Polymorphism of reduced β-nicotinamide mononucleotide disodium salt and its preparation 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 crystal 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 core 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 demonstrated 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). + can synthesize 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 the regulation of NAD, NAM, NR and NMN have already been extensively investigated for their potential therapeutic effects in a number of mouse disease models (Mills et al., 2016), among which NMN is currently the most suitable NAD + It is considered a precursor, and today NMN is sold in markets around the world and is 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. It is the reduced form of NMN and is the NAD + A new precursor that replenishes NAD and is superior to NMN + It has a potent antioxidant effect and other biological functions such as increasing cellular antioxidant capacity, reducing fat accumulation, reducing inflammatory responses and inhibiting tumor cell growth, making it a health-promoting agent with clear commercial potential (WO2021098725A1). [ka]

[0004] The market for the synthesis process of NMNH is still in the laboratory research and development stage, and industrial production has not been realized. The main technical difficulties are: 1) NMNH is the reduced form of NMN, and is easily oxidized by air; 2) Only preparative chromatography can obtain a high-purity aqueous solution in the laboratory, and freeze-drying is required to obtain an amorphous solid, which has no purification effect on the product, and the amorphous solid obtained by freeze-drying is foamy, has poor fluidity, is prone to absorbing moisture and becoming oily, and decomposes quickly; and 3) there is no report of crystal polymorphism. As is well known, freeze-drying consumes a lot of energy and has limited production capacity, so freeze-drying technology is not chosen industrially unless it is 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 crystal 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, the crystal having a crystalline form selected from the group consisting of crystalline form A, crystalline form B, or crystalline 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 said 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 said crystalline form A is essentially characterized by FIG. 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 said 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 the 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°, 25.7°±0.2°, 26.7°±0.2°, 27.7°±0.2°, 28.7°±0.2°, 29.7°±0.2°, 30.7°±0.2°, 31.7°±0.2°, 32.7°±0.2°, 33.7°±0.2°, 34.7°±0.2°, 35.7°±0.2°, 36.7°±0.2°, 37.7°±0.2°, 38.7°±0.2°, 39.7°±0.2°, 40.7°±0.2°, 41.7°±0.2°, 42.7°±0.2°, 43.7°±0.2°, 44.7°±0.2°, 45.7° and 41.4°±0.2°, 43.0°±0.2°, 45.1°±0.2°, and 45.7°±0.2°. 4) The XRPD spectrum of said 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 said 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, a tetrahydrate, a pentahydrate, or a 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 said 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 said 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 said 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 of the present invention, there is provided a method for producing crystals of reduced β-nicotinamide mononucleotide disodium salt, 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) The second solvent is added dropwise under stirring conditions, and crystals are precipitated to obtain crystals of reduced β-nicotinamide mononucleotide disodium salt; the solution is sprayed with nitrogen gas under stirring conditions, and crystals are precipitated to obtain crystals of reduced β-nicotinamide mononucleotide disodium salt; or the solution is concentrated under reduced pressure under stirring conditions, and crystals are precipitated to obtain 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, dimethylsulfoxide, ethyl acetate, isopropyl acetate, a ketone-based solvent, an alcohol-based solvent, or a combination 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 embodiment, when the crystals obtained in step 2) are crystalline form B and / or crystalline form C, step 2) further comprises a sub-step of 2a) converting crystalline form B and / or crystalline form C into crystalline form A by placing the crystalline form B and / or crystalline form C in a gas containing moisture.

[0026] In another preferred embodiment, in step 2a), crystalline form B and / or crystalline form C are converted to crystalline form A by placing 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 pharma- ceutically 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 pharma- ceutically 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, airway 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 a cosmetic for an application 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 of polycystic ovary syndrome patients, preventing / delaying glaucoma, reducing neuroinflammation, reducing cardiotoxicity of anthracycline chemotherapeutic drugs, assisting in recovery from cerebral infarction, preventing / 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 level, improving immunity, improving sub-health, preventing tumors, 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, promoting metabolism, and promoting hair / nail growth.

[0040] Of course, it is understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (for example, 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 description of the drawings]

[0041] [Figure 1]FIG. 1 shows the XRPD spectrum of crystalline form A of the disodium salt of NMNH. [Diagram 2] FIG. 2 shows the TGA graph of crystalline form A of the disodium salt of NMNH. [Diagram 3] FIG. 3 shows a DSC chart of crystalline form A of the disodium salt of NMNH. [Figure 4] FIG. 4 shows the XRPD spectrum of crystalline form B of the disodium salt of NMNH. [Diagram 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 crystal form B of NMNH disodium salt. [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 crystal form C of disodium NMNH. [Figure 10] FIG. 10 shows the amorphous XRPD spectrum of the NMNH disodium salt. [Figure 11] FIG. 11 shows the amorphous TGA graph 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 the disodium salt of NMNH. [Figure 14] FIG. 14 shows the stability of crystalline form A and the amorphous solid when left uncovered 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 PREFERRED EMBODIMENTS

[0042] The present inventors have conducted extensive and in-depth research and have unexpectedly developed a specific salt of reduced NMNH for the first time, said salt being NMNH disodium salt. Research into the present invention has shown that the crystal polymorph of NMNH disodium salt (particularly crystal form A) has excellent stability. Compared with the amorphous solid, the amorphous solid becomes oily or viscous dumpling-like when placed and decomposes quickly. In addition, the crystal polymorph of the present invention has the advantages of high purity, good stability, good fluidity, and low hygroscopicity, and is suitable for use in pharmaceutical compositions, health foods, cosmetics, food additives, etc. 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 "nH 2 In "O", n is any possible number between 2 and 10, including integers and non-integers. 2 "O" is the chemical formula for water and represents the water molecule or water.

[0045] As used herein, when used with a specific exemplified numerical value, the term "about" means that the value may vary within 1% from the specific numerical value exemplified; 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 "containing" or "comprises" 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 with 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," "NMNH-Na 2 " can be used interchangeably and refer to a salt of reduced β-nicotinamide mononucleotide and two sodium ions, the structure of which is represented by Formula I. The terms 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, the molecules are located at sites of 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 crystals having different crystal morphologies, each of which is called a "polymorph." Different polymorphs of a substance may differ from each other in one or more physical properties, such as solubility and dissolution rate, true specific gravity, crystal shape, sediment morphology, flowability and / or solid state stability.

[0051] Polymorphic forms of a compound may 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 overcome the solubility limit of the desired compound. This can be accomplished in a number of ways, for example by 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 desired compound can also be reduced by the inclusion of an anti-solvent or a solvent in which the compound is less soluble, or a mixture of such solvents. Another option is to adjust the pH value to reduce the solubility. For more information on crystallization, see Crystallization, 3rd Edition, JW Mullens, Butterworth-Heineman Ltd., 1993, ISBN 0750611294.

[0054] If simultaneous salt formation and crystallization are desired, the required 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 a synthetic reaction in a medium in which the solubility of the desired form is lower than that of 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, followed by the addition of an appropriate volume of anti-solvent under controlled conditions to bring the system just below the level of saturation. Seeds of the desired morphology (preserving the integrity of the seeds) can then be added and the system cooled to complete the crystallization.

[0056] solvate In the process of contacting a compound or drug molecule 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 crystallization of a compound and a solvent is called a solvate. Types of solvents that are likely to form solvates with active compounds include water, methanol, ethanol, benzene, ether, and heterocyclic aromatic hydrocarbons.

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

[0058] In the present invention, the crystals of the compound represented by formula (I) may be either non-solvated or solvated, but the crystal forms A, B, and C 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, dissolution-precipitation crystallization, temperature and humidity controlled crystallization, vacuum drying crystallization, etc. are used, and the above methods are simple and easy to carry out, and easy to industrialize.

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

[0061] The main advantages of the present invention are: (1) The crystals of the compound of formula (I) of the present invention (including crystal forms A, B and C) have higher purity, better stability, better fluidity and lower hygroscopicity than their amorphous solid counterparts. (2) The preparation method of 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 has high energy consumption and limited production capacity). (3) The crystals of the compound of formula (I) of the present invention (including crystal forms A, B and C) can be used in pharmaceutical compositions, health foods, cosmetics, food additives and the like. (4) The method for producing the crystal 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 explain 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 usually carried out under normal conditions or 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, and any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention.

[0064] All experimental materials and reagents used in the following examples are commercially available unless otherwise specified.

[0065] Test Method: Measurement method of 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 result in measurement differences associated with such X-ray powder diffraction analysis results, including (a) errors in sample preparation (e.g., sample height), (b) instrument errors, (c) calibration errors, (d) operator errors (including errors introduced in measuring peak positions), and (e) material properties (e.g., preferred orientation errors). Calibration errors and sample height errors often result in shifts in all peaks to the same method. When using a horizontal holder, small differences in sample height can result in large shifts in XRPD peak positions. In system studies, a sample height difference of 1 mm can result in shifts in peaks 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 recalibrating the instrument to eliminate the shifts. As mentioned 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 equipment from TA, USA; temperature range: 14.8 to 300°C; heating rate: 10°C / min; nitrogen gas flow rate: 40mL / min.

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

[0069] Moisture content (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: Add 179 mg of MnCl to 2.85 L of Tris-HCl (50 mM) solution. 2 The solution was added with 143 mg of E. coli (EcNADD) NAD+ pyrophosphatase and 10 g of NADH, and stirred for 2 hours. The solution was separated by preparative chromatography to obtain a NMNH solution, which was adjusted to pH 10 with NaOH and concentrated under reduced pressure to obtain 3.8 g of a solid NMNH disodium salt.

[0071] 500 mg of NMNH disodium salt was weighed, dissolved in 1 ml of water, and nitrogen gas was blown onto the solution at 20-40°C to precipitate crystals. The crystals were filtered and the solid was dried in a blow oven. The crystal form of the solid was 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] TIFF2025508874000007.tif83166 The TGA graph of crystalline form A is basically as shown in Figure 2, and the weight loss was 19%-30% at 15℃-200℃.

[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.-80° C.

[0074] Example 2. Kilogram-order expansion and purification effects of crystalline form A Preparation of NMNH disodium salt solution: 1.7 kg of β-NMN, 0.94 kg of Na in 10 liters of saturated sodium bicarbonate solution. 2 S 2 O 4 The mixture was stirred at room temperature overnight and filtered to obtain a clear solution, which was then adjusted to pH 10 with NaOH to obtain an aqueous solution of NMNH disodium salt (HPLC purity: 95.2%).

[0075] The temperature was controlled to 20-40°C, mechanically stirred, and concentrated under reduced pressure. After concentrating to remove some water, 10g of the crystals (crystal form A) prepared in Example 1 was added, and then concentrated under reduced pressure until 1.5-2 liters remained. The concentration was stopped, the temperature was lowered to 0-10°C, filtered, and blow-dried. 2.07kg 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 the 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 crystals of disodium NMNH salt (crystal form A) prepared in Example 2 was weighed and dried in vacuum for 2-4 hours. The crystal form of the obtained solid 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 obtained crystalline form B is basically as shown in FIG. 4, and the diffraction angle data is basically as shown in Table 3 below, where the error range of 2θ value is ±0.2°. [Table 3] TIFF2025508874000010.tif98165The TGA graph of crystalline form B is essentially as shown in FIG. 5, and the weight loss was 12%-23% at 15°C-200°C.

[0079] The DSC chart of crystalline form B was basically as shown in FIG. 6, and had an endothermic peak in the range of 50° C.-80° C.

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

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

[0082] The DSC chart of crystalline form C was basically as shown in FIG. 9, and had an endothermic peak in the range of 50° C.-80° C.

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

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

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

[0086] The resulting 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 basically as shown in FIG. 12, and had an endothermic peak in the range of 50° C.-80° C.

[0089] Example 8. Comparison of the physical stability of crystalline form A and the amorphous solid (1) The stability of both the crystals (crystal form A) of the product in Example 2 and the amorphous solid of the product in 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 (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 crystals (crystal form A) of the product in Example 2 and the amorphous solid of the product in 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 after 18 days, the purity of crystalline form A was 99.33% and remained almost unchanged (still in the form of 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 dumplings), and on the 18th day, the purity dropped to 99.17%. Thus, it can be seen 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 incorporated individually. After reading the above content of the present invention, it will be understood that those skilled in the art can make various changes and modifications to the present invention, and that equivalent forms 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 crystal form is a hydrate of reduced β - nicotinamide mononucleotide disodium salt, and is crystal form A, crystal form B, or crystal form C, 【Chemical 2】 the XRPD spectrum of the crystal form A includes 5 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°, 31.8° ± 0.2°, the XRPD spectrum of the crystal form B includes 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°, the XRPD spectrum of the crystal form C includes 3 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°, 21.5° ± 0.2°, and is characterized by the crystal.

2. The XRPD spectrum of the crystal form A further includes 1 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°, 33.4° ± 0.2°. The crystal of reduced β - nicotinamide mononucleotide disodium salt according to claim 1, characterized in that.

3. The crystal form A further has one or more characteristics selected from the following group, 1) The XRPD spectrum of the crystalline form A includes 2θ values 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°, 27.8° ± 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 graph of the crystalline form A shows a weight loss of 19% - 30% at 15°C - 200°C. 3) The DSC chart of the crystalline form A has an endothermic peak within the range of 50°C - 80°C. The crystal of reduced β - nicotinamide mononucleotide disodium salt according to claim 1 is characterized by this.

4. 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 basically characterized by Figure 1. 2) The TGA graph of the crystalline form A is basically characterized by Figure 2. 3) The DSC of the crystalline form A is basically characterized by Figure 3. The crystal of reduced β - nicotinamide mononucleotide disodium salt according to claim 1 is characterized by this.

5. The crystalline form B has one or more characteristics selected from the following group: 1) The XRPD spectrum of the crystalline form B further includes one or more 2θ values selected from the group consisting of 21.1° ± 0.2°, 23.1° ± 0.2°, 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 DSC chart of the crystalline form B has an endothermic peak within the range of 50°C - 80°C, The crystal of reduced β - nicotinamide mononucleotide disodium salt according to claim 1, characterized in that.

6. The crystalline form B further has one or more characteristics selected from the following group: 1) The XRPD spectrum of the crystalline form B is basically characterized by FIG.

4. 2) The TGA graph of the crystalline form B is basically characterized by FIG.

5. 3) The DSC of the crystalline form B is basically characterized by FIG. 6, The crystal of reduced β - nicotinamide mononucleotide disodium salt according to claim 1, characterized in that.

7. The XRPD spectrum of the crystalline form B includes 2θ values 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°, 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°, The crystal of reduced β - nicotinamide mononucleotide disodium salt according to claim 1, characterized in that.

8. The crystalline form C has one or more characteristics selected from the following group: 1) The XRPD spectrum of the crystalline form C further includes 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°, 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 DSC chart of the crystalline form C has an endothermic peak within the range of 50°C - 80°C. The crystal of the reduced form β - nicotinamide mononucleotide disodium salt according to claim 1 is characterized by this.

9. The crystalline form C further has one or more characteristics selected from the following group: 1) The XRPD spectrum of the crystalline form C is basically characterized by Figure 7. 2) The TGA graph of the crystalline form C is basically characterized by Figure 8. 3) The DSC of the crystalline form C is basically characterized by Figure 9. The crystal of the reduced form β - nicotinamide mononucleotide disodium salt according to claim 1 is characterized by this.

10. It includes the following steps: 1) Put the reduced form β - nicotinamide mononucleotide disodium salt into water to obtain an aqueous solution containing the reduced form β - nicotinamide mononucleotide disodium salt. 2) Under stirring conditions, blow with nitrogen gas to precipitate crystals to obtain the crystalline form A according to claim 1, or add the crystalline form A as a crystal seed under stirring conditions, concentrate under reduced pressure, and precipitate crystals to obtain the crystalline form A according to claim 1. The method for producing the crystal of the reduced form β - nicotinamide mononucleotide disodium salt according to claim 1 is characterized by this.

11. The crystalline form is the crystalline form B, and it includes the following steps: vacuum - dry and crystallize - convert the crystalline form A to obtain the crystalline form B. The method for producing the crystal of the reduced form β - nicotinamide mononucleotide disodium salt according to claim 1 is characterized by this.

12. The crystalline form is the crystalline form C, and it includes the following steps: vacuum - dry and crystallize - convert the crystalline form B to obtain the crystalline form C. The method for producing the crystal of the reduced form β - nicotinamide mononucleotide disodium salt according to claim 1 is characterized by this.

13. A composition comprising (a) the crystal according to claim 1, and (b) a pharmaceutically acceptable adjuvant or a cosmetically acceptable adjuvant.

14. Use of the crystal according to claim 1, characterized in that it is for producing a drug or a food additive.

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