CN111925373A - Preparation method of folic acid EP impurity F - Google Patents
Preparation method of folic acid EP impurity F Download PDFInfo
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- CN111925373A CN111925373A CN202010910277.3A CN202010910277A CN111925373A CN 111925373 A CN111925373 A CN 111925373A CN 202010910277 A CN202010910277 A CN 202010910277A CN 111925373 A CN111925373 A CN 111925373A
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Abstract
The invention provides a preparation method of folic acid EP impurity F, which comprises the following steps: A)2,4,5, 6-tetraaminopyrimidine sulfate and 1,1, 3-trichloroacetone are subjected to condensation reaction in the presence of an antioxidant and a solvent to obtain a compound with a structure shown in a formula (I); B) hydrolyzing the compound with the structure shown in the formula (I) by acid to obtain a folic acid impurity F crude product, and purifying the folic acid impurity F crude product by chromatography to obtain a folic acid EP impurity F with the structure shown in the formula (II). The invention adopts a brand new synthesis route, can prepare the impurity F by only two steps, has higher purity of the obtained product, is suitable for the deep research process of the property and the synthesis mechanism of the folic acid, and lays a foundation for effectively improving the quality control level of the folic acid. The method has the advantages of mild reaction conditions, cheap and easily-obtained starting materials for reaction, short reaction steps, simple post-treatment and high crude product yield which is more than 70%.
Description
Technical Field
The invention relates to the technical field of drug synthesis, in particular to a preparation method of folic acid EP impurity F.
Background
Folic acid, also known as vitamin B9, is a water-soluble vitamin. Experiments show that folic acid plays an important role in protein synthesis, cell division and growth, promotes the formation of normal red blood cells, influences reproductive performance, influences the secretion of pancreas, promotes the growth of human body, improves the immunity of the organism, and plays an important role in the growth, development and metabolism processes. Folate deficiency can lead to reduced hemoglobin production in erythrocytes and impaired cell maturation, resulting in megaloblastic anemia. The lack of folic acid in pregnant women can lead to serious consequences such as fetal birth defects, neural tube malformation and the like, and can increase the risk of pregnancy-induced hypertension, spontaneous abortion and the like of the pregnant women. Therefore, the reasonable use of folic acid has great influence on the maintenance of the physiological condition of pregnant women and the normal development of fetuses. If the mutation of key genes involved in the folate metabolism pathway is generated, the activity of the folate metabolism enzyme is reduced, so that the folate metabolism is disturbed, and the folate deficiency is caused. If the folic acid is excessive, the absorption of zinc element is affected, which may cause abnormal development of fetus and increase the risk of malformation of fetus. The risk type genes exist in Chinese population in a certain proportion, so that different people have different folic acid utilization capacities, and therefore folic acid needs to be supplemented individually according to different populations.
The quality of folic acid raw material medicine is the key and source of folic acid related product quality control, wherein the research on impurities and the control of clinical safety of related medicines become one of the key links of raw material medicine quality control. Adverse reactions in clinical use of a drug product are sometimes closely related to impurities in the drug product in addition to the pharmacological activity of the drug product itself, and thus the impurities in the drug product must be carefully studied and strictly controlled.
Patent document CN105541845 reports that folic acid is composed of three fragments, and although the mechanism is studied for many years, it is still not clear whether the reaction is performed synergistically or stepwise, and one of them is that I is cyclized with II first, and then nucleophilic substitution reaction is performed with a third fragment to generate folic acid.
Based on this reaction mechanism, it is presumed that 7-chloromethylpterin (EP impurities F, IV) and 6-chloromethylpterin (V) are inevitably produced during the reaction. 7-Chloromethylpterin (EP impurities F, IV) and 6-chloromethylpterin (V) have the following structures:
IV is recorded in EP9.5, belongs to a process nonspecific impurity, and is numbered as F, although the chemical structure is simple, the synthetic method and the structural characterization data are not reported in documents so far. In addition, the impurities are not commercially available at present, which adds a little difficulty to the research of related substances of folic acid raw materials.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a method for preparing folic acid EP impurity F, wherein the folic acid EP impurity F prepared by the preparation method provided by the present invention has high yield and purity.
The invention provides a preparation method of folic acid EP impurity F, which comprises the following steps:
A)2,4,5, 6-tetraaminopyrimidine sulfate and 1,1, 3-trichloroacetone are subjected to condensation reaction in the presence of an antioxidant and a solvent to obtain a compound with a structure shown in a formula (I);
B) hydrolyzing the compound with the structure shown in the formula (I) by acid to obtain a folic acid impurity F crude product, and purifying the folic acid impurity F crude product by chromatography to obtain a folic acid EP impurity F with the structure shown in the formula (II);
preferably, the molar ratio of the 2,4,5, 6-tetraaminopyrimidine sulfate, the 1,1, 3-trichloroacetone and the antioxidant in the step A) is 1:1: 2-1: 1: 5.
Preferably, the solvent in the step A) is a mixed solution of water and alcohol; the alcohol is selected from one or more of methanol, ethanol, propanol and isopropanol; the volume ratio of the water to the alcohol is 1: 3-3: 1; the mass ratio of the 2,4,5, 6-tetraaminopyrimidine sulfate to the solvent is 1: (30-80).
Preferably, the antioxidant in the step A) is one or more of L-cysteine, L-pyroglutamic acid, sodium metabisulfite and sodium bisulfite.
Preferably, the reaction in the step A) is carried out under an acidic condition, and the pH value of the reaction is 1-4; the acid for adjusting the pH value is one of hydrochloric acid, sulfuric acid or glacial acetic acid.
Preferably, the reaction temperature in the step A) is 20-80 ℃; the reaction time is 1-36 h.
Preferably, the acid used in the acid hydrolysis in the step B) is one of hydrochloric acid, sulfuric acid or hydrobromic acid; the mass concentration of the acid is 30-50%;
the mass ratio of the compound with the structure of the formula (I) to the acid is 1: (2-8).
Preferably, the temperature of the acid hydrolysis in the step B) is 60-100 ℃; the time of acid hydrolysis is 0.5-5 h.
Preferably, the folic acid EP impurity F with the structure of the formula (II) obtained by chromatographic purification of the crude folic acid impurity F is specifically:
dissolving the folic acid impurity F crude product with a solvent, filtering, feeding the filtrate into a semi-preparative chromatograph, collecting the required component solution, removing the solvent under reduced pressure, freeze-drying, washing the obtained solid with water to remove salt, and drying under reduced pressure to obtain the folic acid impurity F.
Preferably, the chromatographic purification parameters are: the instrument comprises the following steps: semi-preparative chromatograph, SepaBean machine T, column: spherial C18, 50 μm 100A; detection wavelength: 280 nm; mobile phase A: 0.1% phosphoric acid, mobile phase B: acetonitrile; elution gradient: 0-20 min, 90-30% of A.
Compared with the prior art, the invention provides a preparation method of folic acid EP impurity F, which comprises the following steps: A)2,4,5, 6-tetraaminopyrimidine sulfate and 1,1, 3-trichloroacetone are subjected to condensation reaction in the presence of an antioxidant and a solvent to obtain a compound with a structure shown in a formula (I); B) hydrolyzing the compound with the structure shown in the formula (I) by acid to obtain a folic acid impurity F crude product, and purifying the folic acid impurity F crude product by chromatography to obtain a folic acid EP impurity F with the structure shown in the formula (II). The invention adopts a brand new synthesis route, can prepare the impurity F by only two steps, has higher purity of the obtained product, is suitable for the deep research process of the property and the synthesis mechanism of the folic acid, and lays a foundation for effectively improving the quality control level of the folic acid. The method has the advantages of mild reaction conditions, cheap and easily-obtained starting materials for reaction, short reaction steps, simple post-treatment and high crude product yield which is more than 70%.
Drawings
FIG. 1 is a chart of HPLC specific data for crude impurity F synthesized in example 1;
FIG. 2 is a specific data chart of HPLC after the crude product of impurity F in example 1 is prepared and purified;
FIG. 3 is a mass spectrum of impurity F after purification in example 1;
FIG. 4 shows the impurity F purified in example 11HNMR atlas.
Detailed Description
The invention provides a preparation method of folic acid EP impurity F, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a preparation method of folic acid EP impurity F, which comprises the following steps:
A)2,4,5, 6-tetraaminopyrimidine sulfate and 1,1, 3-trichloroacetone are subjected to condensation reaction in the presence of an antioxidant and a solvent to obtain a compound with a structure shown in a formula (I);
B) hydrolyzing the compound with the structure shown in the formula (I) by acid to obtain a folic acid impurity F crude product, and purifying the folic acid impurity F crude product by chromatography to obtain a folic acid EP impurity F with the structure shown in the formula (II);
the invention provides a preparation method of folic acid EP impurity F, which comprises the following steps of carrying out condensation reaction on 2,4,5, 6-tetraaminopyrimidine sulfate and 1,1, 3-trichloroacetone in the presence of an antioxidant and a solvent to obtain a compound with a structure shown in a formula (I).
The compound with the structure shown in the formula (I) is obtained by dropwise adding acid to control the pH value of a reaction system under the condition that an antioxidant and a solvent exist in 2,4,5, 6-tetraaminopyrimidine sulfate and 1,1, 3-trichloroacetone, and carrying out condensation reaction.
The reaction is carried out under an acidic condition, and the pH value of the reaction is preferably 1-4; more preferably 1-2; the acid for adjusting the pH value is one of hydrochloric acid, sulfuric acid or glacial acetic acid.
Wherein the reaction temperature is preferably 20-80 ℃; more preferably 20-60 ℃; the reaction time is preferably 1-36 h; more preferably 1-24 h; most preferably 1-20 h.
The preferable molar ratio of the 2,4,5, 6-tetraaminopyrimidine sulfate, the 1,1, 3-trichloroacetone and the antioxidant is 1:1: 2-1: 1: 5; more preferably 1:1:2 to 1:1: 3.
The mass ratio of the 2,4,5, 6-tetraaminopyrimidine sulfate to the solvent is preferably 1: (30-80); more preferably 1: (40-70); most preferably 1: (40-50). Wherein the solvent is a mixed solution of water and alcohol; the alcohol is selected from one or more of methanol, ethanol, propanol and isopropanol; the volume ratio of the water to the alcohol is preferably 1: 3-3: 1; more preferably 2:1 to 1: 1.
The antioxidant is preferably one or more of L-cysteine, L-pyroglutamic acid, sodium metabisulfite and sodium bisulfite. The present invention is not limited in its source, and may be commercially available.
In a preferred embodiment of the present invention, the reaction formula is as follows:
hydrolyzing the compound with the structure of the formula (I) by acid to obtain a folic acid impurity F crude product. The reaction formula is as follows:
the acid used in the acid hydrolysis is preferably one of hydrochloric acid, sulfuric acid or hydrobromic acid; more preferably hydrobromic acid; the present invention is not limited in its source, and may be commercially available. The mass concentration of the acid is preferably 30-50%; more preferably 35 to 45 percent; most preferably 40%.
Wherein, the mass ratio of the compound with the structure of the formula (I) to the acid is preferably 1: (2-8); more preferably 1: (3-7); most preferably 1: (3-5).
The temperature of acid hydrolysis is preferably 60-100 ℃; more preferably 70-90 ℃; the time for acid hydrolysis is preferably 0.5-5 h; more preferably 1-4 h; most preferably 1-3 h.
And (3) carrying out chromatographic purification on the folic acid impurity F crude product to obtain the folic acid EP impurity F with the structure of the formula (II).
The folic acid EP impurity F with the structure of the formula (II) obtained by chromatographic purification of the folic acid impurity F crude product is specifically as follows:
dissolving the folic acid impurity F crude product with a solvent, filtering, feeding the filtrate into a semi-preparative chromatograph, collecting the required component solution, removing the solvent under reduced pressure, freeze-drying, washing the obtained solid with water to remove salt, and drying under reduced pressure to obtain the folic acid impurity F.
Wherein, the dissolving solvent is preferably DMSO or DMF; the present invention is not limited to the specific filtration method, and may be performed by a conventional method known to those skilled in the art.
Separating the filtrate by semi-preparative chromatograph.
The parameters of the semi-preparative chromatograph preparation and purification are as follows:
semi-preparative chromatography. The instrument comprises the following steps: SepaBean machine T;
a chromatographic column: spherial C18, 50um 100A; the sample injection amount is 10 mL;
detection wavelength: 280 nm;
mobile phase A: 0.1% phosphoric acid, mobile phase B: acetonitrile; elution gradient: 0-20 min, 90-30% of A.
In a preferred embodiment of the present invention, the purity is measured by a high performance liquid chromatograph.
The instrument model is as follows: waters2695, column model:
3 μm Phenyl-Hexyl 100A 150 x 4.6 mm; detector wavelength: 280nm, detector temperature: 30 ℃, flow rate: 0.8ml/min, sample size: 10 μ l.
Mobile phase A: 1.2g/L of monopotassium phosphate, and adjusting the pH value to be 2.8 of mobile phase B by phosphoric acid: methanol
Elution gradients are as follows:
the mass spectrometer model is HP 1100. The conditions ESI source, Positive mode, were tested.
All NMR measurements were performed using a bruker avance model 400 NMR spectrometer with a proton resonance frequency of 400.13 MHz. The solvents used in the experiment are DMSO-d6, TMS is an internal standard substance, and the experiment temperature is 25 ℃.
Then, the effluent is decompressed to remove the solvent, and then is frozen and dried, and the obtained solid is washed by water to remove salt and decompressed and dried to obtain the product.
The specific operation and parameters of the lyophilization, the water washing for desalting and the reduced pressure drying are not limited in the present invention, and are well known to those skilled in the art.
The key point of the patent technology of the invention lies in preparing one impurity in folic acid bulk drugs, the impurity is a chlorinated compound with stable property, the compound is a genetic genotoxic impurity, and the compound needs to be quantitatively controlled strictly according to related guiding principles.
The invention provides a preparation method of folic acid EP impurity F, which comprises the following steps: A)2,4,5, 6-tetraaminopyrimidine sulfate and 1,1, 3-trichloroacetone are subjected to condensation reaction in the presence of an antioxidant and a solvent to obtain a compound with a structure shown in a formula (I); B) hydrolyzing the compound with the structure shown in the formula (I) by acid to obtain a folic acid impurity F crude product, and preparing and purifying the folic acid impurity F crude product by a semi-preparative chromatograph to obtain folic acid EP impurity F with the structure shown in the formula (II). The invention adopts a brand new synthesis route, can prepare the impurity F by only two steps, has higher purity of the obtained product, is suitable for the deep research process of the property and the synthesis mechanism of the folic acid, and lays a foundation for effectively improving the quality control level of the folic acid. The method has the advantages of mild reaction conditions, cheap and easily-obtained starting materials for reaction, short reaction steps, simple post-treatment and high crude product yield which is more than 70%. In the process of preparing the impurity F, the synthesis step and the purification step are systematically optimized, the synthesis step and the purification step are organically combined, and the high purity is realized by controlling the parameters of the preparation process and the operation parameters of the purification process. The whole set of system obtained by the invention is suitable for the method for preparing the high-purity impurity F, and has important practical significance.
In order to further illustrate the present invention, the following examples are provided to describe in detail the preparation of a folic acid EP impurity F.
The various starting materials and reagents used in the examples of the present invention were all commercially available unless otherwise specified.
In the embodiment of the invention, a high performance liquid chromatograph is adopted for purity detection, and the instrument types are as follows: waters2695, column model:
3 μm Phenyl-Hexyl 100A 150 x 4.6 mm; detector wavelength: 280nm, detector temperature: 30 ℃, flow rate: 0.8ml/min, sample size: 10 μ l.
Mobile phase A: 1.2g/L of monopotassium phosphate, and adjusting the pH value to be 2.8 of mobile phase B by phosphoric acid: methanol
In the embodiment of the invention, a semi-preparative chromatograph is adopted for preparation, and the instrument comprises: SepaBean machine T, column: sphere C1850um 100A °, 25g, detection wavelength: 280nm, diluent: DMSO, sample introduction amount: 10 ml.
Mobile phase: a: 0.1% phosphoric acid mobile phase B: acetonitrile
The mass spectrometer model is HP 1100. The conditions ESI source, Positive mode, were tested.
All NMR measurements were performed using a bruker avance model 400 NMR spectrometer with a proton resonance frequency of 400.13 MHz. The solvents used in the experiment are DMSO-d6, TMS is an internal standard substance, and the experiment temperature is 25 ℃.
Example 1:
(1) synthesis of folic acid impurity F crude product
Adding 2,4,5, 6-tetraaminopyrimidine sulfate, 1,1, 3-trichloroacetone and L-cysteine into a reaction bottle, and calculating the mass ratio of the 2,4,5, 6-tetraaminopyrimidine sulfate: 1,1, 3-trichloroacetone I: l-cysteine ═ 1:1: 3. taking water and methanol (volume ratio is 1: 1) as solvents, heating to 50-60 ℃ for reaction for 12h, and adjusting the pH value of the solution to 1-2 by using 1mol/L hydrochloric acid in the reaction process. After the reaction is completed, the reaction solution is cooled to room temperature and stirred for crystallization for 2 hours. Filtering, washing the filter cake with 100ml of water to obtain a wet compound product with the structure of the formula (I);
adding 40% hydrobromic acid with the mass 3 times that of the added 2,4,5, 6-tetraaminopyrimidine sulfate into a wet product of the compound with the structure of the formula (I), and heating to 80-90 ℃ for reaction for 2 hours. The reaction was cooled to room temperature and stirred for 2h, the resulting solid was filtered and dried to give a pale yellow solid with LC purity 80.37% (fig. 1) and yield 83.29%, fig. 1 is a detailed HPLC data chart of crude impurity F synthesized in example 1.
(2) Purification of Folic acid impurity F
Dissolving the folic acid impurity F crude product in DMSO, filtering, separating the filtrate with semi-preparative chromatograph (10 ml each time), collecting the effluent, mixing, concentrating under reduced pressure to vacuum degree of-0.09 MPa or less, and drying to obtain folic acid impurity F.
The purity of the folic acid impurity F is 98.7%, as shown in figure 2, and figure 2 is a specific data diagram of HPLC after the crude product of the impurity F in example 1 is prepared and purified.
FIG. 3 shows the mass spectrometric detection of impurity F in example 1, wherein FIG. 3 shows the mass spectrogram of impurity F purified in example 1, and the positive ion spectrogram of sample F has a strong ion peak at 212.1, which is [ M + H ] of the sample]+And the compound of the formula C7H6ClN5The molecular weights of O are consistent.
It is composed of1HNMR is shown in FIG. 4, and FIG. 4 is a map of 1HNMR of impurity F after purification in example 1. The data are as follows:1HNMR(400MHz，DMSO-d6)8.62(s，1H)，7.59(br，2H)，4.88(s，2H)。
example 2:
(1) synthesis of folic acid impurity F crude product
Adding 2,4,5, 6-tetraaminopyrimidine sulfate, L-cysteine, 1,1, 3-trichloroacetone into a reaction bottle, and calculating the mass ratio of the 2,4,5, 6-tetraaminopyrimidine sulfate: 1,1, 3-trichloroacetone: l-cysteine ═ 1:1: 2. taking water and ethanol (volume ratio is 2:1) as a solvent, heating to 30-40 ℃ for reaction for 24 hours, and adjusting the pH value of the solution to 2-3 by using sulfuric acid in the reaction process. After the reaction is completed, the reaction solution is cooled to room temperature and stirred for crystallization for 2 hours. Filtering, washing the filter cake with 100ml of water to obtain a wet compound product with the structure of the formula (I);
adding 37% hydrochloric acid with the mass 5 times that of the added 2,4,5, 6-tetraaminopyrimidine sulfate into a wet product of the compound with the structure shown in the formula (I), and heating to 90-100 ℃ for reaction for 3 hours. The reaction was allowed to cool to room temperature and stirred for 2h, the resulting solid was filtered and dried to give a pale yellow solid with LC purity 78.68% and yield 81.92%.
(2) Purification of Folic acid impurity F
Dissolving the folic acid impurity F crude product in DMSO, filtering, separating the filtrate with semi-preparative chromatograph (10 ml each time), collecting the effluent, mixing, concentrating under reduced pressure to vacuum degree of-0.09 MPa or less, and drying to obtain folic acid impurity F.
The purity of the folic acid impurity F obtained was 97.9%.
Example 3:
(1) synthesis of folic acid impurity F crude product
Adding 2,4,5, 6-tetraaminopyrimidine sulfate, L-pyroglutamic acid, 1,1, 3-trichloroacetone into a reaction bottle, and calculating the weight ratio of the 2,4,5, 6-tetraaminopyrimidine sulfate based on the substance: 1,1, 3-trichloroacetone: l-pyroglutamic acid ═ 1:1: 4. taking water and isopropanol (volume ratio is 3: 1) as solvents, heating to 40-50 ℃ for reaction for 20h, and adjusting the pH value of the solution to 3-4 by using acetic acid in the reaction process. After the reaction is completed, the reaction solution is cooled to room temperature and stirred for crystallization for 2 hours. Filtering, washing the filter cake with 100ml of water to obtain a wet compound product with the structure of the formula (I);
adding 50% sulfuric acid 4 times the mass of the added 2,4,5, 6-tetraaminopyrimidine sulfate into a wet product of the compound with the structure of the formula (I), and heating to 70-80 ℃ for reaction for 2 hours. The reaction solution was cooled to room temperature and stirred for 2h, the resulting solid was filtered and dried to give a pale yellow solid with an LC purity of 77.45% and a yield of 79.56%.
(2) Purification of Folic acid impurity F
Dissolving the folic acid impurity F crude product with DMF, filtering, separating the filtrate with semi-preparative chromatograph at a volume of 10ml each time, collecting the effluent, combining the effluent, concentrating under reduced pressure at a vacuum degree of less than or equal to-0.09 MPa, and drying to obtain folic acid impurity F.
The purity of the folic acid impurity F was 97.31%.
Example 4:
(1) synthesis of folic acid impurity F crude product
Adding 2,4,5, 6-tetraaminopyrimidine sulfate, sodium metabisulfite and 1,1, 3-trichloroacetone into a reaction bottle, and calculating the weight ratio of the 2,4,5, 6-tetraaminopyrimidine sulfate based on the substances: 1,1, 3-trichloroacetone: sodium metabisulfite 1:1: 5. taking water and isopropanol (volume ratio 1: 3) as solvents, heating to 70-80 ℃, reacting for 1h, and adjusting the pH value of the solution to 3-4 by using sulfuric acid in the reaction process. After the reaction is completed, the reaction solution is cooled to room temperature and stirred for crystallization for 2 hours. Filtering, washing the filter cake with 100ml of water to obtain a wet compound product with the structure of the formula (I);
adding 30% hydrobromic acid with the mass 6 times that of the added 2,4,5, 6-tetraaminopyrimidine sulfate into a wet product of the compound with the structure shown in the formula (I), and heating to 90-100 ℃ for reaction for 4 hours. The reaction was allowed to cool to room temperature and stirred for 2h, the resulting solid was filtered and dried to give a pale yellow solid with LC purity 74.24% and yield 76.47%.
(2) Purification of Folic acid impurity F
Dissolving the folic acid impurity F crude product with DMF, filtering, separating the filtrate with semi-preparative chromatograph at a volume of 10ml each time, collecting the effluent, combining the effluent, concentrating under reduced pressure at a vacuum degree of less than or equal to-0.09 MPa, and drying to obtain folic acid impurity F.
The purity of the folic acid impurity F was 96.62%.
Example 5:
(1) synthesis of folic acid impurity F crude product
Adding 2,4,5, 6-tetraaminopyrimidine sulfate, L-cysteine + sodium metabisulfite and 1,1, 3-trichloroacetone into a reaction bottle, and calculating the mass ratio of the 2,4,5, 6-tetraaminopyrimidine sulfate: 1,1, 3-trichloroacetone: (L-cysteine + sodium metabisulfite) ═ 1:1: 3. heating water and a propanol (volume ratio is 1:2) solvent to 20-30 ℃ for reaction for 36h, and adjusting the pH value of the solution to 2-3 by using hydrochloric acid in the reaction process. After the reaction is completed, the reaction solution is cooled to room temperature and stirred for crystallization for 2 hours. Filtering, washing a filter cake with 100ml of water, and drying to obtain a wet compound product with the structure of the formula (I);
adding 40% sulfuric acid with the mass 4 times that of the added 2,4,5, 6-tetraaminopyrimidine sulfate into a wet product of the compound with the structure shown in the formula (I), and heating to 80-90 ℃ for reaction for 3 hours. The reaction was allowed to cool to room temperature and stirred for 2h, and the resulting solid was filtered to give a pale yellow solid with an LC purity of 75.86% and a yield of 73.19%.
(2) Purification of Folic acid impurity F
Dissolving the folic acid impurity F crude product in DMSO, filtering, carrying out chromatographic separation on the filtrate by 10ml each time, collecting effluent, merging, concentrating under reduced pressure until the vacuum degree is less than or equal to-0.09 MPa, and drying to obtain the folic acid impurity F.
The purity of the folic acid impurity F obtained is 97.35%.
Example 6:
(1) synthesis of folic acid impurity F crude product
Adding 2,4,5, 6-tetraaminopyrimidine sulfate, (sodium metabisulfite + L-pyroglutamic acid), 1,1, 3-trichloroacetone into a reaction bottle, and calculating the weight ratio of the substances, wherein the weight ratio of the 2,4,5, 6-tetraaminopyrimidine sulfate is as follows: 1,1, 3-trichloroacetone: (sodium metabisulfite + L-pyroglutamic acid) ═ 1:1: 4. heating water and isopropanol (volume ratio 1:2) to 60-70 ℃ for reaction for 8h, and adjusting the pH value of the solution to 3-4 by using hydrochloric acid in the reaction process. After the reaction is completed, the reaction solution is cooled to room temperature and stirred for crystallization for 2 hours. Filtering, washing the filter cake with 100ml of water to obtain a wet compound product with the structure of the formula (I);
adding 30% hydrochloric acid with the mass 7 times of that of the added 2,4,5, 6-tetraaminopyrimidine sulfate into a wet product of the compound with the structure of the formula (I), and heating to 70-80 ℃ for reaction for 5 hours. The reaction was allowed to cool to room temperature and stirred for 2h, the resulting solid was filtered and dried to give a pale yellow solid with LC purity 74.96% and yield 71.45%. (2) Purification of folic acid impurity F the crude folic acid impurity F is dissolved by DMF, filtered, the filtrate is separated by a semi-preparative chromatograph, 10ml of the filtrate is separated each time, the effluent liquid is collected, the mixture is decompressed and concentrated, the vacuum degree is less than or equal to-0.09 MPa, and the folic acid impurity F is obtained after drying.
The purity of the folic acid impurity F was 96.71%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for preparing folic acid EP impurity F is characterized by comprising the following steps:
A)2,4,5, 6-tetraaminopyrimidine sulfate and 1,1, 3-trichloroacetone are subjected to condensation reaction in the presence of an antioxidant and a solvent to obtain a compound with a structure shown in a formula (I);
B) hydrolyzing the compound with the structure shown in the formula (I) by acid to obtain a folic acid impurity F crude product, and purifying the folic acid impurity F crude product by chromatography to obtain a folic acid EP impurity F with the structure shown in the formula (II);
2. the preparation method of claim 1, wherein the molar ratio of the 2,4,5, 6-tetraaminopyrimidine sulfate, 1, 3-trichloroacetone and antioxidant in step A) is 1:1:2 to 1:1: 5.
3. The method according to claim 1, wherein the solvent of step a) is a mixed solution of water and alcohol; the alcohol is selected from one or more of methanol, ethanol, propanol and isopropanol; the volume ratio of the water to the alcohol is 1: 3-3: 1; the mass ratio of the 2,4,5, 6-tetraaminopyrimidine sulfate to the solvent is 1: (30-80).
4. The method according to claim 1, wherein the antioxidant in step A) is one or more selected from the group consisting of L-cysteine, L-pyroglutamic acid, sodium metabisulfite and sodium bisulfite.
5. The preparation method according to claim 1, wherein the reaction of step a) is carried out under acidic conditions, and the pH value of the reaction is 1 to 4; the acid for adjusting the pH value is one of hydrochloric acid, sulfuric acid or glacial acetic acid.
6. The preparation method according to claim 1, wherein the temperature of the reaction in the step A) is 20-80 ℃; the reaction time is 1-36 h.
7. The preparation method according to claim 1, wherein the acid used in the acid hydrolysis in step B) is one of hydrochloric acid, sulfuric acid or hydrobromic acid; the mass concentration of the acid is 30-50%;
the mass ratio of the compound with the structure of the formula (I) to the acid is 1: (2-8).
8. The preparation method according to claim 1, wherein the temperature of the acid hydrolysis in the step B) is 60-100 ℃; the time of acid hydrolysis is 0.5-5 h.
9. The preparation method according to claim 1, wherein the folic acid EP impurity F with the structure of formula (II) obtained by chromatographic purification of the crude folic acid impurity F is specifically:
dissolving the folic acid impurity F crude product with a solvent, filtering, feeding the filtrate into a semi-preparative chromatograph, collecting the required component solution, removing the solvent under reduced pressure, freeze-drying, washing the obtained solid with water to remove salt, and drying under reduced pressure to obtain the folic acid impurity F.
10. The method of claim 1, wherein the chromatographic purification parameters are: the instrument comprises the following steps: semi-preparative chromatograph, SepaBean machine T, column: spherial C18, 50 μm 100A; detection wavelength: 280 nm; mobile phase A: 0.1% phosphoric acid, mobile phase B: acetonitrile; elution gradient: 0-20 min, 90-30% of A.
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