Polymorphisms of reduced β-nicotinamide mononucleotide disodium salt, their preparation methods and uses

Abstract

The present invention relates to a compound NMNH disodium salt, and in particular to a polymorph of a reduced β-nicotinamide mononucleotide disodium salt and a preparation method thereof, and their use as pharmaceutical ingredients and cosmetic ingredients, as well as preparations containing these salts, belonging to the fields of medicine and cosmetics. Specifically, the present invention discloses a polymorph of NMNH disodium salt, wherein the crystal has excellent solubility, and is superior to the stability, flowability, anti-hygroscopicity and purification effect of amorphous. The crystal preparation process is simple, easy to control, and suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of chemical raw materials for medicine and cosmetics, and in particular to a polymorph of reduced β-nicotinamide mononucleotide disodium salt and a preparation method and use thereof. Background Art

[0002] As one of the most popular molecules in the field of anti-aging, nicotinamide adenine dinucleotide (NAD + ) has become the core of anti-aging substances in all generations. + It is an important coenzyme required for more than 500 enzymatic reactions and is well known for its role in oxidation and reduction (Ansari and Raghava, 2010; Rajman et al., 2018; Stein and Imai, 2012). More and more studies indicate that increasing NAD + Equivalent amounts can significantly improve multiple organ functions, including liver function, kidney function, cardiac function, and skeletal muscle function (Canto et al., 2012; Mills et al., 2016; Rajman et al., 2018). + It can be synthesized using tryptophan in the de novo biosynthesis pathway, nicotinic acid (NA) in the preiss-handler pathway, and nicotinamide (NAM), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN) in the salvage pathway (Canto et al., 2015; Chiarugi et al., 2012; Johnson and Imai, 2018). In particular, as NAD + The key intermediates, NAM, NR, and NMN, have been extensively studied for their potential therapeutic effects in many mouse disease models (Mills et al., 2016), among which NMN is currently considered the most suitable NAD + Precursor, and currently NMN is hot-selling in the global market and is highly favored by consumers.

[0003] NMNH (molecular structure as shown in formula (A)) is called "reduced nicotinamide mononucleotide" or "reduced β-nicotinamide mononucleotide" in Chinese. It is the reduced form of NMN and is a supplement to NAD. + A new precursor of NAD with better NMN properties + The promoting effect and other biological functions such as increasing cellular antioxidant capacity, reducing fat accumulation, reducing inflammatory response and inhibiting tumor cell growth are health-promoting agents with significant commercial potential (WO2021098725A1).

[0004]

[0005] The synthesis process of NMNH is still in the laboratory research and development stage in the market, and it cannot be realized in industrial production. The main technical difficulties are: 1) NMNH is the reduced form of NMN and is easily oxidized by air; 2) In the laboratory, only the method of preparative chromatography can obtain a high-purity aqueous solution, and the amorphous solid can only be obtained by freeze-drying. This method has no purification effect on the product. The amorphous solid obtained by freeze-drying is foamy, has poor fluidity, easily absorbs moisture and turns into oil, and degrades quickly; 3) There are no reports of polymorphs. It is well known that freeze-drying technology is not used in industry unless necessary, because freeze-drying requires high energy consumption and limited production capacity; amorphous solids have higher energy and are more unstable than crystalline solids.

[0006] Therefore, there is an urgent need in the art to develop new NMNH compounds and their polymorphs that have the advantages of good stability, good fluidity, and suitability for industrialization. Summary of the invention

[0007] The object of the present invention is to provide a novel NMNH compound and its polymorph having the advantages of good stability, good fluidity and suitability for industrialization, namely, a polymorph of NMNH disodium salt and a preparation method and use thereof.

[0008] The first aspect of the present invention provides a crystal of reduced β-nicotinamide mononucleotide disodium salt, wherein the crystal form is selected from the following group: crystal form A, crystal form B, or crystal form C.

[0009] In another preferred embodiment, the structure of the reduced β-nicotinamide mononucleotide disodium salt is as shown in Formula I:

[0010]

[0011] In another preferred embodiment, the crystal of the reduced β-nicotinamide mononucleotide disodium salt is a hydrate.

[0012] In another preferred embodiment, the structural formula of the reduced β-nicotinamide mononucleotide disodium salt is as follows:

[0013]

[0014] Wherein, n is ≥2.

[0015] In another preferred embodiment, n is an integer or a non-integer.

[0016] In another preferred embodiment, n is a positive integer ≥ 2, preferably 2-10, and more preferably 5-9.

[0017] In another preferred embodiment, the XRPD spectrum of the crystalline form A includes 3 or more 2θ values ​​selected from the following group: 12.7°±0.2°, 15.9±0.2°, 18.0°±0.2°, 20.4°±0.2°, 20.9°±0.2°, and 31.8°±0.2°.

[0018] In another preferred embodiment, the XRPD spectrum of the crystalline form A also includes one or more 2θ values ​​selected from the following group: 10.5°±0.2°, 19.8°±0.2°, 22.6°±0.2°, 24.0°±0.2°, 26.1°±0.2°, 28.7°±0.2°, 30.8°±0.2°, and 33.4°±0.2°.

[0019] In another preferred embodiment, the crystalline form A further has one or more characteristics selected from the following group:

[0020] 1) The XRPD spectrum of the crystalline form A comprises 6 or more 2θ values ​​selected from the group consisting of 5.0°±0.2°, 10.5°±0.2°, 12.7°±0.2°, 13.7°±0.2°, 14.9°±0.2°, 15.9°±0.2°, 16.1°±0.2°, 16.6°±0.2°, 18.0°±0.2°, 19.8°±0.2°, 20.4°±0.2°, 20.9°±0.2°, 22.6°±0.2°, 24.0°±0.2°, 24.7°±0.2°, 25.2°±0.2°, 25.7°±0.2°, 26.1°±0.2°, 27. 7.8°±0.2°, 28.7°±0.2°, 29.3°±0.2°, 30.3°±0.2°, 30.8°±0.2°, 31.8°±0.2°, 32.7°±0.2°, 33.4°±0.2°, 34.2°±0.2°, 35.8°±0.2°, 36.4°±0.2°, 37.4°±0.2°, 39.7°±0.2°, 41.2°±0.2°, 41.7°±0.2°, 42.6°±0.2°, 43.9°±0.2°, 44.3°±0.2°, 46.0°±0.2°, 46.4°±0.2°, 49.2°±0.2°;

[0021] 2) The XRPD pattern of the crystalline form A is substantially as follows Figure 1 represented;

[0022] 3) The TGA spectrum of the crystalline form A shows a weight loss of 19%-30% at 15°C-200°C;

[0023] 4) The TGA spectrum of the crystal form A is basically as follows Figure 2 represented;

[0024] 5) The DSC spectrum of the crystal form A has an endothermic peak in the range of 50°C-80°C;

[0025] 6) The DSC spectrum of the crystal form A is substantially as follows Figure 3 represented;

[0026] 7) The crystalline form A is a pentahydrate, a hexahydrate, a heptahydrate, an octahydrate, or a nonahydrate.

[0027] In another preferred embodiment, the crystalline form B has one or more characteristics selected from the following group:

[0028] 1) the XRPD spectrum of the crystalline form B comprises 3 or more 2θ values ​​selected from the following group: 12.0°±0.2°, 14.5°±0.2°, 15.3°±0.2°, 17.5°±0.2°, 19.9°±0.2°, 21.5°±0.2°;

[0029] 2) the XRPD spectrum of the crystalline form B further comprises one or more 2θ values ​​selected from the following group: 21.1°±0.2°, 23.1°±0.2°, 25.5°±0.2°;

[0030] 3) the XRPD pattern of the crystalline form B comprises 6 or more 2θ values ​​selected from the group consisting of 5.2°±0.2°, 7.7°±0.2°, 10.5°±0.2°, 11.5°±0.2°, 12.0°±0.2°, 12.6°±0.2°, 13.7°±0.2°, 14.5°±0.2°, 15.3°±0.2°, 16.5°±0.2°, 17.1°±0.2°, 17.5°±0.2°, 18.5°±0.2°, 19.4°±0.2°, 19.9°±0.2°, 21.1°±0.2°, 21.5°±0.2°, 22.5°±0.2°, 23.1°±0.2° .2°, 24.1°±0.2°, 24.7°±0.2°, 25.5°±0.2°, 26.4°±0.2°, 27.2°±0.2°, 27.7°±0.2°, 28.3°±0.2°, 29.2°±0.2°, 29.5°±0.2°, 31.2°±0.2°, 32.1°±0.2°, 32.6°±0.2°, 34.2°±0.2°, 35.1°±0.2°, 36.6°±0.2°, 38.3°±0.2°, 39.7°±0.2°, 41.4°±0.2°, 43.0°±0.2°, 45.1°±0.2°, 45.7°±0.2°;

[0031] 4) The XRPD pattern of the crystalline form B is substantially as follows Figure 4 represented;

[0032] 5) The TGA spectrum of the crystalline form B shows a weight loss of 12%-23% at 15°C-200°C;

[0033] 6) The TGA spectrum of the crystal form B is basically as follows Figure 5 represented;

[0034] 7) The DSC spectrum of the crystal form B has an endothermic peak in the range of 50°C-80°C;

[0035] 8) The DSC spectrum of the crystal form B is substantially as follows Figure 6 represented;

[0036] 9) The crystalline form B is a trihydrate, a tetrahydrate, a pentahydrate, or a hexahydrate.

[0037] In another preferred embodiment, the crystalline form C has one or more characteristics selected from the following group:

[0038] 1) The XRPD spectrum of the crystalline form C comprises 3 or more 2θ values ​​selected from the following group: 6.3°±0.2°, 15.3°±0.2°, 17.7°±0.2°, 19.9°±0.2°, 20.2°±0.2°, 21.5°±0.2°;

[0039] 2) the XRPD spectrum of the crystalline form C further comprises one or more 2θ values ​​selected from the group consisting of 6.3°±0.2°, 10.0°±0.2°, 12.1°±0.2°, 12.3°±0.2°, 12.8°±0.2°, 15.3°±0.2°, 16.6°±0.2°, 17.7°±0.2°, 19.9°±0.2°, 20.2°±0.2°, 21.5°±0.2°, 23.3°±0.2°, 24.9°±0.2°, 25.6°±0.2°, and 33.7°±0.2°;

[0040] 3) The XRPD pattern of the crystalline form C is substantially as follows Figure 7 represented;

[0041] 4) The TGA spectrum of the crystalline form C shows a weight loss of 8% to 16% at 15° C. to 200° C.;

[0042] 5) The TGA spectrum of the crystal form C is basically as follows Figure 8 represented;

[0043] 6) The DSC spectrum of the crystal form C has an endothermic peak in the range of 50°C-80°C;

[0044] 7) The DSC spectrum of the crystal form C is substantially as follows Fig. 9 represented;

[0045] 8) The crystalline form C is a dihydrate, a trihydrate, or a tetrahydrate.

[0046] The second aspect of the present invention provides a method for preparing a crystal of reduced β-nicotinamide mononucleotide disodium salt, the method comprising the following steps:

[0047] 1) adding reduced β-nicotinamide mononucleotide disodium salt into a first solvent to obtain a solution containing reduced β-nicotinamide mononucleotide disodium salt;

[0048] 2) under stirring conditions, dropwise adding the second solvent to crystallize to obtain crystals of reduced β-nicotinamide mononucleotide disodium salt; or under stirring conditions, purging with nitrogen to crystallize to obtain crystals of reduced β-nicotinamide mononucleotide disodium salt; or under stirring conditions, concentrating under reduced pressure to crystallize to obtain crystals of reduced β-nicotinamide mononucleotide disodium salt.

[0049] In another preferred embodiment, the method further comprises: step 3), drying the crystals.

[0050] In another preferred embodiment, the drying comprises: vacuum drying the obtained crystals for 2-30 hours.

[0051] In another preferred embodiment, the crystal is Form A.

[0052] In another preferred embodiment, when the crystals obtained in step 2) are form B and / or form C, step 2) further comprises a sub-step 2a), placing form B and / or form C in a gas containing moisture, thereby converting them into form A.

[0053] In another preferred embodiment, in step 2a), the crystalline form B and / or the crystalline form C are placed in the air to be converted into the crystalline form A.

[0054] The third aspect of the present invention provides a composition comprising: (a) any one of the crystals described in the first aspect, and (b) a pharmaceutically acceptable excipient or carrier, or a cosmetically acceptable excipient or carrier.

[0055] In another preferred embodiment, the composition is selected from the following group: a pharmaceutical composition and a cosmetic composition.

[0056] In another preferred embodiment, the pharmaceutical composition comprises: (a) any one of the crystals described in the first aspect, and (b) a pharmaceutically acceptable excipient or carrier.

[0057] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: oral preparations, injection dosage forms, respiratory tract administration dosage forms, skin administration dosage forms, mucosal administration dosage forms, cavity administration dosage forms, etc.

[0058] In another preferred embodiment, the cosmetic composition comprises: (a) any one of the crystals described in the first aspect, and (b) cosmetically acceptable excipients or carriers.

[0059] In another preferred embodiment, the cosmetic composition includes cosmetics selected from the following groups: skin cosmetics, hair cosmetics, beauty cosmetics, and special function cosmetics.

[0060] The fourth aspect of the present invention provides a use of a crystal for preparing medicine or cosmetics.

[0061] In another preferred embodiment, the drug is used to protect the optic nerve, improve retinal damage, prevent / treat hair loss, prevent / improve cardiovascular and cerebrovascular diseases, inhibit renal tubular damage and aging, prevent liver fibrosis, improve fatty liver disease, improve dry eye symptoms, repair kidney damage, prevent diabetes / nephropathy, improve sarcopenia symptoms in the elderly, treat chronic inflammation, alleviate the condition of patients with polycystic ovary syndrome, prevent / delay glaucoma, reduce neuroinflammation, reduce the cardiac toxicity of anthracycline chemotherapy drugs, assist in cerebral infarction recovery, prevent and treat heart failure in the elderly, etc.

[0062] In another preferred embodiment, the cosmetics are used to improve the function of damaged cells, improve skin quality / hair quality, prevent / treat skin photoaging, maintain skin softness and elasticity, delay skin aging, etc.

[0063] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The XRPD pattern of NMNH disodium salt Form A is shown.

[0065] Figure 2 The TGA spectrum of NMNH disodium salt Form A is shown.

[0066] Figure 3 The DSC spectrum of NMNH disodium salt Form A is shown.

[0067] Figure 4 The XRPD pattern of NMNH disodium salt Form B is shown.

[0068] Figure 5 The TGA spectrum of NMNH disodium salt Form B is shown.

[0069] Figure 6 The DSC spectrum of NMNH disodium salt Form B is shown.

[0070] Figure 7The XRPD pattern of NMNH disodium salt Form C is shown.

[0071] Figure 8 The TGA spectrum of NMNH disodium salt Form C is shown.

[0072] Fig. 9 The DSC spectrum of NMNH disodium salt Form C is shown.

[0073] Fig.10 The XRPD pattern of NMNH disodium salt amorphous form is shown.

[0074] Fig.11 The TGA spectrum of NMNH disodium salt amorphous form is shown.

[0075] Fig.12 The DSC spectrum of NMNH disodium salt amorphous form is shown.

[0076] Fig.13 The NMNH disodium salt form A is shown 1 H NMR spectrum.

[0077] Fig.14 The stability of the crystalline form A and the amorphous solid under the conditions of 25°C and 65% RH in the open air is shown.

[0078] Fig.15 The stability of the crystalline form A and the amorphous solid under the conditions of 4°C and 75% RH in the open air is shown. DETAILED DESCRIPTION

[0079] Through extensive and in-depth research, the inventors unexpectedly developed a specific salt of reduced NMNH for the first time, which is NMNH disodium salt. The present invention shows that the polymorphs of NMNH disodium salt (especially crystal form A) have excellent stability. Compared with its amorphous solid, the amorphous will change from solid to oily or viscous and agglomerate when placed, and degrade quickly. In addition, the polymorphs of the present invention have the advantages of high purity, good stability, good fluidity, low hygroscopicity, etc., and are suitable for pharmaceutical compositions, cosmetics, etc. On this basis, the inventors completed the present invention.

[0080] Terminology

[0081] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0082] As used herein, n in the term "nH2O" refers to all possible values ​​between 2 and 10, including integers and non-integers; "H2O" is the chemical formula of water, representing a water molecule or water.

[0083] As used herein, the term "about" when used in reference to a specific recited value means that the value may vary by no more than 1% from the specific value recited, for example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0084] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0085] As used herein, the term "n or more 2θ values ​​selected from the following group" refers to any positive integer including n and greater than n (e.g., n, n+1, ...), wherein the upper limit Nup is the number of all 2θ peaks in the group. For example, "3 or more" includes not only 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, ... each positive integer of the upper limit Nup, but also includes "4 or more", "5 or more", "6 or more", etc.

[0086] NMNH disodium salt

[0087] As used herein, the terms "reduced β-nicotinamide mononucleotide disodium salt", "β-dihydronicotinamide mononucleotide disodium salt", "dihydronicotinamide mononucleotide disodium salt", "reduced nicotinamide mononucleotide disodium salt", "reduced NMN disodium salt", and "NMNH-Na2" are used interchangeably and refer to a salt formed by reduced β-nicotinamide mononucleotide and two sodium ions, the structure of which is shown in Formula I. It should be understood that the term includes hydrates and anhydrates.

[0088]

[0089] In the present invention, the preferred reduced NMN disodium salt is a hydrate, and its structure is shown in formula (II):

[0090]

[0091] Polymorph

[0092] Solids exist in either amorphous or crystalline form. In the case of crystalline form, the molecules are positioned in a three-dimensional lattice. When a compound crystallizes from a solution or slurry, it can crystallize in different spatial lattice arrangements (a property known as "polymorphism"), forming crystals with different crystalline forms, which are known as "polymorphs". Different polymorphs of a given substance may differ from one another in one or more physical properties, such as solubility and dissolution rate, true specific gravity, crystal shape, packing mode, flowability and / or solid state stability.

[0093] Polymorphic forms of a compound can exhibit different melting points, hygroscopicity, stability, solubility, bioavailability, biological activity and flowability, which are important factors affecting drugability.

[0094] As used herein, "crystal", "crystal of the present invention" or "polymorph" are used interchangeably and refer to the crystal described in the first aspect of the present invention, whose crystal form is selected from the following group: Form A, Form B, or Form C.

[0095] crystallization

[0096] The solution can be manipulated so that the solubility limit of the target compound is exceeded, thereby completing production-scale crystallization. This can be accomplished in a variety of ways, for example, dissolving the compound at a relatively high temperature and then cooling the solution to below the saturation limit. Alternatively, the liquid volume can be reduced by boiling, atmospheric evaporation, vacuum drying, or by some other method. The solubility of the target compound can be reduced by adding an antisolvent or a solvent in which the compound has a low solubility or a mixture of such solvents. Another alternative is to adjust the pH value to reduce solubility. A detailed description of crystallization can be found in Crystallization, 3rd edition, JW Mullens, Butterworth-Heineman Ltd., 1993, ISBN0750611294.

[0097] If it is desired that salt formation and crystallization occur simultaneously, the addition of an appropriate acid or base may result in direct crystallization of the desired salt if the salt is less soluble in the reaction medium than the starting materials. Likewise, completion of the synthesis reaction in a medium in which the final desired form is less soluble than the reactants may result in direct crystallization of the final product.

[0098] Optimization of crystallization can include seeding the crystallization medium with crystals of the desired form. In addition, many crystallization methods use a combination of the above strategies. One implementation is to dissolve the target compound in a solvent at elevated temperature, followed by controlled addition of an appropriate volume of antisolvent to bring the system just below saturation levels. At this point, seeds of the desired form can be added (while maintaining the integrity of the seeds) and the system cooled to complete crystallization.

[0099] Solvate

[0100] During the contact between compound or drug molecules and solvent molecules, it is inevitable that the solvent molecules and compound molecules form a eutectic and remain in the solid material due to external and internal conditions. The substance formed after the compound and solvent crystallize is called a solvate. The types of solvents that easily form solvates with organic compounds include water, methanol, ethanol, benzene, ether, heterocyclic aromatic hydrocarbons, etc.

[0101] Hydrate

[0102] Hydrate is a special solvate. In the pharmaceutical industry, whether in the synthesis of raw materials, drug preparation, drug storage and drug activity evaluation, hydrate is worth discussing separately because of its particularity.

[0103] In the present invention, the crystal of the compound represented by formula (I) may be a non-solvate or a solvate; the crystal form A, crystal form B and crystal form C of the crystal of the compound represented by formula (I) are all hydrates.

[0104] Preparation method

[0105] When preparing NMNH disodium salt crystals, the present invention uses temperature controlled crystallization, reduced pressure concentrated crystallization, nitrogen purging crystallization, volatilization crystallization, dissolution crystallization, temperature controlled and humidity controlled crystallization, vacuum drying crystallization, etc. The method is simple and easy to implement and is easy to industrialize.

[0106] use

[0107] The present invention provides uses of NMNH disodium salt crystals (including crystal form A, crystal form B, and crystal form C): the crystals are highly effective and broad-spectrum, and can be used in pharmaceutical compositions, cosmetics, and the like.

[0108] The main advantages of the present invention are:

[0109] (1) The crystals of the compound of formula (I) of the present invention (including crystal form A, crystal form B, and crystal form C) have higher purity, better stability, better fluidity, and lower hygroscopicity than their amorphous solids.

[0110] (2) The preparation method of the compound of formula (I) crystals (including crystal form A, crystal form B, and crystal form C) of the present invention is simple and more suitable for industrial production compared with the freeze-drying process (which consumes a lot of energy and has limited production capacity).

[0111] (3) The crystals of the compound of formula (I) of the present invention (including crystal form A, crystal form B, and crystal form C) can be used in pharmaceutical compositions, cosmetics, and the like.

[0112] (4) The polymorph preparation method of the present invention is simple and suitable for industrial production.

[0113] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0114] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to the described content can be applied to the method of the present invention.

[0115] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial channels.

[0116] Test method:

[0117] XRPD (X-ray powder diffraction) pattern determination method: Bruker D2 Phaser X-ray powder diffractometer; radiation source Cu Generator kv: 30 kv; Generator mA: 10 mA; Starting 2θ: 2.000°; Scanning range: 2.0000-50.000°, Scanning step: 0.02°, Scanning speed: 0.1 s / step.

[0118] The measurement differences associated with such X-ray powder diffraction analysis results are caused by a variety of factors including: (a) errors in sample preparation (e.g., sample height), (b) instrument errors, (c) calibration differences, (d) operator errors (including errors that occur when determining peak positions), and (e) the nature of the material (e.g., preferred orientation errors). Calibration errors and sample height errors often result in displacements of all peaks in the same direction. When a flat support is used, small differences in sample height will result in large displacements of XRPD peak positions. Systematic studies have shown that sample height differences of 1 mm can result in peak displacements of up to 1° of 2θ. These displacements can be identified from the XRPD spectrum and can be eliminated by compensating for the displacements (applying the system calibration factor to all peak position values) or recalibrating the instrument. As described above, by applying the system calibration factor to make the peak positions consistent, measurement errors from different instruments can be corrected.

[0119] TGA (thermogravimetric analysis) spectrum determination method: TGA55 instrument from TA Company, USA; temperature range: 14.8~300℃; heating rate: 10℃ / min; nitrogen flow rate: 40mL / min.

[0120] DSC (differential scanning calorimetry) spectrum determination method: TA Q55 instrument from TA Company, USA; temperature range: 20-230°C, heating rate: 10°C / min, nitrogen flow rate: 50mL / min.

[0121] Moisture (KF) determination method: MC-2000 automatic trace moisture analyzer from MinCe Instrument Equipment (Xiamen) Co., Ltd., Karl Fischer reagent from Nanjing Chemical Reagent Co., Ltd.

[0122] Example 1. Preparation of Form A

[0123] Preparation of NMNH disodium salt: Add 179 mg MnCl2 to 2.85 liters of Tris-HCl (50 mM) solution, adjust the solution pH to 8.0, and control the temperature at 37°C. Add 143 mg NAD+ pyrophosphatase from E. coli (EcNADD) to the solution, add 10 g NADH, and stir for 2 hours. Separate the NMNH solution by preparative chromatography, adjust the solution pH to 10 with NaOH, and concentrate under reduced pressure to obtain 3.8 g NMNH disodium salt solid.

[0124] Weigh 500 mg of NMNH disodium salt, dissolve in 1 ml of water, purge with nitrogen at 20-40°C, filter out crystals, and dry the solid in a forced air oven. The obtained solid crystal form is the crystal form A of the compound of formula (I), and the water content (KF) is 27%.

[0125] The obtained solid was subjected to X-ray powder diffraction test, and the XRPD pattern of the obtained crystal form A was basically as follows: Figure 1 As shown, 1 HNMR spectrum Fig.13 As shown, the diffraction angle data are basically as shown in Table 1 below, where the error range of the 2θ value is ±0.2°.

[0126] Table 1 XRPD data of Form A

[0127]

[0128]

[0129] The TGA spectrum of Form A is basically as follows Figure 2 As shown, the weight loss is 19%-30% at 15°C-200°C.

[0130] The DSC spectrum of Form A is basically as follows Figure 3 As shown, there is an endothermic peak in the range of 50°C-80°C.

[0131] Example 2. Kilogram-level scale-up and purification effect of Form A

[0132] Preparation of NMNH disodium salt aqueous solution: Add 1.7 Kg β-NMN and 0.94 Kg Na2S2O4 to 10 liters of saturated sodium bicarbonate aqueous solution, stir at room temperature overnight, filter to obtain a clear solution, adjust the pH of the clear solution to 10 with NaOH, and obtain NMNH disodium salt aqueous solution (HPLC purity is 95.2%).

[0133] The temperature was controlled at 20-40°C, mechanically stirred, and concentrated under reduced pressure; after concentrating to remove part of the water, 10 g of the crystals (crystal form A) obtained in Example 1 was added, and the vacuum concentration was continued until 1.5-2 liters remained, and the concentration was stopped, the temperature was lowered to 0-10°C, filtered, and air-dried. 2.07 kg of NMNH disodium salt crystals were obtained, with a water content (KF) of 26% and a HPLC purity of 99.4%; the obtained crystals were crystal form A of the compound of formula (I).

[0134] Table 2 Purification effect of Form A

[0135]

[0136] It can be seen from Table 2 that Form A has a certain purification effect, while the amorphous solid obtained by freeze-drying has no purification effect.

[0137] Example 3. Preparation of Form B

[0138] Weigh 50 g of NMNH disodium salt crystals (crystal form A) obtained in Example 2 and vacuum dry for 2-4 hours. The obtained solid crystal form is crystal form B of the compound of formula (I), and the water content (KF) is 16%.

[0139] The obtained solid was subjected to X-ray powder diffraction test, and the XRPD pattern of the obtained crystal form B was basically as follows: Figure 4 As shown, the diffraction angle data are basically as shown in Table 3 below, where the error range of the 2θ value is ±0.2°.

[0140] Table 3 XRPD data of Form B

[0141]

[0142]

[0143] The TGA spectrum of Form B is basically as follows Figure 5 As shown, the weight loss is 12%-23% at 15°C-200°C.

[0144] The DSC spectrum of Form B is basically as follows Figure 6 As shown, there is an endothermic peak in the range of 50°C-80°C.

[0145] Example 4. Preparation of Form C

[0146] Weigh 20 g of NMNH disodium salt crystals (crystal form B) obtained in Example 3 and vacuum dry them for 10-20 hours. The obtained solid crystal form is crystal form C of the compound of formula (I), and the water content (KF) is 11%.

[0147] The obtained solid was subjected to X-ray powder diffraction test, and the XRPD pattern of the obtained crystal form C was basically as follows: Figure 7 As shown, the diffraction angle data are basically as shown in Table 4 below, where the error range of the 2θ value is ±0.2°.

[0148] Table 4 XRPD data of Form C

[0149]

[0150]

[0151] The TGA spectrum of Form C is basically as follows Figure 8 As shown, the weight loss is 8%-16% at 15°C-200°C.

[0152] The DSC spectrum of Form C is basically as follows Fig. 9 As shown, there is an endothermic peak in the range of 50°C-80°C.

[0153] Example 5. Crystalline Form B absorbs water from the air and transforms into Crystalline Form A

[0154] Weigh 1 g of NMNH disodium salt crystals (form B) obtained in Example 3, expose to air at 2-8°C with a relative humidity of 70-80%. After 30 days, the solid crystal form obtained is form A of the compound of formula (I) with a moisture content (KF) of 29%.

[0155] Example 6. Form C absorbs water from the air and transforms into Form A

[0156] Weigh 1 g of NMNH disodium salt crystals (form C) obtained in Example 4, expose to air at 2-8°C, with a relative humidity of 70-80%. After 24 hours, the solid crystal form obtained is form A of the compound of formula (I), with a moisture content (KF) of 24%.

[0157] Example 7. Preparation of amorphous solid of compound of formula (I)

[0158] 30 g of NMNH disodium salt crystals prepared in Example 2 were weighed and dissolved in 90 ml of water to obtain a clear solution, which was then frozen into a solid and then freeze-dried. The product obtained after freeze-drying for 24 hours was an amorphous solid, foamy, poorly fluid, and had a water content (KF) of 9%.

[0159] The obtained amorphous solid was subjected to X-ray powder diffraction test, and its XRPD pattern was basically as follows Fig.10 shown.

[0160] The TGA spectrum of amorphous solid is basically as follows Fig.11 As shown, the weight loss is 1%-15% at 15°C-200°C.

[0161] The DSC spectrum of amorphous solid is basically as follows Fig.12 As shown, there is an endothermic peak in the range of 50°C-80°C.

[0162] Example 8. Comparison of stability between crystalline form A and amorphous solid

[0163] (1) The product crystals (crystal form A) in Example 2 and the amorphous solid product in Example 7 were placed in a stability test box at 25° C. and 65% RH to examine their stability. The results are shown in Table 5 and Fig.14 data.

[0164] Table 5 Stability of Form A and Amorphous Solid (25°C, 65% RH)

[0165]

[0166] From Table 5 and Fig.14 It can be seen that after 5 days of storage, the purity of Form A decreased from 99.33% to 99.01% (still a crystalline powder); while after 1 day of storage, the purity of the amorphous solid decreased from 99.30% to 99.02% (the powder absorbed water and turned into an oily substance). It can be seen that the Form A solid of NMNH disodium salt is more stable than the amorphous solid.

[0167] (2) The product crystals (crystal form A) in Example 2 and the amorphous solid product in Example 7 were placed in a stability test box at 4°C and 75% RH to examine their stability. The results are shown in Table 6 and Fig.15 Chinese data.

[0168] Table 6 Stability of Form A and Amorphous Solid (4°C, 75% RH)

[0169]

[0170] From Table 6 and Fig.15 It can be seen that the purity of Form A after 18 days is 99.33%, which is almost unchanged (still a crystalline powder); while the purity of the amorphous solid dropped from 99.30% to 99.26% after 5 days (the powder solid absorbed water and became viscous and agglomerated), and on the 18th day the purity dropped to 99.17% (the color became darker). It can be seen that the Form A solid of NMNH disodium salt is more stable than the amorphous solid.

[0171] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A crystal of reduced β-nicotinamide mononucleotide disodium salt represented by formula (I), characterized in that: The crystal is a reduced β-nicotinamide mononucleotide disodium salt hydrate, and its crystal form is crystal form A, crystal form B, or crystal form C. Wherein, the XRPD spectrum of the crystalline form A includes 5 or more 2θ values ​​selected from the following group: 12.7°±0.2°, 15.9±0.2°, 18.0°±0.2°, 20.4°±0.2°, 20.9°±0.2°, 31.8°±0.2°; The XRPD pattern of the crystalline form B comprises 5 or more 2θ values ​​selected from the group consisting of 12.0°±0.2°, 14.5°±0.2°, 15.3°±0.2°, 17.5°±0.2°, 19.9°±0.2°, 21.5°±0.2°; and The XRPD spectrum of the crystalline form C includes 5 or more 2θ values ​​selected from the following group: 6.3°±0.2°, 15.3°±0.2°, 17.7°±0.2°, 19.9°±0.2°, 20.2°±0.2°, and 21.5°±0.2°.

2. The crystal of reduced β-nicotinamide mononucleotide disodium salt according to claim 1, characterized in that: The XRPD spectrum of the crystalline form A also includes one or more 2θ values ​​selected from the following group: 10.5°±0.2°, 19.8°±0.2°, 22.6°±0.2°, 24.0°±0.2°, 26.1°±0.2°, 28.7°±0.2°, 30.8°±0.2°, and 33.4°±0.2°.

3. The crystal of reduced β-nicotinamide mononucleotide disodium salt according to claim 1, characterized in that: The crystalline form A also has one or more characteristics selected from the following group: 1) The XRPD spectrum of the crystalline form A includes the following 2θ values: 5.0°±0.2°, 10.5°±0.2°, 12.7°±0.2°, 13.7°±0.2°, 14.9°±0.2°, 15.9°±0.2°, 16.1°±0.2°, 16.6°±0.2°, 18.0°±0.2°, 19.8°±0.2°, 20.4°±0.2°, 20.9°±0.2°, 22.6°±0.2°, 24.0°±0.2°, 24.7°±0.2°, 25.2°±0.2°, 25.7°±0.2°, 26.1°±0.2°, 27.8° ±0.2°, 28.7°±0.2°, 29.3°±0.2°, 30.3°±0.2°, 30.8°±0.2°, 31.8°±0.2°, 32.7°±0.2°, 33.4°±0.2°, 34.2°±0.2°, 35.8°±0.2°, 36.4°±0.2°, 37.4°±0.2°, 39.7°±0.2°, 41.2°±0.2°, 41.7°±0.2°, 42.6°±0.2°, 43.9°±0.2°, 44.3°±0.2°, 46.0°±0.2°, 46.4°±0.2°, 49.2°±0.2°; 2) The TGA spectrum of the crystalline form A shows a weight loss of 19%-30% at 15°C-200°C; 3) The DSC spectrum of the crystal form A has an endothermic peak in the range of 50°C-80°C.

4. The crystal of reduced β-nicotinamide mononucleotide disodium salt according to claim 1, characterized in that: The crystalline form A also has one or more characteristics selected from the following group: 1) The XRPD pattern of the crystalline form A is substantially as shown in FIG1 ; 2) The TGA spectrum of the crystalline form A is substantially as shown in FIG2 ; and 3) The DSC spectrum of the crystal form A is basically represented as shown in Figure 3.

5. The crystal of reduced β-nicotinamide mononucleotide disodium salt according to claim 1, characterized in that: The crystalline form B has one or more characteristics selected from the following group: 1) The XRPD spectrum of the crystalline form B further comprises one or more 2θ values ​​selected from the following group: 21.1°±0.2°, 23.1°±0.2°, 25.5°±0.2°; 2) The TGA spectrum of the crystalline form B shows a weight loss of 12%-23% at 15°C-200°C; 3) The DSC spectrum of the crystal form B has an endothermic peak in the range of 50°C-80°C.

6. The crystal of reduced β-nicotinamide mononucleotide disodium salt according to claim 1, characterized in that: The crystalline form B has one or more characteristics selected from the following group: 1) The XRPD pattern of the crystalline form B is substantially as shown in FIG4 ; 2) The TGA spectrum of the crystalline form B is substantially as shown in FIG5 ; and 3) The DSC spectrum of the crystalline form B is basically represented as shown in Figure 6.

7. The crystal of reduced β-nicotinamide mononucleotide disodium salt according to claim 1, characterized in that: The XRPD spectrum of the Form B includes the following group of 2θ values: 5.2°±0.2°, 7.7°±0.2°, 10.5°±0.2°, 11.5°±0.2°, 12.0°±0.2°, 12.6°±0.2°, 13.7°±0.2°, 14.5°±0.2°, 15.3°±0.2°, 16.5°±0.2°, 17.1°±0.2°, 17.5°±0.2°, 18.5°±0.2°, 19.4°±0.2°, 19.9°±0.2°, 21.1°±0.2°, 21.5°±0.2°, 22.5°±0.2°, 23.1°±0.2°, 24. : 4.1°±0.2°, 24.7°±0.2°, 25.5°±0.2°, 26.4°±0.2°, 27.2°±0.2°, 27.7°±0.2°, 28.3°±0.2°, 29.2°±0.2°, 29.5°±0.2°, 31.2°±0.2°, 32.1°±0.2°, 32.6°±0.2°, 34.2°±0.2°, 35.1°±0.2°, 36.6°±0.2°, 38.3°±0.2°, 39.7°±0.2°, 41.4°±0.2°, 43.0°±0.2°, 45.1°±0.2°, 45.7°±0.2°.

8. The crystal of reduced β-nicotinamide mononucleotide disodium salt according to claim 1, characterized in that: The crystalline form C has one or more characteristics selected from the following group: 1) the XRPD spectrum of the crystalline form C further comprises one or more 2θ values ​​selected from the group consisting of 6.3°±0.2°, 10.0°±0.2°, 12.1°±0.2°, 12.3°±0.2°, 12.8°±0.2°, 15.3°±0.2°, 16.6°±0.2°, 17.7°±0.2°, 19.9°±0.2°, 20.2°±0.2°, 21.5°±0.2°, 23.3°±0.2°, 24.9°±0.2°, 25.6°±0.2°, and 33.7°±0.2°; 2) The TGA spectrum of the crystal form C shows a weight loss of 8% to 16% at 15° C. to 200° C.; 3) The DSC spectrum of the crystal form C has an endothermic peak in the range of 50°C-80°C.

9. The crystal of reduced β-nicotinamide mononucleotide disodium salt according to claim 1, characterized in that: The crystalline form C has one or more characteristics selected from the following group: 1) The XRPD pattern of the crystalline form C is substantially as shown in FIG7 ; 2) The TGA spectrum of the crystalline form C is substantially as shown in FIG8 ; and 3) The DSC spectrum of the crystalline form C is basically represented as shown in Figure 9.

10. A method for preparing the crystal of reduced β-nicotinamide mononucleotide disodium salt as claimed in claim 1, characterized in that: The method comprises the following steps: 1) adding reduced β-nicotinamide mononucleotide disodium salt into water to obtain an aqueous solution containing reduced β-nicotinamide mononucleotide disodium salt; 2) Under stirring conditions, purging with nitrogen, crystallizing to obtain the crystal A described in claim 1; or under stirring conditions, adding crystal form A as a seed crystal and concentrating under reduced pressure, crystallizing to obtain the crystal A described in claim 1.

11. A method for preparing the crystal of reduced β-nicotinamide mononucleotide disodium salt as claimed in claim 1, wherein the crystal form is crystal form B, characterized in that: The method comprises the following steps: vacuum drying and crystallizing the crystal form A to obtain the crystal form B.

12. A method for preparing the crystal of reduced β-nicotinamide mononucleotide disodium salt as claimed in claim 1, wherein the crystal form is crystal form C, characterized in that: The method comprises the following steps: vacuum drying and crystallizing the crystal form B to obtain the crystal form C.

13. A composition, characterized in that The composition comprises: (a) the crystal according to any one of claims 1 to 9, and (b) a pharmaceutically acceptable excipient or a cosmetically acceptable excipient.

14. Use of the crystal according to claim 1, characterized in that: For the preparation of medicines or cosmetics.

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