GalNac-oligonucleotide conjugates for delivery to liver and methods of making same

Abstract

The present invention relates to a double-antenna N-acetyl-D-galactosamine (GalNAc) ligand-oligonucleotide conjugate, and a method for preparing the conjugate. The present invention relates to a double-antenna N-acetyl-D-galactosamine (GalNAc) ligand-oligonucleotide conjugate. The present invention provides a conjugate of a double-antenna GalNAc unit and an oligonucleotide having a nucleic acid sequence expected to have a pharmacological action in parenchymal hepatic cells, or a pharmaceutically acceptable salt thereof, and a drug or an analogue containing the conjugate or the pharmaceutically acceptable salt thereof as an active ingredient.

Description

[0001] This application is a divisional application of the PCT application with an international application date of September 9, 2020, an international application number of PCT / JP2020 / 034022, an application number of 202080077913.7 entering the Chinese national phase, and an invention name of “GalNAc-oligonucleotide conjugates for delivery to the liver and preparation methods thereof”. Technical Field

[0002] The present invention relates to biantennary N-acetyl-D-galactosamine (GalNAc) ligand-oligonucleotide conjugates and methods for preparing the conjugates. Background Art

[0003] As a ligand capable of binding to asialoglycoprotein receptor (ASGPR), a conjugate of nucleic acid therapy (e.g., antisense and siRNA) covalently bound to N-acetyl-D-galactosamine (GalNAc) etc. has been reported as delivering oligonucleotides to hepatocytes of the liver (Non-Patent Document 1, Patent Documents 1 to 9). In these reports, one to three GalNAcs are bound to an oligonucleotide. In addition, Non-Patent Document 2 describes an example in which two GalNAcs are bound.

[0004] In the case of using reversed-phase HPLC in the purification step after the solid phase synthesis of oligonucleotides, the retention time of the oligomer with short chain length produced in the solid phase synthesis step is usually close to the retention time of the target oligomer, and it is very difficult to obtain high-purity refined products on a large scale. In the solid phase synthesis of oligonucleotides, a nucleic acid unit in which the 5'-hydroxyl of nucleoside is protected by 4,4'-dimethoxytrityl (DMTr) groups is generally used, and the 5'-hydroxyl of the nucleoside at the 5' end of the oligonucleotide to be synthesized is therefore located with a DMTr group. Non-patent literature 3 and 4 describe the use of highly hydrophobic reversed-phase column purification using DMTr groups (hereinafter, also referred to as DMTr-on reversed-phase column purification; "DMTr-on" means using an oligonucleotide with a DMTr group introduced at its 5' end) and the use of DMTr-on ion exchange column purification (hereinafter, also referred to as DMTr-on ion exchange column purification) examples. However, since the GalNAc unit is usually introduced into the hydroxyl group at the 5' end of the oligonucleotide by deprotecting the DMTr group, the conjugate to be synthesized does not have a DMTr group. Therefore, in order to separate / purify the oligonucleotide without the coupled GalNAc unit, it is necessary to purify by HPLC, which is laborious and time-consuming (Non-Patent Document 5). So far, no examples of methods such as DMTr-on reverse phase column purification or DMTr-on ion exchange column purification for GalNAc unit-oligonucleotide conjugates have been reported.

[0005] Therefore, it is desired to develop a GalNAc unit that can effectively deliver an oligonucleotide to hepatocytes and has an advantageous structure in terms of mass production, a conjugate of the GalNAc unit and an oligonucleotide having a therapeutic effect on a disease, and an efficient production method thereof.

[0006] Reference List Patent Literature Patent Document 1: International Publication No. WO2009 / 073809 Patent Document 2: International Publication No. WO2014 / 076196 Patent Document 3: International Publication No. WO2014 / 179620 Patent Document 4: International Publication No. WO2015 / 006740 Patent Document 5: International Publication No. WO2015 / 105083 Patent Document 6: International Publication No. WO2016 / 055601 Patent Document 7: International Publication No. WO2017 / 023817 Patent Document 8: International Publication No. WO2017 / 084987 Patent Document 9: International Publication No. WO2017 / 131236 Non-patent literature Non-patent document 1: Methods in Enzymology, 1999, Vol. 313, pp. 297-321 Non-patent document 2: Bioorganic & Medicinal Chemistry Letters 26 (2016) 3690-3693 Non-patent literature 3: AKTAdesign Purification-User Manual 18-1124-23 Edition AD Non-patent document 4: Organic Process Research & Development 2005, 9, 212-215 Non-patent literature 5: Molecules 2017, 22, 1356. Summary of the invention

[0007] Problems to be solved by the invention The conventionally used linkers in the study of using the GalNAc unit to deliver synthetic nucleic acids to the liver emphasize keeping the ligand site away from the nucleic acid, using a structure with a long straight chain alkyl and a ring structure in a very long backbone, and also having a complex structure with three or more branches in many cases. However, the inventor's research has newly revealed a problem that when conjugates using such conventional long-chain alkyl linkers are concentrated, micelles are formed in high concentration solutions, and it is difficult to prepare a suitable dose. In addition, in conventional methods, there is no method for column separation of oligonucleotides combined with GalNAc units, and it is difficult to mass produce.

[0008] Means used to solve the problem As a result of in-depth research for solving the above problems, the inventors found that the conjugate of the biantennary GalNAc unit and oligonucleotide with the joint structure of the present invention also shows good solubility even in high-dose solution, while maintaining the ability to be effectively delivered to hepatocytes. It has been found in addition that the oligonucleotides bound to the GalNAc unit with a high hydrophobic protective group (e.g., 4,4'-dimethoxytrityl (DMTr) group) on the 6-hydroxyl of GalNAc can be purified with better efficiency by a hydrophobic resin column, thereby completing the present invention. Therefore, it can be expected that the ion exchange column purification or DMTr-on reverse phase column purification of large-scale conjugates is easy.

[0009] The gist of the present invention is as follows.

[0010] (A1) A conjugate of an oligonucleotide having a nucleic acid sequence expected to have a pharmacological effect in hepatocytes and a biantennary GalNAc unit, or a pharmaceutically acceptable salt thereof, wherein in the conjugate, the biantennary GalNAc unit is represented by the following formula and is bound to the 5' end or the 3' end of the oligonucleotide, and wherein the conjugate is specifically delivered to hepatocytes in vivo: [Formula 1] Wherein W is a group represented by the following formula: [Formula 2] G represents 5-acetylamino-2-hydroxymethyl-3,4-dihydroxytetrahydropyran-6-yl (GalNAc); Z represents an oxygen atom or a sulfur atom; L 1 and L 2 One of the two represents a methylene group (CH 2), and the other represents no intervening atom; o, q, r and s each independently represent 0 or 1; n represents an integer from 1 to 15; m represents an integer from 0 to 5; and v represents an integer from 1 to 7, provided that when n is 1, then m is an integer from 0 to 5, and when n is an integer from 2 to 15, then m is 0.

[0011] (A2) The conjugate according to (A1) or a pharmaceutically acceptable salt thereof, wherein in the formula of the biantennary GalNAc unit, o and q are each 0.

[0012] (A3) The conjugate according to (A1) or (A2) or a pharmaceutically acceptable salt thereof, wherein the biantennary GalNAc unit is bound to the 5′ end of the oligonucleotide.

[0013] (A4) The conjugate according to (A1) or a pharmaceutically acceptable salt thereof, wherein the biantennary GalNAc unit is a group represented by any one of the following formulae: [Formula 3] [Formula 4] [Formula 5] [Formula 6] and [Formula 7] (A5) A medicament comprising the conjugate according to any one of (A1) to (A4) or a pharmaceutically acceptable salt thereof.

[0014] In addition, the present invention provides the following production method and a compound or conjugate containing a GalNac unit having a hydrophobic protective group used as an intermediate in the production method.

[0015] (B1) A method for preparing a conjugate of an oligonucleotide and a GalNAc unit, comprising the following steps: a: synthesizing a conjugate comprising a structure of a GalNAc unit-phosphate group containing a hydrophobic protective group represented by the following formula bound to the 5' end or 3' end of the oligonucleotide: [Formula 8] Where R a , R b and R c At least one of them is a hydrophobic protecting group, and the rest are hydrogen atoms or protecting groups that can be removed under free conditions; X 1 and X 2 Each independently represents a linker structure, which can be 1and X 2 t is the number of branches in the linker structure and is an integer from 1 to 10; and Y represents a bond that is bound to the oligonucleotide through a phosphate group or a phosphorothioate group.

[0016] b: passing the solution containing the product of step a through a column filled with a resin having adsorptive properties due to hydrophobic interactions, and passing an aqueous washing solution through the column; and c: Step of eluting the oligonucleotide from the column after step b.

[0017] (B2) The preparation method according to (B1), wherein the hydrophobic protecting group is a protecting group that is removed under alkaline conditions or acidic conditions, preferably a hydrophobic protecting group that is removed under acidic conditions, more preferably an optionally substituted trityl group or an optionally substituted silyl group.

[0018] (B3) The production method according to (B1), wherein a structure comprising a GalNAc unit-phosphate group containing a hydrophobic protective group is bound to a hydroxyl group at the 5-position of a nucleoside located at the 5′ end of the oligonucleotide.

[0019] (B4) The preparation method according to (B1), wherein in the structure comprising a GalNAc unit-phosphate group containing a hydrophobic protective group, R a is a hydrophobic protecting group, R b and R c Each is a hydrogen atom, and t is an integer of 1 to 3.

[0020] (B5) The preparation method according to (B1), wherein the resin having adsorptivity due to hydrophobic interaction is any one selected from styrene-divinylbenzene adsorptivity resin, styrene-divinylbenzene modified adsorptivity resin, methacrylic adsorptivity resin and phenolic adsorptivity resin.

[0021] (B6) The production method according to (B1), wherein the column filled with the resin having adsorptivity due to hydrophobic interaction is an ion exchange column or a reverse phase column.

[0022] (B7) The preparation method according to (B1), wherein step a comprises the following steps: a1: synthesizing a desired oligonucleotide on a support, the support being capable of extending the oligonucleotide in the direction from the 3' end to the 5' end by repeatedly deprotecting the protecting group of the 5'-hydroxyl group of the nucleoside located at the 5' end and condensing the nucleoside using the support to extend the chain of the oligonucleotide, wherein the protecting group selected is a protecting group other than the protecting group of the 5'-hydroxyl group of the nucleoside used, which is deprotected under conditions that allow cleavage of the bond between the 3' end of the oligonucleotide and the support (hereinafter referred to as "free conditions"), the protecting group is selected as a protecting group other than the protecting group of the 5'-hydroxyl group of the nucleoside used, and a protecting group that is not deprotected under free conditions is selected as a protecting group of the 5'-hydroxyl group of the nucleoside; a2: introducing a GalNAc unit containing a hydrophobic protecting group into the 5'-hydroxyl group of the nucleoside located at the 5' end of the oligonucleotide synthesized on the support, wherein a hydrophobic protecting group that does not undergo deprotection under free conditions is selected; and a3: treating the support obtained in step a2 with a solution under free conditions to obtain a conjugate solution of the GalNAc unit containing a hydrophobic protecting group and the oligonucleotide.

[0023] (B8) The preparation method according to (B1), comprising, after step b and before step c, deprotecting the hydrophobic protecting group of the conjugate adsorbed on the column.

[0024] (B9) The production method according to (B1), wherein the freeing condition is basic, and the hydrophobic protecting group is a protecting group that is removed under acidic conditions.

[0025] (B10) The preparation method according to (B1), wherein the structure comprising the GalNAc unit-phosphate group containing a hydrophobic protective group is represented by any one of the following formulae: [Formula 9] Where R 1 represents a hydrophobic protecting group that is removed under acidic conditions, X represents a linker structure, and Y represents an atomic bond that is bound to the oligonucleotide through a phosphate group or a thiophosphate group, and the same applies below: [Formula 10] [Formula 11] [Formula 12] [Formula 13] [Formula 14] [Formula 15] [Formula 16] [Formula 17] [Formula 18] and [Equation 19] (B11) A compound represented by the following formula: [Formula 20] Where R a , R b and R c At least one of them is a hydrophobic protecting group, and the rest are protecting groups that will be removed under free conditions; X 1 and X 2 Each independently represents a linker structure, which can be 1 and X 2 t is the number of branches in the linker structure and is an integer from 1 to 10; and Y represents a phosphoramidite group (preferably -P(OCH 2 CH 2 CN)N(CH(CH 3 ) 2 ) 2 or -P(OCH 3 )N(CH(CH 3 ) 2 ) 2 ) or an H-phosphonate group (—PH(═O)(OH)); or a salt thereof.

[0026] (B12) The compound according to (B11) or a salt thereof, wherein R a is a hydrophobic protecting group that is removed under acidic conditions, R b and R c Each is an acetyl group, Y represents a phosphoramidite group or an H-phosphonate group, and t is an integer of 1 to 3.

[0027] (B13) The compound according to (B11) or (B12), or a salt thereof, which is represented by any one of the following formulae: [Formula 21] Where R 1 is a hydrophobic protecting group that is removed under acidic conditions, and Y represents a phosphoramidite group or an H-phosphonate group, and the same applies hereinafter: [Formula 22] [Formula 23] [Equation 24] and [Formula 25] (B14) The compound according to (B11) or (B12), or a salt thereof, which is represented by any one of the following formulae: [Equation 26] Where R 1 represents a hydrophobic protecting group that is removed under acidic conditions, X represents a linker structure, and Y represents a phosphoramidite group or an H-phosphonate group, and the same applies hereinafter: [Formula 27] [Equation 28] [Equation 29] [Formula 30] and [Equation 31] (B15) The compound or salt thereof according to any one of (B11) to (B14), wherein the hydrophobic protecting group is an optionally substituted trityl group, and the phosphoramidite group is -P(OCH 2 CH 2 CN)N(CH(CH 3 ) 2 ) 2 or -P(OCH 3 )N(CH(CH 3 ) 2 ) 2 .

[0028] (B16) a conjugate comprising a structure comprising a GalNAc unit-phosphate group containing a hydrophobic protective group represented by the following formula, bound to the 5′ end or 3′ end of an oligonucleotide, or a salt thereof: [Formula 32] Among them, R a , R b and R cAt least one of them is a hydrophobic protecting group, and the rest are hydrogen atoms or protecting groups that can be removed under free conditions; X 1 and X 2 Each independently represents a linker structure, which can be 1 and X 2 t is the number of branches in the linker structure and is an integer from 1 to 10; and Y represents a bond that is bound to the oligonucleotide through a phosphate group or a phosphorothioate group.

[0029] (B17) The conjugate or a salt thereof according to (B16), wherein the structure comprising a GalNAc unit-phosphate group containing a hydrophobic protective group is represented by any one of the following formulae: [Formula 33] Where R 1 represents a hydrophobic protecting group that is removed under acidic conditions, Y represents an atomic bond that is bound to the oligonucleotide through a phosphate group or a thiophosphate group, and the same applies below: [Equation 34] [Formula 35] [Equation 36] and [Equation 37] (B18) The conjugate or a salt thereof according to (B16), wherein the structure comprising a GalNAc unit-phosphate group containing a hydrophobic protecting group is represented by any one of the following formulae: [Equation 38] Where R 1 represents a hydrophobic protecting group that is removed under acidic conditions, X represents a linker structure, and Y represents an atomic bond that is bound to the oligonucleotide through a phosphate group or a thiophosphate group, and the same applies below: [Equation 39] [Formula 40] [Formula 41] [Formula 42] [Formula 43] (B19) The 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 an atomic bond bound to the 5'-hydroxyl group of the nucleoside located at the 5' end of the oligonucleotide via a phosphodiester bond or a phosphorothioate bond.

[0030] Effects of the Invention The conjugate of the present invention realizes the appropriate delivery of various oligonucleotides to the liver and its high water solubility. In addition, it realizes large-scale production by establishing a method for DMTr-on column purification of the conjugate of GalNAc unit and oligonucleotide. These make it possible to increase the dosage to enhance the pharmacological effect and prepare a high-concentration chemical solution for application of long-term continuous preparation, so that the product design of nucleic acid therapy can meet various needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] [ Figure 1 ] Figure 1 It is a graph showing the correction effect of abnormal splicing of G6PC mRNA in the liver when the compounds of Examples 8 to 14 are administered to heterologous knock-in mice. The amount of G6PC mRNA with abnormal splicing in the liver of the corrected heterologous knock-in mice was relatively quantitatively evaluated by qRT-PCR (corrected by the amount of actin mRNA). The Y axis represents the relative value of the normal G6PC mRNA expression amount in the administration group when the physiological saline administration group is 1 (RQ: relative amount; mpk: mg / kg).

[0032] [ Figure 2 ] Figure 2 : is a graph showing the correction effect of abnormal splicing of G6PC mRNA in the liver when the compounds of Examples 15 to 23 are administered to heterologous knock-in mice. The amount of G6PC mRNA with abnormal splicing in the liver of the corrected heterologous knock-in mice was quantitatively evaluated by qRT-PCR (corrected by the amount of actin mRNA). Figure 1 RQ: relative quantity; mpk: mg / kg.

[0033] [ Figure 3 ] Figure 3 It is a graph showing the degradation effect of ApoB mRNA in human primary cultured hepatocytes when ApoB-ASO (white column) and ApoB-ASO conjugate (filled column) are added to the cells. The amount of ApoB mRNA in the cells was relatively quantitatively evaluated by qRT-PCR (corrected by the amount of actin mRNA). The Y axis represents the relative value (%) in the added group when the amount of ApoB mRNA in untreated human primary cultured hepatocytes is 100%.

[0034] [ Figure 4 ] Figure 4It is a correlation diagram of the purification steps and chromatogram of DMTr-on ion exchange column.

[0035] [ Figure 5 ] Figure 5 It is a chromatogram of a conjugate having a DMTr group on the hydroxyl group of GalNAc during purification on a DMTr-on ion exchange column (detection wavelength: 260 nm).

[0036] [ Figure 6 ] Figure 6 It is a chromatogram of the conjugate having a DMTr group near the oligonucleotide during purification on a DMTr-on ion exchange column (detection wavelength: 260 nm). DETAILED DESCRIPTION

[0037] Hereinafter, embodiments of the present invention will be described in detail. In the present specification, the term "conjugate" refers to a compound in which a GalNAc unit is bound to the 5' end and / or 3' end of an oligonucleotide.

[0038] In this specification, the term "GalNAc unit" refers to a partial structure containing N-acetyl-D-galactosamine (GalNAc) that can bind to the asialoglycoprotein receptor (ASGPR) on the hepatocytes of the liver. The GalNAc unit contains a phosphate group or a thiophosphate group for binding a straight chain or branched linker structure to an oligonucleotide. Such a structural unit containing a phosphate group or a thiophosphate group for binding can be referred to as a "GalNAc unit". Unless otherwise specified, the number of GalNac contained in a GalNac unit is not limited, and those known and disclosed in this specification can be used. As long as the ability to bind to ASGPR is maintained, the structure of GalNAc can be modified. In addition, GalNAc with a protective group introduced during the preparation process is also included.

[0039] In the present specification, unless otherwise specified, the biantennary GalNAc unit refers to the GalNAc unit represented by the above formula (A1).

[0040] In this specification, the term "oligonucleotide" refers to a synthetic nucleic acid having a chain length of 100 bases or less, which may be single-stranded or double-stranded. As described later, DNA, RNA, natural nucleic acid, modified nucleic acid, etc. may be selectively used as the nucleic acid to be adopted according to the purpose, and these nucleic acids may be mixed in one oligonucleotide.

[0041] In this specification, the term "freeing condition" refers to the treatment conditions used when the bond between the support and the oligonucleotide is cleaved to free the oligonucleotide from the support during the synthesis of the oligonucleotide. In general solid phase synthesis, the freeing condition is alkaline.

[0042] In the present invention, "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 free conditions. For example, when the free conditions are alkaline, the protecting group is preferably removed under acidic conditions, and when the free conditions are acidic, the protecting group is preferably removed under alkaline conditions.

[0043] In this specification, the term "phosphoramidite group" refers to a amidite reagent used in nucleic acid synthesis consisting of -P(OR)N(CH(CH 3 ) 2 ) 2 (where R is a C1-C3 alkyl or cyano-C1-C3 alkylene) represented by a functional group. Preferably, examples thereof include -P(OCH 2 CH 2 CN)N(CH(CH 3 ) 2 ) 2 and -P(OCH 3 )N(CH(CH 3 ) 2 ) 2 , but are not limited to these examples.

[0044] In the present specification, "H-phosphonate group" is a functional group represented by -PH(=O)(OH) or a salt thereof used in nucleic acid synthesis. A compound having an H-phosphonate group can be combined with -PH(=O)(OH) - ) together to form a salt. Examples of such salts may include metal salts; the metal salts include alkali metal salts such as sodium salts, potassium salts and lithium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, iron salts, zinc salts, copper salts, nickel salts and cobalt salts; amine salts include inorganic salts such as ammonium salts and organic salts such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenylethylamine salts, piperazine salts, tetramethylammonium salts and triethylamine salts. (Hydroxymethyl) aminomethane salt; inorganic acid salts include hydrohalides such as hydrofluoride, hydrochloride, hydrobromide and hydroiodide, nitrate, perchlorate, sulfate and phosphate; organic acid salts include lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate, aryl sulfonates such as benzenesulfonate and p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate and maleate; and amino acid salts such as glycine, lysine, arginine, ornithine, glutamate and aspartate, preferably organic salts, more preferably triethylamine salts. These salts can be prepared by known methods.

[0045] In the present specification, the terms "bound through a phosphate group" and "structure containing a phosphate group" in the binding between nucleotides or the structure of a nucleotide and a conjugate refer to a structure in which two structural units are bound in a binding manner commonly used in nucleic acid synthesis, such as a phosphodiester bond or a modified form thereof (such as a phosphorothioate bond), or a structure containing such a binding manner.

[0046] <1. Oligonucleotide> In the present specification, the term "nucleic acid", "oligonucleotide" or "polynucleotide" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in any of the forms of single-stranded, double-stranded and triple-stranded chains.

[0047] Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses conservatively modified variants (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, and also includes the sequence explicitly indicated.

[0048] 1-1. Types of nucleic acids Nucleic acid of the present invention may include any form known to those skilled in the art. Specific examples of nucleic acid forms may include single-stranded DNA, single-stranded RNA, and single-stranded oligonucleotides in which DNA and RNA are mixed. Specific examples of other forms of nucleic acid may include double-stranded DNA, double-stranded RNA, DNA-RNA hybrid polynucleotides, and double-stranded polynucleotides consisting of two oligonucleotides in which DNA and RNA are mixed.

[0049] Examples of nucleosides or nucleotides constituting the nucleic acid of the present invention include chemically modified modified nucleosides or modified nucleotides in addition to natural nucleosides or nucleotides. Examples of modified nucleosides / nucleotides include sugar-modified nucleosides / nucleotides, core base-modified nucleosides / nucleotides, backbone-modified nucleosides / nucleotides or combinations thereof (e.g., see Nucleic Acid Research, 1997, Vol. 25, No. 22, 4429-4443).

[0050] 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 the nucleoside forms an ester with phosphoric acid. In this specification, oligonucleotide and polynucleotide are used with the same meaning.

[0051] In this specification, 2'-deoxyadenosine may be referred to as A t , 2'-deoxyguanosine can be called G t , 2'-deoxycytidine can be called C t2'-deoxy-5-methylcytidine can be referred to as 5meC t , thymidine can be called T t , 2'-deoxyuridine can be called U t , adenosine can be called A rt , guanosine can be called G rt , cytidine can be called C rt , 5-methylcytidine can be referred to as 5meC rt , and uridine can be called U rt In this specification, 2'-deoxyadenosine nucleotide may be referred to as A p , 2'-deoxyguanosine nucleotide can be called G p , 2'-deoxycytidine nucleotide can be called C p , 2'-deoxy-5-methylcytidine nucleotide can be referred to as 5meC p , thymidine nucleotide can be called T p , 2'-deoxyuridine nucleotide can be called U p , adenosine nucleotide can be called A rp , guanosine nucleotide can be called G rp , cytidine nucleotide can be called C rp , 5-methylcytidine nucleotide can be referred to as 5meC rp , and uracil nucleotides can be referred to as U rp .

[0052] In this manual, p The corresponding phosphothioate salt that replaces the phosphate in the nucleotide can be called A s , and G p The corresponding ones can be called G s , and C p The corresponding ones can be called C s , with 5meC p The corresponding ones can be called 5meC s , and T p The corresponding ones can be called T s , and U p The corresponding ones can be called U s , and A rp The corresponding ones can be called A rs , and G rp The corresponding ones can be called G rs , and C rp The corresponding ones can be called C rs , with 5meC rp The corresponding ones can be called 5meC rs , and with U rp The corresponding ones can be called U rs .

[0053] In the present specification, "sugar-modified nucleosides" refer to nucleosides in which the sugar portion of the nucleoside is modified. Examples of sugar-modified nucleosides include 2'-O-methyl nucleosides, 2'-O,4'-C-ethylene nucleosides (ENA) and 2'-O,4'-C-methylene nucleotides (BNA / LNA).

[0054] Examples of 2'-O-methyl modifications include 2'-O-methyl nucleosides and 2'-O-methyl nucleotides. rt The corresponding ones can be called A m1t , and G rt The corresponding ones can be called G m1t , and C rt The corresponding ones can be called C m1t , with 5meC rt The corresponding ones can be called 5meC m1t , and U rt The corresponding ones can be called U m1t , and A rp The corresponding ones can be called A m1p , and G rp The corresponding ones can be called G m1p , and C rp The corresponding ones can be called C m1p , with 5meC rp The corresponding ones can be called 5meC m1p , and U rp The corresponding ones can be called U m1p , and A rs The corresponding ones can be called A m1s , and G rs The corresponding ones can be called G m1s , and C rs The corresponding ones can be called C m1s , with 5meC s The corresponding ones can be called 5meC m1s , and with U rs The corresponding ones can be called U m1s .

[0055] In the sequence listing attached to this specification, "cm" represents 2'-O-methylcytidine, "um" represents 2'-O-methyluridine, and "gm" represents 2'-O-methylguanosine. <223> middle.

[0056] In the present specification, 2'-0,4'-C-ethylene nucleotide unit and "ENA unit" refer to each of the above nucleosides and nucleotides having ENA. Nucleosides and nucleotides having ENA units can be expressed, for example, with A t The corresponding ones can be called A 2t , and Ap The corresponding ones can be called A e2p , and A s The corresponding ones can be called A e2s , and G t The corresponding ones can be called G 2t , and G p The corresponding ones can be called G e2p , and G s The corresponding ones can be called G e2s , with 5meC t The corresponding ones can be called C 2t , with 5meC p The corresponding ones can be called C e2p , with 5meC s The corresponding ones can be called C e2s , and T t The corresponding ones can be called T 2t , and T p The corresponding ones can be called T e2p , and T s The corresponding ones can be called T e2s .

[0057] In the present specification, 2'-0,4'-C-methylene nucleotide unit and "2',4'-BNA / LNA unit" refer to each of the above nucleosides and nucleotides having 2',4'-BNA / LNA. Nucleosides and nucleotides having 2',4'-BNA / LNA units can be expressed, for example, with A t The corresponding ones can be called A 1t , and A p The corresponding ones can be called A e1p , and A s The corresponding ones can be called A e1s , and G t The corresponding ones can be called G 1t , and G p The corresponding ones can be called G e1p , and G s The corresponding ones can be called G e1s , with 5meC t The corresponding ones can be called C 1t , with 5meC p The corresponding ones can be called C e1p , with 5meC s The corresponding ones can be called C e1s , and T t The corresponding ones can be called T 1t , and T p The corresponding ones can be called T e1p , and Ts The corresponding ones can be called T e1s .

[0058] 1-2. Target genes In this specification, as long as it is RNA in the cell, tissue or individual into which the target gene is introduced (hereinafter, it may be referred to as "introduction object"), "target gene" is not particularly limited, and may be mRNA to be translated into protein or non-coding RNA that is not translated into protein. Examples of non-coding RNA include functional RNA, such as mRNA, tRNA, rRNA, mRNA-type ncRNA (mRNA-type non-coding RNA), long-chain ncRNA (long non-coding RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA) and miRNA (micro RNA) non-translation region. Specifically, the target gene may be endogenous or exogenous to the introduction object, so as to be introduced by technology such as gene transfer. In addition, it may be a gene present on a chromosome or an extrachromosomal gene. Examples of exogenous genes include those derived from viruses, bacteria, fungi or protozoa that can infect the introduction object, but are not limited to these examples. The function of the gene may be known or unknown.

[0059] The example of target gene can include the gene of expression with specific increase in liver and / or the gene of specific mutation in the patient with specific disease in liver.The example of such disease can include cardiovascular disease (for example, hypertension, cardiac hypertrophy, angina pectoris, arteriosclerosis and hypercholesterolemia), cancer (for example, liver cancer), respiratory disease (for example, pneumonia, bronchitis, asthma, chronic obstructive pulmonary disease and pulmonary fibrosis), diabetes, diabetic retinopathy, diabetic nephropathy, anemia (for example, anemia related to chronic disease and iron-refractory iron deficiency anemia), age-related macular degeneration, immune system disease (for example, autoimmune disease, immunodeficiency and leukemia), liver / gallbladder disease (for example, non-alcoholic fatty hepatitis, liver cirrhosis, hepatitis, liver failure, cholestasis and calculus), digestive tract disease (for example, ulcer, enteritis and malabsorption), infection, obesity and fibrosis (for example, liver fibrosis).

[0060] Examples of causative genes for these diseases may include kinesin spindle protein (KSP), vascular endothelial growth factor (VEGF), transthyretin (TTR), proprotein convertase subtilisin / kexin PCSK9, polo kinase 1 (PLK-1), ApoB-100, ribonucleotide reductase M2 subunit (RRM2), clusterin, heat shock protein 27 (Hsp27), survivin, eukaryotic initiation factor-4E (eIF-4E), interleukin-4 receptor alpha subunit (IL-4R-α), factor XI, 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 peptide 3, X-linked), myeloid leukemia sequence 1 (MCL1) gene, PKR (Eif2ak2), Hsp47 ( Serpinh1), hepcidin, activated protein C (APC), signal transducer and activator of transcription (STAT3), collagen, type I, alpha 1 (Col1A1), ApoC-III, angiopoietin type 3 protein (ANGPTL3), growth hormone receptor (GHr), prekallikrein (PKK), transmembrane protease, serine 6 (TMPRSS6), lipoprotein (a), glucagon receptor 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 complement, alpha-1 antitrypsin (AAT), and hepatitis B virus (HBV) genome, but are not limited to these examples.

[0061] 1-3. Double-stranded oligonucleotides In the case where the nucleic acid of the present invention is a nucleic acid having an RNA interference effect on a target gene, as long as it has the RNA interference effect of the nucleic acid, its structure and chemical modification are not limited. Examples thereof may include siRNA (e.g., see International Publication No. WO 2002 / 044321, Current Opinion in Chemical Biology 570-579), AtuRNAi consisting of an oligonucleotide to which RNA and 2'-OMeRNA are alternately bound (e.g., see International Publication No. WO 2004 / 015107), a double-stranded oligonucleotide in which a nucleic acid having different types of sense strands and antisense strands forms a double strand with a Watson-Crick base pair in an oligonucleotide to which DNA and 2'-OMeRNA are alternately bound (which will be described in Section 3-4-1 below (e.g., see International Publication No. WO 2010 / 001909)), a nucleic acid with a modified oligonucleotide end (which will be described in Section 3-4-1 below (e.g., see International Publication No. WO 2010 / 052715)), and a single-stranded oligonucleotide having a single strand by binding through a linker between the 5' end of the antisense strand polynucleotide and the 3' end of the sense strand and having a double-stranded structure by forming a Watson-Crick base pair in the molecule (which will be described in Section 3-4-3 (for example, see International Publication No. WO 2012 / 074038)).

[0062] In this specification, the phrase "consisting of the same nucleotide sequence as the target gene" means that it is composed of the same sequence as at least a portion of the nucleotide sequence of the target gene, including not only completely identical sequences, but also substantially identical nucleotide sequences, as long as RNA interference and / or gene expression inhibition are exerted on the target gene. The phrase "consisting of a nucleotide sequence complementary to the target gene" means that it is composed of a sequence complementary to at least a portion of the nucleotide sequence of the target gene, including not only completely complementary sequences, but also substantially identical sequences, as long as RNA interference and / or gene expression inhibition are exerted on the target gene. In addition, in the case where the SNP in the target gene is known, the sequence with such a mutation is also included in the same nucleotide sequence. In this specification, a polynucleotide containing a nucleotide sequence complementary to the target gene and having RNA interference and / or gene expression inhibition on the target gene is referred to as an oligonucleotide for a target gene.

[0063] 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 the target gene, but can be determined by, for example, using computer software (e.g., GENETYX(R): available from GENETYX COORPORATION) to determine the sequences of the sense strand and the antisense strand based on the sequence expected to have an RNA interference effect on the target gene, or further confirming the RNA interference effect and / or gene expression inhibitory effect of the oligonucleotide generated based on the selected sequence.

[0064] The chain lengths of the sense strand and the antisense strand of the double-stranded oligonucleotide with RNA interference effect can each be any length from 10 nucleotides to the full length of the open reading frame (ORF) of the target gene, as long as the RNA interference effect and / or gene expression inhibition effect is exerted, and are preferably any length from 18 nucleotides to the full length of the open reading frame (ORF) of the target gene, further preferably 10 to 100 nucleotides, and further preferably 15 to 30 nucleotides.

[0065] In the case where a double-stranded oligonucleotide in which a sense strand and an antisense strand of a nucleic acid having different types are used to form a Watson-Crick bound oligonucleotide in which DNA and 2'-OMeRNA are alternately bound (International Publication No. WO 2010 / 001909) as a double-stranded oligonucleotide having an RNA interference effect, the sense strand preferably has a chain length of 18 to 21, and more preferably 18 to 19. The antisense strand preferably has a chain length of 19 to 21, and more preferably 21. In addition, the entire structure does not necessarily have a double-stranded structure, and also includes those that protrude at the 5' and / or 3' end portions. The protruding end includes 1 to 5 nucleotides, preferably 1 to 3 nucleotides, and more preferably 2 nucleotides. In addition, the most preferred example is a polynucleotide having a structure in which the 3' end of the oligonucleotide of the antisense strand protrudes by 2 nucleotides (protruding end structure) and forms 18 base pairs.

[0066] 1-3-1. Oligonucleotides with alternating binding of DNA and 2'-OMeRNA As an example of the nucleic acid of the present invention to be contained in the nucleic acid lipid particle, there can be mentioned a double-stranded oligonucleotide in which nucleic acids having different types of sense and antisense strands form Watson-Crick binding in an oligonucleotide in which DNA and 2′-OMeRNA are alternately bound.

[0067] Specific examples of such a double-stranded oligonucleotide include double-stranded oligonucleotides disclosed in International Publication No. WO 2010 / 001909, International Publication No. WO 2010 / 001909, and the like.

[0068] 1-3-2. Modified double-stranded oligonucleotide In the case of using a nucleic acid with RNA interference as a nucleic acid to be included in the nucleic acid lipid particle, a nucleic acid in which the end of the oligonucleotide is modified can be mentioned as an example thereof, as long as it has RNA interference. For example, a double-stranded oligonucleotide, wherein the phosphate group at the 5' end of the antisense strand in the double-stranded oligonucleotide with RNA interference can be mentioned to be modified to a 5'-aryl phosphate group (e.g., see International Publication No. WO 2010 / 052715), such as a nucleic acid having different types of sense strands and antisense strands, wherein siRNA, AtuRNAi or DNA and 2'-OMeRNA are alternately bound to form a double-stranded oligonucleotide of Watson-Crick binding (e.g., see International Publication No. WO2010 / 001909).

[0069] Specific examples of such modified double-stranded oligonucleotides include modified double-stranded oligonucleotides disclosed in International Publication No. WO 2010 / 052715 and International Publication No. WO 2015 / 005253.

[0070] 1-4. Single-stranded oligonucleotides 1-4-1. RNA interference single-stranded nucleotide Examples of nucleic acids used in the present invention also include oligonucleotides having a sense strand oligonucleotide for a target gene and an antisense strand oligonucleotide having a nucleotide sequence complementary to the sense strand, and having a single-stranded structure bonded by a linker to form a phosphodiester structure at the 5' end of each antisense strand oligonucleotide and the 3' end of the sense strand oligonucleotide (for example, see International Publication No. WO 2012 / 074038), as long as it has an RNA interference effect.

[0071] Specific examples of such compounds may include those described in International Publication No. WO 2012 / 074038 and International Publication No. WO 2015 / 005253.

[0072] 1-4-2. Antisense nucleotides In the treatment of diseases caused by gene mutations, antisense nucleotides are used to treat diseases that can be treated by normalizing or inhibiting the expression of mutant genes. In the case of inhibiting gene expression, a nucleotide having a sequence complementary to a portion of the mRNA of the target gene and a nucleic acid in the middle of the sequence is a DNA nucleotide. Such antisense nucleotides for inhibiting expression form a double strand with the mRNA of the target gene. Since the RNA / DNA double-stranded structure in the formed double strand is specifically decomposed by RNaseH in vivo, the mRNA of the target gene is specifically destroyed, thereby inhibiting the expression of the target gene.

[0073] The example of antisense nucleotides intended to normalize gene expression includes those that suppress abnormal splicing and those that restore the mutation site on the gene to the normal sequence. In the case of restoring the mutation on the gene to the normal sequence, the 3' side of the antisense nucleotide includes the antisense sequence of the 5' side region of the mutation site in the sense strand of the target gene, and the sequence of the hairpin structure that the nuclease associates with it is included on its 5' side (the sequence where the sequences complementary to each other at 2 sites are connected by the intermediate joint / loop sequence). With the help of such nucleotides, the antisense region on the 3' side plays the role of guide RNA, which guides the nuclease to the mutation site (the sense strand of mRNA or genomic DNA) of the target gene. This phenomenon is similar to the genome editing technology known as the CRISPR-Cas system, and is widely considered to be applicable to various gene editing.

[0074] The mechanism of abnormal splicing due to gene mutation is as follows. During the splicing process from pre-mRNA to mature mRNA, the mutation causes the splicing factor to accidentally recognize and bind to a site that does not bind in the normal pre-mRNA, so that the mRNA that undergoes abnormal splicing is expressed, thereby causing the function failure or expression failure of the protein expressed by the target gene. When the antisense nucleotide binds to the region on the mutant pre-mRNA that is misrecognized by the splicing factor, the splicing factor cannot recognize the region and thus inhibits abnormal splicing. Examples of diseases caused by abnormal splicing caused by gene mutations include antisense oligonucleotides that inhibit abnormal splicing caused by the c.648G>T mutation in the G6PC gene of type 1a glycogen storage disease described in detail below, as well as other antisense oligonucleotides that can be mentioned for the treatment of various known diseases.

[0075] The present invention provides an oligonucleotide or a pharmaceutically acceptable salt thereof that can repair the G6PC gene with c.648G>T mutation in patients with type Ia glycogen storage disease at the mRNA level and express normal G6PC protein. The oligonucleotide of the present invention is an oligonucleotide having 15 to 30 bases consisting of a nucleotide sequence complementary to the cDNA of the G6PC gene with c.648G>T mutation, and is composed of a sequence complementary to any one of the 82nd to 92nd sites of the 5' end of exon 5 of the G6PC gene with c.648G>T mutation.

[0076] Among patients with type Ia glycogen storage disease, patients with c.648G>T mutations can be targeted in the present invention. Not only homozygous patients with c.648G>T mutations can be targeted, but also compound heterozygous patients with c.648G>T mutations and other mutations can be targeted.

[0077] The c.648G>T mutation of the G6PC gene is a mutation from G to T at nucleotide position 728 in the Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). It is located at position 86 of the 5' end of exon 5. The oligonucleotide of the present invention may be composed of a sequence complementary to any one of positions 82 to 92 of the 5' end of exon 5 of the G6PC gene having the c.648G>T mutation, preferably composed of a sequence complementary to a region including position 92.

[0078] Examples of oligonucleotides having 15 to 30 bases 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 from the 82nd position to the 92nd position at the 5' end of exon 5 of the G6PC gene having the c.648G>T mutation, may include those containing all or part of any sequence of SEQ ID Nos: 1 to 32, 40 to 42, and 44 to 48 (assuming that t or T may be u or U, or u or U may be t or T in the sequence). In the present invention, the term "part of a sequence" is generally 80% or more of the entire sequence, preferably 85%, further preferably 90%, and most preferably 94% or more. The oligonucleotide of the present invention appropriately has 15 to 30, preferably 15 to 21, and more preferably 15 to 18 bases.

[0079] 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 preferred that at least one of the nucleotides is a modified nucleotide.

[0080] Examples of modified nucleotides in the present invention may include those in which the sugar is modified (e.g., those in which D-ribofuranose is 2'-O-alkylated, those in which D-ribofuranose is 2'-O,4'-C-alkylated, and those in which D-ribofuranose is 2'-,4'-cross-linked), those in which the phosphodiester bond is modified (e.g., thiolated), those in which the base is modified, and combinations thereof. Those in which at least one D-ribofuranose constituting the antisense oligonucleotide is 2'-O-alkylated or 2'-O,4'-C-alkylated can be expected to have a higher therapeutic effect than natural nucleotides (i.e., oligoDNA and oligoRNA) due to their high binding affinity to RNA and high tolerance to nucleases. In addition, those in which at least one phosphodiester bond constituting the oligonucleotide is thiolated can also be expected to have a higher therapeutic effect than natural nucleotides (i.e., oligoDNA and oligoRNA) due to their high tolerance to nucleases. Oligonucleotides containing both the modified sugar and the modified phosphodiester bond described above have higher resistance to nucleases and thus can be expected to have higher therapeutic effects.

[0081] Examples of sugar modifications in the oligonucleotides of the present invention may include 2'-O-alkylation (e.g., 2'-O-methylation, 2'-O-aminoethylation, 2'-O-propylation, 2'-O-allylation, 2'-O-methoxyethylation, 2'-O-butylation, 2'-O-pentylation, and 2'-O-propargylation) of D-ribofuranose, 2'-O,4'-C-alkylidene (e.g., 2'-O,4'-C-ethylidene, 2'-O,4'-C-isopropylation ... methylation, 2'-O,4'-C-propylidene, 2'-O,4'-C-tetramethylidene and 2'-O,4'-C-pentamethyleneation), 2'-deoxy-2'-C,4'-C-methyleneoxymethylidene 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.

[0082] Examples of sugar 2'-, 4'-cross-linking modifications in the oligonucleotides of the present invention may include 2'-O, 4'-C-alkylidene (e.g., 2'-O, 4'-C-ethylidene), 2'-O, 4'-C-methylidene, 2'-O, 4'-C-propylidene, 2'-O, 4'-C-tetramethylidene and 2'-O, 4'-C-pentamethylene) of D-ribofuranose, 2'-deoxy-2'-C, 4'-C-methyleneoxymethylidene, S-cEt (2', 4'-constrained ethyl) and AmNA.

[0083] Examples of modifications of the phosphodiester bond in the oligonucleotide of the present invention may include a phosphorothioate bond, a methylphosphonate bond, a methylphosphonothioate bond, a phosphorodithioate bond, and a phosphoramidate bond.

[0084] In the present invention, examples of base modifications may include 5-methylation, 5-fluorination, 5-bromination, 5-iodination and N4-methylation of cytosine, 5-demethylation (uracil), 5-fluorination, 5-bromination and 5-iodination of thymine, N6-methylation and 8-bromination of adenine, and N2-methylation and 8-bromination of guanine.

[0085] Oligonucleotides of the present invention can be synthesized according to the method described in the literature (Nucleic Acids Research, 12, 4539 (1984)), using a commercially available synthesizer (e.g., Model 392, available from PerkinElmer, Inc., by the phosphoramidite method). As the nucleoside phosphoramidite reagent used therein, commercially available reagents can be used for natural nucleosides and 2'-O-methyl nucleosides (i.e., 2'-O-methylguanosine, 2'-O-methyladenosine, 2'-O-methylcytidine and 2'-O-methyluridine). 2'-O-alkylguanosine, adenosine, cytidine and uridine with an alkyl group containing 2 to 6 carbon atoms can be synthesized as follows.

[0086] 2'-O-aminoethylguanosine, 2'-O-aminoethyladenosine, 2'-O-aminoethylcytidine and 2'-O-aminoethyluridine can be synthesized according to the literature (Blommers et al. Biochemistry (1998), 37, 17714-17725).

[0087] 2'-O-propylguanosine, 2'-O-propyladenosine, 2'-O-propylcytidine and 2'-O-propyluridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838).

[0088] Commercially available reagents can be used for 2'-O-allylguanosine, 2'-O-allyladenosine, 2'-O-allylcytidine, and 2'-O-allyluridine.

[0089] 2'-O-methoxyethylguanosine, 2'-O-methoxyethyladenosine, 2'-O-methoxyethylcytidine and 2'-O-methoxyethyluridine can be synthesized according to patent (US6261840) or literature (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504).

[0090] 2'-O-Butylguanosine, 2'-O-butyladenosine, 2'-O-butylcytidine and 2'-O-butyluridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838).

[0091] 2'-O-pentylguanosine, 2'-O-pentyladenosine, 2'-O-pentylcytidine and 2'-O-pentyluridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838).

[0092] Commercially available reagents can be used for 2'-O-propargylguanosine, 2'-O-propargyladenosine, 2'-O-propargylcytidine, and 2'-O-propargyluridine.

[0093] 2'-O,4'-C-methyleneguanosine, 2'-O,4'-C-methyleneadenosine, 2'-O,4'-C-methylenecytidine, 2'-O,4'-C-methylene-5-methylcytidine and 2'-O,4'-C-methylenethymidine can be prepared according to the method described in International Publication No. WO 99 / 14226, and 2'-O,4'-C-alkyleneguanosine, 2'-O,4'-C-alkyleneadenosine, 2'-O,4'-C-alkylenecytidine, 2'-O,4'-C-alkylene-5-methylcytidine and 2'-O,4'-C-alkylenethymidine having an alkylene group containing 2 to 5 carbon atoms can be prepared according to the method described in International Publication No. WO 00 / 47599.

[0094] Nucleosides in which D-ribofuranose is methylenated with 2'-deoxy-2'-C,4'-C-methyleneoxy can be synthesized according to the literature (Wang, G. et al. Tetrahedron (1999), 55, 7707-7724).

[0095] S-cEt (constrained ethyl group) can be synthesized according to the literature (Seth, PP et al. J. Org. Chem (2010), 75, 1569-1581).

[0096] AmNA can be synthesized according to the literature (Yahara, A. et al. ChemBioChem (2012), 13, 2513-2516.) or International Publication No. WO 2014 / 109384.

[0097] In the present invention, in the nucleic acid nucleotide sequence, adenine can be recorded as (A) or (a), guanine can be recorded as (G) or (g), cytosine can be recorded as (C) or (c), thymine can be recorded as (T) or (t), and uracil can be recorded as (U) or (u). 5-methylcytosine can be used instead of cytosine. In the nucleobase, uracil (U) or (u) and thymine (T) or (t) are compatible with each other. Uracil (U) or (u) and thymine (T) or (t) can both be used to form base pairs with adenine (A) or (a) of a complementary chain.

[0098] Antisense oligonucleotides having phosphorothioate bonds can be synthesized by coupling nucleoside phosphoramidite reagents and then reacting with sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or reagents such as hydroxanthane (Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc. 112, 1253 (1990), PCT / International Publication No. WO 98 / 54198).

[0099] As the controlled pore glass (CPG) used in the synthesizer, a commercially available product to which 2′-O-methyl nucleoside is bound can be used. In addition, for 2'-O,4'-C-methyleneguanosine, 2'-O,4'-C-methyleneadenosine, 2'-O,4'-C-methylene-5-methylcytidine and 2'-O,4'-C-methylenethymidine, nucleosides prepared according to the method described in International Publication No. WO 99 / 14226 can be bound to CPG, and for 2'-O,4'-C-alkyleneguanosine, 2'-O,4'-C-alkyleneadenosine, 2'-O,4'-C-alkylene-5-methylcytidine and 2'-O,4'-C-alkylenethymidine having an alkylene group containing 2 to 5 carbon atoms, nucleosides prepared according to the method described in International Publication No. WO 00 / 47599 can be bound thereto according to the document (Oligonucleotide Synthesis, Edited by MJ Gait, Oxford University Press, 1984). The oligonucleotide in which the 2-hydroxyethyl phosphate group is bound to the 3' end can be synthesized by using modified CPG (disclosed in Example 12b of Japanese Patent Publication No. 7-87982). In addition, the oligonucleotide in which the hydroxyalkyl phosphate group or the aminoalkyl phosphate group is bound to the 3' end can be synthesized by using 3'-amino-modifier C3 CPG, 3'-amino-modifier C7 CPG, GlycerylCPG (Glen Research), 3'-specer C3 SynBase CPG 1000 or 3'-specer C9 SynBase CPG1000 (Link Technology).

[0100] <2. GalNAc unit> In the conjugate of the present invention, the oligonucleotide may have GalNAc bound via a linker and a phosphate group.

[0101] The GalNAc unit in the present invention is a phosphate group that is bound to a linker to which GalNAc is bound, and may have a further bond. The phosphate group of the GalNAc unit may be bound to the 5' end and / or the 3' end of the oligonucleotide, preferably, to the 5' end of the oligonucleotide. Where the GalNAc unit has a bond, the bond may be bound to a hydroxyl, GalNAc, a linker to which GalNAc is bound, a phosphate group of another GalNAc unit, or a phosphate group at the 3' end of the oligonucleotide. A GalNAc unit contains two GalNAcs.

[0102] In the conjugate, one GalNAc unit may be bound to the oligonucleotide, or a plurality of GalNAc units may be bound to the oligonucleotide in series. 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 most preferably 1.

[0103] In the case where two GalNAc units are introduced into the 5' end of the oligonucleotide of the present invention, SymmetricDoubler Phosphoramidite (1,3-bis-[5-(4,4'-dimethoxytrityloxy)pentylamino]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, Glen Research) and the like can be used when synthesizing the oligonucleotide, and in the case where three GalNAc units are introduced into the 5' end of the oligonucleotide, Trebler Phosphoramidite (tris-2,2,2-[3-(4,4'-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, Glen Research), Long Trebler Phosphoramidite (tris-2,2,2-[3-(4,4'-dimethoxytrityloxy)propoxymethyl]methyleneoxypropyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, Glen Research) and the like can be used in the synthesis of oligonucleotides. In the case where 4 GalNAc units are introduced into the 5' end of the oligonucleotide, Symmetric Doubler Phosphoramidite coupled twice and the like can be used in the synthesis of oligonucleotides. In the case where 5 GalNAc units are introduced into the 5' end of the oligonucleotide, Symmetric Doubler Phosphoramidite and Trebler Phosphoramidite or Long Trebler Phosphoramidite each coupled once can be used in the synthesis of oligonucleotides. In the case where 5 or more GalNAc units are introduced into the 5' end of the oligonucleotide, Symmetric Doubler Phosphoramidite, Trebler Phosphoramidite, or Long Trebler Phosphoramidite may be appropriately combined for synthesis.

[0104] In the case where such a GalNAc unit is introduced into the 3' end of the oligonucleotide in the present invention, Asymmetric Doubler (Lev) Phosphoramidite (1-[5-(4,4'-dimethoxytrityloxy)pentylamino]-3-[5-levulinyloxypentylamino]-propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, Glen Research) or the like can be used when synthesizing the oligonucleotide. Specifically, after coupling a commercially available Unylinker Support (e.g., Glen UnySuppor 1000, Glen Research) with Asymmetric Doubler (Lev) Phosphoramidite to remove the DMTr group, the chain of the oligonucleotide of interest is extended. After reaching the desired chain length, the levulinyl group can be deprotected using a reagent such as hydrazine and the GalNAc unit is coupled according to the protocol attached to the Asymmetric Doubler (Lev) Phosphoramidite reagent to introduce it into the 3' end of the oligonucleotide.

[0105] In the present invention, the additional oligonucleotide sequence is a nucleotide sequence different from the nucleotide sequence of the oligonucleotide that exerts a pharmaceutical effect, which is composed of a phosphodiester bond and can be cut in vivo, and can be included in the 5' end and / or 3' end of the oligonucleotide bound to the GalNAc unit. The chain length of the cleavable oligonucleotide is preferably 1 to 6 nucleotides, and further preferably 1 to 3 nucleotides. The oligonucleotide that can be cut is not particularly limited, as long as it can be cut, but examples thereof may include natural oligodeoxynucleotides composed entirely of DNA and natural oligonucleotides composed entirely of RNA. The nucleotide sequence is not particularly limited, as long as it is a sequence that can be cut, but examples thereof may include 5'-TCATCA-3', 5'-CATCA-3', 5'-ATCA-3', 5'-TCA-3', 5'-CA-3' and 5'-A-3'.

[0106] In one aspect, the biantennary GalNAc unit of the present invention is a group represented by the formula: [Formula 44] Wherein W is a group represented by the following formula: [Formula 45] G represents 5-acetylamino-2-hydroxymethyl-3,4-dihydroxytetrahydropyran-6-yl (GalNAc); Z represents an oxygen atom or a sulfur atom; L 1 and L 2 One of the two represents a methylene group (CH2 ), and the other represents no intervening atom. o, q, r and s each independently represent 0 or 1; n represents an integer from 1 to 15; m represents an integer from 0 to 5; and v represents an integer from 1 to 7, provided that when n is 1, then m is an integer from 0 to 5, and when n is an integer from 2 to 15, then m is 0, preferably o and q are each 0.

[0107] In the above formula, preferably L 2 represents methylene (CH 2 ), and L 1 Indicates that L 1 and L 2 No atoms are inserted in it. Preferably, n is an integer of 1 to 10, more preferably an integer of 2 to 7, and further preferably an integer of 2 to 4. Preferably, m is an integer of 0 to 3. Preferably, v is an integer of 1 to 5. More preferably, 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 further preferably, it is a group wherein s is 1, n is 2 or 3, m is 0, and v is an integer of 1 to 3 in the group represented by the above formula.

[0108] The biantennary GalNAc units of the present invention preferably have a biantennary structure, each having a glycerol structure within 3 atoms of the phosphate group, and having GalNAc at each end of the branch, wherein each linker structure from the branch point to the GalNAc is almost the same, the linker backbone connecting the GalNAc portion and the phosphate group portion has a length of 15 atoms or less, a high heteroatom content, and a continuous alkyl chain length of C5 or less, thereby exhibiting good solubility in water.

[0109] Specific examples of the biantennary GalNAc unit of the present invention may include a group represented by the following formula: [Formula 46] [Formula 47] [Formula 48] [Formula 49] and [Equation 50]

[0110] <3. Method for preparing conjugate> The oligonucleotide bound to the GalNAc unit of the present invention can be synthesized according to the method described in the literature (Nucleic Acids Research, 12, 4539 (1984)), using a commercially available synthesizer (e.g., Model 392, available from PerkinElmer, Inc., by the phosphoramidite method) or the like, and by using the phosphoramidite contained in the GalNAc unit in the same manner as the nucleoside phosphoramidite reagent.

[0111] Method for synthesizing phosphoramidites containing GalNAc units Phosphoramidites or H-phosphonates containing the GalNAc unit of the present invention can be prepared according to method A or method B described below. In method A or method B, after completing the reaction according to conventional methods, the target compound of each reaction is collected from the reaction mixture. For example, the target compound is obtained by appropriately neutralizing the reaction mixture, filtering out the insoluble matter (if present), then adding water and an immiscible organic solvent such as ethyl acetate thereto, separating the organic layer containing the target substance, washing it with water, etc., drying it with anhydrous sodium sulfate, and evaporating the solvent. If necessary, the obtained compound can be separated / purified by conventional methods, such as silica gel column chromatography. Alternatively, it can be purified by reprecipitation and recrystallization. The condition is that if r=0, method C is used instead of method A or method B.

[0112] [Method A] Method A is a method for preparing branched compounds (7) and compounds (7') using compound (3). The configuration of compound (1) is not limited. By using compound (3), the protective group of the 6-hydroxyl group of the GalNAc moiety can be appropriately selected. If r = 0, method C is used.

[0113] [Formula 51] Where R 13 is a conventional protecting group for hydroxyl, but is preferably a benzyl group, R 14 is a conventional protecting group for hydroxyl, but is preferably an acetyl group or a hydrophobic protecting group (more preferably 4,4'-dimethoxytrityl), R 15 is a conventional protecting group for the carbonyl group, but is preferably a benzyl group, in -P(R 3 )R 4 In, R 3 and R 4may be the same or different and each represents 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 by an alkyl group having 1 to 4 carbon atoms, X is carbon or oxygen, n is an integer from 1 to 15, m is an integer from 0 to 5, provided that when n is 1, m is an integer from 0 to 5, and when n is an integer from 2 to 15, m is 0, r is independently 0 or 1, and v is an integer from 1 to 7.

[0114] Step A-1 is a carbamate formation step, in which compound (1) is converted into an active ester in an inert solvent in the presence of an activating agent and a base, and then reacted with compound (2) for carbamate formation.

[0115] Examples of the inert solvent used in the reaction include hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons and ethers, preferably dichloromethane.

[0116] The activator used in the 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. Preferably, the activator is 4-nitrophenyl chloroformate.

[0117] Examples of the base used in the reaction include triethylamine, N,N-diisopropylethylamine, pyridine and 4-dimethylaminopyridine, and it is preferred to use pyridine when preparing the active ester and to add N,N-diisopropylethylamine when reacting with an amine thereafter.

[0118] The reaction temperature varies depending on the activating agent, base, inert solvent, etc., but is generally -20°C to reflux temperature. Preferably, the reaction temperature is 10°C to 30°C.

[0119] The reaction time varies depending on the activating agent, the base, the inert solvent, the reaction temperature and the like, but is generally 15 minutes to 24 hours, preferably 1 hour to 4 hours.

[0120] Step A-2 is to remove R as a protecting group. 13 and R 15 The removal of the protecting group varies depending on its type, but can generally be carried out according to methods well known in the field of synthetic organic chemistry, such as those disclosed in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc.

[0121] Step A-3 is a step of conducting amide condensation with compound (3) in an inert solvent in the presence of a condensing agent and a base.

[0122] The inert solvent used in the reaction can be appropriately selected according to the type of the condensing agent to be used. Examples thereof include water, alcohols, and polar aprotic solvents, preferably dichloromethane or N,N-dimethylformamide.

[0123] Examples of the base used in the reaction include triethylamine, N,N-diisopropylethylamine, pyridine and 4-dimethylaminopyridine, with N,N-diisopropylethylamine being preferred.

[0124] Examples of the condensing agent used in the reaction include WSC (1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) and DIC (N,N'-diisopropylcarbodiimide) as carbodiimide condensing agents, CDI (N,N'-carbonyldiimidazole) as imidazole condensing agent, DMT-MM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine) as triazine condensing agent. Chloride n hydrate), HATU (O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) and TSTU (O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate) as urea condensation agents, and PyBOP (1H-benzotriazol-1-yloxytripyrrolidinophosphonium) as phosphonium condensation agents, preferably WSC or HATU. In addition, HOAt (1-hydroxy-7-azabenzotriazole), HOBt (1-hydroxybenzotriazole) and Oxyma (ethyl (hydroxyimino)cyanoacetate) can be added as additives as needed.

[0125] The reaction temperature varies depending on the condensing agent, base, solvent and the like, but is usually -20°C to reflux temperature. Preferably, it is 10°C to 30°C.

[0126] The reaction time varies depending on the condensing agent, base, solvent, reaction temperature and the like, but is generally 15 minutes to 72 hours, preferably 1 hour to 24 hours.

[0127] Step A-4 is a step for preparing compound (7), wherein -P(R 3 )R 4 Introduced into the secondary hydroxyl group of compound (6).

[0128] Examples of the inert solvent used in the reaction include hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers and acetonitrile, preferably dichloromethane.

[0129] Examples of the base used in the reaction include triethylamine, N,N-diisopropylethylamine, pyridine and 4-dimethylaminopyridine, with N,N-diisopropylethylamine being preferred.

[0130] The reaction temperature varies depending on the base, inert solvent and the like, but is usually -20°C to reflux temperature. Preferably, it is 10°C to 30°C.

[0131] The reaction time varies depending on the base, the inert solvent, the reaction temperature and the like, but is usually 15 minutes to 24 hours, preferably 1 hour to 4 hours.

[0132] Step A-5 is a step for synthesizing compound (8) from compound (7), wherein only R as a protecting group is removed. 15 The removal of the protecting group varies depending on its type, but can generally be carried out according to methods well known in the art of synthetic organic chemistry, for example, the method disclosed in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc.

[0133] Step A-6 is a step of subjecting compound (8) to amidation. It can be carried out in the same manner as in the above-mentioned Step A-3.

[0134] Step A-7 is to remove R as a protecting group. 13 The removal of the protecting group varies depending on its type, but can generally be carried out according to a method well known in the field of synthetic organic chemistry, for example, the method disclosed in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc.

[0135] Step A-8 is a step of synthesizing compound (7') as H-phosphonate from compound (6). H-phosphonate can be obtained, for example, by reacting 3-(1,2,4-triazolyl)phosphite (instead of phosphoramidite reagent) prepared in advance from phosphorus trichloride and 1,2,4-triazole according to the literature (BC Freohler, PGNg and MD.Matteucci, Nucleic Acids Res., 145399 (1986)) with compound (6) in an inert solvent, then adding water thereto to terminate the reaction, and post-processing. The solvent used is not particularly limited as long as it does not hinder the reaction, but preferably a halogenated hydrocarbon such as dichloromethane. The reaction temperature is not particularly limited in the range of -20°C to 100°C, but is generally room temperature. The reaction time varies depending on the solvent used and the reaction temperature, but the reaction time is 30 minutes in the case of reaction in dichloromethane at room temperature.

[0136] [Method B] Method B is a method for preparing branched compounds (14) and (14') using compound (3). The configuration of compound (10) is not limited. By using (3), the protective group of the 6-hydroxyl group of the GalNAc moiety can be appropriately selected. If r = 0, method C is used.

[0137] [Formula 52] R 12 is a conventional protecting group for hydroxyl, but is preferably a benzyl group. 3 , R 4 ,X,n,m,r,v,R 14 and R 15 Each has the same meaning as above.

[0138] Step B-1 is a step of subjecting compound (10) to carbamate conversion, and can be performed in the same manner as in the above-mentioned Step A-1.

[0139] Step B-2 is to remove R as a protecting group. 12 and R 15 Step B-2 can be performed in the same manner as the above-mentioned step A-2.

[0140] Step B-3 is a step of subjecting compound (12) to amidation, and can be performed in the same manner as in the above-mentioned Step A-3.

[0141] Step B-4 is to convert -P(R 3 )R 4 This is a step of introducing a hydroxyl group of compound (13), and can be performed in the same manner as in the above-mentioned step A-3.

[0142] Step B-5 is a step for synthesizing compound (15) from compound (11), wherein only R 15 Step B-5 can be performed in the same manner as the above-mentioned step A-5.

[0143] Step B-6 is a step of subjecting compound (15) to amidation. Step B-6 can be carried out in the same manner as in the above-mentioned Step A-6.

[0144] Step B-7 is to remove R as a protecting group. 12 Step B-7 can be performed in the same manner as the above-mentioned step A-7.

[0145] Step B-8 is a step for synthesizing compound (14') from compound (13). Step B-8 can be performed in the same manner as in the above-mentioned step A-8.

[0146] [Method C] Method C is a method for preparing branched compound (19) or compound (22) and compound (19') or compound (22') using compound (3). The configuration of compound (1) and compound (10) is not limited. By using compound (3), the protecting group of the 6-hydroxyl group of the GalNAc moiety can be appropriately selected.

[0147] [Formula 53] Where R 3 , R 4 ,X,n,m,r,R 12 , R 13 and R 14 Each has the same meaning as above.

[0148] Step C-1 is a step of subjecting compound (1) or compound (10) to carbamate conversion, and can be performed in the same manner as in the above-mentioned step A-1 using compound (3) instead of compound (2).

[0149] Step C-2 is to remove R as a protecting group. 12 or R 13 The removal of the protecting group varies depending on its type, but can generally be carried out according to a method well known in the field of synthetic organic chemistry, for example, the method disclosed in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc.

[0150] Step C-3 is to convert -P(R 3 )R 4 This is a step of introducing a hydroxyl group of compound (19) or compound (22), and can be performed in the same manner as in the above-mentioned step A-3.

[0151] Step C-4 is a step of synthesizing compound (19') from compound (18) or synthesizing compound (22') from compound (21). Step C-4 can be performed in the same manner as the above-mentioned Step A-8.

[0152] <4. Preparation method using hydrophobic protecting group> The present invention provides: a method for preparing a conjugate of a GalNAc unit and a synthetic oligonucleotide, by binding the GalNAc unit to the 5' end or 3' end of the oligonucleotide, wherein a highly hydrophobic protecting group is introduced into one or more hydroxyl groups at any of the 3-position, 4-position and 6-position of the GalNAc moiety, and separating the GalNAc unit from impurities by adsorption to a resin having adsorptivity due to hydrophobic interactions; a conjugated compound (e.g., phosphoramidite and H-phosphonate) comprising GalNAc containing a hydrophobic protecting group, used as an intermediate in the preparation method, conjugate, and the like.

[0153] The preparation method of the present invention comprises the following steps: a: synthesizing a conjugate comprising a structure of a GalNAc unit-phosphate group containing a hydrophobic protective group bound to the 5' end or 3' end of an oligonucleotide; b: passing the solution containing the product obtained in step a through a column filled with a resin having adsorptive properties due to hydrophobic interactions, and passing an aqueous washing solution through the column; and c: Elution of the oligonucleotide from the column after step b.

[0154] In this preparation method, various hydrophobic blocking groups can be adopted, as long as they meet the above definition. In normal nucleic acid synthesis, oligonucleotide is freed from support by alkali treatment using ammonia etc., and therefore can use the blocking group removed by treatment under acidic conditions, such as optionally substituted trityl and optionally substituted silyl. The specific example of the optionally substituted trityl includes trityl, monomethoxytrityl and dimethoxytrityl. The example of the optionally substituted silyl includes triphenylsilyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl, preferably 4,4'-dimethoxytrityl (DMTr group).

[0155] As the hydrophobic protecting group to be removed under basic conditions, various ester groups can be used, but preferred examples thereof include acetyl group and benzoyl group.

[0156] In the present invention, the term "structure comprising a GalNAc unit-phosphate group containing a hydrophobic protective group" refers to a structure represented by the following formula.

[0157] [Formula 54] In the formula, R as a substituent of the hydroxyl group on GalNAc is a , R b and R cAt least one of them is a hydrophobic protecting group, and the rest are hydrogen atoms or protecting groups that can be removed under free conditions. All of them can be hydrophobic protecting groups, any two of them can be hydrophobic protecting groups, or only any one of them can be a hydrophobic protecting group. Preferably, R a is a hydrophobic protecting group (more preferably, a hydrophobic protecting group that is removed under acidic conditions, further preferably an optionally substituted trityl group, most preferably a DMTr group), and R b and R c In the case where the substituent is not a hydrophobic protecting group, it may be combined with a protecting group that is removed under free conditions, but these protecting groups are removed during the cleavage of the oligonucleotide from the support so as to become a hydrogen atom (hydroxyl group on GalNAc) at the stage of use in step a.

[0158] X 1 and X 2 Each independently represents a linker structure, which can be 1 and X 2 As the joint structure used in this article, various skeletons such as alkyl chains, PEG chains and peptide chains can be used. The joint structure can contain multiple GalNAc by branching in the middle. The number of branches (t) in the joint structure is about 1 to 10, preferably 1 to 3. In the case of having two or more branches, the joint structure between the branch point and the GalNAc can be the same or different. In the case of containing multiple GalNAc, at least one hydrophobic protecting group is combined with each GalNAc.

[0159] In the above formula, Y represents a bond that is combined with an oligonucleotide by a phosphate group or a thiophosphate group. In the case of combining with the 5' end of an oligonucleotide, a phosphodiester bond or a thiophosphate bond is formed together with the 5'-hydroxyl of the nucleoside at the end. Such a combination can be synthesized by solid phase synthesis of an oligonucleotide, and then a compound (a GalNAc unit amide compound containing a hydrophobic protective group) in which Y (having a structure of a GalNAc unit-phosphate group containing a hydrophobic protective group) forms a phosphoramidite structure or an H-phosphonate structure is synthesized in the same manner as a nucleoside phosphoramidite reagent. The specific example of the GalNAc unit amide compound containing a hydrophobic protective group corresponding to the bisected GalNAc unit of the present invention is described in (B13) above.

[0160] The preparation method of the present invention is not limited to the biantennary GalNAc unit applicable to the present invention described above, and can also be applied to the preparation of conjugates of known GalNAc units (e.g., those described in Patent Documents 1 to 9 and Non-Patent Documents 1 and 2) and oligonucleotides. In this case, a compound having a structure corresponding to a known GalNAc unit is used as a GalNAc unit phosphoramidite or H-phosphonate containing a hydrophobic protective group. Examples of amidite compounds corresponding to known GalNAc units may include the following compounds.

[0161] The following compounds can be found in J. Med. Chem. 2016, 59, 2718-2733 and references cited therein.

[0162] For Tris-based GalNAc units [Formula 55] For Triacid-based GalNAc units [Formula 56] For Pentaerythritol-based GalNAc units [Formula 57] For Lys-based GalNAc units [Formula 58] The GalNAc unit phosphoramidite containing a hydrophobic protective group represented by the following formula can be prepared with reference to International Publication No. WO19027009.

[0163] For Aryl-based GalNAc units [Formula 59] The following compounds can be prepared with reference to J. Med. Chem. 2016, 59, 2718-2733 and cited references.

[0164] For Hydroxyprolinol-based GalNAc units / Piperidine-based GalNAc units [Formula 60] In the above formula of the amido compound corresponding to the known GalNAc unit, R 1represents a hydrophobic protecting group. X represents a linker structure, preferably a C3 to C6 straight chain alkyl group. Y represents a phosphoramidite group or an H-phosphonate group.

[0165] GalNAc unit-phosphoramidite containing a hydrophobic protective group or GalNAc unit-H-phosphonate containing a hydrophobic protective group corresponding to the known GalNAc unit can be synthesized by appropriately using the GalNAc derivative of the above-mentioned compound (3) belonging to the method A to the method C in the process of synthesizing the GalNAc unit. For example, by referring to the method of Example 1, various GalNAc derivatives in which at least one hydroxyl group at the 3-position, 4-position and 6-position is protected by a hydrophobic protective group can be synthesized.

[0166] By introducing the above-mentioned GalNAc unit imide compound containing a hydrophobic protective group into the 5' end or 3' end of an oligonucleotide synthesized in the same manner as the nucleoside imide reagent according to the aforementioned method, a conjugate having a structure including a GalNAc-phosphate group containing a hydrophobic protective group (in the aforementioned formula, Y represents a bond bound to the oligonucleotide through a phosphate group or a thiophosphate group) can be synthesized. Examples of the conjugates thus synthesized may include the conjugates described in (B17) and (B18).

[0167] The solvent used for the reaction of introducing the GalNAc unit-H-phosphonate compound containing a hydrophobic protecting group into the 5' end or 3' end of the synthesized oligonucleotide is not particularly limited, as long as it does not inhibit the reaction. Preferably, anhydrous acetonitrile is used. Examples of reagents used as condensing agents include acid chlorides of carboxylic acids and phosphoric acid, preferably pivaloyl chloride. The oxidant used to oxidize the H-phosphonate bond to a phosphodiester bond is not particularly limited, as long as it is commonly used in oxidation reactions. Examples thereof may include inorganic metal oxidants including 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 ferric chloride; osmium compounds such as osmium tetroxide; silver compounds such as silver oxide; mercury compounds such as mercuric acetate; lead oxide compounds such as lead oxide and lead tetroxide; chromate compounds such as potassium chromate, chromic acid-sulfate complexes, and chromic acid-pyridine complexes; and cerium compounds such as cerium ammonium nitrate (CAN), and inorganic oxidants include halogen molecules such as chlorine molecules, bromine molecules, and iodine molecules; periodic acid such as sodium periodate; ozone; hydrogen peroxide solution; nitrous acid compounds such as nitrous acid; chlorite compounds such as potassium chlorite, sodium chlorite; and persulfate compounds Such as potassium persulfate and sodium persulfate, and organic oxidants, including reagents for DMSO oxidation (dimethyl sulfoxide and dicyclohexylcarbodiimide, oxalyl chloride, acetic anhydride or phosphorus pentoxide, or pyridine-acetic anhydride complex); peroxides such as tert-butyl hydroperoxide; stable cations such as trityl cations; succinimides such as N-bromosuccinimide; hypochlorite compounds such as tert-butyl hypochlorite; azodicarboxylate compounds such as methyl azodicarboxylate; disulfides such as dimethyl disulfide, diphenyl disulfide, and dipyridyl disulfide and triphenylphosphine; nitrites such as methyl nitrite; tetrahalogenated hydrocarbons such as tetrabromomethane; and quinone compounds such as 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), preferably iodine molecules. The example of deoxidizing agent used includes heterocyclic amine such as pyridine, dimethylaminopyridine, and aliphatic amine such as trimethylamine, triethylamine, diisopropylethylamine, preferably heterocyclic amine (especially pyridine). In the case of forming phosphorothioate bond, there is no restriction, as long as it is a reagent that can react H-phosphonate bond to form phosphorothioate bond. Its examples include sulfur, tetraethylthiuram disulfide, Beaucage reagent and phenylacetyl disulfide, preferably sulfur. The synthesized conjugate can be freed from the support in the same manner as the universal DMTr-on purification of oligonucleotides, while retaining the hydrophobic protecting group.

[0168] Such conjugate with the structure that comprises the GalNAc unit-phosphate group that contains hydrophobic blocking group can be free from support in the same mode as the general DMTr-on purification of oligonucleotide, retains hydrophobic blocking group simultaneously.Generally speaking, the key between the 3 ' end of oligonucleotide and support is cut by ammonia, and oligonucleotide is free under alkaline conditions.Therefore, the blocking group that will not be removed by alkalescence, for example the group that is removed under acidic conditions is selected as hydrophobic blocking group.Simultaneously, in adopting the bonding mode that wherein under acidic conditions the bond cutting between the 3 ' end of oligonucleotide and support is as bonding mode, the free condition of oligonucleotide is acidic, and therefore selects the blocking group that will not be removed under acidic conditions, for example the blocking group that is removed under alkaline conditions is as hydrophobic blocking group.

[0169] The thus obtained GalNAc unit-oligonucleotide conjugate containing a hydrophobic protective group is adsorbed to a column filled with a resin having adsorptivity due to hydrophobic interaction, and then the column is washed with an aqueous washing solution, so that impurities without a hydrophobic protective group can be separated.

[0170] The "resin having adsorptivity due to hydrophobic interaction" is not particularly limited as long as it is a resin in which a highly hydrophobic component is used for the base material. Various resins are used for this purpose, and examples thereof include styrene-divinylbenzene adsorptivity resins, modified styrene-divinylbenzene adsorptivity resins, methacrylic adsorptivity resins, or phenolic adsorptivity resins.

[0171] As the filling solution used when the GalNAc unit-oligonucleotide conjugate containing a hydrophobic protecting group is adsorbed onto the column, an alkaline aqueous solution that does not contain a large amount of organic solvent is ideal. For example, concentrated ammonia water used for cleaving oligomers from the support after solid phase synthesis or an alkaline aqueous solution obtained by diluting ammonia water with an appropriate amount of solvent (such as water and a buffer) can be used.

[0172] Various solutions can be used as washing solutions for removing impurities from the column, as long as they are solutions with properties that prevent deprotection of the hydrophobic protecting group and maintain hydrophobic interaction with the resin. The efficiency of removing impurities is improved by using an aqueous solution containing an organic solvent with a concentration low enough to maintain hydrophobic interaction with the resin as a washing solution. Specifically, in the case where the hydrophobic protecting group is a DMTr group, an aqueous solution containing an appropriate amount of salt, an aqueous solution containing acetonitrile, etc. can be used as a washing solution.

[0173] The hydrophobic protecting group combined with GalNAc can be deprotected by treating the conjugate under the deprotection conditions corresponding to the protecting group used. In the case of applying the preparation method after general oligonucleotide synthesis, a hydrophobic protecting group such as the DMTr group that is deprotected under acidic conditions is used, so the deprotection is carried out by treating with an acidic solution.

[0174] By appropriately performing a deprotection treatment on the column to which the conjugate is adsorbed, a deprotected GalNAc-oligonucleotide conjugate can be obtained, while impurities derived from the protecting group are adsorbed on the column.

[0175] Furthermore, impurities derived from the protecting group can be separated from the deprotected conjugate by deprotecting the GalNAc-oligonucleotide conjugate containing a hydrophobic protecting group that has not been deprotected and eluted from the column and passing the deprotected solution again through the same resin column having adsorption force due to hydrophobic interaction.

[0176] The GalNAc-oligonucleotide conjugate can be fractionated with high purity by subjecting the column retaining the conjugate to an appropriate gradient elution method corresponding to the resin employed. In the case of elution from the column without deprotection of the hydrophobic protecting group, the conjugate can be eluted by gradient elution, which increases the content of organic solvent to inhibit the hydrophobic interaction between the hydrophobic protecting group and the adsorption resin.

[0177] In the case where the deprotection treatment is performed in an adsorbed state on a column, appropriate elution conditions can be appropriately selected according to the mode in which the deprotected conjugate is retained on the column.

[0178] In the case of using an ion exchange column with adsorptivity due to hydrophobic interaction, the deprotection treatment is performed while the conjugate is adsorbed to the column, and the GalNAc-oligonucleotide conjugate can be eluted / fractionated from the column according to the conventional method of eluting from the ion exchange column using the salt concentration in the eluent adopted. Specifically, in the case of using RESOURCE Q (available from GE Healthcare Japan Corporation) as a column, a gradient eluent in which the content of sodium chloride in a mixed aqueous solution of sodium hydroxide / sodium chloride is gradually increased from 0 to 2M (2mol / L) can be used.

[0179] When using a reverse phase column, the GalNAc-oligonucleotide conjugate can be eluted / fractionated from the column according to the conventional method of eluting from the reverse phase column using a suitable organic solvent. Specifically, when using Clarity QSP (available from Phenomenex Inc.) as a column, a tris (Tris)-hydrochloric acid buffer containing an appropriate amount of acetonitrile can be used.

[0180] In order to detect the target substance, the absorbance at 260 nm as the nucleic acid detection wavelength is used. The target substance can be obtained by measuring the absorbance at 260 nm of the fluid passing through the column over time and collecting the fraction to be detected in the elution step.

[0181] <5. Drugs> The conjugates of the present invention can be used as drugs for treating diseases targeted by the oligonucleotides contained therein. Treatment includes both preventive treatment and post-treatment.

[0182] The conjugate of the present invention can be used in the form of a pharmaceutically acceptable salt. "Pharmaceutically acceptable salt" refers to a salt of an oligonucleotide, and examples of such salts may include metal salts, including alkali metal salts such as sodium salts, potassium salts and lithium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, iron salts, zinc salts, copper salts, nickel salts and cobalt salts; amine salts, including inorganic salts such as ammonium salts and organic salts such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts , tetramethylammonium salt and tris(hydroxymethyl)aminomethane salt; inorganic acid salts, including hydrohalides such as hydrofluoride, hydrochloride, hydrobromide and hydroiodide, nitrate, perchlorate, sulfate and phosphate; organic acid salts, including lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate, aryl sulfonates such as benzenesulfonate and p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate and maleate; and amino acid salts, such as glycine, lysine, arginine, ornithine, glutamate and aspartate, preferably alkali metal salts, more preferably sodium salts. These salts can be prepared by known methods.

[0183] In addition, the conjugate and its pharmaceutically acceptable salt may also exist as a solvate (eg, hydrate) and such a solvate may be used. In the present invention, such a solvate is considered to be within the scope of the salt of the oligonucleotide or the conjugate.

[0184] When the conjugate of the present invention or a pharmaceutically acceptable salt thereof is used as a pharmaceutical product, the pharmaceutical product itself, or mixed with a suitable pharmaceutically acceptable excipient or diluent, can be orally administered in the form of tablets, capsules, granules, powders or syrups, or parenterally administered in the form of injections, suppositories, patches or external medicines.

[0185] These preparations are prepared by known methods using additives such as excipients (e.g., organic excipients, including sugar derivatives such as lactose, saccharide, dextrose, mannitol and sorbitol; starch derivatives such as corn starch, potato starch, α-starch and dextrin; cellulose derivatives such as crystalline cellulose; gum arabic; dextran; and pullulan, as well as inorganic excipients, including silicate derivatives such as light anhydrous silicate, synthetic aluminum silicate, calcium silicate and magnesium aluminosilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and sulfates such as calcium sulfate), lubricants (e.g., stearic acid; metal stearates such as calcium stearate and magnesium stearate; talc; colloidal silicon dioxide; waxes such as beeswax and gay wax; boric acid; adipic acid; sulfates such as sodium sulfate; ethylene glycol; fumaric acid; sodium benzoate; DL leucine; lauric acid), (e.g., cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium and internally cross-linked carboxymethyl cellulose sodium; and chemically modified starch / cellulose such as carboxymethyl starch, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone), 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 (benzalkonium chloride); chloride); and nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters and sucrose fatty acid esters), stabilizers (parabens such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; and sorbic acid), flavoring agents (e.g., commonly used sweeteners, sour agents and spices) and diluents.

[0186] The drug of the present invention may contain 0.1 to 250 micromol / ml of oligonucleotide (antisense oligonucleotide), preferably 1 to 50 micromol / ml of oligonucleotide or a pharmaceutically acceptable salt thereof, 0.02-10% w / v of carbohydrate or polyol and 0.01-0.4% w / v of a pharmaceutically acceptable surfactant.

[0187] As the carbohydrate, monosaccharide or disaccharide is particularly preferred. Examples of carbohydrates and polyols include glucose, galactose, mannose, lactose, maltose, mannitol and sorbitol. These can be used alone or in combination.

[0188] In addition, preferred examples of the surfactant in the present invention include polyoxyethylene sorbitan monoester to polyoxyethylene sorbitan triester, alkylphenyl polyoxyethylene, sodium taurocholate, sodium cholate and polyol esters. Among them, polyoxyethylene sorbitan monoester to polyoxyethylene sorbitan triester are particularly preferred, and oleate, laurate, stearate, and palmitate are preferred as esters. These can be used alone or in combination.

[0189] In addition, the medicament of the present invention also preferably contains 0.03-0.09 M of a pharmaceutically acceptable neutral salt, such as sodium chloride, potassium chloride, and / or calcium chloride.

[0190] In addition, the drug of the present invention may also preferably contain 0.002-0.05 M of a pharmaceutically acceptable buffer. Preferred examples of the buffer include sodium citrate, sodium glycine, sodium phosphate and tris(hydroxymethyl)aminomethane. These buffers may be used alone or in combination.

[0191] In addition, the medicine can be provided in the form of a solution. However, in the case where it is necessary to store a certain time, it is usually preferred to freeze-dry to stabilize the oligonucleotide to prevent its therapeutic effect from declining. In this case, the medicine can be redissolved (redissolved) with a lysate (e.g., distilled water for injection), that is, redissolved in use as a liquid form to be administered. Therefore, the medicine of the present invention also includes medicines in a freeze-dried state, which are used after being redissolved with a lysate so that each component falls within a predetermined concentration range. For the purpose of improving the solubility of the freeze-dried product, amino acids such as albumin and glycine can also be contained.

[0192] The conjugate of the present invention can be encapsulated using lipids described in International Publication No. WO 2015 / 005253 and the like, and can be administered as nucleic acid lipid nanoparticles or liposomes described in International Publication No. WO 2015 / 005253 and the like.

[0193] When the conjugate of the present invention or a pharmaceutically acceptable salt thereof is administered to a human, the dosage for an adult is about 0.01-100 mg / kg (body weight), preferably 0.1-20 mg / kg (body weight) once or several times a day, for example, by subcutaneous injection, intravenous drip, intravenous injection or intravenous injection. The dosage and frequency of administration can be appropriately changed according to the type of disease, symptoms, age, administration method, etc. Example

[0194] Hereinafter, the present invention will be specifically described by examples. These examples are used to illustrate the present invention rather than to limit the scope of the present invention. "ABI 392 DNA / RNA Synthesizer" (available from Applied Biosystems), "ABI394 DNA / RNA Synthesizer" (available from Applied Biosystems), AKTA Oligopilot (available from GE Healthcare) and nS-8II Synthesizer (Gene Design Inc.) are used as automatic nucleic acid synthesizers, and use phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984)) and the synthesis program corresponding to each synthesis scale.

[0195] Activator solution-3 (0.25 mol / L 5-benzylthio-1H-tetrazole / acetonitrile solution, product number 013-20011, available from Wako Pure Chemical Industries, Ltd.) was used as a condensation activator.

[0196] CAPA for AKTA (1-methylimidazole / acetonitrile solution, product number L040050, available from Sigma-Aldrich Co. LLC), Cap B1 for AKTA (acetic anhydride / acetonitrile solution, product number L050050, available from Sigma-Aldrich Co. LLC), or Cap B2 for AKTA (pyridine / acetonitrile solution, product number L050150, available from Sigma-Aldrich Co. LLC) was used as a capping reagent.

[0197] DCADeblock (dichloroacetate / toluene solution, product number L023050, available from Sigma-Aldrich Co. LLC) or deblocking solution-1 (3 w / v% trichloroacetic acid / dichloromethane solution, product number 042-28921, available from FUJIFILM Wako Pure Chemical Corporation) was used as a deblocking agent.

[0198] Phenylacetyl disulfide (Product No. FP07495, available from Carbosynth Holdings Limited) was dissolved to 0.2 M using a 1:1 (v / v) solution of acetonitrile (dehydrated, Product No. 01837-05, KANTO CHEMICAL CO., INC.) and pyridine (dehydrated, Product No. 11339-05, KANTO CHEMICAL CO., INC.) and was used as a thiolation reagent to form a phosphorothioate bond.

[0199] OXDIZER 0.05M (Product No. L560250-04, available from Sigma-Aldrich Co. LLC) or iodine (Product No. 20035-00, available from KANTO CHEMICAL CO., INC.) was dissolved to 0.02M using a 78:20:2 (v / v / v) solution of tetrahydrofuran (dehydrated, Product No. 40993-05, available from KANTO CHEMICAL CO., INC.), pyridine (dehydrated, Product No. 11339-05, available from KANTO CHEMICAL CO., INC.) and distilled water and used as an oxidant.

[0200] Phosphoramidites of 2'-O-Me nucleosides (adenosine, product number ANP-5751, cytidine, product number ANP-5752, guanosine, product number ANP-5753, or uridine, product number ANP-5754) available from ChemGenes Corporation are used as nucleoside phosphoramidite reagents.

[0201] As the phosphoramidites of the non-natural nucleoside, the phosphoramidites according to Example 14 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylidene-6-N-benzoyladenosine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite) and Example 27 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylidene-2-N-isobutyrylguanosine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite) of Japanese Patent Publication No. 2000-297097 were used. ,N)-diisopropyl) phosphoramidite), Example 22 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylidene-4-N-benzoyl-5-methylcytidine-3'-O-(2-cyanoethyl N,N)-diisopropyl) phosphoramidite) or Example 9 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylidene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl) phosphoramidite).

[0202] NittoPhase UnyLinker 100, 10 μmol (available from NITTO DENKO CORPORATION), Primer Support 5G UnyLinker 350, 10 μmol (available from GE Healthcare), Primer Support 5G Unylinker 350, 100 μmol (available from GE Healthcare) or Glen UnySupport CPG 500, 1 μmol (available from Glen Research) is appropriately selected depending on the synthesis scale as the solid phase support.

[0203] (Example 1) Synthesis of GalNAc containing a hydrophobic protecting group (1A) Synthesis of [(2R,3R,4R,5R)-5-acetylamino-3,4-diacetoxy-6-[3-(benzyloxycarbonylamino)propoxy]tetrahydropyran-2-yl]methyl acetate (Compound 1A) [Formula 61] To N-(3-hydroxypropyl) benzyl carbamate (6.4g, 30.8mmol) and a suspension of [(2R, 3R, 4R, 5R)-5-acetylamino-3,4,6-triacetoxy-tetrahydropyran-2-yl] methyl acetate (10g, 25.7mmol) in dichloromethane (200ml) as a known compound in the literature (International Publication No. WO2011053614) was added trifluoromethanesulfonic acid (450μl, 5.1mmol), and then stirred at 45°C for 4 hours. After the reaction was completed, about half of the solvent was removed by distillation under reduced pressure for concentration. The organic layer was washed with saturated sodium bicarbonate aqueous solution and saturated physiological saline, then dried over anhydrous sodium sulfate, filtered, and the solvent was removed by distillation under reduced pressure to obtain a crude product. Isopropyl ether (100m) was added thereto, followed by stirring for 3 hours. The solid product obtained was filtered to obtain the target substance 1A (11.9g). It can be used for the next step reaction without further purification.

[0204] 1 H-NMR (CDCl 3)δ: 7.43-7.29(5H,m),6.26(1H,d,J=8.5Hz),5.36-5.29(1H,m),5.12(1H,d,J=12.1Hz),5 .08(1H,d,J=12.1Hz),5.03-4.95(2H,m),4.33(1H,d,J=9.1Hz),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).

[0205] C 25 H 34 N 2 O 11 Calculated value: [M+H]+539, found 539.

[0206] (1B) Synthesis of Benzyl N-[3-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxypropyl]carbamate (Compound 1B) [Formula 62] 28% sodium methoxide methanol solution (0.85ml) was added to the suspension of the compound (12.4g, 23mmol) synthesized in step (1A) in ethanol (200ml), and then stirred at room temperature. As the reaction proceeds, the solution becomes a clear solution. A precipitate is obtained by standing for 60 minutes. The precipitate is filtered and washed with ethanol and diisopropyl ether, and then dried under reduced pressure. A crude product (10g) containing the target substance 1B is obtained in solid form. It can be used for the next step reaction without further purification.

[0207] 1 H-NMR (DMSO-D 6 )δ: 7.60(1H,d,J=9.1Hz),7.40-7.17(5H,m),5.01(2H,s),4.62-4.55(2H,m),4.49(1H,d,J=4.2H z), 4.20 (1H, d, J = 8.5Hz), 3.76-3.27 (6H, m), 3.08-2.99 (2H, m), 1.81 (3H, s), 1.66-1.56 (2H, m).

[0208] C 19H 28 N 2 O 8 Calculated value: [M+Na]+435, found 435.

[0209] (1C) Synthesis of N-[(2R,3R,4R,5R,6R)-2-(3-aminopropoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide hydrochloride (Compound 1C) [Formula 63] The compound (22.8g, 55.3mmol) synthesized in step (1B) is dissolved in tetrahydrofuran (280ml) / 1N hydrochloric acid (61ml). 10% palladium / carbon (wet) (5.56g) is added thereto, then vigorously stirred at room temperature for 3.5 hours under a hydrogen atmosphere. After the reaction is complete, the mixture is filtered, and the obtained fluid is removed by distillation under reduced pressure. The solid product produced by adding an appropriate amount of ethanol is filtered, and washed with ethanol and ether, and then dried under reduced pressure. The crude product (17g) containing the target substance 1C is obtained. It can be used for the next step reaction without further purification.

[0210] 1 H-NMR (D 2 O)δ: 4.27(1H,d,J=8.5Hz), 3.90-3.50(8H,m), 2.93(2H,t,J=7.0Hz), 1.89(3H,s), 1.82-1.75(2H,m).

[0211] (1D) Synthesis of 9H-fluoren-9-ylmethyl N-[3-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxypropyl]carbamate (Compound 1D) [Formula 64] The compound synthesized in step (1C) (16.4g, 52.1mmol) was dissolved in tetrahydrofuran (200ml) / pure water (50ml), and N, N-diisopropylethylamine (12.7mL, 72.9mmol) was added thereto. Under ice cooling, N-[(9H-fluorene-9-ylmethoxy)carbonyloxy]succinimide (21.1g, 62.5mmol) was added thereto, and then stirred at room temperature for 1.5 hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure. Ether (100ml) was added thereto, and then stirred for 1 hour. The solid product was washed with ethanol, ethyl acetate and ether in sequence, and then dried under reduced pressure to obtain a crude product (22.9g) containing the target substance 1D.

[0212] 1 H-NMR (DMSO-D 6 )δ: 7.89(2H,d,J=7.3Hz), 7.69(2H,d,J=7.3Hz), 7.61(1H,d,J=9.1Hz), 7.42(2H,t,J=7.3Hz), 7.33(2H,t,J=7.3Hz), 7.24(1H,t,J=5.7 Hz),4.60-4.57(2H,m),4.49(1H,d,J=4.2Hz),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).

[0213] Calculated value for C26H32N2O8: [M+Na]+523, found 523.

[0214] Synthesis of (1E) [(2R,3R,4R,5R,6R)-5-acetylamino-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[3-(9H-fluoren-9-ylmethoxycarbonylamino)propoxy]tetrahydropyran-3-yl]acetate (Compound 1E) [Formula 65] To a suspension of the compound (1.56 g, 3.1 mmol) synthesized in step (1D) in pyridine (20 ml) was added 4,4'-dimethoxytrityl chloride (1.27 g, 3.7 mmol), followed by stirring at room temperature for 30 minutes. Subsequently, acetic anhydride (1.18 ml, 12.5 mmol) was added thereto, followed by stirring at room temperature for 20 hours. After the reaction was completed, N,N-diisopropylethylamine (1.1 mL, 6.2 mmol) and ethanol (2 ml) were added, and the solvent was removed by distillation under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane: ethyl acetate = 75:25-0:100, v / v, containing 1% triethylamine) to obtain an amorphous target substance 1E (2.8 g, quantitative).

[0215] 1 H-NMR (CDCl 3)δ:7.77(2H,d,J=7.3Hz),7.62(2H,d,J=7.3Hz),7.43-7.18(13H,m),6.83-6.79(4H,m ),6.03(1H,d,J=9.1Hz),5.55(1H,d,J=3.0Hz),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).

[0216] C 52 H 55 NO 12 Calculated value: [M+Na]+908, found 909.

[0217] Synthesis of (1F) [(2R,3R,4R,5R,6R)-5-acetylamino-4-acetoxy-6-(3-aminopropoxy)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 1F) [Formula 66] To the dichloromethane (8ml) solution of the compound (2.49g, 2.8mmol) synthesized in step (1E), piperidine (0.45ml, 4.5mmol) was added, followed by stirring at room temperature. If the reaction was incomplete, piperidine was added as appropriate. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain a crude product. It was purified by NH-silica gel column chromatography (dichloromethane: methanol = 100: 0-85: 15, v / v) to obtain an amorphous target substance 1F (1.48g, yield 79%).

[0218] 1 H-NMR (CDCl 3)δ: 7.39-7.18(9H,m),6.83-6.80(4H,m),5.55(1H,d,J=3.0Hz),5.51(1H,d,J=9.1Hz),5. 27(1H,dd,J=11.2,3.3Hz),4.64(1H,d,J=8.5Hz),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=9.1,5.4Hz),3.07(1H,t ,J=8.5Hz),2.82-2.74(4H,m),2.01(3H,s),1.96(3H,s),1.89(3H,s),1.74-1.69(2H,m).

[0219] C 37 H 45 NO 10 Calculated value: [M+Na]+687, found 687.

[0220] (Example 2) with GalNAc unit X 18 Synthesis of the corresponding amidite reagent (compound having a GalNAc-phosphate group containing a hydrophobic protecting group) (2A) Synthesis of 2-[[2-benzyloxy-3-[(2-benzyloxy-2-oxo-ethyl)carbamoyloxy]propoxy]carbonylamino]benzyl acetate (Compound 2A) [Formula 67] 2-Benzyloxy-1,3-propanediol (2.5g, 14mmol) and 4-nitrophenyl chloroformate (5.8g, 29mmol) were dissolved in tetrahydrofuran (100ml), and pyridine (5.0ml, 61.8mmol) was added thereto, followed by stirring at room temperature for 20 minutes. The precipitated solid was filtered, and tetrahydrofuran (70ml) was then added thereto. Subsequently, glycine benzyl ester TFA salt (9.2g, 33mmol) and N,N-diisopropylethylamine (9.6mL, 55mmol) were added thereto, followed by stirring at room temperature overnight. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-40: 60, v / v) to obtain a colloidal target substance 2A (6.5g, yield 84%).

[0221] 1 H-NMR (CDCl 3)δ:7.37-7.33(15H,m),5.48(2H,t,J=5.4Hz),5.18(4H,s),4.64(2H,s),4.23(4H,d,J=5.4Hz), 3.99(4H,d,J=5.4Hz), 3.84(1H,t,J=5.4Hz).

[0222] C 30 H 32 N 2 O 9 Calculated values: [M+H]+565, found 565, [M+Na]+587, found 587 (2B) Synthesis of 2-[[3-(carboxymethylcarbamoyloxy)-2-hydroxy-propyloxy]carbonylamino]acetic acid (Compound 2B) [Formula 68] The 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) (3 g) was added thereto, and then vigorously stirred at room temperature for 5 hours under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered, and the fluid was removed by distillation under reduced pressure to obtain a crude product (3.2 g) containing the target substance 2B. It can be used for the next step reaction without further purification.

[0223] 1 H-NMR (D 2 O)δ: 4.21-4.14(5H,m),3.86(4H,s).

[0224] Synthesis of (2C) [(2R,3R,4R,5R,6R)-5-acetylamino-6-[3-[[2-[[3-[[2-[3-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl 1-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-2-oxo-ethyl]carbamoyloxy]-2-hydroxy-propyloxy]carbonylamino]acetyl]amino]propyloxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 2C) [Formula 69] To a solution of compound 2B (0.31 g, 1.05 mmol) synthesized in step (2B), compound 1F (1.47 g, 2.21 mmol) synthesized in step (1E) and N, N-diisopropylethylamine (0.91 mL, 5.27 mmol) in N, N-dimethylformamide (9 ml), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (11.3 g, 29.6 mmol) was added, followed by stirring at room temperature for 3 hours. After the reaction was completed, the reaction solution was added dropwise to diethyl ether (185 ml in total). The gummy crude product obtained after removing the solvent from the supernatant was purified by silica gel column chromatography (dichloromethane:methanol=100:0-15:85, v / v, containing 1% triethylamine) to obtain the target substance 2C (1.05 g, yield 63%) as a solid.

[0225] 1 H-NMR (CDCl 3 )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=10.9Hz),4.49-3.02(44H,m),2.01(6H,s),1.96(6H,s),1.90(6H,s),1.80-1.63(4H,m).

[0226] C 81 H 98 N 6 O 27 Calculated value: [M+Na]+1611, found 1611 Synthesis of (2D) [(2R,3R,4R,5R,6R)-5-acetylamino-6-[3-[[2-[[3-[[2-[3-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-2-oxo-ethyl]carbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]carbonylamino]acetyl]amino]propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 2D) [Formula 70] The 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 thereto, followed by stirring at room temperature for 1 hour. In addition, 2-cyanoethyldiisopropylchlorophosphoramidite (20 μl, 0.09 mmol) was added thereto, followed by stirring at room temperature for 15 minutes. After the reaction was completed, the solvent was removed by reduced pressure distillation to obtain a crude product. It was purified by silica gel column chromatography (ethyl acetate: methanol = 100: 0-85: 15, v / v, containing 1% triethylamine) to obtain an amorphous target substance 2D (0.34 g, yield 64%).

[0227] 1 H-NMR (CDCl 3 )δ: 7.39-7.17(18H,m),6.87-6.70(8H,m),6.29-6.06(2H,m),5.55(2H,d,J=3.0Hz),5.11(2H,d,J=10.9Hz),4.47-3. 02(47H,m),2.63(2H,t,J=6.3Hz),2.02(6H,s),1.96(6H,s),1.90(6H,s),1.80-1.64(4H,m),1.17(12H,d,J=6.7Hz).

[0228] (Example 3) with GalNAc unit X 19 Synthesis of the corresponding amidite reagent (compound having a GalNAc-phosphate group containing a hydrophobic protecting group) (3A) Synthesis of 2-[[3-benzyloxy-2-[(2-benzyloxy-2-oxo-ethyl)carbamoyloxy]propoxy]carbonylamino]benzyl acetate (Compound 3A) [Formula 71] The target substance 3A (2.8 g, yield 90%) was obtained in the same manner as in step (2A) using (+ / -)-3-benzyloxy-1,2-propanediol (1.0 g, 5.5 mmol) instead of 2-benzyloxy-1,3-propanediol.

[0229] 1 H-NMR (CDCl 3)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=5.4Hz).

[0230] C 30 H 32 N 2 O 9 Calculated value: [M+Na]+587, found 587 (3B) Synthesis of 2-[[2-(carboxymethylcarbamoyloxy)-3-hydroxy-propyloxy]carbonylamino]acetic acid (Compound 3B) [Formula 72] A crude product containing the target substance 3B (1.2 g) was obtained in the same manner as in step (2B) using compound 3A (2.8 g, 5.0 mmol) instead of compound 2A.

[0231] 1 H-NMR (D 2 O)δ: 4.32-4.19(3H,m), 3.94-3.87(4H,m), 3.81-3.71(2H,m).

[0232] Synthesis of (3C) [(2R,3R,4R,5R,6R)-5-acetylamino-6-[3-[[2-[[2-[[2-[3-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-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-1-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 3C) [Formula 73] The target substance 3C (0.74 g, yield 44%) was obtained in the same manner as in step (2C) using compound 3B (0.31 g, 1.05 mmol) instead of compound 2B.

[0233] 1 H-NMR (CDCl 3)δ: 7.40-7.18(18H,m),6.81(8H,dd,J=9.1,3.0Hz),6.70-6.50(1H,m),6.16-6.02(1H,m),5.56-5.54(2 H,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).

[0234] C 81 H 98 N 6 O 27 Calculated value: [M+Na]+1611, found 1611 Synthesis of (3D) [(2R,3R,4R,5R,6R)-5-acetylamino-6-[3-[[2-[[2-[[2-[3-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-2-oxo-ethyl]carbamoyloxy]-3-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]carbonylamino]acetyl]amino]propoxy]-4-acetyloxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 3D) [Formula 74] The target substance 3D (0.60 g, yield 72%) was obtained in the same manner as in step (2D) using compound 3C (0.31 g, 1.05 mmol) instead of compound 2C.

[0235] 1 H-NMR (CDCl 3 )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).

[0236] (Example 4) with GalNAc unit X 20Synthesis of the corresponding amidite reagent (compound having a GalNAc-phosphate group containing a hydrophobic protecting group) (4A) Synthesis of benzyl 3-[[2-benzyloxy-3-[(3-benzyloxy-3-oxo-propyl)carbamoyloxy]propoxy]carbonylamino]propionic acid ester (Compound 4A) [Formula 75] In the same manner as in step (2A), 3-aminopropionic acid benzyl ester TFA salt (2.4 g, 8.17 mmol) was used instead of glycine benzyl ester TFA salt used in step (2A) to obtain the target compound 4A (1.62 g, yield 80%).

[0237] 1 H-NMR (CDCl 3 )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=6.0Hz). C 32 H 36 N 2 O 9 Calculated values: [M+H]+593, found 593, [M+Na]+615, found 615 (4B) Synthesis of 3-[[3-(2-carboxyethylcarbamoyloxy)-2-hydroxy-propyloxy]carbonylamino]propionic acid (Compound 4B) [Formula 76] In the same manner as in step (2B), compound 4A (1.62 g, 2.73 mmol) was used instead of compound 2A used in step (2B) to obtain target substance 4B (0.89 g, quantitative).

[0238] 1 H-NMR(D2O)δ: 4.22-4.04(5H,m), 3.46-3.32(4H,m), 2.57(4H,t,J=6.7Hz).

[0239] Synthesis of (4C) [(2R,3R,4R,5R,6R)-5-acetylamino-6-[3-[3-[[3-[3-[3-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-3-oxo-propyl]carbamoyloxy]-2-hydroxy-propyloxy]carbonylamino]propionylamino]propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 4C) [Formula 77] The target substance 4C (950 mg, yield 63%) was obtained in the same manner as in step (2C) using compound 4B (300 mg, 0.93 mmol) instead of compound 2B used in step (2C).

[0240] 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 =3.0Hz),5.10(2H,d,J=11.5Hz),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).

[0241] C 83 H 102 N 6 O 27 Calculated value: [M+Na]+1638, found 1638 Synthesis of (4D) [(2R,3R,4R,5R,6R)-5-acetylamino-6-[3-[3-[[3-[3-[3-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-3-oxo-propyl]carbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]carbonylamino]propionylamino]propoxy]-4-acetyloxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 4D) [Formula 78] In the same manner as in step (2D), compound 4C (945 mg, 0.58 mmol) was used instead of compound 2C used in step (2D) to obtain the target substance 4D (638 mg, yield 60%).

[0242] 1 H-NMR (CDCl 3 )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=3.0Hz),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=6.0Hz),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).

[0243] (Example 5) with GalNAc unit X 21 Synthesis of the corresponding amidite reagent (compound having a GalNAc-phosphate group containing a hydrophobic protecting group) (5A) Synthesis of benzyl 4-[[2-benzyloxy-3-[(4-benzyloxy-4-oxo-butyl)carbamoyloxy]propoxy]carbonylamino]butyrate (Compound 5A) [Formula 79] In the same manner as in step (2A), 4-aminobutyric acid benzyl ester TFA salt (1.30 g, 4.21 mmol) was used instead of glycine benzyl ester TFA salt used in step (2A) to obtain the target compound 5A (0.85 g, yield 78%).

[0244] 1 H-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 = 7.3Hz), 1.88-1.81 (4H, m).

[0245] C 34 H 40 N 2 O9 Calculated values: [M+H]+621, found 622, [M+Na]+643, found 644 (5B) Synthesis of 4-[[3-(3-carboxypropylcarbamoyloxy)-2-hydroxy-propyloxy]carbonylamino]butyric acid (Compound 5B) [Formula 80] The target substance 5B (0.49 g, quantitative) was obtained in the same manner as in step (2B) using compound 5A (0.85 g, 1.4 mmol) instead of compound 2A used in step (2B).

[0246] 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).

[0247] Synthesis of (5C) [(2R,3R,4R,5R,6R)-5-acetylamino-6-[3-[4-[[3-[[4-[3-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-4-oxo-butyl]carbamoyloxy]-2-hydroxy-propyloxy]carbonylamino]butyrylamino]propyloxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 5C) [Formula 81] The target substance 5C (1.16 g, yield 82%) was obtained in the same manner as in step (2C) using compound 5B (300 mg, 0.86 mmol) instead of compound 2B used in step (2C).

[0248] 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=3.0Hz),5.10(2H,d,J =10.9Hz),4.45(2H,d,J=8.5Hz),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). C85 H 106 N 6 O 27 Calculated value: [M+Na]+1666, found 1667 Synthesis of (5D) [(2R,3R,4R,5R,6R)-5-acetylamino-6-[3-[3-[[3-[3-[3-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-4-oxo-butyl]carbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]carbonylamino]butyrylamino]propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 5D) [Formula 82] In the same manner as in step (2D), compound 5C (1.16 g, 0.71 mmol) was used instead of compound 2C used in step (2D) to obtain the target substance 5D (795 mg, yield 61%).

[0249] 1 H-NMR (CDCl 3 )δ: 7.40-7.17(18H,m),6.85-6.77(8H,m),6.72-5.65(5H,m),5.56(2H,d,J=3.0Hz),5.11-5.03(2H,m),4.46-4.35(2H,m),4.25-3.02(4 2H,m),2.65(2H,t,J=6.0Hz),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=6.7Hz).

[0250] (Example 6) with GalNAc unit X 22 Synthesis of the corresponding amidite reagent (6A) Synthesis of [(2R,3R,4R,5R,6R)-5-acetylamino-3,4-diacetoxy-6-[5-(benzyloxycarbonylamino)pentyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 6A) [Formula 83] To a suspension of N-benzyloxycarbonyl-1-hydroxypentyl-5-amine (8.05 g, 33.9 mmol) as a compound known in the literature (J. Am. Chem. Soc., 2006, 128, 4058-4073.) and acetic acid [(2R, 3R, 4R, 5R) -5-acetylamino-3, 4, 6-triacetoxy-tetrahydropyran-2-yl] methyl ester (12.0 g, 30.8 mmol) as a compound known in the literature (International Publication No. WO 2011053614) in dichloromethane (200 mL) was added trifluoromethanesulfonic acid (450 μL, 5.23 mmol), followed by stirring overnight at 45° C. After the reaction was completed, about half of the solvent was distilled off under reduced pressure for concentration, and the resultant was added to a mixed solution of ethyl acetate / saturated sodium bicarbonate aqueous solution. The organic layer was washed with saturated sodium bicarbonate aqueous solution and saturated physiological saline / phosphate buffer (pH 7.0), 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 (hexane: ethyl acetate = 100: 0-0: 100, v / v) to obtain an amorphous target substance 6A (17.0 g, yield 94%).

[0251] 1 H-NMR (CDCl 3 )δ: 7.37-7.32(5H,m),5.69(1H,d,J=9.1Hz),5.35(1H,d,J=2.4Hz),5.29(1H,d,J=11.5Hz),5.10(2H,s),4.94(1H,br),4.68(1H,d,J=7.9Hz),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). C 27 H 38 N 2 O 11 Calculated value: [M+H] + 567, measured value 567.

[0252] (6B) Synthesis of [(2R,3R,4R,5R,6R)-5-acetylamino-3,4-diacetoxy-6-(5-aminopentyloxy)tetrahydropyran-2-yl]methyl acetate hydrochloride (Compound 6B) [Formula 84] The 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 thereto, and then vigorously stirred at room temperature under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered, and the fluid was removed by distillation under reduced pressure to obtain a crude product (3.1 g, yield 97%) of the target substance 6B. It can be used for the next step reaction without further purification.

[0253] C 19 H 32 N 2 O 9 Calculated value: [M+H] + 433, measured value 433.

[0254] Synthesis of (6C) [(2R,3R,4R,5R,6R)-5-acetylamino-6-[5-[[3-[5-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentylcarbamoyloxy]-2-benzyloxy-propoxy]carbamoylamino]pentyloxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 6C) [Formula 85] 2-Benzyloxy-1,3-propanediol (600mg, 3.29mmol) and 4-nitrophenyl chloroformate (1.39g, 6.91mmol) were dissolved in tetrahydrofuran (22ml), and pyridine (1.1ml, 13.2mmol) was added thereto, followed by stirring at room temperature for 15 minutes. After filtering the precipitated solid, tetrahydrofuran (16ml) was added thereto. Subsequently, 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) was added, followed by stirring at 40°C for 2 hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure. Dichloromethane was added thereto, and the organic layer was washed with 0.5N hydrochloric acid, saturated saline, 0.2N sodium hydroxide aqueous 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. It 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 substance 6C.

[0255] C 50 H 74 N 4 O 23Calculated value: [M+H] + 1099, found 1099. [M+Na] + 1121, measured value 1121.

[0256] Synthesis of (6D) [(2R,3R,4R,5R,6R)-5-acetylamino-6-[5-[[3-[5-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentylcarbamoyloxy]-2-hydroxy-propoxy]carbonylamino]pentyloxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 6D) [Formula 86] The mixture (2.1 g) containing compound 6C obtained in step (6C) is dissolved in tetrahydropyran (20 ml) / pure water (1 ml). 1N hydrochloric acid (0.38 ml) and 10% palladium / carbon (wet) (1 g) are added thereto, followed by vigorous stirring at room temperature under a hydrogen atmosphere. After the reaction is complete, the mixture is filtered, and the obtained fluid is removed by distillation under reduced pressure to obtain a crude product. It is purified by silica gel column chromatography (ethyl acetate: methanol = 100: 0-90: 15, v / v) to obtain a target substance 6D (1.38 g, yield 72%) as a solid.

[0257] 1 H-NMR (CDCl 3 )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).

[0258] C 43 H 68 N 4 O 23 Calculated value: [M+H] + 1009, found 1009. [M+Na] + 1031, measured value 1031.

[0259] Synthesis of (6E)[(2R,3R,4R,5R,6R)-5-acetylamino-6-[5-[[3-[5-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxypentylcarbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]carbamoylamino]pentyloxy]-3,4-diacetyloxy-tetrahydropyran-2-yl]methyl acetate (Compound 6E) [Formula 87] The target substance 6E (850 mg, yield 51%) was obtained in the same manner as in step (2D) using compound 6D (1.38 g, 1.37 mmol) instead of compound 2C used in step (2D).

[0260] 1 H-NMR (CDCl 3 )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=6.4Hz),2.67(2H,t,J=6.3Hz),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=6.7Hz).

[0261] (Example 7) with GalNAc unit X 20 Synthesis of the corresponding amido reagent (7A) Synthesis of [(2R,3R,4R,5R)-5-acetylamino-3,4-diacetoxy-6-(3-aminopropoxy)tetrahydropyran-2-yl]methyl acetate hydrochloride (Compound 7A) [Formula 88] The 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 thereto, followed by vigorous stirring at room temperature for 1 hour under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the fluid was removed by distillation under reduced pressure to obtain a crude product (8.97 g) containing the target substance 7A. It can be used for the next step reaction without further purification.

[0262] C 17 H28 N 2 O 9 Calculated value: [M+H]+405, found 405.

[0263] (7B) Synthesis of [(2R,3R,4R,5R,6R)-5-acetylamino-6-[3-[3-[[3-[3-[3-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxypropylamino]-3-oxo-propyl]carbamoyloxy]-2-hydroxy-propoxy]carbamoylamino]propionylamino]propoxy]-3,4-diacetyloxy-tetrahydropyran-2-yl]methyl acetate (Compound 7B) [Formula 89] Compound 4B (200mg, 0.62mmol) synthesized in step (4B) was added O-(N-succinimidyl)-N, N, N', N'-tetramethyluronium tetrafluoroborate (411mg, 1.37mmol) and N, N-diisopropylethylamine (0.32ml, 1.86mmol) in a solution in N, N-dimethylformamide (2ml), and then stirred at room temperature for 20 minutes. The reaction solution was added to a solution of compound 37B (657mg, 1.49mmol) synthesized in step (37B), and further N, N-diisopropylethylamine (0.32ml, 1.86mmol) was added thereto, and then stirred at room temperature for 60 minutes. After the reaction was completed, the reaction solution was added dropwise to ether (80ml in total). The crude gummy product obtained by removing the solvent from the supernatant was purified by silica gel column chromatography (dichloromethane:methanol=100:0-75:25, v / v) to obtain an amorphous target substance 7B (408 mg, yield 60%).

[0264] 1 H-NMR (CDCl 3 )δ: 6.76(2H,br),6.69-6.63(2H,m),5.96(1H,br),5.36(2H,d,J=3.0Hz),5.13-5.08(2H,m),4.53(2H,d,J=8.5Hz ),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).

[0265] C 45 H70 N 6 O 25 Calculated value: [M+H] + 1095, found 1096. [M+Na] + 1117, measured value 1117.

[0266] Synthesis of (7C) [(2R,3R,4R,5R,6R)-5-acetylamino-6-[3-[3-[[3-[3-[3-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropylamino]-3-oxo-propyl]carbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]carbonylamino]propionylamino]propoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 7C) [Formula 90] In the same manner as in step (2D), compound 7B (1.53 g, 1.40 mmol) was used instead of compound 2C used in step (2D) to obtain the target substance 7C (510 mg, yield 28%).

[0267] 1 H-NMR (CDCl 3 )δ: 6.60-6.44(4H,m),5.76-5.68(1H,m),5.36(2H,d,J=3.0Hz),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=6.3Hz),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=6.7Hz).

[0268] (Example 8) X 18 -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) was synthesized according to the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984)) using ABI 392 DNA / RNA Synthesizer (available from Applied Biosystems) as a nucleic acid automatic synthesizer and Nitto Phase Uny Linker 100, 10 μmol (available from NITTO DENKO CORPORATION) as a solid phase support. Oligonucleotide sequence 15e_001 was then synthesized once using compound 2D as a GalNAc unit amidite synthesized in Example 2. The protected oligonucleotide analog was treated with 6 mL of concentrated ammonia water to cleave the oligomer from the support, and the cyanoethyl group as a protecting group on the phosphorus atom and the protecting group on the nucleobase was removed. Purification was performed using Clarity QSP (available from Phenomenex Inc.) according to the attached manual.

[0269] The nucleotide sequence of the compound is a sequence complementary to the sequence from 92 to 106 at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the nucleotide 728 in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6247.96).

[0270] (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 -G m1s -A m1s -A e2s -G m1t -H (SEQ ID No: 40) was synthesized in the same manner as in Example 8 using the aforementioned sequence 15e_001.5 instead of the sequence used in Example 8.

[0271] The nucleotide sequence of the compound is a sequence complementary to the sequence at positions 92 to 106 at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the nucleotide at position 728 in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6233.97).

[0272] (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) was synthesized in the same manner as in Example 8 using the aforementioned sequence 15e_005.5 instead of the sequence used in Example 8.

[0273] The nucleotide sequence of the compound is a sequence complementary to the 91st to 105th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the 728th nucleotide in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6235.97).

[0274] (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 mlt-H (SEQ ID No: 45) was synthesized in the same manner as in Example 8 using the above sequence 15e_006.5 instead of the sequence used in Example 8.

[0275] The nucleotide sequence of the compound is a sequence complementary to the 90th to 104th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the 728th nucleotide in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6200.94).

[0276] (Example 12) X 18 -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) Synthesis was performed in the same manner as in Example 8 using the aforementioned sequence 16e_001.5 instead of the sequence used in Example 8.

[0277] The nucleotide sequence of the compound is a sequence complementary to the 90th to 107th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the 728th nucleotide in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6593.02).

[0278] (Example 13) X 18 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -Gm1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m12 -C e2t -H (SEQ ID No: 47) Synthesis was performed in the same manner as in Example 8 using the aforementioned sequence 16e_002.5 instead of the sequence used in Example 8.

[0279] The nucleotide sequence of the compound is a sequence complementary to the 91st to 106th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the 728th nucleotide in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6595.01).

[0280] (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 mlt -H (SEQ ID No: 48) Synthesis was performed in the same manner as in Example 8 using the above sequence 16e_003.5 instead of the sequence used in Example 8.

[0281] The nucleotide sequence of the compound is a sequence complementary to the 90th to 105th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the 728th nucleotide in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6571.98).

[0282] (Example 15) X20 -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) using compound 4D prepared in Example 4 as the GalNAc unit imide instead of compound 2D, 18 Replace with X 20 , was synthesized in the same manner as in Example 8. The sequence used was the aforementioned sequence 15e_001.5.

[0283] The nucleotide sequence of the compound is a sequence complementary to the 92nd to 106th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the 728th nucleotide in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6261.98).

[0284] (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) was synthesized in the same manner as in Example 15 using the aforementioned sequence 15e_005.5.

[0285] The nucleotide sequence of the compound is a sequence complementary to the 91st to 105th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the 728th nucleotide in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6264.00).

[0286] (Example 17) X 20 -A mls -A mls -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) Synthesis was performed in the same manner as in Example 15 using the aforementioned sequence 16e_001.5.

[0287] The nucleotide sequence of the compound is a sequence complementary to the 92nd to 107th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the 728th nucleotide in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6621.04).

[0288] (Example 18) X 20 -A mls -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 -Gm1s -C e2t -H (SEQ ID No: 47) Synthesis was performed in the same manner as in Example 15 using the aforementioned sequence 16e_002.5.

[0289] The nucleotide sequence of the compound is a sequence complementary to the 91st to 106th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the 728th nucleotide in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6623.05).

[0290] (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) using compound 5D synthesized in Example 5 as the GalNAc unit imide, 18 Replace with X 21 , was synthesized in the same manner as in Example 8. The sequence used was the aforementioned sequence 15e_001.5.

[0291] The nucleotide sequence of the compound is a sequence complementary to the 92nd to 106th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the 728th nucleotide in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6290.02).

[0292] (Example 20) X 22 -A m1s -A e2s -U m1s -C m1s -Ce2s -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) using compound 6D synthesized in Example 6 as the GalNAc unit imide, 18 Replace with X 22 , was synthesized in the same manner as in Example 8. The sequence used was the aforementioned sequence 15e_001.5.

[0293] The nucleotide sequence of the compound is a sequence complementary to the 92nd to 106th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the 728th nucleotide in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6175.96).

[0294] (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) was synthesized in the same manner as in Example 20 using the aforementioned sequence 15e_005.5.

[0295] The nucleotide sequence of the compound is a sequence complementary to the 91st to 105th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the 728th nucleotide in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6177.98).

[0296] (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) Synthesis was performed in the same manner as in Example 20 using the aforementioned sequence 16e_001.5.

[0297] The nucleotide sequence of the compound is a sequence complementary to the 92nd to 107th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the 728th nucleotide in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6535.03).

[0298] (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) This sequence was replaced with the aforementioned sequence 16e_002.5 and synthesis was carried out in the same manner as in Example 20.

[0299] The nucleotide sequence of the compound is a sequence complementary to the 91st to 106th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the 728th nucleotide in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6537.05).

[0300] (Example 24) X 20 -G e2s -C e2s -A s -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) The sequence complementary to the 10177-10189 nucleic acid sequence of human apolipoprotein B (APOB) GenBank accession number NM_000384.2 was used instead of the sequence used in Example 15 to synthesize a conjugate of a GalNAc unit and an oligonucleotide (ApoB-ASO conjugate) in the same manner as in Example 15. In the aforementioned method, after synthesizing an oligonucleotide having the aforementioned sequence, the synthesis is terminated, followed by excision / collection to synthesize an oligonucleotide (ApoB-ASO) to which a GalNAc unit is not bound. The compound was identified by negative ion ESI mass spectrometry (measured values: 5296.83 (ApoB-ASO conjugate) and 4392.50 (ApoB-ASO)).

[0301] (Experimental Example 1) Evaluation of the Effect of Example Compounds on Repair of Abnormal Splicing Using Cultured Cells (1-1) Cultivation of 293A Cells (Human Fetal Kidney Cells) 293A cells (R705-07 invitrogen) were cultured as follows.

[0302] 293A cells were maintained and cultured in maintenance medium (DMEM, 10% FBS). 5 Cells were seeded in 6-well plates at a density of 1 × 10 5 The cells were seeded at a density of 10 cells / well on a 12-well plate, and as described later, the compound of each example and a plasmid vector were simultaneously introduced on the next day for evaluation.

[0303] (1-2) Preparation of human G6PC full-length plasmid vector The human G6PC full-length plasmid vectors (pcDNAhG6PC, pcDNAhG6PC (c.648G>T)+Int4) were prepared as follows.

[0304] Using Human Multiple Tissuec DNA Panel as template, G6PC cDNA was amplified using the following G6PC cDNA amplification primers, and then further amplified using G6PC cDNA IF primers. The amplified fragment was inserted into the BamHI site of pcDNA3.1 using the InFusion System (pcDNAhG6PC).

[0305] G6PC cDNA amplification primers: Forward primer 5'-ATAGCAGAGCAATCACCACCAAGCC-3' (SEQ ID No: 49) Reverse primer 5'-ATTCCACGACGGCAGAATGGATGGC-3' (SEQ ID No: 50) G6PC IF Primers: Forward primer 5'-TACCGAGCTCGGATCCACCACCAAGCCTGGAATAACTGC-3' (SEQ ID No: 51) Reverse primer 5'-CTGGACTAGTGGATCCTGGCATGGTTGTTGACTTTAAAC-3' (SEQ ID No: 52) Human genomic DNA was used as a template and the following G6PC Int4 amplification primer was used to amplify G6PC Intron4 and a portion of the region containing exon 5, and G6PC Intron4 was further amplified with G6PC Int4 IF primer. In addition, the pcDNA hG6PC generated in step 1-1 was amplified using the following hG6PC vector IF primers. The c.648G>T mutation in exon 5 was introduced into the InFusion primer. The two fragments were connected by the InFusion System, and the G6PC Intron4 sequence was inserted into pcDNAhG6PC (pcDNAhG6PC (c.648G>T)+Int4).

[0306] G6PC Int4 Amplification Primers: Forward primer 5'-TCTGGGCTGTGCAGCTGAATGTCTG-3' (SEQ ID No: 53) Reverse primer 5'-GTAGGGGATGACACTGACGGATGCC-3' (SEQ ID No: 54) G6PC Int4 IF Primers: Forward primer 5'-CTGGAGTCCTGTCAGGTATGGGC-3' (SEQ ID No: 55) Reverse primer 5'-AGCTGAAAAGGAAGAAGGTAATGAG-3' (SEQ ID No: 56) hG6PC vector IF primers: Forward primer 5'-TCTTCCTTTTCAGCTTCGCCATCGG-3' (SEQ ID No: 57) Reverse primer 5'-CTGACAGGACTCCAGCAACAAC-3' (SEQ ID No: 58) (1-3) Co-transfection of Example Compounds and Plasmid Vectors The compound of each example and the plasmid vector were co-transfected as follows.

[0307] Prepare the following solution A and solution B, and then mix them.

[0308] In the case of using a 6-well plate, 250 μL of Opti-MEM medium (Gibco), 0.50 μL of plasmid vector (1 mg / mL) and 4.0 μL (final: 20 nM) of the compound produced in each example (12.5 μM) were prepared per well as solution A, and 250 μL of Opti-MEM medium (Gibco) and 6.0 μL of Lipofectamine 2000 (Invitrogen) were prepared as solution B, and solution A and solution B were mixed. In the case of using a 12-well plate, 125 μL of Opti-MEM medium (Gibco), 0.25 μL of plasmid vector (1 mg / mL) and 2.0 μL (final: 20 nM) of the compound produced in each example (12.5 μM) were prepared per well as solution A, and 125 μL of Opti-MEM medium (Gibco) and 3.0 μL of Lipofectamine 2000 (Invitrogen) were prepared as solution B, and solution A and solution B were mixed.

[0309] The mixed solution was incubated at room temperature for 20 minutes and then added to the cells the day after passaging (co-transfection). The medium was replaced with fresh maintenance medium 6 hours after addition and the mixture was incubated under CO starting from the addition of the mixed solution. 2 Incubate in an incubator for 24 hours.

[0310] (1-4) Evaluation of mRNA by RT-PCR [1] RNA extraction (in vitro) RNA was extracted as follows.

[0311] The cells incubated for 24 hours after co-transfection were washed once with cold PBS. 300 μL of cell lysate of RNeasy micro kit or RNeasy96 kit (QIAGEN KK) was added thereto per well and collected after incubation for 5 minutes at room temperature. RNA was purified according to the kit manual including DNase treatment. RNase-Free DNaseset (QIAGEN KK89254) was used for DNase treatment. The purified / eluted RNA was subjected to the reverse transcription reaction described later.

[0312] [2] Reverse transcription reaction The reverse transcription reaction was performed as follows.

[0313] After adjusting the extracted RNA to 25-100 mg / mL, 10 μL of buffer mixture, 1 μL of enzyme mixture, 9 μL of purified water, and extracted RNA were mixed for each sample using a High Capacity RNA-to-cDNA kit (Applied biosystems) for reverse transcription reaction (37°C for 60 min, 95°C for 5 min, and 4°C hold).

[0314] 20 μL of the reverse transcription reaction product was diluted 5-fold with 80 μL of purified water and stored at −30°C.

[0315] [3]qRT-PCR (SYBR Green) qRTPCR primers (SYBR Green) were designed as follows.

[0316] 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) 5 μL of 2x FAST SYBR Green Master Mix (Applied Biosystems), 2 μL of purified water, 1 μL of primer mixture (10 μM) and 2 μL of cDNA (diluted 5 times) were suspended in each well as a PCR reaction solution to perform PCR reaction using viia7 (Applied Biosystems) (program: SYBR Green Regents, FAST, including melting curve).

[0317] [4]qRT-PCR (Taqman assay) The repaired hG6PC primer set and the total hG6PC primer set were designed as follows, and the 20x primer probe mixture was adjusted (primer concentration: 1000 nM probe concentration: 250 nM). For the hActin primer set and mActin primer set, the stock solutions were used as they were.

[0318] 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'-TCCTGTCAGGCATGC-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 5 μL 2x Taqman Fast Advanced Master Mix (Applied Biosystems), 2.5 μL purified water, 0.5 μL 20x primer probe mixture (10 μM) and 2 μL cDNA (diluted 5 times) were suspended in each well to adjust the PCR reaction solution (per tube) and PCR reaction was performed using viia7 or Quantstadio7 (Applied Biosystems) (program: Taqmanregents, FA).

[0319] (1-5) Evaluation of repair of abnormal splicing of G6PC mRNA by oligonucleotides of the example using a full-length human G6PC plasmid vector (pcDNA hG6PC (c.648G>T) + Int4) A full-length human G6PC plasmid vector (pcDNA hG6PC (c.648G>T) + Int4) and various oligonucleotides (15e_001, 15e_002, and the compound of Example 93 of International Publication No. WO 2004 / 048570 as a control) were co-transfected and evaluated by qRT-PCR (SYBR Green) whether abnormal splicing caused by G6PC (c.648G>T) was repaired. As a result, abnormal splicing of G6PC mRNA was normalized in the compounds of 15e_001 and 15e_002.

[0320] (Experimental Example 2) Evaluation of the Effect of Example Compounds on Repair of Abnormal Splicing Using Model Mice (2-1) Generation of Mice Vector production The G6PC KI vector was prepared according to the following procedure.

[0321] The G6PC 5' arm region was amplified using mouse genomic DNA as a template and the following mG6PC 5' arm amplification primers: Further amplification was performed using the mG6PC 5' arm IF primers and then inserted into the XhoI site of pBluescriptII (+ / -) (G6PC 5' arm vector).

[0322] mG6PC 5' arm amplification primers: Forward primer 5'-GGGAAACATGCATGAAGCCCTGGGC-3' (SEQ ID No: 67) Reverse primer 5'-TCCCTTGGTACCTCAGGAAGCTGCC-3' (SEQ ID No: 68) mG6PC5' arm IF primer: Forward primer 5'-CGGGCCCCCCCTCGAAAACTAGGCCTGAAGAGATGGC-3' (SEQ ID No: 69) Reverse primer 5'-TACCGTCGACCTCGAGGGTTGGCCTTGATCCCTCTGCTA-3' (SEQ ID No: 70) Then, using mouse genomic DNA as a template and using the following mG6PC 3' arm amplification primers, the G6PC 3' arm region was amplified. It was further 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).

[0323] mG6PC 3' arm amplification primers: 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) was used as template using the aforementioned pcDNA hG6PC (c.648G>T)+Int4 and using the following hG6PC+Int4 IF primers to amplify hG6PC (c.648G>T)+Intron4. G6PC 5'+3' arm vector was used as template and the following arm vector IF primers were further amplified. The two fragments were connected using the InFusion System to generate the G6PC KI vector.

[0324] hG6PC+Int4 IF Primers: Forward primer 5'-GGCCAACCCTGGAATAACTGCAAGGGCTCTG-3' (SEQ ID No: 75) Reverse primer 5'-TTGCATGGTTGTTGACTTTAAACACCGAAGA-3' (SEQ ID No: 76) Arm Vector IF Primers: 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 generate pSPgRNA (KI 5') and pSPgRNA (KI 3').

[0325] KI 5'gRNA: 5'-GGGATCAAGGCCAACCGGCTGG-3' (SEQ ID No: 79) KI 3'gRNA: 5'-TAAAGTCAACCGCCATGCAAAGG-3' (SEQ ID No: 80) (2-2) Microinjection Sterile distilled water was used to adjust the final concentrations of various vectors: G6PC KI vector was 10 ng / μL, pSPgRNA (KI 5') was 5 ng / μL, pSPgRNA (KI 3') was 5 ng / μL, and pSPCas9 (addgene) was 5 ng / μL, and injected into the pronucleus of C57BL / 6J mouse fertilized eggs to the extent that the pronucleus was fully expanded (about 2 pL) through a MILLEX-GV syringe filter (Millipore). The fertilized eggs were transplanted into the oviducts of recipient C57BL / 6J mice to obtain F0 mice.

[0326] (2-3) Genotyping and F1 systematization Genotyping for F1 systematization was performed by the following procedure.

[0327] Genomic DNA was extracted from the tail tissue of F0 mice using an automatic nucleic acid extractor (PI-200, available from KURABO INDUSTRIES LTD.) and a special kit. The extracted DNA was amplified using Amplitaq Gold Master mix (Thermo Fisher Scientific) and the following KI screening primers (95°C for 10 minutes, 35 cycles (95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds), 72°C for 2 minutes, and 4°C was maintained).

[0328] KI screening primers: Forward primer 5'-TACGTCCTCTTCCCCATCTG-3' (SEQ ID No: 81), Reverse primer 5'-CTGACAGGACTCCAGCAACA-3' (SEQ ID No: 82) The above-mentioned PCR product was subjected to gel electrophoresis and amplified using the genomic DNA of an individual identified as a band of about 433 bp as a template, PrimeSTAR GXL (Takara Bio Inc.) and the following KI genotyping primers (98°C for 2 minutes, 38 cycles (95°C for 15 seconds, 68°C for 5 minutes), 68°C for 7 minutes, and hold at 15°C).

[0329] KI genotyping primer (5'): Forward primer 5'-TTCCTTCCAAAGCAGGGACTCTCTATGT-3' (SEQ ID No: 83, same as (1)) Reverse primer 5'-CTTGCAGAAGGACAAGACGTAGAAGACC-3' (SEQ ID No: 84, same as (2)) KI genotyping primer (3'): Forward primer 5'-GAGTCTATATTGAGGGCAGGCTGGAGTC-3' (SEQ ID No: 85), Reverse primer 5'-TAGTCTGCCTGCTCACTCAACCTCTCCT-3' (SEQ ID No: 86) The aforementioned PCR product is subjected to gel electrophoresis. The PCR product using the KI genotyping primer (5') of the individual genomic DNA is directly sequenced using a Gentetic analyzer (Lifetechnology) and the following KI sequence primer, the sequence length (4705bp and 4026bp) of the individual genomic DNA being amplified using any one of the KI genotyping primer (5') and the KI genotyping primer (3') as expected. Those with KI as the expected sequence are used as KI positive F0.

[0330] KI sequence primer (5'): 5'-GAGTCTATATTGAGGGCAGGCTGGAGTC-3' (SEQ ID No: 87) KI-positive F0 was mated with C57BL / 6J to obtain F1. Genomic DNA was extracted from the auricle tissue of F1 using DNeasy 96 Blood & Tissue Kit (QIAGEN KK), and amplified using PrimeSTAR GXL (Takara Bio Inc.) and the aforementioned KI genotyping primer (5') (98°C for 2 minutes, 38 cycles (95°C for 15 seconds, 68°C for 5 minutes), 68°C for 7 minutes, 15°C hold).

[0331] The aforementioned PCR products were subjected to gel electrophoresis, and an individual (4705b) having a sequence length amplified as expected was used as a KI-positive F1. One line selected from the KI-positive F1 was propagated to generate the hG6PC (c.648G>T) + Int4 KI line.

[0332] (2-4) Genotyping of hG6PC(c.648G>T)+Int4 line Genomic DNA was extracted from auricle tissue using DNeasy 96 Blood & Tissue Kit (QIAGEN KK), and multiplex amplification was performed using KOD FX (TOYOBO CO., LTD.) and the following KI genotyping primers and mG6PC WT primers (98°C for 2 min, 32 cycles (95°C for 15 sec, 68°C for 5.5 min), 68°C for 5 min, and 4°C hold).

[0333] KI genotyping primer (5'): Forward primer 5'-TTCCTTCCAAAGCAGGGACTCTCTATGT-3' (SEQ ID No: 83, same as (1)) Reverse primer 5'-CTTGCAGAAGGACAAGACGTAGAAGACC-3' (SEQ ID No: 84, same as (2)) mG6PC WT Primers: Forward primer 5'-TAAATTTGACCAATGAGCACTGGAGGGTC-3' (SEQ ID No: 88) Reverse primer 5'-AAAATCATGTGTATGCGTGCCTTTCCTA-3' (SEQ ID No: 89), The aforementioned PCR products were subjected to gel electrophoresis, and the genotype of the offspring (WT, Ht, or Homo) was determined, while the amplification near 4705b was designated as the KI allele, and the amplification near 2536bp was designated as the mG6PC WT allele.

[0334] (2-5) Sampling of mice Sampling of mice was performed as follows.

[0335] On the night before sampling, fasting was started after installing the floor net to avoid fecal ingestion. The next day, the abdomen was opened under anesthesia, and the liver and kidney were removed after sufficient bleeding. Each organ was washed with ice-cold PBS, then trimmed to an appropriate size and stored in a test tube pre-filled with homogenized beads (NIKKATO CORPORATION). Each organ was immediately cooled with liquid nitrogen and then stored at -80°C.

[0336] (2-6) Evaluation of mRNA by qRTPCR [1] RNA extraction (in vivo) RNA was extracted as follows.

[0337] The cell lysate of RNeasy micro kit or Qiacube system (QIAGEN KK) was added to each tissue storage tube in an amount of 600 μL for homogenization at 25kHz for 2 minutes in tissue lyserII (QIAGEN KK). After incubation for 10 minutes under ice cooling, the supernatant was collected by centrifugation at 8000G for 10 minutes at room temperature. RNA purification was performed on the supernatant according to the manual of each kit including DNase treatment. RNase-Free DNase set (QIAGEN KK) was used for DNase treatment. The purified / eluted RNA was subjected to the aforementioned reverse transcription reaction, and the repaired G6PC mRNA was quantified according to the aforementioned qRT-PCR (Taqman assay).

[0338] (2-7) Evaluation of the repair of abnormal splicing by the example compounds using hG6PC (c.648G>T) + Int4 Ht KI mice The compounds of Examples 8 to 14 were each dissolved in Otsuka saline injection and administered subcutaneously to hG6PC (c.648G>T) + Int4 Ht KI mice at 30 mg / kg. Seven days after administration, the liver tissues of the mice were collected under conditions of overnight fasting, and qRT-PCR (Taqman) was used to evaluate whether the abnormal splicing caused by G6PC (c.648G>T) was repaired. As a result, the abnormal splicing of mRNA in the liver of hG6PC (c.648G>T) + Int4 Ht KI mice was normalized by the compounds of Examples 8 to 14, as shown in FIG. Figure 1 As shown in . In addition, the compounds of Examples 15 to 23 were evaluated in the same manner. As a result, the abnormal splicing of mRNA in the liver of hG6PC (c.648G>T) + Int4 Ht KI mice was normalized by the compounds of Examples 15 to 23, as shown in Figure 2 as shown in .

[0339] (Experimental Example 3) Evaluation of Oligonucleotide Delivery to Hepatocytes via GalNAc Unit Conjugates Using ApoB Knockdown Assay (3-1) Cultivation of Human Primary Hepatocytes Human primary hepatocytes were cultured as follows.

[0340] FBS (Gibco) was added to Modified Lanford Medium (Nissui Pharmaceutical Co., Ltd.) to 10% to prepare an adhesion medium. Frozen primary human hepatocytes (Gibco, Hu8105) were taken out of the liquid nitrogen tank, thawed at 37°C, and then immediately poured into CHRM (Invitrogen), gently mixed, and then centrifuged at room temperature (100g, 10 minutes). The supernatant was removed with an aspirator, and then the adhesion medium was heated to 37°C and added to prepare a cell suspension. The number of viable cells was counted by an automatic counter (Bio-Rad Laboratories, Inc.), and then the cells were seeded on a collagen-coated 24-well plate (the number of viable cells seeded: 1.4-2.1×10 5 cells / well). In CO 2 After culturing in an incubator for 4 to 5 hours, adherent cells were confirmed under a microscope, and the following compound addition experiments were performed.

[0341] (3-2) Addition of Example Compounds The compound of each Example was added to human primary hepatocytes as follows.

[0342] The compound of Reference Example 45 not bound to a GalNAc derivative or the compound of Example 208 bound to a GalNAc derivative was added to Modified Lanford Medium containing 10% FBS to prepare a compound-containing medium having a target concentration (which is 100 nM (1) in the evaluation of ApoB mRNA expression and 10 nM and 100 nM (2) in the evaluation of ApoB mRNA expression, as described below). After the culture medium of human primary hepatocytes cultured in each well of a 24-well plate was removed by aspiration, the compound-containing medium was added in an amount of 0.5 mL each to start incubation. After 72 hours, the RNA extraction experiment described below was performed.

[0343] (3-3) Evaluation of mRNA by qRT-PCR [1] RNA extraction (in vitro) RNA was extracted as follows.

[0344] After incubation, the medium containing the compound in each well of the human primary hepatocytes was removed by suction, and the cells were washed with ice-cold PBS (0.5 mL). 1 / 100 amount of 2-mercaptoethanol was added to the RLT buffer of RNeasy Mini QIAcube Kit (QIAGEN KK) to prepare cell lysates. Cell lysates were added in an amount of 400 μL per well and collected after incubation at room temperature for 5 minutes. RNA was purified from 350 μL of the collected solution using QIAcube according to the manual of RNeasy Mini-Animal tissue and cell-DNase digestion. RNase-Free DNase set (QIAGEN KK) was used for DNase treatment. The purified / eluted RNA was subjected to the reverse transcription reaction described later.

[0345] [2] Reverse transcription reaction Reverse transcription reaction was performed as follows. After the extracted RNA was adjusted to 25-100 mg / mL, High Capacity RNA-to-cDNA test kit (Applied biosystems) was used to mix 10 μL buffer mixture, 1 μL enzyme mixture, 9 μL purified water and extracted RNA for each sample for reverse transcription reaction (37°C for 60 minutes, 95°C for 5 minutes, 4°C maintenance). 20 μL reverse transcription reaction products were diluted 5 times with 80 μL purified water and stored at -30°C or evaluated by qRT-PCR as follows.

[0346] [3]qRT-PCR (Taqman assay) The following ApoB primer probe set (x20) and Actinb primer probe set (x20) were used.

[0347] ApoB primer probe set (x20) (Taqman): ABI Hs00181142_m1 FAM Actinb primer probe set (x20) (Taqman): ABI Hs01060665_g1 FAM 5 μL 2x Taqman Fast Advanced Master Mix (Applied Biosystems), 2.5 μL purified water, 0.5 μL 20x primer probe mixture (10 μM) and 2 μL cDNA (diluted 5 times) were suspended in each well to adjust the PCR reaction solution (per tube) and PCR reaction was performed using Quantstadio7 (Applied Biosystems) (95°C 20 seconds, 40 cycles (95°C 1 second, 60°C 20 seconds), 4°C hold). Actinb was used as an endogenous control to evaluate the relative expression level of ApoB mRNA.

[0348] (3-4) Evaluation of the Inhibition of ApoB mRNA Expression by Example Compounds Using Human Liver Cultured Cells (2) The compound synthesized in Example 24 was added at 10 nM and 100 nM, and primary human hepatocytes were inoculated at the same time, and the expression of ApoB mRNA in the cells was evaluated by qRT-PCR (Taqman assay) after 72 hours of culture. Figure 3 As shown in, when the average expression of ApoB mRNA in 4 wells without compound addition was taken as 100%, 10 nM ApoB-ASO inhibited 4.4% of expression and ApoB-ASO conjugate inhibited 30.3% of expression. 100 nM ApoB-ASO inhibited 41.2% of expression and ApoB-ASO conjugate inhibited 68.6% of expression. This indicates that the compound bound to the GalNAc derivative can more strongly inhibit expression than the compound without the GalNAc derivative.

[0349] (Example 25) DMTr-on ion exchange column purification of oligonucleotides bound to a GalNAc unit having a 4,4′-dimethoxytrityl group at the 6-hydroxyl group in the GalNAc moiety (1) Synthesis of oligomers and preparation of mixed solutions The compound described in Example 16 was synthesized. However, AKTA Oligopilot (available from GE Healthcare) was used as an automatic nucleic acid synthesizer, Primer Support 5G Unylinker 350, 390 μmol (available from GE Healthcare) was used as a solid phase support, and a synthesis procedure suitable for scale was used. After the synthesis was completed, a mixture of a conjugate and an oligonucleotide analog having a target oligonucleotide sequence was obtained, wherein the 6-hydroxyl group of the GalNAc portion of the target oligonucleotide sequence was protected by 4,4'-dimethoxytrityl. The resulting mixture was divided into two portions, and each portion was treated with 25 mL of concentrated ammonia to remove the oligonucleotide from the support, while removing the cyanoethyl group as a protecting group on the phosphorus atom and the protecting group on the nucleobase and filtering using a test tube with a filter to obtain an oligonucleotide mixed solution (25 mL) (two bottles) containing the target conjugate. Using AKTA pure150 (available from GE Healthcare) as purification device, with filler SOURCE 15Q (96mL) (available from GE Healthcare) filling column FineLINE Pilot (35mm x 100mm) (available from GE Healthcare), the aforementioned oligonucleotide mixture was purified in two batches by the following method. The UV detection wavelength was set to 260nm, 280nm or 350nm, and the following three steps were used as a purification procedure. Figure 5 Chromatograms detecting absorbance at detection wavelengths of 260 nm and 350 nm are shown because flow through the column was detected during these steps.

[0350] (i) Step of adsorption of DMTr-on oligonucleotide onto column The flow rate was set to 40 ml / min and 1 CV of buffer A1 (10 mM sodium hydroxide solution) was poured. 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 solution) was released. Subsequently, the flow rate was set to 40 ml / min and 1 CV of buffer A1 (10 mM sodium hydroxide solution) was poured. Subsequently, 6 CV of buffer A1 (10 mM sodium hydroxide solution): buffer B1 (10 mM sodium hydroxide solution / 2 M sodium chloride solution) (35:65) was poured to wash away the unbound X 20 Finally, pour in 2CV of buffer A1 (10 mM sodium hydroxide aqueous solution).

[0351] (ii) On-column de-DMTr conversion step The flow rate was set to 40 ml / min and 1 CV of buffer A2 (0.4% trifluoroacetic acid in water) was poured in. Subsequently, the flow rate was changed to 20 ml / min and 6.25 CV was poured in to adjust the bound X adsorbed on the column packing. 20 The oligonucleotide was de-DMTr converted.

[0352] (iii) Step of eluting the target substance The flow rate was set to 40 ml / min, and 3CV of buffer A1 (10 mM sodium hydroxide aqueous solution) was poured for equilibration. After a gradient feed (15CV) of buffer A1 (10 mM sodium hydroxide aqueous solution): buffer B1 (10 mM sodium hydroxide aqueous solution / 2 M sodium chloride aqueous solution) from (75:25) to (25:75), 2CV of buffer B1 (10 mM sodium hydroxide aqueous solution / 2 M sodium chloride aqueous solution) was poured to fractionate the oligonucleotides converted from de-DMTr (the fractionation amount was 48 mL).

[0353] The fractions detected by UV (260 nm) during elution were checked using a microspectrophotometer Xpose (available from Trinean nv) to obtain fraction solutions (first time: 432 ml, and second time: 480 ml) ( Figure 5 ).

[0354] After the column was used once, it was washed with a 2 M sodium chloride aqueous solution containing 30% isopropanol before the next purification.

[0355] (2) Concentration, dialysis and freeze drying The fraction solutions (first time: 432 ml, and second time: 480 ml) were combined into one, and an appropriate amount of acetic acid was added thereto to prepare a mixture with a pH of 7.5 to 8.0. 2 The residue was concentrated using a KR2i TFF system (Repligen Corporation) of GE Healthcare, dialyzed against Otsuka distilled water, and then freeze-dried to obtain a freeze-dried powder of the target substance (1.14 g in total).

[0356] (Comparative Example 1) Purification of the conjugate by DMTr-on ion exchange column with DMTr phase introduced near the oligonucleotide For comparison with Example 25, the following X 13 The GalNAc conjugate was purified by DMTr-on ion exchange column. 13The protective groups of the 3-hydroxyl, 4-hydroxyl and 6-hydroxyl groups on the contained GalNAc can be unified as acetyl groups. This eliminates the need to distinguish the protective groups in the synthesis step and thus facilitates the synthesis of the GalNAc unit portion. In addition, the amount of GalNAc can be easily increased or decreased by adjusting the number of couplings with the 5' end oligonucleotide in the solid phase synthesis step. However, after completing the solid phase synthesis of the oligomer, only one 4,4'-dimethoxytrityl group can be left.

[0357] [Comparative Example 1-1] Synthesis of GalNAc unit imide corresponding to X13 (Compound Ref1F) (Ref1A) Synthesis of 9H-fluoren-9-ylmethyl N-[2-[2-[(2R)-3-benzyloxy-2-hydroxy-propyloxy]ethoxy]ethyl]carbamate (Compound Ref1A) [Formula 91] 9H-fluorene-9-ylmethyl N-[2-(2-hydroxyethoxy)ethyl]carbamate (3.11 g, 10 mmol) and benzyl (S)-(+)-glycidyl ether (0.8 g, 5 mmol), which are known compounds in the literature (Bioconjugate Chemistry, 2000, 11, 755-761), were dissolved in dichloromethane (30 mL), and boron trifluoride-ether complex (0.1 mL, 1 mmol) was added thereto, followed by stirring at room temperature for 16 hours. After the reaction was completed, a saturated aqueous sodium bicarbonate solution was added. The organic layer was washed with a saturated aqueous sodium bicarbonate solution and saturated physiological saline, then dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. Purification was performed by silica gel column chromatography (hexane: ethyl acetate = 80:20-0:100, v / v) to obtain an oily target substance Ref1A (0.74 g, yield 30%).

[0358] 1 H-NMR (CDCl 3 )δ: 7.76(2H,d,J=7.9Hz),7.60(2H,d,J=7.3Hz),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 = 6.7Hz), 4.25-4.19 (1H, m), 4.04-3.97 (1H, m), 3.69-3.35 (12H, m).

[0359] C 29 H 33 NO 6 Calculated value: [M+H] +492, measured value 492.

[0360] (Ref1B) Synthesis of (2R)-1-[2-(2-aminoethoxy)ethoxy]-3-benzyloxy-propane-2-ol, trifluoroacetate (Compound Ref1B) [Formula 92] The compound Ref1A (13.8 g, 28.1 mmol) synthesized in step (Ref1A) was dissolved in ethanol / tetrahydrofuran (50 mL / 50 mL), and 1 N sodium hydroxide aqueous solution (100 mL) was added thereto, followed by stirring at room temperature for 2 hours. After the reaction was completed, the organic solvent was removed by distillation under reduced pressure, and 1 N hydrochloric acid (100 mL) was added. The solid product produced was removed by filtration. The by-product contained in the water layer as the passing fluid was extracted with ethyl acetate and removed by distillation under reduced pressure, and acetonitrile was added to the obtained residue, followed by filtration. The passing fluid was removed by distillation under reduced pressure to obtain a crude product. Using 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution as eluents, separation and purification were performed by reverse phase HPLC (GL Sciences Inc., Inertsil ODS-3), the fractions containing the target substance were combined, and the solvent was removed by distillation under reduced pressure to obtain an oily target substance Ref1B (7.1 g, yield 66%).

[0361] 1 H-NMR (CDCl 3 )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).

[0362] (Ref1C) Synthesis of [(2R,3R,4R,5R,6R)-5-acetylamino-3,4-diacetoxy-6-[2-[2-[2-[(2R)-3-benzyloxy-2-hydroxy-propoxy]ethoxy]ethylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound Ref1C) [Formula 93] 2-[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyacetic acid (6 g, 14.8 mmol), 1-(-3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.4 g, 17.8 mmol), 3H-1,2,3-triazolo[4,5-b]pyridine-3-ol (2.0 g, 14.8 mmol), N,N-diisopropylethylamine (9.0 mL, 51.8 mmol) and compound Ref1B (6.7 g, 17.5 mmol) synthesized in step (Ref1B) were dissolved in dichloromethane (130 mL), followed by stirring at room temperature for 4 hours. After the reaction was completed, the organic layer was washed with 1N hydrochloric acid, saturated sodium bicarbonate aqueous solution and saturated physiological saline, then dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. Purification by silica gel column chromatography (ethyl acetate: methanol = 100: 0-90: 10, v / v) to obtain an amorphous target substance Ref1C (7.9 g, yield 81%).

[0363] 1 H-NMR (CDCl 3 )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=11.5,3.6Hz),4.63- 4.53(3H,m),4.34(1H,d,J=15.1Hz),4.22-3.83(5H,m),3.73-3.26(13H,m),2.15(3H,s),2.05(3H,s),1.99(6H,s).

[0364] C 30 H 44 N 2 O 14 Calculated value: [M+H] + 657, measured value 657.

[0365] (Ref1D) Synthesis of [(2R,3R,4R,5R,6R)-5-acetylamino-3,4-diacetoxy-6-[2-[2-[2-[(2R)-2,3-dihydroxypropoxy]ethoxy]ethylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound Ref1D) [Formula 94] The 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 thereto, followed by vigorous stirring for 8 hours at 50° C. under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the fluid was removed by distillation under reduced pressure. The amorphous target substance Ref1D (6.7 g, yield 98%) was obtained.

[0366] 1 H-NMR (CDCl 3 )δ: 7.03-6.98(1H,m),6.43(1H,d,J=9.1Hz), 5.35(1H,d,J=3.3Hz), 5.15(1H,dd,J=11.2,3.3Hz), 4.58(1H,d,J=7.9Hz ),4.35(1H,d,J=15.7Hz),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).

[0367] C 23 H 38 N 2 O 14 Calculated value: [M+H] + 568, measured value 568.

[0368] (Ref1E) Synthesis of [(2R,3R,4R,5R,6R)-5-acetylamino-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) [Formula 95] An appropriate amount of pyridine is added to the compound Ref1D (6.8 g, 12 mmol) synthesized in step (Ref1D) and azeotroped under reduced pressure. It is dissolved in pyridine (40 mL), and 4,4'-dimethoxytrityl chloride (4.5 g, 13 mmol) is added thereto, followed by stirring at room temperature for 2 hours. After the reaction is completed, the solvent is removed by distillation under reduced pressure. After adding an appropriate amount of toluene and azeotroping, a crude product is obtained. It is purified by silica gel column chromatography (ethyl acetate: ethyl acetate / methanol (5 / 1) = 100:0-50:50, v / v, containing 1% triethylamine) to obtain an amorphous target substance Ref1E (6.9 g, yield 66%).

[0369] 1 H-NMR (CDCl 3 )δ:7.42(2H,d,J=7.9Hz),7.33-7.19(7H,m),7.01-6.94(1H,m),6.83(4H,d,J =9.1Hz), 6.37(1H,d,J=8.5Hz), 5.33(1H,d,J=3.3Hz), 5.15(1H,dd,J=11.2,3. 3Hz),4.58(1H,d,J=7.9Hz),4.32(1H,d,J=15.7Hz),4.22-3.86(5H,m),3.79(6 H,s),3.66-3.12(13H,m),2.13(3H,s),2.03(3H,s),1.97(3H,s),1.95(3H,s).

[0370] C 44 H 56 N 2 O 16 Calculated value: [M+Na] + 893, measured value 892.

[0371] (Ref1F) Synthesis of [(2R,3R,4R,5R,6R)-5-acetylamino-3,4-diacetoxy-6-[2-[2-[2-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]ethoxy]ethylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound Ref1F) [Formula 96] The 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 thereto, followed by stirring at room temperature for 1 hour. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain a crude product. It 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 an amorphous target substance Ref1F (6.6 g, yield 78%).

[0372] 1 H-NMR (CDCl 3)δ:7.47-7.40(2H,m),7.35-7.17(7H,m),6.98-6.91(1H,m),6.85-6.78(4 H,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).

[0373] [Comparative Example 1-2] Synthesis of Conjugate Introduced with 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 amidate to convert X 18 Replace with X 13 , and synthesis was performed in the same manner as in Example 8. However, the addition reaction of the GalNAc unit imide was repeated 3 times to bind three X13.

[0374] The nucleotide sequence of the compound is a sequence complementary to the 92nd to 106th sequence at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the nucleotide 728 in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6878.11).

[0375] [Comparative Example 1-3] DMTr-on ion exchange column purification of oligonucleotide bound to a GalNAc unit having a 4,4'-dimethoxytrityl group at the 5' end (1) Synthesis of oligomers and preparation of mixed solutions 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) AKTA Oligopilot (available from GE Healthcare) was used as a nucleic acid automatic synthesizer, Primer Support 5G Unylinker 350, 390 μmol (available from GE Healthcare) was used as a solid phase support, and a synthesis procedure suitable for scale was used. Using 10 μmol of the obtained solid phase support, GalNAc unit X 13 The oligonucleotides were concentrated three times using an ABI 392 DNA / RNA synthesizer (available from Applied Biosystems). After the synthesis was complete, a mixture of oligonucleotide analogs having the target oligonucleotide sequence and bound to a GalNAc unit having a 4,4'-dimethoxytrityl group at the 5' end was obtained. The oligonucleotides were cut from the support by treatment with 6 mL of concentrated ammonia and the cyanoethyl group as a protecting group on the phosphorus atom and the protecting group on the nucleobase was removed.

[0376] Purification method 1: Clarity QSP (available from Phenomenex Inc.) purification By performing purification in the same manner as in Example 8, the target substance was obtained.

[0377] The nucleotide sequence of the compound is a sequence complementary to the sequence at positions 92 to 106 at the 5' end of exon 5 of the c.648G>T mutant G6PC gene, wherein the nucleotide at position 728 in the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) has a mutation from G to T. The compound was identified by negative ion ESI mass spectrometry (measured value: 6878.11).

[0378] Purification method 2: DMTr-on ion exchange column purification Prior to purification on a DMTr-on ion exchange column, the oligonucleotide mixture was replaced with 10 mM aqueous sodium hydroxide.

[0379] The aforementioned oligonucleotide mixture was purified by the following method using AKTA pure150 (available from GE Healthcare) as a purification apparatus and RESOURCE Q6mL (available from GE Healthcare) as a purification column. The ultraviolet detection wavelength was set to 260 nm, and the following three steps were used as one purification procedure. Figure 6 Chromatograms of the flow through the column during these steps at a detection wavelength of 260 nm are shown.

[0380] (i) Step of adsorbing DMTr-on oligonucleotide on an ion exchange column Pour 2CV buffer A1 (10mM sodium hydroxide aqueous solution) at a flow rate of 3ml / min. Add oligonucleotide mixture (1.5mL, 10mM sodium hydroxide aqueous solution) to the top of the column at a flow rate of 2ml / min, and pour 2.5CV buffer A1 (10mM sodium hydroxide aqueous solution). Subsequently, pour 1CV buffer A1 (10mM sodium hydroxide aqueous solution) at a flow rate of 3ml / min, and then pour buffer A1 (10mM sodium hydroxide aqueous solution): buffer B1 (10mM sodium hydroxide aqueous solution / 2M sodium chloride aqueous solution) (2CV is 40:60 to 25:75 and 4CV is 25:75) at a flow rate of 2.4ml / min.

[0381] (ii) De-DMTr conversion step on ion exchange column 7 CV of buffer A2 (0.4% trifluoroacetic acid in water) was poured in at a flow rate of 1.2 ml / min.

[0382] (iii) Step of eluting the target substance 5CV of buffer A1 (10 mM sodium hydroxide aqueous solution) was poured at a flow rate of 3 ml / min for equilibration. Subsequently, after a gradient feed (10CV) of buffer A1 (10 mM sodium hydroxide aqueous solution): buffer B1 (10 mM sodium hydroxide aqueous solution / 2 M sodium chloride aqueous solution) from (30:70) to (0:100) at a flow rate of 3 ml / min, 2CV of buffer B1 (10 mM sodium hydroxide aqueous solution / 2 M sodium chloride aqueous solution) was poured.

[0383] Depend on Figure 6 As can be seen from the results, since absorption at 260nm was not detected in the elution step after deprotection, the DMT-on conjugate was not adsorbed on the column in the first adsorption step and passed therethrough. Therefore, the target substance cannot be produced by purification by ion exchange column. Thus, it is considered important for the DMTr-on column purification of the conjugate of GalNAc unit and oligonucleotide that the hydrophobic protecting group is bound to GalNAc, and / or the number of hydrophobic protecting groups contained in one GalNAc unit is equal to or greater than the number of GalNAc.

[0384] In this manual, 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 , Ae2p , G e2p , C e2p 、T e2p , A e2s , G e2s , C e2s 、T e2s , 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 and X 22 is a group having the structure shown below.

[0385] [Formula 97] [Formula 98] [Formula 99] [Formula 100] [Formula 101] [Formula 102] [Formula 103] [Formula 104] All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

[0386] Industrial Applicability The present invention can be used to treat type Ia glycogen storage disease.

[0387] Sequence Listing Free Text SEQ ID Nos: 1 to 48 and 93 to 96 each represent the sequence of an antisense oligonucleotide. The nucleotides constituting the antisense oligonucleotide may be any of natural DNA, natural RNA, DNA / RNA chimeras, and modified forms thereof, but preferably at least one of them is a modified nucleotide. SEQ ID Nos: 49 to 65 and 67 to 92 each represent the sequence of a primer. SEQ ID No: 66 represents the sequence of a peptide.

Claims

1. A conjugate of an oligonucleotide having a nucleic acid sequence expected to have a pharmacological effect in hepatocytes and a biantennary GalNAc unit or a pharmaceutically acceptable salt thereof, wherein: In the conjugate, the biantennary GalNAc unit is represented by the following formula and is bound to the 5' end or the 3' end of the oligonucleotide, and Wherein the conjugate is specifically delivered to hepatic parenchymal cells in vivo: [Formula 1] Wherein W is a group represented by the following formula: [Formula 2] G represents 5-acetylamino-2-hydroxymethyl-3,4-dihydroxytetrahydropyran-6-yl (GalNAc); Z represents an oxygen atom or a sulfur atom; L 1 and L 2 One of them represents a methylene group (CH2), and the other represents a non-intervening atom; o, q, r and s each independently represent 0 or 1; n represents an integer from 1 to 15; m represents an integer from 0 to 5; and v represents an integer from 1 to 7, provided that when n is 1, then m is an integer from 0 to 5, and when n is an integer from 2 to 15, then m is 0.

2. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein o and q in the formula of the biantennary GalNAc unit are each 0.

3. The conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the biantennary GalNAc unit is bound to the 5' end of the oligonucleotide.

4. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the biantennary GalNAc unit is a group represented by any one of the following formulae: [Formula 3] [Formula 4] [Formula 5] [Formula 6] and [Formula 7] 5. A medicament comprising the conjugate according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

6. A method for preparing a conjugate of an oligonucleotide and a GalNAc unit, comprising the following steps: a: Synthesizing a conjugate comprising a structure represented by the following formula wherein a GalNAc unit-phosphate group containing a hydrophobic protective group is bound to the 5' end or the 3' end of the oligonucleotide: [Formula 8] in, R a , R b and R c At least one of them is a hydrophobic protecting group, and the rest are hydrogen atoms or protecting groups that are removed under free conditions; X1 and X2 each independently represent a linker structure, which can branch between X1 and X2; t is the number of branches in the linker structure and is an integer from 1 to 10; and Y represents a bond that is bound to the oligonucleotide through a phosphate group or a thiophosphate group; b: passing the solution containing the product of step a through a column filled with a resin having adsorptive properties due to hydrophobic interactions, and passing an aqueous washing solution through the column; and c: Elution of the oligonucleotides from the column after step b.

7. The preparation method according to claim 6, wherein the hydrophobic protecting group is a protecting group that is removed under alkaline conditions or acidic conditions.

8. The preparation method according to claim 6, wherein the structure comprising the GalNAc unit-phosphate group containing a hydrophobic protective group is bound to the 5'-hydroxyl group of the nucleoside located at the 5' end of the oligonucleotide.

9. The preparation method according to claim 6, wherein: In the structure comprising a GalNAc unit-phosphate group containing a hydrophobic protecting group, R a is a hydrophobic protecting group, R b and R c Each is a hydrogen atom, and t is an integer of 1 to 3.

10. The preparation method according to claim 6, wherein the resin having adsorptivity due to hydrophobic interaction is any one selected from styrene-divinylbenzene adsorption resin, styrene-divinylbenzene modified adsorption resin, methacrylic adsorption resin and phenolic adsorption resin.

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