5'-Cytidine monophosphate monohydrate crystal and preparation method thereof

Abstract

The present invention discloses a 5'-cytidine monophosphate monohydrate crystal, the molecular formula of which is C9H 16 N3O9P, belongs to the monoclinic system, the space group is P 21 , unit cell parameters a(Å)=4.858, b(Å)=8.3267, c(Å)=17.0704, β=90.126°, unit cell volume V=690.515Å 3 , the minimum number of asymmetric units in the unit cell Z=2. The present invention also provides a method for preparing the above crystal. The crystallization method of the present invention is simple and easy to control, and the obtained 5'-cytidylic acid monohydrate crystal has good fluidity, good stability, uniform particle size distribution, high yield, and the product can be stored stably for a long time.

Description

Technical Field

[0001] The invention belongs to the field of refining food additives, and specifically relates to a crystal structure of 5'-cytidine monophosphate monohydrate and a crystallization method thereof. Background Art

[0002] 5'-Cytidine monophosphate, alias: 5'-monophosphate cytidine, cytosine (ribose) nucleotide (English: 5'-cytidylicacid, hereinafter referred to as 5'-CMP), polar compound, amphoteric electrolyte; the N-glycosidic bond of 5'-CMP is relatively stable, and only the phosphate group is cleaved under severe hydrolysis conditions. CDP and CTP can be generated by phosphorylation. The main function is to serve as a raw material for nucleic acid biosynthesis and participate in the activation of metabolites (such as choline, riboyl alcohol, etc.). The molecular structure of 5'-CMP is as follows:

[0003]

[0004] 5'-CMP is insoluble in organic solvents such as benzene and ethanol. The 98% UV absorbance ratio is: A250 nm / A260 nm is 0.41-0.49, A280 nm / A260 nm is 2.03-2.17, and the water content is 5.28%.

[0005] 5'-CMP is an important biochemical substance in biological cells and participates in a variety of physiological and biochemical reactions. In industry, 5'-cytidylic acid is mainly used to manufacture drugs such as cytidine diphosphate choline, cytidine triphosphate, cytarabine, and polyinosin. In recent years, it can also be added to milk powder with other nucleotides to enhance the immunity of infants. In 1965, Japan added nucleotides to infant milk powder, and Spain and the United States began to recommend their use in 1983 and 1989 respectively. In 1991, the European Community began to make specific regulations on the level of nucleotide addition in infant food. In the early 1990s, imported brands of infant milk powder containing nucleotides began to appear in the domestic market. There are also relevant patents in China that introduce high-energy milk with added nucleic acids or nucleotides, which is easily absorbed by the human body, can promote blood circulation, improve brain function, promote metabolism, resist fatigue, enhance physical fitness, and improve immunity. 5'-CMP can not only be used in infant formula milk powder, but also in pharmaceutical intermediates and a new generation of health feed additives. The main production methods of 5'-CMP are: RNA (ribonucleic acid) degradation, chemical synthesis, enzyme catalysis synthesis and biological fermentation. At present, the chemical method is mainly used in the industrial synthesis of 5'-CMP. The main disadvantages of this method are high raw material cost, more waste, corrosion equipment, and it is not conducive to the health of personnel and industrial production. There are also reports on enzyme catalysis and biological fermentation methods. For example, in patent CN 202111282072.1, 5'-CMP is extracted from microbial fermentation broth after high-temperature inactivation, acid adjustment, membrane filtration, concentration, decolorization and dissolution crystallization. However, there are problems such as low yield and cumbersome operation. The traditional crystallization method is to freeze below 10°C until crystals are precipitated. Although this method has low equipment requirements, it is easy to cause explosive nucleation, resulting in serious particle aggregation and impurity encapsulation, which ultimately makes the crystal size uneven, the bulk density is small, the crystallinity is low, and the stability is poor. In industrial production, multiple low-temperature alcohol precipitation crystallization is also used, but its process flow is long, the operation is cumbersome, the solvent consumption is large, and the purity and yield of the obtained product are low. In addition, the reported 5'-cytidine monophosphate trihydrate (Acta Cryst. (1979). B35, 2141-2144), belongs to the orthorhombic system, and is obtained by evaporation crystallization in a water-rich isopropanol solution, but has problems such as instability under storage conditions; there are currently few literature reports on 5'-CMP crystallization in China, and process optimization is progressing slowly. Therefore, how to develop high-quality 5'-CMP crystal products suitable for industrial production and their preparation methods is imminent. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a high-quality 5'-cytidylic acid monohydrate crystal and a preparation method thereof in view of the deficiencies in the prior art. The prepared 5'-cytidylic acid monohydrate crystal has good fluidity, good stability, uniform particle size distribution, high yield, and the product can be stored stably for a long time.

[0007] In order to achieve the above technical purpose, the present invention discloses a 5'-cytidine monophosphate monohydrate crystal with good stability and high bulk density, and its molecular formula is C9H 14 N3O8P·H2O, belongs to the monoclinic system, space group is P 21 , unit cell parameters β=90.126°, unit cell volume The minimum asymmetric unit number in the unit cell is Z = 2. In one minimum asymmetric unit, there is one 5'-cytidine monophosphate and one crystal water. The minimum asymmetric unit diagram and unit cell structure diagram are basically as follows Figure 1 and Figure 2 The unit cell stacking diagram is basically as follows Figure 3 , detailed data of its crystal structure - bond angle information such as Figure 8 During the crystal structure determination process, the diffraction data were collected at 298K.

[0008] The monohydrate crystals are as follows Figure 4 The diffraction pattern of the diffraction analysis using CuKa rays as characteristic X-rays has characteristic absorption peaks at diffraction angles 2θ±0.1, such as 5.21°, 10.36°, 11.83°, 15.55°, 18.28°, 18.98°, 21.78°, 23.62°, and 30.13°.

[0009] The crystalline powder of the above 5'-CMP is white or off-white.

[0010] The crystalline powder formed by the above crystals has a bulk density greater than 0.20 g / mL, preferably greater than 0.30 g / mL, and more preferably greater than 0.40 g / mL.

[0011] The crystalline powder formed by the above crystals has a tap density greater than 0.23 g / mL, preferably greater than 0.33 g / mL, and more preferably greater than 0.44 g / mL.

[0012] The crystalline powder formed by the above crystals has a d50 greater than 10 μm, preferably greater than 30 μm, more preferably greater than 50 μm, and most preferably greater than 60 μm.

[0013] The crystalline powder formed by the above crystals has a d10 greater than 3 μm, preferably greater than 6 μm, more preferably greater than 10 μm, and most preferably greater than 15 μm.

[0014] The d50 and d10 described in the present invention are common quantities used to indicate particle size distribution. The d50 described is a value for a particle size such that 50 vol.% of crystals have a size smaller than this value. The d10 described is a value for a particle size such that 10 vol.% of crystals have a size lower than this value.

[0015] The bulk density and tap density are quantities related to the flow characteristics of the powder. In general, high bulk density and tap density values ​​are desired. In short, the bulk density indicates the weight of the powder per volume unit under predetermined conditions. In other words, how many grams of powder can be supplied to a sample holder with a specific volume under predetermined conditions. For this reason, the bulk density is expressed as the weight per volume unit, usually in g / mL. The tap density also indicates the weight per volume unit of powder, in this case, when the holder of the powder is subjected to slapping or vibration under predetermined conditions. The tap density is expressed as the weight per volume unit, usually in g / mL. By slapping or vibrating, more powder can be supplied to the holder. Therefore, for the same powder, its tap density is higher than the bulk density.

[0016] Powders with high bulk density and tap density tend to have a larger specific gravity, which reflects that the crystal product is relatively thick and textured, and its stability is relatively good. From another perspective, products with high bulk density generally have better fluidity of particles, which are also convenient for storage and transportation, and more convenient for efficient mixing with other components.

[0017] Specifically, the particle size distribution, bulk density and tap density are measured as follows:

[0018] Determine the particle size distribution (including d10 and d50) of the sample from the mixer using a Malvern instruments particle size analyzer, dry method;

[0019] The bulk density of the granules was determined according to USP Method II (page 1914);

[0020] The tap density of the particles is measured by FZS4-4 economical tap density tester according to GB / T 5162-2006. Specifically, the measuring conditions are: the vibration stroke of the tapping device is 3±0.1MM, and the vibration frequency is 250±15 times per minute.

[0021] It should be noted that the above-mentioned crystalline powder may show different apparent water contents (including free water and bound water) due to differences in the preparation method, or different drying methods, or different storage environment humidity. Therefore, the crystalline powder may have a water content of 4.5-6%.

[0022] Specifically, the method for determining the water content of the crystalline powder is the Karl Fischer method, which can be determined by using the 870Titrino plus practical volumetric Karl Fischer moisture analyzer produced by Swiss Metrohm.

[0023] When the above crystals are prepared into a 5% by mass aqueous solution, the pH value of the system is between 2 and 5, preferably between 3 and 4.

[0024] The crystalline powder described in the present invention refers to powder with a certain degree of crystallinity, relative to amorphous powder.

[0025] The crystal described in the present invention is a solid having a clear diffraction pattern for X-rays, and its atoms or molecules are repeatedly arranged in space according to a certain regular period.

[0026] The present invention further provides a method for preparing the above-mentioned 5'-cytidine monophosphate monohydrate crystal, which is characterized by comprising the following steps:

[0027] (1) placing an aqueous solution of 5'-cytidine monophosphate in a crystallizer equipped with a probe-type ultrasonic device, adjusting the pH to 4.85 to 5.00, preferably using sodium hydroxide, and stabilizing at 10 to 40° C. for 30 to 40 minutes;

[0028] (2) Turn on the probe ultrasonic device and apply it to the reaction system of step (1) at a power of 20 to 50 W. After maintaining for 20 to 40 minutes, add the reactant until the pH of the system drops to 3.9 to 4.0. If turbidity is observed in the system during the addition, continue ultrasonication for 2 to 5 minutes and then turn off the ultrasonic transmitter. Cultivate the crystals at a stirring rate of 180 to 200 rpm until a large number of crystal particles appear in the system and no longer increase.

[0029] (3) Continue to add the reactant to the system obtained in step (2), and when the pH of the system drops to 0.5-2.0, stop adding the reactant and simultaneously add 0.2 times the volume of the raw material liquid solvent, while maintaining stirring during the addition;

[0030] (4) Stop adding the solvent, maintain the system temperature between 25 and 40 °C, and continue stirring for 3 to 6 hours;

[0031] (5) The slurry is separated into solid and liquid, washed, and dried by air blowing to obtain the slurry.

[0032] In step (1), the reaction concentration of 5'-CMP is 25 g / L to 200 g / L, preferably 100 g / L to 150 g / L.

[0033] In step (2), ultrasound is used to promote nucleation, and the ultrasound device is a probe-type ultrasound, which acts in a continuous (non-pulse) manner.

[0034] In step (2), when the pH of the system is 3.9-4.0, 5'-CMP mainly exists in the form of zwitterion.

[0035] In step (2), the ultrasonic intervention time needs to be stopped before a large number of nuclei are formed. The cavitation effect of ultrasound is used to enable the solute molecules in the system in a supersaturated state to obtain additional energy, reduce the surface energy of aggregation between solute molecules, and promote the irregular solute molecule aggregates in an associated state to overcome the nucleation energy barrier, thereby undergoing regular adjustments to form crystal nuclei and grow into visible grains. However, it is necessary to prevent the crystals from breaking due to excessive ultrasonic time. If turbidity is observed in the system during the process of adding the reactants to pH 3.9-4.0, continue ultrasonication for 2-5 minutes and then turn off the ultrasonic transmitter.

[0036] In step (2), the reactant needs to be slowly added dropwise or pumped in. A fast speed is not conducive to the growth of crystals, and a too slow speed will reduce the efficiency of the crystallization process. Preferably, the addition rate of the reactant is 0.2-0.4 mL / min, preferably 0.2 mL / min. It should be noted that if the system is enlarged or used for industrial production, the flow rate can be adjusted according to the actual situation.

[0037] In step (3), the amount of reactant used must be controlled taking into account the volume of the system and controlling the amount of reactant added, i.e., controlling the pH endpoint of the system so that the final pH falls within the zwitterionic state interval of the 5'-CMP ion distribution curve.

[0038] In steps (3) and (4), the reactant is hydrochloric acid with a concentration of 1 to 3 mol / L. If the concentration of hydrochloric acid is too low, the induction period will be prolonged, and if the concentration is too high, the system will be prone to explosive nucleation.

[0039] The solvent is anhydrous ethanol; the solvent is added to further reduce the solubility of the solute in the system, so that more solute is precipitated and the crystallization yield is improved; the role of the added reactant in the system is to gradually transform 5'-CMP from an ionic state to a molecular state, so that 5'-CMP is precipitated; there is no specific requirement for the addition rate of the solvent, and it is better to add it slowly.

[0040] In step (4), when the pH of the system drops to 0.5-2.0, stop adding the solvent and the reactant, maintain the system temperature unchanged, and continue stirring for 3-6 hours, in order to allow the 5'-CMP crystals to undergo a post-ripening process and make the particle distribution of the crystals more uniform.

[0041] In step (5), the method of solid-liquid separation of the slurry is suction filtration or centrifugation.

[0042] In step (5), washing is necessary, and can be performed using solvents such as ethanol, isopropanol, methanol, propanol, etc. Preferably, washing with 90% by volume ethanol is beneficial for washing away impurities in the slurry.

[0043] The crystallization temperature of the 5'-cytidine monophosphate is 10-40°C.

[0044] In step (5), the drying is carried out by forced air drying at 40 to 50°C. It should be pointed out that too high a drying temperature will easily cause the product to lose part of its crystalline water, thereby causing moisture absorption by subsequent products, and too low a temperature will reduce the drying efficiency. The drying time depends on the amount of the sample. For example, for a wet filter cake less than 1kg, it takes 4 to 8 hours to complete the drying when it is spread out to a thickness of less than 1cm. It is worth noting that the above-mentioned crystalline powder will show different apparent water contents (including free water and bound water) due to different drying methods or different storage environment humidity. Therefore, the crystalline powder may have a water content of 4.00 to 6.50wt%, preferably 4.80 to 5.25wt%.

[0045] The water content of the crystalline powder is determined by the Karl Fischer method, which can be determined by using the 870Titrino plus practical volumetric Karl Fischer moisture meter produced by Swiss Metrohm.

[0046] Beneficial effects: The crystallization method of the present invention is simple and easy to control, and the obtained 5'-cytidylic acid monohydrate crystals have good fluidity, good stability, uniform particle size distribution, high yield, and the product can be stored stably for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is a ball-and-stick diagram of the minimum asymmetric unit of 5'-cytidine monophosphate;

[0048] Figure 2 is the unit cell stacking diagram of 5'-cytidine monophosphate along the b-axis;

[0049] Figure 3 is the 3×3×3 unit cell stacking diagram of 5'-cytidine monophosphate along the b-axis;

[0050] Figure 4 is the x-ray powder diffraction pattern of 5'-cytidine monophosphate;

[0051] Figure 5 is the infrared spectrum of 5'-cytidine monophosphate;

[0052] Figure 6 The humidity stability PXRD pattern of 5'-cytidine monophosphate;

[0053] Figure 7is the bond angle information of 5'-cytidine monophosphate;

[0054] Figure 8 is a particle size analysis diagram of 5'-cytidine monophosphate;

[0055] Fig. 9 is the TG-DSC graph of 5'-cytidine monophosphate;

[0056] Fig.10 The electron microscope images of 5'-cytidine monophosphate, where (a) is without ultrasound and (b) (c) is with ultrasound.

[0057] Fig.11 Figure 2 is a state diagram of 5'-cytidine monophosphate trihydrate.

[0058] Fig.12 This is the distribution curve of 5'-cytidine monophosphate ion. DETAILED DESCRIPTION

[0059] The detection method and instrument of the crystal structure of 5'-CMP of the present invention are as follows:

[0060] Single crystal X-ray diffraction to determine the crystal structure and analysis method: Take the cultured 5'-CMP single crystal with good quality and cut it into about 0.22×0.24×0.26mm 3 The blocks of different sizes were analyzed by Bruker APEX-II CCD diffractometer with Mo Kα radiation source (graphite monochromator, ) to irradiate the sample and collect diffraction data. The diffraction data were reduced by SAINT and then directly analyzed by SHELXTL software. 2 The full matrix least squares refinement was performed, and all non-hydrogen atoms were refined by anisotropic refinement. The final data were mapped using Mercury or Diamond software.

[0061] Powder X-ray diffraction: about 0.100 g of the ground sample was taken and the diffraction data were collected at room temperature using a powder X-ray diffractometer (Rigaku Smartlab or Bruker D8 Advance) with Cu Kα ray as the light source, a scanning step of 0.02°, a scanning voltage of 40 kV, a current of 40 mA, a scanning rate of 0.2 s / 0.02°, and a scanning range of 2θ of 5 to 50°. The data were processed and plotted using JADE software.

[0062] Infrared spectrum: About 0.001 g of the ground sample was taken and background subtraction and data collection were performed using a Fourier transform infrared spectrometer (ThermoFisher scientific, USA) at room temperature with a resolution of 4 cm -1 , the collection range is 4000~500cm -1 .

[0063] Thermogravimetric analysis and differential scanning calorimetry: About 0.05 g of the ground sample was taken and placed in the sample pans of the thermogravimetric analyzer and the differential scanning calorimeter, and the temperature was measured from room temperature to 500°C. The atmosphere used was nitrogen and the heating rate was 20°C / min.

[0064] Particle size analysis: About 0.1 g of 5'-CMP crystal powder was taken and measured by a particle size analyzer (Malverninstruments) at room temperature. The dispersant was ethanol.

[0065] The present invention is described in detail below through specific embodiments and drawings.

[0066] Example 1

[0067] Take 50g of 5'-cytidylic acid amorphous solid powder, prepare it into 1000mL aqueous solution, adjust the pH to 5.0 with 3mol / L sodium hydroxide, put it into a crystallizer with a probe-type ultrasonic transmitter, start stirring at a speed of 200rpm, control the temperature at 10°C throughout the process, and stabilize the system for 30min; turn on the ultrasonic transmitter, set the power to 20W, and pump 1.5mol / L hydrochloric acid at a flow rate of 0.2mL / min to observe the turbidity and pH changes in the system. When the pH drops to 4.0, stop adding hydrochloric acid and continue to maintain the ultrasonic state for 5min. If the system is obviously turbid, turn off the ultrasound and continue to stir for 2h for crystal cultivation; after 2h, continue to pump 1.5mol / L hydrochloric acid at a drip rate of 0.2mL / min, monitor the pH change of the system, stop adding acid when the pH of the system drops to 2.0, and pump anhydrous ethanol at a flow rate of 0.6mL / min. Stop dripping when the system is stable, continue to stir for 6h, and put it into the tank. The crystal slurry was filtered and washed with 60 mL of 90% by volume ethanol, and then dried at 40°C for 6 hours to obtain white crystalline solid particles. The crystallization yield was measured to be 94.5%, the bulk density was 0.41 g / mL, the fluidity was good, the liquid chromatography purity was 99.5%, and the moisture content measured by the Karl Fischer method was 4.9±0.5%. After the product was stored in a sealed container at room temperature for 3 months, there was no obvious change in color, purity, and fluidity.

[0068] Table 1 Single crystal structure data of 5'-cytidylic acid monohydrate

[0069]

[0070] Example 2

[0071] Take 150g of 5'-cytidylic acid amorphous solid powder, prepare it into 1000mL aqueous solution, adjust the pH to 5.2 with sodium hydroxide, put it into a crystallizer with a probe-type ultrasonic transmitter, start stirring, control the temperature at 25°C throughout the process, and stabilize the system for 30min; turn on the ultrasonic transmitter, set the power to 50W, and pump in 1.5mol / L hydrochloric acid at a flow rate of 0.2mL / min to observe the turbidity and pH changes in the system. When the pH drops to 4.0, stop adding hydrochloric acid and continue to maintain the ultrasonic state for 8min. If the system is found to be When the mixture becomes turbid, turn off ultrasound and continue stirring for 2 hours for crystal growth. After 2 hours, continue to pump in 1.5 mol / L hydrochloric acid at a drip rate of 0.2 mL / min, monitor the pH change of the system, and stop adding acid when the pH of the system drops to 2.0. At the same time, pump in anhydrous ethanol at a flow rate of 0.6 mL / min. Stop dripping when the system is stable and continue stirring for 6 hours. Filter the crystal slurry, wash it with 120 mL of 90% ethanol by volume, and dry it in a 40°C oven for 6 hours to obtain a white crystalline product of 5'-cytidylic acid. The crystal morphology is as follows: Fig.10 b, showing a relatively regular lamellar crystal habit. The crystallization yield was measured to be 95.3%, the bulk density was 0.43 g / mL, the fluidity was good, the liquid chromatography purity was 99.5%, and the water content measured by the Karl Fischer method was 4.9±0.5%. The corresponding PXRD spectrum is shown in Figure 4 After the product was stored in a sealed container at room temperature for 3 months, there was no significant change in color, purity and fluidity.

[0072] Example 3

[0073] Take 200g of 5'-cytidylic acid amorphous solid powder, prepare it into 1000mL aqueous solution, adjust the pH to 5.0 with 3mol / L sodium hydroxide, put it into a crystallizer with a probe-type ultrasonic transmitter, start stirring at a speed of 200rpm, control the temperature at 35°C throughout the process, and stabilize the system for 30min; turn on the ultrasonic transmitter, set the power to 30W, and pump 1.5mol / L hydrochloric acid at a flow rate of 0.2mL / min to observe the turbidity and pH changes in the system. When the pH drops to 4.0, stop adding hydrochloric acid and continue to maintain ultrasonic state for 15 minutes. If the system is found to be obviously turbid, turn off the ultrasound and continue stirring for 2 hours for crystal growth. After 2 hours, continue to pump in 1.5 mol / L hydrochloric acid at a drop rate of 0.2 mL / min, monitor the pH change of the system, and when the pH of the system drops to 2.0, stop adding acid and pump in 0.2 times the volume of the feed liquid of anhydrous ethanol at a flow rate of 0.6 mL / min. After the anti-solvent is added, continue to stir for 6 hours and put it into the tank. Filter the slurry, wash it with 60 mL of 90% ethanol by volume, and dry it at 40°C for 6 hours to obtain white crystalline solid particles with good crystal morphology (such as Fig.10c), the crystallization yield was 96.5%, the bulk density was 0.45 g / mL, the fluidity was good, the liquid chromatography purity was 99.5%, the water content measured by Karl Fischer method was 4.9±0.5%, and the corresponding crystal structure was as follows Figure 1-Figure 3 As shown in Table 1, the crystal information data is shown in Table 1 and Figure 7 The corresponding PXRD spectrum is shown in Figure 4 The corresponding infrared spectrum is shown in Figure 5 As shown, the characteristic peak is located at 3560cm -1 、3375cm -1 、3123cm -1 、2948cm -1 The corresponding particle size distribution curve is as follows Figure 8 It can be seen that the median particle size is 63.1 μm. After 3 months of sealed storage at room temperature, there is no obvious change in color, purity, fluidity, and crystal form. Figure 6 As shown, the TG-DSC thermal analysis diagram is as follows Fig. 9 As shown, the water loss weight ratio of the product is 5.20%.

[0074] Example 4

[0075] Take 200g of 5'-cytidylic acid amorphous solid powder, prepare it into 1000mL aqueous solution, adjust the pH to 5.0 with sodium hydroxide, put it into a crystallizer with a probe-type ultrasonic transmitter, start stirring, control the temperature at 40°C throughout the process, and stabilize the system for 30min; turn on the ultrasonic transmitter, set the power to 50W, and pump in 1.5mol / L hydrochloric acid at a flow rate of 0.2mL / min to observe the turbidity and pH changes in the system. When the pH drops to 4.0, stop adding hydrochloric acid and continue to maintain the ultrasonic state for 10min. If The system is obviously turbid, turn off the ultrasound, and continue stirring for 2 hours for crystal growth; after 2 hours, continue to pump in 1.5 mol / L hydrochloric acid at a drop rate of 0.2 mL / min, monitor the pH change of the system, and stop adding acid when the pH of the system drops to 2.0. At the same time, pump in anhydrous ethanol at a flow rate of 0.6 mL / min. Stop dropping when the system is stable and continue stirring for 6 hours; filter the crystal slurry, wash it with 100 mL of 90% ethanol by volume, and dry it in a 40°C oven for 6 hours to obtain a white crystalline product of 5'-cytidylic acid. The crystallization yield was measured to be 95.1%, the bulk density was 0.45 g / mL, the fluidity was good, the liquid chromatography purity was 99.5%, and the moisture content measured by the Karl Fischer method was 4.9±0.5%. The measured ion distribution curve is shown in the figure Fig.12 After the solid was stored in a sealed container at room temperature for 3 months, it was found that its color, purity and fluidity had no obvious changes.

[0076] Comparative Example 1

[0077] The same as Example 3, except that ultrasonic treatment is not used. Specifically, 200 g of 5'-cytidylic acid amorphous solid powder is taken and prepared into 1000 mL of aqueous solution, the pH is adjusted to 5.5 with sodium hydroxide, and it is placed in a crystallizer with a probe-type ultrasonic transmitter, stirring is turned on, and the temperature is controlled at 40°C throughout the process using a circulating water bath; hydrochloric acid with a concentration of 1.5 mol / L is pumped in at a flow rate of 0.2 mL / min to observe the turbidity and pH changes in the system, and when the pH drops to 4.0, the hydrochloric acid is stopped, and stirring is continued for 30 minutes, and the ultrasonic transmitter is kept in a closed state. After continuing for 30 minutes, there is no obvious change in the system and no phase change occurs. Continue to pump in 1.5 mol / L hydrochloric acid at a rate of 0.2 mL / min, monitor the pH change of the system, and when the pH is 3.89, the system has burst nuclei, continue to add acid until the pH is 2.0, stop adding, pump in 0.2 times the volume of the feed liquid anhydrous ethanol at a rate of 0.6 mL / min, continue stirring for 6 hours, and put the tank down. Filter the slurry, wash it with 60 mL of 90% ethanol by volume, and dry it in a 40°C oven for 6 hours. The dried sample has a hardened surface and poor crystal morphology (such as Fig.10 a), the crystallinity is low, the water content is measured to be 5.5±0.5%, and the product turns yellow after being stored in a sealed container at room temperature for 1 month.

[0078] Comparative Example 2

[0079] The same as Example 3, except for the ultrasonic pretreatment method, that is, the probe ultrasonic treatment was performed for 120 minutes before crystallization. Specifically, 200 g of 5'-cytidylic acid amorphous solid powder was prepared into 1000 mL of aqueous solution, the pH was adjusted to 5.5 with sodium hydroxide, and the solution was placed in a crystallizer with a probe-type ultrasonic transmitter. The ultrasound was turned on and the power was set to 50 W, maintained for 120 min, and the temperature of the circulating water bath was controlled to 40 ° C. At the same time, hydrochloric acid with a concentration of 1.5 mol / L was pumped in at a flow rate of 0.2 mL / min to observe the turbidity and pH changes in the system. When the pH of the system dropped to 2.0, the acid was stopped, and anhydrous ethanol was pumped in at a flow rate of 0.6 mL / min. When the system was stable, the dropwise addition was stopped, and stirring was continued for 6 h. It was observed that the crystals in the system agglomerated; the slurry was filtered, washed with 100 mL of ethanol with a volume fraction of 90%, and dried in a 40 ° C oven for 6 h to obtain a 5'-cytidylic acid crystal product. The dried sample showed a hardening phenomenon, and the crystal morphology was poor. The water content was measured to be 5.5±0.5%. After sealed storage at room temperature for 1 month, the product turned yellow.

[0080] Comparative Example 3

[0081] The same as Example 3, except that after adjusting the pH to 1.5-2.5, no ethanol is subsequently added. Specifically, 200 g of 5'-cytidylic acid amorphous solid powder is taken, prepared into 1000 mL of aqueous solution, the pH is adjusted to 5.5 with sodium hydroxide, and it is placed in a crystallizer with a probe-type ultrasonic transmitter, stirring is turned on, and the temperature is controlled at 40°C throughout the process; the ultrasonic transmitter is turned on, the power is set to 50 W, and hydrochloric acid with a concentration of 1.5 mol / L is pumped in at a flow rate of 0.2 mL / min to observe the turbidity and pH changes in the system. When the pH drops to 4.0, stop adding hydrochloric acid, and continue to maintain the ultrasonic state for 10 minutes. If the system is found to be obviously turbid, turn off the ultrasound and continue stirring for 2 hours Used for crystal growth; 2 hours later, continue to pump in 1.5 mol / L hydrochloric acid at a drip rate of 0.2 mL / min, monitor the pH change of the system, and when the pH of the system drops to 2.0, stop adding acid and continue stirring for 6 hours; filter the crystal slurry, wash it with 100 mL of 90% ethanol by volume, and dry it in a 40°C oven for 6 hours to obtain a 5'-cytidylic acid crystal product. The sample after drying shows agglomeration and the crystal morphology is poor. The water content is measured to be 5.5±0.5%. After the product is sealed and stored at room temperature for 1 month, there is no obvious change in color, purity, and fluidity.

[0082] Comparative Example 4

[0083] Same as implementation 3, except that the time of ultrasonic intervention is different. Specifically, take 200g of 5'-cytidylic acid amorphous solid powder, configure it into 1000mL aqueous solution, adjust the pH to 5.5 with sodium hydroxide, put it into a crystallizer with an ultrasonic transmitter, and control the temperature to 40°C throughout the process; pump hydrochloric acid with a concentration of 1.5mol / L at a flow rate of 0.2mL / min to observe the turbidity in the system and monitor the pH change. If the system is found to be turbid, turn on the ultrasonic device and set the power to 50W, maintain the ultrasonic state for 20min, and observe the changes in the crystal nucleus in the system (microscope examination); when a large number of crystals appear in the system, stop ultrasound and continue stirring for 2h for crystal cultivation. After 2h, pump 0.2 times the volume of the feed liquid with anhydrous ethanol at a flow rate of 1mL / min, and at the same time pump 2% hydrochloric acid with a mass fraction at a flow rate of 0.2mL / min, detect the change in pH in the system, and when the pH drops to 1.5, stop the flow addition and continue stirring for about 4h. The crystal slurry was filtered and washed with 100 mL of 90% by volume ethanol, and then dried in a 40° C. oven for 8 h to obtain a white powder product of 5'-cytidine monophosphate. The water content was measured to be 5.5±0.5%. The product became compacted after drying and had a low bulk density.

[0084] Comparative Example 5

[0085] According to the method for preparing 5'-cytidine monophosphate trihydrate reported in the literature (Acta Cryst. (1979). B35, 2141-2144), 5'-cytidine monophosphate crystals were obtained. Specifically, 1.00 g of monohydrate crystals were placed in 5 mL of 1 vol.%, 5 vol.%, and 10 vol.% isopropanol solutions, respectively, stirred for 12 hours, and then placed in a 40°C oven and slowly evaporated until the solvent was completely volatilized. The crystal growth state was observed as follows: Fig.11 The water content determined by Karl Fischer was 13.80% ± 0.5%, which was close to the theoretical trihydrate content. The 5'-cytidine monophosphate obtained by evaporation crystallization was placed at room temperature for 24 hours, and it was found that the crystals lost water. After 15 days of sealed storage at room temperature and pressure, it was found that the surface of the crystals turned yellow, became damp and hardened, and had poor fluidity.

[0086] Comparative Example 6

[0087] According to the method for refining high-purity nucleotides in patent CN 108892699 A, similarly, the obtained 5'-cytidine monophosphate solution is heated to 60°C, kept warm for 2 hours, then ethanol is added at a flow rate of 1 mL / min, kept warm for 30 minutes, slowly cooled to 40-45°C, kept warm for 30 minutes, cooled to 30°C, kept warm for 30 minutes, cooled to 20-25°C, kept warm for 1 hour, and filtered. The product is sequentially washed with 75% ethanol, 85% ethanol, and 95% ethanol by volume, and dried under reduced pressure to obtain a 5'-cytidine monophosphate crystalline white powder product. After drying, crystals aggregate and agglomerate, and the crystals have low fluidity and poor crystal morphology after crushing.

[0088] Comparative Example 7

[0089] According to the 5'-cytidylic acid purification method in patent CN 105348346 A, similarly, 80 g / L of 5'-cytidylic acid monophosphate solution was prepared, and after adjusting the pH to 2.5-3.0 with hydrochloric acid, 1.5 times the volume of ethanol of the original eluent was added to precipitate crystals, and the 5'-cytidylic acid monophosphate product was obtained by filtration, rinsing, and drying. During filtration, the sample adhered to the inner wall of the funnel and was not easy to rinse. After drying, it became hardened and the crystal morphology was poor.

[0090] The present invention provides a 5'-cytidine monophosphate crystal and a method for preparing the same. The above is only a preferred embodiment of the present invention. It should be noted that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be considered as the protection scope of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing 5'-cytidine monophosphate monohydrate crystals, characterized in that: The steps include: Take 50 g of 5'-cytidylic acid amorphous solid powder, prepare it into 1000 mL of aqueous solution, adjust the pH to 5.0 with 3 mol / L sodium hydroxide, put it into a crystallizer with a probe-type ultrasonic transmitter, start stirring at a speed of 200 rpm, control the temperature at 10°C throughout the process, and stabilize the system for 30 min; turn on the ultrasonic transmitter, set the power to 20 W, and pump 1.5 mol / L hydrochloric acid at a flow rate of 0.2 mL / min to observe the turbidity and pH changes in the system. When the pH drops to 4.0, stop adding hydrochloric acid and continue to maintain the ultrasonic state for 5 min. If the system is obviously turbid, turn off the ultrasound and continue to stir for 2 h for crystal cultivation; 2 h later, continue to pump 1.5 mol / L hydrochloric acid at a drop rate of 0.2 mL / min, monitor the pH change of the system, and stop adding acid when the pH of the system drops to 2.

0. At the same time, pump anhydrous ethanol at a flow rate of 0.6 mL / min, stop dropping when the system is stable, and continue to stir for 6 h, put the slurry into the tank, filter it, wash it with 60 mL of 90% ethanol, and dry it at 40 ° C for 6 h to obtain white crystalline solid particles. The molecular formula of the prepared 5'-cytidine monophosphate monohydrate is C9H 14 N3O8P·H2O, belongs to the monoclinic system, space group is P 21 , unit cell parameters a(Å)=4.858, b(Å)=8.3267, c(Å)=17.0704, β=90.126 °, unit cell volume V=690.515 Å 3 The minimum number of asymmetric units in the unit cell is Z = 2. In a minimum asymmetric unit, there is one 5'-cytidine monophosphate and one crystal water.

2. A method for preparing 5'-cytidine monophosphate monohydrate crystals, characterized in that: The steps include: Take 150 g of 5'-cytidylic acid amorphous solid powder, prepare it into 1000 mL aqueous solution, adjust the pH to 5.2 with sodium hydroxide, put it into a crystallizer with a probe-type ultrasonic transmitter, start stirring, control the temperature at 25°C throughout the process, and stabilize the system for 30 min; turn on the ultrasonic transmitter, set the power to 50 W, and pump 1.5 mol / L hydrochloric acid at a flow rate of 0.2 mL / min to observe the turbidity and pH changes in the system. When the pH drops to 4.0, stop adding hydrochloric acid and continue to maintain the ultrasonic state for 8 min. If the system is obviously turbid, turn off the ultrasound and continue to stir for 2 h for crystal cultivation; after 2 h, continue to pump 1.5 mol / L hydrochloric acid at a drop rate of 0.2 mL / min, monitor the pH change of the system, and stop adding acid when the pH of the system drops to 2.

0. At the same time, pump anhydrous ethanol at a flow rate of 0.6 mL / min, stop dropping when the system is stable, and continue to stir for 6 h; filter the crystal slurry and use 120 After washing with 90% ethanol by volume, the mixture was placed in a 40°C oven and dried for 6 h to obtain white crystalline solid particles. The molecular formula of the prepared 5'-cytidine monophosphate monohydrate is C9H 14 N3O8P·H2O, belongs to the monoclinic system, space group is P 21 , unit cell parameters a(Å)=4.858, b(Å)=8.3267, c(Å)=17.0704, β=90.126 °, unit cell volume V=690.515 Å 3 The minimum number of asymmetric units in the unit cell is Z = 2. In a minimum asymmetric unit, there is one 5'-cytidine monophosphate and one crystal water.

3. A method for preparing 5'-cytidine monophosphate monohydrate crystals, characterized in that: The method comprises the following steps: taking 200 g of 5'-cytidylic acid amorphous solid powder, preparing it into 1000 mL of aqueous solution, adjusting the pH value to 5.0 with 3 mol / L sodium hydroxide, placing it in a crystallizer with a probe-type ultrasonic transmitter, starting stirring at a speed of 200 rpm, controlling the temperature to 35°C throughout the process, and stabilizing the system for 30 min; turning on the ultrasonic transmitter, setting the power to 30 W, and simultaneously pumping in 1.5 mol / L hydrochloric acid at a flow rate of 0.2 mL / min to observe the turbidity and pH changes in the system; when the pH drops to 4.0, stopping the flow of hydrochloric acid, and continuing to maintain the ultrasonic state for 15 min; if the system is found to be obviously turbid, turning off the ultrasound, and continuing to stir for 2 h for crystal cultivation; after 2 h, continuing to pump in 1.5 mol / L hydrochloric acid at a drip rate of 0.2 mL / min, monitoring the pH change of the system process, and when the pH of the system drops to 2.0, stopping the addition of acid, and simultaneously pumping in 0.6 mL / min flow rate pumped 0.2 times the volume of the feed liquid anhydrous ethanol, after the anti-solvent addition was completed, continued stirring for 6 h, the tank was put down, the slurry was filtered, and washed with 60 mL of 90% ethanol by volume, and then placed in 40 ° C forced air drying for 6 h to obtain white crystalline solid particles, wherein the prepared 5'-cytidine monophosphate monohydrate molecular formula is C9H 14 N3O8P·H2O, belongs to the monoclinic system, space group is P 21 , unit cell parameters a(Å)=4.858, b(Å)=8.3267, c(Å)=17.0704, β=90.126 °, unit cell volume V=690.515 Å 3 The minimum number of asymmetric units in the unit cell is Z = 2. In a minimum asymmetric unit, there is one 5'-cytidine monophosphate and one crystal water.

4. A method for preparing 5'-cytidine monophosphate monohydrate crystals, characterized in that: The method comprises the following steps: taking 200 g of 5'-cytidylic acid amorphous solid powder, preparing it into 1000 mL of aqueous solution, adjusting the pH to 5.0 with sodium hydroxide, placing it in a crystallizer with a probe-type ultrasonic transmitter, starting stirring, controlling the temperature to 40°C throughout the process, and stabilizing the system for 30 min; starting the ultrasonic transmitter, setting the power to 50 W, and simultaneously pumping in hydrochloric acid with a concentration of 1.5 mol / L at a flow rate of 0.2 mL / min to observe the turbidity and pH changes in the system; when the pH drops to 4.0, stopping the flow of hydrochloric acid, and continuing to maintain the ultrasonic state for 10 min; if the system is found to be obviously turbid, turning off the ultrasound, and continuing to stir for 2 h for crystal cultivation; after 2 h, continuing to pump in 1.5 mol / L hydrochloric acid at a drip rate of 0.2 mL / min, monitoring the pH change of the system process, and when the pH of the system drops to 2.0, stopping the addition of acid, and simultaneously pumping in anhydrous ethanol at a flow rate of 0.6 mL / min, stopping the dripping when the system is stable, and continuing to stir for 6 h; filtering the crystal slurry, and using 100 After washing with 90% ethanol by volume, the mixture was placed in a 40°C oven and dried for 6 h to obtain white crystalline solid particles. The molecular formula of the prepared 5'-cytidine monophosphate monohydrate is C9H 14 N3O8P·H2O, belongs to the monoclinic system, space group is P 21 , unit cell parameters a(Å)=4.858, b(Å)=8.3267, c(Å)=17.0704, β=90.126 °, unit cell volume V=690.515Å 3 The minimum number of asymmetric units in the unit cell is Z = 2. In a minimum asymmetric unit, there is one 5'-cytidine monophosphate and one crystal water.

5. The method for preparing 5'-cytidine monophosphate monohydrate crystals according to any one of claims 1 to 4, characterized in that: The X-ray powder diffraction pattern of the 5'-cytidine monophosphate monohydrate crystal measured using Cu-Kα rays has characteristic absorption peaks at 2θ of 5.21°, 10.36°, 11.83°, 15.55°, 18.28°, 18.98°, 21.78°, 23.62°, and 30.13°.

6. The method for preparing 5'-cytidine monophosphate monohydrate crystals according to any one of claims 1 to 4, characterized in that: The crystals form a crystalline powder having a bulk density of not less than 0.20 g / mL and a d50 of not less than 10 μm.

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