A HPLC analysis method for impurity reference substance of mirabegron

Abstract

The invention discloses an HPLC analysis method for an impurity reference substance of mirabegron. The invention reports the impurity for the first time, and the provided HPLC analysis method can effectively elute and separate mirabegron and the impurity, so that the peak of the impurity is completely separated from the peak of mirabegron, thereby quickly and reliably controlling the impurities of mirabegron products.

Description

Technical Field

[0001] The invention relates to an impurity reference substance of mirabegron and a preparation method and an HPLC analysis method thereof, belonging to the technical field of medicines. Background Art

[0002] Mirabegron (CAS registration number 223673-61-8), chemical name (R)-2-(2-aminothiazol-4-yl)-N-(4-{2-[(2-hydroxy-2-phenylethyl)amino]ethyl}phenyl)acetamide, is a selective β3 adrenergic receptor agonist developed by Astellas Pharmaceuticals of Japan. The drug was first launched in Japan in 2011 for the treatment of overactive bladder (OAB) in adults under the trade name The drug was subsequently approved for marketing by the US FDA in June 2012 and the EU in December 2012, with the marketed dosage form being a coated sustained-release tablet. The molecular formula of mirabegron is: C 21 H 24 N4O2S, molecular weight is 396.51, and its structural formula is:

[0003]

[0004] Patent WO2003037881 reports a method for synthesizing mirabegron, which uses D-mandelic acid and 4-nitrophenylethylamine as starting materials, undergoes amide condensation, borane reduction, catalytic hydrogenation reduction, and finally condenses with 2-aminothiazole-4-acetic acid to obtain mirabegron. The synthetic route is as follows:

[0005]

[0006] Although some mirabegron impurities have been reported in existing literature and data, further research on other impurities produced during the synthesis process of mirabegron or in the subsequent preparation and storage processes can be applied to the quality control of mirabegron, which is of great significance. Summary of the invention

[0007] First, the present invention provides a compound of formula I, which has a chemical structure as shown below:

[0008]

[0009] Secondly, the present invention also provides a method for preparing the compound of formula I, which comprises the following steps:

[0010] The compound of formula II (R-2-hydroxy-N-[2-(4-aminophenyl)ethyl]-2-phenylethylamine) reacts with the compound of formula III or a salt thereof in the presence of a suitable solvent and a base to obtain a compound of formula I;

[0011]

[0012] In some embodiments of the present invention, the compound of formula III or its salt is the compound of formula III or its hydrochloride, preferably the hydrochloride of the compound of formula III.

[0013] In some embodiments of the present invention, the solvent is formamide.

[0014] In some embodiments of the invention, the base is triethylamine.

[0015] In some embodiments of the present invention, the reaction temperature of the reaction is 30-50°C, preferably 35-45°C, and more preferably 40°C.

[0016] In some embodiments of the present invention, the reaction time is 4 to 6 hours, preferably 5 hours.

[0017] On the other hand, the present invention further provides a post-processing method, which comprises the following steps:

[0018] (1) Add water to the reaction solution and stir;

[0019] (2) filtering the reaction solution after stirring in step (1) and washing the filter cake with water;

[0020] (3) Drying the filter cake after elution in step (2).

[0021] In some typical embodiments, in the post-treatment method step (1), water is added to the reaction solution and stirred for 30 minutes; in some typical embodiments, in the post-treatment method step (3), the filter cake after elution in step (2) is dried by forced air at 40°C.

[0022] On the other hand, the present invention also provides an HPLC analysis method for detecting impurities in mirabegron, characterized in that:

[0023] (I) Take an appropriate amount of mirabegron test sample, accurately weigh it, add an appropriate amount of methanol to dissolve it, and then dilute it with mobile phase A to make a 1 mg / mL solution as the test sample solution;

[0024] (II) Take an appropriate amount of pure mirabegron, accurately weigh it, add an appropriate amount of methanol to dissolve it, and then quantitatively dilute it with mobile phase A to make a 1 mg / mL solution as reference solution 1;

[0025] (III) Take an appropriate amount of the reference substance of the compound of formula I, accurately weigh it, add an appropriate amount of methanol to dissolve it, and then quantitatively dilute it with mobile phase A to make a 1 mg / mL solution as the reference substance solution 2;

[0026] (IV) performing gradient elution on a chromatographic column using mobile phase A and / or mobile phase B;

[0027] The mobile phase A is water-acetonitrile-tetrahydrofuran, and the mobile phase B is acetonitrile;

[0028] (V) The content of the compound of formula I in the mirabegron test sample was calculated using the area normalization method.

[0029] In some embodiments, the chromatographic column used in the HPLC analysis method is a chromatographic column filled with octadecylsilane bonded silica gel.

[0030] In some embodiments, the chromatographic column used in the HPLC analysis method has a filler particle size of 2.7 to 4.0 μm, preferably a filler particle size of 3.5 μm.

[0031] In some embodiments, the inner diameter of the chromatographic column used in the HPLC analysis method is 4.5-4.7 mm, and preferably the inner diameter of the chromatographic column is 4.6 mm.

[0032] In some embodiments, the chromatographic column used in the HPLC analysis method has a column length of 90 to 110 mm, preferably a column length of 100 mm.

[0033] In some embodiments, the HPLC analysis method uses a chromatographic column with a filler particle size of 3.5 μm, an inner diameter of 4.6 mm, and a column length of 100 mm. The above model parameters can be abbreviated as 4.6 mm×100 mm, 3.5 um, or 4.6 mm×100 mm×3.5 μm, or other similar abbreviations.

[0034] In some embodiments, the column box temperature of the chromatographic column during separation and analysis in the HPLC analysis method is 20-40°C, preferably 25°C.

[0035] In some embodiments, the volume ratio of water-acetonitrile-tetrahydrofuran in the mobile phase A of the HPLC analysis method is 830-870:90-110:40-60, preferably 850:100:50.

[0036] In some embodiments, the flow rate of the mobile phase in the HPLC analysis method is 0.9-1.1 mL / min, preferably 1.0 mL / min.

[0037] In some embodiments, the detector in the HPLC analysis method is an ultraviolet detector.

[0038] In some embodiments, the detection wavelength used by the ultraviolet detector is 200-220 nm, and the preferred detection wavelength is 210 nm.

[0039] In some embodiments, the HPLC analysis method uses mobile phase A and mobile phase B for gradient elution in a certain ratio, and the gradient ratio is:

[0040] 0min, the volume ratio of mobile phase A to mobile phase B was 100:0;

[0041] 33min, the volume ratio of mobile phase A to mobile phase B was 65:35;

[0042] 38 min, the volume ratio of mobile phase A to mobile phase B was 50:50;

[0043] 40 min, the volume ratio of mobile phase A to mobile phase B was 50:50;

[0044] 40.1min, the volume ratio of mobile phase A to mobile phase B was 100:0;

[0045] 45min, the volume ratio of mobile phase A to mobile phase B was 100:0.

[0046] In yet another aspect, the present invention provides a compound of formula I for use in a method for detecting impurities of mirabegron.

[0047] In another aspect, the present invention provides use of the compound of formula I as a standard reference substance in a method for detecting impurities of mirabegron.

[0048] In the present invention, R-2-hydroxy-N-[2-(4-aminophenyl)ethyl]-2-phenylethylamine can be obtained through commercial sources, including but not limited to Shanghai Bid Pharmaceutical Technology Co., Ltd.; it can also be prepared by referring to the preparation method of patent WO2003037881.

[0049] In the present invention, "HPLC" means high performance liquid chromatography;

[0050] In the present invention, "mL" means milliliter; "μL" means microliter; in the present invention, "mg" means milligram;

[0051] In the present invention, "h" means hour, and "min" means minute;

[0052] In the present invention, "appropriate amount" means, as is easily understood by those skilled in the art, an amount of a solution sufficient to fully dissolve a solute.

[0053] The beneficial effects of the present invention are:

[0054] The present invention discovers a compound of formula I, which is reported for the first time and is a completely new compound. The present invention also provides a method for preparing the compound. At the same time, the present invention provides a high performance liquid chromatography gradient method, which can effectively elute and separate mirabegron and its impurity compound of formula I, so that the peak of the compound of formula I is completely separated from the peak of mirabegron, thereby quickly, simply and reliably controlling the impurities of the mirabegron product. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 LC-MS spectrum of the compound of formula I described in Example 1 of the present invention.

[0056] Figure 2 The hydrogen spectrum of the compound of formula I described in Example 1 of the present invention.

[0057] Figure 3 The carbon spectrum of the compound of formula I described in Example 1 of the present invention.

[0058] Figure 4 HPLC spectrum of the mirabegron test product of the present invention.

[0059] Figure 5 HPLC spectrum of reference solution 1 of the present invention.

[0060] Figure 6 HPLC spectrum of reference solution 2 of the present invention. DETAILED DESCRIPTION

[0061] The present invention is further described below in conjunction with the examples, but the present invention is not limited thereto. The reagents and raw materials used in the examples of the present invention are all commercially available.

[0062] Example 1: Preparation of the compound of formula I

[0063] In a 50 mL reaction bottle, 2.5 g of R-2-hydroxy-N-[2-(4-aminophenyl)ethyl]-2-phenylethylamine, 3.7 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 20 mL of formamide and 3 g of triethylamine were added. The mixture was heated to 40 °C and stirred for 5 h. 100 mL of water was added to the reaction solution, stirred for 30 min, and filtered. The filter cake was rinsed with 20 mL of water and dried with air at 40 °C to obtain the title compound in a yield of 54.8%.

[0064] MS (ESI+): 412.4 (M+H); Figure 1 As shown;

[0065] 1H-NMR (500MHz, DMSO-d6): δ7.43~7.31(m,7H), 7.25~7.23(d,J=8.0Hz,2H), 5.11~5.09(d,J=9.5H z,1H),3.43~3.15(m,14H),2.87(s,2H),2.55(s,6H),1.91(s,2H),1.18~1.15(t,J=6.75Hz,3H); such as Figure 2 As shown;

[0066] 13 C-NMR (125MHz, DMSO-d6): δ158.83, δ154.31, δ142.44, δ135.89, δ135.22, δ130.29, δ128.74, δ128. 05, δ126.32, δ125.55, δ68.76, δ55.11, δ48.62, δ43.21, δ42.65, δ36.92, δ34.51, δ25.84, δ16.10; such as Figure 3 shown.

[0067] Example 2: HPLC analysis method

[0068] The liquid phase detection method was determined in accordance with General Rules 0512 of Part IV of the 2015 edition of the Chinese Pharmacopoeia.

[0069] 1. Chromatographic conditions are as follows:

[0070] Chromatographic column: Waters Xbbridg C18 (4.6 mm × 150 mm, 3.5 μm);

[0071] Mobile phase A: water-acetonitrile-tetrahydrofuran (volume ratio 850:100:50);

[0072] Mobile phase B: acetonitrile;

[0073] Column temperature: 25°C;

[0074] Detection wavelength: 210nm;

[0075] Flow rate: 1.0 mL / min;

[0076] Injection volume: 20.00 μL;

[0077] Mobile phase gradient:

[0078]

[0079]

[0080] 2. Solution Preparation

[0081] Preparation of test solution: Take an appropriate amount of mirabegron test sample, accurately weigh it, add an appropriate amount of methanol to dissolve it, and dilute it with mobile phase A to make a 1 mg / mL solution as the test solution.

[0082] Preparation of reference solution 1: Take an appropriate amount of pure mirabegron, weigh it accurately, add an appropriate amount of methanol to dissolve it, and then add mobile phase A to quantitatively dilute it to make a 1 mg / mL solution.

[0083] Preparation of reference solution 2: Take an appropriate amount of the compound of formula I prepared in Example 1, weigh it accurately, add a small amount of methanol to dissolve it, and then add mobile phase A to quantitatively dilute it to prepare a 1 mg / mL solution.

[0084] 3. Determination

[0085] Accurately measure 20 μl of the test solution, reference solution 1 and reference solution 2, respectively, and inject them into the liquid chromatograph to record the chromatogram.

[0086] Table 1 shows the retention time, peak height, peak area and relative area of ​​the HPLC spectrum of the mirabegron test solution.

[0087] Table 1 HPLC related information of the test products

[0088]

[0089]

[0090] From the data in Table 1 and Figure 4 It can be seen that there is a main peak at the retention time of 16.475min and an impurity peak at the retention time of 21.092min, and the two are well separated.

[0091] Table 2 shows the information such as retention time, peak height, peak area and relative area of ​​the HPLC spectrum of reference solution 1.

[0092] Table 2 HPLC related information of reference solution 1

[0093]

[0094] From the data in Table 2 and Figure 5 It can be seen that the main peak appears at the retention time of 16.479min, corresponding to Figure 4 The main peak position with a retention time of 16.475 min was confirmed Figure 4 The substance eluting at the retention time of 16.475 min is mirabegron.

[0095] Table 3 shows the information such as retention time, peak height, peak area and relative area of ​​the HPLC spectrum of reference solution 2.

[0096] Table 3 HPLC related information of reference solution 2

[0097]

[0098] From the data in Table 3 and Figure 6 It can be seen that the main peak appears at the retention time of 21.093min, corresponding to Figure 4 The peak position with a retention time of 21.092 min was confirmed Figure 4 The substance with a peak at the retention time of 21.092 min is the compound of formula I. The results show that the content of the compound of formula I in the test sample is 4.96%.

Claims

1. An HPLC analysis method for detecting impurities in mirabegron, characterized in that The following steps are involved: (I) Take an appropriate amount of mirabegron test sample, accurately weigh it, add an appropriate amount of methanol to dissolve it, and then dilute it with mobile phase A to make a 1 mg / mL solution as the test sample solution; (II) Take an appropriate amount of pure mirabegron, accurately weigh it, add an appropriate amount of methanol to dissolve it, and then quantitatively dilute it with mobile phase A to make a 1 mg / mL solution as reference solution 1; (III) Take an appropriate amount of the reference substance of the compound of formula I, accurately weigh it, add an appropriate amount of methanol to dissolve it, and then quantitatively dilute it with mobile phase A to make a 1 mg / mL solution as the reference substance solution 2; (IV) performing gradient elution on a chromatographic column using mobile phase A and mobile phase B; The mobile phase A is water-acetonitrile-tetrahydrofuran, and the mobile phase B is acetonitrile; (V) calculating the content of the compound of formula I in the mirabegron test sample by using the area normalization method; The chromatographic column used in the HPLC analysis method is a chromatographic column filled with octadecylsilane bonded silica gel, the filler particle size of the chromatographic column is 3.5 μm, the inner diameter of the chromatographic column is 4.6 mm, and the column length of the chromatographic column is 100 mm; the column box temperature of the chromatographic column during separation and analysis in the HPLC analysis method is 20-40° C.; the volume ratio of water-acetonitrile-tetrahydrofuran in the mobile phase A is 830-870:90-110:40-60; The HPLC analysis method uses mobile phase A and mobile phase B for gradient elution in a certain ratio, and the gradient ratio is: 0min, the volume ratio of mobile phase A to mobile phase B was 100:0; 33min, the volume ratio of mobile phase A to mobile phase B was 65:35; 38 min, the volume ratio of mobile phase A to mobile phase B was 50:50; 40 min, the volume ratio of mobile phase A to mobile phase B was 50:50; 40.1min, the volume ratio of mobile phase A to mobile phase B was 100:0; 45min, the volume ratio of mobile phase A to mobile phase B was 100:

0.

2. The HPLC analysis method according to claim 1, wherein the column box temperature of the chromatographic column during separation and analysis in the HPLC analysis method is 25°C.

3. according to the HPLC analytical method described in claim 1, the volume ratio of water-acetonitrile-tetrahydrofuran in mobile phase A is 850:100:

50.

4. The HPLC analytical method according to claim 1, characterized in that: The preparation method of the reference substance of the compound of formula I comprises the following steps: The compound of formula II (R-2-hydroxy-N-[2-(4-aminophenyl)ethyl]-2-phenylethylamine) reacts with the compound of formula III or a salt thereof in the presence of a suitable solvent and a base to obtain a compound of formula I; 5. The HPLC analysis method according to claim 4, characterized in that: In the preparation method of the reference substance of the compound of formula I, the compound of formula III or its salt is the compound of formula III or its hydrochloride.

6. The HPLC analysis method according to claim 4, characterized in that: In the preparation method of the reference substance of the compound of formula I, the compound of formula III or its salt is the hydrochloride of the compound of formula III.

7. The HPLC analysis method according to claim 4, characterized in that: In the preparation method of the reference substance of the compound of formula I, the solvent is formamide and the base is triethylamine.

8. The HPLC analysis method according to claim 4, characterized in that: In the preparation method of the reference substance of the compound of formula I, the reaction temperature of the reaction is 30-50°C.

9. The HPLC analysis method according to claim 4, characterized in that: In the preparation method of the reference substance of the compound of formula I, the reaction temperature of the reaction is 35-45°C.

10. The HPLC analysis method according to claim 4, characterized in that: In the preparation method of the reference substance of the compound of formula I, the reaction temperature of the reaction is 40°C.

11. The HPLC analysis method according to claim 4, characterized in that: In the preparation method of the reference substance of the compound of formula I, the reaction time of the reaction is 4 to 6 hours.

12. The HPLC analysis method according to claim 4, characterized in that: In the preparation method of the reference substance of the compound of formula I, the reaction time of the reaction is 5 hours.

13. The HPLC analysis method according to claim 4, characterized in that: In the preparation method of the reference substance of the compound of formula I, a post-treatment method is further provided, which comprises the following steps: (1) Add water to the reaction solution and stir; (2) filtering the reaction solution after stirring in step (1) and washing the filter cake with water; (3) Drying the filter cake after elution in step (2).

14. The HPLC analysis method according to claim 13, characterized in that: In the preparation method of the reference substance of the compound of formula I, in the post-treatment method step (1), water is added to the reaction solution and stirred for 30 minutes.

15. The HPLC analysis method according to claim 13, characterized in that: In the preparation method of the reference substance of the compound of formula I, in the post-treatment method step (3), the filter cake after elution in step (2) is dried by forced air at 40°C.

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