Conjugate of a double-stranded SIRNA analog.

Abstract

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

Description

Double-stranded siRNA analog conjugates

[0001] This application claims the following priority:

[0002] CN202010529520.7, application date: June 11, 2020;

[0003] CN202011524835.9, application date December 21, 2020;

[0004] CN202010524584.8, application date: June 10, 2020;

[0005] PCT / CN2020 / 133982, application date December 4, 2020;

[0006] CN202010522407.6, application date: June 10, 2020;

[0007] CN202011524307.3, application date December 21, 2020. Technical Field

[0008] 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

[0009] 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 for global health care and is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma (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, 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 in patients 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 people infected with hepatitis B virus 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.)

[0010] 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.

[0011] 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).

[0012] 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.

[0013] Meanwhile, 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.

[0014] Summary of the Invention

[0015] 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).

[0016] 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 nucleotide residues are replaced by r in the sequence shown in SEQ ID NO: 2, where r is...

[0017]

[0018] Each of the nucleotides and r in the siRNA analog is independently modified or unmodified.

[0019] 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, methoxylation, fluorination, thiophosphate linkage, or replacement of the nucleotide with (S)-glycerol nucleic acid.

[0020] 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, methoxylation, fluorination, thiophosphate linkage, replacement of the nucleotide with (S)-glycerol nucleic acid, or replacement of the nucleotide with (E)-vinyl phosphate, etc.

[0021] In some embodiments, substantially all nucleotides and r in the siRNA analog are modified.

[0022] 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.

[0023] 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.

[0024] 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 other 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.

[0025] In some embodiments, SEQ ID NO: 2 includes a 3' protrusion, preferably derived from modified or unmodified UU. In some embodiments, SEQ ID NO: 2 includes a 3' protrusion, preferably derived from UU. In some embodiments, SEQ ID NO: 2 includes a 3' protrusion UU with two phosphate thioester groups (e.g., c·u·u) linked to an adjacent paired nucleotide.

[0026] In some embodiments, the antisense strand of the double-stranded siRNA analog comprises a sequence in which one or more nucleotide residues are replaced by r in the sequence shown in SEQ ID NO: 2. For example, the antisense strand comprises a sequence in which one nucleotide residue is replaced by r in the sequence shown in SEQ ID NO: 2.

[0027] In some embodiments, the antisense strand of the double-stranded siRNA analog comprises a sequence in which one or more nucleotide residues are replaced by r in the sequence shown in SEQ ID NO: 2. For example, 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.

[0028] In some embodiments, the antisense strand of the double-stranded siRNA analog comprises a sequence in which one or more nucleotide residues 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.

[0029] In some embodiments, the antisense strand of the double-stranded siRNA analog comprises, or is composed of, the sequence 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, the sequence includes further nucleotide modifications, such as methoxy modification, fluorination modification, thiophosphate linkage, or replacement of the nucleotide with (S)-glycerol nucleic acid, etc.

[0030] In some embodiments, the antisense strand of the double-stranded siRNA analog comprises, or consists of, the sequences 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, SEQ ID NO: 32 or SEQ ID NO: 36, SEQ ID NO: 39 or SEQ ID NO: 44, SEQ ID NO: 10 or SEQ ID NO: 45, SEQ ID NO: 40 or SEQ ID NO: 46, SEQ ID NO: 10 or SEQ ID NO: 47, or SEQ ID NO: 10 or SEQ ID NO: 48. In some embodiments, the sequence includes further nucleotide modifications, such as methoxy modification, fluorination modification, thiophosphate linkage, replacement of the nucleotide with (S)-glycerol nucleic acid, or replacement of the nucleotide with (E)-vinyl phosphate modification, etc.

[0031] 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.

[0032] In some embodiments, the positive strand of the double-stranded siRNA analog comprises a sequence in which one or more nucleotide residues are replaced by r in the sequence shown in SEQ ID NO: 1. For example, the positive strand comprises a sequence in which one nucleotide residue is replaced by r in the sequence shown in SEQ ID NO: 1.

[0033] In some embodiments, the positive strand of the double-stranded siRNA analog comprises a sequence in which one or more nucleotide residues are replaced by r in the sequence shown in SEQ ID NO: 1. For example, the positive strand comprises a sequence in which 1, 2, 3, 4, or 5 nucleotide residues are replaced by r in the sequence shown in SEQ ID NO: 1.

[0034] In some embodiments, the positive strand of the double-stranded siRNA analog comprises a sequence in which one or more nucleotide residues are replaced by 'r' in the sequence shown in SEQ ID NO: 1, wherein the 'r' substitution occurs at positions 1 through 19 of the 5' end of SEQ ID NO: 1. For example, the '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, the 'r' substitution occurs at positions 2, 3, 7, 12, 15, 17, or 19 of the 5' end of SEQ ID NO: 1.

[0035] In some embodiments, the sequence of the positive strand of the double-stranded siRNA analog comprises, or consists 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, fluorination modifications, thiophosphate linkages, etc.

[0036] 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 modifications, fluorination modifications, thiophosphate group linkages, replacement of nucleotides with (S)-glycerol nucleic acids, or replacement of nucleotides with (E)-vinyl phosphate modifications, etc.

[0037] In some specific implementations, the sense and antisense strands of the double-stranded siRNA analog contain sequences in which one or more nucleotide residues 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.

[0038] In some implementations, the double-stranded siRNA analog is any one of S18 to S28:

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] In some implementations, the double-stranded siRNA analog is any one of S1 to S17:

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] In some implementations, the double-stranded siRNA analog is any one of S29 to S35:

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] In some embodiments, the double-stranded siRNA analog is selected from: SEQ ID NO: 3 of the positive strand and SEQ ID NO: 4 of the negative strand; SEQ ID NO: 5 of the positive strand and SEQ ID NO: 4 of the negative strand; SEQ ID NO: 3 of the positive strand and SEQ ID NO: 6 of the negative strand; SEQ ID NO: 5 of the positive strand and SEQ ID NO: 6 of the negative strand; SEQ ID NO: 3 of the positive strand and SEQ ID NO: 7 of the positive strand; SEQ ID NO: 5 of the positive strand and SEQ ID NO: 7 of the negative strand; SEQ ID NO: 3 of the positive strand and SEQ ID NO: 8 of the positive strand; SEQ ID NO: 5 of the positive strand and SEQ ID NO: 8 of the negative strand; SEQ ID NO: 3 of the positive strand and SEQ ID NO: 9 of the positive strand; SEQ ID NO: 5 of the positive strand and SEQ ID NO: 9 of the negative strand; SEQ ID NO: 3 of the positive strand and SEQ ID NO: 10 of the positive strand; SEQ ID NO: 5 of the positive strand and SEQ ID NO: 10 of the negative strand. NO: 10, Justice chain is SEQ ID NO: 3 and Antisense chain is SEQ ID NO: 11, Justice chain is SEQ ID NO: 5 and Antisense chain is SEQ ID NO: 11, Justice chain is SEQ ID NO: 12 and Antisense chain is SEQ ID NO: 4, Justice chain is SEQ ID NO: 13 and Antisense chain is SEQ ID NO: 4, Justice chain is SEQ ID NO: 14 and Antisense chain is SEQ ID NO: 4, Justice chain is SEQ ID NO: 1 and Antisense chain is SEQ ID NO: 4, Justice chain is SEQ ID NO: 1 and Antisense chain is SEQ ID NO: 6, Justice chain is SEQ ID NO: 1 and Antisense chain is SEQ ID NO: 7, Justice chain is SEQ ID NO: 1 and Antisense chain is SEQ ID NO: 8, Justice chain is SEQ ID NO: 1 and Antisense chain is SEQ ID NO: 9, Justice chain is SEQ ID NO: 1 and Antisense chain is SEQ ID NO: 10, Justice chain is SEQ ID NO: 1 and Antisense chain is SEQ ID NO: 11, Justice chain is SEQ ID NO:1 and the antisense chain is SEQ ID NO:29, the justice chain is SEQ ID NO:1 and the antisense chain is SEQ ID NO:30, the justice chain is SEQ ID NO:1 and the antisense chain is SEQ ID NO:31, the justice chain is SEQ ID NO:1 and the antisense chain is SEQ ID NO:32, the justice chain is SEQ ID NO:37 and the antisense chain is SEQ ID NO:10, the justice chain is SEQ ID NO:38 and the antisense chain is SEQ ID NO:29.NO: 10, the sense strand is SEQ ID NO: 3 and the antisense strand is SEQ ID NO: 39, the sense strand is SEQ ID NO: 3 and the antisense strand is SEQ ID NO: 10, or the sense strand is SEQ ID NO: 3 and the antisense strand is SEQ ID NO: 40, wherein each of the nucleotides and r in the double-stranded siRNA analog is independently modified or unmodified.

[0077] In some embodiments, the double-stranded siRNA analog is selected from:

[0078]

[0079]

[0080] Each of the nucleotides and r in the double-stranded siRNA analog is independently modified or unmodified.

[0081] In some embodiments, the double-stranded siRNA analog is selected from: SEQ ID NO: 16 of the positive strand and SEQ ID NO: 17 of the negative strand; SEQ ID NO: 18 of the positive strand and SEQ ID NO: 17 of the negative strand; SEQ ID NO: 16 of the positive strand and SEQ ID NO: 19 of the negative strand; SEQ ID NO: 18 of the positive strand and SEQ ID NO: 19 of the negative strand; SEQ ID NO: 16 of the positive strand and SEQ ID NO: 20 of the positive strand; SEQ ID NO: 18 of the positive strand and SEQ ID NO: 20 of the negative strand; SEQ ID NO: 16 of the positive strand and SEQ ID NO: 21 of the positive strand; SEQ ID NO: 18 of the positive strand and SEQ ID NO: 21 of the negative strand; SEQ ID NO: 16 of the positive strand and SEQ ID NO: 22 of the positive strand; SEQ ID NO: 16 of the positive strand and SEQ ID NO: 22 of the negative strand; SEQ ID NO: 16 of the positive strand and SEQ ID NO: 22 of the negative strand. NO: 23, Justice chain is SEQ ID NO: 18 and Antisense chain is SEQ ID NO: 23, Justice chain is SEQ ID NO: 16 and Antisense chain is SEQ ID NO: 24, Justice chain is SEQ ID NO: 18 and Antisense chain is SEQ ID NO: 24, Justice chain is SEQ ID NO: 25 and Antisense chain is SEQ ID NO: 17, Justice chain is SEQ ID NO: 26 and Antisense chain is SEQ ID NO: 17, Justice chain is SEQ ID NO: 27 and Antisense chain is SEQ ID NO: 17, Justice chain is SEQ ID NO: 28 and Antisense chain is SEQ ID NO: 17, Justice chain is SEQ ID NO: 28 and Antisense chain is SEQ ID NO: 19, Justice chain is SEQ ID NO: 28 and Antisense chain is SEQ ID NO: 20, Justice chain is SEQ ID NO: 28 and Antisense chain is SEQ ID NO: 21, Justice chain is SEQ ID NO: 28 and Antisense chain is SEQ ID NO: 22, Justice chain is SEQ ID NO: 28 and Antisense chain is SEQ ID NO: 22, Justice chain is SEQ ID NO: 28 and Antisense chain is SEQ ID NO: 28, Justice chain is SEQ ID NO: 28, ... 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 and the antisense chain is SEQ ID NO: 36, the justice chain is SEQ IDNO: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.

[0082] In some embodiments, the double-stranded siRNA analog is selected from:

[0083]

[0084]

[0085] 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.

[0086] 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.

[0087] In some embodiments, the pharmaceutically acceptable conjugation group in the conjugate of the double-stranded siRNA analog comprises the compound group D.

[0088]

[0089] 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.

[0090] 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.

[0091] In some implementations, the conjugates of the double-stranded siRNA analogs are selected from:

[0092]

[0093]

[0094]

[0095] The term D is as described above.

[0096] In a third aspect, this disclosure provides salts of double-stranded siRNA analogs according to the first aspect of this disclosure or conjugates of double-stranded siRNA analogs according to the second aspect of this disclosure.

[0097] In some embodiments, the salt described above is selected from alkali addition salts, acid addition salts, and combinations thereof.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] Definitions and Explanations

[0107] 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.

[0108] 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.

[0109] 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.

[0110] In some implementations, double-stranded siRNA analogs can target the X opening reading frame (X ORF) of HBV.

[0111] 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 (C) always pairs with the pyrimidine base cytosine (G). 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.

[0112] 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 indicate that the nucleotide represented by the corresponding uppercase letter is modified with a methoxy group; underscores indicate that the nucleotide represented by the uppercase letter is modified with a fluoride group; 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 a (E)-vinyl phosphate modified nucleotide. For example, "a·g" indicates that the residues a and g are linked by a thiophosphate group.

[0113] The “modification” of nucleotides described in this disclosure includes, but is not limited to, methoxy modification, fluorination modification, (E)-vinyl phosphate modification, thiophosphate linkage, or replacement of the nucleotide with (S)-glycerol nucleic acid. The sequences described in this disclosure may include those listed in “Sequences with Further Modifications” in Table 1 below.

[0114] 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.

[0115] The (E)-vinyl phosphate modified nucleotides described in this disclosure represent the following structural units:

[0116]

[0117] Where E is selected from

[0118] X is selected from OCH3 and F.

[0119] The (S)-glycerol nucleic acid (Agn) described in this disclosure represents the following structural unit:

[0120]

[0121] (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.

[0122] 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.

[0123] 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 nucleotide residue in a sequence (such as SEQ ID NO: 2). 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.

[0124] 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 nucleotide residue 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 one or more nucleotides in the sense strand being 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 one or more nucleotides in the antisense strand being replaced by r.

[0125] 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.

[0126] In this disclosure, r represents the following structural unit:

[0127]

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] The compounds disclosed herein may exist in specific geometric or stereoisomeric forms. This disclosure envisions all such compounds, including (R)- and (S)-enantiomers, diastereomers, racemic mixtures, and other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are 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.

[0136] Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

[0137] 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.

[0138] Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration of the center of a solid is represented by a straight solid line key. and straight dashed key The relative configuration of the center of a solid is indicated by a wavy line. Indicates wedge-shaped solid line key or wedge-shaped dashed key Or use wavy lines Indicates a straight solid line key and / or straight dashed key

[0139] 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%.

[0140] 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%.

[0141] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound disclosed herein, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide a pure desired enantiomer. 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 isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and then the pure enantiomer is recovered. 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, compounds can be labeled with radioactive isotopes, 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.

[0142] 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; 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.

[0143] 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.

[0144] 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.

[0145] The solvents used in this disclosure are commercially available.

[0146] Unless otherwise specified, all solvent ratios used in column chromatography and preparative thin-layer silica gel chromatography in this disclosure are volume ratios.

[0147] List of abbreviations

[0148] Acacetyl Boc tert-Butyloxycarbonyl DMSO dimethyl sulfoxide DMT / DMTr 4,4'-dimethoxytriphenylmethyl dsRNA double-stranded ribonucleic acid EC 50Half-maximum effect concentration (SVP) of EDTA disodium ethylenediaminetetraacetate (EDTA), i-Pr isopropyl Me methyl Ms methanesulfonyl Ph phenyl p-HPLC preparative high performance liquid chromatography (HPLC) for compound purification; RNA ribonucleic acid (RNAi) ribonucleic acid interference technology; siRNA small interfering ribonucleic acid (siRNA); t-Bu tert-butyl Tris tris-hydroxymethylaminomethane

[0149] Compounds are named according to conventional naming principles in the field or using Software naming conventions are used; commercially available compounds use supplier catalog names. Attached Figure Description

[0150] Figure 1. Effect of WRG01 on plasma HBsAg in AAV / HBV mice.

[0151] Figure 2. Effect of WRG01 on plasma HBeAg in AAV / HBV mice.

[0152] Figure 3. Effect of WRG01 on plasma HBV DNA in AAV / HBV mice.

[0153] Figure 4. Effect of WRG01 on plasma HBV pgRNA in AAV / HBV mice.

[0154] Figure 5. Changes in mouse body weight after administration of WRG01.

[0155] Figure 6. Effects of WR007 and WR012 on plasma HBsAg in AAV / HBV mice.

[0156] Figure 7. Effects of WR007 and WR012 on plasma HBeAg in AAV / HBV mice.

[0157] Figure 8. Effects of WR007 and WR012 on plasma HBV DNA in AAV / HBV mice.

[0158] Figure 9. Effects of different doses of WRG01 on plasma HBsAg in AAV / HBV mice.

[0159] Figure 10. Concentration of WRG01 in mouse plasma, liver, and kidney. Detailed Implementation

[0160] 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.

[0161] Example 1 Synthesis of phosphoramide monomer

[0162]

[0163] 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, and ethanol (70 mL) was added. The mixture was stirred at 70°C until a homogeneous solution was formed, and then stirring was stopped and the solution was cooled to 50°C. After precipitation, the mixture was allowed to stand and 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 washed with 180 mL of ethanol (60 mL × 3), and then dried under vacuum to obtain 1-2. 1 H NMR (400MHz, CDCl3): ε8.40 (s, 1H), 6.04 (d, J=3.42Hz, 1H), 5.69-5.81 (m, 1H), 5.54 (t , J=5.38Hz, 1H), 4.42-4.51(m, 2H), 4.16-4.30(m, 1H), 3.98(s, 3H), 2.05-2.18(m, 9H).

[0164] 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 obtain 1-3. 1H NMR (400MHz, CD3OD): ε8.87 (s, 1H), 5.93 (d, J=3.42Hz, 1H), 4.48 (dd, J=3.48, 4.83Hz, 1H), 4.33 (t, J=5.2 6Hz, 1H), 4.10-4.16 (m, 1H), 3.95 (s, 3H), 3.84 (dd, J=3.24, 12.29Hz, 1H), 3.70 (dd, J=4.46, 12.29Hz, 1H).

[0165] Step C: The compound shown in Formula 1-3 (10 g, 38.58 mmol) was dissolved in pyridine (250 mL) and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (12.29 g, 38.97 mmol) was added dropwise at 0°C. The mixture was gradually heated to 25°C and stirred for 16 hours. The reaction solution was concentrated under reduced pressure, and the concentrate was suspended in ethyl acetate (250 mL) and filtered through a Buchner funnel. The filtrate was washed with 750 mL (250 mL × 3) of 3M hydrochloric acid and 250 mL (250 mL × 1) of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography (SiO2, petroleum ether / dichloromethane / ethyl acetate = 3 / 1 / 1) yielded 1-4. 1 H NMR (400MHz, CDCl3): ε8.43 (s, 1H), 5.95 (s, 1H), 4.73 (dd, J=4.75, 8.00Hz, 1H), 4.41 (d, J= 4.75Hz, 1H), 4.09-4.19(m, 2H), 3.94-4.03(m, 4H), 2.71-3.34(m, 1H), 1.01-1.15(m, 28H).

[0166] Step D: Iodomethane (11.64 g, 82.02 mmol) was added to a mixture of N,N-dimethylformamide (50 mL) 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), 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 washed with 250 mL of sodium thiosulfate aqueous solution (250 mL × 1), 250 mL of water (250 mL × 1), and 250 mL of saturated brine (250 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1) yielded 1-5. 1H NMR (400MHz, CDCl3): ε8.58 (s, 1H), 5.91 (s, 1H), 4.46 (dd, J=4.22, 9.35Hz, 1H), 4.17-4.28(m, 2H), 3.96-4.06(m, 5H), 3.68(s, 3H), 0.99-1.13(m, 28H).

[0167] 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 (400MHz, CD3OD): ε8.88 (s, 1H), 6.04 (d, J=3.26Hz, 1H), 4.44 (t, J=5.33Hz, 1H), 4.20 (dd, J=3.33, 4.83Hz, 1H ), 4.07-4.14 (m, 1H), 3.96 (s, 3H), 3.84 (dd, J=3.20, 12.36Hz, 1H), 3.69 (dd, J=4.39, 12.30Hz, 1H), 3.52 (s, 3H).

[0168] Step F: At 0°C, 4,4-dimethoxytriphenylmethyl chloride (2.42 g, 7.14 mmol) 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 were washed with 60 mL of water (60 mL × 1) and 60 mL of saturated brine (60 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. Purified by p-HPLC (separation column: Phenomenex luna C18 (size: 250mm×50mm, particle size: 10μm); mobile phase: [water (10mM ammonium bicarbonate)-acetonitrile]; elution gradient: 35%-65%, 20min) to obtain 1-7. 1H NMR (400MHz, CDCl3): ε8.44 (s, 1H), 7.38-7.45 (m, 2H), 7.28-7.34 (m, 5H), 7. 18-7.27(m, 2H), 6.70-6.92(m, 4H), 5.97(d, J=2.88Hz, 1H), 4.37-4.43(m, 1H ), 4.33 (dd, J=2.88, 5.00Hz, 1H), 4.19-4.25 (m, 1H), 3.98 (s, 3H), 3.80 (s, 6H) ), 3.58 (s, 3H), 3.43-3.49 (m, 1H), 3.33-3.40 (m, 1H), 2.55 (d, J=6.88Hz, 1H). LCMS(ESI)m / z; 574.2[MH] - .

[0169] Step G: At 0°C, 2-cyanoethyl-N,N-diisopropylphosphoramide (678.45 mg, 2.87 mmol) and N,N-diisopropylethylamine were added to a dichloromethane (8 mL) solution of the compounds shown in Formulas 1-7 (1.10 g, 1.91 mmol), and the mixture was stirred at 20°C for 0.5 h. 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 the compound of Formula 1. LCMS (ESI) m / z: 776.3 [M+H] + .

[0170] Example 2 Synthesis of D01

[0171]

[0172]

[0173] Step A: Dissolve 25 g of 1,1-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 extract with 800 mL (400 mL × 2) of dichloromethane. Wash the combined organic phases with 400 mL (200 mL × 2) of water and saturated brine, dry to anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain 2-2.

[0174] Step B: The compound shown in Formula 2-3 (20 g, 67.26 mmol) was dissolved in N,N-dimethylformamide (200 mL) at 0°C. Sodium hydride (60% purity, 4.04 g, 100.89 mmol) was added, 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 extracted with 1.6 L (800 mL × 2) of dichloromethane. The combined organic phases were washed with 800 mL (800 mL × 1) of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 2-4. 1 H NMR (400MHz, DMSO-d6): ε7.63-6.89 (m, 10H), 5.64-5.52 (m, 2H), 4.27-4.01 (m, 2H), 3.98- 3.77(m, 2H), 3.72-3.18(m, 4H), 2.23-2.14(m, 2H), 1.98-1.92(m, 1H), 1.54-1.23(m, 16H).

[0175] 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, add 200 mL of chloroform (100 mL × 2), and 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 mixture 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. The reaction solution was diluted with water (500 mL) and extracted with 1 L (500 mL × 2) of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 2-5.

[0176] 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 mixture was diluted with water (500 mL) and extracted with ethyl acetate in 1 L (500 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography (SiO2, petroleum ether / ethyl acetate / ethanol (containing 0.1% ammonia) = 36 / 3 / 1 to 16 / 3 / 1) yielded 2-6. 1 H NMR (400MHz, DMSO-d6): ε3.60-3.54(m, 4H), 3.32(br s, 5H), 3.15 (s, 5H), 2.74-2.66 (m, 1H), 2.40 (t, J=6.0Hz, 4H), 2.18-2.11 (m, 2H), 1.58-1.38 (m, 22H), 1.34-1.23 (m, 12H).

[0177] Step E: Triethylamine (9.15 g, 90.45 mmol) and succinic anhydride (6.79 g, 67.83 mmol) were added to a dichloromethane (250 mL) solution of the compound shown in Formula 2-6 (24.5 g, 45.22 mmol), and 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. The organic phase after separation was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 2-7. 1 H NMR (400MHz, CDCl3): ε6.49-6.37 (m, 1H), 3.72 (s, 2H), 3.70-3.57 (m, 8H), 3.37 (t, J=6.7Hz, 2H), 2.69-2.51 (m, 4H), 2.50-2.3 6(m, 4H), 2.22-2.13(m, 2H), 1.96-1.90(m, 1H), 1.57-1.47(m, 4H), 1.46-1.40(m, 18H), 1.40-1.31(m, 2H), 1.30-1.21(m, 10H).

[0178] 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 solution under reduced pressure, add 300 mL of toluene (150 mL × 2), and concentrate under reduced pressure to obtain 2-8. 1H NMR (400MHz, CDCl3): ε9.79-9.22(m, 3H), 6.44-6.23(m, 1H), 3.88-3.43(m, 10H), 3.39-3.20(m, 2H), 2.77-2.31 (m, 8H), 2.15-2.06 (m, 2H), 1.87 (t, J=2.6Hz, 1H), 1.48-1.28 (m, 6H), 1.26-1.12 (m, 10H).

[0179] 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) were dissolved in N,N-dimethylformamide (250 mL), and N-(3-aminopropyl)carbamate tert-butyl ester (29.74 g, 170.69 mmol) was added. The mixture was stirred at 20 °C for 16 hours. The reaction solution was added with 1 L of dichloromethane and 1 L of hydrochloric acid (1 mol / L). The separated organic phase was washed successively with 1 L of water (1 L × 1), 1 L of sodium bicarbonate aqueous solution (1 L × 1), and 1 L of saturated brine (1 L × 1). After drying with anhydrous sodium sulfate, the solution was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate / ethanol = 40 / 3 / 1 to 10 / 3 / 1) to obtain 2-9. 1 H NMR (400MHz, CDCl3): ε7.22-6.79 (m, 3H), 6.77-6.44 (m, 1H), 5.45-5.00 (m, 3H), 3.86-3 .73(m, 2H), 3.72-3.63(m, 4H), 3.62-3.45(m, 4H), 3.41-3.32(m, 2H), 3.32-3.20(m, 6H) , 3.19-3.03(m, 6H), 2.56-2.47(m, 4H), 2.47-2.39(m, 4H), 2.21-2.12(m, 2H), 1.95-1.9 0 (m, 1H), 1.70-1.57 (m, 6H), 1.56-1.47 (m, 4H), 1.46-1.38 (m, 29H), 1.30-1.25 (m, 10H).

[0180] 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 600 mL (250 mL × 3) of a toluene / acetonitrile = 3 / 1 mixture was added. The solution was then concentrated under reduced pressure to obtain 2-10(((tri(trifluoroacetate))).

[0181] Step I: The 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-azabenzotriazole-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphate (20.53 g, 54.00 mmol) were dissolved in N,N-dimethylformamide (90 mL). A solution of the compound shown in Formula 2-10 (tris(trifluoroacetate), 15.6 g, 15.00 mmol) and N,N-diisopropylethylamine (21.32 g, 165.00 mmol) in N,N-dimethylformamide (120 mL) was added to this mixture. The mixture was stirred at 20°C for 16 hours. The reaction solution was added with 1.2 L of dichloromethane and 1 L of hydrochloric acid (1 mol / L). The separated organic phase was washed successively with 1 L of water (1 L × 1), 1 L of sodium bicarbonate aqueous solution (1 L × 1), and 1 L of saturated brine (1 L × 1). After drying with anhydrous sodium sulfate, the solution was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (SiO2, dichloromethane / methanol = 100 / 1 to 10 / 1 to dichloromethane / ethanol = 1 / 1) to obtain 2-12. 1 H NMR (400MHz, DMSO-d6): ε7.87-7.66 (m, 9H), 7.09 (s, 1H), 5.21 (d, J=3.4Hz, 3H), 4.96 (dd, J=3.4, 11.3Hz, 3H) , 4.48 (d, J=8.5Hz, 3H), 4.06-3.98 (m, 9H), 3.91-3.82 (m, 3H), 3.74-3.66 (m, 3H), 3.58-3.46 (m, 12H), 3.31 (br s, 3H), 3.07-2.98 (m, 12H), 2.71 (t, J=2.6Hz, 1H), 2.33-2.22 (m, 8H), 2.16-2.12 (m, 2H), 2.10 (s, 9H), 2.04 (brt, J=7.1 Hz, 6H), 1.99 (s, 9H), 1.89 (s, 9H), 1.81-1.74 (m, 9H), 1.54-1.39 (m, 22H), 1.32 (brdd, J=4.5, 6.7Hz, 2H), 1.24 (s, 10H).

[0182] Step J: Dissolve the compound shown in Formula 2-12 (1.00 g, 0.50 mmol) and N-methyl-N,N,N-tri-n-octylammonium chloride (20.35 mg, 50.35 μmol) in a mixture of acetic acid (2.7 mL) and n-pentane (6.3 mL). Add a 9 mL solution of potassium permanganate (0.40 g, 2.52 mmol) in water dropwise to this mixture at 0°C. Stir the mixture at 0 to 15°C for 2 hours. Quench the reaction with sodium bisulfite (1.27 g), add hydrochloric acid (2 mol / L, 5 mL) and water (30 mL), and extract with 120 mL (40 mL × 3) of a chloroform / isopropanol 3 / 1 mixture. The combined organic phases were dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and 180 mL (30 mL × 6) of a 1 / 1 toluene / acetonitrile mixture was added. The mixture was then concentrated under reduced pressure to obtain 2-13. 1 H NMR (400MHz, CD3OD): ε5.34 (d, J=2.9Hz, 3H), 5.06 (dd, J=3.3, 11.2Hz, 3H), 4.56 (d,.J=8.4Hz, 3H), 4.19-4.06 (m, 9H), 4.04-3.98 (m, 3H), 3.87 (td, J=5.7, 9.9Hz, 4H), 3.72-3.64 (m, 9H), 3.57-3.50 (m, 3H), 3.39 (brt, J=6.4Hz, 2H), 3.22 (q, J=6.4Hz, 12H), 2.51- 2.40 (m, 9H), 2.21 (brt, J=7.3Hz, 6H), 2.14 (s, 9H), 2.03 (s, 9H), 1.94 (d, J=7.9Hz, 18H), 1.72-1.57 (m, 22H), 1.39 (br s, 12H).

[0183] Step K: Add 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) to a solution of N,N-dimethylformamide (10 mL) of the compound shown in Formula 2-13 (1.00 g, 0.50 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. After separation, wash the organic phase successively with 50 mL of saturated sodium bicarbonate aqueous solution (50 mL × 1), 50 mL of water (50 mL × 1), and 50 mL of saturated brine (50 mL × 1), dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the crude product. After purification by column chromatography (SiO2, dichloromethane / methanol (containing 0.1% triethylamine) = 20 / 1 to 10 / 1), 2-15 was obtained. 1H NMR (400MHz, DMSO-d6): ε7.90-7.82 (m, 6H), 7.78 (br d, J=4.8Hz, 3H), 7.40-7.26 (m, 10H), 6.91 (brdd, J=3.1, 9.0Hz, 4H), 5.26 (d, J=3.4Hz, 3H), 5.03-4.99 (m, 3H), 4.53 (d, J=8.4Hz, 3H), 4.43 (br d, J=3.8Hz, 1H), 4.23-4.14 (m, 1H), 4.12-4.02 (m, 9H), 3.92 (td, J=9.0, 11.0Hz, 3H), 3.78 (s, 6H), 3.7 7-3.71(m, 3H), 3.66-3.51(m, 13H), 3.49-3.41(m, 4H), 3.11-3.01(m, 16H), 2.38-2.37(m, 1H), 2.32(br s, 9H), 2.14 (s, 9H), 2.08 (br t, J=6.9Hz, 7H), 2.04 (s, 9H), 1.93 (s, 9H), 1.82 (s, 9H), 1.57-1.46 (m, 22H), 1.31-1.26 (m, 12H).

[0184] Step L: Triethylamine (67.24 mg, 0.64 mmol), 4-N,N-dimethylaminopyridine (0.12 g, 1.00 mmol), and succinic anhydride (83.13 mg, 0.83 mmol) were added sequentially to a dichloromethane (8 mL) solution of the compound shown in Formula 2-15 (0.80 g, 0.33 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. The separated organic phase was washed sequentially with 30 mL of water (30 mL × 1) and 30 mL of saturated brine (30 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The compound was purified by p-HPLC (separation column: Waters Xbridge C18 (size: 150mm×50mm, particle size: 10μm); mobile phase: [water (10mM ammonium bicarbonate)-acetonitrile]; elution gradient: 27%-57%, 11min) to obtain Example 2 (compound D01). 1H NMR (400MHz, DMSO-d6): ε7.96-7.69 (m, 9H), 7.33-7.09 (m, 10H), 6.90-6.78 (m, 4H), 5.21 (d, J=3.3Hz, 3H), 4.97 (dd, J=3.3, 11.2Hz, 3H), 4.49 (d, J=8.4Hz, 3H), 4.06-3.97 (m, 9H), 3.91-3.83 (m, 3H), 3.79-3.66 (m, 11H), 3.63-3.45 (m, 18H), 3.02 (br d, J=4.6Hz, 14H), 2.46-2.37(m, 4H), 2.35-2.14(m, 12H), 2.10(s, 9H), 2.04(brt, J=7.0Hz, 6H), 1.99 (s, 9H), 1.88 (s, 9H), 1.77 (s, 9H), 1.57-1.37 (m, 22H), 1.22 (br s, 12H).

[0185] Example 3 Synthesis of double-stranded siRNA analogs or their conjugates

[0186] Synthesis of D-containing single-stranded oligonucleotides: Oligonucleotides were synthesized using a phosphoramide solid-phase synthesis technique. This was achieved using a controllable porous glass (aminoCPG) reactor. Synthesized on a solid support prepared by covalently linking 2'-modified RNA phosphoramidite with D01. All 2'-modified RNA phosphoramidite and auxiliary reagents were commercially available. All amides were dissolved in anhydrous acetonitrile and molecular sieves were added. The coupling time using 5-ethylthio-1H-tetrazole (ETT) as an activator was 5 minutes. Thiophosphate 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 minutes. All sequences were synthesized after the final removal of the DMT group.

[0187] Synthesis of D-free single-stranded oligonucleotides: Oligonucleotides were synthesized using a phosphorus amide solid-phase synthesis technique. This was carried out in a universally controllable porous glass CPG system. The synthesis was carried out on [a specific method / procedure]. All 2'-modified RNA phosphoramidite and auxiliary reagents were commercially available. All amides were dissolved in anhydrous acetonitrile and molecular sieves were added. The coupling time using 5-ethylthio-1H-tetrazole (ETT) as an activator was 5 minutes. Thiophosphate 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 minutes. All sequences were synthesized after the final removal of the DMT group.

[0188] 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.

[0189] 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.

[0190] 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: Place 100 μL of the mixture (10 nmol) in a 95°C water bath for 10 minutes (for amounts ≥100 nmol, a 20-minute high-temperature treatment is required) → immediately place in a 60°C water bath and allow to cool naturally → the annealed solution should not be stored at high temperatures. Combine equimolar amounts of the single-stranded oligonucleotide solution to form complementary strands.

[0191] Table 1. Double-stranded siRNA analogs targeting the hepatitis B virus gene, their conjugates, and their corresponding core sequences.

[0192]

[0193]

[0194]

[0195] *: 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:

[0196]

[0197] **: 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.

[0198] ***: When the sequence contains D, D is used to refer to the connection position of the conjugate group D. For example, g·r·guGcACUucgcuucacaD(5'-3') means that the sequence g·r·guGcACUucgcuucaca, as shown in SEQ ID NO: 16, is connected to D at the 3' end.

[0199] Example 3: In vitro HBV testing

[0200] 1. Experimental objective:

[0201] 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.

[0202] 2. Experimental materials:

[0203] 2.1 Cell line: HepG2-NTCP cells

[0204] 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; 2 mM L-glutamine, Invitrogen-25030081; 1 mM sodium pyruvate, Gibco-11360-070; 500 μg / ml geneticin, Invitrogen-10131027)

[0205] 2.2 Reagents:

[0206] 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).

[0207] 2.3 Consumables and Instruments:

[0208] 96-well cell culture plate (Corning-3599); CO2 incubator (HERA-CELL-240)

[0209] Microplate reader (BioTek Synergy 2)

[0210] 3. Experimental steps and methods:

[0211] 3.1 Day 0, seed HepG2-NTCP (7.5×10⁻⁶) 4 Cells (cells / well) were transferred to 48-well plates and incubated overnight at 37°C with 5% CO2.

[0212] 3.2 On day 1, replace the culture medium with one containing 1% DMSO.

[0213] 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).

[0214] 3.4 On the third day, aspirate the infection fluid and add fresh culture medium containing 1% DMSO.

[0215] 3.5 Day 6, according to 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.

[0216] 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.

[0217] 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.

[0218] 3.8 Data Analysis:

[0219] Calculate cell viability percentage:

[0220] %viability = (luminescence value of sample - luminescence value of culture medium control) / (luminescence value of DMSO control - luminescence value of culture medium control) × 100.

[0221] Calculate the inhibition percentages of HBV surface antigen and e antigen:

[0222] %Inh. = (1 - antigen value in sample / DMSO control antigen value) × 100.

[0223] 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.

[0224] 4. Experimental results: see Table 2.

[0225] Table 2 shows the experimental results of the tested sequences in reducing HBsAg and HBeAg levels in cells.

[0226]

[0227] ＊ The sample tested was a conjugate of a double-stranded siRNA analog.

[0228] Example 4: 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)

[0229] Experimental objective:

[0230] 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. Using the AAV / HBV model, HBV DNA can be continuously replicated and HBsAg and HBeAg can be expressed in the mouse liver.

[0231] 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.

[0232] Experimental materials:

[0233] 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 Probe Master (Rox) (Roche, 04914058001), Hepatitis B surface antigen detection kit (Antu Bio, CL0310), Hepatitis B e antigen detection kit (Antu Bio, CL0918), and PureLink. TM The main instruments used included: Pro 96 Viral RNA / DNA kit (Invitrogen, 12280-096A) and FastQuant RT Kit (with gDNase) (TIANGEN, KR106-02). Other instruments included: centrifuge (Beckman Allegra X-15R), multi-functional microplate reader (BioTek, Synergy 2), real-time PCR system (Applied Biosystems, 7900HT Fast Real-time PCR system), and microplate reader (Molecular Devices, SpectraMax 340PC384).

[0234] Experimental methods:

[0235] a) Mice were administered the drug subcutaneously starting on day 34 after viral injection; this day was designated as day 0. Blood plasma was collected from under the jaws of all mice before administration. Specific administration protocols are shown in Table 3.

[0236] b) Plasma was collected from the submandibular vein on days 0, 14, 21, 28 and 32 after administration to mice. The collected blood samples were anticoagulated with K2-EDTA and centrifuged at 4°C, 7000g / min for 10 minutes before plasma collection. Specific blood collection times are shown in Table 3.

[0237] 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.

[0238] d) The pulp sample was sent for testing.

[0239] Table 3 In vivo experimental protocol

[0240]

[0241]

[0242] *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.

[0243] *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.

[0244] *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.

[0245] / : The destination has not yet been reached.

[0246] Sample analysis:

[0247] 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.

[0248] qPCR detection of HBV DNA content in mouse plasma: HBV DNA was extracted from the plasma. The experimental procedure was performed according to the QIAamp 96 DNA Blood Kit instructions. The HBV DNA content in mouse plasma was then detected by qPCR.

[0249] RT-qPCR detection of HBV pgRNA levels in mouse plasma: HBV pgRNA was extracted from plasma, and the experimental procedure was performed according to PureLink. TMFollow the instructions for the Pro 96 Viral RNA / DNA Kit. Digest the DNA and reverse transcribe the RNA into cDNA using 3′ RACE primers containing the hepatitis B virus-specific sequence, following the instructions for the FastQuant RT Kit (with gDNase). Finally, quantify the cDNA content using qPCR, which measures the HBV pgRNA content in mouse plasma.

[0250] Mean ± standard error of each group of mouse samples, unless otherwise specified, n = 5. Statistical analysis was performed using Student's t-test.

[0251] Experimental results:

[0252] a) Serum HBsAg levels were used to evaluate the anti-HBV activity of the test compound in the AAV / HBV mouse model. Results are shown in Tables 4, 4-1, Figure 1, and Figure 6. Mouse plasma HBsAg levels were determined by ELISA. Error bars indicate standard errors. Day 0: All mice were administered the medium or compound for the first time. Day 29: The experimental group WRG01 mice and the blank control group mice were administered the medium or compound for the second time.

[0253] Table 4 Log of mice at different dates after drug administration 10 [HBsAg (IU / mL)]

[0254] Detection days (days) Blank (SC) WRG01 (SC) 0 4.70 4.72 7 4.82 2.90 14 4.43 2.90 21 4.94 3.28 28 4.84 3.77 35 4.78 2.83

[0255] Table 4-1 Log data of mice on different days after drug administration 10 [HBsAg (IU / mL)]

[0256] Detection Days (days) Blank (SC) WR007 (SC) WR012 (SC) 0 4.58 4.19 4.47 7 4.15 1.92 2.00 14 4.57 2.29 2.20 21 4.41 2.63 2.36 28 4.76 2.94 3.10 35 4.62 3.31 3.19

[0257] b) Evaluation of HBeAg levels in mouse plasma to assess the anti-HBV activity of the test compounds in an AAV / HBV mouse model. Results are shown in Tables 5, 5-1, Figures 2 and 7. 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.

[0258] Table 5 Log of mice on different days after drug administration10 [HBeAg (PEIU / mL)]

[0259] Detection days (days) Blank (SC) WRG01 (SC) 0 3.56 3.51 7 3.37 2.89 14 3.56 3.06 21 3.66 3.22

[0260] Table 5-1 Log of mice on different days after drug administration 10 [HBeAg (PEIU / mL)]

[0261] Detection days (days) Blank (SC) WR007 (SC) WR012 (SC) 0 3.44 3.35 3.40 7 3.24 2.49 2.53

[0262] 143.572.802.89213.322.812.82283.382.952.91353.373.093.02

[0263] c) Serum DNA content was used to evaluate the anti-HBV activity of the test compound in the AAV / HBV mouse model. Results are shown in Tables 6, 6-1, Figures 3 and 8. Mouse plasma HBV DNA content was 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.

[0264] Table 6 Log of mice at different dates after drug administration 10 [DNA (copy number / μL)]

[0265] Detection days (days) Blank (SC) WRG01 (SC) 0 5.27 4.84 7 5.39 3.93 14 5.51 3.97 21 5.63 4.37

[0266] Table 6-1 Log of mice on different days after drug administration 10 [DNA (copy number / μL)]

[0267] Detection Days (days) Blank (SC) WR007 (SC) WR012 (SC) 0 5.53 / / 7 4.98 / / 14 5.34 3.44 3.95 21 5.45 3.71 4.21 28 5.63 4.08 4.66 35 5.26 4.42 4.78

[0268] / : No data obtained.

[0269] 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 and Figure 4. The HBV pgRNA content in mouse plasma was determined by quantitative PCR. 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.

[0270] Table 7 Log of mice on different days after drug administration 10 [pgRNA (copy number / μL)]

[0271] Detection days (days) Blank (SC) WRG01 (SC) 0 4.92 4.56 7 4.96 3.28

[0272] 144.933.26215.023.50285.064.13355.173.37

[0273] e) Weight changes are shown in Figure 5. The weight on day 0 was used as a baseline for comparison. According to IACUC guidelines, a 20% weight loss is considered a humane endpoint; any mouse with a weight loss exceeding 20% ​​must be removed from the experiment. No mice were removed from this experiment due to weight loss.

[0274] Experimental conclusion:

[0275] 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.

[0276] Example 5: In vitro HBV test in HepG2.2.15 cells

[0277] 1. Experimental objective:

[0278] 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 CCK8 assay was used to detect the effect of the test compound on cell viability.

[0279] 2. Experimental materials:

[0280] 2.4 Cell line: HepG2.2.15 cells

[0281] HepG2.2.15 cell culture medium (DMEM / F12, Invitrogen-11330032; 10% serum, Hyclone-SV30087.0; 100 units / ml penicillin and 100 μg / ml streptomycin, Hyclone-SV30010; 1% non-essential amino acids, Invitrogen-11140050; 2 mM L-GLUTAMINE, Invitrogen-25030081; 300 μg / ml geneticin, Invitrogen-10131027).

[0282] 2.5 Reagents

[0283] Opti-MEM (Gibco-31985-070): 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, CL0310); Hepatitis B e antigen quantitative detection kit (Antu Bio, CL 0312).

[0284] 2.6 Consumables and Instruments:

[0285] Collagen I 96 Well White / Clear Flat Bottom TC-Treated Microplate (Corning BioCoat-356650); CO2 incubator (HERA-CELL-240); Real-time PCR system (Applied Biosystems-7900 real-time PCR system); Real-time PCR system (Applied Biosystems-QuantStudio 6 Flex); Microplate reader (Molecular Device-SpectraMax M2e); Microplate reader (BioTek-Synergy 2).

[0286] 3. Experimental steps and methods:

[0287] 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 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.

[0288] Table 8 RNAiMAX configuration

[0289]

[0290] siRNA was serially diluted to eight concentrations, 3-fold serially diluted, in duplicate. 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 incubator at 37°C.

[0291] 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.

[0292] 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 the cell viability according to the CCK-8 kit instructions, and detect the absorbance of each well (450nm / 650nm) using a microplate reader (SpectraMax M2e); use the RNeasy 96 kit extraction kit (Qiagen-74182) to extract HBV RNA from the cell culture according to the kit instructions.

[0293] 3.4 The preparation of the PCR reaction solution is shown in Table 9:

[0294] Table 9 Preparation of PCR reaction solution

[0295]

[0296]

[0297] 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.

[0298] 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.

[0299] 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°C 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.

[0300] 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.

[0301] qPCR reaction: 95℃, 10 min; 95℃, 15 s, 60℃, 1 min, 40 cycles. The HBV RNA content in each sample was calculated based on the Ct value.

[0302] 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:

[0303] ΔCT = Average Ct value of target gene - Average Ct value of internal reference gene;

[0304] ΔΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group);

[0305] HBV mRNA relative expression level = 2 - ΔΔCT

[0306] 3.7 Data Analysis:

[0307] Calculate the percentage of inhibition:

[0308] %Inh. = (1 - value in sample / PBS control value) x 100.

[0309] 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

[0310] 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.

[0311] Table 10 shows the experimental results of the sequences tested reducing HBsAg, HBeAg, DNA, and RNA levels in cells.

[0312]

[0313]

[0314] / : Data not yet available.

[0315] ＊ The sample tested was a conjugate of a double-stranded siRNA analog.

[0316] Example 6: Exploration of the effective dosage of anti-hepatitis B virus drugs in an AAV-HBV mouse model

[0317] 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.

[0318] Experimental materials:

[0319] C57BL / 6 mice, PBS (RNase-free) as solvent, test compound, and recombinant virus rAAV8-1.3HBV. The main reagents for this project included the FastStart Universal Probe Master (Rox) (Roche, 04914058001) and a Hepatitis B virus 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).

[0320] Experimental methods:

[0321] 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.

[0322] 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 centrifuged at 4°C, 7000g / min for 10 minutes before plasma collection. The specific blood collection times are shown in Table 11.

[0323] c) On day 42, all mice were euthanized by submandibular vein blood collection, followed by heart blood collection of plasma samples and liver samples.

[0324] d) All plasma samples were sent for testing.

[0325] Table 11 In vivo experimental protocol

[0326]

[0327] *: 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.

[0328] / : The destination has not yet been reached.

[0329] Sample analysis:

[0330] 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.

[0331] Mean ± standard error of each group of mouse samples, unless otherwise specified, n = 5. Statistical analysis was performed using Student's t-test.

[0332] Experimental results:

[0333] 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 and Figure 9. 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.

[0334] Table 12 Log of mice on different days after drug administration 10 [HBsAg (IU / mL)]

[0335]

[0336] Experimental conclusion:

[0337] 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.

[0338] Example 7: Drug concentration tests in mouse plasma, liver, and kidneys

[0339] 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.

[0340] Table 13 In vivo experimental protocol

[0341]

[0342]

[0343] *: 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.

[0344] / : Blood was not drawn at non-endpoint times, only at the end point.

[0345] Experimental results:

[0346] The levels of siRNA in plasma, liver, and kidney of mice at different time points after drug administration were detected by SL-qPCR (Reference: Nair et al. Nucleic Acids Research (2017), 45, 10969-10977). The results are shown in Figure 10.

[0347] Experimental conclusion:

[0348] 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.

[0349] Example 8: Blood biochemical tests in FRG-KO humanized liver mice

[0350] 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.

[0351] 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.

[0352] Table 14 In vivo experimental protocol

[0353]

[0354] *: 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.

[0355] / : The destination has not yet been reached.

[0356] 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

DEPCT6609 / 02 / 25661. An analogue of a double-stranded siRNA, conjugate of that or its salt, comprising a sense strand and an antisense strand, in which the antisense strand comprises a sequence obtained by replacing one or more nucleotide residues in the sequence specified in SEQIDNO:2 with r, and r is (chemical formula) r, in which each nucleotide and r in the siRNA analogue are independently modified or unmodified.

2. An analogue of a double-stranded siRNA, conjugate of that or its salt, according to claim 1, in which nucleotides 70%, 75%, 80%, 85%, 90%, or 95% or more of the nucleotide and r in the double-stranded siRNA analog are modified; selectively, all nucleotides and r in the double-stranded siRNA analog are modified.

3. The double-stranded siRNA analog, conjugate of that or salt of that according to claim 1 or 2, where the modification comprises methoxy modification, fluoromodulation, phosphoothioate linkage, nucleotide replacement with (S)-glycerol nucleic acid or nucleotide replacement with (E)-vinyl phosphate.

4. The double-stranded siRNA analog,A conjugate of that substance or a salt of that substance under any of the claims 1–3 in which the antisense is composed of a sequence obtained by replacing one, two, three, four or five nucleotide residues in the sequence specified in SEQIDNO:2 with r; selectively, the antisense is composed of a sequence obtained by replacing one nucleotide residue in the sequence specified in SEQIDNO:2 with r.

5. A double-stranded siRNA analog, a conjugate of that substance or a salt of that substance under any of the claims 1–4 in which the replacement 7. An analog of a double-stranded siRNA, conjugate of it, or salt of it, according to any of claims 1–5, in which SEQIDNO:2 is optionally included with an overhang at the 5' and / or 3' ends; optionally SEQIDNO:2 is included with an overhang of 0, 1, 2, 3, 4, or 5 nucleotides at the 5' and / or 3' ends; optionally SEQIDNO:2 is included with an overhang at the 3' end and the overhang is selected from a modified or unmodified UU.The conjugate of that thing or the salt of that thing under any of the claims 1–6 in which the antisense is comprised of or contains the sequence specified in SEQIDNO:4 or SEQIDNO:17, SEQIDNO:6 or SEQIDNO:19, SEQIDNO:7 or SEQIDNO:20, SEQIDNO:8 or SEQIDNO:21, SEQIDNO:9 or SEQIDNO:22, SEQIDNO:10 or SEQIDNO:23, SEQIDNO:11 or SEQIDNO:24, SEQIDNO:29 or SEQIDNO:33, SEQIDNO:30 or SEQIDNO:34, SEQIDNO:31 or S EQIDNO:35, SEQIDNO:32 or SEQIDNO:36, SEQIDNO:39 or SEQIDNO:44, SEQIDNO:10 or SEQIDNO:45, SEQIDNO:40 or SEQIDNO:46, SEQIDNO:10 or SEQIDNO:47 or SEQIDNO:10 or SEQIDNO:

488. An analog of a double-stranded siRNA, a conjugate of that or a salt of that according to any one of claims 1–7 in which the sense chain is included or contains the sequence specified in SEQIDNO:1 or SEQIDNO:

289. An analog of a double-stranded siRNA,A conjugate of that substance or a salt of that substance under any of the claims 1–8 in which the sense chain is composed of a sequence obtained by replacing one or more nucleotide residues in the sequence specified in SEQIDNO:1 with r; or selectively, the sense chain is composed of a sequence obtained by replacing one, two, three, four, or five nucleotide residues in the sequence specified in SEQIDNO:1 with r.

10. A double-stranded siRNA analog, a conjugate of that substance or a salt of that substance under any of the claims 1–9. One of the claims 1-10 is that the substitution of r occurs at positions 1-19 of the 5' end of SEQIDNO:

111. An analog of a double-stranded siRNA, a conjugate of that, or a salt of that, according to any one of the claims 1-10, in which the sequence of the sense strand is included with or contains the sequences specified in SEQIDNO:5 or SEQIDNO:18, SEQIDNO:3 or SEQIDNO:16, SEQIDNO:14 or SEQIDNO:27, SEQIDNO:13 or SEQIDNO:26, SEQIDNO:12 or SEQIDNO:25, SEQIDNO:37 or SEQIDNO:42 or SEQIDNO:38 or SEQIDNO:4312. An analog of a double-stranded siRNA,The conjugate of that thing or the salt of that thing according to any one of claims 1-11, where the double-stranded siRNA analog is either S18-S28:S18:Sense strand is SEQIDNO:1 or SEQIDNO:28 and antisense strand is SEQIDNO:4 or SEQIDNO:17,S19:Sense strand is SEQIDNO:1 or SEQIDNO:28 and antisense strand is SEQIDNO:6 or SEQIDNO:19,S20:Sense strand is SEQIDNO:1 or SEQIDNO:28 and antisense strand is SEQIDNO:7 or SEQIDNO:20,S21:Sense strand is SEQIDNO:1 or SEQIDNO:28 and antisense strand The Sense line is SEQIDNO:8 or SEQIDNO:21, S22: The Sense line is SEQIDNO:1 or SEQIDNO:28 and the Anti-Sense line is SEQIDNO:9 or SEQIDNO:22, S23: The Sense line is SEQIDNO:1 or SEQIDNO:28 and the Anti-Sense line is SEQIDNO:10 or SEQIDNO:23, S24: The Sense line is SEQIDNO:1 or SEQIDNO:28 and the Anti-Sense line is SEQIDNO:11 or SEQIDNO:24, S25: The Sense line is SEQIDNO:1 or SEQIDNO:28 and the Anti-Sense line is SEQIDNO:29 or SEQIDNO:33, S26: The Sense line is SEQIDNO:1 or SEQIDNO:28 and the Anti-Sense line is SEQIDNO:30 or SEQIDNO:34.S27: The sense strand is SEQIDNO:1 or SEQIDNO:28 and the antisense strand is SEQIDNO:31 or SEQIDNO:35, and S28: The sense strand is SEQIDNO:1 or SEQIDNO:28 and the antisense strand is SEQIDNO:32 or SEQIDNO:36, or where the double-stranded siRNA analog is any one of S1-S17:S1: The sense strand is SEQIDNO:3 or SEQIDNO:16 and the antisense strand is SEQIDNO:4 or SEQIDNO:17,S2: The sense strand is SEQIDNO:5 or SEQIDNO:18 and the antisense strand is SEQIDNO:4 or SEQIDNO:17,S3: The sense strand is SEQIDNO:3 or SEQIDNO:16 and the antisense strand is SEQIDNO:6 or SEQIDNO:19,S4: ... The Sense line is SEQIDNO:5 or SEQIDNO:18 and the Anti-Sense line is SEQIDNO:6 or SEQIDNO:19, S5: The Sense line is SEQIDNO:3 or SEQIDNO:16 and the Anti-Sense line is SEQIDNO:7 or SEQIDNO:20, S6: The Sense line is SEQIDNO:5 or SEQIDNO:18 and the Anti-Sense line is SEQIDNO:7 or SEQIDNO:20, S7 Sense line is SEQIDNO:3 or SEQIDNO:16 and antisense line is SEQIDNO:8 or SEQIDNO:21, S8: Sense line is SEQIDNO:5 or SEQIDNO:18 and antisense line is SEQIDNO:8 or SEQIDNO:21, S9: Sense line is SEQIDNO:3 or SEQIDNO:16 and antisense line is SEQIDNO:9 or SEQIDNO:22.S10: The Sense line is SEQIDNO:5 or SEQIDNO:18 and the Anti-Sense line is SEQIDNO:9 or SEQIDNO:

22. S11: The Sense line is SEQIDNO:3 or SEQIDNO:16 and the Anti-Sense line is SEQIDNO:10 or SEQIDNO:

23. S12: The Sense line is SEQIDNO:5 or SEQIDNO:18 and the Anti-Sense line is SEQIDNO:10 or SEQIDNO: 23,S13: The Sense line is SEQIDNO:3 or SEQIDNO:16 and the Anti-Sense line is SEQIDNO:11 or SEQIDNO:24,S14: The Sense line is SEQIDNO:5 or SEQIDNO:18 and the Anti-Sense line is SEQIDNO:11 or SEQIDNO:24,S15: The Sense line is SEQIDNO:12 or SEQIDNO:25 and the Anti-Sense line is SEQIDNO:4 or SEQID NO:17,S16: The sense line is SEQIDNO:13 or SEQIDNO:26 and the antisense line is SEQIDNO:4 or SEQIDNO:17 and S17: The sense line is SEQIDNO:14 or SEQIDNO:27 and the antisense line is SEQIDNO:4 or SEQIDNO:17 or where the double-stranded siRNA analog is either S29-S35:S29:S The Sense line is SEQIDNO:37 or SEQIDNO:42 and the Anti-Sense line is SEQIDNO:10 or SEQIDNO:23, S30: The Sense line is SEQIDNO:38 or SEQIDNO:43 and the Anti-Sense line is SEQIDNO:10 or SEQIDNO:23, S31: The Sense line is SEQIDNO:3 or SEQIDNO:16 and the Anti-Sense line is SEQIDNO:39 or SEQIDNO:44.S32: The Sense line is SEQIDNO:3 or SEQIDNO:16 and the Anti-Sense line is SEQIDNO:10 or SEQIDNO:

45. S33: The Sense line is SEQIDNO:3 or SEQIDNO:16 and the Anti-Sense line is SEQIDNO:40 or SEQIDNO:46.S34: The sense chain is SEQIDNO:3 or SEQIDNO:16 and the antisense chain is SEQIDNO:10 or SEQIDNO:47; and S35: The sense chain is SEQIDNO:3 or SEQIDNO:16 and the antisense chain is SEQIDNO:10 or SEQIDNO:

48.

13. Conjugate of a double-stranded siRNA analog or a salt of such under any one of claims 1–12 where the double-stranded siRNA analog is associated with a pharmaceutically acceptable conjugate group and the pharmaceutically acceptable conjugate group contains a GalNAc group; optionally, the pharmaceutically acceptable conjugate group contains a GalNAc group. 1 to 5. Group 14. Conjugate of a double-stranded siRNA analog or salt of such according to any one of claims 1-13 where the double-stranded siRNA analog is linked to a pharmaceutically acceptable conjugate group and the pharmaceutically acceptable conjugate group comprises compound group D (chemical formula) D.

15. Conjugate of a double-stranded siRNA analog or salt of such according to claim 13 or 14 where the pharmaceutically acceptable conjugate group is linked to the 3' end of the sense strand of the double-stranded siRNA analog.

16. Double-stranded siRNA analog,17. A double-stranded siRNA analog, conjugate of that thing, or salt of that thing, under any of the claims 1-16, in which the phosphorothioate moité of the double-stranded siRNA analog or conjugate of that thing is composed of (R)- and (S)-enantiomers, diastereoisomers and / or racimitic mixtures of that thing. Sodium, potassium, calcium, ammonium, organic amines, magnesium, and combinations of those salts and acid-added salts are selected from salts derived from inorganic acids, salts derived from inorganic acids and combinations of those salts; selectively, inorganic acids are selected from hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate radicals, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphoric acid, and combinations of those acids, and organic acids are selected from acetic acid, propionic acid,Isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and combinations of such acids.

18. Pharmaceutical compositions which include a double-stranded siRNA analog, a conjugate of such, or a salt of such pursuant to any one of claims 1-17 and a selectable pharmaceutically acceptable carrier or excipient.

19. Use of a siRNA analog...

20. A method for the treatment of hepatitis B in trial patients which includes the administration of a double-stranded siRNA analog, conjugate of that substance, or salt of that substance under any of the claims 1-17, or a pharmaceutical component under claim 18, to the trial patients for the preparation of a drug for the treatment of hepatitis B.