A method for purifying a methotrexate intermediate

Abstract

The present application belongs to the technical field of medicine, and particularly relates to a refining method of methotrexate intermediate (i.e. methotrexate disodium salt). The finished product of methotrexate contains stubborn impurity C (the limit of impurity C in European Pharmacopoeia EP9.0 is ≤0.5%), which is difficult to remove by ordinary refining method. The inventors surprisingly found that, by adding sodium chloride to the reaction solution of methotrexate disodium salt for crystallization, the content of stubborn impurity C precursor can be greatly reduced, the number and content of impurity peaks are reduced, the pigment in the methotrexate intermediate is removed, and then the content of stubborn impurity C in the methotrexate bulk drug is reduced, so that the purity meets the requirements of European Pharmacopoeia.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a refining method of methotrexate intermediate. BACKGROUND

[0002] Methotrexate (CAS RN: 59-05-2) as an antifolate drug has a good therapeutic effect in the treatment of diseases such as tumors and rheumatoid arthritis, and is a kind of clinical treatment drug. Its mechanism of action is mainly to inhibit dihydrofolate reductase, thereby inhibiting the synthesis of DNA, so as to block mitosis of cells and inhibit cell proliferation.

[0003] Methotrexate disodium salt is a key intermediate in the preparation process of methotrexate. CN107698592A introduces a purification process of methotrexate disodium salt, which is as follows:

[0004]

[0005] It takes 4-methylamino benzoyl glutamic acid zinc (1) as a raw material, reacts with tetraamino pyrimidine sulfate (2) and tribromoacetone (3) in an aqueous solution to obtain a methotrexate reaction solution, then adjusts the pH value by using lye to obtain a methotrexate disodium salt intermediate, and crystallizes by using an organic solvent. Through tracking the patent, it is found that the main purpose of the purification process is to remove methotrexate isomers, but the yield is only 57%, and the purity is 98.5%, which cannot meet the quality requirements of the European Pharmacopoeia (EP9.0).

[0006]

[0007] CN102399224A introduces a preparation method of low-iron methotrexate, which is specifically that the methotrexate disodium salt intermediate is dissolved in water, and then filtered through a silica gel filter column to remove iron ions therein, the purpose of which is to obtain methotrexate with an iron content of less than 1 ppm, and it does not involve the purification of related substances.

[0008] The methotrexate bulk drug recorded in foreign pharmacopoeias has low impurity limits (in the European Pharmacopoeia EP9.0, impurities B and E ≤0.3%, impurity C ≤0.5%, impurities H and I ≤0.2%, and unknown impurities ≤0.05%). Among them, the recalcitrant impurity C is difficult to remove by ordinary refining methods. The inventors found in the research process that by adding sodium chloride to the reaction solution of methotrexate disodium salt to crystallize, the content of the precursor of the recalcitrant impurity C can be greatly reduced, the number and content of impurity peaks can be reduced, the pigment in the methotrexate intermediate can be removed, and thus the content of the recalcitrant impurity C in the methotrexate bulk drug can be reduced, so that the purity meets the requirements of the European Pharmacopoeia.

[0009] The structure of the recalcitrant impurity C and its precursor is as follows:

[0010] SUMMARY

[0011] Specifically, the present application provides a method for purifying methotrexate intermediate of formula I, comprising the following steps:

[0012]

[0013] (1) dissolving methotrexate intermediate crude product with solvent;

[0014] (2) adding sodium chloride solid or sodium chloride aqueous solution into the solution of step (1) and stirring to crystallize;

[0015] (3) filtering, washing the filter cake with acetone and drying under reduced pressure to obtain the purified product of formula I.

[0016] Preferably, the solvent of step (1) is selected from water or organic solvent / water mixed system.

[0017] Preferably, the organic solvent / water mixed system is selected from methanol / water mixed system, ethanol / water mixed system, acetone / water mixed system, isopropanol / water mixed system; wherein the volume ratio of organic solvent to water is (0.01:99.99)~(50:50), preferably (0.01:99.99)~(40:60).

[0018] More preferably, the solvent of step (1) is selected from water, methanol / water mixed system or ethanol / water mixed system.

[0019] Preferably, the mass / volume ratio of methotrexate intermediate crude product to solvent of step (1) is 1:2-1:8 g / ml, preferably 1:3-1:7 g / ml, more preferably 1:4-1:6 g / ml, for example 1:5 g / ml.

[0020] Preferably, the mass / volume ratio of sodium chloride solid to the solvent of step (1) of step (2) is 1:1-1:10 g / ml, preferably 1:3-1:7 g / ml, more preferably 1:4-1:6 g / ml, for example 1:5 g / ml.

[0021] Preferably, the sodium chloride aqueous solution of step (2) is a saturated sodium chloride aqueous solution, and the mass / volume ratio of the saturated sodium chloride aqueous solution to the solvent of step (1) is 1:1-10:1 g / ml, preferably 3:1-6:1 g / ml, more preferably 3:1 g / ml.

[0022] Preferably, the temperature during purification is 10-30℃, preferably 15-25℃, more preferably 20℃.

[0023] Preferably, the stirring crystallization time of step (2) is 0.5-2h, preferably 1-1.5h, more preferably 1h.

[0024] Preferably, the temperature of the reduced pressure drying of step (3) is 30-50℃, preferably 35-45℃, more preferably 45℃.

[0025] Further, the compound of formula I prepared by the above purification method can be hydrolyzed to convert into methotrexate.

[0026] The limit of the precursor of the stubborn impurity C in the compound of formula I prepared by the above preparation method is ≤0.5%, and after hydrolytic conversion into methotrexate, the limit of the stubborn impurity C is also ≤0.5%.

[0027] In another aspect of the present application, a crystal form of the compound of formula I is provided, which has X-ray powder diffraction peaks at 5.38±0.2, 14.06±0.2, 16.25±0.2, 18.78±0.2, 20.44±0.2, 21.00±0.2, 22.41±0.2, 23.57±0.2, 24.79±0.2, 28.37±0.2; preferably, the X-ray powder diffraction pattern of the crystal form is as shown in Figure 2 The X-ray diffraction data is shown in Table 1.

[0028] Table 1 X-ray diffraction data of the crystal form of the compound of formula I of the present application

[0029]

[0030]

[0031] It is found by experiment that, by adding sodium chloride to the reaction solution of methotrexate disodium salt to crystallize, the content of the precursor of the stubborn impurity C in the compound of formula I is greatly reduced, and at the same time, the number and content of impurity peaks can be reduced, and the pigment in the methotrexate intermediate can be removed; after hydrolysis of the compound of formula I, methotrexate drug substance with low content of stubborn impurity C can be obtained. The purification method provided by the present application is simple, the reaction conditions are mild, the solvents and reagents used are low in price, and is suitable for scale-up. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is the HPLC spectrum of the purified product of the compound of formula I prepared in Example 2.

[0033] Figure 2 is the X-ray powder diffraction pattern of the compound of formula I prepared in Example 2. DETAILED DESCRIPTION

[0034] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples, if not specified, are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers.

[0035] Preparation method of methotrexate intermediate

[0036]

[0037] Methotrexate diethyl ester (5 g, 7.32 mmol) and 25 ml purified water were added into a reaction bottle, and aqueous sodium hydroxide (NaOH: 1.11 g, 27.82 mmol; purified water: 10 ml) was added dropwise at 15-25 °C. After the dropwise addition was completed, the reaction was stirred at 20-25 °C for 1.5 h, filtered, and 105 ml of acetone was added dropwise to the filtrate. The mixture was stirred for 0.5 h to precipitate crystals, filtered, and 3.47 g of a yellowish methotrexate intermediate solid was obtained, with a yield of about 95%, a purity of 97.35% as determined by HPLC, and an impurity C content of 1.67% (under the HPLC conditions, the impurity C precursor was converted into impurity C, which was detected as an acid, and the same applied below).

[0038] Example 2

[0039] (1) Purification method of methotrexate intermediate

[0040]

[0041] Methotrexate intermediate (5 g, 10 mmol) and 25 ml of purified water were added into a reaction bottle, and stirred to dissolve at 20 °C. Sodium chloride solid (5 g) was added, and the mixture was stirred at about 20 °C for 1 h to precipitate crystals. The mixture was filtered, and the filtrate was washed with acetone, dried, and placed in a 45 °C vacuum drying oven for 8 h to obtain a product with a mass of about 2.9 g, a yield of 58%, a purity of 99.905% as determined by HPLC, and an impurity C content of 0.037%.

[0042] Figure 1 The HPLC chromatogram of the purified product of the compound of formula I is shown below:

[0043] Chromatographic column: Waters XBridge C18 (250 mm x 4.6 mm, 5 μm) or a chromatographic column with equivalent performance

[0044] Mobile phase A: sodium dihydrogen phosphate solution (pH 6.5)-acetonitrile (95:5)

[0045] Mobile phase B: acetonitrile

[0046] Gradient elution:

[0047]

[0048] Flow rate: 1.0 ml / min; detection wavelength: 220 nm; column temperature: 30 °C; injection volume: 20 μl; diluent: mobile phase A; injection tray temperature: 10 °C.

[0049] Figure 2 X-ray powder diffraction pattern of the compound of formula I, the XRPD of the present application employs the following detection conditions:

[0050] Test method: XRPD test uses Empyream X-ray powder diffractometer of PANalytical company. A proper amount of sample is placed in a single crystal silicon sample disc groove, and a glass slide is pressed to flatten it, and XRPD test is performed using the above test parameters.

[0051] Test parameters are as follows:

[0052]

[0053] (2) Preparation of methotrexate bulk drug

[0054] Methotrexate intermediate refined product (3 g, 6.0 mmol), 30 ml purified water were added to the reaction bottle, and stirred to dissolve. The temperature was raised to 40 °C and maintained at this temperature, and 5% sulfuric acid solution was added dropwise to adjust the pH of the solution to 4.0-4.5. Keep stirring for 30 min, filter, filter cake with 30 ml purified water at 40 °C for 15 min, filter, get orange solid powder, methotrexate, HPLC purity 99.911%, impurity C content 0.026%.

[0055] Example 3: Refining method of methotrexate intermediate

[0056]

[0057] Methotrexate intermediate (5 g, 10 mmol), 15 ml purified water, 10 ml methanol were added to the reaction bottle, stirred to dissolve at 20 °C, 5 g of sodium chloride solid was added, and the stirring was maintained at about 20 °C for 1 h. Filter, filter with acetone, dry, placed in 45 °C under reduced pressure for 8 h, the mass was about 4.43 g, the yield was 88.6%, the HPLC detection purity was 99.752%, and the impurity C content was 0.142%.

[0058] Example 4: Refining method of methotrexate intermediate

[0059]

[0060] Methotrexate intermediate (5 g, 10 mmol), 15 ml purified water, 10 ml ethanol were added into the reaction flask, stirred and dissolved at 20 °C, sodium chloride solid 5 g was added, maintained at about 20 °C, stirred for 1 h. Filtered, the filtrate was washed with acetone, dried, placed in 45 °C, dried under reduced pressure for 8 h, the mass of the product was about 4.7 g, the yield was 94%, the purity was 99.376% by HPLC detection, and the impurity C content was 0.289%.

[0061] Example 5: Purification method of methotrexate intermediate

[0062]

[0063] Methotrexate intermediate (5 g, 10 mmol), 15 ml purified water, 10 ml acetone were added into the reaction flask, stirred and dissolved at 20 °C, sodium chloride solid 5 g was added, maintained at about 20 °C, stirred for 1 h. Filtered, the filtrate was washed with acetone, dried, placed in 45 °C, dried under reduced pressure for 8 h, the mass of the product was about 4.7 g, the yield was 94%, the purity was 99.376% by HPLC detection, and the impurity C content was 0.289%.

[0064] Example 6: Purification method of methotrexate intermediate

[0065]

[0066] Methotrexate intermediate (5 g, 10 mmol), 15 ml purified water, 10 ml isopropanol were added into the reaction flask, stirred and dissolved at 20 °C, sodium chloride solid 5 g was added, maintained at about 20 °C, stirred for 1 h. Filtered, the filtrate was washed with acetone, dried, placed in 45 °C, dried under reduced pressure for 8 h, the mass of the product was about 4.7 g, the yield was 94%, the purity was 99.376% by HPLC detection, and the impurity C content was 0.289%.

[0067] Example 7: Purification method of methotrexate intermediate

[0068]

[0069] Methotrexate intermediate (5 g, 10 mmol), 25 ml purified water were added into the reaction flask, stirred and dissolved at 20 °C, sodium chloride solid 3.75 g was added, maintained at about 20 °C, stirred for 1 h. Filtered, the filtrate was washed with acetone, dried, placed in 45 °C, dried under reduced pressure for 8 h, the mass of the product was about 4.7 g, the yield was 94%, the purity was 99.376% by HPLC detection, and the impurity C content was 0.289%.

[0070] Example 8: Purification method of methotrexate intermediate

[0071]

[0072] The methotrexate intermediate (5 g, 10 mmol) and 25 ml purified water were added into the reaction bottle, stirred and dissolved at 20°C, and then 6.25 g of sodium chloride solid was added. The crystallization was maintained at about 20°C for 1 h. The filtrate was washed with acetone, dried, and then placed in a 45°C vacuum drying oven for 8 h to obtain a product with a mass of about 4.6 g, a yield of 92%, a purity of 99.471% as determined by HPLC, and an impurity C content of 0.368%.

[0073] Example 9: A method for purifying the methotrexate intermediate

[0074]

[0075] The methotrexate intermediate (5 g, 10 mmol) and 25 ml purified water were added into the reaction bottle, stirred and dissolved at 20°C, and then 6.25 g of sodium chloride solid was added. The crystallization was maintained at about 20°C for 1 h. The filtrate was washed with acetone, dried, and then placed in a 45°C vacuum drying oven for 8 h to obtain a product with a mass of about 4.6 g, a yield of 92%, a purity of 99.471% as determined by HPLC, and an impurity C content of 0.368%.

[0076] Example 10: A method for purifying the methotrexate intermediate

[0077]

[0078] The methotrexate intermediate (5 g, 10 mmol) and 25 ml purified water were added into the reaction bottle, stirred and dissolved at 20°C, and then 6.25 g of sodium chloride solid was added. The crystallization was maintained at about 20°C for 1 h. The filtrate was washed with acetone, dried, and then placed in a 45°C vacuum drying oven for 8 h to obtain a product with a mass of about 4.6 g, a yield of 92%, a purity of 99.471% as determined by HPLC, and an impurity C content of 0.368%.

Claims

1. A method for purifying the methotrexate intermediate shown in Formula I, comprising the following steps: (1) Dissolve the crude methotrexate intermediate in a solvent; (2) Add solid sodium chloride or aqueous sodium chloride solution to the solution in step (1) and stir to induce crystallization; (3) Filter, wash the filter cake with acetone, and dry under reduced pressure to obtain the purified product of the compound shown in Formula I; The solvent in step (1) is selected from water or an organic solvent / water mixture; the organic solvent / water mixture is selected from methanol / water mixture, ethanol / water mixture, acetone / water mixture, and isopropanol / water mixture. The mass ratio of the crude methotrexate intermediate to the volume ratio of the solvent in step (1) is 1:5 g / ml; The mass ratio of the sodium chloride solid in step (2) to the volume ratio of the solvent in step (1) is 1:4-1:6 g / ml; The sodium chloride aqueous solution in step (2) is a saturated sodium chloride aqueous solution, and the mass ratio of the saturated sodium chloride aqueous solution to the volume ratio of the solvent in step (1) is 3:1-6:1 g / ml.

2. The refining method according to claim 1, characterized in that, The volume ratio of organic solvent to water in the organic solvent / water mixture is (0.01:99.99) to (50:50).

3. The refining method according to claim 1, characterized in that, The solvent in step (1) is selected from water, methanol / water mixture or ethanol / water mixture.

4. The refining method according to claim 1, characterized in that, The refining temperature is 10-30℃.

5. The refining method according to claim 4, characterized in that, The refining temperature is 15-25℃.

6. The refining method according to claim 1, characterized in that, The stirring and crystallization time in step (2) is 0.5-2h.

7. The refining method according to claim 6, characterized in that, The stirring and crystallization time in step (2) is 1-1.5h.

8. The refining method according to claim 1, characterized in that, The temperature for vacuum drying in step (3) is 30-50℃.

9. The refining method according to claim 8, characterized in that, The temperature for vacuum drying in step (3) is 35-45℃.

10. According to the preparation method of claim 1, further, the compound of formula I can be hydrolyzed to convert into methotrexate.

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