Conjugates of double-stranded siRNA analogs

Abstract

The present disclosure relates to a ribavirin derivative-embedded double-stranded siRNA analogue, a conjugate comprising the same, and salts and uses thereof. The double-stranded siRNA analogue, the conjugate comprising the same, and the salts thereof of the present disclosure can effectively inhibit multiple viral indicators such as hepatitis B virus DNA, pgRNA, S antigen, E antigen, etc., and provide an effective and feasible method for the treatment of hepatitis B.

Description

[0001] This application claims the following priority: CN202010529520.7, application date: June 11, 2020; CN202011524835.9, application date December 21, 2020.

[0002] This application is a divisional application of the invention patent application filed on June 7, 2021, with Chinese application number 202180041587.9 and invention title "Conjugates of Double-Stranded siRNA Analogs". Technical Field

[0003] This disclosure pertains to the field of biomedicine and relates to an r'-intercalated siRNA analog, a double-stranded siRNA analog, a conjugate containing the same, and salts thereof and uses thereof; the uses specifically refer to its use in the preparation of a medicament for treating hepatitis B. Background Technology

[0004] Hepatitis B, or HBV for short, is a disease caused by infection with the hepatitis B virus (HBV). HBV is a hepatotropic virus that primarily resides within hepatocytes and damages them, causing inflammation, necrosis, and fibrosis. HBV is classified into acute and chronic forms. In most adults, acute hepatitis B resolves spontaneously through the body's immune system. However, chronic hepatitis B (CHB) has become a significant challenge to global health care and is a major cause of chronic liver disease, cirrhosis, and liver cancer (HCC) (Edward JG, 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). It is estimated that 2 billion people worldwide are infected with chronic hepatitis B virus (HBV), and more than 350 million have developed hepatitis B. Nearly 600,000 people die annually from complications of chronic hepatitis B (Edward JG, et al., The oral toll-like receptor 7 agonist GS-9620 inpatients with chronic hepatitis B virus infection. Journal of Hepatology (2015)). my country is a high-incidence area for hepatitis B, with a large cumulative number of patients and serious harm. Data shows that there are approximately 93 million HBV carriers in my country, of whom about 20 million are diagnosed with chronic hepatitis B. Of these, 10%-20% may develop cirrhosis, and 1%-5% may develop liver cancer. (Zhang Chunhong, Application of interferon in the treatment of hepatitis B. China Medical Guide (2013); 11: 475-476.) The key to functional cure of hepatitis B is the clearance of HBsAg (hepatitis B virus surface antigen) and the production of surface antibodies. HBsAg quantification is a very important biomarker. In patients with chronic infection, a reduction in HBsAg and seroconversion are rarely observed, which is currently the endpoint of treatment.

[0005] Currently approved anti-HBV drugs mainly include immunomodulators (interferon-α and pegylated interferon-α-2α) and antiviral therapies (lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, clavidine, etc.). Among these, antiviral therapies belong to the nucleotide analogue class, whose mechanism of action is to inhibit HBV DNA synthesis and does not directly reduce HBsAg levels. Similar to prolonged treatment, nucleotide analogues show HBsAg clearance rates similar to natural observations (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.).

[0006] Clinical treatments exist to reduce HBsAg levels, but their efficacy is limited. Therefore, silencing viral gene expression at the gene level, blocking HBV production and replication, especially the production of HBsAg and HBeAg (hepatitis B S antigen and E antigen), could fundamentally reduce viral metabolism and hepatocyte infection. Small interfering RNA (siRNA) can inhibit or block the expression of target genes in a sequence-specific manner based on the RNA interference (RNAi) mechanism, exerting an inhibitory effect at the mRNA-to-protein level, thereby achieving the goal of treating the disease (WO2016077321, WO2018195165). This most ideal hepatitis B treatment requires stabilization modification of siRNA and the use of appropriate delivery systems to target organs and cells, improving metabolic stability. However, current siRNAs cannot effectively reduce the levels of hepatitis B virus S antigen and E antigen.

[0007] Simultaneously, siRNA can regulate the expression of the corresponding gene by partially pairing with certain mRNA fragments. Specifically, the complementary pairing of the 5' seed region of the siRNA antisense strand with a non-target gene can partially or completely silence the gene expression. This phenomenon is the main reason for the off-target effects of siRNA in vivo and in vitro (Jackson et al. RNA (2006), 12, 1179-1187.). This drawback has been observed in both clinical and preclinical stages of siRNA therapy for hepatitis B (WO2020036862). Although some nucleotide modifications can reduce the off-target risk (Iribe et al. ACS Omega (2017), 2, 2055-2064; Janas et al. Nat. Commun. 2018,9, 723-732), the silencing effectiveness is also reduced, and the therapeutic safety window needs to be improved. Summary of the Invention

[0008] This disclosure relates to a ribavirin derivative-embedded double-stranded siRNA analog, a conjugate containing the same, its salt, and its uses. The double-stranded siRNA analog, the conjugate containing the same, and its salt can effectively inhibit multiple viral markers such as hepatitis B virus DNA, S antigen, and E antigen, providing an effective and feasible means for the treatment of hepatitis B, such as chronic hepatitis B (e.g., functional cure).

[0009] Therefore, in a first aspect, this disclosure provides a double-stranded siRNA analog comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence in which one or more nucleotides are replaced by r in the sequence shown in SEQ ID NO: 2, where r is... , Each of the nucleotides and r in the siRNA analog is independently modified or unmodified.

[0010] In some embodiments, one or more of the nucleotides and r in the siRNA analog are modified, while the other nucleotides and r are unmodified. The modifications include, for example, methoxy modification, fluorination modification, thiophosphate linkage, or replacement of the nucleotide with (…). S )-glycerol nucleic acids, etc.

[0011] In some embodiments, one or more of the nucleotides and r in the siRNA analog are modified, while the other nucleotides and r are unmodified. The modifications include, for example, methoxy modification, fluorination modification, thiophosphate linkage, and replacement of the nucleotide with (…). SModifications such as (E)-glycerol nucleic acid or (E)-vinyl phosphate esters.

[0012] In some embodiments, substantially all nucleotides and r in the siRNA analog are modified.

[0013] In some embodiments, 70%, 75%, 80%, 85%, 90%, or more of the nucleotides and r in the double-stranded siRNA analog are modified. In some embodiments, all the nucleotides and r in the double-stranded siRNA analog are modified.

[0014] In some embodiments, SEQ ID NO: 2 optionally includes a protrusion at the 5' end and / or the 3' end. In some embodiments, SEQ ID NO: 2 includes a protrusion of 0, 1, 2, 3, 4, or 5 nucleotides at the 5' end and / or the 3' end.

[0015] In some embodiments, when SEQ ID NO:2 comprises two nucleotide overhangs at the 5' and / or 3' ends, the three terminal nucleotides optionally have two phosphate thioester groups linked together, wherein two of the three nucleotides are overhangs, and the remaining nucleotide is a pairing nucleotide adjacent to the overhang. In some embodiments, the overhang is preferably derived from modified or unmodified UU. In some embodiments, the overhang is preferably derived from UU. In some embodiments, the overhang UU has two phosphate thioester groups linked to its adjacent pairing nucleotide.

[0016] In some embodiments, SEQ ID NO: 2 includes a 3' end overhang, preferably from modified or unmodified UU. In some embodiments, SEQ ID NO: 2 includes a 3' end overhang, preferably from UU. In some embodiments, SEQ ID NO: 2 includes a 3' end overhang, wherein the overhang UU has two phosphate thioester groups (e.g., c•u•u) linked to an adjacent paired nucleotide.

[0017] In some embodiments, the antisense strand of the double-stranded siRNA analog comprises a sequence in which one or more nucleosides are replaced by 'r' in the sequence shown in SEQ ID NO: 2. For example, the antisense strand comprises a sequence in which one nucleoside is replaced by 'r' in the sequence shown in SEQ ID NO: 2.

[0018] In some embodiments, the antisense strand of the double-stranded siRNA analog comprises a sequence in which one or more nucleosides are replaced by r in a sequence as shown in SEQ ID NO: 2. For example, the antisense strand comprises a sequence in which one, two, three, four, or five nucleosides are replaced by r in a sequence as shown in SEQ ID NO: 2.

[0019] In some embodiments, the antisense strand of the double-stranded siRNA analog comprises a sequence in which one or more nucleosides are replaced by 'r' in the sequence shown in SEQ ID NO: 2, said 'r' substitution occurring at any position in SEQ ID NO: 2. Preferably, said 'r' substitution occurs at positions 1 to 21 or 1 to 19 of the 5' end of SEQ ID NO: 2. For example, said 'r' substitution occurs at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 of the 5' end of SEQ ID NO: 2. Preferably, said '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.

[0020] In some embodiments, the antisense strand of the double-stranded siRNA analog comprises, or is composed of, a sequence as shown 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, or SEQ ID NO: 32 or SEQ ID NO: 36. In some embodiments, said sequence includes further nucleotide modifications, such as methoxy modification, fluorination, thiophosphate linkage, or replacement of the nucleotide with ( S )-glycerol nucleic acids, etc.

[0021] In some implementations, the antisense strand of the double-stranded siRNA analog comprises, for example, 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 the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 48, or composed of therein. In some embodiments, the sequence includes further nucleotide modifications, such as methoxy modification, fluorination modification, thiophosphate linkage, or replacement of the nucleotide with ( S Modifications such as (E)-glycerol nucleic acid or (E)-vinyl phosphate esters.

[0022] In some implementations, the positive strand of the double-stranded siRNA analog comprises, or is composed of, the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 28.

[0023] In some embodiments, the positive strand of the double-stranded siRNA analog comprises a sequence in which one or more nucleosides are replaced by r in a sequence as shown in SEQ ID NO: 1. For example, the positive strand comprises a sequence in which one nucleoside is replaced by r in a sequence as shown in SEQ ID NO: 1.

[0024] In some embodiments, the positive strand of the double-stranded siRNA analog comprises a sequence in which one or more nucleosides are replaced by r in a sequence as shown in SEQ ID NO: 1. For example, the positive strand comprises a sequence in which 1, 2, 3, 4, or 5 nucleosides are replaced by r in a sequence as shown in SEQ ID NO: 1.

[0025] In some embodiments, the positive strand of the double-stranded siRNA analog comprises a sequence in which one or more nucleosides are replaced by 'r' in the sequence shown in SEQ ID NO: 1, said 'r' substitution occurring at positions 1 through 19 of the 5' end of SEQ ID NO: 1. For example, said 'r' substitution occurs at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 5' end of SEQ ID NO: 1. Preferably, said 'r' substitution occurs at positions 2, 3, 7, 12, 15, 17, or 19 of the 5' end of SEQ ID NO: 1.

[0026] In some embodiments, the sequence of the positive strand of the double-stranded siRNA analog comprises, or is composed of, the sequence shown 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, or SEQ ID NO: 12 or SEQ ID NO: 25. In some embodiments, the sequence includes further nucleotide modifications, such as methoxy modifications, fluorinated modifications, thiophosphate linkages, etc.

[0027] In some embodiments, the sequence of the positive strand of the double-stranded siRNA analog comprises, or is composed of, the sequences shown 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. In some embodiments, the sequence includes further nucleotide modifications, such as methoxy modification, fluorination modification, thiophosphate linkage, or replacement of the nucleotide with (…). S Modifications such as (E)-glycerol nucleic acid or (E)-vinyl phosphate esters.

[0028] In some specific implementations, the sense and antisense strands of the double-stranded siRNA analog contain sequences in which one or more nucleosides are replaced by r, such as the sequence shown in SEQ ID NO: 2 of the antisense strand, where the r substitution occurs at position 2 at the 5' end of SEQ ID NO: 2, and the sequence shown in SEQ ID NO: 1 of the sense strand, where the r substitution occurs at position 7 at the 5' end.

[0029] In some implementations, the double-stranded siRNA analog is any one of S18 to S28: S18: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 4 or SEQ ID NO: 17. S19: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 6 or SEQ ID NO: 19. S20: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 7 or SEQ ID NO: 20. S21: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 8 or SEQ ID NO: 21. S22: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 9 or SEQ ID NO: 22. S23: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO: 23. S24: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 11 or SEQ ID NO: 24. S25: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 29 or SEQ ID NO: 33. S26: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 30 or SEQ ID NO: 34. S27: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 31 or SEQ ID NO: 35. S28: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 32 or SEQ ID NO: 36.

[0030] In some implementations, the double-stranded siRNA analog is any one of S1 to S17: S1: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 4 or SEQ ID NO: 17. S2: The justice chain is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense chain is SEQ ID NO: 4 or SEQ ID NO: 17. S3: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 6 or SEQ ID NO: 19. S4: The justice chain is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense chain is SEQ ID NO: 6 or SEQ ID NO: 19. S5: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 7 or SEQ ID NO: 20. S6: The justice chain is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense chain is SEQ ID NO: 7 or SEQ ID NO: 20. S7: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 8 or SEQ ID NO: 21. S8: The justice chain is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense chain is SEQ ID NO: 8 or SEQ ID NO: 21. S9: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 9 or SEQ ID NO: 22. S10: The justice chain is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense chain is SEQ ID NO: 9 or SEQ ID NO: 22. S11: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO: 23. S12: The justice chain is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO: 23. S13: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 11 or SEQ ID NO: 24. S14: The justice chain is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense chain is SEQ ID NO: 11 or SEQ ID NO: 24. S15: The justice chain is SEQ ID NO: 12 or SEQ ID NO: 25, and the antisense chain is SEQ ID NO: 4 or SEQ ID NO: 17. S16: The justice chain is SEQ ID NO: 13 or SEQ ID NO: 26, and the antisense chain is SEQ ID NO: 4 or SEQ ID NO: 17. S17: The justice chain is SEQ ID NO: 14 or SEQ ID NO: 27, and the antisense chain is SEQ ID NO: 4 or SEQ ID NO: 17.

[0031] In some implementations, the double-stranded siRNA analog is any one of S29 to S35: S29: The justice chain is SEQ ID NO: 37 or SEQ ID NO: 42, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO: 23. S30: The justice chain is SEQ ID NO: 38 or SEQ ID NO: 43, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO: 23. S31: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 39 or SEQ ID NO: 44. S32: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO: 45. S33: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 40 or SEQ ID NO: 46. S34: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO: 47. S35: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO: 48.

[0032] In some embodiments, the double-stranded siRNA analog is selected from: SEQ ID NO: 3 of the sense strand and SEQ ID NO: 4 of the antisense strand; SEQ ID NO: 5 of the sense strand and SEQ ID NO: 4 of the antisense strand; SEQ ID NO: 3 of the sense strand and SEQ ID NO: 6 of the antisense strand; SEQ ID NO: 5 of the sense strand and SEQ ID NO: 6 of the antisense strand; SEQ ID NO: 3 of the sense strand and SEQ ID NO: 7 of the antisense strand; SEQ ID NO: 5 of the sense strand and SEQ ID NO: 7 of the antisense strand; SEQ ID NO: 3 of the sense strand and SEQ ID NO: 8 of the sense strand; SEQ ID NO: 5 of the sense strand and SEQ ID NO: 8 of the antisense strand; SEQ ID NO: 3 of the sense strand and SEQ ID NO: 9 of the sense strand; SEQ ID NO: 5 of the sense strand and SEQ ID NO: 9 of the antisense strand; SEQ ID NO: 3 of the sense strand and SEQ ID NO: 9 of the antisense strand. 10. The justice chain is SEQ ID NO: 5 and the antisense chain is SEQ ID NO: 10. The justice chain is SEQ ID NO: 3 and the antisense chain is SEQ ID NO: 11. The justice chain is SEQ ID NO: 5 and the antisense chain is SEQ ID NO: 11. The justice chain is SEQ ID NO: 12 and the antisense chain is SEQ ID NO: 4. The justice chain is SEQ ID NO: 13 and the antisense chain is SEQ ID NO: 4. The justice chain is SEQ ID NO: 14 and the antisense chain is SEQ ID NO: 4. The justice chain is SEQ ID NO: 1 and the antisense chain is SEQ ID NO: 4. The justice chain is SEQ ID NO: 1 and the antisense chain is SEQ ID NO: 6. The justice chain is SEQ ID NO: 1 and the antisense chain is SEQ ID NO: 7. The justice chain is SEQ ID NO: 1 and the antisense chain is SEQ ID NO: 8. The justice chain is SEQ ID NO: 1 and the antisense chain is SEQ ID NO: 9. The justice chain is SEQ ID NO: 1. The antisense chain is SEQ ID NO: 10, the justice chain is SEQ ID NO: 1, the antisense chain is SEQ ID NO: 11, the justice chain is SEQ ID NO: 1, the antisense chain is SEQ ID NO: 29, the justice chain is SEQ ID NO: 1, the antisense chain is SEQ ID NO: 30, the justice chain is SEQ ID NO: 1, the antisense chain is SEQ ID NO: 31, the justice chain is SEQ ID NO: 1, the antisense chain is SEQ ID NO: 32, the justice chain is SEQ ID NO:37 and antisense strand is SEQ ID NO: 10, sense strand is SEQ ID NO: 38 and antisense strand is SEQ ID NO: 10, sense strand is SEQ ID NO: 3 and antisense strand is SEQ ID NO: 39, sense strand is SEQ ID NO: 3 and antisense strand is SEQ ID NO: 10, or sense strand is SEQ ID NO: 3 and antisense strand is SEQ ID NO: 40, wherein each of the nucleotides and r in the double-stranded siRNA analog is independently modified or unmodified.

[0033] In some embodiments, the double-stranded siRNA analog is selected from:

[0034] Each of the nucleotides and r in the double-stranded siRNA analog is independently modified or unmodified.

[0035] In some embodiments, the double-stranded siRNA analog is selected from: SEQ ID NO: 16 of the sense strand and SEQ ID NO: 17 of the antisense strand; SEQ ID NO: 18 of the sense strand and SEQ ID NO: 17 of the antisense strand; SEQ ID NO: 16 of the sense strand and SEQ ID NO: 19 of the antisense strand; SEQ ID NO: 18 of the sense strand and SEQ ID NO: 19 of the antisense strand; SEQ ID NO: 16 of the sense strand and SEQ ID NO: 20 of the sense strand; SEQ ID NO: 18 of the sense strand and SEQ ID NO: 20 of the antisense strand; SEQ ID NO: 16 of the sense strand and SEQ ID NO: 21 of the antisense strand; SEQ ID NO: 18 of the sense strand and SEQ ID NO: 21 of the antisense strand; SEQ ID NO: 16 of the sense strand and SEQ ID NO: 22 of the antisense strand; SEQ ID NO: 18 of the sense strand and SEQ ID NO: 22 of the antisense strand; SEQ ID NO: 18 of the sense strand and SEQ ID NO: 23 ... 22. The justice chain is SEQ ID NO: 16 and the antisense chain is SEQ ID NO: 23. The justice chain is SEQ ID NO: 18 and the antisense chain is SEQ ID NO: 23. The justice chain is SEQ ID NO: 16 and the antisense chain is SEQ ID NO: 24. The justice chain is SEQ ID NO: 18 and the antisense chain is SEQ ID NO: 24. The justice chain is SEQ ID NO: 25 and the antisense chain is SEQ ID NO: 17. The justice chain is SEQ ID NO: 26 and the antisense chain is SEQ ID NO: 17. The justice chain is SEQ ID NO: 27 and the antisense chain is SEQ ID NO: 17. The justice chain is SEQ ID NO: 28 and the antisense chain is SEQ ID NO: 17. The justice chain is SEQ ID NO: 28 and the antisense chain is SEQ ID NO: 19. The justice chain is SEQ ID NO: 28 and the antisense chain is SEQ ID NO: 20. The justice chain is SEQ ID NO: 28 and the antisense chain is SEQ ID NO: 21, the justice chain is SEQ ID NO: 28 and the antisense chain is SEQ ID NO: 22, the justice chain is SEQ ID NO: 28 and the antisense chain is SEQ ID NO: 23, the justice chain is SEQ ID NO: 28 and the antisense chain is SEQ ID NO: 24, the justice chain is SEQ ID NO: 28 and the antisense chain is SEQ ID NO: 33, the justice chain is SEQ ID NO: 28 and the antisense chain is SEQ ID NO: 34, the justice chain is SEQ ID NO: 28 and the antisense chain is SEQ ID NO: 35, the justice chain is SEQ ID NO: 28.NO: 28 and the antisense chain is SEQ ID NO: 36, the justice chain is SEQ ID NO: 42 and the antisense chain is SEQ ID NO: 23, the justice chain is SEQ ID NO: 43 and the antisense chain is SEQ ID NO: 23, the justice chain is SEQ ID NO: 16 and the antisense chain is SEQ ID NO: 44, the justice chain is SEQ ID NO: 16 and the antisense chain is SEQ ID NO: 45, the justice chain is SEQ ID NO: 16 and the antisense chain is SEQ ID NO: 46, the justice chain is SEQ ID NO: 16 and the antisense chain is SEQ ID NO: 47, or the justice chain is SEQ ID NO: 16 and the antisense chain is SEQ ID NO: 48.

[0036] In some embodiments, the double-stranded siRNA analog is selected from:

[0037] In a second aspect, this disclosure provides conjugates of double-stranded siRNA analogs comprising a double-stranded siRNA analog according to a first aspect of this disclosure, and pharmaceutically acceptable conjugation groups conjugated to the double-stranded siRNA analog.

[0038] In some embodiments, the pharmaceutically acceptable conjugate in the double-stranded siRNA analog conjugate contains 1 to 5 GalNAc (N-acetylgalactosamine) groups. Preferably, the pharmaceutically acceptable conjugate contains 1, 2, 3, 4, or 5 GalNAc groups. More preferably, the pharmaceutically acceptable conjugate contains 3 or 4 GalNAc groups.

[0039] In some embodiments, the pharmaceutically acceptable conjugate group in the double-stranded siRNA analog conjugate comprises the compound group D. .

[0040] In some implementations, a pharmaceutically acceptable conjugate group in the conjugate of the double-stranded siRNA analog is attached to the 3' end of the positive strand of the double-stranded siRNA analog.

[0041] In some embodiments, the thiophosphate moiety of the double-stranded siRNA analog or its conjugate includes (R)- and (S)-enantiomers, diastereomers, and / or racemic mixtures thereof.

[0042] In some implementations, the conjugates of the double-stranded siRNA analogs are selected from:

[0043] The term D is as described above.

[0044] In a third aspect, this disclosure provides salts of double-stranded siRNA analogs according to a first aspect of this disclosure or conjugates of double-stranded siRNA analogs according to a second aspect of this disclosure.

[0045] In some embodiments, the salt described above is selected from alkali addition salts, acid addition salts, and combinations thereof.

[0046] In some embodiments, 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 inorganic acid salts, organic acid salts and combinations thereof.

[0047] In some embodiments, the inorganic acid is selected from hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, 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, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and combinations thereof.

[0048] In a fourth aspect, this disclosure provides pharmaceutical compositions comprising a double-stranded siRNA analog according to a first aspect of this disclosure, a conjugate of a double-stranded siRNA analog according to a second aspect of this disclosure, or a salt according to a third aspect of this disclosure, and a pharmaceutically acceptable carrier or excipient.

[0049] In a fifth aspect, this disclosure provides the use of a double-stranded siRNA analog according to the first aspect of this disclosure, a conjugate of a double-stranded siRNA analog according to the second aspect of this disclosure, a salt according to the third aspect of this disclosure, or a pharmaceutical composition according to the fourth aspect of this disclosure in the preparation of a medicament for treating hepatitis B.

[0050] In some embodiments, this disclosure provides a double-stranded siRNA analog according to a first aspect of this disclosure, a conjugate of a double-stranded siRNA analog according to a second aspect of this disclosure, a salt according to a third aspect of this disclosure, or a pharmaceutical composition according to a fourth aspect of this disclosure for treating hepatitis B in a subject.

[0051] In a sixth aspect, this disclosure provides a method for treating a subject with hepatitis B virus, comprising the step of administering to the subject a double-stranded siRNA analog according to a first aspect of this disclosure, a conjugate of a double-stranded siRNA analog according to a second aspect of this disclosure, a salt according to a third aspect of this disclosure, or a pharmaceutical composition according to a fourth aspect of this disclosure.

[0052] In a seventh aspect, this disclosure provides a double-stranded siRNA analog according to the first aspect of this disclosure, a conjugate of a double-stranded siRNA analog according to the second aspect of this disclosure, a salt according to the third aspect of this disclosure, or a pharmaceutical composition according to the fourth aspect of this disclosure for treating hepatitis B in subjects.

[0053] In some embodiments of this disclosure, the hepatitis B can be at any stage of the disease, such as acute hepatitis B, chronic hepatitis B, or cirrhosis or liver cancer caused by hepatitis B virus infection. In some embodiments, the hepatitis B is chronic hepatitis B.

[0054] Definitions and Explanations Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with the meaning as understood by one of ordinary skill in the art. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0055] In this disclosure, unless otherwise stated, the terms "comprising, including, and containing" or equivalents are open-ended expressions, meaning that they may cover other unspecified elements, components, or steps in addition to those listed.

[0056] In this disclosure, the HBV gene refers to the gene whose DNA sequence is shown in GenBank accession number NC_003977.1. The gene shown in GenBank accession number NC_003977.1 is the complete HBV genome.

[0057] In some implementations, double-stranded siRNA analogs can target the X opening reading frame (X ORF) of HBV.

[0058] In this disclosure, a double-stranded siRNA analog refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, which have “sense” and “antisense” orientations relative to the target RNA. In this disclosure, “complementary” has the meaning known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand pair complementaryly with the bases of the other strand. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair comprises one purine and one pyrimidine. When adenine on one strand always pairs with uracil on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence.

[0059] In this disclosure, unless otherwise specified, uppercase letters C, G, U, and A represent the base composition of nucleotides. Lowercase letters c, g, u, and a respectively indicate that the nucleotide represented by the corresponding uppercase letter is modified with a methoxy group; underline The uppercase letter indicates that the nucleotide represented by it is fluorinated; the spacer "•" indicates that the two nucleotide residues adjacent to the spacer "•" are linked by a thiophosphate group; VP indicates that the nucleotide to the right of the letter VP is ( E Nucleotides modified with α-vinyl phosphate. For example, "a•g" indicates that the a and g residues are linked by a thiophosphate group.

[0060] The "modification" of nucleotides described in this disclosure includes, but is not limited to, methoxy modification, fluorination modification, and ( E )-vinyl phosphate modification, thiophosphate linkage, or replacement of nucleotides with ( S )-glycerol nucleic acid, etc. The sequences described in this disclosure may include those listed in "Further Modified Sequences" in Table 1 below.

[0061] The fluorinated nucleotides described in this disclosure refer to nucleotides formed by replacing the 2'-hydroxyl group of the ribosome with fluorine, and the methoxylated nucleotides refer to nucleotides formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

[0062] The disclosure describes ( E Nucleotides modified with α-vinyl phosphate represent the following structural units: , Where E is selected from , , , and ; X is selected from OCH3 and F.

[0063] The disclosure describes ( S )-glycerol nucleic acid (Agn) represents the following structural units: ; (Agn) and other nucleotide residues are linked together by phosphate esters or thiophosphate esters. For example, "a•(Agn)" means that the a and (Agn) residues are linked by thiophosphate ester groups, and "a(Agn)" means that the a and (Agn) residues are linked by phosphate ester groups.

[0064] In some embodiments, the double-stranded siRNA analog comprises a sense strand or an r'-intercalated sense strand and an r'-intercalated antisense strand. The sense strand, the r'-intercalated sense strand, and the r'-intercalated antisense strand all contain nucleotide groups as basic structural units. As is known to those skilled in the art, nucleotide groups contain phosphate groups, ribose groups, and bases, which will not be elaborated further here.

[0065] The r'-intercalated sequence described in this disclosure refers to a sequence in which at least one nucleotide residue is linked to r, including sequences in which r replaces a nucleoside. The r'-intercalated sequences described in this disclosure include, but are not limited to: r'-intercalated double-stranded siRNAs, r'-intercalated sense strands, and r'-intercalated antisense strands. For example, 5'-aGUrrA•C-3', 5'-rGgAAC-3', and 5'-AG•UrAAcCuCr-3' are all examples of r'-intercalation.

[0066] The r'-embedded double-stranded siRNA described in this disclosure refers to a double-stranded siRNA in which at least one nucleotide residue is linked to r, including double-stranded siRNA in which r replaces one nucleoside in the sequence of the double-stranded siRNA. The r'-embedded sense strand described in this disclosure refers to a sense strand in which at least one nucleotide residue is linked to r, including if one or more nucleosides in the sense strand are replaced by r. The r'-embedded antisense strand described in this disclosure refers to an antisense strand in which at least one nucleotide residue is linked to r, including if one or more nucleosides in the antisense strand are replaced by r.

[0067] The r' mentioned in this disclosure is (where X is selected from SH and OH), is an analog of natural nucleotide bases, unlike any publicly patented natural nucleotide bases, and brings unpredictable activity when introduced into the nucleic acid sequence.

[0068] In this disclosure, r represents the following structural unit: ; r and other nucleotide residues are linked to each other by phosphate esters or thiophosphate esters. For example, "a•r" means that the a and r residues are linked by thiophosphate ester groups, and "ar" means that the a and r residues are linked by phosphate ester groups.

[0069] The term "multiple" as used in this 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 analogues.

[0070] In this disclosure, the sense or antisense strand of the double-stranded siRNA analog may also include "protrusions," such as unpaired protruding nucleotides that do not directly participate in the RNA double helix structure, which is typically formed by "sense" and "antisense" strand pairs as defined herein. Such protrusions may include one or more modified or unmodified U, T, and A nucleotides. For example, SEQ ID NO: 2 may include a modified or unmodified UU protrusion in the 5' and / or 3' segment.

[0071] In this disclosure, the conjugate of the double-stranded siRNA analog is a compound formed by linking a double-stranded siRNA analog with a pharmaceutically acceptable conjugate group, and the double-stranded siRNA analog and the pharmaceutically acceptable conjugate group are covalently linked.

[0072] In this disclosure, a pharmaceutically acceptable conjugation group may be attached to the 3' end of the positive strand of a double-stranded siRNA analog or an r'-intercalated positive strand.

[0073] Generally, pharmaceutically acceptable conjugates include a pharmaceutically acceptable target molecule and an optional linker. Examples of conjugates, linkers, and target molecules can be found in the disclosure of WO2015006740A2. Exemplary conjugates include, but are not limited to, L96 or compound group D.

[0074] In the context of this disclosure, unless otherwise stated, “conjugation” means the covalent connection between two or more chemical parts, each having a specific function; correspondingly, “conjugated compound” means a compound formed by the covalent connection between the chemical parts.

[0075] The compounds disclosed herein can exist in specific geometric or stereoisomeric forms. This disclosure envisions all such compounds, including ( R )- and( SEnantiomers, diastereomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, are all within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this disclosure.

[0076] Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

[0077] Unless otherwise stated, the term "diastereomer" refers to a stereoisomer of a molecule having two or more chiral centers and being in a non-mirror relationship with each other.

[0078] Unless otherwise specified, the absolute configuration of a solid center is represented by wedge solid key () and wedge dashed key () and the relative configuration of a solid center is represented by straight solid key () and straight dashed key () and the wedge solid key () or wedge dashed key () is represented by wavy line () or straight solid key () and / or straight dashed key () is represented by wavy line ()

[0079] Unless otherwise stated, the terms "rich in one isomer," "isomer enrichment," "rich in one enantiomer," or "enantiomer enrichment" mean that the content of one isomer or 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 greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

[0080] Unless otherwise stated, the terms "isomer excess" or "enantiomer excess" refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if one isomer or enantiomer is 90% and the other isomer or enantiomer is 10%, then the isomer or enantiomer excess (ee value) is 80%.

[0081] Optically active materials can be prepared through chiral synthesis, chiral reagents, or other conventional techniques. R )-and( S )-Isomers and D and LIsomers. To obtain an enantiomer of a compound disclosed herein, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the desired enantiomer in pure form. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric is formed with a suitable optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and then recovery of the pure enantiomer. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., from amines to carbamates). The compounds of this disclosure may contain atomic isotopes in non-natural proportions on one or more atoms constituting the compound. For example, the compound may be labeled with a radioactive isotope, such as tritium ( 3 H), Iodine-125 ( 125 I) or C-14 14 C). For example, deuterium can be used to replace hydrogen to form deuterated drugs. The bond between deuterium and carbon is stronger than that between ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have advantages such as reduced toxicity, increased drug stability, enhanced efficacy, and prolonged drug biological half-life. All isotopic variations of the compounds disclosed herein, regardless of radioactivity, are included within the scope of this disclosure.

[0082] The term "salt" refers to the salt of the compounds disclosed herein, prepared by reacting a compound having specific substituents, as discovered in this disclosure, with a relatively non-toxic acid or base. When the compounds of this disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds 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 salts, or similar salts. When the compounds of this disclosure contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds 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, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; as well as salts of amino acids (such as arginine) and salts of organic acids such as glucuronic acid. Certain specific compounds of this disclosure contain both basic and acidic functional groups, and thus can be converted into either a base or an acid addition salt.

[0083] The salts disclosed herein can be synthesized from parent compounds containing anions or bases using conventional chemical methods. Generally, such salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both.

[0084] The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments disclosed herein.

[0085] The solvents used in this disclosure are commercially available.

[0086] Unless otherwise specified, all solvent ratios used in column chromatography and preparative thin-layer silica gel chromatography in this disclosure are volume ratios.

[0087] List of abbreviations

[0088] Compounds are named according to conventional naming principles in the art or using ChemDraw® software; commercially available compounds are named according to the supplier catalog. Detailed Implementation

[0089] The present disclosure is described in detail below with reference to examples, but this does not imply any adverse limitation thereof. The compounds of the present disclosure can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments 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 thereof.

[0090] Example 1: Synthesis of phosphoramide monomer

[0091] Step A: A solution of (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-macrotriacetate (i.e., Formula 1-1) (30 g, 94.26 mmol) and methyl 1,2,4-triazole-3-carboxylic acid (11.98 g, 94.26 mmol) dissolved in methyl acetate (220 mL) was concentrated to near complete dryness in a 90°C oil bath at 1 bar. A methyl acetate solution (2 mL) of trifluoromethanesulfonic acid (141.46 mg, 0.94 mmol) was added to the mixture, and the mixture was stirred in a 125°C oil bath at 30 mbar for 4 hours. The reaction solution was cooled to 70°C, ethanol (70 mL) was added, and the mixture was stirred at 70°C until a homogeneous solution was formed. Stirring was then stopped and the solution was cooled to 50°C. After precipitation, the mixture was allowed to cool to 25°C and then placed at 0°C for 16 hours. The reaction mixture was filtered through a Buchner funnel, and the filter cake was treated with 180 mL of ethanol (60 mL of ethanol). 3) Rinse and vacuum dry to obtain 1-2. 1 H NMR (400 MHz, CDCl3): 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).

[0092] Step B: The compound shown in Formula 1-2 (15 g, 38.93 mmol) was dissolved in methanol (100 mL) with triethylamine (4.14 g, 40.87 mmol). The mixture was stirred at 50 °C for 17 hours under nitrogen protection. The reaction solution was concentrated under reduced pressure to give 1-3. 1 H NMR (400 MHz, CD3OD): 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).

[0093] Step C: Dissolve the compound shown in Formulas 1-3 (10 g, 38.58 mmol) in pyridine (250 mL) and add 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (12.29 g, 38.97 mmol) dropwise at 0°C. Gradually heat the mixture to 25°C and stir for 16 hours. Concentrate the reaction mixture under reduced pressure, suspend the concentrate in ethyl acetate (250 mL), and filter through a Buchner funnel. Dilute the filtrate with 750 mL (250 mL) of 3 M hydrochloric acid. 3) and 250 ml of saturated saline solution (250 ml) 1) Wash, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain crude product. Purify by column chromatography (SiO2, petroleum ether / dichloromethane / ethyl acetate = 3 / 1 / 1) to obtain 1-4. 1 H NMR (400MHz, CDCl3): δ 8.43 (s, 1H), 5.95 (s, 1H), 4.73 (dd, J = 4.75, 8.00Hz, 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).

[0094] Step D: Add potassium carbonate (11.34 g, 82.02 mmol) and silver oxide (I) (19.01 g, 82.02 mmol) to a solution containing compounds of formulas 1-4 (8.23 g, 16.40 mmol), potassium carbonate (11.34 g, 82.02 mmol), and silver oxide (I) (19.01 g, 82.02 mmol). N , N Iodimethane (11.64 g, 82.02 mmol) was added to a mixture of dimethylformamide (50 mL), and the mixture was stirred at 25°C for 3 hours. The reaction solution was diluted with ethyl acetate (300 mL) and filtered through a Buchner funnel. The filtrate was diluted with 250 mL of sodium thiosulfate aqueous solution. 1) 250 ml of water (250 ml) 1) and 250 ml of saturated saline solution (250 ml) 1) Wash, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain crude product. Purify by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1) to obtain 1-5. 1 H NMR (400 MHz, CDCl3): δ8.58 (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).

[0095] Step E: At 0°C, triethylamine trihydrofluoride (2.25 g, 13.95 mmol) was added dropwise to a tetrahydrofuran (50 mL) solution of the compounds shown in Formulas 1-5 (3.27 g, 6.34 mmol). The mixture was gradually heated to 25°C and stirred for 16 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product. After purification by column chromatography (SiO2, dichloromethane / methanol = 20 / 1), 1-6 were obtained. 1 H NMR (400 MHz, CD3OD): 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).

[0096] Step F: At 0°C, 2.42 g (7.14 mmol) of 4,4-dimethoxytriphenylmethyl chloride was added to a pyridine (20 mL) solution of the compounds shown in Formulas 1-6 (1.30 g, 4.76 mmol), and the mixture was stirred at 25°C for 16 hours. The reaction solution was diluted with ethyl acetate (70 mL), quenched at 25°C with a saturated sodium bicarbonate aqueous solution (20 mL), and diluted with water (40 mL). The combined organic phases after separation were mixed with 60 mL of water (60 mL). 1) and 60 ml of saturated saline solution (60 ml) 1) Wash, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the crude product. After... p -HPLC purification (Separation column: Phenomenexluna C18 (size: 250mm)) 50 mm, particle size: 10 μm); mobile phase: [water (10 mM ammonium bicarbonate) - acetonitrile]; elution gradient: 35%-65%, 20 min) to obtain 1-7. 1 H NMR (400 MHz, CDCl3): 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 [MH] - .

[0097] Step G: At 0 degrees Celsius, add 2-cyanoethyl- to a dichloromethane (8 mL) solution of the compound shown in Formula 1-7 (1.10 g, 1.91 mmol). N , N -Diisopropylphosphonamide (678.45 mg, 2.87 mmol) and N , N -Diisopropylethylamine was reacted and stirred at 20°C for 0.5 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product. Purification by column chromatography (SiO2, petroleum ether / ethyl acetate = 50 / 1 to 1 / 2) yielded compound of formula 1. LCMS (ESI) m / z: 776.3 [M+H] + .

[0098] Synthesis of Example 2D01

[0099]

[0100]

[0101]

[0102]

[0103] Step A: Dissolve 25 g of 11-dodecyn-1-ol (137.14 mmol) and triethylamine (16.65 g, 164.56 mmol) in dichloromethane (250 mL), and add methanesulfonyl chloride (18.85 g, 164.56 mmol) at 0 °C. Stir the mixture at 0 °C for 2 hours. Dilute the reaction mixture with water (400 mL) and add 800 mL of dichloromethane (400 mL) 2) Extraction. The combined organic phases were mixed with 400 mL of water (200 mL of water). 2) Wash with saturated brine, dry with anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain 2-2.

[0104] Step B: The compound shown in Formula 2-3 (20 g, 67.26 mmol) dissolves in N , N- Dimethylformamide (200 mL) was added at 0°C to sodium hydride (60% purity, 4.04 g, 100.89 mmol), followed by the compound shown in formula 2-2 (19.27 g, 73.99 mmol). The mixture was stirred at 25°C for 16 hours. The reaction mixture was quenched with water (1 L) and then with 1.6 L (800 mL) of dichloromethane. 2) Extraction. The combined organic phases were rinsed with 800 ml of saturated brine. 1) Wash, dry with anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain 2-4. 1 H NMR (400 MHz, DMSO-) d 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 (m, 16H).

[0105] Step C: Dissolve the compound shown in Formula 2-4 (48 g, 103.98 mmol) in methanol (870 mL), and add a hydrogen chloride methanol solution (4 mol / L, 400 mL, 1.6 mol). Stir the mixture at 30°C for 2 hours. Add a hydrogen chloride methanol solution (4 mol / L, 350 mL, 1.4 mol) to the reaction mixture. Stir the mixture at 30°C for 16 hours. Concentrate the reaction mixture under reduced pressure, and add 200 mL of chloroform (100 mL) 2) Concentrate under reduced pressure until a white solid appears. Add toluene (130 mL) and petroleum ether (130 mL), and stir the mixture at 15°C for 16 hours. Filter the reaction solution through a Buchner funnel, collect the filter cake, and dry it under vacuum to obtain a white solid. Dissolve the white solid in dichloromethane (50 mL), add an aqueous solution of sodium hydroxide (6.59 g, 164.66 mmol) (50 mL), and stir at 20°C for 1 hour. Dilute the reaction solution with water (500 mL) and then with 1 liter (500 mL) of dichloromethane. 2) Extraction. The combined organic phases were dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 2-5.

[0106] Step D: To a mixture of the compound shown in 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), tert-butyl acrylate (22.72 g, 177.28 mmol) was added, and the mixture was stirred at 25°C for 16 hours under nitrogen protection. The reaction solution was diluted with water (500 mL) and diluted with ethyl acetate (1 L, 500 mL). 2) Extraction. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. After purification by column chromatography (SiO2, petroleum ether / ethyl acetate / ethanol (containing 0.1% ammonia) = 36 / 3 / 1 to 16 / 3 / 1) to obtain 2-6. 1 H NMR (400MHz, DMSO- d 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).

[0107] Step E: Add triethylamine (9.15 g, 90.45 mmol) and succinic anhydride (6.79 g, 67.83 mmol) to a dichloromethane (250 mL) solution of the compound shown in Formula 2-6 (24.5 g, 45.22 mmol), and stir at 20 °C for 16 hours. Add dichloromethane (1 L) and hydrochloric acid (1 mol / L, 1 L) to the reaction mixture. Dry the separated organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain 2-7. 1 H NMR (400 MHz, CDCl3): 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, 1 H), 1.57-1.47 (m, 4H), 1.46-1.40 (m, 18H), 1.40-1.31 (m, 2H), 1.30-1.21 (m, 10H).

[0108] Step F: Dissolve the compound shown in Formula 2-7 (27.4 g, 42.69 mmol) in formic acid (140 mL), and stir the mixture at 20 °C for 16 hours under nitrogen protection. Concentrate the reaction mixture under reduced pressure, and add 300 mL (150 mL) of toluene. 2), concentrated under reduced pressure to obtain 2-8. 1 H NMR (400 MHz, CDCl3): 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).

[0109] Step G: The compound shown in 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 -Tetramethylurea hexafluorophosphate (51.92 g, 136.55 mmol) dissolved in N , N - Dimethylformamide (250 ml) added N 29.74 g (170.69 mmol) of tert-butyl (3-aminopropyl)carbamate. The mixture was stirred at 20°C for 16 hours. Dichloromethane (1 L) and hydrochloric acid (1 mol / L, 1 L) were added to the reaction mixture, and the separated organic phase was diluted with 1 L of water. 1) 1 liter of sodium bicarbonate aqueous solution (1 liter) 1) and 1 liter of saturated saline solution (1 liter) 1) Wash sequentially, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain crude product. Purify by column chromatography (SiO2, petroleum ether / ethyl acetate / ethanol = 40 / 3 / 1 to 10 / 3 / 1) to obtain 2-9. 1 H NMR (400 MHz, CDCl3): 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, 1 H), 1.70-1.57 (m, 6H), 1.56-1.47 (m, 4H), 1.46-1.38 (m, 29H), 1.30-1.25 (m, 10H).

[0110] Step H: The compound shown in 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 hours. The reaction solution was concentrated under reduced pressure, and 750 mL (250 mL) of a toluene / acetonitrile = 3 / 1 mixture was added. 3) Concentrate under reduced pressure to obtain 2-10 (trifluoroacetate).

[0111] Step I: Compound shown in 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) dissolved in N , N - Dimethylformamide (90 mL), add the compound shown in Formula 2-10 (tris(trifluoroacetate), 15.6 g, 15.00 mmol) to the mixture and N , N -Diisopropylethylamine (21.32 g, 165.00 mmol) N , N A solution of dimethylformamide (120 mL) was prepared. The mixture was stirred at 20°C for 16 hours. Dichloromethane (1.2 L) and hydrochloric acid (1 mol / L, 1 L) were added to the reaction mixture, and the separated organic phase was diluted with 1 L of water. 1) 1 liter of sodium bicarbonate aqueous solution (1 liter) 1) and 1 liter of saturated saline solution (1 liter) 1) Wash sequentially, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain crude product. After purification by column chromatography (SiO2, dichloromethane / methanol = 100 / 1 to 10 / 1 to dichloromethane / ethanol = 1 / 1) to obtain 2-12. 1 H NMR (400 MHz, DMSO-) d 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).

[0112] Step J: Mix the compound shown in Formula 2-12 (1.00 g, 0.50 mmol) with... 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 this mixture at 0°C. The mixture was stirred at 0 to 15°C for 2 hours. The reaction was quenched with sodium bisulfite (1.27 g), and hydrochloric acid (2 mol / L, 5 mL) and water (30 mL) were added, followed by 120 mL (40 mL) of a chloroform / isopropanol 3 / 1 mixture. 3) Extraction. The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then 180 mL (30 mL) of a 1 / 1 toluene / acetonitrile mixture was added. 6), concentrated under reduced pressure to obtain 2-13. 1 H NMR (400 MHz, CD3OD): 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).

[0113] Step K: To the compound shown in Formula 2-13 (1.00 g, 0.50 mmol) N , N - Dimethylformamide (10 mL) solution added N , N -Diisopropylethylamine (0.26 g, 1.99 mmol) and O -(7-azabenzotriazol-1-yl)- N , N , N , N -Tetramethylurea hexafluorophosphate (0.23 g, 0.60 mmol). After stirring the mixture, add the compound shown in Formula 2-14 (0.23 g, 0.55 mmol). Stir the mixture at 15°C for 16 hours. Add dichloromethane (50 mL) and water (50 mL) to the reaction mixture, and then separate the organic phase with 50 mL of saturated sodium bicarbonate aqueous solution (50 mL). 1) 50 ml of water (50 ml) 1) and 50 ml of saturated saline solution (50 ml) 1) Wash sequentially, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain crude product. After purification by column chromatography (SiO2, dichloromethane / methanol (containing 0.1% triethylamine) = 20 / 1 to 10 / 1) to obtain 2-15. 1 H NMR (400 MHz, DMSO-) d 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).

[0114] Step L: Triethylamine (67.24 mg, 0.64 mmol) was added sequentially to a dichloromethane (8 mL) solution of the compound shown in Formula 2-15 (0.80 g, 0.33 mmol), 4- N , N - Dimethylaminopyridine (0.12 g, 1.00 mmol) and succinic anhydride (83.13 mg, 0.83 mmol). The mixture was stirred at 10°C for 16 hours. Dichloromethane (50 mL), water (30 mL), and saturated brine (30 mL) were added to the reaction mixture, and the separated organic phase was diluted with 30 mL of water. 1) and 30 ml of saturated saline solution (30 ml) 1) Wash sequentially, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the crude product. After... p -HPLC purification (Separation column: Waters Xbridge C18 (size: 150mm)) 50 mm, particle size: 10 μm); mobile phase: [water (10 mM ammonium bicarbonate) - acetonitrile]; elution gradient: 27%-57%, 11 min) to obtain Example 2 (compound D01). 1 H NMR (400 MHz, DMSO-) d 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).

[0115] Example 3: Synthesis of double-stranded siRNA analogs or their conjugates Synthesis of D-containing single-stranded oligonucleotides: Oligonucleotides were synthesized using a phosphoramidite solid-phase synthesis technique. Synthesis was performed on a solid support made of a controllable porous glass (aminoCPG, 500 Å) covalently linked to DO1. All 2'-modified RNA phosphoramidite and auxiliary reagents were commercially available. All amides were dissolved in anhydrous acetonitrile and added to a molecular sieve (3 Å), with coupling time of 5 min using 5-ethylthio-1H-tetrazole (ETT) as an activator. Phosphothiophosphate bonds were generated using a 50 mM solution of 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazol-3-thione (DDTT) in anhydrous acetonitrile / pyridine (v / v = 1 / 1) for 3 min. All sequences were synthesized after the final removal of the DMT group.

[0116] Synthesis of D-free single-stranded oligonucleotides: Oligonucleotides were synthesized using phosphoramidite solid-phase synthesis. Synthesis was performed on a general-purpose controlled porous glass CPG (500 Å). All 2'-modified RNA phosphoramidite and auxiliary reagents were commercially available. All amides were dissolved in anhydrous acetonitrile and added to a molecular sieve (3 Å), with coupling time of 5 min using 5-ethylthio-1H-tetrazole (ETT) as an activator. Phosphothiophosphate bonds were generated using a 50 mM solution of 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazol-3-thione (DDTT) in anhydrous acetonitrile / pyridine (v / v=1 / 1) for 3 min. All sequences were synthesized after final removal of the DMT group.

[0117] Cleavage and deprotection of oligomers bound to CPG: After termination of solid-phase synthesis, the protecting group was removed by treatment with an acetonitrile solution containing 20% ​​diethylamine for 30 minutes without cleaving the oligonucleotide from the CPG. Subsequently, the dried CPG was treated with concentrated ammonia at 40°C for 18 hours. After centrifugation, the supernatant was transferred to a new tube and the CPG was washed with ammonia. The combined solutions were concentrated to obtain a solid mixture.

[0118] Purification of single-stranded oligonucleotides: Oligomers were purified by HPLC using NanoQ anion exchange. Buffer A consisted of 10 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4, and 20% acetonitrile; buffer B consisted of 500 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4, and 20% acetonitrile. The target product was isolated and desalted using a reverse-phase C18 column.

[0119] Annealing of single-stranded oligonucleotides to produce siRNA: Prepare a 200 μM solution of the single-stranded oligonucleotides to be annealed using sterile RNase-free H2O. Set up the annealing reaction system as follows: Incubate 100 μL of the mixture at 10 nmol in a 95 ℃ water bath for 10 minutes (for amounts ≥100 nmol, a 20-minute high-temperature treatment is required) → Immediately transfer to a 60 ℃ water bath for natural cooling → The annealed solution should not be stored at high temperatures. Combine equimolar amounts of the single-stranded oligonucleotide solution to form complementary strands.

[0120] Table 1. Double-stranded siRNA analogs targeting the hepatitis B virus gene, their conjugates, and their corresponding core sequences.

[0121] *: D represents the residues of the small molecule fragment D01 after a chemical reaction, which bind to nucleic acid via covalent bonds. Its structure is as follows: .

[0122] **: The antisense sequence of the r'-embedded sequence is obtained by r'-embedding the antisense sequence of the core sequence with a 3' end UU. For example, SEQ ID NO: 4 is obtained by r'-embedding SEQ ID NO: 2 with a 3' end UU.

[0123] ***: When the sequence contains D, D refers to the connection position of the conjugate group D. For example, g•r•gu G c ACU ucgcuucacaD (5'-3') represents the sequence g•r•gu as shown in SEQ ID NO: 16. G c ACU ucgcuucaca is connected to D at the 3' end.

[0124] Example 4: In vitro HBV test 1. Experimental objective: The levels of HBV antigens (HBsAg and HBeAg) in the supernatant of HepG2-NTCP cell culture were detected by enzyme-linked immunosorbent assay (ELISA), and the compounds were analyzed by EC50. 50 The values ​​were used as indicators to evaluate the inhibitory activity of the compounds against HBV; at the same time, cell viability was detected by Cell-titer Glo to evaluate the cytotoxicity of the compounds.

[0125] 2. Experimental materials: 2.1 Cell line: HepG2-NTCP cells HepG2-NTCP cell culture medium (DMEM, Invitrogen-11330032; 10% serum, Invitrogen-10099141; 100 units / ml penicillin and 100 µg / ml streptomycin, Hyclone-SV30010; 1% non-essential amino acids, Invitrogen-11140050; 2mM L-glutamine, Invitrogen-25030081; 1mM sodium pyruvate, Gibco-11360-070; 500 μg / ml geneticin, Invitrogen-10131027) 2.2 Reagents: Trypsin (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).

[0126] 2.3 Consumables and Instruments: 96-well cell culture plate (Corning-3599); CO2 incubator (HERA-CELL-240) ELISA reader (BioTek Synergy 2) 3. Experimental steps and methods: 3.1 Day 0, seed HepG2-NTCP (7.5 × 10⁻⁶) 4 Cells / well) cells into a 48-well plate, at 37 Incubate overnight in 5% CO2 at room temperature.

[0127] 3.2 On day 1, replace the culture medium with one containing 1% DMSO.

[0128] 3.3 On day 2, HepG2-NTCP was infected with type D HBV (concentrated from the supernatant of HepG2.2.15 cell culture) (2000 GE / cell).

[0129] 3.4 On the third day, aspirate the infection fluid and add fresh culture medium containing 1% DMSO.

[0130] 3.5 Day 6, according to Lipofectamine ® Instructions for using RNAiMax (Invitrogen): Transfect siRNA conjugates. Perform 5-fold serial dilutions of the conjugate at 7 concentrations, triplet wells, with a final concentration of 0.16 pM. The compound is a combination of sense and antisense strands, a single chemical entity, with a maximum concentration of 2.5 nM.

[0131] 3.6 On day 12, the supernatant from the culture wells was collected, and HBV surface antigen and e antigen were measured by ELISA. After collecting the supernatant, Cell-titer Glo was added to measure cell viability.

[0132] 3.7 ELISA determination of hepatitis B surface antigen (HBsAg) and e antigen (HBeAg): Refer to the product instructions for specific steps. The steps are briefly described as follows: Add 50 μl of sample and standard to each well, then add 50 μl of enzyme conjugate to each well, vortex to mix, incubate at 37°C for 60 minutes, then wash the plate 5 times with washing buffer, then add 50 μl of luminescent substrate to each well, mix, and react at room temperature in the dark for 10 minutes. Finally, use an ELISA reader to detect the chemiluminescence intensity.

[0133] 3.8 Data Analysis: Calculate cell viability percentage: % viability = (luminescence value of sample - luminescence value of culture medium control) / (luminescence value of DMSO control - luminescence value of culture medium control) ×100.

[0134] Calculate the percentage of inhibition of HBV surface antigen and e antigen: % Inh. = (1 - antigen value in sample / DMSO control antigen value) × 100.

[0135] Calculate CC 50 and EC 50 Calculating the C=C of compounds using GraphPad Prism software 50 and 50% inhibitory concentration (EC50) against HBV 50 )value.

[0136] 4. Experimental results: See Table 2.

[0137] Table 2 shows the experimental results of the tested sequences in reducing HBsAg and HBeAg levels in cells.

[0138] * The sample tested was a conjugate of a double-stranded siRNA analog.

[0139] Example 5: Study on anti-hepatitis B virus activity and safety in a mouse model of hepatitis B virus mediated by recombinant adeno-associated virus type 8 vector (AAV-HBV). Experimental objective: AAV vector-mediated HBV transfection in mice is a rapid and efficient HBV model. Utilizing the high hepatotropism of the AAV8 vector, recombinant adeno-associated virus type 8 (rAAV8-1.3HBV) carrying 1.3 copies of the HBV genome is efficiently introduced into hepatocytes via tail vein injection in mice. Due to the characteristics of AAV viral vectors, the mediated vector can be continuously expressed for a long time. The AAV / HBV model allows for the continuous replication of HBV DNA and the expression of HBsAg and HBeAg in the mouse liver.

[0140] Using an AAV / HBV mouse model, the levels of HBsAg, HBeAg, DNA, pgRNA, and body weight in the serum of mice treated with the test compound were measured to evaluate its in vivo anti-HBV efficacy and safety.

[0141] Experimental materials: C57BL / 6 mice, PBS (RNase-free) as solvent, test compound, and recombinant virus rAAV8-1.3HBV. The main reagents used in this project include the QIAamp96 DNA kit (Qiagen, 51162), FastStart Universal ProbeMaster (Rox) (Roche, 04914058001), Hepatitis B surface antigen detection kit (Antu Bio, CL0310), Hepatitis B e antigen detection kit (Antu Bio, CL0918), and PureLink... TMThe equipment used included the Pro 96 Viral RNA / DNA kit (Invitrogen, 12280-096A) and the FastQuant RT Kit (with gDNase) (TIANGEN, KR106-02). Major instruments included: a centrifuge (Beckman Allegra X-15R), a multi-functional microplate reader (BioTek, Synergy 2), a quantitative PCR system (Applied Biosystems, 7900HT Fast Real-time PCR system), and a microplate reader (Molecular Devices, SpectraMax 340PC384).

[0142] Experimental methods: a) Mice were administered the drug subcutaneously starting on day 34 after viral injection; this day was designated as day 0. Blood was collected from the submandibular region of all mice before administration to collect plasma. The specific administration protocol is shown in Table 3.

[0143] b) Plasma was collected from the submandibular vein of mice on days 0, 14, 21, 28, and 32 after drug administration. The collected blood samples were anticoagulated with K2-EDTA and subjected to 4... o Plasma was collected after centrifugation at 7000 g / min for 10 minutes. Specific blood collection times are shown in Table 3.

[0144] c) On day 35 or 42, all mice were euthanized by submandibular vein blood collection, followed by heart blood collection of plasma samples and liver samples.

[0145] d) The pulp sample was sent for testing.

[0146] Table 3 In vivo experimental protocol

[0147] *1 WRG01 is a conjugate with the sense chain being SEQ ID NO: 16, the antisense chain being SEQ ID NO: 23, and the conjugate group being D.

[0148] *2 WR007 is a conjugate with the sense chain being SEQ ID NO: 42, the antisense chain being SEQ ID NO: 23, and the conjugate group being D.

[0149] *3 WR012 is a conjugate with the sense chain being SEQ ID NO: 16, the antisense chain being SEQ ID NO: 47, and the conjugate group being D.

[0150] / : The destination has not yet been reached.

[0151] Sample analysis: The levels of HBsAg and HBeAg in mouse serum were detected by ELISA: The experimental procedures were performed in accordance with the instructions of the HBsAg ELISA (Antu Bio, CL 0310) and HBeAg ELISA (Antu Bio, CL0918) kits.

[0152] qPCR detection of HBV DNA content in mouse plasma: HBV DNA was extracted from plasma, and the experimental procedure was performed according to the QIAamp 96DNA Blood Kit instructions. The HBV DNA content in mouse plasma was then detected by qPCR.

[0153] RT-qPCR detection of HBV pgRNA levels in mouse plasma: HBV pgRNA was extracted from plasma following the PureLink™ Pro 96 Viral RNA / DNA Kit instruction manual. DNA was digested, and RNA was reverse transcribed into cDNA using 3' RACE primers containing the HBV-specific sequence, following the FastQuant RT Kit (with gDNase) instruction manual. Finally, qPCR was used to quantify the cDNA content, thus detecting the HBV pgRNA level in mouse plasma.

[0154] The mean ± standard error of each group of mouse samples is expressed as mean ± standard error, n=5 unless otherwise specified. (Using Student's...) t -test performs statistical analysis.

[0155] Experimental results: a) Serum HBsAg levels evaluated the anti-HBV activity of the test compound in the AAV / HBV mouse model. Results are shown in Tables 4 and 4-1. Mouse plasma HBsAg levels were determined by ELISA. Error bars indicate standard errors. Day 0: First administration of the vector or compound to all mice. Day 29: Second administration of the vector or compound to the experimental group WRG01 mice and the blank control group mice.

[0156] Table 4 Log of mice at different dates after drug administration 10 [HBsAg (IU / mL)]

[0157] Table 4-1 Log data of mice on different days after drug administration 10 [HBsAg (IU / mL)]

[0158] b) Evaluation of HBeAg levels in mouse plasma to assess the anti-HBV activity of the test compounds in the AAV / HBV mouse model. Results are shown in Tables 5 and 5-1. HBeAg levels in mouse plasma were determined by ELISA. Error bars indicate standard error. Day 0: First administration of the drug or compound to all mice.

[0159] Table 5 Log of mice at different dates after drug administration 10 [HBeAg (PEIU / mL)]

[0160] Table 5-1 Log of mice on different days after drug administration 10 [HBeAg (PEIU / mL)]

[0161] c) Serum DNA levels were used to evaluate the anti-HBV activity of the test compounds in the AAV / HBV mouse model. Results are shown in Tables 6 and 6-1. Mouse plasma HBV DNA levels were determined by quantitative PCR. Error bars indicate standard error. Day 0: First administration of the vector or compound to all mice. Day 29: Second administration of the vector or compound to all mice.

[0162] Table 6 Log of mice at different dates after drug administration 10 [DNA (copy number / μL)]

[0163] Table 6-1 Log of mice on different days after drug administration 10 [DNA (copy number / μL)]

[0164] / : No data obtained.

[0165] d) The pgRNA content was used to evaluate the anti-HBV activity of the test compound in the AAV / HBV mouse model. The results are shown in Table 7. The HBV pgRNA content in mouse plasma was determined by quantitative PCR. The error bars show the standard error. Day 0: All mice were given the first administration of the vector or compound. Day 29: All mice were given the second administration of the vector or compound.

[0166] Table 7 Log of mice at different dates after drug administration 10 [pgRNA (copy number / μL)]

[0167] e) Using the body weight on day 0 as a baseline for comparison, according to IACUC guidelines, a 20% body weight loss is considered a humane endpoint, and any mouse with a body weight loss exceeding 20% ​​must be removed from the experiment. No mice were removed from this experiment due to body weight loss.

[0168] Experimental conclusion: In this experiment, the test compound significantly reduced HBsAg, DNA, and pgRNA in an AAV / HBV mouse model. Simultaneously, the test compound also showed some inhibitory effect on HBeAg. During treatment with the test compound, the mice exhibited good tolerance and gradually increased body weight.

[0169] Example 6: In vitro HBV test in HepG2.2.15 cells 1. Experimental objective: The HBV DNA content in the culture supernatant of HepG2.2.15 cells was detected by real-time quantitative qPCR, and the HBV surface antigen and e antigen content were detected by enzyme-linked immunosorbent assay (ELISA). The intracellular HBV RNA content was detected by qRT-PCR. The EC50 value of the compound was used as an indicator to evaluate the inhibitory effect of the compound on HBV. At the same time, the effect of the test compound on cell viability was detected by CCK8 assay.

[0170] 2. Experimental materials: 2.4 Cell line: HepG2.2.15 cells 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; 2mM L-GLUTAMINE, Invitrogen-25030081; 300 µg / ml geneticin, Invitrogen-10131027).

[0171] 2.5 Reagents Opti-MEM (Gibco-31985-070); Lipofectamine® RNAiMAX (Invitrogen-13778-150); CCK8 (Liji-AC11L057); High-throughput DNA purification kit (QIAamp 96 DNA Blood Kit, Qiagen-51162); RNA preparation RNEASY kit (RNeasy 96 Kit (12), Qiagen-74182); Quantitative rapid start universal probe kit (FastStart Universal Probe Master, Roche-04914058001); FastKing cDNA first strand synthesis kit (TianGen-KR106-02); Hepatitis B surface antigen quantitative detection kit (Antu Bio, CL 0310); Hepatitis B e antigen quantitative detection kit (Antu Bio, CL 0312).

[0172] 2.6 Consumables and Instruments: Collagen I 96 Well White / Clear Flat Bottom TC-Treated Microplate (Corning BioCoat-356650); CO2 Incubator (HERA-CELL-240); Real-time PCR System (AppliedBiosystems-7900 real-time PCR system); Real-time PCR System (Applied Biosystems-QuantStudio 6 Flex); Microplate Reader (Molecular Device-SpectraMaxM2e); Microplate Reader (BioTek-Synergy 2).

[0173] 3. Experimental steps and methods: 3.1 On day 1, siRNA transfection and cell plating were performed simultaneously. The procedure is briefly described below: HepG2.2.15 cells were washed with DPBS, digested with 0.05% trypsin, and digestion was terminated with DMEM / F12 medium containing 10% FBS. After centrifugation, the cells were resuspended, and the cells were gently pipetted to single cells before counting. The required volume of transfection reagent was prepared according to the proportions (Table 8), and incubated at room temperature for 15 minutes.

[0174] Table 8 Lipofectamine® RNAiMAX Configuration

[0175] siRNA was serially diluted to eight concentrations (3-fold serial dilution) in duplicate wells. 15 μL of RNAiMAX / Opti-MEM mixture was mixed with 15 μL of siRNA at different concentrations and incubated at room temperature for 15 minutes. 10 μL of the mixture was then added to a 96-well cell culture plate, followed by 90 μL of cell suspension, resulting in a final cell density of 15,000 cells / well and a final volume of 100 μL / well. Cells were then incubated in a 5% CO2, 37 °C incubator.

[0176] 3.2 On the fourth day, replace the culture medium with fresh medium containing the compound, and use the same transfection method as on the first day.

[0177] 3.3 On the seventh day, collect the culture medium from the wells and take a portion of the sample to determine the content of hepatitis B virus S antigen and e antigen by ELISA; take a portion of the sample to extract DNA using a high-throughput DNA purification kit (Qiagen-51162); after collecting the supernatant, determine cell viability according to the CCK-8 kit instructions, and detect the absorbance of each well (450nm / 650nm) using an ELISA reader (SpectraMaxM2e); use the RNeasy 96 kit extraction kit (Qiagen-74182) to extract HBV RNA from the cell culture according to the kit instructions.

[0178] 3.4 The preparation of the PCR reaction solution is shown in Table 9: Table 9 Preparation of PCR reaction solution

[0179] Add 8 µl of reaction mixture to each well of a 96-well PCR plate, and then add 2 µl of sample DNA or HBV DNA standard to each well.

[0180] The PCR reaction conditions were: heating at 95°C for 10 minutes, followed by denaturation at 95°C for 15 seconds, extension at 60°C for 1 minute, for a total of 40 cycles.

[0181] 3.5 ELISA determination of hepatitis B virus S antigen and e antigen content. Specific steps are described in the product instructions. The steps are as follows: Add 50 μL of sample and standard to each well, then add 50 μL of enzyme conjugate to each well. Shake to mix, incubate at 37 ℃ for 60 minutes, then wash the plate 5 times with washing buffer. Add 50 μL of luminescent substrate to each well, mix, and incubate at room temperature in the dark for 10 minutes. Finally, detect the chemiluminescence intensity using an ELISA reader.

[0182] 3.6 HBV RNA was extracted from cell culture using the RNeasy 96 kit (Qiagen, 74182) according to the kit instructions. Cells were lysed with 150 µL of RLT, and RNA was finally washed away with 50 µL of RNase-free water. Following the instructions of the reverse transcription kit (Tiangen, KR106), random primers were added to reverse transcribe the RNA into cDNA. HBV-specific primers were then used to detect total RNA in the sample. Simultaneously, GAPDH primers and probes were used to specifically detect GAPDH cDNA. HBV cDNA in the sample was quantified using qPCR.

[0183] qPCR reaction: 95 °C, 10 min; 95 °C, 15 s, 60 °C, 1 min, 40 cycles. The HBV RNA content in each sample was calculated based on the Ct value.

[0184] The expression level of the target gene HBV mRNA in each sample was calculated using the ΔΔCt relative quantification method. The relative expression level of the target gene is expressed as 2-ΔΔCT, and the calculation formula is as follows: ΔCT = Average Ct value of target gene - Average Ct value of internal reference gene; ΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group); HBV mRNA relative expression level = 2 - ΔΔCT 3.7 Data Analysis: Calculate the percentage of inhibition: % Inh. = (1 - value in sample / PBS control value) x 100.

[0185] Cell viability % = (Sample detection value - Average detection value of culture medium background) / (Average detection value of control group - Average detection value of culture medium background) × 100 Calculate EC 50 and CC 50 The 50% inhibitory concentration (EC50) of the compound against HBV was calculated using GraphPad Prism software. 50 ) value and drug concentration at 50% cell death (CC 50 )value.

[0186] Table 10 shows the experimental results of the sequences tested reducing HBsAg, HBeAg, DNA, and RNA levels in cells.

[0187] / : Data not yet available.

[0188] * The sample tested was a conjugate of a double-stranded siRNA analog.

[0189] Example 7: Exploration of the effective dosage of anti-hepatitis B virus drugs in an AAV-HBV mouse model By using an AAV / HBV mouse model, the serum HBsAg levels in mice treated with different doses of the test compound were detected to evaluate its in vivo anti-HBV effect.

[0190] Experimental materials: C57BL / 6 mice, PBS (RNase-free) as solvent, test compound, and recombinant virus rAAV8-1.3HBV. The main reagents for this project included a FastStart Universal Probe Master (Rox) (Roche, 04914058001) and a Hepatitis B surface antigen detection kit (Antu Bio, CL0310). The main instruments included a centrifuge (Beckman Allegra X-15R), a multi-functional microplate reader (BioTek, Synergy 2), and a microplate reader (Molecular Devices, SpectraMax 340PC384).

[0191] Experimental methods: a) All mice were administered the drug subcutaneously on day 34 post-viral injection, designated as day 0. Plasma was collected from the submandibular region of all mice before administration. The drug was administered once on day 0. See Table 14 for the specific administration regimen.

[0192] b) Plasma was collected from the submandibular vein of all mice on days 0, 14, 21, 28, and 35 after drug administration. The collected blood samples were anticoagulated with K2-EDTA and subjected to 4... o Plasma was collected after centrifugation at 7000 g / min for 10 minutes. Specific blood collection times are shown in Table 11.

[0193] c) On day 42, all mice were euthanized by submandibular vein blood collection, followed by heart blood collection of plasma samples and liver samples.

[0194] d) All plasma samples were sent for testing.

[0195] Table 11 In vivo experimental protocol

[0196] *: WRG01 is a conjugate with the sense chain being SEQ ID NO: 16, the antisense chain being SEQ ID NO: 23, and the conjugate group being D.

[0197] / : The destination has not yet been reached.

[0198] Sample analysis: The HBsAg content in mouse serum was detected by ELISA: The experimental procedure was performed in accordance with the instructions of the HBsAg ELISA (Antu Bio, CL0310) kit.

[0199] The mean ± standard error of each group of mouse samples is expressed as mean ± standard error, n=5 unless otherwise specified. (Using Student's...) t -test performs statistical analysis.

[0200] Experimental results: Serum HBsAg levels were measured to evaluate the anti-HBV activity of the test compounds in an AAV / HBV mouse model. Results are shown in Table 12. Mouse plasma HBsAg levels were determined by ELISA. Error bars indicate standard errors. Day 0: First administration of the drug or compound to all mice.

[0201] Table 12 Log of mice on different days after drug administration 10 [HBsAg (IU / mL)]

[0202] Experimental conclusion: In this experiment, the test compound WRG01 showed good dose-dependent reduction of HBsAg in the AAV / HBV mouse model, that is, the reduction of HBsAg activity increased with increasing drug dose, and it showed long-term inhibitory efficacy against HBsAg.

[0203] Example 8: Drug concentration tests in mouse plasma, liver, and kidneys. In this study, C57BL / 6 mice were administered a single subcutaneous injection of the compound. Plasma and tissue samples were collected at different time points after administration. The metabolic level of the compound in mice was evaluated by detecting the siRNA level in plasma and tissue using SL-qPCR.

[0204] Table 13 In vivo experimental protocol

[0205] *: WRG01 is a conjugate with the sense chain being SEQ ID NO: 16, the antisense chain being SEQ ID NO: 23, and the conjugate group being D.

[0206] / : Blood was not drawn at non-endpoint times, only at the end point.

[0207] Experimental results: The levels of siRNA in plasma, liver, and kidney of mice at different time points after drug administration were detected using the SL-qPCR method (Reference: Nair et al. Nucleic Acids Research (2017), 45, 10969-10977).

[0208] Experimental conclusion: In this experiment, the test compound WRG01 exhibited good tissue distribution and metabolic stability in the C57BL / 6 mouse model. The high liver exposure and long half-life of WR-G01, with a liver-to-blood ratio exceeding 500-fold, demonstrate that WRG01 is metabolically stable and possesses high liver-targeting properties.

[0209] Example 9: Blood Biochemical Tests of FRG-KO Humanized Liver Mice Humanized FRG mice are one of the most commonly used humanized liver models, typically with a humanization rate as high as 70%. This is because human liver cells are colonized in the mouse liver, which can better simulate the natural HBV infection and cccDNA replication process in humans, and at the same time has a good predictive effect on human pharmacokinetics and liver toxicity.

[0210] This study administered the compound to humanized FRG mice multiple times, collecting plasma samples at different time points after administration. The effects of the compound on liver toxicity in mice were evaluated by measuring plasma ALT, AST, and bilirubin levels. In this experiment, the test compound did not induce a significant inflammatory response in the humanized liver, indicating good safety in humans.

[0211] Table 14 In vivo experimental protocol

[0212] *: WRG01 is a conjugate with the sense chain being SEQ ID NO: 16, the antisense chain being SEQ ID NO: 23, and the conjugate group being D.

[0213] / : The destination has not yet been reached.

[0214] This disclosure demonstrates unexpectedly excellent HBsAg and HBeAg inhibitory activity, while effectively inhibiting HBV DNA and pgRNA expression. This indicates that it can suppress the activity of hepatitis B virus, while exhibiting good tissue distribution and metabolic stability, high liver targeting, and is expected to have minimal impact on liver function in mice. This will provide a highly effective treatment for hepatitis B, such as chronic hepatitis B, in clinical practice.

Claims

1. A double-stranded siRNA analog, its conjugate, or a salt thereof, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence in which one or more nucleotide residues are replaced by r in the sequence shown in SEQ ID NO: 2, wherein r is ， Each of the nucleotides and r in the siRNA analog is independently modified or unmodified.

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

3. The double-stranded siRNA analog, its conjugate, or salt thereof according to claim 1 or 2, wherein the modification includes methoxy modification, fluorination modification, thiophosphate group linkage, or replacement of the nucleotide with ( S (E)-glycerol nucleic acid or replace (E)-vinyl phosphate with nucleotides.

4. The double-stranded siRNA analog, its conjugate, or its salt according to any one of claims 1-3, wherein the antisense strand comprises a sequence in which one, two, three, four, or five nucleotide residues are replaced by r in the sequence shown in SEQ ID NO: 2; optionally, the antisense strand comprises a sequence in which one nucleotide residue is replaced by r in the sequence shown in SEQ ID NO:

2.

5. The double-stranded siRNA analog, its conjugate, or its salt according to any one of claims 1-4, wherein the r substitution occurs at any position of SEQ ID NO:

2.

6. The double-stranded siRNA analog, its conjugate, or salt thereof according to any one of claims 1-5, wherein SEQ ID NO: 2 optionally includes a protrusion at the 5' end and / or the 3' end; optionally, SEQ ID NO: 2 includes a protrusion of 0, 1, 2, 3, 4, or 5 nucleotides at the 5' end and / or the 3' end; optionally, SEQ ID NO: 2 includes a protrusion at the 3' end, the protrusion being selected from modified or unmodified UU.

7. The double-stranded siRNA analog, its conjugate, or salt thereof according to any one of claims 1-6, wherein the antisense strand comprises, for example, 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, 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, or the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 47, or SEQ ID NO: 10 or SEQ ID NO: 48, or composed of therein.

8. The double-stranded siRNA analog, its conjugate, or its salt according to any one of claims 1-7, wherein the positive strand comprises, or is composed of, the sequence shown in SEQ ID NO: 1 or SEQ ID NO:

28.

9. The double-stranded siRNA analog, its conjugate, or its salt according to any one of claims 1-8, wherein the sense strand comprises a sequence in which one or more nucleotide residues are replaced by r in the sequence shown in SEQ ID NO: 1; optionally, the sense strand comprises a sequence in which one, two, three, four, or five nucleotide residues are replaced by r in the sequence shown in SEQ ID NO:

1.

10. The double-stranded siRNA analog, its conjugate, or its salt according to any one of claims 1-9, wherein the r substitution occurs at positions 1 to 19 of the 5' end of SEQ ID NO:

1.

11. The double-stranded siRNA analog, its conjugate, or its salt according to any one of claims 1-10, wherein the sequence of the positive strand comprises, or is composed of, the sequence shown 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.

12. The double-stranded siRNA analog, its conjugate, or its salt according to any one of claims 1-11, wherein the double-stranded siRNA analog is any one of S18 to S28: S18: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 4 or SEQ ID NO:

17. S19: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 6 or SEQ ID NO:

19. S20: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 7 or SEQ ID NO:

20. S21: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 8 or SEQ ID NO:

21. S22: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 9 or SEQ ID NO:

22. S23: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO:

23. S24: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 11 or SEQ ID NO:

24. S25: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 29 or SEQ ID NO:

33. S26: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 30 or SEQ ID NO:

34. S27: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 31 or SEQ ID NO:

35. S28: The justice chain is SEQ ID NO: 1 or SEQ ID NO: 28, and the antisense chain is SEQ ID NO: 32 or SEQ ID NO:

36. or, The double-stranded siRNA analogue mentioned above is any one of S1 to S17: S1: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 4 or SEQ ID NO:

17. S2: The justice chain is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense chain is SEQ ID NO: 4 or SEQ ID NO:

17. S3: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 6 or SEQ ID NO:

19. S4: The justice chain is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense chain is SEQ ID NO: 6 or SEQ ID NO:

19. S5: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 7 or SEQ ID NO:

20. S6: The justice chain is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense chain is SEQ ID NO: 7 or SEQ ID NO:

20. S7: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 8 or SEQ ID NO:

21. S8: The justice chain is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense chain is SEQ ID NO: 8 or SEQ ID NO:

21. S9: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 9 or SEQ ID NO:

22. S10: The justice chain is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense chain is SEQ ID NO: 9 or SEQ ID NO:

22. S11: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO:

23. S12: The justice chain is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO:

23. S13: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 11 or SEQ ID NO:

24. S14: The justice chain is SEQ ID NO: 5 or SEQ ID NO: 18, and the antisense chain is SEQ ID NO: 11 or SEQ ID NO:

24. S15: The justice chain is SEQ ID NO: 12 or SEQ ID NO: 25, and the antisense chain is SEQ ID NO: 4 or SEQ ID NO:

17. S16: The justice chain is SEQ ID NO: 13 or SEQ ID NO: 26, and the antisense chain is SEQ ID NO: 4 or SEQ ID NO:

17. S17: The justice chain is SEQ ID NO: 14 or SEQ ID NO: 27, and the antisense chain is SEQ ID NO: 4 or SEQ ID NO:

17. or, The double-stranded siRNA analog is any one of S29 to S35: S29: The justice chain is SEQ ID NO: 37 or SEQ ID NO: 42, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO:

23. S30: The justice chain is SEQ ID NO: 38 or SEQ ID NO: 43, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO:

23. S31: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 39 or SEQ ID NO:

44. S32: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO:

45. S33: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 40 or SEQ ID NO:

46. S34: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO:

47. S35: The justice chain is SEQ ID NO: 3 or SEQ ID NO: 16, and the antisense chain is SEQ ID NO: 10 or SEQ ID NO:

48.

13. The double-stranded siRNA analog, its conjugate, or its salt according to any one of claims 1-12, wherein the double-stranded siRNA analog is selected from: 。 14. The double-stranded siRNA analog, its conjugate, or its salt according to any one of claims 1-13, wherein the double-stranded siRNA analog is selected from: 。 15. The double-stranded siRNA analog, its conjugate, or salt thereof according to any one of claims 1-14, wherein the double-stranded siRNA analog is linked to a pharmaceutically acceptable conjugate group, the pharmaceutically acceptable conjugate group comprising a GalNAc group; optionally, the pharmaceutically acceptable conjugate group contains 1 to 5 GalNAc groups.

16. The double-stranded siRNA analog, its conjugate, or salt thereof according to any one of claims 1-15, wherein the double-stranded siRNA analog is linked to a pharmaceutically acceptable conjugate group, the pharmaceutically acceptable conjugate group comprising compound group D. 。 17. The double-stranded siRNA analog, its conjugate, or salt thereof according to claim 15 or 16, wherein the pharmaceutically acceptable conjugate group is attached to the 3' end of the positive strand of the double-stranded siRNA analog.

18. The double-stranded siRNA analog, its conjugate, or a salt thereof according to any one of claims 1-17, wherein the conjugate of the double-stranded siRNA analog is selected from: Wherein D is 。 19. The double-stranded siRNA analog, its conjugate, or salt thereof according to any one of claims 1-18, wherein the thiophosphate moiety of the double-stranded siRNA analog or its conjugate comprises (R)- and (S)-enantiomers, diastereomers, and / or racemic mixtures thereof.

20. The double-stranded siRNA analog, its conjugate, or its salt according to any one of claims 1-19, 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 inorganic acid salts, organic acid salts, and combinations thereof; optionally, the inorganic acid is selected from hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, 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, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and combinations thereof.

21. A pharmaceutical composition comprising the double-stranded siRNA analog, its conjugate or salt thereof, as described in any one of claims 1-20, optionally comprising a pharmaceutically acceptable carrier or excipient.

22. Use of any double-stranded siRNA analog, its conjugate or salt thereof, or the pharmaceutical composition of claim 21 in the preparation of a medicament for treating hepatitis B.

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