Method for producing maleimide polyethylene glycol derivatives

Abstract

The present invention is a method for producing a maleimide polyethylene glycol derivative which comprises a reaction step for de-protecting a maleimide polyethylene glycol derivative having a protective group by heating in a solvent and in the presence of silica gel.　The present invention makes it possible, when de-protecting a maleimide polyethylene glycol derivative having a protective group, to suppress deterioration of a maleimide group and to produce a maleimide polyethylene glycol derivative which has a high maleimidization rate.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a maleimide polyethylene glycol derivative, which comprises deprotecting a protecting group in a maleimide polyethylene glycol derivative having a protecting group. [Background technology]

[0002] Some drugs are unstable in the bloodstream, others are rapidly excreted from the body, and others exhibit antigenicity. Modification of such drugs with water-soluble polymers such as polyethylene glycol (PEG) can improve their blood retention, leading to active research on PEG in the drug delivery system and pharmaceutical fields. PEG modification can stabilize drugs, reduce antigenicity, and reduce dosage, and further enhance targeting by binding antibodies, etc.

[0003] To modify biologically active substances such as drugs with PEG, it is necessary to convert the end groups of PEG into reactive functional groups. However, if the conversion to reactive functional groups is insufficient, unmodified drugs and PEG with unconverted functional groups will remain as impurities, making it necessary to produce highly pure PEG derivatives.

[0004] One example of a reactive functional group is the maleimide group, which reacts with thiol groups to form stable thioethers, making it a widely used and highly useful reactive functional group in PEGylation reagents.

[0005] However, the double bond site of the maleimide group is highly reactive and prone to degradation. When synthesizing maleimide polyethylene glycol derivatives, the additional steps required after the introduction of the maleimide group can lead to a decrease in the rate of introduction of the maleimide group (maleimidization rate). Therefore, the method of directly introducing the maleimide group in the final step of the synthesis is preferable to prevent degradation.

[0006] Non-Patent Document 1, which describes the reaction of a PEG derivative with maleimide, is an example of a production method for such a compound. In the production method described therein, when maleimide is reacted with the hydroxyl group at the PEG terminal of a PEG derivative at −78° C. in the presence of triphenylphosphine and diisopropyldiazodicarboxylate, the yield of maleimide polyethylene glycol derivative is 31%, and the cause of this low yield is not mentioned.

[0007] Furthermore, a reaction between a maleimide derivative synthesized from a furan derivative and maleimide and a PEG derivative has also been reported (Patent Document 1). In the described production method, the leaving group at the PEG terminal is replaced with a maleimide derivative, and then the maleimide PEG derivative is obtained by deprotection by heating. Although no mention is made of the maleimide conversion rate in this method, one advantage is that a maleimide derivative having a protecting group that protects the highly active double bond of the maleimide is used during the introduction of the maleimide group, which prevents a decrease in the maleimide conversion rate. However, it is difficult to avoid a decrease in the maleimide conversion rate during the deprotection process. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 7,790,835 [Non-patent literature]

[0009] [Non-Patent Document 1] Bioconjugate Chem., vol. 14, No. 2, 377-387(2003) Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to produce a maleimide polyethylene glycol derivative having a high degree of maleimide conversion by suppressing deterioration of the maleimide group when deprotecting the maleimide polyethylene glycol derivative having a protecting group. [Means for solving the problem]

[0011] As a result of extensive research, the present inventors have found that by adding silica gel to the reaction system during deprotection, it is possible to obtain a maleimide polyethylene glycol derivative while suppressing deterioration of the maleimide group compared to conventional methods, and have thus completed the present invention. That is, the present invention relates to the following [1] to [6].

[0012] [1] A method for producing a maleimide polyethylene glycol derivative represented by the following general formula (2), comprising a reaction step of deprotecting a maleimide polyethylene glycol derivative having a protecting group represented by the following general formula (1) by heating it in a solvent in the presence of silica gel. [ka] [ka] (In the formula, R 1 , R 2 , R 3 , and R 4 are each independently a group selected from a hydrogen atom, an alkyl group, a halogen atom, a cyano group, a formyl group, an acyl group, a carboxyl group, an acyloxy group, and an alkylcarbonyloxymethyl group. Z is a residue obtained by removing 2 to 5 active hydrogen groups from a compound having the active hydrogen groups. Y 1 and Y 2 are each independently a single bond, an ether bond, an amide bond, an ester bond, a urethane bond, a carbonate bond, a thioether bond, a disulfide bond, a thioester bond, or an alkylene group containing any of these. W is Ether Based on be. X is a hydrocarbon group having 1 to 7 carbon atoms, a hydroxyl-protecting group, a carboxyl-protecting group, a thiol-protecting group, a cyano group, or an alkylene group containing any of these. m1 and m2 each independently represent 1 or 0. n1 is an integer of 0 or 10 to 2000 that represents the number of repeating units of polyethylene glycol. n2 is an integer of 10 to 2000 that represents the number of repeating units of polyethylene glycol. l is an integer of 0 or 2 to 10 that represents the number of repeating methylene group units. a is an integer of 1 to 5, b is an integer of 0 to 4, and the sum of a and b is an integer of 2 to 5. However, this does not include the case where n1 and b are both 0. ) [2] In the general formula (1), R 1 , R 2 , R 3 , and R 4 are each independently a hydrogen atom or an alkyl group. [ 3 In the general formulas (1) and (2), Z is a residue obtained by removing the active hydrogen groups from a compound having two active hydrogen groups, m1 and m2 are 1, n1 and n2 are integers of 10 to 2000, l is 2, and a and b are 1. or [2] The manufacturing method described in [ 4 In the general formulas (1) and (2), Z is a residue obtained by removing the active hydrogen groups from a compound having three active hydrogen groups, m1 is 0, m2 is 1, n1 is 0, n2 is an integer of 10 to 2000, l is 0, a is 1, and b is 2. or [2] The manufacturing method described in [ 5 In the general formulas (1) and (2), Z is a residue obtained by removing the active hydrogen groups from a compound having four active hydrogen groups, m1 is 1, n1 is 10 to 2000, l is 2, a is 4, and b is 0. or [2] The manufacturing method described in [Effects of the Invention]

[0013] According to the present invention, a method for producing a maleimide polyethylene glycol derivative with a high maleimide conversion rate can be provided. DETAILED DESCRIPTION OF THE INVENTION

[0014] Hereinafter, embodiments of the present invention will be described in detail.

[0015] The method for producing a maleimide polyethylene glycol derivative of the present invention is a method for producing a maleimide polyethylene glycol derivative represented by the following general formula (2), which comprises a reaction step of heating a maleimide polyethylene glycol derivative having a protecting group represented by the following general formula (1) in a solvent in the presence of silica gel to deprotect it. The above-mentioned maleimide polyethylene glycol derivative having a protecting group may be referred to as a "maleimide PEG derivative having a protecting group" or "Compound 1." The above-mentioned maleimide polyethylene glycol derivative may be referred to as a "maleimide PEG derivative." [ka] [ka] (In the formula, R 1 , R 2 , R 3 , and R 4 are each independently a group selected from a hydrogen atom, an alkyl group, a halogen atom, a cyano group, a formyl group, an acyl group, a carboxyl group, an acyloxy group, and an alkylcarbonyloxymethyl group. Z is a residue obtained by removing 2 to 5 active hydrogen groups from a compound having the active hydrogen groups. Y 1 and Y 2 are each independently a single bond, an ether bond, an amide bond, an ester bond, a urethane bond, a carbonate bond, a thioether bond, a disulfide bond, a thioester bond, or an alkylene group containing any of these. W is a group selected from an ether group, a methylene group, and a dimethylvinylidene group. X is a hydrocarbon group having 1 to 7 carbon atoms, a hydroxyl-protecting group, a carboxyl-protecting group, a thiol-protecting group, a cyano group, or an alkylene group containing any of these. m1 and m2 each independently represent 1 or 0. n1 is an integer of 0 or 10 to 2000 that represents the number of repeating units of polyethylene glycol. n2 is an integer of 10 to 2000 that represents the number of repeating units of polyethylene glycol. l is an integer of 0 or 2 to 10 that represents the number of repeating methylene group units. a is an integer from 1 to 5, b is an integer from 0 to 4, and the sum of a and b is an integer from 2 to 5.

[0016] [Maleimide polyethylene glycol derivative having a protecting group represented by general formula (1)] In the production method of the present invention, a maleimide polyethylene glycol derivative having a protecting group represented by general formula (1) is used as a raw material. [ka]

[0017] R in Equation (1) 1 , R 2 , R 3 , and R 4 are each independently any group selected from a hydrogen atom, an alkyl group, a halogen atom, a cyano group, a formyl group, an acyl group, a carboxyl group, an acyloxy group, and an alkylcarbonyloxymethyl group. Among these, R 1 , R 2 , R 3 , and R 4 are preferably each independently a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. Among them, R 1 , R 2 , R 3 and R 4is a hydrogen atom, or R 2 , R 3 and R 4 is a hydrogen atom and R 1 is a methyl group, or R 1 , R 2 , and R 3 is a hydrogen atom and R 4 is a methyl group, or R 2 , R 3 is a hydrogen atom and R 1 , R 4 It is particularly preferred that is a methyl group.

[0018] In formula (1), m1 and m2 each independently represent 1 or 0.

[0019] In formula (1), n1 represents the number of repeating units of polyethylene glycol and is 0 or an integer of 10 to 2000. n1 is preferably an integer of 50 to 1500, and more preferably an integer of 100 to 500.

[0020] In formula (1), n2 represents the number of repeating units of polyethylene glycol and is an integer of 10 to 2000. n2 is preferably an integer of 50 to 1500, and more preferably an integer of 100 to 500.

[0021] In formula (1), l represents the number of repeating methylene groups and is an integer of 0 or 2-10.

[0022] In formula (1), a is an integer of 1 to 5, b is an integer of 0 to 4, and the sum of a and b is an integer of 2 to 5.

[0023] Z in formula (1) is a residue obtained by removing 2 to 5 active hydrogen groups from a compound having the active hydrogen groups. Examples of the active hydrogen group include a hydroxyl group, a carboxyl group, an amino group, and a thiol group, with a hydroxyl group being preferred. Examples of compounds having 2 to 5 active hydrogen groups include compounds having 1 to 20 carbon atoms, preferably 2 to 10 carbon atoms, such as polyol compounds, polycarboxylic acid compounds, polyamine compounds, polythiol compounds, etc. Among these, polyol compounds are preferred. Examples of the polyol compound include ethylene glycol, propylene glycol, diethylene glycol, triethylene glycol, dipropylene glycol, glycerin, trimethylolpropane, 1,3,6-hexanetriol, pentaerythritol, and sorbitol, with ethylene glycol, glycerin, and pentaerythritol being preferred.

[0024] The residue obtained by removing the active hydrogen groups from a compound having 2 to 5 active hydrogen groups refers to the residue obtained by removing the hydroxyl groups, for example, if the active hydrogen groups are hydroxyl groups. For example, if the compound having 2 to 5 active hydrogen groups is ethylene glycol, the group represented by the following formula (a1) corresponds to the residue obtained by removing the active hydrogen groups from the compound having 2 to 5 active hydrogen groups of the present invention. If the compound having 2 to 5 active hydrogen groups is glycerin, the group represented by the following formula (a2) corresponds to the residue obtained by removing the active hydrogen groups from the compound having 2 to 5 active hydrogen groups of the present invention. If the compound having 2 to 5 active hydrogen groups is pentaerythritol, the group represented by the following formula (a3) ​​corresponds to the residue obtained by removing the active hydrogen groups from the compound having 2 to 5 active hydrogen groups of the present invention. In addition, * in formulas (a1), (a2), and (a3) ​​represents a bond. [ka]

[0025] In formula (1), W represents any group selected from an ether group, a methylene group, and a dimethylvinylidene group. Among these, W is preferably an ether group (i.e., an oxygen atom) from the viewpoint of ease of protection and deprotection of the maleimide.

[0026] Y in Equation (1) 1and Y 2 are each independently a single bond, an ether bond, an amide bond, an ester bond, a urethane bond, a carbonate bond, a thioether bond, a disulfide bond, a thioester bond, or an alkylene group containing any of these. From the viewpoint of ease of manufacturing, Y 1 and Y 2 is preferably an ether bond (that is, an oxygen atom) among the above.

[0027] X in formula (1) is a hydrocarbon group having 1 to 7 carbon atoms, a protecting group for a hydroxyl group, a protecting group for a carboxyl group, a protecting group for a thiol group, a cyano group, or an alkylene group containing any of these. Examples of the hydroxyl-protecting group, carboxyl-protecting group, and thiol-protecting group include t-butyl, benzyl, trityl, t-butyldimethylsilyl, and t-butyldiphenylsilyl groups. Among these, X is preferably a hydrocarbon group having 1 to 7 carbon atoms, and more preferably a methyl group.

[0028] In the general formula (1) and the general formula (2) described later, any one of the following embodiments (i) to (iii) is preferred. (i) Z is a residue obtained by removing two active hydrogen groups from a compound having the active hydrogen groups; m1 and m2 are 1; n1 and n2 are integers of 10 to 2000; l is 2; and a and b are 1. (ii) Z is a residue obtained by removing the active hydrogen groups from a compound having three active hydrogen groups, m1 is 0, m2 is 1, n1 is 0, n2 is an integer of 10 to 2000, l is 0, a is 1, and b is 2. (iii) Z is a residue obtained by removing four active hydrogen groups from a compound having the active hydrogen groups, m1 is 1, n1 is 10 to 2000, l is 2, a is 4, and b is 0.

[0029] The method for producing the maleimide polyethylene glycol derivative having a protecting group represented by general formula (1) is not particularly limited, but for example, the derivative can be obtained by preparing the following maleimide protected compound (formula (3)) and PEG derivative (formula (4)) and reacting them. 1 , R 2 , R 3 , R 4 , W, Z, Y 1 , Y 2 , X, m1, m2, n1, n2, l, a and b have the same meanings as those in general formula (1). [ka]

[0030] [Method for producing maleimide polyethylene glycol derivative represented by general formula (2)] The method for producing a maleimide polyethylene glycol derivative represented by the following general formula (2) of the present invention is a production method comprising a reaction step of heating a maleimide polyethylene glycol derivative having a protecting group represented by the above-mentioned general formula (1) in a solvent in the presence of silica gel to deprotect it. [ka] Z and Y in formula (2) 1 , Y 2 , X, m1, m2, n1, n2, l, a and b have the same meanings as those in the general formula (1) above.

[0031] The silica gel used in the production method of the present invention is spherical or crushed silicon dioxide. The average particle size of the silica gel is not particularly limited, but is preferably 5 to 425 μm, and more preferably 50 to 150 μm. The average pore size of the silica gel is not particularly limited, but is preferably 2 to 15 nm, and more preferably 6 to 13 nm. The pore volume of the silica gel is not particularly limited, but is preferably 0.5 to 1.5 mL / g, and more preferably 0.6 to 1.2 mL / g. The specific surface area of ​​the silica gel is not particularly limited, but is preferably 200 to 800 m 2 / g, more preferably 250 to 550m 2 / g. When the average particle size, average pore size, pore volume, and specific surface area are equal to or greater than the lower limit of the above-mentioned ranges, the reaction efficiency is improved. When the average particle size, average pore size, pore volume, and specific surface area are equal to or less than the upper limit of the above-mentioned ranges, the amount of the target substance extracted from the residue after filtering off the silica gel is increased, thereby improving productivity. The average particle size of silica gel is measured by laser diffraction particle size distribution measurement. The average pore size, pore volume, and specific surface area of ​​silica gel are measured by nitrogen gas adsorption. The specific surface area is calculated using the BET equation.

[0032] The amount of silica gel used is preferably 0.1 to 5 times by weight, and particularly preferably 1 to 3 times by weight, relative to the amount of the maleimide PEG derivative (compound 1) having a protecting group represented by general formula (1). When the amount of silica gel used is equal to or greater than these lower limits, deterioration of the maleimide group is easily suppressed, and when it is equal to or less than these upper limits, stirring of the reaction solution becomes easy.

[0033] In the present invention, the deprotection reaction is carried out by heating in a solvent. The temperature of the solvent during heating (i.e., the reaction temperature) in the present invention is preferably 70 to 150° C., and particularly preferably 100 to 120° C. If the solvent temperature is equal to or higher than these lower limits, the reaction will proceed easily, and if it is equal to or lower than these upper limits, deterioration of the maleimide group will be easily suppressed.

[0034] The solvent used in the present invention is not particularly limited as long as it is aprotic and has a boiling point equal to or higher than the reaction temperature, but toluene and p-xylene are preferred. The amount of solvent used is preferably 3 to 50 times, and more preferably 5 to 20 times by weight, the amount of maleimide polyethylene glycol derivative (compound 1) having a protecting group represented by general formula (1). When the amount of solvent used is equal to or higher than these lower limits, stirring of the reaction solution becomes easy, and when it is equal to or lower than these upper limits, economic efficiency is improved.

[0035] The reaction time in the present invention varies depending on the type and amount of the raw materials and solvent used, but is usually 2 to 8 hours. [Example]

[0036] The present invention will now be described in more detail with reference to examples, in which NMR was used to analyze the maleimide ratio.

[0037] < 1 H-NMR analysis method 1 H-NMR analysis was performed using JNM-ECP400 and JNM-ECA600 manufactured by JEOL Datum Co., Ltd. The integrated values ​​in the NMR measurements are theoretical values.

[0038] A maleimide polyethylene glycol derivative (A) (molecular weight: 5,000) having a protecting group was produced according to the following Production Examples (1-1) to (1-3). [ka] n=115

[0039] [Manufacturing Example 1-1] Synthesis of maleimide protected compounds To a 9 ml screw tube were added 157.1 mg (1.61 mmol) of maleimide, 1.5 g of methyl t-butyl ether, and 876.7 mg (12.88 mmol) of furan, and the mixture was reacted for 8 hours at 30°C under a nitrogen atmosphere. After cooling to room temperature, the precipitated crystals were collected by filtration and dried to obtain the maleimide-protected compound (B) (122.9 mg, 0.74 mmol) shown below. [ka]

[0040] [Manufacturing Example 1-2] Synthesis of methoxy PEG (molecular weight: 5000) A 200 ml round-bottom flask equipped with a thermometer, nitrogen inlet, and stirrer was charged with 24 g (0.8 mol) of dehydrated methanol and 0.563 g (24 mmol: 16 mol%) of metallic sodium. The mixture was stirred at room temperature while blowing in nitrogen until the metallic sodium dissolved. This solution was placed in a 5 L autoclave, and after purging the system with nitrogen, the temperature was raised to 80°C. 2300 g (52.3 mol) of ethylene oxide was added at 100-150°C and a pressure of 0.5 MPa or less, and the reaction was continued for another hour. After removing unreacted ethylene oxide gas under reduced pressure, the mixture was cooled to 60°C and the pH was adjusted to 7.5 with 85% aqueous phosphoric acid solution to obtain the following compound (C). [ka] n=115

[0041] [Manufacturing Example 1-3] Introduction of maleimide-protected groups into methoxy PEG derivatives A 20 ml screw tube was charged with 2.00 g (0.40 mmol) of compound (C), 3.5 g of dehydrated toluene, 10 g of chloroform, 231.2 mg (1.40 mmol) of maleimide-protected compound (B), 1.41 g (5.40 mmol) of triphenylphosphine, and 1.09 g (5.40 mmol) of diisopropyl diazodicarboxylate, and the mixture was reacted for 1 hour at room temperature under a nitrogen atmosphere. After the reaction, 10 mg of methanol was added, and the mixture was concentrated at 50 °C. The precipitated crystals were dissolved in 10 g of ethyl acetate by heating at 40 °C, and 8 g of hexane was added to perform crystallization. The precipitated crystals were collected by filtration, and a mixed solvent of 6 g of hexane and 12 g of ethyl acetate was added. This procedure was repeated four times, after which the precipitated crystals were dissolved in 12 g of ethyl acetate by heating at 40 °C, and 6 g of hexane was added to perform crystallization again. Finally, the resulting crystals were washed with 8 g of hexane and dried to obtain the maleimide polyethylene glycol derivative (A) (1.35 g, 0.27 mmol) having the protecting group shown below. [ka] n=115

[0042] A maleimide PEG derivative (D) (molecular weight 20,000) having a protecting group was produced in the same manner as in Production Examples (1-1) to (1-3) above, except that the molecular weight was adjusted by changing the amount of ethylene oxide used in Production Example 1-2. [ka] n=460

[0043] Maleimide polyethylene glycol derivatives (E) (molecular weight: 10,000) having a protecting group were produced according to the following Production Examples (2-1) to (2-7). [ka] n=115

[0044] [Manufacturing Example 2-1] Synthesis of maleimide protected compounds To a 9 ml screw tube were added 157.1 mg (1.61 mmol) of maleimide, 1.5 g of methyl t-butyl ether, and 877.7 mg (10.68 mmol) of 2-methylfuran, and the mixture was reacted for 8 hours at 30°C under a nitrogen atmosphere. The mixture was allowed to cool to room temperature, and the precipitated crystals were collected by filtration and dried to obtain the maleimide-protected product (F) (86.4 mg, 0.48 mmol) shown below. [ka]

[0045] [Production Example 2-2] Benzylation process of isopropylideneglycerol A 1000 mL round-bottom flask equipped with a thermometer, nitrogen inlet, and stirrer was charged with 132.2 g (1.0 mol) of 2,2-dimethyl-1,3-dioxolane-4-methanol, 231.4 g (1.2 mol) of 28% sodium methoxide in methanol, and 500 mL of toluene. The toluene was refluxed under reduced pressure for 1 hour while blowing in nitrogen, and the methanol was distilled off. While maintaining the solution at 80°C, 126.6 g (1.0 mol) of benzyl chloride was added dropwise using a dropping funnel over 2 hours, and the reaction was continued for another 2 hours. The reaction mixture was desolvated and purified by distillation (bp 93-95°C / 266 Pa) to obtain 4-(benzooxymethyl)-2,2-dimethyl-1,3-dioxolane (G). [ka]

[0046] [Manufacturing Example 2-3] Deisopropylidenation process of benzyl-protected isopropylideneglycerol 222 g (1.0 mol) of 4-(benzooxymethyl)-2,2-dimethyl-1,3-dioxolane (G), 250 ml of ethanol, and 400 ml of distilled water were weighed into a 1 L beaker, and the pH was adjusted to 2.0 with phosphoric acid. While blowing in nitrogen, the solution was heated to 70°C and reacted for 1.5 hours. After that, the pH was adjusted to 7.0 with sodium hydroxide, and the salt was adsorbed using a Kyoward 1000 (Kyowa Chemical Industry Co., Ltd.). The solvent was removed, and 3-benzyloxy-1,2-propanediol (H) was obtained. [ka]

[0047] [Manufacturing Example 2-4] PEGylation process of benzyl-protected glycerol (molecular weight: 10,000) A 300 ml round-bottom flask equipped with a thermometer, nitrogen inlet, and stirrer was charged with 27.3 g (0.15 mol) of 3-benzyloxy-1,2-propanediol (H), 127 g (1.2 mol) of dehydrated toluene, and 0.9 g (39 mmol: 26 mol%) of metallic sodium. The mixture was stirred at room temperature while blowing in nitrogen until the metallic sodium dissolved. This solution was placed in a 5 L autoclave, and after purging with nitrogen, the system was heated to 100°C. 1473 g (33.5 mol) of ethylene oxide was added at 100-150°C and a pressure of 1 MPa or less. The reaction was continued for another hour. After removing unreacted ethylene oxide gas under reduced pressure, the mixture was cooled to 60°C and the pH was adjusted to 7.5 with 85% aqueous phosphoric acid solution to obtain the following compound (I). [ka] n=115

[0048] [Manufacturing Example 2-5] Methoxylation of glycerol benzyl-protected PEG A 500 ml round-bottom flask equipped with a thermometer, nitrogen inlet, stirrer, Dean-Stark tube, and condenser was charged with 100 g (10 mmol) of the above compound (I) and 320 g of toluene, and heated to reflux to remove water azeotropically. After cooling to room temperature, 10.12 g (100 mmol) of triethylamine and 6.87 g (60 mmol) of methanesulfonyl chloride were added and the reaction was carried out at 40°C for 6 hours. The reaction solution was filtered, and the filtrate was transferred to a 500 ml round-bottom flask equipped with a thermometer, nitrogen inlet, stirrer, and condenser. 19.3 g (100 mmol) of 28% sodium methoxide in methanol was added and the reaction was carried out at 70°C for 6 hours. Next, 27 g of Kyoward 700 (Kyowa Chemical Industry Co., Ltd.) was added to the reaction solution, and the mixture was further stirred at 70°C for 1 hour to adsorb excess sodium methoxide. The reaction solution was then filtered, and the filtrate was placed in a 1 L beaker, and 300 g of ethyl acetate and 350 g of hexane were added to perform crystallization. The precipitated crystals were filtered into a 1 L beaker, and ethyl acetate was added and the mixture was heated to 40°C to dissolve. 300 g of hexane was added and crystallization was performed again. The precipitated crystals were filtered and dried to obtain the following compound (J). [ka] n=115

[0049] [Production Example 2-6] Debenzylation step of glycerol benzyl-protected PEG A 500 ml round-bottom flask equipped with a thermometer, nitrogen inlet tube, stirrer, and condenser was charged with 15 g of the above compound (J) and 15 g of 5% palladium carbon (50% water content). After replacing the atmosphere with nitrogen, 300 ml of methanol and 150 ml of cyclohexene were added, and the mixture was heated and gently refluxed at 52-55°C for 5 hours. After cooling the reaction mixture to room temperature, the palladium carbon was filtered off, and the filtrate was concentrated. 50 ml of ethyl acetate and 50 ml of hexane were added to the concentrated mixture to cause crystallization. The resulting crystals were filtered and dried to obtain the following compound (K). [ka] n=115

[0050] [Manufacturing Example 2-7] Introduction of maleimide-protected compounds into methoxy PEG A 20 ml screw tube was charged with 2.0 g (0.20 mmol) of compound (K), 3.5 g of dehydrated toluene, 10 g of chloroform, 125.4 mg (0.70 mmol) of maleimide-protected compound (F), 708.2 mg (2.70 mmol) of triphenylphosphine, and 546.0 mg (2.70 mmol) of diisopropyl diazodicarboxylate, and the mixture was reacted at room temperature for 1 hour under a nitrogen atmosphere. After the reaction, 10.0 mg of methanol was added, and the mixture was concentrated at 50 °C. The precipitated crystals were dissolved in 10 g of ethyl acetate by heating at 40 °C, and 8 g of hexane was added to perform crystallization. The precipitated crystals were collected by filtration, and a mixed solvent of 6 g of hexane and 12 g of ethyl acetate was added. This procedure was repeated four times, after which the precipitated crystals were dissolved in 12 g of ethyl acetate by heating at 40 °C, and 6 g of hexane was added to perform crystallization again. Finally, the resulting crystals were washed with 8 g of hexane and dried to obtain the maleimide polyethylene glycol derivative (E) (1.30 g, 0.13 mmol) having the protecting group shown below. [ka] n=115

[0051] A maleimide polyethylene glycol derivative (L) (molecular weight: 40,000) having a protecting group was produced according to the following Production Examples (3-1) to (3-3). [ka] n=230

[0052] [Manufacturing Example 3-1] Synthesis of maleimide protected compounds To a 9 ml screw tube were added 386.9 mg (3.99 mmol) of maleimide, 3.7 g of methyl t-butyl ether, and 3.09 mg (32.14 mmol) of 2,5-dimethylfuran, and the mixture was reacted for 8 hours at 50°C under a nitrogen atmosphere. The mixture was allowed to cool to room temperature, and the precipitated crystals were collected by filtration and dried to obtain the maleimide-protected compound (M) (522.3 mg, 2.70 mmol) shown below. [ka]

[0053] [Manufacturing Example 3-2] Pentaerythritol PEGylation Process A 1000 ml round-bottom flask equipped with a thermometer, nitrogen inlet, and stirrer was charged with 24.5 g (0.18 mol) of pentaerythritol, 420 g (4.6 mol) of dehydrated toluene, and 2.5 g (109 mmol: 73 mol%) of metallic sodium. The mixture was stirred at room temperature while blowing in nitrogen until the metallic sodium dissolved. This solution was placed in a 5 L autoclave, and after purging the system with nitrogen, the temperature was raised to 100°C. 896 g (20.3 mol) of ethylene oxide was added at 100-150°C and a pressure of 1 MPa or less. The reaction was continued for another hour. After removing unreacted ethylene oxide gas under reduced pressure, the mixture was cooled to 60°C and the pH was adjusted to 7.5 with 85% aqueous phosphoric acid solution to obtain the following compound (N). [ka] n=230

[0054] [Manufacturing Example 3-3] Introduction of maleimide-protected compounds into pentaerythritol PEG A 20 ml screw tube was charged with 2.0 g (0.050 mmol) of compound (N), 3.5 g of dehydrated toluene, 10 g of chloroform, 31.4 mg (0.175 mol) of maleimide-protected compound (M), 177.0 mg (0.675 mmol) of triphenylphosphine, and 136.5 mg (0.675 mmol) of diisopropyl diazodicarboxylate, and the mixture was reacted for 1 hour at room temperature under a nitrogen atmosphere. After the reaction, 10.0 mg of methanol was added, and the mixture was concentrated at 50 °C. The precipitated crystals were dissolved in 10 g of ethyl acetate by heating at 40 °C, and 8 g of hexane was added to perform crystallization. The precipitated crystals were collected by filtration, and a mixed solvent of 6 g of hexane and 12 g of ethyl acetate was added. This procedure was repeated four times, after which the precipitated crystals were dissolved in 12 g of ethyl acetate by heating at 40 °C, and 8 g of hexane was added to perform crystallization again. Finally, the resulting crystals were washed with 8 g of hexane and dried to obtain the maleimide polyethylene glycol derivative (L) (1.28 g, 0.032 mmol) having the protecting group shown below. [ka] n=230

[0055] [Example 1] Method for producing maleimide polyethylene glycol derivatives 200 mg (0.04 mmol) of maleimide polyethylene glycol derivative (A) having a protecting group, 10 g of toluene, and 200 mg of silica gel (Fuji Silysia: PSQ100B) were added to a 100 mL side-arm test tube, and the reaction was carried out at 110°C for 5 hours under a nitrogen atmosphere. After decanting the reaction solution, the target product was extracted twice with 4 g of methanol from the silica gel. The reaction solution and the extract were transferred to a 50 mL recovery flask and concentrated to dryness. The mixture was then dried under vacuum for 6 hours to obtain maleimide polyethylene glycol derivative (O) (yield: 189.5 mg, 0.037 mmol, 96% yield, 97% maleimide conversion). The silica gel (Fuji Silysia: PSQ100B) used in this example had an average particle size of 100 μm, an average pore size of 7 nm, a pore volume of 0.8 mL / g, and a specific surface area of ​​500 m 2 / g. [ka] n=115

[0056] The maleimide polyethylene glycol derivative (O) obtained in Example 1 1 H-NMR showed no peaks at 6.51 ppm and 6.42 ppm attributable to the maleimide polyethylene glycol derivative (A) having a protecting group, indicating that deprotection was complete. Furthermore, the integral value of the peak at 6.71 ppm attributable to the vinyl proton of the maleimide group was 1.94, confirming that the target maleimide polyethylene glycol derivative represented by formula (2) was obtained with a maleimidation rate of 97%.

[0057] [Example 2] Method for producing maleimide polyethylene glycol derivatives A 100 mL side-arm test tube was charged with 200 mg (0.01 mmol) of maleimide polyethylene glycol derivative (D) having a protecting group, 10 g of toluene, and 200 mg of silica gel (Fuji Silysia: PSQ100B), and the reaction was carried out at 110°C for 5 hours under a nitrogen atmosphere. After decanting the reaction solution, the target product was extracted twice with 4 g of methanol from the silica gel. The reaction solution and the extract were transferred to a 50 mL recovery flask and concentrated to dryness. The mixture was then dried under vacuum for 6 hours to obtain maleimide polyethylene glycol derivative (P) (yield: 191.4 mg, 0.0095 mmol, 96% yield, 97% maleimide conversion). The silica gel (Fuji Silysia: PSQ100B) used in this example had an average particle size of 100 μm, an average pore size of 7 nm, a pore volume of 0.8 mL / g, and a specific surface area of ​​500 m 2 / g. [ka] n=460

[0058] The maleimide polyethylene glycol derivative (P) obtained in Example 2 1H-NMR showed no peaks at 6.51 ppm and 6.42 ppm attributable to the maleimide polyethylene glycol derivative (D) having a protecting group, indicating that deprotection was complete. Furthermore, the integral value of the peak at 6.71 ppm attributable to the vinyl proton of the maleimide group was 1.94, confirming that the target maleimide polyethylene glycol derivative (P) represented by formula (2) was obtained with a maleimidation rate of 97%.

[0059] [Example 3] Method for producing maleimide polyethylene glycol derivatives A 100 mL test tube was charged with 200 mg (0.02 mmol) of maleimide polyethylene glycol derivative (E) having a protecting group, 10 g of toluene, and 200 mg of silica gel (PSQ100B, manufactured by Fuji Silysia Chemical Industries, Ltd.), and the reaction was carried out at 110°C for 5 hours under a nitrogen atmosphere. After decanting the reaction solution, the target product was extracted twice with 4 g of methanol from the silica gel. The reaction solution and the extract were transferred to a 50 mL recovery flask and concentrated to dryness. The mixture was then dried under vacuum for 6 hours to obtain maleimide polyethylene glycol derivative (Q) (yield: 188.3 mg, 0.019 mmol, 95% yield, 96% maleimide conversion). The silica gel (PSQ100B, manufactured by Fuji Silysia Chemical Industries, Ltd.) used in this example had an average particle size of 100 μm, an average pore size of 7 nm, a pore volume of 0.8 mL / g, and a specific surface area of ​​500 m. 2 / g. [ka] n=115

[0060] The maleimide polyethylene glycol derivative (Q) obtained in Example 3 1H-NMR revealed that the deprotection was complete, as peaks at 6.55 ppm, 6.41 ppm, 6.35 ppm, and 6.24 ppm were absent due to the maleimide polyethylene glycol derivative (E) having a protecting group. Furthermore, the integral value of the peak at 6.71 ppm due to the vinyl proton of the maleimide group was 1.92, confirming that the target maleimide polyethylene glycol derivative (Q) represented by formula (2) was obtained with a maleimidation rate of 96%.

[0061] [Example 4] Method for producing maleimide polyethylene glycol derivatives A 100 mL side-arm test tube was charged with 200 mg (0.005 mmol) of maleimide polyethylene glycol derivative (L) having a protecting group, 10 g of toluene, and 200 mg of silica gel (Fuji Silysia: PSQ100B), and the reaction was carried out at 110°C for 5 hours under a nitrogen atmosphere. After decanting the reaction solution, the target product was extracted twice with 4 g of methanol from the silica gel. The reaction solution and the extract were transferred to a 50 mL recovery flask and concentrated to dryness. The mixture was then dried under vacuum for 6 hours to obtain the maleimide polyethylene glycol derivative (R) (yield: 194.0 mg, 0.0048 mmol, 98% yield, 96% maleimide conversion). The silica gel (Fuji Silysia: PSQ100B) used in this example had an average particle size of 100 μm, an average pore size of 7 nm, a pore volume of 0.8 mL / g, and a specific surface area of ​​500 m 2 / g. [ka] n=230

[0062] H-NMR of the maleimide polyethylene glycol derivative (R) obtained in Example 4 showed no peaks at 6.35 ppm and 6.23 ppm attributable to the maleimide polyethylene glycol derivative (L) having a protecting group, indicating that deprotection was complete. Furthermore, the integral value of the peak at 6.71 ppm attributable to the vinyl proton of the maleimide group was 1.92, confirming that the target maleimide PEG derivative (R) represented by formula (2) was obtained with a maleimidization rate of 96%.

[0063] [Comparative Example 1] Method for producing maleimide polyethylene glycol derivatives 200 mg (0.04 mmol) of maleimide polyethylene glycol derivative (A) having a protecting group and 10 g of toluene were added to a 100 mL three-necked round-bottom flask, and the reaction was carried out at 110°C for 5 hours under a nitrogen atmosphere. The reaction solution was transferred to a 50 mL recovery flask and concentrated to dryness. After that, the mixture was dried in vacuo for 6 hours to obtain maleimide polyethylene glycol derivative (O) (yield: 195.4 g, 0.038 mmol, yield 99%, maleimide conversion rate 85%).

[0064] The maleimide polyethylene glycol derivative (O) obtained in Comparative Example 1 1 H-NMR showed no peaks at 6.51 ppm and 6.42 ppm attributable to the maleimide polyethylene glycol derivative (A) having a protecting group, indicating that deprotection was complete. Furthermore, the integral value of the peak at 6.71 ppm attributable to the vinyl proton of the maleimide group was 1.70, confirming that the desired maleimide PEG derivative (O) represented by formula (2) was obtained with a maleimidation rate of 85%.

[0065] Comparative Example 2 Method for producing maleimide polyethylene glycol derivatives A 100 mL sidearm test tube was charged with 150 mg (0.0037 mmol) of maleimide PEG derivative (L) bearing a protecting group, 7.5 g of toluene, and 150 mg of Kyoward 2000 (aluminum oxide-magnesium oxide solid solution, manufactured by Kyowa Chemical Industry Co., Ltd.), and the reaction was carried out at 110°C for 5 hours under a nitrogen atmosphere. After decanting the reaction mixture, the target product was extracted twice with 3 g of methanol. The reaction mixture and extract were transferred to a 50 mL recovery flask and concentrated to dryness. The mixture was then dried in vacuo for 6 hours to obtain the maleimide polyethylene glycol derivative (R) (yield: 87.5 mg, 0.0021 mmol, 59% yield, 65% maleimide conversion).

[0066] The maleimide polyethylene glycol derivative (R) obtained in Comparative Example 2 1 H-NMR showed no peaks at 6.35 ppm and 6.23 ppm attributable to the maleimide polyethylene glycol derivative (L) having a protecting group, indicating that deprotection was complete. Furthermore, the integral value of the peak at 6.71 ppm attributable to the vinyl proton of the maleimide group was 1.30, confirming that the target maleimide polyethylene glycol derivative (R) represented by formula (2) was obtained with a maleimidation rate of 65%.

[0067] As described above, a method for producing a maleimide polyethylene glycol derivative having a high maleimide conversion rate can be provided by heating a maleimide polyethylene glycol derivative having a protecting group in a solvent in the presence of silica gel.

Claims

1. A method for producing a maleimide polyethylene glycol derivative represented by the following general formula (2), comprising a reaction step of heating a maleimide polyethylene glycol derivative having a protecting group represented by the following general formula (1) in a solvent in the presence of silica gel to deprotect the derivative: 【Chemistry 1】 【Chemistry 2】 (In the formula, R １ , R ２ , R ３ , and R ４ are each independently a group selected from a hydrogen atom, an alkyl group, a halogen atom, a cyano group, a formyl group, an acyl group, a carboxyl group, an acyloxy group, and an alkylcarbonyloxymethyl group. Z is a residue obtained by removing the active hydrogen groups from a compound having 2 to 5 active hydrogen groups. Y １ and Y ２ are each independently a single bond, an ether bond, an amide bond, an ester bond, a urethane bond, a carbonate bond, a thioether bond, a disulfide bond, a thioester bond, or an alkylene group containing any of these. W is an ether group. X is a hydrocarbon group having 1 to 7 carbon atoms, a hydroxyl-protecting group, a carboxyl-protecting group, a thiol-protecting group, a cyano group, or an alkylene group containing any of these. m1 and m2 each independently represent 1 or 0. n1 is 0 or an integer of 10 to 2000, representing the number of repeating units of polyethylene glycol. n2 is an integer of 10 to 2000 representing the number of repeating units of polyethylene glycol. l is an integer of 0 or 2 to 10 representing the number of repeating methylene units. a is an integer from 1 to 5, b is an integer from 0 to 4, and the sum of a and b is an integer from 2 to 5, except when both n1 and b are 0.

2. In the general formula (1), R １ , R ２ , R ３ , and R ４ The method according to claim 1, wherein each of the groups independently represents a hydrogen atom or an alkyl group.

3. The method according to claim 1 or 2, wherein, in the general formulas (1) and (2), Z is a residue obtained by removing the active hydrogen groups from a compound having two active hydrogen groups, m1 and m2 are 1, n1 and n2 are integers of 10 to 2000, 1 is 2, and a and b are 1.

4. 3. The method according to claim 1 or 2, wherein, in the general formulas (1) and (2), Z is a residue obtained by removing the active hydrogen groups from a compound having three active hydrogen groups, m1 is 0, m2 is 1, n1 is 0, n2 is an integer of 10 to 2000, 1 is 0, a is 1, and b is 2.

5. 3. The method according to claim 1 or 2, wherein, in the general formulas (1) and (2), Z is a residue obtained by removing the active hydrogen groups from a compound having four active hydrogen groups, m1 is 1, n1 is 10 to 2000, l is 2, a is 4, and b is 0.

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