Organic acid salt of nicotinamide ribose, crystal form and preparation method thereof

Abstract

The invention relates to the field of chemical raw materials, in particular to an organic acid salt of nicotinamide ribose, a crystal form and a preparation method thereof. The invention discloses an organic acid salt of nicotinamide ribose, and in the organic acid salt of nicotinamide ribose, organic acid is selected from acetic acid, citric acid or fumaric acid; the molar ratio of the nicotinamide ribose to the organic acid is 1: (1-1.5). The invention also discloses a preparation method of the organic acid salt of nicotinamide ribose. The invention also discloses a crystal form A and a crystal form B of the nicotinamide ribose hydrogen fumarate. The nicotinamide ribose hydrogen fumarate in two crystal forms is good in flowability, low in hygroscopicity, good in stability, free of color change, capable of keeping a solid form, low in effective concentration, capable of reducing the use cost, more suitable for batch production and wider in application scene.

Description

[0001] This invention claims the priority of the patent application titled "Organic Acid Salts, Crystal Forms and Preparation Methods Thereof of Nicotinamide Riboside", with the application number 202311781327.2 and filed on December 22, 2023. Technical Field

[0002] This invention relates to the field of chemical raw materials, and particularly to an organic acid salt, crystal form and preparation method thereof of nicotinamide riboside. Background Art

[0003] Nicotinamide riboside (NR) is a derivative of vitamin B3 (also known as niacin). A large number of studies have shown that NR has functions such as enhancing the body's metabolism, preventing stem cell aging, and maintaining stem cell function; in the research of liver cancer, the results show that supplementing NR through diet can prevent the development of liver cancer in mice, induce tumor regression, and no side effects have been found under high-dose use. In addition, β-nicotinamide riboside (β-NR) is phosphorylated to obtain β-nicotinamide mononucleotide (NMN), which is a synthesis substrate of coenzyme I in organisms. Research shows that β-NMN has activities such as anti-aging, regulating insulin secretion, and affecting mRNA expression levels. Therefore, β-NR and β-NMN have become hot compounds in the fields of drug development, regenerative medicine and skin care, and the market demand has great prospects.

[0004] The free form of nicotinamide riboside exists in the form of cations and is actually unstable, and needs to form an ion pair with anions to be stable.

[0005] However, the currently reported NR in the form of chloride salts and other organic acid salts basically have problems such as poor stability requiring refrigeration, high hygroscopicity, poor fluidity, difficult formulation process development, and high usage costs. In addition to chloride salts, many organic acids also have these salts in pharmaceutically unacceptable forms, and these salt forms may be toxic or biologically intolerable in other ways. These problems generally limit its processing and application in downstream fields. Therefore, it is of great significance to develop NR organic acid salts that are more stable, safe and low-cost in water and the environment. Summary of the Invention

[0006] In view of the above-mentioned disadvantages of the prior art, the purpose of this application is to provide an organic acid salt, crystal form and preparation method thereof of nicotinamide riboside.

[0007] The first aspect of this invention provides an organic acid salt of nicotinamide riboside. In the organic acid salt of nicotinamide riboside, the organic acid is selected from acetic acid, citric acid or fumaric acid; the molar ratio of nicotinamide riboside to the organic acid is 1:1 - 1.5.

[0008] In some embodiments of this invention, the molar ratio of nicotinamide riboside to the organic acid is 1:1.

[0009] The second aspect of the present invention provides a preparation method of the organic acid salt of nicotinamide riboside described in the first aspect of the present invention. The preparation method includes the following steps:

[0010] 1) Provide multiple resin columns connected in series, and fill anion resins in each of the resin columns;

[0011] 2) Wash the anion resin with an organic acid salt solution, and then wash the anion resin with water until the pH of the flowing water solution is 7-8;

[0012] 3) Pass an aqueous solution of nicotinamide riboside chloride through the anion resin, then wash the anion resin with water, collect and combine the eluates, and then freeze-dry to obtain the organic acid salt of nicotinamide riboside.

[0013] The third aspect of the present invention provides a crystal form of an organic acid salt of nicotinamide riboside. The organic acid is fumaric acid, and the crystal form of the organic acid salt of nicotinamide riboside is crystal form A of nicotinamide riboside hydrogen fumarate; at least one of the 2θ values of crystal form A measured by X-ray powder diffraction has a characteristic peak at 17.59°±0.2°, 21.52°±0.2°, 22.31°±0.2°, 25.46°±0.2° or 29.17°±0.2°.

[0014] The fourth aspect of the present invention provides another crystal form of an organic acid salt of nicotinamide riboside. The organic acid is fumaric acid, and the crystal form of the organic acid salt of nicotinamide riboside is crystal form B of nicotinamide riboside hydrogen fumarate; at least one of the 2θ values of crystal form B measured by X-ray powder diffraction has a characteristic peak at 13.57°±0.2°, 21.40°±0.2°, 22.64°±0.2° or 24.44°±0.2°.

[0015] The fifth aspect of the present invention provides a preparation method of the crystal forms of the organic acid salts of nicotinamide riboside described in the third and fourth aspects of the present invention. The preparation method includes: dissolving the organic acid salt of nicotinamide riboside in water to obtain a clear solution, adding an organic solvent at room temperature, stirring at low temperature to obtain a white suspension, centrifuging, separating the solid and liquid, and then drying to obtain the crystal form of the organic acid salt of nicotinamide riboside, wherein the organic acid salt of nicotinamide riboside is nicotinamide riboside hydrogen fumarate.

[0016] The beneficial effects of the present invention are as follows: The present invention discloses organic salts of nicotinamide riboside, wherein the organic acid is acetic acid, citric acid or fumaric acid, and the molar ratio of nicotinamide riboside to the organic acid is 1:1 - 1.5, preferably 1:1; the present invention also discloses preparation methods of nicotinamide riboside acetate, nicotinamide riboside dihydrogen citrate and nicotinamide riboside hydrogen fumarate; the present invention also discloses two crystalline forms, crystal form A and crystal form B, of nicotinamide riboside hydrogen fumarate. Among them, the two crystalline forms of nicotinamide riboside hydrogen fumarate have the following advantages: 1. Good fluidity, specifically manifested in a large tapped density (≥0.8 g / mL) and a small angle of repose (30 - 35°); 2. Good stability, and the long-term stability and accelerated stability data show that crystal form B and crystal form A of NR hydrogen fumarate are currently the most stable among the salt forms related to NR; 3. High purity and content, the purity can reach 99.5%, and the content is 99.0%. In addition, it has low hygroscopicity, does not change color and maintains a solid form, has a low effective concentration, can reduce the use cost, and is more suitable for mass production, with a wider application scenario. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It shows the XRPD spectrum of nicotinamide riboside acetate in Example 1 of the present invention.

[0018] Figure 2 It shows the XRPD spectrum of nicotinamide riboside dihydrogen citrate in Example 2 of the present invention.

[0019] Figure 3 It shows the XRPD spectrum of nicotinamide riboside hydrogen fumarate in Example 3 of the present invention.

[0020] Figure 4 It shows the XRPD spectrum of crystal form A of nicotinamide riboside hydrogen fumarate in Example 4 of the present invention.

[0021] Figure 5 It shows the XRPD spectrum of crystal form B of nicotinamide riboside hydrogen fumarate in Example 7 of the present invention.

[0022] Figure 6 It shows the DSC spectrum of crystal form A of nicotinamide riboside hydrogen fumarate in Example 4 of the present invention.

[0023] Figure 7 It shows the DSC spectrum of crystal form B of nicotinamide riboside hydrogen fumarate in Example 7 of the present invention.

[0024] Figure 8 It shows the NMR spectra of crystal form A of nicotinamide riboside hydrogen fumarate in Example 4 and crystal form B of nicotinamide riboside hydrogen fumarate in Example 7 of the present invention.

[0025] Figure 9Shown are the results of the cell proliferation experiment in Example 14 of the present invention.

[0026] Figure 10 Shown are the results of the quantitative measurement of the NAD+ concentration in Example 14 of the present invention by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Detailed implementation manners

[0027] Hereinafter, the implementation manners of the organic acid salts, crystal forms and preparation methods of nicotinamide riboside specifically disclosed will be described in detail.

[0028] The "ranges" disclosed in this application are defined in the form of a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a particular range. The ranges defined in this way can include or exclude the end values, and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, ranges of 60-110 and 80-120 are also contemplated. In addition, if the minimum range values 1 and 2 are listed, and if the maximum range values 3, 4 and 5 are listed, then the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In this application, unless otherwise specified, the numerical range "a-b" represents an abbreviated representation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are fully listed herein, and "0-5" is only an abbreviated representation of these numerical combinations. Additionally, when stating that a certain parameter is an integer ≥2, it is equivalent to disclosing that the parameter is, for example, the integer 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0029] If there is no special instruction, all implementation manners and optional implementation manners of this application can be combined with each other to form a new technical solution.

[0030] If there is no special instruction, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0031] Through a large number of exploratory experiments, the inventors of the present application obtained the acetate of nicotinamide riboside, the dihydrogen citrate of nicotinamide riboside, and the hydrogen fumarate of nicotinamide riboside, and provided corresponding preparation methods. In addition, the present invention also obtained two crystalline forms, crystal form A and crystal form B, of the hydrogen fumarate of nicotinamide riboside. The two crystalline forms of the hydrogen fumarate of nicotinamide riboside have the following advantages: 1. Good fluidity, specifically manifested in a large tapped density (≥0.8 g / mL) and a small angle of repose (30-35°); 2. Good stability. The long-term stability and accelerated stability data show that crystal form B and crystal form A of NR hydrogen fumarate are currently the most stable among the salt forms related to NR; 3. High purity and content. The purity can reach 99.5%, and the content is 99.0%. In addition, it has low hygroscopicity, does not change color and remains in a solid form, has a low effective concentration, that is, excellent use effects can be produced at a lower dosage level or low concentration, can reduce the use cost, is more suitable for mass production, and has a wider application scenario. On this basis, the present application was completed.

[0032] Organic acid salts of nicotinamide riboside

[0033] In the first aspect of the present invention, an organic acid salt of nicotinamide riboside is provided. In the organic acid salt of nicotinamide riboside, the organic acid is selected from acetic acid, citric acid or fumaric acid; the molar ratio of nicotinamide riboside to the organic acid is 1:1-1.5. It can be optionally 1:1-1.3, 1:1.3-1.5, 1:1-1.1, 1:1.1-1.2, 1:1.2-1.3, 1:1.3-1.4 or 1:1.4-1.5. The molar ratio of nicotinamide riboside to the organic acid radical is 1:1-1.5. It can be optionally 1:1-1.3, 1:1.3-1.5, 1:1-1.1, 1:1.1-1.2, 1:1.2-1.3, 1:1.3-1.4 or 1:1.4-1.5. More specifically, for example, the molar ratio of nicotinamide riboside to the acetate radical is 1:1-1.5. It can be optionally 1:1-1.3, 1:1.3-1.5, 1:1-1.1, 1:1.1-1.2, 1:1.2-1.3, 1:1.3-1.4 or 1:1.4-1.5. The molar ratio of nicotinamide riboside to the citrate radical is 1:1-1.5. It can be optionally 1:1-1.3, 1:1.3-1.5, 1:1-1.1, 1:1.1-1.2, 1:1.2-1.3, 1:1.3-1.4 or 1:1.4-1.5. The molar ratio of nicotinamide riboside to the fumarate radical is 1:1-1.5. It can be optionally 1:1-1.3, 1:1.3-1.5, 1:1-1.1, 1:1.1-1.2, 1:1.2-1.3, 1:1.3-1.4 or 1:1.4-1.5.

[0034] In some embodiments of the present invention, in the organic acid salt of nicotinamide riboside, the organic acid is selected from acetic acid, citric acid or fumaric acid; the molar ratio of nicotinamide riboside to the organic acid is 1:1. The molar ratio of nicotinamide riboside to the organic acid radical is 1:1. Further optionally, the molar ratio of nicotinamide riboside to acetate is 1. The molar ratio of nicotinamide riboside to citrate is 1:1. The molar ratio of nicotinamide riboside to fumarate is 1:1.

[0035] When the organic acid is acetic acid, the organic acid salt of nicotinamide riboside is the acetate of nicotinamide riboside.

[0036] When the organic acid is citric acid, the organic acid salt of nicotinamide riboside is the dihydrogen citrate of nicotinamide riboside.

[0037] When the organic acid is fumaric acid, the organic acid salt of nicotinamide riboside is the hydrogen fumarate of nicotinamide riboside. Fumaric acid is the simplest unsaturated dibasic carboxylic acid. It was first discovered in Corydalis yanhusuo, and is also present in a variety of mushrooms and fresh beef. Fumaric acid is a commonly used medicinal acid radical and can also be used as an acidity regulator, acidulant, antioxidant aid, pickling promoter, and spice in the food industry. European Commission, ESIS; IUCLID Dataset, Fumaric acid(110-17-8)p.39(2000 CD-ROM edition) records that 75 inpatients (42 females, 33 males, aged 20 to 91 years) took 500 mg of fumaric acid daily for one year. No effects on various blood and urine parameters were observed, nor were any changes in liver function observed. Therefore, due to its safety, fumaric acid is very suitable as the acid radical of NR.

[0038] In some embodiments of the present invention, the structural formula of the organic acid salt of nicotinamide riboside is wherein, X - is selected from

[0039] Specifically: the acetate of nicotinamide riboside is the citrate of nicotinamide riboside is the hydrogen fumarate of nicotinamide riboside is

[0040] Preparation method of organic acid salts of nicotinamide riboside

[0041] The second aspect of the present invention provides a preparation method of the organic acid salt of nicotinamide riboside described in the first aspect of the present invention, and the preparation method includes the following steps:

[0042] 1) Provide multiple resin columns connected in series, and fill anion resins in each of the resin columns;

[0043] 2) Wash the anion resin with an organic acid salt solution, and then wash the anion resin with water until the pH of the flowing water is 7 - 8;

[0044] 3) Pass the aqueous solution of nicotinamide riboside chloride through the anion resin, then wash the anion resin with water, collect and combine the eluate, and freeze-dry to obtain the organic acid salt of nicotinamide riboside.

[0045] In the preparation method of the organic acid salt of nicotinamide riboside provided by the present invention, step 1) is to provide multiple resin columns connected in series, and anion resins are respectively filled in each of the resin columns. Specifically:

[0046] In step 1) of the present invention, the anion resin is anion resin D301.

[0047] In the preparation method of the organic acid salt of nicotinamide riboside provided by the present invention, in step 2), the anion resin is washed with an organic acid salt solution, and then the anion resin is washed with water until the pH of the flowing water is 7 - 8. Specifically:

[0048] In step 2) of the present invention, the organic acid salt solution is selected from an aqueous solution of sodium acetate, an aqueous solution of sodium citrate monohydrate, or an aqueous solution of sodium fumarate monohydrate.

[0049] In step 2) of the present invention, the concentration of the organic acid salt solution is 3% - 5%. Optionally, the concentration of the organic acid salt solution is 3% - 4% or 4% - 5%, etc.

[0050] In step 2) of the present invention, the dosage of the organic acid salt solution is 380 - 420 mL. Optionally, the dosage of the organic acid salt solution is 380 - 400 mL or 400 - 420 mL, etc.

[0051] In step 2) of the present invention, the organic acid salt solution washes the anion resin at a flow rate of 95 - 105 mL / h. Optionally, the flow rate is 95 - 100 mL / h or 100 - 105 mL / h, etc.

[0052] In step 2) of the present invention, the water for washing the anion resin washes the anion resin at a flow rate of 95 - 105 mL / h. Optionally, the flow rate is 95 - 100 mL / h or 100 - 105 mL / h, etc.

[0053] In step 2) of the present invention, the dosage of the water is 380 - 420 mL. Optionally, the dosage of the water is 380 - 400 mL or 400 - 420 mL, etc.

[0054] In step 2) of the present invention, the water is deionized water.

[0055] In the preparation method of the organic acid salt of nicotinamide riboside provided by the present invention, in step 3), an aqueous solution of nicotinamide riboside chloride is passed through an anion resin, and then the anion resin is washed with water. After collecting and combining the eluate, it is freeze-dried to obtain the organic acid salt of nicotinamide riboside.

[0056] In step 3) of the present invention, the mass of the aqueous solution of nicotinamide riboside chloride is 190 - 210 g.

[0057] In step 3) of the present invention, the mass concentration of the aqueous solution of nicotinamide riboside chloride is 1% - 2%.

[0058] In step 3) of the present invention, the anion resin is washed with water at a flow rate of 95 - 105 mL / h. Optionally, the flow rate is 95 - 100 mL / h or 100 - 105 mL / h, etc.

[0059] In step 3) of the present invention, the amount of water used is 190 - 210 mL. Optionally, the amount of water used is 190 - 200 mL or 200 - 210 mL, etc.

[0060] In step 3) of the present invention, the water is deionized water.

[0061] Crystal form of organic acid salts of nicotinamide riboside( Crystal form A of the hydrogen fumarate of nicotinamide riboside

[0062] The present invention also provides a crystal form of the organic acid salt of nicotinamide riboside, wherein the organic acid is fumaric acid, and the crystal form of the organic acid salt of nicotinamide riboside is crystal form A of the hydrogen fumarate of nicotinamide riboside; under X-ray powder diffraction measurement, crystal form A has characteristic peaks at least at one of 2θ values of 17.59° ± 0.2°, 21.52° ± 0.2°, 22.31° ± 0.2°, 25.46° ± 0.2°, or 29.17° ± 0.2°. At least at one means including one, two, three,... or all. The following explanation of at least at one is the same as above and will not be repeated.

[0063] In some embodiments of the present invention, under X-ray powder diffraction measurement, crystal form A has characteristic peaks at 2θ values selected from 17.59° ± 0.2°, 21.52° ± 0.2°, 22.31° ± 0.2°, 25.46° ± 0.2°, and 29.17° ± 0.2°.

[0064] In some embodiments of the present invention, the crystalline form A has characteristic peaks at least at one of the 2θ values of 12.8°±0.2°, 17.46°±0.2°, 17.59°±0.2°, 21.52°±0.2°, 22.30°±0.2°, 23.36°±0.2°, 25.46°±0.2°, 26.04°±0.2°, 28.29°±0.2° or 29.17°±0.2° as determined by X-ray powder diffraction.

[0065] In some embodiments of the present invention, the crystalline form A has characteristic peaks at the 2θ values of 12.8°±0.2°, 17.46°±0.2°, 17.59°±0.2°, 21.52°±0.2°, 22.30°±0.2°, 23.36°±0.2°, 25.46°±0.2°, 26.04°±0.2°, 28.29°±0.2° and 29.17°±0.2° as determined by X-ray powder diffraction.

[0066] In some embodiments of the present invention, the crystalline form A has characteristic peaks at least at one of the 2θ values of 11.25°±0.2°, 12.8°±0.2°, 12.19°±0.2°, 14.08°±0.2°, 16.47°±0.2°, 17.46°±0.2°, 17.59°±0.2°, 20.50°±0.2°, 21.32°±0.2°, 21.52°±0.2°, 21.50°±0.2°, 22.31°±0.2°, 22.56°±0.2°, 23.36°±0.2°, 24.50°±0.2°, 25.46°±0.2°, 25.7°±0.2°, 26.04°±0.2°, 26.19°±0.2°, 26.39°±0.2°, 27.30°±0.2°, 28.29°±0.2°, 29.17°±0.2°, 31.10°±0.2°, 35.54°±0.2° or 37.89°±0.2° as determined by X-ray powder diffraction.

[0067] In some embodiments of the present invention, the crystalline form A has characteristic peaks at 2θ values of 11.25° ± 0.2°, 12.8° ± 0.2°, 12.19° ± 0.2°, 14.08° ± 0.2°, 16.47° ± 0.2°, 17.46° ± 0.2°, 17.59° ± 0.2°, 20.50° ± 0.2°, 21.32° ± 0.2°, 21.52° ± 0.2°, 22.31° ± 0.2°, 22.56° ± 0.2°, 23.36° ± 0.2°, 24.50° ± 0.2°, 25.46° ± 0.2°, 25.7° ± 0.2°, 26.04° ± 0.2°, 26.19° ± 0.2°, 26.39° ± 0.2°, 27.30° ± 0.2°, 28.29° ± 0.2°, 29.17° ± 0.2°, 31.10° ± 0.2°, 35.54° ± 0.2° and 37.89° ± 0.2° as determined by X-ray powder diffraction.

[0068] In some embodiments of the present invention, the crystalline form A has characteristic peaks at least at one of the 2θ values measured by X-ray powder diffraction at 5.10° ± 0.2°, 11.25° ± 0.2°, 12.19° ± 0.2°, 12.8° ± 0.2°, 13.80° ± 0.2°, 14.08° ± 0.2°, 15.66° ± 0.2°, 16.47° ± 0.2°, 17.46° ± 0.2°, 17.59° ± 0.2°, 18.51° ± 0.2°, 20.50° ± 0.2°, 21.32° ± 0.2°, 21.52° ± 0.2°, 21.95° ± 0.2°, 22.31° ± 0.2°, 22.56° ± 0.2°, 23.36° ± 0.2°, 24.50° ± 0.2°, 24.65° ± 0.2°, 25.11° ± 0.2°, 25.46° ± 0.2°, 25.7° ± 0.2°, 26.04° ± 0.2°, 26.19° ± 0.2°, 26.39° ± 0.2°, 27.09° ± 0.2°, 27.30° ± 0.2°, 27.54° ± 0.2°, 27.66° ± 0.2°, 28.01° ± 0.2°, 28.29° ± 0.2°, 28.50° ± 0.2°, 29.17° ± 0.2°, 29.65° ± 0.2°, 31.10° ± 0.2°, 32.82° ± 0.2°, 32.96° ± 0.2°, 35.47° ± 0.2°, 35.54° ± 0.2°, 35.80° ± 0.2°, 36.39° ± 0.2°, 37.71° ± 0.2°, 37.90° ± 0.2°, 38.15° ± 0.2°, 38.44° ± 0.2°, 38.56° ± 0.2°, 39.05° ± 0.2°, 39.32° ± 0.2°, 41.57° ± 0.2°, 42.18° ± 0.2°, 43.81° ± 0.2°, 45.06° ± 0.2°, 45.23° ± 0.2° or 47.53° ± 0.2°.

[0069] In some embodiments of the present invention, the crystal form A has characteristic peaks at 2θ values of 11.25° ± 0.2°, 12.19° ± 0.2°, 12.8° ± 0.2°, 13.80° ± 0.2°, 14.08° ± 0.2°, 15.66° ± 0.2°, 16.47° ± 0.2°, 17.46° ± 0.2°, 17.59° ± 0.2°, 18.51° ± 0.2°, 20.50° ± 0.2°, 21.32° ± 0.2°, 21.52° ± 0.2°, 21.95° ± 0.2°, 22.31° ± 0.2°, 22.56° ± 0.2°, 23.36° ± 0.2°, 24.50° ± 0.2°, 24.65° ± 0.2°, 25.11° ± 0.2°, 25.46° ± 0.2°, 25.7° ± 0.2°, 26.04° ± 0.2°, 26.19° ± 0.2°, 26.39° ± 0.2°, 27.09° ± 0.2°, 27.30° ± 0.2°, 27.54° ± 0.2°, 27.66° ± 0.2°, 28.01° ± 0.2°, 28.29° ± 0.2°, 28.50° ± 0.2°, 29.17° ± 0.2°, 29.65° ± 0.2°, 31.10° ± 0.2°, 32.82° ± 0.2°, 32.96° ± 0.2°, 35.47° ± 0.2°, 35.54° ± 0.2°, 35.80° ± 0.2°, 36.39° ± 0.2°, 37.71° ± 0.2°, 37.90° ± 0.2°, 38.15° ± 0.2°, 38.44° ± 0.2°, 38.56° ± 0.2°, 39.05° ± 0.2°, 39.32° ± 0.2°, 41.57° ± 0.2°, 42.18° ± 0.2°, 43.81° ± 0.2°, 45.06° ± 0.2°, 45.23° ± 0.2° and 47.53° ± 0.2° as determined by X-ray powder diffraction.

[0070] In some embodiments of the present invention, the crystal form A is also determined by differential scanning calorimetry (DSC), and the crystal form A shows an endothermic peak at least at 113 - 118 °C as determined by DSC.

[0071] In a preferred embodiment, the DSC pattern of the crystal form A shows an endothermic peak near 118 °C, and the pattern is substantially as Figure 6 characterized.

[0072] In some embodiments of the present invention, the crystal form A decomposes at 165 - 200 °C as determined by DSC.

[0073] Crystal form of organic acid salts of nicotinamide riboside( Crystal form B of nicotinamide riboside hydrogen fumarate

[0074] The present invention also provides a crystal form of an organic acid salt of nicotinamide riboside, wherein the organic acid is fumaric acid, and the crystal form of the organic acid salt of nicotinamide riboside is crystal form B of nicotinamide riboside hydrogen fumarate; at least one characteristic peak exists at 2θ values of 13.57° ± 0.2°, 21.40° ± 0.2°, 22.64° ± 0.2° or 24.44° ± 0.2° as determined by X-ray powder diffraction for the crystal form B.

[0075] In some embodiments of the present invention, the crystal form B has characteristic peaks at 2θ values of 13.57° ± 0.2°, 21.40° ± 0.2°, 22.64° ± 0.2° and 24.44° ± 0.2° as determined by X-ray powder diffraction.

[0076] In some embodiments of the present invention, at least one characteristic peak exists at 2θ values of 13.42° ± 0.2°, 13.57° ± 0.2°, 19.15° ± 0.2°, 21.40° ± 0.2°, 22.64° ± 0.2°, 24.44° ± 0.2°, 25.85° ± 0.2° or 29.47° ± 0.2° as determined by X-ray powder diffraction for the crystal form B.

[0077] In some embodiments of the present invention, the crystal form B has characteristic peaks at 2θ values of 13.42° ± 0.2°, 13.57° ± 0.2°, 19.15° ± 0.2°, 21.40° ± 0.2°, 22.64° ± 0.2°, 24.44° ± 0.2°, 25.85° ± 0.2° and 29.47° ± 0.2° as determined by X-ray powder diffraction.

[0078] In some embodiments of the present invention, the crystalline form B has characteristic peaks at least at one of the 2θ values determined by X-ray powder diffraction in the range of 10.52° ± 0.2°, 10.63° ± 0.2°, 12.81° ± 0.2°, 13.42° ± 0.2°, 13.57° ± 0.2°, 13.78° ± 0.2°, 15.19° ± 0.2°, 15.48° ± 0.2°, 17.64° ± 0.2°, 17.80° ± 0.2°, 17.97° ± 0.2°, 19.15° ± 0.2°, 19.86° ± 0.2°, 21.02° ± 0.2°, 21.40° ± 0.2°, 22.64° ± 0.2°, 23.05° ± 0.2°, 24.44° ± 0.2°, 24.85° ± 0.2°, 25.85° ± 0.2°, 26.17° ± 0.2°, 27.12° ± 0.2°, 27.56° ± 0.2°, 27.99° ± 0.2°, 29.47° ± 0.2°, 29.70° ± 0.2°, 30.85° ± 0.2°, 30.94° ± 0.2°, 31.42° ± 0.2°, 31.83° ± 0.2°, 32.17° ± 0.2°, 32.47° ± 0.2°, 34.61° ± 0.2°, 35.29° ± 0.2°, 35.89° ± 0.2°, 36.72° ± 0.2°, 37.48° ± 0.2°, 37.69° ± 0.2°, 38.96° ± 0.2°, 40.45° ± 0.2°, 45.73° ± 0.2°, 46.29° ± 0.2°, or 46.45° ± 0.2°.

[0079] In some embodiments of the present invention, the crystalline form B has characteristic peaks at 2θ values of 10.52° ± 0.2°, 10.63° ± 0.2°, 12.81° ± 0.2°, 13.42° ± 0.2°, 13.57° ± 0.2°, 13.78° ± 0.2°, 15.19° ± 0.2°, 15.48° ± 0.2°, 17.64° ± 0.2°, 17.80° ± 0.2°, 17.97° ± 0.2°, 19.15° ± 0.2°, 19.86° ± 0.2°, 21.02° ± 0.2°, 21.40° ± 0.2°, 22.64° ± 0.2°, 23.05° ± 0.2°, 24.44° ± 0.2°, 24.85° ± 0.2°, 25.85° ± 0.2°, 26.17° ± 0.2°, 27.12° ± 0.2°, 27.56° ± 0.2°, 27.99° ± 0.2°, 29.47° ± 0.2°, 29.70° ± 0.2°, 30.85° ± 0.2°, 30.94° ± 0.2°, 31.42° ± 0.2°, 31.83° ± 0.2°, 32.17° ± 0.2°, 32.47° ± 0.2°, 34.61° ± 0.2°, 35.29° ± 0.2°, 35.89° ± 0.2°, 36.72° ± 0.2°, 37.48° ± 0.2°, 37.69° ± 0.2°, 38.96° ± 0.2°, 40.45° ± 0.2°, 45.73° ± 0.2°, 46.29° ± 0.2° and 46.45° ± 0.2° as determined by X-ray powder diffraction.

[0080] In some embodiments of the present invention, the crystalline form B is also determined by differential scanning calorimetry (DSC). The crystalline form B shows an endothermic peak at least at 110 - 117°C as determined by DSC.

[0081] In a preferred embodiment, the DSC pattern of the crystalline form B shows an endothermic peak near 117°C, and the pattern is substantially as Figure 7 characterized.

[0082] In some embodiments of the present invention, the crystalline form B decomposes at 165 - 185°C as determined by DSC.

[0083] Preparation method of crystal form of organic acid salts of nicotinamide riboside

[0084] The present invention also provides a method for preparing a crystalline form of an organic acid salt of nicotinamide riboside. The preparation method includes: dissolving the organic acid salt of nicotinamide riboside in water to obtain a clear solution, adding an organic solvent at room temperature, stirring at low temperature to obtain a white suspension, centrifuging, separating the solid and liquid, and drying to obtain the crystalline form of the organic acid salt of nicotinamide riboside, wherein the organic acid salt of nicotinamide riboside is the hydrogen fumarate of nicotinamide riboside.

[0085] In some embodiments of the present invention, when the organic solvent is selected from alcohol solvents, crystalline form A of the organic acid salt of nicotinamide riboside is obtained. Optionally, when the organic solvent is selected from methanol and / or ethanol, crystalline form A of the organic acid salt of nicotinamide riboside is obtained.

[0086] In some embodiments of the present invention, when the organic solvent is selected from one or more of acetone, acetonitrile, and ethyl acetate, crystalline form B of the organic acid salt of nicotinamide riboside is obtained. Optionally, when the organic solvent is selected from one of acetone, acetonitrile, and ethyl acetate, crystalline form B of the organic acid salt of nicotinamide riboside is obtained.

[0087] In some embodiments of the present invention, the mass-to-volume ratio of the hydrogen fumarate of nicotinamide riboside to the water is 1.0 - 1.2 g : 4.8 - 5.3 mL. Optionally, the mass-to-volume ratio of the hydrogen fumarate of nicotinamide riboside to the water can be, for example, 1.0 - 1.2 g : 4.8 - 5.0 mL, 1.0 - 1.2 g : 5.0 - 5.3 mL, 1.0 - 1.1 g : 4.8 - 5.3 mL, or 1.1 - 1.2 g : 4.8 - 5.3 mL, etc.

[0088] In some embodiments of the present invention, the mass-to-volume ratio of the hydrogen fumarate of nicotinamide riboside to the organic solvent is 1.0 - 1.2 g : 13.3 - 14.7 mL. Optionally, the mass-to-volume ratio of the hydrogen fumarate of nicotinamide riboside to the organic solvent can be, for example, 1.0 - 1.2 g : 13.3 - 14.0 mL, 1.0 - 1.2 g : 14.0 - 14.7 mL, 1.0 - 1.1 g : 13.3 - 14.7 mL, or 1.1 - 1.2 g : 13.3 - 14.7 mL, etc.

[0089] In some embodiments of the present invention, the low-temperature stirring temperature is 5 - 10 °C. Optionally, the low-temperature stirring temperature is 5 - 8 °C or 8 - 10 °C, etc.

[0090] In some embodiments of the present invention, the low-temperature stirring time is 10 - 12 h. Optionally, the low-temperature stirring time is 10 - 11 h or 11 - 12 h, etc.

[0091] In some embodiments of the present invention, the drying temperature is 40 - 45 °C. Optionally, the drying temperature can be, for example, 40 - 43 °C or 43 - 45 °C, etc.

[0092] Specific embodiments of the present invention are described with reference to the following examples, which are intended to illustrate the present invention and not to limit it in any way. The raw materials can be obtained from public commercial channels unless otherwise specified. The instruments involved in the implementation of this application are common ordinary instruments in the art unless otherwise specified.

[0093] The method for determining the XRPD (X-ray powder diffraction) pattern of the present invention: The sample is collected on a Bruker D2 PHASER X-ray powder diffractometer; the X-ray light source is The voltage is 40 kV; the current is 40 mA; the scanning range: 3.0° to 50°; the scanning step is 0.02°; the scanning speed is 6° / min.

[0094] The method for determining the DSC (differential scanning calorimetry) pattern: The sample is collected on a DSC-25 instrument of TA Company, USA; the scanning speed is 10 °C / min; the protective gas is nitrogen, and the nitrogen flow rate is 50 mL / min; the temperature range is from room temperature to 330 °C.

[0095] Example 1: Preparation method of acetate of nicotinamide riboside (the molar ratio of nicotinamide riboside to acetate in the product is 1:1)

[0096] Load 200 g of anion resin D301 into two 100 mL resin columns, and the two resin columns are connected in series. Wash the resin with an aqueous sodium acetate solution with a mass concentration of 3% at a flow rate of 100 mL / h, and a total of 400 mL is consumed. Then wash the resin with deionized water at a flow rate of 100 mL / h until the pH of the effluent is 7-8, and a total of 400 mL of deionized water is consumed. Pass 200 g of an aqueous solution of nicotinamide riboside chloride with a mass concentration of 1% through the resin at a flow rate of 100 mL / h, and then wash the resin with deionized water at a flow rate of 100 mL / h, consuming 200 g of deionized water. Collect the eluate according to the HPLC results. After combining the eluates, freeze-dry to obtain a powdery solid of nicotinamide riboside acetate. The XRPD pattern of the acetate of nicotinamide riboside is as Figure 1 shown.

[0097] Example 2: Preparation method of dihydrogen citrate of nicotinamide riboside (the molar ratio of nicotinamide riboside to citrate in the product is 1:1)

[0098] Load 200 g of anion resin D301 into two 100 mL resin columns, and connect the two resin columns in series. Wash the resin with a 3% mass concentration aqueous solution of sodium citrate at a flow rate of 100 mL / h, and a total of 400 mL is consumed. Then wash the resin with deionized water at a flow rate of 100 mL / h until the pH of the effluent is 7 - 8, and a total of 400 mL of deionized water is consumed. Pass 200 g of a 1% mass concentration aqueous solution of nicotinamide riboside chloride through the resin at a flow rate of 100 mL / h, then wash the resin with deionized water at a flow rate of 100 mL / h, consuming 200 g of deionized water, and collect the eluate according to the HPLC results. After combining the eluates, freeze-dry to obtain a powdery solid of nicotinamide riboside dihydrogen citrate. The XRPD pattern of the dihydrogen citrate of nicotinamide riboside is as Figure 2 shown.

[0099] Example 3: Preparation method of nicotinamide riboside hydrogen fumarate (the molar ratio of nicotinamide riboside to fumarate in the product is 1:1)

[0100] Load 200 g of anion resin D301 into two 100 mL resin columns, and connect the two resin columns in series. Wash the resin with a 3% mass concentration aqueous solution of sodium fumarate at a flow rate of 100 mL / h, and a total of 400 mL is consumed. Then wash the resin with deionized water at a flow rate of 100 mL / h until the pH of the effluent is 7 - 8, and a total of 400 mL of deionized water is consumed. Pass 200 g of a 1% mass concentration aqueous solution of nicotinamide riboside chloride through the resin at a flow rate of 100 mL / h, then wash the resin with deionized water at a flow rate of 100 mL / h, consuming 200 g of deionized water, and collect the eluate according to the HPLC results. After combining the eluates, freeze-dry to obtain a powdery solid of nicotinamide riboside hydrogen fumarate. The XRPD pattern of the hydrogen fumarate of nicotinamide riboside is as Figure 3 shown.

[0101] Example 4: Preparation method of crystalline form A of nicotinamide riboside hydrogen fumarate

[0102] Take 100 mg of the powdery solid of nicotinamide riboside hydrogen fumarate (amorphous solid of hydrogen fumarate) prepared in Example 3 above in a 3 mL glass vial, add 0.5 mL of pure water, add a polytetrafluoroethylene stir bar, and stir at 35 °C for 1 h to obtain a clear solution. Slowly add 1.4 mL of methanol dropwise at room temperature, then transfer to a biochemical incubator and continue stirring at 5 °C for 10 h to obtain a white suspension. Transfer to a centrifuge tube for centrifugation. After solid-liquid separation, dry the solid in vacuo at 40 °C for 4 h to obtain a solid, and perform XRPD testing. The XRPD results show crystalline form A of nicotinamide riboside hydrogen fumarate. The XRPD pattern of crystalline form A of nicotinamide riboside hydrogen fumarate is basically as Figure 4characterized. The DSC spectrum of the nicotinamide riboside hydrogen fumarate polymorph A shows an endothermic peak near 118°C, and the spectrum is basically as Figure 6 characterized. The NMR spectrum of the nicotinamide riboside hydrogen fumarate polymorph A is as Figure 8 .

[0103] Using Cu-Kα radiation, the X-ray powder diffraction data of the nicotinamide riboside hydrogen fumarate polymorph A at 2θ values are shown in Table 1:

[0104] Table 1

[0105]

[0106]

[0107]

[0108] Example 5: Preparation method of the nicotinamide riboside hydrogen fumarate polymorph A

[0109] Take 100 mg of the powdered solid of nicotinamide riboside hydrogen fumarate (the amorphous solid of hydrogen fumarate) prepared in Example 3 above in a 3 mL glass vial, add 0.5 mL of pure water, add a polytetrafluoroethylene stir bar, and stir at 35°C for 1 h to obtain a clear solution. Slowly add 1.4 mL of ethanol at room temperature, then transfer to a biochemical incubator and continue stirring at 5°C for 10 h to obtain a white suspension. Transfer to a centrifuge tube for centrifugation. After solid-liquid separation, the solid is vacuum dried at 40°C for 4 h to obtain a solid, and XRPD testing is performed. The XRPD results show that it is the nicotinamide riboside hydrogen fumarate polymorph A.

[0110] Example 6: Preparation method of the nicotinamide riboside hydrogen fumarate polymorph A (1.0 kg)

[0111] Take 1.0 kg of the powdered solid of nicotinamide riboside hydrogen fumarate (the amorphous solid of hydrogen fumarate) prepared in Example 3 above in a 20 L jacketed kettle, add 5.0 L of pure water, heat to 35°C and stir mechanically for 1 h to obtain a clear solution. Slowly add 14 L of methanol at room temperature, then cool to 5°C and continue stirring at low temperature for 10 h to obtain a white suspension. Filter. After solid-liquid separation, the solid is vacuum dried at 40°C for 16 h to obtain a solid, and XRPD testing is performed. The XRPD results show that it is the nicotinamide riboside hydrogen fumarate polymorph A.

[0112] Example 7: Preparation method of the nicotinamide riboside hydrogen fumarate polymorph B

[0113] Take 100 mg of the powdered solid of nicotinamide riboside hydrogen fumarate (amorphous solid of hydrogen fumarate) prepared in Example 3 above in a 3 mL glass vial, add 0.5 mL of pure water, add a polytetrafluoroethylene stir bar, and stir at 35 °C for 1 h to obtain a clear solution. Slowly add 1.2 mL of acetone at room temperature, then transfer to a biochemical incubator and continue to stir at 5 °C for 10 h to obtain a white suspension. Transfer to a centrifuge tube for centrifugation. After solid-liquid separation, the solid is dried in vacuo at 40 °C for 4 h to obtain a solid, and XRPD test is carried out. The XRPD result shows it is crystal form B. The XRPD pattern of crystal form B of nicotinamide riboside hydrogen fumarate is basically as Figure 5 characterized. The DSC pattern of crystal form B of nicotinamide riboside hydrogen fumarate shows an endothermic peak near 117 °C, and the pattern is basically as Figure 7 characterized. The NMR spectrum of crystal form B of nicotinamide riboside hydrogen fumarate is as Figure 8 .

[0114] Using Cu-Kα radiation, the X-ray powder diffraction data of crystal form B of nicotinamide riboside hydrogen fumarate at 2theta values are shown in Table 2:

[0115] Table 2

[0116]

[0117]

[0118] Example 8: Preparation method of crystal form B of nicotinamide riboside hydrogen fumarate

[0119] Take 100 mg of the powdered solid of nicotinamide riboside hydrogen fumarate (amorphous solid of hydrogen fumarate) prepared in Example 3 above in a 3 mL glass vial, add 0.5 mL of pure water, add a polytetrafluoroethylene stir bar, and stir at 35 °C for 1 h to obtain a clear solution. Slowly add 1.2 mL of acetonitrile at room temperature, then transfer to a biochemical incubator and continue to stir at 5 °C for 10 h to obtain a white suspension. Transfer to a centrifuge tube for centrifugation. After solid-liquid separation, the solid is dried in vacuo at 40 °C for 4 h to obtain a solid, and XRPD test is carried out. The XRPD result shows it is crystal form B of nicotinamide riboside hydrogen fumarate.

[0120] Example 9: Preparation method of crystal form B of nicotinamide riboside hydrogen fumarate

[0121] Take 100 mg of the powdered solid of nicotinamide riboside hydrogen fumarate (amorphous solid of hydrogen fumarate) prepared in Example 3 above in a 3 mL glass vial, add 0.5 mL of pure water, add a PTFE stir bar and stir at 35 °C for 1 h to obtain a clear solution. Slowly add 1.2 mL of ethyl acetate dropwise at room temperature, then transfer to a biochemical incubator and continue stirring at 5 °C for 10 h to obtain a white suspension. Transfer to a centrifuge tube for centrifugation. After solid-liquid separation, dry the solid in vacuo at 40 °C for 4 h to obtain a solid, and perform XRPD testing. The XRPD results show Form B of nicotinamide riboside hydrogen fumarate.

[0122] Example 10: Preparation method of Form B (1.0 kg) of nicotinamide riboside hydrogen fumarate

[0123] Take 1.0 kg of the powdered solid of nicotinamide riboside hydrogen fumarate (amorphous solid of hydrogen fumarate) prepared in Example 3 above in a 20 L jacketed kettle, add 5.0 L of pure water, heat to 35 °C and stir for 1 h to obtain a clear solution. Slowly add 12 L of acetone dropwise at room temperature, then cool to 5 °C and continue stirring at low temperature for 10 h to obtain a white suspension. Filter. After solid-liquid separation, dry the solid in vacuo at 40 °C for 16 h to obtain a solid, and perform XRPD testing. The XRPD results show Form B.

[0124] Example 11: Flowability experiment

[0125] Adopt the flowability indexes of Form A of nicotinamide riboside hydrogen fumarate (NR hydrogen fumarate Form A) prepared in Example 4, Form B of nicotinamide riboside hydrogen fumarate (NR hydrogen fumarate Form B) prepared in Example 7, NR-chloride Form II (purchased from Hunan Xin'an Biotechnology Co., Ltd., batch number NB240101B, crystallized according to the volume ratio of substrate: water: ethanol = 1:1:10), and NR succinate crystals (self-made, the preparation method is: first prepare anionic resin of succinate type, then pass NR-chloride with batch number NB240101B purchased from Hunan Xin'an Biotechnology Co., Ltd. through the anion resin, and after concentrating the column eluate, crystallize according to the volume ratio of substrate: water: ethanol = 1:3:10):

[0126] Mainly the data of tapped density and angle of repose:

[0127] Tapped density detection method: Accurately weigh about 20 g of the sample with an analytical balance and transfer it to a 50 mL graduated cylinder. Then place the graduated cylinder in a tapped density tester, select Traptimes as 2400 times, control the speed at 80, and press the "start" button to start. After tapping is completed, read the sample volume and calculate the tapped density.

[0128] Angle of repose detection method: Take about 20 g of the sample and slowly add it from above the funnel device. The material leaking from the bottom of the funnel forms a conical accumulation on the horizontal tray. When the horizontal tray is fully covered, use a protractor to measure the acute angle between the inclined surface of the sample accumulation and the bottom horizontal plane, which is the angle of repose.

[0129] Table 3

[0130]

[0131]

[0132] Stability test of Example 12

[0133] (I) Short-term stability test method: Use the acetate prepared in Example 1 [NR-acetate (amorphous)], the dihydrogen citrate of nicotinamide riboside in Example 2 [NR-dihydrogen citrate (amorphous)], the hydrogen fumarate of nicotinamide riboside in Example 3 [NR-hydrogen fumarate (amorphous)], the crystalline form A of the hydrogen fumarate of nicotinamide riboside in Example 4 [NR-hydrogen fumarate (crystalline form A)], and the crystalline form B of the hydrogen fumarate of nicotinamide riboside in Example 7 [NR-hydrogen fumarate (crystalline form B)]. Weigh 30 - 50 mg of each sample and isolate the air by using a 2-layer PE bag (the first layer is tied tightly with a strap, and the second layer is evacuated) plus an aluminum foil bag (evacuated). Place them in a constant temperature and humidity stability chamber with temperatures and humidities of 25°C, 60% RH and 40°C, 75% RH respectively. During the test, take one package of the sample each time and conduct HPLC sample purity test.

[0134] Table 4 shows the purity change of NR organic salts under the conditions of temperature 25°C and humidity 60RH%

[0135] Table 4

[0136]

[0137] Table 5 shows the purity change of NR organic salts under the conditions of temperature 40°C and humidity 75RH%

[0138] Table 5

[0139] Name 0 days 3 days 5 days 7 days NR-dihydrogen citrate (amorphous) 92.13％ 24.16％ 15.58％ 7.76％ NR-hydrogen fumarate (amorphous) 93.83％ 11.91％ 7.58％ 4.26％ NR-acetate (amorphous) 89.55％ 6.97％ 4.20％ 2.26％ NR-hydrogen fumarate (crystal form A) 99.49％ 95.85％ 94.58％ 93.01％ NR-hydrogen fumarate (crystal form B) 99.31％ 96.19％ 94.33％ 91.20％

[0140] For the amorphous forms of NR-hydrogen fumarate, NR-dihydrogen citrate and NR-acetate placed at 25°C, 60% RH, decomposition occurred; the purity of the two crystalline forms of NR-hydrogen fumarate basically did not change and no obvious decomposition occurred, indicating that the stability of the crystalline form is better. Under the conditions of 40°C, 75% RH, the decomposition of the amorphous form is more obvious, while the decomposition rate of the crystalline form sample is relatively slow.

[0141] (2) Long-term stability data

NR-chloride crystal form II, crystal form A of nicotinamide riboside hydrogen fumarate of Example 4 (NR-hydrogen fumarate crystal form A), crystal form B of nicotinamide riboside hydrogen fumarate of Example 7 (NR-hydrogen fumarate crystal form B), NR succinate crystal

[0142] Due to the small sample size in the aforementioned short-term stability experiment, only 30 - 50 mg, the results were easily affected by moisture and oxygen in the air. Therefore, long-term stability experiments were carried out with 2.0 g per package. Among the samples to be tested, NR-chloride crystal form II was purchased from Hunan Xin'an Biotechnology Co., Ltd., batch number NB240101B, and crystallized according to the volume ratio of substrate: water: ethanol = 1:1:10. The NR succinate crystal was self-prepared. The preparation method was as follows: First, prepare anionic resin of succinate type, then pass the NR chloride purchased from Hunan Xin'an Biotechnology Co., Ltd., batch number NB240101B through the anion resin, and after concentrating the column eluent, crystallize according to the volume ratio of substrate: water: ethanol = 1:3:10.

[0143] Testing method: Use NR-chloride crystal form II, crystal form A of nicotinamide riboside hydrogen fumarate of Example 4 (NR-hydrogen fumarate crystal form A), crystal form B of nicotinamide riboside hydrogen fumarate of Example 7 (NR-hydrogen fumarate crystal form B), and NR succinate crystal. Weigh 2.0 g of each sample respectively, and isolate air with 2 layers of PE bags (the first layer tied with a strap, the second layer evacuated) plus an aluminum foil bag (evacuated), and place them in a constant temperature and humidity stability chamber with temperatures and humidities of 25°C, 60% RH and 40°C, 75% RH respectively. During testing, take one package of sample each time, and conduct tests on appearance, pH, moisture, and HPLC sample purity and content (based on dry basis).

[0144] 1. Stability of NR-chloride crystal form II

[0145] Table 6 shows the purity change of NR-chloride crystal form II under the conditions of temperature 25°C and humidity 60% RH

[0146] Table 6

[0147]

[0148] Table 7 shows the purity change of NR-chloride crystal form II under the conditions of temperature 40°C and humidity 75% RH

[0149] Table 7

[0150]

[0151] Note: Stop detection when the purity is lower than 99%

[0152] 2. Stability of NR-hydrogen fumarate crystal form B

[0153] Table 8 shows the purity change of NR hydrogen fumarate crystal form B under the conditions of temperature 25°C and humidity 60% RH.

[0154] Table 8

[0155]

[0156] Table 9 shows the purity change of NR hydrogen fumarate crystal form B under the conditions of temperature 40°C and humidity 75% RH.

[0157] Table 9

[0158]

[0159] 3. Stability of NR succinate crystals

[0160] Table 10 shows the purity change of NR succinate crystals under the conditions of temperature 25°C and humidity 60% RH.

[0161] Table 10

[0162]

[0163] Table 11 shows the purity change of NR succinate crystals under the conditions of temperature 40°C and humidity 75% RH.

[0164] Table 11

[0165]

[0166] 4. Stability of NR hydrogen fumarate crystal form A

[0167] Table 12 shows the purity change of NR hydrogen fumarate crystal form A under the conditions of temperature 25°C and humidity 60% RH.

[0168] Table 12

[0169]

[0170]

[0171] Table 13 shows the purity change of NR hydrogen fumarate crystal form A under the conditions of temperature 40°C and 75% RH.

[0172] Table 13

[0173]

[0174] Combined with Tables 6 - 13, NR-chloride crystal form II has good stability at 25°C and humidity 60% RH, and the purity is still higher than 99% after 6 months; its stability is slightly worse at 40°C / humidity 75% RH, and the purity drops below 99% after 15 days.

[0175] The stability of NR hydrogen fumarate polymorph B and polymorph A is good at 25°C and 60% RH, and the purity is still higher than 99% after 6 months. The stability is good under the condition of 40°C / 75% RH, and it is still higher than 99% after 15 days.

[0176] The stability of NR succinate crystals is slightly poor at 25°C and 60% RH, and the purity drops below 99% after 3 months; the stability is the worst under the condition of 40°C / 75% RH, and the purity drops below 99% after 5 days.

[0177] In summary, through long-term stability experiments, it can be seen that the stability of NR hydrogen fumarate polymorph B and NR hydrogen fumarate polymorph A is the best among the four.

[0178] In summary, the applicant conducted short-term and long-term stability experiments. The main difference lies in the sample dosage. When the sample dosage is very small, its influence by air and moisture will be relatively large, so there are differences in the purity data.

[0179] Example 13 Grinding Stability

[0180] Take appropriate amounts of the NR-hydrogen fumarate polymorph B prepared in Example 7 and the NR-hydrogen fumarate polymorph A solid prepared in Example 4 in an agate mortar, and grind each sample for 3 minutes with approximately the same force. XRPD tests are carried out before and after grinding. According to the test results, there are no obvious changes in the XRPD patterns before and after grinding, indicating that the polymorphs of NR-organic acid salts have strong grinding stability.

[0181] Example 14 Efficacy Results and Methods

[0182] 1. Cell Proliferation Experiment

[0183] HaCat cells were maintained in DMEM solution containing 10% FBS and 1% PenStrep, and the culture medium was changed every 2 - 3 days until the confluence reached 85%. The cells were digested and harvested using 0.25% trypsin solution, and the cells were diluted with DMEM medium containing 0.1% serum. The cells were seeded at 1E4 cells per well in a 96 - well transparent well plate and allowed to adhere and grow overnight. The next day, gradient - diluted crystalline form B of NR - hydrogen fumarate (prepared in Example 7) and crystalline form A of NR - hydrogen fumarate (prepared in Example 4), NR - succinate crystals (self - made, preparation method: first prepare anionic resin of succinate type, then pass NR chloride salt (purchased from Hunan Xin'an Biotechnology Co., Ltd., batch number NB240101B) through the anion resin, and after concentrating the column eluate, crystallize according to the volume ratio of substrate: water: ethanol = 1:3:10), crystalline form II of NR - chloride salt (purchased from Hunan Xin'an Biotechnology Co., Ltd., batch number NB240101B, crystallized according to the volume ratio of substrate: water: ethanol = 1:1:10), and NR - hydrogen malate crystals (Shandong Sihuan Pharmaceutical Co., Ltd., batch number 2024061101) were added. At the same time, cell well controls and blank control wells were set up, shaken and mixed evenly, and the cells were cultured for another 24 h. After 24 h, the culture medium supernatant was removed, and the 96 - well plate was rinsed gently with PBS, and the operation process was gentle and slow to prevent the cells from being washed away. 100 μl of the prepared CCK8 solution was added to each well, and the cells were incubated at 37 °C for 30 min - 1 h. The results were read under the 450 filter of the microplate reader, and the relative cell viability of different samples was calculated. The formula for calculating the relative cell viability is: (As - Ab) / (Ac - Ab)*100%, where As represents the absorbance of the sample well, Ab represents the absorbance of the blank well, and Ac represents the absorbance of the control well. As Figure 9 shown, both crystalline form B and crystalline form A of NR - hydrogen fumarate, NR - hydrogen malate crystals, and NR - succinate crystals significantly promoted the proliferation of HaCat cells, while the effect of crystalline form II of NR - chloride salt was slightly worse. Moreover, the crystalline form B and crystalline form A of NR - hydrogen fumarate in this application had more obvious advantages at lower dosages (0.078 mM and 0.0078 mM), being significantly superior to NR - hydrogen malate crystals, NR - succinate crystals, and crystalline form II of NR - chloride salt.

[0184] 2. Animal experiments

[0185] This experiment was a 7-day repeated-dose experiment to evaluate the effects of crystalline forms A and B of nicotinamide riboside (NR) hydrogen fumarate on NAD+ levels in the blood of male and female C57BL / 6 mice. All test mice underwent a 14-day adaptation period before the start of the experiment. The test samples in this experiment included crystalline form A of NR hydrogen fumarate prepared in Example 4, crystalline form B of NR hydrogen fumarate prepared in Example 7, an NR chloride solution, and nicotinamide mononucleotide (NMN). Each compound was administered to middle-aged mice (64 weeks old) at a dose of 1 mmol / kg / day or 0.33 mmol / kg / day for 7 consecutive days by orally administering an aqueous solution of the test sample. Blood samples were collected from the cheek on day 0 (baseline) and day 7 (after dosing). A positive control was set as young mice (10 weeks old), and blood samples were collected from the cheek on day 0 (without dosing). The blood samples were treated with cell lysis before testing. The internal standard compound used for analysis was purchased from MedChemExpress (MCE). The NAD+ concentration was quantitatively measured by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), as shown in Figure 10 .

[0186] From the above experiment, it was concluded that for the effect on the NAD+ content in the blood of mice, both the crystalline form A solution and the crystalline form B solution of NR-hydrogen fumarate were greater than the NR chloride solution and the NMN solution. Moreover, the crystalline forms B and A of NR-hydrogen fumarate in this application had more obvious advantages at a lower dosage (0.33 mmol / kg / da), being significantly superior to the NR chloride solution and nicotinamide mononucleotide (NMN).

[0187] The above embodiments merely illustrate the principles and effects of the present invention, rather than limiting the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. An organic acid salt of nicotinamide riboside, characterized in that In the organic acid salt of nicotinamide riboside, the organic acid is selected from acetic acid, citric acid or fumaric acid; and the molar ratio of nicotinamide riboside to the organic acid is 1:1-1.

5.

2. The organic acid salt of nicotinamide riboside according to claim 1, characterized in that The molar ratio of nicotinamide riboside to organic acid is 1:

1.

3. The organic acid salt of nicotinamide riboside according to claim 1 or 2, characterized in that The structural formula of the organic acid salt of nicotinamide riboside is: Among them, X - Selected from 4. The method for preparing an organic acid salt of nicotinamide riboside according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: 1) providing a plurality of resin columns connected in series, and filling each of the resin columns with an anion resin; 2) washing the anion resin with an organic acid salt solution, and then washing the anion resin with water until the pH of the flowing water is 7 to 8; 3) passing the nicotinamide ribose chloride aqueous solution through the anion resin, and then washing the anion resin with water, The eluates are collected and combined, and then freeze-dried to obtain the organic acid salt of nicotinamide riboside.

5. The method for preparing an organic acid salt of nicotinamide riboside according to claim 4, characterized in that: Also includes any one or more of the following features: A1) In the step 1), the anion resin is anion resin D301; A2) in step 2), the organic acid salt solution is selected from an aqueous solution of sodium acetate, an aqueous solution of monosodium citric acid or an aqueous solution of monosodium fumarate; A3) In step 2), the concentration of the organic acid salt solution is 3% to 5%; A4) In step 2), the amount of the organic acid salt solution is 380-420 mL; A5) in step 2), the organic acid salt solution is used to wash the anion resin at a flow rate of 95 to 105 mL / h; A6) In step 2), the water washing anion resin is used to wash the anion resin at a flow rate of 95 to 105 mL / h; A7) In step 2), the amount of water is 380-420 mL; A8) in step 3), the mass of the nicotinamide riboside chloride aqueous solution is 190-210 g; A9) In step 3), the mass concentration of the nicotinamide riboside chloride aqueous solution is 1% to 2%; A10) In step 3), the water washing anion resin is used to wash the anion resin at a flow rate of 95 to 105 mL / h; A11) In step 3), the amount of water is 190-210 mL; A12) In steps 2) and 3), the water is deionized water.

6. The crystalline form of an organic acid salt of nicotinamide riboside according to any one of claims 1 to 3, characterized in that The organic acid is fumaric acid, and the crystalline form of the organic acid salt of nicotinamide riboside is crystalline form A of nicotinamide riboside hydrogen fumarate; the crystalline form A has a characteristic peak at at least one of 17.59°±0.2°, 21.52°±0.2°, 22.31°±0.2°, 25.46°±0.2° or 29.17°±0.2° in 2θ value measured by X-ray powder diffraction.

7. The crystalline form of an organic acid salt of nicotinamide riboside according to claim 6, characterized in that: The crystalline form A has a characteristic peak at least one of 12.8°±0.2°, 17.46°±0.2°, 17.59°±0.2°, 21.52°±0.2°, 22.30°±0.2°, 23.36°±0.2°, 25.46°±0.2°, 26.04°±0.2°, 28.29°±0.2° or 29.17°±0.2° in 2θ value determined by X-ray powder diffraction.

8. The crystalline form of an organic acid salt of nicotinamide riboside according to claim 6, characterized in that: The 2θ values ​​of the crystalline form A determined by X-ray powder diffraction are 11.25°±0.2°, 12.8°±0.2°, 12.19°±0.2°, 14.08°±0.2°, 16.47°±0.2°, 17.46°±0.2°, 17.59°±0.2°, 20.50°±0.2°, 21.32°±0.2°, 21.52°±0.2°, 21.50°±0.2°, 22.31°±0.2°, 22.56° ±0.2°, 23.36°±0.2°, 24.50°±0.2°, 25.46°±0.2°, 25.7°±0.2°, 26.04°±0.2°, 26.19°±0.2°, 26.39°±0.2°, 27.30°±0.2°, 28.29°±0.2°, 29.17°±0.2°, 31.10°±0.2°, 35.54°±0.2° or 37.89°±0.2° has a characteristic peak.

9. The crystalline form of an organic acid salt of nicotinamide riboside according to claim 6, characterized in that: The 2θ values ​​of the crystalline form A determined by X-ray powder diffraction are 11.25°±0.2°, 12.19°±0.2°, 12.8°±0.2°, 13.80°±0.2°, 14.08°±0.2°, 15.66°±0.2°, 16.47°±0.2°, 17.46°±0.2°, 17.59°±0.2°, 18.51°±0.2°, 20.50°±0.2°, 21.32°±0.2°, 21.52°±0.2°, 21.95°±0.2°, 22.31° ±0.2°、22.56°±0.2°、23.36°±0.2°、24.50°±0.2°、24.65°±0.2°、25.11°±0.2°、25.46°±0.2°、25.7°±0.2°、26.04°±0.2°、26.19°±0.2°、26.39°±0.2°、27.09°±0.2°、27.30°±0.2°、27.54°±0.2°、27.66°±0.2°、28.01°±0.2°、28.29° ±0.2°、28.50°±0.2°、29.17°±0.2°、29.65°±0.2°、31.10°±0.2°、32.82°±0.2°、32.96°±0.2°、35.47°±0.2°、35.54°±0.2°、35.80°±0.2°、36.39°±0.2°、37.71°±0.2°、37.90°±0.2°、38.15°±0.2°、38.44°±0.2°、38.56°±0.2°、39.05° At least one of the following has a characteristic peak: 39.32°±0.2°, 41.57°±0.2°, 42.18°±0.2°, 43.81°±0.2°, 45.06°±0.2°, 45.23°±0.2° or 47.53°±0.2°.

10. The crystalline form of an organic acid salt of nicotinamide riboside according to claim 6, characterized in that: The crystalline form A has characteristic peaks at 2θ values ​​of 17.59°±0.2°, 21.52°±0.2°, 22.31°±0.2°, 25.46°±0.2° and 29.17°±0.2° as determined by X-ray powder diffraction; preferably, the crystalline form A has characteristic peaks at 2θ values ​​of 12.8°±0.2°, 17.46°±0.2°, 17.59°±0.2°, 21.52°±0.2°, 22.30°±0.2°, 23.36°±0.2°, 25.46°±0.2°, 26.04 ° ± 0.2°, 28.29° ± 0.2° and 29.17° ± 0.2°; more preferably, the crystalline form A has characteristic peaks at 11.25° ± 0.2°, 12.8° ± 0.2°, 12.19° ± 0.2°, 14.08° ± 0.2°, 16.47° ± 0.2°, 17.46° ± 0.2°, 17.59° ± 0.2°, 20.50° ± 0.2°, 21.32° ± 0.2°, 21.52° ± 0.2°, 22.31° ± 0.2°, 22.56° ±0.2°、23.36°±0.2°、24.50°±0.2°、25.46°±0.2°、25.7°±0.2°、26.04°±0.2°、26.19° ± 0.2°, 26.39°±0.2°, 27.30°±0.2°, 28.29°±0.2°, 29.17°±0.2°, 31.10°±0.2°, 35.54°±0.2° and 37.89°±0.2°; further preferably, the crystalline form A has characteristic peaks at 11.25°±0.2°, 12.19°±0.2°, 12.8°±0.2°, 13.80°±0.2°, 14.08°±0.2°, 15.66°±0.2°, 16.47°±0.2°, 17.46°±0.2°, 17.59°±0.2°, 18.51°±0.2°. ±0.2°、20.50°±0.2°、21.32°±0.2°、21.52°±0.2°、21.95°±0.2°、22.31°±0.2°、22.56°±0.2°、23.36°±0.2°、24.50°±0.2°、24.65°±0.2°、25.11°±0.2°、25.46°±0.2°、25.7°±0.2°、26.04°±0.2°、26.19°±0.2°、26.39°±0.2°、27.09° ±0.2°、27.30°±0.2°、27.54°±0.2°、27.66°±0.2°、28.01°±0.2°、28.29°±0.2°、28.50°±0.2°、29.17°±0.2°、29.65°±0.2°、31.10°±0.2°、32.82°±0.2°、32.96°±0.2°、35.47°±0.2°、35.54°±0.2°、35.80°±0.2°、36.39°±0.2°、37.71° There are characteristic peaks at 37.90°±0.2°, 38.15°±0.2°, 38.44°±0.2°, 38.56°±0.2°, 39.05°±0.2°, 39.32°±0.2°, 41.57°±0.2°, 42.18°±0.2°, 43.81°±0.2°, 45.06°±0.2°, 45.23°±0.2° and 47.53°±0.2°.

11. The crystalline form of an organic acid salt of nicotinamide riboside according to any one of claims 6 to 10, characterized in that: Also includes any one or more of the following features: B1) the crystalline form A is also measured by differential scanning calorimetry (DSC), and the crystalline form A exhibits an endothermic peak at least at 113-118° C. when measured by DSC; B2) The crystal form A decomposes at 165-200° C. as determined by DSC.

12. The crystalline form of an organic acid salt of nicotinamide riboside according to any one of claims 1 to 3, characterized in that: The organic acid is fumaric acid, and the crystalline form of the organic acid salt of nicotinamide riboside is crystalline form B of nicotinamide riboside hydrogen fumarate; the crystalline form B has a 2θ value selected from 13.57°±0.2°, 21.40°±0.2°, 22.64° as determined by X-ray powder diffraction. There is a characteristic peak at at least one of ±0.2° or 24.44°±0.2°.

13. The crystalline form of an organic acid salt of nicotinamide riboside according to claim 12, characterized in that: The crystal form B has a 2θ value selected from 13.42°±0.2°, 13.57°±0.2°, 19.15°±0.2°, 21.40° as determined by X-ray powder diffraction. There is a characteristic peak at at least one of 22.64°±0.2°, 24.44°±0.2°, 25.85°±0.2° or 29.47°±0.2°.

14. The crystalline form of an organic acid salt of nicotinamide riboside according to claim 12, characterized in that: The crystal form B has a 2θ value selected from 10.52°±0.2°, 10.63°±0.2°, 12.81°±0.2°, 13.42° as determined by X-ray powder diffraction. ±0.2°、13.57°±0.2°、13.78°±0.2°、15.19°±0.2°、15.48°±0.2°、17.64°±0.2°、17.80°±0.2°、17.97°±0.2°、19.15°±0.2°、19.86°±0.2°、21.02°±0.2°、21.40°±0.2°、22.64°±0.2°、23.05°±0.2°、24.44°±0.2°、24.85°±0.2°、25.85° ±0.2°、26.17°±0.2°、27.12°±0.2°、27.56°±0.2°、27.99°±0.2°、29.47°±0.2°、29.70°±0.2°、30.85°±0.2°、30.94°±0.2°、31.42°±0.2°、31.83°±0.2°、32.17°±0.2°、32.47°±0.2°、34.61°±0.2°、35.29°±0.2°、35.89°±0.2°、36.72° At least one of 37.48°±0.2°, 37.69°±0.2°, 38.96°±0.2°, 40.45°±0.2°, 45.73°±0.2°, 46.29°±0.2° or 46.45°±0.2° has a characteristic peak.

15. The crystalline form of an organic acid salt of nicotinamide riboside according to claim 12, characterized in that: The 2θ values ​​of the crystal form B measured by X-ray powder diffraction are 13.57°±0.2°, 21.40°±0.2°, 22.64°±0.2° and 24.44° ±0.2° has a characteristic peak; preferably, the crystalline form B has a 2θ value selected from 13.42° as determined by X-ray powder diffraction. ± 0.2°, 13.57°±0.2°, 19.15°±0.2°, 21.40°±0.2°, 22.64°±0.2°, 24.44°±0.2°, 25.85°±0.2° and 29.47°±0.2°; more preferably, the crystalline form B has characteristic peaks at 10.52°±0.2°, 10.63°±0.2°, 12.81°±0.2°, 13.42°±0.2°, 13.57°±0.2°, 13.78°±0.2°, 15.19°±0.2°, 15.48°±0.2°, 17.64°±0.2°, 17.80°±0.2°. ±0.2°、17.97°±0.2°、19.15°±0.2°、19.86°±0.2°、21.02°±0.2°、21.40°±0.2°、22.64°±0.2°、23.05°±0.2°、24.44°±0.2°、24.85°±0.2°、25.85°±0.2°、26.17°±0.2°、27.12°±0.2°、27.56°±0.2°、27.99°±0.2°、29.47°±0.2°、29.70° ±0.2°、30.85°±0.2°、30.94°±0.2°、31.42°±0.2°、31.83°±0.2°、32.17°±0.2°、32.47°±0.2°、34.61°±0.2°、35.29°±0.2°、35.89°±0.2°、36.72°±0.2°、37.48°±0.2°、37.69°±0.2°、38.96°±0.2°、40.45°±0.2°、45.73°±0.2°、46.29° There are characteristic peaks at ±0.2° and 46.45°±0.2°.

16. The crystalline form of an organic acid salt of nicotinamide riboside according to any one of claims 12 to 15, characterized in that: Also includes any one or more of the following features: C1) the crystalline form B is also measured by differential scanning calorimetry (DSC), and the crystalline form B exhibits an endothermic peak at least at 110-117° C. when measured by DSC; C2) The crystal form B decomposes at 165-185° C. as determined by DSC.

17. The method for preparing a crystalline form of an organic acid salt of nicotinamide riboside according to any one of claims 6 to 16, characterized in that: The preparation method comprises: dissolving an organic acid salt of nicotinamide riboside in water to obtain a clear solution, adding an organic solvent at room temperature, stirring at low temperature to obtain a white suspension, centrifuging, separating the solid from the liquid, and drying to obtain a crystalline form of the organic acid salt of nicotinamide riboside, wherein the organic acid salt of nicotinamide riboside is hydrogen fumarate of nicotinamide riboside.

18. The method for preparing the crystalline form of the organic acid salt of nicotinamide riboside according to claim 17, characterized in that: Includes any one or more of the following conditions: D1) when the organic solvent is selected from an alcohol solvent, obtaining a crystalline form A of an organic acid salt of nicotinamide riboside; D2) when the organic solvent is selected from one or more of acetone, acetonitrile and ethyl acetate, obtaining Form B of the organic acid salt of nicotinamide riboside; D3) the mass volume ratio of the nicotinamide riboside hydrogen fumarate to the water is 1.0-1.2 g:4.8-5.3 mL; D4) the mass volume ratio of the nicotinamide riboside hydrogen fumarate to the organic solvent is 1.0-1.2 g:13.3-14.7 mL; D5) the low temperature stirring temperature is 5 to 10°C; D6) the low temperature stirring time is 10 to 12 hours; D7) The drying temperature is 40-45°C.

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