Glycyl glutamine potential impurity and preparation method thereof
During the production process of glycylglutamine, the preparation method under mild reaction conditions was adopted to obtain high-purity potential impurity J through silica gel column purification, which solved the problem of impurity generation in the prior art and achieved the goal of high purity Products that support the improvement of glycylglutamine quality standards and pharmacological toxicological analysis.
Patent Information
- Application Number
- CN202411250541.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-06
- Publication Date
- 2025-01-14
AI Technical Summary
During the production process of glycylglutamine, the intermediate N-chloroacetyl-L-glutamine prepared using chloroacetyl chloride undergoes halogenated ammonialysis with glycylglutamine, resulting in by-product impurity J, and The lack of the preparation method of this impurity affects the quality standards and pharmacological toxicological analysis of glycylglutamine.
By using the preparation method under mild reaction conditions, N-chloroacetyl-L-glutamine is formed by reacting glutamine and chloroacetyl chloride in alkaline aqueous solution, and reacting with glycyl glutamine in alkaline aqueous solution. Subsequent purification by silica gel column yields potential impurity J, the reaction temperature and the concentration of alkaline aqueous solution are controlled, and the target product with a specific eluent is achieved with high purity.
The high-purity preparation of potential impurities of glycylglutamine (purity up to more than 98%) was achieved, and it was used for qualitative and quantitative analysis, which improved the quality standards of raw materials and had positive significance for pharmacological and toxicological research.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a glycylglutamine potential impurity and a preparation method thereof. Background Art
[0002] Glycylglutamine is a synthetic polypeptide amino acid. It is one of the main ingredients of 15 kinds of compound amino acid dipeptide injection. Glycine glutamine is mainly used to provide glutamine to promote protein synthesis. Glutamine plays a very important role in parenteral nutrition. It can reduce intestinal mucosal atrophy, enhance the activity of small intestine and colon cells, thereby enhancing intestinal mucosal function and reducing bacteria and endotoxin in the intestine. Therefore, glycyl-L-glutamine has broad development space in the medical field.
[0003] The potential impurity J of glycylglutamine prepared this time is a process impurity in the preparation of glycylglutamine using chloroacetyl chloride as the route. There is currently no report on the preparation method of this impurity. In the production process of preparing glycylglutamine using L-glutamine and chloroacetyl chloride as the route, the intermediate N-chloroacetyl-L-glutamine prepared using chloroacetyl chloride undergoes halogenated aminolysis with glycylglutamine, which inevitably generates the byproduct impurity J. The study of this impurity can be used for the qualitative and quantitative analysis of impurities in glycylglutamine, which has positive significance for improving the quality standard of glycylglutamine and conducting pharmacology and toxicology. Summary of the invention
[0004] This patent provides a method for preparing potential impurities of glycylglutamine. The circuit design is reasonable, the operation is simple, the reaction conditions are mild (water is used as the reaction solvent, and the reaction temperature is mild), the raw materials are easily available (the raw materials for the synthesis of glycylglutamine and itself), and the purity of the obtained target product is more than 98%. It can be used for qualitative and quantitative analysis of impurities, and has positive significance for the quality research of glycylglutamine raw materials.
[0005] The structural formula of the glycylglutamine potential impurity (abbreviated as J for ease of description) disclosed in the present invention is as follows: .
[0006] The reaction process of the potential impurity of glycylglutamine is as follows: .
[0007] Wherein, glutamine (compound 1) and chloroacetyl chloride react in alkaline water-soluble A to obtain compound 2, the pH of the reaction system is ≥9, and the reaction temperature is 0-10°C. Compound 2 (N-chloroacetyl-L-glutamine) reacts with glycylglutamine in alkaline water-soluble B, the reaction temperature is 20-50°C, and after the reaction is completed, the potential impurity J is purified by silica gel column. Wherein, alkaline water-soluble B is selected from sodium carbonate solution or potassium carbonate solution. The concentration of alkaline water-soluble B is 5-20wt%, preferably, the concentration is 10-20wt%. The molar ratio of compound 2, glycylglutamine and base B is 1:0.9-1.0:1.5-3.0, preferably, the molar ratio of compound 2, glycylglutamine and base B is 1:1:2.
[0008] Specifically, the silica gel column purification process is: after the reaction is completed, silica gel is added to the reaction solution, and the reaction solution is concentrated to dryness under reduced pressure to obtain a solid, and the solid is subjected to silica gel column chromatography to obtain potential impurity J. The amount of silica gel used is 0.5-1.5 times the weight of compound 2; preferably, the amount of silica gel used is 1 times the weight of compound 2. Methanol-dichloromethane-acetic acid is used as the eluent, and the volume ratio of methanol: dichloromethane: acetic acid is 2:1:0.01-0.03.
[0009] Wherein, the alkaline aqueous solution A is a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 5-30wt%, preferably 15-20wt%. The molar ratio of L-glutamine, chloroacetyl chloride and sodium hydroxide is 1:1.1-1.5:0.5-1.2. Preferably, the molar ratio of L-glutamine, chloroacetyl chloride and sodium hydroxide is 1:1.1-1.2:1.0-1.2.
[0010] After the reaction of glutamine and chloroacetyl chloride is completed, the pH is adjusted to 1-3, crystallization is performed, and solid-liquid separation is performed to obtain compound 2.
[0011] The method for preparing potential impurities of glycylglutamine provided by the present invention comprises: S1 Chloroacetylation: First dissolve glutamine in water, then drop sodium hydroxide solution; control the dropping temperature at 0-10°C, then drop chloroacetyl chloride, and at the same time drop sodium hydroxide solution to maintain pH ≥ 9; after the dropwise addition is completed, react for 1-2 hours, after the reaction is completed, add concentrated hydrochloric acid to adjust the pH to 1-3, stir and crystallize, and filter to obtain compound 2. Wherein, the molar ratio of L-glutamine, chloroacetyl chloride and sodium hydroxide is 1:1.1-1.5:0.5-1.2, and the concentration of sodium hydroxide solution is 5-30wt%.
[0012] S2 halogenated aminolysis and silica gel column purification: Compound 2 and glycylglutamine are dissolved in alkaline aqueous solution B, reacted at 20-50°C for 6-8 hours, and after the reaction is completed, silica gel is added, and concentrated under reduced pressure (temperature is 50-65°C) to dryness to obtain a solid, and the solid is subjected to silica gel column chromatography, and the eluent is concentrated under reduced pressure (temperature is 50-60°C) to dryness to obtain potential impurity J. Wherein, alkaline aqueous solution B is selected from sodium carbonate solution or potassium carbonate solution, and the concentration of alkaline aqueous solution B is 5-20wt%. The molar ratio of compound 2, glycylglutamine and base B is 1:0.9-1.0:1.5-3.0, the amount of silica gel is 0.5-1.5 times the weight of compound 2, and methanol-dichloromethane-acetic acid is used as the eluent, and the volume ratio of methanol:dichloromethane:acetic acid is 2:1:0.01-0.03.
[0013] Preferably, in step S1, the reaction temperature is 0-5°C.
[0014] Preferably, in step S1, concentrated hydrochloric acid is added to adjust the pH to 1-2.
[0015] Preferably, in step S2, the reaction temperature is 30-40°C. In step S1, glutamine has two amino groups. Under alkaline conditions and low temperatures, amide is not easy to react with acyl chloride, nor is it easy to self-react, and amide is not easy to decompose. The role of adding acid is: first, to convert salt into acid, and second, to allow the product to precipitate.
[0016] In step S2, compound 2 has an amino group, a carboxyl group and a chloro group, and glycylglutamine has two amino groups and a carboxyl group, and each group has the possibility of reaction. The applicant found that under weak alkalinity, the reaction of step S2 is the main reaction (the reaction solution is subjected to liquid chromatography analysis, and the purity of the target product is only 50-60%), allowing other reactions to proceed. Finally, it is purified by a special purification method. Under weak alkaline conditions, the main reaction can be promoted; however, if the alkalinity is too strong, the amide group is easily decomposed, which will make the reaction more complicated, and the alkalinity and concentration of the base are required. For silica gel column purification, since the reaction of this patent is very complicated and the purification difficulty is very high, the requirements for the eluent are very high, which can only be achieved under specific combinations and ratios. Even under optimal conditions, the yield of high-purity products (more than 98%, simple ingredients, suitable for drug analysis, and only one impurity (HPLC detection)) is only about 10%, which is very small, but sufficient for drug analysis. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 It is the HPLC detection result of the potential impurity J of glycylglutamine prepared by the present invention.
[0018] Figure 2This is the MS detection result of the potential impurity J of glycylglutamine prepared by the present invention.
[0019] Figure 3 It is the potential impurity J of glycylglutamine prepared by the present invention. 1 H-NMR test results.
[0020] Figure 4 It is the potential impurity J of glycylglutamine prepared by the present invention. 13 C-NMR test results. DETAILED DESCRIPTION
[0021] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.
[0022] (I) Synthesis of Compound 2 1.1 Add L-glutamine (146g, 1.0mol) and water (50ml) to a 1L reaction bottle, stir evenly, add 220g 20wt% sodium hydroxide aqueous solution (44g, 1.1mol), cool the reaction temperature to 0℃, control the temperature at 0℃-5℃, and add chloroacetyl chloride (124.2g, 1.1mol) and 20wt% sodium hydroxide aqueous solution (control the reaction pH ≥ 9) at the same time. After the addition is complete, control the temperature at 0℃-5℃ and react for 1 hour. After the reaction is complete, adjust the pH to 2 with concentrated hydrochloric acid, stir and crystallize to obtain N-chloroacetyl-L-glutamine (182.5g, purity 98.6%), with a yield of 82.0%.
[0023] 1.2 Add L-glutamine (146g, 1.0mol) and water (50ml) to a 1L reaction bottle, stir evenly, add 200g 20wt% sodium hydroxide aqueous solution (40g, 1.0mol), cool the reaction temperature to 5℃, control the temperature at 5℃-10℃, add chloroacetyl chloride (135.6g, 1.2mol) and 20wt% sodium hydroxide aqueous solution (control the reaction pH ≥ 9), and control the temperature at 5℃-10℃ for 2 hours after the addition. After the reaction is completed, adjust the pH to 1 with concentrated hydrochloric acid, stir and crystallize to obtain N-chloroacetyl-L-glutamine (180.2g, purity 98.1%), with a yield of 80.9%.
[0024] (II) Synthesis of Impurity J 2.1 Compound 2 (22.2 g, 0.1 mol) and glycylglutamine (22.1 g, 0.1 mol) were dissolved in 180 g 15 wt% potassium carbonate (27.6 g, 0.2 mol) aqueous solution and reacted at 50 °C for 8 h. After the reaction was completed, 22.2 g silica gel (1.0 times the amount) was added and concentrated to dryness under reduced pressure at 55-65 °C to obtain a solid for silica gel column purification. The eluent was selected as methanol: dichloromethane: acetic acid, and the ratio was methanol: dichloromethane: acetic acid (volume ratio) = 2:1:0.02. The eluent was concentrated to dryness under reduced pressure at 50 °C to obtain 4 g of solid with a purity of 98.3%. The purity test was as follows: Figure 1 .
[0025] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. Glycylglutamine potential impurity, characterized in that: Its structural formula is as follows: 。 2. The method for preparing the potential impurities of glycylglutamine as claimed in claim 1, characterized in that: The reaction process is as follows: ； Among them, glutamine and chloroacetyl chloride react in alkaline water solution A to obtain compound 2, the pH of the reaction system is ≥9, and the reaction temperature is 0-10°C; compound 2 reacts with glycylglutamine in alkaline water solution B, the reaction temperature is 20-50°C, and after the reaction is completed, silica gel column purification is performed to obtain potential impurity J, the alkaline water solution B is selected from sodium carbonate solution or potassium carbonate solution, the concentration of the alkaline water solution B is 5-20wt%, and the molar ratio of the compound 2, glycylglutamine and base B is 1:0.9-1.0:1.5-3.
0.
3. The preparation method according to claim 2, characterized in that: The process of silica gel column purification is as follows: after the reaction is completed, silica gel is added to the reaction solution, and the solution is concentrated to dryness under reduced pressure to obtain a solid, and the solid is subjected to silica gel column chromatography to obtain potential impurity J; the amount of the silica gel used is 0.5-1.5 times the weight of compound 2, and methanol-dichloromethane-acetic acid is used as the eluent, and the volume ratio of methanol:dichloromethane:acetic acid is 2:1:0.01-0.
03.
4. The preparation method according to claim 2, characterized in that: The alkaline aqueous solution A is a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 5-30wt%.
5. The preparation method according to claim 4, characterized in that: The molar ratio of L-glutamine, chloroacetyl chloride and sodium hydroxide is 1:1.1-1.5:0.5-1.
2.
6. The preparation method according to claim 2, characterized in that: After the reaction of glutamine and chloroacetyl chloride is completed, the pH is adjusted to 1-3, crystallization is performed, and solid-liquid separation is performed to obtain compound 2.
7. The preparation method according to claim 2, characterized in that: The method comprises: S1 Chloroacetylation: dissolve glutamine in a sodium hydroxide solution, cool to 0-10°C, and simultaneously add chloroacetyl chloride and a sodium hydroxide solution dropwise to maintain a pH value of ≥9. After the reaction is completed, add concentrated hydrochloric acid to adjust the pH value to 1-3, stir and crystallize, and filter to obtain compound 2; the molar ratio of L-glutamine, chloroacetyl chloride and sodium hydroxide is 1:1.1-1.5:0.5-1.2, and the concentration of the sodium hydroxide solution is 5-30wt%; S2 halogenated aminolysis and silica gel column purification: Compound 2 and glycylglutamine are dissolved in alkaline water-soluble B, and reacted at 20-50°C. After the reaction is completed, silica gel is added, and the mixture is concentrated to dryness under reduced pressure to obtain a solid. The solid is subjected to silica gel column chromatography, and the eluent is concentrated to dryness under reduced pressure to obtain a potential impurity J. The alkaline water-soluble B is selected from sodium carbonate solution or potassium carbonate solution, and the concentration of the alkaline water-soluble B is 5-20wt%. The molar ratio of the compound 2, glycylglutamine and base B is 1:0.9-1.0:1.5-3.0, and the amount of silica gel used is 0.5-1.5 times the weight of the compound 2. Methanol-dichloromethane-acetic acid is used as the eluent, and the volume ratio of methanol:dichloromethane:acetic acid is 2:1:0.01-0.
03.
8. The preparation method according to claim 7, characterized in that: In step S1, glutamine is first dissolved in water, and then sodium hydroxide solution is added dropwise; then chloroacetyl chloride is added dropwise, and sodium hydroxide solution is added dropwise at the same time to maintain pH ≥ 9; after the addition is completed, the reaction is carried out for 1-2 hours.
9. The preparation method according to claim 7, characterized in that: In step S2, the reaction time is 6-8 hours.
10. The preparation method according to claim 7, characterized in that: In step S2, the temperature for reducing pressure and concentrating the silica gel is 50-65°C, and the temperature for reducing pressure and concentrating the eluent is 50-60°C.