Polyalkylene oxide-bonded lipid derivative production method and lipid derivative mixture

Abstract

Steps (A), (B), and (C) are carried out to produce a lipid derivative to which a polyalkylene oxide of formula (1) described in the specification is bonded. Step (A) is a step for obtaining a compound of formula (3) described in the specification by reacting a compound of formula (2) described in the specification and a solid or liquid alkylating agent in the presence of an alkali metal salt in an organic solvent at 10-40°C. Step (B) is a step for obtaining a compound of formula (4) by reacting the compound of formula (3) in an aqueous solution having a pH adjusted to not more than 3.0. Step (C) is a step for obtaining a lipid derivative of formula (1) by reacting the compound of formula (4) and at least one compound selected from the group consisting of fatty acids, fatty acid anhydrides, and fatty acid chlorides in an organic solvent.

Description

Process for producing a polyalkylene oxide-bound lipid derivative and lipid derivative mixture

[0001] The present invention relates to a process for producing a polyalkylene oxide-bound lipid derivative and a lipid derivative mixture.

[0002] In order to put into practical use nucleic acid therapy using oligonucleic acids such as siRNA and gene therapy using mRNA, pDNA and the like, effective and safe nucleic acid delivery carriers are required. Viral vectors are nucleic acid delivery carriers with high expression efficiency, but have practical problems from the viewpoint of safety. Therefore, development of non-viral nucleic acid delivery carriers that can be used more safely has been promoted.

[0003] Among these, lipid nanoparticles (LNPs), which are carriers using polyalkylene oxide-bound lipid derivatives, are currently the most commonly used non-viral nucleic acid delivery carriers. Polyalkylene oxide-bound lipid derivatives suppress aggregation and control size during carrier particle formation, and also affect the in vivo kinetics of LNPs.

[0004] Representative production methods for such polyalkylene oxide-bound lipid derivatives include, for example, the method described in Patent Document 1.

[0005] In the method described in Patent Document 1, first, isopropylidene glycerol is subjected to an addition reaction with alkylene oxide in a base to obtain a polyalkylene oxide compound having a hydroxy group at a terminal of the polyalkylene oxide. Next, methyl chloride is added to the polyalkylene oxide compound under high temperature and high pressure to methylate the hydroxy group at the terminal of the polyalkylene oxide. Subsequently, after hydrolyzing the product in an acidic aqueous solution, the polyalkylene oxide-bound lipid derivative is synthesized by esterification with a fatty acid, a fatty acid anhydride, a fatty acid chloride or the like.

[0006] In addition, representative production methods for polyalkylene oxide-bound lipid derivatives include, for example, the method described in Patent Document 1.

[0007] Specifically, in the method described in Patent Document 1, first, an alkylene oxide is added to an alcohol, and the resulting polyalkylene oxide compound is reacted with epichlorohydrin in the presence of tin tetrachloride. Then, after treating this reaction product with a base and an acid, it is esterified with a fatty acid, fatty acid anhydride, fatty acid chloride, etc., to synthesize a lipid derivative to which the polyalkylene oxide is bound.

[0008] Japanese Patent Application Publication No. 1994-145341

[0009] "Painting Engineering" 22 (1987), 397-403.

[0010] However, in the manufacturing method described in Patent Document 1, the methylation reaction temperature is 100°C, which is a high temperature condition. Therefore, polyalkylene oxide compounds react with each other during the reaction, producing dimers as a by-product. These dimers are then derivatized in a later step, raising concerns about an increase in the amount of by-products represented by formula (6), which will be described later.

[0011] Furthermore, Non-Patent Literature 1 shows that under high-temperature conditions, the ends of the polyalkylene oxide compound activated by the base are vinylized by a termination reaction. Subsequently, the vinyl group is hydrolyzed in a hydrolysis reaction step in an acidic aqueous solution, returning to the hydroxyl terminus, and is derivatized in a later step, raising concerns about an increase in the amount of by-product shown in formula (6), which will be described later. On the other hand, if the reaction temperature is low, the reaction is not completed, and unreacted raw materials remain, which are derivatized in a later step, raising concerns about an increase in the amount of by-product shown in formula (5), which will be described later.

[0012] Furthermore, in the manufacturing method described in Patent Document 1, when an alkylene oxide is added to an alcohol, a polyalkylene oxide diol compound is produced as a by-product when the alkylene oxide is added to the water in the alcohol. This by-product is then derivatized in a later step, raising concerns about an increase in the amount of the by-product shown in formula (6), which will be described later. In this case, if the number of carbon atoms in the alcohol used as a raw material is large and its boiling point is significantly higher than that of water, the amount of by-product diol compound can be reduced to some extent by removing water from the system by dehydrating under reduced pressure after charging the catalyst. However, if the number of carbon atoms in the raw material alcohol is small, the boiling point of the raw material is close to or lower than that of water, making it impossible to remove water from the system by dehydrating under reduced pressure. Moreover, the raw material alcohols used for polyalkylene oxide compounds used as pharmaceutical raw materials are mostly aliphatic alcohols with 1 to 4 carbon atoms, meaning they have a small number of carbon atoms. Furthermore, the by-products shown in formulas (5) and (6), which will be described later, have similar physical properties to the target product, making it difficult to adjust the amount of by-products through purification.

[0013] The object of the present invention is to provide a manufacturing method that allows for the adjustment of the by-product content to a small amount when producing lipid derivatives to which polyalkylene oxides are bound.

[0014] As a result of diligent research, the inventors have found that by reacting a polyalkylene oxide compound, obtained by adding an alkylene oxide to isopropylideneglycerol, with a solid or liquid alkylating agent in the presence of an alkali metal salt, then hydrolyzing the compound in an aqueous solution adjusted to a specific pH or lower, and reacting it with one or more compounds selected from the group consisting of fatty acids, fatty acid anhydrides, and fatty acid chlorides, a lipid derivative to which a polyalkylene oxide is bound can be obtained, and the content of by-products can be adjusted to a small amount.

[0015] The present invention relates to the following (1) to (8). (1) A method for producing a lipid derivative to which a polyalkylene oxide of the following formula (1) is bound, characterized by carrying out the following steps (A), (B) and (C). (In formula (1), R１ O is an oxyalkylene group having 2 to 4 carbon atoms, and n is an oxyalkylene group R having 2 to 4 carbon atoms. １ The average number of moles of O added is between 10 and 800, and R ２ R is an alkyl group having 1 to 4 carbon atoms. ３ CO and R ４ CO are each independently acyl groups having 4 to 24 carbon atoms. Step (A): A step to obtain a polyalkylene oxide compound of formula (3) by reacting the polyalkylene oxide compound of formula (2) below with a solid or liquid alkylating agent in an organic solvent in the presence of an alkali metal salt at a temperature of 10°C to 40°C. Step (B): A step to obtain a polyalkylene oxide compound of formula (4) below by reacting the polyalkylene oxide compound of formula (3) obtained in step (A) in an aqueous solution adjusted to a pH of 3.0 or less. Step (C): A step to obtain a lipid derivative to which the polyalkylene oxide represented by formula (1) is bound by reacting the polyalkylene oxide compound of formula (4) obtained in step (B) with one or more compounds selected from the group consisting of fatty acids, fatty acid anhydrides, and fatty acid chlorides in an organic solvent. (In equations (2), (3), and (4), R １ O is an oxyalkylene group having 2 to 4 carbon atoms, and n is an oxyalkylene group R having 2 to 4 carbon atoms. １ The average number of moles of O added is between 10 and 800, and R ２ (These are alkyl groups with 1 to 4 carbon atoms.)

[0016] (2) The method for producing a polyalkylene oxide-bonded lipid derivative according to (1), wherein the alkylating agent in the step (A) is one or more alkylating agents selected from the group consisting of bromomethane, iodomethane, N,N-dimethylformamide dimethyl acetal, methyl fluorosulfonate, 1-methyl-3-p-tolyltriazene, dimethyl carbonate, dimethyl sulfate, methyl N,N'-diisopropylcarbamimidate, methyl methanesulfonate, trimethyl orthoformate, tetramethylammonium chloride, methyl p-toluenesulfonate, methyl trifluoromethanesulfonate and trimethyloxonium tetrafluoroborate.

[0017] (3) The method for producing a polyalkylene oxide-bonded lipid derivative according to (1) or (2), wherein the alkali metal salt in the step (A) is one or more alkali metal salts selected from the group consisting of lithium tert-butoxide, sodium tert-butoxide, potassium tert-butoxide, lithium tert-pentoxide, sodium tert-pentoxide, potassium tert-pentoxide, n-butyllithium, sec-butyllithium, tert-butyllithium, lithium diisopropylamide, lithium bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide and potassium bis(trimethylsilyl)amide.

[0018] (4) In the formula (1), R １ O is an oxyethylene group, and R ２ is a methyl group, which is the method for producing a polyalkylene oxide-bonded lipid derivative according to (1) or (2).

[0019] (5) In the formula (1), R ３ CO and R ４ CO each have 12 to 20 carbon atoms, which is the method for producing a polyalkylene oxide-bonded lipid derivative according to (1) or (2).

[0020] (6) A lipid derivative mixture characterized by containing 99.0% by mass or more and 99.999% by mass or less of the lipid derivative of formula (1) below, and containing at least 0.001% by mass or more and 0.5% by mass or less of the lipid derivative of formula (5) and the lipid derivative of formula (6) in total. (In equations (1), (5), and (6), R １ O is an oxyalkylene group having 2 to 4 carbon atoms, and n is an oxyalkylene group R having 2 to 4 carbon atoms. １ The average number of moles of O added is between 10 and 800, and R ２ R is an alkyl group having 1 to 4 carbon atoms. ３ CO, R ４ CO, R ５ CO, R ６ CO and R ７ CO are, independently, acyl groups with 4 to 24 carbon atoms.

[0021] (7) In equations (1), (5), and (6) above, R １ O is an oxyethylene group, R ２ A mixture of lipid derivatives of (6), characterized in that the group is a methyl group.

[0022] (8) In equations (1), (5), and (6) above, R ３ CO, R ４ CO, R ５ CO, R ６ CO and R ７ A lipid derivative mixture of (6) or (7), characterized in that the number of carbon atoms in CO is 12 or more and 20 or less, respectively.

[0023] The present invention's method for producing polyalkylene oxide-bound lipid derivatives allows for the production of small amounts of by-products, making it extremely useful for non-viral nucleic acid delivery carrier applications.

[0024] Furthermore, the present invention provides a lipid derivative mixture to which polyalkylene oxides are bound, successfully reducing the by-products of formulas (5) and (6) to a practically unrealistic level. It was found that further reducing the content of by-products of formulas (5) and (6) is not practical. Moreover, such a mixture would allow for more precise control of the in vivo dynamics of LNPs, for example, in non-viral nucleic acid delivery carrier applications, and thus has significant industrial value.

[0025] The present invention relates to a method for producing a lipid derivative to which a polyalkylene oxide of formula (1) is bound, and is a method that carries out steps (A), (B), and (C).

[0026] (Step (A)) This step involves reacting a polyalkylene oxide compound of formula (2) with a solid or liquid alkylating agent in an organic solvent in the presence of an alkali metal salt at a temperature of 10°C or higher and 40°C or lower to obtain a polyalkylene oxide derivative of formula (3).

[0027]

[0028] Here, in equations (2), (3), and equations (1), (5), and (6) described later, R １ O is an oxyalkylene group having 2 to 4 carbon atoms, and n is an oxyalkylene group R having 2 to 4 carbon atoms. １ The average number of moles of O added is between 10 and 800, and R ２ These are alkyl groups having 1 to 4 carbon atoms.

[0029] R １ The oxyalkylene group represented by O is an oxyalkylene group having 2 to 4 carbon atoms (more preferably 2 or 3 carbon atoms). １Examples of O include oxyethylene groups, oxypropylene groups, oxytrimethylene groups, and oxybutylene groups. Among these, oxyethylene groups and oxypropylene groups are preferred, and oxyethylene groups are particularly preferred. In addition, there are as many oxyalkylene groups as there are n molecules in one molecule, but these oxyalkylene groups may be a single type or a combination of two or more types, and there are no restrictions on the combination. They may also be in a block-like or random arrangement.

[0030] n represents the average number of moles of oxyalkylene groups added and is a number between 10 and 800. When n is 10 or greater, the delivery effect is enhanced when the lipid derivative to which the polyalkylene oxide of the present invention is bound is used in a drug delivery system. From this viewpoint, it is even more preferable that n is 20 or greater. Furthermore, when n is 800 or less, the viscosity of the raw material, the polyalkylene oxide compound of formula (2), does not increase significantly when producing the lipid derivative to which the polyalkylene oxide is bound, thus improving workability. From this viewpoint, it is preferable that n is 500 or less.

[0031] R ２ The alkyl group represented by is an alkyl group having 1 to 4 carbon atoms (preferably 1 carbon atom). ２ Examples of such groups include methyl, ethyl, propyl, isopropyl, butyl, and isobutyl groups.

[0032] The alkylating agent used in step (A) is preferably one that can be dissolved in an organic solvent. Specifically, these include bromomethane, iodomethane, N,N-dimethylformamide dimethyl acetal, methyl fluorosulfonate, 1-methyl-3-p-tolyltriazene, dimethyl carbonate, dimethyl sulfate, N,N'-diisopropylcarbamimoidate methyl, methyl methanesulfonate, trimethyl orthoformate, tetramethylammonium chloride, p-toluenesulfonate methyl, trifluoromethanesulfonate methyl, and trimethyloxonium tetrafluoroborate. Among these, methyl methanesulfonate is particularly preferred.

[0033] It is desirable to set the amount of the polyalkylene oxide compound of formula (2) and the alkylating agent charged in a molar ratio (polyalkylene oxide compound of formula (2):alkylating agent) of 1:1 or greater than 1:1 (excess alkylating agent). Specifically, the molar ratio of the polyalkylene oxide compound of formula (2) and the alkylating agent is preferably 1:1 to 1:5, and more preferably 1:1 to 1:3.

[0034] If there is an excess of alkylating agent relative to the polyalkylene oxide compound of formula (2), it can be easily removed by purification. On the other hand, if there is an excess of polyalkylene oxide compound, unreacted polyalkylene oxide compound will remain after the reaction is complete. The unreacted polyalkylene oxide compound will be derivatized in a later step to become the by-product of formula (5).

[0035] The alkali metal salt used in step (A) is preferably one that can be dissolved in an organic solvent and has low nucleophilicity. Specifically, these include lithium tert-butoxide, sodium tert-butoxide, potassium tert-butoxide, lithium tert-pentoxide, sodium tert-pentoxide, potassium tert-pentoxide, n-butyllithium, sec-butyllithium, tert-butyllithium, lithium diisopropylamide, lithium bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide, and potassium bis(trimethylsilyl)amide. Among these, lithium tert-butoxide, sodium tert-butoxide, and potassium tert-butoxide are preferred, with potassium tert-butoxide being particularly preferred.

[0036] Furthermore, it is desirable to set the amount of the polyalkylene oxide compound of formula (2) and the alkali metal salt charged so that the molar ratio (polyalkylene oxide compound of formula (2):alkali metal salt) is 1:1 or greater than 1:1 (alkali metal salt). Specifically, the molar ratio of the polyalkylene oxide compound of formula (2) and the alkali metal salt is 1:1 to 1:5, preferably 1:1 to 1:3.

[0037] If there is an excess of alkali metal salt relative to the polyalkylene oxide compound of formula (2), the alkali metal salt can be easily removed by purification. On the other hand, if there is an excess of polyalkylene oxide compound, unreacted polyalkylene oxide compound will remain after the reaction is complete. The unreacted polyalkylene oxide compound will be derivatized in a later step to become the by-product of formula (5), so the above molar ratio is preferable.

[0038] The organic solvent used in step (A) can be used without particular limitations, as long as it can dissolve the raw materials and reaction products and does not react with them, or reacts with them very little. Examples include aprotic solvents such as acetonitrile, ethyl acetate, dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, and toluene. Among these, toluene is preferred. Organic solvents having hydroxyl groups, such as ethanol, are undesirable because they react with the alkylating agent.

[0039] Furthermore, the amount of organic solvent used is preferably 1 to 30 times the mass of the compound of formula (2), and more preferably 1 to 10 times the mass.

[0040] The reaction temperature in step (A) is 10 to 40°C, preferably 20 to 30°C. The reaction time is preferably 1 hour or more, preferably 2 to 15 hours. If the reaction temperature is below 10°C, the reaction may not be completed and raw materials may remain. If the temperature exceeds 40°C, the raw materials may react with each other, potentially increasing the amount of dimer produced, which is a precursor to the by-product shown in formula (5).

[0041] (Step (B)) This step involves reacting the polyalkylene oxide compound of formula (3) obtained in step (A) in an aqueous solution adjusted to a pH of 3.0 or lower to obtain the polyalkylene oxide compound of formula (4).

[0042]

[0043] In equation (4), and in equations (1), (5), and (6) described later, R １ O, n, R ２ This is the same as what was mentioned above.

[0044] An "acidic aqueous solution with a pH of 3.0 or lower" refers to an aqueous solution containing an acidic reagent with a pH of 3.0 or lower. Here, the acidic reagent is preferably water-soluble and has a pKa of 1 to 5. Specifically, this includes acetic acid, phosphoric acid, hydrogen chloride, nitric acid, and sulfuric acid. Among these, phosphoric acid is particularly preferred.

[0045] Furthermore, the amount of acidic aqueous solution used is preferably 1 to 30 times the mass of the compound of formula (3), and more preferably 1 to 10 times the mass.

[0046] The amount of acidic reagent added to prepare an acidic aqueous solution with a pH of 3.0 or lower 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 polyalkylene oxide compound.

[0047] If the reaction temperature in step (B) is below 10°C, the reaction may not be completed and raw materials may remain. If the reaction temperature exceeds 80°C, the polyalkylene oxide chain portion may decompose due to increased thermal history, potentially reducing purity. For this reason, the reaction temperature in step (B) is preferably 10 to 80°C, and more preferably 20 to 40°C. The reaction time in step (B) is preferably 1 hour or more, and more preferably 2 to 15 hours.

[0048] (Step (C)) This step involves reacting the polyalkylene oxide compound of formula (4) obtained in step (B) with one or more compounds selected from the group consisting of fatty acids, fatty acid anhydrides, and fatty acid chlorides in an organic solvent to obtain a lipid derivative to which the polyalkylene oxide represented by formula (1) is bound.

[0049]

[0050] In formula (1), R １ O, n, R ２ This is the same as described above. In equation (1), R ３ CO and R ４ Each of the CO groups is independently an acyl group having 4 to 24 carbon atoms.

[0051] R ３ CO, R ４The number of carbon atoms in CO is preferably 12 or more and 20 or less, respectively. ３ CO, R ４ Each CO group may independently be a saturated or unsaturated acyl group, and may be a linear or branched acyl group.

[0052] This acyl group (R ３ CO, R ４ CO) is usually a fatty acid (R ３ COOH, R ４ Derived from COOH. ３ COOH, R ４ Specific examples of COOH include acyl groups derived from saturated and unsaturated linear or branched fatty acids such as lauric acid, tridecyl acid, myristic acid, pentadecyl acid, palmitic acid, margaric acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, vaccenic acid, elaidic acid, linoleic acid, nonadecyl acid, arachidic acid, and arachidonic acid. ３ COOH, R ４ COOH can be the same or different from each other.

[0053] In step (C), the polyalkylene oxide compound of formula (4) is reacted with one or more compounds selected from the group consisting of fatty acids, fatty acid anhydrides, and fatty acid chlorides in an organic solvent to obtain a lipid derivative to which the polyalkylene oxide represented by formula (1) is bound.

[0054] This fatty acid preferably has 4 to 24 carbon atoms, more preferably 12 or more, and even more preferably 20 or less. Furthermore, this fatty acid may be saturated or unsaturated, and may be linear or branched. Examples include lauric acid, tridecyl acid, myristic acid, pentadecyl acid, palmitic acid, margaric acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, vaccenic acid, elaidic acid, linoleic acid, nonadecyl acid, arachidic acid, and arachidonic acid.

[0055] After the reaction of the polyalkylene oxide compound of formula (4) with the fatty acid, unreacted fatty acids can be easily removed by purification. On the other hand, if the polyalkylene oxide compound is in excess, unreacted polyalkylene oxide compounds will remain after the reaction is complete. Removing the remaining polyalkylene oxide compounds is often difficult, making it difficult to obtain lipid derivatives bound to polyalkylene oxides with small amounts of by-products; therefore, the above molar ratio is preferred.

[0056] From this perspective, it is desirable to set the amount of the polyalkylene oxide compound of formula (4) and the fatty acid to be charged in a molar ratio (polyalkylene oxide compound of formula (4):alkylating agent) of 1:2 or greater than 1:2 (excess fatty acid). Specifically, the molar ratio of the polyalkylene oxide compound of formula (4) and the fatty acid is 1:2 to 1:5, preferably 1:2 to 1:4, and more preferably 1:3.

[0057] Furthermore, when carrying out the reaction in step (C) using fatty acids, the reaction can be made more efficient by using a condensing agent and a basic catalyst.

[0058] Examples of such condensing agents include N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, N,N'-di-tert-butylcarbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidemetho-p-toluenesulfonate, N,N'-carbonyldiimidazole, and 1,1'-carbonyldi(1,2,4-triazole). Among these, N,N'-dicyclohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride are preferred.

[0059] It is desirable to set the amount of the polyalkylene oxide compound of formula (4) and the condensing agent charged in a molar ratio (polyalkylene oxide compound of formula (4): condensing agent) of 1:2 or greater than 1:2 (excess condensing agent). Specifically, the molar ratio of the polyalkylene oxide compound of formula (4) and the condensing agent is 1:2 to 1:5, preferably 1:2 to 1:4, and more preferably 1:3.

[0060] If there is an excess of the bonding agent, it can be easily removed by purification. On the other hand, if there is an excess of the polyalkylene oxide compound, unreacted polyalkylene oxide compound will remain after the reaction is complete. Removing the remaining polyalkylene oxide compound is often difficult, and it becomes difficult to obtain a lipid derivative to which polyalkylene oxide is bound in small amounts as a by-product; therefore, the above molar ratio is preferred.

[0061] Examples of basic catalysts used when carrying out the reaction in step (C) using fatty acids include 4-dimethylaminopyridine, 4-dimethylamino-2-methylpyridine, 4-diethylaminopyridine, 4-pyrrolidinopyridine, and 4-pyrrolidino-2-methylpyridine. Among these, 4-dimethylaminopyridine is preferred.

[0062] The amount of basic catalyst used should preferably be 0.1 to 3 mol times, more preferably 0.1 to 1 mol times, the amount of the polyalkylene oxide compound of formula (4) in terms of mole ratio. Using an amount of basic catalyst of 0.1 times the mole or more allows the reaction to proceed more efficiently. Furthermore, using an amount of basic catalyst of 3 times the mole or less makes it easier to remove the basic catalyst by purification, which is preferable.

[0063] In the reaction of step (C), the fatty acid anhydride used in the reaction with the polyalkylene oxide compound of formula (4) preferably has 8 to 48 carbon atoms, more preferably has 24 or more carbon atoms, and even more preferably has 40 or fewer carbon atoms. The fatty acid anhydride used in the reaction of step (C) may be a saturated or unsaturated fatty acid anhydride, and may be a linear or branched fatty acid anhydride. Examples include lauric acid anhydride, tridecyl acid anhydride, myristic acid anhydride, pentadecyl acid anhydride, palmitic acid anhydride, margaric acid anhydride, palmitoleic acid anhydride, stearic acid anhydride, isostearic acid anhydride, oleic acid anhydride, vaccenic acid anhydride, elaidic acid anhydride, linoleic acid anhydride, nonadecyl acid anhydride, arachidic acid anhydride, and arachidonic acid anhydride.

[0064] It is desirable to set the amount of the polyalkylene oxide compound of formula (4) and the fatty acid anhydride to be in a molar ratio (polyalkylene oxide compound of formula (4): fatty acid anhydride) of 1:2 or greater than 1:2 (excess fatty acid anhydride). Specifically, the molar ratio of the polyalkylene oxide compound of formula (4) to the fatty acid anhydride is preferably 1:2 to 1:5, more preferably 1:2 to 1:4, and particularly preferably 1:2 to 1:3.

[0065] If the fatty acid anhydride is in excess relative to the polyalkylene oxide compound of formula (4), it can be easily removed by purification. On the other hand, if the polyalkylene oxide compound is in excess, unreacted polyalkylene oxide compound will remain after the reaction is complete. Removing the remaining polyalkylene oxide compound is often difficult, and it becomes difficult to obtain a lipid derivative to which polyalkylene oxide is bound with a small amount of by-products, so the above molar ratio is preferred.

[0066] Furthermore, when carrying out the reaction in step (C) using fatty acid anhydrides, the reaction can be carried out more efficiently by using a basic catalyst.

[0067] Examples of these basic catalysts include 4-dimethylaminopyridine, 4-dimethylamino-2-methylpyridine, 4-diethylaminopyridine, 4-pyrrolidinopyridine, and 4-pyrrolidino-2-methylpyridine. Among these, 4-dimethylaminopyridine is preferred.

[0068] The amount of basic catalyst used should preferably be 0.1 to 3 mol times, more preferably 0.1 to 1 mol, of the polyalkylene oxide compound represented by formula (4), in terms of mole ratio. Using less than 0.1 moles of basic catalyst is undesirable because the reaction will not proceed efficiently. Using more than 3 moles is also undesirable because it becomes difficult to remove the basic catalyst by purification.

[0069] The fatty acid chloride used in the reaction of step (C) has 4 to 24 carbon atoms, more preferably 12 or more, and even more preferably 20 or less. The fatty acid chloride used in the reaction of step (C) may be saturated or unsaturated fatty acid chloride, and may be a straight-chain or branched-chain fatty acid. Examples include lauric acid chloride, tridecyl acid chloride, myristic acid chloride, pentadecyl acid chloride, palmitic acid chloride, margaric acid chloride, palmitoleic acid chloride, stearate chloride, isostearate chloride, oleic acid chloride, vaccenate chloride, elaidic acid chloride, linoleic acid chloride, nonadecyl acid chloride, arachidic acid chloride, and arachidonic acid chloride.

[0070] It is desirable to set the amount of the polyalkylene oxide compound of formula (4) and fatty acid chloride charged in a molar ratio (polyalkylene oxide compound of formula (4): fatty acid chloride) of 1:2 or greater than 1:2 (excess fatty acid chloride). Specifically, the molar ratio of the polyalkylene oxide compound of formula (4) to fatty acid chloride is 1:2 to 1:5, preferably 1:2 to 1:4, and more preferably 1:2 to 1:3.

[0071] If there is an excess of fatty acid chloride relative to the polyalkylene oxide compound of formula (4), it can be easily removed by purification. On the other hand, if there is an excess of polyalkylene oxide compound, unreacted polyalkylene oxide compound will remain after the reaction is complete. Removing the remaining polyalkylene oxide compound is often difficult, and it becomes difficult to obtain a lipid derivative to which polyalkylene oxide is bound in small amounts as a by-product; therefore, the above molar ratio is preferred.

[0072] The organic solvent used in step (C) is not particularly limited as long as it can dissolve the raw materials and reaction products and does not react with them, or reacts with them very little. Examples include aprotic solvents such as acetonitrile, dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, and toluene. Of these, toluene is preferred. Organic solvents having hydroxyl groups, such as ethanol, are not preferred because they react with fatty acid compounds.

[0073] Furthermore, the amount of organic solvent used is preferably 1 to 30 times the mass of the compound represented by formula (4), and more preferably 1 to 10 times the mass.

[0074] The reaction temperature in step (C) is 20 to 80°C, preferably 40 to 60°C. The reaction time is preferably 1 hour or more, preferably 2 to 15 hours. If the reaction temperature is below 20°C, the reaction may not be completed and raw materials may remain. If the reaction temperature exceeds 80°C, the polyalkylene oxide chain portion may decompose due to increased thermal history, which is undesirable as it may reduce purity.

[0075] (Lipid Derivative Mixture) The lipid derivative mixture to which polyalkylene oxide is bonded according to the present invention is a mixture of the lipid derivative of formula (1) and at least one of the lipid derivative of formula (2) and the lipid derivative of formula (3). The content of the lipid derivative of formula (1) in the mixture is 99.0% by mass or more and 99.999% by mass or less, and the total content of the lipid derivative of formula (2) and the lipid derivative of formula (3) is 0.001% by mass or more and 0.5% by mass or less.

[0076] In equations (1), (5), and (6) above, R ３ CO, R ４ CO, R ５ CO, R ６ CO and R ７ The acyl groups represented by CO are, independently, acyl groups with 4 to 24 carbon atoms. ３ CO, R ４ CO, R ５ CO, R ６ CO and R ７ The number of carbon atoms in CO is preferably 12 or more and 20 or less. This number of carbon atoms is more preferably 14 or more, and even more preferably 18 or less. Also, R ３ CO, R ４ CO, R ５ CO, R ６ CO and R ７ Each CO group may independently be a saturated or unsaturated acyl group, and may be a linear or branched acyl group.

[0077] This acyl group (R ３ CO, R ４ CO, R ５ CO, R ６ CO and R ７ CO) is usually a fatty acid (R ３ COOH, R ４ COOH, R ５ COOH, R ６ COOH and R ７ Derived from COOH. ３ COOH, R ４ COOH, R ５ COOH, R ６ COOH and R ７ Specific examples of COOH include acyl groups derived from saturated and unsaturated linear or branched fatty acids such as lauric acid, tridecyl acid, myristic acid, pentadecyl acid, palmitic acid, margaric acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, vaccenic acid, elaidic acid, linoleic acid, nonadecyl acid, arachidic acid, and arachidonic acid. ３ COOH, R ４ COOH, R ５ COOH, R６ COOH and R ７ COOH can be the same or different from each other.

[0078] (Example 1) (Synthesis of the lipid derivative of formula (1): 1-(methylpolyoxyethylene)-2,3-dimyristoylglycerol)

[0079] (Step (A)) To 1-(hydroxypolyoxyethylene)-2,3-isopropylideneglycerol (polyalkylene oxide compound of formula (2): molecular weight 2000, 250 g, 125 mmol), toluene (1445 mL) was added and dissolved by stirring at 35°C. Then potassium-tert-butoxide (alkali metal salt: 33.2 g, 296 mmol) and methyl methanesulfonate (alkylating agent: 32.7 g, 296 mmol) were added and the mixture was reacted at 30°C for 4 hours.

[0080] (Process (B)) The reaction solution obtained in Process (A) was filtered, and ion-exchanged water (1250 mL) was added to the filtrate and stirred at 25°C for 30 minutes to extract the reactants into the aqueous layer. After allowing the layers to stand and separate, the organic layer was removed, 85% phosphoric acid was added, and the pH of the aqueous layer was adjusted to 1.5. The reaction was carried out at 25°C for 2 hours. 400 g / L sodium hydroxide aqueous solution was added to the reaction solution and the pH was adjusted to 6.7. Sodium chloride (200 g, 3.42 mol) was added and dissolved by stirring at room temperature, and then 400 g / L sodium hydroxide aqueous solution was added to adjust the pH to 7.2. Chloroform (502 mL) was added to the adjusted solution and stirred at 25°C for 20 minutes to extract the target product. After allowing the layers to stand and separate, the organic layer was removed, and the extraction process was repeated twice in the same manner. The organic layer after extraction was concentrated, diluted with ethyl acetate (1665 mL), and then hexane (1517 mL) was added and the mixture was stirred at 25°C for 30 minutes. The precipitated crystals were filtered and dried to obtain 182 g of 1-(methylpolyoxyethylene-oxy)-2,3-propanediol (polyalkylene oxide compound of formula (4): molecular weight 2000, yield 72.4 mol%).

[0081] (Step (C)) To 1-(methylpolyoxyethylene-oxy)-2,3-propanediol (polyalkylene oxide compound of formula (4): molecular weight 2000, 100 g, 50.0 mmol) and myristic anhydride (fatty acid anhydride: 52.6 g, 120 mmol), toluene (organic solvent: 462 mL) was added and dissolved by stirring at 35°C. Then 4-dimethylaminopyridine (2.50 g, 20 mmol) was added and the mixture was reacted at 40°C for 3 hours. The reaction mixture was concentrated and acetonitrile (127 mL) was added to perform azeotropic reaction. After azeotropic reaction, acetonitrile (506 mL) and hexane (607 mL) were added and the mixture was stirred at 25°C for 30 minutes and allowed to stand to separate into layers. After separating into layers, the hexane layer was removed, and hexane (607 mL) was added to the acetonitrile layer. The hexane washing treatment was repeated four times in the same manner.

[0082] The acetonitrile layer after hexane washing was concentrated and diluted with ethyl acetate (555 mL). Then, Kyoward #700 (20.0 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) and Kyoward #1000 (10.0 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) were added as adsorbents, and the mixture was stirred at 25°C for 30 minutes. After filtering the adsorbents, the adsorbent treatment was repeated twice in the same manner. Subsequently, the filtrate was concentrated and dried to obtain 82.5 g of 1-(methylpolyoxyethylene)-2,3-dimyristoylglycerol (lipid derivative of formula (1): yield 66.0 mol%).

[0083] (Example 2) (Synthesis of the lipid derivative of formula (1): 1-(methylpolyoxyethylene)-2,3-distearoylglycerol)

[0084] (Step (C)) To the 1-(methylpolyoxyethylene-oxy)-2,3-propanediol (molecular weight 2000, 40.0 g, 20.0 mmol) and stearic acid (fatty acid: 18.8 g, 66.0 mmol) obtained in Step (B) of Example 1, toluene (organic solvent: 185 mL) was added and stirred at 40°C until dissolved. Then, 4-dimethylaminopyridine (2.50 g, 20.0 mmol) and N,N'-dicyclohexylcarbodiimide (13.6 g, 66.0 mmol) were added and the mixture was reacted at 60°C for 1 hour. After cooling the reaction mixture to 25°C, acetonitrile (506 mL) was added and the mixture was stirred at 25°C for 30 minutes, and the precipitate was removed by filtration. The filtrate was then concentrated, and acetonitrile (102 mL) was added to perform azeotropic reaction. After azeotropic mixing, acetonitrile (305 mL) and hexane (243 mL) were added, and the mixture was stirred at 25°C for 30 minutes, followed by standing to allow the layers to separate. After standing to separate the layers, the hexane layer was removed, and hexane (243 mL) was added to the acetonitrile layer. The hexane washing treatment was then repeated twice in the same manner.

[0085] The acetonitrile layer after hexane washing was concentrated, and ethyl acetate (88.8 mL) was added for azeotropic mixing. After azeotropic mixing, ethyl acetate (222 mL) was added to dilute the mixture, and then Kyoward #700 (8.00 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) and Kyoward #1000 (8.00 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) were added as adsorbents, and the mixture was stirred at 25°C for 30 minutes. After filtering the adsorbents, the adsorbent treatment was repeated twice in the same manner. Subsequently, the filtrate was concentrated, and ethyl acetate (88.8 mL) was added for azeotropic mixing. After azeotropic mixing, ethyl acetate (133 mL) and methyl-tert-butyl ether (378 mL) were added and dissolved, and the mixture was stirred at 6°C for 1 hour. The precipitated crystals were filtered and dried to obtain 35.2 g of 1-(methylpolyoxyethylene)-2,3-distearoylglycerol (lipid derivative of formula (1): yield 70.4 mol%).

[0086] (Example 3) (Synthesis of the lipid derivative of formula (1): 1-(methylpolyoxyethylene)-2,3-dimyristoylglycerol)

[0087] (Step (A)) Toluene (organic solvent: 347 mL) was added to 1-(hydroxypolyoxyethylene)-2,3-isopropylideneglycerol (polyalkylene oxide compound of formula (2): molecular weight 5000, 60.0 g, 12.0 mmol), and the mixture was stirred and dissolved at 35°C. Then potassium-tert-butoxide (alkali metal salt: 3.37 g, 30.0 mmol) and methyl methanesulfonate (alkylating agent: 3.30 g, 30.0 mmol) were added, and the mixture was reacted at 30°C for 6 hours.

[0088] (Process (B)) The reaction solution obtained in Process (A) was filtered, and 300 mL of deionized water was added to the filtrate and stirred at 25°C for 30 minutes to extract the reactants into the aqueous layer. After allowing the layers to stand and separate, the organic layer was removed, 85% phosphoric acid was added, and the pH of the aqueous layer was adjusted to 1.5. The reaction was carried out at 25°C for 2 hours. 400 g / L sodium hydroxide aqueous solution was added to the reaction solution and the pH was adjusted to 6.7. Sodium chloride (80.7 g, 1.38 mol) was added and dissolved by stirring at room temperature, and then 400 g / L sodium hydroxide aqueous solution was added to adjust the pH to 7.2. Chloroform (120 mL) was added to the adjusted solution and stirred at 25°C for 20 minutes to extract the target product. After allowing the layers to stand and separate, the organic layer was removed, and the extraction process was repeated twice in the same manner. The organic layer after extraction was concentrated, diluted with ethyl acetate (400 mL), then hexane (364 mL) was added, and the mixture was stirred at 25°C for 30 minutes. The precipitated crystals were filtered and dried to obtain 57.1 g of 1-(methylpolyoxyethylene-oxy)-2,3-propanediol (polyalkylene oxide compound of formula (4): molecular weight 5000, yield 95.2 mol%).

[0089] (Step (C)) To the 1-(methylpolyoxyethylene-oxy)-2,3-propanediol (polyalkylene oxide compound of formula (4): molecular weight 5000, 10.0 g, 2.00 mmol) and myristic anhydride (fatty acid anhydride: 2.11 g, 4.80 mmol) obtained in Step (B), toluene (organic solvent: 46.2 mL) was added and dissolved by stirring at 35°C. Then, 4-dimethylaminopyridine (2.50 g, 0.80 mmol) was added and the mixture was reacted at 40°C for 3 hours. The reaction mixture was concentrated and acetonitrile (12.7 mL) was added to perform azeotropic reaction. After azeotropic reaction, acetonitrile (50.6 mL) and hexane (60.7 mL) were added and the mixture was stirred at 25°C for 30 minutes and allowed to stand to separate into layers. After standing to separate into layers, the hexane layer was removed, and hexane (60.7 mL) was added to the acetonitrile layer. The hexane washing treatment was repeated four times in the same manner. The acetonitrile layer after hexane washing was concentrated and diluted with ethyl acetate (55.5 mL). Then, Kyoward #700 (2.00 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) and Kyoward #1000 (1.00 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) were added as adsorbents, and the mixture was stirred at 25°C for 30 minutes. After filtering the adsorbents, the adsorbent treatment was repeated twice in the same manner. Subsequently, the filtrate was concentrated and dried to obtain 8.80 g of 1-(methylpolyoxyethylene-oxy)-2,3-propanediol (lipid derivative of formula (1): yield 88.0 mol%).

[0090] (Example 4) (Synthesis of the lipid derivative of formula (1): 1-(methylpolyoxyethylene)-2,3-dipalmitoylglycerol)

[0091] (Step (C)) To the 1-(methylpolyoxyethylene-oxy)-2,3-propanediol (molecular weight 2000, 40.0 g, 20 mmol) and palmitic anhydride (fatty acid anhydride: 31.7 g, 64 mmol) obtained in Step (B) of Example 1, toluene (organic solvent: 185 mL) was added and dissolved by stirring at 35°C. Then, 4-dimethylaminopyridine (0.977 g, 8 mmol) was added and the mixture was reacted at 40°C for 4 hours. The reaction mixture was concentrated and acetonitrile (51.0 mL) was added to perform azeotropy. After azeotropy, acetonitrile (204 mL) and hexane (242 mL) were added and the mixture was stirred at 25°C for 30 minutes and allowed to stand to separate into layers. After separating into layers, the hexane layer was removed, and hexane (242 mL) was added to the acetonitrile layer. The hexane washing treatment was performed four times in the same manner.

[0092] The acetonitrile layer after hexane washing was concentrated and diluted with ethyl acetate (222 mL). Then, Kyoward #700 (8.00 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) and Kyoward #1000 (4.00 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) were added as adsorbents, and the mixture was stirred at 25°C for 30 minutes. After filtering the adsorbents, the adsorbent treatment was repeated twice in the same manner. Subsequently, the filtrate was concentrated and dried to obtain 43.5 g of 1-(methylpolyoxyethylene)-2,3-dipalmitoylglycerol (lipid derivative of formula (1): yield 87.8 mol%).

[0093] (Example 5) (Synthesis of the lipid derivative of formula (1): 1-(methylpolyoxyethylene)-2,3-dioleoilglycerol)

[0094] (Step (C)) To the 1-(methylpolyoxyethylene-oxy)-2,3-propanediol (molecular weight 2000, 2.00 g, 1.00 mmol) and oleic anhydride (fatty acid anhydride: 1.32 g, 2.40 mmol) obtained in Step (B) of Example 1, toluene (organic solvent: 9.25 mL) was added and dissolved by stirring at 35°C. Then, 4-dimethylaminopyridine (48.9 mg, 0.400 mmol) was added and the mixture was reacted at 40°C for 5 hours. The reaction mixture was concentrated and acetonitrile (2.54 mL) was added to perform azeotropy. After azeotropy, acetonitrile (10.2 mL) and hexane (12.1 mL) were added and the mixture was stirred at 25°C for 30 minutes and allowed to stand to separate into layers. After separating into layers, the hexane layer was removed, hexane (12.1 mL) was added to the acetonitrile layer, and the hexane washing treatment was repeated four times in the same manner.

[0095] The acetonitrile layer after hexane washing was concentrated and diluted with ethyl acetate (11.1 mL). Then, Kyoward #700 (0.400 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) and Kyoward #1000 (0.200 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) were added as adsorbents, and the mixture was stirred at 25°C for 30 minutes. After filtering the adsorbents, the adsorbent treatment was repeated twice in the same manner. Subsequently, the filtrate was concentrated and dried to obtain 1.75 g of 1-(methylpolyoxyethylene)-2,3-dioleoylglycerol (lipid derivative of formula (1): yield 69.3 mol%).

[0096] (Example 6) (Synthesis of the lipid derivative of formula (1): 1-(methylpolyoxyethylene)-2,3-dipalmitoylglycerol)

[0097] (Step (C)) To the 1-(methylpolyoxyethylene-oxy)-2,3-propanediol (molecular weight 5000, 10.0 g, 2.00 mmol) and palmitic anhydride (fatty acid anhydride: 2.38 g, 4.80 mmol) obtained in Step (B) of Example 3, toluene (organic solvent: 46.2 mL) was added and dissolved by stirring at 35°C. Then, 4-dimethylaminopyridine (0.144 g, 0.80 mmol) was added and the mixture was reacted at 40°C for 5 hours. The reaction solution was concentrated and acetonitrile (12.7 mL) was added to perform azeotropic reaction. After azeotropic reaction, acetonitrile (50.8 mL) and hexane (60.7 mL) were added and the mixture was stirred at 25°C for 30 minutes and allowed to stand to separate into layers. After standing to separate into layers, the hexane layer was removed, and hexane (60.7 mL) was added to the acetonitrile layer. The hexane washing treatment was repeated four times in the same manner.

[0098] The acetonitrile layer after hexane washing was concentrated and diluted with ethyl acetate (55.5 mL). Then, Kyoward #700 (2.00 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) and Kyoward #1000 (1.00 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) were added as adsorbents, and the mixture was stirred at 25°C for 30 minutes. After filtering the adsorbents, the adsorbent treatment was repeated twice in the same manner. Subsequently, the filtrate was concentrated and dried to obtain 7.80 g of 1-(methylpolyoxyethylene)-2,3-dipalmitoylglycerol (lipid derivative of formula (1): yield 63.4 mol%).

[0099] (Example 7) (Synthesis of the lipid derivative of formula (1): 1-(methylpolyoxyethylene)-2,3-distearoylglycerol)

[0100] (Step (C)) To the 1-(methylpolyoxyethylene-oxy)-2,3-propanediol (molecular weight 5000, 10.0 g, 2.00 mmol) and stearic acid (fatty acid: 2.28 g, 8.00 mmol) obtained in Step (B) of Example 3, toluene (organic solvent: 46.2 mL) was added and stirred at 40°C until dissolved. Then, 4-dimethylaminopyridine (0.977 g, 0.800 mmol) and N,N'-dicyclohexylcarbodiimide (1.65 g, 8.00 mmol) were added and the mixture was reacted at 60°C for 3 hours. After cooling the reaction mixture to 25°C, acetonitrile (127 mL) was added and the mixture was stirred at 25°C for 30 minutes, and the precipitate was removed by filtration. The filtrate was then concentrated, and acetonitrile (25.4 mL) was added to perform azeotropic reaction. After azeotropic mixing, acetonitrile (76.3 mL) and hexane (60.7 mL) were added, and the mixture was stirred at 25°C for 30 minutes, followed by standing to allow the layers to separate. After standing to separate the layers, the hexane layer was removed, and hexane (60.7 mL) was added to the acetonitrile layer. The hexane washing treatment was then repeated three times.

[0101] The acetonitrile layer after hexane washing was concentrated, and ethyl acetate (22.2 mL) was added for azeotropic mixing. After azeotropic mixing, ethyl acetate (55.5 mL) was added for dilution, and then Kyoward #700 (2.00 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) and Kyoward #1000 (1.00 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) were added as adsorbents, and the mixture was stirred at 25°C for 30 minutes. After filtering the adsorbents, the adsorbent treatment was repeated twice in the same manner. Subsequently, the filtrate was concentrated, and ethyl acetate (11.1 mL) was added for azeotropic mixing. After azeotropic mixing, ethyl acetate (99.9 mL) and methyl-tert-butyl ether (54.1 mL) were added and dissolved, and the mixture was stirred at 6°C for 1 hour. The precipitated crystals were filtered and dried to obtain 6.24 g of 1-(methylpolyoxyethylene)-2,3-distearoylglycerol (lipid derivative of formula (1): yield 49.3 mol%).

[0102] (Comparative Example 1) (Synthesis of the lipid derivative of formula (1): 1-(methylpolyoxyethylene)-2,3-dimyristoylglycerol)

[0103] Isopropylideneglycerol (50.0 g, 379 mmol) and potassium hydroxide (0.758 g, 13.5 mmol) were placed in an autoclave, the system was replaced with nitrogen gas, and the temperature was raised to 100°C, then heated to 100-150°C at a rate of 10 kg / cm³. ２ Under the following conditions, ethylene oxide (872 g, 19.8 mol) was added over 3 hours, and the reaction was continued for another hour. Next, the mixture was cooled to 50°C while distilling off unreacted ethylene oxide with nitrogen gas. Then, sodium hydroxide (23.7 g, 0.591 mol) was added, the mixture was purged with nitrogen, and the temperature was raised to 100°C while stirring. Next, an excess amount of methyl chloride was blown in from a cylinder, and the mixture was maintained at 100°C for 3 hours while adjusting the blowing tube to maintain a pressure of 1 atmosphere inside the container. After that, the mixture was cooled to 60°C while distilling off the excess methyl chloride with nitrogen gas.

[0104] The reaction mixture was prepared by adjusting the pH to 1.0 with a 10% hydrochloric acid aqueous solution and stirring at 60°C for 1 hour. Next, the pH of the reaction mixture was adjusted to 6.5 with 50% sodium hydroxide, and the mixture was heated at 100°C and below 100 mmHg for 1 hour. The resulting acetone was removed by distillation along with water, and the precipitated salt was filtered off to obtain 697 g of the intermediate compound (1-(methylpolyoxyethylene-oxy)-2,3-propanediol) (yield 92.0 mol%).

[0105] 3-(methylpolyoxyethylene-oxy)-1,2-propanediol (molecular weight 2000, 200 g, 100 mmol), methyl myristate (60.0 g, 200 mmol), and sodium methylate (0.2 g, 3.70 mmol) were taken and transesterified under reduced pressure of 20 mmHg at 80°C for 3 hours while passing nitrogen through. Then, Kyoward 600 (1.00 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) was added as a silica-based adsorbent, and the mixture was mixed under a nitrogen atmosphere at 80°C for 1 hour before filtration. Subsequently, the filtrate was concentrated and dried to obtain 208 g of 1-(methylpolyoxyethylene)-2,3-dimyristoylglycerol (yield 80.0 mol%).

[0106] (Comparative Example 2) (Synthesis of the lipid derivative of formula (1): 1-(methylpolyoxyethylene)-2,3-dimyristoylglycerol)

[0107] Isopropylideneglycerol (50.0 g, 379 mmol) and potassium hydroxide (0.758 g, 13.5 mmol) were placed in an autoclave, the system was replaced with nitrogen gas, and the temperature was raised to 100°C, then heated to 100-150°C at a rate of 10 kg / cm³. ２ Under the following conditions, ethylene oxide (872 g, 19.8 mol) was added over 3 hours, and the reaction was continued for another hour. Next, the mixture was cooled to 50°C while distilling off unreacted ethylene oxide with nitrogen gas. Then, sodium hydroxide (23.7 g, 0.591 mol) was added, the mixture was purged with nitrogen, and the temperature was raised to 60°C while stirring. Next, an excess amount of methyl chloride was blown in from a cylinder, and the mixture was maintained at 60°C for 3 hours while adjusting the blowing tube to maintain a pressure of 1 atmosphere inside the container. After that, the excess methyl chloride was removed by distilling off with nitrogen gas.

[0108] A 10% aqueous hydrochloric acid solution was added to the reaction mixture to adjust the pH to 1.0, and the mixture was stirred at 60°C for 1 hour. Next, the pH of the reaction mixture was adjusted to 6.5 with 50% sodium hydroxide, and the mixture was heated at 100°C and below 100 mmHg for 1 hour. The resulting acetone was removed by distillation along with water, and the precipitated salt was filtered off to obtain 635 g of the intermediate compound (1-(methylpolyoxyethylene-oxy)-2,3-propanediol) (yield 83.8 mol%).

[0109] 1-(methylpolyoxyethylene-oxy)-2,3-propanediol (molecular weight 2000, 200 g, 100 mmol), methyl myristate (60.0 g, 200 mmol), and sodium methylate (0.2 g, 3.70 mmol) were taken and transesterified under reduced pressure of 20 mmHg at 80°C for 3 hours while passing nitrogen through. Then, Kyoward 600 (1.00 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) was added as a silica-based adsorbent, and the mixture was mixed under a nitrogen atmosphere at 80°C for 1 hour. After filtration, 214 g of 1-(methylpolyoxyethylene)-2,3-dimyristoylglycerol (lipid derivative of formula (1): yield 82.3 mol%) was obtained.

[0110] (Comparative Example 3) (Synthesis of 1-(methylpolyoxyethylene)-2,3-dimyristoylglycerol)

[0111] Methanol (10.0 g, 313 mmol) and sodium hydroxide (1.25 g, 31.3 mmol) were placed in an autoclave, the inside of the system was replaced with nitrogen gas, then the temperature was raised to 100°C, and under the conditions of 100 to 150°C and 10 kg / cm ２ ethylene oxide (719 g, 16.4 mol) was added over 3 hours under the following conditions, and the reaction was further continued for 1 hour. Next, the reaction product was cooled to 80°C, neutralized with dilute hydrochloric acid, dehydrated under reduced pressure with an evaporator, and the generated salt was removed by filtration to obtain 678 g of an intermediate compound (methoxypolyethylene glycol) (yield: 93.0 mol%).

[0112] Methoxypolyethylene glycol (molecular weight 2000, 200 g, 100 mmol) and tin tetrachloride (0.269 g, 1.42 mmol) were placed in an autoclave, the inside of the system was replaced with nitrogen gas, then at 50 to 70°C and 3 kg / cm ２ epichlorohydrin (108 g, 108 mmol) was added over 1 hour under the following conditions, and the reaction was further continued for 30 minutes. Thereafter, 20% aqueous sodium hydroxide solution (30 mL) was added, the mixture was stirred at 70 to 80°C for 2 hours and allowed to stand for 30 minutes, then the separated aqueous layer (lower layer) was removed, water (100 mL) was added, and stirring and washing were performed. After allowing to stand for layer separation, the aqueous layer was drawn off, and the washing treatment was performed once in the same manner. After washing with water, the mixture was transferred to a four-necked flask, 1% aqueous sulfuric acid solution (200 mL) was added, the mixture was stirred at 80 to 90°C for 3 hours and allowed to stand for 30 minutes, then the separated aqueous layer (lower layer) was removed, water (100 mL) was added, and stirring and washing were performed. After allowing to stand for layer separation, the aqueous layer was drawn off, and the washing treatment was performed once in the same manner. After dehydration under reduced pressure with an evaporator, filtration was performed to obtain 178 g of an intermediate compound (1-(methylpolyoxyethylene-oxy)-2,3-propanediol) (yield: 89.0 mol%).

[0113] 3-(methylpolyoxyethylene-oxy)-1,2-propanediol (molecular weight 2000, 100 g, 50 mmol) and pyridine (3.32 g, 42 mmol) were taken and esterified with myristic anhydride (44.9 g, 0.103 mmol) under a nitrogen atmosphere at 90-100 °C. After the reaction, pyridine was removed by distillation under reduced pressure of 30 mmHg or less at 80 °C. Kyoward 600 (1.00 g, manufactured by Kyowa Chemical Industry Co., Ltd., trademark) was added as a silica-based adsorbent, and the mixture was treated at 80 °C under a nitrogen atmosphere for 1 hour. After filtration, 94 g of the target compound (yield 75.2 mol%) was obtained.

[0114] (Analysis of Products) For each product in each example and comparative example, the content of each component was measured by high-performance liquid chromatography (HPLC) analysis using a charged particle detector (CAD). Specifically, each product was quantified using an ultra-high-performance liquid chromatograph / corona charged particle detector (Vanquish Flex UHPLC system, manufactured by Thermo Fisher Scientific Co., Ltd.).

[0115] Specifically, lipid derivatives bound to the polyalkylene oxide shown in formula (5) (triacyl derivative) and lipid derivatives bound to the polyalkylene oxide shown in formula (6) (tetraacyl derivative) were quantified. The analytical results are shown in Tables 1 to 3.

[0116]

[0117]

[0118]

[0119] The results in Tables 1, 2, and 3 show that, according to the examples, lipid derivative mixtures with controlled triacyl and tetraacyl content were obtained.

[0120] In Comparative Example 1, the temperature during the methylation reaction was high at 100°C. As a result, a lipid mixture with high triacyl and tetraacyl content was obtained.

[0121] In Comparative Example 2, the temperature during the methylation reaction was 60°C. As a result, side reactions were suppressed, and a lipid mixture was obtained with a low tetraacyl content but a high triacyl content derived from unreacted raw materials.

[0122] In Comparative Example 3, a lipid mixture with a high tetraacyl content was obtained because a dimer was formed as a by-product due to the water in methanol during the ethylene oxide addition reaction.

[0123] The present invention's method for producing polyalkylene oxide-bound lipid derivatives allows for the production of small amounts of by-products, making it extremely useful for non-viral nucleic acid delivery carrier applications.

[0124] Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. This application is based on Japanese Patent Application No. 2025-049248 filed on 25 March 2025, the contents of which are incorporated herein by reference.

Claims

1. A method for producing a lipid derivative to which a polyalkylene oxide of the following formula (1) is bound, characterized by carrying out the following steps (A), (B), and (C). (In formula (1), R １ O is an oxyalkylene group having 2 to 4 carbon atoms, and n is an oxyalkylene group R having 2 to 4 carbon atoms. １ The average number of moles of O added is between 10 and 800, and R ２ R is an alkyl group having 1 to 4 carbon atoms. ３ CO and R ４ CO are each independently acyl groups having 4 to 24 carbon atoms. Step (A): A step to obtain a polyalkylene oxide compound of formula (3) by reacting the polyalkylene oxide compound of formula (2) below with a solid or liquid alkylating agent in an organic solvent in the presence of an alkali metal salt at a temperature of 10°C to 40°C. Step (B): A step to obtain a polyalkylene oxide compound of formula (4) below by reacting the polyalkylene oxide compound of formula (3) obtained in step (A) in an aqueous solution adjusted to a pH of 3.0 or less. Step (C): A step to obtain a lipid derivative to which the polyalkylene oxide represented by formula (1) is bound by reacting the polyalkylene oxide compound of formula (4) obtained in step (B) with one or more compounds selected from the group consisting of fatty acids, fatty acid anhydrides, and fatty acid chlorides in an organic solvent. (In equations (2), (3), and (4), R １ O is an oxyalkylene group having 2 to 4 carbon atoms, and n is an oxyalkylene group R having 2 to 4 carbon atoms. １ The average number of moles of O added is between 10 and 800, and R ２ (These are alkyl groups with 1 to 4 carbon atoms.) 2. The method for producing a polyalkylene oxide-bound lipid derivative according to claim 1, characterized in that the alkylating agent in step (A) is one or more alkylating agents selected from the group consisting of bromomethane, iodomethane, N,N-dimethylformamide dimethyl acetal, methyl fluorosulfonate, 1-methyl-3-p-tolyltriazene, dimethyl carbonate, dimethyl sulfate, N,N'-diisopropylcarbamimoidate methyl, methyl methanesulfonate, trimethyl orthoformate, tetramethylammonium chloride, p-toluenesulfonate methyl, trifluoromethanesulfonate methyl, and trimethyloxonium tetrafluoroborate.

3. A method for producing a lipid derivative to which a polyalkylene oxide is bound, according to claim 1 or 2, characterized in that the alkali metal salt in step (A) is one or more alkali metal salts selected from the group consisting of lithium tert-butoxide, sodium tert-butoxide, potassium tert-butoxide, lithium tert-pentoxide, sodium tert-pentoxide, potassium tert-pentoxide, n-butyllithium, sec-butyllithium, tert-butyllithium, lithium diisopropylamide, lithium bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide, and potassium bis(trimethylsilyl)amide.

4. In the above formula (1), R １ O is an oxyethylene group, and R ２ is a methyl group, which is the method for producing a lipid derivative bonded with polyalkylene oxide according to claim 1 or 2.

5. In the above formula (1), R ３ CO and R ４ A method for producing a lipid derivative to which a polyalkylene oxide is bonded, characterized in that the number of carbon atoms in CO is 12 or more and 20 or less, respectively, according to claim 1 or 2.

6. A lipid derivative mixture characterized by containing 99.0% by mass or more and 99.999% by mass or less of the lipid derivative of formula (1) below, and containing at least 0.001% by mass or more and 0.5% by mass or less of at least one of the lipid derivative of formula (5) and the lipid derivative of formula (6) in total. (In equations (1), (5), and (6), R １ O is an oxyalkylene group having 2 to 4 carbon atoms, and n is an oxyalkylene group R having 2 to 4 carbon atoms. １ The average number of moles of O added is between 10 and 800, and R ２ R is an alkyl group having 1 to 4 carbon atoms. ３ CO, R ４ CO, R ５ CO, R ６ CO and R ７ CO are, independently, acyl groups with 4 to 24 carbon atoms.

7. In equations (1), (5), and (6) above, R １ O is an oxyethylene group, R ２ The lipid derivative mixture according to claim 6, characterized in that the group is a methyl group.

8. In equations (1), (5), and (6) above, R ３ CO, R ４ CO, R ５ CO, R ６ CO and R ７ The lipid derivative mixture according to claim 6 or 7, characterized in that the number of carbon atoms in CO is 12 or more and 20 or less, respectively.

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