Method for preparing oxidized glutathione, its crystalline form, and impurities.

Abstract

The present invention provides a method for preparing oxidized glutathione and its new crystalline forms and impurities, comprising the steps of: converting reduced glutathione into crude oxidized glutathione using dimethyl sulfoxide (DMSO) as an oxidizing agent; and recrystallizing and purifying in purified water to obtain high purity heptahydrate crystals of oxidized glutathione.

Description

Technical Field

[0001] The present disclosure belongs to the field of chemical synthesis, and particularly relates to a method for preparing oxidized glutathione and its heptahydrate crystal form and impurities.

Background Art

[0002] Oxidized glutathione has the same effects as reduced glutathione, but oxidized glutathione is more stable than reduced glutathione, and thus can be used as an active ingredient in health foods, pharmaceuticals, cosmetics, and other products in place of reduced glutathione.

[0003] All currently reported methods for preparing oxidized glutathione are oxidations of reduced glutathione, for example:

[0004] 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 obtained solution to an appropriate pH value, and oxidizing glutathione with hydrogen peroxide as an oxidizing agent to prepare oxidized glutathione. The disadvantages of this method are that although the rate of the hydrogen peroxide oxidation reaction is relatively fast, the reaction is also relatively intense, so the reaction conditions are relatively harsh, and it is necessary to strictly control process parameters such as temperature, pH, and the amount of hydrogen peroxide in the reaction system. In other respects, further peroxidation or decomposition impurities occur, affecting the purity and yield of the product. Furthermore, the oxidizing agent, hydrogen peroxide, is a harmful compound in China that can easily produce explosives, so the use of hydrogen peroxide is regulated. Moreover, hydrogen peroxide is prone to self-decomposition and needs to be stored in a sealed container at low temperature.

[0005] 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 easy to obtain, and therefore the starting materials are relatively readily available. The disadvantages of this method are: because arginine must be used as a catalyst in the reaction process, residual arginine is likely to remain during the work-up process; and because the reaction must be heated to 50°C, the product, i.e., oxidized glutathione, tends to undergo partial decomposition and racemization under high-temperature conditions, resulting in lower yield and purity.

[0006] 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 disadvantages of this method are that 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.

[0007] Route 4: Diethyl Bromomalonate Method (Chem. Pharm. Bull. 1986, 34, 486-495). The method involves 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. It includes. The disadvantages of this method are: it uses diethyl bromomalonate as the oxidizing agent, which is relatively expensive; diethyl bromomalonate produces more byproducts; and the operation is complex. The reaction process requires a low temperature of -16°C, making it relatively unsuitable for industrialization.

[0008] 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, pp538, 1999). Of 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 in the crystalline form are difficult to remove, resulting in low crystal separation ability; the hexahydrate crystalline form requires pH adjustment during the crystallization process, has a long crystallization time of more than 10 hours, and is therefore relatively difficult to operate and difficult to obtain crystals; 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 viability. Achieving excellent process stability requires an oxidation and crystallization system that is mild, economical, readily available, and produces few by-products.

[0009] Furthermore, detection by liquid chromatography and structural confirmation by mass spectrometry revealed that oxidized glutathione contains the following three impurities: [Table 1]

[0010] There are very few reports on the preparation methods for these three impurities, and the raw materials and reagents used are also unknown. It is not commercially available. Therefore, these preparation methods have no practical value.

[0011] There are no literature reports on the purification process of oxidized glutathione. Since polypeptide compounds readily hydrolyze, racemize, and biodegrade, commonly used purification methods involve separation by ion-exchange resin chromatography or preparative liquid chromatography. These methods suffer from high solvent loss and high production costs.

[0012] In summary, the technology in question requires a method for synthesizing oxidized glutathione that is low-cost, uses mild reaction conditions, has high product purity, and is suitable for industrial production. [Overview of the project]

[0013] Compared to existing methods, the method for synthesizing oxidized glutathione provided in this disclosure can solve the above problems.

[0014] Specifically, this disclosure relates to the following embodiments:

[0015] 1. Equation (I): [ka] Crystals of the heptahydrate of the compound.

[0016] 2. The crystal according to Embodiment 1, characterized by an X-ray powder diffraction pattern obtained using CuKα rays having at least the following characteristic peaks at °2θ: 8.238±0.2, 16.338±0.2, and 24.551±0.2.

[0017] 3. The crystal according to Embodiment 2, characterized by an X-ray powder diffraction pattern obtained using CuKα rays having at least the following characteristic peaks at °2θ: 10.619±0.2, 19.539±0.2, 26.806±0.2, and 34.618±0.2.

[0018] 4. A crystal according to embodiment 3, characterized by an X-ray powder diffraction pattern obtained using a CuKα line having at least the following characteristic peaks at 2θ: 9.750 ± 0.2, 22.738 ± 0.2 and 23.200 ± 0.2.

[0019] 5. A crystal according to embodiment 1, characterized by an X-ray powder diffraction pattern substantially shown in FIG. 5.

[0020] 6. The crystal according to any one of embodiments 1 to 5, further characterized by having a melting point of 6.169 ± 2 °C.

[0021] 7. The crystal according to any one of embodiments 1 to 6, further characterized by a weight loss of about 14 ± 1% at 50 to 100 °C and about 3 ± 1% at 100 to 160 °C in thermogravimetric analysis.

[0022] 8. The crystal according to any one of embodiments 1 to 7, further characterized by having a thermogravimetric analysis diagram shown in FIG. 6.

[0023] 9. A method for preparing a compound of formula (I), comprising the step of oxidizing a compound of formula (II) with DMSO to obtain a compound of formula (I).

Chemical formula

[0024] 10. The method according to embodiment 9, wherein the molar ratio of DMSO to the compound of formula (II) is 2:1 to 25:1, or 2.5:1 to 5:1, or further 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:(原文此处有误,已修正为5:1)

[0025] 11. The method according to Embodiment 9 or 10, wherein a polar solvent is further added to the reaction to promote the dissolution of the compound of formula (II), or the polar solvent is 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, and further or water.

[0026] 12. The method according to Embodiment 11, wherein the ratio of the polar solvent to the compound of formula (II) is 200 to 2000 mL, or 250 mL to 1000 mL, or 250 mL, 300 mL, 500 mL, 750 mL, or 1000 mL of the polar solvent per 100 g of the compound of formula (II).

[0027] 13. The method according to any one of embodiments 9 to 12, wherein the pH of the reaction is adjusted to 2 to 9, or 3 to 7, or further to 5 to 7, for example, 5, 5.5, 6, 6.5, 7, 8, or 9.

[0028] 14. The reaction is carried out at -10°C to 60°C, or at 5°C to 30°C, 40°C, or room temperature, such as 25°C, according to any one of Embodiments 9 to 13.

[0029] 15. The method according to any one of embodiments 9 to 14, wherein the reaction is carried out for 5 to 60 hours, or 10 to 48 hours, for example, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, or 45 hours.

[0030] 16. The method according to any one of embodiments 9 to 15, wherein after the reaction is complete, the pH is adjusted to the isoelectric point of oxidized glutathione (pH 2.75 to 2.90), and crystallization is carried out by stirring for at least 5 hours, or at least 10 hours.

[0031] 17. The method according to Embodiment 16, wherein the crystallization by stirring is carried out at 5°C to 40°C, or at 5°C, 10°C, 15°C, 20°C, or room temperature.

[0032] 18. The method according to any one of Embodiments 9 to 17, wherein the purity of the obtained compound of formula (I) is ≥98%, or ≥98.5%, or ≥99%, or ≥99.7%, or ≥99.8%, or ≥99.9%, and the total content of impurities A, B, and C in the product is less than 1%, or less than 0.5%, or less than 0.3%, or less than 0.1%.

[0033] [Table 2]

[0034] 19.1) 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. 2) After dissolution, the step of filtering the solution while it is hot, and 3) The process includes the step of cooling the filtrate to 10-25°C or 12-20°C for crystallization. A method for purifying oxidized glutathione according to any one of Embodiments 9 to 18.

[0035] 20. The method according to Embodiment 19, wherein in step 1), the temperature of the purified water is 35°C to 55°C, or 40°C to 50°C, and optionally, an iron(II) chloride solution is added to remove residual raw materials, or 100 mL of a 5% iron(II) chloride solution per kilogram of oxidized glutathione is added.

[0036] 21. The method according to Embodiment 19 or 20, wherein in step 3), the crystallization time for recrystallization is 3 to 10 hours or 4 to 6 hours, and optionally gradient cooling is employed with a cooling rate of 10°C per hour.

[0037] 22. The method comprises the step of recrystallizing the oxidized glutathione prepared according to any one of Embodiments 9 to 21 in purified water, wherein the method includes the following steps: 1) Dissolve this oxidized glutathione in purified water and stir until dissolved. 2) After dissolution, the solution is filtered while hot, and then gradient cooled to the target temperature, and 3) Step of crystallization under temperature control. A method for preparing crystals of oxidized glutathione heptahydrate according to any one of Embodiments 1 to 8, including the above.

[0038] 23. The method according to Embodiment 22, wherein in step 1), 2 to 6 L, or 3 to 6 L, or further 3 to 5 L, or further 4 L of purified water is used per kilogram of oxidized glutathione.

[0039] 24. The method according to Embodiment 22 or 23, wherein in step 1), the temperature of the purified water during this dissolution process is 40-60°C, or 40-50°C, or even 50°C.

[0040] 25. The method according to any one of Embodiments 22 to 24, wherein in step 1), an iron(II) chloride solution is added to effectively remove residual raw materials, and optionally, 100 mL of a 5% iron(II) chloride solution per kilogram of oxidized glutathione is added.

[0041] 26. The method according to any one of embodiments 22 to 25, wherein in step 2), the cooling gradient is 5 to 25°C / hour, or 5 to 20°C / hour, or 5 to 15°C / hour, or 10°C / hour.

[0042] 27. The method according to any one of embodiments 22 to 26, wherein in step 2), the target temperature is 5 to 25°C, or 10 to 25°C, or further 15 to 25°C.

[0043] 28. The method according to any one of embodiments 22 to 27, wherein in step 3), the temperature is controlled to 5 to 25°C, or 10 to 25°C, or even 15 to 25°C.

[0044] 29. The method according to any one of embodiments 22 to 28, wherein in step 3), the crystallization time is 6 to 12 hours or 6 to 8 hours.

[0045] 30. [Table 3] A compound selected from the following.

[0046] 31. [ka] This includes oxidizing reduced glutathione and Cys-Gly in a 1:1 molar ratio with DMSO. A method for preparing impurity A as described in Embodiment 30.

[0047] 32. The method according to Embodiment 31, wherein the amount of DMSO is 2 to 10 equivalents, or 3 to 5 equivalents.

[0048] 33.1) Boc-Cys(Trt)-OH and glycine tert-butyl ester in a molar ratio of 1:1 to 1:3, 1.5 to 4 equivalents of a coupling agent (e.g., HATU, HBTU, PyBOP, DEPBT, or HBTU and DEPBT), and 2 equivalents of a tertiary organic amine (e.g., N,N-diisopropylethylamine and triethylamine, or A step of condensation under the catalyst of N,N-diisopropylethylamine, wherein the reaction is carried out in DMF or dichloromethane. 2) The step of removing the Trt, Boc and tBu protecting groups with trifluoroacetic acid to obtain Cys-Gly, and 3) A step comprising oxidizing reduced glutathione and Cys-Gly in a 1:1 molar ratio with 3 to 5 equivalents of DMSO, The method according to Embodiment 31 or 32.

[0049] 34. [ka] This includes oxidizing reduced glutathione and Glu-Cys in a 1:1 molar ratio with DMSO. A method for preparing impurity B as described in Embodiment 30.

[0050] 35. The method according to Embodiment 34, wherein the amount of DMSO is 2 to 10 equivalents, or 3 to 5 equivalents.

[0051] 36.1) 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 a coupling agent (e.g., HATU, HBTU, PyBOP, DEPBT, etc., or HBTU and DEPBT) and 2 equivalents of a tertiary organic amine (e.g., N,N-diisopropylethylamine and triethylamine, or N,N-diisopropylethylamine), 2) The step of removing the Boc, Trt, and tBu protecting groups with trifluoroacetic acid to obtain Glu-Cys, and 3) A step comprising oxidizing reduced glutathione and Glu-Cys in a 1:1 molar ratio with 3 to 5 equivalents of DMSO, The method according to Embodiment 34 or 35.

[0052] The process includes the step of oxidizing reduced glutathione and cysteine ​​in a molar ratio of 37.1:1 with DMSO. A method for preparing impurity C as described in Embodiment 30.

[0053] 38. The method according to Embodiment 37, wherein the amount of DMSO is 2 to 10 equivalents, or 3 to 5 equivalents.

[0054] Favorable effects of this disclosure

[0055] The crude oxidized glutathione obtained by this method is free of peroxidized impurities, has a relatively low content of hydrolyzed impurities, and does not produce other solid by-products. After analyzing and synthesizing the structures of other impurities, it provides a basis and guarantee for subsequent quality control of the oxidized glutathione.

[0056] The inventors have found that a small amount of ferric chloride aqueous solution can be added during the purification process of oxidized glutathione. We discovered by chance that adding it unexpectedly and significantly improved the purification effect and unexpectedly increased the purity of the product, and that oxidized glutathione heptahydrate crystals could be obtained by recrystallizing them in purified water under specific conditions. [Brief explanation of the drawing]

[0057] [Figure 1] Figure 1 is an ESI-MS diagram of oxidized glutathione. [Figure 2] Figure 2 is an ESI-MS diagram of impurity A in oxidized glutathione. [Figure 3] Figure 3 is an ESI-MS diagram of impurity B in oxidized glutathione. [Figure 4] Figure 4 is an ESI-MS diagram of impurity C in oxidized glutathione. [Figure 5] Figure 5 shows the X-ray powder diffraction pattern of oxidized glutathione heptahydrate crystals. [Figure 6] Figure 6 shows the TG and DSC diagrams of the oxidized glutathione heptahydrate crystals. [Modes for carrying out the invention]

[0058] definition

[0059] A "polar solvent" refers to a solvent containing polar groups such as hydroxyl, carbonyl, and carboxyl. In other words, a solvent molecule is a polar molecule because the centers of gravity of the positive and negative charges within the molecule do not coincide, which makes the molecule polar. Polar solvents are 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.

[0060] The isoelectric point is the pH value at which the surface charge of a molecule disappears. At the isoelectric point, there is no mutual repulsion due to the same charge, so the molecules are at their most unstable, have the lowest solubility, and can quickly combine into large aggregates due to electrostatic attraction, thereby causing precipitation.

[0061] The moisture content is calculated as follows:

[0062] Water content = Mass of water molecules * Number of water molecules in a single crystal molecule / (Mass of water molecules * Number of water molecules in a single crystal molecule + Molecular weight of oxidized glutathione) * 100%

[0063] For example, the water content of monohydrate = 18.01*1 / (18.01*1+612.63)*100% = 2.86%, the water content of hexahydrate = 18.01*6 / (18.01*6+612.63) = 14.99%, the water content of heptahydrate = 18.01*7 / (18.01*7+612.63) = 17.07%, and the water content of octahydrate = 18.01*8 / (18.01*8+612.63) = 19.04%.

[0064] Embodiments of the present disclosure are described below clearly and completely with reference to examples. Obviously, the examples described are used only to illustrate the present disclosure, but not to limit it. All other embodiments obtained by those skilled in the art without creative effort based on the embodiments of the present disclosure are included within the scope of the protection of the present disclosure. [Examples]

[0065] (1) Synthesis of oxidized glutathione

[0066] Example 1

[0067] 1.0 kg of reduced glutathione and 10.0 L of water were added to a three-necked flask, followed by 1.0 L of dimethyl sulfoxide. The mixture was reacted at room temperature for 48 hours with stirring. The solid was precipitated, filtered, and dried to obtain 950.2 g of crude oxidized glutathione, with a yield of 95.3% and a purity of 98.4%. ESI-MS[M+H] + = 613.3.

[0068] Example 2

[0069] 1.0 kg of reduced glutathione and 3.0 L of water were added to a three-necked flask. The pH of the mixture was adjusted to 6-7 with NaOH, and then 500 mL of dimethyl sulfoxide was added. The mixture was stirred at room temperature for 10 hours. The pH was adjusted to the isoelectric point (2.75-2.90), and the mixture was stirred at 5°C for 10 hours for crystallization. The mixture was filtered. The filtered cake was rinsed with ethanol and dried to obtain 855.1 g of crude oxidized glutathione, with a yield of 86.0% and a purity of 99.3%. ESI-MS[M+H] + = 613.3.

[0070] Example 3

[0071] 1.0 kg of reduced glutathione and 2.5 L of water were added to a three-necked flask. The pH was adjusted to 6.0 with NaOH, and then 450 mL of dimethyl sulfoxide was added to the three-necked flask. The mixture was stirred at 40°C for 24 hours. The pH was adjusted to the isoelectric point (2.75-2.90), and the mixture was crystallized at 8°C for 10 hours. The mixture was filtered. The filtered cake was rinsed with ethanol and dried to obtain 892.7 g of oxidized glutathione, with a yield of 89.5% and a purity of 99.0%. ESI-MS[M+H] + = 613.3.

[0072] Example 4

[0073] 1.0 kg of reduced glutathione and 3.0 L of water were added to a three-necked flask. The pH was adjusted to 5.5 with NaOH, and then 550 mL of dimethyl sulfoxide was added to the three-necked flask. The mixture was stirred at 25°C for 48 hours. The pH was adjusted to the isoelectric point (2.75-2.90), and the mixture was crystallized at 5°C for 8 hours. The mixture was filtered. The filtered cake was rinsed with ethanol and dried to obtain 912.0 g of oxidized glutathione, with a yield of 91.5% and a purity of 99.2%. ESI-MS[M+H] + = 613.3.

[0074] Comparative Example 1: Oxidation by hydrogen peroxide

[0075] 10 g of reduced glutathione and 30 mL of water were added to a three-necked flask. The pH was adjusted to 5.8 with NaOH, and 3.3 mL of 30% hydrogen peroxide was added to the three-necked flask in a water bath at room temperature. The mixture was reacted for 5 hours. The pH was then adjusted to 3.0, and 50 mL of anhydrous ethanol was added to the system. The mixture was crystallized at 10°C for 10 hours, and then filtered. The filtered cake was rinsed with ethanol and dried to obtain 9.09 g of oxidized glutathione containing peroxide impurities. The yield was 91.2%, and the purity was 96.2%. ESI-MS[M+H] + = 613.3.

[0076] (2) Purification of oxidized glutathione

[0077] Comparative Example 2: Purification and refining using conventional methods

[0078] In the above example (1) of the synthesis of oxidized glutathione, 10 g of crude oxidized glutathione was obtained. Glutathione was dissolved in 200 mL of purified water. The sample was then injected into a strongly acidic cation exchange resin and eluted with purified water. The eluent was collected, and the solid was precipitated by adding anhydrous ethanol to obtain 8.43 g of oxidized glutathione. The one-step yield was 84.3%, and the purity was 99.5%. The purity increase rate was 0.2%.

[0079] Example 5 In the example of synthesis of oxidized glutathione described in (1) above, 1.0 kg of crude oxidized glutathione was added to 4.0 L of purified water at 50°C, and 100 mL of 5% iron(II) chloride solution was added. The mixture was stirred until the solid dissolved. The mixture was filtered while hot, and then gradually cooled to 25°C at a rate of 10°C per hour. Finally, the mixture was crystallized at a controlled temperature of 20°C to 25°C for 8 hours. The mixture was filtered. The filtered cake was rinsed with ethanol and dried.

[0080] In other experimental examples, the parameters in the table below were used, and the above steps were performed sequentially.

[0081] Searching for precipitation time

[0082] [Table 4]

[0083] [Table 5]

[0084] Based on the above results, it can be seen that if the crystallization time is too short, the resulting crystals are a mixture of crystals with unstable water content, and if the crystallization time is too long, impurities precipitate, and the purity of the product decreases. Therefore, the crystallization time should be either 6 to 12 hours or 6 to 8 hours.

[0085] Searching for twice the amount of purified water

[0086] [Table 6]

[0087] [Table 7]

[0088] Based on the above results, it can be seen that when oxidized glutathione is dissolved using purified water, the amount of water has a significant impact on the yield. If the amount of water is too much, the yield will be very low. If the amount of water is too little, the product will not dissolve completely, or after dissolution and reprecipitation, the stirring effect will be insufficient, resulting in a decrease in product purity. Therefore, the amount of water should be either 2 to 6 times, 3 to 6 times, or even 3 to 5 times.

[0089] Searching for dissolution temperature

[0090] [Table 8]

[0091] [Table 9]

[0092] According to the table above, all examples are soluble at temperatures above 40°C, but racemization occurs at temperatures above 60°C. Furthermore, the higher the temperature, the longer the heating time, and the more severe the racemization becomes. Considering the energy consumption of the product, the selected temperature range is 40-60°C, 40-50°C, or even 50°C.

[0093] Search for crystallization retention temperature

[0094] [Table 10]

[0095] [Table 11]

[0096] According to the experimental results above, if the crystallization temperature is too low, the yield is high, but many impurities also precipitate, and the single impurity content exceeds 0.1%. If the crystallization temperature is too high, the yield gradually decreases. Considering the above factors comprehensively, the selected crystallization temperature may be 5-25°C, 10-25°C, or even 15-25°C.

[0097] Searching for a cooling gradient

[0098] [Table 12]

[0099] [Table 13]

[0100] According to the table above: If the cooling rate is too fast, the crystallization rate is fast, but impurities are included, precipitation occurs rapidly, and the purity of the product decreases. If the cooling rate is too slow, the crystallization time is longer, energy consumption and labor costs increase, while the yield does not change much. Therefore, considering all factors, the cooling rate may be 5-20°C / hour, 5-15°C / hour, or even 10°C / hour.

[0101] Example 25 In the above example (1) of the synthesis of oxidized glutathione, 1.0 kg of crude oxidized glutathione was added to 4.0 L of purified water at 40°C, and the mixture was stirred until the solid dissolved. The mixture was filtered while hot, and then gradually cooled to 20°C at a rate of 10°C per hour. Finally, the mixture was crystallized at a controlled temperature of 15°C to 20°C for 8 hours. The mixture was filtered. The filtered cake was rinsed with ethanol and dried to obtain 855.1 g of oxidized glutathione, with a one-step yield of 85.5% and a purity of 99.7%. The purity increase rate was 0.5%.

[0102] Example 26

[0103] In the example of synthesis of oxidized glutathione described in (1) above, 1.0 kg of crude oxidized glutathione was added to 4.0 L of purified water at 45°C, and 100 mL of 5% iron(II) chloride solution was added. The mixture was stirred until the solid dissolved. The mixture was filtered while hot and then gradually cooled to 20°C at a rate of 10°C per hour. Finally, the mixture was crystallized at a controlled temperature of 15°C to 20°C for 7 hours. The mixture was filtered. The filtered cake was rinsed with ethanol and dried to obtain 849.2 g of oxidized glutathione, with a one-step yield of 84.9% and a purity of 99.9%. The purity increase rate was 0.7%.

[0104] Example 27

[0105] In the above example (1) synthesis of oxidized glutathione, 1.0 kg of crude oxidized glutathione was obtained. Glutathione was added to 5.0 L of purified water at 50°C, and the mixture was stirred until the solid dissolved. The mixture was filtered while still hot, and then gradually cooled to 20°C at a rate of 10°C per hour. Finally, the mixture was crystallized at a controlled temperature of 15°C to 20°C for 8 hours. The mixture was filtered. The filtered cake was rinsed with ethanol and dried to obtain 843.3 g of oxidized glutathione, with a one-step yield of 84.3% and a purity of 99.7%. The purity increase rate was 0.5%.

[0106] Example 28

[0107] In the example of synthesis of oxidized glutathione described in (1) above, 1.0 kg of crude oxidized glutathione was added to 5.0 L of purified water at 50°C, and 100 mL of 5% iron(II) chloride solution was added. The mixture was stirred until the solid dissolved. The mixture was filtered while hot and then gradually cooled to 20°C at a rate of 10°C per hour. Finally, the mixture was crystallized at a controlled temperature of 15°C to 20°C for 8 hours. The mixture was filtered. The filtered cake was rinsed with ethanol and dried to obtain 848.6 g of oxidized glutathione, with a one-step yield of 84.9% and a purity of 99.9%. The purity increase rate was 0.7%.

[0108] Example 29

[0109] In the above example (1) synthesis of oxidized glutathione, 1.0 kg of crude oxidized glutathione was added to 4.0 L of purified water at 50°C, and the mixture was stirred until the solid dissolved. The mixture was filtered while hot, and then gradually cooled to 20°C at a rate of 10°C per hour. Finally, the mixture was crystallized at a controlled temperature of 20°C to 25°C for 6 hours. The mixture was filtered. The filtered cake was rinsed with ethanol and dried to obtain 851.6 g of oxidized glutathione, with a one-step yield of 85.2% and a purity of 99.7%. The purity increase rate was 0.5%.

[0110] Example 30

[0111] Preparation of impurity A: 4.6 g of Boc-Cys(Trt)-OH, 1.7 g of glycine tert-butyl hydrochloride, and 7.6 g of HBTU were dissolved in 30 mL of DMF, and 2 mL of DIEA was added dropwise to the mixture. The reaction was carried out at room temperature for 24 hours, and then 100 mL of water was added. The mixture was extracted three times with ethyl acetate, the organic phase was retained, and dried on anhydrous magnesium sulfate. The solvent was evaporated to dryness, and the residue was dissolved in 30 mL of dichloromethane. 30 mL of 30% trifluoroacetic acid aqueous solution was added to remove protecting groups such as Trt, Boc, and tBu. After the reaction was complete, the layers were separated and the aqueous layer was retained. Then 50 mL of ethanol was added to the aqueous layer to precipitate the solid. The mixture was filtered by suction, and the filtered cake was purified by preparative liquid-phase chromatography to obtain Cys-Gly. Then reduced glutathione and Cys-Gly were dissolved in 20 mL of water in a 1:1 molar ratio. For oxidation, 5 equivalents of DMSO were added to the mixture. The reaction was carried out at room temperature for 10 hours, after which 40 mL of ethanol was added to precipitate the solid. The resulting solid was dissolved in 5 mL of 1% trifluoroacetic acid aqueous solution, and the mixture was separated by preparative liquid-phase chromatography. The mobile phases were phase A: 1% trifluoroacetic acid / water; and phase B: 1% trifluoroacetic acid / acetonitrile. Finally, 2.0 g of impurity A was obtained by lyophilization, with an overall yield of 42% and a purity of 99.7%. ESI-MS[M+H] + = 484.1.

[0112] Example 31

[0113] Preparation of impurity B: Dissolve 3.0 g of Boc-Glu-OtBu, 4.2 g of HCl·H-Cys(Trt)-OtBu, and 7.6 g of HBTU in 30 mL of DMF, and add 3 mL of DIEA was added dropwise to the mixture. The reaction was carried out at room temperature for 18 hours, after which 100 mL of water was added. The mixture was extracted three times with ethyl acetate, retaining the organic phase, and dried on anhydrous magnesium sulfate. The solvent was evaporated to dryness, and the residue was dissolved in 30 mL of dichloromethane. 40 mL of 30% trifluoroacetic acid aqueous solution was added to remove protecting groups such as Trt, Boc, and tBu. After the reaction was complete, the aqueous layer was separated. 50 mL of ethanol was then added to the aqueous layer to precipitate the solid. The mixture was filtered by suction, and the filtration cake was purified by preparative liquid-phase chromatography to obtain Glu-Cys. Reduced glutathione and Glu-Cys were then dissolved in 20 mL of water in a 1:1 molar ratio. 5 equivalents of DMSO were added to the mixture for oxidation. The reaction was carried out at room temperature for 12 hours, after which 40 mL of ethanol was added to precipitate the solid. The resulting solid was dissolved in 5 mL of purified water, and the mixture was separated by preparative liquid-phase chromatography. The mobile phases were Phase A: 1% trifluoroacetic acid / water; and Phase B: 1% trifluoroacetic acid / acetonitrile. Finally, 2.3 g of impurity B was obtained by lyophilization, with an overall yield of 41% and a purity of 99.7%. ESI-MS[M+H] + = 555.2.

[0114] Example 32

[0115] Preparation of impurity C: 3.1 g of reduced glutathione and 1.2 g of cysteine ​​were added to 15 mL of water, and 3.9 g of DMSO was added for oxidation. The mixture was reacted at room temperature for 9 hours with stirring. After the reaction was complete, 30 mL of ethanol was added to precipitate the solid. The resulting solid was dissolved in 5 mL of purified water, and the mixture was separated by preparative liquid-phase chromatography. The mobile phases were Phase A: 1% trifluoroacetic acid / water; and Phase B: 1% trifluoroacetic acid / acetonitrile. Finally, 2.1 g of impurity C was obtained by lyophilization, with an overall yield of 49% and a purity of 99.8%. ESI-MS[M+H] + = 427.1.

[0116] (3) Crystallographic properties and analysis

[0117] After testing, the melting point of the obtained crystal was 169°C (microscope-based melting point analyzer X-5, manufacturer: Corey Instrument), which differed from the reported melting point of the crystal, proving that this crystal is a new crystal form. Furthermore, the characteristic peaks (3 high peaks and 8 high peaks) obtained by X-ray powder diffraction testing (specification: D8 ADVANCE, manufacturer: Bruker Company, Germany, equipped with LynxEye detector, 2θ scanning angle 3°~40°, scanning step size 0.02°, scanning speed 0.3 sec / step, and tube voltage and tube current during sample measurement were 40KV and 40mA, respectively) also did not match the reported crystal form. Simultaneously, the associated peak shapes proved that the crystal form is not a mixture of crystals. Furthermore, TG-DSC testing (specification model: STA 449F5, manufacturer: NETZSCH, After heating in Germany, the weight loss of the sample at 50-180°C was measured at a slow heating rate (1.0°C / min). The resulting oxidized glutathione had a water content of 17.2%, which was in agreement with the water content of the sample measured by a Karl Fischer moisture meter (model: V20S, manufacturer: METTLER TOLEDO) (17.2%, results from three parallel runs). Since the water content matched the water content of oxidized glutathione heptahydrate calculated above, the obtained crystals were determined to be oxidized glutathione heptahydrate.

[0118] Table 11 below shows the difference in saturation solubility of the oxidized glutathione heptahydrate crystals and the oxidized glutathione monohydrate and hexahydrate crystals described above.

[0119] [Table 14]

[0120] At the same temperature, the water solubility of the heptahydrate is significantly better than that of the monohydrate and comparable to that of the hexahydrate. Furthermore, compared to the hexahydrate powder crystals, which tend to generate static electricity and suffer significant losses during the packaging process, the resulting heptahydrate granular crystals are more suitable for industrial production.

[0121] [Table 15]

[0122] In summary, this disclosure describes the preparation of oxidized glutathione and its novel crystalline forms and impurities. This method provides a way to simplify the synthesis of oxidized glutathione. The synthesis of impurities ensures the quality of subsequent oxidized glutathione testing. At the same time, using purified water to recrystallize the synthesized oxidized glutathione not only yields high-purity oxidized glutathione but also simplifies existing purification methods. Compared to existing crystalline forms, the newly discovered crystalline form of the heptahydrate has a simpler synthesis method, good water solubility, and good stability, making it more suitable for the industrial production of oxidized glutathione.

Claims

1. Equation (I): 【Chemistry 1】 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) 【Chemistry 2】 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 between 2.5:1 and 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 polar solvent per 100 g of the compound of formula (II); iii) The pH of the above reaction is adjusted to 2-9; iv) The reaction is carried out at a temperature of -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 step of filtering the solution while it is hot, and 3) The step of cooling the filtrate 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, further satisfying 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 step of recrystallizing the oxidized glutathione prepared according to any one of claims 5 to 9 in purified water is included, and the following steps are taken: 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 liters 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. 【Request Item 12】 【Chemistry 3】 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 condensing Boc-Cys(Trt)-OH and glycine tert-butyl ester in a molar ratio of 1:1 to 1:3 under the catalytic conditions of 1.5 to 4 equivalents of HBTU or DEPBT and 2 equivalents of N,N-diisopropylethylamine, wherein the reaction is carried out in DMF or dichloromethane. 2) The step of removing the Trt, Boc, and tBu protecting groups with trifluoroacetic acid to obtain Cys-Gly, and 3) A method comprising the step of oxidizing reduced glutathione and Cys-Gly in a 1:1 molar ratio with 3 to 5 equivalents of DMSO. 【Request Item 13】 【Chemistry 4】 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 condensing Boc-Glu-OtBu and H-Cys(Trt)-OtBu in a molar ratio of 1:1 to 1:2 under the catalytic conditions of 1.5 to 4 equivalents of HBTU or DEPBT and 2 equivalents of N,N-diisopropylethylamine. 2) The step of removing the Boc, Trt, and tBu protecting groups with trifluoroacetic acid to obtain Glu-Cys, and 3) A method comprising the step of 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. 【Transformation 5】

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