Preparation method of duloxetine intermediate

Abstract

The invention discloses a preparation method of a duloxetine intermediate, and belongs to the technical field of biology. According to the invention, 3-dimethylamino-1-(thienyl)-1-acetone hydrochloride is taken as a substrate, and the substrate is converted into the duloxetine intermediate by utilizing specific keto reductase and combining with an optimized preparation process. Compared with other methods, the preparation method provided by the invention has the advantages that the cost is lower, the reaction conditions are more environment-friendly, the substrate concentration can reach 200g / L, a high-purity target product crystal product can be prepared through product extraction and separation after the reaction is finished, and the preparation method has the characteristics of high production efficiency, lower cost, greenness and safety.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing a duloxetine intermediate. Background Art

[0002] Duloxetine, English name (R)-Duloxetine, chemical name (S)-N-methyl-3-(1-naphthyloxy)-3-(2-thienyl)-1-propylamine, trade name Cymbalta, is a dual inhibitor of 5-hydroxytryptamine and norepinephrine reuptake (SNRIs). Its hydrochloride is used clinically to treat depression, stress urinary incontinence, chronic pain associated with depression, and diabetic peripheral neuropathy pain. Among the two isomers of duloxetine with the same chemical composition, only the (S)-type has the above-mentioned pharmacological activity. There are many methods for asymmetric synthesis of chiral duloxetine. Through retrosynthetic analysis, it can be found that (S)-N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1-propylamine (S-DHTP) is an important chiral intermediate for the preparation of duloxetine. The synthesis methods of S-DHTP mainly include chemical method and biological method.

[0003] Chinese patent CN202111662305.5 discloses a method for preparing a duloxetine intermediate, which uses an amine methylation reaction to prepare 3-dimethylamino 1-(2-thienyl)-1-acetone hydrochloride with 2-acetylthiophene as a raw material, and then reduces it with sodium borohydride to obtain (RS)N,N-monomethyl 3-hydroxy 3-(2-thienyl)propylamine, and then uses S-(+)-mandelic acid to split to obtain (S)-(-)-N,N-dimethyl-3-hydroxy-3-(2-thienyl)propylamine, and uses phosphotungstic acid loaded with zirconium oxide-titanium oxide as a catalyst to accelerate the reaction speed and improve the reaction yield. The total yield of the duloxetine intermediate prepared by this method is 67.6%.

[0004] Chinese patent CN201510232218.4 discloses the preparation of duloxetine hydrochloride, which splits (S)-mandelic acid to obtain (R)-N, N-dimethyl-3-hydroxy-3-(2-thiophene)-propylamine and (S)-N, N-dimethyl-3-hydroxy-3-(2-thiophene)propylamine, and then converts (R)-N, N-dimethyl-3-hydroxy-3-(2-thiophene)propylamine into (S)-N, N-dimethyl-3-p-nitrobenzoate-3-(2-thiophene)propylamine, and the S-type reaction obtains duloxetine hydrochloride. By converting the R-configuration compound into the S-type, the total yield is higher than that of preparing duloxetine hydrochloride using the S-configuration compound alone. The yield of preparing (S)-N, N-dimethyl-3-hydroxy-3-(2-thiophene)propylamine using this method is 46.46%.

[0005] Chinese patent CN201610382975.4 discloses a method for asymmetric synthesis of duloxetine intermediates using carbonyl reductase, wherein the reaction system of the method is: 2 mL 0.1 mol / L TEA buffer (pH 9.0) contains 1 g / L DKTP, 4.55 mmol / L coenzyme NAD(P)H and 20 U / L enzyme solution, 1 mmol / L Zn 2+ The reaction was carried out at 40°C and 200 r / min for 8 h. The yield of (S)-DHTP prepared by this method was 67%.

[0006] Chinese patent 201910433914.X discloses a ketoreductase mutant and its use in the preparation of duloxetine chiral alcohol intermediates and analogs thereof. The amount of ketoreductase Rr Kred in the conversion reaction system is 1-10 g / L, the buffer concentration is 50-200 mM, the buffer pH is between 6.0-8.5, the coenzyme concentration is 0.1-0.5 g / L, and the substrate concentration is between 5-20 g / L.

[0007] The above patents use chemical and biological methods to prepare duloxetine intermediates respectively. The chemical method has the problems of low yield or harsh reaction conditions and high cost, such as using S-mandelic acid as a chiral resolving agent or other catalyst; the biological method has the problems of low substrate concentration and low production efficiency, and does not mention the subsequent product separation, extraction and crystallization methods after the conversion is completed.

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] The object of the present invention is to provide a method for preparing a duloxetine intermediate. The preparation method of the present invention can improve production efficiency and reduce production costs.

[0010] The present invention is achieved in that:

[0011] The invention provides a method for preparing a duloxetine intermediate. The method comprises the following steps: using ketoreductase as a catalyst to catalyze a substrate 3-dimethylamino-1-(thienyl)-1-propanone hydrochloride (DKTP) to synthesize (3S)-N,N-dimethyl-3-hydroxy-3-(-2-thiophene)-1-propylamine (S-DHTP).

[0012] In the present invention, the synthetic route of duloxetine intermediate is as follows Figure 1 As shown, the reaction system uses glucose and glucose dehydrogenase (GDH) for coenzyme circulation and ketoreductase for catalysis to efficiently synthesize S-DHTP.

[0013] Among them, ketoreductase is the key enzyme in the reaction. In the research process of the present invention, four initial sequences are first screened from the gene library, and the evolution process is simulated by computer algorithms to screen four sequences from a large number of candidate sequences. The initial sequences are shown in SEQ ID NO: 1-4; the candidate sequences are shown in SEQ ID NO: 5-8. Then, the four candidate sequences are artificially designed, modeled and simulated, and the sequence performance after mutation and recombination is predicted using computer simulation and molecular dynamics simulation. According to the simulation results, the variant sequence with the best performance under the target conditions is screened, and its nucleotide sequence is shown in SEQ ID NO: 9-12. On this basis, according to the 3D model simulation calculation of the binding of protein and substrate ligand, different amino acids are adjusted to obtain three point mutation gene sequences, and the nucleotide sequences after mutation are shown in SEQ ID NO: 13-15. Finally, the three sequences after mutation are subjected to multiple sequence alignment, and a consensus sequence, that is, the final sequence, is generated through the alignment results, and its nucleotide sequence is shown in SEQ ID NO: 16.

[0014] In some embodiments, the ketoreductase used to catalyze the synthesis of S-DHTP from DKTP is selected from any one of the nucleotide sequences shown in SEQ ID NOs: 1-16.

[0015] In some embodiments, the nucleotide sequence of the ketoreductase for catalyzing the synthesis of S-DHTP from DKTP is as shown in SEQ ID NO:16.

[0016] Based on the above ketoreductase, in order to obtain a better preparation effect, the present invention optimizes the reaction conditions for the ketoreductase. Specifically, the preparation method of the duloxetine intermediate provided by the present invention is as follows:

[0017] S1. Construct a recombinant bacterium to obtain the above-mentioned ketoreductase.

[0018] (1) Design primers for the target gene using software.

[0019] (2) After obtaining the target primers, PCR amplification is performed to obtain a PCR product with the expected size consistent with the target sequence.

[0020] (3) The above PCR product was connected to the linearized plasmid, and the recombinant plasmid was heat-transformed into host cells to express the protein.

[0021] Among them, for host cells and plasmids, conventional host cells and plasmids in the art can be selected, and the present invention does not limit this. In some embodiments, the plasmid is pET-19b (+), and the host cell is Escherichia coli.

[0022] (4) Adding the host cells to the culture medium for cultivation and fermentation.

[0023] For the culture and fermentation conditions, those skilled in the art can adjust them according to the selected host cells. In some embodiments, the culture medium is LB culture medium (containing 40 μL of ampicillin), and the fermentation conditions are: at 37°C, shake culture at a speed of 220 rpm for 6-8 hours, control the dissolved oxygen at 20-40%, and the tank pressure is not higher than 0.08 MPa.

[0024] (5) When the OD600 of the fermentation liquid reaches about 25, add the inducer for induction.

[0025] In some embodiments, the induction conditions are: temperature 28°C, inducer IPTG, concentration 0.071 g / L, and release from the tank when the OD value is above 55 after 8-12 hours of induction.

[0026] (6) The fermentation broth obtained after induction is centrifuged, and the supernatant is discarded to obtain wet bacteria, which are then ultrasonically disrupted to obtain a disrupted enzyme solution of ketoreductase.

[0027] S2. Enzyme catalyzes the reaction to obtain a reaction solution.

[0028] (1) Add the substrate and glucose into water, adjust the pH and temperature, and stir to dissolve;

[0029] In some embodiments, the concentration of the substrate is 140-220 g / L; preferably, the concentration of the substrate is 200 g / L.

[0030] In some embodiments, after the substrate and glucose are added to water, the pH of the mixed solution is adjusted to 5.5-6.5 and the temperature is adjusted to 30°C.

[0031] (2) Add ketoreductase crushing enzyme solution, glucose dehydrogenase crushing enzyme solution, zinc acetate and coenzyme NADP+ to the dissolved mixed solution, adjust the pH and start the catalytic reaction.

[0032] In some embodiments, the added amount of glucose dehydrogenase is 5%; the added amount of the recombinant bacteria expressing ketoreductase is 35-55% of the substrate mass, preferably, the added amount of the recombinant bacteria expressing ketoreductase is 40-45%.

[0033] In some embodiments, the amount of glucose in the above reaction system is 1-1.2 times the amount of the substrate, the concentration of zinc acetate is 0.15-0.35 g / L, and the concentration of coenzyme NADP+ is 0.05-0.2 g / L.

[0034] In some embodiments, the conditions for the catalytic reaction are: temperature of 25°C-30°C, pH of 5.8-6.0, and reaction time of 15-24h.

[0035] S3. Extract and purify the reaction solution.

[0036] In some embodiments, the extraction includes: adjusting the pH of the reaction solution and passing it through a ceramic membrane or diatomaceous earth, and then ultrafiltration; cooling and crystallizing the filtrate obtained after ultrafiltration.

[0037] In some embodiments, the pH of the reaction solution is 3.5-4.0 when passing through the ceramic membrane.

[0038] In some embodiments, the ultrafiltration conditions are: filtrate temperature <10°C, pH = 11-12.

[0039] In the present invention, the extraction can also be performed by sequentially subjecting the reaction solution to nanofiltration and concentration treatment, concentrating the reaction solution to a volume of about 35%, and then directly adjusting the base and cooling the reaction solution for crystallization.

[0040] In some embodiments, during the cooling crystallization process, the temperature is maintained at 0-10°C; preferably, the temperature is maintained at 0-5°C.

[0041] When the crystal product obtained after cooling crystallization contains salt, the crystal product is dissolved with twice the weight of dichloromethane, and the refined product is obtained after liquid separation and dehydration and organic phase concentration.

[0042] The present invention has the following beneficial effects:

[0043] The present invention uses 3-dimethylamino-1-(thienyl)-1-propanone hydrochloride as a substrate, utilizes a specific keto-reductase, and combines with an optimized preparation process to convert the substrate into a duloxetine intermediate. Compared with other methods, the preparation method of the present invention has lower cost, more environmentally friendly reaction conditions, and a substrate concentration of 200 g / L. After the reaction is completed, the product is extracted and separated to obtain a high-purity target product crystalline product, which has the characteristics of high production efficiency, lower cost, and green safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0045] Figure 1 This is the synthetic route of the duloxetine intermediate in the present invention;

[0046] Figure 2 The detection spectrum of the reaction product after the catalytic reaction in Example 2;

[0047] Figure 3 This is the purity detection spectrum of the product after passing through the ceramic membrane and ultrafiltration in Example 2;

[0048] Figure 4 This is the chiral purity detection spectrum of the product after passing through the ceramic membrane and ultrafiltration in Example 2. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0050] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0051] Example 1

[0052] The steps for obtaining ketoreductase are as follows:

[0053] 1. First round of screening: 4 initial sequences (SEQ ID NO: 1-4) were screened from the gene library, and 4 sequences (SEQ ID NO: 5-8) were screened from a large number of candidate sequences using a computer algorithm to simulate the evolution process.

[0054] 2. Second round of evolution: The four selected sequences were artificially designed, modeled and simulated, and computer simulation and molecular dynamics simulation were used to predict the sequence performance after mutation and recombination. Based on the simulation results, variant sequences 2-4 (SEQ ID NO: 9-12) with the best performance under the target conditions were selected.

[0055] 3. The third round of evolution: Based on the sequences 2-4 in the second round, the different amino acids were adjusted according to the 3D model simulation calculation of the protein binding to the substrate ligand, and three point mutation gene sequences (SEQ ID NO: 13-15) were obtained.

[0056] 4. Perform multiple sequence alignment on the three mutated sequences, and generate a consensus sequence, i.e., the final sequence (SEQ ID NO: 16), through the alignment results.

[0057] Example 2

[0058] This embodiment is a method for preparing a duloxetine intermediate, which specifically comprises the following steps:

[0059] S1. Preparation of ketoreductase and glucose hydrolase enzyme solutions

[0060] (1) Primers were designed for the target gene shown in SEQ ID NO: 16 using SnapGene as follows:

[0061] 5'-TACTGTGCAC-3'(SEQ ID NO: 17)

[0062] 5'-AGTTCATCAG-3'(SEQ ID NO: 18)

[0063] (2) After obtaining the target primers, PCR amplification is performed to obtain a PCR product with the expected size consistent with the target sequence.

[0064] (3) The PCR product containing the artificially designed gene sequence was connected to the linearized pET-19b(+), and the recombinant expression pET-19b(+) plasmid was heat-transformed into competent cells of Escherichia coli BL21(DE3) to express the protein.

[0065] (4) The transformed competent cells were added to LB medium (containing 40 μL of ampicillin) and cultured in a fermenter at 37°C with shaking at 220 rpm for 6-8 h. The dissolved oxygen was controlled at 30% and the tank pressure was not higher than 0.08 MPa.

[0066] (5) Induction control: When OD600 reaches about 25, cool down to 28°C and add 0.3 mM IPTG at a dosage of 0.071 g / L. After 10 hours of induction, release the cells when the OD value is above 55.

[0067] (6) The fermentation broth was sampled into a centrifuge tube, centrifuged and the supernatant was discarded. The wet bacteria were weighed and diluted in a ratio of 1 g:4 g water. The temperature was cooled to below 20°C and ultrasonically disrupted. The cell disruption solution was set aside.

[0068] (7) The purchased GDH glucose hydrolase (derived from Bacillus amyloliquefaciens) was cultured in the same fermentation tank as described above. After 10 h of induction, the OD value was above 65 and the enzyme solution was obtained and set aside.

[0069] The wet weight of the bacteria is determined by sampling the fermentation liquid into a centrifuge tube (weighing), centrifuging and discarding the supernatant to obtain the bacterial count n g / L. The total wet bacterial count is n*total volume.

[0070] S2. Catalytic reaction

[0071] (1) Weigh 150 g of the raw material 3-dimethylamino-1-(thienyl)-1-propanone hydrochloride, add 0.38 L of water, add 150 g of glucose, adjust the pH to 6 with 30% sodium hydroxide solution, slowly raise the temperature to 30° C., and stir to dissolve.

[0072] (2) After dissolution is completed, add 300 mL of ketoreductase crushing enzyme solution (20% enzyme solution concentration, equivalent to 40% enzyme addition amount), 120 mL of GDH glucose dehydrogenase crushing enzyme solution (5% enzyme addition amount), 0.6 g of zinc acetate, 0.3 g of coenzyme NADP+, adjust pH to 6, and maintain pH reaction with 30% sodium hydroxide solution.

[0073] (3) Start the reaction, control the temperature at 30°C, react for about 24 hours, and control the liquid phase. End the reaction when the reaction rate is above 98%.

[0074] Among them, the liquid phase control conditions are as follows:

[0075] Column: Water Xterra RP18 (250*4.6mm, 5μm)

[0076] Mobile phase: A 0.4% triethylamine (phosphoric acid adjusted pH = 7.0): B acetonitrile, A:B = 75:25%

[0077] Flow rate: 1.00 ml / min; wavelength: 246 nm / 230 nm; injection volume: 5 μL; column temperature: 25 °C.

[0078] Detection spectrum such as Figure 2 shown.

[0079] S3. Extract and purify the reaction solution.

[0080] The reaction solution was adjusted to pH 4.0 with hydrochloric acid and passed through a ceramic membrane (8k-20k Daltons). After filtration, the clear liquid was directly subjected to ultrafiltration (800-1000 molecular weight) to remove protein.

[0081] The ultrafiltration membrane filtrate was adjusted to pH = 11 with saturated sodium hydroxide solution below 10°C, cooled to 5°C for crystallization for 1 hour, filtered to obtain solid crystals, and dried to obtain 113.8 g of the target product with a yield of 90.5%, a purity of 99.2%, and a chiral purity of 99.57%.

[0082] The liquid phase intermediate control conditions in S2 are used to test the purity of the product after passing through the ceramic membrane and ultrafiltration. The test spectrum is as follows: Figure 3 As shown; the chiral purity of the product was tested using the following test conditions:

[0083] Column CHIRALPAK AD-H (250*4.6mm, 5μm);

[0084] Mobile phase: n-hexane: isopropanol: diethylamine = 96:4:0.2;

[0085] Injection volume 10 μL; flow rate 0.6 ml / min; wavelength 230 nm;

[0086] Detection spectrum such as Figure 4 shown.

[0087] Comparative Example

[0088] The difference from Example 2 is that the source of the ketoreductase is different.

[0089] Verification of ketoreductase CR1 (SEQ ID NO: 19): After 24 hours of reaction, the reaction was only 8.7%, and the enzyme activity was not high.

[0090] Experimental Example 1

[0091] This experiment verifies the effect of substrate concentration and ketoreductase dosage on the conversion progress during the conversion process, as follows:

[0092] On the basis of the same steps as in Example 2, the reaction was carried out in a 100 ml reaction system for 24 hours to detect the progress of the reaction (glucose dehydrogenase was mainly used as a coenzyme regeneration system, and the dosage was kept unchanged at 5%).

[0093] Table 1 Effect of substrate concentration and ketoreductase dosage on conversion progress

[0094] Group Substrate concentration (g / L) Ketoreductase (%) Reaction progress (%) 1 80 25 67.5 2 100 30 82.5 3 120 35 95.8 4 140 40 99.2 5 160 40 99.2 6 180 45 99.4 7 200 45 99.3 8 220 50 96.4 9 220 55 97.2

[0095] As can be seen from Table 1, by continuously increasing the substrate concentration and the amount of enzyme, when the ketoreductase dosage is above 40%, the conversion progress can reach above 98% in 24 hours. However, when the substrate concentration continues to be increased, even if the amount of enzyme is further increased, the reaction is not complete. Considering the comprehensive conversion efficiency and cost, the optimal conversion conditions are a substrate concentration of 200 g / L and an enzyme addition of 40%-45%.

[0096] Experimental Example 2

[0097] This experiment verifies the effect of temperature on product purity during the alkali adjustment and crystallization process during product extraction, as follows:

[0098] Table 2 Effect of temperature on product purity during alkali adjustment and crystallization during product extraction

[0099] Group Maintain temperature (℃) purity(%) 1 0 99.5 2 5 99.4 3 10 99.6 4 15 96.3 5 20 94.5

[0100] As can be seen from Table 2, if the temperature is too high during the alkali adjustment crystallization process, salt will be introduced into the crystallization, resulting in unqualified product purity. Therefore, the crystallization process needs to be carried out at a low temperature (0-10°C).

[0101] Experimental Example 3

[0102] In this experiment, the amplification reaction was carried out under the conditions of 200g / L substrate concentration and 45% ketoreductase dosage:

[0103] According to the 3L enzyme reaction system, 600g of substrate 3-dimethylamino-1-(thienyl)-1-propanone hydrochloride was added to 1.5L of water first, and then 660g of glucose was added. The mixture was stirred and heated to 30°C to slowly dissolve. 30% sodium hydroxide solution was used to adjust the pH to about 6.0.

[0104] Then, 1350 mL of ketoreductase crushing enzyme solution (20% enzyme solution concentration, 45% enzyme addition amount) and 120 mL of glucose reductase crushing enzyme solution (5% enzyme addition amount) were added, 0.6 g of zinc acetate, 0.3 g of coenzyme NADP+, and 30% sodium hydroxide were added to adjust and maintain pH = 5.8-6.0, and the reaction was controlled in the liquid phase to more than 98% to terminate the reaction.

[0105] The reaction solution was adjusted to pH = 4 with hydrochloric acid, and 60 g of diatomaceous earth was added. After stirring for 30 minutes, the filter cake layer was stirred and washed twice with 200 mL of 30°C warm water and filtered. The filtrate was passed through an organic membrane to remove protein. The organic membrane filtrate was adjusted to pH = 11-12 with a saturated sodium hydroxide solution, and the target product (3S)-N,N-dimethyl-3-hydroxy-3-(-2-thiophene)-1-propylamine was obtained by filtration. 471.4 g of the product was obtained with a purity of 99.47% and a chiral purity of 99.9%.

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a duloxetine intermediate, characterized in that: Using 3-dimethylamino-1-(thienyl)-1-propanone hydrochloride as a substrate and NADPH as a coenzyme, ketoreductase is used to convert it into the target product; The ketoreductase is selected from any one of the nucleotide sequences shown in SEQ ID NOs: 1-16.

2. The preparation method according to claim 1, characterized in that: In the reaction system, the concentration of the substrate is 140-220 g / L.

3. The preparation method according to claim 1, characterized in that: The ketoreductase is obtained by expression of recombinant bacteria; in the reaction system, the addition amount of the recombinant bacteria expressing the ketoreductase is 35-55% of the substrate mass.

4. The preparation method according to claim 3, characterized in that: In the reaction system, the concentration of the substrate is 200 g / L, and the amount of the recombinant bacteria expressing the ketoreductase added is 40-45% of the substrate mass.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The nucleotide sequence of the ketoreductase is shown in SEQ ID NO:

16.

6. The preparation method according to claim 5, characterized in that: include: Dissolving the substrate and glucose in water, adjusting the pH and temperature, and stirring to obtain a mixed solution; Adding the recombinant bacteria expressing the ketoreductase and glucose dehydrogenase, zinc acetate and coenzyme NADP+ to the mixed solution, controlling the temperature and pH, and performing a catalytic reaction; Preferably, the pH of the mixed solution is 5.5-6.5 and the temperature is 30°C; Preferably, the conditions of the catalytic reaction are: temperature of 25°C-30°C, pH of 5.8-6.0, and reaction time of 15-24h.

7. The preparation method according to claim 6, characterized in that: In the reaction system, the amount of glucose is 1-1.2 times the amount of substrate, the concentration of zinc acetate is 0.15-0.35 g / L, and the concentration of coenzyme NADP+ is 0.05-0.2 g / L.

8. The preparation method according to claim 7, characterized in that: The preparation method further comprises extracting the product after the reaction is completed; The extraction comprises: adjusting the pH of the reaction solution, passing it through a ceramic membrane or diatomaceous earth, and then ultrafiltration; cooling and crystallizing the filtrate obtained after ultrafiltration; Preferably, the pH of the reaction solution is 3-4.5 when passing through the ceramic membrane; Preferably, the ultrafiltration conditions are: filtrate temperature <10°C, pH = 11-12.

9. The preparation method according to claim 8, characterized in that: When performing cooling crystallization treatment, the temperature is maintained at 0-10°C; Preferably, the temperature is maintained at 0-5°C.

10. The preparation method according to claim 9, characterized in that: The preparation method further comprises dissolving the crystallized product with twice the weight of dichloromethane when the crystallized product obtained after cooling crystallization contains salt, separating and dehydrating the liquid, and concentrating the organic phase to obtain a refined product.

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