Method for detecting enantiomer of starting material of nemategravir
Through the combination of high-performance liquid chromatography, CROWNPAK CR(+) chromatography column and aqueous perchloric acid solution, the problem of NMV-C00 enantiomer detection of NMV-C00 starting material was solved, efficient and accurate separation detection was achieved, and the quality control of NMV-C00 was ensured.
Patent Information
- Application Number
- CN202311782187.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-22
- Publication Date
- 2025-06-24
AI Technical Summary
The prior art lacks effective detection separation methods to control the enantiomer content of NMV-C00 in Nematve starting material.
The enantiomers of Compound A or its salt were detected using a CROWNPAK CR(+) chromatography column and 0.4% vol perchloric acid aqueous solution as mobile phase.
The high-efficiency separation of the enantiomers of Compound A or its salt is achieved, with high resolution, sensitivity, accuracy and precision, and can effectively control the quality of NMV-C00.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for detecting enantiomers of the starting material of nirmatrelvir. Background Art
[0002] Nirmatrelvir, chemically named: (1R,2S,5S)-N-[(1S)-1-cyano-2-(2-oxopyrrolidin-3-yl)ethyl]-3-[(S)-3,3-dimethyl-2-(trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, has the following structural formula:
[0003]
[0004] NMV-C00 (with the following structure), as the starting material for synthesizing nirmatrelvir, to ensure the quality of nirmatrelvir, it is necessary to control the content of the enantiomer compound C (with the following structure) of NMV-C00.
[0005]
[0006] Currently, there is no patent or literature reporting a method for detecting and separating the enantiomers of the nirmatrelvir starting material NMV-C00. Therefore, there is still an urgent need for a method for detecting and separating the enantiomers of the nirmatrelvir starting material NMV-C00 with good resolution and high sensitivity. Summary of the Invention
[0007] To solve the above technical problems, the present invention provides a method for detecting the enantiomers of compound A or the enantiomers of the salt of compound A in compound A or its salt.
[0008] A method for detecting the enantiomers of compound A or the enantiomers of the salt of compound A in compound A or its salt, which uses high performance liquid chromatography for detection, uses a CROWNPAK CR(+) chromatographic column as the separation chromatographic column, and uses perchloric acid aqueous solution as the mobile phase for elution; the enantiomer of compound A is as shown in compound B,
[0009]
[0010] In some embodiments, the inner diameter of the separation chromatographic column is 2.0 mm - 10.0 mm. In some embodiments, the inner diameter of the separation chromatographic column is 2.0 mm - 5.0 mm. In some embodiments, the inner diameter of the separation chromatographic column is 2.0 mm - 4.0 mm. In some embodiments, the inner diameter of the separation chromatographic column is 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, 4.5 mm, 4.6 mm, 5.0 mm, 6.0 mm, 7.0 mm, 8.0 mm, 9.0 mm or 10.0 mm.
[0011] In some embodiments, the length of the separation chromatographic column is 100 mm - 250 mm. In some embodiments, the length of the separation chromatographic column is 100 mm, 150 mm, 200 mm or 250 mm. In some embodiments, the length of the separation chromatographic column is 150 mm.
[0012] In some embodiments, the particle size of the packing material of the separation chromatographic column is 3 μm - 5 μm. In some embodiments, the particle size of the packing material of the separation chromatographic column is 3 μm, 3.5 μm, 4.0 μm, 4.5 μm, 4.6 μm or 5 μm.
[0013] In some embodiments, the content of perchloric acid in the perchloric acid aqueous solution is 0.1% vol - 0.5% vol. In some embodiments, the content of perchloric acid in the perchloric acid aqueous solution is 0.1% vol, 0.2% vol, 0.3% vol, 0.4% vol or 0.5% vol. In some embodiments, the content of perchloric acid in the perchloric acid aqueous solution is 0.4% vol.
[0014] In some embodiments, the detection wavelength of the high performance liquid chromatography is 200 nm - 250 nm. In some embodiments, the detection wavelength of the high performance liquid chromatography is 200 nm, 205 nm, 206 nm, 207 nm, 208 nm, 209 nm, 210 nm, 211 nm, 212 nm, 213 nm, 214 nm, 215 nm, 220, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm or 250 nm. In some embodiments, the detection wavelength of the high performance liquid chromatography is 210 nm.
[0015] In some embodiments, the flow rate of the high performance liquid chromatography is 0.1 mL / min - 1.5 mL / min. In some embodiments, the flow rate of the high performance liquid chromatography is 0.5 mL / min - 0.6 mL / min. In some embodiments, the flow rate of the high performance liquid chromatography is 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min or 1.5 mL / min.
[0016] In some embodiments, the column temperature of the high performance liquid chromatography is 20°C - 30°C. In some embodiments, the column temperature of the high performance liquid chromatography is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.
[0017] In some embodiments, the detection method uses high performance liquid chromatography for detection, with a CROWNPAK CR(+) chromatographic column as the separation column and 0.4% vol perchloric acid aqueous solution as the mobile phase for elution; the inner diameter of the separation column is 3.0 mm, the length is 150 mm, and the filler particle size is 5 μm; the detection wavelength of the high performance liquid chromatography is 210 nm, the flow rate is 0.5 mL / min - 0.6 mL / min, and the column temperature is 20°C - 30°C or 25°C.
[0018] In some embodiments, the detection method uses high performance liquid chromatography for detection, with a CROWNPAK CR(+) chromatographic column as the separation column and 0.4% vol perchloric acid aqueous solution as the mobile phase for elution; the inner diameter of the separation column is 3.0 mm, the length is 150 mm, and the filler particle size is 5 μm; the detection wavelength of the high performance liquid chromatography is 210 nm, the flow rate is 0.6 mL / min, and the column temperature is 20°C - 30°C or 25°C.
[0019] In some embodiments, the detection method uses high performance liquid chromatography for detection, with a CROWNPAK CR(+) chromatographic column as the separation column and 0.4% vol perchloric acid aqueous solution as the mobile phase for elution; the inner diameter of the separation column is 3.0 mm, the length is 150 mm, and the filler particle size is 5 μm; the detection wavelength of the high performance liquid chromatography is 210 nm, the flow rate is 0.5 mL / min, and the column temperature is 30°C.
[0020] In some embodiments, the salt of compound A is hydrochloride.
[0021] In some embodiments, the molar ratio of Compound A to hydrochloric acid in the salt of Compound A is 1:1, as shown by Compound NMV-C00; the enantiomers of the salt of Compound A are as shown by Compound C;
[0022]
[0023] Beneficial effects
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] The present invention uses a CROWNPAK CR(+), 150*3.0mm, 5μm chromatographic column as the separation chromatographic column and 0.4% perchloric acid solution as the mobile phase, and can effectively separate Compound A or its salt from the enantiomers of Compound A, or Compound A or its salt from the enantiomers of the salt of Compound A. The detection method has high resolution, high sensitivity, high accuracy, and high precision, and can effectively control the quality of Compound A or its salt.
[0026] The method of the present invention can simply, quickly, and accurately separate and detect the content of the enantiomers in Compound A or its salt.
[0027] Term description
[0028] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.
[0029] The term "%vol" represents volume percentage.
[0030] The term "wt%" represents mass percentage.
[0031] The term "M" represents molar concentration mol / L.
[0032] In the following content, whether or not words such as "about" or "approximate" are used, it means within 1%, 2%, 5%, 7%, 8%, 10%, 15% or 20% of a given value or range, etc. Alternatively, for those of ordinary skill in the art, the term "about" or "approximate" means within the acceptable standard error range of the average value. Whenever a number with a value of N is disclosed, any number with a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15% or N+ / -20% will be explicitly disclosed, where "+ / -" means plus or minus. Description of the Drawings
[0033] Figure 1 It is the chromatogram of the blank solution in Example 1.
[0034] Figure 2 It is the chromatogram of the NMV-C00 solution in Example 1. Among them, Figure 2 the chromatographic peak with a retention time of about 4.6 minutes is the chromatographic peak of NMV-C00.
[0035] Figure 3 It is the chromatogram of the NMV-C00 enantiomer solution in Example 1. Among them, Figure 3 the chromatographic peak with a retention time of about 2.6 minutes is the chromatographic peak of the NMV-C00 enantiomer.
[0036] Figure 4 It is the chromatogram of the blank solution in Example 2.
[0037] Figure 5 It is the chromatogram of the NMV-C00 solution in Example 2. Among them, Figure 5 the chromatographic peak with a retention time of about 4.0 minutes is the chromatographic peak of NMV-C00.
[0038] Figure 6 It is the chromatogram of the NMV-C00 enantiomer solution in Example 2. Figure 6 the chromatographic peak with a retention time of about 2.5 minutes is the chromatographic peak of the NMV-C00 enantiomer.
[0039] Figure 7 It is the chromatogram of the blank solution in Example 3.
[0040] Figure 8 It is the chromatogram of the NMV-C00 solution in Example 3. Figure 8 the chromatographic peak with a retention time of about 4.2 minutes is the chromatographic peak of NMV-C00.
[0041] Figure 9 It is the chromatogram of the NMV-C00 enantiomer solution in Example 3.Figure 9 The chromatographic peak with a retention time of about 2.9 minutes is the chromatographic peak of the NMV-C00 enantiomer. Detailed implementation mode
[0042] To enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below for further detailed description of the present invention.
[0043] All the reagents used in the present invention can be purchased from the market or prepared by the methods described in the present invention.
[0044] Example 1: Column temperature of 20 °C
[0045] Chromatographic conditions:
[0046] Chromatographic column: CROWNPAK CR(+) 150mm * 3.0mm, 5μm;
[0047] Detector: DAD (ultraviolet detector);
[0048] Detection wavelength: 210nm;
[0049] Flow rate: 0.6ml / min;
[0050] Column temperature: 20 °C;
[0051] Sample injection volume: 2μl;
[0052] Mobile phase: 0.4% perchloric acid aqueous solution;
[0053] Running time: 10min.
[0054] Solution preparation:
[0055] Diluent / blank solution: 0.4% vol perchloric acid aqueous solution.
[0056] NMV-C00 solution: Take 43.89mg of NMV-C00 sample, accurately weigh it into a 10mL EP tube, add 1mL of diluent, dissolve it, and shake well to obtain the NMV-C00 solution.
[0057] NMV-C00 enantiomer (Compound C) solution: Take 4.82mg of NMV-C00 enantiomer (Compound C) sample, accurately weigh it into a 10mL EP tube, add 10mL of diluent, dissolve it, and shake well to obtain the NMV-C00 enantiomer solution.
[0058] Operation:
[0059] Take the blank solution, NMV-C00 solution, and NMV-C00 enantiomer solution, perform high-performance liquid chromatography analysis under the above conditions, record the chromatogram, and the results are shown inFigure 1 , Figure 2 , Figure 3 .
[0060] Conclusion:
[0061] From Figure 2 and Figure 3 , it can be seen that this detection method can separate NMV-C00 from its enantiomer. This method can be used for the isomer quality monitoring of Nirmatrelvir NMV-C00.
[0062] Example 2: Column temperature 25°C
[0063] Chromatographic conditions:
[0064] Column temperature: 25°C;
[0065] The remaining chromatographic conditions are the same as those in Example 1.
[0066] Solution preparation:
[0067] The preparation of the blank solution, NMV-C00 solution, and NMV-C00 enantiomer solution is the same as that in Example 1.
[0068] Operation:
[0069] Take the blank solution, NMV-C00 solution, and NMV-C00 enantiomer solution, and perform high-performance liquid chromatography analysis under the above conditions. Record the chromatogram, and the results are shown in Figure 4 , Figure 5 , Figure 6 .
[0070] Conclusion:
[0071] Figure 5 and Figure 6 prove that Nirmatrelvir NMV-C00 and its enantiomer can be separated under the condition of a column temperature of 25°C, and this condition can be used for the isomer quality monitoring of Nirmatrelvir NMV-C00.
[0072] Example 3: Column temperature 30°C
[0073] Chromatographic conditions:
[0074] Column temperature: 30°C;
[0075] Flow rate: 0.5 ml / min;
[0076] The remaining chromatographic conditions are the same as those in Example 1.
[0077] Solution preparation:
[0078] The preparation of the blank solution, NMV-C00 solution, and NMV-C00 enantiomer solution is the same as that in Example 1.
[0079] Operation:
[0080] Take blank, NMV-C00 solution, and NMV-C00 enantiomer solution, perform high performance liquid chromatography analysis under the above conditions, record the chromatogram, and the results are shown in Figure 7 , Figure 8 , Figure 9 .
[0081] Conclusion:
[0082] Figure 8 and Figure 9 It is proved that Nimatrelvir NMV-C00 and its enantiomer can be separated in the method with a column temperature of 30 °C and a flow rate of 0.5 ml / min, and this condition can be used for the quality monitoring of the isomers of Nimatrelvir NMV-C00.
[0083] Comparative Example 1: Investigation of Chromatographic Column
[0084] Chromatographic Conditions:
[0085] Investigate the chromatographic columns in Table 1 respectively, and the other chromatographic conditions are the same as those in Example 1.
[0086] Table 1: Investigation of Chromatographic Column
[0087]
[0088]
[0089] Solution Preparation:
[0090] The preparation of blank solution, NMV-C00 solution, and NMV-C00 enantiomer solution is the same as that in Example 1.
[0091] Operation:
[0092] Take blank, NMV-C00 solution, and NMV-C00 enantiomer solution, perform high performance liquid chromatography analysis under the above conditions, record the chromatogram, and the separation results are shown in Table 1.
[0093] The method of the present invention has been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate modifications and combinations to the methods and applications described herein within the content, spirit, and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
Claims
1. A method for detecting an enantiomer of compound A or a salt thereof in compound A or an enantiomer of a salt of compound A, characterized in that, Detection was carried out by high performance liquid chromatography using a CROWNPAK CR(+) chromatographic column as the separation column and eluting with an aqueous perchloric acid solution; the enantiomers of compound A are shown as compound B.
2. The detection method according to claim 1, wherein the inner diameter of the separation column is 2.0 mm - 10.0 mm or 2.0 mm - 4.0 mm or 3.0 mm; and / or the length of the separation column is 100 mm - 250 mm or 150 mm; and / or the particle size of the packing of the separation column is 3 μm - 5 μm.
3. The detection method according to any one of claims 1 - 2, wherein the content of perchloric acid in the aqueous perchloric acid solution is 0.1% vol - 0.5% vol or 0.4% vol.
4. The detection method according to any one of claims 1 - 3, wherein the detection wavelength of the high performance liquid chromatography is 200 nm - 250 nm or 210 nm.
5. The detection method according to any one of claims 1 - 4, wherein the flow rate of the high performance liquid chromatography is 0.1 mL / min - 1.5 mL / min or 0.5 mL / min - 0.6 mL / min.
6. The detection method according to any one of claims 1 - 5, wherein the column temperature of the high performance liquid chromatography is 20°C - 30°C or 20°C.
7. The detection method according to any one of claims 1-6, characterized in that, Detection was carried out by high performance liquid chromatography using a CROWNPAK CR(+) chromatographic column as the separation column and eluting with an aqueous 0.4% vol perchloric acid solution; the inner diameter of the separation column is 3.0 mm, the length is 150 mm, and the particle size of the packing is 5 μm; the detection wavelength of the high performance liquid chromatography is 210 nm, the flow rate is 0.6 mL / min, and the column temperature is 20°C - 30°C or 25°C; or Detection was carried out by high performance liquid chromatography using a CROWNPAK CR(+) chromatographic column as the separation column and eluting with an aqueous 0.4% vol perchloric acid solution; the inner diameter of the separation column is 3.0 mm, the length is 150 mm, and the particle size of the packing is 5 μm; the detection wavelength of the high performance liquid chromatography is 210 nm, the flow rate is 0.5 mL / min, and the column temperature is 30°C.
8. The detection method according to any one of claims 1 - 7, wherein the salt of compound A is a hydrochloride.
9. The detection method according to claim 8, wherein the molar ratio of compound A to hydrochloric acid in the salt of compound A is 1:1, as shown by compound NMV - C00; the enantiomers of the salt of compound A are shown as compound C.