A method for purifying a methotrexate intermediate

Abstract

The present invention discloses a method for refining methotrexate intermediate Int1, namely diethyl p-(methylamino)benzoylglutamate. The method comprises crystallizing the intermediate Int1 from a mixed solvent of an organic solvent and water. This refining method has a good effect on removing key impurity 1 and impurity 2 in diethyl p-(methylamino)benzoylglutamate, the methotrexate intermediate. The process operation is simple, the yield is high, and it is suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a method for refining an intermediate of methotrexate. Background Art

[0002] Methotrexate is an immunosuppressant that acts by competitive inhibition of dihydrofolate reductase. Since its discovery in the 1940s, it has been used to treat childhood leukemia, and since the 1950s, it has been used to treat psoriasis. Now it is a commonly used anti-cancer drug in clinical practice, mainly applicable to acute leukemia, breast cancer, choriocarcinoma and malignant mole, head and neck tumors, bone tumors, leukemia meningeal spinal cord infiltration, lung cancer, reproductive system tumors, liver cancer, intractable common psoriasis, rheumatoid arthritis / autoimmune diseases.

[0003] European Patent EP 0048001 A1 reported a method for preparing methotrexate, and its chemical reaction process is as follows:

[0004]

[0005] Currently, some domestic production enterprises use this method to synthesize methotrexate from intermediate Int1. At the same time, intermediate Int1 is also a key intermediate for synthesizing folic acid derivative products and plays an important role in the field of pharmaceutical chemistry. However, during the research process, it was found that key impurities 1 and 2 in intermediate Int1 would continuously derivatize into the finished product due to the similarity of their structures and reaction sites, and it was extremely difficult to remove them. It was necessary to reduce the impurity content to a narrow range to avoid risks and ensure that the finished product meets the pharmaceutical requirements. Therefore, based on the defects of the existing technology, it is urgent to invent a method for refining intermediate Int1 to control the quality at the front end of the process.

[0006] The inventor of the present invention found during the research process that by using a mixed solvent system of organic solvent and water to refine intermediate Int1, the contents of key impurities 1 and 2 in this intermediate can be significantly reduced, thereby improving the quality of the intermediate.

[0007] The structures of the two key impurities are as follows:

[0008] . Summary of the Invention

[0009] The purpose of the present invention is to provide a method for refining methotrexate intermediate Int1, which solves the technical problem that the existing technology cannot effectively remove impurities 1 and 2.

[0010] To achieve the purpose of the present invention, the following implementation schemes are provided.

[0011] A method for purifying methotrexate intermediate Int1 of the present invention is characterized in that the intermediate Int1 is crystallized in a mixed solvent of an organic solvent and water. The specific process includes:

[0012] (1) Adding methotrexate intermediate Int1 to a mixed solvent of an organic solvent and water, heating to dissolve, and cooling to crystallize to obtain high-quality methotrexate intermediate Int1; or

[0013] (2) Stirring and dissolving methotrexate intermediate Int1 with an organic solvent, then mixing with water and crystallizing to obtain high-quality methotrexate Int1;

[0014]

[0015] Among them, the organic solvent is selected from one or more of N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, acetonitrile, acetone, isopropanol, and methanol.

[0016] In the purification method, the volume ratio of the organic solvent to water is 1:2 to 1:20. Preferably, the volume ratio of the organic solvent to water is 1:3 to 1:10. The mass (g) / volume (ml) of intermediate Int1 to the organic solvent is 10:1 to 1:20. Preferably, the mass (g) / volume (ml) of intermediate Int1 to the organic solvent is 2:1 to 1:2.

[0017] In the above purification method, preferably, the organic solvent is selected from one or two of acetone, acetonitrile, and isopropanol.

[0018] In the above purification method, the dissolution temperature is 20 to 90 °C, preferably 50 to 70 °C; the crystallization temperature is -10 to 40 °C, preferably 20 to 30 °C; the crystallization time is 1 to 24 hours, preferably 1 to 4 hours.

[0019] In the above purification method, the mixing in process (2) means adding or dropping the dissolved solution into water, or adding water into or dropping it into the dissolved solution.

[0020] In the above purification method, process (2) includes the following two methods:

[0021] 1) Stirring and dissolving methotrexate intermediate Int1 with an organic solvent, then dropping water and crystallizing to obtain high-quality methotrexate Int1;

[0022] 2) Stirring and dissolving methotrexate intermediate Int1 with an organic solvent, then dropping it into water and crystallizing to obtain high-quality methotrexate intermediate Int1.

[0023] As a further improvement of the present invention, the HPLC purity of the intermediate Int1 before purification is not less than 95%.

[0024] The advantages of the method for purifying the methotrexate intermediate Int1 of the present invention are as follows:

[0025] It has excellent impurity removal effect. After purification, the content of key impurities (impurity 1 or impurity 2) in the intermediate Int1 is <0.15%, and the purity of the intermediate Int1 is >99.00%. On the basis of impurity removal, the present invention maintains a high yield, and the process is mild and the operation is simple. This purification method is suitable for industrial production. Brief Description of the Drawings

[0026] Figure 1 It is the HPLC chart of the Int1 intermediate after purification in Example 1,

[0027] Figure 2 It is of the key impurity 1 1 H NMR spectrum,

[0028] Figure 3 It is of the key impurity 2 1 H NMR spectrum. Detailed Description of the Invention

[0029] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate and understand the essence of the present invention, and are not used to limit the scope of the present invention. The reagents or instruments used without indicating the manufacturer can be conventional products purchased through the market. Example 1

[0030] The object processed in this example is the crude product of methotrexate intermediate Int1 (HPLC purity is 98.50%, impurity 1 is 0.27%, and impurity 2 is 0.22%). The purification process is as follows:

[0031] Step 1) Add 20.00 g of the crude product of methotrexate intermediate Int1 and 80 ml of a mixed solvent of acetone and water (V 丙酮 / V 水 = 1:4) to the reaction flask, start stirring, heat up to about 50 °C, and stir until dissolved to obtain a solution.

[0032] Step 2) Cool the solution obtained in Step 1 to about 25 °C, keep the temperature for crystallization for 1 hour. Filter, wash, and dry to obtain 18.85 g of methotrexate intermediate Int1, which is a white solid, and the yield is 94.25%.

[0033] The product purified in this example was detected by an HPLC-VWD detector. The detection chromatographic conditions are as follows:

[0034] Instrument: HPLC-VWD detector (Labsolution CS workstation);

[0035] Chromatographic column: YMC-Triart C18, 4.6 mm × 150 mm, 3 μm;

[0036] Detection wavelength: 230 nm;

[0037] Sample injection volume: 10 μl;

[0038] Flow rate: 1 ml / min;

[0039] Column temperature: 35 °C;

[0040] Sample tray temperature: 5 °C;

[0041] Mobile phase A: Phosphate buffer solution (pH 3.5) - acetonitrile (90:10)

[0042] Mobile phase B: Acetonitrile

[0043] Gradient elution:

[0044]

[0045] Figure 1 This is the HPLC chromatogram of the product obtained in this example. It can be seen from the chromatogram that the HPLC purity of the product of the present invention is 99.60%, impurity 1 is 0.10%, and impurity 2 is 0.10%.

[0046] Example 2

[0047] The purification process of the crude methotrexate intermediate Int1 in this example is as follows:

[0048] Step 1) Add 20.00 g of the crude methotrexate intermediate Int1 and 80 ml of a mixed solvent of acetonitrile and water (V 乙腈 / V 水 = 1:3) to the reaction flask, start stirring, heat up to about 70 °C, and stir to dissolve to obtain a solution.

[0049] Step 2) Cool the solution obtained in Step 1 to about 30 °C, keep the temperature for crystal precipitation for 1 hour. Filter, wash, and dry to obtain 18.47 g of methotrexate intermediate Int1, in the form of a white solid, with a yield of 92.33%.

[0050] The related substances of this product were detected under the same detection conditions as in Example 1, and the HPLC purity was 99.46%, impurity 1 was 0.13%, and impurity 2 was 0.12%.

[0051] Example 3

[0052] The purification process of the crude methotrexate intermediate Int1 in this example is as follows:

[0053] Step 1) Add 20.00 g of the crude methotrexate intermediate Int1, 60 ml of a mixed solvent of isopropanol and water (V 异丙醇 / V 水 = 1:3) into the reaction flask, start stirring, heat up to about 60 °C, and obtain a solution after stirring and dissolving.

[0054] Step 2) Cool the solution obtained in Step 1 to about 20 °C, keep the temperature for crystallization for 1 hour. Filter, wash, and dry to obtain 18.52 g of the methotrexate intermediate Int1, which is a white solid, and the yield is 92.61%.

[0055] Detect the related substances of this product under the same detection conditions as in Example 1, and obtain an HPLC purity of 99.52%, impurity 1 is 0.11%, and impurity 2 is 0.11%.

[0056] Example 4

[0057] The refining process of the crude methotrexate intermediate Int1 in this example is as follows:

[0058] Step 1) Add 150.00 g of the crude methotrexate intermediate Int1, 825 ml of a mixed solvent of acetone and water (V 丙酮 / V 水 = 1:10) into the reaction flask, start stirring, heat up to about 65 °C, and obtain a solution after stirring and dissolving.

[0059] Step 2) Cool the solution obtained in Step 1 to about 20 °C, keep the temperature for crystallization for 4 hours. Filter, wash, and dry to obtain 143.22 g of the methotrexate intermediate Int1, which is a white solid, and the yield is 95.48%.

[0060] Detect the related substances of this product under the same detection conditions as in Example 1, and obtain an HPLC purity of 99.67%, impurity 1 is 0.08%, and impurity 2 is 0.09%.

[0061] Example 5

[0062] The refining process of the crude methotrexate intermediate Int1 in this example is as follows:

[0063] Step 1) Add 20.00 g of the crude methotrexate intermediate Int1 and 20 ml of acetone into the reaction flask, start stirring, and obtain a solution after stirring and dissolving.

[0064] Step 2) Dropwise add 60 ml of water to the solution obtained in Step 1, and keep the temperature for crystallization for 1 hour at about 25 °C after dropping. Filter, wash, and dry to obtain 18.27 g of the methotrexate intermediate Int1, which is a white solid, and the yield is 91.34%.

[0065] The related substances of this product were detected under the same detection conditions as in Example 1, and the HPLC purity was 99.49%, impurity 1 was 0.11%, and impurity 2 was 0.12%.

[0066] Example 6

[0067] The refining process of the crude methotrexate intermediate Int1 in this example is as follows:

[0068] Step 1) Add 20.00 g of the crude methotrexate intermediate Int1 and 20 ml of acetonitrile to the reaction flask, start stirring, and obtain a solution after stirring and dissolving.

[0069] Step 2) Add 80 ml of water dropwise to the solution obtained in Step 1, and keep it at about 25 °C for 1 hour for crystallization. Filter, wash, and dry to obtain 18.67 g of the methotrexate intermediate Int1, which is a white solid, and the yield is 93.37%.

[0070] The related substances of this product were detected under the same detection conditions as in Example 1, and the HPLC purity was 99.43%, impurity 1 was 0.12%, and impurity 2 was 0.12%.

[0071] Example 7

[0072] The refining process of the crude methotrexate intermediate Int1 in this example is as follows:

[0073] Step 1) Add 20.00 g of the crude methotrexate intermediate Int1 and 20 ml of isopropanol to the reaction flask, start stirring, and obtain a solution after stirring and dissolving.

[0074] Step 2) Add 60 ml of water dropwise to the solution obtained in Step 1, and keep it at about 25 °C for 1 hour for crystallization. Filter, wash, and dry to obtain 18.44 g of the methotrexate intermediate Int1, which is a white solid, and the yield is 92.18%.

[0075] The related substances of this product were detected under the same detection conditions as in Example 1, and the HPLC purity was 99.47%, impurity 1 was 0.12%, and impurity 2 was 0.12%.

[0076] Example 8

[0077] The refining process of the crude methotrexate intermediate Int1 in this example is as follows:

[0078] Step 1) Add 20.00 g of the crude methotrexate intermediate Int1 and 40 ml of acetone to the reaction flask, start stirring, and obtain a solution after stirring and dissolving.

[0079] Step 2) The solution obtained in Step 1) was added dropwise to 400 ml of water. After the addition was complete, the mixture was kept at about 25 °C for 1 hour for crystallization. It was filtered, washed, and dried to obtain 18.39 g of methotrexate intermediate Int1, which was a white solid with a yield of 91.97%.

[0080] Using the same detection conditions as in Example 1 to detect the related substances of this product, the HPLC purity was 99.45%, impurity 1 was 0.11%, and impurity 2 was 0.11%.

[0081] Example 9

[0082] The purification process of the crude methotrexate intermediate Int1 in this example is as follows:

[0083] Step 1) 20.00 g of the crude methotrexate intermediate Int1 and 10 ml of acetonitrile were added to a reaction flask, and stirring was started. After stirring and dissolving, a solution was obtained.

[0084] Step 2) The solution obtained in Step 1) was added dropwise to 80 ml of water. After the addition was complete, the mixture was kept at about 25 °C for 1 hour for crystallization. It was filtered, washed, and dried to obtain 18.58 g of methotrexate intermediate Int1, which was a white solid with a yield of 92.88%.

[0085] Using the same detection conditions as in Example 1 to detect the related substances of this product, the HPLC purity was 99.42%, impurity 1 was 0.11%, and impurity 2 was 0.13%.

[0086] Example 10

[0087] The purification process of the crude methotrexate intermediate Int1 in this example is as follows:

[0088] Step 1) 20.00 g of the crude methotrexate intermediate Int1 and 30 ml of isopropanol were added to a reaction flask, and stirring was started. After stirring and dissolving, a solution was obtained.

[0089] Step 2) The solution obtained in Step 1) was added dropwise to 200 ml of water. After the addition was complete, the mixture was kept at about 25 °C for 1 hour for crystallization. It was filtered, washed, and dried to obtain 18.41 g of methotrexate intermediate Int1, which was a white solid with a yield of 92.06%.

[0090] Using the same detection conditions as in Example 1 to detect the related substances of this product, the HPLC purity was 99.41%, impurity 1 was 0.13%, and impurity 2 was 0.11%.

[0091] Example 11

[0092] The purification process of the crude methotrexate intermediate Int1 in this example is as follows:

[0093] Step 1) Add 20.00 g of the crude methotrexate intermediate Int1, 200 ml of a mixed solvent of acetone, isopropanol and water (V 丙酮 / V 异丙醇 / V 水 = 1:1:8) into the reaction flask. Start stirring and heat up to about 60 °C. After stirring and dissolving, a solution is obtained.

[0094] Step 2) Cool the solution obtained in Step 1 to about 25 °C and keep it for crystallization for 1 hour. Filter, wash and dry to obtain 18.63 g of the methotrexate intermediate Int1, in the form of a white solid, with a yield of 93.15%.

[0095] Detect the related substances of this product under the same detection conditions as in Example 1. The HPLC purity is 99.50%, impurity 1 is 0.10%, and impurity 2 is 0.10%.

[0096] Example 12

[0097] The refining process of the crude methotrexate intermediate Int1 in this example is as follows:

[0098] Step 1) Add 20.00 g of the crude methotrexate intermediate Int1, 10 ml of acetone and 10 ml of acetonitrile into the reaction flask. Start stirring and after stirring and dissolving, a solution is obtained.

[0099] Step 2) Dropwise add 60 ml of water to the solution obtained in Step 1. After dropping, keep it for crystallization at about 25 °C for 1 hour. Filter, wash and dry to obtain 18.55 g of the methotrexate intermediate Int1, in the form of a white solid, with a yield of 92.76%.

[0100] Detect the related substances of this product under the same detection conditions as in Example 1. The HPLC purity is 99.48%, impurity 1 is 0.11%, and impurity 2 is 0.12%.

[0101] Comparative Example 1

[0102] The refining process of the crude methotrexate intermediate Int1 in this example is as follows:

[0103] Step 1) Add 20.00 g of the crude methotrexate intermediate Int1, 60 ml of a mixed solvent of ethanol and water (V 乙醇 / V 水 = 1:3) into the reaction flask. Start stirring and heat up to about 60 °C. After stirring and dissolving, a solution is obtained.

[0104] Step 2) Cool the solution obtained in Step 1) to about 25 °C, keep the temperature for crystallization for 1 hour. Filter, wash, and dry to obtain 18.47 g of methotrexate intermediate Int1, in the form of a white solid, with a yield of 92.33%.

[0105] Detect the related substances of this product under the same detection conditions as in Example 1, and obtain an HPLC purity of 98.62%, impurity 1 being 0.23%, and impurity 2 being 0.20%.

[0106] The quality comparisons of the examples, comparative examples and the crude product of methotrexate intermediate Int1 are shown in Table 1.

[0107] Table 1 Quality comparison of the examples and the crude product of methotrexate intermediate Int1

[0108]

[0109] As can be seen from Table 1, for the methotrexate intermediate Int1 prepared by the purification method of the present invention, impurity 1 is 0.08% - 0.13%, impurity 2 is 0.09% - 0.13%, and the HPLC purity is 99.41% - 99.67%, having a good impurity removal effect. At the same time, the yield is 91.34% - 95.48%, the process is mild, the operation is simple, and it is convenient for large-scale application.

[0110] The functions of the above examples are to illustrate the substantial content of the present invention, but do not limit the protection scope of the present invention. Those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and protection scope of the technical solutions of the present invention.

Claims

1. A method for purifying methotrexate intermediate Int1, characterized in that it crystallizes in a mixed solvent of an organic solvent and water, and the specific process includes: (1) adding methotrexate intermediate Int1 to a mixed solvent of an organic solvent and water, heating to dissolve, and cooling to crystallize to obtain high-quality methotrexate intermediate Int1; or (2) stirring and dissolving methotrexate intermediate Int1 with an organic solvent, then mixing with water and crystallizing to obtain high-quality methotrexate Int1; ， the organic solvent is selected from one or two of acetonitrile, acetone and isopropanol.

2. The purification method according to claim 1, wherein the volume ratio of the organic solvent to water is 1:2 to 1:

20.

3. The purification method according to claim 2, wherein the volume ratio of the organic solvent to water is 1:3 to 1:

10.

4. The purification method according to claim 1, wherein the mass (g) / volume (ml) of intermediate Int1 to the organic solvent is 10:1 to 1:

20.

5. The purification method according to claim 4, wherein the mass (g) / volume (ml) of intermediate Int1 to the organic solvent is 2:1 to 1:

2.

6. The purification method according to claim 1, wherein the dissolution temperature is 20 to 90 °C.

7. The purification method according to claim 6, wherein the dissolution temperature is 50 to 70 °C.

8. The purification method according to claim 1, wherein the crystallization temperature is -10 to 40 °C.

9. The purification method according to claim 8, wherein the crystallization temperature is 20 to 30 °C.

10. The purification method according to claim 1, wherein the crystallization time is 1 to 24 hours.

11. The purification method according to claim 10, wherein the crystallization time is 1 to 4 hours.

12. The purification method according to claim 1, wherein the mixing in process (2) means adding or dropping the dissolved solution into water, or adding water into or dropping water into the dissolved solution.

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