Bicyclic structure and oligonucleotide comprising same

Abstract

A bicyclic structure and an oligonucleotide comprising same, the oligonucleotide comprising at least one structure represented by formula (I').

Description

Bicyclic structures and oligonucleotides containing them Technical Field

[0001] This disclosure relates to a bicyclic structure and oligonucleotides containing the same, and also to methods for preparing and using the aforementioned oligonucleotides. Background Technology

[0002] RNA interference (RNAi) is an effective way to silence gene expression. Statistics show that over 80% of disease-related proteins in the human body cannot be targeted by conventional small-molecule drugs and large-molecule biological agents, thus becoming undrugable proteins. Using RNA interference technology, suitable siRNAs can be designed based on the mRNA encoding these proteins, specifically targeting and degrading the target mRNA, thereby inhibiting the production of the related proteins. Therefore, siRNAs hold significant promise for drug development. However, unmodified siRNAs suffer from in vivo instability and non-specific distribution, necessitating the development of specific chemical modifications to further enhance their drug potential. Summary of the Invention

[0003] This disclosure provides an oligonucleotide comprising at least one structure as shown in formula (I'):

[0004] in,

[0005] X1 and X2 are each independently covalently bonded, O or C1-C3 alkylene (e.g., C1, C2 or C3 alkylene);

[0006] X3 is either N or CH;

[0007] R1 and R2 are each independently selected from C1-C6 alkylene groups (e.g., C1, C2, C3, C4, C5, or C6 alkylene groups) or covalently bonded, wherein the C1-C6 alkylene groups are optionally bonded by one or more R1-C2-C6 alkylene groups. a The R that was replaced a Each is independently selected from oxo, C1-C6 alkyl (e.g., C1, C2, C3, C4, C5 or C6 alkyl), C1-C6 alkoxy (e.g., C1, C2, C3, C4, C5 or C6 alkoxy), hydroxy or halogen;

[0008] R3is selected from 3- to 12-membered cycloalkyl (e.g., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered cycloalkyl), 3- to 12-membered heterocyclyl (e.g., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered heterocyclyl), 5- to 12-membered aryl (e.g., 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered aryl), or 5- to 12-membered heteroaryl (e.g., 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered heteroaryl), optionally substituted with one or more R b R b each is independently selected from oxo, C1-C6alkyl (e.g., C1, C2, C3, C4, C5, or C6alkyl), halogen, hydroxyl, cyano, carbonyl, hydroxyl;

[0009] Optionally, one or more hydrogen atoms in the structure of Formula (I’) are replaced with deuterium atoms.

[0010] In some embodiments, R1, R2are each independently selected from C1-C3alkylene (e.g., C1, C2, C3alkylene) or a covalent bond, optionally substituted with one or more R a R a each is independently selected from oxo, C1-C3alkyl (e.g., C1, C2, C3alkyl), C1-C3alkoxy (e.g., C1, C2, C3alkoxy), hydroxyl, or halogen.

[0011] In some embodiments, R3is selected from 5- to 12-membered cycloalkyl, 5- to 12-membered heterocyclyl, 5- to 12-membered aryl, or 5- to 12-membered heteroaryl, optionally substituted with one or more R b R b each is independently selected from oxo, C1-C3alkyl, halogen, hydroxyl, cyano, hydroxyl. In some embodiments, R3is selected from 5- to 8-membered cycloalkyl or 5- to 8-membered heterocyclyl; the 5- to 8-membered heterocyclyl has 1, 2, or 3 heteroatoms.

[0012] In some embodiments, one or more hydrogen atoms in the structure of Formula (I’) are replaced with deuterium atoms, which can be located anywhere in the structure of Formula (I’), e.g., in R1, R2, R3, X1, X2, X3.

[0013] In some embodiments, the structure of Formula (I’) has the structure of Formula (I): In some embodiments, the structure of Formula (I’) has the structure of Formula (I):

[0014] wherein a indicates a direction toward the 3' end of the oligonucleotide sequence, and b indicates a direction toward the 5' end of the oligonucleotide sequence.

[0015] In some embodiments, the structure of Formula (I') is attached to the 5' terminal first nucleoside of any one of the nucleotide sequences in the oligonucleotide.

[0016] In some embodiments, the structure of Formula (I') is attached to the 5' terminal first nucleoside of the nucleotide sequence, wherein R2of the structure of Formula (I') is attached to the 5' end of the 5' terminal first nucleoside via a linking group, and R1is directly attached to the hydroxyl group, or R1is attached to a delivery group via a linking group. The linking groups are each independently selected from -O-, a phosphodiester group, or a phosphodiester group having a modifying group. In some embodiments, the phosphodiester group having a modifying group is a phosphorothioate group.

[0017] In some embodiments, the structure of Formula (I') is attached to the 3' terminal first nucleoside of the nucleotide sequence, wherein R1of the structure of Formula (I') is attached to the 3' terminal first nucleoside via a linking group, and R2is directly attached to the hydroxyl group, or R2is attached to a delivery group via a linking group. The linking groups are each independently selected from -O-, a phosphodiester group, or a phosphodiester group having a modifying group. In some embodiments, the phosphodiester group having a modifying group is a phosphorothioate group.

[0018] In some specific embodiments, the structure of Formula (I) has the structure of Formula (I-1):

[0019] wherein a indicates a direction toward the 3' end of the oligonucleotide sequence, and b indicates a direction toward the 5' end of the oligonucleotide sequence, X1and X2are each independently a methylene group or a covalent bond, indicate the attachment sites of the structure of Formula (I-1) to adjacent groups in the oligonucleotide, and X3and R3are defined and selected as described in any of the embodiments herein.

[0020] In some embodiments, the structure of Formula (I) has the structure of any one of Formulas (I-2) to (I-5):

[0021] wherein a indicates a direction toward the 3' end of the oligonucleotide sequence, and b indicates a direction toward the 5' end of the oligonucleotide sequence.

[0022] In some embodiments, the structure of Formula (I) has a structure of any one of Formulas (I-6) to (I-10):

[0023] wherein a indicates a direction toward the 3’ end of the oligonucleotide sequence and b indicates a direction toward the 5’ end of the oligonucleotide sequence.

[0024] In some embodiments, the structure of Formula (I) is selected from:

[0025] wherein a indicates a direction toward the 3’ end of the oligonucleotide sequence and b indicates a direction toward the 5’ end of the oligonucleotide sequence.

[0026] In some embodiments, the oligonucleotide is a single-stranded, double-stranded oligonucleotide, such as an antisense oligonucleotide (ASO), a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a ribozyme, an RNAi inhibitor molecule, and a Dicer enzyme substrate, etc.

[0027] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide.

[0028] In some embodiments, the single-stranded oligonucleotide is an antisense oligonucleotide (ASO) or a single-stranded RNAi molecule (ssRNAi), which consists of 15-50 nucleotides.

[0029] In some embodiments, the single-stranded oligonucleotide molecule has 16 to 50, 16 to 45, 17 to 45, 17 to 40, 18 to 40, 18 to 35, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides.

[0030] In some embodiments, the oligonucleotide is a double-stranded RNAi agent comprising a sense strand and an antisense strand that are at least partially complementary to form a double-stranded region.

[0031] In some embodiments, the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region.

[0032] In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 mismatch between the sense strand and the antisense strand.

[0033] In some embodiments, the sense strand and the antisense strand are completely reverse complementary.

[0034] In some embodiments, the double stranded RNAi agent molecule comprises one or two blunt ends.

[0035] In some embodiments, the double stranded RNAi agent molecule comprises overhangs having from 1 to 4 unpaired nucleotides, for example 1, 2, 3, or 4 unpaired nucleotide overhangs.

[0036] In some embodiments, the antisense strand 3’ end comprises an overhang.

[0037] In some embodiments, the sense strand is 15-35 nucleotides in length and the antisense strand is 15-35 nucleotides in length.

[0038] In some embodiments, the sense strand and antisense strand each independently have from 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides.

[0039] In some embodiments, the sense strand and antisense strand are the same or different in length, the sense strand is 19-23 nucleotides in length and the antisense strand is 19-26 nucleotides in length.

[0040] The length ratio of the sense strand and antisense strand in the RNAi agents provided by the present disclosure can be 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25, or 23 / 26.

[0041] In some embodiments, the sense strand and antisense strand are 19 / 21, 21 / 21, 21 / 23, 23 / 23, or 23 / 25 in length.

[0042] In some embodiments, the length ratio of the sense strand and antisense strand is 19 / 21.

[0043] In some embodiments, the oligonucleotide of the disclosure further comprises one or more delivery groups attached to any one of the strands of the oligonucleotide.

[0044] In the context of the disclosure, "attached" includes covalent and non-covalent attachment. The delivery groups are capable of delivering the oligonucleotide of the disclosure to a location where the target gene expression exists.

[0045] In some embodiments, the delivery group in the context of the disclosure is a lipophilic group, and the one or more lipophilic groups are attached to any one or more of the nucleotides in the oligonucleotide and / or the structure shown in Formula (I').

[0046] In some embodiments, the lipophilic group is attached to the base of a nucleotide.

[0047] In some embodiments, the lipophilic group is attached to the sugar ring of a nucleotide.

[0048] In some embodiments, the lipophilic group is attached to the internucleoside linking group between two adjacent nucleotides.

[0049] In some embodiments, the lipophilic group is attached to the structure shown in Formula (I') as described above.

[0050] In some embodiments, the oligonucleotide is an RNAi agent, and the one or more lipophilic groups are attached to the sense strand in the RNAi agent. In some embodiments, the one or more lipophilic groups are attached to the antisense strand in the RNAi agent. In some embodiments, at least one of the lipophilic groups is attached to the sense strand in the RNAi agent, and at least one of the lipophilic groups is attached to the antisense strand in the RNAi agent.

[0051] In some embodiments, the delivery group in the context of the disclosure comprises a targeting ligand that targets the liver.

[0052] In some embodiments, the targeting ligand binds to the Asialoglycoprotein Receptor (ASGPR).

[0053] In some embodiments, the targeting ligand comprises a cluster of galactose or a cluster of galactose derivatives selected from N-acetyl-galactosamine, N-trifluoroacetyl galactosamine, N-propionyl galactosamine, N-n-butyryl galactosamine, or N-isobutyryl galactosamine.

[0054] In some embodiments, the delivery group is linked to the 3’ end of either strand of the oligonucleotide. In some embodiments, the delivery group is linked to the oligonucleotide end via a phosphodiester group or a phosphodiester group with a modifying group. In some embodiments, the delivery group is linked to the oligonucleotide end directly via a phosphodiester group.

[0055] In some embodiments, the oligonucleotide is an RNAi agent, and the delivery group is linked to the first position of the 3’ end or 5’ end of the sense strand of the RNAi agent via a phosphodiester group or a phosphodiester group with a modifying group.

[0056] In some embodiments, the delivery group can be a delivery group as shown in PCT application WO2023274395A or WO2014025805A. In some embodiments, the delivery group is selected from:

[0057] In some embodiments, at least one additional nucleotide in the oligonucleotides of the present disclosure is a modified nucleoside. In some embodiments, all nucleotides in the oligonucleotides are modified nucleosides.

[0058] In some embodiments, the modified nucleoside is selected from: 2’-methoxy modified nucleoside, 2’-substituted alkoxy modified nucleoside, 2’-alkyl modified nucleoside, 2’-substituted alkyl modified nucleoside, 2’-amino modified nucleoside, 2’-substituted amino modified nucleoside, 2’-fluoro modified nucleoside, 2’-deoxyribonucleoside, 2’-deoxy-2’-fluoro modified nucleoside, 3’-deoxy-thymine (dT) nucleoside, iso-nucleoside, LNA, ENA, cET, UNA, GNA. In some embodiments, the modified nucleotides are each independently selected from 2’-methoxy modified nucleoside, 2’-fluoro modified nucleoside, or 2’-deoxyribonucleoside.

[0059] In some embodiments, the sense strand contains 3 consecutive 2’-fluoro modified nucleosides, in some embodiments, the 7th, 8th, and 9th nucleosides from the 5’ end to the 3’ end of the sense strand are 2’-fluoro modified nucleosides. In some embodiments, the non-2’-fluoro modified nucleosides are 2’-methoxy modified nucleosides.

[0060] In some embodiments, the antisense strand contains 8 2'-fluoro-modified nucleosides, in the 2nd, 4th, 6th, 10th, 12th, 14th, 16th, and 18th positions of the antisense strand, from the 5' end to the 3' end, each independently is a 2'-fluoro-modified nucleoside, and the nucleotides at the remaining positions are non-2'-fluoro-modified nucleosides. In some embodiments, the non-2'-fluoro-modified nucleosides are 2'-methoxy-modified nucleosides.

[0061] In some embodiments, the nucleosides in the 2nd, 6th, 8th, and 13th positions of the antisense strand, from the 5' end to the 3' end, are 2'-deoxyribonucleosides, the nucleoside in the 15th position is a 2'-fluoro-modified nucleoside, and the nucleosides at the remaining positions are 2'-methoxy-modified nucleosides.

[0062] In some embodiments, the nucleosides in the odd-numbered positions of the antisense strand, from the 5' end to the 3' end, are 2'-methoxy-modified nucleosides, and the nucleosides in the even-numbered positions are 2'-fluoro-modified nucleosides.

[0063] In some embodiments, at least one of the phosphodiester groups in the sense strand and / or the antisense strand is a phosphodiester group with a modification group. In some embodiments, a plurality of the phosphodiester groups in the sense strand and / or the antisense strand comprise a modification group. In some embodiments, a plurality of the phosphodiester groups in both the sense strand and the antisense strand comprise a modification group.

[0064] In some embodiments, the phosphodiester group with a modification group is present at one or more of the positions selected from:

[0065] between the nucleoside at the 1stposition and the nucleoside at the 2ndposition of the 5' terminal end of the sense strand;

[0066] between the nucleoside at the 2ndposition and the nucleoside at the 3rdposition of the 5' terminal end of the sense strand;

[0067] between the nucleoside at the 1stposition and the nucleoside at the 2ndposition of the 3' terminal end of the sense strand;

[0068] between the nucleoside at the 2ndposition and the nucleoside at the 3rdposition of the 3' terminal end of the sense strand;

[0069] between the nucleoside at the 1stposition and the nucleoside at the 2ndposition of the 5' terminal end of the antisense strand;

[0070] between the nucleoside at the 2ndposition and the nucleoside at the 3rdposition of the 5' terminal end of the antisense strand;

[0071] between the nucleoside at the 1stposition and the nucleoside at the 2ndposition of the 3' terminal end of the antisense strand;

[0072] between the nucleoside at the 2ndposition and the nucleoside at the 3rdposition of the 3' terminal end of the antisense strand.

[0073] In some embodiments, the phosphodiester group with a modifying group is a phosphorothioate diester group.

[0074] In some embodiments, the 5' position of the sugar ring of the first nucleotide at the 5' terminus of the sense strand or antisense strand is a 5'-vinyl phosphodiester group (VP). In some embodiments, the 5'-vinyl phosphodiester group is a 5'-trans vinyl phosphodiester group (E-VP).

[0075] In some embodiments, the oligonucleotide is an oligonucleotide targeting INHBE, CTNNB1, ANGPTL3, PCSK9, HBV, APOC3, Lp(a), AGT, or PD-L1. In some embodiments, the oligonucleotide is an oligonucleotide as shown in any one of Table 2.

[0076] The present disclosure also provides a compound as shown in formula (II’):

[0077] wherein W is hydroxyl or -O-R p , R p is a hydroxyl protecting group, and Q is a phosphorus-containing active reactive group;

[0078] Preferably, the R p is selected from methoxyphenyldiphenylmethyl, dimethoxytrityl, or trityl;

[0079] R1, R2, X1, X2, X3, and R3 are defined and selected as described in any one of the schemes in the context of the present disclosure;

[0080] Optionally, one or more hydrogen atoms in the structure as shown in formula (II’) are replaced with deuterium atoms.

[0081] In some embodiments, the structure as shown in formula (II’) has a structure as shown in formula (II):

[0082] wherein W, Q, R1, R2, X1, X2, X3, and R3 are as previously defined.

[0083] In some embodiments, the compound as shown in formula (II) is selected from:

[0084] In some embodiments, the compound as shown in formula (II) is selected from:

[0085] The present disclosure also provides a method of making an oligonucleotide of the present disclosure, comprising the steps of:

[0086] (1) synthesizing a compound represented by formula (II') or a tautomer thereof;

[0087] (2) synthesizing an oligonucleotide of the disclosure using the compound represented by formula (II') or a tautomer thereof obtained in step (1).

[0088] The disclosure also provides a pharmaceutical composition comprising an oligonucleotide of the disclosure;

[0089] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0090] In some embodiments, the pharmaceutically acceptable excipient can be, for example, a carrier, a vehicle, a diluent, and / or a delivery polymer, various drug delivery systems are known and can be used for the oligonucleotide of the disclosure, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the oligonucleotide, receptor-mediated endocytosis, construction of nucleic acids as part of a retrovirus or other vector.

[0091] In some embodiments, the pharmaceutical composition can further comprise a pharmaceutically acceptable adjuvant and / or an adjuvant, which adjuvant can be one or more of various preparations or compounds conventionally employed in the art, for example, the pharmaceutically acceptable adjuvant can include at least one of a pH buffer, a protective agent, and an osmotic pressure adjusting agent.

[0092] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg - 1000 mg.

[0093] In some embodiments, the pharmaceutical composition contains 0.01 - 99.99% of the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof or an isotopically substituted thereof, based on the total weight of the composition.

[0094] In some embodiments, the pharmaceutical composition contains 0.1 - 99.9% of the oligonucleotide of the disclosure or a pharmaceutically acceptable salt thereof or an isotopically substituted thereof. In some embodiments, the pharmaceutical composition contains 0.5% - 99.5% of the oligonucleotide of the disclosure or a pharmaceutically acceptable salt thereof or an isotopically substituted thereof. In some embodiments, the pharmaceutical composition contains 1% - 99% of the oligonucleotide of the disclosure or a pharmaceutically acceptable salt thereof or an isotopically substituted thereof. In some embodiments, the pharmaceutical composition contains 2% - 98% of the oligonucleotide of the disclosure or a pharmaceutically acceptable salt thereof or an isotopically substituted thereof.

[0095] In some embodiments, the pharmaceutical composition contains 0.01-99.99% of a pharmaceutically acceptable excipient, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 0.5-99.5% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 1-99% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 2-98% of a pharmaceutically acceptable excipient.

[0096] In some embodiments, an effective amount or effective dose of the oligonucleotide and / or pharmaceutical composition is about 0.001 mg / kg body weight to about 200 mg / kg body weight, about 0.01 mg / kg body weight to about 100 mg / kg body weight, or about 0.5 mg / kg body weight to about 50 mg / kg body weight.

[0097] In some embodiments, the oligonucleotide or pharmaceutical composition of the present disclosure, when contacted to a cell expressing a target gene, inhibits the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, as determined by, for example, psiCHECK activity screening and luciferase reporter assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence assays, for example, Western Blot or flow cytometry.

[0098] In some embodiments, the oligonucleotide or pharmaceutical composition of the present disclosure, when contacted to a cell expressing a target gene, causes the remaining percentage of mRNA expression of the target gene to be no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10% as determined by, for example, psiCHECK activity screening and luciferase reporter assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence assays, for example, Western Blot or flow cytometry.

[0099] The oligonucleotide and / or pharmaceutical composition of the present disclosure can be administered by local administration (e.g., direct injection, implantation, or topical administration) or systemic administration. The administration can be by any suitable route known in the art, including subcutaneous, intravenous, intraperitoneal, or parenteral routes, for example, intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, buccal, and sublingual administration, etc.

[0100] In some embodiments, the pharmaceutical composition provided by the present disclosure can be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal injection.

[0101] In another aspect, the present disclosure provides a kit comprising the oligonucleotide and / or pharmaceutical composition of the present disclosure. In optional embodiments, the conjugate of the targeting ligand linked to the oligonucleotide can be packaged in the kit.

[0102] The present disclosure also provides use of the oligonucleotide and / or pharmaceutical composition of the present disclosure in the manufacture of a medicament. The medicament can be used for preventing and / or treating a disease associated with the expression of a target gene.

[0103] The present disclosure also provides a method of inhibiting the expression of a target gene or mRNA thereof, comprising administering to a subject an effective amount or dose of the oligonucleotide and / or pharmaceutical composition.

[0104] The present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell, either in vivo or in vitro, comprising the step of introducing into the cell an oligonucleotide and / or pharmaceutical composition according to the present disclosure.

[0105] The present disclosure also provides a method for silencing mRNA of a target gene in a cell, comprising the step of introducing the oligonucleotide and / or pharmaceutical composition of the present disclosure into the cell.

[0106] In another aspect, the present disclosure provides a method for preventing and / or treating a disease associated with expression of a target gene, comprising administering to a subject an effective amount or effective dose of the oligonucleotide and / or pharmaceutical composition of the present disclosure.

[0107] In some embodiments, the target gene is INHBE, CTNNB1, ANGPTL3, PCSK9, HBV, APOC3, Lp(a), AGT, or PD-L1 gene.

[0108] In some embodiments, the target gene is INHBE gene, and the disease is a disease associated with expression of INHBE gene, including but not limited to metabolic disorders.

[0109] In some embodiments, the metabolic disorder is selected from one or more of metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, kidney disease.

[0110] In some embodiments, the target gene is CTNNB1 gene, and the disease is a disease associated with expression of CTNNB1 gene.

[0111] In some embodiments, the disease is cancer, which can be, for example, colorectal cancer, gastric cancer, melanoma, ovarian cancer, endometrial cancer, prostate cancer, hepatoblastoma, hepatocellular carcinoma, lung cancer, breast cancer, medulloblastoma, chronic lymphocytic leukemia, etc.

[0112] In some embodiments, the target gene is ANGPTL3 gene, and the disease is a disease associated with ANGPTL3, including but not limited to lipid metabolism disorders. In some embodiments, the lipid metabolism disorder is hyperlipidemia or hypertriglyceridemia.

[0113] In some embodiments, the subject has been previously identified as having a pathological upregulation of the target gene or mRNA thereof in the targeted cell, cell population, tissue, or subject.

[0114] Unless otherwise specified, the terms "nucleic acid," "oligonucleotide," "single-stranded oligonucleotide," "double-stranded oligonucleotide," "antisense oligonucleotide," "small interfering RNA (siRNA)," "double-stranded RNA (dsRNA)," "micro RNA (miRNA)," "short hairpin RNA (shRNA)," "ribozyme," "RNAi inhibitor molecule," "double-stranded RNAi inhibitor molecule," and "Dicer substrate," and "compound" of the disclosure can exist independently as a salt, a mixed salt, or a non-salt (e.g., free acid or free base). When existing as a salt or a mixed salt, it can be a pharmaceutically acceptable salt.

[0115] In the context of the disclosure, the "compound" includes the oligonucleotides, RNAi agents, chemical structures represented by Formula (I), delivery groups, compounds represented by Formula (II), etc. of the disclosure. Without indicating a configuration, the compounds of the disclosure (e.g., the oligonucleotides, RNAi agents, chemical structures represented by Formula (I), delivery groups, compounds represented by Formula (II), etc. of the disclosure) can exist in particular geometric or stereoisomeric forms. All such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)-isomers, as well as racemic mixtures and other mixtures of the foregoing, are

[0116] Optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the disclosure 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 a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl) is present in the molecule, diastereomeric salts are formed with an appropriate optically active acid or base, and the diastereomeric salt is separated by conventional means, and the pure enantiomer is recovered by treating with base or acid, as the case can be. In addition, the separation of the enantiomers and diastereomers is typically accomplished by chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (e.g., formation of carbamates from amines).

[0117] In the chemical structures of the compounds described herein (e.g., oligonucleotides, siRNAs, chemical structures depicted by Formula (I), targeting ligands, compounds depicted by Formula (II), etc.) where a bond is depicted without an indication of stereochemistry, the bond represents unspecified stereochemistry, i.e., if chiral isomers exist in the chemical structure, the bond may be or both E and Z configurations. In the chemical structures of the compounds described herein, the bond is not specified, i.e., the configuration of the bond may be E or Z, or both E and Z configurations.

[0118] The present disclosure also includes isotopically-labeled compounds of the present disclosure which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I, and 36 Cl.

[0119] Unless otherwise indicated, compounds of the disclosure, when a position is designated specifically as "deuterium" or "D", that position shall be understood to have an isotopic abundance of deuterium that is at least 1000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 15% deuterium incorporation). In some embodiments, the isotopic abundance of deuterium at each designated deuterium atom is at least 1000 times greater than the natural abundance of deuterium (i.e., at least 15% deuterium incorporation). In some embodiments, the isotopic abundance of deuterium at each designated deuterium atom is at least 2000 times greater than the natural abundance of deuterium (i.e., at least 30% deuterium incorporation). In some embodiments, the isotopic abundance of deuterium at each designated deuterium atom is at least 3000 times greater than the natural abundance of deuterium (i.e., at least 45% deuterium incorporation). In some embodiments, the isotopic abundance of deuterium at each designated deuterium atom is at least 3340 times greater than the natural abundance of deuterium (i.e., at least 50.1% deuterium incorporation). In some embodiments, the isotopic abundance of deuterium at each designated deuterium atom is at least 3500 times greater than the natural abundance of deuterium (i.e., at least 52.5% deuterium incorporation). In some embodiments, the isotopic abundance of deuterium at each designated deuterium atom is at least 4000 times greater than the natural abundance of deuterium (i.e., at least 60% deuterium incorporation). In some embodiments, the isotopic abundance of deuterium at each designated deuterium atom is at least 4500 times greater than the natural abundance of deuterium (i.e., at least 67.5% deuterium incorporation). In some embodiments, the isotopic abundance of deuterium at each designated deuterium atom is at least 5000 times greater than the natural abundance of deuterium (i.e., at least 75% deuterium incorporation). In some embodiments, the isotopic abundance of deuterium at each designated deuterium atom is at least 5500 times greater than the natural abundance of deuterium (i.e., at least 82.5% deuterium incorporation). In some embodiments, the isotopic abundance of deuterium at each designated deuterium atom is at least 6000 times greater than the natural abundance of deuterium (i.e., at least 90% deuterium incorporation). In some embodiments, the isotopic abundance of deuterium at each designated deuterium atom is at least 6333.3 times greater than the natural abundance of deuterium (i.e., at least 95% deuterium incorporation). In some embodiments, the isotopic abundance of deuterium at each designated deuterium atom is at least 6466.7 times greater than the natural abundance of deuterium (i.e., at least 97% deuterium incorporation). In some embodiments, the isotopic abundance of deuterium at each designated deuterium atom is at least 6600 times greater than the natural abundance of deuterium (i.e., at least 99% deuterium incorporation). In some embodiments, the isotopic abundance of deuterium at each designated deuterium atom is at least 6633.3 times greater than the natural abundance of deuterium (i.e., at least 99.5% deuterium incorporation). One of skill in the art would be able to synthesize deuterated forms of the compounds of Formula I in reference to the relevant literature. Commercially available deuterated starting materials can be used in the preparation of deuterated forms of the compounds of Formula I, or they can be synthesized using conventional techniques employing deuterated reagents including, but not limited to, deuterated borane, trideuteroborane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane, among others.

[0120] Deuterated drugs have advantages over non-deuterated drugs, such as reduced toxicity, increased drug stability, enhanced efficacy, and prolonged drug biohalf-life. All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are included within the scope of the present disclosure. Each available hydrogen atom attached to a carbon atom can be independently replaced (i.e., substituted) with a deuterium atom, wherein the replacement of hydrogen with deuterium can be partial or complete, with partial replacement referring to at least one hydrogen being replaced with at least one deuterium. Those skilled in the art are able to synthesize deuterated forms of the compounds by reference to the relevant literature. In preparing deuterated forms of the compounds, commercially available deuterated starting materials can be used, or they can be synthesized using conventional techniques employing deuterated reagents, including but not limited to deuterated borane, trideuteroborane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide, and deuterated methyl iodide, among others.

[0121] In addition, unless otherwise indicated, the compounds and intermediates (e.g., oligonucleotides, RNAi agents, chemical structures depicted in Formula (I), delivery groups, compounds depicted in Formula (II), etc.) of the present disclosure can exist in different tautomeric forms, and all such forms are embraced within the scope of the present disclosure. The term “tautomer” or “tautomeric form” refers to structural isomers that can interconvert by a low-energy barrier.

[0122] Terminology

[0123] For the purposes of the present disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise clearly indicated, all other technical and scientific terms used herein have the meanings that would be given to them by one of ordinary skill in the art to which the present disclosure pertains.

[0124] As used herein, “RNAi agent” (also referred to as “RNAi trigger”) means an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that contains a sequence capable of degrading or inhibiting (e.g., under appropriate conditions) the translation of a messenger RNA (mRNA) transcript in a sequence-specific manner. As used herein, an RNAi agent can act through an RNA interference mechanism (i.e., by interacting with the machinery of the RNA interference pathway of a mammalian cell (RNA-induced silencing complex or RISC)) or through any alternative mechanism or pathway. While the term RNAi agent as used herein is believed to act primarily through an RNA interference mechanism, the disclosed RNAi agents are not bound or limited to any particular mechanism or pathway of action. The RNAi agents disclosed herein consist of a sense strand and an antisense strand and include, but are not limited to, short (or small) interfering RNA (siRNA). The antisense strand of the RNAi agent described herein is at least partially complementary to the targeted mRNA. The RNAi agent can comprise one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0125] In the context of the present disclosure, unless otherwise specified, when calculating the length of the sense strand and the antisense strand, the structure as shown in Formula (I') and the inverted abasic modified nucleoside are not counted. For example, for the sense strand as shown in SEQ ID NO: 1 : (NA0255")sUmsCmAmAmAmGmCfUfAfUmUmUmUmCmAmUmAmAmUms(NA0255')NAG0052', wherein both NA0255" and NA0255' comprise the structure as shown in Formula (I'), are not counted in the length of the sense strand, the length of the sense strand as shown in SEQ ID NO: 1 is 19 nucleotides.

[0126] The term "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a target gene, including mRNA that is a processing product of the primary transcript. The portion of the target sequence that is targeted should be long enough to serve as a substrate for iRNA-directed cleavage. In one embodiment, the target sequence is within the protein coding region of the target gene. As used herein, in the context of RNA-mediated gene silencing, the sense strand (also referred to as SS, SS strand, or sense strand) refers to the strand comprising a sequence identical or substantially identical to the sequence of the target mRNA; the antisense strand (also referred to as AS or AS strand) refers to the strand having a sequence complementary to the sequence of the target mRNA.

[0127] In the present disclosure, the "5' region" of a sense strand or an antisense strand, also referred to as "5' end" or "5' terminal" can be used interchangeably. For example, the nucleotides at positions 2 to 10 of the 5' region of an antisense strand can also be referred to as the nucleotides at positions 2 to 10 of the 5' end of the antisense strand. Similarly, the "3' region", "3' terminal" and "3' end" of a sense strand or an antisense strand can be used interchangeably.

[0128] Unless otherwise specified, "G," "C," "A," "T," and "U" in the context of the present disclosure represent nucleotides, respectively comprising the bases of guanine, cytosine, adenine, thymidine, and uracil. It is well known to those skilled in the art that the replacement of bases T and U does not significantly affect the nature of the oligonucleotide sequence, and that U in the sequences of the present disclosure can be replaced by T at will, and the sequence obtained after the replacement is also within the protection scope of the present disclosure. In the sequences of the present disclosure, for the same nucleic acid strand, in the direction from the 5' end to the 3' end, the direction from left to right, the lower case letter m indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; the lower case letter f indicates that the nucleotide adjacent to the left of the letter f is a 2'-fluorinated-modified nucleotide; the lower case letter s indicates that the two nucleotides adjacent to the letter s are connected by a phosphorothioate diester group, and unless otherwise specified, the two nucleotides are connected by a phosphodiester group. Unless otherwise specified, the "RNAi agent," "nucleotide," "compound," "chemical modification," "oligonucleotide," "double-stranded RNAi inhibitor molecule," "siRNA," "dsRNA," "nucleic acid," and "RNAi" in the present disclosure can independently exist in the form of a salt, a mixed salt, or a non-salt (e.g., a free acid or a free base). When it exists in the form of a salt or a mixed salt, it can be a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" includes a pharmaceutically acceptable acid addition salt and a pharmaceutically acceptable base addition salt. When it exists in the form of a salt, some groups can be ionized to form anions / cations, for example, a phosphodiester group and a thiophosphodiester group can exist in the form of an anion, and unless otherwise specified, the structure of the salt form corresponding to each of the following structures is also within the protection scope of the present disclosure. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each strand is a hydroxyl group; and the 5' position of the first nucleotide at the 5' end of each strand is a hydroxyl group.

[0129] The above-mentioned modifications and linking groups respectively have the structures shown in Table 1 below, wherein Base represents the base at the corresponding position:

[0130] Table 1

[0131] A "nucleotide analogue" refers to a group that can replace a nucleotide in a nucleic acid, but is structurally different from an adenine ribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, or a thymine deoxyribonucleotide. Such as an iso-nucleotide, a bridged nucleic acid (BNA) or an acyclic nucleotide. The methoxy modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose sugar is replaced by a methoxy group. The iso-nucleotide refers to a compound in which the base in the nucleotide is changed in position on the ribose ring. In some embodiments, the iso-nucleotide can be a compound in which the base is moved from the 1'-position to the 2'-position or the 3'-position on the ribose ring. The BNA refers to a constrained or inaccessible nucleotide. The BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endo sugar conformation bridged structure. The bridge is usually incorporated at the 2'-, 4'-position of the ribose to provide a 2', 4'-BNA nucleotide. In some embodiments, the BNA can be LNA, ENA, cET BNA, etc. The acyclic nucleotide is a class of nucleotides in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA).

[0132] In the context of the present disclosure, one nucleotide sequence has "nucleotide difference" or "differs by no more than n nucleotides" from another nucleotide sequence, means that the base type of the nucleotide at the same position is changed in the former compared to the latter, for example, when the nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G or T, it is considered that there is a nucleotide difference between the two nucleotide sequences at that position, or differs by one nucleotide. In some embodiments, when a nucleotide at a position that is not a chemical structure represented by formula (I) or a tautomer thereof is replaced by an abasic nucleotide or its equivalent, it is also considered that a nucleotide difference is generated at that position.

[0133] The term "lipophilic group" or "lipophilic moiety" broadly refers to any chemical compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient logK ow , where K ow is the ratio of the concentration of a chemical in the octanol phase to its concentration in the water phase of a two-phase system at equilibrium. In principle, a chemical has lipophilicity when logK ow exceeds 0. Typically, a lipophilic moiety has a logK ow exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10, such as the logK owlogK of cholesteryl N-(hexan-6-ol)carbamate is about 0.7 ow is 10.7.

[0134] The lipophilicity of a molecule can vary relative to the functional groups it carries. For example, the addition of a hydroxyl or amine group at the end of a lipophilic moiety can increase or decrease the partition coefficient (e.g., logK ow ) value of the lipophilic moiety. For example, the lipophilic moiety can be an aliphatic, cyclic such as alicyclic, or polycyclic such as a polyalicyclic compound, such as a steroid (e.g., a sterol) or a straight chain or branched aliphatic hydrocarbon. The lipophilic moiety can generally comprise a hydrocarbon chain, which can be cyclic or acyclic. The hydrocarbon chain can comprise various substituents and / or one or more heteroatoms, such as oxygen or sulfur atoms. Such lipophilic aliphatic moieties include, but are not limited to, saturated or unsaturated C4-C 30 hydrocarbons (e.g., C 10 -C 30 hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monoalcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 terpenes, C 15 sesquiterpenes, C 20 diterpenes, C 30 triterpenes, and C 40 tetra terpenes), and other polyalicyclic hydrocarbons; for example, the lipophilic moiety can be an optionally substituted straight chain alkyl of C 10-30 ; for example, the lipophilic moiety can be an optionally substituted straight chain alkyl of C 14-24 .

[0135] As used herein, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases on the other strand in a complementary manner. In DNA, the purine base adenine always pairs with the pyrimidine base thymine (or, in RNA, uracil); the purine base guanine always pairs with the pyrimidine base cytosine. Each base pair includes one purine and one pyrimidine. When the adenine on one strand always pairs with the thymine (or uracil) on the other strand, and the guanine always pairs with the cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand. Accordingly, "mismatch" in the art means that the bases at the corresponding positions in a double-stranded nucleic acid are not paired in a complementary manner.

[0136] As used herein, the term "inhibit," can be used interchangeably with "reduce," "silence," "down-regulate," "repress," and other similar terms, and includes inhibition at any level. Inhibition can be assessed by a decrease in absolute or relative levels of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-dosing baseline level or a level determined from a subject, cell, or sample that has not been treated or has been treated with a control (e.g., a buffer control or an inert agent control). For example, the degree of inhibition of target gene expression by an oligonucleotide can be characterized by the amount of residual mRNA expression, such as no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%. The rate of inhibition of target gene expression can be characterized by the fold decrease in mRNA expression, such as a decrease of no more than 2-fold, no more than 3-fold, no more than 4-fold, no more than 5-fold, no more than 6-fold, no more than 7-fold, no more than 8-fold, no more than 9-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 35-fold, no more than 40-fold, no more than 45-fold, no more than 50-fold, no more than 55-fold, no more than 60-fold, no more than 65-fold, no more than 70-fold, no more than 75-fold, no more than 80-fold, no more than 85-fold, no more than 90-fold, no more than 95-fold, or no more than 100-fold. Luciferase Assay System, and the firefly chemiluminescence value (Fir) and the Renilla chemiluminescence value (Ren) were read, respectively, and the relative value Ratio = Ren / Fir was calculated. In the present disclosure, the residual mRNA expression ratio (or the residual activity %) = Ratio (RNAi agent treatment group) / Ratio (no RNAi agent control group), and the inhibition rate (%) = 100% - residual mRNA expression (%).

[0137] The term "pharmaceutically acceptable salt" includes a pharmaceutically acceptable acid addition salt and a pharmaceutically acceptable base addition salt.

[0138] A "pharmaceutically acceptable acid addition salt" means a salt that retains the biological effectiveness of the free base and that is not biologically or otherwise undesirable. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like. Organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, mesylate, phenylsulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalene disulfonate, and the like. These salts can be prepared by methods known in the art.

[0139] "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and non-toxicity of the free acids, and which are formed with inorganic or organic bases. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Some embodiments of inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion-exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethyl ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Some embodiments of organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0140] "Effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition, which is necessary or sufficient to result in a desired therapeutic result. For prophylactic use, the desired result includes eliminating or reducing risk, lessening severity, or delaying onset of a disorder, including biochemical, histological, and / or behavioral symptoms of the disorder, its complications, and intermediate pathological phenotypes presenting during progression of the disorder. For therapeutic applications, the desired result includes clinical results such as reducing the incidence of, or ameliorating one or more symptoms of, various target gene, target mRNA, or target protein-related disorders of the disclosure, reducing the dosage of other medications required to treat a disorder, enhancing the effect of another medication, and / or delaying the progression of a target gene, target mRNA, or target protein-related disorder of the disclosure in a patient.

[0141] As used herein, "patient," "subject," or "individual" are used interchangeably and include a human or non-human animal, such as a mammal, e.g., a human or a monkey.

[0142] The oligonucleotides provided by the disclosure can be obtained by methods of preparation conventional in the art, such as methods of solid phase synthesis and liquid phase synthesis. Among them, solid phase synthesis has been commercialized as a subscription service. Methods of preparing nucleoside monomers with corresponding modifications and methods of introducing modified nucleotide groups into oligonucleotides can be used to introduce modified nucleotide groups into the oligonucleotides described by the disclosure, and are well known to those skilled in the art.

[0143] The term "chemical modification" or "modification" includes all changes to a nucleotide by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0144] The term "base" includes any known DNA and RNA bases, base analogs, such as purines or pyrimidines, which also include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs.

[0145] The terms "blunt end" or "blunt ends" are used interchangeably to refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of a double-stranded oligonucleotide, i.e., there is no overhang of nucleotides. Most often, an oligonucleotide that has both ends that are blunt ended will be double-stranded over its entire length.

[0146] The terms "about," "approximately," mean values within an acceptable error range for the specific value being sought to be measured or obtained, said acceptable error range varying with the variability of the measurement or measurements in question (i.e., the limits of error in measuring or measuring systems). For example, "about" can mean within 1 or more than 1 standard deviation, per se. Alternatively, "about" or "approximately" can mean ranges within 20% of an expected value, e.g., within 10% of the expected value, within 1% of the expected value, within 0.5% of the expected value, within 0.1% of the expected value, and the like. Each instance of a number or numerical range in this disclosure that is preceded by the term "about" or "approximately" also includes an implementation of the given number. Unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range of the specific value in question when that specific value occurs in the application and claims.

[0147] Unless otherwise indicated, "optionally," "optional," "optional" or "may" means that the subsequently described event or circumstance can or can not occur, and the description includes instances where the event or circumstance occurs and instances where it does not.

[0148] In the chemical structures of the disclosure, which can be attached to one or more of any of the groups according to the scope of the invention described herein.

[0149] The term "linked" when referring to the association between two molecules means that the two molecules are connected by a covalent bond or that the two molecules are associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond), including direct linkage, indirect linkage.

[0150] The term "directly linked" means that a first compound or group is connected to a second compound or group without any intervening atoms or atom groups.

[0151] The term "indirectly connected" means that a first compound or group is connected to a second compound or group through an intervening group, compound or molecule (e.g., a linker).

[0152] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3, of a group are independently of each other replaced with a corresponding number of substituents. When the substituent is a ketone or oxo (i.e., =0), then two (2) hydrogens on the atom are replaced.

[0153] "Substituted with one or more" means that a single or multiple substituents can be present. When multiple substituents are present, these can be multiple identical substituents or a combination of one or multiple different substituents.

[0154] The prefix "C u-v " indicates that the following group has from u to v carbon atoms. For example, "C 1-6 alkyl" means that the alkyl group has 1 to 6 carbon atoms, in particular it can be an alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms.

[0155] "Alkyl" means a saturated aliphatic hydrocarbon group, including straight-chain and branched-chain groups, containing from 1 to 20 carbon atoms. Alkyl groups contain from 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and various branched isomers of these and the like. Alkyl groups can be substituted or unsubstituted, and when substituted, the substituents can be substituted at any available point of attachment, preferably with one or more groups independently selected from halogen, hydroxyl, oxo, cyano, amino, C 1-6 alkyl, C 1-6 alkoxy, 3- to 6-membered cycloalkyl, or 3- to 6-membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl being optionally substituted with halogen, hydroxyl, nitro, cyano, or amino.

[0156] The term "alkenyl" means an unsaturated aliphatic straight-chain or branched-chain hydrocarbon group and contains one or more carbon-carbon double bonds. Exemplary alkenyl groups include C2-C8, C2-C7, C2-C6, C2-C4, C3-C 12 and C3-C6 alkenyl. Included, but not limited to, are ethenyl (i.e., vinyl), 1-propenyl, 2-propenyl (i.e., allyl), 2-methyl-1-propenyl, 1-butenyl, 2-butenyl (i.e., crotyl), and the like. Alkenyl groups used in any context herein are optionally substituted in the same manner as alkyl groups.

[0157] The term "alkynyl" refers to unsaturated aliphatic straight chain or branched chain hydrocarbon groups and contains one or more carbon-carbon triple bonds. Exemplary alkynyl groups include C2-C8, C2-C7, C2-C6, C2-C4, C3-C6, and C3-C5. Included, but not limited to, ethynyl, prop-1-ynyl, but-1-ynyl, pent-1-ynyl, pent-4-ynyl, and pent-1,4-diynyl. Alkynyl groups used in any context herein are optionally substituted in the same manner as alkyl groups. 12 and C3-C6alkynyl. Included, but not limited to, ethynyl, prop-1-ynyl, but-1-ynyl, pent-1-ynyl, pent-4-ynyl, and pent-1,4-diynyl. Alkynyl groups used in any context herein are optionally substituted in the same manner as alkyl groups.

[0158] The term "cycloalkyl" refers to saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituents, the cycloalkyl ring comprising 3 to 20 carbon atoms, preferably comprising 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like; polycyclic cycloalkyl groups include spiro, fused, and bridged cycloalkyl groups. Cycloalkyl groups can be substituted or unsubstituted, and when substituted, the substituents can be substituted at any available point of attachment, preferably one or more groups independently selected from halogen, hydroxyl, oxo, cyano, amino, C 1-6 alkyl, C 1-6 alkoxy, 3- to 6-membered cycloalkyl, or 3- to 6-membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl optionally substituted with halogen, hydroxyl, nitro, cyano, or amino.

[0159] The term "heterocyclyl" or "heterocycloalkyl" refers to saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituents comprising 3 to 20 ring atoms, one or more of which are heteroatoms selected from nitrogen, oxygen, or S(O) m (wherein m is an integer from 0 to 2) but excluding ring members -O-O-, -O-S-, or -S-S-, the remaining ring atoms being carbon. Preferably, 3 to 12 ring atoms are included, of which 1 to 4 are heteroatoms; more preferably, 3 to 7 ring atoms are included. Non-limiting examples of "heterocyclyl" include:

[0160] and the like.

[0161] The heterocycloalkyl ring can be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is heterocycloalkyl, non-limiting examples of which include:

[0162] and the like.

[0163] Heterocyclyl groups can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from halogen, hydroxyl, oxo, cyano, amino, C 1-6 alkyl, C 1-6Alkoxy, 3- to 6-membered cycloalkyl or 3- to 6-membered heterocyclic group, wherein the alkyl, alkoxy, cycloalkyl or heterocyclic group is optionally substituted with halogen, hydroxyl, nitro, cyano or amino.

[0164] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 12-membered, such as phenyl and naphthyl.

[0165] The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, and non-limiting examples include:

[0166] The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from halogen, hydroxyl, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyloxy group, C 2-6 Alkyne group, 3- to 6-membered cycloalkoxy group, 3- to 6-membered heterocycloalkoxy group, C 3-8 Cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl, wherein the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyloxy group, C 2-6 The alkynyl group, 3 to 6 cycloalkoxy group, 3 to 6 heterocycloalkoxy group, 3 to 8 cycloalkenyl group, 5 to 6 aryl group, or heteroaryl group may be selected from one or more halogens, hydroxyl groups, cyano groups, amino groups, C6 groups, etc. 1-6 Alkyl or C 1-6 Alkyl-substituted.

[0167] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. Heteroaryl groups are preferably 5 to 12-membered, more preferably 5- or 6-membered. For example, non-limiting examples include: imidazolyl, furanyl, thiophene, thiazolyl, pyrazolyl, oxazolyl, pyrroleyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, and pyrazine. wait.

[0168] The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include:

[0169] The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from halogens, hydroxyl groups, cyano groups, amino groups, C4 groups, etc. 1-6 Alkyl or C 1-6 Alkyl group.

[0170] The term "alkoxy" refers to an -O-(alkyl) group, wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propyloxy, butyloxy. The alkoxy group can be optionally substituted or unsubstituted, and when substituted, the substituent(s) are preferably one or more groups independently selected from halogen, hydroxy, oxo, cyano, amino, C 1-6 alkyl, C 1-6 alkoxy, 3- to 7-membered cycloalkyl, or 3- to 7-membered heterocyclyl, said alkyl, alkoxy, cycloalkyl, or heterocyclyl being optionally substituted with halogen, hydroxy, nitro, cyano, or amino.

[0171] The term "hydroxy" refers to an -OH group.

[0172] The term "oxo" refers to an =O substituent.

[0173] The term "cyano" refers to -CN.

[0174] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0175] The term "amino" refers to -NH2.

[0176] The term "protecting group" is used in its conventional chemical sense of a group that reversibly inactivates a functional group under the conditions of a desired reaction. Following the desired reaction, the protecting group can be removed to deprotect the protected functional group. All protecting groups should be removable without degrading a significant proportion of the synthetically produced molecule.

[0177] The present disclosure "hydroxy protecting group" is a group known in the art that can be used to protect a hydroxy group, see the hydroxy protecting groups in the text (Protective Groups in Organic Synthesis, 5 Th Ed. T. W. Greene & P. G. M. Wuts). By way of example, the hydroxy protecting group can be a (C 1-10 alkyl or aryl)3 silyl group, for example: triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and the like; can be a (C 1-10 alkyl or substituted alkyl group, for example: methyl, t-butyl, allyl, benzyl, methoxymethyl, ethoxymethyl, 2-tetrahydropyranyl (THP), and the like; can be a (C 1-10 alkyl or aryl)acyl group, for example: formyl, acetyl, benzoyl, and the like; can be a (C 1-6 alkyl or C 6-10 aryl) sulfonyl group; can also be a (C 1-6 alkoxy or C 6-10Aryloxy)carbonyl groups can be acetyl (Ac), 2-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether (MTM).

[0178] Some abbreviations in the present disclosure are defined as follows: THF: tetrahydrofuran; TFA: trifluoroacetic acid; DMF: dimethylformamide; EDCI: l-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; DMTrCl: 4,4'-dimethoxytrityl chloride; DMTr: dimethoxytrityl protecting group; MMTr: methoxyphenyldiphenylmethyl; Piv: pivaloyl; TBSCl: tert-butyldimethylsilyl chloride; py: pyridine; DMSO: dimethyl sulfoxide; NMI: N-methylimidazole; TBAF: tetra-n-butylammonium fluoride; NMM: N-methylmorpholine; DCI: 4,5-dicyanoimidazole. BRIEF DESCRIPTION OF DRAWINGS

[0179] Figure 1 is a graph showing the inhibition rate of mCTNNB1 mRNA in vivo in mice at day 21 for the TJR105087 and TJR105088 groups in Example 9, where * indicates p<0.05. DETAILED DESCRIPTION

[0180] The present disclosure is further described in conjunction with the following examples, which in no way are intended to limit the scope of the present disclosure. Unless otherwise defined, experimental methods in the Examples of the present disclosure were carried out in accordance with conventional methods or as recommended by the manufacturer of the materials or goods. Unless otherwise specified, reagents were obtained from any supplier of molecular biology reagents in a quality / purity suitable for use in molecular biology applications.

[0181] The structure of the compound was determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (LCMS). NMR shifts (δ) are given in 10 -6 (ppm) units. NMR was measured using a Bruker AVANCE-400 NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD) as the solvent, and tetramethylsilane (TMS) as the internal standard. The spatial configuration of the optical isomers (isomers) of the compound can be further confirmed by measuring single crystal parameters.

[0182] The determination of HPLC uses Waters ACQUITY ultra high performance LC, Shimadzu LC-20A systems, Shimadzu LC-2010HT series or Agilent 1200 LC high pressure liquid chromatograph (ACQUITY UPLC BEH C18 1.7UM 2.1*50MM column, Ultimate XB-C18 3.0*150mm column or Xtimate C18 2.1*30mm column).

[0183] The determination of MS uses Waters SQD2 mass spectrometer, scans in positive / negative ion mode, and the mass scan range is 100-1200.

[0184] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, and the specification of the silica gel plate used in thin layer chromatography is 0.15mm-0.2mm, and the specification of the thin layer chromatography separation and purification product is 0.4mm-0.5mm.

[0185] The flash column purification system uses Combiflash Rf150 (TELEDYNE ISCO) or Isolara one (Biotage).

[0186] The normal phase column chromatography generally uses Yantai Huanghai silica gel 100-200 mesh, 200-300 mesh or 300-400 mesh silica gel as carrier, or uses Changzhou Santai pre-packed ultra-pure normal phase silica gel column (40-63μm, 12g, 25g, 40g, 80g or other specifications).

[0187] The reverse phase column chromatography generally uses Changzhou Santai pre-packed ultra-pure C18 silica gel column (20-45μm, 40g, 80g, 120g, 220g or other specifications).

[0188] The high pressure column purification system uses Waters AutoP, which is used in combination with Waters XBridge BEH C18 OBD Prep Column, 5μm, 19mm X 150mm or Atlantis T3 OBD Prep Column, 5μm, 19mm X 150mm.

[0189] The known starting materials in the present disclosure can be synthesized or purchased from Shanghai Titan Science and Technology, ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Daejeon Chemicals, etc.

[0190] Unless otherwise specified in the examples, the reactions can be carried out under a nitrogen atmosphere.

[0191] An argon or nitrogen atmosphere means that a reaction flask is connected to an argon or nitrogen balloon with a volume of about 1 L.

[0192] A hydrogen atmosphere means that a reaction flask is connected to a hydrogen balloon with a volume of about 1 L.

[0193] A pressurized hydrogenation reaction uses a Parr 3916EKX hydrogenation instrument and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenation instrument.

[0194] A hydrogenation reaction is usually vacuumed and filled with hydrogen, and the operation is repeated 3 times.

[0195] Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20-30°C.

[0196] The reaction progress in the examples is monitored by thin layer chromatography (TLC), and the eluent system for column chromatography used for purifying compounds and the developing agent system for thin layer chromatography include: A: dichloromethane / methanol system, B: n-hexane / ethyl acetate system, C: petroleum ether / ethyl acetate system, D: petroleum ether / ethyl acetate / methanol, E: petroleum ether / tetrahydrofuran system, the volume ratio of the solvents is adjusted according to the polarity of the compound, and a small amount of triethylamine and acetic acid or other basic or acidic reagents can also be added for adjustment.

[0197] The forward column chromatography in the purification process in the examples can use a silica gel column, and the eluent system includes but is not limited to: A: dichloromethane / methanol system, B: petroleum ether / ethyl acetate system, the volume ratio of the solvents is adjusted according to the polarity of the compound, and a small amount of triethylamine or acetic acid or other basic or acidic reagents can also be added for adjustment.

[0198] The C18 reverse phase chromatography used in the purification process in the examples uses a C18 column, and the eluent system includes but is not limited to: acetonitrile / water system (adjust the proportion of the required solvents according to the polarity of the actual compound), and a small amount of ammonium formate or ammonia or formic acid or other basic or acidic reagents can also be added for adjustment.

[0199] Example 1, synthesis of compound 1

[0200] 1-1, Synthesis of compound 1-2

[0201] To a solution of compound 1-1 (10.00 g, 46.7 mmol) (CAS: 1489-97-0) in tetrahydrofuran (100 mL) was added LDA (33 mL, 65.3 mmol) slowly dropwise at -70 °C under nitrogen atmosphere. After stirring for 45 min, benzyl chloromethyl ether (18.30 g, 116.7 mmol) was added. Stirred at room temperature overnight. Quenched by slowly pouring into saturated aqueous ammonium chloride solution (200 mL) under ice bath, extracted with ethyl acetate twice, 100 mL each time, combined organic phase was washed with 50 mL saturated brine, dried over anhydrous sodium sulfate, concentrated, the crude product was purified by normal phase column chromatography to give the title compound 1-2 (10.00 g, yield 64%).

[0202] LCMS: m / z (ESI) = 335.3 [M+H] + .

[0203] 1-2, Synthesis of compound 1-3

[0204] To a solution of compound 1-2 (10.0 g, 29.9 mmol) in tetrahydrofuran (50 mL) was added 1 N aqueous HC1 solution (200 mL) at room temperature. Stirred at room temperature overnight. The reaction was extracted with ethyl acetate twice, 100 mL each time, combined organic phase was washed with 100 mL saturated aqueous sodium bicarbonate solution, dried over anhydrous sodium sulfate, concentrated, the crude product was purified by normal phase column chromatography to give the title compound 1-3 (7.00 g, yield 80%).

[0205] LCMS: m / z (ESI) = 291.4 [M+H] + .

[0206] 1-3, Synthesis of compound 1-4

[0207] To a solution of compound 1-3 (7.00 g, 24.1 mmol) in dichloromethane (70 mL) was added piperidine (3.08 g, 36.2 mmol) and sodium borohydride in acetic acid (10.27 g, 48.2 mmol) sequentially at room temperature. Stirred at room temperature overnight. The reaction was quenched by adding 100 mL water, the organic phase was dried over anhydrous sodium sulfate, concentrated, the crude product was purified by normal phase column chromatography to give the title compound 1-4 (4.10 g, yield 47%).

[0208] LCMS: m / z (ESI) = 360.4 [M+H] + .

[0209] 1-4, synthesis of compound 1-5

[0210] To a solution of compound 1-4 (2.80 g, 7.8 mmol) in dichloromethane (30 mL) was added boron trichloride (27 mL, 27.2 mmol) dropwise at -70 °C under nitrogen atmosphere. The reaction was stirred at 0 °C for 2 h. The reaction was quenched by slow addition of saturated aqueous sodium bicarbonate solution and extracted with dichloromethane and methanol mixture (v:v = 10:1) (4 x 100 mL). The organic phase was combined, washed with a small amount of saturated brine, dried over anhydrous sodium sulfate and concentrated to give the title compound 1-5 (1.00 g, yield 47%).

[0211] LCMS: m / z (ESI) = 270.3 [M+H] +

[0212] 1-5, synthesis of compound 1-6

[0213] To a solution of compound 1-5 (1.00 g, 3.7 mmol) in pyridine (10 mL) was added 4,4'-dimethoxytrityl chloride (2.52 g, 7.4 mmol) at room temperature under nitrogen atmosphere. It was stirred at room temperature overnight. The reaction was concentrated to give the crude product which was purified by normal phase column chromatography to give the title compound 1-6 (1.10 g, yield 52%).

[0214] LCMS: m / z (ESI) = 572.7 [M+H] + .

[0215] 1-6, synthesis of compound 1-7

[0216] To a solution of compound 1-6 (1.20 g, 2.1 mmol) in tetrahydrofuran (12 mL) was added 2.5 M lithium aluminum hydride in tetrahydrofuran (1.85 mL, 4.6 mmol) dropwise at 0 °C under nitrogen atmosphere. It was stirred at room temperature for 2 h. The reaction was quenched by addition of water and extracted with dichloromethane twice, 30 mL each time. The organic phase was combined, washed with a small amount of saturated brine, dried over anhydrous sodium sulfate and concentrated to give the crude product which was separated by normal phase column chromatography to give compound 1-7 and its isomer 1-7-BP. The compound 1-7 was obtained by concentration of the fractions of compound 1-7 (0.52 g, yield 47%).

[0217] LCMS: m / z (ESI) = 530.7 [M+H] + .

[0218] 1H NMR (400 MHz, CDC13) δ 7.45 (d, J = 7.2 Hz, 2H), 7.38 - 7.30 (m, 6H), 7.25 - 7.21 (m, 1H), 6.88 - 6.84 (m, 4H), 3.81 (s, 6H), 3.51 (d, J = 5.6 Hz, 2H), 3.18 (s, 2H), 2.66 (s, 1H), 2.45 (br, 4H), 2.25 (s, 1H), 1.84 - 1.63 (m, 8H), 1.47 (br, 2H), 1.17 - 1.11 (m, 2H), 1.03 - 0.97 (m, 2H).

[0219] The components of compound 1-7-BP obtained were concentrated to obtain compound 1-7-BP

[0220] Isomer 1-7-BP: 1 H NMR (400 MHz, CDC13) δ 7.45 (d, J = 7.2 Hz, 2H), 7.38 - 7.30 (m, 6H), 7.25 - 7.21 (m, 1H), 6.88 - 6.84 (m, 4H), 3.81 (s, 6H), 3.51 (d, J = 5.6 Hz, 2H), 3.18 (s, 2H), 2.66 (s, 1H), 2.45 (br, 4H), 2.25 (s, 1H), 1.84 - 1.63 (m, 8H), 1.47 (br, 2H), 1.17 - 1.11 (m, 2H), 1.03 - 0.97 (m, 2H).

[0221] 1-7, synthesis of compound 1

[0222] Under nitrogen atmosphere, bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.44 g, 1.5 mmol) was added to a solution of compound 1-7 (0.43 g, 0.8 mmol) in dichloromethane (5 mL) at room temperature, followed by the addition of a previously prepared mixture of tetrazole (0.45 M) and 1-methylimidazole (0.18 M) in acetonitrile (1.71 mL) to the above system. Stirring was carried out at room temperature for 2 hours. The reaction was quenched with 5 mL of saturated sodium bicarbonate solution and extracted twice with 10 mL of dichloromethane each time. The obtained organic phases were combined, washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate and concentrated to obtain compound 1 (0.58 g, yield 95%).

[0223] LCMS: m / z (ESI) = 730.9 [M+H] + .

[0224] 1H NMR (400 MHz, CD3CN) δ 7.48 (d, J = 7.2 Hz, 2H), 7.36 - 7.27 (m, 6H), 7.20 (t, J = 7.2 Hz, 1H), 6.87 - 6.83 (m, 4H), 3.81 - 3.77 (m, 2H), 3.75 (s, 6H), 3.67 - 3.60 (m, 2H), 3.51 - 3.45 (m, 2H), 3.05 (m, 2H), 2.75 (t, J = 6.0 Hz, 1H), 2.62 (t, J = 6.0 Hz, 2H), 2.20 (t, J = 4.4 Hz, 4H), 1.95 - 1.92 (m, 4H), 1.70 - 1.61 (m, 2H), 1.43 - 1.41 (m, 4H), 1.37 - 1.31 (m, 4H), 1.24 - 1.19 (m, 12H); 31 P NMR (162 MHz, CD3CN) δ 146.828.

[0225] Example 2, synthesis of compound 2

[0226] 2.1, synthesis of compound 2-4

[0227] Compound 1-3 (10 g, 34.44 mmol) was added to DCM (100 ml) at room temperature, then N-methylpiperazine (5.17 g, 51.66 mmol) and sodium borohydride (14.53 g, 68.88 mmol) were added, and the reaction was carried out at room temperature overnight. Saturated aqueous sodium bicarbonate solution was added to quench, the organic phase was collected, concentrated, and the residue was separated and purified by normal phase column chromatography to obtain the title compound 2-4 (11.3 g, yield 87.6%).

[0228] LCMS: m / z (ESI) = 375.6 [M+H] + .

[0229] 2.2, synthesis of compound 2-5

[0230] Compound 2-4 (11.3 g, 30.17 mmol) was added to DCM (110 ml) at room temperature, and under nitrogen atmosphere, the temperature was lowered to -60°C, BCl3 (105 ml, 105.6 mmol) was slowly added dropwise, and the reaction was carried out at about 0°C for 2 h. Saturated aqueous sodium bicarbonate solution was slowly added to quench, and DCM:MeOH = 10:1 (5 x 200 mL) was used for extraction, the organic phase was combined, washed with a small amount of saturated brine, dried, filtered, and concentrated under reduced pressure to obtain the title compound 2-5 crude (4 g, yield 46.62%).

[0231] LCMS: m / z (ESI) = 285.6 [M+H] + .

[0232] 2.3, Synthesis of compound 2-6

[0233] Compound 2-5 (4 g, 14.07 mmol) was added to pyridine (40 ml) at room temperature, under nitrogen atmosphere, molecular sieves were added, then DMTrCl (9.53 g, 28.13 mmol) was added, and the reaction was allowed to proceed at room temperature overnight. The pyridine was removed under reduced pressure, and the residue was purified by normal phase column chromatography to give the title compound 2-6 (3.6 g, yield 43.64%).

[0234] LCMS: m / z (ESI) = 587.9 [M+H] + .

[0235] 2.4, Synthesis of compound 2-7

[0236] Compound 2-6 (3.6 g, 6.14 mmol) was added to THF (40 ml) solution, under nitrogen atmosphere, the temperature was lowered to about 0°C, LAH (6.1 ml, 15.34 mmol) was added dropwise slowly, and the reaction was allowed to proceed at room temperature for 2 h. Water was added to quench the reaction, and DCM (2 x 100 mL) was added to extract the product, the organic phase was combined and concentrated, and the residue was purified by normal phase column chromatography to give the title compound 2-7 (790 mg, yield 23.64%).

[0237] LCMS: m / z (ESI) = 545.8 [M+H] + .

[0238] 2.5, Synthesis of compound 2

[0239] Bis(diisopropylamino)(2-cyanoethoxy)phosphine (518 mg, 1.72 mmol) was added to a solution of compound 2-7 (520 mg, 0.96 mmol) in dichloromethane (5 mL) at room temperature under nitrogen atmosphere, followed by the addition of a pre-prepared mixture of tetrazole (0.45 M) and 1-methylimidazole (0.18 M) in acetonitrile (2 mL). After the reaction was stirred at room temperature for 2 h, the reaction was quenched with saturated sodium bicarbonate solution (7 ml), and dichloromethane (2 x 10 mL) was added to extract the product, the organic phase was combined and washed with water (10 ml), saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated to give the title compound 2 (880 mg, used directly without purification).

[0240] LCMS: m / z (ESI) = 746.1 [M+H] +.

[0241] 31 P NMR (162 MHz, CDC13) δ 147.28 (s).

[0242] 1 H NMR (400 MHz, CDC13) δ 7.46 (d, J = 7.5 Hz, 2H), 7.34 (d, J = 8.8 Hz, 4H), 7.28 (d, J = 6.7 Hz, 1H), 7.25 (s, 1H), 7.19 (t, J = 7.2 Hz, 1H), 6.81 (d, J = 8.8 Hz, 4H), 4.28 - 4.08 (m, 2H), 3.84 - 3.73 (m, 8H), 3.64 (ddd, J = 10.0, 8.2, 5.2 Hz, 3H), 3.50 (tt, J = 6.8, 5.5 Hz, 3H), 3.06 (s, 2H), 2.75 (tt, J = 10.7, 5.4 Hz, 2H), 2.58 (t, J = 6.5 Hz, 2H), 2.44 (s, 4H), 2.30 (s, 3H), 1.73 (t, J = 11.5 Hz, 3H), 1.50 (s, 2H), 1.19 (dd, J = 9.9, 6.8 Hz, 12H), 0.84 (dd, J = 22.5, 9.8 Hz, 2H).

[0243] Example 3, Synthesis of the oligonucleotide of the disclosure

[0244] The synthesis of the oligonucleotide of the disclosure is no different from the general phosphoramidite solid-phase synthesis method, and the synthesis process is briefly described as follows.

[0245] On the Dr. Oligo 48 synthesizer (Biolytic), starting with a universal CPG carrier, unmodified nucleoside phosphoramidite monomers or nucleoside phosphoramidite monomers with modifications at the corresponding positions in the sequence were sequentially connected according to the synthesis procedure, wherein the phosphoramidite monomer compound 1 prepared in Example 1 was used at the corresponding position of NA0255, and the phosphoramidite monomer compound 2 prepared in Example 2 was used at the corresponding position of NA0282.

[0246] The nucleoside phosphoramidite monomers used in the examples of the disclosure were purchased from Shanghai Zhiwei Company and Suzhou Jimar Company (which need to be adjusted according to the actual supplier). 5-ethylthio-1H-tetrazole (ETT) was used as the activator (0.6M acetonitrile solution), 0.22M PADS dissolved in 1:1 volume ratio of acetonitrile and 3- pyridine (Shanghai Lingjiang) solution was used as the sulfuration reagent, and iodonium / water solution (Shanghai Lingjiang) was used as the oxidizing agent.

[0247] The double-stranded oligonucleotide with NA0052' attached was prepared according to the same method as described in patent application WO2023138663A. The double-stranded oligonucleotide with L96 attached was prepared according to the same method as described in patent application WO2014025805A. The double-stranded oligonucleotide with NA0308 was prepared according to the same method as described in patent application WO2024002006A.

[0248] After the solid phase synthesis was completed, the oligoribonucleotide was cleaved from the solid support by soaking in 28% aqueous ammonia and ethanol solution at 50°C for 16 hours at a ratio of 3:1. Then centrifugation was performed, and the supernatant was transferred to another centrifuge tube. After concentrated evaporation, C18 reverse phase chromatography was used for purification with 0.1M TEAA and acetonitrile as the mobile phase. The target oligonucleotide was collected, freeze-dried, and identified as the target product by LC-MS, and then quantified by UV (260 nm). The obtained single-stranded oligonucleotide was annealed according to the complementary pairing at an equimolar ratio, and the final double-stranded oligonucleotide was dissolved in 1xPBS and adjusted to the required concentration for the experiment. The double-stranded oligonucleotide prepared in this example has the sequence shown in Table 2 below.

[0249] Table 2, oligonucleotide sequences of the present disclosure

[0250] In Table 2, in the same nucleic acid sequence, G, C, A, U represent nucleosides with guanine, cytosine, adenine, and uracil, respectively, from left to right according to the direction from the 5' end to the 3' end, the sequence from left to right represents the direction from the 5' end to the 3' end, the lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside; the lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro-modified nucleoside; the lowercase letter d indicates that the nucleoside adjacent to the right of the letter d is a 2'-deoxy nucleoside; the capital letter VP indicates that the 5' position of the sugar ring of the nucleoside adjacent to the right of the letter is an E-vinyl phosphate group modification; the lowercase letter s indicates that the two nucleosides adjacent to the letter s are connected by a phosphorothioate diester group, and no special instructions, two adjacent nucleosides, bicyclic structures, or delivery groups are connected by a phosphodiester group. NA0255', NA0255", NA0308', and NA0308" respectively indicate that the position has the structure shown in Table 3 below. NAG0052' indicates that the position has the delivery group shown in NAG0052'. L96 indicates that the position has the delivery group shown in L96. IB indicates an inverted abasic modified nucleoside. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each strand is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each strand is a hydroxyl group.

[0251] The structures of the 2'-methoxy modified nucleoside, 2'-fluoro modified nucleoside, phosphorothioate linkage, phosphodiester linkage, inverted abasic modified nucleoside, NA0255', NA0255", NA0308', NA0308", and NAG0052' are shown in Table 3 below. When the oligonucleotide of the present disclosure is in a salt form, for example, in a sodium salt form, the structure of the corresponding salt form of the structure in Table 3 is also within the protection scope of the present disclosure.

[0252] Table 3

[0253] Example 4, INHBE gene psiCHECK verification at target level

[0254] The oligonucleotide of the present disclosure was screened in vitro at the molecular level in HEK293A cells using 9 concentration gradients to simulate the activity at the target.

[0255] The psi-CHECK plasmid used in this example was purchased from Shenguo Bioengineering (Shanghai) Co., Ltd. The in-target sequence corresponding to the oligonucleotide was constructed using the INHBE gene and inserted into the psiCHECK-2 plasmid, i.e., the GSCM in-target plasmid. The plasmid contains a Renilla luciferase gene and a firefly luciferase gene. As a dual reporter gene system, the target sequence of the oligonucleotide is inserted into the 3'UTR region of the Renilla luciferase gene, and the activity of the oligonucleotide on the target sequence can be reflected by the detection of the Renilla luciferase expression calibrated by the firefly luciferase. The detection uses the Dual-Luciferase Reporter Assay System (Promega, E2940).

[0256] HEK293A cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C, 5% CO2. 24h before transfection, HEK293A cells were seeded in a 96-well plate at a seeding density of 1x10 4 cells per well, 100μL of culture medium per well.

[0257] According to the instructions, co-transfect the RNAi agent and corresponding plasmid into cells by Lipofectamine 2000 (ThermoFisher, 11668019), 0.2 μL of Lipofectamine 2000 per well, and 20 ng of plasmid per well. A total of 9 concentration points are set, the highest concentration point is 20 nM, and the 4-fold gradient dilution is 20 nM, 5 nM, 1.25 nM, 0.3125 nM, 0.0781 nM, 0.0195 nM, 0.0049 nM, 0.0012 nM, and 0.0003 nM. 24 hours after transfection, the Dual-Luciferase Reporter Assay System (Promega, E2940) is used to detect the target level.

[0258] The experimental materials and instruments used in this example are shown in Table 4 and Table 5. The dilution scheme used in this example is shown in Table 6. The experimental results are shown in Table 7, and the results in Table 7 show that the oligonucleotide containing the structure represented by formula (I') of the present disclosure has good INHBE inhibitory activity.

[0259] Table 4, psi-CHECK experimental consumables and reagents

[0260] Table 5, psi-CHECK experimental instruments

[0261] Table 6, sample multi-concentration dilution scheme

[0262] Table 7, INHBE target psi-CHECK in-target activity screening results

[0263] Example 5, mouse liver S9 metabolic stability study of oligonucleotides of the present disclosure

[0264] The C57BL / 6 mouse liver S9 was used to evaluate the in vitro metabolic stability of the RNAi agent of the present disclosure.

[0265] Add 10 μL C57BL / 6 mouse liver S9 (20 mg / mL) to 90 μL of metabolic buffer (50 mM sodium phosphate, pH 6.0, containing 1 mM magnesium chloride, 1x antibiotic-antimycotic), add the RNAi agent of the present disclosure (final concentration of 1 μM), and incubate at 37 °C for 0 h and 24 h, respectively. After incubation, add 500 μL lysis solution [5 mM Tris-HCl (1 mM EDTA, 1 mM NaCl, 1.1% SDS, 0.2% Tween 20)] to terminate the reaction, add internal standard (IS, internally synthesized 18-mer single-stranded oligonucleotide) and ammonia water, perform liquid-liquid extraction using phenol / chloroform / isoamyl alcohol (25 / 24 / 1), centrifuge at 4400 rpm for 15 min at 4 °C, and then transfer the supernatant to perform HLB solid-phase extraction, collect the eluate, and perform LC-MS detection analysis. The residual amounts of AS and SS strands are calculated using the following formula, respectively:

[0266] The main experimental materials and reagents used in this example are shown in Table 8, and the main instruments are shown in Table 9. The experimental results of this experiment show that the oligonucleotide containing the structure represented by formula (I’) of the present disclosure has excellent liver metabolic stability.

[0267] Table 8, Main Experimental Materials and Reagents for Liver S9 Metabolic Stability Experiment

[0268] Table 9, Main Experimental Instruments for Liver S9 Metabolic Stability Experiment

[0269] Example 6, Multiple Concentration Point Inhibition Activity of the Oligonucleotide of the Present Disclosure on Human CTNNB1 Gene in Hep3B Cells

[0270] The in vitro molecular level activity of the oligonucleotide TJR105086 of the present disclosure was tested in Hep3B cells (ATCC, HB-8064) using multiple concentration gradients (1 nM, 0.2 nM, 0.04 nM, 0.008 nM, 0.0016 nM, 0.00032 nM, 0.000064 nM).

[0271] Hep3B cells were cultured in DMEM medium containing 10% fetal bovine serum and cultured at 37 °C, 5% CO2.

[0272] According to the product manual, Lipofectamine RNAi MAX (ThermoFisher, 13778150) was used to transfect RNAi, and the gradient final concentration of RNAi transfection was 1 nM, 0.2 nM, 0.04 nM, 0.008 nM, 0.0016 nM, 0.00032 nM, 0.000064 nM, and each concentration was double-replicated. 2 x 104 Hep3B cells were reverse transfected with an RNAi mixture. Twenty-four hours later, total RNA was extracted using a cell RNA magnetic bead extraction kit (Zhiang Bio, MNTR / FX96). RNA reverse transcription assay (Takara, RR037A) and quantitative real-time PCR (Thermo, 4444557) were performed to determine the mRNA level of human CTNNB1. The mRNA level of human CTNNB1 was corrected based on the level of the GAPDH internal reference gene. After the Q-PCR assay, the corresponding Ct value was obtained according to the automatically set threshold. The expression of a gene can be relatively quantified by comparing Ct values. Comparing Ct values ​​refers to calculating the difference in gene expression based on the difference between the Ct value and the internal reference gene value; this is also known as 2-1. -△△Ct △△Ct=[(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)]. 2 -△△Ct This represents the residual expression rate relative to the control group. The half-maximal inhibitory concentration (IC50) is calculated using this value. 50 Fitting and calculation of ).

[0273] The results of this embodiment show that oligonucleotides containing the structure shown in formula (I') of this disclosure have good CTNNB1 inhibitory activity.

[0274] Example 7: Detection of the exogenous knockdown activity of the oligonucleotides disclosed herein in HEK293A cells ANGPTL3

[0275] The RNAi agent TJR104780 disclosed herein was screened for in vitro molecular-level target activity at nine concentration gradients (20 nM, 5 nM, 1.25 nM, 0.3125 nM, 0.0781 nM, 0.0195 nM, 0.0049 nM, 0.0012 nM, and 0.0003 nM) in HEK293A cells.

[0276] The target sequence corresponding to the oligonucleotide was constructed using the ANGPTL3 gene and inserted into the psiCHECK-2 plasmid. This plasmid contains the Renida luciferase gene and the firefly luciferase gene. As a dual reporter gene system, the target sequence of the RNAi agent is inserted into the 3'UTR region of the Renida luciferase gene. The activity of the RNAi agent against the target sequence can be reflected by detecting the expression of Renida luciferase after calibration with firefly luciferase using the Dual-Luciferase Reporter Assay System (Promega, E2940). The psi-CHECK plasmid was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0277] HEK293A cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO2. 16h before transfection, HEK293A cells were seeded in 96-well plates at a density of 1x10 4 cells per well in 100μL medium.

[0278] According to the instructions, the RNAi agent and the corresponding plasmid were co-transfected into the cells by Lipofectamine 2000 (ThermoFisher, 11668019), 0.2μL of Lipofectamine 2000 per well and 20ng of plasmid per well. 24h after transfection, the target level was detected using the Dual-Luciferase Reporter Assay System (Promega, E2940). The half-inhibition rate (IC 50 ) was fitted and calculated by this value.

[0279] The experimental materials and instruments were the same as those in Table 4 and Table 5. The results of this example showed that the RNAi agent containing the structure represented by formula (I’) of the present disclosure had good ANGPTL3 inhibitory activity.

[0280] Example 8, in vivo knockdown verification of the RNAi agent of the present disclosure in mice

[0281] 8-9 week old male Hu-INHBE mice (Saiye (Suzhou) Biotechnology Co., Ltd.) were adaptively fed and divided into an experimental group and a control group, 3 mice in each group. The mice in the experimental group were subcutaneously injected with a test compound at 3mg / kg, and the mice in the control group were given the same volume of normal saline. The mice were euthanized on day 7 (D7) after administration, and the liver tissues were collected in EP tubes containing 10 volumes of solution, stored in a 4°C refrigerator, and then transferred to -80°C. The hINHBE mRNA level in the mouse liver tissue was detected by RT-PCR.

[0282] The grouping information of the in vivo experiment of this example is shown in Table 10, and the primer sequences used in the RT-PCR method are shown in Table 11. The results are shown in Table 12, and the results of Table 12 show that the oligonucleotide TJR103218 containing the structure represented by formula (I’) of the present disclosure has more excellent INHBE inhibitory activity than the positive molecule TJR105327 in mice, and can more effectively inhibit the expression of the INHBE gene in mice.

[0283] Table 10, grouping information of hINHBE in vivo experiment

[0284] Table 11, the sequences of the detection primers are as follows ​

[0285] Table 12, hINHBE mRNA levels in hINHBE rat liver tissues of some compounds (normalized to vehicle group)

[0286] Example 9, in vivo knockdown verification of oligonucleotides of the present disclosure in mice

[0287] 6-8 week old male C57BL / 6J mice (Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were adaptively fed and divided into experimental and control groups, 3 mice per group at each time point. The experimental group of mice was subcutaneously injected with 1 mg / kg of the test compound, and the control group was given the same volume of normal saline. On day 14 (D14) and day 21 (D21) after administration, the mice were euthanized to collect liver tissues, which were placed in EP tubes containing 10 times the volume of RNAiso Plus solution, and stored in a 4°C refrigerator before being transferred to -80°C. The mCTNNB1 mRNA level in the mouse liver tissues was detected by RT-PCR.

[0288] The results of this example are shown in Table 15 and Figure 1. The results of Table 15 and Figure 1 show that the RNAi agent TJR105087 containing the structure shown in Formula (I’) of the present disclosure has more superior CTNNB1 inhibitory activity in mice than the positive molecule TJR105088. The inhibition rate of mCTNNB1 mRNA of the RNAi agent TJR105087 of the present disclosure on day 21 is still as high as 53.78%, and the RNAi agent of the present application can significantly more effectively inhibit the expression of the CTNNB1 gene in mice than the positive molecule.

[0289] Table 13, grouping information of mCTNNB1 in vivo experiment

[0290] Table 14, the sequences of the detection primers are as follows

[0291] Table 15, mCTNNB1 mRNA levels in liver tissues of C57BL / 6J mice

[0292] Example 10, inhibitory activity of oligonucleotides of the present disclosure on ANGPTL3, PCSK9, Lp(a), APOC3, AGT, PD-L1, and HBV genes in mice

[0293] According to a method similar to Example 9, the inhibitory activity of the oligonucleotides of the present disclosure targeting ANGPTL3, PCSK9, Lp(a), APOC3, AGT, PD-L1, and HBV on ANGPTL3, PCSK9, Lp(a), APOC3, AGT, PD-L1, and HBV genes in mice was tested, respectively.​

Claims

1. An oligonucleotide comprising at least one structure as shown in formula (I'): in, X1 and X2 are each independently covalently bonded, O or C1-C3 alkylene; X3 is either N or CH; R1 and R2 are each independently selected from C1-C6 alkylene groups or covalently bonded, wherein the C1-C6 alkylene group is optionally bonded by one or more R1-C6 alkylene groups. a The R that was replaced a Each is independently selected from oxo, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, or halogen; R3 is selected from 3- to 12-membered cycloalkyl, 3- to 12-membered heterocyclic, 5- to 12-membered aryl, or 5- to 12-membered heteroaryl, wherein the 3- to 12-membered cycloalkyl, 3- to 12-membered heterocyclic, 5- to 12-membered aryl, or 5- to 12-membered heteroaryl is optionally surrounded by one or more R3 groups. b The R that was replaced b Each is independently selected from oxo, C1-C6 alkyl, halogen, hydroxyl, cyano, carbonyl, and hydroxyl groups; Optionally, one or more hydrogen atoms in the structure shown in formula (I') are replaced by deuterium atoms.

2. The oligonucleotide according to claim 1, wherein the structure represented by formula (I') has the structure represented by formula (I): In the formula, a represents the direction toward the 3' end of the oligonucleotide sequence, and b represents the direction toward the 5' end of the oligonucleotide sequence.

3. The oligonucleotide according to claim 1 or 2, wherein R1 and R2 are each independently selected from C1-C3 alkylene groups or covalent groups, and the C1-C3 alkylene group is optionally separated by one or more R1-C2 groups. a The R that was replaced a Each is independently selected from oxo, C1-C3 alkyl, C1-C3 alkoxy, hydroxyl, or halogen.

4. The oligonucleotide according to claim 1 or 2, wherein R3 is selected from 5- to 12-membered cycloalkyl, 5- to 12-membered heterocyclic, 5- to 12-membered aryl, or 5- to 12-membered heteroaryl, wherein the 5- to 12-membered cycloalkyl, 5- to 12-membered heterocyclic, 5- to 12-membered aryl, or 5- to 12-membered heteroaryl is optionally surrounded by one or more R3 groups. b The R that was replaced b Each is independently selected from oxo, C1-C3 alkyl, halogen, hydroxyl, cyano, and hydroxyl groups; Preferably, R3 is selected from 5- to 8-membered cycloalkyl or 5- to 8-membered heterocyclic groups; the 5- to 8-membered heterocyclic group has 1, 2 or 3 heteroatoms.

5. The oligonucleotide according to any one of claims 1 to 4, wherein the structure shown in formula (I) has the structure shown in formula (I-1): in, X1 and X2 are each independently methylene or covalently bonded; a indicates the direction toward the 3' end of the oligonucleotide sequence, and b indicates the direction toward the 5' end of the oligonucleotide sequence.

6. The oligonucleotide according to claim 5, wherein the structure shown in formula (I-1) has the structure shown in any one of formulas (I-2) to (I-5): in, a indicates the direction toward the 3' end of the oligonucleotide sequence, and b indicates the direction toward the 5' end of the oligonucleotide sequence; Preferably, the structure shown in formula (I-1) has the structure shown in any one of formulas (I-6) to (I-10): Where a represents the direction toward the 3' end of the oligonucleotide sequence, and b represents the direction toward the 5' end of the oligonucleotide sequence; More preferably, the structure shown in formula (I-1) is selected from: Where a represents the direction toward the 3' end of the oligonucleotide sequence, and b represents the direction toward the 5' end of the oligonucleotide sequence.

7. The oligonucleotide according to any one of claims 1 to 6, wherein, The oligonucleotide is a single-stranded or double-stranded oligonucleotide; Preferably, the oligonucleotide is an ASO or RNAi agent.

8. The oligonucleotide according to any one of claims 1 to 7, wherein at least one additional nucleoside is a modified nucleoside; Preferably, all nucleosides in the oligonucleotide are modified nucleosides.

9. The oligonucleotide according to any one of claims 1 to 8, wherein the structure represented by formula (I') is linked to the adjacent nucleoside via a phosphodiester group or a phosphodiester group having a modifying group.

10. The oligonucleotide according to any one of claims 1 to 9, wherein at least one phosphodiester group in the oligonucleotide is a phosphodiester group having a modifying group; Preferably, the phosphate diester group with the modifying group is a thiophosphate diester group.

11. A compound as shown in formula (II'): in, W represents a hydroxyl group or -OR p R p Q is a hydroxyl protecting group, and Q is a phosphorus-containing reactive group. Preferably, the R p Selected from methoxyphenyl diphenylmethyl, dimethoxytriphenylmethyl, or triphenylmethyl; R1, R2, X1, X2, X3 and R3 as defined in any one of claims 1 to 6; Optionally, one or more hydrogen atoms in the structure shown in formula (II') are replaced by deuterium atoms.

12. The compound according to claim 11, wherein, The compound represented by formula (II') is selected from the compounds represented by formula (II): Wherein, W, Q, R1, R2, X1, X2, X3 and R3 are as defined in claim 11.

13. The compound according to claim 12, wherein, The compound represented by formula (II) is selected from: Preferably, the compound represented by formula (II) is selected from:

14. A method for preparing an oligonucleotide according to any one of claims 1 to 10, comprising the following steps: 1) To synthesize the compound represented by formula (II') according to any one of claims 11 to 12; 2) Synthesize using the compound represented by formula (II') obtained in step 1). The oligonucleotide according to any one of claims 1 to 10.

15. A pharmaceutical composition comprising an oligonucleotide according to any one of claims 1 to 10; preferably, the pharmaceutical composition further comprising one or more pharmaceutically acceptable excipients.

16. Use of an oligonucleotide according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 15 in the preparation of a medicament.

17. A method for inhibiting the expression of a target gene or its mRNA, comprising administering to a subject an effective amount or effective dose of an oligonucleotide according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 15.

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