A method for detecting the content of 2,4-disubstituted-5-fluoropyrimidine derivatives

Abstract

This invention belongs to the field of chemical analysis technology, specifically relating to a method for detecting the content of 2,4-disubstituted-5-fluoropyrimidine derivatives. The invention employs high-performance liquid chromatography (HPLC) for sample analysis. The HPLC conditions are as follows: the chromatographic column is packed with octadecylsilane-bonded silica gel; mobile phase A is a mixed solution of pH 2.5 phosphate buffer and trifluoroacetic acid; mobile phase B is acetonitrile; and gradient elution is used for detection. The method described in this invention has high accuracy and strong specificity, and is currently the only disclosed method for detecting the content of 2,4-disubstituted-5-fluoropyrimidine derivatives.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and specifically relates to a method for detecting the content of 2,4-disubstituted-5-fluoropyrimidine derivatives. Background Art

[0002] The chemical structure of the 2,4-disubstituted-5-fluoropyrimidine derivatives of the present invention is as follows: , whose CAS number is 2968523-32-0, is an innovative drug that can act on the HIPK2 target to fight renal fibrosis, reduce renal damage and renal interstitial fibrosis deposition.

[0003] APIs are the starting materials for drug production, and their quality directly affects the production of subsequent preparations and the quality of the final product. By paying attention to and strengthening content testing during drug production and research and development, the purity, uniformity and stability of APIs can be ensured, thereby meeting the quality requirements of drug production, ensuring the safety of patient medication and the effectiveness of drug efficacy.

[0004] However, at present, only patent CN118707005A discloses a method for detecting the concentration of 2,4-disubstituted-5-fluoropyrimidine derivatives in plasma by liquid chromatography-tandem mass spectrometry, which mainly uses the corresponding relationship between the known concentration of the internal standard (finasteride) and the known concentration of the 2,4-disubstituted-5-fluoropyrimidine derivative, and obtains the concentration / content of the unknown 2,4-disubstituted-5-fluoropyrimidine derivative in plasma from the known concentration of finasteride. There are still no relevant literature and patent reports on the detection method of drug content / purity in 2,4-disubstituted-5-fluoropyrimidine derivative raw materials. Therefore, it is necessary to develop a content analysis method with good specificity and system adaptability to ensure that the main peak and the adjacent peaks are well separated to be suitable for the accurate analysis of the content of the 2,4-disubstituted-5-fluoropyrimidine derivative. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a method for detecting the content of 2,4-disubstituted-5-fluoropyrimidine derivatives.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A method for detecting the content of a 2,4-disubstituted-5-fluoropyrimidine derivative, comprising: injecting and analyzing the 2,4-disubstituted-5-fluoropyrimidine derivative by high performance liquid chromatography to obtain a chromatogram, and determining the content of the 2,4-disubstituted-5-fluoropyrimidine derivative based on the chromatogram, wherein the high performance liquid chromatography adopts the following conditions:

[0008] Chromatographic column: octadecylsilane bonded silica gel column,

[0009] Detection wavelength: 254nm,

[0010] Column temperature: 30~35℃,

[0011] Mobile phase A: a mixture of pH 2.5 phosphate buffer and 0.15% trifluoroacetic acid by volume.

[0012] Mobile phase B: acetonitrile,

[0013] Flow rate: 0.8~1.0mL / min,

[0014] The elution gradient is:

[0015] ,

[0016] Running time: 50min.

[0017] In some embodiments, to ensure effective separation of the main component peak (i.e., main peak) from the interfering peak and a better main peak shape, the chromatographic column is YMC-Pack Pro C18, 150 mm×4.6 mm, 3 μm, or Waters sunfire C18, 150 mm×4.6 mm, 3.5 μm.

[0018] In some embodiments, in order to reduce the interference of ghost peaks during the analysis process, a capture column is added: Ghost-Sniper Column, 50 mm×4.6 mm.

[0019] In some embodiments, the injection volume of the injection analysis is 10 μL.

[0020] In some embodiments, the phosphate in the mobile phase A is dipotassium hydrogen phosphate.

[0021] In some embodiments, the 2,4-disubstituted-5-fluoropyrimidine derivative is provided in the form of a test solution, wherein the test solution is a methanol solution of the 2,4-disubstituted-5-fluoropyrimidine derivative, and based on each milliliter of the test solution, the concentration of the 2,4-disubstituted-5-fluoropyrimidine derivative is about 0.1 mg / mL.

[0022] In some embodiments, the content of the 2,4-disubstituted-5-fluoropyrimidine derivative is determined based on the chromatogram, and the content of the 2,4-disubstituted-5-fluoropyrimidine derivative is determined by calculating the chromatographic peak areas of the test solution and the reference solution obtained from the chromatogram according to the external standard method.

[0023] The present invention further provides a more specific method for detecting the content of 2,4-disubstituted-5-fluoropyrimidine derivatives, comprising:

[0024] (1) Chromatographic conditions

[0025] Chromatographic column: YMC-Pack Pro C18, 150mm×4.6mm, 3μm,

[0026] Capture column: Ghost-Sniper Column, 50mm×4.6mm,

[0027] Detector: PDA detector (i.e. photo-diode array detector, diode array detector),

[0028] Detection wavelength: 254nm,

[0029] Column temperature: 35°C,

[0030] Mobile phase A: a mixture of pH 2.5 potassium hydrogen phosphate buffer and 0.15% trifluoroacetic acid by volume.

[0031] Mobile phase B: acetonitrile,

[0032] Flow rate: 0.8 mL / min,

[0033] The elution gradient is:

[0034] ,

[0035] Running time: 50min,

[0036] Injection volume: 10 μL;

[0037] (2) Diluent

[0038] That is, the blank solution is methanol;

[0039] (3) Preparation of test solution

[0040] Take a certain amount of 2,4-disubstituted-5-fluoropyrimidine derivative, weigh it accurately, add diluent to dissolve and dilute to a concentration of about 0.1 mg / mL;

[0041] (4) Preparation of reference solution

[0042] Take a 2,4-disubstituted-5-fluoropyrimidine derivative reference substance, weigh it accurately, add a diluent to dissolve it and dilute it to a concentration of about 0.1 mg / mL, and prepare two in parallel;

[0043] (5) Sample injection

[0044] Take blank solution, reference solution and test solution and analyze them according to the above chromatographic conditions, and record the chromatogram and data;

[0045] (6) Calculation

[0046] The content of the 2,4-disubstituted-5-fluoropyrimidine derivative is determined by calculating the chromatographic peak areas of the test solution and the reference solution obtained from the chromatogram using the external standard method.

[0047] By means of the above scheme, the present invention has at least the following advantages:

[0048] (1) The detection method provided by the present invention is the only method disclosed so far for detecting the content of the 2,4-disubstituted-5-fluoropyrimidine derivatives. The high performance liquid chromatography analysis is highly efficient and can be used to support the research and development of the new drug, understand the quality and stability of the new drug, and provide a scientific basis for the production and marketing of the new drug.

[0049] (2) The detection method provided by the present invention can achieve effective separation of the main component and the solvent peak and a better main peak shape, has high accuracy and strong specificity, and provides an efficient, stable and reliable solution for the routine determination of raw materials.

[0050] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the blank solution chromatogram under the chromatographic conditions of Example 2;

[0052] Figure 2 This is the chromatogram of the test solution 1-01 described under the chromatographic conditions of Example 2;

[0053] Figure 3 This is the chromatogram of the test solution 1-02 described under the chromatographic conditions of Example 2;

[0054] Figure 4 This is the chromatogram of the test solution 2-01 described under the chromatographic conditions of Example 2;

[0055] Figure 5 This is the chromatogram of the test solution 2-02 described under the chromatographic conditions of Example 2. DETAILED DESCRIPTION

[0056] For ease of understanding, the application will be described more fully below, and preferred embodiments of the application are provided. However, the application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In the embodiment, if specific techniques or conditions are not indicated, the techniques or conditions described in the document in this area or according to the product specification are carried out. Reagents used or instruments that do not indicate manufacturers are conventional products that can be obtained through the market.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0058] 2,4-disubstituted-5-fluoropyrimidine derivative reference substance: with reference to the prior art (the preparation method of compound 7a involved in patent CN116514779A), the purity was measured and calibrated to batch 23081807, with a content of 92.80%, and stored in a refrigerator away from light and moisture.

[0059] The 2,4-disubstituted-5-fluoropyrimidine derivatives to be tested are samples from the homemade 230901 production batch.

[0060] Example 1 Development process of content detection method

[0061] In this example, the development process of the detection method for the content of 2,4-disubstituted-5-fluoropyrimidine derivatives is described in detail. Because the 2,4-disubstituted-5-fluoropyrimidine derivatives are innovative drugs, the detection method is developed and established with the purpose of excellent main peak shape and effective separation of the main peak.

[0062] 1.1 Preliminary determination of chromatographic column

[0063] The method parameters were initially formulated with reference to the characteristics of the compound in the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Rules 0512 High Performance Liquid Chromatography. Because the compound is a free base in the liquid phase, contains amino and benzene ring structures, and has a high polarity, the chromatographic column Waters sunfire C18 (specifications are 150mm×4.6mm, 3.5μm) was selected for development. This chromatographic column has a good peak shape for alkaline compounds. A chromatographic column with equivalent performance can also be selected, replacing the same type of chromatographic column YMC-Pack Pro C18 (specifications are 150mm×4.6mm, 3μm). In order to reduce the interference of ghost peaks during the analysis process, a small trapping column Ghost-Sniper Column (specifications are 50mm×4.6mm) was added.

[0064] 1.2 Selection of detection wavelength

[0065] The maximum absorption wavelength of the 2,4-disubstituted-5-fluoropyrimidine derivative is 223 nm, while 8-hydroxyquinoline is a larger impurity in the test solution, and its maximum absorption wavelength is 239 nm. Based on factors related to the characteristic absorption wavelength, the detection wavelength is set to 254 nm.

[0066] 1.3 Determination of mobile phase

[0067] The LogD value of the 2,4-disubstituted-5-fluoropyrimidine derivative was queried by ACD software, and it was found that the compound was well retained near pH = 2.7. In addition, considering the polarity of the compound and the peak shape of the alkaline compound, 0.15% trifluoroacetic acid by volume was added to the aqueous phase pH 2.5 phosphate buffer. Therefore, a mixed solution of pH 2.5 phosphate buffer and 0.15% trifluoroacetic acid (TFA) by volume was selected as mobile phase A, and acetonitrile was used as mobile phase B.

[0068] 1.4 Preliminary determination of elution gradient

[0069] Considering the elution conditions and separation, a gradient elution method was adopted, and the column temperature was set to 30°C, the elution time to 50 minutes, the flow rate to 1.0 mL / min, and the injection volume to 10 μL.

[0070] Table 1 Chromatographic conditions 1

[0071]

[0072] The blank solution (diluent) and the test solution (about 0.5 mg / mL of the analyte) were injected according to the chromatographic condition 1 in Table 1. It was found that the separation between the main peak and the adjacent peaks was poor, and the method optimization needed to be continued.

[0073] 1.5 Optimization of chromatographic column, elution gradient and flow rate

[0074] In order to increase the elution capacity, the maximum organic phase ratio was further reduced and the elution procedure was adjusted. In addition, the flow rate was reduced from 1.0 mL / min to 0.8 mL / min as chromatographic condition 2 (see Table 2). In addition, under the same gradient, the column temperature was also adjusted from 30°C to 35°C, and the separation effect of each peak was good.

[0075] The blank solution (diluent) and the test solution (about 0.5 mg / mL of the test substance) were injected separately, and it was found that the blank solution did not interfere with the main peak detection, and the separation degree between the main component and the adjacent interference peak and the main peak tailing factor met the requirements.

[0076] Table 2 Chromatographic conditions 2

[0077]

[0078] Example 2 System suitability and specificity test

[0079] In this example, the system applicability and specificity of the method for detecting the content of 2,4-disubstituted-5-fluoropyrimidine derivatives determined in Example 1 were investigated, and the drug content calculated based on the chromatogram obtained under the chromatographic conditions determined in Example 1 was introduced in detail.

[0080] 2.1 Solution preparation

[0081] Diluent: blank solution, methanol.

[0082] Test solution 1: Accurately weigh 5.202 mg of 2,4-disubstituted-5-fluoropyrimidine derivative, place it in a 50 ml volumetric flask, dissolve it in a diluent and dilute it to the scale, shake well, and obtain.

[0083] Test solution 2: Accurately weigh 5.200 mg of 2,4-disubstituted-5-fluoropyrimidine derivative, place it in a 50 ml volumetric flask, dissolve it with diluent and dilute it to the scale, shake well, and obtain.

[0084] Reference solution 1: Accurately weigh 5.136 mg of 2,4-disubstituted-5-fluoropyrimidine derivative reference substance, place it in a 50 ml volumetric flask, dissolve it with a diluent and dilute it to the scale, shake well, and obtain.

[0085] Reference solution 2: Accurately weigh 5.115 mg of 2,4-disubstituted-5-fluoropyrimidine derivative reference substance, place it in a 50 ml volumetric flask, dissolve it with diluent and dilute it to the scale, shake well, and obtain.

[0086] Mobile phase A (pH 2.5 phosphate buffer + 0.15% TFA): Take 4.564 g of potassium dihydrogen phosphate, dissolve it in 2000 ml of water, filter it through 0.22 μm water system, add 3 ml of trifluoroacetic acid, adjust the pH to 2.5 with 5 M sodium hydroxide, and ultrasonicate for 10 min.

[0087] Mobile phase B: Take 1000 ml of acetonitrile and ultrasonicate for 10 min.

[0088] 2.2 Chromatographic conditions

[0089] The detector used in the HPLC was a PDA detector. Other specific chromatographic conditions are shown in Table 2.

[0090] 2.3 Sampling

[0091] Take blank solution, reference solution and test solution and analyze them according to the above chromatographic conditions respectively, and record the chromatograms and data.

[0092] 2.4 Calculation

[0093] The content of the 2,4-disubstituted-5-fluoropyrimidine derivative is determined by calculating the chromatographic peak areas of the test solution and the reference solution obtained from the chromatogram using the external standard method.

[0094] 2.5 System suitability and specificity test results

[0095] After the liquid chromatography system is stable, take the diluent as the blank solution (≥1 injection) and inject the reference solution 1 continuously 5 times (5 injections) to record the chromatogram and data. The results are shown in Table 3 below.

[0096] Table 3 System suitability test results

[0097]

[0098] The results showed that the system was clean and stable, the blank solution had no obvious interference at the peak position of the target peak (i.e., main peak), the relative standard deviation (RSD) of the peak area of ​​5 consecutive target peaks was no more than 2.0%, and the retention time RSD was no more than 1.0%. The system adaptability met the requirements, and this method was suitable for detecting the content of the 2,4-disubstituted-5-fluoropyrimidine derivatives.

[0099] In addition, the blank solution, reference solution 1, reference solution 2, test solution 1, and test solution 2 were injected 1, 5, 2, 2, and 2 times, respectively. The target peak was shown in the reference solution, and the chromatograms of the blank solution and the test solution were shown in Figure 2. Figure 1-5 , showing: (1) blank without interference; (2) the target peak and adjacent peaks in the test solution have good separation; the specificity test meets the requirements, and this method is specific. The final content results of the tested batches of 2,4-disubstituted-5-fluoropyrimidine derivatives are shown in Table 4 below, and the average content is 93.6%.

[0100] Table 4 Content test results

[0101]

[0102] The method for detecting the content of 2,4-disubstituted-5-fluoropyrimidine derivatives described in the present invention has passed system adaptability and specificity tests, and the results are in compliance with the standards, indicating that the method described in the present invention can be used to accurately and quickly detect the content of 2,4-disubstituted-5-fluoropyrimidine derivatives, with strong specificity and high sensitivity.

[0103] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for detecting the content of 2,4-disubstituted-5-fluoropyrimidine derivatives, characterized in that: The 2,4-disubstituted-5-fluoropyrimidine derivative is sampled and analyzed by high performance liquid chromatography to obtain a chromatogram, and the content of the 2,4-disubstituted-5-fluoropyrimidine derivative is determined based on the chromatogram, wherein the high performance liquid chromatography adopts the following conditions: Chromatographic column: octadecylsilane bonded silica gel column, Detection wavelength: 254nm, Column temperature: 30~35℃, Mobile phase A: a mixture of pH 2.5 phosphate buffer and 0.15% trifluoroacetic acid by volume. Mobile phase B: acetonitrile, Flow rate: 0.8~1.0mL / min, The elution gradient is: ， Running time: 50 min; the 2,4-disubstituted-5-fluoropyrimidine derivative is obtained by the preparation method of compound 7a involved in CN116514779A.

2. The detection method according to claim 1, characterized in that: The chromatographic column is selected from YMC-Pack Pro C18, with a specification of 150 mm×4.6 mm, 3 μm, or Waters sunfire C18, with a specification of 150 mm×4.6 mm, 3.5 μm.

3. The detection method according to claim 1, characterized in that: The high performance liquid chromatography also uses a trapping column: Ghost-Sniper Column, 50 mm×4.6 mm.

4. The detection method according to claim 1, characterized in that: The injection volume of the sample analysis was 10 μL.

5. The detection method according to claim 1, characterized in that: The phosphate in the mobile phase A is dipotassium hydrogen phosphate.

6. The detection method according to claim 1, characterized in that: The 2,4-disubstituted-5-fluoropyrimidine derivative is provided in the form of a test solution, wherein the test solution is a methanol solution of the 2,4-disubstituted-5-fluoropyrimidine derivative, and based on each milliliter of the test solution, the concentration of the 2,4-disubstituted-5-fluoropyrimidine derivative is about 0.1 mg / mL.

7. The detection method according to claim 1, characterized in that: The content of the 2,4-disubstituted-5-fluoropyrimidine derivative is determined based on the chromatogram, and the content of the 2,4-disubstituted-5-fluoropyrimidine derivative is determined by calculating the chromatographic peak areas of the test solution and the reference solution obtained from the chromatogram according to the external standard method.

8. A method for detecting the content of 2,4-disubstituted-5-fluoropyrimidine derivatives, characterized in that: include: (1) Chromatographic conditions Chromatographic column: YMC-Pack Pro C18, 150mm×4.6mm, 3μm, Capture column: Ghost-Sniper Column, 50mm×4.6mm, Detector: Diode array detector, Detection wavelength: 254nm, Column temperature: 35°C, Mobile phase A: a mixture of pH 2.5 potassium hydrogen phosphate buffer and 0.15% trifluoroacetic acid by volume. Mobile phase B: acetonitrile, Flow rate: 0.8 mL / min, The elution gradient is: ， Running time: 50min, Injection volume: 10 μL; (2) Diluent That is, the blank solution is methanol; (3) Preparation of test solution Take a certain amount of 2,4-disubstituted-5-fluoropyrimidine derivative, weigh it accurately, add diluent to dissolve and dilute to a concentration of about 0.1 mg / mL; (4) Preparation of reference solution Take a 2,4-disubstituted-5-fluoropyrimidine derivative reference substance, weigh it accurately, add a diluent to dissolve it and dilute it to a concentration of about 0.1 mg / mL, and prepare two in parallel; (5) Sample injection Take blank solution, reference solution and test solution and analyze them according to the above chromatographic conditions, and record the chromatogram and data; (6) Calculation According to the chromatographic peak areas of the test solution and the reference solution obtained from the chromatogram, the content of the 2,4-disubstituted-5-fluoropyrimidine derivative is determined by calculation according to the external standard method; The 2,4-disubstituted-5-fluoropyrimidine derivative is obtained by the preparation method of compound 7a involved in CN116514779A.

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