Melphalan hydrochloride preparation method for removing Melphalan-D-isomer impurities

Abstract

The invention belongs to the field of medicine production, and particularly relates to a melphalan hydrochloride preparation method for removing melphalan-D-isomer impurities, which comprises the following steps of: synthesizing N, N-phthaloyl-4-(dihydroxyethyl amino)-L-phenylalanine ethyl ester, synthesizing N, N-phthaloyl-4-(dihydroxyethyl amino)-L-phenylalanine ethyl ester, synthesizing N, N-phthaloyl-4-(dihydroxyethyl amino)-L- The method comprises the steps of synthesizing N, N-phthaloyl melphalan ethyl ester, preparing melphalan free alkali, crystallizing melphalan hydrochloride, drying and the like, and a tartaric acid aqueous solution is added into a hydrochloric acid-isopropanol crystallization system, so that melphalan-D-isomer impurities are effectively removed, the problem of the isomer impurities is solved, and the purity of the melphalan-D-isomer impurities is improved. The quality of the melphalan hydrochloride product is obviously improved, and the safety risk is reduced.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical production, and particularly relates to a method for preparing melphalan hydrochloride by removing melphalan-D-isomer impurities. Background Art

[0002] Melphalan hydrochloride is widely used and can be used for multiple myeloma, breast cancer, ovarian cancer, chronic lymphocytic and myeloid leukemia, malignant lymphoma, and multiple myeloma. Its chemical structure is shown below:

[0003]

[0004] Melphalan hydrochloride is synthesized from 4-nitro-L-phenylalanine as the raw material. It is converted into melphalan through six steps, including amino protection, carboxyl protection, nitro reduction, epoxy addition, chlorination, and deprotection. During the removal of the amino protecting group, a side reaction occurs in which the amino stereo configuration changes, generating the melphalan-D-isomer impurity. Its structural formula is shown below:

[0005]

[0006] However, testing has revealed that the melphalan-D-isomer impurity is detected in the final product using existing production methods. The document "Melphalan Review Report EMA / CHMP / 624882 / 2020 - European Medicines Agency - ScienceMedicinesHealth" clearly states that due to the presence of a chiral center, melphalan exhibits stereoisomerism, with the active substance in the S(L) configuration. This melphalan-D-isomer impurity has different biological activity from melphalan, posing a safety risk. However, existing production methods do not control the melphalan-D-isomer impurity. This is presumably because the impurity is mirror-image symmetrical to the melphalan hydrochloride molecule, its various physical and chemical properties are nearly identical to those of melphalan hydrochloride, and its chromatographic peaks completely overlap with those of melphalan on a C18 column, leading to its overlooking. This impurity can only be detected using a chiral column. However, the melphalan-D-isomer impurity differs from melphalan hydrochloride in stereoisomerism and biological activity, posing a safety risk. Therefore, its content should be minimized.

[0007] US Patent No. 3032584A discloses a method for synthesizing melphalan using 4-nitro-L-phenylalanine as the raw material. This process uses 4-nitro-L-phenylalanine as the raw material to synthesize N,N-phthaloyl-4-amino-L-phenylalanine ethyl ester, which is then subjected to ethylene oxide addition, chlorination, and deprotection reactions to synthesize melphalan. Hongzhong's melphalan hydrochloride process uses N,N-phthaloyl-4-amino-L-phenylalanine ethyl ester, which is also synthesized through ethylene oxide addition, chlorination, and deprotection reactions. Melphalan is then crystallized from the isoelectric point, dissolved in concentrated hydrochloric acid and filtered to remove inorganic salts, and crystallized using hydrochloric acid-tartaric acid-isopropyl alcohol.

[0008] "CN109422660A" discloses a method for preparing melphalan hydrochloride crystal forms, which comprises mixing melphalan with N,N-dimethylformamide and methyl tert-butyl ether, heating to a temperature not higher than 80°C, adding a hydrochloric acid aqueous solution and stirring to react, and finally preparing two melphalan hydrochloride crystal forms.

[0009] Early experiments tried the process routes of US3032584A, CN109422660A and CN105646258A, and melphalan-D-isomer impurities were detected in the crystalline products. However, the relevant literature did not mention the control methods of isomer impurities. Summary of the Invention

[0010] In order to solve the problem of melphalan-D-isomer impurities generated during the synthesis of melphalan hydrochloride, the melphalan hydrochloride preparation process provided by the present invention can effectively reduce the content of melphalan-D-isomer impurities in the final product and reduce safety risks.

[0011] The specific technical solution adopted is: a method for preparing melphalan hydrochloride, comprising the following steps:

[0012] Step 1. Dissolve N,N-phthaloyl-4-amino-L-phenylalanine ethyl ester in an aqueous acetic acid solution, add ethylene oxide dropwise at 0-5°C, and after the addition is complete, heat to 20-25°C and react until the remaining amount of the raw material is less than 0.5%. Then, concentrate under reduced pressure to remove ethylene oxide, neutralize, and crystallize to obtain N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester;

[0013] Step 2. Dissolve the N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester prepared in Step 1 in toluene. Under nitrogen protection, slowly add phosphorus oxychloride dropwise with stirring. After the addition is complete, reflux for 4 hours under heating conditions at an external temperature of 110-120°C. After reflux, cool the reaction solution and concentrate until no liquid drips out to obtain N,N-phthaloyl melphalan ethyl ester.

[0014] Step 3. Dissolve the N,N-phthaloyl melphalan ethyl ester prepared in Step 2 in hydrochloric acid as a solvent, and stir and reflux under heating conditions at an external temperature of 110-120°C until the remaining melphalan ethyl ester is less than 0.5%. Cool the mixture, add activated carbon, stir, and filter. Add purified water and diethyl ether to the filtrate, control the internal temperature at 0-3°C, add sodium acetate aqueous solution dropwise until the pH reaches 5-6, and filter to obtain melphalan free base containing melphalan-D-isomer impurity.

[0015] Step 4. Dissolve the melphalan free base containing the melphalan-D-isomer impurity obtained in step 3 in concentrated hydrochloric acid as a solvent. After filtering, add an aqueous tartaric acid solution to the filtrate, wherein the tartaric acid is L(+)-tartaric acid. After cooling, add isopropanol dropwise at 0-10° C. for crystallization. Filter the resulting filter cake and dry it at 25-35° C. under a vacuum degree of ≥-0.095 MPa to obtain melphalan hydrochloride.

[0016] Moreover, the conditions for concentrating and removing ethylene oxide in step 1 are a temperature of 20 to 30° C., a vacuum of ≥0.08 MPa, and reduced pressure concentration for 2.5 h; the conditions for neutralization and crystallization are adding water and dichloromethane to the concentrate, cooling to 5 to 10° C., adding sodium bicarbonate dropwise to a pH of pH 6 to 8, and separating the organic phase to dry with anhydrous sodium sulfate, filtering, and concentrating under reduced pressure.

[0017] Furthermore, after refluxing in step 2, the reaction liquid is cooled to below 70° C., and then concentrated under an external temperature of 50 to 70° C. and a vacuum of ≥0.08 MPa until no liquid drops out.

[0018] Moreover, in step 3, the solvent is 9 mol / L hydrochloric acid; after adding sodium acetate aqueous solution dropwise, the filter cake obtained is filtered, slurried with deionized water, and filtered again to obtain melphalan free base containing melphalan-D-isomer impurity.

[0019] Furthermore, the amount of hydrochloric acid solvent used in step 4 is 1.5-1.6 mL / g melphalan free base.

[0020] Moreover, in step 4, the concentration of the tartaric acid aqueous solution is 1.8-2.1 g / L, and the volume ratio of the tartaric acid aqueous solution to the concentrated hydrochloric acid solution is (0.31-0.35):1.

[0021] Moreover, the volume ratio of isopropyl alcohol to concentrated hydrochloric acid is (11.3-11.8):1.

[0022] Compared with the existing technology, the beneficial effects of this technical solution are:

[0023] This method includes the synthesis of N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester, N,N-phthaloyl melphalan ethyl ester, melphalan free base, melphalan hydrochloride crystallization, and drying. The overall synthesis route incorporates portions of US Patent No. 3032584A. During the melphalan hydrochloride crystallization process, it was unexpectedly discovered that the addition of tartaric acid (specifically, L(+)-tartaric acid) to the hydrochloric acid-isopropyl alcohol crystallization system effectively removed the melphalan-D-isomer impurity, resolving the isomer impurity issue, significantly improving the quality of the melphalan hydrochloride product, and reducing safety risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The reaction formula for the synthesis of N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester in step 1;

[0025] Figure 2 is the reaction formula for the synthesis of N,N-phthaloyl melphalan ethyl ester in step 2;

[0026] Figure 3 This is the reaction formula for synthesizing mephalan free base in step 3;

[0027] Figure 4 is the synthesis reaction formula of melphalan hydrochloride in step 4;

[0028] Figure 5 HPLC spectrum of melphalan-D-isomer impurity detection of melphalan free base prepared in Example 1;

[0029] Figure 6 HPLC spectrum of related substance detection of melphalan free base prepared in Example 1;

[0030] Figure 7 This is the HPLC spectrum of melphalan-D-isomer impurity detection of melphalan hydrochloride prepared in Example 3;

[0031] Figure 8 HPLC spectrum of melphalan-related substances detection of melphalan hydrochloride prepared in Example 3;

[0032] Figure 9 HPLC spectrum of melphalan-D-isomer impurity detection of melphalan hydrochloride prepared in Comparative Example 1;

[0033] Figure 10 This is the HPLC spectrum of melphalan-related substances detection of melphalan hydrochloride prepared in Comparative Example 1. DETAILED DESCRIPTION

[0034] The present invention will be described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited to the following embodiments.

[0035] The specific testing method of the obtained product is as follows:

[0036] 1. Melphalan-D-isomer impurity test method:

[0037]

[0038] Mobile phase: n-hexane / anhydrous ethanol / dichloromethane / trifluoroacetic acid / triethylamine = 80:10:10:0.3:0.1 (V / V / V / V / V)

[0039] Test solution: Accurately weigh 25 mg of the sample to be tested, place it in a 25 ml volumetric flask, first add 5 ml of diluent to dissolve it, then add mobile phase to dilute to the scale and mix well.

[0040] 2. Test methods for related substances of melphalan hydrochloride:

[0041]

[0042] Buffer: acetic acid: triethylamine: ammonium acetate: water = 10:2:10:88 (V / V / W / V)

[0043] Mobile phase A: buffer: acetonitrile: water = 10:100:1900 (V / V / V)

[0044] Mobile phase B: buffer: acetonitrile: water = 10:1200:800 (V / V / V)

[0045] Elution gradient:

[0046]

[0047]

[0048] Test solution: Accurately weigh 20 mg of melphalan hydrochloride, place in a 100 ml brown volumetric flask, add methanol to dissolve and dilute to the scale, and mix well.

[0049] The principle and steps of the method for preparing melphalan hydrochloride by removing the melphalan-D-isomer impurity provided in this scheme are as follows:

[0050] Step 1. Synthesis of N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester, reaction formula: Figure 1 shown.

[0051] Dissolve N,N-phthaloyl-4-amino-L-phenylalanine ethyl ester in aqueous acetic acid solution, stir, cool to 0-5°C, add ethylene oxide aqueous solution dropwise, raise the temperature to 20-25°C after the addition is complete, continue stirring, react until the remaining raw material is less than 0.5%, control the temperature at 25±5°C, and concentrate under reduced pressure at a vacuum of ≥0.08 MPa for 2.5 hours to remove ethylene oxide, add water and dichloromethane to the concentrate, cool to 5-10°C, adjust the pH to 6-8 with sodium bicarbonate, separate the liquids, dry the organic phase over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester.

[0052] Step 2. Synthesis of N,N-phthaloyl melphalan ethyl ester, reaction formula: Figure 2 shown.

[0053] Dissolve N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester in toluene at an external temperature of 40-60°C. Under nitrogen protection, slowly add phosphorus oxychloride dropwise with stirring. After the addition is complete, turn on the heat and reflux at an external temperature of 115±5°C for 4 hours. After reflux is complete, cool the reaction solution to below 70°C and then concentrate the reaction solution at an external temperature of 60±10°C under a vacuum of ≥0.08 MPa until no liquid drips out to obtain N,N-phthaloyl melphalan ethyl ester.

[0054] Step 3. Preparation of melphalan free base, reaction formula is as follows: Figure 3 shown.

[0055] Dissolve N,N-phthaloyl melphalan ethyl ester in 9 mol / L hydrochloric acid, stir and reflux at an external temperature of 105±5°C until the remaining melphalan ethyl ester is less than 0.5%, cool to below 70°C, add activated carbon, maintain stirring, continue to cool to 0-3°C and stir for 60 minutes, filter, add purified water and ether to the filtrate, control the internal temperature at 0-3°C, add sodium acetate aqueous solution dropwise to pH 5-6, filter, slurry the filter cake with deionized water, filter to obtain melphalan free base.

[0056] Step 4. Melphalan Hydrochloride Crystallization and Drying

[0057] Melphalan free base was dissolved in concentrated hydrochloric acid and filtered. Tartaric acid aqueous solution was added to the filtrate, wherein the tartaric acid was L(+)-tartaric acid. The temperature was lowered to 5±5°C, and then isopropanol was added dropwise for crystallization. The mixture was filtered and the filter cake was dried under vacuum at 30±5°C with a vacuum degree of ≥-0.095 MPa for 12 h to obtain melphalan hydrochloride.

[0058] Furthermore, the amount of hydrochloric acid used in step 4 is 1.5-1.6 mL / g melphalan free base. The concentration of the tartaric acid aqueous solution is 1.8-2.1 g / L, and the volume ratio of the tartaric acid aqueous solution to the concentrated hydrochloric acid solution is (0.31-0.35):1. The volume ratio of isopropyl alcohol to concentrated hydrochloric acid is (11.3-11.8):1.

[0059] Example 1: Preparation of melphalan free base according to this method

[0060] 200 g of N,N-phthaloyl-4-amino-L-phenylalanine ethyl ester was dissolved in 1240 g of a 68% acetic acid aqueous solution by stirring, and the temperature was lowered to 0-5° C. 520 g of a 25% ethylene oxide aqueous solution precooled to 0-5° C. was added dropwise. After the addition was complete, the temperature was raised to 20-25° C. and stirring was continued. The reaction was continued until the remaining raw material was less than 0.5%. The temperature was controlled at 25±5° C. and a vacuum of ≥0.08 MPa for 2.5 h to remove the ethylene oxide. 1000 mL of deionized water and 1000 mL of dichloromethane were added to the concentrate, and the temperature was lowered to 5-10° C. The pH was adjusted to 6-8 with sodium bicarbonate. The liquids were separated, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester, weighing approximately 260 g and having a purity of 97%.

[0061] The above-mentioned N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester was dissolved in 1500 mL of toluene at an external temperature of 40-60°C. Under nitrogen protection, 440 g of phosphorus oxychloride was slowly added dropwise with stirring. After the addition was complete, the mixture was heated to reflux at an external temperature of 115±5°C for 4 hours. After reflux, the reaction solution was cooled to below 70°C and then concentrated at an external temperature of 60±10°C under a vacuum of ≥0.08 MPa until no more liquid dripped out, to obtain N,N-phthaloyl melphalan ethyl ester weighing 374 g with a purity of 94.13%.

[0062] The above-mentioned N,N-phthaloyl melphalan ethyl ester was dissolved in 600 mL of 9 mol / L hydrochloric acid, stirred and refluxed at an external temperature of 105±5°C until the remaining melphalan ethyl ester was less than 0.5%, cooled to below 70°C, 30 g of activated carbon was added, stirring was maintained, and the temperature was further cooled to 0-3°C and stirred for 60 min. The mixture was filtered, and 600 mL of deionized water and 800 mL of ether were added to the filtrate. The internal temperature was controlled at 0-3°C, and a 32% sodium acetate aqueous solution was added dropwise to a pH of 5-6. The mixture was filtered, and the filter cake was slurried with 1600 mL of deionized water for 30 min. The mixture was filtered to obtain melphalan free base, about 196 g, with a purity of 97.45%. The HPLC spectrum of the related substances of the prepared melphalan free base is shown as follows: Figure 6After testing melphalan free base using the aforementioned test method, it was found that the product contained 2.57% of melphalan-D-isomer impurity. The HPLC spectrum of melphalan-D-isomer impurity detection of the prepared melphalan free base is shown as follows: Figure 5 shown.

[0063] Example 2

[0064] 50 g of melphalan free base prepared in Example 1 was dissolved in 75 mL of concentrated hydrochloric acid and filtered through a 0.45 μm filter membrane. 23.3 mL of a 1.8 g / L aqueous tartaric acid solution was added to the filtrate, and the temperature was cooled to 0°C. 885 mL of isopropyl alcohol was then added dropwise, maintained at 0°C, and stirred for 2 h. The mixture was filtered, and the filter cake was vacuum dried at 30°C with a vacuum of ≥-0.095 MPa for 12 h to yield 35.4 g of melphalan hydrochloride with a purity of 99.71%. The product was tested using the aforementioned assay method, revealing only 0.08% of the melphalan-D-isomer impurity.

[0065] Example 3

[0066] 50 g of melphalan free base prepared in Example 1 was dissolved in 80 mL of concentrated hydrochloric acid and filtered through a 0.45 μm filter membrane. 28 mL of 2.1 g / L tartaric acid aqueous solution was added to the filtrate, and the temperature was lowered to 10° C. Then, 904 mL of isopropanol was added dropwise, maintained at 10° C., and stirred for 2 h. The filter cake was dried under vacuum at 30° C. with a vacuum degree of ≥-0.095 MPa for 12 h to obtain 30.2 g of melphalan hydrochloride with a purity of 99.85%. The HPLC spectrum of the related substances of the prepared melphalan hydrochloride is as follows: Figure 8 As shown. The product was tested by the above-mentioned test method. The melphalan-D-isomer impurity in the product only contained 0.06%, and the actual required melphalan hydrochloride accounted for 98.83% of the product. The melphalan-D-isomer impurity detection HPLC spectrum of the prepared melphalan hydrochloride is shown as follows Figure 7 shown.

[0067] Comparative Example 1

[0068] Take 50g of melphalan free base prepared in Example 1, dissolve it with 75mL of concentrated hydrochloric acid, filter it with a 0.45μm filter membrane, add 24mL of water to the filtrate, cool it to 0℃, then add 885mL of isopropanol dropwise, maintain it at 0℃, continue stirring for 2h, filter it, and dry the filter cake in vacuum at 30℃ with a vacuum degree of ≥-0.095MPa for 12h to obtain 35.9g of melphalan hydrochloride. The HPLC spectrum of the related substances of the prepared melphalan hydrochloride is as follows: Figure 10 As shown, the purity is 99.65%. However, the obtained product was tested and found to contain 2.47% of the melphalan-D-isomer impurity, and the actual required melphalan hydrochloride accounted for 96.44% of the product. The HPLC spectrum of the melphalan-D-isomer impurity test of the prepared melphalan hydrochloride is shown as follows: Figure 9 shown.

[0069] Comparison of experimental results revealed that the impurity content of 2.47% in the melphalan free base of Comparative Example 1, which was not treated with aqueous tartaric acid, was not significantly different from the impurity content of 2.57% in the melphalan free base prepared in Example 1. However, the impurity content of the melphalan free base products of Examples 2 and 3, treated with tartaric acid in the hydrochloric acid-isopropanol crystallization system, was only 0.08% and 0.06%, respectively, significantly reducing the impurity content.

Claims

1. A method for preparing melphalan hydrochloride by removing melphalan-D-isomer impurities, characterized in that The following steps are involved: Step 1. Dissolve N,N-phthaloyl-4-amino-L-phenylalanine ethyl ester in an aqueous acetic acid solution, add ethylene oxide dropwise at 0-5°C, and after the addition is complete, heat to 20-25°C and react until the remaining amount of the raw material is less than 0.5%. Then, concentrate under reduced pressure to remove ethylene oxide, neutralize, and crystallize to obtain N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester; Step 2. Dissolve the N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester prepared in Step 1 in toluene. Under nitrogen protection, slowly add phosphorus oxychloride dropwise with stirring. After the addition is complete, reflux for 4 hours under heating conditions at an external temperature of 110-120°C. After reflux, cool the reaction solution and concentrate until no liquid drips out to obtain N,N-phthaloyl melphalan ethyl ester. Step 3. Dissolve the N,N-phthaloyl melphalan ethyl ester prepared in Step 2 in hydrochloric acid as a solvent, and stir and reflux under heating conditions at an external temperature of 110-120°C until the remaining melphalan ethyl ester is less than 0.5%. Cool the mixture, add activated carbon, stir, and filter. Add purified water and diethyl ether to the filtrate, control the internal temperature at 0-3°C, add sodium acetate aqueous solution dropwise until the pH reaches 5-6, and filter to obtain melphalan free base containing melphalan-D-isomer impurity. Step 4. Dissolve the melphalan free base containing the melphalan-D-isomer impurity obtained in step 3 in concentrated hydrochloric acid as a solvent. After filtering, add an aqueous tartaric acid solution to the filtrate, wherein the tartaric acid is L(+)-tartaric acid. After cooling, add isopropanol dropwise at 0-10° C. for crystallization. Filter the resulting filter cake and dry it at 25-35° C. under a vacuum degree of ≥-0.095 MPa to obtain melphalan hydrochloride.

2. The method for preparing melphalan hydrochloride by removing melphalan-D-isomer impurities according to claim 1, characterized in that: The conditions for concentrating and removing ethylene oxide in step 1 are a temperature of 20-30°C, a vacuum of ≥0.08 MPa, and reduced pressure concentration for 2.5 hours; the conditions for neutralization and crystallization are adding water and dichloromethane to the concentrate, cooling to 5-10°C, adding sodium bicarbonate dropwise to a pH of pH 6-8, separating the organic phase, drying it with anhydrous sodium sulfate, filtering it, and concentrating it under reduced pressure.

3. The method for preparing melphalan hydrochloride by removing melphalan-D-isomer impurities according to claim 1, characterized in that: After refluxing in step 2, the reaction liquid is cooled to below 70° C., and then concentrated at an external temperature of 50 to 70° C. and a vacuum of ≥0.08 MPa until no liquid drops out.

4. The method for preparing melphalan hydrochloride by removing melphalan-D-isomer impurities according to claim 1, characterized in that: In step 3, the solvent is 9 mol / L hydrochloric acid; after adding sodium acetate aqueous solution dropwise, the obtained filter cake is filtered, slurried with deionized water, and filtered again to obtain melphalan free base containing melphalan-D-isomer impurity.

5. The method for preparing melphalan hydrochloride by removing melphalan-D-isomer impurities according to claim 1, characterized in that: The amount of hydrochloric acid solvent used in step 4 is 1.5-1.6 mL / g melphalan free base.

6. The method for preparing melphalan hydrochloride by removing melphalan-D-isomer impurities according to claim 5, characterized in that: In step 4, the concentration of the tartaric acid aqueous solution is 1.8-2.1 g / L, and the volume ratio of the tartaric acid aqueous solution to the concentrated hydrochloric acid solution is (0.31-0.35):

1.

7. The method for preparing melphalan hydrochloride by removing the melphalan-D-isomer impurity according to claim 5, characterized in that: The volume ratio of isopropyl alcohol to concentrated hydrochloric acid is (11.3-11.8):1.

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