A crystal form of PEGylated phospholipid and preparation method thereof

Abstract

The present invention belongs to the field of drug synthesis, and particularly relates to a PEGylated phospholipid crystal form and a preparation method thereof. The crystal form provided by the present invention has an X-ray powder diffraction pattern with diffraction peaks at 2θ of 19.2°±0.2° and 23.4°±0.2°. The preparation method provided by the present invention is simple and easy, the raw material price is low, the reaction time is short, it is suitable for industrial scale-up production, and the prepared crystal form has high purity and low impurities, and can be directly used as a carrier material in the field of liposomes.

Description

Technical Field

[0001] The invention belongs to the field of drug synthesis, and particularly relates to a PEGylated phospholipid crystal form and a preparation method thereof. Background Art

[0002] Dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000 (hereinafter referred to as PEGylated phospholipid A) is a pharmaceutical excipient. It is a polyethylene glycol (PEG) derivative of dipalmitoylphosphatidylethanolamine. Because the end of polyethylene glycol is capped with methoxy, the PEG part is inactivated and cannot continue to react, so it is called MPEG. PEGylated phospholipid A is a PEGylated phospholipid, which is often used as a carrier material in the field of high-end injections represented by liposomes and fat emulsions. Its structure is as follows:

[0003]

[0004] CN1243759C discloses a method for preparing PEGylated phospholipid A. This method produces a large amount of by-products, and the preparation method requires many refining times and has a very low yield, so it is not suitable for industrial production.

[0005] As the basic carrier of liposomes, phospholipids have a decisive influence on the physical and chemical properties of liposomes and are one of the important factors affecting the stability of liposome membranes. Therefore, the preparation of a PEGylated phospholipid A with good stability and high purity has extremely high medicinal research value and good development prospects. Summary of the invention

[0006] The purpose of the present invention is to provide a stable PEGylated phospholipid A crystal form and a preparation method thereof suitable for industrial scale-up production.

[0007] In one aspect, the present invention provides a crystalline form of PEGylated phospholipid A, whose X-ray powder diffraction pattern has diffraction peaks at 2θ of 19.2°±0.2° and 23.4°±0.2°;

[0008] In one embodiment of the present invention, the X-ray powder diffraction pattern of the crystalline form has diffraction peaks at 2θ of 15.1°±0.2°, 18.7°±0.2°, 19.2°±0.2°, 21.2°±0.2°, 22.1°±0.2°, 23.4°±0.2°, 26.2°±0.2° and 26.9°±0.2°.

[0009] In one embodiment of the present invention, the 2θ of the X-ray powder diffraction pattern of the crystal form is shown in the following table:

[0010] Table 1 X-ray powder diffraction data of PEGylated phospholipid A crystal form

[0011]

[0012] In one embodiment of the present invention, the X-ray powder diffraction of the crystalline form expressed in 2θ angle has the following characteristics: Figure 1 The spectrum shown; its DSC has Figure 2 The spectrum shown; its TGA has Figure 3 Spectrum shown.

[0013] On the other hand, the present invention also provides a method for preparing PEGylated phospholipid A.

[0014] In one embodiment of the present invention, the preparation method comprises adding base A to formula I and formula II in an aprotic solvent environment, and performing an esterification reaction at a suitable temperature to generate formula III.

[0015]

[0016] In the present invention, the aprotic solvent in the esterification reaction is selected from one or more of toluene, dichloromethane, chloroform, tetrahydrofuran, acetone, ethyl acetate or acetonitrile.

[0017] In one embodiment of the present invention, the aprotic solvent is toluene.

[0018] In the present invention, the base A in the esterification reaction is selected from one or more of sodium hydroxide, potassium hydroxide, lithium hydroxide, potassium carbonate and sodium bicarbonate.

[0019] In one embodiment of the present invention, the base A is selected from sodium bicarbonate.

[0020] In the present invention, the molar ratio of Formula I to Formula II in the esterification reaction is 1:1-10; preferably 1:2-5.

[0021] In one embodiment of the present invention, the molar ratio of Formula I to Formula II in the esterification reaction is 1:3.5.

[0022] In the present invention, the mass volume ratio of Formula I to the aprotic solvent in the esterification reaction is 1:2-6; preferably 1:3-5.

[0023] In one embodiment of the present invention, the mass volume ratio of Formula I to the aprotic solvent in the esterification reaction is 1:4.

[0024] In the present invention, the molar ratio of Formula I to base A in the esterification reaction is 1:1-20; preferably 1:5-10; more preferably 1:7-9.

[0025] In one embodiment of the present invention, the molar ratio of Formula I to base A in the esterification reaction is 1:8.

[0026] In the present invention, the reaction time of the esterification reaction is 2 to 10 hours, preferably 3 to 7 hours.

[0027] In one embodiment of the present invention, the reaction time of the esterification reaction is 3 hours.

[0028] In one embodiment of the present invention, the reaction time of the esterification reaction is 5 hours.

[0029] In one embodiment of the present invention, the reaction time of the esterification reaction is 7 hours.

[0030] In the present invention, the suitable temperature condition for the esterification reaction is 50°C to 100°C, preferably 60°C to 90°C.

[0031] In one embodiment of the present invention, the suitable temperature condition in the esterification reaction is 70°C to 80°C.

[0032] In one embodiment of the present invention, the preparation method comprises adding base B and formula IV to formula III, causing condensation reaction, filtering, adding n-hexane, cooling and crystallizing to obtain a crude product of PEGylated phospholipid A,

[0033]

[0034] In the present invention, the base B in the condensation reaction is selected from one or more of sodium hydroxide, potassium hydroxide, lithium hydroxide, potassium carbonate and sodium bicarbonate.

[0035] In one embodiment of the present invention, the base B in the condensation reaction is selected from sodium bicarbonate.

[0036] In the present invention, the molar ratio of the compound IV to the base B in the condensation reaction is 1:10-30; preferably 1:15-25.

[0037] In one embodiment of the present invention, the molar ratio of the compound IV to the base B in the condensation reaction is 1:20.

[0038] In the present invention, the reaction time of the condensation step is 3 to 10 hours, preferably 3 to 7 hours.

[0039] In one embodiment of the present invention, the reaction time of the condensation step is 3 hours.

[0040] In the present invention, the condensation step includes a post-treatment step.

[0041] In one embodiment of the present invention, the post-treatment step comprises the steps of adding organic solvent A to dissolve, decolorizing by adsorption with activated carbon, cooling and crystallizing, and column chromatography.

[0042] In the present invention, the organic solvent A is one or more of methanol, ethanol, propanol, acetone, chloroform, tetrahydrofuran, ethyl acetate or acetonitrile.

[0043] In one embodiment of the present invention, the organic solvent A is a mixed solution of ethyl acetate and methanol.

[0044] In the present invention, the volume ratio of ethyl acetate to methanol is 20 to 40:1, preferably 25 to 35:1.

[0045] In one embodiment of the present invention, the volume ratio of ethyl acetate to methanol is 25-30:1.

[0046] In the present invention, the activated carbon is decolorized by adsorption at a temperature of 30 to 80°C, preferably at a temperature of 40 to 70°C.

[0047] In one embodiment of the present invention, the activated carbon is decolorized by adsorption at a temperature of 50-60°C.

[0048] In the present invention, the activated carbon adsorption decolorization is carried out for 0.5-3 hours, preferably 0.5-1.5 hours.

[0049] In one embodiment of the present invention, the activated carbon is adsorbed and decolorized for 1 hour.

[0050] In one embodiment of the present invention, the temperature for crystallization by cooling is selected from -10 to 0°C.

[0051] In one embodiment of the present invention, the preparation method comprises the steps of mixing the crude PEGylated phospholipid A with the organic solvent B and performing crystallization.

[0052] In the present invention, the organic solvent B is one or more of methanol, ethanol, propanol, acetone, chloroform, tetrahydrofuran, ethyl acetate or acetonitrile.

[0053] In one embodiment of the present invention, the organic solvent B is a mixed solution of ethyl acetate and methanol.

[0054] In the present invention, the volume ratio of ethyl acetate to methanol is 25 to 40:1, preferably 30 to 35:1.

[0055] In one embodiment of the present invention, the volume ratio of ethyl acetate to methanol is 32-35:1.

[0056] In the present invention, the mass volume ratio of the crude product to the solvent is 1:1-20; preferably 1:5-15.

[0057] In one embodiment of the present invention, the mass volume ratio of the crude product to the solvent is 1:8-12.

[0058] In the present invention, the purification method includes mixing with a solvent and then heating to dissolve.

[0059] In the present invention, the purification method heats the system to 45-55°C.

[0060] In the present invention, the refining method includes adding activated carbon to the reaction for adsorption and decolorization.

[0061] In the present invention, the crystallization step is crystallization by cooling.

[0062] In the present invention, the temperature for crystallization is selected from -10 to 0°C.

[0063] The present invention also provides a method for preparing a PEGylated phospholipid A crystal form, which specifically comprises the following steps:

[0064] 1) Esterification: Add sodium bicarbonate to toluene with formula I and formula II, raise the temperature to 70°C to 80°C, and stir for 3 to 7 hours to generate formula III, i.e. intermediate 1;

[0065] 2) Condensation: add sodium bicarbonate and formula IV to the intermediate 1, filter the sodium bicarbonate, add n-hexane, cool and crystallize, then add a mixed solution of ethyl acetate and methanol, adsorb on activated carbon, heat to 50-60°C for decolorization, filter while hot, cool and crystallize, and perform column chromatography to obtain a crude product of PEGylated phospholipid A;

[0066] 3) Purification: Add a mixed solution of ethyl acetate and methanol to the crude PEGylated phospholipid A to dissolve it, then add activated carbon, heat to 45-55° C. to decolorize, filter, cool and crystallize, and dry to obtain the PEGylated phospholipid A crystal form.

[0067] Beneficial effects of the present invention:

[0068] The preparation method of the invention is simple and easy, the raw materials are cheap, the reaction time is short, it is suitable for industrial scale-up production, and the prepared crystal has high purity and low impurities, and can be directly used as a carrier material in the field of liposomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 X-ray diffraction pattern of PEGylated phospholipid A crystal form.

[0070] Figure 2 DSC spectrum of PEGylated phospholipid A crystal form.

[0071] Figure 3 TGA spectrum of PEGylated phospholipid A crystal form. DETAILED DESCRIPTION

[0072] The following examples are illustrative and are intended only to suggest methods of practicing the invention.

[0073] Example 1

[0074] Toluene, a compound of formula I, a base and a compound of formula II were added to the reactor, the feed ratio was compound of formula II / compound of formula I=3.5:1.0, and the feed ratio of toluene to the compound of formula I was 4.0V / M. The reaction system was heated to 70°C to 80°C, and the reaction was stirred and kept warm for a certain period of time to obtain a compound of formula III. The purity of the compound of formula III was detected by HPLC. The specific reaction parameters and results are shown in Table 1 below.

[0075] HPLC detection conditions: Chromatographic column: Welch Xtimate C18 (4.6mm×250mm, 5μm); mobile phase A: acetate buffer (take 0.77g of ammonium acetate, add 1ml of formic acid, dissolve in water and dilute to 1000ml); mobile phase B: mobile phase A-isopropanol (5:95); flow rate 0.5mL / min; column temperature 60℃; injection volume 20μL; elution gradient is as follows:

[0076] Time(min) A(％) B(％) 0 80 20 10 68 32 20 20 80 45 20 80 45.1 80 20 55 80 20 .

[0077] Table 1 Effect of alkali type on experimental results

[0078]

[0079]

[0080] As can be seen from the table, when sodium carbonate and sodium phosphate are used in the esterification reaction, the generated product system is unstable and easily degraded into the compound of formula I and an unknown single impurity with RT5.7min; sodium methoxide basically does not react; and when sodium bicarbonate is used, the reaction product has a higher purity and is more stable in the system.

[0081] Example 2

[0082] (1) Esterification: Toluene (320 mL), polyethylene glycol 5000 monomethyl ether (average molecular weight 5000, 80 g, 16 mmol), sodium bicarbonate (10.8 g, 128 mmol) and p-nitrophenyl chloroformate (11.3 g, 56 mmol) were added to a reactor, the reaction system was heated to 70° C. to 80° C., and the mixture was stirred and reacted for 3 h to obtain intermediate 1, which was directly used in the next step;

[0083] (2) Condensation: Sodium bicarbonate (32.2 g, 384 mmol) and dipalmitoylphosphatidylethanolamine (13.3 g, 19.2 mmol) were added to the above reaction system, and the reaction was continued for 3 h. The sodium bicarbonate was filtered, and the filtrate was slowly dripped into n-hexane (800 mL) precooled to -10 to 0°C, and the mixture was stirred and crystallized at -10 to 0°C for 1 h. The mixture was filtered and dried to obtain a yellow solid. Ethyl acetate (800 mL) and methanol (28 mL), Japanese egret activated carbon (37.8 g) were added to the solid, heated to 50-60 ° C for decolorization for 1 h, hot filtered, cooled to -10-0 ° C for crystallization, and filtered to obtain a white solid. The white solid was dissolved in dichloromethane and wet column chromatography (30 μm silica gel, methanol: dichloromethane = 20: 1 (V / V) was used as the eluent to elute until the impurity point appeared, methanol: dichloromethane = 10: 1 (V / V) was used as the eluent to elute until the product point appeared, chloroform-methanol-water (90: 18: 2) was used as the developing agent), the product was collected, and concentrated to dryness at 30-40 ° C to obtain a crude product (50 g, two-step total yield 54.4%), with a purity of 99.0%;

[0084] (3) Purification: Add the crude product (50 g), ethyl acetate (500 mL) and methanol (15 mL), Japanese egret activated carbon (10 g) into a reactor, heat to 45-55°C, decolorize for 1 h, filter while hot, cool to -10-0°C for crystallization, and dry to obtain a refined product (46 g, yield 92.0%) with a purity of 99.7%.

[0085] The XRD (X-ray powder diffractometer, test conditions: light tube voltage and current: 40KV, 40mA; scanning range 3°-40°; scanning step length 0.02°; scanning time per step 0.15s), DSC (differential calorimetry scanner, test conditions: starting temperature 40°C, heating to 250°C at 10°C / min) and TGA (thermogravimetric analyzer, test conditions: starting temperature room temperature, heating to 200°C at 10°C / min) spectra of the samples are shown in the attached Figures 1 to 3 .

[0086] Example 3

[0087] (1) The esterification and condensation steps are the same as in Example 2;

[0088] (2) Purification: Take 1 g of the crude product, add 5 ml of ethanol, heat until dissolved, slowly cool, and filter to obtain a white solid (0.7 g, yield 70%).

[0089] Example 4

[0090] (1) The esterification and condensation steps are the same as in Example 2;

[0091] (2) Purification: Take 1 g of the crude product, add 2 ml of methanol, heat until it becomes clear, slowly cool, and filter to obtain a white solid (0.5 g, yield 50%).

[0092] Example 5 Stability Study Results

[0093] The refined product prepared in Example 2 was stored for a period of time under various conditions (high temperature: 20-30°C, high humidity: 95% RH, light: strong direct light, long term: -20°C, accelerated: 5°C) to examine the stability. The relevant substances were detected by HPLC. The specific HPLC detection parameters are shown in Example 1. The experimental results are shown in Table 2 below.

[0094] Table 2 Stability test data under various influencing factors

[0095]

[0096] The experimental results show that the PEGylated phospholipid A crystal prepared according to the preparation method of the present invention has high purity and good stability. When placed under high temperature, high humidity, light, long-term and accelerated conditions for 10 days, the crystal content is stable, the related substances are not increased, and the known impurities that need to be controlled in the quality standard are not degraded.

[0097] Comparative Example 1: Condensation step without post-treatment

[0098] (3) The esterification step is the same as in Example 2;

[0099] (4) Condensation: Sodium bicarbonate (32.2 g, 384 mmol) and dipalmitoylphosphatidylethanolamine (13.3 g, 19.2 mmol) were added to the above reaction system and reacted for 3 h. The sodium bicarbonate was filtered and the filtrate was slowly dripped into n-hexane (800 mL) precooled at -10 to 0°C. The mixture was stirred and crystallized for 1 h under warm condition. The mixture was filtered and dried to obtain a yellow solid with a purity of 75.2%.

[0100] Comparative Example 2: Condensation step post-treatment with acetone

[0101] (1) The esterification step is the same as in Example 2;

[0102] (2) Condensation: Sodium bicarbonate (32.2 g, 384 mmol) and dipalmitoylphosphatidylethanolamine (13.3 g, 19.2 mmol) were added to the above reaction system, and the reaction was continued for 3 h. The sodium bicarbonate was filtered, and the filtrate was slowly dripped into n-hexane (800 mL) pre-cooled at -10 to 0°C, and the mixture was stirred and crystallized for 1 h. The mixture was filtered to dryness to obtain a yellow solid. Acetone (500 mL) was added, and the mixture was heated to 50°C, and impurities were removed by filtration. A yellow solid was obtained by filtration with a purity of 72.1%.

[0103] Comparative Example 3: Post-treatment of the condensation step: Refining once with acetone and then refining twice with ethyl acetate

[0104] (1) The esterification step is the same as in Example 2;

[0105] (2) Condensation: Sodium bicarbonate (32.2 g, 384 mmol) and dipalmitoylphosphatidylethanolamine (13.3 g, 19.2 mmol) were added to the above reaction system, and the reaction was continued for 3 h. The sodium bicarbonate was filtered and the filtrate was slowly dripped into n-hexane (800 mL) pre-cooled at -10 to 0 °C, and the mixture was stirred and crystallized for 1 h. The mixture was filtered and dried to obtain a yellow solid. Acetone was added, and the mixture was heated to 50 °C and filtered to remove impurities. N-hexane was added to the solution, crystals were precipitated, and the mixture was filtered. Ethyl acetate (900 mL) was added to the solid, and the mixture was dissolved at 65 °C. After stirring for 30 minutes, the mixture was cooled to 5 °C, crystallized, and the precipitated crystals were filtered. The crystallization treatment with ethyl acetate was repeated once, and the mixture was filtered and dried to obtain a yellow solid with a purity of 87.7%.

Claims

1. A crystalline form of dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000, characterized in that: The X-ray powder diffraction pattern of the crystal form has diffraction peaks at 2θ of 15.1°±0.2°, 18.7°±0.2°, 19.2°±0.2°, 21.2°±0.2°, 22.1°±0.2°, 23.4°±0.2°, 26.2°±0.2° and 26.9°±0.2°; preferably, the crystal form has an X-ray powder diffraction pattern as shown in the following table:

2. The crystal form according to claim 1, characterized in that The X-ray powder diffraction of the crystal form expressed in 2θ angle has a spectrum as shown in FIG1 ; its DSC has a spectrum as shown in FIG2 ; and its TGA has a spectrum as shown in FIG3 .

3. A method for preparing dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000, characterized in that: The specific steps include mixing a crude product of dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000 with an organic solvent B, and performing a crystallization step; the organic solvent B is one or more of methanol, ethanol, propanol, acetone, chloroform, tetrahydrofuran, ethyl acetate or acetonitrile.

4. The preparation method according to claim 3, characterized in that: The organic solvent B is a mixed solution of ethyl acetate and methanol; preferably, the volume ratio of ethyl acetate to methanol is 25-40:1; preferably 30-35:1; more preferably 32-35:1; the mass volume ratio of the crude product to the solvent is 1:1-20; preferably 1:5-15; more preferably 1:8-12.

5. The preparation method according to claim 3, characterized in that: Add base B and formula IV to formula III to cause condensation reaction, filter, add n-hexane, cool and crystallize to obtain crude dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000, 6. The preparation method according to claim 5, characterized in that: Base B is selected from one or more of sodium hydroxide, potassium hydroxide, lithium hydroxide, potassium carbonate and sodium bicarbonate; preferably sodium bicarbonate; the molar ratio of formula IV to base B is 1:10-30; preferably 1:15-25; the reaction time is 3 to 10 hours; preferably 3 to 7 hours.

7. The preparation method according to claim 5, characterized in that: The condensation step includes a post-treatment step; the post-treatment step includes the steps of adding an organic solvent A to dissolve, decolorizing by activated carbon adsorption, cooling and crystallizing, and column chromatography; the organic solvent A is one or more of methanol, ethanol, propanol, acetone, chloroform, tetrahydrofuran, ethyl acetate or acetonitrile.

8. The preparation method according to claim 5, characterized in that: The specific steps include adding base A to Formula I and Formula II in an aprotic solvent environment, and causing an esterification reaction under suitable temperature conditions to generate Formula III.

9. The preparation method according to claim 8, characterized in that: The aprotic solvent is selected from one or more of toluene, dichloromethane, chloroform, tetrahydrofuran, acetone, ethyl acetate or acetonitrile; preferably toluene; the base A is selected from one or more of sodium hydroxide, potassium hydroxide, lithium hydroxide, potassium carbonate and sodium bicarbonate; preferably sodium bicarbonate; the molar ratio of formula I to formula II is 1:1-10; preferably 1:2-5; the mass volume ratio of formula I to the aprotic solvent is 1:2-6; preferably 1:3-5; the molar ratio of formula I to base A is 1:1-20; preferably 1:5-10; more preferably 1:7-9; the reaction time is 2-10h; preferably 3-7h; the suitable temperature condition is 50°C-100°C; preferably 60°C-90°C; more preferably 70°C-80°C.

10. A method for preparing dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000, characterized in that: The specific steps include: 1) Esterification: Add sodium bicarbonate to toluene with formula I and formula II, raise the temperature to 70°C to 80°C, and stir for 3 to 7 hours to generate formula III, i.e. intermediate 1; 2) Condensation: add sodium bicarbonate and formula IV to the intermediate 1, filter the sodium bicarbonate, add n-hexane, cool and crystallize, then add a mixed solution of ethyl acetate and methanol, adsorb on activated carbon, heat to 50-60°C for decolorization, filter while hot, cool and crystallize, and perform column chromatography to obtain a crude product of dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000; 3) Purification: A mixed solution of ethyl acetate and methanol is added to the crude product of dipalmitoylphosphatidylethanolamine-methoxy polyethylene glycol 5000 to dissolve it, and then activated carbon is added, heated to 45-55° C. for decolorization, filtered, cooled for crystallization, and dried to obtain dipalmitoylphosphatidylethanolamine-methoxy polyethylene glycol 5000.

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