Method for producing maleimide polyethylene glycol lipids

Abstract

The present invention provides a method for producing a maleimide polyethylene glycol lipid which comprises a reaction step for de-protecting a maleimide polyethylene glycol lipid having a protective group, by heating the maleimide polyethylene glycol lipid having a protective group in a solvent and in the presence of silica gel.　Thus, it is possible to provide a production method that prevents deterioration of a maleimide group and that increases the maleimidization rate.

Description

[Technical Field]

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

[0002] Nucleic acid-encapsulated lipid nanoparticles (LNPs) have been widely studied in the field of drug delivery systems. LNPs are microparticles composed of phospholipids, cholesterol, polyethylene glycol (PEG) lipids, and cationic lipids. The PEG coating protects nucleic acids from rapid enzymatic degradation. As a result, LNPs exhibit high blood retention and interact with and accumulate in various liver cells (hepatocytes, endothelial cells, B cells, and Kupffer cells). Therefore, they are used for nucleic acid delivery targeting hepatocytes. However, while LNPs have been successfully used to deliver nucleic acids to the liver, the delivery of nucleic acids to a wider range of organs requires the introduction of ligands that recognize cell surface receptors, such as antibodies, aptamers, peptides, and glycans, at the PEG end of the LNP surface to improve target organ targeting.

[0003] Since the PEG on the LNP surface is derived from PEG-lipids, which are components of LNPs, introducing an active group capable of conjugating a ligand to the PEG end of the PEG-lipid allows for the immobilization of the ligand on the LNP surface, thereby conferring targeting capabilities to target organs. Therefore, selecting an appropriate active group that reacts with the ligand is important. Thiol groups on cysteine ​​side chains are an example of reactive sites in antibodies, and thiol groups are known to readily react with maleimide groups. Therefore, it is considered effective to use maleimide-PEG-lipids, in which a maleimide group is introduced at the PEG end of the PEG-lipid.

[0004] Patent Document 1 describes a method for producing maleimide-PEG lipids, describing the reaction of a 1,2-diacylglycerol derivative in which the hydroxyl group at the 3-position is substituted with an active group with a maleimide-PEG derivative having a primary amine. In this production method, the hydroxyl group at the 3-position of 1,2-diacylglycerol is converted to an active group, and then a maleimide-PEG derivative having a primary amine is used to condense the amino group with the active group to obtain the maleimide-PEG lipid. Here, the maleimide conversion rate at the PEG lipid end significantly affects the introduction of a ligand into the maleimide-PEG lipid. However, since the aforementioned maleimide-PEG lipid is synthesized through two steps after introducing maleimide into its raw material, the PEG derivative, the highly reactive maleimide group deteriorates with each reaction, resulting in a decrease in the maleimide conversion rate and a decrease in the ligand introduction rate. For this reason, a method of directly introducing the maleimide group in the final synthesis step is preferred to suppress this degradation. Non-Patent Document 1 describes a production method for this, which involves the reaction of a PEG derivative with maleimide. In the production method described, 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, and the yield of the maleimide PEG derivative is 31%, but the cause of this low yield is not mentioned.

[0005] On the other hand, although no examples of maleimide-PEG lipids are given, one synthesis example applicable to the synthesis of maleimide-PEG lipids is described in Patent Document 2, which describes the reaction of a PEG derivative with a maleimide derivative synthesized from a furan derivative and maleimide. In the described production method, the leaving group at the PEG terminal is replaced with a maleimide derivative, followed by deprotection by heating to obtain a maleimide-PEG derivative. Although no mention is made of the maleimide conversion rate, this method uses a maleimide derivative having a protecting group that protects the highly active double bond of the maleimide when introducing the maleimide group. This method has the advantage of being able to suppress the decrease in the maleimide conversion rate compared to Patent Document 1, but it is difficult to avoid the decrease in the maleimide conversion rate during the deprotection process. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2012-214746 [Patent Document 2] U.S. Patent No. 7,790,835 [Non-patent literature]

[0007] [Non-Patent Document 1] Bioconjugate Chem., vol. 14, No. 2, 377-387(2003) Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, an object of the present invention is to provide a production method for producing a maleimide polyethylene glycol lipid by deprotecting a maleimide polyethylene glycol lipid having a protecting group, which suppresses deterioration of the maleimide group and improves the maleimide conversion rate. [Means for solving the problem]

[0009] As a result of extensive research, the present inventors have found that by adding silica gel to the reaction system during deprotection, maleimide polyethylene glycol lipids can be obtained while suppressing deterioration of the maleimide group compared to conventional methods, and have thus completed the present invention. That is, the present invention provides the following [1] to [ 6 ] regarding.

[0010] [1] A method for producing a maleimide polyethylene glycol lipid represented by the following general formula (2), comprising a reaction step of deprotecting a maleimide polyethylene glycol lipid having a protecting group represented by the following general formula (1) by heating it in a solvent in the presence of silica gel. [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. n is an integer of 2 to 10 representing the number of repeating methylene group units. m is an integer of 10 to 2000 representing the number of repeating polyethylene glycol units. Z is an ether The base Y is below Notation (3) It is expressed as Ru base and [ka] A is a group represented by the following general formula (7) or (8). [ka] (R 5 and R 6 are each independently any group selected from a hydrogen atom, a saturated or unsaturated acyl group having 8 to 24 carbon atoms, or a saturated or unsaturated alkyl group having 8 to 24 carbon atoms.) [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), A is a group represented by formula (7), and R 5 and R 6 are each independently selected from a saturated or unsaturated acyl group having 8 to 24 carbon atoms, or a saturated or unsaturated alkyl group having 8 to 24 carbon atoms. [ 4 ] In the general formulas (1) and (2), n is an integer of 2 to 6. 3 ] A manufacturing method described in any one of the above. [ 5 ] In the general formulas (1) and (2), the above [1] to [4 ] A manufacturing method described in any one of the above. [ 6 In the general formulas (1) and (2), m is an integer of 20 to 150. 5 ] A manufacturing method described in any one of the above. [Effects of the Invention]

[0011] According to the present invention, a method for producing a maleimide polyethylene glycol lipid with a high introduction rate of maleimide groups can be provided. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described in detail.

[0013] The method for producing a maleimide polyethylene glycol lipid of the present invention is a method for producing a maleimide polyethylene glycol lipid represented by the following general formula (2), which includes a reaction step of deprotecting a maleimide polyethylene glycol lipid having a protecting group represented by the following general formula (1) by heating it in a solvent in the presence of silica gel. The maleimide polyethylene glycol lipid having the above-mentioned protecting group may be referred to as a "maleimide PEG lipid having a protecting group" or "compound 1." The maleimide polyethylene glycol lipid may be referred to as a "maleimide PEG lipid" or "compound 2." [ka] [In the formula, R 1 , R 2 , R 3 , and R 4are 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. n is an integer of 2 to 10 representing the number of repeating units of a methylene group. m is an integer of 10 to 2000 representing the number of repeating units of polyethylene glycol. Z represents any group selected from an ether group, a methylene group, and a dimethylvinylidene group. Y is any group represented by the following general formulas (3) to (6), [ka] (In formula (5), M represents Na or K, and l represents an integer of 1 to 5 that represents a repeating unit of a methylene group.) A is a group represented by the following general formula (7) or (8). [ka] (R 5 and R 6 are each independently any group selected from a hydrogen atom, a saturated or unsaturated acyl group having 8 to 24 carbon atoms, or a saturated or unsaturated alkyl group having 8 to 24 carbon atoms.)

[0014] [Maleimide PEG lipid having a protecting group represented by general formula (1)] In the production method of the present invention, a maleimide PEG lipid having a protecting group represented by general formula (1) is used as a raw material. [ka]

[0015] 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 each preferably independently a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. 1 , R 2 , R 3 , and R 4 is 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.

[0016] In formula (1), n ​​represents the number of repeating methylene group units and is an integer of 2 to 10. n is preferably an integer of 2 to 6, and more preferably 2.

[0017] In formula (1), m represents the number of repeating units of polyethylene glycol and is an integer of 10 to 2000. m is preferably an integer of 20 to 150, and more preferably an integer of 40 to 50. When m is equal to or greater than the lower limit of the above numerical range, the blood retention of LNP is improved, and when m is equal to or less than the upper limit of the above numerical range, the ligand introduction rate is improved.

[0018] In formula (1), Z represents any group selected from an ether group, a methylene group, and a dimethylvinylidene group. Among these, an ether group (i.e., an oxygen atom) is preferred for Z in view of ease of protection and deprotection of the maleimide.

[0019] Y in formula (1) is any of the groups represented by the following general formulae (3) to (6). [ka] In formula (5), M represents Na or K, and l represents a repeating unit of a methylene group and is an integer of 1 to 5. In each of the groups represented by formulas (3) to (6) above, "-" at both ends represents a bond. From the viewpoint of ease of production, Y is preferably a group represented by formula (3) (that is, an oxygen atom) among the above.

[0020] In formula (1), A is a group represented by the following general formula (7) or (8): Among these, A is preferably a group represented by formula (7). [ka] R 5 and R 6 are each independently any group selected from a hydrogen atom, a saturated or unsaturated acyl group having 8 to 24 carbon atoms, or a saturated or unsaturated alkyl group having 8 to 24 carbon atoms, and among these, a saturated or unsaturated acyl group having 8 to 24 carbon atoms or a saturated or unsaturated alkyl group having 8 to 24 carbon atoms is preferred, and a saturated or unsaturated acyl group having 8 to 24 carbon atoms is particularly preferred. The "-" at the bottom carbon atom in formula (7) and the "-" at the middle carbon atom in formula (8) each represent a bond, specifically a bond to Y in formula (1).

[0021] R 5 and R 6 In the formula (9), the saturated or unsaturated acyl group having 8 to 24 carbon atoms is represented by the following formula (9). [ka] In equation (9), R 7 R is a saturated or unsaturated hydrocarbon group having 7 to 23 carbon atoms, and the hydrocarbon group may be linear or branched. 7is preferably a saturated, linear hydrocarbon group having 10 to 22 carbon atoms, and particularly preferably a saturated, linear hydrocarbon group having 16 to 18 carbon atoms.

[0022] R 5 and R 6 In the formula (I), the saturated or unsaturated alkyl group having 8 to 24 carbon atoms is linear or branched. The saturated or unsaturated alkyl group is preferably a saturated and linear alkyl group having 10 to 22 carbon atoms, and more preferably a saturated and linear alkyl group having 17 to 19 carbon atoms.

[0023] The method for producing a maleimide PEG lipid having a protecting group represented by general formula (1) is not particularly limited. For example, the maleimide protected compound (formula (10)) and the PEG lipid (formula (11)) shown below can be prepared and reacted to obtain the maleimide PEG lipid. 1 , R 2 , R 3 , R 4 , Z, n, m, Y and A have the same meanings as those in general formula (1). [ka] The maleimide-protected compound can be obtained, for example, by the Diels-Alder reaction of maleimide with a five-membered ring compound such as furan or substituted furan. The above-mentioned PEG lipid can be produced, for example, from a raw material such as 1,2-isopropylideneglycerol, and a compound in which the hydroxyl group at the 3-position is modified with polyethylene glycol is used. The hydroxyl group at the end of the PEG is protected with a benzyl group, the isopropylidene is deprotected, and then the remaining two hydroxyl groups of the compound are modified with an acyl group or an alkyl group and debenzylated.

[0024] [Method for producing maleimide PEG lipid represented by general formula (2)] The method for producing a maleimide-PEG lipid represented by the following general formula (2) of the present invention is a production method comprising a reaction step of deprotecting a maleimide-PEG lipid having a protecting group represented by the above-mentioned general formula (1) by heating it in a solvent in the presence of silica gel. [ka] In formula (2), n, m, Y, and A have the same meanings as those in general formula (1) above.

[0025] 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, they are effective for the deprotection reaction. When they 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 increases, 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.

[0026] 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 maleimide-PEG lipid (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 can be suppressed, and when it is equal to or less than these upper limits, stirring of the reaction solution becomes easy.

[0027] 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 more easily, and if it is equal to or lower than these upper limits, deterioration of the maleimide can be suppressed.

[0028] 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, more preferably 5 to 20 times by weight, the amount of maleimide PEG lipid (compound 1) having a protecting group represented by general formula (1). If the amount of solvent used is equal to or higher than these lower limits, stirring of the reaction solution becomes easy, and if it is equal to or lower than these upper limits, economic efficiency is improved.

[0029] 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]

[0030] The present invention will now be described in more detail with reference to examples, in which NMR was used to analyze the maleimide ratio.

[0031] < 1 H-NMR analysis method 1 The 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.

[0032] Maleimide PEG lipids (B) having a protecting group were produced according to the following Production Examples (1-1) to (1-5). [ka] m=45

[0033] [Manufacturing Example 1-1] Synthesis of maleimide protected compound (A) 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 were added to a screw tube and stirred under a nitrogen atmosphere at 30°C for 8 hours. After cooling to room temperature, the precipitated crystals were collected by filtration and dried in vacuo to obtain the maleimide-protected product (A) (122.9 mg, 0.74 mmol) shown below. [ka]

[0034] [Manufacturing Example 1-2] Benzyl protection and deisopropylidenation steps of isopropylideneglycerol PEG A 2 L round-bottom flask equipped with a thermometer, nitrogen inlet, stirrer, and condenser was charged with 100 g (47.3 mmol) of isopropylideneglycerol PEG, 10.6 g (94.6 mmol) of potassium t-butoxide, and 1000 g of toluene. The mixture was stirred at room temperature for 10 minutes while blowing in nitrogen. Then, 12.0 g (94.8 mmol) of benzyl chloride was added and stirred at 80°C for 2 hours. The mixture was allowed to cool to room temperature, and 1930 g of a pH 1.4 aqueous phosphoric acid solution was added and stirred for 20 minutes. After allowing to stand for phase separation, the upper toluene layer was discarded, and 15 g of 85% phosphoric acid was added to bring the aqueous solution to pH 1.5. The mixture was then stirred at room temperature for 2 hours. 50 g of a 400 g / L NaOH aqueous solution was added to adjust the pH to 6.8, and 300 g of sodium chloride was added and dissolved. Then, 33.9 g of a 400 g / L NaOH aqueous solution was added to adjust the pH to 7.1. 300 g of chloroform was added to the mixture, stirred for 20 minutes, and allowed to stand for phase separation. The lower chloroform layer was then extracted. This extraction was repeated twice, and the chloroform layers from the first to third extractions were concentrated at 40°C using a rotary evaporator. 400 g of ethyl acetate was then added to form a solution, and 30 g of magnesium sulfate was added for dehydration. The magnesium sulfate was filtered off using pressure filtration and washed with 200 g of ethyl acetate. The resulting filtrate was cooled to below 5°C. 400 g of hexane was added to the mixture to cause reprecipitation. The precipitated crystals were filtered off using pressure filtration and dried under vacuum to obtain the glycerol benzyl-protected PEG (C) (94 g, 43.48 mmol) shown below. [ka] m=45

[0035] [Manufacturing Example 1-3] Diacylation step of glycerol benzyl-protected PEG A 1 L round-bottom flask equipped with a thermometer, nitrogen inlet, stirrer, and condenser was charged with 50 g (23.1 mmol) of glycerol benzyl-protected PEG(C), 26.3 g (92.5 mmol) of stearic acid, and 200 g of toluene. The mixture was stirred at 40 °C while blowing in nitrogen to form a solution. To this solution, 19.1 g (92.5 mmol) of N,N'-dicyclohexylcarbodiimide and 1.13 g (9.25 mmol) of 4-dimethylaminopyridine were added and stirred at 60 °C for 3 hours. The precipitate was removed by suction filtration, and the filtrate was concentrated at 50 °C on a rotary evaporator. 150 g of acetonitrile and 300 g of hexane were added to form a solution, and the upper hexane layer was discarded. Then, 200 g of hexane was added and the mixture was stirred at 50 °C for 30 minutes. A hexane wash was then performed, discarding the upper hexane layer. After repeating the same hexane washing three times, 150 g of acetonitrile was added to the acetonitrile layer, and 10 g of Kyoward 700 (Kyowa Chemical Industry Co., Ltd.) and 10 g of Kyoward 1000 (Kyowa Chemical Industry Co., Ltd.) were added and stirred at room temperature for 30 minutes. Kyoward 700 and Kyoward 1000 were then filtered off by suction filtration, concentrated at 50 ° C, and vacuum dried to obtain the benzyl-protected PEG lipid (D) (55.4 g, 20.56 mmol) shown below. [ka] m=45

[0036] [Manufacturing Example 1-4] Debenzylation step of benzyl-protected PEG lipids A 1 L round-bottom flask equipped with a thermometer, nitrogen inlet, stirrer, and condenser was charged with 53 g (19.7 mmol) of benzyl-protected PEG lipid (D), 26.5 g of 5% Pd / C, 422.1 g of methanol, and 71.9 g of cyclohexene. The mixture was stirred at 55 °C for 2 hours while blowing in nitrogen. The mixture was allowed to cool to 30 °C, and the 5% Pd / C was filtered off by pressure filtration using Kyoward 300 (Kyowa Chemical Industry Co., Ltd.) as a filter aid. The mixture was then washed four times with 318 g of chloroform. 0.053 g of dibutylhydroxytoluene was added to the filtrate, which was then concentrated at 55 °C on a rotary evaporator and dissolved in 424 g of ethyl acetate. 26.5 g of magnesium sulfate was added, the mixture was dehydrated, suction filtered, and washed with 106 g of ethyl acetate. The resulting filtrate was cooled to below 5 °C, and 954 g of methyl tert-butyl ether was added to perform reprecipitation. The precipitated crystals were collected by filtration and dried in vacuo to obtain the PEG lipid (E) shown below (42.2 g, 16.20 mmol). [ka] m=45

[0037] [Manufacturing Example 1-5] A 500 mL round-bottom flask equipped with a thermometer, nitrogen inlet, stirrer, and condenser was charged with 18.0 g (6.86 mmol) of PEG lipid (E), 45 g of dehydrated toluene, 180 g of chloroform, 1.36 g (8.23 mmol) of maleimide-protected derivative (A), and 9.00 g (34.3 mmol) of triphenylphosphine. The mixture was stirred at room temperature while blowing in nitrogen to form a solution. After cooling the solution to 10 °C, diisopropyl diazodicarboxylate (34.3 mmol) was added and stirred at room temperature for 1 hour. 1.0 g (34.3 mmol) of methanol was added and the mixture was concentrated at 50 °C. The concentrate was purified by column chromatography using silica gel (PSQ100B). Impurities were eluted with hexane / ethyl acetate = 20 / 80, and the target product was eluted with chloroform / methanol = 95 / 5. The fractions containing the target product were concentrated and then vacuum dried to obtain maleimide PEG lipid (B) (18.0 g, 6.49 mmol) having the protecting group shown below. [ka] m=45

[0038] Maleimide PEG lipid (F) having a protecting group was produced according to the following Production Examples (2-1) to (2-2). [ka] m=45

[0039] [Manufacturing Example 2-1] Synthesis of maleimide protected compound (G) 157.1 mg (1.61 mmol) of maleimide, 1.5 g of methyl t-butyl ether, and 877.7 mg (12.89 mmol) of 2-methylfuran were added to a screw tube and stirred under a nitrogen atmosphere at 30°C for 8 hours. After cooling to room temperature, the precipitated crystals were collected by filtration and dried in vacuo to obtain the maleimide-protected product (G) (483.1 mg, 0.48 mmol) shown below. [ka] m=45

[0040] [Manufacturing Example 2-2] Introduction of maleimide-protected compound (G) into PEG lipid 100.0 mg (0.0381 mmol) of PEG lipid (E), 0.25 g of dehydrated toluene, 1.0 g of chloroform, 11.2 mg (0.0580 mmol) of maleimide-protected compound (G), and 50.6 mg (0.193 mmol) of triphenylphosphine were placed in a 9 mL screw tube and stirred at room temperature while blowing in nitrogen to form a solution. After cooling the solution to 10 °C, 41.3 mg (0.204 mmol) of diisopropyl diazodicarboxylate was added and stirred at room temperature for 1 hour. 1.0 g (34.3 mmol) of methanol was added and concentrated at 50 °C. The concentrate was purified by column chromatography using silica gel (PSQ100B). Impurities were eluted with hexane / ethyl acetate = 20 / 80, and the target product was eluted with chloroform / methanol = 95 / 5. The fractions containing the target product were concentrated and then vacuum dried to obtain maleimide PEG lipid (F) (96.3 mg, 0.0346 mmol) having the protecting group shown below. [ka] m=45

[0041] Maleimide PEG lipid (H) having a protecting group was prepared according to the following Preparation Examples (3-1) and (3-2). [ka] m=45

[0042] [Manufacturing Example 3-1] Synthesis of maleimide protected compound (I) 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 were added to a screw tube and stirred under a nitrogen atmosphere at 50°C for 8 hours. After cooling to room temperature, the precipitated crystals were collected by filtration and dried in vacuo to obtain the maleimide-protected product (I) (522.3 mg, 2.70 mmol) shown below. [ka]

[0043] [Manufacturing Example 3-2] Introduction of maleimide-protected compound (I) into PEG lipid (E) A 9 mL screw tube was charged with 151 mg (0.0575 mmol) of PEG lipid (E), 0.37 g of dehydrated toluene, 1.5 g of chloroform, 12.2 mg (0.0680 mmol) of maleimide-protected derivative (I), and 74.6 mg (0.284 mmol) of triphenylphosphine. The mixture was stirred at room temperature while blowing in nitrogen to form a solution. After cooling the solution to 10 °C, 61.0 mg (0.302 mmol) of diisopropyl diazodicarboxylate was added and stirred at room temperature for 1 hour. 27.5 mg (0.859 mmol) of methanol was added and the mixture was concentrated at 50 °C. The concentrate was purified by column chromatography using silica gel (PSQ100B). Impurities were eluted with hexane / ethyl acetate = 20 / 80, and the target product was eluted with chloroform / methanol = 95 / 5. The fractions containing the target product were concentrated and then vacuum dried to obtain maleimide PEG lipid (H) (143.5 mg, 0.0515 mmol) having the protecting group shown below. [ka] m=45

[0044] [Example 1] Method for producing maleimide-PEG lipids 150 mg (0.0554 mmol) of maleimide PEG lipid (B) having a protecting group, 1.5 g of toluene, and 150 mg of silica gel (Fuji Silysia: PSQ100B) were added to a 100 mL side-arm test tube and stirred at 110 °C for 5 hours under a nitrogen atmosphere. After decanting the reaction solution, the target substance was extracted twice with 3 g of methanol from the silica gel. The reaction solution and extract were transferred to a 50 mL recovery flask and concentrated to dryness. After drying in vacuo for 6 hours, maleimide PEG lipid (J) was obtained (yield: 135.3 mg, 0.0512 mmol, 92% yield, 98% maleimidization rate). 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 m2 / g. [ka] m=45 Maleimide PEG lipid (J) obtained in Example 1 1 H-NMR showed no peaks at 6.51 ppm and 6.42 ppm attributable to maleimide-PEG lipid (B), indicating complete deprotection. Furthermore, the integral value of the peak at 6.71 ppm attributable to the vinyl protons of the maleimide group was 1.96, confirming that the desired maleimide-PEG lipid (J) represented by formula (2) was obtained with a maleimide conversion rate of 98%.

[0045] [Example 2] Method for producing maleimide-PEG lipids 100 mg (0.0359 mmol) of maleimide PEG lipid (F) having a protecting group, 1.0 g of toluene, and 100 mg of silica gel (Fuji Silysia: PSQ100B) were added to a 100 mL branched test tube and stirred at 110 °C for 5 hours under a nitrogen atmosphere. After decanting the reaction solution, the target substance was extracted twice with 2 g of methanol from the silica gel. The reaction solution and the extract were transferred to a 50 mL round-bottom flask and concentrated to dryness. After that, the mixture was dried in vacuo for 6 hours to obtain maleimide PEG lipid (J) (yield: 91.0 mg, 0.0327 mmol, yield 91%, maleimide conversion rate 98%). 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.

[0046] Maleimide PEG lipid (J) obtained in Example 2 1H-NMR showed no peaks at 6.55 ppm, 6.41 ppm, 6.35 ppm, or 6.24 ppm attributable to maleimide-PEG lipid (F), indicating that deprotection was complete. Furthermore, the integral value of the peak at 6.71 ppm attributable to the vinyl protons of the maleimide group was 1.96, confirming that the desired maleimide-PEG lipid (J) represented by formula (2) was obtained with a maleimide conversion rate of 98%.

[0047] [Example 3] Method for producing maleimide-PEG lipids 100 mg (0.0357 mmol) of maleimide PEG lipid (H) having a protecting group, 1.0 g of toluene, and 100 mg of silica gel (Fuji Silysia: PSQ100B) were added to a 100 mL side-arm test tube and stirred at 110 °C for 5 hours under a nitrogen atmosphere. After decanting the reaction solution, the target substance was extracted twice with 2 g of methanol from the silica gel. The reaction solution and extract were transferred to a 50 mL recovery flask and concentrated to dryness. After drying in vacuo for 6 hours, maleimide PEG lipid (J) was obtained (yield: 88.7 mg, 0.0336 mmol, yield 94%, maleimide conversion rate 99%). 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.

[0048] According to the H-NMR of the maleimide PEG lipid (J) obtained in Example 3, the peaks at 6.23 ppm and 6.35 ppm derived from the maleimide PEG lipid (H) having a protecting group were not present, indicating that deprotection was complete. In addition, the integral value of the peak at 6.71 ppm derived from the vinyl proton of the maleimide group was 1.98, confirming that the target maleimide PEG lipid (J) represented by formula (2) was obtained with a maleimide conversion rate of 99%.

[0049] [Comparative Example 1] Method for producing maleimide-PEG lipids 12 g (4.4 mmol) of maleimide PEG lipid (B) with a protecting group and 120 g of toluene were added to a 300 mL three-necked round-bottom flask and stirred at 110 °C for 5 hours under a nitrogen atmosphere. The reaction solution was transferred to a 300 mL recovery flask and concentrated to dryness. After that, the mixture was dried in vacuo for 6 hours to obtain maleimide PEG lipid (J) (yield: 11.73 g, 4.4 mmol, 100% yield, 89% maleimidation rate).

[0050] Maleimide PEG lipid (J) obtained in Comparative Example 1 1 H-NMR showed no peaks at 6.51 ppm and 6.42 ppm attributable to maleimide-PEG lipid (B), indicating complete deprotection. Furthermore, the integral value of the peak at 6.71 ppm attributable to the vinyl protons of the maleimide group was 1.78, confirming that the desired maleimide-PEG lipid (J) represented by formula (2) was obtained with a maleimide conversion rate of 89%.

[0051] Comparative Example 2 Method for producing maleimide-PEG lipids A 100 mL sidearm test tube was charged with 150 mg (0.0554 mmol) of maleimide-PEG lipid (B) bearing a protecting group, 1.5 g of toluene, and 150 mg of Kyoward 2000 (aluminum oxide-magnesium oxide solid solution, Kyowa Chemical Industry Co., Ltd.), and the mixture was stirred 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 maleimide-PEG lipid (J) (yield: 89.1 mg, 0.0337 mmol, 61% yield, 65% maleimide conversion).

[0052] Maleimide PEG lipid (J) obtained in Comparative Example 2 1H-NMR showed no peaks at 6.51 ppm and 6.42 ppm attributable to maleimide-PEG lipid (B), indicating complete deprotection. Furthermore, the integral value of the peak at 6.71 ppm attributable to the vinyl protons of the maleimide group was 1.30, confirming that the desired maleimide-PEG lipid (J) represented by formula (2) was obtained with a maleimide conversion rate of 65%.

[0053] As described above, a method for producing a maleimide polyethylene glycol lipid with a high maleimide conversion rate can be provided by heating a maleimide polyethylene glycol lipid having a protecting group in a solvent in the presence of silica gel.

Claims

1. A method for producing a maleimide polyethylene glycol lipid represented by the following general formula (2), comprising a reaction step of deprotecting a maleimide polyethylene glycol lipid having a protecting group represented by the following general formula (1) by heating in a solvent in the presence of silica gel. 【Chemical 1】 [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, a cyano group, a formyl group, an acyl group, a carboxyl group, an acyloxy group, and an alkylcarbonyloxymethyl group. n is an integer of 2 to 10 representing the number of repeating methylene group units. m is an integer of 10 to 2000 representing the number of repeating polyethylene glycol units. Z represents an ether group. Y is a group represented by the following formula (3): 【Chemistry 2】 A is a group represented by the following general formula (7) or (8). 【Chemistry 3】 (R ５ and R ６ are each independently a group selected from a hydrogen atom, a saturated or unsaturated acyl group having 8 to 24 carbon atoms, or a saturated or unsaturated alkyl group having 8 to 24 carbon atoms.

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. In the general formulas (1) and (2), A is a group represented by formula (7), and R ５ and R ６ are each independently selected from a saturated or unsaturated acyl group having 8 to 24 carbon atoms, or a saturated or unsaturated alkyl group having 8 to 24 carbon atoms.

4. The method according to any one of claims 1 to 3, wherein n is an integer of 2 to 6 in the general formulas (1) and (2).

5. The method according to any one of claims 1 to 4, wherein n is 2 in the general formulas (1) and (2).

6. The method according to any one of claims 1 to 5, wherein m in the general formulas (1) and (2) is an integer of 20 to 150.

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