Method for manufacturing polyethylene glycol compound having maleimide group at terminal

Abstract

The present invention comprises: a step (a) in which an amine protecting group represented by formula (2) is deprotected by trifluoroacetic acid to produce a trifluoroacetate salt of an amine represented by formula (3); a step (b) in which the trifluoroacetate salt of said amine is mixed with an organic solvent solution of hydrogen chloride to induce salt exchange and produce an amine hydrochloride represented by formula (4); and a step (c) in which a product that has a terminal activation rate of 95 mass% or more and contains a polyethylene glycol compound represented by formula (1) and containing a maleimide group at the terminal thereof is obtained by using a condensing agent to react a polyethylene glycol compound having a carboxy group represented by formula (5) with a trifluorohydrochloride salt of the amine.

Description

Method for producing polyethylene glycol compound having a maleimide group at the end

[0001] The present invention relates to a method for producing a polyethylene glycol compound having a maleimide group at its terminal.

[0002] In the pharmaceutical field, complexes formed by combining a protein active agent with a water-soluble composition have improved efficacy compared to when bioactive agents are injected into the body, due to increased half-life and evasion of the immune response system.

[0003] One example of a water-soluble composition is polyethylene glycol. Generally, the formation of a polyethylene glycol conjugate with a bioactive agent is achieved by a reaction between the active agent and polyethylene glycol. To achieve this reaction, an activated polyethylene glycol derivative is used, in which the end of the polyethylene glycol is substituted with a reactive functional group. Examples of reactive functional groups include those that chemically bond with functional groups present on the surface of the bioactive agent, such as amino groups, mercapto groups, carboxy groups, and unsaturated bonds. For example, when modifying an amino group, an active group such as a formyl group, epoxy group, p-nitrophenyl ester group, or N-hydroxysuccinimidyl group is attached to the end of the polyethylene glycol chain. For carboxy groups, an active group such as a mercapto group or amino group is attached to the end of the polyethylene glycol chain. For unsaturated bonds, an active group such as a mercapto group is attached to the end of the polyethylene glycol chain. For thiol groups, a maleimide group is used.

[0004] As an approach to producing a polyethylene glycol compound having a maleimide group, Patent Document 1 describes synthesis via polyethylene glycol having an electrophilic group at its terminal. According to the description in Patent Document 1, the production process involves reacting a polyethylene glycol compound having an electrophilic group containing a carboxyl group with a maleimide compound having a nucleophilic group containing a primary amino group to obtain a polyethylene glycol compound having a maleimide group.

[0005] Japanese Patent Publication No. 2006-517600 U.S. Patent No. 8,034,558

[0006] Although the scope of claims in Patent Document 1 includes polyethylene glycol compounds having maleimide groups, it has been found that the purity of the compounds cannot be increased by the method described in Patent Document 1.

[0007] Specifically, as shown in Comparative Example 1 of the present specification, the present inventors attempted to produce a polyethylene glycol compound having a maleimide group at its terminal by reacting a polyethylene glycol compound having an active ester at its terminal with an amine hydrochloride having a maleimide group in accordance with the method of Patent Document 1. However, as a result, it was confirmed that the terminal activation rate was less than 95% by mass.

[0008] Furthermore, the terminal activation rate of a polyethylene glycol compound having a maleimide group at its terminal depends not only on the reactivity of the polyethylene glycol compound with an amine hydrochloride having a maleimide group but also on the purity of the amine hydrochloride having a maleimide group, which is the raw material for maleimide conversion.

[0009] As described in Patent Document 2, amine hydrochlorides having a maleimide group are obtained by deprotecting a t-butoxycarbonyl-protected compound having a maleimide group using a 1,4-dioxane solution of hydrogen chloride. However, this method produces a by-product in which hydrogen chloride is added to the maleimide group (hereinafter abbreviated as "hydrogen chloride adduct"), reducing the purity of the amine hydrochloride having a terminal maleimide group. The present inventors actually reacted a low-purity amine hydrochloride having a maleimide group with a polyethylene glycol compound according to the description of Patent Document 2, as shown in Comparative Example 2 of the present specification, and confirmed that the terminal activation rate of the obtained product was less than 95% by mass.

[0010] The present invention has been made in view of the above problems, and aims to obtain a polyethylene glycol compound having a maleimide group with a high terminal activation rate by efficiently reacting a polyethylene glycol compound having a terminal carboxy group with a high purity amine hydrochloride having a maleimide group at its terminal.

[0011] That is, the present invention is as follows: [1] A method for producing a polyethylene glycol compound having a maleimide group at its terminal and represented by formula (1), comprising: a step (a) of deprotecting a protected amine compound represented by formula (2) with trifluoroacetic acid to obtain a trifluoroacetate salt of an amine represented by formula (3), a step (b) of mixing the trifluoroacetate salt of an amine with an organic solvent solution of hydrogen chloride to perform salt exchange to obtain an amine hydrochloride represented by formula (4), and a step (c) of reacting a polyethylene glycol compound having a carboxy group represented by formula (5) with the amine hydrochloride represented by formula (4) using a condensing agent to obtain a product containing the polyethylene glycol compound having a maleimide group at its terminal and represented by formula (1), wherein the terminal activation rate of the product is 95% by mass or more. (In formula (1), PEG is a polyethylene glycol moiety having a linear or branched structure, and L １ is a divalent linker that binds to the maleimide group. (In formula (2), L １ is a divalent linker that binds to the maleimide group, and R is a protecting group for the amino group. (In formula (3), L １ is a divalent linker that binds to the maleimide group. (In formula (4), L １ is a divalent linker that binds to the maleimide group. (In formula (5), PEG is a polyethylene glycol moiety having a linear or branched structure.)

[0012] [2] The method for producing a polyethylene glycol compound having a maleimide group at its terminal according to [1], wherein the polyethylene glycol compound having a maleimide group at its terminal has a molecular weight of 100 daltons or more and 100,000 daltons or less.

[0013] [3] The method for producing a polyethylene glycol compound having a terminal maleimide group according to [1] or [2], wherein the polyethylene glycol compound having a terminal maleimide group has one or more functional groups selected from the group consisting of a hydroxy group, an azide group, a biotin group, a methoxy group, an acetal-protected aldehyde group, an amino group protected with a 9-fluorenylmethyloxycarbonyl group, and an amino group protected with a t-butoxycarbonyl group.

[0014] [4] The method for producing a polyethylene glycol compound having a maleimide group at its terminal according to [3], characterized in that the protecting group R in formula (2) has a structure represented by formula (6), formula (7), formula (8), formula (9), formula (10), or formula (11).

[0015] [5] The linker L １ The method for producing a polyethylene glycol compound having a maleimide group at its terminal, according to [1] or [2], characterized in that the compound has a structure represented by formula (12). (In formula (12), X １ and X ２ are each independently a divalent hydrocarbon group, and Y is an amide bond.

[0016] [6] X １ and X ２ and each independently represent a divalent hydrocarbon group having 2 to 10 carbon atoms.

[0017] [7] The method for producing a polyethylene glycol compound having a maleimide group at its terminal according to [6], wherein the equivalent of the organic solvent solution of hydrogen chloride used in the step (b) is 1.5 equivalents or more and 12 equivalents or less relative to the amino group of the trifluoroacetate salt of the amine represented by formula (3).

[0018] The present invention can provide a method for producing a polyethylene glycol compound having a maleimide group with a high terminal activation rate, which has been difficult to achieve with conventional techniques, using a high-purity amine hydrochloride having a maleimide group.

[0019] [Polyethylene glycol compound having a maleimide group at the terminal of formula (1)] The following items describe the polyethylene glycol compound produced in the present invention.

[0020] The polyethylene glycol compound of the present invention has a structure of formula (1) and has a terminal activation rate of 95% by mass or more.

[0021] In formula (1), PEG is a polyethylene glycol moiety having a linear or branched structure, and L １ , a divalent linker attached to a maleimide group.

[0022] In a preferred embodiment, the polyethylene glycol compound having a maleimide group at its terminal of formula (1) has one or more functional groups selected from the group consisting of a hydroxy group, an azide group, a biotin group, a methoxy group, an acetal-protected aldehyde group, an amino group protected with a 9-fluorenylmethyloxycarbonyl group, and an amino group protected with a t-butoxycarbonyl group.

[0023] Specific examples of the structure of the polyethylene glycol compound include activated PEGs as shown in formulas (13) to (17). Of these, formulas (13) and (14) are polyethylene glycol compounds having a linear structure, and formulas (15) to (17) are polyethylene glycol compounds having a branched structure.

[0024]

[0025] Here, P １ is a hydrocarbon group having 1 to 7 carbon atoms or a hydrogen atom, and Q １is an atomic group that protects a functional group that reacts with a functional group present in a biofunctional molecule to form a covalent bond, n is an integer of 3 to 2300, m is an integer of 1 to 1200, and A １ , A ２ , A ３ , A ４ are each independently -K １ - (CH ２ ) ｍ１ -, - (CH ２ ) ｍ１ -K １ -, -K １ - (CH ２ ) ｍ１ -K ２ - (CH ２ ) m ２ represents - or a single bond, K １ represents an amide bond, a urethane bond, an ether bond, a secondary amino group, or a single bond; K ２ represents an amide bond, a urethane bond, or an ether bond, m １ and m ２ are each independently an integer of 0 to 5, １ is a carbon atom or a nitrogen atom.

[0026] Q １ is an atomic group that protects a functional group that reacts with a functional group present in a biofunctional molecule to form a covalent bond, and examples of such atomic groups include the following: amino group, formyl group, epoxy group, p-nitrophenyl ester group, and N-hydroxysuccinimidyl group.

[0027] Furthermore, L １ may have a linear structure, a branched structure, or a cyclic structure. １ may contain a hydrocarbon group and / or may have an amide bond. The hydrocarbon group may have no unsaturated bond or may have an unsaturated bond, but the number of unsaturated bonds is preferably 2 or less, more preferably 1 or less. In addition, the linker L １is preferably a substituent having a linear, branched, or cyclic structure containing a hydrocarbon group and an amide bond. The hydrocarbon group preferably has 2 to 20 carbon atoms, more preferably 4 to 16 carbon atoms.

[0028] In a preferred embodiment, L １ has the structure of formula (12).

[0029] Here, X １ and X ２ are each a divalent hydrocarbon group having 2 to 10 carbon atoms, and Y is an amide bond.

[0030] X １ and X ２ The hydrocarbon groups constituting the formula (I) may each have a linear structure, a branched structure, or a cyclic structure. Each hydrocarbon group may have no unsaturated bonds or may have unsaturated bonds, but the number of unsaturated bonds is preferably 2 or less, and more preferably 1 or less. Each hydrocarbon group has 2 to 10 carbon atoms, and more preferably 2 to 6 carbon atoms.

[0031] The molecular weight of the polyethylene glycol compound having a maleimide group at its terminal of formula (1) is preferably 100 to 100,000 daltons, more preferably 2,000 to 80,000 daltons, even more preferably 5,000 to 50,000 daltons, and particularly preferably 10,000 to 40,000 daltons.

[0032] (Step (a)) In this step, the protected amine represented by formula (2) is deprotected with trifluoroacetic acid to give the trifluoroacetate of the amine represented by formula (3).

[0033] (In formula (2), L １ is a divalent linker that binds to the maleimide group, and R is a protecting group for the amino group.

[0034] R in formula (2) is a protecting group for an amino group that can be deprotected with trifluoroacetic acid. Such a protecting group is not limited as long as it is a protecting group that reacts with trifluoroacetic acid, but protecting groups of formulas (6) to (11) are particularly preferred. Among these, the protecting group of formula (6) is the most preferred because only volatile isobutene and carbon dioxide are by-produced during deprotection, making post-treatment easy.

[0035]

[0036] As the protected amine having the maleimide shown in formula (2), for example, protected amines having structures such as those shown in formula (18), formula (19), and formula (20) are particularly preferred. The protected amine of formula (18) can be produced by reacting N-(tert-butoxycarbonyl)-1,2-diaminoethane with N-succinimidyl maleimidopropionate. The protected amine of formula (19) can be produced by reacting N-(tert-butoxycarbonyl)-5-methyl-1,5-diaminoethane with N-succinimidyl maleimidobutanate. The protected amine of formula (20) can be produced by reacting N-(tert-butoxycarbonyl)-1,2-diaminoethane with N-succinimidyl-4-(N-maleimidomethyl)cyclohexanecarboxylate.

[0037]

[0038] Next, the trifluoroacetate of the maleimide group-containing amine obtained in step (a) will be described.

[0039] The trifluoroacetate salt of an amine having a maleimide group has the structure of formula (3). Here, L １ is a divalent linker attached to a maleimide group, as previously described.

[0040] Here, trifluoroacetic acid is used as a method for deprotecting the amine protected compound. Although it is also possible to use hydrochloric acid, the hydrogen chloride contained in hydrochloric acid has a high nucleophilicity of chloride ions, which easily undergo an addition reaction with the maleimide group, resulting in an increase in the hydrogen chloride adduct of formula (21) described below.

[0041] For this reason, in the present invention, trifluoroacetic acid is used when deprotecting a protected amine having a maleimide group, because the trifluoroacetate anion is less nucleophilic than the chloride ion and therefore less likely to undergo an addition reaction with the maleimide group, resulting in a higher purity of the target product.

[0042] Since there is a concern that the amine may add to the maleimide group after step (a), it is preferable to convert the amine salt under acidic conditions. That is, step (a) is a step in which the counter ion of the amine is removed, and purification by, for example, chromatography is not preferable.

[0043] In the deprotection reaction of step (a), the ratio of trifluoroacetic acid to the protected amine having a maleimide group is preferably 2 to 5 times by mass, more preferably 2.2 to 3 times by mass.

[0044] The temperature for the deprotection reaction in step (a) is preferably 15 to 40° C., more preferably 20 to 35° C. The solvent for the deprotection reaction in step (a) is preferably a halogenated solvent such as dichloromethane or chloroform, or an ether solvent such as cyclopentyl methyl ether. During the deprotection in step (a), crystallization of the trifluoroacetate salt of the amine having a maleimide group may be carried out.

[0045] (Step (b)) In this step, the trifluoroacetate of the amine represented by formula (3) is mixed with a solution of hydrogen chloride in an organic solvent to carry out salt exchange, thereby obtaining the amine hydrochloride represented by formula (4). (In formula (4), L １ is a divalent linker that binds to the maleimide group.

[0046] The organic solvent solution of hydrogen chloride used in step (b) is a solvent in which hydrogen chloride gas is soluble and which is liquid under the temperature conditions of step (b), and is preferably a carboxylic acid ester, alcohol, or alkyl ether. Suitable examples of the organic solvents for such carboxylic acid esters, alcohols, and alkyl ethers include the following:

[0047] Carboxylic acid ester: The number of carbon atoms in the alkyl group of the structure derived from carboxylic acid is preferably 1 to 2. The number of carbon atoms in the alkyl group of the structure derived from alcohol is preferably 1 to 4, and more preferably 2 to 3.

[0048] Alcohol: A primary or secondary alcohol, the alkyl group of which preferably has 1 to 3 carbon atoms, more preferably 2 to 3 carbon atoms.

[0049] Alkyl ether: An ether between alkyl groups, an ether between an alkyl group and a cycloalkyl group, or one or two ether bonds, preferably having 4 to 5 carbon atoms.

[0050] The organic solvent for the organic solvent solution of hydrogen chloride used in step (b) is particularly preferably ethyl acetate, ethanol, isopropanol, 1,4-dioxane, or cyclopentyl methyl ether, and most preferably ethyl acetate or 1,4-dioxane.

[0051] When an aqueous solution of hydrochloric acid is used as the hydrogen chloride source in the salt exchange in step (b), the maleimide group-containing amine hydrochloride is highly water-soluble, making it difficult to isolate it as crystals from the aqueous solution. Even if isolation is possible, the number of steps required and the time spent exposed to the hydrogen chloride source are long, resulting in an increase in the amount of by-product impurities such as hydrogen chloride adducts.

[0052] The amount of the organic solvent solution of hydrogen chloride used in the salt exchange in step (b) is preferably reduced as much as possible because hydrogen chloride adds to the maleimide group. On the other hand, if the amount of the organic solvent solution of hydrogen chloride used is too small, the salt exchange from trifluoroacetic acid to hydrogen chloride does not proceed sufficiently. From this perspective, the amount of the organic solvent solution of hydrogen chloride used is preferably 1.5 equivalents or more and 12 equivalents or less, more preferably 1.5 equivalents and 8.2 equivalents or less, and even more preferably 2.0 equivalents or more and 4.0 equivalents or less, relative to the amino group of the target compound (trifluoroacetic acid salt of an amine having a maleimide group). The mass molar concentration of hydrogen chloride in the reaction solution is preferably 0.011 mol / kg or more and 3.2 mol / kg or less, more preferably 0.011 mol / kg or more and 2.19 mol / kg or less, and even more preferably 0.014 mol / kg or more and 1.1 mol / kg or less.

[0053] After step (b), the amine hydrochloride having a maleimide group is recovered from the solution by a process including any one of concentration, crystallization, drying, etc. Since the amine hydrochloride having a maleimide group has low solubility in organic solvents, if it precipitates during salt exchange, it can be recovered by filtration, etc. In this way, a highly pure amine hydrochloride having a maleimide group can be obtained without carrying out complicated processes such as ion exchange chromatography or column chromatography.

[0054] Here, as a side reaction of the reaction in step (b), the amine hydrochloride having a maleimide group contains an impurity shown in formula (21) (hereinafter referred to as a "hydrogen chloride adduct"). The higher the content of the hydrogen chloride adduct, the lower the purity of the amine hydrochloride having a maleimide group, which contributes to a decrease in the terminal activation rate of the polyethylene glycol compound having a maleimide group obtained using the same. For this reason, the content of the hydrogen chloride adduct in the amine hydrochloride having a maleimide group obtained in the present invention is preferably 0.001% by mass or more and 2.5% by mass or less, more preferably 0.001% by mass or more and 1.0% by mass or less, and even more preferably 0.001% by mass or more and 0.5% by mass or less.

[0055]

[0056] (Step (c)) In step (c), a polyethylene glycol compound having a carboxyl group of formula (5) and an amine hydrochloride having a maleimide group of formula (4) are reacted using a condensing agent to obtain a polyethylene glycol compound having a maleimide group at its terminal of formula (1).

[0057]

[0058]

[0059]

[0060] The polyethylene glycol compound having a carboxy group of formula (5) used as the raw material in step (c) has one or more carboxy groups. When a polyethylene glycol compound having one carboxy group is used as the raw material, a polyethylene glycol compound having a monovalent maleimide group is obtained, and when a polyethylene glycol compound having multiple carboxy groups is used as the raw material, a polyethylene glycol compound having divalent or higher maleimide groups is obtained.

[0061] In formula (5), PEG is a polyethylene glycol moiety having a linear or branched structure, and L １ is a divalent linker attached to the maleimide group.

[0062] In a preferred embodiment, the polyethylene glycol compound having a carboxy group of formula (5) has one or more functional groups selected from the group consisting of a hydroxy group, an azide group, a biotin group, a methoxy group, an acetal-protected aldehyde group, an amino group protected with a 9-fluorenylmethyloxycarbonyl group, and an amino group protected with a t-butoxycarbonyl group.

[0063] As the polyethylene glycol compound having a carboxy group of formula (5), polyethylene glycol compounds having a linear or branched structure as shown in formulas (22) to (26) are particularly preferred.

[0064]

[0065] Here, P １ is a hydrocarbon group having 1 to 7 carbon atoms or a hydrogen atom, and Q １ is an atomic group that protects a functional group that reacts with a functional group present in a biofunctional molecule to form a covalent bond, n is an integer of 3 to 2300, m is an integer of 1 to 1200, and A １ , A ２ , A ３ , A ４ are each independently -K １ - (CH ２ ) ｍ１ -, - (CH ２ ) ｍ１ -K １ -, -K １ - (CH ２ ) ｍ１ -K ２ - (CH ２ ) m ２ represents - or a single bond, K １ represents an amide bond, a urethane bond, an ether bond, a secondary amino group, or a single bond; K ２ represents an amide bond, a urethane bond, or an ether bond, m １ and m ２ are each independently an integer of 0 to 5, １ is a carbon atom or a nitrogen atom.

[0066] The molecular weight of the polyethylene glycol compound having a carboxy group of formula (5) is preferably 100 to 100,000 daltons, more preferably 2,000 to 80,000 daltons, even more preferably 5,000 to 50,000 daltons, and particularly preferably 10,000 to 40,000 daltons.

[0067] In step (c), a polyethylene glycol compound having a carboxyl group of formula (5) and an amine hydrochloride having a maleimide group of formula (4) are reacted in a solvent using a condensing agent.

[0068] Exemplary solvents used in step (c) include water, acetonitrile, DMSO, toluene, benzene, alcoholic solvents such as methanol and ethanol, hydrocarbon solvents such as heptane and hexane, and halogenated solvents such as chloroform and dichloromethane. From the viewpoint of the solubility of the polyethylene glycol having a carboxy group of formula (5), water, acetonitrile, DMSO, methanol, ethanol, toluene, chloroform, and dichloromethane are particularly preferred. The necessary conditions for the solvent in step (c) are conditions under which the polyethylene glycol compound and the amine hydrochloride having a maleimide group are partially or completely dissolved and the condensation reaction proceeds.

[0069] The condensing agent used in the present invention is a compound that promotes the condensation reaction between a polyethylene glycol compound having a carboxy group and an amine hydrochloride having a maleimide group. Suitable condensing agents include uronium-based condensing agents and triazine-based condensing agents. Uronium-based condensing agents are condensing agents consisting of a compound containing a uronium structure, and include HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate), HBTU (1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate), TATU (1-[bis(dimethylamino)methylene]- Preferred are 1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide tetrafluoroborate), TBTU (1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide tetrafluoroborate), and COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholinocarbenium hexafluorophosphate), with HATU and COMU being particularly preferred. The triazine-based condensing agent is a condensing agent made of a compound containing a triazine structure, and preferred are DMT-MM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) and (4,6-dimethoxy-1,3,5-triazin-2-yl)-(2-octoxy-2-oxoethyl)dimethylammonium trifluoromethanesulfonate, with DMT-MM being particularly preferred.

[0070] In this product, the condensation reaction can be selectively promoted by adding a base to a solution containing a polyethylene glycol compound having a carboxy group and an amine hydrochloride having a maleimide group. The base used in the reaction can be either an inorganic or organic base. However, the base must be selected based on its solubility in the solvent being used. Exemplary inorganic bases include sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, and disodium hydrogen phosphate. Among the amine compounds commonly used as organic bases, primary and secondary amines may react with the polyethylene glycol compound produced in this reaction, potentially producing impurities as by-products. Therefore, tertiary amines are more preferred. Exemplary tertiary amine bases include triethylamine, N-methylmorpholine, N-phenylmorpholine, N,N-diisopropylethylamine, pyridine, and 2,6-lutidine, with triethylamine and N,N-diisopropylethylamine being preferred.

[0071] The product obtained in step (c) is mainly composed of a polyethylene glycol compound having a terminal maleimide group, and the terminal activation rate is 95% by mass or more, preferably 96% by mass or more, and more preferably 97% by mass or more.

[0072] The terminal activation rate of the product obtained in step (c) is defined as follows: Terminal activation rate = (mass of the compound of formula (1) in the product / (mass of the compound of formula (1) in the product + impurity of the by-produced polyethylene glycol compound + mass of the compound of formula (5))).

[0073] Example 1 (Step (a)) Trifluoroacetic acid (6.0 g, manufactured by Kanto Chemical) was added to a solution of a maleimide-containing amine-protected compound (3.0 g, manufactured by NOF Corp., Compound (A)) in dichloromethane (6.0 g, manufactured by Kanto Chemical Co., Ltd.), and the mixture was stirred under nitrogen at 25°C for 3 hours. After stirring, it was confirmed by thin layer chromatography (hereinafter referred to as "TLC") that the remaining amount of the maleimide-containing amine-protected compound was 5% by mass or less. This was used as a trifluoroacetate (compound (B)) solution.

[0074]

[0075] (Step (b)) Ethyl acetate (Kanto Chemical, 45 g) was added to the obtained solution of compound (B), and the mixture was cooled to 10°C. A 4 M solution of hydrogen chloride in ethyl acetate (Fujifilm Wako Pure Chemical Industries, Ltd., 6.1 g) was added and stirred for 30 minutes. Here, the equivalent of the hydrogen chloride in ethyl acetate solution was 2.75 equivalents relative to the amino group of the maleimide-containing amine-protected compound. The precipitated crystals were filtered and washed with ethyl acetate (Kanto Chemical), yielding crystals of an amine hydrochloride salt having a maleimide group (compound (C)).

[0076]

[0077] The purity of the amine hydrochloride having a maleimide group (compound (C)) obtained in the above manner was 98.3% by mass, and the content of the hydrogen chloride adduct (compound (D)) was 0.03% by mass.

[0078]

[0079] [Method for Analyzing Amine Hydrochloride Having a Maleimide Group] The purity of the amine hydrochloride having a maleimide group is evaluated by High Performance Liquid Chromatography with charged aerosol detection (HPLC-CAD) under the following measurement conditions. Apparatus: Ultimate 3000 Column: Inersil ODS-3 HPLC Column (5 μm, 4.6 × 250 mm) (GL Sciences) Measurement temperature: 40°C Mobile phase: A) 0.05% trifluoroacetic acid aqueous solution, B) 0.05% trifluoroacetic acid methanol solution, A / B = 95 / 5 Flow rate: 1 mL / min Sample concentration: 1 mg / mL Injection volume: 20 μL The content of hydrogen chloride adduct can also be confirmed by Nuclear Magnetic Resonance spectroscopy (NMR). The measurement conditions are as follows: NMR apparatus: JNM-ECA600 (manufactured by JEOL) Measurement temperature: 25°C Measurement solvent: deuterated dimethyl sulfoxide (manufactured by Kanto Chemical Co., Ltd.) Sample concentration: 3 to 7 mg / mL Measurement type: 1 H Number of accumulations: 64 Data analysis: ALICE2 (manufactured by JEOL)

[0080] (Step (c)) A solution of compound (E) (Mw: 4,500, 500 mg), compound (C) (33 mg), N,N-diisopropylethylamine (Kanto Chemical, 22.7 μL), and DMT-MM (Tokyo Chemical Industry, 85 mg) in acetonitrile (Kanto Chemical, 2.5 g) was stirred for 2 hours at 40°C under nitrogen. The reaction solution was diluted with ethyl acetate (100 mL), and crystallized with hexane (100 mL) and filtered. The resulting solid was dried to obtain compound (F).

[0081]

[0082] The terminal activation rate of the polyethylene glycol compound having a maleimide group (compound (F)) obtained in the above manner was 96.7% by mass. The terminal activation rate was calculated as follows: (96.7 [mass of the compound of formula (1) in the product] / (96.7 [mass of the compound of formula (1) in the product] + 2.8 [by-produced polyethylene glycol compound impurity] + 0.5 [mass of the compound of formula (5)]).

[0083] [Method for Analyzing the Terminal Activation Rate of a Polyethylene Glycol Compound Having a Maleimide Group at its Terminal] The terminal activation rate is measured by derivatizing a polyethylene glycol compound having a maleimide group with mercaptopropionic acid and then performing ion exchange chromatography. The derivatization and measurement conditions are as follows. <Derivatization Conditions> 2 mL of an aqueous mercaptopropionic acid solution (concentration: 1 mg / mL) is added to a polyethylene glycol compound having a maleimide group (20 mg) and stirred at room temperature for 3 hours. The mixture is then fractionated using a PD-10 column (GE Healthcare) to obtain a sample for analysis. <Measurement conditions> HPLC apparatus: Alliance 689 Detector: Differential refractive index detector (RI) (manufactured by Waters) Column: Asahipak ES-502N (manufactured by Shodex) Measurement temperature: 30°C Mobile phase: Ammonium formate buffer solution (pH 8.0) Flow rate: 1.0 mL / min Sample concentration: 10 mg / mL Injection volume: 20 μL Data analysis: Empower software (manufactured by Waters)

[0084] Example 2 (Step (a)) Trifluoroacetic acid (manufactured by Kanto Chemical, 6.0 g) was added to a solution of a protected amine having a maleimide group (manufactured by NOF Corp., compound (A), 3.0 g) in dichloromethane (manufactured by Kanto Chemical, 6.0 g), and the mixture was stirred under nitrogen at 25° C. for 3 hours. After stirring, it was confirmed by TLC that the remaining amount of the protected amine having a maleimide group was 5% by mass or less. This was used as a trifluoroacetate (compound (B)) solution.

[0085] (Step (b)) Ethyl acetate (Kanto Chemical, 45 g) was added to the obtained solution of compound (B), and the mixture was cooled to 10°C. A 4 M solution of hydrogen chloride in ethyl acetate (Fujifilm Wako Pure Chemical Industries, Ltd., 6.1 g) was added and stirred for 30 minutes. Here, the equivalent of the hydrogen chloride in ethyl acetate solution was 2.75 equivalents relative to the amino group of the maleimide-containing amine-protected compound. The precipitated crystals were filtered and washed with ethyl acetate (Kanto Chemical), yielding white crystals of a maleimide-containing amine hydrochloride (compound (C)).

[0086] The purity of the amine hydrochloride having a maleimide group (compound (C)) obtained in the above manner was 98.3% by mass, and the content of the hydrogen chloride adduct (compound (D)) was 0.03% by mass.

[0087] (Step (c)) A solution of compound (G) (Mw: 2,000, 300 mg), compound (C) (89 mg), N,N-diisopropylethylamine (61.2 μL), and DMT-MM (288 mg) in acetonitrile (1.5 g) was stirred for 6 hours at 40° C. under nitrogen. The reaction solution was diluted with ethyl acetate (100 mL), and crystallized with hexane (100 mL) and filtered. The obtained solid was dried to obtain compound (H).

[0088]

[0089] The terminal activation rate of the polyethylene glycol compound having a maleimide group (compound (H)) obtained in the above manner was 97.5% by mass.

[0090] Example 3 (Step (a)) Trifluoroacetic acid (manufactured by Kanto Chemical, 6.0 g) was added to a solution of a protected amine having a maleimide group (manufactured by NOF Corp., compound (A), 3.0 g) in dichloromethane (manufactured by Kanto Chemical, 6.0 g), and the mixture was stirred under nitrogen at 25° C. for 3 hours. After stirring, it was confirmed by TLC that the remaining amount of the protected amine having a maleimide group was 5% by mass or less. This was used as a trifluoroacetate (compound (B)) solution.

[0091] (Step (b)) Ethyl acetate (Kanto Chemical, 45 g) was added to the obtained solution of compound (B), and the mixture was cooled to 10°C. A 4 M solution of hydrogen chloride in ethyl acetate (Fujifilm Wako Pure Chemical Industries, Ltd., 6.1 g) was added and stirred for 30 minutes. Here, the equivalent of the hydrogen chloride in ethyl acetate solution was 2.75 equivalents relative to the amino group of the maleimide-containing amine-protected compound. The precipitated crystals were filtered and washed with ethyl acetate (Kanto Chemical), yielding crystals of an amine hydrochloride salt having a maleimide group (compound (C)).

[0092] The purity of the amine hydrochloride having a maleimide group (compound (C)) obtained in the above manner was 98.3% by mass, and the content of the hydrogen chloride adduct (compound (D)) was 0.03% by mass.

[0093] (Step (c)) A solution of compound (I) (Mw: 5,000, 500 mg), compound (C) (30 mg), N,N-diisopropylethylamine (20.4 μL), and DMT-MM (76 mg) in acetonitrile (2.5 g) was stirred for 6 hours at 40°C under nitrogen. After the reaction, the solution was diluted with ethyl acetate (100 mL), and crystallized from hexane (100 mL) and filtered. The resulting solid was dried to obtain compound (J).

[0094]

[0095] The terminal activation rate of the polyethylene glycol compound having a maleimide group (compound (J)) obtained in the above manner was 96.1% by mass.

[0096] Example 4 (Step (a)) Trifluoroacetic acid (manufactured by Kanto Chemical, 6.0 g) was added to a solution of a protected amine having a maleimide group (manufactured by NOF Corp., compound (A), 3.0 g) in dichloromethane (manufactured by Kanto Chemical, 6.0 g), and the mixture was stirred under nitrogen at 25° C. for 3 hours. After stirring, it was confirmed by TLC that the remaining amount of the protected amine having a maleimide group was 5% by mass or less. This was used as a trifluoroacetate (compound (B)) solution.

[0097] (Step (b)) Ethyl acetate (Kanto Chemical, 45 g) was added to the obtained solution of compound (B), and the mixture was cooled to 10°C. A 4 M solution of hydrogen chloride in ethyl acetate (Fujifilm Wako Pure Chemical Industries, Ltd., 6.1 g) was added and stirred for 30 minutes. Here, the equivalent of the hydrogen chloride in ethyl acetate solution was 2.75 equivalents relative to the amino group of the maleimide-containing amine-protected compound. The precipitated crystals were filtered and washed with ethyl acetate (Kanto Chemical), yielding crystals of an amine hydrochloride salt having a maleimide group (compound (C)).

[0098] The purity of the amine hydrochloride having a maleimide group (compound (C)) obtained in the above manner was 98.3% by mass, and the content of the hydrogen chloride adduct (compound (D)) was 0.03% by mass.

[0099] (Step (c)) A solution of compound (K) (Mw: 10,000, 500 mg), compound (C) (62 mg), N,N-diisopropylethylamine (42.5 μL), and DMT-MM (152 mg) in acetonitrile (3.5 g) was stirred for 2 hours at 40°C under nitrogen. The reaction solution was diluted with ethyl acetate (100 mL), and crystallized from hexane (100 mL) and filtered. The resulting solid was dried to obtain compound (L).

[0100]

[0101] The terminal activation rate of the polyethylene glycol compound having a maleimide group (compound (L)) obtained in the above manner was 95.1% by mass.

[0102] Example 5 (Step (a)) Trifluoroacetic acid (Kanto Chemical, 2.0 g) was added to a solution of a protected amine having a maleimide group (compound (A), 1.0 g, purity: 100% by mass) in dichloromethane (Kanto Chemical, 5.0 g), and the mixture was stirred under nitrogen at 25°C for 3 hours. After stirring, it was confirmed by TLC that the remaining amount of the protected amine having a maleimide group was 5% by mass or less. The resulting reaction solution was added dropwise to dichloromethane (300 g) cooled to 5°C to crystallize, and the crystals of the trifluoroacetate salt of the amine (compound (B)) were collected by filtration.

[0103] (Step (b)) Ethyl acetate (Kanto Chemical, 451 mg) was added to the obtained crystals of compound (B) (50 mg), and a 4 M solution of hydrogen chloride in ethyl acetate (Fujifilm Wako Pure Chemical Industries, Ltd., 341 mg) was added at 25°C, followed by stirring for 30 minutes. Here, the equivalent of the hydrogen chloride solution in ethyl acetate was 8.2 equivalents relative to the amino group of the amine-protected compound having a maleimide group. The precipitated crystals were filtered off and washed with ethyl acetate (Kanto Chemical), yielding crystals of an amine hydrochloride salt having a maleimide group (compound (C)).

[0104] The purity of the amine hydrochloride having a maleimide group (compound (C)) obtained in the above manner was 98.5% by mass, and the content of the hydrogen chloride adduct (compound (D)) was 1.3% by mass.

[0105] (Step (c)) A solution of compound (M) (Mw: 40,000, 1.5 g), compound (C) (11 mg), N,N-diisopropylethylamine (7.65 μL), and DMT-MM (26.7 mg) in acetonitrile (7.5 g) was stirred for 4 hours at 40°C under nitrogen. The reaction solution was diluted with ethyl acetate (100 mL), and crystallized from hexane (100 mL) and filtered. The resulting solid was dried to obtain compound (N).

[0106]

[0107] The terminal activation rate of the polyethylene glycol compound having a maleimide group (compound (N)) obtained in the above manner was 97.0% by mass.

[0108] Example 6 (Step (a)) Trifluoroacetic acid (manufactured by Kanto Chemical, 6.0 g) was added to a solution of a protected amine having a maleimide group (manufactured by NOF Corp., compound (A), 3.0 g) in dichloromethane (manufactured by Kanto Chemical, 6.0 g), and the mixture was stirred under nitrogen at 25° C. for 3 hours. After stirring, it was confirmed by TLC that the remaining amount of the protected amine having a maleimide group was 5% by mass or less. This was used as a trifluoroacetate (compound (B)) solution.

[0109] (Step (b)) Ethyl acetate (Kanto Chemical, 45 g) was added to the obtained solution of compound (B), and the mixture was cooled to 10°C. A 4 M solution of hydrogen chloride in ethyl acetate (Fujifilm Wako Pure Chemical Industries, Ltd., 6.1 g) was added and stirred for 30 minutes. Here, the equivalent of the hydrogen chloride in ethyl acetate solution was 2.75 equivalents relative to the amino group of the maleimide-containing amine-protected compound. The precipitated crystals were filtered and washed with ethyl acetate (Kanto Chemical), yielding crystals of an amine hydrochloride salt having a maleimide group (compound (C)).

[0110] The purity of the amine hydrochloride having a maleimide group (compound (C)) obtained in the above manner was 98.3% by mass, and the content of the hydrogen chloride adduct (compound (D)) was 0.03% by mass.

[0111] (Step (c)) A solution of compound (M) (Mw: 40,000, 1.5 g), compound (C) (11 mg), triethylamine (6.3 μL), and DMT-MM (29 mg) in acetonitrile (7.5 g) was stirred for 6 hours at 40°C under nitrogen. The reaction solution was diluted with ethyl acetate (100 mL), and crystallized from hexane (100 mL) and filtered. The resulting solid was dried to obtain compound (N).

[0112] The terminal activation rate of the polyethylene glycol compound having a maleimide group (compound (N)) obtained in the above manner was 97.1% by mass.

[0113] Comparative Example 1 (Step (a)) Trifluoroacetic acid (manufactured by Kanto Chemical, 6.0 g) was added to a solution of a protected amine having a maleimide group (manufactured by NOF Corp., compound (A), 3.0 g) in dichloromethane (manufactured by Kanto Chemical, 6.0 g), and the mixture was stirred under nitrogen at 25° C. for 3 hours. After stirring, it was confirmed by TLC that the remaining amount of the protected amine having a maleimide group was 5% by mass or less. This was used as a trifluoroacetate (compound (B)) solution.

[0114] (Step (b)) Ethyl acetate (Kanto Chemical, 45 g) was added to the obtained solution of compound (B), and the mixture was cooled to 10°C. A 4 M solution of hydrogen chloride in ethyl acetate (Fujifilm Wako Pure Chemical Industries, Ltd., 6.1 g) was added and stirred for 30 minutes. Here, the equivalent of the hydrogen chloride in ethyl acetate solution was 2.75 equivalents relative to the amino group of the maleimide-containing amine-protected compound. The precipitated crystals were filtered and washed with ethyl acetate (Kanto Chemical), yielding crystals of an amine hydrochloride salt having a maleimide group (compound (C)).

[0115] The purity of the amine hydrochloride having a maleimide group (compound (C)) obtained in the above manner was 98.3% by mass, and the content of the hydrogen chloride adduct (compound (D)) was 0.03% by mass.

[0116] A solution of compound (M) (Mw: 40,000, 1.5 g), N-hydroxysuccinimide (Midori Chemical, 13 mg), and N,N'-dicyclohexylcarbodiimide (Tama Chemical, 16 mg) in acetonitrile (7.5 g) was stirred for 3 hours under nitrogen at 40°C to obtain compound (O). Then, triethylamine (10.5 μL) and compound (C) (11 mg) were added to the reaction solution, and the mixture was stirred for 3 hours under nitrogen at 40°C. The reaction solution was diluted with ethyl acetate (100 mL), crystallized with hexane (100 mL), and filtered. The resulting solid was dried to obtain compound (N).

[0117]

[0118] The terminal activation rate of the polyethylene glycol compound having a maleimide group (compound (N)) obtained in the above manner was confirmed to be 92.6% by mass, which was lower than 95% by mass.

[0119] Comparative Example 2 A 4 M solution of hydrogen chloride in 1,4-dioxane (Tokyo Chemical Industry Co., Ltd., 313 mg) was added to 50 mg of a maleimide-containing protected amine (compound (A), purity: 100% by mass) in isopropanol (Kanto Chemical, 1.0 g), and the mixture was stirred at 25°C under nitrogen for 3 hours. Here, the equivalent of the 1,4-dioxane solution of hydrogen chloride was 8.2 equivalents relative to the amino group of the maleimide-containing protected amine (compound (A)). When the remaining amount of the maleimide-containing protected amine remained by TLC, it was found to be 40% by mass.

[0120] Comparative Example 3 A 4 M solution of hydrogen chloride in 1,4-dioxane (7.7 g, manufactured by Tokyo Chemical Industry Co., Ltd.) was added to 500 mg of a maleimide-containing protected amine (compound (A), purity: 88.7% by mass) in isopropanol (5.0 g), and the mixture was stirred at 25°C under nitrogen for 3 hours. The equivalent of the hydrogen chloride solution in 1,4-dioxane was adjusted to 20 equivalents relative to the amino group of the maleimide-containing protected amine (compound (A)). TLC confirmed that the remaining amount of the maleimide-containing protected amine was 5% by mass or less. The mixture was diluted with isopropanol (25.0 g), and hexane (25.5 g) was added. The precipitated crystals were filtered off to obtain a maleimide-containing amine hydrochloride (compound (C)).

[0121] The purity of the amine hydrochloride having a maleimide group (compound (C)) obtained in the above manner was 83.3% by mass, and the content of the hydrogen chloride adduct (compound (D)) was 16.7% by mass.

[0122] A solution of compound (M) (Mw: 40,000, 1.5 g), compound (C) (11 mg), N,N-diisopropylethylamine (7.65 μL), and DMT-MM (22.8 mg) in acetonitrile (7.5 g) was stirred for 4 hours at 40°C under nitrogen. The reaction solution was diluted with ethyl acetate (100 mL), and crystallized from hexane (100 mL) and filtered. The resulting solid was dried to obtain compound (N).

[0123] The terminal activation rate of the polyethylene glycol compound having a maleimide group (compound (N)) obtained in the above manner was confirmed to be 75.1% by mass, which was lower than 95% by mass.

[0124] In Examples 1 to 6, the high-purity amine hydrochloride having a maleimide group obtained through steps (a) and (b) was reacted with various polyethylene glycol compounds having carboxylic acid at the terminal using a condensing agent, and as a result, polyethylene glycol compounds having a maleimide group with a terminal activation rate of 95% by mass or more were obtained.

[0125] In Comparative Example 1, as in Patent Document 1, a polyethylene glycol compound having a terminal carboxy group was converted to an active ester at the terminal using a condensing agent and N-hydroxysuccinimide, and then reacted with a high-purity amine hydrochloride having a maleimide group obtained by carrying out steps (a) and (b) in the presence of a base, but the terminal activation rate of the polyethylene glycol compound having a terminal maleimide group was less than 95% by mass. Therefore, it was found that carrying out step (c) is preferable to the method described in Patent Document 1.

[0126] In Comparative Examples 2 and 3, in the synthesis of amine hydrochloride having a maleimide group, a protected amine having a maleimide group was deprotected using an organic solvent solution of hydrogen chloride. However, in Comparative Example 2, deprotection did not proceed sufficiently, and in Comparative Example 3, 16.7 mass% of a hydrogen chloride adduct was by-produced. In Comparative Example 3, step (c) was carried out using amine hydrochloride having a maleimide group containing 16.7 mass% of a hydrogen chloride adduct, but the terminal activation rate of the obtained polyethylene glycol compound having a maleimide group at its terminal was less than 95 mass%.

[0127] Therefore, it was found that it is preferable to carry out step (c) using the amine hydrochloride having a maleimide group obtained by carrying out steps (a) and (b).

[0128] The production method of the present invention can be used as a method for producing polyethylene glycol compounds having a maleimide group, for which high purity products are required.

[0129] In the present invention, a polyethylene glycol compound having a terminal carboxy group can be efficiently reacted with a highly pure amine hydrochloride having a terminal maleimide group, thereby obtaining a polyethylene glycol compound having a maleimide group with a high terminal activation rate.

[0130] Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a Japanese patent application (Patent Application No. 2024-128933) filed on August 5, 2024, the contents of which are incorporated herein by reference.

Claims

1. A method for producing a polyethylene glycol compound having a terminal maleimide group as shown in formula (1), comprising: a step (a) of deprotecting a protected amine compound as shown in formula (2) with trifluoroacetic acid to obtain the trifluoroacetate salt of the amine as shown in formula (3); a step (b) of mixing the trifluoroacetate salt of the amine with an organic solvent solution of hydrogen chloride to perform salt exchange to obtain the amine hydrochloride as shown in formula (4); and a step (c) of reacting a polyethylene glycol compound having a carboxy group as shown in formula (5) with the amine hydrochloride as shown in formula (4) using a condensing agent to obtain a product containing the polyethylene glycol compound having a terminal maleimide group as shown in formula (1), wherein the terminal activation rate of the product is 95% by mass or more. (In formula (1), PEG is a polyethylene glycol moiety having a linear or branched structure, and L １ is a divalent linker that binds to the maleimide group. (In formula (2), L １ is a divalent linker that binds to the maleimide group, and R is a protecting group for the amino group. (In formula (3), L １ is a divalent linker that binds to the maleimide group. (In formula (4), L １ is a divalent linker that binds to the maleimide group. (In formula (5), PEG is a polyethylene glycol moiety having a linear or branched structure.) 2. A method for producing a polyethylene glycol compound having a maleimide group at its end according to claim 1, characterized in that the polyethylene glycol compound having a maleimide group at its end has a molecular weight of 100 daltons or more and 100,000 daltons or less.

3. A method for producing a polyethylene glycol compound having a terminal maleimide group according to claim 1 or 2, characterized in that the polyethylene glycol compound having a terminal maleimide group has one or more functional groups selected from the group consisting of a hydroxy group, an azide group, a biotin group, a methoxy group, an aldehyde group protected with an acetal, an amino group protected with a 9-fluorenylmethyloxycarbonyl group, and an amino group protected with a t-butoxycarbonyl group.

4. A method for producing a polyethylene glycol compound having a maleimide group at its terminal according to claim 3, characterized in that the protecting group R in formula (2) has a structure represented by formula (6), formula (7), formula (8), formula (9), formula (10) or formula (11).

5. The linker L １ 3. The method for producing a polyethylene glycol compound having a maleimide group at its terminal according to claim 1 or 2, wherein the compound has a structure represented by formula (12): (In formula (12), X １ and X ２ are each independently a divalent hydrocarbon group, and Y is an amide bond.

6. X １ and X ２ and each independently represent a divalent hydrocarbon group having 2 to 10 carbon atoms, 7. The method for producing a polyethylene glycol compound having a maleimide group at its terminal according to claim 6, wherein the amount of hydrogen chloride in the organic solvent solution used in step (b) is 1.5 equivalents or more and 12 equivalents or less relative to the amino group of the trifluoroacetate salt of the amine represented by formula (3).

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