1. A method of preparing a nicotinamide mononucleotide co-crystal comprising: taking nicotinamide mononucleotide as an active ingredient and isonicotin as a eutectic formation substance, mixing the nicotinamide mononucleotide and the isonicotin by a solution synthesis method, and then crystallizing.

2. The method of preparing a nicotinamide mononucleotide co-crystal of claim 1, characterized in that: the mixing ratio of the nicotinamide mononucleotide to the isonicotinine is 1:1 in terms of molar ratio.

3. The method of preparing a nicotinamide mononucleotide co-crystal according to claim 1 or 2, characterized in that: the mixing and the crystallization are both carried out in a mixed system of an organic solvent and water, wherein the organic solvent is tetrahydrofuran, acetonitrile or acetone.

4. The method of preparing a nicotinamide mononucleotide co-crystal of claim 3, characterized in that: in the mixed system of the organic solvent and the water, the volume ratio of the organic solvent to the water is 1: 1-5.

5. The method of preparing a nicotinamide mononucleotide co-crystal of claim 3, characterized in that: during the mixing process, the temperature of the mixed system of the organic solvent and the water is kept between 30 and 55 ℃.

6. The method of preparing a nicotinamide mononucleotide co-crystal of claim 3, characterized in that: the mixed system of the organic solvent and water is obtained by dropping the organic solvent into the water, and the nicotinamide mononucleotide and the isonicotinide are dissolved in the water before dropping the organic solvent.

7. The method of preparing a nicotinamide mononucleotide co-crystal of claim 6, characterized in that: and maintaining the temperature of the mixed system of the organic solvent and the water at 30-55 ℃ in the process of dropwise adding the organic solvent into the water.

8. The method of preparing a nicotinamide mononucleotide co-crystal of claim 3, characterized in that: the crystallization process is carried out in a state that the temperature of the mixed system of the organic solvent and the water is reduced to 4-8 ℃ and then the mixed system is kept still.

Background

Nicotinamide Mononucleotide (NMN) is a biochemical substance inherent in biological cells, and can be adenylated by Nicotinamide nucleotide adenylyl transferase in the cells to be converted into Nicotinamide adenine dinucleotide (NAD, also called coenzyme I) which is an important substance for living of biological cells, exists in all cells, participates in thousands of biocatalytic reactions, and plays an important role in energy generation of biological cells. NMN is a direct precursor of NAD, which acts as an important intermediate in the intracellular NAD salvage synthesis pathway, and the level in the cell directly affects the NAD concentration.

Research shows that in vitro NMN supplementation is the most ideal way to increase intracellular NAD concentration level, and in addition, the in vitro NMN supplementation has been found to achieve various medical and health-care effects such as aging delay, treatment of senile diseases such as Parkinson, insulin secretion regulation, mRNA expression influence and the like, and more new medical applications of NMN are reported continuously. In addition, as the information of the 'non-old medicine' NMN is lost with the plum cardinal investment, the NMN becomes a fragrant pastry at a time and is favored by a plurality of capital, the ordinary people can pursue the NMN medicine or health care product at a normal pace, and the demand of the NMN medicine or health care product on the market is increasing day by day.

Since NMN has insufficient stability and pharmaceutical or health products manufactured using the NMN amorphous powder easily lose pharmaceutical activity during storage and transportation, crystals of NMN were developed, such as two crystal forms of β -nicotinamide mononucleotide, anhydrous crystal (form 1) and dimethylsulfoxide solvate crystal (form 2), as disclosed in chinese patent application CN 108697722A. Nowadays, related enterprises generally adopt NMN in a crystal form to produce NMN medicines or health care products, the stability of the products is obviously improved, but certain problems still exist, such as the poor liquidity of the NMN, larger filling/weight difference and inconsistent quality of the products.

Disclosure of Invention

In view of the above-mentioned shortcomings in the background art, the present invention aims to develop a method for preparing nicotinamide mononucleotide, which can obtain nicotinamide mononucleotide crystals with good fluidity, so as to solve the technical problems of poor fluidity of the existing nicotinamide mononucleotide crystals, large loading/weight difference and inconsistent quality of NMN drugs or health care products.

To achieve the above object, the present invention provides a method for preparing a nicotinamide mononucleotide co-crystal, comprising: taking nicotinamide mononucleotide as an active ingredient and isonicotin as a eutectic formation, mixing the nicotinamide mononucleotide and the isonicotin by a solution synthesis method, and then crystallizing.

Eutectic is defined in the pharmaceutical eutectic regulatory classification guidelines (2011) issued by FDA, and refers to a crystalline substance containing two or more different molecules in the same crystal lattice, and eutectic components are in a neutral state by non-ionic interaction. The eutectic components are divided into two types, one is an Active pharmaceutical Ingredient (API for short), the other is a eutectic Former (CCF for short), and the two eutectic components are combined into a new solid form in a fixed stoichiometric ratio under the action of hydrogen bonds, pi-pi stacking action, van der Waals force or other non-covalent bonds.

Common methods for preparing co-crystals fall into two categories from the morphological aspect of the individual components at the time of preparation: solution synthesis and solid synthesis. The solution synthesis method is that API and CCF are in fluid state during synthesis, and comprises evaporative crystallization, cooling crystallization, suspension method and the like; the solid synthesis method is a method in which both API and CCF are in a solid form during synthesis, and includes sublimation, melting, and milling.

When the eutectic is prepared by a solution synthesis method, the metering ratio of the API and the CCF influences the precipitation amount of the eutectic, and if the metering ratio is not proper, one of the substances may be separated out, so that the precipitation rate of the eutectic is influenced. In the method for preparing a nicotinamide mononucleotide co-crystal provided by the invention, nicotinamide mononucleotide and isonicotinine are preferably mixed according to the molar ratio of 1:1, so that the nicotinamide mononucleotide-isonicotinine co-crystal can be precipitated to the maximum extent.

In the method for preparing nicotinamide mononucleotide cocrystal provided by the invention, the mixing and crystallization processes are carried out in a mixed system of organic solvent and water, the type of the organic solvent plays a key role in whether the crystal can be successfully precipitated, and the organic solvent in the method for preparing nicotinamide mononucleotide cocrystal provided by the invention is preferably tetrahydrofuran, acetonitrile or acetone.

More preferably, in the mixed system of the organic solvent and water in the method for preparing nicotinamide mononucleotide co-crystal provided by the invention, the volume ratio of the organic solvent to the water is 1: 1-5.

Preferably, in the method for preparing the nicotinamide mononucleotide co-crystal provided by the invention, the temperature of a mixed system of the organic solvent and water is kept at 30-55 ℃ in the whole mixing process, so that the preparation is not only for subsequent cooling crystallization, but also the mixing process can be accelerated, and the time is saved.

In the method for preparing nicotinamide mononucleotide co-crystal provided by the invention, the mixed system of the organic solvent and the water can be obtained by directly mixing the organic solvent and the water, or can be obtained by slowly dripping the organic solvent into the water. For the former, nicotinamide mononucleotide and isonicotinine can be added after mixing the organic solvent and water; in the latter case, nicotinamide mononucleotide and isonicotinine should be dissolved in water before the organic solvent is added dropwise to the water.

Preferably, in the method for preparing a nicotinamide mononucleotide co-crystal provided by the invention, the mixed system of the organic solvent and the water is prepared by adopting the latter mode, namely, the nicotinamide mononucleotide and the isonicotin are dissolved in the water, and then the organic solvent is slowly dripped into the water. The method can further improve the bulk density of the crystal, thereby obtaining the crystal with better fluidity.

More preferably, in the method for preparing nicotinamide mononucleotide co-crystal, the temperature of the mixed system of the organic solvent and the water is kept between 30 and 55 ℃ during the process of slowly dripping the organic solvent into the water, so that the advantages of: not only is preparation for subsequent cooling and crystallization, but also can accelerate the mixing process and save time.

Preferably, in the method for preparing a nicotinamide mononucleotide co-crystal provided by the invention, the crystallization process is carried out in a state that a mixed system of an organic solvent and water is cooled to 4-8 ℃ and then stands still.

The inventor finally develops the method for preparing the nicotinamide mononucleotide eutectic provided by the invention through a large amount of long-term experimental groves and creative work, and repeated experiments prove that the method can successfully prepare a new crystal form of nicotinamide mononucleotide presented in a eutectic form, and the success rate is 100%.

Nicotinamide mononucleotide-isonicotine cocrystals prepared by the above-described method for preparing a nicotinamide mononucleotide cocrystal provided by the invention have diffraction peaks at about 9.6 + -0.2 °, about 13.3 + -0.3 °, about 22.8 + -0.2 ° and about 36.5 + -0.2 ° by X-ray powder diffraction using Cu-Ka radiation at 2 θ degrees. And having endothermic peaks at 55.8 + -3 ℃ and 151.9 + -3 ℃ in a differential scanning calorimetry diagram.

Further, the nicotinamide mononucleotide-isonicotine cocrystal prepared by the above-mentioned method for preparing a nicotinamide mononucleotide cocrystal provided by the invention has diffraction peaks at about 9.6 + -0.2 °, about 9.8 + -0.2 °, about 10.6 + -0.2 °, about 13.3 + -0.3 °, about 16.3 + -0.2 °, about 21.3 + -0.2 °, about 22.8 + -0.2 °, about 32.1 + -0.2 ° and about 36.5 + -0.2 ° by X-ray powder diffraction using Cu-Ka radiation and 2 θ.

Experiments show that the nicotinamide mononucleotide-isonicotinine eutectic prepared by the method for preparing the nicotinamide mononucleotide co-crystal provided by the invention not only does not influence the pharmaceutical activity of the nicotinamide mononucleotide, but also has higher bulk density than the existing crystal, thereby obviously improving the flowability of the nicotinamide mononucleotide. The bulk density is related to the characteristics of crystal morphology and particle size, which are determined by the control conditions such as steps, solvent types, temperature, etc. in the preparation process, so the achievement of the crystal with higher bulk density is completely dependent on the selection and setting of specific process conditions in the method for preparing nicotinamide mononucleotide cocrystal provided by the invention.

Has the advantages that:

compared with the prior art, the invention has the following advantages:

1. the invention provides a method for preparing nicotinamide mononucleotide eutectic, which can successfully prepare a novel nicotinamide mononucleotide crystal presented in a eutectic form, fills the blank of the nicotinamide mononucleotide eutectic, and has simple operation and wide application range.

2. Experiments show that the nicotinamide mononucleotide-isonicotine cocrystal prepared by the method for preparing the nicotinamide mononucleotide cocrystal does not affect the pharmaceutical activity of nicotinamide mononucleotide and has higher bulk density than the existing crystal, so that the fluidity of the nicotinamide mononucleotide is obviously improved, and the technical problems of larger loading/weight difference and inconsistent quality of NMN (N-methyl nicotinamide mononucleotide) medicine or health-care product in enterprise production can be well solved.

3. The invention provides a preparation method capable of improving the bulk density of nicotinamide mononucleotide crystals.

Drawings

Figure 1 is an X-ray powder diffraction pattern of a nicotinamide mononucleotide-isonicotin co-crystal provided by the invention;

FIG. 2 is a differential scanning calorimetry trace of a nicotinamide mononucleotide-isonicotine cocrystal provided by the invention.

Detailed Description

The present invention will be described in further detail with reference to the following drawings and specific examples, which are illustrative of the present invention and are not intended to limit the present invention.

The raw materials and reagents used in the following examples were all commercially available unless otherwise specified.

The nicotinamide mononucleotide anhydrous crystal (form 1) is prepared by referring to the method disclosed in example 1 in Chinese patent application CN 108697722A;

nicotinamide mononucleotide dimethyl sulfoxide solvate crystals (form 2) were prepared by referring to the method disclosed in example 4 of chinese patent application CN 108697722A.

Specifically, the method for preparing nicotinamide mononucleotide co-crystal provided by the invention comprises the following two schemes (wherein the organic solvent is tetrahydrofuran, acetonitrile or acetone):

scheme one

Mixing an organic solvent and water according to the volume ratio of 1:1-5 to obtain a mixed system of the organic solvent and the water, adjusting the temperature of the mixed system to be 30-55 ℃, adding the nicotinamide mononucleotide and the isonicotinine into the mixed system according to the molar ratio of 1:1, uniformly mixing, then reducing the temperature of the mixed system to 4-8 ℃, standing, and waiting for crystal precipitation.

Scheme two

Dissolving the nicotinamide mononucleotide and the isonicotinic in water according to the molar ratio of 1:1 of the nicotinamide mononucleotide to the isonicotinic, slowly dropwise adding an organic solvent accounting for 0.2-1 time of the volume of the water into the water in which the nicotinamide mononucleotide and the isonicotinic are dissolved to obtain a mixed system of the organic solvent and the water, keeping the temperature of the mixed system of the organic solvent and the water at 30-55 ℃ in the dropwise adding process, stirring while dropwise adding, reducing the temperature of the mixed system to 4-8 ℃ after the dropwise adding is finished, standing, and waiting for crystal precipitation.

Example 1

The method for preparing the nicotinamide mononucleotide-isonicotine eutectic provided by the invention is adopted to prepare the nicotinamide mononucleotide-isonicotine eutectic

Dissolving 67g of beta-nicotinamide mononucleotide and 24g of isonicotinic acid in 2L of water, slowly dropwise adding 2L of tetrahydrofuran while stirring, keeping the temperature of the solution at 45 ℃ in the dropwise adding process, reducing the temperature of the solution to 6 ℃ after the dropwise adding is finished, standing, waiting for crystal precipitation, and filtering the solution after the crystallization is finished to obtain the nicotinamide mononucleotide-isonicotinic acid eutectic crystal.

Example 2

The method for preparing the nicotinamide mononucleotide-isonicotine eutectic provided by the invention is adopted to prepare the nicotinamide mononucleotide-isonicotine eutectic

Dissolving 67g of beta-nicotinamide mononucleotide and 24g of isonicotinine in 2L of water, then slowly dropwise adding 2L of acetonitrile into the water while stirring, keeping the temperature of the solution at 55 ℃ in the dropwise adding process, reducing the temperature of the solution to 4 ℃ after the dropwise adding is finished, standing, waiting for crystal precipitation, and filtering the solution after the crystallization is finished to obtain the nicotinamide mononucleotide-isonicotinine eutectic.

Example 3

The method for preparing the nicotinamide mononucleotide-isonicotine eutectic provided by the invention is adopted to prepare the nicotinamide mononucleotide-isonicotine eutectic

Dissolving 67g of beta-nicotinamide mononucleotide and 24g of isonicotinine in 2L of water, slowly dropwise adding 2L of acetone into the water while stirring, keeping the temperature of the solution at 30 ℃ in the dropwise adding process, reducing the temperature of the solution to 8 ℃ after the dropwise adding is finished, standing, waiting for crystal precipitation, and filtering the solution after the crystallization is finished to obtain the nicotinamide mononucleotide-isonicotinine eutectic.

Example 4

The method for preparing the nicotinamide mononucleotide-isonicotine eutectic provided by the invention is adopted to prepare the nicotinamide mononucleotide-isonicotine eutectic

Mixing 2L of acetone and 2L of water to obtain a mixed solution of acetone and water, adjusting the temperature of the mixed solution to 30 ℃, adding 67g of beta-nicotinamide mononucleotide and 24g of isonicotinic acid into the mixed solution, stirring to dissolve and uniformly mix, then reducing the temperature of the mixed solution to 8 ℃, standing, waiting for crystal precipitation, and filtering the solution after crystallization is finished to obtain the nicotinamide mononucleotide-isonicotinic acid eutectic.

Example 5

X-ray powder diffraction of nicotinamide mononucleotide-isonicotine cocrystals prepared in examples 1-4:

using a Pasnake X' Pert sharp X-ray powder diffractometer (PW3040/60, Dutch Pasnake analysis Instrument Co., Ltd.), Cu-Ka radiation, wavelength1 degree of divergent slit, 45kV of X-ray light tube voltage, 40mA of X-ray light tube current, 2-40 degrees (2 theta) of scanning range, 0.0130 degree of step size, step size time: 78.7950 s. And flattening the powder sample, placing the powder sample on a micro sample tray, and detecting. The X-ray powder diffraction pattern of the nicotinamide mononucleotide-isonicotin eutectic provided by the invention is shown in figure 1, and the corresponding peak and intensity of the diffraction angle 2 theta are shown in table 1.

TABLE 1

2θ(°)	Relative Strength (%)
		9.6	15.3
9.8	6.8
		10.6	8.9
13.3	100.0
		16.3	16.5
17.1	3.1
		19.4	4.7
20.1	4.3
		21.3	11.9
21.8	5.5
		22.8	28.2
25.7	2.5
		26.2	3.2
26.8	4.9
		31.4	3.7
32.1	15.3
		32.6	4.6
32.9	3.5
		36.2	6.0
36.5	23.4

Example 6

Differential Scanning Calorimetry (DSC) measurements of nicotinamide mononucleotide-isonicotine cocrystals prepared in examples 1-4:

DSC measurements were performed in a TA Instruments Q2000 using a sealed tray apparatus. Samples (approximately 1-3 mg) were weighed in aluminum pans, capped with Tzero, precision recorded to one hundredth of a milligram, and transferred to the instrument for measurement. The instrument was purged with nitrogen at 50 mL/min. Data were collected between room temperature and 220 ℃ at a heating rate of 10 ℃/min. The endothermic peak was plotted downward and the data was analyzed by TA Universal Analysis. A Differential Scanning Calorimetry (DSC) chart of the nicotinamide mononucleotide-isonicotine eutectic provided by the present invention is shown in FIG. 2, wherein the abscissa represents Temperature (deg.C) and the ordinate represents Heat Flow (W/g) released by a substance per unit mass.

Example 7

Bulk Density determination

Appropriate samples of the form 1 crystal, the form 2 crystal, and the nicotinamide mononucleotide-isonicotinine cocrystal prepared in examples 1-4 were separately sieved (1.00mm number 18), precisely weighed, slowly poured into a glass graduated cylinder, scraped to the top, recorded in apparent volume, and calculated in bulk density, with the experimental results shown in table 2.

TABLE 2

Crystal	Bulk density g/ml
		Form 1	0.15
Form 2	0.22
		Co-crystal of example 1	0.67
Co-crystal of example 2	0.68
		Co-crystal of example 3	0.68
Co-crystal of example 4	0.56

Example 8

Measurement of filling amount difference

Taking a proper amount of the form 1 crystal, the form 2 crystal and the nicotinamide mononucleotide-isonicotinine eutectic prepared in the examples 1 to 4 respectively, sieving with a 200-mesh sieve, fixing the capsule shell on a capsule plate, filling powder on the plate, pouring the powder on the plate, scraping the powder back and forth by using a powder scraping plate, and scraping the redundant powder on the plate after the capsule shell is filled with the powder to obtain the capsule. Then, the filled capsules are measured by referring to a method for checking capsules (difference in filling amount) in 0103 capsule general rules of pharmacopoeia of the people's republic of China (2020 edition), the filling amount difference value X (%) after each filling amount corresponding to each group of the form 1 crystal, the form 2 crystal and the cocrystals of examples 1 to 4 is compared with the average filling amount of the group is respectively calculated, then, the absolute value is taken for each filling amount difference value, and the average value of each group is calculated The results are shown in Table 3.

TABLE 3