Method for detecting related substances in acavalopag maleate

Abstract

The invention discloses a method for detecting related substances in acalotrombopag maleate, which comprises the following steps: an octadecyl silane bonded silica gel chromatographic column, a mobile phase A and a mobile phase B are subjected to online mixed gradient elution, the mobile phase A is a water-acid-alcohol mixed solution, the acid is one or more of trifluoroacetic acid, formic acid, acetic acid and phosphoric acid, the mobile phase B is one or more of ethyl acetate, ethyl acetate and ethyl acetate, and the mobile phase C is one or more of ethyl acetate, ethyl acetate and ethyl acetate. The alcohol is methanol and / or ethanol; the mobile phase B is a solution of acid and an organic solvent, the acid is formic acid and / or trifluoroacetic acid, and the organic solvent is one or more of methanol, acetonitrile and tetrahydrofuran; the related substances are impurities A, B, C, D, E, F, G, H and I shown in the following formulas. The method provided by the invention can quickly and effectively separate the avantrombopag maleate raw material medicine from 9 impurities under the same chromatographic condition, the separation degree is greater than 1.2, the method has strong specificity, high sensitivity, good precision and high accuracy, the quality of the raw material medicine can be effectively controlled, and the effectiveness and safety of the medicine are finally ensured. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of drug analysis, and in particular to a method for detecting related substances in avatrombopag maleate. Background Art

[0002] Avatrombopag maleate is the first oral drug for the treatment of thrombocytopenia to enter China. It was developed by AkaRx Inc. of the United States and introduced by Fosun Pharma. It was approved by NMPA for marketing in China in 2020 for the treatment of thrombocytopenia associated with chronic liver disease. The chemical structure is as follows:

[0003]

[0004] The chemical properties of avatrombopag maleate are relatively stable. The synthesis route disclosed in CN201480010396.6 is as follows: In order to ensure the safety and effectiveness of the drug, the relevant substances introduced into its process are detected and monitored.

[0005]

[0006] There are mainly 9 specific impurities and non-specific impurities in the raw material of avatrombopag maleate:

[0007]

[0008]

[0009] The above impurities will affect the effectiveness and safety of avatrombopag maleate, so strict control is required to ensure the quality of the drug.

[0010] Patent CN11047196A provides a liquid phase method for detecting related substances of avatrombopag maleate, using perchloric acid-methanol-acetonitrile as the mobile phase, which can only control 6 process and degradation impurities. There is currently no publicly reported related substance detection method in China that can effectively separate and control the above 9 impurities. Summary of the invention

[0011] The technical problem to be solved by the present invention is to provide a method for determining related substances in avatrombopag maleate raw materials by high performance liquid chromatography, which is completely different from the prior art. The detection method of the present invention can effectively separate the avatrombopag maleate raw materials from 9 impurities under the same chromatographic conditions, and the main peak and the adjacent impurity peaks and the impurity peaks can achieve baseline separation, with a separation degree of ≥1.2. The method has strong specificity, high sensitivity, good precision and high accuracy, can effectively control the quality of the avatrombopag maleate raw materials, ensure the effectiveness and safety of the drug, and is suitable for industrial production and application.

[0012] The invention provides a method for detecting related substances in avatrombopag maleate, which comprises the following steps: using high performance liquid chromatography to detect related substances in avatrombopag maleate raw materials, the test conditions are: octadecylsilane bonded silica gel chromatographic column, the eluent is online mixed gradient elution of mobile phase A and mobile phase B, the mobile phase A is a mixed solution of water-acid-alcohol, the acid is one or more of trifluoroacetic acid, formic acid, acetic acid and phosphoric acid, the alcohol is methanol and / or ethanol; the mobile phase B is acid and an organic solvent, wherein the acid is formic acid and / or trifluoroacetic acid, and the organic solvent is one or more of methanol, acetonitrile and tetrahydrofuran; and the related substances are impurities A (CAS: 2626934-45-8), B (CAS: 2681355-40-6), C, D, E, F (CAS: 2883585-26-8), G (CAS: 570407-42-0), H (CAS: 958457-83-5) and I as shown in the following formula:

[0013]

[0014] In the present invention, the avatrombopag maleate includes the avatrombopag maleate raw material and a pharmaceutical preparation with avatrombopag maleate as an active ingredient, such as an avatrombopag maleate oral pharmaceutical preparation and an avatrombopag maleate injection; the avatrombopag maleate oral pharmaceutical preparation includes avatrombopag maleate tablets and avatrombopag maleate capsules, etc.

[0015] The high performance liquid chromatography detection described in the present invention is carried out in a high performance liquid chromatograph, and the high performance liquid chromatograph is preferably equipped with a diode array detector or an ultraviolet detector. The high performance liquid chromatograph is, for example, an Agilent 1260II high performance liquid chromatograph or a Thermo Fisher U3000 high performance liquid chromatograph.

[0016] In the present invention, the octadecylsilane bonded silica gel column is preferably a YMC Triart C18, 250 mm×4.6 mm, 3 μm chromatographic column.

[0017] In the present invention, the detection column temperature of the HPLC method is preferably 25°C to 35°C, for example 30°C.

[0018] In the present invention, the wavelength for detection by high performance liquid chromatography is preferably 250 nm to 260 nm, for example 254 nm.

[0019] In the present invention, the flow rate of the mobile phase for detection by high performance liquid chromatography is preferably 0.6 mL / min to 1.2 mL / min, and more preferably 0.7 mL / min.

[0020] In the present invention, the injection volume for detection by high performance liquid chromatography is preferably 5 μL to 20 μL, for example 5 μL.

[0021] In the present invention, the running time of the high performance liquid chromatography detection is preferably 100 minutes to

[0022] 200 minutes, more preferably 140 minutes to 180 minutes, for example 160 minutes.

[0023] In the present invention, the alcohol in the mobile phase A is preferably methanol.

[0024] In the present invention, the mobile phase A is preferably a mixed solution of water-methanol-trifluoroacetic acid; a mixed solution of water-methanol-formic acid or a mixed solution of water-methanol-phosphoric acid, and more preferably a mixed solution of water-methanol-trifluoroacetic acid.

[0025] In the mobile phase A, the volume percentages of water, alcohol and acid are preferably (700-950):(250-50):(2-0.5), for example, the volume ratio of water:methanol:trifluoroacetic acid is (900:100:1).

[0026] In the present invention, the acid in the mobile phase B is preferably trifluoroacetic acid; the organic solvent in the mobile phase B is preferably methanol and / or acetonitrile. The mobile phase B is preferably acetonitrile-methanol-trifluoroacetic acid. When the mobile phase B is a mixed solution of acetonitrile-methanol-trifluoroacetic acid, the volume ratio of acetonitrile-methanol-trifluoroacetic acid is preferably (700-950): (250-50): (2-0.5), for example (900:100:1).

[0027] In the mobile phase B, the percentage of the acid is preferably 0.05% to 0.20%, such as 0.10%, and the percentage refers to the percentage of the volume of the acid to the total volume of the acid and the organic solvent solution.

[0028] In the present invention, the diluent used in the high performance liquid chromatography detection is preferably a mixed solution of water, acid and organic solvent, the acid is preferably formic acid and / or trifluoroacetic acid, and the organic solvent is preferably one or more of methanol, acetonitrile and tetrahydrofuran.

[0029] In the present invention, the acid in the diluent is preferably trifluoroacetic acid; the organic solvent in the diluent is preferably methanol and / or acetonitrile. The diluent is preferably water-acetonitrile-trifluoroacetic acid. When the diluent is a mixed solution of water-acetonitrile-trifluoroacetic acid, the volume ratio of water-acetonitrile-trifluoroacetic acid is preferably (700-300): (700-300): (10-1), for example (500:500:2).

[0030] In the present invention, the online mixed gradient elution conditions of the mobile phase A and the mobile phase B can be:

[0031] 0-105min, the volume ratio of mobile phase A to mobile phase B is (80%-30%): (20%-70%);

[0032] 105-140 min, the volume ratio of mobile phase A to mobile phase B is (70%-30%): (60%-40%);

[0033] 140-145 min, the volume ratio of mobile phase A to mobile phase B is (50%-10%): (50%-90%);

[0034] 145.1~160min, the volume ratio of mobile phase A to mobile phase B is (80%~30%):(20%~70%); finally, balance for 10 minutes according to the initial ratio.

[0035] In the present invention, the online mixed gradient elution conditions of mobile phase A and mobile phase B are preferably:

[0036] Time (min) Mobile phase A (%) Mobile phase B (%) 0 60 40 105 50 50 140 30 70 145 10 90 145.1 60 40 160 60 40

[0037] The present invention provides a method for detecting related substances in avatrombopag maleate, wherein the separation degree between each impurity is greater than or equal to 1.2.

[0038] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0039] The reagents and raw materials used in the present invention are commercially available.

[0040] The positive progress of the present invention is that the method of the present invention can quickly and effectively separate the avatrombopag maleate raw material drug from 9 impurities under the same chromatographic conditions, and the main peak and the adjacent impurity peaks and the impurity peaks can achieve baseline separation, and the separation degree is greater than 1.2. The analytical method of the present invention has strong specificity, high sensitivity, good precision and high accuracy, and can effectively control the quality of the avatrombopag maleate raw material drug, and ultimately ensure the effectiveness and safety of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 System suitability solution map;

[0042] Figure 2 Batch 1 avatrombopag maleate API sample test spectrum;

[0043] Figure 3 Chromatogram of the undestroyed avatrombopag maleate API sample test;

[0044] Figure 4Acid-destroyed chromatogram of the Avatrombopag maleate API sample test;

[0045] Figure 5 Alkali-destroyed chromatogram of the Avatrombopag maleate API sample test;

[0046] Figure 6 Chromatogram of the sample of avatrombopag maleate raw material drug tested for oxidation damage;

[0047] Figure 7 High temperature liquid destruction chromatogram of avatrombopag maleate API sample test;

[0048] Figure 8 Chromatogram of the photo-liquid destruction test of avatrombopag maleate raw material sample. DETAILED DESCRIPTION

[0049] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0050] 1. Instrument

[0051] High performance liquid chromatograph: Agilent 1260II, DAD detector or UV detector (diode array detector or UV detector); Thermo Fisher U3000, PDA detector or UV detector (diode array detector or UV detector).

[0052] Chromatographic column: YMC Triart C18, 250 mm × 4.6 mm, 3 μm.

[0053] 2. Chromatographic conditions

[0054] Column temperature: 30℃.

[0055] Detection wavelength: 254nm.

[0056] Flow rate: 0.7mL / min.

[0057] Injection volume: 5 μL.

[0058] Running time: 160 minutes.

[0059] Diluent: water-acetonitrile-trifluoroacetic acid (volume ratio 500:500:2)

[0060] Mobile phase A: water-methanol-trifluoroacetic acid (volume ratio: 900:100:1).

[0061] Mobile phase B: acetonitrile-methanol-trifluoroacetic acid (volume ratio: 900:100:1).

[0062] Gradient elution conditions are shown in Table 1

[0063] Table 1

[0064] Time (min) Mobile phase A (%) Mobile phase B (%) 0 60 40 105 50 50 140 30 70 145 10 90 145.1 60 40 160 60 40

[0065] Example 1

[0066] Preparation of system suitability solution: Accurately weigh appropriate amounts of avatrombopag maleate reference substance, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, and impurity I, and dissolve them by ultrasonication in a diluent to prepare a mixed solution containing 2 mg of avatrombopag maleate and 2 μg of each impurity as the system suitability solution. (The concentration of avatrombopag maleate is about 2.0 mg / mL, and the concentration of a single impurity is 2.0 μg / mL)

[0067] The diluted solution was used as the blank solution.

[0068] Accurately measure 5 μL of system suitability solution and blank solution, operate according to the above chromatographic conditions, record the chromatogram, and the results are shown in Figure 1 , specific parameters are shown in Table 2.

[0069] Table 2

[0070]

[0071] Figure 1 The data showed that the main peak and impurities in the system suitability solution could be effectively separated, with a separation degree greater than 1.2.

[0072] Example 2

[0073] Avatrombopag maleate API sample solution: accurately weigh three batches of avatrombopag maleate API (Batch 1 to Batch 3), add an appropriate amount of diluent to dissolve, and dilute to make a solution containing approximately 2 mg of sample per 1 ml.

[0074] Control solution: Take an appropriate amount of reference solution and add a quantitative diluent to make a solution containing approximately 2 μg of sample per 1 ml.

[0075] Accurately measure 5 μL of the reference solution and three batches of avatrombopag maleate bulk drug sample solutions, respectively, inject them into the liquid chromatograph, and record the chromatograms. The results are shown in Table 3.

[0076] Table 3

[0077]

[0078]

[0079] The impurity profiles of the three batches of avatrombopag maleate API samples were consistent. The detected amounts of specific impurities in each batch were slightly different, but all within the acceptable standards. Figure 2 .

[0080] Impurities D, E, and I were detected in all three batches of avatrombopag maleate raw material samples; each individual unknown impurity was less than 0.10%, and the total impurity was within 0.10%.

[0081] Example 3 Detection method specificity test

[0082] Undamaged API solution: Take the Avatrombopag maleate API and dilute it with solvent to prepare a solution containing 2 mg / ml of this product (Avatrombopag maleate API), shake well, and obtain. Accurately measure 5 μL and inject it into the liquid chromatograph, detect it according to the above chromatographic conditions, record the chromatogram, and the results are shown in Figure 3 .

[0083] Acid destruction solution: Take 20 mg of the raw material drug and place it in a 10 ml volumetric bottle. Add 5 ml of diluent and shake to dissolve. Then add 1 ml of 1 mol / L hydrochloric acid solution. After leaving it for 24 hours, add 1 ml of 1 mol / L sodium hydroxide solution to neutralize it. Then use diluent to make up to the scale and shake well. Accurately measure 5 μL and inject it into the liquid chromatograph. Detect according to the above chromatographic conditions and record the chromatogram. The results are shown in Figure 4 .

[0084] Alkali destruction solution: Take 20 mg of the raw material drug and place it in a 10 ml volumetric bottle. Add 5 ml of diluent and shake to dissolve. Then add 1 ml of 1 mol / L sodium hydroxide solution. After leaving it for 24 hours, immediately add 1 ml of 1 mol / L hydrochloric acid solution to neutralize it. Then use diluent to make up to the mark and shake well. Accurately measure 5 μL and inject it into the liquid chromatograph. Detect it according to the above chromatographic conditions and record the chromatogram. The results are shown in the table. Figure 5 .

[0085] Oxidative destruction solution: Take 20 mg of the raw material and place it in a 10 ml volumetric bottle. Add 5 ml of diluent and shake to dissolve. Then add 1 ml of 10% hydrogen peroxide solution. After leaving it for 24 hours, add diluent to the scale and shake well. Accurately measure 5 μL and inject it into the liquid chromatograph. Detect according to the above chromatographic conditions and record the chromatogram. The results are shown in Figure 6 .

[0086] High temperature destruction: Take 20 mg of the raw material drug and place it in an empty bottle. Add 5 ml of diluent and shake to dissolve. Heat it in a water bath at 100°C for 6 hours. Take it out and place it at room temperature. Transfer it to a 10 ml volumetric bottle and dilute it to the mark with diluent. Shake well. Accurately measure 5 μL and inject it into the liquid chromatograph. Detect it according to the above chromatographic conditions and record the chromatogram. The results are shown in Figure 7 .

[0087] Light damage: Take 20 mg of the raw material and place it in a 10 ml volumetric bottle. Add 5 ml of diluent and shake to dissolve. Then place it in a light box (visible light 2600 lx ± 500 lx, near-ultraviolet energy 100 μw / cm 2 ) for 24 hours, take it out, dilute it to the mark with a diluent, shake it well, and you have it. Accurately measure 5 μL and inject it into the liquid chromatograph, detect it according to the above chromatographic conditions, record the chromatogram, and the results are shown in Figure 8 .

[0088] Depend on Figures 3 to 8 It can be seen that the avatrombopag maleate peak and the degradation peaks produced under various destruction conditions can be effectively separated, and the purity of the main peak is qualified, indicating that the chromatographic conditions of the present invention have good specificity.

[0089] Example 4 Analysis Method Sensitivity Test

[0090] Accurately weigh the avatrombopag maleate reference substance and the above 9 specific impurity reference substances, prepare them into stock solutions with diluents, mix them and dilute them quantitatively step by step to obtain a mixed 0.02% sensitivity solution; then dilute the sensitivity solution by half as the negligible limit solution. Accurately measure 5 μL of each of the two solutions and inject them into the liquid chromatograph, detect them according to the above chromatographic conditions, and record the chromatogram.

[0091] The signal-to-noise ratio summary data is shown in Table 4

[0092] Table 4

[0093]

[0094] From the results in Table 4, it can be seen that the signal-to-noise ratio of each impurity in the sensitive solution is greater than 10, which is above the quantitative limit; the signal-to-noise ratio of each impurity in the negligible limit solution is greater than 3, which is above the detection limit; the sensitivity meets the requirements.

[0095] Example 4 Tests of linearity, range and correction factor of detection method

[0096] Accurately weigh appropriate amounts of avatrombopag maleate reference substance and 9 specific impurity reference substances, dissolve them in diluent to make up the volume, and use them as stock solutions. Accurately measure appropriate amounts of each stock solution, place them in the same volumetric flask, and dilute them with diluent to make up the volume as the mixed stock solution.

[0097] Precisely measure a certain volume of mixed stock solution, place it in 5 volumetric flasks, and use diluent to make up to 20%, 50%, 100%, 150%, 200%, 250% of the impurity limit level, together with the quantitative limit solution, to provide 7 mixed samples with concentration gradients. Precisely measure 5 μL of each of the two solutions and inject them into the liquid chromatograph, detect according to the above chromatographic conditions, perform linear regression on the peak area with concentration, and obtain the linear regression equation. The data are shown in Table 5.

[0098] Table 5

[0099]

[0100] It can be seen from the data in Table 5 that the linear relationship of each impurity is good within the concentration range of 0.04 μg / ml to 5 μg / ml.

[0101] Example 5: Repeatability test of the detection method

[0102] Take 50 mg of avatrombopag maleate sample, accurately weigh it, put it in a 25 mL volumetric flask, add 4 ml of diluent and shake to dissolve, then accurately transfer 1 mL of mixed impurity stock solution, dilute with diluent, shake well, and prepare 6 portions in parallel. Accurately measure 5 μL of each of the six precision solutions and inject them into the liquid chromatograph, and detect according to the above chromatographic conditions. The data are shown in Table 6.

[0103] Table 6

[0104]

[0105] From the data in Table 6, it can be seen that the RSD of the known impurities detected in the six spiked sample solutions is less than 6%, indicating that the repeatability (precision) of this method is good.

[0106] Example 6 Analytical Method Accuracy (Recovery) Experiment

[0107] Weigh an appropriate amount of avatrombopag maleate sample and place it in a corresponding volumetric bottle, add an appropriate amount of diluent, and dissolve it in an appropriate amount; accurately add an appropriate amount of accuracy stock solution, then add solvent to dilute to the scale, and shake well. Prepare spiked samples containing 50%, 100%, and 150% of the impurity limit level, and prepare 3 samples in parallel for each limit. Accurately measure 5μL of each spiked sample solution and inject it into the liquid chromatograph, and detect it according to the above chromatographic conditions. The data is shown in Table 7.

[0108] Table 7

[0109]

[0110] It can be seen from the data in Table 7 that the impurity recoveries at the three limit levels are all between 90% and 110.0%, and the RSDs are all less than 5.0%, which meets the requirements for the accuracy of liquid phase impurity analysis.

[0111] Example 7 Preparation of Reference Impurities A, B, C, D, E, F, G, H and I

[0112] Synthesis of impurity A (CAS: 2626934-45-8, Chinese name: 5-chloro-N-[4-(4-chlorothiophen-2-yl)-5-(4-cyclohexylpiperazine-1-yl)-1,3-thiazol-2-yl]-6-(dimethylamino)pyridine-3-carboxamide)

[0113]

[0114] Add 5,6-dichloro-N-[4-(4-chlorothiophene-2-yl)-5-(4-cyclohexylpiperazine-1-yl)thiazol-2-yl]nicotinamide (Compound 1, 2 g, 3.6 mmol) to a 100 ml three-necked flask, add acetonitrile (20 ml), add tetrahydrofuran (20 ml), add triethylamine (1.82 g, 18 mmol), add dimethylamine hydrochloride (0.59 g, 7.2 mmol), and react at 70-75°C for 16 hours. Add 60 ml of water, filter and rinse with 10 ml of water, and dry the filter cake at 65°C for 16 hours to obtain 1.8 g of impurity A (yellow solid).

[0115] Instrument model: Agilent 1260LC-6120MS, test conditions: ESI, measured molecular ion peak m / z = 565.2 [M+H] + , consistent with its molecular weight.

[0116] Instrument model: Bruker-400 NMR, solvent: DMSO-d6, nuclear magnetic resonance hydrogen spectrum (1H-NMR) and carbon spectrum ( 13 C-NMR).

[0117] δH (ppm, 400MZ, DMSO-d6): 1.46-1.43 (m, 5H), 1.64-1.61 (d, 1H), 1.87-1.84 (d, 2H), 2.14-2.11 (d, 2H), 3.11-3.06 (m, 8H), 3.3 3-3.27(m, 5H), 3.63-3.60(m, 2H), 7.49-7.48(d, 1H), 7.48-7.47(d, 1H), 8.77-8.77(d, 1H), 8.35-8.34(d, 1H), 14.16(s, 1H).

[0118] δC (ppm, 400MZ, DMSO-d6): 26.74, 24.79, 24.64, 48.55, 40.99, 64.75, 51.33, 124.38, 120.74, 199.58, 166.72, 133 .85, 110.99, 146.60, 139.24, 138.93, 137.56, 133.75, 162.81, 159.14, 159.07, 158.75, 158.37, 157.99, 152.78.

[0119] Synthesis of impurity B (CAS: 2681355-40-6, Chinese name: 1-(3-chloro-5-{[4-(4-chlorothiophen-2-yl)-5-(4-cyclohexyl-4-piperazin-1-oxide)-1,3-thiazol-2-yl]carbamoyl}pyridin-2-yl)piperidine-4-carboxylic acid)

[0120]

[0121] Add maleic acid 1-(3-chloro-5-{[4-(4-chlorothiophene-2-yl)-5-(4-cyclohexylpiperazine-1-yl)-1,3-thiazol-2-yl]carbamoyl}pyridin-2-yl)piperidine-4-carboxylic acid (Compound 2, 10 g) to a 250 ml three-necked flask, add methanol (60 ml), add N,N-dimethylacetamide (70 ml), add m-chloroperbenzoic acid (4.5 g, 2 eq.), and react at 40°C for 4 hours. Add 150 ml of water, stir, and filter to obtain a solid crude product. The crude product was separated and purified by column chromatography using dichloromethane:methanol=30:1-10:1 to obtain 3 g of impurity B (light yellow solid).

[0122] Instrument model: Agilent 1260LC-6120MS, test conditions: ESI, measured molecular ion peak m / z = 665.3 [M + H] + , consistent with its molecular weight.

[0123] Instrument model: Bruker-400 NMR, solvent: DMSO-d6, H NMR spectrum ( 1 H-NMR) and carbon spectra ( 13 C-NMR).

[0124] δH (ppm, 400MZ, DMSO-d6): 1.73-1.12 (m, 8H), 1.95-1.91 (d, 4H), 2.29-2.27 (m, 2H), 2.55-2.49 (m, 1H), 3.04-3.02 (t, 2H), 3.37-3.28 (m, 4H), 3.99-3.86 (m, 7H), 6.27 (s, 1H), 8.83-8.82 (d, 1H), 8.39-8.38 (d, 1H), 7.54-7.53 (d, 1H), 7.51-7.51 (d, 1H), 11.86 (br, 1H).

[0125] δC (ppm, 400MZ, DMSO-d6): 27.94, 25.05, 24.69, 24.48, 76.69, 59.36, 48.04, 47.87, 119.71, 119.03, 116.83, 1313.96, 111.08, 137 .42, 133.66, 130.15, 124.24, 120.75, 120.70, 157.95, 152.60, 146.66, 138.80, 175.86, 166.75, 162.68, 159.42, 159.08, 158.71.

[0126] Synthesis of impurity C (Chinese name: 1-(3-chloro-5-{[4-(5-chlorothiophene-2-yl)-5-(4-cyclohexylpiperazin-1-yl)-1,3-thiazol-2-yl]carbamoyl}pyridin-2-yl)piperidine-4-carboxylic acid)

[0127]

[0128] Step 1. Add compound 3 (1.2 g) and dioxane (6 ml) to a 50 ml three-necked flask, add dropwise a solution of NBS (N-bromosuccinimide, 0.985 g) in dioxane (4.8 ml) and DMF (1.2 ml), and react at 15-25° C. for 16 hours. Add 30 ml of water at 15-25° C., filter and rinse with 12 ml of water to obtain a filter cake, which is dried at 60-70° C. for 16 hours to obtain 1.3 g of compound 4.

[0129] Step 2. Add compound 4 (1 g), acetonitrile (20 ml), cyclohexylpiperazine (1.26 g), and triethylamine (1.03 g) into a 250 ml three-necked flask, reflux at 75-80°C for 16 hours, cool to 15-25°C, filter and rinse with 5 ml of water to obtain a filter cake, and dry at 65°C for 16 hours to obtain 1.1 g of compound 5.

[0130] Step 3. Compound 5 (0.7 g) was added to a 50 ml three-necked flask, pyridine (3.5 ml), tetrahydrofuran (5.6 ml), 5,6-dichloronicotinic acid (0.39 g), phosphorus oxychloride (0.34 g) was added dropwise at -10°C, the mixture was reacted at this temperature for 16 hours, 14 ml of a solution of NaOH (5 eq.) in water was added, the pH was adjusted to 7-8, the mixture was filtered and rinsed with 3.5 ml of water to obtain a filter cake, which was dried at 65°C for 16 hours to obtain 0.9 g of compound 6.

[0131] Step 4. Add compound 6 (0.7 g) into a 50 ml three-necked flask, add THF (7 ml), add acetonitrile (7 ml), add ethyl pipecolate (0.39 g), add triethylamine (0.38 g), react at 70-75° C. for 16 hours, add 14 ml of water, add concentrated HCl to adjust the pH to 1, filter and rinse with 3.5 ml of water to obtain a filter cake, and dry at 65° C. for 16 hours to obtain 0.8 g of compound 7.

[0132] Step 5. Add compound 7 (0.5 g), THF (1.5 ml), water (1 ml), lithium hydroxide monohydrate (124 mg) into a 50 ml three-necked flask, react at 20°C for 16 hours, add 5 ml of water, add concentrated HCl to adjust the pH to 1-2, filter and rinse with 5 ml of water to obtain a filter cake, and dry at 65°C for 16 hours to obtain 0.4 g of impurity C.

[0133] Instrument model: Agilent 1260LC-6120MS, test conditions: ESI, measured molecular ion peak m / z = 649.2 [M+H] + , consistent with its molecular weight.

[0134] Instrument model: Bruker-400 NMR, solvent: DMSO-d6, H NMR spectrum ( 1 H-NMR) and carbon spectra ( 13 C-NMR).

[0135] δH (ppm, 400MZ, DMSO-d6): 1.85-1.10 (m, 10H), 3.05-1.93 (m, 9H), 3.34-3.24 (m, 7H), 3.99-3.97 (m, 2H), 7 .12-7.11 (d, 1H), 7.46-7.45 (d, 1H), 8.39-8.38 (d, 1H), 8.83-8.82 (d, 1H), 12.68 (br, 1H), 10.77 (br, 1H).

[0136] δC (ppm, 400MZ, DMSO-d6): 27.85, 26.46, 24.86, 24.55, 64.16, 51.15, 48.21, 47.77, 128.21, 126 .99, 124.04, 120.76, 118.93, 138.61, 135.15, 134.04, 146.53, 175.80, 162.60, 159.31, 152.70.

[0137] Synthesis of impurity D (Chinese name: 1-(3-chloro-5-{[4-(4,5-dichlorothiophene-2-yl)-5-(4-cyclohexylpiperazin-1-yl)-1,3-thiazol-2-yl]carbamoyl}pyridin-2-yl)piperidine-4-carboxylic acid ethyl ester)

[0138]

[0139] Step 1. Add compound 8 (4 g) to a 250 ml three-necked flask, add dioxane (20 ml), add DMF (4 ml), add dropwise a solution of NBS (2.7 g) in dioxane (16 ml) and DMF (4 ml), and react at 15-25° C. for 16 hours. Add 100 ml of water at 15-25° C., filter and rinse with 20 ml of water to obtain a filter cake, which is dried at 40° C. for 16 hours to obtain compound 9.

[0140] Step 2. Add compound 9 (full batch) into a 250 ml three-necked flask, add acetonitrile (100 ml), add cyclohexylpiperazine (3.22 g, 1.2 eq.), add triethylamine (2.42 g), reflux at 75-85°C for 16 hours, cool to 15-25°C, filter to obtain a filter cake, and dry at 60°C to obtain 5.2 g of compound 10.

[0141] Step 3. Add compound 10 (4.2 g) into a 250 ml three-necked flask, add pyridine (20 ml), add tetrahydrofuran (32 ml), add 5,6-dichloronicotinic acid (2.1 g), add phosphorus oxychloride (1.84 g) dropwise at -20°C, react at this temperature for 16 hours, add an aqueous solution of NaOH (5 eq.), adjust the pH to 7-8, filter and rinse with water to obtain a filter cake, and purify it through a column to obtain 0.7 g of compound 11.

[0142] Step 4. Add compound 11 (0.7 g), THF (10 ml), ethyl pipecolate (0.4 g), and triethylamine (0.4 g) into a 50 ml three-necked flask, react at 65-75 °C for 16 hours, add 14 ml of water, add concentrated HCl to adjust the pH to 1, filter and rinse with water to obtain a filter cake, and dry at 60 °C to obtain 0.6 g of compound 12.

[0143] Step 5. Add compound 12 (0.3 g), THF (5 ml), water (5 ml), and lithium hydroxide monohydrate (60 mg) into a 50 ml three-necked flask, react at 20°C for 16 hours, add water, add concentrated HCl to adjust the pH to 5-6, filter and rinse with water to obtain a filter cake, and dry to obtain 180 mg of impurity D.

[0144] Instrument model: Agilent 1260LC-6120MS, test conditions: ESI, measured molecular ion peak m / z = 683.1 [M+H] + , consistent with its molecular weight.

[0145] Instrument model: Bruker-400 NMR, solvent: DMSO-d6, H NMR spectrum ( 1 H-NMR) and carbon spectra ( 13 C-NMR).

[0146] δH (ppm, 400MZ, DMSO-d6): 1.87-1.10 (m, 10H), 3.10-1.91 (m, 9H), 3.64-3.33 (m, 7H), 3.99-3 .96(m, 2H), 7.40(s, 1H), 8.37-8.37(d, 1H), 8.80-8.79(d, 1H), 9.62(br, 1H), 12.68(br, 1H).

[0147] δC (ppm, 400MZ, DMSO-d6): 175.95, 162.78, 159.53, 159.15, 158.76, 158.38, 157.99, 153.23, 146.78, 139.73, 138.78, 134.08, 133.47 , 123.53, 123.47, 123.14, 120.70, 119.58, 119.13, 116.72, 113.86, 110.99, 64.50, 51.50, 48.55, 47.98, 28.05, 26.79, 24.77, 24.62.

[0148] Synthesis of impurity E (Chinese name: 1-(3-chloro-5-{[4-(thiophen-2-yl)-5-(4-cyclohexylpiperazin-1-yl)-1,3-thiazol-2-yl]carbamoyl}pyridin-2-yl)piperidine-4-carboxylic acid)

[0149]

[0150] Step 1. Add compound 13 (2.7 g) and dioxane (14 ml) to a 250 ml three-necked flask, add dropwise a solution of NBS (2.4 g) in dioxane (12 ml) and DMF (3 ml), and react at 25°C for 2 hours. Add 70 ml of water at 25°C, filter and rinse with water to obtain a filter cake, and dry at 40°C for 5 hours to obtain 3 g of compound 14.

[0151] Step 2. Add compound 14 (3 g in total) into a 100 ml three-necked flask, add acetonitrile (75 ml), add cyclohexylpiperazine (3 g), add triethylamine (2.25 g), reflux at 75-85°C for 16 hours, cool to 15-25°C to precipitate solid, filter to obtain a filter cake, and dry at 60°C to obtain 3.55 g of compound 15.

[0152] Step 3. Compound 15 (3 g) was added to a 250 ml three-necked flask, pyridine (15 ml), tetrahydrofuran (24 ml), 5,6-dichloronicotinic acid (1.2 eq.), phosphorus oxychloride (1.2 eq.) was added dropwise at -20°C, the mixture was reacted for 3 hours at this temperature, an aqueous solution of NaOH (5 eq.) was added, the pH was adjusted to 7-8, the mixture was filtered and rinsed with water to obtain a filter cake, which was purified by column chromatography to obtain 3.3 g of compound 16.

[0153] Step 4. Add compound 16 (3.3 g), THF (26 ml), ethyl piperidine carboxylate (2 g), triethylamine (1.92 g) into a 250 ml three-necked flask, react at 65-75° C. for 16 hours, add 30 ml of water, add concentrated HCl to adjust the pH to 1-2, filter and rinse with water to obtain a filter cake, and dry to obtain 4.9 g of compound 17.

[0154] Step 5. Add compound 17 (4.5 g), THF (30 ml), water (15 ml), lithium hydroxide monohydrate (1.2 g) into a 250 ml three-necked flask, react at 20°C for 4 hours, add 20 ml of water, add maleic acid aqueous solution to adjust the pH to 7, filter and rinse with water to obtain a filter cake, and dry to obtain 3.9 g of impurity E.

[0155] Instrument model: Agilent 1260LC-6120MS, test conditions: ESI, measured molecular ion peak m / z = 615.3 [M+H] + , consistent with its molecular weight.

[0156] Instrument model: Bruker-400 NMR, solvent: DMSO-d6, H NMR spectrum ( 1 H-NMR) and carbon spectra ( 13 C-NMR).

[0157] δH (ppm, 400MZ, DMSO-d6): 1.95-1.01 (m, 12H), 2.98-2.70 (m, 5H), 3.04-3.01 (m, 9H), 3.97-3.94 (m, 2H), 7.11-7.09(t, 1H), 7.47-7.45(d, 1H), 8.39-8.38(d, 1H),), 8.83-8.82(d, 1H), 12.48(br, 1H).

[0158] δC (ppm, 400MZ, DMSO-d6): 28.38, 27.87, 25.30, 25.13, 67.02, 54.63, 48.56, 47.80, 127.20, 125.30, 124.38, 121.11, 119.02, 140.36, 138.53, 136.79, 133.47, 146.45, 151.62, 175.33, 162.31, 159.24.

[0159] Synthesis of impurity F (CAS: 2883585-26-8, Chinese name 1-(3-chloro-5-{[4-(4-chloro-5-bromothiophen-2-yl)-5-(4-cyclohexylpiperazin-1-yl)-1,3-thiazol-2-yl]carbamoyl}pyridin-2-yl)piperidine-4-carboxylic acid)

[0160]

[0161] Step 1. Add compound 18 (8 g), acetonitrile (20 ml) and NBS (5.3 g) to a 100 ml three-necked flask and react at 25° C. for 16 hours. After concentration, column chromatography (volume ratio of petroleum ether:ethyl acetate PE:EA=8:1) was performed to obtain 1.5 g of compound 19.

[0162] Step 2. In a 100 ml three-necked flask, compound 19 (1.25 g), acetonitrile (25 ml), cyclohexylpiperazine (0.67 g), and triethylamine (1 g) were added. The mixture was refluxed at 80°C for 16 hours, and the solid was precipitated by cooling to 15-25°C. The filter cake was filtered and dried at 65°C for 16 hours to obtain 1.2 g of compound 20.

[0163] Step 3. Compound 20 (0.8 g) was added to a 50 ml three-necked flask, pyridine (4 ml), tetrahydrofuran (6.4 ml), 5,6-dichloronicotinic acid (0.365 g), phosphorus oxychloride (0.312 g) was added dropwise at -10 to -15°C, the mixture was reacted for 16 hours at this temperature, 16 ml of a solution of NaOH (5 eq.) in water was added, the pH was adjusted to 7 to 8, the mixture was filtered and rinsed with 4 ml of water to obtain a filter cake, which was dried at 65°C for 16 hours to obtain 0.95 g of compound 21.

[0164] Step 4. Add compound 21 (0.8 g) into a 50 ml three-necked flask, add THF (4 ml), add ethyl pipecolate (0.4 g), add triethylamine (0.38 g), react at 70-75°C for 16 hours, cool by 15-25°C, add 8 ml of water, add concentrated HCl to adjust the pH to 1-2, filter and rinse with 4 ml of water to obtain a filter cake, dry at 60°C for 16 hours to obtain 0.85 g of compound 22.

[0165] Step 5. Add compound 22 (0.6 g), THF (3 ml), water (3 ml), and lithium hydroxide monohydrate (167 mg) into a 50 ml three-necked flask, react at 15-25°C for 12 hours, add 6 ml of water, add hydrochloric acid to adjust the pH to 1, filter and rinse with 3 ml of water to obtain a filter cake, and dry to obtain 0.6 g of impurity F.

[0166] Instrument model: Agilent 1260LC-6120MS, test conditions: ESI, measured molecular ion peak m / z = 727.0 [M+H] + , consistent with its molecular weight.

[0167] Instrument model: Bruker-400 NMR, solvent: DMSO-d6, H NMR spectrum ( 1 H-NMR) and carbon spectra ( 13 C-NMR).

[0168] δH (ppm, 400MZ, DMSO-d6): 1.91-1.01 (m, 12H), 3.03-2.15 (m, 5H), 3.35-3.16 (m, 7H), 3.63-3.60 (m, 2H) , 3.98-3.95(m, 2H), 7.36(s, 1H), 8.35-8.34(d, 1H), 8.79-8.78(d, 1H), 12.06(br, 1H), 10.19(br, 1H).

[0169] δC (ppm, 400MZ, DMSO-d6): 28.18, 26.83, 24.92, 24.76, 64.61, 51.50, 48.64, 48.13, 116.75, 113.89, 111.03, 108.95, 126.50, 124 .81, 120.87, 119.61, 119.32, 139.98, 138.94, 137.20, 133.73, 146.84, 153.32, 176.11, 162.89, 159.64, 158.88, 158.49, 158.11.

[0170] Synthesis of impurity G (CAS: 570407-42-0, Chinese name: 4-(4-chlorothiophen-2-yl)-5-(4-cyclohexylpiperazin-1-yl)thiazol-2-amine)

[0171]

[0172] Step 1. Add 4-(4-chloro-2-thienyl)-2-thiazolamine (compound 23, 1 kg) to a 50L reactor, add dioxane (5L), dropwise add NBS (780g) in DMF (1L) and dioxane (4L) solution, and react at 20-30°C for 16 hours. Add water (25L) to precipitate, filter and rinse with 5L of water to obtain a filter cake, which is dried at 40°C for 16 hours to obtain 1.62kg of compound 24 dry product.

[0173] Step 2. Add 1.62 kg (full batch) of compound 24 to a 50 L reactor, add acetonitrile (25 L), add cyclohexylpiperazine (932 g), add triethylamine (700 g), react at 75-85 ° C for 5 hours, cool to 20 ° C, filter and rinse with 5 L of acetonitrile to obtain a filter cake, and dry at 60 ° C for 16 hours to obtain 1.4 kg of impurity G.

[0174] Instrument model: Agilent 1260LC-6120MS, test conditions: ESI, measured molecular ion peak m / z = 383.0 [M+H] + , consistent with its molecular weight.

[0175] Instrument model: Bruker-400 NMR, solvent: DMSO-d6, H NMR spectrum ( 1 H-NMR) and carbon spectra ( 13 C-NMR).

[0176] δH (ppm, 400MZ, DMSO-d6): 1.58-0.95 (m, 6H), 1.78-1.73 (m, 4H), 2.26 (m, 1 H), 2.73-2.65 (m, 8H), 6.99 (s, 2H), 7.31-7.30 (d, 1H), 7.39-7.38 (d, 1H).

[0177] δC (ppm, 400MZ, DMSO-d6): 28.40, 25.90, 25.26, 62.59, 55.08, 48.59, 123.20, 119.54, 134.00, 136.29, 138.17, 162.97.

[0178] Synthesis of impurity H (CAS: 958457-83-5, Chinese name: 6-(4-piperidin-1-yl)-5-chloronicotinic acid)

[0179]

[0180] Step 1. Add compound 25 (10 g), ethyl 4-piperidincarboxylate (16.4 g), triethylamine (26.3 g), and acetonitrile (200 ml) to a 500 ml reaction bottle, stir, react at 80 ° C for 16 hours, cool to room temperature, add the reaction solution to 300 ml of water, add 6N HCl to adjust the pH to 3-4, precipitate solid, filter and rinse with water, and dry the filter cake at 60 ° C for 16 hours to obtain 13.9 g of dry product (compound 26)

[0181] 2. Add compound 26 (6 g), THF (12 ml), water (6 ml), and KOH (4.3 g) into a 100 ml reaction bottle, stir, and react at 60-70 ° C for 16 hours. Add 100 ml of water and adjust the pH to 1-2 with HCl to precipitate solid. Filter and rinse with 15 ml of water. Dry the filter cake at 60 ° C for 16 hours to obtain 1.25 g of dry impurity H.

[0182] Instrument model: Agilent 1260LC-6120MS, test conditions: ESI, measured molecular ion peak m / z = 285.1 [M+H] + , which is consistent with its molecular weight. Instrument model: Bruker-400 NMR instrument, solvent: DMSO-d6, NMR hydrogen spectrum ( 1 H-NMR) and carbon spectra ( 13 C-NMR).

[0183] δH (ppm, 400MZ, DMSO-d6): 1.94-1.71 (m, 2H), 2.54-2.46 (m, 2H), 2.97-2.96 (m, 1H), 3.0 3-3.00 (m, 2H), 3.94-3.91 (m, 2H), 8.04-8.03 (d, 1H), 8.63-8.62 (d, 1H), 12.67 (br, 1H).

[0184] δC (ppm, 400MZ, DMSO-d6): 47.82, 27.84, 119.14, 139.45, 119.98, 147.45, 159.66, 165.38, 175.81.

[0185] Synthesis of impurity I (CAS: 570407-42-0, Chinese name: 1-(1-(3-chloro-5-{[4-(4-chlorothiophen-2-yl)-5-(4-cyclohexylpiperazin-1-yl)thiazol-2-yl]carbamoyl}pyridin-2-yl)piperidine-4-carbonyl)piperidine-4-carboxylic acid)

[0186]

[0187] Step 1. Add compound 27 (5 g), DMF (25 ml), ethyl naphthalene carboxylate (1.33 g), DIEA (2.98 g), and BOP (5.45 g) into a 250 ml three-necked flask, and react at 15-25° C. for 3 hours. Add the reaction solution into 75 ml of water to precipitate a solid, filter it, and rinse it with 10 ml of water to obtain a filter cake, which is dried at 60° C. for 3 hours to obtain 4 g (semi-wet product) of compound 28.

[0188] Step 2. Add THF (10.5 ml), water (7 ml), compound 28 (semi-wet product from the previous step, equivalent to 3.5 g), and lithium hydroxide monohydrate (0.75 g) into a reaction bottle, stir, react at 25-35°C for 2 hours, add 70 ml of water, adjust the pH to 7 with HCl, and then add HCl to adjust the pH to 1 to precipitate a solid, filter and rinse with water, and dry the filter cake at 60°C to obtain 1.24 g of impurity I.

[0189] Instrument model: Agilent 1260LC-6120MS, test conditions: ESI, measured molecular ion peak m / z = 760.2 [M + H] + , consistent with its molecular weight.

[0190] Instrument model: Bruker-400 NMR, solvent: DMSO-d6, H NMR spectrum ( 1 H-NMR) and carbon spectra ( 13 C-NMR).

[0191] δH(ppm,400MZ,DMSO-d6):1.44-1.01(m,8H),1.76-1.71(m,4H),1.87-1.84(m,4H),2.15-2.12(m,2H),3.12-2.60(m,7H),3.32-3.12(m,1H),3.64-3.35(m,7H),4.30-3.90(m,4H),6.27(s,1H),7.49-7.48(d,1H),7.53-7.54(d,1H),8.39-8.38(d,1H),8.83-8.82(d,1H),12.70(br,1H),10.50(br,1H)。

[0192] δC(ppm,400MZ,DMSO-d6):30.45、28.89、28.42、26.56、24.68、24.51、67.08、64.08、51.16、48.36、47.89、44.20、40.63、40.28、40.15、118.78、116.81、113.93、111.05、124.19、124.12、120.70、120.51、119.68、139.19、138.66、137.37、133.51、146.65、152.51、176.11、172.27、166.71、162.66、159.31、158.64、158.27、157.89。

Claims

1. A method for detecting related substances in avatrombopag maleate, characterized in that The following steps are involved: High performance liquid chromatography was used to detect related substances in the avatrombopag maleate raw material drug, and the test conditions were: octadecylsilane bonded silica gel chromatographic column, the eluent was online mixed gradient elution of mobile phase A and mobile phase B, the mobile phase A was a mixed solution of water-acid-alcohol, the acid was one or more of trifluoroacetic acid, formic acid, acetic acid and phosphoric acid, and the alcohol was methanol and / or ethanol; the mobile phase B was a solution of acid and organic solvent, the acid was formic acid and / or trifluoroacetic acid, and the organic solvent was one or more of methanol, acetonitrile and tetrahydrofuran; the related substances were impurities A, B, C, D, E, F, G, H and I as shown in the following formula:

2. The method for detecting related substances in avatrombopag maleate according to claim 1, characterized in that: The high performance liquid chromatography detection is carried out in a high performance liquid chromatography analyzer, and the high performance liquid chromatography analyzer is equipped with a diode array detector or an ultraviolet detector; preferably, the high performance liquid chromatography analyzer is an Agilent 1260II high performance liquid chromatography analyzer or a Thermo Fisher U3000 high performance liquid chromatography analyzer; and / or, The avatrombopag maleate includes the avatrombopag maleate bulk drug and a pharmaceutical preparation with avatrombopag maleate as an active ingredient; preferably, the pharmaceutical preparation with avatrombopag maleate as an active ingredient includes an avatrombopag maleate oral pharmaceutical preparation and an avatrombopag maleate injection; further preferably, the avatrombopag maleate oral pharmaceutical preparation includes avatrombopag maleate tablets and avatrombopag maleate capsules, etc.

3. The method for detecting related substances in avatrombopag maleate according to claim 1, characterized in that: The octadecylsilane bonded silica gel column is a YMC Triart C18, 250 mm×4.6 mm, 3 μm chromatographic column.

4. The method for detecting related substances in avatrombopag maleate according to claim 1, characterized in that: The column temperature of the high performance liquid chromatography detection is 25°C to 35°C; and / or, The wavelength of the high performance liquid chromatography detection is 250nm to 260nm; and / or, The flow rate of the mobile phase detected by high performance liquid chromatography is 0.6 mL / min to 1.2 mL / min; and / or, The injection volume for detection by high performance liquid chromatography is 5 μL to 20 μL; and / or, The running time of the high performance liquid chromatography detection is 100 minutes to 200 minutes.

5. The method for detecting related substances in avatrombopag maleate according to claim 4, characterized in that: The column temperature of the high performance liquid chromatography detection is 30°C; and / or, The wavelength of the high performance liquid chromatography detection is 254nm; and / or, The flow rate of the mobile phase detected by high performance liquid chromatography is 0.7 mL / min; and / or, The injection volume for the high performance liquid chromatography detection is 5 μL; and / or, The running time of the high performance liquid chromatography detection is 140 minutes to 180 minutes.

6. The method for detecting related substances in avatrombopag maleate according to claim 1, characterized in that: The alcohol in the mobile phase A is methanol; and / or, The acid in the mobile phase B is trifluoroacetic acid; and / or, The organic solvent in the mobile phase B is methanol and / or acetonitrile; and / or, In the mobile phase B, the percentage of the acid is 0.05% to 0.20%, and the percentage refers to the percentage of the volume of the acid to the total volume of the acid and organic solvent solution; and / or, The diluent used in the high performance liquid chromatography detection is a mixed solution of water, acid and organic solvent, the acid is formic acid and / or trifluoroacetic acid, and the organic solvent is one or more of methanol, acetonitrile and tetrahydrofuran.

7. The method for detecting related substances in avatrombopag maleate according to claim 6, characterized in that: The mobile phase A is a mixed solution of water-methanol-trifluoroacetic acid; a mixed solution of water-methanol-formic acid or a mixed solution of water-methanol-phosphoric acid water; and / or, The mobile phase B is acetonitrile-methanol-trifluoroacetic acid; and / or, In the mobile phase B, the percentage of the acid is 0.05% to 0.20%, and the percentage refers to the percentage of the volume of the acid to the total volume of the acid and organic solvent solution; The acid in the diluent is trifluoroacetic acid; the organic solvent in the diluent is methanol and / or acetonitrile.

8. The method for detecting related substances in avatrombopag maleate according to claim 7, characterized in that: In the mobile phase A, the volume percentages of water, alcohol and acid are (700-950):(250-50):(2-0.5), for example, the volume ratio of water:methanol:trifluoroacetic acid is (900:100:1); and / or, When the mobile phase B is a mixed solution of acetonitrile-methanol-trifluoroacetic acid, the volume ratio of acetonitrile-methanol-trifluoroacetic acid is (700-950):(250-50):(2-0.5), for example (900:100:1); and / or, The diluent is water-acetonitrile-trifluoroacetic acid; When the diluent is a mixed solution of water-acetonitrile-trifluoroacetic acid, the volume ratio of water-acetonitrile-trifluoroacetic acid is (700-300):(700-300):(10-1), for example (500:500:2).

9. The method for detecting related substances in avatrombopag maleate according to claim 1, characterized in that: The online mixed gradient elution conditions of mobile phase A and mobile phase B are: 0-105min, the volume ratio of mobile phase A to mobile phase B is (80%-30%): (20%-70%); 105-140 min, the volume ratio of mobile phase A to mobile phase B is (70%-30%): (60%-40%); 140-145 min, the volume ratio of mobile phase A to mobile phase B is (50%-10%): (50%-90%); 145.1~160min, the volume ratio of mobile phase A to mobile phase B is (80%~30%):(20%~70%); finally, balance for 10 minutes according to the initial ratio.

10. The method for detecting related substances in avatrombopag maleate according to claim 1, characterized in that: The online mixed gradient elution conditions of mobile phase A and mobile phase B are: 。

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