A new crystalline form of a beta nicotinamide mononucleotide and methods of making and uses thereof

Abstract

The present application relates to a new crystal form of beta nicotinamide mononucleotide shown in formula (I) and a preparation method thereof. The method of the present application does not use toxic and harmful organic solvents such as methanol, is simple to operate, is safe to production personnel, and is friendly to the environment.

Description

Technical Field

[0001] This invention belongs to the field of medicine. Specifically, this invention relates to a novel crystalline form of β-nicotinamide mononucleotide, its preparation method, and its uses. Background Technology

[0002] β-Nicotinamide mononucleotide (NMN) is a substance naturally present in the human body. In the human body, NMN is the most direct precursor to NAD+, and its function is manifested through NAD+. NAD+, also known as coenzyme I, or nicotinamide adenine dinucleotide, is present in every cell and participates in thousands of reactions. NAD+ plays a fundamental role in human health, but with age, the level of NAD+ in the body gradually decreases, communication between mitochondria and the cell nucleus is impaired, and the reduction of NAD+ also damages the cell's ability to produce energy, thus leading to aging and disease.

[0003] Supplementing with β-nicotinamide mononucleotide (NMN) can effectively and rapidly increase the level of NAD+ in the human body, thereby significantly delaying aging and preventing various neurodegenerative diseases such as Alzheimer's disease, and fundamentally regulating and improving various symptoms of aging. Currently, β-nicotinamide mononucleotide (NMN) has been approved for marketing in countries such as Canada and Japan as a food supplement with anti-aging functions. The current recommended daily dose of β-nicotinamide mononucleotide (NMN) is 300mg. As a food supplement, the final crystallization solvent of the product must be non-toxic and harmless; otherwise, residual solvent may have adverse effects on health.

[0004] Crystallization methods for NMN have been reported, such as CN108697722A (PCT / US2016 / 054776, 2016.09.30, Metro Biotechnology Co., Ltd., USA); WO2018 / 047715 A1 (Kyowa Bio-Fermentation Co., Ltd., Japan) and WO2018 / 047715 A1 (The Queen's University of Belfast and ChromaDex.Inc.). However, these three methods mainly use methanol or methanol-water mixtures as crystallization solvents. Since methanol is a toxic and harmful solvent, many countries restrict its use as a final crystallization solvent for food supplements. Furthermore, these methods are complex, time-consuming, and not conducive to production.

[0005] Therefore, there is an urgent need in this field for novel methods for crystallizing β-nicotinamide mononucleotides and for new crystal forms of β-nicotinamide mononucleotides. Summary of the Invention

[0006] The purpose of this invention is to provide a novel β-nicotinamide mononucleotide crystal form.

[0007] The present invention also aims to provide a novel crystallization method for β-nicotinamide mononucleotide, which does not utilize toxic and harmful organic solvents such as methanol and is simple to operate.

[0008] In a first aspect, the present invention provides polymorphs of the compound represented by formula (I),

[0009]

[0010] The X-ray powder diffraction pattern of the polymorph has characteristic peaks at the following 2θ angles (±0.2°): 20.42; 21.65; 22.75 and 24.98.

[0011] In a preferred embodiment, the polymorph is non-solventized, anhydrous, or substantially anhydrous.

[0012] In a specific embodiment, the X-ray powder diffraction pattern of the polymorph further has characteristic peaks at the following 2θ angles (±0.2°): 21.21; 22.26; 23.99; 24.69; and 27.21.

[0013] In a specific embodiment, the X-ray powder diffraction pattern of the polymorph further has characteristic peaks at the following 2θ angles (±0.2°): 7.70; 11.33; 12.43; 16.32; 17.78; 19.06; and 19.93.

[0014] In a specific embodiment, the X-ray powder diffraction pattern of the polymorph further exhibits characteristic peaks at the following 2θ angles (±0.2°): 10.18; 15.25; 19.26; 22.75; 23.34; 25.69; 26.24; 26.70; 27.61; 28.08; 29.88; 31.78; and 38.00.

[0015] In a specific embodiment, the polymorph is characterized by having a generally as follows: Figure 1 The XRPD map shown.

[0016] In a second aspect, the present invention provides a method for preparing the polymorph described in the first aspect, the method comprising the following steps:

[0017] a. Dissolve the compound of formula (I) in water to obtain an aqueous solution of the compound of formula (I); and

[0018] b. Add an antisolvent to the aqueous solution of the compound of formula (I) obtained in step a, so that the crystals of the compound of formula (I) precipitate out of the solution.

[0019] In a specific implementation, the antisolvent is ethanol.

[0020] In a preferred embodiment, the aqueous solution of the compound of formula (I) obtained in step a has a mass percentage concentration of 5%-25%, preferably 8%-20%.

[0021] In a preferred embodiment, the amount (volume) of ethanol added in step b is 1-3 times the volume of water added in step a.

[0022] In a preferred embodiment, the temperature at which ethanol is added in step b is 20°C-40°C, preferably 25°C-35°C.

[0023] In a specific implementation, the amount of ethanol added in step b should be such that the solution remains clear after the ethanol is added. Then, crystallization is induced by stirring to form crystal nuclei or by adding seed crystals.

[0024] In a preferred embodiment, ethanol is added after the crystals of the compound shown in formula (I) precipitate from the solution.

[0025] In a preferred embodiment, the temperature is appropriately reduced after the crystals of the compound shown in formula (I) precipitate from the solution.

[0026] In a third aspect, the present invention provides the use of the polymorph described in the first aspect in the preparation of medicaments for delaying aging and treating or preventing neurodegenerative diseases.

[0027] In a preferred embodiment, the neurodegenerative diseases include, but are not limited to, Alzheimer's disease, ALS, and Parkinson's disease.

[0028] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the polymorph described in the first aspect and optionally a pharmaceutically acceptable excipient.

[0029] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0030] Figure 1 This is the XRPD spectrum of the crystalline compound of formula (I) obtained in the embodiments of the present invention, namely β-nicotinamide mononucleotide (NMN).

[0031] Figure 2 This is the HNMR spectrum of the crystalline compound of formula (I) obtained in the embodiments of the present invention.

[0032] Figure 3 This is the XRPD spectrum of the crystalline compound of formula (I) obtained by reference example in this invention.

[0033] Figure 4 The XRPD comparison spectra are of the crystalline compound of formula (I) obtained in the embodiments of the present invention and the crystalline compound of formula (I) obtained in the reference example.

[0034] Figure 5 This is a comparison of the infrared spectra of the crystalline compound of formula (I) obtained in the embodiments of the present invention and the crystalline compound of formula (I) obtained in the reference example.

[0035] Figure 6 This is the HPLC detection chromatogram of the crystalline compound (I) obtained in the embodiment of the present invention. Detailed Implementation

[0036] Through extensive and in-depth research, the inventors unexpectedly discovered a novel method for crystallizing β-nicotinamide mononucleotide (NMN) and a new crystalline form of NMN obtained using this method. The method of this invention allows for the production of NMN crystals without the use of toxic or harmful organic solvents such as methanol. The polymorphs of this invention not only exhibit significantly improved stability but also show significant improvements in bioavailability, pharmacokinetics, and pharmacodynamics. Based on these findings, this invention was completed.

[0037] The polymorph of the present invention and its preparation method

[0038] In this document, the terms "crystal of the present invention," "polymorph of the present invention," "crystalline compound of the present invention," and "crystalline compound of formula (I)" have the same meaning, referring to the crystalline compound of the compound shown in formula (I).

[0039]

[0040] In specific embodiments, the crystalline compound of formula (I) is non-solventized, anhydrous, or substantially anhydrous.

[0041] In some embodiments, the polymorphism of the crystalline compound is characterized by powder X-ray diffraction (XRD). θ represents the diffraction angle in degrees. In some embodiments, the diffraction angle is measured as twice the diffraction angle θ using an XRD diffractometer. Therefore, the diffraction pattern described herein refers to the X-ray intensity measured relative to angle 2θ.

[0042] In a specific embodiment, the X-ray powder diffraction pattern of the crystalline compound of formula (I) has characteristic peaks at the following 2θ angles (±0.2°): 20.42; 21.65; 22.75 and 24.98; further, it has characteristic peaks at the following 2θ angles (±0.2°): 21.21; 22.26; 23.99; 24.69; and 27.21; even further, it has characteristic peaks at the following 2θ angles (±0.2°). Characteristic peaks: 7.70; 11.33; 12.43; 16.32; 17.78; 19.06; and 19.93; furthermore, characteristic peaks are present at the following 2θ angles (±0.2°): 10.18; 15.25; 19.26; 22.75; 23.34; 25.69; 26.24; 26.70; 27.61; 28.08; 29.88; 31.78; and 38.00. In a preferred embodiment, the crystalline compound of formula (I) has the following characteristics: Figure 1 The XRD pattern shown.

[0043] The polymorph of the present invention is prepared by the following method, which includes: adding an antisolvent to an aqueous solution of the compound of formula (I) to supersaturate the resulting solution, thereby causing the compound of formula (I) to precipitate from the solution. In a specific embodiment, the antisolvent is ethanol. In a specific embodiment, the mass percentage concentration of the aqueous solution of the compound of formula (I) is 5%-25%, preferably 8%-20%. In a specific embodiment, the amount (volume) of ethanol added is 1-3 times the volume of water added to the aqueous solution of the compound of formula (I).

[0044] The inventors have particularly discovered that, in order to obtain the polymorph of the present invention, the amount of ethanol added can be the same or different depending on the concentration of the aqueous solution of the compound of formula (I). The specific principle is that the amount of ethanol added initially should ensure that the solution remains clear after the addition is completed, without the precipitation of oil droplets. After the initial addition of ethanol, crystal nuclei are formed by stirring or crystal seeds obtained in the embodiments of the present invention are added to induce crystallization.

[0045] In some embodiments of the present invention, after the initial addition of ethanol, a clear solution is obtained. Crystal nuclei are formed by stirring or crystal seeds obtained in the embodiments of the present invention are added to induce crystallization. After stirring, a large amount of solid precipitates, and the solution becomes slurry-like. Adding a portion of ethanol will not affect the crystal form of the obtained compound (I) and can also appropriately improve the recovery rate.

[0046] In an embodiment of the present invention, the temperature at which ethanol is added is 20°C-40°C, preferably 25°C-35°C.

[0047] After the addition of ethanol, no additional heating or cooling is required to promote crystallization. Crystal nuclei can be formed by stirring or crystal seeds obtained in the embodiments of the present invention can be added to induce crystallization.

[0048] In some embodiments of the present invention, the addition of ethanol to an aqueous solution of compound (I) under stirring will release heat. The aqueous solution at 20°C-25°C will heat up to 30°C-35°C after the addition of ethanol without cooling. In some embodiments of the present invention, the temperature can be controlled to maintain crystal precipitation at 20°C-25°C, or the temperature can be allowed to rise naturally to 30°C-35°C and maintained at 30°C-35°C to precipitate crystals; the crystal form of the resulting compound (I) will not change.

[0049] In an embodiment of the present invention, after a large amount of crystalline solid is precipitated from the ethanol aqueous solution of compound (I) at 20°C-40°C, the temperature is then lowered, for example, to 0°C-5°C. This does not change the crystal form of the obtained compound (I) and can also appropriately improve the recovery rate.

[0050] Based on the polymorphs provided by this invention, those skilled in the art will understand that the polymorphs of this invention can be prepared into drugs for delaying aging and preventing various neurodegenerative diseases such as Alzheimer's disease. Therefore, this invention also provides a pharmaceutical composition comprising the polymorphs of this invention and optionally pharmaceutically acceptable excipients.

[0051] The specific excipients in the pharmaceutical composition and the specific dosage form of the pharmaceutical composition can be manufactured by those skilled in the art according to specific needs using known methods. For example, the polymorph of the present invention can be manufactured into oral formulations, such as tablets, through conventional mixing, granulation, tableting, dissolving, or freeze-drying processes. Suitable excipients may include fillers, such as sugars like lactose or sucrose, mannitol or sorbitol; cellulose preparations or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate; and binders, such as starch pastes, including corn starch, wheat starch, rice starch, potato starch, gelatin, astragalus gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone. If desired, disintegrants may be added, such as the starches mentioned above, as well as carboxymethyl starch, croscarmellose, agar, or alginate or its salts, such as sodium alginate. Adjuvants, particularly flow regulators and lubricants, such as silica, talc, stearates, such as calcium magnesium stearate, stearic acid, or polyethylene glycol. If desired, a suitable coating resistant to gastric juices can be provided to the tablet core. For this purpose, a concentrated sugar solution can be applied. This solution may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, lacquer solution, and suitable organic solvents or solvent mixtures. To prepare a gastric juice-resistant coating, a suitable cellulose solution, such as cellulose acetate phthalate or hydroxypropyl methylcellulose phthalate, can be used. Dyes or pigments can be added to the coating of the tablet or tablet core, for example, for identification or to characterize the dosage of the active ingredient.

[0052] Based on the aforementioned polymorph, the present invention also provides a method for delaying aging and treating various neurodegenerative diseases such as Alzheimer's disease, the method comprising administering a therapeutically effective amount of the polymorph or pharmaceutical composition of the present invention to a subject in need.

[0053] The main advantages of this invention are:

[0054] 1. The crystallization method of the present invention does not require the use of toxic or harmful organic solvents, such as methanol;

[0055] 2. The crystallization method of the present invention is simple to operate, which is conducive to large-scale production;

[0056] 3. The crystallization method of this invention is safe for production personnel and environmentally friendly;

[0057] 4. The crystallization method of the present invention can obtain higher product purity and the resulting crystals have better stability.

[0058] The technical solution of the present invention is further described below with reference to specific implementation examples. However, the following implementation examples do not constitute a limitation on the present invention. All application methods based on the principles and technical means of the present invention are within the scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0059] Materials and Methods

[0060] The analysis method used for the embodiments and reference examples in this invention is as follows:

[0061] X-ray powder diffraction

[0062] Machine model: Bruker D8 Advance diffractometer, using Cu Kα radiation (40kV, 40Ma);

[0063] Angle range: 3-45 degrees 2θ;

[0064] Step size: 0.02 degrees 2θ;

[0065] Collection rate: 8 degrees / min;

[0066] HPLC

[0067] Shimadzu LC2030C 3D HPLC system, PDA detector, Labsolution workstation

[0068] Chromatographic conditions:

[0069] Chromatographic column: Thermo Hypersil gold 4.6×250mm 5um; (part No. 25005-254630)

[0070] Flow rate: 1.0 ml / min;

[0071] Column temperature: 30℃;

[0072] Sample temperature: 5℃;

[0073] Detection wavelength: 265nm;

[0074] Injection volume: 5 μL;

[0075] Mobile phase: A: 0.005 mol / L ammonium acetate aqueous solution (pH adjusted to 3.0 ± 0.1 with acetic acid); B: methanol

[0076] gradient:

[0077] Time (minutes) 0 5 10 15 15.1 25 Mobile phase A (%) 100 100 0 0 100 100 Mobile phase B (%) 0 0 100 100 0 0

[0078] Infrared analysis (IR)

[0079] Shimadzu RAffinty-1S infrared spectrometer, workstation: Labsolution IR potassium bromide tablet.

[0080] Implementation Column 1.

[0081] 10.0 g of β-nicotinamide mononucleotide (NMN) (purity 98.65%) and 90.0 mL of water were added to a 500 mL reaction flask. After stirring and dissolving, the aqueous solution had a mass percentage of 10.0%. 180 mL of ethanol was added to the aqueous solution at 24 °C with stirring, and the temperature was raised to 32 °C. Initially a clear solution, a white solid gradually precipitated after stirring for 5 minutes. Stirring continued for 3 hours, followed by filtration at 25 °C, rinsing with ethanol, and vacuum drying at 25 °C to constant weight, yielding 8.8 g of white solid.

[0082] After XRPD analysis, the XRPD pattern of the crystals obtained is as follows: Figure 1 As shown;

[0083] The purity was determined to be 99.91% by HPLC. Figure 6 As shown;

[0084] HNMR spectroscopy as follows Figure 2 As shown;

[0085] Loss on drying: Drying under reduced pressure at 60℃ (desiccant: phosphorus pentoxide), loss on drying was 0.05%, and the resulting solid was anhydrous.

[0086] Implement column 2.

[0087] 10.0 g of β-nicotinamide mononucleotide (NMN) and 90.0 mL of water were added to a 500 mL reaction flask. After stirring and dissolving, the aqueous solution had a mass percentage of 10.0%. 180 mL of ethanol was added to the aqueous solution at 24 °C with stirring, and the temperature was raised to 32 °C. Initially a clear solution, a white solid gradually precipitated after stirring for 5 minutes. Stirring continued for 3 hours, followed by the dropwise addition of 90 mL of ethanol. Stirring continued for another 2 hours. The solution was then filtered at 25 °C, washed with ethanol, and dried under vacuum at 25 °C to constant weight, yielding 9.1 g of a white solid.

[0088] After XRPD analysis, the XRPD pattern of the crystals obtained is as follows: Figure 1 As shown.

[0089] Example 3.

[0090] 10.0 g of β-nicotinamide mononucleotide (NMN) and 40.0 mL of water were added to a 250 mL reaction flask. After stirring and dissolving, the aqueous solution had a mass percentage of 20.0%. 60 mL of ethanol was added dropwise to the aqueous solution at 24 °C with stirring. The temperature was raised to 30 °C. After the addition was complete, the solution was initially clear. After stirring for 3 minutes, a white solid gradually precipitated. After stirring for another 2 hours, the solution was filtered to obtain a white solid. The solid was washed with ethanol and dried under vacuum at 25 °C to constant weight, yielding 8.8 g of crystals.

[0091] After XRPD analysis, the XRPD pattern of the crystals obtained is as follows: Figure 1 As shown.

[0092] Example 4.

[0093] 10.0 g of β-nicotinamide mononucleotide (NMN) and 60.0 mL of water were added to a 250 mL reaction flask. After stirring and dissolving, the aqueous solution had a mass percentage of 14.3%. 100 mL of ethanol was added dropwise to the aqueous solution at 24 °C with stirring. The temperature was raised to 31 °C. After the addition was complete, the solution was initially clear, but a solid gradually precipitated out with stirring. After stirring for another hour, the solution was filtered, washed with ethanol, and dried under vacuum at 25 °C to constant weight, yielding 8.6 g of a white solid.

[0094] After XRPD analysis, the XRPD pattern of the crystals obtained is as follows: Figure 1 As shown.

[0095] Example 5.

[0096] 10.0 g of β-nicotinamide mononucleotide (NMN) and 60.0 mL of water were added to a 250 mL reaction flask. After stirring and dissolving, the aqueous solution had a mass percentage of 14.3%. 100 mL of ethanol was added dropwise to the aqueous solution at 24 °C with stirring. The temperature was raised to 31 °C. After the addition was complete, the solution was initially clear, but a solid gradually precipitated out with stirring. After stirring for another hour, the solution was cooled to 0 °C–5 °C in an ice bath and stirred for another hour. The solution was then filtered, washed with ethanol, and dried under vacuum at 25 °C to constant weight, yielding 8.9 g of a white solid.

[0097] After XRPD analysis, the XRPD pattern of the crystals obtained is as follows: Figure 1 As shown.

[0098] Example 6.

[0099] 10.0 g of β-nicotinamide mononucleotide (NMN) and 115.0 mL of water were added to a 250 mL reaction flask. After stirring and dissolving, the aqueous solution had a mass percentage of 8.0%. 250 mL of ethanol was added dropwise to the aqueous solution at 24 °C with stirring. The temperature was raised to 32 °C. After the addition was complete, the solution was initially clear, but a solid gradually precipitated out with stirring. After stirring for another 2 hours, the solution was filtered, washed with ethanol, and dried under vacuum at 25 °C to constant weight, yielding 8.6 g of a white solid.

[0100] After XRPD analysis, the XRPD pattern of the crystals obtained is as follows: Figure 1 As shown.

[0101] Example 7.

[0102] 10.0 g of β-nicotinamide mononucleotide (NMN) and 30 mL of water were added to a 250 mL reaction flask. After stirring and dissolving, the aqueous solution had a mass percentage of 25.0%. 30 mL of ethanol was added dropwise to the aqueous solution at 25 °C with stirring, and the temperature was raised to 30 °C. After the addition was complete, the solution was initially clear. The solid obtained in Example 1 was added as a seed crystal, and a white solid gradually precipitated out with stirring. After stirring for another 2 hours, the solution was filtered to obtain a white solid, washed with ethanol, and dried under vacuum at 25 °C to constant weight, yielding 8.9 g.

[0103] After XRPD analysis, the XRPD pattern of the crystals obtained is as follows: Figure 1 As shown.

[0104] Comparative Example

[0105] Following the methods in CN108697722A and WO2018 / 047715, 50.0 g of β-nicotinamide mononucleotide (NMN) was dissolved in 150 mL of water, and 300 mL of methanol was added with stirring. A white solid precipitated out. After stirring for another 2 hours, the mixture was filtered and dried under vacuum at 25 °C to obtain 46.0 g of white solid.

[0106] After XRPD analysis, the XRPD pattern of the crystals obtained is as follows: Figure 3 As shown. It is largely the same as crystal form 1 reported in CN108697722A.

[0107] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The polymorphism of the compound shown in formula (I), The polymorph has an XRPD pattern generally as shown in Figure 1.

2. The polymorph as described in claim 1, characterized in that, The polymorph is non-solventized or anhydrous.

3. A method for preparing the polymorph of claim 1 or 2, the method comprising the following steps: a. Dissolve the compound shown in formula (I) in water to obtain an aqueous solution of the compound shown in formula (I); and b. Add an antisolvent to the aqueous solution of the compound of formula (I) obtained in step a, so that the crystals of the compound of formula (I) precipitate out of the solution; The antisolvent is ethanol.

4. The method as described in claim 3, characterized in that, The aqueous solution of the compound of formula (I) obtained in step a has a mass percentage concentration of 5%-25%.

5. The method as described in claim 4, characterized in that, The aqueous solution of the compound of formula (I) obtained in step a has a mass percentage concentration of 8%-20%.

6. The method as described in claim 3 or 4, characterized in that, In step b, the volume of ethanol added is 1-3 times the volume of water added in step a.

7. The method as described in claim 3 or 4, characterized in that, The temperature at which ethanol is added in step b is 20℃-40℃.

8. The method as described in claim 7, characterized in that, The temperature at which ethanol is added in step b is 25℃-35℃.

9. The method as described in claim 3 or 4, characterized in that, In step b, the amount of ethanol added should be sufficient to ensure that the solution remains clear after the ethanol is added. Then, crystallization is induced by stirring to form crystal nuclei or by adding seed crystals.

10. The method as described in claim 3 or 4, characterized in that, After the crystals of the compound shown in formula (I) precipitate from the solution, ethanol is added.

11. The method as described in claim 3 or 4, characterized in that, After the crystals of the compound shown in formula (I) precipitate from the solution, the temperature is appropriately reduced.

12. Use of the polymorph of claim 1 or 2 in the preparation of medicaments for delaying aging and treating or preventing neurodegenerative diseases.

13. The use as described in claim 12, characterized in that, The neurodegenerative diseases mentioned are Alzheimer's disease, ALS, or Parkinson's disease.

14. A pharmaceutical composition comprising the polymorph of claim 1 or 2 and optionally a pharmaceutically acceptable excipient.

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