A crystalline form of nervonic acid and methods of preparation

Abstract

The application belongs to the technical field of pharmaceutical crystal, and relates to a crystalline form of nervonic acid and a preparation method thereof. In the X-ray powder diffraction pattern of the crystalline form, Cu-Kalpha radiation has characteristic peaks at diffraction angles 2theta of 5.2+ / -0.2, 8.8+ / -0.2, 12.4+ / -0.2, 16.0+ / -0.2, 19.2+ / -0.2, 23.9+ / -0.2 and 25.2+ / -0.2. The crystalline form provided by the application has good chemical purity and crystal stability, and exhibits excellent physical stability, which is beneficial to the development, production and storage of pharmaceutical preparations. In addition, research shows that the crystalline form also has suitable solubility characteristics, which is beneficial to improving the bioavailability and more beneficial to the related research and development of drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical crystal technology and relates to a crystal form of nervonic acid and its preparation method. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Nervonic acid is a physiologically active compound with an ω-9 long-chain monounsaturated fatty acid structure, chemically named cis-15-tetracosanoic acid (CAS No.: 506-37-6). It is commonly used as a nutritional supplement and infant formula additive, primarily for maintaining nervous system function and as an adjunct treatment for related diseases. Simultaneously, nervonic acid shows broad application potential in cardiovascular protection, anti-inflammation, anti-oxidation, and cosmetic skincare. Furthermore, as a key component of myelin sheath glycolipids and sphingomyelin (accounting for approximately 40% of sphingolipid fatty acids), nervonic acid promotes myelin regeneration and repair of damaged nerve fibers by penetrating the blood-brain barrier, regulates neurotransmitter balance, and simultaneously inhibits cholesterol synthase and promotes its metabolic excretion, thus exerting a dual role in neuroprotection and lipid regulation.

[0004] Currently, the main methods for preparing nervonic acid include bio-fermentation and chemical synthesis. However, existing research on nervonic acid focuses on its sources, extraction and purification, physiological activity, and general applications, lacking research on its solid-state crystalline form. In drug development, the solid form of the active pharmaceutical ingredient (API) requires high quality and stability. This invention, during the drug development process of nervonic acid, revealed that nervonic acid obtained through conventional methods (such as rapid solvent evaporation) is a thermodynamically unstable crystalline form or an amorphous state, which is difficult to meet the requirements of drug development. Therefore, it is necessary to develop a new crystalline form of nervonic acid with stable physicochemical properties suitable for drug development to promote the research, application, and promotion of nervonic acid-related drugs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a crystal form of nervonic acid and a method for its preparation. The crystal form provided by the present invention exhibits good chemical purity and crystal stability, as well as excellent physical stability, which is beneficial for the development, production, and storage of pharmaceutical formulations. Furthermore, research indicates that this crystal form also possesses suitable solubility properties, further facilitating related drug research and development.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: Firstly, a crystalline form of nervonic acid exhibits characteristic peaks in its X-ray powder diffraction pattern at diffraction angles of 5.2°±0.2°, 8.8°±0.2°, 12.4°±0.2°, 16.0°±0.2°, 19.2°±0.2°, 23.9°±0.2°, and 25.2°±0.2° when subjected to Cu-Kα radiation.

[0007] In a second aspect, a method for preparing the crystal form described in the first aspect of the present invention includes the following steps: The nervonic acid raw material is dissolved in a good solvent by heating to form a raw material solution; wherein, the good solvent is one or more selected from N,N-dimethylformamide, ethanol, and dichloromethane; An antisolvent is added to the raw material solution; wherein the antisolvent is one or more of water, methanol, acetonitrile, n-hexane, or petroleum ether. The liquid system after adding the antisolvent is cooled and crystallized at a cooling rate of 0.5~1℃ to obtain the product.

[0008] This invention alters the polarity or composition of the solution system by adding an antisolvent to a good solvent, thereby significantly reducing the solubility of the compound in the mixed solvent and rapidly forming a supersaturated state. Then, under slow stirring and slow cooling conditions, crystal nuclei are induced to form and grow into the aforementioned crystal form.

[0009] The beneficial effects of this invention are as follows: (1) The nervonic acid provided by the present invention has high chemical purity and good crystal stability, and also exhibits excellent physical stability, which is beneficial to the development, production and storage of pharmaceutical preparations.

[0010] (2) Studies have shown that, compared with amorphous nervonic acid, the nervonic acid crystal form provided by the present invention has better solubility properties, which is more conducive to the research and development of related drugs.

[0011] (3) The method for preparing the crystal form of nervonic acid provided by the present invention is simple, mild, and reproducible, with a crystallization yield of 85% to 95%, and the solvent is easy to recover, making it suitable for industrial production. Attached Figure Description

[0012] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0013] Figure 1 The image shows the X-ray powder diffraction (PXRD) pattern of nervonic acid crystal form A prepared in Example 1 of this invention. Figure 2 The gas chromatogram (GC) of nervonic acid crystal form A prepared in Example 1 of this invention; Figure 3 The image shows the X-ray powder diffraction (PXRD) pattern of the nervonic acid product prepared in Comparative Example 1 of this invention. Figure 4 Thermogravimetric analysis (TGA) image of nervonic acid crystal form A prepared in Example 1 of this invention; Figure 5 Differential scanning calorimetry (DSC) images of nervonic acid crystal form A prepared in Example 1 of this invention; Figure 6 This is a scanning electron microscope (SEM) image of nervonic acid crystal form A prepared in Example 1 of the present invention. Detailed Implementation

[0014] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0015] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0016] In view of the lack of suitable pharmaceutical crystal forms of nervonic acid in the prior art, the present invention proposes a crystal form of nervonic acid and a preparation method thereof.

[0017] In a typical embodiment of the present invention, a crystal form of nervonic acid is provided, the X-ray powder diffraction pattern of which shows characteristic peaks at diffraction angles 2θ of 5.2°±0.2°, 8.8°±0.2°, 12.4°±0.2°, 16.0°±0.2°, 19.2°±0.2°, 23.9°±0.2°, and 25.2°±0.2° when subjected to Cu-Kα radiation.

[0018] In some embodiments, the PXRD pattern is basically as follows Figure 1 As shown.

[0019] In some embodiments, the characteristic peak with a diffraction angle of 2θ of 5.2° has a d value of 16.79 and a relative intensity of 100%.

[0020] In some embodiments, the characteristic peak with a diffraction angle of 2θ of 8.8° has a d value of 10.61 and a relative intensity of 48.43%.

[0021] In some embodiments, the characteristic peak with a diffraction angle of 2θ of 12.4° has a d value of 7.13 and a relative intensity of 17.22%.

[0022] In some embodiments, the characteristic peak with a diffraction angle 2θ of 16.0° has a d value of 5.53 and a relative intensity of 5.49%.

[0023] In some embodiments, the characteristic peak with a diffraction angle of 2θ of 19.2° has a d value of 4.62 and a relative intensity of 5.30%.

[0024] In some embodiments, the characteristic peak with a diffraction angle of 2θ of 23.9° has a d value of 3.72 and a relative intensity of 8.10%.

[0025] In some embodiments, the characteristic peak with a diffraction angle of 2θ of 25.2° has a d value of 3.53 and a relative intensity of 7.02%.

[0026] In some embodiments, the melting point is 44±0.5 °C.

[0027] The chemical purity of nervonic acid crystal form A is 99.42%, and the gas phase detection results are as follows: Figure 2 As shown.

[0028] Another embodiment of the present invention provides a method for preparing the above-mentioned crystal form, comprising the following steps: The nervonic acid raw material is dissolved in a good solvent by heating to form a raw material solution; wherein, the good solvent is one or more selected from N,N-dimethylformamide, ethanol, and dichloromethane; An antisolvent is added to the raw material solution; wherein the antisolvent is one or more of water, methanol, acetonitrile, n-hexane, or petroleum ether. The liquid system after adding the antisolvent is cooled and crystallized at a cooling rate of 0.5~1℃ / min to obtain the product.

[0029] In some embodiments, the raw material solution is a saturated solution or a near-saturated solution. Dissolving the nervonic acid raw material in a good solvent to prepare a saturated solution or a near-saturated solution, and using this solution as the raw material solution for subsequent crystallization processes, is beneficial to improving the yield of the target crystal form and increasing the preparation efficiency of the target crystal form.

[0030] In some embodiments, the temperature at which the nervonic acid raw material is dissolved by a good solvent is 30-60°C. Under these conditions, not only can the dissolution of the nervonic acid raw material be accelerated, but the amount of nervonic acid raw material can also be increased, thereby improving the preparation efficiency.

[0031] In some embodiments, the volume ratio of the good solvent to the antisolvent is 1:1 to 5. Studies have shown that this condition is favorable for the preparation of the target crystal form.

[0032] In some embodiments, the antisolvent is added to the raw material solution at a rate of 0.8–1.2 mL / min. Studies have shown that this condition is favorable for the preparation of the target crystal form.

[0033] In some embodiments, an antisolvent is added to the feed solution until the solution becomes turbid or crystals begin to precipitate. Studies have shown that this condition is beneficial for increasing the yield of the target crystal form.

[0034] In some embodiments, slow stirring is performed during the cooling crystallization process. Studies have shown that this condition is more favorable for preparing the target crystal form.

[0035] In some embodiments, the cooling temperature for crystallization is 0~10 °C. Specifically, the temperature is lowered to 0~10 °C and held for 22~26 hours. Under these conditions, it is beneficial to ensure the complete precipitation of the target crystal form.

[0036] In some embodiments, after crystallization, the crystals are filtered, then washed and dried. Specifically, during the washing process, the washing agent is an antisolvent or a mixture of an antisolvent and a good solvent. More specifically, the washing agent is pre-cooled before washing to prevent excessively high washing temperatures from causing a small portion of the target crystal form to re-dissolve, thereby preventing a decrease in yield. Specifically, the drying method is vacuum drying. More specifically, the vacuum drying temperature is 20~30 °C.

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0038] Example 1 A mixture of nervonic acid (1 g) and ethanol (5 mL) was heated to 60 °C and stirred until the solution was clear. Methanol (15 mL, 1 mL / min) was slowly added dropwise while stirring slowly (100 rpm) and cooling down (0.5 °C / min). A solid precipitated during the cooling process. The solid was kept at 10 °C and stirred for 24 h before being filtered. The filter cake was washed with a small amount of pre-cooled methanol and dried under vacuum at 25 °C to obtain nervonic acid crystal form A (0.86 g), with a yield of 86%.

[0039] Example 2 A mixture of nervonic acid (1 g) and ethanol (5 mL) was heated to 60 °C and stirred until the solution was clear. Acetonitrile (25 mL, 1 mL / min) was slowly added dropwise while stirring slowly (100 rpm) and cooling down (0.5 °C / min). A solid precipitated during the cooling process. The solid was kept at 5 °C and stirred for 24 h before being filtered. The filter cake was washed with a small amount of pre-cooled acetonitrile and dried under vacuum at 25 °C to obtain nervonic acid crystal form A (0.90 g), with a yield of 90%.

[0040] Example 3 The mixture of nervonic acid (1 g) and N,N-dimethylformamide (5 mL) was heated to 60 °C and stirred until the solution was clear. Acetonitrile (20 mL, 1 mL / min) was slowly added dropwise while stirring slowly (100 rpm) and cooling down (1 °C / min). A solid precipitated during the cooling process. The solid was kept at 10 °C and stirred for 24 h and then filtered. The filter cake was washed with a small amount of pre-cooled acetonitrile and dried under vacuum at 25 °C to obtain nervonic acid crystal form A (0.85 g), with a yield of 85%.

[0041] Example 4 The mixture of nervonic acid (1 g) and N,N-dimethylformamide (5 mL) was heated to 30 °C and stirred until the solution was clear. Methanol (15 mL, 1 mL / min) was slowly added dropwise while stirring slowly (100 rpm) and cooling down (1 °C / min). A solid precipitated during the cooling process. The solid was kept at 5 °C and stirred for 24 h and then filtered. The filter cake was washed with a small amount of pre-cooled n-hexane and dried under vacuum at 25 °C to obtain nervonic acid crystal form A (0.95 g), with a yield of 95%.

[0042] Example 5 A mixture of nervonic acid (1 g) and dichloromethane (5 mL) was heated to 30 °C and stirred until the solution was clear. Hexane (15 mL, 1 mL / min) was slowly added dropwise while stirring slowly (100 rpm) and cooling down (0.5 °C / min). A solid precipitated during the cooling process. The solid was kept at 0 °C and stirred for 24 h before being filtered. The filter cake was washed with a small amount of pre-cooled hexane and dried under vacuum at 25 °C to obtain nervonic acid crystal form A (0.93 g), with a yield of 93%.

[0043] Example 6 The mixture of nervonic acid (1 g) and dichloromethane (5 mL) was heated to 30 °C and stirred until the solution was clear. Petroleum ether (15 mL, 1 mL / min) was slowly added dropwise while stirring slowly (100 rpm) and cooling down (0.5 °C / min). A solid precipitated during the cooling process. The solid was kept at 0 °C and stirred for 24 h before being filtered. The filter cake was washed with a small amount of pre-cooled petroleum ether and dried under vacuum at 25 °C to obtain nervonic acid crystal form A (0.98 g), with a yield of 98%.

[0044] Comparative Example 1 Dissolve nervonic acid (1g) in dichloromethane (10mL), heat to 35°C while standing to allow the solvent to evaporate, and collect the resulting solid.

[0045] Comparative Example 2 Dissolve nervonic acid (1g) in dichloromethane (5mL), heat to 30℃ while standing, and slowly stir (stirring speed: 100rpm) to cool down (cooling rate: 0.5℃ / min). During the cooling process, the solution is clear and no solid precipitates.

[0046] Comparative Example 3 The mixture of nervonic acid (1g) and ethanol (5mL) was heated to 60℃ and stirred until the solution was clear. The clear solution was added to methanol (15mL) in one go and the mixture was slowly stirred (stirring speed: 100rpm) and cooled (cooling rate: 0.5℃ / min). During the cooling process, a solid precipitated out. The mixture was kept at 10℃ and stirred for 24h and then filtered. The filter cake was washed with a small amount of pre-cooled methanol and dried under vacuum at 25℃.

[0047] Comparative Example 4 The mixture of nervonic acid (1 g) and ethanol (5 mL) was heated to 60 °C and stirred until the solution was clear. Methanol (15 mL, 1 mL / min) was slowly added dropwise while stirring rapidly (1000 rpm) and cooling down (0.5 °C / min). A solid precipitated during the cooling process. The solid was kept at 10 °C and stirred for 24 h before being filtered. The filter cake was washed with a small amount of pre-cooled methanol and dried under vacuum at 25 °C.

[0048] Comparative Example 5 The mixture of nervonic acid (1g) and ethanol (5mL) was heated to 60℃ and stirred until the solution was clear. Methanol (15mL, at a dropping rate of 1mL / min) was slowly added dropwise. After the addition was complete, the mixture was placed directly in an ice-water bath and stirred slowly (stirring speed: 100rpm) for 24h. The mixture was then filtered. The filter cake was washed with a small amount of pre-cooled methanol and dried under vacuum at 25℃.

[0049] X-ray powder diffraction analysis of the nervonic acid products prepared in Examples 1-6 and Comparative Examples 1-5 using Cu-Kα radiation showed that the nervonic acid products prepared in Examples 1-6 had the same crystal structure, namely nervonic acid crystal form A. The PXRD pattern of nervonic acid crystal form A prepared in Example 1 is shown below. Figure 1 As shown, this indicates that the crystal form has characteristic peaks at diffraction angles 2θ of 5.2°, 8.8°, 12.4°, 16.0°, 19.2°, 23.9°, and 25.2°. The peak intensities, d-values ​​of each peak, and relative intensities are shown in Table 1 below.

[0050] Table 1 Peak intensity, d-value of each peak and relative intensity

[0051] The nervonic acid products prepared in Comparative Examples 1-5 were all amorphous nervonic acid; among them, the PXRD pattern of the nervonic acid product prepared in Comparative Example 1 is as follows. Figure 3As shown in the figures, a comparison of the various embodiments and comparative examples reveals the following: First, Comparative Example 1 uses a rapid solvent evaporation method, which produces amorphous nervonic acid, demonstrating that conventional rapid solvent evaporation methods are difficult to use to prepare nervonic acid crystals. Second, Comparative Example 2 uses a conventional heating-dissolution-cooling-crystallization method, which also fails to produce the nervonic acid crystal form A prepared in the examples, proving that the method of the present invention, which uses an anti-solvent to change the polarity and composition of the good solvent solution, plays a crucial role in the preparation of nervonic acid crystal form A. Third, a comparison between Comparative Example 3 and Example 1 demonstrates that the method of adding the anti-solvent also affects the preparation of nervonic acid crystal form A. Fourth, Comparative Examples 4 and 5 demonstrate that the stirring rate and cooling rate during crystallization are also factors affecting the formation of nervonic acid crystal form A.

[0052] Thermal stability test: The alumina crucible was calcined in a muffle furnace at 1100 ℃ for 30 min to remove adsorbed impurities and residual organic matter from the crucible surface. After cooling, it was transferred to a desiccator for later use. 6.306 mg of sample was weighed and evenly spread inside the calcined alumina crucible. The crucible was gently placed in the sample testing position of the thermogravimetric analyzer, and the furnace was closed. High-purity nitrogen was introduced into the furnace as a protective atmosphere, with the nitrogen purging flow rate controlled at 50–100 mL / min. The initial test temperature was set to 30 ℃, and the temperature was increased to 800 ℃ at a constant heating rate of 10 ℃ / min. The entire test was conducted under a nitrogen inert atmosphere. The instrument automatically recorded the relevant data on sample mass changes with temperature in real time and simultaneously acquired thermogravimetric analysis (TGA) data. Figure 4 ) and differential scanning calorimetry (DSC) Figure 5 ) curve. Gas chromatography data ( Figure 2 The results show that the chemical purity of crystal form A is 99.42% and its melting point is 44℃, but TGA shows that the initial decomposition temperature of crystal form A is 196℃, indicating that it has excellent thermal stability.

[0053] Solubility test: Weigh out 0.5, 1, 1.5, 2 and 2.5 g of amorphous and crystalline form A nervonic acid respectively, dissolve them in 5 mL of dichloromethane, and record the solubility. The solubility of nervonic acid in dichloromethane is shown in Table 2 below.

[0054] Table 2. Solubility of amorphous and crystalline form A nervonic acid in dichloromethane

[0055] Among them, "dissolved and clear" means completely dissolved and the solution is clear.

[0056] Table 2 shows that the solubility of nervonic acid crystal form A prepared in this invention in dichloromethane is higher than that of its amorphous form. This means that more nervonic acid crystal form A can be dissolved using the same volume of dichloromethane, greatly improving the efficiency of nervonic acid dissolution, crystallization, and separation, thus facilitating related drug development.

[0057] Scanning electron microscope image of nervonic acid crystal form A prepared in Example 1 is shown below. Figure 6 As shown, the morphology of nervonic acid crystal form A is a plate-like aggregate, while amorphous nervonic acid has an irregular and irregular morphology. The difference in their microscopic morphology may affect their macroscopic solubility.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A crystalline form of nervonic acid, characterized in that, In its X-ray powder diffraction pattern, characteristic peaks were observed at diffraction angles 2θ of 5.2°±0.2°, 8.8°±0.2°, 12.4°±0.2°, 16.0°±0.2°, 19.2°±0.2°, 23.9°±0.2°, and 25.2°±0.2° when Cu-Kα radiation was used.

2. The crystal form as described in claim 1, characterized in that, Its PXRD pattern is basically shown in Figure 1.

3. A method for preparing the crystal form according to claim 1 or 2, characterized in that, Includes the following steps: The nervonic acid raw material is dissolved in a good solvent by heating to form a raw material solution; wherein, the good solvent is one or more selected from N,N-dimethylformamide, ethanol, and dichloromethane; An antisolvent is added to the raw material solution; wherein the antisolvent is one or more of water, methanol, acetonitrile, n-hexane, or petroleum ether. The liquid system after adding the antisolvent is cooled and crystallized at a cooling rate of 0.5~1℃ / min to obtain the product.

4. The preparation method according to claim 3, characterized in that, The raw material solution is a saturated or nearly saturated solution.

5. The preparation method according to claim 3, characterized in that, The optimal temperature for dissolving nervonic acid raw materials with a good solvent is 30~60℃.

6. The preparation method according to claim 3, characterized in that, The volume ratio of good solvent to antisolvent is 1:1 to 5.

7. The preparation method according to claim 3, characterized in that, The rate at which the antisolvent is added to the raw material solution is 0.8~1.2 mL / min.

8. The preparation method according to claim 3, characterized in that, Slow stirring is performed during the cooling and crystallization process.

9. The preparation method according to claim 3, characterized in that, The cooling temperature for crystallization is 0~10 ℃.

10. The preparation method according to claim 3, characterized in that, After crystallization, the mixture is filtered, then washed and dried. Preferably, the washing agent used is an anti-solvent or a mixture of an anti-solvent and a good solvent. Preferably, the washing agent is pre-cooled before washing. Preferably, the drying method is vacuum drying. Preferably, the vacuum drying temperature is 20~30 ℃.

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