WO2023109935A1 - Arn double brin, son procédé de préparation et son utilisation - Google Patents

Arn double brin, son procédé de préparation et son utilisation Download PDF

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WO2023109935A1
WO2023109935A1 PCT/CN2022/139481 CN2022139481W WO2023109935A1 WO 2023109935 A1 WO2023109935 A1 WO 2023109935A1 CN 2022139481 W CN2022139481 W CN 2022139481W WO 2023109935 A1 WO2023109935 A1 WO 2023109935A1
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seq
nucleotide
antisense strand
dsrna
sense strand
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PCT/CN2022/139481
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Chinese (zh)
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侯哲
张建羽
李云飞
张瑱
林晓燕
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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    • 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

  • 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.
  • 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.
  • GalNAc galactosamine molecular clusters
  • Y is selected from O, NH and S;
  • J 2 is H or 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 ring
  • B is a base
  • 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.
  • 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 selected from 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 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.
  • the chemical modification shown in formula (I) is selected from 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 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 ', 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.
  • 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;
  • 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.
  • J 1 and J 2 are independently H;
  • 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.
  • 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.
  • R 3 is selected from H, C 1 -C 6 alkyl, and (CH 2 ) p R 6 .
  • 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;
  • Q 1 is Q 2 is R 2 ; or Q 1 is R 2 and Q 2 is
  • B is selected from the group consisting of purine bases, pyrimidine bases, 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 '), R 4 and R 4 ' are independently 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
  • 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 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 the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Y is O.
  • J 2 is H or methyl. In some embodiments, J is H.
  • R is selected from H and methyl.
  • 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.
  • the chemical modification represented by the formula (I) is selected from any of the following structures:
  • 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.
  • 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 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 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 is selected from the group consisting of the chemical modification represented by formula (I'), its Nucleotides of tautomers or pharmaceutically acceptable salts thereof,
  • 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
  • M is O or S
  • R when X is NH-CO, R is not H.
  • Y is selected from O, NH and S;
  • 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 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.
  • the chemical modification shown in formula (I') is selected from 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-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 ', 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.
  • 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 ;
  • 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' 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;
  • 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.
  • 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:
  • 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.
  • 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 16 is hydrogen or C 1-6 alkyl
  • 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 B1, and the terminal b1 is connected to R11 .
  • 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.
  • 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 a C 1 -C 12 alkyl chain.
  • 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 .
  • 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 , 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,
  • the N- Acetyl-galactosamine moiety is N- 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 is conjugated to the ligand.
  • the 3' end of the sense strand is 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 sense strand of the siRNA.
  • the lipophilic group includes binding with a small interfering nucleic acid with a covalent bond, such as introducing cholesterol, Lipoproteins, vitamin E, etc., in order to facilitate the interaction with intracellular mRNA through the cell membrane composed of lipid bilayers.
  • 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 a pharmaceutically acceptable salt thereof is located at the 5th, 6th, or 5th position of the 5' end of the antisense strand No. 7.
  • the nucleotide comprising the chemical modification represented by formula (I), its tautomer or a pharmaceutically acceptable salt thereof is located at position 7 at 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 when the 5th nucleotide at the 5' end of the antisense strand is unmodified.
  • B is the same as the base when the 6th nucleotide at the 5' end of the antisense strand is unmodified.
  • B is the same as the base when the 7th nucleotide at the 5' end of the antisense strand is unmodified.
  • B is the same as the base when the 8th nucleotide at the 5' end of the antisense strand is unmodified.
  • At least one additional nucleotide in the sense strand and/or antisense strand is a modified nucleotide 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'-deoxynucleotides, 2'-deoxy-2'-fluoro-modified nucleosides acid, 3'-deoxy-thymidine nucleotides, isonucleotides, LNA, ENA, cET, UNA, GNA; in some embodiments, the modified nucleotides are independently selected from: 2'-methoxy modified Nucle
  • 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 nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 of the antisense strand are each independently 2'- Fluoro-modified nucleotides.
  • the antisense strand is at least partially reverse complementary to the target sequence to mediate RNA interference; 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; in some embodiments, the antisense strand is fully reverse complementary to the target sequence.
  • the sense strand and the antisense strand are at least partially reverse complementary 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; 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 nucleotides).
  • 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 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 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, 21
  • the siRNA comprises one or two blunt ends.
  • the siRNA comprises an overhang with 1 to 4 unpaired nucleotides, eg, 1, 2, 3, 4.
  • the siRNA comprises an overhang located 3' to the antisense strand.
  • the sense strand comprises or is selected from 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 comprises or is selected from a nucleotide sequence (5'-3') 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 or is selected from a nucleotide sequence (5'-3') as shown in 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, the base between B and the 7th nucleotide at the 5' end of the antisense strand is not modified same.
  • 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 acid is the same as the base when it 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 sense strand and/or antisense strand include multiple phosphorothioate groups present in:
  • 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;
  • connection between the lowercase letter s and the two nucleotides adjacent to the left and right of the letter s is a phosphorothioate group.
  • 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 modified nucleotide comprising a chemical modification represented by formula (I), its tautomer or a pharmaceutically acceptable salt thereof.
  • 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 at 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 acid is the same as the base when it is not modified.
  • M is S. In some specific embodiments, M is O.
  • the siRNA is an siRNA targeting the 17 ⁇ -hydroxysteroid dehydrogenase type 13 (HSD17B13) gene.
  • the nucleotide sequence of the sense strand of the siRNA comprises at least 15 nucleotide sequences that differ by no more than 3 nucleotides from any of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 8. consecutive nucleotides, and/or,
  • the nucleotide sequence of the antisense strand comprises at least 19 consecutive nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 9 to SEQ ID NO: 12.
  • 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:8, and/or, the nucleosides of the antisense strand
  • the acid sequence comprises any one nucleotide sequence in SEQ ID NO:9 to SEQ ID NO:12;
  • nucleotide sequence of the siRNA is any one of the following schemes:
  • the nucleotide sequence of the sense strand comprises the nucleotide sequence of SEQ ID NO:8, and the nucleotide sequence of the antisense strand comprises the nucleotide sequence of SEQ ID NO:12;
  • the nucleotide sequence of the sense strand comprises the nucleotide sequence of SEQ ID NO:6, and the nucleotide sequence of the antisense strand comprises the nucleotide sequence of SEQ ID NO:11;
  • 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:9;
  • nucleotide sequence of the sense strand comprises the nucleotide sequence of SEQ ID NO:4, and the nucleotide sequence of the antisense strand comprises the nucleotide sequence of SEQ ID NO:10;
  • the nucleotide sequence of the sense strand comprises the nucleotide sequence of SEQ ID NO:7, and the nucleotide sequence of the antisense strand comprises the nucleotide sequence of SEQ ID NO:12;
  • the nucleotide sequence of the sense strand comprises the nucleotide sequence of SEQ ID NO:5, and the nucleotide sequence of the antisense strand comprises the nucleotide sequence of SEQ ID NO:11;
  • 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:9;
  • 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:10.
  • the siRNA is any of the following:
  • the nucleotide sequence of the sense strand is SEQ ID NO:8, and the nucleotide sequence of the antisense strand is SEQ ID NO:12;
  • the nucleotide sequence of the sense strand is SEQ ID NO:6, and the nucleotide sequence of the antisense strand is SEQ ID NO:11;
  • the nucleotide sequence of the sense strand is SEQ ID NO:2, and the nucleotide sequence of the antisense strand is SEQ ID NO:9;
  • nucleotide sequence of the sense strand is SEQ ID NO:4, and the nucleotide sequence of the antisense strand is SEQ ID NO:10;
  • the nucleotide sequence of the sense strand is SEQ ID NO:7, and the nucleotide sequence of the antisense strand is SEQ ID NO:12;
  • nucleotide sequence of the sense strand is SEQ ID NO:5
  • nucleotide sequence of the antisense strand is SEQ ID NO:11;
  • nucleotide sequence of the sense strand is SEQ ID NO: 1
  • nucleotide sequence of the antisense strand is SEQ ID NO: 9; or
  • the nucleotide sequence of the sense strand is SEQ ID NO:3, and the nucleotide sequence of the antisense strand is SEQ ID NO:10.
  • the sense strand of the dsRNA comprises the nucleotide sequence shown in any one of SEQ ID NO:13 to SEQ ID NO:20, and/or, the antisense strand comprises SEQ ID NO: 21 to the nucleotide sequence shown in any one of SEQ ID NO:24.
  • the dsRNA is any of the following: wherein
  • the sense strand comprises the nucleotide sequence shown in SEQ ID NO:20, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:24;
  • the sense strand comprises the nucleotide sequence shown in SEQ ID NO:18, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:23;
  • the sense strand comprises the nucleotide sequence shown in SEQ ID NO:14, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:21;
  • the sense strand comprises the nucleotide sequence shown in SEQ ID NO:16, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:22;
  • the sense strand comprises the nucleotide sequence shown in SEQ ID NO:19, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:24;
  • the sense strand comprises the nucleotide sequence shown in SEQ ID NO:17, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:23;
  • the sense strand comprises the nucleotide sequence shown in SEQ ID NO:13, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:21;
  • the sense strand comprises the nucleotide sequence shown in SEQ ID NO:15
  • the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:22.
  • the dsRNA is any of the following:
  • the sense strand is the nucleotide sequence shown in SEQ ID NO:20, and the antisense strand is the nucleotide sequence shown in SEQ ID NO:24;
  • the sense strand is the nucleotide sequence shown in SEQ ID NO:18, and the antisense strand is the nucleotide sequence shown in SEQ ID NO:23;
  • the sense strand is the nucleotide sequence shown in SEQ ID NO:14, and the antisense strand is the nucleotide sequence shown in SEQ ID NO:21;
  • the sense strand is the nucleotide sequence shown in SEQ ID NO:16, and the antisense strand is the nucleotide sequence shown in SEQ ID NO:22;
  • the sense strand is the nucleotide sequence shown in SEQ ID NO:19, and the antisense strand is the nucleotide sequence shown in SEQ ID NO:24;
  • the sense strand is the nucleotide sequence shown in SEQ ID NO:17, and the antisense strand is the nucleotide sequence shown in SEQ ID NO:23;
  • the sense strand is the nucleotide sequence shown in SEQ ID NO:13, and the antisense strand is the nucleotide sequence shown in SEQ ID NO:21;
  • the sense strand is the nucleotide sequence shown in SEQ ID NO:15
  • the antisense strand is the nucleotide sequence shown in SEQ ID NO:22.
  • the dsRNA is any of the following: wherein
  • the sense strand comprises SEQ ID NO:20, and the antisense strand comprises SEQ ID NO:24;
  • the sense strand comprises SEQ ID NO:18, and the antisense strand comprises SEQ ID NO:23;
  • the sense strand comprises SEQ ID NO:14, and the antisense strand comprises SEQ ID NO:21;
  • the sense strand comprises SEQ ID NO:16, and the antisense strand comprises SEQ ID NO:22;
  • the sense strand comprises SEQ ID NO:19, and the antisense strand comprises SEQ ID NO:24;
  • the sense strand comprises SEQ ID NO:17, and the antisense strand comprises SEQ ID NO:23;
  • the sense strand comprises SEQ ID NO:13, and the antisense strand comprises SEQ ID NO:21;
  • the sense strand comprises SEQ ID NO:15 and the antisense strand comprises SEQ ID NO:22.
  • the dsRNA is any of the following:
  • the sense strand is selected from SEQ ID NO:20, and the antisense strand is selected from SEQ ID NO:24;
  • the sense strand is selected from SEQ ID NO:18, and the antisense strand is selected from SEQ ID NO:23;
  • the sense strand is selected from SEQ ID NO:14, and the antisense strand is selected from SEQ ID NO:21;
  • the sense strand is selected from SEQ ID NO:16, and the antisense strand is selected from SEQ ID NO:22;
  • the sense strand is selected from SEQ ID NO:19, and the antisense strand is selected from SEQ ID NO:24;
  • the sense strand is selected from SEQ ID NO:17, and the antisense strand is selected from SEQ ID NO:23;
  • the sense strand is selected from SEQ ID NO:13, and the antisense strand is selected from SEQ ID NO:21;
  • the sense strand is selected from SEQ ID NO:15 and the antisense strand is selected from SEQ ID NO:22.
  • the dsRNA is any of the following:
  • SEQ ID NO: 13 is
  • SEQ ID NO: 14 is
  • SEQ ID NO:15 is N-(SEQ ID NO:15).
  • SEQ ID NO: 16 is
  • SEQ ID NO: 17 is
  • SEQ ID NO: 18 is
  • SEQ ID NO: 19 is N-(SEQ ID NO: 19
  • SEQ ID NO:20 is N-(SEQ ID NO:20).
  • SEQ ID NO:24 is N-(SEQ ID NO:24)
  • 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 TRD008007, TRD008008, TRD008009, TRD008010, TRD008007-1, TRD008008-1, TRD008009-1, or TRD008010-1;
  • the dsRNA is TRD008007, which has the following structure:
  • the dsRNA is TRD008007-1, which has the following structure:
  • the dsRNA is TRD008008, which has the following structure:
  • the dsRNA is TRD008008-1, which has the following structure:
  • the dsRNA is TRD008009, which has the following structure:
  • the dsRNA is TRD008009-1, which has the following structure:
  • the dsRNA is TRD008010, which has the following structure:
  • the dsRNA is TRD008010-1, which has the following structure:
  • the present disclosure provides a pharmaceutical composition comprising the dsRNA described in the present disclosure.
  • 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 present disclosure provides an application of the dsRNA described in the present disclosure or the pharmaceutical composition described in the present disclosure in the preparation of medicine.
  • the medicament can be used to prevent and/or treat hepatitis, liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), liver cirrhosis, alcoholic steatohepatitis Hepatitis (ASH), alcoholic fatty liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury, or hepatocyte necrosis.
  • NASH nonalcoholic steatohepatitis
  • NAFLD nonalcoholic fatty liver disease
  • ASH alcoholic steatohepatitis Hepatitis
  • ALD alcoholic fatty liver disease
  • HCV-related cirrhosis drug-induced liver injury, or hepatocyte necrosis.
  • the medicament can be used to prevent and/or treat diseases related to HSD17B13 gene expression.
  • the diseases associated with HSD17B13 gene expression can be hepatitis, liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), liver cirrhosis, alcoholic fatty Acute hepatitis (ASH), alcoholic fatty liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury, or hepatocyte necrosis.
  • the disease associated with HSD17B13 gene expression may be chronic fibrotic liver disease.
  • the chronic fibrotic liver disease is associated with accumulation and/or expansion of lipid droplets in the liver.
  • the effective amount or effective dose of the dsRNA or 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.
  • the present disclosure provides a use of the dsRNA described in the present disclosure or the pharmaceutical composition described in the present disclosure in the preparation of a medicament for preventing and/or treating a disease in a subject.
  • the disease may be hepatitis, liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), liver cirrhosis, alcoholic steatohepatitis (ASH), Alcoholic fatty liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury, or hepatocyte necrosis.
  • NASH nonalcoholic steatohepatitis
  • NAFLD nonalcoholic fatty liver disease
  • ASH alcoholic steatohepatitis
  • ALD Alcoholic fatty liver disease
  • HCV-related cirrhosis drug-induced liver injury
  • hepatocyte necrosis hepatocyte necrosis
  • the disease may be a disease associated with HSD17B13 gene expression.
  • the diseases associated with HSD17B13 gene expression can be hepatitis, liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), liver cirrhosis, alcoholic fatty Acute hepatitis (ASH), alcoholic fatty liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury, or hepatocyte necrosis.
  • the disease associated with HSD17B13 gene expression may be chronic fibrotic liver disease.
  • the chronic fibrotic liver disease is associated with accumulation and/or expansion of lipid droplets in the liver.
  • the present disclosure provides a method for preventing and/or treating a disease, which comprises administering to a subject an effective amount or dose of the dsRNA described in the present disclosure or the pharmaceutical composition described in the present disclosure.
  • the disease may be hepatitis, liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), liver cirrhosis, alcoholic steatohepatitis (ASH), Alcoholic fatty liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury, or hepatocyte necrosis.
  • NASH nonalcoholic steatohepatitis
  • NAFLD nonalcoholic fatty liver disease
  • ASH alcoholic steatohepatitis
  • ALD Alcoholic fatty liver disease
  • HCV-related cirrhosis drug-induced liver injury
  • hepatocyte necrosis hepatocyte necrosis
  • the disease may be a disease associated with HSD17B13 gene expression.
  • the diseases associated with HSD17B13 gene expression can be hepatitis, liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), liver cirrhosis, alcoholic fatty Acute hepatitis (ASH), alcoholic fatty liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury, or hepatocyte necrosis.
  • the disease associated with HSD17B13 gene expression may be chronic fibrotic liver disease.
  • the chronic fibrotic liver disease is associated with accumulation and/or expansion of lipid droplets in the liver.
  • the present disclosure provides a method for silencing HSD17B13 or its mRNA in a cell in vivo or in vitro, comprising introducing the dsRNA described in the present disclosure or the pharmaceutical composition described in the present disclosure into the cell step.
  • the present disclosure provides a method for inhibiting the expression of the target gene HSD17B13 or its mRNA, which comprises administering to a subject an effective amount or dose of the dsRNA described in the present disclosure or the pharmaceutical composition described in the present disclosure.
  • 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 a therapeutically effective amount of the dsRNA or pharmaceutical combination described herein to the object substances, thereby inhibiting the expression of the target gene or its mRNA in the subject.
  • the subject has been previously identified as having pathological upregulation of the target gene HSD17B13 or mRNA thereof in the targeted cell, cell population, tissue or subject.
  • the present disclosure provides a method of delivering an oligonucleotide to the liver, which comprises administering to a subject an effective amount or dose of the dsRNA described in the present disclosure or the pharmaceutical composition described in the present disclosure.
  • the present disclosure provides an RNAi (RNA interference) agent comprising the dsRNA described in the present disclosure or the pharmaceutical composition described in the present disclosure.
  • RNAi RNA interference
  • the present disclosure also provides a cell comprising the dsRNA described in the present disclosure or the pharmaceutical composition described in the present disclosure.
  • the present disclosure also provides a kit comprising the dsRNA described in the present disclosure or the pharmaceutical composition described in the present disclosure.
  • the dsRNA or pharmaceutical composition of the present disclosure when contacted with cells expressing the target gene, is detected by, for example: psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or based on branched DNA (bDNA) , or protein-based methods, such as immunofluorescence analysis, such as Western Blot or flow cytometry, the dsRNA or pharmaceutical composition of the present disclosure can 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 dsRNA or pharmaceutical composition of the present disclosure when contacted with cells expressing the target gene, is detected by, for example: psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or based on branched DNA (bDNA) , or protein-based methods, such as immunofluorescence analysis, such as Western Blot or flow cytometry, the remaining expression percentage of the target gene mRNA caused by the dsRNA or the pharmaceutical composition of the present disclosure is not higher than 99%, not high 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 than 45%, not higher 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%.
  • psiCHECK activity screening and luciferase reporter gene assay other methods
  • the dsRNA or pharmaceutical composition of the present disclosure when contacted with cells expressing the target gene, is detected by, for example: psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or based on branched DNA (bDNA) , or protein-based methods, such as immunofluorescence assays, e.g., Western Blot, or flow cytometry, the dsRNA reduces off-target activity by at least 20%, at least 25%, or at least 30%, while maintaining on-target activity , 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%.
  • psiCHECK activity screening and luciferase reporter gene assay other methods such as PCR or based on branched DNA (bDNA) , or protein-based methods, such as immunofluorescence assays, e.g., Western Blot, or flow cytometry
  • the dsRNA or pharmaceutical composition of the present disclosure when contacted with cells expressing the target gene, is detected by, for example: psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or based on branched DNA (bDNA) , or protein-based methods, such as immunofluorescence assays, such as Western Blot, or flow cytometry, dsRNA reduces on-target activity by up to 20%, up to 19%, up to 15%, up to 10%, up to 5% or more than 1%, 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%.
  • psiCHECK activity screening and luciferase reporter gene assay other methods such as PCR or based on branched DNA (bDNA) , or protein-based methods, such as immunofluorescence as
  • dsRNA when the dsRNA or the pharmaceutical composition of the present disclosure contacts the cells expressing the target gene, it can be detected by, for example, psiCHECK activity screening and luciferase reporter gene detection method, other methods such as PCR or based on branched DNA (bDNA) , or protein-based methods, such as immunofluorescence assays, e.g., 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% , 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
  • the present disclosure also provides a method for preparing dsRNA or a pharmaceutical composition, which includes: synthesizing the ligand, siRNA, dsRNA or pharmaceutical composition 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 or both and 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
  • 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.
  • HSD17B13 refers to type 13 17 ⁇ hydroxysteroid dehydrogenase, a member of the 17 ⁇ hydroxysteroid dehydrogenase (hsd17b) family, which catalyzes the conversion between 17-keto- and 17-hydroxysteroids.
  • hsd17b 17 ⁇ hydroxysteroid dehydrogenase
  • Members of this family have various functions including, for example, the reduction or oxidation of sex hormones, fatty acids, and bile acids in vivo.
  • Members of the hsd17b family differ in their tissue distribution, subcellular localization, catalytic priority, and because they also catalyze the conversion of other substrates than steroids (such as lipids and retinoids), they have different substrate specificities .
  • HSD17B13 is mainly expressed in the liver and localized on the surface of lipid droplets (LDs).
  • LDs lipid droplets
  • HSD17B13 protein expression was significantly upregulated in fatty liver LDs.
  • HSD17B13 protein was overexpressed it led to an increase in the number and size of LDs in hepatocytes, and significantly increased hepatic lipogenesis and triglyceride (TG) content.
  • TG hepatic lipogenesis and triglyceride
  • NAFLD nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • HSD17B13 Loss-of-function mutants of HSD17B13 (rs72613567:TA) reduce alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, while reducing inflammation and liver injury in fatty liver patients.
  • HSD17B13 has been shown to be associated with a variety of liver diseases, including but not limited to hepatitis, liver fibrosis, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, cirrhosis, alcoholic steatohepatitis, alcoholic fatty liver disease, HCV- Associated cirrhosis, drug-induced liver injury or hepatocellular necrosis, or chronic fibroinflammatory liver disease.
  • the HSD17B13 sequence includes: GENBANK accession number NM_001136230.3, GENBANK accession number NM_001136230.3,
  • the sense strand also known as SS, SS strand or sense strand
  • the antisense strand also known as AS or AS strand
  • AS or AS strand refers to the strand that has a sequence that is complementary to the target mRNA sequence.
  • the "5' region” of the sense strand or the antisense strand can be used interchangeably.
  • the 2nd to 8th nucleotides in the 5' region 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 one of SEQ ID NO: 1 to SEQ ID NO: 8 by no more than 3 nucleotides and contains at least 15 consecutive Nucleotide” is intended to mean that the siRNA sense strand described herein comprises at least 15 consecutive nucleotides of any sense strand in SEQ ID NO: 1 to SEQ ID NO: 8, or with SEQ ID NO: 1 to SEQ ID NO: At least 15 consecutive nucleotides of any sense strand in ID NO:8 differ by no more than 3 nucleotide sequences (optionally, differ by no more than 2 nucleotide sequences; optionally, differ by 1 nucleoside acid sequences).
  • the siRNA sense strand described herein comprises at least 16 contiguous nucleotides of any sense strand of SEQ ID NO: 1 to SEQ ID NO: 8, or is identical to any of SEQ ID NO: 1 to SEQ ID NO: 8 At least 16 contiguous nucleotides of a 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 any antisense strand of SEQ ID NO: 9 to SEQ ID NO: 12 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: 9 to SEQ ID NO: 12, or with SEQ ID NO: 9 to At least 15 consecutive nucleotides of any antisense strand in SEQ ID NO: 12 differ by no more than 3 nucleotide sequences (optionally, differ by no more than 2 nucleotide sequences, optionally, differ by 1 Nucleotide sequence).
  • G", “C”, “A”, “T” and “U” respectively represent nucleotides, which respectively contain guanine, cytosine, adenine, thymidine
  • the nucleotides are linked by phosphorothioate groups.
  • 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.
  • dsRNA refers to a double-stranded RNA molecule capable of RNA interference, comprising a sense strand and an antisense strand.
  • 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 (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA);
  • the purine base guanine (C) is always paired with the pyrimidine base Cytosine (G) is paired.
  • 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 siRNA and dsRNA provided in the present disclosure can be obtained by conventional preparation methods in the art (such as methods of solid-phase synthesis and liquid-phase synthesis). Among them, solid-phase synthesis has commercialized customized services.
  • a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a correspondingly modified nucleoside monomer, a method for preparing a correspondingly modified nucleoside monomer and introducing a modified nucleotide group
  • the methods of siRNA and dsRNA are also well known to those skilled in the art.
  • the term “comprising” or “comprising” means including stated elements, integers or steps, but not excluding any other elements, integers or steps.
  • the term “comprising” or “comprises” is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps.
  • a reference is made to “comprising” a specific sequence it is also intended to cover the situation consisting of that specific sequence.
  • 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 .
  • a "phosphate group” can be a phosphoric acid monoester group, a phosphoric diester group or a phosphoric acid triester group, preferably a phosphoric diester group; Group" has the same meaning.
  • 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 refers to one or more hydrogen atoms in a group, preferably at most 5, more preferably 1 to 3 hydrogen atoms are independently substituted 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 then exert its biological activity.
  • “Pharmaceutically acceptable excipients” include, but are not limited to, any adjuvants, carriers, glidants, sweeteners, Diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents or emulsifying agents.
  • the term “inhibit”, is used interchangeably with “reduce”, “silence”, “downregulate”, “suppress” and other similar terms, and includes any level of inhibition. 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%.
  • 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 in this disclosure, “subject”, “patient”, “subject” or “individual” are used interchangeably and include humans or non-human animals such as mammals such as humans or monkeys.
  • Figure 1 shows the remaining mRNA expression levels in TTR of conjugates TRD002218 and TRD007205 on day 7 after administration.
  • Figure 2 shows the remaining mRNA expression levels in TTR of conjugates TRD002218 and TRD007205 on day 28 after administration.
  • the present disclosure is further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure.
  • the experimental methods not indicating the specific conditions in the embodiments of the present disclosure are usually in accordance with conventional conditions or in accordance with the conditions suggested by raw materials or commodity manufacturers.
  • Reagents where a specific source is not stated may be obtained from any supplier of molecular biology reagents in quality/purity for molecular biology applications.
  • 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(+).
  • 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).
  • dsRNA dsRNA
  • phosphoramidite monomer synthesized above is used to replace the original nucleotide of the parent sequence.
  • a brief description of the synthesis process is 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.
  • 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(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.
  • the siRNA (sequence shown in Table 4) targeting three different genes (ANGPTL3, HBV-S, HBV-X) mRNA was used, and the compound (+) hmpNA (A), (-) hmpNA ( A) and the GNA (A) compound as a control modify the 7th position of the 5' end of the AS chain (sequence is shown in Table 5), and compare the on-target activity and off-target activity with the parent sequence.
  • uppercase letters G, A, C, and U represent nucleotides containing guanine, adenine, cytosine, and uracil, respectively
  • lowercase letter m represents 2'-methoxy modification
  • lowercase letter f represents 2 '-Fluoro modification
  • when the lowercase letter s is in the middle it means that the two adjacent nucleotides to the left and right of the letter s are connected by a phosphorothioate group
  • the lowercase letter s is the first at the 3' end, it means that the A nucleotide terminal adjacent to the left side of the letter s is a phosphorothioate group; the same below.
  • Embodiment 6 preparation NAG0052, L96
  • 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 (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.
  • NAG0052 is connected to the sequence through solid-phase synthesis, and after aminolysis, the structure of NAG0052 loses some functional groups to become NAG0052'.
  • the prepared dsRNA had the sense and antisense strands shown in Table 8 and Table 9.

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Abstract

La présente invention concerne un ARNdb, son procédé de préparation et son utilisation, et concerne également une composition pharmaceutique comprenant l'ARNdb, ou une cellule ou un kit. L'ARNdb peut interférer avec l'expression du gène HSD17B13, et prévenir et/ou traiter des maladies associées.
PCT/CN2022/139481 2021-12-16 2022-12-16 Arn double brin, son procédé de préparation et son utilisation WO2023109935A1 (fr)

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US20170305956A1 (en) * 2014-12-15 2017-10-26 Dicerna Pharmaceuticals, Inc. Ligand-Modified Double-Stranded Nucleic Acids
CN112020556A (zh) * 2018-03-21 2020-12-01 瑞泽恩制药公司 第13型17β羟基类固醇脱氢酶(HSD17B13)iRNA组成物及其使用方法
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