A process for the preparation and purification of an amino acid amide derivative

Abstract

The application discloses a preparation and purification method of amino acid amide derivatives. Specifically, the application provides a preparation method of a compound of formula I or a compound of formula II, which comprises the following steps: performing a methylation reaction on a compound of formula III in an organic solvent in the presence of a base and a methylating agent to generate the compound of formula I or the compound of formula II; and the molar ratio of the compound of formula III to the base is 1: (2.5-5.5). The method can obtain a mixture of the compound of formula I and the compound of formula II in one step, and the purity of the two compounds can reach more than 95% through gradient elution of a preparation chromatography.

Description

Technical Field

[0001] This invention belongs to the field of drug synthesis technology, specifically relating to a method for preparing and purifying an amino acid amide derivative. Background Technology

[0002] Amino acid derivatives with both N-terminal and C-terminal functionalizations are a class of potent anticonvulsants; these compounds are called functionalized amino acids (FAAs). The N-terminus is an important structural unit of functionalized amino acids. In the initial design of FAAs, the N-terminal amino group is protected as an amide to enhance the lipophilicity of the compound.

[0003] For example, Cécile Béguin et al. reported a series of compounds with this scaffold in Bioorganic & Medicinal Chemistry (2004, 12, 3079–3096) and demonstrated their good anticonvulsant activity. Among them, compound 25 showed improved ED in a maximal electroconvulsive seizure (MES) model. 50 The value was 68 mg / kg, indicating a relatively significant efficacy.

[0004] Furthermore, compound I is a single enantiomer of compound 25, and both compounds I and II belong to the aforementioned functionalized amino acid backbone types. Therefore, these compounds have significant research value in the development of anticonvulsant drugs. However, reports on efficient synthetic methods for this type of compound are still relatively limited, and there is an urgent need to develop simpler and more efficient preparation strategies.

[0005] . Summary of the Invention

[0006] The technical problem to be solved by this invention is that there is no efficient synthesis and purification method for this type of compound in the prior art. Therefore, this invention provides a method for preparing and purifying amino acid amide derivatives. This method can obtain a mixture of N-methyl amino acid amide derivative (the compound shown in Formula I) and N,N-dimethyl amino acid amide derivative (the compound shown in Formula II) in one step. Then, by gradient elution using preparative chromatography, compounds of Formula I and Formula II with a purity greater than 95% can be obtained respectively.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0008] The present invention provides a method for preparing a compound of formula I or a compound of formula II, comprising the following steps: in an organic solvent, in the presence of a base and a methylating agent, a compound of formula III undergoes a methylation reaction to generate a compound of formula I or a compound of formula II; ; The molar ratio of the compound of formula III to the base is 1:(2.5-5.5).

[0009] In some embodiments, the organic solvent is a cyclic ether solvent, preferably tetrahydrofuran, and more preferably anhydrous tetrahydrofuran.

[0010] In some embodiments, the base is an alkali metal hydroxide or an alkali metal hydride; the alkali metal hydroxide may be sodium hydroxide and / or potassium hydroxide; the alkali metal hydride may be sodium hydride; preferably an aqueous solution of sodium hydroxide, more preferably a 30% aqueous solution of sodium hydroxide.

[0011] In some embodiments, the methylating agent is iodomethane or dimethyl sulfate, preferably dimethyl sulfate.

[0012] In some embodiments, the molar volume ratio of the Formula III compound to the organic solvent is 0.5-1 mol / L, preferably 0.83 mol / L.

[0013] In some embodiments, the molar ratio of the Formula III compound to the methylating agent is 1:(1-1.5), for example 1:1.2.

[0014] In some embodiments, the molar ratio of the compound of formula III to the base is 1:4.

[0015] In some embodiments, the reaction temperature is -10°C to 10°C, for example, 0°C.

[0016] In some implementations, the reaction time is 1-3 hours, for example, 2 hours.

[0017] In some embodiments, the post-processing of the reaction includes the following steps: concentrating the reaction solution of the methylation reaction, extracting with an organic solvent, and evaporating to obtain a crude product.

[0018] In some embodiments, the organic solvent in the post-processing is dichloromethane.

[0019] In some embodiments, the post-processing further includes purifying the crude product, the purification comprising the following steps: using high performance liquid chromatography to perform gradient elution on the crude product to obtain a compound of formula I or a compound of formula II; The high-performance liquid chromatography method uses a hydrophilic octadecyl bonded silica column; The mobile phases are mobile phase A and mobile phase B; The mobile phase A is trifluoroacetic acid and water; the volume ratio of trifluoroacetic acid to the total volume of trifluoroacetic acid and water is 0.05-0.15%. The mobile phase B is trifluoroacetic acid, methanol, and acetonitrile; the volume ratio of trifluoroacetic acid, methanol, and acetonitrile is (0.1-0.5): 49.85: 49.85. The gradient elution can be either scheme one or scheme two: In the first scheme, The elution flow rate is 75-85 mL / min; The elution gradient includes a first gradient and a second gradient: In the first gradient, the elution time is 1-3 min, the volume percentage of mobile phase A is 86%, and the volume percentage of mobile phase B is 14%. In the second gradient, the elution time is 10-30 min, the volume percentage of mobile phase A decreases from 86% to 62%, and the volume percentage of mobile phase B increases from 14% to 38%. In the second scheme, The elution flow rate is 45-55 mL / min; The elution gradient includes a first gradient and a second gradient: In the first gradient, the elution time is 3-6 minutes, the volume percentage of mobile phase A is 86%, and the volume percentage of mobile phase B is 14%. In the second gradient, the elution time is 40-60 min, the volume percentage of mobile phase A decreases from 86% to 62%, and the volume percentage of mobile phase B increases from 14% to 38%.

[0020] In some embodiments, the purification process includes a pretreatment of the crude product before gradient elution, which includes the step of mixing the crude product with an alcohol solvent and water.

[0021] In some embodiments, during the pretreatment, the volume ratio of the alcohol solvent to the total volume of water and alcohol solvent is (0.5-1.5):10; preferably 1:10.

[0022] In some embodiments, during the pretreatment, the ratio of the mass of the crude product to the total volume of water and alcohol solvent is 0.01-0.5 g / mL, preferably 0.06 g / mL or 0.1 g / mL.

[0023] In some embodiments, the detector used in the high-performance liquid chromatography is an ultraviolet detector; the detection wavelength of the detector is 190-260 nm, preferably 190-220 nm, for example 210 nm.

[0024] In some embodiments, the hydrophilic octadecyl bonded silica column is model ODS-AQ.

[0025] In some embodiments, the packing material of the hydrophilic octadecyl bonded silica column has a pore size of 10-14 nm, for example, 12 nm.

[0026] In some embodiments, the length of the hydrophilic octadecyl bonded silica column is 250-350 mm, for example, 250 mm.

[0027] In some embodiments, the inner diameter of the hydrophilic octadecyl bonded silica column is 40-60 mm, for example, 50 mm.

[0028] In some embodiments, the hydrophilic octadecyl bonded silica column has a packing particle size of 5-10 μm, for example, 10 μm.

[0029] In some embodiments, the hydrophilic octadecyl-bonded silica column is used at a pressure of 0-10 MPa.

[0030] In some embodiments, the column temperature of the hydrophilic octadecyl bonded silica column is 20-30°C, for example, 25°C.

[0031] In some embodiments, the volume ratio of trifluoroacetic acid to the total volume of trifluoroacetic acid and water in the mobile phase A is 0.1%.

[0032] In some embodiments, the volume ratio of trifluoroacetic acid, methanol, and acetonitrile in the mobile phase B is 0.3:49.85:49.85.

[0033] In some embodiments, in Scheme 1, the elution flow rate is 80 mL / min.

[0034] In some embodiments, in Scheme 2, the elution flow rate is 50 mL / min.

[0035] In some embodiments, in Scheme 1, the elution time in the first gradient is 2 minutes.

[0036] In some embodiments, in Scheme 1, the elution time in the second gradient is 20 minutes.

[0037] In some embodiments, in Scheme 2, the elution time in the first gradient is 4 minutes.

[0038] In some embodiments, in Scheme 2, the elution time in the second gradient is 51 minutes.

[0039] In some embodiments, the elution gradient of Scheme 1 includes the following gradient: .

[0041] In some embodiments, the elution gradient of Scheme 2 includes the following gradient: .

[0043] In some embodiments, the elution gradient of Scheme 1 is as follows: .

[0045] In some embodiments, the elution gradient of Scheme 2 is as follows: .

[0047] The present invention also provides a method for preparing compounds of formula I and formula II, comprising the following steps: in an organic solvent, in the presence of a base and a methylating agent, a compound of formula III undergoes a methylation reaction to generate compounds of formula I and formula II; ; The molar ratio of the compound of formula III to the base is 1:(2.5-5.5).

[0048] In some embodiments, the preparation methods of the compounds of formula I and formula II are any of the preparation methods of the compounds of formula I or formula II as described above.

[0049] The present invention also provides a purification method for a compound of formula I or a compound of formula II, comprising the following steps: using high performance liquid chromatography to perform gradient elution on the crude product to obtain a compound of formula I or a compound of formula II; The crude product contains compounds of formula I, formula II and formula III; The crude product has a purity of 40%-50% for compound of formula I. ; The high-performance liquid chromatography method uses a hydrophilic octadecyl bonded silica column; The mobile phases are mobile phase A and mobile phase B; The mobile phase A is trifluoroacetic acid and water; the volume ratio of trifluoroacetic acid to the total volume of trifluoroacetic acid and water is 0.05-0.15%. The mobile phase B is trifluoroacetic acid, methanol, and acetonitrile; the volume ratio of trifluoroacetic acid, methanol, and acetonitrile is (0.1-0.5): 49.85: 49.85. The gradient elution can be either scheme one or scheme two: In the first scheme, The elution flow rate is 75-85 mL / min; The elution gradient includes a first gradient and a second gradient: In the first gradient, the elution time is 1-3 min, the volume percentage of mobile phase A is 86%, and the volume percentage of mobile phase B is 14%. In the second gradient, the elution time is 10-30 min, the volume percentage of mobile phase A decreases from 86% to 62%, and the volume percentage of mobile phase B increases from 14% to 38%. In the second scheme, The elution flow rate is 45-55 mL / min; The elution gradient includes a first gradient and a second gradient: In the first gradient, the elution time is 3-6 minutes, the volume percentage of mobile phase A is 86%, and the volume percentage of mobile phase B is 14%. In the second gradient, the elution time is 40-60 min, the volume percentage of mobile phase A decreases from 86% to 62%, and the volume percentage of mobile phase B increases from 14% to 38%.

[0050] In some embodiments, the crude product contains a compound of formula II with a purity of 40%-50%; In some embodiments, the crude product contains a compound of formula I with a purity of 44.5%, a compound of formula II with a purity of 45.5%, and a compound of formula III with a purity of 10.1%.

[0051] In some embodiments, the purification method for the compound of formula I is any of the purification schemes described in the preparation methods of compounds of formula I and formula II as above.

[0052] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0053] The reagents and raw materials used in this invention are all commercially available.

[0054] The positive and progressive effects of this invention are as follows: This invention provides a method to obtain N-methyl amino acid amide derivatives (compound of formula I) and N,N-dimethyl amino acid amide derivatives (compound of formula II) with a purity greater than 95% by using a compound of formula III as raw material, undergoing a methylation reaction, and then using efficient preparative liquid phase separation. Detailed Implementation

[0055] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments described herein.

[0056] Example 1: Synthesis of Compounds I and II

[0057] Compound III (19.4 g, 100 mmol, 1.0 equiv.) and 120 mL of anhydrous tetrahydrofuran were added to a two-necked flask and cooled to 0 °C. o C, simultaneously add slowly a 30% NaOH aqueous solution (54.0 g, 400 mmol, 4.0 equiv.) and dimethyl sulfate (15.1 g, 120 mmol, 1.2 equiv.), 0 o After reaction C for 2 h, the reaction solution was concentrated, extracted three times with dichloromethane, and evaporated to dryness to obtain 21.0 g of yellow liquid. HPLC analysis of the reactants showed that the contents of compounds III, I, and II were 10.1%, 44.5%, and 45.5%, respectively.

[0058] Example 2: Separation of Compound I and Compound II

[0059] Take 5.0 g of the yellow liquid obtained in Example 1, dilute it with water, and add methanol to prepare a 10% methanol aqueous solution to 50 mL. Load the crude product using the sample loading system, and then elute from the high performance liquid reverse phase column under the conditions of method A. Collect the eluent with a retention time of 12-14 min (compound of formula I) and the eluent with a retention time of 15-17 min (compound of formula II); HPLC model: LC-20AP.

[0060] The chromatographic conditions are as follows: Column type: ODS-AQ (pore size of packing material: 12nm; column length: 250mm; inner diameter: 50mm; particle size of packing material: 10μm). Mobile phase A is an aqueous solution of trifluoroacetic acid with a volume percentage of 0.1%; mobile phase B is a methanol / acetonitrile mixture (volume ratio of 1:1) containing 0.3% trifluoroacetic acid (i.e., the volume ratio of trifluoroacetic acid, methanol, and acetonitrile is 0.3:49.85:49.85). The flow rate was 80 mL / min; the detection wavelength was 210 nm; and the injection volume was 50 mL. The column temperature is 25°C. o C; The elution gradient is:

[0061] After obtaining the eluent, HPLC was used for detection. Samples with a purity greater than 95% were retained. After concentration to remove methanol and acetonitrile, the samples were lyophilized to obtain yellow oily solids compound I (1.8 g, yield 33.9%, resolution 2.86) and compound II (2.0 g, yield 35.7%, resolution 3.3).

[0062] The high-resolution mass spectra of compound I obtained in this embodiment are shown in Figure C. 12 H 19 N2O2 (M+H) + = 223.1449, its 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (700 MHz, Methanol- d 4) δ 7.35 – 7.28 (m, 4H), 7.28 – 7.24 (m,1H), 4.48 (d, J = 14.7 Hz, 1H), 4.40 (d, J = 14.7 Hz, 1H), 4.02 (dd, J = 5.6,3.5 Hz, 1H), 3.83 (dd, J = 11.2, 3.5 Hz, 1H), 3.76 (dd, J = 11.2, 5.6 Hz, 1H), 3.40 (s, 3H), 2.71 (s, 3H). 13 C NMR (176 MHz, Methanol- d 4) δ 165.4, 137.9, 128.3, 127.2, 127.1,69.2, 61.1, 58.2, 43.0, 31.0. The high-resolution mass spectra of compound II obtained in this embodiment are shown in Figure C. 13 H 22N2O2 (M+H) + = 237.1602, its 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (700 MHz, Methanol- d 4) δ 7.35 – 7.29 (m, 4H), 7.28 – 7.25 (m,1H), 4.50 (d, J = 14.7 Hz, 1H), 4.39 (d, J = 14.7 Hz, 1H), 4.05 (dd, J = 4.9, 3.5 Hz, 1H), 3.93 (dd, J = 11.9, 3.5 Hz, 1H), 3.85 (dd, J = 11.9, 4.9 Hz,1H), 3.39 (s, 3H), 2.93 (s, 6H). 13 C NMR (176 MHz, Methanol- d 4) δ 165.2, 137.8, 128.3, 127.3, 127.2, 68.0, 67.0, 58.2, 43.0. Example 3: Separation of Compound I and Compound II Take 3.0 g of the yellow liquid obtained in Example 1, dilute with water, and add methanol to prepare a 10% methanol-water solution to 50 mL. Load the crude product using the sample loading system, and then elute from a high-performance liquid chromatography reverse-phase column under the conditions of Method B. Collect the eluent with a retention time of 30-33 min (compound I) and the eluent with a retention time of 35-38 min (compound II). HPLC model: LC-20AP.

[0063] The chromatographic conditions are as follows: Column type: ODS-AQ (pore size of packing material: 12nm; column length: 250mm; inner diameter: 50mm; particle size of packing material: 10μm). Mobile phase A is an aqueous solution of trifluoroacetic acid with a volume percentage of 0.1%; mobile phase B is a methanol / acetonitrile mixture (volume ratio of 1:1) containing 0.3% trifluoroacetic acid (i.e., the volume ratio of trifluoroacetic acid, methanol, and acetonitrile is 0.3:49.85:49.85). The flow rate was 50 mL / min; the detection wavelength was 210 nm; and the injection volume was 50 mL. The column temperature is 25°C. o C; The elution gradient is:

[0064] After obtaining the eluent, the sample was analyzed by HPLC. The sample with a purity greater than 98% was retained. After concentration to remove methanol and acetonitrile, the sample was lyophilized to obtain yellow oily solid compound I (2.1 g, yield 39.6%) and compound II (1.3 g, yield 40.7%).

[0065] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A method for preparing a compound of formula I or formula II, characterized in that, It includes the following steps: in an organic solvent, in the presence of a base and a methylating agent, the compound of formula III undergoes a methylation reaction to generate the compound of formula I or the compound of formula II; ； The molar ratio of the compound of formula III to the base is 1:(2.5-5.5).

2. The preparation method according to claim 1, characterized in that, It meets one or more of the following conditions: (1) The organic solvent is a cyclic ether solvent; (2) The base is an alkali metal hydroxide or an alkali metal hydride; (3) The methylating agent is iodomethane or dimethyl sulfate; (4) The molar volume ratio of the compound of formula III to the organic solvent is 0.5-1 mol / L; (5) The molar ratio of the compound of formula III to the methylating agent is 1:(1-1.5); (6) The molar ratio of the compound of formula III to the base is 1:4; (7) The reaction temperature is -10℃ to -10℃; (8) The post-processing of the reaction includes the following steps: concentrating the reaction solution of the methylation reaction, extracting with an organic solvent, and evaporating to obtain the crude product.

3. The preparation method according to claim 1, characterized in that, It meets one or more of the following conditions: (1) The organic solvent is tetrahydrofuran; (2) The alkali is an aqueous solution of sodium hydroxide; (3) The methylating agent is dimethyl sulfate; (4) The molar volume ratio of the compound of formula III to the organic solvent is 0.83 mol / L; (5) The molar ratio of the compound of formula III to the methylating agent is 1:1.2; (6) The reaction temperature is 0℃.

4. The preparation method according to claim 1, characterized in that, It meets one or two of the following conditions: (1) The organic solvent is anhydrous tetrahydrofuran; (2) The alkali is a 30% sodium hydroxide aqueous solution.

5. The preparation method according to claim 2, characterized in that, The post-processing further includes purifying the crude product, the purification comprising the following steps: using high performance liquid chromatography to perform gradient elution on the crude product to obtain compound I or compound II; The high-performance liquid chromatography method uses a hydrophilic octadecyl bonded silica column; The mobile phases are mobile phase A and mobile phase B; The mobile phase A is trifluoroacetic acid and water; the volume ratio of trifluoroacetic acid to the total volume of trifluoroacetic acid and water is 0.05-0.15%. The mobile phase B is trifluoroacetic acid, methanol, and acetonitrile; the volume ratio of trifluoroacetic acid, methanol, and acetonitrile is (0.1-0.5): 49.85: 49.

85. The gradient elution can be either scheme one or scheme two: In the first scheme, The elution flow rate is 75-85 mL / min; The elution gradient includes a first gradient and a second gradient: In the first gradient, the elution time is 1-3 min, the volume percentage of mobile phase A is 86%, and the volume percentage of mobile phase B is 14%. In the second gradient, the elution time is 10-30 min, the volume percentage of mobile phase A decreases from 86% to 62%, and the volume percentage of mobile phase B increases from 14% to 38%. In the second scheme, The elution flow rate is 45-55 mL / min; The elution gradient includes a first gradient and a second gradient: In the first gradient, the elution time is 3-6 minutes, the volume percentage of mobile phase A is 86%, and the volume percentage of mobile phase B is 14%. In the second gradient, the elution time is 40-60 min, the volume percentage of mobile phase A decreases from 86% to 62%, and the volume percentage of mobile phase B increases from 14% to 38%.

6. The preparation method according to claim 5, characterized in that, It meets one or more of the following conditions: (1) The detector used in the high performance liquid chromatography method is an ultraviolet detector; the detection wavelength of the detector is 190-260 nm; (2) The hydrophilic octadecyl bonded silica gel column is model ODS-AQ; (3) The pore size of the packing material of the hydrophilic octadecyl bonded silica column is 10-14 nm; (4) The length of the hydrophilic octadecyl bonded silica gel column is 250-350 mm; (5) The inner diameter of the hydrophilic octadecyl bonded silica gel column is 40-60 mm; (6) The packing material of the hydrophilic octadecyl bonded silica column has a particle size of 5-10 μm; (7) The operating pressure of the hydrophilic octadecyl bonded silica gel column is 0-10 MPa; (8) The column temperature of the hydrophilic octadecyl bonded silica gel column is 20-30℃; (9) In the mobile phase A, the volume ratio of the trifluoroacetic acid to the total volume of trifluoroacetic acid and water is 0.1%; (10) In the mobile phase B, the volume ratio of trifluoroacetic acid, methanol and acetonitrile is 0.3: 49.85: 49.85; (11) In the first scheme, the elution time in the first gradient is 2 min; (12) In the first scheme, the elution time in the second gradient is 20 min; (13) In the second scheme, the elution time in the first gradient is 4 min; (14) In the second scheme, the elution time in the second gradient is 51 min; (15) In Scheme 1, the elution flow rate is 80 mL / min; (16) In the second scheme, the elution flow rate is 50 mL / min; (17) The purification process includes a pretreatment of the crude product before gradient elution, which includes the following steps: mixing the crude product with an alcohol solvent and water.

7. The preparation method according to claim 6, characterized in that, It meets one or more of the following conditions: (1) The detector used in the high performance liquid chromatography method is an ultraviolet detector; the detection wavelength of the detector is 210 nm; (2) The pore size of the packing material of the hydrophilic octadecyl bonded silica column is 12 nm; (3) The length of the hydrophilic octadecyl bonded silica gel column is 250 mm; (4) The inner diameter of the hydrophilic octadecyl bonded silica gel column is 50 mm; (5) The packing material of the hydrophilic octadecyl bonded silica column has a particle size of 10 μm; (6) The column temperature of the hydrophilic octadecyl bonded silica gel column is 25℃; (7) The elution gradient of Scheme 1 includes the following gradients: ； (8) The elution gradient of Scheme 2 includes the following gradients: ； (9) In the pretreatment, the volume ratio of the alcohol solvent to the total volume of water and alcohol solvent is (0.5-1.5):10; (10) In the pretreatment, the ratio of the mass of the crude product to the total volume of water and alcohol solvent is 0.01-0.5 g / mL.

8. The preparation method according to claim 5, characterized in that, It meets one or more of the following conditions: (1) The elution gradient of Scheme 1 is as follows: ； (2) The elution gradient of Scheme 2 is as follows: ； (3) In the pretreatment, the volume ratio of the alcohol solvent to the total volume of water and alcohol solvent is 1:10; (4) In the pretreatment, the ratio of the mass of the crude product to the total volume of water and alcohol solvent is 0.06 g / mL or 0.1 g / mL.

9. A method for purifying a compound of formula I or formula II, characterized in that, It includes the following steps: using high performance liquid chromatography to perform gradient elution on the crude product to obtain compound I or compound II; The crude product contains compounds of formula I, formula II and formula III; The crude product has a purity of 40%-50% for compound of formula I. ； The high-performance liquid chromatography method uses a hydrophilic octadecyl bonded silica column; The mobile phases are mobile phase A and mobile phase B; The mobile phase A is trifluoroacetic acid and water; the volume ratio of trifluoroacetic acid to the total volume of trifluoroacetic acid and water is 0.05-0.15%. The mobile phase B is trifluoroacetic acid, methanol, and acetonitrile; the volume ratio of trifluoroacetic acid, methanol, and acetonitrile is (0.1-0.5): 49.85: 49.

85. The gradient elution can be either scheme one or scheme two: In the first scheme, The elution flow rate is 75-85 mL / min; The elution gradient includes a first gradient and a second gradient: In the first gradient, the elution time is 1-3 min, the volume percentage of mobile phase A is 86%, and the volume percentage of mobile phase B is 14%. In the second gradient, the elution time is 10-30 min, the volume percentage of mobile phase A decreases from 86% to 62%, and the volume percentage of mobile phase B increases from 14% to 38%. In the second scheme, The elution flow rate is 45-55 mL / min; The elution gradient includes a first gradient and a second gradient: In the first gradient, the elution time is 3-6 minutes, the volume percentage of mobile phase A is 86%, and the volume percentage of mobile phase B is 14%. In the second gradient, the elution time is 40-60 min, the volume percentage of mobile phase A decreases from 86% to 62%, and the volume percentage of mobile phase B increases from 14% to 38%.

10. The purification method according to claim 9, characterized in that, It meets one or two of the following conditions: (1) The purification method is as described in any one of claims 6-8; (2) In the crude product, the purity of the compound of formula I is 44.5%, the purity of the compound of formula II is 45.5%, and the purity of the compound of formula III is 10.1%.

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