Detection method of iluaceae intermediate I related substances

Abstract

The invention relates to a method for detecting related substances of an iluaceae intermediate I, and belongs to the technical field of pharmaceutical analysis. According to the method, octadecylsilane chemically bonded silica is adopted as a chromatographic column of a filling agent, a phosphate buffer solution-methanol-acetonitrile is adopted as a mobile phase, a gradient elution mode is adopted, and the established high performance liquid chromatography method can effectively measure the content of the intermediate I, namely the content of 2-(dimethyl phosphoryl)-N-(2, 5-dichloropyrimidine) aniline and related impurities. The method is good in specificity, high in sensitivity and convenient to operate, and provides an effective guarantee for quality control of an iluaceae final product. The invention also provides a novel impurity reference substance compound and a preparation method thereof.

Description

[0001] This application claims the priority of the Chinese patent application filed with the China Patent Office on October 16, 2023, with application number 202311344353.9 and invention name “Method for detecting substances related to Iruak intermediate I”, all contents of which are incorporated by reference into this application. Technical Field

[0002] The present application belongs to the field of drug analysis technology, and specifically relates to a drug analysis method for determining the content of impurities related to Iluak intermediate I, 2-(dimethylphosphoryl)-N-(2,5-dichloropyrimidine)aniline. The present application also relates to a new reference compound QD for the detection method of related substances of Iluak intermediate I and its preparation method. Background Art

[0003] Tumor is one of the major diseases that seriously threatens human health and life. Traditional tumor treatment is achieved by discovering and destroying tumors. Now, with the continuous deepening of research on cell signal transduction pathways, people have a deeper understanding of the role of oncogenes and anti-cancer genes in tumor cells. The design of new anti-tumor drugs targeting specific molecular targets of tumors has attracted more and more attention and has become a hot research area. Targeted anti-tumor drugs have also been used in clinical practice as a new treatment method and have made significant progress in just a few years.

[0004] In recent years, anaplastic lymphoma kinase (ALK) has been a hot target for anti-tumor research and development. Both China and abroad are vigorously developing ALK inhibitors for the treatment of non-small cell lung cancer. Iluak, whose chemical name is (2-((5-chloro-2-((2-methoxy-4-(9-methyl-3,9-diazaspiro[5.5]undecane-3-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide, has the following structural formula:

[0005]

[0006] It is an ALK inhibitor developed by Qilu Pharmaceutical Co., Ltd., which can inhibit the phosphorylation of ALK and ROS1 kinases and block the activation of downstream signaling pathway proteins such as ERK, STAT5 and AKT, thereby inducing tumor cell death (apoptosis). The drug was approved for marketing by the National Medical Products Administration in June 2023, and its trade name is For the treatment of patients with anaplastic lymphoma kinase (ALK)-positive locally advanced or metastatic non-small cell lung cancer (NSCLC) who have progressed after prior treatment with crizotinib or are intolerant to crizotinib.

[0007] The synthetic route of Iluak is as follows:

[0008]

[0009] The quality control of intermediates plays an important role in reaction monitoring and yield improvement, and also affects the quality of the final product. Comprehensive and systematic quality control of the entire process from starting materials to final products, especially the quality control of key intermediates in the synthesis process, has important guiding significance for the quality research of Iluak and the improvement of the synthesis process.

[0010] According to the synthetic route of Iluak intermediate I, the impurities that may be generated during its preparation and storage include raw materials, intermediates, degradation products, etc. In order to improve the intermediate process control, effectively analyze the quality of drugs, and ensure drug safety, it is necessary to develop a convenient and effective analytical method for detecting Iluak intermediate-related substances. Summary of the invention

[0011] In a first aspect, the present application provides a method for analyzing and determining the content of impurities related to Iluak intermediate I, namely 2-(dimethylphosphoryl)-N-(2,5-dichloropyrimidine)aniline, by high performance liquid chromatography. The structure of intermediate I is shown below.

[0012]

[0013] Through structural analysis of intermediate I and its known impurities, a chromatographic column using octadecylsilane bonded silica gel as a filler and a mobile phase of diammonium hydrogen phosphate solution-methanol-acetonitrile system was used.

[0014] Specifically, the analysis method described in this application can be implemented as follows:

[0015] (1) Sample configuration

[0016] Test solution: Take an appropriate amount of the product, weigh accurately, dissolve it in methanol and quantitatively dilute it to make a solution containing about 0.5 mg per 1 ml.

[0017] System suitability solution: Take appropriate amounts of intermediate I, impurity SM1, impurity SM2, impurity SC, impurity SD, impurity SJ, impurity HX, impurity EQ and impurity QD, dissolve and dilute with methanol to make a mixed solution containing approximately 0.5 mg of intermediate I and 0.002 mg of each impurity per 1 ml. The structures of each impurity are shown in Table 1:

[0018] Table 1 Structures of impurities related to intermediate I

[0019]

[0020] (2) Chromatographic conditions

[0021] Instrument: High performance liquid chromatography, UV detector

[0022] Chromatographic column: The chromatographic column is filled with octadecylsilane bonded silica gel, the particle size of the chromatographic column is 5 μm; the length of the chromatographic column is 250 mm; the inner diameter of the chromatographic column is 4.6 mm.

[0023] Mobile phase A: methanol-phosphate buffer with a pH value of 5.5-6.5 (preferably pH 5.8-6.2, most preferably 6.0) (10:90); the phosphate buffer salt is an aqueous solution of diammonium hydrogen phosphate with a concentration of 5-15 mmol / L (preferably 9-11 mmol / L, most preferably 10 mmol / L), and the pH value is adjusted using a phosphoric acid solution of conventional concentration in the art (≥85%).

[0024] Mobile phase B: methanol-acetonitrile (10:30)

[0025] A preferred gradient elution procedure of the present application is as follows:

[0026] Flow rate: preferably 0.8-1.2 ml / min, most preferably 1.0 ml / min;

[0027] Column temperature: preferably 30°C to 40°C, most preferably 35°C;

[0028] Detection wavelength: 220nm;

[0029] Injection volume: 10 μl.

[0030] Preferred gradient elution program

[0031]

[0032] (3) Calculation of impurities

[0033] The impurity peaks in the chromatogram of the test solution were used to calculate the contents of each known impurity and unknown impurity in the intermediate I according to the area normalization method.

[0034] In summary, the analytical method claimed in the present application has the following positive effects: the analytical method can effectively separate the intermediate I and its related impurity system, has many advantages such as good specificity, high sensitivity, strong elution ability for potential impurities, convenient operation, and moderate analysis time. Based on the concept of drug quality control migration, it provides effective guarantee for the quality control of the final product of Iruak.

[0035] The second aspect of the present application provides a new impurity reference compound QD, which has the following structure:

[0036]

[0037] The third aspect of the present application provides a method for preparing the above-mentioned impurity reference compound QD, the method comprising the following steps:

[0038]

[0039] [Step 1] Add 2,5-dichloro-4-hydroxypyrimidine (Compound SC), 2-(dimethylphosphoryl)aniline (Compound SM2) and NMP (N-methylpyrrolidone), and add trifluoromethanesulfonic acid dropwise;

[0040] [Step 2] After heating, react and add sodium hydroxide solution dropwise to adjust the pH to alkaline;

[0041] [Step 3] Add dichloromethane for extraction, and concentrate the aqueous phase by distillation under reduced pressure to obtain a QD intermediate;

[0042] [Step 4] Phosphorus oxychloride is used as a chlorination reagent to undergo a chlorination reaction with the QD intermediate to generate compound QD.

[0043] In certain embodiments of the present application, [Step 1] preferably comprises adding trifluoromethanesulfonic acid dropwise under nitrogen protection.

[0044] In certain embodiments of the present application, [Step 2] is preferably heated to 80-90°C, more preferably to 85°C.

[0045] In certain embodiments of the present application, the pH of [Step 2] is preferably adjusted to 12-14, more preferably to 13.

[0046] In certain embodiments of the present application, [Step 3] preferably, after the aqueous phase is concentrated under reduced pressure, methyl tert-butyl ether is added and concentrated under reduced pressure to obtain a QD intermediate.

[0047] In certain embodiments of the present application, [Step 4] is preferably reacted at 80-90°C; more preferably at 85°C.

[0048] The present application also provides an Iluak intermediate I, which contains an impurity compound QD, and the QD content is not higher than 1.0%.

[0049] The present application also provides a composition, which mainly contains Iluak intermediate I and compound QD with a content not higher than 1.0%. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 System suitability solution profile for the analytical method described in Example 1. DETAILED DESCRIPTION

[0051] The present application is further described below by specific examples. It should be understood that the examples of the present application are only used to illustrate the present application, rather than to limit the present application. The technical solutions obtained by simply improving the present application or by equivalently replacing conventional means or components based on the technical solutions of the present application all belong to the protection scope of the present application.

[0052] The reagents or raw materials used in this application can be prepared through existing literature or existing technologies, or purchased.

[0053] Example 1 Iluak Intermediate I Method Verification

[0054] 1. Chromatographic conditions

[0055] Instrument: High performance liquid chromatography-ultraviolet detector

[0056] Chromatographic column: Octadecylsilane bonded silica gel as filler (Inertsil ODS-3, 4.6mm×250mm, 5μm chromatographic column or chromatographic column with equivalent performance)

[0057] Mobile phase A: methanol-0.01 mol / L diammonium hydrogen phosphate solution (take 1.32 g of diammonium hydrogen phosphate, add 1000 ml of water to dissolve, and adjust the pH value to 6.0 with phosphoric acid) (10:90)

[0058] Mobile phase B: methanol-acetonitrile (10:30)

[0059] Gradient elution program:

[0060]

[0061] Detection wavelength: 220nm

[0062] Flow rate: 1.0ml / min

[0063] Injection volume: 10 μl

[0064] Column temperature: 35°C

[0065] 2. Method Validation

[0066] (1) System applicability

[0067] Positioning solution: Take appropriate amounts of intermediate I, impurity SM1, impurity SM2, impurity SC, impurity SD, impurity SJ, impurity HX, impurity EQ and impurity QD, place them in different volumetric flasks, dissolve and dilute them with methanol to make a mixed solution containing approximately 0.5 mg of intermediate I and 0.1 mg of each impurity per 1 ml.

[0068] System suitability solution: Take appropriate amounts of intermediate I, impurity SM1, impurity SM2, impurity SC, impurity SD, impurity SJ, impurity HX, impurity EQ and impurity QD, dissolve and dilute with methanol to prepare a mixed solution containing approximately 0.5 mg of intermediate I and 0.002 mg of each impurity per 1 ml.

[0069] The above solution was tested according to the chromatographic conditions described in the examples of the present application. The retention time and separation degree of each impurity in the positioning solution and the system suitability solution are shown in Table 2. The system suitability spectrum is shown in Table 2. Figure 1 The results show that all known impurities in the mixed solution can be completely separated from the main component, and the purity angle of the main peak of intermediate I is less than the purity threshold. Therefore, the system suitability meets the requirements.

[0070] Table 2 System suitability results

[0071] (2) Limit of quantification

[0072] Determination of baseline noise: 10 μl of methanol was accurately measured and injected into the liquid chromatograph, and three injections were made continuously. The blank baseline noise within the peak time range of intermediate I and each impurity was recorded, and the average value was calculated.

[0073] Preparation of quantitative limit solution: Take appropriate amounts of intermediate I, impurity SM1, impurity SM2, impurity SC, impurity SD, impurity SJ, impurity HX, impurity EQ and impurity QD, accurately weigh them, dissolve them with methanol and gradually dilute them, and detect them according to the chromatographic conditions described in the examples of this application until the dilution is such that the ratio of the peak height of intermediate I and each impurity to the corresponding noise (i.e., signal-to-noise ratio, S / N) is greater than or equal to 10. At this time, the ratio of the sample concentration to the measured concentration of the test sample is the quantitative limit. 6 consecutive injections are made at this concentration, and the average peak height is calculated.

[0074] The test results show that under the analytical method described in this application, the quantitative limit of each impurity is below 0.05%, that is, all known impurities and unknown impurities with a content of more than 0.05% in the test sample can be detected and accurately quantified. Therefore, the sensitivity of the method meets the requirements.

[0075] Table 3 Intermediate I and the quantitative limits of each known impurity

[0076] name Concentration (μg / ml) Average peak height (μV) S / N Limit of quantitation (%) Impurity SD 0.199 775 287 0.04 Impurity SM2 0.214 283 60 0.04 Impurity HX 0.205 647 13 0.04 Impurity SM1 0.211 590 16 0.04 Impurity SC 0.213 177 38 0.04 Impurity SJ 0.201 770 208 0.04 Impurity EQ 0.202 737 48 0.04 Impurity QD 0.214 296 44 0.04 Intermediate I 0.152 459 24 0.03

[0077] (3) Solution stability

[0078] Test solution: Accurately weigh about 50 mg of the test sample, place it in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well. Place at room temperature (25°C), and inject samples for detection at 0 hours, 2 hours, 4 hours, 6 hours, 17 hours, and 28 hours.

[0079] 10 μl was accurately measured and injected into a liquid chromatograph, and tested according to the chromatographic conditions described in the examples of this application. The change rates of known impurities and total impurities were calculated, and the results are shown in Table 4.

[0080] Table 4 Solution stability results

[0081] Time (hours) Impurity SM1(%) Impurity QD(%) Total impurities (%) 0 0.54 0.07 0.69 2 0.54 0.07 0.65 4 0.54 0.08 0.64 6 0.54 0.07 0.65 17 0.54 0.07 0.64 28 0.53 0.07 0.64

[0082] The test results show that the test sample is stable within 28 hours at 25°C.

[0083] Example 2 Preparation of impurity compound QD

[0084]

[0085] Compound SC, compound SM2, and NMP (N-methylpyrrolidone) were put into a reaction bottle, trifluoromethanesulfonic acid was added under nitrogen protection, the temperature was raised to 85°C for reaction for 6 hours, 1 mol / L sodium hydroxide solution was added to adjust the pH to 13, dichloromethane was added for extraction, the aqueous phase was concentrated under reduced pressure, methyl tert-butyl ether was added and continued to be concentrated under reduced pressure to obtain QD intermediate. QD intermediate and phosphorus oxychloride were put into a reaction bottle, reacted at 85°C, the solvent was removed under reduced pressure, methyl tert-butyl ether was slurried, and after suction filtration, the solid was slurried with water and suction filtered to obtain compound QD.

[0086] 1 H-NMR (400MHz, DMSO, δppm): 1.78-1.82 (d, 6H), 7.12-7.16 (t, 1H), 7.54-7.63 (m, 2H), 8.45-8.47 (q, 1H), 8.68 (s, 1H), 11.61 (s, 1H);

[0087] MS m / z:316.07([M+H] + ).

Claims

1. A method for analyzing and determining the content of related impurities of Iluak intermediate I by high performance liquid chromatography, wherein the intermediate I is 2-(dimethylphosphoryl)-N-(2,5-dichloropyrimidine)aniline, characterized in that: A chromatographic column with octadecylsilane bonded silica as filler was used, phosphate buffer-methanol was used as mobile phase A, methanol-acetonitrile was used as mobile phase B, and gradient elution was adopted.

2. The method according to claim 1, characterized in that The particle size of the chromatographic column is 5 μm; the length of the chromatographic column is 250 mm; and the inner diameter of the chromatographic column is 4.6 mm.

3. The method according to claim 1, characterized in that The pH value of the phosphate buffer in the mobile phase A is 5.8 to 6.2, preferably 6.

0.

4. The method according to claim 1, characterized in that The volume ratio of methanol to phosphate buffer in the mobile phase A is 1:

9.

5. The method according to claim 1, characterized in that The volume ratio of methanol to acetonitrile in the mobile phase B is 1:

3.

6. The method according to claim 1, characterized in that The elution gradient is:

7. The method according to claim 1, characterized in that The flow rate of the gradient elution mobile phase is 0.8-1.2 ml / min, preferably 1.0 ml / min; the column temperature of the chromatographic column is 30° C.-40° C., preferably 35° C.; the detection wavelength is 220 nm; and the injection volume is 10 μl.

8. The analysis method according to claim 1, wherein the phosphate buffer in the mobile phase A is an aqueous solution of diammonium hydrogen phosphate with a concentration of 5 to 15 mmol / L, preferably 9 to 11 mmol / L. 9 . The analysis method according to claim 1 , wherein the phosphate buffer in the mobile phase A is an aqueous solution of diammonium hydrogen phosphate with a concentration of 10 mmol / L.

10. An impurity reference substance QD used in the method of claim 1, having the following structure:

11. A method for preparing an impurity reference substance QD for use in the method of claim 1, the method comprising the following steps: [Step 1] Add 2,5-dichloro-4-hydroxypyrimidine (Compound SC), 2-(dimethylphosphoryl)aniline (Compound SM2) and NMP (N-methylpyrrolidone), and add trifluoromethanesulfonic acid dropwise; [Step 2] After heating, react and add sodium hydroxide solution dropwise to adjust the pH to alkaline; [Step 3] Add dichloromethane for extraction, and concentrate the aqueous phase by distillation under reduced pressure to obtain a QD intermediate; [Step 4] Phosphorus oxychloride is used as a chlorination reagent to undergo a chlorination reaction with the QD intermediate to generate compound QD.

12. The preparation method according to claim 11, wherein [Step 1] Trifluoromethanesulfonic acid is preferably added dropwise under nitrogen protection.

13. The preparation method according to claim 11, wherein: [Step 2] The temperature is preferably raised to 80 to 90°C, more preferably to 85°C.

14. The preparation method according to claim 11, wherein: [Step 2] The pH is preferably adjusted to 12 to 14, more preferably to 13.

15. The preparation method according to claim 11, wherein [Step 3] Preferably, after the aqueous phase is concentrated under reduced pressure, methyl tert-butyl ether is added and the mixture is further concentrated under reduced pressure to obtain a QD intermediate.

16. The preparation method according to claim 11, wherein [Step 4] The reaction is preferably carried out at 80 to 90°C; more preferably at 85°C.

17. Iluak intermediate I, comprising impurity compound QD, wherein the content of QD is not higher than 1.0%.

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