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JP-7857409-B2 - Method for preparing oxidized glutathione, its crystalline form, and impurities.

JP7857409B2JP 7857409 B2JP7857409 B2JP 7857409B2JP-7857409-B2

Inventors

  • リュウ，ジードン
  • ヤン，チィアン
  • レイ，ユ
  • シェン，ジンソン

Assignees

  • シェンヤン シンチ ファーマシューティカル カンパニー， リミテッド

Dates

Publication Date
20260512
Application Date
20221220
Priority Date
20211221

Claims (14)

  1. Equation (I): Crystals of the heptahydrate of the compound.
  2. The crystal according to claim 1, wherein the crystal satisfies any one of the following definitions: i) Characterized by an X-ray powder diffraction pattern obtained using CuKα radiation having at least the following characteristic peaks at °2θ: 8.238±0.2, 16.338±0.2, and 24.551±0.2; ii) Characterized by an X-ray powder diffraction pattern obtained using CuKα radiation having at least the following characteristic peaks at °2θ: 8.238±0.2, 10.619±0.2, 16.338±0.2, 19.539±0.2, 24.551±0.2, 26.806±0.2 and 34.618±0.2; iii) The X-ray powder diffraction pattern obtained using CuKα rays having at least the following characteristic peaks at °2θ: 8.238±0.2, 9.750±0.2, 10.619±0.2, 16.338±0.2, 19.539±0.2, 22.738±0.2, 23.200±0.2, 24.551±0.2, 26.806±0.2 and 34.618±0.2 .
  3. The crystal according to claim 1, further characterized by having a melting point of 169 ± 2°C.
  4. The crystal according to claim 1, further characterized by a weight loss of 14 ± 1% at 50 to 100°C and a weight loss of 3 ± 1% at 100 to 160°C in thermogravimetric analysis.
  5. Formula (II) The step includes oxidizing the compound with DMSO to obtain the compound of formula (I), A method for preparing a compound of formula (I) in which the molar ratio of DMSO to the compound of formula (II) is 2:1 to 25:1 .
  6. The method according to claim 5, wherein the method satisfies one or more of the following definitions: i) The molar ratio of DMSO to the compound of formula (II) is 2.5 :1 to 5: 1 ; ii) To promote the dissolution of the compound of formula (II), a polar solvent is further added to the reaction , the polar solvent being selected from the group consisting of water, formamide, trifluoroacetic acid, acetonitrile, DMF, hexamethylphosphoramide, methanol, ethanol, acetic acid, isopropanol, pyridine, tetramethylethylenediamine, acetone, triethylamine, n-butanol, dioxane, tetrahydrofuran, methyl formate, tributylamine, methyl ethyl ketone, ethyl acetate, chloroform, trioctylamine, dimethyl carbonate, and diethyl ether, or compounds thereof , the ratio of the polar solvent to the compound of formula (II) being 200 to 2000 mL of the polar solvent per 100 g of the compound of formula (II); iii) The pH of the above reaction is adjusted to 2 to 9 ; iv) The above reaction is carried out at -10°C to 60°C ; v) The reaction is carried out for 5 to 60 hours ; vi) After the reaction is complete, the pH is adjusted to the isoelectric point of oxidized glutathione (pH 2.75 to 2.90), and crystallization by stirring is carried out for at least 5 hours , with the crystallization by stirring being carried out at 5°C to 40°C .
  7. The method according to claim 6, wherein the method satisfies one or more of the following definitions: i) The molar ratio of DMSO to the compound of formula (II) is 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1; ii) To promote the dissolution of the compound of formula (II), a polar solvent is further added to the reaction, wherein the polar solvent is water, and the ratio of the polar solvent to the compound of formula (II) is 250 mL, 300 mL, 500 mL, 750 mL, or 1000 mL of polar solvent per 100 g of the compound of formula (II); iii) The pH of the reaction is adjusted to 5, 5.5, 6, 6.5, 7, 8, or 9; iv) The reaction is carried out at 25°C, 40°C, or room temperature; v) The above reaction was carried out for 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, and 4 hours. It will be performed for 0 hours or 45 hours; vi) After the reaction is complete, the pH is adjusted to the isoelectric point of oxidized glutathione (pH 2.75 to 2.90), and crystallization by stirring is carried out for at least 10 hours, with the crystallization by stirring being carried out at 5°C, 10°C, 15°C, 20°C, or room temperature.
  8. 1) A step of dissolving crude oxidized glutathione in purified water, wherein the amount of purified water is 3 to 5 times the weight of the crude oxidized glutathione, and an iron(II) chloride solution is added to effectively remove residual raw materials , 2) After dissolution, the solution is filtered while hot, and 3) the filtrate is cooled to 10 to 25 °C for crystallization. The method according to claim 5, further comprising purifying oxidized glutathione , A method that further satisfies one or more of the following definitions: i) In step 1), the temperature of the purified water is 35°C to 55 °C ; ii) In step 3), the crystallization time for recrystallization is 3 to 10 hours ; iii) In step 3), gradient cooling is employed, and the cooling rate is 10°C per hour.
  9. The method according to claim 8, which further satisfies one or more of the following definitions: i) In step 1), the temperature of the purified water is 40°C to 50°C; ii) In step 1), add 100 mL of 5% iron(II) chloride solution per kilogram of oxidized glutathione; iii) In step 3), the crystallization time for recrystallization is 4 to 6 hours; iv) In step 3), the filtrate is cooled to 12-20°C for crystallization.
  10. The process includes the step of recrystallizing the oxidized glutathione prepared according to any one of claims 5 to 9 in purified water , and the following steps: 1) A step of dissolving the oxidized glutathione in purified water and stirring until dissolved, wherein 2 to 6 liters of purified water are used per kilogram of oxidized glutathione , and the temperature of the purified water during the dissolution process is 40 to 60°C; 2) A step of filtering the solution while it is hot after dissolution, and then gradient cooling to a target temperature, wherein the cooling gradient is 5 to 25°C/hour and the target temperature is 5 to 25°C; and , 3) A step of crystallization under temperature control, wherein the temperature is controlled to 5 to 25°C and the crystallization time is 6 to 12 hours. A method for preparing crystals of oxidized glutathione heptahydrate according to any one of claims 1 to 4, comprising:
  11. The method according to claim 10 , wherein the method satisfies one or more of the following definitions: i) In step 1), use 4 L of purified water per kilogram of oxidized glutathione; ii) In step 1), the temperature of the purified water during the dissolution process is 50 °C; iii) In step 1) , add 100 mL of 5% iron(II) chloride solution per kilogram of oxidized glutathione; iv) In step 2), the cooling gradient is 10 °C/hour; v) In step 2), the target temperature is 15 to 25°C; vi) In step 3), the temperature is controlled to 15 to 25°C; vii) In step 3), the crystallization time is 6 to 8 hours.
  12. A method for preparing impurity A, comprising oxidizing reduced glutathione and Cys-Gly in a 1:1 molar ratio with DMSO , wherein the method further satisfies one or more of the following definitions: i) The amount of DMSO is 3 to 5 equivalents; ii) The above method, 1) A step of catalyzing the condensation of Boc-Cys(Trt)-OH and glycine tert-butyl ester in a molar ratio of 1:1 to 1:3 with 1.5 to 4 equivalents of H BTU or DEPBT and 2 equivalents of N ,N- diisopropylethylamine , wherein the reaction is carried out in DMF or dichloromethane. The method comprises the steps of: 2) removing the Trt, Boc, and tBu protecting groups with trifluoroacetic acid to obtain Cys-Gly; and 3) oxidizing reduced glutathione and Cys-Gly in a 1:1 molar ratio with 3 to 5 equivalents of DMSO.
  13. A method for preparing impurity B , comprising oxidizing reduced glutathione and Glu-Cys in a 1:1 molar ratio with DMSO, wherein the method further satisfies one or more of the following definitions: i) The amount of DMSO is 3 to 5 equivalents; ii) The above method, 1) A step of catalyzing the condensation of Boc-Glu-OtBu and H-Cys(Trt)-OtBu in a molar ratio of 1:1 to 1:2 with 1.5 to 4 equivalents of H BTU or DEPB T and 2 equivalents of N ,N-diisopropylethylamine. The method comprises the steps of: 2) removing the Boc, Trt, and tBu protecting groups with trifluoroacetic acid to obtain Glu-Cys; and 3) oxidizing reduced glutathione and Glu-Cys in a 1:1 molar ratio with 3 to 5 equivalents of DMSO.
  14. A method for preparing impurity C , comprising the step of oxidizing reduced glutathione and cysteine in a 1:1 molar ratio with DMSO, wherein the amount of DMSO is 3 to 5 equivalents.

Description

This disclosure relates to the field of chemical synthesis, and more particularly to methods for preparing oxidized glutathione, its heptahydrate crystalline form, and impurities. Oxidized glutathione has similar effects to reduced glutathione, but it is more stable and can therefore be used as an active ingredient in health foods, pharmaceuticals, cosmetics, and other products instead of reduced glutathione. All currently reported methods for preparing oxidized glutathione involve the oxidation of reduced glutathione, for example: Route 1: Oxidation with Hydrogen Peroxide (Chinese Journal of Pharmaceuticals, 2013, 44, 265). The method includes the following steps: dissolving reduced glutathione in water, adjusting the resulting solution to an appropriate pH, and oxidizing the glutathione with hydrogen peroxide as an oxidizing agent to prepare oxidized glutathione. The drawback of this method is that while the hydrogen peroxide oxidation reaction is relatively fast, the reaction is also relatively vigorous, requiring relatively harsh reaction conditions and necessitating strict control of process parameters such as temperature, pH, and the amount of hydrogen peroxide in the reaction system. Furthermore, further peroxidation or decomposition impurities may occur, affecting the purity and yield of the product. Additionally, the oxidizing agent, hydrogen peroxide, is a hazardous compound in China that can easily be used to create explosives, and therefore its use is regulated. Moreover, hydrogen peroxide is easily self-decomposing and must be stored in a sealed container at low temperatures. Route 2: Catalysis by Arginine (RSC Adv., 2014, 4, 33399-33407). The method includes the following steps: dissolving reduced glutathione in water, and using oxygen gas as an oxidizing agent and arginine as a catalyst. The reaction produces no waste and is environmentally friendly. Furthermore, oxygen gas and arginine are relatively readily available, making the starting materials relatively easy to obtain. The disadvantages of this method are: the need to use arginine as a catalyst during the reaction process, which can lead to residual arginine during the work-up process; and the need to heat the reaction to 50°C, which can cause the product, oxidized glutathione, to undergo partial decomposition and racemization under high-temperature conditions, resulting in lower yield and purity. Route 3: Enzymatic Catalysis (Japanese Patent Application Publication No. 5-146279). This method is a bio-enzyme-catalyzed method that requires the use of a specific bio-oxidase as a catalyst and uses air to oxidize an aqueous solution of reduced glutathione to obtain oxidized glutathione. The drawbacks of this method are: the enzyme is relatively difficult to obtain and store, and is not as good as commonly used chemical reagents. Furthermore, after the reaction is complete, the enzyme needs to be separated from the reaction solution, which requires specific process technology and equipment. It may not be applicable to the existing facilities of typical chemical raw material pharmaceutical companies. Route 4: Diethyl Bromomalonate Method (Chem. Pharma. Bull. 1986, 34, 486-495). The method includes the following steps: dissolving reduced glutathione in an alkaline water/ethanol solution, then adding an ethanol solution of diethyl bromomalonate dropwise, and reacting at -16°C for 1 hour. The disadvantages of this method are: the use of diethyl bromomalonate as an oxidizing agent is relatively expensive; diethyl bromomalonate produces many byproducts; and the operation is complex. The reaction process requires a low temperature of -16°C, making it relatively unsuitable for industrialization. Furthermore, the crystalline forms of oxidized glutathione prepared by the above method mainly include amorphous form (CN102858792A), monohydrate crystalline form (Japanese Patent No. 4401775), hexahydrate crystalline form (CN102869674A), and octahydrate crystalline form (International Union of Crystallography, pp. 538, 1999). In these forms, the amorphous form has low water solubility, limiting its application in the pharmaceutical industry; the monohydrate crystalline form is prone to aggregation in needle-shaped crystals, and impurities are difficult to remove, resulting in low crystal separation ability; the hexahydrate crystalline form requires pH adjustment during crystallization, has a long crystallization time of over 10 hours, and is therefore relatively cumbersome to operate and difficult to obtain crystals from; and the octahydrate crystalline form has uneven water content, low stability, and requires a long time of up to 3-4 days to obtain crystals. Furthermore, existing processes for oxidized glutathione have never achieved large-scale industrial production. The main reason is the difficulty in simultaneously achieving mildness of the reaction (reducing product decomposition and racemization) and economic efficiency. To achieve excellent proces