Preparation method of rotigotine

Abstract

The invention provides a method for preparing rotigotine, and relates to the fields of biotechnology and enzyme engineering. A method for preparing rotigotine comprises the following steps: a) reacting 5-methoxy-2-tetralone with 2-thienylethylamine under the action of a reductive amination enzyme to obtain compound 1; b) performing N-alkylation of compound 1 with a halogenated propane in a solvent to obtain compound 2; c) deprotecting compound 2 under the action of a boronating agent to obtain rotigotine compound 3. The invention utilizes an enzyme catalytic method for amination, and then performs N-alkylation to obtain rotigotine, the reductive amination enzyme has good substrate tolerance, no difficult-to-remove solvent is added in the reaction, the post-processing operation is simple, and it is conducive to industrial production.

Description

Technical Field

[0001] The invention relates to the fields of biotechnology and enzyme engineering, and in particular to a method for preparing rotigotine. Background Art

[0002] Rotigotine was developed by Schwarz Biosciences of Germany and Aderis of the United States. It is used as an adjuvant treatment for early secondary Parkinson's disease and late Parkinson's disease. Its trade name is Neupro. In May 2007, the US FDA approved its marketing. Neupro is the first transdermal patch used to treat Parkinson's syndrome.

[0003] There are several methods for preparing rotigotine molecules:

[0004] The first method is the chiral auxiliary / chiral source synthesis method: Chinese patent CN109574983 reported that 5-methoxy-2-tetralone was used as the raw material, followed by tert-butylsulfenamidation and reduction, bromopropane alkylation, hydrochloric acid deprotection, docking reduction with 2-(2-bromoethyl)thiophene, and finally demethylation with hydrobromic acid to obtain rotigotine. The invention route is too long, the operation is cumbersome, and a chiral catalyst is used, but the conversion rate is low, the cost is expensive, and it is not conducive to industrial production. The specific route is as follows:

[0005]

[0006] The second method is chiral separation: Chinese patent CN113234057 reported that 5-methoxy-2-tetralone was used as the raw material, and was sequentially aminated with R-α-methylbenzylamine, debenzylated, chiral separated by S-mandelic acid, amidated with propionyl chloride, and then reduced with sodium borohydride, and finally reacted with 2-(thiophen-2-yl) 2-nitrobenzenesulfonic acid ethyl ester to obtain rotigotine. This invention has a total of 7 steps, using expensive Pd as a catalyst and using highly toxic boron trifluoride etherate, in which chiral separation was also performed, requiring multiple purifications, with extremely low yields, high costs, and extremely unfriendly environment, which is not conducive to industrialization. The specific route is as follows:

[0007]

[0008] The third method is the asymmetric catalytic method: Chinese patent CN104130238 reported that 5-methoxy-2-tetralone was used as the raw material, amination by n-propylamine, asymmetric reduction of 3-3'-binaphthol phosphoric acid, halogenation of substituted thiophene, and demethoxylation with hydrobromic acid to obtain rotigotine with a yield of 32.8%. This method mostly uses chiral phosphoric acid catalysts, which are expensive, have many reaction steps, and have a low overall yield, which is not suitable for industrial production.

[0009] Summary of the invention

[0010] 1. Technical issues to be resolved

[0011] In view of the deficiencies in the prior art, the present invention provides a method for preparing rotigotine, which solves the technical problems that the prior method for preparing rotigotine has many reaction steps, low overall yield and is not suitable for industrial production.

[0012] (II) Technical solution

[0013] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0014] The present invention provides a method for preparing rotigotine, and its technical route is as follows:

[0015]

[0016] Wherein X is any one of Cl, Br, and I.

[0017] The specific steps are as follows:

[0018] a) 5-methoxy-2-tetralone reacts with 2-thiopheneethylamine under the action of reductive amination enzyme of the recombinant strain to obtain compound 1;

[0019] b) Compound 1 is N-alkylated with a halogenated propane in a solvent to obtain compound 2;

[0020] c) compound 2 is deprotected under the action of a boronating agent to obtain rotigotine compound 3;

[0021] Furthermore, in step a), the reductive aminase is obtained by mutant T67K-I253L, and the mutant T67K-I253L is obtained by single mutation and combined mutation at T67 and I253 sites of mutant RT-L, respectively.

[0022] Furthermore, the host bacteria of the mutant T67K-I253L is Escherichia coli, and the Escherichia coli is Escherichia coli BL21.

[0023] Furthermore, in step a), the reaction system comprises sodium phosphate buffer and a cosolvent, the concentration of the sodium phosphate buffer is 100 mM / L, the pH is 6-9, the cosolvent is isopropyl acetate, the amount of isopropyl acetate accounts for 10-30% of the total volume percentage of the reaction mixture, and the reaction is carried out at 0-50° C. for 1-36 hours to obtain compound 1, preferably the pH of the sodium phosphate buffer is 8, the amount of the cosolvent isopropyl acetate is 15%, and the reaction is carried out at 45° C. for 24 hours.

[0024] Furthermore, in step a), the molar ratio of the substrate 5-methoxy-2-tetralone to the amine donor 2-thiopheneethylamine is 1:1-3, preferably 1:1.2-1.8.

[0025] Furthermore, in step a), the mass volume ratio of the wet weight of the recombinant strain cells having the reductive amination enzyme to the reaction mixture is 5-15 g / L, preferably 7-10 g / L.

[0026] Furthermore, step a) further comprises D-glucose, and the molar ratio of D-glucose to 5-methoxy-2-tetralone is 1-3:1, preferably, 1.2-1.8:1.

[0027] Furthermore, step a) further comprises NADP + , NADP + The dosage is 0.5-1.5mM / L, preferably 0.6-0.9mM / L.

[0028] Furthermore, in step a), the amount of glucose dehydrogenase GDH enzyme powder is 0.5-1.5 mg / L, preferably 0.8-1.1 mg / L.

[0029] Furthermore, in step b), the halogenated propyl ane is one of iodopropyl ane, chloropropyl ane and bromopropyl ane, preferably iodopropyl ane;

[0030] Further, in step b), the solvent is one of acetone and N,N-dimethylformamide (DMF), preferably acetone;

[0031] Furthermore, in step c), the boronating agent is one of boron chloride and boron bromide, preferably boron chloride.

[0032] (III) Beneficial effects

[0033] The present invention provides a method for preparing rotigotine. Compared with the prior art, the method has the following beneficial effects:

[0034] The invention discloses a method for preparing rotigotine, comprising the following steps: a) reacting 5-methoxy-2-tetralone with 2-thienylethylamine under the action of a reductive amination enzyme to obtain a compound 1; b) performing N-alkylation on the compound 1 and a halogenated propane in a solvent to obtain a compound 2; and c) deprotecting the compound 2 under the action of a boronating agent to obtain a rotigotine compound 3. Amination is performed by an enzyme catalysis method, and then N-alkylation is performed to obtain rotigotine. The reductive amination enzyme has a high substrate tolerance concentration, no difficult-to-remove solvent is added in the reaction, and the post-treatment operation is simple, which is conducive to industrial production. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with specific implementation methods.

[0037] Example 1

[0038] This example provides a method for obtaining the mutant T67K-I253L, which specifically includes the following steps:

[0039] The present invention selects 5-methoxy-2-tetralone and 2-thienylethylamine as substrates and amine donors, and uses DiscoveryStudio technology to dock with the active site of the reductive amination enzyme mutant RT-L (amino acid sequence shown in SEQ ID NO: 2, and the nucleotide sequence encoded by it is shown in SEQ ID NO: 1), analyzes the interaction of the docking site, and performs single mutation and combined mutation, wherein T67 is mutated to lysine or alanine, and the selectivity is 85% and 75% respectively, and I253 is mutated to leucine or threonine, and the selectivity is 84% ​​and 80% respectively. The mutant T67K-I253L obtained by combined mutation has a selectivity of 99% (amino acid sequence shown in SEQ ID NO: 4, and the nucleotide sequence encoded by it is shown in SEQ ID NO: 3).

[0040] SEQ ID NO: 1 is as follows:

[0041] atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcgcggcagccatatgccggaat ctaccacccc gagtaccgcc accccggtga ccatcatcggtcttggtgcaatgggcaccg ccctggcaaa cgcattcctc gatgcaggtc atagtaccaccgtttggaatcgtaccgcag cacgcgccac cgcattagcc gcacgcggcg cacatcatgcagaaaccgtgaccgaagcca ttgcagcctc tccgttagtg attgcctgtg tgctggattatgatgcctttcatgaaacct tagccccggc tacagacgcg ctggcaggtc gcgccctggttaatctgaccacaggtaccc cgaaacaggc acgcctaacc gcctcttggg cagccgatcatcgtattgattatctggatg gcaaaattat ggccattccg ccgggtattg caaccccggatagttttattctgtatagcg gtccgttagg tacctttgaa gcacatcgct caaccttagaagtgctgggcgcagcaaatc atgtgggtac cgatgcaggt ttggcgagct tacatgatattgcactgctgaccggtatgt atggcatgat tgcaggcatt ttacaggcct ttgccttaattgatagtgaaggtattccgg caggcgatct ggccccgatg ttaaccaatt ggttaaccggcgcagcacatagcgtggccc attatgccca gcagattgat accggcgatt atgaaaccggtgttgtgtttaatttagcac atcagagcca tggctttgca aaattagttc aggccggtgaagatcagggtgtggatgtgg gcttactgcg tccgctgttt gaactgatgc gtcatcaggttgccgcaggctatggtaatg gtgatgttgc ctcagttattgaactgattc gtcgcgaagaacgtcgtcagccggccaaaa gtccgggcgc agataaaatt acccgtgcac gtcgtccgta a;

[0042] SEQ ID NO: 2 is as follows:

[0043] MGSSHHHHHH SSGLVPRGSH MPESTTPSTA TPVTIIGLGAMGTALANAFL DAGHSTTVWNRTAARATALA ARGAHHAETVTEAIAASPLV IACVLDYDAF HETLAPATDA LAGRALVNLTTGTPKQARLTASWAADHRID YLDGKIMAIP PGIATPDSFILYSGPLGTFE AHRSTLEVLG AANHVGTDAGLASLHDIALLTGMYGMIAGI LQAFALIDSE GIPAGDLAPM LTNWLTGAAHSVAHYAQQID TGDYETGVVFNLAHQSHGFA KLVQAGEDQGVDVGLLRPLF ELMRHQVAAG YGNGDVASVI ELIRREERRQPAKSPGADKITRARRP;

[0044] SEQ ID NO: 3 is as follows:

[0045] atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcgcggcagccatatgccggaat ctaccacccc gagtaccgcc accccggtga ccatcatcggtcttggtgcaatgggcaccg ccctggcaaa cgcattcctc gatgcaggtc atagtaccaccgtttggaatcgtaccgcag cacgcgccaa agcattagcc gcacgcggcg cacatcatgcagaaaccgtgaccgaagcca ttgcagcctc tccgttagtg attgcctgtg tgctggatta tgatgcctttcatgaaaccttagccccggc tacagacgcg ctggcaggtc gcgccctggt taatctgaccacaggtaccccgaaacaggc acgcctaacc gcctcttggg cagccgatca tcgtattgattatctggatggcaaaattat ggccattccg ccgggtattg caaccccgga tagttttatt ctgtatagcggtccgttaggtacctttgaa gcacatcgct caaccttaga agtgctgggc gcagcaaatcatgtgggtaccgatgcaggt ttggcgagct tacatgatat tgcactgctg accggtatgt atggcatgattgcaggcattttacaggcct ttgccttaat tgatagtgaa ggtattccgg caggcgatct ggccccgatgttaaccaattggttaaccgg cgcagcacat agcgtggccc attatgccca gcagattgataccggcgcttatgaaaccgg tgttgtgttt aatttagcac atcagagcca tggctttgca aaattagttcaggccggtgaagatcagggt gtggatgtgg gcttactgcg tccgctgttt gaactgatgcgtcatcaggttgccgcaggc tatggtaatg gtgatgttgc ctcagttattgaactgattcgtcgcgaagaacgtcgtcag ccggccaaaa gtccgggcgc agataaaatt acccgtgcac gtcgtccgtaa;

[0046] SEQ ID NO: 4 is as follows:

[0047] MGSSHHHHHH SSGLVPRGSH MPESTTPSTA TPVTIIGLGAMGTALANAFL DAGHSTTVWNRTAARAKALA ARGAHHAETVTEAIAASPLV IACVLDYDAF HETLAPATDA LAGRALVNLTTGTPKQARLTASWAADHRID YLDGKIMAIP PGIATPDSFILYSGPLGTFE AHRSTLEVLG AANHVGTDAGLASLHDIALLTGMYGMIAGI LQAFALIDSE GIPAGDLAPM LTNWLTGAAHSVAHYAQQID TGLYETGVVFNLAHQSHGFA KLVQAGEDQGVDVGLLRPLF ELMRHQVAAG YGNGDVASVI ELIRREERRQPAKSPGADKITRARRP.

[0048] Example 2

[0049] This embodiment provides a method for preparing bacterial mud, which specifically includes the following steps:

[0050] A recombinant plasmid was constructed using plasmid pET28a and mutant T67K-I253L gene, and transformed into Escherichia coli BL21 by electroporation for expression. A single clone was picked and cultured overnight at 37°C and 220rpm. The bacterial solution was inoculated into LB medium containing 50 μg / mL kanamycin at an inoculum size of 1:100, and cultured at 37°C and 220rpm until the OD600 value was 0.7-1. IPTG (0.2 mM / L) was added to the culture medium to induce gene expression, and the culture was continued at 25°C and 180rpm for 16 hours. The bacterial sludge was then harvested by centrifugation at 5000rpm for 20 minutes, and the bacterial cells were collected after washing twice with sodium phosphate buffer (100 mM / L, pH8) by centrifugation at 5000rpm for 20 minutes.

[0051] Example 3

[0052] This embodiment provides a method for preparing rotigotine, which specifically comprises the following steps:

[0053] a) In a 1 mL reaction system, 5-methoxy-2-tetralinone (50 mM, 1 equivalent), 2-thiopheneethylamine (75 mM / L, 1.5 equivalents), the bacterial sludge in Example 2 (the wet weight of the recombinant strain cells accounted for 8 g / L of the total reaction mixture), D-glucose (1.5 times the ketone equivalent), and NADP were added. + (0.7mM / L), glucose dehydrogenase GDH enzyme powder (0.9mg / mL), sodium phosphate buffer concentration of 100mM / L (pH 8.0) and 15% isopropyl acetate, the mixed solution was reacted at 45°C and 220rpm for 24h, the reaction solution was quenched and diluted with acetonitrile and then centrifuged to obtain the supernatant. The results of HPLC analysis showed that the conversion rate was 97.5% and the ee value was >99%.

[0054] After 24 hours of reaction, the mixture was filtered through a Buchner funnel with diatomaceous earth and extracted with isopropyl acetate (10 mL×3). The solvent was concentrated under reduced pressure to obtain compound 1. The conversion rate, yield and ee of the reaction were 97.5%, 95% and 99.6%, respectively.

[0055] b) Under nitrogen protection, compound 1 (50 mg, 0.17 mmol), iodopropane (34.6 mg, 0.21 mmol), acetone (5 mL), and sodium carbonate (21.6 mg, 0.20 mmol) were added to a three-necked flask, heated to reflux, reacted overnight, and the solvent was concentrated. 20 ml of dichloromethane was added, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain a yellow oil compound 2, weighing 56.2 mg, with a yield of 98% and a purity of 98.5%, which was directly used in the next step.

[0056] c) Compound 2 (50 mg, 0.15 mmol) and dichloromethane (5 mL) were added to a round-bottom flask, the temperature was lowered to 5°C, 30% boron trichloride-dichloromethane solution (117 mg, 0.30 mmol) was added dropwise, and the mixture was reacted at 25°C for 1 hour. After the reaction, 0.1 mol / L sodium citrate aqueous solution was added for washing, and the mixture was extracted with dichloromethane 15 mL×3. The organic phases were combined, the solvent was concentrated, and n-heptane was added for slurry washing. The mixture was filtered to obtain compound 3, weighing 45 mg, with a yield of 95% and a purity of 99.2%.

[0057] Example 4

[0058] This embodiment provides a method for preparing rotigotine, which specifically comprises the following steps:

[0059] a) In a 10 mL reaction system, 5-methoxy-2-tetralinone (50 mM, 1 equivalent), 2-thiopheneethylamine (60 mM / L, 1.2 equivalent), the bacterial sludge in Example 2 (the cell wet weight to total reaction mixture mass volume ratio is 10 g / L), D-glucose (1.8 times the ketone equivalent), NADP + (0.9mM / L), GDH enzyme powder (1.1mg / mL), sodium phosphate buffer with a concentration of 100mM / L (pH 8.0) and 15% isopropyl acetate, the mixed solution was reacted at 45°C and 220rpm for 24h, the reaction solution was quenched and diluted with acetonitrile and then centrifuged to obtain the supernatant. The results of HPLC analysis showed that the conversion rate was 96.8% and the ee value was >99%.

[0060] After 24 hours of reaction, the mixture was filtered through a Buchner funnel with diatomaceous earth and extracted with isopropyl acetate (50 mL×3). The solvent was concentrated under reduced pressure to obtain compound 1. The conversion rate, yield and ee of the reaction were 96.8%, 94.2% and >99.4%, respectively.

[0061] b) Under nitrogen protection, compound 1 (287.4 mg, 1 mmol), bromopropane (246 mg, 2 mmol), DMF (20 mL), and sodium carbonate (212 mg, 2 mmol) were added to a three-necked flask, the temperature was controlled at 90°C, the reaction was allowed to proceed overnight, and the mixture was concentrated at 60°C until there was substantially no fraction. 60 ml of dichloromethane was added, the mixture was washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain compound 2 as a yellow oil, weighing 320 mg, with a yield of 97.2% and a purity of 98.1%, which was directly used in the next step.

[0062] c) Compound 2 (264 mg, 0.8 mmol) and dichloromethane (20 mL) were added to a round-bottom flask, the temperature was lowered to 0°C, 30% boron trichloride-dichloromethane solution (624 mg, 1.6 mmol) was added dropwise, and the mixture was reacted at 25°C for 1 hour. After the reaction, 0.1 mol / L sodium citrate aqueous solution was added for washing, and the mixture was extracted with dichloromethane 25 mL×3. The organic phases were combined, the solvent was concentrated, and n-heptane was added for slurry washing. The mixture was filtered to obtain compound 3, weighing 237 mg, with a yield of 94% and a purity of 99%.

[0063] Example 5

[0064] This embodiment provides a method for preparing rotigotine, which specifically comprises the following steps:

[0065] a) In a 100 mL reaction system, 5-methoxy-2-tetralinone (50 mM, 1 equivalent), 2-thiopheneethylamine (75 mM / L, 1.5 equivalents), the bacterial sludge in Example 2 (the cell wet weight to total reaction mixture mass volume ratio is 8 g / L), D-glucose (1.5 times the ketone equivalent), NADP + (0.7mM / L), GDH enzyme powder (0.9mg / mL), sodium phosphate buffer with a concentration of 100mM / L (pH 8.0) and 15% isopropyl acetate, the mixed solution was reacted at 45°C and 220rpm for 24h, the reaction solution was quenched and diluted with acetonitrile and then centrifuged to obtain the supernatant. The results of HPLC analysis showed that the conversion rate was 97.5% and the ee value was >99%.

[0066] After 24 hours of reaction, the mixture was filtered through a Buchner funnel with diatomaceous earth and extracted with isopropyl acetate (100 mL×3). The solvent was concentrated under reduced pressure to obtain compound 1. The conversion rate, yield and ee of the reaction were 97.2%, 95.0% and 99.2%, respectively.

[0067] b) Under nitrogen protection, compound 1 (28.7 g, 0.1 mol), chloropropane (19.6 g, 0.25 mol), acetone (100 mL), and sodium carbonate (15.9 g, 0.15 mol) were added to a three-necked flask, heated to reflux, reacted overnight, and the solvent was concentrated. 100 ml of dichloromethane was added, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain a yellow oil compound 2, weighing 31.4 g, with a yield of 95.4% and a purity of 98.5%, which was directly used in the next step.

[0068] c) Compound 2 (16.5 g, 0.05 mol) and dichloromethane (100 mL) were added to a round-bottom flask, the temperature was lowered to 5°C, 30% boron tribromide-dichloromethane solution (83.5 g, 0.1 mol) was added dropwise, and the mixture was reacted at 25°C for 1 hour. After the reaction, 0.1 mol / L sodium citrate aqueous solution was added for washing, and the mixture was extracted with dichloromethane 75 mL×3. The organic phases were combined, the solvent was concentrated, and n-heptane was added for slurry washing. The mixture was filtered to obtain compound 3, weighing 14.9 g, with a yield of 94.7% and a purity of 99.2%.

[0069] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing rotigotine, characterized in that: The steps include: a) 5-methoxy-2-tetralone reacts with 2-thiopheneethylamine under the action of reductive amination enzyme to obtain compound 1; b) Compound 1 is N-alkylated with a halogenated propane in a solvent to obtain compound 2; c) Compound 2 is deprotected under the action of a boronating agent to obtain rotigotine compound 3.

2. The preparation method according to claim 1, characterized in that The reductive aminase is obtained through the mutant T67K-I253L, and the mutant T67K-I253L is obtained by subjecting the mutant RT-L to single mutation and combined mutation at the T67 and I253 sites, respectively.

3. The preparation method according to claim 2, characterized in that: The host bacteria of the mutant T67K-I253L is Escherichia coli, and the Escherichia coli is Escherichia coli BL21.

4. The preparation method according to claim 1, characterized in that: Step a) satisfies at least one of the following conditions: Step a) further comprises D-glucose, and the molar ratio of D-glucose to 5-methoxy-2-tetralone is 1-3:1, preferably 1.2-1.8:1; Step a) further comprises NADP+, wherein the amount of NADP+ is 0.5-1.5 mM / L, preferably 0.6-0.9 mM / L; Step a) further comprises glucose dehydrogenase, wherein the amount of glucose dehydrogenase is 0.5-1.5 mg / L, preferably 0.8-1.1 mg / L; Step a) further comprises a sodium phosphate buffer having a concentration of 100 mM / L and a pH of 6-9; Step a) further comprises a co-solvent, which is isopropyl acetate, and the volume of the co-solvent accounts for 10-30% of the total volume of the reaction mixture.

5. The preparation method according to claim 1, characterized in that: The step a) satisfies at least one of the following conditions: The molar ratio of 5-methoxy-2-tetralone to 2-thiopheneethylamine is 1:1-3, preferably 1:1.2-1.8; Step a) The reaction temperature is 0-50°C and the reaction time is 1-36 hours to obtain compound 1.

6. The preparation method according to claim 1, characterized in that: The step b) satisfies at least one of the following conditions: The halogenated propane is selected from any one of iodopropane, chloropropane and bromopropane, preferably iodopropane; The solvent is selected from any one of acetone and N,N-dimethylformamide, preferably acetone.

7. The preparation method according to claim 1, characterized in that: In the step c), the boronating agent is selected from any one of boron chloride and boron bromide, preferably boron chloride.

8. The preparation method according to claim 1, characterized in that: In step a), the mass volume ratio of the wet weight of the recombinant strain cells having reductive amination enzyme to the reaction mixture is 5-15 g / L, preferably 7-10 g / L.

Images