Polymorphic form of reduced β-nicotinamide mononucleotide calcium salt, and preparation method and use therefor

Abstract

A compound NMNH calcium salt, in particular a polymorph of a reduced β-nicotinamide mononucleotide calcium salt and a preparation method therefor, a use thereof as a pharmaceutical ingredient, a health care product ingredient and a cosmetic ingredient or as a food additive, and a formulation containing same, belonging to the field of medicines, health care products, cosmetics and food additives. For the NMNH calcium salt polymorph, the crystal thereof displays long-term continuous stability, and compared with NMNH disodium salt, has significantly superior stability performance and anti-hygroscopic properties, and is more beneficial to long-term storage, promotion and market application. The NMNH calcium salt crystal is simple in preparation process, easy to control and suitable for large-scale production.

Description

In another preferred embodiment, the preparation method of the crystalline form comprises the following steps: 1) Providing a solution of reduced form p-nicotinamide mononucleotide calcium salt formed in a first solvent; 2) Under stirring conditions, dropwise adding a second solvent to the reduced form p-nicotinamide mononucleotide calcium salt solution described in step 1), and crystallizing to obtain crystals of reduced form p-nicotinamide mononucleotide calcium salt. In another preferred embodiment, the first solvent and the second solvent are the same or different, and are independently selected from the group consisting of: water, acetonitrile, tetrahydrofuran, methyl tert-butyl ether, 2-methyltetrahydrofuran, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, N,N-diethylformamide, dimethyl sulfoxide, ethyl acetate, isopropyl acetate, ketone solvents, alcohol solvents, or combinations thereof. In another preferred embodiment, the ketone solvent is selected from the group consisting of: acetone, 2-butanone, methyl isobutyl ketone, methyl tert-butyl ketone, 3-methyl-2-butanone, or combinations thereof. In another preferred embodiment, the alcohol solvent is selected from the group consisting of: methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, n-pentanol, or combinations thereof. In a third aspect of the present invention, a composition is provided, the composition comprises: (a) the crystal according to the first aspect, and (b) a pharmaceutically acceptable excipient or carrier, or a health product acceptable excipient or carrier, or a cosmetically acceptable excipient or carrier, or a food acceptable excipient or carrier. In another preferred embodiment, the composition is selected from the group consisting of: a pharmaceutical composition, a health product composition, a cosmetic composition, and a food composition. In another preferred embodiment, the pharmaceutical composition comprises: (a) any crystal as set forth in the first aspect, and (b) a pharmaceutically acceptable excipient or carrier. In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: oral formulation, injectable formulation, respiratory administration formulation, dermal administration formulation, mucosal administration formulation, cavity administration formulation, and the like. In another preferred embodiment, the health product composition comprises: (a) any crystal as set forth in the first aspect, and (b) an excipient or carrier acceptable for health products. In another preferred embodiment, the cosmetic composition comprises: (a) any crystal as set forth in the first aspect, and (b) an excipient or carrier acceptable for cosmetics. In another preferred embodiment, the cosmetic composition comprises cosmetics used for purposes selected from the group consisting of: skin cosmetics, hair cosmetics, beauty cosmetics, and special function cosmetics. In another preferred embodiment, the food composition comprises: (a) any crystal as set forth in the first aspect, and (b) an excipient or carrier acceptable for food. In a fourth aspect of the present invention, a use of the crystal as set forth in the first aspect for the preparation of a medicament, a health product, a cosmetic or a food additive is provided. In another preferred embodiment, the medicament 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, alleviate 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, mitigate the cardiac toxicity of anthracycline chemotherapeutic drugs, aid in stroke recovery, prevent / treat heart failure in the elderly, and the like. In another preferred embodiment, the health product is used to slow cellular aging, delay female reproductive aging, enhance fertility, improve menopausal symptoms, enhance male sexual function, improve sleep, soothe emotions, boost energy, improve cardiovascular function, enhance cardiovascular health, boost immunity, improve sub-health conditions, prevent tumors, prevent Alzheimer’s disease, and the like. In another preferred embodiment, the cosmetic is used to improve damaged cell function, enhance skin / hair quality, prevent / treat skin photoaging, maintain skin softness and elasticity, delay skin aging, and the like. In another preferred embodiment, the food additive is used to improve appetite, enhance digestive function, promote metabolism, promote hair / nail growth, enhance nutritional value, and the like. It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (e.g., in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they are not exhaustively listed herein. Brief Description of the Drawings FIG. 1 shows an XRPD pattern of NMNH calcium salt crystal form A. FIG. 2 shows an XRPD pattern of NMNH calcium salt crystal form B. FIG. 3 shows an XRPD pattern of NMNH calcium salt crystal form C. FIG. 4 shows an XRPD pattern of NMNH calcium salt crystal form D. FIG. 5 shows an XRPD pattern of NMNH calcium salt crystal form E. FIG. 6 shows a 1H NMR spectrum of NMNH calcium salt hydrate crystal forms A, B, and C. FIG. 7 shows a 1H NMR spectrum of NMNH calcium salt ethanol-hydrate crystal form D. FIG. 8 shows a 1H NMR spectrum of NMNH calcium salt acetone-hydrate crystal form E. Description of the Embodiments Through extensive and in-depth research, the inventors have unexpectedly developed a specific salt of NMNH for the first time, the salt can be NMNH calcium salt. The studies of the present invention show that the polymorphs of NMNH calcium salt exhibit excellent stability. Compared with the crystal form and amorphous form of NMNH disodium salt, the polymorphs of NMNH calcium salt (crystal forms A, B, C, D, and E) demonstrate long-term sustained stability and resistance to moisture absorption, making them more suitable for long-term storage and market promotion. Furthermore, the polymorphs of the present invention meet the shelf-life requirements for commercial products and are suitable for use in pharmaceutical compositions, health supplements, cosmetics, food additives, and the like. On this basis, the inventors have completed the present invention. Terminology Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention belongs. As used herein, the term “nH2O” refers to n as encompassing all possible values between 0 and 6, including integers and non-integers; “H2O” is the chemical formula for water, representing water molecules or water. The x in “xS” refers to all possible values between 0 and 6, including integers and non-integers; “S” herein refers to organic solvent molecules, which can be any type of organic solvent. As used herein, when referring to, for specifically listed numerical values, the term “about” means that the value may vary from the listed specific value by no more than 1%. For example, as used herein, the expression “about 100” includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.). As used herein, the term “containing” or “including (comprising)” may be open-ended, semi-closed, or closed. In other words, the term also includes “substantially composed of” or “composed of.” As used herein, the term “n or more 20 values selected from the group consisting of” refers to n and any positive integer greater than n (e.g., n, n+1, ...), where the upper limit Nup is the total number of 20 peaks in the group. For example, “3 or more” includes not only the positive integers 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, . up to the upper limit Nup, but also ranges such as “4 or more,” “5 or more,” “6 or more,” and so forth. NMNH calcium salt As used herein, the terms “reduced 0-nicotinamide mononucleotide calcium salt,” “0-dihydronicotinamide mononucleotide calcium salt,” “dihydronicotinamide mononucleotide calcium salt,” “reduced nicotinamide mononucleotide calcium salt,” “reduced NMN calcium salt,” “NMNH calcium salt,” and “NMNH-Ca” are used interchangeably and all refer to the salt formed by reduced p-nicotinamide mononucleotide and calcium ions, with the structure shown in Formula (I). It should be understood that this term comprises amorphous forms, hydrates, solvates, solvate-hydrates, and anhydrates. nh2 (I) In the present invention, the preferred reduced form NMNH calcium salt is a hydrate, with a structure as shown in Formula (II): (II) Polymorphs Solids exist either in an amorphous form or in a crystal form. In the case of a crystal form, molecules are positioned within a three-dimensional lattice. When a compound crystallizes from a solution or slurry, it can crystallize with different spatial lattice arrangements (a property referred to as “polymorphism”), forming crystals with different crystalline forms, and these various crystalline forms are called “polymorphs.” Different polymorphs of a given substance may differ from each other in one or more physical properties (such as solubility and dissolution rate, true specific gravity, crystal shape, packing behavior, flowability, and / or solid-state stability). The polymorphic forms of a compound may exhibit different melting points, hygroscopicity, stability, solubility, bioavailability, bioactivity, and flowability, which can be important factors affecting druggability. As used herein, the terms “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, with a crystal form selected from the group consisting of: crystal form A, crystal form B, crystal form C, crystal form D, or crystal form E. Crystallization Production-scale crystallization can be achieved by manipulating a solution so that the solubility limit of the target compound is exceeded. This can be accomplished in various ways, for example, by dissolving the compound at a relatively high temperature and then cooling the solution below the saturation limit. Alternatively, it can be done by boiling, atmospheric evaporation, vacuum drying, or by other methods to reduce the volume of the liquid. The solubility of the target compound can also be reduced by adding an antisolvent or a solvent or a mixture of such solvents in which the compound has low solubility. Another alternative method is to adjust the pH value to reduce solubility. For a detailed description of crystallization, see Crystallization, 3rd Edition, J.W. Mullens, Butterworth-Heinemann Ltd., 1993, ISBN 0750611294. If it is desired that salt formation and crystallization occur simultaneously, and if the salt has lower solubility in the reaction medium than the starting materials, then the addition of an appropriate acid or base can lead to direct crystallization of the desired salt. Similarly, in a medium in which the final desired form has lower solubility than the reactants, completion of the synthesis reaction can result in direct crystallization of the final product. Crystallization optimization may include seeding the crystallization medium with crystals of the desired form. Additionally, many crystallization methods employ a combination of the above strategies. One implementation involves dissolving the target compound in a solvent at high temperature, followed by controlled addition of an appropriate volume of antisolvent such that the system is just below the saturation level. At this point, seed crystals of the desired form may be added (while maintaining the integrity of the seed crystals), and the system is cooled to complete crystallization. Solvates During the contact process between a compound or drug molecule and solvent molecules, it is difficult to avoid the situation where external and internal factors cause the solvent molecules to form a cocrystal with the compound molecules and remain in the solid substance. The substance formed after the compound and the solvent crystallize is called a solvate. Types of solvents that readily form solvates with organic compounds include water, methanol, ethanol, benzene, ether, heterocyclic aromatic hydrocarbons, etc. Hydrates A hydrate is a special type of solvate. In the pharmaceutical industry, whether in the synthesis of active pharmaceutical ingredients, pharmaceutical formulations, drug storage, or drug activity evaluation, hydrates have unique characteristics that merit separate discussion. In the present invention, the crystal of the compound represented by formula (I) can be a non-solvate or a solvate; the crystal forms A, B, and C of the compound represented by formula (I) are all hydrates, crystal form D is an ethanol-hydrate, and crystal form E is an acetone-hydrate. Uses The present invention provides the uses of NMNH calcium salt crystals (including crystal forms A, B, C, D, and E): the crystals are highly efficient and broad-spectrum, and can be used in pharmaceutical compositions, health products, cosmetics, food additives, etc. Compared with the existing technology, the beneficial effects of the present invention are as follows: (1) The crystal of the compound of formula (I) of the present invention (including crystal forms A, B, C, D, and E), compared with the NMNH disodium salt crystalline form and amorphous solid, has better stability, lower hygroscopicity, and is more suitable for long-term storage and market promotion. (2) The crystal of the compound of formula (I) of the present invention (including crystal forms A, B, C, D, and E) has a simple preparation method and is suitable for industrial production. (3) The crystal of the compound of formula (I) of the present invention (including crystal forms A, B, C, D, and E) can be used in pharmaceutical compositions, health products, cosmetics, food additives, etc. The present invention is 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, for which specific conditions are not specified, are generally conducted under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to a person skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. Test Methods: XRPD (X-ray Powder Diffraction) Pattern Determination Method: Bruker D2 Phaser X-ray Powder Diffractometer; Radiation Source Cu (1.54060 A). Measurement variations associated with the results of this type of X-ray powder diffraction analysis arise from several factors, including: (a) errors in sample preparation (e.g., sample height), (b) instrument errors, (c) calibration differences, (d) operator errors (including those occurring during peak position determination), and (e) material properties (e.g., preferred orientation errors). Calibration errors and sample height errors often lead to displacement of all peaks in the same direction. When using a flat holder, small differences in sample height can lead to significant displacement in XRPD peak positions. Systematic studies show that a 1 mm difference in sample height can result in a peak shift of up to 1° in 20. These displacements can be identified from the XRPD pattern and can be corrected by compensating for the displacement (applying a systematic calibration factor to all peak position values) or by recalibrating the instrument. As mentioned above, by applying a systematic calibration factor to align peak positions, measurement errors from different instruments can be corrected. The following examples provide description of the present invention without limiting the present invention. Example 1. Preparation of NMNH Calcium Salt Aqueous Solution To 10 liters of saturated sodium bicarbonate aqueous solution, 1.7 kg of P-NMN and 0.94 kg of sodium dithionite were added, stirred at room temperature overnight, and filtered to obtain a clear solution. The pH of the clear solution was adjusted to 3-4 using 37% hydrochloric acid. The solution was desalted using electrodialysis until the conductivity of the solution decreased to 50-100 ^S. The pH of the solution was adjusted to 10 with calcium hydroxide; alternatively, 0.57 kg of calcium chloride was added, and the pH was adjusted to 10 with sodium hydroxide. An NMNH calcium salt aqueous solution was obtained, containing approximately 1.71 kg of NMNH calcium salt (solution purity determined by HPLC to be 94.5%). Example 2. Preparation of NMNH Calcium Salt crystal Form A 500 ml of NMNH calcium salt aqueous solution was stirred in an oil bath at 40-50°C. 2 L of methanol was slowly added dropwise over more than 1 hour. After the addition was complete, the mixture was stirred at 40-50 °C for 1-2 hours, then filtered and dried at 40-50°C to obtain 79.5 g of NMNH calcium salt crystal. The obtained crystal was crystal form A, with a yield of 84.2% and a purity of 99.83%. The obtained solid was characterized by X-ray powder diffraction testing. The XRPD pattern of crystal form A was substantially as shown in FIG. 1, and the 1H NMR spectrum was as shown in FIG. 6. The diffraction angle data were basically as shown in Table 1 below, in which the error range of the 20 values was ±0.2°. The crystal form A was a hydrate with a water content of 9.2% wt. Table 1 XRPD Data of crystal form A 20 (°) d (A) Relative intensity 6.90 12.80798 100.00% 9.45 9.35343 51.60% 12.59 7.02725 83.20% 13.91 6.36349 8.50% 14.37 6.15884 12.10% 15.58 5.685 63.80% 16.65 5.32022 22.00% 17.15 5.16509 11.30% 17.90 4.95114 67.40% 18.81 4.71318 13.10% 20.96 4.23497 36.80% 21.83 4.06741 39.90% 23.09 3.84858 17.60% 25.40 3.5039 32.40% 26.46 3.3656 7.30% 27.25 3.26985 4.50% 28.12 3.1712 14.30% 31.45 2.84246 17.80% 32.79 2.72933 12.70% 35.55 2.52322 7.30% Example 3. Preparation of NMNH Calcium Salt crystal Form B 500 ml of NMNH calcium salt aqueous solution was stirred in a 20-30C oil bath, 2 L of methanol was added at one time, stirred at 20-30°C for 1-2 hours, filtered, and dried at 20-30°C to obtain 80.3 g of NMNH calcium salt crystals. The obtained crystals were of crystal form B, with a yield of 85.0% and a purity of 99.75%. The obtained solid was characterized by X-ray powder diffraction testing. The XRPD pattern of crystal form B obtained was substantially as shown in FIG. 2, the 1H NMR spectrum as shown in FIG. 6, and the diffraction angle data were basically as shown in Table 2 below, with an error range of ±0.2° for the 20 values. The crystal form B is a hydrate with a water content of 13.3% wt. Table 2 XRPD data of crystal form B 20 (°) d (A) Relative intensity 6.50 13.59751 35.00% 8.07 10.9422 39.50% 11.85 7.46152 39.10% 13.11 6.74933 100.00% 13.50 6.55221 41.30% 14.47 6.11638 19.00% 17.61 5.03257 64.10% 17.98 4.93053 14.90% 18.89 4.69536 43.50% 19.36 4.58022 31.90% 19.73 4.49611 20.90% 20.26 4.37951 7.20% 21.44 4.14153 35.50% 23.66 3.75698 44.60% 24.25 3.66758 30.60% 25.10 3.54465 5.40% 26.44 3.36895 21.40% 26.87 3.31547 76.20% 27.51 3.23951 21.90% 28.70 3.1082 36.80% 29.32 3.0434 7.50% 30.46 2.9327 17.50% 30.87 2.8947 22.40% 31.63 2.82611 11.00% 32.39 2.76185 12.60% 32.86 2.7238 9.90% 34.16 2.62238 10.90% 34.96 2.5646 5.00% 35.41 2.53291 8.80% 36.73 2.44482 2.50% 37.48 2.39762 4.70% 37.92 2.37114 7.70% 38.18 2.3551 7.00% 38.85 2.31647 2.10% 40.21 2.24087 5.80% Example 4. Preparation of NMNH Calcium Salt crystal Form C 500 ml of NMNH calcium salt aqueous solution was stirred in a 20-30C oil bath, and 2 L of methanol was slowly added dropwise, with the dropping time being more than 1 hour. After the addition was complete, stirring was continued for 1-2 hours, filtered, and dried at 40-50C to obtain 80.1 g of NMNH calcium salt crystals. The obtained crystals were crystal form C, with a yield of 84.8% and a purity of 99.78%. The obtained solid was characterized by X-ray powder diffraction testing. The XRPD pattern of crystal form C obtained was substantially as shown in FIG. 3, the 1H NMR spectrum as shown in FIG. 6, and the diffraction angle data were basically as shown in Table 3 below, with an error range of ±0.2° for the 20 values. The crystal form C is a hydrate with a water content of 9.5% wt. Table 3 XRPD data of crystal form C 20 (°) d (A) Relative intensity 6.15 14.36193 19.80% 6.76 13.05853 6.10% 9.66 9.14804 19.80% 11.76 7.51895 21.10% 12.38 7.14644 38.10% 14.20 6.2303 19.00% 15.48 5.72149 24.60% 16.12 5.49534 51.70% 17.14 5.17059 100.00% 17.85 4.96625 20.90% 18.66 4.75074 35.80% 20.14 4.40646 11.00% 20.76 4.27452 25.90% 21.49 4.13203 13.10% 22.85 3.88916 10.50% 23.50 3.78195 17.90% 24.09 3.69144 10.80% 24.46 3.63592 5.90% 25.02 3.55605 5.90% 25.82 3.44726 15.70% 26.59 3.34968 60.60% 27.30 3.26413 10.20% 27.66 3.22297 7.30% 28.28 3.15336 11.10% 28.51 3.12856 13.60% 29.67 3.00887 3.30% 30.31 2.94688 3.90% 30.75 2.90506 4.60% 31.44 2.84307 9.10% 32.75 2.73225 10.50% 33.39 2.68143 9.00% 34.40 2.60519 9.00% 35.31 2.53958 4.20% 35.97 2.49504 3.20% 37.10 2.42147 4.60% 38.02 2.36462 6.30% 39.02 2.30673 1.80% 39.83 2.26136 1.90% Example 5. Preparation of NMNH Calcium Salt crystal Form D 500 ml of NMNH calcium salt aqueous solution was stirred in a 20-30C oil bath, and 2 L of ethanol was slowly added dropwise, with the dropping time being more than 1 hour. After the addition was complete, stirring was continued for 1-2 hours, filtered, and dried at 30-40C to obtain 80.7 g of NMNH calcium salt crystals. The obtained crystals were crystal form D, with a yield of 85.5% and a purity of 99.64%. The obtained solid was characterized by X-ray powder diffraction testing. The XRPD pattern of crystal form C obtained was substantially as shown in FIG. 4, and the diffraction angle data were basically as shown in Table 4 below, with an error range of ±0.2° for the 20 values. The crystal form D is an ethanol hydrate, with a water content of 18.4% wt. Its 1H NMR spectrum is shown in FIG. 7, indicating that each molecule of NMNH calcium salt contains about 0.33 molecules of ethanol. Table 4 XRPD data of crystal form D 20 (°) d (A) Relative intensity 6.96 12.68271 57.90% 8.05 10.98098 64.80% 11.95 7.39903 83.90% 13.50 6.5555 55.10% 14.15 6.25583 40.70% 15.71 5.63829 19.30% 17.78 4.98576 76.00% 19.22 4.61411 22.10% 20.23 4.38555 59.90% 23.49 3.78437 26.30% 24.04 3.69955 48.10% 24.46 3.63676 29.40% 26.05 3.41811 20.30% 26.92 3.30879 100.00% 28.69 3.10959 44.40% 30.53 2.92623 32.30% 33.76 2.65289 9.00% Example 6. Preparation of NMNH Calcium Salt crystal Form E 500 ml of NMNH calcium salt aqueous solution was stirred in a 20-30C oil bath, and 2 L of acetone was slowly added dropwise, with the dropping time being more than 1 hour. After the addition was complete, stirring was continued for 1-2 hours, filtered, and dried at 30-40C to obtain 80.8 g of NMNH calcium salt crystals. The obtained crystals were crystal form E, with a yield of 85.6% and a purity of 99.60%. The obtained solid was characterized by X-ray powder diffraction testing. The XRPD pattern of crystal form C obtained was substantially as shown in FIG. 5, and the diffraction angle data were basically as shown in Table 5 below, with an error range of ±0.2° for the 20 values. The crystal form E is an acetone hydrate, with a water content of 20.3% wt. Its 1H NMR spectrum is shown in FIG. 8, indicating that each molecule of NMNH calcium salt contains about 0.33 molecules of acetone. Table 5 XRPD data of crystal form E 20 (°) d (A) Relative intensity 6.93 12.74914 99.90% 8.03 10.99837 63.00% 11.91 7.42592 100.00% 13.46 6.57313 34.30% 14.24 6.21293 85.30% 15.65 5.65952 20.50% 17.85 4.96424 74.30% 18.75 4.72936 24.30% 19.22 4.61471 22.10% 20.18 4.39593 69.00% 24.12 3.68643 41.30% 25.97 3.42829 24.60% 26.91 3.31089 59.50% 28.65 3.11299 31.70% 30.69 2.91044 26.30% 33.87 2.64479 18.00% Example 7. Stability and Hygroscopicity Comparison of NMNH Calcium Salt Crystal Form A, Crystal Form B, Crystal Form C, Crystal Form D, Crystalline Form E with NMNH Disodium Salt crystal Form A and Amorphous Solid The solid forms of NMNH calcium salt crystal form A, crystal form B, crystal form C, crystal form D, and crystal form E were exposed in a stability test chamber at 25°C and 65% RH to evaluate their stability and hygroscopicity. The obtained data are shown in Table 6 and 7. Table 6 Stability Comparison of NMNH Calcium Salt Crystal Form A, Crystal Form B, Crystal Form C, Crystal Form D, Crystal Form E with Crystal Form A of NMNH Disodium Salt and Amorphous Solid (25°C, 65% RH) Time Purity (HPLC, area%) NMNH d isodium salt NMNH calcium salt Crystal Form A Amorphous Form Crystal Form A Crystal Form B Crystal Form C Crystal Form D Crystal Form E 0 day 99.33 99.30 (powder) 99.83 99.75 99.78 99.64 99.60 1 day 99.27 99.02 (oil) / / / / / 5 days 99.01 / 99.82 99.75 99.78 99.64 99.60 60 days / / 99.82 99.75 99.79 99.64 99.59 Table 7 Hygroscopicity Study of NMNH Calcium Salt Crystal Form A, Crystal Form B, Crystal Form C, Crystal Form D, and Crystal Form E Solids (25°C, 65% RH) Time Water content of NMNH calcium salt crystalline form (KF) Crystal Form A Crystal Form B Crystal Form C Crystal Form D Crystal Form E 0 day 9.2% 13.3% 9.5% 18.4% 20.3% 5 days 9.2% 13.2% 9.5% 18.4% 20.3% 60 days 9.0% 12.9% 9.3% 18.3% 20.0% After being placed for 60 days, the purity and water content of NMNH calcium salt crystal form A, crystal form B, crystal form C, crystal form D, and crystal form E solids showed almost no change, indicating that NMNH calcium salt crystal form A, crystal form B, crystal form C, crystal form D, and crystal form E solids can be exposed under conditions of 25°C and 65% RH for no less than 60 days with continuously stable purity and water content. According to WO2023160405 (A1), when NMNH disodium salt is stored in the air, both crystal forms B and C absorb water and transform into crystal form A. After saturation with water, the water content of crystal form A is 19%-30% (high water content), and the purity drops from 99.33% to 99.01% after 5 days. The amorphous solid of NMNH disodium salt is more unstable in the air. After 1 day of storage, it absorbs water and transforms from a powder into an oil, and the purity drops from 99.30% to 99.02%. It can be seen that both the crystalline and amorphous forms of NMNH disodium salt are unstable and easily absorb moisture, which is extremely unfavorable for storage, do not meet commercial shelf-life requirements, and is difficult to promote for market launch. The stability and moisture resistance of NMNH calcium salt crystalline form A, crystalline form B, crystalline form C, crystalline form D, and crystalline form E solids are significantly improved, facilitating long-term continuous and stable storage, and are conducive to market promotion. All documents mentioned in the present invention are incorporated herein by reference in their entirety. In addition, it should be understood that after reading the above teachings of the present invention, a person 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 appended claims of the present application.

Claims

1. A crystal of a reduced p-nicotinamide mononucleotide calcium salt as shown informula (I),OH(I)wherein the crystal is selected from the group consisting of: crystal form A, crystal formB, crystal form C, crystalline form D, and crystal form E;the crystal form A is a hydrate , and the XRPD pattern of the crystal form A comprises 5 or more 20 values selected from the group consisting of: 6.9°±0.2°, 9.5°±0.2°, 12.6°±0.2°, 15.6°±0.2°, 17.9°±0.2°, 21.0°±0.2°, 21.8°±0.2°, and 25.4°±0.2°;the crystal form B is a hydrate , the XRPD pattern of the crystal form B comprises 3 or more (e.g., 4, 5, 6, 7, or 8) 20 values selected from the group consisting of: 6.5°±0.2°, 8.1°±0.2°, 11.9°±0.2°, 13.1°±0.2°, 17.6°±0.2°, 21.4°±0.2°, 23.7°±0.2°, and 26.9°±0.2°;the crystal form C is a hydrate , and the XRPD pattern of the crystal form C comprises 3 or more (e.g., 4, 5, 6, 7, or 8) 20 values selected from the group consisting of: 6.2°±0.2°, 9.7°±0.2°, 12.4°±0.2°, 16.1°±0.2°, 17.1°±0.2°, 18.7°±0.2°, 20.8°±0.2°, and 26.6°±0.2°;the crystal form D is an ethanol hydrate, and the XRPD pattern of the crystal form D comprises 3 or more 20 values selected from the group consisting of: 7.0°±0.2°, 8.0°±0.2°, 12.0°±0.2°, 17.8°±0.2°, 20.2°±0.2°, 26.9°±0.2°;the crystal form E is an acetone hydrate, and the XRPD pattern of the crystal form E comprises 3 or more 20 values selected from the group consisting of: 6.9°±0.2°, 8.0°±0.2°, 11.9°±0.2°, 17.8°±0.2°, 20.2°±0.2°, 26.9°±0.2°.

2. The crystal according to claim 1, wherein the crystal form A further has one or more features selected from the group consisting of:1) the XRPD pattern of the crystalline form A comprises 20 values selected from the group consisting of: 6.9°±0.2°, 9.5°±0.2°, 12.6°±0.2°, 13.9°±0.2°, 14.4°±0.2°, 15.6°±0.2°, 16.7°±0.2°, 17.2°±0.2°, 17.9°±0.2°, 18.8°±0.2°, 21.0°±0.2°, 21.8°±0.2°, 23.1°±0.2°, 25.4°±0.2°, 26.5°±0.2°, 27.3°±0.2°, 28.1°±0.2°, 31.5°±0.2°, 32.8°±0.2°, and 35.6°±0.2°;2024374738  10 Jun 20262) the XRPD pattern of the crystal form A is substantially characterized as shown in FIG.1;3) the crystal form A is a hydrate with a water content of 4% wt to 13% wt.

3. The crystal according to claim 2, wherein the crystal form A is a 1-3 hydrate.

4. The crystal according to claim 1, wherein the crystal form B further has one or more features selected from the group consisting of:1) the XRPD pattern of the crystalline form B comprises 20 values selected from the group consisting of: 6.5°±0.2°, 8.1°±0.2°, 11.9°±0.2°, 13.1°±0.2°, 13.5°±0.2°, 14.5°±0.2°, 17.6°±0.2°, 18.0°±0.2°, 18.9°±0.2°, 19.4°±0.2°, 19.7°±0.2°, 20.3°±0.2°, 21.4°±0.2°, 23.7°±0.2°, 24.3°±0.2°, 25.1°±0.2°, 26.4°±0.2°, 26.9°±0.2°, 27.5°±0.2°, 28.7°±0.2°, 29.3°±0.2°, 30.5°±0.2°, 30.9°±0.2°, 31.6°±0.2°, 32.4°±0.2°, 32.9°±0.2°, 34.2°±0.2°, 35.0°±0.2°, 35.4°±0.2°, 36.7°±0.2°, 37.5°±0.2°, 37.9°±0.2°, 38.2°±0.2°, 38.9°±0.2°, and 40.2°±0.2°;2) the XRPD pattern of the crystal form B is substantially characterized as shown in FIG. 2;3) the crystal form B is a hydrate with a water content of 4% wt to 20% wt.

5. The crystal according to claim 4, wherein the crystal form B is a 1-5 hydrate.

6. The crystal according to claim 1, wherein the crystal form C further has one or more features selected from the group consisting of:1) the XRPD pattern of the crystal form C comprises 20 values selected from the group consisting of: 6.2°±0.2°, 6.8°±0.2°, 9.7°±0.2°, 11.8°±0.2°, 12.4°±0.2°, 14.2°±0.2°, 15.5°±0.2°, 16.1°±0.2°, 17.1°±0.2°, 17.9°±0.2°, 18.7°±0.2°, 20.1°±0.2°, 20.8°±0.2°, 21.5°±0.2°, 22.9°±0.2°, 23.5°±0.2°, 24.1°±0.2°, 24.5°±0.2°, 25.0°±0.2°, 25.8°±0.2°, 26.6°±0.2°, 27.3°±0.2°, 27.7°±0.2°, 28.3°±0.2°, 28.5°±0.2°, 29.7°±0.2°, 30.3°±0.2°, 30.8°±0.2°, 31.4°±0.2°, 32.8°±0.2°, 33.4°±0.2°, 34.4°±0.2°, 35.3°±0.2°, 36.0°±0.2°, 37.1°±0.2°, 38.0°±0.2°, 39.0°±0.2°, and 39.8°±0.2°;2) the XRPD pattern of the crystal form C is substantially characterized as shown in FIG. 3;3) the crystal form C is a hydrate with a water content of 4% wt to 13% wt.7.The crystal according to claim 6, wherein the crystal form C is a 1-3 hydrate.

8. The crystal according to claim 1, wherein the crystal form D has one or more features selected from the group consisting of:2024374738  10 Jun 20261) the XRPD pattern of the crystal form D further comprises 1 or more 20 values selected from the group consisting of: 13.5°±0.2°, 14.2°±0.2°, 24.0°±0.2°;2) the XRPD pattern of the crystal form D comprises 20 values selected from the group consisting of: 7.0°±0.2°, 8.0°±0.2°, 12.0°±0.2°, 13.5°±0.2°, 14.2°±0.2°, 15.7°±0.2°, 17.8°±0.2°, 19.2°±0.2°, 20.2°±0.2°, 23.5°±0.2°, 24.0°±0.2°, 24.5°±0.2°, 26.1°±0.2°, 26.9°±0.2°, 28.7°±0.2°, 30.5°±0.2°, 33.8°±0.2°;3) the XRPD pattern of the crystal form D is substantially characterized as shown in FIG. 4;4) a 1H NMR spectrum of the crystal form D is substantially characterized as shown in FIG. 7;5) the crystalline form D is an ethanol hydrate, containing about 0.2-0.5 ethanol molecules, with a water content of 16% wt-23% wt.

9. The crystal according to claim 8, wherein the crystal form D is an ethanol hydrate, containing 3-6 molecules of water.

10. The crystal according to claim 1, wherein the crystal form E has one or more features selected from the group consisting of:1) the XRPD pattern of the crystal form E further comprises 1 or more 20 values selected from the group consisting of: 13.5°±0.2°, 14.2°±0.2°, 24.0°±0.2°;2) the XRPD pattern of the crystal form E comprises 20 values selected from the group consisting of: 6.9°±0.2°, 8.0°±0.2°, 11.9°±0.2°, 13.5°±0.2°, 14.2°±0.2°, 15.7°±0.2°, 17.8°±0.2°, 18.8°±0.2°, 19.2°±0.2°, 20.2°±0.2°, 24.1°±0.2°, 26.0°±0.2°, 26.9°±0.2°, 28.7°±0.2°, 30.7°±0.2°, 33.9°±0.2°;3) the XRPD pattern of the crystal form E is substantially characterized as shown in FIG. 5;4) a 1H NMR spectrum of the crystal form D is substantially characterized as shown in FIG. 8;5) the crystalline form E is an acetone hydrate, containing about 0.2-0.5 acetone molecules, with a water content of 16% wt-23% wt.11.The crystal according to claim 10, wherein the crystal form E is an acetone hydrate , containing 3-6 molecules of water.2024374738  10 Jun 202612. A method for preparing the crystal according to claim 1, wherein the method comprises the following steps:(I) Preparation of the crystal form A:I-1) providing a solution formed by the reduced p-nicotinamide mononucleotide calcium salt in a first solvent;I-2) at 40-50°C, dropwise adding a second solvent into the solution of the reduced p-nicotinamide mononucleotide calcium salt in step I-1), crystallizing, filtering and drying at 4050°C to obtain the crystal form A of the reduced p-nicotinamide mononucleotide calcium salt, wherein the first solvent is selected from the group consisting of: water; the second solvent is selected from the group consisting of: methanol, ethanol, n-propanol, isopropanol, acetone, acetonitrile, tetrahydrofuran;(II) Preparation of the crystal form B:II-1) providing a solution formed by the reduced p-nicotinamide mononucleotide calcium salt in a first solvent;II-2) at 20-30°C, dropwise adding a second solvent into the solution of the reduced p-nicotinamide mononucleotide calcium salt in step II-1), crystallizing, filtering and drying at 2030°C to obtain the crystal form B of the reduced p-nicotinamide mononucleotide calcium salt, wherein the first solvent is selected from the group consisting of: water; the second solvent is selected from the group consisting of: methanol;(III) Preparation of the crystal form C:III-1) providing a solution formed by the reduced p-nicotinamide mononucleotide calcium salt in a first solvent;III-2) at 20-30°C, dropwise adding a second solvent into the solution of the reduced p-nicotinamide mononucleotide calcium salt in step III), crystallizing, filtering, and then drying at 40-50°C to obtain the crystal form C of the reduced p-nicotinamide mononucleotide calcium salt, wherein the first solvent is selected from the group consisting of: water; the second solvent is selected from the group consisting of: methanol, and an aqueous solution of methanol;(IV) Preparation of the crystal form D:IV-1) providing a solution formed by the reduced p-nicotinamide mononucleotide calcium salt in a first solvent;IV-2) at 20-30°C, dropwise adding a second solvent into the solution of the reduced p-2024374738  10 Jun 2026nicotinamide mononucleotide calcium salt in step IV-1), crystallizing, filtering, and then drying at 30-40°C to obtain the crystal form D of the reduced p-nicotinamide mononucleotide calcium salt, wherein the first solvent is selected from the group consisting of: water; the second solvent is selected from the group consisting of: ethanol, and an aqueous solution of ethanol;(V) Preparation of the crystal form E:V-1) providing a solution formed by the reduced p-nicotinamide mononucleotide calcium salt in a first solvent;V-2) at 20-30°C, dropwise adding a second solvent into the solution of the reduced p-nicotinamide mononucleotide calcium salt set forth in step V-1), crystallizing, filtering, and then drying at 30-40°C to obtain the crystal form E of the reduced p-nicotinamide mononucleotide calcium salt, wherein the first solvent is selected from the group consisting of: water; the second solvent is selected from the group consisting of: acetone, and an aqueous solution of acetone.

13. The method according to claim 12, the solution formed by the reduced form p-nicotinamide mononucleotide calcium salt is provided by the following method: adding the reduced form p-nicotinamide mononucleotide calcium salt to a first solvent to obtain a solution containing the reduced form p-nicotinamide mononucleotide calcium salt, or generating the reduced form p-nicotinamide mononucleotide calcium salt solution in situ in a reaction solution.

14. A composition, wherein the composition comprises: (a) the crystal according to any one of claims 1-11, and (b) a pharmaceutically acceptable excipient , or a cosmetically acceptable excipient .

15. The composition according to claim 14, wherein the composition is a pharmaceutical composition, and the dosage form of the pharmaceutical composition is selected from the group consisting of: oral formulation, injectable formulation, respiratory administration formulation, dermal administration formulation, mucosal administration formulation, cavity administration formulation.

16. The composition according to claim 14, wherein the composition is a cosmetic composition, and the cosmetic composition comprises cosmetics used for purposes selected from the group consisting of: skin cosmetics, hair cosmetics, beauty cosmetics.

17. The composition according to claim 14, wherein the composition is special function cosmetics.2024374738  10 Jun 202618. A use of the crystal according to any one of claims 1-11, wherein the crystal is used for the preparation of a medicament, a cosmetic.

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