CN108659037B

CN108659037B - Polymorphic substance of valproic acid phospholipid derivative and preparation method thereof

Info

Publication number: CN108659037B
Application number: CN201710208986.5A
Authority: CN
Inventors: 郝超; 马彦琴; 王勇; 周英珍; 陈亮; 司崇静; 张桂森
Original assignee: Nhwa Pharmaceutical Corp
Current assignee: Shanghai Shujing Biotechnology Co ltd
Priority date: 2017-03-31
Filing date: 2017-03-31
Publication date: 2022-01-11
Anticipated expiration: 2037-03-31
Also published as: CN108659037A

Abstract

The present invention relates to 1-palmitoyl-2-valproyl-sn-glycero-3-phosphatidylcholine (C)₁₆-four polymorphs of DP-VPA): crystal form A, crystal form B, crystal form C and crystal form D, and preparation methods thereof. C provided by the invention₁₆The crystal form A and the crystal form B of the-DP-VPA have good stability, are beneficial to clinical storage and use, can meet the requirements of preparations, and are suitable for drug development.

Description

Polymorphic substance of valproic acid phospholipid derivative and preparation method thereof

Technical Field

The invention belongs to the field of medicines, relates to a polymorphic substance of a valproic acid phospholipid derivative and a preparation method thereof, and particularly relates to 1-palmitoyl-2-valproyl-sn-glycero-3-phosphatidylcholine (C)₁₆-DP-VPA) and processes for their preparation.

Background

Epilepsy is a chronic disorder of the brain caused by a variety of etiologies and is characterized by sudden, recurrent and transient central nervous system dysfunction due to excessive firing of neurons in the brain. Seizure is a transient involuntary tic (i.e., partial or generalized) seizure in a part of the body or the entire body, sometimes accompanied by loss of consciousness and urinary incontinence. The epileptic seizure not only brings physical and mental pains to patients, but also increases the burden of medical care, and has become an important social problem. Valproic acid (VPA) is the most frequently reported antiepileptic drug, but it has VPA-induced side effects such as gastro-intestinal irritation, bone marrow suppression (especially manifested as aplastic anemia and thrombocytopenia) and liver dysfunction, and its drug half-life is short, and even if taken as a sustained release formulation, it is required to be taken several times a day.

Chinese patent application 01815173.6 discloses a valproic acid phospholipid derivative and a preparation method thereof, wherein the phospholipid derivative (DP-VPA) is a mixture, and the two components of the phospholipid derivative are respectively 1-palmitoyl-2-valproyl-sn-glycero-3-phosphorusFatty acyl choline (C)₁₆-DP-VPA), 1-stearoyl-2-valproyl-sn-glycero-3-phosphatidylcholine (C)₁₈-DP-VPA), the structural formulae are respectively as follows:

two component ratio C₁₆-DP-VPA:C₁₈The DP-VPA is 15 + -5%: 85 + -5%, and the drug developed by Israel D-Pharm for the treatment of epilepsy is currently in clinical research.

DP-VPA is a potent anticonvulsant agent which is essentially lysophosphatidylcholine as a carrier, and in which VPA (valproic acid), the active ingredient, is ester-linked to a phospholipid at the sn-2 position, and since the phospholipid compound is hydrophobic in nature, it can penetrate biological membranes and barriers, thereby facilitating transport of the drug to cells or organs, e.g., the brain, where its action is desired, and VPA is a phospholipase A at the sn-2 position of the phospholipid via the DP-VPA compound₂Or any other lipase or esterase. The modified release of the valproic acid moiety at the focal site can further improve the therapeutic index of the drug, as the efficacy of the drug is expected to increase while the potential side effects and toxicity are reduced. The compound is at a much lower dose than currently used VPA, and a reduction in the therapeutic dose in turn reduces the toxicological risk and the associated side effects, as well as reducing the risk of unwanted interactions with other drugs. Furthermore, the compound has significantly improved pharmacokinetic properties over VPA (e.g., greatly prolonged half-life in serum and brain tissue). The compound is expected to become an excellent antiepileptic drug.

The polymorphism of the drug is one of factors influencing the quality of the drug, and different crystal forms of the same drug have differences in appearance, solubility, melting point, dissolution rate, biological activity and the like, so that the stability, bioavailability, clinical curative effect and the like of the drug are influenced. Therefore, intensive research has led to the discovery of polymorphs with good chemical stability to improve C₁₆-DP-VPA、C₁₈The properties of the DP-VPA aspects are essential.

Disclosure of Invention

The purpose of the present invention is to provide 1-palmitoyl-2-valproyl-sn-glycero-3-phosphatidylcholine (C)₁₆-four polymorphs of DP-VPA): crystal form A, crystal form B, crystal form C and crystal form D, and preparation methods thereof.

The purpose of the invention can be realized by the following technical scheme:

₁₆C-DP-VPA crystal form A

The invention provides C₁₆Form A of DP-VPA, characterized in that it exhibits characteristic peaks in the X-ray powder diffraction spectrum at 5.09, 7.6, 10.11, 12.63, 15.16, 17.69, 20.24, 22.78, 25.35, expressed in terms of diffraction angles 2 θ ± 0.2 °. Further, said C₁₆The X-ray powder diffraction pattern of form A of-DP-VPA expressed as 2 theta + -0.2 DEG diffraction angle also shows characteristic peaks at 20.73, 21.66, 22.11, 23.9, 24.65, 27.92, 30.53, 34.37, 36.91.

The invention provides C₁₆Form A of DP-VPA, characterized in that it exhibits, in an X-ray powder diffraction spectrum expressed in diffraction angles 2 θ ± 0.2 °, characteristic peaks at 5.089, 7.596, 10.108, 12.625, 15.163, 17.691, 20.237, 22.783, 25.351. Further, said C₁₆The X-ray powder diffraction pattern of form a of-DP-VPA, expressed as 2 θ ± 0.2 ° diffraction angle, also shows characteristic peaks at 20.733, 21.662, 22.112, 23.899, 24.646, 27.922, 30.525, 34.366, 36.908.

Further, said C₁₆The X-ray powder diffraction reflection angle 2 theta +/-0.2 degrees of the crystal form A of the DP-VPA and corresponding d value and relative peak intensity are shown in a table 1:

TABLE 1 form A

Preferably, said C₁₆The X-ray powder diffraction pattern of the form A of the-DP-VPA is substantially as shown in figure 1.

Said C is₁₆Infrared light of form A of DP-VPAThe absorption peaks contained in the spectra are: 3423.66, 3035.60, 2920.53, 2851.68, 1739.55, 1663.90, 1487.67, 1467.34, 1417.65, 1382.33, 1346.50, 1252.02, 1231.33, 1184.69, 1163.74, 1143.86, 1100.85, 1082.30, 1060.12, 1019.19, 970.32, 951.48, 929.89, 875.48, 809.43, 782.21, 745.17, 722.08, 594.57, 512.77 ± 0.5% cm^-1。

Said C is₁₆DSC analysis of form A of DP-VPA shows a first endothermic peak at 88. + -. 1.5 ℃ and a second endothermic peak at 168. + -. 1.5 ℃.

Said C is₁₆Further thermogravimetric analysis of form a of DP-VPA showed: the weight loss ratio of the crystal form A at 25-200 ℃ is 3.15 +/-0.2%.

Said C is₁₆Optical microscopy characterization images of form a of DP-VPA show square, rectangular, irregular platelets.

Said C is₁₆Scanning electron microscopy characterization images of form a of DP-VPA show irregular platelets.

The invention also provides C₁₆A process for the preparation of form a of-DP-VPA, including but not limited to: standing and recrystallizing. The preparation method provided by the invention is used for C which can be used as a raw material₁₆The form in which the-DP-VPA exists is not particularly limited, and any crystalline form or amorphous solid may be used.

Preparation of crystal form A by standing recrystallization method

Specifically, the invention provides C₁₆-a process for the preparation of form a of DP-VPA, a standing recrystallization process comprising the steps of:

1) c is reacted at 40 ℃ to reflux temperature₁₆-DP-VPA dissolved in an organic solvent;

2) and cooling, crystallizing, filtering, washing and drying to obtain the target product.

In a preferred embodiment of the present invention, the organic solvent is selected from ketones, mixed solvents of ketones and inert organic solvents, mixed solvents of alcohols and inert organic solvents, and mixed solvents of alcohols and ketones, and the inert organic solvent is selected from one or more of esters, alkanes, acetonitrile, N-dimethylformamide, methyl tert-butyl ether, and tetrahydrofuran.

Optionally, the ketone is selected from one or more of acetone, butanone and methyl isobutyl ketone, and the alcohol is selected from C_1-8One or more of straight chain or branched chain alkanol, more preferably one or more of methanol, ethanol, n-propanol, isopropanol and n-butanol, the ester is selected from one or more of ethyl formate, butyl formate, ethyl acetate, methyl acetate, butyl acetate and isobutyl acetate, and the alkane is selected from one or more of petroleum ether, n-hexane, cyclohexane and n-heptane.

In a preferred embodiment of the invention, said C₁₆The concentration of the DP-VPA in the organic solvent is 0.05-0.25 g/ml, the volume ratio of the ketone to the inert organic solvent in the combination of the ketone and the inert organic solvent is more than 1:10, the volume ratio of the alcohol to the inert organic solvent in the combination of the alcohol and the inert organic solvent is 1: 5-1: 50, and the volume ratio of the alcohol to the ketone organic solvent in the combination of the alcohol and the ketone is 1: 5-1: 50.

Preferably, the drying is carried out by using a vacuum drying oven, and the preferable vacuum drying temperature is 40-65 ℃.

Another aspect of the invention provides a pharmaceutical composition comprising C₁₆-form a of DP-VPA and a pharmaceutically acceptable carrier.

In still another aspect of the present invention, C is provided₁₆-use of form a of DP-VPA in the manufacture of a medicament for the treatment of epilepsy, migraine, bipolar cell disease and pain.

₁₆C-DP-VPA crystal form B

The invention also provides C₁₆Form B of DP-VPA, characterized in that it exhibits, in an X-ray powder diffraction pattern expressed in diffraction angles 2 θ ± 0.2 °, characteristic peaks at 9.18, 9.92, 11.92, 12.42, 17.51, 19.65, 19.97, 20.56, 21.48, 21.87. Further, said C₁₆The X-ray powder diffraction pattern of the form B of the-DP-VPA expressed as the 2 theta +/-0.2 DEG diffraction angle is also 7.41, 13.40, 14.98, 15.93 and 16.39. Characteristic peaks are shown at 18.30, 18.73, 22.62, 23.7, 24.50.

The invention provides C₁₆Form B of DP-VPA, characterized in that it exhibits, in an X-ray powder diffraction spectrum expressed in diffraction angles 2 θ ± 0.2 °, characteristic peaks at 9.179, 9.923, 11.917, 12.424, 17.511, 19.648, 19.965, 20.560, 21.479, 21.871. Further, said C₁₆The X-ray powder diffraction pattern of form B of-DP-VPA, expressed as 2 θ ± 0.2 ° diffraction angle, also shows characteristic peaks at 7.410, 13.395, 14.975, 15.934, 16.392, 18.299, 18.725, 22.622, 23.702, 24.498.

Further, said C₁₆X-ray powder diffraction reflection angle 2 theta +/-0.2 degrees of the crystal form B of the-DP-VPA, corresponding d value and relative peak intensity are shown in a table 2.

TABLE 2 form B

Preferably, said C₁₆The X-ray powder diffraction pattern of form B of-DP-VPA is substantially as shown in figure 7.

Said C is₁₆-the infrared spectrum of form B of DP-VPA comprises the absorption peaks: 3421.55, 3035.60, 2920.53, 2851.68, 1739.55, 1663.90, 1487.67, 1467.34, 1417.65, 1382.33, 1346.50, 1252.02, 1231.33, 1184.69, 1163.74, 1143.86, 1100.85, 1082.30, 1060.12, 1019.19, 970.32, 951.48, 929.89, 875.48, 809.43, 782.21, 745.17, 722.08, 594.57, 512.77 ± 0.5% cm^-1。

Said C is₁₆DSC analysis of form B of DP-VPA shows a first endothermic peak at 87 + -1.5 deg.C and a second endothermic peak at 167 + -1.5 deg.C.

Said C is₁₆Further thermogravimetric analysis of form B of DP-VPA shows: the weight loss ratio of the crystal form B at 25-200 ℃ is 3.10 +/-0.2%.

Said C is₁₆Scanning electron microscopy characterization images of form B of DP-VPA show irregularly piled powder.

The invention also provides C₁₆A process for the preparation of form B of DP-VPA, including but not limited to: recrystallization and lapping and rotating crystallization.

Recrystallization method for preparing crystal form B

Specifically, the invention provides C₁₆Process for the preparation of form B of DP-VPA, recrystallization, of form C usable as starting material₁₆The form in which the DP-VPA is present is not particularly limited, and any crystalline form or amorphous solid may be used, including the following steps:

1) c is to be₁₆-DP-VPA is made into a suspension with a hydrophobic inert organic solvent;

2) obtaining a clear solution under the condition of 40-reflux temperature;

3) cooling, crystallizing, filtering, washing and drying to obtain the target product.

In a preferred embodiment of the invention, the hydrophobic inert organic solvent is selected from one or more of esters and alkane organic solvents; optionally, the esters are selected from one or more of ethyl formate, butyl formate, ethyl acetate, methyl acetate, butyl acetate and isobutyl acetate, and the alkanes are selected from one or more of petroleum ether, n-hexane, cyclohexane and n-heptane.

In a preferred embodiment of the invention, C₁₆The proportion of the-DP-VPA to the hydrophobic inert organic solvent is 0.01-0.5 g/ml, and the solvents can be combined in any proportion.

Preferably, the drying is carried out by using a vacuum drying oven, and the preferable drying temperature is 40-65 ℃.

Grinding crystal transformation method for preparing crystal form B

The grinding and crystal transformation method comprises mechanical ball milling and manual grinding.

Specifically, the invention provides C₁₆-a process for the preparation of form B of DP-VPA, mechanical ball milling, comprising the steps of: c according to the invention₁₆Putting the crystal form A of the-DP-VPA into an agate mortar of a ball mill, grinding at the speed of 400 revolutions per minute, grinding for 15 minutes, and resting for 15 minutes, grinding for 15 minutesThe rotation directions of the circulating ball mills are also alternately changed, and the grinding time is more than 30 minutes, thus obtaining the target product.

Specifically, the invention also provides a C₁₆-DP-VPA, crystalline form B, a process for the preparation, artificial milling, comprising the steps of: c according to the invention₁₆And (3) placing the crystal form A of the-DP-VPA into a common agate mortar, and forcibly grinding for not less than 1 hour to obtain the target product.

Another aspect of the invention provides a pharmaceutical composition comprising C₁₆-form B of DP-VPA and a pharmaceutically acceptable carrier.

In still another aspect of the present invention, C is provided₁₆-use of form B of DP-VPA in the manufacture of a medicament for the treatment of epilepsy, migraine, bipolar cell disease and pain.

₁₆C-DP-VPA crystal form C

The invention also provides C₁₆Form C of DP-VPA, characterized in that it exhibits, in an X-ray powder diffraction spectrum expressed in diffraction angles 2 θ ± 0.2 °, characteristic peaks at 3.518, 7.425, 9.944, 14.429, 17.528, 18.084, 18.690, 20.080, 20.583. Further, said C₁₆The X-ray powder diffraction pattern of form C of-DP-VPA expressed as 2 theta + -0.2 DEG diffraction angle also shows characteristic peaks at 4.911, 9.198, 11.923, 12.432, 13.410, 14.999, 16.280, 19.638,', 21.514, 21.841, 22.636, 23.684, 24.495. Further, said C₁₆The X-ray powder diffraction pattern of the form C of the-DP-VPA is substantially as shown in figure 12.

Said C is₁₆-the infrared spectrum of form C of DP-VPA comprises the absorption peaks: 3423.82, 3035.50, 2920.29, 2851.57, 1739.50, 1664.31, 1487.48, 1467.29, 1417.38, 1401.25, 1382.28, 1346.45, 1251.91, 1231.29, 1184.80, 1163.72, 1143.90, 1100.87, 1082.49, 1060.18, 1019.28, 970.36, 951.56, 929.91, 875.49, 809.58, 782.27, 745.38, 722.13, 595.41, 512.84 ± 0.5% cm^-1；

Said C is₁₆DSC analysis of form C of DP-VPA with a first peak at 83. + -. 1.5 ℃Endothermic peak, with a second endothermic peak at 166. + -. 1.5 ℃.

Said C is₁₆Further thermogravimetric analysis of form C of DP-VPA shows: the weight loss ratio of the crystal form C at 25-200 ℃ is 1.55 +/-0.2%.

The invention also provides C₁₆A process for the preparation of form C of DP-VPA, including but not limited to: high vacuum solvent removal.

High vacuum solvent removal process for preparing crystal form C

The invention also provides a preparation method of the compound C₁₆Process for the preparation of form C of DP-VPA, high vacuum solvent removal process, for C usable as starting material₁₆The form in which the DP-VPA is present is not particularly limited, and any crystalline form or amorphous solid may be used, including the following steps:

1) c is to be₁₆-DP-VPA is dissolved in a benign organic solvent or a mixture of a benign organic solvent and an inert organic solvent to form a clear solution;

2) removing the solvent from the clear solution under high vacuum condition;

3) slowly cooling, and recovering the solid to obtain the target product.

In a preferred embodiment of the present invention, the benign organic solvent is selected from one or more of methanol, ethanol, n-propanol, isopropanol, chloroform and dichloromethane, and the inert organic solvent is selected from one or more of ketones, esters, alkanes, acetonitrile, methyl tert-butyl ether and tetrahydrofuran; optionally, the ketone is selected from one or more of acetone, butanone and methyl isobutyl ketone; the esters are selected from one or more of ethyl formate, butyl formate, ethyl acetate, methyl acetate, butyl acetate and isobutyl acetate; the alkane is selected from one or more of petroleum ether, n-hexane, cyclohexane and n-heptane.

In a preferred embodiment of the invention, the reduced pressure solvent removal is preferably carried out at a vacuum pressure of less than 20mbar and at a temperature of 73 to 78 ℃.

Another aspect of the invention provides a pharmaceutical composition comprising C₁₆-DP-VPA crystalline formsC and a pharmaceutically acceptable carrier.

In still another aspect of the present invention, C is provided₁₆-use of form C of DP-VPA in the manufacture of a medicament for the treatment of epilepsy, migraine, bipolar cell disease and pain.

₁₆C-DP-VPA crystal form D

The invention also provides C₁₆-form D of DP-VPA, which form D is amorphous, characterized in that the X-ray powder diffraction spectrum expressed in degrees 2 θ using Cu-ka radiation has no sharp diffraction peaks. Further, said C₁₆The X-ray powder diffraction pattern of the form D of-DP-VPA is substantially as shown in figure 16.

The invention also provides a preparation method of the compound C₁₆A process for the preparation of form D of DP-VPA, including but not limited to: vacuum high-temperature melting and cooling method.

Preparation of crystal form D by vacuum high-temperature melting cooling method

The invention also provides a preparation method of the compound C₁₆Process for the preparation of form D of DP-VPA, vacuum high temperature melting cooling process, for C usable as starting material₁₆The form in which the DP-VPA is present is not particularly limited, and any crystalline form or amorphous solid may be used, including the following steps:

1)C₁₆putting a proper amount of DP-VPA solid in a ceramic crucible, and heating and melting under a vacuum condition;

2) and (3) rapidly cooling the product obtained by heating and melting to room temperature to obtain the target product.

In one embodiment of the present invention, the heating and melting conditions under vacuum conditions preferably have a vacuum degree of 0.01 to 0.05Mpa and a temperature of 150 to 168 ℃.

In one embodiment of the invention, the rapid cooling is preferably nitrogen purge cooling.

Another aspect of the invention provides a pharmaceutical composition comprising C₁₆-form D of DP-VPA and a pharmaceutically acceptable carrier.

In still another aspect of the present invention, C is provided₁₆Crystalline form D of DP-VPA for the treatment of epilepsyEpilepsy, migraine, bipolar cell disease and pain.

The invention has the following beneficial effects:

c provided by the invention₁₆The crystal form A and the crystal form B of the-DP-VPA have good stability, are beneficial to clinical storage and use, can meet the requirements of preparations, and are suitable for drug development.

In addition, the preparation process of the crystal form A and the crystal form B is simple to operate, stable, controllable and repeatable, and is suitable for industrial production.

Drawings

FIG. 1C₁₆X-ray powder diffraction pattern of-DP-VPA crystal form A

FIG. 2C₁₆FT-IR spectrum of form A of DP-VPA

FIG. 3C₁₆DSC pattern of form A of DP-VPA

FIG. 4C₁₆TG profile of form A of DP-VPA

FIG. 5C₁₆-optical micrograph of DP-VPA form A

FIG. 6C₁₆Scanning electron micrograph of form A of DP-VPA

FIG. 7C₁₆X-ray powder diffraction pattern of-DP-VPA crystal form B

FIG. 8C₁₆FT-IR spectrum of form B of DP-VPA

FIG. 9C₁₆DSC pattern of form B of DP-VPA

FIG. 10C₁₆TG profile of form B of DP-VPA

FIG. 11C₁₆Scanning electron micrograph of form B of DP-VPA

FIG. 12C₁₆X-ray powder diffraction pattern of-DP-VPA crystal form C

FIG. 13C₁₆FT-IR Spectroscopy of form C of DP-VPA

FIG. 14C₁₆DSC thermogram of form C of DP-VPA

FIG. 15C₁₆TG thermogram of-DP-VPA crystal form C

FIG. 16C₁₆X-ray powder diffraction pattern of form D of DP-VPA

FIG. 17C₁₆Factor influencing stability of (E) -DP-VPA crystal form A at high temperature for 5 days and 10 daysX-ray powder diffraction spectrum of element

FIG. 18C₁₆X-ray powder diffraction spectrum of stability influence factors of-DP-VPA crystal form B at high temperature for 5 days and 10 days

FIG. 19C₁₆X-ray powder diffraction spectrum of stability influence factors of-DP-VPA crystal form A under strong light irradiation for 5 days and 10 days

FIG. 20C₁₆X-ray powder diffraction spectrum of stability influence factors of-DP-VPA crystal form B under strong light irradiation for 5 days and 10 days

FIG. 21C₁₆Time-dependent change of X-ray powder diffraction pattern of-DP-VPA crystal form C under natural condition

FIG. 22C₁₆Time-dependent change of X-ray powder diffraction pattern of-DP-VPA crystal form D under natural condition

Detailed Description

The invention will be further illustrated by the following specific examples, which are not intended to limit the scope of the invention. The skilled person can make modifications to the preparation method and the apparatus used within the scope of the claims, and such modifications should also be considered as the protection scope of the present invention.

In the examples below, unless otherwise indicated, the experimental procedures described are generally carried out according to conventional conditions or conditions recommended by the manufacturer; the raw materials and reagents shown in the figure can be obtained by a commercially available mode.

Test instrument for experiments

X-ray powder diffraction Pattern

The X-ray powder diffractogram of the present invention was collected on a Bruker D8Focus X-ray powder diffractometer. The parameters of the X-ray powder diffraction method are as follows:

x-ray parameters: Cu/Kalpha

Voltage: 40 KV (kV)

Current: 40 milliampere (mA)

Scanning range: from 3.0 to 40 DEG

Sampling step length: 0.02 degree

Sampling pace speed: 0.5 sec/step

DSC spectrum

The Differential Scanning Calorimetry (DSC) analysis chart is detected by a German relaxation-resistant DSC 200F3, the temperature range is 35-200 ℃, and the temperature rise rate is 10K/min; sealing the pricking hole in an aluminum crucible, wherein the purging gas is nitrogen (40ml/min), and the protective gas is nitrogen (20 ml/min).

TGA Spectroscopy

The thermogravimetric analysis (TG) is detected by German relaxation-resistant TG 209F3, the balance is kept at 25 ℃, the temperature range is 35-200 ℃, the heating rate is 5K/min, an aluminum crucible is opened, the purging gas is nitrogen (40ml/min), and the protective gas is nitrogen (20 ml/min).

4. Fourier infrared spectroscopy

The Fourier infrared spectroscopy (FT-IR) of the invention is detected by a NICOLET 330FT-IR infrared spectrophotometer. Weighing 180mg of potassium bromide which is dried and cooled at 120 ℃ in advance, putting the potassium bromide into an agate mortar, grinding the potassium bromide into fine powder, adding about 1.5mg of a test sample, fully mixing the test sample and grinding the test sample into uniform fine powder, and measuring the fine powder by referring to 0402 infrared spectrophotometry in the fourth general rule of Chinese pharmacopoeia 2015 edition.

5. Optically displaying microcrystalline images

The optical microcrystal image is observed on an XPN-203E polarizing hot stage microscope and is obtained by taking a picture by a JVC color camera.

6. Scanning electron microscope images

The scanning electron microscope image is obtained by observing and photographing a desk type scanning electron microscope of a PHENOM in the Netherlands.

7. Determination of moisture content

The moisture content measurement described herein is by the Fischer method in the METTLER TOLEDO V20Volumetric KFTitrator.

Example 1C₁₆Preparation of form A of (DP-VPA)

C is to be₁₆Adding 0.5g of-DP-VPA sample into 3ml of acetone, heating to reflux, clarifying the solution, and continuously heating, stirring and refluxing; stopping stirring after 30min, standing, naturally cooling to room temperature, and separating out a large amount of solids; filtered and washed with 1ml acetoneThe filter cake was collected and dried overnight in a vacuum oven at 60 ℃ to give a white solid. The X-ray powder diffraction pattern of the sample is shown in figure 1, the infrared spectrum is shown in figure 2, the DSC pattern is shown in figure 3, and the TG pattern is shown in figure 4, and the crystal form is defined as a crystal form A.

Example 2C₁₆Preparation of form A of (DP-VPA)

C is to be₁₆0.5g of-DP-VPA sample is added into 2ml of butanone, heated to 40 ℃, the solution is clarified, and the stirring is continued; stopping stirring after 30min, standing, naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with right amount of butanone, collecting filter cake, and drying in vacuum drying oven at 60 deg.C overnight to obtain white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form A.

Example 3C₁₆Preparation of form A of (DP-VPA)

C is to be₁₆Adding 0.5g of DP-VPA sample into a mixed solvent consisting of 2ml of acetone and 2ml of ethyl acetate, heating to reflux, clarifying the solution, and continuously heating, stirring and refluxing; stopping stirring after 30min, standing, naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with a proper amount of the mixed solvent, collecting a filter cake, and drying in a vacuum drying oven at 60 ℃ overnight to obtain a white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form A.

Example 4C₁₆Preparation of form A of (DP-VPA)

C is to be₁₆Adding 0.5g of DP-VPA sample into a mixed solvent consisting of 2ml of acetone and 2ml of acetonitrile, heating to reflux, clarifying the solution, and continuously heating, stirring and refluxing; stopping stirring after 30min, standing, naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with a proper amount of the mixed solvent, collecting a filter cake, and drying in a vacuum drying oven at 60 ℃ overnight to obtain a white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form A.

Example 5C₁₆Preparation of form A of (DP-VPA)

C is to be₁₆Samples of (E) -DP-VPA0.5g of the extract is added into a mixed solvent consisting of 2ml of acetone and 2ml of methyl tert-butyl ether, heated to reflux, clarified, and continuously heated, stirred and refluxed; stopping stirring after 30min, standing, naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with a proper amount of the mixed solvent, collecting a filter cake, and drying in a vacuum drying oven at 60 ℃ overnight to obtain a white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form A.

Example 6C₁₆Preparation of form A of (DP-VPA)

C is to be₁₆Adding 0.5g of DP-VPA sample into a mixed solvent consisting of 2ml of acetone and 2ml of petroleum ether, heating to reflux, clarifying the solution, and continuously heating, stirring and refluxing; stopping stirring after 30min, standing, naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with a proper amount of the mixed solvent, collecting a filter cake, and drying in a vacuum drying oven at 60 ℃ overnight to obtain a white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form A.

Example 7C₁₆Preparation of form A of (DP-VPA)

C is to be₁₆Adding 0.5g of DP-VPA sample into a mixed solvent consisting of 1ml of butanone and 10ml of cyclohexane, heating to reflux, clarifying the solution, and continuously heating, stirring and refluxing; stopping stirring after 30min, standing, naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with a proper amount of the mixed solvent, collecting a filter cake, and drying in a vacuum drying oven at 60 ℃ overnight to obtain a white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form A.

Example 8C₁₆Preparation of form A of (DP-VPA)

C is to be₁₆Adding 0.5g of DP-VPA sample into a mixed solvent consisting of 0.5ml of methanol and 15ml of acetone, heating to reflux, clarifying the solution, and continuously heating, stirring and refluxing; stopping stirring after 30min, standing, naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with appropriate amount of above mixed solvent, collecting filter cake, and vacuum drying at 60 deg.CDrying overnight gave a white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form A.

Example 9C₁₆Preparation of form A of (DP-VPA)

C is to be₁₆Adding 0.5g of DP-VPA sample into a mixed solvent consisting of 0.5ml of methanol and 10ml of ethyl acetate, heating to reflux, clarifying the solution, and continuously heating, stirring and refluxing; stopping stirring after 30min, standing, naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with a proper amount of the mixed solvent, collecting a filter cake, and drying in a vacuum drying oven at 60 ℃ overnight to obtain a white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form A.

Example 10C₁₆Preparation of form A of (DP-VPA)

C is to be₁₆Adding 0.5g of DP-VPA sample into a mixed solvent consisting of 0.5ml of methanol and 10ml of methyl tert-butyl ether, heating to reflux, clarifying the solution, and continuously heating, stirring and refluxing; stopping stirring after 30min, standing, naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with a proper amount of the mixed solvent, collecting a filter cake, and drying in a vacuum drying oven at 60 ℃ overnight to obtain a white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form A.

Example 11C₁₆Preparation of form A of (DP-VPA)

C is to be₁₆Adding 0.5g of DP-VPA sample into a mixed solvent consisting of 0.5ml of methanol and 20ml of n-hexane, heating to reflux, clarifying the solution, and continuously heating, stirring and refluxing; stopping stirring after 30min, standing, naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with a proper amount of the mixed solvent, collecting a filter cake, and drying in a vacuum drying oven at 60 ℃ overnight to obtain a white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form A.

Example 12C₁₆Preparation of form A of (DP-VPA)

C is to be₁₆Adding 0.5g of-DP-VPA sample into a mixed solvent consisting of 1ml of isopropanol and 5ml of ethyl formate, heating to 40 ℃, clarifying the solution, and continuously heating, stirring and refluxing; stopping stirring after 30min, standing, naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with a proper amount of the mixed solvent, collecting a filter cake, and drying in a vacuum drying oven at 60 ℃ overnight to obtain a white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form A.

Example 13C₁₆Preparation of form A of (DP-VPA)

C is to be₁₆Adding 0.5g of a DP-VPA sample into a mixed solvent consisting of 0.2ml of absolute ethyl alcohol and 10ml of n-hexane, heating to reflux, clarifying the solution, and continuing stirring; stopping stirring after 30min, standing, naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with a proper amount of the mixed solvent, collecting a filter cake, and drying in a vacuum drying oven at 60 ℃ overnight to obtain a white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form A.

Example 14C₁₆Preparation of form B of (E) -DP-VPA

C is to be₁₆Adding 0.5g of-DP-VPA sample into 3ml of ethyl acetate, heating to reflux, clarifying the solution, and continuously heating, stirring and refluxing for 30 min; naturally cooling to room temperature, and separating out a large amount of solids; filtration, washing with 1ml ethyl acetate, collection of the filter cake in 60 ℃ vacuum drying oven drying overnight, white solid. The X-ray powder diffraction pattern of the sample is shown in figure 7, the infrared spectrum is shown in figure 8, the DSC pattern is shown in figure 9, and the TG pattern is shown in figure 10, and the crystal form is defined as a crystal form B.

Example 15C₁₆Preparation of form B of (E) -DP-VPA

C is to be₁₆0.5g of-DP-VPA sample is added into 3ml of methyl acetate, heated to 40 ℃, the solution is clarified, and the stirring is continued for 30 min; naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with methyl acetate, collecting filter cake, and vacuum drying at 60 deg.C overnight to obtain white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are researchedComparing and determining that the product is the crystal form B.

Example 16C₁₆Preparation of form B of (E) -DP-VPA

C is to be₁₆Adding 0.5g of-DP-VPA sample into 1ml of ethyl formate, heating to reflux, clarifying the solution, and continuously heating, stirring and refluxing for 30 min; naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with ethyl formate, collecting filter cake, and vacuum drying at 60 deg.c overnight to obtain white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form B.

Example 17C₁₆Preparation of form B of (E) -DP-VPA

C is to be₁₆Adding 0.5g of-DP-VPA sample into 5ml of petroleum ether, heating to reflux, clarifying the solution, and continuously heating, stirring and refluxing for 30 min; naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with appropriate amount of petroleum ether, collecting filter cake, and drying in vacuum drying oven at 60 deg.C overnight to obtain white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form B.

Example 18C₁₆Preparation of form B of (E) -DP-VPA

C is to be₁₆Adding 0.5g of-DP-VPA sample into 50ml of n-hexane, heating to reflux, clarifying the solution, and continuously heating, stirring and refluxing for 30 min; naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with a proper amount of n-hexane, collecting filter cakes, and drying overnight in a vacuum drying oven at 60 ℃ to obtain white solids. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form B.

Example 19C₁₆Preparation of form B of (E) -DP-VPA

C is to be₁₆Adding 0.5g of-DP-VPA sample into a mixed solvent consisting of 2ml of ethyl acetate and 2ml of petroleum ether, heating to reflux, clarifying the solution, and continuously heating, stirring and refluxing for 30 min; naturally cooling to room temperature, and separating out a large amount of solids; filtering, washing with appropriate amount of petroleum ether, collecting filter cake, and drying in vacuum drying oven at 60 deg.C overnight to obtain white solid. Its X-ray powder diffraction patternAnd the infrared spectrum, the DSC spectrum and the TG spectrum are compared by research to determine that the product is the crystal form B.

Example 20C₁₆Preparation of form B of (E) -DP-VPA

C is to be₁₆And (3) putting about 4g of the crystal form A of the DP-VPA into two agate mortars of a ball mill, grinding about 2.0g of each mortar sample at the speed of 400 revolutions per minute, alternately changing the rotation direction of each circulating ball mill every 15 minutes after grinding for 15 minutes, and grinding for 30 minutes to obtain the white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form B.

Example 21C₁₆Preparation of form B of (E) -DP-VPA

C is to be₁₆And (3) placing about 2.0g of the crystal form A of the-DP-VPA into a common agate mortar, grinding with force, continuously grinding in the direction of changing the direction every 15 minutes, and grinding for 60 minutes to obtain a white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form B.

Example 22C₁₆Preparation of form C of (E) -DP-VPA

C is to be₁₆0.5g of a sample DP-VPA was added to 10ml of methanol to give a clear solution, which was evaporated under reduced pressure at a pressure of 20mbar and a temperature of 75 ℃; cooling to room temperature to obtain C₁₆Form C of DP-VPA, which has an X-ray powder diffraction pattern as shown in figure 12, an infrared spectrum as shown in figure 13, a DSC pattern as shown in figure 14, and a TG pattern as shown in figure 15.

Example 23C₁₆Preparation of form C of (E) -DP-VPA

C is to be₁₆0.5g of a sample DP-VPA, added to a mixture of 5ml of methanol and 5ml of acetone to give a clear solution, the solvent is evaporated off under reduced pressure at a pressure of 20mbar and a temperature of 73 ℃; cooling to room temperature to obtain white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form C.

Example 24C₁₆Preparation of form C of (E) -DP-VPA

C is to be₁₆0.5g of a sample of-DP-VPA was added to a mixture of 5ml of chloroform and 5ml of acetonitrileForming a clear solution, and evaporating under reduced pressure to remove solvent at 20mbar and 78 deg.C; cooling to room temperature to obtain white solid. The X-ray powder diffraction pattern, the infrared pattern, the DSC pattern and the TG pattern are compared by research to determine that the product is the crystal form C.

Example 25C₁₆Preparation of form D of (E) -DP-VPA

C₁₆2.0g of DP-VPA solid is placed in a ceramic crucible and melted by heating under vacuum; vacuum degree of 0.01Mpa, temperature of 155 deg.C. Rapidly cooling to room temperature by nitrogen purging to obtain C₁₆-DP-VPA form D.

Test example 1 moisture content measurement test

Measured according to the first method of the 0832 method of water content determination according to the fourth rule of the Chinese pharmacopoeia 2015 edition.

The determination method comprises the following steps: precise weighing of C₁₆And (3) putting a proper amount of the crystal form A and the crystal form B of the DP-VPA into a dry glass bottle with a plug, adding anhydrous methanol, titrating the solution from the Fischer-Tropsch test solution to change the light yellow into reddish brown under stirring, and performing a blank test to calculate the water content from the consumed Fischer-Tropsch test solution.

TABLE 3C₁₆Determination results of water content of crystal form A and crystal form B of DP-VPA

From the data in table 3, the water content of both form a and form B was 2.95%.

Test example 2 stability test of Crystal form A and Crystal form B

(1) High temperature test

The C obtained in example 1₁₆Sample of form A of DP-VPA, C from example 14₁₆The sample of form B of DP-VPA was left open and flat, left at 60. + -. 2 ℃ for 10 days, sampled at day 5 and day 10, and XPRD tested after sampling and compared to the results at day 0, as shown in Table 4, FIGS. 17-18.

(2) Test by intense light irradiation

The C obtained in example 1₁₆Sample of form A of DP-VPA, C from example 14₁₆Samples of form B of DP-VPA were placed open and flat for 10 days at 4500lx + -500 lx, sampled at day 5 and day 10, XPRD tested after sampling, and compared to day 0 results, as shown in Table 5, FIGS. 19-20.

(3) Accelerated test

3 batches of the crystal form A and the crystal form B are respectively packaged by a proper packaging material and placed for 6 months at the temperature of 40 +/-2 ℃ and the relative humidity of 75% +/-5%. Samples were taken at the end of 1 month, 2 months, 3 months and 6 months of the test period for HPLC content measurement, and the results are shown in tables 6 and 7.

TABLE 4 high temperature stability data for form A and form B

TABLE 5 intense light irradiation test data of crystal form A and B

TABLE 6 accelerated test data for form A

TABLE 7 accelerated test data for form B

As is clear from the above results, C provided by the present invention₁₆Of form A or B of DP-VPA at high temperature (60 + -2 deg.C) and under strong light irradiation (irradiation intensity 4500lx + -500 lx)XPRD spectrograms of 5 days and 10 days of placing in the environment are basically consistent with XPRD spectrograms of 0 day of placing, and crystal transformation phenomenon does not occur, which shows that the crystal form A and the crystal form B have good crystal stability under the condition. In addition, in an accelerated test of 6 months, XPRD spectrograms of the crystal form A and the crystal form B respectively sampled at the end of 1 month, 2 months, 3 months and 6 months are basically consistent with the XPRD spectrogram of 0 month, and crystal transformation phenomenon does not occur, so that the crystal provided by the invention has good stability and is beneficial to clinical storage and use.

Test example 3 stability test of Crystal form C

C is to be₁₆Form C DP-VPA was placed in a petri dish and exposed to air at ambient conditions (45% relative humidity, 25 ℃ C.), sampled for XPRD at 2 hours, 6 hours, 24 hours, and 48 hours, respectively, and compared to the 0 hour results, as shown in Table 8, FIG. 21.

TABLE 8C₁₆-DP-VPA form C stability data

From the above results, it is known that form C is an extremely unstable form, which is easily transformed into form B by absorbing moisture in the air when left under ambient conditions.

Test example 4 stability test of Crystal form D

C is to be₁₆Form D of DP-VPA was placed in a petri dish, exposed to air at ambient conditions (45% relative humidity, 25 ℃ C.), sampled for XPRD testing at 1 day, 2 days, 5 days, respectively, and compared to the 0 day results, shown in Table 9, and shown in FIG. 22.

Table 9 stability test of form D

From the above results, it can be seen that form D is an extremely unstable form, which is easily transformed into form a by absorbing moisture in the air when placed under ambient conditions.

Claims

1. C₁₆Form A of DP-VPA, characterized in that it exhibits, in an X-ray powder diffraction pattern expressed in terms of diffraction angles 2 θ ± 0.2 °, characteristic peaks at 5.09, 7.6, 10.11, 12.63, 15.16, 17.69, 20.24, 22.78, 25.35, C₁₆-DP-VPA has the formula

2. C according to claim 1₁₆Form A of DP-VPA, characterized in that it also shows characteristic peaks, expressed in X-ray powder diffraction pattern at diffraction angles 2 θ ± 0.2 °, at 20.73, 21.66, 22.11, 23.9, 24.65, 27.92, 30.53, 34.37, 36.91.

3. C according to claim 1 or 2₁₆A process for the preparation of form a of DP-VPA comprising the steps of:

1) under the condition of reflux temperature, C is added₁₆-DP-VPA dissolved in acetone; said C is₁₆The concentration of DP-VPA in acetone is 0.05-0.25 g/ml;

2) cooling, crystallizing, filtering, washing and drying.