Detection Method for a Key Intermediate and Related Impurities of Relugolix

Abstract

The present invention discloses a method for detecting a key intermediate of relugolix and related impurities. The method uses a chromatographic column filled with octadecylsilane-bonded silica gel, a mixture of phosphate buffer solution and acetonitrile as mobile phase A, and acetonitrile as mobile phase B for gradient elution, and enters a detector for detection. The method provided by the present invention can effectively separate the key intermediate of relugolix and its related impurities, and the method has high sensitivity and resolution, good repeatability and durability, simple operation, stable and reliable results, and is of extremely important significance for realizing the quality control of relugolix.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, and relates to a detection method for a key intermediate of relugolix, in particular an HPLC method for separating and determining a key intermediate of relugolix and its related impurities. Background Art

[0002] Relugolix is an oral non-peptide active gonadotropin-releasing hormone (GnRH) receptor antagonist jointly developed by Takeda, ASKA and Myovant in Japan. It can bind to and block GnRH receptors in the anterior pituitary gland, thereby reducing the estrogen level produced by the ovaries in women; it can reduce the production of testosterone, which is mainly the hormone that stimulates the occurrence and development of prostate cancer; it can also reduce the production of ovarian estradiol, which is known to stimulate the occurrence of uterine fibroids and endometriosis. Relugolix was approved in Japan in January 2019 for the treatment of symptoms related to uterine fibroids. It was approved in the United States in December 2021 for the treatment of advanced prostate cancer. It was also approved for a new indication of endometriosis in December 2021.

[0003] At present, since relugolix has been on the market for a short time, its quality standard has not been included in the latest version of the United States Pharmacopeia, and there are relatively few reports on related analytical methods. Patent CN104703992A discloses a preparation method of thiophenopyrimidine derivatives, and detects 4 starting materials and 7 intermediates for the preparation of relugolix. However, under the conditions of this analytical method, it is difficult to separate the key intermediate and its related impurities, and at the same time, some impurities cannot be eluted completely.

[0004] Ethyl 2-((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-methyl-5-(4-nitrophenyl)thiophene-3-carboxylate is a key starting material of relugolix, and the quality control of this compound plays an important role in the preparation of relugolix raw materials. Therefore, developing an efficient and accurate analytical method for quality analysis of the key starting material of relugolix is of great significance for the subsequent preparation of high-purity relugolix. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for separating and determining a key intermediate of relugolix and its related impurities by HPLC. The method of the present invention can effectively separate the key intermediate of relugolix and its related impurities, and this method has high sensitivity and resolution, good repeatability and durability, simple operation, and stable and reliable results.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] In the first aspect, the present invention provides a detection method for a key intermediate of relugolix and its related impurities, using high performance liquid chromatography, and the chromatographic conditions are as follows:

[0008] Chromatographic column packing: octadecylsilyl-bonded silica gel;

[0009] Flow rate: 0.7 - 1.0 ml / min;

[0010] Mobile phase: Phase A: a mixed solvent of phosphate buffer solution and organic solvent; Phase B: acetonitrile;

[0011] The key intermediate of relugolix is ethyl 2-((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-((dimethylamino)methyl)-5-(4-nitrophenyl)thiophene-3-carboxylate, and its chemical formula is C 26 H 27 F 2 N 3 O 6 S, and its structural formula is shown in formula (I):

[0012]

[0013] The impurities include:

[0014]

[0015] The gradient elution program is set as follows:

[0016]

[0017]

[0018] Furthermore, Phase A of the mobile phase is a mixture of phosphate buffer solution and acetonitrile, and the volume ratio of the phosphate buffer solution to acetonitrile is 70:30.

[0019] Even further, the phosphate buffer solution is an aqueous solution of potassium dihydrogen phosphate with a pH value of 6.8 - 7.2 and a concentration of 15 - 25 mmol / L. Preferably, the concentration is 20 mmol / L.

[0020] Further, the flow rate for elution with the mobile phase is 0.8 mL / min.

[0021] Further, the gradient elution program is set as follows:

[0022]

[0023] Furthermore, the packing particle size of the octadecylsilyl-bonded silica gel chromatographic column is 2 - 5 μm, and the column length is 100 - 250 mm; the column temperature of the chromatographic column is 25 - 35 °C. Preferably, the packing particle size of the octadecylsilyl-bonded silica gel chromatographic column is 3.5 μm, the column length is 150 mm, and the column temperature of the chromatographic column is 30 °C

[0024] Further, the detection wavelength of the detector is 254 ± 2 nm. Preferably, the detection wavelength of the detector is 254 nm.

[0025] Further, the solvent for sample preparation is a mixed solution of 0.05 wt% phosphoric acid aqueous solution and 0.05 wt% acetonitrile phosphoric acid solution with a volume ratio of 10:90.

[0026] Further, the solution concentration for sample preparation is: about 1 mg / mL for the key intermediate of relugolix, and about 1 μg / mL for the impurities.

[0027] In a second aspect, the present invention provides a reagent composition for solid-liquid separation determination of the key intermediate of relugolix and its impurities by the detection method described in the first aspect.

[0028] The composition of the reagent composition is as follows:

[0029] Reagent A: A mixed solvent of phosphate buffer and acetonitrile;

[0030] Reagent B: Acetonitrile.

[0031] Further, the phosphate concentration in the phosphate buffer is 15 - 25 mmol / L.

[0032] Further, the volume ratio of the phosphate buffer to acetonitrile is 70:30.

[0033] The reagent composition provided by the present invention for solid-liquid separation determination of the key intermediate of relugolix and its impurities can effectively separate the key intermediate of relugolix and its impurities, which is of extremely important significance for realizing the quality control of the key intermediate of relugolix and relugolix.

[0034] The beneficial effects of the present invention are as follows:

[0035] 1) The present invention provides a method for separating and determining the key intermediate of relugolix and its related impurities by HPLC. The method of the present invention can effectively separate the key intermediate of relugolix and its related impurities, and the method has high sensitivity and resolution, good repeatability and durability, simple operation, and stable and reliable results.

[0036] 2) It has been found through research that the structures of impurity D, impurity M of the key intermediate of formula I and this key intermediate are very close. If not controlled at this step and carried into the API during continuous reaction, it will be very difficult to remove. Therefore, it is necessary to strictly monitor each impurity during this step of the reaction. That is, the analysis and research of the key intermediate of relugolix in the present invention play a crucial role in the control of the synthesis reaction and the improvement of quality, and also directly affect the quality of the relugolix finished product. Therefore, this method is of extremely important significance for realizing the quality control of the key intermediate of relugolix and relugolix. Description of the Drawings

[0037] Figure 1 Under the conditions of the embodiments of the present invention, the chromatogram of the system suitability solution, and the elution order is impurity N, impurity M, impurity G, impurity D, compound of formula I, impurity A, and impurity B in sequence;

[0038] Figure 2 Chromatogram of the mixed solution under the conditions of Comparative Example 1;

[0039] Figure 3 Chromatogram of the mixed solution under the conditions of Comparative Example 2;

[0040] Figure 4 Chromatogram of the mixed solution under the conditions of Comparative Example 3. Detailed Description of the Invention

[0041] The content of the present invention will be further described below in conjunction with specific embodiments, and the content of the present invention is not limited thereto at all.

[0042] Example 1

[0043] 1 Chromatographic conditions:

[0044] Chromatographic column: YMC-Pack Pro C18 (150 mm × 4.6 mm, 3 μm);

[0045] Mobile phase A: 20 mmol / L potassium dihydrogen phosphate buffer solution (pH 7.0) - acetonitrile (70:30), mobile phase B: acetonitrile; gradient elution was performed, and the gradient elution was set as follows:

[0046]

[0047] Flow rate: 0.8 ml / min, column temperature: 30 °C, detection wavelength: 254 nm, injection volume: 10 μl.

[0048] 2 Method and Results

[0049] 2.1 Solution preparation

[0050] An appropriate amount of the key intermediate of relugolix was accurately weighed, dissolved in the diluent and quantitatively diluted to prepare a solution containing about 1 mg per 1 ml as the test solution; wherein, the diluent was formed by mixing a 0.05 wt% phosphoric acid aqueous solution and a 0.05 wt% phosphoric acid acetonitrile solution in a volume ratio of 10:90, and the same was true for other examples; an appropriate amount of the test solution was accurately measured and diluted with the diluent to prepare a solution containing about 10 μg per 1 ml as the control solution.

[0051] 2.2 Specificity

[0052] Weigh appropriate amounts of the key intermediate of relugolix, impurity A, impurity B, impurity N, impurity M, impurity G, and impurity D accurately, place them in the same volumetric flask, dissolve and dilute with a diluent to prepare a solution containing approximately 1 mg of the compound of formula I and approximately 2 μg of each impurity per 1 ml as the system suitability solution. Pipette 10 μl accurately and inject it into the liquid chromatograph, record the chromatogram. The results of the system suitability solution are shown in the appendix Figure 1 In the system suitability solution, the resolution between the key intermediate of relugolix and the adjacent impurity peak is 2.5, and the retention time is 33.996.

[0053] 2.3 Precision

[0054] Weigh approximately 10 mg of the key intermediate of relugolix accurately, place it in a 10-ml volumetric flask, dissolve and dilute to the mark with a diluent to prepare the test solution; Pipette 1 ml of the test solution accurately and transfer it to a 100-ml volumetric flask, dilute to the mark with a diluent to prepare the control solution. Pipette the above test solution and control solution accurately for injection, record the chromatogram. The results of the detection of related impurities in 6 portions of the test solution are 0.01%, the range is 0%, not greater than 0.02%, meeting the requirements for the detection of related substances by high performance liquid chromatography.

[0055] 3 Conclusion:

[0056] Under this chromatographic condition, the key intermediate of relugolix and its impurities can be completely separated. This method has strong specificity, high precision, good repeatability, and good system suitability, meeting the technical requirements of drug quality research standards, and the obtained results are stable and reliable.

[0057] Comparative Example 1

[0058] 1 Chromatographic conditions:

[0059] Chromatographic column: YMC-Pack-Pro C18, 150 mm × 4.6 mm, 3 μm, mobile phase A: 0.05% phosphoric acid aqueous solution: 0.05% phosphoric acid acetonitrile solution (90:10), mobile phase B: acetonitrile, perform gradient elution, and the gradient elution settings are as follows:

[0060]

[0061] 2 Method and results

[0062] Weigh appropriate amounts of the key intermediate of relugolix and impurity D accurately, place them in the same volumetric flask, dissolve and dilute with a diluent to prepare a solution containing approximately 1 mg of the compound of formula I and approximately 25 μg of impurity D per 1 ml as the mixed solution. Pipette 10 μl accurately and inject it into the liquid chromatograph, record the chromatogram. The results of the detection of the mixed solution are shown in the appendix Figure 2 。

[0063] 3 Conclusion: Under these chromatographic conditions, impurity D and the compound of formula I were not completely separated, affecting the quantitative results and qualitative judgment of impurity D.

[0064] Comparative Example 2

[0065] 1 Chromatographic conditions:

[0066] Chromatographic column: YMC-Pack-Pro C18, 150 mm × 4.6 mm, 3 μm, mobile phase A: 10 mmol / L potassium dihydrogen phosphate solution: acetonitrile (70:30), mobile phase B: acetonitrile, gradient elution was carried out, and the gradient elution settings were as follows:

[0067]

[0068] 2 Methods and results

[0069] An appropriate amount of the key intermediate of relugolix and impurities A, B, and D were accurately weighed and placed in the same volumetric flask. Diluent was added to dissolve and dilute to prepare a solution containing about 2 mg of the compound of formula I and about 2 μg of each impurity per 1 ml as a mixed solution. 10 μl was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded. The test results of the mixed solution are shown in the appendix Figure 3 .

[0070] 3 Conclusion: Under these chromatographic conditions, impurity A and the unknown impurity were not baseline separated, affecting the quantitative results and qualitative judgment of the impurity.

[0071] Comparative Example 3

[0072] 1 Chromatographic conditions:

[0073] Chromatographic column: YMC-Pack-Pro C18, 150 mm × 4.6 mm, 3 μm, mobile phase A: 10 mmol / L potassium dihydrogen phosphate solution (pH 6.8): tetrahydrofuran: acetonitrile (10:3:7), mobile phase B: acetonitrile, gradient elution was carried out, and the gradient elution settings were as follows:

[0074]

[0075] 2 Methods and results

[0076] An appropriate amount of the key intermediate of relugolix and impurities A, B, M, and D were accurately weighed and placed in the same volumetric flask. Diluent was added to dissolve and dilute to prepare a solution containing about 1 mg of the compound of formula I and about 5 μg of each impurity per 1 ml as a system suitability solution. 10 μl was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded. The test results of the mixed solution are shown in the appendix Figure 4 .

[0077] 3 Results: Impurity M elutes earlier and overlaps with the blank solvent peak, affecting the quantitative and qualitative results of the impurity.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

[0079] As mentioned above, the above are only the preferred specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent replacements, and improvements made by those skilled in the art within the technical scope disclosed by the present invention should all be included within the scope of protection of the present invention.

Claims

1. A method for detecting a key intermediate of relugolix and related impurities, which is characterized in that: High performance liquid chromatography is used, and the chromatographic conditions are as follows: Chromatographic column packing: octadecylsilyl bonded silica gel; Flow rate: 0.7 - 1.0 ml / min; Mobile phase: Phase A: a mixed solvent of phosphate buffer solution and acetonitrile, the volume ratio of phosphate buffer solution to acetonitrile is 70:30, the phosphate buffer solution is an aqueous solution of potassium dihydrogen phosphate with a pH value of 6.8 - 7.2 and a concentration of 15 - 25 mmol / L; Phase B: acetonitrile; The key intermediate of relugolix is ethyl 2-(((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-((dimethylamino)methyl)-5-(4-nitrophenyl)thiophene-3-carboxylate, and its chemical formula is C 26 H 27 F 2 N 3 O 6 S, and the structural formula is shown in Formula (I): Formula (I) The impurities include: Impurity A: Ethyl 2-(((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-methyl-5-(4-nitrophenyl)thiophene-3-carboxylate; Impurity B: Ethyl 4-(bromomethyl)-2-(((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-5-(4-nitrophenyl)thiophene-3-carboxylate; Impurity N: 2-(((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-(((dimethylamino)methyl)-5-(4-nitrophenyl)thiophene-3-carboxylic acid; Impurity M: 2-(((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-4-(((dimethylamino)methyl)-5-(4-nitrophenyl)thiophene-3-carboxylic acid; Impurity G: 2-(((2,6-difluorobenzyl)amino)-4-(((dimethylamino)methyl)-5-(4-nitrophenyl)thiophene-3-carboxylic acid; Impurity D: Ethyl 4-(aminomethyl)-2-((((2,6-difluorobenzyl)(ethoxycarbonyl)amino)-5-(4-nitrophenyl)thiophene-3-carboxylate; The gradient elution program is set as follows: 。 2. The detection method according to claim 1, which is characterized in that: The packing particle size of the octadecylsilyl bonded silica gel chromatographic column is 2 - 5 μm, and the column length is 100 - 250 mm; the column temperature of the chromatographic column is 25 - 35 °C.

3. The detection method according to claim 1, which is characterized in that: The detection wavelength of the detector is 254 ± 2 nm.

4. The detection method according to claim 1, which is characterized in that: The solvent for sample preparation is a mixed solution of 0.05 wt% phosphoric acid aqueous solution and 0.05 wt% phosphoric acid acetonitrile solution with a volume ratio of 10:90.

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