Separation and Detection Method for a Key Intermediate of Relugolix and Its Related Impurities

Abstract

The present application discloses a method for separating and detecting a rilugolix key intermediate and its related impurities. The HPLC method is specifically as follows: preparing a test solution: diluting the test sample to obtain it; preparing a control solution: diluting the test solution to obtain it; preparing a system suitability solution: taking the rilugolix key intermediate and its related impurities, mixing them and diluting with a diluent to obtain it; determining the retention time: injecting the system suitability solution, performing HPLC analysis to determine the retention time; detecting the target substance: injecting the test solution and the control solution for separation and detection; this method uses octadecylsilyl silica gel bonded silica as the filler as the stationary phase, a mixed solution of phosphate buffer solution and acetonitrile as mobile phase A, and acetonitrile as mobile phase B for gradient elution. The method of the present application can achieve the separation and content determination of the rilugolix key intermediate and its related impurities, and has good sensitivity, resolution and repeatability.

Description

Technical Field

[0001] This application relates to the field of liquid chromatography analysis, and particularly to a method for separating and detecting a relugolix key intermediate and its related impurities. Background Art

[0002] Relugolix was initially developed by Takeda Pharmaceutical Company Limited in Japan as a small molecule gonadotropin-releasing hormone (GnRH) receptor antagonist, which can rapidly reduce female estrogen and progesterone. In January 2019, relugolix was approved for marketing in Japan under the trade name for improving the following symptoms caused by uterine fibroids: menorrhagia, lower abdominal pain, low back pain, and anemia. On December 18, 2020, the US FDA approved relugolix for the treatment of adult patients with advanced prostate cancer under the trade name

[0003] Ethyl 2-((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-((dimethylamino)methyl)-5-(4-(3-methoxyureido)phenyl)thiophene-3-carboxylate is a key intermediate of relugolix, and the quality control of this compound plays an important role in the preparation of relugolix API. At present, there are few reports on the analytical methods for relugolix key intermediates. In the application document with the patent number CN115073490A, a preparation method of relugolix and its intermediates is disclosed, which records a detection method for relugolix. Using a Sunfire C8 chromatographic column, water (0.1% TFA) is used as mobile phase A, and acetonitrile is used as mobile phase B for gradient elution. However, this method is only used to detect the content of relugolix and cannot detect the content of related impurities in the relugolix intermediate, which is not conducive to monitoring the quality of the relugolix intermediate.

[0004] In view of this, developing an efficient and accurate analytical method for relugolix key intermediates and related impurities and realizing the separation and purification of the intermediates is of great significance for the subsequent preparation of high-purity relugolix. Summary of the Invention

[0005] To achieve the efficient and accurate analysis and separation and purification of the content of relugolix key intermediates and their related impurities, this application provides a method for separating and detecting a relugolix key intermediate and its related impurities.

[0006] The method for separating and detecting a relugolix key intermediate and its related impurities provided by this application uses the HPLC method, and specifically includes the following steps:

[0007] Prepare a test solution: Take the test sample and dissolve it in the diluent to obtain it;

[0008] Prepare the control solution: Take the test solution and dilute it with the diluent to obtain;

[0009] Prepare the system suitability solution: Take the key intermediate of relugolix and its related impurities, mix them and dissolve in the diluent to obtain;

[0010] Retention time determination: Inject the system suitability solution for detection to determine the retention times of the key intermediate of relugolix and its impurities; Target substance separation and detection: Inject the test solution and the control solution for detection, and calculate the content of the impurities in the key intermediate of relugolix in the test solution according to the self-control method of the main component;

[0011] The HPLC method uses octadecylsilane chemically bonded silica as the stationary phase, a mixed solution of phosphate buffer solution and acetonitrile as mobile phase A, and acetonitrile as mobile phase B for gradient elution.

[0012] The key intermediate of relugolix described in this application, ethyl 2-((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-((dimethylamino)methyl)-5-(4-(3-methoxyureido)phenyl)thiophene-3-carboxylate, has the chemical formula C 28 H 32 F2N4O6S, and the structural formula is shown in formula (I).

[0013] Formula (I)

[0014] Preferably, the related impurities include:

[0015] Impurity 03: 2-((2,6-Difluorobenzyl)(ethoxycarbonyl)amino)-4-((dimethylamino)methyl)-5-(4-(3-methoxyureido)phenyl)thiophene-3-carboxylic acid;

[0016] Impurity 01-E: 5-(4-Aminophenyl)-2-((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-((dimethylamino)methyl)thiophene-3-carboxylic acid;

[0017] Impurity 03-C: 2-((2,6-Difluorobenzyl)amino)-4-((dimethylamino)methyl)-5-(4-(3-methoxyureido)phenyl)thiophene-3-carboxylic acid;

[0018] Impurity 02-C: Ethyl 2-((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-((dimethylamino)methyl)-5-(4-ureidophenyl)thiophene-3-carboxylate;

[0019] Impurity 01: Ethyl 5-(4-aminophenyl)-2-((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-((dimethylamino)methyl)thiophene-3-carboxylate;

[0020] Impurity 02-G: Ethyl 2-((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-methyl-5-(4-(3-methoxyureido)phenyl)thiophene-3-carboxylate.

[0021] It is found that the above key intermediate (Formula I) of this application is very close in structure to Impurity 03, Impurity 01-E. If this impurity is introduced into the active pharmaceutical ingredient during the preparation reaction of relugolix, it will be difficult to remove. Therefore, it is necessary to strictly monitor and separate the above impurities during the preparation reaction of the key intermediate. This separation and detection method plays a crucial role in the control of the relugolix synthesis reaction and the improvement of quality, directly affecting the quality of the relugolix finished product. Specifically, in the HPLC analysis of this application, while using the self-control method of the main component, a mixed solution of phosphate buffer and acetonitrile with different solubilities for the key intermediate and each impurity is used as the mobile phase reagent, which can effectively achieve the separation and content determination of the key intermediate and its impurities during the gradient elution process, effectively ensuring the quality of the relugolix product.

[0022] Preferably, in the system suitability solution, the concentration of the key intermediate is 1 mg / ml, and the concentrations of Impurity 03, Impurity 01-E, Impurity 03-C, Impurity 02-C, Impurity 01, and Impurity 02-G are all 5 μg / ml.

[0023] Preferably, the diluent is acetonitrile.

[0024] If the concentration of the test sample is too low, it is easy to cause insufficient signal intensity, making separation and detection difficult and reducing the accuracy and stability of the analysis. If the concentration of the test sample is too high, it is easy to cause instrument overload, affecting the separation effect and detection accuracy. The above range setting can avoid instrument overload while meeting the requirements of detection sensitivity.

[0025] Preferably, the gradient elution is set as follows:

[0026] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75-80 20-25 18 60-65 35-40 30 33-38 62-67 35 33-38 62-67 38 75-80 20-25 45 75-80 20-25

[0027] Preferably, the gradient elution is set as follows:

[0028] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 18 60 40 30 33 67 35 33 67 38 80 20 45 80 20

[0029] In the HPLC analysis of the application, a reasonable gradient elution concentration is set according to the retention time measured by the system suitability solution, which significantly improves the sensitivity and separation of the method.Specifically, corresponding to the retention time measured, the application continuously adjusts the consumption of mobile phase A and mobile phase B in five intervals of 0-18min, 18-30min, 30-35min, 35-38min, 38-45min in the elution process, obtains the mobile phase ratio matched with key intermediate, each related impurity polarity, affinity, so that each component in the test solution is separated successively under the mobile phase effect of different ratios, and the overlap of the elution time is relatively low, and gradient distribution can be presented on the chromatographic column.Finally, the accurate determination of key intermediate and its related impurity content can be completed according to the obtained chromatogram.

[0030] Preferably, in the mobile phase A, the volume ratio of phosphate buffer to acetonitrile is 90:10.

[0031] Preferably, the phosphate buffer is selected from one of sodium dihydrogen phosphate buffer, disodium hydrogen phosphate buffer, potassium dihydrogen phosphate buffer and dipotassium hydrogen phosphate buffer, and more preferably potassium dihydrogen phosphate buffer.

[0032] Preferably, the concentration of the phosphate buffer is 15 to 25 mmol / L; more preferably 20 mmol / L.

[0033] The mobile phase A of the present application adopts a mixed solution of phosphate buffer and acetonitrile to obtain a mobile phase with both buffering capacity and solubility and elution properties, thereby improving the efficiency and accuracy of the separation and detection method.

[0034] Preferably, the flow rate of the mobile phase elution is 0.7 to 1.0 ml / min, more preferably 1.0 ml / min.

[0035] Preferably, in the HPLC analysis, the detection wavelength is 254±2 nm; more preferably 254 nm.

[0036] Preferably, the particle size of the octadecylsilane bonded silica gel as a filler is 2 to 5 μm, and the column temperature of the chromatographic column is 25 to 35°C.

[0037] Preferably, the particle size of the octadecylsilane bonded silica gel as a filler is 5 μm, and the column temperature of the chromatographic column is 30°C.

[0038] Preferably, the injection volume is 5-10 μl, more preferably 5 μl.

[0039] In summary, this application has the following beneficial effects:

[0040] 1. By adopting the principal component self-control method and coordinating with the specific mobile phase composition and gradient concentration setting, the present application effectively realizes the separation and content monitoring of the key intermediate of relugolix and its related impurities, which plays an important role in promoting the production quality of high-purity relugolix.

[0041] 2. The separation and detection method provided by the present application has a high resolution between the key intermediate and impurities 03 and 01-E, and the range of multiple measurement results is not higher than 0.01%. It has the advantages of strong specificity, high precision, good repeatability and good system suitability. Brief Description of the Drawings

[0042] Figure 1 It is the chromatogram of the system suitability solution in Example 1 of the present application.

[0043] Figure 2 It is the chromatogram of the mixed solution in Comparative Example 1 of the present application.

[0044] Figure 3 It is the chromatogram of the mixed solution in Comparative Example 2 of the present application. Detailed Description of the Invention

[0045] Examples

[0046] Example 1, Separation and Detection Method of the Key Intermediate of Relugolix and Its Related Impurities:

[0047] 1. Chromatographic Conditions:

[0048] Chromatographic column: Kromasil 100-5C18, 250 mm × 4.6 mm, 5 μm; Mobile phase A: 20 mmol / L potassium dihydrogen phosphate buffer solution - acetonitrile (90:10), Mobile phase B: acetonitrile, Flow rate: 1.0 ml / min; Column temperature: 30 °C, Detection wavelength: 254 nm, Injection volume: 5 μl. The gradient elution settings are as follows:

[0049]

[0050] 2. Separation and Detection Steps and Detection Results

[0051] Prepare the test solution: Take 10 mg of the relugolix intermediate sample and place it in a 10 ml volumetric flask. Dissolve it with acetonitrile and dilute it to the mark to prepare a 1 mg / ml solution as the test solution.

[0052] Prepare the control solution: Take 1 ml of the test solution and place it in a 100 ml volumetric flask. Dilute it to the mark with acetonitrile to prepare a 10 μg / ml solution to obtain the control solution;

[0053] Prepare the system suitability solution: Weigh appropriate amounts of the key intermediate of relugolix, impurity 03, impurity 01-E, impurity 03-C, impurity 02-C, impurity 01, and impurity 02-G accurately, place them in the same volumetric flask, dissolve with acetonitrile and dilute to prepare a solution with a concentration of 1 mg / ml for the key intermediate and 5 μg / ml for each related impurity as the system suitability solution.

[0054] Retention time determination: Pipette 5 μl of the system suitability solution accurately and inject it into the liquid chromatograph for HPLC analysis, and record the chromatogram. Refer to Figure 1 It can be seen that in the system suitability solution, the resolution between the key intermediate of relugolix and the adjacent impurity peak is 4.8, and the retention time is 18.002.

[0055] Separation and detection of the target substance: Pipette the above-mentioned test solution and reference solution accurately for injection respectively, with an injection volume of 5 μl each, conduct HPLC analysis, and calculate the content of impurities in the key intermediate of relugolix in the test solution by the self-control method of the main component.

[0056] The test results show that the maximum range of the detected related impurity content in the 6 groups of test solutions is 0.01%, which is less than 0.02%.

[0057] Example 2. The method for separating and detecting the key intermediate of relugolix and its related impurities is different from that of Example 1 in that the chromatographic conditions are as follows:

[0058] Chromatographic column: Kromasil 100-5C18, 250 mm × 4.6 mm, 5 μm; Mobile phase A: 15 mmol / L potassium dihydrogen phosphate buffer-acetonitrile (90:10), Mobile phase B: acetonitrile, Flow rate: 1.0 ml / min; Column temperature: 30 °C, Detection wavelength: 254 nm, Injection volume: 5 μl. The gradient elution settings are as follows:

[0059]

[0060]

[0061] Detection results of the content of the key intermediate and related impurities in the relugolix intermediate:

[0062] The results show that the maximum range of the detected related impurity content in the 6 groups of test solutions is 0.01%, which is less than 0.02%.

[0063] Conclusion: It can be seen from Examples 1 and 2 that under the chromatographic conditions of this application, the key intermediate of relugolix and its related impurities can be completely separated. This method has strong specificity, high precision, good repeatability, good system suitability, meets the technical requirements of drug quality research standards, and the obtained results are stable and reliable.

[0064] Comparative Example

[0065] Comparative Example 1

[0066] 1. Chromatographic conditions:

[0067] Chromatographic column: YMC - Pack - Pro C18, 150 mm×4.6 mm, 3 μm; Mobile phase A: 50 mmol / L potassium dihydrogen phosphate buffer - acetonitrile (70:30), Mobile phase B: acetonitrile, Flow rate: 1.0 ml / min; Column temperature: 30 °C, Detection wavelength: 254 nm, Injection volume: 5 μl. The gradient elution settings are as follows:

[0068]

[0069] 2. Separation and detection procedures and detection results

[0070] Take appropriate amounts of Impurity 03, Impurity 01 - E, Impurity 02, Impurity 02 - C, Impurity 02 - G, and Impurity 01, accurately weigh them, place them in the same volumetric flask, dissolve and dilute with diluent to prepare a mixed solution with the concentrations of Impurity 03, Impurity 01 - E, Impurity 02, Impurity 02 - C, Impurity 02 - G, and Impurity 01 all being 20 μg / ml. Accurately measure 5 μl and inject it into the liquid chromatograph. The chromatogram results are as Figure 2 shown.

[0071] Conclusion: Refer to Figure 2 It can be seen that under these chromatographic conditions, Impurity 03 and Impurity 01 - E are not completely separated, affecting the quantitative results and qualitative judgments of Impurity 03 and Impurity 01 - E.

[0072] Comparative Example 2

[0073] 1. Chromatographic conditions:

[0074] Chromatographic column: YMC - Pack - Pro C18, 150 mm×4.6 mm, 3 μm; Mobile phase A: 20 mmol / L potassium dihydrogen phosphate buffer - acetonitrile (70:30), Mobile phase B: acetonitrile, Flow rate: 1.0 ml / min; Column temperature: 30 °C, Detection wavelength: 254 nm, Injection volume: 5 μl. The gradient elution settings are as follows:

[0075]

[0076] 2. Separation and detection procedures and detection results

[0077] Take appropriate amounts of impurity 03, impurity 01-E, impurity 02, impurity 02-C, impurity 02-G, and impurity 01, weigh accurately, place them in the same volumetric flask, dissolve and dilute with a diluent to prepare a mixed solution with the concentrations of impurity 03, impurity 01-E, impurity 02, impurity 02-C, impurity 02-G, and impurity 01 all being 20 μg / ml. Accurately measure 5 μl and inject it into the liquid chromatograph, and the chromatogram results are as Figure 3 shown.

[0078] Conclusion: Referring to Figure 3 it can be seen that under this chromatographic condition, impurity 03 and impurity 01-E are not completely separated, which affects the quantitative results and qualitative judgments of impurity 03 and impurity 01-E.

[0079] This specific embodiment is only an explanation of the present application and does not limit the present application. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A method for the separation and detection of a relugolix key intermediate and its related impurities, characterized in that, The HPLC method is adopted, which specifically includes the following steps: Prepare the test solution: Take the test sample and dissolve it in the diluent to obtain; Prepare the control solution: Take the test solution and dilute it with the diluent to obtain; Prepare the system suitability solution: Take the key intermediate of relugolix and its related impurities, mix them and dissolve in the diluent to obtain; Determine the retention time: Inject the system suitability solution for detection to determine the retention times of the key intermediate of relugolix and its impurities; Separate and detect the target substance: Inject the test solution and the control solution for detection, and calculate the content of impurities in the key intermediate of relugolix in the test solution according to the self-control method of the main component; In the HPLC method, octadecylsilyl silica gel is used as the stationary phase, a mixed solution of phosphate buffer solution and acetonitrile is used as mobile phase A, and acetonitrile is used as mobile phase B for gradient elution; The related impurities include: Impurity 03: 2-(((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-(((dimethylamino)methyl)-5-(4-(3-methoxyureido)phenyl)thiophene-3-carboxylic acid; Impurity 01-E: 5-(4-aminophenyl)-2-(((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-(((dimethylamino)methyl)thiophene-3-carboxylic acid; Impurity 03-C: 2-(((2,6-difluorobenzyl)amino)-4-(((dimethylamino)methyl)-5-(4-(3-methoxyureido)phenyl)thiophene-3-carboxylic acid; Impurity 02-C: Ethyl 2-(((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-(((dimethylamino)methyl)-5-(4-ureidophenyl)thiophene-3-carboxylate; Impurity 01: Ethyl 5-(4-aminophenyl)-2-(((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-(((dimethylamino)methyl)thiophene-3-carboxylate; Impurity 02-G: Ethyl 2-(((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-methyl-5-(4-(3-methoxyureido)phenyl)thiophene-3-carboxylate; The gradient elution is set as follows: 0 - 18 min: 75 - 80% A → 60 - 65% A; 18 - 30 min: 60 - 65% A → 33 - 38% A; 30 - 35 min: 33 - 38% A → 33 - 38% A; 35 - 38 min: 33 - 38% A → 75 - 80% A; 38 - 45 min: 75 - 80% A → 75 - 80% A; In the mobile phase A, the volume ratio of the phosphate buffer solution and acetonitrile is 90:10; The phosphate buffer solution uses potassium dihydrogen phosphate buffer solution; The concentration of the phosphate buffer solution is 15 - 25 mmol / L.

2. The separation and detection method of a relugolix key intermediate and its related impurities according to claim 1, characterized in that, The gradient elution is set as follows: 0 - 18 min: 80% A → 60% A; 18 - 30 min: 60% A → 33% A; 30 - 35 min: 33% A → 33% A; 35 - 38 min: 33% A → 80% A; 38 - 45 min: 80% A → 80% A.

3. The method for separating and detecting a key intermediate of relugolix and its related impurities according to claim 1, characterized in that: The flow rate of the mobile phase elution is 0.7 - 1.0 ml / min.

4. The separation and detection method of a relugolix key intermediate and its related impurities according to claim 1, characterized in that, In the HPLC analysis, the detection wavelength is 254 ± 2 nm.

5. A method for separating and detecting a relugolix key intermediate and its related impurities according to claim 1, characterized in that, The particle size of the octadecylsilyl silica gel used as the filler is 2 - 5 μm, and the column temperature of the chromatographic column is 25 - 35 °C.

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