WO2021249352A1 - 双链siRNA类似物的缀合物 - Google Patents

双链siRNA类似物的缀合物 Download PDF

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WO2021249352A1
WO2021249352A1 PCT/CN2021/098682 CN2021098682W WO2021249352A1 WO 2021249352 A1 WO2021249352 A1 WO 2021249352A1 CN 2021098682 W CN2021098682 W CN 2021098682W WO 2021249352 A1 WO2021249352 A1 WO 2021249352A1
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seq
sense strand
antisense strand
acid
double
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PCT/CN2021/098682
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English (en)
French (fr)
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安可
孙飞
丁照中
陈曙辉
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正大天晴药业集团股份有限公司
南京明德新药研发有限公司
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Priority claimed from PCT/CN2020/133982 external-priority patent/WO2021110148A1/zh
Application filed by 正大天晴药业集团股份有限公司, 南京明德新药研发有限公司 filed Critical 正大天晴药业集团股份有限公司
Priority to CN202180041587.9A priority Critical patent/CN115768439A/zh
Priority to CA3186763A priority patent/CA3186763A1/en
Priority to EP21823093.6A priority patent/EP4166144A4/en
Priority to JP2022575954A priority patent/JP2023528966A/ja
Priority to KR1020227046437A priority patent/KR20230022892A/ko
Priority to US18/001,244 priority patent/US20230235330A1/en
Priority to AU2021288648A priority patent/AU2021288648A1/en
Publication of WO2021249352A1 publication Critical patent/WO2021249352A1/zh

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    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • 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
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    • 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
    • C12N15/1131—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 against viruses
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/312—Phosphonates
    • C12N2310/3125—Methylphosphonates
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/32—Chemical structure of the sugar
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    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • the present disclosure belongs to the field of biomedicine, and relates to an r'-embedded siRNA analog, a double-stranded siRNA analog, a conjugate containing the same, and its salt and use; the use is specifically in the preparation and treatment of viral hepatitis B Use in medicines.
  • Hepatitis B virus referred to as hepatitis B
  • HBV hepatitis B virus
  • Hepatitis B virus is a hepatotropic virus that mainly exists in liver cells and damages liver cells, causing liver cell inflammation, necrosis, and fibrosis.
  • Acute hepatitis B can be self-healed by its own immune mechanism in most adults.
  • CHB chronic hepatitis B
  • HCC liver cancer
  • HBsAg hepatitis B virus surface antigen
  • anti-HBV drugs are mainly immunomodulators (interferon- ⁇ and peginterferon- ⁇ -2 ⁇ ) and antiviral drugs (lamivudine, adefovir dipivoxil, entecavir, Bivudine, tenofovir, clavudine, etc.).
  • antiviral therapy drugs are nucleotide drugs, and their mechanism of action is to inhibit the synthesis of HBV DNA, and cannot directly reduce HBsAg levels.
  • nucleotide drugs show that the clearance rate of HBsAg is similar to the natural observation results (Janssen et al. Lancet (2005), 365, 123-129; Marcellin et al. N. Engl. J. Med. (2004)) , 351, 1206-1217; Buster et al. Hepatology (2007), 46, 388-394.).
  • HBsAg hepatitis B S antigen and E antigen
  • siRNA small interfering RNA
  • RNAi RNA interference
  • the most ideal hepatitis B treatment method requires siRNA to be stabilized and modified with a corresponding delivery system to target target organs and cells to improve metabolic stability.
  • the current siRNA cannot effectively reduce hepatitis B virus S antigen and E Antigen content.
  • siRNA can complement and pair with certain mRNA fragments, thereby regulating the expression of the gene corresponding to the mRNA.
  • the complementary pairing of the 5'-end seed region of the siRNA antisense strand and the non-targeted gene can partially or completely silence the gene expression.
  • This phenomenon is the main reason for the off-target effect of siRNA in vivo and in vitro (Jackson et al. RNA (2006), 12, 1179-1187.).
  • SiRNA for hepatitis B has exposed this shortcoming in both clinical and preclinical stages (WO2020036862).
  • some modifications to nucleotides can reduce the risk of off-target (Iribe et al. ACS Omega (2017), 2, 2055-2064; Janas et al. Nat. Commun. 2018, 9, 723-732)
  • its silence is effective Sex has also decreased, and the treatment safety window needs to be improved.
  • the present disclosure relates to a double-stranded siRNA analog embedded with a ribavirin derivative, a conjugate containing the same, and a salt and use thereof.
  • the double-stranded siRNA analogs, conjugates and salts thereof of the present disclosure can effectively inhibit multiple viral indicators such as hepatitis B virus DNA, S antigen, and E antigen, and are useful for the treatment of hepatitis B such as chronic hepatitis B (e.g., functional Sexual cure) provides an effective and feasible means.
  • the present disclosure provides a double-stranded siRNA analog comprising a sense strand and an anti-sense strand, wherein the anti-sense strand is contained in the sequence shown in SEQ ID NO: 2 with r instead of one Or the sequence of multiple nucleotide residues, r is
  • each of the nucleotide and r in the siRNA analog is independently modified or unmodified.
  • one or more of the nucleotides and r in the siRNA analog are modified, while the other nucleotides and r are unmodified.
  • the modification includes, for example, methoxy modification, fluoro modification, phosphorothioate group attachment, or replacement of nucleotides with (S)-glycerol nucleic acids, and the like.
  • one or more of the nucleotides and r in the siRNA analog are modified, while the other nucleotides and r are unmodified.
  • the modification includes, for example, methoxy modification, fluoro modification, phosphorothioate group attachment, replacement of nucleotides with (S)-glycerol nucleic acid or nucleotides with (E)-vinyl phosphate modification, etc. .
  • substantially all nucleotides and r in the siRNA analog are modified. In some embodiments, all nucleotides and r in the siRNA analog are modified.
  • nucleotides and r in the double-stranded siRNA analog are modified. In some embodiments, all nucleotides and r in the double-stranded siRNA analog are modified.
  • the SEQ ID NO: 2 optionally includes an overhang at the 5'end and/or the 3'end. In some embodiments, the SEQ ID NO: 2 includes an overhang of 0, 1, 2, 3, 4, or 5 nucleotides at the 5'end and/or 3'end.
  • the SEQ ID NO: 2 when the SEQ ID NO: 2 includes an overhang of 2 nucleotides at the 5'end and/or 3'end, there are optionally 2 sulfurs between the 3 nucleotides at the end. Phosphoro-substitute group connection, in which 2 of the 3 nucleotides are overhangs, and the other one is a paired nucleotide adjacent to the overhangs.
  • the overhang is preferably from a modified or unmodified UU.
  • the overhang is preferably from uu.
  • the overhang uu has 2 phosphorothioate linkages between its adjacent paired nucleotide.
  • the SEQ ID NO: 2 includes an overhang at the 3'end, and the overhang is preferably from a modified or unmodified UU. In some embodiments, the SEQ ID NO: 2 includes an overhang at the 3'end, and the overhang is preferably from uu. In some embodiments, the SEQ ID NO: 2 includes an overhang at the 3'end, and the overhang uu and its adjacent pair of nucleotides have two phosphorothioate linkages (for example, c ⁇ u ⁇ u).
  • the antisense strand in the double-stranded siRNA analog includes a sequence in which one or more nucleotide residues are replaced with r in the sequence shown in SEQ ID NO: 2.
  • the antisense strand includes a sequence in which one nucleotide residue is replaced with r in the sequence shown in SEQ ID NO: 2.
  • the antisense strand in the double-stranded siRNA analog includes a sequence in which one or more nucleotide residues are replaced with r in the sequence shown in SEQ ID NO: 2.
  • the antisense strand includes a sequence in which one, two, three, four, or five nucleotide residues are replaced with r in the sequence shown in SEQ ID NO: 2.
  • the antisense strand in the double-stranded siRNA analog includes a sequence in which one or more nucleotide residues are replaced with r in the sequence shown in SEQ ID NO: 2, and the r substitution occurs At any position of SEQ ID NO: 2.
  • the r substitution occurs at positions 1 to 21 or positions 1 to 19 at the 5'end of SEQ ID NO: 2.
  • the r substitution occurs at the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 5'end of SEQ ID NO: 2. 17, 18, 19, 20 or 21 positions.
  • the r substitution occurs at positions 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 16 or 18 of the 5'end of SEQ ID NO: 2.
  • the antisense strand in the double-stranded siRNA analog includes SEQ ID NO: 4 or SEQ ID NO: 17, SEQ ID NO: 6 or SEQ ID NO: 19, SEQ ID NO: 7 or SEQ ID NO: 20, SEQ ID NO: 8 or SEQ ID NO: 21, SEQ ID NO: 9 or SEQ ID NO: 22, SEQ ID NO: 10 or SEQ ID NO: 23, SEQ ID NO: 11 or SEQ ID NO: 24.
  • SEQ ID NO: 29 or SEQ ID NO: 33, SEQ ID NO: 30 or SEQ ID NO: 34, SEQ ID NO: 31 or SEQ ID NO: 35, or SEQ ID NO: 32 or SEQ ID NO: 36 The sequence shown, or consists of it.
  • the sequence includes further nucleotide modifications, such as methoxy modification, fluoro modification, phosphorothioate linkage, or replacement of nucleotides with (S)-glycerol nucleic acids, and the like.
  • nucleotide modifications such as methoxy modification, fluoro modification, phosphorothioate linkage, or replacement of nucleotides with (S)-glycerol nucleic acids, and the like.
  • the antisense strand in the double-stranded siRNA analog includes SEQ ID NO: 4 or SEQ ID NO: 17, SEQ ID NO: 6 or SEQ ID NO: 19, SEQ ID NO: 7 or SEQ ID NO: 20, SEQ ID NO: 8 or SEQ ID NO: 21, SEQ ID NO: 9 or SEQ ID NO: 22, SEQ ID NO: 10 or SEQ ID NO: 23, SEQ ID NO: 11 or SEQ ID NO: 24.
  • the sequence includes further nucleotide modifications, such as methoxy modification, fluoro modification, phosphorothioate linkage, replacement of nucleotides with (S)-glycerol nucleic acids, or nucleotides Replaced by (E)-vinyl phosphate modification, etc.
  • nucleotide modifications such as methoxy modification, fluoro modification, phosphorothioate linkage, replacement of nucleotides with (S)-glycerol nucleic acids, or nucleotides Replaced by (E)-vinyl phosphate modification, etc.
  • the sense strand in the double-stranded siRNA analog comprises or consists of the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 28.
  • the sense strand in the double-stranded siRNA analog includes a sequence in which one or more nucleotide residues are replaced with r in the sequence shown in SEQ ID NO:1.
  • the sense strand includes a sequence in which one nucleotide residue is replaced with r in the sequence shown in SEQ ID NO:1.
  • the sense strand in the double-stranded siRNA analog includes a sequence in which one or more nucleotide residues are replaced with r in the sequence shown in SEQ ID NO:1.
  • the sense strand includes a sequence in which 1, 2, 3, 4, or 5 nucleotide residues are replaced with r in the sequence shown in SEQ ID NO:1.
  • the sense strand in the double-stranded siRNA analog includes a sequence in which one or more nucleotide residues are replaced with r in the sequence shown in SEQ ID NO: 1, and the r substitution occurs in SEQ ID NO: 1 to 19 of the 5'end.
  • the r substitution occurs at the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 5'end of SEQ ID NO: 1. 17, 18, or 19 digits.
  • the r substitution occurs at positions 2, 3, 7, 12, 15, 17 or 19 of the 5'end of SEQ ID NO:1.
  • the sequence of the sense strand in the double-stranded siRNA analog includes SEQ ID NO: 5 or SEQ ID NO: 18, SEQ ID NO: 3 or SEQ ID NO: 16, SEQ ID NO: 14 or SEQ ID NO: 27, SEQ ID NO: 13 or SEQ ID NO: 26, or the sequence shown in SEQ ID NO: 12 or SEQ ID NO: 25, or consisting of them.
  • the sequence includes further nucleotide modifications, such as methoxy modification, fluoro modification, phosphorothioate linkage, and the like.
  • the sequence of the sense strand in the double-stranded siRNA analog includes SEQ ID NO: 5 or SEQ ID NO: 18, SEQ ID NO: 3 or SEQ ID NO: 16, SEQ ID NO: 14 or SEQ ID NO: 27, SEQ ID NO: 13 or SEQ ID NO: 26, SEQ ID NO: 12 or SEQ ID NO: 25, SEQ ID NO: 37 or SEQ ID NO: 42, or SEQ ID NO: 38 or SEQ ID NO: The sequence shown by or consisting of 43.
  • the sequence includes further nucleotide modifications, such as methoxy modification, fluoro modification, phosphorothioate linkage, replacement of nucleotides with (S)-glycerol nucleic acids, or nucleotides Replaced by (E)-vinyl phosphate modification, etc.
  • nucleotide modifications such as methoxy modification, fluoro modification, phosphorothioate linkage, replacement of nucleotides with (S)-glycerol nucleic acids, or nucleotides Replaced by (E)-vinyl phosphate modification, etc.
  • the sense strand and the antisense strand in the double-stranded siRNA analog include a sequence in which one or more nucleotide residues are replaced with r, such as the antisense strand SEQ ID NO: 2
  • the r substitution occurs at position 2 of the 5'end of SEQ ID NO: 2
  • the double-stranded siRNA analog is any one of S18 to S28:
  • S18 The sense strand is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense strand is SEQ ID NO: 4 or SEQ ID NO: 17,
  • the sense strand is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense strand is SEQ ID NO: 6 or SEQ ID NO: 19,
  • S20 The sense strand is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense strand is SEQ ID NO: 7 or SEQ ID NO: 20,
  • S21 The sense strand is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense strand is SEQ ID NO: 8 or SEQ ID NO: 21,
  • S22 The sense strand is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense strand is SEQ ID NO: 9 or SEQ ID NO: 22,
  • S23 The sense strand is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense strand is SEQ ID NO: 10 or SEQ ID NO: 23,
  • S24 The sense strand is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense strand is SEQ ID NO: 11 or SEQ ID NO: 24,
  • the sense strand is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense strand is SEQ ID NO: 29 or SEQ ID NO: 33,
  • S26 The sense strand is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense strand is SEQ ID NO: 30 or SEQ ID NO: 34,
  • the sense strand is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense strand is SEQ ID NO: 31 or SEQ ID NO: 35,
  • S28 The sense strand is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense strand is SEQ ID NO: 32 or SEQ ID NO: 36.
  • the double-stranded siRNA analog is any one of S1 to S17:
  • the sense strand is SEQ ID NO: 3 or SEQ ID NO: 16
  • the antisense strand is SEQ ID NO: 4 or SEQ ID NO: 17,
  • S2 The sense strand is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense strand is SEQ ID NO: 4 or SEQ ID NO: 17,
  • S3 The sense strand is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense strand is SEQ ID NO: 6 or SEQ ID NO: 19,
  • S4 The sense strand is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense strand is SEQ ID NO: 6 or SEQ ID NO: 19,
  • S5 The sense strand is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense strand is SEQ ID NO: 7 or SEQ ID NO: 20,
  • S6 The sense strand is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense strand is SEQ ID NO: 7 or SEQ ID NO: 20,
  • the sense strand is SEQ ID NO: 3 or SEQ ID NO: 16
  • the antisense strand is SEQ ID NO: 8 or SEQ ID NO: 21,
  • S8 The sense strand is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense strand is SEQ ID NO: 8 or SEQ ID NO: 21,
  • the sense strand is SEQ ID NO: 3 or SEQ ID NO: 16
  • the antisense strand is SEQ ID NO: 9 or SEQ ID NO: 22
  • the sense strand is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense strand is SEQ ID NO: 9 or SEQ ID NO: 22,
  • the sense strand is SEQ ID NO: 3 or SEQ ID NO: 16
  • the antisense strand is SEQ ID NO: 10 or SEQ ID NO: 23
  • S12 The sense strand is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense strand is SEQ ID NO: 10 or SEQ ID NO: 23,
  • the sense strand is SEQ ID NO: 3 or SEQ ID NO: 16
  • the antisense strand is SEQ ID NO: 11 or SEQ ID NO: 24,
  • S14 The sense strand is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense strand is SEQ ID NO: 11 or SEQ ID NO: 24,
  • the sense strand is SEQ ID NO: 12 or SEQ ID NO: 25, and the antisense strand is SEQ ID NO: 4 or SEQ ID NO: 17,
  • S16 The sense strand is SEQ ID NO: 13 or SEQ ID NO: 26, and the antisense strand is SEQ ID NO: 4 or SEQ ID NO: 17,
  • S17 The sense strand is SEQ ID NO: 14 or SEQ ID NO: 27, and the antisense strand is SEQ ID NO: 4 or SEQ ID NO: 17.
  • the double-stranded siRNA analog is any one of S29 to S35:
  • S29 The sense strand is SEQ ID NO: 37 or SEQ ID NO: 42, and the antisense strand is SEQ ID NO: 10 or SEQ ID NO: 23,
  • the sense strand is SEQ ID NO: 38 or SEQ ID NO: 43
  • the antisense strand is SEQ ID NO: 10 or SEQ ID NO: 23
  • the sense strand is SEQ ID NO: 3 or SEQ ID NO: 16
  • the antisense strand is SEQ ID NO: 39 or SEQ ID NO: 44
  • S32 The sense strand is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense strand is SEQ ID NO: 10 or SEQ ID NO: 45,
  • S33 The sense strand is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense strand is SEQ ID NO: 40 or SEQ ID NO: 46,
  • S34 The sense strand is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense strand is SEQ ID NO: 10 or SEQ ID NO: 47,
  • S35 The sense strand is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense strand is SEQ ID NO: 10 or SEQ ID NO: 48.
  • the double-stranded siRNA analog is selected from: the sense strand is SEQ ID NO: 3, the antisense strand is SEQ ID NO: 4, the sense strand is SEQ ID NO: 5, and the antisense strand is SEQ ID NO:4, the sense chain is SEQ ID NO: 3 and the antisense strand is SEQ ID NO: 6, the sense strand is SEQ ID NO: 5 and the antisense strand is SEQ ID NO: 6, the sense strand is SEQ ID NO: 3 And the antisense strand is SEQ ID NO: 7, the sense strand is SEQ ID NO: 5, the antisense strand is SEQ ID NO: 7, the sense strand is SEQ ID NO: 3, and the antisense strand is SEQ ID NO: 8, sense The chain is SEQ ID NO: 5, the antisense chain is SEQ ID NO: 8, the sense chain is SEQ ID NO: 3, the antisense chain is SEQ ID NO: 9, the sense chain is SEQ ID NO: 5, and the antisense chain is SEQ ID NO:
  • the sense strand is SEQ ID NO:1 and the antisense strand is SEQ ID NO: 11, the sense strand is SEQ ID NO: 1 and the antisense strand is SEQ ID NO: 29, and the sense strand is SEQ ID NO: 1 and The antisense strand is SEQ ID NO: 30, the sense strand is SEQ ID NO: 1 and the antisense strand is SEQ ID NO: 31, the sense strand is SEQ ID NO: 1 and the antisense strand is SEQ ID NO: 32, the sense strand Is SEQ ID NO: 37 and the antisense strand is SEQ ID NO: 10, the sense strand is SEQ ID NO: 38 and the antisense strand is SEQ ID NO: 1 0.
  • the sense strand is SEQ ID NO: 3 and the antisense strand is SEQ ID NO: 39, the sense strand is SEQ ID NO: 3 and the antisense strand is SEQ ID NO: 10, or the sense strand is SEQ ID NO: 3 and The antisense strand is SEQ ID NO: 40, and each of the nucleotide and r in the double-stranded siRNA analog is independently modified or unmodified.
  • the double-stranded siRNA analog is selected from:
  • Each of the nucleotide and r in the double-stranded siRNA analog is independently modified or unmodified.
  • the double-stranded siRNA analog is selected from: the sense strand is SEQ ID NO: 16, the antisense strand is SEQ ID NO: 17, the sense strand is SEQ ID NO: 18, and the antisense strand is SEQ ID NO: 17, the sense strand is SEQ ID NO: 16 and the antisense strand is SEQ ID NO: 19, the sense strand is SEQ ID NO: 18, and the antisense strand is SEQ ID NO: 19, and the sense strand is SEQ ID NO: 16
  • the antisense strand is SEQ ID NO: 20
  • the sense strand is SEQ ID NO: 18, the antisense strand is SEQ ID NO: 20
  • the sense strand is SEQ ID NO: 16 and the antisense strand is SEQ ID NO: 21
  • the sense The chain is SEQ ID NO: 18 and the antisense strand is SEQ ID NO: 21, the sense chain is SEQ ID NO: 16 and the antisense strand is SEQ ID NO: 22, the sense chain is SEQ ID NO: 18 and
  • the sense strand is SEQ ID NO: 28 and the antisense strand is SEQ ID NO: 24, the sense strand is SEQ ID NO: 28, the antisense strand is SEQ ID NO: 33, and the sense strand is SEQ ID NO: 28 and The antisense strand is SEQ ID NO: 34, the sense strand is SEQ ID NO: 28, the antisense strand is SEQ ID NO: 35, the sense strand is SEQ ID NO: 28, and the antisense strand is SEQ ID NO: 36, the sense strand Is SEQ ID NO: 42 and the antisense strand is SE Q ID NO: 23, the sense strand is SEQ ID NO: 43, the antisense strand is SEQ ID NO: 23, the sense strand is SEQ ID NO: 16 and the antisense strand is SEQ ID NO: 44, the sense strand is SEQ ID NO: 16 and the antisense strand are SEQ ID NO: 45, the sense strand is SEQ ID NO: 16 and the antisense strand is SEQ ID NO
  • the double-stranded siRNA analog is selected from:
  • the present disclosure provides a conjugate of a double-stranded siRNA analog, which comprises the double-stranded siRNA analog according to the first aspect of the present disclosure, and a pharmaceutically acceptable conjugated with the double-stranded siRNA analog Conjugation group.
  • the pharmaceutically acceptable conjugating group in the conjugate of the double-stranded siRNA analog contains 1 to 5 GalNAc (N-acetylgalactosamine) groups.
  • the pharmaceutically acceptable conjugating group contains 1, 2, 3, 4 or 5 GalNAc groups. More preferably, the pharmaceutically acceptable conjugating group contains 3 or 4 GalNAc groups.
  • the pharmaceutically acceptable conjugating group in the conjugate of the double-stranded siRNA analog comprises compound group D
  • the pharmaceutically acceptable conjugating group in the conjugate of the double-stranded siRNA analog is attached to the 3' end of the sense strand of the double-stranded siRNA analog.
  • the phosphorothioate portion of a double-stranded siRNA analog or a conjugate of a double-stranded siRNA analog includes (R)- and (S)-enantiomers, diastereomers, and/ Or its racemic mixture.
  • the conjugate of the double-stranded siRNA analog is selected from:
  • the D is as shown before.
  • the present disclosure provides a double-stranded siRNA analog according to the first aspect of the present disclosure or a salt of a conjugate of the double-stranded siRNA analog according to the second aspect of the present disclosure.
  • the salt as described above is selected from base addition salts, acid addition salts, and combinations thereof.
  • the base addition salt is selected from sodium, potassium, calcium, ammonium, organic amine, magnesium salt, and combinations thereof
  • the acid addition salt is selected from inorganic acid salts, organic acid salts, and combinations thereof.
  • the inorganic acid is selected from hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and Combination
  • organic acid is selected from acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzene Sulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and combinations thereof.
  • the present disclosure provides a pharmaceutical composition comprising a double-stranded siRNA analog according to the first aspect of the present disclosure, a conjugate of a double-stranded siRNA analog according to the second aspect of the present disclosure, or according to the present disclosure
  • the disclosed salt of the third aspect and a pharmaceutically acceptable carrier or excipient.
  • the present disclosure provides a double-stranded siRNA analog according to the first aspect of the present disclosure, a conjugate of a double-stranded siRNA analog according to the second aspect of the present disclosure, or a conjugate according to the third aspect of the present disclosure Salt, or the pharmaceutical composition according to the fourth aspect of the present disclosure, its use in the preparation of a medicament for the treatment of hepatitis B.
  • the present disclosure provides a double-stranded siRNA analog according to the first aspect of the present disclosure, a conjugate of a double-stranded siRNA analog according to the second aspect of the present disclosure, or according to the third aspect of the present disclosure Or the pharmaceutical composition according to the fourth aspect of the present disclosure, which is used to treat hepatitis B in a subject.
  • the present disclosure provides a method for treating hepatitis B virus in a subject, which comprises combining the double-stranded siRNA analog according to the first aspect of the present disclosure to the double-stranded siRNA according to the second aspect of the present disclosure.
  • the present disclosure provides a double-stranded siRNA analog according to the first aspect of the present disclosure for use in the treatment of hepatitis B in a subject, and a conjugate of the double-stranded siRNA analog according to the second aspect of the present disclosure Compound, or a salt according to the third aspect of the present disclosure, or a pharmaceutical composition according to the fourth aspect of the present disclosure.
  • the hepatitis B may be at any stage of the disease.
  • acute hepatitis B chronic hepatitis B, or liver cirrhosis or liver cancer caused by hepatitis B virus infection.
  • the hepatitis B is chronic hepatitis B.
  • the term "comprises, includes, and contains” or equivalents is an open-ended expression, meaning that in addition to the listed elements, components or steps, other unspecified elements, Components or steps.
  • HBV gene refers to a gene whose DNA sequence is as shown in Genbank registration number NC_003977.1.
  • Genbank registration number NC_003977.1 is the complete genome of HBV.
  • double-stranded siRNA analogs can target the X open reading frame (X opening reading frame, X ORF) of HBV.
  • a double-stranded siRNA analog refers to a complex of ribonucleic acid molecules, which has a double-stranded structure and contains two antiparallel and substantially complementary nucleic acid strands, which have "sense” and “sense” relative to the target RNA. Antisense” targeting.
  • “complementary” has a well-known meaning to those skilled in the art, that is, in a double-stranded nucleic acid molecule, bases on one strand are paired with bases on the other strand in a complementary manner.
  • the purine base adenine (A) is always paired with the pyrimidine base uracil (U); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G).
  • Each base pair includes a purine and a pyrimidine.
  • capital letters C, G, U, and A represent the base composition of nucleotides.
  • Lowercase letters c, g, u, and a respectively indicate that the nucleotides represented by their corresponding capital letters are modified by methoxy; underscores indicate that the nucleotides represented by capital letters are modified by fluorine; the interval " ⁇ " indicates the same as the interval " ⁇ ”
  • the two adjacent nucleotide residues on the left and right are connected by phosphorothioate groups;
  • VP indicates that the nucleotide on the right side of the letter VP is (E)-vinyl phosphate modified nucleotide .
  • "a ⁇ g” means that the residues a and g are connected by phosphorothioate groups.
  • the "modifications" of the nucleotides described in the present disclosure include, but are not limited to, methoxy modification, fluoro modification, (E)-vinyl phosphate modification, phosphorothioate linkage, or replacement of nucleotides with (S )-Glycerol nucleic acid, etc.
  • the sequences described in the present disclosure may include the "further modified sequences” listed in Table 1 below.
  • the fluoro-modified nucleotides described in the present disclosure refer to nucleotides formed by substituting fluorine for the hydroxyl group at the 2'position of the ribose group of the nucleotide, and the methoxy-modified nucleotides refer to the 2'-hydroxyl group of the ribose group. Nucleotides formed by substitution with methoxy groups.
  • the (E)-vinyl phosphate modified nucleotides described in the present disclosure represent the following structural units:
  • X is selected from OCH 3 and F.
  • the (S)-glycerol nucleic acid (Agn) described in the present disclosure represents the following structural units:
  • (Agn) and other nucleotide residues are connected to each other through phosphate or phosphorothioate, such as "a ⁇ (Agn)” means that a and (Agn) residues are connected through phosphorothioate group, "a( Agn)” means that a and (Agn) residues are connected by a phosphate group.
  • a double-stranded siRNA analog comprises a sense strand or an r' embedded sense strand and an r' embedded antisense strand.
  • the sense strand, r'-embedded sense strand and r'-embedded antisense strand all contain nucleotide groups as basic structural units. Those skilled in the art know that the nucleotide group contains a phosphate group, a ribose group and a base, which will not be repeated here.
  • the r'-embedded sequence in the present disclosure refers to the presence of at least one nucleotide residue in the sequence that is connected to r, including as in the sequence (such as SEQ ID NO: 2) using r to replace a nucleotide residue The sequence of the base.
  • the r'-embedded sequence described in the present disclosure includes but is not limited to: r'-embedded double-stranded siRNA, r'-embedded sense strand, and r'-embedded antisense strand. For example, 5’-aGUrrA ⁇ C-3’, 5’-rGgAAC-3’ and 5’-AG ⁇ UrAAcCuCr-3’ all belong to the case of r’ embedding.
  • the r'embedded double-stranded siRNA in the present disclosure refers to the presence of at least one nucleotide residue connected to r in the double-stranded siRNA, including, for example, the double-stranded siRNA sequence where r is used to replace one nucleotide residue. Double-stranded siRNA.
  • the r'embedded sense strand in the present disclosure refers to the presence of at least one nucleotide residue connected to r in the sense strand, including, for example, the replacement of one or more nucleotides in the sense strand by r.
  • the r'embedded antisense strand in the present disclosure refers to the presence of at least one nucleotide residue connected to r in the antisense strand, including the replacement of one or more nucleotides in the antisense strand by r.
  • r'is (Wherein X is selected from SH and OH), it is an analog of natural nucleotide base, which is different from any published patent natural nucleotide base, and the introduction of nucleic acid sequence brings unexpected activity.
  • r represents the following structural unit:
  • R and other nucleotide residues are connected to each other through phosphate or phosphorothioate.
  • a ⁇ r means that a and r residues are connected by phosphorothioate
  • ar means a and r residues. They are connected by phosphate groups.
  • the “plurality” in the present disclosure refers to an integer greater than or equal to 2, including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19 or 20, up to the theoretical upper limit of the siRNA analog.
  • the sense strand or antisense strand of the double-stranded siRNA analog may also include "overhangs", for example, unpaired overhanging nucleotides that do not directly participate in the RNA double helix structure, where the RNA double helix structure is usually defined by this article.
  • the defined "sense strand” and “antisense strand” are formed as a pair.
  • Such overhangs may include one or more modified or unmodified U, T, and A.
  • the SEQ ID NO: 2 may include modified or unmodified UU overhangs in the 5'and/or 3'segment.
  • a conjugate of a double-stranded siRNA analog is a compound formed by connecting a double-stranded siRNA analog and a pharmaceutically acceptable conjugating group, and the double-stranded siRNA analog and a pharmaceutically acceptable conjugating group Covalently connected.
  • a pharmaceutically acceptable conjugating group can be attached to the sense strand of a double-stranded siRNA analog or the 3'end of the sense strand with r'embedded.
  • a pharmaceutically acceptable conjugating group includes a pharmaceutically acceptable targeting molecule and an optional linker.
  • exemplary types of conjugating groups, linkers, and targeting molecules can be found in the disclosure of WO2015006740A2.
  • Exemplary conjugating groups include, but are not limited to, L96 or compound group D.
  • conjugate means that two or more chemical moieties each having a specific function are connected to each other in a covalent manner; correspondingly, “conjugate” is Refers to the compound formed by covalent linkage between the various chemical moieties.
  • the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. This disclosure contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomers Body-enriched mixtures, all of these mixtures are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All these isomers and their mixtures are included in the scope of the present disclosure.
  • enantiomers or “optical isomers” refer to stereoisomers that are mirror images of each other.
  • diastereomer refers to a stereoisomer in which a molecule has two or more chiral centers and the relationship between the molecules is non-mirror mirror image.
  • wedge-shaped solid line keys And wedge-shaped dashed key Represents the absolute configuration of a three-dimensional center, with a straight solid line key And straight dashed key Indicates the relative configuration of the three-dimensional center, using wavy lines Represents a wedge-shaped solid line key Or wedge-shaped dashed key Or use wavy lines Represents a straight solid line key And/or straight dashed key
  • the terms “enriched in one isomer”, “enriched in isomers”, “enriched in one enantiomer” or “enriched in enantiomers” refer to one of the isomers or pairs of
  • the content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or 96% or greater, or 97% or greater, or 98% or greater, or 99% or greater, or 99.5% or greater, or 99.6% or greater, or 99.7% or greater, or 99.8% or greater, or greater than or equal 99.9%.
  • the term “isomer excess” or “enantiomeric excess” refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80% .
  • optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one wants to obtain an enantiomer of a compound of the present disclosure, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure The desired enantiomer.
  • the molecule when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), it forms a diastereomeric salt with a suitable optically active acid or base, and then passes through a conventional method known in the art The diastereoisomers are resolved, and then the pure enantiomers are recovered.
  • the separation of enantiomers and diastereomers is usually accomplished through the use of chromatography, which employs a chiral stationary phase and is optionally combined with chemical derivatization (for example, the formation of amino groups from amines). Formate).
  • the compounds of the present disclosure may contain unnatural proportions of atomic isotopes on one or more of the atoms constituting the compound.
  • compounds can be labeled with radioisotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C).
  • deuterium can be substituted for hydrogen to form deuterated drugs.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon.
  • deuterated drugs have reduced toxic side effects and increased drug stability. , Enhance the efficacy, prolong the biological half-life of drugs and other advantages. All changes in the isotopic composition of the compounds of the present disclosure, whether radioactive or not, are included in the scope of the present disclosure.
  • salt refers to a salt of the compound of the present disclosure, which is prepared from a compound with a specific substituent found in the present disclosure and a relatively non-toxic acid or base.
  • a base addition salt can be obtained by contacting the compound with a sufficient amount of base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salt or similar salts.
  • the acid addition salt can be obtained by contacting the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, the organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.) , And salts of organic acids such as glucuronic acid. Certain specific compounds of the present disclosure contain basic and
  • the salt of the present disclosure can be synthesized from a parent compound containing an acid radical or a base by conventional chemical methods.
  • such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of both.
  • the compounds of the present disclosure can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and those well known to those skilled in the art Equivalent alternatives, and preferred implementations include but are not limited to the embodiments of the present disclosure.
  • the solvent used in the present disclosure is commercially available.
  • the ratios of solvents used in column chromatography and thin-layer silica gel chromatography of the present disclosure are all volume ratios.
  • the compound is used according to the conventional naming principle in the field or The software is named, and the commercially available compounds use the supplier catalog name.
  • Figure 7 The effect of WR007 and WR012 on plasma HBeAg in AAV/HBV mice.
  • Figure 8 The effect of WR007 and WR012 on plasma HBV DNA in AAV/HBV mice.
  • FIG. 1 Concentrations of WRG01 in mouse plasma, liver, and kidney.
  • Step A (2S,3R,4R,5R,6R)-3-acetylamino-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-clawyl triacetate ( Formula 1-1) (30 g, 94.26 mmol) and methyl 1,2,4-triazole-3-carboxylate (11.98 g, 94.26 mmol) dissolved in methyl acetate (220 mL) , Concentrate to nearly complete dryness in an oil bath at 90 degrees Celsius at a pressure of 1 bar.
  • a methyl acetate solution (2 mL) of trifluoromethanesulfonic acid (141.46 mg, 0.94 mmol) was added to the mixture and stirred in an oil bath at 125 degrees Celsius under a pressure of 30 mbar for 4 hours.
  • the reaction solution was cooled to 70 degrees Celsius, ethanol (70 ml) was added, stirred at 70 degrees Celsius until a homogeneous solution was formed, and the stirring was stopped and cooled to 50 degrees Celsius. After the precipitate formed, it was allowed to stand and cool to 25 degrees Celsius and the reaction solution was placed at 0 degrees Celsius for 16 hours.
  • Step B The compound represented by formula 1-2 (15 g, 38.93 mmol) and triethylamine (4.14 g, 40.87 mmol) were dissolved in methanol (100 mL). The mixture was stirred at 50 degrees Celsius for 17 hours under the protection of nitrogen. The reaction solution was concentrated under reduced pressure to obtain 1-3.
  • Step C Dissolve the compound represented by formula 1-3 (10 g, 38.58 mmol) in pyridine (250 ml) at 0 degrees Celsius and add dropwise 1,3-dichloro-1,1,3,3-tetraisopropyl Disiloxane (12.29 g, 38.97 mmol). The mixture was gradually heated to 25 degrees Celsius and stirred for 16 hours. The reaction solution was concentrated under reduced pressure, and the concentrate was suspended in ethyl acetate (250 mL) and filtered through a Buchner funnel.
  • Step D To N, N- containing the compound represented by formula 1-4 (8.23 g, 16.40 mmol), potassium carbonate (11.34 g, 82.02 mmol) and silver (I) (19.01 g, 82.02 mmol) Add methyl iodide (11.64 g, 82.02 mmol) to the mixture of dimethylformamide (50 mL), and stir at 25 degrees Celsius for 3 hours. The reaction solution was diluted with ethyl acetate (300 mL) and filtered through a Buchner funnel.
  • Step F Add 4,4-dimethoxytrityl chloride (2.42 g, 7.14 mmol) and stirred at 25 degrees Celsius for 16 hours. After the reaction solution was diluted with ethyl acetate (70 mL), it was quenched with saturated sodium bicarbonate aqueous solution (20 mL) at 25 degrees Celsius and diluted with water (40 mL). After liquid separation, the combined organic phase was washed with 60 ml of water (60 ml ⁇ 1) and 60 ml of saturated brine (60 ml ⁇ 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product.
  • Step A Dissolve 11-dodecyn-1-ol (25 g, 137.14 mmol) and triethylamine (16.65 g, 164.56 mmol) in dichloromethane (250 mL), add methanesulfonyl chloride at 0°C (18.85 g, 164.56 mmol). The mixture was stirred at 0 degrees Celsius for 2 hours. The reaction solution was diluted with water (400 mL) and extracted with 800 mL of dichloromethane (400 mL ⁇ 2). The combined organic phase was washed with 400 ml of water (200 ml ⁇ 2) and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 2-2.
  • Step B The compound represented by formula 2-3 (20 g, 67.26 mmol) was dissolved in N,N-dimethylformamide (200 mL) and sodium hydride (60% purity, 4.04 g, 100.89 mmol) was added at 0°C. ), and then add the compound represented by formula 2-2 (19.27 g, 73.99 mmol). The mixture was stirred at 25 degrees Celsius for 16 hours. The reaction solution was quenched with water (1 liter), and extracted with 1.6 liters of dichloromethane (800 mL ⁇ 2). The combined organic phase was washed with 800 ml (800 ml ⁇ 1) saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 2-4.
  • sodium hydride 50% purity, 4.04 g, 100.89 mmol
  • Step C The compound represented by formula 2-4 (48 g, 103.98 mmol) was dissolved in methanol (870 mL), and methanol solution of hydrogen chloride (4 mol per liter, 400 mL, 1.6 mol) was added. The mixture was stirred at 30 degrees Celsius for 2 hours. A methanol solution of hydrogen chloride (4 moles per liter, 350 ml, 1.4 moles) was added to the reaction solution. The mixture was stirred at 30 degrees Celsius for 16 hours. The reaction solution was concentrated under reduced pressure, 200 mL of chloroform (100 mL ⁇ 2) was added, and concentrated under reduced pressure until a white solid appeared.
  • Step D To a mixture of the compound represented by formula 2-5 (23 g, 80.58 mmol) and sodium hydroxide (322.31 mg, 8.06 mmol) in dimethyl sulfoxide (70 mL) and water (6 mL) Add tert-butyl acrylate (22.72 g, 177.28 mmol), and stir at 25 degrees Celsius for 16 hours under the protection of nitrogen. The reaction solution was diluted with water (500 mL), and extracted with 1 liter of ethyl acetate (500 mL ⁇ 2). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product.
  • Step E To a dichloromethane (250 ml) solution of the compound represented by formula 2-6 (24.5 g, 45.22 mmol) was added triethylamine (9.15 g, 90.45 mmol) and succinic anhydride (6.79 g, 67.83) Millimoles) and stirred at 20 degrees Celsius for 16 hours. Dichloromethane (1 liter) and hydrochloric acid (1 mole per liter, 1 liter) were added to the reaction solution, the separated organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 2-7.
  • Step F The compound represented by formula 2-7 (27.4 g, 42.69 mmol) was dissolved in formic acid (140 ml), and the mixture was stirred at 20 degrees Celsius for 16 hours under the protection of nitrogen. The reaction solution was concentrated under reduced pressure, 300 ml of toluene (150 ml ⁇ 2) was added, and the mixture was concentrated under reduced pressure to obtain 2-8.
  • Step G Combine the compound represented by formula 2-8 (22.6 g, 42.67 mmol), N,N-diisopropylethylamine (33.09 g, 256.03 mmol) and O-(7-azabenzotriazole) -1-yl)-N,N,N,N-tetramethylurea hexafluorophosphate (51.92g, 136.55mmol) was dissolved in N,N-dimethylformamide (250ml), and N-( Tert-Butyl 3-aminopropyl) carbamate (29.74 g, 170.69 mmol). The mixture was stirred at 20 degrees Celsius for 16 hours.
  • Step I The compound represented by formula 2-11 (22.15 g, 49.50 mmol), N,N-diisopropylethylamine (7.75 g, 60.00 mmol), 1-hydroxy-7-azabenzotriazole (6.12 g, 45.00 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphate (20.53 g, 54.00 mmol) Dissolve in N,N-dimethylformamide (90ml), add the compound represented by formula 2-10 (tris(trifluoroacetate), 15.6g, 15.00mmol) and N,N- A solution of diisopropylethylamine (21.32 g, 165.00 mmol) in N,N-dimethylformamide (120 mL).
  • Step J The compound represented by formula 2-12 (1.00 g, 0.50 mmol) and N-methyl-N,N,N-tri-n-octylammonium chloride (20.35 mg, 50.35 micromol) were dissolved in acetic acid ( 2.7 mL) and n-pentane (6.3 mL), and a solution of potassium permanganate (0.40 g, 2.52 mmol) in water (9 mL) was dropped into the mixture at 0 degrees Celsius. The mixture was stirred at 0 to 15 degrees Celsius for 2 hours.
  • Step K Add N,N-diisopropylethylamine (0.26 g, 1.99 Millimoles) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphate (0.23 g, 0.60 mmol). After the mixture was stirred, the compound represented by formula 2-14 (0.23 g, 0.55 mmol) was added. The mixture was stirred at 15 degrees Celsius for 16 hours. Add dichloromethane (50ml) and water (50ml) to the reaction solution, and use 50ml saturated sodium bicarbonate aqueous solution (50ml ⁇ 1), 50ml water (50ml ⁇ 1) and saturated salt for the organic phase after separation.
  • dichloromethane 50ml
  • water 50ml
  • Step L To the dichloromethane (8 mL) solution of the compound represented by formula 2-15 (0.80 g, 0.33 mmol) was sequentially added triethylamine (67.24 mg, 0.64 mmol), 4-N,N-dimethyl Aminopyridine (0.12 g, 1.00 mmol) and succinic anhydride (83.13 mg, 0.83 mmol). The mixture was stirred at 10 degrees Celsius for 16 hours.
  • oligoribonucleotides are synthesized. In controllable porous glass (amino CPG, ) And D01 are synthesized on a solid support made by covalent bonding. All 2'-modified RNA phosphoramidites and auxiliary reagents are commercially available reagents. All amides are dissolved in anhydrous acetonitrile and molecular sieve is added The coupling time using 5-ethylsulfide-1H-tetrazole (ETT) as the activator was 5 minutes.
  • ETT 5-ethylsulfide-1H-tetrazole
  • oligomers purified by HPLC using NanoQ anion exchange oligomers purified by HPLC using NanoQ anion exchange.
  • Buffer A is a 10 mM sodium perchlorate solution, 20 mM Tris, 1 mM EDTA, pH 7.4 and contains 20% acetonitrile
  • buffer B 500 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4 and contains 20% acetonitrile.
  • the target product was separated and desalted with a reversed-phase C18 column.
  • Annealing of single-stranded oligoribonucleotides produces siRNA:
  • the single-stranded oligoribonucleotides to be annealed are formulated to 200 ⁇ M with sterile RNase Free H 2 O (no RNA hydrolase).
  • Set up the annealing reaction system as follows, place the mixed solution with a total volume of 100 ⁇ L and 10nmol in a 95°C water bath for 10 minutes ( ⁇ 100nmol demand requires high temperature for 20 minutes) ⁇ quickly put it in a 60°C water bath and cool down naturally ⁇ the solution after annealing is not allowed Store at high temperature.
  • Complementary strands are formed by combining equimolar solutions of single-stranded oligoribonucleotides.
  • D is the residue after the chemical reaction of the small molecule fragment D01, which binds to the nucleic acid through a covalent bond, and its structure is as follows:
  • the antisense strand sequence of r'embedded sequence is obtained by r'embedding on the basis of the antisense strand sequence with 3'UU of the core sequence.
  • SEQ ID NO: 4 is obtained by r'embedding on the basis of SEQ ID NO: 2 with 3'UU.
  • g ⁇ r ⁇ gu G c ACU ucgcuucacaD ('-3') represents the sequence shown in SEQ ID NO: 16 g ⁇ r ⁇ gu G c ACU ucgcuucaca connected to D at the 3'end.
  • HBV antigens HBsAg and HBeAg
  • ELISA enzyme-linked immunosorbent assay
  • HepG2-NTCP cell culture medium DMEM, Invitrogen-11330032; 10% serum, Invitrogen-10099141; 100units/ml penicillin and 100 ⁇ g/ml streptomycin, Hyclone-SV30010; 1% non-essential amino acids, Invitrogen-11140050; 2mM L- Glutamine, Invitrogen-25030081; 1mM sodium pyruvate, Gibco-11360-070; 500 ⁇ g/ml Geneticin, Invitrogen-10131027)
  • Pancreatin Invitrogen-25300062; DPBS (Corning-21031CVR); DMSO (Sigma-D2650-100ML); Cell-titer Glo (Promega-G7573); Hepatitis B surface antigen quantitative detection kit (Antu Bio-CL 0310 ); Hepatitis B e antigen quantitative detection kit (Antu Bio-CL 0312).
  • HepG2-NTCP 7.5 ⁇ 10 4 cells/well
  • HepG2-NTCP 2000GE/cell
  • type D HBV concentrated from HepG2.2.15 cell culture supernatant
  • RNAiMax RNAiMax
  • transfection of siRNA conjugate was diluted 5 times in 7 concentrations, and the final concentration was 0.16pM.
  • the compound is a combination of the sense strand and the antisense strand, and is a single chemical entity with a maximum concentration of 2.5 nM.
  • hepatitis B virus surface antigen HBsAg
  • e antigen HBeAg
  • the specific steps refer to the product manual, the steps are briefly described as follows: take 50 ⁇ l sample and standard substance into the reaction plate, and then add 50 ⁇ l enzyme binding to each well. Shake and mix, incubate at 37°C for 60 minutes, then wash the plate 5 times with washing solution, then add 50 ⁇ l of luminescent substrate to each well, mix, and react for 10 minutes at room temperature in the dark, and finally detect the chemiluminescence intensity with a microplate reader.
  • %Viability (luminescence value of sample-luminescence value of medium control)/(luminescence value of DMSO control-luminescence value of medium control) ⁇ 100.
  • %Inh. (1-Antigen value in sample/DMSO control antigen value) ⁇ 100.
  • the tested sample is a conjugate of double-stranded siRNA analog.
  • Example 4 Study on anti-hepatitis B virus activity and safety in hepatitis B virus mouse model (AAV-HBV) mediated by recombinant type 8 adeno-associated virus vector
  • AAV vector-mediated HBV transfection mouse model is a fast and efficient HBV model.
  • the recombinant type 8 adeno-associated virus (rAAV8-1.3HBV) carrying 1.3 copies of HBV genome can be injected into the tail vein of mice, and the carried 1.3 copies of HBV genome can be efficiently introduced into hepatocytes. Due to the characteristics of the AAV virus vector, the vector mediated by it can continue to express for a long time.
  • the application of the AAV/HBV model can continuously replicate HBV DNA and express HBsAg and HBeAg in the mouse liver.
  • the HBsAg, HBeAg, DNA, pgRNA and mouse body weight in the serum of the mice treated with the test compound were detected to evaluate the anti-HBV effect and safety in vivo.
  • the main reagents of this project include QIAamp96 DNA kit (Qiagen, 51162), FastStart Universal Probe Master (Rox) (Roche, 04914058001), hepatitis B virus surface antigen detection kit (Antu biological, CL0310), hepatitis B virus e Antigen detection kit (Antu Biologics, CL0918), PureLink TM Pro 96 Viral RNA/DNA kit (Invitrogen, 12280-096A) and FastQuant RT Kit (with gDNase) (TIANGEN, KR106-02).
  • the main instruments include: centrifuge (Beckman Allegra X-15R), multifunctional microplate reader (BioTek, Synergy 2), fluorescent quantitative PCR machine (Applied Biosystems, 7900HT Fast Real-time PCR system), microplate reader (Molecular Devices, SpectraMax 340PC384).
  • mice started subcutaneous injection on the 34th day after the virus injection, and this day was set to day 0. Blood was collected from all mice submandibular before administration to collect plasma. See Table 3 for the specific dosage regimen.
  • the collected blood samples were anticoagulated with K 2 -EDTA and centrifuged for 10 minutes at 4°C and 7000 g/min. Collect plasma. See Table 3 for specific blood collection time.
  • mice On the 35th or 42nd day, all mice were blood collected through the submandibular vein to collect plasma, and then the mice were euthanized by CO 2 inhalation, blood samples were collected from the heart, and liver samples were collected.
  • WRG01 is a conjugate
  • the sense strand is SEQ ID NO: 16
  • the antisense strand is SEQ ID NO: 23
  • the conjugate group is D.
  • WR007 is a conjugate, its sense strand is SEQ ID NO: 42, antisense strand is SEQ ID NO: 23, and the conjugate group is D.
  • WR012 is a conjugate
  • the sense strand is SEQ ID NO: 16
  • the antisense strand is SEQ ID NO: 47
  • the conjugate group is D.
  • ELISA to detect the content of HBsAg and HBeAg in the serum of mice:
  • the experimental procedures refer to the instructions of the HBsAg ELISA (Antu Biological, CL 0310) and HBeAg ELISA (Antu Biological, CL0918) kits.
  • qPCR detection of HBV DNA content in mouse plasma extract HBV DNA from plasma, refer to QIAamp 96 DNA Blood Kit manual for experimental procedures, qPCR detects HBV DNA content in mouse plasma.
  • RT-qPCR detection of HBV pgRNA content in mouse plasma Extract HBV pgRNA from plasma, and refer to the instructions of PureLink TM Pro 96 Viral RNA/DNA Kit for the experimental procedures. Digest the DNA, and use 3'RACE primers containing hepatitis B virus-specific sequences to reverse transcribe RNA into cDNA. The experimental procedures refer to the instructions of the FastQuant RT Kit (with gDNase). Finally, qPCR quantitatively detects the content of cDNA, that is, detects the content of HBV pgRNA in mouse plasma.
  • HBsAg The content of HBsAg in the serum is evaluated for the anti-HBV activity of the test compound in the AAV/HBV mouse model. The results are shown in Table 4, Table 4-1, Figure 1 and Figure 6. The plasma HBsAg content of mice was determined by ELISA. Error bars show standard errors.
  • Day 0 The vehicle or compound is administered to all mice for the first time.
  • Day 29 WRG01 mice in the experimental group and the blank control group mice were inoculated with vehicle or compound for the second time.
  • HBeAg The content of HBeAg in the serum evaluates the anti-HBV activity of the test compound in the AAV/HBV mouse model. The results are shown in Table 5, Table 5-1, Figure 2 and Figure 7. Mouse plasma HBeAg content was determined by ELISA method. Error bars show standard errors. Day 0: The vehicle or compound is administered to all mice for the first time.
  • mice pgRNA content to evaluate the anti-HBV activity of the test compound in the AAV/HBV mouse model.
  • the results are shown in Table 7 and Figure 4.
  • the plasma HBVpgRNA content of mice was determined by quantitative PCR. Error bars show standard errors.
  • Day 0 The vehicle or compound is administered to all mice for the first time.
  • Day 29 All mice were inoculated with vehicle or compound for the second time.
  • test compound can significantly reduce HBsAg, DNA and pgRNA in the AAV/HBV mouse model experiment.
  • the test compound also has a certain inhibitory effect on HBeAg.
  • the mice showed good tolerance and gradually increased their body weight.
  • real-qPCR real-time quantitative qPCR test
  • ELISA enzyme-linked immunosorbent assay
  • detect the intracellular HBV RNA content by qRT-PCR The EC50 value of the compound is used as an index to evaluate the compound's inhibitory effect on HBV, and the CCK8 method is used to detect the effect of the test compound on cell activity.
  • HepG2.2.15 cell culture medium (DMEM/F12, Invitrogen-11330032; 10% serum, Hyclone-SV30087.0; 100units/ml penicillin and 100 ⁇ g/ml streptomycin, Hyclone-SV30010; 1% non-essential amino acids, Invitrogen-11140050 : 2mM L-GLUTAMINE, Invitrogen-25030081: 300 ⁇ g/ml Geneticin, Invitrogen-10131027).
  • DMEM/F12 Invitrogen-11330032
  • 10% serum Hyclone-SV30087.0
  • 100units/ml penicillin and 100 ⁇ g/ml streptomycin Hyclone-SV30010
  • 1% non-essential amino acids Invitrogen-11140050 : 2mM L-GLUTAMINE
  • Invitrogen-25030081 300 ⁇ g/ml Geneticin, Invitrogen-10131027
  • RNAiMAX (Invitrogen-13778-150): CCK8 (Li Ji-AC11L057); High-throughput DNA Purification Kit (QIAamp 96 DNA Blood Kit, Qiagen-51162); RNA Preparation RNEASY Kit (RNeasy 96 Kit (12), Qiagen -74182); Quantitative Quick Start Universal Probe Reagent (FastStart Universal Probe Master, Roche-04914058001); FastKing cDNA First Strand Synthesis Kit (TianGen-KR106-02); Hepatitis B Surface Antigen Quantitative Detection Kit (Antu Biology, CL0310); Hepatitis B e antigen quantitative detection kit (Antu Biology, CL 0312).
  • Collagen I 96 Well White/Clear Flat Bottom TC-Treated Microplate (Corning BioCoat-356650): CO 2 incubator (HERA-CELL-240); fluorescent quantitative PCR instrument (Applied Biosystems-7900 real time PCR system); fluorescent quantitative PCR Microplate reader (Applied Biosystems-QuantStudio 6 Flex); microplate reader (Molecular Device-SpectraMax M2e); microplate reader (BioTek-Synergy 2).
  • siRNA transfection and cell plating were carried out at the same time. The method is briefly described as follows: Take HepG2.2.15 cells, wash them with DPBS, digest with 0.05% trypsin, and terminate with DMEM/F12 medium containing 10% FBS Digest, resuspend after centrifugation, and count the cells after gently pipetting to single cells. Proportionally configure the required transfection reagent volume (Table 8), and incubate at room temperature for 15 minutes.
  • the PCR reaction conditions are: heating at 95°C for 10 minutes, then denaturation at 95°C for 15 seconds, and extension at 60°C for 1 minute, a total of 40 cycles.
  • ELISA measures the content of hepatitis B virus S antigen and e antigen.
  • the steps are briefly described as follows: Take 50 ⁇ L of sample and standard substance into the reaction plate, and then add 50 ⁇ L of enzyme conjugate to each well, shake and mix. Homogenize, incubate at 37°C for 60 minutes, then wash the plate with washing solution 5 times, then add 50 ⁇ L of luminescent substrate to each well, mix well, and react for 10 minutes in the dark at room temperature. Finally, check the chemiluminescence intensity with a microplate reader.
  • RNeasy 96 kit extraction kit Qiagen, 74182
  • kit instructions to extract HBV RNA from the cell culture.
  • the reverse transcription kit Tiangen, KR106
  • add random primers for reverse transcription into cDNA and then HBV-specific primers to detect total RNA in the sample.
  • GAPDH primers and probes specifically detect GAPDH cDNA
  • qPCR method Quantify the HBV cDNA in the sample.
  • the expression level of the target gene HBV mRNA of each sample was calculated by the ⁇ Ct relative quantification method.
  • the relative expression of the target gene is expressed as 2- ⁇ CT, and the calculation formula is as follows:
  • ⁇ CT average Ct value of target gene-average Ct value of internal reference gene
  • ⁇ CT ⁇ CT (medicated group) group- ⁇ CT (RNAiMAX control group);
  • %Inh. (1-value in sample/PBS control value) ⁇ 100.
  • Cell viability% (sample detection value-average detection value of culture fluid background) / (average detection value of control group-average detection value of culture fluid) ⁇ 100
  • the tested sample is a conjugate of double-stranded siRNA analog.
  • Example 6 Exploration of effective dose of anti-hepatitis B virus activity in AAV-HBV mouse model
  • the HBsAg in the serum of mice treated with different doses of the test compound was detected to evaluate its anti-HBV effect in vivo.
  • C57BL/6 mice, PBS (RNase free) is the vehicle, test compound, and recombinant virus rAAV8-1.3HBV.
  • the main reagents of this project include FastStart Universal Probe Master (Rox) (Roche, 04914058001), hepatitis B virus surface antigen detection kit (Antu Biologics, CL0310).
  • the main instruments include: centrifuge (Beckman Allegra X-15R), multi-function microplate reader (BioTek, Synergy 2), microplate reader (Molecular Devices, SpectraMax 340PC384).
  • mice started subcutaneous injection on day 34 after virus injection, and this day was set to day 0. Blood was collected from all mice submandibular before administration to collect plasma. Dosing once on day 0. The specific dosage regimen is shown in Table 14.
  • the collected blood sample was anticoagulated with K 2 -EDTA and centrifuged at 4°C and 7000 g/min for 10 Plasma was collected after minutes. See Table 11 for specific blood collection time.
  • mice On day 42, blood was collected from all mice through the submandibular vein to collect plasma. After that, the mice were euthanized by CO 2 inhalation, plasma samples were collected through heart blood sampling, and liver samples were collected.
  • WRG01 is a conjugate
  • the sense strand is SEQ ID NO: 16
  • the antisense strand is SEQ ID NO: 23
  • the conjugate group is D.
  • ELISA to detect the content of HBsAg in mouse serum The experimental procedures refer to the instructions of the HBsAg ELISA (Antu Biology, CL0310) kit.
  • HBsAg content in the serum to evaluate the anti-HBV activity of the test compound in the AAV/HBV mouse model.
  • the results are shown in Table 12 and Figure 9.
  • the plasma HBsAg content of mice was determined by ELISA. Error bars show standard errors.
  • Day 0 The vehicle or compound is administered to all mice for the first time.
  • test compound WRG01 showed a good dose-dependent effect on reducing HBsAg in the AAV/HBV mouse model experiment, that is, as the dose of the drug increased, the activity of HBsAg decreased and increased, and it exhibited long-term inhibitory effect on HBsAg.
  • Example 7 Test of drug concentration in mouse plasma, liver, and kidney
  • WRG01 is a conjugate
  • the sense strand is SEQ ID NO: 16
  • the antisense strand is SEQ ID NO: 23
  • the conjugate group is D.
  • the SL-qPCR method (reference: Nair et al. Nucleic Acids Research (2017), 45, 10969-10977) was used to detect the levels of siRNA in the plasma, liver, and kidney of mice at different time points after administration. The results are shown in Figure 10.
  • test compound WRG01 has good tissue distribution and metabolic stability in the C57BL/6 mouse model experiment.
  • WR-G01 has a large liver exposure, a long half-life, and a liver-to-blood ratio of more than 500 times, which proves that WRG01 is metabolically stable and has a high degree of liver targeting.
  • the humanized FRG mouse is one of the most commonly used humanized liver models, and the humanized ratio is usually as high as 70%. Because the human liver cells are colonized in the mouse liver, it can better simulate the human HBV natural infection and cccDNA replication process, and has a good predictive effect on human pharmacokinetics and liver toxicity.
  • WRG01 is a conjugate
  • the sense strand is SEQ ID NO: 16
  • the antisense strand is SEQ ID NO: 23
  • the conjugate group is D.
  • the present disclosure exhibits unpredictable and excellent HBsAg and HBeAg inhibitor activity, and can effectively inhibit the expression of HBV DNA and pgRNA, which shows that the activity of hepatitis B virus can be inhibited, and at the same time, it has good tissue distribution and metabolic stability. It has a high degree of liver targeting and is expected to have a small impact on the liver function of mice. It will provide an efficient treatment for hepatitis B in the clinic, such as chronic hepatitis B.

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Abstract

提供了一种利巴韦林衍生物嵌入的双链siRNA类似物、包含其的缀合物，以及其盐和用途。提供的双链siRNA类似物、包含其的缀合物以及其盐可以有效抑制乙肝病毒DNA、pgRNA、S抗原、E抗原等多项病毒指标，为乙型肝炎的治疗提供了有效可行的方法。

Description

双链siRNA类似物的缀合物
本申请主张如下优先权：
CN202010529520.7，申请日2020年06月11日；
CN202011524835.9，申请日2020年12月21日；
CN202010524584.8，申请日2020年06月10日；
PCT/CN2020/133982，申请日2020年12月4日；
CN202010522407.6，申请日2020年6月10日；
CN202011524307.3，申请日2020年12月21日。
技术领域
本公开属于生物医药领域，涉及一种r’嵌入的siRNA类似物、双链siRNA类似物、包含其的缀合物，以及其盐和用途；所述用途具体为在制备治疗乙型病毒性肝炎的药物中的用途。
背景技术
乙型病毒性肝炎，简称乙肝，是一种由乙型肝炎病毒(Hepatitis B Virus，简称HBV)感染机体后所引起的疾病。乙型肝炎病毒是一种嗜肝病毒，主要存在于肝细胞内并损害肝细胞，引起肝细胞炎症、坏死、纤维化。乙型病毒性肝炎分急性和慢性两种。急性乙型肝炎在成年人中大多数可通过其自身的免疫机制而自愈。但是慢性乙型肝炎(CHB)已成为全球健康保健所面临的极大挑战，同时也是引起慢性肝病，肝硬化(cirrhosis)和肝癌(HCC)的主要原因(Edward J.G.，et al.，The oral toll-like receptor-7 agonist GS-9620 in patients with chronic hepatitis B virus infection.Journal of Hepatology(2015)；63：320-328)。据估计，全球有20亿人感染了慢性乙型肝炎病毒，超过3亿5千万人口已发展成为了乙型肝炎，每年近60万人死于慢性乙型肝炎的并发症(Edward J.G.，et al.，The oral toll-like receptor 7 agonist GS-9620 in patients with chronic hepatitis B virus infection.Journal of Hepatology(2015))。我国是乙肝高发区，乙型肝炎累积病人多，危害严重。据资料显示，我国现有乙型肝炎病毒感染者约9300万，而其中约2000万患者确诊为慢性乙型肝炎，当中10％-20％可演变成肝硬化，1％-5％可发展成肝癌。(张春红，干扰素在乙型肝炎治疗中的应用.中国医药指南(2013)；11：475-476.)
乙肝功能性治愈的关键是清除HBsAg(乙型肝炎病毒表面抗原)，产生表面抗体。HBsAg量化是一个非常重要的生物指标。在慢性感染病人中，很少能观察到HBsAg的减少和血清转化，这是目前治疗的终点。
目前被批准上市的抗HBV药物主要是免疫调节剂(干扰素-α和聚乙二醇干扰素-α-2α)和抗病毒治疗药物(拉米夫定、阿德福韦酯、恩替卡韦、替比夫定、替诺福韦、克拉夫定等)。其中，抗病毒治疗药物属于核苷酸类药物，其作用机制是抑制HBV DNA的合成，并不能直接减少HBsAg水平。与延长治疗一样，核苷酸类药物显示HBsAg清除速度类似于自然观察 结果(Janssen et al.Lancet(2005)，365，123-129；Marcellin et al.N.Engl.J.Med.(2004)，351，1206-1217；Buster et al.Hepatology(2007)，46，388-394.)。
临床已有疗法降低HBsAg，但疗效不佳。因此，若能从基因水平沉默病毒的基因表达，阻断HBV的生成和复制，特别是HBsAg和HBeAg(乙型肝炎S抗原和E抗原)的产生，可从根本上降低病毒代谢和对肝细胞的侵染。小干扰RNA(small interfering RNA，siRNA)可基于RNA干扰(RNA interference，RNAi)这一机制，以序列特异性的方式抑制或阻断靶标目的基因的表达，从mRNA翻译至蛋白这一层次发挥抑制作用，从而达到治疗疾病的目的(WO2016077321，WO2018195165)。这一最为理想的乙肝治疗手段，需要对siRNA进行稳定化修饰并辅以相应的递送系统靶向目的器官和细胞，提高代谢稳定性，但目前的siRNA尚不能有效地降低乙肝病毒S抗原和E抗原含量。
同时，siRNA可以通过与某些mRNA片段部分互补配对，从而对该mRNA所对应基因的表达起到调控作用。特别地，siRNA反义链的5’端种子区(seed region)与非靶向基因的互补配对会部分或完全沉默该基因表达，这一现象是siRNA在体内体外出现脱靶效应的主要原因(Jackson et al.RNA(2006)，12，1179-1187.)。治疗乙肝的siRNA在临床与临床前阶段均暴露出该缺点(WO2020036862)。尽管通过一些对核苷酸的修饰可以降低脱靶风险(Iribe etal.ACS Omega(2017)，2，2055-2064；Janas et al.Nat.Commun.2018，9，723-732)，其沉默的有效性也有所降低，治疗安全窗尚待提高。
发明内容
本公开涉及一种利巴韦林衍生物嵌入的双链siRNA类似物、包含其的缀合物，以及其盐和用途。本公开的双链siRNA类似物、包含其的缀合物以及其盐可以有效抑制乙肝病毒DNA、S抗原、E抗原等多项病毒指标，为乙型肝炎例如慢性乙型肝炎的治疗(例如功能性治愈)提供了有效可行的手段。
因此，在第一方面，本公开提供了一种双链siRNA类似物，其包含正义链和反义链，其中反义链包含在如SEQ ID NO：2所示的序列中使用r替换了一个或多个核苷酸残基的序列，r为
Figure PCTCN2021098682-appb-000001
其中所述siRNA类似物中的核苷酸和r中的每一个独立地是经修饰的或未修饰的。
在一些实施方案中，所述siRNA类似物中的核苷酸和r中的一个或多个是经修饰的，而其它核苷酸和r是未修饰的。所述修饰包括例如甲氧基修饰、氟代修饰、硫代磷酸酯基连接或将核苷酸替换为(S)-甘油核酸等。
在一些实施方案中，所述siRNA类似物中的核苷酸和r中的一个或多个是经修饰的，而其它核苷酸和r是未修饰的。所述修饰包括例如甲氧基修饰、氟代修饰、硫代磷酸酯基连接、将核苷酸替换为(S)-甘油核酸或将核苷酸替换为(E)-乙烯基磷酸酯修饰等。
在一些实施方案中，所述siRNA类似物中的基本上所有的核苷酸和r都是经修饰的。在一些实施方案中，所述siRNA类似物中的所有核苷酸和r都是经修饰的。
在一些实施方案中，所述双链siRNA类似物中的70％、75％、80％、85％、90％或95％以上的核苷酸和r都是经修饰的。在一些实施方案中，所述双链siRNA类似物中的所有核苷酸和r都是经修饰的。
在一些实施方案中，所述SEQ ID NO：2任选地包括在5’端和/或3’端的突出端。在一些实施方案中，所述SEQ ID NO：2包括在5’端和/或3’端的0、1、2、3、4或5个核苷酸的突出端。
在一些实施方案中，所述SEQ ID NO：2包括在5’端和/或3’端的2个核苷酸的突出端时，末端的3个核苷酸之间任选地具有2个硫代磷酸酯基连接，其中该3个核苷酸中有2个核苷酸为突出端，另1个核苷酸为邻接突出端的配对核苷酸。在一些实施方案中，所述突出端优选自经修饰的或未修饰的UU。在一些实施方案中，所述突出端优选自uu。在一些实施方案中，所述突出端uu与其邻接的1个配对核苷酸之间具有2个硫代磷酸酯基连接。
在一些实施方案中，所述SEQ ID NO：2包括在3’端的突出端，所述突出端优选自经修饰的或未修饰的UU。在一些实施方案中，所述SEQ ID NO：2包括在3’端的突出端，所述突出端优选自uu。在一些实施方案中，所述SEQ ID NO：2包括在3’端的突出端，所述突出端uu与其邻接的1个配对核苷酸之间具有2个硫代磷酸酯基连接(例如c·u·u)。
在一些实施方案中，双链siRNA类似物中的反义链包含在如SEQ ID NO：2所示的序列中使用r替换了一个或多个核苷酸残基的序列。例如，反义链包含在如SEQ ID NO：2所示的序列中使用r替换了一个核苷酸残基的序列。
在一些实施方案中，双链siRNA类似物中的反义链包含在如SEQ ID NO：2所示的序列中使用r替换了一个或多个核苷酸残基的序列。例如，反义链包含在如SEQ ID NO：2所示的序列中使用r替换了一个、两个、三个、四个或五个核苷酸残基的序列。
在一些实施方案中，双链siRNA类似物中的反义链包含在如SEQ ID NO：2所示的序列中使用r替换了一个或多个核苷酸残基的序列，所述r替换发生在SEQ ID NO：2的任意位置。优选地，所述r替换发生在SEQ ID NO：2的5’端的第1～第21位或第1～第19位。例如，所 述r替换发生在SEQ ID NO：2的5’端的第1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或21位。优选地，所述r替换发生在SEQ ID NO：2的5’端的第2、3、4、5、6、7、8、10、11、12、16或18位。
在一些实施方案中，双链siRNA类似物中的反义链包含如SEQ ID NO：4或SEQ ID NO：17、SEQ ID NO：6或SEQ ID NO：19、SEQ ID NO：7或SEQ ID NO：20、SEQ ID NO：8或SEQ ID NO：21、SEQ ID NO：9或SEQ ID NO：22、SEQ ID NO：10或SEQ ID NO：23、SEQ ID NO：11或SEQ ID NO：24、SEQ ID NO：29或SEQ ID NO：33、SEQ ID NO：30或SEQ ID NO：34、SEQ ID NO：31或SEQ ID NO：35，或SEQ ID NO：32或SEQ ID NO：36所示的序列，或由其组成。在一些实施方案中，所述序列包括进一步核苷酸修饰，例如甲氧基修饰、氟代修饰、硫代磷酸酯基连接或将核苷酸替换为(S)-甘油核酸等。
在一些实施方案中，双链siRNA类似物中的反义链包含如SEQ ID NO：4或SEQ ID NO：17、SEQ ID NO：6或SEQ ID NO：19、SEQ ID NO：7或SEQ ID NO：20、SEQ ID NO：8或SEQ ID NO：21、SEQ ID NO：9或SEQ ID NO：22、SEQ ID NO：10或SEQ ID NO：23、SEQ ID NO：11或SEQ ID NO：24、SEQ ID NO：29或SEQ ID NO：33、SEQ ID NO：30或SEQ ID NO：34、SEQ ID NO：31或SEQ ID NO：35，SEQ ID NO：32或SEQ ID NO：36、SEQ ID NO：39或SEQ ID NO：44、SEQ ID NO：10或SEQ ID NO：45、SEQ ID NO：40或SEQ ID NO：46、SEQ ID NO：10或SEQ ID NO：47、或SEQ ID NO：10或SEQ ID NO：48所示的序列，或由其组成。在一些实施方案中，所述序列包括进一步核苷酸修饰，例如甲氧基修饰、氟代修饰、硫代磷酸酯基连接、将核苷酸替换为(S)-甘油核酸或将核苷酸替换为(E)-乙烯基磷酸酯修饰等。
在一些实施方案中，双链siRNA类似物中的正义链包含如SEQ ID NO：1或SEQ ID NO：28所示的序列，或由其组成。
在一些实施方案中，双链siRNA类似物中的正义链包含在如SEQ ID NO：1所示的序列中使用r替换了一个或多个核苷酸残基的序列。例如，正义链包含在如SEQ ID NO：1所示的序列中使用r替换了一个核苷酸残基的序列。
在一些实施方案中，双链siRNA类似物中的正义链包含在如SEQ ID NO：1所示的序列中使用r替换了一个或多个核苷酸残基的序列。例如，正义链包含在如SEQ ID NO：1所示的序列中使用r替换了1、2、3、4或5个核苷酸残基的序列。
在一些实施方案中，双链siRNA类似物中的正义链包含在如SEQ ID NO：1所示的序列中使用r替换了一个或多个核苷酸残基的序列，所述r替换发生在SEQ ID NO：1的5’端的第1～第19位。例如，所述r替换发生在SEQ ID NO：1的5’端的第1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、或19位。优选地，所述r替换发生在SEQ ID NO：1的5’端的第2、3、7、12、15、17或19位。
在一些实施方案中，双链siRNA类似物中的正义链的序列包含如SEQ ID NO：5或SEQ  ID NO：18、SEQ ID NO：3或SEQ ID NO：16、SEQ ID NO：14或SEQ ID NO：27、SEQ ID NO：13或SEQ ID NO：26，或SEQ ID NO：12或SEQ ID NO：25所示的序列，或由其组成。在一些实施方案中，所述序列包括进一步核苷酸修饰，例如甲氧基修饰、氟代修饰、硫代磷酸酯基连接等。
在一些实施方案中，双链siRNA类似物中的正义链的序列包含如SEQ ID NO：5或SEQ ID NO：18、SEQ ID NO：3或SEQ ID NO：16、SEQ ID NO：14或SEQ ID NO：27、SEQ ID NO：13或SEQ ID NO：26、SEQ ID NO：12或SEQ ID NO：25、SEQ ID NO：37或SEQ ID NO：42、或SEQ ID NO：38或SEQ ID NO：43所示的序列，或由其组成。在一些实施方案中，所述序列包括进一步核苷酸修饰，例如甲氧基修饰、氟代修饰、硫代磷酸酯基连接、将核苷酸替换为(S)-甘油核酸或将核苷酸替换为(E)-乙烯基磷酸酯修饰等。
在一些具体的实施方案中，双链siRNA类似物中的正义链和反义链包含在序列中使用r替换了一个或多个核苷酸残基的序列，如反义链SEQ ID NO：2所示的序列，所述r替换发生在SEQ ID NO：2的5’端的第2位，正义链SEQ ID NO：1所示的序列，所述r替换发生在5’端的第7位。
在一些实施方案中，双链siRNA类似物为S18～S28中的任意一种：
S18：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：4或SEQ ID NO：17，
S19：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：6或SEQ ID NO：19，
S20：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：7或SEQ ID NO：20，
S21：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：8或SEQ ID NO：21，
S22：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：9或SEQ ID NO：22，
S23：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：10或SEQ ID NO：23，
S24：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：11或SEQ ID NO：24，
S25：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：29或SEQ ID NO：33，
S26：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：30或SEQ ID NO：34，
S27：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：31或SEQ ID NO：35，
S28：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：32或SEQ ID NO：36。
在一些实施方案中，双链siRNA类似物为S1～S17中的任意一种：
S1：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：4或SEQ ID NO：17，
S2：正义链为SEQ ID NO：5或SEQ ID NO：18、反义链为SEQ ID NO：4或SEQ ID NO：17，
S3：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：6或SEQ ID NO：19，
S4：正义链为SEQ ID NO：5或SEQ ID NO：18、反义链为SEQ ID NO：6或SEQ ID NO：19，
S5：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：7或SEQ ID NO：20，
S6：正义链为SEQ ID NO：5或SEQ ID NO：18、反义链为SEQ ID NO：7或SEQ ID NO：20，
S7：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：8或SEQ ID NO：21，
S8：正义链为SEQ ID NO：5或SEQ ID NO：18、反义链为SEQ ID NO：8或SEQ ID NO：21，
S9：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：9或SEQ ID NO：22，
S10：正义链为SEQ ID NO：5或SEQ ID NO：18、反义链为SEQ ID NO：9或SEQ ID NO：22，
S11：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：10或SEQ ID NO：23，
S12：正义链为SEQ ID NO：5或SEQ ID NO：18、反义链为SEQ ID NO：10或SEQ ID NO：23，
S13：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：11或SEQ ID NO：24，
S14：正义链为SEQ ID NO：5或SEQ ID NO：18、反义链为SEQ ID NO：11或SEQ ID NO：24，
S15：正义链为SEQ ID NO：12或SEQ ID NO：25、反义链为SEQ ID NO：4或SEQ ID NO：17，
S16：正义链为SEQ ID NO：13或SEQ ID NO：26、反义链为SEQ ID NO：4或SEQ ID NO：17，
S17：正义链为SEQ ID NO：14或SEQ ID NO：27、反义链为SEQ ID NO：4或SEQ ID NO：17。
在一些实施方案中，双链siRNA类似物为S29～S35中的任意一种：
S29：正义链为SEQ ID NO：37或SEQ ID NO：42、反义链为SEQ ID NO：10或SEQ ID NO：23，
S30：正义链为SEQ ID NO：38或SEQ ID NO：43、反义链为SEQ ID NO：10或SEQ ID NO：23，
S31：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：39或SEQ ID NO：44，
S32：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：10或SEQ ID NO：45，
S33：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：40或SEQ ID NO：46，
S34：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：10或SEQ ID NO：47，
S35：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：10或SEQ ID NO：48。
在一些实施方案中，所述双链siRNA类似物选自：正义链为SEQ ID NO：3和反义链为SEQ ID NO：4、正义链为SEQ ID NO：5和反义链为SEQ ID NO：4、正义链为SEQ ID NO：3和反义链为SEQ ID NO：6、正义链为SEQ ID NO：5和反义链为SEQ ID NO：6、正义链为SEQ ID NO：3和反义链为SEQ ID NO：7、正义链为SEQ ID NO：5和反义链为SEQ ID NO：7、正义链为SEQ ID NO：3和反义链为SEQ ID NO：8、正义链为SEQ ID NO：5和反义链为SEQ ID NO：8、正义链为SEQ ID NO：3和反义链为SEQ ID NO：9、正义链为SEQ ID NO：5和反义链为SEQ ID NO：9、正义链为SEQ ID NO：3和反义链为SEQ ID NO：10、正义链为SEQ ID NO：5和反义链为SEQ ID NO：10、正义链为SEQ ID NO：3和反义链为SEQ ID NO：11、正义链为SEQ ID NO：5和反义链为SEQ ID NO：11、正义链为SEQ ID NO：12和反义链为SEQ ID NO：4、正义链为SEQ ID NO：13和反义链为SEQ ID NO：4、正义链为SEQ ID NO：14和反义链为SEQ ID NO：4、正义链为SEQ ID NO：1和反义链为SEQ ID NO：4、正义链为SEQ ID NO：1和反义链为SEQ ID NO：6、正义链为SEQ ID NO：1和反义链为SEQ ID NO：7、正义链为SEQ  ID NO：1和反义链为SEQ ID NO：8、正义链为SEQ ID NO：1和反义链为SEQ ID NO：9、正义链为SEQ ID NO：1和反义链为SEQ ID NO：10、正义链为SEQ ID NO：1和反义链为SEQ ID NO：11、正义链为SEQ ID NO：1和反义链为SEQ ID NO：29、正义链为SEQ ID NO：1和反义链为SEQ ID NO：30、正义链为SEQ ID NO：1和反义链为SEQ ID NO：31、正义链为SEQ ID NO：1和反义链为SEQ ID NO：32、正义链为SEQ ID NO：37和反义链为SEQ ID NO：10、正义链为SEQ ID NO：38和反义链为SEQ ID NO：10、正义链为SEQ ID NO：3和反义链为SEQ ID NO：39、正义链为SEQ ID NO：3和反义链为SEQ ID NO：10、或正义链为SEQ ID NO：3和反义链为SEQ ID NO：40，所述双链siRNA类似物中的核苷酸和r中的每一个独立地是经修饰的或未修饰的。
在一些实施方案中，所述双链siRNA类似物选自：
Figure PCTCN2021098682-appb-000002
Figure PCTCN2021098682-appb-000003
所述双链siRNA类似物中的核苷酸和r中的每一个独立地是经修饰的或未修饰的。
在一些实施方案中，所述双链siRNA类似物选自：正义链为SEQ ID NO：16和反义链为SEQ ID NO：17、正义链为SEQ ID NO：18和反义链为SEQ ID NO：17、正义链为SEQ ID NO：16和反义链为SEQ ID NO：19、正义链为SEQ ID NO：18和反义链为SEQ ID NO：19、正义链为SEQ ID NO：16和反义链为SEQ ID NO：20、正义链为SEQ ID NO：18和反义链为SEQ ID NO：20、正义链为SEQ ID NO：16和反义链为SEQ ID NO：21、正义链为SEQ ID NO：18和反义链为SEQ ID NO：21、正义链为SEQ ID NO：16和反义链为SEQ ID NO：22、正义链为SEQ ID NO：18和反义链为SEQ ID NO：22、正义链为SEQ ID NO：16和反义链为SEQ ID NO：23、正义链为SEQ ID NO：18和反义链为SEQ ID NO：23、正义链为SEQ ID NO：16和反义链为SEQ ID NO：24、正义链为SEQ ID NO：18和反义链为SEQ ID NO：24、正义链为SEQ ID NO： 25和反义链为SEQ ID NO：17、正义链为SEQ ID NO：26和反义链为SEQ ID NO：17、正义链为SEQ ID NO：27和反义链为SEQ ID NO：17、正义链为SEQ ID NO：28和反义链为SEQ ID NO：17、正义链为SEQ ID NO：28和反义链为SEQ ID NO：19、正义链为SEQ ID NO：28和反义链为SEQ ID NO：20、正义链为SEQ ID NO：28和反义链为SEQ ID NO：21、正义链为SEQ ID NO：28和反义链为SEQ ID NO：22、正义链为SEQ ID NO：28和反义链为SEQ ID NO：23、正义链为SEQ ID NO：28和反义链为SEQ ID NO：24、正义链为SEQ ID NO：28和反义链为SEQ ID NO：33、正义链为SEQ ID NO：28和反义链为SEQ ID NO：34、正义链为SEQ ID NO：28和反义链为SEQ ID NO：35、正义链为SEQ ID NO：28和反义链为SEQ ID NO：36、正义链为SEQ ID NO：42和反义链为SEQ ID NO：23、正义链为SEQ ID NO：43和反义链为SEQ ID NO：23、正义链为SEQ ID NO：16和反义链为SEQ ID NO：44、正义链为SEQ ID NO：16和反义链为SEQ ID NO：45、正义链为SEQ ID NO：16和反义链为SEQ ID NO：46、正义链为SEQ ID NO：16和反义链为SEQ ID NO：47、或正义链为SEQ ID NO：16和反义链为SEQ ID NO：48。
在一些实施方案中，所述双链siRNA类似物选自：
Figure PCTCN2021098682-appb-000004
Figure PCTCN2021098682-appb-000005
在第二方面，本公开提供了双链siRNA类似物的缀合物，其包含根据本公开的第一方面的双链siRNA类似物，以及与双链siRNA类似物缀合的药学上可接受的缀合基团。
在一些实施方案中，双链siRNA类似物的缀合物中的药学上可接受的缀合基团含有1至 5个GalNAc(N-乙酰半乳糖胺)基团。优选地，所述药学上可接受的缀合基团含有1、2、3、4或5个GalNAc基团。更优选地，所述药学上可接受的缀合基团含有3或4个GalNAc基团。
在一些实施方案中，双链siRNA类似物的缀合物中的药学上可接受的缀合基团包含化合物基团D
Figure PCTCN2021098682-appb-000006
在一些实施方案中，双链siRNA类似物的缀合物中的药学上可接受的缀合基团连接至双链siRNA类似物的正义链的3’末端。
在一些实施方案中，双链siRNA类似物或双链siRNA类似物的缀合物的硫代磷酸酯部分包括(R)-和(S)-对映体、非对映异构体、和/或其外消旋混合物。
在一些实施方案中，双链siRNA类似物的缀合物选自：
Figure PCTCN2021098682-appb-000007
Figure PCTCN2021098682-appb-000008
Figure PCTCN2021098682-appb-000009
所述D如前所示。
在第三方面，本公开提供了根据本公开的第一方面的双链siRNA类似物或根据本公开的第二方面的双链siRNA类似物的缀合物的盐。
在一些实施方案中，如上所述的盐选自碱加成盐、酸加成盐及其组合。
在一些实施方案中，碱加成盐选自钠、钾、钙、铵、有机胺、镁盐及其组合，酸加成盐选自无机酸盐、有机酸盐及其组合。
在一些实施方案中，无机酸选自盐酸、氢溴酸、硝酸、碳酸、碳酸氢根、磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸及其组合，有机酸选自乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸、甲磺酸及其组合。
在第四方面，本公开提供了药物组合物，其包含根据本公开的第一方面的双链siRNA类似物，根据本公开的第二方面的双链siRNA类似物的缀合物，或根据本公开的第三方面的盐，以及药学上可接受的载体或赋形剂。
在第五方面，本公开提供了根据本公开的第一方面的双链siRNA类似物，根据本公开的第二方面的双链siRNA类似物的缀合物，或根据本公开的第三方面的盐，或根据本公开的第四方面的药物组合物，其在制备用于治疗乙型肝炎的药物中的用途。
在一些实施方案中，本公开提供了根据本公开的第一方面的双链siRNA类似物，根据本公开的第二方面的双链siRNA类似物的缀合物，或根据本公开的第三方面的盐，或根据本公开的第四方面的药物组合物，其用于治疗受试者中的乙型肝炎。
在第六方面，本公开提供了治疗受试者中乙型病毒肝炎的方法，其包括将根据本公开的第一方面的双链siRNA类似物，根据本公开的第二方面的双链siRNA类似物的缀合物，或根据本公开的第三方面的盐，或根据本公开的第四方面的药物组合物施用于受试者的步骤。
在第七方面，本公开提供了用于治疗受试者中的乙型肝炎的根据本公开的第一方面的双链siRNA类似物，根据本公开的第二方面的双链siRNA类似物的缀合物，或根据本公开的第三方面的盐，或根据本公开的第四方面的药物组合物。
在本公开的一些实施方案中，所述乙型肝炎可以处于疾病的任意阶段。例如急性乙型肝炎、慢性乙型肝炎，或由乙型肝炎病毒感染引起的肝硬化或肝癌。在一些实施方案中，所述 乙型肝炎是慢性乙型肝炎。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照本领域普通技术人员所理解的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
本公开中，除非另有说明，否则术语“包含、包括和含有”或等同物为开放式表述，意味着除所列出的要素、组分或步骤外，还可涵盖其他未指明的要素、组分或步骤。
本公开中，HBV基因是指DNA序列如Genbank注册号NC_003977.1所示的基因。如Genbank注册号NC_003977.1所示的基因是HBV的完整基因组。
在一些实施方案中，双链siRNA类似物可以靶向HBV的X开放阅读框(X opening reading frame，X ORF)。
本公开中，双链siRNA类似物是指核糖核酸分子的复合体，其具有双链结构，包含两条反向平行的且基本上互补的核酸链，其相对于靶RNA具有“正义”和“反义”定向。在本公开中，“互补”具有本领域技术人员周知的含义，即，在双链核酸分子中，一条链的碱基与另一条链上的碱基以互补的方式相配对。嘌呤碱基腺嘌呤(A)始终与嘧啶碱基尿嘧啶(U)相配对；嘌呤碱基鸟嘌呤(C)始终与嘧啶碱基胞嘧啶(G)相配对。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的尿嘧啶配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此相互补的，以及从其互补链的序列中可以推断出该链的序列。
在本公开中，如无特别说明，大写字母C、G、U、A表示核苷酸的碱基组成。小写字母c、g、u、a分别表示其相应大写字母所代表的核苷酸被甲氧基修饰； 下划线表示大写字母代表的核苷酸被氟代修饰；间隔号“·”表示与间隔号“·”左右相邻的两个核苷酸残基之间为硫代磷酸酯基连接；VP表示该字母VP右侧的一个核苷酸为(E)-乙烯基磷酸酯修饰的核苷酸。例如，“a·g”表示a和g残基之间通过硫代磷酸酯基连接。
本公开中所述核苷酸的“修饰”包括但不限于甲氧基修饰、氟代修饰、(E)-乙烯基磷酸酯修饰、硫代磷酸酯基连接或将核苷酸替换为(S)-甘油核酸等。本公开所述的序列可以包括如下表1中“进一步修饰的序列”所列。
本公开所述氟代修饰的核苷酸指核苷酸的核糖基2’位的羟基被氟取代形成的核苷酸，所述甲氧基修饰的核苷酸指核糖基的2’-羟基被甲氧基取代而形成的核苷酸。
本公开所述(E)-乙烯基磷酸酯修饰的核苷酸表示以下结构单元：
Figure PCTCN2021098682-appb-000010
其中，E选自
Figure PCTCN2021098682-appb-000011
X选自OCH 3和F。
本公开所述(S)-甘油核酸(Agn)表示以下结构单元：
Figure PCTCN2021098682-appb-000012
(Agn)和其他核苷酸残基通过磷酸酯或硫代磷酸酯互相连接，如“a·(Agn)”表示a和(Agn)残基之间通过硫代磷酸酯基连接，“a(Agn)”表示a和(Agn)残基之间通过磷酸酯基连接。
在一些实施方案中，双链siRNA类似物包含正义链或r’嵌入的正义链和r’嵌入的反义链。正义链、r’嵌入的正义链和r’嵌入的反义链均含有核苷酸基团作为基本结构单元。本领域技术人员公知，核苷酸基团含有磷酸基团、核糖基团和碱基，在此不再赘述。
本公开所述r’嵌入的序列是指所述序列中存在至少一个核苷酸残基与r相连接，包括如在序列(如SEQ ID NO：2)中使用r替换了一个核苷酸残基的序列。本公开所述r’嵌入的序列包括但不限于：r’嵌入的双链siRNA、r’嵌入的正义链和r’嵌入的反义链。例如，5’-aGUrrA·C-3’、5’-rGgAAC-3’和5’-AG·UrAAcCuCr-3’都属于r’嵌入的情形。
本公开所述r’嵌入的双链siRNA是指双链siRNA中存在至少一个核苷酸残基与r相连接，包括如在双链siRNA的序列中使用r替换了一个核苷酸残基的双链siRNA。本公开所述r’嵌入的正义链是指正义链中存在至少一个核苷酸残基与r相连接，包括如正义链中的一个或者多个核苷酸被r替换。本公开所述r’嵌入的反义链是指反义链中存在至少一个核苷酸残基与r相连接，包括如反义链中的一个或者多个核苷酸被r替换。
本公开所述r’为
Figure PCTCN2021098682-appb-000013
(其中，X选自SH和OH)，为天然核苷酸碱基的类似物，不同于任何公开专利的天然核苷酸碱基，在核酸序列的引入带来不可预料的活性。
本公开所述r表示以下结构单元：
Figure PCTCN2021098682-appb-000014
r和其他核苷酸残基通过磷酸酯或硫代磷酸酯互相连接，如“a·r”表示a和r残基之间通过硫代磷酸酯基连接，“ar”表示a和r残基之间通过磷酸酯基连接。
本公开所述“多个”指大于等于2的整数，包括但不限于2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个，至多可达所述siRNA类似物的理论上限。
本公开中，所述双链siRNA类似物的正义链或反义链也可以包括“突出端”，例如不直接参与RNA双螺旋结构的未配对突出核苷酸，其中RNA双螺旋结构通常由本文定义的“正义链”和“反义链”对形成。这样的突出端可以包括一个或多个修饰的或未修饰的U、T和A。例如，所述SEQ ID NO：2可以在5’和/或3’段包括修饰的或未修饰的UU突出端。
本公开中，双链siRNA类似物的缀合物是双链siRNA类似物和药学上可接受的缀合基团连接形成的化合物，并且双链siRNA类似物和药学上可接受的缀合基团共价连接。
本公开中，药学上可接受的缀合基团可连接至双链siRNA类似物的正义链或者r’嵌入的正义链的3’末端。
一般来说，药学上可接受的缀合基团包含药学上可接受的靶向分子和任选的接头(linker)。示例性的缀合基团、接头、靶向分子的种类可参见WO2015006740A2的公开内容。示例性的缀合基团包括但不限于L96或化合物基团D。
在本公开的上下文中，除非另有说明，“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。
本公开的化合物可以存在特定的几何或立体异构体形式。本公开设想所有的这类化合物，包括(R)-和(S)-对映体、非对映异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本公开的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本公开的范围之内。
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，用楔形实线键
Figure PCTCN2021098682-appb-000015
和楔形虚线键
Figure PCTCN2021098682-appb-000016
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2021098682-appb-000017
和直形虚线键
Figure PCTCN2021098682-appb-000018
表示立体中心的相对构型，用波浪线
Figure PCTCN2021098682-appb-000019
表示楔形实线键
Figure PCTCN2021098682-appb-000020
或楔形虚线键
Figure PCTCN2021098682-appb-000021
或用波浪线
Figure PCTCN2021098682-appb-000022
表示直形实线键
Figure PCTCN2021098682-appb-000023
和/或直形虚线键
Figure PCTCN2021098682-appb-000024
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本公开某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。本公开的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本公开的化合物的所有同位素组成的变换，无论放射性与否，都包括在本公开的范围之内。
术语“盐”是指本公开化合物的盐，由本公开发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本公开的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本公开的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸、碳酸氢根、磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲 酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本公开的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本公开的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
本公开的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本公开的实施例。
本公开所使用的溶剂可经市售获得。
如无特殊说明，本公开柱层析、制备薄层硅胶色谱所用溶剂配比均为体积比。
缩略词清单
Ac 乙酰基
Boc 叔丁氧羰基
DMSO 二甲亚砜
DMT/DMTr 4，4’-二甲氧基三苯基甲基
dsRNA 双链核糖核酸
EC 50 半最大效应浓度
EDTA 乙二胺四乙酸二钠
i-Pr 异丙基
Me 甲基
Ms 甲烷磺酰基
Ph 苯基
p-HPLC 制备高效液相色谱，用于化合物的纯化
RNA 核糖核酸
RNAi 核糖核酸干扰技术
siRNA 小干扰核糖核酸
t-Bu 叔丁基
Tris 三羟甲基氨基甲烷
化合物依据本领域常规命名原则或者使用
Figure PCTCN2021098682-appb-000025
软件命名，市售化合物采用供应商目录名称。
附图说明
图1.WRG01对AAV/HBV小鼠血浆HBsAg的影响。
图2.WRG01对AAV/HBV小鼠血浆HBeAg的影响。
图3.WRG01对AAV/HBV小鼠血浆HBV DNA的影响。
图4.WRG01对AAV/HBV小鼠血浆HBV pgRNA的影响。
图5.给予WRG01后小鼠体重变化。
图6.WR007和WR012对AAV/HBV小鼠血浆HBsAg的影响。
图7.WR007和WR012对AAV/HBV小鼠血浆HBeAg的影响。
图8.WR007和WR012对AAV/HBV小鼠血浆HBV DNA的影响。
图9.不同剂量WRG01对AAV/HBV小鼠血浆HBsAg的影响。
图10.WRG01在小鼠血浆、肝脏、肾脏的浓度。
具体实施方式
下面通过实施例对本公开进行详细描述，但并不意味着对本公开任何不利限制。本公开的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本公开的实施例。对本领域的技术人员而言，在不脱离本公开精神和范围的情况下针对本公开具体实施方式进行各种变化和改进将是显而易见的。
实施例1 亚磷酰胺单体的合成
Figure PCTCN2021098682-appb-000026
步骤A：(2S，3R，4R，5R，6R)-3-乙酰氨基-6-(乙酰氧基甲基)四氢-2H-吡喃-2，4，5-爪基三醋酸盐(即式1-1)(30克，94.26毫摩尔)与1，2，4-三氮唑-3-羧酸甲酯(11.98克，94.26毫摩尔)溶于乙酸甲酯(220毫升)的溶液，在90摄氏度油浴中于1bar的压力下浓缩至接近完全干燥。将三氟甲烷磺酸(141.46毫克，0.94毫摩尔)的乙酸甲酯溶液(2毫升)加入混合液中，并于30mbar的压力下在125摄氏度的油浴中搅拌4小时。反应液冷却至70摄氏度，加入乙 醇(70毫升)，在70摄氏度下搅拌直至形成均一的溶液，并停止搅拌冷却至50摄氏度。生成沉淀后，静置冷却至25摄氏度并将反应液在0摄氏度下放置16小时。反应液通过布氏漏斗过滤，滤饼用乙醇180毫升(60毫升×3)淋洗，真空干燥得1-2。 1H NMR(400MHz，CDCl 3)：ε8.40(s，1H)，6.04(d，J＝3.42Hz，1H)，5.69-5.81(m，1H)，5.54(t，J＝5.38Hz，1H)，4.42-4.51(m，2H)，4.16-4.30(m，1H)，3.98(s，3H)，2.05-2.18(m，9H)。
步骤B：将式1-2(15克，38.93毫摩尔)所示化合物与三乙胺(4.14克，40.87毫摩尔)溶于甲醇(100毫升)。混合液在氮气保护下于50摄氏度搅拌17小时。反应液减压浓缩得1-3。 1H NMR(400MHz，CD 3OD)：ε8.87(s，1H)，5.93(d，J＝3.42Hz，1H)，4.48(dd，J＝3.48，4.83Hz，1H)，4.33(t，J＝5.26Hz，1H)，4.10-4.16(m，1H)，3.95(s，3H)，3.84(dd，J＝3.24，12.29Hz，1H)，3.70(dd，J＝4.46，12.29Hz，1H)。
步骤C：将式1-3(10克，38.58毫摩尔)所示化合物溶于吡啶(250毫升)于0摄氏度滴加1，3-二氯-1，1，3，3-四异丙基二硅氧烷(12.29克，38.97毫摩尔)。混合液逐渐升温至25摄氏度搅拌16小时。反应液减压浓缩，将浓缩物悬浮在乙酸乙酯(250毫升)中，通过布氏漏斗过滤。滤液用3M盐酸750毫升(250毫升×3)和饱和食盐水250毫升(250毫升×1)洗涤，无水硫酸钠干燥，过滤后减压浓缩得粗品。经柱层析(SiO 2，石油醚/二氯甲烷/乙酸乙酯＝3/1/1)纯化后得1-4。 1H NMR(400MHz，CDCl 3)：ε8.43(s，1H)，5.95(s，1H)，4.73(dd，J＝4.75，8.00Hz，1H)，4.41(d，J＝4.75Hz，1H)，4.09-4.19(m，2H)，3.94-4.03(m，4H)，2.71-3.34(m，1H)，1.01-1.15(m，28H)。
步骤D：向含有式1-4(8.23克，16.40毫摩尔)所示化合物，碳酸钾(11.34克，82.02毫摩尔)和氧化银(I)(19.01克，82.02毫摩尔)的N，N-二甲基甲酰胺(50毫升)的混合液中加入碘甲烷(11.64克，82.02毫摩尔)，并于25摄氏度下搅拌3小时。反应液用乙酸乙酯(300毫升)稀释，通过布氏漏斗过滤。滤液用硫代硫酸钠水溶液250毫升(250毫升×1)、水250毫升(250毫升×1)和饱和食盐水250毫升(250毫升×1)洗涤，无水硫酸钠干燥，过滤后减压浓缩得粗品。经柱层析(SiO 2，石油醚/乙酸乙酯＝5/1)纯化后得1-5。 1H NMR(400MHz，CDCl 3)：ε8.58(s，1H)，5.91(s，1H)，4.46(dd，J＝4.22，9.35Hz，1H)，4.17-4.28(m，2H)，3.96-4.06(m，5H)，3.68(s，3H)，0.99-1.13(m，28H)。
步骤E：在0摄氏度下，向式1-5(3.27克，6.34毫摩尔)所示化合物的四氢呋喃(50毫升)溶液中滴加三乙胺三氢氟酸盐(2.25克，13.95毫摩尔)，混合液逐渐升温至25摄氏度并搅拌16小时。反应液减压浓缩得粗品。经柱层析(SiO 2，二氯甲烷/甲醇＝20/1)纯化后得1-6。 1H NMR(400MHz，CD 3OD)：ε8.88(s，1H)，6.04(d，J＝3.26Hz，1H)，4.44(t，J＝5.33Hz，1H)，4.20(dd，J＝3.33，4.83Hz，1H)，4.07-4.14(m，1H)，3.96(s，3H)，3.84(dd，J＝3.20，12.36Hz，1H)，3.69(dd，J＝4.39，12.30Hz，1H)，3.52(s，3H)。
步骤F：在0摄氏度下，向式1-6(1.30克，4.76毫摩尔)所示化合物的吡啶(20毫升) 溶液中加入4，4-二甲氧基三苯甲基氯(2.42克，7.14毫摩尔)，并于25摄氏度下搅拌16小时。反应液用乙酸乙酯(70毫升)稀释后，于25摄氏度用饱和碳酸氢钠水溶液(20毫升)淬灭并用水(40毫升)稀释。分液后合并的有机相用水60毫升(60毫升×1)和饱和食盐水60毫升(60毫升×1)洗涤，无水硫酸钠干燥，过滤后减压浓缩得粗品。经p-HPLC纯化(分离柱：Phenomenex luna C18(规格：250mm×50mm，粒径：10μm)；流动相：[水(10mM碳酸氢铵)-乙腈]；洗脱梯度：35％-65％，20min)得1-7。 1H NMR(400MHz，CDCl 3)：ε8.44(s，1H)，7.38-7.45(m，2H)，7.28-7.34(m，5H)，7.18-7.27(m，2H)，6.70-6.92(m，4H)，5.97(d，J＝2.88Hz，1H)，4.37-4.43(m，1H)，4.33(dd，J＝2.88，5.00Hz，1H)，4.19-4.25(m，1H)，3.98(s，3H)，3.80(s，6H)，3.58(s，3H)，3.43-3.49(m，1H)，3.33-3.40(m，1H)，2.55(d，J＝6.88Hz，1H)。LCMS(ESI)m/z；574.2[M-H] -。
步骤G：在0摄氏度下，向式1-7(1.10克，1.91毫摩尔)所示化合物的二氯甲烷(8毫升)溶液中加入2-氰乙基-N，N-二异丙基氯亚磷酰胺(678.45毫克，2.87毫摩尔)和N，N-二异丙基乙胺，并于20摄氏度下搅拌0.5小时。反应液减压浓缩得粗品。经柱层析(SiO 2，石油醚/乙酸乙酯＝50/1至1/2)纯化后得式1化合物。LCMS(ESI)m/z：776.3[M+H] +。
实施例2 D01的合成
Figure PCTCN2021098682-appb-000027
Figure PCTCN2021098682-appb-000028
步骤A：将11-十二炔-1-醇(25克，137.14毫摩尔)和三乙胺(16.65克，164.56毫摩尔)溶于二氯甲烷(250毫升)，于0摄氏度加入甲烷磺酰氯(18.85克，164.56毫摩尔)。混合液在0摄氏度搅拌2小时。反应液用水(400毫升)稀释，二氯甲烷800毫升(400毫升×2)萃取。合并的有机相用水400毫升(200毫升×2)和饱和食盐水洗涤，无水硫酸钠干燥，过滤后减压浓缩得2-2。
步骤B：式2-3(20克，67.26毫摩尔)所示化合物溶于N，N-二甲基甲酰胺(200毫升)于0摄氏度加入氢化钠(60％纯度，4.04克，100.89毫摩尔)，接着加入式2-2(19.27克，73.99毫摩尔)所示化合物。混合液在25摄氏度搅拌16小时。反应液用水(1升)淬灭，并用二氯甲烷1.6升(800毫升×2)萃取。合并的有机相用饱和食盐水800毫升(800毫升×1)洗涤，无水硫酸钠干燥，过滤后减压浓缩得2-4。 1H NMR(400MHz，DMSO-d 6)：ε7.63-6.89(m，10H)，5.64-5.52(m，2H)，4.27-4.01(m，2H)，3.98-3.77(m，2H)，3.72-3.18(m，4H)，2.23-2.14(m，2H)，1.98-1.92(m，1H)，1.54-1.23(m，16H)。
步骤C：将式2-4(48克，103.98毫摩尔)所示化合物溶于甲醇(870毫升)，加入氯化氢甲醇溶液(4摩尔每升，400毫升，1.6摩尔)。混合液在30摄氏度搅拌2小时。向反应液加入氯化氢甲醇溶液(4摩尔每升，350毫升，1.4摩尔)。混合液在30摄氏度搅拌16小时。反应液减压浓缩，加入三氯甲烷200毫升(100毫升×2)，减压浓缩直至出现白色固体。加入甲苯(130毫升)和石油醚(130毫升)，混合液在15摄氏度搅拌16小时。反应液经过布氏漏斗过滤，收集滤饼经真空干燥得白色固体。白色固体溶于二氯甲烷(50毫升)，加入氢氧化钠(6.59克，164.66毫摩尔)的水溶液(50毫升)，在20摄氏度搅拌1小时。反应液用水(500毫升)稀释，并用二氯甲烷1升(500毫升×2)萃取。合并的有机相用无水硫酸钠干燥，过滤后减压浓缩得2-5。
步骤D：向式2-5(23克，80.58毫摩尔)所示化合物和氢氧化钠(322.31毫克，8.06毫摩尔)的二甲亚砜(70毫升)和水(6毫升)的混合液中加入丙烯酸叔丁酯(22.72克，177.28毫摩尔)，在氮气保护下于25摄氏度搅拌16小时。反应液用水(500毫升)稀释，并用乙酸乙酯1升(500毫升×2)萃取。合并的有机相用无水硫酸钠干燥，过滤后减压浓缩得粗品。经柱层析(SiO 2，石油醚/乙酸乙酯/乙醇(含0.1％氨水)＝36/3/1至16/3/1)纯化后得2-6。 1H NMR(400MHz，DMSO-d 6)：ε3.60-3.54(m，4H)，3.32(br s，5H)，3.15(s，5H)，2.74-2.66(m，1H)，2.40(t，J＝6.0Hz，4H)，2.18-2.11(m，2H)，1.58-1.38(m，22H)，1.34-1.23(m，12H)。
步骤E：向式2-6(24.5克，45.22毫摩尔)所示化合物的二氯甲烷(250毫升)溶液中加入三乙胺(9.15克，90.45毫摩尔)和丁二酸酐(6.79克，67.83毫摩尔)，于20摄氏度搅拌16小时。反应液加入二氯甲烷(1升)和盐酸(1摩尔每升，1升)，分液后的有机相用无水硫酸钠干燥，过滤后减压浓缩得2-7。 1H NMR(400MHz，CDCl 3)：ε6.49-6.37(m，1H)，3.72(s，2H)，3.70-3.57(m，8H)，3.37(t，J＝6.7Hz，2H)，2.69-2.51(m，4H)，2.50-2.36(m，4H)，2.22-2.13(m，2H)，1.96-1.90(m，1H)，1.57-1.47(m，4H)，1.46-1.40(m，18H)，1.40-1.31(m，2H)，1.30-1.21(m，10H)。
步骤F：将式2-7(27.4克，42.69毫摩尔)所示化合物溶于甲酸(140毫升)，混合液在氮气保护下于20摄氏度搅拌16小时。反应液减压浓缩，加入甲苯300毫升(150毫升×2)，减压浓缩得2-8。 1H NMR(400MHz，CDCl 3)：ε9.79-9.22(m，3H)，6.44-6.23(m，1H)，3.88-3.43(m，10H)，3.39-3.20(m，2H)，2.77-2.31(m，8H)，2.15-2.06(m，2H)，1.87(t，J＝2.6Hz，1H)，1.48-1.28(m，6H)，1.26-1.12(m，10H)。
步骤G：将式2-8(22.6克，42.67毫摩尔)所示化合物、N，N-二异丙基乙胺(33.09克，256.03毫摩尔)和O-(7-氮杂苯并三唑-1-基)-N，N，N，N-四甲基脲六氟磷酸盐(51.92克，136.55毫摩尔)溶于N，N-二甲基甲酰胺(250毫升)，加入N-(3-氨基丙基)氨基甲酸叔丁酯(29.74克，170.69毫摩尔)。混合液在20摄氏度搅拌16小时。反应液加入二氯甲烷(1升)和盐酸 (1摩尔每升，1升)，分液后的有机相用水1升(1升×1)、碳酸氢钠水溶液1升(1升×1)和饱和食盐水1升(1升×1)依次洗涤，无水硫酸钠干燥，过滤后减压浓缩得粗品。经柱层析(SiO 2，石油醚/乙酸乙酯/乙醇＝40/3/1至10/3/1)纯化后得2-9。 1H NMR(400MHz，CDCl 3)：ε7.22-6.79(m，3H)，6.77-6.44(m，1H)，5.45-5.00(m，3H)，3.86-3.73(m，2H)，3.72-3.63(m，4H)，3.62-3.45(m，4H)，3.41-3.32(m，2H)，3.32-3.20(m，6H)，3.19-3.03(m，6H)，2.56-2.47(m，4H)，2.47-2.39(m，4H)，2.21-2.12(m，2H)，1.95-1.90(m，1H)，1.70-1.57(m，6H)，1.56-1.47(m，4H)，1.46-1.38(m，29H)，1.30-1.25(m，10H)。
步骤H：式2-9(15克，15.03毫摩尔)所示化合物溶于二氯甲烷(114毫升)加入三氟乙酸(38毫升)，混合液于20摄氏度搅拌16小时。反应液减压浓缩，加入甲苯/乙腈＝3/1混合液600毫升(250毫升×3)减压浓缩得2-10(((三(三氟乙酸盐)))。
步骤I：式2-11(22.15克，49.50毫摩尔)所示化合物，N，N-二异丙基乙胺(7.75克，60.00毫摩尔)，1-羟基-7-氮杂苯并三唑(6.12克，45.00毫摩尔)和O-(7-氮杂苯并三唑-1-基)-N，N，N，N-四甲基脲六氟磷酸盐(20.53克，54.00毫摩尔)溶于N，N-二甲基甲酰胺(90毫升)，向该混合液加入式2-10(三(三氟乙酸盐)，15.6克，15.00毫摩尔)所示化合物和N，N-二异丙基乙胺(21.32克，165.00毫摩尔)的N，N-二甲基甲酰胺(120毫升)溶液。混合液在20摄氏度搅拌16小时。反应液加入二氯甲烷(1.2升)和盐酸(1摩尔每升，1升)，分液后的有机相用水1升(1升×1)、碳酸氢钠水溶液1升(1升×1)和饱和食盐水1升(1升×1)依次洗涤，无水硫酸钠干燥，过滤后减压浓缩得粗品。经柱层析(SiO 2，二氯甲烷/甲醇＝100/1至10/1至二氯甲烷/乙醇＝1/1)纯化后得2-12。 1H NMR(400MHz，DMSO-d6)：ε7.87-7.66(m，9H)，7.09(s，1H)，5.21(d，J＝3.4Hz，3H)，4.96(dd，J＝3.4，11.3Hz，3H)，4.48(d，J＝8.5Hz，3H)，4.06-3.98(m，9H)，3.91-3.82(m，3H)，3.74-3.66(m，3H)，3.58-3.46(m，12H)，3.31(br s，3H)，3.07-2.98(m，12H)，2.71(t，J＝2.6Hz，1H)，2.33-2.22(m，8H)，2.16-2.12(m，2H)，2.10(s，9H)，2.04(brt，J＝7.1Hz，6H)，1.99(s，9H)，1.89(s，9H)，1.81-1.74(m，9H)，1.54-1.39(m，22H)，1.32(brdd，J＝4.5，6.7Hz，2H)，1.24(s，10H)。
步骤J：将式2-12(1.00克，0.50毫摩尔)所示化合物和N-甲基-N，N，N-三正辛基氯化铵(20.35毫克，50.35微摩尔)溶于乙酸(2.7毫升)和正戊烷(6.3毫升)的混合液，于0摄氏度向该混合液滴入高锰酸钾(0.40克，2.52毫摩尔)的水(9毫升)溶液。混合液在0至15摄氏度搅拌2小时。反应用亚硫酸氢钠(1.27克)淬灭，加入盐酸(2摩尔每升，5毫升)和水(30毫升)，并用三氯甲烷/异丙醇＝3/1混合液120毫升(40毫升×3)萃取。合并的有机相用无水硫酸钠干燥，过滤后减压浓缩，加入甲苯/乙腈＝1/1混合液180毫升(30毫升×6)，减压浓缩得2-13。 1H NMR(400MHz，CD 3OD)：ε5.34(d，J＝2.9Hz，3H)，5.06(dd，J＝3.3，11.2Hz，3H)，4.56(d，.J＝8.4Hz，3H)，4.19-4.06(m，9H)，4.04-3.98(m，3H)，3.87 (td，J＝5.7，9.9Hz，4H)，3.72-3.64(m，9H)，3.57-3.50(m，3H)，3.39(brt，J＝6.4Hz，2H)，3.22(q，J＝6.4Hz，12H)，2.51-2.40(m，9H)，2.21(brt，J＝7.3Hz，6H)，2.14(s，9H)，2.03(s，9H)，1.94(d，J＝7.9Hz，18H)，1.72-1.57(m，22H)，1.39(br s，12H)。
步骤K：向式2-13(1.00克，0.50毫摩尔)所示化合物的N，N-二甲基甲酰胺(10毫升)溶液加入N，N-二异丙基乙胺(0.26克，1.99毫摩尔)和O-(7-氮杂苯并三唑-1-基)-N，N，N，N-四甲基脲六氟磷酸盐(0.23克，0.60毫摩尔)。混合液搅拌后，加入式2-14(0.23克，0.55毫摩尔)所示化合物。混合液在15摄氏度搅拌16小时。反应液加入二氯甲烷(50毫升)和水(50毫升)，分液后的有机相用饱和碳酸氢钠水溶液50毫升(50毫升×1)、水50毫升(50毫升×1)和饱和食盐水50毫升(50毫升×1)依次洗涤，无水硫酸钠干燥，过滤后减压浓缩得粗品。经柱层析(SiO 2，二氯甲烷/甲醇(含0.1％三乙胺)＝20/1至10/1)纯化后得2-15。 1H NMR(400MHz，DMSO-d 6)：ε7.90-7.82(m，6H)，7.78(br d，J＝4.8Hz，3H)，7.40-7.26(m，10H)，6.91(brdd，J＝3.1，9.0Hz，4H)，5.26(d，J＝3.4Hz，3H)，5.03-4.99(m，3H)，4.53(d，J＝8.4Hz，3H)，4.43(br d，J＝3.8Hz，1H)，4.23-4.14(m，1H)，4.12-4.02(m，9H)，3.92(td，J＝9.0，11.0Hz，3H)，3.78(s，6H)，3.77-3.71(m，3H)，3.66-3.51(m，13H)，3.49-3.41(m，4H)，3.11-3.01(m，16H)，2.38-2.37(m，1H)，2.32(br s，9H)，2.14(s，9H)，2.08(br t，J＝6.9Hz，7H)，2.04(s，9H)，1.93(s，9H)，1.82(s，9H)，1.57-1.46(m，22H)，1.31-1.26(m，12H)。
步骤L：向式2-15(0.80克，0.33毫摩尔)所示化合物的二氯甲烷(8毫升)溶液依次加入三乙胺(67.24毫克，0.64毫摩尔)，4-N，N-二甲基氨基吡啶(0.12克，1.00毫摩尔)和丁二酸酐(83.13毫克，0.83毫摩尔)。混合液在10摄氏度搅拌16小时。反应液加入二氯甲烷(50毫升)，水(30毫升)和饱和食盐水(30毫升)，分液后的有机相用水30毫升(30毫升×1)和饱和食盐水30毫升(30毫升×1)依次洗涤，无水硫酸钠干燥，过滤后减压浓缩得粗品。经p-HPLC纯化(分离柱：Waters Xbridge C18(规格：150mm×50mm，粒径：10μm)；流动相：[水(10mM碳酸氢铵)-乙腈]；洗脱梯度：27％-57％，11min)得实施例2(化合物D01)。 1H NMR(400MHz，DMSO-d 6)：ε7.96-7.69(m，9H)，7.33-7.09(m，10H)，6.90-6.78(m，4H)，5.21(d，J＝3.3Hz，3H)，4.97(dd，J＝3.3，11.2Hz，3H)，4.49(d，J＝8.4Hz，3H)，4.06-3.97(m，9H)，3.91-3.83(m，3H)，3.79-3.66(m，11H)，3.63-3.45(m，18H)，3.02(br d，J＝4.6Hz，14H)，2.46-2.37(m，4H)，2.35-2.14(m，12H)，2.10(s，9H)，2.04(brt，J＝7.0Hz，6H)，1.99(s，9H)，1.88(s，9H)，1.77(s，9H)，1.57-1.37(m，22H)，1.22(br s，12H)。
实施例3 双链siRNA类似物或其缀合物的合成
含D的单链寡核糖核苷酸的合成：按照亚磷酰胺固相合成技术合成寡核糖核苷酸。在可控多孔玻璃(氨基CPG，
Figure PCTCN2021098682-appb-000029
)与D01通过共价键连接制成的固体支持物上进行合成。所 有的2’-修饰的RNA亚磷酰胺(phosphoramidite)和辅助试剂均为商品化可得试剂。所有的酰胺溶于无水乙腈中并且加入分子筛
Figure PCTCN2021098682-appb-000030
使用5-乙基硫-1H-四唑(ETT)作为活化剂的偶合时间为5分钟。使用50mM 3-((二甲基氨基-亚甲基)氨基)-3H-1，2，4-二噻唑-3-硫酮(DDTT)的无水乙腈/吡啶(v/v＝1/1)溶液产生硫代磷酸酯键，反应时间3分钟。所有序列在最后脱除DMT基团后即合成。
不含D的单链寡核糖核苷酸的合成：按照亚磷酰胺固相合成技术合成寡核糖核苷酸。在通用可控多孔玻璃CPG
Figure PCTCN2021098682-appb-000031
上进行合成。所有的2’-修饰的RNA亚磷酰胺(phosphoramidite)和辅助试剂均为商品化可得试剂。所有的酰胺溶于无水乙腈中并且加入分子筛
Figure PCTCN2021098682-appb-000032
使用5-乙基硫-1H-四唑(ETT)作为活化剂的偶合时间为5分钟。使用50mM 3-((二甲基氨基-亚甲基)氨基)-3H-1，2，4-二噻唑-3-硫酮(DDTT)的无水乙腈/吡啶(v/v＝1/1)溶液产生硫代磷酸酯键，反应时间3分钟。所有序列在最后脱除DMT基团后即合成。
CPG上结合的低聚体的切割和去保护：在固相合成终止后，通过用含20％二乙胺的乙腈溶液处理30分钟去除保护基，而没有从CPG上切下寡核苷酸。随后，干燥的CPG在40摄氏度度下用浓氨水处理18小时。在离心之后，上清液被转移至新的管中并且用氨水洗涤CPG。浓缩合并的溶液得到固体混合物。
单链寡核糖核苷酸的纯化：通过使用NanoQ阴离子交换经HPLC纯化的低聚体。缓冲液A是10mM高氯酸钠溶液，20mM Tris，1mM EDTA，pH 7.4和含有乙腈20％，以及缓冲液B，500mM高氯酸钠，20mMTris，1mMEDTA，pH7.4和含有乙腈20％。分离得到目标产物，并用反相C18柱脱盐。
单链寡核糖核苷酸的退火产生siRNA：把待退火的单链寡核糖核苷酸用无菌RNase Free H 2O(无RNA水解酶)配制成200μM。如下设置退火反应体系，将总体积为100μL的混合液，10nmol放置95℃水浴锅10分钟(≥100nmol需求量需要高温20分钟)→迅速放入60℃水浴，自然降温→退火完成后的溶液不可放置在高温中储存。通过合并等摩尔的单链寡核糖核苷酸溶液形成互补链。
表1靶向乙型肝炎病毒基因的双链siRNA类似物、包含其的缀合物和它们对应的核心序列
Figure PCTCN2021098682-appb-000033
Figure PCTCN2021098682-appb-000034
Figure PCTCN2021098682-appb-000035
*：D为小分子片段D01进行化学反应后的残基，通过共价键与核酸结合，其结构如下：
Figure PCTCN2021098682-appb-000036
**：r’嵌入的序列的反义链序列是在核心序列的具有3’端UU的反义链序列的基础上进行r’嵌入得到的。例如，SEQ ID NO：4是在具有3’端UU的SEQ ID NO：2的基础上进行r’嵌入得到的。
***：当序列含有D时，所述D用于指代缀合物基团D的连接位置。例如g·r·gu Gc ACUucgcuucacaD(5’-3’)表示如SEQ ID NO：16所示的序列g·r·gu Gc ACUucgcuucaca在3’端与D连接。
实施例3 HBV体外测试
1.实验目的：
通过酶联免疫吸附测定(ELISA)检测HepG2-NTCP细胞培养上清中HBV抗原(HBsAg和HBeAg)含量，以化合物的EC 50值为指标，来评价化合物对HBV的抑制活性；同时通过Cell-titer Glo检测细胞活力，来评价化合物的细胞毒性。
2.实验材料：
2.1细胞系：HepG2-NTCP细胞
HepG2-NTCP细胞培养基(DMEM，Invitrogen-11330032；10％血清，Invitrogen-10099141；100units/ml青霉素和100μg/ml链霉素，Hyclone-SV30010；1％非必需氨基酸，Invitrogen-11140050；2mM L-谷氨酰胺，Invitrogen-25030081；1mM丙酮酸钠，Gibco-11360-070；500μg/ml Geneticin，Invitrogen-10131027)
2.2试剂：
胰酶(Invitrogen-25300062)；DPBS(Corning-21031CVR)；DMSO(Sigma-D2650-100ML)；Cell-titer Glo(Promega-G7573)；乙型肝炎表面抗原定量检测试剂盒(安图生物-CL 0310)；乙型肝炎e抗原定量检测试剂盒(安图生物-CL 0312)。
2.3耗材与仪器：
96孔细胞培养板(Corning-3599)；CO 2培养箱(HERA-CELL-240)
酶标仪(BioTek Synergy 2)
3.实验步骤和方法：
3.1第0天，种HepG2-NTCP(7.5×10 4细胞/孔)细胞到48孔板，在37℃，5％CO 2培养过夜。
3.2第1天，更换含1％DMSO的培养基。
3.3第2天，用D型HBV(从HepG2.2.15细胞培养上清浓缩)感染HepG2-NTCP(2000GE/细胞)。
3.4第3天，吸掉感染液，加入新鲜的含1％DMSO的培养基。
3.5第6天，根据
Figure PCTCN2021098682-appb-000037
RNAiMax(Invitrogen公司)的使用说明，转染siRNA缀合物。缀合物5倍梯度稀释7个浓度，三复孔，终浓度为0.16pM。化合物为正义链、反义链的组合，为单一化学实体，最大浓度为2.5nM。
3.6第12天，收取培养孔中的上清，ELISA测定HBV表面抗原和e抗原。待收集上清后，加入Cell-titer Glo测定细胞活力。
3.7 ELISA测定乙肝病毒表面抗原(HBsAg)和e抗原(HBeAg)，具体步骤参照该产品说明书，步骤简述如下：取50μl样品和标准品分别加入到反应板中，再每孔分别加入50μl酶 结合物，震荡混匀，37℃孵育60分钟，然后用洗液洗板5次，再每孔加入50μl发光底物，混匀，室温避光反应10分钟，最后用酶标仪检测化学发光强度。
3.8数据分析：
计算细胞活力百分比：
％viability＝(样品的发光值-培养基对照发光值)/(DMSO对照的发光值-培养基对照发光值)×100。
计算HBV表面抗原和e抗原的抑制百分比：
％Inh.＝(1-样品中的抗原值/DMSO对照抗原值)×100。
计算CC 50和EC 50：使用GraphPad Prism软件计算化合物的CC 50和对HBV的50％抑制浓度(EC 50)值。
4.实验结果：见表2。
表2测试的序列在细胞中降低HBsAg和HBeAg水平实验结果
Figure PCTCN2021098682-appb-000038
＊测试的样品是双链siRNA类似物的缀合物。
实施例4：重组8型腺相关病毒载体介导的乙型肝炎病毒小鼠模型(AAV-HBV)中抗乙肝病毒活性与安全性研究
实验目的：
AAV载体介导的HBV转染小鼠模型是一种快速、高效的HBV模型。利用AAV8载体的高度嗜肝性，用携带1.3拷贝HBV基因组的重组8型腺相关病毒(rAAV8-1.3HBV)经小鼠尾静脉注射，可将所携带的1.3拷贝HBV基因组高效导入肝细胞。由于AAV病毒载体的特性，其介导的载体可以持续表达很长时间，应用AAV/HBV模型可以在小鼠肝脏内持续复制HBV DNA并表达HBsAg和HBeAg。
通过运用AAV/HBV小鼠模型，检测受试化合物治疗后的小鼠血清中HBsAg、HBeAg、DNA、pgRNA和小鼠体重，以评价其体内抗HBV效果与安全性。
实验材料：
C57BL/6小鼠、PBS(RNase free)为溶媒、受试化合物、重组病毒rAAV8-1.3HBV。本项目主要试剂包括QIAamp96 DNA试剂盒(Qiagen，51162)、FastStart Universal Probe Master(Rox)(Roche，04914058001)、乙型肝炎病毒表面抗原检测试剂盒(安图生物，CL0310)、乙型肝炎病毒e抗原检测试剂盒(安图生物，CL0918)、PureLink TM Pro 96 Viral RNA/DNA kit(Invitrogen，12280-096A)和FastQuant RT Kit(with gDNase)(TIANGEN，KR106-02)。主要仪器包括：离心机(Beckman Allegra X-15R)、多功能酶标仪(BioTek，Synergy 2)、荧光定量PCR仪(Applied Biosystems，7900HT Fast Real-time PCR system)、酶标仪(Molecular Devices，SpectraMax 340PC384)。
实验方法：
a)鼠在病毒注射后第34天开始皮下注射给药，将该天设为第0天。给药前所有小鼠颌下采血收集血浆。具体给药方案见表3。
b)鼠给药后第0，14，21，28天和32天经颌下静脉采血收集血浆，收集的血样用K 2-EDTA抗凝，在4℃，7000g/分钟条件下离心10分钟后收集血浆。具体采血时间见表3。
c)35天或第42天，所有小鼠经颌下静脉采血收集血浆，之后小鼠经CO 2吸入安乐死，经心脏采血收集血浆样品，并收集肝脏样品。
d)浆样品送至检测。
表3体内实验方案
Figure PCTCN2021098682-appb-000039
Figure PCTCN2021098682-appb-000040
*1：WRG01为缀合物，其正义链为SEQ ID NO：16，反义链为SEQ ID NO：23，缀合物基团为D。
*2：WR007为缀合物，其正义链为SEQ ID NO：42，反义链为SEQ ID NO：23，缀合物基团为D。
*3：WR012为缀合物，其正义链为SEQ ID NO：16，反义链为SEQ ID NO：47，缀合物基团为D。
/：尚未到终点。
样品分析：
ELISA检测小鼠血清中HBsAg、HBeAg的含量：实验步骤参照HBsAg ELISA(安图生物，CL 0310)和HBeAg ELISA(安图生物，CL0918)试剂盒说明书。
qPCR检测小鼠血浆中HBV DNA含量：提取血浆中HBV DNA，实验步骤参照QIAamp 96 DNA Blood Kit说明书，qPCR检测小鼠血浆中HBV DNA的含量。
RT-qPCR检测小鼠血浆中HBV pgRNA含量：提取血浆中HBV pgRNA，实验步骤参照PureLink TM Pro 96 Viral RNA/DNA Kit说明书。消化DNA，并使用含有乙型肝炎病毒特异性序列的3′RACE引物将RNA逆转录为cDNA，实验步骤参照FastQuant RT Kit(with gDNase)的说明书。最后，qPCR定量检测cDNA的含量，即检测小鼠血浆中HBV pgRNA的含量。
每组小鼠样品的平均值±标准误表示，除特别说明外，n＝5。用Student’s t-test进行统计分析。
实验结果：
a)血清中HBsAg含量评价受试化合物在AAV/HBV小鼠模型中抗HBV活性。结果见表4、表4-1、图1和图6。小鼠血浆HBsAg含量由ELISA法测定。误差线显示标准误。第0天：第一次给所有小鼠施用媒介物或化合物。第29天：对实验组WRG01小鼠和该次空白对照组小鼠第二次接种媒介物或化合物。
表4小鼠给药后不同日期的Log 10[HBsAg(IU/mL)]
检测天数(天) 空白(SC) WRG01(SC)
0 4.70 4.72
7 4.82 2.90
14 4.43 2.90
21 4.94 3.28
28 4.84 3.77
35 4.78 2.83
表4-1小鼠给药后不同日期的Log 10[HBsAg(IU/mL)]
检测天数(天) 空白(SC) WR007(SC) WR012(SC)
0 4.58 4.19 4.47
7 4.15 1.92 2.00
14 4.57 2.29 2.20
21 4.41 2.63 2.36
28 4.76 2.94 3.10
35 4.62 3.31 3.19
b)清中HBeAg含量评价受试化合物在AAV/HBV小鼠模型中抗HBV活性。结果见表5、表5-1、图2和图7。小鼠血浆HBeAg含量由ELISA法测定。误差线显示标准误差。第0天：第一次给所有小鼠施用媒介物或化合物。
表5小鼠给药后不同日期的Log 10[HBeAg(PEIU/mL)]
检测天数(天) 空白(SC) WRG01(SC)
0 3.56 3.51
7 3.37 2.89
14 3.56 3.06
21 3.66 3.22
表5-1小鼠给药后不同日期的Log 10[HBeAg(PEIU/mL)]
检测天数(天) 空白(SC) WR007(SC) WR012(SC)
0 3.44 3.35 3.40
7 3.24 2.49 2.53
14 3.57 2.80 2.89
21 3.32 2.81 2.82
28 3.38 2.95 2.91
35 3.37 3.09 3.02
c)血清中DNA含量评价受试化合物在AAV/HBV小鼠模型中抗HBV活性。结果见表6、表6-1、图3和图8。小鼠血浆HBV DNA含量由定量PCR法测定。误差线显示标准误差。第0天：第一次给所有小鼠施用媒介物或化合物。第29天：对所有小鼠第二次接种媒介物或化合物。
表6小鼠给药后不同日期的Log 10[DNA(拷贝数/μL)]
检测天数(天) 空白(SC) WRG01(SC)
0 5.27 4.84
7 5.39 3.93
14 5.51 3.97
21 5.63 4.37
表6-1小鼠给药后不同日期的Log 10[DNA(拷贝数/μL)]
检测天数(天) 空白(SC) WR007(SC) WR012(SC)
0 5.53 / /
7 4.98 / /
14 5.34 3.44 3.95
21 5.45 3.71 4.21
28 5.63 4.08 4.66
35 5.26 4.42 4.78
/：未得到数据。
d)中pgRNA含量评价受试化合物在AAV/HBV小鼠模型中抗HBV活性。结果见表7和图4。小鼠血浆HBVpgRNA含量由定量PCR法测定。误差线显示标准误差。第0天：第一次给所有小鼠施用媒介物或化合物。第29天：对所有小鼠第二次接种媒介物或化合物。
表7小鼠给药后不同日期的Log 10[pgRNA(拷贝数/μL)]
检测天数(天) 空白(SC) WRG01(SC)
0 4.92 4.56
7 4.96 3.28
14 4.93 3.26
21 5.02 3.50
28 5.06 4.13
35 5.17 3.37
e)体重变化见图5。以第0天的体重作为基准进行比较，根据IACUC规定，以20％体重下降作为人道终点，任何小鼠体重下降超过20％，需从实验中移除。此实验中无小鼠因体重下降而移除。
实验结论：
在本实验中，受试化合物在AAV/HBV小鼠模型实验中，能够显著降低HBsAg、DNA和pgRNA。同时，受试化合物对HBeAg也有一定抑制作用。受试化合物治疗过程中，小鼠表现出良好的耐受性，体重逐渐上升。
实施例5 HepG2.2.15细胞HBV体外测试
1.实验目的：
通过实时定量qPCR试验(real time-qPCR)检测HepG2.2.15细胞培养上清的HBV DNA含量，及酶联免疫吸附测定(ELISA)检测HBV表面抗原和e抗原含量；qRT-PCR检测细胞内HBVRNA含量，以化合物的EC50值为指标，来评价化合物对HBV的抑制作用，同时应用CCK8法检测受试化合物对细胞活性影响。
2.实验材料：
2.4胞系：HepG2.2.15细胞
HepG2.2.15细胞培养基(DMEM/F12，Invitrogen-11330032；10％血清，Hyclone-SV30087.0；100units/ml青霉素和100μg/ml链霉素，Hyclone-SV30010；1％非必需氨基酸，Invitrogen-11140050：2mM L-GLUTAMINE，Invitrogen-25030081：300μg/ml Geneticin，Invitrogen-10131027)。
2.5试剂
Opti-MEM(Gibco-31985-070)：
Figure PCTCN2021098682-appb-000041
RNAiMAX(Invitrogen-13778-150)：CCK8(李记-AC11L057)；高通量DNA纯化试剂盒(QIAamp 96 DNA Blood Kit，Qiagen-51162)；RNA制备RNEASY试剂盒(RNeasy 96 Kit(12)，Qiagen-74182)；定量快速启动通用探针试剂(FastStart Universal Probe Master，Roche-04914058001)；FastKing cDNA第一链合成试剂盒(TianGen-KR106-02)；乙型肝炎表面抗原定量检测试剂盒(安图生物，CL0310)；乙型肝炎e抗原定量检测试剂盒(安图生物，CL 0312)。
2.6耗材与仪器：
Collagen I 96 Well White/Clear Flat Bottom TC-Treated Microplate(Corning BioCoat-356650)： CO 2培养箱(HERA-CELL-240)；荧光定量PCR仪(Applied Biosystems-7900 real time PCR system)；荧光定量PCR仪(Applied Biosystems-QuantStudio 6 Flex)；酶标仪(Molecular Device-SpectraMax M2e)；酶标仪(BioTek-Synergy 2)。
3.实验步骤和方法：
3.1第一天，转染siRNA和细胞铺板同时进行，方法简述如下：取HepG2.2.15细胞，先用DPBS洗涤后，0.05％胰蛋白酶进行消化，用含10％FBS的DMEM/F12培养基终止消化，离心后重悬，将细胞轻轻吹打至单个细胞后计数。按比例配置所需转染试剂体积(表8)，室温孵育15分钟。
表8
Figure PCTCN2021098682-appb-000042
RNAiMAX配置
Figure PCTCN2021098682-appb-000043
梯度稀释siRNA，共8个浓度，3倍梯度稀释，双复孔，取15μL RNAiMAX/Opti-MEM的混合液，与15μL不同浓度的siRNA混匀，室温孵育15分钟。先取10μL上述混合溶液加入到96孔细胞培养板中，再加入90μl细胞悬液，细胞终密度为15,000细胞/孔，最终体积为100μL/孔，细胞置于5％CO 2、37℃孵箱中培养。
3.2第四天，更换含有化合物的新鲜培养液，转染方式同第一天。
3.3第七天收取培养孔中的培养液，取部分样品ELISA测定乙肝病毒S抗原和e抗原的含量；取部分样品使用高通量DNA纯化试剂盒(Qiagen-51162)提取DNA；待收集上清后，参照CCK-8试剂盒说明书测定细胞活力，用酶标仪(SpectraMax M2e)检测各孔的吸光值(450nm/650nm)；使用RNeasy 96 kit提取试剂盒(Qiagen-74182)，参照试剂盒说明书提取细胞培养内HBV RNA。
3.4 PCR反应液的配制如表9所示：
表9 PCR反应液的配制
Figure PCTCN2021098682-appb-000044
Figure PCTCN2021098682-appb-000045
在96孔PCR板中每孔加入8μl的反应混合液，然后每孔加入2μL的样品DNA或HBV DNA的标准品。
PCR的反应条件为：95℃加热10分钟，然后95℃变性15秒，60℃延伸1分钟，共40个循环。
3.5 ELISA测定乙肝病毒S抗原和e抗原的含量，具体步骤参照该产品说明书，步骤简述如下：取50μL样品和标准品分别加入到反应板中，再每孔分别加入50μL酶结合物，震荡混匀，37℃温浴60分钟，然后用洗液洗板5次，再每孔加入50μL发光底物，混匀，室温避光反应10分钟，最后用酶标仪检测化学发光强度。
3.6使用RNeasy 96 kit提取试剂盒(Qiagen，74182)，参照试剂盒说明书提取细胞培养内HBV RNA。加150μL RLT裂解细胞，最终用50μL无RNase水洗脱RNA。参照反转录试剂盒(天根，KR106)说明书，加入随机引物反转录成cDNA，然后HBV特异性引物检测样品中的total RNA，同时，GAPDH引物和探针特异性检测GAPDH cDNA，qPCR法定量样品中的HBV cDNA。
qPCR反应：95℃，10分钟；95℃，15秒，60℃，1分钟，40个循环。依据各样品的Ct值计算样品中的HBV RNA含量。
每个样品目的基因HBV mRNA的表达水平通过ΔΔCt相对定量法进行计算。目的基因相对表达量用2-ΔΔCT表示，计算公式如下：
ΔCT＝目的基因平均Ct值-内参基因平均Ct值；
ΔΔCT＝ΔCT(加药组)组-ΔCT(RNAiMAX对照组)；
HBV mRNA相对表达量＝2-ΔΔCT
3.7数据分析：
计算抑制百分比：
％Inh.＝(1-样品中的值/PBS对照值)x100。
细胞活率％＝(样品检测值-培养液本底平均检测值)/(对照组平均检测值-培养液本底平均检测值)×100
计算EC 50和CC 50：使用GraphPad Prism软件计算化合物对HBV的50％抑制浓度(EC 50)值和50％细胞死亡时的药物浓度(CC 50)值。
表10测试的序列在细胞中降低HBsAg、HBeAg、DNA和RNA水平实验结果
Figure PCTCN2021098682-appb-000046
Figure PCTCN2021098682-appb-000047
/：尚未得到数据。
＊测试的样品是双链siRNA类似物的缀合物。
实施例6：AAV-HBV小鼠模型中抗乙肝病毒活性药效剂量探索
通过运用AAV/HBV小鼠模型，检测接受不同剂量受试化合物治疗后的小鼠血清中HBsAg，以评价其体内抗HBV效果。
实验材料：
C57BL/6小鼠、PBS(RNase free)为溶媒、受试化合物、重组病毒rAAV8-1.3HBV。本项目主要试剂包括FastStart Universal Probe Master(Rox)(Roche，04914058001)、乙型肝炎病毒表面抗原检测试剂盒(安图生物，CL0310)。主要仪器包括：离心机(Beckman Allegra X-15R)、多功能酶标仪(BioTek，Synergy 2)、酶标仪(Molecular Devices，SpectraMax 340PC384)。
实验方法：
a)所有小鼠在病毒注射后第34天开始皮下注射给药，将该天设为第0天。给药前所有小鼠颌下采血收集血浆。第0天给药一次。具体给药方案见表14。
b)所有小鼠给药后第0，14，21，28天和35天经颌下静脉采血收集血浆，收集的血样用K 2-EDTA抗凝，在4℃，7000g/分钟条件下离心10分钟后收集血浆。具体采血时间见表11。
c)第42天，所有小鼠经颌下静脉采血收集血浆，之后小鼠经CO 2吸入安乐死，经心脏采血收集血浆样品，并收集肝脏样品。
d)所有血浆样品送至检测。
表11体内实验方案
Figure PCTCN2021098682-appb-000048
*：WRG01为缀合物，其正义链为SEQ ID NO：16，反义链为SEQ ID NO：23，缀合物基团为D。
/：尚未到终点。
样品分析：
ELISA检测小鼠血清中HBsAg的含量：实验步骤参照HBsAg ELISA(安图生物，CL0310)试剂盒说明书。
每组小鼠样品的平均值±标准误表示，除特别说明外，n＝5。用Student’s t-test进行统计分析。
实验结果：
检测血清中HBsAg含量评价受试化合物在AAV/HBV小鼠模型中抗HBV活性。结果见表12和图9。小鼠血浆HBsAg含量由ELISA法测定。误差线显示标准误。第0天：第一次给所有小鼠施用媒介物或化合物。
表12小鼠给药后不同日期的Log 10[HBsAg(IU/mL)]
Figure PCTCN2021098682-appb-000049
实验结论：
在本实验中，受试化合物WRG01在AAV/HBV小鼠模型实验中，对于降低HBsAg展现出良好的剂量依赖性，即随着用药剂量上升降低HBsAg活性增加，并展现出长期抑制HBsAg效力。
实施例7：小鼠血浆、肝脏、肾脏药物浓度测试
本研究对C57BL/6小鼠单次皮下注射给药，在给药后的不同时间点收集血浆和组织样品，通过SL-qPCR检测血浆和组织中siRNA水平评价化合物在小鼠体内的代谢水平。
表13体内实验方案
Figure PCTCN2021098682-appb-000050
Figure PCTCN2021098682-appb-000051
*：WRG01为缀合物，其正义链为SEQ ID NO：16，反义链为SEQ ID NO：23，缀合物基团为D。
/：没有在“非终点”的时间采血，只在终点采血。
实验结果：
使用SL-qPCR法(参考文献：Nair et al.Nucleic Acids Research(2017)，45，10969-10977) 检测小鼠给药后不同时间节点血浆、肝脏、肾脏中siRNA水平，结果见图10。
实验结论：
在本实验中，受试化合物WRG01在C57BL/6小鼠模型实验中具有良好的组织分布与代谢稳定性。WR-G01肝暴露量大、半衰期长，肝血比超过500倍，证明WRG01代谢稳定，具有高度肝靶向性。
实施例8：FRG-KO人源化肝脏小鼠血生化测试
人源化FRG小鼠是最常用的人源化肝脏模型之一，通常人源化比例高达70％。因为在小鼠肝脏中定植的是人类肝脏细胞，可以更好模拟人体HBV自然感染和cccDNA复制过程，同时对于人体药代动力学与肝脏毒性有着良好的预测作用。
本研究对人源化FRG小鼠多次给药，在给药后的不同时间点收集血浆样品，通过检测血浆中ALT、AST和胆红素水平评价化合物对小鼠的肝脏毒副作用影响。在本实验中，受试化合物对人源化肝脏未产生明显的炎症反应，预示人体中安全性良好。
表14体内实验方案
Figure PCTCN2021098682-appb-000052
*：WRG01为缀合物，其正义链为SEQ ID NO：16，反义链为SEQ ID NO：23，缀合物基团为D。
/：尚未到终点。
本公开展现出不可预料的优秀的HBsAg和HBeAg抑制剂活性，同时可以有效抑制HBV DNA和pgRNA表达，其说明了可使乙肝病毒的活跃度受到抑制，同时具有良好的组织分布与代谢稳定性，具有高度肝靶向性，预计对小鼠肝功能影响小，将为临床中乙型肝炎例如慢性乙型肝炎提供了一种高效的治疗手段。

Claims (20)

  1. 一种双链siRNA类似物、其缀合物或其盐，其包含正义链和反义链，其中所述反义链包含在如SEQ ID NO：2所示的序列中使用r替换了一个或多个核苷酸残基的序列，所述r为
    Figure PCTCN2021098682-appb-100001
    其中所述siRNA类似物中的核苷酸和r中的每一个独立地是经修饰的或未修饰的。
  2. 根据权利要求1所述的双链siRNA类似物、其缀合物或其盐，其中所述双链siRNA类似物中的70％、75％、80％、85％、90％或95％以上的核苷酸和r都是经修饰的；任选地，所述双链siRNA类似物中的所有核苷酸和r都是经修饰的。
  3. 根据权利要求1或2所述的双链siRNA类似物、其缀合物或其盐，其中所述修饰包括甲氧基修饰、氟代修饰、硫代磷酸酯基连接、将核苷酸替换为(S)-甘油核酸或将核苷酸替换(E)-乙烯基磷酸酯。
  4. 根据权利要求1-3任一项所述的双链siRNA类似物、其缀合物或其盐，其中反义链包含在如SEQ ID NO：2所示的序列中使用r替换了一个、两个、三个、四个或五个核苷酸残基的序列；任选地，所述反义链包含在如SEQ ID NO：2所示的序列中使用r替换了一个核苷酸残基的序列。
  5. 根据权利要求1-4任一项所述的双链siRNA类似物、其缀合物或其盐，其中所述r替换发生在所述SEQ ID NO：2的任意位置。
  6. 根据权利要求1-5任一项所述的双链siRNA类似物、其缀合物或其盐，其中所述SEQ ID NO：2任选地包括在5’端和/或3’端的突出端；任选地，所述SEQ ID NO：2包括在5’端和/或3’端的0、1、2、3、4或5个核苷酸的突出端；任选地，所述SEQ ID NO：2包括在3’端的突出端，所述突出端选自经修饰的或未修饰的UU。
  7. 根据权利要求1-6任一项所述的双链siRNA类似物、其缀合物或其盐，其中所述反义链包含如SEQ ID NO：4或SEQ ID NO：17、SEQ ID NO：6或SEQ ID NO：19、SEQ ID NO：7或SEQ ID NO：20、SEQ ID NO：8或SEQ ID NO：21、SEQ ID NO：9或SEQ ID NO：22、SEQ ID NO：10或SEQ ID NO：23、SEQ ID NO：11或SEQ ID NO：24、SEQ ID  NO：29或SEQ ID NO：33、SEQ ID NO：30或SEQ ID NO：34、SEQ ID NO：31或SEQ ID NO：35、或SEQ ID NO：32或SEQ ID NO：36、SEQ ID NO：39或SEQ ID NO：44、SEQ ID NO：10或SEQ ID NO：45、SEQ ID NO：40或SEQ ID NO：46、SEQ ID NO：10或SEQ ID NO：47、或SEQ ID NO：10或SEQ ID NO：48所示的序列，或由其组成。
  8. 根据权利要求1-7中任一项所述的双链siRNA类似物、其缀合物或其盐，其中所述正义链包含如SEQ ID NO：1或SEQ ID NO：28所示的序列，或由其组成。
  9. 根据权利要求1-8中任一项所述的双链siRNA类似物、其缀合物或其盐，其中所述正义链包含在如SEQ ID NO：1所示的序列中使用r替换了一个或多个核苷酸残基的序列；任选地，所述正义链包含在如SEQ ID NO：1所示的序列中使用r替换了一个、两个、三个、四个或五个核苷酸残基的序列。
  10. 根据权利要求1-9任一项所述的双链siRNA类似物、其缀合物或其盐，其中所述r替换发生在SEQ ID NO：1的5’端的第1～第19位。
  11. 根据权利要求1-10任一项所述的双链siRNA类似物、其缀合物或其盐，其中所述正义链的序列包含如SEQ ID NO：5或SEQ ID NO：18、SEQ ID NO：3或SEQ ID NO：16、SEQ ID NO：14或SEQ ID NO：27、SEQ ID NO：13或SEQ ID NO：26、SEQ ID NO：12或SEQ ID NO：25、SEQ ID NO：37或SEQ ID NO：42、或SEQ ID NO：38或SEQ ID NO：43所示的序列，或由其组成。
  12. 根据权利要求1-11任一项所述的双链siRNA类似物、其缀合物或其盐，其中所述双链siRNA类似物为S18～S28中的任意一种：
    S18：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：4或SEQ ID NO：17，
    S19：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：6或SEQ ID NO：19，
    S20：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：7或SEQ ID NO：20，
    S21：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：8或SEQ ID NO：21，
    S22：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：9或SEQ ID NO：22，
    S23：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：10或SEQ ID NO：23，
    S24：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：11或SEQ ID NO：24，
    S25：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：29或SEQ ID NO：33，
    S26：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：30或SEQ ID NO：34，
    S27：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：31或SEQ ID NO：35，
    S28：正义链为SEQ ID NO：1或SEQ ID NO：28、反义链为SEQ ID NO：32或SEQ ID NO：36，
    或者，
    其中所述双链siRNA类似物为S1～S17中的任意一种：
    S1：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：4或SEQ ID NO：17，
    S2：正义链为SEQ ID NO：5或SEQ ID NO：18、反义链为SEQ ID NO：4或SEQ ID NO：17，
    S3：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：6或SEQ ID NO：19，
    S4：正义链为SEQ ID NO：5或SEQ ID NO：18、反义链为SEQ ID NO：6或SEQ ID NO：19，
    S5：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：7或SEQ ID NO：20，
    S6：正义链为SEQ ID NO：5或SEQ ID NO：18、反义链为SEQ ID NO：7或SEQ ID NO：20，
    S7：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：8或SEQ ID NO：21，
    S8：正义链为SEQ ID NO：5或SEQ ID NO：18、反义链为SEQ ID NO：8或SEQ ID NO：21，
    S9：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：9或SEQ ID NO：22，
    S10：正义链为SEQ ID NO：5或SEQ ID NO：18、反义链为SEQ ID NO：9或SEQ ID  NO：22，
    S11：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：10或SEQ ID NO：23，
    S12：正义链为SEQ ID NO：5或SEQ ID NO：18、反义链为SEQ ID NO：10或SEQ ID NO：23，
    S13：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：11或SEQ ID NO：24，
    S14：正义链为SEQ ID NO：5或SEQ ID NO：18、反义链为SEQ ID NO：11或SEQ ID NO：24，
    S15：正义链为SEQ ID NO：12或SEQ ID NO：25、反义链为SEQ ID NO：4或SEQ ID NO：17，
    S16：正义链为SEQ ID NO：13或SEQ ID NO：26、反义链为SEQ ID NO：4或SEQ ID NO：17，
    S17：正义链为SEQ ID NO：14或SEQ ID NO：27、反义链为SEQ ID NO：4或SEQ ID NO：17，
    或者，
    其中所述双链siRNA类似物为S29～S35中的任意一种：
    S29：正义链为SEQ ID NO：37或SEQ ID NO：42、反义链为SEQ ID NO：10或SEQ ID NO：23，
    S30：正义链为SEQ ID NO：38或SEQ ID NO：43、反义链为SEQ ID NO：10或SEQ ID NO：23，
    S31：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：39或SEQ ID NO：44，
    S32：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：10或SEQ ID NO：45，
    S33：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：40或SEQ ID NO：46，
    S34：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：10或SEQ ID NO：47，
    S35：正义链为SEQ ID NO：3或SEQ ID NO：16、反义链为SEQ ID NO：10或SEQ ID NO：48。
  13. 根据权利要求1-12中任一项所述的双链siRNA类似物的缀合物或其盐，其中所述双链siRNA类似物和药学上可接受的缀合基团连接，所述药学上可接受的缀合基团包括GalNAc基团；任选地，所述药学上可接受的缀合基团含有1至5个GalNAc基团。
  14. 根据权利要求1-13所述的双链siRNA类似物的缀合物或其盐，其中所述双链siRNA类似物和药学上可接受的缀合基团连接，所述药学上可接受的缀合基团包含化合物基团D
    Figure PCTCN2021098682-appb-100002
  15. 根据权利要求13或14所述的双链siRNA类似物的缀合物或其盐，其中所述药学上可接受的缀合基团连接至所述双链siRNA类似物的正义链的3’末端。
  16. 根据权利要求1-15任一项所述的双链siRNA类似物、其缀合物或其盐，其中所述双链siRNA类似物或其缀合物的硫代磷酸酯部分包括(R)-和(S)-对映体、非对映异构体、和/或其外消旋混合物。
  17. 根据权利要求1-16任一项所述的双链siRNA类似物、其缀合物或其盐，其中所述盐选自碱加成盐、酸加成盐及其组合；任选地，所述碱加成盐选自钠、钾、钙、铵、有机胺、镁盐及其组合，所述酸加成盐选自无机酸盐、有机酸盐及其组合；任选地，所述无机酸选自盐酸、氢溴酸、硝酸、碳酸、碳酸氢根、磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸及其组合，所述有机酸选自乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸、甲磺酸及其组合。
  18. 药物组合物，其包含根据权利要求1-17任一项所述的双链siRNA类似物、其缀合物或其盐，任选地包括药学上可接受的载体或赋形剂。
  19. 权利要求1-17任一项所述的双链siRNA类似物、其缀合物或其盐、或权利要求18所述的药物组合物在制备用于治疗乙型肝炎的药物中的用途。
  20. 治疗受试者中乙型肝炎的方法，其包括将权利要求1-17任一项所述的双链siRNA类似物、其缀合物或其盐、或权利要求18所述的药物组合物施用于所述受试者的步骤。
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