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WO2024217535A1 - 一种寡聚核酸及用于降低脱靶活性的化合物 - Google Patents

一种寡聚核酸及用于降低脱靶活性的化合物 Download PDF

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Publication number
WO2024217535A1
WO2024217535A1 PCT/CN2024/088796 CN2024088796W WO2024217535A1 WO 2024217535 A1 WO2024217535 A1 WO 2024217535A1 CN 2024088796 W CN2024088796 W CN 2024088796W WO 2024217535 A1 WO2024217535 A1 WO 2024217535A1
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stranded
sirna
double
och
modified nucleotides
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PCT/CN2024/088796
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English (en)
French (fr)
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杨志伟
刘楠
黄敏印
朱研
赵勇
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苏州时安生物技术有限公司
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Priority to CN202480000765.7A priority Critical patent/CN119731323A/zh
Publication of WO2024217535A1 publication Critical patent/WO2024217535A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the invention relates to the fields of molecular biology and biochemistry, and in particular to an oligonucleic acid and a compound for reducing off-target activity.
  • siRNA has a very important prospect in drug development. However, in vivo applications, siRNA often has different degrees of off-target effects, and there may be cases where siRNA targets non-target genes. Off-target effects similar to the microRNA pathway are one of the main reasons for siRNA off-target, that is, the inhibitory activity of mRNA generated by the complete or incomplete pairing of the siRNA antisense strand (Antisense Strand, also known as AS strand) seed region (2-8th position at the 5' end) with the target mRNA.
  • the off-target effect of an 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.
  • the antisense strand seed region of siRNA drugs can be chemically modified at specific sites, thereby reducing the off-target toxicity of small nucleic acid drugs.
  • the strength of the interaction between the seed region and the target gene can be adjusted, and efforts can be made to reduce off-target effects without affecting the silencing effect of the target mRNA (Mark K. Schlegel, Chirality Dependent Potency Enhancement and Structural Impact of Glycol Nucleic Acid Modifification on siRNA. J. Am. Chem. Soc. 2017, 139, 8537-8546).
  • the prior art requires a chemical modification method that can reduce the off-target effect of siRNA while not affecting the on-target activity of siRNA to the greatest extent.
  • the purpose of the present invention is to provide an oligonucleic acid modified by reducing off-target activity and a compound for reducing off-target activity.
  • the scheme of the present invention can reduce the off-target effect of the oligonucleic acid while not affecting the on-target activity of the oligonucleic acid to the greatest extent.
  • the present invention can also have the value of improving the stability of single-stranded oligonucleic acid or double-stranded oligonucleic acid.
  • the first aspect of the present invention provides a single-stranded or double-stranded oligonucleic acid, each strand having 15 to 35 nucleotides, wherein at least one nucleotide position other than the first end of the oligonucleic acid comprises a chemical modification represented by formula (I) or a tautomer modification thereof,
  • B is a natural nucleobase, a modified nucleobase, a universal base or an H atom; preferably, B is a natural nucleobase or a modified nucleobase;
  • Z is selected from O, NH and S; preferably, Z is O;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are independently selected from H, OH, halogen, NH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, S-CH 3 , NCH 3 (CH 3 ), OCH 2 CH 2 OCH 3 and -O-alkylamino; preferably, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl;
  • X is O or S
  • n is 1, 2 or 3; preferably, n is 1 or 2.
  • the oligonucleic acid is siRNA, which comprises a sense strand and an antisense strand.
  • the antisense strand comprises a chemical modification represented by formula (I-1) or a tautomer modification thereof at at least one nucleotide position from position 2 to position 8 in the 5' region thereof,
  • B1 and B2 are each independently a natural nucleobase, a modified nucleobase, a universal base or an H atom; preferably, B1 and B2 are each independently a natural nucleobase or a modified nucleobase;
  • Z is selected from O, NH and S; preferably, Z is O;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are independently selected from H, OH, halogen, NH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, S-CH 3 , NCH 3 (CH 3 ), OCH 2 CH 2 OCH 3 and -O-alkylamino; preferably, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are independently H;
  • X is O or S
  • M 1 and M 2 are independently selected from H, OH, F, OCH 3 , OCH 2 CH 2 OCH 3 and -O-alkyl; preferably, M 1 and M 2 are independently selected from H, OH, F and OCH 3 ;
  • n is 1, 2 or 3, preferably, n is 1 or 2.
  • the antisense strand comprises a chemical modification represented by formula (I-2) or a tautomer modification thereof at at least one nucleotide position from position 2 to position 8 in the 5' region of the antisense strand.
  • B1 and B2 are each independently a natural nucleobase, a modified nucleobase, a universal base or an H atom; preferably, B1 and B2 are each independently a natural nucleobase or a modified nucleobase;
  • R 3 and R 4 are independently selected from H, OH, halogen, NH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, S-CH 3 , NCH 3 (CH 3 ), OCH 2 CH 2 OCH 3 and -O-alkylamino; preferably, R 3 and R 4 are independently H;
  • X is O or S; preferably, X is O;
  • M is selected from H, OH, F, OCH 3 , OCH 2 CH 2 OCH 3 and —O-alkyl; preferably, M is selected from H, OH, F and OCH 3 .
  • the antisense strand comprises a chemical modification represented by formula (I-3), (I-4) or (I-5) or a tautomer modification thereof at at least one nucleotide position from position 2 to position 8 in the 5' region thereof,
  • B1 and B2 are each independently a natural nucleobase, a modified nucleobase, a universal base or an H atom; preferably, B1 and B2 are each independently a natural nucleobase or a modified nucleobase;
  • R 3 and R 4 are independently selected from H, OH, halogen, NH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, S-CH 3 , NCH 3 (CH 3 ), OCH 2 CH 2 OCH 3 and -O-alkylamino; preferably, R 3 and R 4 are independently H;
  • X is O or S; preferably, X is O;
  • M is selected from OH, F, OCH 3 , OCH 2 CH 2 OCH 3 and —O-alkyl; preferably, M is selected from OH, F and OCH 3 .
  • the chemical modification is selected from any of the following structures:
  • the antisense strand comprises any one of the chemical modifications defined above or a tautomer modification thereof at at least one nucleotide position from position 5 to position 8 in its 5' region;
  • the antisense strand comprises any one of the chemical modifications defined above or a tautomer modification thereof at at least one nucleotide position in positions 6 and 7 of its 5' region.
  • At least one of the sense strand and/or antisense strand further comprises at least one other modified nucleotide;
  • the remaining nucleotides in the sense strand and/or antisense strand are other modified nucleotides.
  • the other modified nucleotides are independently selected from the group consisting of: 2'-fluoro-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-deoxynucleotides, 2'-amino-modified nucleotides and 2'-substituted amino-modified nucleotides;
  • the other modified nucleotides are independently selected from: 2'-F modified nucleotides and 2'-O-CH 3 modified nucleotides.
  • the single-stranded or double-stranded oligonucleic acid/siRNA has at least one of the following characteristics:
  • the antisense strand comprises 2, 3, 4, 5 or 6 2'-fluoro modifications
  • the antisense strand comprises 1, 2, 3 or 4 phosphorothioate internucleotide linkages
  • the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications
  • the sense strand contains 1, 2, 3 or 4 phosphorothioate internucleotide linkages
  • the dsRNA comprises at least four 2'-fluoro modifications
  • the siRNA comprises a duplex region of 12-40 nucleotide pairs in length.
  • the second aspect of the present invention provides a siRNA conjugate, which comprises the above-mentioned single-stranded or double-stranded oligonucleic acid/siRNA and a conjugated group conjugated to the siRNA.
  • the conjugated group comprises a pharmaceutically acceptable targeting group and a linker, and the single-stranded or double-stranded oligonucleic acid/siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked.
  • the third aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned single-stranded or double-stranded oligonucleic acid/siRNA, or the above-mentioned siRNA conjugate, and a pharmaceutically acceptable carrier.
  • the fourth aspect of the present invention provides a kit comprising the above-mentioned single-stranded or double-stranded oligonucleic acid/siRNA, or the above-mentioned siRNA conjugate, or the above-mentioned pharmaceutical composition.
  • the fifth aspect of the present invention provides a compound represented by the following formula (III) or its tautomer,
  • B1 and B2 are independently a natural nucleobase, a modified nucleobase, a universal base or an H atom;
  • R 3 and R 4 are each independently selected from H, OH, halogen, NH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, S-CH 3 , NCH 3 (CH 3 ), OCH 2 CH 2 OCH 3 and -O-alkylamino;
  • X is O or S
  • M is selected from OH, F, OCH 3 , OCH 2 CH 2 OCH 3 and -O-alkyl;
  • E is a leaving group; preferably, E is MMTr or DMTr;
  • Q is a phosphorus-containing reactive group; preferably, Q is
  • the compound represented by the above formula (III) or (IV) or its tautomer is selected from any of the following structures:
  • the sixth aspect of the present invention provides a method for preparing the above-mentioned single-stranded or double-stranded oligonucleic acid/siRNA or the above-mentioned siRNA conjugate, which comprises the following steps:
  • the seventh aspect of the present invention provides a single-stranded or double-stranded oligonucleic acid, each strand having 15 to 60 nucleotides, wherein at least one nucleotide position other than the first end of the oligonucleic acid comprises a chemical modification represented by formula (V) or a tautomer modification thereof,
  • B1 and B2 are independently selected from natural nucleobases, modified nucleobases, universal bases or H atoms;
  • Z is selected from O, NH and S;
  • R 1 , R 2 , R 5 , R 6 , R 7 , R 8 , R 9 , R8 , R9 , R10 and R11 are each independently selected from H, OH, halogen, NH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, S—CH 3 , NCH 3 (CH 3 ), OCH 2 CH 2 OCH 3 and —O-alkylamino;
  • M 1 and M 2 are independently selected from H, OH, F and OCH 3 ;
  • n 1, 2 or 3;
  • n 1, 2, 3, 4 or 5;
  • y 0, 1, 2, 3;
  • X is O or S.
  • the eighth aspect of the present invention provides a siRNA comprising a sense strand and an antisense strand, each strand having 15 to 60 nucleotides, wherein at least one nucleotide position of the antisense strand comprises a chemical modification represented by formula (V-2), (V-3), (V-4) or (V-5) or a tautomer modification thereof,
  • B1 and B2 are each independently a natural nucleobase, a modified nucleobase, a universal base or an H atom; preferably, B1 and B2 are each independently a natural nucleobase, a modified or unmodified nucleobase;
  • M 1 is selected from H, OH, F and OCH 3 ; preferably, M 1 is selected from F and OCH 3 ;
  • X is O or S.
  • the chemical modification is selected from any one of the following:
  • the antisense strand comprises a chemical modification or a tautomer modification thereof as defined above at at least one nucleotide position from position 5 to position 8 in the 5' region of the antisense strand;
  • the antisense strand comprises a chemical modification or a tautomer modification thereof as defined above at at least one nucleotide position between positions 6 and 7 in the 5' region of the antisense strand.
  • At least one of the sense strand and/or antisense strand further comprises at least one other modified nucleotide;
  • the remaining nucleotides in the sense strand and/or antisense strand are other modified nucleotides.
  • the other modified nucleotides are independently selected from the group consisting of: 2'-fluoro-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-deoxynucleotides, 2'-amino-modified nucleotides and 2'-substituted amino-modified nucleotides;
  • the other modified nucleotides are independently selected from: 2'-F modified nucleotides and 2'-O-CH 3 modified nucleotides.
  • the antisense strand comprises 2, 3, 4, 5 or 6 2'-fluoro modifications
  • the antisense strand comprises 1, 2, 3 or 4 phosphorothioate internucleotide linkages
  • the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications
  • the sense strand contains 1, 2, 3 or 4 phosphorothioate internucleotide linkages
  • the dsRNA comprises at least four 2'-fluoro modifications
  • the siRNA comprises a duplex region of 12-40 nucleotide pairs in length.
  • the ninth aspect of the present invention provides a siRNA conjugate, which comprises the above-mentioned single-stranded or double-stranded oligonucleic acid/siRNA and a conjugated group conjugated to the siRNA.
  • the conjugated group comprises a pharmaceutically acceptable targeting group and a linker, and the single-stranded or double-stranded oligonucleic acid/siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked.
  • the tenth aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned single-stranded or double-stranded oligonucleic acid, or the above-mentioned siRNA conjugate, and a pharmaceutically acceptable carrier.
  • the eleventh aspect of the present invention provides a kit comprising the above-mentioned single-stranded or double-stranded oligonucleic acid, or the above-mentioned siRNA conjugate, or the above-mentioned pharmaceutical composition.
  • the twelfth aspect of the present invention provides a compound represented by the following formula (VI) or its tautomer,
  • B1 and B2 are independently a natural nucleobase, a modified nucleobase, a universal base or an H atom;
  • X is O or S
  • M is selected from H, OH, F, OCH 3 , OCH 2 CH 2 OCH 3 and -O-alkyl;
  • E is a leaving group; preferably, E is MMTr or DMTr;
  • Q is a phosphorus-containing reactive group; preferably, Q is
  • the compound represented by formula (VI) or its tautomer is selected from any of the following structures:
  • the thirteenth aspect of the present invention provides a method for preparing the above-mentioned single-stranded or double-stranded oligonucleic acid or the above-mentioned siRNA conjugate, which comprises the following steps:
  • the fourteenth aspect of the present invention provides a method for inhibiting a target gene in a cell, comprising the step of introducing the above-mentioned single-stranded or double-stranded oligonucleic acid, or the above-mentioned siRNA conjugate into the cell.
  • the fifteenth aspect of the present invention provides a method for modifying positions 2-8 of the antisense chain of siRNA to reduce off-target activity, wherein the above-mentioned chemical modification or its tautomer modification, or the above-mentioned chemical modification or its tautomer modification is introduced into positions 2-8 of the antisense chain of the siRNA molecule.
  • the present invention introduces a special compound into the 5'-end seed region of the antisense strand of siRNA to reduce the off-target effect of siRNA while not affecting the on-target activity of siRNA to the greatest extent, thereby improving the safety of siRNA as a drug.
  • FIG. 1 is a graph showing the results of HBsAg levels before and after administration in Example 5.
  • the structure of the thiophosphate group is shown in formula (1):
  • natural nucleobase refers to a nucleobase that has not been modified from its naturally occurring form in RNA or DNA.
  • natural nucleobases include the purine nucleobases adenine (A) and guanine (G) and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U).
  • A purine nucleobase
  • G guanine
  • T cytosine
  • U uracil
  • many modified nucleobases or nucleobase mimetics known to those skilled in the art are suitable for use in the compounds described herein.
  • modified nucleobase refers to a nucleobase that is structurally very similar to a parent nucleobase, such as 7-deazapurine. uracil, 5-methylcytosine or G-clamp.
  • a universal base refers to a base that can be complementary to at least two commonly used bases, such as hypoxanthine (whose nucleoside is Inosine), which can be paired with any one of A, T, G or C, and its binding ability is I:C>I:A>I:G>I:T; or for example, BrU (5-Bromouridine), which can be paired with A or G; optionally, other universal bases that can be complementary to at least two commonly used bases can also be selected in the present invention, such as 3-nitropyrrole (3-nitropyrrole), 5-nitroindole (5-nitroindole), 7-azaindole (7-Azaindole) and the like.
  • seed region is the region in the antisense strand of an RNAi agent that is responsible for recognizing the target mRNA and responding to, for example, nucleotides 2-8 from the 5' end of the antisense strand.
  • nucleotide position refers to the position of a nucleotide in an oligonucleotide as counted from the nucleotide at the 5' end.
  • nucleotide position 1 refers to the 5' terminal nucleotide of the oligonucleotide.
  • oligonucleotide or “oligonucleic acid” refers to a polymeric form of nucleotides within the range of 2 to 2500 nucleotides. Oligonucleotides may be single-stranded or double-stranded. In certain embodiments, the oligonucleotide has 500 to 1500 nucleotides, typically, for example, wherein the oligonucleotide is used for gene therapy. In certain embodiments, the oligonucleotide is single-stranded or double-stranded and has 7 to 100 nucleotides. In certain embodiments, the oligonucleotide is single-stranded or double-stranded and has 15 to 100 nucleotides.
  • the oligonucleotide is single-stranded or double-stranded with 15 to 50 nucleotides, typically, for example, wherein the oligonucleotide is a nucleic acid inhibitor molecule. In another embodiment, the oligonucleotide is single-stranded or double-stranded with 25 to 40 nucleotides, typically, for example, wherein the oligonucleotide is a nucleic acid inhibitor molecule.
  • the oligonucleotide is single-stranded or double-stranded and has 19 to 40 or 19 to 25 nucleotides, typically, for example, wherein the oligonucleotide is a double-stranded nucleic acid inhibitor molecule and forms a double helix with at least 18 to 25 base pairs.
  • the oligonucleotide is single-stranded and has 15 to 25 nucleotides, typically, for example, wherein the oligonucleotide nucleotide is a single-stranded RNAi inhibitor molecule.
  • the oligonucleotide contains one or more phosphorus-containing internucleotide linking groups.
  • the internucleotide linking group is a phosphoamido group.
  • fluorinated modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and has a structure shown in the following formula (7).
  • the 2'-alkyl modified nucleotide is a methoxy modified nucleotide (2'-OMe), as shown in formula (8).
  • base represents a base, such as A, U, G, C or T.
  • non-fluorinated modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribose group of the nucleotide is replaced by a non-fluorinated group.
  • conjugation refers to the covalent connection between two or more chemical moieties each having a specific function; accordingly, “conjugate” refers to a compound formed by covalent connection between the chemical moieties.
  • siRNA conjugate refers to a compound formed by covalently connecting one or more chemical moieties having a specific function to siRNA.
  • the nucleoside monomer refers to the modified or unmodified nucleoside phosphoramidite monomer (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites are also called Nucleoside phosphoramidites) used in phosphoramidite solid phase synthesis according to the type and order of nucleotides in the siRNA or siRNA conjugate to be prepared.
  • Phosphoramidite solid phase synthesis is a method used in RNA synthesis known to those skilled in the art.
  • the nucleoside monomers used in this application are all commercially available.
  • the present invention provides a single-stranded or double-stranded oligonucleic acid, each strand having 15 to 35 nucleotides, wherein at least one nucleotide position other than the first and last ends of the oligonucleic acid comprises a chemical modification represented by formula (I) or (V) or a tautomer modification thereof,
  • non-first end of an oligonucleotide refers to any position other than the first end and the last end of an oligonucleotide, in other words, any position other than the first nucleotide and the last nucleotide (5'-3' direction) of an oligonucleotide.
  • the siRNA of the present application contains a nucleotide group as a basic structural unit. It is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base.
  • the present invention introduces one of these compounds into the 5'-end seed region of the antisense strand of siRNA for chemical modification, which reduces the off-target effect of siRNA while not affecting the on-target activity of siRNA to the greatest extent, thereby improving the safety of siRNA as a drug.
  • GNA or UNA modification is usually introduced into the 5'-end seed region of the antisense strand of siRNA, which can reduce off-target toxicity, but also cause a significant loss of on-target activity.
  • UNA is shown in formula (3)
  • GNA is shown in formula (4).
  • R is selected from H, OH or alkoxy (O-alkyl).
  • siRNA sequences for multiple targets are designed, and the on-target activity and off-target activity of siRNA are compared when the chemical modification and GNA modification of the present application are introduced at the same position in the 5' end seed region of the antisense chain of siRNA in the siRNA antisense chain composed of the same base sequence.
  • the effect in multiple siRNAs is the same, which also shows that the effect achieved by the siRNA of the present invention is independent of the siRNA sequence itself.
  • the present application provides many chemical modifications, such as Agt, Agt01, Uut, Agt02, Aut, Aut01, Agz, Agy, Agx, etc., and introducing one of these chemical modifications at at least one nucleotide position from the 2nd to the 8th position in the 5' region of the antisense chain can retain the on-target activity basically the same as the original sequence and significantly reduce the off-target activity.
  • siRNA conjugates of the present invention are provided.
  • the present invention provides a siRNA conjugate, which comprises the above-mentioned single-stranded or double-stranded oligonucleic acid/siRNA and a conjugated group conjugated to the siRNA.
  • the conjugated group comprises a pharmaceutically acceptable targeting group and a linker, and the single-stranded or double-stranded oligonucleic acid/siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked.
  • the conjugated group conjugated to the siRNA is TIN, L96 or SA51, etc., wherein the structural formula of TIN is shown in the figure below, and the structural formulas of L96 and SA51 are shown in Example 5.
  • the present application provides a pharmaceutical composition, which contains the single-stranded or double-stranded oligonucleic acid/siRNA or siRNA conjugate as described above as an active ingredient and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be a carrier conventionally used in the field of siRNA administration, such as, but not limited to, magnetic nanoparticles (such as nanoparticles based on Fe3O4 or Fe2O3 ), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly (D&L-lactic/glycolic acid) copolymer (PLGA), poly (2-aminoethyl ethylene phosphate ...
  • magnetic nanoparticles such as nanoparticles based on Fe3O4 or Fe2O3
  • carbon nanotubes mesoporous silicon, calcium phosphate nanoparticles
  • PIM polyethyleneimine
  • PAMAM polyamidoamine dendrimer
  • DOTAP 1,2-d
  • Phosphate Phosphate
  • PPEEA poly(methacrylate-N,N-dimethylaminoethyl ester)
  • poly(2-dimethylaminoethyl methacrylate) and one or more of their derivatives.
  • the pharmaceutical composition there is no particular requirement for the content of the single-stranded or double-stranded oligonucleic acid/siRNA or siRNA conjugate and the pharmaceutically acceptable carrier, and the conventional content of each component may be used.
  • the present disclosure provides a kit comprising the above-mentioned single-stranded or double-stranded oligonucleic acid/siRNA or the above-mentioned siRNA conjugate or the above-mentioned pharmaceutical composition.
  • the kit described herein may provide single-stranded or double-stranded oligonucleic acid/siRNA or siRNA conjugates in one container.
  • the kit described herein may include a container providing a pharmaceutically acceptable excipient.
  • other ingredients such as stabilizers or preservatives, etc., may also be included in the kit.
  • the kit described herein may include at least one other therapeutic agent in other containers other than the container providing single-stranded or double-stranded oligonucleic acid/siRNA or siRNA conjugates described herein.
  • the kit may include instructions for mixing single-stranded or double-stranded oligonucleic acid/siRNA or siRNA conjugates with pharmaceutically acceptable carriers and/or excipients or other ingredients (if any).
  • the single-stranded or double-stranded oligonucleic acid/siRNA, siRNA conjugate and pharmaceutically acceptable carrier and/or adjuvant, and/or pharmaceutical composition, and/or pharmaceutically acceptable carrier and/or adjuvant can be provided in any form, such as liquid form, dry form or lyophilized form.
  • the single-stranded or double-stranded oligonucleic acid/siRNA, siRNA conjugate and pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and pharmaceutically acceptable carrier and/or adjuvant are substantially pure and/or sterile.
  • sterile water can be provided in the kit of the present application.
  • the present invention provides a compound represented by the following formula (III) or formula (VI) or a tautomer thereof:
  • R 3 and R 4 are independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl;
  • X is O or S
  • M is selected from H, OH, F, OCH 3 , OCH 2 CH 2 OCH 3 and -O-alkyl;
  • E is a leaving group; preferably, E is MMTr or DMTr;
  • Q is a phosphorus-containing reactive group; preferably, Q is
  • the above intermediates can be used to synthesize the single-stranded or double-stranded oligonucleic acid/siRNA or siRNA conjugate of the present invention.
  • Compound 9-1 (commercially available, purchased from Shanghai Zhaowei Technology Development Co., Ltd.) (8.0 g, 11.9 mmol, 1.0 equiv) was dissolved in 56 mL of acetonitrile, and the temperature was reduced to 0°C. At this temperature, tert-butyldimethylsilyl chloride (3.6 g, 23.8 mmol, 2.93 mL, 2.0 equiv) and imidazole (3.25 g, 47.7 mmol, 4.0 equiv) were added. After the addition was completed, the temperature was raised to room temperature, and the reaction solution was stirred for 16 hours.
  • N-methylimidazole (11.76 g, 143.27 mmol, 11.4 mL, 12.0 equiv) was added at 0°C, the reaction solution was filtered and the filter cake residue was washed with acetonitrile, the reaction solution and the washing solution were combined, and 10 mL of saturated sodium bicarbonate solution was added.
  • the obtained solution was washed three times with a mixed solvent of n-heptane/methyl tert-butyl ether (4/1), and the aqueous phase was extracted twice with methyl tert-butyl ether.
  • the organic phases were combined and washed twice with water and saturated brine, respectively.
  • the organic phase was dried and the solvent was evaporated to obtain a yellow solid compound 9 (5.0 g, 10.38 mmol, 87% yield), which was directly used in the next step without purification.
  • the remaining aqueous phase was extracted twice with ethyl acetate, and the organic phases were combined.
  • the organic phase was washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure to remove the solvent.
  • Adenine (7.11 g, 52.6 mmol, 1.1 equiv) was dissolved in N-methylformamide, and potassium carbonate (6.61 g, 47.8 mmol, 1.0 equiv) was added at 0°C.
  • the reaction solution was warmed to room temperature and stirred for 2 hours.
  • a solution of compound 2 (18.0 g, 47.8 mmol, 1.0 equiv) in N,N-dimethylformamide was added, the reaction solution was warmed to 90°C, and stirring was continued for 16 hours.
  • the reaction was quenched with saturated ammonium chloride solution at 0°C, and ethyl acetate was added to dilute the reaction solution.
  • the mixed solution was allowed to stand for stratification, and the organic phase was separated.
  • the aqueous phase was extracted twice with ethyl acetate, dried, and the organic phase was concentrated.
  • the crude yellow oil compound 5 was used directly in the next step without purification.
  • Compound 035S2M1 (2.4 g, 3.06 mmol, 1.0 equiv) was placed in a clean dry reaction bottle, and DCA (3.5% V/V, 36 mL, 15.3 mmol, 5.0 equiv) and Cysteine (0.45 g, 3.67 mmol, 1.2 equiv) were added and stirred at room temperature for 16 hours.
  • N6-benzoyl adenine (16.38 g, 68.48 mmol, 1.1 equiv) was placed in a clean dry reaction bottle, 200 mL of DMF was added, the temperature was cooled to 0°C in an ice-salt bath, NaH (3.49 g, 60%, 87.16 mmol, 1.4 equiv) was slowly added, and the mixture was stirred at 0°C for 0.5 hours.
  • a 50 mL DMF solution of 035M1 (18.7 g, 62.26 mmol, 1.0 equiv) was added, the temperature was raised to 60°C and stirred overnight.
  • Compound 035M3 (7.1 g, 21.69 mmol, 1.0 equiv) was placed in a clean dry reaction bottle, 100 mL of pyridine was added, 4,4'-bismethoxytrityl chloride (10.29 g, 30.4 mmol) and DMAP (0.13 g, 1.08 mmol, 0.05 equiv) were added at room temperature, and stirring was continued at room temperature for 16 hours.
  • the special modified compound prepared in the above steps and the commercially purchased conventional modified monomer (purchased from Shanghai Zhaowei Technology Development Co., Ltd.) are used to connect the nucleoside monomers one by one from the 3'-5' direction according to the nucleotide arrangement order.
  • the specially modified anti-off-target compound is placed in the seed region of the antisense chain (any position from the 4th to the 8th position from the 5' end), and each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. Synthesis conditions used for the sense chain and antisense chain.
  • the reagents used to synthesize siRNA conjugates were purchased from Suzhou Kelema.
  • the single-strand synthesis reaction process is extended from 3'-5' direction and completed on a solid phase synthesizer. It includes four main reaction steps:
  • De-DMT reaction Use dichloroacetic acid to remove the DMT protecting group on the nucleotide to obtain the 5'-hydroxyl end;
  • Oxidation reaction Under the action of iodine, the phosphite triester obtained in the previous condensation reaction is converted into a more stable phosphate ester (i.e., trivalent phosphorus is oxidized to pentavalent phosphorus);
  • Capping reaction There may be very few 5'-hydroxyl groups that do not participate in the condensation reaction (less than 2%). Use acetic anhydride and 1-methylimidazole to react with them to form acetate caps that cannot participate in subsequent reactions, thereby preventing further reactions. This short fragment can be separated during purification.
  • the nucleic acid sequence connected to the solid phase carrier is cut, deprotected, purified, desalted, and then freeze-dried to obtain the sense chain and the antisense chain, wherein:
  • Aminolysis is carried out at 50°C in a constant temperature water bath for 16 hours. After 16 hours of aminolysis, the water bath is cooled to warm (25°C ⁇ 2°C), filtered with
  • a small sample of the crude product is sent to the analysis department for detection of the crude product LC-MS.
  • the detection method is as follows: Use Waters Acquity UPLC-LTQ LCMS (column: ACQUITY UPLC BEH C18) to detect the purity of the above-mentioned sense chain and antisense chain and analyze the molecular weight. The measured value is consistent with the theoretical value.
  • the purification and desalting conditions are as follows: purification using an ion exchange column and desalting using a HiPrepTM 26/10 desalting gel column, followed by single-chain freeze-drying. After single-chain freeze-drying, sampling is required for LC-MS.
  • the obtained sense strand and antisense strand need to be annealed into a double strand.
  • Annealing operation is as follows: Dissolve the sense strand and antisense strand obtained by purification in water for injection, prepare 0.1mg/mL-40mg/mL solution, calibrate with thermo Nanodrop Eight to mix in equal molar ratio, heat at 90°C for 5 minutes, and then slowly cool naturally to form a double-stranded structure through hydrogen bonding, take samples and send to test the SEC purity of the product. Lyophilize the double-stranded sample.
  • B may be A, G, C or U.
  • B1 and B2 may be A, G, C or U, respectively.
  • the formed structures are different.
  • the general term Safe02 is used, which includes Agt in Table 1 or Table 2 (when B is base A, the chemically modified base A is called Agt), and its specific structure has been given above.
  • the general term Safe03 is used, which includes Ag147 in Table 1 or Table 2 (when B1 is base G and B2 is base A, the chemically modified base A is called Ag147), and its specific structure has been given above.
  • On-target activity was assessed by co-transfecting cells with 10 ng on-target reporter plasmid (a sequence GSCM that is fully complementary to the antisense strand was inserted into the 3' untranslated region of the Renilla luciferase of the reporter plasmid) and siRNA diluted 3 times from 10 nM to 0.000169 nM using Lipofectamine 2000 (ThermoFisher, 11668019).
  • Off-target activity was assessed by co-transfecting HEK293 cells with 10 ng off-target reporter plasmid (a sequence GSSM containing five tandem sites that are consistent with only the 1-8 sites at the 5' end of the antisense strand was inserted into the 3' untranslated region of the Renilla luciferase of the reporter plasmid) and siRNA diluted 3 times from 90 nM to 0.00152 nM using Lipofectamine 2000.
  • HEK293 cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2 .
  • siRNA modified sequences are shown in Table 1, wherein the sense strand of the naked sequence of the siRNA is 5'-UGACAAGAAUCCUCACAAU-3' (SEQ ID NO: 1), and the antisense strand is 5'-AUUGUGAGGAUUCUU GUCAAC-3' (SEQ ID NO: 2).
  • the dual luciferase assay kit Promega, E1980 was used for detection.
  • the reporter plasmids all contain firefly luciferase as an internal reference. The group transfected with only the plasmid was used as the control group.
  • the Renilla luciferase signal readings in each well were normalized to the firefly luciferase (control) signal, and then compared with cells transfected with the same plasmid but not treated with siRNA to calculate the relative levels at different concentrations, and the IC 50 values were calculated.
  • the test results are shown in Table 2. The results showed that siRNA containing Safe02 and Safe03 retained on-target activity that was basically the same as or even better than the original sequence in vitro, and significantly reduced off-target activity; and the on-target activity of siRNA containing Safe02 and Safe03 was significantly better than that of GNA.
  • C57BL/6-HBV transgenic mice were selected from Beijing Weitongda Biotechnology Co., Ltd.: 1.28 times the length of HBV (type A, GeneBank: AF305422.1) linearized fragments were injected into the pronuclei of C57BL/6NCrl mouse embryos to obtain transgenic positive mice.
  • HBV type A, GeneBank: AF305422.1
  • test sequence is shown in Table 3 below, and the test sequence corresponds to The sense strand of the naked sequence is 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3), and the antisense strand is 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).
  • C57BL/6-HBV transgenic mice were divided into 3 groups, with 5 mice in each group.
  • blood was collected from the animals under the mandible and centrifuged at 5000rpm, 4°C, for 10 minutes.
  • Serum was diluted with PBS, and the dilution factor was determined according to the actual situation.
  • the diluted samples were then sent to Beijing Dian Medical Testing Laboratory Co., Ltd. for HBsAg detection, and the results were back-calculated according to the dilution factor.
  • the HBsAg quantification method was direct electrochemiluminescence. According to the HBsAg level, the animals were subcutaneously injected on day 0, with an injection dose of 3 mg/kg body weight, and injected once. Blood was collected on days 7, 14, 21, and 28 to detect HBsAg levels, and the anti-HBV activity of siRNA containing Safe02 was compared with that of the positive control VIR-2218.
  • conjugated group L96 conjugated to the siRNA is:
  • SA51SA51SA51 means that three compounds SA51 are conjugated consecutively at the 3' end of the sense chain, and the conjugate structure is
  • the synthesis method of SA51 please refer to the invention patent application number 2024100522838.

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Abstract

一种寡聚核酸及用于降低脱靶活性的化合物。一种单链或双链的寡聚核酸，每条链具有15至35个核苷酸，其非首末端至少一个核苷酸位置处包含式（I）所示的化学修饰或其互变异构体修饰。该修饰方法在降低siRNA的脱靶效应的同时，能够最大程度不影响siRNA的在靶活性。

Description

一种寡聚核酸及用于降低脱靶活性的化合物 技术领域
本发明涉及分子生物学和生物化学领域，尤其涉及一种寡聚核酸及用于降低脱靶活性的化合物。
背景技术
siRNA具有非常重要的药物开发前景。但是，siRNA在体内应用中，常常具有不同程度的脱靶作用(Off-target effect)，会存在siRNA靶向非靶标基因的情况，类似microRNA途径的脱靶效应是siRNA脱靶的主要原因之一，即siRNA反义链(Antisense Strand，又称AS链)种子区(5’端第2-8位)与目标mRNA形成的完全或不完全配对所产生的对mRNA的抑制活性。一个siRNA分子的脱靶作用可能影响多个mRNA。因此可能产生不可预测的毒副作用，这是siRNA药物产生毒副作用的主要原因。
为降低脱靶毒性，提升siRNA药物的安全性，可以对siRNA药物的反义链种子区进行特定位点的化学修饰，从而降低小核酸药物的脱靶毒性。通过引入化学修饰，可以调节种子区与靶基因之间的相互作用的强弱，在不影响目标mRNA沉默效应前提下，努力降低脱靶效应(Mark K.Schlegel,Chirality Dependent Potency Enhancement and Structural Impact of Glycol Nucleic Acid Modifification on siRNA.J.Am.Chem.Soc.2017,139,8537-8546)。
现有技术中，在siRNA的反义链5’端种子区引入化学修饰后，例如，在siRNA的反义链5’端种子区引入GNA或UNA修饰，会降低脱靶毒性，但同时也会损失较大的在靶(on-target)活性。因此，现有技术需要一种化学修饰方法，其在降低siRNA的脱靶效应的同时，能够最大程度不影响siRNA的在靶活性。
发明内容
有鉴于此，本发明的目的是提出一种经降低脱靶活性修饰的寡聚核酸及用于降低脱靶活性的化合物，本发明的方案在降低寡聚核酸的脱靶效应的同时，能够最大程度不影响寡聚核酸的在靶活性。同时，本发明能同时具有提高单链寡聚核酸或者双链寡聚核酸的稳定性方面的价值。
基于上述目的，本发明的第一个方面提供了一种单链或双链的寡聚核酸，每条链具有15至35个核苷酸，所述寡聚核酸的非首末端至少一个核苷酸位置处包含式(I)所示的化学修饰或其互变异构体修饰，
其中，B为天然核碱基、修饰的核碱基、通用碱基或H原子；优选地，B为天然核碱基或修饰的核碱基；
Z选自O、NH和S；优选地，Z为O；
R1、R2、R3、R4、R5、R6和R7分别独立地选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3和-O-烷基氨基；优选地，R1、R2、R3、R4、R5、R6和R7分别独立地选自H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；
X为O或者S；
n为1、2或3；优选地，n为1或2。
在本发明的优选的实施方案中，所述寡聚核酸是siRNA，其包含有义链和反义链。
在本发明的优选的实施方案中，所述反义链在其5’区域的第2位至第8位中的至少一个核苷酸位置处包含式(I-1)所示的化学修饰或其互变异构体修饰，
其中：B1和B2分别独立地为天然核碱基、修饰的核碱基、通用碱基或H原子；优选地，B1和B2分别独立地为天然核碱基或修饰的核碱基；
Z选自O、NH和S；优选地，Z为O；
R1、R2、R3、R4、R5、R6和R7分别独立地选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3和-O-烷基氨基；优选地，R1、R2、R3、R4、R5、R6和R7分别独立地为H；
X为O或者S；
M1和M2分别独立地选自H、OH、F、OCH3、OCH2CH2OCH3和-O-烷基；优选地，M1和M2分别独立地选自H、OH、F和OCH3；
n为1、2或3，优选地，n为1或2。
在本发明的优选的实施方案中，所述反义链在其5’区域的第2位至第8位中的至少一个核苷酸位置处包含式(I-2)所示的化学修饰或其互变异构体修饰，
其中，B1和B2分别独立地为天然核碱基、修饰的核碱基、通用碱基或H原子；优选地，B1和B2分别独立地为天然核碱基或修饰的核碱基；
R3和R4分别独立地选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3和-O-烷基氨基；优选地，R3和R4分别独立地为H；
X为O或者S；优选地，X为O；
M选自H、OH、F、OCH3、OCH2CH2OCH3和-O-烷基；优选地，M选自H、OH、F和OCH3。
在本发明的优选的实施方案中，所述反义链在其5’区域的第2位至第8位中的至少一个核苷酸位置处包含式(I-3)、(I-4)或(I-5)所示的化学修饰或其互变异构体修饰，
其中，B1和B2分别独立地为天然核碱基、修饰的核碱基、通用碱基或H原子；优选地，B1和B2分别独立地为天然核碱基或修饰的核碱基；
R3和R4分别独立地选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3和-O-烷基氨基；优选地，R3和R4分别独立地为H；
X为O或者S；优选地，X为O；
M选自OH、F、OCH3、OCH2CH2OCH3和-O-烷基；优选地，M选自OH、F和OCH3。
在本发明的优选的实施方案中，所述化学修饰选自以下任一结构：

在本发明的优选的实施方案中，所述反义链在其5’区域的第5位至第8位中的至少一个核苷酸位置处包含上述定义的任一种化学修饰或其互变异构体修饰；
优选地，所述反义链在其5’区域的第6和7位中的至少一个核苷酸位置处包含上述定义的任一种化学修饰或其互变异构体修饰。
在本发明的优选的实施方案中，除了包含上述定义的任一种化学修饰或其互变异构体修饰的核苷酸之外，所述正义链和/或反义链中的至少一条还包含至少一个其他修饰的核苷酸；
优选地，除了包含上述定义的任一种化学修饰或其互变异构体修饰的核苷酸之外，所述正义链和/或反义链中的其余核苷酸为其他修饰的核苷酸。
在本发明的优选的实施方案中，所述其他修饰的核苷酸相互独立地选自：2’-氟代修饰的核苷酸，2’-烷氧基修饰的核苷酸，2’-取代的烷氧基修饰的核苷酸，2’-烷基修饰的核苷酸，2’-取代的烷基修饰的核苷酸，2’-脱氧核苷酸，2’-氨基修饰的核苷酸和2’-取代的氨基修饰的核苷酸；
优选地，所述其他修饰的核苷酸相互独立地选自：2’-F修饰的核苷酸，2’-O-CH3修饰的核苷酸，2’-O-CH2-CH2-O-CH3修饰的核苷酸，2’-O-CH2-CH＝CH2修饰的核苷酸，2’-CH2-CH2-CH＝CH2修饰的核苷酸和2’-脱氧核苷酸；
更优选地，所述其他修饰的核苷酸相互独立地选自:2’-F修饰的核苷酸和2’-O-CH3修饰的核苷酸。
在本发明的优选的实施方案中，上述单链或双链的寡聚核酸/siRNA具有以下特征中的至少一个：
(i)该反义链包含2、3、4、5或6个2’-氟修饰；
(ii)该反义链包含1、2、3或4个硫代磷酸酯核苷酸间键；
(iv)该正义链包含2、3、4或5个2’-氟修饰；
(v)该正义链包含1、2、3或4个硫代磷酸酯核苷酸间键；
(vi)该dsRNA包含至少四个2’-氟修饰；
(vii)该siRNA包含长度为12-40个核苷酸对的双链体区域。
本发明的第二个方面提供了一种siRNA缀合物，其包含上述单链或双链的寡聚核酸/siRNA及缀合至所述siRNA的缀合基团。
优选地，所述缀合基团包含药学上可接受的靶向基团和接头，并且所述单链或双链的寡聚核酸/siRNA、所述接头和所述靶向基团依次共价或非共价连接。
本发明的第三个方面提供了一种药物组合物，其包含上述单链或双链的寡聚核酸/siRNA，或上述siRNA缀合物，以及药学上可接受的载体。
本发明的第四个方面提供了一种试剂盒，其包含上述单链或双链的寡聚核酸/siRNA，或上述siRNA缀合物，或上述药物组合物。
本发明的第五个方面提供了一种如下式(III)所示的化合物或其互变异构体，
其中，B1和B2分别独立地为天然核碱基、修饰的核碱基、通用碱基或H原子；
R3和R4分别独立地选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3和-O-烷基氨基；
X为O或者S；
M选自OH、F、OCH3、OCH2CH2OCH3和-O-烷基；
E是离去基团；优选地，E为MMTr或DMTr；
Q是含磷活性反应基团；优选地，Q为
在本发明的优选的实施方案中，上述式(III)或者(IV)所示的化合物或其互变异构体，其选自以下任一结构：
本发明的第六个方面提供了一种制备上述单链或双链的寡聚核酸/siRNA或上述SiRNA缀合物的方法，其包括以下步骤：
1)合成根据上述式(III)所示的化合物或其互变异构体；
2)利用步骤1)的化合物或其互变异构体合成所述siRNA或siRNA缀合物。
本发明的第七个方面提供了一种单链或双链的寡聚核酸，每条链具有15至60个核苷酸，所述寡聚核酸的非首末端至少一个核苷酸位置处包含式(V)所示的化学修饰或其互变异构体修饰，
其中，B1和B2独立选自天然核碱基、修饰的核碱基、通用碱基或H原子；
Z选自O、NH和S；
G选自-(CR8R9)m-和-CR10＝CR11-；
R1、R2、R5、R6、R7、R8、R9、R8、R9、R10和R11分别独立地选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3和-O-烷基氨基；
M1和M2分别独立地选自H、OH、F和OCH3；
n为1、2或3；
m为1，2，3，4或5；
y为0，1，2，3；
X为O或者S。
本发明的第八个方面提供了一种siRNA，其包含有义链和反义链，每条链具有15至60个核苷酸，所述反义链至少一个核苷酸位置处包含式(V-2)、(V-3)、(V-4)或(V-5)示的化学修饰或其互变异构体修饰，
其中：B1和B2分别独立地为天然核碱基、修饰的核碱基、通用碱基或H原子；优选地，B1和B2分别独立地为天然核碱基、修饰的或未修饰的核碱基；
M1选自H、OH、F和OCH3；优选地，M1选自F和OCH3；
X为O或者S。
在本发明的优选的实施方案中，所述化学修饰选自以下任一结：

在本发明的优选的实施方案中，所述反义链在其5’区域的第5位至第8位中的至少一个核苷酸位置处包含上述所定义的式(V)、(V-2)、(V-3)或(V-4)所示的化学修饰或其互变异构体修饰；
优选地，所述反义链在其5’区域的第6和7位中的至少一个核苷酸位置处包含上述所定义的式(V)、(V-2)、(V-3)或(V-4)所示的化学修饰或其互变异构体修饰。
在本发明的优选的实施方案中，除了包含上述所定义的式(V)、(V-2)、(V-3)或(V-4)所示的化学修饰或其互变异构体修饰的核苷酸之外，所述正义链和/或反义链中的至少一条还包含至少一个其他修饰的核苷酸；
优选地，除了包含上述所定义的式(V)、(V-2)、(V-3)或(V-4)所示的化学修饰或其互变异构体修饰的核苷酸之外，所述正义链和/或反义链中的其余核苷酸为其他修饰的核苷酸。
在本发明的优选的实施方案中，所述其他修饰的核苷酸相互独立地选自：2’-氟代修饰的核苷酸，2’-烷氧基修饰的核苷酸，2’-取代的烷氧基修饰的核苷酸，2’-烷基修饰的核苷酸，2’-取代的烷基修饰的核苷酸，2’-脱氧核苷酸，2’-氨基修饰的核苷酸和2’-取代的氨基修饰的核苷酸；
优选地，所述其他修饰的核苷酸相互独立地选自：2’-F修饰的核苷酸，2’-O-CH3修饰的核苷酸，2’-O-CH2-CH2-O-CH3修饰的核苷酸，2’-O-CH2-CH＝CH2修饰的核苷酸，2’-CH2-CH2-CH＝CH2修饰的核苷酸和2’-脱氧核苷酸；
更优选地，所述其他修饰的核苷酸相互独立地选自:2’-F修饰的核苷酸和2’-O-CH3修饰的核苷酸。
在本发明的优选的实施方案中，具有以下特征中的至少一个：
(i)该反义链包含2、3、4、5或6个2’-氟修饰；
(ii)该反义链包含1、2、3或4个硫代磷酸酯核苷酸间键；
(iii)该正义链包含2、3、4或5个2’-氟修饰；
(iv)该正义链包含1、2、3或4个硫代磷酸酯核苷酸间键；
(v)该dsRNA包含至少四个2’-氟修饰；
(vi)该siRNA包含长度为12-40个核苷酸对的双链体区域。
本发明的第九个方面提供了一种siRNA缀合物，其包含上述单链或双链的寡聚核酸/siRNA及缀合至所述siRNA的缀合基团。
优选地，所述缀合基团包含药学上可接受的靶向基团和接头，并且所述单链或双链的寡聚核酸/siRNA、所述接头和所述靶向基团依次共价或非共价连接。
本发明的第十个方面提供了一种药物组合物，其包含上述单链或双链的寡聚核酸，或上述siRNA缀合物，以及药学上可接受的载体。
本发明的第十一个方面提供了一种试剂盒，其包含上述单链或双链的寡聚核酸，或上述siRNA缀合物，或上述药物组合物。
本发明的第十二个方面提供了一种如下式(VI)所示的化合物或其互变异构体，
其中，B1和B2分别独立地为天然核碱基、修饰的核碱基、通用碱基或H原子；
G选自-(CR8R9)n-和-CR10＝CR11-；
X为O或者S；
M选自H、OH、F、OCH3、OCH2CH2OCH3和-O-烷基；
E是离去基团；优选地，E为MMTr或DMTr；
Q是含磷活性反应基团；优选地，Q为
在本发明的优选的实施方案中，式(VI)所示的化合物或其互变异构体，其选自以下任一结构：

本发明的第十三个方面提供了一种制备上述单链或双链的寡聚核酸或上述siRNA缀合物的方法，其包括以下步骤：
1)合成根据上述式(VI)所示的化合物或其互变异构体；
2)利用步骤1)的化合物或其互变异构体合成所述siRNA或siRNA缀合物。
本发明的第十四个方面提供了一种用于抑制细胞中靶基因的方法，该方法包括将上述单链或双链的寡聚核酸，或者，上述siRNA缀合物引入该细胞中的步骤。
本发明的第十五个方面提供了一种对siRNA的反义链2-8位进行修饰以降低脱靶活性的方法，将如上述化学修饰或其互变异构体修饰，或者上述化学修饰或其互变异构体修饰引入到siRNA分子的反义链2-8位。
本发明的有益效果为：
本发明通过在siRNA的反义链5’端种子区引入特殊化合物，降低siRNA的脱靶效应的同时，能够最大程度不影响siRNA的在靶活性，从而提升了siRNA作为药物的安全性。
附图说明
图1为实施例5中的给药前后HBsAg水平结果图。
具体实施方式
需要说明的是，除非另外定义，本申请使用的技术术语或者科学术语应当为所属领域的技术人员所理解的通常意义。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的药材原料、试剂材料等，如无特殊说明，均为市售购买产品。当用于本文和所附权利要求书中时，单数形式“一”、“一种”、“另一”和“所述”包括复数指代对象，除非上下文明确地另有指示。
定义
在本发明的上下文中，大写字母A、U、C、G：表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸酯基连接；字母组合VP表示该字母组合VP右侧相邻的一个核苷酸为乙烯基磷酸酯修饰的核苷酸，如下所示。硫代磷酸酯基的结构如式(1)所示：
如本文所用，术语“天然核碱基”是指未从RNA或DNA中的天然存在形式修饰的核碱基。“天然核碱基”的实例包括嘌呤核碱基腺嘌呤(A)和鸟嘌呤(G)以及嘧啶核碱基胸腺嘧啶(T)、胞嘧啶(C)和尿嘧啶(U)。除了“天然核碱基”之外，许多本领域技术人员已知的修饰的核碱基或核碱基模拟物适用于本文所述的化合物。
如本文所用，术语“修饰的核碱基”是指在结构上与母核碱基非常相似的核碱基，例如7-脱氮嘌 呤、5-甲基胞嘧啶或G-钳。
如本文所用，通用碱基(universal nucleic acid base)：可以与至少两种常用碱基进行互补配对的碱基，例如次黄嘌呤(Hypoxanthine，其核苷为Inosine)，可与A、T、G或C中的任一种进行配对，其结合能力为I:C>I:A>I:G>I:T；或是例如嗅尿嘧啶BrU(5-Bromouridine)，可与A或G进行配对；可选地，本发明中还可选用其它具有能够与至少两种常用碱基互补配对的通用碱基，例如3-硝基吡咯(3-nitropyrrole)、5-硝基吲哚(5-nitroindole)、7-氮杂吲哚(7-Azaindole)等等。
如本文所用，术语“种子区”是RNAi试剂的反义链中的负责识别靶mRNA并且响应于例如该反义链的从5’端起的核苷酸2-8的区域。
如本文所用，术语“核苷酸位置”是指如自核苷酸在5′端计数，所述核苷酸在寡核苷酸中的位置。例如，核苷酸位置1是指寡核苷酸的5′端核苷酸。
如本文所用，“寡核苷酸”即“寡聚核酸”，是指在2至2500个核苷酸的范围内的核苷酸的聚合形式。寡核苷酸可为单链或双链的。在某些实施方案中，所述寡核苷酸具有500至1500个核苷酸，通常，例如，其中所述寡核苷酸用于基因疗法。在某些实施方案中，所述寡核苷酸是单链或双链的且具有7至100个核苷酸。在某些实施方案中，所述寡核苷酸是单链或双链的且具有15至100个核苷酸。在另一实施方案中，所述寡核苷酸是具有15至50个核苷酸的单链或双链，通常，例如，其中所述寡核苷酸是核酸抑制剂分子。在另一实施方案中，所述寡核苷酸是具有25至40个核苷酸的单链或双链，通常，例如，其中所述寡核苷酸是核酸抑制剂分子。在又另一实施方案中，所述寡核苷酸是单链或双链的且具有19至40或19至25个核苷酸，通常，例如，其中所述寡核苷酸是双链核酸抑制剂分子并形成具有至少18至25个碱基对的双螺旋。在其他实施方案中，所述寡核苷酸是单链的且具有15至25个核苷酸，通常，例如，其中所述寡核苷酸核苷酸是单链RNAi抑制剂分子。通常，如本文所述，所述寡核苷酸含有一个或多个含磷核苷酸间连接基团。在其他实施方案中，如本文所述，所述核苷酸间连接基团是亚磷酸酰胺基团。
如本文所用，“氟代修饰的核苷酸”是指核苷酸的核糖基2’位的羟基被氟取代形成的核苷酸，其具有以下式(7)所示的结构。在一些实施方案中，2’-烷基修饰的核苷酸为甲氧基修饰的核苷酸(2’-OMe)，如式(8)所示。
其中，base表示碱基，例如A、U、G、C或T。
如本文所用，“非氟代修饰的核苷酸”是指核苷酸的核糖基2’位的羟基被非氟基团取代形成的核苷酸。
如本文所用，“缀合”是指两个或多个各自具有特定功能的化学部分之间以共价连接的方式彼此连接；相应地，“缀合物”是指该各个化学部分之间通过共价连接而形成的化合物。进一步地，“siRNA缀合物”表示一个或多个具有特定功能的化学部分共价连接至siRNA上而形成的化合物。
在本发明的上下文中，特别是在描述本申请的siRNA、含siRNA的组合物或siRNA缀合物的制备方法时，除非特别说明，所述核苷单体(nucleoside monomer)指，根据欲制备的siRNA或siRNA缀合物中核苷酸的种类和顺序，亚磷酰胺固相合成中使用的修饰或未修饰的核苷亚磷酰胺单体(unmodified or modified RNA phosphoramidites，有时RNA phosphoramidites也称为Nucleoside phosphoramidites)。亚磷酰胺固相合成为本领域技术人员所公知的RNA合成中所用的方法。本申请所用的核苷单体均可商购得到。
1.本发明的单链或双链的寡聚核酸/siRNA
本发明提供了单链或双链的寡聚核酸，每条链具有15至35个核苷酸，所述寡聚核酸的非首末端至少一个核苷酸位置处包含式(I)或(V)所示的化学修饰或其互变异构体修饰，
在本发明中，“寡核苷酸的非首末端”是指除了寡核苷酸的首端和末端之外的任何位置，换言之，除了寡核苷酸的第一位核苷酸和最后一位核苷酸(5’-3’方向)之外的任何位置。
本申请的siRNA含有核苷酸基团作为基本结构单元，本领域技术人员公知，所述核苷酸基团含有磷酸基团、核糖基团和碱基。
本发明在siRNA的反义链5’端种子区引入这些化合物中的一种来进行化学修饰，降低siRNA的脱靶效应的同时，能够最大程度不影响siRNA的在靶活性，从而提升siRNA作为药物的安全性。
现有技术中，通常是在siRNA的反义链5’端种子区引入GNA或UNA修饰，会降低脱靶毒性，但同时也会损失较大的在靶活性。
其中，UNA如式(3)所示，GNA如式(4)所示。
上述式(3)和式(4)中，R选自H、OH或烷氧基(O-烷基)。
在本申请设计了针对多个靶标的不同siRNA序列，并比较了在由相同碱基序列构成的siRNA反义链中，并在siRNA的反义链5’端种子区中的相同位置分别引入本申请的化学修饰和GNA修饰时，siRNA的在靶活性和脱靶活性。结果表明，引入本申请的化学修饰和GNA修饰，均能够降低siRNA的脱靶效应，提升了siRNA作为药物的安全性，并且引入本申请的化学修饰在降低脱靶活性上优于GNA修饰；并且引入本申请的化学修饰的siRNA相对于GNA修饰具有更好的在靶活性或者仍能够保证有效的在靶活性，这表明引入本申请的化学修饰能够最大程度不影响siRNA的在靶活性。在多个siRNA中的效果均是如此，这也表明，本发明的siRNA所取得的效果与siRNA序列本身无关。
本申请提供了许多化学修饰，例如Agt、Agt01、Uut、Agt02、Aut、Aut01、Agz、Agy、Agx等，在反义链的5’区域的第2位至第8位中的至少一个核苷酸位置处引入这些化学修饰中的一种，均能够保留基本与原序列相同的在靶活性，并显著降低了脱靶活性。
在本申请的实施例(包括体外和体内实验)中，验证了siRNA反义链5’端种子区的第6位或第7位中的核苷酸位置处包含上述化学修饰，均能够降低siRNA的脱靶效应，同时能够最大程度不影响siRNA的在靶活性。
2.本发明的siRNA缀合物
本发明提供了一种siRNA缀合物，其包含上述单链或双链的寡聚核酸/siRNA及缀合至所述siRNA的缀合基团。优选地，所述缀合基团包含药学上可接受的靶向基团和接头，并且所述单链或双链的寡聚核酸/siRNA、所述接头和所述靶向基团依次共价或非共价连接。
在本发明的优选的实施方案中，所述缀合至所述siRNA的缀合基团为TIN、L96或SA51等，其中，TIN的结构式见下图，L96和SA51的结构式见实施例5。
TIN。
3.本发明的药物组合物和试剂盒
本申请提供了一种药物组合物，所述药物组合物含有如上所述的单链或双链的寡聚核酸/siRNA或siRNA缀合物作为活性成分和药学上可接受的载体。
所述药学上可接受的载体可以是siRNA给药领域常规使用的载体，例如但不限于磁性纳米粒(magnetic nanoparticles，如基于Fe3O4或Fe2O3的纳米粒)、碳纳米管(carbon nanotubes)、介孔硅(mesoporous silicon)、磷酸钙纳米粒(calcium phosphate nanoparticles)、聚乙烯亚胺(polyethylenimine，PEI)、聚酰胺型树形高分子(polyamidoamine(PAMAM)dendrimer)、聚赖氨酸(poly(L-lysine)，PLL)、壳聚糖(chitosan)、1,2-二油酰基-3-三甲铵丙烷(1,2-dioleoyl-3-trimethylammonium-propane，DOTAP)、聚D型或L型乳酸/羟基乙酸共聚物(poly(D&L-lactic/glycolic acid)copolymer，PLGA)、聚(氨乙基乙撑磷酸酯)(poly(2-aminoethyl ethylene phosphate)，PPEEA)和聚(甲基丙烯酸-N,N-二甲氨基乙酯)(poly(2-dimethylaminoethyl methacrylate)，PDMAEMA)以及它们的衍生物中的一种或多种。
所述药物组合物中，对单链或双链的寡聚核酸/siRNA或siRNA缀合物和药学上可接受的载体的含量没有特别要求，可以是各组分常规的含量。
本公开提供了一种试剂盒，其包含上述单链或双链的寡聚核酸/siRNA或上述siRNA缀合物或上述药物组合物。
在一些实施方案中，本文所述的试剂盒可在一个容器中提供单链或双链的寡聚核酸/siRNA或siRNA缀合物。在一些实施方案中，本文所述的试剂盒可包含一个提供药学上可接受的赋形剂的容器。在一些实施方案中，所述试剂盒中还可包含其它成分，如稳定剂或防腐剂等。在一些实施方案中，本文所述的试剂盒可在不同于提供本文所述单链或双链的寡聚核酸/siRNA或siRNA缀合物的容器以外的其它容器中包含至少一种其它治疗剂。在一些实施方案中，所述试剂盒可包含用于将单链或双链的寡聚核酸/siRNA或siRNA缀合物与药学上可接受的载体和/或辅料或其它成分(若有的话)进行混合的说明书。
在本申请的试剂盒中，所述单链或双链的寡聚核酸/siRNA、siRNA缀合物和药学上可接受的载体和/或辅料、和/或药物组合物，和/或药学上可接受的载体和/或辅料可以任何形式提供，例如液体形式、干燥形式或冻干形式。在一些实施方案中，所述单链或双链的寡聚核酸/siRNA、siRNA缀合物和药学上可接受的载体和/或辅料以及所述药物组合物和药学上可接受的载体和/或辅料基本上纯净和/或无菌。在一些实施方案中，可在本申请的试剂盒中提供无菌水。
4.中间体
本发明提供了一种如下式(III)或式(VI)所示的化合物或其互变异构体，
R3和R4分别独立地选自H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基和C2-C6炔基；
X为O或者S；
M选自H、OH、F、OCH3、OCH2CH2OCH3和-O-烷基；
E是离去基团；优选地，E为MMTr或DMTr；
Q是含磷活性反应基团；优选地，Q为
G选自-(CR8R9)n-和-CR10＝CR11-。
利用上述中间体能够合成本发明的单链或双链的寡聚核酸/siRNA或siRNA缀合物。
下面以具体实施例对本发明的技术方案做进一步的说明，但本发明并不限于这些实施例。除 非另有说明，本文使用的制剂和设备均为普通市售品。
实施例1化合物SA000003的制备
本实施例中，化合物SA000003的合成路线如下图所示：
1.1.1中间体9-2的制备
将化合物9-1(市售，购买于上海兆维科技发展有限公司)(8.0g,11.9mmol,1.0equiv)溶于56mL乙腈中，温度降至0℃，在此温度下加入叔丁基二甲基氯硅烷(3.6g,23.8mmol,2.93mL,2.0equiv)，咪唑(3.25g,47.7mmol,4.0equiv)。加料完毕后升至室温，反应液继续搅拌16小时。TLC(展开体系为：石油醚：乙酸乙酯＝1:1)检测，化合物9-1反应完毕消失。反应液温度降至0℃，加入异丙醇(2.87g,47.7mmol,3.66mL,4.0equiv)，搅拌1小时，反应液不经纯化直接用于下步反应。
1.1.2中间体9的制备
将十二硫醇(7.25g,35.8mmol,8.58mL,3.0equiv)与三氟乙酸(13.6g,119.0mmol,8.84mL,10.0equiv)加入到上步反应液中，此反应液在0℃下搅拌2小时，TLC(石油醚：乙酸乙酯＝1:1)检测显示反应原料9-2反应完毕消失。0℃下加入N-甲基咪唑(11.76g,143.27mmol,11.4mL,12.0equiv)，过滤反应溶液并用乙腈洗涤滤饼残留物，合并反应液与洗涤溶液，加入饱和碳酸氢钠溶液10mL。所得溶液用正庚烷/甲基叔丁基醚(4/1)混合溶剂洗涤三次，并用甲基叔丁基醚萃取水相两次，合并有机相，并用水，饱和食盐水分别洗涤有机相两次，干燥有机相，蒸除溶剂，得到黄色固体化合物9(5.0g,10.38mmol,87％收率)，不经纯化，直接用于下步反应。
LC-MS:C21H35N5O6Si，分子量481.2，482.2(M+H).
1H NMR(CDCl3,400MHz):δppm 7.86(s,1H),5.80(d,J＝6.8Hz,1H),4.49-4.47(m,1H),4.29-4.26(m,1H),4.14(d,J＝2.0Hz,1H),3.98-3.95(m,1H),3.74-3.70(m,1H),2.75-2.69(m,1H),1.27-1.18(m,6H),0.93(s,8H),0.13-0.09(m,6H).
1.2.1化合物2的制备
将化合物1(市售，购买于阿达玛斯)(6.0g,81.0mmol,5.36mL,1.0equiv)溶于吡啶中，0℃下加入4,4'-双甲氧基三苯甲基氯(32.9g,97.1mmol,1.2equiv)，继续搅拌1小时。TLC(石油醚/乙酸乙酯＝10/1)检测，化合物1反应完毕消失。0℃下加入200mL饱和碳酸氢钠溶液淬灭反应，加入100mL乙酸乙酯，静置分层，分离有机相，剩余水相用乙酸乙酯萃取两次，合并有机相。有机相用饱和食盐水洗涤两次，并用无水硫酸钠干燥，抽滤，将滤液减压蒸馏蒸除溶剂，所得粗产品经硅胶柱层析(展开体系：石油醚/乙酸乙酯＝20/1到10/1)，得到无色油状物化合物2(20.0g,53.1mmol,65.6％收率)。
LC-MS:C24H24O4分子量376.1,377.2(M+H).
1H NMR(CDCl3,400MHz):δppm 7.49(d,J＝7.2Hz,2H),7.39-7.20(m,8H),6.86(d,J＝8.8Hz,4H),3.82(s,6H),3.37-3.34(m,1H),3.19-3.12(m,2H),2.82-2.80(m,1H),2.67-2.65(m,1H).
1.2.2中间体3的制备
将腺嘌呤(7.11g,52.6mmol,1.1equiv)溶于N-甲基甲酰胺中，0℃下加入碳酸钾(6.61g,47.8mmol,1.0equiv)。反应液升至室温继续搅拌2小时。加入化合物2(18.0g,47.8mmol,1.0equiv)的N,N-二甲基甲酰胺的溶液，将反应液升至90℃，继续搅拌16小时。TLC(石油醚/乙酸乙酯＝3/1)检测显示化合物2消失，反应液降至0℃，加入500mL饱和氯化铵溶液淬灭反应，再加入500mL乙酸乙酯，静置分层，水相用乙酸乙酯萃取两次。合并有机相，并用饱和食盐水洗涤有机相，无水硫酸钠干燥有机相，过滤，减压蒸除滤液中溶剂，所得粗产品直接用于下步反应。
LC-MS:C29H29N5O4,分子量511.2，512.2(M+H).
1H NMR(CDCl3,400MHz):δppm 7.96(d,J＝11.2Hz,3H),7.43-7.38(m,3H),7.33-7.16(m,12H),6.89-6.84(m,6H),5.36(d,J＝5.2Hz,1H),4.32-4.26(m,1H),4.14-4.07(m,2H),3.73(s,8H),2.99-2.96(m,1H),2.88(s,8H),2.72(s,6H).
1.2.3中间体4的制备
氮气保护下化合物3(23.0g,44.9mmol,1.0equiv)与吡啶(100mL x 2)加入到干燥的圆底瓶中，升温蒸除吡啶，移除化合物3中水分，向上述反应瓶中加入160mL吡啶。反应液降温至0-5℃，加入三甲基硅氯(19.5g,179mmol,22.8mL,4.00equiv)，加热至25-30℃搅拌2小时，LCMS检测反应结束。滴加100mL水与200mL浓氨水，搅拌半小时，乙酸乙酯萃取水相，有机相用100mL饱和食盐水洗涤。有机相干燥之后，抽滤，将滤液浓缩。所得残留物柱层析分离纯化(二氯甲烷：甲醇＝50/1到10/1)，得到浅黄色油状物化合物4(9.0g,14.6mmol,33％收率)。
LC-MS:C36H33N5O5，分子量615.2，616.3(M+H).
1HNMR(DMSO-d6,400MHz):δppm 11.13(s,1H),8.72(s,1H),8.36(s,1H),8.06(d,J＝7.2Hz,2H),7.90-7.88(m,2H),7.65-7.20(m,17H),6.90-6.87(m,4H),5.44(d,J＝5.6Hz,1H),4.49-4.45(m,1H),4.30-4.25(m,1H),4.15-4.13(m,1H),3.73(s,6H),3.08-2.87(m,2H).
1.2.4中间体5的制备
将化合物4(7.0g,11.3mmol,1.0equiv)溶于N,N-二甲基甲酰胺中，0℃下加入氢化钠(1.36g,34.1mmol,3.0equiv)。30分钟后将对甲苯磺酰氧甲基膦酸二甲酯(10.0g,34.1mmol,3.0equiv)的N,N-二甲基甲酰胺溶液滴加进上述溶液中。此混合物在25℃下继续搅拌12小时。LC-MS检测显示反应原料化合物4消失。0℃下用饱和氯化铵溶液淬灭反应，加入乙酸乙酯稀释反应液，将混合液静置分层，分离有机相，水相用乙酸乙酯萃取两次，干燥，浓缩有机相，所得黄色油状化合物5粗产物不经纯化，直接用于下步反应。
LC-MS:C39H40N5O8P，分子量737.2，738.3(M+H).
1.2.5中间体6的制备
将化合物5(8.39g,11.3mmol,1.0equiv)溶于48毫升吡啶与32毫升水的混合溶剂中，升温至60℃并搅拌16小时。0℃下加入150毫升饱和碳酸氢钠溶液淬灭反应，再加入150毫升乙酸乙 酯稀释，此混合物静置分层，水相用分别用150毫升乙酸乙酯萃取两次。合并有机相，并用饱和食盐水洗涤两次有机相，无水硫酸钠干燥，抽滤，滤液减压蒸馏浓缩蒸除有机相溶剂，粗产品用硅胶柱层析分离纯化(洗脱体系：二氯甲烷/甲醇＝10/1)，得到褐色固体化合物6(3.8g,4.61mmol,42％收率)。
LC-MS:C38H38N5O8P，分子量723.2,724.3(M+H).
1H NMR(CDCl3,400MHz):δppm 8.68(s,1H),8.42(s,1H),7.97(d,J＝7.6Hz,2H),7.55-7.43(m,3H),7.36-7.29(m,2H),7.23-7.10(m,10H),6.73(d,J＝8.8Hz,4H),4.58-4.53(m,1H),4.37-4.32(m,1H),3.83-3.78(m,2H),3.70-3.65(m,6H),3.52-3.39(m,4H),3.17-3.08(m,2H),2.94-2.89(m,10H),1.29-1.17(m,14H).31P NMR(CDCl3,162MHz):δppm 15.65.
1.2.6中间体7的制备
将化合物6的三乙胺盐(2.8g,3.39mmol,1.0equiv)溶于30mL吡啶中，降温至0℃加入2,4,6-三异丙基苯磺酰氯(3.08g,10.1mmol,3.0equiv)，反应液在0℃下继续搅拌15分钟。加入甲基咪唑(1.39g,16.9mmol,1.35mL,5.0equiv)，随即滴加化合物9(1.55g,3.22mmol,0.95equiv)的吡啶(18mL)溶液。滴加完毕反应液升温至室温，继续搅拌16小时。LCMS检测显示化合物6消失。蒸除反应液溶剂，所得残留物经C18反相柱制备，得到黄色固体化合物7(1.5g,1.26mmol,37％收率).
LC-MS:C59H71N10O13PSi，分子量1186.4，1187.5(M+H).
1H NMR(DMSO-d6,400MHz):δppm 12.10(s,1H),11.58(s,1H),11.12(s,1H),8.67(s,1H),8.33-8.28(m,2H),8.05-8.02(m,2H),7.65-7.51(m,3H),7.39-7.19(m,10H),6.86-6.82(m,4H),5.89-5.86(m,1H),4.49-4.32(m,4H),4.25-3.90(m,6H),3.74-3.71(m,6H),3.58-3.47(m,3H),3.34(s,1H),3.26-3.24(m,3H),3.19-3.16(m,1H),3.03-2.99(m,1H),1.10(d,J＝6.8Hz,6H),0.86-0.85(m,9H),0.07-0.06(m,9H).31P NMR(DMSO-d6,162MHz):δppm 23.17,22.79.
1.2.7中间体8的制备
将化合物7(1.5g,1.26mmol,1.0equiv)溶于四氢呋喃(9.0mL)中，降温至0℃加入三乙胺三氢氟酸盐(1.22g,7.58mmol,1.24mL,6.0equiv)和3mL四氢呋喃。反应液升温至室温，继续搅拌24小时。HPLC显示化合物7仍有约3％残留，反应液中加入50mL乙酸乙酯，所得有机相用饱和碳酸氢钠溶液，饱和食盐水洗涤，无水硫酸钠干燥，有机相抽滤，浓缩滤液。所得残留物制备液相纯化，得到白色固体化合物8(1.0g,931.9umol,74％收率)。
LC-MS:C53H57N10O13P，分子量1072.3,1073.4(M+H).
1H NMR(DMSO-d6,400MHz):δppm 12.16-12.01(m,1H),11.70-11.57(m,1H),11.17-11.01(m,1H),8.66(s,1H),8.33(s,1H),8.24-8.21(m,1H),8.04(d,J＝7.2Hz,2H),7.65-7.48(m,3H), 7.42-7.14(m,9H),6.90-6.79(m,4H),5.93-5.85(m,1H),5.49-5.45(m,1H),4.55-4.47(m,2H),4.35-3.87(m,8H),3.56-3.39(m,3H),3.32-3.24(m,7H),3.20-3.14(m,1H),3.04-2.96(m,1H),2.80-2.66(m,1H),1.17-1.09(m,6H).31P NMR(DMSO-d6,162MHz):δppm 23.08,22.66.
1.2.8化合物SA000003的制备
将化合物8(1.0g,0.93mmol,1.0equiv)溶于10mL二氯甲烷中，加入双(二异丙基氨基)(2-氰基乙氧基)膦(561.0mg,1.86mmol,2.0equiv)与4,5-二氰基咪唑(165.0mg,1.4mmol,1.5equiv)。此反应混合物在25℃反应2小时。LCMS显示化合物8消失。分两批加入40mL饱和碳酸氢钠溶液，静置分层，分离有机相，并用饱和食盐水洗涤有机相，无水硫酸钠干燥有机相，干燥后的有机相过滤，浓缩滤液，粗产品在正己烷/二氯甲烷(100mL/10mL)混合溶剂体系中打浆洗三次。抽滤收集固体，即得到淡黄色固体SA000003产物。
LC-MS:C62H74N12O14P2，分子量1272.4,1273.5(M+H).
实施例2化合物035的制备
本实施例中，化合物035(Ag147)的合成路线如下图所示：
1.1中间体035S2M1的制备
将化合物035S2S1(市售，购买于上海泰坦科技股份有限公司)(2.10g,3.14mmol,1.0equiv)和咪唑(0.64g,9.41mmol,3.0equiv)置于洁净干燥反应瓶中，加入20mL乙腈，室温下缓慢加入叔丁基二甲基氯硅烷(0.95g,6.27mmol,2.0equiv)，随后室温搅拌16小时。反应后向反应液中加入50mL乙酸乙酯，依次用50mL饱和碳酸氢钠溶液和50mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(梯度洗脱：甲醇/二氯甲烷＝0/100-5/100),得到白色固体化合物035S2M1(2.4g,3.06mmol,97.6％收率)。化合物035S2M1分子式:C42H53N5O8Si,分子量:783.3,LC-MS找到784.4(M+H).
1.2中间体035S2的制备
将化合物035S2M1(2.4g,3.06mmol,1.0equiv)置于洁净干燥反应瓶中，加入DCA(3.5％V/V,36mL,15.3mmol,5.0equiv)和Cysteine(0.45g,3.67mmol，1.2equiv)，室温搅拌16小时。反应液用30mL饱和碳酸氢钠溶液洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(梯度洗脱：甲醇/二氯甲烷＝0/100-5/100),得到淡黄色泡沫状化合物035S2(1.21g,2.51mmol,82.3％收率)。化合物035S2分子式:C21H35N5O6S,分子量:481.2,LC-MS找到482.4(M+H).
1.3中间体035M1的制备
将5-羟甲基-2,2-二甲基-1,3-二恶烷(市售，购买于上海泰坦科技股份有限公司)(9.1g,62.2mmol,1.0equiv)置于洁净干燥反应瓶中，加入100mL二氯甲烷和17.3mL三乙胺，冰盐浴降温至0℃，滴加对甲苯磺酰氯(12.46g,65.4mmol，1.05equiv)，室温搅拌5小时。反应后向反应液中加入碳酸钠(7.4g)的80mL水溶液，水相用80mL二氯甲烷萃取，合并有机相，并用50mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品淡黄色固体化合物035M1(18.7g,62.26mmol,100％收率)直接投入下一步。化合物035M1分子式:C14H20O5S,分子量:300.1,LC-MS找到301.3(M+H).
1.4中间体035M2的制备
将N6-苯甲酰基腺嘌呤(16.38g,68.48mmol,1.1equiv)置于洁净干燥反应瓶中，加入200mL DMF，冰盐浴降温至0℃，缓慢加入NaH(3.49g,60％，87.16mmol,1.4equiv)，0℃搅拌0.5小时。加入035M1(18.7g,62.26mmol,1.0equiv)的50mL DMF溶液，升温到60℃搅拌过夜。降温，向反应液中加入500mL乙酸乙酯，并用饱和盐水洗涤三次，每次200mL，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(梯度洗脱：甲醇/二氯甲烷＝0/100-6/100),得到白色固体化合物035M2(11.7g,31.85mmol,51.1％收率)。化合物035M2分子式:C19H21N5O3,分子量:367.1,LC-MS找到366.3(M-H).
1.5中间体035M3的制备
将化合物035M2(11.7g,31.85mmol,1.0equiv)置于洁净干燥反应瓶中，加入125mL 80％乙酸，升温到60℃反应0.5小时。浓缩除去溶剂粗产品经反向柱纯化(梯度洗脱：乙腈/水＝1/20-1/4)得白色固体化合物035M3(7.1g,21.69mmol,68.1％收率)。化合物035M3分子式:C16H17 N5O3,分子量:327.1,LC-MS找到326.1(M-H).
1.6中间体035M4的制备
将化合物035M3(7.1g,21.69mmol,1.0equiv)置于洁净干燥反应瓶中，加入100mL吡啶，室温下加入4,4'-双甲氧基三苯甲基氯(10.29g,30.4mmol)和DMAP(0.13g,1.08mmol,0.05equiv)，室温继续搅拌16小时。反应后向反应液中加入150mL乙酸乙酯，并用150mL饱和碳酸氢钠溶液和150mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(梯度洗脱：甲醇/二氯甲烷＝0/100-2/100),得到淡黄色泡沫化合物035M4(4.8g,7.62mmol,35.1％收率)。化合物035M4分子式:C37H35N5O5,分子量:629.2,LC-MS找到628.4(M-H).
1.7中间体035M5的制备
将化合物035M4(4.8g,7.62mmol,1.0equiv)置于洁净干燥反应瓶中，加入50mL二氯甲烷溶解，室温下加入戴斯-马丁氧化剂(6.47g,15.24mmol,2.0equiv)，随后室温继续搅拌2小时。反应后抽滤，滤饼用20mL二氯甲烷洗涤，合并滤液，浓缩，所得粗产品硅胶柱层析分离纯化(梯度洗脱：甲醇/二氯甲烷＝0/100-5/100)，得到泡沫状固体化合物035M5(3.6g,5.74mmol,75.3％收率)。化合物035M5分子式:C37H33N5O5,分子量:627.2,LC-MS找到628.3(M+H).
1.8中间体035M6的制备
氩气保护下，在洁净干燥反应瓶中，加入50mL甲苯，氢化钠(0.50g,12.62mmol,2.2equiv)，搅拌片刻，缓慢加入四甲基亚甲基二磷酸酯(3.33g,14.34mmol,2.5equiv)，室温搅拌0.5小时。加入035M5(3.6g,5.74mmol,1.0equiv)的50mL甲苯溶液，随后室温继续搅拌2小时。反应后向反应液中加入100mL乙酸乙酯，并依次用100mL饱和碳酸氢钠溶液和100mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(梯度洗脱：甲醇/二氯甲烷＝0/100-4/100),得到泡沫状固体化合物035M6(2.4g,3.27mmol,57.0％收率)。化合物035M6分子式:C40H40N5O7P,分子量:733.2,LC-MS找到734.3(M+H).
1.9中间体035M7的制备
将化合物035M6(1.1g,1.50mmol,1.0equiv)置于洁净干燥反应瓶中，加入6mL吡啶和4mL水，升温到60℃反应16小时。浓缩除去溶剂，粗产品经C18柱纯化(梯度洗脱：乙腈/水＝1/20 -100/0)得白色固体化合物035M7(0.74g,1.03mmol,68.5％收率)。化合物035M7分子式:C39H38N5O7P,分子量:719.2,LC-MS找到720.3(M+H).
1.10中间体035M8的制备
氩气保护下，在洁净干燥反应瓶中，加入5mL乙腈，035M7(540mg,0.75mmol,1.0equiv)，035S2(343.3mg,0.71mmol,0.95equiv)，N,N,N',N'-四甲基氯甲脒六氟磷酸盐(505mg,1.8mmol,2.4equiv)和N-甲基咪唑(431mg,5.25mmol,7.0equiv)，随后室温搅拌16小时。反应后向反应液中加入20mL乙酸乙酯，并依次用20mL饱和碳酸氢钠溶液和20mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(梯度洗脱：甲醇/二氯甲烷＝0/100-5/100),得到泡沫状固体化合物035M8(843mg,0.71mmol,94.9％收率)。化合物035M8分子式:C60H71N10O12PSi,分子量:1182.4,LC-MS找到1183.5(M+H).
1.11中间体035M9的制备
将化合物035M8(843mg,0.71mmol,1.0equiv)置于洁净干燥反应瓶中，加入8mL四氢呋喃，室温下加入四丁基氟化铵(1M,1.4mL,1.4mmol,2.0equiv)，随后室温继续搅拌2小时。反应后向反应液中加入20mL乙酸乙酯，并依次用20mL饱和碳酸氢钠溶液和20mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(梯度洗脱：甲醇/二氯甲烷＝0/100-10/100),并再次用C18柱纯化(梯度洗脱：乙腈/水＝1/20 -100/0)得白色泡沫状固体化合物035M9(482mg,0.45mmol,63.2％收率)。化合物035M9分子式:C54H57N10O12P,分子量:1068.3,LC-MS找到1069.4(M+H).1H NMR(400MHz,DMSO-d6):δ12.08(s,1H),11.63(s,1H),11.14(s,1H),8.70(d,J＝2.3Hz,1H),8.37–8.35(m,1H),8.22–8.18(m,1H),8.03(d,J＝8.0Hz,2H),7.64(t,J＝6.8Hz,1H),7.55(dd,J＝10.5,4.6Hz,2H),7.33–7.25(m,4H),7.17(dt,J＝8.7,4.3Hz,5H),6.84(d,J＝8.9Hz,4H),6.80–6.67(m,1H),5.93–5.91(m,1H),5.88–5.79(m,1H),5.76(s,1H),5.50–5.47(m,1H),4.47–4.41(m,2H),4.33–4.29(m,1H),4.25–4.19(m,1H),4.08–3.94(m,2H),3.72(s,6H),3.50(dd,J＝11.1,9.5Hz,2H),3.40(t,J＝10.7Hz,1H),3.32–3.29(m,2H),3.26–3.22(m,1H),3.16–3.05(m,2H),2.75(dt,J＝13.5,6.9Hz,1H),1.25–1.23(m,1H),1.11(dd,J＝10.7,4.0Hz,6H).31P NMR(162MHz,DMSO-d6):δ19.16(s),18.98(d,J＝8.7Hz).
1.12化合物035的制备
将化合物035M9(471mg,0.44mmol,1.0equiv)置于洁净干燥反应瓶中，加入5mL无水二氯甲烷，室温氩气保护下加入化合物2-氰乙基N,N,N’,N’-四异丙基亚磷酰二胺(265.6mg,0.88mmol,2.0equiv)和4,5-二氰咪唑(78.1mg,0.66mmol,1.5equiv)，室温下继续搅拌1小时。反应后向反应液中加入10mL二氯甲烷，并用20mL饱和碳酸氢钠溶液洗涤，干燥有机相，过滤并浓缩，所得粗产品经C18反相柱(规格:30μm；市售，购买于上海博蕴生物科技有限公司)(乙腈/水＝5/95 -85/15)制备得到白色泡沫状固体化合物035(400mg,0.32mmol,71.5％收率)。化合物035分子式:C63H74N12O13P2,分子量:1268.5,LC-MS找到1269.5(M+H).1H NMR(400MHz,DMSO-d6):δ12.07(s,1H),11.62(s,1H),11.16(s,1H),8.70(t,J＝3.7Hz,1H),8.38–8.33(m,1H),8.28–8.21(m,1H),8.04(d,J＝7.7Hz,2H),7.64(t,J＝7.3Hz,1H),7.55(t,J＝7.6Hz,2H),7.33–7.25(m,4H),7.18–7.14(m,5H),6.84–6.82(m,4H),6.79–6.69(m,1H),5.94–5.73(m,2H),4.56–4.38(m,4H),4.31–3.98(m,3H),3.78(dd,J＝12.8,6.7Hz,2H),3.71(s,6H),3.69–3.57(m,3H),3.54–3.48(m,2H),3.41(dd,J＝12.6,11.3Hz,1H),3.34–3.31(m,1H),3.30–3.26(m,2H),3.16–3.06(m,2H),2.81–2.71(m,3H),1.17–1.07(m,18H).31P NMR(162MHz,DMSO-d6):δ150.36(d,J＝9.8Hz),149.93(d,J＝9.3Hz),19.32(d,J＝15.0Hz),19.16(d,J＝8.0Hz).
实施例3siRNA以及siRNA缀合物的制备
通过固相亚磷酰胺法，利用上述步骤制备的特殊修饰化合物及商业购买的常规修饰的单体(购自上海兆维科技发展有限公司)，按照核苷酸排布顺序自3’-5’方向逐一连接核苷单体。其中特殊修饰的防脱靶化合物置于反义链的种子区(从5’端开始数第4-8位任一位置)，每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化或硫化四步反应。正义链和反义链采用的合成条件。
仪器设备型号：MerMade 12 Oligonucleotide syntheizer固相合成仪，北京海精1ml合成柱，思拓凡SourceTM 15Q 4.6/100PE纯化柱。
合成siRNA缀合物所使用的试剂购自苏州柯乐玛。
合成简介如下：
单链合成反应过程是从3’-5’方向延伸，在固相合成仪上完成。包括四个主要的反应步骤：
a.脱DMT反应：用二氯乙酸脱去核苷酸上的保护基团DMT获得5’-羟基端；
b.偶联反应：保护的核苷酸亚磷酰胺单体，与活化剂乙硫基四氮唑混合，亚磷酰胺基团被活化，5’-羟基仍然被DMT保护，与连接在固相载体上的5’-羟基发生缩合反应，生成亚磷酸三酯；
c.氧化反应：在氧化剂碘的作用下，上步缩合反应所得的亚磷酸三酯转变为更稳定的磷酸酯。(即将三价磷氧化成五价磷)；
d.硫代反应：在硫代试剂PADS的作用下，将上步缩合反应所得的亚磷酸三酯转变为硫代磷酸酯(根据序列设计选择氧化或硫代)。
e.盖帽反应：缩合反应中可能有极少数5’-羟基没有参加反应(少于2％)，用乙酸酐和1-甲基咪唑与其反应，形成不能参与后续反应的乙酸酯封端，阻止其后继续发生反应，这种短片段可以在纯化时分离掉。
重复进行以上四步的循环，直至合成完所需的序列，其主要的化学反应方程式如下
待最后一个核苷单体连接完成后，依次对固相载体上连接的核酸序列进行切割、脱保护、纯化、脱盐，随后冻干获得正义链和反义链，其中：
切割和脱保护条件如下：先配置好氨解溶液(氨水：乙醇＝3:1的混合溶液至体积为2mL)，固相载体加入上述反应瓶中，充分震荡均匀。在恒温水浴内50℃氨解16小时。氨解16小时后，水浴冷却至温(25℃±2℃)，用砂芯漏斗过滤，圆底烧瓶收集滤液，并用50％乙醇水溶液冲洗滤渣，收集滤液，旋转蒸发仪浓缩，再转移至玻璃瓶中，取粗品小样送分析部门检测粗品LC-MS。检测方法如下：使用Waters Acquity UPLC-LTQ LCMS(column：ACQUITY UPLC BEH C18)检测上述正义链和反义链纯度并分析分子量。实测值与理论值相符。
纯化和脱盐条件如下：利用离子交换色谱柱进行纯化并配合使用思拓凡HiPrepTM 26/10desalting凝胶柱脱盐，然后单链冻干。单链冻干后，需要取样测LC-MS。
最后需要需要将获得的正义链和反义链退火成双链。
退火操作如下：将纯化合获得的正义链和反义链分别溶于注射用水中，配制0.1mg/mL-40mg/mL的溶液，用thermo Nanodrop Eight标定等摩尔比混合，90℃加热5分钟，再缓慢自然降温，使它们通过氢键形成双链结构，取样送检测产品的SEC纯度。将双链样品冻干。
实施例4体外评估Safe02以及Safe03的在靶活性及抗脱靶活性
在本实施例中，验证了在不同反义链的5’区域的第7位中的核苷酸位置处包含式(I)或式(V)所示的化学修饰，对siRNA的在靶活性和抗脱靶活性的影响。
在式(I)中，B可以为A、G、C或U。在式(V)中，B1和B2分别可以为A、G、C或U。
当B、B1和B2不同时，所形成的结构不同。为了方便描述本实施例，使用了Safe02这个统称，其包括表1或表2中的Agt(当B为碱基A时，该化学修饰后的碱基A称为Agt)，其具体结构在上文中已经给出。为了方便描述本实施例，使用了Safe03这个统称，其包括表1或表2中的Ag147(当B1为碱基G，且B2为碱基A时，该化学修饰后称为Ag147)，其具体结构在上文中已经给出。
在本实施例中，在碱基A中引入GNA的化学修饰形成Agn。
利用双萤光素酶检测Safe02或Safe03序列中的在靶及抗脱靶活性。在靶活性评估是利用Lipofectamine 2000(ThermoFisher，11668019)将10ng在靶报告基因质粒(在报告子质粒的海肾荧光素酶的3’非翻译区插入与反义链完全互补配对的序列GSCM)和以3倍稀释10nM至0.000169nM的siRNA共转染细胞，脱靶活性评估是利用Lipofectamine 2000将10ng脱靶报告基因质粒(在报告子质粒的海肾荧光素酶的3’非翻译区插入含有仅与反义链5’端1-8位点一致的五个串联的序列GSSM)和以3倍稀释90nM至0.00152nM的siRNA共同转染至HEK293细胞。HEK293细胞培养于含10％胎牛血清的DMEM高糖培养基中，在37℃，5％ CO2条件下培养。siRNA修饰序列见表1，其中该siRNA的裸序列的正义链为5’-UGACAAGAAUCCUCACAAU-3’(SEQ ID NO:1)，反义链为5’-AUUGUGAGGAUUCUU GUCAAC-3’(SEQ ID NO:2)。转染24小时后利用双荧光素酶检测试剂盒(Promega，E1980)进行检测。报告子质粒中都含有萤火虫荧光素酶，作为内参。只转染质粒的组作为对照组。
将各孔的海肾荧光素酶信号读值对萤火虫荧光素酶(对照)信号进行比值均一化，然后与转染有相同质粒但未经siRNA处理的细胞比较计算不同浓度下的相对水平，并计算IC50值，检测结果见表2，结果显示包含Safe02和Safe03的siRNA在体外保留了基本与原序列相同甚至更优的在靶活性的同时，并显著降低了脱靶活性；并且，包含Safe02和Safe03的siRNA的在靶活性明显优于GNA。
表1
表2
实施例5利用C57BL/6-HBV(乙型肝炎病毒)转基因小鼠测评Safe02修饰缀合物的活性
C57BL/6-HBV转基因小鼠选自北京维通达生物技术有限公司：将1.28倍长度的HBV(A型，GeneBank：AF305422.1)线性化片段注入C57BL/6NCrl小鼠胚胎原核，获得转基因阳性小鼠。通过外周血HBV拷贝数分析，保留了拷贝数达到107～108eq/ml的首建鼠，按照半合子配野生C57BL/6NCrl小鼠的方式建立了HBV转基因小鼠品系。测试序列如下表3所示，该测试序列对应 的裸序列的正义链为5’-GUGUGCACUUCGCUUCACA-3’(SEQ ID NO:3)，反义链为5’-UGUGAAGCGAAGUGCACACUU-3’(SEQ ID NO:4)。
C57BL/6-HBV转基因小鼠分为3组，每组5只小鼠，在第-3天对动物进行下颌下取血，取血后离心，离心条件为5000rpm，4℃，离心10分钟。用PBS稀释血清，需按照实际情况确定稀释倍数。然后将稀释后的样品送至北京迪安医学检验实验室有限公司进行检测HBsAg，其测定结果按照稀释倍数进行回算。HBsAg定量方法为直接电化学发光法。根据HBsAg水平分组，在第0天对动物分别皮下注射，注射剂量为3mg/kg体重，共注射一次，在实验进展至第7、14、21、28天取血检测HBsAg水平，比较包含Safe02的siRNA与阳性对照VIR-2218抗乙肝病毒的活性。
给药前后HBsAg水平见图1。结果表明包含Safe02的化合物SD004191显著降低HBsAg水平，优于阳性化合物VIR-2218。
表3
在本实施例中，所述缀合至所述siRNA的缀合基团L96：
在本实施例中，SA51SA51SA51表示3个化合物SA51连续缀合在正义链的3’端，其缀合物结构为SA51的合成方法参见申请号为2024100522838的发明专利。
以上详细描述了本申请的一些实施方式，但是，本申请并不限于上述实施方式中的具体细节，在本申请的技术构思范围内，可以对本申请的技术方案进行多种简单变型，这些简单变型均属于本申请的保护范围。
另外需要说明的是，在上述一些实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本申请对各种可能的组合方式不再另行说明。
此外，本申请的各种不同的实施方式之间也可以进行任意组合，只要其不违背本申请的思想，其同样应当视为本申请所公开的内容。

Claims (37)

  1. 一种单链或双链的寡聚核酸，每条链具有15至35个核苷酸，所述寡聚核酸的非首末端至少一个核苷酸位置处包含式(I)所示的化学修饰或其互变异构体修饰，
    其中，B为天然核碱基、修饰的核碱基、通用碱基或H原子；
    Z选自O、NH和S；
    R1、R2、R3、R4、R5、R6和R7分别独立地选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3和-O-烷基氨基；
    X为O或者S；
    n为1、2或3。
  2. 根据权利要求1所述的单链或双链的寡聚核酸，其中，所述寡聚核酸是siRNA，其包含有义链和反义链；其中：
    B为天然核碱基或修饰的核碱基；
    Z为O；
    R1、R2、R3、R4、R5、R6和R7分别独立地选自H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基；
    X为O或者S；
    n为1或2。
  3. 根据权利要求1所述的单链或双链的寡聚核酸，其中，所述寡聚核酸是siRNA，其包含有义链和反义链；所述反义链在其5’区域的第2位至第8位中的至少一个核苷酸位置处包含式(I-1)所示的化学修饰或其互变异构体修饰，
    其中：B1和B2分别独立地为天然核碱基、修饰的核碱基、通用碱基或H原子；
    Z选自O、NH和S；
    R1、R2、R3、R4、R5、R6和R7分别独立地选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3和-O-烷基氨基；
    X为O或者S；
    M1和M2分别独立地选自H、OH、F、OCH3、OCH2CH2OCH3和-O-烷基；
    n为1、2或3。
  4. 根据权利要求3所述的单链或双链的寡聚核酸，其中，
    B1和B2分别独立地为天然核碱基或修饰的核碱基；
    Z为O；
    R1、R2、R3、R4、R5、R6和R7分别独立地为H；
    X为O或者S；
    M1和M2分别独立地选自H、OH、F和OCH3；
    n为1或2。
  5. 根据权利要求1所述的单链或双链的寡聚核酸，其中，所述寡聚核酸是siRNA，其包含有义链和反义链；所述反义链在其5’区域的第2位至第8位中的至少一个核苷酸位置处包含式(I-2)所示的化学修饰或其互变异构体修饰，
    其中，B1和B2分别独立地为天然核碱基、修饰的核碱基、通用碱基或H原子；
    R3和R4分别独立地选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3和-O-烷基氨基；
    X为O或者S；
    M选自H、OH、F、OCH3、OCH2CH2OCH3和-O-烷基。
  6. 根据权利要求5所述的单链或双链的寡聚核酸，其中，
    B1和B2分别独立地为天然核碱基或修饰的核碱基；
    R3和R4分别独立地为H；
    X为O；
    M选自H、OH、F和OCH3。
  7. 根据权利要求1所述的单链或双链的寡聚核酸，其中，所述寡聚核酸是siRNA，其包含有义链和反义链；所述反义链在其5’区域的第2位至第8位中的至少一个核苷酸位置处包含式(I-3)、(I-4)或(I-5)所示的化学修饰或其互变异构体修饰，
    其中，B1和B2分别独立地为天然核碱基、修饰的核碱基、通用碱基或H原子；
    R3和R4分别独立地选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3和-O-烷基氨基；
    X为O或者S；M选自OH、F、OCH3、OCH2CH2OCH3和-O-烷基；
    M选自OH、F和OCH3。
  8. 根据权利要求7所述的单链或双链的寡聚核酸，其中，
    B1和B2分别独立地为天然核碱基或修饰的核碱基；
    X为O；
    R3和R4分别独立地为H；
    M选自F和OCH3。
  9. 根据权利要求1所述的单链或双链的寡聚核酸，所述化学修饰选自以下任一结构：

  10. 根据权利要求1-9中任一项所述的单链或双链的寡聚核酸，其中，所述寡聚核酸是siRNA， 其包含有义链和反义链；所述化学修饰或其互变异构体修饰在所述反义链的5’区域的第5位至第8位中的至少一个核苷酸位置。
  11. 根据权利要求10所述的单链或双链的寡聚核酸，其中，所述化学修饰或其互变异构体修饰在所述反义链的5’区域的第6和7位中的至少一个核苷酸位置。
  12. 根据权利要求10所述的单链或双链的寡聚核酸，其中，除了包含所述化学修饰或其互变异构体修饰的核苷酸之外，所述正义链和/或反义链中的至少一条还包含至少一个其他修饰的核苷酸；
    优选地，除了包含所述化学修饰或其互变异构体修饰的核苷酸之外，所述正义链和/或反义链中的其余核苷酸为其他修饰的核苷酸。
  13. 根据权利要求12所述的单链或双链的寡聚核酸，其中，所述其他修饰的核苷酸相互独立地选自：2’-氟代修饰的核苷酸，2’-烷氧基修饰的核苷酸，2’-取代的烷氧基修饰的核苷酸，2’-烷基修饰的核苷酸，2’-取代的烷基修饰的核苷酸，2’-脱氧核苷酸，2’-氨基修饰的核苷酸和2’-取代的氨基修饰的核苷酸；
    优选地，所述其他修饰的核苷酸相互独立地选自：2’-F修饰的核苷酸，2’-O-CH3修饰的核苷酸，2’-O-CH2-CH2-O-CH3修饰的核苷酸，2’-O-CH2-CH＝CH2修饰的核苷酸，2’-CH2-CH2-CH＝CH2修饰的核苷酸和2’-脱氧核苷酸；
    更优选地，所述其他修饰的核苷酸相互独立地选自:2’-F修饰的核苷酸和2’-O-CH3修饰的核苷酸。
  14. 根据权利要求10至13中任一项所述的单链或双链的寡聚核酸，其中，具有以下特征中的至少一个：
    (i)该反义链包含2、3、4、5或6个2’-氟修饰；
    (ii)该反义链包含1、2、3或4个硫代磷酸酯核苷酸间键；
    (iii)该正义链包含2、3、4或5个2’-氟修饰；
    (iv)该正义链包含1、2、3或4个硫代磷酸酯核苷酸间键；
    (v)该dsRNA包含至少四个2’-氟修饰；
    (vi)该siRNA包含长度为12-40个核苷酸对的双链体区域。
  15. 一种siRNA缀合物，其包含siRNA及缀合至所述siRNA的缀合基团，其中，所述siRNA为权利要求1-14中任一项所述的单链或双链的寡聚核酸。
  16. 一种药物组合物，其包含权利要求1-14中任一项所述的单链或双链的寡聚核酸或权利要求15所述的siRNA缀合物，以及药学上可接受的载体。
  17. 一种试剂盒，其包含1-14中任一项所述的单链或双链的寡聚核酸，或权利要求15所述的siRNA缀合物，或权利要求16所述的药物组合物。
  18. 一种如下式(III)所示的化合物或其互变异构体，
    其中，B1和B2分别独立地为天然核碱基、修饰的核碱基、通用碱基或H原子；
    R3和R4分别独立地选自H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基和C2-C6炔基；
    X为O或者S；
    M选自H、OH、F、OCH3、OCH2CH2OCH3和-O-烷基；
    E是离去基团；
    Q是含磷活性反应基团。
  19. 根据权利要求18所述的式(III)所示的化合物或其互变异构体，其选自以下任一结构：
  20. 一种制备权利要求1-14中任一项所述的单链或双链的寡聚核酸或权利要求15所述的siRNA缀合物的方法，其包括以下步骤：
    1)合成根据权利要求18-19中任一项所述的式(III)所示的化合物或其互变异构体；
    2)利用步骤1)的化合物或其互变异构体合成所述寡聚核酸或siRNA缀合物。
  21. 一种单链或双链的寡聚核酸，每条链具有15至60个核苷酸，所述寡聚核酸的非首末端至少一个核苷酸位置处包含式(V)所示的化学修饰或其互变异构体修饰，
    其中，B1和B2独立选自天然核碱基、修饰的核碱基、通用碱基或H原子；
    Z选自O、NH和S；
    G选自-(CR8R9)m-和-CR10＝CR11-；
    R1、R2、R5、R6、R7、R8、R9、R8、R9、R10和R11分别独立地选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3和-O-烷基氨基；
    M1和M2分别独立地选自H、OH、F和OCH3；
    n为1、2或3；
    m为1，2，3，4或5；
    y为0，1，2或3；
    X为O或者S。
  22. 根据权利要求21所述的单链或双链的寡聚核酸，其中，所述寡聚核酸为siRNA，其包含有义链和反义链，所述反义链至少一个核苷酸位置处包含式(V-2)、(V-3)、(V-4)或(V-5)所示的化学修饰或其互变异构体修饰，
    其中：B1和B2分别独立地为天然核碱基、修饰的核碱基、通用碱基或H原子；
    M1选自H、OH、F和OCH3；
    X为O或者S。
  23. 根据权利要求22所述的单链或双链的寡聚核酸，其中，B1和B2分别独立地为天然核碱基、修饰的或未修饰的核碱基；
    M1选自F和OCH3；
    X为O或者S。
  24. 根据权利要求22所述的单链或双链的寡聚核酸，所述化学修饰选自以下任一结构：

  25. 根据权利要求21-24中任一项所述的单链或双链的寡聚核酸，其中，所述反义链在其5’区域的第5位至第8位中的至少一个核苷酸位置处包含所述化学修饰或其互变异构体修饰。
  26. 根据权利要求25所述的单链或双链的寡聚核酸，其中，所述反义链在其5’区域的第6和7位中的至少一个核苷酸位置处包含所述化学修饰或其互变异构体修饰。
  27. 根据权利要求25或26所述的单链或双链的寡聚核酸，其中，除了包含所述化学修饰或其互变异构体修饰的核苷酸之外，所述正义链和/或反义链中的至少一条还包含至少一个其他修饰的核苷酸；
    优选地，除了包含所述化学修饰或其互变异构体修饰的核苷酸之外，所述正义链和/或反义链中的其余核苷酸为其他修饰的核苷酸。
  28. 根据权利要求27所述的单链或双链的寡聚核酸，其中，所述其他修饰的核苷酸相互独立地选自：2’-氟代修饰的核苷酸，2’-烷氧基修饰的核苷酸，2’-取代的烷氧基修饰的核苷酸，2’-烷基修饰的核苷酸，2’-取代的烷基修饰的核苷酸，2’-脱氧核苷酸，2’-氨基修饰的核苷酸和2’-取代的氨基修饰的核苷酸；
    优选地，所述其他修饰的核苷酸相互独立地选自：2’-F修饰的核苷酸，2’-O-CH3修饰的核苷酸，2’-O-CH2-CH2-O-CH3修饰的核苷酸，2’-O-CH2-CH＝CH2修饰的核苷酸，2’-CH2-CH2-CH＝CH2修饰的核苷酸和2’-脱氧核苷酸；
    更优选地，所述其他修饰的核苷酸相互独立地选自:2’-F修饰的核苷酸和2’-O-CH3修饰的核苷酸。
  29. 根据权利要求27或28所述的单链或双链的寡聚核酸，其中，具有以下特征中的至少一个：
    (i)该反义链包含2、3、4、5或6个2’-氟修饰；
    (ii)该反义链包含1、2、3或4个硫代磷酸酯核苷酸间键；
    (iii)该正义链包含2、3、4或5个2’-氟修饰；
    (iv)该正义链包含1、2、3或4个硫代磷酸酯核苷酸间键；
    (v)该dsRNA包含至少四个2’-氟修饰；
    (vi)该siRNA包含长度为12-40个核苷酸对的双链体区域。
  30. 一种siRNA缀合物，其包含siRNA及缀合至所述siRNA的缀合基团，其中，所述siRNA为权利要求21-29中任一项所述的单链或双链的寡聚核酸。
  31. 一种药物组合物，其包含权利要求21-29中任一项所述的单链或双链的寡聚核酸或权利要求30所述的siRNA缀合物，以及药学上可接受的载体。
  32. 一种试剂盒，其包含21-29中任一项所述的单链或双链的寡聚核酸，或权利要求30所述的siRNA缀合物，或权利要求31所述的药物组合物。
  33. 一种如下式(VI)所示的化合物或其互变异构体，
    其中，B1和B2分别独立地为天然核碱基、修饰的核碱基、通用碱基或H原子；
    G选自-(CR8R9)n-和-CR10＝CR11-；
    X为O或者S；
    M选自H、OH、F、OCH3、OCH2CH2OCH3和-O-烷基；
    E是离去基团；
    Q是含磷活性反应基团。
  34. 根据权利要求33所述的式(VI)所示的化合物或其互变异构体，其选自以下任一结构：

  35. 一种制备权利要求21-29中任一项所述的单链或双链的寡聚核酸或权利要求30所述的siRNA缀合物的方法，其包括以下步骤：
    1)合成根据权利要求33-34中任一项所述的式(VI)所示的化合物或其互变异构体；
    2)利用步骤1)的化合物或其互变异构体合成所述寡聚核酸或siRNA缀合物。
  36. 一种用于抑制细胞中靶基因的方法，其特征在于：该方法包括将权利要求1至14或权利要求21至29中任一项所述的单链或双链的寡聚核酸，或者，权利要求15或权利要求30所述的siRNA缀合物引入该细胞中的步骤。
  37. 一种对siRNA的反义链2-8位进行修饰以降低脱靶活性的方法，其特征在于：将如权利要求1至9中任一项所述的化学修饰或其互变异构体修饰，或者权利要求21至24中任一项所述的化学修饰或其互变异构体修饰引入到siRNA分子的反义链2-8位。
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