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

Abstract

An organic acid salt of nicotinamide ribose, a crystal form, and a preparation method therefor. The organic acid is selected from acetic acid, citric acid or fumaric acid, and the molar ratio of the nicotinamide ribose to the organic acid is 1:1-1.5. The nicotinamide ribose hydrogen fumarate has two crystalline forms, i.e., crystal form A and crystal form B. A method for preparing an organic acid salt of nicotinamide ribose is provided. The two crystalline forms of the nicotinamide ribose hydrogen fumarate have good fluidity, low hygroscopicity, good stability, and an unchanged color, maintain the solid form, and have a low effective concentration.

Description

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

[0001] The present invention claims priority to the patent application filed on December 22, 2023, with application number 202311781327.2 and invention name “Organic acid salt, crystal form and preparation method of nicotinamide riboside”. Technical Field

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

[0003] Nicotinamide riboside (NR) is a derivative of vitamin B3 (also known as niacin). Numerous studies have shown that NR can enhance the body's metabolism, prevent stem cell aging, and maintain stem cell function. In liver cancer research, results show that dietary NR supplementation can prevent the development of liver cancer in mice and induce tumor regression, and no side effects have been found at high doses. In addition, β-nicotinamide riboside (β-NR) is phosphorylated to produce β-nicotinamide mononucleotide (NMN), which is a synthetic substrate for coenzyme I in organisms. Studies have shown that β-NMN has anti-aging effects, regulates insulin secretion, and affects mRNA expression levels. Therefore, β-NR and β-NMN have become hot compounds in the fields of drug development, regenerative medicine, and skin care, with huge market demand prospects.

[0004] The free form of nicotinamide riboside exists in a cationic form, which is actually unstable and needs to form an ion pair with an anion to be stable.

[0005] However, the currently reported NR salts exist in the form of chloride salts and other organic acid salts, all of which suffer from poor stability, requiring refrigeration, high hygroscopicity, poor flowability, difficulty in formulation development, and high cost. In addition to chloride salts, many organic acids also exist in the form of pharmaceutically unacceptable salts, which may be toxic or otherwise biologically intolerable. These issues generally limit their processing and application in downstream applications. Therefore, the development of NR organic acid salts that are more stable, safe, and cost-effective in terms of water and environmental stability is of great significance. Summary of the Invention

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

[0007] A first aspect of the present invention provides an organic acid salt of nicotinamide riboside, wherein the organic acid in the organic acid salt of nicotinamide riboside 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.

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

[0009] The second aspect of the present invention provides a method for preparing the organic acid salt of nicotinamide riboside according to the first aspect of the present invention, the preparation method comprising the following steps:

[0010] 1) providing a plurality of resin columns connected in series, each of which is filled with an anion resin;

[0011] 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;

[0012] 3) Passing the nicotinamide ribose chloride aqueous solution through an anionic resin, then washing the anionic resin with water, collecting and combining the eluate, and then freeze-drying to obtain an organic acid salt of nicotinamide ribose.

[0013] A third aspect of the present invention provides a crystalline form of an organic acid salt of nicotinamide riboside, wherein 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°, as determined by X-ray powder diffraction.

[0014] A fourth aspect of the present invention provides another crystalline form of an organic acid salt of nicotinamide riboside, wherein the organic acid is fumaric acid, and the crystalline form of the organic acid salt of nicotinamide riboside is Form B of nicotinamide riboside hydrogen fumarate; the Form B has a characteristic peak at at least one 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.

[0015] A fifth aspect of the present invention provides a method for preparing the crystalline form of the organic acid salt of nicotinamide ribose according to the third and fourth aspects of the present invention, the preparation method comprising: dissolving the organic acid salt of nicotinamide ribose 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 the liquid, and drying to obtain the crystalline form of the organic acid salt of nicotinamide ribose, wherein the organic acid salt of nicotinamide ribose is nicotinamide ribose hydrogen fumarate.

[0016] The present invention has the following beneficial effects: The present invention discloses an organic acid salt 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 methods for preparing nicotinamide riboside acetate, nicotinamide riboside dihydrogen citrate, and nicotinamide riboside hydrogen fumarate. The present invention also discloses two crystalline forms of nicotinamide riboside hydrogen fumarate, Form A and Form B. The two crystalline forms of nicotinamide riboside hydrogen fumarate have the following advantages: 1. Good flowability, as demonstrated by a high tap density (≥0.8 g / mL) and a small angle of repose (30-35°); 2. Good stability, with long-term stability and accelerated stability data indicating that Form B and Form A of NR hydrogen fumarate are currently the most stable among NR-related salt forms; and 3. High purity and content, with a purity of up to 99.5% and a content of 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 cost of use, and is more suitable for mass production and has a wider range of application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG1 shows the XRPD spectrum of nicotinamide riboside acetate according to Example 1 of the present invention.

[0018] FIG2 shows the XRPD spectrum of the dihydrogen citrate salt of nicotinamide riboside according to Example 2 of the present invention.

[0019] FIG3 shows the XRPD spectrum of the hydrogen fumarate salt of nicotinamide riboside according to Example 3 of the present invention.

[0020] FIG4 shows the XRPD spectrum of the nicotinamide riboside hydrogen fumarate salt Form A according to Example 4 of the present invention.

[0021] FIG5 shows the XRPD spectrum of the nicotinamide riboside hydrogen fumarate crystal form B according to Example 7 of the present invention.

[0022] FIG6 shows the DSC spectrum of the nicotinamide riboside hydrogen fumarate crystal form A according to Example 4 of the present invention.

[0023] FIG7 shows the DSC spectrum of the nicotinamide riboside hydrogen fumarate crystal form B according to Example 7 of the present invention.

[0024] FIG8 shows the NMR spectra of the nicotinamide riboside hydrogen fumarate crystal form A of Example 4 and the hydrogen fumarate crystal form B of Example 7 of the present invention.

[0025] FIG9 shows the results of the cell proliferation experiment in Example 14 of the present invention.

[0026] FIG10 shows the quantitative measurement results of NAD+ concentration by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) in Example 14 of the present invention. DETAILED DESCRIPTION

[0027] Hereinafter, embodiments of the specifically disclosed organic acid salt of nicotinamide riboside, its crystal form, and its preparation method will be described in detail.

[0028] " range " disclosed in the present application is limited in the form of lower limit and upper limit, and given range is limited by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of special range. The scope limited in this way can be to include end value or not include end value, and can be arbitrarily combined, that is, any lower limit can form a range with any upper limit combination. For example, if the scope of 60-120 and 80-110 is listed for specific parameters, it is understood that the scope of 60-110 and 80-120 is also expected. In addition, if the minimum range value 1 and 2 are listed, and if the maximum range value 3,4 and 5 are listed, then the following range can all be expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In this application, unless otherwise specified, the numerical range " ab " represents the abbreviation of any real number combination between a and b, wherein a and b are all real numbers. For example, a numerical range of "0-5" indicates that all real numbers between "0-5" are listed herein, and "0-5" is simply an abbreviation for these numerical combinations. Furthermore, when a parameter is expressed as an integer ≥ 2, this is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0029] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0030] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0031] After extensive exploratory experiments, the inventors of this application have obtained nicotinamide riboside acetate, nicotinamide riboside dihydrogen citrate, and nicotinamide riboside hydrogen fumarate, and provided corresponding preparation methods. In addition, the present invention also obtained two crystalline forms of nicotinamide riboside hydrogen fumarate, Form A and Form B. The two crystalline forms of nicotinamide riboside hydrogen fumarate have the following advantages: 1. Good fluidity, as demonstrated by a high tap density (≥0.8 g / mL) and a small angle of repose (30-35°); 2. Good stability. Long-term stability and accelerated stability data show that NR hydrogen fumarate Form B and Form A are currently the most stable among NR-related salt forms; 3. High purity and content, with a purity of up to 99.5% and a content of 99.0%. In addition, it has low hygroscopicity, does not change color, and maintains a solid form. The effective concentration is low, that is, it can produce excellent results at lower dosage levels or low concentrations, which can reduce the cost of use, is more suitable for mass production, and has a wider range of application scenarios. On this basis, the present application was completed.

[0032] Organic acid salts of nicotinamide riboside

[0033] In a first aspect, the present invention provides an organic acid salt of nicotinamide riboside, wherein the organic acid in the organic acid salt of nicotinamide riboside 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. Optionally, the molar ratio is 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 is 1:1-1.5. Optionally, the molar ratio is 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. For example, the molar ratio of nicotinamide riboside to acetate is 1:1-1.5. The molar ratio of nicotinamide riboside to citrate can be 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 citrate can be 1:1-1.5. The molar ratio of nicotinamide riboside to fumarate can be 1:1-1.5. The options are 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, the organic acid salt of nicotinamide riboside comprises an organic acid selected from acetic acid, citric acid, or fumaric acid; and 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 radical is 1. The molar ratio of nicotinamide riboside to citrate radical is 1:1. The molar ratio of nicotinamide riboside to fumarate radical is 1:1.

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

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

[0037] When the organic acid is fumaric acid, the organic acid salt of nicotinamide riboside is nicotinamide riboside hydrogen fumarate. Fumaric acid is the simplest unsaturated dicarboxylic acid. It was first discovered in Corydalis yanhusuo and is also found in various mushrooms and fresh beef. Fumaric acid is a commonly used medicinal acid radical and is also used in the food industry as an acidity regulator, acidifier, antioxidant, pickling enhancer, and spice. The European Commission, ESIS; IUCLID Dataset, Fumaric acid (110-17-8), p. 39 (2000 CD-ROM edition) reports that 75 hospitalized patients (42 women, 33 men, 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 changes in liver function. Therefore, fumaric acid is safe and well-suited as a NR acid radical.

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

[0039] Specifically: Nicotinamide riboside acetate is Nicotinamide riboside citrate is Nicotinamide riboside hydrogen fumarate is

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

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

[0042] 1) providing a plurality of resin columns connected in series, each of which is filled with an anion resin;

[0043] 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;

[0044] 3) Passing the nicotinamide ribose chloride aqueous solution through an anionic resin, then washing the anionic resin with water, collecting and combining the eluate, and then freeze-drying to obtain an organic acid salt of nicotinamide ribose.

[0045] In the method for preparing an organic acid salt of nicotinamide riboside provided by the present invention, step 1) is to provide multiple resin columns connected in series, and each of the resin columns is filled with an anionic resin. Specifically:

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

[0047] In the method for preparing an organic acid salt of nicotinamide riboside provided by the present invention, step 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. 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 monosodium citric acid or an aqueous solution of monosodium fumarate.

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

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

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

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

[0053] In step 2) of the present invention, the amount of water used is 380-420 mL. Alternatively, the amount of water used is 380-400 mL or 400-420 mL.

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

[0055] In the method for preparing an organic acid salt of nicotinamide riboside provided by the present invention, step 3) is to pass the aqueous solution of nicotinamide riboside chloride through an anionic resin, then wash the anionic resin with water, collect and combine the eluate, and then freeze-dry to obtain the organic acid salt of nicotinamide riboside.

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

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

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

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

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

[0061] Crystalline form of an organic acid salt of nicotinamide riboside (crystalline form A of nicotinamide riboside hydrogen fumarate)

[0062] The present invention also provides a crystalline form of an organic acid salt of nicotinamide riboside, wherein 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; 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°, as measured by X-ray powder diffraction. "At least one" means including one, two, three, ..., or all of the above. The following explanation regarding at least one is the same as above and will not be repeated here.

[0063] In some embodiments of the present invention, the crystalline 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° as determined by X-ray powder diffraction.

[0064] In some embodiments of the present invention, the crystalline form A has a characteristic peak at at least one of 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 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 2θ values ​​of Form A as 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°, At least one of 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.

[0067] In some embodiments of the present invention, the 2θ values ​​of Form A as 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°, 22.31°±0.2°, 22.5 There are characteristic peaks at 6°±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°.

[0068] In some embodiments of the present invention, the 2θ values ​​of Form A determined by X-ray powder diffraction are 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 .At least one of 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° has a characteristic peak.

[0069] In some embodiments of the present invention, the 2θ values ​​of 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 There are characteristic peaks at 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°.

[0070] In some embodiments of the present invention, the crystalline form A is further 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.

[0071] In a preferred embodiment, the DSC spectrum of the crystalline form A exhibits an endothermic peak near 118° C., and the spectrum is substantially as represented in FIG6 .

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

[0073] Crystalline form of an organic acid salt of nicotinamide riboside (crystalline form B of nicotinamide riboside hydrogen fumarate)

[0074] The present invention also provides a crystalline form of an organic acid salt of nicotinamide riboside, wherein 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 characteristic peak at at least one 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.

[0075] In some embodiments of the present invention, the crystalline 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, the crystalline form B has a characteristic peak at at least one of 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.

[0077] In some embodiments of the present invention, the crystalline 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 2θ values ​​of the crystalline form B as determined by X-ray powder diffraction are 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 At least one of .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° has a characteristic peak.

[0079] In some embodiments of the present invention, the 2θ values ​​of the crystalline form B as determined by X-ray powder diffraction are 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°, 26.1 7°±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°.

[0080] In some embodiments of the present invention, the crystal form B is further measured by differential scanning calorimetry (DSC), and the crystal form B exhibits an endothermic peak at least at 110-117° C. when measured by DSC.

[0081] In a preferred embodiment, the DSC spectrum of Form B exhibits an endothermic peak near 117° C., and the spectrum is substantially as shown in FIG7 .

[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 crystalline form of organic acid salt 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 comprising: 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 the 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 nicotinamide riboside hydrogen fumarate.

[0085] In some embodiments of the present invention, when the organic solvent is selected from an alcohol solvent, the crystalline form A of the organic acid salt of nicotinamide riboside is obtained. Alternatively, when the organic solvent is selected from methanol and / or ethanol, the 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, Form B of an organic acid salt of nicotinamide riboside is obtained. Alternatively, when the organic solvent is selected from one of acetone, acetonitrile, and ethyl acetate, Form B of an organic acid salt of nicotinamide riboside is obtained.

[0087] In some embodiments of the present invention, the mass-to-volume ratio of the nicotinamide riboside hydrogen fumarate to the water is 1.0-1.2 g:4.8-5.3 mL. Alternatively, the mass-to-volume ratio of the nicotinamide riboside hydrogen fumarate 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.

[0088] In some embodiments of the present invention, the mass-to-volume ratio of the nicotinamide riboside hydrogen fumarate to the organic solvent is 1.0-1.2 g:13.3-14.7 mL. Alternatively, the mass-to-volume ratio of the nicotinamide riboside hydrogen fumarate 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.

[0089] In some embodiments of the present invention, the low-temperature stirring temperature is 5-10° C. Alternatively, 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 to 12 hours. Alternatively, the low-temperature stirring time is 10 to 11 hours or 11 to 12 hours.

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

[0092] Specific embodiments of the present invention are described with reference to the following examples, which are intended to illustrate the present invention and are not intended to limit the present invention in any way. The raw materials described are commercially available unless otherwise specified. The instruments used in the practice of this application are, unless otherwise specified, conventional instruments commonly used in the art.

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

[0094] DSC (Differential Scanning Calorimetry) spectrum measurement method: samples were collected on a DSC-25 instrument produced by TA Company, USA; the scanning speed was 10°C / min; the protective gas was nitrogen, and the nitrogen flow rate was 50 mL / min; the temperature range was room temperature to 330°C.

[0095] Example 1: Preparation of Nicotinamide Riboside Acetate (Molar Ratio of Nicotinamide Riboside to Acetate in the Product is 1:1)

[0096] 200g of anionic resin D301 was loaded into two 100mL resin columns, which were connected in series. The resin was washed with a 3% mass concentration sodium acetate aqueous solution at a flow rate of 100mL / h, consuming a total of 400mL. The resin was then washed with deionized water at a flow rate of 100mL / h until the effluent pH reached 7-8, consuming a total of 400mL of deionized water. 200g of a 1% mass concentration nicotinamide riboside chloride aqueous solution was passed through the resin at a flow rate of 100mL / h, and the resin was then washed with deionized water at a flow rate of 100mL / h, consuming 200g of deionized water. The eluate was collected based on the HPLC results. The eluates were combined and lyophilized to obtain a powdered solid of nicotinamide riboside acetate. The XRPD spectrum of the nicotinamide riboside acetate is shown in Figure 1.

[0097] Example 2: Preparation of Nicotinamide Riboside Dihydrogen Citrate (Molar Ratio of Nicotinamide Riboside to Citrate in the Product is 1:1)

[0098] 200 g of anionic resin D301 was loaded into two 100 mL resin columns, which were connected in series. The resin was washed with a 3% aqueous solution of monosodium citrate at a flow rate of 100 mL / h, consuming a total of 400 mL. The resin was then washed with deionized water at a flow rate of 100 mL / h until the effluent pH reached 7-8, consuming a total of 400 mL of deionized water. 200 g of a 1% aqueous solution of nicotinamide riboside chloride was passed through the resin at a flow rate of 100 mL / h, and the resin was then washed with deionized water at a flow rate of 100 mL / h, consuming 200 g of deionized water. The eluate was collected based on the HPLC results. The eluates were combined and lyophilized to obtain a powdered solid of nicotinamide riboside dihydrogen citrate. The XRPD spectrum of the nicotinamide riboside dihydrogen citrate is shown in Figure 2.

[0099] Example 3: Preparation of Nicotinamide Riboside Fumarate (Molar Ratio of Nicotinamide Riboside to Fumarate in the Product is 1:1)

[0100] 200 g of anionic resin D301 was loaded into two 100 mL resin columns, which were connected in series. The resin was washed with a 3% aqueous solution of monosodium fumarate at a flow rate of 100 mL / h, consuming a total of 400 mL. The resin was then washed with deionized water at a flow rate of 100 mL / h until the effluent pH reached 7-8, consuming a total of 400 mL of deionized water. 200 g of a 1% aqueous solution of nicotinamide riboside chloride was passed through the resin at a flow rate of 100 mL / h, and the resin was then washed with deionized water at a flow rate of 100 mL / h, consuming 200 g of deionized water. The eluate was collected based on the HPLC results. The eluates were combined and lyophilized to obtain a powdered solid of nicotinamide riboside hydrogen fumarate. The XRPD spectrum of the nicotinamide riboside hydrogen fumarate is shown in Figure 3.

[0101] Example 4: Preparation of Form A of Nicotinamide Riboside Fumarate

[0102] 100 mg of the powdered solid (amorphous solid of hydrogen fumarate) of nicotinamide riboside fumarate prepared in Example 3 above was placed in a 3 mL glass vial, 0.5 mL of pure water was added, and a polytetrafluoroethylene stirrer was added and stirred at 35°C for 1 hour to obtain a clear solution. 1.4 mL of methanol was slowly added dropwise at room temperature, and the mixture was then transferred to a biochemical incubator at 5°C and stirred at low temperature for 10 hours to obtain a white suspension. The suspension was transferred to a centrifuge tube and centrifuged. After solid-liquid separation, the solid was vacuum-dried at 40°C for 4 hours to obtain a solid. XRPD analysis revealed that the solid was nicotinamide riboside hydrogen fumarate Form A. The XRPD pattern of the nicotinamide riboside hydrogen fumarate Form A is substantially as shown in Figure 4. The DSC spectrum of the nicotinamide riboside hydrogen fumarate Form A exhibits an endothermic peak near 118°C, and the spectrum is substantially as shown in Figure 6. The NMR spectrum of the nicotinamide riboside hydrogen fumarate Form A is shown in Figure 8.

[0103] Using Cu-Ka radiation, the X-ray powder diffraction data of the nicotinamide riboside hydrogen fumarate salt form A at 2theta values ​​are shown in Table 1:

[0104] Table 1

[0105] Example 5: Preparation of Form A of Nicotinamide Riboside Fumarate

[0106] 100 mg of the powdered solid of nicotinamide riboside fumarate (amorphous solid of hydrogen fumarate) prepared in Example 3 was placed in a 3 mL glass vial, 0.5 mL of pure water was added, and a polytetrafluoroethylene stirrer was added, and the mixture was stirred at 35° C. for 1 hour to obtain a clear solution. 1.4 mL of ethanol was slowly added dropwise at room temperature, and the mixture was then transferred to a biochemical incubator at 5° C. and stirred for 10 hours to obtain a white suspension. The suspension was transferred to a centrifuge tube and centrifuged. After solid-liquid separation, the solid was vacuum dried at 40° C. for 4 hours to obtain a solid. XRPD analysis showed that the suspension was nicotinamide riboside fumarate crystalline Form A.

[0107] Example 6: Preparation of Form A (1.0 kg) of Nicotinamide Riboside Fumarate

[0108] 1.0 kg of the powdered solid of nicotinamide riboside fumarate (amorphous solid of hydrogen fumarate) prepared in Example 3 was placed in a 20 L jacketed kettle, 5.0 L of pure water was added, and the temperature was raised to 35° C. and mechanically stirred for 1 hour to obtain a clear solution. 14 L of methanol was slowly added dropwise at room temperature, and the temperature was then lowered to 5° C. and stirred at low temperature for 10 hours to obtain a white suspension. The suspension was filtered, and after solid-liquid separation, the solid was vacuum dried at 40° C. for 16 hours to obtain a solid. XRPD analysis showed that the suspension was nicotinamide riboside fumarate crystalline Form A.

[0109] Example 7: Preparation of Form B of Nicotinamide Riboside Fumarate

[0110] 100 mg of the powdered solid of nicotinamide riboside fumarate (amorphous solid of hydrogen fumarate) prepared in Example 3 above was placed in a 3 mL glass vial, 0.5 mL of pure water was added, and a polytetrafluoroethylene stirrer was added and stirred at 35°C for 1 hour to obtain a clear solution. 1.2 mL of acetone was slowly added dropwise at room temperature, and then the solution was transferred to a biochemical incubator at 5°C and stirred at low temperature for 10 hours to obtain a white suspension. The suspension was transferred to a centrifuge tube and centrifuged. After solid-liquid separation, the solid was vacuum-dried at 40°C for 4 hours to obtain a solid. XRPD analysis showed that the solid was Form B. The XRPD pattern of Form B of the hydrogen fumarate of nicotinamide riboside is substantially as shown in Figure 5. The DSC spectrum of Form B of the hydrogen fumarate of nicotinamide riboside exhibits an endothermic peak near 117°C, and the spectrum is substantially as shown in Figure 7. The NMR spectrum of Form B of the hydrogen fumarate of nicotinamide riboside is shown in Figure 8.

[0111] Using Cu-Ka radiation, the X-ray powder diffraction data of the nicotinamide riboside hydrogen fumarate salt form B at 2theta values ​​are shown in Table 2:

[0112] Table 2

[0113] Example 8: Preparation of Form B of Nicotinamide Riboside Fumarate

[0114] 100 mg of the powdered solid of nicotinamide riboside hydrogen fumarate (amorphous solid of hydrogen fumarate) prepared in Example 3 was placed in a 3 mL glass vial, 0.5 mL of pure water was added, and a polytetrafluoroethylene stirrer was added, and the mixture was stirred at 35° C. for 1 hour to obtain a clear solution. 1.2 mL of acetonitrile was slowly added dropwise at room temperature, and the mixture was then transferred to a biochemical incubator at 5° C. and stirred for 10 hours to obtain a white suspension. The suspension was transferred to a centrifuge tube and centrifuged. After solid-liquid separation, the solid was vacuum dried at 40° C. for 4 hours to obtain a solid. XRPD analysis showed that the suspension was nicotinamide riboside hydrogen fumarate Form B.

[0115] Example 9: Preparation of Form B of Nicotinamide Riboside Fumarate

[0116] 100 mg of the powdered solid of nicotinamide riboside hydrogen fumarate (amorphous solid of hydrogen fumarate) prepared in Example 3 was placed in a 3 mL glass vial, 0.5 mL of purified water was added, and a polytetrafluoroethylene stirrer was added, and the mixture was stirred at 35° C. for 1 hour to obtain a clear solution. 1.2 mL of ethyl acetate was slowly added dropwise at room temperature, and the mixture was then transferred to a biochemical incubator at 5° C. and stirred for 10 hours to obtain a white suspension. The suspension was transferred to a centrifuge tube and centrifuged. After solid-liquid separation, the solid was vacuum dried at 40° C. for 4 hours to obtain a solid. XRPD analysis showed that the suspension was nicotinamide riboside hydrogen fumarate Form B.

[0117] Example 10: Preparation of Form B (1.0 kg) of Nicotinamide Riboside Fumarate

[0118] 1.0 kg of the powdered solid of nicotinamide riboside hydrogen fumarate (amorphous solid of hydrogen fumarate) prepared in Example 3 was placed in a 20 L jacketed kettle, 5.0 L of pure water was added, and the temperature was raised to 35° C. and stirred for 1 hour to obtain a clear solution. 12 L of acetone was slowly added dropwise at room temperature, and the temperature was then lowered to 5° C. and stirred at low temperature for 10 hours to obtain a white suspension. The suspension was filtered, and after solid-liquid separation, the solid was vacuum dried at 40° C. for 16 hours to obtain a solid. XRPD analysis showed that the XRPD results were Form B.

[0119] Example 11: Fluidity Test

[0120] The fluidity indicators of the nicotinamide riboside fumarate form A (NR fumarate form A) prepared in Example 4, the nicotinamide riboside fumarate form B (NR fumarate form B) prepared in Example 7, the NR-chloride salt form II (purchased from Hunan Xin'an Biotechnology Co., Ltd., batch number NB240101B, crystallized in a substrate: water: ethanol = 1:1:10 volume ratio), and the NR succinate salt crystals (prepared in-house, the preparation method is: first prepare a succinate type anion resin, then pass the NR chloride salt purchased from Hunan Xin'an Biotechnology Co., Ltd., batch number NB240101B, through the anion resin, concentrate the column liquid, and then crystallize in a substrate: water: ethanol = 1:3:10 volume ratio) are as follows:

[0121] Mainly the tap density and angle of repose data:

[0122] Tap density test method: Accurately weigh approximately 20g of sample using an analytical balance and transfer it to a 50mL graduated cylinder. Place the cylinder in a tap density meter, set the Traptimes to 2400, the speed to 80, and press the "Start" button. After tapping is complete, read the sample volume and calculate the tap density.

[0123] Angle of repose detection method: Take about 20g of sample and slowly add it from the top of the funnel device. The material leaking out from the bottom of the funnel forms a conical accumulation on the horizontal material tray. When the horizontal material tray is full, use a protractor to measure the acute angle between the inclined surface of the sample accumulation and the horizontal surface at the bottom, which is the angle of repose.

[0124] Table 3

[0125] Example 12 Stability Test

[0126] (I) Short-term stability test method: The acetate prepared in Example 1 [NR-acetate (amorphous)], the nicotinamide riboside dihydrogen citrate [NR-dihydrogen citrate (amorphous)] of Example 2, the nicotinamide riboside hydrogen fumarate [NR-hydrogen fumarate (amorphous)] of Example 3, the crystalline form A of the nicotinamide riboside hydrogen fumarate [NR-hydrogen fumarate (crystalline form A)] of Example 4, and the crystalline form B of the nicotinamide riboside hydrogen fumarate [NR-hydrogen fumarate (crystalline form B)] of Example 7 were used. 30-50 mg of each sample was weighed and sealed with two layers of PE bags (the first layer was tied tightly with a cable tie, and the second layer was vacuumed) and an aluminum foil bag (vacuumed) to isolate the air. The bags were placed in a constant temperature and humidity chamber at 25°C, 60% RH and 40°C, 75% RH, respectively. During testing, one sample was taken at a time and the sample purity was tested by HPLC.

[0127] Table 4 shows the purity change of NR organic acid salt under the conditions of temperature 25℃ and humidity 60RH%

[0128] Table 4

[0129] Table 5 shows the purity change of NR organic acid salt under the conditions of temperature 40℃ and humidity 75RH%

[0130] Table 5

[0131] When placed at 25°C and 60% RH, the amorphous forms of NR-hydrogen fumarate, NR-dihydrogen citrate, and NR-acetate all decomposed. The purity of the two crystalline forms of NR-hydrogen fumarate remained essentially unchanged, with no significant decomposition, indicating the greater stability of the crystalline form. At 40°C and 75% RH, the amorphous form decomposed more significantly, while the crystalline form decomposed relatively slowly.

[0132] (II) Long-term stability data [NR-chloride salt crystal form II, nicotinamide riboside hydrogen fumarate crystal form A of Example 4 (NR-hydrogen fumarate crystal form A), nicotinamide riboside hydrogen fumarate crystal form B of Example 7 (NR hydrogen fumarate crystal form B), NR succinate crystal]

[0133] Because the sample size in the aforementioned short-term stability experiment was relatively small (30-50 mg), the results were easily affected by moisture and oxygen in the air. Therefore, long-term stability experiments were conducted using 2.0 g per package. Among the samples tested, NR-chloride salt Form II was purchased from Hunan Xin'an Biotechnology Co., Ltd., batch number NB240101B, and crystallized using a substrate: water: ethanol ratio of 1:1:10 by volume. NR succinate crystals were prepared in-house by first preparing a succinate-type anion resin. NR chloride salt, purchased from Hunan Xin'an Biotechnology Co., Ltd., batch number NB240101B, was then passed through the anion resin. The column fluid was concentrated and then crystallized using a substrate: water: ethanol ratio of 1:3:10 by volume.

[0134] Testing method: NR-chloride salt Form II, nicotinamide riboside fumarate salt Form A (NR-fumarate salt Form A) of Example 4, nicotinamide riboside fumarate salt Form B (NR-fumarate salt Form B) of Example 7, and NR succinate salt crystals were used. 2.0 g of each sample was weighed and sealed with two layers of PE bags (the first layer was tied tightly with a cable tie, and the second layer was vacuumed) and an aluminum foil bag (vacuumed). The bags were then placed in a constant temperature and humidity chamber at 25°C, 60% RH and 40°C, 75% RH, respectively. During testing, one sample was taken at a time and tested for appearance, pH, moisture, and HPLC sample purity and content (dry basis).

[0135] 1. Stability of NR-chloride salt form II

[0136] Table 6 shows the purity change of NR-chloride salt form II under the conditions of temperature 25°C and humidity 60RH%

[0137] Table 6

[0138] Table 7 shows the purity change of NR-chloride salt form II under the conditions of temperature 40°C and humidity 75% RH%

[0139] Table 7

[0140] Note: Stop testing when purity is lower than 99%

[0141] 2. Stability of NR hydrogen fumarate crystal form B

[0142] Table 8 shows the purity change of NR hydrogen fumarate crystal form B under the conditions of temperature 25°C and humidity 60RH%

[0143] Table 8

[0144] Table 9 shows the purity change of NR hydrogen fumarate crystal form B under the conditions of temperature 40°C and humidity 75% RH%

[0145] Table 9

[0146] 3. NR succinate crystal stability

[0147] Table 10 shows the purity change of NR succinate crystals under the conditions of temperature 25°C and humidity 60RH%

[0148] Table 10

[0149] Table 11 shows the purity change of NR succinate crystals under the conditions of temperature 40°C and humidity 75% RH%

[0150] Table 11

[0151] 4. Stability of NR hydrogen fumarate crystal form A

[0152] Table 12 shows the purity change of NR hydrogen fumarate crystal form A under the conditions of temperature 25°C and humidity 60RH%

[0153] Table 12

[0154] Table 13 shows the purity change of NR hydrogen fumarate crystal form A under the conditions of temperature 40°C and 75% RH%

[0155] Table 13

[0156] Combined with Tables 6 to 13, NR-chloride salt form II has good stability at 25°C and humidity 60RH%, and the purity is still higher than 99% after 6 months; the stability is slightly worse at 40°C / humidity 75RH%, and the purity drops to below 99% after 15 days.

[0157] NR hydrogen fumarate crystal form B and crystal form A showed good stability at 25°C and 60% humidity, with purity still above 99% after 6 months. They showed good stability at 40°C and 75% humidity, with purity still above 99% after 15 days.

[0158] The stability of NR succinate crystals was slightly poor at 25°C and 60RH%, with the purity dropping below 99% after 3 months. The stability was the worst at 40°C and 75RH%, with the purity dropping below 99% after 5 days.

[0159] In summary, through long-term stability experiments, it can be seen that the stability of NR hydrogen fumarate salt form B and NR hydrogen fumarate salt form A is the best among the four types.

[0160] In summary, the difference between the short-term stability and long-term stability experiments conducted by the applicant lies mainly in the sample dosage. When the sample dosage is very small, it will be greatly affected by air and moisture, thus causing differences in purity data.

[0161] Example 13 Grinding stability

[0162] Appropriate amounts of NR-fumarate form B prepared in Example 7 and NR-fumarate form A prepared in Example 4 were respectively placed in an agate mortar, and each sample was ground for 3 minutes with approximately the same force. XRPD tests were performed before and after grinding. According to the test results, there was no obvious change in the XRPD spectrum before and after grinding, which indicates that the NR-organic acid salt crystal form has strong grinding stability.

[0163] Example 14 Efficacy Results and Methods

[0164] 1. Cell proliferation assay

[0165] HaCat cells were maintained in DMEM with 10% FBS and 1% PenStrep, with medium changes every 2-3 days until confluence reached 85%. Cells were harvested using 0.25% trypsin solution and diluted with DMEM supplemented with 0.1% serum. 1E4 cells were seeded per well in a 96-well clear plate and allowed to adhere and grow overnight. The next day, gradient diluted NR-fumarate crystal form B (prepared in Example 7) and NR-fumarate crystal form A (prepared in Example 4) were added, NR-succinate crystals (self-made, preparation method: first prepare a succinate type anion resin, and 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 liquid, crystallize according to the volume ratio of substrate: water: ethanol = 1:3:10), NR-chloride crystal form II (purchased from Hunan Xin'an Biotechnology Co., Ltd., batch number NB240101B, crystallize according to the volume ratio of substrate: water: ethanol = 1:1:10), NR-malate crystals (Shandong Sihuan Pharmaceutical Co., Ltd., batch number 2024061101), and set up cell well control and blank control wells at the same time. Shake and mix, and continue to culture for 24 hours. After 24 hours, remove the culture supernatant and rinse the 96-well plate with PBS, gently and slowly to prevent washing away the cells. Add 100 μl of the prepared CCK8 solution to each well and incubate at 37°C for 30 min-1 h. Read the results using a 450 filter on a microplate reader, and calculate the relative cell viability of each sample. The formula for calculating 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 shown in Figure 9, Forms B and A of NR-fumarate, NR-malate, and NR-succinate all significantly promoted HaCat cell proliferation, while Form II of NR-chloride exhibited a slightly less potent effect. Moreover, the NR-fumarate crystal form B and crystal form A of the present application have more obvious advantages when used in smaller amounts (0.078mM and 0.0078mM), and are significantly better than NR-malate crystals, NR-succinate crystals and NR-chloride crystal form II.

[0166] 2. Animal experiments

[0167] This study was a 7-day repeated dosing study to evaluate the effects of Form A and Form 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 acclimatization period before the start of the experiment. The test samples in this experiment included Form A of NR hydrogen fumarate prepared in Example 4, Form B of NR hydrogen fumarate prepared in Example 7, NR chloride solution, and nicotinamide mononucleotide (NMN). For 7 consecutive days, each compound was orally administered as an aqueous solution of the test sample to middle-aged and elderly mice (64 weeks old) at a dose of 1 mmol / kg / day or 0.33 mmol / kg / day. Blood samples were collected from the cheek on day 0 (baseline) and day 7 (post-dose). A positive control was set up from young mice (10 weeks old), and blood samples were collected from the cheek on day 0 (no dosing). Blood samples were lysed before testing. The internal standard compounds used in the analysis were purchased from MedChemExpress (MCE). NAD+ concentrations were quantitatively measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), as shown in Figure 10.

[0168] The above experiments show that the effects of NR-fumarate crystal form A and crystal form B on NAD+ levels in mouse blood are greater than those of NR chloride solution and NMN solution. Furthermore, the advantages of NR-fumarate crystal form B and crystal form A of the present application are more pronounced at lower dosages (0.33 mmol / kg / da), significantly outperforming NR chloride solution and nicotinamide mononucleotide (NMN).

[0169] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to 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) The nicotinamide ribose chloride aqueous solution is passed through the anion resin, and then the anion resin is washed with water, and the eluate is collected and combined and then freeze-dried to obtain the organic acid salt of nicotinamide ribose.

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°±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° has a characteristic peak.

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° as determined by X-ray powder diffraction; more 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° as determined by X-ray powder diffraction; -X-ray powder diffraction determined that the 2θ values ​​were 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°, 21.5 2°±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°, 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°, 26.1 9°±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°、3 There are characteristic peaks at 8.44°±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 characteristic peak at at least one of the 2θ values ​​selected from 13.57°±0.2°, 21.40°±0.2°, 22.64°±0.2° or 24.44°±0.2° as determined by X-ray powder diffraction.

13. The crystalline form of an organic acid salt of nicotinamide riboside according to claim 12, characterized in that: The crystalline form B has a characteristic peak at at least one of the 2θ values ​​selected from 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.

14. The crystalline form of an organic acid salt of nicotinamide riboside according to claim 12, characterized in that: The crystalline form B has a 2θ value selected from 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°, 18.70°±0.2°, 18.80°±0.2°, 18.90°±0.2°, 19.10°±0.2°, 19.30°±0.2°, 19.70°±0.2°, 19.80°±0.2°, 18.9 ... ±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°, 34.6 At least one of 1°±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° has a characteristic peak.

15. The crystalline form of an organic acid salt of nicotinamide riboside according to claim 12, characterized in that: The crystalline 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; preferably, the crystalline form B has characteristic peaks at 2θ values ​​selected from 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° has a characteristic peak 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°, 17.97°±0.2°, 19.15°±0.2°, 19.20°±0.2°, 19.30°±0.2°, 19.40°±0.2°, 19.50°±0.2°, 19.60°±0.2°, 19.70°±0.2°, 19.80°±0.2°, 19.90°±0.2°, 19.80°±0.2°, 19.80°±0.2°, 19.90°±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°.

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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