High performance liquid chromatography detection method of aboxitinib intermediate and isomer thereof

Abstract

The invention discloses a high performance liquid chromatography detection method of an aboxitinib intermediate (1S, 3S)-3-(methylamino) cyclobutyl) benzyl carbamate and an isomer thereof. According to the method, a Daexid IC-3 chromatographic column is selected, a mixed solution of isopropanol, ethanol and normal hexane added with diethylamine is used as a mobile phase, and isocratic elution is carried out. According to the method, the contents of the aboxitinib intermediate (1S, 3S)-3-(methylamino) cyclobutyl) benzyl carbamate and the isomer thereof can be accurately determined through one-time high performance liquid chromatography detection, and a good separation effect can be achieved.

Description

Technical Field:

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a high performance liquid chromatography detection method for an abrocitinib intermediate and its isomers. Background Art:

[0002] Abrocitinib is a new generation of orally administered Janus kinase 1 (JAK1) selective inhibitor developed by Pfizer, with the trade name Cibinqo. This drug is applicable to adult patients with refractory, moderate to severe atopic dermatitis who have an inadequate response to other systemic therapies such as hormones or biological agents or are unsuitable for such treatments. The US FDA approved the drug for marketing on January 14, 2022, and the National Medical Products Administration of China approved the marketing of the first-class innovative drug abrocitinib tablets through the priority review process on April 11, 2022. The chemical name of abrocitinib is N-((1S,3S)-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)cyclobutyl)-1-propanesulfonamide, and its structure is shown in Formula I.

[0003]

[0004] (1S,3S)-3-(methylamino)cyclobutyl) benzyl carbamate is an important raw material for the synthesis of abrocitinib, and its structure is shown in Formula II. There are 2 chiral centers in the molecular structure of abrocitinib, and both of these chiral centers can be introduced by Compound II. Therefore, the chiral purity of Compound II largely determines the optical purity of abrocitinib and the amount of isomeric impurities, etc.

[0005] There are many optical isomers of the abrocitinib intermediate II, and conventional liquid chromatography columns are prone to peak overlap, making it difficult to achieve effective separation from all its isomers (the information on Compound II and its isomers is shown in Table 1). The separation of optical isomers of compounds containing multiple chiral carbon atoms has always been the focus and difficulty of drug quality control. However, there has been no report on the chiral analysis and determination of the abrocitinib intermediate II and its isomers so far. Therefore, we need to develop a high-efficiency and simple detection method for the detection of the abrocitinib intermediate II.

[0006] Table 1 Basic information of Compound II and its isomers

[0007] Summary of the Invention:

[0008] The purpose of the present invention is to provide a high performance liquid chromatography method for detecting the abrocitinib intermediate II and its isomers in view of the existing technology.

[0009] The technical solution adopted by the present invention is as follows: A Daicel IC-3 chromatographic column is selected, and a mixed solution of isopropanol, ethanol, n-hexane and diethylamine is used as the mobile phase. The volume ratio of the mobile phase is isopropanol / ethanol / n-hexane / diethylamine = 10-5 / 10-5 / 80-90 / 0.1, and isocratic elution is carried out at a flow rate of 0.4-0.8 mL / min.

[0010] Further, the Daicel IC-3 column is packed with silica gel surface-bonded with cellulose tris(3,5-dichlorophenylcarbamate).

[0011] Furthermore, the specifications of the Daicel IC-3 column are 150 mm * 4.6 mm * 3 μm.

[0012] Furthermore, the column temperature of the chiral chromatographic column is 15-30 °C, preferably 20 °C.

[0013] Further, the mobile phase is a mixed solution of isopropanol, ethanol, n-hexane and diethylamine.

[0014] Furthermore, the volume ratio of the mobile phase is isopropanol / ethanol / n-hexane / diethylamine = 10-5 / 10-5 / 80-90 / 0.1, preferably isopropanol / ethanol / n-hexane / diethylamine = 5 / 5 / 90 / 0.1.

[0015] Furthermore, the flow rate of the mobile phase is 0.4-0.8 mL / min, preferably 0.6 mL / min.

[0016] Further, the injection volume is 0.1-10 μL, preferably 1.0 μL.

[0017] Further, methanol and acetonitrile are selected as diluents for preparing the sample to be measured.

[0018] Further, the detection wavelength is 200-220 nm, preferably 217 nm.

[0019] Further, the running time is 30-90 min, preferably 60 min.

[0020] Further, under our detection conditions, the enantiomer II-c of the abrocitinib intermediate elutes first, followed by the abrocitinib intermediate II, and then the diastereomers II-a and II-b of the abrocitinib intermediate elute. The resolution between adjacent isomers is greater than 1.5, fully meeting the requirements of baseline separation.

[0021] The beneficial effects of the present invention are as follows: Through a single high-performance liquid chromatography detection, the present invention can accurately determine the contents of abrocitinib intermediate II and its various isomers, achieving a good separation effect. This method is simple and rapid, and can better control the chiral quality and stability of the product. Description of the Drawings

[0022] Figure 1 High-performance liquid chromatography chart of Example 1

[0023] Figure 2 Ultraviolet absorption chart of Example 1

[0024] Figure 3 High-performance liquid chromatography chart of Example 2

[0025] Figure 4 High-performance liquid chromatography chart of Example 3

[0026] Figure 5 High-performance liquid chromatography chart of Example 4

[0027] Figure 6 High-performance liquid chromatography chart of Example 5 Detailed Description of the Invention

[0028] The technical content of the present invention will be further elaborated below in conjunction with specific embodiments, with the aim of better understanding the content of the present invention, but the protection scope of the present invention is not limited thereto.

[0029] HPLC Analysis of Abrocitinib Intermediate II in Example 1

[0030] 1. Solution Preparation

[0031] a. Preparation of the stock solution of the reference substance

[0032] Weigh approximately 250 mg of abrocitinib intermediate II and its chiral isomer reference substances respectively into 25 mL volumetric flasks, dissolve and dilute to the mark with the diluent to obtain the stock solutions of abrocitinib intermediate II and its chiral isomers respectively. Shake well and set aside.

[0033] b. Preparation of the resolution solution

[0034] Take 2 mL of each chiral isomer solution and add it to 2 mL of the abrocitinib intermediate II solution to obtain the resolution solution. Shake well and set aside.

[0035] 2. Selection of Chromatographic Conditions

[0036] Table 2 Chromatographic Conditions

[0037] Chromatographic parameters Parameter description Chromatographic column Daicel IC-3 column, 150mm * 4.6mm * 3μm Mobile phase Isopropanol / ethanol / n-hexane / diethylamine = (5 / 5 / 90 / 0.1, V / V / V / V) Flow rate 0.6 mL / min Wavelength 217 nm Column temperature 20℃ Diluent 60 min Injection volume Acetonitrile

[0038] Inject the reference substance stock solution and the resolution solution under the above chromatographic conditions, record the chromatograms. The peak with a retention time of approximately 10.594 min is the enantiomer II-c of the abrocitinib intermediate, the peak at 14.249 min is the abrocitinib intermediate II, and the peaks at 25.918 min and 31.736 min are the diastereomers II-a and II-b of the abrocitinib intermediate. The resolution between adjacent peaks is greater than 1.5, meeting the requirements of baseline separation. See Table 3 and the appendix for details. Figure 1 The ultraviolet absorption wavelength is 217 nm. See the appendix for details. Figure 2 .

[0039] Table 3 Summary of Resolution Results

[0040]

[0041] HPLC Analysis of Different Chromatographic Columns in Example 2

[0042] In this example, the effect of chromatographic columns with different packings on the resolution of the method was investigated. Detection was carried out using the same conditions and method as in Example 1, except that the chromatographic column was replaced with a Daicel OD-3 column (cellulose tris(3,5-dimethylphenylcarbamate) coated on the silica surface), with a specification of 150 mm * 4.6 mm * 3 μm. Baseline separation of the abrocitinib intermediate II and its chiral isomers could not be achieved. See the appendix for details. Figure 3 .

[0043] HPLC Analysis of Different Mobile Phase Component Ratios in Example 3

[0044] In this example, the effect of changes in the mobile phase components outside the specified range on the resolution of the method was investigated. Detection was carried out using the same conditions and method as in Example 1, except that the mobile phase was replaced with isopropanol / ethanol / n-hexane / diethylamine (12.5 / 12.5 / 75 / 0.1, V / V / V / V). Baseline separation of all configurations could not be achieved. See the appendix for details. Figure 4 .

[0045] HPLC Analysis of Different Flow Rates in Example 4

[0046] In this example, the effect of fluctuations in the mobile phase flow rate within the specified range on the resolution of the method was investigated. Detection was carried out using the same conditions and method as in Example 1, except that the flow rate was replaced with 0.4 mL / min. Baseline separation of the abrocitinib intermediate II and the chiral isomer derivative products could be achieved. See the appendix for details. Figure 5 .

[0047] HPLC Analysis of Different Column Temperatures in Example 5

[0048] In this example, the influence of the temperature fluctuation of the column oven within the specified range on the resolution of the method was investigated. Detection was carried out using the same conditions and method as in Example 1, except that the column temperature was replaced with 15 °C, and baseline separation of axitinib intermediate II and chiral isomer derivatives could be achieved. For details, see Appendix Figure 6 .

Claims

1. High performance liquid chromatography detection method for abrocitinib intermediate and its isomers, Characterized in that, The abrocitinib intermediate is (1S,3S)-3-(methylamino)cyclobutyl)carbamic acid benzyl ester, and the isomers are (1S,3R)-3-(methylamino)cyclobutyl)carbamic acid benzyl ester, (1R,3S)-3-(methylamino)cyclobutyl)carbamic acid benzyl ester, (1R,3R)-3-(methylamino)cyclobutyl)carbamic acid benzyl ester. The Daicel IC-3 chromatographic column is selected, and a mixed solution of isopropanol, ethanol and n-hexane added with diethylamine is used as the mobile phase. The volume ratio of the mobile phase is isopropanol / ethanol / n-hexane / diethylamine = 10-5 / 10-5 / 80-90 / 0.1, and isocratic elution is carried out at a flow rate of 0.4-0.8 mL / min, the column temperature is 15-30 °C, and the detection wavelength is 200-220 nm.

2. The detection method according to claim 1, Characterized in that, The Daicel IC-3 chromatographic column is packed with silica gel surface-bonded with cellulose-tris(3,5-dichlorophenylcarbamate).

3. The detection method according to claim 1, Characterized in that, The injection volume is 0.1-10 uL.

4. The detection method according to claim 1, Characterized in that, Methanol and acetonitrile solution are selected as the diluent for preparing the sample to be measured.

5. The detection method according to claim 1, Characterized in that, The running time is 30-90 min.

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