A method for preparing impurity A-7-imp3 and its uses

Abstract

This invention relates to a method for preparing impurity A-7-imp3 and its uses. The significance of this invention lies in guiding the improvement of the process for (6-(4-((4-(1H-pyrazol-4-yl)phenyl)amino)pyrimidin-2-yl)-1-methyl-1H-indol-2-yl)(3,3-difluoroazacyclobutane-1-yl)methyl ketone (Formula A), thereby enhancing product quality, making product quality more controllable, and ensuring greater safety of the drug.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to a method for preparing an impurity compound A-7-imp3 and its uses. Background Technology

[0002] ROCK is a Rho-associated protein kinase, a serine-threonine protein kinase belonging to the AGC kinase family. It exerts its biological effects by phosphorylating downstream effector proteins (MLC, Lin-11, Isl-1, LIMK, ERM, MARCKS, CRMP-2, etc.). Studies have shown that various diseases (such as pulmonary fibrosis, cardiovascular and cerebrovascular diseases, neurological disorders, and cancer) are related to the ROCK-mediated pathway. Therefore, ROCK inhibitors are considered an important area of ​​research in drug development. The applicant has discovered that the compound (6-(4-((4-(1H-pyrazol-4-yl)phenyl)amino)pyrimidin-2-yl)-1-methyl-1H-indol-2-yl)(3,3-difluoroazacyclobutane-1-yl) ketone (Formula A) can be used as an effective ROCK inhibitor (see PCT / CN2021 / 115197, which is incorporated herein by reference in its entirety). The compound (6-bromo-1-methyl-1H-indol-2-yl)(3,3-difluoroazacyclobutane-1-yl) ketone (Formula A-7) involved in this invention is a synthetic precursor of the ROCK inhibitor, and the process impurity A-7-imp3 generated during the synthesis of the precursor.

[0003] CN101374834A discloses compounds and methods for regulating FXR. It involves the synthesis of methyl 6-bromo-1-methyl-1H-indole-3-carboxylic acid, using methyl 6-bromo-1H-indole-3-carboxylic acid as the starting material. Iodomethane is chosen as the methylating agent. Iodomethane has acute toxicity, irritant properties, carcinogenicity, easy decomposition in light, and high flammability, making it unsuitable for scale-up synthesis reactions and inconsistent with the concept of green synthesis.

[0004] Therefore, there is an urgent need for a clean, safe, and stable method for preparing A-7-imp3 compounds to guide the improvement of the A process, enhance product quality, make product quality more controllable, and ensure the safety of the drug. Summary of the Invention

[0005] In view of the above-mentioned technical status, this invention provides a method for preparing compound impurity A-7-imp3 and its uses. Impurity A-7-imp3 is a process impurity in the synthetic precursor (A-7) of a novel ROCK inhibitor having formula (A). This impurity affects the quality of A-7 and, through residue, enters the raw material (A-8) of the novel ROCK inhibitor. This ultimately poses a quality risk to the active pharmaceutical ingredient (API) of the novel ROCK inhibitor. Therefore, researching and controlling this impurity is beneficial to improving the quality of A-8 and thus to the safety of the ROCK inhibitor drug. The structures of the compounds involved are shown below:

[0006]

[0007] (6-(4-((4-(1H-pyrazol-4-yl)phenyl)amino)pyrimidin-2-yl)-1-methyl-1H-indol-2-yl)(3,3-difluoroazacyclobutane-1-yl)methyl ketone

[0008]

[0009] (6-bromo-1-methyl-1H-indol-2-yl)(3,3-difluoroazacyclobutan-1-yl)methyl ketone

[0010]

[0011] (3,3-Difluoroazacyclobutane-1-yl)(1-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxoboronacyclopentan-2-yl)-1H-2-indol-2-yl) methyl ketone

[0012]

[0013] Methyl (6-bromo-1-methyl-1H-indol-2-yl)formate

[0014] This invention provides a method for preparing impurity A-7-imp3 of the compound (6-bromo-1-methyl-1H-indol-2-yl)(3,3-difluoroazacyclobutane-1-yl) methyl ketone, the method comprising:

[0015] (1) Place 6-bromo-indole-2-carboxylic acid in 2-8 times its volume of C 1-6 In amide solvents, add 0.1-1 molar equivalent of C 1-10 An amine buffer and 10-18 molar equivalents of a methylating agent are reacted at 20-150°C for 1-15 hours to obtain an A-7-imp3 reaction solution; preferably, the methylating agent is selected from dimethyl sulfate, dimethyl carbonate, methyl p-toluenesulfonate, or methyl trifluoromethanesulfonate.

[0016] Those skilled in the art should know from common sense that "2-8 times the volume of C" 1-6 The amide solvent is based on the mass of 6-bromo-indole-2-carboxylic acid.

[0017] (2) The reaction solution obtained in step (1) is concentrated under reduced pressure at 50-130℃ until no obvious liquid flows out, and the vacuum degree is controlled at 0.085-0.1MPa to obtain A-7-imp3 concentrate.

[0018] (3) Add 5-20 times the volume of water to the concentrate obtained in step (2), the solid precipitates out, cool to 0-30℃ and stir for 0-5 hours, filter, rinse the filter cake with 1-2 times the volume of water, and vacuum dry the filter cake to obtain impurity A-7-imp3.

[0019] The impurity A-7-imp3 has the structure shown in formula (I):

[0020]

[0021] In one embodiment of the method of the present invention, the method further includes, as described in the C... 1-6 The amide solvent is selected from N,N-dimethylformamide and N,N-dimethylacetamide, preferably N,N-dimethylformamide.

[0022] In the method of the present invention, as one embodiment, the method further includes, in step (1), C 1-10 The amine buffer is selected from one of tetramethylethylenediamine, tetramethylpropylenediamine, N,N-diisopropylethylamine, and N,N-diisopropylamine, preferably tetramethylethylenediamine.

[0023] In one embodiment of the present invention, the method further includes that the methylating agent in step (1) is selected from dimethyl carbonate.

[0024] In one embodiment of the method of the present invention, the method further includes, in step (1), placing 6-bromo-indole-2-carboxylic acid in 4-6 times its volume of C24-4000 mol / L water. 1-6 In amide solvents, it is more preferable to place them in 5-6 times their volume of C 1-6 Amide solvents.

[0025] In one embodiment of the method of the present invention, the method further includes adding 0.4-0.6 molar equivalents of C in step (1). 1-10 Amine buffers, preferably 0.5 molar equivalents of C 1-10 Amine buffers.

[0026] In one embodiment of the present invention, the method further includes adding 15 to 18 molar equivalents of methylating agent in step (1), preferably 18 molar equivalents of methylating agent.

[0027] In the method of the present invention, as one of the implementation schemes, the method further includes that the reaction temperature in step (1) is 90-120℃, preferably 115℃, and the reaction time is 8-10h, preferably 10h.

[0028] In the method of the present invention, as one of the embodiments, the method further includes that the vacuum degree of the vacuum concentration in step (2) is controlled at 0.085-0.095 MPa; and the concentration temperature is 80-90℃.

[0029] In the method of the present invention, as one of the embodiments, the method further includes adding 8-10 times the volume, preferably 10 times the volume, of water to the concentrate in step (3).

[0030] In the method of the present invention, as one of the implementation schemes, the method further includes, in step (3), cooling to 25°C for crystallization and stirring for 2 hours.

[0031] In the method of the present invention, as one of the embodiments, the method further includes that the drying temperature in step (3) is 30-80℃, preferably 40-50℃; and the drying time is 5-15h, preferably 8-10h, and more preferably 9h.

[0032] In one embodiment of the method of the present invention, the method further includes:

[0033] (1) Place 6-bromo-indole-2-carboxylic acid in a reaction flask, add 5 times the volume of N,N-dimethylformamide, add 0.5 molar equivalents of tetramethylethylenediamine and 18 molar equivalents of dimethyl carbonate, and react at 115°C for 10 hours to obtain A-7-imp3 reaction solution.

[0034] (2) Transfer the reaction solution obtained in step (1) to a distillation reaction flask, cool it to 85±5℃, concentrate it under reduced pressure until no obvious liquid flows out, vacuum degree 0.085-0.095MPa, concentration temperature 85±5℃, to obtain concentrated solution;

[0035] (3) Slowly add 10 times the volume of water to the concentrate obtained in step (2), precipitate solid, cool to 25°C, stir for 2 hours, filter, rinse the filter cake with 1-2 times the volume of water, and vacuum dry the filter cake at 45±5°C for 9 hours to obtain impurity A-7-imp3 solid.

[0036] The impurity A-7-imp3 has the structure shown in formula (I):

[0037]

[0038] This invention also provides the use of impurity A-7-imp3 prepared according to the foregoing method as a reference standard in the quality control of novel ROCK inhibitor active pharmaceutical ingredient and formulation, wherein the novel ROCK inhibitor has a structure as shown in Formula A:

[0039]

[0040] In one embodiment of the present invention, the impurity A-7-imp3 is used as a reference in the related substances detection of the synthetic precursor A-7 of a novel ROCK inhibitor, wherein the synthetic precursor A-7 has a structure as shown in Formula A-7:

[0041]

[0042] In one embodiment of the present invention, the impurity A-7-imp3 is prepared by the following method:

[0043] (1) Place 6-bromo-indole-2-carboxylic acid in a reaction flask, add 5 times the volume of N,N-dimethylformamide, add 0.5 molar equivalents of tetramethylethylenediamine and 18 molar equivalents of dimethyl carbonate, and react at 115°C for 10 hours to obtain A-7-imp3 reaction solution.

[0044] (2) Transfer the reaction solution obtained in step (1) to a distillation flask, cool it to 85±5℃, concentrate it under reduced pressure until no obvious liquid flows out, vacuum degree 0.085-0.095MPa, concentration temperature 85±5℃, to obtain concentrated solution;

[0045] (3) Slowly add 10 times the volume of water to the concentrate obtained in step (2) to precipitate the solid, cool to 25°C, stir for 2 hours, filter, rinse the filter cake with 1-2 times the volume of water, and vacuum dry the filter cake at 45±5°C for 9 hours to obtain impurity A-7-imp3 solid.

[0046] This invention also provides a method for detecting related substances in synthetic precursor A-7 using impurity A-7-imp3 as a reference standard, the method comprising:

[0047] (1) Prepare impurity A-7-imp3 according to any of the aforementioned methods;

[0048] (2) Using A-7-imp3 as a reference standard, the relevant substances in the synthetic precursor A-7 were detected:

[0049] Weigh 20 mg of A-7 sample to be tested into a 100 mL volumetric flask, add acetonitrile to dilute to volume and mix well. This is recorded as the test solution.

[0050] Weigh 20 mg of impurity A-7-imp3 reference standard into a 100 mL volumetric flask, add acetonitrile to dilute to volume and mix well. This is recorded as the impurity reference standard solution.

[0051] Agilent 1260 series HPLC or similar instruments;

[0052] Chromatographic column: YMC-Triart C18 4.6×150mm, 3.0μm;

[0053] Flow rate 0.8 mL / min, injection volume 5 μL, column temperature 30℃, detection wavelength 234 nm, run time 28 min, gradient table:

[0054]

[0055] Mobile phase A: 0.05% (v / v) aqueous solution of trifluoroacetic acid.

[0056] Mobile phase B: 0.05% trifluoroacetic acid acetonitrile solution (volume concentration).

[0057] The significance of this invention lies in guiding the process improvement of (6-(4-((4-(1H-pyrazol-4-yl)phenyl)amino)pyrimidin-2-yl)-1-methyl-1H-indol-2-yl)(3,3-difluoroazacyclobutane-1-yl)methyl ketone (Formula A), thereby improving product quality, making product quality more controllable, and ensuring greater safety of the drug.

[0058] The beneficial effects of this invention are as follows: Through the study of impurity A-7, this invention obtained an impurity A-7-imp3. High-performance liquid chromatography (HPLC) analysis confirmed its compound purity: relative area percentage ≥ 98.9%. The identification of impurity A-7-imp3 provides more detailed quality standard establishment conditions for the synthetic precursor A-7. It can be used as a reference standard for impurity studies in the related substance detection of novel ROCK inhibitor APIs. According to the quality standards for novel ROCK inhibitor APIs, the control limit for this impurity or its derivatives in API A is less than 0.1%. Spiking experiments confirmed that the content of A-7-imp3 in A-7 should be less than 0.3%, effectively controlling the derivative impurity corresponding to A-7-imp3 in API A to below 0.1%. This further ensures the drug safety of the novel ROCK inhibitor API and formulation. Attached Figure Description

[0059] Figure 1 The image shows the HPLC chromatogram of impurity A-7-imp3.

[0060] Figure 2 The hydrogen spectrum of impurity A-7-imp3 is shown. Detailed Implementation

[0061] The following examples are provided to further illustrate the present invention, but are not intended to limit the scope of the invention in any way.

[0062] Example 1

[0063] Add 5.00 g of 6-bromo-indole-2-carboxylic acid to 25 ml of N,N-dimethylformamide solvent, add 21.44 g of dimethyl carbonate, add 0.92 g of tetramethylethylenediamine, heat to 115 °C, and stir for 10 hours to obtain A-7-imp3 reaction solution.

[0064] The above A-7-imp3 reaction solution was concentrated under reduced pressure (0.095 MPa) and at a concentration temperature of 90°C until no obvious liquid flowed out, thus obtaining the A-7-imp3 concentrate.

[0065] Add 50 ml of water to the above-obtained A-7-imp3 concentrate, precipitate the solid, cool to 20±5℃, and stir for 2 hours. Filter the above reaction solution, rinse the filter cake with 10 ml of water, dry under vacuum, and vacuum dry the wet filter cake at 45℃ for 9 hours to obtain impurity A-7-imp3.

[0066] Yield 94%. HPLC purity 98.09%.

[0067] The purity testing method is as follows:

[0068] Weigh 20 mg of the impurity A-7-imp3 marker obtained in Example 1 into a 100 mL volumetric flask, add acetonitrile and mix well.

[0069] The Agilent 1260 series HPLC column was used: YMC-Triart C18 4.6×150mm, 3.0μm.

[0070] The flow rate was 0.8 mL / min, the injection volume was 5 μL, the column temperature was 30℃, the detection wavelength was 234 nm, and the run time was 28 min. Gradient elution was performed according to the following gradient table:

[0071]

[0072] Example 2

[0073] 50.00 g of 6-bromo-indole-2-carboxylic acid was added to 250 ml of N,N-dimethylformamide solvent, followed by 254.24 g of dimethyl carbonate and 9.22 g of tetramethylethylenediamine. The mixture was heated to 115 °C and stirred for 9 hours to obtain the A-7-imp3 reaction solution.

[0074] The above A-7-imp3 reaction solution was concentrated under reduced pressure (0.095 MPa) and at a concentration temperature of 90°C until no obvious liquid flowed out, thus obtaining the A-7-imp3 concentrate.

[0075] Add 500 ml of water to the above-obtained A-7-imp3 concentrate, precipitate the solid, cool to 20±5℃, and stir for 2 hours. Filter the above reaction solution, rinse the filter cake with 100 ml of water, dry under vacuum, and vacuum dry the filter cake at 40℃ for 10 hours to obtain impurity A-7-imp3.

[0076] Yield 94%; HPLC purity 98.92%, purity detection method referred to the "Purity Detection Method" in "Example 1", and the detection results are attached. Figure 1 , 1 See attached H NMR data. Figure 2 .

[0077] 1 H NMR (400MHz DMSO-d6): δ7.87 (s, 1H), 7.63 (d, 2H), 7.26 (d, d, 2H), 3.99 (s, 3H), 3.86 (s, 3H)

[0078] Example 3

[0079] 100.00 g of 6-bromo-indole-2-carboxylic acid was added to 500 ml of N,N-dimethylformamide solvent, 508.48 g of dimethyl carbonate was added, and 18.44 g of tetramethylethylenediamine was added. The mixture was heated to 120 °C and stirred for 10 hours to obtain the A-7-imp3 reaction solution.

[0080] The above A-7-imp3 reaction solution was concentrated under reduced pressure (0.095 MPa) and at a concentration temperature of 85°C until no obvious liquid flowed out, thus obtaining the A-7-imp3 concentrate.

[0081] Add 1000 ml of water to the above-obtained A-7-imp3 concentrate, precipitate the solid, cool to 25°C, and stir for 3 hours. Filter the above reaction solution, rinse the filter cake with 100 ml of water, dry it under vacuum, and dry the filter cake at 50°C under vacuum for 10 hours to obtain impurity A-7-imp3.

[0082] Yield 96%; HPLC purity 98.50%, purity detection method refers to the "Purity Detection Method" in "Example 1";

[0083] The compound impurity A-7-imp3 in this invention is a process impurity generated during the production of the novel drug A. Its identification and detection are of great significance for 6-(4-((4-(1H-pyrazol-4-yl)phenyl)amino)pyrimidin-2-yl)-1-methyl-1H-indol-2-yl)(3,3-difluoroazacyclobutane-1-yl)methyl ketone (A), and provide important supporting evidence for the quality control of the novel drug A.

[0084] The preparation, identification, and detection of compounds in this invention can guide the optimization of the production process of compound A, thereby improving yield and product quality.

[0085] (1) Preparation of reference impurity A-7-imp3: Refer to the preparation method in Example 2.

[0086] (2) Using A-7-imp3 as a reference standard to detect related substance impurities A-7-imp3 in compound A-7:

[0087] Weigh 20 mg of A-7 sample into a 100 mL volumetric flask, add acetonitrile to dilute to volume and mix well. This is recorded as the test solution.

[0088] Weigh 20 mg of impurity A-7-imp3 reference standard into a 100 mL volumetric flask, add acetonitrile to dilute to volume and mix well. This is recorded as the impurity reference standard solution.

[0089] Agilent 1260 series HPLC or similar instruments;

[0090] Chromatographic column: YMC-Triart C18 4.6×150mm, 3.0μm;

[0091] Flow rate 0.8 mL / min, injection volume 5 μL, column temperature 30℃, detection wavelength 234 nm, run time 28 min, gradient table:

[0092]

[0093] Mobile phase A: 0.05% (v / v) aqueous solution of trifluoroacetic acid.

[0094] Mobile phase B: 0.05% trifluoroacetic acid acetonitrile solution (volume concentration).

[0095] A's process route

[0096]

[0097] (3) The content of impurity A-7-imp3 was determined using the above-mentioned content detection method, and the quality was compared after the process improvement:

[0098]

[0099]

[0100] Production data shows that, after determining the structure of the impurity prepared by this invention, the mechanism of its generation in the actual production process can be inferred, guiding process optimization to improve product yield, while avoiding the generation of the impurity and improving the quality of product A. This has important guiding significance for the production of product A.

[0101] Compared with Example 2, a comparison under different conditions is presented below:

[0102] Comparative Example 1

[0103] Add 50.00 g of 6-bromo-indole-2-carboxylic acid to 250 ml of N,N-dimethylformamide solvent, add 254.24 g of dimethyl carbonate, add 9.22 g of tetramethylethylenediamine, heat to 80±5℃, and stir for 5 hours.

[0104] The above reaction solution was concentrated under reduced pressure until no obvious liquid flowed out, 500 ml of water was added, the temperature was lowered to 20±5℃, and the mixture was stirred for 2 hours.

[0105] The above reaction solution was filtered, the filter cake was rinsed with 100ml of water, dried under vacuum, and the filter cake was vacuum dried at 45℃ for 16 hours to obtain impurity A-7-imp3.

[0106] Yield 78.98%; HPLC purity 58.19%.

[0107] Comparative Example 2

[0108] Add 50.00 g of 6-bromo-indole-2-carboxylic acid to 250 ml of N,N-dimethylformamide solvent, add 127.12 g of dimethyl carbonate, add 9.22 g of tetramethylethylenediamine, heat to 115±5℃, and stir for 5 hours.

[0109] The above reaction solution was concentrated under reduced pressure until no obvious liquid flowed out, 500 ml of water was added, the temperature was lowered to 20±5℃, and the mixture was stirred for 2 hours.

[0110] The above reaction solution was filtered, the filter cake was rinsed with 100ml of water, dried under vacuum, and the filter cake was vacuum dried at 45℃ for 16 hours to obtain impurity A-7-imp3.

[0111] Yield 75.64%; HPLC purity 34.02%.

[0112] Comparative Example 3

[0113] 50.00 g of 6-bromo-indole-2-carboxylic acid was added to 250 ml of N,N-dimethylformamide solvent, along with 85 g of potassium carbonate and 7 g of iodomethane. The mixture was kept at 25°C and stirred for 5 hours. TCL monitoring did not detect the formation of the target compound A-7-imp3. The product obtained was 6-bromo-1-methyl-indole-2-carboxylic acid. The reaction failed to yield the target compound A-7-imp3.

[0114] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or changes made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing impurity A-7-imp3, characterized in that, The method includes: (1) Place 6-bromo-indole-2-carboxylic acid in 2-8 times its volume of C 1-6 In amide solvents, add 0.1-1 molar equivalent of C 1-10 An amine buffer and 15-18 molar equivalents of a methylating agent are reacted at 90-120℃ for 8-10 hours to obtain A-7-imp3 reaction solution, wherein the methylating agent is dimethyl carbonate. (2) The reaction solution obtained in step (1) is concentrated under reduced pressure at 50-130℃ until no obvious liquid flows out, and the vacuum degree is controlled at 0.085-0.1MPa to obtain A-7-imp3 concentrate. (3) Add 5-20 times the volume of water to the A-7-imp3 concentrate obtained in step (2), the solid precipitates out, cool to 0-30℃ and stir for 0-5 hours, filter, rinse the filter cake with 1-2 times the volume of water, and vacuum dry the filter cake to obtain impurity A-7-imp3. In step (1), C 1-10 The amine buffers are selected from tetramethylethylenediamine, tetramethylpropylenediamine, N,N-diisopropylethylamine, or N,N-diisopropylamine; The C 1-6 The amide solvents are selected from N,N-dimethylformamide or N,N-dimethylacetamide; The impurity A-7-imp3 has the structure shown in formula (I):

2. The method according to claim 1, characterized in that, The method further includes, in step (1), C 1-10 The amine buffer is tetramethylethylenediamine.

3. The method according to claim 1, characterized in that, The method further includes, in step (1), placing 6-bromo-indole-2-carboxylic acid in 4-6 times its volume of C24-4000 mol / L. 1-6 In amide solvents.

4. The method according to claim 1, characterized in that, The method further includes adding 0.4-0.6 molar equivalents of C in step (1). 1-10 Amine buffers.

5. The method according to claim 1, characterized in that, The method further includes adding 18 molar equivalents of a methylating agent in step (1).

6. The method according to claim 1, characterized in that, The method further includes that the reaction temperature in step (1) is 115°C and the reaction time is 10h.

7. The method according to claim 1, characterized in that, The method further includes that the vacuum degree of the reduced pressure concentration in step (2) is controlled at 0.085-0.095 MPa; and the concentration temperature is 80-90℃.

8. The method according to claim 1, characterized in that, The method further includes adding 8-10 times the volume of water to the concentrate in step (3).

9. The method according to claim 1, characterized in that, The method further includes cooling the temperature to 25°C for crystallization in step (3) and stirring for 2 hours.

10. The method according to claim 1, characterized in that, The method further includes that the drying temperature in step (3) is 30-80℃ and the drying time is 5-15h.

11. The method according to any one of claims 1-10, characterized in that, The method further includes: (1) Place 6-bromo-indole-2-carboxylic acid in a reaction flask, add 5 times the volume of N,N-dimethylformamide, add 0.5 molar equivalents of tetramethylethylenediamine and 18 molar equivalents of dimethyl carbonate, and react at 115°C for 10 hours to obtain A-7-imp3 reaction solution. (2) Transfer the reaction solution obtained in step (1) to a distillation flask, cool it to 85±5℃, concentrate it under reduced pressure until no obvious liquid flows out, vacuum degree 0.085-0.095MPa, concentration temperature 85±5℃, to obtain concentrated solution; (3) Slowly add 10 times the volume of water to the concentrate obtained in step (2), precipitate solid, cool to 25°C, stir for 2 hours, filter, rinse the filter cake with 1-2 times the volume of water, and vacuum dry the filter cake at 45±5°C for 9 hours to obtain impurity A-7-imp3 solid. The impurity A-7-imp3 has the structure shown in formula (I):

12. The method according to claim 1, characterized in that, The C 1-6 The amide solvent is N,N-dimethylformamide.

13. The method according to claim 3, characterized in that, The method further includes, in step (1), placing 6-bromo-indole-2-carboxylic acid in 5-6 times its volume of C24-44-dimethylformamide. 1-6 In amide solvents.

14. The method according to claim 4, characterized in that, The method further includes adding 0.5 molar equivalents of C in step (1). 1-10 Amine buffers.

15. The method according to claim 8, characterized in that, The method further includes adding 10 times the volume of water to the concentrate in step (3).

16. The method according to claim 10, characterized in that, The method further includes that the drying temperature in step (3) is 40-50℃ and the drying time is 8-10h.

17. The method according to claim 16, characterized in that, The method further includes that the drying time in step (3) is 9 hours.

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