Method for producing compound having two-branched n-acetyl-d-galactosamine structure

Abstract

The present invention addresses the problem of providing: a novel method for producing a N-acetyl-D-galactosamine ligand-oligonucleotide conjugate, particularly a novel method for producing a N-acetyl-D-galactosamine unit; and a novel intermediate.　Studies have been made about the selective deprotection of a benzyl group using a palladium catalyst in a novel method for producing a N-acetyl-D-galactosamine unit. As a result, a novel production method that can achieve high quality and high yield and is industrially advantageous and a novel intermediate are discovered, which leads to the accomplishment of the invention.

Description

Method for producing a compound having a bibranched N-acetyl-D-galactosamine structure

[0001] The present invention relates to a novel method for producing a biantennary N-acetyl-D-galactosamine ligand-oligonucleotide conjugate, and more particularly to an industrially advantageous novel method for producing a biantennary N-acetyl-D-galactosamine unit and novel intermediates.

[0002] A method for delivering oligonucleotides to hepatic parenchymal cells in the liver using conjugates of nucleic acid drugs in which N-acetyl-D-galactosamine (also known as GalNAc) is linked to the oligonucleotide via a linker has been reported (Patent Document 1). A novel biantennary N-acetyl-D-galactosamine unit (also known as biantennary GalNAc unit) that enables hepatic delivery of any oligonucleotide, as well as a method for producing this unit using a protecting group such as an Fmoc group, have also been reported (Patent Document 2). While there is a need for a new method for the mass synthesis of high-quality biantennary GalNAc units, the production of compounds such as biantennary GalNAc units, which generally have complex structures and many reactive sites, is extremely difficult. In particular, the selective introduction and removal of substituents and protecting groups during the production process to obtain high-quality compounds with complex structures and many reactive sites in high yields is difficult. For example, attempts have been made to selectively remove only the benzyl group from compounds containing both trityl and benzyl groups, but the yield is only about 35% (Non-Patent Document 1).

[0003] International Publication No. 2019 / 172286 International Publication No. 2021 / 049504

[0004] J. Chem. Soc., Perkin Trans. 1, 1987, (3), 537-45

[0005] The present invention aims to provide a method for preparing N-acetyl-D-galactosamine ligand-oligonucleotide conjugates, which includes novel steps for selectively introducing and removing substituents and / or protecting groups.

[0006] The present invention relates to a novel method for producing an N-acetyl-D-galactosamine ligand-oligonucleotide conjugate, which solves the above-mentioned problems, and in particular to a novel method for producing an N-acetyl-D-galactosamine unit and a novel intermediate. In particular, the present invention relates to a novel industrially advantageous production method with high quality and high yield, which employs selective deprotection of a benzyl group using a palladium catalyst as a key reaction.

[0007] The present invention includes the following aspects [1] to

[14] .

[0008] [1] Formula (1): [wherein Ac represents an acetyl group, and DMTr represents a 4,4'-dimethoxytrityl group], the method for producing a compound represented by formula (2): [In formula (2), Ac represents an acetyl group, DMTr represents a 4,4'-dimethoxytrityl group, and Bn represents a benzyl group], with a palladium catalyst in a reaction solvent in the presence of a reducing agent.

[0009] [2] The method according to [1], wherein the palladium catalyst is palladium on carbon or palladium hydroxide.

[0010] [3] The production method according to [1] or [2], wherein the reaction solvent is one or more solvents selected from alcohol solvents, ester solvents, ether solvents, and amide solvents.

[0011] [4] The 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.

[0012] [5] The 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.

[0013] [6] Formula (3): [In formula (3), Ac represents an acetyl group, and DMTr represents a 4,4'-dimethoxytrityl group], or a salt thereof, the method comprising the step of reacting the compound represented by formula (1) obtained by the method according to any one of [1] to [5] with an amidite reagent.

[0014] [7] Formula (4): A method for producing a conjugate consisting of an oligonucleotide and a biantennary-D-galactosamine unit, wherein the biantennary-D-galactosamine unit is bound to the 5'-end or 3'-end of the oligonucleotide, and the conjugate is represented by the following formula: [in formula (4), Ac represents an acetyl group, and DMTr represents a 4,4'-dimethoxytrityl group] [in formula (4), Z represents an oxygen atom or a sulfur atom, and the bond at the right end of the structural formula represents a bond to the oligonucleotide], the method comprising the step of reacting the oligonucleotide with the compound represented by formula (3) obtained by the production method described in [6].

[0015] [8] A method for producing a compound represented by formula (2) used in (1), comprising reacting a compound represented by formula (5): [wherein Bn represents a benzyl group] or a salt thereof with a compound represented by formula (6): [wherein Ac represents an acetyl group, and DMTr represents a 4,4'-dimethoxytrityl group], using a condensing agent; and acetylating the hydroxyl group on the pyran ring using an acetylating reagent.

[0016] [9] A method for producing a compound represented by formula (5) or a salt thereof used in [8], comprising reacting a compound represented by formula (7): [in formula (7), Bn represents a benzyl group] with β-alanine or a salt thereof, or with a β-alanine alkyl ester or a salt thereof, and when the process includes a step using a β-alanine alkyl ester or a salt thereof, the process further includes a hydrolysis step.

[0017]

[10] A method for producing the compound represented by formula (7) used in [9], comprising the step of reacting 2-(benzyloxy)propane-1,3-diol with N,N'-disuccinimidyl carbonate in the presence of a base.

[0018]

[11] Formula (2): A compound represented by the following formula (2): wherein Ac represents an acetyl group, DMTr represents a 4,4'-dimethoxytrityl group, and Bn represents a benzyl group.

[0019]

[12] Formula (8): A compound represented by the following formula (8): wherein Ac represents an acetyl group, DMTr represents a 4,4'-dimethoxytrityl group, and Bn represents a benzyl group.

[0020]

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

[0021]

[14] Formula (7): A compound represented by the formula (7): wherein Bn represents a benzyl group.

[0022]

[15] Formula (6): A compound represented by formula (6): wherein Ac represents an acetyl group, and DMTr represents a 4,4'-dimethoxytrityl group, or a salt thereof.

[0023] The present invention provides a novel, industrially advantageous method for producing an N-acetyl-D-galactosamine unit in high yield and high quality using a palladium catalyst, in which selective deprotection of a benzyl group is the key reaction, and provides a novel intermediate. In particular, the present invention provides a simple, inexpensive production method that does not use expensive protecting groups such as the Fmoc group. This provides a novel method for producing an N-acetyl-D-galactosamine ligand-oligonucleotide conjugate.

[0024] Hereinafter, embodiments of the present invention will be described in more detail.

[0025] The present invention provides a method for producing a conjugate comprising an oligonucleotide and a biantennary N-acetyl-D-galactosamine unit, as represented by formula (4), in which a biantennary N-acetyl-D-galactosamine unit is bound to the 5'-end or 3'-end of the oligonucleotide, by reacting a compound represented by formula (3) with an oligonucleotide. The present invention also features a process for obtaining the intermediate compound represented by formula (3). Specifically, the present invention includes a method for producing a compound represented by formula (1) using a compound represented by formula (2), and a method for producing a compound represented by formula (3) or a salt thereof using the compound represented by formula (1) obtained above. Furthermore, the present invention includes a method for producing a compound represented by formula (2) by reacting a compound represented by formula (5) or a salt thereof with a compound represented by formula (6), followed by further reaction with a compound represented by formula (8), and a method for producing a compound represented by formula (5) or a salt thereof using a compound represented by formula (7) or compound (Va) in the following examples. The present invention also relates to intermediates obtained during the above methods.

[0026] The compounds represented by formulas (1) to (7) may be referred to by different names in this specification. These names may also be used together. Table 1 shows the names of the compounds.

[0027] The synthesis process according to one embodiment of the present invention will be described below.

[0028] Method for producing compound (Ib or 6)

[0029] Step A-1: ​​This step involves the β-selective glycosylation 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 to produce compound (IIa). The solvent that can be used is not particularly limited as long as it does not inhibit the reaction, and examples include one or more solvents selected from dichloromethane, acetonitrile, tetrahydrofuran, etc. A preferred solvent is dichloromethane. The acid catalyst that can be used is not particularly limited as long as it is one that is used in glycosylation, and examples include trifluoromethanesulfonic acid, trimethylsilyl trifluoromethanesulfonate, and tin tetrachloride. A preferred acid catalyst is trifluoromethanesulfonic acid. The reaction temperature is typically 25°C to 45°C, preferably 30°C to 40°C. The reaction time varies depending on the type and amount of acid catalyst used, but is typically 1 to 28 hours, and preferably 21 to 23 hours. The resulting compound can be purified by a conventional method such as recrystallization or silica gel chromatography.

[0030] Step A-2: This step involves reacting compound (IIa) with a deprotecting reagent in an organic solvent to deprotect the acetyl (Ac) group, thereby producing compound (IIIa). The deprotecting reagent that can be used is not particularly limited as long as it is a reagent used to deprotect an acetyl group, and examples thereof include inorganic bases such as sodium hydroxide, potassium hydroxide, or sodium carbonate, and organic bases such as ammonia, alkylamines, sodium methoxide, or sodium ethoxide. A preferred deprotecting reagent is sodium methoxide. The solvent that can be used is not particularly limited as long as it does not inhibit the reaction, and examples thereof include one or more solvents selected from methanol and ethanol. A preferred solvent is ethanol. The reaction temperature is usually 30°C to 50°C, preferably 35°C to 45°C. The reaction time varies depending on the type and amount of deprotecting reagent used, but is usually 0.5 to 24 hours, preferably 12 to 14 hours. The resulting compound can be purified using a conventional method such as recrystallization or silica gel chromatography.

[0031] Step A-3: This step involves reacting compound (IIIa) with a tritylation reagent such as 4,4'-dimethoxytrityl chloride or 4,4'-dimethoxytrityl trifluorosulfonate in an organic solvent to selectively protect the primary hydroxyl group with a 4,4'-dimethoxytrityl (DMTr) group, thereby producing compound (IVa). The solvent that can be used is not particularly limited as long as it does not inhibit the reaction, and examples include N,N-dimethylacetamide, N,N-dimethylformamide, and N-methylpyrrolidone. A preferred solvent is N,N-dimethylacetamide. The reaction temperature is typically 15°C to 35°C, preferably 20°C to 30°C. The reaction time varies depending on the type and amount of tritylation reagent used, but is typically 1 hour to 8 hours, preferably 4 hours to 6 hours. The resulting compound can be purified using a conventional method such as recrystallization or silica gel chromatography.

[0032] Step A-4: This step is a step of producing compound (Ib or 6) by selectively deprotecting the benzyloxycarbonyl (Cbz) group of compound (IVa) by reacting compound (IVa) with a deprotecting reagent in an organic solvent. 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. The reducing agent is hydrogen, formic acid, ammonium formate, and the like. A preferred metal catalyst is a palladium on carbon catalyst, and a preferred reducing agent is hydrogen. The solvent that can be used is not particularly limited as long as it does not inhibit the reaction, and examples thereof include one or more solvents selected from methanol, ethanol, tetrahydrofuran, and the like. A preferred solvent is methanol. The reaction temperature is usually 15°C to 35°C, preferably 20°C to 30°C. When the reducing agent is hydrogen, the hydrogen pressure in the reaction system is usually 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 deprotection reagent used, but is usually 0.5 to 6 hours, preferably 2 to 3 hours. The resulting compound can be purified by a conventional method such as recrystallization or silica gel chromatography.

[0033] Method for producing compound (XIIa or 3)

[0034] 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 activated diester (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 condensing compound (Va or 7) with β-alanine in the presence of a base to produce compound (VIa or 5), or a step (Step B-1-2B) of condensing compound (Va or 7) with a β-alanine alkyl ester or a salt thereof in the presence of a base, followed by hydrolysis to produce compound (VIa or 5).

[0035] (B-1-1 process)

[0036] This is a step of converting 2-(benzyloxy)propane-1,3-diol into compound (Va or 7). Examples of bases that can be used include triethylamine and pyridine. A preferred base is pyridine. There are no particular limitations on the solvent that can be used as long as it does not inhibit the reaction, and examples include acetonitrile, tetrahydrofuran, acetone, and N,N'-dimethylacetamide. A preferred solvent is acetonitrile. The reaction temperature is usually −15° C. to 55° C., preferably −5 to 45° C. The reaction time is usually 1 hour to 6 hours, preferably 2 hours to 3 hours.

[0037] (B-1-2A process)

[0038] This is a process for producing compound (VIa or 5) by condensing β-alanine with compound (Va or 7) in the presence of a base. Usable bases include, for example, triethylamine, pyridine, and N,N'-diisopropylethylamine. A preferred base is triethylamine. Any solvent that does not inhibit the reaction may be used, and examples include aqueous acetonitrile, aqueous tetrahydrofuran, and aqueous acetone. A preferred solvent is aqueous acetonitrile. The reaction temperature is typically 15°C to 35°C, preferably 20°C to 30°C. The reaction time varies depending on the type and amount of base used, but is typically 0.25 hours to 6 hours, preferably 0.5 hours to 1.5 hours. After completion of the reaction, compound (VIa or 5) can be mixed with a base to form a salt. A preferred salt of compound (VIa or 5) is the dibenzylamine salt.

[0039] (B-1-2B process)

[0040] In this process, a β-alanine alkyl ester and compound (Va or 7) are condensed in the presence of a base to give compound (Va') (wherein Alkyl represents a linear or branched C1-C6 alkyl group such as a methyl group, an ethyl group, a propyl group, an isopropyl group, or a tert-butyl group), followed by hydrolysis to produce compound (VIa or 7). Usable β-alanine alkyl esters include, for example, β-alanine methyl ester, β-alanine ethyl ester, β-alanine propyl ester, β-alanine isopropyl ester, β-alanine tert-butyl ester, and salts thereof. β-alanine ethyl ester or a salt thereof is preferred. Examples of bases that can be used in the condensation reaction include triethylamine, pyridine, and N,N'-diisopropylethylamine. A preferred base that can be used in the condensation reaction is triethylamine. The base that can be used in the hydrolysis reaction is not particularly limited as long as it is a base that is commonly used in hydrolysis reactions, and examples thereof include lithium hydroxide, sodium hydroxide, and potassium hydroxide. A suitable base for use in the hydrolysis reaction is potassium hydroxide. The solvent that can be used is not particularly limited as long as it does not inhibit the reaction, and examples thereof include acetonitrile, tetrahydrofuran, and acetone. A suitable solvent is tetrahydrofuran. The reaction temperature is usually 15°C to 35°C, preferably 20°C to 30°C. The reaction time varies depending on the type and amount of base used, but is usually 0.25 hours to 6 hours, preferably 0.5 hours to 1.5 hours. Compound (VIa or 5) can also be mixed with a base to form a salt. A suitable salt of compound (VIa or 5) is a dibenzylamine salt.

[0041] Step B-2: This step involves the condensation reaction of compound (Ib or 6) with compound (VIa or 5) or a salt thereof in an organic solvent using a condensing agent to produce compound (VIIa or 8). When a salt of compound (VIa or 5) is used, it is freed by a separation operation or the like before the reaction and then used in the reaction. The condensing agent that can be used is not particularly limited as long as it is a reagent that can be 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-methylmorphonium chloride n-hydrate, and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate. A suitable condensing agent is a combination of 1-hydroxybenzotriazole monohydrate and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. There are no particular limitations on the solvent that can be used, as long as it does not inhibit the reaction, and examples include aqueous tetrahydrofuran, aqueous acetonitrile, and aqueous acetone. A suitable solvent is aqueous tetrahydrofuran. The reaction temperature is usually 10°C to 40°C, preferably 20°C to 30°C. The reaction time varies depending on the type and amount of condensation reagent used, but is usually 5 to 60 hours, preferably 45 to 47 hours. The resulting compound can be purified using a conventional method such as recrystallization or silica gel chromatography.

[0042] Step B-3: This step involves reacting compound (VIIa or 8) with an acetylating reagent in an organic solvent to produce compound (VIIIa or 2). The acetylating reagent that can be used is not particularly limited as long as it is a reagent used in acetylation reactions, and examples thereof include acetic anhydride and acetyl chloride. A preferred acetylating reagent is acetic anhydride, and it is more preferred to use acetic anhydride in combination with 4-dimethylaminopyridine and triethylamine. The solvent that can be used is not particularly limited as long as it does not inhibit the reaction, and examples thereof include one or more solvents selected from ethyl acetate, tetrahydrofuran, acetone, acetonitrile, and the like. A preferred solvent is a mixed solvent of ethyl acetate and tetrahydrofuran. The reaction temperature is usually 10°C to 40°C, preferably 20°C to 30°C. The reaction time varies depending on the type and amount of acetylating reagent used, but is usually 2 to 12 hours, and preferably 7 to 9 hours. The compound (VIIIa or 2) obtained in this step can be used in the next step without isolation or purification, but can also be purified using a conventional method such as recrystallization or silica gel chromatography.

[0043] Step B-4: In this step, compound (VIIIa or 2) is reacted with a benzyl group-deprotecting reagent in an organic solvent in the presence of a reducing agent to selectively deprotect the benzyl group, thereby producing compound (IXa or 1). Examples of the benzyl group-deprotecting reagent include a combination of a palladium catalyst supported on a support and a reducing agent. The palladium catalyst that can be used is not particularly limited as long as it can deprotect the benzyl group, but a palladium-carbon catalyst or a palladium hydroxide-carbon catalyst is preferred. The palladium content in the palladium-carbon catalyst and palladium hydroxide-carbon catalyst is not particularly limited, and catalysts with a palladium content of, for example, 5%, 10%, or 20% can be used. Palladium catalysts with a preferred content are 5% palladium catalyst or 20% palladium hydroxide catalyst. Examples of the reducing agent that can be used include hydrogen, formic acid, and ammonium formate. A preferred reducing agent is hydrogen.

[0044] The solvent that can be used in this step is not particularly limited as long as it does not inhibit the reaction, and may be, for example, one or more solvents selected from alcohol solvents, ester solvents, ether solvents, and amide solvents. For example, one or more solvents selected from 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. Preferred solvents are one or more solvents selected from 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. A 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 usually 10 to 40° C., preferably 20 to 30° C. The reaction time varies depending on the types and amounts of the palladium catalyst and reducing agent used, but is usually 4 to 10 hours, preferably 6 to 8 hours.

[0045] In this step, "selectively deprotecting the benzyl group" means that the 4,4'-dimethoxytrityl group is not deprotected, and a compound in which only the benzyl group is deprotected is produced, meaning that a compound in which only the benzyl group is deprotected is produced as the main product. For example, this means that the compound in which only the benzyl group is deprotected is produced in an amount of 40% or more, preferably 50% or more, and more preferably 60% or more, of the compound in which only the benzyl group is deprotected, in terms of HPLC area ratio.

[0046] Step B-5: This step involves reacting compound (IXa or 1) with an activating agent and an amiditizing reagent in an organic solvent in the presence of a drying agent to produce compound (XIIa or 3). The amiditizing reagent that can be used is not particularly limited, as long as it is a reagent that can be used to introduce a phosphorus-containing group useful for forming 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'-tetraisopropylphosphorodiamidite and 2-cyanoethyldiisopropylchlorophosphoroamidite. A preferred amiditizing reagent is 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite. The amount of the amiditizing reagent that can be used is 1.0 to 1.6 equivalents, preferably 1.2 to 1.4 equivalents, relative to compound (IXa or 1). The activating agent 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 amiditization, and examples thereof include 5-benzylthiotetrazole, 5-phenyltetrazole, dibromoimidazole, 4,5-dicyanoimidazole, and N-alkylimidazole trifluoroacetate. A preferred activating agent is 4,5-dicyanoimidazole. The amount of the activating agent that can be used is 0.1 to 1.0 equivalents, preferably 0.4 to 0.6 equivalents, relative to Compound (IXa or 1). The desiccant that can be used is not particularly limited as long as it is a reagent that can be used to absorb moisture in the reaction solution, and examples thereof include molecular sieve 3A, molecular sieve 4A, and molecular sieve 5A. A preferred desiccant is molecular sieve 4A. The solvent that can be used is not particularly limited as long as it does not inhibit the reaction, and examples thereof include one or more solvents selected from ethyl acetate, dichloromethane, N,N-dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofuran, and methyl isobutyl ketone. A preferred solvent is a mixed solvent of ethyl acetate and dichloromethane. The reaction temperature is usually -5°C to 10°C, and preferably 0°C to 5°C.The reaction time varies depending on the type and amount of reagents used, but when the above-mentioned preferred activating agent, amidite reagent, and desiccant are used, it is usually 12 to 24 hours, preferably 15 to 17 hours.

[0047]

[0043] The conjugate of formula (4), which is composed of an oligonucleotide and a biantennary N-acetyl-D-galactosamine unit and in which the biantennary N-acetyl-D-galactosamine unit is bound to the 5'-end or 3'-end of the oligonucleotide, can be produced by the above-described method, which includes a step of reacting compound (XIIa or 3) with an oligonucleotide. Each step of the above process is described in detail below.

[0048] In the present invention, the term "palladium catalyst" refers to a palladium catalyst used to remove a benzyl group bonded to a hydroxyl group, and is preferably palladium on carbon or palladium hydroxide on carbon.

[0049] In the present invention, examples of the "reducing agent" used in the palladium-catalyzed elimination reaction of the benzyl group include hydrogen, formic acid, ammonium formate, etc. The reducing agent is preferably hydrogen.

[0050] The "reaction solvent" used in the present invention can be any solvent that does not inhibit the reaction. For example, alcohol solvents, ester solvents, ether solvents, amide solvents, etc. can be used in the palladium-catalyzed elimination reaction of the benzyl group.

[0051] Examples of alcoholic solvents that can be used in the step of elimination of the benzyl group using a palladium catalyst of the present invention include methanol, ethanol, 1-propanol, 2-propanol, etc. The alcoholic solvent is preferably 2-propanol.

[0052] Examples of ester solvents that can be used in the step of removing the benzyl group using a palladium catalyst of the present invention include ethyl acetate, propyl acetate, etc. The ester solvent is preferably ethyl acetate.

[0053] Examples of ether solvents that can be used in the step of elimination of the benzyl group using a palladium catalyst of the present invention include tetrahydrofuran, 2-methyltetrahydrofuran, tert-butyl methyl ether, cyclopentyl methyl ether, etc. The ether solvent is preferably 2-methyltetrahydrofuran.

[0054] Examples of amide solvents that can be used in the palladium-catalyzed benzyl group elimination step of the present invention include dimethylacetamide, dimethylformamide, N-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, etc. The amide solvent is preferably dimethylacetamide, N-methyl-2-pyrrolidone, or 1,3-dimethyl-2-imidazolidinone.

[0055] A suitable solvent that can be used in the palladium-catalyzed benzyl group elimination step of the present invention is one or more solvents selected from methanol, 2-propanol, ethyl acetate, 2-methyltetrahydrofuran, dimethylacetamide, N-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone. A more suitable solvent is one or more solvents selected from 2-propanol, dimethylacetamide, N-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone.

[0056] In the palladium-catalyzed benzyl group elimination step of the present invention, a preferred mixed solvent of two or more solvents is a mixed solvent of an amide solvent and ethyl acetate, more preferably a mixed solvent of ethyl acetate and one or more solvents selected from dimethylacetamide, N-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone, and most preferably a mixed solvent of N-methyl-2-pyrrolidone and ethyl acetate.

[0057] When a mixed solvent of N-methyl-2-pyrrolidone and ethyl acetate is used, they can be mixed at any ratio, preferably in a ratio of 1:30 to 1:1, more preferably in a ratio of 1:5 to 1:2.

[0058] In the present invention, the term "amiditizing reagent" refers to a reagent used in the amiditizing step, which is used to introduce a phosphorus-containing group useful for forming a covalent bond between the hydroxyl group of a biantennary N-acetyl-D-galactosamine unit and an oligonucleotide. Examples of the amiditizing reagent include 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite and 2-cyanoethyldiisopropylchlorophosphoroamidite. The amiditizing reagent is preferably 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite.

[0059] The amiditization step in the present invention is a step of producing a compound represented by general formula (3) by reacting a compound represented by formula (1) with an activator and an amiditization reagent in an organic solvent in the presence of a drying agent.

[0060] The solvent that can be used in the amiditization step is, for example, one or more solvents selected from ethyl acetate, dichloromethane, N,N-dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofuran, and methyl isobutyl ketone, and a mixed solvent of ethyl acetate and dichloromethane is preferred.

[0061] The activating agent that can be used in the amiditization step is a reagent used to form an active intermediate of the amiditization reagent, and examples thereof include 5-benzylthiotetrazole, 5-phenyltetrazole, dibromoimidazole, 4,5-dicyanoimidazole, N-alkylimidazole trifluoroacetate, etc. The activating agent is preferably 4,5-dicyanoimidazole.

[0062] The desiccant that can be used in the amiditization step is a reagent used to absorb 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.

[0063] A conjugate consisting of a biantennary N-acetyl-D-galactosamine unit of formula (4) and an oligonucleotide can be produced by coupling the amidite of the biantennary N-acetyl-D-galactosamine unit of formula (3) produced by the production method of the present invention with an oligonucleotide having a desired nucleotide sequence using a conventional method such as the phosphoramidite method. As used herein, the terms "conjugate" and "conjugate" refer to a compound in which an N-acetyl-D-galactosamine unit (GalNAc unit) is attached to the 5'-end and / or 3'-end of an oligonucleotide. The conjugate can be produced using a DNA synthesizer, such as a Perkin-Elmer Model 392 using the phosphoramidite method, according to the method described in Nucleic Acids Research, 12, 4539 (1984), etc. More specifically, a biantennary N-acetyl-D-galactosamine ligand-oligonucleotide conjugate can be produced by the production method disclosed in Patent Document 2 (WO 2021 / 049504).

[0064] The method for producing the oligonucleotides used in the present invention is not particularly limited as long as the desired oligonucleotides can be synthesized, and known chemical synthesis methods (such as the phosphate triester method, phosphoramidite method, or H-phosphonate method) can be used. For example, the oligonucleotides can be synthesized using a commercially available nucleic acid synthesizer and commercially available reagents used for DNA / RNA synthesis.

[0065] By the conventional phosphoramidite method, an oligonucleotide having a desired nucleotide sequence can be synthesized using a DNA synthesizer, such as Perkin-Elmer's Model 392 phosphoramidite method, according to the method described in Nucleic Acids Research, 12, 4539 (1984), etc.

[0066] Amidite reagents corresponding to various nucleosides may be purchased commercially or may be synthesized according to known methods.

[0067] In the present invention, examples of the "acetylating reagent" include acetic anhydride, acetyl chloride, etc. The acetylating reagent is preferably acetic anhydride.

[0068] In the present invention, the term "condensing agent" refers to a reagent used in the condensation reaction of a carboxylic acid with 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-methylmorphonium chloride n-hydrate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, etc. The condensing agent is preferably a combination of 1-hydroxybenzotriazole monohydrate and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0069] In the condensation step with the compound represented by formula (7) of the present invention, β-alanine, a β-alanine alkyl ester, or a salt thereof can be used. Examples of β-alanine alkyl esters include β-alanine methyl ester, β-alanine ethyl ester, β-alanine propyl ester, β-alanine isopropyl ester, β-alanine tert-butyl ester, and salts thereof. In the condensation step, β-alanine or a salt thereof, or β-alanine ethyl ester or a salt thereof is preferred.

[0070] Examples of the base that can be used in the condensation step with the compound represented by formula (7) of the present invention include triethylamine, pyridine, N,N'-diisopropylethylamine, etc. The base is preferably triethylamine.

[0071] The compound represented by formula (1) of the present invention, the compound represented by formula (2) of the present invention, the compound represented by formula (3) of the present invention, the compound represented by formula (4) of the present invention, the compound represented by formula (6) of the present invention or a salt thereof, and the compound represented by formula (8) of the present invention include all isomers (diastereoisomers, optical isomers, geometric isomers).

[0072] The compound represented by formula (3) of the present invention, the compound represented by formula (4) of the present invention, the compound represented by formula (5) of the present invention, and the compound represented by formula (6) of the present invention can be converted into a salt by reacting with a base when they have an acidic group, and can be converted into a salt by reacting with an acid when they have a basic group.

[0073] Examples of salts based on basic groups include hydrohalides such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; alkylsulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as acetate, malate, fumarate, succinate, citrate, ascorbate, tartrate, oxalate, and adipate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate.

[0074] Examples of salts based on acidic groups include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; metal salts such as aluminum salt and iron salt; inorganic salts such as ammonium salt; amine 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, and tris(hydroxymethyl)aminomethane salt; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate.

[0075] A preferred salt of the compound of the present invention represented by formula (8) is a dibenzylamine salt.

[0076] The compound of the present invention or a salt thereof may incorporate water molecules to become a hydrate when left in the air or when recrystallized, and such hydrates are also encompassed in the compound or salt of the present invention.

[0077] The compound of the present invention or a salt thereof may absorb a certain type of solvent when left in a solvent or recrystallized, and become a solvate. Such solvates are also encompassed in the compound or salt of the present invention.

[0078] As used herein, the term "GalNAc unit" refers to a partial structure containing N-acetyl-D-galactosamine (GalNAc) capable of binding to the asialoglycoprotein receptor (ASGPR) on hepatocytes in the liver. The GalNAc unit may contain a linear or branched linker structure and a phosphate group or thiophosphate group for binding to an oligonucleotide. There is no limitation on the number of GalNAc units contained in one GalNAc unit, and unless otherwise specified, publicly known GalNAc units and units disclosed herein may be used. The GalNAc structure may be modified as long as the ability to bind to ASGPR is maintained. Furthermore, units in which a protecting group has been introduced into GalNAc during the production process are also included.

[0079] In addition, in this specification, "oligonucleotide" refers to a synthetic nucleic acid having a chain length of 100 bases or less, and may be single-stranded or double-stranded. As described below, the nucleic acid to be employed may be DNA, RNA, natural nucleic acid, modified nucleic acid, etc., which may be used depending on the purpose, and these may be mixed in a single oligonucleotide. As the GalNAc unit and oligonucleotide, for example, those described in Patent Document 2 (WO 2021 / 049504) may be used.

[0080] The present invention will now be described. The reaction conditions of the present invention should not be construed as being limited thereto. In the present invention, the functional groups of the compounds may be protected with appropriate protecting groups. Examples of such functional groups include hydroxyl groups, carboxyl groups, and amino groups. The types of protecting groups and the conditions for introducing and removing these protecting groups can be found, for example, in Protective Groups in Organic Synthesis (T. W. Green and P. G. M. Wuts, John Wiley & Sons, Inc., New York, 2006).

[0081] The abbreviations used in the examples have the following meanings: g: gram, mL: milliliter, mol: mole, MHz: megahertz, Ac: acetyl group, DMTr: 4,4'-dimethoxytrityl group, Bn: benzyl group, Cbz: benzyloxycarbonyl group, 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. In the following examples, nuclear magnetic resonance (hereinafter referred to as 1 H NMR (500 MHz) spectra were measured using chloroform-d, methanol-d, dimethyl sulfoxide-d, and acetonitrile-d as deuterated solvents, and tetramethylsilane as the standard. Chemical shifts are reported in δ values ​​(ppm). Splitting patterns are indicated by s for singlets, d for doublets, dd for double doublets, t for triplets, q for quartets, m for multiplets, and br for broad peaks.

[0082] Example 1 Preparation of 3-aminopropyl 2-acetamido-6-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β-D-galactopyranoside (Ib or 6)

[0083] Example 1-1: Preparation of benzyl {3-[(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl)oxy]propyl}carbamate (IIa)

[0084]

[0085] 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 concentrated under reduced pressure to obtain a dichloromethane solution (600 mL). Similarly, dichloromethane (300 mL) was added to the resulting dichloromethane solution and concentrated under reduced pressure to obtain a dichloromethane solution (600 mL). The water content of the resulting dichloromethane solution was confirmed to be 0.02% or less using a Karl Fischer water content analyzer (coulometric method). Dichloromethane (300 mL) was added, and benzyl (3-hydroxypropyl)carbamate (33.9 g, 162.0 mmol) and trifluoromethanesulfonic acid (4.6 g, 30.8 mmol) were added in that order, followed by stirring at 35°C for 22 hours. After confirming the completion 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 under reduced pressure to 300 mL. Ethanol (900 mL) was added to the obtained concentrated solution, and the mixture was concentrated under reduced pressure to obtain a 900 mL solution. Subsequently, the same vacuum concentration operation was repeated again, and ethanol (300 mL) was added to the obtained solution (900 mL) to obtain an ethanol solution (1200 mL) of the title compound (IIa).

[0086] The title compound (IIa) was synthesized (28.99 g, yield 83.8%) using 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-D-galactopyranose (25.0 g, 64.21 mmol) in the same manner as in Example 1-1. 1H-NMR(500MHz,CDCl3): δ7.40-7.31(5H,m),6.19(1H,d,J=8.5Hz), 5.32(1H,d,J=2.0Hz), 5.12 and 5.08(2H,d,J=12.0Hz), 5.01-4.96(2H,m), 4.33(1H,d,J=8.5Hz), 4.18-4.09(3H,m), 3.97(1H,ddd,J=10.0,4.0,4.0Hz), 3.77(1H,dd,J=7.0,7.0Hz), 3.59-3.52(1H,m), 3.40(1H,ddd,J=9.5,9.5,3.0Hz), 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)

[0087] (Example 1-2) Preparation of benzyl {3-[(2-acetamido-2-deoxy-β-D-galactopyranosyl)oxy]propyl}carbamate (IIIa)

[0088]

[0089] An ethanol solution (1200 mL) of benzyl {3-[(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl)oxy]propyl}carbamate (IIa) was heated to 40°C, and 28% sodium methoxide methanol solution (5.9 g, 30.8 mmol) was added, followed by stirring for 13 hours. After confirming the completion of the reaction, the reaction solution was cooled to 0°C, and the precipitated solid was filtered, washed with 0°C ethanol (180 mL), and dried under reduced pressure at an external temperature of 40°C to obtain the title compound (IIIa) (56.4 g, area ratio by liquid chromatography analysis: 99.8%, total yield for two steps: 88.7%). 1H-NMR(500MHz,DMSO-d6): δ7.60(1H,d,J=9.5Hz), 7.38-7.29(5H,m), 7.19(1H,dd,J=6.0,6.0Hz),5.01(2H,s), 4.60-4.56(2H,m), 4.49(1H,d,J=4.0Hz), 4.21(1H,d,J=8.0Hz), 3.74-3.67(2H,m), 3.64(1H,dd,J=3.5,3.5Hz), 3.56-3.47(2H,m), 3.42-3.32(2H,m), 3.30(1H,dd,J=5.5,5.5Hz), 3.06-2.99(2H,m), 1.81(3H,s), 1.60(2H,tt,J=6.5,6.5Hz)

[0090] Example 1-3: Preparation of benzyl [3-({2-acetamido-6-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β-D-galactopyranosyl}oxy)propyl]carbamate (IVa)

[0091]

[0092] 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 sequentially to N,N-dimethylacetamide (324 mL) and stirred. Subsequently, 4,4'-dimethoxytrityl chloride (26.6 g, 78.6 mmol) was added, followed by washing with toluene (54 mL) twice, and stirring for 5 hours. After confirming the completion of the reaction, toluene (702 mL) was added, and the mixture was separated with 4% aqueous sodium bicarbonate (540 mL) to obtain a toluene layer. The obtained toluene layer was separated with 8% aqueous sodium bicarbonate (270 mL) to obtain an organic layer. Subsequently, the separation procedure with water (540 mL) was repeated twice. The obtained organic layer was concentrated under reduced pressure to obtain a toluene solution (270 mL) of the title compound (IVa).

[0093] The title compound (IVa) was synthesized (7.29 g, yield 84.1%) using benzyl {3-[(2-acetamido-2-deoxy-β-D-galactopyranosyl)oxy]propyl}carbamate (IIIa) (5.0 g, 12.1 mmol) in the same manner as in Example 1-3. 1 H-NMR(500MHz,CD3CN): δ7.48-7.46(2H,m), 7.39-7.29(11H,m), 7.24-7.21(1H,m), 7.11(1H,d,J=7.0Hz), 6.88-6.85(4H,m), 5.75(1H,dd,J=7.0,4.5Hz), 5.08(1H,d,J=12.5Hz), 5.02(1H,d,J=12.5Hz), 4.74(1H,br s), 4.12(1H,d,J=8.5Hz), 3.91(1H,ddd,J=9.0,4.5,4.5Hz), 3.76(6H,s),3.68(1H,d,J=2.5Hz), 3.63(1H,ddd,J=9.5,8.5,7.0Hz), 3.55(1H,dd,J=7.0,4.5Hz), 3.46-3.37(1H,m), 3.44(1H,dd,J=9.5,2.5Hz), 3.40(1H,ddd,J=9.0,4.0,4.0Hz), 3.27(1H,dd,J=9.0,7.0Hz), 3.11(1H,dd,J=9.0,4.5Hz), 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)

[0094] (Example 1-4) Preparation of 3-aminopropyl 2-acetamido-6-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β-D-galactopyranoside (Ib or 6)

[0095]

[0096] Methanol (270 mL), 5% palladium carbon powder PE type hydrous product (5.4 g dry weight equivalent, manufactured by NE Chemcat Corporation), and 25% aqueous ammonia solution (39.8 g, 655.0 mmol) were added to a toluene solution (270 mL) of benzyl [3-({2-acetamido-6-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β-D-galactopyranosyl}oxy)propyl]carbamate (IVa), and the mixture was stirred for 1 hour at 20 to 30°C using an autoclave. Subsequently, the atmosphere in the reaction system was purged with nitrogen three times, then purged with hydrogen at 0.4 MPa (gauge pressure) three times, and stirred under a hydrogen atmosphere of 0.3 MPa at 20 to 30°C for 4 hours. After confirming the completion of the reaction, the atmosphere in the reaction system was purged with nitrogen three times, and the palladium carbon was filtered off and washed with methanol (270 mL). The resulting filtrate was concentrated under reduced pressure, and water (27 mL) and toluene (540 mL) were added. The mixture was concentrated under reduced pressure to obtain a 540 mL concentrate. Toluene (540 mL) was added to the resulting concentrate, and the mixture was concentrated under reduced pressure to obtain a toluene solution (540 mL). This procedure was repeated three times. The precipitated solid was then 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 by liquid chromatography analysis 99.1%, total yield for two steps 90.1%). 1 H-NMR(500MHz,CD3OD): δ7.45(2H,d,J=7.5Hz), 7.35-7.31(4H,m), 7.27(2H,dd,J=7.5,7.5Hz), 7.19(1H,t,J=7.5Hz), 6.85(4H,d,J=8.0Hz), 4.32(1H,d,J=8.5Hz), 3.98(1H,ddd,J=11.5,6.0,5.0Hz), 3.91(1H,dd,J=10.5,8.5Hz), 3.84(1H,d,J=2.5Hz), 3.77(6H,s), 3.62(1H,ddd,J=6.0,5.0,11.5Hz), 3.57-3.55(1H,m), 3.55(1H,dd,J=10.5,2.5Hz), 3.40(1H,dd,J=9.5,7.0 Hz), 3.31-3.28(1H,m), 2.82(2H,t,J=6.0Hz), 1.98(3H,s), 1.79(2H,m)

[0097] Example 2 Preparation of ([12-({(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}oxy)-5,9,15,19-tetraoxo-10,14-dioxa-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) tetraacetate (XIIa or 3).

[0098] Example 2-1: Preparation of 1,1'-{[2-(benzyloxy)propane-1,3-diyl]bis(oxycarbonyloxy)}di(pyrrolidine-2,5-dione) (Va or 7)

[0099]

[0100] 2-(benzyloxy)propane-1,3-diol (10.0 g, 54.9 mmol) was added to acetonitrile (50 mL), and 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 mixture was stirred at 0°C for 5 hours. After confirming the completion of the reaction, 2-propanol (150 mL) was added to the reaction solution, seed crystals were added, and the mixture was stirred at 0°C for 1 hour. Subsequently, 2-propanol (100 mL) was added, and the 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 in the next step without drying. 1 H-NMR(500MHz,CD3CN): δ7.39-7.30(5H,m), 4.65(2H,s), 4.54(2H,dd,J=11.5,4.0Hz), 4.43(2H,dd,J=11.5,5.5Hz), 4.06(1H,tt,J=5.5,4.0Hz),2.77(8H,s)

[0101] Example 2-1-1: Preparation of 1,1'-{[2-(benzyloxy)propane-1,3-diyl]bis(oxycarbonyloxy)}di(pyrrolidine-2,5-dione) (Va or 7)

[0102]

[0103] 2-(benzyloxy)propane-1,3-diol (1.0 g, 5.49 mmol) was added to acetonitrile (5 mL), and N,N'-disuccinimidyl carbonate (3.2 g, 12.6 mmol) and pyridine (1.1 g, 13.7 mmol) were added, followed by stirring at 0°C for 4 hours. After confirming the completion 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 to the reaction solution, and the mixture was stirred at 0°C for 1 hour. Subsequently, 2-propanol (16 mL) was added, and the 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 by liquid chromatography analysis 99.7%, yield 54.9%, also used as seed crystals). 1 H-NMR(500MHz,CD3CN): δ7.39-7.30(5H,m), 4.65(2H,s), 4.54(2H,dd,J=11.5,4.0Hz), 4.43(2H,dd,J=11.5,5.5Hz), 4.06(1H,tt,J=5.5,4.0Hz), 2.77(8H,s)

[0104] (Example 2-2-1) Preparation of 8-(benzyloxy)-5,11-dioxo-6,10-dioxa-4,12-diazapentadecan-1,15-dioic acid-N-benzyl-1-phenylmethanamine (1 / 1) (VIa or 5)

[0105]

[0106] β-Alanine (14.7 g, 164.6 mmol), acetonitrile (50 mL), and triethylamine (17.2 g, 170.1 mmol) were added sequentially to water (50 mL) and stirred at 20-30°C. Subsequently, wet 1,1'-{[2-(benzyloxy)propane-1,3-diyl]bis(oxycarbonyloxy)}di(pyrrolidine-2,5-dione) (Va or 7) was added over 1.5 hours while controlling the internal temperature to 30°C or less, and the mixture was stirred at 20-30°C for 1 hour. After confirming the completion 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 an organic layer. 20% saline (100 mL) was added to the obtained organic layer and separation was carried out. The same separation procedure was repeated three more times to obtain an organic layer. The resulting organic layer was concentrated under reduced pressure to give a tetrahydrofuran solution (60 mL). Isopropyl acetate (200 mL) was added to the resulting tetrahydrofuran solution, which was then concentrated under reduced pressure to give a 60 mL solution. The same vacuum concentration procedure was repeated two more times to give an isopropyl acetate solution (60 mL). Salts in the resulting isopropyl acetate solution were removed by filtration and washed with 2-propanol (50 mL). 2-Propanol (300 mL) was added to the resulting solution, and the temperature of the resulting solution was adjusted to 35°C. Thereafter, dibenzylamine (11.4 g, 57.6 mmol) was added, seed crystals were inoculated, and the mixture was stirred at 35°C for 1 hour. After stirring at 25°C for 22 hours, the resulting crystals were filtered and washed with 2-propanol (50 mL) to give crude crystals of the wet title compound (VIa) (yield: 32.1 g). The crude crystals obtained were then added to 2-propanol (300 mL), and the temperature was adjusted to 60°C to obtain a 2-propanol solution. The resulting solution was stirred at 60°C for 1 hour, then slowly cooled from 60°C to 25°C over 1 hour, and seed crystals were added to confirm the precipitation of crystals. The slurry was subsequently stirred at 25°C for 18 hours, and the resulting 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 by liquid chromatography analysis 99.7%, total yield for two steps 83.3%).

[0107] 1H-NMR(500MHz,CD3OD): 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)

[0108] (Example 2-2-1-1) Production of diethyl 8-(benzyloxy)-5,11-dioxo-6,10-dioxa-4,12-diazapentadecane-1,15-dioate (Va')

[0109]

[0110] 1,1'-{[2-(benzyloxy)propane-1,3-diyl]bis(oxycarbonyloxy)}di(pyrrolidine-2,5-dione) (Va or 7) (20.0 g, 43.0 mmol), β-alanine ethyl ester hydrochloride (16.5 g, 107.4 mmol), and triethylamine (12.2 g, 120.6 mmol) were added sequentially to tetrahydrofuran (300 mL), and the mixture was stirred at 20 to 30°C for 1 hour. After confirming the completion of the reaction, 7% aqueous sodium bicarbonate (100 mL) was added to the reaction solution and the mixture was separated to obtain an organic layer. 20% saline (100 mL) was added to the obtained organic layer and the mixture was separated to obtain a tetrahydrofuran solution of the title compound (Va').

[0111] The title compound (Va') was obtained (1.58 g, yield 78.4%) using 1,1'-{[2-(benzyloxy)propane-1,3-diyl]bis(oxycarbonyloxy)}di(pyrrolidine-2,5-dione) (Va) (2.0 g, 4.3 mmol) in a similar manner to (Example 2-2-1-1). 1 H-NMR(500MHz,CDCL3): δ7.35-7.34(4H,d,J=4.5Hz), 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=12.5,5.5Hz), 2.53(4H,t,J=6.0Hz), 1.27(6H,t,J=6.5Hz)

[0112] (Example 2-2-1-2) Preparation of 8-(benzyloxy)-5,11-dioxo-6,10-dioxa-4,12-diazapentadecan-1,15-dioic acid-N-benzyl-1-phenylmethanamine (1 / 1) (VIa or 5)

[0113]

[0114] Water (100 mL) and 8N potassium hydroxide (21.6 mL, 172.0 mmol) were added sequentially to a tetrahydrofuran solution of diethyl 8-(benzyloxy)-5,11-dioxo-6,10-dioxa-4,12-diazapentadecane-1,15-dioate (Va'), and the mixture was stirred at 20-30°C for 3.5 hours. After confirming the completion of the reaction, salt (10 g) was added to the reaction solution, and the pH was adjusted to 0.9 using 35% hydrochloric acid (13.2 mL), followed by separation to obtain an organic layer. 20% brine (100 mL) was added to the obtained organic layer, and the layers were separated. 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 sequentially to the obtained tetrahydrofuran solution, and the layers were separated. The obtained organic layer was concentrated under reduced pressure to obtain an isopropyl acetate solution (100 mL). Acetonitrile (300 mL) was added to the resulting solution, and the temperature of the resulting solution was adjusted to 40 ° C. Thereafter, dibenzylamine (2.55 g, 12.9 mmol) was added, seed crystals were inoculated, and the mixture was stirred at 40 ° C. for 4 hours. Subsequently, dibenzylamine (2.55 g, 12.9 mmol) was added, and the mixture was stirred at 40 ° C. for 19 hours. Subsequently, dibenzylamine (3.83 g, 19.4 mmol) was added, and the mixture was stirred at 40 ° C. for 1 hour, and the temperature of the solution was adjusted to 25 ° C. Subsequently, after stirring at 25 ° C. for 2 hours, the obtained crystals were filtered and washed with 2-propanol (50 mL) to obtain crude crystals of the wet title compound (VIa or 5). Next, the obtained crude crystals of the wet title compound (VIa or 5) were added to acetonitrile (300 mL), and the mixture was stirred at 70 ° C. for 1 hour, and then slowly cooled from 70 ° C. to 25 ° C. over 2 hours, and the precipitation of crystals was confirmed. Subsequently, the slurry was stirred at 25°C for 16 hours, and 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 yield for two steps: 88.0%). 1H-NMR(500MHz,CD3OD): 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)

[0115] (Example 2-2-3) Preparation of seed crystals 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)

[0116]

[0117] β-Alanine (19.5 g, 218.5 mmol), acetonitrile (95 mL), and triethylamine (22.9 g, 225.8 mmol) were added sequentially to water (95 mL) and stirred at 20-30°C. Subsequently, 1,1'-{[2-(benzyloxy)propane-1,3-diyl]bis(oxycarbonyloxy)}di(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 less, and the mixture was stirred at 20-30°C for 31 hours. After confirming the completion of the reaction, tetrahydrofuran (570 mL) and sodium chloride (20 g) were added, and the pH was adjusted to 0.1 using 35% hydrochloric acid (37.9 g, 364.1 mmol), followed by separation to obtain an organic layer. 20% brine (125 mL) was added to the resulting organic layer, and the mixture was separated. The same separation operation was performed 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, and the organic layer was obtained after separation. The obtained organic layer was concentrated under reduced pressure to obtain 114 mL of solution. Isopropyl acetate (570 mL) was added to the obtained organic layer, and the solution was concentrated under reduced pressure to obtain 114 mL of solution. Isopropyl acetate (76 mL) was added to the obtained solution, followed by acetonitrile (570 mL), and the temperature of the obtained solution was adjusted to 40 ° C. Thereafter, dibenzylamine (8.6 g, 43.7 mmol) was added and stirred at 40 ° C. for 19 hours. Subsequently, dibenzylamine (6.5 g, 32.8 mmol) was added and stirred at 40 ° C. for 2 hours. Subsequently, after stirring at 25 ° C. for 4 hours, the obtained crystals were filtered and washed with acetonitrile (190 mL) to obtain seed crystals of the title compound (VIa or 5). (41.02 g, area ratio by liquid chromatography analysis 95.8%, yield 86.5%). 1 H-NMR(500MHz,CD3OD): 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)

[0118] (Example 2-3) Preparation of 2-(benzyloxy)propane-1,3-diyl bis({3-[(3-{[(2R,5R)-3-acetamido-6-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4,5-dihydroxyox-2-yl]oxy}propyl)amino]-3-oxopropyl}carbamate) (VIIa or 8)

[0119]

[0120] Water (200 mL), 8-(benzyloxy)-5,11-dioxo-6,10-dioxa-4,12-diazapentadecane-1,15-dioic acid-N-benzyl-1-phenylmethanamine (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 mixture was stirred for 1 hour at 20-30° C. Thereafter, the layers were separated, and the resulting aqueous layer was washed twice with dichloromethane (60 mL). To the resulting aqueous layer, 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-β-D-galactopyranoside (Ib) (37.9 g, 62.3 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15.7 g, 82.0 mmol) were added in that order, and the mixture was stirred at 20-30°C for 5 hours. Thereafter, 3-aminopropyl 2-acetamide-6-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β-D-galactopyranoside (Ib) (2.53 g, 4.16 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.67 g, 8.71 mmol) were added and stirred for 11 hours, and then 3-aminopropyl 2-acetamide-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 stirred for 7 hours, and then 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 stirred for 23 hours. After confirming the completion of the reaction, the mixture was washed with 20% brine (200 mL), and ethyl acetate (600 mL) was added. This was followed by two phases of separation using 8% aqueous sodium bicarbonate (400 mL). The resulting organic layer was concentrated under reduced pressure to give a 300 mL solution.Subsequently, tetrahydrofuran (300 mL) and ethyl acetate (300 mL) were added, and the mixture was concentrated under reduced pressure to a 300 mL solution. This operation was repeated twice. Tetrahydrofuran (1000 mL) was added to the resulting solution, and the mixture was concentrated under reduced pressure to a 200 mL solution. Tetrahydrofuran (1000 mL) was added to the resulting solution, and the mixture was concentrated under reduced pressure to a 244 mL solution. Tetrahydrofuran (56 mL) was added to the resulting solution, and an ethyl acetate / tetrahydrofuran solution (300 mL) of the title compound (VIIa or 8) was obtained.

[0121] The title compound (VIIa or 8) was synthesized using 8-(benzyloxy)-5,11-dioxo-6,10-dioxa-4,12-diazapentadecane-1,15-dioic acid-N-benzyl-1-phenylmethanamine (1 / 1) (VIa or 5) (211.2 mg, 0.4 mmol) in a similar manner to (Example 2-3), and isolated by silica gel column purification (186.4 mg, yield 96.7%). 1 H-NMR(500MHz,CD3OD): δ7.78(2H,t,J=5.5Hz), 7.68(2H,d,J=9.0Hz, 7.41(4H,d,J=7.5Hz), 7.32-7.25(19H,m), 7.21(2H,t,J=7.5Hz), 6.88(8H,d,J=8.5Hz), 4.66(2H,d,J=6.0Hz), 4.57(2H,s), 4.54(2H,d,J=4.0Hz), 4.26(2H,d,J=8.0Hz), 4.10(2H,dd,J=11.5,4.0Hz), 3.99(2H,dd,J=11.5,6.0Hz), 3.76-3.68(5H,m,), 3.73(12H,s), 3.63(2H,dd,J=3.5, 3.5Hz), 3.54(2H,t,J=6.5Hz), 3.47-3.40(4H,m), 3.19-3.10(8H,m), 3.06-2.99(4H,m), 2.23(4H,t,J=7.5Hz), 1.82(6H,s), 1.66-1.57(4H,m)

[0122] (Examples 2-4) Preparation 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)

[0123]

[0124] To an ethyl acetate / tetrahydrofuran solution (300 mL) of 2-(benzyloxy)propane-1,3-diyl bis({3-[(3-{[(2R,5R)-3-acetamido-6-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4,5-dihydroxyox-2-yl]oxy}propyl)amino]-3-oxopropyl}carbamate) (VIIa or 8), tetrahydrofuran (10 mL) and triethylamine (29.9 g, 295.0 mmol) were added and 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 mixture was stirred at 20-30°C for 8 hours. After confirming the completion of the reaction, ethyl acetate (1000 mL) and 8% aqueous sodium bicarbonate solution (500 mL) were added, and the mixture was stirred at 20-30°C for 22 hours, followed by separation of the layers. The resulting organic layer was washed with water (300 mL), a 5% aqueous solution of potassium dihydrogen phosphate (300 mL), an 8% aqueous solution of sodium bicarbonate (300 mL), and water (300 mL), in that order. The resulting organic layer was concentrated under reduced pressure to give an ethyl acetate solution (400 mL). N-methyl-2-pyrrolidone (200 mL) and ethyl acetate (400 mL) were added to the resulting ethyl acetate solution, and the mixture was concentrated under reduced pressure to give an ethyl acetate / N-methyl-2-pyrrolidone solution (600 mL). Subsequently, ethyl acetate (400 mL) was added to the resulting solution, and the mixture was concentrated under reduced pressure to give an ethyl acetate / N-methyl-2-pyrrolidone solution (600 mL). This procedure was repeated three times to give an ethyl acetate / N-methyl-2-pyrrolidone solution (600 mL) of the title compound (VIIIa). The title compound (VIIIa or 2) was synthesized using 2-(benzyloxy)propane-1,3-diyl bis({3-[(3-{[(2R,5R)-3-acetamido-6-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4,5-dihydroxyox-2-yl]oxy}propyl)amino]-3-oxopropyl}carbamate) (VIIa) (33.0 g, 21.46 mmol) in a similar manner to that in (Example 2-4), and isolated by silica gel column purification (38.20 g, yield 104.4%). 1H-NMR(500MHz,CD3OD): δ7.82(2H,d,J=9.5Hz), 7.75(2H,t,J=5.5Hz), 7.34-7.17(25H,m), 6.91-6.86(8H,m), 5.40(2H,d,J=3.5Hz), 4.99(2H,dd,J=11.5,3.5Hz), 4.57(2H,s),4.45(2H,d,J=8.5Hz), 4.10(2H,dd,J=11.5,4.0Hz), 4.08-4.05(2H,m), 3.99(2H,dd,J=11.5,5.5Hz), 7.62(2H,ddd,J=11.5,9.5,8.5Hz), 3.76-3.70(1H,m), 3.74(12H,s), 3.66(2H,ddd,J=10.0,6.5,6.5Hz), 3.37(2H,ddd,J=10.0,6.5,6.5Hz), 3.17-3.11(6H,m), 3.08-3.00(2H,m), 3.02-2.94(2H,m), 2.80(2H,t,J=8.5Hz), 2.22(4H,t,J=7.0Hz),1.89(6H,s), 1.86(6H,s), 1.77(6H,s), 1.56(4H,dddd,J=7.0,7.0,6.5,6.5Hz)

[0125] (Example 2-5) Preparation 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)

[0126]

[0127] To an ethyl acetate / N-methyl-2-pyrrolidone solution (600 mL) 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), 5% palladium-carbon powder (PE type hydrous product) (2.1 g, dry weight equivalent, manufactured by NE Chemcat Corporation) was added, and the system was purged with nitrogen three times using an autoclave, followed by purging with 0.1 MPa hydrogen three times. The mixture was then stirred under a 0.1 MPa hydrogen atmosphere at 20-30°C for 7 hours. After confirming the completion of the reaction, the reaction system was purged with nitrogen three times, the palladium carbon was filtered off, and the mixture was washed with ethyl acetate (200 mL). Ethyl acetate (600 mL) and 2.5% saline (600 mL) were added to the obtained solution, and the layers were separated. The obtained organic layer was separated with a mixed solution of 2.5% saline (600 mL) and N-methyl-2-pyrrolidone (200 mL), and this operation was repeated three times to obtain an organic layer. The obtained organic layer was separated with 2.5% saline (600 mL) three times. Thereafter, ethyl acetate (600 mL) was added, and the mixture was concentrated under reduced pressure to obtain an ethyl acetate solution (800 mL), and this operation was repeated twice. The resulting ethyl acetate solution (800 mL) was purified through a column (800 g of CHROMATOREX DIOL MB100-40 / 75, 16 L of ethyl acetate was passed through, followed by 24 L of a 3% methanol / ethyl acetate solution), and the collected fraction was concentrated under reduced pressure to obtain the title compound (IXa or 1) as an ethyl acetate solution (800 mL) (quantitative value by liquid chromatography analysis: 40.0 g, area ratio: 99.7%, total yield for the three steps: 75.4%). 1H-NMR(500MHz,CD3CN): δ7.40-7.38(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=3.5Hz), 5.01(2H,dd,J=11.5,3.5Hz), 4.45(2H,d,J=8.5Hz), 4.10-3.88(8H,m), 3.85-3.79(1H,m), 3.81(2H,ddd,J=10.5,5.5,5.0Hz), 3.76(12H,s), 3.46(2H,ddd,J=10.5,7.5,5.0Hz), 3.34-3.27(6H,m), 3.23(2H,dd,J=9.0,5.5Hz), 3.07(2H,ddd,J=11.5,11.5,5.5Hz), 2.90(2H,dd,J=9.0,8.5Hz), 2.34-2.25(4H,m), 1.91(6H,s), 1.88(6H,s), 1.86(6H,s), 1.70-1.59(4H,m)

[0128] Example 2-5-1 Examination of deprotection of benzyl group 1 Screening of reaction solvents was carried out in the reaction for obtaining compound (IXa or 1) from compound (VIIIa or 2).

[0129] Reactions were carried out under the same reaction conditions as in Examples 2-5, using 10% palladium carbon powder (AD type hydrous product) (manufactured by Kawaken Fine Chemicals Co., Ltd.) and the organic solvents listed in Table 2 as reaction solvents. The results are shown in Table 2. In the following reaction formula and table, compound (Xa) is an impurity resulting from the elimination of one DMTr group from compound (IXa or 1) due to excessive reduction, and compound (XIa) is an impurity derived from the eliminated protecting group (DMTr group).

[0130] HPLC analysis conditions Detection: 210 nm Column: Xbridge C18 (4.6 mm ID x 150 mm, 3.5 μm) Column temperature: 40°C Mobile phase: A: 10 mM phosphate buffer, B: acetonitrile Gradient conditions: B (conc%) 55 (0-15 min), 55-75 (15-30 min), 75-55 (30-30.01 min), 55 (30.01-40 min) Flow rate: 1.0 mL / min Injection volume: 5 μL

[0131]

[0132]

[0133] (Example 2-5-2) Study on deprotection of benzyl group 2

[0134] To facilitate the separation operation after the reaction and reduce the loss of the product to the aqueous layer, we investigated the use of a mixed solvent of ethyl acetate and various polar solvents. This process is a continuous process, and if the reaction could be carried out in a mixed solvent with ethyl acetate, the post-treatment solvent in the previous process, the process would be simpler. Therefore, when carrying out this reaction in a mixed solvent, we investigated the optimal mixed system of ethyl acetate and various polar solvents. Under the same reaction conditions as in Example 2-5, 5% palladium carbon powder (PE type hydrous product) (manufactured by NE Chemcat Corporation) was used. The results are shown in Table 3.

[0135]

[0136]

[0137] (Example 2-5-3) Study on deprotection of benzyl group 3

[0138] Under the same reaction conditions as in Example 2-5, 5% palladium carbon powder (PE type hydrous product) (manufactured by NE Chemcat Corporation) was used to investigate 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.

[0139]

[0140] HPLC analysis conditions Detection: 210 nm Column: Xbridge C18 (4.6 mm ID x 150 mm, 3.5 μm) Column temperature: 40°C Mobile phase: A: 10 mM ammonium acetate buffer, B: acetonitrile Gradient conditions: B (conc%) 5 (0-3 min), 5-95 (3-30 min), 95 (30-33 min), 95-5 (33-33.1 min), 5 (33.1-42 min) Flow rate: 1.0 mL / min Injection volume: 5 μL

[0141]

[0142] (Example 2-5-4) Study on deprotection of benzyl group 4

[0143] The catalyst amount was investigated using 5% palladium carbon powder (PE type hydrous product) (manufactured by NE Chemcat Corporation) under the same reaction conditions as in Example 2-5. The results are shown in Table 5.

[0144]

[0145] HPLC analysis conditions Detection: 210 nm Column: Xbridge C18 (4.6 mm ID x 150 mm, 3.5 μm) Column temperature: 40°C Mobile phase: A: 10 mM phosphate buffer, B: acetonitrile Gradient conditions: B (conc%) 55 (0-15 min), 55-75 (15-30 min), 75-55 (30-30.01 min), 55 (30.01-40 min) Flow rate: 1.0 mL / min Injection volume: 5 μL

[0146]

[0147] (Example 2-6) Preparation of ([12-({(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}oxy)-5,9,15,19-tetraoxo-10,14-dioxa-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) tetraacetate (XIIa or 3)

[0148]

[0149] An ethyl acetate solution (90 mL) 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) (600 mL, 30.0 g, 18.6 mmol, quantitatively determined by liquid chromatography) was concentrated under reduced pressure to give an ethyl acetate solution (90 mL). Ethyl acetate (900 mL) was added to the resulting solution, and the mixture was concentrated under reduced pressure to 90 mL. Dichloromethane (300 mL) and molecular sieves 4A (15.0 g) were added to the resulting solution, and the mixture was stirred at 20-30°C for 45 minutes. The mixture was then cooled to 0°C, and 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, followed by stirring at 2 to 4°C for 16 hours. After confirming the completion of the reaction, the reaction solution was passed through a pad of CHROMATOREX® PEI MB100-40 / 75 (60.0 g), and the pad was washed with dichloromethane (180 mL). The resulting solution was filtered and washed with dichloromethane (90 mL). The resulting solution was concentrated under reduced pressure to obtain a dichloromethane / ethyl acetate solution (180 mL). Ethyl acetate (600 mL) was added to this solution, and the process of concentrating under reduced pressure was repeated twice to obtain an ethyl acetate solution (300 mL). To the resulting ethyl acetate solution, tert-butyl methyl ether (270 mL) was added to obtain an ethyl acetate / tert-butyl methyl ether solution (570 mL) as solution A. After adding tert-butyl methyl ether (300 mL) and n-heptane (900 mL) to a new container and cooling to 4-5°C, solution A (570 mL) was poured into the container, which was then washed in with a 50% ethyl acetate / tert-butyl methyl ether solution (30 mL), and the mixture was stirred at 2-8°C for 2.5 hours.The obtained slurry was filtered, washed successively with a 25% tert-butyl methyl ether / n-heptane solution (180 mL) pre-cooled to 0°C and n-heptane (300 mL) pre-cooled to 0°C, and then vacuum dried at an external temperature of 40°C to obtain the title compound (XIIa or 3) as a powder (34.3 g, area ratio by liquid chromatography analysis 98.4%, yield 101.7%). 1 H-NMR(500MHz,CD3CN): δ7.39(4H,d,J=8.0Hz), 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=3.5Hz), 5.00(2H,dd,J=11.5Hz), 4.44(2H,d,J=8.5Hz), 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=7.0,6.5Hz), 3.46(2H,ddd,J=10.0,8.0,5.0Hz), 3.33-3.25(6H,m), 3.23(2H,dd,J=9.0,6.0Hz), 3.10-3.04(2H,m), 2.90(2H,dd,J=9.0,8.0Hz), 2.64(1H,dd,J=7.0,5.5Hz), 2.63(1H,dd,J=7.0,5.5Hz), 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=7.0Hz), 1.14(6H,d,J=6.5Hz)

[0150] Compound (XIIa or 3) can be converted into an N-acetyl-D-galactosamine ligand-oligonucleotide conjugate using the methods described in, for example, WO 2019 / 172286 and WO 2021 / 049504.

[0151] The present invention can provide a novel method for producing a biantennary N-acetyl-D-galactosamine ligand-oligonucleotide conjugate that is useful as a pharmaceutical.

Claims

1. Formula (1): 【Chemistry 1】 [In formula (1), Ac represents an acetyl group, and DMTr represents a 4,4'-dimethoxytrityl group.] A method for producing a compound represented by, Formula (2): 【Chemistry 2】 [In formula (2), Ac represents an acetyl group, DMTr represents a 4,4'-dimethoxytrityl group, and Bn represents a benzyl group.] A manufacturing method comprising the step of reacting a compound represented by with a palladium catalyst in a reaction solvent in the presence of a reducing agent.

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

3. The manufacturing method according to claim 1, 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 production method according to claim 1, 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 production method according to claim 1, 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. Formula (3): 【Transformation 3】 [In formula (3), Ac represents an acetyl group, and DMTr represents a 4,4'-dimethoxytrityl group.] A method for producing a compound represented by or a salt thereof, A manufacturing method comprising the step of reacting a compound represented by formula (1), obtained by the manufacturing method described in claim 1, with an amiditating reagent.

7. Formula (4): 【Chemistry 4】 [In formula (4), Ac represents an acetyl group, Z represents an oxygen atom or a sulfur atom, and the bond at the right end of the structural formula indicates a bond with an oligonucleotide.] A method for producing a conjugate comprising an oligonucleotide and a branched N-acetyl-D-galactosamine unit, wherein a branched N-acetyl-D-galactosamine unit is bound to the 5' or 3' end of the oligonucleotide, as shown above, A manufacturing method comprising the step of reacting a compound represented by formula (3), obtained by the manufacturing method described in claim 6, with an oligonucleotide.

8. A method for producing a compound represented by formula (2) as described in claim 1, Formula (5): 【Transformation 5】 [In formula (5), Bn represents a benzyl group.] The compound shown or its salt and Formula (6): 【Transformation 6】 [In formula (6), Ac represents an acetyl group, and DMTr represents a 4,4'-dimethoxytrityl group.] A step of bonding the compounds shown using a condensing agent, and A manufacturing method comprising the step of acetylating the hydroxyl group on the tetrahydropyran ring using an acetylation reagent.

9. A method for producing a compound represented by formula (5) or a salt thereof as described in claim 8, Formula (7): 【Transformation 7】 [In formula (7), Bn represents a benzyl group.] A step of reacting a compound represented by with β-alanine or a salt thereof, or The process includes a step of reacting with a β-alanine alkyl ester or a salt thereof. A manufacturing method that includes a step of hydrolysis if it includes a step of using a β-alanine alkyl ester or a salt thereof.

10. A method for producing the compound represented by formula (7) as described in claim 9, A manufacturing method comprising the step of reacting 2-(benzyloxy)propane-1,3-diol with N,N'-disuccinimidyl carbonate in the presence of a base.

11. Formula (2): 【Transformation 8】 [In formula (2), Ac represents an acetyl group, DMTr represents a 4,4'-dimethoxytrityl group, and Bn represents a benzyl group.] A compound represented by the formula.

12. Formula (8): 【Chemistry 9】 [In formula (8), Ac represents an acetyl group, DMTr represents a 4,4'-dimethoxytrityl group, and Bn represents a benzyl group.] A compound represented by the formula.

13. Formula (5): 【Chemistry 10】 [In formula (5), Bn represents a benzyl group.] A compound represented by or a salt thereof.

14. Formula (7): 【Chemistry 11】 [In formula (7), Bn represents a benzyl group.] A compound represented by the formula.

15. Formula (6): 【Chemistry 12】 [In formula (2), Ac represents an acetyl group, and DMTr represents a 4,4'-dimethoxytrityl group.] A compound represented by or a salt thereof.

16. A method for producing a bibranched N-acetyl-D-galactosamine ligand-oligonucleotide conjugate, wherein the N-acetyl-D-galactosamine unit is bonded to the 5' and / or 3' end of the oligonucleotide, and comprising the method according to any one of claims 1 to 6.

17. The method according to claim 16, wherein the bibranched N-acetyl-D-galactosamine ligand is capable of binding to asialoglycoprotein receptors (ASGPR) on hepatocytes of the liver.

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