A crystalline form of nicotinamide riboflumate, its preparation method and application

Abstract

The invention relates to a nicotinamide ribose malate crystal form and a preparation method and application thereof, and belongs to the technical field of biomedicine. The product prepared by the invention is verified as nicotinamide ribose malate by nuclear magnetic H-NMR, and has obvious characteristic peaks compared with commercially available products, so as to be a nicotinamide ribose malate product of a new crystal form. The preparation method of the invention comprises the following steps: firstly mixing a solvent, DL-malic acid and a base, and then adding nicotinamide ribose chloride to react, and after the reaction, controlling the temperature, reaction time, cooling rate, crystallization time and crystallization temperature to obtain a nicotinamide ribose malate of a new crystal form. The nicotinamide ribose malate of the new crystal form has better air stability and thermal stability, low hygroscopicity and is not easy to decompose.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a nicotinamide ribosylmalate crystal form and a preparation method and application thereof. Background Art

[0002] Nicotinamide riboside can increase tissue NAD concentrations and induce insulin sensitivity and enhance sirtuin function. Its ability to increase NAD production suggests that nicotinamide riboside can also improve mitochondrial health, stimulate mitochondrial function, and induce the production of new mitochondria. Other studies using nicotinamide riboside in Alzheimer's disease models have shown that the molecule is bioavailable to the brain and can provide neuroprotection by stimulating brain NAD synthesis. In liver cancer research, results showed that dietary supplementation with nicotinamide riboside was able to prevent the development of liver cancer in mice and induce tumor regression, and no side effects were found at high doses.

[0003] Currently, the nicotinamide riboside on the market is mainly nicotinamide riboside chloride (3-carbamoyl-1-[(2R, 3R, 4S, 5R)-3, 4-dihydroxy-5-hydroxymethyloxacyclopentane-2-yl]-pyridin-1-ium chloride, also known as 1-(β-D-ribofuranosyl) nicotinamide chloride), and its structure is as follows:

[0004]

[0005] Nicotinamide riboside chloride is very sensitive to light, air, and humidity, and is very easy to degrade. Once degraded, its corresponding efficacy will be lost. Therefore, it is of great significance to find a nicotinamide riboside with a more stable structure.

[0006] Nicotinamide ribosyl malate is a form of nicotinamide riboside. Studies have shown that its stability is higher than that of nicotinamide ribosyl chloride. However, the crystal form of nicotinamide ribosyl malate is greatly affected by the preparation process, crystallization control, crystallization solvent, etc. Different preparation methods result in different crystal forms, and the corresponding product stability and hygroscopicity are greatly different. The existing crystal form of nicotinamide ribosyl malate has strong hygroscopicity and is easily decomposed in the air, especially when the temperature rises. For example, patent CN111454311A discloses a nicotinamide ribosyl malate, which needs to be prepared and filtered under air-tight and low-temperature conditions, and needs to be dried below zero degrees. Summary of the invention

[0007] In view of the problems existing in the background technology, the first object of the present invention is to provide a new crystal form of nicotinamide ribose malate, which has higher stability and low hygroscopicity, still has good chemical stability when exposed to air, basically does not change color, can maintain a solid shape, and has higher thermal stability. The second object of the present invention is to provide a method for preparing the crystal form of nicotinamide ribose malate. The new crystal form of nicotinamide ribose malate provided by the present invention belongs to a type of nicotinamide ribose, and can be used as a pharmaceutical ingredient for increasing the concentration of NAD in tissues, inducing insulin sensitivity, and enhancing the function of sirtuins.

[0008] To achieve the above object, the present invention is implemented through the following technical solutions:

[0009] The nicotinamide ribosylmalate crystalline form has a 2θ value of: 3.1703±0.2, 6.6434±0.2, 12.5953±0.2, 14.7209±0.2, 15.8502±0.2, 16.5144±0.2, 17.0509±0.2, 17.7053±0.2, 18.6569±0.2, 18.9470±0.2, 19.7757±0.2, 21.4535±0.2, 21.9161±0.2, 22.3721±0.2, 22.9562±0.2, 24.0469±0.2, 24.8641±0.2, 25.2183±0 There are characteristic peaks at 25.7679±0 .2, 27.2584±0 .2, 28.5494±0 .2, 36.5968±0 .2, and 36.8355±0 .2.

[0010] Furthermore, the nicotinamide ribosylmalate crystalline form has powder X-ray diffraction data with peaks as shown in Table 1, and the 2θ value error range is ±0.2°.

[0011] Table 1 Nicotinamide ribosylmalate XRPD data

[0012] .

[0013] The method for preparing the nicotinamide ribosylmalate crystalline form is characterized by comprising the following steps:

[0014] (1) First, mix the solvent and DL-malic acid and stir until dissolved;

[0015] (2) adding nicotinamide riboside chloride and a base respectively to react;

[0016] (3) cooling after the reaction and maintaining constant temperature for crystallization to generate nicotinamide ribosylmalate crystals;

[0017] (4) filtering and drying to obtain a new crystalline form of nicotinamide ribosylmalate, which appears as a white solid powder;

[0018] Wherein, in step (1), the solvent is any one of methanol, ethanol, DMF, acetone, DMSO, ethyl acetate, dichloromethane or 1,2-dichloroethane; the base is any one of ammonia water, caustic soda, sodium carbonate, potassium carbonate, triethylamine (TEA) or 1,2-diethylamine DIEA; and the reaction temperature of step (2) is 0°C-60°C.

[0019] Furthermore, the reaction temperature of step (2) is 40-60°C, and the reaction time is 1-3h; the crystallization temperature of step (3) is 0-10°C, and the crystallization time is 1-3h; the drying of step (4) is reduced pressure drying, the drying vacuum is 500Pa, and the drying temperature is 30-60°C.

[0020] Furthermore, the molar ratio of DL-malic acid to nicotinamide riboyl chloride is 1:1.

[0021] Furthermore, the base is DIEA.

[0022] The nicotinamide ribosylmalate crystal form obtained by the present invention is used in drugs for increasing the NAD concentration in tissues, inducing insulin sensitivity, and enhancing the function of sirtuins; it can be used as a substitute for nicotinamide ribosyl chloride.

[0023] Beneficial effects of the present invention:

[0024] The nicotinamide ribose malate crystal form of the present invention has obvious characteristic peaks compared with commercially available products, and is a new crystal form of nicotinamide ribose malate. Performance analysis shows that the nicotinamide ribose malate of the present invention has better air stability and thermal stability, low hygroscopicity, and is not easy to decompose. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the spectrum of nicotinamide ribosylmalate crystal form H obtained in Example 1 of the present invention;

[0026] Figure 2 is the XRD diffraction pattern of the nicotinamide ribosylmalic acid crystal obtained in Example 1 of the present invention;

[0027] Figure 3 is a TGA diagram of the nicotinamide ribosylmalate crystalline form obtained in Example 1 of the present invention;

[0028] Figure 4 is a DSC graph of the nicotinamide ribosylmalic acid crystal obtained in Example 1 of the present invention;

[0029] Figure 5 This is the IR graph of the nicotinamide ribosylmalic acid crystal obtained in Example 1 of the present invention;

[0030] Figure 6 This is the MS image of the nicotinamide ribosylmalate crystal obtained in Example 1 of the present invention;

[0031] Figure 7 This is an XRPD comparison chart of the nicotinamide ribosylmalic acid crystal obtained in Example 1 of the present invention and the nicotinamide ribosylmalic acid crystal purchased from the market. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0033] In order to more clearly illustrate the present invention, it is described in detail through the following examples.

[0034] Example 1

[0035] Add 200ml of anhydrous methanol (unless otherwise specified, all solvents used are anhydrous solvents), 48.43g of DL-malic acid, and 10g of DIEA to a three-necked flask, stir until dissolved, and then add nicotinamide riboside chloride. The molar ratio of nicotinamide riboside chloride to DL-malic acid is 1:1. After adding nicotinamide riboside chloride, start heating to 40°C and continue stirring at 40°C for 2 hours. After 2 hours, continue stirring and start cooling naturally to 25°C, and keep at 25°C for 1 hour. Then continue cooling to 5°C and keep for 2 hours.

[0036] The reaction process is as follows:

[0037]

[0038] The solid phase was dried at 50°C overnight under reduced pressure to obtain a white solid powder of nicotinamide ribosylmalate crystal form. Figure 1 ) verified as nicotinamide ribosylmalate.

[0039] H-NMR characterization data:

[0040]

[0041] The nicotinamide ribosylmalate crystal form obtained in this example was analyzed as follows:

[0042] (1) XRPD analysis results

[0043] The XRPD analysis results are shown in Table 2 and Appendix Figure 2 .

[0044] Table 2 Nicotinamide ribosylmalate XRPD data

[0045]

[0046] (2) TGA analysis

[0047] The analysis results are attached. Figure 3 The nicotinamide riboylmalate crystal loses 1%-33% of its weight at 112°C-200°C.

[0048] (3) DSC analysis

[0049] The analysis results are attached. Figure 4 The nicotinamide riboside malate crystal form has an endothermic peak in the range of 100°C-150°C.

[0050] (4) Infrared spectroscopy analysis,

[0051] IR(KBr)ν max 3360.30,2955.76,1697.79,1582.55,1082.63,619.05cm -1 . The infrared spectrophotometry spectrum is as attached Figure 5 .

[0052] (5) Mass spectrometry analysis, MS (ESI+): 255.10 [M+1], MS (ESI+): 133.05 [M+1]; mass spectrum is as attached Figure 6 .

[0053] (6) Crystal form comparison

[0054] The obtained nicotinamide ribosylmalic acid crystal form was compared with the nicotinamide ribosylmalic acid samples currently purchased on the market (a total of three different samples were purchased, numbered as sample A, sample B and sample C respectively). The comparison results are shown in the attached Figure 7 Attached Figure 7In the figure, line 1 is the XRPD pattern of sample C, line 2 is the XRPD pattern of sample B, line 3 is the XRPD pattern of sample A, and line 4 is the XRPD pattern of nicotinamide ribosylmalate of Example 1 of the present invention. By comparison, it can be found that the nicotinamide ribosylmalic acid crystal form obtained in the present invention has the following 2θ values: 3.1703±0.2, 6.6434±0.2, 12.5953±0.2, 14.7209±0.2, 15.8502±0.2, 16.5144±0.2, 17.0509±0.2, 17.7053±0.2, 18.6569±0.2, 18.9470±0.2, 19.7757±0.2, 21.4535±0.2, 21.9161±0.2, 22.3721±0.2, 22.9562±0.2, 24.0469±0.2, 24.8641±0.2 .2, 25.2183±0.2, 25.7679±0.2, 27.2584±0.2, 28.5494±0.2, 36.5968±0.2, and 36.8355±0.2 have characteristic peaks, indicating that the nicotinamide ribosylmalate crystal form obtained by the present invention is a new crystal form.

[0055] (7) Stability comparison

[0056] Nicotinamide ribosylmalate obtained in this example and purchased nicotinamide ribosylmalate (A, B), each sample was divided into 7 portions, and each sample was individually placed in 7 glass sample dishes, with 1 gram of sample in each sample dish, and sealed with a cover. All samples were stored in a constant temperature and humidity chamber at 25°C and a relative humidity of 40%. The sample purity was tested regularly every day. The purity of each sample on different days and the 6-day decomposition rate data are shown in Table 3. Decomposition rate calculation method: Decomposition rate = (initial sample purity - sample purity) / initial sample purity × 100%

[0057] Product purity analysis method: Test sample solution preparation, take one bag of sample each time, use 5mL or 10mL volumetric flask, prepare 5mL or 10mL solution, HPLC test sample purity.

[0058] Table 3 Comparison of purity and decomposition rate of nicotinamide ribosylmalate obtained in this example and commercially available products

[0059]

[0060] It can be seen from the table that the nicotinamide ribosylmalate crystal stability obtained by the present invention is better than that of the comparative product, and the product of the present invention has better stability.

[0061] Comparative Example 1 (without adding alkali)

[0062] 200 ml of anhydrous methanol and 48.43 g of DL-malic acid were added to a three-necked flask, stirred to dissolve, and then nicotinamide riboside chloride was added, the molar ratio of nicotinamide riboside chloride to DL-malic acid was 1:1, and the temperature was raised to 40°C after the addition of nicotinamide riboside chloride, and stirred continuously at 40°C for 2 hours. Stir continuously and start cooling to 25°C, and keep at 25°C for 1 hour. Then continue to cool to 5°C and keep for 2 hours, and no crystallized product was produced.

[0063] The essence of the present invention is to use nicotinamide riboside chloride as a substrate and replace chloride ions with malic acid anions. Under non-alkaline conditions, the reaction cannot proceed and nicotinamide riboside malate cannot be obtained.

[0064] Comparative Example 2 (adjusting the order of adding malic acid and nicotinamide riboside chloride)

[0065] 200 ml of anhydrous methanol, nicotinamide ribose chloride, and 10 g of DIEA were added to a three-necked flask in sequence, stirred to dissolve, and then 48.43 g of DL-malic acid was added. The molar ratio of nicotinamide ribose chloride to DL-malic acid was 1:1. After adding DL-malic acid, the temperature was raised to 40°C and stirred for 2 hours at 40°C. The temperature was lowered to 25°C while stirring, and maintained at 25°C for 1 hour. The temperature was then lowered to 5°C and maintained for 2 hours. The reaction solution was white and turbid, and the target product, nicotinamide ribose malate, was obtained by centrifugation.

[0066] If nicotinamide riboside chloride is first mixed with DIEA in the absence of DL-malic acid, as the stirring time increases, nicotinamide riboside chloride will be largely decomposed and the product yield will be greatly reduced.

[0067] Example 2

[0068] Add 660L ethanol, 145.29kg DL-malic acid, and nicotinamide ribose chloride to a three-necked flask in sequence, stir to dissolve, then add 30kg DIEA (N,N-diisopropylethylamine), the molar ratio of nicotinamide ribose chloride to DL-malic acid is 1:1, start heating to 40°C, and continue stirring for 2 hours. Continue stirring and start cooling to 25°C, keep for 1 hour. Then continue to cool to 5°C and keep for 2 hours. The reaction solution is white, turbid, and viscous. Centrifuge and dry under reduced pressure at 50°C overnight to obtain a white solid powder, which was verified as nicotinamide ribose malate by H-NMR.

[0069] Example 3

[0070] Add 100 ml of isopropanol, 24 g of DL-malic acid, and 5.2 g of DIEA to a three-necked flask, stir until dissolved, then add nicotinamide ribose chloride, the molar ratio of nicotinamide ribose chloride to DL-malic acid is 1:1, start heating to 40°C, and continue stirring for 2 hours. Continue stirring and start cooling to 25°C, keep for 1 hour. Then continue cooling to 5°C and keep for 2 hours. Filter, dry under reduced pressure at 50°C overnight, and obtain a white solid powder, which is confirmed to be nicotinamide ribose malate by H-NMR.

[0071] Example 4

[0072] Add 500ml 1,4-dioxane, 121g DL-malic acid, and 25g DIEA to a three-necked flask, stir until dissolved, then add nicotinamide ribose chloride, the molar ratio of nicotinamide ribose chloride to DL-malic acid is 1:1, start heating to 40°C, and continue stirring for 2 hours. Continue stirring and start cooling to 25°C, keep for 1 hour. Then continue cooling to 5°C and keep for 2 hours. Filter, dry under reduced pressure at 50°C overnight, and obtain a white solid powder, which is confirmed to be nicotinamide ribose malate by H-NMR.

[0073] Example 5

[0074] Add 1000ml tetrahydrofuran, 242g DL-malic acid, and 50g 9DIEA to a three-necked flask, stir until dissolved, then add nicotinamide ribose chloride, the molar ratio of nicotinamide ribose chloride to DL-malic acid is 1:1, start heating to 40°C, and continue stirring for 2 hours. Continue stirring and start cooling to 25°C, keep for 1 hour. Then continue cooling to 5°C and keep for 2 hours. Filter, dry under reduced pressure at 50°C overnight, and obtain a white solid powder, which is confirmed to be nicotinamide ribose malate by H-NMR.

[0075] Example 6

[0076] Add 3000ml acetonitrile, 300g DL-malic acid, and 62g DIEA to a three-necked flask, stir at 25℃ until dissolved, then add nicotinamide ribose chloride, the molar ratio of nicotinamide ribose chloride to DL-malic acid is 1:1, start heating to 40℃, and continue stirring for 2 hours. Continue stirring and start cooling to 25℃, keep for 1 hour. Then continue cooling to 5℃ and keep for 2 hours. Filter, dry under reduced pressure at 50℃ overnight, and obtain a white solid powder, which is confirmed to be nicotinamide ribose malate by H-NMR.

[0077] Product quality analysis of each embodiment and comparative example

[0078] (1) Stability comparison

[0079] The nicotinamide ribosyl malate obtained in the above examples and comparative examples was divided into sample bags and sealed in PE bags, with 1 gram in each bag. They were then stored in a constant temperature and humidity chamber at 25° C. and a relative humidity of 40%, and the samples were tested regularly.

[0080] Preparation of test sample solution: Take a bottle of sample each time, use a 5mL or 10mL volumetric flask to prepare 5mL or 10mL solution, filter the membrane, prepare HPLC to test the sample purity, and obtain the product purity and decomposition rate data on the 6th day as shown in Table 4.

[0081] Decomposition rate = (initial sample purity - sample purity) / initial sample purity × 100%

[0082] Table 4 Product purity change and decomposition rate (25℃, RH40%)

[0083]

[0084] (2) Comparison of color change and moisture absorption rate

[0085] The nicotinamide ribosylmalate obtained in the above examples and comparative examples were exposed to the air respectively, and the air humidity and temperature were controlled. The color changes of the products on the 0th day and the 6th day were observed, and the moisture analysis was performed. The observation and analysis results are shown in Table 5.

[0086] Table 5 Color and moisture changes of products exposed to air (25°C, RH40%)

[0087]

[0088] Description of the analytical method used in the present invention:

[0089] XRPD (X-ray powder diffraction) spectrum determination method: Bruker D2 Phaser X-ray powder diffractometer; radiation source Cu (1.54184Å) generator kv: 30kv; generator mA: 10mA; starting 2θ: 2.000°; scanning range: 2.0000~50.000°, scanning step 0.02°, scanning speed 0.1s / step.

[0090] The measurement differences associated with such X-ray powder diffraction analysis results are caused by a variety of factors including: (a) errors in sample preparation (e.g., sample height), (b) instrument errors, (c) calibration differences, (d) operator errors (including errors that occur when determining peak positions), and (e) the nature of the material (e.g., preferred orientation errors). Calibration errors and sample height errors often result in displacements of all peaks in the same direction. When a flat support is used, small differences in sample height will result in large displacements of XRPD peak positions. Systematic studies have shown that sample height differences of 1 mm can result in peak displacements of up to 1° of 2θ. These displacements can be identified from the XRPD spectrum and can be eliminated by compensating for the displacements (applying the system calibration factor to all peak position values) or recalibrating the instrument. As described above, by applying the system calibration factor to make the peak positions consistent, measurement errors from different instruments can be corrected.

[0091] TGA (thermogravimetric analysis) spectrum determination method: TGA55 instrument from TA Company, USA; temperature range: 14.8~300℃; heating rate: 10℃ / min; nitrogen flow rate: 40mL / min.

[0092] DSC (differential scanning calorimetry) spectrum determination method: TA Q55 instrument from TA Company, USA; temperature range: 20-230°C, heating rate: 10°C / min, nitrogen flow rate: 50mL / min.

[0093] Moisture (KF) determination method: MC-2000 automatic trace moisture analyzer from MinCe Instrument Equipment (Xiamen) Co., Ltd., Karl Fischer reagent from Nanjing Chemical Reagent Co., Ltd.

[0094] Liquid phase test method:

[0095] Mobile phase: Isocratic elution 5% water (0.1% formic acid) + 95% methanol (0.1% formic acid)

[0096] Wavelength: 254nm

[0097] Temperature and humidity: 23.0℃, 54%RH

[0098] Sample dissolution: methanol dissolution

[0099] Chromatographic column: ODS-2, 4.6*250mm, 5μm, constant pressure at 12-13Mpa

[0100] Flow rate: 1.0mL / min

[0101] Injection volume: 5 μL

[0102] Running time: ≥15min

[0103] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A nicotinamide ribosylmalate crystal form, It is characterized in that In 2θ: 3.1703±0 .2, 6.6434±0 .2, 12.5953±0 .2, 14.7209±0 .2, 15.8502±0 .2, 16.5144±0 .2, 17.0509±0 .2, 17.7053±0 .2, 18.6569±0 .2, 18.9470±0 .2, 19.7757±0 .2, 21.4535±0 .2, 21.9161±0 .2, 22.3721±0 .2, 22.9562±0 .2, 24.0469±0 .2, 24.8641±0 .2, 25.2183±0 .2, 25.7679±0 .2, 27.2584±0 .2, 28.5494±0 .2, 36.5968±0 .2, and 36.8355±0 .2 have characteristic peaks; The preparation method of the nicotinamide ribosylmalate crystalline form comprises the following steps: (1) First, mix the solvent and DL-malic acid and stir until dissolved; (2) adding nicotinamide riboside chloride and a base respectively to react; (3) cooling after the reaction and maintaining constant temperature for crystallization to generate nicotinamide ribosylmalate crystals; (4) filtering and drying to obtain a new crystalline form of nicotinamide ribosylmalate, which appears as a white solid powder; Wherein, in step (1), the solvent is any one of methanol, ethanol, DMF, acetone, DMSO, ethyl acetate, dichloromethane or 1,2-dichloroethane; the base is any one of ammonia water, caustic soda, sodium carbonate, potassium carbonate, triethylamine TEA or 1,2-diethylamine DIEA; The reaction temperature of step (2) is 0°C-60°C.

2. The nicotinamide ribosylmalate crystalline form according to claim 1, It is characterized in that It has characteristic peaks as shown in the following table 2θ±0.2°, 。 3. The nicotinamide ribosylmalate crystalline form according to claim 1, It is characterized in that In the preparation method, the reaction temperature of step (2) is 40-60°C and the reaction time is 1-3h; the crystallization temperature of step (3) is 0-10°C and the crystallization time is 1-3h; the drying of step (4) is reduced pressure drying, the drying vacuum degree is 500Pa, and the drying temperature is 30-60°C.

4. The nicotinamide ribosylmalate crystalline form according to claim 3, It is characterized in that The molar ratio of DL-malic acid to nicotinamide riboyl chloride is 1:

1.

5. The nicotinamide ribosylmalate crystalline form according to any one of claims 1 to 4, It is characterized in that The base is DIEA.

6. Use of the nicotinamide ribomalate crystal form as claimed in claim 1 or 2 in the preparation of a drug for increasing the NAD concentration in tissues or inducing insulin sensitivity or enhancing the function of sirtuins.

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