Method for preparing compound with two-branch N-acetyl-D-galactosamine structure

Abstract

The problem addressed by the present invention is to provide: a novel method for preparing an N-acetyl-D-galactosamine ligand oligonucleotide conjugate, in particular, a novel method for preparing an N-acetyl-D-galactosamine unit; and a novel intermediate. In a novel process for the preparation of N-acetyl-D-galactosamine units, studies have been made regarding the selective deprotection of benzyl groups using a palladium catalyst. As a result, a novel preparation method and a novel intermediate which can achieve high quality and high yield and are industrially advantageous are found, which results in the completion of the invention.

Description

Technical Field

[0001] The present invention relates to a novel method for preparing a biantennary N-acetyl-D-galactosamine ligand-oligonucleotide conjugate, and particularly to a novel industrially advantageous method for preparing a biantennary N-acetyl-D-galactosamine unit, and a novel intermediate. Background Art

[0002] As a method for delivering an oligonucleotide to hepatocytes in the liver, a method using a nucleic acid drug conjugate in which N-acetyl-D-galactosamine (also referred to as GalNAc) is bonded to an oligonucleotide via a linker is known (Patent Document 1), and a novel biantennary N-acetyl-D-galactosamine unit (also referred to as a biantennary GalNAc unit) capable of delivering any oligonucleotide to the liver and a method for preparing the unit using a protecting group such as an Fmoc group have also been reported (Patent Document 2). There is a need to develop a novel preparation method capable of synthesizing a large amount of high-quality biantennary GalNAc units, but it is extremely difficult to generally prepare a compound having a complex structure and many reactive sites (such as a biantennary GalNAc unit). In particular, during the preparation process, it is difficult to selectively introduce and remove substituents and protecting groups to obtain a compound having a complex structure and many reactive sites with high quality and high yield. For example, in a compound containing both a trityl group and a benzyl group, an attempt has been made to selectively remove only the benzyl group, but the yield was about 35% (Non-Patent Document 1).

[0003] Citation List

[0004] Patent Documents

[0005] Patent Document 1: International Publication No. WO 2019 / 172286

[0006] Patent Document 2: International Publication No. WO 2021 / 049504

[0007] Non-Patent Documents

[0008] Non-Patent Document 1: J. Chem. Soc., Perkin Trans. 1, 1987, (3), 537-45 Summary of the Invention

[0009] Technical Problem

[0010] An object of the present invention is to provide a method for preparing an N-acetyl-D-galactosamine ligand-oligonucleotide conjugate, which includes a novel step of selectively introducing and removing substituents and / or protecting groups.

[0011] Solution to the Problem

[0012] The present invention relates to a novel method for preparing an N-acetyl-D-galactosamine ligand-oligonucleotide conjugate for solving the above problems, and particularly to a novel method for preparing an N-acetyl-D-galactosamine unit, and a novel intermediate. In particular, the present invention relates to a novel industrially advantageous preparation method that can achieve high quality and high yield, in which selective deprotection of benzyl using a palladium catalyst is a key reaction.

[0013] The present invention includes the following aspects [1] to

[14] .

[0014] [1] A method for preparing a compound represented by formula (1):

[0015] [Chemical formula 1]

[0016]

[0017] wherein Ac represents an acetyl group, and DMTr represents 4,4'-dimethoxytriphenylmethyl,

[0018] The method includes the step of reacting a compound represented by formula (2) with a palladium catalyst in a reaction solvent in the presence of a reducing agent:

[0019] [Chemical formula 2]

[0020]

[0021] wherein Ac represents an acetyl group, DMTr represents 4,4'-dimethoxytriphenylmethyl, and Bn represents benzyl.

[0022] [2] The preparation method according to [1], wherein the palladium catalyst is palladium on carbon or palladium hydroxide.

[0023] [3] The preparation method according to [1] or [2], wherein the reaction solvent is one or more solvents selected from alcohol-based solvents, ester-based solvents, ether-based solvents, and amide-based solvents.

[0024] [4] The preparation method according to [1] or [2], wherein the reaction solvent is one or more solvents selected from 2-propanol, dimethylacetamide, N-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone.

[0025] [5] The preparation method according to [1] or [2], wherein the reaction solvent is a mixed solvent of ethyl acetate and one or more solvents selected from dimethylacetamide, N-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone.

[0026] [6] A method for preparing a compound represented by formula (3) or a salt thereof:

[0027] [Chemical formula 3]

[0028]

[0029] wherein Ac represents acetyl, and DMTr represents 4,4'-dimethoxytriphenylmethyl,

[0030] The method includes the step of reacting a compound represented by formula (1) obtained by the preparation method according to any one of [1] to [5] with an amidite reagent.

[0031] [7] A method for preparing a conjugate composed of an oligonucleotide and a diantennary N-acetyl-D-galactosamine unit, wherein the diantennary N-acetyl-D-galactosamine unit is bonded to the 5'-end or 3'-end of the oligonucleotide, and the conjugate is represented by formula (4):

[0032] [Chemical formula 4]

[0033]

[0034] wherein Ac represents acetyl, and DMTr represents 4,4'-dimethoxytriphenylmethyl, and

[0035] wherein Z represents an oxygen atom or a sulfur atom, and the bond on the right side of the structural formula represents the bond with the oligonucleotide,

[0036] The method includes the step of reacting a compound represented by formula (3) obtained by the preparation method according to [6] with an oligonucleotide.

[0037] [8] A method for preparing a compound represented by formula (2) used in (1), which includes:

[0038] The step of bonding a compound represented by formula (5) or a salt thereof with a compound represented by formula (6) by using a condensing agent:

[0039] [Chemical formula 5]

[0040]

[0041] wherein Bn represents benzyl,

[0042] [Chemical formula 6]

[0043]

[0044] wherein Ac represents acetyl, and DMTr represents 4,4'-dimethoxytriphenylmethyl, and

[0045] A step of acetylating the hydroxyl group on the pyran ring by using an acetylating agent.

[0046] [9] A method for preparing the compound represented by formula (5) or a salt thereof used in [8], which comprises:

[0047] A step of reacting the compound represented by formula (7) with β-alanine or a salt thereof:

[0048] [Chemical formula 7]

[0049]

[0050] wherein Bn represents benzyl,

[0051] or

[0052] A step of reacting the compound represented by formula (7) with an alkyl β-alaninate or a salt thereof, wherein

[0053] When the method includes a step of using an α-alanine alkyl ester or a salt thereof, the method further includes a hydrolysis step.

[0054]

[10] A method for preparing the compound represented by formula (7) used in [9], which comprises:

[0055] A step of reacting 2-(benzyloxy)propane-1,3-diol with N,N'-disuccinimidyl carbonate in the presence of a base.

[0056]

[11] A compound represented by formula (2):

[0057] [Chemical formula 8]

[0058]

[0059] wherein Ac represents acetyl, DMTr represents 4,4'-dimethoxytriphenylmethyl, and Bn represents benzyl.

[0060]

[12] A compound represented by formula (8):

[0061] [Chemical formula 9]

[0062]

[0063] wherein Ac represents acetyl, DMTr represents 4,4'-dimethoxytriphenylmethyl, and Bn represents benzyl.

[0064]

[13] A compound represented by formula (5) or a salt thereof:

[0065] [Chemical formula 10]

[0066]

[0067] Among them, Bn represents benzyl.

[0068]

[14] A compound represented by formula (7):

[0069] [Chemical formula 11]

[0070]

[0071] Among them, Bn represents benzyl.

[0072]

[15] A compound represented by formula (6) or a salt thereof:

[0073] [Chemical formula 12]

[0074]

[0075] Among them, Ac represents acetyl, and DMTr represents 4,4'-dimethoxytriphenylmethyl.

[0076] Advantageous effects of the invention

[0077] According to the present invention, a novel industrially advantageous method for preparing N-acetyl-D-galactosamine units with high yield and high quality and a novel intermediate can be provided, wherein the selective deprotection of benzyl using a palladium catalyst is the key reaction. In particular, a simple and inexpensive preparation method can be provided, which does not use expensive protecting groups such as the Fmoc group. Thereby, a novel method for preparing N-acetyl-D-galactosamine ligand-oligonucleotide conjugates can be provided.

[0078] Description of the embodiments

[0079] Hereinafter, the embodiments of the present invention will be described in more detail.

[0080] The present invention provides a method for preparing a conjugate composed of an oligonucleotide and a diantennary N-acetyl-D-galactosamine unit represented by formula (4), wherein the diantennary N-acetyl-D-galactosamine unit is bonded to the 5'-end or 3'-end of the oligonucleotide by reacting a compound represented by formula (3) with the oligonucleotide. The present invention is further characterized by a method for obtaining the compound represented by formula (3) as an intermediate.

[0081] That is, the present invention includes a method for preparing a compound represented by formula (1) by using a compound represented by formula (2), and a method for preparing a compound represented by formula (3) or a salt thereof by using the compound represented by formula (1) obtained above.

[0082] In addition, the present invention includes a method for reacting a compound represented by formula (5) or a salt thereof with a compound represented by formula (6) and further with a compound represented by formula (8) to prepare a compound represented by formula (2), and a method for preparing a compound represented by formula (5) or a salt thereof using a compound represented by formula (7) or compound (Va) in the following examples.

[0083] In addition, the present invention relates to intermediates obtained during the above methods.

[0084] Compounds represented by formulas (1) to (7) are sometimes referred to by different names herein. In addition, these names are sometimes listed together. The names of the compounds are shown in Table 1.

[0085] [Table 1]

[0086]

[0087]

[0088] Hereinafter, a synthesis method according to one aspect of the present invention will be shown.

[0089] Method for preparing compound (Ib or 6)

[0090] [Chemical formula 13]

[0091]

[0092] Step A-1: This step is a step of preparing compound (IIa) by carrying out a β-selective glycosylation reaction of 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-D-galactopyranose with an amino alcohol in an organic solvent using an acid catalyst. The solvent that can be used is not particularly limited as long as it is a solvent that does not inhibit the reaction, and examples thereof include one or more solvents selected from dichloromethane, acetonitrile, tetrahydrofuran, etc. The preferred solvent is dichloromethane. The acid catalyst that can be used is not particularly limited as long as it is used for glycosylation, and examples thereof include trifluoromethanesulfonic acid, trimethylsilyl trifluoromethanesulfonate, and tin tetrachloride. The preferred acid catalyst is trifluoromethanesulfonic acid. The reaction temperature is generally 25°C to 45°C, and preferably 30°C to 40°C. The reaction time varies depending on the type and amount of the acid catalyst used, and is generally 1 hour to 28 hours, and preferably 21 hours to 23 hours. The obtained compound can be purified by using conventional methods such as recrystallization or silica gel chromatography.

[0093] Step A-2: This step is to prepare compound (IIIa) by deprotecting the acetyl (Ac) group through reacting compound (IIa) with a deprotecting reagent in an organic solvent. The deprotecting reagent that can be used is not particularly limited as long as it is a reagent for deprotecting acetyl, and examples thereof include inorganic bases such as sodium hydroxide, potassium hydroxide or sodium carbonate, and organic bases such as ammonia, alkylamine, sodium methoxide or sodium ethoxide. The preferred deprotecting reagent is sodium methoxide. The solvent that can be used is not particularly limited as long as it is a solvent that does not inhibit the reaction, and examples thereof include one or more solvents selected from methanol, ethanol, etc. The preferred solvent is ethanol. The reaction temperature is generally 30°C to 50°C, and preferably 35°C to 45°C. The reaction time varies depending on the type and amount of the deprotecting reagent used, and is generally 0.5 hour to 24 hours, and preferably 12 hours to 14 hours. The obtained compound can be purified by using conventional methods such as recrystallization or silica gel chromatography.

[0094] Step A-3: This step is to prepare compound (IVa) by selectively protecting the primary hydroxyl group with a 4,4'-dimethoxytrityl (DMTr) group through reacting compound (IIIa) with a tritylation reagent (such as 4,4'-dimethoxytrityl chloride or 4,4'-dimethoxytrityl trifluoromethanesulfonate) in an organic solvent. The solvent that can be used is not particularly limited as long as it is a solvent that does not inhibit the reaction, and examples thereof include N,N-dimethylacetamide, N,N-dimethylformamide and N-methylpyrrolidone. The preferred solvent is N,N-dimethylacetamide. The reaction temperature is generally 15°C to 35°C, and preferably 20°C to 30°C. The reaction time varies depending on the type and amount of the tritylation reagent used, and is generally 1 hour to 8 hours, and preferably 4 hours to 6 hours. The obtained compound can be purified by using conventional methods such as recrystallization or silica gel chromatography.

[0095] Step A-4: This step is a step of selectively deprotecting the benzyloxycarbonyl (Cbz) group of compound (IVa) by reacting compound (IVa) with a deprotecting reagent in an organic solvent to prepare compound (Ib or 6). The deprotecting reagent that can be used is not particularly limited as long as it can deprotect the benzyloxycarbonyl group, and examples thereof include palladium on carbon or palladium hydroxide on carbon, and examples of the reducing agent include hydrogen, formic acid, or ammonium formate. The preferred metal catalyst is a palladium on carbon catalyst, and the preferred reducing agent is hydrogen. The solvent that can be used is not particularly limited as long as it is a solvent that does not inhibit the reaction, and examples thereof include one or more solvents selected from methanol, ethanol, tetrahydrofuran, etc. The preferred solvent is methanol. The reaction temperature is generally 15°C to 35°C, and preferably 20°C to 30°C. When the reducing agent is hydrogen, the hydrogen pressure in the reaction system is generally 0.1 to 0.5 MPa, and preferably 0.2 to 0.4 MPa. The reaction time varies depending on the type and amount of the deprotecting reagent used, and is generally 0.5 hours to 6 hours, and preferably 2 hours to 3 hours. The obtained compound can be purified by using conventional methods such as recrystallization or silica gel chromatography.

[0096] Method for preparing compound (XIIa or 3)

[0097] [Chemical formula 14]

[0098]

[0099] Step B-1: This step includes a step (Step B-1-1) of converting each of the two hydroxyl groups of 2-(benzyloxy)propane-1,3-diol into an active diester form (compound (Va or 7)) by reacting 2-(benzyloxy)propane-1,3-diol with N,N'-disuccinimidyl carbonate in an organic solvent; and a step (Step B-1-2A) of preparing compound (VIa or 5) by condensing compound (Va or 7) with β-alanine in the presence of a base; or a step (Step B-1-2B) of preparing compound (VIa or 5) by condensing compound (Va or 7) with an alkyl ester of β-alanine or a salt thereof in the presence of a base and then performing hydrolysis.

[0100] (Step B-1-1)

[0101] [Chemical formula 15]

[0102]

[0103] This is the step of converting 2-(benzyloxy)propane-1,3-diol into compound (Va or 7). Examples of the base that can be used include triethylamine and pyridine. The preferred base is pyridine. The solvent that can be used is not particularly limited as long as it is a solvent that does not inhibit the reaction, and examples thereof include acetonitrile, tetrahydrofuran, acetone, and N,N'-dimethylacetamide. The preferred solvent is acetonitrile. The reaction temperature is generally -15°C to 55°C, and preferably -5°C to 45°C. The reaction time is generally 1 hour to 6 hours, and preferably 2 hours to 3 hours.

[0104] (Step B-1-2A)

[0105] [Chemical formula 16]

[0106]

[0107] This is the step of preparing compound (VIa or 5) by condensing β-alanine with compound (Va or 7) in the presence of a base. Examples of the base that can be used include triethylamine, pyridine, and N,N'-diisopropylethylamine. The preferred base is triethylamine. The solvent that can be used is not particularly limited as long as it is a solvent that does not inhibit the reaction, and examples thereof include aqueous acetonitrile, aqueous tetrahydrofuran, and aqueous acetone. The preferred solvent is aqueous acetonitrile. The reaction temperature is generally 15°C to 35°C, and preferably 20°C to 30°C. The reaction time varies depending on the type and amount of the base used, and is generally 0.25 hour to 6 hours, and preferably 0.5 hour to 1.5 hours. After the reaction is completed, compound (VIa or 5) can also be mixed with a base to form a salt. The preferred salt of compound (VIa or 5) is the dibenzylamine salt.

[0108] (Step B-1-2B)

[0109] [Chemical formula 17]

[0110]

[0111] This is a step for preparing compound (VIa or 7) by condensing β-alanine alkyl ester with compound (Va or 7) in the presence of a base to obtain compound (Va'), and then carrying out hydrolysis, wherein the alkyl represents a linear or branched C1-C6 alkyl such as methyl, ethyl, propyl, isopropyl or tert-butyl. Examples of β-alanine alkyl esters that can be used include β-alanine methyl ester, β-alanine ethyl ester, β-alanine propyl ester, β-alanine isopropyl ester, β-alanine tert-butyl ester and their salts. The preferred β-alanine alkyl ester is β-alanine ethyl ester or its salt. Examples of bases that can be used in the condensation reaction include triethylamine, pyridine and N,N'-diisopropylethylamine. The preferred base that can be used in the condensation reaction is triethylamine. There is no particular limitation on the base that can be used in the hydrolysis reaction as long as it is a base used in a conventional hydrolysis reaction, and examples thereof include lithium hydroxide, sodium hydroxide and potassium hydroxide. The preferred base that can be used in the hydrolysis reaction is potassium hydroxide. There is no particular limitation on the solvent that can be used as long as it is a solvent that does not inhibit the reaction, and examples thereof include acetonitrile, tetrahydrofuran and acetone. The preferred solvent is tetrahydrofuran. The reaction temperature is generally 15°C to 35°C, and preferably 20°C to 30°C. The reaction time varies depending on the type and amount of the base used, and is generally 0.25 hours to 6 hours, and preferably 0.5 hours to 1.5 hours. Compound (VIa or 5) can also be mixed with a base to form a salt. The preferred salt of compound (VIa or 5) is the dibenzylamine salt.

[0112] Step B-2: This step is a step of preparing compound (VIIa or 8) by reacting compound (Ib or 6) with compound (VIa or 5) or a salt thereof in an organic solvent using a condensing agent. When using a salt of compound (VIa or 5), before carrying out the reaction, the salt is converted into a free acid by a separation procedure or the like and then used for the reaction. The condensing agent that can be used is not particularly limited as long as it is a reagent used in the condensation reaction of a carboxylic acid and an amino group, and examples thereof include a combination of 1-hydroxybenzotriazole monohydrate and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate, and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate. The preferred condensing agent is a combination of 1-hydroxybenzotriazole monohydrate and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. The solvent that can be used is not particularly limited as long as it is a solvent that does not inhibit the reaction, and examples thereof include aqueous tetrahydrofuran, aqueous acetonitrile, and aqueous acetone. The preferred solvent is aqueous tetrahydrofuran. The reaction temperature is generally 10°C to 40°C, and preferably 20°C to 30°C. The reaction time varies depending on the type and amount of the condensing agent used, and is generally 5 hours to 60 hours, and preferably 45 hours to 47 hours. The obtained compound can be purified by using conventional methods such as recrystallization or silica gel chromatography.

[0113] Step B-3: This step is a step of preparing compound (VIIIa or 2) by reacting compound (VIIa or 8) with an acetylation reagent in an organic solvent. The acetylation reagent that can be used is not particularly limited as long as it is a reagent used in an acetylation reaction, and examples thereof include acetic anhydride and acetyl chloride. The preferred acetylation reagent is acetic anhydride, and more preferably acetic anhydride is used in combination with 4-dimethylaminopyridine and triethylamine. The solvent that can be used is not particularly limited as long as it is a solvent that does not inhibit the reaction, and examples thereof include one or more solvents selected from ethyl acetate, tetrahydrofuran, acetone, acetonitrile, etc. The preferred solvent is a mixed solvent of ethyl acetate and tetrahydrofuran. The reaction temperature is generally 10°C to 40°C, and preferably 20°C to 30°C. The reaction time varies depending on the type and amount of the acetylation reagent used, and is generally 2 hours to 12 hours, and preferably 7 hours to 9 hours. The compound (VIIIa or 2) obtained in this step can be used in the next step without separation and purification, and can also be purified by using conventional methods such as recrystallization or silica gel chromatography.

[0114] Step B-4: This step is a step of selectively deprotecting the benzyl group by reacting compound (VIIIa or 2) with a deprotecting reagent for benzyl in the presence of a reducing agent in an organic solvent to prepare compound (IXa or 1). Examples of the deprotecting reagent for benzyl include a combination of a palladium catalyst supported on a carrier and a reducing agent. The palladium catalyst that can be used is not particularly limited as long as it can deprotect the benzyl group, and preferred examples thereof include palladium on carbon catalyst and palladium hydroxide on carbon catalyst. The palladium content of the palladium catalyst and palladium hydroxide on carbon catalyst is not particularly limited, and for example, catalysts with a palladium content of 5%, 10%, 20%, etc. can be used. The palladium catalyst with a preferred content is 5% palladium catalyst or 20% palladium hydroxide catalyst. Examples of the reducing agent that can be used include hydrogen, formic acid, and ammonium formate. The preferred reducing agent is hydrogen.

[0115] The solvent that can be used in this step is not particularly limited as long as it is a solvent that does not inhibit the reaction, and examples thereof include one or more solvents selected from alcohol-based solvents, ester-based solvents, ether-based solvents, and amide-based solvents. For example, the solvent is selected from one or more solvents such as methanol, ethanol, 1-propanol, 2-propanol, ethyl acetate, propyl acetate, tetrahydrofuran, 2-methyltetrahydrofuran, tert-butyl methyl ether, cyclopentyl methyl ether, dimethylacetamide, dimethylformamide, N-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, etc. The preferred solvent is one or more solvents selected from the following: 2-propanol, dimethylacetamide, N-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone, or a mixed solvent of ethyl acetate and one or more solvents selected from dimethylacetamide, N-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone. The more preferred solvent is a mixed solvent of ethyl acetate and one or more solvents selected from dimethylacetamide, N-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone. The reaction temperature is generally 10°C to 40°C, and preferably 20°C to 30°C. The reaction time varies depending on the type and amount of the palladium catalyst and reducing agent used, and is generally 4 hours to 10 hours, and preferably 6 hours to 8 hours.

[0116] In this step, "selectively deprotecting the benzyl group" means not deprotecting the 4,4'-dimethoxytriphenylmethyl group and generating a compound in which only the benzyl group is deprotected, and means generating a compound in which only the benzyl group is deprotected as the main product. For example, this means generating a compound in which only the benzyl group is deprotected and the HPLC area ratio is 40% or more, preferably 50% or more, and more preferably 60% or more.

[0117] Step B-5: This step is a step of preparing compound (VIIa or 3) by reacting compound (IXa or 1) with an activator and a phosphoramidite reagent in an organic solvent in the presence of a desiccant. The phosphoramidite reagent that can be used is not particularly limited as long as it is a reagent for introducing a phosphorus atom-containing group that can be used to form a covalent bond between the hydroxyl group of compound (IXa or 1) and an oligonucleotide, and examples thereof include 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite and 2-cyanoethyl diisopropylchlorophosphoramidite. The preferred phosphoramidite reagent is 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite. The amount of the phosphoramidite reagent that can be used is 1.0 to 1.6 equivalents based on compound (IXa or 1), and preferably 1.2 to 1.4 equivalents. The activator that can be used is not particularly limited as long as it is a reagent that can be used to form an active intermediate in the formation of phosphoramidite, and examples thereof include 5-benzylthiotetrazole, 5-phenyltetrazole, dibromoimidazole, 4,5-dicyanoimidazole, and N-alkylimidazole trifluoroacetate (salt). The preferred activator is 4,5-dicyanoimidazole. The amount of the activator that can be used is 0.1 to 1.0 equivalent based on compound (IXa or 1), and preferably 0.4 to 0.6 equivalent. The desiccant that can be used is not particularly limited as long as it is a reagent for absorbing moisture in the reaction solution, and examples thereof include molecular sieve 3A, molecular sieve 4A, and molecular sieve 5A. The preferred desiccant is molecular sieve 4A. The solvent that can be used is not particularly limited as long as it is a solvent that does not inhibit the reaction, and examples thereof include one or more solvents selected from ethyl acetate, dichloromethane, N,N-dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofuran, methyl isobutyl ketone, etc. The preferred solvent is a mixed solvent of ethyl acetate and dichloromethane. The reaction temperature is generally -5°C to 10°C, and preferably 0°C to 5°C. The reaction time varies depending on the type and amount of the reagents used, and when the preferred activator, phosphoramidite reagent, and desiccant are used, the reaction time is generally 12 hours to 24 hours, and preferably 15 hours to 17 hours.

[0118] Method for preparing a compound represented by formula (4)

[0119] The conjugate represented by formula (4) consists of an oligonucleotide and a diantennary N-acetyl-D-galactosamine unit, wherein the diantennary N-acetyl-D-galactosamine unit is bonded to the 5'-end or 3'-end of the oligonucleotide, and includes the step of reacting the compound (XIIa or 3) prepared by the above preparation method with the oligonucleotide.

[0120] Hereinafter, each step of the above method will be described in detail.

[0121] In the present invention, the "palladium catalyst" refers to a palladium catalyst for eliminating the benzyl group bonded to a hydroxyl group, and is preferably palladium on carbon or palladium hydroxide on carbon.

[0122] In the present invention, examples of the "reducing agent" used in the benzyl elimination reaction using a palladium catalyst include hydrogen, formic acid, and ammonium formate, etc. The reducing agent is preferably hydrogen.

[0123] As the "reaction solvent" used in the present invention, any solvent that does not inhibit the reaction can be used. Examples of the solvent that can be used in the benzyl elimination reaction using a palladium catalyst include alcohol-based solvents, ester-based solvents, ether-based solvents, and amide-based solvents.

[0124] Examples of the alcohol-based solvent that can be used in the benzyl elimination step using a palladium catalyst according to the present invention include methanol, ethanol, 1-propanol, and 2-propanol. The alcohol-based solvent is preferably 2-propanol.

[0125] Examples of the ester-based solvent that can be used in the benzyl elimination step using a palladium catalyst according to the present invention include ethyl acetate and propyl acetate. The ester-based solvent is preferably ethyl acetate.

[0126] Examples of the ether-based solvent that can be used in the benzyl elimination step using a palladium catalyst according to the present invention include tetrahydrofuran, 2-methyltetrahydrofuran, tert-butyl methyl ether, and cyclopentyl methyl ether. The ether-based solvent is preferably 2-methyltetrahydrofuran.

[0127] Examples of the amide-based solvent that can be used in the benzyl elimination step using a palladium catalyst according to the present invention include dimethylacetamide, dimethylformamide, N-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone. The amide-based solvent is preferably dimethylacetamide, N-methyl-2-pyrrolidone, or 1,3-dimethyl-2-imidazolidinone.

[0128] Preferred solvents that can be used in the benzyl elimination step using a palladium catalyst according to the present invention are one or more solvents selected from methanol, 2-propanol, ethyl acetate, 2-methyltetrahydrofuran, dimethylacetamide, N-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone. More preferred solvents are one or more solvents selected from 2-propanol, dimethylacetamide, N-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone.

[0129] In the benzyl elimination step using a palladium catalyst according to the present invention, the mixed solvent of two or more solvents is preferably a mixed solvent of an amide-based solvent and ethyl acetate, and more preferably a mixed solvent of one or more solvents selected from dimethylacetamide, N-methyl-2-pyrrolidone, or 1,3-dimethyl-2-imidazolidinone and ethyl acetate. The most preferred mixed solvent is a mixed solvent of N-methyl-2-pyrrolidone and ethyl acetate.

[0130] When using a mixed solvent of N-methyl-2-pyrrolidone and ethyl acetate, it can be mixed and used at any ratio. The ratio of N-methyl-2-pyrrolidone:ethyl acetate is preferably 1:30 to 1:1, and more preferably 1:5 to 1:2.

[0131] In the present invention, the "phosphoramidite reagent" is a reagent used in the phosphoramidite formation step, and it is used to introduce a phosphorus atom-containing group for forming a covalent bond between the hydroxyl group of the diantenna N-acetyl-D-galactosamine unit and the oligonucleotide. Examples of the phosphoramidite reagent include 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite and 2-cyanoethyl diisopropylchlorophosphoramidite. The phosphoramidite reagent is preferably 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite.

[0132] The phosphoramidite formation step in the present invention is a step of preparing a compound represented by the general formula (3) by reacting a compound represented by the formula (1) with an activator and a phosphoramidite reagent in an organic solvent in the presence of a desiccant.

[0133] Solvents that can be used in the phosphoramidite formation step are, for example, one or more solvents selected from ethyl acetate, dichloromethane, N,N-dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofuran, and methyl isobutyl ketone, and preferably a mixed solvent of ethyl acetate and dichloromethane.

[0134] The activator that can be used in the phosphoramidite formation step is a reagent for forming an active intermediate of the phosphoramidite reagent, and examples thereof include 5-benzylthiotetrazole, 5-phenyltetrazole, dibromoimidazole, 4,5-dicyanoimidazole, and N-alkylimidazole trifluoroacetate (salt). The activator is preferably 4,5-dicyanoimidazole.

[0135] The desiccants that can be used in the phosphoramidite formation step are reagents for absorbing moisture in the reaction solution, and examples thereof include molecular sieve 3A, molecular sieve 4A, and molecular sieve 5A. The desiccant is preferably molecular sieve 4A.

[0136] By bonding the phosphoramidite form of the diantennary N-acetyl-D-galactosamine unit represented by formula (3) prepared by the preparation method of the present invention to an oligonucleotide having a desired nucleotide sequence using a conventional method (such as the phosphoramidite method), a conjugate form composed of the diantennary N-acetyl-D-galactosamine unit represented by formula (4) and an oligonucleotide can be prepared. As used herein, the terms "conjugate" and "conjugate form" each refer to a compound in which the N-acetyl-D-galactosamine unit (GalNAc unit) is bonded to the 5'-end and / or 3'-end of the oligonucleotide. According to the method disclosed in Nucleic Acids Research, 12, 4539 (1984), etc., its preparation can be carried out using a DNA synthesizer (such as PerkinElmer's Model 392) using the phosphoramidite method. More specifically, the diantennary N-acetyl-D-galactosamine ligand-oligonucleotide conjugate form can be prepared by the preparation method disclosed in Patent Document 2 (International Publication No. WO 2021 / 049504).

[0137] The method for preparing the oligonucleotide used in the present invention is not particularly limited as long as it can synthesize the desired oligonucleotide, and known chemical synthesis methods (such as the phosphotriester method, the phosphoramidite method, or the H-phosphonate method) can be used. For example, a commercially available nucleic acid synthesizer can be used and commercially available reagents for DNA / RNA synthesis can be used to synthesize the oligonucleotide.

[0138] An oligonucleotide having a desired nucleotide sequence can be synthesized by a conventional phosphoramidite method using a DNA synthesizer (such as PerkinElmer's Model 392) using the phosphoramidite method according to the method disclosed in Nucleic Acids Research, 12, 4539 (1984), etc.

[0139] As phosphoramidite reagents corresponding to various nucleosides, commercially available reagents can be purchased and used, or such phosphoramidite reagents can also be synthesized according to known methods.

[0140] In the present invention, examples of the "acetylation reagent" include acetic anhydride and acetyl chloride. The acetylation reagent is preferably acetic anhydride.

[0141] As used herein, "condensing agent" refers to a reagent used in the condensation reaction of a carboxylic acid and an amino group, and examples thereof include the combination of 1-hydroxybenzotriazole monohydrate and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride n-hydrate, and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate. The condensing agent is preferably the combination of 1-hydroxybenzotriazole monohydrate and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0142] In the step of condensing with the compound represented by formula (7) according to the present invention, β-alanine, an alkyl ester of β-alanine, or a salt thereof can be used. Examples of the alkyl ester of β-alanine include methyl β-alaninate, ethyl β-alaninate, propyl β-alaninate, isopropyl β-alaninate, tert-butyl β-alaninate, and salts thereof. In the condensation step, β-alanine or a salt thereof, or ethyl β-alaninate or a salt thereof is preferred.

[0143] Examples of the base that can be used in the condensation step with the compound represented by formula (7) according to the present invention include triethylamine, pyridine, and N,N'-diisopropylethylamine. The base is preferably triethylamine.

[0144] The compound represented by formula (1) according to the present invention, the compound represented by formula (2) according to the present invention, the compound represented by formula (3) according to the present invention, the compound represented by formula (4) according to the present invention, the compound represented by formula (6) according to the present invention or a salt thereof, and the compound represented by formula (8) according to the present invention include all of their isomers (diastereoisomers, optical isomers, and geometric isomers).

[0145] The compound represented by formula (3) according to the present invention, the compound represented by formula (4) according to the present invention, the compound represented by formula (5) according to the present invention, and the compound represented by formula (6) according to the present invention, if they have an acidic group, can each be converted into a salt by reacting with a base, and if they have a basic group, can be converted into a salt by reacting with an acid.

[0146] Examples of salts based on basic groups include: hydrohalides (such as hydrofluorides, hydrochlorides, hydrobromides, or hydroiodides); inorganic acid salts (such as nitrates, perchlorates, sulfates, or phosphates); alkylsulfonates (such as methanesulfonates, trifluoromethanesulfonates, and ethanesulfonates); arylsulfonates (such as benzenesulfonates or p-toluenesulfonates); organic acid salts (such as acetates, malates, fumarates, succinates, citrates, ascorbates, tartrates, oxalates, or adipates); and amino acid salts (such as glycine salts, lysine salts, arginine salts, ornithine salts, glutamate salts, or aspartate salts).

[0147] Examples of salts based on acidic groups include: alkali metal salts (such as sodium salt, potassium salt or lithium salt); alkaline earth metal salts (such as calcium salt or magnesium salt); metal salts (such as aluminum salt or iron salt); inorganic salts (such as ammonium salt); amine salts such as organic salts (such as tert-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzylphenethylamine salt, piperazine salt, tetramethylammonium salt or tris(hydroxymethyl)aminomethane salt); and amino acid salts (such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate salt or aspartate salt).

[0148] A preferred salt of the compound represented by formula (8) according to the present invention is a dibenzylamine salt.

[0149] The compounds or salts thereof of the present invention may combine with water molecules to form hydrates when placed in air or recrystallized, and such hydrates are also included in the compounds or salts thereof of the present invention.

[0150] The compounds or salts thereof of the present invention may absorb a solvent to form solvates when placed in a certain solvent or recrystallized, and such solvates are also included in the compounds or salts thereof of the present invention.

[0151] As used herein, "GalNAc unit" refers to a partial structure containing N-acetyl-D-galactosamine (GalNAc), which can bind to the asialoglycoprotein receptor (ASGPR) on hepatocytes in the liver. The GalNAc unit may include a phosphate group or a phosphorothioate group for bonding between a linear or branched linker structure and an oligonucleotide. The number of GalNAc contained in one GalNAc unit is not limited, and unless otherwise specified, known numbers of GalNAc, numbers of GalNAc disclosed herein, etc. can be adopted. The structure of GalNAc can be modified as long as the ability to bind to ASGPR is maintained. In addition, GalNAc with a protecting group introduced during the preparation method is also included.

[0152] In addition, as used herein, "oligonucleotide" refers to a nucleic acid that can be synthesized and has a chain length of 100 bases or less, and can be single-stranded or double-stranded. As the nucleic acid to be employed, DNA, RNA, natural nucleic acids, modified nucleic acids, etc. can be selectively used according to the purpose, as described later, and they can be mixed in one oligonucleotide. As the GalNAc unit and oligonucleotide, for example, those disclosed in Patent Document 2 (International Publication No. WO 2021 / 049504) can be used. Examples

[0153] Next, the present invention will be described. It should be understood that the reaction conditions of the present invention are not limited to those described herein. In the present invention, the functional groups in the compound can be protected with appropriate protecting groups. Examples of such functional groups can include hydroxyl groups, carboxyl groups, and amino groups, and the types of protecting groups and the conditions for introducing and removing the protecting groups can be determined with reference to those disclosed in, for example, Protective Groups in Organic Synthesis (T.W. Green and P.G.M. Wuts, John Wiley & Sons, Inc., New York, 2006).

[0154] The abbreviations used in the examples have the following meanings.

[0155] g: gram, mL: milliliter, mol: mole, MHz: megahertz, Ac: acetyl, DMTr: 4,4'-dimethoxytriphenylmethyl, Bn: benzyl, Cbz: benzyloxycarbonyl, MeOH: methanol, IPA: 2-propanol, EtOAc: ethyl acetate, 2-MeTHF: 2-methyltetrahydrofuran, DMAc: N,N-dimethylacetamide, NMP: N-methyl-2-pyrrolidone, DMI: 1,3-dimethyl-2-imidazolidinone, DMSO: dimethyl sulfoxide.

[0156] In the following examples, nuclear magnetic resonance (hereinafter, 1 1H NMR: 500 MHz) spectra were measured using chloroform-d, methanol-d4, dimethyl sulfoxide-d6, and acetonitrile-d3 as deuterated solvents, and the chemical shift values measured using tetramethylsilane as a standard substance were represented by δ values (ppm). The splitting patterns were designated as: s for singlet, d for doublet, dd for double doublet, t for triplet, q for quartet, m for multiplet, and br for broad peak.

[0157] (Example 1)

[0158] Preparation of 3-aminopropyl 2-acetamido-6-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β-D-galactopyranoside (Ib or 6)

[0159] (Example 1-1)

[0160] Preparation of Benzyl {3-[(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl)oxy]propyl} Carbamate (IIa)

[0161] [Chemical Formula 18]

[0162]

[0163] 2-Acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-D-galactopyranose (60.0 g, 154.0 mmol) was added to dichloromethane (900 mL), and the resulting mixture was concentrated under reduced pressure to obtain a dichloromethane solution (600 mL). Similarly, dichloromethane (300 mL) was added to the obtained dichloromethane solution, and the resulting mixture was concentrated under reduced pressure to obtain a dichloromethane solution (600 mL). The water content value of the concentrate of the obtained dichloromethane solution was confirmed to be 0.02% or less by using a Karl Fischer moisture meter (coulometric method). Dichloromethane (300 mL) was added, benzyl (3-hydroxypropyl) carbamate (33.9 g, 162.0 mmol) and trifluoromethanesulfonic acid (4.6 g, 30.8 mmol) were added in this order, and the resulting mixture was stirred at 35 °C for 22 hours. After confirming the termination of the reaction, the reaction solution was cooled to 25 °C and separated with 8% aqueous sodium bicarbonate (180 mL). The obtained organic layer was concentrated to 300 mL under reduced pressure. Ethanol (900 mL) was added to the obtained concentrate, and then the resulting mixture was concentrated under reduced pressure to obtain a 900 mL solution. Subsequently, the same procedure of concentration under reduced pressure was repeated again, and ethanol (300 mL) was added to the obtained solution (900 mL) to obtain a solution (1200 mL) of the title compound (IIa) in ethanol.

[0164] In the same manner as in (Example 1-1), the title compound (IIa) (28.99 g, yield: 83.8%) was synthesized by using 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-D-galactopyranose (25.0 g, 64.21 mmol).

[0165] 1 H-NMR (500 MHz, CDCl 3): δ 7.40 - 7.31 (5H, m), 6.19 (1H, d, J = 8.5 Hz), 5.32 (1H, d, J = 2.0 Hz), 5.12 and 5.08 (2H, d, J = 12.0 Hz), 5.01 - 4.96 (2H, m), 4.33 (1H, d, J = 8.5 Hz), 4.18 - 4.09 (3H, m), 3.97 (1H, ddd, J = 10.0, 4.0, 4.0 Hz), 3.77 (1H, dd, J = 7.0, 7.0 Hz), 3.59 - 3.52 (1H, m), 3.40 (1H, ddd, J = 9.5, 9.5, 3.0 Hz), 3.12 - 3.07 (1H, m), 2.15 (3H, s), 2.05 (3H, s), 2.01 (3H, s), 1.94 (3H, s), 1.85 - 1.78 (1H, m), 1.66 - 1.58 (1H, m)

[0166] (Example 1 - 2)

[0167] Preparation of Benzyl {3 - [(2 - Acetamido - 2 - deoxy - β - D - galactopyranosyl)oxy]propyl} Carbamate (IIIa)

[0168] [Chemical Formula 19]

[0169]

[0170] A solution of benzyl {3 - [(2 - acetamido - 3,4,6 - tri - O - acetyl - 2 - deoxy - β - D - galactopyranosyl)oxy]propyl} carbamate (IIa) in ethanol (1200 mL) was heated to 40 °C, and 28% sodium methoxide in methanol solution (5.9 g, 30.8 mmol) was added, and then the resulting mixture was stirred for 13 hours. After confirming the termination of the reaction, the reaction solution was cooled to 0 °C, and the precipitated solid was filtered, washed with ethanol (180 mL) at 0 °C, and dried under reduced pressure at an external temperature of 40 °C to obtain the title compound (IIIa) (56.4 g, area ratio analyzed by liquid chromatography: 99.8%, total two - step yield: 88.7%).

[0171] 1H-NMR(500MHz, DMSO-d6): δ 7.60 (1H, d, J = 9.5 Hz), 7.38 - 7.29 (5H, m), 7.19 (1H, dd, J = 6.0, 6.0 Hz), 5.01 (2H, s), 4.60 - 4.56 (2H, m), 4.49 (1H, d, J = 4.0 Hz), 4.21 (1H, d, J = 8.0 Hz), 3.74 - 3.67 (2H, m), 3.64 (1H, dd, J = 3.5, 3.5 Hz), 3.56 - 3.47 (2H, m), 3.42 - 3.32 (2H, m), 3.30 (1H, dd, J = 5.5, 5.5 Hz), 3.06 - 2.99 (2H, m), 1.81 (3H, s), 1.60 (2H, tt, J = 6.5, 6.5 Hz)

[0172] (Examples 1 - 3)

[0173] Preparation of Benzyl [3 - ({2 - Acetamido - 6 - O - [bis(4 - methoxyphenyl)(phenyl)methyl] - 2 - deoxy - β - D - galactopyranosyl}oxy)propyl] Carbamate (IVa)

[0174] [Chemical Formula 20]

[0175]

[0176] Benzyl {3 - [(2 - Acetamido - 2 - deoxy - β - D - galactopyranosyl)oxy]propyl} carbamate (IIIa) (54.0 g, 131.0 mmol) and pyridine (31.1 g, 393.0 mmol) were added to N,N - dimethylacetamide (324 mL) in this order, and the resulting mixture was stirred. Subsequently, 4,4'-dimethoxytrityl chloride (26.6 g, 78.6 mmol) was added, and then the mixture was rinsed twice with toluene (54 mL), followed by stirring for 5 hours. After confirming the termination of the reaction, toluene (702 mL) was added, and the resulting mixture was separated with 4% aqueous sodium bicarbonate (540 mL) to obtain the toluene layer. The obtained toluene layer was separated with 8% aqueous sodium bicarbonate (270 mL) to obtain the organic layer. Subsequently, the separation operation was repeated twice with water (540 mL), and the resulting organic layer was concentrated under reduced pressure to obtain a solution of the title compound (IVa) in toluene (270 mL).

[0177] In the same manner as in (Examples 1 - 3), by using benzyl {3 - [(2 - acetamido - 2 - deoxy - β - D - galactopyranosyl)oxy]propyl} carbamate (IIIa) (5.0 g, 12.1 mmol), the title compound (IVa) (7.29 g, yield: 84.1%) was synthesized.

[0178] 1 H-NMR (500 MHz, CD 3 CN): δ 7.48 - 7.46 (2H, m), 7.39 - 7.29 (11H, m), 7.24 - 7.21 (1H, m), 7.11 (1H, d, J = 7.0 Hz), 6.88 - 6.85 (4H, m), 5.75 (1H, dd, J = 7.0, 4.5 Hz), 5.08 (1H, d, J = 12.5 Hz), 5.02 (1H, d, J = 12.5 Hz), 4.74 (1H, br s), 4.12 (1H, d, J = 8.5 Hz), 3.91 (1H, ddd, J = 9.0, 4.5, 4.5 Hz), 3.76 (6H, s), 3.68 (1H, d, J = 2.5 Hz), 3.63 (1H, ddd, J = 9.5, 8.5, 7.0 Hz), 3.55 (1H, dd, J = 7.0, 4.5 Hz), 3.46 - 3.37 (1H, m), 3.44 (1H, dd, J = 9.5, 2.5 Hz), 3.40 (1H, ddd, J = 9.0, 4.0, 4.0 Hz), 3.27 (1H, dd, J = 9.0, 7.0 Hz), 3.11 (1H, dd, J = 9.0, 4.5 Hz), 3.09 - 3.02 (1H, m), 3.02 (1H, br s), 1.94 (3H, s), 1.79 - 1.71 (1H, m), 1.69 - 1.61 (1H, m)

[0179] (Example 1 - 4)

[0180] Preparation of 3 - aminopropyl - 2 - acetamido - 6 - O - [bis(4 - methoxyphenyl)(phenyl)methyl] - 2 - deoxy - β - D - galactopyranoside (Ib or 6)

[0181] [Chemical Formula 21]

[0182]

[0183] Methanol (270 mL), 5% palladium on carbon powder type PE moistened with water (5.4 g based on dry weight, manufactured by N.E. CHEMCAT CORPORATION), and 25% aqueous ammonia solution (39.8 g, 655.0 mmol) were added to a solution of benzyl [3-({2-acetamido-6-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β-D-galactopyranosyl}oxy)propyl]carbamate (IVa) in toluene (270 mL), and the resulting mixture was stirred for 1 hour at 20 to 30 °C using an autoclave. Subsequently, the inside of the reaction system was purged with nitrogen three times, and then purged with hydrogen at 0.4 MPa (gauge pressure) three times, and the mixture was stirred at 20 to 30 °C under a hydrogen atmosphere of 0.3 MPa for 4 hours. After confirming the termination of the reaction, the reaction system was purged with nitrogen three times, and the palladium on carbon was filtered off and washed with methanol (270 mL). The resulting filtrate was concentrated under reduced pressure, water (27 mL) and toluene (540 mL) were added, and the resulting mixture was concentrated under reduced pressure to obtain a 540 mL concentrate. The procedure of adding toluene (540 mL) to the obtained concentrate and concentrating the resulting mixture under reduced pressure was repeated three times to obtain a toluene solution (540 mL). Subsequently, the precipitated solid was filtered, washed with toluene (162 mL), and dried under reduced pressure at an external temperature of 40 °C to obtain the title compound (Ib or 6) (68.5 g, area ratio analyzed by liquid chromatography: 99.1%, total two-step yield: 90.1%).

[0184] 1 H-NMR (500 MHz, CD 3 OD): δ 7.45 (2H, d, J = 7.5 Hz), 7.35 - 7.31 (4H, m), 7.27 (2H, dd, J = 7.5, 7.5 Hz), 7.19 (1H, t, J = 7.5 Hz), 6.85 (4H, d, J = 8.0 Hz), 4.32 (1H, d, J = 8.5 Hz), 3.98 (1H, ddd, J = 11.5, 6.0, 5.0 Hz), 3.91 (1H, dd, J = 10.5, 8.5 Hz), 3.84 (1H, d, J = 2.5 Hz), 3.77 (6H, s), 3.62 (1H, ddd, J = 6.0, 5.0, 11.5 Hz), 3.57 - 3.55 (1H, m), 3.55 (1H, dd, J = 10.5, 2.5 Hz), 3.40 (1H, dd, J = 9.5, 7.0 Hz), 3.31 - 3.28 (1H, m), 2.82 (2H, t, J = 6.0 Hz), 1.98 (3H, s), 1.79 (2H, m)

[0185] (Example 2)

[0186] (Preparation of ([12 - ({(2 - cyanoethoxy)[bis(propyl - 2 - yl)amino]phosphoryl}oxy)-5,9,15,19 - tetraoxo - 10,14 - dioxo - 4,8,16,20 - tetraazatricosane - 1,23 - diyl]bis(oxy)(2R,3R,4R,5R,6R)-3 - acetamido - 6 - {[bis(4 - methoxyphenyl)(phenyl)methoxy]methyl}oxane - 2,4,5 - triyl)tetraacetic acid ester (XIIa or 3))

[0187] (Example 2 - 1)

[0188] (Preparation of 1,1'-{[2-(benzyloxy)propane - 1,3 - diyl]bis(oxycarbonyl - oxy)}bis(pyrrolidine - 2,5 - dione) (Va or 7))

[0189] [Chemical formula 22]

[0190]

[0191] 2-(Benzyloxy)propane - 1,3 - diol (10.0 g, 54.9 mmol) was added to acetonitrile (50 mL), and the temperature of the resulting solution was adjusted to 0 °C. Subsequently, N,N'-disuccinimidyl carbonate (32.3 g, 126.2 mmol) and pyridine (10.9 g, 137.2 mmol) were added, and the resulting mixture was stirred at 0 °C for 5 hours. After confirming the termination of the reaction, 2 - propanol (150 mL) was added to the reaction solution, and the resulting mixture was seeded and stirred at 0 °C for 1 hour. Subsequently, 2 - propanol (100 mL) was added, and the resulting mixture was stirred at 0 °C for 16 hours. The precipitated crystals were filtered, washed with 2 - propanol (50 mL), and then washed with tert - butyl methyl ether (50 mL) to obtain wet crystals of the title compound (Va or 7). The obtained wet crystals were used for the next step without a drying procedure.

[0192] 1 H - NMR (500 MHz, CD 3 CN): δ 7.39 - 7.30 (5H, m), 4.65 (2H, s), 4.54 (2H, dd, J = 11.5, 4.0 Hz), 4.43 (2H, dd, J = 11.5, 5.5 Hz), 4.06 (1H, tt, J = 5.5, 4.0 Hz), 2.77 (8H, s)

[0193] (Example 2 - 1 - 1)

[0194] (Preparation of 1,1'-{[2-(benzyloxy)propane - 1,3 - diyl]bis(oxycarbonyl - oxy)}bis(pyrrolidine - 2,5 - dione) (Va or 7))

[0195] [Chemical Formula 23]

[0196]

[0197] 2-(Benzyloxy)propane-1,3-diol (1.0 g, 5.49 mmol) was added to acetonitrile (5 mL), N,N'-disuccinimidyl carbonate (3.2 g, 12.6 mmol) and pyridine (1.1 g, 13.7 mmol) were added, and the resulting mixture was stirred at 0 °C for 4 hours. After confirming the termination of the reaction, the mixture was allowed to stand at 0 °C for 11 days to confirm the precipitation of crystals. 2-Propanol (16 mL) was added, and the resulting mixture was stirred at 0 °C for 1 hour. Subsequently, 2-propanol (16 mL) was added, and the resulting mixture was stirred at 0 °C for 1 hour. The precipitated crystals were filtered, washed with 2-propanol (8 mL), and then dried at an external temperature of 25 °C to obtain the title compound (Va or 7) (1.40 g, area ratio analyzed by liquid chromatography: 99.7%, yield: 54.9%, also used as a seed crystal).

[0198] 1 H-NMR (500 MHz, CD 3 CN): δ 7.39 - 7.30 (5H, m), 4.65 (2H, s), 4.54 (2H, dd, J = 11.5, 4.0 Hz), 4.43 (2H, dd, J = 11.5, 5.5 Hz), 4.06 (1H, tt, J = 5.5, 4.0 Hz), 2.77 (8H, s)

[0199] (Example 2-2-1)

[0200] Preparation of 8-(benzyloxy)-5,11-dioxo-6,10-dioxa-4,12-diazapentadecane-1,15-dioic acid-N-benzyl-1-phenylmethanamine (1 / 1) (VIa or 5)

[0201] [Chemical Formula 24]

[0202]

[0203] β-Alanine (14.7 g, 164.6 mmol), acetonitrile (50 mL), and triethylamine (17.2 g, 170.1 mmol) were added to water (50 mL) in this order, and the resulting mixture was stirred at 20 to 30 °C. Subsequently, 1,1'-{[2-(benzyloxy)propane-1,3-diyl]bis(oxycarbonyl-oxy)}bis(pyrrolidine-2,5-dione) (Va or 7) in the wet form was added over 1.5 hours while controlling the internal temperature to 30 °C or lower, and the resulting mixture was stirred at 20 to 30 °C for 1 hour. After confirming the termination of the reaction, tetrahydrofuran (300 mL) and sodium chloride (50 g) were added, and the pH was adjusted to 0.1 using 35% hydrochloric acid (28.6 g, 274.4 mmol), followed by separation to obtain the organic layer. 20% Brine (100 mL) was added to the obtained organic layer, followed by separation. The same separation procedure was carried out three more times to obtain the organic layer. The obtained organic layer was concentrated under reduced pressure to obtain a tetrahydrofuran solution (60 mL). Isopropyl acetate (200 mL) was added to the obtained tetrahydrofuran solution, and the resulting mixture was concentrated under reduced pressure to obtain a 60 mL solution. The same procedure of concentration under reduced pressure was repeated twice more to obtain an isopropyl acetate solution (60 mL). The salt in the obtained isopropyl acetate solution was removed by filtration, and the filtrate was washed with 2-propanol (50 mL). 2-Propanol (300 mL) was added to the obtained solution, and the temperature of the resulting solution was adjusted to 35 °C. Thereafter, dibenzylamine (11.4 g, 57.6 mmol) was added, and the resulting mixture was seeded and stirred at 35 °C for 1 hour. Subsequently, the mixture was stirred at 25 °C for 22 hours, and then the obtained crystals were filtered and washed with 2-propanol (50 mL) to obtain crude crystals (yield: 32.1 g) of the title compound (VIa) in the wet form.

[0204] Next, the obtained crude crystals were added to 2-propanol (300 mL), and the temperature of the resulting mixture was adjusted to 60 °C to obtain a 2-propanol solution. The obtained solution was stirred at 60 °C for 1 hour, and then slowly cooled from 60 °C to 25 °C over 1 hour and seeded to confirm crystal precipitation. Subsequently, the slurry was stirred at 25 °C for 18 hours, and then the obtained crystals were filtered, washed with 2-propanol (50 mL), and dried under reduced pressure at an external temperature of 40 °C to obtain the title compound (VIa or 5) (28.2 g, area ratio analyzed by liquid chromatography: 99.7%, total two-step yield: 83.3%).

[0205] 1 H-NMR (500 MHz, CD 3OD): δ 7.48 - 7.25 (15H, m), 4.64 (2H, s), 4.18 (2H, dd, J = 12.0, 4.5 Hz), 4.17 (4H, s), 4.10 (2H, dd, J = 12.0, 5.5 Hz), 3.86 - 3.78 (1H, m), 3.34 (4H, t, J = 7.0 Hz), 2.42 (4H, t, J = 7.0 Hz)

[0206] (Example 2 - 2 - 1 - 1) Preparation of Diethyl 8-(benzyloxy)-5,11-dioxo-6,10-dioxa-4,12-diazapentadecane-1,15-dicarboxylate (Va')

[0207] [Chemical Formula 25]

[0208]

[0209] 1,1'-{[2-(Benzyloxy)propane-1,3-diyl]bis(oxycarbonyl oxy)}bis(pyrrolidine-2,5-dione) (Va or 7) (20.0 g, 43.0 mmol), ethyl β-alaninate hydrochloride (16.5 g, 107.4 mmol) and triethylamine (12.2 g, 120.6 mmol) were added to tetrahydrofuran (300 mL) in this order, and the resulting mixture was stirred at 20 to 30 °C for 1 hour. After confirming the termination of the reaction, 7% aqueous sodium bicarbonate (100 mL) was added to the reaction solution, and then separated to obtain the organic layer. 20% brine (100 mL) was added to the obtained organic layer, and then separated to obtain a solution of the title compound (Va') in tetrahydrofuran.

[0210] In the same manner as in (Example 2 - 2 - 1 - 1), by using 1,1'-{[2-(benzyloxy)propane-1,3-diyl]bis(oxycarbonyl oxy)}bis(pyrrolidine-2,5-dione) (Va) (2.0 g, 4.3 mmol), the title compound (Va') (1.58 g, yield: 78.4%) was obtained.

[0211] 1 H-NMR (500 MHz, CDCL 3 ): δ 7.35 - 7.34 (4H, d, J = 4.5 Hz), 7.31 - 7.27 (1H, m), 5.23 (2H, m), 4.64 (2H, s), 4.27 - 4.13 (8H, m), 3.82 - 3.77 (1H, m), 3.44 (4H, dd, J = 12.5, 5.5 Hz), 2.53 (4H, t, J = 6.0 Hz), 1.27 (6H, t, J = 6.5 Hz)

[0212] (Example 2-2-1-2)

[0213] Preparation of 8-(benzyloxy)-5,11-dioxo-6,10-dioxa-4,12-diazapentadecane-1,15-dioic acid-N-benzyl-1-benzylamine (1 / 1) (VIa or 5)

[0214] [Chemical formula 26]

[0215]

[0216] Water (100 mL) and 8N potassium hydroxide (21.6 mL, 172.0 mmol) were added in this order to a solution of diethyl 8-(benzyloxy)-5,11-dioxo-6,10-dioxa-4,12-diazapentadecane-1,15-dicarboxylate (Va') in tetrahydrofuran, and the resulting mixture was stirred at 20 to 30 °C for 3.5 hours. After confirming the termination of the reaction, sodium chloride (10 g) was added to the reaction solution, and the pH was adjusted to 0.9 by using 35% hydrochloric acid (13.2 mL), followed by separation to obtain the organic layer. 20% brine (100 mL) was added to the obtained organic layer, followed by separation. The obtained organic layer was concentrated under reduced pressure to obtain a tetrahydrofuran solution (100 mL). Isopropyl acetate (300 mL) and 20% brine (20 mL) were added in this order to the obtained tetrahydrofuran solution, followed by separation. The obtained organic layer was concentrated under reduced pressure to obtain an isopropyl acetate solution (100 mL). Acetonitrile (300 mL) was added to the obtained solution, and the temperature of the obtained solution was adjusted to 40 °C. Thereafter, dibenzylamine (2.55 g, 12.9 mmol) was added, and the resulting mixture was seeded and stirred at 40 °C for 4 hours. Subsequently, dibenzylamine (2.55 g, 12.9 mmol) was added, and the resulting mixture was stirred at 40 °C for 19 hours. Subsequently, dibenzylamine (3.83 g, 19.4 mmol) was added, the resulting mixture was stirred at 40 °C for 1 hour, and then the temperature of the solution was adjusted to 25 °C. Subsequently, the solution was stirred at 25 °C for 2 hours, and then the obtained crystals were filtered and washed with 2-propanol (50 mL) to obtain crude crystals of the title compound (VIa or 5) in a wet form.

[0217] Next, the crude crystals of the title compound (VIa or 5) obtained in the wet form were added to acetonitrile (300 mL), and the resulting mixture was stirred at 70 °C for 1 hour and then slowly cooled from 70 °C to 25 °C over 2 hours to confirm the precipitation of crystals. Subsequently, the slurry was stirred at 25 °C for 16 hours, and then the resulting crystals were filtered, washed with acetonitrile (100 mL), and dried under reduced pressure at an external temperature of 40 °C to obtain the title compound (VIa or 5) (23.1 g, area ratio by liquid chromatography analysis: 99.98%, total two-step yield: 88.0%).

[0218] 1 H-NMR(500MHz,CD 3 OD):δ7.48-7.25(15H,m),4.64(2H,s),4.18(2H,dd,J=12.0,4.5Hz),4.17(4H,s),4.10(2H,dd,J=12.0,5.5Hz),3.86-3.78(1H,m),3.34(4H,t,J=7.0Hz),2.42(4H,t,J=7.0Hz)

[0219] (Example 2-2-3)

[0220] Preparation of Seed Crystal of 8-(Benzyloxy)-5,11-dioxo-6,10-dioxa-4,12-diazapentadecane-1,15-dioic Acid-N-benzyl-1-benzylamine (1 / 1) (VIa or 5)

[0221] [Chemical Formula 27]

[0222]

[0223] β-Alanine (19.5 g, 218.5 mmol), acetonitrile (95 mL), and triethylamine (22.9 g, 225.8 mmol) were added to water (95 mL) in this order, and the resulting mixture was stirred at 20 to 30 °C. Subsequently, 1,1'-{[2-(benzyloxy)propane-1,3-diyl]bis(oxycarbonyl-oxy)}bis(pyrrolidine-2,5-dione) (Va or 7) (38.0 g, 72.8 mmol) was added over 1 hour while controlling the internal temperature to 30 °C or lower, and the resulting mixture was stirred at 20 to 30 °C for 31 hours. After confirming the termination of the reaction, tetrahydrofuran (570 mL) and sodium chloride (20 g) were added, and the pH was adjusted to 0.1 by using 35% hydrochloric acid (37.9 g, 364.1 mmol), followed by separation to obtain the organic layer. 20% brine (125 mL) was added to the obtained organic layer, followed by separation. The same separation procedure was carried out three more times. The obtained organic layer was concentrated under reduced pressure to obtain a tetrahydrofuran solution (190 mL). Cyclopentyl methyl ether (570 mL) was added to the obtained tetrahydrofuran solution, followed by separation to obtain the organic layer. The obtained organic layer was concentrated under reduced pressure to obtain a 114 mL solution. Isopropyl acetate (570 mL) was added to the obtained organic layer, and the resulting mixture was concentrated under reduced pressure to obtain a 114 mL solution. Isopropyl acetate (76 mL) was added to the obtained solution, followed by the addition of acetonitrile (570 mL), and the temperature of the resulting solution was adjusted to 40 °C. Thereafter, dibenzylamine (8.6 g, 43.7 mmol) was added, and the resulting mixture was stirred at 40 °C for 19 hours. Subsequently, dibenzylamine (6.5 g, 32.8 mmol) was added, and the resulting mixture was stirred at 40 °C for 2 hours. Subsequently, the mixture was stirred at 25 °C for 4 hours, and then the obtained crystals were filtered and washed with acetonitrile (190 mL) to obtain the seed crystals of the title compound (VIa or 5) (41.02 g, area ratio analyzed by liquid chromatography: 95.8%, yield: 86.5%).

[0224] 1 H-NMR(500MHz,CD 3 OD):δ7.48-7.25(15H,m),4.64(2H,s),4.18(2H,dd,J=12.0,4.5Hz),4.17(4H,s),4.10(2H,dd,J=12.0,5.5Hz),3.86-3.78(1H,m),3.34(4H,t,J=7.0Hz),2.42(4H,t,J=7.0Hz)

[0225] (Example 2-3)

[0226] Preparation of 2-(benzyloxy)propane-1,3-diyl bis({3-[(3-{[(2R,5R)-3-acetamido-6-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4,5-dihydroxyoxan-2-yl]oxy}propyl)amino]-3-oxopropyl}carbamate) (VIIa or 8)

[0227] [Chemical formula 28]

[0228]

[0229] Water (200 mL), 8-(benzyloxy)-5,11-dioxo-6,10-dioxa-4,12-diazapentadecane-1,15-dioic acid-N-benzyl-1-benzylamine (1 / 1) (VIa or 5) (20.8 g, 32.8 mmol, content: 96.2%), and triethylamine (9.96 g, 98.4 mmol) were added to dichloromethane (100 mL), and the resulting mixture was stirred at 20 to 30 °C for 1 hour. Thereafter, the mixture was separated, and the resulting aqueous layer was washed twice with dichloromethane (60 mL). In this order, tetrahydrofuran (600 mL), 1-hydroxybenzotriazole monohydrate (10.6 g, 68.9 mmol), 3-aminopropyl-2-acetamido-6-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-1-β-D-galactopyranoside (Ib) (37.9 g, 62.3 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15.7 g, 82.0 mmol) were added to the resulting aqueous layer, and the resulting mixture was stirred at 20 to 30 °C for 5 hours. Thereafter, 3-aminopropyl-2-acetamido-6-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β-D-galactopyranoside (Ib) (2.53, 4.16 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.67 g, 8.71 mmol) were added, and the resulting mixture was stirred for 11 hours; then, 3-aminopropyl-2-acetamido-6-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β-D-galactopyranoside (Ib) (178.0 mg, 293 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (117 mg, 610 μmol) were added, and the resulting mixture was stirred for 7 hours; and furthermore, 3-aminopropyl-2-acetamido-6-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β-D-galactopyranoside (Ib) (51.0 mg, 84 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (34.0 mg, 177 μmol) were added, and the resulting mixture was stirred for 23 hours. After confirming the termination of the reaction, the mixture was washed with 20% brine (200 mL), ethyl acetate (600 mL) was added, and the separation operation was repeated twice with 8% aqueous sodium bicarbonate (400 mL). The resulting organic layer was concentrated under reduced pressure to obtain a 300 mL solution. Subsequently, the procedure of adding tetrahydrofuran (300 mL) and ethyl acetate (300 mL) and concentrating the resulting mixture under reduced pressure to obtain a 300 mL solution was repeated twice. Tetrahydrofuran (1000 mL) was added to the resulting solution, and the resulting mixture was concentrated under reduced pressure to obtain a 200 mL solution.To the resulting solution, tetrahydrofuran (1000 mL) was added, and the resulting mixture was concentrated under reduced pressure to obtain a solution of 244 mL. Tetrahydrofuran (56 mL) was added to the obtained solution to obtain a solution of the title compound (VIIa or 8) in ethyl acetate / tetrahydrofuran (300 mL).

[0230] In the same manner as in (Example 2-3), the title compound (VIIa or 8) was synthesized by using 8-(benzyloxy)-5,11-dioxo-6,10-dioxo-4,12-diazapentadecane-1,15-dioic acid-N-benzyl-1-phenylmethanamine (1 / 1) (VIa or 5) (211.2 mg, 0.4 mmol), and separated by purification through a silica gel column (186.4 mg, yield: 96.7%).

[0231] 1 H-NMR (500 MHz, CD 3 OD): δ 7.78 (2H, t, J = 5.5 Hz), 7.68 (2H, d, J = 9.0 Hz, 7.41 (4H, d, J = 7.5 Hz), 7.32 - 7.25 (19H, m), 7.21 (2H, t, J = 7.5 Hz), 6.88 (8H, d, J = 8.5 Hz), 4.66 (2H, d, J = 6.0 Hz), 4.57 (2H, s), 4.54 (2H, d, J = 4.0 Hz), 4.26 (2H, d, J = 8.0 Hz), 4.10 (2H, dd, J = 11.5, 4.0 Hz), 3.99 (2H, dd, J = 11.5, 6.0 Hz), 3.76 - 3.68 (5H, m,), 3.73 (12H, s), 3.63 (2H, dd, J = 3.5, 3.5 Hz), 3.54 (2H, t, J = 6.5 Hz), 3.47 - 3.40 (4H, m), 3.19 - 3.10 (8H, m), 3.06 - 2.99 (4H, m), 2.23 (4H, t, J = 7.5 Hz), 1.82 (6H, s), 1.66 - 1.57 (4H, m)

[0232] (Example 2-4)

[0233] (Preparation of ([12-(benzyloxy)-5,9,15,19-tetraoxo-10,14-dioxo-4,8,16,20-tetraazatricosane-1,23-diyl]bis(oxy)(2R,5R)-3-acetamido-6-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}oxane-2,4,5-triyl)tetraacetate (VIIIa or 2))

[0234] [Chemical Formula 29]

[0235]

[0236] Tetrahydrofuran (10 mL) and triethylamine (29.9 g, 295.0 mmol) were added to a solution of 2-(benzyloxy)propane-1,3-diyl bis({3-[(3-{[(2R,5R)-3-acetamido-6-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4,5-dihydroxyoxan-2-yl]oxy}propyl)amino]-3-oxopropyl}carbamate) (VIIa or 8) in ethyl acetate / tetrahydrofuran (300 mL), and the resulting mixture was stirred for 10 minutes. Subsequently, 4-dimethylaminopyridine (1.20 g, 9.8 mmol) and acetic anhydride (26.8 g, 263.0 mmol) were added in this order, and the resulting mixture was stirred at 20 to 30 °C for 8 hours. After confirming the termination of the reaction, ethyl acetate (1000 mL) and 8% aqueous sodium hydrogen carbonate (500 mL) were added, and the resulting mixture was stirred at 20 to 30 °C for 22 hours and then separated. The resulting organic layer was washed with water (300 mL), 5% aqueous potassium dihydrogen phosphate (300 mL), 8% aqueous sodium hydrogen carbonate (300 mL) and water (300 mL) in this order. The resulting organic layer was concentrated under reduced pressure to obtain an ethyl acetate solution (400 mL). N-Methyl-2-pyrrolidone (200 mL) and ethyl acetate (400 mL) were added to the obtained ethyl acetate solution, and the resulting mixture was concentrated under reduced pressure to obtain an ethyl acetate / N-methyl-2-pyrrolidone solution (600 mL). Subsequently, the procedure of adding ethyl acetate (400 mL) to the obtained solution and concentrating the resulting mixture under reduced pressure to obtain an ethyl acetate / N-methyl-2-pyrrolidone solution (600 mL) was repeated three times to obtain a solution of the title compound (VIIIa) in ethyl acetate / N-methyl-2-pyrrolidone (600 mL).

[0237] In the same manner as in (Example 2-4), the title compound (VIIIa or 2) was synthesized by using 2-(benzyloxy)propane-1,3-diyl bis({3-[(3-{[(2R,5R)-3-acetamido-6-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4,5-dihydroxyoxan-2-yl]oxy}propyl)amino]-3-oxopropyl}carbamate) (VIIa) (33.0 g, 21.46 mmol), and separated by silica gel column purification (38.20 g, yield: 104.4%).

[0238] 1 H-NMR(500MHz,CD 3OD): δ 7.82 (2H, d, J = 9.5 Hz), 7.75 (2H, t, J = 5.5 Hz), 7.34 - 7.17 (25H, m), 6.91 - 6.86 (8H, m), 5.40 (2H, d, J = 3.5 Hz), 4.99 (2H, dd, J = 11.5, 3.5 Hz), 4.57 (2H, s), 4.45 (2H, d, J = 8.5 Hz), 4.10 (2H, dd, J = 11.5, 4.0 Hz), 4.08 - 4.05 (2H, m), 3.99 (2H, dd, J = 11.5, 5.5 Hz), 7.62 (2H, ddd, J = 11.5, 9.5, 8.5 Hz), 3.76 - 3.70 (1H, m), 3.74 (12H, s), 3.66 (2H, ddd, J = 10.0, 6.5, 6.5 Hz), 3.37 (2H, ddd, J = 10.0, 6.5, 6.5 Hz), 3.17 - 3.11 (6H, m), 3.08 - 3.00 (2H, m), 3.02 - 2.94 (2H, m), 2.80 (2H, t, J = 8.5 Hz), 2.22 (4H, t, J = 7.0 Hz), 1.89 (6H, s), 1.86 (6H, s), 1.77 (6H, s), 1.56 (4H, dddd, J = 7.0, 7.0, 6.5, 6.5 Hz)

[0239] (Example 2 - 5)

[0240] [Preparation of [(12 - hydroxy - 5,9,15,19 - tetraoxo - 10,14 - dioxo - 4,8,16,20 - tetraazatricosane - 1,23 - diyl)bis(oxy)(2R,5R)-3 - acetamido - 6 - {[bis(4 - methoxyphenyl)(phenyl)methoxy]methyl}oxane - 2,4,5 - triyl]tetraacetate (IXa or 1)]

[0241] [Chemical Formula 30]

[0242]

[0243] 5% palladium on carbon powder (PE type moistened with water) (2.1 g, based on dry weight, manufactured by N.E. CHEMCAT CORPORATION) was added to a solution of ([12-(benzyloxy)-5,9,15,19-tetraoxo-10,14-dioxa-4,8,16,20-tetraazatricosane-1,23-diyl]bis(oxy)(2R,5R)-3-acetamido-6-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}oxane-2,4,5-triyl)tetraacetate (VIIIa or 2) in ethyl acetate / N-methyl-2-pyrrolidone (600 mL), and the inside of the system was purged three times with nitrogen using an autoclave, and then purged three times with 0.1 MPa hydrogen. Thereafter, the mixture was stirred at 20 to 30 °C for 7 hours under a 0.1 MPa hydrogen atmosphere. After confirming the termination of the reaction, the reaction system was purged three times with nitrogen, the palladium on carbon was filtered off, and washed with ethyl acetate (200 mL). Ethyl acetate (600 mL) and 2.5% brine (600 mL) were added to the resulting solution, followed by separation. The operation of separating the resulting organic layer with a mixed solution of 2.5% brine (600 mL) and N-methyl-2-pyrrolidone (200 mL) was repeated three times to obtain an organic layer. The operation of separating the obtained organic layer with 2.5% brine (600 mL) was repeated three times. Thereafter, the procedure of adding ethyl acetate (600 mL) and concentrating the resulting mixture under reduced pressure to obtain an ethyl acetate solution (800 mL) was repeated twice. The obtained ethyl acetate solution (800 mL) was purified through a column (CHROMATOREX DIOL MB100-40 / 75 800 g, first through ethyl acetate (16 L), and then through a 3% methanol / ethyl acetate solution (24 L)), and the recovered fractions were concentrated under reduced pressure to obtain a solution of the title compound (IXa or 1) in ethyl acetate (800 mL) (quantitative value analyzed by liquid chromatography: 40.0 g, area ratio: 99.7%, total yield of three steps: 75.4%).

[0244] 1 H-NMR(500MHz,CD 3CN): δ 7.40 - 7.38 (4H, m), 7.31 - 7.21 (14H, m), 6.88 - 6.84 (8H, m), 7.21 - 6.66 (2H, br), 6.59 (2H, br s), 5.91 - 5.87 (2H, br), 5.43 (2H, d, J = 3.5 Hz), 5.01 (2H, dd, J = 11.5, 3.5 Hz), 4.45 (2H, d, J = 8.5 Hz), 4.10 - 3.88 (8H, m), 3.85 - 3.79 (1H, m), 3.81 (2H, ddd, J = 10.5, 5.5, 5.0 Hz), 3.76 (12H, s), 3.46 (2H, ddd, J = 10.5, 7.5, 5.0 Hz), 3.34 - 3.27 (6H, m), 3.23 (2H, dd, J = 9.0, 5.5 Hz), 3.07 (2H, ddd, J = 11.5, 11.5, 5.5 Hz), 2.90 (2H, dd, J = 9.0, 8.5 Hz), 2.34 - 2.25 (4H, m), 1.91 (6H, s), 1.88 (6H, s), 1.86 (6H, s), 1.70 - 1.59 (4H, m)

[0245] (Example 2 - 5 - 1)

[0246] Benzyl Deprotection Study 1

[0247] In the reaction of obtaining compound (IXa or 1) from compound (VIIIa or 2), the reaction solvent was screened.

[0248] Under the same reaction conditions as in Example 2 - 5, reactions were carried out by using 10% palladium - carbon powder (type AD wetted with water) (manufactured by Kawaken Fine Chemicals Co., Ltd.) and using the organic solvents shown in Table 2 as the reaction solvent respectively, and the results are shown in Table 2. In the following reaction formula and table, compound (Xa) is an impurity generated by the loss of 1 DMTr group from compound (IXa or 1) due to over - reduction reaction, and compound (XIa) is an impurity derived from the loss of the protecting group (DMTr group).

[0249] HPLC Analysis Conditions

[0250] Detection: 210 nm

[0251] Column: Xbridge C18 (4.6 mm ID × 150 mm, 3.5 μm)

[0252] Column Temperature: 40 °C

[0253] Mobile Phase: A: 10 mM Phosphate Buffer, B: Acetonitrile

[0254] Gradient conditions: B (concentration %) 55 (0 - 15 minutes), 55 - 75 (15 - 30 minutes), 75 - 55 (30 - 30.01 minutes), 55 (30.01 - 40 minutes)

[0255] Flow rate: 1.0 mL / min

[0256] Injection volume: 5 μL

[0257] [Chemical formula 31]

[0258]

[0259] [Table 2]

[0260]

[0261] (Example 2 - 5 - 2)

[0262] Benzyl deprotection study 2

[0263] In order to facilitate the separation after completion of the reaction and reduce the loss of the product into the aqueous layer, a study was conducted using a mixed solvent of ethyl acetate and any one of various polar solvents. This step is a continuous step, and if the reaction can be carried out in a mixed solvent with ethyl acetate (which is the post - treatment solvent in the previous step), this step will be a simpler step. Therefore, when this reaction is carried out in a mixed solvent, a study was conducted to determine the optimal mixing system of ethyl acetate and any one of various polar solvents. Under the same reaction conditions as in Example 2 - 5, 5% palladium - on - carbon powder (PE type wetted with water) (manufactured by N.E.CHEMCAT CORPORATION) was used. The results are shown in Table 3.

[0264] [Chemical formula 32]

[0265]

[0266] [Table 3]

[0267]

[0268] (Example 2 - 5 - 3)

[0269] Benzyl deprotection study 3

[0270] Under the same reaction conditions as in Example 2 - 5, 5% palladium - on - carbon powder (PE type wetted with water) (N.E.CHEMCAT CORPORATION) was used to study the effect of the solvent composition ratio of N - methyl - 2 - pyrrolidone and ethyl acetate on the reaction yield. The results are shown in Table 4.

[0271] [Chemical Formula 33]

[0272]

[0273] HPLC analysis conditions

[0274] Detection: 210 nm

[0275] Column: Xbridge C18 (4.6 mm ID × 150 mm, 3.5 μm)

[0276] Column temperature: 40 °C

[0277] Mobile phase: A: 10 mM ammonium acetate buffer, B: acetonitrile

[0278] Gradient conditions: B (concentration %) 5 (0 - 3 minutes), 5 - 95 (3 - 30 minutes), 95 (30 - 33 minutes), 95 - 5 (33 - 33.1 minutes), 5 (33.1 - 42 minutes)

[0279] Flow rate: 1.0 mL / min

[0280] Injection volume: 5 μL

[0281] [Table 4]

[0282]

[0283] (Example 2 - 5 - 4)

[0284] Benzyl deprotection study 4

[0285] Under the same reaction conditions as in Example 2 - 5, 5% palladium on carbon powder (PE type wetted with water) (manufactured by N.E. CHEMCAT CORPORATION) was used to study the amount of catalyst. The results are shown in Table 5.

[0286] [Chemical Formula 34]

[0287]

[0288] HPLC analysis conditions

[0289] Detection: 210 nm

[0290] Column: Xbridge C18 (4.6 mm ID × 150 mm, 3.5 μm)

[0291] Column temperature: 40 °C

[0292] Mobile phase: A: 10 mM phosphate buffer, B: acetonitrile

[0293] Gradient conditions: B (concentration %) 55 (0 - 15 minutes), 55 - 75 (15 - 30 minutes), 75 - 55 (30 - 30.01 minutes), 55 (30.01 - 40 minutes)

[0294] Flow rate: 1.0 mL / min

[0295] Injection volume: 5 μL

[0296] [Table 5]

[0297]

[0298] (Examples 2 - 6)

[0299] (Preparation of (12 - ({(2 - cyanoethoxy)[bis(propan - 2 - yl)amino]phosphoryl}oxy)-5,9,15,19 - tetraoxo - 10,14 - dioxo - 4,8,16,20 - tetraazatricosane - 1,23 - diyl)bis(oxy)(2R,3R,4R,5R,6R)-3 - acetamido - 6 - {[bis(4 - methoxyphenyl)(phenyl)methoxy]methyl}oxane - 2,4,5 - triyl)tetraacetic acid ester (XIIa or 3))

[0300] [Chemical Formula 35]

[0301]

[0302] The ethyl acetate solution (600 mL, quantitative value by liquid chromatography: 30.0 g, 18.6 mmol) of [(12-hydroxy-5,9,15,19-tetraoxo-10,14-dioxa-4,8,16,20-tetraazatricosane-1,23-diyl)bis(oxy)(2R,5R)-3-acetamido-6-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}oxane-2,4,5-triyl]tetraacetate (IXa or 1) was concentrated under reduced pressure to obtain an ethyl acetate solution (90 mL). Ethyl acetate (900 mL) was added to the obtained solution, and the resulting mixture was concentrated to 90 mL under reduced pressure. Dichloromethane (300 mL) and molecular sieve 4A (15.0 g) were added to the obtained solution, and the resulting mixture was stirred at 20 to 30 °C for 45 minutes. Thereafter, the mixture was cooled to 0 °C, 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (7.27 g, 24.1 mmol) and 4,5-dicyanoimidazole (1.10 g, 9.31 mmol) were added, and the resulting mixture was stirred at 2 to 4 °C for 16 hours. After confirming the termination of the reaction, the reaction solution was passed through a CHROMATOREX (registered trademark) PEI MB100-40 / 75 (60.0 g) pad, and the pad was washed with dichloromethane (180 mL). The obtained solution was filtered and washed with dichloromethane (90 mL). The obtained solution was concentrated under reduced pressure to obtain a dichloromethane / ethyl acetate solution (180 mL). The procedure of adding ethyl acetate (600 mL) to this solution and concentrating the resulting mixture under reduced pressure was repeated twice to obtain an ethyl acetate solution (300 mL). tert-Butyl methyl ether (270 mL) was added to the obtained ethyl acetate solution to obtain an ethyl acetate / tert-butyl methyl ether solution as solution A (570 mL).

[0303] tert-Butyl methyl ether (300 mL) and n-heptane (900 mL) were added to a new container and cooled to 4 to 5 °C, and then solution A (570 mL) was poured into it, followed by rinsing with a 50% ethyl acetate / tert-butyl methyl ether solution (30 mL) and stirring at 2 to 8 °C for 2.5 hours. The resulting slurry was filtered, washed in this order with a pre-cooled to 0 °C 25% tert-butyl methyl ether / n-heptane solution (180 mL) and pre-cooled to 0 °C n-heptane (300 mL), and dried under vacuum at an external temperature of 40 °C to obtain a powder (34.3 g, area ratio by liquid chromatography analysis: 98.4%, yield: 101.7%) of the title compound (XIIa or 3).

[0304] 1 H-NMR(500MHz,CD 3CN): δ 7.39 (4H, d, J = 8.0 Hz), 7.31 - 7.21 (14H, m), 6.87 - 6.83 (8H, m), 6.71 - 6.62 (2H, br), 6.57 (2H, brs), 5.97 - 5.83 (2H, br), 5.42 (2H, d, J = 3.5 Hz), 5.00 (2H, dd, J = 11.5 Hz), 4.44 (2H, d, J = 8.5 Hz), 4.20 - 3.97 (5H, m), 3.95 - 3.88 (4H, m), 3.83 - 3.70 (16H, m), 3.64 - 3.54 (2H, qq, J = 7.0, 6.5 Hz), 3.46 (2H, ddd, J = 10.0, 8.0, 5.0 Hz), 3.33 - 3.25 (6H, m), 3.23 (2H, dd, J = 9.0, 6.0 Hz), 3.10 - 3.04 (2H, m), 2.90 (2H, dd, J = 9.0, 8.0 Hz), 2.64 (1H, dd, J = 7.0, 5.5 Hz), 2.63 (1H, dd, J = 7.0, 5.5 Hz), 2.36 - 2.23 (4H, m), 1.91 (6H, s), 1.88 (6H, s), 1.86 (6H, s), 1.70 - 1.58 (4H, m), 1.15 (6H, d, J = 7.0 Hz), 1.14 (6H, d, J = 6.5 Hz)

[0305] The compound (XIIa or 3) can be converted into an N - acetyl - D - galactosamine ligand - oligonucleotide conjugate, for example, by using the methods disclosed in International Publication No. WO 2019 / 172286, International Publication No. WO 2021 / 049504, etc.

[0306] Industrial Applicability

[0307] By using the present invention, a novel method for preparing a di - antennal N - acetyl - D - galactosamine ligand - oligonucleotide conjugate that can be used as a drug can be provided.

Claims

1. A method for preparing a compound represented by formula (1): [Chemical formula 1] wherein Ac represents acetyl and DMTr represents 4,4'-dimethoxytriphenylmethyl, the method comprising the step of reacting a compound represented by formula (2) with a palladium catalyst in a reaction solvent in the presence of a reducing agent: [Chemical formula 2] wherein Ac represents acetyl, DMTr represents 4,4'-dimethoxytriphenylmethyl, and Bn represents benzyl.

2. The preparation method according to claim 1, wherein the palladium catalyst is palladium on carbon or palladium hydroxide on carbon.

3. The preparation method according to claim 1 or 2, wherein the reaction solvent is one or more solvents selected from alcohol-based solvents, ester-based solvents, ether-based solvents, and amide-based solvents.

4. The preparation method according to claim 1 or 2, wherein the reaction solvent is one or more solvents selected from 2-propanol, dimethylacetamide, N-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone.

5. The preparation method according to claim 1 or 2, wherein the reaction solvent is a mixed solvent of ethyl acetate and one or more solvents selected from dimethylacetamide, N-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone.

6. A method for preparing a compound represented by formula (3) or a salt thereof: [Chemical formula 3] wherein Ac represents acetyl and DMTr represents 4,4'-dimethoxytriphenylmethyl, the method comprising the step of reacting the compound represented by formula (1) obtained by the preparation method according to any one of claims 1 to 5 with an amidite reagent.

7. A method for preparing a conjugate composed of an oligonucleotide and a biantennary N-acetyl-D-galactosamine unit, the biantennary N-acetyl-D-galactosamine unit being bonded to the 5'-end or 3'-end of the oligonucleotide, the conjugate being represented by formula (4): [Chemical formula 4] wherein Ac represents acetyl, Z represents an oxygen atom or a sulfur atom, and the bond on the right side of the structural formula represents a bond with the oligonucleotide, the method comprising the step of reacting the compound represented by formula (3) obtained by the preparation method according to claim 6 with the oligonucleotide.

8. A method for preparing the compound represented by formula (2) according to claim 1, which comprises: the step of bonding a compound represented by formula (5) or a salt thereof to a compound represented by formula (6) by using a condensing agent: [Chemical formula 5] wherein Bn represents benzyl, [Chemical formula 6] wherein Ac represents acetyl and DMTr represents 4,4'-dimethoxytriphenylmethyl, and the step of acetylating the hydroxyl group on the pyran ring by using an acetylation reagent.

9. A method for preparing the compound represented by formula (5) or a salt thereof according to claim 8, which comprises: the step of reacting a compound represented by formula (7) with β-alanine or a salt thereof: [Chemical formula 7] wherein Bn represents benzyl, or A step of reacting the compound represented by formula (7) with a β-alanine alkyl ester or a salt thereof, wherein When the method includes a step of using a β-alanine alkyl ester or a salt thereof, the method further includes a hydrolysis step.

10. A method for preparing the compound represented by formula (7) according to claim 9, which comprises: A step of reacting 2-(benzyloxy)propane-1,3-diol with N,N'-disuccinimidyl carbonate in the presence of a base.

11. A compound represented by formula (2): [Chemical formula 8] wherein Ac represents acetyl, DMTr represents 4,4'-dimethoxytriphenylmethyl, and Bn represents benzyl.

12. A compound represented by formula (8): [Chemical formula 9] wherein Ac represents acetyl, DMTr represents 4,4'-dimethoxytriphenylmethyl, and Bn represents benzyl.

13. A compound represented by formula (5) or a salt thereof: [Chemical formula 10] wherein Bn represents benzyl.

14. A compound represented by formula (7): [Chemical formula 11] wherein Bn represents benzyl.

15. A compound represented by formula (6) or a salt thereof: [Chemical formula 12] wherein Ac represents acetyl, and DMTr represents 4,4'-dimethoxytriphenylmethyl.

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