A method for high-performance liquid chromatography separation of ticagliptin hydrobromide intermediates and their impurities

Abstract

This invention discloses a high-performance liquid chromatography (HPLC) method for the separation of ticagliptin hydrobromide intermediates and their impurities. The method employs an HPLC system with a Kromasil 100-5-C18 column (5 μm, 4.6 × 250 mm), a mobile phase of a mixture of phosphoric acid and acetonitrile, and maintains a flow rate of 1.0 mL / min. The column temperature is 30 °C, the injection volume is 10 μL, and the detection wavelength is 210 nm. This method effectively separates and determines ticagliptin hydrobromide intermediates and their impurities. It is simple, inexpensive, highly sensitive, and provides good separation results. Furthermore, it allows for the simultaneous detection of the intermediate and four impurities, enabling quality control of the ticagliptin hydrobromide intermediate.

Description

Technical Field

[0001] This invention pertains to pharmaceutical analysis and detection methods, and specifically relates to a high-performance liquid chromatography (HPLC) method for the separation of ticagliptin hydrobromide intermediates and their impurities. Background Technology

[0002] (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazolidin-3-carbonyl)pyrrolidine (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazolidin-3-carbonyl)pyrrolidine is a key intermediate in the synthesis of ticagliptin hydrobromide, with the chemical formula C2. 27 H 38 N6O3S, English name: 2-methylpropan-2-yl(2S,4S)-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(1,3-thiazolidin-3-ylcarbonyl)pyrrolidine-1-carboxylate.

[0003] Tilgliptin hydrobromide was approved for marketing in Japan by the Pharmaceuticals and Medical Devices Agency in 2012, primarily for the treatment of type 2 diabetes in adults. Clinical studies have found that ticagliptin hydrobromide, as a dipeptidyl peptidase-4 inhibitor, effectively inhibits the activity of DPP-4 enzyme, thereby increasing the level of glucagon-like peptide-1 (GLP-1), producing effects such as promoting insulin secretion, inhibiting glucagon release, improving glycemic control, and reducing glycated hemoglobin (HbA1c). Tilgliptin hydrobromide is not a controlled substance and has no known abuse or dependence. (2S,4S)-1-tert-butyloxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazolidin-3-carbonyl)pyrrolidine is an important intermediate in the synthesis of ticagliptin hydrobromide, and its purity directly affects the purity of the final product, ticagliptin hydrobromide. Therefore, the study of (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazolyl-3-carbonyl)pyrrolidine and its impurity separation methods has important industrial value.

[0004] During the synthesis of this intermediate, some byproducts were generated due to incomplete reaction, including four impurities, with the following structural formula.

[0005]

[0006] Four impurity structures in the intermediate synthesis process Impurities introduced during the synthesis of the intermediate (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazoline-3-carbonyl)pyrrolidine require strict quality control. Therefore, selecting a specific, sensitive, accurate, and reproducible detection method to control potential impurities in (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazoline-3-carbonyl)pyrrolidine is of great significance. Summary of the Invention

[0007] Purpose of the Invention: To overcome the shortcomings of existing technologies, the purpose of this invention is to provide a high-performance liquid chromatography (HPLC) method for the separation of ticagliptin hydrobromide intermediates and their impurities. This method exhibits good peak symmetry, high sensitivity, good resolution, and accurate and reliable results. It can effectively, rapidly, and sensitively detect and separate the ticagliptin intermediate (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazolyl-3-carbonyl)pyrrolidine and its four impurities ((2S,4R)-4-hydroxy-2-(3-thiazolylcarbonyl)-1-pyrrolidinecarboxylic acid tert-butyl ester (Compound 3-IMP-2), (2S,4S)-1-(tert-butoxycarbonyl)-4-(4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl)pyrrolidine-2-carboxylic acid (Compound 3-IMP-2), and (2S,4S)-1-(tert-butoxycarbonyl)-4-(4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl)pyrrolidine-2-carboxylic acid (Compound 3-IMP-2). 1-IMP-6), (2S,4S)-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-[(1-oxoylide-1λ4-1,3-thiazacyclopentan-3-yl)carbonyl]pyrrolidine-1-carboxylic acid-2-methylpropyl-2-yl ester (Compound 11-IMP-3), (2S,4S)-2-[(1,1-dioxoylide-1λ6-1,3-thiazacyclopentan-3-yl)carbonyl]-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]pyrrolidine-1-carboxylic acid-2-methylpropyl-2-yl ester (Compound 11-IMP-4)).

[0008] Technical Solution: This invention provides a method for separating and detecting (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazoline-3-carbonyl)pyrrolidine and impurities using high-performance liquid chromatography (HPLC). The method includes: preparing a detection solution of (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazoline-3-carbonyl)pyrrolidine containing impurities using a diluent; using a Kromasil 100-5-C18 column (5μm, 4.6×250mm); and using phosphoric acid and acetonitrile as the mobile phase, with gradient elution for separation.

[0009] Preferred, The mobile phase flow rate was 1.0 mL / min; The column temperature was 30℃. The injection volume was 10 μL; The detection wavelength is 210nm; Mobile phase A: 10 mmol / L KH2PO4, pH adjusted to 6.0 (v / v) with TEA; Mobile phase B: Acetonitrile; The gradient elution procedure is as follows:

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The method of the present invention can not only effectively detect (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazolidin-3-carbonyl)pyrrolidine, but also effectively separate and detect the four impurities generated in the process.

[0011] 2. The separation method of the present invention has good peak shape and separation degree, high sensitivity, and accurate and reliable results; it is also low in cost, simple and convenient, and has strong practicality. Attached Figure Description

[0012] Figure 1 This is a superimposed image of the blank solution and the system suitability solution in Example 1; Figure 2 This is the chromatogram of the system suitability solution in Example 1; Figure 3 The chromatograms are for solutions with the limits of quantitation for each known impurity in Example 2. Figure 4 The chromatogram of the Compound 11 limit-of-quantitation solution in Example 2 is shown below. Figure 5 This is a linear relationship diagram for Example 4. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described below.

[0014] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0015] The models and manufacturers of the instruments used in each embodiment are shown in Table 1: Table 1: Instrument Models and Manufacturers Used

[0016] The batch numbers and manufacturers of the reagents used in the examples are shown in Table 2: Table 2: Batch Numbers and Manufacturers of Reagents Used

[0017] Information on the reference standards and samples used in the examples is shown in Table 3: Table 3: Information on reference standards and samples used

[0018] Example 1 The system suitability verification method for the high-performance liquid chromatography (HPLC) separation of the ticagliptin hydrobromide intermediate (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazolidin-3-carbonyl)pyrrolidine and its impurities in this embodiment is as follows: 600 mL of acetonitrile and 400 mL of pure water are mixed uniformly to prepare a diluent (acetonitrile:water = 60:40); 5 mg each of the impurities Compound 3-IMP-2, Compound 11-IMP-3, Compound 11-IMP-4, and Compound 1-IMP-6 working reference standards are accurately weighed and placed in a 10 mL volumetric flask, diluted thoroughly with the diluent, and mixed evenly to prepare an impurity stock solution; 25 mg of the Compound 11 positioning sample is then accurately weighed and placed in a 50 mL volumetric flask, and 0.075 mL of the impurity stock solution is added. Dissolve thoroughly in diluent and bring to volume, mix well, and prepare the system adaptability solution. Inject the blank and system adaptability solution into the syringe; results are shown in [Figure number missing]. Figure 1Accurately weigh 25 mg COMPOUND 11 test sample into a 50 mL volumetric flask, add diluent to dissolve completely and dilute to the mark, mix well, and prepare test solutions labeled as Test Sample 1 and Test Sample 2; then accurately transfer 75 μL of Test Sample Solution 1 into a 50 mL volumetric flask, dissolve completely with diluent and dilute to the mark, mix well, and prepare sensitivity solutions. Accurately measure 10 μL each of blank, system suitability solution, reference solution, test sample solution, and sensitivity solution, and inject them into the liquid chromatograph. The results are shown in the figure. Figure 2 .

[0019] The gradient elution procedure is as follows:

[0020] The mobile phase flow rate was 1.0 mL / min; The column temperature was 30℃. Injection volume: 10 μL; The detection wavelength is 210nm.

[0021] The image results show that in this example, the separation between the main peak and its adjacent peaks is no less than 1.5, and the theoretical plate number of the main peak is no less than 5000. The separation between the impurities and their adjacent counterparts is also no less than 1.5, meeting the system adaptability requirements.

[0022] Example 2 The specificity verification method under system suitability verification in the high performance liquid chromatography separation of ticagliptin hydrobromide intermediate (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazolidin-3-carbonyl)pyrrolidine and its impurities in this embodiment is as follows: Accurately weigh 5 mg each of impurities Compound 3-IMP-2, Compound 11-IMP-3, Compound 11-IMP-4, and Compound 1-IMP-6 working reference standards and place them in a 10 mL volumetric flask. Add diluent to dissolve them completely and bring the volume to the mark. Mix well to prepare an impurity stock solution. Accurately weigh 25 mg of Compound 11 positioning sample and place it in a 50 mL volumetric flask. Add 0.075 mL of the impurity stock solution, add diluent to dissolve them completely and bring the volume to the mark. Mix well to prepare a system suitability solution. Injection blank and system suitability solution; specificity test results are shown in Table 4.

[0023] Table 4: Results of Specificity Tests

[0024] As shown in Table 4, the blank did not interfere in this embodiment. In the system suitability chromatogram, the resolution between the main peak and adjacent peaks was not less than 2.7, and the theoretical plate number of the main peak was 149,579. The resolution between the impurities of interest and adjacent impurities was not less than 2.2, meeting the specificity acceptance criteria, indicating good specificity. The results are consistent with those of the previous embodiment. Figure 2 .

[0025] Example 3 The validation method for the limit of quantitation and limit of detection under system suitability verification in the high performance liquid chromatography (HPLC) separation of ticagliptin hydrobromide intermediate (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazolidin-3-carbonyl)pyrrolidine and its impurities is as follows: Accurately weigh 20 mg of Compound 11 positioning sample into a 10 mL volumetric flask, dissolve it completely in the diluent and bring the volume to the mark, mix well, and prepare Compound 11 stock solution; then accurately transfer 0.5 mL of the impurity stock solution from Example 1 and 0.025 mL of the Compound 11 stock solution into the same 100 mL volumetric flask. Dilute to the mark in a volumetric flask, mix thoroughly, and prepare a mixed mother liquor; take the mixed mother liquor and dilute it stepwise to obtain the quantitation limit and detection limit concentration solutions of each impurity and main component; accurately measure the stepwise dilution of the mixed mother liquor and inject it into the liquid chromatograph. The detection limit verification results are shown in Table 5; the quantitation limit verification results are shown in Table 6.

[0026] Table 5: Detection Limit Validation Results

[0027] Table 6: Results of Limit of Quantitation Validation

[0028] As can be seen from Tables 5 and 6, in this embodiment, the signal-to-noise ratios (SNRs) for the quantitation limits of the main component and each impurity are all greater than 10, and the SNRs for the detection limits are all greater than 3. Simultaneously, the detection requirement of the 0.15% control limit is met, indicating good validation. The results are shown in Tables 5 and 6. Figure 3 , Figure 4 .

[0029] Example 4 The method for verifying the linearity and range of the system suitability of the ticagliptin hydrobromide intermediate (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazolidin-3-carbonyl)pyrrolidine and its impurities in high performance liquid chromatography (HPLC) is as follows: The Compound 11 stock solution from Example 3 was used, and the Compound 11 stock solution was diluted to a series of concentrations (n≥5) with a LOQ of 120%; the above series of solutions were accurately measured and injected into the HPLC instrument, and the peak area of ​​the principal component was plotted against the impurity concentration. Linear regression was performed using the least squares method, and the results of the linearity and range verification are shown in Table 7.

[0030] Table 7: Linearity and Range Validation Results

[0031] As can be seen from the results in Table 7, Compound 11 exhibits good linearity in the range of 0.0001 mg / mL to 0.6153 mg / mL (0.02%–120%), with r = 0.9999, meeting the linearity and range acceptance criteria, and demonstrating good validation.

[0032] Example 5 The method for verifying the stability of the solution under system suitability verification in the high performance liquid chromatography separation of ticagliptin hydrobromide intermediate (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazolidin-3-carbonyl)pyrrolidine and its impurities is as follows: Using the system suitability solution in Example 1, i.e., the spiked solution of the test sample, 10 μL of the spiked solution of the test sample, which has been placed at room temperature for 0 h, 2 h, 4 h, 8 h, and 22 h, is accurately measured and injected into the liquid chromatograph. The results of the stability verification of the spiked solution of the test sample are shown in Table 8.

[0033] Table 8: Results of Stability Verification of Spiked Solutions of Test Samples

[0034] As shown in Table 8, under room temperature conditions, the spiked solution of the test sample showed no new impurities within 22 hours. The RSD of the main peak area was 0.73%, and the RSD of other impurity peak areas was no greater than 6.23%, indicating good solution stability.

[0035] In summary, this invention provides a ticagliptin hydrobromide intermediate (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazolidin-3-carbonyl)pyrrolidine and its four impurities ((2S,4R)-4-hydroxy-2-(3-thiazolidinylcarbonyl)-1-pyrrolidinecarboxylic acid tert-butyl ester (Compound 3-IMP-2), (2S,4S)-1-(tert-butoxycarbonyl)-4-(4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl)pyrrolidine-2-carboxylic acid (Compound 3-IMP-2), and (2S,4S)-1-(tert-butoxycarbonyl)-4-(4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl)pyrrolidine-2-carboxylic acid (Compound 3-IMP-2). 1-IMP-6), (2S,4S)-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-[(1-oxoylide-1λ4-1,3-thiazacyclopentan-3-yl)carbonyl]pyrrolidine-1-carboxylic acid-2-methylpropyl-2-yl ester (Compound 11-IMP-3) and (2S,4S)-2-[(1,1-dioxoylide-1λ6-1,3-thiazacyclopentan-3-yl)carbonyl]-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]pyrrolidine-1-carboxylic acid-2-methylpropyl-2-yl ester (Compound 11-IMP-3) The high performance liquid chromatography (HPLC) method of 11-IMP-4 can effectively separate the intermediate (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazolidin-3-carbonyl)pyrrolidine from four impurities, with a resolution of 2.2-6.2.

[0036] The above are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. Any equivalent substitutions or modifications made by those skilled in the art to the technical solutions and content disclosed in the present invention without departing from the scope of the present invention shall be deemed to have remained within the protection scope of the present invention.

Claims

1. A method for high-performance liquid chromatography separation of ticagliptin hydrobromide intermediates and their impurities, characterized in that, High-performance liquid chromatography (HPLC) was used, with a mixture of mobile phases A and B as the mobile phase, and gradient elution was employed for separation. Mobile phase A: 10 mmol / L KH2PO4, pH adjusted to 6.0 (v / v) with TEA. Mobile phase B: Acetonitrile; The gradient elution procedure is as follows: ； The method simultaneously separates (2S,4S)-1-tert-butoxycarbonyl-4-[4-(5-methyl-2-phenylpyrazol-3-yl)piperazin-1-yl]-2-(thiazolyl-3-carbonyl)pyrrolidine and its four impurities: Compound 3-IMP-2, Compound 1-IMP-6, Compound 11-IMP-3, and Compound 11-IMP-4.

2. The method according to claim 1, characterized in that, The chromatographic column was a Kromasil 100-5-C18 (5μm, 4.6×250mm).

3. The method according to claim 1, characterized in that, The mobile phase flow rate was 1.0 mL / min, the column temperature was 30 °C, and the detection wavelength was 210 nm.

4. The method according to claim 1, characterized in that, The injection volume is 10 μL.

5. The method according to claim 1, characterized in that, The separation degree between the intermediate and the four impurities is not less than 2.

0.

6. The method according to claim 1, characterized in that, The high-performance liquid chromatograph used is an Agilent 1260 high-performance liquid chromatograph.

7. The method according to claim 1, characterized in that, The method exhibits linearity in the determination of the intermediate within a concentration range of 0.0001 mg / mL to 0.6153 mg / mL, with a correlation coefficient R0. 2 Not less than 0.999.

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