Method for producing phospholipids

Abstract

To provide a method for more efficiently producing a phospholipid represented by a specific formula.SOLUTION: A method for producing a phospholipid represented by formula (1) according to reaction formula I includes mixing, in a flow path, an organic solvent solution containing a compound represented by formula (A) with an aqueous solvent solution containing phospholipase D and a compound represented by formula (B), and then allowing the mixture to react at an enzymatic reaction temperature of the phospholipase D.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing a phospholipid. [Background technology]

[0002] In recent years, RNA interference agents, including small interfering RNA (siRNA), have attracted great expectations as attractive pharmaceutical seeds. While promising seeds have been discovered one after another, highly sophisticated delivery systems are required for exogenously administered RNA to demonstrate its intended activity in vivo. This is due to the fact that RNA is rapidly degraded by enzymes and rarely crosses cell membranes. Therefore, the practical application of RNA interference agents inevitably requires the development of a delivery system.

[0003] A known delivery system for drugs such as RNA involves administering the drug encapsulated in lipid particles. However, when administering negatively charged nucleic acids, positively charged lipids are usually used to induce electrostatic interactions, which raises concerns about cytotoxicity (Patent Document 1).

[0004] Under such circumstances, Patent Document 2 reports on a phospholipid (a phospholipid represented by the general formula (1) described below) that is not positively charged at the pH of body fluids (usually in the neutral range) and can form lipid particles that can more efficiently exert the effects of the encapsulated drug. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2016-023147 [Patent Document 2] International Publication No. 2018 / 190017 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for more efficiently producing a phospholipid represented by general formula (1). [Means for solving the problem]

[0007] In view of the above problems, the present inventors have conducted extensive research and have found that by mixing two types of solutions (an organic solvent solution and an aqueous solvent solution) containing raw materials for the phospholipid represented by general formula (1) in a channel and then reacting them at the enzymatic reaction temperature of phospholipase D, the reaction time can be shortened and the phospholipid represented by general formula (1) can be produced more efficiently. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects.

[0008] Item 1. A method for producing a phospholipid represented by general formula (1) according to reaction formula I,

[0009] [ka] [In the formula, R 1 and R 2 are the same or different and represent a chain hydrocarbon group. a , R b and R c are the same or different and represent a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms; R a and R b may be linked to each other to form a ring containing the adjacent nitrogen atom. m represents 1 or 2. n represents 1 or 2. p represents an integer of 1 to 4.] A method comprising mixing an organic solvent solution containing a compound represented by general formula (A) with an aqueous solvent solution containing phospholipase D and a compound represented by general formula (B) in a flow path, and then reacting the mixture at the enzymatic reaction temperature of phospholipase D.

[0010] Item 2. The cross-sectional area of ​​the flow path is 0.1 mm 2 ~10mm 2 The method according to Item 1,

[0011] Item 3. The cross-sectional area of ​​the flow path is 1 mm 2 Flow rate per 50cm 3 / h~1500cm 3 Item 3. The method according to Item 1 or 2, wherein the serotonin concentration is 100 mg / h.

[0012] Item 4. The method according to any one of Items 1 to 3, wherein the mixing in the flow channel is performed using a static mixer.

[0013] Item 5. The method according to any one of Items 1 to 4, wherein the organic solvent solution contains an ether solvent.

[0014] Item 6. The method according to any one of Items 1 to 5, wherein the aqueous solvent solution contains a buffer.

[0015] Item 7. The method according to any one of Items 1 to 6, wherein the reaction time is 240 minutes or less.

[0016] Item 8. The method according to any one of Items 1 to 7, wherein at least a part of the reaction is carried out in a tubular reactor.

[0017] Item 9. The method according to any one of Items 1 to 8, wherein the chain hydrocarbon group is an unsaturated chain hydrocarbon group.

[0018] Item 10. The method according to any one of Items 1 to 9, wherein the chain hydrocarbon group has 12 to 24 carbon atoms.

[0019] Item 11. The method according to any one of Items 1 to 10, wherein both m and n are 2.

[0020] Item 12. The method according to any one of Items 1 to 11, wherein p is 1 or 2.

[0021] Section 13. Said R a , the R b and the R c 13. The method according to any one of Items 1 to 12, wherein is hydrogen.

[0022] Section 14. Said R a , the Rb and the R c are the same or different and are hydrocarbon groups having 1 to 5 carbon atoms, and R a and R b and may be linked to each other to form a ring containing the adjacent nitrogen atom. [Effects of the Invention]

[0023] According to the present invention, a method for more efficiently producing a phospholipid represented by general formula (1) can be provided. [Brief explanation of the drawings]

[0024] [Figure 1] 1 shows the results of thin-layer chromatography analysis of the reaction solutions of Example 1 and Comparative Example 1. The spot showing DOPC is indicated by an arrow. DETAILED DESCRIPTION OF THE INVENTION

[0025] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0026] In one aspect, the present invention provides a method for producing a phospholipid represented by general formula (1) according to reaction formula I,

[0027] [ka] The present invention relates to a method (sometimes referred to herein as the "production method of the present invention") that comprises mixing an organic solvent solution containing a compound represented by general formula (A) with an aqueous solvent solution containing phospholipase D and a compound represented by general formula (B) in a flow channel, and then reacting the mixture at the enzymatic reaction temperature of phospholipase D. This method is described below.

[0028] R 1 and R 2 are the same or different and represent a chain hydrocarbon group.

[0029] R 1 or R 2 The chain hydrocarbon group represented by the formula (I) is not particularly limited as long as it is a monovalent chain hydrocarbon group, and includes both straight-chain and branched-chain (preferably straight-chain) ones. The number of carbon atoms in the chain hydrocarbon group is not particularly limited as long as it is a number that allows the formation of lipid particles, and is, for example, 4 to 30, preferably 8 to 26, more preferably 12 to 22, even more preferably 14 to 20, and still more preferably 15 to 19. The chain hydrocarbon group includes both saturated chain hydrocarbon groups and unsaturated hydrocarbon groups, but is preferably an unsaturated chain hydrocarbon group, more preferably an unsaturated chain hydrocarbon group containing a double bond, and even more preferably an unsaturated chain hydrocarbon group having only one double bond. Examples of chain hydrocarbon groups include butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, tridecyl, tetradecyl, pentadecyl, 9-pentadecenyl, hexadecyl, heptadecyl, cis-9-heptadecenyl, 11-heptadecenyl, cis,cis-9,12-heptadecadienyl, 9,12,15-heptadecanthrienyl, 6,9,12-heptadecanthrienyl, 9,11,13-heptadecanthrienyl, nonadecyl, 8,11-nonadecadienyl, 5,8,11-nonadecatrienyl, 5,8,11,14-nonadecatetraenyl, henicosyl, tricosyl, cis-15-tricosenyl, pentacosyl, heptacosyl, and nonacosyl.

[0030] R 1 and R 2 At least one of the groups is preferably an unsaturated chain hydrocarbon group, and it is more preferable that both of the groups are unsaturated chain hydrocarbon groups.

[0031] R a , R b and R c are the same or different and represent a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms; R a and R b may be linked to each other to form a ring containing the adjacent nitrogen atom.

[0032] The hydrocarbon group having 1 to 5 carbon atoms is a monovalent hydrocarbon group, and is not particularly limited thereto. The hydrocarbon group is preferably a chain hydrocarbon group (more preferably linear), and more preferably an alkyl group. The number of carbon atoms in the hydrocarbon group is preferably 1 to 3, more preferably 1 to 2, and even more preferably 1. Specific examples of the hydrocarbon group include methyl, ethyl, propyl, butyl, and pentyl, and among these, methyl is particularly preferred.

[0033] R a and R b and R are linked to each other to form a ring containing the adjacent nitrogen atom, in other words, R a and R b and (B) are linked to form a divalent chain hydrocarbon group, and one end of the divalent chain hydrocarbon group is R a and the other end is connected to the nitrogen atom adjacent to R in general formula (1) or (B). b indicates that the nitrogen atom is connected to the nitrogen atom adjacent to the nitrogen atom.

[0034] The divalent chain hydrocarbon group is more preferably linear, and is more preferably an alkyl group. The chain hydrocarbon group preferably has 1 to 8 carbon atoms, more preferably 1 to 5 carbon atoms, even more preferably 1 to 3 carbon atoms, still more preferably 2 to 3 carbon atoms, and particularly preferably 2 carbon atoms.

[0035] R a and R b and are linked to each other to form a ring, and p is 2 to 4 (i.e., R a When there are multiple R a is any one of, preferably -NR b R c The R closest to a is.

[0036] In one embodiment of the present invention, preferably R a , R b and R c are all hydrogen atoms.

[0037] In one embodiment of the present invention, preferably R a , R b and R c are all alkyl groups, particularly preferably methyl groups.

[0038] In one embodiment of the present invention, preferably R a , R b and R c are the same or different and are hydrocarbon groups having 1 to 5 carbon atoms, and R a and R b may be linked to each other to form a ring containing the adjacent nitrogen atom.

[0039] m represents 1 or 2. m is preferably 2.

[0040] n represents 1 or 2. n is preferably 2.

[0041] Preferably, m and n are both 2.

[0042] p represents an integer of 1 to 4. p is preferably 1 or 2. From the viewpoint of the cytotoxicity of the phospholipid, p is more preferably 1. Furthermore, from the viewpoint of the encapsulation rate of the drug in the lipid particles obtained from the phospholipid, p is more preferably 2.

[0043] The following partial structures in general formulae (1) and (B): [ka] Preferably, the following four: [ka] and particularly preferably the following two: [ka] is.

[0044] The organic solvent solution is not particularly limited as long as it contains a compound represented by general formula (A) and an organic solvent as the main solvent (for example, the organic solvent accounts for 70% by volume or more, preferably 80% by volume or more, more preferably 90% by volume or more, even more preferably 95% by volume or more, still more preferably 99% by volume or more, and particularly preferably 100% by volume, based on 100% by volume of the total solvents constituting the organic solvent solution).

[0045] The organic solvent is not particularly limited as long as it can dissolve the compound represented by general formula (A) and has a boiling point that is equal to or higher than the temperature range at which phospholipase D can exhibit its enzymatic activity. Examples of organic solvents include ether solvents, ester solvents, halogenated solvents, hydrocarbon solvents, and aromatic hydrocarbon solvents. Among these, ether solvents are preferred, and hydrophobic ether solvents are more preferred. These solvents may be used alone or in combination.

[0046] Examples of ether solvents include cyclopentyl methyl ether, 4-methyltetrahydropyran, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, diethyl ether, diisopropyl ether, t-butyl methyl ether, tetrahydropyran, etc. Among these, cyclopentyl methyl ether and 4-methyltetrahydropyran are particularly preferred.

[0047] Examples of the ester solvent include ethyl acetate, methyl acetate, butyl acetate, propyl acetate, and methyl lactate.

[0048] Examples of halogen-based solvents include chloroform, dichloromethane, and carbon tetrachloride.

[0049] Examples of hydrocarbon solvents include hexane, pentane, octane, nonane, and decane.

[0050] Examples of aromatic hydrocarbon solvents include benzene, toluene, and xylene.

[0051] The concentration of the compound represented by general formula (A) in the organic solvent solution is not particularly limited, but is, for example, 0.01 g / mL to 1 g / mL, preferably 0.03 g / mL to 0.5 g / mL, and more preferably 0.08 g / mL to 0.2 g / mL.

[0052] The organic solvent solution can be obtained by mixing the compound represented by general formula (A) with an organic solvent.

[0053] The aqueous solvent solution is not particularly limited, as long as it contains phospholipase D and a compound represented by general formula (B) and water as the main solvent (for example, 70% by volume or more, preferably 80% by volume or more, more preferably 90% by volume or more, even more preferably 95% by volume or more, still more preferably 99% by volume or more, and particularly preferably 100% by volume of water, based on 100% by volume of the total solvent constituting the aqueous solvent solution).

[0054] Phospholipase D is an enzyme that can hydrolyze phosphatidylcholine into phosphatidic acid and choline, and is, for example, an enzyme classified as phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4.

[0055] The organism from which phospholipase D is derived is not particularly limited, but is preferably a bacterium, and particularly preferably a bacterium of the genus Streptomyces.

[0056] From the viewpoint of yield and the like, the amount of phospholipase D used is preferably 100 to 1500 U, more preferably 250 to 1000 U, and even more preferably 300 to 600 U per 1 mmol of the compound represented by general formula (A). 1 U is defined as the amount of enzyme (1 micromole per minute) that can convert 1 micromole (μmol) of substrate per minute under optimal conditions (a temperature of 30°C and an acidity at which the chemical reaction proceeds most rapidly).

[0057] From the viewpoint of yield and the like, the amount of the compound represented by general formula (B) used is preferably 2 to 20 mol, more preferably 4 to 16 mol, and even more preferably 5 to 10 mol, per 1 mol of the compound represented by general formula (A).

[0058] The concentration of phospholipase D in the aqueous solvent solution is not particularly limited, but is, for example, 0.05 mg / mL to 5 mg / mL, preferably 0.15 mg / mL to 2 mg / mL, and more preferably 0.20 mg / mL to 1 mg / mL.

[0059] The concentration of the compound represented by general formula (B) in the aqueous solvent solution is not particularly limited, but is, for example, 0.02 g / mL to 2 g / mL, preferably 0.05 g / mL to 1 g / mL, and more preferably 0.08 g / mL to 0.5 g / mL.

[0060] The aqueous solvent solution preferably contains a buffer. Examples of the buffer include acetate buffer, phosphate buffer, citrate buffer, succinate buffer, and phthalate buffer. Among these, acetate buffer is particularly preferred.

[0061] The pH of the aqueous solvent solution is preferably 4-7, more preferably 5-6.

[0062] In addition to the above components, the organic solvent solution and the aqueous solvent solution of the present reaction may contain additives as appropriate to the extent that the reaction progress is not significantly impaired. The content of the additive in each solution is, for example, 10 parts by mass or less, 5 parts by mass or less, 2 parts by mass or less, 1 part by mass or less, 0.1 parts by mass or less, 0.01 parts by mass or less, 0.001 parts by mass or less, 0.0001 parts by mass or less, or 0.00001 parts by mass or less, relative to 100 parts by mass of the compound represented by general formula (A).

[0063] In the production method of the present invention, an organic solvent solution and an aqueous solvent solution are mixed in a flow channel, which allows the organic solvent solution and the aqueous solvent solution to be mixed well while maintaining the activity of phospholipase D at a certain level or higher due to turbulence, backflow, crossflow, and the like that occur in the flow channel, thereby achieving high reaction efficiency.

[0064] From the viewpoint of reaction efficiency, the cross-sectional area of ​​the channel is preferably 0.01 mm 2 ~10mm 2 , more preferably 0.1 mm 2 ~10mm 2 , and more preferably 0.3 mm 2 ~5mm 2 , and even more preferably 0.7 mm 2 ~1.8mm 2 is.

[0065] Cross-sectional area of ​​flow path: 1mm 2 From the viewpoint of reaction efficiency, the flow rate per 3 / h~1000000cm 3 / h, more preferably 50cm 3 / h~100000cm 3 / h, more preferably 50 cm 3 / h~10000cm 3 / h, particularly preferably 50 cm 3 / h~5000cm 3 / h, especially preferably 50 cm 3 / h~1500cm 3 / h, especially more preferably 150 cm 3 / h~1000cm 3 / h, particularly preferably 300 cm 3 / h~600cm 3 / h.

[0066] The ratio (b / a) of the length (b) of the channel to the diameter (a) of the channel is preferably 5 to 200, more preferably 10 to 100, and even more preferably 15 to 35, from the viewpoint of reaction efficiency.

[0067] From the viewpoint of reaction efficiency, it is preferable that an obstacle to the flow of the solution is placed in the channel, which makes it possible to effectively generate turbulent flow, backflow, crossflow, etc. Mixing in the channel is particularly preferably performed using a static mixer.

[0068] The mixing ratio of the organic solvent solution to the aqueous solvent solution is not particularly limited, but it is preferable to set it so that the amounts of phospholipase D and the compound represented by general formula (B) used are within the above ranges.

[0069] In the production method of the present invention, after the above mixing, the mixture is reacted at the enzymatic reaction temperature of phospholipase D. The temperature is not particularly limited as long as it is a temperature at which the activity of phospholipase D can be exerted, and is usually 20 to 70°C, preferably 30 to 60°C, and more preferably 40 to 55°C.

[0070] The reaction time is not particularly limited. According to the production method of the present invention, the reaction can be carried out efficiently, so that a larger amount of the target product can be obtained even with a short reaction time. From this viewpoint, the reaction time is preferably 240 minutes or less, more preferably 30 to 240 minutes, even more preferably 50 to 200 minutes, and even more preferably 70 to 150 minutes.

[0071] The reaction can be carried out using various reactors. The reaction can be carried out in a storage tank capable of storing the mixed liquid, or in a tubular reactor. When a storage tank is used, it is preferable to stir the mixed liquid in the tank as necessary. When a tubular reactor is used, the reaction is usually carried out while moving the mixed liquid in the tube. When the reaction is carried out in a continuous system, it is preferable to adjust the flow rate of the mixed liquid in the reactor so that the total residence time in the reactor is within the above-mentioned reaction time range.

[0072] After the reaction is complete, the solvent is distilled off, and the product can be isolated and purified by conventional methods such as chromatography and recrystallization. The structure of the product can be determined by elemental analysis, MS (FD-MS), IR analysis, 1 H-NMR, 13It can be identified by C-NMR or the like. [Example]

[0073] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0074] Example 1 280 mL of a solution of 30 g of dioleoylphosphatidylcholine (DOPC) in cyclopentyl methyl ether (CPME) and 180 mL of a mixture of 26 g of 2-(2-aminoethylamino)ethanol and 60 mg of phospholipase D (PLDP, Asahi Kasei Pharma, 262 U / mg) in acetate buffer (pH 5.5, 0.5 M) were mixed in a static mixer (cross-sectional area of ​​the flow path: 0.8 mm). 2 , cross-sectional area of ​​the flow channel is 1 mm 2 Flow rate per: 450cm 3 After mixing at a flow rate of 1000 kJ / h (ratio of flow path length (b) to flow path diameter (a) (b / a): 20), the mixture was transferred to a tubular reactor (50°C) and then to a two-stage tank reactor (50°C) to carry out a continuous reaction. The total residence time in the reactors was 100 minutes. After the raw material solution was consumed, the reaction mixture was pumped through the tubular reactor and then the two-stage tank reactor using CPME, yielding a total of 467 mL of reaction mixture. The reaction mixture was analyzed by thin-layer chromatography (TLC) (developing solvent: chloroform / methanol / water = 60 / 30 / 5, TLC plate used: NH2 silica gel 60 F 254 When the sample was collected using a Wako plate (layer thickness 0.25 mm), the disappearance of DOPC was confirmed (Figure 1).

[0075] The reaction mixture was washed with water, extracted, converted to a hydrochloride salt, and reprecipitated to give 11 g of a solid (yield: 40%). NMR analysis confirmed that the target product (the compound represented by the following formula (DOP-DEDA)) was obtained.

[0076] [ka]

[0077] Comparative Example 1 6.7 mL of a CPME solution of DOPC prepared to the same concentration as in Example 1 and 4.5 mL of 2-(2-aminoethylamino)ethanol and PLDP in acetate buffer were added to a 20 mL screw tube and stirred for 100 minutes at 700 rpm and an internal temperature of 50°C using a tapered stir bar (PTFE, length 15 mm, diameter 5 mm). After 100 minutes, stirring was stopped and the organic layer was analyzed by TLC in the same manner as in Example 1, confirming a DOPC spot (Figure 1).

[0078] Example 2 The static mixer was replaced with another mixer (cross-sectional area of ​​the flow path: 0.04 mm) that can mix two liquids in the flow path. 2 , cross-sectional area of ​​the flow channel is 1 mm 2 Flow rate per unit: 15000 cm 3 / h~750000 cm 3 The procedure was carried out in the same manner as in Example 1, except that the flow rate was adjusted appropriately within the range of / h, and the ratio of the length (b) of the flow path to the diameter (a) of the flow path (b) (b / a): 150), and 11 g of DOP-DEDA solid was obtained.

[0079] Example 3 225 mL of a 4-methyltetrahydropyran (MTHP) solution containing 30 g of DOPC and 225 mL of a mixture of 47 g of 2-[[2-(dimethylamino)ethyl]methylamino]ethanol and 50 mg of phospholipase D (PLDP, Asahi Kasei Pharma, 260 U / mg) in acetate buffer (pH 6.0, 0.5 M) were mixed in a static mixer (cross-sectional area of ​​the flow path: 0.8 mm). 2 , cross-sectional area of ​​the flow channel is 1 mm 2 Flow rate per: 450cm 3 After mixing at a flow rate of 1000 kJ / h and a ratio of the length (b) of the flow channel to the diameter (a) of the flow channel (b) (b / a: 20), the mixture was sent to a tubular reactor (50°C) and then a two-stage tank reactor (50°C) to carry out a continuous reaction. The total residence time in the reactors was 100 minutes. After the raw material solution was consumed, the reaction mixture was pushed out of the tubular reactor and then the two-stage tank reactor using MTHP, yielding a total of 450 mL of reaction mixture.

[0080] The reaction mixture was washed with water, extracted, converted to a hydrochloride salt, and reprecipitated to give 16 g of a solid (yield: 46%). NMR analysis confirmed that the target product (compound represented by the following formula (compound a)) was obtained.

[0081] [ka]

Claims

1. A method for producing a phospholipid represented by general formula (1) according to reaction formula I, 【Chemistry 1】 [In the formula, R 1 and R 2 are the same or different and represent a chain hydrocarbon group. a , R b and R c are the same or different and represent a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms; R a and R b may be linked to each other to form a ring containing the adjacent nitrogen atom. m represents 1 or 2. n represents 1 or 2. p represents an integer of 1 to 4.] A method comprising mixing an organic solvent solution containing a compound represented by general formula (A) with an aqueous solvent solution containing phospholipase D and a compound represented by general formula (B) in a flow path, and then reacting the mixture at the enzymatic reaction temperature of phospholipase D.

2. The cross-sectional area of ​​the flow path is 0.1 mm 2 ~10mm 2 The method of claim 1, wherein

3. The cross-sectional area of ​​the flow path is 1 mm 2 Flow rate per 50cm 3 / h~1500cm 3 10. The method of claim 1, wherein the heating time is 100 min. / h.

4. The method according to claim 1 , wherein the mixing in the flow path is performed using a static mixer.

5. The method of claim 1 , wherein the organic solvent solution comprises an ethereal solvent.

6. The method of claim 1 , wherein the aqueous solvent solution contains a buffer.

7. 10. The method of claim 1, wherein the reaction time is 240 minutes or less.

8. 10. The process of claim 1, wherein at least a portion of the reaction is carried out in a tubular reactor.

9. The method according to any one of claims 1 to 8, wherein the chain hydrocarbon group is an unsaturated chain hydrocarbon group.

10. The method according to any one of claims 1 to 8, wherein the chain hydrocarbon group has 12 to 24 carbon atoms.

11. The method according to any one of claims 1 to 8, wherein m and n are both 2.

12. The method according to any one of claims 1 to 8, wherein p is 1 or 2.

13. R a , the R b and the R c The method according to any one of claims 1 to 8, wherein is a hydrogen atom.

14. R a , the R b and the R c are the same or different and are hydrocarbon groups having 1 to 5 carbon atoms, and R a and R b The method according to any one of claims 1 to 8, wherein the and may be linked to each other to form a ring containing adjacent nitrogen atoms.

Images