomers.
  • the isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or the preferred isomers may be separated by Prepared by asymmetric synthesis.
  • HPLC high pressure liquid chromatography
  • the present invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described in formula (I), except that one or more atoms are surrounded by atoms having an atomic mass or mass number different from that common in nature. replaced.
  • isotopes that may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 respectively. O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 Cl.
  • isotopically labeled compounds of the present invention such as those incorporating radioactive isotopes (eg, 3 H and 14 C), may be used in drug and/or substrate tissue distribution assays.
  • Tritium, ie 3 H, and carbon-14, ie 14 C isotopes are particularly preferred because they are easy to prepare and detect. In turn, replacement by heavier isotopes, such as deuterium, i.e.
  • the isotope-labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by replacing non-isotopes with readily available isotope-labeled reagents when performing the following processes and/or the processes disclosed in the Examples and Preparation Examples. Labeled reagents.
  • the present invention relates to an oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, the 5' end of the oligonucleotide comprising the structure represented by formula (I):
  • the present invention also relates to an oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, the 5' end of the oligonucleotide comprising the structure represented by formula (II):
  • the present invention also relates to an oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, the 5' end of the oligonucleotide comprising formula (III), formula (IV) Or the nucleotide monomer represented by formula (V):
  • the present invention also relates to compounds represented by formula (VI), formula (VII) or formula (VIII) or their pharmaceutically acceptable salts, tautomers or stereoisomers:
  • oligonucleotide represents the remainder linked to the oligonucleotide; in another embodiment, Indicates attachment to a modified or unmodified nucleoside.
  • X is CR a R b , preferably CH 2 ; in another embodiment, X is (CR a R b ) 2 .
  • Y is CR a R b , preferably CH 2 ; in another embodiment, Y is (CR a R b ) 2 .
  • Z is CR a , preferably CH.
  • X and Y are optionally replaced by 1 or 2 deuterium atoms, and Z is optionally replaced by 1 deuterium atom.
  • L 1 is a chemical bond; in another embodiment, L 1 is O; in another embodiment, L 1 is S; in another embodiment, L 1 is C 1-4 alkyl.
  • L 2 is C 1-4 alkylene, preferably C 1-2 alkylene.
  • L 3 is C 1-4 alkylene, preferably C 1-2 alkylene.
  • L 2 and L 3 are connected, together with L 1 and its adjacent carbon atoms, to form a C 5-6 cycloalkyl group; in another embodiment, L 2 and L 3 are connected, together with L 1 Together with its adjacent carbon atoms, it forms a 5-6 membered heterocyclyl group, preferably U, L 1 , L 2 and L 3 and its adjacent carbon atoms together form ribose or deoxyribose, which is optionally substituted by R 5 .
  • L 1 is unsubstituted; in another embodiment, L 1 is further substituted with 1 R#; in another embodiment, L 1 is further substituted with 2 independently selected R#; in In another embodiment, L 1 is further substituted with 3 independently selected R#; in another embodiment, L 1 is further substituted with 4 independently selected R#; in another embodiment, L 1 is further substituted with 5 An independently selected R# is further substituted.
  • L 2 is unsubstituted; in another embodiment, L 2 is further substituted by 1 R#; in another embodiment, L 2 is further substituted by 2 independently selected R#; in In another embodiment, L 2 is further substituted by 3 independently selected R#; in another embodiment, L 2 is further substituted by 4 independently selected R#; in another embodiment, L 2 is further substituted by 5 An independently selected R# is further substituted.
  • L 3 is unsubstituted; in another embodiment, L 3 is further substituted with 1 R#; in another embodiment, L 3 is further substituted with 2 independently selected R#; in another embodiment, L 3 is further substituted by 3 independently selected R#; in another embodiment, L 3 is further substituted by 4 independently selected R#; in another embodiment, L 3 is further substituted by 5 An independently selected R# is further substituted.
  • L 1 , L 2 and L 3 are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms until completely deuterated.
  • U is O; in another embodiment, U is S; in another embodiment, U is CR a R b , such as CH 2 ; in another embodiment, U is NR c , such as NH.
  • Q is H; in another embodiment, Q is D; in another embodiment, Q is halogen; in another embodiment, Q is OH; in another embodiment, Q is C 1-6 alkyl, such as C 1-4 alkyl; in another embodiment, Q is C 1-6 haloalkyl, such as C 1-4 haloalkyl; in another embodiment, Q is C 1-6 alkoxy, such as C 1-4 alkoxy; in another embodiment, Q is C 1-6 haloalkoxy, such as C 1-4 haloalkoxy; in another embodiment, Q is C 2-6 alkenyl, such as C 2-4 alkenyl; in another embodiment, Q is C 2-6 alkynyl, such as C 2-4 alkynyl.
  • R 1 is O; in another embodiment, R 1 is S.
  • R 2 is OH; in another embodiment, R 2 is SH; in another embodiment, R 2 is NH 2 ; in another embodiment, R 2 is C 1-6 Alkyl; in another embodiment, R 2 is C 1-6 haloalkyl; in another embodiment, R 2 is C 1-6 alkoxy; in another embodiment, R 2 is C 1 -6 haloalkoxy.
  • R2 is ORd ; in another embodiment, R2 is OP1 ; in another embodiment, R2 is SRd ; in another embodiment, R2 is SP1 ; In another embodiment, R 2 is NR e R f .
  • R 2 is unsubstituted; in another embodiment, R 2 is further substituted by 1 R#; in another embodiment, R 2 is further substituted by 2 independently selected R#; in In another embodiment, R 2 is further substituted by 3 independently selected R#; in another embodiment, R 2 is further substituted by 4 independently selected R#; in another embodiment, R 2 is further substituted by 5 An independently selected R# is further substituted.
  • R 3 is OH; in another embodiment, R 3 is SH; in another embodiment, R 3 is NH 2 ; in another embodiment, R 3 is C 1-6 Alkyl; in another embodiment, R 3 is C 1-6 haloalkyl; in another embodiment, R 3 is C 1-6 alkoxy; in another embodiment, R 3 is C 1 -6 haloalkoxy.
  • R3 is ORd ; in another embodiment, R3 is OP1 ; in another embodiment, R3 is SRd ; in another embodiment, R3 is SP1 ; In another embodiment, R 3 is NR e R f .
  • R 3 is unsubstituted; in another embodiment, R 3 is further substituted with 1 R#; in another embodiment, R 3 is further substituted with 2 independently selected R#; in another embodiment, R 3 is further substituted by 3 independently selected R#; in another embodiment, R 3 is further substituted by 4 independently selected R#; in another embodiment, R 3 is further substituted by 5 An independently selected R# is further substituted.
  • R 2 and R 3 are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms until completely deuterated.
  • R 4 is H; in another embodiment, R 4 is D; in another embodiment, R 4 is halogen; in another embodiment, R 4 is OH; in another In one embodiment, R 4 is C 1-6 alkyl, such as C 1-4 alkyl; in another embodiment, R 4 is C 1-6 haloalkyl, such as C 1-4 haloalkyl; in another In one embodiment, R 4 is C 1-6 alkoxy, such as C 1-4 alkoxy; in another embodiment, R 4 is C 1-6 haloalkoxy, such as C 1-4 haloalkoxy. .
  • R 5 is H; in another embodiment, R 5 is D; in another embodiment, R 5 is halogen; in another embodiment, R 5 is OH; in another In one embodiment, R 5 is C 1-6 alkyl, such as C 1-4 alkyl; in another embodiment, R 5 is C 1-6 haloalkyl, such as C 1-4 haloalkyl; in another In one embodiment, R 5 is C 1-6 alkoxy, such as C 1-4 alkoxy; in another embodiment, R 5 is C 1-6 haloalkoxy, such as C 1-4 haloalkoxy. .
  • R 6 is H; in another embodiment, R 6 is D; in another embodiment, R 6 is halogen; in another embodiment, R 6 is OH; in another In one embodiment, R 6 is C 1-6 alkyl, such as C 1-4 alkyl; in another embodiment, R 6 is C 1-6 haloalkyl, such as C 1-4 haloalkyl; in another In one embodiment, R 6 is C 1-6 alkoxy, such as C 1-4 alkoxy; in another embodiment, R 6 is C 1-6 haloalkoxy, such as C 1-4 haloalkoxy. .
  • R 7 is H; in another embodiment, R 7 is D; in another embodiment, R 7 is halogen; in another embodiment, R 7 is OH; in another In one embodiment, R 7 is C 1-6 alkyl, such as C 1-4 alkyl; in another embodiment, R 7 is C 1-6 haloalkyl, such as C 1-4 haloalkyl; in another In one embodiment, R 7 is C 1-6 alkoxy, such as C 1-4 alkoxy; in another embodiment, R 7 is C 1-6 haloalkoxy, such as C 1-4 haloalkoxy. .
  • R 4 , R 5 , R 6 and R 7 are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms until completely deuterated.
  • Ra is H; in another embodiment, Ra is D; in another embodiment, Ra is halogen; in another embodiment, Ra is OH; in another In one embodiment, Ra is NH 2 ; in another embodiment, Ra is CN; in another embodiment, Ra is C 1-6 alkyl, such as C 1-4 alkyl; in another In one embodiment, Ra is C 1-6 haloalkyl, such as C 1-4 haloalkyl; in another embodiment, Ra is C 1-6 alkoxy, such as C 1-4 alkoxy; in In another embodiment, Ra is C 1-6 haloalkoxy, such as C 1-4 haloalkoxy; in another embodiment, Ra is C 2-6 alkenyl, such as C 2-4 alkenyl ; In another embodiment, R a is C 2-6 alkynyl, such as C 2-4 alkynyl.
  • Ra is unsubstituted; in another embodiment, Ra is further substituted by 1 R#; in another embodiment, Ra is further substituted by 2 independently selected R#; in In another embodiment, Ra is further substituted with 3 independently selected R#; in another embodiment, Ra is further substituted with 4 independently selected R#; in another embodiment, Ra is further substituted with 5 An independently selected R# is further substituted.
  • R b is H; in another embodiment, R b is D; in another embodiment, R b is halogen; in another embodiment, R b is OH; in another In one embodiment, R b is NH 2 ; in another embodiment, R b is CN; In another embodiment, R b is C 1-6 alkyl, such as C 1-4 alkyl; in another embodiment, R b is C 1-6 haloalkyl, such as C 1-4 haloalkyl; In another embodiment, R b is C 1-6 alkoxy, such as C 1-4 alkoxy; in another embodiment, R b is C 1-6 haloalkoxy, such as C 1-4 Haloalkoxy; in another embodiment, R b is C 2-6 alkenyl, such as C 2-4 alkenyl; in another embodiment, R b is C 2-6 alkynyl, such as C 2- 4 alkynyl.
  • R b is unsubstituted; in another embodiment, R b is further substituted by 1 R#; in another embodiment, R b is further substituted by 2 independently selected R#; in In another embodiment, R b is further substituted by 3 independently selected R#; in another embodiment, R b is further substituted by 4 independently selected R#; in another embodiment, R b is further substituted by 5 An independently selected R# is further substituted.
  • R c is H; in another embodiment, R c is C 1-6 alkyl; in another embodiment, R c is C 1-6 haloalkyl; in another embodiment In another embodiment, R c is C 2-6 alkenyl; in another embodiment, R c is C 2-6 alkynyl.
  • R a , R b and R c are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms until completely deuterated.
  • Rd is H; in another embodiment, Rd is C 1-6 alkyl, such as C 1-4 alkyl; in another embodiment, Rd is C 1-6 Haloalkyl, for example, C 1-4 haloalkyl; in another embodiment, R d is C 2-6 alkenyl; in another embodiment, R d is C 2-6 alkynyl.
  • Rd is unsubstituted; in another embodiment, Rd is optionally substituted with 1 or more Ds, up to fully deuterated; in another embodiment, Rd is substituted with halogen ; In another embodiment, R d is substituted with C 1-6 alkyl; in another embodiment, R d is substituted with C 1-6 haloalkyl.
  • Re is H; in another embodiment, Re is C 1-6 alkyl, such as C 1-4 alkyl; in another embodiment, Re is C 1-6 Haloalkyl, for example C 1-4 haloalkyl.
  • Re is unsubstituted; in another embodiment, Re is optionally substituted with 1 or more Ds, up to fully deuterated; in another embodiment, Re is substituted with halogen ; In another embodiment, Re is substituted with C 1-6 alkyl; in another embodiment, Re is substituted with C 1-6 haloalkyl.
  • R f is H; in another embodiment, R f is C 1-6 alkyl, such as C 1-4 alkyl; in another embodiment, R f is C 1-6 Haloalkyl, for example C 1-4 haloalkyl.
  • Rf is unsubstituted; in another embodiment, Rf is optionally substituted with 1 or more Ds, up to fully deuterated; in another embodiment, Rf is substituted with halogen ; In another embodiment, R f is substituted with C 1-6 alkyl; in another embodiment, R f is substituted with C 1-6 haloalkyl.
  • R d , Re and R f are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms until completely deuterated.
  • P1 is a protecting group; in another embodiment, P1 is a hydroxyl protecting group.
  • P2 is a reactive phosphorus group, such as -P( OCH2CH2CN )(N( iPr ) 2 ).
  • Base is H; in another embodiment, Base is a modified or unmodified base or leaving group; in another embodiment, Base is selected from modified or unmodified A, U, T, G and C, for example
  • Base' is H; in another embodiment, Base' is a modified or unmodified base or leaving group; in another embodiment, Base' is selected from modified or unmodified A ,U,T,G and C, for example
  • R# is H; in another embodiment, R# is D; in another embodiment, R# is halogen; in another embodiment, R# is C 1-6 alkane base; in another embodiment, R# is C 1-6 haloalkyl; in another embodiment, R# is C 2-6 alkenyl; in another embodiment, R# is C 2-6 Alkynyl;
  • R# is optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms until completely deuterated.
  • GalNAc is a conjugation group represented by formula (X):
  • Q G is independently H
  • L G1 is a chemical bond, -CH 2 -, -CH 2 CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-( CH 2 NHC(O)) a -;
  • L G2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L G3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L G4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • A is a chemical bond, -CH 2 O- or -NHC(O)-;
  • A' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • B is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
  • R G1 and R G2 together form -CH 2 CH 2 O- or -CH 2 CH(R G )-O-, and R G3 is H;
  • R G1 and R G3 together form -C 1-2 alkylene-, and R G2 is H;
  • RG is -OR G ', -CH 2 OR G ' or -CH 2 CH 2 OR G ', wherein RG ' is H, a hydroxyl protecting group or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O )CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n1 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • GalNAc is a conjugated group of formula (I'):
  • Q G is independently H
  • L G1 is a chemical bond, -CH 2 -, -CH 2 CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-( CH 2 NHC(O)) a -;
  • L G2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L G3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L G4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • A is -CH 2 O- or -NHC(O)-;
  • A’ is a chemical bond, -C(O)NH- or -NHC(O)-;
  • R G1 and R G2 together form -CH 2 CH 2 O- or -CH 2 CH(R G )-O-, and R G3 is H;
  • R G1 and R G3 together form -C 1-2 alkylene-, and R G2 is H;
  • RG is -OR G ', -CH 2 OR G ' or -CH 2 CH 2 OR G ', wherein RG ' is H, a hydroxyl protecting group or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O )CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n1 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • GalNAc is a conjugation group of formula (X), wherein,
  • Q G is independently H
  • L G1 is a chemical bond, -CH 2 -, -CH 2 CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-( CH 2 NHC(O)) a -;
  • L G2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L G3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L G4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • A is a chemical bond, -CH 2 O- or -NHC(O)-;
  • A' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • B is a chemical bond, -CH 2 -, -M-, -CH 2 -M- or -C(O)-M-;
  • R G1 and R G2 together form -CH 2 CH 2 O- or -CH 2 CH(R G )-O-, and R G3 is H;
  • R G1 and R G3 together form -C 1-2 alkylene-, and R G2 is H;
  • RG is -OR G ', -CH 2 OR G ' or -CH 2 CH 2 OR G ', wherein RG ' is H, a hydroxyl protecting group or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O )CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n1 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • GalNAc is a conjugation group of formula (X), wherein:
  • Q G is independently H
  • L G1 is a chemical bond, -CH 2 -, -CH 2 CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-( CH 2 NHC(O)) a -;
  • L G2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L G3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L G4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • A is a chemical bond, -CH 2 O- or -NHC(O)-;
  • A' is -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • B is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
  • R G1 and R G2 together form -CH 2 CH 2 O- or -CH 2 CH(R G )-O-, and R G3 is H;
  • R G1 and R G3 together form -C 1-2 alkylene-, and R G2 is H;
  • RG is -OR G ', -CH 2 OR G ' or -CH 2 CH 2 OR G ', wherein RG ' is H, a hydroxyl protecting group or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O )CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n1 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • any technical solution or any combination thereof in any of the above specific embodiments may be combined with any technical solution or any combination thereof in other specific embodiments.
  • any technical solution of X or any combination thereof can be combined with Y, Z, L1 , L2 , L3 , R1 , R2 , R3 , R4 , R5 , R6 , R7 , U , Q, P 1 , P 2 , Base and Base', etc., or any combination thereof.
  • the present invention is intended to include combinations of all these technical solutions, and due to space limitations, they will not be listed one by one.
  • the invention also provides a vector comprising a nucleotide sequence encoding the siRNA of the invention.
  • the vector of the present invention can amplify or express the nucleotide encoding the siRNA of the present invention connected thereto.
  • siRNA targeting the PCSK9 gene can be expressed from a transcription unit inserted into a DNA or RNA vector. Expression can be transient (within hours to weeks) or sustained (weeks to months or longer), depending on the specific construct used and the target tissue or cell type.
  • the siRNA encoding nucleotides can be introduced into linear constructs, circular plasmids, or viral vectors.
  • the siRNA nucleotides can be integrated into the cell genome for stable expression, or can be stably inherited and expressed extrachromosomally.
  • siRNA expression vectors are usually DNA plasmids or viral vectors.
  • Viral vector systems containing siRNA coding sequences include, but are not limited to: (a) adenovirus vectors; (b) retroviral vectors; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vector; (f) polyomavirus vector; (g) papillomavirus vector; (h) picornavirus vector; (i) poxvirus vector; and (j) helper virus-dependent adenovirus or gutless adenovirus.
  • the invention also provides cells containing the siRNA or vector of the invention, wherein the siRNA or vector of the invention is capable of being transcribed in the cell.
  • the invention relates to an oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, the 5' end of the oligonucleotide comprising: structure:
  • oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, the 5' end of the oligonucleotide comprising the structure represented by formula (I):
  • oligonucleotide means linked to the remainder of the oligonucleotide, preferably to a modified or unmodified nucleoside;
  • X and Y are each independently selected from CR a R b or (CR a R b ) 2 , preferably CR a R b ;
  • Z is CR a ;
  • R a and R b are independently selected from H, D, halogen, OH, NH 2 , CN, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy group, C 2-6 alkenyl or C 2-6 alkynyl; the R a and R b are optionally further substituted by 1, 2, 3, 4 or 5 independently selected R#;
  • R 1 is selected from O or S
  • R 2 and R 3 are independently selected from OH, SH, NH 2 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy; the R 2 and R3 optionally further substituted by 1, 2, 3, 4 or 5 independently selected R#;
  • R# is selected from H, D, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl;
  • Each of the above defined groups is optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms until completely deuterated.
  • oligonucleotide means linked to the remainder of the oligonucleotide, preferably to a modified or unmodified nucleoside;
  • X and Y are each independently selected from CR a R b or (CR a R b ) 2 , preferably CR a R b ;
  • Z is CR a ;
  • R a and R b are independently selected from H, D, halogen, OH, NH 2 , CN, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy base;
  • R 1 is selected from O or S
  • R 2 and R 3 are independently selected from OH, SH, C 1-6 alkoxy or C 1-6 haloalkoxy; the R 2 and R 3 are optionally selected from 1, 2 or 3 independently R# is further replaced;
  • R# is selected from H, D, halogen, C 1-6 alkyl and C 1-6 haloalkyl.
  • oligonucleotide means linked to the remainder of the oligonucleotide, preferably to a modified or unmodified nucleoside;
  • X and Y are each independently selected from CR a R b or (CR a R b ) 2 , preferably CR a R b ;
  • Z is CR a ;
  • R a and R b are independently selected from H, D, halogen, C 1-4 alkyl or C 1-4 haloalkyl;
  • R 1 is selected from O or S
  • R 2 and R 3 are independently selected from OH or SH, preferably OH.
  • oligonucleotide represents attachment to the 5' end group of the oligonucleotide, preferably to a modified or unmodified nucleoside;
  • X and Y are CH 2 ;
  • R 1 is O
  • R 2 and R 3 are OH.
  • X and Y are CR a R b ;
  • Z is CR a ;
  • L 1 is selected from chemical bond, O, S or C 1-4 alkylene
  • L 2 and L 3 are independently selected from C 1-4 alkylene
  • L 1 , L 2 and L 3 are optionally independently replaced by 1, 2, 3, 4 or 5 independently selected R#;
  • U is selected from O, S, CR a R b or NR c ;
  • R a and R b are independently selected from H, D, halogen, OH, NH 2 , CN, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy base, C 2-6 alkenyl or C 2-6 alkynyl;
  • R c is independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl;
  • R a , R b and R c are optionally further substituted by 1, 2, 3, 4 or 5 independently selected R#;
  • L 2 and L 3 are connected to form a C 5-6 cycloalkyl or 5-6 membered heterocyclyl group together with L 1 and its adjacent carbon atoms, preferably U, L 1 , L 2 and L 3 and their Adjacent carbon atoms together form ribose or deoxyribose, which are optionally substituted by R;
  • R 1 is selected from O or S
  • R 2 and R 3 are independently selected from OH, SH, NH 2 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy; the R 2 and R3 optionally substituted by 1, 2, 3, 4 or 5 independently selected R#;
  • R 5 is selected from H, D, halogen, C 1-6 alkoxy or C 1-6 haloalkoxy;
  • R# is selected from H, D, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl;
  • Base is selected from H, modified or unmodified bases
  • Each of the above defined groups is optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms until completely deuterated.
  • X and Y are CR a R b ;
  • Z is CR a ;
  • R a and R b are independently selected from H, D, halogen, OH, NH 2 , CN, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy base;
  • L 1 is selected from chemical bond, O or C 1-4 alkylene
  • L 2 and L 3 are independently selected from C 1-4 alkylene
  • L 1 , L 2 and L 3 are optionally independently replaced by 1, 2 or 3 independently selected R#;
  • U is selected from O, S, NH or CH 2 ;
  • L 2 and L 3 are connected to form a C 5-6 cycloalkyl or 5-6 membered heterocyclyl group together with L 1 and its adjacent carbon atoms, preferably U, L 1 , L 2 and L 3 and their Adjacent carbon atoms together form ribose or deoxyribose, which are optionally substituted by R;
  • R 1 is selected from O or S
  • R 2 and R 3 are independently selected from OH, SH, C 1-6 alkoxy or C 1-6 haloalkoxy; the R 2 and R 3 are optionally selected from 1, 2 or 3 independently Replaced by R#;
  • R 5 is selected from H, D, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy;
  • R# is selected from H, D, halogen, C 1-6 alkyl and C 1-6 haloalkyl;
  • Base is selected from H, modified or unmodified bases.
  • X and Y are CR a R b ;
  • Z is CR a ;
  • R a and R b are independently selected from H, D, halogen, C 1-4 alkyl or C 1-4 haloalkyl;
  • L 1 is selected from chemical bond or C 1-4 alkylene
  • L 2 and L 3 are independently selected from C 1-4 alkylene
  • U is selected from O or S
  • R 1 is selected from O or S
  • R 2 and R 3 are independently selected from OH or SH, preferably OH;
  • X and Y are CH 2 ;
  • L 1 is a chemical bond
  • L 2 and L 3 are independently selected from C 1-2 alkylene
  • R 1 is O
  • R 2 and R 3 are OH
  • the oligonucleotide of technical solution 1 or its pharmaceutically acceptable salt, tautomer or stereoisomer, the 5' end of the oligonucleotide contains formula (III), formula (IV) Or the nucleotide monomer represented by formula (V):
  • Q is selected from H, D, halogen, OH, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 2-6 alkenyl or C 2 -6 alkynyl;
  • R 4 , R 5 , R 6 and R 7 are independently selected from H, D, halogen, OH, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkyl Oxygen group, C 2-6 alkenyl group or C 2-6 alkynyl group;
  • Base is selected from H, modified or unmodified bases
  • L 1 , L 3 , X, Y, Z, R 1 , R 2 , R 3 and U are as defined in any one of technical solutions 5-8;
  • Each of the above defined groups is optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms until completely deuterated.
  • Q is selected from H, D, halogen, OH, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy;
  • R 4 , R 5 , R 6 and R 7 are independently selected from H, D, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl;
  • Base is selected from H, modified or unmodified bases, preferably
  • L 1 , L 3 , X, Y, Z, R 1 , R 2 , R 3 and U are as defined in any one of technical solutions 5-8.
  • Q is selected from H, halogen or C 1-4 alkoxy, preferably H, F or methoxy;
  • R 4 , R 5 , R 6 and R 7 are independently selected from H, halogen, C 1-4 alkyl or C 1-4 haloalkyl, preferably H;
  • Base is selected from Preferably
  • L 1 , L 3 , X, Y, Z, R 1 , R 2 , R 3 and U are as defined in any one of technical solutions 5-8.
  • oligonucleotide of any one of technical solutions 9-11 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5' end of the oligonucleotide contains Nucleotide monomers of the following structure:
  • each group is as defined in any one of technical solutions 9-11.
  • oligonucleotide of any one of technical solutions 9-12 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5' end of the oligonucleotide contains Nucleotide monomers of the following structure:
  • Base is selected from
  • R 1 is selected from O or S
  • R 2 and R 3 are independently selected from OR d , OP 1 , SR d , SP 1 or NR e R f ;
  • R d is selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally replaced by D, halogen, C 1-6 alkyl or C 1-6 haloalkyl substituted until completely deuterated;
  • R e and R f are independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl, and the R e and R f may be optionally replaced by D, halogen, C 1-6 alkyl or C 1 -6 haloalkyl substitution until completely deuterated;
  • P 1 is selected from protective groups, preferably hydroxyl protective groups
  • P 2 is selected from reactive phosphorus groups, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 );
  • Base' is independently selected from H, modified or unmodified bases or leaving groups
  • L 1 , L 3 , X, Y, Z, U, Q, R 4 , R 5 , R 6 and R 7 are as defined in any one of technical solutions 5-11;
  • Each of the above defined groups is optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms until completely deuterated.
  • R 1 is selected from O or S
  • R 2 and R 3 are independently selected from OR d , OP 1 , SR d , SP 1 or NR e R f ;
  • R d is selected from H, C 1-4 alkyl or C 1-4 haloalkyl, which is optionally substituted by D or halogen until completely deuterated;
  • R e and R f are independently selected from H, C 1-4 alkyl or C 1-4 haloalkyl, and the R e and R f may be optionally substituted by D or halogen until completely deuterated;
  • P 1 is selected from protective groups, preferably hydroxyl protective groups
  • P 2 is selected from reactive phosphorus groups, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 );
  • Base' is selected from
  • L 1 , L 3 , X, Y, Z, U, Q, R 4 , R 5 , R 6 and R 7 are as defined in any one of technical solutions 5-11.
  • R 1 is selected from O or S, preferably O;
  • R 2 and R 3 are independently selected from OR d or OP 1 ;
  • R d is selected from H, C 1-4 alkyl or C 1-4 haloalkyl
  • R e and R f are independently selected from H, C 1-4 alkyl or C 1-4 haloalkyl;
  • P 1 is selected from protective groups, preferably hydroxyl protective groups
  • P 2 is selected from reactive phosphorus groups, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 );
  • Base' is selected from More preferably
  • L 1 , L 3 , X, Y, Z, U, Q, R 4 , R 5 , R 6 and R 7 are as defined in any one of technical solutions 5-11.
  • oligonucleotide according to any one of technical solutions 1-13, wherein the oligonucleotide is a single strand having 14 to 30 nucleotides.
  • oligonucleotide of any one of technical solutions 1-13 wherein the oligonucleotide is a double-stranded RNA, which includes a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides.
  • the antisense strand has a sequence that is fully complementary to the sense strand and target mRNA.
  • RNA includes the formula (I), (II), (III), ( The structure of IV), (V), (wyin'c), (IIIb), (IVa), (IVb), (Va) or (Vb) or the nucleotide monomer described in technical scheme 13.
  • the ligand includes one or more GalNAc.
  • a nucleic acid molecule, the nucleotide sequence of the nucleic acid molecule contains one or more nucleotide monomers as described in any one of technical solutions 9-13.
  • the nucleic acid molecule of technical solution 23 which is single-stranded or double-stranded.
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • Vector which contains a nucleotide sequence encoding the double-stranded RNA described in any one of the preceding technical solutions 19-21.
  • a pharmaceutical composition comprising the double-stranded RNA molecule as described in any one of technical solutions 19-21, and a pharmaceutically acceptable carrier or excipient.
  • kits comprising the double-stranded RNA molecule as described in any one of technical solutions 19-21.
  • a method for inhibiting the expression of a target gene in a cell comprising the step of introducing the double-stranded RNA molecule according to any one of technical solutions 19-21 into the cell.
  • formula (IIIa), formula (IVa) and formula (Va) can make the oligonucleotide of the invention (especially the phosphonate group at its 5' end) better It effectively binds to the Ago2 protein, thereby increasing the activity of the oligonucleotide.
  • Dissolve compound 1b (310g, 1.52mol, 1.00eq), imidazole (206g, 3.02mol, 2.00eq), triphenylphosphine (476g, 1.82mol, 1.20eq) in toluene (2.1L), add iodine in batches at room temperature Elemental substance (446g, 1.76mmol, 1.16eq). After the addition, the temperature was raised to 70°C and stirred for 1.5 hours. TLC showed the reaction was complete.
  • Disperse zinc-copper reagent (90.0g, 1.37mol, 5.50eq) into diethyl ether (200mL), add compound 1d (60g, 241mmol, 1.00eq) at 25°C, and add trichloroacetyl chloride (61.5g, 338mmol, 1.00eq) dropwise. 1.40 eq) in diethyl ether (200 mL) and stirred for 1 hour.
  • reaction solution was poured into saturated sodium bicarbonate aqueous solution (1.50L), extracted with dichloromethane (700mL) three times, the organic phases were combined, washed with saturated brine (500mL), dried over anhydrous sodium sulfate, filtered, concentrate.
  • the crude product was purified by silica gel column chromatography (eluent: dichloromethane/methanol) to obtain compound 1n (14.4 g, 84.3%).
  • the crude compound 1q (10.9g, 20.7mmol, 1.00eq) was purified by C 18 reverse-phase column chromatography (ammonium bicarbonate aqueous solution/acetonitrile) to obtain compound 1q-1 (1.50g, 11.5%) and compound 1q-2 (1.50g, 11.5%).
  • HPLC retention time 3.065min (column: Chiralpak AD-3, 150 ⁇ 4.6mm ID, 3um; mobile phase: A: CO 2 , B: IPA (0.2% NH 3 (7M methanol solution), v/v); gradient: 0 -0.5min 10%B, 0.5-3.5min 10-50%B, 3.5-4.5min 50%B, 4.5-5.0min, 50%-10%B; flow rate: 2.5mL/min; column temperature: 35°C)
  • HPLC retention time 3.626min (Column: Chiralpak AD-3, 150 ⁇ 4.6mm ID, 3um; mobile phase: A: CO 2 , B: IPA (0.2% NH 3 (7M methanol solution), v/v); gradient: 0 -0.5min 10%B, 0.5-3.5min 10-50%B, 3.5-4.5min 50%B, 4.5-5.0min, 50%-10%B; flow rate: 2.5mL/min; column temperature: 35°C)
  • siRNA of the invention is prepared using the solid-phase phosphoramidite method, which is well known in the art. Specific methods can be found, for example, in PCT publication numbers WO2016081444 and WO2019105419, and are briefly described below.
  • a blank CPG solid-phase carrier or a solid-phase carrier connected with L96 is used as the starting cycle, and the nucleosides are connected one by one from the 3'-5' direction in the order of the sense strand nucleotides. monomer.
  • Each connected nucleoside monomer includes four-step reactions of deprotection, coupling, capping, oxidation or sulfation.
  • the synthesis conditions for an oligonucleotide with a synthesis scale of 5umol are as follows:
  • the nucleoside monomer is provided with a 0.05 mol/L acetonitrile solution.
  • the reaction conditions for each step are the same, that is, the temperature is 25°C.
  • the temperature is 25°C.
  • the capping agent used 10% acetic anhydride-acetonitrile and pyridine/N-methylimidazole/acetonitrile (10:14:76, v/v/ v), capping 2 times; oxidation using 0.05mol/L iodine/tetrahydrofuran/pyridine/water (70/20/10, v/v/v), oxidation 2 times; sulfide using 0.2mol/L PADS acetonitrile/ 3-methylpyridine (1/1, v/v), sulfide substituted 2 times.
  • a blank CPG solid-phase carrier is used as the initial cycle, and the nucleoside monomers or the core of the present invention are connected one by one from the 3'-5' direction according to the sequence of the antisense strand nucleotides. glycoside dimer.
  • Each connection of a nucleoside monomer or nucleotide dimer of the present invention includes a four-step reaction of deprotection, coupling, capping, oxidation or sulfation, oligonucleotide synthesis conditions of 5 ⁇ mol of the antisense strand and oligonucleotide synthesis conditions of the sense strand. same.
  • a strong anion packing column can be used, a sodium chloride-sodium hydroxide system can be used for elution and purification, and the products can be collected and tubed.
  • a gel packing purification column can be used for desalting, and the elution system is pure water.
  • siRNA sequences used in the present invention are as follows:
  • the A, U, G, and C distributions represent natural adenine ribonucleotides, uracil ribonucleotides, guanine ribonucleotides, and cytosine ribonucleotides.
  • m indicates that the adjacent nucleotide to its left is a 2'-OCH 3 modified nucleotide.
  • Am, Um, Gm and Cm represent 2'-OCH 3 modified A, U, G and C.
  • f indicates that the adjacent nucleotide to its left is a 2'-F modified nucleotide.
  • Af, Uf, Gf and Cf represent 2′-F modified A, U, G and C respectively.
  • s or "s-" means that two adjacent nucleotides and/or delivery vectors are connected through phosphorothioate.
  • L96 represents a GalNAc delivery vector of the following structure, which is well known in the art, wherein Indicates the position of attachment to siRNA via a phosphate group or a phosphorothioate group, see for example PCT Publication Nos. WO2009073809 and WO2009082607.
  • VP indicates that the nucleotide adjacent to the right side is a vinylphosphonate-modified nucleotide, which is well known in the art, see, for example, PCT Publication Nos. WO2011139702, WO2013033230 and WO2019105419.
  • SCP1a, SCP1b, SCP2a and SCP2b represent nucleotide substitutions of the structure described above, where Base can be any base, for example, SCP1a-U represents Base as uracil.
  • C57BL/6 wild-type mouse liver primary cells were isolated, counted, and plated on a 24-well plate at 900 ⁇ L/well, 8 ⁇ 10 4 cells/well; and on a 96-well plate, 100 ⁇ L/well, 1 ⁇ 10 4 cells/well. Then choose ad libitum uptake or transfection.
  • Free uptake Add 10 ⁇ L of diluted compound to 90 ⁇ L of Opti-MEM, mix well, add to the corresponding well, and incubate in a 37°C, 5% CO2 incubator for 24 hours. No siRNA was added to the control group.
  • ⁇ Ct [(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)].
  • the target gene is mAPOB or mFXII, and the control gene is mGAPDH.
  • C57BL/6 wild-type mouse primary hepatocytes were selected. After plating the cells on a 24-well plate, the starting concentration of the compound was selected to be 40nM, and diluted 10 times into 5 concentration points (40nM, 4nM, 0.4nM, 0.04nM). , 0.004nM) for 5-point IC50 activity screening of C57BL/6 wild-type mouse primary liver cells. The experimental results are shown in Table 2.
  • C57BL/6 wild-type mouse primary hepatocytes were selected. After the cells were plated on a 96-well plate, the starting concentration of the compound was selected as 100nM, and diluted 10 times into 5 concentration points (100nM, 10nM, 1nM, 0.1nM, 0.01nM) 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 3.
  • mice Male, 18-21g, 6-8 weeks, Speford (Suzhou) Biotechnology Co., Ltd.
  • the dosage was calculated according to the body weight of each animal and administered as a single subcutaneous injection.
  • the siRNA conjugate was administered in a 10 mg/mL solution (0.9% sodium chloride aqueous solution as the solvent). ) provided to the CRO company; specifically, before the experiment, the siRNA conjugate was dissolved with 0.9% sodium chloride aqueous solution and diluted to the required solution concentration and volume.
  • the administration volume of physiological saline and siRNA conjugate was 5 mL. /kg.
  • Blood tests were taken before administration (recorded as day -2), and grouped according to LDL on day 2, and the remaining samples were retained for detection of target proteins; and on days 7 and 14 after administration (recorded as day 0)
  • day 21 and 21 blood was collected from the orbital venous plexus of the mice (starved for 5 hours before each blood collection), and serum LDL concentrations were detected by Suzhou Anling using the direct method (Neusoft fully automatic biochemical analyzer, NT-1000) at each time point.
  • the compounds of the present invention can lock the phosphonic acid in an ap conformation that is highly similar to unmodified 5'-phosphate-bound Ago2, in which the phosphonate group is perfectly aligned with Arg812, Lys570, Lys566 and Lys533 Matching, multiple salt bridge bonds and hydrogen bonds are formed, and one hydrogen bond is also formed with Tyr529 and Cys526.
  • the base (uracil) in the spirocyclic phosphonic acid compound forms one hydrogen bond each with the backbone amide of Thr526 and Gly524. The combination of these structures stabilizes the binding of the compound of the invention to the Ago2 protein.

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Abstract

本发明提供具有环膦酸酯修饰的寡核苷酸。本发明的寡核苷酸显示增强的稳定性、降低的脱靶毒性和增强的有效性中的一种或多种。

Description

环膦酸酯修饰的核苷酸 发明领域
本发明属于医药领域，具体涉及具有环膦酸酯结构的双链RNA。
背景技术
RNA干扰是一种由双链RNA(double-stranded RNA，dsRNA，也称siRNA)诱发的靶标mRNA高效特异性降解的现象。
siRNA中引导链(反义链)的5'-磷酸盐通过与Ago2蛋白MID及PIWI结构域界面附近的阳离子氨基酸残基形成静电相互作用，可稳定引导链和Ago2形成的复合物，因此，siRNA引导链的5'-磷酸盐对于基于RNAi的基因沉默至关重要。然而，5'-磷酸盐可被内源性磷酸酶切割，从而失去5'末端磷酸基团，引起siRNA活性的显著降低。
通过在小干扰RNA(siRNA)的末端，特别是反义链的5’端引入膦酸酯修饰的核苷酸，可以显著提高siRNA的体内活性。例如，WO2011139702A2和WO2013033230A1公开了一种包含5’-乙烯基膦酸酯(简称E-VP或VP)的核苷酸，5’-E-VP可以替代5'-磷酸与Ago2结合，并且对内源性磷酸酶具有高度抗性，从而了siRNA的活性和/或稳定性。
WO2017214112A1公开了一种包含5’-环-膦酸酯的核苷酸。
然而，发明人发现，5'-E-VP与糖环通过C-C单键连接，该C-C单键仍可自由旋转，从而导致膦酸基团相对于糖环可处于不同位置(见下图，其中ap位置即未修饰的5’-磷酸盐与Ago2蛋白结合的构象)。
其中的相当部分构象(例如+sc和-sc)可能不利于与Ago2阳离子区域结合。因此，本领域需要开发可以锁定Ago2结合构象的膦酸酯修饰。
发明内容
发明人出乎意料地发现，通过刚性的环结构限制末端膦酸转动，将其锁定在Ago2结合构象，有利于进一步提高siRNA活性。
在一个方面中，本发明涉及一种寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，所述寡核苷酸的5’末端包含式(I)所示的结构：
其中，
表示连接至所述寡核苷酸的剩余部分，优选连接至修饰或未修饰的核苷；
X和Y各自独立地选自CRaRb或(CRaRb)2，优选为CRaRb；
Z为CRa；
Ra和Rb独立地选自H、D、卤素、OH、NH2、CN、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C2-6烯基或C2-6炔基；所述Ra和Rb任选地被1个、2个、3个、4个或5个独立选择的R#进一步取代；
R1选自O或S；
R2和R3独立地选自OH、SH、NH2、C1-6烷基、C1-6卤代烷基、C1-6烷氧基或C1-6卤代烷氧基；所述R2和R3任选地被1个、2个、3个、4个或5个独立选择的R#进一步取代；
R#选自H、D、卤素、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基；
其中上述各基团定义任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
不受具体理论的约束，本发明的结构中包含四元碳环，这一相对刚性结构能够使得本发明的寡核苷酸(特别是其5’末端的膦酸酯基团)更好地与Ago2蛋白结合，从而提高寡核苷酸的活性。
通过分子模拟，我们观察到本发明的化合物可将膦酸锁定在与未修饰的5’-磷酸盐结合Ago2高度相似的ap构象，其中的膦酸盐基团与Arg812，Lys570，Lys566及Lys533完美匹配，形成了多个盐桥键和氢键，同时还与Tyr529及Cys526各形成了一个氢键。此外，该螺环膦酸化合物中的碱基(尿嘧啶)与Thr526和Gly524的骨架酰胺各形成一个氢键。这些结构的共同左右稳定了本发明的化合物与Ago2蛋白的结合。
分子模拟显示，与螺环化合物类似，糖环稠合高度刚性的芳香环(包括五元芳香环和六元芳香环)后，将5’末端膦酸盐锁定在了特定的构象上，该构象高度模拟了天然5’-磷酸盐的ap构象，膦酸盐基团与Arg812，Lys570，Lys566及Lys533完美匹配，形成了多个盐桥键和氢键，同样还与Tyr529及Cys526各形成了一个氢键。糖环上碱基尿嘧啶同样与Thr526和Gly524的骨架酰胺各形成一个氢键。这些结构的共同左右稳定了本发明的化合物与Ago2蛋白的结合。
发明详述
定义
化学定义
下面更详细地描述具体官能团和化学术语的定义。
当列出数值范围时，既定包括每个值和在所述范围内的子范围。例如“C1-6烷基”包括C1、C2、C3、C4、C5、C6、C1-6、C1-5、C1-4、C1-3、C1-2、C2-6、C2-5、C2-4、C2-3、C3-6、C3-5、C3-4、C4-6、C4-5和C5-6烷基。
“C1-6烷基”是指具有1至6个碳原子的直链或支链饱和烃基团。在一些实施方案中，C1-4烷基和C1-2烷基是优选的。C1-6烷基的例子包括：甲基(C1)、乙基(C2)、正丙基(C3)、异丙基(C3)、正丁基(C4)、叔丁基(C4)、仲丁基(C4)、异丁基(C4)、正戊基(C5)、3-戊基(C5)、戊基(C5)、新戊基(C5)、3-甲基-2-丁 基(C5)、叔戊基(C5)和正己基(C6)。术语“C1-6烷基”还包括杂烷基，其中一或多个(例如，1、2、3或4个)碳原子被杂原子(例如，氧、硫、氮、硼、硅、磷)替代。烷基基团可以被一或多个取代基任选取代，例如，被1至5个取代基、1至3个取代基或1个取代基取代。常规烷基缩写包括：Me(-CH3)、Et(-CH2CH3)、iPr(-CH(CH3)2)、nPr(-CH2CH2CH3)、n-Bu(-CH2CH2CH2CH3)或i-Bu(-CH2CH(CH3)2)。
“C2-6烯基”是指具有2至6个碳原子和至少一个碳碳双键的直链或支链烃基团。在一些实施方案中，C2-4烯基是优选的。C2-6烯基的例子包括：乙烯基(C2)、1-丙烯基(C3)、2-丙烯基(C3)、1-丁烯基(C4)、2-丁烯基(C4)、丁二烯基(C4)、戊烯基(C5)、戊二烯基(C5)、己烯基(C6)，等等。术语“C2-6烯基”还包括杂烯基，其中一或多个(例如，1、2、3或4个)碳原子被杂原子(例如，氧、硫、氮、硼、硅、磷)替代。烯基基团可以被一或多个取代基任选取代，例如，被1至5个取代基、1至3个取代基或1个取代基取代。
“C2-6炔基”是指具有2至6个碳原子、至少一个碳-碳叁键以及任选地一个或多个碳-碳双键的直链或支链烃基团。在一些实施方案中，C2-4炔基是优选的。C2-6炔基的例子包括但不限于：乙炔基(C2)、1-丙炔基(C3)、2-丙炔基(C3)、1-丁炔基(C4)、2-丁炔基(C4)，戊炔基(C5)、己炔基(C6)，等等。术语“C2-6炔基”还包括杂炔基，其中一或多个(例如，1、2、3或4个)碳原子被杂原子(例如，氧、硫、氮、硼、硅、磷)替代。炔基基团可以被一或多个取代基任选取代，例如，被1至5个取代基、1至3个取代基或1个取代基取代。
“卤代”或“卤素”是指氟(F)、氯(Cl)、溴(Br)和碘(I)。
因此，“C1-6卤代烷基”是指上述“C1-6烷基”，其被一个或多个卤素基团取代。在一些实施方案中，C1-4卤代烷基是特别优选的，更优选C1-2卤代烷基。示例性的所述卤代烷基包括但不限于：-CF3、-CH2F、-CHF2、-CHFCH2F、-CH2CHF2、-CF2CF3、-CCl3、-CH2Cl、-CHCl2、2,2,2-三氟-1,1-二甲基-乙基，等等。卤代烷基基团可以在任何可用的连接点上被取代，例如，1至5个取代基、1至3个取代基或1个取代基。
“C1-6烷氧基”是指-O-R基团，其中R如上文的“C1-6烷基”所定义。
“C1-6卤代烷氧基”是指-O-R基团，其中R如上文的“C1-6卤代烷基”所定义。
“C5-6环烷基”是指具有5至6个环碳原子和零个杂原子的非芳香环烃基团。环烷基还包括其中上述环烷基环与一个或多个芳基或杂芳基稠合的环体系，其中连接点在环烷基环上，且在这样的情况中，碳的数目继续表示环烷基体系中的碳的数目。示例性的所述环烷基包括但不限于：环戊基(C5)、环戊烯基(C5)、环己基(C6)、环己烯基(C6)、环已二烯基(C6)，等等。环烷基基团可以被一或多个取代基任选取代，例如，被1至5个取代基、1至3个取代基或1个取代基取代。
“5-6元杂环基”是指具有环碳原子和1至3个环杂原子的5至6元非芳香环系的基团，其中，每个杂原子独立地选自氮、氧、硫、硼、磷和硅。在包含一个或多个氮原子的杂环基中，只要化合价允许，连接点可为碳或氮原子。杂环基还包括其中上述杂环基环与一个或多个环烷基稠合的环体系，其中连接点在环烷基环上，或其中上述杂环基环与一个或多个芳基或杂芳基稠合的环体系，其中连接点在杂环基环上；且在这样的情况下，环成员的数目继续表示在杂环基环体系中环成员的数目。示例性的含有一个杂原子的5元杂环基包括但不限于：四氢呋喃基、二氢呋喃基、四氢噻吩基、二氢噻吩基、 吡咯烷基、二氢吡咯基和吡咯基-2,5-二酮。示例性的包含两个杂原子的5元杂环基包括但不限于：二氧杂环戊烷基、氧硫杂环戊烷基(oxasulfuranyl)、二硫杂环戊烷基(disulfuranyl)和噁唑烷-2-酮。示例性的包含三个杂原子的5元杂环基包括但不限于：三唑啉基、噁二唑啉基和噻二唑啉基。示例性的包含一个杂原子的6元杂环基包括但不限于：哌啶基、四氢吡喃基、二氢吡啶基和硫杂环己烷基(thianyl)。示例性的包含两个杂原子的6元杂环基包括但不限于：哌嗪基、吗啉基、二硫杂环己烷基、二噁烷基。示例性的包含三个杂原子的6元杂环基包括但不限于：六氢三嗪基(triazinanyl)。杂环基基团可以被一或多个取代基任选取代，例如，被1至5个取代基、1至3个取代基或1个取代基取代。
本文定义的烷基、烯基、炔基、环烷基和杂环基等为任选取代的基团。
示例性的碳原子上的取代基包括但不局限于：卤素、-CN、-NO2、-N3、-SO2H、-SO3H、-OH、-ORaa、-ON(Rbb)2、-N(Rbb)2、-N(Rbb)3 +X-、-N(ORcc)Rbb、-SH、-SRaa、-SSRcc、-C(＝O)Raa、-CO2H、-CHO、-C(ORcc)2、-CO2Raa、-OC(＝O)Raa、-OCO2Raa、-C(＝O)N(Rbb)2、-OC(＝O)N(Rbb)2、-NRbbC(＝O)Raa、-NRbbCO2Raa、-NRbbC(＝O)N(Rbb)2、-C(＝NRbb)Raa、-C(＝NRbb)ORaa、-OC(＝NRbb)Raa、-OC(＝NRbb)ORaa、-C(＝NRbb)N(Rbb)2、-OC(＝NRbb)N(Rbb)2、-NRbbC(＝NRbb)N(Rbb)2、-C(＝O)NRbbSO2Raa、-NRbbSO2Raa、-SO2N(Rbb)2、-SO2Raa、-SO2ORaa、-OSO2Raa、-S(＝O)Raa、-OS(＝O)Raa、-Si(Raa)3、-OSi(Raa)3、-C(＝S)N(Rbb)2、-C(＝O)SRaa、-C(＝S)SRaa、-SC(＝S)SRaa、-SC(＝O)SRaa、-OC(＝O)SRaa、-SC(＝O)ORaa、-SC(＝O)Raa、-P(＝O)2Raa、-OP(＝O)2Raa、-P(＝O)(Raa)2、-OP(＝O)(Raa)2、-OP(＝O)(ORcc)2、-P(＝O)2N(Rbb)2、-OP(＝O)2N(Rbb)2、-P(＝O)(NRbb)2、-OP(＝O)(NRbb)2、-NRbbP(＝O)(ORcc)2、-NRbbP(＝O)(NRbb)2、-P(Rcc)2、-P(Rcc)3、-OP(Rcc)2、-OP(Rcc)3、-B(Raa)2、-B(ORcc)2、-BRaa(ORcc)、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代；
或者在碳原子上的两个偕氢被基团＝O、＝S、＝NN(Rbb)2、＝NNRbbC(＝O)Raa、＝NNRbbC(＝O)ORaa、＝NNRbbS(＝O)2Raa、＝NRbb或＝NORcc取代；
Raa的每个独立地选自烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基，或者两个Raa基团结合以形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代；
Rbb的每个独立地选自：氢、-OH、-ORaa、-N(Rcc)2、-CN、-C(＝O)Raa、-C(＝O)N(Rcc)2、-CO2Raa、-SO2Raa、-C(＝NRcc)ORaa、-C(＝NRcc)N(Rcc)2、-SO2N(Rcc)2、-SO2Rcc、-SO2ORcc、-SORaa、-C(＝S)N(Rcc)2、-C(＝O)SRcc、-C(＝S)SRcc、-P(＝O)2Raa、-P(＝O)(Raa)2、-P(＝O)2N(Rcc)2、-P(＝O)(NRcc)2、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基，或者两个Rbb基团结合以形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代；
Rcc的每个独立地选自氢、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基，或者两个Rcc基团结合以形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代；
Rdd的每个独立地选自：卤素、-CN、-NO2、-N3、-SO2H、-SO3H、-OH、-ORee、-ON(Rff)2、-N(Rff)2,、 -N(Rff)3 +X-、-N(ORee)Rff、-SH、-SRee、-SSRee、-C(＝O)Ree、-CO2H、-CO2Ree、-OC(＝O)Ree、-OCO2Ree、-C(＝O)N(Rff)2、-OC(＝O)N(Rff)2、-NRffC(＝O)Ree、-NRffCO2Ree、-NRffC(＝O)N(Rff)2、-C(＝NRff)ORee、-OC(＝NRff)Ree、-OC(＝NRff)ORee、-C(＝NRff)N(Rff)2、-OC(＝NRff)N(Rff)2、-NRffC(＝NRff)N(Rff)2、-NRffSO2Ree、-SO2N(Rff)2、-SO2Ree、-SO2ORee、-OSO2Ree、-S(＝O)Ree、-Si(Ree)3、-OSi(Ree)3、-C(＝S)N(Rff)2、-C(＝O)SRee、-C(＝S)SRee、-SC(＝S)SRee、-P(＝O)2Ree、-P(＝O)(Ree)2、-OP(＝O)(Ree)2、-OP(＝O)(ORee)2、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基、杂芳基，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rgg基团取代，或者两个偕Rdd取代基可结合以形成＝O或＝S；
Ree的每个独立地选自烷基、卤代烷基、烯基、炔基、环烷基、芳基、杂环基和杂芳基，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rgg基团取代；
Rff的每个独立地选自氢、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基，或者两个Rff基团结合形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rgg基团取代；
Rgg的每个独立地是：卤素、-CN、-NO2、-N3、-SO2H、-SO3H、-OH、-OC1-6烷基、-ON(C1-6烷基)2、-N(C1-6烷基)2、-N(C1-6烷基)3 +X-、-NH(C1-6烷基)2 +X-、-NH2(C1-6烷基)+X-、-NH3 +X-、-N(OC1-6烷基)(C1-6烷基)、-N(OH)(C1-6烷基)、-NH(OH)、-SH、-SC1-6烷基、-SS(C1-6烷基)、-C(＝O)(C1-6烷基)、-CO2H、-CO2(C1-6烷基)、-OC(＝O)(C1-6烷基)、-OCO2(C1-6烷基)、-C(＝O)NH2、-C(＝O)N(C1-6烷基)2、-OC(＝O)NH(C1-6烷基)、-NHC(＝O)(C1-6烷基)、-N(C1-6烷基)C(＝O)(C1-6烷基)、-NHCO2(C1-6烷基)、-NHC(＝O)N(C1-6烷基)2、-NHC(＝O)NH(C1-6烷基)、-NHC(＝O)NH2、-C(＝NH)O(C1-6烷基)、-OC(＝NH)(C1-6烷基)、-OC(＝NH)OC1-6烷基、-C(＝NH)N(C1-6烷基)2、-C(＝NH)NH(C1-6烷基)、-C(＝NH)NH2、-OC(＝NH)N(C1-6烷基)2、-OC(NH)NH(C1-6烷基)、-OC(NH)NH2、-NHC(NH)N(C1-6烷基)2、-NHC(＝NH)NH2、-NHSO2(C1-6烷基)、-SO2N(C1-6烷基)2、-SO2NH(C1-6烷基)、-SO2NH2、-SO2C1-6烷基、-SO2OC1-6烷基、-OSO2C1-6烷基、-SOC1-6烷基、-Si(C1-6烷基)3、-OSi(C1-6烷基)3、-C(＝S)N(C1-6烷基)2、C(＝S)NH(C1-6烷基)、C(＝S)NH2、-C(＝O)S(C1-6烷基)、-C(＝S)SC1-6烷基、-SC(＝S)SC1-6烷基、-P(＝O)2(C1-6烷基)、-P(＝O)(C1-6烷基)2、-OP(＝O)(C1-6烷基)2、-OP(＝O)(OC1-6烷基)2、C1-6烷基、C1-6卤代烷基、C2-C6烯基、C2-C6炔基、C3-C7环烷基、C6-C10芳基、C3-C7杂环基、C5-C10杂芳基；或者两个偕Rgg取代基可结合形成＝O或＝S；其中，X-为反离子。
示例性的氮原子上取代基包括但不局限于：氢、-OH、-ORaa、-N(Rcc)2、-CN、-C(＝O)Raa、-C(＝O)N(Rcc)2、-CO2Raa、-SO2Raa、-C(＝NRbb)Raa、-C(＝NRcc)ORaa、-C(＝NRcc)N(Rcc)2、-SO2N(Rcc)2、-SO2Rcc、-SO2ORcc、-SORaa、-C(＝S)N(Rcc)2、-C(＝O)SRcc、-C(＝S)SRcc、-P(＝O)2Raa、-P(＝O)(Raa)2、-P(＝O)2N(Rcc)2、-P(＝O)(NRcc)2、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基，或者连接至氮原子的两个Rcc基团结合形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代，且其中Raa、Rbb、Rcc和Rdd如上所述。
其他定义
本文术语“siRNA”是一类双链RNA分子，其可以介导与其互补的靶RNA(例如mRNA，例如，编码蛋白质的基因的转录物)的沉默。siRNA通常是双链的，包括与靶RNA互补的反义链，和与该反义链互补的正义链。为方便起见，这样的mRNA在此也被称为有待被沉默的mRNA。这样的基因也称为靶基因。通常，有待被沉默的RNA是内源基因或病原体基因。另外，除了mRNA以外的RNA(例如tRNA)以及病毒RNA也可以被靶向。
术语“反义链”是指siRNA的这样一条链，所述链包含与靶序列完全、充分或基本互补的区域。术语“正义链”是指siRNA的这样一条链，所述链包括与作为在此定义的术语反义链的区域完全、充分或基本互补的区域。
术语“互补区域”是指反义链上与靶mRNA序列完全、充分或基本互补的区域。在互补区域与靶序列不完全互补的情况下，错配可以位于分子的内部或末端区域中。通常，最耐受的错配位于末端区域中，例如，在5’和/或3’端的5、4、3、2或1个核苷酸内。对错配最敏感的反义链部分被称为“种子区”。例如，在包含19nt的链的siRNA中，第19个位置(从5’向3’)可以耐受一些错配。
术语“互补”是指第一多核苷酸在某些条件例如严格条件下与第二多核苷酸杂交的能力。例如，严格条件可包括400mM NaCl、40mM PIPES pH 6.4、1mM EDTA在50℃或70℃下持续12-16小时。就满足以上相对于它们杂交的能力而言的要求来说，“互补”序列还可以包括或完全形成自非沃森-克里克碱基对和/或从非天然的以及经修饰的核苷酸形成的碱基对。此类非沃森-克里克碱基对包括但不限于G:U摇摆碱基配对或Hoogstein碱基配对。
与信使RNA(mRNA)的“至少部分互补”、“充分互补”或“基本上互补”的多核苷酸是指与感兴趣的mRNA的连续部分基本互补的多核苷酸。例如，如果序列与编码PCSK9的mRNA的非中断部分基本上互补，则多核苷酸与PCSK9mRNA的至少部分互补。在此的术语“互补”、“完全互补”、“充分互补”和“基本上互补”可以相对于siRNA的正义链与反义链之间，或siRNA试剂的反义链与靶序列之间的碱基配对使用。
“充分互补”是指为了维持分子的整体双链特征，正义链仅需要与反义链互补的程度。换言之，虽然通常需要完美的互补性，但在一些情况下，特别是在反义链中，可以包括一个或多个，例如6个、5个、4个、3个、2个或1个的错配(相对于靶标mRNA)，但是正义链与反义链仍可以维持分子的整体双链特征。
“shRNA”是指短发夹RNA。shRNA包括两个短反向重复序列。克隆到shRNA表达载体中的shRNA包括两个短反向重复序列，中间由一茎环(loop)序列分隔的，组成发夹结构，由polⅢ启动子控制。随后再连上5-6个T作为RNA聚合酶Ⅲ的转录终止子。
“核苷”是由嘌呤碱或嘧啶碱、以及核糖或脱氧核糖两种物质组成的化合物，“核苷酸”则是由嘌呤碱或嘧啶碱、核糖或脱氧核糖以及磷酸三种物质组成的化合物，“寡核苷酸”是指例如具有少于100、200、300或400个核苷酸长度的核酸分子(RNA或DNA)，可以是单链或双链的。
“碱基”是合成核苷、核苷酸和核酸的基本组成单位，其组成元素中含有氮，也称“含氮碱基”。本文中，如无特别说明，大写字母A、U、T、G和C表示核苷酸的碱基组成，分别为腺嘌呤、尿嘧啶、 胸腺嘧啶、鸟嘌呤和胞嘧啶。
本文中所述核苷酸的“修饰”包括但不限于甲氧基修饰、氟代修饰、硫代磷酸酯基连接或常规保护基保护等。例如，所述氟代修饰的核苷酸指核苷酸的核糖基2’位的羟基被氟取代形成的核苷酸，所述甲氧基修饰的核苷酸指核糖基的2’-羟基被甲氧基取代而形成的核苷酸。
本文中“修饰的核苷酸”包括但不限于2'-O-甲基修饰的核苷酸、2'-氟代修饰的核苷酸、2'-脱氧-修饰的核苷酸、肌苷核糖核苷酸、脱碱基核苷酸、反向无碱基脱氧核糖核苷酸、包含硫代磷酸酯基团的核苷酸、乙烯基磷酸酯修饰的核苷酸、锁核苷酸、2'-氨基-修饰的核苷酸、2'-烷基-修饰的核苷酸、吗啉代核苷酸、氨基磷酸酯、包含核苷酸的非天然碱基、以及连接到胆固醇基衍生物或十二烷酸二癸酰胺基团上的末端核苷酸、脱氧核糖核苷酸或常规保护基保护等。例如，所述2'-氟代修饰的核苷酸指核苷酸的核糖基2’位的羟基被氟取代形成的核苷酸。所述2'-脱氧-修饰的核苷酸指核糖基的2’-羟基被甲氧基取代而形成的核苷酸。
“反应性磷基团”是指包含在核苷酸单元中或核苷酸类似物单元中的含磷基团，其可以通过亲核攻击反应，与包含在另一个分子中、尤其是另一个核苷酸单元中或另一个核苷酸类似物中的羟基或胺基反应。通常，这样的反应产生将所述第一核苷酸单元或所述第一核苷酸类似物单元与所述第二核苷酸单元或所述第二核苷酸类似物单元连接的酯型核苷间键。反应性磷基团可选自亚磷酰胺，H-膦酸酯，烷基-膦酸酯，磷酸酯或磷酸酯模拟物，包括但不限于：天然磷酸酯、硫代磷酸酯、二硫代磷酸酯、硼烷磷酸酯、硼烷硫代磷酸酯、膦酸酯、卤素取代的膦酸酯和磷酸酯、氨基磷酸酯、磷酸二酯、磷酸三酯、硫代磷酸二酯、硫代磷酸三酯、二磷酸酯和三磷酸酯，优选-P(OCH2CH2CN)(N(iPr)2)。
“保护基”又称“保护基团”，是指被添加到分子中以防止分子中现有基团进行不期望的化学反应的任何原子或原子团。“保护基”可为本领域已知的不稳定的化学部分，其用于保护反应性基团，例如羟基、氨基和硫醇基团，以防止在化学合成过程中发生不期望的或不合时宜的反应。保护基通常在其它反应性位点的反应期间选择性地和/或正交地用于保护位点，然后可以被去除以留下未受保护的基团保持原样或可用于进一步的反应。
保护基团的非限制性列表包括苄基；取代的苄基；烷基羰基和烷氧基羰基(例如，叔丁氧基羰基(BOC)、乙酰基或异丁酰基)；芳基烷基羰基和芳基烷氧基羰基(例如，苄基氧基羰基)；取代的甲基醚(例如甲氧基甲基醚)；取代的乙醚；取代的苄基醚；四氢吡喃基醚；甲硅烷基(例如，三甲基甲硅烷基、三乙基甲硅烷基、三异丙基甲硅烷基、叔丁基二甲基甲硅烷基、三-异丙基甲硅烷基氧基甲基、[2-(三甲基甲硅烷基)乙氧基]甲基或叔丁基二苯基甲硅烷基)；酯类(例如苯甲酸酯)；碳酸酯类(例如碳酸甲氧基甲基酯)；磺酸酯类(例如甲苯磺酸酯或甲磺酸酯)；非环缩酮(例如二甲基乙缩醛)；环缩酮(例如，1,3-二噁烷、1,3-二氧戊环以及本文所述的那些)；非环乙缩醛；环乙缩醛(例如，本文所述的那些)；非环半缩醛；环半缩醛；环二硫缩酮(例如，1,3-二噻烷或1,3-二硫戊环)；原酸酯(例如，本文所述的那些)以及三芳基甲基基团(例如，三苯甲基；单甲氧基三苯甲基(MMTr)；4,4′-二甲氧基三苯甲基(DMTr)；4,4′,4″-三甲氧基三苯甲基(TMTr)；以及本文所述的那些)。优选的保护基团选自乙酰基(Ac)、苯甲酰基(Bzl)、苄基(Bn)、异丁酰基(iBu)、苯基乙酰基、苄基氧基甲基乙缩醛(BOM)、β-甲氧基乙氧基甲基醚(MEM)、甲氧基甲基醚(MOM)、对-甲氧基苄基醚(PMB)、甲基硫代甲基醚、新戊酰基(Piv)、四氢 吡喃基(THP)、三苯基甲基(Trt)、甲氧基三苯甲基[(4-甲氧基苯基)二苯基甲基](MMT)、二甲氧基三苯甲基、[双-(4-甲氧基苯基)苯基甲基(DMT)、三甲基甲硅烷基醚(TMS)、叔丁基二甲基甲硅烷基醚(TBDMS)、三-异-丙基甲硅烷基氧基甲基醚(TOM)、三-异丙基甲硅烷基醚(TIPS)、甲基醚、乙氧基乙醚(EE)N,N-二甲基甲脒和2-氰基乙基(CE)。
“羟基保护基”是指能够避免羟基遭受化学反应，又可以在特定条件下脱除以恢复羟基的基团。主要包括硅烷型保护基、酰基型保护基或醚型保护基，优选以下：
三甲基硅基(TMS)、三乙基硅基(TES)、二甲基异丙基硅基(DMIPS)、二乙基异丙基硅基(DEIPS)、叔丁基二甲基硅基(TBDMS)、叔丁基二苯基硅基(TBDPS)、三异丙基硅基(TIPS)、乙酰基(Ac)、氯乙酰基、二氯乙酰基、三氯乙酰基、三氟乙酰基(TFA)、苯甲酰基、对甲氧基苯甲酰基、9-芴基甲氧基羰基(Fmoc)、烯丙氧羰基(Alloc)、2,2,2-三氯乙氧羰基(Troc)、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、苯甲基(Bn)、对甲氧基苄基(PMB)、烯丙基、三苯基甲基(Tr)、双对甲氧基三苯甲基(DMTr)、甲氧基甲基(MOM)、苯氧基甲基(BOM)、2,2,2-三氯乙氧基甲基、2-甲氧基乙氧基甲基(MEM)、甲硫基甲基(MTM)、对甲氧基苄氧基甲基(PMBM)、-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基，优选-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基，更优选-C(O)CH2CH2C(O)OH。
本文所用的术语“药学上可接受的盐”表示本发明化合物的那些羧酸盐、氨基酸加成盐，它们在可靠的医学判断范围内适用于与患者组织接触，不会产生不恰当的毒性、刺激作用、变态反应等，与合理的益处/风险比相称，就它们的预期应用而言是有效的，包括(可能的话)本发明化合物的两性离子形式。
本发明包括互变异构体，其为分子中某一原子在两个位置迅速移动而产生的官能团异构体。在不同的互变异构形式存在的化合物，一个所述化合物并不局限于任何特定的互变异构体，而是旨在涵盖所有的互变异构形式。
本发明化合物可包括一个或多个不对称中心，且因此可以存在多种立体异构体形式，例如，对映异构体和/或非对映异构体形式。例如，本发明化合物可为单独的对映异构体、非对映异构体或几何异构体(例如顺式和反式异构体)，或者可为立体异构体的混合物的形式，包括外消旋体混合物和富含一种或多种立体异构体的混合物。异构体可通过本领域技术人员已知的方法从混合物中分离，所述方法包括：手性高压液相色谱法(HPLC)以及手性盐的形成和结晶；或者优选的异构体可通过不对称合成来制备。
本发明还包括同位素标记的化合物(同位素变体)，它们等同于式(I)所述的那些，但一个或多个原子被原子质量或质量数不同于自然界常见的原子质量或质量数的原子所代替。可以引入本发明化合物中的同位素的实例包括氢、碳、氮、氧、磷、硫、氟和氯的同位素，分别例如2H、3H、13C、11C、14C、15N、18O、17O、31P、32P、35S、18F和36Cl。含有上述同位素和/或其它原子的其它同位素的本发明化合物、其前体药物和所述化合物或所述前体药物的药学上可接受的盐都属于本发明的范围。某些同位素标记的本发明化合物、例如引入放射性同位素(例如3H和14C)的那些可用于药物和/或底物组织分布测定。氚、即3H和碳-14、即14C同位素是特别优选的，因为它们容易制备和检测。进而，被更重的同位素取代，例如氘、即2H，由于代谢稳定性更高可以提供治疗上的益处，例如延长体内半衰期或 减少剂量需求，因而在有些情况下可能是优选的。同位素标记的本发明式(I)化合物及其前体药物一般可以这样制备，在进行下述流程和/或实施例与制备例所公开的工艺时，用容易得到的同位素标记的试剂代替非同位素标记的试剂。
本发明化合物
本发明涉及一种寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，所述寡核苷酸的5’末端包含式(I)所示的结构：
其中，各基团如上下文中所定义。
本发明还涉及一种寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，所述寡核苷酸的5’末端包含式(II)所示的结构：
其中，各基团如上下文中所定义。
本发明还涉及一种寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，所述寡核苷酸的5’末端包含式(III)、式(IV)或式(V)所示的核苷酸单体：
其中，各基团如上下文中所定义。
本发明还涉及式(VI)、式(VII)或式(VIII)所示的化合物或其药学上可接受的盐、互变异构体或立体异构体：
其中，各基团如上下文中所定义。
在一个实施方案中，表示连接至所述寡核苷酸的剩余部分；在另一个实施方案中，表示连接至修饰或未修饰的核苷。
X、Y和Z
在一个实施方案中，X为CRaRb，优选为CH2；在另一个实施方案中，X为(CRaRb)2。
在一个实施方案中，Y为CRaRb，优选为CH2；在另一个实施方案中，Y为(CRaRb)2。
在一个实施方案中，Z为CRa，优选为CH。
其中X和Y任选地被1个或2个氘原子取代，Z任选地被1个氘原子取代。
L1、L2和L3
在一个实施方案中，L1为化学键；在另一个实施方案中，L1为O；在另一个实施方案中，L1为S；在另一个实施方案中，L1为C1-4亚烷基。
在一个实施方案中，L2为C1-4亚烷基，优选为C1-2亚烷基。
在一个实施方案中，L3为C1-4亚烷基，优选为C1-2亚烷基。
在一个实施方案中，L2和L3连接，与L1及其相邻的碳原子一起形成C5-6环烷基；在另一个实施方案中，L2和L3连接，与L1及其相邻的碳原子一起形成5-6元杂环基，优选U、L1、L2和L3及其相邻的碳原子一起形成核糖或脱氧核糖，其任选地被R5取代。
在一个实施方案中，L1未被取代；在另一个实施方案中，L1被1个R#进一步取代；在另一个实施方案中，L1被2个独立选择的R#进一步取代；在另一个实施方案中，L1被3个独立选择的R#进一步取代；在另一个实施方案中，L1被4个独立选择的R#进一步取代；在另一个实施方案中，L1被5个独立选择的R#进一步取代。
在一个实施方案中，L2未被取代；在另一个实施方案中，L2被1个R#进一步取代；在另一个实施方案中，L2被2个独立选择的R#进一步取代；在另一个实施方案中，L2被3个独立选择的R#进一步取代；在另一个实施方案中，L2被4个独立选择的R#进一步取代；在另一个实施方案中，L2被5个独立选择的R#进一步取代。
在一个实施方案中，L3未被取代；在另一个实施方案中，L3被1个R#进一步取代；在另一个实施方案中，L3被2个独立选择的R#进一步取代；在另一个实施方案中，L3被3个独立选择的R#进一步取代；在另一个实施方案中，L3被4个独立选择的R#进一步取代；在另一个实施方案中，L3被5个独立选择的R#进一步取代。
其中上述L1、L2和L3任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
U
在一个实施方案中，U为O；在另一个实施方案中，U为S；在另一个实施方案中，U为CRaRb，例如CH2；在另一个实施方案中，U为NRc，例如NH。
其中上述U任选地被1个或更多个氘原子取代，直至完全氘代。
Q
在一个实施方案中，Q为H；在另一个实施方案中，Q为D；在另一个实施方案中，Q为卤素；在另一个实施方案中，Q为OH；在另一个实施方案中，Q为C1-6烷基，例如C1-4烷基；在另一个实施方案中，Q为C1-6卤代烷基，例如C1-4卤代烷基；在另一个实施方案中，Q为C1-6烷氧基，例如C1-4烷氧基；在另一个实施方案中，Q为C1-6卤代烷氧基，例如C1-4卤代烷氧基；在另一个实施方案中，Q为C2-6烯基，例如C2-4烯基；在另一个实施方案中，Q为C2-6炔基，例如C2-4炔基。
其中上述Q任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
R1
在一个实施方案中，R1为O；在另一个实施方案中，R1为S。
R2和R3
在一个实施方案中，R2为OH；在另一个实施方案中，R2为SH；在另一个实施方案中，R2为NH2；在另一个实施方案中，R2为C1-6烷基；在另一个实施方案中，R2为C1-6卤代烷基；在另一个实施方案中，R2为C1-6烷氧基；在另一个实施方案中，R2为C1-6卤代烷氧基。
在一个实施方案中，R2为ORd；在另一个实施方案中，R2为OP1；在另一个实施方案中，R2为SRd；在另一个实施方案中，R2为SP1；在另一个实施方案中，R2为NReRf。
在一个实施方案中，R2未被取代；在另一个实施方案中，R2被1个R#进一步取代；在另一个实施方案中，R2被2个独立选择的R#进一步取代；在另一个实施方案中，R2被3个独立选择的R#进一步取代；在另一个实施方案中，R2被4个独立选择的R#进一步取代；在另一个实施方案中，R2被5个独立选择的R#进一步取代。
在一个实施方案中，R3为OH；在另一个实施方案中，R3为SH；在另一个实施方案中，R3为NH2；在另一个实施方案中，R3为C1-6烷基；在另一个实施方案中，R3为C1-6卤代烷基；在另一个实施方案中，R3为C1-6烷氧基；在另一个实施方案中，R3为C1-6卤代烷氧基。
在一个实施方案中，R3为ORd；在另一个实施方案中，R3为OP1；在另一个实施方案中，R3为SRd；在另一个实施方案中，R3为SP1；在另一个实施方案中，R3为NReRf。
在一个实施方案中，R3未被取代；在另一个实施方案中，R3被1个R#进一步取代；在另一个实施方案中，R3被2个独立选择的R#进一步取代；在另一个实施方案中，R3被3个独立选择的R#进一步取代；在另一个实施方案中，R3被4个独立选择的R#进一步取代；在另一个实施方案中，R3被5个独立选择的R#进一步取代。
其中上述R2和R3任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
R4、R5、R6和R7
在一个实施方案中，R4为H；在另一个实施方案中，R4为D；在另一个实施方案中，R4为卤素；在另一个实施方案中，R4为OH；在另一个实施方案中，R4为C1-6烷基，例如C1-4烷基；在另一个实施方案中，R4为C1-6卤代烷基，例如C1-4卤代烷基；在另一个实施方案中，R4为C1-6烷氧基，例如C1-4烷氧基；在另一个实施方案中，R4为C1-6卤代烷氧基，例如C1-4卤代烷氧基。
在一个实施方案中，R5为H；在另一个实施方案中，R5为D；在另一个实施方案中，R5为卤素；在另一个实施方案中，R5为OH；在另一个实施方案中，R5为C1-6烷基，例如C1-4烷基；在另一个实施方案中，R5为C1-6卤代烷基，例如C1-4卤代烷基；在另一个实施方案中，R5为C1-6烷氧基，例如C1-4烷氧基；在另一个实施方案中，R5为C1-6卤代烷氧基，例如C1-4卤代烷氧基。
在一个实施方案中，R6为H；在另一个实施方案中，R6为D；在另一个实施方案中，R6为卤素；在另一个实施方案中，R6为OH；在另一个实施方案中，R6为C1-6烷基，例如C1-4烷基；在另一个实施方案中，R6为C1-6卤代烷基，例如C1-4卤代烷基；在另一个实施方案中，R6为C1-6烷氧基，例如C1-4烷氧基；在另一个实施方案中，R6为C1-6卤代烷氧基，例如C1-4卤代烷氧基。
在一个实施方案中，R7为H；在另一个实施方案中，R7为D；在另一个实施方案中，R7为卤素；在另一个实施方案中，R7为OH；在另一个实施方案中，R7为C1-6烷基，例如C1-4烷基；在另一个实施方案中，R7为C1-6卤代烷基，例如C1-4卤代烷基；在另一个实施方案中，R7为C1-6烷氧基，例如C1-4烷氧基；在另一个实施方案中，R7为C1-6卤代烷氧基，例如C1-4卤代烷氧基。
其中上述R4、R5、R6和R7任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
Ra、Rb和Rc
在一个实施方案中，Ra为H；在另一个实施方案中，Ra为D；在另一个实施方案中，Ra为卤素；在另一个实施方案中，Ra为OH；在另一个实施方案中，Ra为NH2；在另一个实施方案中，Ra为CN；在另一个实施方案中，Ra为C1-6烷基，例如C1-4烷基；在另一个实施方案中，Ra为C1-6卤代烷基，例如C1-4卤代烷基；在另一个实施方案中，Ra为C1-6烷氧基，例如C1-4烷氧基；在另一个实施方案中，Ra为C1-6卤代烷氧基，例如C1-4卤代烷氧基；在另一个实施方案中，Ra为C2-6烯基，例如C2-4烯基；在另一个实施方案中，Ra为C2-6炔基，例如C2-4炔基。
在一个实施方案中，Ra未被取代；在另一个实施方案中，Ra被1个R#进一步取代；在另一个实施方案中，Ra被2个独立选择的R#进一步取代；在另一个实施方案中，Ra被3个独立选择的R#进一步取代；在另一个实施方案中，Ra被4个独立选择的R#进一步取代；在另一个实施方案中，Ra被5个独立选择的R#进一步取代。
在一个实施方案中，Rb为H；在另一个实施方案中，Rb为D；在另一个实施方案中，Rb为卤素；在另一个实施方案中，Rb为OH；在另一个实施方案中，Rb为NH2；在另一个实施方案中，Rb为CN； 在另一个实施方案中，Rb为C1-6烷基，例如C1-4烷基；在另一个实施方案中，Rb为C1-6卤代烷基，例如C1-4卤代烷基；在另一个实施方案中，Rb为C1-6烷氧基，例如C1-4烷氧基；在另一个实施方案中，Rb为C1-6卤代烷氧基，例如C1-4卤代烷氧基；在另一个实施方案中，Rb为C2-6烯基，例如C2-4烯基；在另一个实施方案中，Rb为C2-6炔基，例如C2-4炔基。
在一个实施方案中，Rb未被取代；在另一个实施方案中，Rb被1个R#进一步取代；在另一个实施方案中，Rb被2个独立选择的R#进一步取代；在另一个实施方案中，Rb被3个独立选择的R#进一步取代；在另一个实施方案中，Rb被4个独立选择的R#进一步取代；在另一个实施方案中，Rb被5个独立选择的R#进一步取代。
在一个实施方案中，Rc为H；在另一个实施方案中，Rc为C1-6烷基；在另一个实施方案中，Rc为C1-6卤代烷基；在另一个实施方案中，Rc为C2-6烯基；在另一个实施方案中，Rc为C2-6炔基。
其中上述Ra、Rb和Rc任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
Rd、Re和Rf
在一个实施方案中，Rd为H；在另一个实施方案中，Rd为C1-6烷基，例如C1-4烷基；在另一个实施方案中，Rd为C1-6卤代烷基，例如C1-4卤代烷基；在另一个实施方案中，Rd为C2-6烯基；在另一个实施方案中，Rd为C2-6炔基。
在一个实施方案中，Rd未被取代；在另一个实施方案中，Rd任选地被1个或多个D取代，直至完全氘代；在另一个实施方案中，Rd被卤素取代；在另一个实施方案中，Rd被C1-6烷基取代；在另一个实施方案中，Rd被C1-6卤代烷基取代。
在一个实施方案中，Re为H；在另一个实施方案中，Re为C1-6烷基，例如C1-4烷基；在另一个实施方案中，Re为C1-6卤代烷基，例如C1-4卤代烷基。
在一个实施方案中，Re未被取代；在另一个实施方案中，Re任选地被1个或多个D取代，直至完全氘代；在另一个实施方案中，Re被卤素取代；在另一个实施方案中，Re被C1-6烷基取代；在另一个实施方案中，Re被C1-6卤代烷基取代。
在一个实施方案中，Rf为H；在另一个实施方案中，Rf为C1-6烷基，例如C1-4烷基；在另一个实施方案中，Rf为C1-6卤代烷基，例如C1-4卤代烷基。
在一个实施方案中，Rf未被取代；在另一个实施方案中，Rf任选地被1个或多个D取代，直至完全氘代；在另一个实施方案中，Rf被卤素取代；在另一个实施方案中，Rf被C1-6烷基取代；在另一个实施方案中，Rf被C1-6卤代烷基取代。
其中上述Rd、Re和Rf任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
P1和P2
在一个实施方案中，P1为保护基；在另一个实施方案中，P1为羟基保护基。
在一个实施方案中，P2为反应性磷基团，例如-P(OCH2CH2CN)(N(iPr)2)。
Base和Base’
在一个实施方案中，Base为H；在另一个实施方案中，Base为修饰或未修饰的碱基或离去基；在另一个实施方案中，Base选自修饰或未修饰的A、U、T、G和C，例如
在一个实施方案中，Base’为H；在另一个实施方案中，Base’为修饰或未修饰的碱基或离去基；在另一个实施方案中，Base’选自修饰或未修饰的A、U、T、G和C，例如
R#
在一个实施方案中，R#为H；在另一个实施方案中，R#为D；在另一个实施方案中，R#为卤素；在另一个实施方案中，R#为C1-6烷基；在另一个实施方案中，R#为C1-6卤代烷基；在另一个实施方案中，R#为C2-6烯基；在另一个实施方案中，R#为C2-6炔基；
其中上述R#任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
GalNAc
在一个实施方案中，GalNAc为式(X)所示的缀合基团：
其中，
表示与生物分子连接的位置；
QG独立地为H、
其中LG1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；
LG2为化学键或-CH2CH2C(O)-；
LG3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；
LG4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；
其中a＝0、1、2或3；
b＝1、2、3、4或5；
c＝1、2、3、4或5；
d＝1、2、3、4、5、6、7或8；
A为化学键、-CH2O-或-NHC(O)-；
A’为化学键、-C(O)NH-、-NHC(O)-或-O(CH2CH2O)e-；
其中e为1、2、3、4或5；
B为化学键、-CH2-、-C(O)-、-M-、-CH2-M-或-C(O)-M-；
其中M为
RG1和RG2一起形成-CH2CH2O-或-CH2CH(RG)-O-，并且RG3为H；
或者RG1和RG3一起形成-C1-2亚烷基-，并且RG2为H；
其中RG为-ORG’、-CH2ORG’或-CH2CH2ORG’，其中RG’为H、羟基保护基或固相载体，所述羟基保护基优选-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基；
m1＝0、1、2、3、4、5、6、7、8、9或10；
n1＝0、1、2、3、4、5、6、7、8、9或10。
在另一个实施方案中，GalNAc为式(I’)所示的缀合集团：
其中，
表示与生物分子连接的位置；
QG独立地为H、
其中LG1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；
LG2为化学键或-CH2CH2C(O)-；
LG3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；
LG4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；
其中a＝0、1、2或3；
b＝1、2、3、4或5；
c＝1、2、3、4或5；
d＝1、2、3、4、5、6、7或8；
A为-CH2O-或-NHC(O)-；
A’为化学键、-C(O)NH-或-NHC(O)-；
RG1和RG2一起形成-CH2CH2O-或-CH2CH(RG)-O-，并且RG3为H；
或者RG1和RG3一起形成-C1-2亚烷基-，并且RG2为H；
其中RG为-ORG’、-CH2ORG’或-CH2CH2ORG’，其中RG’为H、羟基保护基或固相载体，所述羟基保护基优选-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基；
m1＝0、1、2、3、4、5、6、7、8、9或10；
n1＝0、1、2、3、4、5、6、7、8、9或10。
在另一个实施方案中，GalNAc为式(X)所示的缀合基团，其中，
QG独立地为H、
其中LG1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；
LG2为化学键或-CH2CH2C(O)-；
LG3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；
LG4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；
其中a＝0、1、2或3；
b＝1、2、3、4或5；
c＝1、2、3、4或5；
d＝1、2、3、4、5、6、7或8；
A为化学键、-CH2O-或-NHC(O)-；
A’为化学键、-C(O)NH-、-NHC(O)-或-O(CH2CH2O)e-；
其中e为1、2、3、4或5；
B为化学键、-CH2-、-M-、-CH2-M-或-C(O)-M-；
其中M为
RG1和RG2一起形成-CH2CH2O-或-CH2CH(RG)-O-，并且RG3为H；
或者RG1和RG3一起形成-C1-2亚烷基-，并且RG2为H；
其中RG为-ORG’、-CH2ORG’或-CH2CH2ORG’，其中RG’为H、羟基保护基或固相载体，所述羟基保护基优选-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基；
m1＝0、1、2、3、4、5、6、7、8、9或10；
n1＝0、1、2、3、4、5、6、7、8、9或10。
在另一个实施方案中，GalNAc为式(X)所示的缀合基团，其中：
QG独立地为H、
其中LG1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；
LG2为化学键或-CH2CH2C(O)-；
LG3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；
LG4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；
其中a＝0、1、2或3；
b＝1、2、3、4或5；
c＝1、2、3、4或5；
d＝1、2、3、4、5、6、7或8；
A为化学键、-CH2O-或-NHC(O)-；
A’为-O(CH2CH2O)e-；
其中e为1、2、3、4或5；
B为化学键、-CH2-、-C(O)-、-M-、-CH2-M-或-C(O)-M-；
其中M为
RG1和RG2一起形成-CH2CH2O-或-CH2CH(RG)-O-，并且RG3为H；
或者RG1和RG3一起形成-C1-2亚烷基-，并且RG2为H；
其中RG为-ORG’、-CH2ORG’或-CH2CH2ORG’，其中RG’为H、羟基保护基或固相载体，所述羟基保护基优选-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基；
m1＝0、1、2、3、4、5、6、7、8、9或10；
n1＝0、1、2、3、4、5、6、7、8、9或10。
以上任一具体实施方案中的任一技术方案或其任意组合，可以与其它具体实施方案中的任一技术方案或其任意组合进行组合。例如，X的任一技术方案或其任意组合，可以与Y、Z、L1、L2、L3、R1、R2、R3、R4、R5、R6、R7、U、Q、P1、P2、Base和Base’等的任一技术方案或其任意组合进行组合。本发明旨在包括所有这些技术方案的组合，限于篇幅，不再一一列出。
本发明还提供了载体，其包含编码本发明所述的siRNA的核苷酸序列。本发明的载体能够扩增或表达与其连接的编码本发明所述的siRNA的核苷酸。
例如，靶向PCSK9基因的siRNA可以从插入DNA或RNA载体中的转录单位表达。表达可以是短暂的(数小时至数星期内)或持续的(数星期至数个月或更久)，取决于所使用的特定建构体及靶组织或细胞类型。可以将siRNA的编码核苷酸引入线性建构体、环状质体或病毒载体中。siRNA的核苷酸可以被整合到细胞基因组中稳定表达，或者在染色体外稳定遗传而表达。一般来说，siRNA表达载体通常是DNA质粒或病毒载体。
包含siRNA的编码序列的病毒载体系统包括但不局限于：(a)腺病毒载体；(b)逆转录病毒载体；(c)腺伴随病毒载体；(d)单纯疱疹病毒载体；(e)SV40载体；(f)多瘤病毒载体；(g)乳头瘤病毒载体；(h)微小核糖核酸病毒载体；(i)痘病毒载体；以及(j)辅助病毒依赖性腺病毒或无肠腺病毒。
本发明还提供了细胞，其含有本发明所述的siRNA或载体，其中本发明所述的siRNA或载体能够在细胞中转录。
在另一个方面，本发明涉及一种寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，所述寡核苷酸的5’末端包含下式所示的结构：
其中表示连接至所述寡核苷酸的剩余部分，Base如上下文所定义。
本发明具体涉及以下技术方案：
1.一种寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，所述寡核苷酸的5’末端包含式(I)所示的结构：
其中，
表示连接至所述寡核苷酸的剩余部分，优选连接至修饰或未修饰的核苷；
X和Y各自独立地选自CRaRb或(CRaRb)2，优选为CRaRb；
Z为CRa；
Ra和Rb独立地选自H、D、卤素、OH、NH2、CN、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C2-6烯基或C2-6炔基；所述Ra和Rb任选地被1个、2个、3个、4个或5个独立选择的R#进一步取代；
R1选自O或S；
R2和R3独立地选自OH、SH、NH2、C1-6烷基、C1-6卤代烷基、C1-6烷氧基或C1-6卤代烷氧基；所述R2和R3任选地被1个、2个、3个、4个或5个独立选择的R#进一步取代；
R#选自H、D、卤素、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基；
其中上述各基团定义任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
2.技术方案1的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
表示连接至所述寡核苷酸的剩余部分，优选连接至修饰或未修饰的核苷；
X和Y各自独立地选自CRaRb或(CRaRb)2，优选为CRaRb；
Z为CRa；
Ra和Rb独立地选自H、D、卤素、OH、NH2、CN、C1-6烷基、C1-6卤代烷基、C1-6烷氧基或C1-6卤代烷氧基；
R1选自O或S；
R2和R3独立地选自OH、SH、C1-6烷氧基或C1-6卤代烷氧基；所述R2和R3任选地被1个、2个或3个独立选择的R#进一步取代；
R#选自H、D、卤素、C1-6烷基和C1-6卤代烷基。
3.技术方案1或2的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
表示连接至所述寡核苷酸的剩余部分，优选连接至修饰或未修饰的核苷；
X和Y各自独立地选自CRaRb或(CRaRb)2，优选为CRaRb；
Z为CRa；
Ra和Rb独立地选自H、D、卤素、C1-4烷基或C1-4卤代烷基；
R1选自O或S；
R2和R3独立地选自OH或SH，优选为OH。
4.技术方案1-3中任一项的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
表示连接至所述寡核苷酸的5’末端基团，优选连接至修饰或未修饰的核苷；
X和Y为CH2；
Z为CH；
R1为O；
R2和R3为OH。
5.技术方案1的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其具有式(II)的结构：
其中，
表示连接至所述寡核苷酸的剩余部分；
X和Y为CRaRb；
Z为CRa；
L1选自化学键、O、S或C1-4亚烷基；
L2和L3独立地选自C1-4亚烷基；
所述L1、L2和L3任选独立地被1个、2个、3个、4个或5个独立选择的R#取代；
U选自O、S、CRaRb或NRc；
Ra和Rb独立地选自H、D、卤素、OH、NH2、CN、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C2-6烯基或C2-6炔基；
Rc独立地选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基；
所述Ra、Rb和Rc任选地被1个、2个、3个、4个或5个独立选择的R#进一步取代；
或者，L2和L3连接，与L1及其相邻的碳原子一起形成C5-6环烷基或5-6元杂环基，优选U、L1、L2和L3及其相邻的碳原子一起形成核糖或脱氧核糖，其任选地被R5取代；
R1选自O或S；
R2和R3独立地选自OH、SH、NH2、C1-6烷基、C1-6卤代烷基、C1-6烷氧基或C1-6卤代烷氧基；所述R2和R3任选地被1个、2个、3个、4个或5个独立选择的R#取代；
R5选自H、D、卤素、C1-6烷氧基或C1-6卤代烷氧基；
R#选自H、D、卤素、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基；
Base选自H、修饰或未修饰的碱基；
其中上述各基团定义任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
6.技术方案5的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
表示连接至所述寡核苷酸的剩余部分；
X和Y为CRaRb；
Z为CRa；
Ra和Rb独立地选自H、D、卤素、OH、NH2、CN、C1-6烷基、C1-6卤代烷基、C1-6烷氧基或C1-6卤代烷氧基；
L1选自化学键、O或C1-4亚烷基；
L2和L3独立地选自C1-4亚烷基；
所述L1、L2和L3任选独立地被1个、2个或3个独立选择的R#取代；
U选自O、S、NH或CH2；
或者，L2和L3连接，与L1及其相邻的碳原子一起形成C5-6环烷基或5-6元杂环基，优选U、L1、L2和L3及其相邻的碳原子一起形成核糖或脱氧核糖，其任选地被R5取代；
R1选自O或S；
R2和R3独立地选自OH、SH、C1-6烷氧基或C1-6卤代烷氧基；所述R2和R3任选地被1个、2个或3个独立选择的R#取代；
R5选自H、D、C1-6烷基、C1-6卤代烷基、C1-6烷氧基或C1-6卤代烷氧基；
R#选自H、D、卤素、C1-6烷基和C1-6卤代烷基；
Base选自H、修饰或未修饰的碱基。
7.技术方案5或6的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
表示连接至所述寡核苷酸的剩余部分；
X和Y为CRaRb；
Z为CRa；
Ra和Rb独立地选自H、D、卤素、C1-4烷基或C1-4卤代烷基；
L1选自化学键或C1-4亚烷基；
L2和L3独立地选自C1-4亚烷基；
U选自O或S；
R1选自O或S；
R2和R3独立地选自OH或SH，优选为OH；
Base选自
8.技术方案5-7中任一项的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
表示连接至所述寡核苷酸的剩余部分；
X和Y为CH2；
Z为CH；
L1为化学键；
L2和L3独立地选自C1-2亚烷基；
U为O；
R1为O；
R2和R3为OH；
Base选自
9.技术方案1的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，所述寡核苷酸的5’末端包含式(III)、式(IV)或式(V)所示的核苷酸单体：
其中，
Q选自H、D、卤素、OH、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C2-6烯基或C2-6炔基；
R4、R5、R6和R7独立地选自H、D、卤素、OH、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C2-6烯基或C2-6炔基；
Base选自H、修饰或未修饰的碱基；
L1、L3、X、Y、Z、R1、R2、R3、U如技术方案5-8中任一项所定义；
其中上述各基团定义任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
10.技术方案9的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
Q选自H、D、卤素、OH、C1-6烷基、C1-6卤代烷基、C1-6烷氧基或C1-6卤代烷氧基；
R4、R5、R6和R7独立地选自H、D、卤素、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基；
Base选自H、修饰或未修饰的碱基，优选
L1、L3、X、Y、Z、R1、R2、R3、U如技术方案5-8中任一项所定义。
11.技术方案9或10的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
Q选自H、卤素或C1-4烷氧基，优选为H、F或甲氧基；
R4、R5、R6和R7独立地选自H、卤素、C1-4烷基或C1-4卤代烷基，优选为H；
Base选自优选为
L1、L3、X、Y、Z、R1、R2、R3、U如技术方案5-8中任一项所定义。
12.技术方案9-11中任一项的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中，所述寡核苷酸的5’末端包含具有以下结构的核苷酸单体：
其中，各基团如技术方案9-11中任一项所定义。
13.技术方案9-12中任一项的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中，所述寡核苷酸的5’末端包含具有以下结构的核苷酸单体：
其中，Base选自
14.式(VI)、式(VII)或式(VIII)所示的化合物或其药学上可接受的盐、互变异构体或立体异构体：
其中，
R1选自O或S；
R2和R3独立地选自ORd、OP1、SRd、SP1或NReRf；
Rd选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被D、卤素、C1-6烷基或C1-6卤代烷基取代，直至完全氘代；
Re和Rf独立地选自H、C1-6烷基或C1-6卤代烷基，所述Re和Rf可任选地被D、卤素、C1-6烷基或C1-6卤代烷基取代，直至完全氘代；
P1选自保护基，优选为羟基保护基；
P2选自反应性磷基团，优选-P(OCH2CH2CN)(N(iPr)2)；
Base’独立地选自H、修饰或未修饰的碱基或离去基；
L1、L3、X、Y、Z、U、Q、R4、R5、R6和R7如技术方案5-11中任一项所定义；
其中上述各基团定义任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
15.技术方案14的化合物或其药学上可接受的盐、互变异构体或立体异构体，其中：
R1选自O或S；
R2和R3独立地选自ORd、OP1、SRd、SP1或NReRf；
Rd选自H、C1-4烷基或C1-4卤代烷基，其任选地被D或卤素取代，直至完全氘代；
Re和Rf独立地选自H、C1-4烷基或C1-4卤代烷基，所述Re和Rf可任选地被D或卤素取代，直至完全氘代；
P1选自保护基，优选为羟基保护基；
P2选自反应性磷基团，优选-P(OCH2CH2CN)(N(iPr)2)；
Base’选自
L1、L3、X、Y、Z、U、Q、R4、R5、R6和R7如技术方案5-11中任一项所定义。
16.技术方案14或15的化合物或其药学上可接受的盐、互变异构体或立体异构体，其中：
R1选自O或S，优选为O；
R2和R3独立地选自ORd或OP1；
Rd选自H、C1-4烷基或C1-4卤代烷基；
Re和Rf独立地选自H、C1-4烷基或C1-4卤代烷基；
P1选自保护基，优选为羟基保护基；
P2选自反应性磷基团，优选-P(OCH2CH2CN)(N(iPr)2)；
Base’选自更优选为
L1、L3、X、Y、Z、U、Q、R4、R5、R6和R7如技术方案5-11中任一项所定义。
17.技术方案14-16中任一项的化合物或其药学上可接受的盐、互变异构体或立体异构体，其中，所述化合物选自：
18.技术方案1-13中任一项的寡核苷酸，其中，所述寡核苷酸为单链，其具有14至30个核苷酸。
19.技术方案1-13中任一项的寡核苷酸，其中，所述寡核苷酸为双链RNA，其包含正义链和反义链，其中各链具有14至30个核苷酸，所述反义链具有与所述正义链和靶标mRNA充分互补的序列。
20.技术方案19的寡核苷酸，其中，所述双链RNA在反义链中包含技术方案1-12中任一项所述的式(I)、(II)、(III)、(IV)、(V)、(wyin’c)、(IIIb)、(IVa)、(IVb)、(Va)或(Vb)的结构或者技术方案13所述的核苷酸单体。
21.技术方案19-20中任一项的寡核苷酸，其中所述双链RNA进一步偶联至配体，优选地，该配体包含一个或多个GalNAc。
22.一种核酸分子，所述核酸分子的核苷酸序列中包含一个或多个如技术方案9-13中任一项所述的核苷酸单体。
23.技术方案22的核酸分子，其中所述核酸选自DNA、RNA和DNA/RNA杂合体。
24.技术方案23的核酸分子，所述核酸分子是单链的或双链的。
25.技术方案22-24中任一项的核酸分子，其中所述核酸分子选自小干扰RNA(siRNA)和短发夹RNA(shRNA)。
26.载体，其包含编码前述技术方案19-21中任一项所述的双链RNA的核苷酸序列。
27.细胞，其含有如技术方案19-21中任一项所述的双链RNA或如技术方案24所述的载体。
28.一种药物组合物，其包含如技术方案19-21中任一项所述的双链RNA分子，和药学上可接受的载体或赋形剂。
29.一种试剂盒，其包含如技术方案19-21中任一项所述的双链RNA分子。
30.一种用于抑制细胞中靶基因的表达的方法，包括将技术方案19-21中任一项所述的双链RNA分子引入该细胞的步骤。
不受具体理论的约束，式(IIIa)、式(IVa)和式(Va)的立体构型可以使本发明的寡核苷酸(特别是其5’末端的膦酸酯基团)更好地与Ago2蛋白结合，从而提高寡核苷酸的活性。
实施例
以下实施例用于例示本发明而非限制本发明的范围。
实施例1 化合物E1-1和E1-2的制备
1.化合物1b的制备
将化合物1a(200g，1.33mol,1.00eq)悬浮于无水丙酮(1.00L)与无水甲醇(1.00L)的混合溶液中，逐滴加入浓硫酸(20.0mL，0.27eq)，在25℃反应24小时。反应液用饱和碳酸氢钠中和，浓缩后，得到的残渣用乙酸乙酯溶解，用饱和食盐水(500mL)洗涤三次。有机相用无水硫酸钠干燥，浓缩，得到化合物1b(242g，88.9％)。
1H NMR:400MHz CDCl3δ4.97(s,1H),4.84(d,J＝6.0Hz,1H),4.59(d,J＝6.0Hz,1H),4.44-4.43(m,1H),3.72-3.59(m,2H),3.44(s,3H),1.49(s,3H),1.32(s,3H).
2.化合物1c的制备
将化合物1b(310g，1.52mol，1.00eq)，咪唑(206g，3.02mol，2.00eq)，三苯基膦(476g，1.82mol，1.20eq)溶于甲苯(2.1L)，室温分批加入碘单质(446g，1.76mmol，1.16eq)。加完升温到70℃搅拌1.5小时。TLC表明反应完全。向反应液中加入甲醇(60mL)进行淬灭，然后降温，加入饱和硫代硫酸钠水溶液(2.1L)，分液收集有机相，用饱和食盐水(1.5L)洗涤两次，用无水硫酸钠干燥，过滤浓缩，得到的残渣用甲基叔丁基醚打浆过滤，滤液浓缩所得粗品用硅胶柱层析纯化(洗脱液：石油醚/乙酸乙酯)得到化合物1c(401g，1.27mol，91.1％)。
1H NMR:400MHz CDCl3δ4.98(s,1H),4.69(d,J＝6.0Hz,1H),4.56(d,J＝6.0Hz,1H),4.39-4.35(m,1H),3.30(s,3H),3.24-3.20(m,1H),3.09(t,J＝10.0Hz,1H),1.41(s,3H),1.26(s,3H).
3.化合物1d的制备
将化合物1c(203g，646mmol，1.00eq)溶于四氢呋喃(2.03L)，在0℃分批加入叔丁醇钾(145g，1.29mol，1.29eq)。在25℃搅拌16小时。降温至5℃用冰水(1.1L)淬灭，甲基叔丁基醚萃取(2.0L)，饱和食盐水洗涤(2.0L)，无水硫酸钠干燥、过滤、浓缩。粗品硅胶柱层析纯化(洗脱液：石油醚/乙酸乙酯)得到化合物1d(160g，91.3％)。
1H NMR:400MHz CDCl3δ5.11(s,1H),5.02(d,J＝5.6Hz,1H),4.06(d,J＝1.6Hz,1H),4.50(d,J＝5.6Hz,1H),4.39(d,J＝1.6Hz,1H),3.41(s,3H),3.24-3.20(m,1H),3.09(t,J＝10.0Hz,1H),1.47(s,3H),1.35(s,3H).
4.化合物1e的制备
将锌铜试剂(90.0g，1.37mol，5.50eq)分散到乙醚(200mL)中，在25℃加入化合物1d(60g，241mmol，1.00eq)，逐滴加入三氯乙酰氯(61.5g，338mmol，1.40eq)的乙醚(200mL)溶液，并继续搅拌1小时。过滤，用甲基叔丁基醚(500mL)冲洗滤饼，滤液倒入饱和碳酸氢钠水溶液(2.50L)中，过滤，滤液用饱和食盐水(500mL)洗涤三次，无水硫酸钠干燥、过滤、浓缩得到粗品化合物1e(71.8g)。
1H NMR:400MHz CDCl3δ5.12(d,J＝5.6Hz,1H),5.09(s,1H),4.70(d,J＝6.0Hz,1H),3.70-3.55(m,2H),3.54(s,3H),1.45(s,3H),1.36(s,3H).
5.化合物1f的制备
将化合物1e(71.8g，242mmol，1.00eq)溶于四氢呋喃(1.60L)，加入冰醋酸(69.1mL，1.20mol，5.00eq)，分批加入锌粉(142g，2.17mol，9.00eq)。25℃搅拌18小时。反应完毕后过滤，滤液浓缩得到粗品，用甲基叔丁基醚溶解后，倒入饱和碳酸氢钠水溶液(2.0L)中，过滤，滤液用饱和食盐水洗涤(500mL)两次，用无水硫酸钠干燥、过滤、浓缩，粗品用硅胶柱层析纯化(洗脱液：石油醚/乙酸乙酯/二氯甲烷)得到化合物1f(31g，两步收率56.3％)。
1H NMR:400MHz CDCl3δ4.97(s,1H),4.70-4.67(m,2H),3.54-3.48(m,2H),3.37-3.31(m,4H),3.16-3.10(m,1H),3.09-3.03(m,1H),1.43(s,3H),1.34(s,3H).
6.化合物1g的制备
将化合物1f(50g，219mmol，1.00eq)和亚磷酸二乙酯(33.2g，240mmol，1.20eq)溶于二氯甲烷(100mL)中，在0℃加入1,8-二氮杂二环十一碳-7-烯(DBU，6.67g，43.8mmol，0.20eq)，25℃搅拌18小时。反应液用饱和NH4Cl(100mL)洗涤三次，饱和食盐水(100mL)洗涤一次，无水硫酸钠干燥，浓缩，粗品经硅胶柱层析纯化(洗脱液：石油醚/二氯甲烷)得到化合物1g(70g，87.2％)
1H NMR:400MHz CDCl3δ5.27(s,1H),5.01(brs,1H),4.78(s,1H),4.65(d,J＝5.6Hz,1H),4.49(d, J＝6.0Hz,1H),4.20-4.05(m,5H),3.31(s,1H),3.29-3.05(m,1H),2.75-2.68(m,1H),2.50-2.40(m,1H),2.35-2.25(m,1H),1.36-1.25(m,12H).
7.化合物1h的制备
将化合物1g(80.2g，219mmol，1.00eq)、DMAP(40.1g，328mmol，1.50eq)溶于乙腈(562mL)，在5℃加入草酰氯单甲酯(40.2g，328mmol，1.50eq)。25℃搅拌半小时。将反应液浓缩，粗品用乙酸乙酯溶解，用饱和氯化铵(300mL)洗涤五次，水(200mL)洗涤一次，食盐水(200mL)洗涤一次，用无水硫酸钠干燥、过滤、浓缩得到粗品化合物1h(99g)。
1H NMR:400MHz CDCl3δ4.88-4.80(m,2H),4.67-4.53(m,2H),4.24-4.12(m,4H),3.89(s,3H),3.51-3.43(m,1H),3.35-3.33(m,3H),3.15-3.07(m,1H),2.92-2.80(m,1H),2.69-2.61(m,1H),1.39-1.29(m,12H).
8.化合物1i的制备
将化合物1h(99g，218mmol，1.00eq)溶于无水甲苯(1.00L)，加入三正丁基锡氢(76.4g，262mmol，1.20eq)和偶氮二异丁腈(AIBN，1.08g，6.56mmol，0.03eq)。回流2小时。将反应液浓缩，粗品用硅胶柱层析纯化(洗脱液：石油醚/乙酸乙酯)得到粗品化合物1i(146g)。
1H NMR:400MHz CDCl3δ4.75-4.49(m,3H),4.05-3.95(m,4H),3.26-3.24(m,3H),2.76-2.07(m,5H),1.39-1.29(m,12H).
9.化合物1j的制备
将化合物1i(141g，201mmol，1.00eq)溶于甲醇(1.41L)中，加入HCl(704mL，1.40mol，2M，7.00eq)水溶液，反应液于60℃搅拌1小时。反应液用甲基叔丁基醚/石油醚混合液萃取。取水相，用饱和碳酸氢钠调节pH到8，将水相浓缩，向所得残留物中加入四氢呋喃并过滤，滤液浓缩 得到粗品化合物1j(62.4g,201mmol)。
10.化合物1k的制备
将化合物1j(62.4g，201mmol，1.00eq)溶于吡啶(300mL)，加入乙酸酐(47.4mL，502mmol，2.50eq)，25℃搅拌12小时。反应液用饱和碳酸氢钠(1.00L)稀释，用乙酸乙酯(600mL)萃取两次，用无水硫酸钠干燥，过滤、浓缩。粗品用硅胶柱层析纯化(洗脱液：石油醚/乙酸乙酯)得到化合物1k(74g，93.3％)。
1H NMR:400MHz CDCl3δ5.38-5.10(m,3H),4.07-4.03(m,4H),3.36-3.32(m,3H),2.41-2.05(m,5H),2.04-1.98(m,9H),1.27-1.23(m,6H).
11.化合物1l的制备
将化合物1k(79.3g，201mmol，1.00eq)溶于乙酸乙酯(476mL)，加入乙酸酐(62.6mL，663mmol，3.30eq)和浓硫酸(5.38mL，100mmol，0.50eq)。25℃搅拌3小时。反应液用饱和碳酸氢钠水溶液中和，用乙酸乙酯萃取(1.00L)两次，合并有机相，用饱和食盐水洗涤(500mL)洗涤两次，用无水硫酸钠干燥、过滤、浓缩。粗品用硅胶柱层析纯化得到化合物1l(43g，50.6％)。
1H NMR:400MHz CDCl3δ6.11(d,J＝2.8Hz,1H),5.44-5.39(m,1H),5.33-5.32(m,1H),4.10-4.05(m,5H),2.80-2.35(m,4H),2.12-2.02(m,9H),1.32-1.23(m,6H).
12.化合物1n的制备
将化合物1m(8.55g，76.2mmol，2.30eq)和双(三甲基硅基)乙酰胺(BSA，34.3g，169mmol，5.10eq)悬浮于乙腈(200mL)中，85℃搅拌1小时。降温后加入化合物1l(14.0g，33.1mmol，1.00eq)的乙腈溶液(50.0mL)，再加入四氯化锡(37.1g，142mmol，4.30eq。将混合液在25℃ 搅拌15分钟后85℃油浴搅拌45分钟。降温后，将反应液倒入饱和碳酸氢钠水溶液(1.50L)，用二氯甲烷萃取(700mL)三次，合并有机相，用饱和食盐水(500mL)洗涤，用无水硫酸钠干燥、过滤、浓缩。粗品用硅胶柱层析纯化(洗脱液：二氯甲烷/甲醇)得到化合物1n(14.4g，84.3％)。
1H NMR:400MHz CDCl3δ9.70(brs,0.46H),9.60(brs,0.60H),7.17(d,J＝8.0Hz,0.60H),7.12(d,J＝8.0Hz,0.49H),5.98(d,J＝6.4Hz,0.43H),5.93(d,J＝5.2Hz,0.55H),5.78(dt,J1＝8.0Hz,J2＝2.0Hz,1H),5.50-5.45(m,2H),4.45-4.02(m,5H),2.77-2.42(m,6H),2.18(s,1.22H),2.16(s,1.74H),2.04(s,1.31H),2.02(s,1.67H),1.32-1.28(m,6H).
13.化合物1o的制备
将化合物1n(8.30g，17.4mmol，1.00eq)溶于DMF(40.0mL)，加入DBU(2.93g，19.2mmol，1.10eq)，降温后加入苄氧甲基氯(BOMCl，3.01g，19.2mmol，1.10eq)。混合液在0～5℃下搅拌2.5小时。反应液用饱和氯化铵(160mL)稀释，用二氯甲烷(80.0mL)萃取两次，合并有机相用饱和食盐水(80mL)洗涤，用无水硫酸钠干燥、过滤、浓缩。粗品用硅胶柱层析纯化(洗脱液：二氯甲烷/甲醇)得到化合物1o(12.0g，92.3％)。
1H NMR:400MHz CDCl3δ7.36-7.24(m,5H),7.15-7.09(m,1H),5.96-5.92(m,1H),5.81-5.79(m,1H),5.52-5.42(m,4H),4.67(s,2H),4.15-4.05(m,4H),2.80-2.45(m,5H),2.19-2.17(m,3H),2.05-2.03(m,3H),1.34-1.30(m,6H).
14.化合物1p的制备
将化合物1o(10.4g，17.4mmol，1.00eq)溶于氨甲醇溶液(29.9mL，7M)，21℃搅拌1小时。反应液浓缩得到粗品，经硅胶柱层析纯化(洗脱液二氯甲烷/甲醇)得到化合物1p(8.00g，62.7％)。
1H NMR:400MHz CDCl3δ7.34-7.06(m,5H),5.83(s,0.52H),5.71(d,J＝8.4Hz,0.41H),5.661(d,J＝8.4Hz,0.53H),5.60(d,J＝2.0Hz,0.59H),5.53(d,J＝5.2Hz,0.41H),5.42-5.36(m,2H),4.62(s,2H),4.51(d,J＝3.6Hz,0.45H),3.72(d,J＝2.8Hz,0.36H),3.54(d,J＝2.0Hz,0.48H),2.93-2.40(m,5H),2.24-2.13(m,1H),1.27-1.23(m,6H).
15.化合物1q的制备
将化合物1p(1g，1.95mmol，1.00eq)溶于丙酮(19.6mL)，加入氧化银(3.63g，15.6mmol，10.0eq)和碘甲烷(1.22mL，19.5mmol，10.0eq)，在25℃搅拌24小时。反应液过滤、浓缩得到粗品化合物1q(1.36g)。
1H NMR:400MHz CDCl3δ5.11(s,1H),5.02(d,J＝5.6Hz,1H),4.06(d,J＝1.6Hz,1H),4.50(d,J＝5.6Hz,1H),4.39(d,J＝1.6Hz,1H),3.41(s,3H),3.24-3.20(m,1H),3.09(t,J＝10.0Hz,1H),1.47(s,3H),1.35(s,3H).
16.化合物1q-1和1q-2的制备
将粗品化合物1q(10.9g,20.7mmol，1.00eq)用C18反相柱层析纯化(碳酸氢铵水溶液/乙腈)纯化，得到化合物1q-1(1.50g,11.5％)和化合物1q-2(1.50g,11.5％)。
化合物1q-1：
m/z：ES+[M+H]+525.2
HPLC:保留时间0.820min(Column:XBridge C18 2.1*50mm,5um；Mobile phase:A:10mM NH4HCO3aqueous solution B:Acetonitrile；Gradient:0-0.01min 5％B,0.01-0.7min 5-95％B,0.7-1.16min 95％B,1.16-1.5min,95％-5％B；Flow rate:1.5mL/min；Column temp.:40℃)
1H NMR:400MHz CDCl3δ7.37-7.08(m,5H),7.09(d,J＝8.0Hz,1H),5.76-5.74(m,2H),5.49-5.44(m,2H),4.73-4.67(m,2H),4.15-4.06(m,4H),4.02(d,J＝4.8Hz,1H),3.87-3.85(m,1H),3.56(s,3H),2.81-2.64(m,2H),2.50-2.39(m,2H),2.36-2.27(m,1H),1.34-1.30(m,6H).
化合物1q-2：
m/z：ES+[M+H]+525.2
HPLC:保留时间0.836min(Column:XBridge C18 2.1*50mm,5um；Mobile phase:A:10mM NH4HCO3aqueous solution B:Acetonitrile；Gradient:0-0.01min 5％B,0.01-0.7min 5-95％B,0.7-1.16min 95％B,1.16-1.5min,95％-5％B；Flow rate:1.5mL/min；Column temp.:40℃)
1H NMR:400MHz CDCl3δ7.37-7.27(m,5H),7.08(d,J＝8.0Hz,1H),5.75-5.73(m,2H),5.50-5.45(m,2H),4.73-4.67(m,2H),4.18-4.08(m,4H),4.06(d,J＝4.8Hz,1H),3.88-3.86(m,1H),3.56(s,3H),3.06-2.95(m,1H),2.83-2.41(m,5H),1.34-1.31(m,6H).
17.化合物1r-1的制备
将化合物1q-1(1.00g,1.90mmol，1.00eq)溶于无水二氯甲烷DCM(20.0mL)，降温至-60℃。加入三氯化硼(7.62mL,7.62mmol，1M二氯甲烷溶液，4.00eq)，在-20℃搅拌1小时。将反应液用无水乙醇(10.0mL)淬灭，用浓氨水中和至pH约为7，浓缩。粗品加入二氯甲烷和乙醇的混合溶液并过滤，滤液浓缩，所得残留物经C18反相柱层析纯化(碳酸氢铵水溶液/乙腈)纯化，得到化合物1r-1(400mg，51.9％)。
m/z：ES+[M+H]+405.3
1H NMR:400MHz CDCl3δ8.51-8.43(m,1H),7.15(d,J＝8.4Hz,1H),5.78(d,J＝3.2Hz,1H),5.76(dd,J1＝8.0Hz,J2＝2.4Hz,1H),4.16-4.07(m,5H),3.94(dd,J1＝4.8Hz,J2＝3.2Hz,1H),3.55(s,3H),2.96-2.94(m,1H),2.79-2.66(m,2H),2.52-2.41(m,2H),2.35-2.26(m,1H),1.34-1.31(m,6H).
18.化合物1r-2的制备
采用与化合物1r-1相同合成方法得到化合物1r-2。
1H NMR:400MHz CDCl3δ9.16-8.81(m,1H),7.15(d,J＝8.0Hz,1H),5.78(d,J＝2.8Hz,1H),5.76(dd,J1＝8.0Hz,J2＝2.0Hz,1H),4.65(brs,1H),4.18-4.08(m,5H),3.96(dd,J1＝4.8Hz,J2＝3.2Hz,1H),3.53(s,3H),3.05-2.95(m,1H),2.81-2.41(m,2H),1.34-1.30(m,6H).
19.化合物E1-1的制备
将化合物1r-1(500mg，1.23mmol，1.00eq)溶于二氯甲烷(5.00mL)，加入化合物1s(0.59mL，1.85mmol，1.50eq)，降温至0℃后加入4,5-二氰基咪唑(160mg，1.36mmol，1.10eq)，15℃搅拌4小时。将反应液用饱和碳酸氢钠淬灭(10.0mL)，用二氯甲烷萃取(10.0mL)两次。浓缩得到粗品，经硅胶柱层析纯化(洗脱液：二氯甲烷/丙酮)得到化合物E1-1(530mg，70.9％)。
1H NMR:400MHz CDCl3δ9.17(brs,1H),7.24(d,J＝8.4Hz,1H),5.83-5.79(m,1H),5.64-5.61(m,1H),4.43-4.33(m,1H),4.09-3.62(m,9H),3.43-3.38(m,3H),2.86-2.66(m,3H),2.57-2.21(m,4H),1.28-1.18(m,18H).
20.化合物E1-2的制备
将化合物1r-2(500mg，1.23mmol，1.00eq)溶于二氯甲烷(5.00mL)，加入化合物1s(0.59mL，1.85mmol，1.50eq)，降温至0℃后加入4,5-二氰基咪唑(160mg，1.36mmol，1.10eq)，15℃搅拌4小时。将反应液用饱和碳酸氢钠淬灭(10.0mL)，用二氯甲烷萃取(10.0mL)两次。浓缩得到粗品，经硅胶柱层析纯化(洗脱液：二氯甲烷/丙酮)得到化合物E1-2(480mg，64.0％)。
1H NMR:400MHz CDCl3δ9.06(brs,1H),7.33-7.31(m,1H),5.89-5.88(m,1H),5.66-5.64(m,1H),4.49-4.40(m,1H),4.08-3.64(m,9H),3.40-3.36(m,3H),3.00-2.19(m,7H),1.29-1.18(m,18H).
实施例2 化合物E2-1和E2-2的制备
1.化合物2c的制备
氮气气氛下，在-70至-65℃下将化合物2a(86.0g,493mmol)的THF(250mL)溶液滴入LDA的THF溶液(271mL,543mmol,2M)中，30分钟后，加入化合物2b(142g,543mmol)的THF(250mL)溶液搅拌1小时。反应完成后将反应液倾入饱和氯化铵水溶液(2L)中，甲基叔丁基醚萃取三次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，减压真空浓缩后，所得残留物经硅胶柱层析纯化(洗脱液：石油醚/乙酸乙酯)得到化合物2c(113g，70.6％)。
1H NMR:400MHz DMSO-d6δ7.35-7.31(m,2H),7.28-7.24(m,3H),4.36(s,2H),3.52(s,3H),3.39-3.36(m,2H),3.03(s,3H),3.00(s,3H),2.46-2.45(m,1H),2.44-2.43(m,1H),2.12-2.11(m,1H),2.10-2.09(m,1H),2.03-2.00(m,2H).
2.化合物2d的制备
15℃下，将1M盐酸(217mL，217mmol)加入到化合物2c(13.4g,43.4mmol)的THF(200mL)中，搅拌24小时。向反应液中加入乙酸乙酯萃取三次，合并有机相，饱和碳酸氢钠水溶液洗涤两次，饱和食盐水洗涤，无水硫酸钠干燥，减压真空浓缩后，所得残留物经硅胶柱层析纯化(洗脱液：石油醚/乙酸乙酯)得到化合物2d(9.9g，76.7％)。
1H NMR:400MHz CDCl3δ7.36-7.26(m,5H),4.45(s,2H),3.68(s,3H),3.59-3.56(m,3H),3.53-3.49(m,1H),3.10-3.08(m,1H),3.06-3.04(m,1H),2.26(t,J＝6.0Hz,2H).
3.化合物2f的制备
在0至5℃下，将DBU(1.15g，7.54mmol)加入到化合物2d(9.90g,37.7mmol)和化合物2e((6.25g,45.2mmol)的二氯甲烷(20mL)中，15℃下搅拌24小时。将反应液减压浓缩，所得残留物经硅胶柱层析纯化(洗脱液：石油醚/乙酸乙酯/二氯甲烷)得到化合物2f(15.5g,92.3％)。
1H NMR:400MHz CDCl3δ7.34-7.26(m,5H),4.42-4.41(m,2H),4.20-4.11(m,5H),3.64-3.60(m,3H),3.49-3.45(m,1H),3.44-3.41(m,1H),3.11-3.04(m,1H),2.70-2.56(m,2H),2.28-2.25(m,1H),2.20-2.04(m,2H),1.38-1.30(m,6H).
4.化合物2g的制备
将化合物2f(14.5g,36.2mmol)、DMAP(6.64g,54.3mmol)溶于乙腈(100mL)，在5℃加入草酰氯单甲酯(5.00mL,54.3mmol)。15℃搅拌半小时。将反应液浓缩，粗品用乙酸乙酯溶解，依次用饱和氯化铵洗涤、食盐水洗涤，用无水硫酸钠干燥，减压浓缩得到粗品化合物2g(16.4g)
5.化合物2h的制备
将化合物2g(17.5g,35.9mmol)溶于无水甲苯(175mL)，加入三正丁基锡氢(12.5g,43.1mmol)和偶氮二异丁腈(AIBN，0.30g,1.79mmol)。回流2小时。将反应液浓缩，粗品用硅胶柱层析纯化(洗脱液：石油醚/乙酸乙酯)得到化合物2h(11.3g，79.9％(两步))。
1H NMR:400MHz CDCl3δ7.34-7.24(m,5H),4.43-4.41(m,2H),3.63-3.60(m,3H),3.49-3.40(m,2H),2.79-2.59(m,3H),2.39-2.28(m,1H),2.17-2.13(m,2H),1.32-1.29(m,6H).
6.化合物2i的制备
将化合物2h(8.80g,22.8mmol)溶于THF(176mL)和甲醇(3.1mL，76mmol)中，在0℃加入硼氢化锂(2.49g,114mmol)，15℃搅拌2小时。向反应液中加入水(100mL)，1M盐酸调节pH至3，二氯甲烷萃取三次。合并有机相，依次用饱和碳酸氢钠水溶液和饱和食盐水洗涤，无水硫酸钠干燥，浓缩，粗品经硅胶柱层析纯化(洗脱液：二氯甲烷/甲醇)得到化合物2i(7.86g,52.4％)。
1H NMR:400MHz DMSO-d6δ7.35-7.25(m,5H),4.70-4.68(m,1H)4.44-4.42(m,2H),3.97-3.92(m,4H),3.49-3.26(m,3H),2.60-2.50(m,1H),1.96-1.67(m,5H),1.21-1.18(m,6H).
7.化合物2j的制备
将化合物2i(7.86g,22.0mmol)溶于吡啶(47mL)，在5℃加入苯甲酰氯(2.81mL,24.2mmol)。15℃搅拌1小时。将反应液浓缩，残留物用乙酸乙酯(40mL)溶解，依次用2M盐酸、饱和碳酸氢 钠水溶液、饱和食盐水洗涤，无水硫酸钠干燥、过滤、浓缩得到粗品化合物2j(10g)。
1H NMR:400MHz CDCl3δ8.03-7.98(m,2H),7.54-7.43(m,1H),7.42-7.39(m,2H),7.27-7.23(m,5H),4.45-4.44(m,2H),4.33-4.22(m,2H),4.11-4.03(m,4H),3.56-3.51(m,2H),2.72-2.62(m,1H),2.45-2.05(m,4H),1.97-1.93(m,1H),1.30-1.27(m,6H).
8.化合物2k的制备
将化合物2j(8.00g,17.3mmol)溶于二氯甲烷(160mL)，-60℃下加入三氯化硼(69.4mL,69.4mmol)的二氯甲烷溶液(1M)，-20℃下反应1小时。向反应液中加入乙醇(40.5mL,694mmol)，再加入饱和碳酸氢钠水溶液。取有机相依次用饱和碳酸氢钠水溶液、饱和食盐水洗涤，无水硫酸钠干燥、过滤、浓缩，所得残留物用硅胶柱层析纯化(洗脱液：石油醚/乙酸乙酯，二氯甲烷/甲醇)得到化合物2k(6.10g,85.3％)。
1H NMR:400MHz CDCl3δ8.06-7.99(m,2H),7.59-7.53(m,1H),7.47-7.42(m,2H),4.35-4.28(m,2H),4.16-4.03(m,4H),3.83-3.74(m,2H),2.78-2.65(m,1H),2.50-2.33(m,2H),2.21-2.06(m,2H),2.00-1.97(m,1H),1.86-1.82(m,1H),1.37-1.27(m,6H).
9.化合物2m的制备
将化合物2k(5.80g,15.6mmol)、化合物2l(3.55g,16.4mmol)和三苯基膦(4.93g,18.7mmol)溶于四氢呋喃(58mL)中，5℃下加入偶氮二甲酸二乙酯DEAD(3.27g,18.7mmol)，反应液在15℃下搅拌2小时。向反应液中加入水，乙酸乙酯萃取两次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥、过滤，减压浓缩，所得残留物得到用甲基叔丁基醚/石油醚混合液萃取。取水相，用饱和碳酸氢钠调节pH到8，将水相浓缩，所得残留物用硅胶柱层析纯化(洗脱液：石油醚/乙酸乙酯，二氯甲烷/甲醇)得到化合物2m(7.40g,61.9％)。
1H NMR:400MHz CDCl3δ8.06-8.00(m,2H),7.94-7.91(m,2H),7.66-7.51(m,2H),7.42-7.32(m,5H),7.32-7.29(m,1H),5.80-5.77(m,1H),4.39-4.38(m,2H),4.12-4.02(m,5H),3.87-3.83(m,2H),2.79-2.67(m,1H),2.44-1.95(m,6H),1.33-1.25(m,6H).
10.化合物2n的制备
将化合物2m(3.00g,5.27mmol)溶于氨甲醇溶液(150mL,7M)中，60℃下反应48小时。将反应液浓缩，残留物用硅胶柱层析纯化(洗脱液：二氯甲烷/甲醇)得到粗品化合物2n(3.10g)。
1H NMR:400MHz DMSO-d6δ11.21(brs,1H),7.69-7.63(m,1H),5.56-5.52(m,1H),4.80-4.77(m,1H),3.98-3.93(m,4H),3.60-3.69(m,2H),3.42(d,J＝5.2HZ,1H),2.75-2.55(m,1H),1.93-1.87(m,6H),1.22-1.18(m,6H).
11.化合物2n-1和2n-2的制备
化合物2n(3.70g,10.2mmol)通过SFC纯化得到化合物2n-1(2.00g,50.7％)和化合物2n-2(1.00g,24.1％)。纯化柱：DAICEL CHIRALPAK AD(250mm*30mm,10um)；流动相：[A:CO2；B:IPA]；B％:40.00％-40.00％,3.00min。
化合物2n-1：
HPLC:保留时间3.065min(柱:Chiralpak AD-3,150×4.6mm I.D.,3um；流动相:A：CO2,B:IPA(0.2％NH3(7M甲醇溶液),v/v)；梯度:0-0.5min 10％B,0.5-3.5min 10-50％B,3.5-4.5min 50％B,4.5-5.0min,50％-10％B；流速:2.5mL/min；柱温度:35℃)
1H NMR:400MHz DMSO-d6δ11.20(brs,1H),7.64(d,J＝8.0Hz,1H),5.53(d,J＝8.0Hz,1H),4.78(t,J＝5.2Hz,1H),3.99-3.93(m,4H),3.55-3.75(m,2H),3.42(d,J＝5.6Hz,2H),2.70-2.60(m,1H),1.93-1.87(m,4H),1.68-1.67(m,2H),1.22-1.18(m,6H).
化合物2n-2：
HPLC:保留时间3.626min(Column:Chiralpak AD-3,150×4.6mm I.D.,3um；流动相:A：CO2,B:IPA(0.2％NH3(7M甲醇溶液),v/v)；梯度:0-0.5min 10％B,0.5-3.5min 10-50％B,3.5-4.5min 50％B,4.5-5.0min,50％-10％B；流速:2.5mL/min；柱温度:35℃)
1H NMR:400MHz DMSO-d6δ11.22(brs,1H),7.68(d,J＝8.0Hz,1H),5.56-5.53(m,1H),4.68(t,J＝5.2Hz,1H),4.00-3.91(m,4H),3.68-3.64(m,2H),3.31-3.30(m,2H),2.74-2.62(m,1H),2.03-1.93(m, 2H),1.88-1.75(m,4H),1.22-1.19(m,6H).
12.化合物E2-1的制备
将化合物2n-1(1.00g,2.77mmol)溶于二氯甲烷(10mL)，加入化合物1s(1.32mL,4.16mmol)，降温至0℃后加入4,5-二氰基咪唑DCI(360mg,3.05mmol)，15℃搅拌1小时。将反应液用饱和碳酸氢钠淬灭(20.0mL)，用二氯甲烷萃取(20.0mL),饱和食盐水洗涤，无水硫酸钠干燥，浓缩所得粗品经硅胶柱层析纯化(洗脱液：二氯甲烷/丙酮)得到化合物E2-1(1.41g，89.5％)。
1H NMR:400MHz DMSO-d6δ11.21(brs,1H),7.63(d,J＝8.0Hz,1H),5.55(d,J＝8.0Hz,1H),3.98-3.94(m,4H),3.80-3.62(m,8H),2.79-2.76(m,3H),2.06-1.93(m,4H),1.80-1.75(m,2H),1.21-1.14(m,18H).
13.化合物E2-2的制备
将化合物2n-2(780mg,2.16mmol)溶于二氯甲烷(7.8mL)，加入化合物1s(936mg,3.24mmol)，降温至0℃后加入4,5-二氰基咪唑(280mg,2.37mmol)，15℃搅拌1小时。将反应液用饱和碳酸氢钠淬灭(20.0mL)，用二氯甲烷萃取(20.0mL),饱和食盐水洗涤，无水硫酸钠干燥，浓缩所得粗品经硅胶柱层析纯化(洗脱液：二氯甲烷/丙酮)得到化合物E2-2(860mg，60.7％)。
1H NMR:400MHz DMSO-d6δ11.21(brs,1H),7.66(d,J＝8.0HZ,1H),5.55(d,J＝8.0Hz,1H),4.03-3.90(m,4H),3.81-3.66(m,4H),3.61-3.31(m,4H),2.80-2.67(m,3H),2.05-1.84(m,6H),1.21(t,J＝7.2Hz,6H),1.15-1.13(m,12H).
实施例3 化合物E3的制备
化合物E3按照上述步骤进行制备。
实施例4 siRNA的制备
使用本领域熟知的固相亚磷酰胺法制备本发明的siRNA。具体方法可参见例如PCT公开号WO2016081444和WO2019105419，并简述如下。
1正义链(SS链)的合成
通过固相亚磷酰胺合成法，利用空白的CPG固相载体或连接有L96的固相载体做为起始循环，按照正义链核苷酸排布顺序自3’-5’方向逐一连接核苷单体。每连接一个核苷单体都包含了脱保护、偶联、盖帽、氧化或硫代四步反应，合成规模为5umol的寡核酸合成条件如下：
核苷单体提供的是0.05mol/L的乙腈溶液，每一步反应的条件相同，即温度为25℃，脱保护使用3％的三氯乙酸-二氯甲烷溶液，脱保护3次；偶联反应使用的活化剂为0.25mol/L的ETT-乙腈溶液，偶联2次；盖帽使用10％醋酐-乙腈和吡啶/N-甲基咪唑/乙腈(10:14:76,v/v/v)，盖帽2次；氧化使用0.05mol/L的碘/四氢呋喃/吡啶/水(70/20/10,v/v/v)，氧化2次；硫代使用0.2mol/L PADS的乙腈/3-甲基吡啶(1/1,v/v)，硫代2次。
2反义链(AS链)的合成
通过固相亚磷酰胺合成法，利用空白的CPG固相载体做为起始循环，按照反义链核苷酸排布顺序自3’-5’方向逐一连接核苷单体或本发明的核苷酸双聚体。每连接一个核苷单体或本发明的核苷酸双聚体都包含了脱保护、偶联、盖帽、氧化或硫代四步反应，反义链的5μmol的寡核酸合成条件和正义链的相同。
3寡核苷酸的纯化与退火
3.1氨解
将合成好的固相载体(正义链或者反义链)加入到5mL的离心管中，加入3％的二乙胺/氨水(v/v)，35℃(或者55℃)恒温水浴下反应16小时(或者8小时)，过滤，固相载体用乙醇/水洗涤三次，每次1mL，滤液离心浓缩后粗品进行纯化。
3.2纯化
纯化和脱盐的方法是本领域人员所熟知的。例如，可采用强阴离子填料装柱，氯化钠-氢氧化钠体系进行洗脱纯化，产品收集并管，可采用凝胶填料纯化柱进行脱盐，洗脱体系是纯水。
3.3退火
根据下表将正义链(SS链)链与反义链(AS链)以摩尔比(SS链/AS链＝1/1.05)混合，水浴锅加热至70-95℃，保持3-5min，自然冷却至室温，将体系冻干得到产品。
本发明中使用的siRNA序列如下：

本文中，各缩写的意义如下：
A、U、G和C分布表示天然的腺嘌呤核糖核苷酸、尿嘧啶核糖核苷酸、鸟嘌呤核糖核苷酸和胞嘧啶核糖核苷酸。
m表示其左侧相邻的核苷酸是2‘-OCH3修饰的核苷酸。例如，Am、Um、Gm和Cm表示2‘-OCH3修饰的A、U、G和C。
f表示其左侧相邻的核苷酸是2‘-F修饰的核苷酸。例如，Af、Uf、Gf和Cf分别表示2‘-F修饰的A、U、G和C。
“s”或“s-”表示其左右相邻的两个核苷酸和/或递送载体通过硫代磷酸酯连接。
L96表示本领域熟知的以下结构的GalNAc递送载体，其中表示通过磷酸酯基团或硫代磷酸酯基团与siRNA连接的位置，可参见例如PCT公开号WO2009073809和WO2009082607。
VP表示其右侧相邻的核苷酸是乙烯基膦酸酯修饰的核苷酸，是本领域熟知的，可参见例如PCT公开号WO2011139702、WO2013033230和WO2019105419。
SCP1a、SCP1b、SCP2a和SCP2b表示上文所述结构的核苷酸替代物，其中Base可以是任何碱基，例如SCP1a-U表示Base为尿嘧啶。
实施例5 C57BL/6野生型小鼠原代肝细胞(PMH)活性筛选实验
1.自由摄取或转染
分离C57BL/6野生型小鼠肝原代细胞，计数，24孔板铺板，900μL/孔，8×104cells/孔；96孔板铺板，100μL/孔，1×104cells/孔。之后选择自由摄取或转染。
自由摄取：将10μL稀释化合物加入90μL Opti-MEM中混匀，加入对应孔中，37℃、5％CO2培养箱中培养24小时。对照组不加siRNA。
转染：将10μL稀释化合物加入40μL Opti-MEM中混匀，3μL RNAiMAX加入47μL Opti-MEM中混匀，孵育5min后，与稀释好的化合物混匀，室温静置10min，加入对应孔中，37℃、5％CO2培养箱中培养24小时。对照组不加siRNA。
2.荧光定量PCR
使用高通量核酸提取仪-磁珠法(凡知医疗，FG0412；杭州奥盛，Auto-pure96)提取总RNA，反转录(PrimeScriptTM II 1st Strand cDNA Synthesis Kit(Takara，6210B))后进行荧光定量PCR检测(TaqManTM Fast Advanced Master Mix(ABI，4444965))。
表1 引物信息
3.数据统计
计算2-△△Ct值并换算成百分比以得到剩余抑制率；
△△Ct＝[(Ct实验组目的基因-Ct实验组内参)-(Ct对照组目的基因-Ct对照组内参)]。
其中目的基因为mAPOB或mFXII，对照基因为mGAPDH。
4.mAPOB基因实验结果
使用转染的方法，选取C57BL/6野生型小鼠原代肝细胞，24孔板铺板细胞后选择化合物起始浓度为40nM，10倍稀释5个浓度点(40nM、4nM、0.4nM、0.04nM、0.004nM)进行C57BL/6野生型小鼠原代肝细胞5点IC50活性筛选，实验结果见表2。
表2 携带本发明的核苷酸的靶向mAPOB基因的siRNA化合物PMH 5点IC50活性筛选实验结果
5.mFXII基因实验结果
使用自由摄取的方法，选取C57BL/6野生型小鼠原代肝细胞，96孔板铺板细胞后选择化合物起始浓度为100nM，10倍稀释5个浓度点(100nM、10nM、1nM、0.1nM、0.01nM)进行C57BL/6野生型小鼠原代肝细胞5点IC50活性筛选-自由摄取，实验结果见表3。
表3 携带本发明的核苷酸的靶向mFXII基因的siRNA化合物PMH 5点IC50活性筛选实验结果
实施例6 C57BL/6小鼠模型的化合物药效验证
将C57BL/6小鼠(雄性，18～21g,6～8周，斯贝福(苏州)生物技术有限公司)进行随机分组，每组9只动物(其中第14天，第28天会安乐死小鼠(N＝3/组)取肝脏)，每只动物根据体重计算给药剂量，采用皮下注射方式单次给药，siRNA缀合物以10mg/mL的溶液(0.9％氯化钠水溶液作为溶剂)提供给CRO公司；具体地，在实验前，用0.9％氯化钠水溶液将siRNA缀合物溶解且定容至所需溶液浓度和体积，生理盐水和siRNA缀合物的给药体积为5mL/kg。
分别于给药前(记为第-2天)取血检测，以第2天的LDL进行分组，剩余样本留存备用于检测目的蛋白；及给药(记为第0天)后第7、14和21天对小鼠眼眶静脉丛取血(每次取血前均进行饥饿处理5小时)，苏州安领在各个时间点直接法(东软全自动生化分析仪，NT-1000)检测血清LDL浓度(美康品牌，低密度脂蛋白胆固醇检测试剂)，剩余样本留存备用于ELISA方法检测目的蛋白(Mouse ApoB ELISA Kit，Abcam，ab230932)；其中第14、28和42天取肝脏10mg(n＝3/组/时间点)放于RNAlater溶液中，-80℃冻存，干冰寄送用于抽提和检测肝脏mRNA表达(检测引物见表4，需要后续时间点更新)。实验结果见表5。
表4 引物信息
表5 3条siRNA化合物C57BL/6小鼠模型的化合物药效

实施例7 C57BL/6野生型小鼠原代肝细胞(PMH)活性筛选实验
根据实施例3的方法，测试了更多的本发明的化合物。
表6 携带本发明的化合物的靶向mAPOB基因的siRNA化合物PMH 5点IC50活性筛选实验结果
表7 携带本发明的化合物的靶向mFXII基因的siRNA化合物PMH 5点IC50活性筛选实验结果
实施例8 分子模拟
通过分子模拟，我们观察到本发明的化合物可将膦酸锁定在与未修饰的5’-磷酸盐结合Ago2高度相似的ap构象，其中的膦酸盐基团与Arg812，Lys570，Lys566及Lys533完美匹配，形成了多个盐桥键和氢键，同时还与Tyr529及Cys526各形成了一个氢键。此外，该螺环膦酸化合物中的碱基(尿嘧啶)与Thr526和Gly524的骨架酰胺各形成一个氢键。这些结构的共同左右稳定了本发明的化合物与Ago2蛋白的结合。
分子模拟显示，与螺环化合物类似，糖环稠合高度刚性的芳香环(包括五元芳香环和六元芳香环)后，将5’末端膦酸盐锁定在了特定的构象上，该构象高度模拟了天然5’-磷酸盐的ap构象，膦酸盐基团与Arg812，Lys570，Lys566及Lys533完美匹配，形成了多个盐桥键和氢键，同样还与Tyr529及Cys526各形成了一个氢键。糖环上碱基尿嘧啶同样与Thr526和Gly524的骨架酰胺各形成一个氢键。这些结构的共同左右稳定了本发明的化合物与Ago2蛋白的结合。

Claims (29)

  1. 一种寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，所述寡核苷酸的5’末端包含式(I)所示的结构：
    其中，
    表示连接至所述寡核苷酸的剩余部分，优选连接至修饰或未修饰的核苷；
    X和Y各自独立地选自CRaRb或(CRaRb)2，优选为CRaRb；
    Z为CRa；
    Ra和Rb独立地选自H、D、卤素、OH、NH2、CN、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C2-6烯基或C2-6炔基；所述Ra和Rb任选地被1个、2个、3个、4个或5个独立选择的R#进一步取代；
    R1选自O或S；
    R2和R3独立地选自OH、SH、NH2、C1-6烷基、C1-6卤代烷基、C1-6烷氧基或C1-6卤代烷氧基；所述R2和R3任选地被1个、2个、3个、4个或5个独立选择的R#进一步取代；
    R#选自H、D、卤素、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基；
    其中上述各基团定义任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
  2. 权利要求1的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
    表示连接至所述寡核苷酸的剩余部分，优选连接至修饰或未修饰的核苷；
    X和Y各自独立地选自CRaRb或(CRaRb)2，优选为CRaRb；
    Z为CRa；
    Ra和Rb独立地选自H、D、卤素、OH、NH2、CN、C1-6烷基、C1-6卤代烷基、C1-6烷氧基或C1-6卤代烷氧基；
    R1选自O或S；
    R2和R3独立地选自OH、SH、C1-6烷氧基或C1-6卤代烷氧基；所述R2和R3任选地被1个、2个或3个独立选择的R#进一步取代；
    R#选自H、D、卤素、C1-6烷基和C1-6卤代烷基。
  3. 权利要求1或2的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
    表示连接至所述寡核苷酸的剩余部分，优选连接至修饰或未修饰的核苷；
    X和Y各自独立地选自CRaRb或(CRaRb)2，优选为CRaRb；
    Z为CRa；
    Ra和Rb独立地选自H、D、卤素、C1-4烷基或C1-4卤代烷基；
    R1选自O或S；
    R2和R3独立地选自OH或SH，优选为OH。
  4. 权利要求1-3中任一项的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
    表示连接至所述寡核苷酸的5’末端基团，优选连接至修饰或未修饰的核苷；
    X和Y为CH2；
    Z为CH；
    R1为O；
    R2和R3为OH。
  5. 权利要求1的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其具有式(II)的结构：
    其中，
    表示连接至所述寡核苷酸的剩余部分；
    X和Y为CRaRb；
    Z为CRa；
    L1选自化学键、O、S或C1-4亚烷基；
    L2和L3独立地选自C1-4亚烷基；
    所述L1、L2和L3任选独立地被1个、2个、3个、4个或5个独立选择的R#取代；
    U选自O、S、CRaRb或NRc；
    Ra和Rb独立地选自H、D、卤素、OH、NH2、CN、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C2-6烯基或C2-6炔基；
    Rc独立地选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基；
    所述Ra、Rb和Rc任选地被1个、2个、3个、4个或5个独立选择的R#进一步取代；
    或者，L2和L3连接，与L1及其相邻的碳原子一起形成C5-6环烷基或5-6元杂环基，优选U、L1、L2和L3及其相邻的碳原子一起形成核糖或脱氧核糖，其任选地被R5取代；
    R1选自O或S；
    R2和R3独立地选自OH、SH、NH2、C1-6烷基、C1-6卤代烷基、C1-6烷氧基或C1-6卤代烷氧基；所述R2和R3任选地被1个、2个、3个、4个或5个独立选择的R#取代；
    R5选自H、D、卤素、C1-6烷氧基或C1-6卤代烷氧基；
    R#选自H、D、卤素、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基；
    Base选自H、修饰或未修饰的碱基；
    其中上述各基团定义任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
  6. 权利要求5的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
    表示连接至所述寡核苷酸的剩余部分；
    X和Y为CRaRb；
    Z为CRa；
    Ra和Rb独立地选自H、D、卤素、OH、NH2、CN、C1-6烷基、C1-6卤代烷基、C1-6烷氧基或C1-6卤代烷氧基；
    L1选自化学键、O或C1-4亚烷基；
    L2和L3独立地选自C1-4亚烷基；
    所述L1、L2和L3任选独立地被1个、2个或3个独立选择的R#取代；
    U选自O、S、NH或CH2；
    或者，L2和L3连接，与L1及其相邻的碳原子一起形成C5-6环烷基或5-6元杂环基，优选U、L1、L2和L3及其相邻的碳原子一起形成核糖或脱氧核糖，其任选地被R5取代；
    R1选自O或S；
    R2和R3独立地选自OH、SH、C1-6烷氧基或C1-6卤代烷氧基；所述R2和R3任选地被1个、2个或3个独立选择的R#取代；
    R5选自H、D、C1-6烷基、C1-6卤代烷基、C1-6烷氧基或C1-6卤代烷氧基；
    R#选自H、D、卤素、C1-6烷基和C1-6卤代烷基；
    Base选自H、修饰或未修饰的碱基。
  7. 权利要求5或6的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
    表示连接至所述寡核苷酸的剩余部分；
    X和Y为CRaRb；
    Z为CRa；
    Ra和Rb独立地选自H、D、卤素、C1-4烷基或C1-4卤代烷基；
    L1选自化学键或C1-4亚烷基；
    L2和L3独立地选自C1-4亚烷基；
    U选自O或S；
    R1选自O或S；
    R2和R3独立地选自OH或SH，优选为OH；
    Base选自
  8. 权利要求5-7中任一项的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
    表示连接至所述寡核苷酸的剩余部分；
    X和Y为CH2；
    Z为CH；
    L1为化学键；
    L2和L3独立地选自C1-2亚烷基；
    U为O；
    R1为O；
    R2和R3为OH；
    Base选自
  9. 权利要求1的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，所述寡核苷酸的5’末端包含式(III)、式(IV)或式(V)所示的核苷酸单体：
    其中，
    Q选自H、D、卤素、OH、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C2-6烯基或C2-6炔基；
    R4、R5、R6和R7独立地选自H、D、卤素、OH、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C2-6烯基或C2-6炔基；
    Base选自H、修饰或未修饰的碱基；
    L1、L3、X、Y、Z、R1、R2、R3、U如权利要求5-8中任一项所定义；
    其中上述各基团定义任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
  10. 权利要求9的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
    Q选自H、D、卤素、OH、C1-6烷基、C1-6卤代烷基、C1-6烷氧基或C1-6卤代烷氧基；
    R4、R5、R6和R7独立地选自H、D、卤素、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基；
    Base选自H、修饰或未修饰的碱基，优选
    L1、L3、X、Y、Z、R1、R2、R3、U如权利要求5-8中任一项所定义。
  11. 权利要求9或10的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中：
    Q选自H、卤素或C1-4烷氧基，优选为H、F或甲氧基；
    R4、R5、R6和R7独立地选自H、卤素、C1-4烷基或C1-4卤代烷基，优选为H；
    Base选自优选为
    L1、L3、X、Y、Z、R1、R2、R3、U如权利要求5-8中任一项所定义。
  12. 权利要求9-11中任一项的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中，所述寡核苷酸的5’末端包含具有以下结构的核苷酸单体：
    其中，各基团如权利要求9-11中任一项所定义。
  13. 权利要求9-12中任一项的寡核苷酸或其药学上可接受的盐、互变异构体或立体异构体，其中，所述寡核苷酸的5’末端包含具有以下结构的核苷酸单体：
    其中，Base选自
  14. 式(VI)、式(VII)或式(VIII)所示的化合物或其药学上可接受的盐、互变异构体或立体异构体：
    其中，
    R1选自O或S；
    R2和R3独立地选自ORd、OP1、SRd、SP1或NReRf；
    Rd选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被D、卤素、C1-6烷基或C1-6卤代烷基取代，直至完全氘代；
    Re和Rf独立地选自H、C1-6烷基或C1-6卤代烷基，所述Re和Rf可任选地被D、卤素、C1-6烷基或C1-6卤代烷基取代，直至完全氘代；
    P1选自保护基，优选为羟基保护基；
    P2选自反应性磷基团，优选-P(OCH2CH2CN)(N(iPr)2)；
    Base’独立地选自H、修饰或未修饰的碱基或离去基；
    L1、L3、X、Y、Z、U、Q、R4、R5、R6和R7如权利要求5-11中任一项所定义；
    其中上述各基团定义任选地被1个、2个、3个、4个、5个或更多个氘原子取代，直至完全氘代。
  15. 权利要求14的化合物或其药学上可接受的盐、互变异构体或立体异构体，其中：
    R1选自O或S；
    R2和R3独立地选自ORd、OP1、SRd、SP1或NReRf；
    Rd选自H、C1-4烷基或C1-4卤代烷基，其任选地被D或卤素取代，直至完全氘代；
    Re和Rf独立地选自H、C1-4烷基或C1-4卤代烷基，所述Re和Rf可任选地被D或卤素取代，直至完全氘代；
    P1选自保护基，优选为羟基保护基；
    P2选自反应性磷基团，优选-P(OCH2CH2CN)(N(iPr)2)；
    Base’选自
    L1、L3、X、Y、Z、U、Q、R4、R5、R6和R7如权利要求5-11中任一项所定义。
  16. 权利要求14或15的化合物或其药学上可接受的盐、互变异构体或立体异构体，其中：
    R1选自O或S，优选为O；
    R2和R3独立地选自ORd或OP1；
    Rd选自H、C1-4烷基或C1-4卤代烷基；
    Re和Rf独立地选自H、C1-4烷基或C1-4卤代烷基；
    P1选自保护基，优选为羟基保护基；
    P2选自反应性磷基团，优选-P(OCH2CH2CN)(N(iPr)2)；
    Base’选自更优选为
    L1、L3、X、Y、Z、U、Q、R4、R5、R6和R7如权利要求5-11中任一项所定义。
  17. 权利要求14-16中任一项的化合物或其药学上可接受的盐、互变异构体或立体异构体，其中，所述化合物选自：
  18. 权利要求1-13中任一项的寡核苷酸，其中，所述寡核苷酸为单链，其具有14至30个核苷酸。
  19. 权利要求1-13中任一项的寡核苷酸，其中，所述寡核苷酸为双链RNA，其包含正义链和反义链，其中各链具有14至30个核苷酸，所述反义链具有与所述正义链和靶标mRNA充分互补的序列。
  20. 权利要求19的寡核苷酸，其中，所述双链RNA在反义链中包含权利要求1-12中任一项所述的式(I)、(II)、(III)、(IV)、(V)、(IIIa)、(IIIb)、(IVa)、(IVb)、(Va)或(Vb)的结构或者权利要求13所述的核苷酸单体。
  21. 权利要求19-20中任一项的寡核苷酸，其中所述双链RNA进一步偶联至配体，优选地，该配体包含一个或多个GalNAc。
  22. 一种核酸分子，所述核酸分子的核苷酸序列中包含一个或多个如权利要求9-13中任一项所述的核苷酸单体。
  23. 权利要求22的核酸分子，其中所述核酸选自DNA、RNA和DNA/RNA杂合体。
  24. 权利要求23的核酸分子，所述核酸分子是单链的或双链的。
  25. 权利要求22-24中任一项的核酸分子，其中所述核酸分子选自小干扰RNA(siRNA)和短发夹RNA(shRNA)。
  26. 细胞，其含有如权利要求19-21中任一项所述的双链RNA。
  27. 一种药物组合物，其包含如权利要求19-21中任一项所述的双链RNA分子，和药学上可接受的载体或赋形剂。
  28. 一种试剂盒，其包含如权利要求19-21中任一项所述的双链RNA分子。
  29. 一种用于抑制细胞中靶基因的表达的方法，包括将权利要求19-21中任一项所述的双链RNA分子引入该细胞的步骤。
PCT/CN2023/099987 2022-06-14 2023-06-13 环膦酸酯修饰的核苷酸 WO2023241587A1 (zh)

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Citations (2)

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US20160046939A1 (en) * 2010-04-28 2016-02-18 Isis Pharmaceuticals, Inc. 5' modified nucleosides and oligomeric compounds prepared therefrom
CN109526222A (zh) * 2016-06-06 2019-03-26 箭头药业股份有限公司 5’-环膦酸酯修饰核苷酸

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