Glycylglutamine process impurities, methods of making and uses thereof

Abstract

The application discloses a glycyl glutamine process impurity and a preparation method and application thereof, and belongs to the technical field of analytical chemistry. The preparation method comprises the following steps: dissolving compound 1 in 4-10 times weight of water, adding 20-35 wt% of hydrogen peroxide, controlling the concentration of the hydrogen peroxide to be 1-3 wt%, and reacting at 40-60 DEG C for 4-6 hours; after the reaction is completed, the temperature is lowered to room temperature, saturated sodium sulfite solution is added for quenching; then, vacuum concentration is carried out at 45-55 DEG C, and the concentration of the target product is concentrated to 30-60 wt%; hydrochloric acid is added until the pH is 3.0-4.0, ethanol is added until the volume ratio of water to ethanol is 1:0.5-2.0, the temperature is lowered to 0-5 DEG C, and stirring crystallization is carried out for 2-3 hours; solid-liquid separation is carried out, and the solid ethanol is eluted; the wet product is vacuum dried at 40-50 DEG C for 5-6 hours, and the process impurity K is obtained.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis technology, and specifically relates to a process impurity of glycylglutamine, its preparation method and application. The impurity is a trace product generated by the oxidation and destruction of glycylglutamine, with a content of about 0.1-1.0 wt%. Background Technology

[0002] Glycylglutamine is a synthetic polypeptide amino acid and one of the main components of a 15-amino acid dipeptide injection. Glycylglutamine primarily provides glutamine to promote protein synthesis. Glutamine plays a crucial role in parenteral nutrition, reducing intestinal mucosal atrophy, enhancing the activity of small and colonic cells, thereby improving intestinal mucosal function and reducing bacterial and endotoxin ectopic growth in the intestines. Therefore, glycylglutamine has broad prospects for development in the medical field.

[0003] The glycylglutamine process impurity K prepared in this patent is based on the cyclization and deamination of glycylglutamine. Currently, there are no reports on the preparation method of this impurity. The conditions for the generation of impurity K are affected by factors such as solution pH and temperature, and it is difficult to occur during normal storage. However, the study of this impurity can be used for the qualitative and quantitative analysis of impurities in glycylglutamine (such as oxidative degradation), which is of positive significance for improving the quality standards of glycylglutamine and for pharmacological and toxicological studies. Summary of the Invention

[0004] On one hand, embodiments of the present invention provide a glycylglutamine process impurity (referred to as K for ease of description), the structural formula of which is as follows:

[0005] .

[0006] This impurity is a white to off-white solid powder with a melting point of about 190°C. It is hygroscopic and should be stored in a sealed container in an inert gas environment, protected from light, under frozen (<-20°C) conditions. It is soluble in water and can precipitate in a mixture of water and ethanol.

[0007] On the other hand, this embodiment of the invention also provides an application of glycylglutamine process impurities in glycylglutamine impurity analysis, specifically for oxidative damage analysis.

[0008] In another aspect, embodiments of the present invention also provide a method for preparing process impurities of glycylglutamine, the reaction process of which is as follows: .

[0009] In this process, compound 1 (glycyl-L-glutamine) reacts with hydrogen peroxide in water to produce impurity K at a reaction temperature of 40-60℃. The concentration of compound 1 in the reaction system is 10-20 wt%, and the concentration of hydrogen peroxide is 1-3 wt%. After the reaction is complete, impurity K is obtained by solvent crystallization at a pH of 3.0-4.0. In this patent, the concentration of compound 1 must be controlled within a suitable range. A high concentration will result in a high dissolution temperature, leading to the formation of impurities such as cyclic-(glycyl-L-glutamine). A low concentration will result in high energy consumption for subsequent concentration. As for hydrogen peroxide, its concentration cannot be too high; otherwise, the hydrogen peroxide will exhibit oxidizing properties, leading to numerous side reactions.

[0010] Further, after the reaction is complete, the product is quenched with a saturated sodium sulfite solution. After quenching, the solution is concentrated under reduced pressure to a concentration of 30-60 wt% (usually concentrated to 1 / 4-1 / 2 volume). The concentrated solution is then adjusted to pH 3.0-4.0 with acid, and ethanol is added before cooling and crystallization to obtain impurity K. In the crystallization system, the volume ratio of water to ethanol is 1:0.5-2.0. Specifically, if there is too little ethanol, no precipitation or a low yield will occur; if there is too much ethanol, an oil will form.

[0011] During the reaction, the endpoint was determined by thin-layer chromatography; the developing solvent for thin-layer chromatography was n-butanol:acetic acid:water in a volume ratio of 4:1:1.

[0012] In the quenching process, saturated sodium sulfite solution is added until no more bubbles are produced. The quenching endpoint is then determined by testing with starch-potassium iodide test paper. If the test paper does not turn blue, the quenching endpoint is reached.

[0013] The reaction time is 4-6 hours.

[0014] The concentration of hydrogen peroxide is 10-35 wt%.

[0015] The concentration under reduced pressure is carried out at a temperature of 45-55℃. In this patent, it is necessary to control the concentration temperature to avoid side reactions. If the temperature is too high, the product yield will decrease.

[0016] In this patent, both the reaction temperature and the concentration temperature are required to be low; if they are high (especially the reaction temperature), the product yield will be low. This can be seen from the subsequent examples. Figure 1 The reaction process in this patent is very complex (one-step reaction, yield less than 50%), and the crystalline product contains a large number of byproducts (more than 10, and the impurities in the reaction product can only be more), requiring strict control of the reaction conditions. Furthermore, although this patent has many side reactions and complex products, a high purity (>97%) product can be obtained through a simple purification method (but strict control of the water-to-ethanol ratio in the system is required).

[0017] Furthermore, after crystallization, solid-liquid separation is performed, followed by ethanol rinsing and drying to obtain impurity K.

[0018] Specifically, the method for preparing glycylglutamine process impurities provided in this embodiment of the invention includes the following steps: S1 catalytic reaction: Dissolve compound 1 in 4-10 times its weight of water, add 10-35 wt% hydrogen peroxide and control the concentration of hydrogen peroxide to 1-3 wt% and the concentration of compound 1 to 10-20 wt%, react at 40-60℃ for 4-6 hours, and determine the reaction endpoint by thin-layer chromatography.

[0019] S2 Quenching and Concentration: After the reaction is complete, cool to room temperature and add saturated sodium sulfite solution for quenching. During quenching, add saturated sodium sulfite solution until no more bubbles are produced. Detect the quenching endpoint using starch-potassium iodide test paper. If the test paper does not turn blue, the quenching endpoint is reached. Then concentrate under reduced pressure at 45-55℃ until the concentration of the target product is 30-60 wt%.

[0020] S3 Crystallization and Purification: Add hydrochloric acid to the concentrated solution to pH 3.0-4.0, add ethanol to make the volume ratio of water to ethanol 1:0.5-2.0, cool to 0-5℃, stir to precipitate crystals for 2-3 hours, separate solid and liquid, and wash the solid with ethanol.

[0021] S4 Drying: The wet product is vacuum dried at 40-50℃ for 5-6 hours to obtain impurity K.

[0022] After obtaining impurity K according to this patent, thin-layer chromatography revealed that the glycylglutamine oxidation degradation product had a band corresponding to impurity K. Using the glycylglutamine oxidation degradation product as the test solution and impurity K as the impurity reference standard, the content of impurity K was determined by high-performance liquid chromatography. The results showed that the content of impurity K in the glycylglutamine oxidation degradation product was 0.1-1.0 wt%.

[0023] The patented preparation method features a rationally designed circuit, simple operation, mild reaction conditions, and readily available raw materials (this patent uses the analyte as the raw material). The resulting target product has a purity of over 95%, which can be used for qualitative and quantitative analysis of impurities, and is of positive significance for the quality research of glycylglutamine raw materials. Specifically, in this patent, a product with a purity of over 97% can be obtained solely through ethanol crystallization at a relatively low yield. Attached Figure Description

[0024] Figure 1 This is the HPLC detection result of impurity K in the glycylglutamine prepared by this invention; Figure 2 This is the MS detection result of impurity K in the glycylglutamine prepared by this invention; Figure 3 It is the process impurity K in the glycylglutamine prepared by this invention. 1 H-NMR detection results; Figure 4 It is the process impurity K in the glycylglutamine prepared by this invention. 13 C-NMR detection results. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0026] Example 1 Compound 1 (22.1 g, 0.1 mol) was dissolved in 90 ml of water, and 3 ml of 30 wt% hydrogen peroxide was added. The reaction was carried out at 50 °C for 5 h. After the reaction was complete, the temperature was lowered to 20 °C, and saturated sodium sulfite solution was added until no bubbles were produced. The reaction was tested with starch-potassium iodide test paper; the paper did not turn blue, indicating the reaction was complete. The solution was concentrated under reduced pressure at 50 °C, and approximately 61 ml of water was distilled off. The concentrated solution was acidified to pH 3.5 with hydrochloric acid, and then 26 ml of ethanol was added. The mixture was stirred and cooled to 0 °C, and crystallization was allowed to occur for 2 hours. The crystals were filtered to obtain 12.6 g of solid, which was dried under reduced pressure at 50 °C to obtain 9.8 g of solid with a purity of 97.1%. The purity was determined as follows: Figure 1-4 .

[0027] Example 2 Compound 1 (22.1 g, 0.1 mol) was dissolved in 85 ml of water, and 5 ml of 30 wt% hydrogen peroxide was added. The reaction was carried out at 55 °C for 5 h. After the reaction was completed, the temperature was lowered to 20 °C, and quenching was performed with saturated sodium sulfite solution. After quenching, the solution was concentrated under reduced pressure at 55 °C, and about 65 ml of water was distilled off. The concentrated solution was acidified to pH 3.8 with hydrochloric acid, and then 25 ml of ethanol was added. The mixture was stirred and cooled to 0 °C for 2 hours to allow crystals to precipitate. The solution was filtered to obtain 12.0 g of solid, which was dried under reduced pressure at 50 °C to obtain 9.2 g of solid.

[0028] Comparative Example 1 Compound 1 (22.1 g, 0.1 mol) was dissolved in 90 ml of water, and 3 ml of 30 wt% hydrogen peroxide was added. The reaction was carried out at 50 °C for 5 h. After the reaction was completed, the temperature was lowered to 20 °C, and saturated sodium sulfite solution was added until no bubbles were produced. The reaction was tested with starch-potassium iodide test paper. The test paper did not turn blue, indicating the reaction was complete. The solution was concentrated under reduced pressure at 60 °C, and about 61 ml of water was distilled off. The concentrated solution was acidified to pH 3.5 with hydrochloric acid, and then 26 ml of ethanol was added. The mixture was stirred and cooled to 0 °C. Crystallization was allowed to occur for 2 hours. The solution was filtered to obtain 10.6 g of solid, which was dried under reduced pressure at 50 °C to obtain 8.8 g of solid.

[0029] As can be seen from Comparative Example 1, higher concentration temperatures reduce the yield of the target product.

[0030] Comparative Example 2 Compound 1 (22.1 g, 0.1 mol) was dissolved in 90 ml of water, and 3 ml of 30 wt% hydrogen peroxide was added. The reaction was carried out at 80 °C for 4 h. After the reaction was completed, the temperature was lowered to 20 °C, and saturated sodium sulfite solution was added until no bubbles were produced. The reaction was tested with starch-potassium iodide test paper. The test paper did not turn blue, indicating the reaction was complete. The solution was concentrated under reduced pressure at 50 °C, and about 74 ml of water was distilled off. The concentrated solution was acidified to pH 3.5 with hydrochloric acid, and then 14 ml of ethanol was added (the ratio of ethanol to water was controlled to be similar to that in Example 1 and also similar to that of the product). The mixture was stirred and cooled to 0 °C. Crystallization was carried out for 2 hours, and the solution was filtered to obtain 6.8 g of solid. The solid was dried under reduced pressure at 50 °C to obtain 5.6 g of solid.

[0031] Comparative Example 2 shows that higher reaction temperatures significantly reduce the yield of the target product.

[0032] Comparative Example 3 Compound 1 (22.1 g, 0.1 mol) was dissolved in 90 ml of water, and 20 ml of 30 wt% hydrogen peroxide was added. The reaction was carried out at 50 °C for 5 h. After the reaction was completed, the temperature was lowered to 20 °C, and saturated sodium sulfite solution was added until no bubbles were produced. The reaction was tested with starch-potassium iodide test paper. The test paper did not turn blue, indicating the reaction was complete. The solution was concentrated under reduced pressure at 50 °C, and about 91 ml of water was distilled off. The concentrated solution was acidified to pH 3.5 with hydrochloric acid, and then 12 ml of ethanol was added (the ratio of ethanol to water was controlled to be similar to that in Example 1 and also similar to that of the product). The solution was stirred and cooled to 0 °C. Crystallization was allowed to occur for 2 hours. The solution was filtered to obtain 5.6 g of solid, which was dried under reduced pressure at 50 °C to obtain 4.7 g of solid with a purity of 90.6%.

[0033] Comparative Example 3 shows that reacting under higher concentrations of hydrogen peroxide significantly reduces the quality and yield of the target product.

[0034] Comparative Example 4 Compound 1 (22.1 g, 0.1 mol) was dissolved in 90 ml of water, and 3 ml of 30 wt% hydrogen peroxide was added. The reaction was carried out at 50 °C for 5 h. After the reaction was completed, the temperature was lowered to 20 °C, and saturated sodium sulfite solution was added until no bubbles were produced. The reaction was tested with starch-potassium iodide test paper. The test paper did not turn blue, indicating the reaction was complete. The solution was concentrated under reduced pressure at 50 °C, and about 60 ml of water was distilled off. The concentrated solution was acidified to pH 3.5 with hydrochloric acid, and then 100 ml of ethanol was added. Crystallization occurred under stirring to form an oil, and no solid was obtained.

[0035] As can be seen from Comparative Example 4, the crystallization of the target product needs to be carried out under a specific solvent ratio.

[0036] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Glycylglutamine process impurities, characterized in that, Its structural formula is as follows: 。 2. The application of glycylglutamine process impurities as described in claim 1 in the analysis of glycylglutamine impurities.

3. The method for preparing process impurities of glycylglutamine as described in claim 1, characterized in that, The reaction process is as follows: ； In this process, compound 1 reacts in water under the catalysis of hydrogen peroxide to obtain impurity K at a reaction temperature of 40-60℃. In the reaction system, the concentration of compound 1 is 10-20wt%, and the concentration of hydrogen peroxide is 1-3wt%. After the reaction is completed, impurity K is obtained by solvent crystallization under pH 3.0-4.0 conditions.

4. The preparation method according to claim 3, characterized in that, After the reaction is completed, the product is quenched with a saturated sodium sulfite solution. After quenching, the product is concentrated under reduced pressure to a concentration of 30-60 wt%. The pH of the concentrate is adjusted to 3.0-4.0 by adding acid. Then, ethanol is added and the mixture is cooled to crystallize and impurity K is obtained. In the crystallization system, the volume ratio of water to ethanol is 1:0.5-2.

0.

5. The preparation method according to claim 3, characterized in that, During the reaction, the reaction endpoint was determined by thin-layer chromatography; the developing solvent for thin-layer chromatography was n-butanol:acetic acid:water in a volume ratio of 4:1:

1.

6. The preparation method according to claim 4, characterized in that, During quenching, add saturated sodium sulfite solution until no more bubbles are produced. Test with starch-potassium iodide test paper; if the test paper does not turn blue, the quenching endpoint is reached.

7. The preparation method according to claim 3, characterized in that, The reaction time is 4-6 hours.

8. The preparation method according to claim 3, characterized in that, The concentration of hydrogen peroxide is 10-35 wt%.

9. The preparation method according to claim 4, characterized in that, The temperature for vacuum concentration is 45-55℃.

10. The preparation method according to claim 3, characterized in that, The method includes: S1 catalytic reaction: Dissolve compound 1 in 4-10 times its weight of water, add 10-35 wt% hydrogen peroxide and control the concentration of hydrogen peroxide to 1-3 wt%, react at 40-60℃ for 4-6 hours, and determine the reaction endpoint by thin-layer chromatography. S2 quenching and concentration: After the reaction is complete, cool to room temperature and add saturated sodium sulfite solution for quenching; then concentrate under reduced pressure at 45-55℃ until the concentration of the target product is 30-60 wt%. S3 Crystallization and Purification: Add hydrochloric acid to the concentrated solution to pH 3.0-4.0, add ethanol to make the volume ratio of water to ethanol 1:0.5-2.0, cool to 0-5℃, stir to precipitate crystals for 2-3 hours, separate solid and liquid, and wash the solid with ethanol; S4 Drying: The wet product is vacuum dried at 40-50℃ for 5-6 hours to obtain impurity K.

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