Preparation method of high-purity dimyristoyl phosphatidylcholine

Abstract

The invention belongs to the technical field of pharmaceutical chemicals, and discloses a preparation method of high-purity dimyristoyl phosphatidylcholine. The myristic acid, the glycerophosphorylcholine, the alkali and the condensing agent are subjected to condensation reaction in the solvent to obtain a crude product, then two-step recrystallization is performed by adopting a single or mixed solvent, the purification efficiency is high, the controllability is good, the operability is high, the cost is greatly reduced, the defects of high cost and complicated operation in the prior art are overcome, large-scale industrial production is easy, and the method is suitable for industrial production. The method has very high economic benefits, the yield can reach 75% or above, and the purity is greater than or equal to 99.0%. The prepared high-purity dimyristoyl phosphatidylcholine product is stable in quality and meets the standards of medical injection and pharmaceutic adjuvants after sterilization and vacuum drying.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical chemical industry, and in particular to a method for preparing high-purity dimyristoylphosphatidylcholine. Background Art

[0002] Phospholipids are important components of biological membranes. The polar end containing phosphate groups is hydrophilic, and the two longer non-polar ends of hydrocarbon chains are lipophilic. This unique physical and chemical property enables it to spontaneously form a closed bilayer in an aqueous medium and become the skeleton of biological membranes. Phospholipids play a vital role in liposome technology. Liposomes were first discovered by British scholar Bangham when he dispersed phospholipids in water and observed them under an electron microscope. In 1971, British scholars such as Lehman used liposomes as drug carriers. However, natural phospholipids as liposome materials have the disadvantages of short life, easy oxidation, and poor stability, which limits their application.

[0003] Dimyristoylphosphatidylcholine is an important synthetic phospholipid, mainly used in the preparation of liposomes. It is an indispensable additive in liposome drug preparations. Synthetic dimyristoylphosphatidylcholine has high purity, good stability, and stronger antioxidant capacity than natural phospholipids. It is very ideal for preparing liposomes.

[0004] There are many common methods for preparing phosphatidylcholine in the prior art, among which there is natural countercurrent extraction, which has relatively high purity after extraction, but low yield, and the extracting agent is usually acetonitrile, ethanol, etc., with low selectivity and serious material waste; the purity of phosphatidylcholine obtained by supercritical extraction is about 70%, but there are problems such as low purity, low yield, high equipment requirements, and large investment; the method of separating phosphatidylcholine by column chromatography and membrane has the disadvantages of non-renewable, high cost, and small processing capacity. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing high-purity dimyristoylphosphatidylcholine, so as to solve the problems that the existing method for preparing dimyristoylphosphatidylcholine has harsh conditions, complicated post-treatment procedures, serious environmental pollution, low purity, high production cost and is not suitable for industrial production.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing high-purity dimyristoylphosphatidylcholine, comprising the following steps:

[0008] The crude product of dimyristoylphosphatidylcholine is obtained by condensing myristic acid, glycerophosphatidylcholine, a base and a condensing agent in an organic solvent;

[0009] The crude dimyristoylphosphatidylcholine is recrystallized using a mixed solvent of solvent 1 and solvent 2, the temperature is lowered to precipitate a solid, and after filtering and drying, the solid is dissolved by heating with solvent 3, and the insoluble matter is removed by filtering, and solvent 1 is added to the filtrate, the temperature is lowered to precipitate a solid, and the solid is filtered and dried to obtain high-purity dimyristoylphosphatidylcholine.

[0010] Preferably, in the above method for preparing high-purity dimyristoylphosphatidylcholine, the mass ratio of myristic acid, glycerophosphatidylcholine, base and condensing agent is 1-5:1-2:0.1-0.5:3-7.

[0011] Preferably, in the above-mentioned method for preparing high-purity dimyristoylphosphatidylcholine, the base is one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, potassium acetate, triethylamine, 4-dimethylaminopyridine, and N,N-diisopropylethylamine.

[0012] Preferably, in the above-mentioned method for preparing high-purity dimyristoylphosphatidylcholine, the condensing agent is one or more of 2-(7-azobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate, and N,N-dicyclohexylcarbodiimide.

[0013] Preferably, in the above-mentioned method for preparing high-purity dimyristoylphosphatidylcholine, the organic solvent is one or more of dichloromethane, chloroform, 1,2-dichloroethane, tetrahydrofuran, dioxane, ethyl acetate, and N,N-dimethylformamide.

[0014] Preferably, in the above method for preparing high-purity dimyristoylphosphatidylcholine, the temperature of the condensation reaction is 50-70° C.; and the time of the condensation reaction is 16-24 hours.

[0015] Preferably, in the above-mentioned method for preparing high-purity dimyristoylphosphatidylcholine, the solvent 1, solvent 2, and solvent 3 are independently one or more of methanol, ethanol, acetone, tetrahydrofuran, ethyl acetate, dichloromethane, chloroform, N,N-dimethylformamide, and methyl tert-butyl ether.

[0016] Preferably, in the above method for preparing high-purity dimyristoylphosphatidylcholine, the solvent 1 is acetone; the solvent 2 is dichloromethane; and the solvent 3 is ethanol.

[0017] Preferably, in the above method for preparing high-purity dimyristoylphosphatidylcholine, the temperature for cooling to precipitate solids is independently 0 to 30°C.

[0018] Preferably, in the above method for preparing high-purity dimyristoylphosphatidylcholine, the temperature for heating and dissolving the solid using solvent 3 is 20-70°C.

[0019] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention directly reacts glycerophosphorylcholine and myristic acid under the action of alkali and condensing agent to obtain crude dimyristoylphosphatidylcholine, with cheap raw materials, mild reaction conditions and safe operation. Further, the purification process of crude dimyristoylphosphatidylcholine adopts two-step recrystallization of single or mixed solvents, with high purification efficiency, good controllability, strong operability and greatly reduced cost, overcoming the disadvantages of high cost and complicated operation of the prior art, being easy to large-scale industrial production, having high economic benefits, and having a yield of more than 75% and a purity of greater than or equal to 99.0%.

[0021] (2) The high-purity dimyristoylphosphatidylcholine product prepared by the present invention has stable quality and meets the standards of medical injection and pharmaceutical excipients after sterilization and vacuum drying. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the description of the embodiments or the prior art are briefly introduced below.

[0023] Figure 1 is a liquid chromatogram of the refined dimyristoylphosphatidylcholine obtained in Example 1;

[0024] Figure 2 IR spectrum of the refined dimyristoylphosphatidylcholine obtained in Example 1;

[0025] Figure 3 This is the MS spectrum of the refined dimyristoylphosphatidylcholine prepared in Example 1. DETAILED DESCRIPTION

[0026] The present invention provides a method for preparing high-purity dimyristoylphosphatidylcholine, comprising the following steps:

[0027] The crude product of dimyristoylphosphatidylcholine is obtained by condensing myristic acid, glycerophosphatidylcholine, a base and a condensing agent in an organic solvent;

[0028] The crude dimyristoylphosphatidylcholine is recrystallized using a mixed solvent of solvent 1 and solvent 2, the temperature is lowered to precipitate a solid, and after filtering and drying, the solid is dissolved by heating with solvent 3, and the insoluble matter is removed by filtering, and solvent 1 is added to the filtrate, the temperature is lowered to precipitate a solid, and the solid is filtered and dried to obtain high-purity dimyristoylphosphatidylcholine.

[0029] In the present invention, the preparation process of the crude dimyristoylphosphatidylcholine is as follows:

[0030]

[0031] Among them, compound 1 is myristic acid, compound 2 is glycerophosphatidylcholine, and compound 3 is dimyristoylphosphatidylcholine.

[0032] In the present invention, the mass ratio of myristic acid, glycerophosphorylcholine, base and condensing agent is preferably 1-5:1-2:0.1-0.5:3-7, more preferably 3-4:1-1.5:0.1-0.2:3.5-5, and more preferably 3.65:1:0.11:4.

[0033] In the present invention, the usage ratio of the myristic acid to the organic solvent is preferably 1-5 g:2-10 mL, more preferably 3-4 g:5-8 mL, and even more preferably 3.65 g:7.5 mL.

[0034] In the present invention, the base is preferably one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, potassium acetate, triethylamine, 4-dimethylaminopyridine, and N,N-diisopropylethylamine, further preferably one or more of sodium carbonate, triethylamine, 4-dimethylaminopyridine, and N,N-diisopropylethylamine, and more preferably 4-dimethylaminopyridine.

[0035] In the present invention, the condensing agent is preferably one or more of 2-(7-azobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate, and N,N-dicyclohexylcarbodiimide, further preferably benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate or N,N-dicyclohexylcarbodiimide, and more preferably N,N-dicyclohexylcarbodiimide.

[0036] In the present invention, the organic solvent is preferably one or more of dichloromethane, chloroform, 1,2-dichloroethane, tetrahydrofuran, dioxane, ethyl acetate, and N,N-dimethylformamide, more preferably one or more of chloroform, 1,2-dichloroethane, and N,N-dimethylformamide, and more preferably chloroform.

[0037] In the present invention, the temperature of the condensation reaction is preferably 50-70°C, more preferably 55-65°C, and more preferably 60°C; the time of the condensation reaction is preferably 16-24h, more preferably 17-20h, and more preferably 18h.

[0038] In the present invention, the solvent 1 is preferably one or more of methanol, ethanol, acetone, tetrahydrofuran, ethyl acetate, dichloromethane, chloroform, N,N-dimethylformamide, and methyl tert-butyl ether, more preferably one or more of acetone, ethyl acetate, and methyl tert-butyl ether, and more preferably acetone.

[0039] In the present invention, the solvent 2 is preferably one or more of methanol, ethanol, acetone, tetrahydrofuran, ethyl acetate, dichloromethane, chloroform, N,N-dimethylformamide, and methyl tert-butyl ether, further preferably one or more of tetrahydrofuran, dichloromethane, chloroform, and N,N-dimethylformamide, and more preferably dichloromethane.

[0040] In the present invention, the solvent 3 is preferably one or more of methanol, ethanol, acetone, tetrahydrofuran, ethyl acetate, dichloromethane, chloroform, N,N-dimethylformamide, and methyl tert-butyl ether, more preferably one or more of methanol, ethanol, and methyl tert-butyl ether, and more preferably ethanol.

[0041] In the present invention, the temperature for precipitating a solid by cooling is independently preferably 0 to 30°C, more preferably 0 to 10°C, and even more preferably 0°C.

[0042] In the present invention, the temperature for heating and dissolving the solid using the solvent 3 is preferably 20 to 70°C, more preferably 30 to 50°C, and even more preferably 40°C.

[0043] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Example 1

[0045] This embodiment provides a method for preparing high-purity dimyristoylphosphatidylcholine, comprising the following steps:

[0046] (1) Add 100 g of glycerophosphorylcholine, 365 g of myristic acid, 11 g of 4-dimethylaminopyridine, and 500 mL of chloroform to a clean and dry 3000 mL three-necked flask, and stir evenly with a magnetic stirrer; dissolve 400 g of N,N-dicyclohexylcarbodiimide in 250 mL of chloroform, and dropwise add the chloroform solution of N,N-dicyclohexylcarbodiimide to the reaction system; react at 60° C. for 18 h; TLC detection shows no further progress in the reaction, stop the reaction, cool and filter, and concentrate the mother liquor to dryness to obtain a brown solid, which is a crude product of dimyristoylphosphatidylcholine;

[0047] (2) Using 600 mL of solvent 1 (acetone) and 300 mL of solvent 2 (dichloromethane), the mixture was heated to 40° C. and stirred to dissolve the crude dimyristoylphosphatidylcholine. The mixture was cooled to 0° C., a large amount of solid precipitated, and the mixture was filtered and dried to obtain a solid. The solid was heated to 40° C. and stirred to dissolve with 300 mL of solvent 3 (ethanol). The insoluble matter was removed by filtration while hot. The filtrate was cooled to 0° C., and 1500 mL of solvent 1 (acetone) was slowly added dropwise. The system gradually became turbid. The mixture was slowly stirred and cooled to 0° C., a large amount of solid precipitated, and the refined dimyristoylphosphatidylcholine was filtered to obtain a qualified purity by TLC detection. The mixture was vacuum dried to obtain 207 g of dry solid with a yield of 79%.

[0048] The above-prepared dimyristoylphosphatidylcholine product was subjected to HPLC detection under the following conditions: using octylsilane bonded silica gel as filler (25cm×4.6mm, 5μm); using 0.1% formic acid in water as mobile phase A, and using 0.1% formic acid in methanol as mobile phase B; performing gradient elution according to Table 1; column temperature of 40°C, flow rate of 1.0mL / min, and detecting with an evaporative light scattering detector (reference conditions: drift tube temperature of 40°C; nitrogen flow rate of 2.0L / min; gain of 4). The liquid chromatogram is shown in Table 1. Figure 1 shown.

[0049] Table 1 Gradient elution program

[0050] Time (min) Mobile phase A (%) Mobile phase B (%) 0 10 90 10 0 100 20 0 100 20.5 10 90 30 10 90

[0051] Depend on Figure 1 It can be seen that the purity of the refined dimyristoylphosphatidylcholine is 99.95%.

[0052] The IR spectrum and MS spectrum of the above-prepared dimyristoylphosphatidylcholine are as follows: Figure 2 and Figure 3 As shown. Figure 2 and Figure 3 It can be seen that the final product obtained is dimyristoylphosphatidylcholine.

[0053] Embodiments 2 to 7

[0054] Based on Example 1, the type of organic solvent in step (1) was adjusted to investigate the effect of different organic solvents on the yield in step (1). The results are shown in Table 2.

[0055] Table 2 Effect of different organic solvents on yield in step (1)

[0056] Example Organic solvents Yield (%) Example 2 Dichloromethane 35.82 Example 3 1,2-Dichloroethane 51.79 Example 4 Tetrahydrofuran 22.38 Example 5 Dioxane 11.42 Example 6 Ethyl acetate 16.33 Example 7 N,N-Dimethylformamide 33.98

[0057] Embodiments 8 to 16

[0058] Based on Example 1, the type of base in step (1) was adjusted to investigate the effect of different bases in step (1) on the yield. The results are shown in Table 3.

[0059] Table 3 Effect of different bases on yield in step (1)

[0060]

[0061]

[0062] Embodiments 17-18

[0063] Based on Example 1, the type of condensing agent in step (1) was adjusted to investigate the effect of different condensing agents on the yield in step (1). The results are shown in Table 4.

[0064] Table 4 Effect of different condensing agents on yield in step (1)

[0065]

[0066] Examples 19 to 24

[0067] Based on Example 1, the types of solvent 1, solvent 2, and solvent 3 in step (2) were adjusted to investigate the effect of different crystallization solvents on the yield in step (2). The results are shown in Table 5.

[0068] Table 5 Effect of different crystallization solvents on yield in step (2)

[0069]

[0070]

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing high-purity dimyristoylphosphatidylcholine, characterized in that: The following steps are involved: The crude product of dimyristoylphosphatidylcholine is obtained by condensing myristic acid, glycerophosphatidylcholine, a base and a condensing agent in an organic solvent; The crude dimyristoylphosphatidylcholine is recrystallized using a mixed solvent of solvent 1 and solvent 2, the temperature is lowered to precipitate a solid, and after filtering and drying, the solid is dissolved by heating with solvent 3, and the insoluble matter is removed by filtering, and solvent 1 is added to the filtrate, the temperature is lowered to precipitate a solid, and the solid is filtered and dried to obtain high-purity dimyristoylphosphatidylcholine.

2. The method for preparing high-purity dimyristoylphosphatidylcholine according to claim 1, characterized in that: The mass ratio of the myristic acid, glycerophosphorylcholine, base and condensing agent is 1-5:1-2:0.1-0.5:3-7.

3. The method for preparing high-purity dimyristoylphosphatidylcholine according to claim 2, characterized in that: The base is one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, potassium acetate, triethylamine, 4-dimethylaminopyridine, and N,N-diisopropylethylamine.

4. A method for preparing high-purity dimyristoylphosphatidylcholine according to claim 1 or 3, characterized in that: The condensing agent is one or more of 2-(7-azobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate, and N,N-dicyclohexylcarbodiimide.

5. The method for preparing high-purity dimyristoylphosphatidylcholine according to claim 1 or 2, characterized in that: The organic solvent is one or more of dichloromethane, chloroform, 1,2-dichloroethane, tetrahydrofuran, dioxane, ethyl acetate, and N,N-dimethylformamide.

6. The method for preparing high-purity dimyristoylphosphatidylcholine according to claim 4, characterized in that: The temperature of the condensation reaction is 50-70° C.; the time of the condensation reaction is 16-24 hours.

7. The method for preparing high-purity dimyristoylphosphatidylcholine according to any one of claims 1 to 3, characterized in that: The solvent 1, solvent 2 and solvent 3 are independently one or more of methanol, ethanol, acetone, tetrahydrofuran, ethyl acetate, dichloromethane, chloroform, N,N-dimethylformamide and methyl tert-butyl ether.

8. The method for preparing high-purity dimyristoylphosphatidylcholine according to claim 7, characterized in that: The solvent 1 is acetone; the solvent 2 is dichloromethane; and the solvent 3 is ethanol.

9. The method for preparing high-purity dimyristoylphosphatidylcholine according to claim 8, characterized in that: The temperature for cooling and precipitating solid is independently 0 to 30°C.

10. The method for preparing high-purity dimyristoylphosphatidylcholine according to claim 7, characterized in that: The temperature for heating and dissolving the solid using the solvent 3 is 20-70°C.

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