CONJUGATE OF DOUBLE-STRANDED SIRNA ANALOGUE

Abstract

Provided are a double-stranded siRNA analogue embedded with a ribavirin derivative, a conjugate containing same, and a salt and the use thereof. The provided double-stranded siRNA analogue, the conjugate containing same and the salt thereof can effectively inhibit multiple viral indicators such as hepatitis B virus DNA, pgRNA, S antigen, and E antigen, which provide an effective and feasible method for treating hepatitis B.

Claims

1. A double-stranded siRNA analogue, a conjugate thereof or a salt thereof, comprising: a sense strand and an antisense strand, wherein the antisense strand comprises a sequence obtained by replacing one or more nucleotide residues in a sequence set forth in SEQ ID NO: 2 with r, and the r is ##STR00016##  wherein each of nucleotides and r in the siRNA analogue is independently modified or unmodified.

2.-20. (canceled)

21. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1, wherein 70%, 75%, 80%, 85%, 90% or 95% or more of the nucleotides and r in the double-stranded siRNA analogue are modified; optionally, all the nucleotides and r in the double-stranded siRNA analogue are modified.

22. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1, wherein the modification comprises methoxy modification, fluoro modification, phosphorothioate linkage, replacement of a nucleotide with (S)-glycerol nucleic acid or replacement of a nucleotide with (E)-vinyl phosphate.

23. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1, wherein the antisense strand comprises a sequence obtained by replacing one, two, three, four or five nucleotide residues in the sequence set forth in SEQ ID NO: 2 with r; optionally, the antisense strand comprises a sequence obtained by replacing one nucleotide residue in the sequence set forth in SEQ ID NO: 2 with r.

24. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1, wherein the r replacement occurs at any position of the SEQ ID NO: 2.

25. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1, wherein the SEQ ID NO: 2 optionally comprises an overhang at the 5′ end and/or 3′ end; optionally, the SEQ ID NO: 2 comprises an overhang of 0, 1, 2, 3, 4 or 5 nucleotides at the 5′ end and/or 3′ end; optionally, the SEQ ID NO: 2 comprises an overhang at the 3′ end, and the overhang is selected from modified or unmodified UU.

26. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1, wherein the antisense strand comprises or consists of a sequence set forth in 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, SEQ ID NO: 32 or SEQ ID NO: 36, SEQ ID NO: 39 or SEQ ID NO: 44, SEQ ID NO: 10 or SEQ ID NO: 45, SEQ ID NO: 40 or SEQ ID NO: 46, SEQ ID NO: 10 or SEQ ID NO: 47, or SEQ ID NO: 10 or SEQ ID NO: 48.

27. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1, wherein the sense strand comprises or consists of a sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 28.

28. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1, wherein the sense strand comprises a sequence obtained by replacing one or more nucleotide residues in the sequence set forth in the SEQ ID NO: 1 with r; optionally, the sense strand comprises a sequence obtained by replacing one, two, three, four or five nucleotide residues in the sequence set forth in the SEQ ID NO: 1 with r.

29. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 28, wherein the r replacement occurs at positions 1-19 of the 5′ end of the SEQ ID NO: 1.

30. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1, wherein the sequence of the sense strand comprises or consists of a sequence set forth in 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: 43.

31. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1, wherein the double-stranded siRNA analogue is any one of S18-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, S19: 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, S25: 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, S27: 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, and 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, or, wherein the double-stranded siRNA analogue is any one of S1-S17: S1: the sense strand is SEQ ID NO: 3 or SEQ ID NO: 16, and 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, S7: the sense strand is SEQ ID NO: 3 or SEQ ID NO: 16, and 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, S9: the sense strand is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense strand is SEQ ID NO: 9 or SEQ ID NO: 22, S10: 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, S11: 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: 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, S13: the sense strand is SEQ ID NO: 3 or SEQ ID NO: 16, and 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, S15: 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, and 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, or, wherein the double-stranded siRNA analogue is any one of S29-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, S30: the sense strand is SEQ ID NO: 38 or SEQ ID NO: 43, and the antisense strand is SEQ ID NO: 10 or SEQ ID NO: 23, S31: the sense strand is SEQ ID NO: 3 or SEQ ID NO: 16, and 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, or 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.

32. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1, wherein the double-stranded siRNA analogue is linked to a pharmaceutically acceptable conjugate group, and the pharmaceutically acceptable conjugate group comprises a GalNAc group; optionally, the pharmaceutically acceptable conjugate group comprises 1 to 5 GalNAc groups.

33. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 32, wherein the double-stranded siRNA analogue is linked to a pharmaceutically acceptable conjugate group, and the pharmaceutically acceptable conjugate group comprises a compound group D: ##STR00017##

34. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 32, wherein the pharmaceutically acceptable conjugate group is linked to the 3′ end of the sense strand of the double-stranded siRNA analogue.

35. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1, wherein a phosphorothioate moiety of the double-stranded siRNA analogue or the conjugate thereof comprises (R)- and (S)-enantiomers, diastereoisomers, and/or racemic mixtures thereof, or, wherein the salt is selected from base addition salts, acid addition salts and combinations thereof; optionally, the base addition salt is selected from sodium, potassium, calcium, ammonium, organic amine, magnesium salts and combinations thereof, and the acid addition salt is selected from salts derived from inorganic acids, salts derived from inorganic acids and combinations thereof; optionally, the inorganic acid is selected from hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate radical, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid and combinations thereof, and the 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, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid or combinations thereof.

36. A pharmaceutical composition, comprising the double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1, and optionally a pharmaceutically acceptable carrier or excipient.

37. A method for treating hepatitis B in a subject, comprising administering to the subject the double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1 or the pharmaceutical composition thereof.

38. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 1, wherein the double-stranded siRNA analogue is selected from the following: TABLE-US-00022 Serial SEQ ID Sequence of sense SEQ ID Sequence of antisense number NO strand (5′-3′) NO strand (5′-3′)  1  3 GrGUGCACUUCGCUUCACA  4 UGUGArGCGAAGUGCACACUU  2  5 GUrUGCACUUCGCUUCACA  4 UGUGArGCGAAGUGCACACUU  3  3 GrGUGCACUUCGCUUCACA  6 UrUGAAGCGAAGUGCACACUU  4  5 GUrUGCACUUCGCUUCACA  6 UrUGAAGCGAAGUGCACACUU  5  3 GrGUGCACUUCGCUUCACA  7 UGrGAAGCGAAGUGCACACUU  6  5 GUrUGCACUUCGCUUCACA  7 UGrGAAGCGAAGUGCACACUU  7  3 GrGUGCACUUCGCUUCACA  8 UGUrAAGCGAAGUGCACACUU  8  5 GUrUGCACUUCGCUUCACA  8 UGUrAAGCGAAGUGCACACUU  9  3 GrGUGCACUUCGCUUCACA  9 UGUGrAGCGAAGUGCACACUU 10  5 GUrUGCACUUCGCUUCACA  9 UGUGrAGCGAAGUGCACACUU 11  3 GrGUGCACUUCGCUUCACA 10 UGUGAArCGAAGUGCACACUU 12  5 GUrUGCACUUCGCUUCACA 10 UGUGAArCGAAGUGCACACUU 13  3 GrGUGCACUUCGCUUCACA 11 UGUGAAGrGAAGUGCACACUU 14  5 GUrUGCACUUCGCUUCACA 11 UGUGAAGrGAAGUGCACACUU 15 12 GUGUGCrCUUCGCUUCACA  4 UGUGArGCGAAGUGCACACUU 16 13 GUGUGCACUUCGCUUCrCA  4 UGUGArGCGAAGUGCACACUU 17 14 GUGUGCACUUCGCUUCACr  4 UGUGArGCGAAGUGCACACUU 18  1 GUGUGCACUUCGCUUCACA  4 UGUGArGCGAAGUGCACACUU 19  1 GUGUGCACUUCGCUUCACA  6 UrUGAAGCGAAGUGCACACUU 20  1 GUGUGCACUUCGCUUCACA  7 UGrGAAGCGAAGUGCACACUU 21  1 GUGUGCACUUCGCUUCACA  8 UGUrAAGCGAAGUGCACACUU 22  1 GUGUGCACUUCGCUUCACA  9 UGUGrAGCGAAGUGCACACUU 23  1 GUGUGCACUUCGCUUCACA 10 UGUGAArCGAAGUGCACACUU 24  1 GUGUGCACUUCGCUUCACA 11 UGUGAAGrGAAGUGCACACUU 25  1 GUGUGCACUUCGCUUCACA 29 UGUGAAGCGrAGUGCACACUU 26  1 GUGUGCACUUCGCUUCACA 30 UGUGAAGCGArGUGCACACUU 27  1 GUGUGCACUUCGCUUCACA 31 UGUGAAGCGAAGUGCrCACUU 28  1 GUGUGCACUUCGCUUCACA 32 UGUGAAGCGAAGUGCACrCUU 29 37 GrGUGCACUUCGCUrCACA 10 UGUGAArCGAAGUGCACACUU 30 38 GrGUGCACUUCrCUUCACA 10 UGUGAArCGAAGUGCACACUU 31  3 GrGUGCACUUCGCUUCACA 39 UGUrAArCGAAGUGCACACUU 32  3 GrGUGCACUUCGCUUCACA 10 UGUGAArCGAAGUGCACACUU 33  3 GrGUGCACUUCGCUUCACA 40 UGUGAAGCGAArUGCACACUU or, wherein the double-stranded siRNA analogue is selected from the following: TABLE-US-00023 Serial SEQ ID Sequence of sense SEQ ID Sequence of antisense number NO strand (5′-3′) NO strand (5′-3′)  1 16 g•r•guGcACUucgcuucaca 17 u•G•ugargCGaaguGcAcac•u•u  2 18 g•u•ruGcACUucgcuucaca 17 u•G•ugargCGaaguGcAcac•u•u  3 16 g•r•guGcACUucgcuucaca 19 u•r•ugaAgCGaaguGcAcac•u•u  4 18 g•u•ruGcACUucgcuucaca 19 u•r•ugaAgCGaaguGcAcac•u•u  5 16 g•r•guGcACUucgcuucaca 20 u•G•rgaAgCGaaguGcAcac•u•u  6 18 g•u•ruGcACUucgcuucaca 20 u•G•rgaAgCGaaguGcAcac•u•u  7 16 g•r•guGcACUucgcuucaca 21 u•G•uraAgCGaaguGcAcac•u•u  8 18 g•u•ruGcACUucgcuucaca 21 u•G•uraAgCGaaguGcAcac•u•u  9 16 g•r•guGcACUucgcuucaca 22 u•G•ugrAgCGaaguGcAcac•u•u 10 18 g•u•ruGcACUucgcuucaca 22 u•G•ugrAgCGaaguGcAcac•u•u 11 16 g•r•guGcACUucgcuucaca 23 u•G•ugaArCGaaguGcAcac•u•u 12 18 g•u•ruGcACUucgcuucaca 23 u•G•ugaArCGaaguGcAcac•u•u 13 16 g•r•guGcACUucgcuucaca 24 u•G•ugaAgrGaaguGcAcac•u•u 14 18 g•u•ruGcACUucgcuucaca 24 u•G•ugaAgrGaaguGcAcac•u•u 15 25 g•u•guGcrCUucgcuucaca 17 u•G•ugargCGaaguGcAcac•u•u 16 26 g•u•guGcACUucgcuucrca 17 u•G•ugargCGaaguGcAcac•u•u 17 27 g•u•guGcACUucgcuucacr 17 u•G•ugargCGaaguGcAcac•u•u 18 28 g•u•guGcACUucgcuucaca 17 u•G•ugargCGaaguGcAcac•u•u 19 28 g•u•guGcACUucgcuucaca 19 u•r•ugaAgCGaaguGcAcac•u•u 20 28 g•u•guGcACUucgcuucaca 20 u•G•rgaAgCGaaguGcAcac•u•u 21 28 g•u•guGcACUucgcuucaca 21 u•G•uraAgCGaaguGcAcac•u•u 22 28 g•u•guGcACUucgcuucaca 22 u•G•ugrAgCGaaguGcAcac•u•u 23 28 g•u•guGcACUucgcuucaca 23 u•G•ugaArCGaaguGcAcac•u•u 24 28 g•u•guGcACUucgcuucaca 24 u•G•ugaAgrGaaguGcAcac•u•u 25 28 g•u•guGcACUucgcuucaca 33 u•G•uga(Agn)gCGraguGcAcac•u•u 26 28 g•u•guGcACUucgcuucaca 34 u•G•uga(Agn)gCGarguGcAcac•u•u 27 28 g•u•guGcACUucgcuucaca 35 u•G•uga(Agn)gCGaaguGcrcac•u•u 28 28 g•u•guGcACUucgcuucaca 36 u•G•uga(Agn)gCGaaguGcAcrc•u•u 29 42 g•r•guGcACUucgcurcaca 23 u•G•ugaArCGaaguGcAcac•u•u 30 43 g•r•guGcACUucrcuucaca 23 u•G•ugaArCGaaguGcAcac•u•u 31 16 g•r•guGcACUucgcuucaca 44 u•G•uraArCGaaguGcAcac•u•u 32 16 g•r•guGcACUucgcuucaca 45 u•G•uga(Agn)rCGaaguGcAcac•u•u 33 16 g•r•guGcACUucgcuucaca 46 u•G•uga(Agn)gCGaaruGcAcac•u•u 35 16 g•r•guGcACUucgcuucaca 47 VPu•G•ugaArCGaaguGcAcac•u•u 36 16 g•r•guGcACUucgcuucaca 48 VPu•G•uga(Agn)rCGaaguGcAcac•u•u

39. The double-stranded siRNA analogue, the conjugate thereof or the salt thereof according to claim 33, wherein the conjugate of the double-stranded siRNA analogue is selected from the following: TABLE-US-00024 Serial Sequence of sense strand SEQ ID Sequence of antisense SEQ ID number (5′-3′)-conjugate group NO: strand (5′-3′) NO:  1 g•r•guGcACUucgcuucacaD 16 u•G•ugargCGaaguGcAcac•u•u 17  2 g•u•ruGcACUucgcuucacaD 18 u•G•ugargCGaaguGcAcac•u•u 17  3 g•r•guGcACUucgcuucacaD 16 u•r•ugaAgCGaaguGcAcac•u•u 19  4 g•u•ruGcACUucgcuucacaD 18 u•r•ugaAgCGaaguGcAcac•u•u 19  5 g•r•guGcACUucgcuucacaD 16 u•G•rgaAgCGaaguGcAcac•u•u 20  6 g•u•ruGcACUucgcuucacaD 18 u•G•rgaAgCGaaguGcAcac•u•u 20  7 g•r•guGcACUucgcuucacaD 16 u•G•uraAgCGaaguGcAcac•u•u 21  8 g•u•ruGcACUucgcuucacaD 18 u•G•uraAgCGaaguGcAcac•u•u 21  9 g•r•guGcACUucgcuucacaD 16 u•G•ugrAgCGaaguGcAcac•u•u 22 10 g•u•ruGcACUucgcuucacaD 18 u•G•ugrAgCGaaguGcAcac•u•u 22 11 g•r•guGcACUucgcuucacaD 16 u•G•ugaArCGaaguGcAcac•u•u 23 12 g•u•ruGcACUucgcuucacaD 18 u•G•ugaArCGaaguGcAcac•u•u 23 13 g•r•guGcACUucgcuucacaD 16 u•G•ugaAgrGaaguGcAcac•u•u 24 14 g•u•ruGcACUucgcuucacaD 18 u•G•ugaAgrGaaguGcAcac•u•u 24 15 g•u•guGcrCUucgcuucacaD 25 u•G•ugargCGaaguGcAcac•u•u 17 16 g•u•guGcACUucgcuucrcaD 26 u•G•ugargCGaaguGcAcac•u•u 17 17 g•u•guGcACUucgcuucacrD 27 u•G•ugargCGaaguGcAcac•u•u 17 18 g•u•guGcACUucgcuucacaD 28 u•G•ugargCGaaguGcAcac•u•u 17 19 g•u•guGcACUucgcuucacaD 28 u•r•ugaAgCGaaguGcAcac•u•u 19 20 g•u•guGcACUucgcuucacaD 28 u•G•rgaAgCGaaguGcAcac•u•u 20 21 g•u•guGcACUucgcuucacaD 28 u•G•uraAgCGaaguGcAcac•u•u 21 22 g•u•guGcACUucgcuucacaD 28 u•G•ugrAgCGaaguGcAcac•u•u 22 23 g•u•guGcACUucgcuucacaD 28 u•G•ugaArCGaaguGcAcac•u•u 23 24 g•u•guGcACUucgcuucacaD 28 u•G•ugaAgrGaaguGcAcac•u•u 24 25 g•u•guGcACUucgcuucacaD 28 u•G•uga(Agn)gCGraguGcAcac•u•u 33 26 g•u•guGcACUucgcuucacaD 28 u•G•uga(Agn)gCGarguGcAcac•u•u 34 27 g•u•guGcACUucgcuucacaD 28 u•G•uga(Agn)gCGaaguGcrcac•u•u 35 28 g•u•guGcACUucgcuucacaD 28 u•G•uga(Agn)gCGaaguGcAcrc•u•u 36 29 g•r•guGcACUucgcurcacaD 42 u•G•ugaArCGaaguGcAcac•u•u 23 30 g•r•guGcACUucrcuucacaD 43 u•G•ugaArCGaaguGcAcac•u•u 23 31 g•r•guGcACUucgcuucacaD 16 u•G•uraArCGaaguGcAcac•u•u 44 32 g•r•guGcACUucgcuucacaD 16 u•G•uga(Agn)rCGaaguGcAcac•u•u 45 33 g•r•guGcACUucgcuucacaD 16 u•G•uga(Agn)gCGaaruGcAcac•u•u 46 34 g•r•guGcACUucgcuucacaD 16 VPu•G•ugaArCGaaguGcAcac•u•u 47 35 g•r•guGcACUucgcuucacaD 16 VPu•G•uga(Agn)rCGaaguGcAcac•u•u 48

Description

DETAILED DESCRIPTION

[0143] The present disclosure is described in detail below by way of examples. However, this is by no means disadvantageously limiting the scope of the present disclosure. The compounds of the present disclosure can be prepared using a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art. The preferred embodiments include, but are not limited to, the examples of the present disclosure. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present disclosure without departing from the spirit and scope of the present disclosure.

Example 1: Synthesis of Phosphoramidite Monomer

[0144] ##STR00008##

[0145] Step A: a solution of (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (i.e., formula 1-1) (30 g, 94.26 mmol) and methyl 1,2,4-triazole-3-carboxylate (11.98 g, 94.26 mmol) in methyl acetate (220 mL) was concentrated to almost complete dryness in an oil bath at 90° C. under a pressure of 1 bar. A solution of trifluoromethanesulfonic acid (141.46 mg, 0.94 mmol) in methyl acetate (2 mL) was added to the mixture, and the resulting mixture was stirred in an oil bath at 125° C. for 4 h under a pressure of 30 mbar. The reaction solution was cooled to 70° C., and ethanol (70 mL) was added. The mixture was stirred at 70° C. until a homogeneous solution was formed, and then the stirring was stopped and the solution was cooled to 50° C. After the precipitate was generated, the reaction solution was left to stand and cooled to 25° C., and then the reaction solution was left to stand at 0° C. for 16 h. The reaction solution was filtered through a Buchner funnel, and the filter cake was rinsed with 180 mL (60 mL×3) of ethanol and dried under vacuum to give formula 1-2. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.40 (s, 1H), 6.04 (d, J=3.42 Hz, 1H), 5.69-5.81 (m, 1H), 5.54 (t, J=5.38 Hz, 1H), 4.42-4.51 (m, 2H), 4.16-4.30 (m, 1H), 3.98 (s, 3H), 2.05-2.18 (m, 9H).

[0146] Step B: the compound of 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° C. for 17 h under nitrogen atmosphere. The reaction solution was concentrated under reduced pressure to give formula 1-3. .sup.1H NMR (400 MHz, CD.sub.3OD): δ 8.87 (s, 1H), 5.93 (d, J=3.42 Hz, 1H), 4.48 (dd, J=3.48, 4.83 Hz, 1H), 4.33 (t, J=5.26 Hz, 1H), 4.10-4.16 (m, 1H), 3.95 (s, 3H), 3.84 (dd, J=3.24, 12.29 Hz, 1H), 3.70 (dd, J=4.46, 12.29 Hz, 1H).

[0147] Step C: the compound of formula 1-3 (10 g, 38.58 mmol) was dissolved in pyridine (250 mL), and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (12.29 g, 38.97 mmol) was added dropwise at 0° C. The mixture was gradually warmed to 25° C. and stirred for 16 h. The reaction solution was concentrated under reduced pressure, and the concentrate was suspended in ethyl acetate (250 mL). The mixture was filtered through a Buchner funnel. The filtrate was washed with 750 mL (250 mL×3) of 3 M hydrochloric acid and 250 mL (250 mL×1) of saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (SiO.sub.2, petroleum ether/dichloromethane/ethyl acetate=3/1/1) to give formula 1-4. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.43 (s, 1H), 5.95 (s, 1H), 4.73 (dd, J=4.75, 8.00 Hz, 1H), 4.41 (d, J=4.75 Hz, 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).

[0148] Step D: iodomethane (11.64 g, 82.02 mmol) was added to a mixed solution of the compound of formula 1-4 (8.23 g, 16.40 mmol), potassium carbonate (11.34 g, 82.02 mmol) and silver(I) oxide (19.01 g, 82.02 mmol) in N,N-dimethylformamide (50 mL), and the mixture was stirred at 25° C. for 3 h. The reaction solution was diluted with ethyl acetate (300 mL) and filtered through a Buchner funnel. The filtrate was washed with 250 mL (250 mL×1) of aqueous sodium thiosulfate solution, 250 mL (250 mL×1) of water and 250 mL (250 mL×1) of saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (SiO.sub.2, petroleum ether/ethyl acetate=5/1) to give formula 1-5. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.58 (s, 1H), 5.91 (s, 1H), 4.46 (dd, J=4.22, 9.35 Hz, 1H), 4.17-4.28 (m, 2H), 3.96-4.06 (m, 5H), 3.68 (s, 3H), 0.99-1.13 (m, 28H).

[0149] Step E: triethylamine trihydrofluoride (2.25 g, 13.95 mmol) was added dropwise to a solution of the compound of formula 1-5 (3.27 g, 6.34 mmol) in tetrahydrofuran (50 mL) at 0° C., and the mixture was gradually warmed to 25° C. and stirred for 16 h. The reaction solution was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (SiO.sub.2, dichloromethane/methanol=20/1) to give formula 1-6. .sup.1H NMR (400 MHz, CD.sub.3OD): δ 8.88 (s, 1H), 6.04 (d, J=3.26 Hz, 1H), 4.44 (t, J=5.33 Hz, 1H), 4.20 (dd, J=3.33, 4.83 Hz, 1H), 4.07-4.14 (m, 1H), 3.96 (s, 3H), 3.84 (dd, J=3.20, 12.36 Hz, 1H), 3.69 (dd, J=4.39, 12.30 Hz, 1H), 3.52 (s, 3H).

[0150] Step F: 4,4-dimethoxytrityl chloride (2.42 g, 7.14 mmol) was added to a solution of the compound of formula 1-6 (1.30 g, 4.76 mmol) in pyridine (20 mL) at 0° C., and the mixture was stirred at 25° C. for 16 h. The reaction solution was diluted with ethyl acetate (70 mL), quenched with saturated aqueous sodium bicarbonate solution (20 mL) at 25° C. and then diluted with water (40 mL). After liquid separation, the organic phases were combined, washed with 60 mL (60 mL×1) of water and 60 mL (60 mL×1) of saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by p-HPLC (separation column: Phenomenex luna C18 (specification: 250 mm×50 mm, particle size: 10 μm); mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]; elution gradient: 35%-65%, 20 min) to give formula 1-7. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.44 (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=2.88 Hz, 1H), 4.37-4.43 (m, 1H), 4.33 (dd, J=2.88, 5.00 Hz, 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=6.88 Hz, 1H). LCMS (ESI) m/z: 574.2 [M−H].sup.−.

[0151] Step G: 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (678.45 mg, 2.87 mmol) and N,N-diisopropylethylamine were added to a solution of the compound of formula 1-7 (1.10 g, 1.91 mmol) in dichloromethane (8 mL) at 0° C., and the mixture was stirred at 20° C. for 0.5 h. The reaction solution was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (SiO.sub.2, petroleum ether/ethyl acetate=5011 to 1/2) to give the compound of formula 1. LCMS (ESI) m/z: 776.3 [M+H].sup.+.

Example 2: Synthesis of D01

[0152] ##STR00009## ##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014##

[0153] Step A: 11-dodecyn-1-ol (25 g, 137.14 mmol) and triethylamine (16.65 g, 164.56 mmol) were dissolved in dichloromethane (250 mL), and methanesulfonyl chloride (18.85 g, 164.56 mmol) was added at 0° C. The mixture was stirred at 0° C. for 2 h. The reaction solution was diluted with water (400 mL) and extracted with 800 mL (400 mL×2) of dichloromethane. The organic phases were combined, washed with 400 mL (200 mL×2) of water and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give formula 2-2.

[0154] Step B: the compound of formula 2-3 (20 g, 67.26 mmol) was dissolved in N,N-dimethylformamide (200 mL), and sodium hydride (60% pure, 4.04 g, 100.89 mmol) was added at 0° C., followed by the addition of the compound of formula 2-2 (19.27 g, 73.99 mmol). The mixture was stirred at 25° C. for 16 h. The reaction solution was quenched with water (1 L) and extracted with 1.6 L (800 mL×2) of dichloromethane. The organic phases were combined, washed with 800 mE (800 mL×1) of saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give formula 2-4. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.63-6.89 (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 (in, 16H).

[0155] Step C: the compound of formula 2-4 (48 g, 103.98 mmol) was dissolved in methanol (870 mL), and a solution of hydrogen chloride in methanol (4 mol/L, 400 mL, 1.6 mol) was added. The mixture was stirred at 30° C. for 2 h. A solution of hydrogen chloride in methanol (4 mol/L, 350 mE, 1.4 mol) was added to the reaction solution. The mixture was stirred at 30° C. for 16 h. The reaction solution was concentrated under reduced pressure, and 200 mL (100 mL×2) of chloroform was added. The mixture was concentrated under reduced pressure until a white solid appeared. Toluene (130 mL) and petroleum ether (130 mL) were added, and the mixture was stirred at 15° C. for 16 h. The reaction solution was filtered through a Buchner funnel, and the filter cake was collected and dried under vacuum to give a white solid. The white solid was dissolved in dichloromethane (50 mL), and an aqueous solution (50 mL) of sodium hydroxide (6.59 g, 164.66 mmol) was added, and the mixture was stirred at 20° C. for 1 h. The reaction solution was diluted with water (500 mL) and extracted with 1 L (500 mL×2) of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give formula 2-5.

[0156] Step D: tert-butyl acrylate (22.72 g, 177.28 mmol) was added to a mixed solution of the compound of 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), and the mixture was stirred at 25° C. for 16 h under nitrogen atmosphere. The reaction solution was diluted with water (500 mL) and extracted with 1 L (500 mL×2) of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (SiO.sub.2, petroleum ether/ethyl acetate/ethanol (containing 0.1% ammonia water)=36/3/1 to 16/3/1) to give formula 2-6. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 3.60-3.54 (m, 4H), 3.32 (br s, 5H), 3.15 (s, 5H), 2.74-2.66 (m, 1H), 2.40 (t, J=6.0 Hz, 4H), 2.18-2.11 (m, 2H), 1.58-1.38 (m, 22H), 1.34-1.23 (m, 12H).

[0157] Step E: triethylamine (9.15 g, 90.45 mmol) and succinic anhydride (6.79 g, 67.83 mmol) were added to a solution of the compound of formula 2-6 (24.5 g, 45.22 mmol) in dichloromethane (250 mL), and the mixture was stirred at 20° C. for 16 h. Dichloromethane (1 L) and hydrochloric acid (1 mol/L, 1 L) were added to the reaction solution, and after liquid separation, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give formula 2-7. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 6.49-6.37 (m, 1H), 3.72 (s, 2H), 3.70-3.57 (m, 8H), 3.37 (t, J=6.7 Hz, 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).

[0158] Step F: the compound of formula 2-7 (27.4 g, 42.69 mmol) was dissolved in formic acid (140 mL), and the mixture was stirred at 20° C. for 16 h under nitrogen atmosphere. The reaction solution was concentrated under reduced pressure, and 300 mL (150 mL×2) of toluene was added. The mixture was concentrated under reduced pressure to give formula 2-8. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 9.79-9.22 (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=2.6 Hz, 1H), 1.48-1.28 (m, 6H), 1.26-1.12 (m, 10H).

[0159] Step G: the compound of formula 2-8 (22.6 g, 42.67 mmol), N,N-diisopropylethylamine (33.09 g, 256.03 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (51.92 g, 136.55 mmol) were dissolved in N,N-dimethylformamide (250 mL), and tert-butyl N-(3-aminopropyl)carbamate (29.74 g, 170.69 mmol) was added. The mixture was stirred at 20° C. for 16 h. Dichloromethane (1 L) and hydrochloric acid (1 mol/L, 1 L) were added to the reaction solution, and after liquid separation, the organic phase was washed successively with 1 L (1 L×1) of water, 1 L (1 L×1) of aqueous sodium bicarbonate solution and 1 L (1 L×1) of saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (SiO.sub.2, petroleum ether/ethyl acetate/ethanol=40/3/1 to 10/3/1) to give formula 2-9. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.22-6.79 (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).

[0160] Step H: the compound of formula 2-9 (15 g, 15.03 mmol) was dissolved in dichloromethane (114 mL), and trifluoroacetic acid (38 mL) was added. The mixture was stirred at 20° C. for 16 h. The reaction solution was concentrated under reduced pressure, and 600 mL (250 mL×3) of a mixture of toluene/acetonitrile=3/1 was added. The mixture was concentrated under reduced pressure to give formula 2-10 (tris(trifluoroacetate)).

[0161] Step I: the compound of 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-tetramethyluronium hexafluorophosphate (20.53 g, 54.00 mmol) were dissolved in N,N-dimethylformamide (90 mL), and a solution of the compound of formula 2-10 (tris(trifluoroacetate), 15.6 g, 15.00 mmol) and N,N-diisopropylethylamine (21.32 g, 165.00 mmol) in N,N-dimethylformamide (120 mL) was added to the mixture. The mixture was stirred at 20° C. for 16 h. Dichloromethane (1.2 L) and hydrochloric acid (1 mol/L, 1 L) were added to the reaction solution, and after liquid separation, the organic phase was washed successively with 1 L (1 L×1) of water, 1 L (1L×1) of aqueous sodium bicarbonate solution and 1 L (1 L×1) of saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (SiO.sub.2, dichloromethane/methanol=100/1 to 10/1 to dichloromethane/ethanol=1/1) to give formula 2-12. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.87-7.66 (m, 9H), 7.09 (s, 1H), 5.21 (d, J=3.4 Hz, 3H), 4.96 (dd, J=3.4, 11.3 Hz, 3H), 4.48 (d, J=8.5 Hz, 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=2.6 Hz, 1H), 2.33-2.22 (m, 8H), 2.16-2.12 (m, 2H), 2.10 (s, 9H), 2.04 (br t, J=7.1 Hz, 6H), 1.99 (s, 9H), 1.89 (s, 9H), 1.81-1.74 (m, 9H), 1.54-1.39 (m, 22H), 1.32 (br dd, J=4.5, 6.7 Hz, 2H), 1.24 (s, 10H).

[0162] Step J: the compound of formula 2-12 (1.00 g, 0.50 mmol) and N-methyl-N,N,N-tri-n-octylammonium chloride (20.35 mg, 50.35 μmol) was dissolved in a mixture of 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 added dropwise to the mixture at 0° C. The mixture was stirred at 0-15° C. for 2 h. The reaction was quenched with sodium bisulfite (1.27 g), and hydrochloric acid (2 mol/L, 5 mL) and water (30 mL) were added. The mixture was extracted with 120 mL (40 mL×3) of a chloroform/isopropanol (3/1) mixture. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and then 180 mL (30 mL×6) of a toluene/acetonitrile (1/1) mixture was added. The resulting mixture was concentrated under reduced pressure to give formula 2-13. .sup.1H NMR (400 MHz, CD.sub.3OD): δ 5.34 (d, J=2.9 Hz, 3H), 5.06 (dd, J=3.3, 11.2 Hz, 3H), 4.56 (d, J=8.4 Hz, 3H), 4.19-4.06 (m, 9H), 4.04-3.98 (m, 3H), 3.87 (td, J=5.7, 9.9 Hz, 4H), 3.72-3.64 (m, 9H), 3.57-3.50 (m, 3H), 3.39 (br t, J=6.4 Hz, 2H), 3.22 (q, J=6.4 Hz, 12H), 2.51-2.40 (m, 9H), 2.21 (br t, J=7.3 Hz, 6H), 2.14 (s, 9H), 2.03 (s, 9H), 1.94 (d, J=7.9 Hz, 18H), 1.72-1.57 (m, 22H), 1.39 (br s, 12H).

[0163] Step K: N, N-diisopropylethylamine (0.26 g, 1.99 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (0.23 g, 0.60 mmol) were added to a solution of the compound of formula 2-13 (1.00 g, 0.50 mmol) in N,N-dimethylformamide (10 mL). After the mixture was stirred, the compound of formula 2-14 (0.23 g, 0.55 mmol) was added. The mixture was stirred at 15° C. for 16 h. Dichloromethane (50 mL) and water (50 mL) were added to the reaction solution, and after liquid separation, the organic phase was washed successively with 50 mL (50 mL×1) of saturated aqueous sodium bicarbonate solution, 50 mL (50 mL×1) of water and 50 mL (50 mL×1) of saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product.

[0164] The crude product was purified by column chromatography (SiO.sub.2, dichloromethane/methanol (containing 0.1% triethylamine)=20/1 to 10/1) to give formula 2-15. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.90-7.82 (m, 6H), 7.78 (br d, J=4.8 Hz, 3H), 7.40-7.26 (m, 10H), 6.91 (br dd, J=3.1, 9.0 Hz, 4H), 5.26 (d, J=3.4 Hz, 3H), 5.03-4.99 (m, 3H), 4.53 (d, J=8.4 Hz, 3H), 4.43 (br d, J=3.8 Hz, 1H), 4.23-4.14 (m, 1H), 4.12-4.02 (m, 9H), 3.92 (td, J=9.0, 11.0 Hz, 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=6.9 Hz, 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).

[0165] Step L: triethylamine (67.24 mg, 0.64 mmol), 4-N,N-dimethylaminopyridine (0.12 g, 1.00 mmol) and succinic anhydride (83.13 mg, 0.83 mmol) were added successively to a solution of the compound of formula 2-15 (0.80 g, 0.33 mmol) in dichloromethane (8 mL). The mixture was stirred at 10° C. for 16 h. Dichloromethane (50 mL), water (30 mL) and saturated brine (30 mL) were added to the reaction solution, and after liquid separation, the organic phase was washed successively with 30 mL (30 mL×1) of water and 30 mL (30 mL×1) of saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by p-HPLC (separation column: Waters Xbridge C18 (specification: 150 mm×50 mm, particle size: 10 μm); mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]; elution gradient: 27%-57%, 11 min) to give Example 2 (compound D01). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.96-7.69 (m, 9H), 7.33-7.09 (m, 10H), 6.90-6.78 (m, 4H), 5.21 (d, J=3.3 Hz, 3H), 4.97 (dd, J=3.3, 11.2 Hz, 3H), 4.49 (d, J=8.4 Hz, 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=4.6 Hz, 14H), 2.46-2.37 (m, 4H), 2.35-2.14 (m, 12H), 2.10 (s, 9H), 2.04 (br t, J=7.0 Hz, 6H), 1.99 (s, 9H), 1.88 (s, 9H), 1.77 (s, 9H), 1.57-1.37 (m, 22H), 1.22 (br s, 12H).

Example 3: Synthesis of Double-Stranded siRNA Analogue or Conjugates Thereof

[0166] Synthesis of D-containing single-stranded oligoribonucleotides: oligoribonucleotides were synthesized according to the phosphoramidite solid-phase synthesis technique. Synthesis was performed on a solid support made by covalently linking controlled porous glass (amino CPG, 500 Å) to D01. All 2′-modified RNA phosphoramidites and ancillary reagents were commercially available reagents. All amides were dissolved in anhydrous acetonitrile and a molecular sieve (3 Å) was added, and the coupling time when using 5-ethylthio-1H-tetrazole (ETT) as the activating agent was 5 min. Phosphorothioate bonds were generated using a 50 mM solution of 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile/pyridine (v/v=1/1), and the reaction time was 3 min. The sequences were synthesized after the removal of the DMT group at last.

[0167] Synthesis of D-free single-stranded oligoribonucleotides: oligoribonucleotides were synthesized according to the phosphoramidite solid-phase synthesis technique. The synthesis was performed on universal controlled porous glass CPG (500 Å). All 2′-modified RNA phosphoramidites and ancillary reagents were commercially available reagents. All amides were dissolved in anhydrous acetonitrile and a molecular sieve (3 Å) was added, and the coupling time when using 5-ethylthio-1H-tetrazole (ETT) as the activating agent was 5 min. Phosphorothioate bonds were generated using a 50 mM solution of 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile/pyridine (v/v=1/1), and the reaction time was 3 min. The sequences were synthesized after the removal of the DMT group at last.

[0168] Cleavage and deprotection of bound oligomers on CPG: after the solid-phase synthesis was terminated, the protecting group was removed by treatment with a solution of 20% diethylamine in acetonitrile for 30 min, without cleaving the oligonucleotide from the CPG. Subsequently, the dried CPG was treated with concentrated ammonia water at 40° C. for 18 h. After centrifugation, the supernatant was transferred to a new tube and the CPG was washed with ammonia water. The combined solution was concentrated to give a solid mixture.

[0169] Purification of single-stranded oligoribonucleotides: the oligomers purified by HPLC were exchanged by using NanoQ anions. Buffer A was a 10 mM sodium perchlorate solution, 20 mM Tris, 1 mM EDTA, pH 7.4 and containing 20% acetonitrile, and buffer B was 500 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4 and containing 20% acetonitrile. The desired product was separated out and desalted using a reverse phase C18 column.

[0170] Annealing of single-stranded oligoribonucleotides for siRNA production: the single-stranded oligoribonucleotides to be annealed were formulated to 200 μM using sterile RNase Free H.sub.2O (no RNA hydrolase). The annealing reaction system was set as follows: a total of 100 μL of 10 nmol mixed solution was placed in 95° C. water bath for 10 min (20 min at high temperature was required for 100 nmol or more).fwdarw.the solution was quickly placed in 60° C. water bath, and naturally cooled.fwdarw.the solution after annealing cannot be placed at high temperature for storage. Complementary strands were formed by combining equimolar solutions of single-stranded oligoribonucleotides.

TABLE-US-00005 TABLE 1 Double-stranded siRNA analogues targeting hepatitis B virus genes, conjugates comprising the same, and their corresponding core sequences Core sequences r′-embedded sequences** Sequence of Sequence Sequence of Further modified sequences SEQ Sequence of SEQ antisense SEQ of sense SEQ antisense SEQ Sequence of  SEQ Sequence of ID sense strand ID strand ID strand ID strand ID sense strand ID antisense NO (5′-3′) NO (5′-3′) NO (5′-3′)** NO (5′-3′) NO (5′-3′)*** NO strand (5′-3′) 1 GUGUGCA 2 UGUGAAG  3 GrGUGCA  4 UGUGArG 16 g•r•guGcA 17 u•G•ugargCG CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc aaguGcAcac• UCACA CACAC UCACAD* CACACUU acaD* u•u 1 GUGUGCA 2 UGUGAAG  5 GUrUGCA  4 UGUGArG 18 g•u•ruGcA 17 u•G•ugargCG CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc aaguGcAcac• UCACA CACAC UCACAD CACACUU acaD u•u 1 GUGUGCA 2 UGUGAAG  3 GrGUGCA  6 UrUGAAG 16 g•r•guGcA 19 u•r•ugaAgCG CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc aaguGcAcac• UCACA CACAC UCACAD CACACUU acaD u•u 1 GUGUGCA 2 UGUGAAG  5 GUrUGCA  6 UrUGAAG 18 g•u•ruGcA 19 u•r•ugaAgCG CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc aaguGcAcac• UCACA CACAC UCACAD CACACUU acaD u•u 1 GUGUGCA 2 UGUGAAG  3 GrGUGCA  7 UGrGAAG 16 g•r•guGcA 20 u•G•rgaAgC CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc GaaguGcAca UCACA CACAC UCACAD CACACUU acaD c•u•u 1 GUGUGCA 2 UGUGAAG  5 GUrUGCA  7 UGrGAAG 18 g•u•ruGcA 20 u•G•rgaAgC CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc GaaguGcAca UCACA CACAC UCACAD CACACUU acaD c•u•u 1 GUGUGCA 2 UGUGAAG  3 GrGUGCA  8 UGUrAAG 16 g•r•guGcA 21 u•G•uraAgC CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc GaaguGcAca UCACA CACAC UCACAD CACACUU acaD c•u•u 1 GUGUGCA 2 UGUGAAG  5 GUrUGCA  8 UGUrAAG 18 g•u•ruGcA 21 u•G•uraAgC CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc GaaguGcAca UCACA CACAC UCACAD CACACUU acaD c•u•u 1 GUGUGCA 2 UGUGAAG  3 GrGUGCA  9 UGUGrAG 16 g•r•guGcA 22 u•G•ugrAgC CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc GaaguGcAca UCACA CACAC UCACAD CACACUU acaD c•u•u 1 GUGUGCA 2 UGUGAAG  5 GUrUGCA  9 UGUGrAG 18 g•u•ruGcA 22 u•G•ugrAgC CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc GaaguGcAca UCACA CACAC UCACAD CACACUU acaD c•u•u 1 GUGUGCA 2 UGUGAAG  3 GrGUGCA 10 UGUGAAr 16 g•r•guGcA 23 u•G•ugaArC CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc GaaguGcAca UCACA CACAC UCACAD CACACUU acaD c•u•u 1 GUGUGCA 2 UGUGAAG  5 GUrUGCA 10 UGUGAAr 18 g•u•ruGcA 23 u•G•ugaArC CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc GaaguGcAca UCACA CACAC UCACAD CACACUU acaD c•u•u 1 GUGUGCA 2 UGUGAAG  3 GrGUGCA 11 UGUGAAG 16 g•r•guGcA 24 u•G•ugaAgrG CUUCGCU CGAAGUG CUUCGCU rGAAGUG CUucgcuuc aaguGcAcac• UCACA CACAC UCACAD CACACUU acaD u•u 1 GUGUGCA 2 UGUGAAG  5 GUrUGCA 11 UGUGAAG 18 g•u•ruGcA 24 u•G•ugaAgrG CUUCGCU CGAAGUG CUUCGCU rGAAGUG CUucgcuuc aaguGcAcac• UCACA CACAC UCACAD CACACUU acaD u•u 1 GUGUGCA 2 UGUGAAG 12 GUGUGCr  4 UGUGArG 25 g•u•guGcrC 17 u•G•ugargCG CUUCGCU CGAAGUG CUUCGCU CGAAGUG Uucgcuuca aaguGcAcac• UCACA CACAC UCACAD CACACUU caD u•u 1 GUGUGCA 2 UGUGAAG 13 GUGUGC  4 UGUGArG 26 g•u•guGcA 17 u•G•ugargCG CUUCGCU CGAAGUG ACUUCGC CGAAGUG CUucgcuuc aaguGcAcac• UCACA CACAC UUCrCAD CACACUU rcaD u•u 1 GUGUGCA 2 UGUGAAG 14 GUGUGC  4 UGUGArG 27 g•u•guGcA 17 u•G•ugargCG CUUCGCU CGAAGUG ACUUCGC CGAAGUG CUucgcuuc aaguGcAcac• UCACA CACAC UUCACrD CACACUU acrD u•u 1 GUGUGCA 2 UGUGAAG  1 GUGUGC  4 UGUGArG 28 g•u•guGcA 17 u•G•ugargCG CUUCGCU CGAAGUG ACUUCGC CGAAGUG CUucgcuuc aaguGcAcac• UCACA CACAC UUCACA CACACUU acaD u•u D 1 GUGUGCA 2 UGUGAAG  1 GUGUGC  6 UrUGAAG 28 g•u•guGcA 19 u•r•ugaAgCG CUUCGCU CGAAGUG ACUUCGC CGAAGUG CUucgcuuc aaguGcAcac• UCACA CACAC UUCACA CACACUU acaD u•u D 1 GUGUGCA 2 UGUGAAG  1 GUGUGC  7 UGrGAAG 28 g•u•guGcA 20 u•G•rgaAgC CUUCGCU CGAAGUG ACUUCGC CGAAGUG CUucgcuuc GaaguGcAca UCACA CACAC UUCACA CACACUU acaD c•u•u D 1 GUGUGCA 2 UGUGAAG  1 GUGUGC  8 UGUrAAG 28 g•u•guGcA 21 u•G•uraAgC CUUCGCU CGAAGUG ACUUCGC CGAAGUG CUucgcuuc GaaguGcAca UCACA CACAC UUCACA CACACUU acaD c•u•u D 1 GUGUGCA 2 UGUGAAG  1 GUGUGC  9 UGUGrAG 28 g•u•guGcA 22 u•G•ugrAgC CUUCGCU CGAAGUG ACUUCGC CGAAGUG CUucgcuuc GaaguGcAca UCACA CACAC UUCACA CACACUU acaD c•u•u D 1 GUGUGCA 2 UGUGAAG  1 GUGUGC 10 UGUGAAr 28 g•u•guGcA 23 u•G•ugaArC CUUCGCU CGAAGUG ACUUCGC CGAAGUG CUucgcuuc GaaguGcAca UCACA CACAC UUCACA CACACUU acaD c•u•u D 1 GUGUGCA 2 UGUGAAG  1 GUGUGC 11 UGUGAAG 28 g•u•guGcA 24 u•G•ugaAgrG CUUCGCU CGAAGUG ACUUCGC rGAAGUG CUucgcuuc aaguGcAcac• UCACA CACAC UUCACA CACACUU acaD u•u D 1 GUGUGCA 2 UGUGAAG  1 GUGUGC 29 UGUGAAG 28 g•u•guGcA 33 u•G•uga(Agn) CUUCGCU CGAAGUG ACUUCGC CGrAGUG CUucgcuuc gCGraguGc UCACA CACAC UUCACA CACACUU acaD Acac•u•u D 1 GUGUGCA 2 UGUGAAG  1 GUGUGC 30 UGUGAAG 28 g•u•guGcA 34 u•G•uga(Agn) CUUCGCU CGAAGUG ACUUCGC CGArGUG CUucgcuuc gCGarguGc UCACA CACAC UUCACA CACACUU acaD Acac•u•u D 1 GUGUGCA 2 UGUGAAG  1 GUGUGC 31 UGUGAAG 28 g•u•guGcA 35 u•G•uga(Agn) CUUCGCU CGAAGUG ACUUCGC CGAAGUG CUucgcuuc gCGaaguGcr UCACA CACAC UUCACA CrCACUU acaD cac•u•u D 1 GUGUGCA 2 UGUGAAG  1 GUGUGC 32 UGUGAAG 28 g•u•guGcA 36 u•G•uga(Agn) CUUCGCU CGAAGUG ACUUCGC CGAAGUG CUucgcuuc gCGaaguGc UCACA CACAC UUCACA CACrCUU acaD Acrc•u•u D 1 GUGUGCA 2 UGUGAAG 37 GrGUGCA 10 UGUGAAr 42 g•r•guGcA 23 u•G•ugaArC CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcurc GaaguGcAca UCACA CACAC rCACAD   CACACUU acaD c•u•u 1 GUGUGCA 2 UGUGAAG 38 GrGUGCA 10 UGUGAAr 43 g•r•guGcA 23 u•G•ugaArC CUUCGCU CGAAGUG CUUCrCU CGAAGUG CUucrcuuc GaaguGcAca UCACA CACAC UCACAD CACACUU acaD c•u•u 1 GUGUGCA 2 UGUGAAG 3 GrGUGCA 39 UGUrAArC 16 g•r•guGcA 44 u•G•uraArCG CUUCGCU CGAAGUG CUUCGCU GAAGUGC CUucgcuuc aaguGcAcac• UCACA CACAC UCACAD ACACUU acaD u•u 1 GUGUGCA 2 UGUGAAG 3 GrGUGCA 10 UGUGAAr 16 g•r•guGcA 45 u•G•uga(Agn) CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc rCGaaguGc UCACA CACAC UCACAD CACACUU acaD Acac•u•u 1 GUGUGCA 2 UGUGAAG 3 GrGUGCA 40 UGUGAAG 16 g•r•guGcA 46 u•G•uga(Agn) CUUCGCU CGAAGUG CUUCGCU CGAArUG CUucgcuuc gCGaaruGc UCACA CACAC UCACAD CACACUU acaD Acac•u•u 1 GUGUGCA 2 UGUGAAG 3 GrGUGCA 10 UGUGAAr 16 g•r•guGcA 47 VPu•G•ugaA CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc rCGaaguGcA UCACA CACAC UCACAD CACACUU acaD cac•u•u 1 GUGUGCA 2 UGUGAAG 3 GrGUGCA 10 UGUGAAr 16 g•r•guGcA 48 VPu•G•uga CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuuc (Agn)rCGaagu UCACA CACAC UCACAD CACACUU acaD GcAcac•u•u 1 GUGUGCA 2 UGUGAAG 3 GrGUGCA 10 UGUGAAr 15 g•u•guGcA 41 VPu•G•uga CUUCGCU CGAAGUG CUUCGCU CGAAGUG CUucgcuur (Agn)gCGarg UCACA CACAC UCACAD CACACUU acaD uGcAcac•u•u *: D is a residue obtained after chemical reaction of the small molecular fragment DO1, is combined with nucleic acid through a covalent bond, and has the following structure: **: The sequence of antisense strand in the r′-embedded sequences is obtained by r′ embedment on the basis of the sequence of antisense strand with UU at the 3′ end in the core sequences. For example, SEQ ID NO: 4 is obtained by r′ embedment on the basis of SEQ ID NO: 2 with UU at the 3′ end. ***: When a sequence contains D, the D is used to refer to the linking position of the conjugate group D. For example, g•r•guGcACUucgcuucacaD (5′-3′) indicates that the sequence set forth in SEQ ID NO. 16, g•r•guGcACUucgcuucaca, is linked to D at the 3′ end.

##STR00015##

Example 3: In Vitro HBV Assay

[0171] 1. Experimental Objective:

[0172] The content of HBV antigens (HBsAg and HBeAg) in HepG2-NTCP cell culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA), and the inhibitory activity of the compound on HBV was evaluated by taking the EC.sub.50 value of the compound as an index; meanwhile, the cell viability was detected by Cell-titer Glo to evaluate the cytotoxicity of the compound.

[0173] 2. Experimental Materials:

[0174] 2.1. Cell Line: HepG2-NTCP Cells

[0175] HepG2-NTCP cell culture medium (DMEM, Invitrogen-11330032; 10% serum, Invitrogen-10099141; 100 units/mL penicillin and 100 μg/mL streptomycin, Hyclone-SV30010; 1% nonessential amino acids, Invitrogen-11140050; 2 mM L-glutamine, Invitrogen-25030081; 1 mM sodium pyruvate, Gibco-11360-070; 500 μg/mL Geneticin, Invitrogen-10131027)

[0176] 2.2. Reagents:

[0177] Pancreatin (Invitrogen-25300062); DPBS (Corning-21031CVR); DMSO (Sigma-D2650-100 ML); Cell-titer Glo (Promega-G7573); HBsAg quantitative assay kit (Autobio-CL 0310); HBeAg quantitative assay kit (Autobio-CL 0312).

[0178] 2.3. Consumables and Instrument:

[0179] 96-well cell culture plates (Corning-3599); CO.sub.2 incubator (HERA-CELL-240) microplate reader (BioTek Synergy 2)

[0180] 3. Experimental Procedures and Method:

[0181] 3.1. On day 0, HepG2-NTCP (7.5×10.sup.4 cells/well) cells were plated onto a 48-well plate and incubated overnight at 37° C. and 5% CO.sub.2.

[0182] 3.2. On day 1, the medium containing 1% DMSO was used for medium change.

[0183] 3.3. On day 2, HepG2-NTCP (2000 GE/cell) was infected with HBV/D (concentrated from HepG2.2.15 cell culture supernatant).

[0184] 3.4. On day 3, the infection solution was pipetted off and fresh medium containing 1% DMSO was added.

[0185] 3.5. On day 6, the siRNA conjugate was transfected according to instructions of Lipofectamine® RNAiMax (Invitrogen). The conjugate was subjected to 5-fold gradient dilution to obtain 7 concentrations, triplicate wells were set, and the final concentration was 0.16 pM. The compound was a combination of sense and antisense strands and was a single chemical entity, with a maximum concentration of 2.5 nM.

[0186] 3.6. On day 12, supernatant from the culture wells was collected and assayed for HBsAg and HBeAg by ELISA. After the supernatant was collected, Cell-titer Glo was added to measure cell viability.

[0187] 3.7. Reference was made to the instructions of the product for specific procedures of ELISA assay for HBsAg and HBeAg, and the brief procedures are as follows: 50 L of sample and 50 μL of standard substance were each added into a reaction plate, then enzyme conjugate was added at 50 μL/well, and the mixture was well mixed by shaking and incubated at 37° C. for 60 min; the plate was washed 5 times by using a washing solution, luminescent substrate was then added at 50 μL/well, and the mixture was well mixed and reacted at room temperature in the dark for 10 min, and finally the chemiluminescence intensity was detected by using a microplate reader.

[0188] 3.8. Data Analysis:

[0189] Calculation of percentage cell viability:

% viability=(luminescence value of sample−luminescence value of medium control)/(luminescence value of DMSO control−luminescence value of medium control)×100.

[0190] Calculation of the inhibition percentage for HBsAg and HBeAg:

% Inh.=(1−antigen value in sample/antigen value in DMSO control)×100.

[0191] Calculation of CC.sub.50 and EC.sub.50: CC.sub.50 and 50% inhibitory concentration for HBV (EC.sub.50) values of compounds were calculated using GraphPad Prism software.

[0192] 4. Experimental Results: See Table 2.

TABLE-US-00006 TABLE 2 Results of test sequences in reducing HBsA g and HBeAg levels in cells Experimental results Test sequences Cell SEQ ID Sequence of sense SEQ ID Sequence of antisense HBsAg HBeAg viability NO strand (5′-3′) NO strand (5′-3′) EC.sub.50 (pM) EC.sub.50 (pM) CC.sub.50 (nM) 16 g•r•guGcACUucgcuuc 17 u•G•ugargCGaaguGcA 13.75 20.84 >2.5 acaD cac•u•u 16 g•r•guGcACUucgcuuc 20 u•G•rgaAgCGaaguGcA 17.40 34.21 >2.5 acaD cac•u•u 16 g•r•guGcACUucgcuuc 23 u•G•ugaArCGaaguGcA 12.44 21.07 >2.5 acaD cac•u•u 28 g•u•guGcACUucgcuuc 17 u•G•ugargCGaaguGcA 13.09 22.68 >2.5 acaD cac•u•u 28 g•u•guGcACUucgcuuc 20 u•G•rgaAgCGaaguGcA 14.69 28.90 >2.5 acaD cac•u•u 28 g•u•guGcACUucgcuuc 23 u•G•ugaArCGaaguGcA 14.99 34.72 >2.5 acaD cac•u•u 28 g•u•guGcACUucgcuuc 34 u•G•uga(Agn)gCGargu 30.72 52.56 >2.5 acaD GcAcac•u•u *The test samples were conjugates of double-stranded siRNA analogues.

Example 4: Anti-Hepatitis B Virus Activity and Safety Research in Recombinant 8-Type Adeno-Associated Virus Vector-Mediated Hepatitis B Virus Mouse Model (AAV-HBV)

[0193] Experimental Objective:

[0194] The AAV vector-mediated HBV transfected mouse model is a rapid and efficient HBV model. By utilizing the high hepatotropism of the AAV8 vector, the recombinant 8-type adeno-associated virus carrying 1.3 copies of HBV genome (rAAV8-1.3HBV) is injected via tail vein of mice, which can efficiently introduce the carried 1.3 copies of HBV genome into liver cells. Due to the characteristics of AAV viral vector, the vector mediated by it can express continuously for a long period of time, and HBV DNA can be continuously replicated and HBsAg and HBeAg can be expressed in the liver of mice by applying the AAV/HBV model.

[0195] By using the AAV/HBV mouse model, HBsAg, HBeAg, DNA and pgRNA in the serum of mice and the weight of mice were detected after treating the mice with the test compound, thus evaluating the in vivo anti-HBV effect and safety of the test compound.

[0196] Experimental Materials:

[0197] C57BL/6 mice, PBS (RNase free) as vehicle, test compounds, recombinant virus rAAV8-1.3HBV. Main reagents of the project include QIAamp96 DNA Kit (Qiagen, 51162), FastStart Universal Probe Master (Rox) (Roche, 04914058001), HBsAg assay kit (Autobio-CLO310); HBeAg assay kit (Autobio-CL0918), PureLink™ Pro 96 Viral RNA/DNA kit (Invitrogen, 12280-096A) and FastQuant RT Kit (with gDNase) (TIANGEN, KR106-02). Main instruments include centrifuge (Beckman Allegra X-15R), multifunctional microplate reader (BioTek, Synergy 2), fluorescent quantitative PCR instrument (Applied Biosystems, 7900HT Fast Real-time PCR system) and microplate reader (Molecular Devices, SpectraMax 340PC384).

[0198] Experimental Method:

[0199] a) Mice were subjected to subcutaneous injection on day 34 after virus injection, and this day was set as day 0. Before administration, all the mice were subjected to submaxillary blood sampling for plasma collection. The specific administration regimen is shown in Table 3.

[0200] b) The mice were subjected to blood sampling via submaxillary vein on days 0, 14, 21, 28 and 32 after administration for plasma collection, and the blood samples were anticoagulated with K.sub.2-EDTA and centrifuged at 4° C. and 7000 g/min for 10 min to collect plasma. The specific time for blood sampling is shown in Table 3.

[0201] c) On day 35 or 42, all the mice were subjected to blood sampling via submaxillary vein for plasma collection, after which the mice were euthanized by C02 inhalation. Plasma samples were collected by blood sampling from the heart, and liver samples were collected.

[0202] d) The plasma samples were sent for detection.

TABLE-US-00007 TABLE 3 Scheme for in vivo experiment Administration design Administration Administration Non-endpoint Number Test amount volume Administration blood sampling Endpoint of of mice compound (mg/kg) (mL/kg) regimen scheme experiment 5 Vehicle / 5 Day 34 after Day 34 after virus On day 35 after 5 WRG01.sup.*1 3 virus injection injection was set administration, the was set as day 0, as day 0, and the mice were subjected and drug blood sampling to blood sampling via administration time was days 0, submaxillary venous was performed 7, 14, 21, 28, 32 plexus for plasma once via and 35. collection, after which subcutaneous the mice were injection on day euthanized by CO.sub.2 0 and day 29. inhalation. Plasma samples were collected by blood sampling from the heart, and liver samples were collected. 5 WR007.sup.*2 3 Day 34 after / 5 WR012.sup.*3 3 virus injection was set as day 0, and on day 0, drug administration was performed once by subcutaneous injection. .sup.*1: WRG01 is a conjugate, in which the sense strand is SEQ ID NO: 16, the antisense strand is SEQ ID NO: 23, and the conjugate group is D. .sup.*2: WR007 is a conjugate, in which the sense strand is SEQ ID NO: 42, the antisense strand is SEQ ID NO: 23, and the conjugate group is D. .sup.*3: WR012 is a conjugate, in which the sense strand is SEQ ID NO: 16, the antisense strand is SEQ ID NO: 47, and the conjugate group is D. /: the endpoint has not been reached.

[0203] Sample Analysis:

[0204] ELISA assay for the content of HBsAg and HBeAg in the serum of mice: reference was made to the instructions of the HBsAg ELISA kit (Autobio, CL 0310) and HBeAg ELISA kit (Autobio, CL0918) for experimental procedures.

[0205] qPCR assay for the content of HBV DNA in the plasma of mice: HBV DNA in plasma was extracted, and reference was made to the instructions of QIAamp 96 DNA Blood Kit for experimental procedures, thus detecting the content of HBV DNA in the plasma of mice by qPCR. RT-qPCR assay for the content of HBV pgRNA in the plasma of mice: HBV pgRNA was extracted from plasma and reference was made to the instructions of PureLink™ Pro 96 Viral RNA/DNA Kit for experimental procedures. The DNA was digested and the RNA was reverse transcribed into cDNA using a 3′RACE primer containing hepatitis B virus specific sequence, and reference was made to the instructions of FastQuant RT Kit (with gDNase) for experimental procedures. Finally, the content of cDNA was quantitatively detected by qPCR, namely detecting the content of HBV pgRNA in the plasma of mice.

[0206] Mean±standard error of mean was used to express the value of each group of mouse samples, and n=5 unless otherwise specified. Statistical analysis was performed using Student's t-test.

[0207] Experimental Results:

[0208] a) The anti-HBV activity of the test compounds in AAV/HBV mouse models was evaluated according to the content of HBsAg in serum. The results are shown in Table 4, Table 4-1, FIG. 1 and FIG. 6. The content of HBsAg in the plasma of mice was determined by ELISA. Error bars represent the standard error. Day 0: all mice were subjected to administration of vehicle or compound for the first time. Day 29: the mice in the experimental group WRG01 and mice in the corresponding blank control group were inoculated with vehicle or compound for the second time.

TABLE-US-00008 TABLE 4 Log.sub.10 [HBsAg (IU/mL) ] of mice on different days after administration Days of detection (day) Blank (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

TABLE-US-00009 TABLE 4-1 Log.sub.10 [HBsAg (IU/mL)] of mice on different days after administration Days of detection (day) Blank (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

[0209] b) The anti-HBV activity of the test compounds in AAV/HBV mouse models was evaluated according to the content of HBeAg in serum. The results are shown in Table 5, Table 5-1, FIG. 2 and FIG. 7. The content of HBeAg in the plasma of mice was determined by ELISA. Error bars represent the standard error. Day 0: all mice were subjected to administration of vehicle or compound for the first time.

TABLE-US-00010 TABLE 5 Log.sub.10 [HBeAg (PEIU/mL)] of mice on different days after administration Days of detection (day) Blank (SC) WRG01 (SC) 0 3.56 3.51 7 3.37 2.89 14 3.56 3.06 21 3.66 3.22

TABLE-US-00011 TABLE 5-1 Log.sub.10 [HBeAg (PEIU/mL)] of mice on different days after administration Days of detection (day) Blank (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

[0210] c) The anti-HBV activity of the test compounds in AAV/HBV mouse models was evaluated according to the content of DNA in serum. The results are shown in Table 6, Table 6-1, FIG. 3 and FIG. 8. The content of HBV DNA in the plasma of mice was determined by quantitative PCR. Error bars represent the standard error. Day 0: all mice were subjected to administration of vehicle or compound for the first time. Day 29: all the mice were inoculated with vehicle or compound for the second time.

TABLE-US-00012 TABLE 6 Log.sub.10 [DNA (copy number/μL)] of mice on different days after administration Days of detection (day) Blank (SC) WRG01 (SC) 0 5.27 4.84 7 5.39 3.93 14 5.51 3.97 21 5.63 4.37

TABLE-US-00013 TABLE 6-1 Log.sub.10 [DNA (copy number/μL)] of mice on different days after administration Days of detection (day) Blank (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 /: no data were obtained.

[0211] d) The anti-HBV activity of the test compounds in AAV/HBV mouse models was evaluated according to the content of pgRNA in serum. The results are shown in Table 7 and FIG. 4. The content of HBV pgRNA in the plasma of mice was determined by quantitative PCR. Error bars represent the standard error. Day 0: all mice were subjected to administration of vehicle or compound for the first time. Day 29: all the mice were inoculated with vehicle or compound for the second time.

TABLE-US-00014 TABLE 7 Log.sub.10 [pgRNA (copy number/μL)] of mice on different days after administration Days of detection (day) Blank (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

[0212] e) The change in body weight is shown in FIG. 5. The comparison was performed with the body weight on day 0 used as a baseline. As per IACUC regulation, losing of 20% body weight is considered as a humane endpoint, and any mouse that loses more than 20% of its body weight should be removed from the experiment. None of the mice in this experiment was removed due to weight loss.

[0213] Experimental Conclusion:

[0214] In this experiment, the test compounds were able to significantly reduce HBsAg, DNA and pgRNA in AAV/HBV mouse models. Meanwhile, the test compounds also had a certain inhibiting effect on HBeAg. During the treatment with the test compounds, the mice showed good tolerance and the body weight gradually increased.

Example 5: HBV In Vitro Assay of HepG2.2.15 Cells

[0215] 1. Experimental Objective:

[0216] The content of HBV DNA in the HepG2.2.15 cell culture supernatant was detected using real-time qPCR, and the content of HBsAg and HBeAg was detected using ELISA; the content of HBV RNA in cells was detected using qRT-PCR, the EC.sub.50 value of the compound was used as an index to evaluate the inhibitory effect of the compound on HBV, and the influence of the test compound on the cell viability was detected using the CCK8 method.

[0217] 2. Experimental Materials:

[0218] 2.4. Cell Line: HepG2.2.15 Cells

[0219] HepG2.2.15 cell culture medium (DMEM/F12, Invitrogen-11330032; 10% serum, Hyclone-SV30087.0; 100 units/mL penicillin and 100 μg/mL streptomycin, Hyclone-SV30010; 1% non-essential amino acids, Invitrogen-11140050; 2 mM L-glutamine, Invitrogen-25030081; 300 μg/mL Geneticin, Invitrogen-10131027).

[0220] 2.5. Reagents

[0221] Opti-MEM (Gibco-31985-070); Lipofectamine® RNAiMAX (Invitrogen-13778-150); CCK8 (Life-iLab-AC11L057); high-throughput DNA purification kit (QIAamp 96 DNA Blood Kit, Qiagen-51162); RNA preparation RNEASY Kit (RNeasy 96 Kit (12), Qiagen-74182); quantitative fast start universal probe reagent (FastStart Universal Probe Master, Roche-04914058001); FastKing cDNA first strand synthesis kit (TianGen-KR106-02); HBsAg quantitative assay kit (Autobio-CL 0310); HBeAg quantitative assay kit (Autobio-CL 0312).

[0222] 2.6. Consumables and Instrument:

[0223] Collagen I 96 Well White/Clear Flat Bottom TC-Treated Microplate (Coming BioCoat-356650); C02 incubator (HERA-CELL-240); fluorescent quantitative PCR instrument (Applied Biosystems-7900 real time PCR system); fluorescent quantitative PCR instrument (Applied Biosystems-QuantStudio 6 Flex); microplate reader (Molecular Device-SpectraMax M2e); microplate reader (BioTek-Synergy 2).

[0224] 3. Experimental Procedures and Method:

[0225] 3.1. On day one, transfection of siRNA and cell plating were performed simultaneously, and the brief procedures are as follows: HepG2.2.15 cells were washed with DPBS and digested with 0.05% trypsin, and then the digestion was terminated with DMEM/F12 medium containing 10% FBS; the cells were then centrifuged, resuspended, gently pipetted into single cells and counted. The volume of desired transfection reagent was set according to certain ratio (Table 8), and the cells were incubated for 15 min at room temperature.

TABLE-US-00015 TABLE 8 Allocation of Lipofectamine ® RNAiMAX Reagent Ratio (allocation for one well as an example) Lipofectamine ® RNAiMAX 1.5 Opti-MEM 23.5

[0226] The siRNA was subjected to 3-fold gradient dilution to get 8 concentrations, and two duplicate wells were set. 15 μL of RNAiMAX/Opti-MEM mixture was well mixed with 15 μL of siRNA at different concentrations, and the mixture was incubated for 15 min at room temperature. 10 μL of the above mixed solution was added into a 96-well cell culture plate, then 90 μL of cell suspension was added, and the final cell density was 15,000 cells/well and the final volume was 100 μL/well. The cells were then incubated in an incubator at 37° C. and 5% C02.

[0227] 3.2. On day four, the original culture medium was replaced with a fresh culture medium containing the compound, and the transfection procedure was the same as that of day one.

[0228] 3.3. On day seven, the culture solution in the culture well was collected and sampling was performed. A part of the samples were used for ELISA assay of the content of HBsAg and HBeAg; a part of the samples were used for DNA extraction by using a high-throughput DNA purification kit (Qiagen-51162); after the supernatant was collected, the cell viability was detected according to instructions of the CCK-8 kit, and the absorbance (450 nm/650 nm) of each well was detected with a microplate reader (SpectraMax M2e); HBV RNA was extracted from the cell culture using the RNeasy 96 kit extraction kit (Qiagen-74182) with reference to the kit instructions. 3.4. The preparation of the PCR reaction solution is shown in Table 9:

TABLE-US-00016 TABLE 9 Preparation of PCR reaction solution Volume required for 1 Volume required for 100 Items well (μL) wells (μL) Quantitative fast start universal 5 500 probe reagent Forward primer (10 μmol) 0.4 40 Reverse primer (10 μmol) 0.4 40 Probe (10 μmol) 0.2 20 AE 2 200

[0229] 8 μL of the reaction mixture was added into each well of the 96-well PCR plate, and then 2 μL of sample DNA or HBV DNA standard substance was added into each well.

[0230] The reaction conditions of PCR are as follows: heating for 10 min at 95° C., then denaturing for 15 s at 95° C. and extending for 1 min at 60° C., 40 cycles in total.

[0231] 3.5. Reference was made to the instructions of the product for specific procedures of ELISA assay for content of HBsAg and HBeAg, and the brief procedures are as follows: 50 VL of sample and 50 μL of standard substance were each added into a reaction plate, then enzyme conjugate was added at 50 L/well, and the mixture was well mixed by shaking and incubated at 37° C. for 60 min in a warm bath; the plate was washed 5 times by using a washing solution, luminescent substrate was then added at 50 L/well, and the mixture was well mixed and reacted at room temperature in the dark for 10 min, and finally the chemiluminescence intensity was detected by using a microplate reader.

[0232] 3.6. The HBV RNA in cell culture was extracted by using the RNeasy 96 kit extraction kit (Qiagen, 74182) with reference to the kit instructions. Cells were lysed with 150 μL of RLT, and finally RNA was eluted with 50 μL of RNase-free water. A random primer was added according to the instructions of the reverse transcription kit (Tiangen, KR106) for reverse transcription into cDNA, then an HBV specific primer was used for detecting total RNA in the sample; meanwhile, GAPDH primers and probes were used for specifically detecting GAPDH cDNA, and the qPCR method was used for quantifying the HBV cDNA in the sample.

[0233] qPCR reaction: 95° C., 10 min; 95° C., 15 s; 60° C., 1 min, 40 cycles in total. The content of HBV RNA in each sample was calculated according to the Ct value of the sample.

[0234] The expression level of HBV mRNA, the target gene of each sample, was calculated by the relative quantification method of ΔΔCt. The relative expression level of the target gene was expressed by 2-ΔΔCT, and the calculation formula is as follows:

ΔCT=mean Ct value of target gene−mean Ct value of reference gene;

ΔΔCT=ΔCT(treatment group)−ΔCT(RNAiMAX control group);

Relative expression level of HBV mRNA=2−ΔΔCT

[0235] 3.7. Data Analysis:

[0236] Calculation of percentage inhibition:

% Inh.=(1−value in sample/PBS control value)×100.

Cell viability %=(detection value of sample−background average detection value of culture solution)/(average detection value of control group−background average detection value of culture solution)×100

[0237] Calculation of EC.sub.50 and CC.sub.50: the 50% inhibitory concentration (EC.sub.50) of the compound for HBV and the drug concentration at 50% cell death (CC.sub.50) were calculated using GraphPad Prism software.

TABLE-US-00017 TABLE 10 Results of test sequences in reducing HBsAg, HBeAg, DNA and RNA levels in cells Test sequences Experimental results Sequence of Sequence of HBsAg HBeAg DNA RNA Cell SEQ sense strand SEQ antisense strand EC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50 viability ID NO (5-3) ID NO (5′-3) (nM) (nM) (nM) (nM) CC.sub.50 (nM) 16 g•r•guGcACUuc 23 u•G•ugaArCGaag 0.179 0.755 0.14 0.87 >50 gcuucacaD uGcAcac•u•u 28 g•u•guGcACUuc 17 u•G•ugargCGaag 0.105 0.37 0.16 0.567 >50 gcuucacaD uGcAcac•u•u 28 g•u•guGcACUuc 23 u•G•ugaArCGaag 0.079 0.587 0.135 3.197 >50 gcuucacaD uGcAcac•u•u 28 g•u•guGcACUuc 34 u•G•uga(Agn)gC 0.263 1.268 0.553 0.963 >50 gcuucacaD GarguGcAcac•u• u 42 g•r•guGcACUuc 23 u•G•ugaArCGaag 0.07 0.389 0.019 0.65 >50 gcurcacaD uGcAcac•u•u 43 g•r•guGcACUucr 23 u•G•ugaArCGaag 0.231 / / / >50 cuucacaD uGcAcac•u•u 16 g•r•guGcACUuc 44 u•G•uraArCGaag 0.072 2.202 0.06 / >50 gcuucacaD uGcAcac•u•u 16 g•r•guGcACUuc 45 u•G•uga(Agn)rC 0.054 3.707 0.15 / >50 gcuucacaD GaaguGcAcac•u• u 16 g•r•guGcACUuc 46 u•G•uga(Agn)gC 0.497 / / / >50 gcuucacaD GaaruGcAcac•u• u 16 g•r•guGcACUuc 47 VPu•G•ugaArCG 0.082 0.205 0.037 0.68 >50 gcuucacaD aaguGcAcac•u•u 16 g•r•guGcACUuc 48 VPu•G•uga(Agn) 0.104 3.83 0.105 / >50 gcuucacaD rCGaaguGcAcac• u•u /: no data were obtained. * The test samples were conjugates of double-stranded siRNA analogues.

Example 6: Dose Exploration for Effective Anti-Hepatitis B Virus Activity in AAV-HBV Mouse Models

[0238] By using the AAV/HBV mouse models, HBsAg in the serum of mice was detected after treating the mice with the test compound at different doses, thus evaluating the in vivo anti-HBV effect of the test compound.

[0239] Experimental Materials:

[0240] C57BL/6 mice, PBS (RNase free) as vehicle, test compounds, recombinant virus rAAV8-1.3HBV.

[0241] Main reagents of the project include FastStart Universal Probe Master (Rox) (Roche, 04914058001) and HBsAg assay kit (Autobio, CL0310). Main instruments include centrifuge (Beckman Allegra X-15R), multifunctional microplate reader (BioTek, Synergy 2) and microplate reader (Molecular Devices, SpectraMax 340PC384).

[0242] Experimental Method:

[0243] a) All the mice were subjected to subcutaneous injection on day 34 after virus injection, and this day was set as day 0. Before administration, all the mice were subjected to submaxillary blood sampling for plasma collection. Drug administration was performed once on day 0. The specific administration regimen is shown in Table 14.

[0244] b) All the mice were subjected to blood sampling via submaxillary vein on days 0, 14, 21, 28 and 35 after administration for plasma collection, and the blood samples were anticoagulated with K.sub.2-EDTA and centrifuged at 4° C. and 7000 g/min for 10 min to collect plasma. The specific time for blood sampling is shown in Table 11.

[0245] c) On day 42, all the mice were subjected to blood sampling via submaxillary vein for plasma collection, after which the mice were euthanized by CO.sub.2 inhalation. Plasma samples were collected by blood sampling from the heart, and liver samples were collected.

[0246] d) All the plasma samples were sent for detection.

TABLE-US-00018 TABLE 11 Scheme for in vivo experiment Administration design Non-endpoint Administration Administration blood Number Test amount volume Administration sampling Endpoint of mice compound (mg/kg) (mL/kg) regimen scheme of experiment 5 Vehicle / 5 Day 34 after Day 34 after virus 5 WRG01* 0.3 virus injection injection was set On day 42 after 5 1 was set as day as day 0, and the administration, the mice 0, and on day 0, blood sampling were subjected to blood drug time was days 0, sampling via submaxillary administration 7, 14, 21, 28 and venous plexus for plasma was performed 35. collection, after which the once by mice were euthanized by subcutaneous CO.sub.2 inhalation. Plasma injection. samples were collected by blood sampling from the heart, and liver samples were collected. 5 3 / 5 10 / *WRG01 is a conjugate, in which the sense strand is SEQ ID NO: 16, the antisense strand is SEQ ID NO: 23, and the conjugate group is D. /: the endpoint has not been reached.

[0247] Sample Analysis:

[0248] ELISA assay for the content of HBsAg in the serum of mice: reference was made to the instructions of the HBsAg ELISA kit (Autobio, CL 0310) for experimental procedures.

[0249] Mean±standard error of mean was used to express the value of each group of mouse samples, and n=5 unless otherwise specified. Statistical analysis was performed using Student's t-test.

[0250] Experimental Results:

[0251] The anti-HBV activity of the test compound in AAV/HBV mouse models was evaluated by detecting the content of HBsAg in serum. The results are shown in Table 12 and FIG. 9. The content of HBsAg in the plasma of mice was determined by ELISA. Error bars represent the standard error.

[0252] Day 0: all mice were subjected to administration of vehicle or compound for the first time.

TABLE-US-00019 TABLE 12 Log.sub.10 [HBsAg (IU/mL)] of mice on different days after administration Days of WRG01, WRG01, WRG01, WRG01, detection Blank 0.3 1 mpk 3 mpk 10 mpk (day) (SC) mpk (SC) (SC) (SC) (SC) 0 4.58 4.49 4.53 4.31 4.56 7 4.15 3.67 2.93 2.19 1.98 14 4.57 4.18 3.60 2.26 2.12 21 4.41 4.46 3.80 2.48 2.17 28 4.76 4.76 4.25 3.22 2.99 35 4.62 4.65 4.31 3.40 3.12

[0253] Experimental Conclusion:

[0254] In this experiment, the test compound WRG01 exhibited good dose dependence for reducing HBsAg in AAV/HBV mouse models; that is, its activity for reducing HBsAg increased along with the increase in the drug dose, and it exhibited long-term efficacy in inhibiting HBsAg.

Example 7: Drug Concentration Test in Mouse Plasma, Liver and Kidney

[0255] In this study, C57BL/6 mice were subjected to a single administration via subcutaneous injection, plasma and tissue samples were collected at various time points after drug administration, and metabolic levels of the compound in the mice were evaluated by SL-qPCR detection for siRNA levels in plasma and tissues.

TABLE-US-00020 TABLE 13 Scheme for in vivo experiment Non-endpoint Administration design peripheral Administration Administration blood Number Test amount volume Administration collection of mice compound (mg/kg) (mL/kg) regimen scheme Endpoint of experiment 3 WRG01* 3 5 Drug The blood At 0.5 h after administration, administration sampling time the mice were subjected to was performed was 0.083 h blood sampling via once via after submaxillary venous plexus for subcutaneous administration. plasma collection, after which injection on the mice were euthanized by day 0. CO.sub.2 inhalation, and liver and kidney samples were collected. 3 3 The blood At 1 h after administration, the sampling time mice were subjected to blood was 0.25 h sampling via submaxillary after venous plexus for plasma administration. collection, after which the mice were euthanized by CO.sub.2 inhalation, and liver and kidney samples were collected. 3 3 / At 2 h after administration, the mice were subjected to blood sampling via submaxillary venous plexus for plasma collection, after which the mice were euthanized by CO.sub.2 inhalation, and liver and kidney samples were collected. 3 3 / At 4 h after administration, the mice were subjected to blood sampling via submaxillary venous plexus for plasma collection, after which the mice were euthanized by CO.sub.2 inhalation, and liver and kidney samples were collected. 3 3 / At 8 h after administration, the mice were subjected to blood sampling via submaxillary venous plexus for plasma collection, after which the mice were euthanized by CO.sub.2 inhalation, and liver and kidney samples were collected. 3 3 / At 32 h after administration, the mice were subjected to blood sampling via submaxillary venous plexus for plasma collection, after which the mice were euthanized by CO.sub.2 inhalation, and liver and kidney samples were collected. 3 3 The blood At 168 h after administration, sampling time the mice were subjected to was 48 h and blood sampling via 96 h after submaxillary venous plexus for administration. plasma collection, after which the mice were euthanized by CO.sub.2 inhalation, and liver and kidney samples were collected. *: WRG01 is a conjugate, in which the sense strand is SEQ ID NO: 16, the antisense strand is SEQ ID NO: 23, and the conjugate group is D. /: blood sampling was not performed at “non-endpoint” time, only at endpoint.

[0256] Experimental Results:

[0257] The siRNA levels in plasma, liver and kidney of mice at different time points after administration were detected using the SL-qPCR method (reference: Nair et al., Nucleic Acids Research (2017), 45, 10969-10977) and the results are shown in FIG. 10.

[0258] Experimental Conclusion:

[0259] In this experiment, the test compound WRG01 had good tissue distribution and metabolic stability in the C57BL/6 mouse models. WR-G01 has large liver exposure, long half-life period and liver-to-blood ratio of more than 500 times, which proves WRG01 has metabolic stability and high liver-targeting property.

Example 8: Blood Biochemical Test in Mice with FRG-KO Humanized Liver

[0260] The humanized FRG mouse is one of the most commonly used humanized liver models, usually with a humanization rate as high as 70%. Because human liver cells are planted in the liver of the mouse, the natural HBV infection and cccDNA replication process of a human body can be better simulated, and meanwhile, this model can well predict the pharmacokinetics and hepatotoxicity of the human body.

[0261] In this study, the humanized FRG mice were subjected to multiple times of drug administration, plasma samples at different time points after administration were collected, and the toxic and side effect of the compound on the liver of the mice was evaluated by detecting ALT, AST and bilirubin levels in the plasma. In this experiment, the test compound did not cause significant inflammatory response of the humanized liver, indicating good safety in human body.

TABLE-US-00021 TABLE 14 Scheme for in vivo experiment Administration design Administration Administration Non-endpoint Number Test amount volume Administration peripheral blood Endpoint of of mice compound (mg/kg) (mL/kg) regimen collection scheme experiment 3 Vehicle / 5 Drug administration The blood sampling / 3 WRG01* 15 was performed once time was 1 day before 3 50 via subcutaneous administration, and injection on days 0, days 7, 14, 21, 28, 35 21, 28, 35 and 42. and 42 after administration. *: WRG01 is a conjugate, in which the sense strand is SEQ ID NO: 16, the antisense strand is SEQ ID NO: 23, and the conjugate group is D. /: the endpoint has not been reached.

[0262] The present disclosure exhibits unpredictably excellent inhibitory activity on HBsAg and HBeAg while effectively inhibiting expression of HBV DNA and pgRNA, which demonstrates that the present disclosure can inhibit the activity of hepatitis B virus. Meanwhile, the present disclosure has good tissue distribution and metabolic stability, and it has high liver-targeting property and is expected to have little effect on mouse liver function. It will provide an efficient treatment means for hepatitis B in clinic, such as chronic hepatitis B.