CN110684066B

CN110684066B - Cytophosphocholine medicinal preparation and new application thereof in cerebral infarction acute-stage disturbance of consciousness

Info

Publication number: CN110684066B
Application number: CN201910429990.3A
Authority: CN
Inventors: 马庆双; 傅苗青
Original assignee: Guangdong Jincheng Jinsu Pharmacy Co ltd
Current assignee: Guangdong Jincheng Jinsu Pharmacy Co ltd
Priority date: 2019-05-22
Filing date: 2019-05-22
Publication date: 2020-12-08
Anticipated expiration: 2039-05-22
Also published as: CN110684066A

Abstract

The invention provides sodium citicoline or a composition thereof, which comprises the sodium citicoline with the content of more than 99.0 percent and the application of the sodium citicoline in the acute period of cerebral infarction and also provides a preparation method of the sodium citicoline or the composition thereof, comprising the steps of dispersing 5' -cytidylic acid and choline chloride phosphate in an aprotic organic solvent, adding 1-propylphosphoric cyclic anhydride for reaction, filtering insoluble substances in reaction liquid, and collecting filtrate; cooling the filtrate to separate out crystals, and filtering to obtain the citicolic acid; the citicoline acid reacts with the sodium base/salt to generate the citicoline sodium. By selecting proper raw materials and condensing agents, the invention avoids the use of a large amount of strong acid condensing agents and high-toxicity solvents such as pyridine, benzene and the like in the traditional chemical method process, and improves the product quality.

Description

Cytophosphocholine medicinal preparation and new application thereof in cerebral infarction acute-stage disturbance of consciousness

Technical Field

The invention relates to preparation of citicoline, in particular to a preparation method of citicoline with short synthetic process route and high product quality, a citicoline medicinal preparation and a new application thereof in cerebral infarction acute-stage disturbance of consciousness.

Background

Citicoline (citicoline sodium or citicoline sodium) is brain metabolism activator, and can promote brain cell respiration, improve brain function, enhance function of ascending net structure activation system, promote revival, and reduce cerebrovascular resistance. The structural formula of citicoline is as follows:

citicoline is the nerve activator with the largest clinical dosage, and the citicoline is obtained by a biological fermentation method and a chemical synthesis method at present. The biological fermentation method has the advantages of long fermentation period, low unit yield, complex components in reaction liquid, high difficulty in post-treatment of the process, complex operation, column chromatography purification and the like required for post-treatment, a large amount of equipment for treatment, and no advantages in efficiency and yield compared with a chemical synthesis method.

The traditional chemical synthesis method generally takes choline phosphocholine chloride calcium salt as a starting material, takes benzene as a solvent, removes crystal water by refluxing, removes calcium ions by using carbonate or oxalic acid through forming insoluble calcium carbonate or calcium oxalate, activates the choline phosphochloride by using acetyl chloride or solid phosgene in an organic amine solvent without active hydrogen, finally reacts with cytidylic acid to generate citicoline, and then carries out corresponding post-treatment steps such as recrystallization and the like to obtain a finished product. In the conventional chemical synthesis process, a large amount of strong acidic condensing agent (such as oxalyl chloride and the like) and high-toxicity solvents such as pyridine, benzene and the like are used, so that the following obvious disadvantages are brought:

1. the choline phosphocholine chloride calcium salt is adopted as an initial raw material, a class 1 solvent benzene is required for dehydration, the process flow is complex, and meanwhile, the solvent residue of benzene is brought, so that the production of a subsequent preparation is not facilitated;

2. a large amount of acidic wastewater, calcium carbonate, sodium chloride and other waste salts are generated in the synthesis process, the environmental protection treatment difficulty is high, special means (removing organic amine solvents without active hydrogen) such as reduced pressure distillation and the like are needed in the post-treatment process, the requirements on the corrosion resistance of equipment and the like in production are high, and the cost is high;

3. the intermediate after being treated by acetyl chloride and solid phosgene is a highly active substance, and is easy to generate side reaction, thus causing incomplete reaction of raw materials, low yield and low product content.

In order to solve the problems in the prior art, the inventor researches a synthesis method for preparing high-quality citicoline with mild reaction conditions and environmental friendliness on the basis of the prior art, thereby providing citicoline sodium or a composition thereof, and a preparation method, a preparation and an application thereof.

Disclosure of Invention

In order to solve the above problems, the present inventors have conducted intensive studies and, as a result, have found that: by selecting proper raw materials and condensing agents, the use of a large amount of strong acid condensing agents (such as oxalyl chloride and the like) and high-toxicity solvents such as pyridine, benzene and the like in the traditional chemical process is avoided; the method has the advantages of mild reaction conditions, short route, low energy consumption and simple and convenient post-treatment, thereby completing the invention.

The object of the present invention is to provide the following:

in a first aspect, the invention provides citicoline sodium or a composition thereof, which comprises the citicoline sodium with the mass content of more than 99.0%.

The citicoline sodium or the composition thereof also comprises an impurity A,

the content is below 0.2 percent; and/or the presence of a gas in the gas,

furthermore, the composite material also comprises impurities B,

the content is below 0.1%.

The citicoline sodium or the composition thereof also comprises an impurity C,

the content is below 0.1%.

In a second aspect, the present invention provides a method for preparing the above citicoline sodium or the composition thereof, comprising the following steps:

step 1: dispersing 5' -cytidylic acid and choline chloride phosphate in an aprotic organic solvent, adding 1-propylphosphoric cyclic anhydride for reaction, filtering insoluble substances in a reaction solution, and collecting filtrate;

step 2: cooling the filtrate obtained in the step 1, separating out crystals, and filtering to obtain the citicolic acid;

and step 3: the citicoline acid reacts with the sodium base/salt to generate the citicoline sodium.

Wherein the aprotic organic solvent is selected from one or a combination of acetonitrile, dimethylformamide, dimethyl sulfoxide, benzene, diethyl ether, carbon tetrachloride and acetone, and is preferably acetonitrile, diethyl ether or acetone;

the sodium alkali/salt is alkaline and is selected from any one or combination of sodium hydroxide, sodium carbonate, sodium bicarbonate, sodium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium acetate and sodium lactate, and preferably sodium hydroxide, sodium carbonate and/or sodium bicarbonate.

In the process for the preparation of the above-mentioned,

adding 1-propylphosphoric cyclic anhydride, stirring and reacting for 0.5-3 h, heating the reaction system to reflux, and continuing to react for 3-5 h;

and after the reaction is finished, cooling the reaction system to 40-70 ℃, and removing insoluble substances while the reaction system is hot.

Preferably, the citicoline sodium prepared in step 3 can be subjected to recrystallization.

In a third aspect, the present invention provides a citicoline preparation containing the citicoline sodium or the composition thereof, or the citicoline sodium or the composition thereof prepared by the above method, wherein the citicoline sodium is an active ingredient in the preparation.

Wherein, the preparation is used for injection or oral administration, and preferably the preparation is an injection or an oral preparation.

In a fourth aspect, the invention provides an application of the citicoline sodium or the composition thereof, and the citicoline sodium prepared by the method or the composition thereof in preparing a medicine for recovering cerebral ischemic neurological function, and further an application in preparing a medicine for treating cerebral infarction acute-stage disturbance of consciousness.

According to the citicoline sodium or the composition thereof, the preparation method and the application thereof, the beneficial effects are as follows:

(1) compared with the traditional synthesis method, the preparation method provided by the invention has the advantages of higher conversion rate, less side reaction and no use of reactive organic amine solvent; the reaction solvent and the recrystallization solvent can be recycled after simple treatment, the complexity of the reaction system is reduced, and the product can be purified by adopting a simple recrystallization process to obtain high-content citicoline sodium;

(2) in the preparation method provided by the invention, the 1-propyl phosphoric cyclic anhydride which is an efficient condensing agent is used, so that the environmental protection problems of a large amount of strong acid condensing agents (such as oxalyl chloride and the like) and toxic solvents such as pyridine, benzene and the like in the traditional chemical process technology, and a large amount of process wastewater and the like caused by the use of the high-efficiency condensing agents are avoided;

(3) meanwhile, the process has the advantages of mild reaction conditions, short route, low energy consumption and simple and convenient post-treatment, and the high-content product is obtained only by mixed solvent recrystallization, thereby solving the problems of difficult post-treatment and low yield in the traditional chemical method and fermentation method processes.

Detailed Description

The features and advantages of the present invention will become more apparent and appreciated from the following detailed description of the invention, as illustrated in the accompanying drawings.

The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

In the description of the present invention, it should be noted that the terms "first", "second", "third" and "fourth" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

The present invention is described in detail below.

The traditional chemical synthesis method of citicoline sodium generally takes choline chloride calcium salt as an initial raw material and benzene as a solvent, removes crystal water by refluxing, then removes calcium ions by using carbonate or oxalic acid through forming insoluble calcium carbonate or calcium oxalate, activates the choline chloride by using acetyl chloride or solid phosgene in an organic amine solvent without active hydrogen, finally reacts with cytidylic acid to generate citicoline, and then carries out corresponding post-treatment steps such as recrystallization and the like to obtain a finished product.

The reaction equation of the chemical synthesis process is as follows:

the traditional chemical synthesis process is very easy to generate side reaction, and the content of the prepared product is low. Through careful study, the following impurities (impurities) A, B, C, D and E are generally produced in the process,

because the synthesis process is long in period, complex in treatment and rich in impurities in the product, on the basis of multiple tests, the invention optimally selects starting raw materials, a condensing agent, purification process and the like, takes choline chlorophosphate as the starting raw material, skips the processes of benzene reflux dehydration and acid radical addition to remove calcium ions in the traditional process from the source, shortens the process flow, avoids the residue of class 1 solvents and reduces the generation of solid wastes; simultaneously selects a mild condensing agent 1-propyl phosphoric cyclic anhydride (T)₃P) is condensed with cytidylic acid, so that side reactions caused by high activity of the traditional acyl chloride reagents and intermediates are avoided, the raw material reaction is more complete, less impurities are generated, and the product content is higher.

Specifically, the invention provides a preparation method of citicoline sodium or a composition thereof, which comprises the following steps:

step 1: mixing 5' -cytidylic acid and phosphorylcholine chloride (C)₅H₁₅ClNO₄P) is dispersed in an aprotic organic solvent, 1-propylphosphoric acid cyclic anhydride (T) is added₃P) reacting, filtering insoluble substances in the reaction solution, and collecting filtrate;

and step 3: the citicoline acid reacts with the soda alkali/salt to generate citicoline sodium, namely the citicoline.

The synthetic route of the invention is schematically shown as follows:

in step 1, the added 1-propylphosphoric cyclic anhydride has the function of a condensing agent, so that the 5' -cytidylic acid dispersed in the aprotic organic solvent and the phosphorylcholine chloride are subjected to condensation reaction. The 1-propyl phosphoric cyclic anhydride has good activation capability and can easily react with active hydrogen, and the aprotic organic solvent can avoid the side reaction of the 1-propyl phosphoric cyclic anhydride and the solvent.

The aprotic organic solvent is selected from one or a combination of acetonitrile, dimethylformamide, dimethyl sulfoxide, benzene, diethyl ether, carbon tetrachloride and acetone, preferably acetonitrile, diethyl ether or acetone, and more preferably acetonitrile with a relatively high boiling point and good solubility to raw materials is used as a reaction solvent.

The dosage of the aprotic organic solvent is generally 8 to 15 times of that of 5' -cytidylic acid, and the dosage is preferably 8 to 13 times by weight.

The 1-propyl phosphoric cyclic anhydride can improve the reactivity of 5 '-cytidylic acid and choline chloride phosphate, and is different from acyl chloride or solid phosgene in the traditional synthesis process, and the 1-propyl phosphoric cyclic anhydride has a synergistic condensation effect on the 5' -cytidylic acid and the choline chloride phosphate without an intermediate transition state; the acyl chloride or the solid phosgene needs to be firstly generated into an intermediate acylate and then converted into a target product, so that side reactions related to intermediate transition states, such as impurity E commonly existing in the traditional process, are easily generated in the reaction process.

In addition, the acyl chloride or the solid phosgene has high reaction activity, and can easily activate the active hydrogen of the amino group in the 5' -cytidylic acid to generate the impurity D. The activation activity of the 1-propyl phosphoric cyclic anhydride is lower than that of acyl chloride or solid phosgene, and the activation activity of the active hydrogen of the amino group in the 5' -cytidylic acid is insufficient, so that the impurity D is not generated.

The generation of impurities D and E is avoided by selecting a mild condensing agent, so that the generation of side reactions in the reaction is reduced, and the generation of a target product is promoted.

It has also been found that in the conventional synthesis process of citicoline sodium, the activating agent used is acyl chloride or phosgene in solid form, which has too high activity and can even react with water, and in order to avoid the side reaction of acyl chloride or solid light and water, benzene is used to dehydrate the solvent and reagent used in the reaction to ensure the progress of the target reaction.

In the field of chemicals, according to the technical guidelines on the study of residual solvents from chemical drugs, the first class of solvents refers to organic solvents that are carcinogens in humans, suspected carcinogens in humans, or environmental hazards, and benzene is specifically specified as the first class of solvents, which have unacceptable toxicity or cause environmental pollution and should be avoided as much as possible.

In the invention, the condensing agent is 1-propyl phosphoric cyclic anhydride, the reaction activity is relatively low, and the condensing agent can not react with water, so that the water in the reaction system is not required to be specially removed in the synthesis process of the sodium citicoline. This avoids the use of benzene as the first solvent and shortens the synthetic route and production cycle.

In order to make the reactivity of the 1-propylphosphoric cyclic anhydride in the reaction system milder, it is preferable to control the reaction temperature of the reaction system when the condensing agent is added, so as to avoid the increase of the activity of the 1-propylphosphoric cyclic anhydride at high temperature and the resulting increase of the activity of the non-target group.

The condensing agent is generally added at temperatures below 50 deg.C based on the reactivity of the 1-propylphosphoric acid cyclic anhydride. Preferably, the reaction is carried out by adding a condensing agent at room temperature (10 to 30 ℃).

When 1-propylphosphoric cyclic anhydride is added at room temperature, 5 '-cytidylic acid and choline chloride phosphate react mainly by kinetics under the condensation activation action of 1-propylphosphoric cyclic anhydride due to the high concentration of the 1-propylphosphoric cyclic anhydride at the beginning of the reaction, and specific reaction is carried out according to the activation difficulty of active hydrogen in 5' -cytidylic acid.

In the lactone ring of 5 '-cytidylic acid, because the hydroxyl group on the ring has steric hindrance, 1-propyl phosphoric cyclic anhydride with a cyclic structure rarely activates the cyclic hydroxyl groups (3' position and 4 'position) of 5' -cytidylic acid, but easily activates hydrogen phosphate with smaller steric hindrance on the cyclic hydroxyl groups, so that the target reaction in a reaction system becomes a dominant reaction.

Further, it is also necessary to control the addition rate of the 1-propylphosphoric cyclic anhydride. The addition rate of the 1-propylphosphoric cyclic anhydride is 0.5-5 ml/min, preferably 1-3 ml/min, and most preferably 1.5-2 ml/min.

It is preferable to continuously stir the 1-propylphosphoric cyclic anhydride during the addition of the 1-propylphosphoric cyclic anhydride so that the 1-propylphosphoric cyclic anhydride can be rapidly dispersed after being added to the reaction system.

The reaction system immediately starts to react after the 1-propylphosphoric cyclic anhydride is added, and the 1-propylphosphoric cyclic anhydride is continuously stirred to react after the addition is finished, so that the 5' -cytidylic acid and the choline chloride are fully reacted under the condition of kinetic reaction, and side reactions which are easy to occur in the thermodynamic reaction under the heating condition can be avoided.

After the 1-propylphosphoric cyclic anhydride is added, the stirring reaction is continued for 0.5 to 3 hours, preferably 0.5 to 2 hours, and more preferably 0.75 to 1.5 hours.

At this time, most of 5' -cytidylic acid and phosphorylcholine chloride in the reaction system have reacted, and in order to improve the reactivity of the remaining reaction raw materials, the reaction yield is improved by increasing the thermal power of the molecules of the reaction substance after heating the reaction system.

In a preferred embodiment, the reaction system is heated to reflux, and the reaction is continued for 3 to 5 hours to fully react the reaction raw materials.

In the step 1, the molar ratio of the 5' -cytidylic acid to the phosphorylcholine chloride can be 1:1, preferably 1:1 to 1.1, and more preferably 1:1 to 1.05.

Wherein the molar ratio of the 1-propylphosphoric cyclic anhydride to the 5' -cytidylic acid is preferably 1: 1-1.1, and more preferably 1: 1-1.05.

After the reflux reaction was completed, the heating was stopped, the temperature of the reaction system was lowered, and the insoluble matter was removed while it was hot. Considering the volatility of the organic solvent, the high temperature of the solution during the filtration is easy to generate hidden troubles, and the safety of a production workshop is influenced; and is easy to be inhaled by the operator, which affects the health of the operator. When the temperature of the reaction system is too low, products or other substances are separated out, and the purpose of removing impurities cannot be realized.

Through a plurality of researches, the temperature of a reaction system is preferably reduced to 40-70 ℃, more preferably 50-60 ℃, the volatilization speed of a reaction solvent is relatively slow, and soluble substances are not precipitated under the temperature condition. The content of the product can be increased primarily by removing insoluble substances in the reaction system through hot filtration.

In the reaction process of the step 1, due to mild reaction conditions, the filtrate does not contain impurities D and E which are usually generated in the traditional synthetic process, but only contains the impurity A and a very small amount of the impurity B. Wherein A is a raw material impurity, and B is a reaction impurity.

In the reaction method provided by the invention, because the 3 ' site and 4 ' site cyclic hydroxyl groups on 5 ' -cytidylic acid are weakly activated by the condensing agent, and because of the influence of steric hindrance on the ring and the multiple actions of mild reaction conditions of the invention, the content of impurities B in the thermal reaction is very low and is rarely generated, the difficulty of subsequent treatment is reduced in the step 1, and the improvement of the quality of the final product of the citicoline sodium is facilitated.

In step 2, the filtrate obtained in step 1 is cooled to precipitate citicolic acid.

When the filtrate is cooled, the filtrate is cooled at a too high temperature, and precipitates can be embedded in the filtrate, so that the precipitate impurity content is high.

In a preferred embodiment, the filtrate obtained in step 1 may be cooled naturally. The natural cooling means that the temperature of the filtrate is not reduced, and the filtrate is slowly reduced only by the temperature difference between the filtrate and the ambient temperature.

When naturally cooled, more product can be precipitated as the ambient temperature is lower, but the precipitation period is correspondingly longer. The environment temperature is preferably 10 to 30 ℃, more preferably 10 to 25 ℃, and most preferably 10 to 20 ℃.

In another preferred embodiment, the filtrate is cooled at a cooling rate of 5-15 ℃/h to precipitate the citicolic acid. Preferably 6-12 ℃/h, more preferably 7-10 ℃/h, high precipitation speed and less embedded impurities.

And filtering and collecting the precipitate to obtain the citicoline acid.

The filtrate for filtering the citicolic acid can be used for the next production, so that the utilization rate of the reaction solvent is effectively improved, and the production cost and the treatment capacity of the chemical waste liquid are reduced.

In step 3, the citicoline sodium is prepared by the salifying reaction of the citicoline acid prepared in the step 2 and alkaline sodium alkali/salt. Preferably, the citicolic acid is dissolved in purified water to carry out salt-forming reaction with water-soluble sodium alkali/salt.

In order to increase the dissolution rate of the citicolic acid, heating may be used if necessary.

Wherein the alkaline sodium alkali/salt is selected from any one or combination of sodium hydroxide, sodium carbonate, sodium bicarbonate, sodium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium acetate and sodium lactate; preferably the sodium base/salt is sodium hydroxide, sodium carbonate and/or sodium bicarbonate; more preferably, sodium carbonate or bicarbonate, which is relatively weakly basic, is used to reduce hydrolysis of the ester bond during salt formation.

In order to ensure that the citicolic acid is completely converted into sodium salt, preferably, sodium base/salt is added to adjust the pH value of the aqueous solution to 7-8, preferably 7.5-8, so that the citicolic acid can fully react to form the citicolic sodium. The sodium base/salt is preferably dissolved and added dropwise to the solution in which the citicolic acid is dissolved.

To increase the reaction rate of the citicolinic acid and the sodium base/salt, the aqueous solution in which the citicolinic acid is dissolved may be heated. However, when the temperature of the solution is too high, hydrolysis of the reaction product may be caused, particularly under alkaline aqueous conditions.

It is found that during the salt-forming reaction, the phosphate group in 5' -cytidylic acid is hydrolyzed in alkaline aqueous solution to form impurity C. And the P-O ester bond in the citicoline sodium is not easy to hydrolyze due to the influence of steric hindrance.

After multiple researches, the sodium alkali/salt is added after the solution is heated to 30-40 ℃, so that salt forming reaction is facilitated, and hydrolysis reaction can be reduced.

After the salt-forming reaction is finished, adding a poor solvent into the solution under stirring to separate out the sodium citicoline. The poor solvent is preferably ethanol, methanol, isopropanol, acetone and/or dimethylformamide, preferably acetone or ethanol, more preferably ethanol.

Preferably, the poor solvent is added while the solution is hot, and when the dissolving performance of the salification reaction solution on the sodium citicoline is gradually deteriorated, a gradually decreasing process also exists at a relatively high solution temperature, so that the impurities A and B with low content are continuously dissolved without precipitation, and the target product sodium citicoline is gradually precipitated along with the saturation of the solution dissolving capacity.

Wherein the volume of the poor solvent is 0.1-0.5 times, preferably 0.1-0.4 times, and most preferably 0.1-0.3 times of the volume of the aqueous solution in the salt forming reaction. When the addition amount of the poor solvent is excessive, the precipitation speed of the citicoline sodium is too high, excessive impurities are easily embedded, and the purity and the content of the product are reduced.

The adding speed of the poor solvent is not easy to be too fast so as to prevent the citicoline sodium from being locally separated out. Preferably, the poor solvent is added in 3 to 5 times under stirring.

When the poor solvent is added, the stirring speed is 50-300 r/min, preferably 100-150 r/min, so that the poor solvent can be rapidly dispersed.

After the poor solvent is added, the citicoline sodium is separated out.

Further, after the poor solvent is added, the temperature of the solution can be reduced to 10 +/-2 ℃, and the preferable cooling rate is 0.1-0.5 ℃/min; more preferably 0.1 to 0.3 ℃/min.

Keeping the temperature for 30-60 min to gradually precipitate crystal nuclei in the solution, preferably for 45-60 min; keeping the stirring speed at 40-80 r/min; preferably 40-50 r/min, so that the crystal particles are uniform in size.

In the precipitation process, the slow crystal growth speed is beneficial to the growth of dominant crystals and is beneficial to improving the stability of the product.

And filtering and collecting the precipitate, and drying to obtain the citicoline sodium (citicoline), wherein the citicoline sodium only contains impurities A, B and C.

In a preferred embodiment, the prepared citicoline can be subjected to recrystallization treatment to further increase the content of citicoline and reduce the content of impurities.

In the recrystallization treatment, the prepared sodium citicoline is preferably dissolved in a hot crystallization solvent until the solution is saturated, cooled and crystallized.

The crystallization solvent is one or more aqueous solutions of ethanol, methanol, isopropanol, acetone and dimethylformamide, and in order to reduce the toxicity of the reaction, the ethanol/aqueous solution is preferably used as the crystallization solvent.

The physical properties of the impurities A, B and C are similar to those of the product sodium citicoline, and both impurities have good water solubility. In order to increase the removal amount of the impurities A, B and C and to keep the impurities in the recrystallization mother liquor as much as possible, an aqueous solution having an ethanol volume concentration of 15 to 25% is preferably used as the crystallization solvent.

In order to reduce the heat source, microorganisms, and the like in the recrystallized product, water for injection or a sterilized water for injection crystallization solvent may also be used.

During the recrystallization treatment, the citicoline sodium prepared in the step 2 is dissolved in a crystallization solvent, and the solubility is improved by heating so as to reduce the dosage of the solvent. In order to reduce ester bond hydrolysis of the product prepared in the step 2 in recrystallization, the recrystallization solvent is preferably heated to 35-45 ℃ to dissolve the sodium citicoline to saturation.

Filtering with microporous membrane while hot, removing insoluble substances, sterilizing, and cooling to reduce the content of impurity C in sodium citicoline.

During recrystallization, when the cooling rate is too high, the product is quickly separated out and is easy to embed and mix with impurities in the mother solution, so that the impurity content of the product is too high; when the cooling rate is too slow, the precipitation time is too long, and the crystallization efficiency is low.

Preferably, the cooling rate is 5-10 ℃/h, preferably 6-8 ℃/h, the solution is cooled to 5-8 ℃, and the temperature is maintained to separate out crystals. Preferably, the temperature is kept for 5-10 h to fully grow the crystal.

The stirring speed is controlled to be 80-120 r/min (revolution/minute) in the recrystallization process, and preferably 80-100 r/min.

Filtering and collecting filter cakes, and drying in vacuum or drying to obtain the citicoline sodium (also called citicoline).

Impurities A, B and C are retained in the mother liquor by recrystallization, and the purity and content of the product sodium citicoline are further improved. The preparation method of the citicoline sodium provided by the invention can obtain the citicoline sodium with the content of more than 99.0%, particularly more than 99.3%, and preferably more than 99.6%.

In the preparation method of the sodium citicoline provided by the invention, because the reaction condition is mild, impurities D and E cannot be generated, the reaction raw materials are easy to react completely, and the amount of the impurities A and B is greatly reduced.

Compared with the traditional synthesis method, the preparation method provided by the invention has the advantages of higher conversion rate, less side reaction and no use of reactive organic amine solvent; the reaction solvent and the recrystallization solvent can be recycled after simple treatment, the complexity of a reaction system is reduced, the product can be purified by adopting a simple recrystallization process, the content of impurities A, B and C is reduced, and the high-content citicoline sodium is obtained.

In the preparation process, the solvents crystallized twice can be recycled after simple distillation and recovery, so that the use amount of the organic solvent is effectively reduced, and the production cost and the difficulty of reagent recovery are reduced; the method is environment friendly.

In addition, in the preparation method provided by the invention, the 1-propyl phosphoric cyclic anhydride which is an efficient condensing agent is used, so that the environmental protection problems of a large amount of strong acid condensing agents (such as oxalyl chloride and the like) and toxic solvents such as pyridine, benzene and the like in the traditional chemical method process, and a large amount of process wastewater and the like caused by the use of the toxic solvents are avoided; meanwhile, the process has the advantages of mild reaction conditions, short route, low energy consumption and simple and convenient post-treatment, and the high-content product is obtained only by mixed solvent recrystallization, thereby solving the problems of difficult post-treatment and low yield in the traditional chemical method and fermentation method processes.

In a second aspect, the present invention also provides citicoline sodium prepared by the above method or a composition thereof, which comprises citicoline sodium in an amount of 99% by mass or more, preferably 99.5% by mass or more.

Further, the citicoline sodium or the composition thereof also contains impurity A, and the content of the impurity A is below 0.2%, preferably below 0.1%;

the citicoline sodium or the composition thereof also contains impurity B, and the content of the impurity B is below 0.1 percent, preferably below 0.05 percent;

the citicoline sodium or the composition thereof also contains impurity C, and the content of the impurity C is below 0.1%, preferably below 0.05%.

In particular, the citicoline sodium or the composition thereof is placed under the condition of long-term test (25 ℃ +/-2 ℃/65% RH +/-5% RH) for 18 months, the content of the citicoline sodium is not lower than 99.0%, the content of the impurity A is not higher than 0.2%, the content of the impurity B is not higher than 0.1%, and the content of the impurity C is not higher than 0.1%.

In a third aspect, the present invention provides a citicoline preparation of citicoline sodium or a composition thereof prepared by the above method, wherein the preparation contains citicoline sodium as an active ingredient.

The preparation is used for injection or oral administration, and preferably the preparation is an injection or oral preparation.

Wherein, the injection can be a freeze-dried preparation, a solution or a sterile subpackaged preparation; the oral preparation can be tablet, capsule, sustained release preparation or granule.

The preparation can also be used together with other active ingredients, such as piracetam, edaravone or thromboxane, to prepare the citicoline compound preparation.

In addition, the invention also provides application of the citicoline sodium or the composition thereof, the citicoline preparation and the citicoline compound preparation in preparing the cerebral ischemia nerve function recovery medicine. Further, the application in preparing the medicine for treating the cerebral infarction acute-stage disturbance of consciousness.

Examples

Example 1

Preparation of sodium citicoline:

adding 50g of 5' -cytidylic acid, 500ml of acetonitrile and 36g of choline chloride phosphate into a reaction bottle, starting stirring, adding 52g of 1-propylphosphoric acid cyclic anhydride at the temperature of 25 ℃ at the speed of 2.0ml/min, stirring for 1 hour, heating to reflux the reaction system, carrying out reflux reaction for 3 hours, cooling the reaction system to 55 ℃, filtering while the reaction system is hot, and collecting filtrate;

naturally cooling the filtrate to 25 deg.C, precipitating a large amount of crystals, filtering to obtain citicolic acid, and mechanically applying the filtrate to the next batch;

dissolving the collected citicolic acid in 1000ml of purified water, heating to 35 ℃, stirring for dissolving, adding sodium bicarbonate to adjust the pH value to 7.5, adding 200ml of ethanol while stirring, cooling the solution to 10 ℃ at the cooling rate of 2 ℃/min, preserving the temperature for 45min, filtering and collecting citicolic sodium;

dissolving sodium citicoline into ethanol/water solution with ethanol volume concentration of 15% at about 40 deg.C until saturation, filtering with 0.2 μm microporous membrane, cooling the filtrate to 5 deg.C at 6 deg.C/h, stirring at 80r/min, and maintaining the temperature for 5 h.

The filter cake was collected by filtration and dried in vacuo to give 72.08g of sodium citicoline in 91.30% yield.

Example 2

Preparation of sodium citicoline:

adding 50g of 5' -cytidylic acid, 500ml of acetonitrile and 34.5g of choline chloride phosphate into a reaction bottle, starting stirring, adding 49.5g of 1-propylphosphoric acid cyclic anhydride at the temperature of 30 ℃ at the speed of 1.5ml/min, stirring for 0.75 hour after the addition, heating to a reaction system for reflux, carrying out reflux reaction for 5 hours, cooling the reaction system to 60 ℃, filtering while the reaction system is hot, and collecting filtrate;

cooling the filtrate to 15 ℃, precipitating a large amount of crystals, filtering to obtain the citicolic acid, and mechanically applying the filtrate to the next batch;

dissolving the collected citicolic acid in 1000ml of purified water, heating to 40 ℃, stirring for dissolving, adding sodium bicarbonate to adjust the pH value to 7, adding 300ml of ethanol while stirring, cooling the solution to 10 ℃ at the cooling rate of 1 ℃/min, preserving the temperature for 60min, filtering and collecting citicolic sodium;

dissolving sodium citicoline into 25% ethanol/water solution at 50 deg.C, filtering with 0.2 μm microporous membrane, cooling to 8 deg.C at 8 deg.C/h, stirring at 100r/min, and maintaining for 5 h.

The filter cake was collected by filtration and dried in vacuo to give 73.06g of sodium citicoline in 92.54% yield.

Example 3

Preparation of sodium citicoline:

adding 50g of 5' -cytidylic acid, 500ml of acetonitrile and 35.5g of choline chloride phosphate into a reaction bottle, starting stirring, adding 51g of 1-propylphosphoric acid cyclic anhydride at the temperature of 20 ℃ at the speed of 3.0ml/min, stirring for 1.5 hours after the addition, heating to a reaction system for reflux, carrying out reflux reaction for 5 hours, cooling the reaction system to 50 ℃, filtering while hot, and collecting filtrate;

dissolving the collected citicolic acid in 1000ml of purified water, heating to 30 ℃, stirring and dissolving, adding sodium carbonate to adjust the pH value to 8, adding 200ml of ethanol while stirring, cooling the solution to 10 ℃ at the cooling rate of 3 ℃/min, preserving the temperature for 60min, filtering and collecting citicolic sodium;

dissolving sodium citicoline into 20% ethanol/water solution at 45 deg.C, filtering with 0.2 μm microporous membrane, cooling to 7 deg.C at 5 deg.C/h, stirring at 100r/min, and maintaining for 5 h.

The filter cake was collected by filtration and dried in vacuo to give 71.41g of sodium citicoline in 90.45% yield.

Example 4

62.5g of citicoline sodium prepared in the embodiment 1-3 and 4.5g of sodium chloride are respectively weighed, dissolved in 500ml of water for injection, added with 1g of active carbon for injection to be adsorbed for 30min, filtered by a 0.2 mu m microporous membrane, 2ml of the solution is measured by citicoline sodium, the content is 250mg, and the preparation A, B and C are prepared by moist heat sterilization.

Example 5

Cytophosphocholine sodium tablet

The prescription is as follows:

the preparation process comprises the following steps:

mixing the above materials uniformly, making into soft material by conventional process, oven drying, and tabletting with 100mg standard of sodium citicoline.

Comparative example

Comparative example 1

The same preparation method as in example 1 is adopted, except that:

the temperature of the reaction system was adjusted to 50 ℃ when 1-propylphosphoric acid cyclic anhydride was added.

Comparative example 2

The same preparation method as in example 1 is adopted, except that:

in the preparation of the sodium salt, sodium hydroxide was used to adjust the pH of the solution to 9.0.

Comparative example 3

The same preparation method as in example 1 is adopted, except that:

the sodium citicoline is not subjected to recrystallization.

Examples of the experiments

Experimental example 1

The residual solvent of citicoline prepared in examples 1 to 3 was measured according to the rules of the pharmacopoeia of the 2015 edition. The results are as follows:

	ethanol/ppm	Acetonitrile/ppm
			Example 1	88	6
Example 2	125	12
			Example 3	106	8

The embodiment 1-3 avoids the use of a class 1 solvent, so that toxic and harmful organic solvents hardly exist in the product; in addition, the organic solvent residue in examples 1 to 3 was very small, and the long-term use safety was high.

Experimental example 2

The content of citicoline prepared in examples 1 to 3 and comparative examples 1 to 3 was measured by high performance liquid chromatography (external standard method).

Examples	Content/%	Comparative example	Content/%
				Example 1	99.6	Comparative example 1	98.3
Example 2	99.7	Comparative example 2	98.6
				Example 3	99.7	Comparative example 3	98.6

Experimental example 3

The mass contents of citicoline prepared in examples 1 to 3 and comparative examples 1 to 3, and the mass contents of impurities A, B and C were measured by high performance liquid chromatography (external standard method). The test samples were placed under long-term test conditions (25 ℃. + -. 2 ℃/65% RH. + -. 5% RH) for 0, 6, 12, 18 months, respectively. The results are as follows:

as can be seen from the above table, after long-term testing, the content of citicoline sodium in examples 1 to 3 is stable, while the content of impurity A is not more than 0.2%, the content of impurity B is not more than 0.05%, and the content of impurity C is not more than 0.1%.

Experimental example 4

The mass contents of citicoline (in percent of the labeled amount) and impurities A, B and C in the citicoline preparation A, B, C prepared in example 4 were determined by high performance liquid chromatography (external standard method). The detection samples are respectively placed for 1, 3 and 6 months under accelerated test conditions (40 ℃ +/-2 ℃/75% RH +/-5% RH). Acidity and clarity of the solution were measured according to the rules of the 2015 pharmacopoeia.

The results are as follows:

therefore, after an accelerated test, the content of citicoline in the preparations A to C is very stable and only a small amount of citicoline is degraded; the content of the impurity A is not more than 0.3 percent, the content of the impurity B is not more than 0.3 percent, and the content of the impurity C is not more than 0.1 percent; the clarity of the citicoline preparation after the accelerated test also meets the injection requirement.

Experimental example 5

80 healthy male mice with weight of 25-30g are selected, normally bred for 3 days, weighed and randomly divided into 5 groups of 16 mice, wherein the groups are respectively a pseudo-operation group (negative control group), a model group, a nimodipine group, and a high-dose and low-dose citicoline sodium group. 30mg/kg of nimodipine intravenous injection suspension (positive control drug), high and low doses of citicoline sodium (administration dose is 5g/kg of body weight and 2.5g/kg of body weight respectively), and the pseudo-operation group and the model group are administered with the same volume of water for injection for intravenous injection, 1 time per day and 7 days of continuous administration.

On the 6 th day, 8 pm, fasting in batches without water feeding is started, on the 7 th day, 8 am, weighing and dosing in batches for 1h, carrying out intraperitoneal injection on an anesthetized mouse by using 10% chloral hydrate (0.03ml/10g), after the mouse is anesthetized, cutting a left incision in the center of the neck, separating and exposing a left common carotid artery, an external carotid artery and an internal carotid artery layer by layer, ligating the common carotid artery and the external carotid artery, clamping the internal carotid artery by an artery, cutting a small opening with the width of about 0.2mm at the position 1mm away from the bifurcation of the common carotid artery, inserting a thread plug, not entering the internal carotid artery through the bifurcation of the common carotid artery, extending upwards to the bifurcation by 8-10mm only until resistance exists, blocking an entrance of a middle cerebral artery, ligating an incision and a thread of the internal carotid artery, and slightly drawing out the thread after 2h, realizing reperfusion, and creating artery obstruction in the middle cerebral artery and reperfusion. Only the left blood vessel was exposed in the sham group without any treatment for a patch.

After 22h of reperfusion of all mice, the molded mice were scored for loss of neurological function: the Longa standard was used. Grading standard: 0 minute: no symptom of neurological deficit; 1 minute: the contralateral anterior paw cannot be fully extended; and 2, dividing: hemiplegic paralysis turns when crawling; and 3, dividing: when walking, the body falls over to the hemiplegic side; and 4, dividing: the person cannot walk spontaneously and consciousness is lost; and 5, dividing: and death.

Note: p < 0.01, P < 0.05

The invention has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to be construed in a limiting sense. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, which fall within the scope of the present invention. The scope of the invention is defined by the appended claims.

Claims

1. A method for preparing a citicoline sodium composition is characterized in that,

the citicoline sodium composition comprises citicoline sodium with the content of more than 99.0 percent,

also comprises an impurity A which is a nitrogen-containing impurity,

the content is below 0.2 percent;

the impurity B is a nitrogen-containing impurity,

the content of the organic acid is below 0.1 percent,

the impurity C is the impurity C which is the impurity,

the content of the organic acid is below 0.1 percent,

the preparation method comprises the following steps:

step 1: dispersing 5' -cytidylic acid and choline chloride phosphate in an aprotic organic solvent, adding 1-propylphosphoric acid cyclic anhydride for reaction, filtering insoluble substances in a reaction solution, and collecting filtrate, wherein the aprotic organic solvent is acetonitrile, diethyl ether or acetone, and the adding speed of the 1-propylphosphoric acid cyclic anhydride is 1-3 ml/min;

step 2: cooling the filtrate obtained in the step 1, precipitating crystals, and filtering to obtain the citicolic acid, wherein the cooling rate is 7-10 ℃/h; and step 3: the citicoline acid reacts with sodium base/salt to generate the citicoline sodium, and the sodium base/salt is sodium hydroxide, sodium carbonate and/or sodium bicarbonate.

2. The production method according to claim 1,

3. The method according to claim 1, wherein the sodium citicoline obtained in step 3 is subjected to recrystallization.