GalNAc derivatives

Abstract

To provide a construct comprising an oligonucleotide strand having a GalNAc moiety.SOLUTION: The present invention provides a construct, comprising at least one oligonucleotide strand, having at least one GalNAc moiety, conjugated either directly or via a linker, wherein each GalNAc moiety, bound to the oligonucleotide strand, is independently selected from GalNAc1 to GalNAc13. In one embodiment, the GalNAc moiety is GalNAc-2.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is incorporated herein by reference in its entirety. U.S. Patent Application Claiming Benefit of Priority to U.S. Provisional Patent Application No. 62 / 558,773 is. [Background technology]

[0002] Antisense oligonucleotide therapy is a promising treatment for viral, neurological, and neurodegenerative diseases. It has been investigated for the treatment or prevention of various diseases and conditions, including fibrotic and hyperproliferative diseases. .

[0003] Certain viral diseases, such as Hepatitis B virus (HBV), remain intractable with conventional treatments. It is difficult to treat and an estimated 240 million people are infected (HBV surface antigen positive for at least 6 months). The virus continues to cause over 686,000 deaths each year. and other antiviral drugs, including treatment with oral antiviral nucleotide analogs such as entecavir or entecavir. Conventional treatment only suppresses viral replication and does not cure HBV infection. Even if treated with current HBV therapy, treatment must be continued for life. do not have.

[0004] Oligonucleotides are capable of binding to complementary RNA or DNA sequences. Intracellular processes such as metabolism, differentiation, proliferation, and replication, as well as viral replication, e.g., HBV The oligonucleotides are bound to specific nucleic acid targets that are involved in specific aspects of replication of V. This makes it possible to

[0005] N-acetylgalactosamine (GalNAc) binds oligonucleotides to their intended target. This helps guide the oligonucleotide to its target cell and improves its uptake into the cell. To achieve this, the oligonucleotide is conjugated to the oligonucleotide either directly or via a linker. Summary of the Invention [Problem to be solved by the invention]

[0006] The art is advancing the discovery and development of novel therapies with improved specificity and efficiency in their administration. There is a need to develop and [Means for solving the problem]

[0007] The present disclosure provides GalNAc moieties, methods for making them, and oligonucleotides to which they are conjugated. Methods for use in targeting nucleotides to cells such as hepatocytes - Patent Application 20070122997 .

[0008] The present disclosure provides a construct comprising at least one oligonucleotide strand, at least one GalNAc moiety attached to said chain, each GalNAc moiety comprising: Independently, the constructs are selected from GalNAc1 to GalNAc13. In some embodiments, the GalNAc moiety is GalNAc-2. In embodiments, the GalNAc moiety is GalNAc-6. At least one GalNAc moiety is attached to the oligonucleotide chain via a linker. In some embodiments, the linker is conjugated to at least one C6 In some embodiments, the oligonucleotide sequence is an HBV It has affinity for or is substantially complementary to the RNA transcript. In some embodiments, the construct comprises a single-stranded oligonucleotide. In some embodiments, the construct comprises a double-stranded oligonucleotide. GalNAc moieties are chemically attached to the 3' and 5' ends of the oligonucleotide chain. is doing.

[0009] The present disclosure also relates to compounds having the structure of formula (I) or (II):

[0010] [ka] [Wherein R1 is

[0011] [ka] and R8 is C3 to C 10 Alkyl moiety, C3-C1 with 1-5 oxygen atoms 0 alkyl oxide moiety, or

[0012] [ka] and R2 is C3 to C 10 C3-C with alkyl moiety or 1-5 oxygen atoms 10 is an alkyloxide moiety, and R3 is H, PG',

[0013] [ka] where PG' is a protecting group, R4 is H, and R5 is

[0014] [ka] wherein R6 is a protecting group or an optional linker and / or phosphoramidator. R7 is attached to the oligonucleotide via a phosphate or phosphodiester bond; H, a solid support linker (e.g., succinate), or -PH(O)CHCHCN or R4 and R5 are 5 or 6 optionally substituted by CH2OPG. together form a six-membered ring, PG is an alcohol protecting group, and L is a linker moiety. , a is an integer from 1 to 3, and b is an integer from 1 to 4. In some embodiments, R2 is a C3, C4, C5, C6, C7, C8, C9, or C10 alkyl. In some embodiments, R8 is a C3-C10 alkyl group containing 2-5 ethylene oxide moieties. In some embodiments, the linker is a C6-NH2 linker. In some embodiments, a is 1. In some embodiments, a is 3 In some embodiments, b is 2 or 3. In some embodiments, P G is DMTr. In some embodiments, the compound is a compound of Formula (I). In some embodiments, the compound is a compound of formula (II). , R8 is .

[0015] [ka] In some embodiments, PG is tert-butyldimethylsilyl ether (TBM DS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl Trityl ether (TIPS), monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl In some embodiments, R6 is selected from dimethylaminobenzoate (DMTr), tritolyl, or tritolyl. Linker and / or oligonucleotides via phosphoramidate or phosphodiester bonds In some embodiments, R6 is a bond to a phosphoramidate bond. In some embodiments, R6 comprises a phosphodiester bond. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present disclosure provides one or more GalNAc moieties conjugated to an oligonucleotide. The present disclosure further provides a method for the preparation of a GalNAc moiety and its attachment to an oligonucleotide. The present invention relates to methods of using and preparing jugation.

[0017] Some embodiments of the GalNAc moiety include compounds of formula (I) or (II):

[0018] [ka] [In the formula, R1 is

[0019] [ka] and R8 is C2~C 10 Alkyl moiety, C3-C with 1-5 oxygen atoms 10 Archi peroxide moiety,

[0020] [ka] and R2 is C2~C 10 C3-C with alkyl moiety or 1-5 oxygen atoms 10 Al is an alkyl oxide moiety, R3 is H, PG',

[0021] [ka] and R4 is H, R5 is

[0022] [ka] (wherein: R6 is a protecting group or an optional linker and / or phosphoramidate or phosphonate. Attachment to the oligonucleotide via a phosphodiester bond, R7 is H, a solid support linker (e.g., succinate), or -PH(O)CH2 CHCN), R9 is C2~C 10 C3-C with alkyl moiety or 1-5 oxygen atoms 10 Al is an alkyl oxide moiety, R 10 is C2~C 10 C3-C with alkyl moiety or 1-5 oxygen atoms 10 a is an alkyl oxide moiety, Alternatively, R4 and R5 are a 5- or 6-membered ring optionally substituted with CH2OPG. together to form PG is an alcohol protecting group, PG' is a protecting group, L is a linker moiety, a is an integer from 1 to 3, b is an integer from 1 to 4, c is an integer from 3 to 7; and d is an integer from 0 to 4.

[0023] In some embodiments, R, R, and / or R 10 are C2, C3, C4, and C5 , C6, C7, C8, C9, or C 10 In some embodiments, C3 ~C10 The alkyl oxide moiety contains 2 to 5 ethylene oxide moieties. For example, C 3~C 10 The alkyl oxide moiety is a repeating moiety of:

[0024] [ka] and optionally containing additional alkylene moieties (wherein e is 1 to 5). In some embodiments, e is 1, 2, 3, 4, or 5. In some embodiments, R 8 is C2, C3, C4, C5, C6, C7, C8, C9, or C 10 It is alkyl. In some embodiments, C3 to C 10 The alkyloxide portion is composed of 2 to 5 ethylene oxide units. Contains oxide moieties, e.g., C3-C 10 The alkyl oxide moiety consists of the following repeating moiety: :

[0025] [ka] and optionally containing additional alkylene moieties (wherein e is 1 to 5). In some embodiments, a is 1. In some embodiments, a is 3. In some embodiments, b is 2 or 3. In some embodiments, e is 1, 2, 3 , 4, or 5.

[0026] In some embodiments, R8 is

[0027] [ka] wherein the alkylene chain contains the following number of carbon atoms (c, d): (3, 0 ), (3, 1), (3, 2), (3, 3), (3, 4), (4, 0), (4, 1), (4 ,2),(4,3),(4,4),(5,0),(5,1),(5,2),(5,3), (5, 4), (6, 0), (6, 1), (6, 2), (6, 3), (6, 4), (7, 0 ), (7, 1), (7, 2), (7, 3), or (7, 4).

[0028] In some embodiments, PG is an alcohol protecting group, for example, a silyl protecting group (e.g., , tert-butyldimethylsilyl ether (TBMDS), tert-butyldiphenyl triisopropylsilyl ether (TBDPS), triisopropylsilyl ether (TIPS), or monomethylsilyl 4,4'-dimethoxytrityl (MMTr), or 4,4'-dimethoxytrityl (DMTr), or trimethylsilyl Litholyl, or Wuts, Peter G.M., and Theodora W. Gree ne.Greene's protective groups in organic synthesis. John Wiley & Sons, 2006 It may contain any other suitable protecting group.

[0029] In some embodiments, PG' is a protecting group, e.g., in some instances, PG' teeth,

[0030] [ka] where f is an integer from 1 to 5. In some embodiments, f is 1, 2, 3, 4, or 5.

[0031] In some embodiments, R3 is

[0032] [ka] The oligo is not particularly limited, and may be an antisense oligonucleotide or an siRNA. The oligonucleotide may be any oligonucleotide, such as an oligonucleotide. In the formula, the optional linker is a C1-C8-NH2 linker, for example, a C6-NH2 linker is.

[0033] In some embodiments, R6 is a linker and / or a phosphoramidate or phosphine In some embodiments, the attachment to the oligonucleotide is via a phosphodiester bond. In some embodiments, R6 comprises a phosphoramidate linkage. Contains a diester bond.

[0034] In some embodiments, the GalNAc moiety is one of the following, as understood: The squiggled lines represent couplings with nucleotides or oligonucleotides. represents the final cleavage point after the final GalNAc It is not present in the substitution oligonucleotide, as shown in the examples.

[0035] [ka]

[0036] [ka]

[0037] [ka]

[0038] [ka]

[0039] In some embodiments, the GalNAc moiety is a succinate moiety or a solid support phosphorus. In some embodiments, the GalNAc moiety comprises a fluorophenyl ester. (e.g., pentafluorophenyl ester) and the 3' or 5' end of the oligonucleotide The termini may be chemically linked. In some embodiments, the GalNAc moiety , H-phosphonate, and the 3' or 5' end of the oligonucleotide is chemically bonded Further details of these moieties can be found in the Examples.

[0040] Conjugated GalNAc In embodiments, the oligonucleotide may include an aminoalkyl linker (e.g., C6-NH 2) is linked to the targeting moiety via a linker such as GalNAc1 to 13 or formula (I) or (II) can be linked to an oligonucleotide via such a linker For example, in embodiments, the 3' end of the oligonucleotide may be linked to the following construct: As shown in the structure, GalNAc such as GalNAc1-13 or formula (I) or The C6-amino linker further connected to (II) is a phosphoramidate or phosphodiamine. The bond is via an ester bond.

[0041] [ka]

[0042] Oligonucleotides containing GalNAc moieties of the present disclosure are incorporated herein by reference in their entirety. Modifications or modifications such as those described in WO 2018 / 053185, which is incorporated herein by reference. may comprise an unmodified oligonucleotide sequence.

[0043] Exemplary oligonucleotides containing a GalNAc moiety of the present disclosure include those listed in the Examples. Examples include:

[0044] In aspects of the present disclosure, the oligonucleotide sequences described herein may optionally include a linker. The GalNAc moiety of the present disclosure is linked via a carboxyl group at one or both ends. In some embodiments, the oligonucleotide strand is optionally conjugated or modified. GalN of the present disclosure conjugated at the 5' and / or 3' end via a linker In some embodiments, the GalNAc moiety of the present disclosure comprises an oligonucleotide. The linking moiety of the present disclosure is a HEG linker or It may also include a C6 amino linker.

[0045] In some embodiments, the GalNAc moiety is cleaved by specific cell types, such as hepatocytes. The activity, intracellular distribution, or cellular uptake of the oligonucleotide is enhanced.

[0046] In certain embodiments of the compositions and methods of the present invention, the ligand comprises one or more GalNAc derivatives, e.g., oligonucleotides, can be linked via bivalent or trivalent branched linkers, respectively. Two or three GalNAc derivatives are attached to the leutide.

[0047] composition The present disclosure also includes pharmaceutical compositions comprising the GalNAc-substituted oligonucleotides of the present disclosure. One embodiment is a method for preparing a pharmaceutical composition comprising an oligonucleotide of the present disclosure and a pharmaceutically acceptable diluent or carrier. and a pharmaceutical composition comprising:

[0048] In some embodiments, pharmaceutical compositions containing GalNAc-substituted oligonucleotides of the present disclosure are The pharmaceutical composition is formulated for systemic administration by parenteral delivery. Parenteral administration can be by intravenous, intraarterial, or Intravenous, subcutaneous, intraperitoneal, or intramuscular injection or infusion, and also includes, for example, implantable devices. In a preferred embodiment, the oligonucleotides of the present disclosure are administered subcutaneously via Pharmaceutical compositions containing the compound may be formulated for subcutaneous (SC) or intravenous (IV) delivery. Formulations for oral administration may include sterile aqueous solutions, which may contain buffers, diluents, and Other pharmaceutically acceptable additives may be included as will be understood by those skilled in the art. In addition, the total concentration of solutes can be controlled to render the formulation isotonic.

[0049] Pharmaceutical compositions containing GalNAc-substituted oligonucleotides of the present disclosure can be prepared, for example, by administering to a subject in need thereof a pharmaceutical composition comprising HB They are useful for treating diseases or disorders associated with V gene expression or activity.

[0050] How to use One aspect of the present technology is to provide a method for treating HBV infection and / or HBV-related diseases, including the treatment of ... In therapeutic applications, the GalV of the present technology can be used to treat subjects diagnosed as being at risk for the disease. Compositions containing NAc-substituted oligonucleotides may be used to treat or treat a range of conditions, including those suspected of or suffering from such diseases. already suffering from such a disease (e.g., HBV antigen in the serum and / or liver of the subject) Surface and envelope antigens (e.g., HBsAg and / or HBeAg) are present, or Complications in disease development in subjects with high HBV DNA or HBV viral load levels and curing or at least partially curing the symptoms of the disease, including the inflammatory and intermediate pathological phenotypes. It is administered in an amount sufficient to inhibit.

[0051] In some embodiments, the GalNAc-substituted oligonucleotides of the present technology are those listed in the table below. A. It shows affinity for at least one of the regions in A or the HBV RNA transcript.

[0052] [Table 1]

[0053] Subjects suffering from HBV infection and / or HBV-related diseases are treated with any of the methods known in the art. The disease can be identified by any or a combination of known diagnostic or prognostic assays. For example, typical symptoms of HBV infection and / or HBV-related disease include elevated serum and / or liver Hb levels. Presence of BV antigens (e.g., HBsAg and / or HBeAg), elevated ALT, elevated AST Increased, absence or low concentration of anti-HBV antibodies, liver damage, cirrhosis, hepatitis D, acute hepatitis B, acute fulminant hepatitis B, chronic hepatitis B, liver fibrosis, end-stage liver disease, hepatocellular carcinoma, serum sickness-like syndrome, foodborne illness Loss of appetite, nausea, vomiting, slight fever, muscle pain, fatigue, abnormal taste and smell (to food and tobacco) aversion to food), right upper quadrant and epigastric pain (intermittent, mild to moderate), hepatic encephalopathy, somnolence, sleep Pattern disorder, mental confusion, coma, ascites, gastrointestinal bleeding, coagulation disorders, jaundice, hepatomegaly (slowly and enlargement, soft liver), splenomegaly, palmar erythema, spider nevi, muscle wasting, spider angiomas, vasculitis, venous Bleeding, peripheral edema, gynecomastia, testicular atrophy, abdominal collateral veins (caput medusae), alani ALT and aspartate aminotransferase ( High levels of AST (1000-2000 IU / mL), AL higher than AST T levels, gamma-glutamyl transpeptidase (GGT) and / or alkaline phosphatase Increased alkaline phosphatase (ALP) concentration, decreased albumin concentration, increased serum iron concentration, decreased white blood cells (i.e. agranulocytosis), lymphocytosis, increased erythrocyte sedimentation rate (ESR), and shortened red blood cell life span. thrombocytopenia, hemolysis, thrombocytopenia, prolonged international normalized ratio (INR), presence of serum HBV DNA, Elevated aminotransferases (less than 5 times the ULN), elevated bilirubin levels, protease activity, Prolonged thrombin time (PT), hyperglobulinemia, anti-smooth muscle antibodies (ASMA) or antinuclear antibodies The presence of tissue-nonspecific antibodies, such as antibodies against the thyroid gland (ANA), and tissue-specific antibodies, such as antibodies against the thyroid gland The presence of rheumatoid factor (RF) elevated levels, hyperbilirubinemia, platelet and white blood cell counts low levels, higher AST levels than ALT levels, lobular inflammation with degenerative and neoplastic hepatocyte changes, and large These include, but are not limited to, centrilobular necrosis.

[0054] In some embodiments, subjects treated with the oligonucleotide compositions of the present technology include: conditions or symptoms, i.e., serum and / or liver HBV antigens (e.g., HBsAg and / or The presence of HBeAg, the absence or low concentration of anti-HBV antibodies, liver damage, cirrhosis, hepatitis D, Acute hepatitis B, acute fulminant hepatitis B, chronic hepatitis B, liver fibrosis, end-stage liver disease, hepatocellular carcinoma, blood Symptoms like illness, loss of appetite, nausea, vomiting, slight fever, muscle pain, fatigue, abnormal taste and smell ( aversion to food and tobacco), right upper quadrant and epigastric pain (intermittent, mild to moderate), hepatic Encephalopathy, somnolence, disturbed sleep patterns, mental confusion, coma, ascites, gastrointestinal bleeding, coagulopathy, jaundice, Hepatomegaly (slowly enlarging, soft liver), splenomegaly, palmar erythema, spider nevi, muscle wasting, spider veins edema, vasculitis, varicose vein bleeding, peripheral edema, gynecomastia, testicular atrophy, abdominal collateral veins (mesu) ALT levels higher than AST levels, leukopenia (i.e., granulocytopenia), albumin Decreased serum iron concentration, increased serum iron concentration, lymphocytosis, increased erythrocyte sedimentation rate (ESR), red blood cell count Shortened blood cell life span, hemolysis, thrombocytopenia, prolonged international normalized ratio (INR), serum HBV D The presence of NA, prolonged prothrombin time (PT), hyperglobulinemia, and anti-smooth muscle antibodies (A The presence of tissue-nonspecific antibodies, such as SMA or antinuclear antibodies (ANA), antibodies against the thyroid gland, Presence of tissue-specific antibodies, hyperbilirubinemia, low platelet and white blood cell counts, ALT levels Higher AST levels, lobular inflammation with degenerative and neoplastic hepatocyte changes, and predominantly centrilobular Demonstrates improvement or elimination of one or more of the necrosis.

[0055] In some embodiments, subjects treated with the oligonucleotide compositions of the present technology Alanine alanine compared with untreated subjects with BV infection and / or HBV-related disease aminotransferase (ALT), aspartate aminotransferase (AST) ), γ-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP ), bilirubin, and rheumatoid factor (RF). The results show a decrease in the expression level of β-actin.

[0056] The present disclosure provides a method for treating a cancer, comprising administering to a subject an effective amount of an oligonucleotide composition of the present technology, Treating subjects diagnosed with or suspected of having HBV infection and / or HBV-related disease The present invention provides a method for treating

[0057] The oligonucleotides and compositions of the present disclosure can be used in antisense therapy. For example, The oligonucleotides are designed to target known viral DNA or RNA sequences, e.g., HBV. It may contain a nucleobase sequence that is complementary to or hybridizes to the nucleic acid sequence.

[0058] In some embodiments, the target nucleic acid is converted to an antisense oligonucleotide comprising the oligonucleotides of the present disclosure. In some embodiments, the method includes contacting a target with a compound to modulate expression of the target. In the present case, the target nucleic acid is present intracellularly in an animal, such as a human.

[0059] In some embodiments, antisense compounds comprising oligonucleotides of the present disclosure are administered to animals. The present invention also includes a method for inhibiting expression of a target RNA in an animal, comprising administering to an animal a nucleic acid sequence comprising: The nucleotide is complementary to, or capable of hybridizing to, a portion of the target RNA. .

[0060] Some embodiments provide a therapeutically effective amount of an oligonucleotide or composition of the present disclosure in a viral vector. to a subject in need of reducing the viral load, thereby Methods for reducing viral load in a subject infected with a virus, The oligonucleotide is complementary to a portion of the target RNA in the virus, i.e. It can hybridize.

[0061] Some embodiments involve contacting a cell with an oligonucleotide or composition of the disclosure. or administering a therapeutically effective amount of an oligonucleotide or composition of the present disclosure to a viral gene. Virus in a cell or subject, including administering to a subject in need of inhibition of gene expression. The method involves inhibiting gene expression. The oligonucleotide binds to a portion of the target RNA in the virus. They are complementary, i.e., capable of hybridizing.

[0062] Other embodiments provide a therapeutically effective amount of an oligonucleotide or composition of the disclosure for use in an antiviral agent. to a subject in need of a reduction in viral levels, thereby reducing viral load in the subject. Method for reducing viral antigen levels in a subject infected with a virus The oligonucleotide is complementary to a portion of the target RNA in the virus, i.e. It can hybridize.

[0063] The oligonucleotides and compositions of the present disclosure can be used to treat, for example, hepatitis B virus (HBV ) gene expression or inhibiting HBV viral replication; or , treating a subject with HBV, or preventing hepatitis B virus infection in a subject infected with HBV. In embodiments, the disclosed chimeras can reduce the viral load of Hepatitis B virus (HBV). Oligonucleotides are used to induce RNase H activity in the target gene.

[0064] The oligonucleotides and compositions of the present disclosure can be used to, for example, identify targets for HCV RNA. They can compete for microRNA binding sites, thereby inhibiting replication.

[0065] The present disclosure is also directed to a method of stabilizing an oligonucleotide for delivery to a subject. The stabilization of the oligonucleotide is herein defined as the melting temperature of the oligonucleotide, i.e. temperature T m It is characterized by [quantified as] increasing

[0066] The disclosed oligonucleotide constructs can be used alone or for targeted disease treatment. The disclosed oligonucleotides may be administered in combination with one or more additional therapeutic agents for the treatment of The oxidase construct may be administered alone or in combination with one or more additional therapeutic agents for HBV infection. In combination therapy, the oligonucleotide construct and H One or more additional therapeutic agents for BV infection may be administered simultaneously in the same or separate compositions. It is understood that the compounds may be administered in separate, simultaneous or sequential doses. do.

[0067] In some embodiments, the disclosed oligonucleotide constructs are directed against HBV complexes. in combination with an HBV replication inhibitor or immunomodulator, or in combination with both an HBV replication inhibitor and an immunomodulator In some embodiments, the anti-HBV oligonucleotide agent is administered in a combined regimen. The disclosed oligonucleotide constructs may be combined with standard treatments for HBV infection. The standard treatment for HBV infection is nucleotide / nucleotide adenosine monophosphate (nucleotide adenosine monophosphate). analogs (e.g., lamivudine, telbivudine, entecavir, adefovir, tenofovir, and clevudine, tenofovir alafenamide (TAF), CMX157, and AGX- 1009) and interferons (e.g., Peg-IFN-2a and IFN-a-2b and interferon lambda). In embodiments, the disclosed oligonucleotide constructs are administered simultaneously (in combination) or in combination. is administered in combination with one or more oligonucleotides, either after sequential administration. The oligonucleotides used were ALN-HBV, ARB-1467, ARC-520, and siRNA such as ARC-521, RG6004 (LNA HBV), Ionis-HB V Rx and Ionis-HBV-L RxAntisense oligonucleotides such as miR NA mimics or inhibitors, aptamers, steric blockers, saRNA, shRNA, immunoassays Modulators and / or REP 2139 and REP 2165 oligonucleotides, etc. In embodiments, the disclosed oligonucleotides can be used as HBsAg release inhibitors. The octide construct may be combined with one or more antiviral agents, such as a viral replication inhibitor. In embodiments, the disclosed oligonucleotide constructs are administered in combination with HB It is administered in combination with an HBV capsid inhibitor, NVR 3. -778, AB-423, GLS-4, Bayer 41-4109, HAP-1, and In embodiments, the disclosed oligonucleotide constructs The lactate is administered in combination with one or more immunomodulatory agents, such as a TLR agonist. LR agonists: GS-9620, ARB-1598, ANA975, and RG779 5 (ANA773), MEDI9197, PF-3512676, and IMO-2055 In embodiments, the disclosed oligonucleotide constructs include , and is administered in combination with the HBV vaccine. In embodiments, the disclosed The oligonucleotide constructs are administered in combination.

[0068] Some embodiments involve contacting a cell with an oligonucleotide or composition of the disclosure. or administering a therapeutically effective amount of an oligonucleotide or composition of the present disclosure to an HBV gene targeting and administering to a subject in need thereof an HBV gene expression inhibitor. This includes the inhibition of offspring expression.

[0069] Some embodiments provide a therapeutically effective amount of an oligonucleotide or composition of the present disclosure in a HB and administering the compound to a subject in need of treatment for a disease or disorder associated with V gene expression or activation. and (iii) treating a disease or disorder associated with HBV gene expression or activation.

[0070] In some embodiments, a therapeutically effective amount of an oligonucleotide or composition of the present disclosure is administered to a patient of type B administering to a subject in need of a reduction in viral load of hepatitis virus (HBV), thereby reducing the HBV viral load in the subject. Some embodiments also include methods for reducing the viral load of HBV in a patient receiving hepatitis D virus (HBV) therapy. Also provided is a method for reducing HDV viral load in a subject infected with HDV.

[0071] Other embodiments include administering a therapeutically effective amount of an oligonucleotide or composition of the disclosure to a patient suffering from hepatitis B virus. to a subject in need of a reduction in HBV antigen levels, thereby HBV antigen levels in a subject, Some embodiments also include methods for reducing BV viral antigen levels. Also provided are methods for reducing HDV antigen levels in a subject infected with HDV. In this embodiment, the HBV antigen is HBsAg or HBeAg.

[0072] In one embodiment, the oligonucleotide or composition of the present disclosure targeting HBV is HBV infection or both HBV and HDV infection, and / or HBV-related disease When the oligonucleotide or composition of the disclosure is administered to a subject, e.g., Expression of one or more HBV genes in cells, tissues, blood, or other tissues or fluids, H BV cccDNA level, HBV antigen level, HBV viral load level, ALT, and and / or AST is at least about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 5 1%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 4 1%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 5 1%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 6 1%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 7 1%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 8 1%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least The reduction is greater than or equal to about 99%, or between two of these numbers. In some embodiments, the HBV antigen level is reduced by the above amount. In some embodiments, the HBV viral load level is HBV Ag or HBeAg. Reduce the amount of writing.

[0073] In one embodiment, the oligonucleotide or composition of the present disclosure targeting HBV is HBV infection or both HBV and HDV infection, and / or HBV-related disease When an oligonucleotide or composition of the disclosure is administered to a subject, e.g., the subject The level of anti-HBV antibodies in the cells, tissues, blood, or other tissues or fluids of Also about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% %, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44 %, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54 %, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64 %, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74 %, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, or at least about 99% or more, or It rises between two of the numbers.

[0074] Administration of the disclosed oligonucleotides or compositions according to the disclosed methods and uses may be used to treat HBV In patients with HBV infection, or both HBV and HDV infection, and / or HBV-related disease resulting in a reduction in the severity, signs, symptoms, and / or markers of such disease or disorder. "Reduce" in this context means a statistically significant decrease in such levels. The reduction may be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, or about 100%, or a value between two of these numbers. do.

[0075] The amount of the oligonucleotide or composition of the present disclosure can be determined by a medical professional. The daily dose of this product is 0.001 to 1,000 mg per day for an adult human, or For oral administration, the dose is preferably adjusted to the dose of the patient to be treated. For symptomatic adjustment of dosage to patients, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5 , 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 200, 250 The composition is provided in the form of a tablet containing 500 milligrams of the active ingredient. The effective dose is usually about 0.01 mg / kg body weight to about 100 mg / kg body weight per day, or Preferably, the range is from about 0.5 to 100 mg / day. 0.1 to about 50.0 mg / kg body weight, or any range therein. More preferably, The daily dose is about 0.01 to about 10.0 mg / kg body weight, or any range therein. Preferably, the dose is about 0.01 to about 1.0 mg / kg body weight per day, or any range therein. The oligonucleotide may be administered on a regimen of 1 to 4 times per day. For example, the oligonucleotides of the present disclosure may be administered at a single dose of about 0.1 mg / kg to about 100 mg / kg. For example, the disclosed oligonucleotides may be administered in doses of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1. 2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3. 3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5. 4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7. 5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9. 6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13 , 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18 , 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23 , 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28 , 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37 , 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, Doses of 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100 mg / kg Values ​​and ranges intermediate to the listed values ​​are also part of this disclosure. These values ​​may apply to intravenous infusion and / or subcutaneous delivery. Other forms of delivery as described may also be administered in these doses. Doses may be adjusted based on the needs of the patient. The therapeutic effect of the present invention may vary depending on the therapeutic requirements, the severity of the condition being treated, and the oligonucleotide used. Either daily or intermittent dosing may be used.

[0076] The oligonucleotides of the present disclosure may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about 25 minutes, etc. The administration can be by intravenous infusion over a period of time, for example, 1 month, 2 months, or 3 months. For example, once a week, once every two weeks (i.e., once a month, three months, four months, or more) After the initial treatment regimen, the frequency of treatment may be reduced. For example, once weekly or once every two weeks for three months followed by six months Or, the dose may be repeated once a month for a year or more.

[0077] The oligonucleotides of the present disclosure can also be administered by subcutaneous delivery. For example, once a week, once every two weeks, for one, two, three, four, or more months. After the initial treatment regimen, the frequency may be increased. Therapeutic doses may be reduced, for example, to once a week or once every two weeks for three months. After administration, monthly administration may be repeated for six months or a year or more.

[0078] Effectiveness in treating or preventing a disease can be measured, for example, by measuring the progression of the disease, the remission of the disease, the severity of symptoms, pain, and the like. Pain reduction, quality of life, amount of medication needed to maintain treatment effect, and disease marker levels or any other measurable variable appropriate for the disease being targeted for treatment or prevention. It can be evaluated by measuring various parameters. Treatment or prevention can be achieved by measuring any one or any combination of parameters. Monitoring the effectiveness of CH The effectiveness of treatment of B can be assessed by, for example, regular monitoring of viral load and transaminase levels. By comparing initial and subsequent values, the effectiveness of the treatment can be assessed. An indication of whether the method is effective is provided.

[0079] definition The terminology used herein is used only for the purpose of describing particular embodiments and is not intended to limit the scope of the present invention. It should be understood that these definitions are not intended to limit the scope of the present invention. This shall apply as far as possible.

[0080] As used herein, the terms "complementary" or "complementarity" refer to a polynucleotide (i.e., base pairing with respect to a sequence of nucleotides, such as an oligonucleotide or a target nucleic acid As used herein, the complement of a nucleic acid sequence refers to the sequence in which the 5' end of one sequence is aligned with the 5' end of the other. When aligned with a nucleic acid sequence so that it pairs with the 3' end of the For example, the sequence "5'-AGT-3'" refers to the sequence "3'-T- CA-5'. Certain bases not commonly found in naturally occurring nucleic acids are These may include, for example, inosine, 7-deoxyribonucleotides, and the like. These include nucleic acids, locked nucleic acids (LNA), and peptide nucleic acids (PNA). A stable duplex does not have to be perfect, and may contain mismatched base pairs, denatured or mismatched bases. Those skilled in the art of nucleic acid technology will appreciate that, for example, the length, base composition, and Many variables, such as the sequence of the oligonucleotide, ionic strength, and frequency of mismatches, were considered. Empirical considerations can be used to determine the stability of the duplex. It may be an RNA sequence complementary to the sequence or its complementary sequence, or it may be cDNA.

[0081] As used herein, the term "hybridize" refers to the formation of two substantially complementary nucleic acid strands. (At least about 65% complementary over at least 14-25 nucleotides, at least about 75%, or at least about 90% complementary) to each other under appropriately stringent conditions. annealing to each other, forming a duplex or heteroduplex by the formation of hydrogen bonds between complementary base pairs. Hybridization typically and preferably refers to the process of forming a A nucleic acid molecule of length 18 to 200 nucleotides, preferably 15 to 100 nucleotides in length, more preferably 18 to 200 nucleotides in length. Nucleic acid hybridization techniques are known in the art. See, e.g., Sambrook, et al., 1989, Molecular Biology, vol. lar Cloning:A Laboratory Manual,Second E dition,Cold Spring Harbor Press,Plainvie See, NY, Hybridization and Hybridization Intensity (i.e., the strength of the association between nucleic acids) depends on the degree of complementarity between the nucleic acids, the stringency of the conditions used, and The thermal melting point (T m ) and other factors. Those skilled in the art will appreciate that sequences with at least the desired level of complementarity will stably hybridize. The hybridization is performed so that the hybrids are matched, while those with lower complementarity do not hybridize. Understand how to estimate and adjust the stringency of the hybridization conditions. For examples of redox conditions and parameters, see, e.g., Sambrook, et al. al.,1989,Molecular Cloning:A Laboratory Manual,Second Edition,Cold Spring Harbo r Press,Plainview,NY;Ausubel,FMet al .1994,Current Protocols in Molecular Bio logy, John Wiley & Sons, Secaucus, NJ In some embodiments, specific hybridization is stringent. Under hybridization conditions, the target nucleic acid is bound to an oligonucleotide or polymer. A oligonucleotide (e.g., a probe or primer) can be "haploidized" to a target nucleic acid under suitable conditions. "Hybridize."

[0082] The term "stringent hybridization conditions" as used herein , refers to hybridization conditions that are at least as stringent as: 50 % formamide, 5xSSC, 50mM NaH2PO4, pH 6.8, 0.5% SD S, 0.1 mg / mL sonicated salmon sperm DNA, and 5x Denhardt's solution overnight hybridization at 42°C with 2x SSC, 0.1% SDS; and a wash with 0.2×SSC, 0.1% SDS at 45° C. Stringent hybridization conditions are defined as those that require 20 consecutive nucleotides. hybridization of two nucleic acids that differ by more than three bases .

[0083] As used herein, the term "substantially complementary" means that two sequences are stringently It means that the hybridization occurs under suitable hybridization conditions. It is understood that substantially complementary sequences need not hybridize along their entire length. Specifically, substantially complementary sequences will exhibit stringent hybridization. 3' or 5' to a contiguous sequence of bases that hybridizes to the target sequence under cleavage conditions The target sequence may comprise a contiguous sequence of bases positioned between the bases that do not hybridize to the target sequence.

[0084] "Pharmaceutically acceptable" means a material that is not undesirable for biological or other reasons. This refers to the fact that the material can be used without causing or containing undesirable biological effects. The pharmaceutical composition can be administered to a patient without adversely interacting with any of the other components of the composition. The term "pharmaceutically acceptable" refers to a pharmaceutical carrier or When used to refer to an excipient, the carrier or excipient is meets the required standards or the carrier is approved by the U.S. Food and Drug Administration This means that the ingredient is included in the tive Ingredient Guide.

[0085] An oligonucleotide "construct" is an oligonucleotide of the present disclosure, and For example, (1) a conjugate moiety, such as those described herein (targeting moiety) or ( 2) Domains of modified / unmodified nucleotides, e.g., some chimeric oligonucleotides It is possible.

[0086] "Chimeric oligonucleotides" are exemplified, for example, by formulas (VI) and (VII): An oligonucleotide that has two or more domains, such as a chimeric oligonucleotide. The leutides may contain additional components, such as a ligand targeting group or a pharmacophore or additional It may also contain nucleotides, linkers, etc.

[0087] A "modified nucleoside" has, independently, a modified sugar moiety and / or a modified nucleobase. Nucleosides refer to nucleosides that have an intersubunit bond, e.g., a phosphodiesterase Intersubunit bond, phosphate intersubunit bond, phosphoramidate intersubunit bond The modified nucleoside is linked via intersubunit bonds and thiophosphoramidate intersubunit bonds. It is understood that "nucleotide" can refer to both a nucleoside and an intersubunit linkage.

[0088] The "unmodified" or "natural" nucleobases are the purine bases adenine (A) and guanine (G). thionine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U) "Modified nucleobases" include 5-methylcytosine (5-me-C), 5- Hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine 6-methyl and other alkyl derivatives of adenine and guanine, 2-proton derivatives of adenine and guanine pyridinium and other alkyl derivatives, 2-thiouracil, 2-thiothymine, and 2-thiocytosine uracil, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3)uracil and Cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and Thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino , 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and Anions, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-adenine Amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7- Other synthetic and Natural nucleobases include: phenoxazine cytidine (1H -pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenotypic Azincytidine (1H-pyrimido[5,4-b][1,4]benzothiazine-2(3H) -one), G-clamp, e.g., substituted phenoxazine cytidine (e.g., 9-(2- am-oelhoxy)-H-pyrimido[5,4-b][1,4]benzoxazine-2 (3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indole- 2-one), pyridoindole cytidine (H-pyrido[3,2,5]pyrrolo[2,3-d ]pyrimidin-2-one). Modified nucleobases also include The purine or pyrimidine base is replaced by another heterocycle, e.g., 7-deaza- May include adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. stomach.

[0089] In some embodiments, the modified nucleobase is 5-methylcytosine, 2,6-diaminopropyl Some are selected from the group consisting of phosphorus, 5-methyluracil, and g-clamp. In an embodiment, the G-clamp is

[0090] [ka] is.

[0091] The "ligand targeting group" targets the oligonucleotide to HBV-infected hepatocytes via receptor binding. These groups include the A receptor, which is a cell surface receptor, and the A receptor, which is a nucleotide that enhances delivery of the nucleotide. GalNAc, which targets SGPR and LDL receptors on the cell surface, respectively. These receptors on the cell surface are targeted to other receptors. Body-targeting ligands are also within the scope of this term.

[0092] The "pharmacophore" is a compound that binds to HBV DNA or HBV in HBV / HDV or HBV-infected cells. It refers to an oligonucleotide drug sequence that interacts with an RNA molecule and triggers an antiviral response. vinegar.

[0093] "Conformationally restricted nucleosides" have a bridged or bicyclic sugar structure. It refers to a nucleoside, the conformation of which may be fixed in a particular configuration. For example, a conformationally restricted nucleoside may be a glycoside with a fixed C3' end. Exemplary embodiments include bridged nucleic acids (BNAs), e.g. For example, α-L-methyleneoxy(4'-CH2-O-2')LNA, β-D-methyleneoxy oxy(4'-CH2-O-2')LNA, ethyleneoxy(4'-(CH2)2-O-2 ')ENA, 2',4'-BNA NC [NH], 2',4'-BNA NC [NMe], 2 ',4'-BNA NC [NBn], aminooxy(4'-CH2-ON(R)-2') 2',4' such as BNA and oxyamino (4'-CH2-N(R)-O-2') BNA Another exemplary BNA structure is a nucleoside with a 4'- and 2'-sugar bond. and oligonucleotides having at least one bridge between the ' and ' positions. and the like, wherein each of the bridges is independently -[C(R1)(R2)] n - , -C(R1)=C(R2)-, -C(R1)=N-, -C(=NR1)-, -C(=O )-, -C(=S)-, -O-, -Si(R1)2-, -S(=O) x - and -N(R1 )-, wherein x is 0, 1, or is 2, n is 1, 2, 3, or 4, and each R1 and R2 is independently H, a protecting group, Group, hydroxyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 a Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 a Rucynyl, C5-C 20 Aryl, substituted C5-C 20 Aryl, heterocyclic radicals, substituted heterocyclic radicals Aromatic radicals, heteroaryls, substituted heteroaryls, C5-C7 alicyclic radicals, substituted C 5~C7 alicyclic radicals, halogens, OJ1, NJ1J2, SJ1, N3, COOJ1, a acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(═O)—J1), where each J1 and J2 is independently H, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Ants C5-C substitution 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical C1-C, substituted heterocyclic radicals 12 Aminoalkyl, substituted C1-C 12 Aminoal Certain BNAs have been described in the patent and scientific literature as being prepared and developed. (See, e.g., U.S. Pat. No. 7,055,056, which is incorporated herein by reference in its entirety. No. 3,207, No. 6,268,490, No. 6,770,748, No. 6,794 , No. 499, No. 7,034,133, No. 6,525,191, No. 7,696, No. 345, No. 7,569,575, No. 7,314,923, No. 7,217,8 (See US Patent Nos. 7,084,125 and 7,084,125.) "do" is a conformationally restricted nucleoside linked via an intersubunit bond Refers to...

[0094] In some embodiments, the conformationally restricted nucleoside is an optionally substituted Selected from LNA or optionally substituted ENA. Optionally substituted LNA or ENA is a group in which one of the -CH2- moieties is replaced by an alkyl moiety, e.g., methyl or ethyl. may be replaced.

[0095] "Inhibit expression" refers to a decrease or blockage of expression or activity, and does not necessarily mean an increase in expression or activity. It does not indicate a complete disappearance of

[0096] "Inhibiting viral replication" refers to a reduction or blocking of viral replication, and does not necessarily mean It does not indicate a complete abolition of viral replication.

[0097] "Subject" refers to mammals, including humans and non-human mammals. In the present case, the subject is a human, for example an adult.

[0098] "Treating" a disease or "treatment" of a disease in a subject means (1) reducing the susceptibility to that disease; Preventing disease from occurring in subjects who are not yet symptomatic of the disease or (2) inhibiting the disease, i.e., stopping its onset, or (3) alleviating the disease. It refers to causing dissolution or regression.

[0099] "Therapeutically effective amount" means an amount of a pharmaceutical agent that provides a therapeutic effect to a subject.

[0100] "Pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the present disclosure; That is, to retain the desired biological activity of the parent oligonucleotide / compound and to eliminate any unwanted By "aromatic" is meant a salt that does not impart any significant toxic effects.

[0101] The following abbreviations are used in this disclosure: 2'-H (deoxyribose) nucleosides, nucleic acid salts The corresponding groups are designated by capital letters, e.g., A, C, G, and T. 2'-OH (ribose) Nucleosides are designated by a lowercase r and the capital letter corresponding to the nucleobase, e.g., rA, rC, rG, and 2'-O-Me nucleosides are designated by a lowercase m and the capital letter corresponding to the nucleobase. They are referred to symbolically as, for example, mA, mC, mG, and mU. 2'-MOE nucleosides are The lowercase "moe" and the uppercase corresponding nucleic acid base, e.g., moeA, moeC, moe 2'-ribo-F nucleosides are those that are linked to a lowercase "f" and a nucleobase. They are referred to by their corresponding capital letters, e.g., fA, fC, fG, and fU. Nucleosides are written with a lowercase "af" and the corresponding capital letter for the nucleobase, e.g., afA, afC , afG and afU. mA * is 3'-amino-2'-OMe-2,6-diazo It is minopurin. A * is 3'-amino-2-deoxy-2,6-diaminopurine fA * is 3'-amino-2'-F-2,6-diaminopurine. Osides are designated by the capital letter "L" and the corresponding nucleic acid base, e.g., LA, LC, LG, LTA. will be done.

[0102] For nucleotide backbones or intersubunit bonds, phosphodiester subunits Inter-linkages are referred to as "PO" or are generally not included in the sequence specification. Intersubunit bonds are abbreviated as "ps" in lower case. The thiophosphoramidate intersubunit linkage is abbreviated as "np" in lower case. It is abbreviated as "nps" in lowercase.

[0103] N3'→P5' means that the 3' portion contains an N (e.g., NH) and is linked via a P; It refers to a modified nucleotide having an intersubunit bond. For example, the following structure: N3' → With P5' linkage:

[0104] [ka]

[0105] As used in this specification and the appended claims, unless otherwise specified: Please note that the singular forms "a," "an," and "the" include plural referents. It is further noted that the claims may be drafted to exclude any optional element. Therefore, this statement should be read "only" in connection with the recitation of claim elements. , the use of exclusive language such as "only," or the use of "negative" limitations. It is intended to serve as a basis.

[0106] The term "about" will be understood by those of ordinary skill in the art and will vary depending on the context in which the term is used. The terms used may differ to some extent in some contexts where they are used and may not be clear to a person skilled in the art. When used herein, "about" shall mean up to plus or minus 10% of the particular term. Ranges of are indicated herein by numerical values ​​preceded by the term "about." The term "about" means As used herein, the exact number that it precedes, as well as the approximate or approximate number that it precedes, are used interchangeably. Used to provide a literal basis for a number. In determining whether a particular item is a approximation or approximation, the approximation or approximation listed shall be used. Any number not listed must, in the context in which it is presented, substantially correspond to the number specifically recited. It can be a number that provides.

[0107] The "linker portion" is, for example, C2 to C 10 alkyl moiety or 1 to 5 oxygen atoms C3~C 10 The linker may also contain an alkyl oxide moiety. At the terminal end or within the linker, e.g., C2-C 10 Alkyl moiety or C3-C 10 a The alkyl oxide moiety may contain one or more amine moieties.

[0108] A "solid support linker" is a chemical moiety that links a compound to a solid support, such as a resin. It will be understood by those skilled in the art that the solid support linker is readily available under certain conditions at the end of the synthesis. A common linker is a succinyl linker. which can be easily cleaved by treatment with concentrated ammonium hydroxide. The term solid support linker refers to embodiments in which a chemical entity is attached to a solid support, and and embodiments where the solid support linker does not comprise a solid support.

[0109] The various modes of treatment or prevention of a disease or condition described herein may be used to treat or prevent a complete cure or prophylaxis. prevention, but falls short of complete treatment or prevention, resulting in some degree of biologically or medically relevant consequences It should also be understood that the term "substantial" is intended to mean that a "substantial" result is achieved. can be used for continuous long-term treatment for chronic conditions or for single-use treatment for acute conditions. Or it may be administered in several doses.

[0110] When a range of values ​​is provided, each value in between (unless the context clearly indicates otherwise) is to the tenth of the unit of the lower limit between the upper and lower limits of the range and any other stated range, i.e. It is understood that all values ​​within the stated ranges are encompassed by the present invention. The upper and lower limits of smaller ranges may independently be included in the smaller ranges and It is to be understood that any limit may be specifically excluded from any stated range, and that the scope of the invention remains the same. Where the stated range includes one or both of the limits, Ranges excluding either or both of the included limits are also intended to be included in the invention.

[0111] The present disclosure is not limited to the particular embodiments described and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only. and is not intended to be limiting, the scope of the present invention being limited only by the appended claims. It should be understood that:

[0112] As will be apparent to those skilled in the art upon reading this disclosure, the Each individual embodiment may be combined with any number of other embodiments without departing from the scope and spirit of the present disclosure. It is easily separable from or combined with any of the features of the embodiments. Any method described may be implemented by any method that includes distinct components and features that can be implemented by any method that includes any sequence of events described. The steps may be carried out in order, in sequence, or in any other order which is logically possible.

[0113] All publications and patents cited herein are to be construed as though each individual publication or patent were incorporated by reference. All such references are incorporated herein by reference as if specifically and individually indicated to be incorporated herein by reference. and to disclose and describe in connection with the methods and / or materials in which the documents are cited. The citation of any publication is hereby incorporated by reference for its disclosure prior to the filing date. and that the present invention is not entitled to antedate such publication by virtue of prior invention. Nothing in this document should be construed as an admission that the invention does not have the date of publication. The dates may be different from the actual publication dates, which may need to be independently confirmed. do. [Example]

[0114] The following examples are provided to aid those skilled in the art in practicing the present disclosure in accordance with certain embodiments of the present disclosure. Therefore, the examples should not be construed as limiting the scope of the present disclosure in any way. .

[0115] Manufacturing method All monomers were dried in a vacuum desiccator using desiccants (KOH and P2 (05, RT, 24 h). A synthetic solid support (CPG) bound to the first 5' residue was prepared from a commercially available All other synthetic reagents and solvents were obtained from commercial sources and used as such. Chemicals and solvents for the post-synthesis workflow were purchased from commercial sources and purified or It was used without any treatment. During the synthesis, the solvent (acetonitrile) and solution (amidite) and activator) were stored over molecular sieves.

[0116] The control nuclease-stabilized 3'-GalNAc conjugate used in this study Antisense oligonucleotides are shown, for example, in the table below. The ABI-394 compound was tested using the standard 93-step cycle provided by the manufacturer. The solid support was controlled pore glass, and the monomers had standard protecting groups. Each oligonucleotide was prepared using commercially available 5'-O-(4,4'-dimethoxytrimethylsilyl) ethyl)-3'-O-(2-cyanoethyl-N,N-diisopropyl)DNA, and or , 6-N-benzoyl adenosine (A Bz ), 4-N-acetylcytidine (C Ac ), 2 -N-isobutyrylguanosine (G iBu ), and 2'-O-Me phosphate of thymidine (T). Standard solid-phase oligonucleotide synthesis protocols were followed using phosphoamidite monomers. The phosphoramidites were purchased from commercial sources. -2,6-Diaminopurine phosphoramidite was purchased from a commercial source. ((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazoline-3-thiazoline as sulfur transfer agents for the synthesis of oligoribonucleotide phosphorothioates In the presence of 5-(ethylthio)-1H-tetrazole activator in CH3CN, Extension coupling of 0.1M solutions of phosphoramidites to solid-bound oligonucleotides followed by standard capping, oxidation and deprotection to form the modified oligonucleotide. The stepwise coupling efficiency of all modified phosphoramidites was greater than 98%. The oligonucleotide-supported solid support was purified by precipitation using an aqueous solution of ammonia / ethanol (3:1). The mixture was heated at 55°C for 8 hours to remove the base-labile protecting groups.

[0117] GalNAc-conjugated ASOs are prepared by binding to hydroxyprolinol-GalNAc solid supports. GalNAc was trans-4 linked via a 6-aminohexanoate linkage. -hydroxyprolinol to obtain the hydroxyprolinol-GalNAc moiety , which was then attached to functionalized controlled pore glass (CPG) to obtain a solid support.

[0118] Unconjugated and GalNAc-modified oligonucleotides were purified by anion exchange HPLC. The buffer was 20 mM sodium phosphate in 10% CH3CN, pH 8. 5 (Buffer A) and 20 mM sodium phosphate in 10% CH3CN, 1.8 M NaBr The fractions containing the full-length oligonucleotide were pooled. It was then desalted and freeze-dried.

[0119] [ka]

[0120] GalNAc synthesis

[0121] [ka]

[0122] Oxane-2,6-dione (1000 g, 8.76 mol, 1.00 equiv.), 4-dimethyl Dichloromethylaminopyridine (53.5 g, 437.9 mmol, 0.05 equiv.) To a solution of phenylmethylsulfonyl ether (10000 mL) was added phenylmethylsulfonyl ether (10000 mL) at room temperature under an inert atmosphere of nitrogen with stirring. To the resulting solution was added ethanol (900 g, 8.32 mol, 0.95 eq.) dropwise. The mixture was stirred overnight at room temperature, and the resulting mixture was washed with saturated sodium bicarbonate solution. The pH value was adjusted to 1 with 10% hydrochloric acid. The resulting solution was diluted with 3 x 2000 mL of ethyl acetate. The resulting mixture was extracted with 2×3000 mL of saturated sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. This gave 1240 g (64%) of G-1 as a colorless oil. MS m / z [M+H ]+(ESI):223.

[0123] [ka]

[0124] G-1 (58.5 g, 263.23 mmol, 1.20 equivalents), N,N-diisopropyl N,N-dimethylformamide (34 g, 263.57 mmol, 1.20 equiv.) To a solution of O-benzotriazole- N,N,N',N'-Tetramethyl-uronium-hexafluorophosphate (100 g, 263.69 mmol, 1.20 equiv.) was added at room temperature. The resulting solution was stirred at room temperature for 1 The mixture was stirred for 2 hours. Subsequently, (2R)-3-aminopropane-1,2-diol (20 g, The resulting solution was stirred at room temperature. The reaction was allowed to proceed at room temperature overnight. The resulting solution was diluted with 2000 mL of ethyl acetate. The mixture was washed with 2 x 1000 mL of saturated sodium bicarbonate solution. The mixture was dried over sodium hydroxide and concentrated under reduced pressure. The residue was applied to a silica gel column. This gave 38.7 g (60%) of G-2 as a light yellow solid. MS m / z [M +H]+(ESI):296.

[0125] [ka]

[0126] A solution of G-2 (10 g, 33.86 mmol, 1.00 equiv.) in pyridine (100 mL) 1-[chloro(4-methoxyphenyl)benzyl]-4- Methoxybenzene (12.63 g, 37.28 mmol, 1.10 equiv) was added at room temperature. The resulting solution was stirred overnight at room temperature. Then, methanol (10 mL) was added to The reaction was stopped by adding 1000 ml of ethanol. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with 2 x 500 mL of saturated sodium bicarbonate. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The mixture was applied to a silica gel column, whereby 10.2 g (50%) of G-3 was obtained as a light yellow oil. MS m / z [M+Na]+ (ESI): 620.

[0127] [ka]

[0128] Dissolve G-3 (10 g, 16.73 mmol, 1.00 equiv.) in methanol (100 mL). To the solution, 10% palladium on activated carbon (1 g) was added at room temperature. The flask was evacuated and purged with hydrogen five times. The resulting solution was stirred at room temperature for 4 hours. The solid was filtered off. The mixture was concentrated under reduced pressure to give 7.6 g (89%) of G-4 as a white solid. MS m / z [M+Na]+ (ESI): 530.

[0129] [ka]

[0130] G-4 (8.90 g, 17.53 mmol, 1.05 equiv.) in N,N-dimethylform To a solution of amide (300 mL) under an inert atmosphere of nitrogen, N,N-diisopropylethyl The amine (6.47 g, 50.16 mmol, 3.00 equiv.) was added at room temperature. -benzotriazole-N,N,N-etramethyl-uronium-hexa Fluorophosphate (7.10 g, 18.73 mmol, 1.12 equiv.) was added at room temperature. The resulting solution was stirred for 15 minutes at room temperature. To this mixture was added G-5 Ref(Nuc leic Acids Research,2014,42,(13)8796-880 7), (30 g, 16.72 mmol, 1.00 equiv.) was added at room temperature. The resulting solution was The reaction was allowed to proceed overnight at room temperature with stirring, and the resulting mixture was concentrated under reduced pressure. This was purified by flash preparative HPLC, yielding 20.1 g (53%) of G-6. Obtained as a white solid. MS m / z [M+H] + (ESI):2283.

[0131] [ka]

[0132] G-6 (25 g, 10.96 mmol, 1.00 equiv.) in dichloromethane (750 mL ) solution under an inert atmosphere of nitrogen, triethylamine (4.98 g, 49.21 mmol) 1.33 g of 4-dimethylaminopyridine (1.33 g, 4.49 equivalents) was added at room temperature. , 10.89 mmol, 0.99 equiv.) was added at room temperature. ,5-dione (3.29 g, 32.88 mmol, 3.00 equiv.) was added at room temperature. The resulting solution was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure. The crude product was This was followed by purification by preparative HPLC to give 15.83 g (61%) of G-7. Obtained as a yellow solid as the ammonium salt. MS m / z [M / 2+NH4]+ (ES I):1210.

[0133] [ka]

[0134] Using HBTU / TEA, Biotechniques., 1988 Sep;6( G-7 was loaded onto CPG according to the procedure described in 8):768-75, followed by GalNAc- 2-CPG (53 μmol / g) was obtained.

[0135] [ka]

[0136] G3-0 (12.8 g, 24.57 mmol, 1.00 equiv.) N,N-dimethylformamide N,N-diisopropylethylamine (9.0 g, 69 mL) was added to a solution of methyl amide (500 mL). 0.64 mmol, 3.00 equiv), O-benzotriazole-N,N,N-tetramethyl -Uronium hexafluorophosphate (9.9 g, 27.03 mmol, 1.10 The resulting solution was stirred at room temperature for 2 hours. G3-1 (44 g, The resulting solution was stirred at room temperature for 16 hours. The resulting mixture was concentrated under reduced pressure, and the crude product was purified by flash preparative HPLC. This gave 22 g (41%) of G3-2 as a light yellow solid. MS m / z[MH] - (ESI):2295.H-NMR(DMSO,400MHz):7.7 9-7.83(m,6H),7.70-7.73(m,4H),7.33-7.35(m ,2H),7.26-7.30(m,2H),7.19-7.22(m,5H),7.0 1(s,1H),6.84-6.87(m,4H),5.19-5.20(d,J=4. 0Hz,3H),4.93-4.97(m,3H),4.58-4.59(d,J=4. 0Hz,1H),4.46-4.48(d,J=8.0Hz,3H),3.95-4.0 4(m,9H),3.82-3.89(m,3H),3.67-3.71(m,9H), 3.45-3.61(m,12H),3.36-3.41(m,3H),2.94-3. 09(m,16H),2.24-2.27(m,6H),2.00-2.08(m,29 H),1.87(s,9H),1.75(s,9H),1.63-1.69(m,3H) ,1.41-1.51(m,19H).

[0137] G3-2 (22g, 9.58mmol, 1.00 equivalent), TEA (4.4g, 4.50 eq.), 4-dimethylaminopyridine (1.15 g, 1.00 eq.) in dichloromethane ( 220 mL) solution of oxolane-2,5-dione (2.87 g, 28.68 mmol, (3.00 equivalents) was added at room temperature. The resulting solution was stirred at room temperature for 18 hours. The mixture was concentrated in vacuo and the crude product was purified by flash preparative HPLC. From this, 15.01 g (65%) of G3-3 was obtained as a white solid. MS m / z [M- H] - (ESI):2395.H-NMR(DMSO,400MHz):7.96-8. 16(m,10H),7.28-7.36(m,4H),7.21-7.23(m,6H ),6.86-6.89(m,4H),5.21-5.22(d,J=3.2Hz,3H ),4.97-5.03(m,4H),4.51-4.53(d,J=8Hz,3H), 4.03(m,9H),3.85-3.92(m,3H),3.69-3.74(m,9 H),3.52-3.59(m,12H),3.38-3.44(m,4H),2.95 -3.04(m,15H),2.23-2.29(m,10H),2.10(s,9H) ,2.04-2.07(m,9H),2.00(s,9H),1.89(s,9H),1 .78(s,10H),1.63-1.68(m,3H),1.45-1.54(m,1 8H).

[0138] [ka]

[0139] G4-0 (10.13 g, 17.53 mmol, 1.05 equiv.) N,N-dimethylphosphine To a solution of N,N-diisopropyl ether (300 mL) under an inert atmosphere of nitrogen, Cetylamine (6.47 g, 50.06 mmol, 2.99 equiv.) was added at room temperature. , O-benzotriazole-N,N,N-tetramethyl-uronium-hexafluorophosphate Sulfate (7.10 g, 18.72 mmol, 1.12 equiv.) was added at room temperature. The resulting solution was stirred for 15 minutes at room temperature. To this mixture was added G4-1 (30 g, 16.72 mm The resulting solution was stirred at room temperature overnight. The resulting mixture was concentrated under reduced pressure. The crude product (30 g) was purified by flash preparative HPLC. This gave 22.3 g (57%) of G4-2 as a white solid. Ta.

[0140] G4-2 (15 g, 6.38 mmol, 1.00 equiv.) in dichloromethane (450 mL ) solution under an inert atmosphere of nitrogen, triethylamine (2.90 g, 28.66 mmol) 1, 4.49 equivalents) was added at room temperature. , 6.36 mmol, 1.00 equiv.) was added at room temperature. 5-dione (1.91 g, 19.09 mmol, 2.99 equiv.) was added at room temperature. The resulting solution was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure to give the crude product (15 g). ) was purified by flash preparative HPLC to give 10.8047 g (69%) G4-3 was obtained as a light yellow solid. MS m / z [M+H]+ (ESI): 2453 . 1H NMR(DMSO-d6,400Hz,ppm):8.10-7.92(m,1 0H),7.35-7.30(m,4H),7.24-7.22(m,5H),7.10 -7.01(m,1H),6.89-6.87(m,4H),5.22-5.21(d, J=3.2Hz,3H),4.00-4.97(m,4H),.4.53-4.51(m ,3H),4.04-3.90(m,9H),3.87-3.3.80(m,3H),3 .74-3.70(m,10H),3.69-3.39(m,16H),3.05-3. 02(m,16H),2.51-2.50(m,2H),2.30-2.27(m,8H ),2.11-1.99(m,29H),1.89(s,9H),1.77(s,9H) ,1.52-1.32(m,22H),1.20(s,9H).

[0141] [ka]

[0142] G5-0 (10 g, 16.90 mmol, 1.06 eq) N,N-dimethylformamide To a solution of N,N-diisopropylethyl ether under an inert atmosphere of nitrogen was added amine (6.18 g, 47.96 mmol, 3.0 equiv.), O-benzotriazole-N, N,N-tetramethyl-uronium-hexafluorophosphate (6.84 g, 18. 04 mmol, 1.13 eq) was added. The resulting solution was stirred at room temperature for 5 minutes. , G5-1 (28.68 g, 15.99 mmol, 1.00 equiv.) N,N-dimethylphosphine A solution of methyl methyl ether (300 mL) was added, and the resulting solution was stirred at room temperature overnight. The resulting mixture was concentrated under reduced pressure. The crude product (20 g) was purified by flash preparative HPPE. This gave 20.44 g (54%) of G5-2 as a white solid. I got it.

[0143] G5-2 (12.5 g, 5.28 mmol, 1.00 equiv.) in dichloromethane (375 4-dimethylaminopyridine (650 mg, 5 mL) was added to the solution under an inert atmosphere of nitrogen. 0.32 mmol, 1.01 equiv.), triethylamine (2.4 g, 23.76 mmol, 4.50 equiv.), and oxolane-2,5-dione (1.59 g, 15.89 mmol, The resulting solution was stirred overnight at room temperature. After concentration under reduced pressure, the crude product (15 g) was purified by flash preparative HPLC. This gave 9.0 g (73%) of G5-3 as a white solid. MS m / z [MH ]-(ESI):2465.1H-NMR(DMSO-d6,300Hz):8.10- 7.90(m,10H),7.36-7.20(m,9H),7.10(s,1H),6 .88-6.85(m,4H),5.22-5.20(d,J=3.0Hz,3H),4 .99-4.95(m,4H),4.52-4.49(m,3H),4.02-3.89 (s,9H),3.85-3.73(m,3H),3.70-68(m,9H),3.6 5-3.52(m,12H),3.52-3.38(m,6H),3.02-2.94( m,15H),2.30-2.25(m,10H),2.09-1.99(M,29H) ,1.88-(s,9H),1.77(s,11H),1.52-1.45(m,22H ), 1.23-1.19(m,9H).

[0144] [ka]

[0145] [ka]

[0146] Decanedioic acid (100 g, 494.4 mmol, 1.00 equiv.) in dichloromethane (20 00 mL) solution, 4-dimethylaminopyridine (18.1 g, 148.2 mmol, 0 To this was added N-(3-dimethylaminopropyl)-N'-ethoxybenzoate (30 equivalents) at room temperature. Add ethylcarbodiimide hydrochloride (114 g, 594.7 mmol, 1.20 equiv.) at room temperature. The resulting solution was stirred at room temperature for 1 hour. To the mixture was added benzyl alcohol (64. 1g) was added dropwise with stirring at 0°C. The resulting solution was reacted overnight at room temperature with stirring. The resulting mixture was washed with saturated aqueous sodium chloride. The crude product (100 g) was purified by flash preparative HPLC. C. This gave 60.7 g (42%) of G-8 as a white solid. MS m / z [M+H]+ (ESI): 293.

[0147] [ka]

[0148] G-8 (4.48 g, 15.32 mmol, 1.50 equiv.) in acetonitrile (320 mL) solution of O-benzotriazole-N,N,N-tetramethyl-uronium-hexyl N,N-difluorophosphate (5.84 g, 15.40 mmol, 1.50 equiv.) Diisopropylethylamine (3.96 g, 30.64 mmol, 3.00 equiv.) was added. The resulting solution was stirred at 25°C for 1 hour. The resulting solution was stirred at 25°C for 16 hours, then The crude product was purified by flash chromatography. This gave 12 g (5 G-10 (7%) was obtained as a white solid. H-NMR (DMSO, 400 MHz, pp m):7.74-7.83(m,9H), 7.31-7.37(m,5H), 6.97( s,1H), 5.21(d,J=3.3Hz,3H), 5.07(s,2H), 4.98 (dd,J=11.2Hz,3.4Hz,3H),4.49(d,J=8.4Hz,3H ), 4.04(s,9H), 3.83-3.99(m,3H), 3.67-3.72(m ,3H), 3.52-3.55(m,12H), 3.37-3.43(m,3H), 2. 99-3.05 (m, 12H), 2.25-2.35 (m, 8H), 2.12 (s, 9H) ), 1.99-2.11(m,17H), 1.92(s,9H), 1.77(s,9H) , 1.40-1.53(m,22H), 1.19-1.25(m,8H).

[0149] [ka]

[0150] G-10 (5 g, 2.45 mmol, 1.00 equiv.) in methanol / ethyl acetate (10 10% palladium on carbon (1.5 g, 10%) was added to the solution. The flask was evacuated and flushed with hydrogen five times. The mixture was stirred at room temperature under a hydrogen atmosphere for 2 The mixture was stirred for 1 hour, and the solid matter was filtered off. The resulting mixture was concentrated in vacuo to give 4 g (82%) of G-11 was obtained as a white solid.

[0151] [ka]

[0152] G-11 (6.3 g, 3.18 mmol, 1.00 equiv.) of N,N-dimethylformamide In a solution of 1.0 g of N,N-diisopropylethylamine (7.95 m mol, 2.50 equivalents) was added. Then, with stirring at 0°C, pentafluorophenyl 2,2,2-trifluoroacetate (1.33 g, 4.77 mmol, 1.50 equiv.) ) was added dropwise. The resulting solution was stirred at 25°C for 3 hours. The resulting mixture was The crude product was purified by flash chromatography using the following conditions: C18 gel column, eluent Eluent A: water, Eluent B: acetonitrile; Gradient: 20% to 80% in 15 min, 3 min The detector was kept at 100% for a period of time, and UV was set at 210 nm. This resulted in 5 g (73%) of GalNA c-6 was obtained as a white solid. MS m / z [M / 2+H] + (ESI):1073; H-NMR (DMSO, 300MHz, ppm): 7.71-7.80 (m, 9H), 6 .98(s,1H), 5.22(d,J=3.3Hz,3H), 4.99(dd,J=1 1.1Hz,3.3Hz,3H), 4.50(d,J=8.4Hz,3H), 4.02( s,9H), 3.82-3.92(m,3H), 3.69-3.74(m,3H), 3. 52-3.56(m,12H), 3.39-3.44(m,3H), 3.03(s,12 H), 2.75-2.79(m,2H), 2.28(t,J=6.3Hz,6H), 2. 00-2.10(m,26H), 1.89(s,9H), 1.77(s,9H), 1.6 4-1.68(m,2H), 1.25-1.53(m,28H);F-NMR(DMSO ,162MHz,ppm):-153.60,-153.67,-153.68,-15 3.69, -158.05, -158.14, -158.22, -162.53, -16 2.60, -162.62, -162.69, -162.70.

[0153] [ka]

[0154] In a 5000 mL four-neck round bottom flask purged and maintained with an inert atmosphere of nitrogen, Dichloromethane (250 g, 2.19 mol, 1.00 equiv.) (2.5 L) solution, 4-dimethylaminopyridine (11.1 g, 90.91 mmol, 0 0.05 equiv.) was added. After this, phenylmethanol (225 g, 2.08 mol, 0. 95 equivalents) was added dropwise with stirring. The resulting solution was stirred at 25°C for 16 hours. The resulting solution was extracted with saturated aqueous sodium bicarbonate solution, and the aqueous layers were combined. The pH was adjusted to 1 with hydrogen chloride (1 mol / L). The resulting solution was extracted with ethyl acetate. The organic layers were combined, dried over sodium sulfate, filtered and concentrated in vacuo. 230 g (47%) of G7-1 was obtained.

[0155] In a 5 L three-neck round bottom flask purged and maintained under an inert atmosphere of nitrogen, G7-1( 100 g, 449.97 mmol, 1.00 equiv.) in tetrahydrofuran (2.0 L). After this, (dimethyl-3-sulfanyl)boranide (41 g, 539.6 9 mmol, 1.20 equiv.) was added dropwise with stirring. The resulting solution was heated at 25°C for 3 hours. The reaction was then stopped by adding 200 mL of methanol and the mixture was evaporated under vacuum. The crude product was purified by recrystallization from hexane. 58 g (62%) of G7-2 was obtained.

[0156] Into a 10000 mL four-neck round bottom flask purged and maintained with an inert atmosphere of nitrogen (3R,4R,5R,6R)-3-amino-6-(hydroxymethyl)oxane-2,4 ,5-triol hydrochloride (350 g, 1.62 mol, 1.00 equiv.) After this, acetic anhydride (1246 g, 12.2 ml) solution was added at 25°C. The resulting solution was stirred at 25°C for 16 hours. The resulting mixture was concentrated in vacuo. The crude product was purified by recrystallization from water / ice. The solid was collected by filtration, yielding 507 g (80%) of G7-3.

[0157] Into a 50 L three-neck round bottom flask purged and maintained under an inert atmosphere of nitrogen, add G7-3 (220 g, 565 mmol, 1.00 equiv.) in dichloromethane (22 L) After this, ferric chloride (275 g) was added in portions at 25° C. The resulting solution was The mixture was stirred at 5°C for 2 hours. The reaction was then stopped by adding 17 L of water / ice. The resulting solution was washed with water and saturated aqueous sodium chloride. The mixture was dried over ice, filtered and concentrated under vacuum to give 121 g (65%) of G7- I got 4.

[0158] In a 2000 mL round-bottom flask purged and maintained under an inert atmosphere of nitrogen, add G7-4 (111 g, 337.1 mmol, 1.00 equivalents) and benzyl 5-hydroxypentanoate ester (70.2 g, 438.2 mmol, 1.3 equiv.) of 1,2-dichloroethane (1 100 mL) solution and 22 g of molecular sieves type 4A. The mixture was stirred at 20°C for 30 minutes. After this, trimethylsilyl trifluoromethanesulfonate (2 2.48 g, 101.12 mmol, 0.3 equivalents) was added dropwise with stirring over 10 minutes. The resulting solution was stirred at 25° C. for 15 hours. The resulting solution was diluted with dichloromethane and The mixture was washed with water, saturated aqueous sodium bicarbonate solution, and saturated aqueous sodium chloride solution, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. This gave 110 g (57%) of G7-5.

[0159] In a 5000 mL round-bottom flask, add G7-5 (330 g, 614 mmol, 1.00 equiv.) ) in ethyl acetate (3300 mL), 10% anhydrous palladium on carbon (100 g) (30% The flask was evacuated and flushed with hydrogen five times. The mixture was placed under a hydrogen atmosphere. The mixture was stirred at room temperature under air for 3 hours, the solid matter was removed by filtration, and the filtrate was concentrated under vacuum. This gave 184 g (67%) of G7-6. MS m / z [M+H] + ( ESI):448;H-NMR(CD3Cl,300Hz,ppm):δ 5.99(d ,J=8.7Hz,1H),5.36(d,J=3.0Hz,1H),5.29(dd, J=10.6,3.0Hz,1H),4.69(d,J=8.4Hz,1H),3.91 -4.21(m,5H),3.51-3.56(m,1H),2.31-2.52(m, 2H), 1.91-2.21(m, 12H), 1.71(s, 4H).

[0160] In a 100 mL three-neck round-bottom flask purged and maintained with an inert atmosphere of nitrogen, add G7 A solution of 6 (5 g, 11.17 mmol, 1.00 equiv) in dichloromethane (50 mL) DIEA (2.17 g, 16.79 mmol, 1.50 equivalents) was added. While stirring at 40°C, pentafluorophenyl 2,2,2-trifluoroacetate (4.6 The resulting solution was stirred at 25°C for 3 hours. The resulting mixture was concentrated in vacuo and the residue was applied to a silica gel column. This gave 4 g (58%) of G7-8. MS m / z [M+H] + (ESI) :614;H-NMR(DMSO,400Hz,ppm):δ 7.85(d,J=9. 2Hz,1H),5.23(d,J=3.2Hz,1H),4.99(dd,J=11. 2,3.2Hz,1H),4.52(d,J=8.4Hz,1H),4.04(s,3H ),3.87-3.94(m,1H),3.74.3.79(m,1H),3.45-3 .51(m,1H),2.78-2.81(m,2H),2.11(s,3H),2.0 6(s,3H),1.90(s,3H),1.84(s,3H),1.66-1.77( m,4H);F-NMR(DMSO,400Hz,ppm):δ-153.61,-15 3.66,-158.04,-158.16,-162.58,-162.64,-16 2.71.

[0161] [ka]

[0162] Decane-1,10-diol (125 g, 717.23 mmol, 5.00 equiv.) In a solution of chloromethane / N,N-dimethylformamide (250 mL / 250 mL), Ethylamine (21.8 g, 215.44 mmol, 1.50 equiv) was added, followed by 4-Toluolsulfonyl chloride (27.3 g, 143.19 mmol, 1.00 equiv.) was added at 25° C. The resulting solution was stirred at 25° C. for 16 hours. The resulting mixture was vacuum The mixture was concentrated under reduced pressure and diluted with 500 mL of dichloromethane. The solid matter was filtered off. The resulting solution The organic layer was washed with 3 x 500 mL of water and 3 x 500 mL of saturated sodium chloride. The residue was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. This gave 41 g (87%) of G9-1 as a colorless oil.

[0163] G9-1 (32.8 g, 99.86 mmol, 1.00 equiv.) N,N-dimethylformamide A solution of sodium azide (13.0 g, 199.97 mmol) in ethanolamide (150 mL) was , 2.00 equivalents) was added. The resulting solution was stirred in an oil bath at 90°C for 3 hours. The resulting solution was diluted with 500 mL of ethyl acetate and washed with 3 x 500 mL of saturated bicarbonate. The organic layer was washed with 500 mL of saturated sodium chloride and 3×500 mL of anhydrous sodium sulfate. The mixture was dried over sodium, filtered, and concentrated in vacuo, and the residue was applied to a silica gel column. This gave 18 g (83%) of G9-2 as a yellow oil.

[0164] G9-2 (10 g, 50.18 mmol, 1.00 equiv.) in tetrahydrofuran (10 0 mL) solution, triphenylphosphine (14.4 g, 54.90 mmol, 1.10 The resulting solution was stirred at 25° C. for 16 hours. The resulting mixture was concentrated in vacuo The resulting solution was diluted with 50 mL of water and washed with 3×50 mL of toluene. The aqueous layer was concentrated under vacuum and applied to a silica gel column, which gave 8 g (86%) of methylcellulose. ) to give G9-3 as a light yellow solid.

[0165] G9-3 (9.9 g, 22.13 mmol, 1.00 equiv.) in tetrahydrofuran (1 00 mL) solution, 1-hydroxybenzotriazole (3 g, 22.20 mmol, 1. 00 equivalents) and N,N-diisopropylcarbodiimide (5.6 g, 44.37 mmol) , 2.00 equivalents) was added. After that, 10-aminodecan-1-ol was added with stirring at 0°C. The resulting solution was stirred at 25°C for 1 hour. The mixture was stirred for 6 hours, concentrated in vacuo, and the crude product was purified by flash. This gave 9.3 g (69%) of G9-4 as a white solid. / z[M+H] + (ESI):603,1H NMR(DMSO,400Hz,ppm) :δ 7.81(d,J=9.2Hz,1H),7.69(t,J=5.6Hz,1H) ,5.22(s,1H),4.98(dd,J=11.2Hz,3.2Hz,1H),4 .49(d,J=8.4Hz,1H),4.33(t,J=5.2Hz,1H),4.0 2(s,3H),3.86-3.88(m,1H),3.69-3.72(m,1H), 3.34-3.41(m,3H),2.97-3.02(m,2H),2.11(s,3 H),2.00-2.04(m,5H),1.89(s,3H),1.77(s,3H) ,1.36-1.50(m,8H),1.24(s,12H).

[0166] To a solution of G9-4 (3 g, 4.98 mmol, 1.00 equiv.) in acetone (30 mL), Jones reagent (3.6 mL, 1.87 equiv.) was added dropwise with stirring at 0°C. The resulting solution was stirred at 0° C. for 2 hours. The pH value of the solution was adjusted to 6 with saturated aqueous sodium bicarbonate solution. The solid was filtered off and the filtrate was washed with 3 x 500 mL of saturated sodium chloride. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. This gave 2 g (65%) of G9-5 as a white solid.

[0167] G9-5 (4 g, 6.49 mmol, 1.00 equiv.) dissolved in dichloromethane (40 mL) solution, N,N-diisopropylethylamine (2.1 g, 16.25 mmol, 2.00 equivalents) amount), pentafluorophenyl 2,2,2-trifluoroacetate (2.73 g, 9 0.75 mmol, 1.50 equiv.) was added dropwise with stirring at 0°C. The mixture was stirred at 5°C for 3 hours. The resulting mixture was concentrated in vacuo. The crude product was purified by flash filtration. This gave 3 g (59%) of G9-6 as a white solid. / z[M+H] + (ESI):783.1H NMR(DMSO,400Hz,ppm) δ 7.81(d,J=9.2Hz,1H),7.68(t,J=5.6Hz,1H), 5.21(s,1H),4.98(dd,J=11.2Hz,3.2Hz,1H),4. 49(d,J=8.8Hz,1H),4.02(s,3H),3.83-3.90(m, 1H),3.67-3.73(m,1H),3.37-3.43(m,1H),2.97 -3.02(m,2H),2.75-2.79(m,2H),2.10(s,3H),1 .99-2.04(m,5H),1.89(s,3H),1.77(s,3H),1.6 2-1.69(m,2H),1.25-1.50(m,16H);F NMR(DMSO ,400Hz,ppm):δ-153.65,-153.66,-153.71,-15 8.09,-158.15,-158.21,-162.58,-162.63,-16 2.64,-162.70,-162.71.

[0168] [ka]

[0169] 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethane-1-ol Dissolve 194 g (998.84 mmol, 10.00 equiv.) in dichloromethane (120 mL ) solution, triethylamine (15.15 g, 149.72 mmol, 1.50 equiv.) After this, 4-toluenesulfonyl chloride (19.1 g, 100.18 mmol) (1.00 equiv.) was added at 25° C. The resulting solution was stirred at 16° C. overnight. The resulting solution was diluted with 100 mL of water. The resulting solution was diluted with 3 x 200 mL of dichloromethane. The resulting mixture was extracted with 3 x 100 mL of 5% aqueous citric acid solution. The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was applied to a silica gel column, and 35 g (93%) of G10-1 was obtained as a white solid. Obtained as a material object.

[0170] G10-1 (34.8 g, 99.88 mmol, 1.00 equiv.) N,N-dimethylphosphine A solution of sodium azide (13.0 g, 199.97 mmol) in 180 mL of methyl benzoate was added. 1, 2.00 equiv.) was added. The resulting solution was stirred in an oil bath at 90° C. for 3 hours. The resulting solution was diluted with 500 mL of ethyl acetate and added to 3 x 500 mL of saturated aqueous sodium hydroxide. The organic layer was washed with sodium carbonate and 3 x 500 mL of saturated sodium chloride. The mixture was dried over sodium, filtered, and concentrated in vacuo. The residue was applied to a silica gel column. This gave 17 g (71%) of G10-2 as a light yellow oil.

[0171] G10-2 (2.2 g, 10.03 mmol, 1.00 equiv.) in tetrahydrofuran ( 20 mL) solution of triphenylphosphine (2.88 g, 10.98 mmol, 1.1 0 equivalents) was added. The resulting solution was stirred at 25°C for 12 hours. The mixture was diluted with 1 mL of water and washed with 3 x 50 mL of toluene. The aqueous layer was concentrated in vacuo. This gave 1.7 g (81%) of G10-3 as a light yellow oil.

[0172] G10-3 (10 g, 22.35 mmol, 1.00 equiv.) in tetrahydrofuran (1 1-hydroxybenzotriazole (3 g, 22.20 mmol, 1 0.00 equiv.), N,N-diisopropylcarbodiimide (5.62 g, 44.53 mmol) 1, 2.00 equivalents) was added. Then, 2-[2-[2-(2-aza- [Ethoxy]ethoxy]ethan-1-ol (5.6g, 28.98mmol) The resulting solution was stirred at 25°C for 16 hours. The mixture was concentrated in vacuo, and the residue was applied to a silica gel column. This gave 8 g (58%) of G10-4 as a light yellow oil. MS m / z[M+H]+(ESI):623.,1H NMR(DMSO,400Hz ,ppm):δ 7.82(d,J=4.4Hz,2H),5.22(s,1H),4. 98(dd,J=11.2Hz,3.6Hz,1H),4.58(t,J=5.2Hz, 1H),4.49(d,J=8.4Hz,1H),4.01(s,3H),3.83-3 .91(m,1H),3.67-3.73(m,1H),3.48-3.51(m,10 H),3.37-3.42(m,5H),3.19-3.20(m,2H),2.11( s,3H),2.08-2.10(m,2H),2.08(s,3H),1.89(s, 3H), 1.77(s, 3H), 1.40-1.60(m, 4H).

[0173] A solution of G10-4 (6 g, 9.64 mmol, 1.00 equiv.) in acetone (60 mL) Jones reagent (6.7 mL) was added dropwise with stirring at 0° C. The resulting solution was The mixture was stirred for 3 hours at 0 C. The pH value of the solution was adjusted to 5 with a saturated aqueous solution of sodium bicarbonate. The solids were filtered off and the filtrate was washed with 3 x 500 mL of saturated sodium chloride. It was dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by flash. This gave 3 g (49%) of G10-5 as a solid.

[0174] G10-5 (1 g, 1.57 mmol, 1.00 equiv) in dichloromethane (10 mL) The solution was treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. (360 mg, 1.88 mmol, 1.20 equiv.), 2,3,5,6-tetrafluoroethylene Phenol (310 mg, 1.87 mmol, 1.20 equiv.). The resulting solution was heated at 25°C. The mixture was stirred at rt for 3 h. The resulting mixture was concentrated in vacuo. The crude product was obtained by flash. The resulting solution was extracted with chloroform, and the organic layers were combined. The extract was dried over sodium, filtered, and concentrated under reduced pressure to give 0.5 g (41%) of methyl methylcellulose. The product from other batches was combined to give 5.0422 g of G10-6 as a light yellow oil. MS m / z[M+H] + (ESI): 785. 1H NMR (CD3CN, 400Hz,ppm):δ 7.30-7.40(m,1H),6.48-6.60(m ,2H),5.30(s,1H),5.04(dd,J=11.2Hz,3.6Hz,1 H),4.52-4.59(m,3H),4.08-4.13(m,2H),3.95- 3.99(m,2H),3.76-3.78(m,3H),3.60-3.67(m,6 H),3.48-3.59(m,3H),3.30-3.48(m,2H),2.14- 2.16(m,5H),2.12(s,3H),1.93(s,3H),1.88(s, 3H),1.50-1.60(m,4H).F NMR(CD3CN,400Hz,pp m):δ-140.76,-140.79,-140.82,-140.84,-154 .52,-154.54,-154.57,-154.60.

[0175] [ka]

[0176] [ka]

[0177] Oxane-2,6-dione (1000 g, 8.76 mol, 1.00 equiv.), 4-dimethyl Dichloromethylaminopyridine (53.5 g, 437.9 mmol, 0.05 equiv.) To a solution of phenylmethylsulfonyl ether (10000 mL) was added phenylmethylsulfonyl ether (10000 mL) at room temperature under an inert atmosphere of nitrogen with stirring. To the resulting solution, ethanol (900 g, 8.32 mol, 0.95 equiv.) was added dropwise. The mixture was stirred at room temperature, and the resulting mixture was washed with saturated sodium bicarbonate solution. The value was adjusted to 1 with 10% hydrochloric acid. The resulting solution was extracted with ethyl acetate, and the organic layers were combined. The resulting mixture was washed with saturated sodium chloride. The organic layer was extracted with anhydrous sodium sulfate. The mixture was dried at 77°C, filtered, and concentrated under reduced pressure to give 1240 g (64%) of G11- 1 was obtained as a colorless oil. MS m / z [M+H]+ (ESI): 223.

[0178] G11-1 (500 g, 2.24 mol, 1.00 equiv.) in tetrahydrofuran (40 00mL) solution was stirred at 0°C and boron-methyl sulfide complex (270mL, The resulting solution was stirred at room temperature for 2 hours. The reaction was quenched by adding 1 L of methanol. The resulting mixture was concentrated under reduced pressure. The residue was applied to a silica gel column, which gave 312 g (67%) of G11-2. was obtained as a yellow oil. MS m / z [M+H]+ (ESI): 209.

[0179] (3R,4R,5R,6R)-2,4,5-trihydroxy-6-(hydroxymethyl ) oxan-3-aminium chloride (1000 g, 4.63 mol, 1.00 equiv.) Acetic anhydride (3560 g, 34.9 mol, 7.50 mL) was added to a solution of pyridine (10000 mL). (equivalent) was added at room temperature. The resulting solution was stirred overnight at room temperature. The resulting mixture was heated under reduced pressure. The crude product was purified by recrystallization from ice / water, which gave 14 Obtained 50 g (80%) of G11-3 as a white solid. MS m / z [M+H]+ (E SI):390.

[0180] G11-3 (100 g, 256.84 mmol, 1.00 equiv.) in dichloromethane (1 0 L) solution under an inert atmosphere of nitrogen, iron(III) chloride (125 g, 771.60 m mol, 3.00 equivalents) was added at room temperature. The resulting solution was stirred at room temperature for 3 hours. The resulting mixture was washed with water and saturated sodium chloride. The organic layer was extracted with anhydrous sodium sulfate. The mixture was dried, filtered, and concentrated under reduced pressure to give 52 g (61%) of G11-4. The product was used directly in the next step without further purification.

[0181] G11-4 (52 g, 157.91 mmol, 1.00 equiv.), benzyl 5-hydroxybenzoate Cypentanoate (42.7 g, 205.04 mmol, 1.30 equiv.) 1,2-dichloro Trimethylsilane (500 mL) was added to a solution of 1,000 chloroethane (500 mL) under an inert atmosphere of nitrogen at 0°C with stirring. Tylsilyl trifluoromethanesulfonate (10.5 g, 47.24 mmol, 0.3 0 equiv.) was added dropwise. The resulting solution was stirred at room temperature for 1 hour. The reaction was stopped by adding 100 ml of ethyl acetate. The resulting solution was extracted with dichloromethane, and the organic layers were combined. The resulting mixture was washed with water and saturated sodium chloride. The organic layer was extracted with anhydrous sodium sulfate. The mixture was dried over 1000 kJ / kg, filtered, and concentrated under reduced pressure. The residue was applied to a silica gel column. The crude product was purified by flash preparative HPLC to give 46.4 g (55%) of G11 -5 was obtained as a light yellow oil. MS m / z [M+H]+ (ESI): 538.

[0182] G11-5 (10 g, 18.60 mmol, 1.00 equiv.) in ethyl acetate (100 mL To the solution was added 10% palladium on activated carbon (1 g). The flask was evacuated and flushed with hydrogen five times. The resulting solution was stirred at room temperature for 3 hours. The solid was filtered and washed with methanol. The mixture was washed and dried under high vacuum overnight, yielding 6.82 g (82%) of G11-6. Obtained as a white solid.

[0183] 2-amino-2-(hydroxymethyl)propane-1,3-diol (300 g, 2. A solution of 48 mol (1.00 equiv.) in DMSO (500 mL) was placed in an inert atmosphere of nitrogen at 15°C. 5.0 M sodium hydroxide (49.5 mL, 0.248 mol, 0.1 equiv.) under atmosphere After this, tert-butyl acrylate (1079 g, 8.4 mol, 3.4 The resulting solution was stirred at 25°C for 24 hours. The resulting solution was extracted with 4 x 3000 mL of ethyl acetate and the organic layers were combined. The organic layer was washed with water and saturated sodium chloride. The organic layer was dried over anhydrous sodium sulfate and The mixture was filtered and concentrated under reduced pressure. The residue was applied to a silica gel column. This gave 526 g (42%) of G11-7 was obtained as a light yellow oil. MS m / z [M+H]+ (ESI ):506.6.

[0184] G11-7 (50 g, 99.0 mmol, 1.00 equiv.) in CHCl (750 mL ) solution, 365 mL of 25% aqueous Na2CO3 solution was added with stirring. Dichloroformate (50.5 g, 0.297 mmol, 3.00 equiv.) was added to the flask and stirred at room temperature. The resulting solution was stirred overnight at room temperature. The organic layers were combined and washed with saturated aqueous sodium chloride solution. The mixture was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was applied to a silica gel column. This gave 42.4 g (67%) of G11-8 as a light yellow oil. m / z[M+H]+(ESI):640.4.

[0185] A solution of G11-8 (300 g, 468.9 mmol, 1.00 equiv.) was added to formic acid (3 L, 96%) was added. The resulting solution was stirred at 25°C for 16 hours. The reaction mixture was then heated under reduced pressure. The pH value of the solution was adjusted to 12 with sodium hydroxide (1 mol / L). The resulting solution was extracted with 3 x 6 L of ether, and the aqueous layers were combined. The pH of the aqueous layer was adjusted to 4 using ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 198.96 g (90%) of G11-9 was obtained as a yellow oil. MS m / z [M+ H]+(ESI):472.

[0186] G11-9 (264 g, 560.51 mmol, 1.00 equiv.) in dichloromethane / aq. A solution of acetonitrile (1400 mL / 1400 mL) was added to N,N-diisopropylethyl ether. amine (578.4 g, 4.484 mol, 8 equiv.), O-benzotriazole-N,N, N-tetramethyl-uronium-hexafluorophosphate (848g, 2.231m The resulting solution was stirred at 25°C for 30 minutes. Then, tert-butyl N-(3-aminopropyl)carbamate (390 g, 2.238 (mmol, 4.00 equiv.) was added at 25° C. The resulting solution was stirred at 25° C. for 6 hours. The resulting solution was concentrated under reduced pressure. The residue was diluted with 3000 mL of dichloromethane. The organic layer was washed with saturated sodium bicarbonate solution, saturated ammonium chloride, and saturated sodium chloride. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was applied to a silica gel column, which gave 336 g (70%) of G11- 10 was obtained as a white foam. MS m / z [M+H]+ (ESI): 941.

[0187] G11-10 (350 g, 372.3 mmol, 1.00 equiv.) in dichloromethane (1 To the resulting solution (0.4 L) was added trifluoroacetic acid (350 mL). The mixture was stirred for 4 hours and concentrated under reduced pressure to give 470 g (crude) of G 11-11 was obtained as a red oil. MS m / z [M+H]+ (ESI): 641.

[0188] G11-6 (66 g, 147.51 mmol, 3.50 equivalents) and N,N-diisopropyl Pyroethylamine (37 g, 286.29 mmol, 6.78 equiv.) in dichloromethane / In an acetonitrile (200 mL / 200 mL) solution, 1-hydroxybenzoate was added under an inert atmosphere of nitrogen. hydroxybenzotriazole (24.5 g, 181.48 mmol, 4.30 equiv.) and O -Benzotriazole-N,N,N-etramethyl-uronium hexafluoride Fluorophosphate (56 g, 147.66 mmol, 3.50 equiv) was added at room temperature. The resulting solution was stirred for 30 minutes at room temperature. After this, benzyl G11-11 (41.4 g) , 42.20 mmol, 1.00 equiv.) was added at room temperature. The resulting solution was stirred. The mixture was concentrated under reduced pressure and the resulting solution was diluted with dichloromethane. The resulting mixture was diluted with saturated sodium bicarbonate and saturated sodium chloride. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude material was purified by flash preparative HPLC. This gave 41.2 g (51 %) of G11-12 was obtained as a light yellow solid. MS m / z [M / 2+H]+ (ESI ):965.

[0189] G11-12 (20 g, 10.37 mmol, 1.00 equiv.) in methanol (200 m To the solution was added 10% palladium on activated carbon (2 g) at room temperature. The flask was evacuated and hydrogen The resulting solution was stirred at room temperature for 4 hours. The solid matter was filtered off. The resulting mixture was concentrated under reduced pressure to give 16.5 g (89%) of G11-13. Obtained as a white solid. MS m / z[M / 2+H] + (ESI):898.

[0190] G11-0 (12.8 g, 24.57 mmol, 1.00 equiv.) N,N-dimethylphosphine In a solution of 500 mL of methyl methyl ether, N,N-diisopropylethylamine (9.0 g, 6 9.64 mmol, 3.00 equiv), O-benzotriazole-N,N,N-etramethyl 1-Uronium hexafluorophosphate (9.9 g, 27.03 mmol, 1.1 0 equiv.) was added. The resulting solution was stirred at room temperature for 2 hours. G11-13 (4 The resulting solution was stirred at room temperature for 16 hours. The resulting mixture was concentrated under reduced pressure and the crude product was purified by flash preparative HPLC. This gave 22 g (41%) of G11-14 as a light yellow solid. MS m / z[MH] - (ESI): 2295. H-NMR (DMSO, 400 M H z):7.79-7.83(m,6H),7.70-7.73(m,4H),7.33- 7.35(m,2H),7.26-7.30(m,2H),7.19-7.22(m,5 H),7.01(s,1H),6.84-6.87(m,4H),5.19-5.20( d,J=4.0Hz,3H),4.93-4.97(m,3H),4.58-4.59( d,J=4.0Hz,1H),4.46-4.48(d,J=8.0Hz,3H),3. 95-4.04(m,9H),3.82-3.89(m,3H),3.67-3.71( m,9H),3.45-3.61(m,12H),3.36-3.41(m,3H),2 .94-3.09(m,16H),2.24-2.27(m,6H),2.00-2.0 8(m,29H),1.87(s,9H),1.75(s,9H),1.63-1.69 (m,3H), 1.41-1.51(m,19H).

[0191] G11-14 (2 g, 0.87 mmol, 1.00 equiv.) in dichloromethane (20 mL ) solution under an inert atmosphere of nitrogen, xi) Phosphine (786.7 mg, 1.62 mmol, 3.00 equivalents) was added at room temperature. After this, pyridine trifluoroacetate (336.3 mg, 1.74 mmol, 2.0 0 equivalents) was added at room temperature. The resulting solution was stirred at room temperature for 2 hours. Concentration under reduced pressure gave 2.0 g (crude) of G11-15 as a yellow oil. The product was used directly in the next step without further purification.

[0192] G11-15 (2 g, 0.80 mmol, 1.00 equiv.) in acetonitrile (20 mL ) solution under an inert atmosphere of nitrogen, water (43 mg, 2.4 mmol, 3.00 equiv.) and and 4,5-dicyanoimidazole (113 mg, 0.96 mmol, 1.20 equiv.) The resulting solution was stirred at room temperature for 2 hours. The resulting mixture was concentrated under reduced pressure. The crude material was purified by flash preparative HPLC. The product fractions were purified by dichloromethane. The resulting mixture was washed with saturated sodium chloride and the organic layer was extracted with anhydrous sodium sulfate. The mixture was dried over sodium, filtered, and concentrated under reduced pressure to give 1.025 g (53%) of G11-16 was obtained as a white solid. MS m / z [MH] - (ESI):2412 .1H NMR(DMSO-d6,400Hz):8.10-7.90(m,1H),7 .85-7.7.81(m,6H),7.75-7.72(m,3H),7.35-7. 22(m,9H),7.05-7.00(m,1H),6.89-6.87(m,4H) ,5.21-5.20(d,J=3.2Hz,3H),4.98-4.95(m,3H) ,4.50(s,1H),4.47-4.40(m,3H),4.04-4.01(s, 11H),3.88-3.85(m,3H),3.73-3.69(m,9H),3.5 5-3.52(m,12H),3.41-3.39(m,3H),3.20(s,2H) ,3.04-3.00(m,14H),2.85-2.75(m,2H),2.49-2 .29(m,6H),2.10-1.99(m,28H),1.89-1.77(m,1 1H),1.77-1.70(m,9H),1.70-1.65(m,2H),1.52 -1.45(m,18H).P NMR(CD3OD,400Hz):7.957,7. 927.

[0193] [ka]

[0194] G12-1 (4.0 g, 1.70 mmol, 1.00 equiv.) in dichloromethane (40 m L) solution under an inert atmosphere of nitrogen, (xy)phosphine (922.6 mg, 3.06 mmol, 1.80 equiv.) was added at room temperature. To this, pyridine trifluoroacetate (525 mg, 2.72 mmol, 1.60 (equivalent) was added at room temperature. The resulting solution was stirred at room temperature for 1 hour. The resulting mixture was heated under reduced pressure. The mixture was concentrated under reduced pressure to give 3.0 g (crude) of G12-2 as a yellow oil.

[0195] G12-2 (3.0 g, 1.18 mmol, 1.00 equiv.) in acetonitrile (30 m L) solution under an inert atmosphere of nitrogen was added water (166 mg, 9.22 mmol, 7.85 equiv. Amount) was added at room temperature. mol, 0.46 equivalents) was added at room temperature. The resulting solution was stirred at room temperature for 1 hour. The resulting mixture was concentrated under reduced pressure. The crude product (2.5 g) was purified by flash preparative HPLC. This gave 2.0542 g (71%) of G12-3 as a white solid. . MS m / z[MH]-(ESI):2468.1H-NMR(DMSO-d6,4 00Hz):7.91-7.80(m,7H),7.74-7.71(m,3H),7. 38-7.31(m,4H),7.29-7.23(m,5H),6.97(s,1H) ,6.87-6.81(m,4H),5.21(s,3H),4.98-4.94(m, 3H),4.60(s,1H),4.49-4.47(d,J=8.4Hz,3H),4 .25-4.10(m,2H),4.02(s,9H),3.88-3.85(m,3H ),3.73-3.68(m,9H),3.55-3.52(m,11H),3.52- 3.41(m,3H),3.20(s,3H),3.04-2.92(m,13H),2 .90-2.89(m,2H),2.29-2.10(m,6H),2.09-1.99 (m,28H),1.89-1.80(m,9H),1.77-1.71(m,9H), 1.52-1.37(m,23H),1.19(s,9H).P-NMR(DMSO-d 6,400Hz):8.456.

[0196] [ka]

[0197] G13-1 (4.0 g, 1.70 mmol, 1.00 equiv.) in dichloromethane (40 m L) solution under an inert atmosphere of nitrogen, (oxy)phosphine (690 mg, 2.29 mmol, 1.80 equiv.) was added at room temperature. In addition, pyridine trifluoroacetate (390 mg, 2.02 mmol, 1.60 equiv. ) was added at room temperature. The resulting solution was stirred at room temperature for 2 hours. The resulting mixture was heated under reduced pressure. Concentration gave 2.5 g (crude) of G13-2 as a yellow oil.

[0198] G13-2 (2.3 g, 0.90 mmol, 1.00 equiv.) in acetonitrile (22 m L) solution and water (50 mg, 2.78 mmol, 3.00 equiv.) Midazole (0.12 g, 101.69 mmol, 1.20 equiv.) was added at room temperature. The resulting solution was stirred at room temperature for 2 hours. The resulting mixture was concentrated under reduced pressure. 0.3g) was purified by flash preparative HPLC, which gave 2.076g (94% ) G13-3 was obtained as an off-white solid. MS m / z [M+Na]+ (ESI ):2506.1H-NMR(DMSO-d6,300Hz):8.05-7.90(m ,1H),7.80-7.71(m,9H),7.38-7.22(m,9H),6.9 5(s,1H),6.89-6.86(m,4H),5.21(s,3H),4.99- 4.94(m,3H),4.65-4.40(m,4H),4.02(s,11H),3 .88-3.85(m,3H),3.73-3.68(m,9H),3.56-3.51 (m,12H),3.42-3.38(m,3H),3.32(s,2H),3.04( s,14H),2.84-2.82(m,2H),2.29-2.22(m,6H),2 .10-1.99(m,28H),1.89(s,10H),1.77(s,10H), 1.52-1.45(m,22H),1.22(s,9H).P-NMR(DMSO-d 6,300Hz):7.867.

[0199] GalNAc conjugation The following modifications: 2'-F-NPS-PS-2'-F-NPS; 2'-F-NP-PS-2' -F-NP;2'-OMe-NP-PS-2'-OMe-NP;2'-OMe-NPS- DNA-PS-2'-OMe-NPS, 2'-OEt-NPS-DNA-PS-2'-O Et-NPS, and 2'-MOE-NPS-DNA-PS-2'-MOE-NPS For the preparation of 5'GalNAc-conjugated oligomers, a scale of 10-200 μM was used. 5'-phosphoramide diluted to a concentration of 0.1 M in anhydrous CH3CN in the 5' to 3' direction. The midite monomer was used in the presence of 5-(benzylthio)-1H-tetrazole activator. (coupling time 2.0-4.0 min) to synthesize GalNac 2-CPG. The modified coupling cycle was followed by standard capping, acid The modified oligonucleotide was obtained by cleavage and deprotection. The recovery rate was over 98%. DDTT (Dimethylamino-methylidene)amino)-3H-1, 2,4-Dithiazoline-3-thione was used to synthesize oligoribonucleotide phosphorothioates. 0.2% lutidine in acetonitrile (1:1) was used as a sulfur transfer agent for the synthesis. Large-scale synthesis of phenylacetyl disulfide (PADS) (Akta OP-100) The oligonucleotide-carrying solid support was heated in a shaker with ammonia. The product was cleaved from the support and desalted by heating in a 1:1 dimethylammonium chloride / methylamine solution at room temperature for 3 hours. The group-labile protecting groups were deprotected.

[0200] [ka]

[0201] 3'-C6NH2-NPS-PS-NPS- (precursor) synthesis The following modifications: 2'-F-NPS-PS-2'-F-NPS; 2'-F-NP-PS-2' -F-NP;2'-OMe-NP-PS-2'-OMe-NP;2'-OMe-NPS- DNA-PS-2'-OMe-NPS, 2'-OEt-NPS-DNA-PS-2'-O Et-NPS, and 2'-MOE-NPS-DNA-PS-2'-MOE-NPS To prepare the 3'GalNAc-conjugated oligomer, a general-purpose support (loading 6 ASO was synthesized on a 10 μmol scale using 5 μmol / g of PEG. The synthesis procedure was as follows: Same as above. To introduce a C6-NH2 linker at the 3' end, 0.1M 6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl) N,N-diisopropyl)-phosphoramidite, with a coupling time of 10 min. The oligonucleotide-loaded solid support was heated in a shaker with ammonia / methylamine. The base-labile cleavage was performed by heating in aqueous ethanol (1:1) at room temperature for 3 hours. After IEX purification and desalting, the C6-NH2 modified ASO was used to deprotect the post-synthesis Conjugation can be performed.

[0202] [ka]

[0203] 3'-GalNAc NPS-PS-NPS-ASO synthesis (post-synthesis conjugation) ) The 3'-C6-NH2 modified ASO was dissolved in 0.2 M sodium bicarbonate buffer, pH 8.5 (0 0.015 mM) and 5-7 molar equivalents of GalNAc-6 ester dissolved in DMSO. The reaction mixture was stirred at room temperature for 4 hours. A sample was analyzed to determine any unreacted The presence of amino-modified ASO was confirmed. The reaction mixture was concentrated under reduced pressure and the residue was The residue was dissolved in water and purified by HPLC on a strong anion exchange column.

[0204] [ka]

[0205] [Table 2]

[0206] Quantitative or raw analysis of crude oligomers The samples were dissolved in deionized water (1.0 mL) and quantified as follows: first, water; A blank was run using 1.0 mL of the sample, 20 μL of the sample, and 980 μL of water. Mix thoroughly in a microfuge tube, transfer to a cuvette, and obtain an absorbance reading at 260 nm The crude material is dried and stored at -20°C.

[0207] Crude HPLC / LC-MS analysis 0.1 OD of the crude sample was subjected to crude MS analysis. After confirming the crude LC-MS data, the purified The process was carried out.

[0208] HPLC purification Phosphoramidate (NP) and thiol with and without GalNAc conjugates Phosphoramidate (NPS) modified oligonucleotides were purified by anion exchange HPLC. The buffer was 20 mM sodium phosphate in 10% CH3CN, pH 8.5 (buffer Buffer A) and 10% CH3CN, 20 mM sodium phosphate in 1.8 M NaBr, pH 8.5 (Buffer B). Fractions containing full-length oligonucleotides were pooled and desalted. and freeze-dried.

[0209] Desalting of purified oligomers Then, Sephadex G-25 M (Amersham Bioscience The purified dried oligomer was desalted using a 10 mL desalting column. The mixture was conditioned three times with ionized water. Finally, the mixture was conditioned in 2.5 mL of RNAse-free water. Completely dissolved purified oligomers are applied to the cartridge and eluted dropwise at extremely slow flow rates. The salt-free oligomer was directly poured into a screw cap using 3.5 mL of deionized water. The solution was eluted into a drop vial.

[0210] Stability testing of conjugated oligonucleotides In embodiments, the disclosed oligonucleotides are unmodified oligonucleotides of the same sequence. These antibodies exhibit increased affinity for the target nucleic acid sequence compared to the original antibody. For example, In the disclosed oligonucleotides, the oligonucleotides are more easily modified than unmodified oligonucleotides of the same sequence. having a nucleobase sequence that is complementary to or hybridizes with a target nucleic acid sequence with high affinity In embodiments, the oligonucleotide of the present disclosure complexed with the complementary target nucleic acid sequence comprises: Melting temperature T >37°C m The complex is stored in physiological conditions or in phosphate buffered saline (PBS). In embodiments, the complex can be formed under near-physiological conditions, such as in PBS. In embodiments, the Tm of the complex is >50 In an embodiment, the Tm of the complex is 50 to 100°C. The oligonucleotides of the present disclosure are duplexed with a target nucleic acid sequence under physiological or near physiological conditions. The Tm of this compound is >50°C.

[0211] In certain embodiments, the target nucleic acid sequence is a known viral DNA, such as the HBV genome, or It may be selected from RNA sequences.

[0212] In embodiments, the oligonucleotides of the present disclosure are directed to the HBV genome or its RNA equivalent. and / or HBV, It exhibits stability by complexing with at least one of the genome or its RNA equivalent. In embodiments, the oligonucleotide complexed with the complementary HBV genome is melted at >37°C. The HBV genome contains the RNA sequences of DR-1 and / or DR-2. The complex may be any RNA sequence. The complex may be stored in physiological conditions or in phosphate buffered saline (PBMS). In embodiments, the complex can be formed under near physiological conditions, such as in PBS (S). In embodiments, the Tm of the complex is >50°C. In one embodiment, the Tm of the complex is 50 to 100°C. The oligonucleotides of the present disclosure are duplexed with HBV RNA under physiological or near physiological conditions. The Tm of the nucleotide is >50°C.

[0213] In vitro testing of oligonucleotides Two HBV cell lines, HepG2.2.15 (2215) and HepG2.117 ( 2117) was used to evaluate the in vitro potency of oligonucleotides. G2.2.15 cells were used to reduce HBsAg in tissue culture supernatants (sup), and Cytotoxicity was measured. The reduction of HBV DNA in the supernatant and intracellular fractions was .117 cells.

[0214] The HepG2.2.15 cell line was a stable cell line harboring four integrated HBV genomes. 10% FCS, 100 IU / mL penicillin, 100 μg / mL streptomycin The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 2% glutamine and 5% CO₂ at 37°C. The cells were grown in a CO atmosphere. The day before administration, 2.5 × 10 cells were 4 pcs / well, The cells were plated onto a 96-well plate coated with IgG and incubated overnight. On the day of the test, Lipofectamine RNAiMax (Thermo Fisher Scientific) was used. Serially diluted oligonucleotides were prepared using a 100% RT-PCR kit (Saltham, MA) according to the manufacturer's protocol. The cells were transfected with the oligomer. Two experiments were performed for each drug concentration, and the E Both C50 and CC50 measurements were performed. Three days after transfection, the supernatant ( sup) and used for HBsAg ELISA (AutoBio , China). For CC50 measurement, CellTiter-Glo (registered trademark) ) (Promega, Madison, WI) was used in the assay according to the manufacturer's instructions. did.

[0215] HepG2.117 is under the control of TetOFF (tetracycline or its homologs) In the absence of doxycycline, 1.05 copies of HBV were integrated. This is a stable hepatoma cell line carrying the V genome (ayw subtype). 10% FCS, 100IU / mL penicillin, 100μg / mL streptomycin, 2% glutamine, DMEM / supplemented with 250 μg / mL G418 and 2 μg / mL tetracycline Cells were grown in F12 medium at 37°C in a 5% CO2 atmosphere. The cell culture medium containing the tetracycline is removed, and the cells are washed to remove any residual tetracycline. Treatment medium (Tet system approved) 2% FBS 100 IU / mL penicillin, 100 with DMEM / F12 containing 2 μg / mL streptomycin and 2% glutamine 2.5 x 10 4 Cells / well were plated on a collagen-coated 96-well plate. The cells were then incubated overnight. On the day of the experiment, Lipofectamine was added. ne RNAiMax (Thermo Fisher, Waltham, MA) Cells were transfected with serially diluted oligomers according to the protocol of the author. Each drug concentration was tested twice, and both EC50 and CC50 measurements were performed. Four days after transfection, HBV DNA was directly analyzed by qPCR. The supernatant was collected for further use. HBV DNA from the cells was purified using MagMAX™. Total Nucleic Acid Isolation Kit(Thermo After isolation, HBV subtypes were analyzed using a qPCR kit (Fisher). The forward primer was -(5'-TTG CCT TCT GAC TTC TTT CCT TCT-3') , reverse primer (5'-TGC CTG AGT GCT GTA TGG TG AG-3'), and 5' with FAM (6-carboxyfluorescein) and 3' with Fluorescent Taq labeled with TAMRA (6-carboxytetramethylrhodamine) Man® probe (5'-TCG GGA AGC CTT AGA GTC TCC TGA-3') was designed (Primer Express, Thermo Scientific). These primers and probes were used to identify the AmpliTaq Go ld DNA polymerase (Perkin-Elmer Life Science, W Quantitative real-time PCR was performed using a 100-kDa antibody (Altham, MA). The reaction conditions were as follows: The cycle consisted of 10 minutes at 95°C, followed by 50 cycles of denaturation (9 5°C for 15 seconds) and annealing / polymerization (59°C for 1 minute).

[0216] Infectious HBV strains in primary human hepatocytes Cryopreserved primary human hepatocytes (PHH) were thawed and plated at 200,000 cells / well for 24 hours. The cells were seeded onto well plates. The cells were allowed to recover overnight at 37°C and 5% CO2. The cells were infected with HBV at 50-100 μg / mL overnight at 37°C / 5% CO2. Remove the virus inoculum and wash the cells three times with prewarmed wash medium. Then, inoculate with fresh P Re-supplement the HH culture medium. Replace the medium with 450 μL of fresh medium. Add the infecting mixture. The oligomers were then added to Opti-MEM I (Life Tech Dilute to a final concentration of 20x with Liposome (Liposome Technology, Catalog #31985-070). ofectamine RNAiMAX (Invitrogen, Catalog #: 1377 Mix with an equal volume of Opti-MEM I containing 8-150 ml of PBS and pipette up and down three times. Add 50 μL of the oligo:RNAiMAX mixture and incubate at room temperature for 10-20 minutes. Add the mixture to the wells and tap the plate several times with your hand. Place the plate in the incubator. On the day of the assay, for HBsAg and HBeAg ELISA assays, The supernatant was collected and the cells were harvested for cell viability assay. HBsAg ELISA was performed as described above. For HBeAg, the Autobio Diagnostics method (C L0312-2) was used.

[0217] In vivo testing of oligonucleotides AAV / HBV is a recombinant AAV that carries a replication-competent HBV genome. Taking advantage of the high liver tropism of AAV 8, we have developed a method to efficiently transduce the HBV genome into mouse hepatocytes. Immunocompetent mice were infected with AAV / HBV, This may result in prolonged HBV viremia, reproducing chronic HBV infection in patients. Using the AAV / HBV model, we investigated the in vivo activity of various anti-HBV drugs. Mice were infected with AAV-HBV on day -28 of the study. Test substance or negative control (PBS) was administered subcutaneously at the indicated doses three times on days 0, 2, and 4. or injected as a single dose at the specified dose on Day 0 (unless otherwise specified). Entecavir (E), a positive control for HBV DNA (not HBV antigen), The serum HBV S antigen (HBsAg) and E antigen (HBeAg) were measured. g) was assessed by ELISA and HBV DNA by real-time PCR. The LISA and qPCR methods are described above in the in vitro assays section.

[0218] The following describes how the data in Tables 1-22 was obtained. For all in vitro HBsAg cell line EC50 and CC50 data, Use the method in pG2.2.15, and therefore, enter "" in the column or row where the data is presented. 2215". In vitro HBV DNA cell line EC50 and CC50 For all data, the HepG2.117 method was used, and therefore the data shown The column or row where the number is listed is indicated as "2117." All EC50 data for HBsAg and HBeAg in vitro are available from PHH The method used was "PHH" and therefore the column or row where the data is presented is designated "PHH." For the results of the AAV-HBV mouse model in vivo, see the in vivo The maximum reduction in HBsAg (or HBeAg) was measured in nadir (unit: The column or row where the data is presented is labeled nadir. Two types of ASOs are often compared based on their nadir. If other values ​​were compared, they will be indicated in the text.

[0219] Treatment method Administering a therapeutically effective compound of the present disclosure, e.g., a compound listed in Tables 1-2, to an adult suffering from HBV infection. The compound selected from the group consisting of HBV, HBV-1, HBV-2, HBV-3, HBV-4, HBV-5, HBV-6, HBV-7, HBV-8, HBV-9, HBV-10, HBV-11, HBV-12, HBV-13, HBV-14, HBV-15, HBV-16, HBV-17, HBV-1 For example, serum HBV S antigen (HBsAg) and / or E antigen (HBeAg) levels Treatment is continued until the level subsides.

[0220] Administering a therapeutically effective compound of the present disclosure, e.g., a compound listed in Tables 1-2, to an adult suffering from HBV infection. 2. A compound selected from the group consisting of: For example, serum HBV S antigen (HBsAg) and / or E antigen (HBeAg) levels Continue treatment until the level drops.

[0221] [Table 3] * Sequences 260 and 261 were also tested with similar results.

[0222] [Table 4] * 5'-GalNac2-moeGnpsmoeCnpsmoeAnpsmoeGnp smoeAnpsGpsGpsTpsGpsApsApsGps(5m)CpsGpsA psmoeAnpsmoeGnpsmoeUnpsmoeGnpsmoeCn-3' and 5'-GalNac1-moeGnpsmoeCnpsmoeAnpsmoeGn psmoeAnpsGpsGpsTpsGpsApsApsGpsCpsGpsApsm oeAnpsmoeGnpsmoeUnpsmoeGnpsmoeCn-3' was tested and gave similar results.

[0223] [Table 5] * 5'-GalNAc2-mGnpsmCnpsmAnpsmGnpsmAnpsGp sGpsTpsGpsApsApsGps(5m)CpsGpsApsmAnpsmGn psmUnpsmGnpsmCn-3' was also tested, and similar results were obtained.

[0224] As can be seen from the above, MOE NPS oligomers are involved in the formation of MOE PS in vivo. The activity of OMe NPS was higher than that of MOE PS oligomer, and OMe NPS had similar activity to that of MOE PS oligomer.

[0225] Two oligonucleotides were prepared, one containing an OEt NPS substitution and the other containing an MOE NPS. The nucleotides were tested in vitro and in vivo. Table 4 below summarizes the results of the tests. The results are summarized.

[0226] [Table 6] As can be seen from the above, MOE NPS oligomers have the same structure as OEt NPS oligomers. It had similar activity.

[0227] The first contains a MOE PS substitution, the second has a MOE NPS substitution, and the third is OM Four oligonucleotides were prepared: one with an EPS substitution and the other with a OME NPS. The results of the studies are summarized in Table 5 below.

[0228] [Table 7]

[0229] [Table 8] The first contains a 5'GalNAc-2'-MOE NPS substitution and the second contains a 5'-Ga Two oligonucleotides with lNAc-6:MOE PS substitutions were analyzed in vivo. The results of the tests are summarized in Table 6 below.

[0230] [Table 9]

[0231] [Table 10]

[0232] The first contains a 3'-GalNAc-2'-MOE NPS substitution, and the second contains a 3'-G Two oligonucleotides with alNAc-2'-MOE PS substitutions were analyzed in vivo. The results of the study are summarized in Table 7 below.

[0233] [Table 11]

[0234] [Table 12]

[0235] The first contains an OME NPS substitution, and the second contains two OME PS substitutions. The oligonucleotides were tested in vivo.

[0236] [Table 13]

[0237] The following sequences were tested in the HBV mouse model: 3'GalNac MOE NPS is 0.8 l longer than 5'GalNac MOE PS. og (6-fold) maintained good efficacy, with superiority maintained over most of the 21-day study. 5'GalNac MOE NPS was more stable than 5'GalNac MOE PS. The efficacy of 0.4 log (2.5 times) was maintained throughout the 21-day study. At a dose of 3 × 3.3 mg / kg, 3'GalNac MO E NPS and 5'GalNac MOE NPS act similarly, both with 5'Gal It maintained 0.6 log (4-fold) better efficacy than Nac MOE PS, with a 2-fold advantage. This was maintained for most of the 1-day test.

[0238] [Table 14]

[0239] The following sequences were tested in the HBV mouse model:

[0240] [Table 15]

[0241] The following sequences were tested in the HBV mouse model. Values ​​in the right column are 3 x 10 mg / kg The maximum LOG reduction in HBsAg is shown in g administered on days 0, 2, and 4.

[0242] [Table 16]

[0243] The following sequences were tested in the HBV mouse model. Values ​​in the right column are 3 x 10 mg / kg The maximum LOG reduction in HBsAg is shown in g administered on days 0, 2, and 4.

[0244] [Table 17]

[0245] [Table 18]

[0246] [Table 19]

[0247] [Table 20]

[0248] [Table 21]

[0249] [Table 22]

[0250] [Table 23]

[0251] [Table 24]

[0252] Three oligonucleotides containing F NPS chemistry were tested in vivo. The first oligonucleotide has a tri-GalNac moiety at the 5' end, and the second oligonucleotide The oxidase has two tri-GalNac moieties, one at the 5' end and one at the 3' end. The third oligonucleotide has a tri-GalNac moiety at the 5' end and a 3' end. It has a mono-GalNac at the end. Table 21 below summarizes the results of the test.

[0253] [Table 25]

[0254] 3x5mkp has GalNac conjugation at both the 5' and 3' ends. Conjugation at the 5' end is more evident than conjugation at the 5' end only at a certain time point. It is excellent, with in vivo activity 0.6 logs better.

[0255] One contains a tri-GalNac moiety at the 5' end and the second contains a tri-GalNa Two oligonucleotides containing a c moiety and a tocopherol moiety at the 3' end were 3x A dose of 10 mpk was tested in vivo.

[0256] [Table 26]

[0257] In some embodiments, the oligonucleotides of the present disclosure also include those listed in Tables 1-43. An oligonucleotide selected from the nucleobase sequences listed in Tables 1 to 43, regardless of any modifications of the sequence. The oligonucleotides of the present disclosure also include the sequences listed in Tables 1-43. and at least one nucleobase sequence selected from the sequences listed in Tables 1 to 43, regardless of modification. In some embodiments, the oligonucleotide also includes an oligonucleotide comprising a sequence that is 90% identical to the sequences listed in Tables 1 to 43, regardless of any modifications of the sequences listed in Tables 1 to 43; They differ in 2, 3, 4, or 5 nucleobases.

[0258] In some embodiments, the oligonucleotides of the present disclosure also include those listed in Tables 1-43. nucleotide sequences selected from those listed in Tables 1-43, regardless of the nucleic acid base of the sequence. The oligonucleotides of the present disclosure also include those listed in Tables 1-43. nucleobases selected from the sequences listed in Tables 1 to 43, regardless of the nucleobases of the sequences Also included are oligonucleotides containing sequences that are at least 90% identical to the sequence. In embodiments, the sequences listed in Tables 1-43 are used without regard to modifications of the sequences listed in Tables 1-43. The sequence differs by 1, 2, 3, 4, or 5 nucleobases.

Claims

1. Formula (I) or (II): 【Chemistry 1】 [where, R １ is 【Chemistry 2】 and R ８ is C ３ , C ４ , C ５ , C ６ , C ７ , C ８ , C ９ , or C １０ C having an alkyl moiety or 1, 2, 3, 4, or 5 ethylene oxide moieties ３ , C ４ , C ５ , C ６ , C ７ , C ８ , C ９ , or C １０ Contains an alkyl oxide moiety or R ８ teeth, 【Transformation 3】 and R ２ is C ３ , C ４ , C ５ , C ６ , C ７ , C ８ , C ９ , or C １０ C having an alkyl moiety or 1, 2, 3, 4, or 5 ethylene oxide moieties ３ , C ４ , C ５ , C ６ , C ７ , C ８ , C ９ , or C １０ Contains an alkyl oxide moiety, R ３ teeth, 【Chemistry 4】 and R ４ is H, R ５ teeth 【Transformation 5】 is (In the formula, R ６ is the bond to the oligonucleotide, and R ７ is H and the linkage contains a phosphoramidate or phosphodiester moiety, or R ６ is an alcohol protecting group selected from the group consisting of tert-butyldimethylsilyl (TBMDS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), monomethoxytrityl (MMTr), and tritolyl; R ７ is a solid support linker), Or, R ４ and R ５ together form a 5- or 6-membered ring, and the 5- or 6-membered ring is unsubstituted or has one —CH ２ is substituted with an OPG group, PG is an alcohol protecting group; a is 3, b is 3; c is 4, 5, 6, or 7; d is 0, 1, 2, 3, or 4. A compound having the structure:

2. R ２ But C ３ , C ４ , C ５ , C ６ , C ７ , C ８ , C ９ , or C １０ The compound of claim 1 containing an alkyl moiety.

3. R ２ C having 2, 3, 4, or 5 ethylene oxide moieties ３ , C ４ , C ５ , C ６ , C ７ , C ８ , C ９ , or C １０ The compound of claim 1 containing an alkyl oxide moiety.

4. R4 and R ５ is one -CH ２ 2. The compound of claim 1, which together form a 5- or 6-membered ring substituted by OPG groups, PG being 4,4'-dimethoxytrityl (DMTr).

5. The compound according to any one of claims 1 to 4, which is a compound of formula (I).

6. The compound according to any one of claims 1 to 4, which is a compound of formula (II):

7. R ８ but, 【Transformation 6】 2. The compound of claim 1, wherein:

8. R4 and R ５ is one -CH ２ 2. The compound of claim 1, wherein together they form a 5- or 6-membered ring substituted by an OPG group, PG being selected from the group consisting of tert-butyldimethylsilyl (TBMDS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr), and tritolyl.

9. R4 is H, R ５ teeth 【Transformation 7】 is (In the formula, R ６ is a bond to an oligonucleotide containing a phosphoramidate or phosphodiester moiety, and R ７ is H), The compound of claim 1.

10. R4 is H, R ５ teeth 【Transformation 8】 is (In the formula, R ６ is the bond to an oligonucleotide containing a phosphoramidate moiety, and R ７ is H), The compound of claim 9.

11. R4 is H, R ５ teeth 【Chemistry 9】 is (In the formula, R ６ is the bond to an oligonucleotide containing a phosphodiester moiety, and R ７ is H), The compound of claim 9.

12. R4 is H, R ５ teeth 【Chemistry 10】 is (In the formula, R ６ is an alcohol protecting group selected from the group consisting of tert-butyldimethylsilyl (TBMDS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), monomethoxytrityl (MMTr), and tritolyl; R ７ is a solid support linker), The compound of claim 1.

13. R ７ The compound of claim 12, wherein is a succinate ester.

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