Crystalline polymorphism of reduced β-nicotinamide mononucleotide disodium salt and its manufacturing method and use

Abstract

The present invention relates to a compound NMNH disodium salt, in particular to a crystal polymorph of reduced β-nicotinamide mononucleotide disodium salt and a method for preparing the same, as well as to the use of the same as a drug ingredient, a health food ingredient, a cosmetic ingredient or a food additive, and to preparations containing the same, which belong to the fields of medicine, health food, cosmetics and food additives. Specifically, the present invention relates to a crystal polymorph of NMNH disodium salt, which has excellent solubility, and better stability, flowability, anti-hygroscopicity and purification effect than the amorphous form. The preparation process of the crystal form 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 medicines, health foods and cosmetics, and in particular to a crystalline polymorph of reduced β-nicotinamide mononucleotide disodium salt and its preparation method and use. [Background technology]

[0002] Nicotinamide adenine dinucleotide (NAD) is one of the most popular molecules in the anti-aging field. + ) has been at the center of anti-aging substances for many generations. + NAD is an important coenzyme required for over 500 enzyme-catalyzed reactions and is well known for its role in oxidation and reduction (Ansari and Raghava, 2010; Rajman et al., 2018; Stein and Imai, 2012). + A growing body of research has shown that increasing NAD can clearly improve the function of many organs, including liver, kidney, heart, and skeletal muscle (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, NAD + As key intermediates in NAD, NAM, NR, and NMN have already been extensively investigated for their potential therapeutic effects in many mouse disease models (Mills et al., 2016), among which NMN is currently the most relevant NAD + It is considered a precursor, and NMN is now sold in markets around the world and is widely used by consumers.

[0003] NMNH (the molecular structure is represented by formula (A)) is called "reduced nicotinamide mononucleotide" or "reduced β-nicotinamide mononucleotide" in Japanese, and is the reduced form of NMN, which is a soluble form of NAD. + A new precursor to replenish NAD, which is superior to NMN + It has a beneficial effect on the immune system and other biological functions such as increasing the antioxidant capacity of cells, reducing fat accumulation, reducing inflammatory responses, and inhibiting the growth of tumor cells, making it a health-promoting agent with clear commercial potential (WO2021098725A1). [ka]

[0004] The market for the synthesis of NMNH is still in the laboratory research and development stage, and industrial production has not yet been realized. The main technical difficulties are: 1) NMNH is the reduced form of NMN and is easily oxidized by air; 2) high-purity aqueous solutions can only be obtained in laboratories using preparative chromatography; and freeze-drying is required to obtain amorphous solids, which does not purify the product. The amorphous solids obtained by freeze-drying are foamy, have poor fluidity, are prone to absorbing moisture and becoming oily, and decompose quickly; and 3) no reports of crystalline polymorphism have yet been reported. As is well known, freeze-drying consumes a lot of energy and has limited production capacity, so freeze-drying technology is not used industrially unless it is absolutely necessary. Amorphous solids are higher-energy and more unstable than crystalline solids.

[0005] Therefore, in this field, there is a strong demand for the development of new NMNH-based compounds and their crystalline polymorphs that have advantages such as good stability, good fluidity, and suitability for industrialization. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a new NMNH-based compound having advantages such as good stability, good fluidity, and suitability for industrialization, and a crystalline polymorph thereof, i.e., a crystalline polymorph of NMNH disodium salt, and a production method and use thereof. [Means for solving the problem]

[0007] In a first aspect, the present invention provides a crystal of reduced β-nicotinamide mononucleotide disodium salt, wherein the crystal form is selected from the group consisting of crystal form A, crystal form B, and crystal form C.

[0008] In another preferred embodiment, the reduced β-nicotinamide mononucleotide disodium salt has the structure represented by Formula I: [ka]

[0009] In another preferred embodiment, the crystal of reduced β-nicotinamide mononucleotide disodium salt is a hydrate.

[0010] In another preferred embodiment, the reduced β-nicotinamide mononucleotide disodium salt is represented by structural formula II: [ka] (In the formula, n is ≧2.)

[0011] In another preferred embodiment, n is an integer or a non-integer.

[0012] In another preferred embodiment, n is a positive integer ≧2, preferably 2-10, more preferably 5-9.

[0013] In another preferred embodiment, the XRPD spectrum of crystalline form A comprises three or more 2θ values ​​selected from the group consisting of 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°.

[0014] In another preferred embodiment, the XRPD spectrum of crystalline form A further comprises one or more 2θ values ​​selected from the group consisting of 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°.

[0015] In another preferred embodiment, the crystalline form A further has one or more characteristics selected from the following group: 1) The XRPD spectrum of the crystalline form A is 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°, and 49.2°±0.2°. 2) The XRPD spectrum of the crystalline form A is essentially characterized by FIG. 3) The TGA graph of the crystalline form A shows a weight loss of 19%-30% at 15°C-200°C. 4) The TGA graph of the crystalline form A is essentially characterized by FIG. 2. 5) The DSC chart of the crystalline form A has an endothermic peak in the range of 50°C to 80°C. 6) The DSC chart of the crystalline form A is basically characterized by FIG. 7) The crystalline form A is a pentahydrate, hexahydrate, heptahydrate, octahydrate, or nonahydrate.

[0016] In another preferred embodiment, the crystalline form B has one or more characteristics selected from the following group: 1) The XRPD spectrum of crystalline form B comprises three 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°, and 21.5°±0.2°. 2) The XRPD spectrum of crystalline form B further comprises one or more 2θ values ​​selected from the group consisting of 21.1°±0.2°, 23.1°±0.2°, and 25.5°±0.2°. 3) The XRPD spectrum of crystalline form B is 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°, 24.1°±0.2°, 24.7°±0.2° and 41.4°±0.2°, 43.0°±0.2°, 45.1°±0.2°, and 45.7°±0.2°. 4) The XRPD spectrum of the crystalline form B is essentially characterized by FIG. 5) The TGA graph of the crystalline form B shows a weight loss of 12%-23% at 15°C-200°C. 6) The TGA graph of the crystalline form B is essentially characterized by FIG. 7) The DSC chart of the crystalline form B has an endothermic peak in the range of 50°C to 80°C. 8) The DSC chart of the crystalline form B is essentially characterized by Figure 6. 9) The crystalline form B is a trihydrate, tetrahydrate, pentahydrate, or hexahydrate.

[0017] In another preferred embodiment, the crystalline form C has one or more characteristics selected from the following group: 1) The XRPD spectrum of crystalline form C comprises three or more 2θ values ​​selected from the group consisting of 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 XRPD spectrum of 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°. 3) The XRPD spectrum of the crystalline form C is essentially characterized by FIG. 4) The TGA graph of the crystalline form C shows a weight loss of 8%-16% at 15°C-200°C. 5) The TGA graph of the crystalline form C is essentially characterized by FIG. 6) The DSC chart of the crystalline form C has an endothermic peak in the range of 50°C to 80°C. 7) The DSC chart of the crystalline form C is essentially characterized by FIG. 8) The crystalline form C is a dihydrate, trihydrate, or tetrahydrate.

[0018] In a second aspect, the present invention provides a method for producing crystals of reduced β-nicotinamide mononucleotide disodium salt, the method comprising the steps of: 1) Reduced β-nicotinamide mononucleotide disodium salt is placed in a first solvent to obtain a solution containing reduced β-nicotinamide mononucleotide disodium salt. 2) A second solvent is added dropwise under stirring conditions, causing crystals to precipitate, thereby obtaining crystals of reduced β-nicotinamide mononucleotide disodium salt; a nitrogen gas is sprayed over the solution under stirring conditions, causing crystals to precipitate, thereby obtaining crystals of reduced β-nicotinamide mononucleotide disodium salt; or a reduced β-nicotinamide mononucleotide disodium salt is concentrated under reduced pressure under stirring conditions, causing crystals to precipitate, thereby obtaining crystals of reduced β-nicotinamide mononucleotide disodium salt.

[0019] In another preferred embodiment, the first solvent and the second solvent are the same or different and are each independently selected from the group consisting of water, acetonitrile, tetrahydrofuran, methyl t-butyl ether, 2-methyltetrahydrofuran, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, N,N-diethylformamide, dimethyl sulfoxide, ethyl acetate, isopropyl acetate, ketone-based solvents, alcohol-based solvents, or combinations thereof.

[0020] In another preferred embodiment, the ketone solvent is selected from the group consisting of acetone, 2-butanone, methyl isobutyl ketone, methyl t-butyl ketone, 3-methyl-2-butanone, or a combination thereof.

[0021] In another preferred embodiment, the alcohol solvent is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, and n-pentanol.

[0022] In another preferred embodiment, the method further comprises the step of 3) drying the crystals.

[0023] In another preferred embodiment, the drying comprises vacuum drying the obtained crystals for 2-30 hours.

[0024] In another preferred embodiment, the crystal is crystalline form A.

[0025] In another preferred example, when the crystals obtained in step 2) are crystalline form B and / or crystalline form C, step 2) further comprises the sub-step of 2a) converting crystalline form B and / or crystalline form C into crystalline form A by placing them in a gas containing moisture.

[0026] In another preferred embodiment, in step 2a), crystalline form B and / or crystalline form C are converted into crystalline form A by placing them in air.

[0027] In a third aspect of the present invention, there is provided a composition comprising (a) any of the crystals according to the first aspect, and (b) a pharmaceutically acceptable adjuvant or carrier, or a health food acceptable adjuvant or carrier, or a cosmetically acceptable adjuvant or carrier, or a food acceptable adjuvant or carrier.

[0028] In another preferred embodiment, the composition is selected from the group consisting of a drug composition, a health food composition, a cosmetic composition, or a food composition.

[0029] In another preferred embodiment, the pharmaceutical composition comprises (a) any of the crystals according to the first aspect, and (b) a pharmaceutically acceptable adjuvant or carrier.

[0030] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of oral administration preparations, injection dosage forms, respiratory administration dosage forms, skin administration dosage forms, mucosal administration dosage forms, and luminal administration dosage forms.

[0031] In another preferred embodiment, the health food composition comprises (a) any of the crystals according to the first aspect, and (b) a health food acceptable adjuvant or carrier.

[0032] In another preferred embodiment, the cosmetic composition comprises (a) any of the crystals according to the first aspect, and (b) a cosmetically acceptable adjuvant or carrier.

[0033] In another preferred embodiment, the cosmetic composition includes cosmetics for use selected from the group consisting of skin cosmetics, hair cosmetics, beauty cosmetics, and special functional cosmetics.

[0034] In another preferred embodiment, the food composition comprises (a) any of the crystals according to the first aspect, and (b) a food-acceptable adjuvant or carrier.

[0035] In a fourth aspect of the present invention, there is provided a use of the crystals for producing a drug, health food, cosmetic, or food additive.

[0036] In another preferred embodiment, the drug is used for protecting the optic nerve, improving retinal damage, preventing / treating hair loss, preventing / improving cardio-cerebrovascular diseases, inhibiting renal tubule damage and aging, preventing liver fibrosis, improving fatty liver disease, improving symptoms of dry eye disease, repairing kidney damage, preventing diabetes / kidney diseases, improving symptoms of sarcopenia in the elderly, treating chronic inflammation, alleviating symptoms in patients with polycystic ovary syndrome, preventing / delaying glaucoma, reducing neuroinflammation, reducing cardiotoxicity of anthracycline chemotherapy drugs, assisting in recovery from cerebral infarction, preventing and treating heart failure in the elderly, etc.

[0037] In another preferred embodiment, the health food is used for delaying cellular aging, delaying female reproductive aging, improving fertility, improving menopause, enhancing male sexual function, improving sleep, easing emotions, improving vitality, improving cardiovascular function, improving cardiovascular health, improving immunity, improving general health, preventing tumors, and preventing dementia, etc.

[0038] In another preferred embodiment, the cosmetic product is used to improve the function of damaged cells, improve skin / hair quality, prevent / treat photoaging of the skin, maintain skin softness and elasticity, delay skin aging, etc.

[0039] In another preferred embodiment, the food additive is used to improve nutritional value, such as improving appetite, improving digestive function, accelerating metabolism, and promoting hair / nail growth.

[0040] Of course, it is 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 (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be described here one by one due to space limitations. [Brief explanation of the drawings]

[0041] [Figure 1]FIG. 1 shows the XRPD spectrum of crystalline form A of the disodium salt of NMNH. [Figure 2] FIG. 2 shows the TGA graph of crystalline form A of the disodium salt of NMNH. [Figure 3] FIG. 3 shows a DSC chart of crystalline form A of disodium salt of NMNH. [Figure 4] FIG. 4 shows the XRPD spectrum of crystalline form B of the disodium salt of NMNH. [Figure 5] FIG. 5 shows the TGA graph of crystalline form B of the disodium salt of NMNH. [Figure 6] FIG. 6 shows a DSC chart of crystalline form B of disodium salt of NMNH. [Figure 7] FIG. 7 shows the XRPD spectrum of crystalline form C of the disodium salt of NMNH. [Figure 8] FIG. 8 shows the TGA graph of crystalline form C of the disodium salt of NMNH. [Figure 9] FIG. 9 shows a DSC chart of crystalline form C of disodium salt of NMNH. [Figure 10] FIG. 10 shows the XRPD spectrum of the amorphous form of the disodium salt of NMNH. [Figure 11] FIG. 11 shows the TGA graph of the amorphous form of NMNH disodium salt. [Figure 12] FIG. 12 shows a DSC chart of the amorphous form of disodium salt of NMNH. [Figure 13] FIG. 13 shows the 1H NMR spectrum of crystalline form A of NMNH disodium salt. [Figure 14] FIG. 14 shows the stability of crystalline form A and the amorphous solid when left open at 25° C. and 65% RH. [Figure 15] FIG. 15 shows the stability of crystalline form A and the amorphous solid when left uncovered at 4° C. and 75% RH. DETAILED DESCRIPTION OF THE INVENTION

[0042] After extensive and in-depth research, the present inventors have unexpectedly developed a specific salt of reduced NMNH, which is NMNH disodium salt. Research into the present invention has shown that the crystalline polymorph of NMNH disodium salt (particularly crystalline form A) has excellent stability. Compared to the amorphous solid, the amorphous form becomes oily or viscous, lumpy, and decomposes quickly upon standing. Furthermore, the crystalline polymorph of the present invention has the advantages of high purity, good stability, good fluidity, and low hygroscopicity, making it suitable for use in pharmaceutical compositions, health foods, cosmetics, food additives, and the like. Based on this, the inventors have completed the present invention.

[0043] term Unless otherwise defined, 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.

[0044] As used herein, the term "nH2O" refers to all possible numbers between 2 and 10, including integers and non-integers, and "H2O" refers to the chemical formula for water, or the water molecule.

[0045] As used herein, when used with a specific exemplified numerical value, the term "about" means that the value may vary within 1% of the specific exemplified numerical value; for example, as used herein, the expression "about 100" includes all values ​​between 99 and 100 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0046] As used herein, the terms "comprise" or "include" may refer to open, semi-closed, and closed systems. In other words, the terms also include "consisting essentially of" or "consisting of."

[0047] As used herein, the term "n or more 2θ values ​​selected from a group" includes n and any positive integer greater than n (e.g., n, n+1, ...), where the upper limit Nup is the number of all 2θ peak values ​​in the group. For example, "three 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 in the upper limit Nup, but also ranges such as "four or more," "five or more," "six or more," etc.

[0048] NMNH disodium salt 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" can be used interchangeably and all refer to a salt consisting of reduced β-nicotinamide mononucleotide and two sodium ions, the structure of which is represented by Formula I. These terms also include hydrates and anhydrates. [ka]

[0049] In the present invention, a suitable reduced NMN disodium salt is a hydrate, the structure of which is represented by formula (II). [ka]

[0050] Polymorph Solids exist in either amorphous or crystalline form. In crystalline form, molecules are located at sites in a three-dimensional crystal lattice. When a compound crystallizes from a solution or solution, it may be arranged in different spatial lattices (a phenomenon called "polymorphism"), resulting in the formation of crystals with different crystalline forms, each of which is called a "polymorph." Different polymorphs of a substance may differ from each other in one or more physical properties (e.g., solubility and dissolution rate, true specific gravity, crystal shape, sediment morphology, flowability, and / or solid-state stability).

[0051] Polymorphic forms of a compound exhibit different melting points, hygroscopicity, stability, solubility, bioavailability, bioactivity and flowability, which are factors that affect drug-likeness.

[0052] As used herein, the terms "crystal," "crystal of the present invention," or "crystalline polymorph" can be used interchangeably and refer to a crystal according to the first aspect of the present invention, the crystalline form of which is selected from the group consisting of crystalline form A, crystalline form B, or crystalline form C.

[0053] Crystallization Production-scale crystallization can be accomplished by manipulating the solution to exceed the solubility limit of the target compound. This can be accomplished in a number of ways, including dissolving the compound at a relatively high temperature and then cooling the solution below the saturation limit. Alternatively, the volume of the liquid can be reduced by boiling, atmospheric evaporation, vacuum drying, or other methods. The solubility of the target compound can also be reduced by adding an antisolvent, a solvent in which the compound has low solubility, or a mixture of such solvents. Another option is to adjust the pH to reduce solubility. For more information on crystallization, see Crystallization, Third Edition, J.W. Mullens, Butterworth-Heineman Ltd., 1993, ISBN 0750611294.

[0054] If simultaneous salt formation and crystallization are desired, the desired salt will crystallize directly upon addition of an appropriate acid or base if the salt has a lower solubility in the reaction medium than the starting materials. Similarly, the final product will crystallize directly upon completion of the synthesis reaction in a medium in which the desired form has a lower solubility than the reactants.

[0055] Optimizing crystallization involves seeding the crystallization medium with crystals of the desired morphology. Many crystallization methods also use a combination of the above strategies. One implementation involves dissolving the compound of interest in a solvent at high temperature, then adding an appropriate volume of antisolvent under controlled conditions to bring the system just below saturation. At this point, seed crystals of the desired morphology (while preserving the integrity of the seed crystals) can be added, and the system can be cooled to complete the crystallization.

[0056] solvate When a compound or drug molecule comes into contact with a solvent molecule, it is inevitable that the solvent molecule and the compound molecule will form a co-crystal and remain in the solid substance due to external and internal conditions. The substance formed by the crystallization of a compound and a solvent is called a solvate. Types of solvents that easily form solvates with active compounds include water, methanol, ethanol, benzene, ether, and heterocyclic aromatic hydrocarbons.

[0057] hydrate Hydrates are a special type of solvate. Their unique properties make them worthy of separate consideration in the pharmaceutical industry, whether in drug substance synthesis, drug formulation, drug storage, or drug activity evaluation.

[0058] In the present invention, the crystals of the compound represented by formula (I) may be either non-solvates or solvates, but crystalline forms A, B, and C of the crystals of the compound represented by formula (I) are all hydrates.

[0059] Manufacturing method In the present invention, when producing crystals of disodium salt of NMNH, temperature-controlled crystallization, reduced pressure concentration crystallization, nitrogen gas blowing crystallization, volatilization crystallization, solution-precipitation crystallization, temperature and humidity controlled crystallization, vacuum drying crystallization, etc. are used, and these methods are simple, easy to implement, and easy to industrialize.

[0060] Purpose The present invention provides uses of NMNH disodium salt crystals (including crystal forms A, B, and C), which are effective and have a wide range of applications, and can be used in pharmaceutical compositions, health foods, cosmetics, food additives, etc.

[0061] The main advantages of the present invention are as follows: (1) The crystals of the compound of formula (I) of the present invention (including crystalline form A, crystalline form B, and crystalline form C) have higher purity, better stability, better fluidity, and lower hygroscopicity than their amorphous solid counterparts. (2) The method for preparing the crystals of the compound of formula (I) of the present invention (including crystalline form A, crystalline form B, and crystalline form C) is simple and more suitable for industrial production than the freeze-drying process (which consumes high energy and has limited production capacity). (3) The crystals of the compound of formula (I) of the present invention (including crystalline forms A, B, and C) can be used in pharmaceutical compositions, health foods, cosmetics, food additives, etc. (4) The method for producing the crystalline polymorph of the present invention is simple and suitable for industrial production.

[0062] The present invention will be further described below with reference to specific examples. It is understood that these examples are only used to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods for which no specific conditions are described were generally carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention.

[0064] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available products.

[0065] Test Method: Measurement method for XRPD (X-ray powder diffraction) spectrum: Bruker D2 Phaser X-ray powder diffractometer; radiation source Cu (1.54060 A); generator kv: 30 kv; generator mA: 10 mA; starting 2θ: 2.000°; scanning range: 2.0000~50.000°, scanning step width: 0.02°, scanning speed: 0.1 s / step.

[0066] Many factors contribute to measurement differences associated with such X-ray powder diffraction analysis results, including (a) errors in sample preparation (e.g., sample height), (b) instrument error, (c) calibration error, (d) operator error (including errors introduced in measuring peak positions), and (e) material properties (e.g., preferred orientation error). Calibration and sample height errors often result in shifts in all peaks toward the same direction. When using a horizontal holder, small differences in sample height can result in large shifts in XRPD peak positions. In system studies, a 1 mm sample height difference can result in peak shifts as high as 1° 2θ. These shifts can be identified from the XRPD spectrum and corrected for (applying a system calibration factor to all peak position values) or the instrument can be recalibrated to eliminate the shifts. As noted above, applying a system calibration factor can unify peak positions and correct for measurement errors from different instruments.

[0067] Measurement method for TGA (thermogravimetric analysis) graph: TGA55 type apparatus from TA Corporation, USA; temperature range: 14.8 to 300°C; heating rate: 10°C / min; nitrogen gas flow rate: 40 mL / min.

[0068] DSC (differential scanning calorimetry) chart measurement method: TA Q55 type device from TA Corporation, USA; temperature range: 20 to 230°C, heating rate: 10°C / min, nitrogen gas flow rate: 50 mL / min.

[0069] Moisture (KF) measurement method: Fujian Survey Instruments and Equipment (XIAMEN) Co., Ltd. MC-2000 automatic trace moisture analyzer, Nanjing Chemical Reagent Co., Ltd. Karl Fischer reagent.

[0070] Example 1. Preparation of Crystalline Form A Preparation of NMNH disodium salt: 179 mg of MnCl was added to 2.85 L of Tris-HCl (50 mM) solution, the pH of the solution was adjusted to 8.0, and the temperature was maintained at 37°C. 143 mg of NAD pyrophosphatase from Escherichia coli (EcNADD) and 10 g of NADH were added to the solution, and the mixture was stirred for 2 hours. The NMNH solution was separated by preparative chromatography, the pH of the solution was adjusted to 10 with NaOH, and the solution was concentrated under reduced pressure to obtain 3.8 g of a solid NMNH disodium salt.

[0071] 500 mg of NMNH disodium salt was weighed and dissolved in 1 ml of water, and nitrogen gas was blown into the solution at 20-40°C to precipitate crystals. The crystals were filtered and the resulting solid was dried in a blow oven. The crystalline form of the resulting solid was crystalline form A of the compound of formula (I), and the moisture content (KF) was 27%.

[0072] The obtained solid was subjected to powder X-ray diffraction measurement, and the XRPD spectrum of the obtained crystalline form A was basically as shown in Figure 1. 1The 1 H NMR spectrum is shown in FIG. 13, and the diffraction angle data is essentially as shown in Table 1 below, where the error range of the 2θ values ​​is ±0.2°. [Table 1] The TGA graph of crystalline form A was essentially as shown in Figure 2, and showed a weight loss of 19%-30% at 15°C-200°C.

[0073] The DSC chart of crystalline form A was basically as shown in FIG. 3, and had an endothermic peak in the range of 50°C to 80°C.

[0074] Example 2. Kilogram-order expansion and purification effects of crystalline form A Preparation of NMNH disodium salt aqueous solution: 1.7 kg of β-NMN and 0.94 kg of NaSO were added to 10 liters of saturated sodium bicarbonate aqueous solution, stirred overnight at room temperature, and filtered to obtain a clear solution, which was then adjusted to pH 10 with NaOH to obtain an NMNH disodium salt aqueous solution (HPLC purity: 95.2%).

[0075] The temperature was controlled to 20-40°C, and the mixture was mechanically stirred and concentrated under reduced pressure. After concentration to remove some of the water, 10 g of the crystals prepared in Example 1 (crystal form A) was added, and the mixture was concentrated under reduced pressure until 1.5-2 liters remained. The concentration was stopped, the temperature was lowered to 0-10°C, and the mixture was filtered and blow-dried. 2.07 kg of crystals of NMNH disodium salt were obtained, with a moisture content (KF) of 26% and an HPLC purity of 99.4%, and the obtained crystals were crystal form A of compound of formula (I).

[0076] [Table 2] From Table 2, it can be seen that crystalline form A had some purification effect, but the amorphous solid obtained by freeze-drying had no purification effect.

[0077] Example 3. Preparation of Crystalline Form B 50 g of the NMNH disodium salt crystals (crystal form A) prepared in Example 2 was weighed and dried under vacuum for 2-4 hours. The resulting solid crystal form was crystal form B of the compound of formula (I), and the moisture content (KF) was 16%.

[0078] The resulting solid was subjected to powder X-ray diffraction measurement. The XRPD spectrum of the resulting crystalline form B was essentially as shown in FIG. 4, and the diffraction angle data was essentially as shown in Table 3 below, with an error range of ±0.2° for 2θ values. [Table 3] The TGA graph of crystalline form B was essentially as shown in Figure 5, and showed a weight loss of 12%-23% at 15°C-200°C.

[0079] The DSC chart of crystalline form B was essentially as shown in FIG. 6, with an endothermic peak in the range of 50°C to 80°C.

[0080] Example 4. Preparation of crystalline form C 20 g of the NMNH disodium salt crystals (crystal form B) prepared in Example 3 was weighed and dried under vacuum for 10-20 hours. The resulting solid was crystalline form C of the compound of formula (I) and had a moisture content (KF) of 11%.

[0081] The resulting solid was subjected to powder X-ray diffraction measurement. The XRPD spectrum of the resulting crystalline form C was essentially as shown in FIG. 7, and the diffraction angle data was essentially as shown in Table 4 below, with an error range of ±0.2° for 2θ values. [Table 4] The TGA graph of crystalline form C is essentially as shown in Figure 8, and it showed a weight loss of 8%-16% at 15°C-200°C.

[0082] The DSC chart of crystalline form C was essentially as shown in Figure 9, with an endothermic peak in the range of 50°C to 80°C.

[0083] Example 5. Crystalline Form B absorbs water from the air and becomes Crystalline Form A 1 g of the crystals of disodium salt of NMNH (crystal form B) prepared in Example 3 was weighed and exposed to air at 2-8°C with a relative humidity of 70-80% for 30 days. The resulting solid crystal form was crystal form A of the compound of formula (I) with a moisture content (KF) of 29%.

[0084] Example 6. Crystalline Form C absorbs water from the air and becomes Crystalline Form A 1 g of the crystals of disodium salt of NMNH (crystal form C) prepared in Example 4 was weighed and exposed to air at 2-8°C with a relative humidity of 70-80%. After 24 hours, the obtained solid crystal form was crystal form A of the compound of formula (I) with a moisture content (KF) of 24%.

[0085] Example 7. Preparation of amorphous solid of compound of formula (I) 30 g of the NMNH disodium salt crystals prepared in Example 2 were weighed and dissolved in 90 ml of water to obtain a clear solution. This solution was frozen into a solid and then freeze-dried. After freeze-drying for 24 hours, the product obtained was an amorphous solid, foamy, and had poor fluidity, with a moisture content (KF) of 9%.

[0086] The obtained amorphous solid was subjected to powder X-ray diffraction measurement, and the XRPD spectrum was essentially as shown in FIG.

[0087] The TGA graph of the amorphous solid was essentially as shown in Figure 11, with a weight loss of 1%-15% from 15°C to 200°C.

[0088] The DSC chart of the amorphous solid was essentially as shown in FIG. 12, with an endothermic peak in the range of 50°C to 80°C.

[0089] Example 8. Comparison of the stability of crystalline form A and the amorphous solid (1) The stability of both the crystalline product of Example 2 (crystal form A) and the amorphous solid product of Example 7 was examined in an open stability test box at 25°C and 65% RH, and the data shown in Table 5 and Figure 14 were obtained. [Table 5] From Table 5 and Figure 14, it can be seen that the purity of crystalline form A decreased from 99.33% to 99.01% after 5 days (it was still a crystalline powder), while the purity of the amorphous solid decreased from 99.30% to 99.02% after 1 day (the powder absorbed water and became an oil). Thus, it can be seen that the crystalline form A solid of NMNH disodium salt is more stable than the amorphous solid.

[0090] (2) The stability of both the crystalline product of Example 2 (crystalline form A) and the amorphous solid product of Example 7 was examined in an open stability test box at 4°C and 75% RH, and the data shown in Table 6 and Figure 15 were obtained. [Table 6] From Table 6 and Figure 15, it can be seen that the purity of crystalline form A remained almost unchanged at 99.33% after 18 days (it was still a crystalline powder), while the purity of the amorphous solid decreased from 99.30% to 99.26% after 5 days (the powder solid absorbed water and became sticky and lumpy), and on day 18 the purity decreased to 99.17%. This shows that the crystalline form A solid of NMNH disodium salt is more stable than the amorphous solid.

[0091] All documents related to the present invention are incorporated herein by reference as if each document were individually incorporated by reference. After reading the above content of the present invention, it should be understood that those skilled in the art can make various changes and modifications to the present invention, and that equivalents thereof are within the scope of the claims of the present invention.

Claims

1. A crystal of reduced β-nicotinamide mononucleotide disodium salt represented by formula (I), wherein the crystalline form is a hydrate of reduced β-nicotinamide mononucleotide disodium salt, and is crystalline form A, crystalline form B, or crystalline form C; 【Chemistry 2】 the XRPD spectrum of crystalline form A comprises five or more 2θ values ​​selected from the group consisting of 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°; the XRPD spectrum of crystalline form B comprises five 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°, and 21.5°±0.2°; The XRPD spectrum of crystalline form C comprises five or more 2θ values ​​selected from the group consisting of 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, wherein the XRPD spectrum of crystalline form A further contains one or more 2θ values ​​selected from the group consisting of 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 crystalline form A further has one or more characteristics selected from the group consisting of: 1) The XRPD spectrum of crystalline form A has the following ranges: 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°, 28.0°±0.2°, 29.0°±0.2°, 30.0°±0.2°, 31.0°±0.2°, 32.0°±0.2°, 33.0°±0.2°, 34.0°±0.2°, 35.0°±0.2°, 36.0°±0.2°, 37.0°±0.2°, 38.0°±0.2°, 39.0°±0.2°, 40.0°±0.2°, 41.0°±0.2°, 42.0°±0.2°, 43.0°±0.2°, 44.0°±0.2°, 45.0°±0.2°, 46.0°±0.2°, 47.0°±0.2°, 48.0°±0.2°, 49.0°±0.2°, and 2θ values ​​consisting of: 2θ values ​​of 37.7°±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°, and 49.2°±0.2°; 2) The TGA graph of the crystalline form A shows a weight loss of 19%-30% at 15°C-200°C. 3) The crystal of reduced β-nicotinamide mononucleotide disodium salt according to claim 1, wherein the DSC chart of the crystalline form A has an endothermic peak in the range of 50°C to 80°C.

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

5. The XRPD spectrum of crystalline form B shows the following angles: 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°, 24.1°±0.2°, 24.7°±0.2°, 25.5°±0.2°, 2. The crystal of reduced β-nicotinamide mononucleotide disodium salt according to claim 1, wherein the crystal has 2θ values ​​consisting of: 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°, and 45.7°±0.2°.

6. The crystalline form C has one or more characteristics selected from the group consisting of: 1) the XRPD spectrum of 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 graph of the crystalline form C shows a weight loss of 8%-16% at 15°C-200°C. 3) The crystal of reduced β-nicotinamide mononucleotide disodium salt according to claim 1, wherein the DSC chart of the crystalline form C has an endothermic peak in the range of 50°C to 80°C.

7. The method includes the following steps: 1) adding reduced β-nicotinamide mononucleotide disodium salt to water to obtain an aqueous solution containing reduced β-nicotinamide mononucleotide disodium salt; 2) A method for producing the crystal of reduced β-nicotinamide mononucleotide disodium salt according to claim 1, characterized in that the method comprises blowing nitrogen gas onto the mixture under stirring conditions to precipitate crystals and obtain the crystal form A according to claim 1, or adding the crystal form A as a crystal seed under stirring conditions, concentrating under reduced pressure to precipitate crystals and obtain the crystal form A according to claim 1.

8. The method for producing crystals of reduced β-nicotinamide mononucleotide disodium salt according to claim 1, wherein the crystalline form is crystalline form B, and the method comprises the steps of: converting crystalline form A into a vacuum-dried crystal to obtain crystalline form B.

9. The method for producing crystals of reduced β-nicotinamide mononucleotide disodium salt according to claim 1, wherein the crystalline form is crystalline form C, and the method comprises the steps of: converting crystalline form B into a vacuum-dried crystal to obtain crystalline form C.

10. A composition comprising (a) a crystal according to any one of claims 1 to 6, and (b) a pharmaceutically acceptable adjuvant or a cosmetically acceptable adjuvant.

11. 10. Use of the crystals according to claim 1 for producing drugs or cosmetics.

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