WO2022028462A1 - 脱靶活性降低的修饰sirna - Google Patents

脱靶活性降低的修饰sirna Download PDF

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WO2022028462A1
WO2022028462A1 PCT/CN2021/110509 CN2021110509W WO2022028462A1 WO 2022028462 A1 WO2022028462 A1 WO 2022028462A1 CN 2021110509 W CN2021110509 W CN 2021110509W WO 2022028462 A1 WO2022028462 A1 WO 2022028462A1
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sirna
nucleotide
methoxy
alkyl
alkynyl
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French (fr)
Chinese (zh)
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黄金宇
罗敏
尹科
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Tuojie Biotech Shanghai Co Ltd
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Tuojie Biotech Shanghai Co Ltd
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Priority to BR112023002080A priority Critical patent/BR112023002080A2/pt
Priority to CN202180048996.1A priority patent/CN115955973A/zh
Priority to KR1020237006847A priority patent/KR20230043195A/ko
Priority to CA3190097A priority patent/CA3190097A1/en
Priority to EP21852482.5A priority patent/EP4194553A4/en
Priority to JP2023507494A priority patent/JP7849353B2/ja
Priority to US18/019,763 priority patent/US20230287418A1/en
Publication of WO2022028462A1 publication Critical patent/WO2022028462A1/zh
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Priority to ZA2023/03275A priority patent/ZA202303275B/en
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Definitions

  • the present disclosure relates to siRNAs with inhibited target gene expression, and in particular to modified siRNAs with reduced off-target activity.
  • 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 belong to undruggable proteins. Using RNA interference technology, according to the mRNA encoding these proteins, appropriate siRNA can be designed to specifically target the target mRNA and degrade the target mRNA, thereby inhibiting the production of related proteins. Therefore, siRNA has a very important prospect for drug development.
  • siRNA often has different degrees of off-target effects.
  • One off-target effect is the off-target effect of miRNA-like, that is, the complete or incomplete pairing formed by the seed region (positions 2-8 at the 5' end) of the siRNA antisense strand (Antisense Strand, also known as the AS strand) and the target mRNA.
  • the resulting inhibitory activity on mRNA Off-target effects of one siRNA molecule may affect multiple mRNAs. Therefore, unpredictable toxic side effects may occur, which is the main reason for the toxic side effects of siRNA drugs (Janas, MM, Schlegel, MK, Harbison, CE et al. Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat Commun 9, 723 (2016)).
  • the present disclosure provides an siRNA with chemical modifications introduced into its seed region, thereby inhibiting or reducing siRNA off-target activity while maintaining (or even enhancing) siRNA on-target activity.
  • the present disclosure provides an siRNA comprising a sense strand and an antisense strand, each strand having 15 to 35 nucleotides, the antisense strand at positions 2 to 8 in its 5' region (eg, At least one of the 2nd, 3rd, 4th, 5th, 6th, 7th, and 8th nucleotide positions comprises the chemical modification represented by formula (I) or its mutual Variant modification:
  • 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 each independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • Q1 is Q 2 is R 2 ; or Q 1 is R 2 and Q 2 is
  • J 1 is H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is a base or a base analog
  • the antisense strand is at positions 2 to 8 of its 5' region (eg, position 2, position 3, position 4, position 5, position 6, position 7, position 8
  • At least one of the nucleotide positions in the formula (I-1) contains the chemical modification represented by the formula (I-1) or its tautomer modification:
  • 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 each independently H or C 1 -C 6 alkyl;
  • Each J 1 , J 2 is independently H or C 1 -C 6 alkyl
  • R1 and R2 are directly connected to form a ring.
  • the antisense strand is at positions 2 to 8 of its 5' region (eg, position 2, position 3, position 4, position 5, position 6, position 7, position 8
  • At least one nucleotide position in the formula (I-2) contains the chemical modification represented by formula (I-2) or its tautomer modification:
  • 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 each independently H or C 1 -C 6 alkyl;
  • Each J 1 , J 2 is independently H or C 1 -C 6 alkyl
  • R1 and R2 are directly connected to form a ring.
  • the nucleotide comprising the chemical modification shown in formula (I) or its tautomer modification is a nucleoside comprising the chemical modification shown in formula (I') or its tautomer modification acid,
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 ′), S, NR 5 and NH-CO, wherein R 4 , R 4 ′, R 5 are each independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • Q1 ' is Q 2' is R 2 ; or Q 1' is R 2 and Q 2' is
  • J 1 is H or C 1 -C 6 alkyl
  • M is O or S
  • R 1 and R 2 are directly connected to form a ring
  • B is a base or a base analog
  • the antisense strand is at positions 2 to 8 of its 5' region (eg, position 2, position 3, position 4, position 5, position 6, position 7, position 8
  • At least one of the nucleotide positions in the formula (I-3) contains the chemical modification represented by the formula (I-3) or its tautomer modification:
  • 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 each independently H or C 1 -C 6 alkyl;
  • Each J 1 , J 2 is independently H or C 1 -C 6 alkyl
  • M is O or S
  • R1 and R2 are directly connected to form a ring.
  • the antisense strand is at positions 2 to 8 of its 5' region (eg, position 2, position 3, position 4, position 5, position 6, position 7, position 8
  • At least one nucleotide position in the formula (I-4) contains the chemical modification represented by formula (I-4) or its tautomer modification:
  • 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 each independently H or C 1 -C 6 alkyl;
  • Each J 1 , J 2 is independently H or C 1 -C 6 alkyl
  • M is O or S
  • R1 and R2 are directly connected to form a ring.
  • R1 is not H.
  • 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;
  • Each J 1 , J 2 is independently H or C 1 -C 3 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.
  • 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, methyl, B radical, n-propyl or isopropyl;
  • Each J 1 , J 2 is independently H or methyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2- Aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2- Thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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, methyl, B radical, n-propyl or isopropyl;
  • Each J 1 , J 2 is independently H or methyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, 7-deazapurine, cytosine Pyrimidine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • Each J 1 , J 2 is 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;
  • R3 is selected from H, OH, NH2 , methyl and CH2OH ;
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • Each J 1 , J 2 is independently H;
  • R 1 is selected from H, methyl and CH 2 OH;
  • R 2 is selected from H, methyl and CH 2 OH;
  • R3 is selected from H, OH, NH2 , methyl and CH2OH ;
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine, 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:
  • B is a base or base analog, eg, selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethyl aminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole , 5-nitroindole and 3-nitropyrrole.
  • B is the antisense strand at positions 2 to 8 in its 5' region (e.g., positions 2, 3, 4, 5, 6, 7) , the base at the corresponding position in the 8th position).
  • B is the antisense strand at positions 2 to 8 in its 5' region (e.g., position 2, position 3, position 4, position 5, position 6, position 8). 7 and 8) the natural base at the corresponding position.
  • the chemical modification represented by the formula (I) is selected from:
  • B is a base or base analog, eg, selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethyl aminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from the group consisting of purine, 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:
  • B is a base or base analog, eg, selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethyl aminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole , 5-nitroindole and 3-nitropyrrole.
  • B is selected from the group consisting of adenine, guanine, cytosine, uracil, and thymine.
  • the chemical modification represented by the formula (I) is selected from:
  • M is O or S
  • B is a base or base analog, eg, selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethyl aminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from the group consisting of purine, 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:
  • M is O or S
  • B is a base or base analog, eg, selected from purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethyl aminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from the group consisting of purine, 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:
  • M is O or S
  • B is a base or base analog, eg, selected from purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethyl aminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole , 5-nitroindole and 3-nitropyrrole.
  • B is selected from the group consisting of adenine, guanine, cytosine, uracil, and thymine.
  • the chemical modification represented by the formula (I) includes but is not limited to:
  • adenine in their structure is replaced by a guanine, cytosine, uracil or thymine.
  • the antisense strand is at positions 2-8, 3-8, 4-8, 5-8, 5 in its 5' region
  • the chemical modification represented by the above-mentioned formula (I) or the modification of the tautomer thereof is contained in at least one nucleotide position from the 7th position.
  • the antisense strand comprises a chemical modification of formula (I) above or a tautomer thereof at the nucleotide position at position 5, 6 or 7 in its 5' region retouch.
  • the antisense strand comprises a chemical modification of formula (I) above, or a tautomeric modification thereof, at the nucleotide position at position 7 in its 5' region.
  • 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.
  • the sense and antisense strands are the same or different in length, the sense strand being 19-23 nucleotides in length and the antisense strand 19-26 nucleotides in length.
  • the length ratio of the sense strand and antisense strand of the siRNA 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 length ratio of the sense and antisense strands of the siRNA is 19/21, 21/23, or 23/25. In some embodiments,
  • 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, between the antisense strand and the target sequence , 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 and antisense strands are at least partially reverse complementary to form a double-stranded region; in some embodiments, there are no more than 5, no more than 5, between the sense and antisense strands 4, no more than 3, no more than 2, no more than 1 mismatch; in some embodiments, the sense and antisense strands are fully reverse complementary.
  • the present disclosure also provides a siRNA, which is the above-mentioned modified siRNA, in addition to comprising nucleotides modified by the chemical modification represented by the above-mentioned formula (I) or its tautomer, the sense strand and /or at least one of the antisense strands further comprises at least one other modified nucleotide.
  • the remaining nucleotides in the sense strand and/or antisense strand except for the nucleotides modified by the chemical modification shown in the above formula (I) or tautomers thereof, are other modified nucleotides.
  • the other modified nucleotides are independently selected from deoxy-nucleotides, 3'-terminal deoxy-thymine nucleotides, 2'-O-methyl modified nucleotides, 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, abasic Nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural base-containing
  • the other modified nucleotides are independently selected from the group consisting of: 2'-alkoxy-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'-fluoromodified nucleotides, 3'-deoxy-thymidine nucleotides, isonucleotides, LNA, ENA, cET, UNA and GNA.
  • the other modified nucleotides are independently selected from the group consisting of: 2'-methoxy-modified nucleotides, 2'-fluoro-modified nucleotides, and 2'-deoxy-modified nucleotides Nucleotides.
  • a fluoro-modified nucleotide refers to a nucleotide formed by substituting the hydroxyl group at the 2' position of the ribosyl group of the nucleotide with fluorine.
  • the 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe).
  • a 2'-substituted alkoxy-modified nucleotide for example, can be a 2'-O-methoxyethyl-modified nucleotide (2'-MOE) or a 2'-amino Modified Nucleotides (2'- NH2 ).
  • the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are each independently 2'-deoxynucleotides or 2'-fluoronucleotides in the 5'-end to 3'-end direction Substitute modified nucleotides.
  • the nucleotides at positions 2, 6, 9, 12, and 14 of the antisense strand are each independently 2'-deoxynucleotides or 2', in the 5'-end to 3'-end orientation - Fluorinated modified nucleotides.
  • the nucleotides at positions 2, 4, 6, 9, 12, 14, and 18 of the antisense strand are each independently a 2'-deoxynucleoside in the 5'-end to 3'-end orientation Acid or 2'-fluoro modified nucleotides.
  • the nucleotides at positions 2, 4, 6, 9, 12, 14, 16, and 18 of the antisense strand are each independently 2'-deoxy, in the 5' to 3' direction Nucleotides or 2'-fluoromodified nucleotides.
  • At least one phosphate group in the sense strand and/or antisense strand is a phosphate group with a modifying group.
  • the modifying groups provide increased stability of the siRNA in a biological sample or environment.
  • the phosphate group having the modifying group is a phosphorothioate group.
  • a phosphorothioate group refers to a phosphodiester group in which a non-bridging oxygen atom is replaced by a sulfur atom.
  • the phosphorothioate group is present in at least one position selected from the group consisting of:
  • the sense strand has a nucleotide sequence of the formula:
  • each of Na and Nb independently represents a modified nucleotide or an unmodified nucleotide, and the modifications on Na and Nb are different;
  • the antisense strand has the nucleotide sequence of the formula:
  • each Na ' and Nb ' independently represents a modified nucleotide or an unmodified nucleotide, wherein the modifications on Na' and Nb ' are different; each X ' is independently Na ' or N b ';Y' is Na ' or N b ' ; W' represents a nucleotide containing the chemical modification represented by the above formula (I) or a tautomer modification thereof.
  • the sense strand has a nucleotide sequence of the formula:
  • each of Na and Nb independently represents a modified nucleotide or an unmodified nucleotide, and the modifications on Na and Nb are different;
  • the antisense strand has the nucleotide sequence of the formula:
  • each Na ' and Nb ' independently represents a modified nucleotide or an unmodified nucleotide, wherein the modifications on Na' and Nb ' are different; each X ' is independently Na ' or N b ';Y' is Na ' or N b ' ; W' represents a nucleotide containing the chemical modification represented by the above formula (I) or a tautomer modification thereof.
  • the sense strand has a nucleotide sequence of the formula:
  • each of Na and Nb independently represents a modified nucleotide or an unmodified nucleotide, and the modifications on Na and Nb are different;
  • the antisense strand has the nucleotide sequence of the formula:
  • each Na ' and Nb ' independently represents a modified nucleotide or an unmodified nucleotide, wherein the modifications on Na' and Nb ' are different; each X ' is independently Na ' or N b ';Y' is Na ' or N b ' ; W' represents a nucleotide containing the chemical modification represented by the above formula (I) or a tautomer modification thereof.
  • Na is a 2'-methoxy-modified nucleotide and Nb is a 2'-fluoro-modified nucleotide or a 2'-deoxy-modified nucleotide.
  • Na' is a 2'-methoxy-modified nucleotide and Nb ' is a 2'-fluoro-modified nucleotide or a 2'-deoxy-modified nucleotide.
  • At least one phosphate group in the sense strand and/or antisense strand is a phosphate group with a modifying group that provides increased stability of the siRNA in a biological sample or environment sex.
  • the phosphate group having the modifying group is a phosphorothioate group.
  • a phosphorothioate group refers to a phosphodiester group in which one non-bridging oxygen atom is replaced by a sulfur atom.
  • the phosphorothioate group is present in at least one position selected from the group consisting of:
  • the sense strand has a nucleotide sequence of the formula:
  • Nm represents any nucleotide modified by methoxy, such as C, G, U, A, T modified by methoxy
  • Nf represents any nucleotide modified by fluorine, such as C, G, U, A, T
  • lowercase s indicates a phosphorothioate linkage between two nucleotides adjacent to the letter s
  • the antisense strand has the nucleotide sequence of the formula:
  • Nm' represents any nucleotide modified by methoxy group, such as methoxy-modified C, G, U, A, T
  • Nf' represents any nucleotide modified by fluorine, such as fluorine-modified C, G, U, A, T
  • the lowercase letter s indicates that the two nucleotides adjacent to the letter s are connected by phosphorothioate groups
  • W' indicates that the chemical modification represented by the above formula (I) or its tautomer-modified nucleotides.
  • the nucleotides at positions 2, 6, and 14 of the antisense strand are each independently a 2'-deoxynucleotide or a 2'-fluoromodification in the 5'-end to 3'-end direction nucleotides.
  • the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are each independently 2'-deoxynucleotides or 2'-fluoronucleotides in the 5'-end to 3'-end direction Substitute modified nucleotides.
  • the nucleotides at positions 2, 6, 9, 12, and 14 of the antisense strand are each independently 2'-deoxynucleotides or 2', in the 5'-end to 3'-end orientation - Fluorinated modified nucleotides.
  • the nucleotides at positions 2, 6, 10, 12, and 14 of the antisense strand are each independently 2'-deoxynucleotides or 2', in the 5'-end to 3'-end orientation - Fluorinated modified nucleotides.
  • the nucleotides at positions 2, 4, 6, 9, 12, 14, and 18 of the antisense strand are each independently a 2'-deoxynucleoside in the 5'-end to 3'-end orientation Acid or 2'-fluoro modified nucleotides.
  • the nucleotides at positions 2, 4, 6, 10, 12, 14, and 18 of the antisense strand are each independently a 2'-deoxynucleoside in the 5' to 3' direction Acid or 2'-fluoro modified nucleotides.
  • the nucleotides at positions 2, 4, 6, 9, 12, 14, 16, and 18 of the antisense strand are each independently 2'-deoxy, in the 5' to 3' direction Nucleotides or 2'-fluoromodified nucleotides.
  • the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 of the antisense strand are each independently 2'-deoxy, in the 5' to 3' direction Nucleotides or 2'-fluoromodified nucleotides.
  • the nucleotides at positions 2, 4, 6, 9, 10, 12, 14, 16 and 18 of the antisense strand are each independently 2' in the 5' to 3' direction - Deoxynucleotides or 2'-fluoromodified nucleotides.
  • nucleotides 2, 6, and 14 of the antisense strand are each independently 2'-fluoromodified nucleotides in the 5'-end to 3'-end orientation.
  • the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are each independently 2'-fluoro-modified nucleotides in the 5'-end to 3'-end direction.
  • the nucleotides at positions 2, 6, 12, and 14 of the antisense strand are each independently 2'-fluoro-modified nucleotides in the 5'-end to 3'-end direction.
  • the nucleotides at positions 2, 4, 6, 12, 14, 16, and 18 of the antisense strand are each independently 2'-fluoro-modified in the 5'-end to 3'-end direction nucleotides.
  • the nucleotides at positions 2, 4, 6, 9, 12, 14, 16, and 18 of the antisense strand are each independently 2'-fluoro in the 5'-end to 3'-end orientation Substitute modified nucleotides.
  • the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 of the antisense strand are each independently 2'-fluoro, in the 5' to 3' direction Substitute modified nucleotides.
  • the siRNA sense strand of the present disclosure has a nucleotide sequence of the formula:
  • each of Na and Nb independently represents a modified nucleotide or an unmodified nucleotide, and the modifications on Na and Nb are different; each X is independently Na or Nb .
  • the siRNA antisense strand of the present disclosure has a nucleotide sequence of the formula:
  • each Na ' and Nb ' independently represents a modified nucleotide or an unmodified nucleotide, wherein the modifications on Na' and Nb ' are different; each X ' is independently Na ' Or N b ', Y' is Na ' or N b ' , W' represents a nucleotide containing the chemical modification represented by any of the formula (I) of the present disclosure or a tautomer modification thereof.
  • the modifications on X' and Y' are different.
  • Na is a 2'-methoxy-modified nucleotide and Nb is a 2'-fluoro-modified nucleotide or a 2'-deoxy-modified nucleotide.
  • Na' is a 2'-methoxy-modified nucleotide and Nb ' is a 2'-fluoro-modified nucleotide or a 2'-deoxy-modified nucleotide.
  • Na is a 2'-methoxy-modified nucleotide and Nb is a 2'-fluoro-modified nucleotide.
  • Na' is a 2'-methoxy-modified nucleotide and Nb ' is a 2'-fluoro-modified nucleotide.
  • the siRNA antisense strand of the present disclosure has a nucleotide sequence of the formula:
  • each X' is independently Na ' or Nb '
  • Y' is Na ' or Nb '
  • the modifications on X' and Y' are different
  • Na' is a 2'-methoxy modification
  • the nucleotides of , N b ' is a 2'-fluoro modified nucleotide
  • W' represents a nucleotide containing the chemical modification shown in any of the formula (I) of the present disclosure or a tautomer modification thereof.
  • the siRNA sense strand of the present disclosure has a nucleotide sequence of the formula:
  • Na is a 2'-methoxy-modified nucleotide
  • N b is a 2'-fluoro-modified nucleotide
  • the siRNA antisense strand of the present disclosure has a nucleotide sequence of the formula:
  • Na is a 2'-methoxy-modified nucleotide
  • N b is a 2'-fluoro-modified nucleotide
  • Na ' is a 2'-methoxy-modified nucleotide
  • Nb ' is a 2'-fluoromodified nucleotide
  • W' represents a nucleotide containing a chemical modification represented by any of the formula (I) of the present disclosure or a tautomer modification thereof.
  • W' represents a nucleotide containing a chemical modification or a tautomer modification thereof; the chemical modification is selected from:
  • B is selected from guanine, adenine, cytosine or uracil; in some specific embodiments, B is selected from the base at the corresponding position in the 7th position of the 5' region of the antisense strand.
  • W' represents a nucleotide containing a chemical modification or a tautomer modification thereof; the chemical modification is selected from:
  • M is O or S; wherein: B is selected from guanine, adenine, cytosine or uracil; in some specific embodiments, B is selected from the antisense strand corresponding to the 7th position in its 5' region position of the base.
  • M is S. In some specific embodiments, M is O.
  • At least one phosphate group in the sense strand and/or antisense strand is a phosphate group with a modifying group that provides increased stability of the siRNA in a biological sample or environment property; in some embodiments, the phosphate group with the modifying group is a phosphorothioate group.
  • a phosphorothioate group refers to a phosphodiester group in which a non-bridging oxygen atom is replaced by a sulfur atom.
  • the phosphorothioate group is present in at least one position selected from the group consisting of:
  • a plurality of phosphorothioate groups are included in the sense and/or antisense strands, the phosphorothioate groups being present in:
  • the first nucleotide end of the 3' end of the sense strand and/or,
  • the sense strand is selected from a nucleotide sequence of the formula:
  • Nm represents any nucleotide modified by 2'-methoxy group, such as C, G, U, A, T modified by 2'-methoxy group
  • Nf represents any nucleotide modified by 2'-fluoro, For example, 2'-fluoro-modified C, G, U, A, T;
  • the antisense strand has a nucleotide sequence of the formula:
  • Nm' represents any nucleotide modified by 2'-methoxy, such as C, G, U, A, T modified by 2'-methoxy
  • Nf' represents any nucleotide modified by 2'-fluoro Acids such as 2'-fluoro-modified C, G, U, A, T;
  • W' represents a nucleotide containing chemical modification or its tautomer modification; the chemical modification is selected from:
  • B is selected from guanine, adenine, cytosine or uracil, and in some embodiments is selected from the base of the antisense strand corresponding to position 7 in its 5' region.
  • W' represents a nucleotide containing a chemical modification or a tautomer modification thereof; the chemical modification is selected from:
  • M is O or S; wherein: B is selected from guanine, adenine, cytosine or uracil; in some specific embodiments, B is selected from the antisense strand corresponding to the 7th position in its 5' region position of the base.
  • M is S. In some specific embodiments, M is O.
  • the siRNA comprises a sense strand selected from Table 5.
  • the siRNA comprises any antisense strand selected from Table 5.
  • the siRNA comprises any sense strand selected from Table 8.
  • the siRNA comprises any antisense strand selected from Table 8.
  • the siRNA comprises any antisense strand selected from Table 9.
  • the siRNA comprises any sense strand selected from Table 13.
  • the siRNA comprises any antisense strand selected from Table 13.
  • the siRNA comprises any sense strand selected from Table 15.
  • the siRNA comprises any antisense strand selected from Table 15.
  • the siRNA comprises any sense strand selected from Table 24.
  • the siRNA comprises any antisense strand selected from Table 24.
  • the siRNA comprises any sense strand selected from Table 25.
  • the siRNA comprises any antisense strand selected from Table 25.
  • the siRNA comprises any sense strand selected from Table 26.
  • the siRNA comprises any antisense strand selected from Table 26.
  • the siRNA comprises any sense strand selected from Table 66.
  • the siRNA comprises any antisense strand selected from Table 66.
  • the siRNA described above when contacted with cells expressing the target gene, is screened for activity by, for example, psiCHECK and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based Methods, as determined by immunofluorescence assays such as Western Blot or flow cytometry, the above siRNAs 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%.
  • psiCHECK and luciferase reporter gene assays other methods
  • the siRNA described above when contacted with cells expressing the target gene, is screened for activity by, for example, psiCHECK and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based Methods, such as immunofluorescence analysis, such as Western Blot or flow cytometry, the percentage of residual expression of target gene mRNA caused by the above siRNA is not higher than 99%, not higher than 95%, not higher than 90%, not higher at 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45% %, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%.
  • psiCHECK and luciferase reporter gene assays other methods such as PCR or branched DNA (bDNA)-based methods,
  • siRNAs comprising chemical modifications of the present disclosure, when contacted with cells expressing the target gene, are screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or branched DNA (bDNA) based methods , or protein-based methods, such as assayed by immunofluorescence assays, such as Western Blot, or flow cytometry, comprising chemical modifications of the present disclosure, such as chemically modified siRNAs of formula (I) or formula (II) maintained at 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 and luciferase reporter gene assays others such as PCR or branched DNA (bDNA) based methods , or protein-based methods, such as assayed by immunofluorescence assays
  • siRNAs comprising chemical modifications of the present disclosure, when contacted with cells expressing the target gene, are screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or branched DNA (bDNA) based methods , or protein-based methods, such as assayed by immunofluorescence assays, such as Western Blot, or flow cytometry, comprising chemical modifications of the present disclosure, such as chemically modified siRNAs of formula (I) or formula (II) to enable targeting 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 and luciferase reporter gene assays others such as PCR or branched DNA (bDNA) based methods , or protein-based methods, such as assayed by immunofluorescence assay
  • siRNAs comprising chemical modifications of the present disclosure when contacted with cells expressing the target gene, are screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or branched DNA (bDNA) based methods , or protein-based methods, such as assayed by immunofluorescence assays, such as Western Blot, or flow cytometry, comprising chemical modifications of the present disclosure, such as chemically modified siRNAs of formula (I) or formula (II) to enable targeting Increased activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, At least 60%, at least 65%, at least 70%, at least 75%, or at least 80% while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% , at
  • the present disclosure also provides an siRNA conjugate comprising any one of the siRNAs described above and a conjugation group attached to the siRNA.
  • the conjugation group comprises a pharmaceutically acceptable targeting ligand and an optional linker
  • the siRNA, the linker and the targeting ligand are sequentially covalent or non-covalent price connection.
  • the linker is attached to the 3' end of the sense strand of the siRNA.
  • the present disclosure also provides an siRNA conjugate comprising any one of the siRNAs described above and a targeting ligand linked to the siRNA.
  • the siRNA and the targeting ligand are covalently or non-covalently linked.
  • the targeting ligand is attached to the 3' end of the sense strand of the siRNA.
  • the targeting ligand targets the liver.
  • the targeting ligand binds the asialoglycoprotein receptor (ASGPR).
  • ASGPR asialoglycoprotein receptor
  • the targeting ligand is selected from: a cluster of galactose or a cluster of galactose derivatives selected from the group consisting of N-acetyl-galactosamine, N-trifluoroacetylgalactose amine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine.
  • a lipophilic group such as cholesterol can be introduced at the end of the siRNA sense strand, and the lipophilic group can be combined with the small interfering nucleic acid by covalent bond, such as cholesterol, lipid and other lipophilic groups are introduced at the end.
  • Protein, 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, cationic polymers, etc. through hydrophobic bonds or ionic bonds to increase stability and biological activity.
  • the targeting ligand is attached to the end of the siRNA through a phosphate group, phosphorothioate group, or phosphonate group.
  • the targeting ligand is indirectly attached to the end of the siRNA through a phosphate group, phosphorothioate group, or phosphonate group.
  • the targeting ligand is directly attached to the end of the siRNA through a phosphate group, phosphorothioate group, or phosphonate group.
  • the targeting ligand is directly attached to the end of the siRNA through a phosphate group or a phosphorothioate group.
  • the targeting ligand is directly attached to the 3' end of the siRNA sense strand through a phosphate group or a phosphorothioate group.
  • the targeting ligand structure is shown in the following formula (IV),
  • T is the targeting moiety
  • E is the branching group
  • L1 is the linker moiety
  • L2 is the tethering moiety between the targeting moiety and the branching group, wherein i is selected from an integer from 1 to 10, such as 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10.
  • i is selected from an integer from 2 to 8.
  • i is selected from an integer from 3 to 5.
  • L 1 is N
  • R 9 and R 10 are each independently selected from -S-, -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O) -, -C(O)NH-, -CH2- , -CH2NH-, -CH2O- , -NH-C(O) -CH2- , -C(O) -CH2 - NH- , -NH(CO)NH-, 3-12-membered heterocyclic group, the -CH 2 - is optionally substituted by a substituent selected from halogen, alkyl, alkoxy, and alkylamino, and the alkyl is optionally substituted is further substituted with a substituent selected from hydroxy, amino, halogen;
  • R 11 is selected from deuterium, halogen, alkyl, amino, cyano, nitro, alkenyl, alkynyl, carboxyl, hydroxyl, mercapto, alkylmercapto, alkoxy, alkylamino, -C(O)-alkyl, -C(O)-O-Alkyl, -CONH 2 , -CONH-Alkyl, -OC(O)-Alkyl, -NH-C(O)-Alkyl, -S(O)O-Alkyl , -S(O)ONH 2 , -S(O)ONH-alkyl, said alkyl, alkenyl, alkynyl, alkylmercapto, alkyloxy, -C(O)-alkyl, - C(O)-O-Alkyl, -CONH-Alkyl, -OC(O)-Alkyl, -NH-C(
  • the k is selected from 0, 1, 2, 3, 4;
  • the j is selected from integers from 1 to 20 (eg 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).
  • L 1 is N
  • R 11 is selected from deuterium, halogen, alkyl, amino, cyano, nitro, alkenyl, alkynyl, carboxyl, hydroxyl, mercapto, alkylmercapto, alkoxy, alkylamino, -C(O)-alkyl , -C(O)-O-Alkyl, -CONH 2 , -CONH-Alkyl, -OC(O)-Alkyl, -NH-C(O)-Alkyl, -S(O)O-Alkyl base, -S(O)ONH 2 , -S(O)ONH-alkyl, said alkyl, alkenyl, alkynyl, carboxyl, alkylmercapto, alkyloxy, -C(O)-alkane Alkyl, -C(O)-O-Alkyl, -CONH-Alkyl, -OC(O)-Al,
  • the k is selected from 0, 1, 2, 3, 4;
  • L 1 is N
  • L 1 is N
  • L 1 is N
  • E in the targeting ligand is
  • the R 12 , R 13 , R 14 and R 15 are each independently selected from -C(O)NH-, -C(O)-, the carbonyl group is optionally further substituted by an alkyl group, and the alkyl group optionally further substituted by the group consisting of alkyl, hydroxy, -C(O)O-, -C(O)O-alkyl-, -C(O)NH-;
  • the X 2 , X 3 , X 4 and X 5 are each independently an integer selected from 0 to 10 (eg, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).
  • E in the targeting ligand is
  • R 12 , R 13 , R 14 and R 15 are each independently selected from -C(O)NH-, -C(O)-, said -C(O)NH-, -C(O)- Optionally further substituted with an alkyl group optionally further selected from the group consisting of alkyl, hydroxyl, -C(O)O-, -C(O)O-alkyl-, -C(O)NH- replace;
  • the X 2 , X 3 , X 4 and X 5 are each independently an integer selected from 0 to 10 (eg, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).
  • E in the targeting ligand is
  • the R 12 , R 13 , R 14 and R 15 are each independently selected from -C(O)NH-, -C(O)-,
  • the X 2 , X 3 , X 4 and X 5 are each independently an integer selected from 0 to 10 (eg, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).
  • E in the targeting ligand is
  • E in the targeting ligand is selected from
  • E in the targeting ligand is selected from
  • E in the targeting ligand is selected from
  • E in the targeting ligand is
  • L 1 is selected from the following structures:
  • R 9 and R 10 are each independently selected from -S-, -NH-, -O-, -S-, -C(O)-, -OC(O)-, -C(O)O-, - NHC(O)-, -C(O)NH-, -CH2- , -CH2NH-, -CH2O- , -NH-C(O) -CH2- , -C (O)-CH 2 -NH-, -NH(CO)NH-, 3-12-membered heterocyclic group, the -CH 2 - is optionally substituted by a substituent selected from halogen, alkyl, alkoxy, alkylamino, the The alkyl group is optionally further substituted with a substituent selected from hydroxy, amino, halogen;
  • R 11 is selected from deuterium, halogen, alkyl, amino, cyano, nitro, alkenyl, alkynyl, carboxyl, hydroxyl, mercapto, alkylmercapto, alkoxy, alkylamino, -C(O)-alkyl, -C(O)-O-Alkyl, -CONH 2 , -CONH-Alkyl, -OC(O)-Alkyl, -NH-C(O)-Alkyl, -S(O)O-Alkyl , -S(O)ONH 2 , -S(O)ONH-alkyl, said alkyl, alkenyl, alkynyl, alkylmercapto, alkyloxy, -C(O)-alkyl, - C(O)-O-Alkyl, -CONH-Alkyl, -OC(O)-Alkyl, -NH-C(
  • the k is selected from 0, 1, 2, 3, 4;
  • Said j is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.
  • E in the targeting ligand is selected from:
  • L1 is selected from :
  • E in the targeting ligand is selected from:
  • L1 is selected from : That is, EL 1 is
  • E in the targeting ligand is selected from:
  • L1 is selected from : That is, EL 1 is
  • E in the targeting ligand is selected from L1 is selected from That is, EL 1 is
  • E in the targeting ligand is selected from L1 is selected from That is, EL 1 is
  • L 2 is the tethering moiety between the targeting moiety and the branching group, and L 2 plays the role of linking and spacing between the branching group and each targeting moiety.
  • one end of L2 is directly attached to the targeting ligand and the other end is directly attached to the branching group E.
  • one end of L 2 is directly attached to the targeting ligand and the other end is indirectly attached to the branching group E.
  • one end of L2 is indirectly linked to the targeting ligand and the other end is indirectly linked to the branching group E.
  • the targeting ligands disclosed herein include 2 L2 and 2 targeting moieties.
  • the targeting ligands disclosed herein include 3 L2 and 3 targeting moieties.
  • the targeting ligands disclosed herein include 4 L2 and 4 targeting moieties.
  • the targeting ligands disclosed herein include multiple L 2s and multiple targeting moieties.
  • L in the present disclosure is selected from 1 or a combination of 2-20 covalently linked groups (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20):
  • Substituted or unsubstituted cycloalkyl eg cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclooctyl, etc.
  • substituted or unsubstituted cycloalkenyl eg cyclohexene base, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.
  • substituted or unsubstituted aryl eg phenyl, naphthyl, binaphthyl, anthracenyl, etc.
  • substituted or unsubstituted heteroaryl eg pyridyl, pyrimidinyl,
  • L in the present disclosure is selected from 1 or a combination of 2-20 covalently linked groups (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20):
  • the targeting ligand comprises L 2 having the structure shown below,
  • x6 is an integer from 1 to 20 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).
  • the targeting ligand comprises L2 having the structure shown below,
  • the targeting ligand comprises L2 having the structure shown below,
  • the targeting ligand comprises L2 having the structure shown below,
  • the targeting ligand comprises L2 having the structure shown below,
  • the targeting ligand comprises L2 having the structure shown below,
  • the targeting ligand comprises L2 having the structure shown below,
  • x 7 is an integer from 1 to 20 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and Z is
  • the targeting ligand has L2 of the structure shown below,
  • the targeting ligand has L2 of the structure shown below,
  • x8 is an integer from 1 to 20 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19 or 20), and Z is
  • the targeting ligand has L2 of the structure shown below,
  • x 9 and X 10 are each independently selected from integers from 1 to 20 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and Z is
  • the targeting ligand has L2 of the structure shown below,
  • the targeting ligand has L2 of the structure shown below, wherein x7 and X8 are each independently selected from integers from 1 to 20 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, or 20), and Z is
  • the targeting ligand has the following structure:
  • the targeting ligand has the following structure:
  • the targeting ligand has the following structure:
  • the targeting ligand has the following structure:
  • the targeting moiety of the targeting ligand is comprised of one or more targeting moieties that assist in directing delivery of the therapeutic agent attached thereto to the desired target site.
  • the targeting moiety can bind to a cell or cellular receptor and initiate endocytosis to facilitate entry of the therapeutic agent into the cell.
  • the targeting moiety may comprise a compound having affinity for a cell receptor or cell surface molecule or antibody.
  • Various targeting ligands containing targeting moieties can be linked to therapeutic agents and other compounds to target the agents to cells and specific cellular receptors.
  • the types of targeting moieties include carbohydrates, cholesterol and cholesteryl groups or steroids.
  • Targeting moieties that can bind to cellular receptors include carbohydrates such as galactose, galactose derivatives (eg N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine), mannose and mannose derivatives.
  • Targeting moieties known to bind the asialoglycoprotein receptor are particularly useful for directing delivery of oligomeric compounds to the liver.
  • the asialoglycoprotein receptor is abundantly expressed on liver cells (hepatocytes).
  • Cell receptor targeting moieties targeting ASCPR include galactose and galactose derivatives. Specifically, clusters of galactose derivatives, including clusters consisting of 2, 3, 4, or more than 4 N-acetyl-galactosamines (GalNAc or NAG), can promote the uptake of certain compounds in hepatocytes.
  • GalNAc clusters coupled to oligomeric compounds were used to direct the composition to the liver, where N-acetyl-galactosamine sugars were able to bind to asialoglycoprotein receptors on the surface of liver cells. Binding of the asialoglycoprotein receptor is thought to initiate receptor-mediated endocytosis, thereby facilitating the entry of compounds into the cell interior.
  • the targeting ligand may include 2, 3, 4, or more than 4 targeting moieties.
  • the targeting ligands disclosed herein can include 1, 2 , 3, 4, or more than 4 targeting moieties linked to a branching group through L2.
  • the targeting ligand is in the form of a galactose cluster.
  • each targeting moiety includes a galactosamine derivative, which is N-acetyl-galactosamine.
  • Other sugars that can be used as targeting moieties and have affinity for the asialoglycoprotein receptor can be selected from galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine and N-isobutyryl-galactosamine, etc.
  • the targeting ligands of the present disclosure include N-acetylgalactosamine as a targeting moiety
  • the targeting ligand includes three terminal galactosamine or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has an affinity for the sialoglycoprotein receptor sex.
  • the targeting ligand includes a three-terminal N-acetyl-galactosamine (GalNAc or NAG) as a targeting moiety.
  • the targeting ligand includes four terminal galactosamine or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for the asialoglycoprotein receptor and sex. In some embodiments, the targeting ligand includes four terminal N-acetyl-galactosamine (GalNAc or NAG) as targeting moieties.
  • galactosamine or galactosamine derivatives such as N-acetyl-galactosamine
  • NAG N-acetyl-galactosamine
  • the targeting ligands of the present disclosure have the following structure,
  • the targeting ligands provided by the present disclosure have the following structures,
  • the targeting ligands provided by the present disclosure have the following structures,
  • the targeting ligands provided by the present disclosure have the following structures,
  • the siRNA of the present disclosure is linked to a targeting ligand of the present disclosure to form a siRNA conjugate having the following,
  • T is the targeting moiety
  • E is the branching group
  • L1 is the linker moiety
  • L2 is the tethering moiety between the targeting moiety and the branching group
  • x is an integer from 1 to 10
  • D is any of the above Protocol for siRNA.
  • D is an siRNA targeting ApoC3.
  • D is an siRNA targeting HBV-X.
  • D is an siRNA targeting F11.
  • D is an siRNA targeting HBV-S.
  • D is an siRNA targeting angiopoietin-like protein-3 (ANGPTL3).
  • D is an siRNA targeting the thyroxine transporter (TTR) gene.
  • TTR thyroxine transporter
  • D is any siRNA of the present disclosure.
  • the L1 is linked to the 3 ' end of the sense strand of the siRNA.
  • the targeting ligand is attached to the end of the siRNA through a phosphate group, phosphorothioate group, or phosphonate group.
  • the targeting ligand is indirectly attached to the end of the siRNA through a phosphate group, phosphorothioate group, or phosphonate group.
  • the targeting ligand is directly attached to the end of the siRNA through a phosphate group, phosphorothioate group, or phosphonate group.
  • the targeting ligand is directly attached to the end of the siRNA through a phosphate group, a phosphorothioate group.
  • the targeting ligand is directly attached to the 3' end of the siRNA sense strand through a phosphate group, a phosphorothioate group.
  • siRNA conjugates described in the present disclosure are as follows,
  • D is the siRNA of any of the above-mentioned schemes.
  • D is an siRNA targeting ApoC3.
  • D is an siRNA targeting HBV-X.
  • D is an siRNA targeting F11.
  • D is an siRNA targeting HBV-S.
  • D is an siRNA targeting angiopoietin-like protein-3 (ANGPTL3).
  • D is an siRNA targeting the thyroxine transporter (TTR) gene.
  • TTR thyroxine transporter
  • the targeting ligand is directly attached to the 3' end of the siRNA sense strand through a phosphate group or a phosphorothioate group.
  • siRNA conjugates described in the present disclosure are as follows,
  • D is the siRNA of any of the above-mentioned schemes.
  • D is an siRNA targeting ApoC3.
  • D is an siRNA targeting HBV-X.
  • D is an siRNA targeting F11.
  • D is an siRNA targeting HBV-S.
  • D is an siRNA targeting angiopoietin-like protein-3 (ANGPTL3).
  • D is an siRNA targeting the thyroxine transporter (TTR) gene.
  • TTR thyroxine transporter
  • the targeting ligand is directly attached to the 3' end of the siRNA sense strand through a phosphate group or a phosphorothioate group.
  • siRNA conjugates described in the present disclosure are as follows,
  • D is the siRNA of any of the above-mentioned schemes.
  • D is an siRNA targeting ApoC3.
  • D is an siRNA targeting HBV-X.
  • D is an siRNA targeting F11.
  • D is an siRNA targeting HBV-S.
  • D is an siRNA targeting angiopoietin-like protein-3 (ANGPTL3).
  • D is an siRNA targeting the thyroxine transporter (TTR) gene.
  • TTR thyroxine transporter
  • the targeting ligand is directly attached to the 3' end of the siRNA sense strand through a phosphate group or a phosphorothioate group.
  • siRNA conjugates described in the present disclosure are as follows,
  • D is the siRNA of any of the above-mentioned schemes.
  • D is an siRNA targeting ApoC3.
  • D is an siRNA targeting HBV-X.
  • D is an siRNA targeting F11.
  • D is an siRNA targeting HBV-S.
  • D is an siRNA targeting angiopoietin-like protein-3 (ANGPTL3).
  • D is an siRNA targeting the thyroxine transporter (TTR) gene.
  • TTR thyroxine transporter
  • the targeting ligand is directly attached to the 3' end of the siRNA sense strand through a phosphate group or a phosphorothioate group.
  • L 1 and D are linked through a phosphate group, a phosphorothioate group, or a phosphonic acid group.
  • L 1 is linked to the 3' end of the D sense strand through a phosphate group, a phosphorothioate group, or a phosphonic acid group.
  • the L 1 and D sense strand 3' ends are directly linked through a phosphate group or a phosphorothioate group.
  • L 1 is indirectly linked to the 3' terminus of the D sense strand through a phosphate group or a phosphorothioate group.
  • a lipophilic group such as cholesterol can be introduced at the end of the siRNA sense strand, and the lipophilic group includes covalent bonding with small interfering nucleic acid, such as introduction of cholesterol at the end, Lipoprotein, 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, cationic polymers, etc. through hydrophobic bonds or ionic bonds to increase stability and biological activity.
  • compositions comprising a conjugate as claimed above, and one or more pharmaceutically acceptable excipients such as a carrier, vehicle, diluent, and/or delivery polymer thing.
  • the present disclosure also provides a pharmaceutical composition comprising the siRNA or siRNA conjugate of the present disclosure.
  • the pharmaceutical composition may also contain pharmaceutically acceptable adjuvants and/or adjuvants, and the adjuvants may be one or more various formulations or compounds conventionally used in the art.
  • the pharmaceutically acceptable adjuvant may include at least one of pH buffering agents, protecting agents and osmotic pressure adjusting agents.
  • Another aspect of the present disclosure provides use of the above conjugate or a composition containing the conjugate in the manufacture of a medicament for treating a disease in a subject, in some embodiments the disease is selected from liver-derived diseases.
  • Another aspect of the present disclosure provides a method of treating a disease in a subject, comprising administering to the subject the above-described conjugate, or composition.
  • Another aspect of the present disclosure provides a method of inhibiting mRNA expression in a subject, the method comprising administering to the subject the above-described conjugate, or composition.
  • Another aspect of the present disclosure provides a method of delivering an expression-inhibiting oligomeric compound to the liver in vivo by administering the above-described conjugate, or composition, to a subject.
  • the conjugates, compositions and methods disclosed herein can reduce the level of a target mRNA in a cell, cell population, cell population, tissue or subject, including administering to the subject a therapeutically effective amount of the expression described herein An inhibitory oligomer linked to a targeting ligand, thereby inhibiting the expression of a target mRNA in a subject.
  • the subject has been previously identified as having pathogenic upregulation of the target gene in the targeted cell or tissue.
  • a subject referred to in the present disclosure refers to a subject suffering from a disease or disorder that would benefit from reduction or inhibition of target mRNA expression.
  • Delivery can be by local administration (eg, direct injection, implantation, or topical administration), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (eg, intraventricular, parenchymal) intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.
  • local administration eg, direct injection, implantation, or topical administration
  • systemic administration eg., systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (eg, intraventricular, parenchymal) intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.
  • compositions provided by the present disclosure may be administered by injection, eg, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal injection.
  • the conjugate can be packaged in a kit.
  • the siRNA conjugates or pharmaceutical compositions described above when contacted with cells expressing the target gene, are screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or based on branched DNA (bDNA)
  • the above-described siRNA conjugate or pharmaceutical composition inhibits 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 siRNA conjugates or pharmaceutical compositions described above when contacted with cells expressing the target gene, are screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or based on branched DNA (bDNA) method, or a protein-based method, such as immunofluorescence assay, such as Western Blot or flow cytometry, the residual expression percentage of target gene mRNA caused by the above-mentioned siRNA conjugate or pharmaceutical composition is not higher than 99%, not higher than 95%, not higher than 90%, not higher than 85%, not higher than 80%, not higher than 75%, not higher than 70%, not higher than 65%, not higher than 60%, not higher over 55%, up to 50%, up to 45%, up to 40%, up to 35%, up to 30%, up to 25%, up to 20%, up to 15 %, or not higher than 10%.
  • psiCHECK activity and luciferase reporter gene assays others such as PCR or based on
  • the siRNA conjugate or pharmaceutical composition when contacted with cells expressing the target gene, is screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or branched DNA (bDNA) based assays.
  • Methods, or protein-based methods, such as immunofluorescence assays, such as Western Blot, or flow cytometry the siRNA conjugate reduces off-target activity by at least 20%, at least 25% while maintaining on-target activity , at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.
  • the siRNA conjugate or pharmaceutical composition when contacted with cells expressing the target gene, is screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or branched DNA (bDNA) based assays.
  • the siRNA conjugate reduces on-target activity by up to 20%, up to 19%, up to 15%, up to 10% , at least 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%.
  • the siRNA conjugate or pharmaceutical composition when contacted with cells expressing the target gene, is screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or branched DNA (bDNA) based assays.
  • the siRNA conjugate increases on-target activity by at least 1%, at least 5%, at least 10%, at least 15% , at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, At least 70% or at least 75%.
  • the present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell, the method comprising the step of introducing the siRNA, siRNA conjugate and/or pharmaceutical composition of the present disclosure into the cell.
  • the present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell in vivo or in vitro, the method comprising introducing into the cell an siRNA, siRNA conjugate and/or pharmaceutical composition according to the present disclosure steps in .
  • the present disclosure also provides a method for inhibiting a target gene or mRNA expression of a target gene, the method comprising administering to a subject in need thereof an effective amount or an effective dose of the siRNA, siRNA conjugate and /or pharmaceutical composition.
  • administration is by means of administration including intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, cerebrospinal, or combinations thereof.
  • the effective amount or effective dose of the siRNA, siRNA conjugate and/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 target gene is a hepatitis B virus (HBV) gene, an angiopoietin-like protein-3 (ANGPTL3) gene, or a thyroxine transporter (TTR) gene.
  • HBV hepatitis B virus
  • ANGPTL3 angiopoietin-like protein-3
  • TTR thyroxine transporter
  • the present disclosure also provides use of the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate in the manufacture of a medicament for preventing and/or treating pathological conditions and diseases caused by hepatitis B virus.
  • the present disclosure also provides the use of the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate in the preparation of a medicament for preventing and/or treating hepatitis B.
  • the present disclosure also provides a method of treating hepatitis B, the method comprising administering the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate to a patient in need thereof.
  • the present disclosure also provides a method of inhibiting HBV gene expression in chronic HBV-infected hepatitis cells, the method comprising introducing an effective amount or an effective dose of the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate into the infection Hepatitis cells of chronic HBV.
  • the present disclosure also provides the aforementioned siRNA and/or pharmaceutical compositions and/or siRNA conjugates prepared for the prevention and/or treatment of pathological conditions caused by abnormal expression of ANGPTL3 gene or TTR gene in mammals (eg, humans) and use in medicines for diseases.
  • the present disclosure also provides a method of treating pathological conditions and diseases caused by abnormal expression of ANGPTL3 gene or TTR gene, comprising administering an effective amount or an effective dose of the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugation thing.
  • Pathological conditions and diseases caused by abnormal expression of the ANGPTL3 gene include cardiovascular disease and/or metabolic disease, such as hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, obesity, diabetes and/or ischemia heart disease.
  • Pathological conditions and diseases caused by abnormal TTR gene expression include sensory neuropathy (eg, distal limb paresthesia, hypoesthesia), autonomic neuropathy (eg, gastrointestinal disorders such as gastric ulcer, or orthostatic hypotension), motor neuropathy, Seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic deficits, cardiomyopathy, vitreous opacity, renal insufficiency, nephropathy, parenchymal mBMI (change in body mass index), cranial nerve dysfunction, and lattice corneal dystrophy .
  • sensory neuropathy eg, distal limb paresthesia, hypoesthesia
  • autonomic neuropathy eg, gastrointestinal disorders such as gastric ulcer, or orthostatic hypotension
  • motor neuropathy Seizures
  • dementia myelopathy
  • polyneuropathy polyneuropathy
  • carpal tunnel syndrome autonomic deficits
  • cardiomyopathy vitreous opacity
  • renal insufficiency nephropathy
  • the aforementioned siRNAs and/or pharmaceutical compositions and/or siRNA conjugates are excellent in modulating genes expressed in the liver, or in treating pathological conditions or diseases caused by abnormal gene expression in hepatocytes on-target activity and reduced off-target activity.
  • the genes expressed in the liver include, but are not limited to, ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, HCV and other genes.
  • the specific gene is selected from the hepatitis B virus gene, the angiopoietin-like protein 3 gene, or the apolipoprotein C3 gene.
  • the disease is selected from chronic liver disease, hepatitis, liver fibrotic disease, liver proliferative disease and dyslipidemia.
  • the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.
  • the aforementioned siRNAs, siRNA conjugates, and/or pharmaceutical compositions may also be used to treat other liver diseases, including diseases characterized by unwanted cell proliferation, blood diseases, metabolic diseases, and inflammation disease.
  • Proliferative diseases of the liver can be benign or malignant diseases such as cancer, hepatocellular carcinoma (HCC), liver metastases or hepatoblastoma.
  • Liver hematological or inflammatory diseases may be diseases involving coagulation factors, complement-mediated inflammation or fibrosis.
  • Metabolic diseases of the liver include dyslipidemia and irregularities in glucose regulation.
  • the present disclosure also provides a cell comprising the siRNA or siRNA conjugate of the present disclosure.
  • the present disclosure also provides a kit comprising the siRNA or siRNA conjugate of the present disclosure.
  • the present disclosure also provides a compound represented by formula (II) or a tautomer thereof,
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 ′), S, NR 5 and NH-CO, wherein R 4 , R 4 ′, R 5 are each independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • Q' 1 is Q 2 is R 2 ; or Q 1 is R 2 and Q' 2 is
  • J 1 is H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is a base or a base analog
  • W is a leaving group and Z is a phosphorus-containing reactive reactive group.
  • W is MMTr or DMTr.
  • Z is
  • the above-mentioned compounds are not
  • R1 is not H.
  • the above compound is not:
  • the above compound is not:
  • the compound represented by formula (II) or its tautomer is specifically: the compound represented by formula (II-1) or its tautomer,
  • 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 each independently H or C 1 -C 6 alkyl;
  • Each J 1 , J 2 is independently H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is a base or a base analog
  • W is a leaving group and Z is a phosphorus-containing reactive reactive group.
  • W is MMTr or DMTr.
  • Z is
  • the above-mentioned compounds are not
  • R1 is not H.
  • the above compound is not:
  • the above compound is not:
  • the compound represented by formula (II) or its tautomer is specifically: the compound represented by formula (II-2) or its tautomer,
  • 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 each independently H or C 1 -C 6 alkyl;
  • Each J 1 , J 2 is independently H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is a base or a base analog
  • W is a leaving group and Z is a phosphorus-containing reactive reactive group.
  • W is MMTr or DMTr.
  • Z is
  • the above-mentioned compounds are not
  • R1 is not H.
  • the above compound is not:
  • the above compound is not:
  • 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;
  • Each J 1 , J 2 is independently H or C 1 -C 3 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.
  • 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, methyl, B radical, n-propyl or isopropyl;
  • Each J 1 , J 2 is independently H or methyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2- Aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2- Thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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, methyl, B radical, n-propyl or isopropyl;
  • Each J 1 , J 2 is independently H or methyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, 7-deazapurine, cytosine Pyrimidine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • Each J 1 , J 2 is 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;
  • R3 is selected from H, OH, NH2 , methyl and CH2OH ;
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • Each J 1 , J 2 is independently H;
  • R 1 is selected from H, methyl and CH 2 OH;
  • R 2 is selected from H, methyl and CH 2 OH;
  • R3 is selected from H, OH, NH2 , methyl and CH2OH ;
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from the group consisting of adenine, guanine, cytosine, uracil, and thymine.
  • the above-mentioned compounds include, but are not limited to:
  • the present disclosure also provides a compound of formula (III) or a tautomer thereof,
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 ′), S, NR 5 and NH-CO, wherein R 4 , R 4 ′, R 5 are each independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • Q” 1 is Q 2 is R 2 ; or Q 1 is R 2 and Q" 2 is
  • J 1 is H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is a base or a base analog
  • W is a leaving group
  • W is MMTr or DMTr.
  • the above-mentioned compounds are not
  • R1 is not H.
  • the above-mentioned compounds are not one or more of the following compounds:
  • the compound represented by formula (III) or its tautomer is specifically: the compound represented by formula (III-1) or its tautomer,
  • 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 each independently H or C 1 -C 6 alkyl;
  • Each J 1 , J 2 is independently H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is a base or a base analog
  • W is a leaving group
  • W is MMTr or DMTr.
  • the above-mentioned compounds are not
  • R1 is not H.
  • the compound of formula (III) is not one or more of the following compounds:
  • the compound represented by formula (III) or its tautomer is specifically: the compound represented by formula (III-2) or its tautomer,
  • 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 each independently H or C 1 -C 6 alkyl;
  • Each J 1 , J 2 is independently H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is a base or a base analog
  • W is a leaving group
  • W is MMTr or DMTr.
  • the above-mentioned compounds are not
  • R1 is not H.
  • the above-mentioned compounds are not one or more of the following compounds:
  • 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;
  • Each J 1 , J 2 is independently H or C 1 -C 3 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.
  • 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, methyl, B radical, n-propyl or isopropyl;
  • Each J 1 , J 2 is independently H or methyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2- Aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2- Thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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, methyl, B radical, n-propyl or isopropyl;
  • Each J 1 , J 2 is independently H or methyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, 7-deazapurine, cytosine Pyrimidine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • Each J 1 , J 2 is 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;
  • R3 is selected from H, OH, NH2 , methyl and CH2OH ;
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • Each J 1 , J 2 is independently H;
  • R 1 is selected from H, methyl and CH 2 OH;
  • R 2 is selected from H, methyl and CH 2 OH;
  • R3 is selected from H, OH, NH2 , methyl and CH2OH ;
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from the group consisting of adenine, guanine, cytosine, uracil, and thymine.
  • the above-mentioned compounds include, but are not limited to:
  • the present disclosure also provides a siRNA or siRNA conjugate, which is characterized in that it is modified with a 2'-methoxy group to replace the chemical modification shown in formula (I) of the antisense strand of any siRNA or siRNA conjugate of the present disclosure or its tautomer modification.
  • the present disclosure also provides an siRNA or siRNA conjugate, characterized in that the chemical modification represented by the formula (I) contained in the antisense strand of any siRNA or siRNA conjugate of the present disclosure is a 2'-methoxy modification .
  • the present disclosure also provides an siRNA or siRNA conjugate, the antisense strand comprising a modification in at least one nucleotide in positions 2 to 8 of its 5' region, the modification being of formula (I) Chemical modifications or tautomeric modifications as indicated.
  • the present disclosure also provides an siRNA or siRNA conjugate, characterized in that a base T is used to replace one or more bases U of any siRNA or siRNA conjugate of the present disclosure, such as 1, 2, 3 1, 3, 5, 6, 7, 8, 9, 10.
  • the present disclosure also provides a method for preparing the aforementioned siRNA or siRNA conjugate, comprising the following steps: (1) synthesizing the compound represented by formula (II) or its tautomer; (2) utilizing step (1) ) or its tautomer to synthesize the aforementioned siRNA or siRNA conjugate.
  • the compound of formula (III) or its tautomer is used to synthesize the compound of formula (II) or its tautomer.
  • the present disclosure also provides the use of the above-mentioned compound represented by formula (II) or its tautomer in inhibiting or reducing off-target activity of siRNA.
  • the present disclosure also provides the use of the above-mentioned compound represented by formula (II) or its tautomer in preparing siRNA.
  • the 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 racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which belong to within the scope of this disclosure.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl. All such isomers, as well as mixtures thereof, are included within the scope of this disclosure.
  • tautomer or “tautomeric form” refers to structural isomers of different energies that are interconvertible via a low energy barrier.
  • proton tautomers also known as proton tautomers
  • proton transfer such as keto-enol and imine-enamine, lactam-lactam isomerizations .
  • An example of a lactam-lactam equilibrium is between A and B as shown below.
  • the compounds of the present disclosure may be asymmetric, eg, have one or more stereoisomers. Without specifying configuration, all stereoisomers are included, such as enantiomers and diastereomers.
  • 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 chiral reagents.
  • Optically active (R)- and (S)-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 a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide pure desired enantiomer.
  • a diastereomeric salt is formed with an appropriate optically active acid or base, followed by conventional methods known in the art
  • the diastereoisomers were resolved and the pure enantiomers recovered.
  • separation of enantiomers and diastereomers is usually accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (eg, from amines to amino groups) formate).
  • the present disclosure also includes certain isotopically-labeled compounds of the present disclosure which are identical to those described herein, except that one or more atoms have been replaced by an atom having an atomic weight or mass number different from that normally found in nature.
  • isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2H, 3H , 11C , 13C , 14C , 13 , respectively N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl and the like.
  • deuterium when a position is specifically designated as deuterium (D), the position is understood to have an abundance of deuterium (ie, at least 1000 times greater than the natural abundance of deuterium (which is 0.015%)) % of deuterium incorporated).
  • Exemplary compounds having natural abundance greater than deuterium may 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 4000 times more abundant 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 in various deuterated forms.
  • Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom.
  • Those skilled in the art can synthesize compounds of formula I in deuterated form with reference to the relevant literature.
  • Commercially available deuterated starting materials can be used in preparing deuterated forms of the compounds of formula I, or they can be synthesized using conventional techniques using deuterated reagents including, but not limited to, deuterated borane, trideuterated borane Tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane, etc.
  • C 1-6 alkyl optionally substituted by halogen or cyano means that halogen or cyano may but need not be present, and the description includes the case where the alkyl is substituted by halogen or cyano and the case where the alkyl is not substituted by halogen and cyano substitution.
  • the bond Indicates an unspecified configuration, i.e. if a chiral isomer exists in the chemical structure, the bond can be or both Two configurations.
  • the bond no configuration is specified, i.e. the bond The configuration can be E or Z, or both E and Z configurations.
  • the "chemical modifications", “compounds”, “ligands”, “conjugates” and “nucleic acids” of the present disclosure can be independently expressed as salts, mixed salts or non-salts (such as free acids or free bases) ) in the form of.
  • a salt or mixed salts it can be a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt includes 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 which retains the biological availability of the free base without other side effects.
  • Inorganic acid salts include but are not limited to hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; organic acid salts include but are not limited to formate, acetate, 2,2-dichloroacetate , trifluoroacetate, propionate, caproate, caprylate, caprate, undecylenate, glycolate, gluconate, lactate, sebacate, hexamethylene Acid, 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 salts” refers to salts with inorganic or organic bases that retain the biological availability of the free acid without other adverse 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 derived from organic bases include, but are not limited to, the following: 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, dicyclohe
  • connection when referring to a connection between two molecules, refers to the connection of two molecules by covalent bonds or the association of two molecules via non-covalent bonds (eg, hydrogen bonds or ionic bonds), including direct connection, indirect connect.
  • non-covalent bonds eg, hydrogen bonds or ionic bonds
  • directly attached refers to the attachment of a first compound or group to a second compound or group through an intermediate group, compound or molecule (eg, a linking group).
  • the sense strand also known as the sense strand, SS or SS strand
  • the antisense strand also called AS or AS strand
  • siRNA refers to the strand having a sequence complementary to the target mRNA sequence.
  • the terms "complementary” or “reverse complementary” are used interchangeably and have the meaning well known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand are 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) pairs.
  • 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 corresponding positions are not paired in complementary form.
  • base includes any known DNA and RNA bases, base analogs such as purines or pyrimidines, which also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogues.
  • base analogs are generally purine or pyrimidine bases, excluding common bases: guanine (G), cytosine (C), adenine (A), thymine (T), and uracil ( U).
  • bases include hypoxanthine (I), xanthine (X), 3 ⁇ -D-ribofuranosyl-(2,6-diaminopyrimidine) (K), 3- ⁇ -D-ribofuranose yl-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione) (P), isocytosine (iso-C), isoguanine (iso -G), 1- ⁇ -D-ribofuranosyl-(5-nitroindole), 1- ⁇ -D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2-amino Purine, 4-thio-dT, 7-(2-thienyl)-
  • the term "universal base” refers to a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety in a modified nucleotide or an equivalent position in a nucleotide sugar moiety substitution, the heterocyclic moiety When present in a nucleic acid duplex, more than one base can be positioned relative to one another without altering the duplex structure (eg, the structure of the phosphate backbone). Furthermore, the universal base does not disrupt the ability of the single-stranded nucleic acid in which it resides to form a duplex with the target nucleic acid.
  • a single-stranded nucleic acid containing a universal base to form a duplex with a target nucleic acid can be determined by methods apparent to those skilled in the art (eg, UV absorbance, circular dichroism, gel shift, single-stranded nuclease sensitivity, etc. ). Additionally, the conditions under which duplex formation is observed can be varied to determine duplex stability or formation, eg, temperature, such as melting temperature (Tm), correlates with nucleic acid duplex stability.
  • Tm melting temperature
  • the Tm of the duplex formed by the single-stranded nucleic acid containing the universal base with the target nucleic acid is lower than that of the duplex formed with the complementary nucleic acid.
  • the Tm of the duplex formed by the single-stranded nucleic acid containing the universal base and the target nucleic acid is higher than that of the single-stranded nucleic acid with the mismatched base. nucleic acid duplexes.
  • Some universal bases can be formed by base pairing between universal bases and all bases guanine (G), cytosine (C), adenine (A), thymine (T) and uracil (U) hydrogen bonds for base pairing.
  • Universal bases are not bases that only form base pairs with a single complementary base.
  • the universal base may not hydrogen bond, form one hydrogen bond, or form more than one hydrogen bond with each of the G, C, A, T, and U opposite it on the opposite strand of the duplex.
  • the universal base does not interact with the opposite base on the opposite strand of the duplex.
  • base pairing with universal bases does not alter the double helix structure of the phosphate backbone.
  • Universal bases can also interact with bases in adjacent nucleotides on the same nucleic acid strand via stacking interactions. This stacking interaction can stabilize the duplex, especially if the universal base does not form any hydrogen bonds with bases positioned opposite it on the opposite strand of the duplex.
  • Non-limiting examples of universal binding nucleotides include inosine, 1-beta-D-ribofuranosyl-5-nitroindole, and/or 1-beta-D-ribofuranosyl-3-nitropyrrole.
  • 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.
  • group middle A moiety can be replaced with any group that enables linkage to adjacent nucleotides.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, such as an alkyl group containing 1 to 12 carbon atoms, an alkyl group containing 1 to 6 carbon atoms alkyl.
  • Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1- Dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethyl butyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl or 2,3-dimethylbutyl base.
  • alkoxy refers to -O-alkyl, wherein alkyl is as defined above.
  • alkoxy groups include, but are not limited to: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • C 1 -C 6 alkoxy can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, Alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy , cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.
  • alkenyl refers to a hydrocarbon group containing at least one double bond.
  • alkenyl groups 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 groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, or 2-butynyl, and their various branched chain isomers.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • the "ring” in “R 1 and R 2 are directly connected to form a ring” may be “cycloalkyl” or “heterocycloalkyl”.
  • cycloalkyl may be referred to as “carbocycle” and refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, in some embodiments is selected from containing from 3 to 7 carbon atoms, and in some embodiments from 5 to 6 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like; polycyclic cycloalkyl groups include spiro Ring, fused and bridged cycloalkyl groups.
  • Cycloalkyl groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, and in some embodiments are selected from one or more of the following groups, independently selected from halogen, deuterium, hydroxyl, oxo, nitro, cyano, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 3-6 Cycloalkoxy, 3- to 6-membered heterocycloalkoxy, C 3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl, the C 1-6 alkyl, C 1-6 alkoxy base, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 3-6 cycloalkoxy, 3- to 6-membered heterocycloalkoxy, C 3-8 cycloalkenyloxy, 5- to 6-membered Aryl or heteroaryl groups are optionally substituted with one or more
  • the cycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthyl, benzo rings Heptyl, etc.
  • Cycloalkyl groups can be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups independently selected from halogen, deuterium, hydroxy, oxo, Nitro, cyano, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 3-6 cycloalkoxy, 3-6 membered Heterocycloalkoxy, C 3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl, the C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyloxy , C 2-6 alkynyloxy, C 3-6 cycloalkoxy, 3- to 6-membered heterocycloalkoxy, C 3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl optionally One or more substituted by halogen, deuterium, hydroxyl,
  • heterocycloalkyl also known as “heterocycle” or “heterocyclyl” refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms, one of which or Multiple ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) m (where m is an integer from 0 to 2), excluding ring moieties of -OO-, -OS- or -SS-, the remaining ring atoms for carbon. In some embodiments selected from containing 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; in some embodiments selected from containing 3 to 7 ring atoms.
  • Non-limiting examples of monocyclic heterocycloalkyl include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piper pyridyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc.
  • Polycyclic heterocycloalkyl groups include spiro, fused and bridged ring heterocycloalkyl groups.
  • Non-limiting examples of "heterocycloalkyl" include:
  • heterocycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a heterocycloalkyl, non-limiting examples of which include:
  • Heterocycloalkyl can be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups independently selected from halogen, deuterium, hydroxy, oxo , nitro, cyano, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 3-6 cycloalkoxy, 3 to 6 Membered heterocycloalkoxy, C 3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl, the C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyloxy group, C 2-6 alkynyloxy, C 3-6 cycloalkoxy, 3- to 6-membered heterocycloalkoxy, C 3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl optional Substituted with one or more selected from halogen, de
  • Bz represents a benzoyl protecting group
  • MMTr represents a methoxyphenyldiphenylmethyl group
  • DMTr represents a dimethoxytrityl protecting group
  • capital letters C, G, U, A, T represent the base composition of nucleotides; lower case letter d represents that the adjacent nucleotide to the right of the letter d is deoxyribose Nucleotides; lowercase letter m indicates that a nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates that a nucleotide adjacent to the left of the letter f is a fluorine modified The lowercase letter s indicates that the two nucleotides adjacent to the letter s are connected by phosphorothioate groups.
  • fluoro-modified nucleotide refers to a nucleotide formed by substituting the hydroxyl group at the 2' position of the ribosyl of a nucleotide with fluorine
  • non-fluoro-modified nucleotide refers to a nucleoside Nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group of an acid with a non-fluorine group.
  • Nucleotide analog means capable of replacing a nucleotide in a nucleic acid, but differing in structure from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymus A group of pyrimidine deoxyribonucleotides. Such as isonucleotides, bridged nucleotides (bridged nucleic acid, BNA for short) or acyclic nucleotides.
  • the methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group.
  • Isonucleotides refer to compounds formed by changing the position of the base in the nucleotide on the ribose ring.
  • the isonucleotide may be a compound formed by moving the base from the 1'-position to the 2'-position or the 3'-position of the ribose ring.
  • BNA refers to constrained or inaccessible nucleotides.
  • the BNA may contain a five-membered, six-membered, or seven-membered ring bridged structure with "fixed" C3'-endoglycan constriction.
  • the bridge is typically incorporated at the 2'-,4'-position of the ribose sugar to provide a 2',4'-BNA nucleotide.
  • the BNA can be an LNA, ENA, cET BNA, or the like.
  • Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide.
  • an acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA).
  • the term “inhibit”, is used interchangeably with “reduce,””silence,””down-regulate,””repression,” and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in absolute or relative levels 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 controlled (eg buffer only control or inert control) treated subject, cell , or the level determined by the sample.
  • the residual expression of mRNA can be used to characterize the degree of inhibition of target gene expression by siRNA, such as the residual expression of mRNA is not higher than 99%, not higher than 95%, not higher than 90%, not higher than 85%, not higher than 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%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%.
  • an “effective amount” or “effective dose” refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results.
  • beneficial or desired results include elimination or reduction of risk, reduction in severity, or delay in onset of disorders, including biochemical, tissue academic and/or behavioral symptoms.
  • beneficial or desired outcomes include clinical outcomes, such as reducing the incidence of or ameliorating one or more symptoms of the various target gene, target mRNA or target protein-related disorders of the present disclosure, reducing treatment disorders
  • the dosage of the other agent is required to enhance the therapeutic effect of the other agent, and/or delay the progression of the target gene, target mRNA or target protein-related disorder of the present disclosure in the patient.
  • angiopoietin-like protein-3 can refer to any nucleic acid or protein of ANGPTL3.
  • sequence accession number for human ANGPTL3 is NP_055310.
  • ANGPTL3 expression refers to the level of mRNA transcribed from the gene encoding ANGPTL3 or the level of protein translated from mRNA.
  • TTR thyroxine transporter
  • HsT2651 HsT2651
  • PALB prealbumin
  • TBPA transthyretin
  • TTR expression refers to the level of mRNA transcribed from the gene encoding TTR or the level of protein translated from mRNA.
  • a “pharmaceutical composition” comprises the siRNA or siRNA conjugate of the present disclosure and a pharmaceutically acceptable adjuvant and/or adjuvant, which may be one or more of various formulations or compounds conventionally used in the art.
  • the pharmaceutically acceptable adjuvant may include at least one of pH buffering agents, protecting agents and osmotic pressure adjusting agents.
  • patient As used herein, “patient”, “subject” or “individual” are used interchangeably and include humans or non-human animals, eg, mammals, eg, humans or monkeys.
  • siRNA or siRNA conjugates 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 acid as part of a retrovirus or other vector.
  • the siRNA provided by the present disclosure can be obtained by conventional preparation methods in the art (eg, solid-phase synthesis and liquid-phase synthesis methods). Among them, solid-phase synthesis has already had commercial customized services.
  • Modified nucleotide groups can be introduced into siRNAs described in the present disclosure by using nucleoside monomers with corresponding modifications, methods of preparing nucleoside monomers with corresponding modifications, and introduction of modified nucleotide groups Methods for siRNA are also well known to those skilled in the art.
  • Figures 1A to 1L show the experimental results of off-target activity of siRNAs containing different test compounds.
  • Figures 2A to 2G show the experimental results of off-target activity of siRNA2 containing different test compounds.
  • Figures 3A to 3G are the experimental results of off-target activity of siRNA3 containing the compounds to be tested.
  • Figure 4 shows the inhibitory activity of galactosamine cluster-conjugated siRNA on mTTR gene in murine primary hepatocytes.
  • Figure 5 shows the in vivo inhibitory activity of galactosamine cluster-conjugated siRNA on mouse mTTR gene.
  • Figure 6 shows the long-term inhibitory activity of galactosamine cluster-conjugated siRNA on mouse mTTR gene in vivo.
  • Figure 7 shows the effect of siRNA reagents on total cholesterol levels in Apoc3 transgenic mice.
  • Figure 8 shows the effect of siRNA reagents on triglyceride levels in Apoc3 transgenic mice.
  • Figure 9 shows the effect of siRNA reagents on Apoc3 protein levels in Apoc3 transgenic mice.
  • the present disclosure is further described below in conjunction with the examples, but these examples do not limit the scope of the present disclosure.
  • the experimental methods without specific conditions are generally in accordance with conventional conditions or in accordance with conditions suggested by raw material or commodity manufacturers.
  • Reagents that do not specify a specific source are available from any supplier of molecular biology reagents in quality/purity for molecular biology applications.
  • the obtained residue was purified by reverse column (C 18 , acetonitrile+H2O, 50%), and after lyophilization, the target product 5a (730 mg, yield 17.6%) and target product 5b (1.1 g, 26.6%) were obtained.
  • reaction solution was extracted with ethyl acetate (200 mL) and water (200 mL), the organic phase was dried and spin-dried, and the mixed sample was purified with a forward column (PE:EtOAc was passed through the column, peak at 84%) to obtain compound 7 as a yellow oil (12g).
  • the obtained racemic compound was SFC resolved to obtain the target compound 7A(-)(3.9g) and the target compound 7B(+)(3.8g).
  • siRNA is no different from the general phosphoramidite solid-phase synthesis method.
  • the phosphoramidite monomer synthesized above is used to replace the original nucleotide of the parent sequence.
  • nucleoside phosphoramidite monomers are linked one by one according to the synthesis procedure. Except for the nucleoside phosphoramidite monomer at the 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 were purchased from From Shanghai Zhaowei or Suzhou Jima.
  • ETT 5-Ethylthio-1H-tetrazole
  • 0.6M acetonitrile solution 0.22M PADS was dissolved in a 1:1 volume ratio of acetonitrile and melidine (Suzhou Kelema) solution
  • iodopyridine/water solution Coldema
  • the oligoribonucleotides were cleaved from the solid support, and soaked in a 3:1 solution of 28% ammonia water and ethanol at 50° C. for 16 hours. After centrifugation, the supernatant was transferred to another centrifuge tube, concentrated and evaporated to dryness, and purified using C18 reverse-phase chromatography with 0.1 MTEAA and acetonitrile as mobile phases, and DMTr was removed using 3% trifluoroacetic acid solution. The target oligonucleotides were collected and lyophilized, identified as the target product by LC-MS, and then quantified by UV (260 nm).
  • the obtained single-stranded oligonucleotides were annealed according to equimolar ratio and complementary pairing, and finally the obtained double-stranded siRNA was dissolved in 1 ⁇ PBS, and adjusted to the concentration required by the experiment for use.
  • siRNA sample synthesis was described above, and the plasmid was obtained from Sangon Bioengineering (Shanghai) Co., Ltd. Consumables, reagents and instruments for psiCHECK experiments are shown in Tables 1 and 2.

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BR112023002080A BR112023002080A2 (pt) 2020-08-04 2021-08-04 Sirna modificado com atividade fora do alvo reduzida
CN202180048996.1A CN115955973A (zh) 2020-08-04 2021-08-04 脱靶活性降低的修饰siRNA
KR1020237006847A KR20230043195A (ko) 2020-08-04 2021-08-04 오프-타겟 활성이 감소된 변형된 siRNA
CA3190097A CA3190097A1 (en) 2020-08-04 2021-08-04 Modified sirna with reduced off-target activity
EP21852482.5A EP4194553A4 (en) 2020-08-04 2021-08-04 MODIFIED SIRNA WITH REDUCED OFF-TARGET ACTIVITY
JP2023507494A JP7849353B2 (ja) 2020-08-04 2021-08-04 オフターゲット活性が低減された修飾ｓｉＲＮＡ
US18/019,763 US20230287418A1 (en) 2020-08-04 2021-08-04 Modified sirna with reduced off-target activity
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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022206946A1 (zh) * 2021-04-02 2022-10-06 上海拓界生物医药科技有限公司 乙型肝炎病毒siRNA和siRNA缀合物
WO2023109932A1 (zh) * 2021-12-16 2023-06-22 上海拓界生物医药科技有限公司 一种dsRNA、其制备方法及应用
WO2023138659A1 (zh) 2022-01-20 2023-07-27 上海拓界生物医药科技有限公司 一种dsRNA、其应用及制备方法
WO2024125556A1 (zh) * 2022-12-13 2024-06-20 上海拓界生物医药科技有限公司 包含亲脂性单体的寡核苷酸以及其在非肝递送中的应用
WO2024175013A1 (zh) * 2023-02-21 2024-08-29 成都先导药物开发股份有限公司 核苷酸类似物及其制备方法和用途
WO2024179573A1 (zh) 2023-03-02 2024-09-06 上海拓界生物医药科技有限公司 靶向抑制素βE的siRNA、siRNA缀合物及其医药用途
WO2024217535A1 (zh) * 2023-04-21 2024-10-24 苏州时安生物技术有限公司 一种寡聚核酸及用于降低脱靶活性的化合物
WO2024222826A1 (zh) 2023-04-27 2024-10-31 上海森辉医药有限公司 用于制备寡核苷酸类似物或其盐的中间体及其制备方法
WO2024245410A1 (zh) 2023-05-31 2024-12-05 上海拓界生物医药科技有限公司 一种靶向LPA的dsRNA及其应用
WO2024255759A1 (zh) 2023-06-13 2024-12-19 上海拓界生物医药科技有限公司 靶向MMP7的siRNA、siRNA缀合物及其医药用途
WO2025007907A1 (zh) * 2023-07-04 2025-01-09 大睿生物 包含肽的寡核苷酸递送配体
WO2025026390A1 (zh) 2023-08-01 2025-02-06 上海拓界生物医药科技有限公司 靶向MAPT的dsRNA及其医药用途
CN119410644A (zh) * 2025-01-08 2025-02-11 北京安龙生物医药有限公司 修饰的双链寡核苷酸及其缀合物和组合物
WO2025040164A1 (zh) 2023-08-24 2025-02-27 上海拓界生物医药科技有限公司 靶向RAGE的RNAi剂及其医药用途
CN119528986A (zh) * 2025-01-23 2025-02-28 北京悦康科创医药科技股份有限公司 一种核苷类似物及其在降低寡核苷酸药物脱靶活性中的应用
WO2025067317A1 (zh) * 2023-09-26 2025-04-03 上海拓界生物医药科技有限公司 一种脂质修饰的核酸化合物、其制备方法及用途
EP4328310A4 (en) * 2021-04-22 2025-10-15 Tuojie Biotech Shanghai Co Ltd SIRNA TARGETING 17β-HYDROXYSTEROID DEHYDROGENASE TYPE 13 AND SIRNA CONJUGATE
WO2026002138A1 (zh) * 2024-06-27 2026-01-02 成都新泽利医药科技有限公司 特定修饰的双链寡核苷酸及其组合物

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019006455A1 (en) 2017-06-30 2019-01-03 Solstice Biologics, Ltd. AUXILIARIES OF CHIRAL PHOSPHORAMIDITIS AND METHODS OF USE THEREOF
TW202526017A (zh) * 2023-09-14 2025-07-01 美商Ionis製藥公司 用於減少ａｐｏｃｉｉｉ表現的化合物及方法
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009102427A2 (en) * 2008-02-11 2009-08-20 Rxi Pharmaceuticals Corp. Modified rnai polynucleotides and uses thereof
WO2014025805A1 (en) 2012-08-06 2014-02-13 Alnylam Pharmaceuticals, Inc. Carbohydrate conjugated rna agents and process for their preparation
CN107201367A (zh) * 2016-08-18 2017-09-26 广州市锐博生物科技有限公司 用于抑制TP53靶基因mRNA表达的寡核酸分子及其成套组合物
CN108367022A (zh) * 2015-12-13 2018-08-03 日东电工株式会社 具有高活性和降低脱靶的sirna结构
CN111378656A (zh) * 2018-12-28 2020-07-07 苏州瑞博生物技术有限公司 一种抑制埃博拉病毒的核酸、含有该核酸的药物组合物及其用途
CN111378655A (zh) * 2018-12-28 2020-07-07 苏州瑞博生物技术有限公司 抑制CTGF基因表达的siRNA、含有该siRNA的药物组合物及其用途
CN111378657A (zh) * 2018-12-28 2020-07-07 苏州瑞博生物技术有限公司 抑制COL1A1基因表达的siRNA、含有该siRNA的药物组合物及其用途

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1798225A1 (en) * 2004-09-16 2007-06-20 Gifu University Nucleoside analogues or salts thereof
SG171879A1 (en) * 2008-12-03 2011-07-28 Marina Biotech Inc Usirna complexes
HK1244843A1 (zh) * 2014-11-17 2018-08-17 Alnylam Pharmaceuticals, Inc. 载脂蛋白c3(apoc3)irna组合物及其使用方法
KR102645243B1 (ko) * 2016-11-23 2024-03-11 알닐람 파마슈티칼스 인코포레이티드 표적외 효과가 감소된 변형 rna 작용제
WO2019222479A1 (en) * 2018-05-16 2019-11-21 Alnylam Pharmaceuticals, Inc. Modified rna agents with reduced off-target effect
US20240229037A1 (en) * 2021-04-22 2024-07-11 Tuojie Biotech(Shanghai) Co., Ltd. SIRNA TARGETING 17Beta-HYDROXYSTEROID DEHYDROGENASE TYPE 13 AND SIRNA CONJUGATE

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009102427A2 (en) * 2008-02-11 2009-08-20 Rxi Pharmaceuticals Corp. Modified rnai polynucleotides and uses thereof
WO2014025805A1 (en) 2012-08-06 2014-02-13 Alnylam Pharmaceuticals, Inc. Carbohydrate conjugated rna agents and process for their preparation
CN108367022A (zh) * 2015-12-13 2018-08-03 日东电工株式会社 具有高活性和降低脱靶的sirna结构
CN107201367A (zh) * 2016-08-18 2017-09-26 广州市锐博生物科技有限公司 用于抑制TP53靶基因mRNA表达的寡核酸分子及其成套组合物
CN111378656A (zh) * 2018-12-28 2020-07-07 苏州瑞博生物技术有限公司 一种抑制埃博拉病毒的核酸、含有该核酸的药物组合物及其用途
CN111378655A (zh) * 2018-12-28 2020-07-07 苏州瑞博生物技术有限公司 抑制CTGF基因表达的siRNA、含有该siRNA的药物组合物及其用途
CN111378657A (zh) * 2018-12-28 2020-07-07 苏州瑞博生物技术有限公司 抑制COL1A1基因表达的siRNA、含有该siRNA的药物组合物及其用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JANAS, M.M.SCHLEGEL, M.K.HARBISON, C.E. ET AL.: "Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity", NAT COMMUN, vol. 9, 2018, pages 723
MOLECULAR THERAPY, vol. 26, no. 3, March 2018 (2018-03-01)
See also references of EP4194553A4
SEVERGINI ET AL., CYTOTECHNOLOGY, vol. 64, no. 2, 2012, pages 187 - 195

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EP4328310A4 (en) * 2021-04-22 2025-10-15 Tuojie Biotech Shanghai Co Ltd SIRNA TARGETING 17β-HYDROXYSTEROID DEHYDROGENASE TYPE 13 AND SIRNA CONJUGATE
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WO2024179573A1 (zh) 2023-03-02 2024-09-06 上海拓界生物医药科技有限公司 靶向抑制素βE的siRNA、siRNA缀合物及其医药用途
EP4674965A1 (en) 2023-03-02 2026-01-07 Tuojie Biotech (Shanghai) Co., Ltd. Sirna targeting inhibin ?e, sirna conjugate, and medical use thereof
WO2024217535A1 (zh) * 2023-04-21 2024-10-24 苏州时安生物技术有限公司 一种寡聚核酸及用于降低脱靶活性的化合物
WO2024217152A3 (zh) * 2023-04-21 2024-12-05 苏州时安生物技术有限公司 一种寡聚核酸及其制备方法和应用
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