Double-stranded siRNA analogues including R and E and conjugates thereof

Abstract

Provided are a series of double-stranded siRNA analogues comprising R and / or E, conjugates thereof, salts of the conjugates thereof, and uses thereof.

Description

Double-stranded siRNA analogs containing R and E and conjugates thereof

[0001] The present invention claims the following priority:

[0002] CN2023101774460, application date: February 24, 2023;

[0003] CN2023101914409, application date: February 28, 2023;

[0004] CN2023104534435, application date: April 23, 2023;

[0005] CN202311511833X, application date: November 13, 2023;

[0006] CN 2024100843576, application date: January 19, 2024. Technical Field

[0007] The present invention relates to the field of biomedicine, and in particular to a double-stranded siRNA analog comprising E and R, a conjugate thereof, a salt of the conjugate thereof, and uses thereof. Background Art

[0008] 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 in and damages liver cells, causing inflammation, necrosis, and fibrosis. Hepatitis B can be classified as acute or chronic. Acute HBV in adults is generally self-resolved through the body's own immune system. However, chronic hepatitis B (CHB) has become a significant challenge to global healthcare and a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC). An estimated 2 billion people worldwide are infected with chronic HBV, over 350 million have developed HBV, and nearly 600,000 people die annually from complications of CHB. my country is a high-incidence area for HBV, with a high cumulative number of patients and severe consequences. According to data, there are about 93 million people infected with hepatitis B virus in my country, and about 20 million of them are diagnosed with chronic hepatitis B. Among them, 10% to 20% may develop into cirrhosis and 1% to 5% may develop into liver cancer.

[0009] The key to a functional cure for hepatitis B is clearance of HBsAg (hepatitis B virus surface antigen) and the production of surface antibodies. In chronically infected patients, HBsAg reduction and seroconversion are rarely observed. Currently approved anti-HBV drugs are primarily immunomodulators (interferon-α and pegylated interferon-α-2α) and antiviral drugs (lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, and clavulanate). These antiviral drugs belong to the nucleotide class, whose mechanism of action is to inhibit HBV DNA synthesis and do not directly reduce HBsAg levels. Existing clinical therapies for lowering HBsAg levels have limited efficacy. Therefore, if viral gene expression could be silenced at the genetic level, blocking HBV production and replication, particularly the production of HBsAg and HBeAg (hepatitis B S and E antigens), viral metabolism and infection of hepatocytes could be fundamentally reduced.

[0010] 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 level of mRNA translation to protein, thereby achieving the goal of treating the disease. This ideal approach to treating hepatitis B requires stabilization and modification of siRNAs and the use of appropriate delivery systems to target target organs and cells, improving metabolic stability. However, current siRNAs are not yet able to effectively reduce the levels of HBV S and E antigens. Furthermore, siRNAs can partially complement certain mRNA fragments, thereby regulating the expression of the genes corresponding to these mRNAs. In particular, complementary pairing of the 5'-terminal seed region of the siRNA antisense strand with non-targeted genes can partially or completely silence the expression of these genes. This phenomenon is the main cause of off-target effects of siRNAs in vitro and in vivo. This shortcoming has been exposed in both clinical and preclinical studies of siRNAs used for the treatment of hepatitis B. Although some nucleotide modifications can reduce the risk of off-target effects, the effectiveness of silencing is also reduced, and the therapeutic safety window needs to be improved.

[0011] Summary of the Invention

[0012] The present invention provides a double-stranded siRNA analog, a conjugate thereof, a salt thereof, or a salt of its conjugate, comprising a sense strand and an antisense strand capable of forming a double-stranded region, wherein the double-stranded siRNA analog is selected from any double-stranded strand shown in Table 1, and each nucleotide on the double-stranded strand is independently and optionally modified.

[0013] In some embodiments of the present invention, the double-stranded siRNA analog is selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21, S22, S23, S24, S25, S26, S27 and S28.

[0014] In some embodiments of the present invention, the double-stranded siRNA analog is selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11.

[0015] The present invention provides a double-stranded siRNA analog, a conjugate thereof, a salt thereof, or a salt of its conjugate, which is selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21, S22, S23, S24, S25 and S26.

[0016] The unmodified double-stranded siRNA analogs provided by the present invention are shown in Table 1.

[0017] Table 1 Unmodified double-stranded siRNA analogs

[0018] The R and E of the present invention are

[0019] In some embodiments of the present invention, when the R or E is located at the 5' end of the AS or SS of the siRNA sequence, the 5' position of R or E can be a hydroxyl group, a phosphate bond or a phosphorothioate bond, and the 2' position of R or E can be optionally modified.

[0020] In some embodiments of the present invention, the sense strand of the double-stranded siRNA analog comprises 0, 1, 2, 3, 4, 5 or 6 unmodified nucleotides.

[0021] In some embodiments of the present invention, the antisense strand of the double-stranded siRNA analog comprises 0, 1, 2, 3, 4, 5 or 6 unmodified nucleotides.

[0022] In some embodiments of the present invention, the sense strand of the double-stranded siRNA analog comprises 0, 1, 2 or 3 nucleosides substituted with R nucleotides.

[0023] In some embodiments of the present invention, the antisense strand of the double-stranded siRNA analog comprises 0, 1, 2 or 3 nucleosides substituted with R nucleotides.

[0024] In some embodiments of the present invention, the sense strand of the double-stranded siRNA analog comprises 0, 1, 2 or 3 nucleosides substituted with E nucleotides.

[0025] In some embodiments of the present invention, the antisense strand of the double-stranded siRNA analog comprises 0, 1, 2 or 3 nucleosides substituted with E nucleotides.

[0026] In some embodiments of the present invention, the double-stranded siRNA analog is selected from any double-stranded siRNA shown in Table 2.

[0027] Table 2 Modified double-stranded siRNA analogs

[0028] In some embodiments of the present invention, the above mR is The intermediate for synthesizing the mR is shown in compound 2:

[0029] In some embodiments of the present invention, the above MOER is The intermediate for synthesizing the MOER is shown in compound 1:

[0030] In some embodiments of the present invention, the above-mentioned E is The intermediate for synthesizing the E is shown in compound 3:

[0031] In some embodiments of the present invention, the conjugate of the double-stranded siRNA analog or the salt of the conjugate is formed by conjugating the double-stranded siRNA analog to a pharmaceutically acceptable conjugation group.

[0032] In some embodiments of the present invention, the pharmaceutically acceptable conjugated group in the conjugate of the double-stranded siRNA analog or the salt of the conjugate contains 1 to 5 GalNAc groups.

[0033] In some embodiments of the present invention, the pharmaceutically acceptable conjugated group is linked to any position of the double-stranded siRNA analog.

[0034] In some embodiments of the present invention, the pharmaceutically acceptable conjugated group is linked to the 3' end of the sense strand of the double-stranded siRNA analog.

[0035] In some embodiments of the present invention, the pharmaceutically acceptable conjugated group in the above-mentioned double-stranded siRNA analog conjugate or its salt is selected from D1, D2, D3 and L96,

[0036] In some embodiments of the present invention, the above-mentioned conjugated groups The representative conjugated group is linked to the double-stranded siRNA analog at this site via a phosphodiester bond or a phosphorothioate bond.

[0037] Some other solutions of the present invention are obtained by arbitrarily combining the above solutions.

[0038] The present invention also provides a conjugate of a double-stranded siRNA analog or a salt thereof, which is selected from Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16.

[0039] The conjugates of the double-stranded siRNA analogs provided by the present invention are shown in Table 3.

[0040] Table 3 Conjugates of double-stranded siRNA analogs

[0041] The present invention also provides use of the double-stranded siRNA analog, its conjugate, its salt or the salt of its conjugate in preparing a drug for treating hepatitis B.

[0042] Technical Effects

[0043] The double-stranded siRNA analogs, conjugates, salts thereof, or salts of their conjugates of the present invention exhibit excellent anti-HBV biological activity. The compounds of the present invention exhibit excellent anti-HBsAg activity both in vitro and in vivo, particularly in vivo anti-HBV DNA activity in an AAV-HBV mouse model. Furthermore, the compounds of the present invention exhibit good in vitro liver S9 stability, low immunogenicity risk, and low off-target risk. Preliminary safety studies have shown that the compounds of the present invention are highly safe.

[0044] Definition and Description

[0045] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. The absence of a specific definition for a particular term or phrase should not be construed as undefined or ambiguous, but rather should be understood as meaning understood by one of ordinary skill in the art. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0046] Unless otherwise stated, the terms "comprises," "includes," and "contains" or equivalents herein are open-ended expressions, meaning that in addition to the listed elements, components, or steps, other unspecified elements, components, or steps may also be included.

[0047] The terms "optional" or "optionally" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0048] Unless otherwise indicated, the term "nucleic acid" as used herein refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in single-stranded or double-stranded form, and includes DNA and RNA. A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are linked together by the phosphate group. A "base" includes purines and pyrimidines, which also include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs.

[0049] Unless otherwise indicated, the term "oligonucleotide" or "oligonucleotide" herein refers to a polymer or oligomer of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars, and intersugar (backbone) linkages, and also includes polymers or oligomers containing non-naturally occurring monomers or portions thereof, which function similarly.

[0050] Unless otherwise specified, the "oligonucleotide" or "oligonucleotide" of the present invention is a nucleotide sequence containing 10 to 50 nucleotides or nucleotide base pairs. In some embodiments of the present invention, the oligonucleotide has a nucleobase sequence that is at least partially complementary to a coding sequence in a target nucleic acid or target gene expressed in a cell. The nucleotides may be optionally modified. In some embodiments of the present invention, after the oligonucleotide is delivered to a cell expressing a gene, the oligonucleotide is able to inhibit or block the expression of the gene in vitro or in vivo. "Oligonucleotide" includes, but is not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and Dicer enzyme substrates.

[0051] Unless otherwise specified, "short interfering RNA (siRNA)" as used herein refers to a class of RNA molecules, 14-30 base pairs in length, similar to miRNA, that operate within the RNA interference (RNAi) pathway, interfering with the translation of mRNA of a specific gene with a complementary nucleotide sequence, leading to mRNA degradation. Short interfering RNA (siRNA) as used herein includes double-stranded siRNA (including both sense and antisense strands) and single-stranded siRNA (e.g., including only the antisense strand).

[0052] Unless otherwise specified, "inhibit" as used herein, when referring to the expression of a given gene, means that gene expression is reduced when the cell, cell population or tissue is treated with the oligonucleotides of the invention, compared to cells, cell populations or tissues that have not been so treated.

[0053] Unless otherwise indicated, the term "double-stranded siRNA analog" as used herein refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleotide strands that have a "sense" and "antisense" orientation relative to the target RNA. In the present invention, "complementary" has the meaning known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with bases on the other strand in a complementary manner. 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 includes a purine and a 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 to be complementary to each other, and the sequence of that strand can be inferred from the sequence of its complementary strand.

[0054] Unless otherwise indicated, the term "substantially complementary" in the present invention means that the corresponding positions of the two sequences can be completely complementary or there can be one or more mismatches. When mismatches exist, there are usually no more than 5, 4, 3, 2 or 1 mismatched base pairs.

[0055] Unless otherwise specified, a "sequence" or "nucleotide sequence" as used herein refers to an order or sequence of nucleobases or nucleotides described by a sequence of letters using standard nucleotide nomenclature.

[0056] Unless otherwise specified, the "antisense strand" (AS), template strand or "guide strand" of the present invention refers to the strand in the oligonucleotide compound that is substantially complementary to the corresponding region of the target sequence (eg, AGT mRNA).

[0057] Unless otherwise specified, the "sense strand", "sense strand" or "sense strand" (SS), coding strand or "passenger strand" described in the present invention refers to a strand that can form a substantially complementary region with the antisense strand. The "substantially complementary" means that the corresponding positions of the two sequences can be completely complementary, or there can be one or more mismatches. When there are mismatches, there are usually no more than 3, 2 or 1 mismatched base pairs. In a double-stranded nucleic acid molecule, the bases of one chain are paired with the bases on the other chain in a complementary manner. 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).

[0058] In the present invention, the nucleotide monomers "A", "U", "G" and "C" represent adenosine-3'-phosphate, uridine-3'-phosphate, guanosine-3'-phosphate and cytidine-3'-phosphate, respectively.

[0059] Unless otherwise indicated, the term "conjugation" in the present invention refers to the covalent linkage of two or more chemical moieties, each with a specific function, to each other; accordingly, "conjugate" refers to a compound formed by covalent linkage of the chemical moieties.

[0060] Unless otherwise indicated, the term "conjugate of double-stranded siRNA analog" of the present invention refers to a compound formed by linking a double-stranded siRNA analog and a pharmaceutically acceptable conjugating group, and the double-stranded siRNA analog and the pharmaceutically acceptable conjugating group are covalently linked.

[0061] Unless otherwise indicated, the "pharmaceutically acceptable conjugate groups" of the present invention facilitate the in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use. In some embodiments, the "pharmaceutically acceptable conjugate groups" of the present invention facilitate enhancing the affinity of nucleic acids and compositions suitable for in vivo therapeutic use for their target (target tissue / cell). Exemplary conjugate groups include, but are not limited to, L96, compound group D1, compound group D2, and compound group D3.

[0062] Unless otherwise indicated, the "modification" of the present invention refers to the modification of the bases, sugar rings, nucleotides and / or linkages between groups (such as R, E, etc.) of nucleotides or replacement nucleotides. Exemplarily, the "modification" of the present invention includes but is not limited to MOE modification, methoxy modification, fluoro modification, (E)-vinyl phosphate modification, thiophosphate linkage or replacement of nucleotides with GNA (glycerol nucleic acid), etc. Exemplarily, the "modification" of the present invention may include one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with a modified ribose moiety, wherein the ribose moiety includes an additional bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in a 3'-endo conformation.

[0063] Unless otherwise specified, the "nucleotides are optionally modified" described in the present invention means that each nucleotide can independently be an unmodified nucleotide or a modified nucleotide, and the modification on each modified nucleotide is also independent. The "modification" includes but is not limited to modifications to nucleobases, modifications to ribose, and modifications to phosphates. The "unmodified nucleotides" refer to nucleotides composed of naturally occurring nucleobases, natural sugar rings, and natural phosphates. The "modified nucleotides" refer to nucleotides containing at least one of modified nucleobases, modified sugar rings, and modified phosphates. In some embodiments of the present invention, "modified nucleotides" refer to nucleotides composed of modified nucleobases, and / or modified sugar rings, and / or modified phosphates. In some embodiments of the present invention, a "modified nucleotide" is composed of a modified nucleobase, a natural sugar ring, and a natural phosphate; in some embodiments of the present invention, a "modified nucleotide" is composed of a natural nucleobase, a modified sugar ring, and a natural phosphate; in some embodiments of the present invention, a "modified nucleotide" is composed of a natural nucleobase, a natural sugar ring, and a modified phosphate; in some embodiments of the present invention, a "modified nucleotide" is composed of a natural nucleobase, a modified sugar ring, and a modified phosphate; in some embodiments of the present invention, a "modified nucleotide" is composed of a modified nucleobase, a natural sugar ring, and a modified phosphate; in some embodiments of the present invention, a "modified nucleotide" is composed of a modified nucleobase, a modified sugar ring, and a natural phosphate; in some embodiments of the present invention, a "modified nucleotide" is composed of a modified nucleobase, a modified sugar ring, and a natural phosphate; in some embodiments of the present invention, a "modified nucleotide" is composed of a modified nucleobase, a modified sugar ring, and a modified phosphate. Unless otherwise specified, the "natural sugar ring" of the present invention is selected from a 2'-OH five-membered sugar ring and a 2'-deoxy five-membered sugar ring.

[0064] Unless otherwise specified, the "natural bases" of the present invention are selected from the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0065] Unless otherwise specified, the "modified nucleobase" of the present invention refers to a 5-12 membered saturated, partially unsaturated or aromatic heterocycle other than a natural base, including a monocyclic or condensed ring, specific examples of which include but are not limited to thiophene, thianthrene, furan, pyran, isobenzofuran, benzothiazine, pyrrole, imidazole, substituted or unsubstituted triazole, pyrazole, isothiazole, isoxazole, pyridazine, indolizine, indole, isoindole, isoquinoline, quinoline, naphthopyridine, quinazoline, carbazole, phenanthridine, piperidine, phenazine, phenazine, phenothiazine, furane, phenoxazine, pyrrolidine, pyrroline, imidazolidine, imidazoline, pyrazolidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 2-aminoadenine, 2-aminoguanine, 2 -propyl adenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (parauracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine, etc.

[0066] Unless otherwise specified, the "modified sugar ring" of the present invention may include, but is not limited to, one of the following modifications at the 2' position: H; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, wherein n and m are from 1 to 10. In other embodiments, the 2' position includes but is not limited to one of the following modifications: substituted or unsubstituted C1 to C 10Lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA, or group for improving the pharmacodynamic characteristics of iRNA, and other substituents with similar properties. In some embodiments, the modification includes but is not limited to 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as '-O-(2-methoxyethyl) or 2'-MOE).

[0067] Unless otherwise specified, the "modified phosphate" of the present invention includes but is not limited to: phosphorothioate modification, and the "phosphorothioate" includes (R)- and (S)-isomers and / or mixtures thereof.

[0068] In some embodiments of the present invention, the modified nucleotides may comprise one or more locked nucleic acids (LNAs). LNAs are nucleotides with a modified ribose moiety, wherein the ribose moiety contains an additional bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in a 3'-endo conformation.

[0069] In some embodiments of the present invention, the modified nucleotides include one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which any sugar bonds have been removed, thereby forming an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the bond between C1'-C4' has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., a covalent carbon-carbon bond between the C2' and C3' carbons) has been removed.

[0070] In some embodiments of the present invention, the modified nucleotide comprises one or more monomers of GNA (glycerol nucleic acid) nucleotides. GNA includes GNA-A, GNA-T, GNA-C, GNA-G and GNA-U. The structure of GNA-A is The structure of GNA-T is The structure of GNA-C is The structure of GNA-G is The structure of GNA-U is

[0071] In some embodiments of the present invention, the nucleotide of modification also can comprise one or more bicyclic sugar moieties." bicyclic sugar " is furanyl (furanosyl) ring modified by the bridge connection of two atoms." bicyclic nucleoside " (" BNA ") is the nucleoside with sugar moiety, and described sugar moiety comprises the bridge of two carbon atoms connecting sugar ring, forms bicyclic ring system thus.In specific embodiments, the 4 '-carbon and 2 '-carbon of bridge connection sugar ring.

[0072] Unless otherwise indicated, the "covalent linkage" described in the present invention includes but is not limited to "phosphate bond" (ie, "phosphodiester bond"), "phosphorothioate bond" and the like.

[0073] The "modification" of nucleotides in the present invention includes modification of nucleobases, modification of sugar rings, modification of internucleotide linkages, etc. Exemplarily, the "modification" of nucleotides in the present invention includes, but is not limited to, methoxy modification, fluorination modification, (E)-vinyl phosphate modification, phosphorothioate linkage, or replacement of nucleotides with (S)-glycerol nucleic acids, etc.

[0074] In the present invention, "m" before "R", "E", "A", "U", "G" and "C" represents that the nucleotide is modified with 2'-O-methyl, and "f" represents that the nucleotide is modified with 2'-fluoro nucleotide.

[0075] Unless otherwise specified, the "R", "E", "A", "U", "G" and "C" described in the present invention are connected by phosphate groups. For example, the chemical structure of 5'-fAmRmC-3' is as follows:

[0076] In the present invention, "s" between "R", "E", "A", "U", "G" and "C" represents that the nucleotides are connected through a phosphorothioate group.

[0077] In the present invention, "R", "E", "A", "U", "C" and "G" are marked with " MOE " represents that a nucleotide or a group replacing a nucleotide is modified with a 2'-O-methoxyethyl group.

[0078] In some embodiments of the present invention, the "m" before the "R", "E", "A", "U", "C" and "G" represents that the nucleotide or the group replacing the nucleotide is modified by 2'-O-methyl (belonging to methoxy modification), the "f" represents that the nucleotide or the group replacing the nucleotide is modified by 2'-fluoro nucleotide (belonging to fluorinated modification), and the " MOE" represents that the nucleotide or the group replacing the nucleotide is modified by 2'-O-methoxyethyl (belonging to MOE modification). In some embodiments of the present invention, between the "R", "E", "A", "U", "C" and "G", the "s" marked represents that the nucleotides are connected by thiophosphate groups. Exemplary, 5'-mRs MOE The chemical structure of GsmCmAfC-3' is as follows:

[0079] Unless otherwise specified, the term "multiple" and "multivalent" herein refer 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 number of GalNAc derivatives to which the siRNA analogs or branched linkers are attached.

[0080] The sense or antisense strands of the double-stranded siRNA analogs of the present invention may also include "overhangs," e.g., unpaired protruding nucleotides that are not directly involved in the RNA double helix structure, wherein the RNA double helix structure is typically formed by a "sense strand" and an "antisense strand" pair as defined herein. Exemplarily, such overhangs may include one or more modified or unmodified U, T, and A residues.

[0081] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are encompassed within the scope of the present invention.

[0082] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0083] Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.

[0084] Unless otherwise specified, use a solid wedge key. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond and straight dashed key Indicate the relative configuration of stereocenters with a wavy line Indicates a wedge-shaped solid key or dotted wedge key Or use a wavy line Indicates a straight solid bond and / or straight dashed key

[0085] Unless otherwise specified, when a group has one or more linkable sites, any one or more sites of the group can be linked to other groups via chemical bonds. The chemical bonds linking the sites to other groups can be represented by straight solid bonds. Straight dotted key or wavy lines express.

[0086] Unless otherwise indicated, the terms "enriched in one isomer", "isomerically enriched", "enriched in one enantiomer" or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100%, and the content of that 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%.

[0087] Unless otherwise indicated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.

[0088] Optically active (R)- and (S)-isomers as well as D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, 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 the 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 diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are split by conventional methods known in the art and then recovered to obtain the pure enantiomer. In addition, the separation of enantiomers and diastereoisomers is typically accomplished by using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate generated from an amine). The compounds of the present invention may contain non-natural ratios of atomic isotopes on one or more atoms constituting the compound. For example, the compound may be labeled with a radioactive isotope, such as tritium ( 3H), iodine-125( 125 I) or C-14( 14 C). For example, deuterated drugs can be formed by replacing hydrogen with heavy hydrogen. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and prolonged drug biological half-life. All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of this invention.

[0089] The term "salt" refers to salts of the compounds of the present invention, prepared by reacting the compounds of the present invention with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the compounds with a sufficient amount of base in a neat 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 the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the compounds with a sufficient amount of acid in a neat 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, bisulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as 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, and methanesulfonic acid; and salts of amino acids (such as arginine) and organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and can be converted into either base or acid addition salts.

[0090] The salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two.

[0091] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0092] The structures of the compounds of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compounds, the absolute configuration can be confirmed by conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data on the cultured single crystal using a Bruker D8 venture diffractometer, using CuKα radiation as the light source and scanning mode: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0093] The solvent used in the present invention is commercially available.

[0094] Unless otherwise specified, the solvent ratios used in the column chromatography and preparative thin-layer silica gel chromatography of the present invention are all volume ratios.

[0095] Table 4 List of abbreviations used in this invention

[0096] Compounds are named according to the conventional nomenclature in the art or using The software named the commercially available compounds using the supplier's catalog name. DETAILED DESCRIPTION

[0097] The present invention is described in detail below by examples, but it is not intended to limit the present invention in any way. The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement modes well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications will be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0098] Example 1 Synthesis of Compound 1

[0099] Step A: Compound 1-1 (30.00 g, 94.26 mmol) and compound A (11.98 g, 94.26 mmol) were dissolved in methyl acetate (220 mL) and concentrated to near dryness at 90°C under 1 bar pressure. A solution of trifluoromethanesulfonic acid (141.46 mg, 0.94 mmol) in methyl acetate (2 mL) was then added. After the addition, the reaction mixture was stirred at 125°C under 30 mbar pressure for 4 hours. The reaction mixture was cooled to 70°C, ethanol (70 mL) was added, and the mixture was stirred at 70°C until a homogeneous solution was formed. Stirring was then stopped and the mixture was cooled to 50°C. The mixture was allowed to stand and naturally cooled to 25°C. The reaction mixture was then allowed to stand at 0°C for 16 hours. Filter the mixture, and rinse the filter cake with 180 mL of ethanol (60 mL x 3). The filter cake was collected and dried under vacuum to yield compound 1-2. 1 H NMR (400MHz, CDCl3): δ=8.40 (s, 1H), 6.04 (d, J=3.42Hz, 1H), 5.81-5.69 (m, 1H), 5.54 (t , J=5.38Hz, 1H), 4.51-4.42(m, 2H), 4.30-4.16(m, 1H), 3.98(s, 3H), 2.18-2.05(m, 9H).

[0100] Step B: Compound 1-2 (15.00 g, 38.93 mmol) and triethylamine (4.14 g, 40.87 mmol) were dissolved in methanol (100 ml), and the mixture was stirred at 50°C under nitrogen for 17 hours. The reaction mixture was concentrated under reduced pressure to obtain compound 1-3. 1 H NMR (400MHz, CD3OD): δ=8.87 (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), 4.16-4.10 (m, 1H), 3.95 (s, 3H), 3.84 (dd, J=3.24, 12.29Hz, 1H), 3.70 (dd, J=4.46, 12.29Hz, 1H).

[0101] Step C: Dissolve compound 1-3 (10.00 g, 38.58 mmol) in pyridine (250 mL). Cool the mixture to 0°C and add 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (12.29 g, 38.97 mmol) dropwise. After addition, warm the reaction mixture to 25°C and continue stirring for 16 hours. Concentrate the reaction mixture under reduced pressure, and thoroughly resuspend the concentrate in ethyl acetate (250 mL). Filter the mixture, and wash the filtrate three times with 3 mol / L hydrochloric acid (250 mL x 3) and once with 250 mL of saturated brine. The organic phase is dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate is concentrated under reduced pressure. The residue is purified by silica gel column chromatography (eluent: v / v / v petroleum ether / dichloromethane / ethyl acetate = 3 / 1 / 1) to provide compound 1-4. 1 H NMR (400MHz, CDCl3): δ=8.43 (s, 1H), 5.95 (s, 1H), 4.73 (dd, J=4.75, 8.00Hz, 1H), 4.41 (d, J =4.75Hz, 1H), 4.19-4.09(m, 2H), 4.03-3.94(m, 4H), 3.34-2.71(m, 1H), 1.15-1.01(m, 28H).

[0102] Step D: Compound 1-4 (20.00 g, 39.86 mmol) was dissolved in anhydrous toluene (200 ml). Silver oxide (92.38 g, 398.63 mmol) and 2-iodoethyl methyl ether (22.24 g, 119.56 mmol) were added sequentially. The reaction mixture was stirred at 130°C for 24 hours. After cooling to room temperature, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain crude compound 1-5, which was used directly in the next reaction. LC-MS (ESI) m / z: 604.3 [M+H] + .

[0103] Step E: Dissolve compound 1-5 (20.00 g, 33.12 mmol) in anhydrous methanol (200 ml), then add triethylamine (3.38 g, 33.45 mmol). The reaction mixture is stirred at 25 degrees Celsius for 12 hours. After cooling to room temperature, it is concentrated under reduced pressure to obtain crude compound 1-6, which is used directly in the next reaction. LC-MS (ESI) m / z: 560.3 [M+H] + .

[0104] Step F: Compound 1-6 (18.50 g, 33.12 mmol) was dissolved in anhydrous tetrahydrofuran (185 ml) at 0°C, followed by the addition of triethylamine trihydrofluoride (11.72 g, 72.70 mmol). The reaction mixture was stirred at 25°C for 12 hours. After cooling to room temperature, it was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: v / v dichloromethane / methanol = 40 / 1 to 10 / 1) to obtain compound 1-7. 1 H NMR (400MHz, CD3OD) δ = 8.87 (s, 1H), 6.04 (d, J = 3.3Hz, 1H), 4.45-4.36 (m, 2H), 4. 15-4.09 (m, 1H), 3.96 (s, 3H), 3.87-3.67 (m, 4H), 3.59-3.54 (m, 2H), 3.34 (s, 3H).

[0105] Step G: Compound 1-7 (6.00 g, 18.91 mmol) was dissolved in anhydrous pyridine (20 mL) at 0°C, followed by the addition of DMTr-Cl (7.69 g, 22.69 mmol). The reaction mixture was allowed to warm naturally and stirred at 20-25°C for 12 hours. After concentration under reduced pressure, the residue was purified by silica gel column chromatography (eluent: v / v petroleum ether / ethyl acetate = 30 / 1 to 0 / 1, containing 0.2% triethylamine) to yield compound 1-8. 1 H NMR (400MHz, DMSO-d6) δ = 8.95 (s, 1H), 7.33 (d, J = 7.4Hz, 2H), 7.26-7.17 (m, 7H), 6.82 (t, J = 7.9Hz, 4H), 6.12 (d, J = 1.8Hz, 1H), 5.23 (d, J = 6.1Hz, 1H), 4 .43-4.29(m, 2H), 4.12-4.03(m, 1H), 3.83(s, 3H), 3.79-3.75(m, 1H), 3.72 (s, 6H), 3.67 (brs, 1H), 3.48-3.42 (m, 2H), 3.20 (s, 3H), 3.16-3.08 (m, 2H).

[0106] Step H: Compound 1-8 (1.10 g, 1.77 mmol) was dissolved in anhydrous dichloromethane (8 ml) at 0°C, followed by the addition of compound B (0.62 g, 2.61 mmol) and 4,5-dicyanoimidazole (0.10 g, 0.89 mmol). After the addition was complete, the reaction mixture was stirred at 20°C for 0.5 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: v / v petroleum ether / ethyl acetate = 50 / 1 to 1 / 2) to obtain compound 1. LCMS (ESI) m / z: 820.3 [M+H]+ .

[0107] Example 2 Synthesis of Compound 2

[0108] Step A: 1-4 (8.23 g, 16.40 mmol), potassium carbonate (11.34 g, 82.02 mmol), and silver oxide (19.01 g, 82.02 mmol) were added sequentially to N,N-dimethylformamide (50 mL) at room temperature, followed by iodomethane (11.64 g, 82.02 mmol). The reaction mixture was stirred at room temperature for 3 hours, then diluted with ethyl acetate (300 mL) and filtered. The filtrate was washed once with saturated aqueous sodium thiosulfate (250 mL), once with water (250 mL), and once with saturated brine (250 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 5 / 1, v / v) to provide 1-9. 1 H NMR (400MHz, CDCl3): δ=8.58 (s, 1H), 5.91 (s, 1H), 4.46 (dd, J=4.22, 9.35Hz, 1H), 4.28-4.17(m, 2H), 4.06-3.96(m, 5H), 3.68(s, 3H), 1.13-0.99(m, 28H).

[0109] Step B: At 0°C, 1-9 (3.27 g, 6.34 mmol) was added to tetrahydrofuran (50 mL) and stirred to dissolve. Triethylamine trihydrofluoride (2.25 g, 13.95 mmol) was then added dropwise. After the addition, the reaction mixture was allowed to warm to room temperature and stirred for 16 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: v / v dichloromethane / methanol = 20 / 1) to afford 1-10. 1 H NMR (400MHz, CD3OD): δ=8.88 (s, 1H), 6.04 (d, J=3.26Hz, 1H), 4.44 (t, J=5.33Hz, 1H), 4.20 (dd, J=3.33, 4.83Hz, 1 H), 4.14-4.07 (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).

[0110] Step C: At 0°C, 1-10 (1.30 g, 4.76 mmol) was added to anhydrous pyridine (20 mL). After stirring to dissolve, DMTr-Cl (2.42 g, 7.14 mmol) was added. After complete addition, the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with ethyl acetate (70 mL), followed by addition of saturated aqueous sodium bicarbonate (20 mL) and water (40 mL). After uniform stirring, the mixture was allowed to stand for separation. The organic phase was washed once with water (60 mL) and once with saturated brine (60 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (Phenomenex luna C18 column (size: 250 mm × 50 mm, particle size: 10 μm); mobile phase: phase A: 10 mM aqueous ammonium bicarbonate; phase B: acetonitrile; elution gradient: 35% to 65% over 20 minutes) to afford 1-11. 1 H NMR (400MHz, CDCl3): δ=8.44 (s, 1H), 7.45-7.38 (m, 2H), 7.34-7.28 (m, 5H), 7.27-7. 18(m, 2H), 6.92-6.70(m, 4H), 5.97(d, J=2.88Hz, 1H), 4.43-4.37(m, 1H), 4.33(dd, J =2.88, 5.00Hz, 1H), 4.25-4.19 (m, 1H), 3.98 (s, 3H), 3.80 (s, 6H), 3.58 (s, 3H), 3.49 -3.43 (m, 1H), 3.40-3.33 (m, 1H), 2.55 (d, J = 6.88Hz, 1H). LCMS (ESI) m / z: 574.2 [MH] - .

[0111] Step D: At 0°C, 1-11 (1.10 g, 1.91 mmol) was added to anhydrous dichloromethane (8 ml), followed by the addition of compound B (678.45 mg, 2.87 mmol) and 4,5-dicyanoimidazole (0.11 g, 0.96 mmol). After the addition was complete, the reaction mixture was stirred at room temperature under nitrogen for 0.5 hours. After concentration under reduced pressure, the residue was purified by silica gel column chromatography (eluent: v / v petroleum ether / ethyl acetate = 50 / 1 to 1 / 2) to obtain compound 2. LCMS (ESI) m / z: 776.3 [M+H] + .

[0112] Example 3: Synthesis of Compound 3

[0113] Step A: Dissolve 1-12 (10 g, 36.06 mmol) in anhydrous pyridine (200 mL) and add trimethylsilyl chloride (48.98 g, 450.81 mmol). The reaction mixture is stirred at 25°C for 2 hours, then isobutyric anhydride (71.32 g, 450.81 mmol) is added and stirring is continued at 25°C for 12 hours. The reaction mixture is cooled and 28% aqueous ammonia (109.37 g, 873.85 mmol) is slowly added dropwise at 0°C. After the addition is complete, stirring is continued at 0°C for 15 minutes. Filter, and the filtrate is concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: v / v dichloromethane / methanol = 1 / 0 to 30 / 1, containing 0.2% triethylamine) and preparative HPLC (chromatographic column: Kromasil Eternity XT 250×80 mm×10 μm; mobile phase: phase A: 0.05% ammonia solution; phase B: 5% to 35% acetonitrile; 20 minutes) to give 1-13. 1 H NMR (400MHz, DMSO-d6) δ = 7.97 (s, 1H), 5.47 (dd, J = 7.9, 10.3Hz, 1H), 5.13 (br s, 1H), 4.58 (t, J=2.1Hz, 1H), 4.334.21 (m, 1H), 3.55 (d, J=6.6Hz, 2H), 2.84-2.69 (m, 1H), 2.58-2.52 (m, 1H), 2.43-2.33 (m, 1H), 2.28 (br s, 1H), 2.07 (s, 1H), 1.11 (d, J=6.8Hz, 6H). LC-MS (ESI) m / z: 348.1[M+H] + .

[0114] Step B: Compound 1-13 (11.9 g, 34.26 mmol) was dissolved in anhydrous pyridine (120 mL) and DMTr-C1 (13.93 g, 41.11 mmol) was added at 0°C. The reaction mixture was stirred at 20°C for 12 hours. After filtration, the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (eluent: v / v dichloromethane / methanol = 1 / 0 to 30 / 1, containing 0.2% triethylamine) and preparative HPLC (column: Kromasil Eternity XT 250 × 80 mm × 10 μm; mobile phase: phase A: 0.05% ammonia solution; phase B: 35% to 65% acetonitrile; 21 minutes) to obtain compound 1-14. 1H NMR (400MHz, DMSO-d6) δ = 12.27-11.39 (m, 2H), 7.74 (s, 1H), 7.43-7.22 (m, 9 H), 6.90 (d, J=8.6Hz, 4H), 5.46 (t, J=8.8Hz, 1H), 5.11-4.89 (m, 2H), 4.56 (br s, 1H), 4.22 (br s, 1H), 3.74 (s, 6H), 3.22 (d, J = 6.4Hz, 2H), 2.74 (s, 1H), 2.63 (br s, 1H), 2.24-2.05 (m, 2H), 1.11 (d, J=6.8Hz, 6H). LC-MS (ESI) m / z: 650.3[M+H] + .

[0115] Step C: Compound 1-14 was dehydrated three times with anhydrous acetonitrile for later use. At 0°C under nitrogen, compound 1-14 (5.10 g, 7.85 mmol) was dissolved in anhydrous dichloromethane (50 mL), followed by the addition of compound B (3.55 g, 11.8 mmol, 3.74 mL) and 4,5-dicyanoimidazole (1.21 g, 10.2 mmol). After the addition, the reaction was stirred at 20°C for 2 hours, then diluted with dichloromethane (20 mL) and saturated aqueous sodium bicarbonate (20 mL) was added. The organic phase was separated by extraction, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1 to 1 / 1) to yield compound 3. 1 H NMR (400MHz, CD3CN) δ=7.55 (d, J=12.0Hz, 1H), 7.51-7.44 (m, 2H), 7.39-7. 19 (m, 7H), 6.91-6.80 (m, 4H), 5.43-5.37 (m, 1H), 5.05 (m, 1H), 4.68 (m, 1H), 4.53(m, 1H), 3.78(m, 1H), 3.76(s, 6H), 3.75-3.43(m, 3H), 3.37-3.26(m, 2 H), 2.87(m, 1H), 2.68-2.55(m, 2H), 2.54-2.25(m, 3H), 1.21-1.09(m, 18H). 31 P NMR (162MHz, CD3CN) δ = -147.46 (s, 1P), -146.79 (s, 1P).

[0116] Example 4: Synthesis of D01

[0117] Step A: Dissolve 11-dodecyne-1-ol (2-1, 25 g, 137.14 mmol) and triethylamine (16.65 g, 164.56 mmol) in dichloromethane (250 mL). 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 x 2) of dichloromethane. Wash the combined organic phases with 400 mL (200 mL x 2) of water and saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to yield 2-2.

[0118] Step B: 2-3 (20 g, 67.26 mmol) was dissolved in N,N-dimethylformamide (200 mL). Sodium hydride (w / w = 60%, 4.04 g, 100.89 mmol) was added at 0°C, followed by 2-2 (19.27 g, 73.99 mmol). The reaction mixture was stirred at 25°C for 16 hours, then quenched with water (1 L) and extracted with 1.6 L of dichloromethane (800 mL x 2). The combined organic phases were washed once with 800 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to provide 2-4. 1 H NMR (400MHz, DMSO-d6): δ=7.63-6.89 (m, 10H), 5.64-5.52 (m, 2H), 4.27-4.01 (m, 2H), 3.98- 3.77(m, 2H), 3.72-3.18(m, 4H), 2.23-2.14(m, 2H), 1.98-1.92(m, 1H), 1.54-1.23(m, 16H).

[0119] Step C: 2-4 (48 g, 103.98 mmol) was dissolved in methanol (870 ml), followed by the addition of methanolic hydrogen chloride solution (4 mol / L, 400 ml). The reaction mixture was stirred at 30 degrees Celsius for 2 hours, and then methanolic hydrogen chloride solution (4 mol / L, 350 ml) was added. Stirring was continued at 30 degrees Celsius for 16 hours. After the reaction solution was concentrated under reduced pressure, 200 ml of chloroform was added and the mixture was concentrated under reduced pressure until a white solid appeared. Toluene (130 ml) and petroleum ether (130 ml) were added to the residue and stirring was continued at 15 degrees Celsius for 16 hours. The mixture was then filtered through a Buchner funnel, the filter cake was collected, and vacuum dried to obtain a white solid. The white solid was dissolved in dichloromethane (50 ml), and an aqueous solution of sodium hydroxide (6.59 g, 164.66 mmol) (50 ml) was added. The mixture was stirred at 20 degrees Celsius for 1 hour, then diluted with water (500 ml) and extracted with 1 liter of dichloromethane (500 ml x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 2-5.

[0120] Step D: 2-5 (23 g, 80.58 mmol) and sodium hydroxide (322.31 mg, 8.06 mmol) were added to a mixture of dimethyl sulfoxide (70 mL) and water (6 mL), followed by the addition of tert-butyl acrylate (22.72 g, 177.28 mmol). The reaction mixture was stirred at 25°C under nitrogen for 16 hours, then diluted with water (500 mL) and extracted with 1 L of ethyl acetate (500 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: v / v / v petroleum ether / ethyl acetate / ethanol (containing 0.1% ammonia) = 36 / 3 / 1 to 16 / 3 / 1) to afford 2-6. 1 H NMR (400MHz, DMSO-d6): δ=3.60-3.54 (m, 4H), 3.32 (br s, 5H), 3.15 (s, 5H), 2.74-2.66 (m, 1H), 2.40 (t, J=6.0Hz, 4H), 2.18-2.11 (m, 2H), 1.58 -1.38(m,22H),1.34-1.23(m,12H).

[0121] Step E: To a solution of 2-6 (24.5 g, 45.22 mmol) in dichloromethane (250 mL) were added triethylamine (9.15 g, 90.45 mmol) and succinic anhydride (6.79 g, 67.83 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 solution, stirred, and allowed to stand for separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to provide 2-7. 1 H NMR (400MHz, CDCl3): δ=6.49-6.37 (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).

[0122] Step F: Dissolve 2-7 (27.4 g, 42.69 mmol) in formic acid (140 mL). Stir the solution under nitrogen at 20°C for 16 hours. After concentration under reduced pressure, add toluene (150 mL) to the residue and concentrate to dryness under reduced pressure. Add toluene (150 mL) again and concentrate to dryness under reduced pressure to afford 2-8. 1H NMR (400MHz, CDCl3): δ=9.79-9.22(m, 3H), 6.44-6.23(m, 1H), 3.88-3.43(m, 10H), 3.39-3.20(m, 2H) , 2.77-2.31(m, 8H), 2.15-2.06(m, 2H), 1.87(t, J=2.6Hz, 1H), 1.48-1.28(m, 6H), 1.26-1.12(m, 10H).

[0123] Step G: Dissolve 2-8 (22.6 g, 42.67 mmol), N,N-diisopropylethylamine (33.09 g, 256.03 mmol), and HATU (51.92 g, 136.55 mmol) in N,N-dimethylformamide (250 mL). Add tert-butyl N-(3-aminopropyl)carbamate (29.74 g, 170.69 mmol). The reaction mixture is stirred at 20°C for 16 hours. Dichloromethane (1 L) and hydrochloric acid (1 mol / L, 1 L) are then added. After stirring, allow to stand and separate. The organic phase is washed sequentially with water (1 L), saturated aqueous sodium bicarbonate (1 L), and saturated brine (1 L), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: V / V / V petroleum ether / ethyl acetate / ethanol = 40 / 3 / 1 to 10 / 3 / 1) to obtain 2-9. 1 H NMR (400MHz, CDCl3): δ=7.22-6.79 (m, 3H), 6.77-6.44 (m, 1H), 5.45-5.00 (m, 3H), 3.86- 3.73(m, 2H), 3.72-3.63(m, 4H), 3.62-3.45(m, 4H), 3.41-3.32(m, 2H), 3.32-3.20(m, 6H) , 3.19-3.03(m, 6H), 2.56-2.47(m, 4H), 2.47-2.39(m, 4H), 2.21-2.12(m, 2H), 1.95-1.9 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).

[0124] Step H: Dissolve 2-9 (15 g, 15.03 mmol) in dichloromethane (114 mL), then add trifluoroacetic acid (38 mL). The reaction mixture is stirred at 20°C for 16 hours. After concentration under reduced pressure, a mixture of toluene / acetonitrile (v / v = 3 / 1, 200 mL) is added and the mixture is concentrated under reduced pressure to dryness. Repeat the above concentration step three times to obtain 2-10.

[0125] Step I: 2-11 (22.15 g, 49.50 mmol), N,N-diisopropylethylamine (7.75 g, 60.00 mmol), HOAt (6.12 g, 45.00 mmol) and HATU (20.53 g, 54.00 mmol) were dissolved in N,N-dimethylformamide (90 mL), followed by the addition of a solution of 2-10 (15.6 g, 15.00 mmol) and N,N-diisopropylethylamine (21.32 g, 165.00 mmol) in N,N-dimethylformamide (120 mL). The mixture was stirred at 20°C for 16 h, followed by the addition of dichloromethane (1.2 L) and hydrochloric acid (1 mol / L, 1 L). After stirring and separating the liquids, the organic phase was washed sequentially with 1 L of water, 1 L of aqueous sodium bicarbonate solution, and 1 L of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: v / v / v dichloromethane / methanol = 100 / 1 to 10 / 1 to dichloromethane / ethanol = 1 / 1) to obtain 2-12. 1 H NMR (400MHz, DMSO-d6): δ=7.87-7.66 (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 (br t, J=7.1Hz, 6H), 1.99 (s, 9H), 1.89 (s, 9H), 1.81-1.74 (m, 9H), 1.54-1.39 (m, 22H), 1.32 (dd, J=4.5, 6.7Hz, 2H), 1.24 (s, 10H).

[0126] Step J: Dissolve 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 potassium permanganate (0.40 g, 2.52 mmol) in water (9 mL) dropwise to the mixture at 0°C. Stir the mixture at 0-15°C for 2 hours. The reaction is quenched with sodium bisulfite (1.27 g), hydrochloric acid (2 mol / L, 5 mL) and water (30 mL) are added, and the mixture is extracted with 120 mL of a mixture of chloroform / isopropanol (v / v = 3 / 1, 40 mL x 3). The combined organic phases are dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure. Add 30 mL of a mixture of toluene and acetonitrile to the residue, and the mixture is concentrated again under reduced pressure to dryness. The above concentration step was repeated 6 times to obtain 2-13. 1 H NMR (400MHz, CD3OD): δ=5.34 (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 (br t, J=6.4Hz, 2H), 3.22(q, J=6.4Hz, 12H), 2.51-2.40(m, 9H), 2.21(br t, 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).

[0127] Step K: To a stirred solution of 2-13 (1.00 g, 0.50 mmol) in N,N-dimethylformamide (10 mL) were added N,N-diisopropylethylamine (0.26 g, 1.99 mmol) and HATU (0.23 g, 0.60 mmol), followed by the addition of 2-14 (0.23 g, 0.55 mmol). The reaction mixture was stirred at 15°C for 16 hours, after which dichloromethane (50 mL) and water (50 mL) were added. After stirring, the mixture was separated. The organic phase was washed sequentially with saturated aqueous sodium bicarbonate (50 mL), water (50 mL), and saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: v / v dichloromethane / methanol = 20 / 1 to 10 / 1, containing 0.1% triethylamine) to provide 2-15. 1H NMR (400MHz, DMSO-d6): δ=7.90-7.82 (m, 6H), 7.78 (d, J=4.8Hz, 3H), 7.40-7.26 (m, 10H), 6.91 (dd, 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 (d, J=3.8 Hz, 1H), 4.23-4.14 (m, 1H), 4.12-4.02 (m, 9H), 3.92 (td, J=9.0, 11.0Hz, 3H), 3.78 (s, 6H), 3.77-3. 71(m, 3H), 3.66-3.51(m, 13H), 3.49-3.41(m, 4H), 3.11-3.01(m, 16H), 2.38-2.37(m, 1H), 2.32(br s, 9H), 2.14 (s, 9H), 2.08 (br t, J=6.9Hz, 7H), 2.04 (s, 9H), 1.93 (s, 9H), 1.82 (s, 9H), 1.57-1.46 (m, 22H), 1.31-1.26 (m, 12H).

[0128] Step L: To a solution of 2-15 (0.80 g, 0.33 mmol) in dichloromethane (8 mL) were added 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) in sequence. The mixture was stirred at 10°C for 16 hours, followed by the addition of dichloromethane (50 mL), water (30 mL), and saturated brine (30 mL). The organic phase was separated and washed with water (30 mL) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (separation column: Waters Xbridge C18 (specifications: 150 mm×50 mm, particle size: 10 μm); mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]; elution gradient: 27%-57%, 11 minutes) to give compound D01. 1H NMR (400MHz, DMSO-d6): δ=7.96-7.69 (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(t, 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).

[0129] Example 5: Synthesis of D02

[0130] Step A: Compound 3-1 (50 g, 271.27 mmol), trimethylamine hydrochloride (2.59 g, 27.13 mmol), and p-toluenesulfonyl chloride (77.98 g, 406.91 mmol) were added sequentially to dichloromethane (500 ml). Triethylamine (32.94 g, 325.53 mmol) was slowly added dropwise at 0°C. After the addition, the reaction mixture was allowed to warm to room temperature and stirred for 12 hours. The reaction mixture was diluted with dichloromethane (500 ml) and washed once with sodium hydroxide solution (1 mol / L, 500 ml), dilute hydrochloric acid (1 mol / L, 500 ml), water (500 ml), and saturated brine (500 ml). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 3-2. 1 H NMR (400MHz, DMSO-d6) δ = 7.77 (d, J = 8.2Hz, 2H), 7.48 (d, J = 8.2Hz, 2H), 5.79-5.77 (m, 1H), 5.04-4.90 (m, 2H), 3.99 (t, J=6.2Hz, 2H), 2.42 (s, 3H), 2.00 (q, J=6.75Hz, 2H), 1.59-1.47 (m, 2H), 1.35-1.13 (m, 14H).

[0131] Step B: To a solution of compound 3-2 (50 g, 147.71 mmol) in tetrahydrofuran (500 ml) was added potassium hydroxide (41.14 g, 738.54 mmol) and 2-3 (46.12 g, 155.09 mmol). The mixture was stirred at 80 degrees Celsius for 12 hours. After the reaction was complete, the reaction mixture was quenched by adding ice water (1000 ml), then washed twice with 2000 ml of methyl tert-butyl ether (1000 ml / time). The organic phase was washed once with water (1000 ml) and once with saturated brine (1000 ml), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain 3-3, which was used directly in the next step.

[0132] Step C: Compound 3-3 (65.53 g, 141.34 mmol) was dissolved in a mixed solution of methanol (230 ml) and water (77 ml), followed by the addition of concentrated hydrochloric acid (12 mol / L, 40 ml). The reaction mixture was stirred at 50 degrees Celsius for 12 hours and then concentrated under reduced pressure until about 10 ml of liquid remained. Dilute hydrochloric acid (2 mol / L, 500 ml), methyl tert-butyl ether (500 ml) and petroleum ether (500 ml) were added to the residue, stirred evenly, and allowed to stand for a long time before separating into three layers. The intermediate liquid phase was separated, and then sodium hydroxide solution (1 mol / L) was added to adjust the pH to >11, and then extracted twice with dichloromethane (500 ml x 2). The combined organic phase was washed once with saturated brine (200 ml), dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure. Acetonitrile (90 mL) was added to the residue, and the mixture was heated to 60°C and stirred to dissolve. The mixture was then slowly cooled to 0°C and stirred for 30 minutes to precipitate a solid. The filter cake was collected by low-temperature filtration and dried in vacuo to obtain compound 3-4. 1 H NMR (400MHz, DMSO-d6) δ = 5.80-5.76 (m, 1H), 5.07-4.83 (m, 2H), 4.34 (t, J = 5.44Hz, 2H), 3.21 (d, J = 5 .26Hz, 4H), 3.16 (s, 2H), 2.03-1.97 (m, 2H), 1.46 (t, J=6.36Hz, 2H), 1.32 (d, J=6.72Hz, 2H), 1.24 (br s, 12H).

[0133] Step D: To a solution of compound 3-4 (17.91 g, 62.31 mmol) in dimethyl sulfoxide (100 ml) were added sodium hydroxide (224.3 mg, 5.61 mmol) and water (10 ml), followed by the addition of tert-butyl acrylate (17.57 g, 137.08 mmol) under nitrogen. After the addition, the reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with 0.2% aqueous sodium chloride solution (250 ml) and extracted twice with 400 ml of dichloromethane (250 ml x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (eluent: v / v / v petroleum ether / ethyl acetate / ethanol = 80 / 3 / 1 to 32 / 3 / 1) to obtain 3-5. 1 H NMR (400MHz, CDCl3) δ = 5.83-5.77 (m, 1H), 5.10-4.83 (m, 2H), 3.64 (t, J = 6.42Hz, 5H), 3.40-3.24 (m, 8H), 2. 44(t, J=6.36Hz, 5H), 2.02(q, J=7.01Hz, 2H), 1.54-1.48(m, 2H), 1.43(s, 18H), 1.38-1.33(m, 2H), 1.25(br s, 12H).LC-MS(ESI)m / z: 544.4[M+H] + .

[0134] Step E: Compound 3C (10.47 g, 22.07 mmol) was dissolved in N,N-dimethylformamide (140 mL), followed by the addition of HATU (6.70 g, 17.65 mmol) and triethylamine (4.10 mL). The mixture was stirred at 35°C for 5 minutes, followed by the addition of compound 3-5 (8.00 g, 14.71 mmol). After the addition was complete, stirring was continued at 35°C for 12 hours. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (300 mL). The organic phase was washed twice with water (100 mL) and once with saturated brine (50 mL). After concentration under reduced pressure, the residue was purified by preparative HPLC (separation column: Agela Innoval ODS-2250 mm×100 mm×10 μm; mobile phase: phase A: 10 mM trifluoroacetic acid aqueous solution, phase B: acetonitrile; elution gradient: 90% to 100%, 20 minutes) to obtain compound 3-6. 1H NMR (400MHz, CDCl3) δ=9.08 (s, 1H), 8.34 (s, 2H), 8.29 (s, 1H), 7.73 (d, J=7.60Hz, 2H), 7.57 (d, J=7.60Hz, 2H), 7.36 (t, J=7. 20Hz, 2H), 7.28-7.24 (m, 1H), 6.76 (s, 1H), 5.94-5.93 (m, 1H), 5.86-5.76 (m, 1H), 5.01-4.92 (m, 2H), 4.38 (d, J=7.20Hz, 2H) , 4.19-4.16 (m, 1H), 3.91-3.83 (m, 6H), 3.75-3.72 (m, 4H), 3.63-3.59 (m, 2H), 3.46 (t, J=6.80Hz, 2H), 2.74-2.71 (m, 2H), 2. 51(t, J=6.00Hz, 4H), 2.04-2.01(m, 2H), 1.56-1.54(m, 2H), 1.42(s, 18H), 1.30-1.25(m, 16H). LC-MS (ESI) m / z: 1014.6[M+H] + .

[0135] Step F: Compound 3-6 (2.55 g, 2.55 mmol) was dissolved in N,N-dimethylformamide (50 mL), followed by the addition of HATU (1.07 g, 2.80 mmol) and triethylamine (0.35 mL). The reaction mixture was stirred at 25-35°C for 5 minutes, followed by the addition of compound 3D (0.81 g, 2.55 mmol) and triethylamine (0.35 mL). Stirring was continued for 3 hours after the addition was complete. The reaction mixture was diluted with water (50 mL) and then extracted with ethyl acetate (150 mL). The organic phase was washed once with water (50 mL) and once with saturated brine (50 mL). After concentration under reduced pressure, compound 3-7 was obtained. 1H NMR (400MHz, CDCl3) δ = 8.40 (s, 1H), 8.20 (s, 1H), 8.04 (s, 2H), 8.00 (s, 1H), 7.83 (s, 1H), 7.75-7.73 (m, 4H), 7.59-7.57 (m, 4H), 7.4 7(brs, 1H), 7.39-7.35(m, 4H), 7.26-7.24(m, 2H), 6.69(s, 1H), 5.83-5.79(m, 2H), 5.70(brs, 1H), 5.02-4.93(m, 2H), 4.40-4.36(m, 3H), 4.21-4.17(m, 2H), 3.90-3.88(m, 2H), 3.80-3.78(m, 4H), 3.73-3.70(m, 4H), 3.62-3.54(m, 3H), 3.47-3.40(m, 4H), 2.62(brs, 2H), 2.48(t, J=6.00Hz, 4H), 2.05-2.01(m, 2H), 1.55-1.52(m, 2H), 1.38(s, 18H), 1.30-1.26(m, 16H). LC-MS (ESI) m / z: 1265.0[M+H] + .

[0136] Step G: Compound 3-7 (9 g, 7.12 mmol) was dissolved in anhydrous dioxane (15 ml), and then a hydrochloric acid / dioxane solution (4 mol / L, 71.17 ml) was added. The reaction mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure to obtain compound 3-8. 1 H NMR (400MHz, DMSO-d6) δ = 10.22 (s, 1H), 8.56 (t, J = 5.3Hz, 1H), 8.24 (s, 1H), 8.0 6(s, 1H), 7.95-7.81(m, 5H), 7.69(d, J=7.4Hz, 4H), 7.47-7.24(m, 10H), 5.76(t dd, J=6.6, 10.3, 17.0Hz, 1H), 5.05-4.82 (m, 2H), 4.42-4.27 (m, 4H), 4.22 (d, J= 6.4Hz, 2H), 3.71 (s, 3H), 3.68-3.60 (m, 6H), 3.36 (dd, J=6.8, 13.4Hz, 6H), 3.22- 3.16 (m, 2H), 2.57-2.53 (m, 2H), 2.45 (t, J=6.3Hz, 3H), 2.10-1.86 (m, 2H), 1.58-1.01 (m, 18H). LC-MS (ESI) m / z: 1152.6 [M+H] + .

[0137] Step H: Compound 3-8 (9 g, 7.81 mmol) was dissolved in N,N-dimethylformamide (100 ml), followed by the addition of HATU (6.83 g, 17.96 mmol), N,N-diisopropylethylamine (1.01 g, 7.81 mmol, 1.36 ml) and tert-butyl (3-aminopropyl)carbamate (2.99 g, 17.18 mmol). The reaction mixture was stirred at 25 degrees Celsius for 4 hours and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: v / v petroleum ether / ethyl acetate = 30 / 1 to 0 / 1) to provide compound 3-9.

[0138] Step I: Compound 3-9 (15 g, 10.24 mmol) was dissolved in anhydrous dichloromethane (110 mL), followed by the addition of tetrahydropyrrole (1.82 g, 25.60 mmol, 2.14 mL). The reaction mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure. The residue was purified by preparative HPLC (Separation column: Waters Xbridge C18 (size: 250 mm × 100 mm, particle size: 10 μm); mobile phase: Phase A: 10 mM trifluoroacetic acid aqueous solution; Phase B: acetonitrile; elution gradient: 35% to 55% over 25 minutes) to obtain a colorless oil. This colorless oil (5 g) was dissolved in anhydrous dioxane (35 mL), and a hydrochloric acid / dioxane solution (4 mol / L, 38 mL) was added. The mixture was stirred at 25°C for 12 hours. The mixture was concentrated under reduced pressure to obtain compound 3-10. 1 H NMR (400MHz, DMSO-d6) δ = 10.66 (s, 1H), 9.04 (t, J = 5.1Hz, 1H), 8.35 (s, 1H), 8.24-8. 18(m, 3H), 7.42(s, 1H), 5.92-5.82(m, 1H), 5.04-4.94(m, 2H), 3.83-3.57(m, 13H), 3. 48-3.39 (m, 2H), 3.20-3.02 (m, 8H), 2.91-2.76 (m, 6H), 2.06 (q, J=6.8Hz, 2H), 1.73 ( q, J=6.9Hz, 4H), 1.58-1.47 (m, 2H), 1.45-1.18 (m, 16H). LC-MS (ESI) m / z: 821.0 [M+H] + .

[0139] Step J: Compound 3-10 (9.37 g, 20.94 mmol), N,N-diisopropylethylamine (2.71 g, 20.94 mmol), and HATU (9.27 g, 20.94 mmol) were added sequentially to N,N-dimethylformamide (50 mL). The reaction mixture was stirred at 10-15°C for 2 hours. Compound 2-11 (4.6 g, 4.76 mmol) and N,N-diisopropylethylamine (2.46 g, 19.04 mmol) were then slowly added to the reaction mixture. After the addition was complete, the reaction mixture was stirred at 10-15°C for 12 hours. The reaction mixture was slowly poured into water (500 mL) and extracted with dichloromethane (500 mL). The organic phase was washed once with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (chromatographic column: Welch Ultimate XB-CN, 250 mm×70 mm×10 μm; mobile phase: phase A: n-hexane; phase B: ethanol; 15 minutes) to obtain compound 3-11. 1 H NMR (400MHz, CDCl3) δ = 8.62 (br s, 1H), 8.20 (s, 1H), 8.07 (s, 1H), 7.96-7.88 (m, 2H), 7.82 (br d, J = 9.0Hz, 6H), 7.71 (t, J = 5.3Hz, 1H), 7.27 (br s, 1H), 5.22 (d, J=3.3Hz, 4H), 5.01-4.94 (m, 4H), 4.48 (br d, J=8.5Hz, 4H), 4.03 (s, 12H), 3.95-3.83 (m, 4H), 3.78-3.58 (m, 14H), 3.38-3.27 (m, 19H), 3.00 (d, J=5.6Hz, 7H), 2.38-2.27 (m, 4H) , 2.11 (s, 12H), 2.04 (d, J = 6.8Hz, 8H), 2.00 (s, 11H), 1.89 (s, 12H), 1.77 (d, J = 1.5Hz, 12H), 1.57-1.42 (m, 20H), 1.27-1.15 (m, 16H).

[0140] Step K: Compound 3-11 (2.0 g, 788.10 μmol) and ruthenium trichloride trihydrate (4.12 mg, 15.76 μmol) were added to a mixture of dichloromethane (6 mL), acetonitrile (6 mL), and water (9 mL) at room temperature. Sodium periodate (1.26 g, 5.9 mmol) and sodium bicarbonate (132.41 mg, 1.58 mmol) were then added. The reaction mixture was stirred at room temperature for 16 hours, then slowly poured into water (50 mL), and extracted with dichloromethane (50 mL). The organic phase was washed once with saturated sodium sulfite solution (20 mL), then once with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure to provide compound 3-12.

[0141] Step L: Compound 3-12 (1.3 g, 508.66 μmol) was dissolved in dichloromethane (15 mL), followed by the addition of HATU (232.09 mg, 610.40 μmol) and N,N-diisopropylethylamine (65.74 mg, 508.66 μmol). The reaction mixture was stirred at 10-15°C for 15 minutes, followed by the addition of compound 3E (234.73 mg, 559.53 μmol), and stirring was continued at 10-15°C for 1 hour. The reaction mixture was added to a 5% aqueous sodium bicarbonate solution (50 mL) and extracted with dichloromethane (50 mL). The organic phase was washed once with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (chromatographic column: Welch Ultimate XB-SiOH, 250 mm×70 mm×10 μm; mobile phase: phase A: n-hexane; phase B: ethanol; 15 minutes) to obtain compound 3-13. 1H NMR (400MHz, CDCl3) δ=8.69-8.57 (m, 1H), 8.20 (s, 1H), 8.07 (s, 1H), 7.91 (q, J=5.5Hz, 2H), 7.83 (d, J=9.2Hz, 6H), 7.72 (t, J=5 .3Hz, 2H), 7.37-7.26 (m, 5H), 7.22-7.15 (m, 4H), 6.92-6.83 (m, 4H), 5.21 (d, J=3.3Hz, 4H), 4.99-4.95 (m, 4H), 4.48 (d, J=8.4H z, 4H), 4.02 (s, 12H), 3.93-3.82 (m, 4H), 3.76-3.55 (m, 21H), 3.42-3.34 (m, 22H), 3.25-3.14 (m, 3H), 3.04-2.91 (m, 9H), 2.33- 2.27 (m, 4H), 2.10 (s, 12H), 2.05-1.99 (m, 21H), 1.89 (s, 12H), 1.77 (d, J=1.7Hz, 12H), 1.51-1.42 (m, 20H), 1.28-1.16 (m, 16H).

[0142] Step M: Compound 3-13 (1 g, 338.16 μmol), triethylamine (119.76 mg, 1.18 mmol), and 4-dimethylaminopyridine (20.66 mg, 169.08 μmol) were added sequentially to dichloromethane (10 mL), followed by succinic anhydride (101.52 mg, 1.01 mmol). The reaction mixture was stirred at room temperature for 48 hours. The reaction solution was diluted with water (20 mL) and extracted with dichloromethane (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Waters Xbridge C18, 150 mm × 50 mm × 10 μm; mobile phase: phase A: 10 mM aqueous ammonium bicarbonate; phase B: acetonitrile; 10 minutes) to afford compound D02. 1H NMR (400MHz, CD3CN) δ = 9.68 (br s, 1H), 8.29 (br s, 1H), 8.15 (d, J=2.5Hz, 2H), 7.87 (s, 1H), 7.43-7.19 (m, 13H), 7.15-6.97 (m, 7 H), 6.93-6.80 (m, 4H), 5.47-5.26 (m, 5H), 5.04 (dd, J=2.7, 11.2Hz, 4H), 4.62-4 .48(m, 4H), 4.25-3.92(m, 18H), 3.87-3.65(m, 22H), 3.54-3.34(m, 13H), 3.10- 3.01(m, 8H), 2.44-2.38(m, 6H), 2.32-2.28(m, 2H), 2.20-2.09(m, 22H), 2.00(br s, 12H), 1.97 (d, J=2.4Hz, 4H), 1.96-1.92 (m, 12H), 1.87 (s, 12H), 1.64-1.41 (m, 24H), 1.34-1.13 (m, 12H). LC-MS (ESI) m / z: 1529.3[M+2H] 2+ .

[0143] Preparation of intermediate 3C:

[0144] Compound 3B (42.97 g, 138.01 mmol) was dissolved in N,N-dimethylformamide (500 ml) and cooled to 0-10 degrees Celsius. HATU (57.72 g, 151.81 mmol) and N,N-diisopropylethylamine (21.40 g, 165.61 mmol) were then added. The reaction mixture was stirred at room temperature for 0.5 hours, followed by the addition of compound 3A (25.00 g, 138.01 mmol). After the addition was complete, stirring was continued at room temperature for 1 hour. The reaction mixture was poured into a dilute aqueous hydrochloric acid solution (23 ml of concentrated hydrochloric acid + 2.5 L of water) and stirred for 0.5 hours. After filtration, the filter cake was added to ethanol (650 ml) and stirred at 60 degrees Celsius for 1 hour. After cooling naturally to room temperature, the mixture was filtered under reduced pressure, the filter cake was collected, and dried under reduced pressure to obtain compound 3C. 1H NMR (400MHz, DMSO-d6) δ = 10.34 (s, 1H), 8.46 (s, 2H), 8.15 (s, 1H), 7.95 (s, 1H), 7.87 (d, J = 7.50Hz, 2H), 7.67 (br d, J=7.38Hz, 2H), 7.45 (t, J=5.50Hz, 1H), 7.39 (t, J=7.44Hz, 2H), 7.26-7.33 (m, 2H ), 4.26-4.30(m, 2H), 4.18-4.24(m, 1H), 4.04-4.04(m, 1H), 2.54(t, J=5.57Hz, 2H).

[0145] Example 6: Synthesis of D03

[0146] Step A: To a toluene solution (150 mL) of compound 4-2 (13.24 g, 50.93 mmol) at 25°C were added triethylamine (10.10 g, 99.86 mmol, 13.90 mL) and a toluene solution (100 mL) of compound 4-1 (12 g, 49.93 mmol, 11.76 mL). The reaction was stirred at 100°C for 16 hours. Saturated aqueous sodium carbonate (200 mL) was added to the reaction solution, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was separated by preparative HPLC (column: Agela Innoval ODS-2 250 mm x 100 mm x 10 μm; mobile phase: [water (0.225% formic acid)-acetonitrile]; acetonitrile percentage: 15%-35%, 25 min) to obtain compound 4-3.

[0147] Step B: A solution of compound 4-3 (12.6 g, 31.04 mmol), palladium on carbon (5 g, 10% content), and di-tert-butyl carbonic anhydride (14.90 g, 68.28 mmol, 15.69 ml) in methanol (120 ml) was purged with argon three times and then with hydrogen three times. The reaction mixture was stirred at 25°C under a hydrogen atmosphere at atmospheric pressure for 16 hours. The reaction mixture was filtered through celite and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to yield compound 4-4.

[0148] Step C: Compound 4-4 (7.5 g, 18.66 mmol) was dissolved in hydrochloric acid ethyl acetate solution (4 mol / L, 18.66 ml), replaced with nitrogen three times, and stirred at 25°C for 1 hour under nitrogen protection. The mixture was concentrated under reduced pressure to obtain compound 4-5.

[0149] Step D: A solution of compound 4-5 (2.24 g, 9.68 mmol), compound 4-6 (2.2 g, 5.69 mmol), triethylamine (2.88 g, 28.47 mmol, 3.96 mL), and 1-propylphosphonic anhydride (5.06 g, 7.95 mmol, 4.73 mL, 50% content) in N,N-dimethylformamide (25 mL) was purged with nitrogen three times and stirred at 25°C under nitrogen for 1 hour. Water (50 mL) was added to the reaction solution, which was then extracted with ethyl acetate (80 mL x 2). The combined organic phases were washed with saturated brine (100 ml * 1), dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by preparative HPLC (column: Phenomenex luna C18 150 * 40 mm * 15 μm; mobile phase: [water (0.225% formic acid) - acetonitrile]; acetonitrile%: 14%-44%, 15 minutes) to give compound 4-7.

[0150] Step E: A solution of compound 4-7 (2.2 g, 4.06 mmol), compound 4-8 (1.50 g, 6.09 mmol), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (2.22 g, 6.90 mmol), and triethylamine (1.23 g, 12.18 mmol, 1.69 ml) in N,N-dimethylformamide (20 ml) was purged with nitrogen three times and stirred at 25°C under nitrogen for 1 hour. Water (80 ml) was added to the reaction solution, which was then extracted with ethyl acetate (80 ml x 2). The combined organic phases were washed with saturated brine (100 ml x 1), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, gradient elution) to obtain compound 4-9.

[0151] Step F: To a mixed solution of compound 4-9 (2.5 g, 3.43 mmol) in methanol (22 ml) and water (7 ml) was added lithium hydroxide monohydrate (504.37 mg, 12.02 mmol). The reaction solution was stirred at 25°C for 12 hours. Water (50 ml) was added to the reaction solution. The aqueous phase was adjusted to pH 4-5 with 2M hydrochloric acid and extracted with ethyl acetate (100 ml * 2). The combined organic phases were washed with saturated brine (50 ml). After drying over anhydrous sodium sulfate, the filtrate was filtered and concentrated to obtain compound 4-10.

[0152] Step G: A solution of compound 4-10 (500 mg, 710.49 μmol), compound 4-11 (406.18 mg, 781.54 μmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (485.01 mg, 1.28 mmol), and triethylamine (215.68 mg, 2.13 mmol) in N,N-dimethylformamide (5 mL) was purged with nitrogen three times and stirred at 25°C under a nitrogen atmosphere for 1 hour. Water (80 mL) was added to the reaction mixture, followed by extraction with dichloromethane (80 mL x 2). The combined organic phase was washed with saturated brine (100 ml * 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. Preparative HPLC (column: Waters Xbridge C18 150 * 50 mm * 10 μm; mobile phase: [water (10 mM ammonium bicarbonate) - acetonitrile]; acetonitrile%: 57%-87%, 10 minutes) was used to obtain compound 4-12.

[0153] Step H: To a solution of compound 4-12 (320 mg, 203.93 μmol) in methanol (10 mL) was added palladium on carbon (100 mg, 10% content). The atmosphere was then replaced with argon three times and then with hydrogen three times. The reaction mixture was stirred at 25°C under a hydrogen atmosphere at atmospheric pressure for 16 hours. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to provide compound 4-13.

[0154] Step I: A solution of compound 4-13 (178 mg, 221.66 μmol), compound 4-14 (413.14 mg, 775.80 μmol), benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (336.25 mg, 886.63 μmol), and triethylamine (179.43 mg, 1.77 mmol) in N-dimethylformamide (4 mL) was purged with nitrogen three times. The reaction mixture was stirred at 25°C under a nitrogen atmosphere for 1 hour. Water (100 mL) was added to the reaction mixture and extracted twice with 50 mL of DCM / i-PrOH (3 / 1). The combined organic phases were washed with saturated brine (150 mL x 1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by preparative HPLC (column: Waters Xbridge C18150*50 mm*10 μm; mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]; acetonitrile%: 36%-66%, 10 minutes) to give compound 4-15.

[0155] Step J: A dichloromethane solution (5 mL) of compound 4-15 (240 mg, 97.49 μmol), compound 4-16 (34.15 mg, 341.21 μmol), 4-dimethylaminopyridine (11.91 mg, 97.49 μmol), and triethylamine (9.86 mg, 97.49 μmol) was purged with nitrogen three times and stirred at 25°C under nitrogen for 16 hours. The reaction mixture was concentrated and purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]; acetonitrile percentage: 19%-49%, 10 minutes) to afford compound D03. LC-MS (ESI) m / z: 1172 [M-2H] 2- . 1 H NMR (400MHz, DMSO-d6) δ = 7.65-8.20 (m, 11H) 7.28-7.39 (m, 4H) 7.17-7.28 (m, 5H) 6.78-7.0 3(m, 4H) 5.13-5.33 (m, 3H) 4.89-5.10 (m, 3H) 4.39-4.60 (m, 3H) 3.94-4.15 (m, 15H) 3.82-3. 92(m,5H)3.64-3.81(m,12H)2.83-3.19(m,16H)2.08-2.19(m,14H)1.96-2.07(m,19H)1.8 6-1.92 (m, 9H) 1.74-1.81 (m, 10H) 1.54-1.65 (m, 7H) 1.34-1.54 (m, 16H) 1.03-1.33 (m, 16H).

[0156] Example 7: Synthesis of double-stranded siRNA analogs or their conjugates

[0157] Synthesis of single-stranded oligoribonucleotides containing D1, D2 or D3: oligoribonucleotides were synthesized using phosphoramidite solid phase synthesis technology. ) and D01 or D02 or D03 by covalent bonding and synthesis was carried out on a solid support. All 2'-modified RNA phosphoramidites and auxiliary reagents were commercially available reagents. All amides were dissolved in anhydrous acetonitrile and molecular sieves were added. Coupling time was 5 minutes using 5-ethylthio-1H-tetrazole (ETT) as an activator. Alternatively, phosphorothioate bonds were generated using a 50 mM solution of 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile / pyridine (v / v = 1 / 1) for 3 minutes. All sequences were synthesized after the final removal of the DMTr group.

[0158] Synthesis of single-stranded oligoribonucleotides without D1, D2 or D3: Based on the sequence design, oligoribonucleotides were synthesized using phosphoramidite solid phase synthesis technology. All 2'-modified RNA phosphoramidites and auxiliary reagents are commercially available reagents. Modified R and E are obtained by Examples 1, 2 and 3. All phosphoramidites are dissolved in anhydrous acetonitrile and molecular sieves are added. Coupling time was 5 minutes using 5-ethylthio-1H-tetrazole (ETT) as an activator. Alternatively, phosphorothioate bonds were generated using a 50 mM solution of 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile / pyridine (v / v = 1 / 1) for 3 minutes. All sequences were synthesized after the final removal of the DMTr group.

[0159] Cleavage and Deprotection of CPG-Bound Oligomers: After solid-phase synthesis, the protecting groups were removed by treatment with 20% diethylamine in acetonitrile for 30 minutes, without cleaving the oligonucleotide from the CPG. Subsequently, the dried CPG was treated with concentrated aqueous ammonia at 40 degrees Celsius for 18 hours. After centrifugation, the supernatant was transferred to a new tube and the CPG was washed with aqueous ammonia. The combined solution was concentrated to yield a solid mixture.

[0160] Purification of single-stranded oligoribonucleotides: Oligomers were purified by HPLC using a NanoQ anion exchange column. Buffer A was 10 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4, and 20% acetonitrile, and buffer B was 500 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4, and 20% acetonitrile. The desired product was isolated and desalted using a reversed-phase C18 column.

[0161] Annealing of single-stranded oligoribonucleotides to produce double-stranded siRNA: The single-stranded oligoribonucleotides to be annealed are prepared to 200 μM using sterile RNase Free H2O (no RNA hydrolase). Complementary chains are formed by combining equimolar single-stranded oligoribonucleotide solutions. The annealing reaction system is set up as follows: a total volume of 100 μL of the mixture, 10 nmol, is placed in a 95°C water bath for 10 minutes (amounts ≥100 nmol require high temperature for 20 minutes), then quickly placed in a 60°C water bath, and annealing is completed after natural cooling. The purity of the obtained double-stranded siRNA is detected by HPLC, and the molecular weight of the sense and antisense chains is detected by HRMS to confirm the molecular weight of the double-stranded siRNA and identify it as the target product.

[0162] Experimental Example 1: Evaluation of the inhibitory activity of the test compound against HBV using HepG2.2.15 cells

[0163] 1. Experimental purpose:

[0164] The purpose of this study was to evaluate the inhibitory activity of test compounds against HBV in HepG2.2.15 cells.

[0165] 2. Experimental Materials:

[0166] 1) Cells and main reagents

[0167] HepG2.2.15 cells were provided by Shanghai WuXi AppTec Co., Ltd.

[0168] Transfection reagent: Lipofectamine RNAiMAX (Invitrogen-13778-150).

[0169] Hepatitis B virus surface antigen quantitative detection kit (Antu Bio-CL-0310).

[0170] Hepatitis B virus e antigen quantitative detection kit (Antu Bio-CL-0312).

[0171] Fluorescent probe FastStart Universal Probe Master (Roche, 04914058001).

[0172] 2) Main instruments

[0173] 79001 Real Time PCR system (Applied Biosystems), Synergy 2 (BioTek) and cell counter (Vi-cell™ XR).

[0174] 3. Experimental steps and methods:

[0175] siRNA was transfected into HepG2.2.15 cells as follows:

[0176] On day 0, siRNA was serially diluted into 8 concentrations using PBS. HepG2.2.15 cells were harvested, washed with DPBS, and digested with trypsin. The cells were adjusted to an appropriate cell density and transfected with siRNA using Lipofectamine RNAiMax. 15,000 cells were seeded into 96-well plates in 150 μL of culture medium per well. The cells were incubated in a 5% CO2, 37°C incubator for 3 days. Eight 3-fold dilutions of the test siRNA were prepared, with duplicate wells.

[0177] On the third day, fresh culture medium was replaced and culture was continued for another 3 days.

[0178] On day 6, the cell culture supernatant was collected and tested for HBsAg and HBeAg according to the instructions of the hepatitis B virus surface antigen and e antigen quantitative detection kit, and HBV DNA was detected by qPCR.

[0179] The steps for detecting HBsAg or HBeAg are briefly described as follows: take 50 μL of sample and standard and add them to the reaction plate respectively, then add 50 μL of enzyme conjugate to each well, shake and mix, incubate at 37°C for 60 minutes, then wash the plate 5 times with washing solution, then add 50 μL of luminescent substrate to each well, mix, react in the dark at room temperature for 10 minutes, and finally detect the chemiluminescence intensity with an enzyme reader.

[0180] The steps for HBV DNA detection are briefly described as follows: qPCR reaction system was set up and either the supernatant sample or a plasmid containing a full-length HBV standard was added. The PCR reaction program was: 95°C for 10 minutes, followed by cycling mode: 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles. The HBV DNA content in each sample was calculated based on the Ct value.

[0181] Data Analysis:

[0182] HBsAg or HBeAg or HBV DNA inhibition percentage = (1-sample HBsAg or HBeAg or HBV DNA content / RNAiMAX transfection reagent control HBsAg or HBeAg or HBV DNA average) × 100%

[0183] GraphPad Prism software was used to calculate the 50% inhibitory concentration (EC 50 )value.

[0184] 4. Experimental results: see Table 5.

[0185] Table 5 EC values ​​of compounds for reducing HBsAg, HBeAg and HBV DNA in Hep2.2.15 cells 50 result

[0186] Experimental conclusion: The compound of the present invention has the biological activity of significantly reducing HBV HBsAg, HBeAg and HBV DNA in HepG2.2.15 cells.

[0187] Experimental Example 2: Evaluation of the inhibitory activity of the test compound against HBV using primary human hepatocytes (PHH)

[0188] 1. Experimental purpose:

[0189] The HBV DNA content in the supernatant of human primary hepatocytes (PHH) was detected by real-time quantitative qPCR, the hepatitis B surface antigen and e antigen content were detected by ELISA, and the total RNA content was detected. 50 The value is used as an indicator to evaluate the inhibitory effect of the compound on HBV.

[0190] 2. Experimental Materials:

[0191] 1) Cells and reagents:

[0192] Frozen primary human hepatocytes (PHH)

[0193] Hepatitis B virus surface antigen quantitative detection kit (Antu Bio-CL-0310).

[0194] Hepatitis B virus e antigen quantitative detection kit (Antu Bio-CL-0312).

[0195] RNA extraction kit (Qiagen).

[0196] FastKing cDNA First Strand Synthesis Kit (TianGen).

[0197] Fluorescent probe FastStart Universal Probe Master (Roche, 04914058001).

[0198] 2) Main instruments:

[0199] 79001 Time PCR system (Applied Biosystems), Synergy 2 (BioTek) and cell counter (Vi-cell™ XR).

[0200] 3. Experimental steps and methods:

[0201] On day 0, the compound was serially diluted with PBS to 7 concentrations and added to the cell plate (22 μL / well). Frozen PHH were revived and the density was adjusted to 6.67×10 5 Cells / mL were seeded into 48-well plates (198 μL / well). The total volume per well was 220 μL. After mixing, the cells were placed in an incubator and cultured overnight at 37°C, 5% CO2. Each compound was tested at seven 3-fold dilutions in duplicate.

[0202] On day 1, the culture medium in the culture plate was aspirated and type D HBV (concentrated from the culture supernatant of HepG2.2.15 cells) was added to infect PHH (100 GE / cell). The final concentration of DMSO in the culture medium was 2%.

[0203] On days 2, 4, and 6, fresh culture medium (without compound) was replaced.

[0204] On day 8, the cell culture supernatant was collected and assayed for HBsAg and HBeAg according to the instructions of the hepatitis B virus surface antigen and e antigen quantitative detection kits, and HBV DNA was assayed by qPCR. Finally, cells were collected for HBV RNA testing.

[0205] The steps for detecting HBsAg or HBeAg are briefly described as follows: take 50 μL of sample and standard and add them to the reaction plate respectively, then add 50 μL of enzyme conjugate to each well, shake to mix, incubate at 37°C for 60 minutes, then wash the reaction plate 5 times with washing solution, then add 50 μL of luminescent substrate to each well, mix, react in the dark at room temperature for 10 minutes, and finally detect the chemiluminescence intensity with a microplate reader.

[0206] The steps for HBV DNA detection are briefly described as follows: Prepare a qPCR reaction system and add either the supernatant sample or a full-length HBV plasmid standard. The PCR reaction program is as follows: heat at 95°C for 30 seconds; then denature at 95°C for 5 seconds and extend at 60°C for 34 seconds for 40 cycles. Finally, perform a final cycle at 95°C for 15 seconds and 60°C for 1 minute. Calculate the HBV DNA content in each sample based on the Ct value.

[0207] The steps for HBV RNA detection are briefly described as follows: harvest cells, extract RNA according to the Qiagen-74182 RNA Extraction Kit instructions, and reverse transcribe RNA into cDNA according to the FastKing cDNA First-Strand Synthesis Kit instructions. Quantitative PCR is used to detect the target gene cDNA, using GAPDH as an internal reference gene.

[0208] The target gene RNA expression level in each sample was calculated based on the Ct value of each sample using the ΔΔCt relative quantification method. The relative expression of the target gene was calculated using 2 -ΔΔCT express.

[0209] The calculation formula is as follows:

[0210] ΔCT = average Ct value of target gene - average Ct value of reference gene;

[0211] ΔΔCT = ΔCT (drug-addition group) - ΔCT (control group); relative expression of target gene mRNA = 2 -ΔΔCT

[0212] 4. Data Analysis:

[0213] HBsAg or HBeAg or HBV DNA inhibition percentage = (1-sample HBsAg or HBeAg or HBV DNA content / average HBsAg or HBeAg or HBV DNA in the culture medium control group) × 100%

[0214] GraphPad Prism software was used to calculate the 50% inhibitory concentration (EC) of the compounds for HBsAg, HBeAg or HBV DNA. 50 ).

[0215] 5. Experimental results: see Table 6.

[0216] Table 6 EC values ​​of compounds for reducing HBsAg, HBeAg, HBV DNA and Total RNA in PHH cells 50 result

[0217] Experimental conclusion: The compounds of the present invention have the biological activity of significantly reducing HBV HBsAg, HBeAg, HBV DNA and RNA in PHH.

[0218] Experimental Example 3. Evaluation of the Competitive Efficiency of Non-Fluorescently Labeled siRNA with GalNac3-Cy5 in PHH

[0219] 1. Research Objectives

[0220] The purpose of this study was to evaluate the competitive efficiency of non-fluorescently labeled test products with GalNac3-Cy5 in PHH using flow cytometry.

[0221] 2. Control compound GalNac3 and competitive compound:

[0222] The control compound was GalNac3 (EE(ahGalNAc)3 in Komilova AY, Algayer B, Breslin M, Uebele V. Anal Biochem. 2012; 425(1): 43-46), and the competitive compound was GalNac3 labeled with Cy5 fluorescence.

[0223] 3. Main Reagents and Cells

[0224] Table 7

[0225] 4. Experimental Plan

[0226] On day 0, the revived PHH suspension was adjusted to an appropriate density and the cells were seeded into 48-well plates.

[0227] On the first day, pre-mixed diluted test products and GalNac3 labeled with Cy5 fluorescence (final DMSO concentration is 2%) were added, starting at 20 μM of the test products, diluted 3-fold, 11 concentration points, single well.

[0228] After incubating the test products with the cells for 4 hours, the cells were digested and fixed and the corresponding fluorescence intensity of PHH was detected by flow cytometry.

[0229] The control compound is unlabeled GalNac3, and both the test compound and the control compound have 11 concentration points. The test is set up in a single well. The competitive compound is GalNac3 labeled with Cy5 fluorescence, and its final concentration is 0.05 μM.

[0230] Graphpad Prism software was used to analyze the EC (four parameter logistic equations). 50 value.

[0231] Table 8 EC of the competition efficiency of compounds with GalNac3-Cy5 in PHH 50 result

[0232] Experimental conclusion: The compound of the present invention has a good competitive efficiency relative to GalNac3-Cy5 in PHH.

[0233] Experimental Example 4. Study on Anti-HBV Activity and Safety in a Recombinant Adeno-Associated Virus Type 8 Vector-Mediated Hepatitis B Virus Mouse Model (AAV-HBV)

[0234] 1. Experimental purpose:

[0235] The AAV vector-mediated HBV transfection mouse model is a rapid and efficient HBV model. Leveraging the high hepatotropism of the AAV8 vector, a recombinant adeno-associated virus type 8 (rAAV8-1.3HBV) carrying 1.3 copies of the HBV genome can be efficiently introduced into hepatocytes using tail vein injection of the AAV8 vector. Due to the properties of AAV viral vectors, the vectors they mediate can be expressed for extended periods of time. The AAV / HBV model allows for sustained HBV DNA replication and expression of HBsAg and HBeAg in the mouse liver.

[0236] By using the AAV-HBV mouse model, HBsAg, HBeAg, DNA in the serum of mice and the weight of mice after treatment with the test compound were detected to evaluate its anti-HBV effect and safety in vivo.

[0237] 2. Experimental Materials:

[0238] C57BL / 6 mice, PBS (RNase free) as solvent, test compound, recombinant virus rAAV8-1.3HBV. The main reagents of this project include QIAamp96 DNA kit (Qiagen, 51162), FastStart Universal Probe Master (Rox) (Roche, 04914058001), Hepatitis B virus surface antigen detection kit (Antu Biotechnology, CL0310), Hepatitis B virus e antigen detection kit (Antu Biotechnology, CL0918), PureLink TM Pro 96 Viral RNA / DNA kit (Invitrogen, 12280-096A) and FastQuant RT Kit (with gDNase) (TIANGEN, KR106-02) were used. Key instruments included a centrifuge (Beckman Allegra X-15R), a multifunctional microplate reader (BioTek Synergy 2), a fluorescence quantitative PCR instrument (Applied Biosystems, 7900HT Fast Real-time PCR system), and a microplate reader (Molecular Devices, SpectraMax 340PC384).

[0239] 3. Experimental methods:

[0240] a) Mice were administered subcutaneously starting on day 34 after virus injection at a dose of 3 or 30 mg / kg, which was designated as day 0. Before administration, all mice were bled submandibular to collect plasma.

[0241] b) Blood was collected from the submandibular vein of mice on days 0, 14, and 21 after administration, and plasma was collected. The collected blood samples were anticoagulated with K2-EDTA and centrifuged at 4°C, 7000 g / min for 10 minutes.

[0242] c) All mice were bled through the submandibular vein to collect plasma, and then the mice were euthanized by CO2 inhalation, and plasma samples were collected through cardiac bleeding, and liver samples were collected.

[0243] d) Send the sample for testing.

[0244] 4. Sample analysis:

[0245] ELISA was used to detect the levels of HBsAg and HBeAg in mouse serum: the experimental steps were based on the instructions of the HBsAg ELISA (Antu Biotech, CL 0310) and HBeAg ELISA (Antu Biotech, CL0918) kits.

[0246] Detection of HBV DNA content in mouse plasma by qPCR: HBV DNA was extracted from plasma according to the QIAamp 96 DNA Blood Kit instructions, and HBV DNA content in mouse plasma was detected by qPCR.

[0247] The data of each group of mice are expressed as mean ± standard error, unless otherwise specified, n = 5. Statistical analysis was performed using Student's t-test.

[0248] 5. Experimental results:

[0249] a) Serum HBsAg Content: The anti-HBV activity of the test compounds in the AAV-HBV mouse model was evaluated. Plasma HBsAg levels in mice were measured by ELISA. The results are shown in Table 9.

[0250] Table 9 Percentage reduction of HBsAg on days 7, 14 and 21 after administration to mice relative to day 0

[0251] b) Serum HBeAg Content: The anti-HBV activity of the test compounds in the AAV-HBV mouse model was evaluated. Mouse plasma HBeAg levels were measured by ELISA. The results are shown in Table 10.

[0252] Table 10 The percentage reduction of HBeAg on days 7, 14 and 21 after administration to mice relative to day 0

[0253] c) Serum DNA Content: The anti-HBV activity of the test compounds in the AAV-HBV mouse model was evaluated. HBV DNA levels in mouse plasma were measured by quantitative PCR. The results are shown in Table 11.

[0254] Table 11 Percentage reduction of HBV DNA on days 7, 14, and 21 after administration to mice relative to day 0

[0255] d) Body weight change. Day 0 body weight was used as the baseline for comparison. According to IACUC guidelines, a 20% body weight loss was considered the humane endpoint. Any mouse with a body weight loss exceeding 20% ​​was removed from the experiment. No mice were removed due to weight loss in this experiment.

[0256] Experimental conclusion:

[0257] In this study, the test compound significantly reduced HBsAg, HBeAg, and HBV DNA in an AAV-HBV mouse model. The mice showed good tolerance during the experiment.

[0258] Experimental Example 5: Evaluation of the Immunogenicity of Compounds Using hPBMCs

[0259] Objective: To evaluate the immunogenicity of the test compound using hPBMC

[0260] Experimental materials and instruments:

[0261] hPMBC cells: freshly isolated peripheral blood mononuclear cells (PBMCs) purchased from Shanghai Saili Biotechnology Co., Ltd.

[0262] Compound:

[0263] Table 12

[0264] Reagents:

[0265] Table 13

[0266] Experimental instruments:

[0267] Table 14

[0268] Experimental steps:

[0269] Day 1: hPBMC cells cultured at high density overnight

[0270] Prepare RPMI-1640 cell culture medium containing 10% FBS, 10% FBS, and 1% Penicillin-streptomycin solution in RPMI Medium 1640 media (1×) according to the following ratios;

[0271] Centrifuge the two newly purchased batches of hPBMCs together with 50 mL centrifuge tubes at 400 × g and 25°C for 5 minutes;

[0272] The supernatant was discarded, and hPBMCs were resuspended in 10 mL of culture medium and counted;

[0273] Adjust the cell density to 10 7 / mL and transferred to 25cm 2 Cell culture flasks were cultured in a 5% CO2, 37°C incubator overnight;

[0274] Day 2: hPBMC plating and compound treatment

[0275] Compound dilution

[0276] The test compound was diluted 3-fold starting at 15 μg / mL to four concentration points in a 96-well V-bottom plate to prepare a 10× serial dilution of the compound according to the following dilution scheme. The reference compound GS9688 was diluted 20 mM starting at 0.5 μM to prepare a 10× serial dilution of the compound according to the following dilution scheme in a 96-well V-bottom plate. The reference compound LPS_TLR4 activator, 25 mg / tube, was diluted to a 20 mg / mL stock solution by adding 1.25 mL of endotoxin-free physiological water. Starting at 1 μg / mL, the compound was diluted 4-fold to prepare two concentration points in a 96-well V-bottom plate to prepare a 10× serial dilution of the compound according to the following dilution scheme.

[0277] Collect hPBMC cultured overnight the previous day into a 50 mL centrifuge tube, centrifuge at 400 × g for 5 minutes, remove the supernatant, and add 15 mL of RPMI-1640 medium containing 10% FBS to the cell pellet to resuspend the cells;

[0278] Count the cells using a cell counter and adjust the cell density of the resuspended cells to 2.2 × 10 6 cells / mL;

[0279] Transfer 10 μL of the compound in step 1 to a 96-well cell plate, and then add 90 μL of the cell suspension adjusted in step 3 to each well. The total volume of each well is 100 μL, and the number of cells is 2.0 × 10 6 indivual;

[0280] The cell plate was placed in a 5% CO2, 37°C constant temperature incubator and cultured for 1 day.

[0281] Day 3: hPBMC cytokine detection

[0282] The Human Procarta Plex Mix & Match 4-plex Kit was used to detect the levels of IFN alpha, IFN beta, IL-6, and TNF alpha in the hPBMC supernatant. The specific procedures were as follows, referring to the kit instructions:

[0283] 1. Label the cytokine assay plate according to the hPBMC plating map;

[0284] 2. Vortex the Magnetic Beads for 1 minute and dispense 50 μL of Magnetic Beads into each well of the assay plate using a dispenser.

[0285] 3. Place the test plate on a magnetic plate and let it sit for 2 minutes to collect the magnetic beads. Pour off the liquid and add 150 μL of washing solution. Let it sit for 30 seconds and then pour off the liquid to wash the magnetic beads in the test plate. Repeat the washing process twice.

[0286] 4. Add 50 μL of standard and sample to each well of the test plate;

[0287] 5. Seal the test plate with the sealing film provided in the kit, cover with a black microplate cover, and incubate on a shaker at 500 rpm at 4°C overnight.

[0288] Day 4: hPBMC cytokine assay plate reading

[0289] Transfer the assay plate that was incubated overnight to room temperature and continue shaking at 500 rpm for 30 minutes. Wash the plate using a magnetic plate as before. Protect from light throughout the following steps:

[0290] Add 25 μL of Detection Antibody Mixture to each well of the test plate, apply sealing film, cover with a black microplate cover, and incubate at room temperature at 500 rpm on a shaker for 30 minutes;

[0291] Repeat the previous washing steps;

[0292] Add 50 μL of SAPE solution to each well, apply sealing film, cover with a black microplate lid, and incubate at room temperature at 500 rpm for 30 min;

[0293] Repeat the previous washing steps;

[0294] Add 120 μL of Reading Buffer to each well, apply the sealing film, cover the black test plate, and incubate at room temperature at 500 rpm on a shaker for 5 min;

[0295] Remove the sealing film on the test plate and place the test plate on the Luminex instrument for reading;

[0296] Luminex experiments were set up and concentration data were determined using the Bio-plex™ 200 system Bio-plex Manager version 5.0 software. A 5-parameter (5-PL) regression model was used to fit the standard curve of the target factor (Luminex automatic fitting). Fluorescence values ​​measured by the instrument were used to analyze the target factor content in the sample.

[0297] Result analysis:

[0298] Luminex was used to detect the levels of IL-6, TNF-α, IFN-α, and IFN-beta in the supernatant of hPBMCs treated with compounds to evaluate the immunogenicity of the compounds to hPBMCs.

[0299] After donor 1 and donor 2 hPBMCs were treated with different doses of the test compound, the levels of IFN-α, IFN-beta, IL-6 and TNF-α in the cell supernatant were detected. After donor 3 hPBMCs were treated with different doses of the test compound, the levels of IL-6 and TNF-α in the cell supernatant were detected.

[0300] Experimental results:

[0301] Table 15 IFN-α, IFN-beta, IL-6 and TNF-α contents in the cell supernatant after treatment of donor 1 hPBMC with different doses of test compounds

[0302] *Data for the PBS group: INF-alpha: 0.81 pg / mL; INF-beta: 4.48 pg / mL; IL-6: 2270.91 pg / mL; TNF-alpha: 33.24 pg / mL

[0303] Table 16 IFN-α, IFN-beta, IL-6 and TNF-α contents in the cell supernatant of donor 2 hPBMC after treatment with different doses of test compounds

[0304] *Data for the PBS group: INF-alpha: 0.81 pg / mL; INF-beta: 4.48 pg / mL; IL-6: 120.43 pg / mL; TNF-alpha: 5.66 pg / mL

[0305] Table 17 IL-6 and TNF-α levels in the supernatant of donor 3hPBMC after treatment with different doses of test compounds

[0306] Experimental conclusion: The compound of the present invention has a low immunogenicity risk to hPBMC.

[0307] Experimental Example 6. Study on Anti-HBV Activity and Safety in a Recombinant Adeno-Associated Virus Type 8 Vector-Mediated Hepatitis B Virus Mouse Model (AAV-HBV)

[0308] 1. Experimental purpose:

[0309] The AAV vector-mediated HBV transfection mouse model is a rapid and efficient HBV model. Leveraging the high hepatotropism of the AAV8 vector, a recombinant adeno-associated virus type 8 (rAAV8-1.3HBV) carrying 1.3 copies of the HBV genome can be efficiently introduced into hepatocytes using tail vein injection of the AAV8 vector. Due to the properties of AAV viral vectors, the vectors they mediate can be expressed for extended periods of time. The AAV / HBV model allows for sustained HBV DNA replication and expression of HBsAg and HBeAg in the mouse liver.

[0310] By using the AAV-HBV mouse model, HBsAg, HBeAg, DNA in the serum of mice and the weight of mice after treatment with the test compound were detected to evaluate its anti-HBV effect and safety in vivo.

[0311] 2. Experimental Materials:

[0312] C57BL / 6 mice, PBS (RNase free) as solvent, test compound, recombinant virus rAAV8-1.3HBV. The main reagents of this project include QIAamp96 DNA kit (Qiagen, 51162), FastStart Universal Probe Master (Rox) (Roche, 04914058001), Hepatitis B virus surface antigen detection kit (Antu Biotechnology, CL0310), Hepatitis B virus e antigen detection kit (Antu Biotechnology, CL0918), PureLink TM Pro 96 Viral RNA / DNA kit (Invitrogen, 12280-096A) and FastQuant RT Kit (with gDNase) (TIANGEN, KR106-02) were used. Key instruments included a centrifuge (Beckman Allegra X-15R), a multifunctional microplate reader (BioTek Synergy 2), a fluorescence quantitative PCR instrument (Applied Biosystems, 7900HT Fast Real-time PCR system), and a microplate reader (Molecular Devices, SpectraMax 340PC384).

[0313] 3. Experimental methods:

[0314] a) Mice were administered subcutaneously starting on day 34 after virus injection at a dose of 2, 6, or 12 mg / kg, and this day was designated as day 0. Before administration, all mice were bled submandibular to collect plasma.

[0315] b) Blood was collected from the submandibular vein of mice on days 0, 14, 21, and 28 after administration, and plasma was collected. The collected blood samples were anticoagulated with K2-EDTA and centrifuged at 4°C, 7000 g / min for 10 minutes.

[0316] c) All mice were bled through the submandibular vein to collect plasma, and then the mice were euthanized by CO2 inhalation, and plasma samples were collected through cardiac bleeding, and liver samples were collected.

[0317] d) Send the sample for testing.

[0318] 4. Sample analysis:

[0319] ELISA was used to detect the levels of HBsAg and HBeAg in mouse serum: the experimental steps were based on the instructions of the HBsAg ELISA (Antu Biotech, CL 0310) and HBeAg ELISA (Antu Biotech, CL0918) kits.

[0320] Detection of HBV DNA content in mouse plasma by qPCR: HBV DNA was extracted from plasma according to the QIAamp 96 DNA Blood Kit instructions, and HBV DNA content in mouse plasma was detected by qPCR.

[0321] The data of each group of mice are expressed as mean ± standard error, unless otherwise specified, n = 5. Statistical analysis was performed using Student's t-test.

[0322] 5. Experimental results:

[0323] a) Serum HBsAg Content: The anti-HBV activity of the test compounds in the AAV-HBV mouse model was evaluated. Plasma HBsAg levels in mice were measured by ELISA. The results are shown in Table 18.

[0324] Table 18 The percentage reduction of HBsAg on days 7, 14, 21 and 28 relative to day 0 after administration of Z13 to mice

[0325] b) Serum HBeAg Content: The anti-HBV activity of the test compounds in the AAV-HBV mouse model was evaluated. Mouse plasma HBeAg levels were measured by ELISA. The results are shown in Table 19.

[0326] Table 19 The percentage reduction of HBeAg on days 7, 14, 21 and 28 relative to day 0 after administration of Z13 to mice

[0327] c) Serum DNA Content: The anti-HBV activity of the test compounds in the AAV-HBV mouse model was evaluated. HBV DNA content in mouse plasma was measured by quantitative PCR. The results are shown in Table 20.

[0328] Table 20 The percentage reduction of HBV DNA on days 7, 14, 21, and 28 relative to day 0 after administration of Z13 to mice

[0329] d) Body weight change. Day 0 body weight was used as the baseline for comparison. According to IACUC guidelines, a 20% body weight loss was considered the humane endpoint. Any mouse with a body weight loss exceeding 20% ​​was removed from the experiment. No mice were removed due to weight loss in this experiment.

[0330] Experimental conclusion:

[0331] In this experiment, the compound of the present invention significantly reduced HBsAg, HBeAg, and HBV DNA in an AAV-HBV mouse model, showing a significant dose-response relationship. During the experiment, the mice showed good tolerance.

[0332] Experimental Example 7 In vitro plasma stability study

[0333] 1. Experimental Materials

[0334] a) CD-1 mouse plasma, SD rat plasma, and human plasma;

[0335] b) Test compound: 50 μL of the test compound (1 mg / mL, solvent: nuclease-free water) was diluted to 100 μL / mL with 450 μL of nuclease-free water.

[0336] c) Control compound: Patisiran, supplier: MedChemExpress

[0337] 2. Experimental Procedure

[0338] a) Thaw frozen plasma in cold water for 10-20 minutes and then centrifuge at 3220 × g for 5 minutes.

[0339] b) Prepare working solutions of the test compound and the control compound. For each time point, take 2 μL of the working solution and mix it with 98 μL of blank plasma in duplicate.

[0340] c) For T0 samples, add the stop solution immediately after adding blank plasma and the working solutions of the test compound and control compound.

[0341] d) Except for T0, samples at each time point (0.5, 1, 2, 4, 6, 8, and 24 hours) were incubated in a 37°C water bath.

[0342] e) At the end of each time point, add 100 μL of an aqueous solution (containing 100 mM ammonium acetate (pH 10.0), 2 mM tris(2-carboxyethyl)phosphine hydrochloride, 1 mM ethylenediaminetetraacetic acid, and 750 ng / mL internal standard) and vortex for 60 seconds.

[0343] f) Add 100 μL of PCL (phenol / chloroform / isoamyl alcohol = 25:24:1) reagent and 200 μL of dichloromethane to each sample well, mix thoroughly, and then centrifuge at 3220×g for 20 minutes.

[0344] g) The aqueous layer was transferred to a new 96-well plate and stored at 4°C for LC-MS analysis.

[0345] 3. Data Analysis

[0346] The remaining percentage of the test compound after incubation in plasma was calculated by the following formula: Remaining percentage (%) = 100*(PAR at incubation time point / PAR at T0), where PAR is the peak area ratio of the analyte to the internal standard.

[0347] The incubation time points were T0 (0 min) and Tn (n=0, 0.5, 1, 2, 4, 6, 8, and 24 hours).

[0348] 4. Experimental results: see Table 21

[0349] Table 21 Plasma stability of compounds

[0350] T 1 / 2 Represents half-life

[0351] Experimental conclusion: The compound of the present invention is metabolized rapidly in the plasma of rats, mice and humans.

[0352] Experimental Example 8 In vitro liver S9 stability study

[0353] 1. Experimental Materials

[0354] a) Liver S9: Human and animal S9 were purchased from qualified suppliers such as BioIVT and stored in a -80°C freezer.

[0355] b) Control compound: Patisiran, supplier: MedChemExpress, batch number: 155113

[0356] 2. Experimental Procedure

[0357] 2.1 Preparation of buffer and working solution

[0358] Buffer: Prepare the buffer using nuclease-free ultrapure water containing 100 mM tris(hydroxymethyl)aminomethane hydrochloride, 1 mM magnesium chloride, and 1× penicillin-streptomycin dual antibody solution. Adjust the pH of the solution to 6.00 ± 0.10 using hydrochloric acid.

[0359] S9 working solution: dilute liver S9 solution to 1.05 mg / mL with buffer.

[0360] Working solution of test compound or control compound: dilute 1.00 mg / mL test compound or control compound to 2.00 μg / mL with nuclease-free water.

[0361] 2.2 Experimental steps

[0362] a) Prepare seven 96-well incubation plates, designated T0, T1, T4, T8, T24, T48, and Blank48. The first six plates correspond to reaction time points of 0, 1, 4, 8, 24, and 48 hours, respectively. In the Blank48 plate, no test compound or control compound is added, and the reaction is terminated after 48 hours of incubation. Samples at all time points are collected in duplicate.

[0363] b) Add 190 μL of S9 working solution (protein concentration: 1.05 mg / mL) to each of the T0, T1, T4, T8, T24, T48, and Blank48 plates. Pre-incubate all plates except T0 in a 37°C water bath for approximately 10 minutes.

[0364] c) After the preincubation period, add 10 μL of the test compound or control stock solution to each of the T0, T1, T4, T8, T24, and T48 plates, and 10 μL of water to the Blank48 plate. Place the incubation plates (T1, T4, T8, T24, T48, and Blank48, except T0) in a 37°C water bath to initiate the reaction. The final reaction volume is 200 μL. For wells containing liver S9, the liver S9 protein concentration is 1.00 mg / mL; for wells containing test compound or control stock solution, the final reaction concentration is 2.00 μg / mL.

[0365] d) Add 200 μL of stop solution (an aqueous solution containing 2.00 mM tris(2-carboxyethyl)phosphine hydrochloride, 100 mM ammonium acetate, 1.00 mM ethylenediaminetetraacetic acid, and 750 ng / mL internal standard) to the T0 sample and shake thoroughly. Then, add 200 μL of a mixed solution (phenol:chloroform:isoamyl alcohol = 25:24:1) and shake thoroughly for 10 minutes. Then, add 400 μL of dichloromethane, shake thoroughly, and centrifuge at 4°C, 3220 × g for 20 minutes.

[0366] e) At the end of each incubation time point (1, 4, 8, 24, and 48 hours), remove the corresponding incubation plate from the water bath and process according to the T0 sample processing method. 100 μL of supernatant from each sample was used for LC-MS / MS analysis.

[0367] 3. Sample Analysis

[0368] The antisense strands of the test compounds in this study were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Retention times of the analytes and internal standards, chromatogram acquisition, and chromatogram integration were performed using Analyst software (Sciex, Framingham, MA, USA).

[0369] 4. Data Analysis

[0370] The in vitro elimination rate constant k of the test compound and the control compound was obtained by converting the ratio of the compound to the internal standard peak area into the residual percentage in the following formula: e :

[0371] when

[0372] By k e Calculation of in vitro liver S9 intrinsic clearance (CL int(S9) ) and hepatic intrinsic clearance (CL int(liver) )

[0373] CL int(S9) =0.693 / T 1 / 2 / S9 protein content (S9 concentration during incubation mg / mL)

[0374] CL int(liver) =CL int(S9) × S9 protein content in liver (mg / g) × liver weight to body weight ratio

[0375] The parameters used in the formula are shown in Table 22 below.

[0376] Table 22. Liver weight to body weight ratio and corresponding S9 protein content in different species

[0377] 5. Experimental results: see Table 23

[0378] Table 23 Liver S9 stability of compounds

[0379] *T 1 / 2 Represents half-life

[0380] Experimental conclusion: The antisense chain of the compound of the present invention has good stability in the liver S9 of three species: mouse, rat and human.

[0381] Experimental Example 9 RNA sequence off-target study of the compounds of the present invention

[0382] 1. Experimental Introduction:

[0383] The transcriptome refers to the sum of all RNA transcribed by a specific tissue or cell at a specific time or state, primarily including mRNA and non-coding RNA. Transcriptome sequencing, based on the Illumina sequencing platform, studies all mRNA transcribed by a specific tissue or cell at a specific time. It is fundamental to the study of gene function and structure and plays a crucial role in understanding biological development and disease. With the advancement of gene sequencing technology and the reduction in sequencing costs, RNA-seq has become a leading method for transcriptome research due to its high throughput, high sensitivity, and wide range of applications. The RNA-seq technical process primarily consists of two parts: library construction and sequencing, and bioinformatics analysis.

[0384] 2. Research Methods

[0385] After incubating HepG2.2.15 cells with siRNA or PBS for 24 hours, the cells were lysed and RNA was collected. RNA-seq was then used to analyze differentially expressed genes between each siRNA group and the PBS group. Enrichment of differentially expressed genes was determined by comparison with various databases. HepG2.2.15 cells were transfected with 100 nM siRNA using Lipofectamine RNAiMax. After 24 hours of incubation in a 5% CO2, 37°C incubator, the cells were harvested and RNA was extracted. RNA samples were then rigorously quality-controlled, primarily using an Agilent 2100 bioanalyzer to accurately measure RNA integrity.

[0386] 3. Library construction and quality control:

[0387] There are two main methods for obtaining mRNA: 1. Taking advantage of the structural characteristic of most eukaryotic mRNAs with polyA tails, mRNA with polyA tails can be enriched using Oligo(dT) magnetic beads. 2. mRNA can be obtained by removing ribosomal RNA from total RNA. The resulting mRNA is then randomly fragmented with divalent cations in NEB Fragmentation Buffer and constructed using either the NEB standard library construction method or the strand-specific library construction method.

[0388] NEB general library construction: Using fragmented mRNA as a template and random oligonucleotides as primers, the first cDNA chain is synthesized in the M-MuLV reverse transcriptase system, followed by RNaseH degradation of the RNA chain, and the second cDNA chain is synthesized using dNTPs as raw materials in the DNA polymerase I system. The purified double-stranded cDNA is end-repaired, A-tailed, and ligated to sequencing adapters. AMPure XP beads are used to screen cDNA of about 250-300bp, PCR amplification is performed, and the PCR product is purified again using AMPure XP beads to finally obtain the library. The kit used for library construction is Ultra TM RNA Library Prep Kit for

[0389] Chain-specific library construction: The method for reverse transcription synthesis of the first chain of cDNA is the same as the NEB general library construction method. The difference is that when synthesizing the second chain, dTTP in dNTPs is replaced by dUTP. After that, the cDNA end repair, A tail addition, sequencing adapter connection and length screening are also performed. Then, the second chain of cDNA containing U is degraded with USER enzyme and PCR amplification is performed to obtain the library. Chain-specific libraries have many advantages, such as obtaining more effective information with the same amount of data; obtaining more accurate gene quantification, positioning and annotation information; and providing the expression level of antisense transcripts and a single exon in each isoform. The kit used for library construction is Ultra TM Directional RNA Library Prep Kit for

[0390] Note: Sequencing adapters consist of three parts: P5 / P7, index, and Rd1 / Rd2SP. P5 / P7 are the PCR amplification primers and the primer binding sites on the flow cell. The index provides information to distinguish different libraries. Rd1 / Rd2SP, or read1 / read2 sequence primers, is the sequencing primer binding region. Theoretically, sequencing proceeds backward from Rd1 / Rd2SP.

[0391] After library construction, a Qubit2.0 Fluorometer was used for preliminary quantification, and the library was diluted to 1.5 ng / μL. The insert size of the library was then detected using an Agilent 2100 bioanalyzer. Once the insert size was in line with expectations, qRT-PCR was used to accurately quantify the effective concentration of the library (the effective concentration of the library was greater than 2 nM) to ensure library quality.

[0392] 4. Sequencing:

[0393] After passing the library inspection, different libraries are pooled according to effective concentration and target data volume requirements before Illumina sequencing. The basic principle of sequencing is sequencing by synthesis. Four fluorescently labeled dNTPs, DNA polymerase, and adapter primers are added to the sequencing flow cell for amplification. As each sequencing cluster extends the complementary chain, each added fluorescently labeled dNTP releases corresponding fluorescence. The sequencer captures the fluorescent signal and converts it into sequencing peaks through computer software, thereby obtaining the sequence information of the fragment to be tested.

[0394] 5. Experimental results:

[0395] The differential gene analysis used DESeq2 padj≤0.05|log2FoldChange|≥1.0 as the threshold, and the statistical results are shown in Figure 24 below.

[0396] Table 24. Statistical results of differentially expressed genes

[0397] Experimental conclusion: The off-target risk of the compounds of the present invention is relatively low.

[0398] Experimental Example 10 Preliminary safety study of the compound of the present invention in rats

[0399] 1. Experimental Materials

[0400] a) Test compound: Compound of the present invention

[0401] b) Male rats (3), SPF grade, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0402] 2. Experimental Procedure

[0403] a) Preparation of dosing formulation: The solvent is enzyme-free phosphate buffered saline (pH 7.4, 1X). The test compound is prepared as a 15 mg / mL clear solution;

[0404] b) Weigh the animal before dosing and calculate the dosing volume based on the body weight (based on the 150 mpk dosing volume). Observe the animal's overall health and appearance on the day of dosing;

[0405] c) A single subcutaneous injection of the test compound solution was given on the first day, and cage-side observation was performed once a day;

[0406] d) At 336 hours, blood (approximately 1.5 mL) was collected by jugular venipuncture and placed in blood collection tubes containing ethylenediaminetetraacetic acid dipotassium salt anticoagulant, sodium citrate anticoagulant, and silica coagulant, respectively. The tubes were kept on wet ice for routine blood test, coagulation analysis, and blood biochemistry testing.

[0407] 3. Experimental Results

[0408] The experimental results of compound Z13 are as follows:

[0409] a) Weight and behavior monitoring: No abnormalities

[0410] b) Routine blood count: White blood cell (WBC), neutrophil (NEUT), lymphocyte (LYMPH), monocyte (MONO), eosinophil (EOS), basophil (BASO), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), hemoglobin (MCH), hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelet count (PLT), and platelet volume (MPV) were normal; reticulocyte (RET) was slightly elevated in one rat.

[0411] c) Blood coagulation: prothrombin time (PT), activated partial prothrombin time (APTT), and fibrinogen (FIB) were normal.

[0412] d) Blood biochemistry: Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were normal.

[0413] 4. Experimental conclusion: The compound of the present invention has good preliminary safety.

Claims

1. A double-stranded siRNA analog, a conjugate thereof, a salt thereof or a salt of a conjugate thereof, comprising a sense strand and an antisense strand capable of forming a double-stranded region, wherein: The double-stranded siRNA analog is selected from any double-stranded one shown in Table 1, and each nucleotide on the double-stranded one is optionally modified independently.

2. The double-stranded siRNA analog, its conjugate, its salt or the salt of its conjugate according to claim 1, wherein The double-stranded siRNA analog is selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21, S22, S23, S24, S25, S26, S27 and S28.

3. The double-stranded siRNA analog, its conjugate, its salt or the salt of its conjugate according to claim 2, wherein: The double-stranded siRNA analog is selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11.

4. The double-stranded siRNA analog, its conjugate, its salt or the salt of its conjugate according to claim 1, wherein The double-stranded siRNA analog is selected from any of the double-stranded siRNAs shown in Table 2.

5. The double-stranded siRNA analog, its conjugate, its salt or the salt of its conjugate according to claim 1, wherein The conjugate of the double-stranded siRNA analog or the salt of the conjugate thereof is formed by conjugating the double-stranded siRNA analog according to any one of claims 1 to 4 with a pharmaceutically acceptable conjugation group.

6. The double-stranded siRNA analog, its conjugate, its salt or the salt of its conjugate according to claim 5, wherein: The pharmaceutically acceptable conjugated group contains 1 to 5 GalNAc groups.

7. The double-stranded siRNA analog, its conjugate, its salt or the salt of its conjugate according to claim 6, wherein: The pharmaceutically acceptable conjugating group is linked to the 3' end of the sense strand of the double-stranded siRNA analog.

8. The double-stranded siRNA analog, its conjugate, its salt or the salt of its conjugate according to claim 5, wherein: The pharmaceutically acceptable conjugated group is selected from, 9. A conjugate of a double-stranded siRNA analog or a salt thereof, which is selected from Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16.

10. Use of the double-stranded siRNA analog, its conjugate, its salt or the salt of its conjugate according to any one of claims 1 to 9 in the preparation of a medicament for treating hepatitis B.