A method for preparing melphalan hydrochloride free from melphalan-d-isomer impurities

Abstract

The application belongs to the field of medicine production, and particularly relates to a preparation method of melphalan hydrochloride for removing melphalan-D-isomer impurities, which comprises the following steps: synthesizing N,N-o-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester, synthesizing N,N-o-phthaloyl melphalan ethyl ester, preparing melphalan free base, crystallizing melphalan hydrochloride and drying, and adding a tartaric acid aqueous solution into a hydrochloric acid-isopropyl alcohol crystallization system, which can effectively remove melphalan-D-isomer impurities, solve the problem of the isomer impurities, and significantly improve the product quality of melphalan hydrochloride and reduce the safety risk.

Description

Technical Field

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

[0002] Melphalan hydrochloride has a wide range of applications and can be used to treat multiple myeloma, breast cancer, ovarian cancer, chronic lymphocytic and granulocytic leukemia, malignant lymphoma, and multiple myeloma. Its chemical structure is shown below:

[0003]

[0004] The synthesis of melphalan hydrochloride begins with 4-nitro-L-phenylalanine, involving six steps: amino protection, carboxyl protection, nitro reduction, epoxide addition, chlorination, and deprotection. During the removal of the amino protecting group, a side reaction occurs that alters the stereoconfiguration of the amino group, generating the melphalan-D-isomer impurity, the structural formula of which is shown below:

[0005]

[0006] However, experiments have revealed that the melphalan-D-isomer impurity is detected in the final product when produced using existing methods. The literature "Melphalan Review Report EMA / CHMP / 624882 / 2020 - European Medicines Agency - ScienceMedicines Health" clearly states that due to the presence of a chiral center, melphalan exhibits stereoisomerism, with the active substance configured in S(L). This melphalan-D-isomer impurity has different biological activities than melphalan and poses a safety risk. Currently, existing formulations do not control for the melphalan-D-isomer impurity. The presumed reason is that this impurity is mirror-symmetric to the melphalan hydrochloride molecule, its various physicochemical properties are almost identical to those of melphalan hydrochloride, and its chromatographic peaks completely overlap with melphalan on a C18 column, thus it is overlooked. This impurity can only be detected using a chiral column. However, given that the melphalan-D-isomer impurity has a different stereoconfiguration and biological activity than melphalan hydrochloride and poses a safety risk, its content should be minimized.

[0007] US3032584A discloses a method for synthesizing melphalan using 4-nitro-L-phenylalanine as a starting material. The process involves synthesizing N,N-phthaloyl-4-amino-L-phenylalanine ethyl ester from 4-nitro-L-phenylalanine, followed by ethylene oxide addition, chlorination, and deprotection reactions to synthesize melphalan.

[0008] CN109422660A discloses a method for preparing melphalan hydrochloride crystal forms. Melphalan is mixed with N,N-dimethylformamide and methyl tert-butyl ether, heated to a temperature not exceeding 80°C, and hydrochloric acid aqueous solution is added and stirred to react, ultimately preparing two melphalan hydrochloride crystal forms.

[0009] Previous experiments explored the process routes described in US3032584A, CN109422660A, and CN105646258A, and melphalan-D-isomer impurities were detected in the crystalline products. However, none of the relevant literature mentioned methods for controlling isomer impurities. Summary of the Invention

[0010] To address the issue of melphalan-D-isomer impurities generated during the synthesis of melphalan hydrochloride, the melphalan hydrochloride preparation process provided by this invention can effectively reduce the content of melphalan-D-isomer impurities in the final product, thereby lowering safety risks.

[0011] The specific technical solution adopted is: a method for preparing melphalan hydrochloride, including 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, raise the temperature to 20-25°C to react until the remaining amount of 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 obtained in Step 1 in toluene as solvent. Under nitrogen protection, stir and slowly add phosphorus oxychloride. After the addition is complete, reflux for 4 hours at an external temperature of 110-120°C. After reflux, cool the reaction solution and concentrate until no more liquid drips out to obtain N,N-phthaloyl melphalan ethyl ester.

[0014] Step 3. Dissolve the N,N-phthaloyl melphalan ethyl ester obtained in Step 2 in hydrochloric acid. 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 down, add activated carbon and stir, filter, add purified water and diethyl ether to the filtrate, and add sodium acetate aqueous solution dropwise to the pH of 5-6 while controlling the internal temperature at 0-3°C. Filter to obtain melphalan free base containing melphalan-D-isomer impurities.

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

[0016] Furthermore, the conditions for concentrating and removing ethylene oxide in step 1 are a temperature of 20–30°C and vacuum concentration at ≥0.08 MPa for 2.5 h; the conditions for neutralization and crystallization are to add water and dichloromethane to the concentrate, cool to 5–10°C, add sodium bicarbonate dropwise until the pH is 6–8, dry the organic phase obtained by separation with anhydrous sodium sulfate, filter, and concentrate under reduced pressure.

[0017] Furthermore, after reflux in step 2, the reaction solution is cooled to below 70°C, and then concentrated under external temperature of 50-70°C and vacuum ≥0.08MPa until no more droplets are expelled.

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

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

[0020] Furthermore, 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 isopropanol to concentrated hydrochloric acid is (11.3~11.8):1.

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

[0023] This method includes the synthesis of N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester, the synthesis of N,N-phthaloyl melphalan ethyl ester, the synthesis of melphalan free base, crystallization of melphalan hydrochloride, and drying. The overall synthetic route adopts part of the content of US3032584A. During the crystallization of melphalan hydrochloride, it was unexpectedly discovered that the addition of tartaric acid (specifically L(+)-tartaric acid) to the hydrochloric acid-isopropanol crystallization system can very effectively remove the melphalan-D-isomer impurity, solving the problem of this isomer impurity, significantly improving the quality of melphalan hydrochloride product, and reducing safety risks. Attached Figure Description

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

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

[0026] Figure 3 The reaction formula for the synthesis of free base in step 3 is as follows;

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

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

[0029] Figure 6 HPLC chromatogram of related substances for the melphalan free base prepared in Example 1;

[0030] Figure 7 The HPLC chromatogram of melphalan-D-isomer impurity detection in melphalan hydrochloride prepared in Example 3;

[0031] Figure 8 The HPLC chromatogram of related substances in melphalan hydrochloride prepared in Example 3;

[0032] Figure 9 HPLC chromatogram for the detection of melphalan-D-isomer impurities in melphalan hydrochloride prepared in Comparative Example 1;

[0033] Figure 10 The HPLC chromatogram of related substances in melphalan hydrochloride prepared for Comparative Example 1 is shown. Detailed Implementation

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

[0035] The specific testing methods for the obtained product are as follows:

[0036] I. Test method for impurities in melphalan-D-isomer:

[0037] Column: Daicel Chiralpak IC normal phase column; 4.6 mm × 250 mm; 5 μm;

[0038] Column temperature: 25℃; Injection volume: 5μL

[0039] Detector: Ultraviolet detector; Detection wavelength: 260nm

[0040] Flow rate: 1.0 mL / min Run time: 20 min

[0041] Mobile phase: n-hexane / anhydrous ethanol / dichloromethane / trifluoroacetic acid / triethylamine = 80:10:10:0.3:0.1 (V / V / V / V / V)

[0042] Test solution: Accurately weigh 25 mg of the sample to be tested, place it in a 25 ml volumetric flask, add 5 ml of diluent to dissolve it, then add the mobile phase to dilute to the mark and mix well.

[0043] II. Test methods for related substances in melphalan hydrochloride:

[0044] Chromatographic column: C18 4.6×150mm; 5µm; Supelco Discovery column;

[0045] Column temperature: 40℃; Injection volume: 10μL; Injection tray temperature: 5℃

[0046] Detector: Ultraviolet detector; Detection wavelength: 260nm

[0047] Flow rate: 1.5 mL / min Run time: 40 minutes

[0048] Buffer solution: Acetic acid: Triethylamine: Ammonium acetate: Water = 10:2:10:88 (V / V / W / V)

[0049] Mobile phase A: Buffer: Acetonitrile: Water = 10:100:1900 (V / V / V)

[0050] Mobile phase B: Buffer: Acetonitrile: Water = 10:1200:800 (V / V / V)

[0051] Elution gradient:

[0052] time Mobile phase A Mobile phase B 0 100.0％ 0.0％ 25.0 50.0％ 50.0％ 300 0.0％ 100.0％ 35.0 0.0％ 100.0％ 38.0 100.0％ 0.0％ 40 100.0％ 0.0％

[0053] Test solution: Accurately weigh 20 mg of melphalan hydrochloride, place it in a 100 ml brown volumetric flask, add methanol to dissolve and dilute to the mark, and mix well.

[0054] The principle and steps of the method for preparing melphalan hydrochloride with the removal of melphalan-D-isomer impurities provided in this solution are as follows:

[0055] Step 1. Synthesis of N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester, reaction formula as follows: Figure 1 As shown.

[0056] N,N-phthaloyl-4-amino-L-phenylalanine ethyl ester was dissolved in an aqueous acetic acid solution, stirred, and cooled to 0–5°C. An aqueous ethylene oxide solution was added dropwise. After the addition was complete, the temperature was raised to 20–25°C, and stirring was continued until the remaining raw material was less than 0.5%. The mixture was concentrated under reduced pressure for 2.5 h at 25±5°C and a vacuum of ≥0.08 MPa to remove the ethylene oxide. Water and 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 mixture was separated, and the organic phase was dried with anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure to obtain N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester.

[0057] Step 2. Synthesis of N,N-phthaloyl melphalan ethyl ester, reaction formula as follows: Figure 2 As shown.

[0058] N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester was dissolved in toluene at an external temperature of 40–60 °C. Under nitrogen protection, 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, yielding N,N-phthaloyl melphalan ethyl ester.

[0059] Step 3. Preparation of melphalan free base, reaction formula as follows: Figure 3 As shown.

[0060] N,N-phthaloyl melphalan ethyl ester was dissolved in 9 mol / L hydrochloric acid and stirred under reflux at an external temperature of 105±5℃ until the remaining melphalan ethyl ester was less than 0.5%. The temperature was then lowered to below 70℃, activated carbon was added, and stirring was maintained. The temperature was further lowered to 0-3℃ and stirred for 60 min. The mixture was filtered, and purified water and diethyl ether were added to the filtrate. The internal temperature was controlled at 0-3℃. Sodium acetate aqueous solution was added dropwise until the pH reached 5-6. The mixture was filtered, and the filter cake was slurried with deionized water and filtered again to obtain melphalan free base.

[0061] Step 4. Crystallization and drying of melphalan hydrochloride

[0062] Melphalan free base was dissolved in concentrated hydrochloric acid, filtered, and tartaric acid aqueous solution (L(+)-tartaric acid) was added to the filtrate. The solution was cooled to 5±5℃, and then isopropanol was added dropwise to induce crystallization. The solution was filtered, and the filter cake was dried under vacuum at 30±5℃ and a vacuum degree ≥-0.095MPa for 12 hours to obtain mephalan hydrochloride.

[0063] Furthermore, in step 4, the amount of hydrochloric acid used is 1.5–1.6 mL / g of 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 isopropanol to concentrated hydrochloric acid is (11.3–11.8):1.

[0064] Example 1: Preparation of melphalan free base according to this method

[0065] 200g of N,N-phthaloyl-4-amino-L-phenylalanine ethyl ester was dissolved in 1240g of 68% acetic acid aqueous solution by stirring. The solution was cooled to 0-5℃, and 520g of 25% ethylene oxide aqueous solution pre-cooled to 0-5℃ was added dropwise. After the addition was complete, the temperature was raised to 20-25℃, and stirring was continued until the remaining raw material was less than 0.5%. The solution was concentrated under reduced pressure for 2.5h at 25±5℃ and a vacuum of ≥0.08Mpa to remove ethylene oxide. 1000mL of deionized water and 1000mL of dichloromethane were added to the concentrate, and the solution was cooled to 5-10℃. The pH was adjusted to 6-8 with sodium bicarbonate. The solution was separated, and the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain N,N-phthaloyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester, weighing approximately 260g with a purity of 97%.

[0066] 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 h. 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 droplets were expelled, yielding N,N-phthaloyl melphalan ethyl ester, weighing 374 g with a purity of 94.13%.

[0067] The above-mentioned N,N-phthaloyl melphalan ethyl ester was dissolved in 600 mL of 9 mol / L hydrochloric acid and refluxed with stirring at an external temperature of 105±5℃ until the remaining melphalan ethyl ester was less than 0.5%. The temperature was then lowered to below 70℃, 30 g of activated carbon was added, and stirring was maintained. The temperature was further lowered to 0–3℃ and stirred for 60 min. The mixture was filtered, and 600 mL of deionized water and 800 mL of diethyl ether were added to the filtrate. The internal temperature was controlled at 0–3℃, and a 32% sodium acetate aqueous solution was added dropwise until the pH reached 5–6. The mixture was filtered, and the filter cake was slurried with 1600 mL of deionized water for 30 min and then filtered again to obtain approximately 196 g of free melphalan base with a purity of 97.45%. The HPLC chromatogram of the prepared free melphalan base for related substances is shown below. Figure 6As shown. After testing the free melphalan base using the aforementioned methods, it was found that the product contained 2.57% melphalan-D-isomer impurity. The HPLC chromatogram of the prepared free melphalan base for melphalan-D-isomer impurity detection is shown below. Figure 5 As shown.

[0068] Example 2

[0069] 50g of melphalan free base prepared in Example 1 was dissolved in 75mL of concentrated hydrochloric acid and filtered through a 0.45μm filter membrane. 23.3mL of a 1.8g / L tartaric acid aqueous solution was added to the filtrate, and the solution was cooled to 0℃. Then, 885mL of isopropanol was added dropwise, and the temperature was maintained at 0℃. Stirring was continued for 2 hours, followed by filtration. The filter cake was vacuum dried at 30℃ under a vacuum of ≥-0.095MPa for 12 hours to obtain 35.4g of melphalan hydrochloride with a purity of 99.71%. The product was tested using the aforementioned methods, and the melphalan-D-isomer impurity was found to be only 0.08%.

[0070] Example 3

[0071] 50g of melphalan free base prepared in Example 1 was dissolved in 80mL of concentrated hydrochloric acid and filtered through a 0.45μm filter membrane. 28mL of 2.1g / L tartaric acid aqueous solution was added to the filtrate, and the solution was cooled to 10℃. Then, 904mL of isopropanol was added dropwise, and the solution was maintained at 10℃ with stirring for 2 hours. The solution was filtered, and the filter cake was vacuum dried at 30℃ under a vacuum of ≥-0.095MPa for 12 hours to obtain 30.2g of melphalan hydrochloride with a purity of 99.85%. The HPLC chromatogram of related substances in the prepared melphalan hydrochloride is shown below. Figure 8 As shown in the figure. The product was tested using the aforementioned methods, and the melphalan-D-isomer impurity was found to be only 0.06%, with the actual required melphalan hydrochloride comprising 98.83% of the product. The HPLC chromatogram of the melphalan-D-isomer impurity in the prepared melphalan hydrochloride is shown in the figure. Figure 7 As shown.

[0072] Comparative Example 1

[0073] 50g of melphalan free base prepared in Example 1 was dissolved in 75mL of concentrated hydrochloric acid, filtered through a 0.45μm filter membrane, 24mL of water was added to the filtrate, the temperature was lowered to 0℃, and then 885mL of isopropanol was added dropwise. The temperature was maintained at 0℃, and stirring was continued for 2 hours. The mixture was then filtered, and the filter cake was vacuum dried at 30℃ under a vacuum of ≥-0.095MPa for 12 hours to obtain 35.9g of melphalan hydrochloride. The HPLC chromatogram of the related substances of the prepared melphalan hydrochloride is shown below. Figure 10 As shown, the purity is 99.65%. However, testing of the obtained product revealed that the melphalan-D-isomer impurity content was 2.47%, and the actual required melphalan hydrochloride accounted for 96.44% of the product. The HPLC chromatogram of the melphalan-D-isomer impurity of the prepared melphalan hydrochloride is shown below. Figure 9 As shown.

[0074] Comparative experiments revealed that the impurity content of the melphalan free base in Comparative Example 1 (2.47%), which was not treated with tartaric acid aqueous solution, was not significantly different from the impurity content of the melphalan free base prepared in Example 1 (2.57%). The melphalan free bases in Examples 2 and 3, treated with tartaric acid in a hydrochloric acid-isopropanol crystallization system, contained only 0.08% and 0.06% impurities, respectively, significantly reducing the impurity content.

Claims

1. A process for the preparation of meiphalan hydrochloride free from meiphalan-D-isomer impurities, characterized in that Comprising the following steps: Step 1. Dissolve N,N-terephthalyl-4-amino-L-phenylalanine ethyl ester in aqueous acetic acid, add oxirane dropwise at 0-5℃, then warm to 20-25℃ until the residual amount of the starting material is less than 0.5%, then remove oxirane by concentration under reduced pressure, neutralize and crystallize to obtain N,N-terephthalyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester; Step 2. Dissolve N,N-terephthalyl-4-(dihydroxyethylamino)-L-phenylalanine ethyl ester obtained in step 1 in toluene, add phosphorus oxychloride dropwise under nitrogen protection, stir and reflux at an external temperature of 110-120℃ for 4h, then cool the reaction solution and concentrate to drop out no liquid drops to obtain N,N-terephthalyl melphalan ethyl ester; Step 3. Dissolve N,N-terephthalyl melphalan ethyl ester obtained in step 2 in hydrochloric acid, stir and reflux at an external temperature of 110-120℃ until the residual amount of melphalan ethyl ester is less than 0.5%, cool, add activated carbon, stir, filter, add purified water and diethyl ether to the filtrate, control the internal temperature at 0-3℃, add sodium acetate aqueous solution dropwise until the pH is 5-6, and filter to obtain melphalan free base containing melphalan-D-isomer impurities; Step 4. Dissolve the melphalan free base containing melphalan-D-isomer impurities obtained in step 3 in concentrated hydrochloric acid, filter, add tartaric acid aqueous solution to the filtrate, cool to 0-10℃, add isopropyl alcohol dropwise to crystallize, filter the filter cake, and dry at 25-35℃ under a vacuum degree of ≥-0.095MPa to obtain melphalan hydrochloride.

2. The process for preparing meiphalan hydrochloride free from D-isomer impurities according to claim 1, characterized by: In step 1, the conditions for removing oxirane by concentration are a temperature of 20-30℃, a vacuum of ≥0.08MPa, and concentration under reduced pressure for 2.5h; the conditions for neutralization and crystallization are adding water and dichloromethane to the concentrated solution, cooling to 5-10℃, adding sodium bicarbonate dropwise until the pH is 6-8, drying the organic phase obtained by liquid separation with anhydrous sodium sulfate, filtering, and concentrating under reduced pressure.

3. The method of claim 1, wherein the method is characterized by: In step 2, after refluxing, the reaction solution is cooled to below 70℃, and then concentrated to drop out no liquid drops under a vacuum of ≥0.08MPa at an external temperature of 50-70℃.

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

5. The method of claim 1, wherein the method is for removing the D-isomer impurity of melphalan from melphalan hydrochloride. In step 4, the amount of hydrochloric acid used as the solvent is 1.5-1.6mL / g of melphalan free base.

6. The method of claim 5, wherein the method is for removing the D-isomer impurity of melphalan from melphalan hydrochloride. In step 4, the concentration of the tartaric acid aqueous solution is 1.8-2.1g / 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 of claim 5, wherein the method is for removing the D-isomer impurity of melphalan from melphalan hydrochloride. The volume ratio of isopropyl alcohol to concentrated hydrochloric acid is (11.3-11.8):1.

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