Edoxaban intermediate and its manufacturing method

Abstract

The present invention discloses an edoxaban intermediate and a method for preparing the same. The general structural formula of the edoxaban intermediate is: [Formula 1] TIFF2024543705000030.tif27170 or [Case 2] TIFF2024543705000031.tif27170, where R1 is OH, an alkoxy group or an N,N-dialkyl group, and R2 is hydrogen, an alkoxycarbonyl group or an amino protecting group. [C3] and mixing the compound of formula II, transaminase, transaminase coenzyme and phosphate buffer solution for enzyme catalysis or further amine derivatization to obtain the compound of formula I. Compared with the chemical synthesis methods of the prior art, the key intermediate of edoxaban and the preparation method thereof provided by the present invention have a novel structure, mild reaction conditions, high yield and good industrial value.

Description

[Technical Field]

[0001] The present invention relates to the technical field of biocatalysis, and more particularly to a key intermediate of edoxaban and an enzyme-catalyzed method for its preparation. [Background technology]

[0002] Edoxaban p-toluenesulfonate monohydrate, developed by Daiichi Sankyo Co., Ltd., is a small molecule drug and a blood coagulation factor Xa inhibitor. Currently, the drug is undergoing final R&D to obtain marketing approval. It treats pulmonary embolism, venous thromboembolism, stroke, venous thrombosis, and embolism. On April 22, 2011, edoxaban toluenesulfonate was approved by the Pharmaceuticals and Medical Devices Agency (PMDA) and is marketed by Daiichi Sankyo Co., Ltd. On January 8, 2015, edoxaban toluenesulfonate was approved by the U.S. Food and Drug Administration (FDA) and is marketed by Daiichi Sankyo Inc. under the trade name Savaysa® (NDA 206316). On June 19, 2015, edoxaban toluenesulfonate was approved by the European Medicines Agency (EMA) and is marketed by Daiichi Sankyo Europe GmbH under the trade name Lixiana®. (EMEA / H / C / 002629) On December 25, 2018, edoxaban toluenesulfonate was approved by the China National Medical Products Administration (NMPA) and is sold by Daiichi Sankyo Europe GmbH under the trade name Lixianan®.

[0003] Edoxaban has three chiral centers and has a total of eight isomers, but only one configuration has good activity. The structure of edoxaban is as follows: [ka]

[0004] The synthetic route developed by Daiichi Sankyo Co., Ltd. and disclosed in WO2008156159A1 is as follows: Racemic compound S-1 was decomposed in the presence of chiral amine S-2, followed by intramolecular nucleophilic attack under the action of an electrophilic bromination reagent to obtain bridged ring compound S-4. S-4 was then aminolyzed and ring-opened via propylene oxide intermediate S-6 under the action of NH4OH to obtain amino alcohol compound S-7. S-7 was then protected with Boc, the alcohol hydroxyl group was protected with Ms, and the N3 group was introduced by attack with NaN3 to obtain compound S-10. The amino group was then obtained by hydrogenation reduction, which was then condensed with S-13 to obtain compound S-14. Compound S-14 was then condensed with carboxylic acid S-16 under acidic conditions to obtain the final compound S-17, which could be converted into a TsOH salt to obtain edoxaban. The synthetic route for this process is as follows: [ka]

[0005] The difficulty in synthesizing this compound is the preparation of chiral substrates such as compound S-10.

[0006] In the synthetic route disclosed by Daiichi Sankyo Co., Ltd. in Patent CN101263110, the substitution of methanesulfonate with sodium azide produces more than 10% racemate. The synthetic route for this process is as follows: [ka]

[0007] Daiichi Sankyo Co., Ltd. has published the following in CN105008325A, DOI: 10.1021 / acs.oprd.8b00413. The process route was optimized using Burgess reagent, avoiding the racemic issue of the first-generation route and improving yield by 20%. However, the reagent generates significant heat during use and has a significant amplification effect during the amplification process. The synthetic route for this process is as follows: [ka]

[0008] CN106866452 discloses a method for constructing an amino chiral center by chemical methods, which is difficult to reproduce. The synthetic route for this process is as follows: [ka] Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a method for producing a key intermediate of edoxaban by an enzyme catalysis.

[0010] In order to solve the above problems, the present invention uses the following technical means.

[0011] Edoxaban intermediates have the general structural formula represented by Formula I or Formula II. [ka] [ka] wherein R1 is OH, an alkoxy group, or an N,N-dialkyl group, and R2 is hydrogen, an alkoxycarbonyl group, or other amino-protecting group.

[0012] Preferably, in the general structural formula, R1 is N,N-dimethyl or ethoxy group, and R2 is hydrogen, tert-butoxycarbonyl group, benzyloxycarbonyl group, ethoxycarbonyl group, benzyl group, or benzoyl group.

[0013] The present invention further provides a method for producing the above-mentioned edoxaban intermediate, which comprises the following steps 1) and 2):

[0014] In step 1), a compound of formula II is obtained by oxidizing a compound of formula III. [ka]

[0015] The process route for step 1) is as follows: [ka]

[0016] In step 2), the compound of formula II, transaminase, transaminase coenzyme and phosphate buffer solution are mixed together to undergo an enzyme-catalyzed reaction or further an amine derivatization reaction, after which the compound of formula I is obtained.

[0017] The process route for step 2) is as follows: [ka]

[0018] Preferably, the oxidizing agent in step 1) is John's reagent, PCC reagent or TEMPO reagent, preferably TEMPO reagent.

[0019] Preferably, the transaminase in step 2) is selected from the transaminase library of Shangke Biotechnology (Shanghai) Co., Ltd.

[0020] Preferably, in the system of step 2), the substrate concentration is 10 to 100 g / L, the transaminase is involved in the reaction in the form of one or a combination of wet cells and liquid enzymes, and when the transaminase is added in the form of a liquid enzyme, the mass ratio of the liquid enzyme is 1 to 20%, the reaction temperature is 0 to 30°C, the reaction pH value is 6.5 to 7.0, and the reaction time is 12 to 30 hours.

[0021] Preferably, in step 2), the concentration of the compound of formula II is 10 to 100 g / L, the concentration of the transaminase is 5 to 20 g / L, the concentration of the transaminase coenzyme is 1 to 10 mg / L, and the concentration of the phosphate buffer is 10 to 100 mM.

[0022] Preferably, after obtaining the amine product by the amine derivatization reaction in the step 2), the characteristic reaction of the amine functional group protecting group is to directly add a protecting group reagent to the reaction solution of the enzyme-catalyzed reaction and carry out the reaction.

[0023] Preferably, the method further comprises a post-treatment step of adding a filter aid to the reaction solution after the reaction of step 2), and then subjecting the solution to filtration, extraction, concentration and crystallization to obtain the compound of formula I.

[0024] More preferably, the filter aid is diatomaceous earth.

[0025] The process route of the present invention is as follows: [ka]

[0026] Furthermore, the compound of formula III can be prepared by the following method. [ka]

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The enzyme-catalyzed method for preparing the compound of formula I provided by the present invention can achieve high yields and chiral purity, requires a small amount of environmentally friendly organic solvent, has mild reaction conditions, and is simple in process operation, making it suitable for industrial production.

[0029] In order to further confirm the chiral configuration of the amino chiral center constructed by the transaminase in the key step described in the present invention, the compound of formula I-2 obtained is derivatized as follows to obtain the compound of formula V, and the single crystal X-ray diffraction pattern of the compound of formula V is obtained experimentally to further confirm that the absolute configuration of the key intermediate obtained in the present invention is correct. [ka] [Brief explanation of the drawings]

[0030] [Figure 1] HPLC spectrum of the compound of formula V ((1R,2S,5S)-2-(((benzyloxy)carbonyl)amino)-5-(dimethylcarbamoyl)cyclohexyl)-14-azocarboxylic acid tert-butyl ester. [Figure 2] HPLC spectra of the compound of formula V ((1R,2S,5S)-2-(((benzyloxy)carbonyl)amino)-5-(dimethylcarbamoyl)cyclohexyl)-14-azocarboxylic acid tert-butyl ester and its chiral isomers. [Figure 3] 1 is a single crystal X-ray diffraction spectrum of the compound of formula V ((1R,2S,5S)-2-(((benzyloxy)carbonyl)amino)-5-(dimethylcarbamoyl)cyclohexyl)-14-azocarboxylic acid tert-butyl ester). DETAILED DESCRIPTION OF THE INVENTION

[0031] In order that the present invention may be more clearly understood, a preferred embodiment will now be described in detail with reference to the drawings.

[0032] Example 1 Preparation of (1S,4S,5S)-4-bromo-6-oxabicyclo[3.2.1]octan-7-one (IV) [ka] (S)-3-Cyclohexenecarboxylic acid (126.2 g, 1.0 mol), 200 mL of water, and potassium hydroxide (41 g, 1.05 mol) were added sequentially to a reaction flask. The reaction mixture was stirred and cooled to approximately -10 °C. NBS (196 g, 1.1 mol) was gradually added. After the addition was complete, the reaction mixture was maintained at approximately -5 to 5 °C and stirred for 1 hour. The reaction mixture was monitored by TLC. After the reaction was complete, sodium sulfite (25.2 g, 0.2 mol) was added and stirred for 0.5 hours. The mixture was then filtered to obtain 198.8 g of compound III in 97% yield. The proton nuclear magnetic resonance spectral data of the resulting product was as follows:

[0033] 1 H NMR(400 MHz,Chloroform-d)δ4.79(d,J=5.1 Hz,1H),4.39(d,J=4.7 Hz,1H),2.65(d,J=12.3 Hz,2H),2.46-2.35(m,1H),2.35-2.27(m,1H),2.13(dd,J=16.4,5.4 Hz,1H),1.93(dt,J=12.9,5.5,2.2 Hz,1H),1.84(dt,J=12.1,11.0,4.9,3.1 Hz,1H).

[0034] A series of one-pot methods for preparing compound III are exemplified where R is OCH2CH3 and N(CH3)2.

[0035] Example 2 Preparation of (1S,3R,4R)-3-azido-4-hydroxycyclohexane-1-carboxylic acid ethyl ester (III-1) [ka] Compound IV (20.5 g, 0.1 mol) and 20 mL of ethanol were added to a 100 mL three-neck flask and cooled to 0-5 ° C. Sodium ethoxide (10.2 g, 0.15 mol) was gradually added to the reaction mixture, and the mixture was stirred for 1-2 hours while maintaining the temperature at 0-5 ° C. 30 mL of water and sodium azide (9.7 g, 0.15 mol) were added to the reaction mixture, and the mixture was heated to 15-20 ° C. and stirred for 3 hours. 50 mL of dichloromethane was added and the mixture was separated. The dichloromethane layer was concentrated under reduced pressure to remove the dichloromethane, and compound III-1 (18.7 g) was obtained in an 88.4% yield. The proton nuclear magnetic resonance spectral data of the obtained product are as follows:

[0036] 1 H NMR(400 MHz,CDCl3)δ4.16(dd,J=7.2,1.2 Hz,2H),3.52(dt,J=24.7,8.5,4.0 Hz,2H),2.72(d,J=4.8 Hz,1H),2.59-2.48(m,1H),2.36(dt,J=14.0,4.4 Hz,1H),2.14-2.05(m,1H),1.95-1.85(m,1H),1.63-1.46(m,3H),1.26(dt,J=8.9,5.1,1.4 Hz,3H).

[0037] Example 3 Preparation of (1S,3R,4R)-3-azido-4-hydroxy-N,N-dimethylcyclohexane-1-carboxamide (III-2) [ka] Compound IV (20.5 g, 0.1 mol) and 20 mL of acetonitrile were added to a 250 mL three-neck flask and cooled to 0-5 °C. 40% aqueous dimethylamine solution (16.8 g, 0.15 mol) was added, and the mixture was stirred at 0-5 °C for 10-11 hours. Dimethylamine and acetonitrile were removed by distillation under reduced pressure. 30 mL of water and sodium hydroxide (6 g, 0.15 mol) were added, and the mixture was stirred at 0-10 °C for 5-6 hours. Sodium azide (9.7 g, 0.15 mol) was weighed and added to the mixture. The mixture was heated to 15-20 °C and stirred for 3-4 hours. 50 mL of dichloromethane was added and the mixture was separated. The dichloromethane layer was concentrated under reduced pressure to remove the dichloromethane, yielding compound III-1 (19.1 g). The yield was 90.1%. The proton nuclear magnetic resonance spectral data of the resulting product are as follows:

[0038] 1 HNMR(400 MHz,CDCl3)δ3.98(dd,J=10.0,6.0 Hz,1H),3.64(s,1H),3.43(s,1H),3.03(s,3H),2.91(s,3H),2.88(d,J=3.9 Hz,1H),2.23-2.09(m,1H),1.95-1.77(m,2H),1.76-1.65(m,1H),1.54(dt,J=16.4,5.8 Hz,2H). 13 C NMR (101 MHz, CDCl3) δ174.93,69.31,62.25,37.34,35.70,34.58,29.01,28.11,23.48.

[0039] Example 4 Preparation of (1S,3R)-4-azido-4-oxocyclohexane-1-carboxylic acid ethyl ester (II-1) [ka] Compound III-1 (2.1 g, 0.01 mol), sodium bicarbonate (1.9 g, 0.02 mol), sodium bromide (0.1 g, 0.001 mol), and TEMPO (0.08 g, 4%, w / w) were added to a 100 mL three-neck flask, and 15 mL of dichloromethane was added and stirred to dissolve. The temperature was lowered to -5 to 0 °C. Aqueous sodium hypochlorite solution (15.8 g, 0.015 mol) was weighed and slowly added dropwise to the reaction system, maintaining the temperature below 0 °C. After the addition was complete, the mixture was stirred for 0.5 to 1.0 hours, and then allowed to stand for separation. The dichloromethane layer was concentrated under reduced pressure to remove the solvent, yielding compound II-1 (1.9 g) in 90.5% yield. The proton nuclear magnetic resonance spectral data of the resulting product are as follows:

[0040] 1 H NMR(400 MHz,CDCl3)δ4.16(dd,J=7.2,1.2 Hz,2H),3.9(d,J=4.8 Hz,1H),2.72(d,J=4.8 Hz,1H),2.36(dd,J=14.0,4.4 Hz,1H),2.14-2.05(m,1H),1.95-1.85(m,1H),1.63-1.46(m,3H),1.26(dd,J=8.9,5.1,1.4 Hz,3H). 13 C NMR (125 MHz, CDCl3) δ209.75,174.15,60.85,59.37,39.24,36.44,34.75,27.08,14.26.

[0041] Example 5 Preparation of (1S,3R)-3-azido-N,N-dimethyl-4-oxocyclohexane-1-carboxamide (II-2) [ka] Compound III-2 (2.1 g, 0.01 mol), sodium bicarbonate (1.9 g, 0.02 mol), sodium bromide (0.1 g, 0.001 mol), and TEMPO (0.08 g, 4%, w / w) were added to a 100 mL three-neck flask, and 15 mL of dichloromethane was added and stirred to dissolve. The temperature was lowered to -5 to 0 °C. Sodium hypochlorite aqueous solution (15.8 g, 0.015 mol) was weighed and slowly added dropwise to the reaction system, maintaining the temperature below 0 °C. After the addition was complete, the mixture was stirred for 0.5 to 1.0 hours, and the mixture was allowed to stand for separation. The dichloromethane layer was concentrated under reduced pressure to remove the solvent, yielding compound II-2 (1.8 g) in 86.5% yield. The proton nuclear magnetic resonance spectral data of the resulting product are as follows:

[0042] 1 H NMR(400 MHz,CDCl3)δ4.68-4.56(m,1H),3.17(dd,J=11.2,6.7 Hz,1H),3.07(s,3H),2.95(s,3H),2.76- 2.63(m,1H),2.49(dd,J=11.7,4.9 Hz,1H),2.32(dd,J=13.4,5.5 Hz,1H),2.14-2.02(m,1H),2.02-1.91(m,1H),1.91-1.79(m,1H). 13 C NMR(101 MHz, CDCl3) d 205.55,173.40,63.68,37.21,37.06,35.27,34.27,33.94,27.86.

[0043] Example 6 Preparation of (1S,3R,4S)-4-amino-3-azidocyclohexane-1-carboxylic acid ethyl ester (I-1) [ka] Compound II-1 (5 g), PBS buffer (pH 7.0, 1 M, 50 mL), water (350 mL), PLP (2.6 mg, 5.3 mg / L), isopropylamine (100 mL, 50 g / L), and ATA101 (5.0 g) were added to a 500 mL reaction flask and reacted at 25-30 °C with mechanical stirring for 25-30 hours. After the reaction was completed, the mixture was filtered through diatomaceous earth, and the mother liquor was extracted with dichloromethane to obtain compound I-1 (4.3 g) in an 86.5% yield.

[0044] Example 7 Preparation of (1S,3R,4S)-4-amino-3-azido-N,N-dimethylcyclohexane-1-carboxamide (I-2) [ka] Compound II-2 (5 g), PBS buffer (pH 7.0, 1M, 50 mL), water (350 mL), PLP (2.6 mg, 5.3 mg / L), isopropylamine (50 mL, 100 g / L), and ATA101 (5.0 g) were added to a 500 mL reaction flask and reacted for 25-30 hours with mechanical stirring at 25-30 °C. After completion of the reaction, the mixture was filtered through diatomaceous earth, and the mother liquor was extracted with dichloromethane to obtain compound I-2. The proton nuclear magnetic resonance spectral data of the resulting product are as follows:

[0045] 1 H NMR(400 MHz,CDCl3)δ3.82(d,J=2.3 Hz,1H),2.99(s,3H),2.85(s,3H),2.79-2.63(m,2H),1.92(dd,J=14.3,2.7 Hz, 1H), 1.87-1.72 (m, 1H), 1.72-1.56 (m, 2H), 1.54-1.18 (m, 4H). 13 C NMR (101 MHz, CDCl3) δ174.62,64.49,52.00,37.08,35.53,33.66,32.15,30.05,27.55.

[0046] Example 8 Preparation of (1S,3R,4S)-3-azido-4-(tert-butoxycarbonyl)amino)cyclohexane-1-carboxylic acid ethyl ester (I-3) [ka] Compound I-1 (2.1 g, 10 mmol), BocO (2.6 g, 12 mmol), potassium carbonate (2.1 g, 15 mmol), and 50 mL of water were added all at once to a 100 mL three-neck flask, heated to 40-50°C, and stirred for 3-4 hours to react. A white solid precipitated and was filtered under reduced pressure to obtain 2.8 g of compound I-3 in a 90% yield.

[0047] Example 9 Preparation of ((1S,2R,4S)-2-azido-4-(dimethylcarbamoyl)cyclohexyl)carbamic acid tert-butyl ester (I-4) [ka] Compound I-2 (2.1 g, 10 mmol), BocO (2.6 g, 12 mmol), potassium carbonate (2.1 g, 15 mmol), and 50 mL of water were added to a 100 mL three-neck flask at once, heated to 40-50 °C, and stirred for 3-4 hours. A white solid precipitated and was filtered under reduced pressure to obtain 2.9 g of compound I-4 in 94% yield. The proton nuclear magnetic resonance spectral data of the resulting product are as follows:

[0048] 1 H NMR(400 MHz,CDCl3)δ4.87(d,J=7.2 Hz,1H),4.03(s,1H),3.54(s,1H),2.95(s,3H),2.82(s,3H),2.70(t,J=11.1 Hz,1H),1.85(dd,J=31.3,13.5 Hz, 2H), 1.66 (t, J=13.1 Hz, 2H), 1.57 - 1.40 (m, 2H), 1.33 (s, 9H). 13C NMR (101 MHz, CDCl3) δ174.20,154.89,79.42,61.16,50.94,37.00,35.48,33.51,31.83,28.24,27.41,26.40.

[0049] Example 10 Preparation of ((1S,2R,4S)-2-azido-4-(dimethylcarbamoyl)cyclohexyl)carbamic acid methyl ester (I-5) [ka] Compound I-2 (2.0 g) was added to 2-methyltetrahydrofuran (80 mL), sodium bicarbonate (1.6 g, 2.0 eq) was added, and the mixture was stirred at room temperature. Methyl chloroformate (1.2 g, 1.3 eq) was added, and the mixture was stirred for 2-3 hours. After the conversion of the raw materials was complete, the insoluble matter was removed by filtration. The filter cake was washed with 2-methyltetrahydrofuran, and the filtrate was separated. The organic phase was concentrated and recrystallized with PE / EA to give a white solid I-5 in 84% yield. The proton nuclear magnetic resonance spectral data of the resulting product are as follows:

[0050] 1 H NMR(400 MHz,CDCl3)δ4.87(d,J=8.5 Hz,1H),4.13(d,J=4.2 Hz,1H),3.69(s,4H),3.08(s,3H),2.96(s,3H),2.83(d,J=11.7,3.6 Hz, 1H), 2.09-1.92 (m, 2H), 1.84-1.75 (m, 2H), 1.67-1.45 (m, 2H).

[0051] Example 11 Preparation of ((1S,2R,4S)-2-azido-4-(dimethylcarbamoyl)cyclohexyl)carbamic acid benzyl ester (compound I-6) [ka] Compound I-2 (2.0 g) was added to 2-methyltetrahydrofuran (80 mL), sodium bicarbonate (1.6 g, 2.0 eq) was added, and the mixture was stirred at room temperature. Benzyl chloroformate (2.1 g, 1.3 eq) was added, and the mixture was stirred for 2-3 hours. After the conversion of the raw materials was complete, the insoluble matter was removed by filtration. The filter cake was washed with 2-methyltetrahydrofuran, and the filtrate was separated. The organic phase was concentrated and recrystallized with PE / EA to obtain a white solid I-6 in 86% yield. The proton nuclear magnetic resonance spectral data of the resulting product are as follows:

[0052] 1 H NMR(400 MHz,CDCl3)δ7.41-7.30(m,5H),5.12(s,2H),4.95(d,J=8.5 Hz,1H),4.14(s,1H),3.83-3.69(m,1H),3.08(s,3H),2.96(s,3H),2.90-2.75(m,1H),2.10-1.91(m,2H),1.81(d,J=12.6 Hz, 2H), 1.69 (s, 1H), 1.58-1.46 (m, 1H).

[0053] Example 12 Preparation of ((1R,2S,5S)-2-(((benzyloxy)carbonyl)amino)-5-(dimethylcarbamoyl)cyclohexyl)-14-azocarboxylic acid tert-butyl ester (V) [ka] Compound I-6 (1.05 g) was added to triphenylphosphine (2.4 g, 3.0 eq), tetrahydrofuran (30 mL), and deionized water (7 mL). After the reaction was completed, 1N HCl was added to adjust the pH to 2-3, and the mixture was concentrated to remove tetrahydrofuran. The aqueous phase was extracted with dichloromethane, retained, and adjusted to pH 8-9 by adding sodium hydroxide. Boc anhydride (1.0 g, 1.5 eq) was added and stirred overnight. The mixture was suction filtered and washed to obtain a white solid, V, in an 85% yield. The proton nuclear magnetic resonance spectral data of the resulting product are as follows:

[0054] 1 H NMR(400 MHz,cdcl3)δ7.38 - 7.26(m,5H),5.22(s,1H),5.09(t,J=10.3 Hz,2H),4.12(s,1H),3.70(s,1H),3.02(s,3H),2.92(s,3H),2.62(s,1H),1.99(d,J=37.6 Hz,2H),1.72(d,J=18.7 Hz,5H),1.43(s,9H)。

Claims

1. An edoxaban intermediate having a general structural formula represented by Formula I or Formula II. 【Chemistry 1】 【Chemistry 2】 (In the formula, R １ is OH, an alkoxy group, or an N,N-dialkylamino group, and R ２ is hydrogen, an alkoxycarbonyl group, or a tert-butoxycarbonyl group.

2. In the general structural formula, R １ is an N,N-dimethylamino or ethoxy group, and R ２ is hydrogen, tert-butoxycarbonyl, benzyloxycarbonyl, ethoxycarbonyl, benzyl or benzoyl; The edoxaban intermediate according to claim 1 .

3. Step 1) oxidizing a compound of formula III to obtain a compound of formula II; 【Transformation 3】 (wherein R is an alkoxy group or an N,N-dialkylamino group) and step 2) mixing the compound of formula II, transaminase (ATA101 provided by Shangke Bio (Shanghai) Co., Ltd.), transaminase coenzyme and phosphate buffer solution to perform an enzyme-catalyzed reaction or further an amine derivatization reaction, thereby obtaining the compound of formula I. The method for producing the edoxaban intermediate according to claim 1 or 2.

4. The oxidizing agent in step 1) is John's reagent, PCC reagent, or TEMPO reagent. The method for producing an edoxaban intermediate according to claim 3 .

5. In the system of step 2), the substrate concentration is 10 to 100 g / L, the transaminase is involved in the reaction in the form of one or a combination of wet cells and a liquid enzyme, and when the transaminase is added in the form of a liquid enzyme, the mass ratio of the liquid enzyme is 1 to 20%, the reaction temperature is 0 to 30°C, the reaction pH value is 6.5 to 7.0, and the reaction time is 12 to 30 hours. The method according to claim 3 .

6. In the step 2), the concentration of the compound of formula II is 10-100 g / L, the concentration of the transaminase is 5-20 g / L, the concentration of the transaminase coenzyme is 1-10 mg / L, and the concentration of the phosphate buffer is 10-100 mM. The method according to claim 3 .

7. After obtaining the amine product by the amine derivatization reaction in the step 2), the characteristic reaction of the amine functional group protecting group is to directly add a protecting group reagent to the reaction solution of the enzyme-catalyzed reaction, and then react. The method according to claim 3 .

8. The method further comprises a post-treatment step of adding a filter aid to the reaction solution after the reaction of step 2), and then filtering, extracting, concentrating, and crystallizing the solution to obtain the compound of formula I. The method according to claim 3 .

9. The filter aid is diatomaceous earth. The method according to claim 8 .

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