WO2024032608A1 - Sirna molecule for regulating angptl3 gene activity - Google Patents

Sirna molecule for regulating angptl3 gene activity Download PDF

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WO2024032608A1
WO2024032608A1 PCT/CN2023/111739 CN2023111739W WO2024032608A1 WO 2024032608 A1 WO2024032608 A1 WO 2024032608A1 CN 2023111739 W CN2023111739 W CN 2023111739W WO 2024032608 A1 WO2024032608 A1 WO 2024032608A1
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seq
sirna
antisense strand
nhc
chain
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PCT/CN2023/111739
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French (fr)
Chinese (zh)
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黄金宇
岳明
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大睿生物医药科技(上海)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • This disclosure relates to the field of RNA interference.
  • Angiopoietins are a family of secreted growth factors. Together with its corresponding endothelial-specific receptors, angiopoietin plays an important role in angiogenesis.
  • Angiopoietin-like 3 also known as angiopoietin-like 3, ANGPTL3 or angiopoietin 5, ANGPT5
  • ANGPTL3 angiopoietin-like 3
  • ANGPT5 angiopoietin 5
  • Angiopoietin-like 3 or ANGPTL3 is a lipid metabolism regulator that can regulate VLDL triglycerides (TG) by inhibiting the catalytic activity of lipoprotein lipase (LPL).
  • TG VLDL triglycerides
  • LPL lipoprotein lipase
  • the APOEKO mouse model with hypl mutation (apoEKO-hypl) has reduced Angptl3 expression.
  • This mouse model exhibited significant reductions in VLDL TG, VLDL cholesterol, and plasma apoB levels, and post-heparin plasma LPL and hepatic lipase activities were significantly increased in apoEKO-hypl mice, indicating enhanced lipid metabolism (Ando et al., (2003) J. Lipid Res, 44: 1216-1223).
  • Human ANGPTL3 plasma concentrations are positively correlated with plasma HDL cholesterol and HDL phospholipid levels (Shimamura et al., (2007) Arterioscler. Thr
  • siRNA has great potential for development as a new treatment method.
  • siRNA acts on intracellular mRNA. Compared with traditional small molecule drugs, it can directly silence target genes, so it can fundamentally prevent the occurrence and development of diseases more efficiently. develop.
  • due to the poor stability of siRNA it is easily degraded by nucleases in the body, is not easily absorbed by tissues, is difficult to be taken up by cells, and is prone to off-target effects, which limits its clinical application.
  • siRNA that can effectively inhibit the expression of ANGPTL3 gene in cells.
  • the present disclosure provides new small interfering RNA (siRNA), kits and pharmaceutical compositions for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, and the siRNA, kits or pharmaceutical compositions are effective in inhibiting the expression of angiopoietin-like 3 (ANGPTL3). Or methods for reducing ANGPTL3 gene expression or treating diseases or symptoms related to ANGPTL3 expression.
  • the present disclosure provides a small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, the siRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the length of the sense strand and the antisense strand is each independently 15-30 nucleotides, and the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 16-29 A nucleotide sequence of at least 15 consecutive nucleotides.
  • the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-14.
  • the length of the sense strand and the antisense strand is each independently 17-27 nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides.
  • the double-stranded region is 15-25 nucleotide pairs in length, preferably 17-23 nucleotide pairs, more preferably 19-21 nucleotide pairs.
  • one or both of the sense strand and the antisense strand comprise a 3' overhang and/or a 5' overhang having at least 1 nucleotide, e.g., the sense strand and the One or both antisense strands contain a 3' overhang and/or a 5' overhang of at least 1 nucleotide.
  • the antisense strand has a 3' overhang and/or a 5' overhang of at least 2 nucleotides, preferably the antisense strand comprises a 3' overhang and/or a 5' overhang of 2 nucleotides. end.
  • the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 16-29, and at least 17 contiguous nucleotides.
  • the sense strand includes the nucleotide sequence shown in any one of SEQ ID NO:16-29.
  • the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-14, and at least 17 contiguous nucleotides
  • the nucleotide sequence of the acid, the nucleotide sequence of at least 18 consecutive nucleotides, preferably the antisense strand includes the nucleotide sequence shown in any one of SEQ ID NO: 1-14.
  • the siRNA comprises paired sense and antisense strand sequences as shown in Table 3.
  • substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, or all of the nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.
  • the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, phosphorothioates Ester internucleotide linkage modification, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholine nucleotides, phosphoramidates, non-natural bases containing nucleotides, terminal nucleotides attached to cholesterol-based derivatives or dodecyl dodecylamide groups, deoxyribonucleotides and STM1.
  • the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, reverse abasic deoxyribonucleotides, phosphorothioate internucleotide linkage modifications, and STM1.
  • the sense strand and/or the antisense strand comprises at least 2 2'-fluoro modified nucleotides.
  • the sense strand and/or the antisense strand comprises at least 8 2'-O-methyl modified nucleotides.
  • the 3' end and/or the 5' end of the sense strand and/or the antisense strand comprise 1-5 phosphorothioate groups, preferably 2-3 phosphorothioate groups ester group.
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the siRNA is further conjugated to a ligand moiety comprising N-acetylgalactosamine, preferably the sense strand of the siRNA is conjugated to the ligand moiety.
  • the 3' end of the sense strand is conjugated to the ligand moiety.
  • the 5' end of the sense strand is conjugated to the ligand moiety.
  • the ligand moiety includes a conjugation group represented by formula (X'):
  • L 1 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 2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 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;
  • L is a chemical bond, -CH 2 O- or -NHC(O)-;
  • L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • T is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the conjugation ligand targets asialoglycoprotein receptor (ASGPR).
  • ASGPR asialoglycoprotein receptor
  • the conjugation group is selected from Table 1:
  • the conjugation group is selected from Table 2:
  • the ligand comprised in the siRNA has the following structure:
  • the ligand comprised in the siRNA has the following structure:
  • the ligand comprised in the siRNA of the present disclosure has the following structure:
  • the ligand comprised in the siRNA of the present disclosure has the following structure:
  • the present disclosure provides a cell containing the siRNA described in the present disclosure.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the siRNA or cells of the disclosure, and optionally a pharmaceutically acceptable carrier or excipient.
  • the present disclosure provides a kit comprising the siRNA, cells or pharmaceutical composition of the present disclosure.
  • the present disclosure provides a method of reducing ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels, cholesterol levels, glucose levels, and fat pad weight in a subject, the method comprising: The subject is administered a siRNA, cell, or pharmaceutical composition of the present disclosure.
  • the present disclosure also provides a method of treating a disease or condition associated with ANGPTL3 expression in a subject, the method comprising the step of administering to the subject a siRNA, cell, or pharmaceutical composition described in the present disclosure.
  • the disease associated with ANGPTL3 expression is the metabolic disease or cardiovascular disease.
  • the metabolic disease or the cardiovascular disease is selected from obesity, diabetes, atherosclerosis, dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease (NAFLD), hyperfattyemia or metabolic syndrome or a combination thereof.
  • NAFLD non-alcoholic fatty liver disease
  • the disease associated with ANGPTL3 expression is a dyslipidemia, and the dyslipidemia is hyperlipidemia.
  • the hyperlipidemia is hypercholesterolemia, hypertriglyceridemia, or a combination thereof.
  • the disease associated with ANGPTL3 expression is NAFLD, and the NAFLD is hepatic steatosis or steatohepatitis.
  • the disease associated with ANGPTL3 expression is diabetes, which is type 2 diabetes or type 2 diabetes with dyslipidemia.
  • methods of the present disclosure for treating a disease or condition associated with ANGPTL3 expression in a subject include administering the siRNA, cells, or pharmaceutical composition to the subject by subcutaneous administration, topical administration, or intravenous administration.
  • the subject is a human subject.
  • 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.
  • RNA 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 or substantially complementary to a target sequence.
  • the term "complementary region” refers to a region on the antisense strand that is completely 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').
  • 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 processed base pairs.
  • Base pairs formed by modified nucleotides include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.
  • a polynucleotide that is “at least partially complementary” or “substantially complementary” to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding ANGPTL3). glycosides.
  • a polynucleotide is complementary to at least a portion of ANGPTL3 mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding ANGPTL3.
  • sense strand refers to a strand of siRNA that includes a region that is substantially complementary to a region that is the antisense strand as the term is defined herein.
  • Nucleoside is a compound composed of two substances: purine base or pyrimidine base, and ribose or deoxyribose.
  • Nucleoside is a compound composed of three substances: purine base or pyrimidine base, ribose or deoxyribose, and phosphate.
  • Olionucleotide refers to, for example, a nucleic acid molecule (RNA or DNA) having a length of less than 100, 200, 300 or 400 nucleotides.
  • Base is the basic unit for the synthesis of nucleosides, nucleotides and nucleic acids. Its constituent elements contain nitrogen, also known as “nitrogen-containing bases”.
  • the capital letters A, U, T, G and C represent the base composition of nucleotides, which are adenine, uracil, thymine, guanine and cytosine respectively.
  • nucleotide overhang refers to at least one unpaired nucleotide that protrudes from the duplex structure of an siRNA (eg, siRNA). Nucleotide overhangs exist, for example when the 3'-end of one strand of siRNA extends beyond the 5'-end of the other strand or vice versa.
  • the siRNA can comprise an overhang having at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. many.
  • Nucleotide overhangs may comprise or consist of nucleotide/nucleoside analogs (including deoxynucleotides/nucleosides). One or more overhangs can be on the sense strand, the antisense strand, or any combination thereof. Additionally, the overhanging nucleotide or nucleotides may be present on the 5'-end, 3'-end, or both ends of the antisense or sense strand of the siRNA.
  • siRNAs of the present disclosure include siRNAs with nucleotide overhangs at one end (i.e., agents with one overhang and one blunt end) or with nucleotide overhangs at both ends.
  • nucleotides of the iRNAs of the present disclosure are modified.
  • All nucleotides are modified nucleotides, and/or substantially all nucleotides in the antisense strand are modified nucleotides, and/or substantially all nucleotides in both the sense and antisense strands are Acids are all modified nucleotides.
  • all nucleotides of the iRNA of the disclosure are modified nucleotides.
  • nucleotides of the sense strand are modified nucleotides
  • all nucleotides of the antisense strand are modified nucleotides
  • all nucleotides of both the sense strand and the antisense strand are all modified nucleotides.
  • substantially all nucleotides are modified means that the siRNAs of the present disclosure are mostly, but not entirely, modified and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.
  • Modified nucleotides herein include, but are not limited to, 2'-O-alkyl modified nucleotides (e.g., 2'-O-methyl modified nucleotides, 2'-methoxyethyl modified nucleic acids).
  • nucleotides 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides , nucleotides containing phosphorothioate groups, phosphorothioate internucleotide linkage modifications, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleosides Acids, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases containing nucleotides, linked to cholesteryl derivatives or dodecyl dodecyl amido groups Terminal nucleotides on the group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, conformationally restricted nucleo
  • 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.
  • Nucleotide containing a phosphorothioate group refers to a nucleotide in which one or more oxygen atoms on the phosphate are replaced by sulfur atoms.
  • Modification of phosphorothioate inter-nucleotide bonding refers to the modification in which two adjacent nucleotides on the left and right are connected through phosphorothioate.
  • a "ligand moiety” refers to a chemical moiety that conjugates to an siRNA and is capable of altering the distribution, targeting, or lifetime of the siRNA.
  • ligand is a selected target (e.g. molecule, cell or cell type, compartment (e.g. cell or organ compartment, tissue, organ or area of the body) provides enhanced affinity.
  • the ligand moiety targets the asialoglycoprotein receptor (ASGPR) on hepatocytes. Binding of the ligand moiety to ASGPR mediates internalization via clathrin-coated vesicles. Maturation of endosomes results in a decrease in lysosomal pH, which promotes dissociation of ligand-receptor complexes, thereby releasing siRNA. Conjugation of a ligand moiety targeting the asialoglycoprotein receptor (ASGPR) on hepatocytes results in siRNA efficacy and stability in vivo or intracellularly. This facilitates subcutaneous administration of the siRNA.
  • ASGPR asialoglycoprotein receptor
  • inhibitortion is used interchangeably with “reduction,” “silencing,” “downregulation,” and other similar terms and includes any level of inhibition.
  • the phrase "inhibiting the expression of ANGPTL3" is intended to mean inhibiting the expression of any ANGPTL3 gene as well as variants or mutants of the ANGPTL3 gene.
  • the ANGPTL3 gene may be a wild-type ANGPTL3 gene, a mutant ANGPTL3 gene, or in the case of a genetically manipulated cell, cell population, or organism, a transgenic ANGPTL3 gene.
  • “Inhibition of ANGPTL3 gene expression” includes any level of inhibition of the ANGPTL3 gene, such as at least partial inhibition of ANGPTL3 gene expression.
  • ANGPTL3 gene expression can be assessed based on the level or change in level of any variable associated with ANGPTL3 gene expression, such as ANGPTL3 mRNA levels, ANGPTL3 protein levels, or lipid levels. This level can be assessed in individual cells or in a group of cells (including, for example, a sample derived from a subject).
  • Inhibition can be assessed by a reduction in absolute or relative levels of one or more variables associated with ANGPTL3 expression compared to control levels.
  • the control level may be any type of control level utilized in the art, such as a pre-dose baseline level or from a similar untreated or control (e.g., buffer only control or inert control) subject, cell , or the level determined by the sample.
  • “Hydroxy 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.
  • Mainly include silane-type protective groups, acyl-type protective groups or ether-type protective groups, preferably the following: trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), Diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac ), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc
  • 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. In some embodiments, 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 alkylene refers to a divalent group formed by removing another hydrogen of C 1-6 alkyl, and may be substituted or unsubstituted. In some embodiments, C 1-4 alkylene, C 2-4 alkylene, and C 1-2 alkylene are preferred.
  • the unsubstituted alkylene group includes, but is not limited to: methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene (-CH 2 CH 2 CH 2 -), ethylene Base (-CH 2 CH 2 CH 2 CH 2 -), pentylene (-CH 2 CH 2 CH 2 CH 2 CH 2 -), hexylene (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -) ,etc.
  • alkylene groups substituted by one or more alkyl (methyl) include, but are not limited to: substituted methylene (-CH(CH 3 )- , -C(CH 3 ) 2 -), substituted ethylene (-CH(CH 3 )CH 2 -, -CH 2 CH(CH 3 )-, -C(CH 3 ) 2 CH 2 -, -CH 2 C(CH 3 ) 2- ), substituted propylene (-CH(CH 3 )CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 -, -CH 2 CH 2 CH(CH 3 ) -, -C(CH 3 ) 2 CH 2 CH 2 -, -CH 2 C(CH 3 ) 2 CH 2 -, -CH 2 CH 2 C(CH 3 ) 2 -), etc.
  • treatment refers to administering an agent or performing a procedure in order to obtain an effect. These effects may be prophylactic in the sense of completely or partially preventing the disease or its symptoms, and/or may be therapeutic insofar as affecting the partial or complete cure of the disease and/or the symptoms of the disease.
  • treatment may include treatment of a disease or condition (eg, cancer) in a mammal, particularly a human, and includes: (a) prophylaxis in a subject susceptible to the disease but who has not yet been diagnosed with the disease.
  • the occurrence of the disease or disease symptoms (for example, including diseases that may be related to or caused by the primary disease); (b) inhibit the disease, that is, prevent its progression; (c) alleviate the disease, that is, cause the regression of the disease.
  • Treatment may refer to any indication of success in treating or ameliorating or preventing cancer, including any objective or subjective parameter, such as elimination; remission; reduction of symptoms or making disease symptoms more tolerable for the patient; slowing of progression or decline ; or reduce the endpoint of worsening frailty.
  • Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician's examination.
  • treating includes administering an siRNA or pharmaceutical composition disclosed in the present disclosure to prevent or delay, alleviate, or arrest or inhibit the development of symptoms or conditions associated with a disease (eg, cancer).
  • a disease eg, cancer
  • therapeutic effect refers to the reduction, elimination, or prevention of disease, disease symptoms, or disease side effects in a subject.
  • terapéuticaally effective amount refers to an amount that, when administered to a subject to treat a disease, is sufficient to effect treatment of a disease.
  • the term "subject” refers to any mammalian subject for whom diagnosis, treatment, or therapy is desired.
  • "Mammal” for therapeutic purposes means any animal classified as a mammal, including humans, domestic animals, and laboratory, zoo, sporting or pet animals, such as dogs, horses, cats, cattle, sheep, Goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.
  • the present disclosure provides a small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, the siRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the length of the antisense strand is each independently 15-30 nucleotides, and the antisense strand includes at least 15 consecutive nucleotide sequences of the nucleotide sequence shown in any one of SEQ ID NO: 16-29 The nucleotide sequence of a nucleotide.
  • siRNA small interfering RNA
  • the double-stranded region formed by the sense strand and antisense strand is completely complementary. In other embodiments, the double-stranded region formed by the sense strand and the antisense strand is substantially complementary and may contain 1, 2, 3, 4, or 5 non-complementary sites.
  • the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-14.
  • the length of the sense strand and the antisense strand is each independently 17-27 nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides.
  • the double-stranded region is 15-25 nucleotide pairs in length, preferably 17-23 nucleotide pairs, more preferably 19-21 nucleotide pairs.
  • the sense strand and the antisense strand comprise a 3' overhang and/or a 5' overhang having at least 1 nucleotide, for example one or both of the sense strand and the antisense strand or Both contain 3' overhangs and/or 5' overhangs with at least 1 nucleotide.
  • the antisense strand has a 3' overhang and/or a 5' overhang of at least 2 nucleotides, preferably the antisense strand includes a 3' overhang of 2 nucleotides. Overhangs and/or 5' overhangs.
  • the sense strand and the antisense strand are the same length.
  • the entire length of the sense strand is complementary to the entire length of the antisense strand to form a double strand, ie, has blunt ends.
  • the sense strand and the antisense strand are the same length, and a part of the sense strand is complementary to a part of the antisense strand, that is, both the sense strand and the antisense strand have a 5' overhang.
  • the sense strand and the antisense strand are different lengths.
  • the 5' end of the antisense strand has an overhang of at least 1 nucleotide, more preferably 2 or 3 nucleotides.
  • the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 16-29, and at least 17 contiguous nucleotides.
  • the sense strand includes the nucleotide sequence shown in any one of SEQ ID NO:16-29.
  • the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-14, at least 17 contiguous nucleotides acid nucleotide sequence, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, a nucleotide sequence of at least 20 consecutive nucleotides, preferably the reverse
  • the sense strand includes the nucleotide sequence shown in any one of SEQ ID NO: 1-1093.
  • the siRNA comprises paired sense strand sequences and reverse strand sequences as shown in Table 3 sense strand sequence.
  • substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the sense strand are modified nucleotides, and/or at least 80% of the nucleotides of the antisense strand are modified nucleotides.
  • all nucleotides of the sense strand and/or all nucleotides of the antisense strand are modified nucleotides.
  • Modifications of nucleotides described in the present disclosure may be modifications on the phosphate group, ribose group and/or base group of the nucleotide.
  • the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, phosphorothioates Ester internucleotide linkage modification, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholine nucleotides, phosphoramidates, non-natural bases containing nucleotides, terminal nucleotides attached to cholesterol-based derivatives or dodecyl dodecylamide groups, deoxyribonucleotides and STM1.
  • the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, reverse abasic deoxyribonucleotides, and phosphorothioate internucleotide linkage modifications.
  • the sense strand and/or the antisense strand comprises at least 2 2'-fluoro modified nucleotides.
  • the sense strand and/or the antisense strand comprises at least 8 2'-O-methyl modified nucleotides.
  • the 3' end and/or the 5' end of the sense strand and/or the antisense strand comprise 1-5 phosphorothioate groups, preferably 2-3 phosphorothioate groups ester group.
  • the sense strand and/or antisense strand comprise adenine deoxyribonucleotides, thymine deoxyribonucleotides, guanine deoxyribonucleotides and/or cytosine deoxyribonucleotides glycosides.
  • the sense strand and/or antisense strand comprise thymidine deoxyribonucleotides.
  • the sense strand comprises thymidine deoxyribonucleotides.
  • the antisense strand comprises a modified nucleotide sequence shown in any one of Table 5 of the specification, and/or the sense strand comprises a modified nucleotide sequence shown in any one of Table 4 of the specification. Modified nucleotide sequence.
  • the siRNA includes the paired modified sense strand sequence and the modified antisense strand sequence shown in any one of Table 6 of the specification.
  • the siRNAs of the present disclosure are further conjugated to a ligand moiety comprising N-acetylgalactosamine.
  • the sense strand of the siRNA is conjugated to the ligand moiety.
  • the 3' end of the sense strand is conjugated to the ligand moiety.
  • the 5' end of the sense strand is conjugated to the ligand moiety.
  • the ligand moiety includes a conjugation group represented by formula (X'):
  • L 1 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 2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 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;
  • L is a chemical bond, -CH 2 O- or -NHC(O)-;
  • L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • T is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the conjugating group is represented by Formula (I'):
  • L 1 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 2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 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 ) 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;
  • L is -CH 2 O- or -NHC(O)-;
  • L’ is a chemical bond, -C(O)NH- or -NHC(O)-;
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • L 1 is -CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;
  • L 2 is -CH 2 CH 2 C(O)-
  • L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
  • L 4 is -(OCH 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;
  • L is -CH 2 O-
  • L’ is a chemical bond
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the conjugation group is represented by Formula (I'-1), Formula (I'-2), or Formula (I'-3):
  • L 1 is -CH 2 O- or -NHC(O)-;
  • L 2 is -CH 2 CH 2 C(O)-
  • L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
  • L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is -CH 2 O-
  • R' 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.
  • L 1 is -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;
  • L 2 is -CH 2 CH 2 C(O)-
  • L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 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;
  • L is -CH 2 O- or -NHC(O)-;
  • L’ is a chemical bond or -C(O)NH-
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the conjugation group is represented by Formula (II'-1) or Formula (II'-2):
  • L 1 is -CH 2 O- or -CH 2 O-CH 2 CH 2 O-;
  • L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is -NHC(O)-
  • L’ is a chemical bond or -C(O)NH-
  • R' 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;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • L 1 is -CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a - ;
  • L 2 is a chemical bond
  • L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 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;
  • L is -CH 2 O- or -NHC(O)-;
  • L’ is a chemical bond or -C(O)NH-
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • L 1 is -CH 2 - or -C(O)-;
  • L 3 is -(NHCH 2 CH 2 ) b -;
  • L 4 is -(OCH 2 CH 2 ) c -;
  • c 1, 2, 3, 4 or 5;
  • L is -CH 2 O- or -NHC(O)-;
  • R' 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.
  • L 1 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 2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 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;
  • L is a chemical bond, -CH 2 O- or -NHC(O)-;
  • L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • T is a chemical bond, -CH 2 -, -M-, -CH 2 -M- or -C(O)-M-;
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • T is -M-, -CH 2 -M- or -C(O)-M-, where M is
  • L 1 is -CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;
  • L 2 is -CH 2 CH 2 C(O)-
  • L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
  • L 4 is -(OCH 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;
  • L is a chemical bond or -CH 2 O-;
  • L' is a chemical bond or -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • T is as defined in the embodiment above.
  • conjugation group is represented by Formula (III'-1), Formula (III'-2) or Formula (III'-3):
  • L 1 is -CH 2 O- or -NHC(O)-;
  • L 2 is -CH 2 CH 2 C(O)-
  • L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
  • L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is a chemical bond or -CH 2 O-;
  • R' 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;
  • T is as defined in the embodiment above.
  • L 1 is -CH 2 -, -CH 2 O- or -C(O)-;
  • L 2 is a chemical bond
  • L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is a chemical bond or -NHC(O)-
  • L’ is a chemical bond
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • T is as defined in the embodiment above.
  • L 1 is -CH 2 -, -CH 2 O- or -C(O)-;
  • L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is a chemical bond or -NHC(O)-
  • L’ is a chemical bond
  • R' 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;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • T is as defined in the embodiment above.
  • L 1 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 2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 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;
  • L is a chemical bond, -CH 2 O- or -NHC(O)-;
  • L' is -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • T is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • conjugating group is selected from the following:
  • conjugating group is selected from the following:
  • the ligand targets asialoglycoprotein receptor (ASGPR).
  • ASGPR asialoglycoprotein receptor
  • said ligand has the following structure:
  • said ligand has the following structure:
  • said ligand has the following structure:
  • said ligand has the following structure:
  • the siRNA of the present disclosure can inhibit ANGPTL3 gene expression by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, At least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, At least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
  • Inhibition of ANGPTL3 gene expression may be manifested by a decrease in the amount of mRNA expressed by a first cell or population of cells (such cells may be present, for example, in a sample derived from the subject) in which the ANGPTL3 gene is transcribed and the cells or these cells have been treated (e.g., by contacting the cell or cells with an siRNA of the present disclosure, or by administering an siRNA of the present disclosure to a subject in which these cells are present or previously present, such that contact with the first cell or cells ANGPTL3 gene expression is inhibited compared to a second group or group of cells (one or more control cells) that are substantially the same but have not been so treated.
  • the inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells using the following formula.
  • inhibition of ANGPTL3 gene expression may be assessed in terms of reduction in parameters functionally related to ANGPTL3 gene expression, such as lipid levels, cholesterol levels, e.g., LDLc levels.
  • ANGPTL3 gene silencing can be determined in any cell that expresses ANGPTL3 constitutively or by genome engineering and by any assay known in the art.
  • the liver is the main site of ANGPTL3 expression.
  • Other important sites of expression include the pancreas, kidney, and intestine.
  • Inhibition of expression of ANGPTL3 protein may be manifested by a decrease in the level of ANGPTL3 protein expressed by a cell or population of cells (eg, the level of protein expressed in a sample derived from a subject).
  • a cell or population of cells eg, the level of protein expressed in a sample derived from a subject.
  • inhibition of protein expression levels in treated cells or populations of cells can similarly be expressed as a percentage of the levels of the protein in control cells or populations of cells.
  • Control cells or cell populations that may be used to assess inhibition of ANGPTL3 gene expression include cells or cell populations that have not been contacted with the siRNA of the present disclosure.
  • the control cells or population of cells may be derived from an individual subject (eg, a human or animal subject) prior to treatment of the subject with siRNA.
  • the present disclosure provides cells containing the siRNA of the present disclosure, wherein the siRNA of the present disclosure is capable of being transcribed in the cell.
  • compositions comprising the siRNA or cells of the present disclosure, and optionally a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms that, within the scope of sound medical judgment, are suitable for contact with tissues of human subjects and animal subjects without undue toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • a pharmaceutically acceptable carrier refers to a pharmaceutical carrier that facilitates the administration of siRNA or cells containing the siRNA to the human body and/or facilitates its absorption or effect.
  • diluents excipients such as water, fillers such as starch, sucrose, etc.
  • binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone
  • wetting agents such as glycerin
  • disintegrants such as agar, carbonic acid Calcium and sodium bicarbonate
  • absorption accelerators such as quaternary ammonium compounds
  • surfactants such as cetyl alcohol
  • adsorption carriers such as kaolin and bentonite
  • lubricants such as talc, calcium/magnesium stearate, polyethylene glycol, etc.
  • other auxiliary agents such as flavoring agents, sweeteners, etc. can also be added to the composition.
  • a pharmaceutical composition containing the siRNA or cells of the present disclosure may include a pharmaceutically acceptable diluent or sustained-release matrix in which the siRNA of the present disclosure is embedded.
  • kits comprising the siRNA or cells described in the present disclosure.
  • kits for using the siRNA described in the present disclosure and/or performing the methods of the present disclosure include one or more siRNAs or cells described in the present disclosure and may further include instructions for use. Instructions for inhibiting the expression of ANGPTL3 in the cell by contacting the cell with the siRNA described in the present disclosure in an amount effective to inhibit the expression of ANGPTL3 may be recorded in the instructions for use.
  • the kit of the present disclosure may also include means (e.g., an injection device) for contacting the cells with the siRNA of the present disclosure or with Tools for measuring the inhibitory effect of ANGPTL3 (e.g., a device for measuring inhibition of ANGPTL3 mRNA or protein).
  • a device for measuring inhibition of ANGPTL3 may comprise a device for obtaining a sample (eg, like a plasma sample) from a subject.
  • the kit of the present disclosure may also optionally include a method for administering the siRNA or cells of the present disclosure to the subject.
  • Device or device for determining a therapeutically effective amount or a prophylactically effective amount may also optionally include a method for administering the siRNA or cells of the present disclosure to the subject.
  • the present disclosure provides a method of reducing ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels, cholesterol levels, glucose levels, fat pad weight in a subject, the method comprising administering to the subject siRNA, cells, or pharmaceutical compositions of the present disclosure.
  • the present disclosure provides a method of treating a disease or condition associated with ANGPTL3 expression in a subject, the method comprising the step of administering to the subject a siRNA, cell, or pharmaceutical composition described in the present disclosure.
  • the disease associated with ANGPTL3 expression is cardiovascular disease.
  • the cardiovascular disease is selected from obesity, diabetes, atherosclerosis, dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease (NAFLD), hyperlipidemia or metabolic syndrome or its combination.
  • the disease associated with ANGPTL3 expression is a dyslipidemia, and the dyslipidemia is hyperlipidemia.
  • hyperlipidemia is hypercholesterolemia, hypertriglyceridemia, or a combination thereof.
  • the disease associated with ANGPTL3 expression is NAFLD, and the NAFLD is hepatic steatosis or steatohepatitis.
  • the disease associated with ANGPTL3 expression is diabetes, and the diabetes Is type 2 diabetes or type 2 diabetes with dyslipidemia.
  • methods of the present disclosure for treating a disease or condition associated with ANGPTL3 expression in a subject include administering the siRNA or pharmaceutical composition to the subject including subcutaneous administration or intravenous administration.
  • the subject is a human patient.
  • the present disclosure also relates to the siRNA, cells or pharmaceutical compositions of the present disclosure for treating a disease or condition associated with ANGPTL3 expression in a subject.
  • the present disclosure also relates to the use of the siRNA, cells, or pharmaceutical compositions of the present disclosure in the preparation of a medicament for treating a disease or symptom associated with ANGPTL3 expression in a subject.
  • the medicine of the present disclosure can be prepared into emulsions, microemulsions, and microparticles.
  • RNA sequence provided by the present disclosure targets the human ANGPTL3 gene (or target gene, target mRNA sequence, target sequence).
  • Tables 4 to 6 show modified RNA sequences used in the present disclosure.
  • the A, U, G, and C distributions represent natural adenine ribonucleotides, uracil ribonucleotides, guanine ribonucleotides, and cytosine ribonucleotides.
  • d indicates that the nucleotide adjacent to the right is a deoxyribonucleotide.
  • dA, dT, dG and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide and cytosine deoxyribonucleotide respectively.
  • i inosine ribonucleotide
  • m 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.
  • 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.
  • IB stands for reverse abasic deoxyribonucleotide, which can include the following three structures depending on its position/connection method in siRNA (respectively for the 5' end, middle and 3' end of the nucleic acid chain):
  • 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.
  • NAG37 represents a GalNAc delivery vector of the structure well known in the art, wherein Indicates the position of attachment to siRNA via a phosphate group or phosphorothioate group, see for example PCT Publication No. WO2018044350
  • GL6 represents a GalNAc delivery vector of the following structure, where Indicates the position where the phosphate group or phosphorothioate group is attached to the siRNA
  • GL12 represents a GalNAc delivery vector of the following structure, where Indicates the position where the phosphate group or phosphorothioate group is attached to the siRNA
  • STM1 represents the nucleotide substitution of the following structure. Depending on the position of STM1 in the nucleic acid chain, Can be linked to adjacent nucleotides, 3' end structures or 5' end structures
  • Huh7 cell line was purchased from Nanjing Kebai, product number CBP60202;
  • Hep3B cell line was purchased from Nanjing Kebai, product number CBP60197;
  • PHH cells were purchased from Shanghai Xuanyi, product number QYLF-HPMC;
  • HEK293A cell line was purchased from Nanjing Kebai, product number CBP60436;
  • Balb/c mice were from Zhejiang Vitong Lever, product number Balb/c.
  • the filter cake was ground with water (1.45 L) at 25°C for 30 minutes. The mixture was filtered and the filter cake was washed with water (175 mL x 3), and the filter cake was collected to obtain compound 2A as a white solid (about 580 g).
  • reaction solution was concentrated under reduced pressure, and the crude product obtained was prepared by preparative high-performance liquid chromatography (preparative-HPLC, column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: water-ACN; B%: 17% -57%, 5min) to obtain white solid compound E7 (53.0mg, yield 17.08%, purity 78.94%).
  • reaction mixture was slowly poured into a stirred cold 0.5 M aqueous HCl solution (230 mL), stirred for 10 minutes, a white solid formed and filtered, and the aqueous phase was extracted twice with DCM (1.50 L).
  • the combined organic phases were washed with 5% NaHCO 3 (aq.) (200 mL), dried (Na 2 SO 4 ), and concentrated by evaporation under pressure.
  • compound 1-7 (8.00g, 24.4mmol) was added to HCl (100mL, 12M), and the reaction was carried out at 50°C for 2 hours. The reaction solution was concentrated under reduced pressure to obtain compound 1-8 (about 3.60g) as a colorless oil. ,HCl salt),
  • siRNAs of the present disclosure are 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.
  • the blank CPG solid-phase carrier is used as the starting cycle, and the nucleoside monomers or nucleotide analogues are connected one by one from the 3'-5' direction according to the sequence of the sense strand nucleotides. monomer.
  • Each connection of a nucleoside monomer or nucleotide analog monomer includes a four-step reaction of deprotection, coupling, capping, oxidation or sulfation.
  • the synthesis conditions for an oligonucleotide with a synthesis scale of 5 ⁇ mol are as follows:
  • the nucleoside monomer or nucleotide analog monomer is provided with a 0.05 mol/L acetonitrile solution.
  • the reaction conditions of each step are the same, that is, the temperature is 25°C, and 3% trichloroacetic acid-dichloromethane solution is used for deprotection.
  • the activator used in the coupling reaction was 0.25 mol/L ETT-acetonitrile solution, coupled 2 times; the capping reaction used 10% acetic anhydride-acetonitrile and pyridine/N-methylimidazole/acetonitrile (10: 14:76, v/v/v), blocked twice; oxidized using 0.05mol/L iodine/tetrahydrofuran/pyridine/water (70/20/10, v/v/v), oxidized twice; used sulfide 0.2mol/L PADS acetonitrile/3-methylpyridine (1/1, v/v), sulfide 2 times.
  • the nucleotide monomer of IB was purchased from Shanghai Zhaowei Technology Development Co., Ltd., product number OP-040.
  • the blank CPG solid-phase carrier is used as the starting cycle, and the nucleoside monomers or nucleotide analogues are connected one by one from the 3'-5' direction according to the sequence of the antisense strand nucleotides. single object.
  • Each linkage of a nucleoside monomer or nucleotide analog monomer involves deprotection, coupling, capping, oxidation or sulfation.
  • the synthesis conditions of 5 ⁇ mol of oligonucleotide for the antisense strand are the same as those for the sense strand.
  • 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.
  • Compound E13 was connected to the CPG carrier in a manner similar to the method for connecting compound E7 to the CPG carrier.
  • the GL6 solid-phase carrier prepared in Section 2.1.1 of Example 4 is used as the initial cycle, and the nucleosides are connected one by one from the 3'-5' direction in the sequence of the sense strand nucleotides. monomer.
  • Each connection of a nucleoside monomer includes four-step reactions of deprotection, coupling, capping, oxidation or sulfation.
  • the synthesis conditions for an oligonucleotide with a synthesis scale of 5 ⁇ mol 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), cap twice; use 0.05mol/L iodine for oxidation /tetrahydrofuran/pyridine/water (70/20/10, v/v/v), oxidized twice; for sulfide, use 0.2 mol/L PADS acetonitrile/3-methylpyridine (1/1, v/v). Thiogeneration 2 times.
  • a blank CPG solid-phase carrier is used as the starting cycle, and the nucleoside monomers are connected one by one from the 3'-5' direction in the order of the antisense strand nucleotide arrangement.
  • Each connection of a nucleoside monomer involves a four-step reaction of deprotection, coupling, capping, oxidation or sulfation.
  • the synthesis conditions of 5 ⁇ mol of oligonucleotide of the antisense strand are the same as those of the sense strand.
  • a strong anion packing column can be used, a sodium chloride-sodium hydroxide system can be used for elution and purification, the products can be collected and tubed, and a gel packing purification column can be used for desalting.
  • the elution system is pure water.
  • L96-conjugated siRNA and NAG37-conjugated siRNA were obtained in a similar manner.
  • the Huh7 cell line (Nanjing Kebai, Cat. No. CBP60202) was digested, resuspended, counted, and plated in a 96-well plate at 100 ⁇ L/well and 1 ⁇ 10 4 cells/well. Transfection was performed 18 hours later.
  • RNAiMAX (Thermo, 13778150)
  • 14.1 ⁇ L Opti-MEM and dilute 0.9 ⁇ L RNAiMAX (Thermo, 13778150)
  • mix gently by pipetting and let stand at room temperature for 5 minutes.
  • Cell RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the operating procedures of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412).
  • Denaturation reaction mixture To prepare the denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 1 ⁇ L of Oligo dT Primer, 1 ⁇ L of dNTP Mixture, and 12.5 ⁇ L of template RNA. Denaturation reaction was performed by incubating at 65°C for 5 min in a conventional PCR machine. Place the mixture Cool quickly on ice for 2 minutes.
  • PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 ⁇ L of 5 ⁇ Prime Script II Buffer, 0.5 ⁇ L of RNase Inhibitor, and 1 ⁇ L of PrimeScript II RTase.
  • reaction program is: (50°C, 2min) ⁇ 1Cycle; (95°C, 20s) ⁇ 1Cycle; (95°C, 1s; 60°C, 24s) ⁇ 40Cycles.
  • ⁇ Ct [(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)].
  • the final concentration of siRNA was 1 nM for high-throughput screening of cell line activity of siRNA compounds.
  • the experimental screening results are shown in Table 8.
  • the starting concentration of siRNA was 10 nM, and 10-fold gradient dilution was performed to obtain 5 concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM), and the Hep3B cell line activity of siRNA was screened.
  • the screening results are shown in Table 9, in which columns 2-6 are the remaining inhibition rates and column 7 is the IC50 value.
  • PHH cells (Shanghai Xuanyi, Cat. No. QYLF-HPMC) were revived at 37°C, added to the recovery medium, centrifuged, resuspended and counted. PHH cells were plated in a 96-well plate at 90 ⁇ L/well, 2 ⁇ 10 4 cells/well; complete medium was replaced after 4 hours, and transfection was performed after 18 hours.
  • siRNA stock solution was diluted with Opti-MEM. Add 198 ⁇ L Opti-MEM to 2 ⁇ L siRNA stock solution, mix by pipetting, and use it as the first concentration point. Perform corresponding gradient dilutions according to actual experimental needs.
  • RNAiMAX (Thermo, 13778150)
  • Opti-MEM dilute 0.9 ⁇ L RNAiMAX
  • 15 ⁇ L of the prepared RNAi-MAX mixture and 15 ⁇ L of the diluted compound by gently pipetting and mixing without bringing in air bubbles. Let it stand at room temperature for 10 minutes.
  • Cell RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the operating procedures of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412).
  • Denaturation reaction mixture To prepare the denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 1 ⁇ L of Oligo dT Primer, 1 ⁇ L of dNTP Mixture, and 12.5 ⁇ L of template RNA. Denaturation reaction was performed by incubating at 65°C for 5 min in a conventional PCR machine. Place the mixture on ice to cool quickly for 2 minutes.
  • PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 ⁇ L of 5 ⁇ Prime Script II Buffer, 0.5 ⁇ L of RNase Inhibitor, and 1 ⁇ L of PrimeScript II RTase.
  • reaction program is: (50°C, 2min) ⁇ 1Cycle; (95°C, 20s) ⁇ 1Cycle; (95°C, 1s; 60°C, 24s) ⁇ 40Cycles.
  • ⁇ Ct [(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)].
  • the starting concentration of siRNA was 10 nM, and it was diluted 10 times to obtain 5 concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM).
  • the activity of siRNA in human liver primary cells was screened. The screening results are shown in Table 11. , where columns 2-6 are the remaining inhibition rates and column 7 is the IC50 value.
  • the corresponding antisense strand off-target plasmid was designed based on the siRNA sequence, and the psiCHECK2 GSSM-5Hits recombinant plasmid was prepared by Sangon Bioengineering (Shanghai) Co., Ltd., and the recombinant plasmid was diluted to 1000ng/ ⁇ L for later use.
  • HEK293A cell (Nanjing Kebai, Cat. No. CBP60436) resuspension was plated, with 8 ⁇ 10 3 cells/well.
  • siRNA preparation Dilute siRNA 3 times starting from a final concentration of 40nM, with a total of 11 concentration points (10nM, 3.3333nM, 1.1111nM, 0.37037nM, 0.12346nM, 0.04115nM, 0.01372nM, 0.00457nM, 0.00152nM, 0.00051nM, 0.0 0017 nM).
  • the preparation volume for a single well is 0.01 ⁇ L/well for plasmid and 8.99 ⁇ L/well for Opti-MEM.
  • Lipo mixture Preparation of Lipo mixture: Add 0.2 ⁇ L of Lipo 2000 and 9.8 ⁇ L of Opti-MEM to each well to dilute Lipo 2000 (Lipofectamine TM 2000 transfection reagent, Thermo, 11668019) with Opti-MEM to obtain a Lipo mixture, and let it stand at room temperature for 5 minutes.
  • Lipo 2000 Lipofectamine TM 2000 transfection reagent, Thermo, 11668019
  • the fluorescence activity is measured by a microplate reader, and the collected Renilla signals are normalized by the Firefly signal standard.
  • the inhibitory effect of siRNA is obtained by comparing the unprocessed results (residual inhibitory activity). The calculation process is as follows:
  • Ratio Renilla (Renilla luciferase)/Firefly (firefly luciferase).
  • the remaining inhibition rate (RatiosiRNA/Ratiocontrol) * 100%, take the average of two duplicate wells: Ratiocontrol is the Ratio value of the control well (excluding siRNA) (take the average of two duplicate wells).
  • IC50 Half maximal inhibitory concentration
  • results of the off-target activity screening of siRNA's psiCHECK2 GSSM-5Hits are shown in Table 12, in which columns 2-12 are the remaining inhibition rates and column 13 is the IC50 value.
  • the results show that DR000405, DR000424, DR000430, DR000442, DR000447, DR000619, and DR000621 of the present disclosure have lower off-target activity against HEK293A cells compared to the positive control DR001483.
  • mice Using high-pressure tail vein injection, six- to eight-week-old female Balb/c mice were treated with double-gene stabilized
  • the transfection system performs in vivo transfection modeling. Piggy-Bac transposon plasmids containing target gene cDNA sequences (Genbank registration number NM_014495.2) with different mass ratios are transferred through the tail vein through a 27-gauge needle within 5-7 seconds.
  • the delivery solution (total volume is 10% of the animal body weight, Mirusbio-MIR 5240) was injected into the mice, and after injection, they were returned to the cage for observation for 30 min. Taking the day of modeling as day 0, serum was obtained at various time points after modeling (day 7 to day 35) for detecting SEAP expression levels.
  • Dual-gene stable transfection system including Piggy-Bac auxiliary plasmid and Piggy-Bac transposon plasmid, in which Piggy-Bac auxiliary plasmid provides Piggy-Bac transposase; Piggy-Bac transposon plasmid uses Piggy-Bac transposon Based on it, it contains a dual-gene expression element, which contains the secreted alkaline phosphatase gene (SEAP) and the target gene (ANGPTL3). SEAP and ANGPTL3 are co-expressed.
  • SEAP secreted alkaline phosphatase gene
  • ANGPTL3 target gene
  • CSPD substrate and reaction buffer diluent at a ratio of 1:20 to form a reaction solution.
  • siRNA compounds The effect of siRNA compounds on inhibiting the expression of target genes was evaluated by measuring SEAP expression levels in serum. The lower the SEAP chemiluminescence value, the better the siRNA compound is at inhibiting the expression of the target gene. Select the test sample that can inhibit the expression level of SEAP as a nucleic acid drug.
  • each mouse was given a single subcutaneous administration according to Table 13: 200 ⁇ l of physiological saline containing 3 mg/kg (mpk) RNAi reagent; or 200 ⁇ l of physiological saline without RNAi reagent was used as a control (vehicle) .
  • the HDI model screening results are shown in Table 14.

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Abstract

Provided are a small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) protein in a cell, a cell comprising the siRNA, and a method for treating a disease or symptom associated with ANGPTL3 expression in a subject using the siRNA or the cell.

Description

调控ANGPTL3基因活性的siRNA分子siRNA molecules that regulate ANGPTL3 gene activity 技术领域Technical field
本公开涉及RNA干扰领域。This disclosure relates to the field of RNA interference.
背景技术Background technique
血管生成素是分泌型生长因子家族。与其相应内皮特异性受体一起，血管生成素在血管发生中起着重要的作用。血管生成素样3(也称为angiopoietin-like 3、ANGPTL3或血管生成素5、ANGPT5)是血管生成素家族的一个成员，主要在肝中表达，并且被认为在调节脂质代谢中起作用。Angiopoietins are a family of secreted growth factors. Together with its corresponding endothelial-specific receptors, angiopoietin plays an important role in angiogenesis. Angiopoietin-like 3 (also known as angiopoietin-like 3, ANGPTL3 or angiopoietin 5, ANGPT5) is a member of the angiopoietin family that is primarily expressed in the liver and is thought to play a role in regulating lipid metabolism.
血管生成素样3(angiopoietin-like 3或ANGPTL3)是一种脂质代谢调节剂，能够通过抑制脂蛋白脂肪酶(LPL)的催化活性而调节VLDL甘油三酯(TG)。具有hypl突变的APOEKO小鼠(apoEKO-hypl)模型具有降低的Angptl3表达。该小鼠模型表现出显著的VLDL TG、VLDL胆固醇和血浆apoB水平的降低，并且肝素后血浆中LPL和肝脂肪酶活性在apoEKO-hypl小鼠中显著增加，表明具有增强的脂质代谢(Ando等人，(2003)J.Lipid Res，44：1216-1223)。人类的ANGPTL3血浆浓度与血浆HDL胆固醇和HDL磷脂水平正相关(Shimamura等人，(2007)Arterioscler.Thromb.Vasc.Biol.，27：366-372)。Angiopoietin-like 3 or ANGPTL3 is a lipid metabolism regulator that can regulate VLDL triglycerides (TG) by inhibiting the catalytic activity of lipoprotein lipase (LPL). The APOEKO mouse model with hypl mutation (apoEKO-hypl) has reduced Angptl3 expression. This mouse model exhibited significant reductions in VLDL TG, VLDL cholesterol, and plasma apoB levels, and post-heparin plasma LPL and hepatic lipase activities were significantly increased in apoEKO-hypl mice, indicating enhanced lipid metabolism (Ando et al., (2003) J. Lipid Res, 44: 1216-1223). Human ANGPTL3 plasma concentrations are positively correlated with plasma HDL cholesterol and HDL phospholipid levels (Shimamura et al., (2007) Arterioscler. Thromb. Vasc. Biol., 27:366-372).
近年来，以ANGPTL3为靶点的抑制剂成为这些疾病的新型治疗药物。siRNA作为一种新的治疗方法具有巨大的发展潜力，siRNA作用于细胞内的mRNA,相对于传统的小分子药物，它能够直接沉默靶基因，因此可以从根本上更高效地阻止疾病的发生和发展。但由于siRNA稳定性较差，在体内容易被核酸酶降解，不易被组织吸收，难以被细胞摄取，易产生脱靶效应等缺陷，使得其在临床应用上受到局限。目前亟需一种能够有效抑制细胞内ANGPTL3基因表达的siRNA。In recent years, inhibitors targeting ANGPTL3 have become new therapeutic drugs for these diseases. siRNA has great potential for development as a new treatment method. siRNA acts on intracellular mRNA. Compared with traditional small molecule drugs, it can directly silence target genes, so it can fundamentally prevent the occurrence and development of diseases more efficiently. develop. However, due to the poor stability of siRNA, it is easily degraded by nucleases in the body, is not easily absorbed by tissues, is difficult to be taken up by cells, and is prone to off-target effects, which limits its clinical application. There is an urgent need for an siRNA that can effectively inhibit the expression of ANGPTL3 gene in cells.
发明内容Contents of the invention
本公开提供了新的用于抑制细胞中血管生成素样3(ANGPTL3)的表达的小干扰RNA(siRNA)、试剂盒及其药物组合物，以及所述siRNA、试剂盒或药物组合物在抑制或降低ANGPTL3基因表达或治疗ANGPTL3表达相关的疾病或症状的方法。The present disclosure provides new small interfering RNA (siRNA), kits and pharmaceutical compositions for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, and the siRNA, kits or pharmaceutical compositions are effective in inhibiting the expression of angiopoietin-like 3 (ANGPTL3). Or methods for reducing ANGPTL3 gene expression or treating diseases or symptoms related to ANGPTL3 expression.
在第一方面，本公开提供了一种用于抑制细胞中血管生成素样3(ANGPTL3)的表达的小干扰RNA(siRNA)，所述siRNA包含形成双链区的正义链和反义链，其中所述正义链和所述反义链的长度各自独立地为15-30个核苷酸，并且所述反义链包含SEQ ID NO:16-29中任一项所示的核苷酸序列的至少15个连续核苷酸的核苷酸序列。在一些具体的实施方案中，所述正义链包含SEQ ID NO:1-14中任一项所示的核苷酸序列的至少15个连续的核苷酸的核苷酸序列。In a first aspect, the present disclosure provides a small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, the siRNA comprising a sense strand and an antisense strand forming a double-stranded region, Wherein the length of the sense strand and the antisense strand is each independently 15-30 nucleotides, and the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 16-29 A nucleotide sequence of at least 15 consecutive nucleotides. In some specific embodiments, the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-14.
在一些实施方案中，所述正义链和所述反义链的长度各自独立地为17-27个核苷酸，优选19-25个核苷酸，更优选19-23个核苷酸。In some embodiments, the length of the sense strand and the antisense strand is each independently 17-27 nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides.
在一些实施方案中，所述双链区的长度为15-25个核苷酸对，优选17-23个核苷酸对，更优选19-21个核苷酸对。In some embodiments, the double-stranded region is 15-25 nucleotide pairs in length, preferably 17-23 nucleotide pairs, more preferably 19-21 nucleotide pairs.
在一些实施方案中，所述正义链和所述反义链之一或两者包含具有至少1个核苷酸的3’突出端和/或5’突出端，例如所述正义链和所述反义链之一或两者包含具有至少1个核苷酸的3’突出端和/或5’突出端。在一些优选的实施方案中， 所述反义链具有至少2个核苷酸的3’突出端和/或5’突出端，优选地所述反义链包含具有2个核苷酸的3’突出端和/或5’突出端。In some embodiments, one or both of the sense strand and the antisense strand comprise a 3' overhang and/or a 5' overhang having at least 1 nucleotide, e.g., the sense strand and the One or both antisense strands contain a 3' overhang and/or a 5' overhang of at least 1 nucleotide. In some preferred embodiments, The antisense strand has a 3' overhang and/or a 5' overhang of at least 2 nucleotides, preferably the antisense strand comprises a 3' overhang and/or a 5' overhang of 2 nucleotides. end.
在一些实施方案中，所述反义链包含SEQ ID NO:16-29中任一项所示的核苷酸序列的至少16个连续核苷酸的核苷酸序列，至少17个连续核苷酸的核苷酸序列，至少18个连续核苷酸的核苷酸序列，至少19个连续核苷酸的核苷酸序列，至少20个连续核苷酸的核苷酸序列，优选所述反义链包含SEQ ID NO:16-29中任一项所示的核苷酸序列。In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 16-29, and at least 17 contiguous nucleotides. A nucleotide sequence of an acid, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, a nucleotide sequence of at least 20 consecutive nucleotides, preferably the reverse The sense strand includes the nucleotide sequence shown in any one of SEQ ID NO:16-29.
在一些实施方案中，所述正义链包含与SEQ ID NO:1-14中所示的核苷酸序列任一项的至少16个连续核苷酸的核苷酸序列，至少17个连续核苷酸的核苷酸序列，至少18个连续核苷酸的核苷酸序列，优选所述反义链包含SEQ ID NO:1-14中任一项所示的核苷酸序列。In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-14, and at least 17 contiguous nucleotides The nucleotide sequence of the acid, the nucleotide sequence of at least 18 consecutive nucleotides, preferably the antisense strand includes the nucleotide sequence shown in any one of SEQ ID NO: 1-14.
在一些实施方案中，所述siRNA包含如表3所示的配对的正义链序列和反义链序列。In some embodiments, the siRNA comprises paired sense and antisense strand sequences as shown in Table 3.
在一些实施方案中，所述正义链的基本上所有的核苷酸和所述反义链的基本上所有的核苷酸是修饰的核苷酸，或者所述正义链的所有的核苷酸和所述反义链的所有的核苷酸是修饰的核苷酸。In some embodiments, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, or all of the nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.
在一些具体的实施方案中，所述正义链和所述反义链各自独立地包含选自下组的一种或多种核苷酸修饰：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、无碱基核苷酸、反向无碱基脱氧核糖核苷酸、硫代磷酸酯核苷酸间键联修饰、乙烯基膦酸酯修饰的核苷酸、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、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、脱氧核糖核苷酸和STM1。In some specific embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, phosphorothioates Ester internucleotide linkage modification, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholine nucleotides, phosphoramidates, non-natural bases containing nucleotides, terminal nucleotides attached to cholesterol-based derivatives or dodecyl dodecylamide groups, deoxyribonucleotides and STM1.
在一些优选的实施方案中，所述正义链和所述反义链各自独立地包含选自下组的一种或多种核苷酸修饰：2'-O-甲基修饰的核苷酸、2'-氟代修饰的核苷酸、反向无碱基脱氧核糖核苷酸、硫代磷酸酯核苷酸间键联修饰、和STM1。在一些优选的实施方案中，所述正义链和/或所述反义链包含至少2个2'-氟代修饰的核苷酸。在一些优选的实施方案中，所述正义链和/或所述反义链包含至少8个2'-O-甲基修饰的核苷酸。在一些优选的实施方案中，所述正义链和/或所述反义链的3’末端和/或5’末端包含1-5个硫代磷酸酯基团，优选2-3个硫代磷酸酯基团。In some preferred embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, reverse abasic deoxyribonucleotides, phosphorothioate internucleotide linkage modifications, and STM1. In some preferred embodiments, the sense strand and/or the antisense strand comprises at least 2 2'-fluoro modified nucleotides. In some preferred embodiments, the sense strand and/or the antisense strand comprises at least 8 2'-O-methyl modified nucleotides. In some preferred embodiments, the 3' end and/or the 5' end of the sense strand and/or the antisense strand comprise 1-5 phosphorothioate groups, preferably 2-3 phosphorothioate groups ester group.
在一些实施方案中，在本公开的siRNA中，In some embodiments, in the siRNA of the present disclosure,
(a)所述正义链包含(a) The chain of justice includes
CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm(SEQ ID NO:31)，CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm(SEQ ID NO:31),
所述反义链包含The antisense strand contains
UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:61)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:61);
(b)所述正义链包含(b) The chain of justice includes
CmsAmsAmUmUmAmAfGfCfUmCmCmUmUmCmUmUmUmUm(SEQ ID NO:32)，CmsAmsAmUmUmAmAfGfCfUmCmCmUmUmCmUmUmUmUm(SEQ ID NO:32),
所述反义链包含The antisense strand contains
AmsAfsAmAfGmAfAmGfGmAfGmCfUmUfAmAfUmUfGmsUfsGm(SEQ ID NO:62)；AmsAfsAmAfGmAfAmGfGmAfGmCfUmUfAmAfUmUfGmsUfsGm(SEQ ID NO:62);
(c)所述正义链包含 (c) The chain of justice includes
UmsAmsUmUmGmUmUfCfCfUmCmUmAmGmUmUmAmUmUm(SEQ ID NO:33)，UmsAmsUmUmGmUmUfCfCfUmCmUmAmGmUmUmAmUmUm(SEQ ID NO:33),
所述反义链包含The antisense strand contains
AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm(SEQ ID NO:63)；AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm(SEQ ID NO:63);
(d)所述正义链包含(d) The chain of justice includes
GmsUmsUmAmUmUmUfCfCfUmCmCmAmGmAmAmUmUmAm(SEQ ID NO:34)，GmsUmsUmAmUmUmUfCfCfUmCmCmAmGmAmAmUmUmAm(SEQ ID NO:34),
所述反义链包含The antisense strand contains
UmsAfsAmUfUmCfUmGfGmAfGmGfAmAfAmUfAmAfCmsUfsAm(SEQ ID NO:64)；UmsAfsAmUfUmCfUmGfGmAfGmGfAmAfAmUfAmAfCmsUfsAm(SEQ ID NO:64);
(e)所述正义链包含(e) The chain of justice includes
AmsGmsAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUm(SEQ ID NO:35)，AmsGmsAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUm(SEQ ID NO:35),
所述反义链包含The antisense strand contains
AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:65)；AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:65);
(f)所述正义链包含(f) The chain of justice includes
AmsUmsCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUm(SEQ ID NO:36)，AmsUmsCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUm(SEQ ID NO:36),
所述反义链包含The antisense strand contains
AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm(SEQ ID NO:66)；AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm(SEQ ID NO:66);
(g)所述正义链包含(g) The justice chain includes
GmsAmsGmCmCmAmAfAfAfUmCmAmAmGmAmUmUmUmAm(SEQ ID NO:37)，GmsAmsGmCmCmAmAfAfAfUmCmAmAmGmAmUmUmUmAm(SEQ ID NO:37),
所述反义链包含The antisense strand contains
UmsAfsAmAfUmCfUmUfGmAfUmUfUmUfGmGfCmUfCmsUfsGm(SEQ ID NO:67)；UmsAfsAmAfUmCfUmUfGmAfUmUfUmUfGmGfCmUfCmsUfsGm(SEQ ID NO:67);
(h)所述正义链包含(h) The justice chain includes
UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUm(SEQ ID NO:38)，UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUm(SEQ ID NO:38),
所述反义链包含The antisense strand contains
AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:68)；AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:68);
(i)所述正义链包含(i) The chain of justice includes
AmsGmsAmGmCmAmAfCfUfAmAmCmUmAmAmCmUmUmAm(SEQ ID NO:39)，AmsGmsAmGmCmAmAfCfUfAmAmCmUmAmAmCmUmUmAm(SEQ ID NO:39),
所述反义链包含The antisense strand contains
UmsAfsAmGfUmUfAmGfUmUfAmGfUmUfGmCfUmCfUmsUfsCm(SEQ ID NO:69)；UmsAfsAmGfUmUfAmGfUmUfAmGfUmUfGmCfUmCfUmsUfsCm(SEQ ID NO:69);
(j)所述正义链包含(j) The chain of justice includes
GmsCmsAmAmCmUmAfAfCfUmAmAmCmUmUmAmAmUmUm(SEQ ID NO:40)，GmsCmsAmAmCmUmAfAfCfUmAmAmCmUmUmAmAmUmUm(SEQ ID NO:40),
所述反义链包含 The antisense strand contains
AmsAfsUmUfAmAfGmUfUmAfGmUfUmAfGmUfUmGfCmsUfsCm(SEQ ID NO:70)；AmsAfsUmUfAmAfGmUfUmAfGmUfUmAfGmUfUmGfCmsUfsCm(SEQ ID NO:70);
(k)所述正义链包含(k) The justice chain includes
CmsUmsAmAmCmUmAfAfCfUmUmAmAmUmUmCmAmAmAm(SEQ ID NO:41)，CmsUmsAmAmCmUmAfAfCfUmUmAmAmUmUmCmAmAmAm(SEQ ID NO:41),
所述反义链包含The antisense strand contains
UmsUfsUmGfAmAfUmUfAmAfGmUfUmAfGmUfUmAfGmsUfsUm(SEQ ID NO:71)；UmsUfsUmGfAmAfUmUfAmAfGmUfUmAfGmUfUmAfGmsUfsUm(SEQ ID NO:71);
(l)所述正义链包含(l) The justice chain includes
CmsUmsAmAmCmUmUfAfAfUmUmCmAmAmAmAmUmCmAm(SEQ ID NO:42)，CmsUmsAmAmCmUmUfAfAfUmUmCmAmAmAmUmCmAm(SEQ ID NO:42),
所述反义链包含The antisense strand contains
UmsGfsAmUfUmUfUmGfAmAfUmUfAmAfGmUfUmAfGmsUfsUm(SEQ ID NO:72)；UmsGfsAmUfUmUfUmGfAmAfUmUfAmAfGmUfUmAfGmsUfsUm(SEQ ID NO:72);
(m)所述正义链包含(m) The justice chain includes
UmsAmsAmCmUmUmAfAfUfUmCmAmAmAmAmUmCmAmAm(SEQ ID NO:43)，UmsAmsAmCmUmUmAfAfUfUmCmAmAmAmAmUmCmAmAm(SEQ ID NO:43),
所述反义链包含The antisense strand contains
AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm(SEQ ID NO:73)；或AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm(SEQ ID NO:73); or
(n)所述正义链包含(n) The justice chain includes
GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAm(SEQ ID NO:44)，GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAm(SEQ ID NO:44),
所述反义链包含The antisense strand contains
UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:74)。UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm (SEQ ID NO:74).
在一些实施方案中，在本公开的siRNA中，In some embodiments, in the siRNA of the present disclosure,
(a)所述正义链包含(a) The chain of justice includes
UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmsUm(SEQ ID NO:131)，UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmsUm(SEQ ID NO:131),
并且所述反义链包含and the antisense strand contains
AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:132)；AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:132);
(b)所述正义链包含(b) The chain of justice includes
GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmsAm(SEQ ID NO:133)，GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmsAm(SEQ ID NO:133),
并且所述反义链包含and the antisense strand contains
UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:134)；UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:134);
(c)所述正义链包含(c) The chain of justice includes
CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm(SEQ ID NO:135)，CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm(SEQ ID NO:135),
并且所述反义链包含and the antisense strand contains
UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:136)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:136);
(d)所述正义链包含 (d) The chain of justice includes
STM1s-CmsUmUmGmAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUms-STM1(SEQ ID NO:137)，STM1s-CmsUmUmGmAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUms-STM1 (SEQ ID NO: 137),
并且所述反义链包含and the antisense strand contains
AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:138)；AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:138);
(e)所述正义链包含(e) The chain of justice includes
STM1s-AmsUmGmGmAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAms-STM1(SEQ ID NO:139)，STM1s-AmsUmGmGmAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAms-STM1 (SEQ ID NO: 139),
并且所述反义链包含and the antisense strand contains
UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:140)；UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:140);
(f)所述正义链包含(f) The chain of justice includes
STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-STM1(SEQ ID NO:141)，STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-STM1(SEQ ID NO:141),
并且所述反义链包含and the antisense strand contains
UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:142)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:142);
(g)所述正义链包含(g) The justice chain includes
STM1-STM1-STM1-STM1-
UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1(SEQ ID NO:143)，UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1 (SEQ ID NO: 143),
并且所述反义链包含and the antisense strand contains
UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:144)；或UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:144); or
(h)所述正义链包含(h) The justice chain includes
IBs-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-IB(SEQ ID NO:145)，IBs-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-IB (SEQ ID NO: 145),
并且所述反义链包含and the antisense strand contains
UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:146)。UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 146).
在一些实施方案中，在本公开的siRNA中，In some embodiments, in the siRNA of the present disclosure,
(a)所述正义链包含(a) The chain of justice includes
STM1-STM1-STM1-STM1-
UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm-STM1-STM1(SEQ ID NO:147)，UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm-STM1-STM1 (SEQ ID NO: 147),
并且所述反义链包含and the antisense strand contains
UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:148)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:148);
(b)所述正义链包含(b) The chain of justice includes
STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1(SEQ ID NO:149)，STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1 (SEQ ID NO: 149),
并且所述反义链包含and the antisense strand contains
UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:150)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:150);
(c)所述正义链包含 (c) The chain of justice includes
STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUms-STM1(SEQ ID NO:151)，STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUms-STM1 (SEQ ID NO: 151),
并且所述反义链包含and the antisense strand contains
AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:152)；AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:152);
(d)所述正义链包含(d) The chain of justice includes
STM1-STM1-STM1-STM1-
CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmsUm-STM1-STM1(SEQ ID NO:153)，CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmsUm-STM1-STM1 (SEQ ID NO:153),
并且所述反义链包含and the antisense strand contains
AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:154)；AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:154);
(e)所述正义链包含(e) The chain of justice includes
STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUm-STM1-STM1(SEQ ID NO:155)，STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUm-STM1-STM1 (SEQ ID NO: 155),
并且所述反义链包含and the antisense strand contains
AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:156)；AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:156);
(f)所述正义链包含(f) The chain of justice includes
STM1s-UmsGmGmUmAmUmUmAmAfAfUfCmCmUmUmAmAmGmAmGmAms-STM1(SEQ ID NO:157)，STM1s-UmsGmGmUmAmUmUmAmAfAfUfCmCmUmUmAmAmGmAmGmAms-STM1 (SEQ ID NO: 157),
并且所述反义链包含and the antisense strand contains
UmsCfsUmCfUmUfAmAfGmGfAmUfUmUfAmAfUmAfCmsCfsAm(SEQ ID NO:158)；UmsCfsUmCfUmUfAmAfGmGfAmUfUmUfAmAfUmAfCmsCfsAm(SEQ ID NO:158);
(g)所述正义链包含(g) The justice chain includes
STM1s-CmsCmAmGmAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUms-STM1(SEQ ID NO:159)，STM1s-CmsCmAmGmAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUms-STM1 (SEQ ID NO: 159),
并且所述反义链包含and the antisense strand contains
AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:160)；或AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:160); or
(h)所述正义链包含(h) The justice chain includes
STM1s-UmsGmAmUmCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUms-STM1(SEQ ID NO:161)，STM1s-UmsGmAmUmCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUms-STM1 (SEQ ID NO: 161),
并且所述反义链包含and the antisense strand contains
AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm(SEQ ID NO:162)。AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm (SEQ ID NO: 162).
在一些实施方案中，所述siRNA进一步与包含N-乙酰半乳糖胺的配体部分缀合，优选所述siRNA的正义链与所述配体部分缀合。在一些优选的实施方案中，所述正义链的3’端与所述配体部分缀合。在另一些优选的实施方案中，所述正义链的5’端与所述配体部分缀合。In some embodiments, the siRNA is further conjugated to a ligand moiety comprising N-acetylgalactosamine, preferably the sense strand of the siRNA is conjugated to the ligand moiety. In some preferred embodiments, the 3' end of the sense strand is conjugated to the ligand moiety. In other preferred embodiments, the 5' end of the sense strand is conjugated to the ligand moiety.
在一些实施方案中，所述配体部分包含式(X’)所示的缀合基团：
In some embodiments, the ligand moiety includes a conjugation group represented by formula (X'):
其中， in,
表示与生物分子连接的位置； Indicates the location of attachment to biomolecules;
Q独立地为H、 Q is independently H,
其中L1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 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 -;
L2为化学键或-CH2CH2C(O)-；L 2 is a chemical bond or -CH 2 CH 2 C(O)-;
L3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
L4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；L 4 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 -;
其中a＝0、1、2或3；where a=0, 1, 2 or 3;
b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
L为化学键、-CH2O-或-NHC(O)-；L is a chemical bond, -CH 2 O- or -NHC(O)-;
L’为化学键、-C(O)NH-、-NHC(O)-或-O(CH2CH2O)e-；L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
其中e为1、2、3、4或5； where e is 1, 2, 3, 4 or 5;
T为化学键、-CH2-、-C(O)-、-M-、-CH2-M-或-C(O)-M-；T is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
其中M为 where M is
R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
在一些实施方案中，所述缀合配体靶向去唾液酸糖蛋白受体(ASGPR)。In some embodiments, the conjugation ligand targets asialoglycoprotein receptor (ASGPR).
在一些优选的实施方案中，所述缀合基团选自表1：In some preferred embodiments, the conjugation group is selected from Table 1:
表1缀合基团的结构



Table 1 Structure of conjugated groups



在一些优选的实施方案中，所述缀合基团选自表2：In some preferred embodiments, the conjugation group is selected from Table 2:
表2缀合基团的结构




Table 2 Structure of conjugated groups




在一些实施方案中，所述siRNA中包含的所述配体具有以下结构：
In some embodiments, the ligand comprised in the siRNA has the following structure:
其中表示通过磷酸酯基团或硫代磷酸酯基团与所述siRNA的正义链连接的位置。in Indicates the position connected to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.
在一些实施方案中，所述siRNA中包含的所述配体具有以下结构：
In some embodiments, the ligand comprised in the siRNA has the following structure:
其中表示通过磷酸酯基团或硫代磷酸酯基团与所述siRNA的正义链连接的位置。in Indicates the position connected to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.
在一些实施方案中，本公开的siRNA中包含的配体具有以下结构：
In some embodiments, the ligand comprised in the siRNA of the present disclosure has the following structure:
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。in Indicates the position of attachment to the siRNA via a phosphate group or phosphorothioate group.
在一些实施方案中，本公开的siRNA中包含的配体具有以下结构：
In some embodiments, the ligand comprised in the siRNA of the present disclosure has the following structure:
其中表示通过磷酸酯基团或硫代磷酸酯基团与siRNA的正义链连接的位置。in Indicates the position of attachment to the sense strand of siRNA via a phosphate group or phosphorothioate group.
本公开本公开在第二方面，本公开提供了一种细胞，其含有本公开所述的siRNA。The present disclosure In a second aspect, the present disclosure provides a cell containing the siRNA described in the present disclosure.
在第三方面，本公开提供了一种药物组合物，其包含本公开所述的siRNA或细胞，以及任选的药学上可接受的载剂或赋形剂。In a third aspect, the disclosure provides a pharmaceutical composition comprising the siRNA or cells of the disclosure, and optionally a pharmaceutically acceptable carrier or excipient.
在第四方面，本公开提供了一种试剂盒，其包含本公开所述的siRNA、细胞或药物组合物。 In a fourth aspect, the present disclosure provides a kit comprising the siRNA, cells or pharmaceutical composition of the present disclosure.
在第五方面，本公开提供了一种减少受试者中ANGPTL3水平、LDL水平、apoC-III水平、甘油三酯水平、胆固醇水平、葡萄糖水平、脂肪垫重的方法，所述方法包括向所述受试者施用本公开所述的siRNA、细胞、或药物组合物的步骤。In a fifth aspect, the present disclosure provides a method of reducing ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels, cholesterol levels, glucose levels, and fat pad weight in a subject, the method comprising: The subject is administered a siRNA, cell, or pharmaceutical composition of the present disclosure.
本公开还提供了一种治疗受试者中与ANGPTL3表达相关的疾病或症状的方法，所述方法包括向所述受试者施用本公开所述的siRNA、细胞、或药物组合物的步骤。The present disclosure also provides a method of treating a disease or condition associated with ANGPTL3 expression in a subject, the method comprising the step of administering to the subject a siRNA, cell, or pharmaceutical composition described in the present disclosure.
在一些实施方案中，所述ANGPTL3表达相关的疾病为所述代谢疾病或心血管疾病。在一些优选的实施方案中，所述代谢疾病或所述心血管疾病选自肥胖症、糖尿病、动脉粥样硬化、血脂障碍、冠心病、非酒精性脂肪肝疾病(NAFLD)、高脂肪酸血症或代谢综合征或其组合。In some embodiments, the disease associated with ANGPTL3 expression is the metabolic disease or cardiovascular disease. In some preferred embodiments, the metabolic disease or the cardiovascular disease is selected from obesity, diabetes, atherosclerosis, dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease (NAFLD), hyperfattyemia or metabolic syndrome or a combination thereof.
在一些实施方案中，所述ANGPTL3表达相关的疾病为血脂障碍，所述血脂障碍是高脂血症。在一些实施方案中，所述高脂血症是高胆固醇血症、高甘油三酯血症、或其组合。In some embodiments, the disease associated with ANGPTL3 expression is a dyslipidemia, and the dyslipidemia is hyperlipidemia. In some embodiments, the hyperlipidemia is hypercholesterolemia, hypertriglyceridemia, or a combination thereof.
在一些实施方案中，所述ANGPTL3表达相关的疾病为NAFLD，所述NAFLD是肝脂肪变性或脂肪肝炎。In some embodiments, the disease associated with ANGPTL3 expression is NAFLD, and the NAFLD is hepatic steatosis or steatohepatitis.
在一些实施方案中，所述ANGPTL3表达相关的疾病为糖尿病，所述糖尿病是2型糖尿病或具有血脂障碍的2型糖尿病。In some embodiments, the disease associated with ANGPTL3 expression is diabetes, which is type 2 diabetes or type 2 diabetes with dyslipidemia.
在一些实施方案中，本公开的治疗受试者中与ANGPTL3表达相关的疾病或症状的方法包括通过皮下施用、局部施用或静脉内向所述受试者施用所述siRNA、细胞或药物组合物。在一些实施方案中，所述受试者是人受试者。In some embodiments, methods of the present disclosure for treating a disease or condition associated with ANGPTL3 expression in a subject include administering the siRNA, cells, or pharmaceutical composition to the subject by subcutaneous administration, topical administration, or intravenous administration. In some embodiments, the subject is a human subject.
发明详述Detailed description of the invention
以下通过特定的具体实例说明本公开的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本公开的其他优点与功效。本公开还可以通过另外不同的具体实施方式实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本公开的精神下进行各种修饰或改变。The following describes the embodiments of the present disclosure through specific examples. Those skilled in the art can easily understand other advantages and effects of the present disclosure from the content disclosed in this specification. The present disclosure can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present disclosure.
应理解，本公开的保护范围不局限于下述特定的具体实施方案；还应当理解，本公开的实施方案中使用的术语是为了描述特定的具体实施方案，而不是为了限制本公开的保护范围。It should be understood that the protection scope of the present disclosure is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present disclosure are for describing specific embodiments, rather than limiting the protection scope of the present disclosure. .
在本公开说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。In this disclosure and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
当实施方案给出数值范围时，应理解，除非本公开另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本公开中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本公开的记载，还可以使用与本公开实施方案中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本公开，均属于本公开的保护范围。下文更具体详细说明本公开的实施方式。When embodiments give numerical ranges, it should be understood that both endpoints of each numerical range and any number between the two endpoints may be selected unless otherwise stated in the disclosure. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the examples, those skilled in the art can also use methods, equipment, and materials described in the embodiments of the present disclosure based on their understanding of the prior art and the description of the present disclosure. Any methods, equipment and materials similar or equivalent to the prior art to implement the present disclosure shall fall within the protection scope of the present disclosure. Embodiments of the present disclosure are described in greater detail below.
定义definition
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。The term "siRNA" herein 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. For convenience, such mRNA is also referred to herein as the mRNA to be silenced. Such genes are also called target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA other than mRNA (e.g. tRNA) as well as viral RNA can also be targeted.
如在此使用的，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或基本互补的区域。As used herein, the term "antisense strand" refers to a strand of siRNA that contains a region that is completely or substantially complementary to a target sequence.
如在此使用的，术语“互补区域”是指反义链上与靶mRNA序列完全或基本互补的区域。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。As used herein, the term "complementary region" refers to a region on the antisense strand that is completely 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').
如此处所使用，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。As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA at 50°C or 70°C for 12-16 hours.
如此处所使用，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。As used herein, "complementary" sequences, to the extent that they meet the above requirements with respect to their ability to hybridize, may also include or be formed entirely from non-Watson-Crick base pairs and/or from non-natural and processed base pairs. Base pairs formed by modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.
如在此使用的，与信使RNA(mRNA)的“至少部分互补”或“基本上互补”的多核苷酸是指与感兴趣的mRNA(例如，编码ANGPTL3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ANGPTL3的mRNA的非中断部分基本上互补，则多核苷酸与ANGPTL3mRNA的至少部分互补。As used herein, a polynucleotide that is "at least partially complementary" or "substantially complementary" to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding ANGPTL3). glycosides. For example, a polynucleotide is complementary to at least a portion of ANGPTL3 mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding ANGPTL3.
在此的术语“互补”、“完全互补”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。The terms "complementary," "completely complementary," and "substantially complementary" as used herein may be used with respect to base pairing between the sense strand and the antisense strand of an siRNA, or between the antisense strand of an siRNA agent and a target sequence.
如在此使用的，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基本互补的区域。As used herein, the term "sense strand" refers to a strand of siRNA that includes a region that is substantially complementary to a region that is the antisense strand as the term is defined herein.
“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)。"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. "Oligonucleotide" refers to, for example, a nucleic acid molecule (RNA or DNA) having a length of less than 100, 200, 300 or 400 nucleotides.
“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。"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". In this article, unless otherwise specified, the capital letters A, U, T, G and C represent the base composition of nucleotides, which are adenine, uracil, thymine, guanine and cytosine respectively.
如在此使用的，术语“核苷酸突出端”是指至少一个未配对的核苷酸，其从siRNA的双链体结构(例如，siRNA)突出。例如当siRNA的一条链的3'-末端延伸超过另一条链的5'-末端时或反之亦然，存在核苷酸突出端。siRNA可以包含具有至少一个核苷酸的突出端；替代地，该突出端可以包含至少两个核苷酸、至少三个核苷酸、至少四个核苷酸、至少五个核苷酸或更多。核苷酸突出端可以包含核苷酸/核苷类似物(包括脱氧核苷酸/核苷)或由其组成。一个或多个突出端可以处于正义链、反义链或其任何组合上。另外，突出端的一个或多个核苷酸可以存在于siRNA的反义或正义链的5'-末端、3'-末端或两个末端上。As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of an siRNA (eg, siRNA). Nucleotide overhangs exist, for example when the 3'-end of one strand of siRNA extends beyond the 5'-end of the other strand or vice versa. The siRNA can comprise an overhang having at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. many. Nucleotide overhangs may comprise or consist of nucleotide/nucleoside analogs (including deoxynucleotides/nucleosides). One or more overhangs can be on the sense strand, the antisense strand, or any combination thereof. Additionally, the overhanging nucleotide or nucleotides may be present on the 5'-end, 3'-end, or both ends of the antisense or sense strand of the siRNA.
“平端”或“平末端”或“钝端”意指在该双链siRNA的该端处不存在不成对的核苷酸，即无核苷酸突出端。“平端”siRNA是在其整个长度上为双链的siRNA，即，在分子的任一端处没有核苷酸突出端。本公开的siRNA包括在一端处具有核苷酸突出端(即，具有一个突出端和一个平端的试剂)或在两端处都具有核苷酸突出端的siRNA。"Blunt end" or "blunt end" or "blunt end" means that there are no unpaired nucleotides at the end of the double-stranded siRNA, ie, no nucleotide overhangs. A "blunt-ended" siRNA is a siRNA that is double-stranded throughout its length, ie, has no nucleotide overhangs at either end of the molecule. siRNAs of the present disclosure include siRNAs with nucleotide overhangs at one end (i.e., agents with one overhang and one blunt end) or with nucleotide overhangs at both ends.
本公开的iRNA的基本上所有的核苷酸是修饰的。例如，正义链的基本上所 有核苷酸都是修饰的核苷酸，和/或反义链的基本上所有核苷酸都是修饰的核苷酸，和/或正义链和反义链两者的基本上所有核苷酸都是修饰的核苷酸。在本公开其他实施方案中，本公开iRNA的所有核苷酸都为修饰的核苷酸。例如，正义链的所有核苷酸都是修饰的核苷酸，和/或反义链的所有核苷酸都是修饰的核苷酸，和/或正义链和反义链两者的全部核苷酸都是修饰的核苷酸。“基本上所有核苷酸是修饰的”表示本公开的siRNA是大部分但不是全部修饰的，并且可以包括不多于5、4、3、2或1个未修饰的核苷酸。Substantially all nucleotides of the iRNAs of the present disclosure are modified. For example, basically the chain of justice All nucleotides are modified nucleotides, and/or substantially all nucleotides in the antisense strand are modified nucleotides, and/or substantially all nucleotides in both the sense and antisense strands are Acids are all modified nucleotides. In other embodiments of the disclosure, all nucleotides of the iRNA of the disclosure are modified nucleotides. For example, all nucleotides of the sense strand are modified nucleotides, and/or all nucleotides of the antisense strand are modified nucleotides, and/or all nucleotides of both the sense strand and the antisense strand The nucleotides are all modified nucleotides. "Substantially all nucleotides are modified" means that the siRNAs of the present disclosure are mostly, but not entirely, modified and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.
本文中“修饰的核苷酸”包括但不限于2'-O-烷基修饰核苷酸(例如2'-O-甲基修饰的核苷酸、2'-甲氧基乙基修饰的核苷酸)、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、无碱基核苷酸、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、硫代磷酸酯核苷酸间键联修饰、乙烯基膦酸酯修饰的核苷酸、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、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、3'-末端脱氧胸腺嘧啶(dT)核苷酸、构象限制核苷酸、限制性乙基核苷酸、2'-羟基修饰的核苷酸、包含甲基膦酸基团的核苷酸、包含5'-磷酸的核苷酸、包含5'-磷酸模拟物的核苷酸、二醇修饰核苷酸(GNA)、以及2-O-(N-甲基乙酰胺)修饰的核苷酸、和STM1，或常规保护基保护等。"Modified nucleotides" herein include, but are not limited to, 2'-O-alkyl modified nucleotides (e.g., 2'-O-methyl modified nucleotides, 2'-methoxyethyl modified nucleic acids). nucleotides), 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides , nucleotides containing phosphorothioate groups, phosphorothioate internucleotide linkage modifications, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleosides Acids, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases containing nucleotides, linked to cholesteryl derivatives or dodecyl dodecyl amido groups Terminal nucleotides on the group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-hydroxy modified nucleosides Acids, nucleotides containing a methylphosphonic acid group, nucleotides containing a 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, diol-modified nucleotides (GNA), and 2-O -(N-methylacetamide) modified nucleotides, and STM1, or conventional protecting group protection, etc.
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。For example, 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.
“包含硫代磷酸基团的核苷酸”是指核苷酸中的磷酸上的一个或多个氧原子被硫原子取代的核苷酸。“硫代磷酸酯核苷酸间键合的修饰”是指左右相邻的两个核苷酸通过硫代磷酸酯相连的修饰。"Nucleotide containing a phosphorothioate group" refers to a nucleotide in which one or more oxygen atoms on the phosphate are replaced by sulfur atoms. "Modification of phosphorothioate inter-nucleotide bonding" refers to the modification in which two adjacent nucleotides on the left and right are connected through phosphorothioate.
如本文所使用的，“配体部分”是指与siRNA缀合的化学部分，其能够改变siRNA的分布、靶向或寿命。在优选的实施方案中，与例如不存在这样一个配体的siRNA相比，这种配体为选择的靶标(例如分子、细胞或细胞类型、区室(例如细胞或器官区室、组织、器官或身体的区域)提供增强的亲和力。As used herein, a "ligand moiety" refers to a chemical moiety that conjugates to an siRNA and is capable of altering the distribution, targeting, or lifetime of the siRNA. In preferred embodiments, such ligand is a selected target (e.g. molecule, cell or cell type, compartment (e.g. cell or organ compartment, tissue, organ or area of the body) provides enhanced affinity.
在一些实施方案中，该配体部分靶向肝细胞上的去唾液酸糖蛋白受体(ASGPR)。配体部分与ASGPR的结合介导了经由网格蛋白包被的囊泡的内化。核内体的成熟导致溶酶体的pH降低，这促进配体-受体复合物的解离，从而释放siRNA。靶向肝细胞上的去唾液酸糖蛋白受体(ASGPR)的配体部分的缀合导致siRNA在体内或细胞内的功效和稳定性。这有利于皮下施用该siRNA。In some embodiments, the ligand moiety targets the asialoglycoprotein receptor (ASGPR) on hepatocytes. Binding of the ligand moiety to ASGPR mediates internalization via clathrin-coated vesicles. Maturation of endosomes results in a decrease in lysosomal pH, which promotes dissociation of ligand-receptor complexes, thereby releasing siRNA. Conjugation of a ligand moiety targeting the asialoglycoprotein receptor (ASGPR) on hepatocytes results in siRNA efficacy and stability in vivo or intracellularly. This facilitates subcutaneous administration of the siRNA.
如在此使用的，术语“抑制”与“减少”、“沉默”、“下调”、以及其他类似术语可互换使用，并且包括任何水平的抑制。As used herein, the term "inhibition" is used interchangeably with "reduction," "silencing," "downregulation," and other similar terms and includes any level of inhibition.
短语“抑制ANGPTL3的表达”旨在指抑制任何ANGPTL3基因以及ANGPTL3基因的变体或突变体的表达。因此，该ANGPTL3基因可以是野生型ANGPTL3基因、突变ANGPTL3基因、或在遗传操作的细胞、细胞群组或生物体的情形下的转基因ANGPTL3基因。The phrase "inhibiting the expression of ANGPTL3" is intended to mean inhibiting the expression of any ANGPTL3 gene as well as variants or mutants of the ANGPTL3 gene. Thus, the ANGPTL3 gene may be a wild-type ANGPTL3 gene, a mutant ANGPTL3 gene, or in the case of a genetically manipulated cell, cell population, or organism, a transgenic ANGPTL3 gene.
“抑制ANGPTL3基因表达”包括ANGPTL3基因的任何水平的抑制，例如ANGPTL3基因表达的至少部分阻抑。基于与ANGPTL3基因表达相关的任何变量的水平或水平变化，例如ANGPTL3mRNA水平、ANGPTL3蛋白水平、或脂质水平，可以评估ANGPTL3基因表达。此水平可以在个体细胞中或在一组细胞中(包括例如来源于受试者的样品)进行评估。 "Inhibition of ANGPTL3 gene expression" includes any level of inhibition of the ANGPTL3 gene, such as at least partial inhibition of ANGPTL3 gene expression. ANGPTL3 gene expression can be assessed based on the level or change in level of any variable associated with ANGPTL3 gene expression, such as ANGPTL3 mRNA levels, ANGPTL3 protein levels, or lipid levels. This level can be assessed in individual cells or in a group of cells (including, for example, a sample derived from a subject).
可以通过与对照水平相比的一个或多个与ANGPTL3表达相关的变量的绝对或相对水平的降低来评估抑制。对照水平可以是本领域中利用的任何类型的对照水平，例如给药前基线水平或从类似的未经处理或经对照(例如，仅缓冲液对照或惰性剂对照)处理的受试者、细胞、或样品确定的水平。Inhibition can be assessed by a reduction in absolute or relative levels of one or more variables associated with ANGPTL3 expression compared to control levels. The control level may be any type of control level utilized in the art, such as a pre-dose baseline level or from a similar untreated or control (e.g., buffer only control or inert control) subject, cell , or the level determined by the sample.
“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)。"Hydroxy 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. Mainly include silane-type protective groups, acyl-type protective groups or ether-type protective groups, preferably the following: trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), Diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac ), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyl Oxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl ( PMB), allyl, triphenylmethyl (Tr), di-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2 , 2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM).
“卤代”或“卤素”是指氟(F)、氯(Cl)、溴(Br)和碘(I)。"Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).
“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。"C 1-6 haloalkyl" refers to the above-mentioned "C 1-6 alkyl" which is substituted by one or more halogen groups. In some embodiments, 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.
“C1-6亚烷基”是指除去C1-6烷基的另一个氢而形成的二价基团，并且可以是取代或未取代的。在一些实施方案中，C1-4亚烷基、C2-4亚烷基和C1-2亚烷基是优选的。未取代的所述亚烷基包括但不限于：亚甲基(-CH2-)、亚乙基(-CH2CH2-)、亚丙基(-CH2CH2CH2-)、亚丁基(-CH2CH2CH2CH2-)、亚戊基(-CH2CH2CH2CH2CH2-)、亚己基(-CH2CH2CH2CH2CH2CH2-)，等等。示例性的取代的所述亚烷基，例如，被一个或多个烷基(甲基)取代的所述亚烷基，包括但不限于：取代的亚甲基(-CH(CH3)-、-C(CH3)2-)、取代的亚乙基(-CH(CH3)CH2-、-CH2CH(CH3)-、-C(CH3)2CH2-、-CH2C(CH3)2-)、取代的亚丙基(-CH(CH3)CH2CH2-、-CH2CH(CH3)CH2-、-CH2CH2CH(CH3)-、-C(CH3)2CH2CH2-、-CH2C(CH3)2CH2-、-CH2CH2C(CH3)2-)，等等。"C 1-6 alkylene" refers to a divalent group formed by removing another hydrogen of C 1-6 alkyl, and may be substituted or unsubstituted. In some embodiments, C 1-4 alkylene, C 2-4 alkylene, and C 1-2 alkylene are preferred. The unsubstituted alkylene group includes, but is not limited to: methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene (-CH 2 CH 2 CH 2 -), ethylene Base (-CH 2 CH 2 CH 2 CH 2 -), pentylene (-CH 2 CH 2 CH 2 CH 2 CH 2 -), hexylene (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -) ,etc. Exemplary substituted alkylene groups, for example, the alkylene groups substituted by one or more alkyl (methyl) include, but are not limited to: substituted methylene (-CH(CH 3 )- , -C(CH 3 ) 2 -), substituted ethylene (-CH(CH 3 )CH 2 -, -CH 2 CH(CH 3 )-, -C(CH 3 ) 2 CH 2 -, -CH 2 C(CH 3 ) 2- ), substituted propylene (-CH(CH 3 )CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 -, -CH 2 CH 2 CH(CH 3 ) -, -C(CH 3 ) 2 CH 2 CH 2 -, -CH 2 C(CH 3 ) 2 CH 2 -, -CH 2 CH 2 C(CH 3 ) 2 -), etc.
如本公开所用，术语“治疗”、“处理”等，指施用药剂或进行程序，以便获得效果。这些效果可以就完全或部分地预防疾病或其症状而言是预防性的，和/或可以就影响疾病和/或疾病症状的部分或完全治愈而言是治疗性的。如本公开所用，“治疗”可包括治疗哺乳动物，特别是人类的疾病或病症(例如癌症)，并且包括：(a)在对疾病易感而尚未被诊断为患病的受试者中预防该疾病或疾病症状的发生(例如，包括可能与原代疾病相关或由其引起的疾病)；(b)抑制疾病，即阻止其发展；(c)缓解疾病，即导致疾病的消退。治疗可指在治疗或改善或预防癌症方面取得成功的任何指代，包括任何客观或主观参数，例如消除；缓解；减少症状或使疾病病症对患者而言更容易忍受；减慢恶化或衰退速度；或使恶化的终点衰弱减少。症状的治疗或改善基于一个或多个客观或主观参数；包括医生检查的结果。因此，术语“治疗”包括施用本公开公开的siRNA或药物组合物，以预防或延迟、缓解或阻止或抑制与疾病(例如癌症)相关的症状或病症的发展。术语“治疗效果”是指受试者中疾病、疾病症状或疾病副作用的减少、消除或预防。 As used in this disclosure, the terms "treatment,""treatment," etc., refer to administering an agent or performing a procedure in order to obtain an effect. These effects may be prophylactic in the sense of completely or partially preventing the disease or its symptoms, and/or may be therapeutic insofar as affecting the partial or complete cure of the disease and/or the symptoms of the disease. As used in this disclosure, "treatment" may include treatment of a disease or condition (eg, cancer) in a mammal, particularly a human, and includes: (a) prophylaxis in a subject susceptible to the disease but who has not yet been diagnosed with the disease. The occurrence of the disease or disease symptoms (for example, including diseases that may be related to or caused by the primary disease); (b) inhibit the disease, that is, prevent its progression; (c) alleviate the disease, that is, cause the regression of the disease. Treatment may refer to any indication of success in treating or ameliorating or preventing cancer, including any objective or subjective parameter, such as elimination; remission; reduction of symptoms or making disease symptoms more tolerable for the patient; slowing of progression or decline ; or reduce the endpoint of worsening frailty. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician's examination. Accordingly, the term "treating" includes administering an siRNA or pharmaceutical composition disclosed in the present disclosure to prevent or delay, alleviate, or arrest or inhibit the development of symptoms or conditions associated with a disease (eg, cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of disease, disease symptoms, or disease side effects in a subject.
本公开所用的术语“治疗有效量”是指当施用至受试者以治疗疾病时足以实现这种疾病的治疗的量。The term "therapeutically effective amount" as used in this disclosure refers to an amount that, when administered to a subject to treat a disease, is sufficient to effect treatment of a disease.
如本文所用，术语“受试者”是指期望诊断、医治或治疗的任何哺乳动物受试者。用于治疗目的的“哺乳动物”是指任何归类为哺乳动物的动物，包括人、家畜、以及实验室动物、动物园动物、运动动物或宠物动物，如狗、马、猫、牛、绵羊、山羊、猪、小鼠、大鼠、兔、豚鼠、猴子等。As used herein, the term "subject" refers to any mammalian subject for whom diagnosis, treatment, or therapy is desired. "Mammal" for therapeutic purposes means any animal classified as a mammal, including humans, domestic animals, and laboratory, zoo, sporting or pet animals, such as dogs, horses, cats, cattle, sheep, Goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.
I.siRNAI.siRNA
本公开提供了一种用于抑制细胞中血管生成素样3(ANGPTL3)的表达的小干扰RNA(siRNA)，所述siRNA包含形成双链区的正义链和反义链，其中所述正义链和所述反义链的长度各自独立地为15-30个核苷酸，并且所述反义链包含SEQ ID NO:16-29中任一项所示的核苷酸序列的至少15个连续核苷酸的核苷酸序列。The present disclosure provides a small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, the siRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the length of the antisense strand is each independently 15-30 nucleotides, and the antisense strand includes at least 15 consecutive nucleotide sequences of the nucleotide sequence shown in any one of SEQ ID NO: 16-29 The nucleotide sequence of a nucleotide.
在一些实施方案中，正义链和反义链形成的双链区是完全互补的。在另一些实施方案中，正义链和反义链形成的双链区是基本上互补的，其中可以包含1个、2个、3个、4个或5个非互补位点。In some embodiments, the double-stranded region formed by the sense strand and antisense strand is completely complementary. In other embodiments, the double-stranded region formed by the sense strand and the antisense strand is substantially complementary and may contain 1, 2, 3, 4, or 5 non-complementary sites.
在一些具体的实施方案中，所述正义链包含SEQ ID NO:1-14中任一项所示的核苷酸序列的至少15个连续的核苷酸的核苷酸序列。In some specific embodiments, the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-14.
在一些实施方案中，所述正义链和所述反义链的长度各自独立地为17-27个核苷酸，优选19-25个核苷酸，更优选19-23个核苷酸。In some embodiments, the length of the sense strand and the antisense strand is each independently 17-27 nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides.
在一些实施方案中，所述双链区的长度为15-25个核苷酸对，优选17-23个核苷酸对，更优选19-21个核苷酸对。In some embodiments, the double-stranded region is 15-25 nucleotide pairs in length, preferably 17-23 nucleotide pairs, more preferably 19-21 nucleotide pairs.
所述正义链和所述反义链之一或两者包含具有至少1个核苷酸的3’突出端和/或5’突出端，例如所述正义链和所述反义链之一或两者包含具有至少1个核苷酸的3’突出端和/或5’突出端。在一些优选的实施方案中，所述反义链具有至少2个核苷酸的3’突出端和/或5’突出端，优选地所述反义链包含具有2个核苷酸的3’突出端和/或5’突出端。在一些实施方案中，所述正义链和所述反义链的长度相同。在一些实施方案方案中，所述正义链的全长与所述反义链的全长互补形成双链，即具有平末端。在另一些实施方案中，所述正义链和所述反义链长度相同，正义链的一部分与反义链的一部分互补，即正义链和反义链均具有5’突出端。在一些实施方案中，所述正义链和所述反义链的长度不同。在优选的实施方案中，反义链的5’端具有至少1个核苷酸的突出端，更优选2个或3个核苷酸的突出端。One or both of the sense strand and the antisense strand comprise a 3' overhang and/or a 5' overhang having at least 1 nucleotide, for example one or both of the sense strand and the antisense strand or Both contain 3' overhangs and/or 5' overhangs with at least 1 nucleotide. In some preferred embodiments, the antisense strand has a 3' overhang and/or a 5' overhang of at least 2 nucleotides, preferably the antisense strand includes a 3' overhang of 2 nucleotides. Overhangs and/or 5' overhangs. In some embodiments, the sense strand and the antisense strand are the same length. In some embodiments, the entire length of the sense strand is complementary to the entire length of the antisense strand to form a double strand, ie, has blunt ends. In other embodiments, the sense strand and the antisense strand are the same length, and a part of the sense strand is complementary to a part of the antisense strand, that is, both the sense strand and the antisense strand have a 5' overhang. In some embodiments, the sense strand and the antisense strand are different lengths. In a preferred embodiment, the 5' end of the antisense strand has an overhang of at least 1 nucleotide, more preferably 2 or 3 nucleotides.
在一些实施方案中，所述反义链包含SEQ ID NO:16-29中任一项所示的核苷酸序列的至少16个连续核苷酸的核苷酸序列，至少17个连续核苷酸的核苷酸序列，至少18个连续核苷酸的核苷酸序列，至少19个连续核苷酸的核苷酸序列，至少20个连续核苷酸的核苷酸序列，优选所述反义链包含SEQ ID NO:16-29中任一项所示的核苷酸序列。In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 16-29, and at least 17 contiguous nucleotides. A nucleotide sequence of an acid, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, a nucleotide sequence of at least 20 consecutive nucleotides, preferably the reverse The sense strand includes the nucleotide sequence shown in any one of SEQ ID NO:16-29.
在一些实施方案中，所述正义链包含与SEQ ID NO:1-14中所示的核苷酸序列任一项的至少16个连续核苷酸的核苷酸序列，至少17个连续核苷酸的核苷酸序列，至少18个连续核苷酸的核苷酸序列，至少19个连续核苷酸的核苷酸序列，至少20个连续核苷酸的核苷酸序列，优选所述反义链包含SEQ ID NO:1-1093中任一项所示的核苷酸序列。In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-14, at least 17 contiguous nucleotides acid nucleotide sequence, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, a nucleotide sequence of at least 20 consecutive nucleotides, preferably the reverse The sense strand includes the nucleotide sequence shown in any one of SEQ ID NO: 1-1093.
在一些实施方案中，所述siRNA包含如表3所示的配对的正义链序列和反 义链序列。In some embodiments, the siRNA comprises paired sense strand sequences and reverse strand sequences as shown in Table 3 sense strand sequence.
II.核苷酸的修饰II. Modification of Nucleotides
在一些实施方案中，所述正义链的基本上所有的核苷酸和所述反义链的基本上所有的核苷酸是修饰的核苷酸。在一些实施方案中，所述正义链的至少80％的核苷酸是修饰的核苷酸，和/或所述反义链的至少80％的核苷酸是修饰的核苷酸。In some embodiments, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the sense strand are modified nucleotides, and/or at least 80% of the nucleotides of the antisense strand are modified nucleotides.
在一些实施方案中，所述正义链的所有的核苷酸和/或所述反义链的所有的核苷酸是修饰的核苷酸。In some embodiments, all nucleotides of the sense strand and/or all nucleotides of the antisense strand are modified nucleotides.
本公开所述的核苷酸的修饰可以是在核苷酸的磷酸基团、核糖基团和/或碱基基团上的修饰。Modifications of nucleotides described in the present disclosure may be modifications on the phosphate group, ribose group and/or base group of the nucleotide.
在一些具体的实施方案中，所述正义链和所述反义链各自独立地包含选自下组的一种或多种核苷酸修饰：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、无碱基核苷酸、反向无碱基脱氧核糖核苷酸、硫代磷酸酯核苷酸间键联修饰、乙烯基膦酸酯修饰的核苷酸、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、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、脱氧核糖核苷酸和STM1。In some specific embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, phosphorothioates Ester internucleotide linkage modification, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholine nucleotides, phosphoramidates, non-natural bases containing nucleotides, terminal nucleotides attached to cholesterol-based derivatives or dodecyl dodecylamide groups, deoxyribonucleotides and STM1.
在一些优选的实施方案中，所述正义链和所述反义链各自独立地包含选自下组的一种或多种核苷酸修饰：2'-O-甲基修饰的核苷酸、2'-氟代修饰的核苷酸、反向无碱基脱氧核糖核苷酸、和硫代磷酸酯核苷酸间键联修饰。在一些优选的实施方案中，所述正义链和/或所述反义链包含至少2个2'-氟代修饰的核苷酸。在一些优选的实施方案中，所述正义链和/或所述反义链包含至少8个2'-O-甲基修饰的核苷酸。在一些优选的实施方案中，所述正义链和/或所述反义链的3’末端和/或5’末端包含1-5个硫代磷酸酯基团，优选2-3个硫代磷酸酯基团。在一些优选的实施方案中，所述正义链和/或反义链包含腺嘌呤脱氧核糖核苷酸、胸腺嘧啶脱氧核糖核苷酸、鸟嘌呤脱氧核糖核苷酸和/或胞嘧啶脱氧核糖核苷酸。在更优选的实施方案中，所述正义链和/或反义链包含胸腺嘧啶脱氧核糖核苷酸。在最优选的实施方案中，所述正义链包含胸腺嘧啶脱氧核糖核苷酸。In some preferred embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, reverse abasic deoxyribonucleotides, and phosphorothioate internucleotide linkage modifications. In some preferred embodiments, the sense strand and/or the antisense strand comprises at least 2 2'-fluoro modified nucleotides. In some preferred embodiments, the sense strand and/or the antisense strand comprises at least 8 2'-O-methyl modified nucleotides. In some preferred embodiments, the 3' end and/or the 5' end of the sense strand and/or the antisense strand comprise 1-5 phosphorothioate groups, preferably 2-3 phosphorothioate groups ester group. In some preferred embodiments, the sense strand and/or antisense strand comprise adenine deoxyribonucleotides, thymine deoxyribonucleotides, guanine deoxyribonucleotides and/or cytosine deoxyribonucleotides glycosides. In a more preferred embodiment, the sense strand and/or antisense strand comprise thymidine deoxyribonucleotides. In the most preferred embodiment, the sense strand comprises thymidine deoxyribonucleotides.
在一些优选的实施方案中，所述反义链包含说明书表5中任一项所示的经修饰的核苷酸序列，和/或所述正义链包含说明书表4中任一项所示的经修饰的核苷酸序列。在一些优选的实施方案中，所述siRNA包含说明书表6中任一项所示的配对的经修饰的正义链序列和经修饰的反义链序列。In some preferred embodiments, the antisense strand comprises a modified nucleotide sequence shown in any one of Table 5 of the specification, and/or the sense strand comprises a modified nucleotide sequence shown in any one of Table 4 of the specification. Modified nucleotide sequence. In some preferred embodiments, the siRNA includes the paired modified sense strand sequence and the modified antisense strand sequence shown in any one of Table 6 of the specification.
III.配体III. Ligand
本公开所述的siRNA进一步与包含N-乙酰半乳糖胺的配体部分缀合。在优选的实施方案中，所述siRNA的正义链与所述配体部分缀合。在一些优选的实施方案中，所述正义链的3’端与所述配体部分缀合。在另一些优选的实施方案中，所述正义链的5’端与所述配体部分缀合。The siRNAs of the present disclosure are further conjugated to a ligand moiety comprising N-acetylgalactosamine. In a preferred embodiment, the sense strand of the siRNA is conjugated to the ligand moiety. In some preferred embodiments, the 3' end of the sense strand is conjugated to the ligand moiety. In other preferred embodiments, the 5' end of the sense strand is conjugated to the ligand moiety.
在一些实施方案中，所述配体部分包含式(X’)所示的缀合基团：
In some embodiments, the ligand moiety includes a conjugation group represented by formula (X'):
其中， in,
表示与生物分子连接的位置； Indicates the location of attachment to biomolecules;
Q独立地为H、 Q is independently H,
其中L1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 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 -;
L2为化学键或-CH2CH2C(O)-；L 2 is a chemical bond or -CH 2 CH 2 C(O)-;
L3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
L4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；L 4 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 -;
其中a＝0、1、2或3；where a=0, 1, 2 or 3;
b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
L为化学键、-CH2O-或-NHC(O)-；L is a chemical bond, -CH 2 O- or -NHC(O)-;
L’为化学键、-C(O)NH-、-NHC(O)-或-O(CH2CH2O)e-；L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
其中e为1、2、3、4或5； where e is 1, 2, 3, 4 or 5;
T为化学键、-CH2-、-C(O)-、-M-、-CH2-M-或-C(O)-M-；T is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
其中M为 where M is
R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
在一些实施方案中，所述缀合基团如式(I’)所示：
In some embodiments, the conjugating group is represented by Formula (I'):
其中，in,
表示与生物分子连接的位置； Indicates the location of attachment to biomolecules;
Q独立地为H、 Q is independently H,
其中L1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 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 -;
L2为化学键或-CH2CH2C(O)-；L 2 is a chemical bond or -CH 2 CH 2 C(O)-;
L3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
L4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、- (OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；L 4 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 -;
其中a＝0、1、2或3；where a=0, 1, 2 or 3;
b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
L为-CH2O-或-NHC(O)-；L is -CH 2 O- or -NHC(O)-;
L’为化学键、-C(O)NH-或-NHC(O)-；L’ is a chemical bond, -C(O)NH- or -NHC(O)-;
R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
在一些具体的实施方案中，其中，In some specific embodiments, wherein,
Q独立地为H或 Q is independently H or
其中L1为-CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 is -CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;
L2为-CH2CH2C(O)-；L 2 is -CH 2 CH 2 C(O)-;
L3为-(NHCH2CH2)b-或-(NHCH2CH2CH2)b-；L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
其中a＝0、1、2或3；where a=0, 1, 2 or 3;
b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
L为-CH2O-；L is -CH 2 O-;
L’为化学键；L’ is a chemical bond;
R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
在一些实施方案中，所述缀合基团如式(I’-1)、式(I’-2)或式(I’-3)所示：
In some embodiments, the conjugation group is represented by Formula (I'-1), Formula (I'-2), or Formula (I'-3):
其中，in,
表示与生物分子连接的位置； Indicates the location of attachment to biomolecules;
Q为 Q is
其中L1为-CH2O-或-NHC(O)-；Where L 1 is -CH 2 O- or -NHC(O)-;
L2为-CH2CH2C(O)-；L 2 is -CH 2 CH 2 C(O)-;
L3为-(NHCH2CH2)b-或-(NHCH2CH2CH2)b-；L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
其中b＝1、2、3、4或5；Where b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
L为-CH2O-；L is -CH 2 O-;
R’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；R' 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或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
在一些具体的实施方案中，其中，In some specific embodiments, wherein,
Q独立地为H、 Q is independently H,
其中L1为-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 is -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;
L2为-CH2CH2C(O)-；L 2 is -CH 2 CH 2 C(O)-;
L3为-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
其中a＝0、1、2或3；where a=0, 1, 2 or 3;
b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
L为-CH2O-或-NHC(O)-；L is -CH 2 O- or -NHC(O)-;
L’为化学键或-C(O)NH-；L’ is a chemical bond or -C(O)NH-;
R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
在一些实施方案中，所述缀合基团如式(II’-1)或式(II’-2)所示：
In some embodiments, the conjugation group is represented by Formula (II'-1) or Formula (II'-2):
其中，in,
表示与生物分子连接的位置； Indicates the location of attachment to biomolecules;
Q独立地为 Q is independently
其中L1为-CH2O-或-CH2O-CH2CH2O-；Where L 1 is -CH 2 O- or -CH 2 O-CH 2 CH 2 O-;
L3为-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
其中b＝1、2、3、4或5；Where b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
L为-NHC(O)-；L is -NHC(O)-;
L’为化学键或-C(O)NH-；L’ is a chemical bond or -C(O)NH-;
R’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；R' 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;
m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
在一些具体的实施方案中，其中，In some specific embodiments, wherein,
Q独立地为H、 Q is independently H,
其中L1为-CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；where L 1 is -CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a - ;
L2为化学键；L 2 is a chemical bond;
L3为-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
其中a＝0、1、2或3；where a=0, 1, 2 or 3;
b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
L为-CH2O-或-NHC(O)-；L is -CH 2 O- or -NHC(O)-;
L’为化学键或-C(O)NH-；L’ is a chemical bond or -C(O)NH-;
R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
在一些实施方案中，其中所述缀合基团如式(II’-2)所示：
In some embodiments, wherein the conjugating group is represented by Formula (II'-2):
其中，in,
表示与生物分子连接的位置； Indicates the location of attachment to biomolecules;
Q独立地为 Q is independently
其中L1为-CH2-或-C(O)-；Where L 1 is -CH 2 - or -C(O)-;
L3为-(NHCH2CH2)b-；L 3 is -(NHCH 2 CH 2 ) b -;
L4为-(OCH2CH2)c-；L 4 is -(OCH 2 CH 2 ) c -;
其中b＝1、2、3、4或5；Where b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
L为-CH2O-或-NHC(O)-；L is -CH 2 O- or -NHC(O)-;
R’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；R' 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或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
在一些具体的实施方案中，其中：In some specific embodiments, wherein:
Q独立地为H、 Q is independently H,
其中L1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 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 -;
L2为化学键或-CH2CH2C(O)-；L 2 is a chemical bond or -CH 2 CH 2 C(O)-;
L3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
L4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；L 4 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 -;
其中a＝0、1、2或3；where a=0, 1, 2 or 3;
b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
L为化学键、-CH2O-或-NHC(O)-；L is a chemical bond, -CH 2 O- or -NHC(O)-;
L’为化学键、-C(O)NH-、-NHC(O)-或-O(CH2CH2O)e-；L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
其中e为1、2、3、4或5；where e is 1, 2, 3, 4 or 5;
T为化学键、-CH2-、-M-、-CH2-M-或-C(O)-M-；T is a chemical bond, -CH 2 -, -M-, -CH 2 -M- or -C(O)-M-;
其中M为 where M is
R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
在一些具体的实施方案中，其中，In some specific embodiments, wherein,
T为-M-、-CH2-M-或-C(O)-M-，其中M为 T is -M-, -CH 2 -M- or -C(O)-M-, where M is
在一些具体的实施方案中，其中，In some specific embodiments, wherein,
Q独立地为H或 Q is independently H or
其中L1为-CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 is -CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;
L2为-CH2CH2C(O)-；L 2 is -CH 2 CH 2 C(O)-;
L3为-(NHCH2CH2)b-或-(NHCH2CH2CH2)b-；L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
其中a＝0、1、2或3； where a=0, 1, 2 or 3;
b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
L为化学键或-CH2O-；L is a chemical bond or -CH 2 O-;
L’为化学键或-O(CH2CH2O)e-；L' is a chemical bond or -O(CH 2 CH 2 O) e -;
其中e为1、2、3、4或5；where e is 1, 2, 3, 4 or 5;
R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
n＝0、1、2、3、4、5、6、7、8、9或10；n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
其中T如以上实施方案所定义。where T is as defined in the embodiment above.
在一些实施方案中，其中所述缀合基团如式(III’-1)、式(III’-2)或式(III’-3)所示：
In some embodiments, wherein the conjugation group is represented by Formula (III'-1), Formula (III'-2) or Formula (III'-3):
其中，in,
Q为 Q is
其中L1为-CH2O-或-NHC(O)-；Where L 1 is -CH 2 O- or -NHC(O)-;
L2为-CH2CH2C(O)-；L 2 is -CH 2 CH 2 C(O)-;
L3为-(NHCH2CH2)b-或-(NHCH2CH2CH2)b-；L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
其中b＝1、2、3、4或5；Where b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
L为化学键或-CH2O-；L is a chemical bond or -CH 2 O-;
其中R’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；Wherein R' 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或10；n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
其中T如以上实施方案所定义。where T is as defined in the embodiment above.
在一些具体的实施方案中，其中， In some specific embodiments, wherein,
Q独立地为H、 Q is independently H,
其中L1为-CH2-、-CH2O-或-C(O)-；Where L 1 is -CH 2 -, -CH 2 O- or -C(O)-;
L2为化学键；L 2 is a chemical bond;
L3为-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
其中b＝1、2、3、4或5；Where b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
L为化学键或-NHC(O)-；L is a chemical bond or -NHC(O)-;
L’为化学键；L’ is a chemical bond;
R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
m＝0、1、2、3、4、5、6、7、8、9或10； m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
n＝0、1、2、3、4、5、6、7、8、9或10；n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
其中T如以上实施方案所定义。where T is as defined in the embodiment above.
在一些实施方案中，其中所述缀合基团如式(IV-1)或式(IV-2)所示：
In some embodiments, wherein the conjugating group is represented by Formula (IV-1) or Formula (IV-2):
其中，in,
Q独立地为 Q is independently
其中L1为-CH2-、-CH2O-或-C(O)-；Where L 1 is -CH 2 -, -CH 2 O- or -C(O)-;
L3为-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
其中b＝1、2、3、4或5；Where b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
L为化学键或-NHC(O)-；L is a chemical bond or -NHC(O)-;
L’为化学键；L’ is a chemical bond;
其中R’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；Wherein R' 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;
m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
n＝0、1、2、3、4、5、6、7、8、9或10；n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
其中T如以上实施方案所定义。 where T is as defined in the embodiment above.
在一些具体的实施方案中，其中：In some specific embodiments, wherein:
Q独立地为H、 Q is independently H,
其中L1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 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 -;
L2为化学键或-CH2CH2C(O)-；L 2 is a chemical bond or -CH 2 CH 2 C(O)-;
L3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
L4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；L 4 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 -;
其中a＝0、1、2或3；where a=0, 1, 2 or 3;
b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
L为化学键、-CH2O-或-NHC(O)-；L is a chemical bond, -CH 2 O- or -NHC(O)-;
L’为-O(CH2CH2O)e-； L' is -O(CH 2 CH 2 O) e -;
其中e为1、2、3、4或5；where e is 1, 2, 3, 4 or 5;
T为化学键、-CH2-、-C(O)-、-M-、-CH2-M-或-C(O)-M-；T is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
其中M为 where M is
R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
在一些优选的实施方案中，其中所述缀合基团选自以下：



In some preferred embodiments, wherein said conjugating group is selected from the following:



在一些优选的实施方案中，其中所述缀合基团选自以下：




In some preferred embodiments, wherein said conjugating group is selected from the following:




在一些实施方案中，所述配体靶向去唾液酸糖蛋白受体(ASGPR)。In some embodiments, the ligand targets asialoglycoprotein receptor (ASGPR).
在一个优选的实施方案中，其中所述配体具有以下结构：
In a preferred embodiment, wherein said ligand has the following structure:
其中表示通过磷酸酯基团或硫代磷酸酯基团与siRNA的正义链连接的位置。in Indicates the position of attachment to the sense strand of siRNA via a phosphate group or phosphorothioate group.
在一个优选的实施方案中，其中所述配体具有以下结构：
In a preferred embodiment, wherein said ligand has the following structure:
其中表示通过磷酸酯基团或硫代磷酸酯基团与siRNA的正义链连接的位置。in Indicates the position of attachment to the sense strand of siRNA via a phosphate group or phosphorothioate group.
在一个优选的实施方案中，其中所述配体具有以下结构：
In a preferred embodiment, wherein said ligand has the following structure:
其中表示通过磷酸酯基团或硫代磷酸酯基团与所述siRNA的正义链连接的位置。in Indicates the position connected to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.
在一个优选的实施方案中，其中所述配体具有以下结构：
In a preferred embodiment, wherein said ligand has the following structure:
其中表示通过磷酸酯基团或硫代磷酸酯基团与所述siRNA的正义链连接的位置。in Indicates the position connected to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.
IV.ANGPTL3基因表达抑制IV.ANGPTL3 gene expression inhibition
本公开的siRNA能够将ANGPTL3基因表达抑制至少约5％、至少约10％、至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％或至少约99％。The siRNA of the present disclosure can inhibit ANGPTL3 gene expression by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, At least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, At least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
ANGPTL3基因表达的抑制可以通过由第一细胞或细胞群组(这样的细胞可以存在于例如来源于受试者的样品中)表达的mRNA的量的降低来显现，其中ANGPTL3基因被转录并且该细胞或这些细胞已经被处理(例如通过使该细胞或这些细胞与本公开的siRNA接触，或通过向现在存在或以前存在这些细胞的受试者给予本公开的siRNA，使得与该第一细胞或细胞群组基本上相同但尚未被如此处理的第二细胞或细胞群组(一种或多种对照细胞)相比，ANGPTL3基因表达被抑制。Inhibition of ANGPTL3 gene expression may be manifested by a decrease in the amount of mRNA expressed by a first cell or population of cells (such cells may be present, for example, in a sample derived from the subject) in which the ANGPTL3 gene is transcribed and the cells or these cells have been treated (e.g., by contacting the cell or cells with an siRNA of the present disclosure, or by administering an siRNA of the present disclosure to a subject in which these cells are present or previously present, such that contact with the first cell or cells ANGPTL3 gene expression is inhibited compared to a second group or group of cells (one or more control cells) that are substantially the same but have not been so treated.
在优选的实施方案中，通过使用下式将被处理的细胞中的mRNA的水平表示为对照细胞中的mRNA的水平的百分比来评估该抑制。在一些具体的实施方案中，计算2-△△Ct值以比较实验组和对照组之间的差异，其中△△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)]。In a preferred embodiment, the inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells using the following formula. In some specific embodiments, the 2 -ΔΔCt value is calculated to compare the difference between the experimental group and the control group, where ΔΔCt=[(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group Target gene-Ct control group (internal reference)].
可替代地，可以就与ANGPTL3基因表达在功能上有关的参数(如脂质水平、胆固醇水平，例如LDLc水平)的降低而言来评估ANGPTL3基因表达例如ANGPTL3蛋白表达的抑制。可以组成性地或者通过基因组工程化而表达ANGPTL3的任何细胞中并且通过本领域中已知的任何测定来确定ANGPTL3基因沉默。肝脏是ANGPTL3表达的主要部位。其他重要表达部位包括胰腺、肾脏以及肠。Alternatively, inhibition of ANGPTL3 gene expression, e.g., ANGPTL3 protein expression, may be assessed in terms of reduction in parameters functionally related to ANGPTL3 gene expression, such as lipid levels, cholesterol levels, e.g., LDLc levels. ANGPTL3 gene silencing can be determined in any cell that expresses ANGPTL3 constitutively or by genome engineering and by any assay known in the art. The liver is the main site of ANGPTL3 expression. Other important sites of expression include the pancreas, kidney, and intestine.
ANGPTL3蛋白的表达的抑制可以通过由细胞或细胞群组表达的ANGPTL3蛋白水平(例如来源于受试者的样品中表达的蛋白质水平)的降低来显现。如以上关于mRNA阻抑的评估所解释，被处理的细胞或细胞群组中的蛋白质表达水平的抑制可以类似地表示为对照细胞或细胞群组中的蛋白质的水平的百分比。Inhibition of expression of ANGPTL3 protein may be manifested by a decrease in the level of ANGPTL3 protein expressed by a cell or population of cells (eg, the level of protein expressed in a sample derived from a subject). As explained above with respect to the assessment of mRNA repression, inhibition of protein expression levels in treated cells or populations of cells can similarly be expressed as a percentage of the levels of the protein in control cells or populations of cells.
可以用来评估ANGPTL3基因表达的抑制的对照细胞或细胞群组包括尚未与本公开的siRNA接触的细胞或细胞群组。例如，该对照细胞或细胞群组可以来源于在用siRNA处理受试者之前的个体受试者(例如人类或动物受试者)。Control cells or cell populations that may be used to assess inhibition of ANGPTL3 gene expression include cells or cell populations that have not been contacted with the siRNA of the present disclosure. For example, the control cells or population of cells may be derived from an individual subject (eg, a human or animal subject) prior to treatment of the subject with siRNA.
V.细胞V. cells
本公开提供了细胞，其含有本公开所述的siRNA，其中本公开所述的siRNA能够在细胞中转录。The present disclosure provides cells containing the siRNA of the present disclosure, wherein the siRNA of the present disclosure is capable of being transcribed in the cell.
VI.药物组合物VI. Pharmaceutical compositions
本公开提供了药物组合物，其包含本公开所述的siRNA或细胞，以及任选的药学上可接受的载剂或赋形剂。The present disclosure provides pharmaceutical compositions comprising the siRNA or cells of the present disclosure, and optionally a pharmaceutically acceptable carrier or excipient.
本文使用的“药学上可接受的”是指那些化合物、材料、组合物和/或剂型，其在正确医学判断范围内，适合于接触人类受试者和动物受试者的组织而没有过度 的毒性、刺激性、过敏反应或其他问题或并发症，与合理的效益/风险比相称。As used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that, within the scope of sound medical judgment, are suitable for contact with tissues of human subjects and animal subjects without undue toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
在本文中，药学上可接受的载剂是指有助于将siRNA或包含所述siRNA的细胞施用至人体和/或有利于其吸收或发挥作用的药物载剂。例如：稀释剂、赋形剂如水等，填充剂如淀粉、蔗糖等；粘合剂如纤维素衍生物、藻酸盐、明胶和聚乙烯吡咯烷酮；湿润剂如甘油；崩解剂如琼脂、碳酸钙和碳酸氢钠；吸收促进剂如季铵化合物；表面活性剂如十六烷醇；吸附载体如高岭土和皂粘土；润滑剂如滑石粉、硬脂酸钙/镁、聚乙二醇等。另外还可以在组合物中加入其它辅剂如香味剂、甜味剂等。As used herein, a pharmaceutically acceptable carrier refers to a pharmaceutical carrier that facilitates the administration of siRNA or cells containing the siRNA to the human body and/or facilitates its absorption or effect. For example: diluents, excipients such as water, fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; wetting agents such as glycerin; disintegrants such as agar, carbonic acid Calcium and sodium bicarbonate; absorption accelerators such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium/magnesium stearate, polyethylene glycol, etc. In addition, other auxiliary agents such as flavoring agents, sweeteners, etc. can also be added to the composition.
例如，包含本公开所述的siRNA或细胞的药物组合物可以包含药学上可接受的稀释剂或缓释基质，本公开所述的siRNA被嵌入该缓释基质中。For example, a pharmaceutical composition containing the siRNA or cells of the present disclosure may include a pharmaceutically acceptable diluent or sustained-release matrix in which the siRNA of the present disclosure is embedded.
VIII.试剂盒VIII. Kit
本公开提供了试剂盒，其包含本公开所述的siRNA或细胞。The present disclosure provides kits comprising the siRNA or cells described in the present disclosure.
本公开还提供了用于使用本公开所述的siRNA和/或执行本公开的方法的试剂盒。这样的试剂盒包括一种或多种本公开所述的siRNA或细胞，还可以进一步包括使用说明书。该使用说明书中可以记录用于通过使细胞与本公开所述的siRNA接触以有效抑制ANGPTL3表达的量接触来抑制该细胞中的该ANGPTL3表达的说明书。The present disclosure also provides kits for using the siRNA described in the present disclosure and/or performing the methods of the present disclosure. Such kits include one or more siRNAs or cells described in the present disclosure and may further include instructions for use. Instructions for inhibiting the expression of ANGPTL3 in the cell by contacting the cell with the siRNA described in the present disclosure in an amount effective to inhibit the expression of ANGPTL3 may be recorded in the instructions for use.
在本公开所述的siRNA在体外与细胞接触的情况下，任选地，本公开的试剂盒还可以包括用于使细胞与本公开所述的siRNA接触的工具(例如，注射装置)或用于测量ANGPTL3的抑制效果的工具(例如，用于测量ANGPTL3mRNA或蛋白质的抑制的装置)。这样的用于测量ANGPTL3的抑制的装置可以包含用于从受试者获得样品(例如像，血浆样品)的装置。Where the siRNA of the present disclosure is contacted with cells in vitro, optionally, the kit of the present disclosure may also include means (e.g., an injection device) for contacting the cells with the siRNA of the present disclosure or with Tools for measuring the inhibitory effect of ANGPTL3 (e.g., a device for measuring inhibition of ANGPTL3 mRNA or protein). Such a device for measuring inhibition of ANGPTL3 may comprise a device for obtaining a sample (eg, like a plasma sample) from a subject.
在将本公开所述的siRNA或已经在体外导入siRNA的细胞施用至体内的情况下，本公开的试剂盒还可以任选地包括用于将本公开所述siRNA或细胞给予至受试者的装置或用于确定治疗有效量或预防有效量的装置。In the case where the siRNA of the present disclosure or the cells into which the siRNA has been introduced in vitro are administered into the body, the kit of the present disclosure may also optionally include a method for administering the siRNA or cells of the present disclosure to the subject. Device or device for determining a therapeutically effective amount or a prophylactically effective amount.
IX.治疗方法、制药用途IX. Treatment methods and pharmaceutical uses
本公开提供了一种减少受试者中ANGPTL3水平、LDL水平、apoC-III水平、甘油三酯水平、胆固醇水平、葡萄糖水平、脂肪垫重的方法，所述方法包括向所述受试者施用本公开所述的siRNA、细胞、或药物组合物的步骤。The present disclosure provides a method of reducing ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels, cholesterol levels, glucose levels, fat pad weight in a subject, the method comprising administering to the subject siRNA, cells, or pharmaceutical compositions of the present disclosure.
本公开提供了一种治疗受试者中与ANGPTL3表达相关的疾病或症状的方法，所述方法包括向所述受试者施用本公开所述的siRNA、细胞、或药物组合物的步骤。The present disclosure provides a method of treating a disease or condition associated with ANGPTL3 expression in a subject, the method comprising the step of administering to the subject a siRNA, cell, or pharmaceutical composition described in the present disclosure.
在一些实施方案中，所述ANGPTL3表达相关的疾病为心血管疾病。在一些优选的实施方案中，所述心血管疾病选自肥胖症、糖尿病、动脉粥样硬化、血脂障碍、冠心病、非酒精性脂肪肝疾病(NAFLD)、高脂肪酸血症或代谢综合征或其组合。In some embodiments, the disease associated with ANGPTL3 expression is cardiovascular disease. In some preferred embodiments, the cardiovascular disease is selected from obesity, diabetes, atherosclerosis, dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease (NAFLD), hyperlipidemia or metabolic syndrome or its combination.
在一些实施方案中，所述ANGPTL3表达相关的疾病为血脂障碍，所述血脂障碍是高脂血症。在一些实施方案中，高脂血症是高胆固醇血症、高甘油三酯血症、或其组合。In some embodiments, the disease associated with ANGPTL3 expression is a dyslipidemia, and the dyslipidemia is hyperlipidemia. In some embodiments, hyperlipidemia is hypercholesterolemia, hypertriglyceridemia, or a combination thereof.
在一些实施方案中，所述ANGPTL3表达相关的疾病为NAFLD，所述NAFLD是肝脂肪变性或脂肪肝炎。In some embodiments, the disease associated with ANGPTL3 expression is NAFLD, and the NAFLD is hepatic steatosis or steatohepatitis.
在一些实施方案中，所述ANGPTL3表达相关的疾病为糖尿病，所述糖尿病 是2型糖尿病或具有血脂障碍的2型糖尿病。In some embodiments, the disease associated with ANGPTL3 expression is diabetes, and the diabetes Is type 2 diabetes or type 2 diabetes with dyslipidemia.
在一些实施方案中，本公开的治疗受试者中与ANGPTL3表达相关的疾病或症状的方法包括向所述受试者施用所述siRNA或药物组合物包括皮下施用或静脉内施用。在一些实施方案中，所述受试者是人患者。In some embodiments, methods of the present disclosure for treating a disease or condition associated with ANGPTL3 expression in a subject include administering the siRNA or pharmaceutical composition to the subject including subcutaneous administration or intravenous administration. In some embodiments, the subject is a human patient.
本公开还涉及用于治疗受试者中与ANGPTL3表达相关的疾病或症状的本公开所述的siRNA、细胞或药物组合物。The present disclosure also relates to the siRNA, cells or pharmaceutical compositions of the present disclosure for treating a disease or condition associated with ANGPTL3 expression in a subject.
本公开还涉及本公开所述的siRNA、细胞、或药物组合物在制备用于治疗受试者中与ANGPTL3表达相关的疾病或症状的药物中的用途。本公开的药物可以制备成乳剂、微乳剂、微颗粒。The present disclosure also relates to the use of the siRNA, cells, or pharmaceutical compositions of the present disclosure in the preparation of a medicament for treating a disease or symptom associated with ANGPTL3 expression in a subject. The medicine of the present disclosure can be prepared into emulsions, microemulsions, and microparticles.
序列sequence
本公开提供的RNA序列靶向人ANGPTL3基因(或靶基因、靶mRNA序列、靶序列)。The RNA sequence provided by the present disclosure targets the human ANGPTL3 gene (or target gene, target mRNA sequence, target sequence).
表3靶向ANGPTL3基因的正义链和反义链的核苷酸序列
Table 3 Nucleotide sequences of the sense and antisense strands targeting the ANGPTL3 gene
表4至表6示出了本公开使用的修饰的RNA序列。Tables 4 to 6 show modified RNA sequences used in the present disclosure.
本文中，各缩写的意义如下：In this article, the meanings of each abbreviation are as follows:
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。The A, U, G, and C distributions represent natural adenine ribonucleotides, uracil ribonucleotides, guanine ribonucleotides, and cytosine ribonucleotides.
d表示其右侧相邻的核苷酸是脱氧核糖核苷酸。例如dA、dT、dG和dC分别表示腺嘌呤脱氧核糖核苷酸、胸腺嘧啶脱氧核糖核苷酸、鸟嘌呤脱氧核糖核苷酸和胞嘧啶脱氧核糖核苷酸。d indicates that the nucleotide adjacent to the right is a deoxyribonucleotide. For example, dA, dT, dG and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide and cytosine deoxyribonucleotide respectively.
i表示肌苷核糖核苷酸。i represents inosine ribonucleotide.
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。m indicates that the adjacent nucleotide to its left is a 2'-OCH 3 modified nucleotide. For example, Am, Um, Gm and Cm represent 2'-OCH 3 modified A, U, G and 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。 f indicates that the adjacent nucleotide to its left is a 2'-F modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.
“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。"s" or "s-" means that two adjacent nucleotides and/or delivery vectors are connected through phosphorothioate.
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。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.
IB表示反向无碱基脱氧核糖核苷酸，根据其在siRNA中所在位置/连接方式的不同可包括以下三种结构(分别用于核酸链的5’端，中间和3’端)：
IB stands for reverse abasic deoxyribonucleotide, which can include the following three structures depending on its position/connection method in siRNA (respectively for the 5' end, middle and 3' end of the nucleic acid chain):
IB是本领域熟知的，参见例如F.Czauderna,Nucleic Acids Res.,2003,31(11),2705-16以及PCT公开号WO2016011123和WO2019051402。IB is well known in the art, see for example F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16 and PCT Publication Nos. WO2016011123 and WO2019051402.
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。
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.
NAG37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WO2018044350
NAG37 represents a GalNAc delivery vector of the structure well known in the art, wherein Indicates the position of attachment to siRNA via a phosphate group or phosphorothioate group, see for example PCT Publication No. WO2018044350
GL6表示以下结构的GalNAc递送载体，其中表示磷酸酯基团或硫代磷酸酯基团与siRNA连接的位置
GL6 represents a GalNAc delivery vector of the following structure, where Indicates the position where the phosphate group or phosphorothioate group is attached to the siRNA
GL12表示以下结构的GalNAc递送载体，其中表示磷酸酯基团或硫代磷酸酯基团与siRNA连接的位置
GL12 represents a GalNAc delivery vector of the following structure, where Indicates the position where the phosphate group or phosphorothioate group is attached to the siRNA
STM1表示以下结构的核苷酸替代物。根据STM1在核酸链中位置的不同，可连接至相邻核苷酸、3’端结构或5’端结构 STM1 represents the nucleotide substitution of the following structure. Depending on the position of STM1 in the nucleic acid chain, Can be linked to adjacent nucleotides, 3' end structures or 5' end structures
表4靶向ANGPTL3基因的经修饰的siRNA正义链序列

Table 4 Modified siRNA sense strand sequence targeting ANGPTL3 gene

表5靶向ANGPTL3基因的经修饰的siRNA反义链序列

Table 5 Modified siRNA antisense strand sequences targeting ANGPTL3 gene

表6靶向ANGPTL3基因的配对的siRNA正义链和反义链

Table 6 Paired siRNA sense and antisense strands targeting the ANGPTL3 gene

实施例Example
如无特别说明，实施例中使用的材料来源如下：Unless otherwise stated, the sources of materials used in the examples are as follows:
Huh7细胞系购自南京科佰，货号CBP60202；Huh7 cell line was purchased from Nanjing Kebai, product number CBP60202;
Hep3B细胞系购自南京科佰，货号CBP60197；Hep3B cell line was purchased from Nanjing Kebai, product number CBP60197;
PHH细胞购自上海轩一，货号QYLF-HPMC；PHH cells were purchased from Shanghai Xuanyi, product number QYLF-HPMC;
HEK293A细胞系购自南京科佰，货号CBP60436；HEK293A cell line was purchased from Nanjing Kebai, product number CBP60436;
Balb/c小鼠来自浙江维通利华，货号Balb/c。Balb/c mice were from Zhejiang Vitong Lever, product number Balb/c.
实施例1化合物E7的制备Example 1 Preparation of Compound E7
1.中间体3-4的制备1. Preparation of intermediate 3-4
1.1化合物2的制备
1.1 Preparation of compound 2
在15℃下向化合物1(300g,2.01mol)的DCM(1.80L)中缓慢加入苄基(2,5-二氧代吡咯烷-1-基)碳酸酯(600g,2.40mol)并逐滴加入TEA(203g,2.01mol,280mL)。加入后，将混合物在25℃下搅拌16小时。TLC(二氯甲烷:甲醇＝10:1)显示反应物1(Rf＝0.32)被保留且检测到一个重要新点(Rf＝0.52)。使用饱和碳酸氢钠溶液洗涤反应混合物(1.00L x 2)，使用盐水(1.00L)洗涤有机相，用无水Na2SO4干燥并真空浓缩。不经纯化，化合物2(约385g)为黄色油状。To compound 1 (300 g, 2.01 mol) in DCM (1.80 L) was added slowly dropwise benzyl (2,5-dioxopyrrolidin-1-yl) carbonate (600 g, 2.40 mol) at 15°C. Add TEA (203g, 2.01mol, 280mL). After addition, the mixture was stirred at 25°C for 16 hours. TLC (dichloromethane:methanol=10:1) showed that reactant 1 (R f =0.32) was retained and an important new spot was detected (R f =0.52). The reaction mixture was washed with saturated sodium bicarbonate solution (1.00L x 2), the organic phase was washed with brine (1.00L), dried over anhydrous Na2SO4 and concentrated in vacuo. Without purification, compound 2 (approximately 385 g) was a yellow oil.
1.2化合物2A的制备
1.2 Preparation of compound 2A
在0-15℃下向化合物4(350g,1.62mol,HCl)和Ac2O(994g,9.74mol,912mL)的吡啶(1.75L)溶液中一次性加入DMAP(19.8g,162mmol)并逐滴加入TEA(164g,1.62mol,226mL)。将混合物在25℃下搅拌16小时。LCMS(产物:RT＝0.687min)显示起始反应物消耗完全。在25℃下向混合物中添加EtOAc(1.40L)并搅拌30分钟，随后过滤混合物并使用EtOAc(300mL)清洗滤饼。在25℃下用水(1.45L)磨碎滤饼30分钟。过滤混合物并使用水(175mL x 3)清洗滤饼，收集滤饼以获得白色固体状的化合物2A(约580g)。To a solution of compound 4 (350 g, 1.62 mol, HCl) and Ac 2 O (994 g, 9.74 mol, 912 mL) in pyridine (1.75 L) at 0-15°C, DMAP (19.8 g, 162 mmol) was added dropwise in one portion Add TEA (164g, 1.62mol, 226mL). The mixture was stirred at 25°C for 16 hours. LCMS (product: RT = 0.687 min) showed complete consumption of starting reactants. EtOAc (1.40 L) was added to the mixture at 25°C and stirred for 30 minutes, then the mixture was filtered and the filter cake was washed with EtOAc (300 mL). The filter cake was ground with water (1.45 L) at 25°C for 30 minutes. The mixture was filtered and the filter cake was washed with water (175 mL x 3), and the filter cake was collected to obtain compound 2A as a white solid (about 580 g).
1.3化合物2B的制备
1.3 Preparation of compound 2B
三次反应平行进行。Three reactions were carried out in parallel.
在10-15℃下经0.5小时向化合物2A(200g,514mmol)的DCM(800mL)溶液中逐滴加入TMSOTf(137g,616mmol,111mL)。随后将混合物在25℃下搅拌3小时。TLC(二氯甲烷:甲醇＝20:1)显示化合物2A(Rf＝0.54)消耗完全且新点(Rf＝0.24)形成。合并三次反应。将混合物冷却至0-15℃，在0-5℃下缓慢倒入NaHCO3的溶液(300g溶解在3.00L水)中，分离有机相并用DCM(1.00L x 3)萃取水相，合并有机层，用Na2SO4,干燥，过滤并真空浓缩。不经纯化，获得黄色油状的化合物2B(约507g)用于下一步骤。To a solution of compound 2A (200 g, 514 mmol) in DCM (800 mL) was added dropwise TMSOTf (137 g, 616 mmol, 111 mL) at 10-15 °C over 0.5 h. The mixture was then stirred at 25°C for 3 hours. TLC (dichloromethane:methanol=20:1) showed that compound 2A (R f =0.54) was completely consumed and a new spot (R f =0.24) was formed. Three reactions were combined. Cool the mixture to 0-15°C, slowly pour into a solution of NaHCO 3 (300g dissolved in 3.00L water) at 0-5°C, separate the organic phase and extract the aqueous phase with DCM (1.00L x 3), combine the organic layers , dried over Na 2 SO 4 , filtered and concentrated in vacuo. Without purification, compound 2B (approximately 507 g) was obtained as a yellow oil and used in the next step.
1.4化合物3的制备
1.4 Preparation of compound 3
在0-10℃下向在DCM(1.00L)中的化合物2B(250g,759mmol)和化合物2(151g,531mmol)的混合物中逐滴加入TMSOTf(84.4g,380mmol,69.0mL)并将混合物在20℃搅拌12小时。TLC(二氯甲烷:甲醇＝20:1)显示化合物2(Rf＝0.33)消耗完全且形成新点(Rf＝0.03)。将合并的反应物冷却至0-5℃，随后将反应物倒入NaHCO3(水溶液，100g溶于1L水)中并在5-10℃下搅拌10分钟，分离相。使用DCM(500mL x 2)萃取水相，合并的有机相用Na2SO4干燥，过滤，真空浓缩滤液。不经纯化，化合物3(约360g)为黄色油状。To a mixture of compound 2B (250 g, 759 mmol) and compound 2 (151 g, 531 mmol) in DCM (1.00 L) was added dropwise TMSOTf (84.4 g, 380 mmol, 69.0 mL) at 0-10 °C and the mixture was added Stir at 20°C for 12 hours. TLC (dichloromethane:methanol=20:1) showed complete consumption of compound 2 (R f =0.33) and the formation of new spots (R f =0.03). The combined reactants were cooled to 0-5°C, then the reaction was poured into NaHCO3 (aq, 100 g in 1 L water) and stirred at 5-10°C for 10 min and the phases separated. The aqueous phase was extracted with DCM (500 mL x 2), the combined organic phases were dried over Na2SO4 , filtered, and the filtrate was concentrated in vacuo. Without purification, compound 3 (approximately 360 g) was a yellow oil.
1H NMR:(400MHz,DMSO). 1 H NMR: (400MHz, DMSO).
δ＝7.79-7.37(m,1H),7.35-7.26(m,5H),5.21-5.20(m,1H),5.00-4.95(m,3H),4.55-4.53(m,1H),4.03-3.86(m,3H),3.61-3.59(m,1H),3.59-3.57(m,1H),3.48-3.40(m,6H),3.39-3.31(m,2H),3.14-3.13(m,2H),2.09(s,3H),1.99(s,3H),1.88(s,3H),1.76-1.74(m,3H).δ＝7.79-7.37(m,1H),7.35-7.26(m,5H),5.21-5.20(m,1H),5.00-4.95(m,3H),4.55-4.53(m,1H),4.03-3.86 (m,3H),3.61-3.59(m,1H),3.59-3.57(m,1H),3.48-3.40(m,6H),3.39-3.31(m,2H),3.14-3.13(m,2H) ,2.09(s,3H),1.99(s,3H),1.88(s,3H),1.76-1.74(m,3H).
1.5中间体3-4(TFA盐)的制备
1.5 Preparation of intermediate 3-4 (TFA salt)
三次反应平行进行。Three reactions were carried out in parallel.
在氩气气氛下向在THF(1.80L)中的Pd/C(18.0g,16.3mmol,10％含量)的混合物中加入化合物3(180g,293mmol)和TFA(33.5g,293mmol,21.8mL)。对混 悬液排气并使用氢气通气三次。在H2(50Psi)和30℃下将混合物搅拌2小时。LCMS(产物:RT＝0.697min)显示化合物3消耗且检测到产物峰。合并三次反应。通过celite过滤混合物，减压浓缩滤液以除去溶剂。不经纯化，获得黄色固体状的中间体3-4(TFA盐)(393g,660mmol,74.8％产率,99.6％纯度,TFA)。To a mixture of Pd/C (18.0 g, 16.3 mmol, 10% content) in THF (1.80 L) was added compound 3 (180 g, 293 mmol) and TFA (33.5 g, 293 mmol, 21.8 mL) under an argon atmosphere. . Mix up The suspension was vented and vented three times with hydrogen gas. The mixture was stirred under H2 (50 Psi) and 30°C for 2 hours. LCMS (Product: RT = 0.697 min) showed consumption of compound 3 and a product peak was detected. Three reactions were combined. The mixture was filtered through celite and the filtrate was concentrated under reduced pressure to remove the solvent. Without purification, intermediate 3-4 (TFA salt) was obtained as a yellow solid (393 g, 660 mmol, 74.8% yield, 99.6% purity, TFA).
1H NMR:(400MHz,DMSO-d6) 1 H NMR: (400MHz, DMSO-d6)
δ＝7.92(d,J＝9.1Hz,4H),5.27-5.17(m,1H),5.03-4.91(m,1H),4.60-4.50(m,1H),4.09-3.97(m,4H),3.85(s,2H),3.65-3.46(m,10H),3.04-2.92(m,2H),2.10(s,3H),2.00(s,3H),1.94-1.86(m,3H),1.82-1.71(m,4H).δ=7.92(d,J=9.1Hz,4H),5.27-5.17(m,1H),5.03-4.91(m,1H),4.60-4.50(m,1H),4.09-3.97(m,4H), 3.85(s,2H),3.65-3.46(m,10H),3.04-2.92(m,2H),2.10(s,3H),2.00(s,3H),1.94-1.86(m,3H),1.82- 1.71(m,4H).
2.中间体3-5的制备2. Preparation of intermediate 3-5
2.1化合物5的制备
2.1 Preparation of compound 5
在25℃下向化合物4B(10.0g,35.5mmol,1.00eq)和以上制备的化合物3-4(46.3g,78.2mmol,2.20eq,TFA)的DCM(1.00L)溶液中一次性加入DIEA(30.3g,234mmol,40.8mL,6.60eq)。25℃搅拌半小时。向混合物中加入HBTU(30.3g,234mmol,40.8mL,6.60eq)。25℃搅拌16小时。LCMS(产物:RT＝0.681mins)显示反应完成，真空浓缩混合物。在20℃下向混合物中加入0.50N HCl(200mL x 2)，并用DCM(3x 500mL)萃取，合并有机层并用饱和NaHCO3(3x 800mL)清洗直至pH＝8，用盐水(3x 500mL)清洗，用Na2SO4干燥并真空浓缩纯化。通过柱色谱(SiO2,DCM:MeOH＝50:1-15:1)纯化残留物。在40℃下真空浓缩残留物并通过制备型-MPLC(柱:800g Agela C18；流动相:[水-ACN]；15-45％25min；45％10min)纯化。真空干燥以获得黄色固体状的化合物5(约180g+75.0g+87.0g+40.0g+38.0g)。DIEA ( 30.3g, 234mmol, 40.8mL, 6.60eq). Stir at 25°C for half an hour. HBTU (30.3g, 234mmol, 40.8mL, 6.60eq) was added to the mixture. Stir at 25°C for 16 hours. LCMS (Product: RT = 0.681 mins) showed the reaction was complete and the mixture was concentrated in vacuo. 0.50N HCl (200mL Dry over Na2SO4 and concentrate in vacuo for purification. The residue was purified by column chromatography (SiO 2 , DCM:MeOH=50:1-15:1). The residue was concentrated in vacuo at 40°C and purified by preparative-MPLC (column: 800 g Agela C18; mobile phase: [water-ACN]; 15-45% 25 min; 45% 10 min). Dry under vacuum to obtain compound 5 as a yellow solid (approximately 180g+75.0g+87.0g+40.0g+38.0g).
417.0g的化合物3-4通过9批次被转换为化合物5。417.0 g of compound 3-4 was converted to compound 5 in 9 batches.
2.2中间体3-3的制备
2.2 Preparation of intermediate 3-3
在氩气气氛下向Pd/C(3.00g,10％含量)的THF(300mL)中加入化合物5(73.0g,61.7mmol,1.00eq)和TFA(7.04g,61.7mmol,4.57mL,1.00eq)。对悬浮液脱气并使用氢气通气三次。在H2(20Psi)和20℃下搅拌16小时。TLC(二氯甲烷:甲醇＝8:1,Rf＝0.0)显示反应完成。通过celite过滤混合物，加压浓缩滤液以除去溶剂以获得白色固体状的化合物3-3(约33.4g+129g+75.0g)。To Pd/C (3.00g, 10% content) in THF (300mL), compound 5 (73.0g, 61.7mmol, 1.00eq) and TFA (7.04g, 61.7mmol, 4.57mL, 1.00eq) were added under an argon atmosphere. ). The suspension was degassed and vented three times with hydrogen gas. Stir in H 2 (20 Psi) and 20°C for 16 hours. TLC (dichloromethane:methanol=8:1, R f =0.0) showed that the reaction was complete. The mixture was filtered through celite, and the filtrate was concentrated under pressure to remove the solvent to obtain compound 3-3 (approximately 33.4g+129g+75.0g) as a white solid.
1H NMR:(400MHz,DMSO) 1 H NMR: (400MHz, DMSO)
δ＝8.53(t,J＝5.2Hz,1H),8.18(d,J＝2.4Hz,3H),8.03(t,J＝5.2Hz,1H),7.84(dd,J＝3.6Hz,2H),5.22(d,J＝3.2Hz,2H),4.96(dd,J＝3.2Hz,2H),4.55(d,J＝8.4 Hz,2H),4.02(t,J＝8.8Hz,6H),3.77-3.59(m,5H),3.58-3.45(m,21H),3.40-3.20(m,4H),2.18(t,J＝7.6Hz,2H),2.17(d,J＝8.0Hz,6H),2.10(s,6H),1.99(s,6H),1.90-1.80(m,8H),1.77(s,6H).δ＝8.53(t,J＝5.2Hz,1H),8.18(d,J＝2.4Hz,3H),8.03(t,J＝5.2Hz,1H),7.84(dd,J＝3.6Hz,2H), 5.22(d,J＝3.2Hz,2H),4.96(dd,J＝3.2Hz,2H),4.55(d,J＝8.4 Hz,2H),4.02(t,J＝8.8Hz,6H),3.77-3.59(m,5H),3.58-3.45(m,21H),3.40-3.20(m,4H),2.18(t,J＝ 7.6Hz,2H),2.17(d,J＝8.0Hz,6H),2.10(s,6H),1.99(s,6H),1.90-1.80(m,8H),1.77(s,6H).
3.化合物E7的制备3. Preparation of compound E7
3.1化合物3的制备
3.1 Preparation of compound 3
在室温下将化合物1(2.00g,1.87mmol，根据上述中间体3-3的方法制备)溶解到DCM(20.0mL)中，向溶液中依次加入DIEA(0.135mL,0.814mmol)，化合物2(0.550g,0.814mmol)，置换3次氮气。反应混合物在25℃下搅拌16小时。液质联用检测到产物的MS响应，薄层色谱(二氯甲烷/甲醇＝5/1)显示原料消失且有新点生成。反应液在减压下浓缩，所得的粗产品经柱层析纯化(二氯甲烷/甲醇＝5/1)得到白色固体化合物3(约780mg)。Compound 1 (2.00g, 1.87mmol, prepared according to the method of intermediate 3-3 above) was dissolved in DCM (20.0mL) at room temperature, DIEA (0.135mL, 0.814mmol), compound 2 ( 0.550g, 0.814mmol), replace nitrogen 3 times. The reaction mixture was stirred at 25°C for 16 hours. Liquid mass spectrometry detected the MS response of the product, and thin layer chromatography (dichloromethane/methanol=5/1) showed that the raw material disappeared and new spots were generated. The reaction solution was concentrated under reduced pressure, and the obtained crude product was purified by column chromatography (dichloromethane/methanol=5/1) to obtain compound 3 (approximately 780 mg) as a white solid.
1H NMR(400MHz,CD3OD) 1 H NMR (400MHz, CD 3 OD)
δ＝7.28-7.42(m,5H),5.30-5.34(m,4H),5.04-5.14(m,6H),4.63-4.67(m,4H),4.36-4.44(m,2H),4.00-4.20(m,23H),3.91-3.95(m,4H),3.69-3.77(m,9H),3.52-3.67(m,32H),3.34-3.43(m,9H),2.29-2.31(m,4H),2.14(s,12H),2.03(s,12H),1.92-1.96(m,24H).LCMS:m/z＝1221.6(M/2+H)+.δ＝7.28-7.42(m,5H),5.30-5.34(m,4H),5.04-5.14(m,6H),4.63-4.67(m,4H),4.36-4.44(m,2H),4.00-4.20 (m,23H),3.91-3.95(m,4H),3.69-3.77(m,9H),3.52-3.67(m,32H),3.34-3.43(m,9H),2.29-2.31(m,4H) ,2.14(s,12H),2.03(s,12H),1.92-1.96(m,24H).LCMS:m/z＝1221.6(M/2+H) + .
3.2化合物4的制备
3.2 Preparation of compound 4
在室温下将化合物3(1.10g,0.451mmol)溶解到MeOH(10.0mL)中，向该溶液中加入质量分数为10％的湿Pd/C(0.050g,0.451mmol)，置换3次氢气，反应混合物在氢气氛围下(14.696psi)、25℃下搅拌18小时。液质联用检测到产物的MS响应，薄层色谱(二氯甲烷/甲醇＝10/1，磷钼酸显色)显示原料已完全消耗且有新点生成。反应液过滤，滤液在减压下浓缩得到白色固体化合物4(约840mg)。Dissolve compound 3 (1.10g, 0.451mmol) in MeOH (10.0mL) at room temperature, add 10% mass fraction of wet Pd/C (0.050g, 0.451mmol) to the solution, and replace hydrogen gas three times. The reaction mixture was stirred at 25°C for 18 hours under a hydrogen atmosphere (14.696 psi). The MS response of the product was detected by liquid mass spectrometry, and thin layer chromatography (dichloromethane/methanol=10/1, phosphomolybdic acid color development) showed that the raw materials had been completely consumed and new spots were generated. The reaction liquid was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 4 (approximately 840 mg) as a white solid.
1H NMR(400MHz,CD3OD) 1 H NMR (400MHz, CD 3 OD)
δ＝5.32-5.34(m 4H),5.06-5.10(m,4H),4.63-4.65(m,4H),4.38-4.40(m,2H),3.99-4.20(m,20H),3.90-3.97(m,4H),3.69-3.76(m,6H),3.50-3.68(m,36H),3.35-3.44(m,11H),2.28-2.38(m,4H),2.15(s,12H),2.03(s,12H),1.90-1.94(m,24H).LCMS:m/z＝1154.7(M/2+H)+.δ＝5.32-5.34(m 4H),5.06-5.10(m,4H),4.63-4.65(m,4H),4.38-4.40(m,2H),3.99-4.20(m,20H),3.90-3.97( m,4H),3.69-3.76(m,6H),3.50-3.68(m,36H),3.35-3.44(m,11H),2.28-2.38(m,4H),2.15(s,12H),2.03( s,12H),1.90-1.94(m,24H).LCMS:m/z＝1154.7(M/2+H) + .
3.3化合物6的制备
3.3 Preparation of compound 6
在室温下将化合物5(232mg,0.364mmol)溶解到DCM(10.0mL)中，向溶液中依次加入HBTU(207mg,0.546mmol)，DIEA(0.181mL,1.09mmol))和化合物4(840mg,0.364mmol)，置换3次氮气。反应混合物在25℃下搅拌1小时。液质联用检测到原料消失，薄层色谱(二氯甲烷/甲醇＝5/1)显示原料消失且有新点生成。反应液在减压下浓缩，所得的粗产品经柱层析纯化(二氯甲烷/甲醇＝8/1～5/1)得到白色固体化合物6(约620mg)。Compound 5 (232mg, 0.364mmol) was dissolved in DCM (10.0mL) at room temperature, and HBTU (207mg, 0.546mmol), DIEA (0.181mL, 1.09mmol)) and compound 4 (840mg, 0.364) were added to the solution in sequence. mmol), replaced with nitrogen three times. The reaction mixture was stirred at 25°C for 1 hour. Liquid mass spectrometry detected the disappearance of the raw material, and thin layer chromatography (dichloromethane/methanol=5/1) showed that the raw material disappeared and new spots were generated. The reaction solution was concentrated under reduced pressure, and the obtained crude product was purified by column chromatography (dichloromethane/methanol = 8/1 to 5/1) to obtain compound 6 (approximately 620 mg) as a white solid.
1H NMR(400MHz,CD3OD) 1 H NMR (400MHz, CD 3 OD)
δ＝7.41-7.43(m,2H),7.23-7.34(m,7H),6.83-6.90(m,4H),5.31-5.35(m,4H),5.01-5.12(m,4H),4.63-4.65(m,4H),4.41-4.45(m,2H),4.31-4.33(m,1H),3.99-4.22(m,22H),3.87-3.97(m,6H),3.58-3.81(m,45H),3.34-3.43(m,10H),2.19-2.40(m,10H),2.14(s,12H),2.02(s,12H),1.92-1.96(mz,24H),1.48-1.63(m,4H),1.28-1.38(m,8H).LCMS:m/z＝1460.0(M/2+H)+.δ＝7.41-7.43(m,2H),7.23-7.34(m,7H),6.83-6.90(m,4H),5.31-5.35(m,4H),5.01-5.12(m,4H),4.63-4.65 (m,4H),4.41-4.45(m,2H),4.31-4.33(m,1H),3.99-4.22(m,22H),3.87-3.97(m,6H),3.58-3.81(m,45H) ,3.34-3.43(m,10H),2.19-2.40(m,10H),2.14(s,12H),2.02(s,12H),1.92-1.96(mz,24H),1.48-1.63(m,4H) ,1.28-1.38(m,8H).LCMS:m/z＝1460.0(M/2+H) + .
4.化合物E7的制备
4. Preparation of compound E7
在室温下将化合物6(300mg,0.103mmol)溶解到DCM(10.0mL)中，向溶液中依次加入DIEA(0.102mL,0.618mmol)，化合物7(10.3mg,0.103mmol)和DMAP(12.6mg,0.103mmol)，置换3次氮气。反应混合物在25℃下搅拌2小时。液质联用检测到原料消失。反应液在减压下浓缩，所得的粗产品经制备型高效液相色谱制备(制备型-HPLC,柱：Waters Xbridge BEH C18 100*30mm*10um；流动相:水-ACN；B％:17％-57％,5min)分离得到白色固体化合物E7(53.0mg,收率17.08％,纯度78.94％)。Compound 6 (300 mg, 0.103 mmol) was dissolved in DCM (10.0 mL) at room temperature, and DIEA (0.102 mL, 0.618 mmol), compound 7 (10.3 mg, 0.103 mmol) and DMAP (12.6 mg) were added to the solution in sequence. 0.103mmol), replacing nitrogen three times. The reaction mixture was stirred at 25°C for 2 hours. The disappearance of raw material was detected by LC/MS. The reaction solution was concentrated under reduced pressure, and the crude product obtained was prepared by preparative high-performance liquid chromatography (preparative-HPLC, column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: water-ACN; B%: 17% -57%, 5min) to obtain white solid compound E7 (53.0mg, yield 17.08%, purity 78.94%).
1H NMR(400MHz,CD3OD) 1 H NMR (400MHz, CD 3 OD)
δ＝7.41-7.45(m,2H),7.17-7.34(m,7H),6.85-6.89(m,4H),5.32-5.36(m,4H),5.03-5.13(m,4H),4.63-4.67(m,4H),4.38-4.47(m,2H),4.32-4.34(m,1H),4.01-4.26(m,22H),3.88-4.00(m,6H),3.77-3.81(m,7H),3.49-3.76(m,45H),3.33-3.47(m,10H),2.56-2.62(m,2H),2.45-2.55(m,3H),2.21-2.38(m,7H),2.14(s,12H),2.05-2.11(m,2H),2.02(s,12H),1.92-1.96(m,24H),1.47-1.68(m,4H),1.28-1.34(m,8H)δ＝7.41-7.45(m,2H),7.17-7.34(m,7H),6.85-6.89(m,4H),5.32-5.36(m,4H),5.03-5.13(m,4H),4.63-4.67 (m,4H),4.38-4.47(m,2H),4.32-4.34(m,1H),4.01-4.26(m,22H),3.88-4.00(m,6H),3.77-3.81(m,7H) ,3.49-3.76(m,45H),3.33-3.47(m,10H),2.56-2.62(m,2H),2.45-2.55(m,3H),2.21-2.38(m,7H),2.14(s, 12H),2.05-2.11(m,2H),2.02(s,12H),1.92-1.96(m,24H),1.47-1.68(m,4H),1.28-1.34(m,8H)
MS:m/z＝3022.36(M+H)+. MS: m/z＝3022.36(M+H) + .
实施例2化合物E13的制备Example 2 Preparation of Compound E13
1.中间体2-2的制备
1. Preparation of intermediate 2-2
1.1化合物3的制备
1.1 Preparation of compound 3
4次反应平行进行。Four reactions were carried out in parallel.
向化合物1(250g,741mmol)的THF(1.75L)溶液中加入4-甲基吗啉(434g,4.30mol,472mL)，随后冷却至0℃。在10分钟内向反应混合物中加入氯甲酸异丁酯(243g,1.78mol,233mL)，维持反应温度小于4.0℃。加入后，将混合物搅拌40分钟或更久，在10分钟内向反应混合物中逐次加入化合物2(526g,1.78mol)，维持反应温度小于4.0℃。加入后，移除冰浴，随后允许反应物升温至室温，持续2小时。TLC(石油醚/乙酸乙酯＝1/1,化合物1Rf＝0.43)显示化合物1消耗完全且形成新点。合并4次反应。将反应溶液倒入搅拌中的冷的(0℃)0.50M HCl(aq.)(12.0L)溶液中并搅拌约10分钟。随后加入EtOAc(4.00L x 3)，搅拌一段时间，分层，并使用盐水(10.0L)洗涤有机相，用Na2SO4干燥，真空浓缩，产生稠的无色油状物。向搅拌的油状物中加入己烷(1.20L)。溶液中出现白色烟雾，并随后在进一步搅拌后消失。加入晶种(1.20g,0.10wt％)，此时白色晶体缓慢形成。在20分钟内，悬浮液足够稠以阻碍搅拌，此时加入额外的己烷(6.00L)，搅拌12小时。过滤悬浮液，使用己烷(1.20L)清洗并干燥以获得白色固体状的化合物3(约1.20kg)。To a solution of compound 1 (250 g, 741 mmol) in THF (1.75 L) was added 4-methylmorpholine (434 g, 4.30 mol, 472 mL), followed by cooling to 0°C. Isobutyl chloroformate (243g, 1.78mol, 233mL) was added to the reaction mixture within 10 minutes, maintaining the reaction temperature below 4.0°C. After addition, the mixture was stirred for 40 minutes or more, and compound 2 (526g, 1.78mol) was gradually added to the reaction mixture within 10 minutes, maintaining the reaction temperature less than 4.0°C. After addition, the ice bath was removed and the reaction was allowed to warm to room temperature for 2 hours. TLC (petroleum ether/ethyl acetate=1/1, compound 1R f =0.43) showed that compound 1 was completely consumed and new spots were formed. The 4 reactions were combined. The reaction solution was poured into a stirring cold (0°C) 0.50 M HCl (aq.) (12.0 L) solution and stirred for about 10 minutes. EtOAc ( 4.00L Hexane (1.20L) was added to the stirring oil. White smoke appeared in the solution and subsequently disappeared after further stirring. Seed crystals (1.20g, 0.10wt%) were added, and white crystals slowly formed. Within 20 minutes, the suspension was thick enough to prevent stirring, at which time additional hexane (6.00 L) was added and stirred for 12 hours. The suspension was filtered, washed with hexane (1.20 L) and dried to obtain compound 3 as a white solid (approximately 1.20 kg).
1H NMR:(400MHz DMSO) 1 H NMR: (400MHz DMSO)
δ＝8.10-8.08(m,1H),7.62-7.60(m,1H),7.38-7.31(m,5H),5.11-4.94(m,2H),4.12-4.09(m,1H),3.92-3.87(m,1H),2.25-2.19(m,4H),1.90-1.88(m,2H),1.73-1.67(m,2H),1.40-1.39(m,27H).δ＝8.10-8.08(m,1H),7.62-7.60(m,1H),7.38-7.31(m,5H),5.11-4.94(m,2H),4.12-4.09(m,1H),3.92-3.87 (m,1H),2.25-2.19(m,4H),1.90-1.88(m,2H),1.73-1.67(m,2H),1.40-1.39(m,27H).
1.2化合物4的制备
1.2 Preparation of compound 4
5次反应平行进行。Five reactions were performed in parallel.
将化合物3(240g,415mmol)的HCOOH(2.40L)溶液在45℃下搅拌2小时。LCMS(化合物4＝0.570min)显示化合物3消耗完全且出现所需m/z的主峰。合并5次反应。使用甲苯和ACN(各1.50L)稀释并浓缩。使用1:1ACN和甲苯(500mL)和ACN三次(各500mL)共沸除去甲酸。高真空干燥化合物4。随后使用DCM(500mL)搅拌残留物并倒去有机层，随后使用ACN(400mL)共沸干燥两次，真空干燥，随后使用甲苯(400mL)共沸9次以得到白色固体状的化合物4(约800g)。A solution of compound 3 (240 g, 415 mmol) in HCOOH (2.40 L) was stirred at 45°C for 2 hours. LCMS (compound 4 = 0.570 min) showed that compound 3 was completely consumed and the main peak of the desired m/z appeared. Five reactions were combined. Dilute and concentrate using toluene and ACN (1.50 L each). Formic acid was azeotropically removed using 1:1 ACN and toluene (500 mL) and ACN three times (500 mL each). Dry compound 4 under high vacuum. The residue was then stirred with DCM (500 mL) and the organic layer was poured off, followed by azeotropic drying with ACN (400 mL) twice, vacuum drying, and then azeotroping with toluene (400 mL) 9 times to obtain compound 4 as a white solid (approximately 800g).
1H NMR:(400MHz DMSO) 1 H NMR: (400MHz DMSO)
δ＝12.4-12.2(m,2H),8.12-8.10(m,1H),7.62-7.60(m,1H),7.38-7.34(m,5H),5.07-5.02(m,2H),4.22-4.17(m,1H),3.99-3.96(m,1H),2.31-2.21(m,4H),2.00-1.93(m,2H),1.76-1.75(m,2H).δ＝12.4-12.2(m,2H),8.12-8.10(m,1H),7.62-7.60(m,1H),7.38-7.34(m,5H),5.07-5.02(m,2H),4.22-4.17 (m,1H),3.99-3.96(m,1H),2.31-2.21(m,4H),2.00-1.93(m,2H),1.76-1.75(m,2H).
1.3化合物6的制备
1.3 Preparation of compound 6
向化合物4(80.0g,194mmol)和(384g,700mmol,TFA)的搅拌的DMF(2.00L)溶液中依次加入HOBT(103g,760mmol)、EDCI(146g,760mmol)和DIEA(113g,877mmol,153mL)。将反应物在20℃下搅拌2小时。TLC(二氯甲烷/甲醇＝5/1,化合物6Rf＝0.43)显示化合物4消耗完全并形成新点。将反应混合物缓慢倒入搅拌的冷的0.5M HCl水溶液(230mL)中，搅拌10分钟，形成白色固体并过滤，使用DCM(1.50L)萃取水相两次。使用5％NaHCO3(aq.)(200mL)清洗合并的有机相，之后干燥(Na2SO4)，然后加压蒸发浓缩。通过柱色谱(SiO2,二氯甲烷/甲醇＝5/1,化合物6Rf＝0.43)纯化残留物以得到黄色固体状的化合物6(约180g)。To a stirred DMF (2.00L) solution of compound 4 (80.0g, 194mmol) and (384g, 700mmol, TFA), HOBT (103g, 760mmol), EDCI (146g, 760mmol) and DIEA (113g, 877mmol, 153mL) were added successively ). The reaction was stirred at 20°C for 2 hours. TLC (dichloromethane/methanol=5/1, compound 6R f =0.43) showed that compound 4 was completely consumed and new spots formed. The reaction mixture was slowly poured into a stirred cold 0.5 M aqueous HCl solution (230 mL), stirred for 10 minutes, a white solid formed and filtered, and the aqueous phase was extracted twice with DCM (1.50 L). The combined organic phases were washed with 5% NaHCO 3 (aq.) (200 mL), dried (Na 2 SO 4 ), and concentrated by evaporation under pressure. The residue was purified by column chromatography (SiO 2 , dichloromethane/methanol=5/1, compound 6R f =0.43) to obtain compound 6 as a yellow solid (approximately 180 g).
1H NMR:(400MHz DMSO) 1 H NMR: (400MHz DMSO)
δ7.95-7.91(m,3H),7.82-7.80(m,4H),7.39-7.31(m,6H),5.21-5.01(m,3H),5.00-4.96(m,5H),4.56-4.53(m,3H),4.02(s,1H),3.88(s,9H),3.85(s,4H),3.76(s,3H),3.50-349(m,9H),3.39-3.36(m,6H),3.19-3.15(m,6H),2.15-2.05(m,12H),1.99(s,9H),1.89-1.77(m,21H). δ7.95-7.91(m,3H),7.82-7.80(m,4H),7.39-7.31(m,6H),5.21-5.01(m,3H),5.00-4.96(m,5H),4.56-4.53 (m,3H),4.02(s,1H),3.88(s,9H),3.85(s,4H),3.76(s,3H),3.50-349(m,9H),3.39-3.36(m,6H ),3.19-3.15(m,6H),2.15-2.05(m,12H),1.99(s,9H),1.89-1.77(m,21H).
1.4中间体2-2的制备
1.4 Preparation of intermediate 2-2
在氩气气氛下向干燥的氢化瓶中加入Pd(OH)2/C(4.94g,3.52mmol,10％含量)，加入MeOH(350mL)并依次加入化合物6(47.4g,28.5mmol)、TFA(3.26g,28.5mmol,2.11mL)，在H2(50psi)和20℃下搅拌3小时。通过TLC(二氯甲烷/甲醇＝5/1,中间体2-2Rf＝0.25)监测反应，显示化合物6消耗。过滤反应混合物并真空浓缩滤液以获得白色固体状的中间体2-2(TFA盐)(46.3g,94.6％产率,95.7％纯度,TFA)。Add Pd(OH) 2 /C (4.94g, 3.52mmol, 10% content) to the dry hydrogenation bottle under an argon atmosphere, add MeOH (350mL), and add compound 6 (47.4g, 28.5mmol) and TFA in sequence. (3.26g, 28.5mmol, 2.11mL), stirred in H2 (50psi) and 20°C for 3 hours. The reaction was monitored by TLC (dichloromethane/methanol=5/1, intermediate 2-2R f =0.25), showing consumption of compound 6. The reaction mixture was filtered and the filtrate was concentrated in vacuo to obtain Intermediate 2-2 (TFA salt) as a white solid (46.3 g, 94.6% yield, 95.7% purity, TFA).
1H NMR:(400MHz DMSO) 1 H NMR: (400MHz DMSO)
δ＝8.56-8.50(m,1H),8.14-8.05(m,4H),7.99(s,1H),7.88-7.82(m,4H),5.22-5.21(m,3H),4.99-4.96(m,3H),4.55-4.53(m,3H),4.09(s,1H),4.03(s,9H),3.87-3.79(m,6H),3.59-3.52(m,1H),3.51-347(m,9H),3.39-3.37(m,5H),3.17-3.16(m,6H),2.22-2.10(m,12H),1.99(s,9H),1.93-1.73(m,21H).δ＝8.56-8.50(m,1H),8.14-8.05(m,4H),7.99(s,1H),7.88-7.82(m,4H),5.22-5.21(m,3H),4.99-4.96(m ,3H),4.55-4.53(m,3H),4.09(s,1H),4.03(s,9H),3.87-3.79(m,6H),3.59-3.52(m,1H),3.51-347(m ,9H),3.39-3.37(m,5H),3.17-3.16(m,6H),2.22-2.10(m,12H),1.99(s,9H),1.93-1.73(m,21H).
2.化合物E13的制备

2. Preparation of compound E13

2.1化合物Int1的制备2.1 Preparation of compound Int1
2.1.1化合物1-2的制备
2.1.1 Preparation of compound 1-2
室温条件下，将化合物1-1(38.0g,231mmol)，TsNHBoc(75.4g,278mmol)，K2CO3(6.40g,46.3mmol)，TEBA(5.27g,23.1mmol)加入到三口瓶中。在95℃下反应2小时，随后降温到25℃，TLC(PE/EA＝2/1,UV 254nm)显示反应完成，反应液加水(500mL)稀释，二氯甲烷(200mL x 3)萃取，有机相减压浓缩，粗产品柱分离纯化(PE/EA＝10/1-3/1)得到无色油状化合物1-2(40.0g)。At room temperature, compound 1-1 (38.0g, 231mmol), TsNHBoc (75.4g, 278mmol), K 2 CO 3 (6.40g, 46.3mmol), and TEBA (5.27g, 23.1mmol) were added to a three-necked flask. React at 95°C for 2 hours, then cool to 25°C. TLC (PE/EA=2/1, UV 254nm) shows that the reaction is complete. The reaction solution is diluted with water (500mL), extracted with dichloromethane (200mL x 3), and organically The phase was concentrated under reduced pressure, and the crude product was separated and purified by column (PE/EA=10/1-3/1) to obtain compound 1-2 (40.0g) as a colorless oil.
1H NMR(400MHz,CDCl3) 1 H NMR (400MHz, CDCl 3 )
δ7.72(d,J＝8.4Hz,2H),7.28-7.39(m,7H),4.72-4.86(m,2H),4.42-4.56(m,2H),3.52-3.61m,2H),3.14-3.39(m,2H),2.43(s,3H),1.47(s,9H)δ7.72(d,J=8.4Hz,2H),7.28-7.39(m,7H),4.72-4.86(m,2H),4.42-4.56(m,2H),3.52-3.61m,2H),3.14 -3.39(m,2H),2.43(s,3H),1.47(s,9H)
2.1.2化合物1-4的制备
2.1.2 Preparation of compounds 1-4
将化合物1-2(79.0g,181mmol)，化合物1-3(4.21g,30.5mmol)，TEBA(3.47g,15.2mmol)，95℃反应3小时，TLC(PE/EA＝2/1,UV 254nm)显示反应完成， 反应液加水(500mL)稀释，二氯甲烷(200mL x 3)萃取，有机相减压浓缩，粗产品柱分离纯化(PE/EA＝10/1-3/1)得到无色油状化合物1-4(约40.0g)。Compound 1-2 (79.0g, 181mmol), compound 1-3 (4.21g, 30.5mmol), TEBA (3.47g, 15.2mmol) were reacted at 95°C for 3 hours, TLC (PE/EA=2/1, UV 254nm) indicates that the reaction is complete, The reaction solution was diluted with water (500mL), extracted with dichloromethane (200mL (approximately 40.0g).
1H NMR(400MHz,CDCl3) 1 H NMR (400MHz, CDCl 3 )
δ＝7.64-7.77(m,2H),7.27-7.38(m,13H),5.05-5.16(m,1H),4.50-4.59(m,4H),4.03-4.11(m,1H),3.62-3.76(m,2H),3.44-3.58(m,3H),3.25-3.33(m,2H),3.15-3.20(m,1H),2.42(s,3H),1.46(s,9H)δ＝7.64-7.77(m,2H),7.27-7.38(m,13H),5.05-5.16(m,1H),4.50-4.59(m,4H),4.03-4.11(m,1H),3.62-3.76 (m,2H),3.44-3.58(m,3H),3.25-3.33(m,2H),3.15-3.20(m,1H),2.42(s,3H),1.46(s,9H)
2.1.3化合物1-5的制备
2.1.3 Preparation of compounds 1-5
冰浴条件下，将化合物1-4(77.0g,128mmol)，Et3N(28.6mL,205mmol)，依次加入到二氯甲烷(800mL)中，缓慢滴加甲烷磺酰氯(24.2g,212mmol)。在25℃下反应2小时，LCMS显示反应完成。反应液加入水(800mL)洗涤，有机相减压浓缩得到无色油状化合物1-5(约90.0g)。Under ice bath conditions, compound 1-4 (77.0g, 128mmol) and Et 3 N (28.6mL, 205mmol) were sequentially added to dichloromethane (800mL), and methanesulfonyl chloride (24.2g, 212mmol) was slowly added dropwise. . After 2 hours of reaction at 25°C, LCMS showed that the reaction was complete. The reaction solution was washed with water (800 mL), and the organic phase was concentrated under reduced pressure to obtain compound 1-5 (approximately 90.0 g) as a colorless oil.
LCMS(ESI):m/z＝700.1(M+Na)+；LCMS (ESI): m/z=700.1(M+Na) + ;
1H NMR(400MHz,CDCl3)δ7.68(d,J＝8.4Hz,2H),7.28-7.39(m,12H),5.10-5.20(m,1H),4.93-5.02(m,1H),4.52-4.61(m,4H),3.72-3.88(m,2H),3.52-3.67(m,4H),3.27-3.36(m,1H),3.15-3.22(m,1H),3.04(s,3H),2.43(s,3H),1.43(s,9H).LCMS:m/z＝700.1(M+Na)+. 1 H NMR (400MHz, CDCl 3 ) δ7.68 (d, J = 8.4Hz, 2H), 7.28-7.39 (m, 12H), 5.10-5.20 (m, 1H), 4.93-5.02 (m, 1H), 4.52-4.61(m,4H),3.72-3.88(m,2H),3.52-3.67(m,4H),3.27-3.36(m,1H),3.15-3.22(m,1H),3.04(s,3H ),2.43(s,3H),1.43(s,9H).LCMS: m/z＝700.1(M+Na) + .
2.1.4化合物1-6的制备
2.1.4 Preparation of compounds 1-6
室温条件下，将化合物1-5(90.0g,133mmol)，碳酸钾(91.8g,664mmol)加入到MeOH(900mL)中。在66℃下反应2小时，TLC(PE/EA＝2/1,UV 254nm)显示有新点生成，有机相减压浓缩，反应液加水(200mL)稀释，二氯甲烷(200mL x 3)萃取,有机相减压浓缩，粗产品柱分离纯化(PE/EA＝30/1-2/1)得到白色固体化合物1-6(约64.0g)。Compound 1-5 (90.0 g, 133 mmol) and potassium carbonate (91.8 g, 664 mmol) were added to MeOH (900 mL) at room temperature. React for 2 hours at 66°C. TLC (PE/EA=2/1, UV 254nm) shows the formation of new spots. The organic phase is concentrated under reduced pressure. The reaction solution is diluted with water (200mL) and extracted with dichloromethane (200mL x 3). , the organic phase was concentrated under reduced pressure, and the crude product was separated and purified by column (PE/EA=30/1-2/1) to obtain white solid compound 1-6 (about 64.0g).
1H NMR(400MHz,CDCl3)δ7.62(d,J＝8.4Hz,2H),7.27-7.39(m,12H),4.48-4.62(m,4H),3.93-4.08(m,2H),3.55-3.69(m,4H),2.84-3.10(m,4H),2.44(s,3H) 1 H NMR (400MHz, CDCl 3 ) δ7.62 (d, J = 8.4Hz, 2H), 7.27-7.39 (m, 12H), 4.48-4.62 (m, 4H), 3.93-4.08 (m, 2H), 3.55-3.69(m,4H),2.84-3.10(m,4H),2.44(s,3H)
1.1.5化合物1-7的制备
1.1.5 Preparation of compounds 1-7
室温条件下，将化合物1-6(69.0g,143mmol)，镁屑(54.7g,2.28mol)加入到MeOH(400mL)中，在66℃下反应1小时。TLC(DCM/MeOH＝10/1,UV 254nm)显示原料反应完全且有新点生成。反应液加水(3000mL)和饱和氯化铵水溶液(3000mL)稀释,二氯甲烷萃取(1000mL x3)，有机相使用饱和碳酸氢钠(300mL X 3)洗涤，有机相减压浓缩得到无色油状化合物1-7(约32.0g)。 At room temperature, compound 1-6 (69.0g, 143mmol) and magnesium shavings (54.7g, 2.28mol) were added to MeOH (400mL), and the mixture was reacted at 66°C for 1 hour. TLC (DCM/MeOH=10/1, UV 254nm) showed that the raw material reaction was complete and new spots were generated. The reaction solution was diluted with water (3000mL) and saturated aqueous ammonium chloride solution (3000mL), extracted with dichloromethane (1000mL x 3), the organic phase was washed with saturated sodium bicarbonate (300mL x 3), and the organic phase was concentrated under reduced pressure to obtain a colorless oily compound. 1-7 (about 32.0g).
1H NMR(400MHz,CDCl3)δ7.27-7.40(m,10H),4.57(s,4H),3.90-4.00(m,2H),3.59-3.69(m,4H),2.77-3.04(m,4H) 1 H NMR (400MHz, CDCl 3 ) δ7.27-7.40(m,10H),4.57(s,4H),3.90-4.00(m,2H),3.59-3.69(m,4H),2.77-3.04(m ,4H)
1.1.6化合物1-8的制备
1.1.6 Preparation of compounds 1-8
室温条件下，将化合物1-7(8.00g,24.4mmol)加入到HCl(100mL,12M)中，50℃下反应2小时，反应液减压浓缩得到无色油状化合物1-8(约3.60g,HCl盐)，At room temperature, compound 1-7 (8.00g, 24.4mmol) was added to HCl (100mL, 12M), and the reaction was carried out at 50°C for 2 hours. The reaction solution was concentrated under reduced pressure to obtain compound 1-8 (about 3.60g) as a colorless oil. ,HCl salt),
1H NMR(400MHz,CDCl3)δ4.12-4.22(m,2H),3.78(d,J＝4.4Hz,4H),3.17-3.30(m,4H) 1 H NMR (400MHz, CDCl 3 ) δ4.12-4.22 (m, 2H), 3.78 (d, J = 4.4Hz, 4H), 3.17-3.30 (m, 4H)
1.1.7化合物Int-1的制备
1.1.7 Preparation of compound Int-1
室温下，将化合物1-8(8.29g,24.5mmol)加到吡啶(50mL,618mmol)中，再加入DMTrCl(4.77g,12.3mmol)。该反应体系在25℃反应18小时，TLC(DCM/MeOH＝10/1,UV 254nm)显示产物生成。反应液减压浓缩，加饱和氯化铵(200mL)稀释，DCM(100mLx3)萃取，有机相减压浓缩。粗品柱层析(DCM/MeOH＝99/1-10/1),分离得到黄色固体Int-1(约2.6g,5.78mmol,23.6％)。Compound 1-8 (8.29g, 24.5mmol) was added to pyridine (50mL, 618mmol) at room temperature, and then DMTrCl (4.77g, 12.3mmol) was added. The reaction system reacted at 25°C for 18 hours, and TLC (DCM/MeOH=10/1, UV 254nm) showed that the product was generated. The reaction solution was concentrated under reduced pressure, diluted with saturated ammonium chloride (200 mL), extracted with DCM (100 mLx3), and the organic phase was concentrated under reduced pressure. The crude product was subjected to column chromatography (DCM/MeOH=99/1-10/1) to obtain yellow solid Int-1 (about 2.6g, 5.78mmol, 23.6%).
1H NMR(400MHz,CDCl3)δ7.38-7.50(m,2H),7.25-7.36(m,6H),7.18-7.25(m,1H),6.87(d,J＝8.8Hz,4H),4.26-4.40(m,1H),3.94-4.05(m,1H),3.85(d,J＝4.4Hz,2H)3.73-3.82(m,6H),3.34-3.41(m,1H),3.23-3.30(m,3H),3.05-3.23(m,2H) 1 H NMR (400MHz, CDCl 3 ) δ7.38-7.50 (m, 2H), 7.25-7.36 (m, 6H), 7.18-7.25 (m, 1H), 6.87 (d, J = 8.8Hz, 4H), 4.26-4.40(m,1H),3.94-4.05(m,1H),3.85(d,J＝4.4Hz,2H)3.73-3.82(m,6H),3.34-3.41(m,1H),3.23-3.30 (m,3H),3.05-3.23(m,2H)
2.2化合物Int6的制备 2.2 Preparation of compound Int6
2.2.1化合物3的制备
2.2.1 Preparation of compound 3
在25℃下将化合物1(1.00g,3.05mmol)溶解到无水N,N-二甲基甲酰胺中(10.0mL)，依次加入HOBt(0.54g,3.97mmol)，EDCI(0.76g,3.97mmol)，DIEA(1.51mL,9.16mmol)，之后将化合物2(0.79g,3.66mmol)加入其中。混合液在25℃搅拌2小时。液质联用显示原料反应完全，产物生成。薄层色谱板(二氯甲烷：甲醇＝10：1)显示原料反应完全，产物生成。向混合液中加入二氯甲烷(200ml),混合液用饱和柠檬酸溶液(20ml x 3)，饱和碳酸氢钠溶液(20ml x 3)，饱和盐水分别洗涤三次(20ml x 3)。有机相用无水硫酸钠干燥，在减压条件下浓缩得到粗产品。粗品经柱层析纯化(二氯甲烷:甲醇＝10:1)得到黄色油状化合物3(约1.00g)。 Dissolve compound 1 (1.00g, 3.05mmol) into anhydrous N,N-dimethylformamide (10.0mL) at 25°C, and add HOBt (0.54g, 3.97mmol) and EDCI (0.76g, 3.97 mmol), DIEA (1.51 mL, 9.16 mmol), and then compound 2 (0.79 g, 3.66 mmol) was added. The mixture was stirred at 25°C for 2 hours. Liquid mass spectrometry showed that the raw material reacted completely and the product was produced. The thin layer chromatography plate (methylene chloride: methanol = 10:1) showed that the raw material reaction was complete and the product was generated. Add methylene chloride (200ml) to the mixture, and wash the mixture three times with saturated citric acid solution (20ml x 3), saturated sodium bicarbonate solution (20ml x 3), and saturated brine (20ml x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:methanol=10:1) to obtain compound 3 (approximately 1.00g) as a yellow oil.
1H NMR(400MHz,CD3OD)δ7.21-7.41(m,10H),4.47-4.60(m,4H),3.88-4.01(m,2H),3.75(dd,J＝3.2,13.2Hz,1H),3.63-3.65(m,3H),3.40-3.63(m,7H),2.27-2.34(m,4H),1.50-1.60(m,4H),1.28-1.34(m,8H) 1 H NMR (400MHz, CD 3 OD) δ7.21-7.41 (m, 10H), 4.47-4.60 (m, 4H), 3.88-4.01 (m, 2H), 3.75 (dd, J＝3.2, 13.2Hz, 1H),3.63-3.65(m,3H),3.40-3.63(m,7H),2.27-2.34(m,4H),1.50-1.60(m,4H),1.28-1.34(m,8H)
LCMS:m/z＝526.8(M+H)+.LCMS: m/z＝526.8(M+H) + .
2.2.2化合物4的制备
2.2.2 Preparation of compound 4
在25℃下将化合物3(380mg,0.20mmol)溶于无水甲醇中(10.0mL)，将质量分数为10％的湿Pd/C(500mg,4.70mmol)加入到其中。反应液在50℃下搅拌12小时。液质联用显示产物生成。反应液通过硅藻土过滤，滤液在减压条件下浓缩得到黄色油状化合物4(约600mg)。Compound 3 (380 mg, 0.20 mmol) was dissolved in anhydrous methanol (10.0 mL) at 25°C, and 10% wet Pd/C (500 mg, 4.70 mmol) with a mass fraction of 10% was added thereto. The reaction solution was stirred at 50°C for 12 hours. LC/MS showed product formation. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain compound 4 (approximately 600 mg) as a yellow oil.
1H NMR(400MHz,CD3OD)δ3.79-3.89(m,2H),3.73(dd,J＝3.6,13.2Hz,1H),3.64-3.68(m,5H),3.57-3.62(m,2H),3.48-3.56(m,2H),3.38-3.47(m,1H),1.58-1.62(m,5H),1.34(d,J＝2.8Hz,11H),1.30-1.37(m,1H).LCMS:m/z＝346.5(M+H)+. 1 H NMR (400MHz, CD 3 OD) δ3.79-3.89 (m, 2H), 3.73 (dd, J = 3.6, 13.2Hz, 1H), 3.64-3.68 (m, 5H), 3.57-3.62 (m, 2H),3.48-3.56(m,2H),3.38-3.47(m,1H),1.58-1.62(m,5H),1.34(d,J＝2.8Hz,11H),1.30-1.37(m,1H) .LCMS: m/z＝346.5(M+H) + .
2.2.3化合物5的制备
2.2.3 Preparation of compound 5
在25℃下将化合物4(600mg,1.74mmol)溶于吡啶(3.00mL)中，将DMTrCl(883mg,2.61mmol)加入其中。反应液在25℃下搅拌1小时。液质联用显示产物生成。薄层色谱(二氯甲烷：甲醇＝10：1)显示原料反应完全，产物生成。向混合液中加入二氯甲烷(200ml),混合液用饱和碳酸氢钠溶液(20ml x 3)，盐水分别洗涤三次(20ml x 3)。有机相用无水硫酸钠干燥，在减压条件下浓缩得到粗产品。粗品经柱层析纯化(石油醚:乙酸乙酯＝3:1)得到无色液体化合物5(约475mg)。Compound 4 (600 mg, 1.74 mmol) was dissolved in pyridine (3.00 mL) at 25°C, and DMTrCl (883 mg, 2.61 mmol) was added thereto. The reaction solution was stirred at 25°C for 1 hour. LC/MS showed product formation. Thin layer chromatography (dichloromethane: methanol = 10:1) showed that the raw material reaction was complete and the product was generated. Add dichloromethane (200ml) to the mixture, and wash the mixture three times with saturated sodium bicarbonate solution (20ml x 3) and brine (20ml x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate=3:1) to obtain colorless liquid compound 5 (about 475 mg).
1H NMR(400MHz,CD3OD)δ7.41-7.43(m,2H),7.16-7.34(m,7H),6.83-6.87(m,4H),3.88-4.04(m,2H),3.77-4.09(m,7H),3.61-3.68(m,4H),3.43-3.58(m,3H),3.33-3.38(m,1H),3.14-3.20(m,1H),3.05(dd,J＝8.4,13.2Hz,1H),2.20-2.42(m,4H),1.51-1.60(m,4H),1.25-1.39(m,8H).LCMS:m/z＝648.3(M+H)+. 1 H NMR (400MHz, CD 3 OD) δ7.41-7.43(m,2H),7.16-7.34(m,7H),6.83-6.87(m,4H),3.88-4.04(m,2H),3.77- 4.09(m,7H),3.61-3.68(m,4H),3.43-3.58(m,3H),3.33-3.38(m,1H),3.14-3.20(m,1H),3.05(dd,J＝8.4 ,13.2Hz,1H),2.20-2.42(m,4H),1.51-1.60(m,4H),1.25-1.39(m,8H).LCMS:m/z＝648.3(M+H) + .
2.2.4化合物Int-6的制备
2.2.4 Preparation of compound Int-6
在25℃下将化合物5(475mg,0.73mmol)溶于四氢呋喃(8.00mL)和H2O(2.00mL)中，向其中加入一水合氢氧化锂(36.9mg,0.89mmol)。反应液在25 摄氏度下搅拌12小时。薄层色谱(二氯甲烷：甲醇＝10：1)显示原料反应完全，产物生成。将反应液减压条件下浓缩后，在冻干机上直接冻干得到白色固体产物化合物6(约430mg)。Compound 5 (475 mg, 0.73 mmol) was dissolved in tetrahydrofuran (8.00 mL) and H 2 O (2.00 mL) at 25°C, and lithium hydroxide monohydrate (36.9 mg, 0.89 mmol) was added thereto. The reaction solution is at 25 Stir for 12 hours at Celsius. Thin layer chromatography (dichloromethane: methanol = 10:1) showed that the raw material reaction was complete and the product was generated. After the reaction solution was concentrated under reduced pressure, it was directly lyophilized on a freeze dryer to obtain compound 6 (approximately 430 mg) as a white solid product.
2.3化合物E13的制备 2.3 Preparation of compound E13
2.3.1化合物7的制备
2.3.1 Preparation of compound 7
在25℃下将化合物Int-6(100mg,0.156mmol)溶于无水N,N-二甲基甲酰胺(3.00mL)中，依次加入HBTU试剂(89.0mg,0.234mmol)，DIEA(0.078mL,0.47mmol)，中间体C(254mg,0.156mmol，根据实施例2中间体2-2的方法合成)。反应液在25℃下搅拌12小时。薄层色谱(二氯甲烷：甲醇＝10：1)显示新点生成。向混合液中加入二氯甲烷(200ml)，混合液用饱和碳酸氢钠溶液洗涤三次(20ml x 3)，盐水洗涤三次(20ml x 3)。有机相用无水硫酸钠干燥，在减压条件下浓缩得到粗产品。粗产品经柱层析纯化(二氯甲烷:甲醇＝10:1)得到黄色油状产物化合物7(约140mg)。Compound Int-6 (100 mg, 0.156 mmol) was dissolved in anhydrous N, N-dimethylformamide (3.00 mL) at 25°C, and HBTU reagent (89.0 mg, 0.234 mmol) and DIEA (0.078 mL) were added in sequence. , 0.47mmol), intermediate C (254mg, 0.156mmol, synthesized according to the method of intermediate 2-2 in Example 2). The reaction solution was stirred at 25°C for 12 hours. Thin layer chromatography (dichloromethane:methanol=10:1) showed the formation of new spots. Dichloromethane (200ml) was added to the mixture, and the mixture was washed three times with saturated sodium bicarbonate solution (20ml x 3) and three times with brine (20ml x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:methanol=10:1) to obtain compound 7 (approximately 140 mg) as a yellow oily product.
1H NMR(400MHz,CD3OD)δ7.43(d,J＝8.4Hz,2H),7.18-7.34(m,7H),6.81-6.91(m,4H),5.34(d,J＝2.8Hz,3H),5.09-5.11(m,3H),4.62-4.66(m,3H),4.27-4.35(m,2H),4.07-4.17(m,9H),4.00-4.07(m,4H),3.89-3.98(m,5H),3.79(d,J＝2.4Hz,6H),3.69-3.75(m,5H),3.60-3.64(m,9H),3.52-3.56(m,10H),3.43-3.50(m,4H),3.39-3.40(m,1H),2.25-2.40(m,8H),2.13-2.15(m,9H),2.02(s,9H),1.94-1.96(m,18H),1.48-1.66(m,4H),1.21-1.36(m,11H) 1 H NMR (400MHz, CD 3 OD) δ7.43 (d, J = 8.4Hz, 2H), 7.18-7.34 (m, 7H), 6.81-6.91 (m, 4H), 5.34 (d, J = 2.8Hz ,3H),5.09-5.11(m,3H),4.62-4.66(m,3H),4.27-4.35(m,2H),4.07-4.17(m,9H),4.00-4.07(m,4H),3.89 -3.98(m,5H),3.79(d,J＝2.4Hz,6H),3.69-3.75(m,5H),3.60-3.64(m,9H),3.52-3.56(m,10H),3.43-3.50 (m,4H),3.39-3.40(m,1H),2.25-2.40(m,8H),2.13-2.15(m,9H),2.02(s,9H),1.94-1.96(m,18H),1.48 -1.66(m,4H),1.21-1.36(m,11H)
2.3.2 E13的制备
2.3.2 Preparation of E13
将化合物7(400mg,0.187mmol)溶于无水二氯甲烷(5.00mL)中，依次加入DIEA(0.18mL,1.12mmol)，DMAP(5.71mg,0.047mmol)，最后将化合物8(112mg,1.12mmol)加入其中。混合液在25℃下搅拌3小时。薄层色谱(二氯甲烷：甲醇＝10：1)显示新点生成。向混合液中加入二氯甲烷(200ml)，用饱和食盐水洗涤三次(15.0ml x 3),有机相经无水硫酸钠干燥后，在减压条件下浓缩得到粗产品。粗产品经高效液相色谱分离(色谱柱：Welch Xtimate C18 150*25mm*5um；流动相：水-ACN；梯度：27％-57％/11min；流速：25ml/min)得到白色固体化合物E13(74.8mg,0.033mmol,收率17.9％)。Compound 7 (400mg, 0.187mmol) was dissolved in anhydrous dichloromethane (5.00mL), DIEA (0.18mL, 1.12mmol), DMAP (5.71mg, 0.047mmol) were added in sequence, and finally compound 8 (112mg, 1.12 mmol) was added. The mixture was stirred at 25°C for 3 hours. Thin layer chromatography (dichloromethane:methanol=10:1) showed the formation of new spots. Add methylene chloride (200ml) to the mixture, wash with saturated brine three times (15.0ml x 3), dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a crude product. The crude product was separated by high performance liquid chromatography (chromatographic column: Welch Xtimate C18 150*25mm*5um; mobile phase: water-ACN; gradient: 27%-57%/11min; flow rate: 25ml/min) to obtain white solid compound E13 ( 74.8 mg, 0.033 mmol, yield 17.9%).
LCMS(ESI):m/z＝1149.5(M/2+H)+；1H NMR(400MHz,CD3OD)δ7.42-7.44(m,2H),7.18-7.34(m,7H),6.83-6.90(m,4H),5.33-5.34(m,3H),5.08-5.11(m,3H),4.61-4.68(m,3H),4.27-4.36(m,2H),4.19-4.27(m,1H),4.10-4.19(m,7H),4.08(s,2H),4.00-4.07(m,4H),3.90-3.94(m,4H),3.83-3.89(m,1H),3.79(d,J＝2.4Hz,6H), 3.70-3.72(m,4H),3.53-3.54(m,15H),3.44-3.48(m,3H),3.35-3.40(m,4H),3.10-3.23(m,2H),2.53-2.63(m,4H),2.22-2.40(m,8H),2.11-2.16(m,9H),2.05-2.10(m,2H),2.02(s,9H),1.90-1.97(m,19H),1.48-1.65(m,4H),1.29-1.32(m,8H)LCMS (ESI): m/z=1149.5 (M/2+H) + ; 1 H NMR (400MHz, CD 3 OD) δ7.42-7.44 (m, 2H), 7.18-7.34 (m, 7H), 6.83 -6.90(m,4H),5.33-5.34(m,3H),5.08-5.11(m,3H),4.61-4.68(m,3H),4.27-4.36(m,2H),4.19-4.27(m, 1H),4.10-4.19(m,7H),4.08(s,2H),4.00-4.07(m,4H),3.90-3.94(m,4H),3.83-3.89(m,1H),3.79(d, J＝2.4Hz,6H), 3.70-3.72(m,4H),3.53-3.54(m,15H),3.44-3.48(m,3H),3.35-3.40(m,4H),3.10-3.23(m,2H),2.53-2.63(m ,4H),2.22-2.40(m,8H),2.11-2.16(m,9H),2.05-2.10(m,2H),2.02(s,9H),1.90-1.97(m,19H),1.48-1.65 (m,4H),1.29-1.32(m,8H)
实施例3化合物E2的制备
Example 3 Preparation of Compound E2
1.化合物2b的制备
1. Preparation of compound 2b
室温条件下，将化合物2a(38.0g,231mmol)，TsNHBoc(75.4g,278mmol)，K2CO3(6.40g,46.3mmol)，TEBA(5.27g,23.1mmol)加入到三口瓶中。在95℃下反应2小时，随后降温到25℃，TLC(PE/EA＝2/1,UV 254nm)显示反应完成，反应液加水(500mL)稀释，二氯甲烷(200mL x 3)萃取，有机相减压浓缩，粗产品柱分离纯化(PE/EA＝10/1-3/1)得到标题化合物2b(40.0g)。1H NMR(400MHz,CDCl3)δ7.72(d,J＝8.4Hz,2H),7.28-7.39(m,7H),4.72-4.86(m,2H),4.42-4.56(m,2H),3.52-3.61m,2H),3.14-3.39(m,2H),2.43(s,3H),1.47(s,9H)At room temperature, compound 2a (38.0g, 231mmol), TsNHBoc (75.4g, 278mmol), K 2 CO 3 (6.40g, 46.3mmol), and TEBA (5.27g, 23.1mmol) were added to the three-necked flask. React at 95°C for 2 hours, then cool to 25°C. TLC (PE/EA=2/1, UV 254nm) shows that the reaction is complete. The reaction solution is diluted with water (500mL), extracted with dichloromethane (200mL x 3), and organically The phase was concentrated under reduced pressure, and the crude product was separated and purified by column (PE/EA=10/1-3/1) to obtain the title compound 2b (40.0g). 1 H NMR (400MHz, CDCl 3 ) δ7.72 (d, J = 8.4Hz, 2H), 7.28-7.39 (m, 7H), 4.72-4.86 (m, 2H), 4.42-4.56 (m, 2H), 3.52-3.61m,2H),3.14-3.39(m,2H),2.43(s,3H),1.47(s,9H)
2.化合物2d的制备
2. Preparation of compound 2d
将化合物2b(79.0g,181mmol)，化合物2c(24.7g,150.8mmol)，碳酸钾(4.21g,30.5mmol)，TEBA(3.47g,15.2mmol)，95℃反应3小时。TLC(PE/EA＝2/1,UV 254nm)显示反应完成，反应液加水(500mL)稀释，二氯甲烷(200mL x 3)萃取，有机相减压浓缩，粗产品柱分离纯化(PE/EA＝10/1-3/1)得到化合物2d(40.0g,收率43.8％)。Compound 2b (79.0g, 181mmol), compound 2c (24.7g, 150.8mmol), potassium carbonate (4.21g, 30.5mmol), TEBA (3.47g, 15.2mmol) were reacted at 95°C for 3 hours. TLC (PE/EA=2/1, UV 254nm) showed that the reaction was completed. The reaction solution was diluted with water (500mL), extracted with dichloromethane (200mL x 3), the organic phase was concentrated under reduced pressure, and the crude product was separated and purified by column (PE/EA =10/1-3/1) to obtain compound 2d (40.0g, yield 43.8%).
1H NMR(400MHz,CDCl3)δ7.64-7.77(m,2H),7.27-7.38(m,13H),5.05-5.16(m,1H),4.50-4.59(m,4H),4.03-4.11(m,1H),3.62-3.76(m,2H),3.44-3.58(m,3H),3.25-3.33(m,2H),3.15-3.20(m,1H),2.42(s,3H),1.46(s,9H)1H NMR(400MHz, CDCl3)δ7.64-7.77(m,2H),7.27-7.38(m,13H),5.05-5.16(m,1H),4.50-4.59(m,4H),4.03-4.11(m ,1H),3.62-3.76(m,2H),3.44-3.58(m,3H),3.25-3.33(m,2H),3.15-3.20(m,1H),2.42(s,3H),1.46(s ,9H)
3.化合物2e的制备
3. Preparation of compound 2e
冰水浴条件下，将化合物2d(77.0g,128mmol)，三乙胺(28.6mL,205mmol)，依次加入到二氯甲烷(800mL)中，缓慢滴加甲基磺酰氯(24.2g,212mmol)。在25℃下反应2小时，LCMS显示反应完成。反应液加入水(800mL)洗涤，有机相减压浓缩得到粗品化合物2e(90.0g)。Under ice-water bath conditions, compound 2d (77.0g, 128mmol) and triethylamine (28.6mL, 205mmol) were sequentially added to dichloromethane (800mL), and methylsulfonyl chloride (24.2g, 212mmol) was slowly added dropwise. After 2 hours of reaction at 25°C, LCMS showed that the reaction was complete. The reaction solution was washed with water (800 mL), and the organic phase was concentrated under reduced pressure to obtain crude compound 2e (90.0 g).
m/z：ES+[M+Na]+700.1m/z：ES+[M+Na]+700.1
1H NMR(400MHz,CDCl3)δ7.68(d,J＝8.4Hz,2H),7.28-7.39(m,12H),5.10-5.20(m,1H),4.93-5.02(m,1H),4.52-4.61(m,4H),3.72-3.88(m,2H),3.52-3.67(m,4H),3.27-3.36(m,1H),3.15-3.22(m,1H),3.04(s,3H),2.43(s,3H),1.43(s,9H). 1 H NMR (400MHz, CDCl 3 ) δ7.68 (d, J = 8.4Hz, 2H), 7.28-7.39 (m, 12H), 5.10-5.20 (m, 1H), 4.93-5.02 (m, 1H), 4.52-4.61(m,4H),3.72-3.88(m,2H),3.52-3.67(m,4H),3.27-3.36(m,1H),3.15-3.22(m,1H),3.04(s,3H ),2.43(s,3H),1.43(s,9H).
4.化合物2f的制备
4. Preparation of compound 2f
室温条件下，将化合物2e(90.0g,133mmol)，碳酸钾(91.8g,664mmol)加入到甲醇(900mL)中。在66℃下反应2小时，TLC(PE/EA＝2/1,UV 254nm) 显示有新点生成，有机相减压浓缩，反应液加水(200mL)稀释，二氯甲烷(200mL x 3)萃取,有机相减压浓缩，粗产品柱分离纯化(PE/EA＝30/1-2/1)得到化合物2f(64.0g，收率99.9％)。Compound 2e (90.0g, 133mmol) and potassium carbonate (91.8g, 664mmol) were added to methanol (900mL) at room temperature. React at 66°C for 2 hours, TLC (PE/EA=2/1, UV 254nm) It shows that new spots are generated, the organic phase is concentrated under reduced pressure, the reaction solution is diluted with water (200mL), extracted with dichloromethane (200mL x 3), the organic phase is concentrated under reduced pressure, and the crude product is separated and purified by column (PE/EA=30/1- 2/1) to obtain compound 2f (64.0 g, yield 99.9%).
1H NMR(400MHz,CDCl3)δ7.62(d,J＝8.4Hz,2H),7.27-7.39(m,12H),4.48-4.62(m,4H),3.93-4.08(m,2H),3.55-3.69(m,4H),2.84-3.10(m,4H),2.44(s,3H) 1 H NMR (400MHz, CDCl 3 ) δ7.62 (d, J = 8.4Hz, 2H), 7.27-7.39 (m, 12H), 4.48-4.62 (m, 4H), 3.93-4.08 (m, 2H), 3.55-3.69(m,4H),2.84-3.10(m,4H),2.44(s,3H)
5.化合物2g的制备
5. Preparation of compound 2g
室温条件下，将化合物2f(69.0g,143mmol)，镁屑(54.7g,2.28mol)加入到甲醇(400mL)中，在66℃下反应1小时。TLC(DCM/MeOH＝10/1,UV 254nm)显示原料反应完全且有新点生成。反应液加水(3000mL)和饱和氯化铵水溶液(3000mL)稀释,二氯甲烷萃取(1000mL x3)，有机相使用饱和碳酸氢钠(300mL X 3)洗涤，有机相减压浓缩得到粗品化合物2g(32.0g)。At room temperature, compound 2f (69.0g, 143mmol) and magnesium chips (54.7g, 2.28mol) were added to methanol (400mL), and the mixture was reacted at 66°C for 1 hour. TLC (DCM/MeOH=10/1, UV 254nm) shows that the raw material reaction is complete and new spots are generated. The reaction solution was diluted with water (3000mL) and saturated aqueous ammonium chloride solution (3000mL), extracted with dichloromethane (1000mL x 3), the organic phase was washed with saturated sodium bicarbonate (300mL x 3), and the organic phase was concentrated under reduced pressure to obtain 2g of crude compound ( 32.0g).
1H NMR(400MHz,CDCl3)δ7.27-7.40(m,10H),4.57(s,4H),3.90-4.00(m,2H),3.59-3.69(m,4H),2.77-3.04(m,4H) 1 H NMR (400MHz, CDCl 3 ) δ7.27-7.40(m,10H),4.57(s,4H),3.90-4.00(m,2H),3.59-3.69(m,4H),2.77-3.04(m ,4H)
6.化合物2h的制备
6. Preparation of compound 2h
室温条件下，将化合物2g(3.00g,9.16mmol)，化合物1b(1.90mL,18.3mmol)，醋酸(1.01mL,18.3mmol)加入到甲醇(30mL)中。在25℃下反应18小时，随后加入氰基硼氢化钠(2.30g,36.7mmol)，50℃下反应4小时，TLC(DCM/MeOH＝10/1)显示有新点生成。反应液加水(50mL)稀释,二氯甲烷萃取(30mL x3)，有机相减压浓缩。粗产品通过柱层析纯化(二氯甲烷/甲醇＝99/1-5/1)得到化合物2h(3.00g,收率79.9％)。At room temperature, compound 2g (3.00g, 9.16mmol), compound 1b (1.90mL, 18.3mmol), and acetic acid (1.01mL, 18.3mmol) were added to methanol (30mL). React at 25°C for 18 hours, then add sodium cyanoborohydride (2.30g, 36.7mmol), and react at 50°C for 4 hours. TLC (DCM/MeOH=10/1) shows that new spots are generated. The reaction solution was diluted with water (50mL), extracted with dichloromethane (30mL x3), and the organic phase was concentrated under reduced pressure. The crude product was purified by column chromatography (dichloromethane/methanol=99/1-5/1) to obtain compound 2h (3.00g, yield 79.9%).
1H NMR(400MHz,CDCl3)δ7.28-7.41(m,10H),4.51-4.71(m,4H),4.00-4.35(m,2H),3.49-3.77(m,4H),2.69-2.98(m,2H),1.63-2.12(m,7H),1.16-1.44(m,6H) 1 H NMR (400MHz, CDCl3) δ7.28-7.41(m,10H),4.51-4.71(m,4H),4.00-4.35(m,2H),3.49-3.77(m,4H),2.69-2.98( m,2H),1.63-2.12(m,7H),1.16-1.44(m,6H)
7.化合物2i的制备
7. Preparation of Compound 2i
室温条件下，将化合物2h(3.00g,7.32mmol)加入到浓盐酸(10mL,12M)中，50℃下反应18小时，TLC(DCM/MeOH＝10/1)显示有新点生成，反应液减压浓缩得到粗品化合物2i(2.00g)。At room temperature, compound 2h (3.00g, 7.32mmol) was added to concentrated hydrochloric acid (10mL, 12M), and the reaction was carried out at 50°C for 18 hours. TLC (DCM/MeOH=10/1) showed that new spots were formed, and the reaction solution Concentrate under reduced pressure to obtain crude compound 2i (2.00g).
1H NMR(400MHz,CD3OD)δ4.27-4.38(m,1H),4.11-4.20(m,1H),3.96-4.04(m,2H),3.60-3.68(m,3H),3.36-3.43(m,1H),3.19-3.29(m,2H),3.11(s,1H),2.02-2.17(m,2H),1.90-2.00(m,2H),1.69-1.78(m,1H),1.57-1.69(m,1H),1.35-1.52(m,3H),1.20-1.31(m,1H) 1 H NMR (400MHz, CD3OD) δ4.27-4.38(m,1H),4.11-4.20(m,1H),3.96-4.04(m,2H),3.60-3.68(m,3H),3.36-3.43( m,1H),3.19-3.29(m,2H),3.11(s,1H),2.02-2.17(m,2H),1.90-2.00(m,2H),1.69-1.78(m,1H),1.57- 1.69(m,1H),1.35-1.52(m,3H),1.20-1.31(m,1H)
8.化合物2j的制备
8. Preparation of compound 2j
室温下，将化合物2i(2.00g,8.72mmol)溶解到吡啶(40mL)中，再加入DMTrCl(2.96g,8.72mmol)，25℃反应18小时，TLC(PE/EA＝1/1,UV 254nm)显示反应完成。反应液减压浓缩，加饱和氯化铵水溶液(200mL)，DCM(100mLx3)萃取，有机相减压浓缩，粗产品通过柱层析纯化(二氯甲烷/甲醇＝99/1-10/1)得到化合物2j(1.40g,收率30.2％)。Dissolve compound 2i (2.00g, 8.72mmol) into pyridine (40mL) at room temperature, then add DMTrCl (2.96g, 8.72mmol), react at 25°C for 18 hours, TLC (PE/EA=1/1, UV 254nm) ) indicates that the reaction is complete. The reaction solution was concentrated under reduced pressure, added with saturated aqueous ammonium chloride solution (200mL), extracted with DCM (100mLx3), the organic phase was concentrated under reduced pressure, and the crude product was purified by column chromatography (dichloromethane/methanol=99/1-10/1) Compound 2j (1.40g, yield 30.2%) was obtained.
9.化合物E2的制备
9. Preparation of compound E2
将化合物2j(660mg,1.24mmol)，化合物1h(374mg,1.24mmol)，DCI(73.3mg,0.621mmol)加入到DCM(10mL)，25℃反应1小时，TLC(PE/EA＝5/1,PMA)显示原料反应完全。加入饱和碳酸氢钠(50mL)，DCM(30mL x 3)萃取，有机相减压浓缩，粗品柱分离(PE/EA＝99/1-30/1)得到化合物E2(350mg,收率19.3％)。Add compound 2j (660mg, 1.24mmol), compound 1h (374mg, 1.24mmol), DCI (73.3mg, 0.621mmol) to DCM (10mL), react at 25°C for 1 hour, TLC (PE/EA=5/1, PMA) shows that the raw material reaction is complete. Add saturated sodium bicarbonate (50mL), extract with DCM (30mL .
m/z:ES+[M+H]+732.2m/z:ES+[M+H]+732.2
1H NMR(400MHz,CDCl3)δ7.40-7.50(m,2H),7.28-7.37(m,6H),7.21(d,J＝7.2Hz,1H),6.77-6.89(m,4H),3.67-4.07(m,11H),3.56-3.66(m,2H),3.04-3.33(m,2H),2.30-2.96(m,6H),2.10-2.28(m,1H),1.63-1.88(m,4H),1.47-1.52(m,1H),1.24-1.31(m,3H),1.15-1.23(m,12H),1.11-1.14(m,1H) 1 H NMR (400MHz, CDCl 3 ) δ7.40-7.50 (m, 2H), 7.28-7.37 (m, 6H), 7.21 (d, J = 7.2Hz, 1H), 6.77-6.89 (m, 4H), 3.67-4.07(m,11H),3.56-3.66(m,2H),3.04-3.33(m,2H),2.30-2.96(m,6H),2.10-2.28(m,1H),1.63-1.88(m ,4H),1.47-1.52(m,1H),1.24-1.31(m,3H),1.15-1.23(m,12H),1.11-1.14(m,1H)
实施例4 siRNA的制备Example 4 Preparation of siRNA
使用本领域熟知的固相亚磷酰胺法制备本公开的siRNA。具体方法可参见例如PCT公开号WO2016081444和WO2019105419，并简述如下。The siRNAs of the present disclosure are 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.
1.未连接配体的siRNA的制备1. Preparation of siRNA without ligand attached
1.1正义链(SS链)的合成1.1 Synthesis of justice chain (SS chain)
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，按照正义链核苷酸排布顺序自3’-5’方向逐一连接核苷单体或核苷酸类似物单体。每连接一个核苷单体或核苷酸类似物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5μmol的寡核酸合成条件如下：Through the solid-phase phosphoramidite synthesis method, the blank CPG solid-phase carrier is used as the starting cycle, and the nucleoside monomers or nucleotide analogues are connected one by one from the 3'-5' direction according to the sequence of the sense strand nucleotides. monomer. Each connection of a nucleoside monomer or nucleotide analog monomer includes a four-step reaction of deprotection, coupling, capping, oxidation or sulfation. The synthesis conditions for an oligonucleotide with a synthesis scale of 5 μmol are as follows:
核苷单体或核苷酸类似物单体提供的是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。The nucleoside monomer or nucleotide analog monomer is provided with a 0.05 mol/L acetonitrile solution. The reaction conditions of each step are the same, that is, the temperature is 25°C, and 3% trichloroacetic acid-dichloromethane solution is used for deprotection. , deprotection 3 times; the activator used in the coupling reaction was 0.25 mol/L ETT-acetonitrile solution, coupled 2 times; the capping reaction used 10% acetic anhydride-acetonitrile and pyridine/N-methylimidazole/acetonitrile (10: 14:76, v/v/v), blocked twice; oxidized using 0.05mol/L iodine/tetrahydrofuran/pyridine/water (70/20/10, v/v/v), oxidized twice; used sulfide 0.2mol/L PADS acetonitrile/3-methylpyridine (1/1, v/v), sulfide 2 times.
IB的核苷酸单体购自上海兆维科技发展有限公司，产品编号OP-040。The nucleotide monomer of IB was purchased from Shanghai Zhaowei Technology Development Co., Ltd., product number OP-040.
1.2反义链(AS链)的合成1.2 Synthesis of antisense chain (AS chain)
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核苷酸类似物单体。每连接一个核苷单体或核苷酸类似物单体都包含了脱保护、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。Through the solid-phase phosphoramidite synthesis method, the blank CPG solid-phase carrier is used as the starting cycle, and the nucleoside monomers or nucleotide analogues are connected one by one from the 3'-5' direction according to the sequence of the antisense strand nucleotides. single object. Each linkage of a nucleoside monomer or nucleotide analog monomer involves deprotection, coupling, capping, oxidation or sulfation. For a four-step reaction, the synthesis conditions of 5 μmol of oligonucleotide for the antisense strand are the same as those for the sense strand.
1.3寡核苷酸的纯化与退火1.3 Purification and annealing of oligonucleotides
1.3.1氨解1.3.1 Ammonolysis
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。Add the synthesized solid phase carrier (sense strand or antisense strand) into a 5mL centrifuge tube, add 3% diethylamine/ammonia water (v/v), and react in a constant temperature water bath at 35°C (or 55°C) for 16 hour (or 8 hours), filter, and wash the solid phase carrier three times with ethanol/water, 1 mL each time. The filtrate is centrifuged and concentrated and the crude product is purified.
1.3.2纯化1.3.2 Purification
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。Methods of purification and desalting are well known to those in the art. For example, 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.
1.3.3退火1.3.3 Annealing
根据表6将正义链(SS链)链与反义链(AS链)以摩尔比(SS链/AS链＝1/1.05)混合，水浴锅加热至70-95度，保持3-5min，自然冷却至室温，将体系冻干得到产品。According to Table 6, mix the sense strand (SS chain) and the antisense strand (AS chain) at a molar ratio (SS chain/AS chain = 1/1.05), heat the water bath to 70-95 degrees, keep for 3-5 minutes, naturally Cool to room temperature and freeze-dry the system to obtain the product.
2.正义链与配体连接的siRNA的制备2. Preparation of siRNA with sense strand connected to ligand
2.1配体与CPG载体的连接2.1 Connection of ligand to CPG carrier
2.1.1化合物E7与CPG载体的连接2.1.1 Connection of compound E7 to CPG carrier
将化合物E7(53mg，0.018mmol)和HBTU(13.3mg，0.035mmol)混合后加入乙腈(5mL)震荡溶解，随后加入DIEA(9.0mg，0.07mmol)和DMAP(2.1mg，0.018mmol)震荡溶解直至变得清澈。称取空白载体Resin(550mg，CPG孔径)加入到反应液中，控温20℃，摇床反应过夜。取样并监测，进行薄层色谱TLC，结果显示反应完全，其中展开剂为DCM/甲醇＝4/1，用磷钼酸显色。使用砂芯漏斗进行过滤，滤饼用无水乙腈洗涤(20mL*5)，取滤饼，使用油泵减压抽滤6h，得到类白色固体530mg。Compound E7 (53 mg, 0.018 mmol) and HBTU (13.3 mg, 0.035 mmol) were mixed, acetonitrile (5 mL) was added with shaking to dissolve, and then DIEA (9.0 mg, 0.07 mmol) and DMAP (2.1 mg, 0.018 mmol) were added with shaking until dissolved. become clear. Weigh the blank carrier Resin (550 mg, CPG pore size ) was added to the reaction solution, the temperature was controlled at 20°C, and the reaction was carried out overnight on a shaking table. Samples were taken and monitored, and thin-layer chromatography and TLC were performed. The results showed that the reaction was complete. The developing agent was DCM/methanol=4/1, and phosphomolybdic acid was used to develop the color. Use a sand core funnel to filter, wash the filter cake with anhydrous acetonitrile (20 mL*5), take the filter cake, use an oil pump to filter under reduced pressure for 6 hours, and obtain 530 mg of an off-white solid.
将上述缩合后的产品530mg放于50mL的圆底瓶中，依次加入CapC(DMAP/乙腈)，CapB(N-甲基咪唑/吡啶/乙腈)，CapA(醋酐/乙腈)，室温下摇床过夜。过滤，滤饼用乙腈洗涤，20mL*4)，取滤饼，油泵减压抽滤8h后得到类白色固体200mg，用于固相合成。Put 530 mg of the above-mentioned condensation product into a 50 mL round-bottomed bottle, add CapC (DMAP/acetonitrile), CapB (N-methylimidazole/pyridine/acetonitrile), CapA (acetic anhydride/acetonitrile) in sequence, and shake at room temperature. overnight. Filter, wash the filter cake with acetonitrile, 20mL*4), take the filter cake, and filter under reduced pressure with an oil pump for 8 hours to obtain 200 mg of off-white solid, which is used for solid phase synthesis.
2.1.2化合物E13与CPG载体的连接2.1.2 Connection of compound E13 to CPG carrier
以与化合物E7与CPG载体的连接方法相似的方法将化合物E13与CPG载体连接。Compound E13 was connected to the CPG carrier in a manner similar to the method for connecting compound E7 to the CPG carrier.
2.2正义链(SS链)的合成2.2 Synthesis of justice chain (SS chain)
通过固相亚磷酰胺合成法，利用实施例4的2.1.1节制备的GL6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5μmol的寡核酸合成条件如下：Through the solid-phase phosphoramidite synthesis method, the GL6 solid-phase carrier prepared in Section 2.1.1 of Example 4 is used as the initial cycle, and the nucleosides are connected one by one from the 3'-5' direction in the sequence of the sense strand nucleotides. monomer. Each connection of a nucleoside monomer includes four-step reactions of deprotection, coupling, capping, oxidation or sulfation. The synthesis conditions for an oligonucleotide with a synthesis scale of 5 μmol are as follows:
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。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. For deprotection, use 3% trichloroacetic acid-dichloromethane solution, and deprotect 3 times; coupling The activator used in the reaction was 0.25 mol/L ETT-acetonitrile solution, coupled twice; the capping agent used 10% acetic anhydride-acetonitrile and pyridine/N-methylimidazole/acetonitrile (10:14:76, v/v/ v), cap twice; use 0.05mol/L iodine for oxidation /tetrahydrofuran/pyridine/water (70/20/10, v/v/v), oxidized twice; for sulfide, use 0.2 mol/L PADS acetonitrile/3-methylpyridine (1/1, v/v). Thiogeneration 2 times.
2.3反义链(AS链)的合成2.3 Synthesis of antisense chain (AS chain)
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。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，反义链的5μmol的寡核酸合成条件和正义链的相同。Through the solid-phase phosphoramidite synthesis method, a blank CPG solid-phase carrier is used as the starting cycle, and the nucleoside monomers are connected one by one from the 3'-5' direction in the order of the antisense strand nucleotide arrangement. Each connection of a nucleoside monomer involves a four-step reaction of deprotection, coupling, capping, oxidation or sulfation. The synthesis conditions of 5 μmol of oligonucleotide of the antisense strand are the same as those of the sense strand.
2.4寡核苷酸的纯化与退火2.4 Purification and annealing of oligonucleotides
2.4.1氨解2.4.1 Ammonolysis
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。Add the synthesized solid phase carrier (sense strand or antisense strand) into a 5mL centrifuge tube, add 3% diethylamine/ammonia water (v/v), and react in a constant temperature water bath at 35°C (or 55°C) for 16 hour (or 8 hours), filter, and wash the solid phase carrier three times with ethanol/water, 1 mL each time. The filtrate is centrifuged and concentrated and the crude product is purified.
2.4.2纯化2.4.2 Purification
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，采用凝胶填料纯化柱进行脱盐，洗脱体系是纯水。Methods of purification and desalting are well known to those in the art. For example, a strong anion packing column can be used, a sodium chloride-sodium hydroxide system can be used for elution and purification, the products can be collected and tubed, and a gel packing purification column can be used for desalting. The elution system is pure water.
2.4.3退火2.4.3 Annealing
根据表6将正义链(SS链)链与反义链(AS链)以摩尔比(SS链/AS链＝1/1.05)混合，水浴锅加热至70-95℃，保持3-5min，自然冷却至室温，将体系冻干得到产品。According to Table 6, mix the sense strand (SS chain) and the antisense strand (AS chain) at a molar ratio (SS chain/AS chain = 1/1.05), heat the water bath to 70-95°C, keep for 3-5 minutes, and naturally Cool to room temperature and freeze-dry the system to obtain the product.
以相似的方法获得缀合有L96的siRNA以及缀合有NAG37的siRNA。L96-conjugated siRNA and NAG37-conjugated siRNA were obtained in a similar manner.
实施例5 Huh7细胞系活性筛选Example 5 Huh7 cell line activity screening
细胞转染cell transfection
第一天，消化Huh7细胞系(南京科佰，货号CBP60202)后重悬，计数，以100μL/孔，1×104个细胞/孔铺板在96孔板中，18h后进行转染操作。On the first day, the Huh7 cell line (Nanjing Kebai, Cat. No. CBP60202) was digested, resuspended, counted, and plated in a 96-well plate at 100 μL/well and 1×10 4 cells/well. Transfection was performed 18 hours later.
第二天，20μM的实施例4制备的siRNA母液用Opti-MEM稀释，取198μL Opti-MEM加入2μLsiRNA母液，吹吸混匀，待用。每次实验时，根据不同实验需求进行相应的稀释操作。The next day, 20 μM of the siRNA stock solution prepared in Example 4 was diluted with Opti-MEM, 198 μL of Opti-MEM was added to 2 μL of the siRNA stock solution, piped and mixed, and set aside. In each experiment, corresponding dilution operations are performed according to different experimental needs.
第二天，取14.1μL Opti-MEM稀释0.9μL RNAiMAX(Thermo，13778150)，轻轻吹吸混匀，室温下静置5min。然后将配置好的RNAi-MAX混合液取15μL、稀释好的化合物15μL轻轻吹吸混匀，不要带入气泡，室温静置10min，然后以10μL/孔加入96孔板。37℃、5％CO2培养箱培养24h后提取RNA。The next day, take 14.1 μL Opti-MEM and dilute 0.9 μL RNAiMAX (Thermo, 13778150), mix gently by pipetting, and let stand at room temperature for 5 minutes. Then take 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted compound by gently pipetting and mixing without bringing in air bubbles. Let stand at room temperature for 10 minutes, and then add 10 μL/well to a 96-well plate. After culturing for 24 hours in a 37°C, 5% CO2 incubator, RNA was extracted.
RNA提取RNA extraction
按照高通量细胞RNA提取试剂盒(凡知医疗，FG0412)的操作程序使用核酸提取仪(杭州奥盛，Auto-pure96)进行细胞RNA提取。Cell RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the operating procedures of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412).
RNA反转录RNA reverse transcription
变性反应混合液配制，参考PrimeScriptTM II 1st Strand cDNA Synthesis Kit(Takara，6210B)。每孔中含有Oligo dT Primer 1μL，dNTP Mixture 1μL，模板RNA 12.5μL。在常规PCR仪中在65℃下孵育5min进行变性反应。将混合液置 于冰上迅速冷却2min。To prepare the denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. Denaturation reaction was performed by incubating at 65°C for 5 min in a conventional PCR machine. Place the mixture Cool quickly on ice for 2 minutes.
反转录反应液配制，参考PrimeScriptTM II 1st Strand cDNA Synthesis Kit(Takara，6210B)。每孔中含有5×Prime Script II Buffer 4μL，RNase Inhibitor 0.5μL，PrimeScript II RTase 1μL。For the preparation of reverse transcription reaction solution, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 μL of 5× Prime Script II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.
将变性后的反应液14.5μL与反转录反应液缓慢混匀，在常规PCR仪中在42℃孵育45min进行反转录，在95℃孵育5min使酶失活，4℃将反转录产物(cDNA)冷却。Slowly mix 14.5 μL of the denatured reaction solution with the reverse transcription reaction solution, incubate in a conventional PCR machine at 42°C for 45 minutes to perform reverse transcription, incubate at 95°C for 5 minutes to inactivate the enzyme, and remove the reverse transcription product at 4°C. (cDNA) cooling.
反转录结束后，向每个孔的cDNA样品中加入无DNA酶和RNA酶的蒸馏水30μL。After reverse transcription, add 30 μL of DNase- and RNase-free distilled water to the cDNA sample in each well.
荧光定量PCRFluorescent quantitative PCR
参考TaqManTM Fast Advanced Master Mix(ABI，4444965)的操作流程，以20μL体系进行荧光定量PCR反应(ABI，QuantStudio3)。反应程序为：(50℃，2min)×1Cycle；(95℃，20s)×1Cycle；(95℃，1s；60℃，24s)×40Cycles。Refer to the operating procedure of TaqMan TM Fast Advanced Master Mix (ABI, 4444965), and perform a fluorescence quantitative PCR reaction (ABI, QuantStudio3) with a 20 μL system. The reaction program is: (50°C, 2min)×1Cycle; (95°C, 20s)×1Cycle; (95°C, 1s; 60°C, 24s)×40Cycles.
表7引物信息
Table 7 Primer information
数据统计Statistics
计算2-△△Ct值并换算成百分比以得到剩余抑制率。Calculate the 2 -ΔΔCt value and convert to percentage to obtain the remaining inhibition rate.
△△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)]。△△Ct=[(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)].
siRNA的终浓度为1nM进行siRNA化合物细胞系活性高通量筛选，实验筛选结果如表8所示。The final concentration of siRNA was 1 nM for high-throughput screening of cell line activity of siRNA compounds. The experimental screening results are shown in Table 8.
表8使用1nM的siRNA进行Huh7细胞系活性高通量筛选的结果

Table 8 Results of high-throughput screening of Huh7 cell line activity using 1 nM siRNA

实施例6使用10倍梯度稀释的5个浓度的siRNA进行Huh7细胞系活性筛选Example 6 Screening of Huh7 cell line activity using 10-fold gradient dilutions of 5 concentrations of siRNA
与以上实施例5的活性筛选相似，使用Huh7细胞系进行进一步活性筛选。Similar to the activity screening in Example 5 above, further activity screening was performed using the Huh7 cell line.
siRNA的起始浓度为10nM，10倍梯度稀释获得5个浓度点(10nM，1nM，0.1nM，0.01nM，0.001nM)，进行siRNA的Hep3B细胞系活性筛选。筛选结果如表9所示，其中第2-6列为剩余抑制率，第7列为IC50值。The starting concentration of siRNA was 10 nM, and 10-fold gradient dilution was performed to obtain 5 concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM), and the Hep3B cell line activity of siRNA was screened. The screening results are shown in Table 9, in which columns 2-6 are the remaining inhibition rates and column 7 is the IC50 value.
表9使用5个浓度的siRNA进行Huh7细胞系活性筛选的结果
Table 9 Results of Huh7 cell line activity screening using 5 concentrations of siRNA
实施例7人原代肝细胞(PHH细胞)活性筛选Example 7 Activity Screening of Primary Human Hepatocytes (PHH Cells)
细胞转染cell transfection
取鼠尾胶原蛋白溶液(Sigma，C3867)1.4mL，加入40.6mL无DNA酶和RNA酶的蒸馏水中，混匀，96孔培养板中每孔加入40μL，4℃包被过夜，第二天去包被液。Take 1.4 mL of rat tail collagen solution (Sigma, C3867), add 40.6 mL of DNase- and RNase-free distilled water, mix well, add 40 μL to each well of a 96-well culture plate, coat overnight at 4°C, and remove the mixture the next day. Coating fluid.
第二天，使用前，包被的细胞板用DPBS润洗后吸去DPBS，将PHH细胞(上海轩一，货号QYLF-HPMC)37℃复苏，加入复苏培养基中，离心重悬并计数。将PHH细胞铺板在96孔板中，90μL/孔，2×104个细胞/孔；4h后更换完全培养基，18h后进行转染操作。The next day, before use, the coated cell plate was rinsed with DPBS and the DPBS was aspirated off. PHH cells (Shanghai Xuanyi, Cat. No. QYLF-HPMC) were revived at 37°C, added to the recovery medium, centrifuged, resuspended and counted. PHH cells were plated in a 96-well plate at 90 μL/well, 2×10 4 cells/well; complete medium was replaced after 4 hours, and transfection was performed after 18 hours.
第三天，20μM的siRNA母液用Opti-MEM进行稀释，取198μL Opti-MEM加入2μLsiRNA母液，吹吸混匀，作为第1个浓度点，按照实际实验需要进行相应梯度稀释。On the third day, 20 μM siRNA stock solution was diluted with Opti-MEM. Add 198 μL Opti-MEM to 2 μL siRNA stock solution, mix by pipetting, and use it as the first concentration point. Perform corresponding gradient dilutions according to actual experimental needs.
第三天，取14.1μL Opti-MEM稀释0.9μL RNAiMAX(Thermo，13778150)，轻轻吹吸混匀，室温下静置5min。然后将配置好的RNAi-MAX混合液取15μL、稀释好的化合物15μL轻轻吹吸混匀，不要带入气泡，室温静置10min，加入96孔板，10μL/孔。37℃、5％CO2培养箱培养24h后提取RNA。 On the third day, take 14.1 μL Opti-MEM and dilute 0.9 μL RNAiMAX (Thermo, 13778150), mix gently by pipetting, and let stand at room temperature for 5 minutes. Then take 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted compound by gently pipetting and mixing without bringing in air bubbles. Let it stand at room temperature for 10 minutes. Add 10 μL/well to a 96-well plate. After culturing for 24 hours in a 37°C, 5% CO2 incubator, RNA was extracted.
RNA提取RNA extraction
按照高通量细胞RNA提取试剂盒(凡知医疗，FG0412)的操作程序使用核酸提取仪(杭州奥盛，Auto-pure96)进行细胞RNA提取。Cell RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the operating procedures of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412).
RNA反转录RNA reverse transcription
变性反应混合液配制，参考PrimeScriptTM II 1st Strand cDNA Synthesis Kit(Takara，6210B)。每孔中含有Oligo dT Primer 1μL，dNTP Mixture 1μL，模板RNA 12.5μL。在常规PCR仪中在65℃下孵育5min进行变性反应。将混合液置于冰上迅速冷却2min。To prepare the denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. Denaturation reaction was performed by incubating at 65°C for 5 min in a conventional PCR machine. Place the mixture on ice to cool quickly for 2 minutes.
反转录反应液配制，参考PrimeScriptTM II 1st Strand cDNA Synthesis Kit(Takara，6210B)。每孔中含有5×Prime Script II Buffer 4μL，RNase Inhibitor 0.5μL，PrimeScript II RTase 1μL。For the preparation of reverse transcription reaction solution, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 μL of 5× Prime Script II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.
将变性后的反应液14.5μL与反转录反应液缓慢混匀，在常规PCR仪中在42℃孵育45min进行反转录，在95℃孵育5min使酶失活，4℃将反转录产物(cDNA)冷却。Slowly mix 14.5 μL of the denatured reaction solution with the reverse transcription reaction solution, incubate in a conventional PCR machine at 42°C for 45 minutes to perform reverse transcription, incubate at 95°C for 5 minutes to inactivate the enzyme, and remove the reverse transcription product at 4°C. (cDNA) cooling.
反转录结束后，向每个孔的cDNA样品中加入无DNA酶和RNA酶的蒸馏水30μL。After reverse transcription, add 30 μL of DNase- and RNase-free distilled water to the cDNA sample in each well.
荧光定量PCRFluorescent quantitative PCR
参考TaqManTM Fast Advanced Master Mix(ABI，4444965)的操作流程，以20μL体系进行荧光定量PCR反应(ABI，QuantStudio3)。反应程序为：(50℃，2min)×1Cycle；(95℃，20s)×1Cycle；(95℃，1s；60℃，24s)×40Cycles。Refer to the operating procedure of TaqMan TM Fast Advanced Master Mix (ABI, 4444965), and perform a fluorescence quantitative PCR reaction (ABI, QuantStudio3) with a 20 μL system. The reaction program is: (50°C, 2min)×1Cycle; (95°C, 20s)×1Cycle; (95°C, 1s; 60°C, 24s)×40Cycles.
表10引物信息
Table 10 Primer information
数据统计Statistics
计算2-△△Ct值并换算成百分比以得到剩余抑制率。Calculate the 2 -ΔΔCt value and convert to percentage to obtain the remaining inhibition rate.
△△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)]。△△Ct=[(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)].
siRNA起始浓度为10nM，10倍梯度稀释，获得5个浓度点(10nM，1nM，0.1nM，0.01nM，0.001nM)，进行siRNA的人肝原代细胞活性筛选，筛选结果如表11所示，其中第2-6列为剩余抑制率，第7列为IC50值。The starting concentration of siRNA was 10 nM, and it was diluted 10 times to obtain 5 concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM). The activity of siRNA in human liver primary cells was screened. The screening results are shown in Table 11. , where columns 2-6 are the remaining inhibition rates and column 7 is the IC50 value.
表11使用5个浓度的siRNA进行人原代肝细胞筛选的结果

Table 11 Results of human primary hepatocyte screening using 5 concentrations of siRNA

实施例8 psiCHECK2 GSSM-5Hits脱靶活性筛选Example 8 Screening of off-target activity of psiCHECK2 GSSM-5Hits
质粒制备Plasmid preparation
根据siRNA序列设计相应的反义链脱靶质粒，由生工生物工程(上海)股份有限公司制备psiCHECK2 GSSM-5Hits重组质粒，并将重组质粒稀释至1000ng/μL备用。The corresponding antisense strand off-target plasmid was designed based on the siRNA sequence, and the psiCHECK2 GSSM-5Hits recombinant plasmid was prepared by Sangon Bioengineering (Shanghai) Co., Ltd., and the recombinant plasmid was diluted to 1000ng/μL for later use.
细胞转染cell transfection
在96孔板的每个孔中，用100μL HEK293A细胞(南京科佰，货号CBP60436)重悬液铺板，8×103个细胞/孔。In each well of a 96-well plate, 100 μL of HEK293A cell (Nanjing Kebai, Cat. No. CBP60436) resuspension was plated, with 8 × 10 3 cells/well.
第二天，首先将孔中的完全培养基吸弃，换成Opti-MEM培养基80μL/孔，饥饿处理大约1.5h。The next day, first remove the complete culture medium in the well, replace it with 80 μL/well of Opti-MEM culture medium, and starve for about 1.5 h.
siRNA配制：将siRNA从40nM终浓度开始3倍稀释，共11个浓度点(10nM，3.3333nM，1.1111nM，0.37037nM，0.12346nM，0.04115nM，0.01372nM，0.00457nM，0.00152nM，0.00051nM，0.00017nM)。siRNA preparation: Dilute siRNA 3 times starting from a final concentration of 40nM, with a total of 11 concentration points (10nM, 3.3333nM, 1.1111nM, 0.37037nM, 0.12346nM, 0.04115nM, 0.01372nM, 0.00457nM, 0.00152nM, 0.00051nM, 0.0 0017 nM).
质粒混合物的配制：单孔配制量为质粒0.01μL/孔，Opti-MEM 8.99μL/孔。Preparation of plasmid mixture: The preparation volume for a single well is 0.01 μL/well for plasmid and 8.99 μL/well for Opti-MEM.
Lipo混合物的配制：每孔中加入Lipo 2000 0.2μL和Opti-MEM 9.8μL从而用Opti-MEM稀释Lipo 2000(LipofectamineTM 2000转染试剂，Thermo，11668019)获得Lipo混合物，室温静置5min。Preparation of Lipo mixture: Add 0.2 μL of Lipo 2000 and 9.8 μL of Opti-MEM to each well to dilute Lipo 2000 (Lipofectamine TM 2000 transfection reagent, Thermo, 11668019) with Opti-MEM to obtain a Lipo mixture, and let it stand at room temperature for 5 minutes.
将已配制好的Lipo混合物22μL、siRNA 2.2μL、质粒混合物19.8μL，分别分装到对应的同一孔中，命名为well A混合物。吹打混匀后，在室温孵育20min后进行共转染。将well A混合物加入每孔细胞中，20μL/孔，加上原有80μL Opti-MEM，每孔终体积为100μL。37℃5％CO2培养箱培养4h后，每孔补加100μL含20％胎牛血清的DMEM培养基。37℃5％CO2培养箱培养24h后检测。Dispense 22 μL of prepared Lipo mixture, 2.2 μL of siRNA, and 19.8 μL of plasmid mixture into the corresponding wells and name them well A mixture. After mixing by pipetting, incubate at room temperature for 20 minutes before co-transfection. Add the well A mixture to the cells in each well, 20μL/well, plus the original 80μL Opti-MEM, the final volume of each well is 100μL. After culturing for 4 hours in a 37°C 5% CO2 incubator, 100 μL of DMEM medium containing 20% fetal calf serum was added to each well. Test after 24 hours of incubation in a 37°C 5% CO2 incubator.
结果检测Result detection
在实验前先将混合好的(Luciferase Assay System，Promega，E2940)进行复融，等平衡到室温后每管按1:1加入DMEM配制成底物I，现配现用。将Stop&Buffer进行复融，等平衡到室温后与Stop&100:1配制成底物II，现配现用。用真空泵吸去96孔培养板中原有的培养基。每孔加入150μL底物I，在摇床上室温孵育10min。取120μL底物I，转移到96孔酶标板上，在酶标仪(Tecan，Infinite 200)上读取Firefly化学发光值。再向每孔加入60μL底物II，在摇床上室温孵育10min，在酶标仪上读取Renilla化学发光值。Before the experiment, mix the ( Luciferase Assay System, Promega, E2940) was remelted, and after it was equilibrated to room temperature, DMEM was added to each tube at a ratio of 1:1 to prepare substrate I, which was ready for use. Will Stop& Remelt the Buffer and wait until it equilibrates to room temperature. Stop& Prepared as Substrate II at 100:1, ready for immediate use. Use a vacuum pump to remove the original culture medium in the 96-well culture plate. Add 150 μL of substrate I to each well and incubate on a shaker at room temperature for 10 min. Take 120 μL of substrate I, transfer it to a 96-well microplate, and read the Firefly chemiluminescence value on a microplate reader (Tecan, Infinite 200). Then add 60 μL of substrate II to each well, incubate at room temperature on a shaker for 10 min, and read the Renilla chemiluminescence value on a microplate reader.
数据分析处理Data analysis and processing
荧光活性由酶标仪测定,收集得到的Renilla信号由Firefly信号标准均一化，siRNA的抑制效果由未经过处理的结果比较得出(剩余抑制活性)，其计算过程见下：The fluorescence activity is measured by a microplate reader, and the collected Renilla signals are normalized by the Firefly signal standard. The inhibitory effect of siRNA is obtained by comparing the unprocessed results (residual inhibitory activity). The calculation process is as follows:
均一化Ren/Fir比值：Ratio＝Renilla(海肾荧光素酶)/Firefly(萤火虫荧光素酶)。Normalized Ren/Fir ratio: Ratio=Renilla (Renilla luciferase)/Firefly (firefly luciferase).
剩余抑制率＝(RatiosiRNA/Ratiocontrol)*100％，取两个复孔的均值：其中Ratiocontrol为对照孔(不含siRNA)Ratio值(取两个复孔的均值)。The remaining inhibition rate = (RatiosiRNA/Ratiocontrol) * 100%, take the average of two duplicate wells: Ratiocontrol is the Ratio value of the control well (excluding siRNA) (take the average of two duplicate wells).
作图：利用Graphpad Prism作图Drawing: Drawing using Graphpad Prism
半数抑制浓度(Half maximal inhibitory concentration，IC50)：本次实验以Top和Bottom作图，IC50值根据公式Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))求得，其中Y＝50，X＝log(浓度)。Half maximal inhibitory concentration (IC50): This experiment is plotted with Top and Bottom. The IC50 value is based on the formula Y=Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope)) Obtained, where Y=50, X=log (concentration).
siRNA的psiCHECK2 GSSM-5Hits脱靶活性筛选结果如表12所示，其中第2-12列为剩余抑制率，第13列为IC50值。结果表明，本公开的DR000405、DR000424、DR000430、DR000442、DR000447、DR000619、DR000621相对于阳性对照DR001483，针对HEK293A细胞具有更低的脱靶活性。The results of the off-target activity screening of siRNA's psiCHECK2 GSSM-5Hits are shown in Table 12, in which columns 2-12 are the remaining inhibition rates and column 13 is the IC50 value. The results show that DR000405, DR000424, DR000430, DR000442, DR000447, DR000619, and DR000621 of the present disclosure have lower off-target activity against HEK293A cells compared to the positive control DR001483.
表12 psiCHECK2 GSSM-5Hits脱靶活性筛选结果
Table 12 Screening results of psiCHECK2 GSSM-5Hits off-target activity
实施例9小鼠HDI模型活性筛选Example 9 Mouse HDI model activity screening
HDI动物造模HDI animal modeling
使用尾静脉高压注射的方法，将六至八周龄雌性Balb/c小鼠用双基因稳定 转染***进行体内转染造模，经由尾静脉，在5-7秒内通过27规格针头将含有不同质量比例的靶标基因cDNA序列(Genbank注册号NM_014495.2)的Piggy-Bac转座子质粒(购自苏州邦业)和Piggy-Bac辅助质粒(购自苏州邦业)(质量比为1:1，质粒总量100ug)的递送溶液(总体积为10％动物体重，Mirusbio-MIR 5240)注射至小鼠体内，注射后放回笼中观察30min。以造模日为第0天，在造模之后的各个时间点(第7天-第35天)获得血清用于检测SEAP表达水平。Using high-pressure tail vein injection, six- to eight-week-old female Balb/c mice were treated with double-gene stabilized The transfection system performs in vivo transfection modeling. Piggy-Bac transposon plasmids containing target gene cDNA sequences (Genbank registration number NM_014495.2) with different mass ratios are transferred through the tail vein through a 27-gauge needle within 5-7 seconds. (purchased from Suzhou Bangye) and Piggy-Bac helper plasmid (purchased from Suzhou Bangye) (mass ratio 1:1, total plasmid amount 100ug) The delivery solution (total volume is 10% of the animal body weight, Mirusbio-MIR 5240) was injected into the mice, and after injection, they were returned to the cage for observation for 30 min. Taking the day of modeling as day 0, serum was obtained at various time points after modeling (day 7 to day 35) for detecting SEAP expression levels.
双基因稳定转染***，包括Piggy-Bac辅助质粒和Piggy-Bac转座子质粒，其中Piggy-Bac辅助质粒提供Piggy-Bac转座酶；Piggy-Bac转座子质粒以Piggy-Bac转座子为基础，含有双基因表达元件，该双基因表达元件含有分泌型碱性磷酸酶基因(SEAP)、目的基因(ANGPTL3)，SEAP与ANGPTL3共同表达。Dual-gene stable transfection system, including Piggy-Bac auxiliary plasmid and Piggy-Bac transposon plasmid, in which Piggy-Bac auxiliary plasmid provides Piggy-Bac transposase; Piggy-Bac transposon plasmid uses Piggy-Bac transposon Based on it, it contains a dual-gene expression element, which contains the secreted alkaline phosphatase gene (SEAP) and the target gene (ANGPTL3). SEAP and ANGPTL3 are co-expressed.
检测SEAP表达Detection of SEAP expression
试剂盒(Phospha-LightTMSEAP报告基因检测***，Invitrogen，T1016)标准品以15mU/mL为初始浓度进行2倍稀释，获得7个浓度点。The standard of the kit (Phospha-Light TM SEAP Reporter Gene Detection System, Invitrogen, T1016) was diluted 2-fold with an initial concentration of 15mU/mL to obtain 7 concentration points.
将CSPD底物按1:20比例与反应缓冲液稀释液混合成反应液。用无DNA酶和RNA酶的蒸馏水将5×稀释缓冲液稀释至1×稀释缓冲液。在离心管中将血清与1×稀释缓冲液混合成样本稀释液，将样本稀释液在65℃孵育30min，然后冷却至室温。将50μL样本稀释液加入96孔板中，然后每孔加入50μL测定缓冲液，室温孵育5min。每孔加入50μL反应液，室温孵育20min，在酶标仪(Tecan，Infinite 200)上读取SEAP化学发光值。Mix CSPD substrate and reaction buffer diluent at a ratio of 1:20 to form a reaction solution. Dilute the 5× dilution buffer to 1× dilution buffer with DNase- and RNase-free distilled water. Mix the serum and 1× dilution buffer in a centrifuge tube to form a sample dilution, incubate the sample dilution at 65°C for 30 minutes, and then cool to room temperature. Add 50 μL of sample dilution into the 96-well plate, then add 50 μL of assay buffer to each well, and incubate at room temperature for 5 minutes. Add 50 μL reaction solution to each well, incubate at room temperature for 20 min, and read the SEAP chemiluminescence value on a microplate reader (Tecan, Infinite 200).
通过测量血清中的SEAP表达水平评价siRNA化合物抑制目的基因表达的效果。SEAP化学发光值越低，siRNA化合物抑制目的基因表达的效果越好。选择能够抑制SEAP表达水平的待测样品，作为核酸药物。The effect of siRNA compounds on inhibiting the expression of target genes was evaluated by measuring SEAP expression levels in serum. The lower the SEAP chemiluminescence value, the better the siRNA compound is at inhibiting the expression of the target gene. Select the test sample that can inhibit the expression level of SEAP as a nucleic acid drug.
造模后在第15天，根据表13向各小鼠给予单次皮下施用：200μl含有3mg/kg(mpk)RNAi试剂的生理盐水；或200μl不含RNAi试剂的生理盐水用作对照(vehicle)。HDI模型筛选结果如表14所示。On day 15 after modeling, each mouse was given a single subcutaneous administration according to Table 13: 200 μl of physiological saline containing 3 mg/kg (mpk) RNAi reagent; or 200 μl of physiological saline without RNAi reagent was used as a control (vehicle) . The HDI model screening results are shown in Table 14.
表13小鼠单次皮下施用RNAi试剂剂量

Table 13 Single subcutaneous administration of RNAi reagent dosage to mice

表14 HDI稳转质粒小鼠模型的实验结果
Table 14 Experimental results of HDI stably transfected plasmid mouse model
实施例10小鼠HDI模型活性筛选Example 10 Mouse HDI model activity screening
根据上文实施例9的方法，对表15的序列进行进一步活性筛选，结果如表16所示。According to the method of Example 9 above, the sequences in Table 15 were further screened for activity, and the results are shown in Table 16.
表15.siRNA序列
Table 15.siRNA sequences
表16.HDI稳转质粒小鼠模型的实验结果

Table 16. Experimental results of HDI stably transfected plasmid mouse model

实施例11小鼠HDI模型活性筛选Example 11 Mouse HDI model activity screening
根据实施例9的方法，对表17中的序列进行进一步活性筛选，结果如表18所示。According to the method of Example 9, the sequences in Table 17 were further screened for activity, and the results are shown in Table 18.
表17.siRNA的序列

Table 17. Sequence of siRNA

表18.HDI稳转质粒小鼠模型的实验结果
Table 18. Experimental results of HDI stably transfected plasmid mouse model

Claims (48)

  1. 一种用于抑制细胞中血管生成素样3(ANGPTL3)蛋白的表达的小干扰RNA(siRNA)，所述siRNA包含形成双链区的正义链和反义链，其中所述正义链和所述反义链的长度各自独立地为15-30个核苷酸，并且所述反义链包含SEQ ID NO:16-29中任一项所示的核苷酸序列的至少15个连续核苷酸的核苷酸序列。A small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) protein in cells, the siRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the The length of the antisense strands is each independently 15-30 nucleotides, and the antisense strands comprise at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 16-29 nucleotide sequence.
  2. 权利要求1的siRNA，其中所述正义链包含SEQ ID NO:1-14中任一项所示的核苷酸序列的至少15个连续的核苷酸的核苷酸序列。The siRNA of claim 1, wherein the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-14.
  3. 权利要求1-2中任一项的siRNA，其中所述双链区的长度为15-25个核苷酸对，优选17-23个核苷酸对，更优选19-21个核苷酸对。The siRNA of any one of claims 1-2, wherein the length of the double-stranded region is 15-25 nucleotide pairs, preferably 17-23 nucleotide pairs, more preferably 19-21 nucleotide pairs .
  4. 权利要求1-3中任一项的siRNA，其中所述反义链包含SEQ ID NO:16-29中任一项所示的核苷酸序列的至少16个连续核苷酸的核苷酸序列，至少17个连续核苷酸的核苷酸序列，至少18个连续核苷酸的核苷酸序列，至少19个连续核苷酸的核苷酸序列，至少20个连续核苷酸的核苷酸序列，优选所述反义链包含SEQ ID NO:16-29中任一项所示的核苷酸序列。The siRNA of any one of claims 1-3, wherein the antisense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO: 16-29 , a nucleotide sequence of at least 17 contiguous nucleotides, a nucleotide sequence of at least 18 contiguous nucleotides, a nucleotide sequence of at least 19 contiguous nucleotides, a nucleoside of at least 20 contiguous nucleotides Acid sequence, preferably the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 16-29.
  5. 权利要求1-4中任一项的siRNA，其中所述正义链包含与SEQ ID NO:1-14中所示的核苷酸序列任一项的至少16个连续核苷酸的核苷酸序列，至少17个连续核苷酸的核苷酸序列，至少18个连续核苷酸的核苷酸序列，优选所述反义链包含SEQ ID NO:1-14中任一项所示的核苷酸序列。The siRNA of any one of claims 1-4, wherein the sense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides with any one of the nucleotide sequences shown in SEQ ID NO: 1-14 , a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, preferably the antisense strand includes the nucleoside shown in any one of SEQ ID NO: 1-14 acid sequence.
  6. 权利要求1-5中任一项的siRNA，其中所述siRNA包含如表3所示的配对的正义链序列和反义链序列。The siRNA of any one of claims 1-5, wherein the siRNA comprises paired sense strand sequences and antisense strand sequences as shown in Table 3.
  7. 权利要求1-6中任一项的siRNA，其中所述正义链的基本上所有的核苷酸和所述反义链的基本上所有的核苷酸是修饰的核苷酸，或者所述正义链的所有的核苷酸和所述反义链的所有的核苷酸是修饰的核苷酸。The siRNA of any one of claims 1-6, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, or the sense strand All nucleotides of the strand and all nucleotides of the antisense strand are modified nucleotides.
  8. 权利要求7的siRNA，其中所述正义链和所述反义链各自独立地包含选自下组的一种或多种核苷酸修饰：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、无碱基核苷酸、反向无碱基脱氧核糖核苷酸、硫代磷酸酯核苷酸间键联修饰、乙烯基膦酸酯修饰的核苷酸、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、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、脱氧核糖核苷酸和STM1。The siRNA of claim 7, wherein the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2 '-Fluoromodified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, phosphorothioates Internucleotide linkage modification, vinylphosphonate-modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino Nucleotides, phosphoramidates, non-natural bases containing nucleotides, terminal nucleotides linked to cholesteryl derivatives or dodecyl dodecylamide groups, deoxyribonucleotides and STM1.
  9. 权利要求7的siRNA，其中所述正义链和所述反义链各自独立地包含选自下组的一种或多种核苷酸修饰：2'-O-甲基修饰的核苷酸、2'-氟代修饰的核苷酸、反向无碱基脱氧核糖核苷酸、硫代磷酸酯核苷酸间键联修饰、和STM1。The siRNA of claim 7, wherein the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2 '-fluorinated modified nucleotides, reverse abasic deoxyribonucleotides, phosphorothioate internucleotide linkage modifications, and STM1.
  10. 权利要求7所述的siRNA，其中， siRNA according to claim 7, wherein,
    (a)所述正义链包含(a) The chain of justice includes
    CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm(SEQ ID NO:31)，CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm(SEQ ID NO:31),
    所述反义链包含The antisense strand contains
    UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:61)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:61);
    (b)所述正义链包含(b) The chain of justice includes
    CmsAmsAmUmUmAmAfGfCfUmCmCmUmUmCmUmUmUmUm(SEQ ID NO:32)，CmsAmsAmUmUmAmAfGfCfUmCmCmUmUmCmUmUmUmUm(SEQ ID NO:32),
    所述反义链包含The antisense strand contains
    AmsAfsAmAfGmAfAmGfGmAfGmCfUmUfAmAfUmUfGmsUfsGm(SEQ ID NO:62)；AmsAfsAmAfGmAfAmGfGmAfGmCfUmUfAmAfUmUfGmsUfsGm(SEQ ID NO:62);
    (c)所述正义链包含(c) The chain of justice includes
    UmsAmsUmUmGmUmUfCfCfUmCmUmAmGmUmUmAmUmUm(SEQ ID NO:33)，UmsAmsUmUmGmUmUfCfCfUmCmUmAmGmUmUmAmUmUm(SEQ ID NO:33),
    所述反义链包含The antisense strand contains
    AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm(SEQ ID NO:63)；AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm(SEQ ID NO:63);
    (d)所述正义链包含(d) The chain of justice includes
    GmsUmsUmAmUmUmUfCfCfUmCmCmAmGmAmAmUmUmAm(SEQ ID NO:34)，GmsUmsUmAmUmUmUfCfCfUmCmCmAmGmAmAmUmUmAm(SEQ ID NO:34),
    所述反义链包含The antisense strand contains
    UmsAfsAmUfUmCfUmGfGmAfGmGfAmAfAmUfAmAfCmsUfsAm(SEQ ID NO:64)；UmsAfsAmUfUmCfUmGfGmAfGmGfAmAfAmUfAmAfCmsUfsAm(SEQ ID NO:64);
    (e)所述正义链包含(e) The chain of justice includes
    AmsGmsAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUm(SEQ ID NO:35)，AmsGmsAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUm(SEQ ID NO:35),
    所述反义链包含The antisense strand contains
    AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:65)；AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:65);
    (f)所述正义链包含(f) The chain of justice includes
    AmsUmsCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUm(SEQ ID NO:36)，AmsUmsCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUm(SEQ ID NO:36),
    所述反义链包含The antisense strand contains
    AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm(SEQ ID NO:66)；AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm(SEQ ID NO:66);
    (g)所述正义链包含(g) The justice chain includes
    GmsAmsGmCmCmAmAfAfAfUmCmAmAmGmAmUmUmUmAm(SEQ ID NO:37)，GmsAmsGmCmCmAmAfAfAfUmCmAmAmGmAmUmUmUmAm(SEQ ID NO:37),
    所述反义链包含The antisense strand contains
    UmsAfsAmAfUmCfUmUfGmAfUmUfUmUfGmGfCmUfCmsUfsGm(SEQ ID NO:67)；UmsAfsAmAfUmCfUmUfGmAfUmUfUmUfGmGfCmUfCmsUfsGm(SEQ ID NO:67);
    (h)所述正义链包含(h) The justice chain includes
    UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUm(SEQ ID NO:38)，UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUm(SEQ ID NO:38),
    所述反义链包含 The antisense strand contains
    AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:68)；AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:68);
    (i)所述正义链包含(i) The chain of justice includes
    AmsGmsAmGmCmAmAfCfUfAmAmCmUmAmAmCmUmUmAm(SEQ ID NO:39)，AmsGmsAmGmCmAmAfCfUfAmAmCmUmAmAmCmUmUmAm(SEQ ID NO:39),
    所述反义链包含The antisense strand contains
    UmsAfsAmGfUmUfAmGfUmUfAmGfUmUfGmCfUmCfUmsUfsCm(SEQ ID NO:69)；UmsAfsAmGfUmUfAmGfUmUfAmGfUmUfGmCfUmCfUmsUfsCm(SEQ ID NO:69);
    (j)所述正义链包含(j) The chain of justice includes
    GmsCmsAmAmCmUmAfAfCfUmAmAmCmUmUmAmAmUmUm(SEQ ID NO:40)，GmsCmsAmAmCmUmAfAfCfUmAmAmCmUmUmAmAmUmUm(SEQ ID NO:40),
    所述反义链包含The antisense strand contains
    AmsAfsUmUfAmAfGmUfUmAfGmUfUmAfGmUfUmGfCmsUfsCm(SEQ ID NO:70)；AmsAfsUmUfAmAfGmUfUmAfGmUfUmAfGmUfUmGfCmsUfsCm(SEQ ID NO:70);
    (k)所述正义链包含(k) The justice chain includes
    CmsUmsAmAmCmUmAfAfCfUmUmAmAmUmUmCmAmAmAm(SEQ ID NO:41)，CmsUmsAmAmCmUmAfAfCfUmUmAmAmUmUmCmAmAmAm(SEQ ID NO:41),
    所述反义链包含The antisense strand contains
    UmsUfsUmGfAmAfUmUfAmAfGmUfUmAfGmUfUmAfGmsUfsUm(SEQ ID NO:71)；UmsUfsUmGfAmAfUmUfAmAfGmUfUmAfGmUfUmAfGmsUfsUm(SEQ ID NO:71);
    (l)所述正义链包含(l) The justice chain includes
    CmsUmsAmAmCmUmUfAfAfUmUmCmAmAmAmAmUmCmAm(SEQ ID NO:42)，CmsUmsAmAmCmUmUfAfAfUmUmCmAmAmAmUmCmAm(SEQ ID NO:42),
    所述反义链包含The antisense strand contains
    UmsGfsAmUfUmUfUmGfAmAfUmUfAmAfGmUfUmAfGmsUfsUm(SEQ ID NO:72)；UmsGfsAmUfUmUfUmGfAmAfUmUfAmAfGmUfUmAfGmsUfsUm(SEQ ID NO:72);
    (m)所述正义链包含(m) The justice chain includes
    UmsAmsAmCmUmUmAfAfUfUmCmAmAmAmAmUmCmAmAm(SEQ ID NO:43)，UmsAmsAmCmUmUmAfAfUfUmCmAmAmAmAmUmCmAmAm(SEQ ID NO:43),
    所述反义链包含The antisense strand contains
    AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm(SEQ ID NO:73)；或AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm(SEQ ID NO:73); or
    (n)所述正义链包含(n) The justice chain includes
    GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAm(SEQ ID NO:44)，GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAm(SEQ ID NO:44),
    所述反义链包含The antisense strand contains
    UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:74)。UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm (SEQ ID NO:74).
  11. 权利要求7所述的siRNA，其中：The siRNA of claim 7, wherein:
    (a)所述正义链包含(a) The chain of justice includes
    UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmsUm(SEQ ID NO:131)，UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmsUm(SEQ ID NO:131),
    并且所述反义链包含 and the antisense strand contains
    AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:132)；AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:132);
    (b)所述正义链包含(b) The chain of justice includes
    GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmsAm(SEQ ID NO:133)，GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmsAm(SEQ ID NO:133),
    并且所述反义链包含and the antisense strand contains
    UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:134)；UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:134);
    (c)所述正义链包含(c) The chain of justice includes
    CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm(SEQ ID NO:135)，CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm(SEQ ID NO:135),
    并且所述反义链包含and the antisense strand contains
    UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:136)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:136);
    (d)所述正义链包含(d) The chain of justice includes
    STM1s-CmsUmUmGmAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUms-STM1(SEQ ID NO:137)，STM1s-CmsUmUmGmAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUms-STM1 (SEQ ID NO: 137),
    并且所述反义链包含and the antisense strand contains
    AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:138)；AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:138);
    (e)所述正义链包含(e) The chain of justice includes
    STM1s-AmsUmGmGmAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAms-STM1(SEQ ID NO:139)，STM1s-AmsUmGmGmAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAms-STM1 (SEQ ID NO: 139),
    并且所述反义链包含and the antisense strand contains
    UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:140)；UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:140);
    (f)所述正义链包含(f) The chain of justice includes
    STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-STM1(SEQ ID NO:141)，STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-STM1(SEQ ID NO:141),
    并且所述反义链包含and the antisense strand contains
    UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:142)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:142);
    (g)所述正义链包含(g) The justice chain includes
    STM1-STM1-STM1-STM1-
    UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1(SEQ ID NO:143)，UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1 (SEQ ID NO: 143),
    并且所述反义链包含and the antisense strand contains
    UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:144)；或UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:144); or
    (h)所述正义链包含(h) The justice chain includes
    IBs-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-IB(SEQ ID NO:145)，IBs-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-IB (SEQ ID NO: 145),
    并且所述反义链包含and the antisense strand contains
    UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:146)。 UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 146).
  12. 权利要求7所述的siRNA，其中：The siRNA of claim 7, wherein:
    (a)所述正义链包含(a) The chain of justice includes
    STM1-STM1-STM1-STM1-
    UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm-STM1-STM1(SEQ ID NO:147)，UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm-STM1-STM1 (SEQ ID NO: 147),
    并且所述反义链包含and the antisense strand contains
    UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:148)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:148);
    (b)所述正义链包含(b) The chain of justice includes
    STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1(SEQ ID NO:149)，STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1 (SEQ ID NO: 149),
    并且所述反义链包含and the antisense strand contains
    UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:150)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:150);
    (c)所述正义链包含(c) The chain of justice includes
    STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUms-STM1(SEQ ID NO:151)，STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUms-STM1 (SEQ ID NO: 151),
    并且所述反义链包含and the antisense strand contains
    AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:152)；AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:152);
    (d)所述正义链包含(d) The chain of justice includes
    STM1-STM1-STM1-STM1-
    CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmsUm-STM1-STM1(SEQ ID NO:153)，CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmsUm-STM1-STM1 (SEQ ID NO:153),
    并且所述反义链包含and the antisense strand contains
    AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:154)；AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:154);
    (e)所述正义链包含(e) The chain of justice includes
    STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUm-STM1-STM1(SEQ ID NO:155)，STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUm-STM1-STM1 (SEQ ID NO: 155),
    并且所述反义链包含and the antisense strand contains
    AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:156)；AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:156);
    (f)所述正义链包含(f) The chain of justice includes
    STM1s-UmsGmGmUmAmUmUmAmAfAfUfCmCmUmUmAmAmGmAmGmAms-STM1(SEQ ID NO:157)，STM1s-UmsGmGmUmAmUmUmAmAfAfUfCmCmUmUmAmAmGmAmGmAms-STM1 (SEQ ID NO: 157),
    并且所述反义链包含and the antisense strand contains
    UmsCfsUmCfUmUfAmAfGmGfAmUfUmUfAmAfUmAfCmsCfsAm(SEQ ID NO:158)；UmsCfsUmCfUmUfAmAfGmGfAmUfUmUfAmAfUmAfCmsCfsAm(SEQ ID NO:158);
    (g)所述正义链包含(g) The justice chain includes
    STM1s-CmsCmAmGmAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUms-STM1(SEQ ID NO:159)，STM1s-CmsCmAmGmAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUms-STM1 (SEQ ID NO: 159),
    并且所述反义链包含and the antisense strand contains
    AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:160)；或AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:160); or
    (h)所述正义链包含 (h) The justice chain includes
    STM1s-UmsGmAmUmCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUms-STM1(SEQ ID NO:161)，STM1s-UmsGmAmUmCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUms-STM1 (SEQ ID NO: 161),
    并且所述反义链包含and the antisense strand contains
    AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm(SEQ ID NO:162)。AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm (SEQ ID NO: 162).
  13. 权利要求1-12中任一项的siRNA，其中所述siRNA进一步与包含N-乙酰半乳糖胺的配体部分缀合，优选所述siRNA的正义链的3’端与所述配体部分缀合。The siRNA of any one of claims 1-12, wherein the siRNA is further conjugated with a ligand moiety comprising N-acetylgalactosamine, preferably the 3' end of the sense strand of the siRNA is conjugated with the ligand moiety. combine.
  14. 权利要求13的siRNA，其中所述配体部分包含式(X’)所示的缀合基团：
    The siRNA of claim 13, wherein the ligand moiety comprises a conjugation group represented by formula (X'):
    其中，in,
    表示与生物分子连接的位置； Indicates the location of attachment to biomolecules;
    Q独立地为H、 Q is independently H,
    其中L1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 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 -;
    L2为化学键或-CH2CH2C(O)-；L 2 is a chemical bond or -CH 2 CH 2 C(O)-;
    L3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
    L4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；L 4 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 -;
    其中a＝0、1、2或3；where a=0, 1, 2 or 3;
    b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
    c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
    d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
    L为化学键、-CH2O-或-NHC(O)-；L is a chemical bond, -CH 2 O- or -NHC(O)-;
    L’为化学键、-C(O)NH-、-NHC(O)-或-O(CH2CH2O)e-；L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
    其中e为1、2、3、4或5；where e is 1, 2, 3, 4 or 5;
    T为化学键、-CH2-、-C(O)-、-M-、-CH2-M-或-C(O)-M-；T is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
    其中M为 where M is
    R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
    或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
    其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
    m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  15. 权利要求14所述的siRNA，其中所述缀合基团如式(I’)所示：
    The siRNA of claim 14, wherein the conjugation group is represented by formula (I'):
    其中，in,
    表示与生物分子连接的位置； Indicates the location of attachment to biomolecules;
    Q独立地为H、 Q is independently H,
    其中L1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 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 -;
    L2为化学键或-CH2CH2C(O)-；L 2 is a chemical bond or -CH 2 CH 2 C(O)-;
    L3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
    L4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；L 4 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 -;
    其中a＝0、1、2或3；where a=0, 1, 2 or 3;
    b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
    c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
    d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
    L为-CH2O-或-NHC(O)-；L is -CH 2 O- or -NHC(O)-;
    L’为化学键、-C(O)NH-或-NHC(O)-；L’ is a chemical bond, -C(O)NH- or -NHC(O)-;
    R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
    或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
    其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
    m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  16. 权利要求15所述的siRNA，其中，siRNA according to claim 15, wherein,
    Q独立地为H或 Q is independently H or
    其中L1为-CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 is -CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;
    L2为-CH2CH2C(O)-； L 2 is -CH 2 CH 2 C(O)-;
    L3为-(NHCH2CH2)b-或-(NHCH2CH2CH2)b-；L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
    L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
    其中a＝0、1、2或3；where a=0, 1, 2 or 3;
    b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
    c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
    d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
    L为-CH2O-；L is -CH 2 O-;
    L’为化学键；L’ is a chemical bond;
    R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
    或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
    其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
    m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  17. 权利要求16的siRNA，其中所述缀合基团如式(I’-1)、式(I’-2)或式(I’-3)所示：
    The siRNA of claim 16, wherein the conjugation group is represented by formula (I'-1), formula (I'-2) or formula (I'-3):
    其中，in,
    表示与生物分子连接的位置； Indicates the location of attachment to biomolecules;
    Q为 Q is
    其中L1为-CH2O-或-NHC(O)-；Where L 1 is -CH 2 O- or -NHC(O)-;
    L2为-CH2CH2C(O)-；L 2 is -CH 2 CH 2 C(O)-;
    L3为-(NHCH2CH2)b-或-(NHCH2CH2CH2)b-；L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
    L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
    其中b＝1、2、3、4或5；Where b=1, 2, 3, 4 or 5;
    c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
    d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
    L为-CH2O-；L is -CH 2 O-;
    R’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；R' 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或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  18. 权利要求15的siRNA，其中， The siRNA of claim 15, wherein,
    Q独立地为H、 Q is independently H,
    其中L1为-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 is -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;
    L2为-CH2CH2C(O)-；L 2 is -CH 2 CH 2 C(O)-;
    L3为-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
    L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
    其中a＝0、1、2或3；where a=0, 1, 2 or 3;
    b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
    c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
    d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
    L为-CH2O-或-NHC(O)-；L is -CH 2 O- or -NHC(O)-;
    L’为化学键或-C(O)NH-；L’ is a chemical bond or -C(O)NH-;
    R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
    或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
    其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
    m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  19. 权利要求18的siRNA，其中所述缀合基团如式(II’-1)或式(II’-2)所示：
    The siRNA of claim 18, wherein the conjugation group is represented by formula (II'-1) or formula (II'-2):
    其中，in,
    表示与生物分子连接的位置； Indicates the location of attachment to biomolecules;
    Q独立地为 Q is independently
    其中L1为-CH2O-或-CH2O-CH2CH2O-；Where L 1 is -CH 2 O- or -CH 2 O-CH 2 CH 2 O-;
    L3为-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
    L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
    其中b＝1、2、3、4或5；Where b=1, 2, 3, 4 or 5;
    c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
    d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
    L为-NHC(O)-；L is -NHC(O)-;
    L’为化学键或-C(O)NH-；L’ is a chemical bond or -C(O)NH-;
    R’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；R' 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;
    m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  20. 权利要求15的siRNA，其中， The siRNA of claim 15, wherein,
    Q独立地为H、 Q is independently H,
    其中L1为-CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；where L 1 is -CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a - ;
    L2为化学键；L 2 is a chemical bond;
    L3为-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
    L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
    其中a＝0、1、2或3；where a=0, 1, 2 or 3;
    b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
    c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
    d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
    L为-CH2O-或-NHC(O)-；L is -CH 2 O- or -NHC(O)-;
    L’为化学键或-C(O)NH-；L’ is a chemical bond or -C(O)NH-;
    R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
    或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
    其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
    m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  21. 权利要求20的siRNA，其中所述缀合基团如式(II’-2)所示：
    The siRNA of claim 20, wherein the conjugation group is represented by formula (II'-2):
    其中，in,
    表示与生物分子连接的位置； Indicates the location of attachment to biomolecules;
    Q独立地为 Q is independently
    其中L1为-CH2-或-C(O)-；Where L 1 is -CH 2 - or -C(O)-;
    L3为-(NHCH2CH2)b-；L 3 is -(NHCH 2 CH 2 ) b -;
    L4为-(OCH2CH2)c-；L 4 is -(OCH 2 CH 2 ) c -;
    其中b＝1、2、3、4或5；Where b=1, 2, 3, 4 or 5;
    c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
    L为-CH2O-或-NHC(O)-；L is -CH 2 O- or -NHC(O)-;
    R’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；R' 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或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  22. 权利要求14的siRNA，其中：The siRNA of claim 14, wherein:
    Q独立地为H、 Q is independently H,
    其中L1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 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 -;
    L2为化学键或-CH2CH2C(O)-；L 2 is a chemical bond or -CH 2 CH 2 C(O)-;
    L3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
    L4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；L 4 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 -;
    其中a＝0、1、2或3；where a=0, 1, 2 or 3;
    b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
    c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
    d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
    L为化学键、-CH2O-或-NHC(O)-；L is a chemical bond, -CH 2 O- or -NHC(O)-;
    L’为化学键、-C(O)NH-、-NHC(O)-或-O(CH2CH2O)e-；L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
    其中e为1、2、3、4或5；where e is 1, 2, 3, 4 or 5;
    T为化学键、-CH2-、-M-、-CH2-M-或-C(O)-M-；T is a chemical bond, -CH 2 -, -M-, -CH 2 -M- or -C(O)-M-;
    其中M为 where M is
    R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
    或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
    其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
    m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  23. 权利要求22的siRNA，其中， The siRNA of claim 22, wherein,
    T为-M-、-CH2-M-或-C(O)-M-，其中M为 T is -M-, -CH 2 -M- or -C(O)-M-, where M is
  24. 权利要求22或23的siRNA，其中，The siRNA of claim 22 or 23, wherein,
    Q独立地为H或 Q is independently H or
    其中L1为-CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 is -CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;
    L2为-CH2CH2C(O)-；L 2 is -CH 2 CH 2 C(O)-;
    L3为-(NHCH2CH2)b-或-(NHCH2CH2CH2)b-；L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
    L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
    其中a＝0、1、2或3；where a=0, 1, 2 or 3;
    b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
    c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
    d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
    L为化学键或-CH2O-；L is a chemical bond or -CH 2 O-;
    L’为化学键或-O(CH2CH2O)e-；L' is a chemical bond or -O(CH 2 CH 2 O) e -;
    其中e为1、2、3、4或5；where e is 1, 2, 3, 4 or 5;
    R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
    或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
    其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
    m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    n＝0、1、2、3、4、5、6、7、8、9或10；n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    其中T如权利要求22或23中所定义。wherein T is as defined in claim 22 or 23.
  25. 权利要求24的siRNA，其中所述缀合基团如式(III’-1)、式(III’-2)或式(III’-3)所示：
    The siRNA of claim 24, wherein the conjugation group is represented by formula (III'-1), formula (III'-2) or formula (III'-3):
    其中，in,
    Q为 Q is
    其中L1为-CH2O-或-NHC(O)-；Where L 1 is -CH 2 O- or -NHC(O)-;
    L2为-CH2CH2C(O)-； L 2 is -CH 2 CH 2 C(O)-;
    L3为-(NHCH2CH2)b-或-(NHCH2CH2CH2)b-；L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
    L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
    其中b＝1、2、3、4或5；Where b=1, 2, 3, 4 or 5;
    c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
    d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
    L为化学键或-CH2O-；L is a chemical bond or -CH 2 O-;
    其中R’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；Wherein R' 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或10；n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    其中T如权利要求33或34中所定义。wherein T is as defined in claim 33 or 34.
  26. 权利要求22或23的siRNA，其中，The siRNA of claim 22 or 23, wherein,
    Q独立地为H、 Q is independently H,
    其中L1为-CH2-、-CH2O-或-C(O)-；Where L 1 is -CH 2 -, -CH 2 O- or -C(O)-;
    L2为化学键； L 2 is a chemical bond;
    L3为-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
    L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
    其中b＝1、2、3、4或5；Where b=1, 2, 3, 4 or 5;
    c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
    d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
    L为化学键或-NHC(O)-；L is a chemical bond or -NHC(O)-;
    L’为化学键；L’ is a chemical bond;
    R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
    或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
    其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
    m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    n＝0、1、2、3、4、5、6、7、8、9或10；n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    其中T如权利要求22或23中所定义。wherein T is as defined in claim 22 or 23.
  27. 权利要求22或23的siRNA，其中所述缀合基团如式(IV-1)或式(IV-2)所示：
    The siRNA of claim 22 or 23, wherein the conjugation group is represented by formula (IV-1) or formula (IV-2):
    其中，in,
    Q独立地为 Q is independently
    其中L1为-CH2-、-CH2O-或-C(O)-； Where L 1 is -CH 2 -, -CH 2 O- or -C(O)-;
    L3为-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
    L4为-(OCH2CH2)c-或-NHC(O)-(CH2)d-；L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
    其中b＝1、2、3、4或5；Where b=1, 2, 3, 4 or 5;
    c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
    d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
    L为化学键或-NHC(O)-；L is a chemical bond or -NHC(O)-;
    L’为化学键；L’ is a chemical bond;
    其中R’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；Wherein R' 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;
    m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    n＝0、1、2、3、4、5、6、7、8、9或10；n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    其中T如权利要求22或23中所定义。wherein T is as defined in claim 22 or 23.
  28. 权利要求14的siRNA，其中：The siRNA of claim 14, wherein:
    Q独立地为H、 Q is independently H,
    其中L1为化学键、-CH2-、-CH2CH2-、-C(O)-、-CH2O-、-CH2O-CH2CH2O-或-NHC(O)-(CH2NHC(O))a-；Where L 1 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 -;
    L2为化学键或-CH2CH2C(O)-；L 2 is a chemical bond or -CH 2 CH 2 C(O)-;
    L3为化学键、-(NHCH2CH2)b-、-(NHCH2CH2CH2)b-或-C(O)CH2-；L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
    L4为-(OCH2CH2)c-、-(OCH2CH2CH2)c-、-(OCH2CH2CH2CH2)c-、-(OCH2CH2CH2CH2CH2)c-或-NHC(O)-(CH2)d-；L 4 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 -;
    其中a＝0、1、2或3；where a=0, 1, 2 or 3;
    b＝1、2、3、4或5；b=1, 2, 3, 4 or 5;
    c＝1、2、3、4或5；c=1, 2, 3, 4 or 5;
    d＝1、2、3、4、5、6、7或8；d=1, 2, 3, 4, 5, 6, 7 or 8;
    L为化学键、-CH2O-或-NHC(O)-；L is a chemical bond, -CH 2 O- or -NHC(O)-;
    L’为-O(CH2CH2O)e-；L' is -O(CH 2 CH 2 O) e -;
    其中e为1、2、3、4或5；where e is 1, 2, 3, 4 or 5;
    T为化学键、-CH2-、-C(O)-、-M-、-CH2-M-或-C(O)-M-；T is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
    其中M为 where M is
    R1和R2一起形成-CH2CH2O-或-CH2CH(R)-O-，并且R3为H；R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
    或者R1和R3一起形成-C1-2亚烷基-，并且R2为H；Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
    其中R为-OR’、-CH2OR’或-CH2CH2OR’，其中R’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；Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
    m＝0、1、2、3、4、5、6、7、8、9或10；m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    n＝0、1、2、3、4、5、6、7、8、9或10。n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  29. 权利要求14的siRNA，其中所述缀合基团选自以下：



    The siRNA of claim 14, wherein said conjugation group is selected from the following:



  30. 权利要求14的siRNA，其中所述缀合基团选自以下：




    The siRNA of claim 14, wherein said conjugation group is selected from the following:




  31. 权利要求13-30中任一项的siRNA，其中所述配体靶向去唾液酸糖蛋白受体 (ASGPR)。The siRNA of any one of claims 13-30, wherein the ligand targets the asialoglycoprotein receptor (ASGPR).
  32. 权利要求13的siRNA，其中所述配体具有以下结构：
    The siRNA of claim 13, wherein said ligand has the following structure:
    其中表示通过磷酸酯基团或硫代磷酸酯基团与siRNA的正义链连接的位置。in Indicates the position of attachment to the sense strand of siRNA via a phosphate group or phosphorothioate group.
  33. 权利要求13的siRNA，其中所述配体具有以下结构：
    The siRNA of claim 13, wherein said ligand has the following structure:
    其中表示通过磷酸酯基团或硫代磷酸酯基团与siRNA的正义链连接的位置。in Indicates the position of attachment to the sense strand of siRNA via a phosphate group or phosphorothioate group.
  34. 权利要求13的siRNA，其中所述配体具有以下结构：
    The siRNA of claim 13, wherein said ligand has the following structure:
    其中表示通过磷酸酯基团或硫代磷酸酯基团与所述siRNA的正义链连接的位置。 in Indicates the position connected to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.
  35. 权利要求13的siRNA，其中The siRNA of claim 13, wherein
    (a)所述正义链包含(a) The chain of justice includes
    UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmsUms-GL6(SEQ ID NO:91)，UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmsUms-GL6 (SEQ ID NO:91),
    所述反义链包含The antisense strand contains
    AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:92)；AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:92);
    (b)所述正义链包含(b) The chain of justice includes
    GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmsAms-GL6(SEQ ID NO:93)，GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmsAms-GL6 (SEQ ID NO:93),
    所述反义链包含The antisense strand contains
    UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:94)；UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:94);
    (c)所述正义链包含(c) The chain of justice includes
    CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAms-GL6(SEQ ID NO:95)，CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAms-GL6 (SEQ ID NO:95),
    所述反义链包含The antisense strand contains
    UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:96)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:96);
    (d)所述正义链包含(d) The chain of justice includes
    STM1s-CmsUmUmGmAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUms-STM1s-GL6(SEQ ID NO:97)，STM1s-CmsUmUmGmAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUms-STM1s-GL6 (SEQ ID NO:97),
    所述反义链包含The antisense strand contains
    AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:98)；AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:98);
    (e)所述正义链包含(e) The chain of justice includes
    STM1s-AmsUmGmGmAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAms-STM1s-GL6(SEQ ID NO:99)，STM1s-AmsUmGmGmAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAms-STM1s-GL6 (SEQ ID NO:99),
    所述反义链包含The antisense strand contains
    UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:100)；UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:100);
    (f)所述正义链包含(f) The chain of justice includes
    STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-STM1s-GL6(SEQ ID NO:101)，STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-STM1s-GL6 (SEQ ID NO:101),
    所述反义链包含The antisense strand contains
    UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:102)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:102);
    (g)所述正义链包含(g) The justice chain includes
    STM1-STM1-STM1-STM1-
    UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1-GL6(SEQ ID NO:103)，UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1-GL6 (SEQ ID NO: 103),
    所述反义链包含The antisense strand contains
    UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:104)；或UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:104); or
    (h)所述正义链包含(h) The justice chain includes
    IBs-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-IBs- GL6(SEQ ID NO:105)，IBs-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms- GL6(SEQ ID NO:105),
    所述反义链包含The antisense strand contains
    UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:106)。UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 106).
  36. 权利要求13的siRNA，其中The siRNA of claim 13, wherein
    (a)所述正义链包含(a) The chain of justice includes
    STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-STM1s-GL6(SEQ ID NO:107)，STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-STM1s-GL6 (SEQ ID NO: 107),
    所述反义链包含The antisense strand contains
    UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:108)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:108);
    (b)所述正义链包含(b) The chain of justice includes
    STM1-STM1-STM1-STM1-
    UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm-STM1-STM1s-GL6(SEQ ID NO:109)，UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm-STM1-STM1s-GL6(SEQ ID NO:109),
    所述反义链包含The antisense strand contains
    UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:110)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:110);
    (c)所述正义链包含(c) The chain of justice includes
    STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1s-GL6(SEQ ID NO:111)，STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1s-GL6 (SEQ ID NO: 111),
    所述反义链包含The antisense strand contains
    UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:112)；UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:112);
    (d)所述正义链包含(d) The chain of justice includes
    STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUms-STM1s-GL6(SEQ ID NO:113)，STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUms-STM1s-GL6 (SEQ ID NO: 113),
    所述反义链包含The antisense strand contains
    AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:114)；AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:114);
    (e)所述正义链包含(e) The chain of justice includes
    STM1-STM1-STM1-STM1-
    CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmsUm-STM1-STM1s-GL6(SEQ ID NO:115)，CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmsUm-STM1-STM1s-GL6 (SEQ ID NO:115),
    所述反义链包含The antisense strand contains
    AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:116)；AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:116);
    (f)所述正义链包含(f) The chain of justice includes
    STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUm-STM1-STM1s-GL6(SEQ ID NO:117)，STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUm-STM1-STM1s-GL6 (SEQ ID NO: 117),
    所述反义链包含The antisense strand contains
    AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:118)；AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:118);
    (g)所述正义链包含(g) The justice chain includes
    STM1s-UmsGmGmUmAmUmUmAmAfAfUfCmCmUmUmAmAmGmAmGmAms- STM1s-GL6(SEQ ID NO:119)，STM1s-UmsGmGmUmAmUmUmAmAfAfUfCmCmUmUmAmAmGmAmGmAms- STM1s-GL6 (SEQ ID NO:119),
    所述反义链包含The antisense strand contains
    UmsCfsUmCfUmUfAmAfGmGfAmUfUmUfAmAfUmAfCmsCfsAm(SEQ ID NO:120)；UmsCfsUmCfUmUfAmAfGmGfAmUfUmUfAmAfUmAfCmsCfsAm(SEQ ID NO:120);
    (h)所述正义链包含(h) The justice chain includes
    STM1s-CmsCmAmGmAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUms-STM1s-GL6(SEQ ID NO:121)，STM1s-CmsCmAmGmAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUms-STM1s-GL6 (SEQ ID NO: 121),
    所述反义链包含The antisense strand contains
    AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:122)；或AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:122); or
    (i)所述正义链包含(i) The chain of justice includes
    STM1s-UmsGmAmUmCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUms-STM1s-GL6(SEQ ID NO:123)，STM1s-UmsGmAmUmCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUms-STM1s-GL6(SEQ ID NO:123),
    所述反义链包含The antisense strand contains
    AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm(SEQ ID NO:124)。AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm (SEQ ID NO: 124).
  37. 细胞，其含有如权利要求1-36中任一项所述的siRNA。Cells containing the siRNA of any one of claims 1-36.
  38. 药物组合物，其包含如权利要求1-36中任一项所述的siRNA、或如权利要求37所述的细胞，以及任选的药学上可接受的载剂或赋形剂。A pharmaceutical composition comprising the siRNA according to any one of claims 1 to 36, or the cell according to claim 37, and optionally a pharmaceutically acceptable carrier or excipient.
  39. 试剂盒，其包含如权利要求1-36中任一项所述的siRNA、如权利要求37所述的细胞、或权利要求38所述的药物组合物。A kit comprising the siRNA according to any one of claims 1-36, the cell according to claim 37, or the pharmaceutical composition according to claim 38.
  40. 治疗受试者中与ANGPTL3表达相关的疾病或症状的方法，所述方法包括向所述受试者施用权利要求1-36中任一项的siRNA、如权利要求37所述的细胞、或如权利要求38的药物组合物的步骤。A method of treating a disease or symptom associated with ANGPTL3 expression in a subject, the method comprising administering to the subject the siRNA of any one of claims 1-36, the cell of claim 37, or as 38. The step of the pharmaceutical composition of claim 38.
  41. 如权利要求40所述的方法，其中所述与ANGPTL3表达相关的疾病或症状是代谢疾病或心血管疾病。The method of claim 40, wherein the disease or condition associated with ANGPTL3 expression is a metabolic disease or a cardiovascular disease.
  42. 如权利要求41所述的方法，其中所述代谢疾病或心血管疾病是肥胖症、糖尿病、动脉粥样硬化、血脂障碍、冠心病、非酒精性脂肪肝疾病(NAFLD)、高脂肪酸血症或代谢综合征或其组合。The method of claim 41, wherein the metabolic disease or cardiovascular disease is obesity, diabetes, atherosclerosis, dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, or Metabolic syndrome or combination thereof.
  43. 如权利要求42所述的方法，其中所述血脂障碍是高脂血症。The method of claim 42, wherein said dyslipidemia is hyperlipidemia.
  44. 如权利要求43所述的方法，其中所述高脂血症是高胆固醇血症、高甘油三酯血症、或其组合。The method of claim 43, wherein the hyperlipidemia is hypercholesterolemia, hypertriglyceridemia, or a combination thereof.
  45. 如权利要求42所述的方法，其中所述NAFLD是肝脂肪变性或脂肪肝炎。The method of claim 42, wherein said NAFLD is hepatic steatosis or steatohepatitis.
  46. 如权利要求42所述的方法，其中所述糖尿病是2型糖尿病或具有血脂障碍的2型糖尿病。 The method of claim 42, wherein the diabetes is type 2 diabetes or type 2 diabetes with dyslipidemia.
  47. 一种减少受试者中ANGPTL3水平、LDL水平、apoC-III水平、甘油三酯水平、胆固醇水平、葡萄糖水平、脂肪垫重的方法，所述方法包括向所述受试者施用权利要求1-36中任一项的siRNA、如权利要求37所述的细胞、或如权利要求38的药物组合物的步骤。A method of reducing ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels, cholesterol levels, glucose levels, and fat pad weight in a subject, the method comprising administering to the subject claims 1- The siRNA of any one of 36, the cell of claim 37, or the pharmaceutical composition of claim 38.
  48. 如权利要求40-47中任一项所述的方法，其中向所述受试者施用所述siRNA、细胞或药物组合物包括皮下施用、局部施用或静脉内施用。 The method of any one of claims 40-47, wherein administering the siRNA, cell or pharmaceutical composition to the subject comprises subcutaneous administration, topical administration or intravenous administration.
PCT/CN2023/111739 2022-08-08 2023-08-08 Sirna molecule for regulating angptl3 gene activity WO2024032608A1 (en)

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Citations (6)

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US20130023579A1 (en) * 2010-01-08 2013-01-24 Isis Pharmaceuticals, Inc. Modulation of angiopoietin-like 3 expression
US20180087054A1 (en) * 2015-04-13 2018-03-29 Alnylam Pharmaceuticals, Inc. ANGIOPOIETIN-LIKE 3 (ANGPTL3) iRNA COMPOSITIONS AND METHODS OF USE THEREOF
WO2020099482A2 (en) * 2018-11-13 2020-05-22 Lipigon Pharmaceuticals Ab Angptl 3 oligonucleotides influencing the regulation of the fatty acid metabolism
CN111343994A (en) * 2017-09-14 2020-06-26 箭头药业股份有限公司 RNAi agents and compositions for inhibiting expression of angiopoietin-like 3(ANGPTL3) and methods of use
WO2022068923A1 (en) * 2020-09-30 2022-04-07 纳肽得(青岛)生物医药有限公司 Sirna of angiopoietin-like 3 (angptl3) and use thereof
WO2022079222A1 (en) * 2020-10-16 2022-04-21 Sanofi Novel rna compositions and methods for inhibiting angptl3

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130023579A1 (en) * 2010-01-08 2013-01-24 Isis Pharmaceuticals, Inc. Modulation of angiopoietin-like 3 expression
US20180087054A1 (en) * 2015-04-13 2018-03-29 Alnylam Pharmaceuticals, Inc. ANGIOPOIETIN-LIKE 3 (ANGPTL3) iRNA COMPOSITIONS AND METHODS OF USE THEREOF
CN111343994A (en) * 2017-09-14 2020-06-26 箭头药业股份有限公司 RNAi agents and compositions for inhibiting expression of angiopoietin-like 3(ANGPTL3) and methods of use
WO2020099482A2 (en) * 2018-11-13 2020-05-22 Lipigon Pharmaceuticals Ab Angptl 3 oligonucleotides influencing the regulation of the fatty acid metabolism
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