GalNAc-oligonucleotide conjugates for liver delivery and methods of manufacture

Abstract

The present invention provides: a conjugate of an oligonucleotide having a nucleotide sequence that is expected to have a pharmacological effect in a hepatic parenchymal cell and a biantennary GalNAc unit, or a pharmaceutically acceptable salt thereof; a drug containing the conjugate or a pharmaceutically acceptable salt thereof as an active ingredient; and others.

Description

[Technical field]

[0001] The present invention relates to biantennary N-acetyl-D-galactosamine (GalNAc) ligand-oligonucleotide conjugates and methods for producing the conjugates. [Background technology]

[0002] Conjugates of nucleic acid drugs (antisense, siRNA, etc.) covalently bound to N-acetyl-D-galactosamine (GalNAc) or the like as a ligand capable of binding to the asialoglycoprotein receptor (ASGPR) have been reported as a method for delivering oligonucleotides to hepatic parenchymal cells in the liver (Non-Patent Document 1 and Patent Documents 1-9). One to three GalNAc are bound to one oligonucleotide. An example in which two GalNAc are bound is described in Non-Patent Document 2.

[0003] When reverse-phase HPLC is used in the purification process after solid-phase synthesis of oligonucleotides, the retention time of the short oligomers produced in the solid-phase synthesis process is often close to that of the target oligomer, making it very difficult to obtain a high-purity purified product on a large scale. In solid-phase synthesis of oligonucleotides, nucleic acid units in which the 5'-hydroxyl group of the nucleoside is protected with a 4,4'-dimethoxytrityl (DMTr) group are usually used, so that the 5'-hydroxyl group of the nucleoside located at the 5' end of the synthesized oligonucleotide has a DMTr group. Examples of reverse-phase column purification utilizing the high hydrophobicity of this DMTr group (hereinafter also referred to as DMTr-on reverse-phase column purification. "DMTr-on" indicates that an oligonucleotide with a DMTr group introduced at the 5' end is used) and ion-exchange column purification using DMTr-on (hereinafter also referred to as DMTr-on ion-exchange column purification) are described in Non-Patent Documents 3 and 4. However, since the GalNAc unit is usually introduced to the hydroxyl group at the 5' end of the oligonucleotide by deprotecting the DMTr group, the synthesized conjugate does not have a DMTr group. Therefore, separation and purification from oligonucleotides not conjugated with the GalNAc unit must be performed by HPLC, which is laborious and time-consuming (Non-Patent Document 5). Up to now, there have been no reports of the use of DMTr-on reversed-phase column purification or DMTr-on ion-exchange column purification methods for conjugates of GalNAc units and oligonucleotides.

[0004] Thus, there is a need for the development of a GalNAc unit that can efficiently deliver oligonucleotides to hepatic parenchymal cells and has an advantageous structure in terms of mass production, as well as a conjugate of the GalNAc unit with an oligonucleotide that has a therapeutic effect on a disease, and an efficient method for producing the same. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2009 / 073809 [Patent Document 2] International Publication No. 2014 / 076196 [Patent Document 3] International Publication No. 2014 / 179620 [Patent Document 4] International Publication No. 2015 / 006740 [Patent Document 5] International Publication No. 2015 / 105083 [Patent Document 6] International Publication No. 2016 / 055601 [Patent Document 7] International Publication No. 2017 / 023817 [Patent Document 8] International Publication No. 2017 / 084987 [Patent Document 9] International Publication No. 2017 / 131236 [Non-patent literature]

[0006] [Non-Patent Document 1] Methods in Enzymology, 1999, 313, 297-321 [Non-Patent Document 2] Bioorganic & Medicinal Chemistry Letters 26 (2016) 3690-3693 [Non-Patent Document 3] AKTAdesign Purification - User Manual 18-1124-23 Edition AD [Non-Patent Document 4] Organic Process Research & Development 2005, 9, 212-215 [Non-Patent Document 5] Molecules 2017, 22, 1356. Summary of the Invention [Problem to be solved by the invention]

[0007] In the research on the delivery of synthetic nucleic acids to the liver using GalNAc units, the linkers used in the past placed importance on keeping the ligand site away from the nucleic acid, and structures with long linear alkyl or ring structures in a very long backbone were adopted, and in many cases, complex structures with three or more branches were also adopted. However, when conjugates using such conventional long-chain alkyl linkers were concentrated, the inventors' research newly discovered a problem that micelles were formed in high-concentration solutions, making it difficult to prepare appropriate doses. In addition, in the conventional method, a method for column separation of oligonucleotides bound to GalNAc units had not been established, making mass production difficult. [Means for solving the problem]

[0008] In order to solve the above problems, the present inventors have conducted extensive research and have found that a conjugate of an oligonucleotide and a biantennary GalNAc unit having a linker structure of the present invention exhibits good solubility even in a high volume of solution while retaining the ability to be efficiently delivered to hepatocytes. Furthermore, the present inventors have found that an oligonucleotide bound to a GalNAc unit having a highly hydrophobic protecting group (e.g., a 4,4'-dimethoxytrityl (DMTr) group) on the 6-hydroxyl group of GalNAc can be purified efficiently using a hydrophobic resin column, thereby completing the present invention. This is expected to facilitate large-scale ion exchange column purification or DMTr-on reversed phase column purification of the conjugate.

[0009] The gist of the present invention is as follows. (A1) A conjugate of an oligonucleotide having a nucleic acid sequence expected to have a pharmacological effect in hepatic parenchymal cells and a bibranched GalNAc unit, the conjugate being characterized in that a bibranched GalNAc unit of the following formula is bound to the 5'-end or 3'-end of the oligonucleotide, and the conjugate is specifically delivered to hepatic parenchymal cells in vivo, or a pharma- ceutical acceptable salt thereof;

[0010] [ka]

[0011] [Wherein W is a compound represented by the formula

[0012] [ka]

[0013] G represents a 5-acetamido-2-hydroxymethyl-3,4-dihydroxytetrahydropyran-6-yl group (GalNAc), Z represents an oxygen atom or a sulfur atom, L 1 and L 2 one represents a methylene group (CH2) and the other represents no atom between them, o, q, r, and s each independently represent 0 or 1, n represents an integer of 1 to 15, m represents an integer of 0 to 5, and v represents 1 to 7. However, when n is 1, m is an integer of 0 to 5, and when n is an integer of 2 to 15, m is 0.

[0014] (A2) The conjugate according to (A1), wherein o and q in the biantennary GalNAc unit are 0, or a pharma- ceutically acceptable salt thereof.

[0015] (A3) A conjugate of (A1) or (A2), or a pharma- ceutically acceptable salt thereof, wherein a bisecting GalNAc unit is bound to the 5' end of the oligonucleotide.

[0016] (A4) The 2-branched GalNAc unit has any one of the following formulas:

[0017] [ka]

[0018] [ka]

[0019] [ka]

[0020] [ka]

[0021] [ka]

[0022] The conjugate according to (A1), or a pharma- ceutical acceptable salt thereof, wherein R is a group represented by the formula:

[0023] (A5) A medicine comprising a conjugate of any one of (A1) to (A4) or a pharma- ceutically acceptable salt thereof.

[0024] The present invention also provides the following production methods, as well as compounds, conjugates and the like containing a GalNac unit having a hydrophobic protecting group added thereto, which are used as intermediates in the production methods.

[0025] (B1) A method for producing a conjugate of an oligonucleotide and a GalNAc unit, comprising the steps of: a: A structure containing a hydrophobic protecting group-added GalNAc unit-phosphate group represented by the following formula at the 5'-end or 3'-end of an oligonucleotide:

[0026] [ka]

[0027] [In the formula, R a , R b and R c at least one of them is a hydrophobic protecting group, and the others are hydrogen atoms or protecting groups which are removable under liberating conditions. X1 and X2 each independently represent a linker structure, and may be branched between X1 and X2. t represents the number of branched chains in the linker structure and is an integer of 1 to 10. Y represents a bond which bonds to an oligonucleotide via a phosphate group or a thiophosphate group. b: passing the solution containing the product of step a through a column packed with a resin having adsorptivity by hydrophobic interaction, and passing an aqueous washing solution through the column; and c: Eluting the oligonucleotide from the column after step b.

[0028] (B2) The method for producing (B1), wherein the hydrophobic protecting group is a protecting group which can be removed under basic or acidic conditions, preferably a hydrophobic protecting group which can be removed under acidic conditions, more preferably an optionally substituted trityl group or an optionally substituted silyl group.

[0029] (B3) The method of (B1), characterized in that a structure containing a hydrophobic protecting group-added GalNAc unit-phosphate group is bound to the 5-hydroxyl group of the nucleoside located at the 5'-end of an oligonucleotide.

[0030] (B4) In the structure containing a hydrophobic protecting group-added GalNAc unit and a phosphate group, R a is a hydrophobic protecting group, R b and R c is a hydrogen atom; and t is an integer of 1 to 3.

[0031] (B5) The manufacturing method of (B1), in which the resin having adsorption properties due to hydrophobic interactions is any one selected from a styrene-divinylbenzene-based adsorption resin, a styrene-divinylbenzene-based modified adsorption resin, a methacrylic-based adsorption resin, and a phenol-based adsorption resin.

[0032] (B6) The production method of (B1), wherein the column packed with a resin having adsorptivity due to hydrophobic interaction is an ion exchange column or a reverse phase column.

[0033] (B7) The method for producing (B1), wherein step a comprises the following steps: a1: a process of synthesizing a desired oligonucleotide on a support capable of extending an oligonucleotide in the 3'-terminal to 5'-terminal direction by repeatedly deprotecting the protecting group of the 5'-hydroxyl group of the nucleoside located at the 5'-terminal and condensing the nucleoside to extend the oligonucleotide chain; in this process, for the protecting groups other than the protecting group of the 5'-hydroxyl group of the nucleoside used, a protecting group that is deprotected under the conditions for cleaving the bond between the 3'-terminal of the oligonucleotide and the support (hereinafter referred to as "release conditions") is selected, and for the protecting group of the 5'-hydroxyl group of the nucleoside, a protecting group that is not deprotected under release conditions is selected. a2: a step of introducing a hydrophobic protecting group-added GalNAc unit into the 5'-hydroxyl group of the nucleoside located at the 5'-end of the oligonucleotide synthesized on the support, wherein the hydrophobic protecting group is selected to be one that is not deprotected under release conditions, and a3: A step of treating the support obtained in step a2 with a solution under releasing conditions to obtain a solution of a conjugate of a hydrophobic protecting group-added GalNAc unit and an oligonucleotide.

[0034] (B8) The method for producing (B1), characterized in that after the step b, the hydrophobic protective group of the conjugate adsorbed on the column is deprotected, and then the step c is carried out.

[0035] (B9) A method for producing (B1), wherein the releasing conditions are basic and the hydrophobic protecting group is a protecting group that can be removed with acid.

[0036] (B10) A structure containing a hydrophobic protecting group-added GalNAc unit-phosphate group is represented by the following formula:

[0037] [ka]

[0038] (In the formula, R1 represents a hydrophobic protecting group that is removable under acidic conditions, X represents a linker structure, and Y represents a bond that bonds to an oligonucleotide via a phosphate group or a thiophosphate group. The same applies hereinafter in the same paragraph.)

[0039] [ka]

[0040] [ka]

[0041] [ka]

[0042] [ka]

[0043] [ka]

[0044] [ka]

[0045] [ka]

[0046] [ka]

[0047] [ka]

[0048] [ka]

[0049] The method for producing (B1) represented by any one of the following:

[0050] (B11) The following formula

[0051] [ka]

[0052] [In the formula, R a , R b and R c At least one of them is a hydrophobic protecting group, and the others are protecting groups which are removed under release conditions. X1 and X2 each independently represent a linker structure, and may be branched between X1 and X2. t represents the number of branches in the linker structure and is an integer of 1 to 10. Y represents a phosphoramidite group (preferably a group represented by -P(OCH2CH2CN)N(CH(CH3)2)2 or -P(OCH3)N(CH(CH3)2)2) or an H-phosphonate group (-PH(=O)(OH)). A compound represented by the following formula (I) or a salt thereof:

[0053] (B12)R a is a hydrophobic protecting group that is removed under acidic conditions, and R b and R c The compound (B11) or a salt thereof, wherein: is an acetyl group; Y is a phosphoramidite group or an H-phosphonate group; and t is an integer of 1 to 3.

[0054] (B13) The following formula

[0055] [ka]

[0056] [In the formula, R1 is a hydrophobic protecting group that is removable under acidic conditions, and Y is a phosphoramidite group or an H-phosphonate group. The same applies hereinafter in the same paragraph.]

[0057] [ka]

[0058] [ka]

[0059] [ka]

[0060] [ka]

[0061] The compound (B11) or (B12) represented by any one of the following formulae:

[0062] (B14) The following formula

[0063] [ka]

[0064] [In the formula, R1 represents a hydrophobic protecting group that is removable under acidic conditions, X represents a linker structure, and Y represents a phosphoramidite group or an H-phosphonate group. The same applies hereinafter in the same paragraph.]

[0065] [ka]

[0066] [ka]

[0067] [ka]

[0068] [ka]

[0069] [ka]

[0070] The compound (B11) or (B12) represented by any one of the following formulae:

[0071] (B15) Any one of the compounds (B11) to (B14) or a salt thereof, wherein the hydrophobic protecting group is an optionally substituted trityl group, and the phosphoramidite group is -P(OCH2CH2CN)N(CH(CH3)2)2 or -P(OCH3)N(CH(CH3)2)2.

[0072] (B16) A structure containing a hydrophobic protecting group-added GalNAc unit-phosphate group represented by the following formula at the 5'-end or 3'-end of an oligonucleotide:

[0073] [ka]

[0074] [In the formula, R a , R b and R c at least one of them is a hydrophobic protecting group, and the others are hydrogen atoms or protecting groups which are removable under free conditions. X1 and X2 each independently represent a linker structure, and may be branched between X1 and X2. t represents the number of branched chains in the linker structure and is an integer of 1 to 10. Y represents a bond which bonds to the oligonucleotide via a phosphate group or a thiophosphate group. A conjugate or a salt thereof having] bonded thereto.

[0075] (B17) A structure containing a hydrophobic protecting group-added GalNAc unit-phosphate group is represented by the following formula:

[0076] [ka]

[0077] [In the formula, R1 represents a hydrophobic protecting group that is removable under acidic conditions, and Y represents a bond that bonds to an oligonucleotide via a phosphate group or a thiophosphate group. The same applies hereinafter in the same paragraph.]

[0078] [ka]

[0079] [ka]

[0080] [ka]

[0081] [ka]

[0082] A conjugate of (B16) or a salt thereof, represented by any one of the following:

[0083] (B18) The structure containing a hydrophobic protecting group-added GalNAc unit-phosphate group is represented by the following formula:

[0084] [ka]

[0085] [In the formula, R1 represents a hydrophobic protecting group that is removable under acidic conditions, X represents a linker structure, and Y represents a bond that bonds to an oligonucleotide via a phosphate group or a thiophosphate group. The same applies hereinafter in the same paragraph.]

[0086] [ka]

[0087] [ka]

[0088] [ka]

[0089] [ka]

[0090] [ka]

[0091] A conjugate of (B16) or a salt thereof, represented by any one of the following:

[0092] (B19) A conjugate or a salt thereof according to any one of (B16) to (B18), wherein the hydrophobic protecting group is an optionally substituted trityl group, and Y represents a bond that bonds to the 5'-hydroxyl group of the nucleoside located at the 5'-end of the oligonucleotide via a phosphodiester bond or a phosphothioate bond. Effect of the Invention

[0093] The conjugate of the present invention achieved appropriate delivery to the liver and high water solubility for various oligonucleotides. In addition, the establishment of a DMTr-on column purification method for the conjugate of GalNAc units and oligonucleotides made it possible to produce it on a large scale. This makes it possible to increase the dosage to enhance the pharmacological effect and to prepare high-concentration drug solutions for long-lasting formulations, enabling the product design of nucleic acid medicines that meet various needs. [Brief description of the drawings]

[0094] [Figure 1]FIG. 1 shows the effect of administering the compounds of Examples 8 to 14 to hetero knock-in mice on correcting abnormal splicing of G6PC mRNA in the liver. The amount of G6PC mRNA in which abnormal splicing has been corrected in the liver of hetero knock-in mice was evaluated by relative quantification using qRT-PCR (corrected by the amount of actin mRNA). The Y-axis shows the relative value of the expression level of normal G6PC mRNA in each administration group, with the saline administration group set at 1 (RQ: Relative Quantification; mpk: mg / kg). [Diagram 2] FIG. 1 shows the effect of administering the compounds of Examples 15 to 23 to heterozygous knock-in mice on correcting abnormal splicing of G6PC mRNA in the liver. The amount of G6PC mRNA in which abnormal splicing has been corrected in the liver of heterozygous knock-in mice was quantitatively evaluated by qRT-PCR (corrected by the amount of actin mRNA). The Y axis is the same as in FIG. 1. RQ: Relative Quantification; mpk: mg / kg [Diagram 3] Figure showing the effect of degrading ApoB mRNA in cells when ApoB-ASO (open columns) and ApoB-ASO conjugates (filled columns) were added to primary cultured human hepatocytes. The amount of ApoB mRNA in the cells was assessed quantitatively by relative RT-PCR (corrected for the amount of actin mRNA). The Y-axis shows the relative value (%) in each addition group when the amount of ApoB mRNA in untreated primary cultured human hepatocytes is set to 100%. [Figure 4] Correlation diagram between each process and chromatogram during DMTr-on ion exchange column purification. [Diagram 5] Chromatogram of the conjugate with a DMTr group on the hydroxyl group of GalNAc purified with a DMTr-on ion exchange column (detection wavelength 260 nm) [Figure 6] Chromatogram (detection wavelength 260 nm) of a conjugate having a DMTr group near the oligonucleotide purified using a DMTr-on ion exchange column. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0095] Hereinafter, the embodiment of the present invention will be described in more detail. As used herein, "conjugate" refers to a compound in which a GalNAc unit is attached to the 5' and / or 3' end of an oligonucleotide.

[0096] In the present specification, the term "GalNAc unit" refers to a partial structure containing N-acetyl-D-galactosamine (GalNAc) capable of binding to the asialoglycoprotein receptor (ASGPR) on hepatocytes in the liver. The GalNAc unit contains a linear or branched linker structure and a phosphate group or a thiophosphate group for binding to an oligonucleotide. A structural unit containing one phosphate group or one thiophosphate group for such binding may be referred to as a "GalNAc unit". There is no limit to the number of GalNAcs contained in one GalNAc unit, and unless otherwise specified, publicly known ones and those disclosed in the present specification can be used. As long as the binding ability to ASGPR is maintained, the structure of GalNAc may be modified. It also includes those in which a protecting group has been introduced into GalNAc during the production process. As used herein, unless otherwise specified, the biantennary GalNAc unit refers to a GalNAc unit represented by the above formula (A1).

[0097] In the present specification, the term "oligonucleotide" refers to a synthetic nucleic acid having a chain length of 100 bases or less, and may be single-stranded or double-stranded. As described later, the nucleic acid to be used may be DNA, RNA, natural nucleic acid, modified nucleic acid, etc., depending on the purpose, and these may be mixed in one oligonucleotide.

[0098] In this specification, the term "release conditions" refers to the treatment conditions employed in the process of oligonucleotide synthesis when the bond between the support and the oligonucleotide is cleaved and the oligonucleotide is released from the support. The release conditions in general solid phase synthesis are basic.

[0099] In the present invention, the "hydrophobic protecting group" refers to a protecting group that can be used as a protecting group for a hydroxyl group, has a highly hydrophobic structure, and is not removed under release conditions. For example, when the release conditions are basic, a protecting group that is removed under acidic conditions is preferred, and when the release conditions are acidic, a protecting group that is removed under basic conditions is preferred.

[0100] As used herein, the term "phosphoramidite group" refers to a functional group used in an amidite reagent in nucleic acid synthesis, represented by -P(OR)N(CH(CH3)2)2 (wherein R is a C1-C3 alkyl group or a cyano-C1-C3 alkylene group). Suitable examples include, but are not limited to, -P(OCH2CH2CN)N(CH(CH3)2)2, -P(OCH3)N(CH(CH3)2)2, and the like.

[0101] As used herein, the term "H-phosphonate group" refers to a functional group employed in nucleic acid synthesis represented by -PH(=O)(OH) or a salt thereof. A compound having an H-phosphonate group is represented by -PH(=O)(OH). -) can form a salt. Examples of such salts include metal salts such as alkali metal salts (such as sodium salt, potassium salt, and lithium salt), alkaline earth metal salts (such as calcium salt and magnesium salt), aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts (such as ammonium salt), t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris(hydrogen) salt, and the like. Examples of suitable salts include amine salts such as organic salts such as dimethylaminomethane salts; hydrohalides such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate. The preferred salts are organic salts, and more preferred are triethylamine salts. These salts can be prepared by known methods.

[0102] As used herein, in relation to a bond between nucleotides or a structure of a conjugate between a nucleotide and a nucleotide, terms such as "a bond via a phosphate group" and "a structure containing a phosphate group" refer to a bond between two structural units that is commonly used in nucleic acid synthesis, such as a phosphodiester bond or a modified form thereof (e.g., a phosphorothioate bond), or a structure that includes such a bond.

[0103] <1. Oligonucleotides> As used herein, the terms "nucleic acid," "oligonucleotide," or "polynucleotide" refer to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded, double-stranded, or triple-stranded form.

[0104] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variations thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as the sequence explicitly indicated.

[0105] 1-1.Types of Nucleic Acids The nucleic acids of the present invention include any form known to those of skill in the art. Specific examples of such nucleic acid forms include single-stranded DNA, single-stranded RNA, and single-stranded oligonucleotides that are a mixture of DNA and RNA. Specific examples of other nucleic acid forms include double-stranded DNA, double-stranded RNA, DNA-RNA hybrid polynucleotides, and double-stranded polynucleotides that are a mixture of two oligonucleotides that are a mixture of DNA and RNA.

[0106] The nucleosides or nucleotides constituting the nucleic acid of the present invention include not only natural nucleosides or nucleotides but also chemically modified nucleosides or nucleotides. Modified nucleosides / nucleotides include, for example, sugar-modified nucleosides / nucleotides, nucleic acid base-modified nucleosides / nucleotides, backbone-modified nucleosides / nucleotides, or combinations thereof (see, for example, Nucleic Acid Research, 1997, Vol. 25, No. 22, 4429-4443).

[0107] In this specification, "natural nucleoside" refers to 2'-deoxynucleosides such as 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, and thymidine, and ribonucleosides such as adenosine, guanosine, cytidine, 5-methylcytidine, and uridine. In addition, "oligonucleotide" refers to an oligonucleotide composed of a compound in which the sugar portion of a nucleoside forms an ester with phosphoric acid. In this specification, oligonucleotide and polynucleotide are used with the same meaning.

[0108] As used herein, 2'-deoxyadenosine is A t , 2'-deoxyguanosine to G t , 2'-deoxycytidine C t , 2'-deoxy-5-methylcytidine 5meC t , thymidine to T t , 2'-deoxyuridine to U t , adenosine A rt , guanosine to G rt , cytidine to C rt , 5-methylcytidine 5meC rt , uridine to U rt In the present specification, 2'-deoxyadenosine nucleotide is also referred to as A p , 2'-deoxyguanosine nucleotide G p , 2'-deoxycytidine nucleotide C p , 2'-deoxy-5-methylcytidine nucleotides at 5meC p , thymidine nucleotide T p , 2'-deoxyuridine nucleotide U p , adenosine nucleotide A rp , guanosine nucleotide G rp , cytidine nucleotide C rp , 5-methylcytidine nucleotide 5meC rp , uracil nucleotide U rp It can also be expressed as:

[0109] In the present specification, the term "phosphorothioate ester" refers to a phosphorothioate ester in place of the phosphate ester of a nucleotide. p Corresponding to A s , G p As the corresponding s , C p As a counterpart to C s , 5meC p Corresponding to 5meC s , T p As the corresponding s , U p As the corresponding s , A rp Corresponding to A rs , G rp As the corresponding rs , C rp As a counterpart to C rs , 5meC rp Corresponding to 5meC rs , U rp As the corresponding rs It can also be expressed as:

[0110] As used herein, the term "sugar-modified nucleoside" refers to a nucleoside in which the sugar moiety of the nucleoside has been modified. Examples of sugar-modified nucleosides include 2'-O-methyl nucleosides. Examples of such nucleosides include 2'-O,4'-C-ethylene nucleosides (ENA), and 2'-O,4'-C-methylene nucleotides (BNA / LNA).

[0111] Examples of 2'-O-methyl modifications include 2'-O-methyl nucleosides and 2'-O-methyl nucleotides. rt Corresponding to A m1t , G rt As the corresponding m1t , C rt As a counterpart to C m1t , 5meC rt Corresponding to 5meC m1t , U rt As the correspondingm1t , A rp Corresponding to A m1p , G rp As the corresponding m1p , C rp Corresponding to C m1p , 5meC rp Corresponding to 5meC m1p , U rp As the corresponding m1p , A rs Corresponding to A m1s , G rs As the corresponding m1s , C rs Corresponding to C m1s , 5meC s Corresponding to 5meC m1s , U rs As the corresponding m1s It can also be expressed as:

[0112] In the sequence listing attached to this specification, <223> In the above items, "cm" stands for 2'-O-methylcytidine, "um" stands for 2'-O-methyluridine, and "gm" stands for This indicates 2'-O-methylguanosine.

[0113] In the present specification, the term "2'-O,4'-C-ethylene nucleotide unit" and "ENA unit" refer to the above-mentioned nucleosides and nucleotides each having ENA. t Corresponding to A 2t , A p Corresponding to A e2p , A s For A e2s , G t As the corresponding 2t , G p As the corresponding e2p , G s For G e2s , 5meC tAs a counterpart to C 2t , 5meC p As a counterpart to C e2p , 5meC s For C e2s , T t As the corresponding 2t , T p As the corresponding e2p , T s For T e2s Thus, it can also refer to nucleosides and nucleotides having ENA units.

[0114] As used herein, the term "2'-O,4'-C-methylene nucleotide unit" and "2',4'-BNA / LNA unit" refer to the above-mentioned nucleosides and nucleotides each having 2',4'-BNA / LNA. t Corresponding to A 1t , A p Corresponding to A e1p , A s For A e1s , G t As the corresponding 1t , G p As the corresponding e1p , G s For G e1s , 5meC t As a counterpart to C 1t , 5meC p As a counterpart to C e1p , 5meC s Against is C e1s , T t As the corresponding 1t , T p As the corresponding e1p , T s For T e1s Thus, a nucleotide having a 2',4'-BNA / LNA unit is It can also refer to oxides and nucleotides.

[0115] 1-2.Target gene In the present specification, the term "target gene" is not particularly limited as long as it is RNA in a cell, tissue, or individual (hereinafter sometimes referred to as a "recipient") to which it is introduced, and may be either mRNA that is translated into a protein or non-coding RNA that is not translated into a protein. Examples of non-coding RNA include functional RNA, such as the non-translated region of mRNA, tRNA, rRNA, mRNA-like non-coding RNA (ncRNA), long non-coding RNA (ncRNA), small nuclear RNA (snRNA), small nuclear RNA (snoRNA), and microRNA (miRNA). Specifically, the target gene may be endogenous to the recipient to be introduced, or exogenous introduced by a method such as gene introduction. In addition, the target gene may be a gene present on a chromosome or an extrachromosomal gene. Examples of exogenous genes include, but are not limited to, those derived from viruses, bacteria, fungi, or protozoa that can infect the recipient. The function of the gene may be known or unknown.

[0116] Examples of such target genes include genes whose expression is specifically increased and / or mutated in the liver in patients with a particular disease in the liver, such as cardiovascular diseases (e.g., hypertension, cardiac hypertrophy, angina pectoris, arteriosclerosis, hypercholesterolemia, etc.), cancer (e.g., liver cancer, etc.), respiratory diseases (e.g., pneumonia, bronchitis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, etc.), diabetes, diabetic retinopathy, diabetic nephropathy, anemia (e.g., anemia associated with chronic disease, iron-refractory iron deficiency anemia, etc.), age-related macular degeneration, immune system diseases (e.g., autoimmune diseases, immunodeficiencies, leukemia, etc.), liver and gallbladder diseases (e.g., nonalcoholic steatohepatitis, liver cirrhosis, hepatitis, liver failure, cholestasis, stones, etc.), gastrointestinal diseases (e.g., ulcers, enteritis, malabsorption, etc.), infectious diseases, obesity, and fibrosis (e.g., hepatic fibrosis, etc.).

[0117] Causative genes of these diseases include, for example, kinesin spindle protein (KSP), vascular endothelial growth factor (VEGF), transthyretin (TTR), proprotein convertase subtilisin / kexin type 9 (PCSK9), polo-like kinase 1 (PLK-1), ApoB-100, ribonucleotide reductase M2 subunit (RRM2), clusterin, heat shock protein 27 (Hsp27), survivin, eukaryotic initiation factor-4E (eIF-4E), alpha subunit of the interleukin 4 receptor (IL-4R-alpha), Factor XI, and Factor VII, N-ras, H-ras, K-ras, bcl-2, bcl-xL, Her-1, Her-2, Her-3, Her-4, MDR-1, human β-catenin gene, DDX3 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked), Myeloid Cell Leukemia Sequence 1 (MCL1) gene, PKR (Eif2ak2), Hsp47 (Serpinh1), Hepcidin, activated protein C (APC), signal tranducer and activator of transcription (STAT3), Collagen, type I, alpha 1 (Col1A1), ApoC-III, angiopoietin-like 3 protein (ANGPTL3), growth hormone receptor (GHr), prekallikrein (PKK), transmembrane protease,Serine 6 (TMPRSS6), lipoprotein(a), glucagon receptors, or diacylglycerol acyltransferase 2 (DGAT2), angiotensinogen, complement factor B (FB), aminolevulinic acid synthase 1 (ALAS1), antithrombin (AT), Primary Hyperoxaluria Type 1 (PH1), C5 component of the complement, alpha-1 antitrypsin (AAT), and hepatitis B virus (HBV) genome can be mentioned, but are not limited to these.

[0118] 1-3.Double-stranded oligonucleotides When the nucleic acid of the present invention is a nucleic acid having an RNA interference effect on a target gene, there is no limitation on its structure and chemical modification as long as the nucleic acid has an RNA interference effect. For example, siRNA (see, for example, WO2002 / 044321, Current Opinion in Chemical Biology 570-579), AtuRNAi consisting of an oligonucleotide in which RNA and 2'-OMeRNA are alternately linked (see, for example, WO2004 / 015107), double-stranded oligonucleotides in which DNA and 2'-OMeRNA are alternately linked and the sense and antisense strands of different nucleic acids form a double strand by Watson-Crick base pairing (see, for example, WO2010 / 001909), as described in the following section 3-4-1, nucleic acids in which the ends of the oligonucleotides are modified (see, for example, WO2010 / 052715), as described in the following section 3-4-2, and single-stranded oligonucleotides in which the 5' end of the antisense strand polynucleotide and the 3' end of the sense strand oligonucleotide are linked via a linker to form a single strand and further form Watson-Crick base pairs within the molecule to form a double-stranded structure (see, for example, WO2012 / 074038), as described in the following section 3-4-3.

[0119] In the present specification, "comprising the same nucleotide sequence as the target gene" refers to comprising the same sequence as at least a part of the nucleotide sequence of the target gene, and includes not only a completely identical sequence, but also a substantially identical sequence as long as it has an RNA interference effect and / or gene expression suppression effect on the target gene. "Comprising a nucleotide sequence complementary to the target gene" refers to comprising a sequence complementary to at least a part of the nucleotide sequence of the target gene, and includes not only a completely complementary sequence, but also a substantially identical sequence as long as it has an RNA interference effect and / or gene expression suppression effect on the target gene. In addition, when SNPs or the like are known in the target gene, sequences having these mutations are also included in the same nucleotide sequence. In the present specification, a polynucleotide that contains a nucleotide sequence complementary to the target gene and has an RNA interference effect and / or gene expression suppression effect on the target gene is called an oligonucleotide for the target gene.

[0120] The nucleotide sequence of the nucleic acid of the present invention is not particularly limited as long as it has an RNA interference effect and / or a gene expression inhibitory effect on a target gene, but can be determined, for example, by determining the sequences of the sense strand and the antisense strand based on a sequence predicted to have an RNA interference effect on a target gene using computer software (e.g., GENETYX (registered trademark): manufactured by GENETYX CORPORATION, etc.), or can be determined by further confirming the RNA interference effect and / or gene expression inhibitory effect of an oligonucleotide prepared based on the selected sequence.

[0121] The lengths of the sense and antisense strands of a double-stranded oligonucleotide having RNA interference activity may be any length from 10 nucleotides to the full length of the open reading frame (ORF) of the target gene, preferably any length from 18 nucleotides to the full length of the open reading frame (ORF) of the target gene, more preferably 10 to 100 nucleotides, and even more preferably 15 to 30 nucleotides, so long as the double-stranded oligonucleotide has RNA interference activity and / or gene expression inhibitory activity.

[0122] When a double-stranded oligonucleotide (WO2010 / 001909) in which DNA and 2'-OMeRNA are alternately bound and the sense and antisense strands are different nucleic acids bound by Watson-Crick bonds is used as the double-stranded oligonucleotide having an RNA interference effect, the sense strand is preferably 18 to 21 in length, more preferably 18 to 19 in length. The antisense strand is preferably 19 to 21 in length, more preferably 21 in length. In addition, it is not necessary for the whole to be a double-stranded structure, and it may include those in which the 5' and / or 3' ends are partially overhanging, and the overhanging ends are 1 to 5 nucleotides, preferably 1 to 3 nucleotides, more preferably 2 nucleotides. In addition, the most preferred example is a polynucleotide in which the 3' end of the antisense strand has a structure overhanging 2 nucleotides (overhang structure) and forms 18 base pairs.

[0123] 1-3-1. Oligonucleotides with alternating DNA and 2'-OMeRNA An example of the nucleic acid contained in the nucleic acid-lipid particle of the present invention is an oligonucleotide in which DNA and 2'-OMeRNA are alternately linked, and in which the sense strand and antisense strand are different types of nucleic acid and are bonded via Watson-Crick bonds.

[0124] Specific examples of such double-stranded oligonucleotides include the double-stranded oligonucleotides described in WO2010 / 001909 and WO2010 / 001909.

[0125] 1-3-2. Modified double-stranded oligonucleotides When using the nucleic acid having RNA interference effect as the nucleic acid contained in the nucleic acid-lipid particle, as long as it has RNA interference effect, the nucleic acid of which the end of the oligonucleotide is modified can be mentioned as an example.For example, in the double-stranded oligonucleotide having RNA interference effect such as siRNA, AtuRNAi, and the double-stranded oligonucleotide in which DNA and 2'-OMeRNA are alternately bound, the sense strand and the antisense strand are different kinds of nucleic acids and are bound by Watson-Crick (see, for example, WO2010 / 001909), the double-stranded oligonucleotide in which the phosphate group at the 5' end of the antisense strand is modified to a 5' aryl phosphate group (see, for example, WO2010 / 052715) can be mentioned.

[0126] Specific examples of such modified double-stranded oligonucleotides include the modified double-stranded oligonucleotides described in WO2010 / 052715 and WO2015 / 005253.

[0127] 1-4. Single-stranded oligonucleotides 1-4-1. RNA interference single-stranded nucleotide The nucleic acid used in the present invention includes, as long as it has an RNA interference effect, an oligonucleotide having a sense strand oligonucleotide for a target gene and an antisense strand oligonucleotide having a base sequence complementary to the sense strand oligonucleotide, and also includes an oligonucleotide having a single-stranded structure in which the 5' end of the antisense strand oligonucleotide and the 3' end of the sense strand oligonucleotide are linked by a linker forming a phosphodiester structure (see, for example, WO2012 / 074038).

[0128] Specific examples of such compounds include those described in WO2012 / 074038 and WO2015 / 005253.

[0129] 1-4-2. Antisense nucleotides Antisense nucleotides are used for the purpose of treating diseases caused by gene mutations, which can be treated by normalizing or suppressing the expression of the mutant gene. When suppressing gene expression, nucleotides are used that have a sequence complementary to a part of the mRNA of the target gene, and the nucleic acid in the middle of the sequence is DNA. Such expression-suppressing antisense nucleotides form a double strand with the mRNA of the target gene. The RNA / DNA double-stranded structure in the formed double strand is specifically degraded by RNase H in the body, so that the mRNA of the target gene is specifically destroyed, and as a result, the expression of the target gene is suppressed.

[0130] Antisense nucleotides aimed at normalizing gene expression include those that suppress abnormal splicing and those that restore a mutated site on a gene to a normal sequence. When restoring a mutated site on a gene to a normal sequence, the 3' side of the antisense nucleotide has an antisense sequence to the 5' region of the mutated site on the sense strand of the target gene, and the 5' side has a sequence that forms a hairpin structure (a sequence in which two mutually complementary sequences are connected by an intermediate linker / loop sequence) with which a nuclease associates. With such nucleotides, the 3' antisense region functions as a guide RNA that guides the nuclease to the mutated site of the target gene (sense strand of mRNA or genomic DNA). This phenomenon is similar to the genome editing technology known as the CRISPR-Cas system, and is widely known to be applicable to various gene editing techniques.

[0131] The mechanism by which abnormal splicing occurs due to gene mutation is that, during the splicing process from pre-mRNA to mature mRNA, the splicing factor mistakenly recognizes and binds to a site that is not bound by normal pre-mRNA, resulting in the expression of abnormally spliced ​​mRNA, causing the protein expressed from the target gene to malfunction or be expressed poorly. When an antisense nucleotide binds to the region on the mutant pre-mRNA that is mistakenly recognized by the splicing factor, the splicing factor cannot recognize the region, and abnormal splicing is suppressed. Examples of diseases caused by abnormal splicing due to such gene mutation include antisense oligonucleotides that suppress abnormal splicing caused by the c.648G>T mutation in the G6PC gene in glycogen storage disease type 1a, which will be described in detail below, and other antisense oligonucleotides for treating various known diseases.

[0132] The present invention provides an oligonucleotide or a pharma- ceutical acceptable salt thereof that can repair a G6PC gene having a c.648G>T mutation at the mRNA level in patients with glycogen storage disease type Ia and allow normal G6PC protein to be expressed. The oligonucleotide of the present invention is an oligonucleotide having 15 to 30 bases and consisting of a nucleotide sequence complementary to the cDNA of a G6PC gene having a c.648G>T mutation, and consisting of a sequence complementary to a region including any one of the 82nd to 92nd sites from the 5' end of exon 5 of the G6PC gene having a c.648G>T mutation.

[0133] In the present invention, among patients with glycogen storage disease type Ia, patients with the c.648G>T mutation can be targeted. Not only patients who are homozygous for the c.648G>T mutation, but also patients who are compound heterozygous for the c.648G>T mutation and other mutations can be targeted.

[0134] The c.648G>T mutation in the G6PC gene is a mutation from G to T at nucleotide number 728 in Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). It is located at the 86th position from the 5' end of exon 5. The oligonucleotide of the present invention may comprise a sequence complementary to a region including any one of the 86th to 92nd positions from the 5' end of exon 5 of the G6PC gene having the c.648G>T mutation, and preferably comprises a sequence complementary to a region including the 92nd position.

[0135] An oligonucleotide having 15 to 30 bases and consisting of a nucleotide sequence complementary to the cDNA of the G6PC gene having the c.648G>T mutation, and consisting of a sequence complementary to a region including any one of the 82nd to 92nd sites from the 5' end of exon 5 of the G6PC gene having the c.648G>T mutation, can be exemplified by an oligonucleotide containing all or a part of any one of the sequences of SEQ ID NOs: 1 to 32, 40 to 42, and 44 to 48 (wherein t or T may be u or U, and u or U may be t or T). In the present invention, a "part of the sequence" is usually 80% or more of the entire sequence, preferably 85%, more preferably 90%, and most preferably 94%. The number of bases of the oligonucleotide of the present invention is suitably 15 to 30, preferably 15 to 21, and more preferably 15 to 18.

[0136] The nucleotides constituting the oligonucleotide of the present invention may be any of natural DNA, natural RNA, DNA / RNA chimera, and modified forms thereof, but it is preferable that at least one of them is a modified nucleotide.

[0137] The modified nucleotides in the present invention include those in which the sugar is modified (for example, those in which D-ribofuranose is 2'-O-alkylated, those in which D-ribofuranose is 2'-O,4'-C-alkylenated, and those in which D-ribofuranose is 2'-,4'-bridged), those in which the phosphodiester bond is modified (for example, thioated), those in which the base is modified, and those in which they are combined. Antisense oligonucleotides in which at least one D-ribofuranose is 2'-O-alkylated or 2'-O,4'-C-alkylenated have high binding strength to RNA and high resistance to nuclease, and therefore can be expected to have a higher therapeutic effect than natural nucleotides (i.e., oligoDNA, oligoRNA). In addition, oligonucleotides in which at least one phosphodiester bond is thioated also have high resistance to nuclease, and therefore can be expected to have a higher therapeutic effect than natural nucleotides (i.e., oligoDNA, oligoRNA). Oligonucleotides containing both modified sugars and modified phosphodiester linkages as described above are more resistant to nucleases and therefore are expected to have greater therapeutic efficacy.

[0138] For the oligonucleotide of the present invention, examples of sugar modifications include 2'-O-alkylation of D-ribofuranose (e.g., 2'-O-methylation, 2'-O-aminoethylation, 2'-O-propylation, 2'-O-allylation, 2'-O-methoxyethylation, 2'-O-butylation, 2'-O-pentylation, 2'-O-propargylation, etc.), 2'-O,4'- Examples of such alkylation include C-alkylenation (e.g., 2'-O,4'-C-ethylenation, 2'-O,4'-C-methylenation, 2'-O,4'-C-propylenation, 2'-O,4'-C-tetramethylenation, and 2'-O,4'-C-pentamethyleneation), 2'-deoxy-2'-C,4'-C-methyleneoxymethylenation of D-ribofuranose, S-cEt (2',4'-constrained ethyl), AmNA (amide-bridged nucleic acid), 3'-deoxy-3'-amino-2'-deoxy-D-ribofuranose, and 3'-deoxy-3'-amino-2'-deoxy-2'-fluoro-D-ribofuranose.

[0139] For the oligonucleotides of the present invention, examples of 2'-,4'-bridged modifications of sugars include 2'-O,4'-C-alkylenation of D-ribofuranose (e.g., 2'-O,4'-C-ethylenation, 2'-O,4'-C-propylenation, 2'-O,4'-C-tetramethylenation, 2'-O,4'-C-pentamethyleneation, etc.), 2'-deoxy-2'-C,4'-C-methyleneoxymethylenation of D-ribofuranose, S-cEt (2',4'-constrained ethyl), AmNA, etc.

[0140] For the oligonucleotides of the present invention, examples of modifications of phosphodiester bonds include phosphorothioate bonds, methylphosphonate bonds, methylthiophosphonate bonds, phosphorodithioate bonds, phosphoramidate bonds, and the like.

[0141] In the present invention, examples of base modifications include 5-methylation, 5-fluoroation, 5-bromination, 5-iodination, and N4-methylation of cytosine; 5-demethylation (uracil), 5-fluoroation, 5-bromination, and 5-iodination of thymine; N6-methylation and 8-bromination of adenine; and N2-methylation and 8-bromination of guanine.

[0142] The oligonucleotide of the present invention can be synthesized using a commercially available synthesizer (e.g., Perkin Elmer's Model 392 using the phosphoramidite method) according to the method described in the literature (Nucleic Acids Research, 12, 4539 (1984)). The nucleoside phosphoramidite reagent used in this case can be a commercially available reagent for natural nucleosides and 2'-O-methyl nucleosides (i.e., 2'-O-methyl guanosine, 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl uridine). The following is a list of 2'-O-alkylguanosine, adenosine, cytidine, and uridine with alkyl groups having 2 to 6 carbon atoms.

[0143] 2'-O-aminoethylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Blommers et al. Biochemistry (1998), 37, 17714-17725.).

[0144] 2'-O-propylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838.).

[0145] Commercially available reagents can be used for 2'-O-allylguanosine, adenosine, cytidine, and uridine.

[0146] 2'-O-Methoxyethylguanosine, adenosine, cytidine, and uridine can be synthesized according to the patent (US6261840) or the literature (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504.

[0147] 2'-O-Butylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838.).

[0148] 2'-O-pentylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838.).

[0149] Commercially available reagents can be used for 2'-O-propargylguanosine, adenosine, cytidine, and uridine.

[0150] 2'-O,4'-C-methyleneguanosine, adenosine, cytidine, 5-methylcytidine and thymidine can be produced according to the method described in WO99 / 14226, and 2'-O,4'-C-alkyleneguanosine, adenosine, cytidine, 5-methylcytidine and thymidine having an alkylene group with 2 to 5 carbon atoms can be produced according to the method described in WO00 / 47599.

[0151] 2'-Deoxy-2'-C,4'-C-methyleneoxymethylenated nucleosides of D-ribofuranose can be synthesized according to the literature (Wang, G. et al. Tetrahedron (1999), 55, 7707-7724).

[0152] S-cEt (constrained ethyl) can be synthesized according to the literature (Seth, PP et al. J. Org. Chem (2010), 75, 1569-1581.).

[0153] AmNA can be synthesized according to the literature (Yahara, A. et al. ChemBioChem (2012), 13, 2513-2516.) or WO2014 / 109384.

[0154] In the present invention, the nucleic acid base sequence can be described as adenine (A) or (a), guanine (G) or (g), cytosine (C) or (c), thymine (T) or (t), and uracil (U) or (u). 5-methylcytosine can be used instead of cytosine. Among the nucleic acid bases, uracil (U) or (u) and thymine (T) or (t) are interchangeable. Either uracil (U) or (u) or thymine (T) or (t) can be used to form a base pair with adenine (A) or (a) in the complementary strand.

[0155] Antisense oligonucleotides having phosphorothioate bonds can be synthesized by coupling a nucleoside phosphoramidite reagent and then reacting with a reagent such as sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or xanthan hydride (Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc. 112, 1253 (1990), PCT / WO98 / 54198).

[0156] The controlled pore glass (CPG) used in the synthesizer can be a commercially available product bound to a 2'-O-methyl nucleoside. For 2'-O,4'-C-methylene guanosine, adenosine, 5-methyl cytidine and thymidine, the nucleoside produced according to the method described in WO99 / 14226 can be bound to the CPG, and for 2'-O,4'-C-alkylene guanosine, adenosine, 5-methyl cytidine and thymidine with an alkylene group having 2 to 5 carbon atoms, the nucleoside produced according to the method described in WO00 / 47599 can be bound to the CPG according to the literature (Oligonucleotide Synthesis, Edited by MJGait, Oxford University Press, 1984). By using a modified CPG (described in Example 12b of JP-A-7-87982), an oligonucleotide bound to a 2-hydroxyethyl phosphate group at the 3' end can be synthesized. In addition, 3'-amino-Modifier C3 CPG, 3'-amino-Modifier C7 CPG, Glyceryl CPG, (Glen Research), 3'-specer C3 SynBase CPG 1000, and 3'-specer C9 SynBase CPG 1000 (link technologies) can be used to synthesize oligonucleotides with a hydroxyalkyl phosphate group or an aminoalkyl phosphate group attached to the 3' end.

[0157] <2.GalNAc unit> In the conjugate of the present invention, the oligonucleotide may have GalNAc bound via a linker and a phosphate group.

[0158] The GalNAc unit in the present invention is a phosphate group to which a linker having GalNAc is bound, and may further have one bond. The phosphate group of the GalNAc unit can be bound to the 5'-end and / or 3'-end of an oligonucleotide, and is preferably bound to the 5'-end of an oligonucleotide. When the GalNAc unit has a bond, the bond can be bound to a hydroxyl group, GalNAc, a linker to which GalNAc is bound, a phosphate group of another GalNAc unit, or a phosphate group at the 3'-end of an oligonucleotide. The number of GalNAcs contained in one GalNAc unit is two.

[0159] In the conjugate, one GalNAc unit may be bound to the oligonucleotide, or multiple GalNAc units may be bound consecutively to the oligonucleotide. The number of GalNAc units bound to one oligonucleotide is preferably 1 to 7, more preferably 1 to 5, particularly preferably 1 to 3, and the optimal number is 1.

[0160] In the present invention, when two GalNAc units are introduced to the 5' end of an oligonucleotide, Symmetric Doubler Phosphoramidite (1,3-bis-[5-(4,4'-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, Glen Research) or the like is used in the synthesis of the oligonucleotide, and when three GalNAc units are introduced to the 5' end of an oligonucleotide, Trebler Phosphoramidite (Tris-2,2,2-[3-(4,4'-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, Glen Research) or Long Trebler Phosphoramidite (Glen Research) are used. (Tris-2,2,2-[3-(4,4'-dimethoxytrityloxy)propyloxymethyl]methyleneoxypropyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, Glen Research) or the like can be used during oligonucleotide synthesis. When four GalNAc units are to be introduced into the 5'-end of an oligonucleotide, synthesis can be performed by using Symmetric Doubler Phosphoramidite or the like, which are coupled twice during oligonucleotide synthesis. When five GalNAc units are to be introduced into the 5'-end of an oligonucleotide, synthesis can be performed by using Symmetric Doubler Phosphoramidite, Trebler Phosphoramidite, or Long Trebler Phosphoramidite, which are each coupled once during oligonucleotide synthesis.When five or more GalNAc units are introduced at the 5' end of an oligonucleotide, an appropriate combination of Symmetric Doubler Phosphoramidite, Trebler Phosphoramidite, or Long Trebler Phosphoramidite can be used for synthesis.

[0161] In the present invention, when a GalNAc unit is introduced into the 3'-end of an oligonucleotide, it can be synthesized by using Asymmetric Doubler (Lev) Phosphoramidite (1-[5-(4,4'-dimethoxytrityloxy)pentylamido]-3-[5-levulinyloxypentylamido]-propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, Glen Research) or the like during oligonucleotide synthesis. Specifically, Asymmetric Doubler (Lev) Phosphoramidite is coupled to a commercially available Unylinker Support (e.g., Glen UnySuppor 1000, Glen Research), the DMTr group is removed, and then the chain of the target oligonucleotide is elongated. After the desired chain length is reached, the levulinyl group can be deprotected using a reagent such as hydrazine according to the protocol provided with the Asymmetric Doubler (Lev) Phosphoramidite reagent, and a GalNAc unit can be coupled to introduce it into the 3' end of the oligonucleotide.

[0162] In the present invention, the 5'-end and / or 3'-end of the oligonucleotide bound to the GalNAc unit may have an additional oligonucleotide sequence that is different from the oligonucleotide that exerts a medicinal effect, is composed of a phosphodiester bond, and can be cleaved in vivo. The chain length of the cleavable oligonucleotide is preferably 1 to 6 nucleotides, more preferably 1 to 3 nucleotides. The cleavable oligonucleotide is not particularly limited as long as it can be cleaved, and examples of the cleavable oligonucleotide include natural oligodeoxynucleotides that are entirely composed of DNA and natural oligonucleotides that are entirely composed of RNA. The base sequence is not particularly limited as long as it can be cleaved, and examples of the cleavable sequence include 5'-TCATCA-3', 5'-CATCA-3', 5'-ATCA-3', 5'-TCA-3', 5'-CA-3', 5'-A-3', etc.

[0163] In one embodiment, the biantennary GalNAc unit of the present invention has the formula

[0164] [ka]

[0165] [Wherein W is a compound represented by the formula

[0166] [ka]

[0167] wherein G represents a 5-acetamido-2-hydroxymethyl-3,4-dihydroxytetrahydropyran-6-yl group (GalNAc), Z represents an oxygen atom or a sulfur atom, and L 1 and L 2one of which represents a methylene group (CH2) and the other represents no atom between them, o, q, r and s each independently represent 0 or 1, n represents an integer of 1 to 15, m represents an integer of 0 to 5, and v represents 1 to 7. However, when n is 1, m is an integer of 0 to 5, and when n is an integer of 2 to 15, m is 0.], and preferably o and q are 0.

[0168] In the above formula, L 1 and L 2 is preferably L 2 indicates a methylene group (CH2), and L 1 does not sandwich any atom. n is preferably an integer of 1 to 10, more preferably an integer of 2 to 7, and even more preferably an integer of 2 to 4. m is preferably an integer of 0 to 3. v is preferably an integer of 1 to 5. More preferably, in the above formula, s is 1, n is an integer of 2 to 7, m is 0, 1 or 2, and v is an integer of 1 to 5, and even more preferably, in the above formula, s is 1, n is 2 or 3, m is 0, and v is an integer of 1 to 3.

[0169] A preferred bibranched GalNAc unit of the present invention has a bibranched structure, each of which has a glycerol structure within 3 atoms of the phosphate group, and each branched chain has GalNac at the end, the linker structures from the branch points to the GalNac are almost identical, the linker backbone connecting the GalNAc portion and the phosphate group is 15 atoms or less in length, has a high heteroatom content, and the length of the consecutive alkyl chains is C5 or less, thereby exhibiting good solubility in water.

[0170] Specific examples of the biantennary GalNAc unit of the present invention include groups represented by the following formulas:

[0171] [ka]

[0172] [ka]

[0173] [ka]

[0174] [ka]

[0175] [ka]

[0176] 3. Method for producing conjugates The oligonucleotides having GalNAc units linked thereto of the present invention can be synthesized in accordance with the method described in the literature (Nucleic Acids Research, 12, 4539 (1984)) using a commercially available synthesizer (e.g., Perkin-Elmer Model 392 using the phosphoramidite method) by using the phosphoramidite contained in the GalNAc unit in the same manner as the phosphoramidite reagent for the nucleoside.

[0177] Synthesis of GalNAc unit-containing phosphoramidites The GalNAc unit-containing phosphoramidite or H-phosphonate of the present invention can be produced according to Method A or Method B described below. In Method A or Method B, after the completion of each reaction, the target compound of each reaction is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if any insoluble matter is present, it is removed by filtration, and then an immiscible organic solvent such as water and ethyl acetate is added, and the organic layer containing the target product is separated, washed with water, etc., dried with anhydrous sodium sulfate, etc., and the solvent is distilled off to obtain the product. If necessary, the obtained compound can be separated and purified by a conventional method, for example, silica gel column chromatography. Alternatively, it can be purified by reprecipitation or recrystallization. However, when r = 0, Method C is used instead of Method A or Method B.

[0178] [A method] Method A is a method for producing branched compounds (7) and (7') using compound (3). The configuration of compound (1) is not limited. By using compound (3), the protecting group for the 6-hydroxyl group of the GalNAc moiety can be appropriately selected. When r=0, Method C is used.

[0179] [ka]

[0180] (R in the formula 13 is a common protecting group for a hydroxyl group, and is preferably a benzyl group. 14 is a common protecting group for a hydroxyl group, but is preferably an acetyl group or a hydrophobic protecting group (more preferably a 4,4'-dimethoxytrityl group). 15 is a common protecting group for a carbonyl group, preferably a benzyl group. 3 )R 4 [R 3 and R 4 are the same or different and represent a hydroxyl group, a protected hydroxyl group, a mercapto group, a protected mercapto group, an amino group, an alkoxy group having 1 to 4 carbon atoms, an alkylthio group having 1 to 4 carbon atoms, a cyanoalkoxy group having 1 to 5 carbon atoms, or an amino group substituted with an alkyl group having 1 to 4 carbon atoms. In the formula, X is carbon or oxygen. In the formula, n is an integer of 1 to 15, and m is an integer of 0 to 5, provided that when n is 1, m is an integer of 0 to 5, and when n is an integer of 2 to 15, m is 0. In the formula, r is independently 0 or 1. In the formula, v is 1 to 7.

[0181] Step A-1 is a carbamate formation step in which compound (1) is converted to an active ester in the presence of an activator and a base in an inert solvent, and then reacted with compound (2) to form a carbamate.

[0182] The inert solvent used in the above reaction includes, for example, hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers, etc., and preferably dichloromethane.

[0183] The activating agent used in the above reaction is not particularly limited as long as it forms an activated ester, and examples thereof include bis(pentafluorophenyl) carbonate, bis(4-nitrophenyl) carbonate, and 4-nitrophenyl chloroformate. Of these, 4-nitrophenyl chloroformate is preferred.

[0184] Examples of the base used in the above reaction include triethylamine, N,N-diisopropylethylamine, pyridine, and 4-dimethylaminopyridine. Preferably, pyridine is used when preparing an active ester, and N,N-diisopropylethylamine is added when reacting with an amine.

[0185] The reaction temperature varies depending on the activating agent, base, inert solvent, etc., but is usually from -20°C to the reflux temperature, preferably from 10°C to 30°C.

[0186] The reaction time varies depending on the activating agent, base, inert solvent, reaction temperature, etc., but is usually from 15 minutes to 24 hours, preferably from 1 hour to 4 hours.

[0187] In step A-2, a protecting group R 13 and R 15 The removal of the protecting group varies depending on the type of the protecting group, but can generally be carried out according to a conventional method such as a method well known in the art of organic synthetic chemistry, for example, the method described in TH Greene, PG Wuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc.

[0188] Step A-3 is a step of carrying out amide condensation with compound (3) in an inert solvent in the presence of a condensing agent and a base.

[0189] The inert solvent used in the above reaction can be appropriately selected depending on the type of condensation agent used, and examples thereof include water, alcohols, aprotic polar solvents, and the like, and preferably dichloromethane or N,N-dimethylformamide.

[0190] Examples of the base used in the above reaction include triethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine, and the like, with N,N-diisopropylethylamine being preferred.

[0191] Condensing agents used in the above reaction include, for example, carbodiimide-based condensing agents such as WSC (1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) and DIC (N,N'-diisopropylcarbodiimide), imidazole-based condensing agents such as CDI (N,N'-carbonyldiimidazole), triazine-based condensing agents such as DMT-MM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate), uronium-based condensing agents such as HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) and TSTU (O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), and the phosphonium condensing agent PyBOP (1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate), and preferably WSC or HATU. If necessary, additives HOAt (1-hydroxy-7-azabenzotriazole), HOBt (1-hydroxybenzotriazole), and Oxyma (ethyl (hydroxyimino) cyanoacetate) may be added.

[0192] The reaction temperature varies depending on the condensing agent, base, solvent, etc., but is usually from -20°C to the reflux temperature, preferably from 10°C to 30°C.

[0193] The reaction time varies depending on the condensing agent, base, solvent, reaction temperature, etc., but is usually from 15 minutes to 72 hours, preferably from 1 hour to 24 hours.

[0194] Step A-4 is a step for producing compound (7), in which -P(R 3 )R 4 Introduce the following.

[0195] The inert solvent used in the above reaction includes, for example, hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers, acetonitrile and the like, and preferably dichloromethane.

[0196] Examples of the base used in the above reaction include triethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine, and the like, with N,N-diisopropylethylamine being preferred.

[0197] The reaction temperature varies depending on the base, the inert solvent, etc., but is usually from -20°C to the reflux temperature, preferably from 10°C to 30°C.

[0198] The reaction time varies depending on the base, inert solvent, reaction temperature, etc., but is usually from 15 minutes to 24 hours, preferably from 1 hour to 4 hours.

[0199] Step A-5 is a step of synthesizing compound (8) from compound (7), in which a protecting group R 15 The removal of the protecting group varies depending on the type of the protecting group, but can generally be carried out according to a conventional method such as a method well known in the art of organic synthetic chemistry, for example, the method described in TH Greene, PG Wuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc.

[0200] Step A-6 is a step of amidating compound (8), which can be carried out in the same manner as in the above-mentioned step A-3.

[0201] In step A-7, a protecting group R 13 The removal of the protecting group varies depending on the type of the protecting group, but can generally be carried out according to a conventional method such as a method well known in the art of organic synthetic chemistry, for example, the method described in TH Greene, PG Wuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc.

[0202] Step A-8 is a step of synthesizing the H-phosphonate compound (7') from the compound (6). In an inert solvent, the compound (6) is reacted with tris-(1,2,4-triazolyl)phosphite prepared from phosphorus trichloride and 1,2,4-triazole in advance according to the literature (BCFreohler, PGNg and MD. Matteucci, Nucleic Acids Res., 14 5399 (1986)) instead of a phosphoramidite reagent, and then water is added to stop the reaction, followed by post-treatment to obtain the H-phosphonate. The solvent used is not particularly limited as long as it does not inhibit the reaction, but is preferably a halogenated hydrocarbon such as methylene chloride. The reaction temperature is not particularly limited within the range of -20° to 100° C., but is usually carried out at room temperature. The reaction time varies depending on the solvent used and the reaction time, but is 30 minutes when reacted at room temperature in methylene chloride.

[0203] [B method] Method B is a method for producing branched compounds (14) and (14') using compound (3). The configuration of compound (10) is not limited. By using compound (3), the protecting group for the 6-hydroxyl group of the GalNAc moiety can be appropriately selected. When r=0, Method C is used.

[0204] [ka]

[0205] R 12 is a common protecting group for a hydroxyl group, and is preferably a benzyl group. 3 , R 4 ,X,n,m,r,v,R 14 and R 15 has the same meaning as above.

[0206] Step B-1 is a step of converting compound (10) into a carbamate, and can be carried out in the same manner as in the above-mentioned step A-1.

[0207] In step B-2, a protecting group R 12 and R 15 This step can be carried out in the same manner as in the above-mentioned step A-2.

[0208] Step B-3 is a step of amidating compound (12), and can be carried out in the same manner as in the above-mentioned step A-3.

[0209] In step B-4, the hydroxyl group of compound (13) is reacted with -P(R 3 )R 4 This is a step of introducing the above-mentioned compound, which can be carried out in the same manner as in step A-3.

[0210] Step B-5 is a step of synthesizing compound (15) from compound (11), in which a protecting group R 15 This can be carried out in the same manner as in step A-5 above.

[0211] Step B-6 is a step of amidating compound (15), which can be carried out in the same manner as in the above-mentioned step A-6.

[0212] In step B-7, a protecting group R 12 This step can be carried out in the same manner as in step A-7 above.

[0213] Step B-8 is a step of synthesizing compound (14') from compound (13), which can be carried out in the same manner as in step A-8 described above.

[0214] [C method] Method C is a method for producing a branched compound (19) or compound (22), and compound (19') or compound (22') using compound (3). The configurations of compound (1) and compound (10) are not limited. By using compound (3), the protecting group for the 6-hydroxyl group of the GalNAc moiety can be appropriately selected.

[0215] [ka]

[0216] R in the formula 3 , R 4 , X, n, m, r, R 12 , R 13 and R 14 has the same meaning as above.

[0217] Step C-1 is a step of converting compound (1) or compound (10) into a carbamate, and can be carried out in the same manner as in the above-mentioned step A-1, except that compound (3) is used instead of compound (2).

[0218] In step C-2, a protecting group R 12 Or R 13 The removal of the protecting group varies depending on the type of the protecting group, but can generally be carried out according to a conventional method such as a method well known in the art of organic synthetic chemistry, for example, the method described in TH Greene, PG Wuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc.

[0219] In step C-3, a hydroxyl group of compound (19) or compound (22) is reacted with -P(R 3 )R 4This is a step of introducing the above-mentioned compound, which can be carried out in the same manner as in step A-3.

[0220] Step C-4 is a step of synthesizing compound (19') from compound (18) or compound (22') from compound (21), which can be carried out in the same manner as in step A-8 described above.

[0221] <4. Production method using hydrophobic protecting groups> The present invention provides a method for producing a conjugate of a GalNAc unit and an oligonucleotide by binding a GalNAc unit having a highly hydrophobic protecting group introduced on one or more of the 3rd, 4th and 6th hydroxyl groups of the GalNAc site to the 5' or 3' end of a synthetic oligonucleotide and separating the GalNAc unit from impurities by adsorbing the GalNAc unit to a resin having adsorptivity through hydrophobic interaction; and provides binding compounds (phosphoramidites, H-phosphonates) and conjugates containing hydrophobic protecting groups to be used as intermediates in the production method.

[0222] The manufacturing method of the present invention includes the following steps. a: synthesizing a conjugate in which a structure containing a hydrophobic protecting group-added GalNAc unit-phosphate group is bound to the 5'-end or 3'-end of an oligonucleotide; b: passing the solution containing the product of step a through a column packed with a resin having adsorptivity by hydrophobic interaction, and passing an aqueous washing solution through the column; and c: Eluting the oligonucleotide from the column after step b.

[0223] In the present production method, various hydrophobic protective groups can be used as long as they meet the above definition. In normal nucleic acid synthesis, oligonucleotides are released from the support by basic treatment with ammonia water or the like, so protective groups that can be removed by treatment under acidic conditions, such as an optionally substituted trityl group and an optionally substituted silyl group, can be used. Specific examples of such optionally substituted trityl groups include a trityl group, a monomethoxytrityl group, and a dimethoxytrityl group. Examples of optionally substituted silyl groups include a triphenylsilyl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, and the like, and preferably a 4,4'-dimethoxytrityl group (DMTr group).

[0224] As the hydrophobic protecting group which can be removed under basic conditions, various kinds of ester groups can be used, and preferred examples include an acetyl group and a benzoyl group.

[0225] In the present invention, the term "structure containing a hydrophobic protecting group-added GalNAc unit-phosphate group" refers to a structure represented by the following formula:

[0226] [ka]

[0227] In the above formula, R which is a substituent of the hydroxyl group on GalNAc a , R b and R c At least one of R is a hydrophobic protecting group, and the others are hydrogen atoms or protecting groups that are removed under liberating conditions. All of R may be hydrophobic protecting groups, any two of R may be hydrophobic protecting groups, or only one of R may be a hydrophobic protecting group. a is a hydrophobic protecting group (more preferably a hydrophobic protecting group that can be removed under acidic conditions, even more preferably an optionally substituted trityl group, and most preferably a DMTr group), and R b and R cis a hydrogen atom. When this substituent is not a hydrophobic protecting group, a protecting group that is removed under release conditions may be bonded, but these protecting groups are removed during the process of cleaving the oligonucleotide from the support, and become a hydrogen atom (a hydroxyl group on GalNAc) at the stage used in step a.

[0228] X1 and X2 each independently represent a linker structure, and may be branched between X1 and X2. As the linker structure employed here, various skeletons such as an alkyl chain, a PEG chain, a peptide chain, etc. may be employed. This linker structure may contain multiple GalNAc by branching in the middle. The number (t) of branched chains in the linker structure is about 1 to 10, preferably 1 to 3. When two or more branched chains are present, the linker structures between the branch points and GalNAc may be the same or different. When multiple GalNAc are contained, at least one hydrophobic protecting group is bound to each GalNAc.

[0229] In the above formula, Y represents a bond that is bonded to an oligonucleotide via a phosphate group or a thiophosphate group. When bonded to the 5'-terminus of an oligonucleotide, it is bonded to the 5'-hydroxyl group of the nucleoside at the terminus via a phosphodiester bond or a phosphorothioate bond. Such a bond can be synthesized by using a compound (a hydrophobic protecting group-added GlcNAc unit amidite-like compound) in which Y is a phosphoramidite structure or an H-phosphonate structure containing a hydrophobic protecting group-added GalNAc unit-phosphate group in the same manner as a nucleoside phosphoramidite reagent following solid-phase synthesis of an oligonucleotide. Specific examples of hydrophobic protecting group-added GalNAc unit amidite-like compounds corresponding to the biantennary GalNAc unit of the present invention are described above in (B13).

[0230] The production method of the present invention is not limited to the biantennary GalNAc unit of the present invention described above, but can also be applied to the production of conjugates of known GalNAc units (e.g., those described in Patent Documents 1 to 9, Non-Patent Documents 1 and 2, etc.) with oligonucleotides. In this case, a compound having a structure corresponding to a known GlcNAc unit is used as a hydrophobic protecting group-added GlcNAc unit phosphoramidite or H-phosphonate. Examples of these amidite-like compounds corresponding to known GalNAc units include, for example, the following compounds.

[0231] The following compounds can be produced with reference to J. Med. Chem. 2016, 59, 2718-2733 and the references cited therein.

[0232] For Tris-based GalNAc unit [ka]

[0233] For Triacid-based GalNAc unit [ka]

[0234] For Pentaerythritol based GalNAc unit

[0235] [ka]

[0236] For Lys-based GalNAc unit

[0237] [ka]

[0238] The hydrophobic protecting group-added GalNAc unit phosphoramidite represented by the following formula can be produced with reference to WO19027009.

[0239] For Aryl-based GalNAc unit

[0240] [ka]

[0241] The following compounds can be produced with reference to J. Med. Chem. 2016, 59, 2718-2733 and the references cited therein.

[0242] For Hydroxyprolinol based GalNAc unit / Piperidine based GalNAc unit

[0243] [ka]

[0244] In the formula of the amidite-like compound corresponding to the above known GlcNAc unit, R1 represents a hydrophobic protecting group, X represents a linker structure, preferably a C3-C6 alkyl linear chain, and Y represents a phosphoramidite group or an H-phosphonate group.

[0245] A hydrophobic protecting group-added GalNAc unit-phosphoramidite or a hydrophobic protecting group-added GalNAc unit-H-phosphonate corresponding to a known GlcNAc unit can be synthesized by appropriately using, in the synthesis process of the GalNAc unit, a GalNAc derivative corresponding to compound (3) described in the above Methods A to C. Various GalNAc derivatives in which at least one of the 3-, 4-, and 6-position hydroxyl groups is protected with a hydrophobic protecting group can be synthesized, for example, by referring to the method in Example 1.

[0246] A conjugate having a structure containing a hydrophobic protecting group-added GalNAc-phosphate group (wherein Y represents a bond that bonds to the oligonucleotide via a phosphate group or a thiophosphate group) can be synthesized by introducing the hydrophobic protecting group-added GalNAc unit amidite-like compound into the 5'-end or 3'-end of an oligonucleotide synthesized according to the above-mentioned method in the same manner as a nucleoside amidite reagent. Examples of the conjugates thus synthesized include the conjugates described in (B17) and (B18).

[0247] The solvent used in the reaction for introducing the hydrophobic protecting group-added GalNAc unit-H-phosphonate compound into the 5'-end or 3'-end of the synthesized oligonucleotide is not particularly limited as long as it does not inhibit the reaction, but anhydrous acetonitrile is preferably used. The reagent used as the condensation agent is an acid chloride of a carboxylic acid or phosphoric acid, but pivaloyl chloride is preferably used. The oxidizing agent for oxidizing the H-phosphonic acid bond to a phosphoric diester bond is not particularly limited as long as it is one that is usually used in oxidation reactions, and examples of the oxidizing agent include manganese oxides such as potassium permanganate and manganese dioxide; ruthenium oxides such as ruthenium tetroxide; selenium compounds such as selenium dioxide; iron compounds such as iron chloride; osmium compounds such as osmium tetroxide; silver compounds such as silver oxide; mercury compounds such as mercury acetate; lead oxide compounds such as lead oxide and lead tetroxide; inorganic metal oxidizing agents such as potassium chromate, chromate-sulfuric acid complex, chromate-pyridine complex, and cerium compounds such as cerium ammonium nitrate (CAN); halogen molecules such as chlorine molecules, bromine molecules, and iodine molecules; periodic acids such as sodium periodate; ozone; hydrogen peroxide solution; nitrous acid compounds; chlorite compounds such as potassium chlorite and sodium chlorite; potassium persulfate, sodium persulfate, and the like. Examples of the oxidizing agent include inorganic oxidizing agents such as persulfuric acid compounds such as sodium; reagents used in DMSO oxidation (complexes of dimethyl sulfoxide with dicyclohexylcarbodiimide, oxalyl chloride, acetic anhydride or phosphorus pentoxide, or pyridine-acetic anhydride complex); peroxides such as t-butyl hydroperoxide; stable cations such as triphenylmethyl cation; succinimides such as N-bromosuccinimide; hypochlorite compounds such as t-butyl hypochlorite; azodicarboxylate compounds such as methyl azodicarboxylate; disulfides and triphenylphosphine such as dimethyl disulfide, diphenyl disulfide, and dipyridyl disulfide; nitrite esters such as methyl nitrite; tetrahalogenated carbons such as tetrabromomethane; and organic oxidizing agents such as quinone compounds such as 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), with molecular iodine being preferred.Examples of the deoxidizing agent used include heterocyclic amines such as pyridine and dimethylaminopyridine, and aliphatic amines such as trimethylamine, triethylamine, and diisopropylethylamine, and are preferably heterocyclic amines (especially pyridine). When forming a phosphorothioate bond, the agent is not particularly limited as long as it is a reagent capable of reacting with an H-phosphonic acid bond to form a phosphorothioate bond, and includes sulfur, tetraethylthiuram disulfide, Beaucage reagent, and phenylacetyl disulfide, and is preferably sulfur. The synthesized conjugate can be released from the support while retaining the hydrophobic protecting group by a method similar to that of DMTr-on purification of ordinary oligonucleotides.

[0248] A conjugate having a structure containing such a hydrophobic protecting group-added GalNAc unit-phosphate group can be released from the support while retaining the hydrophobic protecting group by a method similar to that of DMTr-on purification of a normal oligonucleotide. Usually, the bond between the 3' end of the oligonucleotide and the support is cleaved with aqueous ammonia, and the oligonucleotide is released under basic conditions. Therefore, as the hydrophobic protecting group, a protecting group that is not removed under basic conditions, for example, a protecting group that is removed under acidic conditions, is selected. On the other hand, when a bonding mode that is cleaved under acidic conditions is adopted as the bonding mode between the 3' end of the oligonucleotide and the support, the release condition of the oligonucleotide becomes acidic, so as the hydrophobic protecting group, a protecting group that is not removed under acidic conditions, for example, a protecting group that is removed under basic conditions, is selected.

[0249] The thus obtained hydrophobic protecting group-added GalNAc unit-oligonucleotide conjugate can be adsorbed onto a column packed with a resin having adsorptivity through hydrophobic interaction, and impurities not having a hydrophobic protecting group can then be separated by washing the column with an aqueous washing solution.

[0250] The "resin having adsorptivity due to hydrophobic interactions" is not particularly limited as long as it is a resin in which a highly hydrophobic component is used as the base material, and various types can be used, for example, styrene-divinylbenzene-based adsorption resins, styrene-divinylbenzene-based modified adsorption resins, methacrylic-based adsorption resins, or phenol-based adsorption resins.

[0251] The packing solution used for adsorbing the hydrophobic protecting group-added GalNAc unit-oligonucleotide conjugate to the column is preferably a basic aqueous solution that does not contain a large amount of organic solvent. For example, concentrated aqueous ammonia used for cleaving the oligomer from the support after solid-phase synthesis, or a basic aqueous solution obtained by diluting the aqueous ammonia with an appropriate amount of solvent (water, buffer, etc.) can be used.

[0252] As the washing solution used for removing impurities from the column, various solutions can be used as long as they do not deprotect the hydrophobic protecting group and maintain the hydrophobic interaction with the resin. As such a washing solution, an aqueous solution containing an organic solvent at a low concentration enough to maintain the hydrophobic interaction with the resin can be used to improve the efficiency of removing impurities. Specifically, when the hydrophobic protecting group is a DMTr group, an aqueous solution containing an appropriate amount of salt or an aqueous solution containing acetonitrile can be used as the washing solution.

[0253] The hydrophobic protecting group attached to GalNAc can be deprotected by treating the conjugate under deprotection conditions that depend on the protecting group employed. When applying this production method following a general oligonucleotide synthesis, a hydrophobic protecting group that is deprotected under acidic conditions, such as a DMTr group, is employed, and therefore deprotection is performed by treatment with an acidic solution.

[0254] By appropriately carrying out the deprotection treatment on the column to which the conjugate is adsorbed, a deprotected GalNAc-oligonucleotide conjugate can be obtained while leaving impurities derived from the protecting group adsorbed on the column.

[0255] In addition, the hydrophobic protecting group-added GalNAc-oligonucleotide conjugate eluted from the column without deprotection can be deprotected and the treated solution can be passed through a similar hydrophobic interaction adsorption resin column again to separate the deprotected conjugate from impurities derived from the protecting groups.

[0256] The column retaining the conjugate can fractionate the GalNAc-oligonucleotide conjugate with a high degree of purification by using an appropriate gradient elution method depending on the resin used. When eluting from the column without deprotecting the hydrophobic protecting group, the conjugate can be eluted by gradient elution with increasing organic solvent content by disrupting the hydrophobic interaction between the hydrophobic protecting group and the adsorption resin.

[0257] When the deprotection treatment is carried out while the conjugate is adsorbed on the column, appropriate elution conditions can be appropriately selected depending on the retention mode of the deprotected conjugate on the column.

[0258] When using an ion exchange column with adsorptivity due to hydrophobic interactions, the conjugate is deprotected while adsorbed on the column, and the GalNAc-oligonucleotide conjugate can be eluted from the column by the usual method of elution from an ion exchange column, utilizing the salt concentration in the eluent. Specifically, when using a RESOURCE Q (GE Healthcare) column, a gradient eluent can be used in which the sodium chloride content in a sodium hydroxide / sodium chloride aqueous solution mixture is gradually increased from 0 to 2M (2mol / L).

[0259] When using a reversed-phase column, GalNAc-oligonucleotide conjugates can be eluted from the column by using an appropriate organic solvent and fractionating according to the usual method used for elution from reversed-phase columns. Specifically, when using a Clarity QSP (Phenomenex) column, a trishydroxymethylaminomethane (Tris)-HCl buffer containing an appropriate amount of acetonitrile can be used.

[0260] The absorbance at 260 nm, which is the detection wavelength for nucleic acids, is utilized to detect the target substance. The absorbance at 260 nm of the liquid passing through the column is measured over time, and the target substance can be obtained by collecting the fraction detected in the elution step.

[0261] <5. Pharmaceuticals> The conjugate of the present invention can be used as a medicine for treating a disease targeted by the oligonucleotide contained therein. Treatment includes both prevention and post-treatment.

[0262] The conjugate of the present invention may be used in the form of a pharma- ceutically acceptable salt. The term "pharma-ceutically acceptable salt" refers to a salt of an oligonucleotide, and examples of such salts include metal salts such as alkali metal salts, such as sodium salt, potassium salt, and lithium salt, alkaline earth metal salts, such as calcium salt and magnesium salt, aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts, such as ammonium salt, t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylamine ... Examples of the salts include amine salts such as organic salts such as ammonium salts and tris(hydroxymethyl)aminomethane salts; hydrohalides such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate, and preferably alkali metal salts, and more preferably sodium salts. These salts can be produced by known methods.

[0263] In addition, the conjugate and its pharma- ceutically acceptable salt may exist as a solvate (e.g., hydrate). In the present invention, such a solvate is treated as being included in the salt of the oligonucleotide or conjugate.

[0264] When the conjugate of the present invention or a pharma- ceutical acceptable salt thereof is used as a pharmaceutical, it can be administered orally in the form of tablets, capsules, granules, powders, syrups, or the like, or parenterally in the form of injections, suppositories, patches, external preparations, or the like, either by itself or mixed with an appropriate pharma- ceutical acceptable excipient, diluent, or the like.

[0265] These preparations may contain excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, bayerian starch, alpha starch, and dextrin; cellulose derivatives such as crystalline cellulose; organic excipients such as gum arabic, dextran, and pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and sulfates such as calcium sulfate), lubricants, and the like. lubricants (e.g., stearic acid; metal stearates such as calcium stearate and magnesium stearate; talc; colloidal silica; waxes such as beeswax and gay wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL-leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; the above-mentioned starch derivatives, etc.), binders (e.g., hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, etc.), lydon, macrogol, compounds similar to the above excipients, etc.), disintegrants (e.g., cellulose derivatives such as low-substituted hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, and internally crosslinked sodium carboxymethylcellulose; chemically modified starches and celluloses such as carboxymethyl starch, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone, etc.), emulsifiers (e.g., colloidal clays such as bentonite and veegum; metal hydroxides such as magnesium hydroxide and aluminum hydroxide; anionic surfactants such as sodium lauryl sulfate and calcium stearate; cationic surfactants such as benzalkonium chloride; nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, and sucrose fatty acid esters, etc.), stabilizers (paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal;It is produced by a known method using additives such as dehydroacetic acid, sorbic acid, flavoring agents (e.g., commonly used sweeteners, acidulants, flavors, etc.), diluents, etc.;

[0266] The therapeutic agent of the present invention may contain 0.1 to 250 μmoles / mL of oligonucleotide (antisense oligonucleotide), and preferably may contain 1 to 50 μmoles / mL of oligonucleotide or a pharma- ceutically acceptable salt thereof, 0.02 to 10% w / v of carbohydrate or polyhydric alcohol, and 0.01 to 0.4% w / v of a pharma- ceutically acceptable surfactant.

[0267] As the carbohydrate, monosaccharides or disaccharides are particularly preferred. Examples of these carbohydrates and polyhydric alcohols include glucose, galactose, mannose, lactose, maltose, mannitol and sorbitol. These may be used alone or in combination.

[0268] In addition, preferred examples of the surfactant in the present invention include polyoxyethylene sorbitan mono- to tri-esters, alkylphenyl polyoxyethylenes, sodium taurocholate, sodium cholate, and polyhydric alcohol esters. Of these, polyoxyethylene sorbitan mono- to tri-esters are particularly preferred, and oleate, laurate, stearate, and palmitate are particularly preferred as esters. These may be used alone or in combination.

[0269] Furthermore, the therapeutic agent of the present invention may further preferably contain 0.03 to 0.09 M of a pharma- ceutical acceptable neutral salt, such as sodium chloride, potassium chloride and / or calcium chloride.

[0270] Furthermore, the therapeutic agent of the present invention may more preferably contain a pharma- ceutically acceptable buffer at 0.002 to 0.05 M. Preferred examples of the buffer include sodium citrate, sodium glycinate, sodium phosphate, and tris(hydroxymethyl)aminomethane. These buffers may be used alone or in combination.

[0271] Furthermore, the above-mentioned therapeutic agent may be supplied in a solution state. However, in cases where it is necessary to store the agent for a certain period of time, it is usually preferable to freeze-dry the agent in order to stabilize the oligonucleotide and prevent a decrease in therapeutic effect. In this case, the agent may be reconstituted with a dissolving solution (such as distilled water for injection) at the time of use, that is, in a liquid state to be administered. Therefore, the therapeutic agent of the present invention also includes a freeze-dried agent that is used by reconstituting the agent with a dissolving solution so that each component has a predetermined concentration range. In order to promote the solubility of the freeze-dried agent, an amino acid such as albumin or glycine may be further contained.

[0272] The conjugates of the invention may also be encapsulated using lipids, such as those described in WO 2015 / 005253, and administered as nucleic acid-lipid nanoparticles or liposomes, such as those described in WO 2015 / 005253.

[0273] When the conjugate of the present invention or a pharma- ceutical acceptable salt thereof is administered to a human, it may be administered, for example, at a daily dose of about 0.01 to 100 mg / kg (body weight), preferably 0.1 to 20 mg / kg (body weight) for an adult, once or several times as subcutaneous injection, intravenous drip injection, or intravenous injection; however, the dose and number of administrations may be appropriately changed depending on the type of disease, symptoms, age, administration method, etc. EXAMPLES

[0274] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of explaining the present invention and are not intended to limit the scope of the present invention. As an automatic nucleic acid synthesizer, either "ABI 392 DNA / RNA Synthesizer" (Applied Biosystems), "ABI 394 DNA / RNA Synthesizer" (Applied Biosystems), AKTA Oligopilot (GE Healthcare), or nS-8II Synthesizer (Gene Design Inc.) was used, and the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984)) was used with a synthesis program appropriate for the synthesis scale.

[0275] The condensation activator used was activator solution-3 (0.25 mol / L 5-benzylthio-1H-tetrazole·acetonitrile solution, Wako Pure Chemical Industries, product No. 013-20011).

[0276] The capping reagents used were CAP A for AKTA (1-methylimidazole / acetonitrile solution, Sigma-Aldrich, product No. L040050), Cap B1 for AKTA (acetic anhydride / acetonitrile solution, Sigma-Aldrich, product No. L050050), and Cap B2 for AKTA (pyridine / acetonitrile solution, Sigma-Aldrich, product No. L050150).

[0277] For deblocking, DCA Deblock (dichloroacetic acid / toluene solution, Sigma-Aldrich, product No. L023050) or Deblocking Solution-1 (3 w / v% trichloroacetic acid / dichloromethane solution, Fujifilm Wako Pure Chemical Industries, product No. 042-28921) was used.

[0278] As a thiolation reagent for forming phosphorothioate bonds, phenylacetyl disulfide (Carbosynth, product No. FP07495) was dissolved to a concentration of 0.2 M in a 1:1 (v / v) solution of acetonitrile (anhydrous, Kanto Chemical, product No. 01837-05) and pyridine (anhydrous, Kanto Chemical, product No. 11339-05).

[0279] As an oxidizing agent, OXDIZER 0.05M (Sigma-Aldrich, product No. L560250-04) or iodine (Kanto Chemical, product No. 20035-00) was dissolved to a concentration of 0.02M in a solution of tetrahydrofuran (anhydrous, Kanto Chemical, product No. 40993-05), pyridine (anhydrous, Kanto Chemical, product No. 11339-05), and distilled water 78:20:2 (v / v / v) and used.

[0280] As nucleoside phosphoramidite reagents, 2'-O-Me nucleoside phosphoramidites (adenosine product No. ANP-5751, cytidine product No. ANP-5752, guanosine product No. ANP-5753, uridine product No. ANP-5754) manufactured by ChemGenes were used.

[0281] The phosphoramidite of the non-natural nucleoside is Example 14 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-6-N-benzoyl adenosine-3'-O-(2-cyanoethyl N,N-diisopropyl) phosphoramidite), Example 27 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-2-N-isobutyrylguanosine-3'-O-(2-cyanoethyl N,N-diisopropyl) phosphoramidite), Example 22 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-4-N-benzoyl-5-methylcytidine-3'-O-(2-cyanoethyl) phosphoramidite) of JP-A 2000-297097. The compounds used were those of Example 1 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite) and Example 9 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite).

[0282] As the solid phase support, NittoPhase UnyLinker 100, 10 μmol (manufactured by Nitto Denko), Primer Support 5G UnyLinker 350, 10 μmol (manufactured by GE Healthcare), Primer Support 5G Unylinker 350, 100 μmol (manufactured by GE Healthcare), or Glen UnySupport CPG 500, 1 μmol (manufactured by GlenResearch) was appropriately selected according to the synthesis scale.

[0283] Example 1: Synthesis of GalNAc with a hydrophobic protecting group (1A) Synthesis of [(2R,3R,4R,5R)-5-acetamido-3,4-diacetoxy-6-[3-(benzyloxycarbonylamino)propoxy]tetrahydropyran-2-yl]methyl acetate (Compound 1A)

[0284] [ka]

[0285] N-(3-hydroxypropyl)benzyl carbamate (6.4g, 30.8mmol) and the literature-known compound [(2R,3R,4R,5R)-5-acetamido-3,4,6-triacetoxy-tetrahydropyran-2-yl]methyl acetate (WO2011053614) (10g, 25.7mmol) were added to a dichloromethane (200ml) suspension, and the mixture was stirred at 45°C for 4 hours. After the reaction was completed, the solvent was evaporated under reduced pressure to about half, and the mixture was concentrated. The organic layer was washed with a saturated aqueous solution of sodium bicarbonate and saturated saline, then dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain a crude product. Isopropyl ether (100m) was added, and the mixture was stirred for 3 hours. The solid obtained was filtered to obtain the target product 1A (11.9g). This was used in the next reaction without further purification. 1 H-NMR (CDCl3) δ: 7.43-7.29 (5H, m), 6.26 (1H, d, J = 8.5 Hz), 5.36-5.29 (1H, m), 5.12 (1H, d, J = 12.1 Hz), 5.08 (1H, d, J = 12.1 Hz), 5.03-4.95 (2H, m), 4.33 (1H, d, J = 9.1 Hz), 4.21-4.06 (3H, m), 4.01-3.93 (1H, m), 3.80-3.73 (1H, m), 3.62-3.49 (1H, m), 3.44-3.35 (1H, m), 3.14-3.04 (1H, m), 2.15 (3H, s), 2.05 (3H, s), 2.01 (3H, s), 1.95 (3H, s), 1.88-1.75 (1H, m), 1.69-1.56 (1H, m). Calcd for C 25 H 34 N2O 11 : [M+H]+ 539, Found 539.

[0286] (1B) Synthesis of benzyl N-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxypropyl]carbamate (Compound 1B)

[0287] [ka]

[0288] A 28% sodium methoxide methanol solution (0.85 ml) was added to a suspension of the compound (12.4 g, 23 mmol) synthesized in step (1A) in ethyl alcohol (200 ml) and stirred at room temperature. As the reaction proceeded, the solution became clear at first. After leaving it for 60 minutes, a precipitate was obtained. The precipitate was filtered, washed with ethyl alcohol and diisopropyl ether, and then dried under reduced pressure. A crude product (10 g) containing the target product 1B was obtained as a solid. It was used in the next reaction without further purification. 1 H-NMR (DMSO-D6) δ: 7.60 (1H, d, J = 9.1 Hz), 7.40-7.17 (5H, m), 5.01 (2H, s), 4.62-4.55 (2H, m), 4.49 (1H, d, J = 4.2 Hz), 4.20 (1H, d, J = 8.5 Hz), 3.76-3.27 (6H, m), 3.08-2.99 (2H, m), 1.81 (3H, s), 1.66-1.56 (2H, m). Calcd for C 19 H 28 N2O8: [M+Na]+ 435, Found 435.

[0289] (1C) Synthesis of N-[(2R,3R,4R,5R,6R)-2-(3-aminopropoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide hydrochloride (Compound 1C)

[0290] [ka]

[0291] The compound (22.8 g, 55.3 mmol) synthesized in step (1B) was dissolved in tetrahydrofuran (280 ml) / 1N hydrochloric acid (61 ml). 10% palladium / carbon (wet) (5.56 g) was added, and the mixture was vigorously stirred at room temperature for 3.5 hours under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered, and the filtrate was distilled under reduced pressure. An appropriate amount of ethyl alcohol was added, and the resulting solid was filtered, washed with ethyl alcohol and diethyl ether, and then dried under reduced pressure. A crude product (17 g) containing the target product 1C was obtained. This was used in the next reaction without further purification. 1 H-NMR (D2O) δ: 4.27 (1H, d, J = 8.5 Hz), 3.90-3.50 (8H, m), 2.93 (2H, t, J = 7.0 Hz), 1.89 (3H, s), 1.82-1.75 (2H, m).

[0292] (1D) Synthesis of 9H-fluoren-9-ylmethyl N-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxypropyl]carbamate (Compound 1D)

[0293] [ka]

[0294] The compound (16.4 g, 52.1 mmol) synthesized in step (1C) was dissolved in tetrahydrofuran (200 ml) / pure water (50 ml), and N,N-diisopropylethylamine (12.7 mL, 72.9 mmol) was added. Under ice cooling, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide (21.1 g, 62.5 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. Diethyl ether (100 ml) was added, and the mixture was stirred for 1 hour. The solid matter was washed with ethyl alcohol, ethyl acetate, and diethyl ether in that order, and then dried under reduced pressure to obtain a crude product (22.9 g) containing the target product 1D. 1 H-NMR (DMSO-D6) δ: 7.89 (2H, d, J = 7.3 Hz), 7.69 (2H, d, J = 7.3 Hz), 7.61 (1H, d, J = 9.1 Hz), 7.42 (2H, t, J = 7.3 Hz), 7.33 (2H, t, J = 7.3 Hz), 7.24 (1H, t, J = 5.7 Hz), 4.60-4.57 (2H, m), 4.49 (1H, d, J = 4.2 Hz), 4.31-4.18 (4H, m), 3.76-3.28 (8H, m), 3.05-2.97 (2H, m), 1.81 (3H, s), 1.62-1.57 (2H, m). Calcd for C26H32N2O8: [M+Na]+ 523, Found 523.

[0295] (1E) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[3-(9H-fluoren-9-ylmethoxycarbonylamino)propoxy]tetrahydropyran-3-yl] acetate (compound 1E)

[0296] [ka]

[0297] 4,4'-Dimethoxytrityl chloride (1.27 g, 3.7 mmol) was added to a suspension of the compound (1.56 g, 3.1 mmol) synthesized in step (1D) in pyridine (20 ml) and stirred at room temperature for 30 minutes. Then, acetic anhydride (1.18 ml, 12.5 mmol) was added and stirred at room temperature for 20 hours. After the reaction was completed, N,N-diisopropylethylamine (1.1 mL, 6.2 mmol) and ethyl alcohol (2 ml) were added, and the solvent was distilled off under reduced pressure to obtain a crude product. This was purified by silica gel column chromatography (hexane:ethyl acetate=75:25-0:100, v / v, containing 1% triethylamine) to obtain the amorphous target product 1E (2.8 g, quant.). 1 H-NMR (CDCl3) δ: 7.77 (2H, d, J = 7.3 Hz), 7.62 (2H, d, J = 7.3 Hz), 7.43-7.18 (13H, m), 6.83-6.79 (4H, m), 6.03 (1H, d, J = 9.1 Hz), 5.55 (1H, d, J = 3.0 Hz), 5.14-5.03 (2H, m), 4.46-4.43 (3H, m), 4.24-4.04 (2H, m), 3.97-3.91 (1H, m), 3.85-3.82 (1H, m), 3.77 (6H, s), 3.72-3.71 (1H, m),3.54-3.33 (3H, m), 3.09-3.04 (2H, m), 2.03 (3H, s), 1.89 (6H, s), 1.65-1.56 (2H, m). Calcd for C 52 H 55 NO 12 : [M+Na]+ 908, Found 909.

[0298] (1F) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-4-acetoxy-6-(3-aminopropoxy)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl] acetate (Compound 1F)

[0299] [ka]

[0300] Piperidine (0.45 ml, 4.5 mmol) was added to a dichloromethane (8 ml) solution of the compound (2.49 g, 2.8 mmol) synthesized in step (1E), and the mixture was stirred at room temperature. If the reaction was not completed, piperidine was added as needed. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. This was purified by NH-silica gel column chromatography (dichloromethane:methanol = 100:0-85:15, v / v) to obtain the amorphous target product 1F (1.48 g, 79% yield). 1 H-NMR (CDCl3) δ: 7.39-7.18 (9H, m), 6.83-6.80 (4H, m), 5.55 (1H, d, J = 3.0 Hz), 5.51 (1H, d, J = 9.1 Hz), 5.27 (1H, dd, J = 11.2, 3.3 Hz), 4.64 (1H, d, J = 8.5 Hz), 3.98-3.90 (2H, m), 3.84-3.82 (1H, m), 3.79 (6H, s), 3.75-3.74 (1H, m), 3.58-3.56 (1H, m), 3.35 (1H, dd, J = 9.1, 5.4 Hz), 3.07 (1H, t, J = 8.5 Hz), 2.82-2.74 (4H, m), 2.01 (3H, s), 1.96 (3H, s), 1.89 (3H, s), 1.74-1.69 (2H, m). Calcd for C 37 H 45 NO 10 : [M+Na]+ 687, Found 687.

[0301] Example 2 GalNAc unit X 18 Synthesis of the corresponding amidite reagent (a compound having a hydrophobic protecting group-added GalNAc-phosphate group) (2A) Synthesis of benzyl 2-[[2-benzyloxy-3-[(2-benzyloxy-2-oxo-ethyl)carbamoyloxy]propoxy]carbonylamino]acetate (compound 2A)

[0302] [ka]

[0303] 2-benzyloxy-1,3-propanediol (2.5g, 14mmol) and 4-nitrophenyl chloroformate (5.8g, 29mmol) were dissolved in tetrahydrofuran (100ml), pyridine (5.0ml, 61.8mmol) was added, and the mixture was stirred at room temperature for 20 minutes. The precipitated solid was filtered, and then tetrahydrofuran (70ml) was added. Then, glycine benzyl ester TFA salt (9.2g, 33mmol) and N,N-diisopropylethylamine (9.6mL, 55mmol) were added, and the mixture was stirred at room temperature overnight. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. This was purified by silica gel column chromatography (hexane:ethyl acetate=100:0-40:60, v / v) to obtain the gum-like target product 2A (6.5g, yield 84%). 1 H-NMR (CDCl3) δ: 7.37-7.33 (15H, m), 5.48 (2H, t, J = 5.4 Hz), 5.18 (4H, s), 4.64 (2H, s), 4.23 (4H, d, J = 5.4 Hz), 3.99 (4H, d, J = 5.4 Hz), 3.84 (1H, t, J = 5.4 Hz). Calcd for C 30 H 32 N2O9: [M+H]+ 565, Found 565, [M+Na]+ 587, Found 587

[0304] (2B) Synthesis of 2-[[3-(carboxymethylcarbamoyloxy)-2-hydroxy-propoxy]carbonylamino]acetic acid (compound 2B)

[0305] [ka]

[0306] Compound 2A (7.3 g, 2.8 mmol) synthesized in step (2A) was dissolved in tetrahydrofuran (200 ml) / pure water (50 ml). 20% palladium hydroxide-activated carbon (about 50% water content) (3 g) was added and vigorously stirred at room temperature for 5 hours under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the effluent was distilled under reduced pressure to obtain a crude product (3.2 g) containing the target compound 2B. This was used in the next reaction without further purification. 1 H-NMR (D2O) δ: 4.21-4.14 (5H, m), 3.86 (4H, s).

[0307] (2C) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-6-[3-[[2-[[3-[[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-l-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-2-oxo-ethyl]carbamoyloxy]-2-hydroxy-propoxy]carbonylamino]acetyl]amino]propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl] acetate (compound 2C)

[0308] [ka]

[0309] Compound 2B (0.31g, 1.05mmol) synthesized in step (2B), compound 1F (1.47g, 2.21mmol) synthesized in step (1E), and N,N-diisopropylethylamine (0.91mL, 5.27mmol) were added to a solution of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (11.3g, 29.6mmol) in N,N-dimethylformamide (9ml), and stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was added dropwise to diethyl ether (total 185ml). The jelly-like crude product obtained after removing the supernatant solvent was purified by silica gel column chromatography (dichloromethane:methanol=100:0-15:85, v / v, containing 1% triethylamine) to obtain the target product 2C (1.05 g, yield 63%) as a solid. 1 H-NMR (CDCl3) δ: 7.39-7.17 (18H, m), 6.83-6.80 (8H, m), 6.70-6.27 (2H, m), 5.56-5.54 (2H, m), 5.10 (2H, d, J = 10.9 Hz), 4.49-3.02 (44H, m), 2.01 (6H, s), 1.96 (6H, s), 1.90 (6H, s), 1.80-1.63 (4H, m). Calcd for C 81 H 98 NO 27 : [M+Na]+ 1611, Found 1611

[0310] (2D) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-6-[3-[[2-[[3-[[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-2-oxo-ethyl]carbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxy-propoxy]carbonylamino]acetyl]amino]propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl] acetate (compound 2D)

[0311] [ka]

[0312] Compound 2C (0.47 g, 0.30 mmol) synthesized in step (2C) was azeotroped with an appropriate amount of ethyl acetate / toluene (1 / 1) and then dissolved in dichloromethane (3 ml). N,N-diisopropylethylamine (210 μl, 1.18 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (79 μl, 0.36 mmol) were added and stirred at room temperature for 1 hour. Furthermore, 2-cyanoethyldiisopropylchlorophosphoramidite (20 μl, 0.09 mmol) was added and stirred at room temperature for 15 minutes. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. This was purified by silica gel column chromatography (ethyl acetate:methanol=100:0-85:15, v / v, containing 1% triethylamine) to obtain the amorphous target product 2D (0.34 g, yield 64%). 1H-NMR (CDCl3) δ: 7.39-7.17 (18H, m), 6.87-6.70 (8H, m), 6.29-6.06 (2H, m), 5.55 (2H, d, J = 3.0 Hz), 5.11 (2H, d, J = 10.9 Hz), 4.47-3.02 (47H, m), 2.63 (2H, t, J = 6.3 Hz), 2.02 (6H, s), 1.96 (6H, s), 1.90 (6H, s), 1.80-1.64 (4H, m), 1.17 (12H, d, J = 6.7 Hz).

[0313] Example 3 GalNAc unit X 19 Synthesis of the corresponding amidite reagent (a compound having a hydrophobic protecting group-added GalNAc-phosphate group) (3A) Synthesis of benzyl 2-[[3-benzyloxy-2-[(2-benzyloxy-2-oxo-ethyl)carbamoyloxy]propoxy]carbonylamino]acetate (Compound 3A)

[0314] [ka]

[0315] The target compound 3A (2.8 g, 90% yield) was obtained in the same manner as in step (2A) except that (+ / -)-3-benzyloxy-1,2-propanediol (1.0 g, 5.5 mmol) was used instead of 2-benzyloxy-1,3-propanediol. 1 H-NMR (CDCl3) δ: 7.39-7.30 (15H, m), 5.37-5.35 (2H, m), 5.17-5.12 (5H, m), 4.56-4.51 (2H, m), 4.33-4.30 (2H, m), 4.03-3.94 (4H, m), 3.61 (2H, d, J = 5.4 Hz). Calcd for C 30 H 32 N2O9: [M+Na]+ 587, Found 587

[0316] (3B) Synthesis of 2-[[2-(carboxymethylcarbamoyloxy)-3-hydroxy-propoxy]carbonylamino]acetic acid (compound 3B)

[0317] [ka]

[0318] Compound 3A (2.8 g, 5.0 mmol) was used instead of compound 2A, and the procedure was carried out in the same manner as in step (2B) to give a crude product (1.2 g) containing the target compound 3B. 1 H-NMR (D2O) δ: 4.32-4.19 (3H, m), 3.94-3.87 (4H, m), 3.81-3.71 (2H, m).

[0319] (3C) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-6-[3-[[2-[[2-[[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-2-oxo-ethyl]carbamoyloxy]-3-hydroxy-propoxy]carbonylamino]acetyl]amino]propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-l-methoxy]methyl]tetrahydropyran-3-yl] acetate (compound 3C)

[0320] [ka]

[0321] Compound 3B (0.31 g, 1.05 mmol) was used instead of compound 2B, and the procedure was carried out in the same manner as in step (2C) to obtain the target compound 3C (0.74 g, yield 44%). 1H-NMR (CDCl3) δ: 7.40-7.18 (18H, m), 6.81 (8H, dd, J = 9.1, 3.0 Hz), 6.70-6.50 (1H, m), 6.16-6.02 (1H, m), 5.56-5.54 (2H, br), 5.11-5.00 (2H, m), 4.53-2.99 (44H, m), 2.01 (6H, s), 1.95 (6H, s), 1.88 (6H, s), 1.77-1.68 (4H, m). Calcd for C 81 H 98 NO 27 : [M+Na]+ 1611, Found 1611

[0322] Synthesis of (3D) [(2R,3R,4R,5R,6R)-5-acetamido-6-[3-[[2-[[2-[[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-2-oxo-ethyl]carbamoyloxy]-3-[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxy-propoxy]carbonylamino]acetyl]amino]propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl] acetate (compound 3D)

[0323] [ka]

[0324] Compound 3C (0.31 g, 1.05 mmol) was used instead of compound 2C, and the procedure was carried out in the same manner as in step (2D) to obtain the target compound 3D (0.60 g, yield 72%). 1H-NMR (CDCl3) δ: 7.38-7.18 (18H, m), 7.13-6.97 (1H, m), 6.83-6.81 (8H, m), 6.58-6.36 (1H, m), 5.56-5.52 (2H, m), 5.10-5.04 (2H, m), 4.46-2.97 (47H, m), 2.65-2.63 (2H, m), 2.02 (6H, s), 1.95 (3H, s), 1.94 (3H, s), 1.90 (3H, s), 1.89 (3H, s), 1.77-1.67 (4H, m), 1.20-1.16 (12H, m).

[0325] Example 4 GalNAc unit X 20 Synthesis of the corresponding amidite reagent (a compound having a hydrophobic protecting group-added GalNAc-phosphate group) (4A) Synthesis of 3-[[2-benzyloxy-3-[(3-benzyloxy-3-oxopropyl)carbamoyloxy]propoxy]carbonylamino]propanoic acid benzyl ester (compound 4A)

[0326] [ka]

[0327] The target compound 4A (1.62 g, 80% yield) was obtained in the same manner as in step (2A) except that 3-aminopropanoic acid benzyl ester TFA salt (2.4 g, 8.17 mmol) was used instead of the glycine benzyl ester TFA salt used in step (2A). 1 H-NMR (CDCl3) δ: 7.39-7.27 (15H, m), 5.21 (2H, br s), 5.14 (4H, s), 4.63 (2H, s), 4.24-4.10 (4H, m), 3.79-3.74 (1H, m), 3.48-3.44 (4H, m), 2.59 (4H, t, J = 6.0 Hz).Calcd for C 32 H 36N2O9: [M+H]+ 593, Found 593, [M+Na]+ 615, Found 615

[0328] (4B) Synthesis of 3-[[3-(2-carboxyethylcarbamoyloxy)-2-hydroxy-propoxy]carbonylamino]propanoic acid (compound 4B)

[0329] [ka]

[0330] Compound 4A (1.62 g, 2.73 mmol) was used instead of compound 2A used in step (2B), and the same procedure as in step (2B) was followed to obtain the target compound 4B (0.89 g, quant). 1 H-NMR (D2O) δ: 4.22-4.04 (5H, m), 3.46-3.32 (4H, m), 2.57 (4H, t, J = 6.7 Hz).

[0331] (4C) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-6-[3-[3-[[3-[[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-3-oxo-propyl]carbamoyloxy]-2-hydroxy-propoxy]carbonylamino]propanoylamino]propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (compound 4C)

[0332] [ka]

[0333] Compound 4B (300 mg, 0.93 mmol) was used instead of compound 2B used in step (2C) and the procedure was repeated in the same manner as in step (2C) to obtain the target compound 4C (950 mg, yield 63%). 1 H-NMR (CDCl3) δ: 7.32-7.24 (18H, m), 6.82-6.80 (8H, m), 6.64-6.61 (1H, br), 6.27-6.24 (1H, br), 5.91-5.88 (1H, br), 5.56 (2H, d, J = 3.0 Hz), 5.10 (2H, d, J = 11.5 Hz), 4.46-3.05 (43H, m), 2.47-2.37 (4H, m), 2.02 (6H, s), 1.98 (6H, s), 1.90 (6H, s), 1.78-1.67 (4H, m). Calcd for C 83 H 102 NO 27 : [M+Na]+ 1638, Found 1638

[0334] (4D) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-6-[3-[3-[[3-[[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-3-oxo-propyl]carbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino) phosphanyl]oxy-propoxy]carbonylamino]propanoylamino]propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl] acetate (compound 4D)

[0335] [ka]

[0336] Compound 4C (945 mg, 0.58 mmol) was used instead of compound 2C used in step (2D), and the same procedure as in step (2D) was repeated to obtain the target compound 4D (638 mg, yield 60%). 1 H-NMR (CDCl3) δ: 7.39-7.18 (18H, m), 6.85-6.78 (8H, m), 6.73-6.64 (1H, m), 6.40-6.23 (1H, m), 5.83-5.72 (1H, m), 5.56 (2H, d, J = 3.0 Hz), 5.13-5.04 (2H, m), 4.45-4.35 (2H, m), 4.22-3.30 (40H, m), 3.22-3.03 (4H, m), 2.64 (2H, t, J = 6.0 Hz), 2.54-2.32 (4H, m), 2.02 (6H, s), 2.00-1.95 (6H, m), 1.90 (6H, s), 1.83-1.59 (4H, m), 1.20-1.10 (12H, m).

[0337] Example 5 GalNAc unit X 21 Synthesis of the corresponding amidite reagent (a compound having a hydrophobic protecting group-added GalNAc-phosphate group) (5A) Synthesis of 4-[[2-benzyloxy-3-[(4-benzyloxy-4-oxo-butyl)carbamoyloxy]propoxy]carbonylamino]butanoic acid benzyl ester (compound 5A)

[0338] [ka]

[0339] The target compound 5A (0.85 g, 78% yield) was obtained in the same manner as in step (2A) except that 4-aminobutanoic acid benzyl ester TFA salt (1.30 g, 4.21 mmol) was used instead of the glycine benzyl ester TFA salt used in step (2A). 1H-NMR (CDCl3) δ: 7.38-7.27 (15H, m), 5.11 (4H, s), 4.80 (2H, br s), 4.63 (2H, s), 4.19-4.15 (4H, m), 3.77-3.75 (1H, m), 3.23-3.18 (4H, m), 2.40 (4H, t, J = 7.3 Hz), 1.88-1.81 (4H, m). Calcd for C 34 H 40 N2O9: [M+H]+ 621, Found 622, [M+Na]+ 643, Found 644

[0340] (5B) Synthesis of 4-[[3-(3-carboxypropylcarbamoyloxy)-2-hydroxy-propoxy]carbonylamino]butanoic acid (compound 5B)

[0341] [ka]

[0342] Compound 5A (0.85 g, 1.4 mmol) was used instead of compound 2A used in step (2B), and the same procedure as in step (2B) was followed to obtain the target compound 5B (0.49 g, quant). 1 H-NMR (D2O) δ: 4.19-4.05 (5H, m), 3.19-3.12 (4H, m), 2.36-2.27 (4H, m), 1.83-1.73 (4H, m).

[0343] (5C) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-6-[3-[4-[[3-[[4-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl] tetrahydropyran-2-yl]oxypropylamino]-4-oxo-butyl] carbamoyloxy]-2-hydroxy-propoxy]carbonylamino]butanoylamino] propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (compound 5C)

[0344] [ka]

[0345] Compound 5B (300 mg, 0.86 mmol) was used instead of compound 2B used in step (2C) and the procedure was repeated in the same manner as in step (2C) to obtain the target compound 5C (1.16 g, yield 82%). 1 H-NMR (CDCl3) δ: 7.31-7.24 (18H, m), 6.82-6.80 (8H, m), 6.68-6.65 (3H, m), 5.91-5.84 (1H, m), 5.56 (2H, d, J = 3.0 Hz), 5.10 (2H, d, J = 10.9 Hz), 4.45 (2H, d, J = 8.5 Hz), 4.23-3.12 (40H, m), 2.31-2.22 (4H, m), 2.01 (6H, s), 1.98 (6H, s), 1.90 (6H, s), 1.81-1.68 (8H, m). Calcd for C 85 H 106 NO 27 : [M+Na]+ 1666, Found 1667

[0346] (5D) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-6-[3-[3-[[3-[[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-4-oxo-butyl]carbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino) phosphanyl]oxy-propoxy]carbonylamino]butanoylamino]propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl] acetate (compound 5D)

[0347] [ka]

[0348] Compound 5C (1.16 g, 0.71 mmol) was used instead of compound 2C used in step (2D), and the procedure was carried out in the same manner as in step (2D) to obtain the target compound 5D (795 mg, yield 61%). 1 H-NMR (CDCl3) δ: 7.40-7.17 (18H, m), 6.85-6.77 (8H, m), 6.72-5.65 (5H, m), 5.56 (2H,d, J = 3.0 Hz), 5.11-5.03 (2H, m), 4.46-4.35 (2H, m), 4.25-3.02 (42H, m), 2.65 (2H, t, J = 6.0 Hz), 2.39-2.16 (4H, m), 2.02 (6H, s), 1.99-1.94 (6H, m), 1.90 (6H, s), 1.87-1.59 (8H, m), 1.18 (12H, d, J = 6.7 Hz).

[0349] Example 6 GalNAc unit X 22 Synthesis of the corresponding amidite reagent (6A) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[5-(benzyloxycarbonylamino)pentoxy]tetrahydropyran-2-yl]methyl acetate (Compound 6A)

[0350] [ka]

[0351] A suspension of N-benzyloxycarbonyl-1-hydroxypentyl-5-amine (J. Am. Chem. Soc., 2006, 128, 4058-4073.) (8.05 g, 33.9 mmol) and acetic acid [(2R, 3R, 4R, 5R)-5-acetamido-3,4,6-triacetoxy-tetrahydropyran-2-yl] methyl (WO2011053614) (12.0 g, 30.8 mmol) in dichloromethane (200 mL) was added with trifluoromethanesulfonic acid (450 μL, 5.23 mmol) and stirred overnight at 45 ° C. After the reaction was completed, about half of the solvent was distilled off under reduced pressure to concentrate, and the mixture was added to a mixed solution of ethyl acetate / saturated aqueous sodium bicarbonate solution. The organic layer was washed with saturated aqueous sodium bicarbonate and saturated saline / phosphate buffer (pH 7.0), then dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (hexane:ethyl acetate=100:0-0:100, v / v) to obtain the amorphous target product 6A (17.0 g, yield 94%). 1H-NMR (CDCl3) δ: 7.37-7.32 (5H, m), 5.69 (1H, d, J = 9.1 Hz), 5.35 (1H, d, J = 2.4 Hz), 5.29 (1H, d, J = 11.5 Hz), 5.10 (2H, s), 4.94 (1H, br), 4.68 (1H, d, J = 7.9 Hz), 4.20-4.09 (2H, m), 3.97-3.86 (3H, m), 3.49-3.44 (1H, m), 3.20-3.17 (2H, m), 2.14 (3H, s), 2.05 (3H, s), 2.01 (3H, s), 1.93 (3H, s), 1.66-1.34 (6H, m). Calcd for C 27 H 38 N2O 11 : [M+H] + 567, Found 567.

[0352] (6B) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-(5-aminopentoxy)tetrahydropyran-2-yl]methyl acetate hydrochloride (compound 6B)

[0353] [ka]

[0354] Compound 6A (3.87 g, 6.83 mmol) synthesized in step (6A) was dissolved in tetrahydropyran (32 ml) / 1N hydrochloric acid (8 ml). 10% palladium / carbon (wet) (2 g) was added and vigorously stirred at room temperature under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the effluent was distilled under reduced pressure to obtain the crude product of the target compound 6B (3.1 g, 97% yield). This was used in the next reaction without further purification. Calcd for C 19 H 32 N2O9: [M+H] + 433, Found 433.

[0355] (6C) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-6-[5-[[3-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentylcarbamoyloxy]-2-benzyloxy-propoxy]carbamoylamino]pentoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (compound 6C)

[0356] [ka]

[0357] 2-benzyloxy-1,3-propanediol (600mg, 3.29mmol) and 4-nitrophenyl chloroformate (1.39g, 6.91mmol) were dissolved in tetrahydrofuran (22ml), pyridine (1.1ml, 13.2mmol) was added, and the mixture was stirred at room temperature for 15 minutes. The precipitated solid was filtered, and then tetrahydrofuran (16ml) was added. Next, a solution of compound (6B) (3.09g, 6.59mmol) synthesized in step (6B) in tetrahydrofuran (15ml) / dichloromethane (5ml) and N,N-diisopropylethylamine (3.4mL, 19.8mmol) were added, and the mixture was stirred at 40°C for 2 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. Dichloromethane was added, and the organic layer was washed with 0.5N hydrochloric acid, saturated saline, 0.2N aqueous sodium hydroxide solution, and saturated saline, then dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (dichloromethane:methanol=100:0-90:10, v / v) to obtain a mixture (2.1 g) mainly containing the target compound 6C. Calcd for C 50 H 74 N4O 23 : [M+H] + 1099, Found 1099. [M+Na] + 1121, Found 1121.

[0358] (6D) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-6-[5-[[3-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentylcarbamoyloxy]-2-hydroxy-propoxy]carbonylamino]pentoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (compound 6D)

[0359] [ka]

[0360] The mixture (2.1 g) containing 6C obtained in step (6C) was dissolved in tetrahydropyran (20 ml) / pure water (1 ml). 1N hydrochloric acid (0.38 ml) and 10% palladium / carbon (wet) (1 g) were added and vigorously stirred at room temperature under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the effluent was distilled under reduced pressure to obtain a crude product. This was purified by silica gel column chromatography (ethyl acetate:methanol = 100:0-90:15, v / v) to obtain the target product 6D (1.38 g, yield 72%) as a solid. 1 H-NMR (CDCl3) δ: 6.10-5.96 (1H, m), 5.38-4.94 (6H, m), 4.70-4.64 (2H, m), 4.23-3.44 (19H, m), 3.20-3.15 (4H, m), 2.15 (6H, s), 2.05 (6H, s), 2.01 (6H, s), 1.97 (6H, s), 1.67-1.38 (12H, m). Calcd for C 43 H 68 N4O 23 : [M+H] + 1009, Found 1009. [M+Na] + 1031, Found 1031.

[0361] (6E) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-6-[5-[[3-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentylcarbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxy-propoxy]carbamoylamino]pentoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (compound 6E)

[0362] [ka]

[0363] Compound 6D (1.38 g, 1.37 mmol) was used instead of compound 2C used in step (2D), and the procedure was carried out in the same manner as in step (2D) to obtain target compound 6E (850 mg, yield 51%). 1 H-NMR (CDCl3) δ: 5.95-5.89 (1H, m), 5.38-5.26 (4H, m), 5.20-5.16 (1H, m), 5.00-4.95 (1H, m), 4.71-4.68 (2H, m), 4.36-3.44 (22H, m), 3.15 (4H, q, J = 6.4 Hz), 2.67 (2H, t, J = 6.3 Hz), 2.15 (6H, s), 2.05 (6H, s), 2.01 (6H, s), 1.96 (6H, s), 1.67-1.33 (12H, m), 1.18 (12H, d, J = 6.7 Hz).

[0364] Example 7 GalNAc unit X 20 Synthesis of the corresponding amidite reagent (7A) Synthesis of [(2R,3R,4R,5R)-5-acetamido-3,4-diacetoxy-6-(3-aminopropoxy)tetrahydropyran-2-yl]methyl acetate hydrochloride (compound 7A)

[0365] [ka]

[0366] Compound 1A (10 g, 18.6 mmol) synthesized in step (1A) was dissolved in tetrahydrofuran (200 ml) / 1N hydrochloric acid (20 ml). 10% palladium / carbon (wet) (1 g) was added and vigorously stirred at room temperature for 1 hour under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the effluent was distilled under reduced pressure to obtain a crude product (8.97 g) containing the target compound 7A. This was used in the next reaction without further purification. Calcd for C 17 H 28 N2O9: [M+H]+ 405, Found 405.

[0367] (7B) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-6-[3-[3-[[3-[[3-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropylamino]-3-oxo-propyl]carbamoyloxy]-2-hydroxy-propoxy]carbonylamino]propanoylamino]propoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (compound 7B)

[0368] [ka]

[0369] To a solution of compound 4B (200 mg, 0.62 mmol) synthesized in step (4B) in N,N-dimethylformamide (2 ml), O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (411 mg, 1.37 mmol) and N,N-diisopropylethylamine (0.32 ml, 1.86 mmol) were added and stirred at room temperature for 20 minutes. This reaction solution was added to a solution of compound 37B (657 mg, 1.49 mmol) synthesized in step (37B) in N,N-dimethylformamide (2 ml), and then N,N-diisopropylethylamine (0.32 ml, 1.86 mmol) was added and stirred at room temperature for 60 minutes. After the reaction was completed, the reaction solution was added dropwise to diethyl ether (total 80 ml). The jelly-like crude product obtained after removing the supernatant solvent was purified by silica gel column chromatography (dichloromethane:methanol=100:0-75:25, v / v) to obtain the amorphous target product 7B (408 mg, yield 60%). 1 H-NMR (CDCl3) δ: 6.76 (2H, br), 6.69-6.63 (2H, m), 5.96 (1H, br), 5.36 (2H, d, J = 3.0 Hz), 5.13-5.08 (2H, m), 4.53 (2H, d, J= 8.5 Hz), 4.25-3.07 (27H, m), 2.51-2.44 (4H, m), 2.17 (6H, s), 2.06 (6H, s), 2.02 (6H, s), 2.00 (6H, s), 1.86-1.73 (4H, m). Calcd for C 45 H 70 NO 25 : [M+H] + 1095, Found 1096. [M+Na] + 1117, Found 1117.

[0370] (7C) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-6-[3-[3-[[3-[[3-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropylamino]-3-oxo-propyl]carbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxy-propoxy]carbonylamino]propanoylamino]propoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (compound 7C)

[0371] [ka]

[0372] Compound 7B (1.53 g, 1.40 mmol) was used instead of compound 2C used in step (2D), and the same procedure as in step (2D) was repeated to obtain the target compound 7C (510 mg, yield 28%). 1 H-NMR (CDCl3) δ: 6.60-6.44 (4H, m), 5.76-5.68 (1H, m), 5.36 (2H, d, J = 3.0 Hz), 5.09-5.05 (2H, m), 4.50-4.45 (2H, m), 4.26-3.37 (28H, m), 3.17 (2H, br), 2.67 (2H, t, J = 6.3 Hz), 2.52-2.42 (4H, m), 2.17 (6H, s), 2.05 (6H, s), 2.02 (6H, s), 1.99 (6H, s), 1.81-1.70 (4H, m), 1.18 (12H, d, J = 6.7 Hz).

[0373] Example 8 X 18 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -Gm1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (SEQ ID NO: 40) The oligonucleotide sequence 15e_001 was synthesized by the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984)) using an ABI 392 DNA / RNA Synthesizer (Applied Biosystems) as an automatic nucleic acid synthesizer and NittoPhase UnyLinker 100, 10 μmol (Nitto Denko) as a solid phase support, and then condensed once using 2D synthesized in Example 2 as a GlcNAc unit amidite. The protected oligonucleotide analog was treated with 6 mL of concentrated aqueous ammonia to cleave the oligomer from the support and remove the cyanoethyl protecting group on the phosphorus atom and the protecting group on the nucleic acid base. The product was purified using a Clarity QSP (Phenomenex) according to the attached protocol. The base sequence of this compound is complementary to the 92nd to 106th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a mutation from G to T at nucleotide number 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6247.96).

[0374] Example 9 X 18 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -Gm1s -A m1s -A e2s -G m1t -H (SEQ ID NO: 40) Synthesis was carried out in the same manner as in Example 8, except that the above sequence 15e_001.5 was used in place of the sequence used in Example 8. The base sequence of this compound is complementary to the 92nd to 106th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a mutation from G to T at nucleotide number 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6233.97).

[0375] Example 10 X 18 -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (SEQ ID NO: 44) Synthesis was carried out in the same manner as in Example 8, except that the above sequence 15e_005.5 was used in place of the sequence used in Example 8. The base sequence of this compound is complementary to the 91st to 105th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a G to T mutation at nucleotide 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6235.97).

[0376] Example 11 X 18 -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2s -U m1t -H (SEQ ID NO: 45) Synthesis was carried out in the same manner as in Example 8, except that the above sequence 15e_006.5 was used in place of the sequence used in Example 8. The base sequence of this compound is complementary to the 90th to 104th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a G to T mutation at nucleotide 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6200.94).

[0377] Example 12 X 18 -A m1s -A m1s -Ae2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (SEQ ID NO: 42) Synthesis was carried out in the same manner as in Example 8, except that the above sequence 16e_001.5 was used in place of the sequence used in Example 8. The base sequence of this compound is complementary to the 92nd to 107th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a G to T mutation at nucleotide 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion electrospray ionization mass spectrometry. (Actual value: 6593.02)

[0378] (Example 13) X 18 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (SEQ ID NO: 47) Synthesis was carried out in the same manner as in Example 8, except that the above sequence 16e_002.5 was used in place of the sequence used in Example 8. The base sequence of this compound is complementary to the 91st to 106th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a G to T mutation at nucleotide 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6595.01).

[0379] Example 14 X 18 -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2s -U m1t -H (SEQ ID NO: 48) Synthesis was carried out in the same manner as in Example 8, except that the above sequence 16e_003.5 was used in place of the sequence used in Example 8. The base sequence of this compound is complementary to the 90th to 105th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a G to T mutation at nucleotide 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6571.98).

[0380] Example 15 X 20 -A m1s -Ae2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (SEQ ID NO: 40) By using compound 4D prepared in Example 4 instead of compound 2D as the GalNAc unit amidite, X 18 Change the part to X 20 The synthesis was carried out in the same manner as in Example 8, with the substitution of 15e_001.5. The sequence used was the above sequence 15e_001.5. The base sequence of this compound is complementary to the 92nd to 106th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a mutation from G to T at nucleotide number 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6261.98).

[0381] (Example 16) X 20 -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (SEQ ID NO: 44) The synthesis was carried out in the same manner as in Example 15, except that the sequence was 15e_005.5. The base sequence of this compound is complementary to the 91st to 105th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a mutation from G to T at nucleotide 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6264.00).

[0382] (Example 17) X 20 -A m1s -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (SEQ ID NO: 42) The synthesis was carried out in the same manner as in Example 15, except that the sequence was 16e_001.5. The base sequence of this compound is complementary to the 92nd to 107th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a mutation from G to T at nucleotide number 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6621.04).

[0383] (Example 18) X 20 -A m1s -A e2s -Um1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (SEQ ID NO: 47) The sequence was changed to the above sequence 16e_002.5, and synthesis was carried out in the same manner as in Example 15. The base sequence of this compound is complementary to the 91st to 106th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a mutation from G to T at nucleotide number 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6623.05).

[0384] (Example 19) X 21 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (SEQ ID NO: 40) By using the compound 5D synthesized in Example 5 as the GalNAc unit amidite, X 18 Change the part to X 21 The synthesis was carried out in the same manner as in Example 8, with the substitution of 15e_001.5. The sequence used was the above sequence 15e_001.5. The base sequence of this compound is complementary to the 92nd to 106th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a mutation from G to T at nucleotide number 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6290.02).

[0385] (Example 20) X 22 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (SEQ ID NO: 40) By using compound 6D synthesized in Example 6 as the GalNAc unit amidite, X 18 Change the part to X 22 The synthesis was carried out in the same manner as in Example 8, with the substitution of 15e_001.5. The sequence used was the above sequence 15e_001.5. The base sequence of this compound is complementary to the 92nd to 106th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a G to T mutation at nucleotide 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6175.96).

[0386] Example 21 X 22 -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (SEQ ID NO: 44) The synthesis was carried out in the same manner as in Example 20, except that the sequence was 15e_005.5. The base sequence of this compound is complementary to the 91st to 105th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a mutation from G to T at nucleotide 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6177.98).

[0387] Example 22 X 22 -A m1s -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (SEQ ID NO: 42) The synthesis was carried out in the same manner as in Example 20, except that the sequence was 16e_001.5. The base sequence of this compound is complementary to the 92nd to 107th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a mutation from G to T at nucleotide number 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6535.03).

[0388] Example 23 X 22 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (SEQ ID NO: 47) The synthesis was carried out similarly to Example 20, substituting the sequence shown above for sequence 16e_002.5. The base sequence of this compound is complementary to the 91st to 106th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a mutation from G to T at nucleotide 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6537.05).

[0389] (Example 24) X 20 -G e2s -C e2s -As -T s -T s -G s -G s -T s -A s -T s -T e2s -C e2s -A e2t -H (SEQ ID NO: 96) Instead of the sequence used in Example 15, the above sequence complementary to the nucleic acid sequence 10177-10189 of human apolipoprotein B (APOB) GenBank accession No. NM_000384.2 was used to synthesize a conjugate of a GalNAc unit and an oligonucleotide (ApoB-ASO conjugate) in the same manner as in Example 15. In addition, in the same method, synthesis was terminated at the stage where an oligonucleotide of the above sequence was synthesized, and an oligonucleotide (ApoB-ASO) to which a GalNAc unit was not bound was synthesized by excision and recovery. The compound was identified by negative ion ESI mass spectrometry (measured value: 5296.83 (ApoB-ASO conjugate), 4392.50 (ApoB-ASO)).

[0390] (Test Example 1) Evaluation of the repair of abnormal splicing by example compounds using cultured cells (1-1) Cultivation of 293A cells (human fetal kidney cells) 293A cells (R705-07 Invitrogen) were cultured as follows. 293A cells were maintained and cultured in maintenance medium (DMEM, 10% FBS). In the experiment, 2x10 5 cells / well 1x10 in 12 well plate 5 The cells were seeded at a density of 100 cells / well, and the next day, a compound of the example and a plasmid vector were simultaneously introduced as described below, and the cells were used for evaluation.

[0391] (1-2) Construction of full-length human G6PC plasmid vector Human G6PC full-length plasmid vectors (pcDNA hG6PC, pcDNA hG6PC (c.648G>T) + Int4) were prepared as follows. Using the Human Multiple Tissue cDNA Panel as a template, G6PC cDNA was amplified using the G6PC cDNA Amplified primer described below, and then further amplified using the G6PC cDNA IF primer. The amplified fragment was inserted into the BamHI site of pcDNA3.1 using the InFusion System (pcDNA hG6PC).

[0392] G6PC cDNA Amplified primer: Forward primer 5'-ATAGCAGAGCAATCACCACCAAGCC-3' (SEQ ID NO: 49) Reverse primer 5'-ATTCCACGACGGCAGAATGGATGGC-3' (SEQ ID NO:50) G6PC IF primer: Forward primer 5'-TACCGAGCTCGGATCCACCACCAAGCCTGGAATAACTGC-3' (SEQ ID NO:51), Reverse primer 5'-CTGGACTAGTGGATCCTGGCATGGTTGTTGACTTTAAAC-3' (SEQ ID NO: 52)

[0393] Using human genome DNA as a template, a region including G6PC Intron4 and a part of Exon5 was amplified using the G6PC Int4 Amplified primer described below, and G6PC Intron4 was further amplified using the G6PC Int4 IF primer. In addition, pcDNA hG6PC created in STEP1-1 was amplified using the hG6PC vector IF primer described below. The c.648G>T mutation in Exon5 was introduced into the InFusion primer. Both fragments were ligated using the InFusion System, and the G6PC Intron4 sequence was inserted into pcDNA hG6PC (pcDNA hG6PC (c.648G>T) + Int4).

[0394] G6PC Int4 Amplified primer: Forward primer 5'-TCTGGGCTGTGCAGCTGAATGTCTG-3' (SEQ ID NO:53) Reverse primer 5'-GTAGGGGATGACACTGACGGATGCC-3' (SEQ ID NO:54)

[0395] G6PC Int4 IF primer: Forward primer 5'-CTGGAGTCCTGTCAGGTATGGGC-3' (SEQ ID NO:55) Reverse primer 5'-AGCTGAAAAGGAAGAAGGTAATGAG-3' (SEQ ID NO:56)

[0396] hG6PC vector IF primer: Forward primer 5'-TCTTCCTTTTCAGCTTCGCCATCGG-3' (SEQ ID NO:57) Reverse primer 5'-CTGACAGGACTCCAGCAACAAC-3' (SEQ ID NO:58)

[0397] (1-3) Cotransfection of the compound of the embodiment and a plasmid vector The compound of the example and the plasmid vector were cotransfected as follows. The following solutions A and B were prepared and then mixed. When performing the experiment in a 6-well plate, 250 μL Opti-MEM Medium (gibco), 0.50 μL plasmid vector (1 mg / mL), and 4.0 μL (final: 20 nM) of the compound produced in the examples (12.5 μM) were prepared per well as solution A, and 250 μL Opti-MEM Medium (gibco) and 6.0 μL Lipofectamine2000 (Invitrogen) were prepared as solution B, and solutions A and B were mixed. When performing the experiment in a 12-well plate, 125 μL Opti-MEM Medium (Gibco), 0.25 μL plasmid vector (1 mg / mL), and 2.0 μL (final: 20 nM) of the compound produced in the Example (12.5 μM) were prepared as solution A per well. 125 μL Opti-MEM Medium (Gibco) and 3.0 μL Lipofectamine 2000 (Invitrogen) were prepared as solution B, and solutions A and B were mixed. The above mixture was incubated at room temperature for 20 minutes and then added to the cells the day after subculture (cotransfection). Six hours after addition, the medium was replaced with fresh maintenance medium, and the mixture was incubated in a CO2 incubator for 24 hours.

[0398] (1-4) Evaluation of mRNA by RT-PCR [1] RNA extraction (in vitro) RNA extraction was carried out as follows. After 24 hours of incubation following cotransfection, the cells were washed once with cold PBS. 300 μL of cell lysate from the RNeasy mini kit or RNeasy 96 kit (Qiagen) was added per well and incubated at room temperature for 5 minutes before collection. RNA was purified from the collected solution according to the kit's protocol, including DNase treatment. RNase-Free DNase set (Qiagen 89254) was used for DNase treatment. The purified and eluted RNA was subjected to reverse transcription as described below.

[0399] [2] Reverse transcription reaction The reverse transcription reaction was carried out as follows. After adjusting the extracted RNA to 25-100 mg / mL, reverse transcription reaction was performed using a High Capacity RNA-to-cDNA kit (Applied biosystems) by mixing 10 μL of Buffer mix, 1 μL of Enzyme mix, and 9 μL of purified water + extracted RNA per sample (37°C 60 min, 95°C 5 min, 4°C hold). 20 μL of the reverse transcription reaction product was diluted 5-fold with 80 μL of purified water and stored at -30°C.

[0400] [3] qRT-PCR (SYBR Green) qRT PCR primers (SYBR Green) were designed as follows. Repaired hG6PC primer (SYBR): Forward primer 5'-TTGTGGTTGGGATTCTGGGC-3' (SEQ ID NO:59) Reverse primer 5'-ATGCTGTGGATGTGGCTGAA-3' (SEQ ID NO:60) hActin primer (SYBR): Forward primer 5'-TGGCACCCAGCACAATGAA-3' (SEQ ID NO:61) Reverse primer 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3' (SEQ ID NO:62) The PCR reaction mixture was prepared by suspending 5 μL of 2x FAST SYBR Green Master Mix (Applied Biosystems), 2 μL of purified water, 1 μL of primer mix (10 μM), and 2 μL of cDNA (diluted 5-fold) per well, and performing the PCR reaction using viia7 (Applied Biosystems) (program: SYBR Green Regents, FAST, include Melt curve).

[0401] [4] qRT-PCR (TaqMan assay) Repaired hG6PC primer set and Total hG6PC primer set were designed as follows, and 20x primer probe mix (primer concentration: 1000nM, probe concentration: 250nM) was prepared. hActin primer set and mActin primer set were used as undiluted solutions. Repaired hG6PC primer set (Taqman): Forward primer 5'-GCTGCTCATTTTCCTCATCAAGTT-3' (SEQ ID NO:63) Reverse primer 5'-TGGATGTGGCTGAAAGTTTCTGTA-3' (SEQ ID NO:64) Probe 5'-TCCTGTCAGGCATTGC-3' FAM (SEQ ID NO:65) hActin primer set(Taqman):ABI Hs01060665_g1 FAM mActin primer set(Taqman): ABI Mm02619580_g1 FAM 18s primer set(Taqman): ABI Hs99999901_s1 FAM In addition, 5 μL of 2x Taqman Fast Advanced Master Mix (Applied Biosystems), 2.5 μL of purified water, 0.5 μL of 20x Primer probe mix (10 μM), and 2 μL of cDNA (diluted 5 times) were suspended per well to prepare a PCR reaction solution (per tube), and PCR reactions were performed using Viia7 or Quantstadio7 (Applied Biosystems) (program: Taqman regents, FA

[0402] (1-5) Evaluation of repair of abnormal splicing of G6PC mRNA by the oligonucleotides of the present embodiment using the full-length human G6PC plasmid vector (pcDNA hG6PC (c.648G>T) + Int4) Human G6PC full-length plasmid vector (pcDNA hG6PC (c.648G>T) + Int4) and oligonucleotides (15e_001, 15e_002, and the compound in Example 93 of WO 2004 / 048570 as a control) were co-transfected, and the abnormal splicing caused by G6PC (c.648G>T) was evaluated by qRT-PCR (SYBR Green). As a result, the abnormal splicing of G6PC mRNA was normalized by compounds 15e_001 and 15e_002.

[0403] (Test Example 2) Evaluation of the repair of abnormal splicing by example compounds using model mice (2-1) Generation of mice Creating a Vector The G6PC KI vector was prepared according to the following procedure. The G6PC 5' arm region was amplified using the following mG6PC 5' arm amplified primer with mouse genome DNA as a template, and then amplified with the mG6PC 5' arm IF primer and inserted into the XhoI site of pBluescriptII(+ / -) (G6PC 5' arm vector). mG6PC 5' arm Amplified primer: Forward primer 5'-GGGAAACATGCATGAAGCCCTGGGC-3' (SEQ ID NO:67) Reverse primer 5'-TCCCTTGGTACCTCAGGAAGCTGCC-3' (SEQ ID NO:68) mG6PC 5' arm IF primer: Forward primer 5'-CGGGCCCCCCCTCGAAAACTAGGCCTGAAGAGATGGC-3' (SEQ ID NO: 69) Reverse primer 5'-TACCGTCGACCTCGAGGGTTGGCCTTGATCCCTCTGCTA-3' (SEQ ID NO: 70)

[0404] Next, the G6PC 3' arm region was amplified using the mouse genome DNA as a template with the mG6PC 3' arm amplified primer described below, and then amplified with the mG6PC 3' arm IF primer and inserted into the NotI site of the G6PC 5' arm vector (G6PC 5'+3' arm vector). mG6PC 3' arm Amplified primer: Forward primer 5'-GGTTGAGTTGATCTTCTACATCTTG-3' (SEQ ID NO:71) Reverse primer 5'-GCAAGAGAGCCTTCAGGTAGATCCC-3' (SEQ ID NO:72) mG6PC 3' arm IF primer: Forward primer 5'-AGTTCTAGAGCGGCCGCCCATGCAAAGGACTAGGAACAAC-3' (SEQ ID NO: 73) Reverse primer 5'-ACCGCGGTGGCGGCCAATGTTGCCTGTCTTCCTCAATC-3' (SEQ ID NO:74)

[0405] Using the above-mentioned pcDNA hG6PC (c.648G>T) + Int4 as a template, hG6PC (c.648G>T) + Intron4 was amplified using the hG6PC + Int4 IF primer below. In addition, using the G6PC 5'+3' arm vector as a template, it was amplified using the Arm vector IF primer below. Both fragments were ligated using the InFusion System to create the G6PC KI vector. hG6PC + Int4 IF primer: Forward primer 5'-GGCCAACCCTGGAATAACTGCAAGGGCTCTG-3' (SEQ ID NO: 75) Reverse primer 5'-TTGCATGGTTGTTGACTTTAAACACCGAAGA-3' (SEQ ID NO: 76) Arm vector IF Primer: Forward primer 5'-TCAACAACCATGCAAAGGACTAGGAACAAC-3' (SEQ ID NO: 77) Reverse primer 5'-ATTCCAGGGTTGGCCTTGATCCCTCTGCTA-3' (SEQ ID NO:78) The following KI 5' gRNA and KI 3' gRNA sequences were introduced into the gRNA sequence introduction region of pSPgRNA (addgene) to prepare pSPgRNA(KI 5') and pSPgRNA(KI 3'). KI 5' gRNA: 5'-GGGATCAAGGCCAACCGGCTGG-3' (SEQ ID NO: 79) KI 3' gRNA: 5'-TAAAGTCAACCGCCATGCAAAGG-3' (SEQ ID NO: 80)

[0406] (2-2) Microinjection The vectors were adjusted with sterile distilled water to a final concentration of 10ng / μL for G6PC KI vector, 5ng / μL for pSPgRNA(KI 5'), 5ng / μL for pSPgRNA(KI 3'), and 5ng / μL for pSPCas9(addgene), and then passed through a MILLEX-GV syringe filter (Millipore) and injected into C57BL / 6J mouse fertilized eggs until the pronuclei were sufficiently expanded (approximately 2pL). The fertilized eggs were transplanted into the oviducts of recipient C57BL / 6J mice to obtain F0 mice.

[0407] (2-3) Genotyping, F1 systematics Genotyping was carried out and F1 lineages were generated according to the following procedure. Genomic DNA was extracted from the tail tissue of F0 mice using an automatic nucleic acid extractor (PI-200 Kurabo) and a dedicated kit. The extracted DNA was amplified using Amplitaq Gold Master mix (Thermo Fisher) and the following KI screening primers (95°C 10 min, (95°C 30 sec, 60°C 30 sec, 72°C 30 sec) 35 cycles, 72°C 2 min, 4°C hold). KI screening primer: Forward primer 5'-TACGTCCTCTTCCCCATCTG-3' (SEQ ID NO:81), Reverse primer 5'-CTGACAGGACTCCAGCAACA-3' (SEQ ID NO:82) The PCR products were subjected to gel electrophoresis, and the genomic DNA of the individual that showed a band around 433 bp was used as a template and amplified using PrimeSTAR GXL (Takara) and the following KI genotyping primers (98°C 2 min, (95°C 15 sec, 68°C 5 min) 38 cycles, 68°C 7 min, 15°C hold). KI genotyping primer (5'): Forward primer 5'-TTCCTTCCAAAGCAGGGACTCTCTATGT-3' (same as SEQ ID NO: 83 (1)) Reverse primer 5'-CTTGCAGAAGGACAAGACGTAGAAGACC-3' (same as SEQ ID NO: 84 (2)) KI genotyping primer (3'): Forward primer 5'-GAGTCTATATTGAGGGCAGGCTGGAGTC-3' (SEQ ID NO:85), Reverse primer 5'-TAGTCTGCCTGCTCACTCAACCTCTCCT-3' (SEQ ID NO:86) The PCR products were subjected to gel electrophoresis. PCR products from genomic DNA of individuals that showed amplification of the expected sequence length (4705bp, 4026bp) using both the KI genotyping primer (5') and the KI genotyping primer (3') were subjected to direct sequencing using a Gentetic analyzer (Lifetechnology) and the following KI sequence primers. F0s in which the expected sequence KI was confirmed were considered KI positive. KI sequence primer (5'): 5'-GAGTCTATATTGAGGGCAGGCTGGAGTC-3' (SEQ ID NO:87) The KI-positive F0 was crossed with C57BL / 6J to obtain the F1. Genomic DNA was extracted from the ear tissue of the F1 using the DNeasy 96 Blood & Tissue Kit (Qiagen) and amplified using PrimeSTAR GXL (Takara) and the aforementioned KI genotyping primer (5') (98℃ 2 min, (95℃ 15 sec, 68℃ 5 min) 38 cycles, 68℃ 7 min, 15℃ hold). The PCR products were subjected to gel electrophoresis, and individuals that showed amplification of the expected sequence length (4705b) were designated as KI-positive F1. One line selected from the KI-positive F1 was bred to become the hG6PC (c.648G>T) + Int4 KI line.

[0408] (2-4) Genotyping of hG6PC (c.648G>T) + Int4 lineage Genomic DNA was extracted from ear tissue using the DNeasy 96 Blood & Tissue Kit (Qiagen) and multiplex amplified using KOD FX (TOYOBO) and the following KI genotyping primer and mG6PC WT primer (98°C 2 min, (95°C 15 sec, 68°C 5.5 min) 32 cycles, 68°C 5 min, 4°C hold). KI genotyping primer (5'): Forward primer 5'-TTCCTTCCAAAGCAGGGACTCTCTATGT-3' (same as SEQ ID NO: 83 (1)) Reverse primer 5'-CTTGCAGAAGGACAAGACGTAGAAGACC-3' (same as SEQ ID NO: 84 (2)) mG6PC WT primer: Forward primer 5'-TAAATTTGACCAATGAGCACTGGAGGTC-3' (SEQ ID NO:88) Reverse primer 5'-AAAATCATGTGTATGCGTGCCTTTCCTA-3' (SEQ ID NO:89)' The PCR products were subjected to gel electrophoresis, and the amplification near 4705 bp was identified as the KI allele, and the amplification near 2536 bp was identified as the mG6PC WT allele, and the genotyping of the offspring (WT, Ht, Homo) was determined.

[0409] (2-5) Mouse sampling Mice were sampled as follows. The evening before sampling, a floor net was placed to prevent eating feces, and fasting was started. The next day, the abdomen was opened under anesthesia, and after sufficient blood was removed, the liver and kidneys were removed. Each organ was washed with ice cold PBS, trimmed to an appropriate size, and stored in a tube containing homogenized beads (Nikkato). Each organ was flash-cooled with liquid nitrogen and then stored at -80°C.

[0410] (2-6) Evaluation of mRNA by qRTPCR [1] RNA extraction (in vivo) RNA extraction was carried out as follows. RNeasy mini kit or Qiacube system (Qiagen) cell lysis solution (600 μL) was added per tissue storage tube, and the tissue was homogenized at 25 kHz for 2 min using a tissue lyser II (Qiagen). After 10 min of incubation on ice, the tissue was centrifuged at 8000G for 10 min at room temperature to collect the supernatant. RNA was purified from the supernatant according to the protocol of each kit, including DNase treatment. RNase-Free DNase set (Qiagen) was used for DNase treatment. The purified and eluted RNA was subjected to reverse transcription as described above, and repaired G6PC mRNA was quantified according to the qRT-PCR (Taqman assay) described above.

[0411] (2-7) Evaluation of abnormal splicing repair by example compounds using hG6PC (c.648G>T) + Int4 Ht KI mice The compounds of Examples 8 to 14 were dissolved in Otsuka saline and subcutaneously administered to hG6PC (c.648G>T) + Int4 Ht KI mice at 30 mg / kg. Liver tissues were collected from the mice under overnight fasting conditions 7 days after administration, and abnormal splicing caused by G6PC (c.648G>T) was evaluated by qRT-PCR (Taqman) to see if it could be repaired. As shown in Figure 1, the compounds of Examples 8 to 14 normalized abnormal splicing of mRNA in the liver of hG6PC (c.648G>T) + Int4 Ht KI mice. Furthermore, when the compounds of Examples 15 to 23 were similarly evaluated, as shown in FIG. 2, the compounds of Examples 15 to 23 were found to normalize abnormal splicing of mRNA in the liver of hG6PC (c.648G>T) + Int4 Ht KI mice.

[0412] (Test Example 3) Evaluation of oligonucleotide delivery to liver cells by GalNAc unit conjugates using ApoB knock down test (3-1) Cultivation of human primary hepatocytes Human primary hepatocytes were cultured as follows. Adhesion medium was prepared by adding FBS (Gibco) to 10% of modified Rampford medium (Nissui Pharmaceutical). Human frozen primary hepatocytes (Gibco, Hu8105) were removed from the liquid nitrogen tank and thawed at 37°C. They were immediately poured into CHRM (Invitrogen), gently mixed, and centrifuged at room temperature (100 g, 10 min). The supernatant was removed with an aspirator, and adhesion medium preheated to 37°C was added to prepare a cell suspension. The number of viable cells was counted with an automatic counter (BioRAD), and the cells were seeded on a collagen-coated 24-well plate (number of viable cells seeded: 1.4-2.1 x 10 5 After culturing for 4 to 5 hours in a CO2 incubator, the adhesion of the cells was confirmed under a microscope, and then a compound addition experiment, which will be described later, was performed.

[0413] (3-2) Addition of Example Compound Example compounds were added to human primary hepatocytes as follows. The compound of Reference Example 45, which is not bound to a GalNAc derivative, or the compound of Example 208, which is bound to a GalNAc derivative, was added to a modified Rumford medium containing 10% FBS to prepare a compound-containing medium at a target concentration (100 nM in ApoB mRNA expression evaluation (1), and 10 nM and 100 nM in ApoB mRNA expression evaluation (2), as described below). After removing the medium from each well of human primary hepatocytes cultured in a 24-well plate by suction, 0.5 mL of compound-containing medium was added to each well to start incubation. After 72 hours, an RNA extraction experiment, which will be described later, was performed.

[0414] (3-3) Evaluation of mRNA by qRT-PCR [1] RNA extraction (in vitro) RNA extraction was carried out as follows. After incubation, the compound-containing medium was removed from each well of human primary hepatocytes by aspiration, and the cells were washed with ice-cold PBS (0.5 mL). The RLT buffer of the RNeasy Mini QIAcube Kit (Qiagen) was supplemented with 1 / 100 volume of 2-Mercaptoethanol to prepare a cell lysis solution. 400 μL of the cell lysis solution was added to each well, and the cells were incubated at room temperature for 5 minutes before recovery. RNA was purified from 350 μL of the recovered solution using the QIAcube with the RNeasy Mini - Animal tissues and cells - DNase digest protocol. RNase-Free DNase set (Qiagen) was used for DNase treatment. The purified and eluted RNA was subjected to reverse transcription as described below.

[0415] [2] Reverse transcription reaction The reverse transcription reaction was carried out as follows. After adjusting the extracted RNA to 25-100 mg / mL, reverse transcription was performed using the High Capacity RNA-to-cDNA kit (Applied biosystems) by mixing 10 μL Buffer mix, 1 μL Enzyme mix, 9 μL purified water, and extracted RNA per sample (37°C 60 min, 95°C 5 min, 4°C hold). 20 μL of the reverse transcription product was diluted 5-fold with 80 μL of purified water and stored at -30°C or subjected to the following qRT-PCR evaluation.

[0416] [3] qRT-PCR (TaqMan assay) The following ApoB primer probe set (x20) and Actinb primer probe set (x20) were used. ApoB primer probe set(x20) (Taqman):ABI Hs00181142_m1 FAM Actinb primer probe set(x20) (Taqman):ABI Hs01060665_g1 FAM In addition, 5μL 2x Taqman Fast Advanced Master Mix (Applied Biosystems), 2.5μL purified water, 0.5μL 20x Primer probe mix (10μM), and 2μL cDNA (5-fold diluted) were suspended per well to prepare a PCR reaction solution (per tube), and PCR reaction was performed using Quantstadio7 (Applied Biosystems) (95℃ 20sec, (95℃ 1sec, 60℃ 20sec) 40 cycles, 4℃ Hold). Actinb was used as an endogenous control, and the expression level of ApoB mRNA was evaluated relative to the control.

[0417] (3-4) Evaluation of ApoB mRNA expression suppression by example compounds using cultured human hepatocytes (2) The compound synthesized in Example 24, adjusted to 10 nM and 100 nM, was added at the same time as seeding human primary hepatocytes, and the expression of ApoB mRNA in the cells after 72 hours of culture was evaluated by qRT-PCR (Taqman assay). When the average expression of ApoB mRNA in 4 wells without compound was taken as 100%, as shown in FIG. 3, when 10 nM was used, ApoB-ASO showed 4.4% expression inhibition, and ApoB-ASO conjugate showed 30.3% expression inhibition. When 100 nM was used, ApoB-ASO showed 41.2% expression inhibition, and ApoB-ASO conjugate showed 68.6% expression inhibition. This indicates that the compound bound to the GalNAc derivative can inhibit expression more strongly than the compound not bound to the GalNAc derivative.

[0418] (Example 25) DMTr-on ion exchange column purification of oligonucleotides bound to GalNAc units having a 4,4'-dimethoxytrityl group on the 6-hydroxyl group of the GalNAc moiety (1) Synthesis of oligomers and preparation of mixture The compound described in Example 16 was synthesized. However, AKTA Oligopilot (GE Healthcare) was used as the nucleic acid automatic synthesizer, Primer Support 5G Unylinker 350, 390 μmol (GE Healthcare) was used as the solid phase carrier, and a synthesis program suitable for the scale was used. After completion of the synthesis, a mixture of conjugates and oligonucleotide analogs having the desired oligonucleotide sequence and in which the 6-position hydroxyl group of the GalNAc site was protected with a 4,4'-dimethoxytrityl group was obtained. The resulting mixture was divided into two portions, and each portion was treated with 25 mL of concentrated ammonia water to cut out the oligonucleotide from the support, and the protecting group cyanoethyl group on the phosphorus atom and the protecting group on the nucleic acid base were removed, and the mixture was filtered using a filter tube to obtain a mixture of oligonucleotides (25 mL) (2 tubes) containing the desired conjugate. The purification apparatus was AKTA pure150 (GE Healthcare), and the packing material SOURCE 15Q (96mL) (GE Healthcare) was packed in a column FineLINE Pilot (35mm x 100mm) (GE Healthcare), and the above oligonucleotide mixture was purified in two separate runs by the following method. The UV detection wavelengths were set to (260nm, 280nm, 350nm), and the following three steps were used as one purification program. During these steps, the column effluent was detected at 260nm and 350nm. The chromatogram is shown in Figure 5.

[0419] (i) Adsorption step of DMTr-on oligonucleotide onto a column The flow rate was set to 40 ml / min, and 1 CV of buffer A1 (10 mM sodium hydroxide aqueous solution) was run. The flow rate was changed to 10 ml / min, the oligonucleotide mixture (25 mL) was added to the top of the column, and 170 mL of buffer A1 (10 mM sodium hydroxide aqueous solution) was run. Next, the flow rate was set to 40 ml / min, and 1 CV of buffer A1 (10 mM sodium hydroxide aqueous solution) was run. Next, 6 CV of buffer A1 (10 mM sodium hydroxide aqueous solution): buffer B1 (10 mM sodium hydroxide / 2 M sodium chloride aqueous solution) (35:65) was run, resulting in a total of X 20 Unbound oligonucleotides and impurities were washed away. Finally, 2 CV of buffer A1 (10 mM sodium hydroxide solution) was run.

[0420] (ii) On-column DMTr removal step The flow rate was set to 40 ml / min, and 1 CV of buffer A2 (0.4% trifluoroacetic acid aqueous solution) was run. Then, the flow rate was changed to 20 ml / min, and 6.25 CV was run to remove the X adsorbed on the column packing material. 20 The oligonucleotides to which β-D-glucosamine was bound were subjected to demethylation.

[0421] (iii) Extraction of the target substance The flow rate was set to 40 ml / min, and 3 CV of buffer A1 (10 mM sodium hydroxide aqueous solution) was run to equilibrate. After a gradient (15 CV) of Buffer A1 (10 mM sodium hydroxide aqueous solution):Buffer B1 (10 mM sodium hydroxide / 2 M sodium chloride aqueous solution) (75:25) to (25:75), 2 CV of buffer B1 (10 mM sodium hydroxide / 2 M sodium chloride aqueous solution) was run to fractionate the de-DMTr-modified oligonucleotides (fraction volume was 48 mL).

[0422] The fractions detected by UV (260 nm) during elution were confirmed using a microspectrophotometer Xpose (manufactured by Trinean), and fraction solutions (first time: 432 ml, second time: 480 ml) were obtained (FIG. 5). After each use, the column was washed with 2M aqueous sodium chloride solution containing 30% isopropyl alcohol before the next purification.

[0423] (2) Concentration, dialysis, freeze-drying The fraction solutions (first: 432 ml, second: 480 ml) were combined and an appropriate amount of acetic acid was added to adjust the pH to 7.5-8.0. 2 The concentrate was concentrated using a KR2i TFF system (Repligen) equipped with a filter (GE Healthcare), then dialyzed against Otsuka distilled water and freeze-dried to obtain a freeze-dried powder of the indicated product (total 1.14 g).

[0424] (Comparative Example 1) DMTr-on ion exchange column purification of a conjugate in which a DMTr group was introduced near an oligonucleotide For comparison with Example 25, the following X 13 The GalNAc conjugate composed of was purified using a DMTr-on ion exchange column. 13The protecting groups at the 3-, 4-, and 6-positions of GalNAc in the oligonucleotide can be unified to acetyl groups. This makes it easier to synthesize the GalNAc unit, since there is no need to distinguish between protecting groups in the synthesis process. Furthermore, in the solid-phase synthesis process, the number of GalNAc units can be easily increased or decreased by adjusting the number of couplings with the 5'-end of the oligonucleotide. However, only one 4,4'-dimethoxytrityl group can remain after the solid-phase synthesis of the oligomer is completed.

[0425] [Comparative Example 1-1] Synthesis of GalNAc unit amidite corresponding to X13 (compound Ref1F) (Ref1A) Synthesis of 9H-fluoren-9-ylmethyl N-[2-[2-[(2R)-3-benzyloxy-2-hydroxy-propoxy]ethoxy]ethyl]carbamate (compound Ref1A)

[0426] [ka]

[0427] The literature-known compound 9H-fluoren-9-ylmethyl N-[2-(2-hydroxyethoxy)ethyl]carbamate (Bioconjugate Chemistry, 2000, 11, 755-761) (3.11g, 10mmol) and benzyl (S)-(+)-glycidyl ether (0.8g, 5mmol) were dissolved in dichloromethane (30mL), boron trifluoride-diethyl ether complex (0.1mL, 1mmol) was added, and the mixture was stirred at room temperature for 16 hours. After the reaction was completed, a saturated aqueous solution of sodium bicarbonate was added. The organic layer was washed with a saturated aqueous solution of sodium bicarbonate and saturated saline, then dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. The product was purified by silica gel column chromatography (hexane:ethyl acetate=80:20-0:100, v / v) to obtain the oily target product Ref1A (0.74g, 30% yield). 1H-NMR (CDCl3) δ: 7.76 (2H, d, J = 7.9 Hz), 7.60 (2H, d, J= 7.3 Hz), 7.42-7.37 (2H, m), 7.35-7.27 (7H, m), 5.40 (1H, s), 4.53 (2H, s), 4.39 (2H, d, J = 6.7 Hz), 4.25-4.19 (1H, m), 4.04-3.97 (1H, m), 3.69-3.35 (12H, m). Calcd for C 29 H 33 NO6: [M+H] + 492, Found 492.

[0428] (Ref1B) Synthesis of (2R)-1-[2-(2-aminoethoxy)ethoxy]-3-benzyloxy-propan-2-ol, trifluoroacetate (Compound Ref1B)

[0429] [ka]

[0430] Compound Ref1A (13.8 g, 28.1 mmol) synthesized in step (Ref1A) was dissolved in ethanol / tetrahydrofuran (50 mL / 50 mL), 1N aqueous sodium hydroxide solution (100 mL) was added, and the mixture was stirred at room temperature for 2 hours. After the reaction was completed, the organic solvent was distilled off under reduced pressure, and 1N hydrochloric acid (100 mL) was added. The resulting solid matter was removed by filtration. The by-product contained in the aqueous layer, which was the effluent, was extracted with ethyl acetate, and acetonitrile was added to the residue obtained after distillation under reduced pressure, and the residue was filtered. The effluent was distilled off under reduced pressure to obtain a crude product. Separation and purification were performed using reverse phase HPLC (GL Science, Inertsil ODS-3) using 0.1% aqueous trifluoroacetic acid solution and 0.1% trifluoroacetic acid acetonitrile solution as eluents, and the fractions containing the target product were combined, the solvent was distilled off under reduced pressure, and the oily target product Ref1B (7.1 g, yield 66%) was obtained. 1H-NMR (CDCl3) δ: 7.38-7.27 (5H, m), 4.55 (2H, s), 4.07-3.99 (1H, m), 3.77-3.48 (10H, m), 2.96-2.88 (2H, m).

[0431] (Ref1C) Synthesis of Acetate [(2R,3R,4R,5R,6R)-5-Acetamido-3,4-diacetoxy-6-[2-[2-[2-[(2R)-3-benzyloxy-2-hydroxy-propoxy]ethoxy]ethylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl (Compound Ref1C)

[0432] [ka]

[0433] The literature-known compound 2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyacetic acid (WO2012037254) (6g, 14.8mmol), 1-(-3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.4g, 17.8mmol), 3H-1,2,3-triazole[4,5-b]pyridin-3-ol (2.0g, 14.8mmol), N,N-diisopropylethylamine (9.0mL, 51.8mmol), and compound Ref1B (6.7g, 17.5mmol) synthesized in step (Ref1B) were dissolved in dichloromethane (130mL) and stirred at room temperature for 4 hours. After the reaction was completed, the organic layer was washed with 1N hydrochloric acid, saturated aqueous sodium bicarbonate solution, and saturated saline, then dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. The solvent was distilled off under reduced pressure to obtain a crude product. The product was purified by silica gel column chromatography (ethyl acetate:methanol = 100:0-90:10, v / v) to obtain the amorphous target product Ref1C (7.9 g, yield 81%). 1H-NMR (CDCl3) δ: 7.39-7.28 (5H, m), 7.00-6.93 (1H, m), 6.50-6.44 (1H, m), 5.33-5.29 (1H, m), 5.16 (1H, dd, J = 11.5, 3.6 Hz), 4.63-4.53 (3H, m), 4.34 (1H, d, J = 15.1 Hz), 4.22-3.83 (5H, m), 3.73-3.26 (13H, m), 2.15 (3H, s), 2.05 (3H, s), 1.99 (6H, s). Calcd for C 30 H 44 N2O 14 : [M+H] + 657, Found 657.

[0434] (Ref1D) Synthesis of acetic acid [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[2-[2-[2-[(2R)-2,3-dihydroxypropoxy]ethoxy]ethylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl acetate (compound Ref1D)

[0435] [ka]

[0436] Compound Ref1C (7.9 g, 12 mmol) synthesized in step (Ref1C) was dissolved in ethanol (80 mL). 20% palladium hydroxide / carbon (wet) (2.0 g) was added and vigorously stirred at 50°C for 8 hours under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the filtrate was distilled under reduced pressure. Amorphous target product Ref1D (6.7 g, 98% yield) was obtained. 1H-NMR (CDCl3) δ: 7.03-6.98 (1H, m), 6.43 (1H, d, J = 9.1 Hz), 5.35 (1H, d, J = 3.3 Hz), 5.15 (1H, dd, J = 11.2, 3.3 Hz), 4.58 (1H, d, J = 7.9 Hz), 4.35 (1H, d, J = 15.7 Hz), 4.28-3.82 (5H, m), 3.77-3.34 (13H, m), 2.18 (3H, s), 2.06 (3H, s), 2.03 (3H, s), 2.01 (3H, s). Calcd for C 23 H 38 N2O 14 : [M+H] + 568, Found 568.

[0437] (Ref1E) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[2-[2-[2-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propoxy]ethoxy]ethylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl acetate (compound Ref1E)

[0438] [ka]

[0439] An appropriate amount of pyridine was added to the compound Ref1D (6.8 g, 12 mmol) synthesized in step (Ref1D), and the mixture was azeotroped under reduced pressure. The compound was dissolved in pyridine (40 mL), and 4,4'-dimethoxytrityl chloride (4.5 g, 13 mmol) was added, followed by stirring at room temperature for 2 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. An appropriate amount of toluene was added and the mixture was azeotroped to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate:ethyl acetate / methanol (5 / 1) = 100:0-50:50, v / v, containing 1% triethylamine) to obtain the amorphous target product Ref1E (6.9 g, yield 66%). 1H-NMR (CDCl3) δ: 7.42 (2H, d, J = 7.9 Hz), 7.33-7.19 (7H, m), 7.01-6.94 (1H, m), 6.83 (4H, d, J = 9.1 Hz), 6.37 (1H, d, J = 8.5 Hz), 5.33 (1H, d, J = 3.3 Hz), 5.15 (1H, dd, J = 11.2, 3.3 Hz), 4.58 (1H, d, J = 7.9 Hz), 4.32 (1H, d, J = 15.7 Hz), 4.22-3.86 (5H, m), 3.79 (6H, s), 3.66-3.12 (13H, m), 2.13 (3H, s), 2.03 (3H, s), 1.97 (3H, s), 1.95 (3H, s). Calcd for C 44 H 56 N2O 16 : [M+Na] + 893, Found 892.

[0440] (Ref1F) Synthesis of Acetate [(2R,3R,4R,5R,6R)-5-Acetamido-3,4-diacetoxy-6-[2-[2-[2-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxy-propoxy]ethoxy]ethylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl (Compound Ref1F)

[0441] [ka]

[0442] Compound Ref1E (6.9 g, 7.9 mmol) synthesized in step (Ref1E) was azeotroped with an appropriate amount of toluene, and then dissolved in dichloromethane (80 mL). N,N-diisopropylethylamine (5.5 mL, 32 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (2.1 mL, 9.5 mmol) were added and stirred at room temperature for 1 hour. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. This was purified by silica gel column chromatography (ethyl acetate:ethyl acetate / methanol (5 / 1) = 100:0-50:50, v / v, containing 1% triethylamine) to obtain the amorphous target product Ref1F (6.6 g, yield 78%). 1 H-NMR (CDCl3) δ: 7.47-7.40 (2H, m), 7.35-7.17 (7H, m), 6.98-6.91 (1H, m), 6.85-6.78 (4H, m), 6.05-5.89 (1H, m), 5.37-5.33 (1H, m), 5.17-5.07 (1H, m), 4.59-4.49 (1H, m), 4.37-4.30 (1H, m), 4.23-3.06 (28H, m), 2.69-2.42 (2H, m), 2.15 (3H, s), 2.05 (3H, s), 2.02-1.98 (3H, m), 1.97-1.94 (3H, m), 1.30-1.00 (12H, m).

[0443] [Comparative Example 1-2] Synthesis of conjugates incorporating X13 HO-X 13 -X 13 -X 13 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (SEQ ID NO:40) Compound Ref1F was used as the GalNAc unit amidite to obtain X 18 Change the part to X 13 The synthesis was carried out in the same manner as in Example 8, except that the addition reaction of the GalNAc unit amidite was repeated three times to bind three X13s. The base sequence of this compound is complementary to the 92nd to 106th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a mutation from G to T at nucleotide 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6878.11).

[0444] [Comparative Example 1-3] DMTr-on ion exchange column purification of oligonucleotides bound to GalNAc units having a 4,4'-dimethoxytrityl group on the 5'-terminus (1) Synthesis of oligomers and preparation of mixture HO-X 13 -X 13 -X 13 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (SEQ ID NO:40) The automated nucleic acid synthesizer used was AKTA Oligopilot (GE Healthcare), the solid phase support was Primer Support 5G Unylinker 350, 390 μmol (GE Healthcare), and a synthesis program suitable for scale was used. Using 10 μmol of the obtained solid support, GalNAc unit X13 was condensed three times using an ABI 392 DNA / RNA Synthesizer (Applied Biosystems). After completion of the synthesis, a mixture of oligonucleotide analogs having the desired oligonucleotide sequence and bound to a GalNAc unit having a 4,4'-dimethoxytrityl group at the 5' end was obtained. The oligonucleotide was cleaved from the support by treating with 6 mL of concentrated aqueous ammonia, and the protecting group cyanoethyl group on the phosphorus atom and the protecting group on the nucleic acid base were removed.

[0445] Purification method 1: Clarity QSP (Phenomenex) purification Purification in the same manner as in Example 8 gave the title target product. The base sequence of this compound is complementary to the 92nd to 106th bases from the 5' end of exon 5 of the c.648G>T mutant G6PC gene, which is a mutation from G to T at nucleotide 728 of Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession No. NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6878.11).

[0446] Purification method 2: DMTr-on ion exchange column purification Before purifying the oligonucleotide mixture through a DMTr-on ion exchange column, the mixture was replaced with a 10 mM aqueous sodium hydroxide solution.

[0447] The above oligonucleotide mixture was purified by the following method using an AKTA pure150 (GE Healthcare) purification apparatus and a RESOURCE Q 6mL purification column (GE Healthcare). The UV detection wavelength was set to (260 nm), and the following three steps were used as one purification program. The chromatograms of the column effluent during these steps at a detection wavelength of 260 nm are shown in Figure 6.

[0448] (i) Adsorption of DMTr-on oligonucleotides to an ion exchange column 2 CV of buffer A1 (10 mM sodium hydroxide aqueous solution) was applied at a flow rate of 3 ml / min. The oligonucleotide mixture (1.5 mL, 10 mM sodium hydroxide aqueous solution) was added to the top of the column at a flow rate of 2 ml / min, and 2.5 CV of buffer A1 (10 mM sodium hydroxide aqueous solution) was applied. Next, 1 CV of buffer A1 (10 mM sodium hydroxide aqueous solution) was applied at a flow rate of 3 ml / min, and then buffer A1 (10 mM sodium hydroxide aqueous solution): buffer B1 (10 mM sodium hydroxide / 2 M sodium chloride aqueous solution) (40:60 to 25:75 2 CV, 25:75 4 CV) was applied at a flow rate of 2.4 ml / min.

[0449] (ii) DMTr removal step on an ion exchange column 7 CV of buffer A2 (0.4% aqueous trifluoroacetic acid solution) was run at a flow rate of 1.2 ml / min.

[0450] (iii) Extraction of the target substance Equilibration was performed by passing 5 CV of Buffer A1 (10 mM sodium hydroxide aqueous solution) at a flow rate of 3 ml / min. Then, a gradient (10 CV) of Buffer A1 (10 mM sodium hydroxide aqueous solution):Buffer B1 (10 mM sodium hydroxide / 2 M sodium chloride aqueous solution) (30:70) to (0:100) at a flow rate of 3 ml / min was performed, followed by passing 2 CV of Buffer B1 (10 mM sodium hydroxide / 2 M sodium chloride aqueous solution).

[0451] As shown in Figure 6, no absorption at 260 nm was detected in the elution step after deprotection, indicating that the DMT-on conjugate was not adsorbed to the column in the first adsorption step and passed through. Therefore, the target product could not be purified using an ion exchange column. From this, it is considered important that a hydrophobic protecting group is bonded to GalNAc and / or that the number of hydrophobic protecting groups contained in one GalNAc unit is equal to or greater than the number of GalNAcs in order to purify a conjugate of a GalNAc unit and an oligonucleotide using a DMTr-on column.

[0452] In this specification, A t , G t , 5meC t , C t , T t , U t , A p , G p , 5meC p , C p , T p , U p , A s , G s , 5meC s , C s , T s , U s , A m1t , G m1t , C m1t , 5meC m1t , U m1t , A m1p , G m1p , C m1p , 5meC m1p , U m1p , A m1s , G m1s , C m1s , 5meC m1s , U m1s , A 2t , G 2t , C 2t , T 2t , A e2p , G e2p , C e2p , T e2p , A e2s , G e2s , C e2s , Te2s , A 1t , G 1t , C 1t , T 1t , A e1p , G e1p , C e1p , T e1p , A e1s , G e1s , C e1s , T e1s , A m2t , G m2t , 5meC m2t , T m2t , A m2p , G m2p , 5meC m2p , T m2p , A m2s , G m2s , 5meC m2s , T m2s , X, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 is a group having the structure shown below.

[0453] [ka]

[0454] [ka]

[0455] [ka]

[0456] [ka]

[0457] [ka]

[0458] [ka]

[0459] [ka]

[0460] [ka]

[0461] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. [Industrial Applicability]

[0462] The present invention can be used to treat glycogen storage disease type Ia. [Sequence List Free Text]

[0463] SEQ ID NOs: 1 to 48, 93 to 96 show the sequences of antisense oligonucleotides. The nucleotides constituting the antisense oligonucleotides may be natural DNA, natural RNA, DNA / RNA chimera, or modified versions thereof, but it is preferable that at least one of them is a modified nucleotide. SEQ ID NOs: 49 to 65, 67 to 92 show primer sequences. SEQ ID NO: 66: Shows the sequence of the peptide.

Claims

1. Oligonucleotides having a nucleic acid sequence that is expected to have a pharmacological effect in hepatocytes (excluding antisense oligonucleotides against the pre-mRNA of c.64 8G>T mutant G6PC) and a biantennary GalNAc unit conjugate, wherein the biantennary GalNAc unit of the following formula is bound to the 5'-end or 3'-end of the oligonucleotide, and the conjugate specifically binds to hepatic parenchymal cells in vivo. The conjugate or a pharma- ceutically acceptable salt thereof, characterized in that it is delivered. 【Chemistry 1】 [Wherein W is a group represented by the formula 【Chemistry 2】 G represents a 5-acetamido-2-hydroxymethyl-3,4-dihydroxytetrahydropyran-6-yl group (GalNAc), Z represents an oxygen atom or a sulfur atom, L １ and L ２ One of them is a methylene group (CH ２ ), and the other does not sandwich an atom between them, o, q, r, and s each independently represent 0 or 1, n represents an integer of 1 to 15, m represents an integer of 0 to 5, and v represents 1 to 7. However, when n is 1, m is an integer of 0 to 5, and when n is an integer of 2 to 15, m is 0.

2. The conjugate or copolymer of claim 1, wherein o and q in the biantennary GalNAc unit are 0. and a pharma- ceutical acceptable salt thereof.

3. 3. The conjugate of claim 1 or 2, wherein the biantennary GalNAc unit is attached to the 5' end of the oligonucleotide, or a pharma- ceutically acceptable salt thereof.

4. The biantennary GalNAc unit has one of the following formulas: 【Chemistry 3】 【Chemistry 4】 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 The conjugate according to claim 1 , wherein the R 1 is a group represented by the formula:

5. Any of claims 1 to 4, wherein the oligonucleotide is an RNA interference oligonucleotide.

2. The conjugate according to claim 1, or a pharma- ceutically acceptable salt thereof.

6. A medicine comprising the conjugate of any one of claims 1 to 5 or a pharma- ceutically acceptable salt thereof.

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