A process for the preparation of (s)-5-formyltetrahydropteroyl acid

Abstract

The application belongs to the technical field of organic synthesis, and particularly relates to a preparation method of (S)-5-formyl tetrahydropteroyl acid, which comprises the following steps: (1) under nitrogen protection, a pteroyl acid shown in formula I is subjected to a reduction reaction with a reducing agent in an alkaline aqueous solution to obtain a reaction solution containing tetrahydropteroyl acid shown in formula II; (2) under nitrogen protection, the reaction solution obtained in step (1) is added into a mixed solution of formic acid and hydrochloric acid, and reaction is performed to obtain 5,10-methyl tetrahydropteroyl acid chloride shown in formula III; (3) the compound shown in formula III obtained in step (2) is dissolved in formic acid, and a sodium chloride solution or a hydrochloric acid solution is added, and crystallization is performed to obtain (S)-5,10-methyl tetrahydropteroyl acid chloride shown in formula IV; (4) under nitrogen protection, the product obtained in step (3) is subjected to hydrolysis to obtain (S)-5-formyl tetrahydropteroyl acid shown in formula V. The preparation method of (S)-5-formyl tetrahydropteroyl acid provided in the application has the advantages of simple process, mild conditions, high yield and high purity.

Description

Technical Field

[0001] This invention belongs to the technical field of organic synthesis, specifically relating to a method for preparing (S)-5-formyltetrahydropteroic acid. Background Technology

[0002] Levofolinate is a drug primarily used to treat symptoms associated with folic acid antagonism after high-dose methotrexate treatment for osteosarcoma. It can also be used to treat megaloblastic anemia caused by folic acid deficiency, and can be used in combination with fluorouracil to prolong the survival of patients with advanced colorectal cancer or relieve symptoms.

[0003] The European Pharmacopoeia (EP) mandates quantitative studies of (S)-5-formyltetrahydropteroic acid (EP impurity E), an impurity in levofolate calcium, and includes it in quality standards. The chemical name of this impurity is 4-(((2-amino-5-formyl-4-oxo-1,4,5,6,7,8-hexahydropterodin-6-yl)methyl)amino)benzoic acid.

[0004] However, there are few reports in the existing literature on the synthesis methods of this impurity, making it difficult to obtain impurity reference standards. Therefore, developing a method for preparing (S)-5-formyltetrahydroptergic acid to obtain a stable compound with high quantification content is of guiding significance for the control of drug quality. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing (S)-5-formyltetrahydropteroic acid, which features a simple process, mild conditions, and high yield and purity.

[0006] To solve the above-mentioned technical problems, the objective of this invention is achieved through the following technical solution: A method for preparing (S)-5-formyltetrahydropteranoic acid includes the following steps: (1) Under nitrogen protection, the pteridine of Formula I is reduced in an alkaline aqueous solution with a reducing agent. After the reaction is completed, the pH is adjusted to 6-7 with an inorganic acid and an antioxidant is added to obtain a reaction solution containing tetrahydropteridine of Formula II. (2) Under nitrogen protection, the reaction solution obtained in step (1) was added to a mixed solution of formic acid and hydrochloric acid, and the reaction was carried out at room temperature. The solid was separated to obtain 5,10-methylenetetrahydropteroic acid chloride as shown in Formula III. (3) Dissolve the compound of formula III obtained in step (2) in formic acid, add sodium chloride solution or hydrochloric acid solution, stir to crystallize, separate the solid, and obtain (S)-5,10-methylenetetrahydropteroic acid chloride as shown in formula IV; (4) Under nitrogen protection, the compound IV obtained in step (3) was suspended in water, and an alkali and antioxidant were added to adjust the pH to 6-8. The temperature was raised to react, and after the reaction was completed, the temperature was lowered and the pH was adjusted to 2-4. Crystallization was carried out, and the solid was separated to obtain (S)-5-formyltetrahydropteroic acid as shown in formula V. The structures of compounds of formula I to V are as follows: Formula I: ; Formula II: ; Formula III: ; Formula IV: ; Formula V: .

[0007] As a further improvement of the present invention, in the reduction reaction of step (1): The reducing agent is selected from one or more of sodium borohydride, potassium borohydride, sodium bisulfite, and sodium metabisulfite; among them, sodium borohydride / potassium borohydride has strong reducing power, few side reactions, and high yield, and is the preferred reducing agent; The molar ratio of pteric acid to reducing agent is 1:2 to 5, and the molar ratio of pteric acid to base is 1:1.5 to 4.5. The reduction reaction is carried out at a temperature of 60–80°C for 1–3 hours. Within the above temperature and time range, TLC detection shows that pteric acid disappears, and the reaction can achieve complete conversion.

[0008] After the reduction reaction is complete, the temperature is lowered to below 10°C, and then quenched dropwise with an inorganic acid (formic acid, hydrochloric acid, or acetic acid) until the pH reaches 6-7. The quenching process releases a large amount of heat; localized overheating can lead to the oxidative degradation of tetrahydropteranoic acid and may also damage the product structure. Controlling the quenching temperature to no more than 20°C can effectively protect the stability of tetrahydropteranoic acid. Among the inorganic acids, formic acid can also serve as a raw material for subsequent cyclization reactions, making it a preferred quenching acid in this invention.

[0009] After the reduction reaction is complete, an antioxidant is added, selected from one or more of vitamin C, 2-mercaptoethanol, and sodium hydrosulfite. The addition of the reducing agent significantly improves the stability of tetrahydropteric acid.

[0010] As a further improvement of the present invention, in the cyclization reaction of step (2): The amount of formic acid used is 1 to 2 times the mass of pteroic acid in step (1), and the amount of hydrochloric acid used is 1.5 to 3 times the mass of pteroic acid. The reaction time at room temperature is 4 to 8 hours.

[0011] As a further improvement of the present invention, in the chiral resolution reaction in step (3): The 6-carbon atom of 5,10-methylenetetrahydropteroic acid chloride is a chiral center, exhibiting both (6R) and (6S) configurations. In formic acid solvent, the (S)- and (R)-configuration isomers show different association abilities with chloride ions. When chloride ions are provided by sodium chloride or hydrochloric acid, the (S)-configuration product preferentially precipitates as crystals, thus achieving chiral separation. This method eliminates the need for expensive chiral resolution reagents or chiral chromatographic columns, resulting in low production costs and simple operation.

[0012] The amount of formic acid used is 2.5 to 5 times the amount of 5,10-methylenetetrahydropteranoic acid chloride. The concentration of the sodium chloride solution or hydrochloric acid solution is 1N to 6N, and the amount used is 1 to 6 times the amount of 5,10-methylenetetrahydropteroic acid chloride. Chloride ions are the key factor inducing chiral selective crystallization. Within the above concentration and amount range, (S)-configuration products with ideal optical purity can be obtained.

[0013] The stirring crystallization temperature is 20–30°C, and the time is 4–8 hours.

[0014] As a further improvement of the present invention, in the hydrolysis reaction of step (4): Under alkaline conditions, the CN bond in the 5,10-methylene-bridged ring of (S)-5,10-methylenetetrahydropteroic acid chloride breaks, allowing water molecules to nucleophilically attack the 5,10-methylene group while simultaneously protonating the nitrogen atom at position 10, ultimately yielding 5-formyltetrahydropteroic acid. The regioselectivity of this hydrolysis reaction determines that the product is 5-formylated (rather than 10-formylated) tetrahydropteroic acid.

[0015] The alkali is selected from one or more of sodium hydroxide, potassium hydroxide, piperazine, morpholine and ammonia water; among which, piperazine is an organic weak base, with mild alkalinity, low destructive effect on the product, and high yield after hydrolysis, and is the preferred alkali of the present invention.

[0016] The antioxidant is selected from one or more of vitamin C and 2-mercaptoethanol; the addition of the antioxidant can significantly inhibit oxidation side reactions and ensure the high purity of the hydrolysis product. Among them, vitamin C is non-toxic and has good water solubility, making it the preferred antioxidant of this invention; 2-mercaptoethanol has stronger antioxidant capacity and is suitable for occasions with higher purity requirements.

[0017] The heating reaction is carried out at a temperature of 60–80°C for 2–4 hours; within the above temperature and time range, TLC or HPLC detection shows that the raw material is completely converted.

[0018] The cooling process involves lowering the temperature to 0–5°C, and the pH adjustment to 2–4 followed by crystallization over 1–3 hours. Formyltetrahydropteridine exhibits significantly reduced solubility under acidic conditions; by controlling the temperature and pH, it can precipitate as high-purity crystals. Low-temperature crystallization reduces the product's solubility in solution, increases the crystallization yield, and simultaneously improves crystal purity.

[0019] After crystallization, the solid is separated, slurried with anhydrous ethanol for 10-15 minutes, filtered again, and dried under vacuum at 25-50°C.

[0020] The present invention also provides a method for preparing (S)-5-formyltetrahydropteroate calcium. When it is necessary to prepare calcium salt, after the hydrolysis reaction in step (4) is completed, acidification crystallization is not performed. Instead, the reaction solution is cooled to 20-30°C, a calcium salt aqueous solution is added and the pH is adjusted to 8-9. The mixture is stirred for 4-8 hours to crystallize, the solid is separated, washed with ethanol, and dried under vacuum to obtain (S)-5-formyltetrahydropteroate calcium as shown in Formula VI.

[0021] Preferably, the calcium salt is calcium chloride, and the amount of calcium chloride used is 1 to 4 times the mass of (S)-5,10-methylenetetrahydropteroic acid chloride.

[0022] This invention also provides the application of (S)-5-formyltetrahydropteridine or (S)-5-formyltetrahydropteridine calcium as impurity reference standards in the quality control of levofolinic acid calcium. Using the (S)-5-formyltetrahydropteridine or (S)-5-formyltetrahydropteridine calcium prepared according to this invention as reference standards, and employing analytical methods such as high-performance liquid chromatography, the content of this impurity in levofolinic acid calcium raw materials or preparations can be qualitatively and / or quantitatively detected, thereby effectively controlling drug quality.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1. The preparation method of (S)-5-formyltetrahydropteroate calcium provided by the present invention fills the gap in the prior art. The target product is obtained by reducing, cyclizing, chiral resolution, hydrolyzing and salting pteridine as raw material. The process is simple, the conditions are mild and the equipment requirements are low. The obtained product has high purity and standard content and can be used as a reference for key impurities, providing reliable support for the quality control of folic acid drugs such as levofloxacin calcium.

[0024] 2. This invention uses pteroic acid as a starting material to directly construct the target impurity molecule framework, avoiding the cumbersome step of additional glutamic acid cleavage required when starting from folic acid. Precise construction of the target impurity molecule is achieved through the chemically selective reduction of the pteroic acid-pteridine ring and the regioselective hydrolysis ring-opening under alkaline conditions. The reactions are tightly linked, and the intermediates can be used in subsequent reactions without complex purification. Attached Figure Description

[0025] Figure 1 This is the liquid phase spectrum of (S)-5-formyltetrahydropteroic acid prepared in Example 1; Figure 2 This is the liquid phase spectrum of (S)-5-formyltetrahydropteroic acid prepared in Example 2; Figure 3 This is the liquid phase spectrum of (S)-5-formyltetrahydropteroic acid prepared in Example 3; Figure 4 This is the mass spectrum of (S)-5-formyltetrahydropteroic acid prepared in this invention; Detailed Implementation

[0026] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0027] All raw materials used in the examples were commercially available.

[0028] In this example, high-performance liquid chromatography (HPLC) was used to determine the purity of the obtained product, and the chromatographic conditions were as follows: Detector: Ultraviolet absorbance detector, detection wavelength 287nm; Chromatographic column: 2.0 mm inner diameter, 10 cm long stainless steel column, packed with octadecylsilane-modified silica gel (5 μm particle size). Flow rate: 0.5 mL / min; Injection volume: 10 μL; Column temperature: 40℃; Injector temperature: 5℃; Mobile phase A: Dissolve 1.15 g potassium trihydrogen phosphate monohydrate and 1.7 g tetrabutylammonium hydrogen sulfate in 950 mL of water, adjust the pH to 6.5 with phosphoric acid, and add water to 1000 mL; Mobile phase B: Methanol; Gradient elution procedure: Example 1 (1) Preparation of tetrahydropteric acid Under nitrogen protection, an aqueous solution of sodium hydroxide (2.5 g, 62.5 mmol, dissolved in 10 ml of purified water) was added to the reaction flask, followed by the slow addition of sodium borohydride (3.5 g, 92.5 mmol) while stirring until dissolved. Pteric acid (6.7 g, 21.5 mmol) was suspended in 20 ml of purified water and slowly added dropwise to the reaction flask. After the addition was complete, the temperature was slowly raised to 80 °C and reacted for 1 h. TLC analysis showed the disappearance of pteroic acid. The temperature was then lowered to below 10 °C, and formic acid was added dropwise to quench the reaction until the pH reached 6-7. The temperature was controlled to not exceed 20 °C. Vitamin C (0.15 g) was added, and the mixture was filtered to obtain the tetrahydropteroic acid reaction solution.

[0029] (2) Preparation of 5,10-methylenetetrahydropteric acid chloride Under nitrogen protection, formic acid (8 g, 173.8 mmol) and hydrochloric acid (12 g, 329.1 mmol) were added to a reaction flask, and the tetrahydropteranoic acid reaction solution obtained in the first step was slowly added dropwise, with stirring until homogeneous. The reaction was stirred at 25 °C for 6 hours, filtered, washed once with water and once with ethanol, and dried under vacuum at 50 °C for 8 hours to obtain 7.1 g of 5,10-methylenetetrahydropteranoic acid chloride, with a two-step yield of 91.3%.

[0030] (3) Preparation of (S)-5,10-methylenetetrahydropteric acid chloride 7.1 g of 5,10-methylenetetrahydropteranoic acid chloride was dissolved in 25 g of formic acid, and a prepared sodium chloride solution (1.75 g, 30 mmol, dissolved in 15 ml of purified water) was added dropwise. The mixture was stirred at 25 °C for 4 hours to induce crystallization. After filtration, the solution was washed once with water and once with ethanol, and then dried under vacuum at 50 °C for 8 hours to obtain 2.9 g of (S)-5,10-methylenetetrahydropteranoic acid chloride, with a yield of 40.8%.

[0031] (4) Preparation of (S)-5-formyltetrahydropteroic acid.

[0032] Under nitrogen protection, (S)-5,10-methylenetetrahydropteranoic acid chloride (2.9 g, 8 mmol) was suspended in 30 ml of water. Potassium hydroxide solution was added and stirred until dissolved. The pH was adjusted to 7. Vitamin C (0.1 g) was added, and the reaction was carried out at 80 °C for 2 h. The temperature was lowered to 0–5 °C, and the pH was adjusted to 2.5 with 4N hydrochloric acid. Crystallization was carried out for 2 h with stirring. The mixture was filtered, stirred in 10 ml of ethanol for 10 min, filtered again, and dried under vacuum at 30 °C for 12 h to obtain 1.5 g of (S)-5-formyltetrahydropteranoic acid (54.5% yield). HPLC analysis was performed as shown in the attached figure. Figure 1 As shown, the purity is 96.6%; Example 2 (1) Preparation of tetrahydropteric acid Under nitrogen protection, an aqueous solution of sodium hydroxide (2.0 g, 50 mmol, dissolved in 8 ml of purified water) was added to the reaction flask, followed by the slow addition of sodium borohydride (2.6 g, 68.5 mmol) while stirring until dissolved. Pteric acid (5 g, 16 mmol) was suspended in 15 ml of purified water and slowly added dropwise to the reaction flask. After the addition was complete, the temperature was slowly raised to 80 °C and reacted for 1 h. TLC analysis showed the disappearance of pteroic acid. The mixture was then cooled to below 10 °C, and formic acid was added dropwise to quench the reaction until the pH reached 6-7. The temperature was controlled to not exceed 20 °C. Vitamin C (0.1 g) was added, and the mixture was filtered to obtain the tetrahydropteroic acid reaction solution.

[0033] (2) Preparation of 5,10-methylenetetrahydropteric acid chloride Under nitrogen protection, formic acid (6.0 g, 130.4 mmol) and hydrochloric acid (8.5 g, 233.6 mmol) were added to a reaction flask, and the tetrahydropteranoic acid reaction solution obtained in the first step was slowly added dropwise, with stirring until homogeneous. The reaction was stirred at 25 °C for 6 hours, filtered, washed once with water and once with ethanol, and dried under vacuum at 50 °C for 8 hours to obtain 5.25 g of 5,10-methylenetetrahydropteranoic acid chloride, with a two-step yield of 90.5%.

[0034] (3) Preparation of (S)-5,10-methylenetetrahydropteric acid chloride 5.25 g of 5,10-methylenetetrahydropteranoic acid chloride was dissolved in 18.5 g of formic acid, and a prepared hydrochloric acid solution (1.0 g, 27.4 mmol, dissolved in 10 ml of purified water) was added dropwise. The mixture was stirred at 25 °C for 4 hours to induce crystallization. After filtration, the solution was washed once with water and once with ethanol, and dried under vacuum at 50 °C for 8 hours to obtain 2.06 g of (S)-5,10-methylenetetrahydropteranoic acid chloride, with a yield of 39.2%.

[0035] (4) Preparation of (S)-5-formyltetrahydropteroate calcium.

[0036] Under nitrogen protection, (S)-5,10-methylenetetrahydropteranoic acid chloride (2.06 g, 5.7 mmol) was suspended in 15 ml of water. Piperazine solution was added and stirred until dissolved. The pH was adjusted to 7.5. Vitamin C (0.08 g) was added, and the reaction was carried out at 80 °C for 2 h. The temperature was then lowered to 20–30 °C, and calcium chloride solution (4.1 g, 36.9 mmol, dissolved in 6 ml of purified water) was added. The pH was adjusted to 8.5, and the mixture was stirred to crystallize for 6 h. The crystals were filtered, washed with ethanol, and dried under vacuum to obtain 1.3 g of (S)-5-formyltetrahydropteranoic acid calcium, with a yield of 62.9%. HPLC analysis was performed as shown in the attached figure. Figure 2 As shown, the purity is 98.6%.

[0037] Example 3 (1) Preparation of tetrahydropteric acid Under nitrogen protection, an aqueous solution of sodium hydroxide (3.6 g, 90 mmol, dissolved in 12 ml of purified water) was added to the reaction flask, followed by the slow addition of sodium borohydride (3.4 g, 90 mmol) while stirring until dissolved. Pteric acid (6.7 g, 21.5 mmol) was suspended in 20 ml of purified water and slowly added dropwise to the reaction flask. After the addition was complete, the temperature was slowly raised to 70 °C and reacted for 3 hours. TLC analysis showed the disappearance of pteroic acid. The temperature was then lowered to below 10 °C, and hydrochloric acid was added dropwise to quench the reaction until the pH reached 6-7. The temperature was controlled to not exceed 20 °C. Vitamin C (0.15 g) was added, and the mixture was filtered to obtain the tetrahydropteroic acid reaction solution.

[0038] (2) Preparation of 5,10-methylenetetrahydropteric acid chloride Under nitrogen protection, formic acid (13.4 g, 291.3 mmol) and hydrochloric acid (20.1 g, 552.8 mmol) were added to a reaction flask, and the tetrahydropteranoic acid reaction solution obtained in the first step was slowly added dropwise, with stirring until homogeneous. The reaction was stirred at 25 °C for 6 hours, filtered, washed once with water and once with ethanol, and dried under vacuum at 50 °C for 8 hours to obtain 7.4 g of 5,10-methylenetetrahydropteranoic acid chloride, with a two-step yield of 95.2%.

[0039] (3) Preparation of (S)-5,10-methylenetetrahydropteric acid chloride 7.4 g of 5,10-methylenetetrahydropteranoic acid chloride was dissolved in 22.2 g of formic acid, and a prepared hydrochloric acid solution (1.6 g, 43.9 mmol, dissolved in 11 ml of purified water) was added dropwise. The mixture was stirred at 25 °C for 4 hours to induce crystallization. After filtration, the solution was washed once with water and once with ethanol, and dried under vacuum at 50 °C for 8 hours to obtain 3.1 g of (S)-5,10-methylenetetrahydropteranoic acid chloride, with a yield of 41.9%.

[0040] (4) Preparation of (S)-5-formyltetrahydropteroate calcium.

[0041] Under nitrogen protection, (S)-5,10-methylenetetrahydropteranoic acid chloride (3.1 g, 8.56 mmol) was suspended in 31 ml of water. Piperazine solution was added and stirred until dissolved. The pH was adjusted to 7. 2-Mercaptoethanol (0.1 g) was added, and the reaction was carried out at 60 °C for 4 h. After the reaction was completed, the temperature was lowered to 0–5 °C, and the pH of the system was slowly adjusted to 2.5 with 2N hydrochloric acid. Crystallization was carried out at low temperature for 3 h with stirring. The solid was collected by vacuum filtration, slurryed with 10 ml of anhydrous ethanol for 15 min, filtered again, and dried under vacuum at 30 °C for 12 h to obtain 2.03 g of (S)-5-formyltetrahydropteranoic acid, with a yield of 62.2%. HPLC analysis was performed as shown in the attached figure. Figure 3 As shown, the product purity is 97.9%.

[0042] Mass spectrometry analysis was performed on (S)-5-formyltetrahydropteridine obtained in Example 1, as shown in the attached figure. Figure 4As shown, ESI-MS m / z[M+H] + The measured value was consistent with its theoretical molecular weight, confirming that the obtained product was (S)-5-formyltetrahydropteroic acid, and the structure was correct.

Claims

1. A method for preparing (S)-5-formyltetrahydropteranoic acid, characterized in that, Includes the following steps: (1) Under nitrogen protection, the pteridine of Formula I is reduced in an alkaline aqueous solution with a reducing agent. After the reaction is completed, the pH is adjusted to 6-7 with an inorganic acid and an antioxidant is added to obtain a reaction solution containing tetrahydropteridine of Formula II. (2) Under nitrogen protection, the reaction solution obtained in step (1) was added to a mixed solution of formic acid and hydrochloric acid, and the reaction was carried out at room temperature. The solid was separated to obtain 5,10-methylenetetrahydropteroic acid chloride as shown in Formula III. (3) Dissolve the compound of formula III obtained in step (2) in formic acid, add sodium chloride solution or hydrochloric acid solution, stir to crystallize, separate the solid, and obtain (S)-5,10-methylenetetrahydropteroic acid chloride as shown in formula IV; (4) Under nitrogen protection, the compound IV obtained in step (3) was suspended in water, and an alkali and antioxidant were added to adjust the pH to 6-8. The temperature was raised to react, and after the reaction was completed, the temperature was lowered and the pH was adjusted to 2-4. Crystallization was carried out, and the solid was separated to obtain (S)-5-formyltetrahydropteroic acid as shown in formula V. The structures of compounds of formula I to V are as follows: Formula I: ; Formula II: ; Formula III: ; Formula IV: ; Formula V: .

2. The method for preparing (S)-5-formyltetrahydropteranoic acid according to claim 1, characterized in that, The reducing agent in step (1) is selected from one or more of sodium borohydride, potassium borohydride, sodium bisulfite and sodium metabisulfite; the molar ratio of pteric acid to reducing agent is 1:2 to 5, and the molar ratio of pteric acid to base is 1:1.5 to 4.5; the temperature of the reduction reaction is 60 to 80°C, and the time is 1 to 3 hours.

3. The method for preparing (S)-5-formyltetrahydropteranoic acid according to claim 1, characterized in that, After the reduction reaction in step (1) is completed, the temperature is lowered to below 10°C and the pH is adjusted. During the adjustment process, the temperature is controlled to not exceed 20°C.

4. The method for preparing (S)-5-formyltetrahydropteranoic acid according to claim 1, characterized in that, The amount of formic acid used in step (2) is 1 to 2 times the mass of pteroic acid in step (1), and the amount of hydrochloric acid used is 1.5 to 3 times the mass of pteroic acid; the reaction time at room temperature is 4 to 8 hours.

5. The method for preparing (S)-5-formyltetrahydropteranoic acid according to claim 1, characterized in that, In step (3), the amount of formic acid used is 2.5 to 5 times the amount of 5,10-methylenetetrahydropteroic acid chloride; the concentration of the sodium chloride solution or hydrochloric acid solution is 1N to 6N, and the amount used is 1 to 6 times the amount of 5,10-methylenetetrahydropteroic acid chloride; the stirring and crystallization temperature is 20 to 30°C, and the time is 4 to 8 hours.

6. The method for preparing (S)-5-formyltetrahydropteranoic acid according to claim 1, characterized in that, The alkali in step (4) is selected from one or more of sodium hydroxide, potassium hydroxide, piperazine, morpholine and ammonia water; the antioxidant is selected from one or more of vitamin C and 2-mercaptoethanol; the temperature of the heating reaction is 60-80°C and the time is 2-4 hours.

7. The method for preparing (S)-5-formyltetrahydropteranoic acid according to claim 1, characterized in that, The cooling in step (4) is to cool to 0-5°C, and the pH is adjusted to 2-4 and crystallization occurs after 1-3 hours.

8. The method for preparing (S)-5-formyltetrahydropteranoic acid according to claim 1, characterized in that, After the reaction in step (4) is completed, the temperature is lowered to 20-30°C, a calcium salt aqueous solution is added and the pH is adjusted to 8-9, and the mixture is stirred for 4-8 hours to crystallize and obtain (S)-5-formyltetrahydropteroate calcium.

9. The method for preparing (S)-5-formyltetrahydropteranoic acid according to claim 8, characterized in that, The calcium salt is calcium chloride, and the amount of calcium chloride used is 1 to 4 times the mass of (S)-5,10-methylenetetrahydropteroic acid chloride.

10. The use of (S)-5-formyltetrahydropteroic acid or (S)-5-formyltetrahydropteroic acid calcium prepared by any one of claims 1 to 9 as an impurity reference standard in the quality control of leucovorin calcium.

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