阅读说明：本技术 一种3′,5′-环化腺苷酸盐的制备方法 (Preparation method of 3',5' -cyclic adenylate ) 是由 张国全 许怡学 赵琛 于 2019-11-22 设计创作，主要内容包括：一种3’,5’-环化腺苷酸盐的制备方法,包括以下步骤：3’,5’-环化腺苷酸与水配成3’,5’-环化腺苷酸混悬液,在搅拌的条件下加入强碱或强碱弱酸盐或金属氧化物,调节pH值；过滤,取清液,加入活性炭,搅拌；脱碳,除菌过滤,除去不溶物,得反应液；从反应液中析晶,收集析出的湿晶,干燥,粉碎,过筛,混合,即成。本发明方法操作简单,所得3’,5’-环化腺苷酸盐产品,尤其是3’,5’-环化腺苷酸钙、3’,5’-环化腺苷酸钠,其金属离子含量适中,纯度高、收率高。(A method of preparing a 3',5' -cyclic adenylate salt comprising the steps of: preparing 3',5' -cyclic adenylic acid and water into 3',5' -cyclic adenylic acid suspension, adding strong base or strong base weak acid salt or metal oxide under stirring, and adjusting pH value; filtering, collecting clear liquid, adding active carbon, and stirring; decarbonizing, sterilizing and filtering, and removing insoluble substances to obtain reaction liquid; crystallizing from the reaction solution, collecting the wet crystal, drying, pulverizing, sieving, and mixing. The method is simple to operate, and the obtained 3',5' -cyclic adenylate products, particularly 3',5' -cyclic calcium adenylate and 3',5' -cyclic sodium adenylate, have moderate metal ion content, high purity and high yield.)

1. A method of preparing a 3',5' -cyclic adenylate salt comprising the steps of:

(1) preparing 3',5' -cyclic adenylic acid and water into 3',5' -cyclic adenylic acid suspension, adding strong base or strong base weak acid salt or metal oxide under stirring, and adjusting pH value;

(2) filtering, collecting clear liquid, adding active carbon, and stirring;

(3) decarbonizing, sterilizing and filtering, and removing insoluble substances to obtain reaction liquid;

(4) crystallizing from the reaction liquid, carrying out solid-liquid separation, and collecting wet crystals separated out;

(5) drying, pulverizing, sieving, and mixing;

in the step (1) and the step (4), the pH value adjustment, the crystallization and the solid-liquid separation adopt the following methods:

method A

Adjusting the pH value: adding strong base or strong base weak acid salt or metal oxide until the pH value is 6.0-7.0, preferably 6.5-6.9;

crystallization and solid-liquid separation: adopting a method of evaporating solvent, solvent crystallization or combining the evaporating solvent and the solvent crystallization; the solvent crystallization adopts an organic solvent; the organic solvent is preferably one or more of methanol, tetrahydrofuran, ethanol, propanol, isopropanol, acetone, isobutanol, diethyl ether and petroleum ether; when an alcohol or ketone solvent is used in combination with an ether solvent, the amount of the alcohol or ketone solvent used is preferably 1/5 or more based on the total amount of the solvent used; the ether solvent is preferably used in combination with an alcohol or ketone solvent;

or method B

Adjusting the pH value: adding strong base or strong base weak acid salt or metal oxide until the pH value is more than 7.0, preferably 7.1-7.5;

crystallization and solid-liquid separation: a method of combining solvent crystallization or evaporation solvent with solvent crystallization is adopted; the solvent crystallization adopts an organic solvent, and the organic solvent is preferably one or more of methanol, ethanol, propanol, isopropanol, acetone and isobutanol;

or method C

Adjusting the pH value: adding strong base or strong base weak acid salt or metal oxide until the pH value is more than 7.0, preferably 7.1-7.5;

crystallization and solid-liquid separation: a method of combining solvent crystallization or evaporation solvent with solvent crystallization is adopted; the solvent crystallization adopts an organic solvent, and the organic solvent is preferably acetone;

or method D

Adjusting the pH value: adding strong base or strong base weak acid salt or metal oxide until the pH value is more than 7.1, adding acid liquor, and adjusting the pH value to 6.0-7.0, preferably 6.5-6.9;

crystallization and solid-liquid separation: a method of combining solvent crystallization or evaporation solvent with solvent crystallization is adopted; the solvent crystallization adopts an organic solvent, and the organic solvent is preferably one or more of methanol, ethanol, propanol, isopropanol, acetone and isobutanol.

2. The method of preparing a 3',5' -cyclic adenylate salt according to claim 1, wherein: in the step (1), the purity of the 3',5' -cyclic adenylic acid is 98.5% or more, preferably 99.5% or more; the temperature of the water is 10-100 ℃, preferably 40-60 ℃; the mass percentage concentration of the 3',5' -cyclic adenylic acid suspension is 5-40%, preferably 10-20%; the purity of the strong base or strong base weak acid salt or metal oxide is more than 98.0%, preferably more than 99.0%.

3. The method for producing a 3',5' -cyclic adenylate salt according to claim 1 or 2, wherein in the step (1), the acid solution used in the method D for adjusting the pH is hydrochloric acid or nitric acid; the concentration of the acid liquor is 1-6 mol/L, preferably 2-3 mol/L.

4. The method for producing a 3',5' -cyclic adenylate salt according to any one of claims 1 to 3, wherein in the step (1), when the strong base or the weak acid salt or the metal oxide or the acid solution is added to adjust the pH, the mixture is stirred for 5 minutes or more, preferably 10 to 40 minutes, more preferably 20 to 30 minutes after each addition, and the pH is measured.

5. The method for preparing a 3',5' -cyclic adenylate salt according to any one of claims 1 to 4, wherein in the step (2), the activated carbon is added in an amount of 0.1 to 1 wt%, preferably 0.3 to 0.5 wt%, based on the total amount of the supernatant; the stirring time after adding the activated carbon is more than 10min, preferably 20 min-30 min.

6. The method for producing a 3',5' -cyclic adenylate salt according to any one of claims 1 to 5, wherein the step (3) is performed by a filtration membrane having a pore size of 0.22 to 0.45 μm.

7. The method for producing a 3',5' -cyclic adenylate salt according to any one of claims 1 to 6, wherein in the step (4), the method for evaporating the solvent is vacuum concentration, spray drying or freeze drying, and the vacuum concentration is preferably: taking reaction liquid, and placing the reaction liquid in a water bath at the temperature of 60-100 ℃, preferably 90-95 ℃; concentrating under reduced pressure until water is basically volatilized, and separating out precipitate as wet crystal.

8. The method for producing a 3',5' -cyclic adenylate salt according to any one of claims 1 to 7, wherein in the step (4), the method A or B or C or D, and the step of solvent crystallization are: cooling the reaction liquid to room temperature, adding an organic solvent in times or at one time, repeatedly shaking or fully stirring until a large amount of white crystals are separated out, placing the mixture below room temperature, preferably below 10 ℃, for more than 5min, preferably 30 min-2 h, filtering, and washing the obtained precipitate to obtain wet crystals; the washing frequency is more than one time, preferably 2-3 times; the dosage of the organic solvent for solvent crystallization is 1 to 40 times, preferably 3 to 4 times of the reaction solution.

9. The method for producing a 3',5' -cyclic adenylate salt according to any one of claims 1 to 8, wherein in the step (4), the method A or B or C or D, the step of evaporating the solvent and crystallizing the solvent in combination comprises: placing the reaction solution in a water bath at 60-100 ℃, preferably 90-95 ℃, concentrating under reduced pressure until the volume of the concentrated solution is 1/4-1/5 of the volume of the original reaction solution, and removing the concentrated solution; washing the inner wall of the concentrator with water or an organic solvent, and washing the material residue on the inner wall of the concentrator to obtain a washing solution; mixing the concentrated solution and the washing solution to obtain a mixed solution, adding an organic solvent, repeatedly shaking or fully stirring to separate out a precipitate, filtering, and washing the precipitate to obtain wet crystals; the washing frequency is more than one time, preferably 2-3 times; the amount of the organic solvent added to the combined solution is 1 to 40 times, preferably 3 to 4 times of the combined solution.

10. The method for producing a 3',5' -cyclic adenylate salt according to any one of claims 1 to 9, wherein in the step (5), the drying is performed at a temperature of 40 ℃ to 130 ℃ for 2 hours to 20 hours under normal pressure or reduced pressure; the drying times are more than or equal to 2 times; the screening is performed by using a screen mesh of 80-120 meshes; the mixing time is more than 10min, preferably 20 min-30 min.

11. The method for producing a 3',5' -cyclic adenylate salt according to any one of claims 1 to 10, wherein in the step (5), the drying, the pulverization, the sieving, the redrying at a temperature of 40 ℃ to 130 ℃ for 2 hours to 20 hours, and the redrying are carried out under normal pressure or reduced pressure.

12. The method for preparing a 3',5' -cyclic adenylate salt according to any one of claims 1 to 11, wherein in step (1), the alkali or weak acid salt is sodium, and the addition is performed by adding a solid or adding an alkali solution, preferably an alkali solution with a concentration of 1 to 6mol/L, more preferably an alkali solution with a concentration of 2 to 3 mol/L.

13. The method for preparing a 3',5' -cyclic adenylate salt according to any one of claims 1 to 11, wherein in step (1), the alkali or weak acid salt or metal oxide is calcium alkali or salt or metal oxide, and the addition means for adding the alkali or salt or metal oxide to the suspension is solid addition.

14. The method for producing a 3',5' -cyclic adenylate salt according to any one of claims 1 to 12, wherein in steps (1) and (4), the weak acid and strong base is sodium bicarbonate; regulating pH value, crystallizing and solid-liquid separating by method A.

15. The method for producing a 3',5' -cyclic adenylate salt according to any one of claims 1 to 12, wherein in steps (1) and (4), the strong base and weak acid salt is sodium carbonate, and method a or method B is used for pH adjustment, crystallization, and solid-liquid separation.

16. The method for producing a 3',5' -cyclic adenylate salt according to any one of claims 1 to 12, wherein the strong base is sodium hydroxide in steps (1) and (4), and method a or method C is used for pH adjustment, crystallization, and solid-liquid separation.

17. The method for preparing a 3',5' -cyclic adenylate salt according to any one of claims 1 to 11 and 13, wherein in steps (1) and (4), the strong base or the strong base weak acid salt or the metal oxide is calcium hydroxide, calcium bicarbonate or calcium oxide, and the method A or the method D is used for pH value adjustment, crystallization and solid-liquid separation.

Technical Field

The invention relates to a production method of a chemical, in particular to a preparation method of a cyclic adenylate of the chemical.

Background

Calcium 3',5' -cyclic adenylate and sodium 3',5' -cyclic adenylate are two important metal salts of 3',5' -cyclic adenylate, which contain not only 3',5' -cyclic adenylate but also two important alkali metals: calcium and sodium, have multiple physiological functions, and detailed information is as follows:

chinese name: 3',5' -Cyclic calcium adenylate

Molecular weight: 348.21

The molecular formula is as follows: c₁₀H₁₁N₅O₆P·1/2Ca

Structural formula (xvi):

TABLE 1 theoretical values of calcium element content in calcium 3',5' -cyclic adenylate

Element name	Number of	Molecular weight	Total amount of each element	Ratio (%)
					C	10	12	120	34.48
H	11	1	11	3.16
					N	5	14	70	20.11
Ca	0.5	40	20	5.75
					O	6	16	96	27.59
P	1	31	31	8.91
					Total up to	-	-	348	-

Chinese name: 3',5' -Cyclic adenosine sodium salt

Molecular weight: 351.21

The molecular formula is as follows: c₁₀H₁₁N₅O₆P·Na

Structural formula (xvi):

TABLE 2 theoretical values of the sodium content in the sodium 3',5' -cyclic adenylate

Element name	Number of	Molecular weight	Total amount of each element	Ratio (%)
					C	10	12	120	34.19
H	11	1	11	3.13
					N	5	14	70	19.94
Na	1	23	23	6.55
					O	6	16	96	27.35
P	1	31	31	8.83
					Total up to	-	-	351	-

Both the electrophysiological and mechanical activity of the heart is influenced by intracellular 3',5' -cyclic adenylate levels, which is achieved primarily by altering intracellular calcium concentrations. When intracellular Ca²⁺When the concentration rises above the micromolar level, feedback inhibits adenylyl cyclase and activates cyclic nucleotide phosphodiesterase to begin to decompose 3',5' -cyclic adenylate. At the same time, through Na⁺/Ca²⁺Exchange mechanism, and the calcium pump for activation of the sarcoplasmic reticulum, to supply Ca in the cytoplasm²⁺The concentration decreases. Thus, the metabolic process of 3',5' -cyclic adenylate is essentially a dynamic process of signal extinction. Thus, calcium 3',5' -cyclic adenylate not only acts as a cellular second messenger, directly playing a pharmacological role, itself, but also via another, more extensive intracellular messenger Ca^{2 +}Activate and regulate numerous physiological functions within the cell. Therefore, the content of calcium ion in the 3',5' -cyclic calcium adenylate should be considered important clinically.

Songbeiying, as mentioned in the article "rational calcium supplement in shallow talk" from Nei Mongolia Chinese medicine "4 months 2011, excessive calcium can also cause a series of adverse reactions in the body, such as: calcium deposition in the kidney also causes frequent urination, hematuria, urinary stones, repeated urinary infections and even renal failure.

Liu Zhiyi et al reported in the text of "inspection analysis of adverse reactions in sodium chloride injection" of Yangtze pharmacy, 4 th phase, volume 18, 2006 that children, the elderly and patients with specific constitutions could have adverse reactions caused by excessive sodium ions due to excessive overquick input, and the authors also considered that the above reactions are likely to occur in the presence of compatible sodium salts of antibiotics, petadine, sodium chloride and other drugs.

Since the cost of 3',5' -cyclic adenosine (abbreviated as cAMP) is high, it is usually economical to use a slight excess of strong base or weak base to fully salify 3',5' -cyclic adenosine. However, if the excess metal ions are not removed by filtration, extraction with an organic solvent, or by adjusting the pH of the reaction solution by adding an acid and then extracting with an organic solvent, there is a high possibility that the metal ion impurity salts and the metal salt of 3',5' -cyclic adenosine co-precipitate.

Although methods for producing calcium 3',5' -cyclic adenylate and sodium 3',5' -cyclic adenylate have been reported, there has been no disclosure of the problem of the limitation of the content of calcium and sodium ions contained in calcium 3',5' -cyclic adenylate and sodium 3',5' -cyclic adenylate, and how to control the production process effectively.

Huttin et al reported in the paper of "preparation of dibutyryl 3',5' -cyclic adenylate, content thereof and determination of related substances" of volume 35 < J > China journal of Biochemical medicine "of 2 nd phase 2015, using 3',5' -cyclic adenylate to drop sodium hydroxide aqueous solution, adjusting pH to 7-8 with HCl solution, and precipitating crystals with anhydrous ethanol to prepare sodium 3',5' -cyclic adenylate.

CN106589031A discloses a method for preparing sodium dibutyryl 3',5' -cyclic adenylate, which comprises the steps of dripping sodium hydroxide aqueous solution into 3',5' -cyclic adenylate, adjusting the pH value to 7-12, preferably 8, by using HCl solution, and precipitating crystals by using absolute ethyl alcohol to prepare the sodium 3',5' -cyclic adenylate.

In the above two methods, in order to sufficiently salify 3',5' -cyclic adenylic acid, excess NaOH is added, and the pH of the reaction solution needs to be adjusted back by using an HCl solution, excess NaCl may be generated in the reaction solution. Since NaCl is slightly soluble in organic solvents, NaCl is very likely to be embedded in the crystals of the sodium 3',5' -cyclic adenylate and form a coprecipitation phenomenon with the sodium 3',5' -cyclic adenylate, so that the sodium ions in the sodium 3',5' -cyclic adenylate exceed the limit.

CN106336439B discloses a method for preparing calcium dibutyryl cyclic adenosine monophosphate, which is to add cyclic adenosine monophosphate and calcium hydroxide into water according to the molar ratio of 1: 1-1.2, and precipitate crystals by using ethanol to prepare calcium 3',5' -cyclic adenosine monophosphate. However, since calcium hydroxide is slightly soluble in water and hardly soluble in ethanol, saturated calcium hydroxide dissolved in water is completely precipitated in ethanol and coprecipitated with calcium 3',5' -cyclic adenylate, resulting in Ca in calcium 3',5' -cyclic adenylate prepared by the method²⁺The ions are overrun.

CN102250179B discloses a compound entity of adenosine cyclophosphate salt and application thereof, wherein a hydrate of the adenosine cyclophosphate salt is prepared by a method of reacting adenosine cyclophosphate with metal oxide or hydroxide or metal salt or organic base thereof according to a reaction molar ratio. The method determines the dosage of reactants according to the reaction molar ratio, and because the 3',5' -cyclic adenylic acid is slightly soluble in water, the solubility of partial metal salt is low, the incomplete reaction is easily caused, and the content of metal ions in the 3',5' -cyclic adenylic acid salt is low or the purity of the product is low.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects in the prior art and provide a preparation method of 3',5' -cyclic adenylate. The method is simple to operate, and the obtained 3',5' -cyclic adenylate, especially calcium 3',5' -cyclic adenylate and sodium 3',5' -cyclic adenylate, has moderate metal ion content, high product purity and high yield.

The technical scheme adopted by the invention for solving the technical problems is as follows: a method of preparing a 3',5' -cyclic adenylate salt comprising the steps of:

(1) preparing 3',5' -cyclic adenylic acid and water into 3',5' -cyclic adenylic acid suspension, adding strong base or strong base weak acid salt or metal oxide under stirring, and adjusting pH value;

(2) filtering, collecting clear liquid, adding active carbon, and stirring;

(3) decarbonizing, sterilizing and filtering, and removing insoluble substances to obtain reaction liquid;

(4) crystallizing from the reaction liquid, carrying out solid-liquid separation, and collecting wet crystals separated out;

(5) drying, pulverizing, sieving, and mixing;

in the step (1) and the step (4), the pH value adjustment, the crystallization and the solid-liquid separation adopt the following methods:

method A.

Adjusting the pH value: adding strong base or strong base weak acid salt or metal oxide until the pH value is 6.0-7.0, preferably 6.5-6.9;

crystallization and solid-liquid separation: adopting a method of evaporating solvent, solvent crystallization or combining the evaporating solvent and the solvent crystallization; the solvent crystallization adopts an organic solvent; the organic solvent is preferably one or more of methanol, tetrahydrofuran, ethanol, propanol, isopropanol, acetone, isobutanol, diethyl ether and petroleum ether; when an alcohol or ketone solvent is used in combination with an ether solvent, the amount of the alcohol or ketone solvent used is preferably 1/5 or more based on the total amount of the solvent used; the ether solvent is preferably used in combination with an alcohol or ketone solvent;

or method B.

Adjusting the pH value: adding strong base or strong base weak acid salt or metal oxide until the pH value is more than 7.0, preferably 7.1-7.5;

crystallization and solid-liquid separation: a method of combining solvent crystallization or evaporation solvent with solvent crystallization is adopted; the solvent crystallization adopts an organic solvent, and the organic solvent is preferably one or more of methanol, ethanol, propanol, isopropanol, acetone and isobutanol;

or method C.

Adjusting the pH value: adding strong base or strong base weak acid salt or metal oxide until the pH value is more than 7.0, preferably 7.1-7.5;

crystallization and solid-liquid separation: a method of combining solvent crystallization or evaporation solvent with solvent crystallization is adopted; the solvent crystallization adopts an organic solvent, and the organic solvent is preferably acetone;

or method D

Adjusting the pH value: adding strong base or strong base weak acid salt or metal oxide until the pH value is more than 7.1, adding acid liquor, and adjusting the pH value to 6.0-7.0, preferably 6.5-6.9;

crystallization and solid-liquid separation: a method of combining solvent crystallization or evaporation solvent with solvent crystallization is adopted; the solvent crystallization adopts an organic solvent, and the organic solvent is preferably one or more of methanol, ethanol, propanol, isopropanol, acetone and isobutanol.

Preferably, in the step (1), the purity of the 3',5' -cyclic adenylic acid is 98.5% or more, more preferably 99.5% or more.

Preferably, in the step (1), the temperature of the water is 10 to 100 ℃, more preferably 40 to 60 ℃.

Preferably, in the step (1), the 3',5' -cyclic adenylic acid suspension has a mass percentage concentration of 5% to 40%, more preferably 10% to 20%.

Preferably, in the step (1), the purity of the strong base or the weak acid salt of strong base or the metal oxide is 98.0% or more, and more preferably 99.0% or more.

Preferably, in the step (1), the acid solution used for adjusting the pH value in the method D is hydrochloric acid or nitric acid; the concentration of the acid solution is 1-6 mol/L, and more preferably 2-3 mol/L.

Preferably, in the step (1), when adding the strong base or the strong base and the weak acid salt or the metal oxide or the acid solution to adjust the pH value, stirring for more than 5min, more preferably 10min to 40min, further preferably 20min to 30min after each addition, and testing the pH value. The 3',5' -cyclic adenosine is slightly soluble in water, and part of strong base and weak acid salt is not easily soluble in water, so that stirring is needed to fully react the reactants.

Preferably, in the step (2), the adding amount of the activated carbon is 0.1 wt% to 1 wt% of the total amount of the clear liquid (wt% represents mass percentage), and more preferably 0.3 wt% to 0.5 wt% of the total amount of the clear liquid.

Preferably, in the step (2), the time for stirring after adding the activated carbon is more than 10min, and more preferably 20min to 30 min.

Preferably, in the step (3), the sterilization filtration adopts a filtration membrane with the pore size of 0.22 um-0.45 um.

Preferably, in step (4), the solvent is evaporated by method A, such as vacuum concentration, spray drying or freeze drying; the step of concentration under reduced pressure is more preferably: taking reaction liquid, and placing the reaction liquid in a water bath at the temperature of 60-100 ℃, and further preferably selecting the reaction liquid at the temperature of 90-95 ℃; concentrating under reduced pressure until water is basically volatilized, and separating out precipitate as wet crystal.

Preferably, in step (4), the method A or B or C or D, the solvent crystallization step is: cooling the reaction solution to room temperature, adding an organic solvent in several times or once, repeatedly shaking or fully stirring until a large amount of white crystals are separated out, placing the mixture below room temperature, more preferably below 10 ℃, for more than 5min, more preferably 30 min-2 h, filtering, and washing the obtained precipitate to obtain wet crystals; the washing frequency is more than one time, preferably 2-3 times; the amount of the organic solvent for solvent crystallization is 1 to 40 times, more preferably 3 to 4 times of the reaction solution.

Preferably, in step (4), method A or B or C or D, the step of evaporating the solvent and crystallizing and combining with the solvent is: placing the reaction solution in a water bath at the temperature of 60-100 ℃, preferably 90-95 ℃, concentrating under reduced pressure until the volume of the concentrated solution is 1/4-1/5 of the volume of the original reaction solution, and removing the concentrated solution; washing the inner wall of the concentrator with water or an organic solvent, and washing the material residue on the inner wall of the concentrator to obtain a washing solution; mixing the concentrated solution and the washing solution to obtain a mixed solution, adding an organic solvent, repeatedly shaking or fully stirring to separate out a precipitate, filtering, and washing the precipitate to obtain wet crystals; the washing frequency is more than one time, and more preferably 2-3 times; the amount of the organic solvent added to the combined solution is 1 to 40 times, and more preferably 3 to 4 times of the combined solution.

Preferably, in the step (5), the drying temperature is 40-130 ℃, the drying time is 2-20 h, and the drying is carried out under the normal pressure or reduced pressure condition; the drying times are more than or equal to 2 times.

Preferably, in the step (5), a sieve of 80-120 meshes is selected for sieving; the mixing time is 10min or more, more preferably 20min to 30 min.

Preferably, in the step (5), drying, crushing, sieving, re-drying at the temperature of 40-130 ℃, the re-drying time of 2-20 h, and re-drying under normal pressure or reduced pressure.

Preferably, in the step (1), the strong base or the strong base weak acid salt is sodium base or sodium salt, and the adding mode when the strong base or the strong base weak acid salt is added into the suspension is solid adding or alkali liquor adding, more preferably alkali liquor with the concentration of 1-6 mol/L is added, and further preferably alkali liquor with the concentration of 2-3 mol/L is added.

Preferably, in the step (1), the alkali or alkali weak acid salt or metal oxide is calcium alkali or salt or metal oxide, and the addition mode when adding into the suspension is solid addition.

Preferably, in the steps (1) and (4), the strong base and weak acid salt is sodium bicarbonate; regulating pH value, crystallizing and solid-liquid separating by method A.

Preferably, in the steps (1) and (4), the strong base and weak acid salt is sodium carbonate, and the method A or the method B is adopted for pH value adjustment, crystallization and solid-liquid separation.

Preferably, in the steps (1) and (4), the strong base is sodium hydroxide, and the method A or the method C is adopted for pH value adjustment, crystallization and solid-liquid separation.

Preferably, in the steps (1) and (4), the strong base or the strong base weak acid salt or the metal oxide is calcium hydroxide or calcium bicarbonate or calcium oxide, and the method A or the method D is adopted for adjusting the pH value, crystallizing and separating solid and liquid.

The key to the practice of the present invention is the proper selection of acidic and basic pH modifiers, the proper control of the pH of the reaction solution, and the proper selection of organic solvents.

The production precision required by controlling the content of metal ions in the product is higher, and the inventor selects to judge whether the charging ratio is proper or not by adjusting the pH value of the salified solution in consideration of the problem that the raw materials in actual production may have inconsistent purity or absorb moisture. In actual production, stable products are difficult to obtain at a fixed feed ratio, and the content of metal ions in the obtained 3',5' -cyclic adenosine finished products is often higher or lower.

Since 3',5' -cyclic adenylate is acidic, the metal ion selected is calcium, sodium ion salt should be able to form water-soluble sodium 3',5' -cyclic adenylate and calcium 3',5' -cyclic adenylate with 3',5' -cyclic adenylate, and it should be excluded that the metal ion which does not form a salt with 3',5' -cyclic adenylate in an aqueous solution is calcium, sodium ion salt (see table 3) and the metal ion which does form a salt with 3',5' -cyclic adenylate but which is acidic in pH of the reaction solution after salt formation is calcium, sodium ion salt of organic acid (see table 4).

TABLE 3 name details of strong base or weak base salt in which the cation not forming a salt with 3',5' -cyclic adenosine in aqueous solution is calcium or sodium ion

TABLE 4 partial organic acid salts which form salts with 3',5' -cyclic adenosine in aqueous solution, but have acidic pH, and calcium and sodium ions as cations

The results of the study on the dissolution of the solid base in which part of the metal ions are sodium ions in 95% ethanol and 95% acetone are shown in tables 5, 6 and 7:

TABLE 5.8% aqueous sodium bicarbonate solution, dissolution of sodium bicarbonate solids in 95% ethanol

The results in Table 5 show that sodium bicarbonate is insoluble in 95% ethanol and 95% acetone.

TABLE 6.5% aqueous solution of sodium carbonate, solid sodium carbonate dissolved in 95% ethanol, 95% acetone

According to the results of table 6, although solid sodium carbonate was insoluble in 95% ethanol and 95% acetone, 5% aqueous sodium carbonate solution was soluble in 95% ethanol and 95% acetone. Therefore, even if an excessive amount of sodium carbonate is added to the reaction, the solvent can be removed by using ethanol or acetone, and when sodium carbonate is used as a reactant, the pH of the reaction solution can be adjusted to 7.0 or more.

TABLE 7.10% NaOH solution in water and NaOH solid dissolved in 95% ethanol and 95% acetone

From the results in Table 7, it is shown that a 10% aqueous solution of sodium hydroxide was dissolved in a predetermined proportion of 95% ethanol, and the solubility decreased conversely as the amount of ethanol increased; aqueous sodium hydroxide is readily soluble in 95% acetone. The sodium hydroxide solid is insoluble in 95% ethanol and 95% acetone. Therefore, when the crystallization is carried out using an ethanol solvent, the coprecipitation tends to occur with the 3',5' -cyclic adenosine disodium salt. In addition, in view of the fact that sodium hydroxide is readily soluble in acetone, acetone is preferred as a solvent for removing excess sodium hydroxide.

It is noted that the 10% NaOH in acetone solution was pale yellow after standing for one day. Because, at higher temperatures and longer reaction times, sodium hydroxide tends to form ketals from acetone, which turn yellow in color. Therefore, when the alkali is sodium hydroxide and the solvent is acetone, the crystallization temperature should be reduced as much as possible to shorten the crystallization time.

When the metal ion which is easily soluble in water is selected from strong base or strong base weak acid salt (such as sodium bicarbonate)Sodium carbonate, sodium hydroxide) with acid (HCl, HNO)₃) When the alkali (pH 7.0 or more) is excessively present in the neutralization reaction solution, sodium chloride and sodium nitrate, which are insoluble or poorly soluble in organic solvents such as ethanol and acetone, are formed, and are easily coprecipitated with 3',5' -cyclic adenosine disodium salt, resulting in an excessive sodium ion. Therefore, after the alkali is excessively added, the 3',5' -cyclic adenosine sodium salt can be prepared by a method of adjusting back the reaction liquid by using acid liquor.

According to the relevant documents, the dissolution of calcium bicarbonate, calcium hydroxide and calcium carbonate in water is summarized as follows:

according to the data of the text of experimental exploration on the solubility of calcium hydroxide changing along with the temperature of the 1 st phase of chemistry teaching of middle school in 2019 of Yanxiping et al, 0.16g of calcium hydroxide is dissolved in 100g of water at 25 ℃, so that the calcium hydroxide is slightly soluble in the water.

According to general calculation of Zhangyi Fan in 257 th edition of 2017, namely, literature navigation: according to the data in Yi Wen of saturated solubility of calcium bicarbonate, 100g of water can dissolve 0.116g of calcium bicarbonate, so that the calcium bicarbonate is slightly soluble in water.

According to data of a sentence law of calculation of solubility and pH value of an aqueous solution of calcium carbonate in Li Yongxin at volume 22, volume 1, 1999, in university of Anhui province (Nature science edition), the solubility of calcium carbonate in water is as follows: 9.3X 10^-5mol/L, corresponding to 9.3X 10^-4g/100ml, calcium carbonate is hardly soluble or poorly soluble in water.

According to the test of the present inventors, the dissolution of calcium bicarbonate and calcium hydroxide in 95% ethanol at room temperature is listed below (table 8):

TABLE 8 dissolution of calcium bicarbonate and calcium hydroxide in 95% ethanol at room temperature

It can be seen that calcium bicarbonate or calcium hydroxide is used as a calcium source, water or ethanol is used as a solvent, and if the added calcium hydroxide is excessive, the excessive calcium hydroxide and calcium bicarbonate and 3',5' -cyclic adenylic acid calcium form coprecipitation, so that the content limit of calcium ions in the 3',5' -cyclic adenylic acid exceeds the standard; if the amount of calcium hydroxide added is insufficient, the salt formation with 3',5' -cyclic adenylic acid cannot be completed.

The results of the tests conducted by the present inventors on the dissolution of calcium chloride in 95% ethanol and 95% acetone are shown in Table 9:

TABLE 9 solubility of calcium chloride in 95% ethanol, 95% acetone

Item	Solid body	Solid body	Aqueous solution	Aqueous solution
					Name (R)	CaCl2	CaCl2	CaCl2	CaCl2
Sample weighing (g)	0.1	0.1	0.1	0.1
					Water (ml)	-	-	1	1
Name of added solvent	95% ethanol	95% acetone	95% ethanol	95% acetone
					Volume of solubiliser (ml)	1	1	1	1
Whether or not to dissolve	Is that	Whether or not	Is that	Is that

The metal ion slightly soluble in water is strong base of calcium ion or weak acid salt of strong base (such as calcium hydroxide, calcium oxide, calcium bicarbonate). Such bases are difficult to dissolve in water, but if acids (HCl, HNO) are used₃) Neutralizing the excessive alkali in the salt solution (pH of the salt solution is above 7.0) to generate CaCl which is soluble in organic solvent such as ethanol and acetone₂、Ca(NO₃)₂. Therefore, in order to ensure that the alkali in the reaction solution is not excessive, the salt forming solution needs to use acid solution to neutralize the excessive alkali in the salt forming solution, and the pH value of the salt forming solution should be controlled to be 6.0-7.0, preferably 6.5-6.9. Filtering to remove insoluble substance, extracting with organic solvent such as ethanol and acetone to remove CaCl₂、Ca(NO₃)₂Impurities.

For hardly soluble or difficultThe metal ion dissolved in water is strong base or weak base acid salt (such as calcium carbonate) of calcium ion. At room temperature, CaCO₃The presence of an ionization equilibrium (solubility product K at 20 ℃) of poorly soluble electrolytes in aqueous solutions_sp,CaCO₃＝8.7×10^-9(ii) a Solubility of 0.00093g/100ml), if H is present in the reaction mixture⁺High ion concentration (meaning that most of the 3',5' -cyclic adenosine is not salified), CaCO₃Hydrolyzed to OH^-The hydrolysis speed of the compound is higher, and the salt formation speed with 3',5' -cyclic adenosine is also higher. With H in the reaction solution⁺Reduction of the ion concentration (i.e., most of the 3',5' -cyclic adenosine in the reaction solution has become sodium salt), in which case CaCO₃Hydrolyzed to OH^-The speed of (c) is significantly reduced. However, the solubility of cAMP in water is still far greater than that of CaCO₃(cAMP solubility in water at 25 ℃ 0.4g/100ml), therefore, H in solution⁺Since the concentration of (A) is high, the pH value of the salt-forming solution near the end point is weakly acidic (about 6.1).

Thus, at room temperature, for CaCO₃OH dissociated in solution^-H whose ion can be dissociated by cAMP⁺Complete neutralization is required to ensure that the salified solution is fully stirred, and the stirring time is longer (more than 20 min).

However, when the temperature is raised to 40 ℃ or higher, the solubility of cAMP in water is 1.3%, and as the temperature of water is increased, H in the solution⁺The concentration of (A) is significantly increased with the increase in the amount of cAMP dissolved, and therefore, CaCO is broken₃The presence of the ionization balance of the poorly soluble electrolyte in the aqueous solution significantly accelerates the rate of calcium formation by cAMP (i.e., increasing the reaction temperature will favor calcium formation by cAMP), and the salified solution will be more basic than the salt solution obtained at room temperature.

The invention has the beneficial effects that: the method is simple to operate, and the obtained product has high purity, proper metal ion content and high yield; the purity of the prepared 3',5' -cyclic calcium adenylate is more than 99.5 percent, the content of calcium ions is 5.6 to 5.9 percent, and the yield is more than 93 percent; the prepared 3',5' -cyclic sodium adenylate has the purity of more than 99.5 percent, the sodium ion content of 6.4 to 6.7 percent and the yield of more than 95 percent.

Detailed Description

The present invention is further illustrated by the following examples.

The method for measuring the contents of calcium and sodium ions in calcium and sodium 3',5' -cyclic adenylate used in each example of the present invention is as follows.

Determination of the limits of the calcium ion content

According to the 2015 edition of Chinese pharmacopoeia, the conventional method for measuring the content of calcium ions in a test sample is complexometric titration, and the complexometric titration is mainly carried out through titration operation. Considering that the measurement error is inevitable, the relative deviation of the primary standard average value and the complex standard average value of the titration solution is not more than +/-0.1% according to the 2010 version of Chinese drug inspection standard operating specification.

Since the weighing error of the sample is 0.1% (the error of the one-hundred-thousand balance is 0.1%); because the volume error of the volumetric flask is 0.1%, the preparation error in the solution preparation process is 0.1%; the error of the burette volume is 0.1%, so the error of the titration is 0.1% (above all refer to A-grade glass instruments). Therefore, the vector summation of the errors is often greater than 0.1% of the actual titration solution calibration value.

The error in the calibration process of the actual titration solution is often larger. According to the standard content limit range of calcium gluconate of the second part of the Chinese pharmacopoeia of 2015 edition, the content limit range of calcium ions in the calcium cyclophosphate is temporarily set to be +/-5% according to the actual situation.

According to the actual situation, the deviation rate of the content of calcium ions in the product is set as +/-5%. The theoretical value of calcium element content in the 3',5' -cyclic calcium adenylate is 5.75%, so the theoretical value of calcium ion in the product is 5.5% -6.0%.

Calcium ion content determination method

0.3g of the product is taken, precisely weighed and placed in a conical flask, a small amount of water is firstly used for wetting, 3ml of 2mmol of hydrochloric acid and 25ml of water are then added, 1 drop of 0.025% methyl red ethanol solution is added after dissolution, ammonia test solution is dripped until the solution is yellowish, 25ml of water and 10ml of ammonia-ammonium chloride buffer solution (pH 10.0) are added, a proper amount of chrome black T indicator is added, and the solution is dripped until the solution is changed from purple to pure blue. And the titration results were corrected for a blank test. Each 1ml of disodium EDTA titrate (0.05mol/L) corresponds to 2.004mg of calcium.

Determination of sodium ion content limits

With reference to the theoretical 6.55% of the sodium ion content of the sodium 3',5' -cyclic adenylate, the deviation was set to ± 5%, i.e. the limit ranges: 6.3 to 6.9 percent.

Method for measuring sodium ion content

Preparation of sodium Single element reference solution

Precisely measuring 2ml of sodium single element standard solution (1000 mu g/ml), placing the solution in a 100ml measuring flask, diluting with water to scale, and shaking up to obtain a reference solution;

taking 25mg of a test sample, accurately weighing, placing in a 100ml measuring flask, adding water to dissolve and dilute to a scale, shaking up, accurately weighing 3ml, placing in a 25ml measuring flask, diluting with water to a scale, and shaking up to obtain a test sample solution.

Determination method comprises precisely measuring 4ml, 5ml, 6ml, 7ml and 8ml of reference solution, respectively placing in 100ml measuring flask, diluting with water to scale, shaking, measuring the solution and the test solution at 589nm wavelength by atomic absorption spectrophotometry (first method 0406 in the four parts of pharmacopoeia 2015 edition).

The following examples are intended to facilitate the understanding of the present invention by those skilled in the art and should not be construed as limiting the invention as described in the claims.

The feeding materials in the embodiment and the comparative example are calculated according to the dry basis.