Method for producing polyethylene glycol compound

Abstract

To provide an industrially feasible method for producing an activated polyethylene glycol compound capable of purifying an activated polyethylene glycol compound efficiently and with good purity.SOLUTION: In producing an activated polyethylene glycol compound having a carbamate bond between a functional group reactive with a bio-related substance or a precursor of the functional group and a polyethylene glycol moiety, the following steps (1), (2), (3) are carried out. Step (1): a polyethylene glycol raw material having a nitrophenyl group at the terminal is reacted with an amine compound having a functional group reactive with a bio-related substance or a precursor of a functional group in a solution containing a non-protonic solvent to obtain a reaction mixture containing an activated polyethylene glycol compound and 4-nitrophenol. Step (2): a basic aqueous solution having a pH of 10 or more and 14 or less is added to the reaction mixture obtained in the Step (1) to obtain a two-phase mixture. Step (3): an organic layer of the two-phase mixture is separated from the water layer to recover the activated polyethylene glycol compound from the organic layer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing a polyethylene glycol compound suitable for medical use. [Background technology]

[0002] When administered into the body, pharmaceuticals that use bio-related substances such as hormones, cytokines, and enzymes are usually rapidly excreted from the body through glomerular filtration in the kidneys or uptake by macrophages in the liver, spleen, etc. Therefore, the half-life of the bio-related substances in the blood is short, and it is often difficult to obtain a sufficient pharmacological effect.

[0003] Activated polyethylene glycol is widely used as a means to solve this problem. Activated polyethylene glycol is a polyethylene glycol whose end is replaced with a reactive functional group in order to react with biological substances. The biological substances bound with activated polyethylene glycol can extend the half-life in blood of the biological substances by increasing the molecular weight and forming a hydration layer. It is also known that modifying biological substances with activated polyethylene glycol (PEGylation) can reduce the toxicity and antigenicity of the biological substances and improve their coagulation properties.

[0004] Between the polyethylene glycol moiety and the terminal functional group of the activated polyethylene glycol, a linker not derived from the polyethylene glycol moiety is generally included. The chain length, steric hindrance and bond of this linker may affect the stability of the activated polyethylene glycol compound itself and the stability of the modified product after modification to a biologically relevant substance. For example, ester bonds and carbonate bonds are susceptible to hydrolysis. Therefore, if these bonds are used as linkers, the linker part is gradually hydrolyzed by the water in the aqueous solution or the system, and the activated polyethylene glycol compound or its modified product is cleaved, which is not preferable. On the other hand, activated polyethylene glycol compounds using a carbamate bond as a linker are not easily affected by hydrolysis. Therefore, activated polyethylene glycol compounds having a carbamate bond are known as highly stable PEGylation reagents and are also used in Cimzia (registered trademark), a drug for treating rheumatoid arthritis.

[0005] Such an activated polyethylene glycol compound having a carbamate bond can be obtained by reacting a polyethylene glycol compound having an active carbonate group such as 4-nitrophenyl carbonate with an amine compound, or by reacting a polyethylene glycol compound having an amino group with an active carbonate compound. These reactions produce 4-nitrophenol as a by-product along with the target product. If even a small amount of 4-nitrophenol remains in the activated polyethylene glycol compound, the activated polyethylene glycol compound, which is originally white to off-white, may turn yellow in appearance. For this reason, it is desirable to reduce the amount of 4-nitrophenol.

[0006] In general, when removing low molecular weight compounds as impurities from an amphiphilic polymer compound such as polyethylene glycol, purification methods such as adsorption and crystallization can be used.

[0007] In Patent Document 1, a white powder is obtained by using a solvent in an amount 200 times or more by volume of an activated polyethylene glycol compound obtained by reacting a polyethylene glycol compound having an active carbonate group with an amine compound.

[0008] In Patent Document 2, polyethylene glycol having an amino group is reacted with 4-nitrophenyl carbonate, and 4-nitrophenol produced as a by-product as an impurity is purified by being adsorbed onto a resin to obtain a pale yellow solid. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] JP 2008-545371 A [Patent Document 2] JP 2020-7562 A Summary of the Invention [Problem to be solved by the invention]

[0010] However, when the production method of Patent Document 1 is used for large-scale production, the amount of solvent used is large, and the amount that can be produced at one time is limited due to equipment restrictions, making it unsuitable for industrial production.

[0011] Moreover, the product obtained by the manufacturing method of Patent Document 2 is light yellow. Even a trace amount of 4-nitrophenol will color the product, so the method described in Patent Document 2 cannot remove 4-nitrophenol to a level where it does not color the product. On the other hand, it is possible to remove 4-nitrophenol to a level where it does not color the product by repeating the adsorption. However, in this case, the activated PEG is also adsorbed together with 4-nitrophenol, resulting in a low yield. In addition, a large amount of adsorbent is ultimately produced as waste, which is problematic from an industrial standpoint.

[0012] In view of the above problems, an object of the present invention is to provide an industrially practicable method for producing an activated polyethylene glycol compound, which can purify an activated polyethylene glycol compound efficiently and with high purity. [Means for solving the problem]

[0013] The present invention provides the following. [1] A method for producing an activated polyethylene glycol compound having a carbamate bond between a functional group capable of reacting with a biologically relevant substance or a precursor of the functional group and a polyethylene glycol moiety, comprising the steps of: A method for producing an activated polyethylene glycol compound, comprising the following steps (1), (2) and (3): Process (1): A step of reacting a polyethylene glycol raw material having a nitrophenyl group at its end with an amine compound having the functional group or the precursor of the functional group capable of reacting with the biologically relevant substance in a solution containing an aprotic solvent to obtain a reaction mixture containing the activated polyethylene glycol compound and 4-nitrophenol. Process (2): A step of obtaining a two-phase mixture by adding a basic aqueous solution having a pH of 10 or more and 14 or less to the reaction mixture obtained in the step (1). Process (3): separating the organic layer of the two-phase mixture from the aqueous layer and recovering the activated polyethylene glycol compound from the organic layer.

[0014] [2] The method for producing an activated polyethylene glycol compound according to [1], characterized in that the activated polyethylene glycol compound has the carbamate bond between the precursor and the polyethylene glycol moiety, and the precursor is a protecting group for the functional group.

[0015] [3] The method for producing an activated polyethylene glycol compound according to [2], characterized in that a deprotection step of deprotecting the protecting group is carried out after the step (1) and before the step (2).

[0016] [4] The method for producing an activated polyethylene glycol compound according to [3], characterized in that in the deprotection step, a deprotection reagent comprising an organic base having an amino group is added to the reaction mixture containing the activated polyethylene glycol compound and 4-nitrophenol.

[0017] [5] The method for producing an activated polyethylene glycol compound according to any one of [1] to [4], wherein the activated polyethylene glycol compound is represented by the following formula (1): [ka] (In formula (1), PEG is the polyethylene glycol moiety; X is the functional group capable of reacting with the biologically relevant substance or the precursor of the functional group, A is a divalent linker that connects the functional group or the precursor to the carbamate group. Effect of the Invention

[0018] As a result of intensive research to solve the above problems, the present inventors have focused on a method of separating an activated polyethylene glycol compound and 4-nitrophenol by extraction to efficiently produce a polyethylene glycol compound with high purity, and have specifically studied the method. In general, in extraction, it is necessary that the solubility of the target substance and impurities in water or an organic solvent is significantly different. Amphiphilic polyethylene glycol has an extremely rare physical property that it can be selectively distributed into either the organic layer or the aqueous layer by using appropriate conditions in a two-phase extraction. Therefore, when the impurity is a water-soluble compound, purification is possible by distributing the polyethylene glycol into the organic layer and the impurity into the aqueous layer. However, although the water-octanol coefficient of 4-nitrophenol is 1.91, which is somewhat hydrophobic, its solubility in water is 1.6 g / 100 mL, and it is soluble in both the organic layer and the aqueous layer. Therefore, even if polyethylene glycol is distributed into the aqueous layer and 4-nitrophenol is extracted with an organic solvent, it was thought that efficient purification would be difficult.

[0019] However, the present inventors have discovered a method for producing an activated polyethylene glycol compound by adding a basic aqueous solution having a pH of 10 to 14 to a reaction mixture containing an activated polyethylene glycol compound and 4-nitrophenol to form a two-phase mixture and separating the mixture into an organic layer containing the activated polyethylene glycol compound and an aqueous layer containing 4-nitrophenol. This invention is characterized in that the reaction mixture is washed and distributed with a basic aqueous solution having a pH of 10 to 14, and the method is suitable for industrial use since it does not require a large amount of adsorbent and can be carried out in large-scale production.

[0020] Therefore, the production method of the present invention is epoch-making in that it can be easily carried out industrially, has excellent productivity, does not produce waste materials such as adsorbents, and is capable of recovering a specific activated polyethylene glycol compound having a carbamate bond in high yield. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] [Activated polyethylene glycol compounds] The activated polyethylene glycol compound produced by the production method of the present invention is an activated polyethylene glycol compound having a carbamate bond between a functional group capable of reacting with a biologically relevant substance or a precursor of the functional group and the polyethylene glycol moiety. The polyethylene glycol moiety may have a linear or branched structure.

[0022] In a preferred embodiment, the activated polyethylene glycol compound has the following formula (1): [ka]

[0023] In formula (1), PEG is a polyethylene glycol moiety having a linear or branched structure, X is a functional group capable of reacting with a biologically relevant substance or a precursor of the functional group, and A is a divalent linker that bonds the carbamate group and X.

[0024] The molecular weight of the polyethylene glycol moiety in the activated polyethylene glycol compound is preferably 1,000 to 100,000 daltons, more preferably 1,000 to 80,000 daltons, and even more preferably 1,000 to 60,000 daltons.

[0025] In the activated polyethylene glycol compound, the polyethylene glycol moiety having a branched structure is a polyethylene glycol chain which is branched into two or more chains, and may have a plurality of branching points in the polyethylene glycol chain. A preferred example is a polyethylene glycol chain which is branched into two or more chains, with a polyhydric alcohol such as glycerin as a branching point, as shown in the following formula (2).

[0026] [ka]

[0027] In formula (2), the polyethylene glycol moiety (CH2CH2O) n The molecular weight of the polypeptide is preferably 1,000 to 100,000 daltons, more preferably 1,000 to 80,000 daltons, and further preferably 1,000 to 60,000 daltons.

[0028] The term "functional group capable of reacting with a biologically relevant substance or a precursor of said functional group" refers to a functional group or a precursor thereof that reacts with a functional group present in a biologically relevant substance, including a nucleic acid compound including a protein drug, a polypeptide, an enzyme, an antibody, an antibody drug, a gene, an oligonucleic acid, etc., a nucleic acid drug, an anticancer drug, a low molecular weight drug, etc., to form a covalent bond. Specific examples of this functional group or the precursor of said functional group include an amino group, an amino group protected with a phthalimidyl group, a thiol group, a formyl group, a formyl group protected with an acetal, a carboxyl group, a maleimidyl group, a substituted maleimidyl group, a p-nitrophenyl ester group, an N-hydroxysuccinimidyl ester group, an azide group, an alkynyl group, etc., and preferably an amino group, an amino group protected with a phthalimidyl group, a formyl group, a formyl group protected with an acetal, an azide group, or an alkynyl group.

[0029] The divalent linker that connects the carbamate group to the functional group or the precursor X is preferably an alkylene group having 1 to 10 carbon atoms or -(CH2CH2O) n -R-, where n is an integer, preferably 1 to 4000, more preferably 1 to 3000, and further preferably 1 to 2000. R is an alkylene group having 1 to 10 carbon atoms.

[0030] [Method of producing activated polyethylene glycol compound] The present invention relates to a method for producing an activated polyethylene glycol compound, comprising the following steps (1), (2) and (3). Each step will be described below.

[0031] [Regarding step (1)] In step (1), a polyethylene glycol raw material having a terminal nitrophenyl group is reacted with an amine compound having the functional group or the precursor of the functional group capable of reacting with the biologically relevant substance in a solution containing an aprotic solvent to obtain a reaction mixture containing the activated polyethylene glycol compound and 4-nitrophenol.

[0032] The starting polyethylene glycol having a terminal nitrophenyl group has the following formula (3): [ka] In formula (3), PEG is a polyethylene glycol moiety having a linear or branched structure.

[0033] The polyethylene glycol moiety having a branched structure is a polyethylene glycol chain that is branched into two or more chains, and may have multiple branching points. A suitable example is a polyethylene glycol chain that is branched into two or more chains, with a polyhydric alcohol such as glycerin as the branching point, as shown in the following formula (4). [ka]

[0034] The polyethylene glycol portion of the polyethylene glycol raw material is monodisperse polyethylene glycol or polydisperse polyethylene glycol. As described in Japanese Patent No. 6,638,970, monodisperse polyethylene glycol is a polyethylene glycol characterized by a polyethylene glycol purity of 90% or more and an impurity content of 2% or less, and is a polyethylene glycol of a single molecular weight without molecular weight distribution. Polydisperse polyethylene glycol is a polymer of ethylene glycol, and is a polyethylene glycol having a molecular weight distribution compared to monodisperse polyethylene glycol. The polydisperse polyethylene glycol in the present invention is a polymer having a molecular weight distribution of preferably 1.2 or less, more preferably 1.1 or less, and most preferably 1.03 or less. The polyethylene glycol in the present invention refers to a compound synthesized by a polymerization reaction of ethylene glycol or ethylene oxide.

[0035] The molecular weight of the polyethylene glycol moiety of the polyethylene glycol raw material is preferably 1,000 to 100,000 daltons, more preferably 1,000 to 80,000 daltons, and even more preferably 1,000 to 60,000 daltons.

[0036] The term "aprotic solvent" refers to a solvent that does not have proton-donating properties, and examples thereof include hydrocarbon solvents such as toluene, benzene, hexane, and heptane, and chlorine-containing hydrocarbon solvents such as chloroform and dichloromethane. From the viewpoint of the solubility of polyethylene glycol and the phase separation with water, toluene, chloroform, and dichloromethane are particularly preferred. The amount of the aprotic solvent is 1 to 50 times by weight, preferably 1 to 20 times by weight, and more preferably 1 to 10 times by weight, the weight of the polyethylene glycol raw material.

[0037] The "amine compound having a functional group capable of reacting with a biologically relevant substance or a precursor of the functional group" refers to a compound having a functional group capable of reacting with a biologically relevant substance or a precursor of the functional group at one end and an amino group at the other end, and is preferably represented by the following formula (5). [ka]

[0038] In formula (5), the amino group (NH2) is converted to hydrochloride (NH3 + Cl - ), in which case an amine reagent such as triethylamine is added to carry out the reaction. X is a functional group capable of reacting with a biologically relevant substance or a precursor of the functional group. A is a divalent linker that bonds the amino group and X.

[0039] The term "functional group capable of reacting with a biologically relevant substance or a precursor of said functional group" refers to a functional group or a precursor thereof that reacts with a functional group present in a biologically relevant substance, including a protein drug, a polypeptide, an enzyme, an antibody, an antibody drug, a gene, a nucleic acid compound including an oligonucleic acid, etc., a nucleic acid drug, an anticancer drug, a low molecular weight drug, etc., to form a covalent bond. Specifically, the functional group is an amino group, an amino group protected with a phthalimidyl group, a thiol group, a formyl group, a formyl group protected with an acetal, a carboxyl group, a maleimidyl group, a substituted maleimidyl group, a p-nitrophenyl ester group, an N-hydroxysuccinimidyl ester group, an azide group, an alkynyl group, etc., and is preferably an amino group, an amino group protected with a phthalimidyl group, a formyl group, a formyl group protected with an acetal, an azide group, or an alkynyl group.

[0040] The divalent linker A that bonds the amino group to the functional group or the precursor X is preferably an alkylene group having 1 to 10 carbon atoms or -(CH2CH2O) n -R-, where n is an integer, preferably 1 to 4000, more preferably 1 to 3000, and further preferably 1 to 2000. R is an alkylene group having 1 to 10 carbon atoms.

[0041] (Deprotection step) When the precursor is a protecting group for the functional group, a deprotection step may be included between step (1) and step (2). The deprotection step is a step of deprotecting the protecting group of the activated polyethylene glycol compound using a deprotection reagent.

[0042] The type of the protecting group and the type of the deprotecting reagent are not particularly limited and can be selected from various types depending on the type of chemically reactive functional group to be protected, the conditions used, and the presence of other functional groups or protecting groups in the molecule, and specific examples can be found in many general textbooks (Greene's Protective Groups in Organic Synthesis Fifth edition, Peter GM Wuts). For example, examples of the protecting group for an amino group include a tert-butoxycarbonyl group, a benzyloxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, an allyloxycarbonyl group, a phthalimidyl group, a p-toluenesulfonyl group, and a 2-nitrobenzenesulfonyl group, and the phthalimidyl group is preferred. The "deprotection reagent" is a reagent that deprotects a protecting group and converts it into a functional group that reacts with a biologically relevant substance. The "deprotection reagent" is, for example, an organic base having an amino group. The organic base having an amino group is not particularly limited as long as it is a low molecular weight amine compound having a primary amino group, but is preferably a polyvalent amine compound having two or more primary amino groups. Specific examples include ethylenediamine, trimethylenediamine, diethylenetriamine, tris(2-aminoethyl)amine, triethylenetetramine, etc., and particularly preferably ethylenediamine.

[0043] [Regarding step (2)] The term "basic aqueous solution having a pH of 10 or more and 14 or less" refers to an aqueous solution containing a basic reagent and having a pH of 10 or more and a pH of 14 or less. Examples of the basic reagent include sodium hydroxide, potassium hydroxide, primary amines, secondary amines, and tertiary amines, preferably primary amines, secondary amines, and tertiary amines, more preferably compounds having a pKa of 7 to 11 and having 1 to 2 nitrogen atoms, further preferably methylamine, ethylamine, ethylenediamine, dimethylamine, diethylamine, ethylpropylamine, triethylamine, and N,N-diisopropylethylamine, and most preferably ethylenediamine and diethylamine. The amount of the basic reagent added to prepare a basic aqueous solution having a pH of 10 or more and 14 or less is preferably 0.1 to 30 equivalents, more preferably 0.3 to 20 equivalents, and particularly preferably 0.5 to 10 equivalents relative to the activated polyethylene glycol compound. When a deprotection step is included between step (1) and step (2), and the basic reagent is used in excess of the amount of the deprotection reagent consumed in the deprotection reaction, the amount of the basic reagent added in step (2) may be reduced according to the excess amount. This is because the reaction mixture after the deprotection step contains basic reagent that was not consumed in the deprotection reaction.

[0044] A two-phase mixture of an organic phase mainly composed of an aprotic solvent and an aqueous phase can be obtained by adding a basic aqueous solution having a pH of 10 or more and 14 or less to the reaction mixture obtained in step (1). The weight ratio of the organic phase mainly composed of an aprotic solvent to the aqueous phase is preferably 1:0.001 to 1:50, more preferably 1:0.005 to 1:30, and particularly preferably 1:0.01 to 1:10. The aprotic solvent may be added to the reaction mixture obtained in step (1) to achieve the above weight ratio. The combination of the organic phase mainly composed of an aprotic solvent and the aqueous phase is not particularly limited, but is preferably a combination in which the organic phase mainly composed of an aprotic solvent is toluene, chloroform or dichloromethane, and the aqueous phase is ethylenediamine, diethylamine or saline containing sodium hydroxide.

[0045] [Regarding step (3)] In step (3), the organic layer of the two-phase mixture is separated from the aqueous layer. During this process, the organic layer containing the activated polyethylene glycol compound, 4-nitrophenol, and the aprotic solvent carried over from step (1) is stirred with the aqueous layer containing the basic aqueous solution having a pH of 10 to 14 added in step (2), and 4-nitrophenol is transferred to the aqueous layer, followed by layer separation and removal of the aqueous layer. The time required for stirring or layer separation is not particularly limited, but is preferably 1 minute to 6 hours, and more preferably 10 minutes to 3 hours. The temperature during stirring and layer separation is not particularly limited, but is preferably 10 to 80° C. Step (3) of separating the organic layer from the aqueous layer can be carried out repeatedly to improve purification efficiency, and therefore may be carried out not only once but multiple times. When carrying out step (3), it is preferable to further include a water-washing step for removing the base component in the basic aqueous solution contained in the organic layer. The "water-washing step" referred to here is a step of adding 5 to 25% saline to the organic layer from which the aqueous layer has been removed, stirring and separating the layers, and removing the 5 to 25% saline. This water-washing step may be carried out not only once, but multiple times, preferably once or twice.

[0046] "Recovering the activated polyethylene glycol compound from the organic layer" means recovering the activated polyethylene glycol compound as a solid or liquid from the organic layer. Specifically, after separating the organic layer from the aqueous layer, the organic layer contains water, which is removed by distillation through a concentration operation. After removing the water, if salt precipitation is observed, filtration is performed. When the aprotic solvent used in the organic layer is an aprotic solvent in which the activated polyethylene glycol compound is highly soluble, such as chloroform or dichloromethane, the aprotic solvent is distilled off by a concentration operation.

[0047] When the obtained activated polyethylene glycol compound is in a liquid state, the solvent is entirely removed by a concentration operation to recover the liquid activated polyethylene glycol compound. On the other hand, when the obtained activated polyethylene glycol compound is a solid and the aprotic solvent is not distilled off from the organic layer, crystallization is carried out to obtain the solid activated polyethylene glycol compound. When the obtained activated polyethylene glycol compound is a solid and the aprotic solvent such as chloroform or dichloromethane is distilled off from the organic layer, toluene or ethyl acetate is added to the organic layer from which the aprotic solvent has been distilled off, and then crystallization is carried out to obtain the solid activated polyethylene glycol compound. Crystallization means adding the organic layer to a poor solvent for the polyethylene glycol compound, or adding the poor solvent to the organic layer. Examples of poor solvents for activated polyethylene glycol compounds include diethyl ether, methyl tert-butyl ether (MTBE), hexane, and heptane. EXAMPLES

[0048] Example 1 In a 50 mL three-neck round-bottom flask equipped with a Dimroth condenser, a thermometer, and a nitrogen inlet tube, a polyethylene glycol raw material having a terminal nitrophenyl group represented by formula (6) (Mw: 2,000, 2.0 g, 1.0 mmol) and toluene (8.0 g) were charged and dissolved at about 50° C. using a water bath while stirring under nitrogen. Then, N-aminoethoxyphthalimide hydrochloride (1.0 g) and triethylamine (0.64 g) were added and reacted at about 50° C. for 4 hours to obtain a reaction mixture (step (1)).

[0049] Next, insoluble N-aminoethoxyphthalimide hydrochloride was removed by filtration. At this time, the filter was washed with toluene (7.4 g). Ethylenediamine monohydrate (0.47 g) was added to the filtrate and reacted at about 50° C. for 2 hours to deprotect the phthalimide group (deprotection step).

[0050] Next, toluene (2.6 g) and 25 wt % saline (5.3 g) previously adjusted to pH 10 by adding ethylenediamine (0.07 g) were added to the reaction mixture after the deprotection step to obtain a two-phase mixture (step (2)).

[0051] The two-phase mixture was stirred at about 75°C, then separated, and the aqueous layer of the two-phase mixture was removed. 25% by weight saline (5.3g) to which ethylenediamine (0.07g) had been added in advance to adjust the pH to 10 was added to the remaining organic layer to obtain a two-phase mixture, and the mixture was stirred and separated at about 75°C, and the organic layer of the two-phase mixture was separated from the aqueous layer. This operation was repeated four times. 25% by weight saline (5.3g) was added to the separated organic layer, and the mixture was stirred and separated at about 75°C, and the 25% by weight saline was removed to separate the organic layer (water washing process). The separated organic layer was then concentrated under reduced pressure to remove water, and toluene (6.0g) was added to the organic layer after the water had been removed, and filtration was performed. The filtrate was added to hexane (12 g) and crystallized at 10° C., then suction filtered to obtain crystals. Toluene (12 g) was added to the obtained crystals, the crystals were dissolved at 40° C., and the crystals were added to hexane (12 g) and crystallized at 10° C. The procedure was repeated, and the crystals were dried under reduced pressure to obtain crystals of the activated polyethylene glycol compound represented by formula (7) (step (3)).

[0052] The crystals of the activated polyethylene glycol compound were analyzed using an ultraviolet-visible spectrophotometer (hereinafter referred to as a "UV-Vis spectrophotometer"), and the 4-nitrophenol content was confirmed to be 11 ppm.

[0053] [ka]

[0054] [ka]

[0055] Example 2 In a 50 mL three-neck round-bottom flask equipped with an L-shaped tube, a thermometer, and a nitrogen inlet tube, the polyethylene glycol raw material having a terminal nitrophenyl group represented by the above formula (6) (Mw: 2,000, 2.0 g, 1.0 mmol) and dichloromethane (20 g) were charged and dissolved by stirring under nitrogen. Then, N-aminoethoxyphthalimide hydrochloride (1.3 g) and triethylamine (0.64 g) were added and reacted at room temperature for 5 hours to obtain a reaction mixture (step (1)).

[0056] Next, insoluble N-aminoethoxyphthalimide hydrochloride was removed by filtration. Ethylenediamine monohydrate (0.47 g) was added to the filtrate and reacted at room temperature for 2 hours to deprotect the phthalimide group (deprotection step).

[0057] To the resulting reaction mixture, 10 wt% saline solution containing sodium hydroxide (pH 11, 8.0 g) was added to obtain a two-phase mixture (step (2)). Specifically, the 10 wt% saline solution containing sodium hydroxide (pH 11) was prepared as follows. 3 g of table salt and 27 g of distilled water were mixed to obtain 10% saline solution. Next, 0.09 g of 400 g / L sodium hydroxide solution was added to the 10% saline solution to obtain 10 wt% saline solution containing sodium hydroxide (pH 11).

[0058] The two-phase mixture was stirred at about 25°C, then separated, and the aqueous layer of the two-phase mixture was removed. 10% by weight of sodium hydroxide-containing saline (pH 11, 8.0 g) was added to the remaining organic layer to obtain a two-phase mixture, and the mixture was stirred at about 25°C, separated, and the organic layer of the two-phase mixture was separated from the aqueous layer. This operation was repeated four times. 10% by weight of saline (8.0 g) was added to the separated organic layer, then stirred at about 25°C, separated, and the 10% by weight of saline was removed to separate the organic layer (water washing process). The separated organic layer was then concentrated under reduced pressure to remove water and dichloromethane. Toluene (12 g) was then added to the organic layer after the water and dichloromethane were removed, and the organic layer was dissolved at about 40°C and filtered. The filtrate was added to hexane (12 g) and crystallized at 10°C, and the crystals were obtained by suction filtration. The obtained crystals were dissolved in toluene (12 g) at 40° C., and then added to hexane (12 g) and crystallized at 10° C. This procedure was repeated, and the crystals were then dried under reduced pressure to obtain crystals of the activated polyethylene glycol compound represented by formula (7) (step (3)).

[0059] The crystals of the activated polyethylene glycol compound were analyzed using a UV-Vis spectrophotometer, and the 4-nitrophenol content was confirmed to be 413 ppm.

[0060] Example 3 In a 50 mL three-neck round-bottom flask equipped with an L-shaped tube, a thermometer, and a nitrogen inlet tube, the polyethylene glycol raw material having a terminal nitrophenyl group represented by the above formula (6) (Mw: 2,000, 2.0 g, 1.0 mmol) and chloroform (18 g) were charged and dissolved by stirring under nitrogen. Then, N-aminoethoxyphthalimide hydrochloride (1.3 g) and triethylamine (0.64 g) were added and reacted at room temperature for 5 hours to obtain a reaction mixture (step (1)).

[0061] Next, insoluble N-aminoethoxyphthalimide hydrochloride was removed by filtration. Ethylenediamine monohydrate (0.47 g) was added to the filtrate and reacted at room temperature for 2 hours to deprotect the phthalimide group (deprotection step).

[0062] Next, to the reaction mixture was added 25 wt % saline (5.3 g) previously adjusted to pH 12 by adding diethylamine (0.07 g), to obtain a two-phase mixture (step (2)).

[0063] The two-phase mixture was stirred at about 25°C, then separated, and the aqueous layer of the two-phase mixture was removed. 25% by weight saline (5.3g) that had been adjusted to pH 12 by adding diethylamine (0.07g) to the remaining organic layer was added to obtain a two-phase mixture, and the two-phase mixture was stirred and separated at about 25°C, and the organic layer of the two-phase mixture was separated from the aqueous layer. This operation was repeated four times. 25% by weight saline (5.3g) was added to the separated organic layer, then stirred and separated at about 25°C, and the 25% by weight saline was removed to separate the organic layer (water washing step). The separated organic layer was then concentrated under reduced pressure to remove water and chloroform. Toluene (12g) was then added to the organic layer after the water and chloroform were removed, and the solution was dissolved at about 40°C and filtered. The filtrate was added to hexane (12g) and crystallized at 10°C, and the crystals were obtained by suction filtration. The obtained crystals were dissolved in toluene (12 g) at 40° C., and then added to hexane (12 g) and crystallized at 10° C. This procedure was repeated, and the crystals were then dried under reduced pressure to obtain crystals of the activated polyethylene glycol compound represented by the above formula (7) (step (3)).

[0064] The crystals of the activated polyethylene glycol compound were analyzed using a UV-Vis spectrophotometer, and the 4-nitrophenol content was confirmed to be 209 ppm.

[0065] Example 4 A branched polyethylene glycol raw material having a terminal nitrophenyl group represented by the following formula (8) (Mw: 60,000, 2.0 g, 0.033 mmol) and toluene (8.0 g) were charged into a 50 mL three-neck round-bottom flask equipped with a Dimroth condenser, a thermometer, and a nitrogen inlet tube, and dissolved in a water bath at about 50° C. while stirring under nitrogen. Then, 3-aminopropionaldehyde diethyl acetal (14.7 mg) was added and reacted at about 50° C. for 2 hours to obtain a reaction mixture (step (1)).

[0066] Next, toluene (10.0 g) and 25 wt % saline (5.3 g) previously adjusted to pH 12 by adding ethylenediamine (0.08 g) were added to the reaction mixture obtained in step (1) to obtain a two-phase mixture (step (2)).

[0067] The two-phase mixture was stirred at about 75°C, then separated, and the aqueous layer of the two-phase mixture was removed. 25% by weight saline (5.3 g) to which ethylenediamine (0.08 g) had been added in advance to adjust the pH to 12 was added to the remaining organic layer to obtain a two-phase mixture, which was then stirred and separated at about 75°C to separate the organic layer of the two-phase mixture from the aqueous layer. 25% by weight saline (5.3 g) was added to the separated organic layer, which was then stirred and separated at about 75°C to remove the 25% by weight saline to separate the organic layer (water washing step). The separated organic layer was then concentrated under reduced pressure to remove water, and then filtered. Hexane (12 g) was added to the filtrate to perform crystallization, and the crystals were obtained by suction filtration. Ethyl acetate (20 g) was added to the obtained crystals, which were dissolved at 40°C, and hexane (10 g) was added to perform crystallization. The operation was repeated, and the mixture was dried under reduced pressure to obtain crystals of an activated polyethylene glycol compound represented by the following formula (9) (step (3)).

[0068] The crystals of the activated polyethylene glycol compound were analyzed using a UV-Vis spectrophotometer, and the 4-nitrophenol content was confirmed to be 2 ppm.

[0069] [ka]

[0070] [ka]

[0071] Example 5 In a 20 mL screw tube, polyethylene glycol raw material having a nitrophenyl group at the end represented by formula (1) (Mw: 1,000, 0.5 g, 0.5 mmol) and dichloromethane (1.0 g) were charged and dissolved by stirring under nitrogen. Then, Azido-dPEG-Amine (0.16 g) was added and reacted at room temperature for 3 hours to obtain a reaction mixture (Step (1)).

[0072] Next, to the reaction mixture was added 25 wt % saline (2.5 g) previously adjusted to pH 12 by adding ethylenediamine (0.04 g) to obtain a two-phase mixture (step (2)).

[0073] The two-phase mixture was stirred at about 25°C, then separated, and the aqueous layer of the two-phase mixture was removed. 25% by weight saline (2.5g) that had been adjusted to pH 12 by adding ethylenediamine (0.04g) to the remaining organic layer was added to obtain a two-phase mixture, and the two-phase mixture was stirred and separated at about 25°C, and the organic layer of the two-phase mixture was separated from the aqueous layer. This operation was repeated four times. 25% by weight saline (2.5g) was added to the separated organic layer, then stirred and separated at about 25°C, and the 25% by weight saline was removed to separate the organic layer (water washing process). The separated organic layer was then concentrated under reduced pressure to remove water and dichloromethane. Toluene (6g) was then added to the organic layer after the water and dichloromethane were removed, and the organic layer was dissolved at about 40°C and filtered. The filtrate was added to hexane (6g) and crystallized at 10°C, and the crystals were obtained by suction filtration. The obtained crystals were dissolved in toluene (6 g) at 40° C., and then added to hexane (6 g) and crystallized at 10° C. This procedure was repeated, and the crystals were then dried under reduced pressure to obtain crystals of an activated polyethylene glycol compound represented by the following formula (10) (step (3)).

[0074] The crystals of the activated polyethylene glycol compound were analyzed using a UV-Vis spectrophotometer, and the 4-nitrophenol content was confirmed to be 233 ppm.

[0075] [ka]

[0076] Comparative Example 1 In a 50 mL three-neck round-bottom flask equipped with a Dimroth condenser, a thermometer, and a nitrogen inlet tube, the polyethylene glycol raw material having a terminal nitrophenyl group represented by the above formula (6) (Mw: 2,000, 2.0 g, 1.0 mmol) and toluene (8.0 g) were charged and dissolved at about 50° C. using a water bath while stirring under nitrogen. Then, N-aminoethoxyphthalimide hydrochloride (1.0 g) and triethylamine (0.64 g) were added and reacted at about 50° C. for 4 hours to obtain a reaction mixture (step (1)).

[0077] Next, insoluble N-aminoethoxyphthalimide hydrochloride was removed by filtration. At this time, the filter was washed with toluene (7.4 g). Ethylenediamine monohydrate (0.47 g) was added to the filtrate and reacted at about 50° C. for 2 hours to deprotect the phthalimide group (deprotection step).

[0078] Then, toluene (2.6 g) and 20 wt % saline (5.3 g) were added to the reaction mixture after the deprotection step to obtain a two-phase mixture.

[0079] The two-phase mixture was stirred at about 75°C, then separated, and the aqueous layer of the two-phase mixture was removed. 20% by weight saline (5.3 g) was added to the remaining organic layer to obtain a two-phase mixture, and the mixture was stirred at about 75°C, separated, and the organic layer of the two-phase mixture was separated from the aqueous layer. This operation was repeated five times. The separated organic layer was concentrated under reduced pressure to remove water. Next, toluene (6.0 g) was added to the organic layer after the water was removed, and filtration was performed. The filtrate was added to hexane (12 g) and crystallized at 10°C, and then suction filtration was performed to obtain crystals. Toluene (12 g) was added to the obtained crystals, dissolved at 40°C, added to hexane (12 g), and crystallized at 10°C. The operation was repeated, and then dried under reduced pressure to obtain crystals of the activated polyethylene glycol compound represented by the above formula (7).

[0080] The crystals of the activated polyethylene glycol compound were analyzed using a UV-Vis spectrophotometer, and the 4-nitrophenol content was confirmed to be 755 ppm.

[0081] The results of Examples 1 to 5 and Comparative Example 1 are shown in Table 1. These results show that 4-nitrophenol could be removed more effectively in Examples 1 to 5 than in Comparative Example 1. As a result, the production method of the present invention can be easily carried out industrially, has excellent productivity, and is capable of recovering a specific activated polyethylene glycol compound in high yield without generating waste materials such as adsorbents.

[0082] [Table 1]

Claims

1. 1. A method for producing an activated polyethylene glycol compound having a carbamate bond between a polyethylene glycol moiety and a functional group capable of reacting with a biologically relevant substance or a precursor of said functional group, comprising: A method for producing an activated polyethylene glycol compound, comprising the following steps (1), (2) and (3): Process (1): A step of reacting a polyethylene glycol raw material having a terminal nitrophenyl group with an amine compound having the functional group or the precursor of the functional group capable of reacting with the biologically relevant substance in a solution containing an aprotic solvent to obtain a reaction mixture containing the activated polyethylene glycol compound and 4-nitrophenol. Process (2): A step of adding a basic aqueous solution having a pH of 10 or more and 14 or less to the reaction mixture obtained in the step (1) to obtain a two-phase mixture. Process (3): separating the organic layer of the two-phase mixture from the aqueous layer and recovering the activated polyethylene glycol compound from the organic layer.

2. 2. The method for producing an activated polyethylene glycol compound according to claim 1, wherein the activated polyethylene glycol compound has the carbamate bond between the precursor and the polyethylene glycol moiety, and the precursor is a protecting group for the functional group.

3. The method for producing an activated polyethylene glycol compound according to claim 2, characterized in that a deprotection step of deprotecting the protecting group is carried out after the step (1) and before the step (2).

4. The method for producing an activated polyethylene glycol compound according to claim 3, wherein in the deprotection step, a deprotection reagent comprising an organic base having an amino group is added to the reaction mixture containing the activated polyethylene glycol compound and 4-nitrophenol.

5. The method for producing an activated polyethylene glycol compound according to any one of claims 1 to 4, wherein the activated polyethylene glycol compound is represented by the following formula (1): 【Chemistry 1】 (In formula (1), PEG is the polyethylene glycol moiety; X is the functional group capable of reacting with the biologically relevant substance or the precursor of the functional group, A is a divalent linker that connects the functional group or the precursor to the carbamate group.

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