A method for preparing folate ep impurity f

Abstract

The application provides a preparation method of folic acid EP impurity F, comprising: 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 the structure of formula (I); B) the compound with the structure of formula (I) is subjected to acid hydrolysis to obtain a folic acid impurity F crude product, and the folic acid impurity F crude product is subjected to chromatographic purification to obtain folic acid EP impurity F with the structure of formula (II). The application adopts a brand-new synthetic route, and impurity F can be prepared in only two steps, the obtained product has high purity, is suitable for in-depth research on the properties and synthetic mechanism of folic acid, and lays a foundation for effectively improving the folic acid quality control level. The reaction condition is mild, the starting materials used in the reaction are cheap and easy to obtain, the reaction steps are short, the post-treatment is simple, the crude product yield is high, and reaches more than 70 %.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical synthesis technology, and in particular to a method for preparing folic acid EP impurity F. Background Technology

[0002] Folic acid, also known as vitamin B9, is a water-soluble vitamin. Experiments have shown that folic acid plays a crucial role in protein synthesis and cell division and growth, promotes the formation of normal red blood cells, affects reproductive performance, influences pancreatic secretion, promotes human growth, and enhances immunity. It plays a vital role in growth, development, and metabolism. Folic acid deficiency can lead to reduced hemoglobin production in red blood cells and inhibited cell maturation, resulting in megaloblastic anemia. Folic acid deficiency in pregnant women can lead to serious consequences such as birth defects and neural tube defects in the fetus, and may also increase the risk of preeclampsia and miscarriage in the mother. Therefore, the proper use of folic acid has a significant impact on maintaining the physiological condition of pregnant women and the normal development of the fetus. Mutations in key genes involved in the folic acid metabolic pathway can lead to reduced activity of folic acid metabolic enzymes, resulting in folic acid metabolic disorders and folic acid deficiency. Excessive folic acid can affect zinc absorption, potentially leading to fetal developmental abnormalities and increasing the risk of birth defects. A certain proportion of risk-prone genes exist in the Chinese population, resulting in varying abilities to utilize folic acid among individuals; therefore, folic acid supplementation needs to be personalized according to different population groups.

[0003] The quality of folic acid raw materials is the key and starting point for the quality control of folic acid-related products. The research and control of impurities is crucial to the clinical safety of drugs, thus becoming a key aspect of raw material quality control. Adverse reactions in clinical use of drugs depend not only on the pharmacological activity of the drug itself but also sometimes on impurities within the drug. Therefore, meticulous research and strict control of impurities in drugs are essential.

[0004] Patent document CN105541845 reports that folic acid is composed of three fragments. Although the mechanism has been studied for many years, it is still not certain whether the reaction proceeds in a concerted or stepwise manner. One possibility is that fragment I first cyclizes with fragment II, and then undergoes a nucleophilic substitution reaction with fragment III to generate folic acid.

[0005]

[0006] Based on this reaction mechanism, it can be inferred that 7-chloromethylpterin (EP impurities F, IV) and 6-chloromethylpterin (V) will inevitably be produced during the reaction. The structures of 7-chloromethylpterin (EP impurities F, IV) and 6-chloromethylpterin (V) are as follows:

[0007]

[0008] Folic acid impurity IV, listed in EP9.5, is a process-unspecified impurity with the designation F. Although its chemical structure is simple, no synthetic method or structural characterization data has been reported in the literature to date. Furthermore, this impurity is currently not commercially available, significantly increasing the difficulty of research on related substances in folic acid raw materials. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing folic acid EP impurity F, which has high yield and purity.

[0010] This invention provides a method for preparing folic acid EP impurity F, comprising:

[0011] A) 2,4,5,6-tetraaminopyrimidine sulfate and 1,1,3-trichloroacetone undergo a condensation reaction in the presence of an antioxidant and a solvent to give a compound with the structure of formula (I).

[0012] B) The compound of formula (I) was acid hydrolyzed to obtain crude folic acid impurity F, and the crude folic acid impurity F was purified by chromatography to obtain folic acid EP impurity F of formula (II);

[0013]

[0014] Preferably, the molar ratio of 2,4,5,6-tetraaminopyrimidine sulfate, 1,1,3-trichloroacetone and antioxidant in step A) is 1:1:2 to 1:1:5.

[0015] Preferably, the solvent in 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 water to alcohol is 1:3 to 3:1; and the mass ratio of 2,4,5,6-tetraaminopyrimidine sulfate to solvent is 1:(30 to 80).

[0016] Preferably, the antioxidant in step A) is one or more of L-cysteine, L-pyroglutamic acid, sodium metabisulfite, and sodium bisulfite.

[0017] Preferably, the reaction in step A) is carried out under acidic conditions, and the pH value of the reaction is 1 to 4; the acid used to adjust the pH value is one of hydrochloric acid, sulfuric acid or glacial acetic acid.

[0018] Preferably, the temperature of the reaction in step A) is 20–80°C; and the reaction time is 1–36 h.

[0019] Preferably, 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% to 50%.

[0020] The mass ratio of the compound and acid in the structure of formula (I) is 1:(2-8).

[0021] Preferably, the acid hydrolysis temperature in step B) is 60–100°C; and the acid hydrolysis time is 0.5–5 h.

[0022] Preferably, the crude folic acid impurity F is purified by chromatography to obtain folic acid EP impurity F with the structure of formula (II), specifically as follows:

[0023] The crude folic acid impurity F was dissolved in a solvent, filtered, and the filtrate was fed into a preparative chromatograph to collect the desired component solution. After removing the solvent under reduced pressure, the solution was lyophilized, and the resulting solid was washed with water to remove salts and dried under reduced pressure to obtain folic acid impurity F.

[0024] Preferably, the parameters for the chromatographic purification are as follows: instrument: semi-preparative chromatograph, SepaBean machine T; column: Spherical C 18, 50 μm 100A; detection wavelength: 280 nm; mobile phase A: 0.1% phosphoric acid; mobile phase B: acetonitrile; elution gradient: 0–20 min, 90% A–30% A.

[0025] Compared with existing technologies, this invention provides a method for preparing folic acid EP impurity F, comprising: A) a condensation reaction of 2,4,5,6-tetraaminopyrimidine sulfate and 1,1,3-trichloroacetone in the presence of an antioxidant and a solvent to obtain a compound of formula (I); B) acid hydrolysis of the compound of formula (I) to obtain crude folic acid impurity F, which is then purified by chromatography to obtain folic acid EP impurity F of formula (II). This invention employs a novel synthetic route, requiring only two steps to prepare impurity F, and yields a product with high purity. This method is suitable for in-depth research on the properties and synthetic mechanism of folic acid, laying the foundation for effectively improving the quality control level of folic acid. The reaction conditions are mild, the starting materials used are inexpensive and readily available, the reaction steps are short, the post-processing is simple, and the crude product yield is high, reaching over 70%. Attached Figure Description

[0026] Figure 1 This is a detailed HPLC data graph of the crude impurity F synthesized in Example 1;

[0027] Figure 2 This is a chromatogram of HPLC data for the crude impurity F from Example 1 after preparation and purification.

[0028] Figure 3 The mass spectrum of impurity F after purification in Example 1;

[0029] Figure 4 The purified impurity F from Example 1 1 HNMR spectrum. Detailed Implementation

[0030] This invention provides a method for preparing folic acid EP impurity F. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0031] This invention provides a method for preparing folic acid EP impurity F, comprising:

[0032] A) 2,4,5,6-tetraaminopyrimidine sulfate and 1,1,3-trichloroacetone undergo a condensation reaction in the presence of an antioxidant and a solvent to give a compound with the structure of formula (I).

[0033] B) The compound of formula (I) was acid hydrolyzed to obtain crude folic acid impurity F, and the crude folic acid impurity F was purified by chromatography to obtain folic acid EP impurity F of formula (II);

[0034]

[0035] This invention provides a method for preparing folic acid EP impurity F. First, 2,4,5,6-tetraaminopyrimidine sulfate and 1,1,3-trichloroacetone undergo a condensation reaction in the presence of an antioxidant and a solvent to obtain a compound with the structure of formula (I).

[0036] 2,4,5,6-Tetraaminopyrimidine sulfate and 1,1,3-trichloroacetone, under the presence of antioxidants and solvents, were reacted by adding acid dropwise to control the pH of the reaction system, and the resulting compound with the structure of formula (I) was obtained through a condensation reaction.

[0037] The reaction described in this invention is carried out under acidic conditions, and the pH value of the reaction is preferably 1 to 4; more preferably 1 to 2; the acid used to adjust the pH value is one of hydrochloric acid, sulfuric acid or glacial acetic acid.

[0038] The reaction temperature is preferably 20–80°C; more preferably 20–60°C; the reaction time is preferably 1–36 h; more preferably 1–24 h; and most preferably 1–20 h.

[0039] The molar ratio of 2,4,5,6-tetraaminopyrimidine sulfate, 1,1,3-trichloroacetone and antioxidant in this invention is preferably 1:1:2 to 1:1:5; more preferably 1:1:2 to 1:1:3.

[0040] The mass ratio of the 2,4,5,6-tetraaminopyrimidine sulfate to the solvent is preferably 1:(30-80); more preferably 1:(40-70); and most preferably 1:(40-50). The solvent is a mixture of water and alcohol; the alcohol is selected from one or more of methanol, ethanol, propanol, and isopropanol; and the volume ratio of water to alcohol is preferably 1:3 to 3:1; more preferably 2:1 to 1:1.

[0041] The antioxidant described in this invention is preferably one or more of L-cysteine, L-pyroglutamic acid, sodium metabisulfite, and sodium bisulfite. This invention does not limit the source of the antioxidant; commercially available products are acceptable.

[0042] In a preferred embodiment of the present invention, the reaction formula is as follows:

[0043]

[0044] The compound with structure (I) was subjected to acid hydrolysis to yield crude folic acid impurity F. The reaction formula is as follows:

[0045]

[0046] The acid used in the acid hydrolysis described in this invention is preferably one of hydrochloric acid, sulfuric acid, or hydrobromic acid; more preferably, hydrobromic acid; the source of the acid is not limited, and commercially available acid is acceptable. The mass concentration of the acid is preferably 30% to 50%; more preferably 35% to 45%; and most preferably 40%.

[0047] The mass ratio of the compound and acid in the formula (I) structure is preferably 1:(2-8); more preferably 1:(3-7); and most preferably 1:(3-5).

[0048] The preferred temperature for acid hydrolysis in this invention is 60–100°C; more preferably 70–90°C; the preferred time for acid hydrolysis is 0.5–5 h; more preferably 1–4 h; and most preferably 1–3 h.

[0049] The crude folic acid impurity F was purified by chromatography to obtain folic acid EP impurity F with the structure of formula (II).

[0050] The crude folic acid impurity F of the present invention, after chromatographic purification, yields folic acid EP impurity F with the structure of formula (II), specifically:

[0051] The crude folic acid impurity F was dissolved in a solvent, filtered, and the filtrate was fed into a preparative chromatograph to collect the desired component solution. After removing the solvent under reduced pressure, the solution was lyophilized, and the resulting solid was washed with water to remove salts and dried under reduced pressure to obtain folic acid impurity F.

[0052] The preferred solvent is DMSO or DMF; the present invention does not limit the specific filtration method, and conventional methods known to those skilled in the art are acceptable.

[0053] The filtrate was then separated using a semi-preparative chromatograph.

[0054] The parameters for preparation and purification using the semi-preparative chromatograph described in this invention are as follows:

[0055] Semi-preparative chromatography. Instrument: SepaBean machine T;

[0056] Chromatographic column: Spherical C 18, 50µm 100A; injection volume: 10mL;

[0057] Detection wavelength: 280nm;

[0058] Mobile phase A: 0.1% phosphoric acid; Mobile phase B: acetonitrile; Elution gradient: 0–20 min, 90% A–30% A.

[0059] In a preferred embodiment of the present invention, a high-performance liquid chromatograph is used for purity detection.

[0060] Instrument model: Waters 2695, Column model: 3μm Phenyl-Hexyl 100A 150*4.6mm; Detector wavelength: 280nm, Detector temperature: 30℃, Flow rate: 0.8ml / min, Injection volume: 10μl.

[0061] Mobile phase A: Potassium dihydrogen phosphate 1.2 g / L, pH adjusted to 2.8 with phosphoric acid. Mobile phase B: Methanol.

[0062] The elution gradient is shown in the table below:

[0063]

[0064] The mass spectrometer was an HP1100. Testing conditions: ESI source, Positive mode.

[0065] All NMR measurements were performed using a Bruker Avance 400 nuclear magnetic resonance spectrometer, with a proton resonance frequency of 400.13 MHz. DMSO-d6 was used as the solvent, TMS was used as the internal standard, and the experimental temperature was 25 °C.

[0066] The effluent was then desolventized under reduced pressure, freeze-dried, and the resulting solid was washed with water to remove salt, then dried under reduced pressure to obtain the final product.

[0067] The present invention does not limit the specific operations and parameters of the freeze-drying, water washing and desalination, and vacuum drying, which are well known to those skilled in the art.

[0068] The key technology of this invention lies in the preparation of an impurity in folic acid raw materials. This impurity is relatively stable and is a chlorinated compound. This compound is a genotoxic impurity and needs to be strictly controlled quantitatively according to relevant guidelines. This invention uses 2,4,5,6-tetraaminopyrimidine sulfate and 1,1,3-trichloroacetone as starting materials. Under specific temperature, specific time, specific pH, specific inorganic acid and antioxidant conditions, a cyclization reaction is first carried out, followed by an acidic hydrolysis reaction, and finally the pure product is prepared by semi-preparative chromatographic separation.

[0069] This invention provides a method for preparing folic acid EP impurity F, comprising: A) a condensation reaction of 2,4,5,6-tetraaminopyrimidine sulfate and 1,1,3-trichloroacetone in the presence of an antioxidant and a solvent to obtain a compound of formula (I); B) acid hydrolysis of the compound of formula (I) to obtain crude folic acid impurity F, wherein the crude folic acid impurity F is prepared and purified by semi-preparative chromatography to obtain folic acid EP impurity F of formula (II). This invention employs a novel synthetic route, requiring only two steps to prepare impurity F, yielding a product with high purity, suitable for in-depth research on the properties and synthetic mechanism of folic acid, and laying the foundation for effectively improving the quality control level of folic acid. The reaction conditions are mild, the starting materials used are inexpensive and readily available, the reaction steps are short, the post-processing is simple, and the crude product yield is high, reaching over 70%. In the preparation of impurity F, this invention systematically optimizes the synthetic and purification steps, organically combining them, and achieving high purity by controlling the parameters of the preparation process and the operating parameters of the purification process. The complete system obtained by this invention is applicable to the preparation of high-purity impurity F and has important practical significance.

[0070] To further illustrate the present invention, the following describes in detail a method for preparing folic acid EP impurity F provided by the present invention with reference to embodiments.

[0071] Unless otherwise specified, all raw materials and reagents used in the embodiments of this invention are commercially available.

[0072] In this embodiment of the invention, high-performance liquid chromatography (HPLC) was used for purity detection. Instrument model: Waters 2695; chromatographic column model: 3μm Phenyl-Hexyl 100A 150*4.6mm; Detector wavelength: 280nm, Detector temperature: 30℃, Flow rate: 0.8ml / min, Injection volume: 10μl.

[0073] Mobile phase A: Potassium dihydrogen phosphate 1.2 g / L, pH adjusted to 2.8 with phosphoric acid. Mobile phase B: Methanol.

[0074]

[0075] In this embodiment of the invention, a semi-preparative chromatograph was used for preparation. The instrument was a SepaBean machine T, and the chromatographic column was a Spherical C2000. 18 50um 100A°, 25g, detection wavelength: 280nm, diluent: DMSO, injection volume: 10ml.

[0076] Mobile phase: A: 0.1% phosphoric acid; B: acetonitrile

[0077]

[0078] The mass spectrometer was an HP1100. Testing conditions: ESI source, Positive mode.

[0079] All NMR measurements were performed using a Bruker Avance 400 nuclear magnetic resonance spectrometer, with a proton resonance frequency of 400.13 MHz. DMSO-d6 was used as the solvent, TMS was used as the internal standard, and the experimental temperature was 25 °C.

[0080] Example 1:

[0081] (1) Synthesis of crude folic acid impurity F

[0082] 2,4,5,6-Tetraaminopyrimidine sulfate, 1,1,3-trichloroacetone, and L-cysteine ​​were added to a reaction flask. Based on the molar ratio, the ratio of 2,4,5,6-tetraaminopyrimidine sulfate: 1,1,3-trichloroacetone I: L-cysteine ​​was 1:1:3. Water and methanol (volume ratio 1:1) were used as solvents, and the mixture was heated to 50-60℃ and reacted for 12 hours. During the reaction, the pH of the solution was adjusted to 1-2 using 1 mol / L hydrochloric acid. After the reaction was complete, the reaction solution was cooled to room temperature and stirred to allow crystals to precipitate for 2 hours. The mixture was filtered, and the filter cake was washed with 100 ml of water to obtain the wet product of the compound with structure (I).

[0083] Add 40% hydrobromic acid (3 times the mass of the added 2,4,5,6-tetraaminopyrimidine sulfate) to a wet sample of the compound with structure (I), and heat to 80–90 °C for 2 h. Cool the reaction solution to room temperature and stir for 2 h. Filter the resulting solid, dry it, and obtain a light yellow solid with an LC purity of 80.37%. Figure 1 The yield was 83.29%. Figure 1 This is a detailed HPLC data graph of the crude impurity F synthesized in Example 1.

[0084] (2) Purification of folic acid impurity F

[0085] The crude folic acid impurity F was dissolved in DMSO, filtered, and the filtrate was separated in a semi-preparative chromatograph. 10 ml was injected each time, and the eluent was collected, combined, concentrated under reduced pressure (≤-0.09 MPa), and dried to obtain folic acid impurity F.

[0086] The purity of the obtained folic acid impurity F was 98.7%, such as... Figure 2 As shown, Figure 2 This is a HPLC data graph showing the specific data of the crude impurity F in Example 1 after preparation and purification.

[0087] Mass spectrometry analysis, such as Figure 3 As shown, Figure 3 The image shows the mass spectrum of impurity F after purification in Example 1. In the positive ion spectrum of sample F, there is a strong ion peak at 212.1, which represents the [M+H] ion of the sample. + The molecular weight is consistent with that of the compound with the molecular formula C7H6ClN5O.

[0088] That 1 HNMR see Figure 4 , Figure 4 The image shows the 1H NMR spectrum of impurity F purified in Example 1. The data are as follows: 1 HNMR (400MHz, DMSO-d6) 8.62 (s, 1H), 7.59 (br, 2H), 4.88 (s, 2H).

[0089] Example 2:

[0090] (1) Synthesis of crude folic acid impurity F

[0091] 2,4,5,6-Tetraaminopyrimidine sulfate, L-cysteine, and 1,1,3-trichloroacetone were added to a reaction flask. Based on the molar ratio, the ratio of 2,4,5,6-tetraaminopyrimidine sulfate:1,1,3-trichloroacetone:L-cysteine ​​was 1:1:2. The mixture was reacted at 30–40°C for 24 hours using water and ethanol (volume ratio 2:1) as solvents. During the reaction, the pH of the solution was adjusted to 2–3 with sulfuric acid. After the reaction was complete, the reaction solution was cooled to room temperature and stirred to allow crystals to precipitate for 2 hours. The mixture was filtered, and the filter cake was washed with 100 ml of water to obtain the wet product of the compound with structure (I).

[0092] Add 37% hydrochloric acid (5 times the mass of the added 2,4,5,6-tetraaminopyrimidine sulfate) to a wet sample of the compound with structure (I), and heat to 90–100 °C for 3 h. Cool the reaction solution to room temperature and stir for 2 h. Filter the resulting solid, dry it, and give a light yellow solid with an LC purity of 78.68% and a yield of 81.92%.

[0093] (2) Purification of folic acid impurity F

[0094] The crude folic acid impurity F was dissolved in DMSO, filtered, and the filtrate was separated in a semi-preparative chromatograph. 10 ml was injected each time, and the eluent was collected, combined, concentrated under reduced pressure (≤-0.09 MPa), and dried to obtain folic acid impurity F.

[0095] The purity of the obtained folic acid impurity F was 97.9%.

[0096] Example 3:

[0097] (1) Synthesis of crude folic acid impurity F

[0098] 2,4,5,6-Tetraaminopyrimidine sulfate, L-pyroglutamic acid, and 1,1,3-trichloroacetone were added to a reaction flask. Based on the molar ratio, the ratio of 2,4,5,6-tetraaminopyrimidine sulfate:1,1,3-trichloroacetone:L-pyroglutamic acid was 1:1:4. Water and isopropanol (volume ratio 3:1) were used as solvents, and the mixture was heated to 40–50°C and reacted for 20 h. During the reaction, the pH of the solution was adjusted to 3–4 using acetic acid. After the reaction was complete, the reaction solution was cooled to room temperature and stirred to allow crystallization for 2 h. The mixture was filtered, and the filter cake was washed with 100 ml of water to obtain the wet product of the compound with structure (I).

[0099] Add 50% sulfuric acid (4 times the mass of the added 2,4,5,6-tetraaminopyrimidine sulfate) to a wet sample of the compound with structure (I), and heat to 70–80 °C for 2 h. Cool the reaction solution to room temperature and stir for 2 h. Filter the resulting solid, dry it, and give a light yellow solid with an LC purity of 77.45% and a yield of 79.56%.

[0100] (2) Purification of folic acid impurity F

[0101] The crude folic acid impurity F was dissolved in DMF, filtered, and the filtrate was separated in a semi-preparative chromatograph. 10 ml was injected each time, and the eluent was collected, combined, concentrated under reduced pressure (≤-0.09 MPa), and dried to obtain folic acid impurity F.

[0102] The purity of the obtained folic acid impurity F was 97.31%.

[0103] Example 4:

[0104] (1) Synthesis of crude folic acid impurity F

[0105] 2,4,5,6-Tetraaminopyrimidine sulfate, sodium metabisulfite, and 1,1,3-trichloroacetone were added to a reaction flask. Based on the molar ratio, the ratio of 2,4,5,6-tetraaminopyrimidine sulfate:1,1,3-trichloroacetone:sodium metabisulfite was 1:1:5. The mixture was heated to 70–80°C for 1 hour using water and isopropanol (volume ratio 1:3) as solvents. During the reaction, the pH of the solution was adjusted to 3–4 using sulfuric acid. After the reaction was complete, the reaction solution was cooled to room temperature and stirred to allow crystals to precipitate for 2 hours. The mixture was filtered, and the filter cake was washed with 100 ml of water to obtain the wet product of the compound with structure (I).

[0106] Add 30% hydrobromic acid (6 times the mass of the added 2,4,5,6-tetraaminopyrimidine sulfate) to a wet sample of the compound with structure (I), and heat to 90–100 °C for 4 h. Cool the reaction solution to room temperature and stir for 2 h. Filter the resulting solid, dry it, and give a light yellow solid with an LC purity of 74.24% and a yield of 76.47%.

[0107] (2) Purification of folic acid impurity F

[0108] The crude folic acid impurity F was dissolved in DMF, filtered, and the filtrate was separated in a semi-preparative chromatograph. 10 ml was injected each time, and the eluent was collected, combined, concentrated under reduced pressure (≤-0.09 MPa), and dried to obtain folic acid impurity F.

[0109] The purity of the obtained folic acid impurity F was 96.62%.

[0110] Example 5:

[0111] (1) Synthesis of crude folic acid impurity F

[0112] 2,4,5,6-Tetraaminopyrimidine sulfate, L-cysteine ​​+ sodium metabisulfite, and 1,1,3-trichloroacetone were added to a reaction flask. Based on the molar ratio, the ratio of 2,4,5,6-tetraaminopyrimidine sulfate:1,1,3-trichloroacetone:(L-cysteine ​​+ sodium metabisulfite) was 1:1:3. The mixture was reacted with water and propanol (volume ratio 1:2) at 20–30 °C for 36 h. During the reaction, the pH of the solution was adjusted to 2–3 with hydrochloric acid. After the reaction was complete, the reaction solution was cooled to room temperature and stirred to allow crystals to precipitate for 2 h. The mixture was filtered, the filter cake was washed with 100 ml of water, and dried to obtain the wet product of the compound with structure (I).

[0113] Add 40% sulfuric acid (4 times the mass of the added 2,4,5,6-tetraaminopyrimidine sulfate) to a wet sample of the compound with structure (I), and heat to 80–90 °C for 3 h. Cool the reaction solution to room temperature and stir for 2 h. Filter the resulting solid to obtain a light yellow solid with an LC purity of 75.86% and a yield of 73.19%.

[0114] (2) Purification of folic acid impurity F

[0115] The crude folic acid impurity F was dissolved in DMSO, filtered, and the filtrate was subjected to preparative chromatography. 10 ml was injected each time, and the eluent was collected, combined, concentrated under reduced pressure (≤-0.09 MPa), and dried to obtain folic acid impurity F.

[0116] The purity of the obtained folic acid impurity F was 97.35%.

[0117] Example 6:

[0118] (1) Synthesis of crude folic acid impurity F

[0119] 2,4,5,6-Tetraaminopyrimidine sulfate, (sodium metabisulfite + L-pyroglutamic acid), and 1,1,3-trichloroacetone were added to a reaction flask. Based on the molar ratio, the ratio of 2,4,5,6-tetraaminopyrimidine sulfate:1,1,3-trichloroacetone:(sodium metabisulfite + L-pyroglutamic acid) was 1:1:4. The mixture was heated to 60–70 °C with water and isopropanol (volume ratio 1:2) for 8 hours. During the reaction, the pH of the solution was adjusted to 3–4 with hydrochloric acid. After the reaction was complete, the reaction solution was cooled to room temperature and stirred to allow crystals to precipitate for 2 hours. The mixture was filtered, and the filter cake was washed with 100 ml of water to obtain the wet product of the compound with structure (I).

[0120] Add 30% hydrochloric acid (7 times the mass of the added 2,4,5,6-tetraaminopyrimidine sulfate) to the wet product of the compound with structure (I), and heat to 70-80℃ for 5 h. Cool the reaction solution to room temperature and stir for 2 h, filter the obtained solid, dry it, and obtain a light yellow solid with LC purity of 74.96% and yield of 71.45%. (2) Purification of folic acid impurity F: Crude folic acid impurity F was dissolved in DMF, filtered, and the filtrate was separated by semi-preparative chromatography. 10 ml was used each time, and the eluent was collected, combined, concentrated under reduced pressure (vacuum degree ≤ -0.09 MPa), and dried to obtain folic acid impurity F.

[0121] The purity of the obtained folic acid impurity F was 96.71%.

[0122] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing folic acid EP impurity F, characterized in that, include: A) 2,4,5,6-Tetraaminopyrimidine sulfate and 1,1,3-trichloroacetone undergo a condensation reaction in the presence of an antioxidant and a solvent to yield a compound of formula (I); the solvent is a mixed solution of water and an alcohol; the alcohol is selected from one or more of methanol, ethanol, propanol, and isopropanol; the antioxidant is one or more of L-cysteine, L-pyroglutamic acid, sodium metabisulfite, and sodium bisulfite; the reaction is carried out under acidic conditions, and the pH of the reaction is 1 to 4; the acid used to adjust the pH is one of hydrochloric acid, sulfuric acid, or glacial acetic acid. B) The compound of formula (I) was acid hydrolyzed to obtain crude folic acid impurity F, and the crude folic acid impurity F was purified by chromatography to obtain folic acid EP impurity F of formula (II); The crude folic acid impurity F was purified by chromatography to obtain folic acid EP impurity F with the structure of formula (II), specifically: The crude folic acid impurity F was dissolved in a solvent, filtered, and the filtrate was fed into a preparative chromatograph to collect the desired component solution. After removing the solvent under reduced pressure, the solution was lyophilized, and the resulting solid was washed with water to remove salts and dried under reduced pressure to obtain folic acid impurity F. The parameters for the chromatographic purification were as follows: Instrument: Semi-preparative chromatograph, SepaBean machine T; Column: Spherical C 18, 50 μm 100A; Detection wavelength: 280 nm; Mobile phase A: 0.1% phosphoric acid; Mobile phase B: acetonitrile; Elution gradient: 0–20 min, 90% A–30% A.

2. The preparation method according to claim 1, characterized in that, In step A), the molar ratio of 2,4,5,6-tetraaminopyrimidine sulfate, 1,1,3-trichloroacetone, and antioxidant is 1:1:2 to 1:1:

5.

3. The preparation method according to claim 1, characterized in that, In step A), the volume ratio of water to alcohol is 1:3 to 3:1; the mass ratio of 2,4,5,6-tetraaminopyrimidine sulfate to solvent is 1:(30 to 80).

4. The preparation method according to claim 1, characterized in that, The reaction temperature in step A) is 20–80°C; the reaction time is 1–36 h.

5. The preparation method according to claim 1, characterized in that, The acid used in the acid hydrolysis described in step B) is one of hydrochloric acid, sulfuric acid, or hydrobromic acid; the mass concentration of the acid is 30% to 50%. The mass ratio of the compound and acid in the structure of formula (I) is 1:(2-8).

6. The preparation method according to claim 1, characterized in that, The acid hydrolysis temperature in step B) is 60–100°C; the acid hydrolysis time is 0.5–5 h.

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