A method for separating enantiomers of N-Boc-L-pyroglutamic acid ethyl ester and its application

Abstract

This invention discloses a method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester and its application, comprising the following steps: taking a test sample containing the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester, and separating them by liquid chromatography. The liquid chromatography conditions include: using a Daicel AD-H column as the stationary phase, and using a mixed solution containing n-hexane and anhydrous ethanol as the mobile phase for isocratic elution, wherein the volume ratio of n-hexane to anhydrous ethanol in the mobile phase is 3-9:1. The chromatographic conditions of this invention can achieve effective separation of N-Boc-L-pyroglutamic acid ethyl ester and its enantiomers, with a resolution of 9.77 or higher. Furthermore, the short retention time of N-Boc-L-pyroglutamic acid ethyl ester in this invention enables rapid and effective separation, providing technical assurance for controlling the quality of N-Boc-L-pyroglutamic acid ethyl ester.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for separating enantiomers of N-Boc-L-pyroglutamic acid ethyl ester and its application. Background Technology

[0002] L-pyroglutamate esters are an important and promising class of chiral inducing agents. They possess a five-membered nitrogen-containing heterocycle and a bifunctional structure with ester carbonyl and amide groups, and each of the five carbons exhibits different reactivity. Therefore, L-pyroglutamate esters can serve as ideal chiral raw materials for the synthesis of a large number of natural or non-natural chiral compounds. Furthermore, due to their natural thermal insulation properties, they are widely used as raw materials / intermediates in the daily chemical and pharmaceutical industries. N-Boc-L-pyroglutamate ethyl ester has become one of the most commonly used L-pyroglutamate esters due to its relatively mild preparation conditions and simple operation. However, the proportion of its enantiomer, N-Boc-D-pyroglutamate ethyl ester, directly affects subsequent processes and, consequently, the efficacy of the drug. Therefore, isolating the enantiomers of N-Boc-L-pyroglutamate ethyl ester is of great significance for improving the quality control of N-Boc-L-pyroglutamate ethyl ester.

[0003] However, there are no reports on methods for separating enantiomers of N-Boc-L-pyroglutamic acid ethyl ester in the existing technology. Summary of the Invention

[0004] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester.

[0005] This invention also proposes a method for detecting the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester.

[0006] The present invention also proposes applications of the above separation or detection methods.

[0007] According to one aspect of the present invention, a method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester is provided, comprising the following steps: taking a test sample containing the enantiomer of N-Boc-L-pyroglutamic acid ethyl ester, and separating it by liquid chromatography, wherein the chromatographic conditions of the liquid chromatography include: using a Daicel column AD-H as the stationary phase, and using a mixed solution containing n-hexane and anhydrous ethanol as the mobile phase for isocratic elution, wherein the volume ratio of n-hexane to anhydrous ethanol in the mobile phase is 3 to 9:1, preferably 5.67:1.

[0008] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved: the chromatographic conditions of the present invention enable effective separation of N-Boc-L-pyroglutamic acid ethyl ester and its enantiomers, with a separation degree of 9.77 or higher. Simultaneously, the short retention time of N-Boc-L-pyroglutamic acid ethyl ester in the present invention allows for rapid and effective separation, providing technical assurance for controlling the quality of N-Boc-L-pyroglutamic acid ethyl ester.

[0009] In some embodiments of the present invention, the specifications of the Daicel AD-H chromatographic column include: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0010] In some embodiments of the present invention, the chromatographic conditions of the liquid chromatography further include: a flow rate of 0.5 ml / min to 1.5 ml / min; preferably 1 ml / min.

[0011] In some embodiments of the present invention, the chromatographic conditions of the liquid chromatography further include: the column temperature is room temperature.

[0012] In some embodiments of the present invention, the room temperature is 20°C to 35°C; preferably 25°C to 35°C.

[0013] In some embodiments of the present invention, the chromatographic conditions of the liquid chromatography further include: an injection volume of 10 μl to 30 μl, preferably 20 μl.

[0014] In some embodiments of the present invention, diethylamine is added to the mobile phase and / or diluent. The addition of diethylamine improves the peak shape.

[0015] In some embodiments of the present invention, the volume percentage of diethylamine in the mobile phase and / or diluent is 0.1% to 2%.

[0016] In some embodiments of the present invention, the test sample is a test sample treated with a diluent, wherein the diluent is n-hexane-anhydrous ethanol-diethylamine. The present invention allows for the addition of the diluent only, thus saving costs.

[0017] In some embodiments of the present invention, the test sample is a test sample treated with a diluent, wherein the diluent is n-hexane-anhydrous ethanol-diethylamine, and the volume ratio of the three is 85:15:(0.1-2).

[0018] In some preferred embodiments of the present invention, the test sample is a test sample treated with a diluent, wherein the diluent is n-hexane-anhydrous ethanol-diethylamine, and the volume ratio of the three is 85:15:1.

[0019] According to another aspect of the present invention, a method for detecting the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester is provided, the method comprising the following steps: after separation by the above separation method, the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester are detected by a detector.

[0020] The detection method according to a preferred embodiment of the present invention has at least the following beneficial effects: after separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester by the scheme of the present invention, when detected by the detector, the chromatographic peak shape is good, the baseline is stable, and the main peak position of N-Boc-L-pyroglutamic acid ethyl ester and its enantiomers can be accurately located. It has the advantages of strong specificity, high precision, high accuracy and good robustness.

[0021] In some embodiments of the present invention, the detector is an ultraviolet detector.

[0022] In some embodiments of the present invention, the detection wavelength of the ultraviolet detector is 210 nm.

[0023] In some embodiments of the present invention, the detection method further includes a step of qualitative analysis of the detection results by retention time.

[0024] In some embodiments of the present invention, the detection method further includes a step of quantitatively analyzing the detection results using the area normalization method.

[0025] According to another aspect of the present invention, the application of the above-described separation or detection method in the quality control of N-Boc-L-pyroglutamic acid ethyl ester is proposed.

[0026] The quality control method according to a preferred embodiment of the present invention has at least the following beneficial effects: the separation method and detection method of the present invention have good application prospects in the quality control of N-Boc-L-pyroglutamic acid ethyl ester.

[0027] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0028] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0029] Figure 1 This is the chromatogram of the blank solution detected in Example 1 of the present invention;

[0030] Figure 2 This is a chromatogram of the solution used to detect the suitability of the detection system in Embodiment 1 of the present invention;

[0031] Figure 3 This is the chromatogram of the test sample G22070401-1 detected in Example 1 of the present invention;

[0032] Figure 4 This is the chromatogram of the test sample G22070401-2 detected in Example 1 of the present invention;

[0033] Figure 5 This is the chromatogram of the test sample G22090501-1 detected in Example 1 of the present invention;

[0034] Figure 6 This is the chromatogram of the test sample G22090501-2 detected in Example 1 of the present invention;

[0035] Figure 7 This is the chromatogram of the test sample G22100802-1 detected in Example 1 of the present invention;

[0036] Figure 8 This is the chromatogram of the test sample G22100802-2 detected in Example 1 of the present invention;

[0037] Figure 9 This is the chromatogram of the test sample G22100803-1 detected in Example 1 of the present invention;

[0038] Figure 10 This is the chromatogram of the test sample G22100803-2 detected in Example 1 of the present invention;

[0039] Figure 11 This is a chromatogram of the solution used to detect the suitability of the detection system in Embodiment 2 of the present invention;

[0040] Figure 12 This is a chromatogram of the solution used to detect the suitability of the detection system in Embodiment 3 of the present invention. Detailed Implementation

[0041] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. Unless otherwise specified, the same parameter value is the same in each embodiment. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0042] Example 1

[0043] This embodiment provides a method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester. The specific process is as follows: Take the test sample solution (preparation process as follows: accurately weigh approximately 0.02 g of the test sample, place it in a 20 ml volumetric flask, and dissolve it with diluent [n-hexane: anhydrous ethanol: diethylamine = 85:15:(0.1~2)]), and separate it using a high-performance liquid chromatograph (Thermoscientific UltiMate 3000). The chromatographic conditions of the high-performance liquid chromatograph are as follows:

[0044] Chromatographic column: AD-H, (4.6*250mm, 5μm)

[0045] Mobile phase: n-hexane: anhydrous ethanol = 85:15 (elution method: isocratic elution)

[0046] Diluent: n-hexane: anhydrous ethanol: diethylamine = 85:15:1

[0047] Column temperature: room temperature

[0048] Flow rate: 1.0 ml / min

[0049] Injection volume: 20 μl

[0050] This embodiment also provides a detection method in which the test sample separated under the above conditions is detected at 210 nm by an ultraviolet detector built into a high-performance liquid chromatograph, and the detection results are quantitatively analyzed by the area normalization method.

[0051] Specifically, the quantitative analysis process is as follows: First, take an appropriate amount of diluent, accurately measure 20 μl and inject it into the liquid chromatograph, record the chromatogram. After removing the blank test chromatographic peak from the chromatogram of the test solution, proceed as follows: Calculate the mass percentage of the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester.

[0052] The above detection method also includes a step of verifying the system's suitability using reference standards: Weigh 0.02 g each of N-Boc-L-pyroglutamic acid ethyl ester and N-Boc-D-pyroglutamic acid ethyl ester working reference standards, place them in a 20 ml volumetric flask, add an appropriate amount of diluent to dissolve them, and dilute to the mark with the diluent to obtain the final product. (Each 1 ml contains approximately 1 mg each of N-Boc-L-pyroglutamic acid ethyl ester and N-Boc-D-pyroglutamic acid ethyl ester).

[0053] The results of the blank test are as follows: Figure 1 As shown, the system adaptability results are as follows: Figure 2As shown in the figure. During the testing process, four batches of N-Boc-L-pyroglutamic acid ethyl ester (G22070401, G22090501, G22100802, and G22100803) produced during the production of (2S,5R)-5-[(phenylmethoxy)amino]-2-piperidinecarboxylic acid ethyl ester oxalate in a certain factory were randomly selected. Two test solutions were prepared for each batch according to the steps described above, and the test results are shown in the figure. Figures 3-10 As shown.

[0054] from Figure 1 As can be seen, the blank solution does not interfere with the detection process.

[0055] from Figure 2 As can be seen, the resolution between the main peaks of N-Boc-L-pyroglutamic acid ethyl ester and N-Boc-D-pyroglutamic acid ethyl ester is 5.72, the theoretical plate number of the N-Boc-L-pyroglutamic acid ethyl ester main peak is 11085, and the theoretical plate number of the N-Boc-D-pyroglutamic acid ethyl ester main peak is 4190. Both are greater than 3000, and the peak shapes are good. This meets the industry requirements for the suitability test of the N-Boc-L-pyroglutamic acid ethyl ester system (the theoretical plate number calculated based on the N-Boc-L-pyroglutamic acid ethyl ester peak should not be less than 1500; the resolution between N-Boc-L-pyroglutamic acid ethyl ester and N-Boc-D-pyroglutamic acid ethyl ester should be greater than 1.5). Therefore, this invention meets the relevant industry requirements and has good application prospects.

[0056] from Figures 3-10 As can be seen, when the present invention is actually applied to the separation of N-Boc-L-pyroglutamic acid ethyl ester enantiomers, it can effectively separate N-Boc-L-pyroglutamic acid ethyl ester from its enantiomers, and the baseline is stable with good peak shape.

[0057] Summary Figure 3-10 The detection results are shown in Table 1 below:

[0058] Table 1

[0059]

[0060]

[0061] As shown in the table above, the resolution of N-Boc-L-pyroglutamic acid ethyl ester and its enantiomers in randomly selected batches of samples using the method of the present invention reached above 9.77. The test results for samples from the same batch were basically consistent, indicating that the test results of the method of the present invention are accurate and reliable. Based on N-Boc-L-pyroglutamic acid ethyl ester, the theoretical plate number was above 10273 and the retention time of N-Boc-L-pyroglutamic acid ethyl ester was within 9.5 min, indicating that the separation method of the present invention is rapid and efficient. Therefore, the method of the present invention has good application prospects in the quality control of N-Boc-L-pyroglutamic acid ethyl ester.

[0062] Example 2

[0063] The difference between Example 2 and Example 1 of this invention is as follows: the mobile phase: hexane: anhydrous ethanol volume ratio is 75:25 (elution method is isocratic elution); the diluent: hexane: anhydrous ethanol: diethylamine = 75:25:1.

[0064] The test results of the system suitability solution (prepared in the same manner as in Example 1, a mixed sample of N-Boc-L-pyroglutamic acid ethyl ester (batch number: G22070401) and N-Boc-D-pyroglutamic acid ethyl ester (batch number: 20220607) produced during the production of (2S,5R)-5-[(phenylmethoxy)amino]-2-piperidinecarboxylic acid ethyl oxalate in a certain factory) are as follows: Figure 11 As shown.

[0065] Example 3

[0066] The difference between Example 3 and Example 1 of this invention is as follows: the mobile phase: hexane: anhydrous ethanol volume ratio is 90:10 (elution method is isocratic elution); the diluent: hexane: anhydrous ethanol: diethylamine = 90:10:1.

[0067] The test results of the system suitability solution (prepared in the same manner as in Example 1, a mixed sample of N-Boc-L-pyroglutamic acid ethyl ester (batch number: G22070401) and N-Boc-D-pyroglutamic acid ethyl ester (batch number: 20220607) produced during the production of (2S,5R)-5-[(phenylmethoxy)amino]-2-piperidinecarboxylic acid ethyl oxalate in a certain factory) are as follows: Figure 12 As shown.

[0068] from Figure 11 and 12It can be seen that when the volume percentage of n-hexane is 75%, the retention time of N-Boc-L-pyroglutamic acid ethyl ester and its enantiomer increases, with a resolution of 6.07; when the volume percentage of n-hexane is 90%, the retention time of N-Boc-L-pyroglutamic acid ethyl ester and its enantiomer decreases, with a resolution of 4.13. In summary, when the volume percentage of n-hexane is 75%–90%, it exhibits good separation performance for both N-Boc-L-pyroglutamic acid ethyl ester and its enantiomer. The room temperature mentioned in the above examples is 25 degrees Celsius.

[0069] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester, characterized in that: The procedure includes the following steps: A test sample containing the enantiomer of N-Boc-L-pyroglutamic acid ethyl ester is taken and separated by liquid chromatography. The chromatographic conditions of the liquid chromatography include: using a Daicel AD-H column as the stationary phase and a mixed solution containing n-hexane and anhydrous ethanol as the mobile phase for isocratic elution, wherein the volume ratio of n-hexane to anhydrous ethanol in the mobile phase is 75:25 to 90:10; the test sample is a test sample treated with a diluent, wherein the diluent is n-hexane-anhydrous ethanol-diethylamine, and the volume ratio of the three components is 75:25:1 to 90:10:

1.

2. The method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester according to claim 1, characterized in that: The volume ratio of n-hexane to anhydrous ethanol in the mobile phase is 5.67:

1.

3. The method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester according to claim 1, characterized in that: The specifications of the Daicel AD-H chromatographic column include: column length 250 mm, inner diameter 4.6 mm, and particle size 5 µm.

4. The method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester according to claim 1, characterized in that: The chromatographic conditions for the liquid chromatography also include a flow rate of 0.5 ml / min to 1.5 ml / min.

5. The method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester according to claim 1, characterized in that: The chromatographic conditions for liquid chromatography also include a flow rate of 1 ml / min.

6. The method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester according to claim 1, characterized in that: The chromatographic conditions for liquid chromatography also include: column temperature at room temperature.

7. The method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester according to claim 6, characterized in that: The room temperature is 20℃~35℃.

8. The method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester according to claim 6, characterized in that: The room temperature is 25℃~35℃.

9. The method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester according to claim 1, characterized in that: The chromatographic conditions for liquid chromatography also include an injection volume of 10µl to 30µl.

10. The method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester according to claim 1, characterized in that: The chromatographic conditions for liquid chromatography also include an injection volume of 20 µl.

11. The method for separating the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester according to claim 10, characterized in that: The volume ratio of n-hexane-anhydrous ethanol-diethylamine is 85:15:

1.

12. A method for detecting the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester, characterized in that: The detection method includes the following steps: after separation by the separation method according to any one of claims 1 to 11, the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester are detected by a detector.

13. The method for detecting the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester according to claim 12, characterized in that: The detector is an ultraviolet detector.

14. The method for detecting the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester according to claim 13, characterized in that: The detection wavelength of the ultraviolet detector is 210 nm.

15. The method for detecting the enantiomers of N-Boc-L-pyroglutamic acid ethyl ester according to claim 12, characterized in that: The detection method further includes a step of qualitative analysis of the detection results by retention time or a step of quantitative analysis of the detection results by area normalization.

16. The application of the separation method as described in any one of claims 1 to 11 or the detection method as described in any one of claims 12 to 15 in the quality control of N-Boc-L-pyroglutamic acid ethyl ester.

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