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WO2023109945A1 - 一种dsRNA、其制备方法及应用 - Google Patents

一种dsRNA、其制备方法及应用 Download PDF

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Publication number
WO2023109945A1
WO2023109945A1 PCT/CN2022/139585 CN2022139585W WO2023109945A1 WO 2023109945 A1 WO2023109945 A1 WO 2023109945A1 CN 2022139585 W CN2022139585 W CN 2022139585W WO 2023109945 A1 WO2023109945 A1 WO 2023109945A1
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Prior art keywords
nucleotide
dsrna
seq
antisense strand
group
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PCT/CN2022/139585
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English (en)
French (fr)
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李云飞
张瑱
林晓燕
侯哲
张建羽
耿俊
黄龙飞
周雅琴
吕珍珍
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上海拓界生物医药科技有限公司
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Priority to CN202280080982.2A priority Critical patent/CN118355121A/zh
Publication of WO2023109945A1 publication Critical patent/WO2023109945A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present disclosure relates to a dsRNA that can be targeted and delivered into cells to play the role of RNA interference.
  • the present disclosure also relates to preparation methods and applications of dsRNA.
  • RNA interference is an effective way to silence gene expression. According to statistics, more than 80% of the disease-related proteins in the human body cannot be targeted by current conventional small molecule drugs and biological macromolecular preparations, and are non-druggable proteins. Using RNA interference technology, we can design appropriate siRNA based on the mRNA encoding these proteins, specifically target the target mRNA and degrade the target mRNA, so as to inhibit the production of related proteins. Therefore, siRNA has a very important prospect of drug development. However, in order to achieve the RNA interference effect for therapeutic purposes in vivo, it is necessary to deliver siRNA molecules to specific cells in vivo.
  • Conjugate siRNA with a targeting ligand and use the targeting ligand to bind to the receptor on the cell membrane surface, thereby endocytosis into the cell, which is an effective drug delivery method.
  • asialoglycoprotein receptor is a receptor specifically expressed in hepatocytes, which has high abundance on the surface of hepatocytes and is characterized by rapid intracellular and extracellular transitions.
  • Monosaccharide and polysaccharide molecules such as galactose, galactosamine, and N-acetylgalactosamine have high affinity for ASGPR.
  • siRNA can be effectively delivered to hepatocytes using galactosamine molecular clusters (GalNAc), and GalNAc molecules designed as trivalent or tetravalent molecular clusters can significantly increase the concentration of monovalent or divalent GalNAc.
  • GalNAc galactosamine molecular clusters
  • the present disclosure provides a double-stranded ribonucleic acid (dsRNA) comprising siRNA and one or more ligands conjugated thereto, the siRNA comprising a sense strand and an antisense strand, the antisense strand At least one nucleotide position from the 2nd to the 8th position of the 5' end contains a chemical modification represented by formula (I), its tautomer or a pharmaceutically acceptable salt thereof:
  • dsRNA double-stranded ribonucleic acid
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • Q 1 is Q 2 is R 2 ;
  • Q 1 is R 2
  • Q 2 is
  • J 1 is H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is a base
  • the ligand is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof,
  • R 13 is a single or double bond, and when When it is a single bond, R 13 is independently CR 17 R 18 , NR 16 , O or S, when When it is a double bond, R 13 is independently CR 19 or N;
  • R 14 is independently CR 19 or N;
  • Ring A is cycloalkyl, heterocycloalkyl, aryl or heteroaryl, present or absent, and when ring A exists, R 15 is independently CR 19 or N, and when ring A does not exist, R 15 independently CR 17 R 18 , NR 16 or O;
  • R' and R" are independently hydrogen, deuterium, hydroxyl, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, the alkyl, alkoxy, cycloalkyl, Heterocycloalkyl, aryl or heteroaryl is optionally substituted by one or more substituents selected from halogen, hydroxy, oxo, nitro and cyano;
  • n1, p1 and q1 are independently 0, 1, 2, 3 or 4;
  • z1, z2, z3, z4, z5, z6, z7, z8 and z9 are independently an integer of 0-10;
  • r1 is an integer of 1-10.
  • R when X is NH-CO, R is not H.
  • the chemical modification represented by formula (I), its tautomer, or a pharmaceutically acceptable salt thereof is replaced with a 2'-methoxy modification.
  • the chemical modification shown in formula (I) is the chemical modification shown in formula (I-1):
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • Each J 1 and J 2 are independently H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine , 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine Pyrimidine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymus Pyrimidine, indole, 5-nitroindole and 3-nitropyrrole.
  • B is the same as the base of the antisense strand when the nucleotide at this position is not modified.
  • the chemical modification shown in formula (I) is the chemical modification shown in formula (I-2):
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • Each J 1 and J 2 are independently H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine , 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine Pyrimidine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymus Pyrimidine, indole, 5-nitroindole and 3-nitropyrrole.
  • B is the same as the base of the antisense strand when the nucleotide at this position is not modified.
  • each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are each independently H or C 1 -C 3 alkyl groups;
  • Each J 1 and J 2 are independently H or C 1 -C 3 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • each X is independently selected from CR 4 (R 4 '), S, NR 5 , and NH-CO, wherein R 4 , R 4 ', and R 5 are each independently H, methyl, ethyl radical, n-propyl or isopropyl;
  • Each J 1 and J 2 are independently H or methyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • J 1 and J 2 are independently H;
  • R 1 is selected from H, methyl and CH 2 OH;
  • R 2 is selected from H, OH, NH 2 , methyl and CH 2 OH;
  • R 3 is selected from H, OH, NH 2 , methyl and CH 2 OH;
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • J 1 and J 2 are independently H;
  • R 1 is selected from H, methyl and CH 2 OH;
  • R 2 is selected from H, methyl and CH 2 OH;
  • R 3 is selected from H, OH, NH 2 , methyl and CH 2 OH;
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Y is O or NH
  • Each X is independently selected from CR 4 (R 4 '), NR 5 and NH-CO, R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • R 3 is selected from H, OH, NH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy and (CH 2 ) p R 6 ;
  • Q 1 is Q 2 is R 2 ; or Q 1 is R 2 and Q 2 is
  • R 1 is selected from H, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy and (CH 2 ) q R 7 ;
  • J 1 is H or C 1 -C 6 alkyl
  • R 2 is selected from H, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy and (CH 2 ) r R 8 ;
  • R 1 and R 2 are directly connected to form a 3-6 membered ring;
  • B is a base
  • X is independently selected from CR 4 (R 4 ′) and NH—CO.
  • X is independently selected from CR 4 (R 4 ′).
  • R 3 is selected from H, C 1 -C 6 alkyl, and (CH 2 ) p R 6 .
  • R 3 is selected from H and C 1 -C 6 alkyl.
  • R 1 is selected from H, C 1 -C 6 alkyl, and (CH 2 ) q R 7 .
  • R 1 is selected from H and C 1 -C 6 alkyl.
  • R 2 is selected from H, OH, C 1 -C 6 alkyl, and (CH 2 ) r R 8 .
  • R 2 is selected from H, C 1 -C 6 alkyl, and (CH 2 ) r R 8 .
  • Y is O
  • Each X is independently selected from CR 4 (R 4 ') and NH-CO, R 4 and R 4 ' are independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • R 3 is selected from H, C 1 -C 6 alkyl and (CH 2 ) p R 6 ;
  • Q 1 is Q 2 is R 2 ; or Q 1 is R 2 and Q 2 is
  • R 1 is selected from H, C 1 -C 6 alkyl and (CH 2 ) q R 7 ;
  • J 1 is H or C 1 -C 6 alkyl
  • R 2 is selected from H, OH, C 1 -C 6 alkyl and (CH 2 ) r R 8 ;
  • R 1 and R 2 are directly connected to form a 5-6 membered ring;
  • B is a base
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Y is O
  • Each X is independently selected from CR 4 (R 4 '), R 4 and R 4 ' are independently H or C 1 -C 6 alkyl;
  • R 3 is selected from H and C 1 -C 6 alkyl
  • Q 1 is Q 2 is R 2 ; or Q 1 is R 2 and Q 2 is
  • R 1 is selected from H and C 1 -C 6 alkyl
  • J 1 is H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a 5-6 membered ring;
  • B is a base
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Y is O.
  • X is independently selected from CR 4 (R 4 '), NR 5 and NH-CO, R 4 , R 4 ', and R 5 are independently H, methyl, ethyl, n-propyl or isopropyl.
  • X is independently selected from NH—CO, CH 2 and NH.
  • X is independently selected from NH—CO and CH 2 .
  • X is CH2 .
  • J 2 is H or methyl. In some embodiments, J is H.
  • R is selected from H and methyl.
  • R 1 is selected from H and methyl.
  • R2 is selected from H, methyl, and CH2OH .
  • R1 and R2 are directly linked to form a 5-6 membered ring. In some embodiments, R and R are directly connected to form a 3-6 membered cycloalkyl. In some embodiments, R 1 and R 2 are directly connected to form cyclopentyl or cyclohexyl.
  • the chemical modification represented by the formula (I) is selected from any of the following structures:
  • B is selected from purine base, pyrimidine base, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the chemical modification represented by the formula (I) is selected from any of the following structures:
  • B is selected from purine base, pyrimidine base, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the chemical modification represented by the formula (I) is selected from any of the following structures:
  • B is selected from purine base, pyrimidine base, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the chemical modification represented by the formula (I) is selected from any of the following structures:
  • B is selected from purine base, pyrimidine base, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the nucleotide comprising the chemical modification represented by formula (I), its tautomer or a pharmaceutically acceptable salt thereof comprises the chemical modification represented by formula (I'), its tautomer Nucleotides of variants or pharmaceutically acceptable salts thereof,
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • Q 1' is Q 2' is R 2 ; or Q 1' is R 2 and Q 2' is
  • J 1 is H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is a base
  • M is O or S
  • R when X is NH-CO, R is not H.
  • the chemical modification shown in formula (I') is the chemical modification shown in formula (I'-1):
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • Each J 1 and J 2 are independently H or C 1 -C 6 alkyl
  • M is O or S
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diamino Purine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudo Cytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidines, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, Thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • B is identical to the base when the nucleotide at this position of the antisense strand is not modified.
  • the chemical modification shown in formula (I') is the chemical modification shown in formula (I'-2):
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • Each J 1 and J 2 are independently H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • M is O or S
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diamino Purine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudo Cytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidines, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, Thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • B is identical to the base when the nucleotide at this position of the antisense strand is not modified.
  • each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are each independently H or C 1 -C 3 alkyl groups;
  • Each J 1 and J 2 are independently H or C 1 -C 3 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • each X is independently selected from CR 4 (R 4 '), S, NR 5 , and NH-CO, wherein R 4 , R 4 ', and R 5 are each independently H, methyl, ethyl radical, n-propyl or isopropyl;
  • Each J 1 and J 2 are independently H or methyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • J 1 and J 2 are independently H;
  • R 1 is selected from H, methyl and CH 2 OH;
  • R 2 is selected from H, OH, NH 2 , methyl and CH 2 OH;
  • R 3 is selected from H, OH, NH 2 , methyl and CH 2 OH;
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • J 1 and J 2 are independently H;
  • R 1 is selected from H, methyl and CH 2 OH;
  • R 2 is selected from H, methyl and CH 2 OH;
  • R 3 is selected from H, OH, NH 2 , methyl and CH 2 OH;
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Y is O or NH
  • Each X is independently selected from CR 4 (R 4 '), NR 5 and NH-CO, R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • R 3 is selected from H, OH, NH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy and (CH 2 ) p R 6 ;
  • Q 1' is Q 2' is R 2 ; or Q 1' is R 2 and Q 2' is
  • R 1 is selected from H, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy and (CH 2 ) q R 7 ;
  • J 1 is H or C 1 -C 6 alkyl
  • R 2 is selected from H, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy and (CH 2 ) r R 8 ;
  • R 1 and R 2 are directly connected to form a 3-6 membered ring;
  • M is O or S
  • B is a base
  • X is independently selected from CR 4 (R 4 ′) and NH—CO.
  • X is independently selected from CR 4 (R 4 ′).
  • R 3 is selected from H, C 1 -C 6 alkyl, and (CH 2 ) p R 6 .
  • R 3 is selected from H and C 1 -C 6 alkyl.
  • R 1 is selected from H, C 1 -C 6 alkyl, and (CH 2 ) q R 7 .
  • R 1 is selected from H and C 1 -C 6 alkyl.
  • R 2 is selected from H, OH, C 1 -C 6 alkyl, and (CH 2 ) r R 8 .
  • R 2 is selected from H, C 1 -C 6 alkyl, and (CH 2 ) r R 8 .
  • Y is O
  • Each X is independently selected from CR 4 (R 4 ') and NH-CO, R 4 and R 4 ' are independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • R 3 is selected from H, C 1 -C 6 alkyl and (CH 2 ) p R 6 ;
  • Q 1' is Q 2' is R 2 ; or Q 1' is R 2 and Q 2' is
  • R 1 is selected from H, C 1 -C 6 alkyl and (CH 2 ) q R 7 ;
  • J 1 is H or C 1 -C 6 alkyl
  • R 2 is selected from H, OH, C 1 -C 6 alkyl and (CH 2 ) r R 8 ;
  • R 1 and R 2 are directly connected to form a 5-6 membered ring;
  • M is O or S
  • B is a base
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Y is O
  • Each X is independently selected from CR 4 (R 4 '), R 4 and R 4 ' are independently H or C 1 -C 6 alkyl;
  • R 3 is selected from H and C 1 -C 6 alkyl
  • Q 1' for Q 2' is R 2 ; or Q 1' is R 2 and Q 2' is
  • R 1 is selected from H and C 1 -C 6 alkyl
  • J 1 is H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a 5-6 membered ring;
  • M is O or S
  • B is a base
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Y is O.
  • X is independently selected from CR 4 (R 4 '), NR 5 and NH-CO, R 4 , R 4 ', and R 5 are independently H, methyl, ethyl, n-propyl or isopropyl.
  • X is independently selected from NH—CO, CH 2 and NH.
  • X is independently selected from NH—CO and CH 2 .
  • X is CH2 .
  • J 2 is H or methyl. In some embodiments, J is H.
  • R is selected from H and methyl.
  • R 1 is selected from H and methyl.
  • R2 is selected from H, methyl, and CH2OH .
  • R1 and R2 are directly linked to form a 5-6 membered ring. In some embodiments, R and R are directly connected to form a 3-6 membered cycloalkyl. In some embodiments, R 1 and R 2 are directly connected to form cyclopentyl or cyclohexyl.
  • the chemical modification represented by the formula (I') is selected from any of the following structures:
  • M is O or S
  • B is selected from purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the chemical modification represented by the formula (I') is selected from any of the following structures:
  • M is O or S
  • B is selected from purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the chemical modification represented by the formula (I') is selected from any of the following structures:
  • M is O or S
  • B is selected from purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the chemical modification represented by the formula (I') is selected from any of the following structures:
  • adenine in the structure is replaced by guanine, cytosine, uracil, or thymine.
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudourine Pyrimidine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5- Nitroindole and 3-nitropyrrole.
  • B is the same as the base of the antisense strand when the nucleotide at this position is unmodified.
  • the ligand is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof,
  • R 13 is CR 17 R 18 , NR 16 , O or S;
  • R 14 is CR 19 ;
  • R 15 is independently CR 17 R 18 , NR 16 or O;
  • R 16 to R 19 are independently hydrogen, deuterium or alkyl
  • m1, p1 and q1 are independently 0, 1, 2, 3 or 4;
  • z5, z6, z7, z8 and z9 are independently an integer of 0-10;
  • r1 is an integer of 1-10.
  • R 13 is CR 17 R 18 or O
  • R 14 is CR 19 ;
  • R 15 is independently CR 17 R 18 or O;
  • R 16 to R 19 are independently hydrogen or alkyl
  • n 1;
  • z8 and z9 are independently an integer of 0-10;
  • L 2 is -(CH 2 ) j15 -(OCH 2 CH 2 ) 1-4 -(CH 2 ) j16 - or
  • j15 and j16 are independently an integer of 0-4;
  • r1 is 3, 4, 5 or 6.
  • j11, j12, j13, and j14 are independently integers from 0-2 or 4-10. In some embodiments, j11, j12, j13, and j14 are independently 0, 1, 2, 6, 7, 8, 9, or 10.
  • L can be The definition of j12 is the same as that described in the previous scheme, wherein the terminal a1 is connected to B, and the terminal b1 is connected to R1 .
  • L can be Wherein, terminal a1 is connected to B1 , and terminal b1 is connected to R11 .
  • R 11 can be a chemical bond and R 12 can be NR 16 , and the definition of R 16 is the same as described in any of the previous schemes.
  • R 16 can be hydrogen or C 1-6 alkyl.
  • R 16 can be hydrogen, methyl, ethyl, propyl, or isopropyl.
  • R 16 can be hydrogen
  • R 17 and R 18 can be hydrogen.
  • R 19 can be hydrogen
  • ring A when present, can be a C 6-12 aryl.
  • ring A can be phenyl
  • m1 can be 0 or 1.
  • m1 can be 3.
  • n1 can be 0 or 1.
  • p1 and q1 are independently 0 or 1.
  • z1, z2, z3, z4, z5, z6, z7, z8, and z9 can independently be an integer from 0-4. In some embodiments, z1, z2, z3, z4, z5, z6, z7, z8, and z9 can be 0, 1, or 2 independently.
  • B 1 can be any organic compound
  • B 1 can be any organic compound
  • the definitions of z5, z6, z7, z8 and z9 are the same as those described in the previous scheme.
  • B 1 can be any organic compound
  • L 2 can be -(CH 2 ) j15 -(OCH 2 CH 2 ) 1-4 -(CH 2 ) j16 -, j15 and j16 are as defined in the previous scheme.
  • L2 can be In some embodiments, L2 can be Among them, the left side is connected with O atom, and the right side is connected with B1 .
  • L 2 can be a C 1 -C 12 alkyl chain.
  • L2 can be any organic compound
  • L2 can be In some embodiments, L2 can be In some embodiments, L2 can be In some embodiments, L2 can be In some embodiments, L2 can be Among them, the a3 end is connected to the O atom, and the b3 end is connected to the B1 .
  • L2 can be Among them, the a3 end is connected to the O atom, and the b3 end is connected to the B1 .
  • r1 can be 3, 4, 5 or 6. In some embodiments, r1 can be 3.
  • Q3 can be In some embodiments, Q3 can be Wherein, the definitions of R 13 , R 14 , R 15 and n1 are the same as those described in the previous scheme.
  • R 13 , R 14 , R 15 , p1 and q1 are the same as those described in the previous scheme.
  • R 13 , R 14 , R 15 , p1 and q1 are the same as those described in the previous scheme.
  • Can be The definitions of p1 and q1 are the same as those described in the previous scheme.
  • R 13 , R 14 , n1, p1 and q1 are the same as those described in the previous scheme.
  • R 13 , R 14 , n1, p1 and q1 are the same as those described in the previous scheme.
  • Can be The definitions of n1, p1 and q1 are the same as those described in the previous scheme.
  • the ligand can be any of the following structures or a pharmaceutically acceptable salt thereof,
  • the ligand can be any of the following structures or a pharmaceutically acceptable salt thereof,
  • the ligand can be the following structure or a pharmaceutically acceptable salt thereof,
  • the chemical modification represented by the formula (I) is B is selected from guanine, adenine, cytosine and uracil; and the ligand is any of the following structures or a pharmaceutically acceptable salt thereof,
  • the chemical modification represented by the formula (I) is B is selected from guanine, adenine, cytosine and uracil, and the ligand is any of the following structures or a pharmaceutically acceptable salt thereof,
  • the chemical modification represented by the formula (I) is B is selected from guanine, adenine, cytosine and uracil; and, the ligand is the following structure or a pharmaceutically acceptable salt thereof,
  • N in the above ligands can be replaced with N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine - the acetyl-galactosamine moiety.
  • the siRNA and the ligand are covalently or non-covalently linked.
  • the 3' end and/or the 5' end of the sense strand may be conjugated to the ligand.
  • the 3' end of the sense strand can be conjugated to the ligand.
  • the ligand is attached to the end of the siRNA via a phosphate group or a phosphorothioate group.
  • the ligand is attached to the end of the siRNA via a phosphodiester group or a phosphorothioate group.
  • the ligand is attached to the end of the siRNA via a phosphodiester group.
  • the ligand is indirectly linked to the end of the siRNA via a phosphate group or a phosphorothioate group.
  • the ligand is directly attached to the end of the siRNA via a phosphate group or a phosphorothioate group.
  • the ligand is directly linked to the 3' end of the sense strand of the siRNA via a phosphate group or a phosphorothioate group.
  • the phosphate group is a phosphate monoester group or a phosphodiester group. In some embodiments, the phosphate group is a phosphodiester group.
  • the phosphorothioate group is a phosphorothioate monoester group or a phosphorothioate diester group. In some embodiments, the phosphorothioate group is a phosphorothioate diester group.
  • a lipophilic group such as cholesterol can be introduced at the end of the siRNA sense strand.
  • the lipophilic group includes binding to siRNA with a covalent bond, such as introducing cholesterol , lipoprotein, vitamin E, etc., in order to facilitate the interaction with intracellular mRNA through the cell membrane composed of lipid bilayer.
  • siRNA can also be modified by non-covalent bonds, such as binding phospholipid molecules, polypeptides, and cationic polymers through hydrophobic bonds or ionic bonds to increase stability and biological activity.
  • the nucleotide comprising the chemical modification represented by formula (I), its tautomer or its pharmaceutically acceptable salt is located at the 5th and 6th positions from the 5' end of the antisense strand or number 7.
  • the nucleotide comprising the chemical modification represented by formula (I), its tautomer or a pharmaceutically acceptable salt thereof is located at the 7th position from the 5' end of the antisense strand.
  • B is selected from adenine, guanine , 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from adenine, guanine , 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from adenine, guanine , 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from adenine, guanine , 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • B is the same as the base of the antisense strand when the 5th nucleotide from its 5' end is not modified.
  • B is the same as the base when the 6th nucleotide from the 5' end of the antisense strand is unmodified.
  • B is the same as the base when the 7th nucleotide from the 5' end of the antisense strand is unmodified.
  • B is the same as the base when the 8th nucleotide from the 5' end of the antisense strand is not modified.
  • At least one additional nucleotide in the sense strand and/or antisense strand is a modified nucleotide at other positions other than the chemical modification shown in formula (I)
  • the Modified nucleotides are selected from the group consisting of: 2'-methoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'- Substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxynucleosides acid, 2'-deoxy-2'-fluoro-modified nucleotides, 3'-deoxy-thymidine nucleotides, isonucleotides, LNA, ENA, cET, UNA,
  • the sense strand contains three consecutive nucleotides with the same modification. In some embodiments, the three nucleotides with the same modification are 2'-fluoro-modified nucleotides.
  • the 2'- Fluoro-modified nucleotides according to the direction from the 5' end to the 3' end, the 2'- Fluoro-modified nucleotides.
  • the antisense strand is at least partially reverse complementary to the target sequence. In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 mismatches between the antisense strand and the target sequence; In some embodiments, the antisense strand is fully reverse complementary to the target sequence.
  • the sense strand is at least partially reverse complementary to the antisense strand to form a double-stranded region. In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 mismatch between the sense strand and the antisense strand. In some embodiments, the sense strand is fully reverse complementary to the antisense strand.
  • the sense and antisense strands each independently have 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides (eg, 19, 20, 21, 22, 23).
  • the sense strand and the antisense strand are the same or different in length, the sense strand is 19-23 nucleotides in length, and the antisense strand is 19-26 nucleotides in length acid.
  • the length ratio of the sense strand and the antisense strand in the dsRNA provided by the present disclosure can be 19/19, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26 , 20/19, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21 /24, 21/25, 21/26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22 , 23/23, 23/24, 23/25 or 23/26.
  • the sense and antisense strands have a length ratio of 19/21
  • the siRNA comprises one or two blunt ends.
  • each strand of the siRNA independently comprises an overhang formed by 1 to 2 unpaired nucleotides.
  • the siRNA comprises an overhang located 3' to the antisense strand.
  • the sense strand contains a nucleotide sequence (5'-3') represented by the following formula:
  • each X is independently Na or N b ; Na is a 2'-methoxy-modified nucleotide, and N b is a 2'-fluoro-modified nucleotide.
  • the sense strand contains a nucleotide sequence represented by the following formula:
  • N a is a 2'-methoxy-modified nucleotide
  • N b is a 2'-fluoro-modified nucleotide
  • the antisense strand contains a nucleotide sequence represented by the following formula:
  • N a ' is a 2'-methoxy-modified nucleotide
  • N b ' is a 2'-fluoro-modified nucleotide
  • W' means that it contains the chemical modification shown in formula (I), its interconversion Isomer-modified nucleotides or pharmaceutically acceptable salts thereof.
  • W' represents a nucleotide comprising a chemical modification represented by formula (I), a tautomer or a pharmaceutically acceptable salt thereof.
  • the chemical modification shown in formula (I) is selected from:
  • B is selected from guanine, adenine, cytosine and uracil. In some specific embodiments, B is the same as the base of the antisense strand when the 7th nucleotide from the 5' end is not modified.
  • the chemical modification shown in formula (I) is selected from:
  • M is O or S; wherein: B is selected from guanine, adenine, cytosine or uracil. In some specific embodiments, B is the same as the base of the antisense strand when the 7th nucleotide from the 5' end is not modified.
  • M is S. In some specific embodiments, M is O.
  • At least one phosphate group in the sense strand and/or the antisense strand is a phosphate group with a modification group that allows the siRNA to have an increased stability; in some embodiments, the phosphate group having a modifying group is a phosphorothioate group. In some embodiments, the phosphate group with a modifying group is a phosphorothioate group.
  • the phosphorothioate group is present in at least one of the following positions:
  • the 5' end of the sense strand starts between the first nucleotide and the second nucleotide
  • the 5' end of the antisense strand starts between the first nucleotide and the second nucleotide
  • the 3' end of the antisense strand starts between the second nucleotide and the third nucleotide.
  • the sense strand and/or antisense strand include multiple phosphorothioate groups present in:
  • the 3' end of the antisense strand starts between the second nucleotide and the third nucleotide.
  • the sense strand comprises a nucleotide sequence represented by the following formula:
  • Nm represents any nucleotide modified by 2'-methoxy, such as C, G, U, A modified by 2'-methoxy
  • Nf represents any nucleotide modified by 2'-fluoro, such as 2 '- Fluoro-modified C, G, U, A;
  • the antisense strand comprises a nucleotide sequence represented by the following formula:
  • Nm' represents any nucleotide modified by 2'-methoxy group, such as C, G, U, A modified by 2'-methoxy group
  • Nf' represents any nucleotide modified by 2'-fluoro group, For example, 2'-fluoro modified C, G, U, A;
  • W' represents a 2'-methoxy modified nucleotide or a modified nucleotide comprising a chemical modification represented by formula (I), its tautomer or a pharmaceutically acceptable salt thereof.
  • W' represents a 2'-methoxy modified nucleotide.
  • the chemical modification represented by formula (I) is selected from:
  • B is selected from guanine, adenine, cytosine and uracil; in some embodiments, B is the same as the base when the 7th nucleotide from the 5' end of the antisense strand is not modified .
  • the chemical modification shown in formula (I) is selected from:
  • M is O or S;
  • B is selected from guanine, adenine, cytosine or uracil;
  • the bases are the same when the nucleotides are not modified.
  • M is S. In some specific embodiments, M is O.
  • the siRNA is an siRNA targeting the factor XI (FXI) gene.
  • the nucleotide sequence of the sense strand of the siRNA comprises no more than 3 nucleotides different from the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3, and comprises at least 15 consecutive nucleotides (in some embodiments, at least 19), and/or,
  • the nucleotide sequence of the antisense strand comprises no more than 3 nucleotides different from any nucleotide sequence in SEQ ID NO: 4 or SEQ ID NO: 5, and comprises at least 19 consecutive nucleotides (some implementations program, at least 21).
  • the nucleotide sequence of the sense strand of the siRNA comprises any one of the nucleotide sequences of SEQ ID NO:1 to SEQ ID NO:3, and/or, the nucleosides of the antisense strand
  • the acid sequence comprises any one nucleotide sequence in SEQ ID NO:4 or SEQ ID NO:5.
  • the sense and antisense strands of the siRNA are selected from any of the following groups:
  • the nucleotide sequence of the sense strand comprises the nucleotide sequence of SEQ ID NO:3, and the nucleotide sequence of the antisense strand comprises the nucleotide sequence of SEQ ID NO:5;
  • the nucleotide sequence of the sense strand comprises the nucleotide sequence of SEQ ID NO:2, and the nucleotide sequence of the antisense strand comprises the nucleotide sequence of SEQ ID NO:5;
  • the nucleotide sequence of the sense strand comprises the nucleotide sequence of SEQ ID NO:1
  • the nucleotide sequence of the antisense strand comprises the nucleotide sequence of SEQ ID NO:4.
  • the sense and antisense strands of the siRNA are selected from any of the following groups:
  • the nucleotide sequence of the sense strand is SEQ ID NO:3, and the nucleotide sequence of the antisense strand is SEQ ID NO:5;
  • the nucleotide sequence of the sense strand is SEQ ID NO:2, and the nucleotide sequence of the antisense strand is SEQ ID NO:5;
  • the nucleotide sequence of the sense strand is SEQ ID NO:1
  • the nucleotide sequence of the antisense strand is SEQ ID NO:4.
  • SEQ ID NO: 1 is CUUGCAACAAAGACAUUUA
  • SEQ ID NO: 2 is UCAGGAUGAUUUUCUUAUU;
  • SEQ ID NO: 3 is UAAAUGUCUUUGUUGCAAGCG
  • SEQ ID NO:4 is UAUAAGAAAAUCAUCCUGAAA.
  • the sense strand of the dsRNA comprises any one of SEQ ID NO:6 to SEQ ID NO:10, and/or, the antisense strand comprises SEQ ID NO:14 or SEQ ID NO:15 any of the .
  • the sense and antisense strands of the dsRNA are selected from any of the following groups:
  • the sense strand comprises SEQ ID NO:10, and the antisense strand comprises SEQ ID NO:15;
  • the sense strand comprises SEQ ID NO:9, and the antisense strand comprises SEQ ID NO:15;
  • the sense strand comprises SEQ ID NO:7
  • the antisense strand comprises SEQ ID NO:14;
  • the sense strand comprises SEQ ID NO:8, and the antisense strand comprises SEQ ID NO:15;
  • the sense strand comprises SEQ ID NO:6
  • the antisense strand comprises SEQ ID NO:14.
  • the sense and antisense strands of the dsRNA are selected from any of the following groups:
  • Group 2 the sense strand is shown in SEQ ID NO:9, and the antisense strand is shown in SEQ ID NO:15;
  • the dsRNA is selected from any of the following groups:
  • Group 1 comprising or selected from the sense strand shown in SEQ ID NO: 10 and the antisense strand shown in SEQ ID NO: 15;
  • Group 2 comprising or selected from the sense strand shown in SEQ ID NO: 9 and the antisense strand shown in SEQ ID NO: 15;
  • Group 3 comprising or selected from the sense strand shown in SEQ ID NO: 7 and the antisense strand shown in SEQ ID NO: 14;
  • Group 4 comprising or selected from the sense strand shown in SEQ ID NO: 8 and the antisense strand shown in SEQ ID NO: 15;
  • Group 5 comprising or selected from the sense strand shown in SEQ ID NO:6 and the antisense strand shown in SEQ ID NO:14.
  • SEQ ID NO:10 is N-(SEQ ID NO:10).
  • SEQ ID NO: 14 is
  • SEQ ID NO:15 is N-(SEQ ID NO:15).
  • s means that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate groups
  • the dsRNA is selected from the following structures or pharmaceutically acceptable salts thereof:
  • the pharmaceutically acceptable salts can be conventional salts in the art, including but not limited to: sodium salts, potassium salts, ammonium salts, amine salts and the like.
  • the dsRNA is selected from TJR100407, TRD008003-1, TRD008002-1, TRD008003, or TRD008002.
  • the dsRNA is TRD008002, which has the following structure:
  • the dsRNA is TRD008002-1, which has the following structure:
  • the dsRNA is TRD008003, which has the following structure:
  • the dsRNA is TRD008003-1, which has the following structure:
  • the dsRNA is TJR100407, which has the following structure:
  • the present disclosure provides a pharmaceutical composition comprising the above-mentioned dsRNA.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.
  • Various delivery systems are known and can be used for the dsRNA or pharmaceutical compositions of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated intracellular Endocytosis, construction of nucleic acids as part of retroviral or other vectors.
  • the administration of the dsRNA or pharmaceutical composition of the present disclosure is conventional, and may be administered locally (e.g., by direct injection or implantation) or systemically, or orally, rectally, or gastrointestinally.
  • the parenteral route includes but not limited to subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, transdermal administration, inhalation administration (such as aerosol), mucosal administration (such as sublingual, nasal intracranial administration), intracranial administration, etc.
  • a dsRNA or pharmaceutical composition provided herein can be administered by injection, eg, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal injection.
  • the dsRNA or pharmaceutical compositions provided by the present disclosure can be packaged in kits.
  • the effective amount or effective dose of the dsRNA or the pharmaceutical composition is about 0.001 mg/kg body weight to about 200 mg/kg body weight, about 0.01 mg/kg body weight to about 100 mg/kg body weight, or about 0.5 mg/kg Body weight to about 50 mg/kg body weight.
  • dsRNA dsRNA
  • pharmaceutical composition is an effective amount or an effective dosage.
  • the present disclosure provides an application of the above-mentioned dsRNA or the above-mentioned pharmaceutical composition in the preparation of medicine.
  • the medicament is useful for preventing and/or treating thromboembolic complications.
  • the thromboembolic complication is selected from any one or combination of deep vein thrombosis, pulmonary embolism, myocardial infarction, or stroke.
  • the medicament can be used to prevent and/or treat diseases related to the expression of blood coagulation factor XI gene.
  • the disease associated with the expression of coagulation factor XI gene may be a thromboembolic complication.
  • the thromboembolic complication is selected from any one or combination of deep vein thrombosis, pulmonary embolism, myocardial infarction, or stroke.
  • the present disclosure provides a method for preventing and/or treating a disease, which comprises administering an effective amount or dose of the above-mentioned dsRNA or the above-mentioned pharmaceutical composition to a subject.
  • the disease may be a thromboembolic complication.
  • the thromboembolic complication is selected from any one or combination of deep vein thrombosis, pulmonary embolism, myocardial infarction, or stroke.
  • the disease may be a disease associated with the expression of the blood coagulation factor XI gene.
  • the disease associated with the expression of coagulation factor XI gene may be a thromboembolic complication.
  • the thromboembolic complication is selected from any one or combination of deep vein thrombosis, pulmonary embolism, myocardial infarction, or stroke.
  • the present disclosure provides a method for silencing a target gene FXI or its mRNA in a cell in vivo or in vitro, which includes the step of introducing the above-mentioned dsRNA or the above-mentioned pharmaceutical composition into the cell.
  • the present disclosure provides a method for inhibiting the expression of the target gene FXI or its mRNA, which comprises administering to a subject an effective amount or dose of the above-mentioned dsRNA or the above-mentioned pharmaceutical composition.
  • the dsRNA or pharmaceutical composition of the present disclosure can reduce the expression level of the target gene or its mRNA in cells, cell groups, tissues or subjects, including: administering to the subject a therapeutically effective amount of the dsRNA described herein or A pharmaceutical composition whereby expression of a target gene or mRNA thereof is inhibited in a subject.
  • the subject has been previously identified as having pathological upregulation of the target gene FXI or mRNA thereof in the targeted cell, cell population, tissue or subject.
  • the present disclosure provides a method for delivering oligonucleotides to the liver, which comprises administering to a subject an effective amount or dose of the above-mentioned dsRNA or the above-mentioned pharmaceutical composition.
  • RNAi RNA interference agent
  • dsRNA dsRNA or the above-mentioned pharmaceutical composition.
  • the present disclosure also provides a cell comprising the aforementioned dsRNA or the aforementioned pharmaceutical composition.
  • the present disclosure also provides a kit or kit, which comprises the above dsRNA or the above pharmaceutical composition.
  • the above-mentioned dsRNA or pharmaceutical composition when the above-mentioned dsRNA or pharmaceutical composition is contacted with cells expressing the target gene, as determined (for example, by psiCHECK activity screening, luciferase reporter gene assay, PCR or branched DNA-based methods, or protein-based method, such as immunofluorescence analysis, Western Blot or flow cytometry), the above-mentioned dsRNA or pharmaceutical composition will inhibit the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.
  • the remaining expression percentage of target gene mRNA caused by the above-mentioned dsRNA or pharmaceutical composition is not higher than 99%, not higher than 95%, not higher than 90%, Not higher than 85%, not higher than 80%, not higher than 75%, not higher than 70%, not higher than 65%, not higher than 60%, not higher than 55%, not higher than 50%, not higher More than 45%, not more than 40%, not more than 35%, not more than 30%, not more than 25%, not more than 20%, not more than 15%, or not more than 10%.
  • the dsRNA when the above-mentioned dsRNA or pharmaceutical composition is contacted with cells expressing the target gene, as determined (for example, by psiCHECK activity screening, luciferase reporter gene assay, PCR or branched DNA-based methods, or protein-based methods such as immunofluorescence analysis, Western Blot, or flow cytometry), the dsRNA reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, or at least 40%, while maintaining on-target activity , at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.
  • the dsRNA when the above-mentioned dsRNA or pharmaceutical composition is contacted with cells expressing the target gene, as determined (for example, by psiCHECK activity screening, luciferase reporter gene assay, PCR or branched DNA-based methods, or protein-based methods such as immunofluorescence analysis, Western Blot, or flow cytometry), the dsRNA reduces target activity by at most 20%, at most 19%, at most 15%, at most 10%, at most 5%, or by more than 1%. Off-target activity is reduced by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% %.
  • dsRNA when the above-mentioned dsRNA or pharmaceutical composition is contacted with cells expressing the target gene, as determined (for example, by psiCHECK activity screening, luciferase reporter gene assay, PCR or branched DNA-based methods, or protein-based methods such as immunofluorescence analysis, Western Blot or flow cytometry), dsRNA increases on-target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30% , at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, while reducing off-target activity by at least 20% %, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.
  • the present disclosure also provides a method for preparing dsRNA or a pharmaceutical composition, which includes: synthesizing the ligand, siRNA, and dsRNA described in the present disclosure.
  • Compounds of the present disclosure may exist in particular geometric or stereoisomeric forms. This disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and their racemic and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of this disclosure. Compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or reagents.
  • Optically active (R)- and (S)-isomers as well as D and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer.
  • a diastereoisomeric salt is formed with an appropriate optically active acid or base, and then a diastereomeric salt is formed by a conventional method known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally in combination with chemical derivatization methods (e.g. amines to amino groups formate).
  • the bond Indicates unassigned configuration, i.e. if chiral isomers exist in the chemical structure, the bond can be or both Two configurations.
  • the bond configuration is not specified, i.e. the key The configuration of can be E type or Z type, or contain both E and Z configurations.
  • tautomer or "tautomeric form” refers to structural isomers of different energies that can interconvert via a low energy barrier.
  • proton tautomers also known as prototropic tautomers
  • interconversions via migration of a proton such as keto-enol and imine-enamine, lactam-lactim isomerization .
  • An example of a lactam-lactim equilibrium is between A and B as shown below.
  • the present disclosure also includes certain isotopically labeled compounds of the disclosure that are identical to those described herein, but wherein one or more atoms are replaced by an atom of an atomic mass or mass number different from that normally found in nature.
  • isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.
  • deuterium when a position is specifically designated as deuterium (D), the position is understood to have an abundance of deuterium (i.e., at least 10 % deuterium incorporation).
  • exemplary compounds having a natural abundance greater than deuterium can be at least 1000 times more abundant deuterium, at least 2000 times more abundant deuterium, at least 3000 times more abundant deuterium, at least 4000 times more abundant deuterium, at least 5000 times more abundant deuterium, at least 6000 times more abundant deuterium, or more abundant deuterium.
  • the present disclosure also includes compounds of Formula (I), Formula (I'), Formula (II) in various deuterated forms. Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom.
  • deuterated starting materials can be used when preparing deuterated forms of compounds of formula (I), formula (I'), and formula (II), or they can be synthesized using conventional techniques using deuterated reagents, including But not limited to deuterated borane, trideuterioborane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide and deuterated methyl iodide, etc.
  • a target refers to the object to which a dsRNA of the present disclosure (or a conjugate thereof) is directed; a target may be a nucleic acid (gene, mRNA, etc.) or a protein (precursor, mature protein, isoform, variant, etc.). In the present disclosure, target especially refers to the factor XI gene or its expression product.
  • factor XI should be interpreted broadly, referring to the factor XI gene itself and its expression products in various forms in various stages, such as but not limited to gene amplification, replication, transcription, splicing, processing, translation, Molecules produced during modification, such as cDNA, mRNA, precursor proteins, mature proteins, natural variants, modified forms, and fragments thereof.
  • a Factor XI nucleic acid refers to any nucleic acid that encodes Factor XI.
  • a Factor XI nucleic acid includes a DNA sequence encoding Factor XI, e.g., a "Factor XI gene", an RNA sequence (including genomic DNA comprising introns and exons) transcribed from DNA encoding Factor XI and the mRNA sequence encoding Factor XI.
  • Vector XI gene is any one of the following sequences: GENBANK Accession No. NM_000128.4; GENBANK Accession No. NT_022792.17, 19598000 to 19624000 deletion; GENBANK Accession No. NM_028066.3, exons 1-15; GENBANK Accession No. XM_006253144 .4, XM_006253145.3; GENBANK accession numbers XM_015139652.2, XM_015139653.2; GENBANK accession numbers XM_005556483.3, XM_005556484.3; GENBANK accession numbers NW_001118167.1.
  • “Factor XI mRNA” refers to mRNA encoding Factor XI protein.
  • sense strand also known as SS, SS strand or sense strand
  • antisense strand also known as AS or AS strand
  • the "5' region” of the sense strand or the antisense strand can be used interchangeably.
  • the 2nd to 8th nucleotides at the 5' end of the antisense strand can also be replaced with the 2nd to 8th nucleotides at the 5' end of the antisense strand.
  • the "3' region”, "3' end” and “3' end” of the sense strand or the antisense strand can also be used interchangeably.
  • the term "differs from the nucleotide sequence of any of SEQ ID NO: 1 to SEQ ID NO: 3 by no more than 3 nucleotides and contains at least 15 "Contiguous nucleotides” is intended to mean that the siRNA sense strand described herein comprises at least 15 consecutive nucleotides of any sense strand of SEQ ID NO: 1 to SEQ ID NO: 3, or with SEQ ID NO: 1 At least 15 consecutive nucleotides to any sense strand of SEQ ID NO: 3 differ by no more than 3 nucleotide sequences (optionally, differ by no more than 2 nucleotide sequences; optionally, differ by 1 nucleotide sequence).
  • the siRNA sense strand described herein comprises at least 16 consecutive nucleotides of any sense strand of SEQ ID NO:1 to SEQ ID NO:3, or with SEQ ID NO:1 to SEQ ID NO: 3 At least 16 consecutive nucleotides of any sense strand differ by no more than 3 nucleotide sequences (optionally, differ by no more than 2 nucleotide sequences, optionally, differ by 1 nucleotide sequence) .
  • the term "differs from either antisense strand of SEQ ID NO: 4 or SEQ ID NO: 5 by no more than 3 nucleotide sequences and contains at least 15 consecutive core Nucleotide” is intended to mean that the siRNA antisense strand described herein comprises at least 15 consecutive nucleotides of any antisense strand in SEQ ID NO: 4 or SEQ ID NO: 5, or with SEQ ID NO: 4 or SEQ ID NO: At least 15 consecutive nucleotides of any antisense strand in ID NO:5 differ by no more than 3 nucleotide sequences (optionally, differ by no more than 2 nucleotide sequences, optionally, differ by 1 nucleus nucleotide sequence).
  • dsRNA refers to a double-stranded RNA molecule capable of RNA interference, comprising a sense strand and an antisense strand.
  • the term "inhibiting the expression of coagulation factor XI” includes inhibiting the expression of coagulation factor XI gene and variants (such as naturally occurring variants) or mutants of coagulation factor XI gene, inhibiting the expression of coagulation factor XI mRNA, And/or inhibit the expression of coagulation factor XI protein.
  • the factor XI gene can be a wild-type human factor XI gene, a mutant human factor XI gene, or a transgenic human factor XI gene in the context of a genetically manipulated cell, group of cells or organism.
  • Inhibition of Factor XI gene expression includes inhibition of Factor XI gene at any level, for example at least partial inhibition of Factor XI gene expression, such as inhibition of 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%.
  • at least partial inhibition of Factor XI gene expression such as inhibition of 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
  • Factor XI gene expression can be assessed based on the level of any variable associated with factor XI gene expression, such as factor XI mRNA levels or factor XI protein levels. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to control levels.
  • the control level can be any type of control level used in the art, such as a pre-dose baseline level or from a similar untreated or control (eg buffer only or inert control) treated subject, cell , or sample-determined levels.
  • the residual expression of mRNA can be used to characterize the inhibition degree of siRNA on target gene expression, such as the remaining expression of mRNA is not higher than 99%, not higher than 95%, not higher than 90%, not higher than 85%, not higher than More than 80%, not more than 75%, not more than 70%, not more than 65%, not more than 60%, not more than 55%, not more than 50%, not more than 45%, not more than 40%, not higher than 35%, not higher than 30%, not higher than 25%, not higher than 20%, not higher than 15%, or not higher than 10%.
  • G", “C”, “A”, “T” and “U” respectively represent nucleotides, which respectively contain guanine, cytosine, adenine, thymidine
  • the base with uracil indicates that the nucleotide adjacent to the downstream of the letter d is deoxyribonucleotide
  • the lowercase letter m indicates that the nucleotide adjacent to the upstream of the letter m is methoxy modified Nucleotide
  • the lowercase letter f indicates that the nucleotide adjacent to the upstream of the letter f is a fluorinated nucleotide
  • the lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are sulfur-substituted Phosphodiester linkage.
  • 2'-fluoro (2'-F) modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine
  • non-fluorine Modified nucleotides refer to nucleotides or nucleotide analogues in which the hydroxyl group at the 2' position of the ribose group of a nucleotide is replaced by a non-fluorine group.
  • 2'-methoxy (2'-OMe) modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is substituted with a methoxy group.
  • nucleotide difference exists between a nucleotide sequence and another nucleotide sequence, which means that the base type of the nucleotide at the same position has changed between the former and the latter, For example, when a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G or T, it is recognized as a difference between the two nucleotide sequences. There is a nucleotide difference at this position. In some embodiments, a nucleotide difference at a position is also considered to have occurred when an abasic nucleotide or its equivalent is substituted for the nucleotide at that position.
  • the terms "complementary” or “reverse complementary” are used interchangeably and have the meaning known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand interact with the other. The bases on the strand pair up in a complementary fashion.
  • the purine base adenine always pairs with the pyrimidine base thymine (or, in RNA, uracil); the purine base guanine always pairs with the pyrimidine base cytosine (G).
  • Each base pair consists of a purine and a pyrimidine.
  • mismatch in the art means that in a double-stranded nucleic acid, the bases at the corresponding positions are not paired in a complementary form.
  • chemical modification includes all alterations of nucleotides by chemical means, such as the addition or removal of chemical moieties, or the substitution of one chemical moiety for another.
  • base includes any known DNA and RNA base, base analogs such as purine or pyrimidine, which also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and Natural analogs. Base analogs can also be universal bases.
  • blunt end or blunt end are used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of an siRNA, ie, no nucleotide overhangs. In most cases, siRNAs with both blunt-ended ends will be double-stranded throughout their entire length.
  • the "compound”, “chemical modification”, “ligand”, “dsRNA”, “nucleic acid” and “RNAi” of the present disclosure can be independently salt, mixed salt or non-salt (such as free acid or in the form of the free base).
  • a salt or mixed salt it may be a pharmaceutically acceptable salt.
  • “Pharmaceutically acceptable salt” may be selected from inorganic salts or organic salts, and may also include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to a salt formed with an inorganic or organic acid that retains the biological effectiveness of the free base without other side effects.
  • Inorganic acid salts include but not limited to hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.
  • organic acid salts include but not limited to formate, acetate, 2,2-dichloroacetate , Trifluoroacetate, Propionate, Caproate, Caprylate, Caprate, Undecylenate, Glycolate, Gluconate, Lactate, Sebacate, Hexanoate glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate , cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, mesylate, benzenesulfonate, p
  • “Pharmaceutically acceptable base addition salt” refers to a salt formed with an inorganic base or an organic base that can maintain the biological effectiveness of the free acid without other side effects.
  • Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like.
  • Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts, preferably sodium salts.
  • Salts derived from organic bases include, but are not limited to, those of primary, secondary, and tertiary amines, substituted amines, including natural substituted amines, cyclic amines, and basic ion exchange resins , such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, bicyclic Hexylamine, Lysine, Arginine, Histidine, Caffeine, Procaine, Choline, Betaine, Ethylenediamine, Glucosamine, Methylglucamine, Theobromine, Purine, Piperazine, Piperazine Pyridine, N-ethylpiperidine, polyamine resin, etc.
  • Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine,
  • Alkyl refers to a saturated aliphatic hydrocarbon group, such as straight chain and branched chain groups (C 1 -C 30 alkyl groups) including 1 to 30 carbon atoms, and for example, alkyl groups containing 1 to 6 carbon atoms (C 1 -C 6 alkyl), another example is an alkyl (C 1 -C 3 alkyl) having 1 to 3 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl and various branched isomers, etc.
  • alkenyl refers to a hydrocarbon group containing at least one double bond.
  • alkenyl include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, or 2-butenyl, and various branched isomers thereof.
  • alkynyl refers to a hydrocarbon group containing at least one triple bond.
  • alkynyl include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, or 2-butynyl, and various branched isomers thereof.
  • alkoxy refers to -O-(alkyl), wherein alkyl is as defined above.
  • alkoxy include: methoxy, ethoxy, propoxy, butoxy.
  • Cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring contains 3 to 20 carbon atoms, preferably contains 3 to 6 carbon atoms, more preferably contains 5-6 carbon atom.
  • monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, etc.; multicyclic cycloalkyls include spiro Cycloalkyls of rings, parallel rings and bridged rings.
  • Heterocycloalkyl means a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2), but excluding ring portions of -OO-, -OS- or -SS-, the remaining ring atoms being carbon. Preferably it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably it contains 3 to 7 ring atoms.
  • Non-limiting examples of “heterocycloalkyl” include:
  • heterocycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring bonded to the parent structure is a heterocycloalkyl, non-limiting examples of which include:
  • Aryl means a 6 to 14 membered all-carbon monocyclic or fused polycyclic (that is, rings sharing adjacent pairs of carbon atoms) group, preferably 6 to 12 membered, having a conjugated pi-electron system, such as phenyl and naphthyl.
  • the aryl ring may be fused to a heteroaryl, heterocycloalkyl, or cycloalkyl ring, where the ring bonded to the parent structure is an aryl ring, non-limiting examples of which include:
  • Heteroaryl refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen.
  • the heteroaryl group is preferably 6 to 12 membered, more preferably 5 or 6 membered.
  • Non-limiting examples thereof include: imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl , Thiadiazole, pyrazinyl, triazolyl, indazolyl, benzimidazolyl, wait.
  • the heteroaryl ring may be fused to an aryl, heterocycloalkyl or cycloalkyl ring, wherein the ring bonded to the parent structure is a heteroaryl ring, non-limiting examples of which include:
  • hydroxyl refers to a -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • cyano refers to -CN.
  • amino refers to -NH2 .
  • nitro refers to -NO2 .
  • the "phosphate group” may be a phosphoric monoester group, a phosphoric diester group or a phosphoric triester group, preferably a phosphoric diester group.
  • phosphorothioate group refers to a phosphodiester group modified by replacing a non-bridging oxygen atom with a sulfur atom, which can be used (M is an S atom) are used interchangeably.
  • substitution means that one or more hydrogen atoms in a group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, are independently replaced by a corresponding number of substituents.
  • two (2) hydrogens on the atom are replaced.
  • group middle A moiety can be replaced by any group that enables linkage to adjacent nucleotides.
  • linked when referring to a link between two molecules, means that two molecules are connected by a covalent bond or that two molecules are associated by a non-covalent bond (for example, a hydrogen bond or an ionic bond), including direct connection, indirect connect.
  • a non-covalent bond for example, a hydrogen bond or an ionic bond
  • directly linked means that a first compound or group is linked to a second compound or group without any intervening atoms or groups of atoms.
  • directly linked means that a first compound or group is linked to a second compound or group through an intervening group, compound or molecule (eg, a linking group).
  • “Pharmaceutical composition” means a mixture containing one or more compounds described herein, or a physiologically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiologically acceptable carriers and excipients. Forming agent.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.
  • “Pharmaceutically acceptable excipients” include, but are not limited to, any adjuvants, carriers, glidants, sweeteners, diluents, preservatives, dyes/colorants that have been approved for use in humans or livestock animals agent, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier.
  • an “effective amount” or “effective dose” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to permit or facilitate diagnosis.
  • Effective amounts for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the method, route and dosage of administration, and the severity of side effects.
  • An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • subject As used herein, “subject”, “patient”, “subject” or “individual” are used interchangeably and include humans or non-human animals such as mammals such as humans or monkeys.
  • the dsRNA or siRNA provided in the present disclosure can be obtained by conventional preparation methods in the art (such as solid-phase synthesis and liquid-phase synthesis methods). Among them, solid-phase synthesis has commercialized customized services.
  • the modified nucleotide group can be introduced into the dsRNA or siRNA described in the present disclosure by using the correspondingly modified nucleoside monomer, the method for preparing the correspondingly modified nucleoside monomer and the modified nucleotide group Methods for introducing groups into dsRNA or siRNA are also well known to those skilled in the art.
  • Figure 1 shows the remaining mRNA expression levels in TTR of TRD002218 and TRD007205 on the 7th day after administration.
  • Figure 2 shows the remaining mRNA expression levels in TTR of TRD002218 and TRD007205 on the 28th day after administration.
  • Figure 3 shows the inhibition of humanized mouse (hF11) serum FXI protein expression, # means TRD0008003-1 compared with TJR100362 P ⁇ 0.05; & means TRD0008002-1 compared with TJR100364 P ⁇ 0.05; * means each group compared with TJR100364 Compared with the blank control, P ⁇ 0.05; **** indicates that each group is P ⁇ 0.0001 compared with the blank control; ns indicates that each group has no statistical difference compared with the blank control.
  • Embodiment 1 Preparation of chemical modification
  • Racemate compound 6 was passed through a chiral column (Daicel IE 250*4.6mm, 5 ⁇ m, A: n-hexane, B: ethanol) resolved to obtain 410mg 6A(-) and 435mg 6B(+).
  • compound 5 (6.8 g, 18.581 mmol) was dissolved in pyridine (80 mL), and TMSCl (14.250 mL, 111.489 mmol) was slowly added at 0° C., and stirred for 2 h. Then isobutyryl chloride (2.044 mL, 19.511 mmol) was added at 0°C and stirred at 25°C for 1 h. LCMS showed the reaction was complete.
  • reaction solution was extracted with ethyl acetate (200mL) and water (200mL), the organic phase was dried and spin-dried, and the mixed sample was purified by a forward column (PE:EtOAc was passed through the column, and the peak was at 84%) to obtain yellow oily compound 7 (12g).
  • compound 1 (5g, 23.1272mmol), compound 2 (6.76g, 46.254mmol) and triphenylphosphine (7.28g, 27.753mmol) were dissolved in 30mL of dioxane, and slowly dropped at 0°C DEAD (5.502 mL, 27.753 mmol) was added. After the dropwise addition was completed, the temperature of the reaction was slowly raised to 25° C. to continue the reaction for 1 h.
  • Embodiment 2 the synthesis of siRNA
  • siRNA is the same as the usual phosphoramidite solid-phase synthesis method.
  • the phosphoramidite monomer synthesized above is used to replace the original nucleotide of the parent sequence.
  • the synthesis process is briefly described as follows: On Dr. Oligo48 synthesizer (Biolytic), start with Universal CPG carrier, and link nucleoside phosphoramidite monomers one by one according to the synthesis procedure.
  • nucleoside phosphoramidite monomer at the 5' 7th position of the AS chain described above, other nucleoside monomer raw materials such as 2'-F RNA, 2'-O-methyl RNA and other nucleoside phosphoramidite monomers can be purchased From Shanghai Zhaowei or Suzhou Jima.
  • ETT 5-ethylthio-1H-tetrazole
  • PADS 0.22M PADS dissolved in acetonitrile and collidine
  • the oligoribonucleotide is cleaved from the solid support, and soaked at 50° C. for 16 hours using a 3:1 28% ammonia water and ethanol solution. Then centrifuged, the supernatant was transferred to another centrifuge tube, concentrated and evaporated to dryness, purified by C18 reverse chromatography, the mobile phase was 0.1M TEAA and acetonitrile, and 3% trifluoroacetic acid solution was used to remove DMTr.
  • the target oligonucleotides were collected and freeze-dried, identified as the target product by LC-MS, and then quantified by UV (260nm).
  • the obtained single-stranded oligonucleotides were annealed according to the equimolar ratio and complementary pairing, and finally the obtained dsRNA was dissolved in 1 ⁇ PBS and adjusted to the required concentration for the experiment.
  • Lipo 0.2 ⁇ L/well
  • plasmid 0.05 ⁇ L/well
  • Opti-MEM 10 ⁇ L/well.
  • Embodiment 4 Characterization of different chemical modifications
  • hmpNA from nucleotides synthesized from 2-hydroxymethyl-1,3-propanediol as the starting material
  • (+)hmpNA(A) is obtained by solid-phase synthesis of the nucleoside phosphoramidite monomer 1-1b in Example 1.1, and the absolute configuration is (S)-hmpNA(A);
  • (-)hmpNA(A) is obtained by solid-phase synthesis of nucleoside phosphoramidite monomer 1-1a in Example 1.1, and its absolute configuration is (R)-hmpNA(A);
  • (+)hmpNA(G) the absolute configuration is (S)-hmpNA(G);
  • (+)hmpNA(C) the absolute configuration is (S)-hmpNA(C);
  • (+)hmpNA(U) the absolute configuration is (R)-hmpNA(U);
  • TJ-NA067 The detection crystal is a colorless block (0.30 ⁇ 0.10 ⁇ 0.04mm3), which belongs to the monoclinic crystal system P21 space group.
  • the detection crystal is a colorless block (0.30 ⁇ 0.20 ⁇ 0.10mm3), belonging to the monoclinic crystal system P21 space group.
  • TJ-NA048 The detected crystal is colorless needle-shaped (0.30 ⁇ 0.04 ⁇ 0.04mm3), belonging to the monoclinic P1 space group.
  • GNA modification is known to be siRNA sequence dependent, so the inventors tested the experimental compounds of the present disclosure on a number of different sequences.
  • siRNA targeting different gene (ANGPTL3, HBV-S, HBV-X) mRNAs (sequences are shown in Table 4), using the compounds (+)hmpNA(A) and (-)hmpNA(A) of Example 1
  • the GNA (A) compound as a control modified the 7th position of the 5' end of the AS chain (the sequence is shown in Table 5), and then compared with the parent sequence on the target activity and off-target activity.
  • the starting material Compound 1 was purchased from Jiangsu Beida Pharmaceutical Technology Co., Ltd.
  • TMSCN (13.5mL, 101mmol) was added to a solution of compound 2 (13.0g, 33.6mmol) in DCM (300mL) at one time, followed by dropwise addition of TMSOTf (9.14mL, 50.5mmol) in DCM (30 mL) solution. The reaction solution was stirred at 20°C for 15 hours.
  • the compound NAG0024 (271 mg, 0.151 mmol) was dissolved in anhydrous THF (2 mL) and anhydrous DMF (4 mL), and 3A molecular sieves were added, followed by addition of compound 12 (100 mg, 0.151 mmol), HOBt ( 25mg, 0.181mmol), DCC (38mg, 0.181mmol) and DIEA (39mg, 0.302mmol).
  • the reaction solution was reacted at 45°C for 16h. After LC-MS showed that the reaction was complete, it was quenched with water and filtered. After the filtrate was concentrated, it was purified by C18 reverse phase column (H 2 O/MeCN) to obtain compound 13 (210 mg, yield 57%).
  • the compound NAG0052 is connected to the sequence through solid-phase synthesis, and after aminolysis, the structure of NAG0052 loses some functional groups to become the aforementioned NAG0052'.
  • the compound NAG0052 (157mg, 0.062mmol) containing a carboxylic acid group was dissolved in anhydrous DMF (3mL). After the substrate was completely dissolved, anhydrous acetonitrile (4mL), DIEA (0.03mL, 0.154mmol, 2.5eq ) and HBTU (35mg, 0.093mmol, 1.5eq). After the reaction solution was mixed evenly, macroporous amine methyl resin (476mg, blank load was 0.41mmol/g, target load was 0.1mmol/g) was added. The reaction solution was placed on a shaker (temperature: 25° C., rotation speed: 200 rpm) and shaken overnight. The reaction liquid was filtered, and the filter cake was washed successively with DCM and anhydrous acetonitrile, and the solid was collected and dried overnight in vacuum.
  • anhydrous acetonitrile 4mL
  • DIEA 0.03mL, 0.154mmol, 2.5eq
  • HBTU
  • the above solid was dispersed in anhydrous acetonitrile (5 mL), and pyridine (0.18 mL), DMAP (3 mg), NMI (0.12 mL) and CapB1 (2.68 mL) were added sequentially.
  • the reaction solution was placed on a shaker (temperature: 25° C., rotation speed: 200 rpm) and shaken for 2 h.
  • the reaction liquid was filtered, and the filter cake was washed with anhydrous acetonitrile, and the solid was collected and vacuum-dried overnight to obtain a resin with a carrier.
  • the loading capacity was determined to be 0.1 mmol/g.
  • NAG0052 For the NAG0052 that has been connected to the resin, use the resin as a starting point, and connect the nucleoside monomers one by one from the 3'-5' direction according to the sequence of nucleotide arrangement. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The operation is conventional in the art.
  • the prepared dsRNA had the sense and antisense strands shown in Table 8 and Table 9.
  • TRD002218 is used as a reference positive compound
  • Z represents siRNA
  • Example 8 Inhibition of dsRNA on target gene mRNA expression in vivo
  • mice Male 6-8 week-old C57BL/6 mice were randomly divided into 6 groups, 3 at each time point, and TRD007205 of the present disclosure, reference positive TRD002218 and PBS were given to each group of mice.
  • mice All animals were dosed according to their total body weight, and were administered once by subcutaneous injection.
  • the dose of dsRNA (based on the amount of siRNA) was 1 mg/kg, and the volume of administration was 5 mL/kg.
  • the mice were sacrificed, the liver was collected, and preserved with RNA later (Sigma Aldrich); then the liver tissue was homogenized with a tissue homogenizer, and then the tissue RNA extraction kit (Fanzhi Medical Technology, FG0412) was used The total RNA of the liver tissue was extracted according to the operation steps marked in the operation manual.
  • the total RNA was reverse-transcribed into cDNA, and the expression of TTR mRNA in liver tissue was detected by real-time fluorescent quantitative PCR method.
  • glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference gene, and Taqman probe primers for TTR and GAPDH were used to detect the mRNA expression levels of TTR and GAPDH, respectively.
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • TTR mRNA expression was calculated according to the following equation:
  • TTR mRNA expression [(TTR mRNA expression of test group/GAPDH mRNA expression of test group)/(TTR mRNA expression of control group/GAPDH mRNA expression of control group)] ⁇ 100%.
  • TRD007205 After 7 days and 28 days of administration, the in vivo inhibition efficiencies of dsRNA conjugated with different structures of the present disclosure on target gene mRNA expression are shown in Figure 1 and Figure 2, respectively. It can be seen from the results in Figure 1 that TRD007205 has a good effect on the expression inhibition of TTR mRNA 7 days after administration. It can be seen from Figure 2 that after 28 days of administration, TRD007205 has a better inhibitory effect on target gene mRNA expression than TRD002218.
  • each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. Specifically refer to the synthetic method of embodiment 2.
  • the prepared dsRNA had the sense and antisense strands shown in Table 13 and Table 14.
  • the naked sequences corresponding to the dsRNA are shown in Table 15.
  • NAG0052' The structure of NAG0052' is:
  • NAG1 The structure of NAG1 is
  • the target sequence corresponding to the siRNA was constructed with the human FXI gene and inserted into the psiCHECK-2 plasmid.
  • the plasmid contains Renilla luciferase gene and Firefly luciferase gene.
  • the target sequence of dsRNA is inserted into the 3'UTR region of the Renilla luciferase gene, and the activity of dsRNA on the target sequence can be detected by the detection of the expression of Renilla luciferase calibrated by firefly luciferase For reflection, Dual-Luciferase Reporter Assay System (Promega, E2940) was used for detection.
  • HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO 2 . 24 hours before transfection, HEK293A cells were seeded in a 96-well plate at a seeding density of 8 ⁇ 10 cells per well and 100 ⁇ L of medium per well.
  • dsRNA has a total of 11 concentration points, the final concentration of the highest concentration point is 20nM, 3-fold serial dilution, 20nM, 6.6667nM, 2.2222nM, 0.7407nM, 0.2469nM, 0.0823nM, 0.0274nM, 0.0091nM, 0.0030nM, 0.0010nM and 0.0003nM. 24h after transfection, the target level was detected by Dual-Luciferase Reporter Assay System (Promega, E2940). The on-target activities of the detected sequences are shown in Table 16.
  • N/A means: not applicable
  • TJR100407, TRD008003-1, TRD008002-1, TRD008003 and TRD008002 compounds had high level of on-target inhibitory activity against FXI gene in psiCHECK system.
  • Example 11 Inhibition of human FXI by dsRNA in primary human hepatocytes (PHH)
  • the dsRNA was screened for PHH activity in primary human hepatocytes (PHH) using 7 concentration gradients. Each dsRNA sample was transfected with a constant concentration of 20 nM, 5-fold serial dilution and 7 concentration points.
  • PHH cells (Novabiosis, nHP Hepatocytes) were cryopreserved in liquid nitrogen. 24 hours before transfection, the PHH cells were resuscitated and seeded in 96-well plates at a seeding density of 3 ⁇ 10 4 cells per well and 80 ⁇ L of medium per well.
  • Lipofectamine RNAi MAX (ThermoFisher, 13778150) to transfect dsRNA, and the gradient final concentration of dsRNA transfection is 10nM, 2nM, 0.4nM, 0.08nM, 0.016nM, 0.0032nM and 0.00064nM.
  • the high-throughput cell RNA extraction kit (Fanzhi, FG0417) was used to extract total cellular RNA, RNA reverse transcription experiment (Takara, 6210B) and quantitative real-time PCR detection (ThermoFisher, 4444557) to determine human FXI
  • ThermoFisher 4444557
  • ThermoFisher 4444557
  • the probe Q-PCR detection experiment is used, and the primer information is shown in Table 17.
  • Results are expressed as percent remaining mRNA expression of human FXI relative to control dsRNA-treated cells. See Table 18 for the IC50 results of the inhibition rate.
  • TRD008002 and TRD008003 compounds had a high level of on-target inhibitory activity against FXI gene in PHH cells.
  • Example 12 dsRNA inhibits FXI in cynomolgus monkey primary hepatocytes (PCH)
  • dsRNA with 7 concentration gradients of FXI was used to screen for reverse transfection activity.
  • Each dsRNA sample was transfected at a constant concentration of 10 nM, with 5-fold serial dilutions and 7 concentration points.
  • PCH cells were cryopreserved in liquid nitrogen, resuscitated before transfection and inoculated in 96-well plates at a seeding density of 3 ⁇ 104 cells per well and 90uL medium per well (Miaoshun Biology, HEP044, HEP024, HEP054 , HEP064).
  • RNAi MAX Lipofectamine RNAi MAX (ThermoFisher, 13778150) to transfect dsRNA, and the gradient final concentration of dsRNA transfection is 10nM, 2nM, 0.4nM, 0.08nM, 0.016nM, 0.0032nM and 0.00064nM.
  • a high-throughput cell RNA extraction kit to extract total cellular RNA, perform RNA reverse transcription experiments, and detect quantitative real-time PCR to measure the mRNA level of monkey FXI. Correction.
  • Results are expressed as percent remaining monkey FXI mRNA expression relative to control-treated cells. See Table 20 for the IC50 results of the inhibition rate. The results showed that TRD008002 and TRD008003 had good inhibitory activity against FXI in PCH cells.
  • Example 13 Determination of dsRNA in vivo (in vivo) activity in humanized mice (hF11)
  • the humanized mouse (hF11) used in this example was purchased from Saiye (Suzhou) Biotechnology Co., Ltd., product number: C001272, 40ul of serum was collected, anticoagulated with EDTAK2, and Human Coagulation Factor XI ELISA kit (Sigma company, batch number 0309J2350, article number RAB1385-1KT) to test the FXI protein content in the serum of mice from the above-mentioned groups.
  • serum FXI protein According to the content of serum FXI protein, they were evenly divided into groups, with 6 rats in each group (2 males and 4 females), a total of 5 groups, and the five groups were given to the control group (normal saline) by subcutaneous injection, and the dsRNA administration volume was 10 ⁇ l/g, TJR100362, TRD008003-1, TJR100364, and TRD008002-1 groups were administered at a dose of 10 mg/kg.

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Abstract

一种dsRNA、其制备方法及应用，以及包含该dsRNA的药物组合物、细胞或试剂盒，所述的dsRNA可以干扰FXI基因的表达，预防和/或治疗相关疾病。

Description

一种dsRNA、其制备方法及应用
本公开要求申请日为2021年12月16日的中国专利申请202111542323.X的优先权，本公开引用上述中国专利申请的全文。
技术领域
本公开涉及一种dsRNA，该dsRNA可以被靶向递送到细胞内，发挥RNA干扰的作用。本公开还涉及dsRNA的制备方法及应用。
背景技术
RNA干扰(RNAi)是一种有效的沉默基因表达的方式。据统计，在人体内的疾病相关蛋白中，大约超过80％的蛋白质不能被目前常规的小分子药物以及生物大分子制剂所靶向，属于不可成药蛋白。利用RNA干扰技术，可以根据编码这些蛋白的mRNA，设计合适的siRNA，特异性靶向目标mRNA并降解目标mRNA，从而达到抑制相关的蛋白生成。因此siRNA具有非常重要的药物开发前景。然而要实现体内的治疗目的RNA干扰效应，需要向体内特定的细胞递送siRNA分子。
采用靶向配体缀合siRNA，利用靶向配体与细胞膜表面的受体结合，从而内吞进入到细胞内，是一种有效的药物递送方式。例如，去唾液酸糖蛋白受体(ASGPR)是肝细胞特异性表达的受体，在肝细胞表面具有高丰度，胞内外转换快速的特点。半乳糖、半乳糖胺、N-乙酰半乳糖胺等单糖和多糖分子对ASGPR有高亲和性。文献报道(10.16476/j.pibb.2015.0028)使用氨基半乳糖分子簇(GalNAc)可以有效递送siRNA到肝细胞，GalNAc分子被设计成三价或四价的分子簇可以显著提高单价或二价的GalNAc分子靶向肝细胞的能力。
不同分子簇结构，和与siRNA之间不同的连接方式会明显的影响siRNA在体内的活性，更高的活性意味着更好的治疗效果，或更低的给药剂量，在同等药效下，更低的给药剂量也意味着更低的毒性反应。
发明内容
第一方面，本公开提供了一种双链核糖核酸(dsRNA)，其包含siRNA和一个或多个与其缀合的配体，所述siRNA包含有义链和反义链，所述反义链在其5’端起第2位至第8位中的至少一个核苷酸位置处包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐：
Figure PCTCN2022139585-appb-000001
其中：Y选自O、NH和S；
每个X独立地选自CR 4(R 4’)、S、NR 5和NH-CO，其中R 4、R 4’、R 5分别独立地为H或C 1-C 6烷基；
J 2为H或C 1-C 6烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
R 3选自H、OH、卤素、NH 2、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) pR 6；其中R 6选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，p＝1、2或3；
Q 1为
Figure PCTCN2022139585-appb-000002
Q 2为R 2；或者
Q 1为R 2，Q 2为
Figure PCTCN2022139585-appb-000003
其中：
R 1选自H、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基和(CH 2) qR 7；其中R 7选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，q＝1、2或3；
J 1为H或C 1-C 6烷基；
R 2选自H、OH、卤素、NH 2、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) rR 8；其中R 8选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，r＝1、2或3；
任选地，R 1和R 2直接相连成环；
B是碱基；
所述式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰不是
Figure PCTCN2022139585-appb-000004
所述配体为如式(II)所示化合物或其药学上可接受的盐，
Figure PCTCN2022139585-appb-000005
其中，L 1为C 1-C 30烷基链、或包含被一个或多个氧、硫、氮原子或C＝O间断的C 1-C 30烷基链；
R 11和R 12独立地为化学键、NR 16、C＝O或-OC(＝O)-；
Q 3为
Figure PCTCN2022139585-appb-000006
Figure PCTCN2022139585-appb-000007
为单键或双键，且当
Figure PCTCN2022139585-appb-000008
为单键时，R 13独立地为CR 17R 18、NR 16、O或S，当
Figure PCTCN2022139585-appb-000009
为双键时，R 13独立地为CR 19或N；
R 14独立地为CR 19或N；
环A为存在或不存在的环烷基、杂环烷基、芳基或杂芳基，且当环A存在时，R 15独立地为CR 19或N，当环A不存在时，R 15独立地为CR 17R 18、NR 16或O；
R 16和R 19独立地为氢、氘、烷基、烷氧基、环烷基、杂环烷基、芳基、杂芳基、SR'、S(＝O)R'、S(＝O) 2R'、S(＝O) 2NR'(R”)、NR'(R”)、C(＝O)R'、C(＝O)OR'或C(＝O)NR'(R”)，所述烷基、烷氧基、环烷基、杂环烷基、芳基或杂芳基任选被一个或多个选自卤素、羟基、氧代、硝基、氰基、C 1-6烷基、C 1-6烷氧基、C 3-7环烷基、3-12元杂环烷基、6-12元芳基、5-12元杂芳基、SR'、S(＝O)R'、S(＝O) 2R'、S(＝O) 2NR'(R”)、NR'(R”)、C(＝O)R'、C(＝O)OR'和C(＝O)NR'(R”)中的基团所取代；
R 17和R 18独立地为氢、氘、烷基、烷氧基、环烷基、杂环烷基、芳基、杂芳基、SR'、S(＝O)R'、S(＝O) 2R'、S(＝O) 2NR'(R”)、NR'(R”)、C(＝O)R'、C(＝O)OR'或C(＝O)NR'(R”)，所述烷基、烷氧基、环烷基、杂环烷基、芳基或杂芳基任选被一个或多个选自卤素、羟基、氧代、硝基、氰基、C 1-6烷基、C 1-6烷氧基、C 3-7环烷基、3-12元杂环烷基、6-12元芳基、5-12元杂芳基、SR'、S(＝O)R'、S(＝O) 2R'、S(＝O) 2NR'(R”)、NR'(R”)、C(＝O)R'、C(＝O)OR'和C(＝O)NR'(R”)中的基团所取代；
R'和R”独立地为氢、氘、羟基、烷基、烷氧基、环烷基、杂环烷基、芳基或杂芳基，所述烷基、烷氧基、环烷基、杂环烷基、芳基或杂芳基任选被一个或多个选自卤素、羟基、氧代、硝基和氰基中的取代基所取代；
m1、n1、p1和q1独立地为0、1、2、3或4；
B1为
Figure PCTCN2022139585-appb-000010
R b1、R b2、R b3、R b4、R b5、R b6和R b7独立地为-C(＝O)-、-NHC(＝O)-、-C(＝O)O-、-C(＝O)-(CH 2) z8-O-或-NHC(＝O)-(CH 2) z9-O-；
z1、z2、z3、z4、z5、z6、z7、z8和z9独立地为0-10的整数；
L 2为C 1-C 30烷基链、或包含被一个或多个氧、硫、氮原子或C＝O间断的C 1-C 30烷基链；
r1为1-10的整数。
在一些实施方案中，当X为NH-CO时，R 1不是H。
在一些实施方案中，以2’-甲氧基修饰替换式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐。
在一些实施方案中，式(I)所示的化学修饰是式(I-1)所示的化学修饰：
Figure PCTCN2022139585-appb-000011
其中：Y选自O、NH和S；
每个X独立地选自CR 4(R 4’)、S、NR 5和NH-CO，其中R 4、R 4’、R 5分别独立地为H或C 1-C 6烷基；
每个J 1、J 2分别独立地为H或C 1-C 6烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
R 3选自H、OH、卤素、NH 2、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) pR 6；其中R 6选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，p＝1、2或3；
R 1选自H、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基和(CH 2) qR 7；其中R 7选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，q＝1、2或3；
R 2选自H、OH、卤素、NH 2、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) rR 8；其中R 8选 自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，r＝1、2或3；
任选地，R 1和R 2直接相连成环；
B如式(I)中所定义。
在式(I-1)的一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在式(I-1)的一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在式(I-1)的一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在式(I-1)的一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，式(I)所示的化学修饰是式(I-2)所示的化学修饰：
Figure PCTCN2022139585-appb-000012
其中Y选自O、NH和S；
每个X独立地选自CR 4(R 4’)、S、NR 5和NH-CO，其中R 4、R 4’、R 5分别独立地为H或C 1-C 6烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
每个J 1、J 2分别独立地为H或C 1-C 6烷基；
R 3选自H、OH、卤素、NH 2、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) pR 6；其中R 6选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，p＝1、2或3；
R 1选自H、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基和(CH 2) qR 7；其中R 7选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，q＝1、2或3；
R 2选自H、C 1-C 6烷基、C 1-C 6烷氧基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) rR 8；其中R 8选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基；r＝1、2或3；
任选地，R 1和R 2直接相连成环；
B如式(I)中所定义。
在式(I-2)的一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在式(I-2)的一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在式(I-2)的一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在式(I-2)的一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，每个X独立地选自CR 4(R 4’)、S、NR 5和NH-CO，其中R 4、R 4’、R 5分别独立地为H或C 1-C 3烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
每个J 1、J 2分别独立地为H或C 1-C 3烷基；
R 3选自H、OH、卤素、NH 2、C 1-C 3烷基、C 1-C 3烷氧基、C 2-C 4烯基、C 2-C 4炔基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) pR 6；其中R 6选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，p＝1、2或3；
R 1选自H、C 1-C 3烷基、C 1-C 3烷氧基、C 2-C 4烯基、C 2-C 4炔基和(CH 2) qR 7；其中R 7选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 4烯基和C 2-C 4炔基，q＝1、2或3；
R 2选自H、OH、卤素、NH 2、C 1-C 3烷基、C 1-C 3烷氧基、C 2-C 4烯基、C 2-C 4炔基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) rR 8；其中R 8选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 4烯基和C 2-C 4炔基，r＝1、2或3；
任选地，R 1和R 2直接相连成环；
B如式(I)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌 呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，每个X独立地选自CR 4(R 4’)、S、NR 5和NH-CO，其中R 4、R 4’、R 5分别独立地为H、甲基、乙基、正丙基或异丙基；
n＝0、1或2；m＝0、1或2；s＝0或1；
每个J 1、J 2分别独立地为H或甲基；
R 3选自H、OH、F、Cl、NH 2、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-甲基氨基、-O-乙基氨基和(CH 2) pR 6；其中R 6选自OH、F、Cl、甲氧基、乙氧基、N 3、乙烯基、烯丙基、乙炔基和炔丙基，p＝1或2；
R 1选自H、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基和(CH 2) qR 7；其中R 7选自OH、F、Cl、甲氧基、乙氧基、N 3、乙烯基、烯丙基、乙炔基和炔丙基，q＝1或2；
R 2选自H、OH、F、Cl、NH 2、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-甲基氨基、-O-乙基氨基和(CH 2) rR 8；其中R 8选自OH、F、Cl、甲氧基、乙氧基、N 3、乙烯基、烯丙基、乙炔基和炔丙基，r＝1或2；
任选地，R 1和R 2直接相连成环；
B如式(I)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O或NH；每个X独立地选自NH-CO、CH 2和NH；
n＝0或1；m＝0或1；s＝0或1；
每个J 1、J 2分别独立地为H；
R 1选自H、甲基和CH 2OH；
R 2选自H、OH、NH 2、甲基和CH 2OH；
R 3选自H、OH、NH 2、甲基和CH 2OH；
任选地，R 1和R 2直接相连成环；
B如式(I)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O或NH；每个X独立地选自NH-CO、CH 2和NH；
n＝0或1；m＝0或1；s＝0或1；
每个J 1、J 2分别独立地为H；
R 1选自H、甲基和CH 2OH；
R 2选自H、甲基和CH 2OH；
R 3选自H、OH、NH 2、甲基和CH 2OH；
任选地，R 1和R 2直接相连成环；
B如式(I)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O或NH；
每个X独立地选自CR 4(R 4’)、NR 5和NH-CO，R 4、R 4’、R 5分别独立地为H或C 1-C 6烷基；
J 2为H或C 1-C 6烷基；
n＝0或1；m＝0或1；s＝0或1；
R 3选自H、OH、NH 2、C 1-C 6烷基、C 1-C 6烷氧基和(CH 2) pR 6；R 6选自OH、甲氧基和乙氧基，p＝1、2或3；
Q 1为
Figure PCTCN2022139585-appb-000013
Q 2为R 2；或者Q 1为R 2，Q 2为
Figure PCTCN2022139585-appb-000014
R 1选自H、OH、C 1-C 6烷基、C 1-C 6烷氧基和(CH 2) qR 7；R 7选自OH、甲氧基和乙氧基，q＝1、2或3；
J 1为H或C 1-C 6烷基；
R 2选自H、OH、C 1-C 6烷基、C 1-C 6烷氧基和(CH 2) rR 8；R 8选自OH、甲氧基和乙氧基，r＝1、2或3；
任选地，R 1和R 2直接相连成3-6元环；
B是碱基；
所述式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰不是
Figure PCTCN2022139585-appb-000015
在一些实施方案中，X独立地选自CR 4(R 4’)和NH-CO。
在一些实施方案中，X独立地选自CR 4(R 4’)。
在一些实施方案中，R 3选自H、C 1-C 6烷基和(CH 2) pR 6。
在一些实施方案中，R 3选自H和C 1-C 6烷基。
在一些实施方案中，R 1选自H、C 1-C 6烷基和(CH 2) qR 7。
在一些实施方案中，R 1选自H和C 1-C 6烷基。
在一些实施方案中，R 2选自H、OH、C 1-C 6烷基和(CH 2) rR 8。
在一些实施方案中，R 2选自H、C 1-C 6烷基和(CH 2) rR 8。
在一些实施方案中，Y为O；
每个X独立地选自CR 4(R 4’)和NH-CO，R 4和R 4’分别独立地为H或C 1-C 6烷基；
J 2为H或C 1-C 6烷基；
R 3选自H、C 1-C 6烷基和(CH 2) pR 6；R 6选自OH，p＝1、2或3；
Q 1为
Figure PCTCN2022139585-appb-000016
Q 2为R 2；或者Q 1为R 2，Q 2为
Figure PCTCN2022139585-appb-000017
R 1选自H、C 1-C 6烷基和(CH 2) qR 7；R 7选自OH，q＝1、2或3；
J 1为H或C 1-C 6烷基；
R 2选自H、OH、C 1-C 6烷基和(CH 2) rR 8；R 8选自OH，r＝1、2或3；
任选地，R 1和R 2直接相连成5-6元环；
B是碱基。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O；
每个X独立地选自CR 4(R 4’)，R 4和R 4’分别独立地为H或C 1-C 6烷基；
J 2为H；
R 3选自H和C 1-C 6烷基；
Q 1为
Figure PCTCN2022139585-appb-000018
Q 2为R 2；或者Q 1为R 2，Q 2为
Figure PCTCN2022139585-appb-000019
R 1选自H和C 1-C 6烷基；
J 1为H或C 1-C 6烷基；
R 2选自H、C 1-C 6烷基和(CH 2) rR 8；R 8选自OH，r＝1、2或3；
任选地，R 1和R 2直接相连成5-6元环；
B是碱基。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O。
在一些实施方案中，X独立地选自CR 4(R 4’)、NR 5和NH-CO，R 4、R 4’、R 5分别独立地为H、甲基、乙基、正丙基或异丙基。在一些实施方案中，X独立地选自NH-CO、CH 2和NH。在一些实施方案中，X独立地选自NH-CO和CH 2。在一些实施方案中，X为CH 2。
在一些实施方案中，J 2为H或甲基。在一些实施方案中，J 2为H。
在一些实施方案中，R 3选自H、OH、NH 2、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基和(CH 2) pR 6，R 6选自OH、甲氧基和乙氧基，p＝1或2。在一些实施方案中，R 3选自H、甲基、乙基、正丙基、异丙基和(CH 2) pR 6，R 6选自OH，p＝1或2。在一些实施方案中，R 3选自H和甲基。
在一些实施方案中，R 1选自H、OH、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基和(CH 2) qR 7，R 7选自OH，q＝1或2。在一些实施方案中，R 1选自H、甲基、乙基、正丙基、异丙基和(CH 2) qR 7，R 7选自OH，q＝1或2。在一些实施方案中，R 1选自H和甲基。
在一些实施方案中，R 2选自H、OH、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基和(CH 2) rR 8，R 8选自OH，r＝1或2。在一些实施方案中，R 2选自H、OH、甲基、乙基、正丙基、异丙基和(CH 2) rR 8，R 8选自OH，r＝1或2。在一些实施方案中，R 2选自H、甲基和CH 2OH。
在一些实施方案中，R 1和R 2直接相连成5-6元环。在一些实施方案中，R 1和R 2直接相连形成3-6元环烷基。在一些实施方案中，R 1和R 2直接相连形成环戊基或环己基。
在一些实施方案中，所述式(I)所示的化学修饰选自以下任一结构：
Figure PCTCN2022139585-appb-000020
其中：B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、 胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述式(I)所示的化学修饰选自以下任一结构：
Figure PCTCN2022139585-appb-000021
其中：B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述式(I)所示的化学修饰选自以下任一结构：
Figure PCTCN2022139585-appb-000022
Figure PCTCN2022139585-appb-000023
其中：B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯中。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述式(I)所示的化学修饰选自以下任一结构：
Figure PCTCN2022139585-appb-000024
其中：B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸是包含式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸，
Figure PCTCN2022139585-appb-000025
其中：Y选自O、NH和S；
每个X独立地选自CR 4(R 4’)、S、NR 5和NH-CO，其中R 4、R 4’、R 5分别独立地为H或C 1-C 6烷基；
J 2为H或C 1-C 6烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
R 3选自H、OH、卤素、NH 2、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) pR 6；其中R 6选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，p＝1、2或3；
Q 1’为
Figure PCTCN2022139585-appb-000026
Q 2’为R 2；或者Q 1’为R 2，Q 2’为
Figure PCTCN2022139585-appb-000027
其中：
R 1选自H、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基和(CH 2) qR 7；其中R 7选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，q＝1、2或3；
J 1为H或C 1-C 6烷基；
R 2选自H、OH、卤素、NH 2、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) rR 8；其中R 8选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，r＝1、2或3；
任选地，R 1和R 2直接相连成环；
B是碱基；
M为O或S；
所述式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐不是
Figure PCTCN2022139585-appb-000028
在一些实施方案中，当X为NH-CO时，R 1不是H。
在一些实施方案中，式(I’)所示的化学修饰是式(I’-1)所示的化学修饰：
Figure PCTCN2022139585-appb-000029
其中：Y选自O、NH和S；
每个X独立地选自CR 4(R 4’)、S、NR 5和NH-CO，其中R 4、R 4’、R 5分别独立地为H或C 1-C 6烷基；
每个J 1、J 2分别独立地为H或C 1-C 6烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
R 3选自H、OH、卤素、NH 2、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) pR 6；其中R 6选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，p＝1、2或3；
R 1选自H、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基和(CH 2) qR 7；其中R 7选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，q＝1、2或3；
R 2选自H、OH、卤素、NH 2、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) rR 8；其中R 8选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，r＝1、2或3；
M为O或S；
任选地，R 1和R 2直接相连成环；
B如式(I’)中所定义。
在式(I’-1)的一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在式(I’-1)的一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在式(I’-1)的一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在式(I’-1)的一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱 基相同。
在一些实施方案中，式(I’)所示的化学修饰是式(I’-2)所示的化学修饰：
Figure PCTCN2022139585-appb-000030
其中，Y选自O、NH和S；
每个X独立地选自CR 4(R 4’)、S、NR 5和NH-CO，其中R 4、R 4’、R 5分别独立地为H或C 1-C 6烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
每个J 1、J 2分别独立地为H或C 1-C 6烷基；
R 3选自H、OH、卤素、NH 2、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) pR 6；其中R 6选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，p＝1、2或3；
R 1选自H、C 1-C 6烷基、C 1-C 6烷氧基、C 2-C 6烯基、C 2-C 6炔基和(CH 2) qR 7；其中R 7选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，q＝1、2或3；
R 2选自H、C 1-C 6烷基、C 1-C 6烷氧基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) rR 8；其中R 8选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基；r＝1、2或3；
任选地，R 1和R 2直接相连成环；
M为O或S；
B如式(I’)中所定义。
在式(I’-2)的一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在式(I’-2)的一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在式(I’-2)的一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚 和3-硝基吡咯。
在式(I’-2)的一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，每个X独立地选自CR 4(R 4’)、S、NR 5和NH-CO，其中R 4、R 4’、R 5分别独立地为H或C 1-C 3烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
每个J 1、J 2分别独立地为H或C 1-C 3烷基；
R 3选自H、OH、卤素、NH 2、C 1-C 3烷基、C 1-C 3烷氧基、C 2-C 4烯基、C 2-C 4炔基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) pR 6；其中R 6选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 6烯基和C 2-C 6炔基，p＝1、2或3；
R 1选自H、C 1-C 3烷基、C 1-C 3烷氧基、C 2-C 4烯基、C 2-C 4炔基和(CH 2) qR 7；其中R 7选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 4烯基和C 2-C 4炔基，q＝1、2或3；
R 2选自H、OH、卤素、NH 2、C 1-C 3烷基、C 1-C 3烷氧基、C 2-C 4烯基、C 2-C 4炔基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-烷基氨基和(CH 2) rR 8；其中R 8选自OH、卤素、甲氧基、乙氧基、N 3、C 2-C 4烯基和C 2-C 4炔基，r＝1、2或3；
任选地，R 1和R 2直接相连成环；
B如式(I’)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，每个X独立地选自CR 4(R 4’)、S、NR 5和NH-CO，其中R 4、R 4’、R 5分别独立地为H、甲基、乙基、正丙基或异丙基；
n＝0、1或2；m＝0、1或2；s＝0或1；
每个J 1、J 2分别独立地为H或甲基；
R 3选自H、OH、F、Cl、NH 2、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-甲基氨基、-O-乙基氨基和(CH 2) pR 6；其中R 6选自OH、F、Cl、甲氧基、乙氧基、N 3、乙烯基、烯丙基、乙炔基和炔丙基，p＝1或2；
R 1选自H、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基和(CH 2) qR 7；其中R 7选自OH、F、Cl、甲氧基、乙氧基、N 3、乙烯基、烯丙基、乙炔基和炔丙基，q＝1或2；
R 2选自H、OH、F、Cl、NH 2、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基、S-CH 3、NCH 3(CH 3)、OCH 2CH 2OCH 3、-O-甲基氨基、-O-乙基氨基和(CH 2) rR 8；其中R 8选自OH、F、Cl、甲氧基、乙氧基、N 3、乙烯基、烯丙基、乙炔基和炔丙基，r＝1或2；
任选地，R 1和R 2直接相连成环；
B如式(I’)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O或NH；每个X独立地选自NH-CO、CH 2和NH；
n＝0或1；m＝0或1；s＝0或1；
每个J 1、J 2分别独立地为H；
R 1选自H、甲基和CH 2OH；
R 2选自H、OH、NH 2、甲基和CH 2OH；
R 3选自H、OH、NH 2、甲基和CH 2OH；
任选地，R 1和R 2直接相连成环；
B如式(I’)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O或NH；每个X独立地选自NH-CO、CH 2和NH；
n＝0或1；m＝0或1；s＝0或1；
每个J 1、J 2分别独立地为H；
R 1选自H、甲基和CH 2OH；
R 2选自H、甲基和CH 2OH；
R 3选自H、OH、NH 2、甲基和CH 2OH；
任选地，R 1和R 2直接相连成环；
B如式(I’)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O或NH；
每个X独立地选自CR 4(R 4’)、NR 5和NH-CO，R 4、R 4’、R 5分别独立地为H或C 1-C 6烷基；
J 2为H或C 1-C 6烷基；
n＝0或1；m＝0或1；s＝0或1；
R 3选自H、OH、NH 2、C 1-C 6烷基、C 1-C 6烷氧基和(CH 2) pR 6；R 6选自OH、甲氧基和乙氧基，p＝1、2或3；
Q 1’为
Figure PCTCN2022139585-appb-000031
Q 2’为R 2；或者Q 1’为R 2，Q 2’为
Figure PCTCN2022139585-appb-000032
R 1选自H、OH、C 1-C 6烷基、C 1-C 6烷氧基和(CH 2) qR 7；R 7选自OH、甲氧基和乙氧基，q＝1、2或3；
J 1为H或C 1-C 6烷基；
R 2选自H、OH、C 1-C 6烷基、C 1-C 6烷氧基和(CH 2) rR 8；R 8选自OH、甲氧基 和乙氧基，r＝1、2或3；
任选地，R 1和R 2直接相连成3-6元环；
M为O或S；
B是碱基；
所述式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐不是
Figure PCTCN2022139585-appb-000033
在一些实施方案中，X独立地选自CR 4(R 4’)和NH-CO。
在一些实施方案中，X独立地选自CR 4(R 4’)。
在一些实施方案中，R 3选自H、C 1-C 6烷基和(CH 2) pR 6。
在一些实施方案中，R 3选自H和C 1-C 6烷基。
在一些实施方案中，R 1选自H、C 1-C 6烷基和(CH 2) qR 7。
在一些实施方案中，R 1选自H和C 1-C 6烷基。
在一些实施方案中，R 2选自H、OH、C 1-C 6烷基和(CH 2) rR 8。
在一些实施方案中，R 2选自H、C 1-C 6烷基和(CH 2) rR 8。
在一些实施方案中，Y为O；
每个X独立地选自CR 4(R 4’)和NH-CO，R 4和R 4’分别独立地为H或C 1-C 6烷基；
J 2为H或C 1-C 6烷基；
R 3选自H、C 1-C 6烷基和(CH 2) pR 6；R 6选自OH，p＝1、2或3；
Q 1’为
Figure PCTCN2022139585-appb-000034
Q 2’为R 2；或者Q 1’为R 2，Q 2’为
Figure PCTCN2022139585-appb-000035
R 1选自H、C 1-C 6烷基和(CH 2) qR 7；R 7选自OH，q＝1、2或3；
J 1为H或C 1-C 6烷基；
R 2选自H、OH、C 1-C 6烷基和(CH 2) rR 8；R 8选自OH，r＝1、2或3；
任选地，R 1和R 2直接相连成5-6元环；
M为O或S；
B是碱基。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O；
每个X独立地选自CR 4(R 4’)，R 4和R 4’分别独立地为H或C 1-C 6烷基；
J 2为H；
R 3选自H和C 1-C 6烷基；
Q 1’为
Figure PCTCN2022139585-appb-000036
Q 2’为R 2；或者Q 1’为R 2，Q 2’为
Figure PCTCN2022139585-appb-000037
R 1选自H和C 1-C 6烷基；
J 1为H或C 1-C 6烷基；
R 2选自H、C 1-C 6烷基和(CH 2) rR 8；R 8选自OH，r＝1、2或3；
任选地，R 1和R 2直接相连成5-6元环；
M为O或S；
B是碱基。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O。
在一些实施方案中，X独立地选自CR 4(R 4’)、NR 5和NH-CO，R 4、R 4’、R 5分别独立地为H、甲基、乙基、正丙基或异丙基。在一些实施方案中，X独立地选自NH-CO、CH 2和NH。在一些实施方案中，X独立地选自NH-CO和CH 2。在一些实施方案中，X为CH 2。
在一些实施方案中，J 2为H或甲基。在一些实施方案中，J 2为H。
在一些实施方案中，R 3选自H、OH、NH 2、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基和(CH 2) pR 6，R 6选自OH、甲氧基和乙氧基，p＝1或2。在一些实施方案中，R 3选自H、甲基、乙基、正丙基、异丙基和(CH 2) pR 6，R 6选自OH，p＝1或2。在一些实施方案中，R 3选自H和甲基。
在一些实施方案中，R 1选自H、OH、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基和(CH 2) qR 7，R 7选自OH，q＝1或2。在一些实施方案中，R 1选自H、甲基、乙基、正丙基、异丙基和(CH 2) qR 7，R 7选自OH，q＝1或2。在一些实施方案中，R 1选自H和甲基。
在一些实施方案中，R 2选自H、OH、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基和(CH 2) rR 8，R 8选自OH，r＝1或2。在一些实施方案中，R 2选自H、OH、甲基、乙基、正丙基、异丙基和(CH 2) rR 8，R 8选自OH，r＝1或2。在一些实施方案中，R 2选自H、甲基和CH 2OH。
在一些实施方案中，R 1和R 2直接相连成5-6元环。在一些实施方案中，R 1和R 2直接相连形成3-6元环烷基。在一些实施方案中，R 1和R 2直接相连形成环戊基或环己基。
在一些实施方案中，所述式(I’)所示的化学修饰选自以下任一结构：
Figure PCTCN2022139585-appb-000038
Figure PCTCN2022139585-appb-000039
其中：M为O或S；
B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述式(I’)所示的化学修饰选自以下任一结构：
Figure PCTCN2022139585-appb-000040
其中：M为O或S；
B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述式(I’)所示的化学修饰选自以下任一结构：
Figure PCTCN2022139585-appb-000041
其中：M为O或S；
B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述式(I’)所示的化学修饰选自以下任一结构：
Figure PCTCN2022139585-appb-000042
Figure PCTCN2022139585-appb-000043
以及它们
结构中的腺嘌呤被置换为鸟嘌呤、胞嘧啶、尿嘧啶或胸腺嘧啶的那些。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与所述反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述配体为如式(II)所示化合物或其药学上可接受的盐，
Figure PCTCN2022139585-appb-000044
其中，L 1为C 1-C 30烷基链、或包含被一个或多个氧、硫、氮原子或C＝O间断的C 1-C 30烷基链；
R 11和R 12独立地为化学键、NR 16或C＝O；
Q 3为
Figure PCTCN2022139585-appb-000045
R 13为CR 17R 18、NR 16、O或S；
R 14为CR 19；
R 15独立地为CR 17R 18、NR 16或O；
R 16至R 19独立地为氢、氘或烷基；
m1、p1和q1独立地为0、1、2、3或4；
B1为
Figure PCTCN2022139585-appb-000046
R b5、R b6和R b7独立地为-C(＝O)-、-NHC(＝O)-、-C(＝O)O-、-C(＝O)-(CH 2) z8-O-或-NHC(＝O)-(CH 2) z9-O-；
z5、z6、z7、z8和z9独立地为0-10的整数；
L 2为C 1-C 30烷基链、或包含被一个或多个氧、硫、氮原子或C＝O间断的C 1-C 30烷基链；
r1为1-10的整数。
在一些实施方案中，L 1为-(CH 2) j11-C(＝O)-(CH 2) j12-；
R 11和R 12独立地为化学键、NR 16或C＝O；
Q 3为
Figure PCTCN2022139585-appb-000047
R 13为CR 17R 18或O；
R 14为CR 19；
R 15独立地为CR 17R 18或O；
R 16至R 19独立地为氢或烷基；
m1、p1和q1独立地为0或1；
B1为
Figure PCTCN2022139585-appb-000048
R b5、R b6和R b7独立地为-C(＝O)-(CH 2) z8-O-或-NHC(＝O)-(CH 2) z9-O-；
z8和z9独立地为0-10的整数；
L 2为-(CH 2) j15-(OCH 2CH 2) 1-4-(CH 2) j16-或
Figure PCTCN2022139585-appb-000049
j15和j16独立地为0-4的整数；
r1为3、4、5或6。
在一些实施方案中，L 1可为L 3或L 3-R 110-R 111-L 3，其中，L 3独立地为C 1-C 12烷基链、-(CH 2) j11-C(＝O)-(CH 2) j12-或-(CH 2) j13-(CH 2CH 2O) 1-4-(CH 2) j14-，R 110和R 111独立地为化学键、-NR 112-、-C(＝O)-或-OC(＝O)-，R 112为氢或C 1-C 12烷基，j11、j12、j13和j14独立地为0-10的整数。在一些实施方案中，j11、j12、j13和j14独立地为0-2或4-10的整数。在一些实施方案中，j11、j12、j13和j14独立地为0、1、2、6、7、8、9或10。
在一些实施方案中，L 1可为-(CH 2) j11-C(＝O)-(CH 2) j12-，j11和j12的定义同前任一方案所述。
在一些实施方案中，L 1可为
Figure PCTCN2022139585-appb-000050
j12的定义同前任一方案所述，其中，a1端与B相连，b1端与R 1相连。
在一些实施方案中，L 1可为
Figure PCTCN2022139585-appb-000051
Figure PCTCN2022139585-appb-000052
其中，a1端与B 1相连，b1端与R 11相连。
在一些实施方案中，R 11可为化学键且R 12可为C＝O。
在一些实施方案中，R 11可为化学键且R 12可为NR 16，R 16的定义同前任一方案所述。
在一些实施方案中，R 11可为化学键且R 12可为-OC(＝O)-。
在一些实施方案中，R 11可为NR 16且R 12可为C＝O，R 16的定义同前任一方案所述。
在一些实施方案中，R 1可为NR 16且R 12可为-OC(＝O)-，R 16的定义同前任一方案所述。
在一些实施方案中，R 12可为NR 16且R 11可为C＝O，R 16的定义同前任一方案所述。
在一些实施方案中，R 12可为NR 16且R 11可为-OC(＝O)-，R 16的定义同前任一方案所述。
在一些实施方案中，R 11可为NH且R 12可为C＝O。
在一些实施方案中，R 12可为NH且R 11可为C＝O。
在一些实施方案中，R 16可为氢或C 1-6烷基。
在一些实施方案中，R 16可为氢、甲基、乙基、丙基或异丙基。
在一些实施方案中，R 16可为氢。
在一些实施方案中，R 17和R 18可为氢。
在一些实施方案中，R 19可为氢。
在一些实施方案中，环A存在时，环A可为C 6-12芳基。
在一些实施方案中，环A可为苯基。
在一些实施方案中，m1可为0或1。
在一些实施方案中，m1可为3。
在一些实施方案中，n1可为0或1。
在一些实施方案中，p1和q1独立地为0或1。
在一些实施方案中，p1＝1且q1＝1。
在一些实施方案中，p1＝1且q1＝0。
在一些实施方案中，p1＝0且q1＝1。
在一些实施方案中，p1＝0且q1＝0。
在一些实施方案中，z1、z2、z3、z4、z5、z6、z7、z8和z9可独立地为0-4的整数。在一些实施方案中，z1、z2、z3、z4、z5、z6、z7、z8和z9可独立地为 0、1或2。
在一些实施方案中，B 1可为
Figure PCTCN2022139585-appb-000053
R b1、R b2、R b3和R b4独立地为-C(＝O)-或-NHC(＝O)-，N原子与L 1相连，z1、z2、z3和z4的定义同前任一方案所述。
在一些实施方案中，B 1可为
Figure PCTCN2022139585-appb-000054
R b1、R b2、R b3和R b4独立地为-C(＝O)-或-NHC(＝O)-，N原子与L 1相连，R b1、R b3和R b4相同，z1、z2、z3和z4的定义同前任一方案所述。
在一些实施方案中，B 1可为
Figure PCTCN2022139585-appb-000055
在一些实施方案中，B 1可为
Figure PCTCN2022139585-appb-000056
在一些实施方案中，B 1可为
Figure PCTCN2022139585-appb-000057
R b5、R b6和R b7独立地为-C(＝O)-(CH 2) z8-O-或-NHC(＝O)-(CH 2) z9-O-，N原子与L 1相连，z5、z6、z7、z8和z9的定义同前任一方案所述。
在一些实施方案中，B 1可为
Figure PCTCN2022139585-appb-000058
R b5、R b6和R b7独立地为-C(＝O)-(CH 2) z8-O-或-NHC(＝O)-(CH 2) z9-O-，N原子与L 1相连，R b5、R b6和R b7相同，z5、z6、z7、z8和z9的定义同前任一方案所述。
在一些实施方案中，B 1可为
Figure PCTCN2022139585-appb-000059
在一些实施方案中，L 2可为L 4或L 4-R 13-R 14-L 4，其中，L 4独立地为C 1-C 12烷基链或-(CH 2) j15-(OCH 2CH 2) 1-4-(CH 2) j16-，R 113和R 114独立地为化学键、-NR 115-、-C(＝O)-或-OC(＝O)-，R 115独立地为氢或C 1-C 12烷基，j15和j16独立地为0-10的整数。在一些实施方案中，j15和j16独立地为0-6的整数。在一些实施方案中，j15和j16独立地为0、1、2、3或4。
在一些实施方案中，L 2可为-(CH 2) j15-(OCH 2CH 2) 1-4-(CH 2) j16-，j15和j16的定义同前任一方案所述。
在一些实施方案中，L 2可为
Figure PCTCN2022139585-appb-000060
在一些实施方案中，L 2可为
Figure PCTCN2022139585-appb-000061
其中，左侧与O原子相连，右侧与B 1相连。
在一些实施方案中，L 2可为C 1-C 12烷基链。
在一些实施方案中，L 2可为
Figure PCTCN2022139585-appb-000062
Figure PCTCN2022139585-appb-000063
在一些实施方案中，L 2可为
Figure PCTCN2022139585-appb-000064
在一些实施方案中，L 2可为
Figure PCTCN2022139585-appb-000065
在一些实施方案中，L 2可为
Figure PCTCN2022139585-appb-000066
在一些实施方案中，L 2可为
Figure PCTCN2022139585-appb-000067
其中，a3端与O原子相连，b3端与B 1相连。
在一些实施方案中，L 2可为
Figure PCTCN2022139585-appb-000068
其中，a3端与O原子相连，b3端与B 1相连。
在一些实施方案中，r1可为3、4、5或6。在一些实施方案中，r1可为3。
在一些实施方案中，Q 3可为
Figure PCTCN2022139585-appb-000069
在一些实施方案中，Q 3可为
Figure PCTCN2022139585-appb-000070
其中，R 13、R 14、R 15和n1的定义同前任一方案所述。
在一些实施方案中，
Figure PCTCN2022139585-appb-000071
可为
Figure PCTCN2022139585-appb-000072
其中，R 13、R 14、R 15、p1和q1的定义同前任一方案所述。
在一些实施方案中，
Figure PCTCN2022139585-appb-000073
可为
Figure PCTCN2022139585-appb-000074
Figure PCTCN2022139585-appb-000075
其中，R 13、R 14、R 15、p1和q1的定义同前任一方案所述。
在一些实施方案中，
Figure PCTCN2022139585-appb-000076
可为
Figure PCTCN2022139585-appb-000077
在 一些实施方案中，
Figure PCTCN2022139585-appb-000078
可为
Figure PCTCN2022139585-appb-000079
在一些实施方案中，
Figure PCTCN2022139585-appb-000080
可为
Figure PCTCN2022139585-appb-000081
p1和q1的定义同前任一方案所述。
在一些实施方案中，
Figure PCTCN2022139585-appb-000082
可为
Figure PCTCN2022139585-appb-000083
Figure PCTCN2022139585-appb-000084
Figure PCTCN2022139585-appb-000085
在一些实施方案中，
Figure PCTCN2022139585-appb-000086
可为
Figure PCTCN2022139585-appb-000087
Figure PCTCN2022139585-appb-000088
在一些实施方案中，
Figure PCTCN2022139585-appb-000089
可为
Figure PCTCN2022139585-appb-000090
Figure PCTCN2022139585-appb-000091
p1和q1的定义同前任一方案所述。
在一些实施方案中，
Figure PCTCN2022139585-appb-000092
可为
Figure PCTCN2022139585-appb-000093
其中，R 13、R 14、n1、p1和q1的定义同前任一方案所述。
在一些实施方案中，
Figure PCTCN2022139585-appb-000094
可为
Figure PCTCN2022139585-appb-000095
其中，R 13、R 14、n1、p1和q1的定义同前任一方案所述。
在一些实施方案中，
Figure PCTCN2022139585-appb-000096
可为
Figure PCTCN2022139585-appb-000097
在一些实施方案中，
Figure PCTCN2022139585-appb-000098
可为
Figure PCTCN2022139585-appb-000099
n1、p1和q1的定义同前任一方案所述。
在一些实施方案中，
Figure PCTCN2022139585-appb-000100
可为
Figure PCTCN2022139585-appb-000101
n1、p1和q1的定义同前任一方案所述。
在一些实施方案中，所述的配体可为以下任一结构或其药学上可接受的盐，
Figure PCTCN2022139585-appb-000102
Figure PCTCN2022139585-appb-000103
Figure PCTCN2022139585-appb-000104
Figure PCTCN2022139585-appb-000105
Figure PCTCN2022139585-appb-000106
在一些实施方案中，所述的配体可为以下任一结构或其药学上可接受的盐，
Figure PCTCN2022139585-appb-000107
Figure PCTCN2022139585-appb-000108
Figure PCTCN2022139585-appb-000109
Figure PCTCN2022139585-appb-000110
Figure PCTCN2022139585-appb-000111
Figure PCTCN2022139585-appb-000112
Figure PCTCN2022139585-appb-000113
Figure PCTCN2022139585-appb-000114
Figure PCTCN2022139585-appb-000115
Figure PCTCN2022139585-appb-000116
Figure PCTCN2022139585-appb-000117
Figure PCTCN2022139585-appb-000118
Figure PCTCN2022139585-appb-000119
Figure PCTCN2022139585-appb-000120
Figure PCTCN2022139585-appb-000121
在一些实施方案中，所述的配体可为以下结构或其药学上可接受的盐，
Figure PCTCN2022139585-appb-000122
在一些实施方案中，所述式(I)所示的化学修饰为
Figure PCTCN2022139585-appb-000123
Figure PCTCN2022139585-appb-000124
B选自鸟嘌呤、腺嘌呤、胞嘧啶和尿嘧啶；且所述配体为如下任一结构或其药学上可接受的盐，
Figure PCTCN2022139585-appb-000125
Figure PCTCN2022139585-appb-000126
在一些实施方案中，所述式(I)所示的化学修饰为
Figure PCTCN2022139585-appb-000127
Figure PCTCN2022139585-appb-000128
B选自鸟嘌呤、腺嘌呤、胞嘧啶和尿嘧啶，且，所述配体为如下任一结构或其药学上可接受的盐，
Figure PCTCN2022139585-appb-000129
在一些实施方案中，所述式(I)所示的化学修饰为
Figure PCTCN2022139585-appb-000130
Figure PCTCN2022139585-appb-000131
B选自鸟嘌呤、腺嘌呤、胞嘧啶和尿嘧啶；且，所述配体为如下结构 或其药学上可接受的盐，
Figure PCTCN2022139585-appb-000132
在一些实施方案中，可以用N-三氟乙酰基半乳糖胺、N-丙酰基半乳糖胺、N-正丁酰基半乳糖胺或N-异丁酰基半乳糖胺替换以上配体中的N-乙酰基-半乳糖胺部分。
在一些实施方案中，所述siRNA和所述配体共价或非共价连接。
在一些实施方案中，所述有义链的3’端和/或5’端可与所述配体缀合。
在一些实施方案中，所述有义链的3’端可与所述配体缀合。
在一些实施方案中，所述配体通过磷酸酯基团或硫代磷酸酯基团与所述siRNA末端连接。
在一些实施方案中，所述配体通过磷酸二酯基团或硫代磷酸二酯基团与所述siRNA末端连接。
在一些实施方案中，所述配体通过磷酸二酯基团与所述siRNA末端连接。
在一些实施方案中，所述配体通过磷酸酯基团或硫代磷酸酯基团与所述siRNA末端间接连接。
在一些实施方案中，所述配体通过磷酸酯基团或硫代磷酸酯基团与所述siRNA末端直接连接。
在一些实施方案中，所述配体通过磷酸酯基团或硫代磷酸酯基团与所述siRNA的有义链3’末端直接连接。
在一些实施方案中，所述磷酸酯基团为磷酸一酯基团或磷酸二酯基团。在一些实施方案中，所述磷酸酯基团为磷酸二酯基团。
在一些实施方案中，所述硫代磷酸酯基团为硫代磷酸一酯基团或硫代磷酸二酯基团。在一些实施方案中，所述硫代磷酸酯基团为硫代磷酸二酯基团。
在一些可选的实施方案中，为了促进siRNA进入细胞，可以在siRNA有义链的末端引入胆固醇等亲脂性的基团，亲脂性的基团包括以共价键与siRNA结合，如末端引入胆固醇、脂蛋白、维生素E等，以利于通过由脂质双分子层构成的细胞膜与细胞内的mRNA发生作用。同时，siRNA也可以进行非共价键修饰，如通过疏水键或离子键结合磷脂分子、多肽、阳离子聚合物等增加稳定性和生物学活性。
在一些实施方案中，包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸位于反义链5’端起第5位、第6位或第7位。
在一些实施方案中，包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸位于反义链5’端起第7位。
在一些实施方案中，式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰在其5’端起第5位时，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰在其5’端起第6位时，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰在其5’端起第7位时，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰在其5’端起第8位时，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与所述反义链在其5’端起第5位核苷酸未被修饰时的碱基相同。
在一些实施方案中，B与所述反义链在其5’端起第6位核苷酸未被修饰时的碱基相同。
在一些实施方案中，B与所述反义链在其5’端起第7位核苷酸未被修饰时的碱基相同。
在一些实施方案中，B与所述反义链在其5’端起第8位核苷酸未被修饰时的碱基相同。
一些实施方案中，在包含式(I)所示化学修饰以外的其余位置处，所述的有义链和/或反义链中至少一个另外的核苷酸为修饰的核苷酸，所述修饰的核苷酸选自：2'-甲氧基修饰的核苷酸、2'-经取代的烷氧基修饰的核苷酸、2'-烷基修饰的核苷酸、2'-经取代的烷基修饰的核苷酸、2'-氨基修饰的核苷酸、2'-经取代的氨基修饰的核苷酸、2'-氟代修饰的核苷酸、2'-脱氧核苷酸、2'-脱氧-2'-氟代修饰的核苷酸、3'-脱氧-胸腺嘧啶核苷酸、异核苷酸、LNA、ENA、cET、UNA、GNA；一些实施方案中，修饰的核苷酸相互独立地选自：2'-甲氧基修饰的核苷酸或2'-氟代修饰的核苷酸。
在一些实施方案中，所述的有义链含有连续三个具有相同修饰的核苷酸。在一些实施方案中，所述的三个具有相同修饰的核苷酸为2'-氟代修饰的核苷酸。
在一些实施方案中，按照5'端到3'端的方向，所述反义链起第2、4、6、10、12、14、16和18位的核苷酸各自独立地为2'-氟代修饰的核苷酸。
在一些实施方案中，所述反义链与靶序列至少部分地反向互补。在一些实施方案中，所述反义链与靶序列之间存在不多于5个、不多于4个、不多于3个、不多于2个、不多于1个错配；在一些实施方案中，所述反义链与靶序列完全反向互补。
在一些实施方案中，所述有义链与反义链至少部分地反向互补以形成双链区。在一些实施方案中，所述有义链与反义链之间存在不多于5个、不多于4个、不多于3个、不多于2个、不多于1个错配。在一些实施方案中，所述有义链与反义链完全反向互补。
在一些实施方案中，所述有义链和反义链各自独立地具有16至35个、16至34个、17至34个、17至33个、18至33个、18至32个、18至31个、18至30个、18至29个、18至28个、18至27个、18至26个、18至25个、18至24个、18至23个、19至25个、19至24个、或19至23个核苷酸(例如19、20、21、22、23个)。
在一些实施方案中，所述有义链和反义链长度相同或不同，所述有义链的长度为19-23个核苷酸，所述反义链的长度为19-26个核苷酸。本公开提供的dsRNA中的有义链和反义链的长度比可以是19/19、19/20、19/21、19/22、19/23、19/24、19/25、19/26、20/19、20/20、20/21、20/22、20/23、20/24、20/25、20/26、21/20、21/21、21/22、21/23、21/24、21/25、21/26、22/20、22/21、22/22、22/23、22/24、22/25、22/26、23/20、23/21、23/22、23/23、23/24、23/25或23/26。在一些实施方案中，所述有义链和反义链的长度比为19/21、21/23或23/25。在一些实施方案中，所述有义链和反义链的长度比为19/21。
在一些实施方案中，所述siRNA包含一个或两个平端。
一些具体的实施方案中，siRNA的每条链各自独立地包含由1至2个未配对核苷酸而形成的突出端。
在一些实施方案中，所述siRNA包含位于所述反义链3’端的突出端。
在一些实施方案中，所述有义链含有如下式所示的核苷酸序列(5’-3’)：
N aN aN aN aXN aN bN bN bN aN aN aN aN aN aN aN aN aN a
其中，每个X独立地为N a或N b；N a为2'-甲氧基修饰的核苷酸，N b为2'-氟代修饰的核苷酸。
在一些实施方案中，所述有义链含有如下式所示的核苷酸序列：
5’-N aN aN aN aN aN aN bN bN bN aN aN aN aN aN aN aN aN aN a-3’；或，
5’-N aN aN aN aN bN aN bN bN bN aN aN aN aN aN aN aN aN aN a-3’；
其中，N a为2'-甲氧基修饰的核苷酸，N b为2'-氟代修饰的核苷酸。
在一些实施方案中，所述反义链含有如下式所示的核苷酸序列：
5’-N a’N b’N a’N b’N a’N b’W’N a’N a’N b’N a’N b’N a’N b’N a’N b’N a’N b’N a’N a’N a’-3’；
其中，N a’为2'-甲氧基修饰的核苷酸，N b’为2'-氟代修饰的核苷酸；W’表示包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰的核苷酸。
在一些具体的实施方案中，W’表示包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸。
在一些具体的实施方案中，式(I)所示的化学修饰选自：
Figure PCTCN2022139585-appb-000133
其中：B选自鸟嘌呤、腺嘌呤、胞嘧啶和尿嘧啶。在一些具体的实施方案中，B与所述反义链在其5’端起第7位核苷酸未被修饰时的碱基相同。
在一些具体的实施方案中，式(I)所示的化学修饰选自：
Figure PCTCN2022139585-appb-000134
其中：M为O或S；其中：B选自鸟嘌呤、腺嘌呤、胞嘧啶或尿嘧啶。在一些具体的实施方案中，B与所述反义链在其5’端起第7位核苷酸未被修饰时的碱基相同。
在一些具体的实施方案中，M为S。一些具体的实施方案中，M为O。
在一些实施方案中，所述有义链和/或反义链中至少一个磷酸酯基为具有修饰基团的磷酸酯基，所述修饰基团使得所述siRNA在生物样品或环境中具有增加的稳定性；在一些实施方案中，所述具有修饰基团的磷酸酯基为硫代磷酸酯基。在一些实施方案中，所述具有修饰基团的磷酸酯基为硫代磷酸二酯基。
在一些实施方案中，所述硫代磷酸二酯基存在于以下位置中的至少一处：
所述有义链的5'端起第1个核苷酸和第2个核苷酸之间；
所述有义链的5'端起第2个核苷酸和第3个核苷酸之间；
所述反义链的5'端起第1个核苷酸和第2个核苷酸之间；
所述反义链的5'端起第2个核苷酸和第3个核苷酸之间；
所述反义链的3'端起第1个核苷酸和第2个核苷酸之间；以及
所述反义链的3'端起第2个核苷酸和第3个核苷酸之间。
在一些实施方案中，所述有义链和/或反义链中包括多个硫代磷酸二酯基，所述硫代磷酸二酯基存在于：
所述有义链的5'端起第1个核苷酸和第2个核苷酸之间；和，
所述有义链的5'端起第2个核苷酸和第3个核苷酸之间；和，
所述反义链的5'端起第1个核苷酸和第2个核苷酸之间；和，
所述反义链的5'端起第2个核苷酸和第3个核苷酸之间；和，
所述反义链的3'端起第1个核苷酸和第2个核苷酸之间；和，
所述反义链的3'端起第2个核苷酸和第3个核苷酸之间。
在一些实施方案中，所述有义链包含如下式所示的核苷酸序列：
5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’，或，
5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’，
其中，Nm表示2'-甲氧基修饰的任意核苷酸，例如2'-甲氧基修饰的C、G、U、A；Nf表示2'-氟代修饰的任意核苷酸，例如2'-氟代修饰的C、G、U、A；
小写字母s在中间时表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接。
在一些实施方案中，所述反义链包含如下式所示的核苷酸序列：
5’-Nm’sNf’sNm’Nf’Nm’Nf’W’Nm’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nf’Nm’sNm’sNm’-3’；
其中，Nm’表示2'-甲氧基修饰的任意核苷酸，例如2'-甲氧基修饰的C、G、U、A；Nf’表示2'-氟代修饰的任意核苷酸，例如2'-氟代修饰的C、G、U、A；
小写字母s在中间时表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接；
W’表示2'-甲氧基修饰的核苷酸或包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰的核苷酸。
在一些实施方案中，W’表示2'-甲氧基修饰的核苷酸。
在一些实施方案中，式(I)所示的化学修饰选自：
Figure PCTCN2022139585-appb-000135
其中：B选自鸟嘌呤、腺嘌呤、胞嘧啶和尿嘧啶；在一些实施方案中，B与所述反义链在其5’端起第7位核苷酸未被修饰时的碱基相同。
一些实施方案中，式(I)所示的化学修饰选自：
Figure PCTCN2022139585-appb-000136
其中：M为O或S；其 中：B选自鸟嘌呤、腺嘌呤、胞嘧啶或尿嘧啶；在一些具体的实施方案中，B与所述反义链在其5’端起第7位核苷酸未被修饰时的碱基相同。
在一些实施方案中，M为S。在一些具体的实施方案中，M为O。
靶向凝血因子XI(FXI)基因的dsRNA
在一些实施方案中，所述siRNA为靶向凝血因子XI(FXI)基因的siRNA。
在一些实施方案中，所述siRNA的有义链的核苷酸序列包含与SEQ ID NO:1至SEQ ID NO:3中任一的核苷酸序列相差不超过3个核苷酸，且包含至少15个连续核苷酸(一些实施方案中，至少19个)，和/或，
反义链的核苷酸序列包含与SEQ ID NO:4或SEQ ID NO:5中任一的核苷酸序列相差不超过3个核苷酸，且包含至少19个连续核苷酸(一些实施方案中，至少21个)。
在一些实施方案中，所述siRNA的有义链的核苷酸序列包含SEQ ID NO:1至SEQ ID NO:3中的任一项核苷酸序列，和/或，反义链的核苷酸序列包含SEQ ID NO:4或SEQ ID NO:5中的任一项核苷酸序列。
在一些实施方案中，所述siRNA的有义链和反义链选自以下任一组方案：
有义链的核苷酸序列包含SEQ ID NO:3的核苷酸序列，反义链的核苷酸序列包含SEQ ID NO:5的核苷酸序列；
有义链的核苷酸序列包含SEQ ID NO:2的核苷酸序列，反义链的核苷酸序列包含SEQ ID NO:5的核苷酸序列；
有义链的核苷酸序列包含SEQ ID NO:1的核苷酸序列，反义链的核苷酸序列包含SEQ ID NO:4的核苷酸序列。
在一些实施方案中，所述siRNA的有义链和反义链选自以下任一组：
有义链的核苷酸序列是SEQ ID NO:3，反义链的核苷酸序列是SEQ ID NO:5；
有义链的核苷酸序列是SEQ ID NO:2，反义链的核苷酸序列是SEQ ID NO:5；
有义链的核苷酸序列是SEQ ID NO:1，反义链的核苷酸序列是SEQ ID NO:4。
本公开中，按照5’-3’方向，
SEQ ID NO:1是CUUGCAACAAAGACAUUUA；
SEQ ID NO:2是UCAGGAUGAUUUUCUUAUU；
SEQ ID NO:3是UAAAUGUCUUUGUUGCAAGCG；
SEQ ID NO:4是UAUAAGAAAAUCAUCCUGAAA。
在一些实施方案中，所述dsRNA的有义链包含SEQ ID NO:6至SEQ ID NO:10中的任一项，和/或，反义链包含SEQ ID NO:14或SEQ ID NO:15中的任一项。
在一些实施方案中，所述dsRNA的有义链和反义链选自以下任一组：
组1)：有义链包含SEQ ID NO:10，反义链包含SEQ ID NO:15；
组2)：有义链包含SEQ ID NO:9，反义链包含SEQ ID NO:15；
组3)：有义链包含SEQ ID NO:7，反义链包含SEQ ID NO:14；
组4)：有义链包含SEQ ID NO:8，反义链包含SEQ ID NO:15；
组5)：有义链包含SEQ ID NO:6，反义链包含SEQ ID NO:14。
在一些实施方案中，所述dsRNA的有义链和反义链选自以下任一组：
组1)：有义链是SEQ ID NO:10所示，反义链是SEQ ID NO:15所示；
组2)：有义链是SEQ ID NO:9所示，反义链是SEQ ID NO:15所示；
组3)：有义链是SEQ ID NO:7所示，反义链是SEQ ID NO:14所示；
组4)：有义链是SEQ ID NO:8所示，反义链是SEQ ID NO:15所示；
组5)：有义链是SEQ ID NO:6所示，反义链是SEQ ID NO:14所示。
在一些实施方案中，所述dsRNA选自以下任一组：
组1)：包含或选自SEQ ID NO：10所示的有义链和SEQ ID NO：15所示的反义链；
组2)：包含或选自SEQ ID NO：9所示的有义链和SEQ ID NO：15所示的反义链；
组3)：包含或选自SEQ ID NO：7所示的有义链和SEQ ID NO：14所示的反义链；
组4)：包含或选自SEQ ID NO：8所示的有义链和SEQ ID NO：15所示的反义链；
组5)：包含或选自SEQ ID NO：6所示的有义链和SEQ ID NO：14所示的反义链。
本公开中，按照5’-3’方向，
SEQ ID NO:6是
CmsUmsUmGmCfAmAfCfAfAmAmGmAmCmAmUmUmUmAm-NAG0052’；
SEQ ID NO:7是
CmsUmsUmGmCmAmAfCfAfAmAmGmAmCmAmUmUmUmAm-NAG0052’；
SEQ ID NO:8是
UmsCmsAmGmGfAmUfGfAfUmUmUmUmCmUmUmAmUmUm-NAG0052’；
SEQ ID NO:9是
UmsCmsAmGmGmAmUfGfAfUmUmUmUmCmUmUmAmUmUm-NAG0052’；
SEQ ID NO:10是
UmsCmsAmGmGmAmUfGfAfUmUmUmUmCmUmUmAmUmAm-NAG0052’
SEQ ID NO:14是
UmsAfsAmAfUmGf(-)hmpNA(U)CmUmUfUmGfUmUfGmCfAmAfGmsCmsGm；
SEQ ID NO:15是
UmsAfsUmAfAmGf(-)hmpNA(A)AmAmAfUmCfAmUfCmCfUmGfAmsAmsAm；
其中，Af＝腺嘌呤2'-F核糖核苷(adenine 2'-F ribonucleoside)；Cf＝胞嘧啶2'-F核糖核苷(cytosine 2'-F ribonucleoside)；Uf＝尿嘧啶2'-F核糖核苷(uracil 2'-F  ribonucleoside)；Gf＝鸟嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；Am＝腺嘌呤2'-OMe核糖核苷(adenine 2'-OMe ribonucleoside)；Cm＝胞嘧啶2'-OMe核糖核苷(cytosine 2'-OMe ribonucleoside)；Gm＝鸟嘌呤2'-OMe核糖核苷(guanine2'-OMe ribonucleoside)；Um＝尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)；
s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接；
NAG0052’表示
Figure PCTCN2022139585-appb-000137
(-)hmpNA(A)表示
Figure PCTCN2022139585-appb-000138
(-)hmpNA(U)表示
Figure PCTCN2022139585-appb-000139
在一些实施方案中，所述dsRNA选自如下结构或其药学上可接受的盐：
Figure PCTCN2022139585-appb-000140
Figure PCTCN2022139585-appb-000141
其中，Af＝腺嘌呤2'-F核糖核苷(adenine 2'-F ribonucleoside)；Cf＝胞嘧啶2'-F核糖核苷(cytosine 2'-F ribonucleoside)；Uf＝尿嘧啶2'-F核糖核苷(uracil 2'-F ribonucleoside)；Am＝腺嘌呤2'-OMe核糖核苷(adenine 2'-OMe ribonucleoside)；Cm＝胞嘧啶2'-OMe核糖核苷(cytosine 2'-OMe ribonucleoside)；Gf＝鸟嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；Gm＝鸟嘌呤2'-OMe核糖核苷(guanine 2'-OMe ribonucleoside)；Um＝尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)；
Figure PCTCN2022139585-appb-000142
表示硫代磷酸二酯基，
Figure PCTCN2022139585-appb-000143
表示磷酸二酯基，
NAG0052’表示
Figure PCTCN2022139585-appb-000144
(-)hmpNA(U)表示
Figure PCTCN2022139585-appb-000145
(-)hmpNA(A)表示
Figure PCTCN2022139585-appb-000146
在一些实施方案中，所述药学上可接受的盐可为本领域常规的盐，包括但不限于：钠盐、钾盐、铵盐、胺盐等。
在一些实施方案中，所述dsRNA选自TJR100407、TRD008003-1、TRD008002-1、TRD008003、或TRD008002。
在一些实施方案中，所述dsRNA为TRD008002，其为如下结构：
Figure PCTCN2022139585-appb-000147
在一些实施方案中，所述dsRNA为TRD008002-1，其为如下结构：
Figure PCTCN2022139585-appb-000148
在一些实施方案中，所述dsRNA为TRD008003，其为如下结构：
Figure PCTCN2022139585-appb-000149
在一些实施方案中，所述dsRNA为TRD008003-1，其为如下结构：
Figure PCTCN2022139585-appb-000150
在一些实施方案中，所述dsRNA为TJR100407，其为如下结构：
Figure PCTCN2022139585-appb-000151
其中，Af＝腺嘌呤2'-F核糖核苷(adenine 2'-F ribonucleoside)；Cf＝胞嘧啶2'-F 核糖核苷(cytosine 2'-F ribonucleoside)；Uf＝尿嘧啶2'-F核糖核苷(uracil 2'-F ribonucleoside)；Am＝腺嘌呤2'-OMe核糖核苷(adenine 2'-OMe ribonucleoside)；Cm＝胞嘧啶2'-OMe核糖核苷(cytosine 2'-OMe ribonucleoside)；Gf＝鸟嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；Gm＝鸟嘌呤2'-OMe核糖核苷(guanine 2'-OMe ribonucleoside)；Um＝尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)。
Figure PCTCN2022139585-appb-000152
表示硫代磷酸二酯基，
Figure PCTCN2022139585-appb-000153
表示磷酸二酯基，
NAG0052’表示
Figure PCTCN2022139585-appb-000154
(-)hmpNA(U)表示
Figure PCTCN2022139585-appb-000155
(-)hmpNA(A)表示
Figure PCTCN2022139585-appb-000156
药物组合物
另一方面，本公开提供了一种药物组合物，其包含上述的dsRNA。
在一些实施方案中，所述的药物组合物还包含一种或多种药学上可接受的赋形剂。各种递药系统是已知的并且可以用于本公开的dsRNA或药物组合物，例如封装在脂质体中、微粒、微囊、能够表达该化合物的重组细胞、受体介导的细胞内吞作用、构建核酸作为逆转录病毒或其他载体的一部分。
在一些实施方案中，本公开的dsRNA或药物组合物的给药方式是常规的，可通过局部给药(例如，直接注射或植入)或全身给药，也可通过口服、直肠或胃 肠外途径进行给药，所述肠胃外途径包括但不限于皮下注射、静脉注射、肌肉注射、腹腔注射、透皮给药、吸入给药(如气溶胶)、粘膜给药(如舌下、鼻内给药)、颅内给药等。
在一些实施方案中，本公开提供的dsRNA或药物组合物可以通过注射给予，例如，静脉内、肌内、皮内、皮下、十二指肠内或腹膜内注射。
在一些实施方案中，本公开提供的dsRNA或药物组合物可被包装在试剂盒中。
本公开中，所述的dsRNA或药物组合物的有效量或有效剂量为约0.001mg/kg体重至约200mg/kg体重、约0.01mg/kg体重至约100mg/kg体重或约0.5mg/kg体重至约50mg/kg体重。
本公开中，dsRNA、药物组合物是有效量或有效剂量的。
用途和治疗方法
另一方面，本公开提供了一种上述的dsRNA或上述的药物组合物在制备药物中的应用。
在一些实施方案中，所述的药物可用于预防和/或治疗血栓栓塞性并发症。在一些实施方案中，所述的血栓栓塞性并发症选自以下任一项或其组合：深静脉血栓形成、肺栓塞、心肌梗塞或中风。
在一些实施方案中，所述的药物可用于预防和/或治疗与凝血因子XI基因表达相关的疾病。在一些实施方案中，所述的凝血因子XI基因表达相关的疾病可为血栓栓塞性并发症。在一些实施方案中，所述的血栓栓塞性并发症选自以下任一项或其组合：深静脉血栓形成、肺栓塞、心肌梗塞或中风。
另一方面，本公开提供了一种预防和/或治疗疾病的方法，其包括向受试者给予有效量或有效剂量的上述的dsRNA或上述的药物组合物。
在一些实施方案中，所述的疾病可为血栓栓塞性并发症。在一些实施方案中，所述的血栓栓塞性并发症选自以下任一项或其组合：深静脉血栓形成、肺栓塞、心肌梗塞或中风。
在一些实施方案中，所述的疾病可为与凝血因子XI基因表达相关的疾病。在一些实施方案中，所述的凝血因子XI基因表达相关的疾病可为血栓栓塞性并发症。在一些实施方案中，所述的血栓栓塞性并发症选自以下任一项或其组合：深静脉血栓形成、肺栓塞、心肌梗塞或中风。
调节靶基因表达的方法
另一方面，本公开提供了一种用于在体内或在体外沉默细胞中靶基因FXI或其mRNA的方法，其包括将上述的dsRNA或上述的药物组合物引入该细胞中的步骤。
另一方面，本公开提供了一种抑制靶基因FXI或其mRNA表达的方法，其包括向受试者给予有效量或有效剂量的上述的dsRNA或上述的药物组合物。
本公开的dsRNA或药物组合物可以在细胞、细胞群、组织或受试者等对象中 降低靶基因或其mRNA的表达水平，包括：向受试者给予治疗有效量的本文所述的dsRNA或药物组合物，从而抑制靶基因或其mRNA在对象中的表达。
在一些实施方案中，所述受试者已在先前被鉴定为在靶向的细胞、细胞群、组织或受试者中具有靶基因FXI或其mRNA的病理性上调。另一方面，本公开提供了一种递送寡核苷酸至肝脏的方法，其包括向受试者给予有效量或有效剂量的上述的dsRNA或上述的药物组合物。
另一方面，本公开提供了一种RNAi(RNA干扰)试剂，其包含上述的dsRNA或上述的药物组合物。
另一方面，本公开还提供了一种细胞，其包含上述的dsRNA或上述的药物组合物。
另一方面，本公开还提供了一种试剂盒或药盒，其包含上述的dsRNA或上述的药物组合物。
本公开中，上述dsRNA或药物组合物当接触到表达靶基因的细胞时，如所测定的(例如通过psiCHECK活性筛选、荧光素酶报告基因检测法、PCR或基于分支DNA的方法、或基于蛋白质的方法，如免疫荧光分析、Western Blot或流式细胞术)，上述dsRNA或药物组合物会抑制靶基因的表达至少5％、至少10％、至少15％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％。
本公开中，上述dsRNA或药物组合物当接触到表达靶基因的细胞时，如所测定的(例如通过psiCHECK活性筛选、荧光素酶报告基因检测法、PCR或基于分支DNA的方法、或基于蛋白质的方法，如免疫荧光分析、Western Blot或流式细胞术)，上述dsRNA或药物组合物引起的靶基因mRNA剩余表达百分比为不高于99％、不高于95％、不高于90％、不高于85％、不高于80％、不高于75％、不高于70％、不高于65％、不高于60％、不高于55％、不高于50％、不高于45％、不高于40％、不高于35％、不高于30％、不高于25％、不高于20％、不高于15％、或不高于10％。
本公开中，上述dsRNA或药物组合物当接触到表达靶基因的细胞时，如所测定的(例如通过psiCHECK活性筛选、荧光素酶报告基因检测法、PCR或基于分支DNA的方法、或基于蛋白质的方法，如免疫荧光分析、Western Blot或流式细胞术)，dsRNA在保持在靶活性的同时，将脱靶活性减少了至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％或至少75％。
本公开中，上述dsRNA或药物组合物当接触到表达靶基因的细胞时，如所测定的(例如通过psiCHECK活性筛选、荧光素酶报告基因检测法、PCR或基于分 支DNA的方法、或基于蛋白质的方法，如免疫荧光分析、Western Blot或流式细胞术)，dsRNA使在靶活性降低至多20％、至多19％、至多15％、至多10％、至多5％或超过1％的同时，将脱靶活性减少了至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％或至少75％。
本公开中，上述dsRNA或药物组合物当接触到表达靶基因的细胞时，如所测定的(例如通过psiCHECK活性筛选、荧光素酶报告基因检测法、PCR或基于分支DNA的方法、或基于蛋白质的方法，如免疫荧光分析、Western Blot或流式细胞术)，dsRNA使在靶活性提高至少1％、至少5％、至少10％、至少15％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％或至少80％的同时，将脱靶活性减少了至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％或至少75％。
本公开还提供了一种制备dsRNA或药物组合物的方法，其包括：合成本公开所述的配体、siRNA、dsRNA。
本公开引入WO2022028457A1全文。
本公开化合物可以存在特定的几何或立体异构体形式。本公开设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本公开的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本公开的范围之内。本公开的含有不对称碳原子的化合物可以以光学活性纯的形式或外消旋形式被分离出来。光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本公开某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本公开所述化合物的化学结构中，键
Figure PCTCN2022139585-appb-000157
表示未指定构型，即如果化学结构中存在手性异构体，键
Figure PCTCN2022139585-appb-000158
可以为
Figure PCTCN2022139585-appb-000159
或者同时包含
Figure PCTCN2022139585-appb-000160
两种构型。本公开所述化合物的化学结构中，键
Figure PCTCN2022139585-appb-000161
并未指定构型，即键
Figure PCTCN2022139585-appb-000162
的构 型可以为E型或Z型，或者同时包含E和Z两种构型。
在本公开的化学结构式中，
Figure PCTCN2022139585-appb-000163
可以根据本文所述发明范围连接一个或多个任何基团；星号“*”表示手性中心。
在不指明构型的情况下，本公开的化合物和中间体还可以以不同的互变异构体形式存在，并且所有这样的形式包含于本公开的范围内。
术语“互变异构体”或“互变异构体形式”是指可经由低能垒互变的不同能量的结构异构体。例如，质子互变异构体(也称为质子转移互变异构体)包括经由质子迁移的互变，如酮-烯醇及亚胺-烯胺、内酰胺-内酰亚胺异构化。内酰胺-内酰亚胺平衡实例是在如下所示的A和B之间。
Figure PCTCN2022139585-appb-000164
本公开中的所有化合物可以被画成A型或B型。所有的互变异构形式在本发明的范围内。化合物的命名不排除任何互变异构体。
本公开还包括一些与本文中记载的那些相同的，但一个或多个原子被原子量或质量数不同于自然中通常发现的原子量或质量数的原子置换的同位素标记的本公开化合物。可结合到本公开化合物的同位素的实例包括氢、碳、氮、氧、磷、硫、氟、碘和氯的同位素，诸如分别为 2H、 3H、 11C、 13C、 14C、 13N、 15N、 15O、 17O、 18O、 31P、 32P、 35S、 18F、 123I、 125I和 36Cl等。
除另有说明，当一个位置被特别地指定为氘(D)时，该位置应理解为具有大于氘的天然丰度(其为0.015％)至少1000倍的丰度的氘(即，至少10％的氘掺入)。示例中化合物的具有大于氘的天然丰度可以是至少1000倍的丰度的氘、至少2000倍的丰度的氘、至少3000倍的丰度的氘、至少4000倍的丰度的氘、至少5000倍的丰度的氘、至少6000倍的丰度的氘或更高丰度的氘。本公开还包括各种氘化形式的式(I)、式(I’)、式(II)化合物。与碳原子连接的各个可用的氢原子可独立地被氘原子替换。本领域技术人员能够参考相关文献合成氘化形式的式(I)、式(I’)、式(II)化合物。在制备氘代形式的式(I)、式(I’)、式(II)化合物时可使用市售的氘代起始物质，或它们可使用常规技术采用氘代试剂合成，氘代试剂包括但不限于氘代硼烷、三氘代硼烷四氢呋喃溶液、氘代氢化锂铝、氘代碘乙烷和氘代碘甲烷等。
除另有说明，“任选地”、“任选”、“可选的”或“可选”是指意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如“任选地，R 1和R 2直接相连成环”是指R 1和R 2直接相连成环可以发生但不必须存在，该说明包括R 1和R 2直接相连成环的情形和R 1和R 2不成环的情形。
术语解释
为了更容易理解本公开，以下具体定义了一些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
靶标是指本公开的dsRNA(或其缀合物)所针对的客体；靶标可以是核酸(基因、mRNA等)，也可以是蛋白(前体、成熟蛋白、同种型、变体等)。在本公开中，靶标尤其是指因子XI基因或其表达产物。
本公开中因子XI应作做广泛的解读，是指因子XI基因本身及其在各阶段中各种形式的表达产物，例如但不限于基因在扩增、复制、转录、剪接、加工、翻译、修饰过程中所产生的分子，例如cDNA、mRNA、前体蛋白、成熟蛋白、天然变体、修饰形式、及其片段。
本公开上下文中，术语“因子XI”、“因子11”、“凝血因子XI”、“凝血因子11”、“FXI”和“F11”可互换使用。“因子XI核酸”是指编码因子XI的任意核酸。例如，在一些实施方案中，因子XI核酸包括编码因子XI的DNA序列，例如“因子XI基因”，由编码因子XI的DNA转录的RNA序列(包括含内含子和外显子的基因组DNA)以及编码因子XI的mRNA序列。“因子XI基因”是下列序列中的任一个：GENBANK登录号NM_000128.4；GENBANK登录号NT_022792.17，19598000至19624000缺失；GENBANK登录号NM_028066.3，外显子1-15；GENBANK登录号XM_006253144.4，XM_006253145.3；GENBANK登录号XM_015139652.2,XM_015139653.2；GENBANK登录号XM_005556483.3，XM_005556484.3；GENBANK登录号NW_001118167.1。“因子XI mRNA”是指编码因子XI蛋白的mRNA。
技术人员应当理解，尽管在具体实施方案中给出了具体的登录号，但是因子XI不限于特定数据库中的编号，还意图涵盖现有技术中任何文献、书籍、数据库中的等同指代物。当dsRNA靶向因子XI或其表达产物的特定靶点时，技术人员能够确定该靶点在不同数据库中的等同位置。如本文所使用的，有义链(又称SS、SS链或正义链)是指包含与靶mRNA序列相同或基本上相同的序列的链；反义链(又称AS或AS链)是指具有与靶mRNA序列互补或部分互补的序列的链。
本公开中，有义链或反义链的“5’区域”也即“5’端”、“5’末端”，可替换使用。例如反义链5’端起第2位至第8位的核苷酸，也可替换为反义链5’端的第2位至第8位的核苷酸。同理，有义链或反义链的“3’区域”、“3’末端”和“3’端”也可替换使用。
在描述本公开所述的有义链的上下文中，术语“与SEQ ID NO:1至SEQ ID NO:3任一的核苷酸序列相差不超过3个核苷酸序列，且包含至少15个连续核苷酸”旨在表示本文所述的siRNA有义链包含SEQ ID NO:1至SEQ ID NO:3中任一有义链的至少15个连续核苷酸，或与SEQ ID NO:1至SEQ ID NO:3中任一有义链的至少15个连续核苷酸相差不超过3个核苷酸序列(任选地，相差不超过2个核苷酸序列；任选地，相差1个核苷酸序列)。任选地，本文所述的siRNA有义链包 含SEQ ID NO:1至SEQ ID NO:3任一有义链的至少16个连续核苷酸，或与SEQ ID NO:1至SEQ ID NO:3任一有义链的至少16个连续核苷酸相差不超过3个核苷酸序列(任选地，相差不超过2个核苷酸序列，任选地，相差1个核苷酸序列)。
在描述本公开所述的反义链的上下文中，术语“与SEQ ID NO:4或SEQ ID NO:5任一反义链相差不超过3个核苷酸序列，且包含至少15个连续核苷酸”旨在表示本文所述的siRNA反义链包含SEQ ID NO:4或SEQ ID NO:5中任一反义链的至少15个连续核苷酸，或与SEQ ID NO:4或SEQ ID NO:5中任一反义链的至少15个连续核苷酸相差不超过3个核苷酸序列(任选地，相差不超过2个核苷酸序列，任选地，相差1个核苷酸序列)。
术语“dsRNA”是指能够进行RNA干扰的双链RNA分子，包含有义链和反义链。
如本文所使用的，术语“抑制凝血因子XI表达”包括抑制凝血因子XI基因以及凝血因子XI基因的变体(例如天然存在的变体)或突变体的表达，抑制凝血因子XI mRNA的表达，和/或抑制凝血因子XI蛋白的表达。凝血因子XI基因可以是野生型人凝血因子XI基因、突变人凝血因子XI基因、或在遗传操纵的细胞、细胞群组或生物体情形下的转基因人凝血因子XI基因。抑制凝血因子XI基因表达包括任何水平的凝血因子XI基因的抑制，例如至少部分抑制凝血因子XI基因的表达，如抑制至少约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％。基于与凝血因子XI基因表达相关的任何变量水平，例如凝血因子XI的mRNA水平或凝血因子XI的蛋白水平，可以评估凝血因子XI基因的表达。抑制可通过这些变量中的一个或多个与对照水平相比的绝对或相对水平的减少来评估。该对照水平可以是本领域中使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。例如，可以采用mRNA剩余表达量来表征siRNA对靶基因表达的抑制程度，如mRNA剩余表达量为不高于99％、不高于95％、不高于90％、不高于85％、不高于80％、不高于75％、不高于70％、不高于65％、不高于60％、不高于55％、不高于50％、不高于45％、不高于40％、不高于35％、不高于30％、不高于25％、不高于20％、不高于15％、或不高于10％。靶基因表达的抑制率可以采用
Figure PCTCN2022139585-appb-000165
Luciferase Assay System检测，分别读取萤火虫化学发光值(Fir)和海肾化学发光值(Ren)，计算相对值Ratio＝Ren/Fir，抑制率(％)＝1-(Ratio+siRNA/仅报告基因时的Ratio)*100％；本公开中，剩余mRNA表达量比例＝100％-抑制率(％)。
如无特别说明，在本公开上下文中，“G”、“C”、“A”、“T”与“U”分别代表核苷 酸，其分别包含鸟嘌呤、胞嘧啶、腺嘌呤、胸苷与尿嘧啶的碱基；小写字母d表示该字母d下游相邻的一个核苷酸为脱氧核糖核苷酸；小写字母m表示该字母m上游相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f上游相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接。
如本公开所使用的，术语“2'-氟代(2’-F)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，“非氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物。
如本公开所使用的，术语“2'-甲氧基(2’-OMe)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。
在本公开的上下文中，一个核苷酸序列与另外一个核苷酸序列存在“核苷酸差异”，是指前者与后者相比，相同位置的核苷酸的碱基种类发生了改变，例如，在后者中一个核苷酸碱基为A时，在前者的相同位置处的对应核苷酸碱基为U、C、G或者T的情况下，认定为两个核苷酸序列之间在该位置处存在核苷酸差异。在一些实施方案中，以无碱基核苷酸或其等同物代替原位置的核苷酸时，也可认为在该位置处产生了核苷酸差异。
如本文所使用的，术语“互补”或“反向互补”一词可互相替代使用，并具有本领域技术人员周知的含义，即，在双链核酸分子中，一条链的碱基与另一条链上的碱基以互补的方式相配对。在DNA中，嘌呤碱基腺嘌呤始终与嘧啶碱基胸腺嘧啶(或者在RNA中为尿嘧啶)相配对；嘌呤碱基鸟嘌呤始终与嘧啶碱基胞嘧啶(G)相配对。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的胸腺嘧啶(或尿嘧啶)配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此相互补的，以及从其互补链的序列中可以推断出该链的序列。与此相应地，“错配”在本领域中意指在双链核酸中，对应位置上的碱基并未以互补的形式配对存在。
术语“化学修饰”或“修饰”包括核苷酸经化学手段的所有改变，例如化学部分的添加或去除、或以一个化学部分取代另一个化学部分。
术语“碱基”包含任何已知的DNA和RNA碱基、碱基类似物，例如嘌呤或嘧啶，其还包括天然化合物腺嘌呤、胸腺嘧啶、鸟嘌呤、胞嘧啶、尿嘧啶、次黄苷和天然类似物。碱基类似物还可以是通用碱基。
术语“平端”或“平末端”可互换使用，是指在siRNA的给定的末端没有未配对的核苷酸或核苷酸类似物，即，没有核苷酸突出。大多数情况下，两个末端都是平末端的siRNA将在其整个长度范围内是双链的。
术语“约”、“大约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％ 的范围，例如1％至15％之间、在1％至10％之间、在1％至5％之间、在0.5％至5％之间、在0.5％至1％之间变化，本公开中，数字或数值范围之前有术语“约”的每种情况也包括给定数的实施方案。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
本公开中，术语“包含”可替换为“由……组成”。
如无特殊说明，本公开的“化合物”、“化学修饰”、“配体”、“dsRNA”、“核酸”和“RNAi”均可独立地以盐、混合盐或非盐(例如游离酸或游离碱)的形式存在。当以盐或混合盐的形式存在时，其可为药学上可接受的盐。
“药学上可接受的盐”可选自无机盐或有机盐，也可包括药学上可接受的酸加成盐和药学上可接受的碱加成盐。
“药学上可接受的酸加成盐”是指能够保留游离碱的生物有效性而无其它副作用的，与无机酸或有机酸所形成的盐。无机酸盐包括但不限于盐酸盐、氢溴酸盐、硫酸盐、硝酸盐、磷酸盐等；有机酸盐包括但不限于甲酸盐、乙酸盐、2,2-二氯乙酸盐、三氟乙酸盐、丙酸盐、己酸盐、辛酸盐、癸酸盐、十一碳烯酸盐、乙醇酸盐、葡糖酸盐、乳酸盐、癸二酸盐、己二酸盐、戊二酸盐、丙二酸盐、草酸盐、马来酸盐、琥珀酸盐、富马酸盐、酒石酸盐、柠檬酸盐、棕榈酸盐、硬脂酸盐、油酸盐、肉桂酸盐、月桂酸盐、苹果酸盐、谷氨酸盐、焦谷氨酸盐、天冬氨酸盐、苯甲酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐、海藻酸盐、抗坏血酸盐、水杨酸盐、4-氨基水杨酸盐、萘二磺酸盐等。这些盐可通过本领域已知的方法制备。
“药学上可接受的碱加成盐”是指能够保持游离酸的生物有效性而无其它副作用的、与无机碱或有机碱所形成的盐。衍生自无机碱的盐包括但不限于钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐、锌盐、铜盐、锰盐、铝盐等。优选的无机盐为铵盐、钠盐、钾盐、钙盐及镁盐，优选钠盐。衍生自有机碱的盐包括但不限于以下的盐：伯胺类、仲胺类及叔胺类，被取代的胺类，包括天然的被取代胺类、环状胺类及碱性离子交换树脂，例如氨、异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、乙醇胺、二乙醇胺、三乙醇胺、二甲基乙醇胺、2-二甲氨基乙醇、2-二乙氨基乙醇、二环己胺、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、胆碱、甜菜碱、乙二胺、葡萄糖胺、甲基葡萄糖胺、可可碱、嘌呤、哌嗪、哌啶、N-乙基哌啶、聚胺树脂等。优选的有机碱包括异丙胺、二乙胺、乙醇胺、三甲胺、二环己基胺、胆碱及咖啡因。这些盐可通过本领域已知的方法制备。
“烷基”指饱和的脂族烃基团，例如包括1至30个碳原子的直链和支链基团(C 1-C 30烷基)，又例如含有1至6个碳原子的烷基(C 1-C 6烷基)，又例如1至3个碳原子的烷基(C 1-C 3烷基)。非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基及其各种支链异构体等。
术语“烯基”是指含有至少一个双键的烃基。烯基的非限制性实例包括但不限于：乙烯基、1-丙烯基、2-丙烯基、1-丁烯基或2-丁烯基及其各种支链异构体。
术语“炔基”指含有至少一个三键的烃基。炔基的非限制性实例包括但不限于：乙炔基、1-丙炔基、2-丙炔基、1-丁炔基或2-丁炔基及其各种支链异构体。
术语“烷氧基”指-O-(烷基)，其中烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基。
“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至6个碳原子，更优选包含5-6个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基等；多环环烷基包括螺环、并环和桥环的环烷基。
“杂环烷基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1至4个是杂原子；更优选包含3至7个环原子。“杂环烷基”非限制性实例包括：
Figure PCTCN2022139585-appb-000166
Figure PCTCN2022139585-appb-000167
等等。
所述杂环烷基环可以稠合于芳基或杂芳基环上，其中与母体结构连接在一起的环为杂环烷基，其非限制性实例包括：
Figure PCTCN2022139585-appb-000168
等。
“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为6至12元，例如苯基和萘基。所述芳基环可以稠 合于杂芳基、杂环烷基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性实例包括：
Figure PCTCN2022139585-appb-000169
“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为6至12元，更优选为5元或6元。例如。其非限制性实例包括：咪唑基、呋喃基、噻吩基、噻唑基、吡唑基、噁唑基(oxazolyl)、异噁唑基(isoxazolyl)、吡咯基、四唑基、吡啶基、嘧啶基、噻二唑、吡嗪基、三唑基、吲唑基、苯并咪唑基、
Figure PCTCN2022139585-appb-000170
等。
所述杂芳基环可以稠合于芳基、杂环烷基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性实例包括：
Figure PCTCN2022139585-appb-000171
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氰基”指-CN。
术语“氨基”指-NH 2。
术语“硝基”指-NO 2。
术语“氧代”指＝O取代基。
本公开中，“磷酸酯基团”可为磷酸一酯基团、磷酸二酯基团或磷酸三酯基团，优选磷酸二酯基团。
本公开中，硫代磷酸二酯基是指一个非桥接氧原子被硫原子替代而修饰的磷 酸二酯基，可用
Figure PCTCN2022139585-appb-000172
(M为S原子)互换使用。
“取代”指基团中的一个或多个氢原子，优选为最多5个，更优选为1至3个氢原子彼此独立地被相应数目的取代基取代。当取代基是酮或氧代(即，＝O)时，则原子上有两个(2个)氢被替代。
本公开上下文中，基团
Figure PCTCN2022139585-appb-000173
中的
Figure PCTCN2022139585-appb-000174
部分可以替换为能够与相邻核苷酸实现连接的任意基团。
术语“连接”，当表示两个分子之间的联系时，指两个分子通过共价键连接或者两个分子经由非共价键(例如，氢键或离子键)关联，包括直接连接、间接连接。
术语“直接连接”指第一化合物或基团与第二化合物或基团在没有任何间插原子或原子基团的情况下连接。
术语“间接连接”指第一化合物或基团与第二化合物或基团通过中间基团、化合物或分子(例如，连接基团)连接。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
“药学上可接受的赋形剂”包括但不限于任何已经被批准对于人类或家畜动物使用可接受的任何助剂、载体、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、增香剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂。
“有效量”或“有效剂量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
如本文所使用的，“对象”、“患者”、“受试者”或“个体”可互换使用，包括人类或者非人类动物，例如哺乳动物，例如人或猴。
本公开提供的dsRNA或siRNA可以通过本领域常规的制备方法(例如固相合成和液相合成的方法)得到。其中，固相合成已经有商业化订制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的dsRNA或siRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入dsRNA或siRNA的方法也是本领域技术人员所熟知的。
附图说明
图1为TRD002218和TRD007205在给药后第7天TTR中mRNA的剩余表达量。
图2为TRD002218和TRD007205在给药后第28天TTR中mRNA的剩余表达量。
图3为对人源化小鼠(hF11)血清FXI蛋白表达的抑制，#表示TRD0008003-1与TJR100362相比P<0.05；&表示TRD0008002-1与TJR100364相比P<0.05；*表示各组与空白对照相比P<0.05；****表示各组与空白对照相比P<0.0001；ns表示各组与空白对照相比无统计学差异。
具体实施方式
以下结合实施例进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，则该试剂可自任意分子生物学试剂的供应商以用于分子生物学应用的质量/纯度而获得。
实施例1：化学修饰的制备
1.1合成化合物1-1a和化合物1-1b
Figure PCTCN2022139585-appb-000175
将化合物1(500mg,3.42mmol)和三乙胺(Et 3N，692mg,6.84mmol,0.95mL)溶于二氯甲烷(DCM，10mL)中，冰浴下滴加4-甲苯磺酰氯(TsCl,717mg,3.76mmol)的二氯甲烷(10mL)溶液，滴加完毕后反应在室温下搅拌过夜，待反应完毕后，用水淬灭，水相用二氯甲烷(15mL)提取三次，合并的有机相先用饱和碳酸氢钠水溶液(10mL)洗涤，再用饱和食盐水(20mL)洗涤，随后减压蒸干溶剂得到粗品2(820mg,80％)，直接用于下一步反应。MS m/z：C 14H 21O 5S，[M+H] +理论：301.10实测：301.2。
Figure PCTCN2022139585-appb-000176
将化合物3(239mg,1.22mmol)溶解于二甲基甲酰胺(DMF，10mL)中，冰浴下加入NaH(60％溶解在矿物油中,93mg,2.33mmol)溶液，该反应下搅拌30分钟，然后滴加化合物2(350mg,1.16mmol)，滴加完毕后反应在60℃下搅拌5小时，反应完毕后，加水淬灭，水相用乙酸乙酯(15mL)提取三次，合并的有机相先用水(10mL)洗涤三次，再用饱和食盐水(10mL)洗涤，随后减压蒸干溶剂，经反相制备HPLC(C 18,条件:5-50％(A:H 2O,B:CH 3CN),流速:70mL/min)，冻干后得到220mg化合物4。MS m/z：C 19H 21N 5O 3Na，[M+Na] +理论：390.16，实测：390.3。
Figure PCTCN2022139585-appb-000177
室温下将化合物4(1.50g,4.08mmol)溶解于20mL的醋酸和水(4:1)的混合溶液中，60℃下搅拌30分钟，待反应完毕后减压蒸干溶剂，经反相制备HPLC(C 18,条件:5-25％(A:H 2O,B:CH 3CN),流速:70mL/min)，冻干后得到1.10g化合物5。MS m/z：C 16H 18N 5O 3，[M+H] +理论：328.13，实测：328.4。
Figure PCTCN2022139585-appb-000178
将化合物5(1.00g,3.05mmol)溶于吡啶(Py，10mL)中，冰浴下滴4,4'-双甲氧基三苯甲基氯(DMTrCl,1.50g,4.58mmol)的吡啶(5mL)溶液，滴加完毕后反应在室温下搅拌过夜，待反应完毕后，用水淬灭，减压蒸干溶剂，经反相制备HPLC(C 18,条件:5-80％(A:H 2O,B:CH 3CN),流速:70mL/min)，冻干后得到1.00g化合物6。MS m/z：C 37H 36N 5O 5，[M-H] +理论：630.26，实测：630.5。消旋体化合物6经手 性柱(Daicel
Figure PCTCN2022139585-appb-000179
IE 250*4.6mm,5μm,A:正己烷,B:乙醇)拆分得410mg 6A(-)和435mg 6B(+)。
Figure PCTCN2022139585-appb-000180
将化合物6A(-)(200mg,0.32mmol)，四氮唑(11mg,0.16mmol)，N-甲基咪唑(5mg,0.06mmol)，3A分子筛(500mg)溶于10mL的乙腈中，室温下加入化合物7(144mg,0.48mmol)，在室温下搅拌过夜。反应完毕后，将分子筛过滤掉，加入二氯甲烷(30mL)，饱和碳酸氢钠水溶液(10mL)洗涤三次，再用饱和食盐水(20mL)洗涤，滤液旋干并经反相制备HPLC(C 18,条件:5-100％(A:水,B:CH 3CN),流速:70mL/min)，冻干后得到200mg化合物1-1a。MS m/z：C 40H 39N 6O 7P，[M-二异丙基+OH] +理论：747.26，实测：747.6。1H NMR(400MHz,乙腈-d 3)δ7.56,7.54(2s,1H),7.36-7.27(m,2H),7.24-7.21(m,7H),6.83-6.80(m,4H),4.12-4.10(m,2H),3.75-3.68(m,10H),3.20-2.80(m,2H),2.68-2.54(m,4H),1.22-1.04(m,18H)。
Figure PCTCN2022139585-appb-000181
将化合物6B(+)(200mg,0.32mmol)，四氮唑(11mg,0.16mmol)，N-甲基咪唑(5mg,0.06mmol)，3A分子筛(500mg)溶于10mL的乙腈中，室温下加入化合物7(144mg,0.48mmol)，在室温下搅拌过夜。反应完毕后，将分子筛过滤掉，加入二氯甲烷(30mL)，饱和碳酸氢钠水溶液(10mL)洗涤三次，再用饱和食盐水(20mL)洗涤，滤液旋干并经反相制备HPLC(C 18,条件:5-100％(A:水,B:CH 3CN),流速:70mL/min)，冻干后得到200mg化合物1-1b。MS m/z：C 40H 39N6O 7P，[M-二异丙基+OH] +理论：747.26，实测：747.5。
1.2合成化合物1-6a
Figure PCTCN2022139585-appb-000182
将化合物1(10g,68.404mmol),化合物2(15g,62.186mmol)和三苯基膦(32.62g,124.371mmol)溶于无水THF(30mL),于0℃下缓慢滴加DIAD(24.656mL，124.371mmol)。该反应液在25℃下反应12h.LCMS显示反应完成。将该反应液用乙酸乙酯(200mL)和水(200mL)萃取，有机相干燥将滤液浓缩，得到的残留物用正向柱纯化(DCM/MeOH＝10/1)得目标产物3(20g)。
Figure PCTCN2022139585-appb-000183
将化合物3(20g,28.585mmol)溶于醋酸(24mL,426.016mmol)和H 2O(12mL)中，60℃搅拌1小时。之后将反应液旋干加入THF(12mL)和H 2O(12mL)，80℃搅拌7小时。LCMS显示反应完成。将反应液加入乙酸乙酯(200mL)和水(100mL)萃取，水相加入碳酸钠固体直到水相有大量固体析出。将固体过滤，用水洗涤，将滤饼用油泵拉干，得到目标化合物5(9g)。
Figure PCTCN2022139585-appb-000184
在氮气保护下,将化合物5(6.8g,18.581mmol)溶于吡啶(80mL)中，于0℃下缓慢加入TMSCl(14.250mL,111.489mmol)，搅拌2h。之后在0℃下加入异丁酰氯(2.044mL,19.511mmol)，于25℃下搅拌1h.LCMS显示反应完成。用二氯甲烷(200mL)和水(200mL)萃取，有机相干燥旋干后拌样，用正向柱纯化(DCM:MeOH＝10:1)过柱，在4.8％处出峰)，得到黄色油状化合物6(12g).
Figure PCTCN2022139585-appb-000185
在氮气保护下,将化合物6(5.5g,12.392mmol)溶于吡啶(30mL)，加入MOLECULAR SIEVE 4A 1/16(7g,12.392mmol)，然后在0℃下分批加入DMTrCl(5.04g,14.870mmol)固体，25℃反应2h.TLC(PE:EtOAc＝1:1,Rf＝0.69)显示反应已经完成。该反应液和TJN200879-040-P1合并一起处理。将反应液用乙酸乙酯(200mL)和水(200mL)萃取，有机相干燥旋干后拌样用正向柱纯化(PE:EtOAc过柱，在84％处出峰)，得到黄色油状化合物7(12g)。
Figure PCTCN2022139585-appb-000186
将化合物7(12g,15.389mmol)溶于EtOAc(140mL)，加入湿钯碳Pd/C(7g,15.389mmol)该反应液在25℃，氢气(15Psi)下反应2小时。TLC(PE:EtOAc＝0:1,Rf＝0.09)显示反应已经完成。将反应液过滤，滤饼用乙酸乙酯(30mL)冲洗三遍后，收集滤液。滤液旋干后加入50mL二氯甲烷和2mL三乙胺拌样用正向柱纯化(DCM:MeOH＝10:1过柱，在0.5％处出峰)，得到9g(黄色泡沫状固体).将所得消旋化合物SFC拆分，得到产品目标化合物7A(-)(3.9g)和目标化合物7B(+)(3.8g)。
Figure PCTCN2022139585-appb-000187
将化合物7A(-)(3.30g,5.40mmol)，四氮唑(190mg,2.70mmol)，1-甲基咪唑(90mg,1.10mmol)，3A分子筛(500mg)溶于30mL的乙腈中，室温下加入化合物8(2.50g,8.10mmol)，在室温下搅拌2h。反应完毕后，将分子筛过滤掉，加入DCM(150mL)，饱和碳酸氢钠水溶液洗涤(30mL*3)，再用饱和食盐水(30mL) 洗涤，滤液旋干并经反相制备HPLC(C18，条件：5-100％(A：水，B:CH3CN),流速:70mL/min)，冻干后得到1-6a(2.9g,66％)。MS m/z：C43H55N7O7P[M+H]+，理论：812.38，实测：812.5。1H NMR(400MHz,乙腈-d3)δ7.56,7.54(2s,1H),7.36-7.27(m,2H),7.24-7.21(m,7H),6.83-6.80(m,4H),4.12-4.10(m,2H),3.75-3.68(m,10H),3.20-2.80(m,2H),2.68-2.54(m,4H),1.22-1.04(m,18H)。
1.3合成化合物1-7a
Figure PCTCN2022139585-appb-000188
在氮气保护下，将化合物1(5g,23.1272mmol),化合物2(6.76g,46.254mmol)和三苯基磷(7.28g,27.753mmol)溶于30mL二氧六环中，于0℃缓慢滴加入DEAD(5.502mL,27.753mmol)。滴加完成后，反应缓慢升温至25℃继续反应1h。在反应液里加入100mL H 2O和100mL EtOAc萃取，有机相合并干燥过滤浓缩后拌样过柱，用正向柱纯化(PE:EtOAc＝1:1过柱得目标产物(4g)。
Figure PCTCN2022139585-appb-000189
将化合物3(3.3g)溶于HOAc(16mL)和H 2O(4mL),油浴60℃加热0.5h.将反应液旋干得到的残留物用正向柱纯化(PE:EtOAc＝0:1过柱)，得到目标产物4(3g)。
Figure PCTCN2022139585-appb-000190
将化合物4(3g,8.873mmol)溶于5mL吡啶中，在氮气保护下于0℃缓慢滴加DMTrCl(3.91g,11.535mmol)的10mL吡啶的溶液。滴加完毕后反应升温至 25℃并继续反应1h。在反应液中加入50mL水和100mL乙酸乙酯萃取。水相再用100mL乙酸乙酯萃取三次，有机相合并干燥过滤浓缩用正向柱纯化(用PE:EtOAc＝2:1)。得到目标产物5(4g)。
Figure PCTCN2022139585-appb-000191
将化合物5(4g,5.769mmol)溶于甲醇(10mL),加入饱和的NH3甲醇溶液(40mL),0℃反应6h.将反应液旋干用正向柱纯化(用PE:EtOAc＝0:1)得消旋化合物2.4g SFC拆分，得到目标产物6A(750mg,100％纯度)和目标产物6B(400mg,99.16％纯度)。
Figure PCTCN2022139585-appb-000192
将化合物6A(-)(700mg,1.40mmol)，四氮唑(50mg,0.70mmol)，1-甲基咪唑(23mg,0.28mmol)，3A分子筛(500mg)溶于10mL的乙腈中，室温下加入化合物7(630mg,2.10mmol)，在室温下搅拌2h。反应完毕后，将分子筛过滤掉，加入DCM(50mL)，饱和碳酸氢钠水溶液洗涤(10mL*3)，再用饱和食盐水(20mL)洗涤，滤液旋干并经反相制备HPLC(C18，条件：5-100％(A:水B:CH 3CN),流速:70mL/min)，冻干后得到1-7a(700mg,72％)。MS m/z：C38H47N4O7PNa[M+Na]+，理论：725.32，实测：725.5。
1.4合成化合物1-8a
Figure PCTCN2022139585-appb-000193
将化合物1(8.5g,76.508mmol),化合物2(30.64g,91.809mmol)溶于DMF(150mL),加入CS2CO3(29.91g,91.809mmol)，反应于氮气保护下，90℃反应12h。LCMS检测反应完成。将反应液过滤，油泵旋干，正向柱分离纯化(80g,DCM/MeOH＝10/1至5/1)得到目标产物3(13.5g,80％纯度)。
Figure PCTCN2022139585-appb-000194
将化合物3(10.5g,35.105mmol)溶于吡啶(65mL)和CH 3CN(65mL)，向溶液中滴加BzCl(4.894mL,42.126mmol)，于25℃反应2h。LCMS检测大部分原料反应完成，加H 2O(100mL)淬灭，EtOAc(100mL X 3)萃取，干燥旋干，柱分离(合并TJN200872-101)纯化(80g,PE/EtOAc＝10/1至0/1,DCM/MeOH＝10/1)得到目标产物4(14g,90％纯度)。
Figure PCTCN2022139585-appb-000195
将化合物4(14g,36.694mmol)溶于HOAc(56mL,314.796mmol)和H 2O(14mL)，于60℃反应2h,LCMS显示反应完成。油泵浓缩，正向柱分离(40g,DCM/MeOH＝1/0至5/1)得到目标产物5(8.4g,90％纯度&2.4g,80％纯度)。
Figure PCTCN2022139585-appb-000196
将化合物5(7.4g,21.957mmol),DMAP(0.54g,4.391mmol),MOLECULAR SIEVE 4A(11.1g,2.967mmol)溶于吡啶(60mL)，冰浴下搅拌10min，然后加入DMTrCl(8.93g,26.348mmol)，反应搅拌1.8h.LCMS检测约19％原料剩余，约60％目标MS。合并(TJN200872-105&106)一起纯化。向反应液中加入H 2O(50mL)，经DCM(50mL×3)萃取，干燥，旋干，柱分离(120g,PE/(EA:DCM:TEA＝1:1:0.05)＝1/0至0/1至DCM/MeOH＝10/1)得到目标化合物6(11g,89％纯度,TJN200872-105&106&107),回收原料(3.0g,70％纯度)。
Figure PCTCN2022139585-appb-000197
化合物6(15g,22.041mmol)经SFC(DAICEL CHIRALPAK AD(250mm*50mm,10um)；0.1％NH 3H 2O EtOH，B:45％-45％；200ml/min)分离得到目标产物6A(5.33g,94.29％纯度),目标产物6B(6.14g,97.91％纯度),化合物6回收1.0g。
Figure PCTCN2022139585-appb-000198
将化合物6B(-)(5.4g,8.92mmol)，四氮唑(312mg,4.46mmol)，1-甲基咪唑(146mg,1.78mmol)，3A分子筛(500mg)溶于40mL的乙腈中，室温下加入化合物7(4g,13.4mmol)，在室温下搅拌2h。反应完毕后，将分子筛过滤掉，加入DCM(200mL)，饱和碳酸氢钠水溶液洗涤(30mL*3)，再用饱和食盐水(50mL)洗 涤，滤液旋干并经反相制备HPLC(C18，条件：5-100％(A：水，B:CH 3CN),流速:70mL/min)，冻干后得到1-8a(5.8g,80％)。MS m/z：C45H51N5O7P，[M+H]+，理论：804.36，实测：804.4。
实施例2：siRNA的合成
siRNA的合成与通常的亚磷酰胺固相合成法无异，在合成AS链5’第7位修饰的核苷酸时，使用上述合成的亚磷酰胺单体替换母序列原核苷酸。合成过程简要描述如下：于Dr.Oligo48合成器(Biolytic)上，以Universal CPG载体为起始，根据合成程序逐个连接核苷亚磷酰胺单体。除上述描述的AS链5’第7位的核苷亚磷酰胺单体外，其余核苷单体原料2’-F RNA、2’-O-甲基RNA等核苷亚磷酰胺单体购自上海兆维或苏州吉玛。采用5-乙基硫-1H-四唑(ETT)作为活化剂(0.6M乙腈溶液)，使用0.22M的PADS溶于1:1体积比的乙腈和三甲基吡啶(苏州柯乐玛)溶液作为硫化试剂，使用碘吡啶/水溶液(柯乐玛)作为氧化剂。
固相合成完成后，寡核糖核苷酸自该固体支撑物裂解，采用3:1的28％氨水和乙醇溶液在50℃条件下浸泡16小时。然后离心，将上清液转移到另一个离心管中，浓缩蒸发干后，使用C18反向色谱纯化，流动相为0.1M TEAA和乙腈，并使用3％三氟乙酸溶液脱除DMTr。目标寡核苷酸收集后冻干，并经LC-MS鉴定为目标产物，再经过UV(260nm)定量。
所得到的单链寡核苷酸，根据等摩尔比，按照互补配对，退火，最后所得到的dsRNA溶于1×PBS中，并调整至实验所需浓度备用。
实施例3：psiCHECK活性筛选实验
dsRNA合成见前述，质粒来源于生工生物工程(上海)股份有限公司。psiCHECK实验耗材如表1所示。
表1.psiCHECK实验耗材和试剂
Figure PCTCN2022139585-appb-000199
实验步骤：细胞铺板、细胞转染，其中，转染复合物具体配制量如表2所示。
表2. 96孔板每孔所需转染复合物用量
Figure PCTCN2022139585-appb-000200
Figure PCTCN2022139585-appb-000201
注：Lipo：0.2μL/孔；质粒：0.05μL/孔；Opti-MEM：10μL/孔。
依照表3，根据不同的实验需求稀释至不同浓度作为工作液备用，现用现配。转染24h后，按照
Figure PCTCN2022139585-appb-000202
Luciferase Assay System检测试剂盒的实验操作方案进行检测。计算相对值Ratio＝Ren/Fir(海肾/萤火虫比值)；计算抑制率1-(Ratio+siRNA/仅报告基因时的Ratio)*100％＝抑制率(％)；本公开中，剩余活性％(也称为mRNA剩余表达量％或mRNA剩余表达比例)＝100％-抑制率(％)。
表3.多浓度点siRNA稀释方案
终浓度(nM) 加水与dsRNA
40 4μL siRNA(20μM)+96μL H 2O
13.33333333 30μL dsRNA+60μL H 2O
4.444444444 30μL dsRNA+60μL H 2O
1.481481481 30μL dsRNA+60μL H 2O
0.49382716 30μL dsRNA+60μL H 2O
0.164609053 30μL dsRNA+60μL H 2O
0.054869684 30μL dsRNA+60μL H 2O
0.018289895 30μL dsRNA+60μL H 2O
0.006096632 30μL dsRNA+60μL H 2O
0.002032211 30μL dsRNA+60μL H 2O
0.000677404 30μL dsRNA+60μL H 2O
实施例4：不同化学修饰表征
Figure PCTCN2022139585-appb-000203
其中：我们将由2-羟甲基-1,3-丙二醇为起始原料合成的核苷酸定义hmpNA；
(+)hmpNA(A)为实施例1.1节中核苷亚磷酰胺单体1-1b通过固相合成获得，绝对构型为(S)-hmpNA(A)；
(-)hmpNA(A)为实施例1.1节中核苷亚磷酰胺单体1-1a通过固相合成获得，绝对构型为(R)-hmpNA(A)；
类似的，替换hmpNA的碱基种类，通过固相合成获得以下结构并确认绝对构型：
(+)hmpNA(G)，绝对构型为(S)-hmpNA(G)；
(-)hmpNA(G)，绝对构型为(R)-hmpNA(G)；
(+)hmpNA(C)，绝对构型为(S)-hmpNA(C)；
(-)hmpNA(C)，绝对构型为(R)-hmpNA(C)；
(+)hmpNA(U)，绝对构型为(R)-hmpNA(U)；
(-)hmpNA(U)，绝对构型为(S)-hmpNA(U)。
(S)-hmpNA(G)，(R)-hmpNA(G)，(S)-hmpNA(C)，(R)-hmpNA(C)，(S)-hmpNA(U)和(R)-hmpNA(U)的绝对构型由其中间体或衍生物经X-Ray衍射而确认。
中间体或衍生物的结构为：
Figure PCTCN2022139585-appb-000204
TJ-NA067:检测晶体为无色块状(0.30×0.10×0.04mm3)，属于单斜晶系P21空间群。晶胞参数
Figure PCTCN2022139585-appb-000205
α＝90°,β＝118.015(4)°,γ＝90°,
Figure PCTCN2022139585-appb-000206
Z＝4。计算密度Dc＝1.389g/cm3，单胞中电子数F(000)＝504.0，单胞的线性吸收系数μ(Cu Kα)＝0.840mm–1，衍射实验温度T＝150.00(11)K。
Figure PCTCN2022139585-appb-000207
6A(+):检测晶体为无色块状(0.30×0.20×0.10mm3)，属于单斜晶系P21空间群。晶胞参数
Figure PCTCN2022139585-appb-000208
α＝90°,β＝113.876(3)°,γ＝90°,
Figure PCTCN2022139585-appb-000209
Z＝2。计算密度Dc＝0.999g/cm3，单胞中电子数F(000)＝1318.0，单胞的线性吸收系数μ(Cu Kα)＝0.570mm–1，衍射实验温度T＝100.01(18)K。
Figure PCTCN2022139585-appb-000210
TJ-NA048:检测晶体为无色针状(0.30×0.04×0.04mm3)，属于单斜晶系P1空间群。晶胞参数
Figure PCTCN2022139585-appb-000211
α＝85.007(4)°,β＝88.052(4)°,γ＝70.532(4)°,
Figure PCTCN2022139585-appb-000212
Z＝2。计算密度Dc＝1.366g/cm3，单胞中电子数F(000)＝620.0，单胞的线性吸收系数μ(Cu Kα)＝0.856mm–1，衍射实验温度T＝150.00(13)K。
Figure PCTCN2022139585-appb-000213
TJ-NA092:检测晶体为无色棱柱状(0.30×0.10×0.10mm3)，属于三斜晶系P1空间群。晶胞参数
Figure PCTCN2022139585-appb-000214
α＝93.146(2)°,β＝101.266(2)°,γ＝96.134(2)°,
Figure PCTCN2022139585-appb-000215
Z＝2。计算密度Dc＝1.412g/cm3，单胞中电子数F(000)＝228.0，单胞的线性吸收系数μ(Cu Kα)＝0.945mm–1，衍射实验温度T＝100.00(10)K。
实施例5：包含不同化学修饰的siRNA的序列依赖性实验
已知GNA修饰具有siRNA序列依赖性，因此发明人在多条不同序列上测试了本公开的实验化合物。使用了靶向不同基因(ANGPTL3、HBV-S、HBV-X)mRNA的siRNA(序列如表4所示)，使用实施例1的化合物(+)hmpNA(A)、(-)hmpNA(A)和作为对照的GNA (A)化合物修饰AS链5’端第7位(序列如表5所示)，再与母序列比较在靶活性和脱靶活性。实验方法参照实施例3。
表4.靶向不同基因的siRNA序列
Figure PCTCN2022139585-appb-000216
Figure PCTCN2022139585-appb-000217
表5.靶向不同基因的包含化学修饰的siRNA序列
Figure PCTCN2022139585-appb-000218
在靶活性实验结果参见表6，GNA (A)显现出明显的序列依赖性，不同序列的在靶活性差异明显。本公开的实验化合物没有显示出明显的序列依赖性，普遍适用性更强。
脱靶活性实验结果参见表7，可以看出，相对于母序列，本公开的修饰明显降低了siRNA的脱靶活性。
表6.针对不同靶序列的siRNA的在靶活性结果
Figure PCTCN2022139585-appb-000219
Figure PCTCN2022139585-appb-000220
表7.针对不同靶序列的siRNA的脱靶活性结果
Figure PCTCN2022139585-appb-000221
实施例6：配体的制备(NAG0052、L96)
化合物NAG0024、NAG0026购买自天津药明康德新药开发有限公司。除非特别说明，以下实施例中所用的试剂均为市售商品。
化合物NAG0052的合成
起始原料化合物1采购自江苏倍达医药科技有限公司。
Figure PCTCN2022139585-appb-000222
Figure PCTCN2022139585-appb-000223
化合物2
在0℃以及氮气保护下，往化合物1(12.3mL,101mmol)的THF(300mL)溶液中分批加入NaH(12.2g,304mmol，纯度60％)。该混合物在20℃下搅拌1小时之后再次冷却到0℃，接着往体系中逐滴加入苄溴(36.3mL,304mmol)，并且在20℃搅拌12小时。将该反应液用H 2O(100mL)淬灭后，用EtOAc(200mL×2)萃取。合并后的有机相用饱和食盐水(100mL)洗涤,Na 2SO 4干燥，过滤，浓缩得到的残留物经过硅胶柱层析分离后得到目标化合物2(20.0g,51.8mmol,产率51％)。
LCMS:t R＝2.615and 2.820min in 30-90AB_7min_220&254_Shimadzu.lcm(Xtimate C18,3um,2.1*30mm),MS(ESI)m/z＝351.2[M+Na] +。
1H NMR:(400MHz,CDCl 3)δppm 7.35-7.12(m,10H),5.06-4.95(m,1H),4.51-4.39(m,4H),4.24-3.87(m,2H),3.50-3.40(m,2H),3.38-3.20(m,3H),2.20-1.91 (m,2H)。
化合物3和4
在20℃以及氮气保护下，往化合物2(13.0g,33.6mmol)的DCM(300mL)溶液中一次性加入TMSCN(13.5mL,101mmol)，接着逐滴加入TMSOTf(9.14mL,50.5mmol)的DCM(30mL)溶液。该反应液在20℃下搅拌15小时。反应结束之后用饱和NaHCO 3水溶液(80mL)淬灭该体系，并且用DCM(150mL×2)萃取，合并后的有机相用饱和食盐水(80mL)洗涤，Na 2SO 4干燥，过滤以及浓缩后通过硅胶柱层析分离后得到目标化合物3(3.30g,9.18mmol,产率27％)以及淡黄色油状液体化合物4(8.50g,9.18mmol,产率70％)。
化合物3
1H NMR:(400MHz,CDCl 3)δppm 7.42-7.29(m,10H),4.81(t,J＝7.8Hz,1H),4.65-4.49(m,4H),4.30-4.21(m,2H),3.65-3.57(m,1H),3.57-3.49(m,1H),2.49-2.40(m,2H)。
化合物4
1H NMR:(400MHz,CDCl 3)δppm 7.42-7.26(m,10H),4.93-4.87(m,1H),4.65-4.48(m,4H),4.43-4.38(m,1H),4.21-4.17(m,1H),3.79-3.70(m,1H),3.54(d,J＝4.0Hz,1H),2.45-2.37(m,2H)。
化合物5
在0℃及氮气保护下将化合物4(3.00g,9.28mmol)的THF(15mL)溶液，滴加到LiAlH 4(0.79g,20.9mmol)的THF(15mL)溶液中，滴加完后体系在0℃反应1小时。TLC(PE:EtOAc＝3:1)监测到原料完全消失。向反应液中缓慢加入十水硫酸钠，加至不冒泡为止。之后将反应液过滤，滤饼用二氯甲烷(60mL)洗涤三次后，收集滤液旋干，得目标化合物5(3.00g,产率90％).
1H NMR:(400MHz,DMSO-d 6)δppm 7.40-7.14(m,10H),4.54-4.38(m,4H),4.06-3.99(m,2H),3.91(q,J＝6.4Hz,1H),3.48-3.37(m,2H),2.67-2.52(m,2H),2.21-2.18(m,1H),1.77-1.73(m,1H)。
化合物6
在氮气保护下，将化合物5(3.00g,8.25mmol)溶于DCM(30mL)，加入TEA(3.44mL,24.7mmol)和CbzCl(1.76mL,12.4mmol)，20℃反应2小时。LCMS显示反应完成。将反应液加入二氯甲烷(30mL)和水(60mL)萃取。有机相用水(60mL×3)洗涤三次，无水硫酸钠干燥，浓缩用正向柱纯化(PE:EtOAc＝1:1)，得到目标化合物6(2.5g,产率90％)。
LCMS:t R＝0.810min in 5-95AB_1min,MS(ESI)m/z＝462.2[M+H] +。
1H NMR:(400MHz,CDCl 3)δppm 7.39-7.29(m,15H),5.35(s,1H),5.15-5.01(m,2H),4.72(d,J＝6.0Hz,1H),4.54-4.40(m,3H),4.26(s,1H),4.23-4.18(m,1H),4.11-4.04(m,1H),3.54-3.41(m,3H),3.37-3.25(m,1H),2.34-2.23(m,1H),1.85-1.79(m,1H)。
化合物7
在氮气保护下，将化合物6(2.00g,3.90mmol)溶于DCM(5mL)，在-78℃下加入BCl 3的THF溶液(1M,27.3mL)，反应1小时。TLC(DCM:MeOH＝10:1)监测到原料完全消失。将反应液在-78℃下加入甲醇(20mL)淬灭，浓缩，用正向柱纯化(DCM:MeOH＝10:1)，得到目标化合物7(2.00g,产率60％)。
1H NMR:(400MHz,CD 3OD)δppm 7.41-7.23(m,5H),5.08(s,2H),4.25-4.07(m,2H),3.85-3.75(m,1H),3.63-3.56(m,1H),3.54-3.48(m,1H),3.30-3.27(m,2H),2.34-2.21(m,1H),1.71-1.64(m,1H)。
化合物8
在氮气保护下，将化合物7(0.50g,1.78mmol)溶于吡啶(5mL)中，在0℃下加入4A分子筛(500mg)和DMTrCl(0.66mL,2.13mmol)，之后升温至20℃反应1.5小时。TLC(PE:EtOAc＝2:1)监测到原料完全消失。将反应液加入乙酸乙酯(60mL)和水(60mL)萃取，有机相用水(60mL×3)洗涤三次后用无水硫酸钠干燥，浓缩，用正向柱纯化(PE:EtOAc＝1:1)，得到目标化合物8(800mg,产率90％)。
1H NMR:(400MHz,CDCl 3)δppm 7.44(d,J＝7.6Hz,2H),7.37-7.23(m,11H),7.22-7.15(m,1H),6.84(d,J＝8.8Hz,4H),5.09(s,2H),4.31-4.17(m,2H),4.02-3.91(m,1H),3.84-3.73(m,6H),3.33(s,1H),3.28(s,1H),3.19-3.01(m,2H),2.34-2.25(m,1H),1.70-1.62(m,1H)。
化合物9
将化合物8(800mg,1.234mmol)溶于EtOAc(5mL)，加入Pd/C 10％(800mg,7.517mmol)，反应在H 2条件(15Psi)，20℃下反应1小时。LCMS显示反应已经完成。反应液过滤，滤饼用二氯甲烷(100mL)和甲醇(100mL)洗涤三次，浓缩，经过反相柱分离得到化合物9(300mg,54％)。
LCMS:t R＝2.586min in 10-80CD_3min MS(ESI)m/z＝450.2[M+H] +。
化合物11
将化合物10(435mg,1.780mmol)溶于DCM(10mL)，加入DIEA(0.441mL,2.67mmol)和HATU(677mg,1.78mmol)后,再加入化合物9(400mg,0.890mmol)，20℃反应1小时。TLC(DCM:MeOH＝10:1)监测反应完成。将反应液加入二氯甲烷(60mL)和水(60mL)萃取，有机相用水(60mL x 3)洗涤三次,无水硫酸钠干燥，浓缩用正向柱纯化(PE:EtOAc＝0:1过柱，在100％处出产品峰),得到目标化合物11(600mg,产率90％)。
LCMS:t R＝2.745min in 30-90CD_3min,MS(ESI)m/z＝698.4[M+Na] +。
1H NMR:(400MHz,CD 3OD)δppm 7.46-7.38(m,2H),7.35-7.24(m,6H),7.22-7.16(m,1H),6.90-6.78(m,4H),4.29-4.21(m,2H),4.02-3.95(m,1H),3.77(s,6H),3.66-3.62(m,3H),3.41(s,1H),3.18-3.04(m,2H),2.36-2.17(m,5H),1.71-1.50(m,5H),1.39-1.25(m,14H)。
化合物12
将化合物11(600mg,0.799mmol)溶于THF(3mL)和H 2O(1mL)，加入LiOH.H 2O(134mg,3.20mmol)，20℃反应12小时。TLC(DCM:MeOH＝10:1)显示反应完成。将反应液旋干，用水(5mL)和甲醇(5mL)溶解，用反向柱纯化(H 2O:CH 3CN＝1:1,在35％左右出峰)，得到目标化合物12(460mg,产率100％，锂盐)。
LCMS:t R＝1.346min in 10-80CD_3min,MS(ESI)m/z＝684.3[M+Na] +。
HPLC:t R＝1.879min in 10-80CD_6min。
1H NMR:(400MHz,CD 3OD)δppm 7.47-7.39(m,2H),7.35-7.24(m,6H),7.22-7.15(m,1H),6.91-6.79(m,4H),4.31-4.18(m,2H),4.02-3.95(m,1H),3.78(s,6H),3.44-3.33(m,2H),3.18-3.04(m,2H),2.35-2.27(m,1H),2.24-2.10(m,4H),1.70-1.51(m,5H),1.31-1.23(m,12H)。
化合物13
室温环境，氮气保护下，将化合物NAG0024(271mg,0.151mmol)溶解于无水THF(2mL)和无水DMF(4mL)，加入3A分子筛，再依次加入化合物12(100mg,0.151mmol),HOBt(25mg,0.181mmol),DCC(38mg,0.181mmol)和DIEA(39mg,0.302mmol)。反应液45℃反应16h.LC-MS显示反应完全后，加水淬灭，过滤。滤液浓缩后，经C18反相柱纯化(H 2O/MeCN)，得到化合物13(210mg，产率57％)。
化合物NAG0052
室温环境下，化合物13(230mg，0.094mmol)溶于吡啶(5mL)，加入分子筛，加入DMAP(12mg，0.283mmol)，丁二酸酐(28mg，0.283mmol)。氮气保护，50℃搅拌16小时。LCMS检测反应完全，过滤旋干。过C18反相柱纯化后，由制备HPLC二次纯化，得到目标化合物NAG0052(123mg，0.048mmol，产率51％)。
MS(ESI)m/z＝2535.3[M-1] -.理论：2536.2。
1H NMR(400MHz,乙腈-d 3)δ7.48-7.43(m,2H),7.37-7.12(m,11H),7.00-6.85(m,10H),6.66(s,1H),5.31(dd,J＝3.4,1.1Hz,3H),5.20-5.13(m,1H),5.05(dd,J＝11.3,3.4Hz,3H),4.56(d,J＝8.5Hz,3H),4.30(dd,J＝7.7,5.3Hz,1H),4.18-3.93(m,14H),3.79(s,10H),3.65(q,J＝4.7,3.6Hz,13H),3.56-3.07(m,24H),2.56(s,6H),2.37(t,J＝5.8Hz,10H),2.17(t,J＝7.5Hz,9H),2.02-1.96(m,20H),1.88(s,8H),1.82-1.73(m,2H),1.60(dt,J＝15.0,7.3Hz,16H),1.27(s,13H)。
NAG0052’
化合物NAG0052经固相合成连接到序列上，再经过胺解后，NAG0052结构脱去一部分官能团成为前述NAG0052’。
L96的合成
Figure PCTCN2022139585-appb-000224
按照专利申请WO2014025805A1记载的方法制备获得。
实施例7：dsRNA的合成
1.自制带有载体的树脂
将含有羧酸基团的化合物NAG0052(157mg,0.062mmol)溶于无水DMF(3mL)，待底物完全溶解后，依次加入无水乙腈(4mL),DIEA(0.03mL,0.154mmol，2.5eq)和HBTU(35mg,0.093mmol，1.5eq)。反应液混合均匀后，再加入大孔胺甲基树脂(476mg,空白载量为0.41mmol/g,目标载量为0.1mmol/g)。将反应液放入摇床上(温度：25℃，转速：200rpm)振摇过夜。反应液过滤，滤饼依次分别用DCM，无水乙腈洗涤，收集固体，真空干燥过夜。
将上步固体分散于无水乙腈(5mL)，依次加入吡啶(0.18mL)，DMAP(3mg)，NMI(0.12mL)和CapB1(2.68mL)。将反应液放入摇床上(温度：25℃，转速：200rpm)振摇2h。反应液过滤，滤饼用无水乙腈洗涤，收集固体，真空干燥过夜，得到带有载体的树脂。载量经过测定为0.1mmol/g。
2.对于已经连接在树脂上的NAG0052，使用该树脂作为起始，按照核苷酸排布顺序自3’-5’方向逐一连接核苷单体。每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化或硫化四步反应。操作为本领域常规。
制得的dsRNA具有表8和表9中所示的有义链和反义链。
表8.dsRNA列表
dsRNA编号 有义链编号 反义链编号
TRD002218 TJR4373-SS TJR0414-AS
TRD007205 TJR013485S TJR0414-AS
表9.有义链和反义链的核酸列表
Figure PCTCN2022139585-appb-000225
表10.dsRNA的结构
Figure PCTCN2022139585-appb-000226
其中，TRD002218作为参比阳性化合物，Z表示siRNA。
实施例8：dsRNA在体内对靶基因mRNA表达量的抑制
本实验考察本公开的缀合不同结构的dsRNA在体内对靶基因mRNA表达量的抑制效率。
将雄性6-8周龄C57BL/6小鼠随机分组，每组共6只，每个时间点各3只，分别向每组小鼠给予本公开的TRD007205、参比阳性TRD002218以及PBS。
所有动物依据体总计算给药量，采用皮下注射方式单次给药，dsRNA给药剂量(以siRNA的量计)为1mg/kg，给药体积为5mL/kg。给药7天、28天后处死小鼠，收集肝脏，用RNA later(Sigma Aldrich公司)保存；随后用组织匀浆仪匀浆肝组织，再用组织RNA提取试剂盒(凡知医疗科技，FG0412)根据操作说明书标注的操作步骤提取得到肝组织总RNA。将总RNA反转录成cDNA并采用实时荧光定量PCR方法检测肝组织中的TTR mRNA的表达量。在该荧光定量PCR法中，以甘油醛3-磷酸脫氫酶(GAPDH)基因作为内参基因，使用针对TTR和GAPDH的Taqman探针引物分别检测TTR和GAPDH的mRNA表达量。
表11.小鼠体内实验化合物分组信息：
Figure PCTCN2022139585-appb-000227
表12.检测引物的序列参见如下：
Figure PCTCN2022139585-appb-000228
TTR mRNA表达量按照如下等式计算：
TTR mRNA表达量＝【(测试组TTR mRNA表达量/测试组GAPDH mRNA表达量)/(对照组TTR mRNA表达量/对照组GAPDH mRNA表达量)】x 100％。
给药7天、28天后，本公开的缀合不同结构的dsRNA的在体内对靶基因mRNA表达量的抑制效率分别见图1和图2。由图1的结果可知，TRD007205在给药后7天对于TTR mRNA的表达抑制具有良好的效果。由图2可知，给药28天后，TRD007205对靶基因mRNA表达量的抑制作用优于TRD002218。
实施例9：合成dsRNA
1.自制带有载体的树脂
具体操作同实施例7。
2.使用带有NAG0052的树脂作为起始，按照核苷酸排布顺序自3’-5’方向逐一连接核苷单体。每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化或硫化四步反应。具体参照实施例2的合成方法。
制得的dsRNA具有表13和表14中所示的有义链和反义链。dsRNA所对应的裸序列如表15所示。
表13.dsRNA列表
Figure PCTCN2022139585-appb-000229
Figure PCTCN2022139585-appb-000230
表14.dsRNA的序列
Figure PCTCN2022139585-appb-000231
表15.dsRNA有义链和反义链对应的裸序列
Figure PCTCN2022139585-appb-000232
Figure PCTCN2022139585-appb-000233
其中，(-)hmpNA(A)、(-)hmpNA(G)、(-)hmpNA(C)、(-)hmpNA(U)的结构参见实施例4。
NAG0052’的结构为：
Figure PCTCN2022139585-appb-000234
NAG1的结构为
Figure PCTCN2022139585-appb-000235
实施例10：dsRNA的在靶活性
在HEK293A细胞中采用11个浓度梯度对dsRNA进行体外分子水平模拟在靶活性筛选。
以人FXI基因构建siRNA对应的在靶序列，插入到psiCHECK-2质粒中。该质粒包含海肾荧光素酶基因及萤火虫荧光素酶基因。作为双报告基因系统，dsRNA的靶序列插入到海肾荧光素酶基因的3’UTR区域，dsRNA对于靶标序列的活性可以通过经萤火虫荧光素酶校准后的海肾荧光素酶表达情况的检测来反映，检测使用Dual-Luciferase Reporter Assay System(Promega，E2940)。
HEK293A细胞培养于含10％胎牛血清的DMEM高糖培养基中，在37℃，5％CO 2条件下培养。转染前24h，将HEK293A细胞接种于96孔板，接种密度为每孔8×10 3个细胞，每孔100μL培养基。
按照说明书，使用Lipofectamine2000(ThermoFisher，11668019)对细胞共转染dsRNA及对应质粒，Lipofectamine2000每孔使用0.2μL，质粒转染量为20ng 每孔。对于在靶序列质粒，dsRNA共设置11个浓度点，最高浓度点终浓度为20nM，3倍梯度稀释，20nM，6.6667nM，2.2222nM，0.7407nM，0.2469nM，0.0823nM，0.0274nM，0.0091nM，0.0030nM，0.0010nM和0.0003nM。转染后24h，采用Dual-Luciferase Reporter Assay System(Promega，E2940)检测在靶水平。检测序列的在靶活性如表16所示。
表16.dsRNA的psi-CHECK在靶活性筛选结果
Figure PCTCN2022139585-appb-000236
N/A表示：不适用；
以上结果表明，TJR100407、TRD008003-1、TRD008002-1、TRD008003和TRD008002化合物在psiCHECK系统针对FXI基因具有高水平的在靶抑制活性。
实施例11：dsRNA对人原代肝细胞(PHH)中人FXI的抑制
在人原代肝细胞(PHH)中采用7个浓度梯度对dsRNA进行PHH活性筛选。各个dsRNA样品转染起始终浓度为20nM，5倍梯度稀释和7个浓度点。
PHH细胞(Novabiosis,nHPHepatocytes)冻存于液氮中，转染前24h，将PHH细胞复苏后接种于96孔板，接种密度为每孔3×10 4个细胞，每孔80μL培养基。
参照产品说明手册，使用Lipofectamine RNAi MAX(ThermoFisher，13778150)转染dsRNA，dsRNA转染的梯度终浓度为10nM，2nM，0.4nM，0.08nM，0.016nM，0.0032nM和0.00064nM。在处理24小时后，使用高通量细胞RNA提取试剂盒(凡知，FG0417)进行细胞总RNA提取、RNA逆转录实验(Takara，6210B)和定量实时PCR检测(ThermoFisher，4444557)，测定人FXI的mRNA水平，根据GAPDH内参基因水平对人FXI、的mRNA水平进行校正。
其中，在定量实时PCR检测时，采用的是探针Q-PCR检测实验，其引物信息如表17所示。
表17.Taqman引物信息表
引物名称 SEQ ID NO 引物序列
hFXI-PF 40 TTTGCTGGGAGAGGGTGTTG
hFXI-PR 41 TACAAACACCAAGCCCCTTCA
hFXI-P 42 CCAGCATGCTTCCTCCACAGTAACACG
hGAPDH-PF1-MGB 43 GACCCCTTCATTGACCTCAACTAC
hGAPDH-PR1-MGB 44 TTGACGGTGCCATGGAATTT
hGAPDH-P1-MGB 45 TTACATGTTCCAATATGATTCC
结果分析方法
Q-PCR检测实验完毕后，按照系统自动设定的阈值获取相应的Ct值，可以通过Ct值比较，相对定量某个基因的表达：比较Ct指的是通过与内参基因Ct值之间的差值来计算基因表达差异,也称之是2 -△△Ct，△△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)]。抑制率(％)＝(1-目的基因表达剩余量)*100％。
结果以相对于经过对照dsRNA处理的细胞的人FXI的mRNA表达剩余百分比来表示。抑制率的IC 50结果见表18。
结果表明，TRD008002、TRD008003化合物在PHH细胞针对FXI基因具有高水平的在靶抑制活性。
表18.dsRNA在PHH细胞中的抑制活性
Figure PCTCN2022139585-appb-000237
实施例12：dsRNA对食蟹猴原代肝细胞(PCH)中FXI的抑制
在PCH细胞(妙顺生物，CCH100CY-V09201)中采用7个浓度梯度FXI的dsRNA进行反向转染活性筛选。各个dsRNA样品转染起始终浓度为10nM，5倍梯度稀释和7个浓度点。
PCH细胞冻存于液氮中，转染前将PCH细胞复苏后接种于96孔板，接种密度为每孔3×10 4个细胞，每孔90uL培养基(妙顺生物，HEP044，HEP024，HEP054，HEP064)。
参照产品说明手册，使用Lipofectamine RNAi MAX(ThermoFisher，13778150)转染dsRNA，dsRNA转染的梯度终浓度为10nM，2nM，0.4nM，0.08nM，0.016nM，0.0032nM和0.00064nM。在处理24小时后，使用高通量细胞RNA提取试剂盒进行细胞总RNA提取、RNA逆转录实验和定量实时PCR检测，测定猴FXI的mRNA水平，根据GAPDH内参基因水平对猴FXI的mRNA水平进行校正。
表19.猴Taqman探针引物信息表
引物名称 SEQ ID NO 引物序列
mkFXI-V1-PF1 46 CTGGATATTGTTGCTGTGAAAGGT
mkFXI-V1-PR1 47 CCTTCGTTGCAAGATGCTTGA
mkFXI-V1-P1 48 CTGTGCACCAATGCCGTCCGC
mkGAPDH-PF1-MGB 49 AGTCAGCCGCATTTTCTCTTG
mkGAPDH-PR1-MGB 50 AAATCCGTTGACTCCGACCTT
mkGAPDH-P1-MGB 51 ATCGCCAGCGCATC
结果以相对于经过对照处理的细胞的猴FXI mRNA表达剩余百分比表示。抑制率的IC 50结果见表20。结果表明，TRD008002、TRD008003在PCH细胞针对FXI有很好的抑制活性。
表20.dsRNA在PCH中多剂量抑制活性
Figure PCTCN2022139585-appb-000238
实施例13：dsRNA在人源化小鼠(hF11)体内(in vivo)活性的测定
本实施例中使用的人源化小鼠(hF11)购自赛业(苏州)生物科技有限公司，产品编号：C001272，采集血清40ul，使用EDTAK2抗凝，使用Human Coagulation Factor XI ELISA试剂盒(Sigma公司，批号0309J2350，货号RAB1385-1KT)测试自上述各组小鼠血清中的FXI蛋白含量。
根据血清FXI蛋白含量均匀分组，每组6只(2雄，4雌)，共5组，五组分别通过皮下注射的方式给予对照组(生理盐水)，dsRNA给药体积10μl/g，TJR100362，TRD008003-1，TJR100364，TRD008002-1组给药剂量均为10mg/kg。给药当天采集血清40μl，给药后第8天，第15天采集血清40μl，使用Human Coagulation Factor XI ELISA试剂盒测定血浆FXI蛋白含量，计算dsRNA对人源化小鼠(hF11)血清FXI蛋白表达的抑制。实验结果见表21和图3。
给药当天，以空白对照组(生理盐水)给药前采血的血清FXI含量平均值作为基线。第8天，第15天，第22天，计算各组每只小鼠血清FXI含量和基线的比值，采用Two-way ANOVA(第8天，第15天)/One-way ANOVA(第22天)进行统计，得到图3。
根据图3结果可见，TRD008003-1与TJR100362相比，第22天时，TJR100362与空白对照组(生理盐水)相比无显著性差异(图3中以ns表示)，而TRD008003-1与空白对照组(生理盐水)相比P<0.0001(图3中以****表示)，即表明第22天，TJR100362已无治疗效果，而TRD008003-1仍保持治疗作用。
TRD008002-1与TJR100364相比，第15天时，TJR100364与空白对照组(生 理盐水)相比无显著性差异(图3中以ns表示)，而TRD008002-1与空白对照组(生理盐水)相比P<0.0001(图3中以****表示)，即表明第15天，TJR100364已无治疗效果，而TRD008002-1仍保持治疗作用。
同时，在第15天和第22天，TRD008003-1抑制率显著优于TJR100362(图3中以#表示，#，P<0.05)；在第15天，TRD008002-1抑制率显著优于TJR100364(图3中以&表示，&，P<0.05)。
ELISA检测
使用Human Coagulation Factor XI ELISA试剂盒(Sigma公司，货号RAB1385-1KT)测试各组小鼠血清中的FXI蛋白含量。
按说明书稀释试剂盒中的样品，得到各稀释液A、B、C、D，备用，小鼠血清用样品稀释液A，进行5000倍稀释，作为待测样本留存备用。
按说明书操作，终止反应，立即用全自动酶标仪(Perkin Elmer公司，Envision2015)在450nm波长处读取光密度值。
根据对标准浓度梯度溶液所测得光密度值，拟合标准曲线，根据标准曲线计算待测血清中蛋白浓度，拟合获得的标准曲线符合以下计算公式：
Y＝slope X+intercept
式中：Y是在450nm处读取的相应光密度值；X为标准曲线中浓度值(pg/mL)，intercept是标准曲线的截距，而slope是曲线的斜率。
由获得的标准曲线，代入各血清样品测定的光密度值，获得各样品对应的浓度数值X，计算获得各给予不同样品的血清FXI蛋白浓度值＝5000X(pg/mL)。
根据血浆清FXI蛋白浓度值，相对于对照组的蛋白浓度，计算FXI蛋白抑制率＝(对照组蛋白浓度-测试组蛋白浓度)/对照组蛋白浓度×100％。抑制率如表21所示。
表21.dsRNA在人源化小鼠(hF11)体内抑制活性
Figure PCTCN2022139585-appb-000239
实施例14：dsRNA在人源化小鼠(hF11)体内(in vivo)活性的测定
本实施例中使用的人源化小鼠(hF11)购自赛业(苏州)生物科技有限公司，产品编号：C001272，采集血清40ul，使用EDTAK2抗凝，使用 Human Coagulation Factor XI ELISA试剂盒(Sigma公司，批号0309J2350，货号RAB1385-1KT)测试自上述各组小鼠血清中的FXI蛋白含量。
根据血清FXI蛋白含量均匀分组，每组6只(2雄，4雌)，分别通过皮下注射的方式给予对照组(生理盐水)，TJR100407给药体积10μl/g，TJR100407组给药剂量降低至3mg/kg。给药当天采集血清40μl，给药后第8天采集血清40μl，使用Human Coagulation Factor XI ELISA试剂盒测定血浆FXI蛋白含量，计算dsRNA对人源化小鼠(hF11)血清FXI蛋白表达的抑制。ELISA检测和抑制率计算，同实施例13，实验结果见表22。
表22结果表明，当给药剂量降低到3mg/kg时，在较低的给药剂量下，TJR100407对于人FXI蛋白表达仍具有高抑制率。
表22.dsRNA在人源化小鼠(hF11)体内抑制活性
Figure PCTCN2022139585-appb-000240

Claims (20)

  1. 一种双链核糖核酸(dsRNA)，其包含：
    siRNA和
    一个或多个与其缀合的配体；
    所述siRNA包含有义链和反义链，
    所述反义链在其5’端起第2位至第8位中的至少一个核苷酸位置处包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐；
    所述式(I)所示的化学修饰选自以下任一结构：
    Figure PCTCN2022139585-appb-100001
    B各自独立地选自反义链5’端起第2位至第8位对应位置的碱基；
    所述配体是以下结构所示或其药学上可接受的盐：
    Figure PCTCN2022139585-appb-100002
    所述siRNA为靶向凝血因子XI(FXI)基因的siRNA。
  2. 如权利要求1所述的dsRNA，其中：
    所述有义链包含至少15个连续核苷酸，且与SEQ ID NO:1至SEQ ID NO:3中任一的核苷酸序列相差不超过3个核苷酸，和/或，
    所述反义链包含至少19个连续核苷酸，且与SEQ ID NO:4或SEQ ID NO:5中任一的核苷酸序列相差不超过3个核苷酸；
    优选地，
    所述有义链包含SEQ ID NO:1至SEQ ID NO:3中的任一项所示的核苷酸序列，和/或，
    所述反义链包含SEQ ID NO:4或SEQ ID NO:5中的任一项所示的核苷酸序列；
    更优选地，所述siRNA为选自以下任一组：
    有义链包含SEQ ID NO:2所示的核苷酸序列，反义链包含SEQ ID NO:5所示的核苷酸序列；
    有义链包含SEQ ID NO:1所示的核苷酸序列，反义链包含SEQ ID NO:4所示的核苷酸序列；
    有义链包含SEQ ID NO:3所示的核苷酸序列，反义链包含SEQ ID NO:5所示的核苷酸序列。
  3. 如权利要求1或2所述的dsRNA，其中：
    所述有义链的3’端与所述配体缀合。
  4. 如权利要求1-3中任一项所述的dsRNA，其中：
    所述配体通过磷酸酯基团或硫代磷酸酯基团与所述siRNA末端连接；
    优选，通过磷酸二酯基团或硫代磷酸二酯基团连接，
    更优选，通过磷酸二酯基团连接。
  5. 如权利要求1-4中任一项所述的dsRNA，其中：
    所述反义链在其5’端起第5位、第6位或第7位的核苷酸位置处包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐；优选位于第7位。
  6. 如权利要求1-5中任一项所述的dsRNA，其中：
    除了式(I)所示化学修饰的位置之外，所述有义链和/或反义链中至少一个另外的核苷酸为修饰的核苷酸。
  7. 如权利要求1-6中任一项所述的dsRNA，其中：
    所述有义链含有如下式所示的核苷酸序列：
    5’-N aN aN aN aN aN aN bN bN bN aN aN aN aN aN aN aN aN aN a-3’；或，
    5’-N aN aN aN aN bN aN bN bN bN aN aN aN aN aN aN aN aN aN a-3’；
    其中，N a为2'-甲氧基修饰的核苷酸，N b为2'-氟代修饰的核苷酸。
  8. 如权利要求1-7中任一项所述的dsRNA，其中：
    所述反义链含有如下式所示的核苷酸序列：
    5’-N a’N b’N a’N b’N a’N b’W’N a’N a’N b’N a’N b’N a’N b’N a’N b’N a’N b’N a’N a’N a’-3’；
    N a’为2'-甲氧基修饰的核苷酸，N b’为2'-氟代修饰的核苷酸；
    W’表示式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰的核苷酸，所述式(I)所示的化学修饰选自：
    Figure PCTCN2022139585-appb-100003
    其中：B与所述反义链在其5’端起第7位核苷酸未被修饰时的碱基相同。
  9. 如权利要求1-8中任一项所述的dsRNA，其中：
    所述有义链和/或反义链中至少一个磷酸酯基为具有修饰基团的磷酸酯基；
    优选地，所述具有修饰基团的磷酸酯基为硫代磷酸二酯基。
  10. 如权利要求9所述的dsRNA，其中：
    所述硫代磷酸二酯基存在于以下位置中的至少一处：
    所述有义链的5'端起第1个核苷酸和第2个核苷酸之间；
    所述有义链的5'端起第2个核苷酸和第3个核苷酸之间；
    所述反义链的5'端起第1个核苷酸和第2个核苷酸之间；
    所述反义链的5'端起第2个核苷酸和第3个核苷酸之间；
    所述反义链的3'端起第1个核苷酸和第2个核苷酸之间；以及
    所述反义链的3'端起第2个核苷酸和第3个核苷酸之间；
    优选地，
    所述硫代磷酸二酯基存在于：
    所述有义链的5'端起第1个核苷酸和第2个核苷酸之间；和，
    所述有义链的5'端起第2个核苷酸和第3个核苷酸之间；和，
    所述反义链的5'端起第1个核苷酸和第2个核苷酸之间；和，
    所述反义链的5'端起第2个核苷酸和第3个核苷酸之间；和，
    所述反义链的3'端起第1个核苷酸和第2个核苷酸之间；和，
    所述反义链的3'端起第2个核苷酸和第3个核苷酸之间。
  11. 一种dsRNA，其中：
    所述dsRNA选自以下任一组方案：
    组1)包含SEQ ID NO:9所示的有义链和SEQ ID NO:15所示的反义链；
    组2)包含SEQ ID NO:7所示的有义链和SEQ ID NO:14所示的反义链；
    组3)包含SEQ ID NO:10所示的有义链和SEQ ID NO:15所示的反义链；
    组4)包含SEQ ID NO:8所示的有义链和SEQ ID NO:15所示的反义链；
    组5)包含SEQ ID NO:6所示的有义链和SEQ ID NO:14所示的反义链。
  12. 如权利要求1-11中任一项所述的dsRNA，其中：
    所述dsRNA选自以下任一结构或其药学上可接受的盐：
    Figure PCTCN2022139585-appb-100004
    其中，
    Af＝腺嘌呤2'-F核糖核苷；
    Cf＝胞嘧啶2'-F核糖核苷；
    Uf＝尿嘧啶2'-F核糖核苷；
    Gf＝鸟嘌呤2'-F核糖核苷；
    Am＝腺嘌呤2'-OMe核糖核苷；
    Cm＝胞嘧啶2'-OMe核糖核苷；
    Gm＝鸟嘌呤2'-OMe核糖核苷；
    Um＝尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)；
    Figure PCTCN2022139585-appb-100005
    表示硫代磷酸二酯基，
    Figure PCTCN2022139585-appb-100006
    表示磷酸二酯基，
    NAG0052’表示
    Figure PCTCN2022139585-appb-100007
    (-)hmpNA(U)表示
    Figure PCTCN2022139585-appb-100008
    (-)hmpNA(A)表示
    Figure PCTCN2022139585-appb-100009
  13. 一种药物组合物，其包含：
    权利要求1-12中任一项所述的dsRNA；
    任选地，还包含一种或多种药学上可接受的赋形剂。
  14. 权利要求1-12中任一项所述的dsRNA或权利要求13所述的药物组合物在制备药物中的用途；
    所述药物用于预防和/或治疗血栓栓塞性并发症，
    优选地，所述的血栓栓塞性并发症选自以下任一项：深静脉血栓形成、肺栓塞、心肌梗塞或中风。
  15. 一种抑制凝血因子XI表达的方法，其包括：
    向受试者给予有效量或有效剂量的权利要求1-12中任一项所述的dsRNA或权利要求13所述的药物组合物。
  16. 一种递送siRNA至肝脏的方法，其包括：
    向受试者给予有效量或有效剂量的权利要求1-12中任一项所述的dsRNA或权利要求13所述的药物组合物。
  17. 一种细胞，其包含权利要求1-12中任一项所述的dsRNA。
  18. 一种药盒，其包含至少一个容器：
    所述容器各自独立地包含权利要求1-12中任一项所述的dsRNA或权利要求13所述的药物组合物。
  19. 一种载体，包含权利要求1-12中任一项所述的dsRNA。
  20. 一种制备dsRNA或药物组合物的方法，其包括：
    合成权利要求1-12中任一项所述的dsRNA。
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